Exploring adamantine-like scaffolds for a wide range of therapeutic targets Elena Valverde Murillo Aquesta tesi doctoral est? subjecta a la llic?ncia Reconeixement- CompartIgual 3.0. Espanya de Creative Commons. Esta tesis doctoral est? sujeta a la licencia Reconocimiento - CompartirIgual 3.0. Espa?a de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution-ShareAlike 3.0. Spain License. UNIVERSITAT DE BARCELONA FACULTAT DE FARM?CIA EXPLORING ADAMANTANE?LIKE SCAFFOLDS FOR A WIDE RANGE OF THERAPEUTIC TARGETS Elena VALVERDE MURILLO 2015 UNIVERSITAT DE BARCELONA FACULTAT DE FARM?CIA PROGRAMA DE DOCTORAT de Qu?m ica Org?nica Experimental i Industrial EXPLORING ADAMANTANE?LIKE SCAFFOLDS FOR A WIDE RANGE OF THERAPEUTIC TARGETS Mem?ria presentada per Elena VALVERDE MURILLO per o ptar al t?tol de Doctor per la U niversitat de Barcelona Co - Director i Tutor: Dr. Santiago V?quez Cruz Co - Director: Dr. Manuel V?zquez Carrera Doctoranda: Elena Valverde Murillo Elena VALVERDE MURILLO 2015 A mi familia Al meu petit estimat ?La lluna ?s bella com tu, nineta meva Mira?m amb els teus ullets verds i jo ser? el teu sant que va cap al cel Vine amb mi, a veure la lluna brillant i blanca, com la teva ?nima Vine amb mi, amor meu, fes que et pugui estimar i besar cada dia? -?ngela Hitos Murillo- El treball experimental recollit en aquesta Mem?ria s?ha realitzat al Departament de Farmacologia i Qu?mica Terap?utica de la Facultat de Farm?cia de la Universitat de Barcelona. Aquest treball ha estat finan?at pel Ministerio de Ciencia e Innovaci?n (Projectes CTQ2008-03768, CTQ2011-22433 i SAF2014-57094-R). Per la realitzaci? de la present Tesi Doctoral he gaudit d?una beca predoctoral D?Ajuts de Tercer Cicle atorgada per l?Institut de Biomedicina de la Universitat de Barcelona (2011-2015). Agra?ments Desprps de moltes hores d?escriptura i llesta per enquadernar aquesta tesi, arriba un dels moments m?s esperats perz Tue a l?hora resulta el mps diItcil d?escriure 6yn moltes les persones les Tue han contribuwt d?alguna manera en la realitzaciy d?aTuesta tesi directament o indirecta i des d?aTuestes pjgines voldria e[pressar el meu mps sincer agra?ment a totes elles, i a les que segur se?m passen d?anomenar. Primero de todo, a mi co-director y tutor de tesis el Dr. Santiago V?zquez Cruz, Professor agregat del Departament de Farmacologia i Qu?mica Terap?utica. Ha sido largo el viaje desde que me abriste las puertas del laboratorio hace ya m?s de 8 a?os. Tu dedicaci?n y pasi?n por la qu?mica, as? como la buena persona que eres, me han empujado siempre a ser mejor y seguir aprendiendo. Muchas gracias por haber pensado en m? al escoger estudiante para la beca predoctoral, por estar siempre dispuesto a escucharme y ayudarme en lo necesario, y por haberme dado la suficiente libertad a la hora de llevar a cabo la tesis. Se me va hacer raro eso de que no seas mi jefe a partir de ahora, pero aun as? espero que sigas siendo mi consejero y amigo. Gracias Santi! En segona posici?, per? no per aix? menys important, al meu co-director de tesi el Dr. Manel V?zquez Carrera, Professor titular del Departament de Farmacologia i Qu?mica Terap?utica. Ha estat m?s curt el temps que he estat al teu laboratori, per? suficient per veure el gran professional i encara millor persona que ets. Gr?cies per ajudar-me quan ho he necessitat, i per acceptar-me en el teu grup de treball sense pr?cticament con?ixer-me. Et desitjo molta sort a tu i a la teva fam?lia. Quisiera agradecer tambi?n al Dr. Pelayo Camps, Professor em?rit del Departament de Farmacologia i Qu?mica Terap?utica. Gracias por acogerme en su grupo de investigaci?n y por poner toques de cordura cuando ?sta faltaba en el laboratorio. Voldria agrair al grup de cient?fics que han col?laborat en aquest treball. Al Dr. Francesc X. Sureda, de la Universitat Rovira i Virgili (Reus), per la determinaci? de l?activitat antagonista contra el receptor de l?10DA dels compostos sintetitzats Al Dr David Soto, de la Universitat de Barcelona, pels estudis electrofisiol?gics i per explicar-me amb molta paci?ncia els seus experiments. Al grup de recerca del Dr. Scott Webster, de la University of Edinburgh (Regne Unit), per la determinaci? de la inhibici? dels nostres compostos contra l?enzim 11?-HSD1. Al grup de recerca del Professor Dr. Francisco J. Luque, per la realitzaci? dels c?lculs de docking i din?miques moleculars. Al grup de recerca del Professor Dr. Bruce Hammock i al Dr. Christophe Morisseau pels seus consells en quant a la realitzaci? dels assajos enzim?tics i de solubilitat. A l?Institut de Biomedicina de la Universitat de Barcelona i especialment a la Judit Agust?, per l?ajuda prestada en Tuant a la beca predoctoral. Tamb?, a la M? ?ngels Barcel? de la 6ecretaria d?(studiants i Docqncia de la )acultat de )armjcia per la paciqncia mostrada durant el dip?sit de la tesi. Al personal Cient?fico-T?cnic de la Universitat de Barcelona, concretament a la Dra. Ana Linares, a la M? Ant?nia Molins i a la Vicky Mu?oz-Torrero de la Unitat de RMN, per la realitzaci? dels espectres de RMN, i a la Dra. Asunci?n Mar?n i a la Laura Ortiz per la realitzaci? dels espectres de masses. Tamb? a la Pilar Dom?nech, del Servei de Microan?lisi del Centre d?Investigaciy i Desenvolupament (CID C6IC de Barcelona per la realitzaciy dels an?lisis elementals. Al Dr. Diego Mu?oz-Torrero, Professor titular del Departament de Farmacologia i Qu?mica Terap?utica, por su amabilidad y cordialidad, y su disposici?n de aconsejar a los doctorandos cuando es necesario. A la Dra. Carmen Escolano por amenizar los mediod?as, por ser una gran anfitriona en sus fiestas de la piscina, y por prestarme su ayuda en el HPLC. A la Maite, per la seva efic?cia en el tema administratiu i per resoldre?ns els dubtes de paperassa. A la Pilar per mantenir el laboratori net i polit i pels ?bon dies? tan agradables cada mat?. A Javier por llenar el agua destilada, llevar la muestras al PCB, CSIC y qu?micas, realizar los pedidos y hacer los partes. A Armando por ser el manitas m?s eficiente del laboratorio, y probablemente de toda la facultad. Y al Josep Gald?n per mantenir la facultat en ordre i estar sempre alerta. A tota la gent que ha passat i estat pel laboratori de Qu?mica Farmac?utica durant aquest temps i que sense ells no hagu?s estat lo mateix. Als que considero els mestres del lab, que han ensenyat a les generacions venidores les ?bones? practiTues de laboratori A Loli, porque fuiste t? quien me ense?? c?mo trabajar en el laboratorio de la mejor forma posible. A l?(va per ser un e[emple a seguir de constjncia i per les teves ganes contagioses de saber-ne m?s i m?s. Ara tens un nou repte, el petit Marc, segur que ho far?s incre?blement b?! A la T?nia, pel teu saber fer i per ajudar-nos als m?s despistats en la recerca de material i reactius A l?(li per ensenyar que el temps ?s relatiu i que es pot treballar molt m?s r?pid que la resta. Al Carles, per confirmar que el temps ?s relatiu i que el dia d?na per molt, per parlar, cotillejar, ensenyar i, tamb?, treballar ;). Als dos, per rebre?m tan bp a Dundee i per acollir-me a Cambridge al vostre mega-super sof?! Als que les nostres tesis han coincidit m?s temps i amb els que tamb? hem compartit moments molt divertits A l?Ane per haver sigut la meva primera cordada a escalada (te?n recordes quins nervis?) i per haver-te convertit amb el temps en una gran companya. A la Rosana, per la gran hard-worker que ets, pel teu inter?s en la recerca del grup i pels moments on compart?em vitrina. Al David, per les converses tan interessants que hem tingut i per ser el creador del Daily Lab. A la Irene, per rebre?m a 0echelen i per les rialles tan divertides que hem tingut des de la carrera. A Javi, por haber introducido palabras desconocidas por entonces en nuestro vocabulario, y que ahora son tan ?tiles. Ahora que eres mi relevo en el piso, cuida de mi habitaci?n ;). A Ornella, por sus risas incontroladas y por compartir habitaci?n en Valencia (qu? tiempos aquellos!). A la Marta B., per descobrir-me grups de m?sica que desconeixia. A l?Arnau per tot el Tue hem aprps amb tu quan estaves amb nosaltres. A la gent que ha estat de pas pel laboratori, per? que ha deixat empremta. Al Toni, per compartir gustos musicals i per la teva perseveran?a en les curses. A l?Albert per recolzar- me en discussions pol?tiques i ser tan eixerit. A l?(nric per ensen\ar-me a ensenyar i pels teus riures i converses. A la Marta F., per acollir-nos a Stevenage i per les teves classes de ball. A M? Eugenia, por ser como sos, tan divertida y alegre, y por ense?arnos parte de C?rdoba que desconoc?a. Al Ra?l, pel bon rotllo que desprens. A Juanlo y Lorena, por los consejos de qu?mica y por el buen ambiente que hac?ais en el lab. Encara que s?n molts m?s els que han passat per les nostres vitrines i que han fet que els dies fossin m?s amens: Marta M., Maria, Deborah, Salva, Jordi B., Agn?s, Ester, Natalia, Mattia, Al?xia, Daniela, Alicja, i molts que segur em deixo. A les noves incorporacions, Sandra, Andreaa, Eug?nia i Katia, molta sort i paci?ncia en aquesta nova etapa. A mis compa?eros del laboratorio de Farmacolog?a, Tue aunTue mi visita ?abajo? ha sido fugaz, he podido conocer a gente que merece mucho la pena. En especial, a Emma B., mi maestra y una gran persona. Gracias por haberme ense?ado tan bien y con tanta paciencia, pero sobre todo por las conversaciones y risas compartidas. S? que me llevo una amiga del laboratorio 6 ;). A Gaia, por tus clases de cultivos y tus regalitos de USA. A Mohammad y Xevi, por haberme ayudado cuando me hac?a falta. Al resto de compa?eros del laboratorio, por las conversaciones espontaneas que alegraban el d?a. A Silvia y a Mar, por los apoyos t?cnicos y log?sticos. A la gente del laboratorio de Qu?mica Org?nica, por el intercambio de reactivos y de material, y por las birras y salidas en grupo. En particular, a S?nia (gr?cies per l?ajuda amb l?+3/C  a *uillaume (merci pour ta compagnie ? des conf?r?nces), a Elena, a Francesco, a Alex, a Juan Andr?s, a Claudio, a Celeste, a Guilhem y a Caroline. A los compa?eros de master, sobre todo con los que compartimos casa en Valencia y horas fat?dicas de estudio. Gracias a Manu, Alberto y Fabrizio. I would like to thank the people I did my placement with in GSK, especially to Dr. Simon MacDonald and John Pritchard for welcoming me in their team. Also, to Dr. Niall Anderson, who was really helpful and kind to me, I really appreciate what you taught me. Thanks also to Ana?s, without you that year would have been really different, to Katharina for our exchange trips, and to Richard, for your Afrikaans and Khoisan?s classes. I would also like to thank Dr. Allan Watson, my supervisor in Glasgow and lab mate in GSK. I am really happy for how your group is expanding, and to have been part of it even it was for a short period of time. I am also thankful to my lab mates in Scotland, for welcoming me and showing me the Scottish lifestyle, and in particular to Carolina for the climbing evenings and for your visit to Barcelona. A mis compa?eros de piso Irene, Sonia, David y Ana por alegrarme las tardes-noches despu?s de un duro d?a de trabajo. Y en especial a H?ctor, por las risas, las imitaciones y el dise?o de la portada! Als meus companys de viatges filipins, la Maria, la Luc?a el 3edro el -on l?Anna i l?Anaht pels moments d?evasiy Tue he tingut amb vosaltres per les hist?ries tan divertides Tue hem viscut junts i Tue segur no oblidarem A en 0acedo n?(lisa en 0arc i n?Aina per acollir-nos de luxe al vostre santuari menorqu?. A mis amigas de siempre Sara, Melania y Clara, como decimos nosotras, por las historias acumuladas, por estar dispuestas a escuchar mis aventuras, y por intentar entender qu? hago realmente. A l?(mma ( pels ~ltims an\s de carrera juntes i per la teva visita a Esc?cia (i tamb? al nostre beb? ;) ). A mi familia argentina-andorrana, por ser tan buen anfitriones cuando os he visitado, y por haberme aceptado como miembro de vuestra familia. A los animales de la familia, y con esto me refiero a Bolo, a Miel y a Joy, por su compa??a y su amor devoto. A mis padres Jos? y ?ngela, por ser tan luchadores, por vuestros sabios consejos y por preocuparos por m? en todo momento. A mi tata Lidia, por ser tan divertida, por cuidarme tanto desde peque?ita y por sacrificarse como lo hace. A mis t?os Lidia, Antonio y Milagros, y mis primas Irene y ?ngela, por vuestro apoyo incondicional. Gracias tambi?n a mi familia de Barcelona. A todos vosotros por quererme tal como soy, por aceptar sin rechistar mis locas decisiones y por estar dispuestos a ayudarme siempre. Yo tambi?n lo estoy, os quiero! I finalment, al Mat?as, per dedicar-te tant a mi, a cuidar-me i a treure?m un somriure de la cara sempre que pots i vols. Moltes gr?cies per haver llegit i corregit la tesi, que no ha sigut tasca f?cil. I com diu aquella can??, meu riso ? t?o feliz contigo, o meu melhor amigo ? o meu amor. RESUL TS The present thesis has led to the following scientific publications, symposium communications and awards: Scientific publications: 1. Valverde, E.; Barroso, E.; V?zquez-Carrera, M.; V?zquez, S. Scaffold-hopping for new soluble epoxide hydrolase with improved lipophilic ligand efficiency. Scientific paper, writing in progress. 2. V?zquez, S.; Leiva, R.; Valverde, E. New inhibitors of the 11?-HSD1 with potential use in therapeutics. European patent application, writing in progress. 3. Valverde, E.; Seira, C.; Bidon-Chanal, A.; McBride, A.; Luque, F. J.; Webster, S. P.; V?zquez, S. Searching for novel applications of the benzohomoadamantane scaffold in medicinal chemistry: synthesis of new 11?-HSD1 inhibitors. Bioorganic and Medicinal Chemistry 2015 , accepted. 4. V?zquez, S.; Valverde, E.; V?zquez-Carrera, M. Analogues of adamantyl ureas as soluble epoxide hydrolase inhibitors. European patent application, EP15178618.3. 5. Barniol-Xicota, M.; Escandell, A.; Valverde, E.; Juli?n, E.; Torrents, E.; V?zquez, S. Antibacterial activity of novel benzopolycyclic amines. Bioorganic and Medicinal Chemistry 2015 , 23, 290-293. 6. Valverde, E.; Sureda, F. X.; V?zquez, S. Novel benzopolycyclic amines with NMDA receptor antagonist activity. Bioorganic and Medicinal Chemistry 2014 , 22, 2678-2683. Symposium communications: 1. Valverde, E.; Barroso, E.; V?zquez-Carrera, M.; V?zquez, S. Novel soluble epoxide hydrolase inhibitors with enhanced lipophilic ligand efficiency via scaffold-hopping approaches. XXXV Reuni?n Bienal de la Real Sociedad Espa?ola de Qu?mica (RSEQ), A Coru?a (Spain), Flash communication, 2015 . 2. Valverde, E.; Seira, C.; Bidon-Chanal, A.; McBride, A.; Luque, F. J.; Webster, S. P.; V?zquez, S. A quest for further uses of adamantane-like scaffolds in medicinal chemistry: discovery of new 11?-HSD1 inhibitors. 14th Belgian Organic Synthesis Symposium, Louvain-la-Neuve (Belgium), Poster, 2014 . 3. Valverde, E.; Seira, C.; Bidon-Chanal, A.; McBride, A.; Luque, F. J.; Webster, S. P.; V?zquez, S. Further applications of a novel scaffold as an adamantane surrogate: synthesis of new 11?-HSD1 inhibitors. I Simposio de J?venes Investigadores de la Sociedad Espa?ola de Qu?mica Terap?utica, Madrid (Spain), Poster, 2014 . 4. V?zquez, S.; Valverde, E.; Torres, E.; Sureda, F. X. Novel analogues of memantine with potent activity as glutamate N-methyl-D-aspartate receptor antagonists. XVIIth National 0eeting on ?Advances in Drug Discover\ 6uccesses, Trends, and Future Challenges, Madrid (Spain), Poster, 2013 . 5. Valverde, E.; Lafuente, M.; Sureda, F. X.; V?zquez, S. New benzopolycyclic cage amines with NMDA receptor antagonist activity. XXXIV Reuni?n Bienal de la Real Sociedad Espa?ola de Qu?mica, Santander (Spain), Flash communication, 2013 . 6. V?zquez, S.; Duque, M. D.; Torres, E.; Valverde, E.; Lafuente, M.; Sureda, F. X. New benzopolycyclic cage amines with NMDA receptor antagonist activity. VI Mediterranean Organic Chemistry Meeting, Granada (Spain), Poster, 2013 . Awards: 1. Valverde, E.; V?zquez-Carrera, M.; V?zquez, S. Exploring adamantane-like scaffolds for a wide range of therapeutic targets. )inalist oI the (li /ill\?s aZards Ior the e[cellence in the research of Ph.D. students, Lilly, Alcobendas (Spain), Poster, 2015 . Research stays: 1. Development of new methods for organic synthesis based on control of boron solution speciation. Dr. Allan J. B :atson?s group Department oI 3ure and Applied Chemistry, University of Strathclyde, Glasgow (United Kingdom), September- December 2014 . Other scientific publications: 1. Fyfe, J. W. B.; Valverde, E.; Seath, C. P.; Kennedy, A. R.; Redmond, J. M.; Anderson, N. A.; Watson, A. J. B. Speciation control during Suzuki-Miyaura cross- coupling of haloaryl and haloalkenyl MIDA boronic esters. Chemistry, a European Journal 2015 , 21, 8951-8964. 2. Anderson, N. A.; Fallon, B. J.; Valverde, E.; MacDonald, S. J. F.; Pritchard, J. M.; Suckling, C. J.; Watson, A. J. B. Asymmetric rhodium-catalysed addition of arylboronic acids to acyclic unsaturated esters containing a basic ?-amino group. Synlett 2012 , 23, 2817- 2821. 3. Valverde, E.; Torres, E.; Guardiola, S.; Naesens, L.; V?zquez, S. Synthesis and antiviral evaluation of bisnoradamantane sulfites and related compounds. Medicinal Chemistry 2011 , 7, 135-140. 4. Duque, M. D.; Torres, E.; Valverde, E.; Barniol, M.; Guardiola, S.; Rey, M.; V?zquez, S. Inhibitors of the M2 channel of influenza A virus. Recent Advances in Pharmaceutical Sciences 2011 , 35-64 ISBN: 978-81-7895-528-5, book chapter. 5. Duque, M. D.; Camps, P.; Torres, E.; Valverde, E.; Sureda, F. X.; L?pez-Querol, M.; Camins, A.; Radhika Prathalingam, S.; Kelly, J. M.; V?zquez, S. New oxapolycyclic cage amines with NMDA receptor antagonist and trypanocidal activities. Bioorganic and Medicinal Chemistry 2010 , 18, 46-57. Abbreviation list ACU: N -adamantyl-1?-cyclohexylurea AD: Alzheimer?s disease ADME: Absorption, Distribution, Metabolism and Excretion process ADMET: Absorption, Distribution, Metabolism, Excretion and Toxicology AEPU: N -adamantanyl-1?-(5-(2-(2- ethoxyethoxy)ethoxy)pentyl)urea AIBN: azobisisobutyronitrile AMCU: 1-adamantan-1-yl-3-(4-(3- morpholinopropoxy)cyclohexyl)urea AMPA: ?-amino-3-hydroxy -5-methyl-4- isoxazolepropionic acid APAU: N -(1-acetylpiperidin-4-yl)-1?- adamantylurea API: active pharmaceutical ingredient ARA: arachidonic acid ATF3: activating transcription factor 3 ATF4: activating transcription factor 4 ATF6: activating transcription factor 6 AUDA: N -adamantanyl-1?-dodecanoic acid urea AUSM: 4-(3-adamantan-1-yl-ureido)-2- hydroxyl -benzoic acid methyl ester BBB: blood-brain barrier BiP (or GRP78): immunoglobulin-heavy- chain-binding protein BSA: bovine serum albumin cAMP: cyclic adenosine monophosphate CDI: N, 1?-carbonyldiimidazole CGC: cerebellar granule cells CHOP (or GADD153): CCAAT enhancer-binding protein (C/EBP) homologous protein CNS: central nervous system COX: cyclooxygenase CT: control CV: cardiovascular CYP450: cytochrome P -450 DAST: (diethylamino)sulfur trifluoride DCE: 1,2-dichloroethane DCM: dichloromethane DCU: 11?-dicyclohexylurea DHET: dihydroeicosatrienoic acid DIPEA: 11?-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMF: dimethylformamide DMSO: dimethylsulfoxide DNA: deoxyribonucleic acid DPPA: diphenylphosphoryl azide DPP-IV: dipeptidyl peptidase IV DTBP: di-tert-butyl peroxide EDC: 1-ethyl-3-(?- dimethylaminopropyl)carbodiimide EDG: electron-donating group EET: epoxyeicosatrienoid acid eIF2: eukaryotic translation initiation factor 2 EMA: European Medicines Agency ER: endoplasmic reticulum ERAD: ER-associated degradation EWG: electron-withdrawing group FDA: Food & Drugs Administration Fura-2 AM: Fura-2-acetoxymethyl ester GABA: ?-aminobutiric acid GAPDH: glyceraldehyde 3 -phosphate dehydrogenase GC: glucocorticoid GPCR: G protein-coupled receptor GR: glucocorticoid receptor GSK: GlaxoSmithKline HATU: 1- [ bis(dimethylamino)methylene] -1H -1,2,3- triazolo[4,5 -b]pyridinium 3 -oxid hexafluorophosphate HDL: high-density lipoprotein hERG: human Ether-? -go-go-Related Gene HMPA: hexamethylphosphoramide HOAt: 7-aza-1-hydroxybenzotriazole HOBt: h ydroxybenzotriazole HPA: hypothalamic-pituitary-adrenal HPLC: high performance liquid chromatography HRMS: high resolution -mass spectrometry 11?-HSD1 : 11?-hydroxysteroid dehydrogenase type 1 11?-HSD1: 11?-hydroxysteroid dehydrogenase type 2 17?-HSD : 17?-hydroxysteroid dehydrogenase HTS: high-throughput screening IC50: half-maximal inhibitory concentration IKK: inhibitor of ?B kinase IR: infrared IRE1: inositol-requiring enzyme 1 JNK: c-Jun amino-terminal kinase LC-MS: liquid chromatography-mass spectrometry LDA: lithim diisopropylamide LE: ligand efficiency LHS: left -hand side LipE: lipophilic ligand efficiency LKT: leukotriene LOX: lipoxygenase MD: molecular dynamics mEH: microsomal epoxide hydrolase MetS: metabolic syndrome mp: melting point MR: mineralocorticoid receptor mRNA: messenger ribonucleic acid MS: mass spectrometry MW: molecular weight NADPH : nicotinamide adenine dinucleotide phosphate ND: not determined NEPC: 4-nitrophenyl-trans-2,3-epoxy -3- phenylpropyl carbonate NF-?B: nuclear factor-?B NMDA: N -methyl-D-aspartic acid NMDAR: N -methyl-D-aspartic acid receptor NMR: nuclear magnetic resonance NSAID: nonsteroidal anti-inflammatory drug PAL: palmitate PBA: 4-phenyl butyric acid PCR: polymerase chain reaction PD: pharmacodynamics PDB: Protein Data Bank PERK: PKR-like ER-regulated kinase PG: prostaglandin PHOME: (3-phenyl-oxiranyl) -acetic acid cyano-(6-methoxy -naphthalen-2-yl)-methyl ester PK: pharmacokinetic PP: primary pharmacophore PPAR: peroxisome proliferator -activated receptor PSA: polar surface area PVT: polyvinyl toluene RHS: right -hand side ROS: reactive oxygen species RT: room temperature S: solubility SAR: structure-activity relationship SD: Standard deviation SEM: Standard error of the mean SET: single electron transfer sEH: soluble epoxide hydrolase SP: secondary pharmacophore SPA: scintillation proximity assay t-AUCB: trans-4-[4 -(3-adamantan-1- ylureido)cyclohexyloxy]benzoic acid T2DM: type 2 Diabetes Mellitus t-DPPO: trans-diphenyl-propene oxide TEA: triethylamine TFA: trifluoroacetic acid TFAA: trifluoroacetic anhydride THF: tetrahydrofuran TNF-?: tumor necrosis factor-? TP: tertiary pharmacophore t-SO: trans-stilbene oxide UPR: unfolded protein response UV: ultraviolet vs: versus WHO: World Health Organization XBP1: X-box -binding protein 1 ?W: microwave apparatus INDEX Index 2 4 3 INTRODUCTION 1. Adamantane: the precious nucleus 3 1.1 Origin of adamantane and first synthesis 3 12 Adamantane?s ph\sicochemical properties and its multidimensional value 6 1.2.1 Physicochemical properties 6 1.2.2 Adamantane ring as a pharmacophore 8 1.2.3 ADME properties of adamantane-containing compounds 8 1.3 Medicinal chemistry of adamantane: the promiscuous lipophilic pellet 10 1.3.1 Clinically approved adamantane-based drugs 10 1.3.2 Adamantane-containing candidates in development 13 1.4 Not all that glitters is gold: a moot point of perfection 16 1.4.1 High lipophilicity compromises PK properties 16 1.4.2 Ready access to intermediates that prevent scaffold optimization 19 1.4.3 Adamantane group as an imperfect space-filling pharmacophore 20 1.5 Adamantane alternatives: previous work of the group 20 CHAPTER 1: NMDA receptor antagonism INTRODUCTION 1. NMDA receptor antagonism by adamantane-like scaffolds 29 1.1 The glutamatergic neurotransmitter system 29 1.1.1 NMDA receptor and its physiological function 29 1.1.2 Glutamate and related pathological states 31 1.2 NMDA as a therapeutic target 33 1.2.1 Competitive NMDAR antagonists 33 2 4 4 Index 1.2.2 Uncompetitive NMDAR antagonists 34 1.2.3 Non-competitive NMDAR antagonists 36 1.3 The neuroprotector memantine 37 1.4 Previous work of the group: new antagonists of the NMDAR 40 OBJECTIVES 45 RESULTS & DISCUSSION 1. Effect of the C-9 substitution in new benzopolycyclic compounds 49 1.1 Synthesis of the C9-demethylated compound, 23 49 1.2 Exploration of the C -9 substitution of the benzo-homoadamantane scaffold 67 2. Pharmacological evaluation of new benzo-homoadamantane derivatives 73 2.1 Assessment of the NMDAR antagonistic activity 73 2.2 Electrophysiological measurements 77 CONCLUSIONS 83 CHAPTER 2: 11?-HSD1 inhibition INTRODUCTION 1. 11?-HSD1 inhibition by adamantane-based derivatives 89 1.1 The glucocorticoid system and its physiological actions 89 1.1.1 GC regulation by 11?-HSD enzymes 89 1.2 11?-HSD1 as a pleiotropic therapeutical target 91 1.2.1 11?-HSD1 inhibition and type 2 diabetes mellitus, obesity and metabolic syndrome 93 1.2.2 11?-HSD1 inhibition and inflammation 96 1.2.2 11?-HSD1 inhibition and cognitive dysfunction with aging 97 1.2.3 11?-HSD1 inhibition for other diseases 99 1.3 Crystal structure of 11?-HSD1 and its binding site 100 1.4 11?-HSD1 inhibitors in development 102 Index 2 4 5 1.4.1 Non-selective 11?-HSD1 inhibitors as tools 103 1.4.2 Studies with selective 11?-HSD1 inhibitors 104 1.4.3 Adamantane-based inhibitors of 11?-HSD1 107 OBJECTIVES 113 RESULTS & DISCUSSION 1. Application of the benzopolycyclic scaffold as an adamantane analogue for 11?-HSD1 inhibition 117 1.1 Synthesis of the C-9 substituted 6,7,8,9,10,11- hexahydro -5,7:9,11-dimethano-5H-benzocyclononen-7-yl derivatives 117 1.1.1 Preparation of amide compounds 120 1.1.2 Synthesis of the thiazolone scaffold 128 1.1.3 Urea group formation 129 1.2 Pharmacological evaluation of the C-9 substituted 6,7,8,9,10,11-hexahydro -5,7:9,11-dimethano-5H- benzocyclononen-7-yl derivatives 130 1.3 Synthesis of the fluoro-benzohomoadamantane derivative of PF-877423 134 1.4 Pharmacological evaluation of the fluoro-benzohomoadamantane derivative of PF-877423 135 1.5 Computational studies of the first family of 11?-HSD1 inhibitors 136 2. Application of the hexacyclic scaffold as an adamantane analogue in 11?-HSD1 inhibitors 138 2.1 Synthesis of the 3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15]pentadecane derivatives 138 2.2 Pharmacological evaluation and computational studies of the 3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15 ]pentadecane derivatives 146 CONCLUSIONS 153 CHAPTER 3: sEH inhibition INTRODUCTION 2 4 6 Index 1. sEH inhibition by adamantane-based derivatives 159 1.1 Epoxyeicosatrienoic acids and their biological role 159 1.1.1 Oxidative pathways of arachidonic acid metabolism 159 1.1.2 Biological relevance of EETs and importance of the sEH management 160 1.2 Targeting sEH: an overview of its pharmacology 162 1.2.1 Regulation of inflammation by sEH inhibitors 162 1.2.2 Effects of sEH inhibitors on pain 164 1.2.3 sEH and cardiovascular disease 164 1.2.4 Role of sEH in the development of diabe tes and metabolic syndrome. Involvement of ER stress 165 1.2.5 sEH inhibition for other clinical applications 166 1.3 sEH: crystal structure, catalytic mechanism, tissue expression and regulation 167 1.4 Discovery of sEH inhibitors 169 OBJECTIVES 179 RESULTS & DISCUSSION 1. Scaffold-hopping approach for the development of new sEH inhibitors 183 1.1 Synthesis of N-adamantane-like-N?-(2,3,4-trifluorophenyl)ureas 186 1.1.1 Preparation of intermediate scaffolds 187 1.1.2 Preparation of pentacyclic urea 157 197 1.1.3 Synthesis of final ureas 170-183 201 1.2 Pharmacological evaluation of final ureas. IC50 determination 202 1.2.1 A little of background 202 1.2.2 Tune-up of the screening assay 205 1.2.3 IC50 determination of ureas 157 and 170-183 208 1.3 Water solubility determination 211 Index 2 4 7 1.4 Lipophilic ligand efficiency: a new metric for drug discovery 216 2. New ureas with selected scaffolds 220 2.1 Synthesis of new ureas with selected scaffolds 221 2.2 Pharmacological evaluation of new ureas with selected scaffolds 226 2.3 Water solubility, melting points and LipE 229 3. ER stress amelioration with selected sEH inhibitors. In vitro studies 231 CONCLUSIONS 241 MATERIALS & METHODS General methods 245 CHAPTER 1: NMDA receptor antagonism o Preparation of 5,6,8,9-tetrahydro-5,9-propanebenzocycloheptane- 7,11-dione, 27 and 7,11-epoxi -6,7,8,9-tetrahydro-5,9- propane-5H-benzocycloheptane-7,11-diol, 28 249 o Preparation of 5,6,8,9-tetrahydro-5,9-propanebenzocycloheptane-11- ene-7-one, 25 249 o Preparation of 2-chloro-N-(9-hydroxy -5,6,8,9,10,11- hexahydro -7H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)acetamide, 34 250 o Preparation of 9-amino-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-ol hydrochloride, 46?HCl 251 o Preparation of 9-(dimethylamino)-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-ol hydrochloride, 50?HCl 252 o Preparation of 9-bromo-5,6,8,9,10,11- hexahydro -7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine, 47 253 o Preparation of 5,6,8,9,10,11-hexahydro -7H-5,9:7,11- dimethanobenzo[9]annulen -7-amine hydrochloride, 23?HCl 254 o Preparation of N,N-dimethyl-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine hydrochloride, 24?HCl 255 o Preparation of 2-chloro-N-(9-chloro-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)acetamide, 40 256 o Preparation of 9-chloro-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine hydrochloride, 42?HCl 257 o Preparation of N,N-dimethyl-9-chloro-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine hydrochloride, 49?HCl 258 o Preparation of 9-fluoro-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine hydrochloride, 48?HCl 259 2 4 8 Index o Preparation of 9-fluoro-N,N-dimethyl-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amine hydrochloride, 51?HCl 260 o Preparation of 9-methoxy -5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-ol, 53 261 o Preparation of 2-chloro-N-(9-methoxy -5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)acetamide, 54 262 o Preparation of 9-methoxy -5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]an nulen-7-amine, 52 263 CHAPTER 2: 11?-HSD1 inhibition 1. Benzo-homoadamantane scaffolds o Preparation of 5,6,8,9-tetrahydro-5,9-propanebenzocycloheptane- 7,11-diene, 29 269 o Preparation of N-(6,7,8,9,10,11-hexahydro -9-methyl- 5,7:9,11-dimethano-5H-benzocyclononen-7-yl)chloroacetamide, 55 269 o Preparation of 6,7,8,9,10,11-hexahydro -9-methyl- 5,7:9,11-dimethano-5H-benzocyclononen-7-amine, 19 270 o Preparation of (2R)-tert-butyl 2-[(9 -hydroxy -6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)carbamoyl]pyrrolidine -1- carboxylate, 71 270 o Preparation of (2R)-N-(9-hydroxy -6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2- carboxamide, 72 271 o Preparation of (2R)-1-ethyl-N-(9-hydroxy -6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2- carboxamide, 56 272 o Preparation of (2R)-tert-butyl-2-[(9 -methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)carbamoyl]pyr rolidine-1- carboxylate, 73 273 o Preparation of (2R)-N-(9-methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2- carboxamide, 74 274 o Preparation of (2R)-1-ethyl-N-(9-methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2-carboxamide, 57?tartrate 275 o Preparation of 4-amino-3,5-dichloro-N-(9-hydroxy -6,7,8,9,10,11- hexahydro -5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)benzamide, 58 277 o Preparation of 4-amino-3,5-dichloro-N-(9-methyl-6,7,8,9,10,11- hexahydro -5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)benzamide, 59 278 o Preparation of N-(9-methyl-6,7,8,9,10,11-hexahydro - Index 2 4 9 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)cyclohexanecarboxamide, 60 279 o Preparation of N-(9-methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)thiourea, 76 280 o Preparation of 5,5-dimethyl-2-[(9 -methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)amino]thiazol -4(5H) -one, 61 281 o Preparation of N-(9-hydroxy -6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)piperidine-1- carboxamide, 62 282 o Preparation of N-(9-methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)piperidine-1- carboxamide, 63 283 o Preparation of N-(9-fluoro-5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)piperidine-1- carboxamide, 64 284 o Preparation of N-(9-methoxy -5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)piperidine-1- carboxamide, 65 285 o Preparation of (2R)-tert-butyl-2-[(9 -fluoro-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)carbamoyl]pyrrolidine - 1-carboxylate, 78 286 o Preparation of (2R)-N-(9-fluoro-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2- carboxamide, 79 287 o Preparation of (2R)-1-ethyl-N-(9-fluoro-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)pyrrolidine-2-carboxamide, 77?tartrate 287 2. Hexacyclic scaffolds o Preparation of dimethyl pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca - 5,11-diene-8,9-dicarboxylate , 82 291 o Preparation of pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca -5,11- diene-8,9-dicarboxylic acid , 88 292 o Preparation of 3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15]pentadeca - 7,13-diene-2,4-dione, 89 293 o Preparation of 3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15]pentadeca - 7,13-diene, 90 293 o Preparation of 3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15] pentadecane hydrochloride, 95?HCl 294 o Preparation of tert-butyl (R)-2-(3-azahexacyclo [7.6.0.0 1,5.05,12.06,10.011,15] -pentadecane-3-carbonyl)pyrrolidine-1- carboxylate , 96 294 2 5 0 Index o Preparation of 3-[ (2R)-prolyl] -3- azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15 ]pentadecane , 97 295 o Preparation of 3-[(2 R)-N-ethylprolyl] -3- azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15 ]pentadecane , 98?tartrate 295 o Preparation of (4-amino-3,5-dichlorophenyl) (3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15 ]pentadeca -7,13-dien-3-yl) methanone, 99 296 o Preparation of (4-amino-3,5-dichlorophenyl) (3- azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15 ]pentadecan -3-yl) methanone, 100 297 o Preparation of (3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15] pentadeca-7,13-dien-3-yl)(cyclohexyl)methanone , 101 298 o Preparation of (3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15] pentadecane-3-yl)(cyclohexyl)methanone , 102 299 o Preparation of (3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15] pentadeca-7,13-diene-3-yl)(piperidin-1-yl)methanone, 103 300 o Preparation of (3-azahexacyclo[7.6.0.0 1,5.05,12.06,10.011,15] pentadeca -3-yl)(piperidin-1-yl)methanone, 104 301 CHAPTER 3: sEH inhibition 1. Chemistry o Preparation of N-benzyl(2-oxaadamant -1-yl)amine hydrochloride, 126?HCl 307 o Preparation of (2-oxaadamant -1-yl)amine hydrochloride, 120?HCl 307 o Preparation of N-benzyl(4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11 ] dodec-3-yl)amine, 129 308 o Preparation of 4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11 ] dodec-3-yl)amine hydrochloride, 121?HCl 308 o Preparation of tetramethyl (1?,4?,5?,8?)-3,7-hydroxy -bicyclo [3.3.0]octa -2,6-diene-2,4,6,8-tetracarboxylate , 135 309 o Preparation of cis-bicyclo[3.3.0]octane -3,7-dione, 133 309 o Preparation of the stereoisomeric mixture of cis-3,7-dihydroxybicyclo [3.3.0]octa -3,7-dicarbonitrile, 136 310 o Preparation of the mixture of cis-bicyclo[3.3.0]octa -2,7-diene- 3,7-dicarbonitrile, 137, and cis-bicyclo[3.3.0]octa -2,6-diene-3,7- dicarbonitrile, 138 310 o Preparation of the stereoisomeric mixture of cis-bicyclo[3.3.0] octane-3,7-dicarbonitrile, 139 311 o Preparation of the stereoisomeric mixture of dimethyl cis-bicyclo [3.3.0]octane -3,7-dicarboxylate , 141 311 o Preparation of dimethyl tricyclo[3.3.0.0 3,7 ]octane -1,5-dicarboxylate , 131 312 Index 2 5 1 o Preparation of tricyclo[3.3.0.0 3,7 ]octane -1,5-dicarboxylic acid , 141 313 o Preparation of 3-oxatetracyclo[5.2.1.1 5,8.01,5 ]undeca -2,4-dione, 144 313 o Preparation of 5-(methoxycarbonyl)tricyclo[3.3.0.0 3,7]octane -1- carboxylic acid , 146 314 o Preparation of tricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid , 155 314 o Preparation of (tricyclo[3.3.0.0 3,7 ]octa -1-yl)amine hydrochloride, 122?HCl 315 o Preparation of 3,7-dimethyltricyclo[3.3.0.0 3,7 ]octane -1,5- dicarboxylic acid , 143 316 o Preparation of 7,8-dimethyl-3-oxatetracyclo[5.2.1.1 5,8.01,5] undeca-2,4-dione, 145 316 o Preparation of 3,7-dimethyl-5-(methoxycarbonyl)tricyclo [3.3.0.0 3,7]octane -1-carboxylic acid , 147 317 o Preparation of methyl 3,7-dimethyltricyclo[3.3.0.0 3,7 ]octane -1- carboxylate , 149 317 o Preparation of 3,7-dimethyltricyclo[3.3.0.0 3,7 ]octane -1- carboxylic acid , 156 318 o Preparation of 3,7-dimethyl(tricyclo[3.3.0.0 3,7 ]octa -1-yl)amine hydrochloride, 123?HCl 318 o Preparation of 1-adamantanecarbonyl azide, 164 319 o Preparation of 1-(1-adamantyl)-3-(2,3,4-trifluorophenyl)urea, 166 320 o Preparation of pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca -5,11-diene- 8,9-dicarboxylic anhydride , 160 321 o Preparation of 9-methoxycarbonilpentacyclo[6.4.0 2,10.03,7.04,9] dodeca-5,11-diene-8-carboxylic acid , 161 321 o Preparation of methyl pentacyclo[6.4.0 2,10.03,7.04,9 ] dodeca-5,11-diene-8-carboxylate , 162 322 o Preparation of pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca -5,11-diene- 8-carboxylic acid , 159 322 o Preparation of pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca -5,11-diene- 8-carbonyl azide, 169 323 o Preparation of 1-(pentacyclo[6.4.0.0 2,10.03,7.04,9 ]dodeca - 5,11-diene-8-yl)-3-(2,3,4-trifluorophenyl)urea, 157 324 o General method for the synthesis of urea compounds 108-109 and 170-183 325 o Preparation of 1-(1-adamantyl)-3-(2,3,4-trifluorophenyl)urea, 108 326 o Preparation of 1-(2-adamantyl)-3-(2,3,4-trifluorophenyl)urea, 109 326 o Preparation of 1-(2-oxaadamant -1-yl)-3-(2,3,4-trifluorophenyl) urea, 170 326 o Preparation of 1-(3-methyl-2-oxaadamant -1-yl)-3- (2,3,4-trifluorophenyl)urea, 171 327 o Preparation of 1-(3-ethyl-2-oxaadamant -1-yl)-3- 2 5 2 Index (2,3,4-trifluorophenyl)urea, 172 328 o Preparation of 1-(3-cyclohexyl -2- oxaadamant -1-yl)-3-(2,3,4-trifluorophenyl)urea, 173 329 o Preparation of 1-(3-phenyl-2-oxaadamant -1-yl)-3- (2,3,4-trifluorophenyl)urea, 174 330 o Preparation of 1-(2-oxaadamant -5-yl)-3-(2,3,4-trifluorophenyl) urea, 175 331 o Preparation of 1-(4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11] dodecan-3-yl)-3-(2,3,4-trifluorophenyl)urea, 176 332 o Preparation of 1-(tricyclo[3.3.0.0 3,7 ]oct -1-yl)-3- (2,3,4-trifluorophenyl)urea, 177 333 o Preparation of 1-(3,7-dimethyl(tricyclo[3.3.0.0 3,7 ] oct-1-yl))-3-(2,3,4-trifluorophenyl)urea, 178 334 o Preparation of 1-[(3,7 -dimethyl(tricyclo[3.3.0.0 3,7 ] oct-1-yl)methyl] -3-(2,3,4-trifluorophenyl)urea, 179 335 o Preparation of 1-(?) -[(tricyclo[3.3.1.0 3,7 ]non -3-yl)ethyl] -3- (2,3,4-trifluorophenyl)urea, 180 336 o Preparation of 1-(pentacyclo[6.4.0.0 2,10.03,7.04,9 ] dodec-8-yl)-3-(2,3,4-trifluorophenyl)urea, 181 337 o Preparation of 1-(9-methyl-6,7,8,9,10,11-hexahydro - 5H-5,9:7,11-dimethanobenzo[9]annulen -7-yl)-3- (2,3,4-trifluorophenyl)urea, 182 338 o Preparation of 1-( 1,2,3,5,6,7-hexahydro -5-methyl-1,5:3,7- dimethano-4-benzoxonin -3-yl)-3-(2,3,4-trifluorophenyl)urea, 183 339 o Preparation of (2-oxaadamant -1-yl)isocyanate, 185 340 o Preparation of N-(1-acetylpiperidin-4-yl)-1?-(oxaadamant -1-yl) urea, 184 341 o Preparation of N-(1-acetylpiperidin-4-yl)-1H -imidazole- 1-carboxamide , 187 342 o Preparation of N-(1-acetylpiperidin-4-yl)-1?-(oxaadamant -1-yl) urea, 184 342 o Preparation of 4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11 ]dodec -3-yl) isocyanate, 189 343 o Preparation of 1-(1-acetylpiperidin-4-yl)-3-(4-oxahexacyclo [5.4.1.0 2,6.03,10.05,9.08,11 ]dodec -3-yl)urea, 188 343 o Preparation of trans-4-(4-amino-cyclohexyloxy) -benzonitrile, 191 344 o Preparation of trans-4-(4-amino-cyclohexyloxy) -benzoic acid, 190?HCl 345 o Preparation of trans-4-[4 -(3-oxaadamant -1-yl-ureido)- cyclohexyloxy] -benzoic acid, 192 345 o Preparation of trans-4-[4 -[ 3-(4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11] dodec-3-yl)ureido]cyclohexyloxy] -benzoic acid, 193 346 o Preparation of N-(1-acetylpiperidin-4-yl)-1?-(3,7- Index 2 5 3 dimethyl(tricyclo[3.3.0.0 3,7 ]octa -1-yl)urea, 195 348 2. IC50 assay determination 353 3. Apparent or semi-equilibrium solubility measurements 357 4. In vitro cell cultures 361 REFERENCES 365 INTRODUCTION Adamantane nucleus: Introduction 3 1. ADAMANTANE: THE PRECIOUS NUCLEUS For over eighty years, the adamantane nucleus has interested organic chemists for its simplicity and symmetry.1 ,2 The rigid tricyclo[3.3.1.1 3,7]decane skeleton has provided a unique structural template for evaluating important theoretical concepts in chemistry (Fig. 1).3,4,5,6,7 From its discovery, adamantanes have been particularly useful for proving through- bond and through-space electronic effects of different substituents in saturated systems and have significantly contributed to our understanding of hyperconjugation and degenerate isomerization. Furthermore they have provided molecular scaffolds for investigating the chemical reactivity and the diastereoselectivity of addition/elimination reactions in saturated polycyclic cages. Fig. 1. Structural representations of tricyclo[3.3.1.1 3,7]decane, widely referred as adamantane ring. Hydrogen atoms are shown in blue. Representations done by ChemBio3D Ultra. Energy minimized through MM2. The physical and chemical properties of the adamantane nucleus have largely overshadoZed the structure?s contribution to the discover\ oI human therapeutics Nevertheless in the last decades its application has become more and more significant in medicinal chemistry, where the adamantane ring is identified as a key structural subunit in several synthetic drugs for multiple targets. 8 , 9 , 10 But prior to an in-depth analysis of adamantane?s role in drug discover\ it is worth to introduce it from its onset. 1.1 Origin of a damantane and first synthesis Adamantane belongs to the lower diamondoids, which are a group of saturated cage- like hydrocarbon compounds with a diamond structure present in crude oil in highly variable concentrations (Fig. 2).11 Adamantane owes its name to its structural relation with 1 Land a, S. Chem. Listy 1933, 27, 415 - 4 18 an d 1933, 27, 443 - 448. 2 Fort , R. C., Jr.; Sch l ey er, P. vo n R. Chem. Rev. 1964, 277 - 30 0. 3 Adcock , W.; Kok , G. B. J. Org. Chem. 1987, 52, 356 - 36 4. 4 Dudd eck, H. Tetrahedron 1978 , 34, 247 - 2 51. 5 Kaselj, M.; Adcock , J. L.; Lu o, H.; Zh an g, H.; Li , H.; William, J. le N. J. Am. Chem. Soc. 1995, 117, 7088 - 7091. 6 Adcock , W .; Tro u t, N. A. Chem. Rev. 1999, 99, 141 5 - 1 435. 7 Gleich er, G. J. ; Schley er, P. vo n R. J. Am. Chem. Soc. 1967, 89 , 582 - 5 93. 8 Wish n o k, J. J. Chem. Educ. 1973 , 50, 780 - 781. 9 Wanka , L.; Iq b al, K.; Sch rein er, P. R. Chem. Rev. 2013, 113, 3516 - 3604. 10 Lamou reu x, G.; Art a via, G. Curr. Med. Chem. 2010, 17, 296 7 - 297 8. 11 Dah l, J. E.; Liu , S. G.; Carl so n , R. M. K. Science 2003, 299, 96 - 99. 4 INTRODUCTION the diamond, as it is derived from the Greek adamantinos (of steel, of diamond).12 The smallest diamondoid, adamantane (C10H 16), was the first isolated from petroleum oil in the early 1930s and its amount was estimated to be 0.0004% by volume. 13 Fig. 2. The relation between the face-centered cubic diamond lattice and diamondoid structures.11 Nowadays diamandoids are of current interest because of their ready accessibility and the ease of selective functionalizations, yielding building blocks highly employed in nanotechnology and electronics.14 Nature is the oldest contributor to adamantane ring-containing substances and several natural products with biological activity incorporate an adamantane motif. Tetrodotoxin, the potent toxic principle from puffer fish ( Tora fugu ), contains an oxygenated adamantane framework in its structure,15 and Nature-inspired heteroadamantane antiviral ansabananin incorporates an azaadamantane moiet\ plus a ?trio[aadamantane? ring ()ig  16 Besides, other natural compounds enclose the adamantane hydrocarbon system itself, such as sampsonione I and hyperibone K, both of them showing moderate cytotoxicity against 12 Senning, A. Elsevier?s Dictionary of Chemoetymology; Elsevier: Oxford, 2007 . 13 Mair, B. J.; Shamai en gar, M.; Krou sko p , N. C.; Ros sin i, F. D. Anal. Chem. 1959, 31, 208 2 - 2 083. 14 Schrein er, P. R.; Fok in a, N. A. ; Tka ch en ko , B. A. ; Hau s man n , H.; Sera fin , M.; Dah l, J. E. P.; Li u , S.; Carl so n , R. M. K.; Fokin , A. A. J. Org. Chem . 2006, 71, 6709 - 67 20. 15 Wood war d , R. B.; Gou gou ta s, J. Z. J. Am. Chem. Soc. 1964, 86 , 5030 - 5030. 16 T an n er, J. A.; Zh en g, B. - J. ; Zh ou , J.; Watt , R. M.; Jian g, J. - Q.; Won g, K. - L.; Lin , Y. - P.; Lu , L. - Y.; He, M. - L.; Kun g, H. - F.; Ke s el, A. J.; Huan g , J. - D. Chem. Biol. 2005, 12, 30 3 - 311 . Adamantane nucleus: Introduction 5 different tumoral cell lines.17,18 Even though adamantane is present in Nature, it is not considered as a ?natural product? nor a ?native b ological substrate? but it is classiIied as a ?mineral? Fig. 3. Nature-occurring and Nature-inspired adamantane-containing products. Albeit adamantane was isolated from petroleum oil and synthesized chemically in earliest approaches,19 its availabilit\ Zas limited until the 6chle\er?s s\nthesis which allowed the wide study of adamantane and its functionalization.20,21 The synthetic route entailed a Lewis-acid induced rearrangement of the C10H 16 precursor tetrahydrodicyclopentadiene (Scheme 1). Scheme 1. 6chle\er?s s\nthesis. This ready access to the adamantane scaffold constituted its birth as a precursor in pharmaceutical sciences, while its derivatives expanded the application in medicinal chemistry thereof, and they are still doing so to date. 17 Hu, L. H.; Sim, K. Y. Org. Lett . 1999, 1, 879 - 882. 18 Tan ak a, N.; Tak aish i, Y.; Shiki sh ima, Y. ; Nak an ish i, Y.; Basto w, K.; Le e, K. - H . ; Hon d a, G.; It o, M.; Tak ed a, Y.; Kod zh imat o v, O. K.; A sh u rmeto v, O . J. Nat. Prod . 2004, 67 , 1870 - 187 5. 19 Prelo g, V.; S eiw erth , R. Ber. Dtsch. Chem. Ges . 1941, 74, 1 6 44 - 16 48. 20 Schleyer, P. v. R. J. Am. Chem. Soc . 1957, 79, 3292 - 3 292. 21 Schleyer, P. v. R. ; Don ald so n , M. M.; Nicho las, R. D.; Cup as, C. Org. Synth. Coll. Vol. V 1973 , 16 - 19. 6 INTRODUCTION 1.2 Adamantane?s physicochemical properties and its multidimensional value The closing section will provide the physicochemical profile of the adamantane moiety, the structural relationships between adamantane-bearing compounds and protein targets as well as the general aspects in the pharmacokinetics of the adamantane group, with the aim of supplying an overview of the role of this valuable ring. 1.2.1 Physicochemical properties The four cyclohex anes in chair conformation contained within the adamantane ring are the source of its uniqueness in that it is both rigid and virtually stress-free. If we bear in mind the three-dimensional structure in figure 1, adamantane possesses a spherical shape with a diameter of about 6.4 ? .22 Compared to a benzene molecule, the adamantane ring displays a larger volume not depending on its orientation, whereas the benzene ring is planar (Fig. 4).10 Fig. 4 . Structural representations of adamantane and benzene in space-filling mode and their dimensions. H ydrogen bonds are shown in blue. Representations done by ChemBio3D Ultra. Energy minimized through MM2. Polarizability is another property that affects the binding between the ligand or drug and its target, not only influencing the hydrophobicity but also the biological activity. Polarization is the phenomenon whereby the charge distribution in a molecule is modified due to the presence of an external field. 23 Whereas benzene shows a quadrupole, which forms ?-type interactions, adamantane presents an octapole, with a higher polarizability value than benzene, which indicates that the electrostatic forces between adamantane and its target could have a greater effect on the binding than previously thought. It is worth to 22 Mo rel - D e sro si ers, N.; Mor el, J. - P . J. Solution Chem . 1979, 8 , 579 - 59 2. 23 Leach , A. R. Compr. Med. Chem. II 2007, 4, 87 - 1 18. Adamantane nucleus: Introduction 7 highlight that the interest in the polarizable force fields for small molecules in organic and biochemical systems is currently increasing, drawing the attention of many scientists.24,25 Nevertheless, the most significant property that characterizes the adamantane group is its high lipophilicity. Considered the most important drug-like physical property, lipophilicity is one of the main criteria relevant for oral bioavailability, included in the well- NnoZn /ipinsNi?s rules26 The lipophilic value is commonly estimated by the logarithm of the octanol/water partition coefficient (log P), which is defined as the ratio of non-ionized drug distributed between octanol and water phases at equilibrium. Higher val ues imply greater lipophilicity and often a log P value of 5 or higher is considered as an upper limit of desired lipophilicity in drug discovery.27 Generally, an adamantane-bearing compound will be more lipophilic than the des-adamantyl analogue. Calculation of the hydrophobic substituent constant determined by non-empirical methods for the adamantane group, the clog P (calculated log P) measures of the known adamantane-containing compounds and the des-adamantyl derivatives, revealed a ?adamantyl value of 3.1.28 That is, the incorporation of an adamantane motif will increase by about 3.1 log units the log P value of any given drug. As a consequence adamantane is considered as a ?lipophilic carrier? that alloZs poorly absorbed drugs to slipping across cell membranes, such as the Blood-Brain Barrier (BBB) enhancing Central Nervous System (CNS) access.29,30 Inasmuch as clog P is a composite property dependent on molecular size, polarity and hydrogen bonding,31 it is related to solubility. In almost all cases the adamantane group lowers the clog P compared to the linear constitutional isomer (Fig. 5). This effect is partly due to the fact that the adamantane-based compounds feature a close lateral packing, with the interstitial space present in solution increasing the propensity towards solvation. Hence molecules hosting an adamantane moiety will have solubility properties more tractable in medicinal chemistry setting compared to other C10-based frameworks.28 Fig. 5. clog P values of adamantane derivatives and their linear analogues, calculated using Bio- Loom? . 24 Kamin ski, G. A .; Stern , H. A .; Bern e, B. J.; Fri esn e r, R. A . J. Phys. Chem. A 2004, 108, 621 - 6 27. 25 Maple, J. R.; Cao, Y. ; Dam m, W.; Halgren, T. A.; Ka min ski, G. A.; Zh an g, L. Y.; Fri esn er, R. A. J. Chem. Theory Comput. 2005, 1, 69 4 - 715. 26 Lip in ski, C. A; Lomb ar d o , F.; D omin y , B. W.; Fe en ey , P. J. Adv. Drug Deliv. Rev. 1997, 23, 3 - 25. 27 Leeso n , P. D.; Sprin gth o rp e, B . Nat. Rev. Drug Discov. 2007, 6, 881 - 8 90. 28 Liu , J.; Oban d o , D.; Lia o , V.; Lifa, T .; Cod d , R. Eur. J. Med. Chem. 2011, 46, 1949 - 19 63. 29 Terasak i, T.; Pard rid ge, W. M. J. Drug Targets 2000, 8, 35 3 - 355. 30 Tsuzu ki, N.; Hama, T.; Ka wad a, M.; Hasui, A.; Kon ish i, R.; S hiwa, S.; Och i, Y.; Futa ki, S.; Kita gawa, K. J. Pharm. Sci. 1994, 83, 481 - 4 84 . 31 Abrah am, M. H., Chad h a, H. S., Wh itin g, G. S. ; Mitch ell, R. C . J. Pharm. Sci. 1994, 83, 1085 - 1100 . 8 INTRODUCTION Worth to bear in mind, is the fact that, notwithstanding the increase in the solubility of the adamantane-containing compounds with respect to their linear structural isomers as a result of the above mentioned, incorporation of an adamantane ring in any given structure will compromise the water solubility of the molecule since the log P is enlarged by about 3.1 log units, which represents a considerable increment in drug design. 1.2.2 Adamantane ring as a pharmacophore The three-dimensional structure is essential for the sheer activity of a drug, exerted by the fit into the receptor?s binding site. The target-ligand interaction was generally described as the ?Ne\-locN? model but currentl\ the ?induced Iit? principle has been more accepted, which express es a more dynamic understanding of the receptor?ligand interactions. 32 Accordingly, the adamantane core has been used to organize the spatial position of the functional groups that form the pharmacophore of a lead molecule, providing an ideal skeleton for this purpose. That is, to fix the molecular fea tures that are essential for molecular recognition.10 Thus adamantane scaffold plays a decisive role in the three- dimensional adjustment of the pharmacophores to its protein target. The favourable chemical and geometric properties of adamantane make it possible to introduce several functional groups, consisting drug, targeting part, linker, or similar, without undesirable interactions between such groups.33 Adamantane?s physicochemical properties and structural uniqueness are in consequence directly related to its binding mode with specific therapeutic targets. Firstly, adamantane moiety can bind to hydrophobic pockets of enzymes, and secondly it can affect ion channels by disrupting the transmembrane flow. Worth to highlight is the difference betZeen the term ?blocNer? and the term ?antagonist? The Iirst locution concerns the alteration of the permeability of the ion channels by the interaction of the ligand with these. The second term refers to these drugs binding to an enzyme or receptor, which can control an ion channel, preventing the binding of the natural mediator and thus eliminating the physiological response related to the enzyme/ion channel. A deeper but brief look into adamantane?s role in medicinal chemistry will be discussed later on. 1.2.3 ADME properties of adamantane-containing compounds As most part of the drugs that are administered, compounds incorporating adamantane as a building block suffer from metabolism once they are inside the organism. With regard to Absorption and Distribution, incorporation of the cage-shaped adamantane nucleus into medicinal agents has provided a viable approach for designing molecules that can access lipophilic cell membranes, as aforementioned. Therefore, targets of the CNS are being addressed today with, remarkably structurally simple adamantane derivatives, such as 32 Spyra kis, F.; Bid o n - Cha n al, A.; Barril, X.; Lu q u e, F. J. Curr. Top. Med. Chem. 2011, 11, 192 - 210. 33 Manso o ri, G. A.; Georg e, T. F. ; As so u fid , L.; Zh an g, G. Molec u lar buildin g block s for nan o tech n o logy . From diamon d o id s to nan o sc ale mat erial s and app licat ion s . Sprin ger: Ne w York , 2007 . Adamantane nucleus: Introduction 9 amantadine and memantine, with anti-Parkinson and anti-Alzheimer activities respectively (Fig. 6). Fig. 6 . Structures of amantadine and memantine. In terms of Metabolism and Excretion, the cytochrome P-450 is believed to control the oxidative metabolism for a rapid excretion of the adamantane derivatives,34 mainly through hydroxylation with mono - and dihydroxylated de rivatives as common metabolites. As it can be deduced from the crystal structure of CYP450 CAM (bacterial camphor hydroxylase P-450) complexed with adamantane, this process is generally not selective and, in the absence of further functional groups in the drug that could orientate the cage hydrocarbon, several sites are being hydroxylated, as can be seen wi th, for example, amantadine,35 memantine,36 rimantadine,37,38 and saxagliptin 39 (Fig. 7). Fig. 7. Main metabolites of amantadine, memantine, rimantadine, and saxaglipti n. 34 Raags, R.; Pou los, T. L. Biochemistry 1991, 30, 26 74 - 2684. 35 Sucko w, R. F. J. Chromatogr. B Biomed. Sci. Appl . 2001, 764 , 313 - 32 5. 36 Stu rm, G.; Sch o ll mey er, J. D.; Wes eman n , W. IRCS Medical Science: Library Compendium , 1976 , 4, 55. 37 Hay d en , F. G.; Min o ch a, A. ; S py ker, D. A.; Hof fman , H. E. Antimicrob. Agents Chemother. 1985 , 28, 216 - 221. 38 Rubio, F. A.; Cho ma, N.; Fuk u d a, E. K. J. Chromatogr. 1989, 497, 147 - 157. 39 Su, H.; Bou lto n , D. W.; Barro s , A . J r .; Wan g, L.; Cao , K.; Bon aco rsi, S. J. J r .; Iy er, R. A. ; Hump h rey s, W. G.; Chri sto p h er, L. J. Drug Metab. Dispos. 2012, 40, 1 345 - 13 56. 10 INTRODUCTION Metabolism studies have shown that the bridgehead hydroxylation ( tertiary carbon) is favoured over the secondary carbon positions, producing water-soluble hydroxyadamantane derivatives in the liver , which are then easily excreted. This hydroxylation mechanism is prevalent for electron -rich adamantane cores.40 As an example, while amantadine is not metabolized by hydroxylation of the adamantane nucleus, cage hydroxylation is the main metabolic degradat ion for its dimethyl derivative memantine. In addition, the steric bulk of the adamantane motif can reduce the amidase or esterase enzyme activity in the regions proximal to the scaffold, especially where the adamantyl group has been appended to the parent drug via an amide or an ester bond. So the rigid hydrocarbon skeleton protects functional groups in its proximity from metabolic cleavage, enhancing the duration of action of peptide-derived drugs, among others, that feature an adamantane ring.41 1.3 Medicinal c hemistry of a damantane : the promiscuous lipophilic pellet 1.3.1 Clinically approved adamantane-based drugs At the time of writing this thesis, eight drugs are marketed featuring the adamantane motif. A brief introduction of each one will be commented below. ? Amantadine: considered the birth of the adamantane derivatives in medicinal chemistry, amantadine constituted the structurally simplest monofunctionalized adamantane drug with application in medicinal chemistry. Discovered in the early 1960s,42 1-aminoadamantane was found to be a selective antiviral, especially against influenza A and rubella virus. Related to influenza A virus, the mechanism of action consists in the blockade of the M2 ion channel. This protein is essential for viral replication and is involved in the endosomal uptake of protons, thus acidifying the interior of the virus that leads to the uncoating of the viral genetic material.43,44 The inhibitory activity of amantadine is by different mechanisms; the adamantane hydrophobic sphere physically occludes the transmembrane flux of protons , locks the protein conformation in a single state hampering its plasticity, and perturbs the p K a of the His37 tetrad by H -bonding through the entry of water cluster.45 Furthermore, amantadine was found to be useIul Ior the treatment oI 3arNinson?s disease when in 1968 a patient who was taking amantadine to prevent the flu, experienced a remarkably remission in her symptoms of rigidity, tremor and akinesia.46 This fortuitous 40 Fokin , A. A.; Schr ein er, P. R. Chem. Rev. 2002, 102, 1551 - 15 93. 41 Naga sawa, H. T.; Elb erlin g, J. A.; Shirot a, F. N. J. Med. Chem. 1975, 18, 826 - 83 0 . 42 Davie s, W. L.; Gru n ert, R. R. ; Ha ff, R. F.; McGah en , J. W.; N eu may er, E. M.; Pau l sh o ck, N.; Watt s, J. C.; Wood , T. R.; Herman n , E. C.; Hoffman , C. E. Science 1964, 144 , 862 - 863. 43 De Cle rcq , E. Nat. Rev. Drug Discov. 2006, 5, 101 5 - 1 025. 44 Cady , S. D.; Lu o, W.; Hu, F.; Hon g, M. Biochemistry 2009, 48 , 7356 - 7364. 45 Cady , S. D.; Wan g, J.; Wu , Y.; DeGra d o , W. F.; Hon g, M. J. Am. Chem. Soc. 2011, 133, 4274 - 428 4. 46 Schwab , R. S.; Englan d , A. C. Jr.; Poska n zer, D. C.; You n g, R. R. J. Am. Med. Assoc. 1969, 208, 1168 - 1 170. Adamantane nucleus: Introduction 11 finding marked the beginning of medicinal chemistry of adamantane derivatives in the context of diseases affecting the CNS, accounting for its lipophilicity and BBB-penetration, enhancing properties of the adamantane motif. In 3arNinson?s disease the Food and Drugs Administration (FDA) approved amantadine has been demonstrated to act directly on the D2 dopamine receptors, resulting in enhanced dopamine release, while inhibiting post- synaptic uptake. Besides, amantadine has antiglutamatergic properties via the non-competitive antagonism of N-methyl-D-aspartic acid (NMDA) receptors,47,48 and displays trypanocidal activity along with rimantadine and memantine.49,50 However, amantadine is not clinically used exploiting these properties. ? Memantine: the 3,5-dimethyl-1-aminoadamantane was approved in 2002 by the European Medicines Agency (EMA) and in 2003 by the FDA for use in treatment of patients with moderate-to-severe Alzheimer?s disease (AD  Memantine is an non- competitive, low- to moderate-affinity NMDA receptor (NMDAR) antagonist, with strong voltage dependency and rapid blocking and unblocking kinetics.51,52 Memantine is efficient in neurological diseases that are mediated, at least in part, by overactivation of NMDARs, producing excessive Ca 2+ inIlu[ through the receptor?s associated ion channel and consequent free-radical formation, leading to cellular damage and death.53 The NMDA receptor antagonism is a topic of the present thesis, hence it will be discussed further on. ? Rimantadine: soon after amantadine introduction in clinic, several compounds were developed with the insertion of a bridge of one or more carbon atoms between the 1- adamantyl and the amino group, which led to compounds with generally high antiviral activity, with rimantadine outperforming amantadine in terms of activity (Fig. 8).54 The ?- methyl adamantanemethanamine have been found to be active among a wider range of viruses, although its main use is for the treatment and prophylaxis of influenza A infection.43 Compared to amantadine, rimantadine has a comparable or higher oral availability, produces fewer side effects and is absorbed well from the gastrointestinal tract.55 As well as amantadine, rimantadine is also a trypanocidal agent, whose activity is generally associated with a blockade of a membrane ion channel. Trypanosoma brucei is carried by the Tse-tse fly 47 Korn h u b er , J.; Borman n , J.; H ub ers , M.; Rus ch e , K .; Ri ed er e r , P . J Pharmacol. 1991, 206, 2 97 - 30 0. 48 Blan p ied , T. A. ; Clar k e, R. J.; Joh n so n , J. W. J. Neurosci. 2005 , 25, 3312 - 3322. 49 Kelly, J. M.; Mi le s, M. A.; Skin n er, A. C. Antimicrob. Agents Chemother . 1999, 43, 985 - 98 7. 50 Kelly, J. M.; Quack , G. ; Mile s, M. A. Antimicrob. Agents Chemother. 2001, 45, 1360 - 13 66. 51 Ramm es, G. ; D an y sz, W.; Par so n s, C. G. Curr. Neuropharmacol. 2008, 6, 55 - 78 . 52 Herrman n , N.; Li, A.; Lan ct? t, K. Expert Opin. Pharmacother. 2011, 12, 787 - 80 0. 53 Lip to n , S. A . Nat. Rev. Drug Discov. 2006, 5, 160 - 170. 54 Tsun o d a, A.; Maas sab , H. F.; Coch ra n , K. W.; Evelan d , W. C. Antimicrob. Agents Chemother . 1965, 5 , 553 - 5 60. 55 Hay d en , F. G.; Min o ch a, A. ; S py ker, D. A.; Hof fman , H. E. Antimicrob. Agents Chemother. 1985 , 28, 216 - 221. 12 INTRODUCTION and causes the so-called sleeping sickness, which is a serious health issue in many areas of the sub-Saharan Africa, considered as a ?neglected disease?56 Fig. 8. Structures of the adamantane-based drugs rimantadine, saxagliptin , vildagliptin, tromantadine, adapalene and arterolane. ? Saxagliptin and vildagliptin: in the multibillion dollar market of type 2 Diabetes Mellitus (T2DM), big pharmaceutical companies have initiated drug development programs around dipeptidyl peptidase IV (DPP-IV) inhibitors.57 The activity of DDP-IV negatively affects glucose homeostasis, and its inhibition increases the levels of incretin hormones, such as glucagon-like peptide-1. This target is successfully hit by adamantane compounds; saxagliptin and vildagliptin, which have been approved in recent years.58,59 The adamantyl group in saxagliptin and vildagliptin serves a dual role in terms of directing inter- and intramolecular interactions in the DPP-IV binding site and in reducing the propensity towards intramolecular cyclisation reactions, by means of the encumbrance supplied by the adamantane moiety (Fig. 9).60 The production of the inactive cyclic amidine form of saxagliptin has been observed during process-scale production. 56 Barret, M. P.; Burch mo r e, R. J. S.; Stich , A.; Lazz ar i, J. O.; F rasch , A. C. ; Cazz u lo, J. J. ; Kri s h n a, S. Lancet 2003, 362, 14 69 - 1480. 57 von Geld e rn , T. W.; Tr e villy an , J. M. Drug Dev. Res. 2006, 67 , 627 - 64 2. 58 Villh au er, E. B.; Brink man , J. A.; Nad eri, G. B.; Burk ey , B. F. ; Dun n in g, B. E.; Pra sad , K.; Man gold , B. L.; Russ ell, M. E.; Hugh es, T. E. J. Med. Chem. 2003, 46, 2774 - 2 789 . 59 Augeri, D. J.; Rob l, J. A. ; Bet eb en n er, D. A.; Magn in , D. R .; Khan n a, A.; Rob ertson , J. G.; Wan g, A. ; Simp kin s, L. M.; Tau n k, P.; Hua n g, Q.; Han , S. - P. ; Abb o a - O ff ei , B.; Cap , M .; Xin, L. ; Tao , L.; To zz o , E.; Welzel, G. E.; Egan , D. M.; Marcin ke vi cien e, J.; Cha n g, S. Y.; Bill er, S. A.; Kirb y , M. S. ; Park er, R. A. ; Haman n , L. G. J. Med. Chem. 2005, 48, 5025 - 5 037 . 60 Metz ler, W. J. ; Yan ch u n as, J. ; Weigelt, C. ; Ki sh , K.; Kl ei, H. E.; Xie, D.; Zh an g, Y. ; Corb ett , M.; Ta mu ra , J. K.; He, B.; Haman n , L. G.; Kirb y , M. S.; Marcin kevi cien e, J. Protein Sci . 2008, 17, 240 - 250. Adamantane nucleus: Introduction 13 Fig. 9. Internal cyclization can play a decisive role in inactivating cyanopyrrolidine-based DPP- IV inhibitors. ? Tromantadine : this aminoadamantane marks the third adamantane derivative that was successfully introduced to the market, even though its identification as an anti-Herpes simplex agent was not so much the result of research specifically addressing this issue. Tromantadine is a derivative of amantadine that is used topically for the treatment of the herpes virus, being inactive against influenza A.61 Its precise mechanism of action is still unknown, despite numerous studies taking aim at it. However, tromantad ine apparently exerts, at least in part, its activity through influencing the fusion of lipid membranes. 62 Tromantadine is particularly interesting for the treatment of aciclovir-resistant strains since it does not require a viral kinase to become activated, unlike aciclovir. . Adapalene: this adamantyl retinoid is used for the topical treatment of Acne vulgaris sold as hydrogel in combination with the antimicrobial benzoyl peroxide. 63 Its pharmacological and chemical properties include photochemical stability, local activity, high stability, and as expected, low local side -effect profile. The lipophilicity of the adamantyl group contributes to the low percutaneous flux of adapalene and an improved penetration into the pilosebaceous follicles.64 ? Arterolane: this adamantyl ozonide has been recently approved for the treatment of Plasmodium falciparum infection. It constitutes the first Indian new molecular entity that has reached the market. Since 2014, this anti-malaria drug is available combined with piperaquine phosphate in African and Asian countries.65 1.3.2 Adamantane-containing candidates in development The quest for new applications of the adamantane ring is an issue on the rise in medicinal chemistry. Thanks to the great efforts from the academy and industry, many 61 Rosen th al, K. S.; Sok o l, M. S.; In gram, R. L.; Sub ra man ian , R.; Fort , R. C. Antimicrob. Agents Chemother . 1982, 22, 103 1 - 1 036. 62 Ickes, D. E.; Ven ett a, T. M.; P hon p h o k, Y.; Rosen th al, K. S. Antiviral Res . 1990, 14, 75 - 85 . 63 Shro o t, B.; Mich el, S. J. Am. Acad. Dermatol . 1997, 36 (Sup p l. ) , S96 - S 103. 64 Pi?ra rd , G. E.; Pi?ra rd - Fran c h imo n t, C.; Paq u et, P.; Qu at reso o z, P. Expert Opin. Drug Metab. Toxicol. 2009, 5, 1565 - 15 75 . 65 Wells, T. N.; Alon so , P. L.; Gut teridge, W. E. Nat. Rev. Drug Discovery 2009, 8, 879 - 891. 14 INTRODUCTION adamantyl-based compounds have been studied as potential therapeutics for conditions including cancer,66 neurological conditions, hypertension,67 malaria,68,69 and tuberculosis.70 For the sake of brevity, table 1 encloses some of the main targets that are hit by adamantane-bearing compounds that are still under development, as well as the pharmacodynamic (PD) and pharmacokinetic (PK) modulation exert ed by adamantane. For a deeper study on the subject, the reading of the reviews by Wanka, Iqbal and Schreiner,9 Lamoureux and Artavia, 10 and Codd and coworkers, is recommended.28 Table 1. Representative adamantane-based biologically active compounds. CCK-B: cholecystokinins B; ROS: reactive oxygen species. Compound Target Condition Main features 171 CCK-B receptor Anxiety Neuropeptide with better penetration of the BBB 2 (top) and 3 (bottom)72,73 Plasmodium falciparum Malaria Improved pharmacological profile, whether displaying greater oral bioavailability or higher activity against resistant strains 66 Glen n o n , R. A. Drug Dev. Res . 1992, 26, 251 - 27 4. 67 Imig, J. D.; Zh ao , X. ; Zah ar is, C. Z.; Olear czy k, J. J.; Pollock, D. M.; Newman , J. W.; Kim, I. H.; Wata n ab e, T.; Ham mo ck, B. D. Hypertension 2005, 46, 975 - 981. 68 Fies er, L. F.; Naz er, M. Z.; Ar c h er, S.; Berb erian , D. A.; Sligh t er, R. G. J. Med. Chem . 1967, 10, 517 - 521 . 69 Wang , X.; Don g, Y.; Witt lin, S.; Creek, D. ; Cho ll et, J.; Cha rman , S. A.; San to To mas, J.; S ch eu r er, C.; Sny d er, C.; Venn er stro m, J. L. J. Med. Chem. 2007, 50, 584 0 - 5 847. 70 Proto p o p o va, M.; Han ra h an, C.; Niko n en ko , B.; Samala, R.; Chen , P.; Gear h ar t, J.; Ein ck, L.; Nacy , C. A. J. Antimicrob. Chemother . 2005 , 56, 968 - 9 74. 71 Triv ed i, B. K.; Pad ia, J. K. ; Holme s, A.; Ros e, S.; Wri gh t, D. S.; Hin to n , J. P.; Pritch ar d , M. C.; Eden , J. M.; Kneen , C.; W eb d ale, L.; Su ma n - Cha u h an , N.; Bod en , P.; Sin gh , L.; Field , M. J.; Hill, D. J. Med. Chem . 1998, 41, 38 - 4 5. 72 Sola ja , B. A.; Op sen ica, D. ; S mith , K. S.; Milho u s, W. K. ; Te rzic, N.; Op sen ica, I.; Burn ett , J. C.; Nus s, J.; Gus sio, R.; Ba var i, S. J. Med. Chem . 2008, 51, 4388 - 439 1. 73 Moeh rle, J. J.; D up ar c, S.; Si et h o ff, C.; van Gier sb erg en , P. L.; Craft, J. C. ; Arb e - Barn e s, S.; Cha rman , S. A.; Gutierr ez, M.; Witt lin, S.; V en n ertr o m, J. L. Br. J. Clin. Pharmacol. 2012, 75, 524 - 537. Adamantane nucleus: Introduction 15 450 T rypanosoma brucei Sleeping sickness Improvement in the anti-parasitic activity 570 Mycobacterium tuberculosis Tuberculosis Improved in vivo potency and limited in vitro and in vivo toxicity 6 (adaphostin)74,75 Tyrosine kinase and /or ROS Cancer Less susceptible of hydrolysis by means of a superior lipophilicity and encumbrance 776 DNA Cancer Cisplatin-analogue with improved therapeutic index and oral bioavailability 877, 78 P2X7 receptor Inflammation Good balance between potency, molecular weight and lipophilicity 74 Le, S. B.; Hail er, M. K.; Buh r o w, S.; Wan g, Q.; Flat t en , K.; Pedia d ita kis, P.; Bible, K. C . ; Lewi s, L. D.; Sau svil le, E. A .; Pan g, Y. P.; Ame s, M. M.; Lemaster s, J. J.; Holmu h amed o v, E. L.; Kau fm an n , S. H. J. Biol. Chem. 2007, 282, 88 60 - 8872. 75 Kau r, G.; Naray an an , V. L.; Risb o o d , P. A . ; Hollin gsh ead , M. G.; Stin so n , S. F .; Varma, R. K.; Sau svill e, E. A . Bioorg. Med. Chem. 2005, 13, 1749 - 176 1. 76 Kellan d , L. R.; Barn ar d , F. J.; Evan s, I. G.; Mu rrer, B. A.; Theo b ald , B. R. C.; Wy er, S. B.; God d ar d , P. M.; Jon es, M. ; Valent i, M.; B ry an t, A.; Roger s, P. M.; Harra p , K. R . J. Med. Chem . 1995, 38, 30 1 6 - 302 4 . 77 Guile, S. D.; Alcar az , L.; Birkin sh aw, T. N.; Bow ers, K . C. ; Eb d en , M. R.; Furb er, M. ; Sto ck s , M. J. J. Med. Chem. 2009, 52, 312 3 - 3 141. 78 Mehta , N.; Kau r, M.; Singh , M.; Cha n d , S.; Vyas, B.; Silak ar i, P.; Bah ia, M. S.; Silak ar i, O. Bioorg. Med. Chem. 2014, 22, 54 - 88. 16 INTRODUCTION 979, 80 ?/? -opioid receptors Analgesia Modification of the selectivity pattern, and better in crossing the BBB Other protein targets than the already introduced are hit by adamantane-based compounds. An emerging field is the inhibition of enzymes with therapeutically interesting profiles, such as the 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) and the soluble epoxide hydrolase (sEH). These targets will be two independent topics of this manuscript, hence they will be discussed in the next chapt ers. All these examples prove that the value of the adamantyl group in drug design is multifunctional, and that its study is of major importance to understand the influence of this simple, yet potent, structural element. 1.4 Not all that glitters is gold : a moot point of perfection In the previous sections we have gone through the value of the adamantane nucleus in medicinal chemistry, and the early examples of adamantane -based compounds have shown that this precious ring modulates different properties essential for drug behaviour within the organism. Despite all the advantages the adamantane offers, a few drawbacks are identified, which render the adamantane group not as a magic bullet but as a ring whose introduction should be given careful considerations. The following section encloses these disadvantages and the way they affect to the drug discovery process. 1.4.1 High lipophilicity compromises PK properties Lipophilicity is the most critical property Irom the Iamous /ipinsNi?s rule or ?rule oI Iive?26 which states that drug permeability and absorption are threatened if: i. clog P is > 5; ii. molecular weight is > 500 Da; iii. number of hydrogen-bond donors is > 5; iv. number of hydrogen-bond acceptors is > 10. Since its appearance, several studies have highlighted the polar surface area (PSA) and the number of rotatable bonds as other key parameters that control permeability and 79 Horvat , S.; Varga - D eft erd ar o v ic, L.; Hor vat , J. ; Jukic, R.; Kan to ci, D.; Chu n g, N. N.; S ch ill er, P. W.; Bie sert , L.; Pfu tz n er, A.; Suh ar to n o , H.; Rub sam en - Wa ig man n , H . J. Pept. Sci. 1995, 1, 303 - 310. 80 Loveka mp , T.; Coop er, P. S.; Hard iso n , J.; Bry an t, S. D.; Gue rrin i, R.; Balb o n i, G.; Sa lvad o ri , S.; Laza ru s, L . H. Brain Res. 2001 , 902, 131 - 1 34. Adamantane nucleus: Introduction 17 absorption.81 Thus, overall oral bioavailability can be simply predicted by number of rule- of-five violations, PSA and number of rotatable bonds. Although these physical properties play an important role in the assessment of the drug bioavailability, lipophilicity stands out among them since it is decisive to avoid later attrition during the discovery of drug candidates, as numerous studies have confirmed it.82 During the last two decades, clog P has barely changed for approved oral drugs. However, there is a noted trend that lipophilicity increases as candidate molecules progress through clinical trials. A recent study published in 2007 compared the physicochemical properties of more than 500 marketed drugs and compounds in development from big pharmaceutical companies such as GlaxoSmithKline (GSK), Merck , AstraZeneca and Pfizer.27 The authors observed that the average clog P value was higher than 4.1 in the patent literature, whereas the more recent drugs have a median clog P of 3.1 (Fig. 10). Fig. 10. Mean molecular weight and clog P values of oral drugs according to time of publication.83 On the other hand, a comparison of marketed oral drugs with compounds in early stages of the drug discovery shows that high lipophilicity (clog P > 4) leads to compounds with: i. poor solubility; ii. rapid metabolic turnover; iii. low bioavailability; iv. off-target promiscuity. 81 Lu, J. J. ; Cr imin , K .; Good win , J. T.; Crivo r i, P.; Orreniu s, C.; Xing, L.; Tan d ler, P. J.; Vid mar , T. J.; Amor e, B. M.; Wil so n , A. G. E.; Sto u te n , P. F. W.; Burto n , P. S. J. Med. Chem. 2004, 47, 6104 - 61 07 . 82 Arnott , J. A . ; Plan ey , S. L. Expert Opin. Drug Discov. 2012, 7 , 863 - 87 5. 83 (a) Lee so n , P. D.; You n g, R. J. ACS Med. Chem. Lett. 2015, 6 , 722 - 7 25. (b) Warin g, M. J. ; Ar ro ws mith , J. ; Leach , A. R.; Leeso n , P. D.; Man d rell, S.; Owen , R. M.; Pairau d eau , G.; Penn ie, W. D.; Pickett, S. D.; Wan g, J.; Wallac e, O.; Weir, A. Nat. Rev. Drug Discov. 2015, 14, 475 - 486. 18 INTRODUCTION The later refers to the increase in the likelihood of binding to multiple targets, such as the human Ether-? -go-go-Related Gene (hERG) ion channel, that can result in the appearance of side effects and toxicology. A deeper study performed by GSK from the ADMET profile and physicochemical data of ~ 30,000 molecules revealed that compounds that display a clog P < 4 appear to be optimal for achieving appropriate physicochemical characteristics to ensure downstream drug success.84 Table 2 summarizes the effects of the molecular weight and the clog P in the main set of PK assays applied in industry. Because of approximately two -thirds of all existing drug entities are classified as basic or acidic molecules,85 these two types are the ones disclosed below. Table 2. Influence of the key molecular properties on the ADMET parameters.84 Basic molecules MW < 400 and clog P < 4 MW > 400 and clog P > 4 Solubility high/average low/average Permeability high/average average Bioavailability average low Volume of distribution high/average high Plasma protein binding low average CNS penetration high/average average/low Brain tissue binding low high P-gp efflux average high/average in vivo clearance average high/average hERG inhibition average/high high CY P450 inhibition low 1A2, 2C9 & 2C19 inhibition low 1A2 inhibition CYP 450 inhibition average 2D6 & 3A4 inhibition average 2C9 & 2C19 inhibition P-450 inhibition high 2D6 & 3A4 inhibition 84 Glee so n , M. P. J. Med. Chem. 2008, 51, 817 - 83 4. 85 Charifso n , P. S.; Walt ers, W. P. J. Med. Chem. 2014, 57, 97 01 - 97 17. Adamantane nucleus: Introduction 19 Acidic molecules MW < 400 and clog P < 4 MW > 400 and clog P > 4 Solubility high average/high Permeability low average/low Bioavailability average average Volume of distribution low low Plasma protein binding average/h igher high CNS penetration low low Brain tissue binding low high P-gp efflux low low in vivo clearance low/average average hERG inhibition low low CYP 450 inhibition low 1A2, 2C9, 2C19, 2D6 & 3A4 inhibition low 1A2, 2C19, 2D6 & 3A4 inhibition CYP 450 inhibition high 2C9 inhibition As mentioned in section 1.2.1, having an adamantane present in a test drug gives a molecule of significantly higher lipophilicity compared to a molecule with just a proton or a methyl group instead. Specifically, the log P increases 3.1 log units when an adamantane is incorporated into a molecule. An increase in lipophilicity can be beneficial, but there is obviousl\ a ?size limit? oI the lipophilic add-on. Considering the above mentioned, it seems evident that the adamantane ring does not always possess the optimal properties as a scaffold in medicinal chemistry. A decrease in the overall lipophilicity will lead to compounds with an improved PK profile, which will thereIore be more ?drug-liNe?86 In this sense, there is an urgent need for the development of new scaffolds that could provide compounds with more optimal physicochemical properties to avoid attrition in later stages of the drug discovery process. 1.4.2 Ready access to intermediates that prevent scaffold opt imization Taking into account what it has been stated so far, it is logical to wonder why the adamantane ring is widely used in medicinal chemistry. And the answer is simple. Apart from the fact that in some cases the adamantane ring is the more suitable structure for any taken target, the easy access to adamantyl intermediates restrains the use of similar, yet disparate, analogues. The adamantane-based pharmaceuticals are derived from a small library of adamantane derivatives, mostly bearing simple functional groups, like amines or carboxylic 86 Zhan g, M . - Q . Methods Mol. Biol. 2012, 803, 29 7 - 3 07. 20 INTRODUCTION acids. These intermediates are commercially available from common suppliers, such as Sigma-Aldrich, Fluorochem or TCI. In particular, from 100 to 150 derivatives can be easily purchased from their webpage in a timely and cost effective way, at least for the developed countries. For this reason, the adamantane ring is considered as a standard lipophilic building block in test drug library syntheses. Frequently, the industry, with its desire to make more and more compounds for testing in a fast turnaround time, makes no efforts in the optimization of this kind of polycycles, since medicinal chemistry strategies around other functionalities and structures of parent molecules are more straightforward. However, the employment of similar polycycles as adamantane-like analogues can overcome the related issues to the use of the adamantane itself. Although some of the alternative cores entail tedious and long synthetic routes, others can be prepared in a synthetically feasible way. 1.4 .3 Adamantane group as an imperfect space-filling pharmacophore The lipophilic cage structure of adamantane has been used to rigidify the pharmacophore. True is the fact that in some cases the adamantane ring possesses the optimum molecular dimensions to functionally hit its target. Notwithstanding that, the adamantane does not always fill perfectly the active site of the desired target. Its dimensions are limited and the adamantane moiety can exceed or lose in filling the space of the target cavity. The binding constant for the ligand-receptor interaction can be optimized by varying the shape, orientation and flexibility of the scaffold. Since adamantane is a rigid structure with a well-defined geometry, the manipulation of this structure can give rise to several different scaffolds with diverse effects on its interaction to the target. Several research groups have worked, and still are, in the development of different polycycles for their use in diverse targets as surrogates of the adamantane ring with promising results. These adamantane-like scaffolds have been applied in targets such as influenza A and vaccinia viruses,87 or against CNS disorders, targeting the ?-aminobutiric acid (GABA) or 5-HT 2 receptors of serotonin, among others. 88 , 89 This issue will be examined in detail in the section that follows. 1.5 Adamantane alternatives: previous work of the group Over the last few years, the research group headed by Dr. Santiago V?zquez Cruz has gained a wide expertise in the synthesis of adamantane-like polycyclic scaffolds and their application to different therapeutical targets. In this regard, ring-expanded, ring -contracted, oxa -derivatives and related cage compounds have been explored by the group as analogues 87 Jord an , R.; Bailey , T. R.; R ip p in , S. R.; Dai, D. WO 20081 303 48, 2008. 88 Zoid is, G.; Pap an astasio u , I.; Dot sika s, I.; San d o val, A. ; D os San to s, R. G. ; Pap ad o p o u lou - Daifoti, Z.; Vamvak id es, A.; Kolo cou ris, N .; Felix, R. Bioorg. Med. Chem. 2005, 13, 279 1 - 2 798. 89 Becker, D. P.; Flyn n , D. L.; Shon e, R. L.; Gullikson , G. Bioorg. Med. Chem. Lett. 2004, 14, 55 09 - 55 12. Adamantane nucleus: Introduction 21 to the drugs amantadine, memantine and rimantadine. Figure 11 summarizes the mentioned polycycles. Fig. 11 . Polycyclic scaffolds applied in different therapeutic targets by the group. The potential of these polycycles to substitute the adamantane ring in known bioactive compounds has been proven in multiple cases for a few targets. At the beginning of this thesis, three main targets had been hit with these cage structures: ? the M2 channel of influenza A virus90,91,92,93,94 90 Duqu e, M. D.; Ma, C.; Torre s, E.; Wan g, J.; Nae sen s, L.; Ju?r ez - Ji m?n ez, J.; Camp s, P. ; Lu q u e, F. J.; DeGra d o , W. F.; La mb , R. A.; P in to , L. H.; V?zq u ez, S. J. Med. Chem. 2011, 54, 264 6 - 26 57. 91 Rey - Carrizo , M.; Torre s, E.; Ma, C.; Barn iol - X icot a, M.; W an g , J.; Wu , Y.; Naes en s, L.; Degrad o , W. F.; Lamb , R. A.; Pin to , L. H.; V?zq u ez, S. J. Med. Chem. 2013, 56 , 9265 - 9 274. 92 Torre s, E.; Lei va, R .; Gaz za rrin i, S.; R ey - C ar rizo , M .; Fri gol? - Viva s, M.; Moro n i, A.; Nae sen s, L.; V?z q u ez, S. ACS Med. Chem. Lett. 2014 , 5, 831 - 83 6. 93 Rey - Carrizo , M.; Barn iol - Xico t a, M.; Ma, C.; Frigol? - Vi vas, M .; Torre s, E.; Naesen s, L.; Lla b r?s, S.; Ju?r ez - Jim ?n ez, J. ; Lu q u e, F. J.; Degra d o , W. F.; Lamb , R. A.; Pin to , L. H.; V?z q u ez, S. J. Med. Chem. 2014 , 57, 57 38 - 5747. 94 Rey - Carrizo , M.; Gaz za rrin i, S.; Lla b r?s, S.; Frigol ? - V iva s , M.; Ju?r ez - J im ?n ez, J.; Fo n t - Bard ia, M.; Naesen s, L.; Moro n i, A.; Lu q u e, F. J.; V?zq u ez, S. Eur. J. Med. Chem. 2015, 96, 318 - 329 . 22 INTRODUCTION ? and the NMDA receptor of glutamate and the parasite Trypanosoma brucei.95,96,97,98 The particular and unique shape and size of each scaffold has given new insights into the binding mode of the different targets, as well as a better understanding of how to rationally design compounds with improved activities and physicochemical properties. The chief idiosyncrasy of the new polycycles is the different space-filling that they provide within their target. The results that the group has obtained have evidenced the unsuitable fit of the adamantane ring into the different active sites of the considered targets. Probably, the research around the inhibition of the M2 channel is the most clear and obvious example of this i nappropriate space-filling of the adamantane. Briefly, inhibition of M2 channel by amantadine became futile with the appearance of mutant strains, namely L26F, V27A and S31N.99 These amantadine-insensitive M2 channels display a different pore size, rendering the aminoadamantane ineffective. The design and synthesis of a wide array of adamantane-like scaffolds have provided molecules with greater activities against the wild-type than amantadine, establishing that the latter was not fully optimized inside the M2 channel lumen. Furthermore, enlarged derivatives have displayed interesting inhibitory activities against both wild-type and the V27A M2 mutant protein (Fig. 12). 95 Camp s, P.; Duq u e, M. D.; V?zq u ez, S.; Naesen s, L.; De Clerc q , E.; Sured a, F. X.; L?p ez - Qu er o l, M.; Camin s, A.; Pall? s, M.; Pra th alin gam, S . R.; Kelly, J. M.; Rom ero , V. ; Iv orra , D.; Cort ?s, D. Bioorg. Med. Chem. 2008 , 16, 9925 - 993 6. 96 Duqu e, M. D.; Camp s, P.; Pro fire, L.; Mon ta n er, S.; V?z q u ez, S.; Sured a, F. X.; Ma llol, J.; L?p ez - Qu ero l , M.; Naes en s, L.; D e Cl ercq , E.; Pra th alin gam, S. R.; Ke lly, J. M . Bioorg. Med. Chem. 2009, 17 , 3198 - 3 206. 97 Duqu e, M. D.; Camp s, P.; Torre s, E.; Valverd e, E.; Sured a, F. X.; L?p ez - Qu ero l, M .; Camin s, A. ; Pra th alin gam, S. R.; Ke lly, J. M .; V?zq u ez, S. Bioorg. Med. Chem. 2010, 18, 46 - 57. 98 Torre s, E.; Du q u e, M. D.; L?p ez - Qu ero l, M.; Tay lor, M. C.; Naesen s, L.; Ma, C.; Pin to , L. H.; Sured a, F. X.; Kelly, J. M.; V?z q u ez, S. Bioorg. Med. Chem. 2012, 20, 942 - 9 48. 99 Gu, R. - X.; Liu , L. A.; W an g, Y. - H .; Xu, Q.; Wei, D. - Q . J. Phys. Chem. B 2013, 117, 6 042 - 605 1. Adamantane nucleus: Introduction 23 Fig. 1 2. Representation of the predicted binding mode (down, up) of amantadine (shown as gray sticks) and two different polycyclic scaffolds (shown as orange and yellow sticks) in the interior of the wild type M2 channel and its V27A variant.94 With these encouraging results in hand, much is to be expected for adamantane-like scaffolds as modifiers or enhancers of active pharmacophores. This uncharted territory certainly holds great promise for the pharmaceutical industry. CHAPTER 1 : NMDA receptor antagonis m Introduct ion Introduction 29 1. NMDA receptor antagonism by adamanta ne-like scaffolds The following section will introduce the glutamatergic system and the NMDA receptor (NMDAR), as well as their role in the development of neurodegenerative disorders. More in detail, the adamantane-based compound memantine will be discussed as NMDAR antagonist along Zith its application in the Alzheimer?s disease (AD). Finally, a review of the previous work of the group related to the discovery of new NMDAR antagonists will be covered. 1.1 The glutamatergic neurotransmitter system The amino acid glutamate is the main excitatory neurotransmitter and is involved in almost all CNS functions, especially in cortical and hippocampal regions, hence being crucial for the normal functioning of the brain (Fig. 14a).100 It is released upon depolarization of the nerve terminals, which accumulate the glutamate in its inactive form glutamine.101 Once in the extracellular fluid, the glutamate exerts its signalling function by binding to three different ionotropic receptors, which are classified according to their selective, synthetic agonists (Fig. 14b): ?-amino-3-hydroxy -5-methyl-4-isoxazolepropi onic acid (AMPA), kainate, and NMDA receptors. These glutamate-gated ion channels are permeable to Ca2+ , Na+ and/or K+ ions.102 Fig. 1 4. Glutamic acid (a) and its analogues (b). The compounds are represented in their neutral form, and named accordingly. Worth to mention is that apart from the ionotropic receptors, glutamate binds also to metabotropic receptors, which are coupled to G-proteins and are divided into three major groups, I-III.103 1.1.1 NMDA receptor and its physiological function The tetrameric assembly of the NMDA receptor confers a three-dimensional channel that allows the influx of Ca 2+ and Na+ ions and the efflux of K + ions when opened. The NMDA receptor is unique among the ionotropic receptors since it needs the presence of two agonists for its activation; glutamate and glycine.104 Albeit these are the two essential 100 Watk in s, J. C.; Evan s, R. H. Annu. Rev. Pharmacol. Toxicol. 1981, 21, 165 - 204. 101 Dan b o lt, N. C. Prog. Neurobiol. 2001, 65, 1 - 105. 102 Tray n eli s, S. F.; Wol lmu th , L. P.; McBa in , C. J.; Men n iti, F. S .; Van ce, K. M. ; Ogd en , K. K. ; Han sen , K. B. ; Yuan , H.; My ers, S. J. ; Dingl ed in e, R. Pharmacol. Rev. 2010, 62 , 405 - 496. 103 Niswen d er, C. M.; Con n , P. J. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 295 - 32 2. 104 Kleckn er, N. W.; Din gl ed in e, R. Science 1988, 241, 835 - 837 . 30 CHA PTER 1: NMDA receptor antagonism ligands, the NMDA receptor features several modulating binding sites for, for instance, polyamines.105 The endogenous channel blocker is the Mg2+ ion, whose binding site is within the pore (Fig. 15).106 The activation of the receptor is voltage-dependent, whereupon the Mg2+ ion is displaced. Thus, for the permeation of Ca2+ ions the two following conditions need to be fulfilled: activation by endogenous ligands, and depolarization of the neuron, for example prior activation of AMPA or kainate receptors. Fig. 15. Schematic representation of the NMDA receptor, including the topology and pharmacological recognition sites.107 The X-ray crystal structures of the intact heterodimeric and heterotetrameric NMDA receptor have been published recently almost simultaneously in two of the most distinguished journals; Science and Nature. 108,109 The NMDA receptor can be formed by combination of three different subunits designated as GluN1, GluN2A-D, and GluN3 (also referred as NR1, NR2 and NR3 in former literature). However, the heterotetramers are chiefly composed of two copies each of GluN1 and GluN2, where glycine binds to GluN1 and glutamate to GluN2.110 The overall structure of the NMDA receptor possesses a modular domain architecture, with extracellular amino -terminal domains (ATDs) and ligand-binding domains (LBDs), a transmembrane domain (TMD) and an intracellular carboxy -terminal domain (CTD) (Fig. 16). The discovery of the crystal structure of the NMDA receptor offers new insights into the ion channel function and the allosteric binding of modulators and antagonists. 105 Chris Parso n s w eb page. Projects : NMDA re cep to rs. htt p :// ww w.ch ri sp ar so n s.d e/Ch ri s/n md a. h tm (acce ss ed on 11 th Septemb er 2015). Dr. Christo p h er Par so n s is the head of the In vitro Ph ar maco logy at Merz, the phar maceut ical co mp an y that develop ed me ma n tin e. 106 Bleich, S.; Ro mer, K. ; Wiltfan g , J.; Korn h u b er, J. Int. J. Geriatr. Psychiatry 2003, 18, S 33 - S4 0. 107 Parso n s, C. G. ; St? f fler, A.; Da n y sz, W. Neuropharmacology 2007, 53, 699 - 72 3. 108 Kara ka s, E.; Furu ka wa, H. Science 2014, 344, 992 - 997. 109 Lee, C. - H . ; L? , W.; Mich el, J. C.; Goeh rin g, A.; Du, J. ; Son g, X. ; Gou au x, E. Nature 2014, 511, 191 - 19 7. 110 Kara ka s, E.; Regan , M. C.; Fu r u ka wa, H. Trends Biochem. Sci. 2015, 40, 328 - 33 7. Introduction 31 Fig. 16. Overall structure of the heterotetrameric GluN1a-GluN2B NMDA receptor. Protein Data Bank (PDB) code: 4PE5.108 A further significant characteristic of the NMDA receptor is its slow gating kinetics, which control the postsynaptic Ca2+ levels in physiological conditions. The influx of calcium ions triggers signal transduction cascades that control the strength of neural connectivity and neuroplasticity.111 The specific attributes of the activation of the NMDA receptors by glutamate are accepted to be an intrinsic synaptic mechanism for cognition, learning and memory processes.112 1.1.2 Glutamate and related pathological states Normal glutamate receptor activity mediates, in large measure, physiological excitatory synaptic transmission in the brain, as just mentioned. The maintenance of low extracellular levels of glutamate is essential for controlling the CNS functions.101 However, in a variety of pathological conditions, including various neurodegenerative disorders, there is an excessive activation of the NMDA receptor by the glutamate that leads to an increase in the intracellular calcium concentration. This contributes to the subsequent formation of 111 Dan y sz, W.; Parso n s, C. G. Br. J. Pharmacol. 2012, 167, 324 - 352. 112 Butt erfield , D. A. ; Pocern ich, C. B. CNS Drugs 2003, 17, 64 1 - 652. 32 CHA PTER 1: NMDA receptor antagonism harmful free radicals and activation of proteolytic cascades which eventually cause cell damage or death (Fig. 17).113,114 Fig. 17. Cellular apoptotic pathways triggered by excessive NMDAR activation. a) NMDAR hyperactivation; b) activation of the p38 mitogen-activated kinase (MAPK)-MEF2C pathway; c) toxic effects of free radicals such as ROS; and d) activation of apoptosis -inducing enzymes. 115 This overactivation of the NMDAR is attributable to a combination of different occurrences. Firstly, glutamate is not cleared properly and/or can even be inappropriately released during acute and chronic neurodegenerative disorders.115 Secondly, under these conditions, neurons become depolarized in a sustained manner and the cells cannot maintain their ionic homeostasis. This contributes to the permanent liberation of the Mg2+ by NMDAR.116 The combination of some or all of these events leads to the so-called excitotoxicity, which is defined as cell death from the toxic ity of an excessive action of excitatory amino acids, such as glutamate. 117 113 Lip to n , S. A.; Rosen b erg, P. A. N. Engl. J. Med. 1994, 330, 613 - 622 . 114 Lip to n , S. A.; Nicoreta, P. Cell Calcium 1998, 23, 165 - 17 1. 115 Lip to n , S. A. Nat. Rev. Drug Discov. 2006, 5, 160 - 170. 116 Zeevalk , G. D.; Nickla s, W. J. J. Neurochem. 1992, 59, 1211 - 1220. 117 Herrman n , N.; Li, A.; Lan ct? t, K. Expert Opin. Pharmacother. 2011, 12, 787 - 80 0. Introduction 33 According to the aforementioned, disorders including Alzheimer?s 3arNinson?s and +untington?s diseases, depression, schizophrenia, ischemic injuries associated with stroke , HIV -associated dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain and glaucoma share a final common pathway to neuronal damage and death, i.e. overstimulation of the NMDAR and excitotoxicity .112,118,119,120 NMDAR antagonism could therefore potentially be of therapeutic interest in a number of neurological disorders. 1.2 NMDA as a therapeutic target The pursuit of strategies for the development of neuroprotective agents that are both effective and well tolerated has been bourgeoning in the last decades.121 Theoretically, any disorder of the CNS characterized by glutamate excitotoxicity -induced neuronal death, should be relieved by a treatment with NMDA receptor antagonists. In this sense, several NMDAR antagonists have been developed disclosing different binding modes and clinical tolerations.122,123,124 1.2.1 Competitive NMDAR antagonists NMDAR antagonists that compete directly with glutamate at its binding site block the natural neuronal communication. Since the desired therapy must re-establish the normal function of this excitatory neurotransmitter, the development o f competitive NMDAR blockers was dismissed due to the appearance of intolerable side effects. A few compounds have been identified as competitive NMDAR antagonist, although their use has been reduced only to comparative studies and target validation (Fig. 18).125,126,127,128,129 118 Lange, K. W.; Korn h u b er, J.; R ied erer, P. Neurosci. Biobehav. Rev. 1997, 21, 393 - 40 0 . 119 Cioffi, C. L. Bioorg. Med. Chem. Lett. 2013, 23, 5034 - 5044 . 120 Ramm es, G. ; Dan y sz, W.; Par so n s, C. G. Curr. Neuropharmacol. 2008, 6, 55 - 78. 121 Lip to n , S. A. Nat. Rev. Neurosci. 2007, 8, 803 - 808. 122 Kemp , J. A . ; McK ern an , R. M. Nat. Neurosci. 2002, 5 (Sup p l.) , 1039 - 1042. 123 Koller, M.; Ur wy ler, S. Expert Opin. Ther. Pat. 2010 , 20, 168 3 - 170 2. 124 Stro n g, K. L.; Jin g, Y. ; Pro s ser, A. R.; Tray n e lis, S. F.; L iot ta , D. C. Expert Opin. Ther. Pat. 2014 , 24, 134 9 - 1366. 125 Abrah am, W. C.; Mason , S. E. Brain Res. 1988, 462, 40 - 46. 126 Lehman n , J.; Schn eid er, J.; McPh erso n , S.; Mu rp h y , D. E.; Bern ar d , P.; Tsai, C.; Ben n ett , D. A . ; Pasto r, G.; Ste el, D. J. ; Boeh m, C. J. Pharmacol. Exp. Ther. 1987, 240 , 737 - 74 6. 127 Svein b jo rn sd o tt ir, S.; San d er, J. W.; Upto n , D.; Thomp so n , P. J.; Pat salo s, P. N. ; Hirt, D.; Em re, M.; Lo we , D.; Dun can , J. S. Epilepsy Res. 1993, 16, 165 - 17 4. 128 Davis, S. ; Butch er, S. P.; Mo rr i s, G. M. J. Neurosci. 1992, 12 , 21 - 34 . 129 Yenar i, M. A. ; Bell, T. E.; Kot a ke, A. N.; Pow ell, M.; Stein b er g, G. K. Clin. Neuropharmacol. 1998 , 21, 2 8 - 34. 34 CHA PTER 1: NMDA receptor antagonism Fig. 18 . Structures of competitive NMDAR antagonists. CPP: 3-(2-carboxypiperazin -4-yl)propyl- 1-phosphonic acid; ?-AP5: ? -2-amino-5-phosphonopentanoate. Recently, novel competitive antagonists with preference for the glycine-binding site in the GluN3B subunit have appeared, such as TK80 (Fig. 19).130 Their discovery have clarified the structural differences between the orthosteric binding site of GluN1 and GluN3. Fig. 1 9. Structure of a GluN3B subunit antagonist. 1.2.2 Uncompetitive NMDAR antagonists Before explaining this type of antago nism, it is worth distinguishing it from the non- competitive one. The latter refers to the allosteric binding of the antagonist whose availability is not affected by the concentration of agonist ? in this case glutamate ? whereas in the uncompetitive antagonism the inhibitory effect depends on prior activation of the receptor by the agonist.121,122 The majority of the uncompetitive NMDAR antagonists block the ion channel by occluding the pore, in a similar way to the Mg2+ ion. Among them, two classes of antagonists are differentiated depending on their off-rate constant: - High-affinity blockers: these antagonists show slow ?oII? constants and loZ voltage dependency. These features render the NMDA receptor virtually almost fully occupied by the blocker, which cannot be stimulated under physiological conditions. The inability of the receptor to recover from the inhibition is reflected in the appearance of side effects on the CNS, such as hallucination, agitation, catatonia, centrally-mediated increase in the blood pressure and anaesthesia. Indeed, some of these antagonists have been developed as anaesthetics only, like ketamine and phencyclidine (Fig. 20).131,132 130 Kvist, T. ; Gre en wo o d , J. R.; Han sen , K. B.; Tray n elis, S. F. ; Br?u n er - O sb o rn e, H. Neuropharmacology 2013, 75, 324 - 33 6. 131 Son ku sar e, S. K.; Kau l, C. L.; R amara o , P. Pharmacol. Res. 2005 , 51, 1 - 1 7. 132 Michau d , M.; Warren , H.; Drian , M. J.; Ramb au d , J.; Cerr u ti, P.; Nicolas, J. P.; Vigno n , J.; Privat , A. ; Kamen ka , J. M. Eur. J. Med. Chem. 1994, 29, 869 - 876. Introduction 35 Fig. 20 . High -affinity uncompetitive NMDAR antagonists. PCP: N-(1- phenylcyclohexyl)piperidine; TCP: N-[1 -(2-thienyl)cyclohexyl]piperidine. Ketamine is represented as racemate. Besides, ketamine, dizocilpine and analogues of phencyclidine were studied as anticonvulsants, albeit their high neurobehavioural toxicity. 133,134 - Low- and moderate-affinity blockers: offset kinetics are inversely related to affinity, and low- and moderate-affinity compounds show faster relief of blockade upon removal of the antagonist or upon physiological glutamatergic activation than high-affinity inhibitors.135 This critical characteristic allows the inhibition only in pathological conditions, leaving physiological functions unaltered. Moreover, low- and moderate- affinity blockers have a better therapeutic window than high-affinity blockers.122 For these reasons, low- and moderate-affinity antagonists have been more successful in clinical studies than the ones displaying high affinity. A few number of compounds have been identified as low- and moderate-affinity NMDAR antagonists in the last decades (Fig. 21).136,137,138,139,140 Recently, propellanamines have appeared with an affinity similar to amantadine and memantine to the PCP binding site.141 133 White , J . M .; Ryan , C . F . Drug Alcohol Rev. 1996, 15, 145 - 15 5 . 134 Dora n d eu , F.; Dh ote, F.; Barb i er, L.; Baccu s, B. ; Testy li er, G. CNS Neurosci. Ther. 2013, 19, 411 - 4 27. 135 Parso n s, C. G. ; Dan y sz, W.; Q uack , G. Amino Acids 2000, 19 , 157 - 16 6. 136 Blan p ied , T. A; Clark e, R. J.; Jo h n so n , J. W. J. Neurosci. 2005 , 25, 3312 - 3322. 137 Ramm es, G. Expert Rev. Clin. Pharmacol. 2009, 2, 231 - 2 38. 138 Matt ia, C.; Colu zz i, F. Drugs 2007, 10, 636 - 644 . 139 Kiewert, C.; Hartman n , J.; Sto ll, J.; Thekk u mka ra , T. J.; Van der Schy f, C. J.; Klein , J. Neurochem. Res. 2006, 31, 395 - 39 9. 140 Roga wski, M. A. Amino Acids 2000, 19, 133 - 14 9. 141 Torres - G? mez, H.; Leh mku h l, K.; Freh lan d , B.; Dan iliu c, C.; Schep man n , D.; Ehrh ar d t, C.; W? n sch , B. Bioorg. Med. Chem. 2015, 23 , 4277 - 4285. 36 CHA PTER 1: NMDA receptor antagonism Fig. 21. Structures of low- and moderate-affinity uncompetitive NMDAR antagonists. 1.2.3 Non-competitive NMDAR antagonists Whilst competitive antagonists bind to the same binding site as glutamate or glycine, and the uncompetitive antagonists we have seen so far exert their interactions within the ion channel, other types of blockers owe their activity by binding to allosteric sites. This is the case for the compounds ifenprodil and eliprodil, whose binding site is located in the GluN2B subunit (Fig. 22).142,143 Ifenprodil has shown considerable attenuation of the neurotoxicity in animal models. 144 Ifenprodil and ifenprodil-like inhibitors have a reduced affinity and unbind from inactivated receptors leaving transiently activated receptors relatively unaffected.145 Other high-affinity GluN2B ligands that modulate NMDAR activity have appeared recently, such as benzo[7] annulen-7-amines.146 Similar to ifenprodil, TCN-201 binds also to allosteric sites, but in this case to GluN2A.147 Many other allosteric modulators have emerged with distinct binding regions all along the ion channel.148 142 William s, K. Curr. Drug Targets 2001, 2, 285 - 29 8. 143 Parso n s, C. G. ; Dan y sz, W.; Q uack , G. Neuropharmacology 1999, 38, 735 - 76 7. 144 Gott i, B. Duverg er, D; Berti n , J.; Carter, C.; Dup o n t, R.; Frost, J.; Gau d illi ere, B.; Ma c Ken zie, E. T.; Rou ss eau , J.; Scatt o n , B.; Wick , A. J. Pharmacol. Exp. Ther. 1988 , 247, 121 1 - 1 221 . 145 Kew, J. N. C. ; Tru b e, G .; Ke mp , J. A. J. Physiol . 1996, 497, 7 6 1 - 772 . 146 Gawaska r, S. ; Schep man n , D.; Bon ifaz i, A.; W? n sch , B. Bioorg. Med. Chem . 2014, 22, 6638 - 6646 . 147 Bett in i, E.; Sava, A. ; Gri ffan te, C.; Cari gn an i, C.; Buso n , A. ; Ca p elli, A. M.; N egri, M.; And reet ta , F.; Sen ar - S an ch o , S. A.; Guiral, L.; Card u llo, F. J. Pharmacol. Exp. Ther. 2010 , 335, 63 6 - 6 44. 148 Regan , M. C.; Ro mero - H ern a n d ez, A.; Furu ka wa, H. Curr. Opin. Struct. Biol. 2015, 33, 68 - 75. Introduction 37 Fig. 22 . NMDAR antagonists with allosteric binding mode. Inasmuch as the sole compound that has reached the market as NMDAR antagonist is memantine, a broader analysis will be performed thereof. 1.3 The neuroprotector memantine Memantine is a low- to moderate-affinity uncompetitive NMDAR antagonist, with strong voltage dependency and rapid blocking and unblocking kinetics.120 Thanks to these particular qualities, memantine shows the effect known as partial trapping, where around one sixth of the ion channels remain free of the antagonist under resting conditions and are directly available for physiological transmission, unlike in the presence of high-affinity antagonists.149 Thus, memantine blocks the neurotoxicity of glutamate in pathological conditions without interfering with its physiological actions required for learning and memory (Fig. 23).111 149 Blan p ied , T . A.; Boe ckman , F . A.; Aizen man , E.; Joh n so n , J . W. J. Neurophysiology 1997, 77, 309 - 3 23. 38 CHA PTER 1: NMDA receptor antagonism Fig. 2 3. Schematic illustration of the glutamatergic system involving the NMDA receptor based on the signal-noise hypothesis. A) Normal physiological transmission of glutamate resulting in a sufficient signal-to-noise ratio. B) In neurodegenerative disorders, like in AD, there is a sustained activation of the NMDARs, which leads to the rise of the synaptic noise. The impaired detection of the relevant synaptic signal hampers the learning and plasticity processes. C) Synaptic plasticity recovery by antagonism with memantine.111 Further studies indicate that memantine exer ts its effect by binding at or near the Mg2+ site within the ion channel (Fig. 24).150,151 More precisely, it is believed that memantine is placed with the charged nitrogen close to the critical NMDAR channel asparagines. At the time of writing this thesis, there is any resolved structure of the NMDAR with a channel blocker. However, a recent computational study has revealed that the excellent affinity of memantine is due to the presence of hydrophobic binding pockets in the binding site for the two methyl groups of memantine. The removal of these two groups, leading to amantadine, or the addition of a third methyl group diminished affinity.152 150 Chen, H. S. V.; Lip to n , S. A. J. Pharmacol. Exp. Ther. 2005, 314 , 961 - 971. 151 John so n , J. W.; Gla sgo w, N. G.; Povy sh e va, N. V. Curr. Opin. Pharmacol. 2015, 20, 54 - 6 3. 152 Limap icha t, W.; Yu, W. Y.; Bran igan , E. ; Lester, H. A.; Dou gh erty , D. A . ACS Chem. Neurosci. 2013, 4, 255 - 2 60. Introduction 39 Fig. 2 4. Images of NMDAR channel blocked by memantine, representing the likely location of memantine binding site. PDB code: 4TLM.109 Memantine was approved by the EMA in 2002 and by the FDA in 2003 for the treatment of moderate to severe AD.153,154 AD is the most common type of dementia, and there is no eIIective cure The American Alzheimer?s association estimated current cost to the healthcare system of $250 billion, and the number of patients to rise by three times by 2050.155 In consequence, there is an urgent need to discover new therapeutics for the treatment of AD. Memantine has shown modest efficacy in randomized controlled trials in improving cognition, function, and global status, either as monotherapy or in combination with acetylcholinesterase inhibitors.117,156,157 Memantine has also been shown to improve behavioural disturbances such as agitation and aggression.158 In other dementias, memantine has not yet been shown to be beneficial, although an intense research is being conducted around that matter.159 Besides, memantine has been studied for different conditions, such as +untington?s disease160 depression,161 DoZn?s s\ndrome162 and drug addiction.163 153 Euro p ean Med icin es Ag en cy , Ebixa (meman tin e ). htt p ://ww w.e ma. eu ro p a. eu / e ma/in d ex.j sp ?cu rl =p age s/m e d icin es/h u man / med i cine s/00 0463/h u man _ med _ 0007 50.jsp & mid =W C0b 01ac058 001d12 4 (acc es sed o n 21 st May 2015). 154 FDA ap p ro ve s me man tin e dru g for treat in g AD. Am. J. Alzheimers Dis. Other Demen. 2003, 18 , 329 - 3 30 . 155 Al?heimer?s association. h tt p : //w ww.a lz.o rg / (acc es sed on 31 st Augu st 2 015). 156 Anan d , R.; Gill, K. D.; Mah d i, A. A. Neuropharmacology 2014 , 76, 27 - 50 . 157 Parso n s, C. G. ; Dan y sz, W.; D eku n d y , A.; Pu lte, I. Neurotox. Res. 2013, 24, 35 8 - 3 69. 158 Herrman n , N.; Cap p ell, J.; Ery avec, G. M.; Lan ct? t, K. L. CNS Drugs 2011 , 25, 425 - 433. 159 Peng, D. ; Yuan , X.; Zh u , R. J. Clin. Neurosci. 2013, 20, 1482 - 1485. 160 Broca rd o , P. S.; Gil - Mo h ap el, J. M. Curr. Physicopharmacol. 2012, 1, 137 - 154 . 161 Dan g, Y. - H .; Ma, X. - C.; Zh an g, J. - C.; Ren , Q.; Wu , J.; Gao , C. - G.; Hashimot o , K. Curr. Pharm. Des. 2014, 20, 5151 - 515 9. 162 Costa , A. C. S. CNS Neurol. Disord. Drug Targets 2014, 13, 1 6 - 25. 163 Tomek, S. E.; La Cro s se, L. A.; Nemiro vsky , N. E.; Foster Ol iv e, M. Pharmaceuticals 2013, 6, 251 - 26 8. M em antine a) b) 40 CHA PTER 1: NMDA receptor antagonism On account to the outstanding mechanism of action of memantine and its success in the treatment of moderate to severe AD, numerous research groups from industry and academia have developed new NMDAR antagonist that are currently in distinct stages of the drug discovery process.124 Related to this, the group of Dr. Santiago V?zquez Cruz has discovered several compounds with activity as NMDAR antagonists. The following section will cover them. 1.4 Previous work of the group: new antagonists of the NMDAR As stated in the previous introduction, the group has a wide expertise in the synthesis of cage compounds and their application as bioactive molecules. In 2008, a new research line targeting the NMDA receptor emerged with the initial results of the polycyclic amines shown in Figure 25.95,96,164 Fig. 2 5. Memantine analogues with moderate to good activity as NMDAR antagonists prepared by the group. R1 and R2 are H, alkyl, methylene -spaced aryl, guanidine or acetamidine. R3 is H, alkyl or phenyl. Functional data were obtained from primary cultures of cerebellar granule neurons by measuring the intracellular calcium concentration. Cells were challenged with glutamate NMDA (100 ?M). Data shown are means of at least three separate experiments carried out on three different batches of cultured cells. Thereafter, several (1,2,3,5,6,7-hexahydro -1,5:3,7-dimethano-4-benzoxonin -3- yl)amines with general structure I, and 6,7,8,9,10,11-hexahydro -9-methyl-5,7:9,11- dimethano-5H-benzocyclononen-7-amines with general structure II, were synthesized by Dr. M. D. Duque and Dr. E. Torres and their activity against NMDAR was evaluated.97,98 Table 3 includes the more potent polycyclic compounds of the two series. 164 Duqu e, M. D. Ph .D. Diss erta ti o n , Univer sity of Barcelon a, 2 010. Introduction 41 Table 3. Benzo-homoxaadamantane , I, and benzo-homoadamantane, II, derivatives active as NMDAR antagonists. Functional data were obtained from primary cultures of cerebellar granule neurons by measuring the intracellular calcium concentration. Cells were challenged with glutamate NMDA (100 ?M). Data shown are means ? SEM of at least three separate experiments carried out on three different batches of cultured cells. Comp. R1 R2 R3 IC50 (?M) Comp. R1 R2 IC50 (?M) 10 H H H 35 ? 6.8 19 H H 13.6 ? 3.4 11 H Me H 6.0 ? 1.3 20 H Me 19.4 ? 3.3 12 H Me Me 3.8 ? 0.3 21 Me Me 11.8 ? 3.1 13 H Et Et 14 ? 1.1 22 -(CH 2)5- 101 ? 25 14 H -(CH 2)5- 30 ? 1.0 15 Me H H 98 ? 26 16 Me Me Me 3.9 ? 0.4 17 Me Et Et 7.7 ? 1.0 18 Et H H > 200 Overall, a few trends were noted: - Generally, tertiary amines were slightly more potent than their parent secondary amines, and the latter were more potent than the primary ones. For instance, compare 12 vs 11, and 11 vs 10. Also, small groups, such as methyl group, were more tolerated than large groups; see compound 12 vs 13, and compound 16 vs 17. - Furthermore, 8-oxapolycyclic primary amines were less potent than their corresponding 8-carba analogues. For example, compound 19 showed a 7-fold increase in potency compared to its oxapolycyclic analogue 15. - Of note, while memantine (IC50 = 1.5 ? 0.1 ? M), which features two methyl groups in its structure, is much more potent than amantadine (IC50 = 92 ? 29 ? M), compound 10 was more active than its methyl analogue, 15. - The introduction of an oxygen atom was deleterious for the activity, as well as bearing an alkyl group at the bridgehead position (Fig. 26). 42 CHA PTER 1: NMDA receptor antagonism Fig. 2 6. Observed trend in the benzopolycyclic scaffolds. The increase in potency goes from left to right. Objetive s Objectives 45 Bearing in mind what has been mentioned, it seems logical that derivative 23 which combines the best findings of our previously studies on benzo-adamantanic amines, that is a hydrogen atom at C-9 position and a methylene group in C-8 position (Fig. 27), may display a better inhibitory activity against the NMDA receptor than its predecessors. Fig. 27 . Previous results suggest compound 23 as a potential NMDAR antagonist. Considering this fact, we established the following goals of this chapter: 1. Synthesis and pharmacological evaluation of compound 23 and its corresponding methylated tertiary amine 24 (Fig. 28). The preparation of the secondary amine was discarded due to the preceding results, where tertiary amines proved to be the most potent derivatives. With this objective in mind, we aimed to confirm our hypothesis that both compounds would show higher potencies compared to previous derivatives. Fig. 2 8. Amines to synthesize as potential NMDAR antagonists. 2. Exploration of a structure-activity relationship (SAR) around the C-9 position, with the introduction of different groups such as halogens or a hydroxyl group (Fig. 29). This study would include the preparation of 5,6,8,9,10,11-hexahydro - 7H-5,9:7,11-dimethanobenzo[9]annulen -7-amines with general structure III, as well as their corresponding tertiary amines. We aimed to find the effect of the different substituents at the C-9 position on the inhibitory activity. 46 CHA PTER 1: NMDA receptor antagonism Fig. 29. Amines with general structure III. Results & Discussion Results & Discussion 49 1. Effect of the C -9 substitution in new benzopolycyclic compounds 1.1 Synthesis of the C9-demethylated compound, 23 According to the previous work of the group related to the synthesis of benzopolycyclic cage amines, a benzo-homoadamantane with a hydrogen atom at the C-9 position would fulfil the gap existing between the former families. H ence, following the SAR studies we envisaged compound 23 as a potential antagonist of the NMDAR with hypothetical improved potency (Fig. 30). Fig. 30 . Compound to prepare from rational design. The synthesis was designed based on prior synthetic routes described by our group. We conceived enone 25 as the starting point for the preparation of compound 23 (Scheme 2). This enone is a homoconjugated system that allows the 1,4-like addition of nucleophiles to the alkene, whereupon a transannular cyclization occurs with the attack to the carbonyl. This intramolecular through-space interaction betZeen the tZo ?-orbitals of 25 has been exploited in many transformation s, such as in the intramolecular cyclization through a Ritter transformation, as it will be discussed later on.165,166 Scheme 2. Enone 25 as a precursor of amine 23. In this way, we decided to prepare enone 25 following the procedure firstly reported by F?hlisch et al.167,168 and widely employed by our group. Starting from commercially available o-phthaldialdehyde and dimethyl-1,3-acetonedicarboxylate, a Weiss-Cook condensation took place affording tetraester intermediate 26. Taking advantage of the keto-enol tautomerism of dimethyl-1,3-acetonedicarboxylate, t he reaction proceeded through a double aldol condensation and subsequent dehydration, under basic catalysis. 165 Amin i; Bi sh o p , R. Aust. J. Chem. 1983, 36, 2465 - 24 72. 166 Amin i; Bish o p , R.; Burg es s, G.; Craig, D. C.; Dan ce, I. G.; S c u d d er, M. L. Aust. J. Chem. 1989 , 42, 1919 - 1928 . 167 F? hlisch , B.; Wid man n , E.; Sch u p p , E. Tetrahedron Lett . 1969 , 28, 2355 - 235 8. 168 F ? hlisch , B.; Du kek, U. ; Grae s sle, I.; Novo tn y , B.; Sch u p p , E.; Sch waig er, G.; Wid man n , E. Justus Liebigs Ann. Chem. 1973, 1839 - 18 50. 50 CHAPTER 1: NMDA receptor antagonism The second equivalent of the ?-keto ester underwent a double Michael addition to the ?,?- unsaturated ketone leading to the dienol-tetraester (Scheme 3). Scheme 3. Weiss-Cook condensation of o-phthaldialdehyde and two equivalents of dimethyl-1,3- acetondicarboxylate . Acid hydrolysis of the ester groups rendered ?-keto acids which decarboxylated spontaneously to furnish a mixture of diketone 27 and its hydrate 28 in a 1:3 ratio (Scheme 4). Of note, the amount of the hydrate increased over time because of the atmospheric moisture, and even in a short period of time the equilibrium was utterly shifted to the hydrate. In order to procure the pure ketone, the mixture was refluxed in toluene using a Dean-Stark apparatus to finally provide pure diketone 27 in 64% overall yield. 169 Scheme 4. Acid hydrolysis and decarboxylation of tetraester 26. 169 The prev iou s meth o d used fo r deh y d ra tio n of the hyd rate was to sub li mat e th e mi xt u re at 160 ? C an d 0.5 Torr. Results & Discussion 51 The next step was a Wittig reaction of a single ketone to form enone 25. This conversion was inspired from the Corey procedure for the Wittig reaction,170,171 and needed sodium hydride in anhydrous DMSO and methyltriphenylphosphonium iodide as the ylide counterpart. The double Wittig transformation of diketone 27 to form the corresponding diene 29 was earlier optimized by Dr. E. Torres, although using a large excess (8 equivalents) of NaH and of the Wittig reagent. 172 We believed that the reaction was poorly efficient due to the water content of the self-prepared anhydrous DMSO. Based on that, we aimed to optimize the mono-Wittig reaction adjusting the equivalents of both reagents and usi ng a fresh bottle of anhydrous DMSO purchased from a chemical supplier. A short optimization process was performed (Table 4). Worth to note is the formation of diene 29 with only 2.1 equivalents of each reagent (entry 3), compared with the 8 equivalents needed in the previous procedure. Table 4. Optimization process for the preparation of enone 25. Yields are of isolated products. Entry Ph3PCH 3I (eq.) NaH (eq.) 25 (% yield) 29 (% yield) 1 1.1 1.1 63 - 2 1.25 1.25 84 - 3 2.1 2.1 - 80 In this transformation, a mixture of NaH in anhydrous DMSO was heated to 75 ?C for 45 minutes so as to form the conjugate base of DMSO, which is the actual species that deprotonated the phosphonium salt upon its addition in order to obtain the ylide. Twenty minutes after, a solution of diketone 27 in anhydrous DMSO was added to the mixture and heated to 75 ?C overnight. The best conditions were the ones from entry 2 in table 4, with 1.25 equivalents of the base and of the ylide that furnished desired enone 25 in 84% yield. Apart from the reaction optimization, an easy and efficient purification process was required in order to remove the phosphine oxide, which in many cases can be tedious. With the aim of avoiding the purification by column chromatography, we came up with a handy method by means of packing the impure mixture with silica gel followed by extraction with an appropriate mixture of petroleum ether and diethyl ether. 170 Green wald , R.; Cha y ko v sky , M.; Corey, E. J. J. Org. Chem. 1963 , 28, 1128 - 11 29. 171 Coxo n , J. M.; Ma clagan , R. G. A. R.; McDon ald , D. Q.; St eel, P. J. J. Org. Chem. 1991 , 56, 2 542 - 2 549. 172 Torre s, E. Ph .D. Diss erta tio n , Unive rsity of Bar celon a, 2013. 52 CHAPTER 1: NMDA receptor antagonism Noteworthy, all the reactions so far were carried out in a multigram scale. With pure enone 25 in hand, we envisaged the preparation of amine 23 from alcohol 30 (Scheme 5). A bridgehead alcohol can be replaced by an amino group via different procedures, such as the already mentioned Ritter reaction,173 the metal-catalysed addition of ammonia to alcohols,174,175 or the nucleophilic substitution prior functional group interconversion to a better leaving group, like a halide or a mesylate group. Inspired by the work of Itoh et al.,176 where 1-adamantanol was obtained from 7- methylenebicyclo[3.3.1]nonan -3-one through an electroreductive transannular reaction, and by the boron chemistry for the 1,4-enone reduction,177 a 1,4-conjugated -like reduction of enone 25 would afford desired alcohol 30. Scheme 5. Plausible formation of alcohol 30 from enone 25 by a 1,4-conjugated -like reduction. Many hydrides have been applied as reductive agents in the 1,4-reduction of ?,?- unsaturated ketones. Especially, alkali metal trialkylborohydride reagents are excellent choices for enone reduction that have been proved to give very high regio- and stereoselectivities.178 Organoboranes such as lithium tri-sec-butylborohydride (L-selectride ? ) or lithium n-butylborohydride have been largely employed in multiple reductions, although 173 Fokin , A. A.; Merz, A.; Fok in a, N. A.; Schwertf eg er, H.; Liu , S. L.; Dah l, J. E. P.; Carlso n , R. K. M.; Schr ein er , P. R. Synthesis 2009, 6, 909 - 9 12. 174 Shimizu , K. - I.; Kan n o , S.; Kon , K.; Hak im Sid d iki, S. M. A.; Ta n ak a, H.; Sak at a, Y . Catal. Today 2014, 232, 134 - 1 38. 175 Sigl, M.; H eid e man n , T. Pro c es s for pr ep ar in g a primar y a min e with a tertiar y alp h a ca rb o n atom by react in g a tertia ry alcoh o l wit h ammo n ia. WO 2009 /053 27 5 A1. 176 Itoh , H.; Kat o , I.; Uno u ra , K.; Sen d a, Y. Bull. Chem. Soc. Jpn. 2001, 74, 339 - 34 5. 177 Burkh ar d t, E. R.; Mato s, K. Chem. Rev. 2006, 106, 261 7 - 2 65 0. 178 Fort u n at o , J. M.; Gan em, B. J. Org. Chem. 1976 , 41, 2194 - 2 200. Results & Discussion 53 1,2- or 1,4-hydride additions are mainly controlled by substrate structure.179,180,181 Hence, we treated enone 25 with L-selectride ? in anhydrous THF with or without tri ethylamine (TEA), and under short or longer reaction times, without success under any condition (Table 5). Table 5. Attempt to prepare alcohol 30 through reduction with lithium tri-sec-butylborohydride. Entry TEA Temp. (?C) Time (h) 30 (% yield) 1 -78 0.5 - 2 25 18 - On the other hand, sodium hydrogen telluride (NaHTe) was successfully employed in the selective 1,4-reduction of ?,?-unsaturated carbonyl compounds.182,183,184 However, the requirement of its in situ formation from telluride powder and sodium borohydride was reason enough to preclude its use. Then, we explored the 1,4 -conjugated -like reduction of enone 25 with other hydride sources such as sodium borohydride, which has been proved as an efficient reducing agent in several conjugated systems. 185,186 Even though in most cases the 1,2-reduction prevails, we wondered if in our homoconjugated system the 1,4 -reduction would overcome the 1,2- reduction (Scheme 6). Unfortunately, no expected compound was recovered but the 1,2- reduced product 31. Scheme 6. Reduction of the ketone after treatment of enone 25 with NaBH 4. 179 Martin , H. J.; Dres ch er, M.; M ulzer, J. Angew. Chem. Int. Ed. 2000, 39, 581 - 58 3. 180 thite, D. Z. ?? - Alky lsp ectin o my cins. U S 4532 3 36 A. 181 Kim, S.; Cho o n Moon , Y.; Han Ah n , K. J. Org. Chem. 1982 , 47 , 3311 - 3315. 182 Yamash ita , M.; Kato , Y.; Su e mitsu , R. Chem. Lett. 1980, 84 7 - 848 . 183 Bart o n , D. H. R.; Boh ?, L.; Lu si n ch i, X. Tetrahedron 1990, 46 , 5273 - 5284. 184 Yamash ita , M.; Tan ak a, Y.; Ari ta , A.; Nish id a, M. J. Org. Chem. 1994, 59, 3500 - 35 02. 185 March an d , A. P.; La Roe, W. D.; Shar ma, G. V. M. ; Suri, S. C.; Redd y , D. S. J. Org. Chem. 1986 , 51, 1622 - 1625. 186 Jack so n , W. R.; Zu rq iy ar , A. J. Chem. Soc. 1965, 5280 - 5 2 87. 54 CHAPTER 1: NMDA receptor antagonism Searching for an alternative approach for the preparation of alcohol 30, different publications reached our hands reporting the catalytic hydrogenation at atmospheric pressure of 7-methylenebicyclo[3.3.1]nonan -3-one to afford 1-adamantanol in good yields.187,188 To our disappointment, when this conditions were undertaken with enone 25, we observed the double bond migration of enone 25 to furnish ketone 32, as well as the dihydroxylated compound 33 (Table 6). The formation of the latter can be reasoned via the addition of a water molecule, coming from the moisture in the Pd/C , to the conjugated - like enone. The ratio of the resulting mixture depended on the reaction time and on the equivalents of the palladium catalyst. Table 6. Catalytic hydrogenation of enone 25 to provide different mixtures of 32 and 33. Entry Pd/C (eq.) Time (h) Solvent 25 (% yield) 32 (% yield) 33 (% yield) 1 0.1 64 DCM 43 - 21 2 0.1 64 Toluene 60 - 30 3 0.2 72 Toluene - 71 - 4 0.1 120 Toluene 50 - 50 5 0.4 64 Toluene 22 - 58 6 1 64 Toluene - 24 15 All these failures did not discourage us to persist in the quest for amine 23, so we pursued its synthesis through a different approach. Instead of a conjugated -like addition to the well-known enone, we aimed to proceed by means of functional group replacement. To do so, the synthesis continued with a Prins-Ritter transannular cyclization with chloroacetonitrile in the presence of sulphuric acid from the same enone 25.165,166,189 The reaction mechanism for the formation of chloroacetamide 34 is displayed in scheme 7. The reaction involves the protonation of the carbonyl group followed by the attack of the ?- electrons of the alkene (Prins reaction) to generate a stable carbenium ion. The nitrile?s nitrogen then adds to the carbocation to give a nitrilium ion intermediate, which undergoes hydrolysis upon addition of water during the work-up affording the desired chloroacetamide (Ritter reaction). 187 Ishiy ama, J.; S en d a, Y.; Imaiz u mi, S. Chem . Lett. 1983, 771 - 774. 188 Ishiy ama, J.; S en d a, Y.; Imaiz u mi, S. Chem . Lett. 1983, 1243 - 1244 . 189 Bish o p , R. Ritter- Type Reactions, Comprehensive Organic Synthesis II ; Else vie r Ltd ., 2014. Results & Discussion 55 Scheme 7. Mechanism of the Prins-Ritter transannulation. Other preparations of similar chloroacetamides entailed the combination of glacial acetic acid and sulphuric acid.98,190 The first attempts of the procedure led to the formation of the corresponding acetate 35, whose formation can be explained by the protonation of the hydroxyl group of 34, the formation of a stable carbenium ion with the loss of a molecule of water, and finally the nucleophilic addition of the acetate (Scheme 8). In the absence of acetic acid, the conjugate base of sulphuric acid is not nucleophilic enough to add to the carbocation. Hence, the hydroxyl group remains unaltered. 190 Schwert fe ger, H.; W? rt ele, C. ; Se far in , M.; Hau s man n , H.; Carlso n , R. M. K. ; Dah l, J. E. P. ; Schr ein er, P. R. J. Org. Chem. 2008, 73, 7 78 9 - 779 2. 56 CHAPTER 1: NMDA receptor antagonism Scheme 8. Addition of an acetate to the carbocation generated during the course of the Prins- Ritter transformation. Once chloroacetamide 34 was prepared in reasonable yield (49 %), we sought the known Barton-McCombie deoxygenation for the preparation of the chloroacetamide 36. This type of transformation consists in the radical deoxygenation of previously-modified alcohols.191,192,193 First, the alcohol needs to be temporally converted to an O- alkylthiocarbonyl derivative, whose sulphur atom reacts with a radical 0?capable oI forming a stable bond with it. The new bond fragments into a carbonyl compound and an alkyl radical, followed by a hydrogen atom abstraction to afford the reduced product (Scheme 9). The driving force of the reaction is the energy gained by the transition from the C=S to a C=O bond (C=S bond: 138 kcal mol -1, C=O bond: 178 kcal mol -1).194 On the basis of thermochemical data, trialkyltin hydrides seemed to be particularly suitable because of their exceptional behaviour as hydrogen -donors and the high stability of the Sn-S bond (Sn-S bond: 110 kcal mol-1). He nce, the reaction mechanism normally implies a radical initiator, like azobisisobutyronitrile (AIBN), to initiate the radical process. However, due to the inherent toxicity of tin hydrides, the development of tin -free Barton-McCombie deoxygenations ha s been rising since its discovery.195,196,197 Regarding O-alkylthiocarbonyl compounds, secondary and in a lesser extent primary and tertiary alcohols , are usually 191 Bart o n , D. H. R.; McCo mb i e, S . W. J. Chem. Soc., Perkin Trans. 1975, 1, 157 4 - 1 585. 192 McComb i e, S. W.; Mo th er wel l, W. B.; Toz er, M. J. Org. React. 2012, 77, 161 - 591. 193 Zard , S. Z. Xanthates and Related Derivatives as Radical Precursors, in Encyclopedia of Radicals in Chemistry, Biology and Materials ; Joh n Wiley & Son s, Ltd ., C hichester, UK, 2012 . 194 Kerr, J. A. Chem. Rev. 1966 , 66, 465 - 5 00. 195 Chenn eb erg, L.; Bara lle, A. ; Dan iel, M.; F en st erb an k, L.; God d ar d , J. - P.; Olli vier, C. Adv. Synth. Catal. 2014, 356, 27 56 - 2762. 196 Chatgilialoglu , C.; F erreri, C. Res. Chem. Intermed. 1993, 19 , 755 - 7 75. 197 Schu mm er, D.; Hoef le, G. Synlett 1990, 11, 705 - 706. Results & Discussion 57 transformed to thiocarbonates, xanthates and thioesters, wh ose reactivity in Barton- McCombie deox ygenations has been attested several times. Scheme 9. Mechanism of the radical Barton-McCombie deoxygenation. Encouraged by the literature precedents, we decided to convert 34 into phenylthiocarbonate 37 by applying the conditions reported in the literature (Table 7). Treatment of 34 with O-phenyl chlorothionoformate with no base added did not yield the expect ed product.198 Neither did the reaction with addition of a base such as NaH, pyridine or 4-dimethylaminopyridine (DMAP).199,200,201,202,203 In all the cases the starting material was recovered. 198 Xu, J.; Yad an , J. C. Tetrahedron Lett. 1996, 37, 2421 - 2424. 199 Padwa, A.; Harrin g, S. R.; Se m o n es, M. A. J. Org. Chem. 1998 , 63, 44 - 54 . 200 Marin o , J. P.; Osterh o u t, M. H.; Pad wa, A. J. Org. Chem. 1995 , 60, 2704 - 271 3. 201 Bou ssagu et, P.; Del mo n d , B.; Du mar tin , G.; Per ey re, M. Tetrahedron Lett. 2000, 41, 3377 - 3380 . 202 Kan eko , S.; Wata n ab e, T. ; Od a, K.; Moh an , R.; Sch weig er, E . J.; Martin , R. Fus ed - rin g pyri mid in - 4( 3H) - o n e derivat i ve s, pro ce ss es for the prep ar at ion and uses thereo f. WO 03 /106 435 A1. 203 Luzz io, F. A.; Fitch , R. W. J. Org. Chem. 1999, 64, 5485 - 54 93 . 58 CHAPTER 1: NMDA receptor antagonism Table 7. No reaction observed with the treatment of 34 with O-phenyl chlorothionoformate. Entry O-phenyl chlorothionoformate (eq.) Base Solvent Temp. (?C) Time (h) 37 (% yield) 1 1.2 DMAP (1.5 eq.) DCM 25 21 - 2 1.1 NaH (3 eq.) THF 25 3 - 3 2 NaH (4.4 eq.) THF reflux 21 - 4 1.5 Pyridine (excess) DCM 25 24 - 5 2 - THF From -70 to 25 20 - In accordance with radical deoxygenations, Mark? and co -workers published a procedure using toluates as simple and versatile radical precursors for the reduction of alcohols to alkanes.204 We contemplated hence the preparation of the corresponding toluate of 34 for its subsequent treatment with samarium(II) iodide and hexamethylphosphoramide (HMPA) (Table 8). The two different attempts for the formation of 38 did not afford the toluate and the starting material was again recovered. Thus, this approximation was abandoned. 204 Lam, K.; Mark ? , I. E. Org. Lett. 2008, 10, 277 3 - 2 776. Results & Discussion 59 Table 8. Attempted reactions of 34 with toluolyl chloride and a base. Entry Base Solvent 38 (% yield) 1 TEA (2 eq.) DCM - 2 NaH (2 eq.) THF - A similar procedure appeared in 2001 dealing with the radical deoxygenation of alcohols via their trifluoroacetate derivatives with diphenylsilane and di-tert-butyl peroxide (DTBP).205 In this case, the hydroxyl group of 34 needed to be turned into a trifluoroacetate group (Scheme 10). Two different pathways were essayed: 1) formation of a carbocation with conc. H 2SO4 and ensuing addition of trifluoroacetate; or 2) deprotonation of the alcohol and nucleophilic attack to trifluoroacetic anhydride (TFAA). Unfortunately, none of the tested conditions yielded the desired product 39 nor the starting material but only a mixture of unknown compounds. Specifically, we observed by 1H -NMR the lack of proton signals for the methylene in C-8 position. This fact can be explained through a putative retro-Ritter reaction, although a mechanism cannot be postulated without having identified the product. 205 Jan g, D. O.; Kim, J.; Cho, D. H. ; Chu n g, C. - M. Tetrahedron Lett. 2001, 42, 1073 - 10 75. 60 CHAPTER 1: NMDA receptor antagonism Scheme 10. Attempt to prepare compound 36 through a radical deoxygenation from a trifluoroacetate group. This large list of negative results did not dishearten us in our pursuit of deoxygenated compound 36. Further bibliographic search led us to the widely applied ionic deoxygenation triggered by organosilicon hydrides, which are used as a source of ionic hydride thanks to the presence of at least one Si-H bond. 206 For alcohols that can form a sufficiently stabilized carbenium ion, treatment with a Br?nsted or Lewis acid in the presence of triethylsilane or similar leads to their reduction. Tertiary alcohols are more effective in the transformation into the corresponding alkanes than secondary and primary alcohols. The mechanism of reaction entails the coordination of the oxygen atom to the acid, formation of a carbenium ion prior addition of the hydride. Taking this into account, compound 36 could be prepared from hydroxylated compound 34 in just one step by addition of Et3SiH and an acid .207,208,209,210 Different conditions were tested, using trifluoroacetic acid (TFA) or boron trifluoride etherate as Br?nsted or Lewis acids respectively, and changing the equivalents of both partners as well as the reaction time (Table 9). Regrettably, even the best conditions, i.e. a large excess of Et 3SiH and of BF3?Et2O, in anhydrous DCM at 75 ?C for 3 days, only gave a mixture of the intended product, unreacted starting material and unidentified products. 206 Larso n , G. L. ; Fry , J. L. Ionic and organometallic- catalyzed organosilane reductions, in Organic Reactions ; Joh n Wiley & Son s, Ltd ., Chichester, UK, 2008. 207 Gevorgy an , V.; Rub in , M.; Be n so n , S.; Liu , J. - X. ; Yama mo to , Y. J. Org. Chem. 2000 , 65, 617 9 - 618 6. 208 Dudd eck, H.; Rosen b au m, D. J. Org. Chem. 1991, 56, 17 07 - 1713. 209 Ban id e, E. V. ; Molloy , B. C. ; O rtin , Y.; M? ller - Bu n z, H.; M cGl in ch ey , M. J. Eur. J. Org. Chem. 2007 , 2611 - 2622. 210 Ito, M.; Kon n o , F. ; Kun amot o , T.; Suzu ki, N.; Kawah at a, M.; Ya magu ch i, K. ; I s h ika wa, T. Tetrahedron 2011, 67, 804 1 - 8 049. Results & Discussion 61 Table 9. Failed ionic hydrogenation of alcohol 34. Entry Et3SiH (eq.) Acid Temp. (?C) Time (h) 34 (% yield) 36 (% yield) 1 excess TFA (excess) 25 1 Quant. - 2 4 BF3?Et2O (1 eq.) 25 1 86 - 3 excess BF3?Et2O (excess) 25 18 Quant. - 4 4 BF3?Et2O (1 eq.) reflux 18 Quant. - 5 excess BF3?Et2O (excess) reflux 6 Quant. - 6 excess TFA (excess) reflux 15 Quant. - 7 excess TFA (excess) reflux 24 40 - 8 excess BF3?Et2O (excess) reflux 24 32 25 9 excess BF3?Et2O (excess) reflux 48 26 45 10 excess BF3?Et2O (excess) reflux 72 16 50 Considering the high reactivity of bridgehead hydroxyl groups in traditiona l reductions, the difficulty of transforming alcohol 34 can be reasoned by the presence of the chloroacetamide in the molecule, which may facilitate the retro-Ritter reaction on one side, and possesses a high reactive chlorine atom on the other. In spite of all the efforts in performing Barton-McCombie and ionic deoxygenation reactions, we decided to tackle the synthesis of amine 23 by applying a different approach. Exploring alternative methodologies, dehalogenation seemed at first glance a feasible way to remove a tertiary alcohol through its prior interconversion to a halide. Different publications have appeared reporting the dechlorination of adamantane groups through diverse procedures, such as the use of a hydride source (e.g. lithium aluminium hydride),211,212 the radical dehalogenation with phenylsilane,213 or the photoinduced reduction of organic halides with samarium(II) iodide.214 On that account, we needed to transform the hydroxyl group to a chloride. Alkyl chlorides are most easily prepared by reaction with thionyl chloride or phosphorous 211 Ashb y , E. C. ; Desh p an d e, A. K . J. Org. Chem. 1994, 59, 37 98 - 3805 . 212 Ban ister, S. D.; Yoo, D. T.; Ch u a, S. W.; Cui, J. ; Mach , R. H.; Kass iou , M. Bioorg. Med. Chem. Lett. 2011 , 21, 5289 - 529 2. 213 Jan g, D. O. Synth. Commun. 1997, 27, 1023 - 10 27. 214 Sumin o , Y.; Harat o , N.; Tomi s ak a, Y.; Ogawa, A. Tetrahedron 2003, 59, 1049 9 - 1 0508. 62 CHAPTER 1: NMDA receptor antagonism trichloride, among other reagents. Hence, we treated chloroacetamide 34 with neat thionyl chloride for one hour under reflux, giving desired chloride 40 in 56% yield after purification by column chromatography (Scheme 11). Scheme 11. Replacement of the hydroxyl group by a chloride. Afterwards, reaction of chloro compound 40 with LiAlH 4 in anhydrous dioxane for 18 hours at reflux afforded a complex mixture of products. By gas chromatography -mass spectroscopy, the corresponding ene-imine product 41 was observed, whose formation can be explained by the attack of the hydride to the carbonyl group, which expelled the nitrogen atom generating an electron cascade movement with the final release of the chloride (Scheme 12). Scheme 12. Formation of ene-imine 41 after treatment of 40 with LiAlH 4. Regardless of the unfortunate result of the aforementioned reaction, we wanted to further explore the potential of chlor o derivative 40 as a precursor of unsubstituted scaffold in amine 23. Among all the reagents for the formation of radicals from halides, samarium(II) iodide is possible the most versatile and widely used after tributyltin hydride.215 In 2003, a publication came out describing the radical dechlorination of 1- chloroadamantane using SmI2 in anhydrous THF under irradiation with a tungsten lamp to afford the adamantane ring in 86% yield. 214 But because there is a potential coordination of samarium with the carboxyl group in a Lewis acid manner, we determined to hydrolyse the amide group prior to the radical dehalogenation in order to avoid side reactions. A previous exploration of the aqueous hydrolysis of related compounds gave either low yields in acid media, or high yields at the expense of drastic basic conditions. 98 Searching for a substitutive method, it was found that the chloroacetyl group could be cleaved by thiourea in an efficient way for the synthesis of tert-alkylamines.216 Applying these conditions to our scaffold, chloroacetamide 40 was transformed into chloro-amine 42 in 73% yield (Scheme 13). The deprotection mechanism entails the nucleophilic substitution of the chloride by the attack of the sulphur atom, followed by the formation of a thiazolidine ring as a by- 215 Rowlan d s, G. J. Tetrahedron 2009, 65, 860 3 - 8 655. 216 Jirgen so n s, A.; Kau ss, V.; Kalvi n sh , I.; Gold , M. R. Synthesis 2000, 12, 1709 - 17 12. Results & Discussion 63 product. Subsequent elimination of the amine provided compound 42 and pseudothiohydantoin as a by-product. The reaction is also amenable in DCM. Scheme 13. Deprotection of chloroacetamide with thiourea. The proton migrations have been obviated for the sake of clarity. Once deprotected, chloro-amine 42 was reacted with SmI2 and irradiated with a 100 W lamp until the mixture shifted from dark blue to yellow. 217 To our surprise, desired amine 23 was recovered along with the starting material 42 in an optimized 3:1 ratio (Scheme 14). Regrettably, the mixture was inseparable through column chromatography. Scheme 14. Partial dehalogenation of chloro-amine 42 with samarium(II) iodide. Based on the partial success of the previous reaction, we believed that the use of organotin reagents would facilitate the radical dehalogenation of our scaffold because of their proven reactivity and versatility.192,215,218,219 In addition, it is well-known that the bond dissociation enthalpy of the halogen strongly determines the dehalogenation rate. Accordingly, bond enthalpies decrease as we move down the periodic table, i.e. Cl > Br > I. So, instead of working with the chloro derivative, we decided to replace the hydroxyl group of 34 by a bromide following the aforementioned procedure but in this case with thionyl bromide (Scheme 15). Albeit the excellence conversion of the alcohol, we observed 217 The blu e colo u r i s di stin ctiv e of s amariu m(II) , wherea s the yellow colou r of samariu m(III) . 218 Jasch , H. Hein rich , M. R. Tin hydrides and functional group transformations , in Encyclopedia of Radicals in Chemistry, Biology and Materials ; Joh n Wi ley & Son s, Ltd ., Chicheste r, UK, 201 2 . 219 Renau d , P.; Sib i, M. P. Radicals in Organic Synthesis Vol. 1 ; Wiley - V CH, Wein h ei m, G erma n y , 2001. 64 CHAPTER 1: NMDA receptor antagonism by gas chromatography-mass spectroscopy and 1 H -NMR, the concomitant formation of bromoacetamide 44 by nucleophilic substitution of the chloro atom, as well as, to a lesser degree, the bromination of the aromatic ring as either chloro- or bromoacetamide, 45, whose rate of formation varied from one reaction to another. Scheme 15. Functional group interconversion of a hydroxyl group to a bromide . In order to avoid the chloro-bromo exchange of the acetamide, we ended up moving one-step forward and deprotecting chloroacetamide 34 to obtain the corresponding aminoalcohol 46. In this way, applying the previous conditions with thiourea and glacial acetic acid in absolute ethanol, primary amine 46 was prepared in quantitative yield (Scheme 16a). Afterwards, transformation of 46 to the bromo derivative with thionyl bromide in toluene afforded the desired halide 47 in 79% yield (Scheme 16 b). Scheme 16. Thiourea-mediated deprotection of chloroacetamide 34, followed by bromination with thionyl bromide. Surprisingly, purification of bromo-amine 47 either by column chromatography or by an acid-base extraction led to the formation of enone 25. This fact can be explained through the elimination of the bromide pushed by the amino group. Final hydrolysis of the intermediate imine provides the former enone (Scheme 17). The formation of an enone from a cage-annulated fragmentation has been described, using in these cases different bases as the promoter of the ring-opening.220,221 Regardless of the vain efforts put on the purification of compound 47, we continued with the synthetic route without any purification. On the other hand, amine 47 was found to be bench-top unstable in a short period of time, so it had to be used right after its preparation in the next step. 220 Kumar , K.; Tep p er, R. J.; Z en g , Y.; Zim mt, M. B. J. Org. Chem. 1995, 60, 405 1 - 4 066. 221 March an d , A. P.; Namb o o th iri, I. N. N. Heterocycles 2000, 52, 451 - 4 57. Results & Discussion 65 Scheme 17. Formation of enone 25 from bromo-amine 47. The impure and unstable amine 47 was subjected to radical debromination conditions with tri-n-butyltin hydride and AIBN as the radical initiator (Scheme 18). To our delight, amine 23 was finally secured as the hydrochloride salt after its purification by column chromatography, despite of the low overall yield (12%). The mechanism of the reaction entails similar pathways to those shown in scheme 9, although the driving force in this case is provided by the formation of a C-H bond at the expense of a Sn-H (C-H bond: 80 kcal mol-1, Sn-H bond: 64 kcal mol -1), and then a Sn-Br at the expense of a C-Br (Sn-Br bond: 81 kcal mol-1, C-Br bond: 67 kcal mol.1). Scheme 18. Synthesis of amine 23 via radical dehalogenation. Table 10 recapitulates the attempts for the synthesis of unsubstituted amine 23. 66 CHAPTER 1: NMDA receptor antagonism Table 10. Summary of the different routes applied for the preparation of amine 23. Entry Starting material R1 R2 Reagents Product (% yield) 1 25 - - L-Selectride - 2 25 - - NaBH 4 1,2-addition product 3 25 - - H 2, Pd/C Isomerization + diol 4 34 OH Chloroacetamide O-phenyl chlorothiono- formate 34 5 34 OH Chloroacetamide Toluolyl chloride 34 6 34 OH Chloroacetamide TFA, conc. H 2SO4 Unknown products 7 34 OH Chloroacetamide TFAA, TEA Unknown products 8 34 OH Chloroacetamide Et3SiH, TFA or BF3?Et2O Complex mixture 9 40 Cl Chloroacetamide LiAlH 4 Ene-imine 10 42 Cl NH 2 SmI2, h? 23 : 42 (3:1 ratio) 11 47 Br NH 2 Bu3SnH, AIBN 23 (12%) Once the desired amine 23 was prepared in reasonable amounts, we wished to appraise the effect on the alkylation on the amine group. As already mentioned in the introduction, the antagonist activity increased as the amine went from primary to secondary, and therefrom to tertiary. Moreover, it was observed that it was preferably to incorporate small alkyl groups, such as the methyl group. Taking into account the aforementioned, we settled the synthesis of dimethylated amine 24 following the described procedures.98 We proceeded through a reductive alkylation of amine 23 with formaldehyde and sodium cyanoborohydride (Scheme 19). The latter was chosen as hydride donor because its noted selectivity for the reduction of the formed iminium ion over the ketone or aldehyde, which remains unaltered and free to react with the amine.222,223 This phenomenon is due to the electron-withdrawing effect of the cyano moiety that stabilizes the negative charge on the boron, thus rendering it less reactive and more inclined to reduce the more unstable iminium ion than the carbonyl. Hence, this transformation can be performed in a one -pot 222 Borch , R. F.; Bern stein, M. D.; Du rst, H. D. J. Am. Chem. Soc. 1971, 93, 289 7 - 2 904. 223 Lane, C. F. Synthesis 1975 , 1975, 135 - 146. Results & Discussion 67 fashion, where the amine is mixed with the desired ketone or aldehyde in the presence of NaBH 3CN in mild acidic conditions. Scheme 19. Reductive alkylation of amine 23 and schematic representation of the kinetics of sodium cyanoborohydride-mediated reduction of imines versus ketones or aldehydes. With compound 24 we expected an inc rease in the potency compared with primary amine 23, in agreement with the observed trend in former families of compounds. 1.2 Explor ation of the C -9 substitution of the benzo -homoadamantane scaffold As discussed earlier, in relation with the metabolism of the adamantane group, a bridgehead hydroxylation is favoured over the secondary carbon positions, producing water-soluble 1-hydroxyadamantane derivatives in the liver, which are then readily excreted. Hence, one of the most applied strategies to avoid this issue is to prolong the pharmacokinetic stability by blocking the potential sites of metabolic activity with a fluorine or a hydroxyl group.224,225 In order to expand the SAR studies, we were encouraged to investigate the effect of the different substitution around the C-9 position of the benzo-homoadamantane polycycle that may lead to more metabolically stable compounds. Similar to the adamantane group, the benzo-homoadamantane scaffold is easily functionalized at the tertiary positions, allowing subsequent substitutions with other functionalities. Hitherto, we have synthesized a few C-9-substituted derivatives thanks to the large exploration carried out during the preparation of amine 23. Specifically, the different synthetic routes have led us to obtain in sufficient amounts the chloro 42, hydroxyl 46, and bromo 47 derivatives (Fig. 31). 224 Roh d e, J. J.; Pliu sh ch e v, M. A. ; Soren s en , B. K.; Wod ka , D.; S hu ai, Q.; Wan g, J.; Fun g, S.; M on zo n , K. M.; Chio u , W. J.; Pan , L.; Den g, X.; Cho van , L. E.; Ramaiy a, A.; M ullally , M.; Hen ry , R. F.; Sto lari k, D. F.; Imad e, H. M.; Mar sh , K. C.; Ben o , D. W. A. ; Fey , T. A. ; Droz , B . A.; Bru n e, M. E.; Camp , H. S.; Sham, H. L.; Fre v ert, E. U.; Jaco b so n , P. B.; Lin k, J. T. J. Med. Chem. 2007, 50, 14 9 - 164. 225 Jasys, V. J. ; Lomb ar d o , F.; App leto n , T. A.; Bord n er, J.; Zili o x, M.; Volk man n , R. A. J. Am. Chem. Soc. 2000, 122, 46 6 - 4 73. 68 CHAPTER 1: NMDA receptor antagonism Fig. 31 . Amines prepared from the previous synthetic routes. The high instability observed in amine 47 prompted its exclusion for the screening as potential antagonist of the NMDA receptor. In accordance to the most common strategy for the blockade of potential sites of metabolic activity, i.e. the introduction of a fluorine atom, we contemplated the formation of fluorinated compound 48 (Fig. 32). Fig. 3 2. New fluorinated derivative to prepare. Incorporation of a fluorine atom has become in the last years a key designer component in the structure of modern biologically active compounds, agrochemicals and functional materials.226,227,228,229 As fluorine is the most electronegative and the second- smallest atom in the periodic table, fluorine-containing molecules display different chemical, physical and biological properties from their parent compounds. Hence, the high carbon-fluorine bond strength (C-F bond: 105.4 kcal mol-1; C-H bond: 98.8 kcal mol -1)230 provides the molecule high thermal and oxidative stability. Moreover, some fluorine effects are of special interest in pharmaceutical sciences: the increased lipophilicity enhances bioavailability, fluorine can block key metabolic positions and it can mimic enzyme substrates.231 Because of its characteristics, fluorine is considered as a bioisostere of hydrogen.232 Taking into account the intrinsic characteristics of fluorine, it is plain to recognize that its introduction is in need of sophisticated synthetic procedures far from the conventional organic synthetic methods. During the last decade, organic chemists have developed new strategies to create carbon-fluorine (C-F) bonds on aromatic and aliphatic molecules.233 In most cases, a single fluorine is successfully introduced using either nucleophilic agents, such 226 Hagman n , W. K. J. Med. Chem. 2008, 51, 4359 - 43 69. 227 M?ller, K.; Faeh , C. ; Died erich , F. Science 2007, 317, 1881 - 1 886. 228 Furu y a, T.; Kamlet, A. S.; Ritte r, T. Nature 2011, 473, 470 - 47 7. 229 Jes ch ke, P. ChemBioChem 2004, 5, 570 - 5 89. 230 O?,agan, D. Chem. Soc. Rev . 2008, 37, 308 - 31 9 . 231 ChemBioChem 2004, 5, 557 - 7 26 (sp e cial is su e : Fluo rin e in Li fe Sci en ce s). 232 Mean wel l, N. A . J. Med. Chem. 2011, 54, 2529 - 25 91. 233 Shimizu , M.; Hiy ama, T. Angew. Chem. Int. Ed. Engl. 2004, 44, 214 - 2 31. Results & Discussion 69 as (diethylamino)sulfur trifluoride (DAST)234 or bis(2-methoxyethyl) aminosulfur trifluoride (Deoxofluor), 235 or electrophilic agents, such as 1-chloromethyl-4- fluorodiazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor)236,237 (Fig. 33). Fig. 33 . Efficient nucleophilic and electrophilic fluorinating agents. Following the procedure described by Johnson,238 we first tested our chances in the preparation of amine 48 via nucleophilic substitution of the hydroxyl group of 46 using DAST as fluorinating agent. DAST is an aminosulfurane reagent that selectively exchanges hydroxyl groups for fluorine, and has proven highly applicable since its discovery in 1970.239 The main advantage of DAST over previous fluorinating agents is that it is easily handled and versatile, unlike the more classical gaseous sulphur tetrafluoride. Luckily enough, we managed to obtain the desired fluorinated amine 48 in 44% yield under these conditions. The reaction goes through a deoxyfluorination mechanism where the oxygen atom of the substrate reacts with the sulfur, with elimination of a hydrogen fluoride molecule. The alkoxyaminosulfur difluoride intermediate eliminates a fluoride ion that attacks the sulfanylidene intermediate either by an SN1 or SN2 pathway, affording the desired product (Scheme 20). If the SN2 mechanism prevails over the SN1, chiral alcohols predominantly suffer from the Walden inversion of configuration. Obviously, due to steric issues, in our case only a SN1 pathway can operate. 234 Midd leto n , W. J. J. Org. Chem. 1975, 40, 574 - 57 8. 235 Lal, G. S.; Pez, G. P.; P esar es i, R. J.; Pro zo n ic, F. M.; Chen g, H. J. Org. Chem. 1999, 64, 70 48 - 7054 . 236 Lal, G. S.; Pez, G. P.; Syvret, R. G. Chem. Rev. 1996, 96, 1 737 - 1756 . 237 Sod eo ka , M. Science 2011 , 334, 1651 - 1652. 238 John so n , A. L. J. Org. Chem. 1982, 47, 5220 - 52 22. 239 Singh , R. P.; Shree ve, J. M. Synthesis 2002, 25 61 - 2578. 70 CHAPTER 1: NMDA receptor antagonism Scheme 20. Deoxyfluorination of alkanolamine 46 with DAST. As in the case of unsubstituted amine 23 and its dimethylated analogue 24, the chloro 42, hydroxy 46, and fluoro 48 derivatives were subjected to reductive alkylation conditions to obtain their corresponding dimethylated tertiary amines 49, 50 and 51, respectively (Scheme 21). Scheme 21. Dimethylation of earlier amines by reductive alkylation. For the purpose of further inspecting the effect of the substitution on the C-9 position, and bearing in mind the intrinsic characteristic of enone 25 in relation to the 1,4- conjugated -like addition, our next goal was the synthesis of methoxy derivative 52, which would add one extra group in our array of compounds (Fig. 34). Fig. 34 . The methoxy derivative closes this family of compounds. Results & Discussion 71 Thus, enone 25 was submitted to the 1,4-conjugated -like addition of methanol with p- toulenesulfonic acid, as reported by Bishop et al.165 The reaction provided alcohol 53 which was transformed to the corresponding chloroacetamide 54 in 31% yield via the already mentioned Ritter reaction (Scheme 22). In this case, the procedure entails the protonation of the bridgehead hydroxyl group, formation of a tertiary carbocation and later addition of the nitrile nitrogen. The mechanism continues as indicated in scheme 7. Likewise, the methoxy group was prone to eliminat ion and chloroacetamide 34 was recovered in 37% yield. Furthermore, with the purification by column chromatography the starting material was recovered in 16% yield. Scheme 22. Formation of alcohol 53 followed by the Ritter reaction, affording a mixture of 54, 34 and the starting material. The pure methoxy derivative 54 was deprotected under the previous conditions with thiourea and glacial acetic acid in absolute ethanol, to get final amine 52 in 59% yield (Scheme 23). Scheme 23. Synthesis of amine 52 with thiourea under acid conditions. To sum up, scheme 24 discloses the synthetic routes followed for the new derivatives. Globally, we have prepared unsubstituted amines 23 and 24, along with C-9 substituted benzo-homoadamantanes bearing different substituents (hydroxyl and methoxy groups , and chloro and fluoro atoms). 72 CHAPTER 1: NMDA receptor antagonism Results & Discussion 73 2. Pharmacological evaluation of new benzo -homoadamantane derivatives 2.1 Assessment of the NMDAR antagonistic activity The synthesized compounds were first evaluated as NMDAR antagonists by the determination of their respective half-maximal inhibitory concentration or IC 50. The IC50 represents the concentration of a drug that is required to produce 50% inhibition of a n enzymatic reaction at a specific substrate concentration.240 This work was carried out by the research group of Dr. Francesc Xavier Sureda (University Rovira i Virgili, Reus, Spain). The group of Dr. Sureda is highly specialized in testing the ability of a given ligand to prevent neurodegenerative changes in several animal models of AD, both in vivo and in vitro. Specifically, to demonstrate the interactions of the new analogues with glutamate physiological actions, Dr. Sureda evaluated the competence of the compounds to block the increase of intracellular calcium evoked by NMDA in cultured rat cerebellar granule cells (CGC). Since the 1990s, different protocols for the determination of the intracellular calcium concentration have been established. Most part of the glutamatergic receptors are coupled to signal transduction processes that are dependent on the levels of this secondary messenger. In consequence, the study of its modulation is a valuable tool for the assessment of new molecules as NMDAR agonists or antagonists. The assay involves the addition of Fura-2-acetoxymethyl ester (Fu ra-2 AM) to the cultured CGCs, as previously reported.241 Fura-2 AM is a membrane-permeable analogue of the ratiometric fluorescent probe calcium indicator Fura-2.242 Once Fura-2 AM enters the cell, it is hydrolysed by nonspecific cytoplasmic esterases to Fura-2, which binds to free intracellular calcium and whose fluorescence depends on the concentration levels of the latter (Fig. 35). Both calcium-bound and calcium-free species fluoresce quite strongly, at 340 and 380 nm respectively, and the ratio of emissions at those wavelengths is directly related to the amount of intracellular calcium. Regardless of the levels of Ca2+ , Fura-2 emits fluorescence at 510 nm.243 240 Yung - Chi, C. ; Pru so f f, W. H. Biochem. Pharmacol. 1973, 22 , 3099 - 3108. 241 Verd agu er, E.; Gar c?a - Jo rd ?, E.; Ji m?n ez, A.; Stra n ges, A.; S ured a, F. X.; Can u d as, A. M.; Escu b ed o , E.; Camar asa, J.; Pall?s, M. ; Cam i n s, A. Br. J. Pharmacol. 2002, 135 , 1297 - 13 07. 242 Gryn kiewicz, G.; Poen i e, M.; Tsi en , R. Y. J. Biol. Chem. 1985 , 260, 3440 - 3450. 243 Life tech n o logi es web page. I on in d icato rs & ion o p h o res: Fura - 2. htt p s://w ww. lif etechn o logi es. com/ord er/ cat alo g/p ro d u ct/F 1200 (acc e ss ed on 14 th June 2 015). 74 CHAPTER 1: NMDA receptor antagonism Fig. 35 . Hydrol ysis of Fura-2 AM to Fura-2. Therefore, the determination of the calcium levels is based on the measurement of the fluorescence at 510 nm when excited at 340 (F 340) and 380 (F380) nm, followed by the calculation of the ratio F340 /F 380 (R). For the assessment of the effect of the newly synthesized compounds against glutamate- induced Ca2+ mobilization, the procedure is as follows. A single administration of the agonists glutamate (100 ?M) and glycine (10 ?M) leads to a significant increase in R value. After this stimulation, increasing cumulative concentrations of the compound to be tested are added, represented in blue arrows in figure 36. Fig. 36 . Fluorescence detection after addition of the agonists glutamate and glycine prior administration of increasing concentrations of a given antagonist. Final adjustment of the data to a concentration -response sigmoidal curve using a non- linear regression curve fitting (variable slope) gives the percentage of inhibition at every tested concentration of each compound, which in turn can be used for the calculation of the IC50 values. It is worth to highlight that the use of glutamate as agonist in the assay may lead to a calcium mobilization originated from the activation of other receptors different from the NMDAR, such as kainate. Therefore, for a better estimation of the effect of a plausible NMDAR antagonist, in most assays NMDA is used as the stimulator, allowing a 100% inhibition to be possible. Results & Discussion 75 Evaluation of the ability of the new compounds to block the NMDAR by this technique revealed some interesting results. The data shown in table 11 are means of IC50 values ? SEM of at least three separate experiments carried out on three different batches of cultured cells. Amantadine and memantine were used as standard controls. Table 11. IC50 values (?M) for 5,6,8,9,10,11-hexahydro -7H-5,9:7,11-dimethanobenzo[9]annulen -7- amines as NMDAR antagonists. Comp. R1 R2 IC50 (NMDA 100 ?M) 23 H H 0.70 ? 0.12 24 H Me 2.30 ? 0.10 42 Cl H 4.99 ? 1.53 49 Cl Me 17.0 ? 9.3 46 OH H 16.2 ? 3.3 50 OH Me 71.1 ? 9.9 48 F H 1.93 ? 0.21 51 F Me 16.5 ? 2.4 52 OMe H 24.3 ? 2.0 19 Me H 13.6 ? 3.4 Amantadine - - 92 ? 29 Memantine - - 1.5 ? 0.1 As aforementioned, the IC50 values were obtained by the measure of the antagonistic activity at varied concentrations of each compound, providing a concentration-response semi-logarithmic curve with the familiar sigmoidal shape. The plot in figure 37 includes the most potent compounds. 76 CHAPTER 1: NMDA receptor antagonism Fig. 37 . IC50 curves of compounds 23 and 48 and their dimethylated analogues 24 and 51 respectively, compared with memantine as a standard. Gratifyingly, rationalisation of the results shown in table 11 led to some attractive outcomes and a SAR could be established around the C-9 substitution: ? All the new compounds have IC50 values lower than that of amantadine, with amines 23, 42, 24 and 48 in the low micromolar range. ? Confirming our first hypothesis, compound 23 was the most potent compound with an IC50 lower than that of memantine (0.70 and 1.5 ?M, respectively). Pleasingly, primary amine 23 and its bioisostere 48 (1.93 ?M) were clearly more potent than our previous synthesized families I and II (Fig. 38). Of note, the observed trend of going from 23 to its methylated analogue 19 (13.6 ?M) is the opposite of the one observed in going from amantadine to its dimethylated derivative memantine. ? Considering solely primary amines, smaller substituents such as a hydrogen (23) or a fluorine (48) are better tolerated than larger groups, like hydroxyl (46), methoxy (52) or methyl (19). Particularly, either polar (46 or 52), or lipophilic electron-donating groups (EDG) as in the previously reported methylated compound 19 led to a significant reduction of the activity. Introduction of the electron-withdrawing chlorine (42) and fluorine (48) resulted in an increase on the potency compared with compounds bearing an EDG. ? Interestingly, within this novel series of benzo-homoadamantanes, all the tertiary amines were clearly less potent than the corresponding primary amines (23 vs 24, 42 vs 49, 46 vs 50, 48 vs 51), in contrast to the behaviour observed in former families I and II. Results & Discussion 77 Fig. 38 . Confirmation of our first hypothesis from the former families. These results suggest that the combination of the size, the ability to form specific interactions, such as hydrophobic interactions or hydrogen bonds, and the inductive effects of the substituted group, alters greatly the activity as NMDAR antagonist of the molecule. The lack of a crystal structure of the ion channel resolved with an antagonist renders difficult the reasoning of the observed trends. 2.2 Electrophysiological measurements In order to appraise if the new NMDA antagonists displayed a similar blocking mode to that of memantine, i.e. characterized by rapid and strongly voltage-dependent kinetics, Dr. David Soto from IBIDELL, performed a patch-clamp experiment with the more promising compounds, 23 and 48. Briefly, the patch-clamp technique permits the study of membrane current contributions of individual ionic channels from cells of any size and particularly the study of small cells in culture.244 It consists of the insertion of a micro-pipette into the cell membrane in a way that the connection is sealed. This tight seal isolates the membrane patch electrically, which means that all ions passing through the ion channels flow into the pipette. An electrode connected to a highly sensitive differential amplifier can record currents flowing through the membrane patch (Fig. 39). This technique has been widely used for NMDAR blockers.245,246,247,248,249 244 Sakman n , B.; Neh er, E. Ann. Rev. Physiol. 1984 , 46, 455 - 47 2. 245 Chen, H. S.; Pellegrin i, J. W.; A ggarwal, S. K.; Lei, S. Z.; Wara c h , S.; Jen sen , F. E.; Lip to n , S. A . J. Neurosci. 1992, 12, 442 7 - 4 436. 246 Parso n s, G. R.; Gru n er, R.; Ro zen ta l, J.; Millar, J. ; Lod ge, D. Neuropharmacol. 1993, 32, 1337 - 1 350. 247 Blan p ied , T. A ; Clar k e, R. J.; Jo h n so n , J. W. J. Neurosci. 2005 , 25, 3312 - 3322. 248 Gillin g, K.; Jat zk e, C.; Wollen b u rg, C.; Van eje vs, M.; Kau ss, V.; Jirg en so n s, A.; Par so n s, C. G. J. Neural Transm. 2007, 114, 1 529 - 153 7. 249 Koller, M.; Ur wy ler, S. Expert Opin. Ther. Pat. 2010 , 20, 168 3 - 170 2. 78 CHAPTER 1: NMDA receptor antagonism A B Fig. 39 . (A) Schematic representation of the patch-clamp principle and (B) image of a patch pipette attached to the membrane of a neuron.250 During the experiment, the ion channels undergo different stages, which affect the current that flows through the membrane patch: 1. Baseline conditions: voltage of -60 mV with the ion channels closed. 2. Addition to the media of agonists (glutamate and glycine): ion channels are now open with the entrance of cations until normalization. Reduction on the intensity. 3. Addition of antagonist to the former media: some ion channels are now blocked. Increase on the intensity. 4. Application of a positive pulse of +60 mV: release of the blockers. 5. Stop of the positive pulse and addition of agonists + antagonist: recovery of the state in step 3. 250 Scien c e Lab web sit e. Th e pat ch - clamp te ch n iq u e. htt p ://w ww.l eica - mic ro sy st em s .com/ scien c e - lab /th e - p at ch - cla mp - te ch n iq u e/ (acc es sed on 18 t h Septe mb er 2015 ). Results & Discussion 79 6. Withdrawal of the antagonist, but with agonists: recovery of the state in step 2. 7. Application of a positive pulse of +60 mV : to check if the antagonist is an open-channel blocker or, in other words, voltage-dependent blocker. If the intensity of the second positive pulse is higher than the first one, it means that the antagonist is a non-voltage-dependent blocker. Following thus this protocol, amine 23 and 48 were assayed along with memantine for comparison (Fig. 40). Fig. 40 . Results from electrophysiological studies with (A) memantine, (B) amine 48 and (C) amine 23. 80 CHAPTER 1: NMDA receptor antagonism As shown in figure 40, amine 48, which bears a fluorine atom in its structure, compares well with memantine, with a similar kinetic profile after running the experiment. However, this is not the case for amine 23, which presents a low-binding kinetic profile. Specifically, when 23 enters in the media, there is a rapid blockade of the NMDARs, and this blockade is slightly more intense than for the other two antagonists. In contrast, the recovery of the baseline levels is slower than memantine and amine 48. Altogether, the data suggests that amine 23 shows a similar association rate constant (kon) but a smaller dissociation rate constant (koff) than memantine. On the other hand, when applying the second positive pulse, the intensity in this case is higher than the one from the first pulse. This fact indicates that actually amine 23 is a non-voltage-dependent and non-open channel blocker. Therefore, amine 23 and 48, despite containing two bioisostere groups, bind to the channel in a distinct way. One hypothesis is that amine 23 behaves as a high-affinity NMDA receptor antagonist. At the time of writing this thesis, work is still ongoing in order to further study the electrophysiological behaviour of our NMDAR antagonists. Conclusi ons Conclusions 83 In this first part of the thesis, 12 new compounds, revolving around the benzo- homoadamantane structure, have been synthesized and fully characterized by their spectroscopic and analytical data. Among them, 10 were pharmacologically evaluated as NMDA receptor antagonists. From the data of the first chapter of this dissertation, we can conclude the following: 1. Amine 23 was prepared following a radical dehalogenation in low yield, after several synthetic attempts. 2. A SAR study has been developed around the substitution pattern at the C-9 position, with polar and non-polar substituents, partly thanks to the synthetic exploration performed for the synthesis of amine 23. 3. The activity displayed by compound 23 confirmed our first hypothesis that an unsubstituted benzo-homoadamantane would exhibit a higher activity than the previous series of compounds. Indeed, amine 23 displayed the lowest IC50 value ever described to date for the benzo-homoadamantane scaffold as surrogate of the adamantane group of memantine (IC50 of 0.70 ?M for 23 v s 1.5 ?M for memantine, Fig. 41). Fig. 41. Best compound identified from this family of benzo- homoadamantanes. 4. The second most active structure discovered with this series of compounds was the fluoro derivative 48, bioisostere of 23 and with an equipotent activity compared with memantine (IC50 of 1.93 ?M). 5. Halides were better tolerated than hydroxyl and methoxy groups . Thus, compounds bearing a fluorine (48, IC50 of 1.93 ?M) or a chlorine ( 42, IC50 of 4.99 ?M) were more potent as NMDA receptor antagonists than compounds with a hydroxyl ( 46, IC50 of 16.2 ?M) or a methoxy ( 52, IC50 of 24.3 ?M). 6. All the tertiary amines were less active than their corresponding primary amines. 7. Electrophysiological studies revealed that amine 48 possessed a similar kinetic profile than memantine in a patch-clamp assay, with a voltage-dependent binding mode, whereas amine 23 tended to stay longer time within the channel, in a non-voltage-dependent mode. Hence, amine 23 could not be considered as an open-channel blocker but, probably, as a high-affinity antagonist. CHAPTER 2 : 11? - HSD 1 inhibition Introduct ion Introduction 89 1. 11?-HSD1 inhibition by adamantane -based derivatives The following section will discuss the glucocorticoid effects in the organism and the regulation of these stress hormones by the 11?-HSD1 enzyme, which has been found to be relevant in the development of multiple diseases, such as the metabolic syndrome (MetS), inflammation and cognitive dysfunction. A brief disclosure of the developed 11?-HSD1 inhibitors will be covered. Particularly, the adamantane-based compounds will be studied in order to understand their binding mode in the active site of the enzyme as well as their progress throughout the drug discovery process. 1.1 The glucocorticoid system and its physiological actions Glucocorticoids (GCs) are well-known ubiquitous hormones playing a key role in modulating immune and inflammatory responses, regulating energy metabolism and cardiovascular homeostasis and the bod\?s responses to stress Opposing the action of insulin, GCs promote gluconeogenesis, inhibit ?-cell insulin secretion and peripheral glucose uptake. Moreover, GCs contribute to lipolysis, with subsequent fatty acid mobilization, and proteolysis. Thus, overall, GCs prompt catabolic states.251,252 The active human GC cortisol, is synthesized by the adrenal cortex with a strong circadian rhythm. Its secretion is controlled by the hypothalamic-pituitary-adrenal (HP A) axis and induced in stressful conditions. Serum cortisol readily passes cell membranes and exerts its int racellular functions by binding to GC receptors (GRs), which are ligand- activated nuclear receptors. GRs regulate, directly or indirectly, the expression of a plethora of genes involved in various physiological processes, including peroxisome proliferator - activated receptor ? (PPAR-?), CCAAT enhancer-binding protein and nuclear factor-?B (NF-?B), inter alia.253,254 Furthermore, GCs bind to mineralocorticoid receptors (MRs), which regulate blood pressure and sodium balance in the kidney.255 1.1.1 GC regulation by 11?-HSD enzymes Along Zith the +3A a[is *C?s homeostasis is controlled b\ the 11?-HSD enzyme. Two isoforms of this enzyme, 11?-HSD type 1 (11 ?-HSD1) and type 2 (11 ?-HSD2), catalyse the interconversion between active 11-hydroxy forms ( cortisol in humans, and corticosterone in rodents) and inert 11-ketosteroids (cortisone in humans, and 11- dehydrocorticosterone in rodents), with the concomitant conversion of NADPH cofactor to NAD+ (Fig. 42). 11?-HSD1 is anchored to the membrane of the endoplasmic reticulum (ER) by the N-terminus, with the catalytic domain located within the lumen of the ER. 251 Dallman , M. F.; Stra ck, A. M.; Aka n a, S. F.; Brad b u ry , M. J.; H an so n , E. S.; Sc rib n er, K. A.; S m ith , M. Front. Neuroendocrinol. 1993, 14, 303 - 347 . 252 Sap olsky , R. M.; Rom ero , L. M.; Mu n ck, A. U. Endocr. Rev. 2000, 21, 55 - 89. 253 Rosen , E. D.; MacDou gald , O. A. Nat. Rev. Mol. Cell Bio. 2006 , 7, 885 - 89 6. 254 Smoak , K. A.; Cid lo wski, J. A. Mech. Ageing Dev. 2004, 125 , 697 - 7 06. 255 Arriza , J. L.; W ein b erg er, C.; Cerelli , G.; Glas e, T. M.; Han d eli n , B. L.; Hou sman , D. E.; E van s, R. M. Science 1987, 237, 26 8 - 2 75. 90 CHAPTER 2: 11 ?-HSD1 inhibition H exose -6-phosphate dehydrogenase, also located in the ER, is thought to supply adequate concentrations of NADPH cofactor , which favour the behaviour of the 11?-HSD1 as a reductase in vivo.256,257 Besides, the higher affinity of 11?-HSD1 for cortisone than cortisol enhances this oxoreductive activity. In in vitro conditions, though, 11?-HSD1 is bidirectional and can also act as a dehydrogenase. Fig. 42 . Interconversion of cortisone to cortisol in humans catalyzed by 11?-HSD enzyme . 11?-HSD1 is mainly expressed in liver, adipose tissue, bone, pancreas, vasculature and brain, whilst 11?-HSD2 is found chiefly in mineralocorticoid target tissues such as the kidney, colon, pancreas and the salivary gland.258 In mineralocorticoid tissues, the MR have the same binding affinity for cortisol as aldosterone; 11?-HSD2 protects the MR from inappropriate activation by cortisol and allows aldosterone to act as the ligand, by inactivating cortisol to cortisone.259 Figure 43 summarizes what has been commented hitherto. 256 Dzy ak an ch u k, A. A.; Bal?z s, Z.; Nashev, L. G.; Amrein , K. E.; Od ermat t, A. Mol. Cell. Endocrinol. 2009, 301, 137 - 141. 257 Atan asov, A. G.; Nashev, L. G.; Ge lman , L.; Legeza , B.; Sa ck, R.; Portman n , R.; Od ermat t, A . Biochim. Biophys. Acta 2008, 1783 , 153 6 - 154 3. 258 Hard y , R. S.; Seib el, M. J.; Coo p er, M. S. Curr. Opin. Pharmacol. 2013, 13, 440 - 444. 259 Edwar d s, C. R.; Ste war t, P. M.; Burt, D.; Brett , L. ; McIn ty re, M. A.; Suta n to , W. S. d e Klo et, E. R.; Mon d er, C. Lancet 1988, 2, 986 - 98 9. Introduction 91 Fig. 43 . Biosynthesis of cortisol by adrenal gland under HPA axis control, and tissue -specific metabolism of GC by 11?-HSD enzymes. CRH: corticotropin -releasing hormone; ACTH: adrenocorticotrophic hormone.260,261 It is worth to highlight that cortisone is not the only substrate of 11?-HSD1, but several additional substrates have been identified such as 7-oxygenated steroids, 7 -ketocholesterol and non-steroidal unspecific carbonyl compounds, thus endowing the enzyme with an important role in the detoxification of exogenous substances .262,263,264 1.2 11?-HSD1 as a pleiotropic therapeutical target The rationale for the inhibition of 11?-HSD1 came from the striking resemblances betZeen the phenot\pe oI h\percortisolism also NnoZn as Cushing?s s\ndrome and the symptoms of the MetS The Cushing?s s\ndrome reveals a set of phenotypical characteristics such as insulin resistance, hypertension, dyslipidaemia, and osteoporosis (Fig. 44).265 While the Cushing?s s\ndrome is rare it does e[empliI\ the pathological eIIects associated Zith imbalances in circulating cortisol levels. 260 Hollis, G.; Hub er, R. Diabetes Obes. Metab. 2011, 13, 1 - 6. 261 Staab , C. A; Maser, E. J. Steroid Biochem. Mol. Biol. 2010, 119 , 56 - 7 2. 262 Odermat t, A.; Nashe v, L. G. J. Steroid Biochem. Mol. Biol. 2010 , 119, 1 - 1 3. 263 Diti?, d.; Andre?, Z.; tal?er, . Z.; ,ado?e, W. t. &. Biochimie 2013, 95, 548 - 555. 264 Odermat t, A.; Klu so n o va, P. J. Steroid Biochem. Mol. Biol. 2015, 151, 85 - 92. 265 Gath ercole, L. L.; Lavery , G. G.; Morgan , S. A ; Coop er, M. S.; Sin clair, A. J.; Tomlinson , J. W.; Stewar t, P . M. Endocr. Rev. 2013, 34, 525 - 555. Physiological metabolic effects: Gluconeog enesi s Lipolys is Mobili zation of am ino acids 92 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 44 . The pleiotropic eIIects oI *C e[cess in Cushing?s s\ndrome Endogenous GC production and metabolism have been highlighted as having a pivotal role in the aetiology of several disorders. While this production underlies the attractiveness of pharmacological inhibition of 11?-HSD1 to modulate local GC action, it requires to inhibit selectively this specific isoform to avoid undesired effects associated with the binding to 11?-HSD2, or other hydroxysteroid dehydrogenase enzymes, such as 17 ?- HSD. 266 It has been demonstrated that a deficient 11?-HSD2 activity (as a result of gene mutation, or by inhibition of this isoform) results in an impaired cortisol inactivation and apparent mineralocorticoid excess , leading to hypokalemia and hypernatremia, eventually inducing hypertension and related cardiovascular problems.265,267 On the other hand, inhibition of 17?-HSD affects local estrogen metabolism. Worth to note is that 11?-HSD1 is also expressed in areas of the brain relevant to metabolic control. 268 Although it has been demonstrated that 11?-HSD1 activity in brain does not contribute to systemic cortisol/cortisone turnover, 269 inhibition of 11?-HSD1 must be considered with caution since it may result in the risk of compensatory cortisol production via up-regulation of the 266 Klein , T.; Hen n , C.; N egri, M.; Frot sch er, M. PLoS One 2011 , 6, 1 - 13. 267 Kotelevt se v , Y.; Bro wn , R. W.; Flemin g, S.; Ken y o n , C.; Edwar d s, C. R.; Seckl, J. R.; Mu llin s, J. J. J. Clin. Invest. 1999, 103, 68 3 - 6 89. 268 Bissch o p , P. H.; D ekk er, M. J. H. J. ; Ost erth u n , W.; Kwak kel, J.; Anin k, J. J.; Boel en , A.; Unm eh o p a, U. A . ; Kop er, J. W.; Lamb ert s, S. W. J.; Ste war t, P. M.; Swaa b , D. F .; Flier s, E . J. Neuroendocrinol. 2013 , 25, 425 - 432. 269 Kilgou r, A. H. M.; Se mp le, S. ; Marsh all, I.; And re ws, P. ; A nd rew, R.; Walker, B. R. J. Clin. Endocrinol. Metab. 2015, 100, 483 - 489. Introduction 93 HPA axis .270 11?-HSD1 is considered as a pre-receptor target since it controls the binding of active cortisol to GR by modulating its metabolism. In recent years, there has been a rapid growth in publications concerning 11?-HSD1 as a promising target.271 Many research groups in academia and in the pharmaceutical industry have put forth great efforts to determine the role of 11?-HSD1 in the development of multiple diseases, and hence to discover suitable inhibitors to overcome these disorders. As just seen, selectivity and tissue-targeting specificity are the ultimate criteria in the discovery of potent 11?-HSD1 inhibitors. 1.2.1 11?-HSD1 inhibition and type 2 diabetes mellitus, obesity and metabolic syndrome Type 2 diabetes mellitus (T2DM) is characterized by increased cellular insulin resistance with an initial phase of enhanced insulin secretion followed by pancreatic ?-cell failure. Regarding obesity, a person with a body mass index of 30 or more is considered obese, entailing health risks. T2DM and obesity are considered as first-world epidemics and are nowadays great healthcare challenges in the developed countries. According to the World Health Organization (WHO), in 2014 , 9% of the worldwide adult population had diabetes.272 That same year, the percentage of total health expenditure in T2DM reached US $ 612 billion. The expected increase on the diabetic population by 2035 is about 205 million, which will elevate the total health cost still higher.273 Concerning obesity, in 2014 more than 1.9 billion adults were overweight, and of these, 600 million were obese.274 From another standpoint, the MetS is a cluster of metabolic disorders and it is diagnosed in a given patient if visceral obesity and two or more risk factors are present, including elevated fasting plasma glucose, elevated triglycerides, elevated blood pressure and reduced high-density lipoprotein (HDL), as it has been recently redefined by the International Diabetes Foundation (Fig. 45). Conversely, the WHO was the first to tie together the key components of insulin resistance, obesity, dyslipidaemia and hypertension.275,276 270 Rob b , G. R.; Boy d , S.; Davie s, C. D.; Do ss ett er, A. G. ; Gold b e rg, F. W.; Ke mmitt , P. D.; S cot t, J. S.; Swal e s , J. G. Med. Chem. Commun. 2015 , 6, 926 - 9 34. 271 Wamil, M.; S eckl, J. R. Drug Discov. Today 2007, 12, 50 4 - 5 2 0. 272 WHO web page. Diab ete s Fact She et. htt p ://w w w. wh o .in t/med iac en tre/ fact sh e ets/ fs3 12/ en / (acce ss ed on 26 th Jun e 2015 ). 273 Euro p ean Comiss ion web pag e. Majo r an d ch ro n ic diseas es : Diab ete s. htt p ://ec.eu ro p a. eu /h ealt h /m aj o r_ch ro n ic_d i sea se s/d is eas es/d iab et es /in d ex_ en .h tm# fr agmen t7 (acce ss ed on 2 nd July 20 15). 274 WHO web page. Ob e sity and over wei gh t Fact Sheet. htt p ://ww w.wh o .in t/ med iac e n tre/fact sh eet s/f s31 1/en / (a c ces sed on 26 t h June 20 15). 275 Gru n d y , S. M.; Brew er, H. B.; Clee man , J. I.; S mith , S. C.; L e n fan t, C. Circulation 2004, 109 , 433 - 43 8. 276 Huan g, P. L. Dis. Model Mech. 2009, 2, 231 - 237. 94 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 4 5. Combination of the above risk factors leads to the MetS. Currently, the predominant therapeutic strategy for preventing or managing T2DM and MetS is the individual treatment of the set of risk factors instead of treating the disorder as a whole. The increase in the prevalence of T2DM and MetS over the last few decades in the developed countries has propelled a growing need for the development of novel therapies to treat MetS and its associated disorders. Unlike the Cushing?s s\ndrome Zhere elevated circulating cortisol levels cause the phenotypic changes, in MetS circulating GC levels are not usually elevated, despite the fact that, until recently, the extended belief was that the major contributor to the symptomatology was the level of free cortisol in plasma and the densities of GR and MR in target tissues. Therefore, it has been speculated that inter- and intracellular local levels of GC regulated by the 11?-HSD enzyme are responsible for metabolic abnormalities. The impact of tissue-specific GC activation by 11?-HSD1 in metabolic homeostasis has been examined using several transgenic mouse models. Experiments in mice with adipose tissue-targeted 11?-HSD1 overexpression developed a phenotype similar to the one oI Cushing?s s\ndrome including insulin resistance visceral obesit\ d\slipidaemia and hypertension, without an increase in the GC plasmatic levels.277,278 On the other hand, overexpression of 11 ?-HSD1 in liver tissue led to the appearance of MetS but without impaired glucose tolerance and obesity.279 In contrast, 11?-HSD1 knock-out [ 11?-HSD1(-/ - )] mice had reduced triglyceride and non-esterified fatty acids, improved glucose tolerance 277 Masu za ki , H.; Pat erso n , J.; S hin y ama, H.; Morto n , N. M.; Mu llin s, J. J. ; S eckl, J. R. ; Fli er , J. S. Science 2001, 294, 21 66 - 2170. 278 Masu za ki, H.; Yama mo to , H.; Ken y o n , C. J.; El mq u ist, J. K.; Morto n , N. M.; Pater so n , J. M .; Shin y ama, H.; Shar p , M. G. F.; Fl emin g, S. ; Mu llin s, J. J.; S eckl, J. R.; Flier , J. S. J. Clin. Invest. 2003, 112 5, 83 - 90. 279 Paterso n , J.; Morto n , N. ; Fie vet, C.; Ken y o n , C.; Holme s, M. ; Sta el s, B.; Seckl, J. R. ; Mu llin s, J. J. Proc. Natl. Acad. Sci. USA 2004, 101 , 7088 - 7 093 . Metabolic Syndrome Visce ral Obesity Introduction 95 and higher HDLs on a high -fat diet.280,281,282 Parallelly, overexpression of adipose-specific 11?-HSD2 result ed in reduced fat mass, and increased glucose tolerance and insulin sensitivity.283 Several studies have appeared over the recent years proving the relationship between tissue-specific alterations in 11?-HSD1 expression in human T2DM, obesity and MetS.284 For instance, the expression of 11 ?-HSD1 in human adipose tissue is positively correlated with the degree of obesity.285,286,287 In view of all these studies, it is likely that an aberrant 11?-HSD1 expression/activity in several key metabolic tissues underlies the pathogenesis of these diseases (Fig. 46).288 280 Kotele vt se v , Y. ; Hol me s, M. C.; Burch ell, A.; Hou sto n , P. M.; Sch mo ll, D.; Jami eso n , P. ; Bes t, R.; Bro wn , R.; Edwar d s, C. R.; Seckl, J. R.; Mu llin s, J. J. Proc. Natl. Acad. Sci. USA 1997, 94, 14924 - 14 9 29. 281 Morto n , N . M.; Paterso n , J . M .; Masu za ki , H.; Holm es , M . C.; Sta els , B .; Fi ev et , C.; Wa lker , B . R.; Fl ier , J . S.; Mu llin s , J . J.; S eckl , J . R. Diabetes 2004, 53, 931 - 938 . 282 Morto n , N . M.; Holmes , M . C.; Fie vet , C. ; Sta el s , B.; Tai lleu x , A.; Mu llin s , J . J.; S eckl , J . R. J. Biol. Chem. 2001, 276, 41 293 - 413 00. 283 Kersh a w, E. E.; Morto n , N. M.; Dhillo n , H.; Ramag e, L.; Se c kl, J. R.; Fli er, J. S. Diabetes 2005 , 54, 1023 - 1031. 284 Morgan , S. A.; Tom linson , J. W. Expert Opin. Investig. Drugs 2010, 19, 10 67 - 1076. 285 Wake, D. J.; Ra sk, E.; Li vin gsto n e, D. E.; Sod erb erg , S.; Ol sso n , T.; Walk er, B. R. J. Clin. Endocrinol. Metab . 2003, 88, 398 3 - 3 988. 286 Valsamak i s, G.; An war , A.; To mlinson , J. W. ; Shack leto n , C. H. L.; McT ern an , P. G.; Chett y , R.; Wood , P. J.; Ban erje e, A. K.; Hold er, G.; Barn ett , A. H.; Stewar t, P. M.; Kum ar , S. J. Clin. Endocrinol. Metab. 2004, 89, 4755 - 4761. 287 Wang, M. Drug Dev. Res. 2006 , 67, 567 - 5 69. 288 Coop er, M. S.; Ste war t, P. M. J. Clin. Endocrinol. Metab. 2009 , 94, 4645 - 4654. 96 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 46 . Role of 11?-HSD1 in the pathogenesis of the MetS and related disorders. Hence, modulation of 11 ?-HSD enzyme , and in particular inhibition of 11?-HSD1 in adipose tissue and liver, are of special interest as pharmacotherapeutic strategies in the medical treatment of the MetS and associated disorders.289,290,291 1.2.2 11?-HSD1 inhibition and inflammation The anti-inflammatory effects of pharmacological levels of GCs are well known and widely exploited. 292 Short-term therapy with GCs, chiefly cortisone, is broadly described to treat acute inflammation. However, endogenous GCs are immunomodulatory rather than simply anti-inflammatory agents.293,294 GCs both enhance or suppress immune responses depending on concentration and timing, by affecting the migration of immune cells, their differentiated states, and the production of a range of inflammatory cytokines.288 Ultimately, GCs shape an ongoing inflammatory response as it progresses, depending on the local environment. With respect to11?-HSD1 , it is expressed in certain immune c ells, 289 Morto n , N. M. Mol. Cell. Endocrinol. 2010, 316, 154 - 16 4. 290 Johar ap u rk ar , A.; Dhan esh a, N.; Shah , G.; Kh ar u l, R.; Jain , M. Pharmacol. reports 2012, 64, 1055 - 106 5. 291 Anderso n , A.; Walker, B. R. Drugs 2013, 73, 1385 - 139 3. 292 McEwen , B. S.; Biron , C. A.; Bru n so n , K. W.; Bulloch , K.; Chamb ers, W. H.; Dhab h ar , F. S.; Gold far b , R. H.; Kitso n , R. P.; Mill er, A. H.; Spen cer, R. L.; W eis s, J. M. Brain Res. Rev. 1997, 23, 79 - 13 3 . 293 Sap olsky , R. M.; Rom ero , L. M.; Mu n ck, A. U. Endocr. Rev. 2000, 21, 55 - 89. 294 Yeag er, M. P.; Guy re, P. M.; Mu n ck, A. U. Acta Anaesthesiol. Scand. 2004, 48, 799 - 813. Introduction 97 such as the differentiated monocytes and in most lymphocyte populations.295 Regulation of 11?-HSD1 at this cellular level provides a further mechanism to fine-tune cellular immune responses. In chronic inflammatory processes, such as rheumatoid arthritis, atherosclerosis and rhinitis, there is an imbalance on the expression/activity of the two isoforms of the 11 ?- HSD. 296,297,298 It has been suggested that dysregulation of the normal reciprocal control of 11?-HSD1 and 11 ?-HSD2 may contribute to the failure of resolution during chronic inflammatory states. Therefore, the control oI *C?s bioavailabilit\ Zithin immune cells b\ 11?-HSD1 provides a checkpoint for the attenuation of unwanted immune responses. 299,300 Hence, m odulation of 11?-HSD1 expressed in immune cells may offer an alternative approach for the treatment of rheumatoid arthritis, rhinitis and atherosclerosis, diseases highly linked to MetS. Of note, metabolic disorders such as obesity and T2DM are associated with a dysregulated immune response,301 thereby underpinning the potential of the inhibition of 11?-HSD1 as a new therapeutic strategy for such conditions. 1.2.2 11?-HSD1 inhibition and cognitive dysfunction with aging The hippocampus region of the brain is an area that plays an important role in the formation of long-lasting memories and wields an executive level of control over the HPA axis. 300,302 In this particular region, 11?-HSD1 but not 11 ?-HSD2 is greatly expressed , together with GRs and MRs. As already discussed, GCs are known to deeply influence metabolism, immune system and neuronal function.292 Bearing in mind the phenotype of Cushing?s s\ndrome depicted in figure 3, deficient regulation of the HPA axis with resultant chronically elevated levels of GCs exerts deleterious effects on specific areas of the brain, eventually resulting in cognitive dysfunction. Specifically, excessive GC concentrations in the brain enhance neuronal death due to a series of conditions such as increase of the levels of amyloid precursor protein, hypoxia and excitotoxic concentrations of glutamate (therefore connected with the previous chapter).303,304 There is mounting evidence from human and rodent studies that this GC-related cognitive decline is apparent 295 Chap man , K. E.; Cou tin h o , A. E.; Gray , M.; Gil mo u r, J. S. ; S avill, J. S.; Seckl, J. R. Mol. Cell. Endocrinol. 2009, 301, 12 3 - 1 31. 296 Hard y , R. S.; Seib el, M. J.; Coo p er, M. S. Curr. Opin. Pharmacol. 2013, 13, 440 - 444. 297 Had oke, P. W. F.; Kip ar i, T.; S eckl, J. R.; Cha p man , K. E. Curr. Atheroscler. Rep. 2013, 15, 3 20 - 33 0. 298 Jun, Y. J.; Park , S. J.; Hwan g, J. W.; Kim, T. H.; Jun g, K. J.; Jun g, J. Y.; H wan g, G. H.; L ee, S. H.; Lee, S. H . Clin. Exp. Allergy 2014, 44, 19 7 - 211 . 299 Chap man , K. E.; Gil mo u r, J. S. ; Cou tin h o , A. E.; Savill, J. S.; S eckl, J. R. Mol. Cell. Endocrinol. 2006, 248 , 3 - 8. 300 Chap man , K. E.; Seckl, J. R. Neurochem. Res. 2008, 33, 624 - 6 36. 301 Hota misligi l, G. S. Nature 2006 , 444, 860 - 867. 302 Squire, L. R. Psychol. Rev. 1992 , 99, 195 - 2 31. 303 Sap olsky , R. M.; Pu lsin el li, W. A. Science 1985, 229, 1397 - 1 400. 304 Beraki, S. ; Litr u s, L.; Soria n o , L.; Mon b u reau , M.; To, L. K. ; Braith wait e, S. P. ; Niko lich , K.; Ur fer, R. ; Oksen b erg, D. ; Shamloo , M. PLoS One 2013, 8, 1 - 13. 98 CHAPTER 2: 11 ?-HSD1 inhibition with aging.305,306,307 Indeed, GCs are strongly implicated in the pathogenesis of age-related cognitive impairment and AD.308,309,310 It is believed that low intracellular concentration of GCs in hippocampus activates MRs, whose stimulation lead to increased memory, whilst high concentration of GCs saturates MRs and over-activates GRs, thus affecting memory processes.311 Moreover, inhibition of 11?-HSD1 reduces A ? plaque burden and plasma A? in AD animal models.312,313 In a similar way as for the previous disorders, influence on the metabolism of GCs within target tissues may offer a route for tissue-selective control of GC exposure. In this sense, 11?-HSD1 inhibition may assist in keeping the GC levels low throughout life, which may prevent the appearance of cognitive deficits with aging. This fact has been confirmed in aged [ 11?-HSD1(-/ -)] mice, which are protected from GC-associated hippocampal dysfunction with aging.314 In contrast to aged wild type mice, [ 11?-HSD1(-/ -)] counterparts learn as young mice, avoiding the normal cognitive decline seen with aging. On the other hand, it has been demonstrated that inhibition of 11?-HSD1 ameliorates ag e-related cognitive impairments in mice, suggesting that 11?-HSD1 is a potential target for the treatment of cognitive dysfunction.315,316 A study carried out by Seckl and co-workers proved that the use of a non-selective 11?-HSD inhibitor for 4 -6 weeks improved the cognitive functions in healthy and diabetic elderly.305 T2DM is a risk factor of dementia, whose pathogenesis is triggered by an overactivation of the HPA axis. 317 305 San d eep , T. C.; Yau , J. L. W.; Maclullich , A. M. J.; Nob le, J.; Dear y , I. J.; Walker, B. R.; Seckl, J. R. Proc. Natl. Acad. Sci. USA 2004, 101 , 6734 - 6 739. 306 Deaney, D. :.; O?Donnell, D.; Zo?e, t.; dannen?a?m, .; Steverman , A.; Walker, M. ; Nair, N. P. ; Lu p ien , S. Exp. Gerontol. 1995 , 30, 229 - 25 1. 307 Yau , J. L. W.; Wh eelan , N.; Nob le, J.; Walk er, B. R. ; Webst er, S. P.; Ken y o n , C. J.; Lu d wig, M .; Seckl, J. R . Neurobiol. Aging 2015, 36, 334 - 343 . 308 de Quervain , D. J. - F.; Poir ier, R.; Woll mer, M. A.; Grimald i, L. M. E.; Tsolak i, M.; Stref fer, J. R.; Hock, C. ; Nitsch , R. M.; Moh aj eri, M. H. ; Pap asso tiro p o u los, A. Hum. Mol. Genet. 2004, 13, 47 - 52. 309 Marto cchia , A.; Stefan elli, M .; Fala sch i, G. M.; Tou s san , L.; Fer ri, C.; Fala s ch i, P. Aging Clin. Exp. Res. 2015, ahead of print. DOI: 10. 1007/ s405 20 - 015 - 035 3 - 0 . 310 Katz, D. A. ; Liu , W.; Locke, C.; Jaco b so n , P.; Barn e s, D. M.; Basu, R.; An, G.; Rie ser, M. J.; D aszko wski, D . ; Gro v es, F. ; Hen e gh an , G.; Sha h , A.; Ge vo rk y an , H.; Jhee, S . S .; Eresh e fsky , L.; Mar ek, G. J. Transl. Psychiatry 2013, 3, e29 5. 311 Yau , J. L. W.; Nob le, J.; S eckl, J. R. J. Neurosci. 2011, 31, 418 8 - 419 3. 312 Gre en , K . N.; Billing s , L . M.; R oozen d aa l , B.; McGau gh , J . L. ; LaFerla , F . M . J. Neurosci . 2006 , 26, 9047 - 9056. 313 Sooy , K.; Nob le, J.; McBrid e, A.; Binn ie, M.; Yau , J. L. W.; Seckl, J. R.; Walk er, B. R.; W eb ster, S. P . Endocrinology 2015, ahead of print . DOI: 10.1 210/ en .201 5 - 1395. 314 Yau , J. L. W.; Nob le, J.; Ken y o n , C. J.; Hib b erd , C.; Kot el evt se v, Y. ; Mu llin s, J. J.; Seckl, J. R. Proc. Natl. Acad. Sci. USA 2001, 98, 4716 - 4721 . 315 Sooy , K.; Webster, S. P.; Nob le, J.; Binn ie, M.; Walker, B. R.; Seckl, J. R.; Yau , J. L. W. J. Neurosci. 2010, 30, 13867 - 13 872. 316 Mohler, E. G.; Brow man , K. E. ; Rod er wald , V. A ; Cro n in , E. A ; Mark o sy an , S.; Bitn er, R. S. ; S tra kh o va, M. I.; Dre sch er, K. U.; Horn b erge r, W.; Roh d e, J. J.; Bru n e, M. E.; Jaco b so n , P. B.; Ruet er, L. E. J. Neurosci. 2011, 31, 540 6 - 5 413. 317 Stra ch an , M. W. J.; Rey n o ld s, R. M.; Frier, B. M. ; Mitch ell, R. J.; P rice, J. F. Diabetes Obes. Metab. 2009 , 11, 407 - 414. Introduction 99 1.2.3 11?-HSD1 inhibition for other diseases 11?-HSD1 inhibition has been investigated also for other indications, such as glaucoma,318 osteoporosis,319 wound healing and age-associated skin damage.320,321 For the sake of brevity, these disorders are not discussed any further, the reader is referred to the titled references if desired. Fig. 47 . Plausible therapeutic indications arising from 11?-HSD1 inhibition. 271 318 Anderso n , S.; Carreiro, S.; Q uen zer, T.; Gal e, D.; Xian g, C.; Guka sy an , H.; Lafo n ta in e, J.; Chen g, H.; Krau ss, A.; Pra san n a, G. J. Ocul. Pharmacol. Ther. 2009, 25, 215 - 2 22. 319 Park, J. S.; Bae, S. J.; Cho i, S. - W.; Son , Y. H.; Park , S. B.; Rhee, S. D.; Kim, H. Y.; Jun g, W. H.; Kan g, S. K.; Ahn , J. H.; Kim, S. H.; Ki m, K. Y. J. Mol. Endocrinol. 2014, 52, 191 - 2 02. 320 Tigan es cu , A.; Tah ra n i, A. A.; Morgan , S. A.; Otra n to , M.; Desmo u l i ?re, A.; Abra h am s, L.; Hassan - S mith , Z.; Walker, E. A.; Rabb itt , E. H.; Coop er, M. S.; Amrein , K.; Lavery , G. G.; Ste war t, P. M. J. Clin. Invest. 2013, 123, 3051 - 30 60. 321 Terao , M.; Mu ro ta , H.; Ki mu r a, A.; Kato , A.; Ish ika wa, A.; Ig awa, K.; Miy o sh i, E.; K at ay am a, I. PLoS One 2011, 6, 1 - 1 1. 100 CHAPTER 2: 11 ?-HSD1 inhibition With the data provided so far, it would be logical to consider 11?-HSD1 inhibition as a potential panacea for the most prevalent diseases of the developed countries originated by HPA axis overactivation and subsequent chronically elevated GCs. In the light of the current insights of the role of 11?-HSD1 in tissue-specific reactivation of cortisone to cortisol and the pathological effects of an excess of GCs , perhaps we are in a position to re- evaluate the use of cortisone as anti-inflammatory agent for the treatment of arthritis and other inflammatory conditions, which was the subject of the Noble Prize in Physiology and Medicine in 1950.322 1.3 Crystal structure of 11 ?-HSD1 and i ts binding site 11?-HSD1 is a 34 kD protein of 292 amino acids that forms homodimers and tetramers in solution, which are believed to be the functional unit in vivo.323,324 The enzyme is comprised of 4 main regions, i.e. (i) a transmembrane domain at the N-terminus linked to the ER membrane; (ii) a cofactor binding domain, characterized by a Rossman fold; (iii) a cluster of key residues that constitute the catalytic site; and (iv) a region essential for enzyme dimerization at the C-terminus. The Rossman fold is highly conserved in the so-called short-chain dehydrogenase/reductase family of enzymes, whereof 11?-HSD1 is member. This particular three-dimensional structure consists of seven-stranded parallel ?-sheets flanked by three ?-helices on the left and right sides each.325 In 11?-HSD1, the cofactor-binding site is placed in between the Rossman fold, and close to the location of the catalytic site of the substrate (Fig. 48). 322 Hench , P. S.; Ken d all, E. C.; Sl ocu mb , C. H.; Polley , H. F. Ann. Rheum. Dis. 1949, 8, 97 - 104. 323 San d eep , T. C.; Walk er, B. R. Trends Endocrinol. Metab. 2001 , 12, 446 - 453. 324 Zhan g, J.; Osslu n d , T. D. ; Plan t, M. H.; Clogsto n , C. L.; Nyb o , R. E.; Xion g, F.; Delan ey , J. M. ; Jord an , S. R. Biochemistry 2005 , 44, 694 8 - 695 7. 325 Schu ster, D.; Mau r er, E. M.; Laggner, C. ; Nashe v, L. G.; Wi lcken s, T.; Lan g er, T. ; Od er ma tt , A. J. Med. Chem. 2006, 49, 345 4 - 3 466. Introduction 101 Fig. 48 . X-ray crystal structure of 11?-HSD1 bound to cofactor NADPH and a novel inhibitor . PDB code: 3CH6. 326 The substrate catalytic site shows the amino acid sequence Tyr-X-X-X-Lys (specifically Tyr183 and Lys187, wherein X-X-X is a certain combination of amino acids). The X-X-X motif often includes a conserved serine residue (Ser170), that participates in ligand binding, stabilizes the orientation of the substrate within the catalytic site, and catalyses the proton transfer to and from reduced and oxidized intermediates. 327 In contrast, Lys187 forms hydrogen bonds with the nicotinamide ribose of NADP(H) and lowers the p Ka of the hydroxy l group of the neighbouring Tyr183, which also binds to the substrate and cofactor. The also known as catalytic triad Tyr183 ? Ser170 ? Lys187, interacts thus with the substrate and promotes the proton transfer to the reactive keto-oxygen of cortisone to furnish the active GC cortisol (Fig. 49). 326 Wang, H.; Ruan , Z.; Li, J. J.; S imp kin s, L. M .; S mirk, R. A; W u, S. C.; Hutch in s, R. D. ; Nir sc h l, D. S.; Van Kirk , K.; Coo p e r, C. B. ; Sutt o n , J. C.; Ma, Z.; Golla, R.; S eeth al a, R.; Saly an , M. E. K.; Nay ee m, A.; Kry stek, S. R.; Sheri ff, S. ; Ca mac, D. M. ; Morin , P. E.; C arp en ter, B. ; Ro b l, J. A; Zah ler, R. ; Gord o n , D. A ; Haman n , L. G. Bioorg. Med. Chem. Lett. 2008 , 18, 3168 - 3172. 327 Hosfield , D. J.; Wu , Y.; Sk en e , R. J.; Hilger s, M.; Jen n in g s, A.; Snell, G. P.; A ertge erts, K. J. Biol. Chem. 2005, 280, 46 39 - 4648. Ross man fold NADP(H) In h ib ito r 102 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 49 . Binding mode of a steroid ligand (carbenoxolone) and cofactor NADPH with key residues highlighted similar to the endogenous substrate. PDB code: 2BEL.328 Despite the residues that establish key hydrogen bond interactions, the catalytic pocket is largely hydrophobic.329 Furthermore, both ends of the catalytic site are open to the solvent, thus ligands that are too long to fit within the pocket can extend out of it and contact with surface residues. The distinctive binding site of the steroid substrate and the conformational flexibility in crystal structures assert 11?-HSD1 as an ideal target for rational drug design. It has been suggested that the murine structure can provide a reliable model for the design of inhibitors for the human enzyme.330 Notwithstanding that, the preferred structure for the computer- aided drug design is the human protein, as it can be seen in most resolved crystal structures so far. 1.4 11?-HSD1 inhibitors in development Since the discovery of 11?-HSD1, there has been a rapid growth in small molecules that inhibit this enzyme as potential therapeutics for the treatment of multiple conditions. Many pharmaceutical companies have pursued development programmes targeting 11?- HSD1 , which still represent an important area. The patent literature surrounding the development of novel 11?-HSD1 inhibitors has been extremely rich in the last decade, with 328 Scott , J. S.; Cho o ra mu n , J. 11?-hydroxysteroid dehydrogenase Type 1 (11?-HSD1) inhibitors in development, in RSC Drug Discovery Series No. 27 , The Roy al Society of Che mi stry , 2012. 329 Thomas, M. P.; Pott er, B. V. L . Future Med. Chem. 2011, 3, 367 - 3 90. 330 Zh an g, J.; O s slu n d , T. D. ; Plan t, M. H.; Clogsto n , C. L.; Nyb o , R. E.; Xion g, F.; Delan ey , J. M. ; Jord an , S. R. Biochemistry 2005 , 44, 6948 - 6957. Introduction 103 more than 250 patent applications published, as well as several publications from academia and industry covering biologically active inhibitors.331,332,333,334,335 1.4.1 Non-selective 11?-HSD1 inhibitors as tools The Tibetan remedy liquorice root was used for the treatment of disorders in respiratory and digestive systems, biliary tracts and kidneys, paralysis with nervous origin, anaemia and as antidote in snake and wild dog bites.336 More than 250 active molecules have been identified from the extracts of this ancient medicine, and among them, the 18?- glycyrrhetinic acid was isolated. This naturally occurring steroid is a potent, non-selective inhibitor of 11?-HSD s with IC50 values in the nano-molar range (Fig. 50).337 Interestingly, structural modification of the 18?-glycyrrhetinic acid led to inhibitors with a reverse selectivity towards 11?-HSD2. 338,339 Later on, its hemisuccinate ester derivative carbenoxolone, which also inhibits both 11?-HSD1 and 11 ?-HSD2, was approved in the 1960s in UK for the treatment of gastric ulcers and inflammation.340 Fig. 50 . Structure of 18?-glycyrrhetinic acid and its synthetically derived carbenoxolone, a clinically used inhibitor of 11?-HSDs . Despite inhibiting both isoforms, carbenoxolone has been used in animal and human studies as a proof-of-concept during the target validation of 11?-HSD1 . Several studies have confirmed the increase of hepatic insulin sensitivity in humans with carbenoxolone, and 331 Webster, S. P.; Pal lin, T. D. Expert Opin. Ther. Pat. 2007 , 17 , 1407 - 1422. 332 Hugh es, K. A.; Web ster, S. P.; Walker, B. R. Expert Opin. Investig. Drugs 2008, 17, 481 - 49 6. 333 St. Jean Jr., D. J.; Wan g, M.; F otsch , C. Curr. Top. Med. Chem. 2008, 8, 1508 - 152 3. 334 Boy le, C. D.; Ko walski, T. J. Expert Opin. Ther. Pat. 2009 , 80 1 - 826 . 335 Scott , J. S.; Gold b er g, F. W.; Turn b u ll, A. V. J. Med. Chem. 2014 , 57, 4466 - 44 86. 336 Pavlova, S. I.; Ute sh ev, B. S.; S erge ev, A. V. Pharm. Chem. J. 2003, 37, 314 - 31 7. 337 B?hler, H.; Pers ch el, F. H.; Hi erh o lzer, K. Biochim. Biophys. Acta 1991, 1075, 206 - 21 2. 338 Su, X.; La wren c e, H.; Gan e sh ap illai, D.; Cru tt en d en , A.; Pu ro h it, A.; Re ed , M. J.; Vicker, N.; Pott er, B. V . L. Bioorg. Med. Chem. 2004, 12, 4439 - 4457. 339 Pand y a, K.; Dietrich , D.; Seib ert, J.; Ved era s, J. C.; Od er ma tt , A. Bioorg. Med. Chem. 2013 , 21, 6274 - 6281. 340 Wang, M. Inhibitors of 11?-hydroxysteroid dehydrogenase type 1 in antidiabetic therapy, in Diabetes ? Perspectives in Drug Therapy , Handbook of Experimental Pharmacology, 203 , Sp rin ger - V erlag Ber lin Heid elb erg, 2 011. 104 CHAPTER 2: 11 ?-HSD1 inhibition attenuation of the symptoms of MetS and atherogenesis in obese mice.341,342,343 Moreover, carbenoxolone improved cognitive function in healthy elderly men and type 2 diabetics, as commented before.305 However, the therapeutical potential of carbenoxolone is limited due to the hypokalemia and hypertension derived from 11?-HSD2 inhibition. 1.4.2 Studies with selective 11?-HSD1 inhibitors Since the discovery of the latter dual inhibitors of 11?-HSDs, the surge of research and clinical interest in this area has resulted in many companies and academic groups developing selective 11?-HSD1 inhibitors from a variety of structural classes. The large majority of 11?-HSD1 inhibitors feature a hydrogen bond acce ptor functionality such as amides, lactams, ureas, carbamates, sulphones, sulphonamides or heterocycles flanked by bulky lipophilic radicals such as cyclohexane s, adamantanes or benzene rings. A great part of the candidates can be sorted in a few groups: arylsulphonamides, triazoles, piperazine sulphonamides, piperidine carboxamides, thiazolones, heterocyclic and adamantyl amides, ureas, etc. From the available X-ray crystal structures of different inhibitors resolved with the protein,344 a few points can be drawn for the assistance when designing potential ligands: ? the H -bond acceptor unit mimics the reducible keto function of the endogenous substrate cortisone by interacting with the residues of the active site. ? The bulky lipophilic groups fit the buried hydrophobic pockets of the catalytic site, generally near the binding region of the nicotinamide unit of NADP(H) or the steroid substrate. ? The catalytic site is open to the solvent region, which allows the binding pocket to accommodate extended ligands. In this area, tyrosine 177 plays an important role in interacting with the hydrophobic regions of the molecules. 341 Walker, B. R.; Con n ach er , A. A.; Lin d say , R. M.; Webb , D. J. ; Edwar d s, C. R. J. Clin. Endocrinol. Metab. 1995, 80, 315 5 - 3 159. 342 Andrews, R. C.; Rooy ack er s, O.; Walker, B. R. J. Clin. Endocrinol. Metab. 2003, 88, 285 - 2 91. 343 Nuotio - An ta r, A. M.; Hach ey , D. L.; Hasty , A. H. Am. J. Physiol. Endocrinol. Metab. 2007, 293 , E1517 - E 1528. 344 PDB cod es : 2BEL, 3D4N , 3D3 E , 2IL T, 3BYZ, 3CZ R, 3CH 6 , am on g oth ers. Se e also re fer en c e 329 for a deta iled analy s e s of the cry st al stru ctu res o f 11 ? - H SD1 . Introduction 105 Table 12 includes the most relevant candidates that have reached clinical trials in the last few years.345,346,347,348,349 The positive data from human studies validate 11?-HSD1 as a target for the treatment of T2DM, MetS, related disorders and AD. Table 12. List of drug candidates that have achieved clinical trials as inhibitors of the 11?-HSD1 . Compound Company Status MK -0916 Merck No longer in company pipeline (achieved Phase II in 2004) MK -0736 Merck No longer in company pipeline (achieved Phase II in 2005) PF -915275 Pfizer Discontinued Phase II (2007) 345 Stefan , N.; Ra msau er, M.; Jor d an , P.; Nowo tn y , B.; Kan ta rt zis, K.; Ma ch an n , J.; Hwan g, J. H .; Nowo tn y , P.; Kah l, S.; Harreiter, J.; Horn eman n , S.; San y al, A. J.; Stewar t, P. M.; Pfeif fer, A. F.; Ka u tz ky - W i ller, A.; Rod en , M.; H?rin g, H. U.; F?rs t - Reckt en wald , S. Lancet Diabetes Endocrinol. 2014, 2, 40 6 - 416. 346 Siu, M.; Joh n so n , T. O. ; Wan g, Y.; Nair, S. K.; Tay lor, W. D. ; Crip p s, S. J. ; Matt h e ws, J. J.; Ed war d s, M. P. ; Pau ly , T. A; Ermo lief f, J.; Castr o , A.; Hosea, N. A; LaP aglia, A.; Fan ju l, A. N.; Vogel, J. E. Bioorg. Med. Chem. Lett. 2009, 19, 3493 - 349 7. 347 Feig, P. U.; Shah , S.; Herman o wski - Vo sat ka , A; Plotk in , D.; Sprin ger, M. S.; Don ah u e, S.; Thach , C.; Klein , E. J.; La i, E.; Kau fman , K. D. Diabetes Obes. Metab. 2011, 13 , 498 - 50 4. 348 Shah , S.; Herman o w ski - Vosat ka , A.; Gib so n , K.; Ru ck, R. A.; Jia, G.; Zh an g, J. ; Hwan g, P. M . T.; Ryan , N. W.; Lan gd o n , R. B.; Feig, P. U. J. Am. Soc. Hypertens. 2011, 5 , 166 - 17 6. 349 Clin ical trials web site. 11 ?- H SD1 . htt p s:// w ww.cl in icaltria l s.go v / (acc es sed on 28 t h July 2 015). 106 CHAPTER 2: 11 ?-HSD1 inhibition HSD-016 Wyeth Discontinued Phase I (2008) AMG -221/BVT -83370 Amgen/Biovitrum Discontinued Phase I (2011) AZD -8329 AstraZeneca Discontinued Phase I (2011) INCB13739 Structure not released yet Incyte Phase I completed RO5093151 Structure not released yet Hoffman -La Roche Phase I completed BI 135585 Boehringer Ingelheim Phase I completed BMS -770767 Bristol-Myers Squibb Phase II completed Introduction 107 ABT -384 Abbott Phase II completed P2202 Structure not released yet Eli Lilly & Piramal Phase II completed AZD -4017 AstraZeneca Phase II (recruiting) UE2343 Structure not released yet University of Edinburgh - Actinogen Phase II (recruiting) ASP3662 Structure not released yet Astellas Pharma Phase II (recruiting) In the interest of this project, 11?-HSD1 inhibitors that are adamantane-based compounds will be discussed in more detail. 1.4.3 Adamantane-based inhibitors of 11?-HSD1 The adamantyl group appears to be a privileged scaffold present in many 11?-HSD1 inhibitors disclosed in the patent and scientific literature, and a few of them have reached clinical trials (see Table 12). As in the previous examples, the inhibitors bearing an adamantane moiety feature an H -bond acceptor functionality attached at one end to an adamantane group, which fits one of the hydrophobic pockets of the catalytic site. Despite of these general binding characteristics, different subclasses of adamantane-based compounds have been revealed, from amides and ureas to triazoles and thiazolones. A brief 108 CHAPTER 2: 11 ?-HSD1 inhibition compilation of the most significant adamantanyl 11?-HSD1 inhi bitors is contained in figure 51.350.351,352,353,354,355,356,357,358,359,360 350 Cheng, H.; Hoff man , J.; Le, P. ; Na ir, S. K.; Crip p s, S.; Matt h ew s, J.; S mith , C.; Yan g, M.; Kup ch in sky , S.; Dres s, K.; Edwar d s, M.; Cole, B.; Walt ers, E.; Lo h , C.; Ermo lieff, J.; Fan ju l, A.; Bhat , G. B.; Herrera , J.; Pau ly , T.; Hos ea, N.; Pad ere s, G.; R ej to , P. Bioorg. Med. Chem. Lett. 2010, 20, 289 7 - 2 902. 351 Johan sso n , L.; Fot sch , C. ; Bar tb erger, M. D. ; Ca stro , V. M.; Chen , M.; Em ery , M.; Gu sta f s so n , S.; Hale , C.; Hickman , D.; Homan , E.; Jord an , S. R.; Komoro wski, R.; Li, A.; McRae, K.; Mon iz, G.; Matsu mo to , G.; Orih u ela, C.; Palm, G.; Veni an t, M.; Wan g, M.; Williams, M.; Zh an g, J. J. Med. Chem. 2008, 51, 2933 - 2 943. 352 Tic e, C. M. ; Zh ao , W.; Xu, Z.; Cacat ian , S. T. ; Si mp so n , R. D.; Ye, Y. J.; Singh , S. B. ; Mc K ee ver, B. M. ; Lin d b lom, P.; Guo , J. ; Kro sky , P. M.; Kru k, B. A ; Berb au m , J. ; Harriso n , R. K.; Joh n so n , J. J. ; Bukh tiy ar o v, Y.; Pan eman galo re, R.; Scott , B. B.; Zhao , Y.; Bru n o , J. G.; Zh u ang, L.; McGeeh a n , G. M.; He, W.; Claremo n , D. A . Bioorg. Med. Chem. Lett. 2010 , 20, 881 - 886. 353 Olson , S. ; A ster, S. D. ; Bro wn , K.; Carb in , L.; Grah am, D . W.; Herman o wski - Vo sat ka , A.; L e Gra n d , C. B. ; Mu n d t, S. S.; Rob b in s, M. A ; S ch aeff er, J. M.; S los sb erg, L. H .; Szy mo n ifka , M. J.; Thier in ge r, R.; Wrigh t, S. D.; Balk o v ec, J. M. Bioorg. Med. Chem. Lett. 2005, 15, 4359 - 4362. 354 Roh d e, J. J.; Pli u sh ch e v , M. A. ; Soren s en , B. K.; Wod ka , D.; S hu ai, Q.; Wan g, J.; Fun g, S.; M on zo n , K. M.; Chio u , W. J.; Pan , L.; Den g, X.; Cho van , L. E.; Ramaiy a, A.; M ullally , M.; Hen ry , R. F.; Sto lari k, D. F.; Imad e, H. M.; Mar sh , K. C.; Ben o , D. W. A. ; Fey , T. A . ; Droz , B . A . ; Bru n e, M. E.; Camp , H. S.; Sham, H. L.; Fre v ert, E. U.; Jaco b so n , P. B.; Lin k, J. T. J. Med. Chem. 2007, 50, 14 9 - 164. 355 Rich ar d s, S.; Soren sen , B.; Jae , H. - S.; Win n , M.; Chen , Y.; W an g, J.; Fun g, S.; Mon zo n , K.; Frev ert, E. U. ; Jaco b so n , P.; Sham, H.; Lin k, J. T. Bioorg. Med. Chem. Lett. 2006 , 16, 6241 - 62 45. 356 Venier, O.; Pa scal, C. ; Brau n , A.; Naman e, C.; Mou gen o t, P.; Cre sp in , O.; Pacq u et, F.; Mou gen o t, C. ; Mon seau , C.; On ofr i, B.; Dad ji - Fa?h u n , R.; Lege r, C.; Ben - H as si n e, M.; Van - Ph am, T.; Rago t , J . L.; Ph ilip p o , C.; Farj o t, G.; Noah , L.; Man i an i, K.; Bou ta rfa, A.; Nicola? , E.; Guillo t, E.; Pru n iau x, M. P. ; G?s sr e gen , S. ; Engel, C.; Cou ta n t, A. L.; de Mi gu el, B.; Ca stro , A. Bioorg. Med. Chem. Lett. 2013, 23, 2414 - 2421. 357 Webster, S. P.; Ward , P.; Binn ie, M.; Craigie, E.; McCo n n el l, K. M. M.; Soo y , K.; Vinter, A.; Seckl, J. R. ; Walker, B. R. Bioorg. Med. Chem. Lett. 2007, 17, 2838 - 2843 . 358 Okaz ak i, S.; Tak ah ashi, T.; Iwamu ra , T.; Nak ak i, J.; Sekiy a, Y.; Yagi, M.; Kumaga i, H.; Sat o , M.; Sak ami, S.; N itt a, A.; Kawai, K. J. Pharmacol. Exp. Ther. 2014, 351, 1 81 - 18 9. 359 Ryu, J. H.; Kim, S.; Lee, J. A.; Han , H. Y.; Son , H. J.; Lee, H. J.; Kim, Y. H.; Kim, J. - S. ; Park , H. Bioorg. Med. Chem. Lett. 2015, 15, 1679 - 16 83. 360 Kim, Y. H. ; Kan g , S. K. ; Lee, G. Bin; Lee, K. M.; Ku mar , J. A. ; Ki m, K. Y. ; Rhe e, S. D.; Joo, J.; Ba e, M. A.; L ee, W. K.; Ah n , J. H. Med. Chem. Commun. 2015, 6, 1360 - 1 369. Introduction 109 Fig. 51 . Selection of adamantane-based inhibitors of 11?-HSD1 in development . The preponderance of the adamantane ring in many 11?-HSD1 inhibitors suggests that this precious polycycle forms tight-binding interactions within the hydrophobic binding pockets. Regardless of the good match that the adamantane moiety exerts in the binding site of the enzyme, the high lipophilicity furnished by the adamantane leads to potential solubility and metabolic stability problems, as seen in the introductory part of the present thesis. The initial pharmacological results imply that increasing the hydrophobicity of the inhibitors generally entails an improvement of 11?-HSD1 inhibitory potency, but this progression is often counterbalanced by a poor performance in the PD assay. With the goal of improving PK and PD profiles, academic and industrial research groups have carried 110 CHAPTER 2: 11 ?-HSD1 inhibition out different approaches. On one hand, the introduction of polar groups at key positions, such as amides, sulphonamides and hydroxyl groups, with diverse orientations on the ring stabilize the hydrophobic ring from metabolism and increase water solubility. On the other hand, replacement of the adamantane group by other scaffolds, such as bicyclo[2.2.2]octane, provides candidates that possess excellent PK and PD profiles in target tissues. Merck followed this strategy in a development programme with success, leading to MK-0736 (see table 12).361,362,363 These findings suggest that it is possible to optimize the lipophilic adamantane moiety effectively for clinical administration targeting the promising 11?-HSD1 enzyme. 361 Wadd ell, S. T.; Balko v ec, J. M .; Kev in , N. J.; Gu, X. Prepa ra tio n of tria zo les deri vat iv es a s inh i b ito rs o f 11 ?- h y d ro xy ster o id deh y d ro g en ase - 1. WO 2 00704 7625 . 362 Wang, H.; Rob l, J. A. ; Haman n , L. G.; Si mp kin s, L. M. ; Golla , R.; Li, X.; Se eth ala , R.; Zvy ag a, T.; Gord o n , D. A.; Rob l, J. A.; Li, J. L . Bioorg. Med. Chem. Lett. 2011, 21, 4146 - 4149. 363 Bau man , D. R.; Wh itehead , A.; Con tin o , L. C.; Cui, J.; Garcia - Cal vo , M.; Gu, X.; Ke vin , N.; Ma, X.; Pai, L.; Shah , K.; Shen , X.; Strib l in g, S. ; Zokia n , H. J.; M etz ger, J.; Sh ev ell, D. E.; Wad d ell, S. T. Bioorg. Med. Chem. Lett. 2013, 23, 3650 - 365 3. Objetive s Objectives 113 At the time of the present dissertation, no 11?-HSD1 inhibitor had reached the market, what illustrates the difficultness of developing drug candidates with optimal PK and PD profiles and the need for discovering new active pharmaceutical ingredients (API) that may overcome these issues. From the available data drawn from the X-ray crystal structures of different inhibitors bound to the 11?-HSD1 catalytic site, a general trend can be deduced. For the most part, two hydrophobic moieties are linked by an H -bond acceptor, which fill the buried hydrophobic pockets and interact with the key residues of the catalytic site, respectively. These crucial pieces constitute the pharmacophore of the 11?-HSD1 inhibitors. From this observation, we hypothesized that different scaffolds can be placed as hydrophobic moieties instead of the adamantane group in order to achieve structures with more appropriate profiles. Taking advantage of the extended expertise of our research group in the synthesis of polycyclic scaffolds, we started a new project related to the discovery of novel 11 ?-HSD1 inhibitors bearing adamantane-like scaffolds as hydrophobic counterparts. We hypothesized that the substitution of the adamantane moiety for other polycyclic cage structures may offer compounds with improved PK and PD profiles whilst keeping good binding affinity to the target. Under these premises, we determined the following goals of this chapter: 1. Synthesis and pharmacological evaluation of a family of compounds with the general structure of the benzo-homoadamantane IV (Fig. 52). This scaffold, already seen in the previous chapter of the present thesis, embodies a phenyl ring in its structure and lets the introduction of different substituents at the C-9 position, which may alter the binding mode along with PK properties. Fig. 52. Replacement of the adamantane group by a scaffold with general structure IV. RHS: right-hand side. 2. Synthesis and pharmacological evaluation of a family of compounds with general structure V (Fig. 53). This hexacyclic structure provides a larger lipophilic scaffold than the adamantane group. Derived from the synthetic route, both dienes and alkanes can be prepared. We aimed to determine the effect of an expanded adamantane analogue in the binding affinity of the new compounds. 114 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 53. Incorporation of hexacycles V as surrogates of the adamantane moiety. In order to test our polycycles we designed the compounds based on the literature. In this manner, we chose as the right-hand side of the molecules a diversity of already proven subunits stemming from the work of a few pharmaceutical companies (Fig. 54).350,351,352,355 Fig. 54. Right-hand sides (RHS) applied in the synthesis of the new compounds. Results & Discussion Results & Discussion 117 1. Application of the benzo polycyclic scaffold as an adamantane analogue for 11?-HSD1 inhibition 1.1 Synthesis of the C-9 substituted 6,7,8,9,10,11 -hexahydro -5,7:9,11 -dimethano -5H- benzocyclononen -7-yl derivatives For the design of the new compounds, we have adopted a hybrid strategy whereby the inhibitor is partitioned into a hydrophobic adamantane-like unit (left-hand side of the molecule) and a cyclic structure (right-hand side), linked by an amido- or urea-like unit (Fig. 55). The adamantane moiety has been replaced by the benzo-homoadamantane polycycle IV . This strategy obeys a twofold purpose. First, the introduction of the phenyl ring, together with polar and non-polar groups attached to the polycycle (OH, OMe, F, Me) is intended to modify the PK properties of the compounds, and particularly the metabolism at specific ring positions. Second, the incorporation of the phenyl moiety is expected to establish new binding interactions in the hydrophobic cage. In addition, this insertion involves a size expans ion of the carbocyclic structure. At this point, it is worth noting that the active site of the enzyme seems to be wide enough as to accommodate this polycycle, as suggested upon inspection of the available X-ray structures. On the other hand, a series of carbocyclic and heterocyclic subunits previously tested as 11?-HSD1 inhibitors are integrated into the final polar moiety as the RHS . Finally, the amido- or urea-like unit present in the linker should enable the formation of key hydrogen bonds at the binding site. Fig. 55 . Design of the new 11?-HSD1 inhibitors with general structure IV . The selection of the different moieties as the RHS was in accordance to a balance between the ease on their synthesis and the reasonable inhibitory activity. 350,351,352,355 Figure 56 includes the IC50 values of the parent inhibitors. Worth to consider is the fact that the IC50 oI 9itae?s inhibitor Zas determined in an enz\matic assa\ Zhich may differ from the value obtained by a cellular assay (not available). Thus, a direct comparison cannot be made 118 CHAPTER 2: 11 ?-HSD1 inhibition with the rest of the IC50 values, but it provides some indications of how potent is the compound. Fig. 56. Biological activity of the 11?-HSD1 inhibitors taken as templates. Moreover, we decided to reduce the complexity of the Amgen?s analogue b\ placing two identical substituents in C-5 position, i.e. two methyl groups as shown in figure 55. In this manner, any stereochemistry derived from the substitution pattern is avoided. Taking advantage of the C-9-substitution patterns obtained from the previous chapter of the thesis, we sought to explore the different combinations derived from the substitution at the C-9 position of the adamantane-like polycycle and each one of the selected polar units as RHS . First, we decided to use four of our amines as starting points (Fig. 57). Amine 23 was not included due to its low-yielding synthetic procedure. Fig. 57 . Amines 46, 19, 48 and 52 used as left-hand side of the putative new 11?-HSD1 inhibitors. Amine 19 preparation was similar to the synthesis of hydroxyl derivative 46. Likewise, Prins-Ritter transannulation of diene 29 with chloroacetonitrile and sulphuric acid in neat glacial acetic acid, followed by thiourea-mediated deprotection of the chloroacetamide group gave the desired intermediate, amine 19, in 56% yield (two steps) (Scheme 25 ). Scheme 25. Prins-Ritter transannular cyclization of 29, followed by thiourea-mediated deprotection of chloroacetamide. To optimize the synthesis, we applied the following strategy to detect the perfect match between the C-9 substituent and the polar unit: Results & Discussion 119 ? Explore the effects on the C -9 substitution, essentially with polar and non-polar groups. We chose piperidine-1-carboxamide as the right -hand side of the molecule due to the ease of preparation of the final compounds. ? Identify the polar unit from figure 55 that performs best with the scaffold at issue. To do so, we used amines 46 and 19 featuring a hydroxyl and a methyl group, respectively, so as to have one polar and one non-polar substituent to compare. With this in mind, the compounds that needed to be synthesized are depicted in figure 58. Fig. 58. Compounds to be made to assess the effect of the different substitutions in benzo- homoadamantane as a scaffold for 11?-HSD1 inhibitors . Compound 60, which contains a cyclohexyl amide attached to the p olycyclic structure, was also included in the list of polar units. We intended to analyse whether the replacement of a urea by an amide, a common functional group in most 11?-HSD1 inhibitors, might affect the inhibitory activity. This structure was previously tested by our group in a different series of compounds.364 The syntheses will be organized according to the different functionalities of the core moiety. 364 Leiva, R. ; Se ira, C.; M cBrid e, A.; Binn ie, M. ; Lu q u e, F. J. ; Bi d o n - Cha n al, A.; Webst er , S. P .; V?z q u ez, S . Bioorg. Med. Chem. Lett. 2015 , 25, 4250 - 4253. Exploration of the right-hand side Identification of the best C-9 substituent 120 CHAPTER 2: 11 ?-HSD1 inhibition 1.1.1 Preparation of amide compounds Chemical reactions for the formation of amide bonds are among the most executed transformations in organic and medicinal chemistry.365 The current methods for amide formation are remarkably general but at the same time widely regarded as expensive and inelegant.366 Acylation of amines with activated carboxylic acids is the most frequent reaction performed in the synthesis of pharmaceuticals and in peptide chemistry, representing more than 70% of all acylations and related processes executed in medicinal chemistry (Scheme 26).367,368 Scheme 26. Carboxylic acid activation for subsequent amine attack and final amide bond formation. Based on the success and broad use of the activating or coupling agents, the titled amides were prepared following two different procedures. In these cases, the acylating agent is generated in situ from the acid in presence of the amine, by the addition of a coupling agent. The first approach implied the use of carbodiimides as coupling agents, which were the first activating reagents to be used in synthesis.369 Different carbodiimides are currently employed, such as dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) and 1-ethyl-3-(?-dimethylaminopropyl)carbodiimide (EDC), each of one showing its own pros and cons. As for the reaction mechanism, the first step of the process involves the attack of the carboxylic aci d to the carbodiimide to form O-acylisourea 66. This intermediate can yield a number of different products, depending on which species reacts with it: i) formation of the desired amide 67 via coupling with the amine; ii) formation of anhydride 68, which can lead to amide 67 after attack of the amine (it requires two equivalent of the carboxylic acid ); and iii) generation of N-acylurea 69 by rearrangement of the common intermediate. To exemplify the mechanism of this coupling it is represented in scheme 27 using EDC. This carbodiimide was selected as the coupling agent due to its high water solubility, facilitating its removal by aqueous washes. 365 Rou gh ley , S. D.; Jord an , A. M. J. Med. Chem. 2011, 54, 345 1 - 3479 . 366 Patt ab iraman , V. R.; Bod e, J. W. Nature 2011, 480, 471 - 479 . 367 Monta lb ett i, C. A. G. N.; Falq u e, V. Tetrahedron 2005, 61, 10827 - 108 52. 368 Valeur, E.; Brad ley , M. Chem. Soc. Rev. 2009, 38, 606 - 6 31. 369 Sheeh an , J. C.; He s s, G. P. J. Am. Chem. Soc. 1955, 77, 1067 - 1068. Results & Discussion 121 Scheme 27. One-pot coupling of carboxylic acids with amines using EDC . Inasmuch as part of the acid partner is lost with the formation of the N-acylurea 69, it was reasoned that the addition of a selected nucleophile that reacted faster than the competing acyl transfer, generating an intermediate still active enough to couple with the amine might avoid this side reaction. Hydroxybenzotriazole or HOBt is one of those nucleophiles.370 Hence, the established procedures consist in the addition of EDC as coupling agent, HOBt as additive, as shown in scheme 28. Scheme 28. Avoidance of the formation of N-acylurea 69 by addition of HOBt . 370 Chan , L. C.; Cox, B. G. J. Org. Chem. 2007, 72, 886 3 - 8 869. 122 CHAPTER 2: 11 ?-HSD1 inhibition As the evolution in the coupling agents progressed, novel activators appeared with the purpose of preventing the use of two reagent species and increasing the reactivity. Onium salts based upon 7-aza-1-hydroxybenzotriazole (HOAt) have replaced the predominant carbodiimide and HOBt technique. 371,372 Among them, 1-[ bis(dimethylamino)methylene] - 1H-1,2,3-triazolo[4,5 -b]pyridi nium 3-oxid hexafluorophosphate (HATU) has become one the most popular coupling agent. When first reported, HATU was described as an uronium salt (O-isomer), but X-ray crystal structure revealed that the preferred state is as guanidinium salt (N-isomer) (Fig. 59).373 However, there is an equilibrium between the two forms in solution. Fig. 5 9. Different isomers of HATU, the N-isomer being the predominant form in the crystal structure. The coupling mechanism entails first the generation of both an activated O-acylisourea 70 and a HOAt unit. The negatively charged oxygen atom of this same benzotriazole reacts with the activated acid in a similar manner to the previous mechanism. Elimination of tetramethylurea, prior to the coupling of the amine to the highly reactive ester-like species, affords the desired amide compound (Scheme 29). The generation of the urea by-product is the driving force of the reaction. 371 Carp in o , L. A. J. Am. Chem. Soc. 1993, 115, 4397 - 43 98. 372 Albericio , F.; Bo fill, J. M . El - Fa h am, A.; Kat e s, S. A. J. Org. Chem. 1998, 63, 9678 - 96 83. 373 Carp in o , L. A.; Imazu mi, H.; El - Fah am, A.; Ferr er, F. J.; Zh ang, C.; Lee, Y.; Foxman , B. M.; Hen klein , P. ; Han ay , C.; M? gge, C.; Wens c h u h , H.; Klose, J.; Bey erman n , M.; Bien ert, M. Angew. Chem. Int. Ed. 2002, 41, 441 - 445. Results & Discussion 123 Scheme 29. Activation process of carboxylic acids using uronium/guanidinium type coupling agents. Despite the high efficiency of HATU, especially in difficult sterically hindered couplings, the amine can react with the coupling agent to give a guanidinium by-product as a side reaction (Scheme 30). Scheme 30. Possible guanidinium side-product when using HATU. Having introduced these two approaches, i.e. the use of EDC/HOBt or HATU as coupling agents, starting from amines 46, 19, 48 and 52, the synthetic procedure towards the obtaining of final amides is outlined in scheme 31. 124 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 31. Amide coupling of amines 46, 19, 48 and 52 and selected carboxylic acids applied in this thesis. The first attempt was using HATU as the activating reagent, because of the known high efficiency of this agent in difficult sterically hindered couplings. Beginning with the preparation of proline derivative 56, the synthetic route started with the amide coupling between amine 46 and the commercially available N-Boc-D-proline (Scheme 32). A slightly excess of the amine was used over the non -natural amino acid, in order to prevent the guanidinium side reaction, as well as for HATU and the base. The reaction afforded the desired Boc-protected pyrrolidine 71 in 50% yield after purification by column chromatography. Scheme 32. Coupling of amine 46 with N-Boc-D-proline gave amide 71. The synthetic sequence towards amide 56 continued with the deprotection of the t- butyl carbamate group following the procedure reported by Li et al.374 The method consisted in the use of aqueous phosphoric acid, which is an acid whose conjugate base is non-nucleophilic. This hampers the dehydration of the hydroxyl group at the tertiary position and subsequent introduction of the conjugate base. After neutralization of the media, deprotected pyrrolidine 72 was secured in 87% yield as a free base (Scheme 33 ). These two simple steps furnished the first compound for pharmacological testing that was also central to access the next final compound. 374 Li, B.; Be mish , R.; Buzo n , R. A. ; Chiu , C. K. F.; Col gan , S. T.; Ki ss el, W.; Le, T.; Le eman , K. R.; Newel l, L.; Rot h , J. Tetrahedron Lett. 2003 , 44, 8113 - 8115. Results & Discussion 125 Scheme 33. Deprotection of boc-protected pyrrolidine 71 to afford free amine 72. The reaction entails the formation of isobutene and carbon dioxide. With amine 72 in hand, we proceeded to the reductive alkylation following our previously mentioned methodology. We treated the free amine with acetaldehyde and sodium cyanoborohydride under the same conditions as scheme 19 (NMDA chapter), but regrettably no alkylated amine was observed (Scheme 34), and the starting material was fully recovered. Scheme 34. Attempt of reductive alkylation of amine 72 with acetaldehyde and NaBH 3CN. Unhappy with the result, we applied the procedure described in the literature for the preparation of PF-877423 derivative, which involved the N-alkylation with ethyl bromide in the presence of a base and potassium iodide. The latter was used in catalytic amounts in order to displace the bromide in an SN2 fashion and promote the attack of the amine to the alkylating agent, transformation known as Finkelstein reaction (Scheme 35). Pleasingly, amide 56 was obtained in 72% yield without need of column chromatography. 126 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 35. N-alkylation of amine 72 with ethyl iodide after halogen displacement. Once hydroxyl derivative 56 was prepared, we next moved to the synthesis of its C -9 methylated analogue 57. Despite the avoidance of extra additives when using HATU as coupling agent, the moderate yield obtained in the preparation of 71 (50% yield) showed a partial success of the reaction. In consequence, we essayed the peptide-like coupling using EDC and HOBt as activating agents. Based on standard protocols,375,376,377 we subjected amine 19 to a coupling process with 1.5 equivalents of both agents, along with TEA as a base in ethyl acetate (Scheme 36). After several aqueous washes, pure amide 73 was obtained in 84% yield without any purification needed. Scheme 36. Formation of an amide bond with EDC and HOBt . Amide 73 was in turn submitted to Boc-deprotection with 85% aqueous ortho - phosphoric acid to provide free amine 74 in quantitative yield. Primary amine 74 was purified and fully characterized for pharmacological evaluation. Last, N-alkylation with 375 Drago vich, P. S.; Prin s, T. J.; Zh ou , R.; Joh n so n , T. O.; Hua, Y.; Lu u , H. T.; Sak at a, S. K.; Brown, E. L.; Mald o n ad o , F. C.; Tun tla n d , T.; Lee, C. A.; Fuh rman , S. A .; Zalman , L. S .; Patick, A. K.; Matt hew s, D. A .; Wu , E. Y.; Guo, M.; Bor er, B. C.; N ay y ar , N. K.; Mora n , T.; Chen , L.; Rejt o , P. A .; Ro se, P. W.; Guzman , M. C. ; Doval san to s, E. Z.; L ee, S .; M c Ge e, K.; Moh aj eri, M.; Lie se, A.; Tao , J.; Kosa, M. B.; Liu , B. ; Batu go, M. R. ; Gle eso n , J. P. R. ; Wu , Z. P.; Liu , J.; Mead o r, J. W.; Ferr e, R. A. J. Med. Chem. 2003, 46, 457 2 - 45 85 . 376 Nikit en ko , A.; Alimar d an o v, A.; Afra gola , J.; Schmid, J.; Kri sto fo va, L.; Evra rd , D.; Hat zen b u h ler, N. T.; Mara th ias, V.; Sta ck, G.; L en ic ek, S.; Potski, J. Org. Process Res. Dev. 2009, 13, 91 - 97. 377 Sasak i, N. A.; Garcia - ?lvar ez, M. C.; Wan g, Q.; Erm o len ko , L.; Fran ck, G.; Nhiri, N.; Martin M. T.; Aud ic, N.; Potier, P. Bioorg. Med. Chem. 2009, 17, 2310 - 23 20. Results & Discussion 127 ethyl bromide, KI and TEA afforded final compound 57 in moderate yield (44%) , which was isolated as the L-(+) -tartrate salt after column chromatography (Scheme 37). Previous attempts to precipitate the resulting amine as its hydrochloride salt yielded a hygroscopic solid, which did not meet the required physical properties for pharmacological evaluation. Scheme 37. Boc-deprotection and N-alkylation reaction to provide 57?tartrate. In line with our objectives, aryl amides 58 and 59 were synthesized according to the best conditions obtained for the amide coupling, that is the combination of EDC and HOBt (84% vs 50% yield with HATU). Therefore, a slightly excess of amines 46 and 19 were added to a mixture of commercially available 4 -amino-3,5-dichlorobenzoic acid, EDC, HOBt and TEA in ethyl acetate to produce the corresponding final amides 58 and 59 in 95% and 53% yield, respectively (Scheme 38). The significant differences in the yields indicate that the substitution on the C-9 position may affect directly the reactivity of the amine group, or indirectly by changes in solubility. Scheme 38. Amide coupling between amines 46 and 19 with 4-amino-3,5-dichlorobenzoic acid. The synthesis of the last amide 60 was performed by the same protocol, in this case with cyclohexanecarboxylic acid. Satisfyingly, reaction with amine 19 furnished the corresponding final amide 60 in excellent yield (Scheme 39 ). 128 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 39. Reaction of amine 19 with cyclohexanecarboxylic acid under amide coupling conditions provided 60 in 96% yield. 1.1.2 Synthesis of the thiazolone scaffold The 2-amino-1,3-thiazol-4(5H)-one skeleton was built following the described procedures.378,379 Starting from amine 19, the intermediate thiourea 76 was prepared via a two-step protocol. First, treatment with benzoyl isothiocyanate provided benzamide 75 that was subsequently hydrolysed under basic conditions to give 76 in 52% ove rall yield (Scheme 40). Scheme 40. Transformation of amine 19 to thiourea 76 after reaction with benzoyl isothiocyanate and later basic hydrolysis. The construction of the thiazolone ring system was accomplished by a substitution/c yclization reaction between thiourea 76 and ethyl 2-bromoisobutyrate in the presence of 11?-diisopropylethylamine (DIPEA) under microwave irradiation (Scheme 41). Researchers from Biovitrum found that this process required long reaction times (3-8 days) heating to 90-105 ?C by conventional methods. Nevertheless, the reaction time was substantially reduced by microwave-assisted heating to 130 ?C, but at the expense of lowering the yield (13%). 378 Jean , D. J. S.; Yuan , C.; Ber c o t, E. A ; Cup p les, R.; Chen , M.; Fretla n d , J. ; Hale, C. ; Hun gat e, R. W. ; Komoro w ski, R.; V en ian t, M.; Wan g, M.; Z han g, X.; Fot sch , C. J. Med. Chem. 2007 , 50, 42 9 - 432 . 379 Johan sso n , L.; Fot sch , C. ; Bar tb erger, M. D. ; Ca stro , V. M.; Chen , M.; Em ery , M.; Gu sta f s so n , S.; Hale , C.; Hickman , D.; Homan , E.; Jord an , S. R.; Komoro wski, R.; Li, A.; McRae, K.; Mon iz, G.; Matsu mo to , G.; Orih u ela, C.; Palm, G.; Venian t, M.; Wan g, M.; Williams, M.; Zh an g, J. J. Med. Chem. 2008, 51, 2933 - 2 943. Results & Discussion 129 Scheme 41. Substitution/cyclization sequence to assemble the thiazolone ring with a thiourea and an ?-bromoester. For the sake of clarity, the proton exchanges have been omitted . 1.1.3 Urea group formation Different approaches have been described for the synthesis of the urea moiety of many 11?-HSD1 inhibitors, from direct coupling of amines with isocyanates to the use of amine- activating agents such as triphosgene or carbonyldiimidazole (CDI).380,381 Some of them will be reviewed in the last chapter of the thesis. Alternatively, another method to install a urea functionality concerns the reaction of an amine with a carbamoyl chloride in the presence of a base, in a similar way than with an isocyanate.382 Since we aimed to prepare a urea embodied in a piperidine ring, we pursued its synthesis following this last approach, where amines 46, 19, 48 and 52 were reacted with 1-piperidinecarbonyl chloride in TEA to afford the corresponding final ureas 62-65 in low to medium yields (Scheme 42). In all cases, the final compounds were purified by column chromatography and/or crystallization in order to eliminate the e xcess of the starting material. The reaction mechanism is simple, and entails the addition of the amine to the carbonyl, with the subsequent elimination of the chloride, which is trapped by the base as the hydrochloride salt. 380 Tice, C. M.; Zh ao , W.; Xu, Z.; Cacat ian , S. T.; Simp so n , R. D.; Ye, Y. J. ; Singh , S. B.; McKee ver, B. M.; Lin d b lom, P.; Gu o, J. ; Kro sky , P. M .; Kru k, B. A ; Berb au m, J. ; Harriso n , R. K.; Joh n so n , J. J. ; Bukh tiy ar o v, Y.; Pan eman galo re, R.; Scott , B. B.; Zhao , Y.; Bru n o , J. G.; Zh u ang, L.; McGeeh a n , G. M.; He, W.; Claremo n , D. A . Bioorg. Med. Chem. Lett. 2010 , 20, 881 - 886. 381 Claremo n t, D. A.; Zh u a n g, L.; Ye, Y.; Singh , S. B.; Tice, C. M. Carb amat e an d urea inh ib itors of 11beta - h y d ro xy steroid deh y d ro gen ase 1. US 2 011/0 1120 62 A1. 382 Jim en ez, H. N.; Ma, G.; L i, G.; Gren o n , M.; Doller, D. Adam an ty l amid e deri vat iv es an d u se s of sam e . WO 2011/ 0877 58 A1. 130 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 42. Formation of ureas 62-65 via reaction of amine 46, 19, 48 and 52 with a carbamoyl chloride. 1.2 Pharmacological evaluation of the C -9 substituted 6, 7,8,9,10,11 -hexahydro -5,7:9,11 - dimethano -5H-benzocyclononen -7-yl der ivatives A preliminary in vitro screening was performed to evaluate if the synthesized compounds were able to inhibit human 11?-HSD1. The research group of Dr. Scott Webster at the University of Edinburgh (United Kingdom) carried out these assays. Screening for 11?-HSD1 potency was accomplished using a functional scintillation proximity assay or SPA employing microsomes isolated from human embryonic kidney cells (HEK 293) overexpressing human 11 ?-HSD1. We should clarify at this point that this procedure measures the blockade of 11?-HSD1 isoform exclusively. The SPA is a well-stablished high throughput screening assay for the evaluation of the ligand binding affinity to 11?-HSD1. 383,384 The principle of this technique consists of microscopic beads that contain scintillant compounds. There are two types of beads: i) plastic beads such as polyvinyl toluene (PVT) beads; ii) crystalline beads such as yttrium silicate beads. In the PVT beads, the more common ones, the scintillator is diphenylanthracine.385 This molecule emits light when radiolabelled cortisol (3 H -cortisol) binds to specific anti-cortisol antibodies that are exposed in the surface of the bead. The generation of light comes from the activation of the scintillator through energy transfer derived from the emitted radiation after binding. The amount of light is proportional to the amount of [ 3H ] -cortisol molecules that binds to the beads. The emitted light is quantified by a scintillation detector and converted into concentration of cortisol present in the media. Thus, the assay measures the production of [ 3H ] -cortisol from [ 3 H ] -cortisone by the action of 11?-HSD1 contained in the microsomes. Figure 60 illustrates the SPA principle. 383 Mund t, S.; Solly , K.; Thieri n g er, R.; Her man o w ski - Vo sat ka , A . Assay Drug Dev. Technol. 2005 , 3, 367 - 375. 384 Solly , K.; Mu n dt, S. S.; Zokia n , H. J.; Ding, G. J.; Herman o w ski - vo sat ka , A.; Stru lovici, B.; Zh en g, W. Assay Drug Dev. Technol. 2005, 3, 3 77 - 38 4. 385 Perkin E lmer web s ite. Rad iom etric as say s and detection , SP A bead Tech n o logy . htt p ://ww w.p erk in e lm er.co m /Re so u rce s/T e ch n icalR eso u rc es/Ap p li cat ion Su p p o rtKn o wle d geb ase/rad io metric /sp a_b ead .xh tml#to p ( acces s ed on 11 th Augu st 2 015 ). Results & Discussion 131 Fig. 6 0. Schematic representation of the SPA procedure. When an inhibitor is added in the media in high concentrations, the production of [ 3H ] -cortisol is reduced with a resultant decrease on the amount of emitted light from the SPA beads. In order to assess the binding affinity of each inhibitor, a first screening was carried out with a fixed concentration of every inhibitor (10 ?M) . Compounds with 70% or higher of inhibition were then further evaluated by determination of their IC50 values. The enzyme activity was measured with increasing concentrations of each compound, providing a dose-response plot obeying a Langmuir isotherm. Normalization to the signal from the activity with no inhibitor added and further mathematical calculations furnished the IC50 values, which are included in table 13. 132 CHAPTER 2: 11 ?-HSD1 inhibition Table 13. Percentage of inhibition of the tested compounds at 10 ?M and the IC 50 value of amide 57. ND: not determined. PF-877423 was used as a standard. Comp. R RHS % inhi bition at 10 ?M IC50 (?M) 72 OH 15 ND 56 OH 24 ND 74 Me 57 ND 57 Me 77 1.0 ? 0.3 58 OH 31 ND 59 Me 36 ND 60 Me 52 ND 61 Me 35 ND 62 OH 30 ND 63 Me 36 ND 64 F 62 ND 65 OMe 41 ND PF -877423 - - 93 0.004 From the above results, some conclusions can be tentatively drawn for this first series of compounds: Results & Discussion 133 ? Four compounds (74, 57, 60 and 64) were found to have an inhibitory effect larger than 50% at a single dose of 10 ?M. ? Comparing the C-9-methylated derivatives, the N-ethyl-D-proline subunit behaved better than the other tested right-hand sides. Within this family of compounds, the alkylation of the proline ring led to an increase in the inhibitory activity (compare 72 vs 56; 74 vs 57). ? The replacement of the nitrogen atom in 63 by a methine group in 60 caused an enhancement of the potency against 11?-HSD1. ? The introduction of a polar group at the C-9 position (hydroxyl in 72, 56, 58 and 62; or methoxide in 65) was deleterious for the inhibitory activity, whereas lipophilic groups (methyl in 74, 57, 59, 60, 61, and 63; or fluorine in 64) were generally better tolerated. Thus, the inhibitory activity improved as the lipophilicity increased. For a better understanding of this fact, compare 56 vs 57, and 62 vs 65. ? Considering the urea compounds 62-65, the fluorinated derivative 64 displayed the higher percentage of inhibition compared to the rest of substituents. ? The substitution of the adamantane ring in PF-877423 by the benzo- homoadamantane scaffold in 56 and 57 was detrimental to the inhibitory activity, with a 250-fold lower affinity. Taking into account the aforementioned, we identified the N-ethyl-D-proline subunit as the best right-hand side for the new scaffold (compound 57) when considering only the C-9-methylated derivatives. On the other hand, the introduction of a fluorine atom at the C-9 position performed better in the assay than the other substitution patterns. According to that, we hypothesized that the compound featuring both substituents would afford an inhibitor with improved potency (Fig. 61). This highlights the relevant role played by the polar moiety at the right-hand side as well as by the C-9 substituent. 134 CHAPTER 2: 11 ?-HSD1 inhibition Fig. 61. Compound 77 combines the best findings from the previous series of analogues. 1.3 Synthesis of the fluoro -benzohomoadamantane analogu e of PF -877423 For the preparation of the fluorinated analogue 77 the same synthetic route as for the compounds 56 and 57 was applied. The amide coupling reaction of amine 48 with (R)- pyrrolidine-2-carboxylic acid under EDC and HOBt conditions afforded the desired Boc - protected pyrrolidine 78 in 92% yield (Scheme 43 ). Next, the product was subjected to deprotection with aqueous phosphoric acid to provide proline 79 in quantitative yield. Final N-alkylation of the free amine with ethyl bromide under the already described conditions produced the final amide 77 in moderate yield. Similar to compound 57, the hydrochloride salt produced a hydroscopic solid. In order to have a compound suitable for characterization and pharmacological evaluation, PF-2?s analogue 77 was prepared as its L-(+) -tartrate salt. From the exploration of the right-hand side From the identification of the best C-9 substituent Results & Discussion 135 Scheme 43. Full synthetic route for the preparation of amide 77 from intermediate 48. 1.4 Pharmacological evaluation of the fluoro -benzohomoadamantane derivative of PF - 877423 The research group of Dr. Scott Webster (University of Edinburgh) tested the (R)- proline 77 as an 11?-HSD1 inhibitor in the SPA . At a concentration of 10 ?M, the compound inhibited 76% of the total enzyme, not far away from the percentage of inhibition of its methylated analogue 57 (Table 14). Pleasingly, in the IC50 determination, the threefold reduction on its value confirmed our first assumption, that the compound bearing a fluorine atom at the C-9 position and the N-ethyl-D-proline as the polar unit would result in an 11?-HSD1 inhibit or with enhanced potency, compared to the previous series of compounds. Table 14. Results of the pharmacological evaluation of amide 77 and its comparison with the methylated analogue 57 and the standard PF-877423. Comp. R % inhi bition at 10 ?M IC50 (?M) 57 Me 77 1.0 ? 0.3 77 F 76 0.35 ? 0.2 0 PF -877423 - 93 0.004 136 CHAPTER 2: 11 ?-HSD1 inhibition However, compound 77 is around ~100-fold less potent than the adamantyl analogue PF-877423. Therefore, it can be argued that the substitution of the adamantane moiety by the size-expanded benzopolycycl e might be detrimental for the binding to the enzyme. To check this hypothesis, molecular modelling studies were performed. 1.5 Computational studies of the first family of 11 ?-HSD1 inhibitors To gain insight on the mode of inhibition of the most potent compounds 57 and 77, the research group of Prof. Dr. F. Javier Luque of the University of Barcelona examined the binding of both prolines 57 and 77 on the 11?-HSD1 enzyme through docking and molecular dynamics simulations. Preliminary docking calculations performed with Glide supported the ability of the ring-expanded benzopolycycle to fill the binding cavity of human 11 ?-HSD1. 386 The docking scores of 57 (-8.1 kcal/mol) and 77 (-8.2 kcal/mol) compared well with PF -877423 (-7.8 kcal/mol). Moreover, t he best scored poses of both inhibitors indicated that, on one hand, the polar units mimicked the binding mode of PF-877423, retaining the hydrogen bond formed between the amide carbonyl oxygen and the hydroxyl group of Ser170. On the other hand, the hydrophobic cage filled the site occupied by the adamantane unit in PF-877423. Despite the tolerable binding of new compounds 57 and 77 observed in the docking studies, we aimed to investigate further the reason for that difference on the inhibitory activity. With this in mind, the structural integrity of the best poses of compounds 57 and 77 was explored by means of molecular dynamics (MD) simulations. Three independent 50 ns MD simulations were run for each ligand-receptor complex, yielding the following characteristics: ? All the simulations were stable, as noted in the root-mean square deviation profiles ranging from 1.5 to 2.4 ? for the protein backbone, and from 2.5 to 3.5 ? for the residues in the binding site. This fact reflected the enhanced flexibility of the loops that enclose the binding pocket. ? The ligand pose was preserved along the simulations. Particularly, the hydrogen bond formed between the inhibitor and Ser170 residue was maintained in all cases, with an average distance of 2.8 ? 0.2 ? (Fig. 62). ? The relative binding affinities were estimated from solvent interaction energy calculations, which revealed that compounds 57 and 77 should be stronger binders, by 0.5 and 1.1 kcal/mol, respectively, than PF -877423. 386 Frie sn er, R. A . ; Mu rp h y , R. B.; Repasky , M. P.; Fry e, L. L.; Gr een wo o d , J. R.; Halgr en , T. A . ; San sch agrin , P. C.; Main z, D. T. J. Med. Chem. 2006, 49, 6177 - 61 96. Results & Discussion 137 Fig. 62. Representative snapshots taken from the MD simulations of the complexes between human 11?-HSD1 and compounds PF -877423 (top; green sticks) and 77 (bottom; pink sticks). Residues Tyr183 and Ser170 are shown as gray sticks, the NADP cofactor as yellow-based sticks and the protein backbone as blue cartoon. For the sake of clarity only polar hydrogens are shown. The shape of the binding cavity is shown as a white contour. While the preceding results support the assumption that the insertion of a phenyl ring should lead to an enhanced binding affinity, the reduction of inhibitory potency may alternatively be ascribed to a lower effective concentration due to the increase in the hydrophobicity of the polycycle. In order to confirm this hypothesis, the log P was determined using quantum mechanical IEF/MST calculations for the most favourable 138 CHAPTER 2: 11 ?-HSD1 inhibition conformations of compounds 57, 77 and PF-877423.387 The study indicated that the introduction of a phenyl ring in the polycyclic structure increases the log P value by 1.5 units for amide 57 and 0.9 units for amide 77 relative to the adamantyl parent compound PF-877423. Taken together, the in silico results suggest that the ring-expansion strategy performed with the benzopolycyclic scaffold might lead to effective inhibitors of human 11?-HSD1 enzyme. Nevertheless, the data from log P calculations along with the pharmacological results have demonstrated that a proper balance of hydrophobicity, furnished chiefly by the adamantane-like scaffold, is necessary to attain an optimum pharmacological profile. 2. Application of the hexacyclic scaffold as an adamantane analogue in 11 ?-HSD1 inhibitors Before starting with this section, it is worth to mention that the work presented henceforth was developed simultaneously with the previous family of compounds. Consequently, an iterative process could not be achieved to reshape the design of the new family of compounds. 2.1 Synthesis of the 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadecane derivatives Following the analogy-based approach applied in the former section, and bearing in mind the flexibility of the binding pocket of the 11 ?-HSD1 protein arisen from the available X-ray data, we pursued the preparation of a new family of compounds featuring a size-expanded ring in l ieu of the adamantane group. Specifically, we decided to assemble the pentacyclic pyrrolidine scaffold V and amido- or urea-like units so as to assess the effect of this hydrophobic adamantane-like structure in the inhibition of 11?-HSD1 (Fig. 63 ). The polar groups attached to this polycycle consisted of the already tested moieties, again referred as right-hand sides (RHS). These functionalities should enable the formation of key hydrogen bonds at the binding site, whilst the polycyclic structure should fill one of the hydrophobic pockets of the catalytic site. 387 Curu tch et, C.; Oro zco , M.; Lu q u e, F. J. J. Comput. Chem. 2001 , 22, 1180 - 119 3. Results & Discussion 139 Fig. 63 . Design of the new 11?-HSD1 inhibitors with general structure V . The preparation of the hexacycles V has been described and applied multiple times by our research group.90,164,172 The synthetic pathway started with an elegant domino Diels- Alder reaction between 9,10-dihydrofulvalene and dimethyl acetylenedicarboxylate , originally published by two independent groups; L. A. Paquette and E. Hedaya .388,389 In the first step, cyclopentadiene (prior to distillation from commercially available dicyclopentadiene) was deprotonated with sodium hydride, whose anion 80 self-coupled through low-temperature iodine-promoted oxidation, to generate in situ 9,10- dihydrofulvalene 81 (Scheme 44). The latter underwent a double Diels-Alder reaction after the addition of one equivalent of dimethyl acetylenedicarboxylate , yielding a mixture of monoadducts 82 and 83, which are referred as pincer and domino products, respectively.390 Along with them, diadducts 84 and 85 were formed as side-products. 388 Paqu ett e, L. A.; Wy vra tt , M. J. J. Am. Chem. Soc. 1974, 42, 4671 - 4 673 . 389 McNeil, D.; Vogt , B. R.; Sud o l, J. J.; Theo d o ro p u los, S.; H e d ay a, E. J. Am. Chem. Soc. 1974 , 96, 4673 - 4674 . 390 Domingo , L. R.; Arn ? , M.; And r?s, J. Tetrahedron Lett. 1996 , 37, 7573 - 7576 . 140 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 44. Domino Diels-Alder reaction of 9,10-dihydrofulvalene 81 and dimethyl acetylenedicarboxylate . The earlier procedures applied a tedious purification that implied difficult distillations. Fortunately, a few years later a more straightforward purification method appeared, allowing the separation of the pair of monoadducts from the pair of diadducts.391 Taking advantage of the solubility properties of the products, 82 and 83 were isolated by precipitation of the side-products 84 and 85 with diethyl ether and subsequent filtration. This experimental -related improvement permitted to scale-up the reaction up to 40 g of final mixture. Separation of monoadducts 82 and 83 was achieved by selective hydrolysis of the less sterically hindered diester 83. Treatment with aqueous potassium hydroxide solution in methanol at room temperature for a short period of time afforded diacid 87, which was 391 Tay lor, R. T.; Pelt er, M. W.; P aq u ett e, L. A. Org. Synth. Coll. Vol. VIII 1993, 298 - 30 2 . Results & Discussion 141 isolated from the intact diester 82 in the aqueous work-up (Scheme 45). In this way, we prepared 20 g of the desired pentacyclic diester 82 in 7% overall yield. Scheme 45. Selective hydrolysis of 83 vs 82 due to steric hindrance. Employing harsher conditions, diester 82 could be hydrolysed providing its corresponding pentacyclic diacid 88 in 81% yield (Scheme 46). Next , diacid 88 was subjected to imide formation with neat urea at 220 ?C to afford imide 89 in 86% yield . At this temperature, the already melt urea undergoes thermal decomposition to supply cyanic acid and ammonia, which is the actual nucleophile that adds to each one of the carboxylic acids.392 Scheme 46. Basic hydrolysis of diester 82 and later imide formation. Afterwards, the imide was transformed to the corresponding pentacyclic pyrrolidine 90 based on the protocol formerly described by our research group.393 Unlike the more common reductive agents, such as lithium aluminium hydride, sodium bis(2- methoxyethoxy)aluminium hydride (Vitride ? or Red-Al? ) is soluble in most of the common organic solvents, which facilitates the handling of the substance, and reacts less strongly with water than LiAlH 4, although it is still moisture sensitive.394 Besides, Red-Al ? is highly efficient in the reduction of multiple functional groups, such as nitriles, isocyanates and sulfonamides, inter alia. Thus, imide 89 was reduced with Red-Al ? to produce secondary amine 90 in 92% yield (Scheme 47). 392 For furth er stu d y on th e pyroly sis of urea: Scha b er, P. M.; Colso n , J.; Higgin s, S.; Thie len , D.; Ansp ach , B.; Bra u er, J. Thermochim. Acta 2004, 424, 131 - 14 2. 393 Rey - Carrizo , M. Ph .D. Di s sert at ion , Univers ity of Barcelon a, 2014. 394 Gugelchu k, M.; Sil va, L. F. Jr. ; Vascon ce los, R. S. ; Qu in tilian o , S. A. P. e-EROS 2007, 1 - 8. 142 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 47. Imide reduction with Red-Al? . With key intermediate 90 in hand, we proceeded to the coupling of this pentacyclic pyrrolidine to the different polar units showed in figure 63. Applying the same procedures as in section 1, we started with the amide coupling with N-Boc-D-proline using EDC and HOBt as activating agen ts to provide hexacyclic amide 91 in 90% y ield (Scheme 48). Scheme 48. Amide bond formation with (R)-N-boc-pyrrolidine-2-carboxilyc acid. Regrettably, when the Boc-protected pyrrolidine 91 was subjected to acid conditions for its deprotection, only a complex was obtained, from which the desired amine 92 was could not be isolated. Illustrative computational studies revealed the feasibility that one part of the product might have been lost in the intramolecular attack of the amine to one of the double bonds of the polycyclic system, giving compound 94 as a plausible side- product (Scheme 49).395 Scheme 49. Possible explanation for the absence of desired amine 92. 395 Data fro m comp u ta tio n al stu d ies i s not shown . Results & Discussion 143 To avoid this issue, we decided to reduce the olefinic double bonds prior to the amide coupling, even though it meant to skip the no preparation of the final compound 93. For the provision of saturated pentacyclic pyrrolidine 95 for further uses, diene 90 was hydrogenated under atmospheric pressure and at room temperature for 4 hours, whereupon amine 95 was recovered after filtration of the catalyst in 86% yield (Scheme 50). Scheme 50. Catalytic hydrogenation of pentacyclo 90 at atmospheric pressure. Starting now from saturated pentacyclo pyrrolidine 95, final ethylated proline 98 was synthesized after amide bond formation with N-Boc-D-proline, followed by deprotection of the carbamate group with aqueous phosphoric acid and subsequent N-alkylation with ethyl bromide and catalytic amounts of potassium iodide, as shown in the scheme below. Of note were the requirement of column chromatography after the acid deprotection affording compound 97 in 36% yield, and the isolation of the final amide 98 as tartrate salt for its full characterization and pharmacological evaluation. Scheme 51. Synthetic pathway for the preparation of amide 98 from saturated pyrrolidine 95. The substituted phenyl analogue 99 and its saturated derivative 100 were prepared applying the same conditions as before, i.e. amide coupling with EDC and HOBt, and 144 CHAPTER 2: 11 ?-HSD1 inhibition catalytic hydrogenation with palladium on carbon, affording the corresponding products in 47% and 89 % yield, respectively (Scheme 52 ). Scheme 52. Amide coupling between diene 90 with 4-amino-3,5-dichlorobenzoic acid, then hydrogenation at 1 atm. Similarly, the pair of cyclohexyl amides 101 and 102 were obtained under the same procedures in moderate and excellent yields, respectively (Scheme 53). Scheme 53. Synthesis of saturated and unsaturated cyclohexyl amides 101 and 102. Next, we moved to the hexacyclic derivatives bearing a urea unit as the polar core of the molecule. In analogy with the previous benzopolycyclic scaffold, secondary amine 90 was reacted with 1-piperidinecarbonyl chloride in the presence of a base to provide Results & Discussion 145 unsaturated urea 103 in 71% yield (Scheme 54). Albeit multiple attempts to purify the product, from different gradient elution systems in column chromatography to several solvent mixtures in crystallization techniques, urea 103 did not achieve the adequate purity for pharmacological testing. Because of that, diene 103 was subjected to catalyti c hydrogenation, furnishing 104 in 72% yield and pure enough for its biological evaluation. Scheme 54. Urea formation with a carbamoyl chloride prior to reduction of the double bonds. For the synthesis of the last analogue of this family, which featured a thiazolone ring in the right-hand side of the molecule, pyrrolidine 90 was reacted with benzoyl isothiocyanate in chloroform to give carbonyl thiourea derivative 105 in quantitative yield (Sheme 55). Basic hydrolysis with potassium carbonate and water did not afford the desired thiourea 106, and the starting material was fully recovered. An alternative for the formation of thiourea 106 was adapted from the literature and consisted in the hydrazine-promoted debenzoylation under solvent-free conditions at room temperature.396 Worth the mention is the fact that if the reaction was carried out in chloroform at reflux, triazole -ring formation takes place instead of the debenzoylation. Nevertheless, neither thiourea 106 nor the corresponding triazole ring were formed, but again the starting material was recovered. We reasoned the lack of reactivity was coming from the steric hindrance furnished by the ring- expanded scaffold. 396 Kod omar i, M.; Suzu ki, M.; Ta n igawa, K.; Aoy ama, T. Tetrahedron Lett. 2005, 46, 5841 - 58 43. 146 CHAPTER 2: 11 ?-HSD1 inhibition Scheme 55. Conversion of pyrrolidine 90 to benzoylthiourea 105, and failed attempts of hydrolysis. In the end, this family of hexacyclic derivatives comprehended 6 new compounds that were tested as 11?-HSD1 inhibitors. 2.2 Phar macological evaluation and computational studies of the 3- azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]penta decane derivatives Compounds 98-102 and 104 were tested, using the SPA technique, by Dr. Scott Webster in the University of Edinburgh. As for the previous family, compounds with a percentage of inhibition of 70% or higher at 10 ?M were further evaluated w ith their IC50 determination (Table 15). Results & Discussion 147 Table 15. Inhibition of the 11?-HSD1 by the new hexacyclic compounds. ND: not determined. PF-877423 was used as a standard. Comp. No. of double bonds RHS % inhibition at 10 ?M IC50 (?M) 98 0 56 ND 99 2 98 2.77 100 0 98 0.414 101 2 95 1.076 102 0 103 0.289 104 0 101 0.320 PF -877423 - - 93 0.004 These data allowed us to deduce the following: ? Five compounds (98-102 and 104) inhibited almost totally the 11?-HSD1 enzyme in the SPA at 10 ?M concentration. ? Generally, aliphatic amides 101-102 and urea 104 were slightly more potent than aromatic amides 99-100. ? In contrast with the previous benzopolycyclic family, the PF-2?s analogue 98 showed the lowest activity for this series of compounds. ? As a common trend, saturated pentacyclic pyrrolidines were more potent that their parent unsaturated analogues (compare 100 vs 99 and 102 vs 101). ? The replacement of the methine group in 102 by a nitrogen atom in 104 did not greatly improved the activity, unlike the previous family. 148 CHAPTER 2: 11 ?-HSD1 inhibition ? The substitution of the adamantane ring in PF-877423 for the hexacyclic scaffold led to a significant reduction in the inhibitory activity. The fact that proline derivative 98 was the least potent compound of this series, but the most potent analogue for the benzo-homoadamantane series, reflected that the lipophilic scaffold oriented the polar units in a way that could alter their binding mode within the catalytic site. Thus, a specific right-hand side should not necessarily behave similarly for any given scaffold. Consequently, for each scaffold a screening of polar subunits should be performed in order to identify the best combination of fragments. In order to shed light on the reasoning of the binding mode of the new structures, Prof. Luque performed preliminary MD studies of the most active compounds 102 and 104, and simultaneously, the adamantane derivative 107, synthesized by our group, was evaluated (Fig. 64).364 Fig. 64. Three selected compounds for MD simulations. From the preliminary results, two main conclusions are depicted: ? The hexacyclic scaffold occupies a larger space than the adamantane, being practically the upper-limit the size the lipophilic scaffold can reach. In other words, the scaffold is too big to properly fit within the hydrophobic pocket (Fig. 65). ? There is no significant difference between 102 and 104 in their binding mode. Thus, the nitrogen does not seem to affect the potency, as reflected by their very similar IC50 values. Results & Discussion 149 Fig. 65. Representative snapshots taken from the MD simulations of the complexes between human 11?-HSD1 and compounds 102 (top left; purple sticks), 104 (top right, yellow sticks) and 107 (bottom; blue sticks). Residues Tyr183 and Ser170 are shown as green sticks, the NADP cofactor as orange-based sticks and the protein backbone as green cartoon. For the sake of clarity only polar hydrogens are shown. The shape of the binding cavity is shown as a white contour. As a final remark, the larger inhibitory potency of saturated compounds 100 and 102 (also 104, although it was not possible to test its parent compound 103) could also reflect the increase in hydrophobicity resulting from saturation of the double bonds in 99 and 101, respectively, as expected from the higher hydrophobicity of ethane relative to ethylene (experimental log P of 1.8 and 1.1, respectively).397 The decreased activity of the benzopolycyclic and hexacyclic compounds compared to the adamantyl analogue underlined the requirement of both a perfect match and the reduction of the overall lipophilicity of a bulky group in this pocket to achieve high potency. Hen ce, the incorporation of nuclei with enhanced polarity and reduced size appears to be a suitable strategy for developing novel inhibitors. 397 Han sch , C. ; Leo, A. ; Hoekman , D. Exp lorin g QSAR: Hyd ro p h o b ic, Electro n ic and Steric Con sta n ts, Ameri can Chem ical Soci ety , Washin gto n , DC, 1995 . 150 CHAPTER 2: 11 ?-HSD1 inhibition It is worth to mention that taking advantage of the conclusions drawn from the two sets of compounds reported in this manuscript, the Ph.D. student Rosana Leiva, in her thesis, has synthesized novel 11?-HSD1 inhibitors with low nanomolar IC50 values. Conclusi ons Conclusions 153 In the second chapter of the thesis, 19 new final compounds were prepared and thoroughly characterized by means of spectroscopic and analytical techniques. Concretely, the families prepared in this work and the conclusion derived from them are: ? 6, 7, 8, 9, 10, 11 - hexa hydro - 5,7:9, 11 - dimethan o - 5H - b e n zocyclon one n - 7 - yl derivatives: 1. From the synthetic point of view: since the intermediate scaffolds were already prepared in the previous chapter, only procedures described in the literature were followed for the final reactions. 2. From a pharmacological activity point of view: only two derivatives, 57 and 77 , displayed enough percentage of inhibition at 10 ?M (77 and 76% , respectively) for their IC50 determination (Fig. 66). The methylated compound 57 showed low inhibitory activity in the SPA (IC50 of 1.0 ? M), and this activity was slightly improved with the fluorinated analogue 77 (IC50 of 0.35 ?M). None of the others benzo- homoadamantane compounds were active as 11?-HSD1 inhibitors. The substitution of the adamantane group for the benzo-homoadamantane nucleus is unfavourable. Fig. 66 . Active compounds from the first family. 3. From a computational point of view: docking and MD simulations revealed that the insertion of a phenyl ring in the bulky group should not be detrimental for the activity, compared with the standard PF- 877423. However, the lack of inhibitory activity of the benzo- homoadamantane derivatives may be due to an improper balance of lipophilicity. ? 3- azahe xac ycl o[7.6.0.0 1,5 .0 5,12 .0 6,1 0 .0 11,15 ]pe n tade can e derivatives: 1. From the synthetic point of view: their preparation entailed the synthesis first of the scaffold, through a 5-step sequence, implying an elegant domino Diels-Alder reaction. The subsequent reaction conditions were taken from the previous family of compounds. 154 CHAPTER 2: 11 ?-HSD1 inhibition However, not all the reactions were efficient, an d a few of the targeted products were abandoned. 2. From a pharmacological activity point of view: except for compound 98 , all the tested compounds were active as inhibitors of the 11?-HSD1 , showing moderate potency. Of note is the increase on the potency with the saturation of the double bonds, resulting in the most potent compounds of the series 100 , 102 and 104 (IC50 of 0.414, 0.289 and 0.320 ?M , respectively) (Fig. 67). The different RHSs do not alter significantly the activity between them. The substitution of the adamantane ring for the hexacylic nucleus is unfavourable. Fig. 67 . Active compounds from the second family. 3. From a computational point of view: preliminary MD simulations of 102 and 104 revealed that the hexacyclic scaffold fills the hydrophobic cavity to a larger extent than the adamantane standard 107 , diminishing the activity. No significant difference was observed between the binding mode of 102 and 104 . Finally, the increase on the activity with saturated compounds 100 , 102 and 104 can be due to changes on the lipophilicity from their parent compounds. CHAPTER 3 : sEH inhib ition Introduct ion Introduction 159 1. sEH inhibition by adamantane -based derivatives The last chapter of the present thesis will deal with the discovery of sEH as a pleiotropic target for drug development, as well as the manifold therapeutic benefits that its inhibition may lead. A summary of the sEH inhibitors that are currently in development will be included, most of which integrate an adamantane ring as the bulky moiety. Finally, the issues that have arisen over the drug discovery process will be discussed in order to comprehend the current needs for the development of future drug candidates. 1.1 Epoxyeicosatrienoic acids and their biological role 1. 1. 1 Oxi dati ve pathw ays of arachi doni c ac i d me tabol i sm One of the principal metabolic pathways in the organism is the arachidonate cascade, which revolves around the arachidonic acid (ARA), an ?-6, 20-carbon polyunsaturated fatty acid that is produced from membrane phospholipids of activated cells upon various stimuli.398 ARA is transformed to lipid chemical mediators with pro-inflammatory properties, such as prostaglandins (PGs) and leukotrienes (LKTs), by the action of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes respectively, which are well-known drug targets against inflammation (Fig. 68).399 The most recently discovered cytochrome P450 (CYP450) branch of the cascade, which has not been exploited as a pharmaceutical target yet, also generates physiologically important eicosanoids.400 Among them, epoxyeicosatrienoid acids (EETs) are formed after epoxidation of ARA by mainly CYP2C and CYP2J, and possess an opposite effect than their counterpart products from COX and LOX metabolism.401 Detailed discussions on each of these three pathways are beyond the scope of this thesis. 398 Harizi, H.; Corcuff, J. B.; Gualde, N. Trends Mol. Med. 2008, 14, 461-469. 399 Funk, C. D. Science 2001, 294, 1871-1875. 400 Zeldin, D. C. J. Biol. Chem. 2001, 276, 36059-36062. 401 Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Spiecker, M.; Ley, K.; Zeldin, D. C.; Liao, J. K. Science 1999, 285, 1276-1279. 160 CHAPTER 3: sEH inhibition Fig. 68 . Three metabolic pathways of ARA: the COX, LOX and CYP450 enzymatic routes, resulting in an array of chemical mediators involved in inflammatory processes. Inhibitors of COX (COX-1 and COX-2) (NSAIDs) are used for the treatment of pain and inflammation. Leukotriene antagonists are indicated in asthmatic and allergic states. NSAIDs: nonsteroidal anti-inflammatory drugs. 1. 1. 2 Bi ol ogi c al rel evanc e of E E T s and im po rtance of the sEH manageme nt Generally, EETs are lipid mediators that move biological processes towards a homeostatic or status quo condition. EETs are autocrine and paracrine endothelium-derived factors that display attractive effects particularly in vascular and renal systems. To begin with, EETs are potent vasodilatory and anti-inflammatory agents.402 The vasodilatory properties of EETs are associated with an increased open-state probability of calcium- activated potassium channels, which lead to hyperpolarization of the vascular smooth muscle.403 Of note, EETs seem to encourage the resolution of inflammation rather than prevent it vi a inhibition of the nuclear factor ?B (NF-?B), activation of subfamilies of nuclear peroxisome proliferator-activated receptors (PPARs), and decreasing the expression 402 Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 37-58. 403 Fleming, I. Circ. Res. 2001, 89, 753-762. Inf lammator y eicosano ids Anti - inf lammator y e icosano ids Introduction 161 of inducible COX-2. Besides, EETs exhibit other beneficial effects on biological systems, such as analgesic, anti-hypertensive, anti-platelet aggregation, fibrinolytic, pro-angiogenic and anti-apoptotic effects.404,405 In addition, some metabolic events are mediated by EETs, such as control of insulin release and modulation of insulin sensitivity.406,407 Taken together, EETs are considered important chemical mediators that interact with a plethora of proteins to generate a set of wholesome effects. Experimental studies have revealed that EETs act through an intracellular mechanism by binding to fatty-acid-binding proteins and PPARs, i nter al i a .408 However, the cascade processes derived from the action of EETs are still unknown for some of the abovementioned effects. For the moment, an EET receptor has yet to be identified. Figure 69 includes a schematic representation of the potential mechanism for the anti-inflammatory, cardiovascular and analgesic effects of EETs.409 Fig. 69 . Plausible mode of action of EETs in cardiovascular system, inflammation and pain. GPCR: G protein-coupled receptor; cAMP: cyclic adenosine monophosphate. The metabolic fate of the EETs is under control of the sEH, an enzyme that belongs to the ?/? hydrolase family and facilitates the addition of water to an epoxide, leading to the formation of a vicinal diol.410 Thus, EETs are subjected to epoxide-ring opening to 404 El-Sherbeni, A. A.; El-Kadi, A. O. S. Arch. Toxicol. 2014, 88, 2013-2032. 405 Duflot, T.; Roche, C.; Lamoureux, F.; Guerrot, D.; Bellien, J. Expert Opin. Drug Discov. 2014, 9, 1-15. 406 Falck, J. R.; Manna, S.; Moltz, J.; Chacos, N.; Capdevila, J. Biochem. Biophys. Res. Commun. 1983, 114, 743-749. 407 Skepner, J. E.; Shelly, L. D.; Ji, C.; Reidich, B.; Luo, Y. Prostaglandins Other Lipid Mediat. 2011, 94, 3-8. 408 Imig, J. D.; Hammock, B. D. Nat. Rev. Drug Discov. 2009, 8, 794-805. 409 Pillarisetti, S. Inflamm. Allergy - Drug Targets 2012, 11, 143-158. 410 Harris, T. R.; Hammock, B. D. Gene 2013, 526, 61-74. 162 CHAPTER 3: sEH inhibition afford dihydroeicosatrienoic acids (DHETs), whereby the biological effects of EETs are diminished, eliminated or altered (Fig. 70).411 Fig. 70 . Transformation of active EETs to inactive DHETs through the sEH enzyme. Consequently, since the discovery of the biological role of EETs and their metabolism, sEH has emerged as a promising therapeutic strategy for the treatment of multiple conditions. Inhibition of sEH stabilizes the levels of EETs for the maintenance of the cellular homeostasis. Because other metabolic routes different from the sEH have been described that ensure the control oI ((Ts? concentration in plasma and tissues even in the absence of sEH activity, EETs levels can only increase moderately with the inhibition of sEH.412 Hence, limited target-related side effects are predicted with the administration of potent sEH inhibitors. 1.2 Targeting sEH: an overview of its pharmacology Over the past decade, the appreciation of the importance of EETs and their regulation by sEH has greatly accelerated due to the huge therapeutical potential derived from the inhibition of sEH. Since the first studies performed 30 years ago, the value of the sEH inhibition in various animal models of diseases has been demonstrated. The contribution of these studies will be covered in order to understand the beneficial outcomes from sEH inhibition. 1. 2. 1 Regul ati on o f i nfl am mati on by sEH inhi bi tors The anti - i nf l amm atory role of EETs has been demonstrated using multiple strategies including overexpression of CYP450, deletion of sEH in animal models or use of sEH 411 Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 311-333. 412 Imig, J. D. Physiol. Rev. 2012, 92, 101-130. E poxy e icosatr ie no ic a cids (EE Ts) Dihy dr oxy e ic osatr ie noi c acids ( DHE Ts) Introduction 163 inhibitors and directly monitoring EET effects.413,414,415 Reduced activation of the NF-?B is a key event in the anti-inflammatory activity of EETs, specially of 11,12-EET and 14,15- EET, as previously indicated. This reduction seems to follow three complementary cellular mechanisms (Fig. 71): i) decrease of the activation of NF-?B induced by tumor necrosis factor-? (TNF-?); ii) activation of PPAR-? activity; iii) reduction of the prostaglandin E2 (PGE2).402,416 Fig. 71 . Action of epoxy-fatty acids (EpFAs) in the inflammatory process. PLA2: phospholipase A2. FAs: fatty acids. DiHFAs: dihydroxy-fatty acids. IKK: inhibitor of ?B kinase. iNOS: inducible nitric oxide synthase. VCAM-1: vascular cell adhesion molecule 1. IL: interleukin. NO: nitric oxide. Furthermore, the agents that elevate cAMP are known to be anti-inflammatory, also by inhibiting NF-?B transcription.417 Thus, it is likely that the elevation of cAMP, pri or activation of GPCR, is another major mechanism behind the anti-inflammatory effects of EETs. All in all, these data indicate that sEH is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. 413 Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Spiecker, M.; Ley, K.; Zeldin, D. C.; Liao, J. K. Science 1999, 285, 1276-1279. 414 Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I.; Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci. USA 2005, 102, 9772-9777. 415 Deng, Y.; Edin, M. L.; Theken, K. N.; Schuck, R. N.; Flake, G. P.; Kannon, M. A.; DeGraff, L. M.; Lih, F. B.; Foley, J.; Bradbury, J. A.; Graves, J. P.; Tomer, K. B.; Falck, J. R.; Zeldin, D. C.; Lee, C. R. FASEB J. 2011, 25, 703-713. 416 Liu, Y.; Zhang, Y.; Schmelzer, K.; Lee, T. S.; Fang, X.; Zhu, Y.; Spector, A. A.; Gill, S.; Morisseau, C.; Hammock, B. D.; Shyy, J. Y. J. Proc. Natl. Acad. Sci. USA 2005, 102, 16747-16752. 417 Ollivier, V.; Parry, G. C. N.; Cobb, R. R.; de Prost, D.; Mackman, N. J. Biol. Chem. 1996, 271, 20828-20835. 164 CHAPTER 3: sEH inhibition 1. 2 . 2 E ffec ts of sEH inhi bi tors on p ai n Neuropathic pain is a component of many disease states, such as T2DM, and there is no current treatment to suppress it. Despite the intensive efforts and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches has been attained. A recently identified, non-channel, non-neurotransmitter therapeutic target for pain is the enzyme sEH.418 Given the effectiveness in reducing inflammation in some rodent models, it was not surprising that the inhibition of sEH also reduced i nfl am m atory and neurop athi c pai n . This effect has been subsequently detected in multiple animal models of pain in which sEH inhibitors and EETs reduced pain perception.419,420 The mechanism by which the sEH intervenes in the nociception process has been confirmed to imply opioid and GABAergic pathways, as well as PPARs.421 As it will be discussed later on, the sEH has also been associated with the endoplasmic reticulum (ER) stress, a causative agent of neuropathic pain.422,423 Interestingly, the sEH inhibitors are more effective than the opioid morphine in relieving some pain conditions, with no change in behaviour or sedation, unlike many opiates. Some studies have demonstrated that the co-administration of sEH inhibitors and NSAIDs produced a valuable analgesic and anti-inflammatory effect with a reduction of cardiovascular toxicity derived from the NSAID treatment.424 The efficacy of sEH inhibitors against diabetic neuropathic pain is noteworthy because of its potential to address the unmet therapeutic need of relieving this chronic pain condition. 1. 2 . 3 sEH and c ardio vasc ular dise ase Another therapeutic area of interest for sEH inhibitors is cardiovascular diseases. EETs are putative endothelium-derived hyperpolarizing factors (EDHP) that increase open-state frequency of calcium ion channels leading to vasodilatation of vascular smooth muscle by activation of potassium channels, through a cAMP/protein kinase A-dependent mechanism that involves the intracellular translocation of transient receptor potential channels.425 The identification of their vasodilatory properties suggested a role of the sEH in blo od pressure regulati on . EETs also promote natriuresis, regulating indirectly the blood 418 Hammock, B. D.; Wagner, K.; Inceoglu, B. Pain Manag. 2011, 1, 383-386. 419 Wagner, K.; Inceoglu, B.; Dong, H.; Yang, J.; Hwang, S. H.; Jones, P.; Morisseau, C.; Hammock, B. D. Eur. J. Pharmacol. 2013, 700, 93-101. 420 Sing, K.; Lee, S.; Liu, J.; Wagner, K. M.; Pakhomova, S.; Dong, H.; Morisseau, C.; Fu, S. H.; Yang, J.; Wang, P.; Ulu, A.; Mate, C. A.; Nguyen, L. V; Hwang, S. H.; Edin, M. L.; Mara, A. A.; Wul, H.; Newcomer, M. E.; Zeldin, D. C.; Hammock, B. D. J. Med. Chem. 2014, 57, 7016-7030. 421 Pillarisetti, S.; Khanna, I. Drug Discov. Today 2015, In press. DOI: 10.1016/j.drudis.2015.07.017. 422 Bettaieb, A.; Nagata, N.; Aboubechara, D.; Chahed, S.; Morisseau, C.; Hammock, B. D.; Haj, F. G. J. Biol. Chem. 2013, 288, 14189-14199. 423 Inceoglu, B.; Bettaieb, A.; Trindade da Silva, C. A.; Lee, K. S. S.; Haj, F. G.; Hammock, B. D. Proc. Natl. Acad. Sci. USA 2015, 112, 9082-9087. 424 Schmelzer, K. R.; Inceoglu, B.; Kubala, L.; Kim, I.; Jinks, S. L.; Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci. USA 2006, 103, 13646-13651. 425 Fleming, I.; Rueben, A.; Popp, R.; Fisslthaler, B.; Schrodt, S.; Sander, A.; Haendeler, J.; Falck, J. R.; Morisseau, C.; Hammock, B. D.; Busse, R. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 2612-2618. Introduction 165 pressure.426 This hypothesis has been confirmed in several studies using inhibitors of sEH, which led to an increase in the ((Ts? levels resulting in a reduction oI blood pressure in angiotensin-driven hypertensive rodent models.427 Ulu et al. showed that inhibition of sEH in a mammalian model of atheroscl erosi s by an orally administered agent reduced the formation of aortic plaques.428 A recent study performed with mice treated with a sEH inhibitor revealed a reduction in the size of atherosclerotic plaques, along with an improvement in the cholesterol levels.429 Numerous studies with sEH inhibitors have also shown cardiovascular-protective effects in cerebral and cardiac ischaemia, arrhythmia, hypertrophy and blood clotting, 430,431,432,433 proving once more the great potential of the sEH as a c ar dio vasc ular regulator . The data regarding the cardiovascular effects of sEH inhibition has been compiled in several reviews.408,412,434,435 These finding encourage the use of sEH inhibitors in patients who have cardiovascular problems. 1. 2 . 4 Rol e of sEH in the devel op m ent o f diabetes and metaboli c syndrom e . I nvol vement o f E R stress Metabolic processes are coordinately regulated by lipids, where EETs display a significant role in the pathophysiology of the endocrine system in relation to glucose homeostasis.406 Whilst EETs and sEH inhibitors do not alter the levels of glucose and insulin in healthy patients, they have proven efficient in the regulation of glycemic states in some models of diabetes and MetS. Luo et al . demonstrated that sEH inhibitors or sEH knockout [EHPX2 ( - / - ) ] control glucose homeostasis in streptozotocin-treated mice. Specifically, they observed reduction of the glycemic levels, increase of insulin secretion and islet apoptosis reduction.436 These alterations of the pancreatic islets and the improvement of the glucose homeostasis have been also confirmed in an animal model of insulin resistance.437 426 Imig, J. D. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004, 287, R3-5 427 Loch, D.; Hoey, A.; Morisseau, C.; Hammock, B. O.; Brown, L. Cell Biochem. Biophys. 2007, 47, 87-98. 428 Ulu, A.; Davis, B. B.; Tsai, H.; Kim, I.; Morisseau, C.; Inceoglu, B.; Fiehn, O.; Hammock, B. D.; Weiss, R. H. J. Cardiovasc. Pharmacol. 2008, 52, 314-323. 429 Shen, L.; Peng, H.; Peng, R.; Fan, Q.; Zhao, S.; Xu, D.; Morisseau, C.; Chiamvimonvat, N.; Hammock, B. D. Atherosclerosis 2015, 239, 557-565. 430 Shrestha, A.; Krishnamurthy, P. T.; Thomas, P.; Hammock, B. D.; Hwang, S. H. J. Pharm. Pharmacol. 2014, 66, 1251-1258. 431 Xu, D.; Davis, B. B.; Wang, Z.; Zhao, S.; Wasti, B.; Liu, Z.; Li, N.; Morisseau, C.; Chiamvimonvat, N.; Hammock, B. D. Int. J. Cardiol. 2013, 167, 1298-1304. 432 Zhang, W.; Otsuka, T.; Sugo, N.; Ardeshiri, A.; Alhadid, Y. K.; Iliff, J. J.; DeBarber, A. E.; Koop, D. R.; Alkayed, N. J. Stroke 2008, 39, 2073-2078. 433 Xu, D.; Li, N.; He, Y.; Timofeyev, V.; Lu, L.; Tsai, H.; Kim, I.; Tuteja, D.; Mateo, R. K. P.; Singapuri, A.; Davis, B. B.; Low, R.; Hammock, B. D.; Chiamvimonvat, N. Proc. Natl. Acad. Sci. USA 2006, 103, 18733-18738. 434 Duflot, T.; Roche, C.; Lamoureux, F.; Guerrot, D.; Bellien, J. Expert Opin. Drug Discov. 2014, 9, 1-15. 435 Deng, Y.; Thenken, K. N.; Lee, C. R. J. Mol. Cell. Cardiol. 2010, 48, 331-353. 436 Luo, P.; Chang, H. H.; Zhou, Y.; Zhang, S.; Hwang, S. H.; Morisseau, C.; Wang, C.-Y.; Inscho, E. W.; Hammock, B. D.; Wang, M. H. J. Pharmacol. Exp. Ther. 2010, 334, 430-438. 437 Luria, A.; Bettaieb, A.; Xi, Y.; Shieh, G.-J.; Liu, H.-C.; Inoue, H.; Tsai, H.-J.; Imig, J. D.; Haj, F. G.; Hammock, B. D. Proc. Natl. Acad. Sci. USA 2011, 108, 9038-9043. 166 CHAPTER 3: sEH inhibition EETs also regulate the levels of lipids and adipogenesis by activation of PPARs.438 In addition, the diet-induced MetS in rats was ameliorated with the administration of sEH inhibitors, with a notable reduction of the blood pressure and the body weight gain, an increase of the insulin sensitivity and an improvement of the inflammation markers.439 Moreover, sEH has been related to the ER stress in liver and adipose tissue, whose dysfunction is a contributor to metabolic diseases.422 Perturbation of the ER function and chronic ER stress are also associated with many other pathologies ranging from neurodegenerative diseases to cancer and inflammation.440 ER stress will be covered in more depth later on in the present chapter. The (patho)physiological importance of the sEH in the glucose and lipid homeostasis indicates that treatment with sEH inhibitors may address major comorbidities of diabetes, obesity and MetS. 1. 2 . 5 sEH inhi bi ti on for other po tenti al c l ini c al app li c ations The inhibition of the sEH has been studied for other conditions with positive results. In brief, inhibitors of the sEH display a protective effect on renal damage induced by hypertension or inflammation.441,442 Furthermore, the inhibition of sEH has resulted effective in the treatment of chronic obstructive pulmonary disease, especially when cigarette smoke is a risk factor, and pulmonary fibrosis.443,444,445 On the other hand, because of the structural similarity of some inhibitors of sEH with sorafenib, the only FDA- approved small molecule used for the treatment of advanced hepatocellular carcinoma, their cytotoxicities were also similar to sorafenib in various human cancer cell lines.446,447 438 De Taeye, B. M.; Morisseau, C.; Coyle, J.; Covington, J. W.; Luria, A.; Yang, J.; Murphy, S. B.; Friedman, D. B.; Hammock, B. B.; Vaughan, D. E. Obesity 2010, 18, 489-498. 439 Iyer, A.; Kauter, K.; Alam, M. A.; Hwang, S. H.; Morisseau, C.; Hammock, B. D.; Brown, L. Exp. Diabetes Res. 2012, 2012, 14-16. 440 Wang, S.; Kaufman, R. J. J. Cell Biol. 2012, 197, 857-867. 441 Zhao, X.; Yamamoto, T.; Newman, J. W.; Kim, I. H.; Watanabe, T.; Hammock, B. D.; Stewart, J.; Pollock, J. S.; Pollock, D. M.; Imig, J. D. J. Am. Soc. Nephrol. 2004, 15, 1244-1253. 442 Olearczyk, J. J.; Quigley, J. E.; Mitchell, B. C.; Yamamoto, T.; Kim, I.-H.; Newman, J. W.; Luria, A.; Hammock, B. D.; Imig, J. D. Clin. Sci. 2009, 116, 61-70. 443 Wang, L.; Yang, J.; Guo, L.; Uyeminami, D.; Dong, H.; Hammock, B. D.; Pinkerton, K. E. Am. J. Respir. Cell Mol. Biol. 2012, 46, 614-622. 444 Podolin, P. L.; Bolognese, B. J.; Foley, J. F.; Long, E.; Peck, B.; Umbrecht, S.; Zhang, X.; Zhu, P.; Schwartz, B.; Xie, W.; Quinn, C.; Qi, H.; Sweitzer, S.; Chen, S.; Galop, M.; Ding, Y.; Belyanskaya, S. L.; Israel, D. I.; Morgan, B. A; Behm, D. J.; Marino, J. P.; Kurali, E.; Barnette, M. S.; Mayer, R. J.; Booth-Genthe, C. L.; Callahan, J. F. Prostaglandins Other Lipid Mediators 2013, 104- 105, 25-31. 445 Zhou, Y.; Sun, G. Y.; Liu, T.; Duan, J. X.; Zhou, H. F.; Lee, K. S.; Hammock, B. D.; Fang, X.; Jiang, J. X.; Guan, C. X. Cell Tissue Res. 2015, Ahead of print. DOI: 10.1007/s00441-015-2262-0. 446 Wecksler, A. T.; Hwang, S. H.; Wettersten, H. I.; Gilda, J. E.; Patton, A.; Leon, L. J.; Carraway, K. L.; Gomes, A. V; Baar, K.; Weiss, R. H.; Hammock, B. D. Anticancer. Drugs 2014, 25, 433-446. 447 Wecksler, A. T.; Hwang, S. H.; Liu, J. Y.; Wettersten, H. I.; Morisseau, C.; Wu, J.; Weiss, R. H.; Hammock, B. D. Cancer Chemother. Pharmacol. 2014, 75, 161-171. Introduction 167 Besides, schizophrenia, seizures and hepatic fibrosis has been recently related to sEH.448,449,450 The potential clinical indications of sEH inhibitors are shown in Figure 72. Fig. 72 . Potential clinical indications for the use of sEH inhibitors. 1.3 sEH: crystal structure, catalytic mechanism, tissue expression and regulation There are several mammalian epoxide hydrolases, including microsomal EH (mEH) and sEH, which are named according to their subcellular location. They can be also differentiated by their substrate specificity. Whereas mEH activity is focused on the metabolism of xenobiotics, where it degrades preferentially epoxides on cyclic systems, such as arenes, sEH is prone to hydrolyze endogenous epoxide-fatty acids, such as the oxidation products of linoleic acid, linolenic acid or the EETs themselves.451 14,15-EET is the preferred substrate of sEH with the highest V m a x and lowest K m , followed by 11,12-EET, 8,9- EET and 5,6-EET.405 Other epoxide hydrolases have been identified and studied, such as cholesterol and hepoxilin epoxide hydrolases, but they are beyond the purpose of the present thesis.452 The E PHX - 2 gene, located on the short arm of chromosome 8, with nearly 45 kb and 19 axons, encodes the sEH.410 Human sEH is a homodimer formed by two ~ 62 kDa monomeric subunits arranged in an anti-parallel fashion and separated by a short proline- rich linker (Fig. 73).453 The epoxide hydrolase activity resides in the C-terminal domain, whereas the N-terminal domain exhibits a phosphatase activity that apparently acts on lysophosphatidic acids.454 448 Ma, M.; Ren, Q.; Fujita, Y.; Ishima, T.; Zhang, J. C.; Hashimoto, K. Pharmacol. Biochem. Behav. 2013, 110, 98-103. 449 Inceoglu, B.; Zolkowska, D.; Yoo, H. J.; Wagner, K. M.; Yang, J.; Hackett, E.; Hwang, S. H.; Lee, K. S. S.; Rogawski, M. A.; Morisseau, C.; Hammock, B. D. PLoS One 2013, 8, 8-17. 450 Harris, T. R.; Bettaieb, A.; Kodani, S.; Dong, H.; Myers, R.; Chiamvimonvat, N.; Haj, F. G.; Hammock, B. D. Toxicol. Appl. Pharmacol. 2015, 286, 102-111. 451 Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog. Lipid Res. 2005, 44, 1-51. 452 Fretland, A. J.; Omiecinski, C. J. Chem. Biol. Interact. 2000, 129, 41-59. 453 Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Biochemistry 2004, 43, 4716-4723. 454 Morisseau, C.; Schebb, N. H.; Dong, H.; Ulu, A.; Aronov, P. A.; Hammock, B. D. Biochem. Biophys. Res. Commun. 2012, 419, 796-800. s EH inhi bi ti on In flam m ation Pain Can c e r CV disease s Pulm o n ar y and ren al disease s Diab e te s an d MetS 168 CHAPTER 3: sEH inhibition A prerequisite for the development of potent and selective inhibitors is the understanding of the sEH mechanism of action. The catalytic mechanism of the sEH has been elucidated in the last few years with the aim of defining the key residues involved in the hydrolysis. As a result, X-ray crystallographic data has revealed that the hydrolase catalytic pocket consists of two tyrosine residues (Tyr382 and Tyr465) which act as hydrogen bond donors to promote epoxide-ring opening by the backside attack vi a an SN2- type reaction of an aspartic acid residue (Asp334) to form a hydroxyl alkyl-enzyme intermediate (Fig. 74).411 In this step, the nucleophilic Asp334 is oriented and activated by a histidine residue (His523). Then, different proton shifts, which have yet to be proven, occur and a water molecule attacks the intermediate releasing the diol product and the original enzyme. Fig. 7 4. Schematic representation of the polarization of the epoxide by the two tyrosine residues and subsequent attack of the aspartic acid residue. Catalytic triad (blue), long branch (purple) and short branch (green). 455 Gorman, J.; Shapiro, L. Acta Crystallogr. Sect. D 2004, 60, 1600-1605. Fig. 73. X-ray crystal structure of the sEH homodimer. PDB code: 1S80.455 Introduction 169 Extensive enzymological and structural studies revealed an ?L -?shaped hydrophobic tunnel of the binding site, whose branches measure 10 and 15 ?, respectively, and with the catalytic triad placed between them (Fig. 75). Albeit being largely hydrophobic, each branch of the pocket features residues that are involved in specific interactions, such as hydrogen bonds, Van der Waals and ?-stacking interactions, and are open to the solvent. Fig. 75 . Overview of the catalytic pocket, with representative fragments bound to each site (catalytic triad: cyan; long branch: purple; short branch: orange).456 Although at different levels, sEH is expressed in all organs and tissues and differs from the expression of mEH. The specific activity of sEH is highest in the liver, followed by kidney, heart, lung and brain.457,458 In a lesser extent, sEH activity has been detected in spleen, adrenals, intestine, urinary bladder, vascular endothelium and smooth muscle, placenta, skin, mammary gland, testicles and leucocytes.451 Concerning the regulation of the sEH, both PPAR-? and PPAR-? agonists, e.g. fibrates and glitazones, have proven to alter sEH expression, underlying the close relationship between PPARs and sEH.416,459,460 Angiotensin II and homocysteine, which appears to depend on activating transcription factor 6 (ATF6), are inducers of sEH.461,462 1.4 Discovery of sEH inhibitors In an effort to explore sEH inhibition as an avenue for the development of therapeutic agents, many academic and industrial groups have entered the arena of the discovery of potent sEH inhibitors. The research group of Prof. Dr. Hammock has been the major contributor to sEH inhibitors.463,464 A detailed understanding of the catalytic mechanism of the enzyme and later multiple crystal structures helped in the identification of a diversity of functional groups that can be considered for the ligand design. The structural types of 456 Amano, Y.; Yamaguchi, T.; Tanabe, E. Bioorg. Med. Chem. 2014, 22, 2427-2434. 457 Gill, S. S.; Hammock, B. D. Biochem. Pharmacol. 1980, 29, 389-395. 458 Enayetallah, A. E.; French, R. A.; Thibodeau, M. S.; Grant, D. F. J. Histochem. Cytochem. 2004, 52, 447- 454. 459 Fang, X.; Hu, S.; Watanabe, T.; Weintraub, N. L.; Snyder, G. D.; Yao, J.; Liu, Y.; Shyy, J. J. Y.; Hammock, B. D.; Spector, A. A. J. Pharmacol. Exp. Ther. 2005, 314, 260-270. 460 Wong, S. L.; Huang, Y. Clin. Exp. Pharmacol. Physiol. 2011, 38, 356-357. 461 Ai, D.; Fu, Y.; Guo, D.; Tanaka, H.; Wang, N.; Tang, C.; Hammock, B. D.; Shyy, J. J. Y.; Zhu, Y. Proc. Natl. Acad. Sci. USA 2007, 104, 9018-9023. 462 Zhang, D.; Xie, X.; Chen, Y.; Hammock, B. D.; Kong, W.; Zhu, Y. Circ. Res. 2012, 110, 808-817. 463 Shen, H. C. Expert Opin. Ther. Pat. 2010, 20, 941-956. 464 Shen, H. C.; Hammock, B. D. J. Med. Chem. 2012, 55, 1789-1808. 170 CHAPTER 3: sEH inhibition sEH inhibitors are extremely broad, which is consistent with the wide binding pocket of the enzyme. From a historical point of view, the first generation of sEH inhibitors were potent competitive epoxide molecules and included chalcone oxides and trans -3-phenylglycidols (Fig. 76).465,466 These compounds were generally transient inhibitors with low turnover rates. Also, these substrates were rapidly inactivated by glutathione transferases, rendering them ineffective in i n vi tro and i n vi vo assays. Fig. 76. Early inhibitors of sEH bearing an epoxide moiety. 5 and 5? are various substituents Just to mention, heavy metals such as Zn, Hg, Cu and Cd have been reported also to inhibit sEH activity vi a interactions with the phosphatase domain.467 Since the early 2000s, several pharmacophores have emerged and major advances in the potency and pharmacokinetic properties of the inhibitors have been made. It was demonstrated that functional groups such as ureas, carbamates, amides, thioesters, carbonates, esters, thioureas, guanidines, i nter ali a , fit well into the active site and display a similar binding to that of endogenous epoxides.468,469,470,471 Therefore, in recent years there has been a significant and rapid development of compounds featuring mainly a urea or amide group as reversible sEH inhibitors. The urea group, which is the most exploited functionality for the design of sEH inhibitors, mimics the endogenous epoxide substrate by binding to the key residues of the catalytic triad. Thus, the carbonyl oxygen is involved in a hydrogen bonding interaction with both tyrosines, Tyr382 and Tyr465, whereas the NH groups act as hydrogen bond donors to the aspartic acid residue Asp334 (Fig. 77).472 These specific interactions are responsible for their consideration as tight-binding sEH inhibitors reIerred as ?transit on state competitive inhibitors?473 465 Morisseau, C.; Du, G.; Newman, J. W.; Hammock, B. D. Arch. Biochem. Biophys. 1998, 356, 214-228. 466 Dietze, E. C.; Kuwano, E.; Casas, J.; Hammock, B. D. Biochem. Pharmacol. 1991, 42, 1163-1175. 467 Draper, A. J.; Hammock, B. D. Toxicol. Sci. 1999, 52, 23-32. 468 Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.; Greene, J. F.; Sanborn, J. R.; Hammock, B. D. Proc. Natl. Acad. Sci. USA 1999, 96 , 8849-8854. 469 Nakagawa, Y.; Wheelock, C. E.; Morisseau, C.; Goodrow, M. H.; Hammock, G.; Hammock, B. D. Bioorg. Med. Chem. 2000, 8, 2663-2675. 470 McElroy, N. R.; Jurs, P. C.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2003, 46, 1066-1080. 471 Anandan, S. K.; Do, Z. N.; Webb, H. K.; Patel, D. V; Gless, R. D. Bioorg. Med. Chem. Lett. 2009, 19, 1066- 1070. 472 Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Protein Sci. 2006, 15, 58-64. 473 Kodani, S. D.; Hammock, B. D. Drug Metab. Dispos. 2015, 43, 788-802. Introduction 171 Figure 77 . General binding mode of urea-based sEH inhibitors and key residues of the binding pocket of sEH: catalytic triad (blue), long branch (purple) and short branch (green). LHS: left- hand side; RHS: right-hand side. 1,3-Disubstituted ureas have undergone a remarkable evolution since their discovery. The earliest symmetrical 11?-disubstituted ureas displayed good inhibitory activities. Their design was based on the fact that the catalytic triad is in between two hydrophobic pockets, which could be filled with cycloalkyl groups for the establishment of Van der Waals-like interactions. Thus, the side chains of the urea were selected according to these premises and DCU (11?-dicyclohexylurea) appeared as a promising inhibitor with proven efficacy lowering blood pressure in hypertensive rats.474 Other carbocyclic substituents were introduced, such as phenyl, cyclooctyl and adamantyl groups.475 Despite the fact that this type of compounds showed potent inhibitory activities, they lacked optimal physicochemical properties for good oral absorption. These dialkylureas had high crystal lattice energies as indicated by their high melting points, and limited solubility in water. From this first series of compounds, the adamantane nucleus afforded a superior enzyme inhibition. Taking into account the characteristics of the binding site of sEH and the overall high hydrophobicity of the tunnel, the introduction of an adamantane ring has proven a successful strategy for the space-filling of the cavity. Thus, several sEH inhibitors incorporate an adamantane moiety on the LHS exhibiting high affinity and potency. Since the bulky adamantane ring can occupy one of two sides of the active site tunnel, it is possible that 1-adamantyl-urea-based inhibitors bind in the active site of human sEH with different orientations.476 Figure 78a highlights the binding mode of an adamantane-based inhibitor. In the present introduction, only the adamantane-based sEH inhibitors will be examined, despite the fact that further development has been done regarding other scaffolds, mainly substituted phenyl rings (Fig. 78b).477,478 474 Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.; Newman, J. W.; Parker, C.; Graham, L.; Engler, M. M.; Hammock, B. D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res. 2000, 87, 992-998. 475 Morisseau, C.; Goodrow, M. H.; Newman, J. W.; Wheelock, C. E.; Dowdy, D. L.; Hammock, B. D. Biochem. Pharmacol. 2002, 63, 1599-1608. 476 Chen, H.; Zhang, Y.; Ye, C.; Feng, T. T.; Han, J. G. J. Biomol. Struct. Dyn. 2014, 32, 1231-1247. 477 Anandan, S.-K.; Webb, H. K.; Do, Z. N.; Gless, R. D. Bioorg. Med. Chem. Lett. 2009, 19, 4259-4263. 478 Rose, T. E.; Morisseau, C.; Liu, J.-Y.; Inceoglu, B.; Jones, P. D.; Sanborn, J. R.; Hammock, B. D. J. Med. Chem. 2010, 53, 7067-7075. 172 CHAPTER 3: sEH inhibition a) b) Figure 78 . (a) Crystal structure of t -AUCB ( vi de i nfra ) bound to sEH. PDB code: 3WKE;456 (b) Structure of GlaxoSmithKline?s candidate that entered phase I clinical trials in 2013.444,473 Structural refinement of the first generation of inhibitors led to the discovery of adamantyl ureas attached to long alkyl chains featuring a terminal polar functionality, so as to resemble the carboxylic acid in a putative endogenous substrate, in an attempt to render these compounds more druggable. In this manner, N -adamantanyl-1?-dodecanoic acid urea (AUDA) emerged as an improved inhibitor with excellent inhibition of murine and human sEH and enhanced water solubility.479 AUDA has been extensively used as a tool molecule to validate efficacy in models of hypertension, cardio-protection and inflammation.431,480,481 Addition of polar ethylene glycol linkers in the alkyl chain afforded new compounds with similar properties as AUDA, such as N -adamantanyl-1?-(5-(2-(2- ethoxyethoxy)ethoxy)pentyl)urea (AEPU).482 Although improving water solubility, these compounds were rapidly metabolized through ?-oxidation of the alkyl chain.483 To avoid this issue, the incorporation of a conformationally restricted unit on the RHS of the molecule, such as an heterocycle or a phenyl ring, led to sEH inhibitors with improved metabolic stability whilst maintaining the potency.484,478 Here is where came into pla\ the ?pharmacophore model?, which may consist oI three parts the ?primar\ pharmacophore or PP? the ?secondar\ pharmacophore r SP? and the ?tertiar\ pharmacophore or TP ? (Fig. 79).485,486 33 reIers to the urea amide carbamate ? group 479 Morisseau, C.; Goodrow, M. H.; Newman, J. W.; Wheelock, C. E.; Dowdy, D. L.; Hammock, B. D. Biochem. Pharmacol. 2002, 63, 1599-1608. 480 Dorrance, A. M.; Rupp, N.; Pollock, D. M.; Newman, J. W.; Hammock, B. D.; Imig, J. D. J. Cardio. Pharm. 2005, 46, 842-848. 481 Imig, J. D.; Zhao, Z.; Zaharis, C. Z.; Olearczyk, J. J.; Pollock, D. M.; Newman, J. W.; Kim, I. H.; Watanabe, T.; Hammock, B. D. Hypertension 2005, 46, 975-981. 482 Hammock, B. D.; Kim, I. H.; Morisseau, C.; Watanabe, T.; Newman, J. W. Inhibitors of the soluble epoxide hydrolase. US 2005/0164951 A1. 483 Hwang, S. H.; Tsai, H.; Liu, J.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2007, 50, 3825-3840. 484 Jones, P. D.; Tsai, H. J.; Do, Z. N.; Morisseau, C.; Hammock, B. D. Bioorg. Med. Chem. Lett. 2006, 16, 5212-5216. 485 Kim, I.; Morisseau, C.; Watanabe, T.; Hammock, B. D. J. Med. Chem. 2004, 47, 2110-2122. 486 Kim, I.; Tsai, H.; Nishi, K.; Kasagami, T.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2007, 50, 5217- 5226. Introduction 173 bearing the bulky hydrophobic adamantane nucleus on one side. SP is a polar group, e.g. ester, ether, amide, sulphonamide, alcohol or ketone, normally positioned approximately 7 ? from the carbonyl group of the PP.479,487 In addition, a polar TP such as ester, ether, carboxylic acid, or amide that is 13 atoms or ~ 17 ? away from the urea carbonyl has also been identified and applied. Hydrophobic linkers join the PP to the SP, and the SP to the TP, where the presence of cyclic groups seem to increase oral bioavailability compared to linear alkyl chains.488 Apart from improving the metabolic stability of the RHS, the incorporation of the SP and TP enhanced the water solubility and lowered the melting points. Figure 79 . 5epresentation oI the ?pharmacophore model? Table 16 summarizes the evolution of the adamantane-based sEH inhibition over the course of their development. Table 16. Representative adamantane-based sEH inhibitors and their evolution in chronological order. sEH inhibitor Pharmacophore model & Structural features Characteristics N-adamantyl -1?-cyclohexylurea (ACU) PP; two hydrophobic side chains High potency, low solubility, poor PK 487 Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H. J.; Hammock, B. D. J. Med. Chem. 2005, 48, 3621-3629. 488 Kasagami, T.; Kim, I. H.; Tsai, H. J.; Nishi, K.; Hammock, B. D.; Morisseau, C. Bioorg. Med. Chem. Lett. 2009, 19, 1784-1789. 174 CHAPTER 3: sEH inhibition AUDA ?33;? long carbon chain with a terminal carboxyclic acid High potency, improved solubility, poor PK AEPU ?33;? long carbon chain with ethylene glycol units High potency, improved solubility, poor PK 4-(3 -adamantan -1-yl -ureido) -2-hydroxyl -benzoic acid methyl ester (AUSM ) SP; benzoic acid methyl ester High potency, improved solubility, enhanced PK N-(1 -acetylpiperidin -4-yl) -1?-adamantylurea (APAU, AR9281) SP; conformationally restricted amide High potency, improved solubility, enhanced PK 1-adamantan -1-yl -3-(4 -(3 - morpholinopropoxy)cyclohexyl)urea (AMCU) TP; ether and morpholine ring groups as pharmacophores High potency, improved solubility, enhanced PK Introduction 175 trans-4-[4 -(3 -adamantan -1- ylureido)cyclohexyloxy]benzoic acid ( t-AUCB) TP; ether and carboxylic acid groups as pharmacophores High potency, improved solubility, enhanced PK Among these compounds, APAU or AR9281, developed by Ar?te Therapeutics, was the only adamantane-based sEH inhibitor that reached phase II clinical trials. It showed a high level of safety but failed to show efficacy in early-stage hypertension and impaired glucose tolerance. This failure was attributed to poor PK properties or variable effects of the inhibitor.405,489 Regardless of the efforts made to procure drug candidates, it still remains a significant challenge to develop sEH inhibitors with excellent inhibitory activities and optimum solubility and PK profiles. A vast number of sEH inhibitors in development enclose an adamantane moiety, which possess particular properties that determine the behaviour of the future drug candidates: ? Its pronounced lipophilicity compromises negatively the overall water solubility of the molecule, which is an important physicochemical parameter in the early stages of drug discovery.490,491 In addition, the general stability of the adamantyl compounds? crystal structures, indicated by their high melting points, leads to a general lack of solubility even in organic solvents. ? Besides, the adamantane nucleus is prone to rapid metabolism i n vi vo giving rise to a variety of inactive, hydroxylated derivatives and leading to low drug concentrations in blood and short i n vi vo half-life. ? Again, the high lipophilicity of the inhibitors furnished chiefly by the adamantyl unit results in an increased likelihood of binding to multiple targets, leading to apparent toxicity.492 Considering the above mentioned, it seems evident that the adamantane ring does not possess the optimal properties as a scaffold for sEH inhibition. A decrease in the overall lipophilicity will lead to more soluble and metabolically stable compounds, which will thereIore be more ?drug-liNe?29-30 In this sense, there is an urgent need for the development 489 Clinical trials web site. AR9281. https://www.clinicaltrials.gov/ (accessed on 5th September 2015). 490 Bhattachar, S. N.; Deschenes, L. A; Wesley, J. A. Drug Discov. Today 2006, 11, 1012-1018. 491 Di, L.; Fish, P. V; Mano, T. Drug Discov. Today 2012, 17, 486-495. 492 Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Discov. 2007, 6, 881-890. 176 CHAPTER 3: sEH inhibition of new scaffolds that provide compounds with more optimal physicochemical properties to avoid attrition in later stages of the drug discovery process. Objetive s Objectives 179 Regardless of the gigantic effort made from academic and industrial research groups in the development of drug candidates, only a few sEH inhibitors have entered into clinical trials. At the time of writing the present dissertation, no sEH inhibitor had got into the market, and only two compounds have reached human clinical trials, one of which contains an adamantane group, as mentioned previously. This points out that the drug discovery process of sEH inhibitors is complex and highly challenging. Some development limitations are: inappropriate physicochemical properties, especially high lipophilicity and low water solubility, and chemical and metabolic instability. Therefore, there is a demand for the development of new sEH inhibitors that, having an acceptable inhibitory activity, will overcome these issues. The adamantane nucleus seems to be a privileged scaffold in sEH inhibitors since it is the most exploited bulky group when designing a new drug candidate. This precious ring, together with the urea group as primary pharmacophore, fits into one of the hydrophobic pockets of the catalytic site of the enzyme. However, considering that the adamantane does not possess the optimum properties (i.e. lipophilicity, water solubility, etc? for a urea- based sEH inhibitor, we hypothesized that the replacement of the adamantane moiety by other polycyclic cage structures may afford compounds with improved PK and PD profiles by changes in the physicochemical properties, whilst maintaining a good binding affinity for the target. In line with the previous chapter of the thesis, and taking advantage of the extended expertise of our rese arch group in the synthesis of polycyclic scaffolds, we started a new project related to the discovery of novel sEH inhibitors bearing adamantane-like scaffolds as hydrophobic counterparts. Thus, these were the main goals of this chapter: 1. Synthesis of a variety of scaffolds, including ring-contracted, ring-expanded and oxa - derivatives of the adamantane group, attached to an already reported substituted urea (Fig. 80). 180 CHAPTER 3: sEH inhibition Fig. 80. Replacement of the adamantane group by a variety of scaffolds. 2. Pharmacological evaluation of these scaffolds to provide useful information of the threshold of tolerance of the catalytic site to accept a diversity of structures. Determination of water solubility as well as calculation of clog P of each new compound to assess the changes in physicochemical properties. 3. Determination of the ability to ameliorate the ER stress in human hepatocytes of selected inhibitors, according to the results from 2. With the in vitro assays, the cell permeation and cytotoxicity will be also evaluated. Results & Discussion Results & Discussion 183 1. Scaffold -hopping approach for the development of new sEH inhibitors From the available data regarding sEH and its inhibition by small molecules, we identified a few relevant issues: ? The adamantane ring is predominantly chosen as the bulky group for the space- filling of the hydrophobic pockets of the sEH catalytic site. ? On the other hand, the adamantane group provides a remarkable increase in the overall lipophilicity of the molecule, compromising the water solubility and the pharmacokinetic profile. ? From the crystal structure of sEH, it has been suggested that, while the catalytic tunnel is restricted around the catalytic residues, it enlarges as one moves away from the site of reaction to yield relatively large cavities. These structural features show that the enzyme can accommodate larger groups on both sides of the urea moiety. ? Additionally, polar groups can be incorporated into one of the alkyl substituents without loss of activity if they are placed at an appropriate distance from the urea function. Considering the aforementioned, we aimed to pursue a small and medium-step scaffold-hopping approach via the replacement of the adamantane nucleus by diverse polycycles that possess different physicochemical properties and varied shapes and sizes (Fig. 81). Scaffold - hopping is a strategy widely applied in medicinal chemistry whose purpose is to identify novel structures that are active against a given target with acceptable pharmacological properties defined by marketed drugs.493 Fig. 81 . Design of new sEH inhibitors by scaffold-hopping approach. 493 Sun, H.; Tawa, G.; Wallq vist, A. Drug Discov. Today 2012, 17 , 310 - 3 24. 184 CHAPTER 3: sEH inhibition The design of these compounds was carefully studied in order to fulfil most of the established criteria (potency, molecular weight and lipophilicity), wherein the adamantane- like ring plays a crucial role. Because of the shape and size of the active site tunnel as well as its hydrophobicity, varying the polycyclic cages may affect how the urea moiety of the molecule sits within the tunnel, thereby affecting inhibition. Bearing in mind the existence oI residues capable oI establishing h\drogen bonds and ?-stacking interactions in the long branch of the binding site of sEH , we hypothesized that the introduction of an heteroatom, such as an oxygen atom, and/or an aromatic ring on t he polycyclic structure may be tolerated. In addition, the size and shape of that branch may assist the adjustment of the dimensions of the lipophilic group. Table 17 outlines the selected scaffolds, including their structural contributions and antecedent applications in medicinal chemistry. Table 17. Selected polycycles for the design of novel sEH inhibitors. CB: cannabinoid receptor. Entry Scaffold Structural features Previous uses in medicinal chemistry 1 Replacement of a methylene group by an oxygen atom at C-2 position. Introduction of non-polar groups at C-3 position NMDA receptor antagonism, anti- trypanosome,95 CB agonism,494 cancer495 2 Replacement of a methylene group by an oxygen atom at C -5 position No described 494 Dixon , D. D.; Seth u mad h avan , D.; Ben n ech e, T. ; Ban aa g, A. R.; Tiu s, M. A .; Thak u r, G. A.; Bowman , A. ; Wood , K. T.; Makr iy an n is, A. J. Med. Chem. 2010, 53, 5656 - 5 666 . 495 Wang, L.; Doh erty , G. ; Wan g, X.; Tao , Z. F.; Bru n ko , M.; Ku n zer, A. R.; W en d t, M. D.; Son g, X.; Fr ey , R.; Han sen , T. M.; Sulli van , G. M.; Jud d , A.; Sou ers, A. 8 - Carbamoy l - 2 - (2,3 - d isu b stit u ted p y r id - 6 - y l) - 1,2, 3,4 - tetra h y d ro iso q u in o line deriva tive s as ap op to si s - in d u cing ag en ts for th e treat men t of can c er an d immu n e an d auto immu n e disea se. WO 201305 5897 A 1 . Results & Discussion 185 3 ?Bird cage? similar shape to adamantane Incorporation of an oxygen atom. Racemic mixture NMDA receptor antagonism496 4 Double ring-contraction of adamantane group. Addition of methyl groups to increase molecular volume and/or a small linker to space out the urea moiety from the bulky group NMDA receptor antagonism, anti- trypanosome,95 M2 channel blocker91 5 Single ring-contraction of rimantadine M2 channel blocker, NMDA receptor antagonism 95 6 Ring-expansion and rearrangement of adamantane nucleus. Presence of double bonds M2 channel blocker90 7 Insertion of a phenyl ring into the adamantane-like group. Introduction of a methyl group, and possibility to replace bridged methylene by an oxygen atom NMDA receptor antagonism, anti- trypanosome97, 98 In order to assess the effect of each new scaffold in the sEH inhibition, we placed them as the left-hand side (LHS) of the urea group and a selected unit from the literature was chosen as the right-hand side (RHS) (Fig. 82). Particularly, 2,3,4-trifluorophenyl group was selected due to its facile synthesis and high inhibitory activity against sEH. In fact, both 1 - (1-adamantyl)-3-(2,3,4-trifluorophenyl)urea 108 and 1-(2-adamantyl)-3-(2,3,4- trifluorophenyl)urea 109 were firstly reported as anti-tuberculosis agents whose mechanism 496 Geld en h u y s, W. J. ; Malan , S. F.; Bloo mq u i st, J. R.; Mar ch an d , A. P.; Van der Schy f, C. J. Med. Res. Rev. 2005, 25, 21 - 48 . 186 CHAPTER 3: sEH inhibition of action consists in the inhibition of the epoxide hydrolase family of enzymes ( Mycobacterium tuberculosis epoxide hydrolase s B and E, but also human sEH). 497,498 Fig. 82. 2,3,4-Trifluorophenyl unit as RHS . 1.1 Synthesis of N-adamantane -like -1?-(2,3,4 -trifluor ophenyl)ureas The synthetic plan of the final ureas was simple and straightforward, and consisted in the coupling of an amine with an isocyanate. Because of the handy experience of our research group in the preparation of polycyclic cage amines, and that 2,3,4-trifluorophenyl isocyanate was commercially available, we prepared the adamantyl-based ureas as showed in scheme 56. Scheme 56. General procedure for the preparation of adamantyl-like ureas. In order to save time and resources, in certain cases we started the synthesis from intermediate amines already prepared by former group members or myself in earlier time (Fig. 83). 497 Brown, J. R.; North , E. J.; H urd le, J. G. ; Mori ss eau , C.; S carb o ro u gh , J. S.; Sun , D.; Ko rd u l?k o v?, J.; Scherman , M. S.; Jon e s, V.; Gr zegorz e wicz, A.; Cre w, R. M.; Jack so n , M.; McN eil, M. R .; Le e, R. E. Bioorg. Med. Chem. 2011, 19, 5585 - 5 595. 498 Scherman , M. S.; North , E. J.; Jon es, V.; He s s, T. N.; Grze gorz ewi c z, A. E.; Kasaga mi, T.; Kim, I. H. ; Merzlik in , O.; L en aert s, A. J.; L ee, R. E.; Jack so n , M.; Mor is se au , C.; McN ei l, M. R. Bioorg. Med. Chem. 2012, 20, 3255 - 356 2. Results & Discussion 187 Fig. 83. Intermediate amines formerly synthesized by the group. The rest of the amines were prepared from different intermediates, and some of them starting from scratch (Fig. 84). Fig. 84. Intermediate amines to prepare. 1.1.1 Preparation of intermediate scaffolds 1.1.1.1 Synthesis of (2-oxaadamantan -1-yl)amine, 120: The oxa -derivative of amantadine 120 was prepared following the procedure firstly described by Gagneux and Meier in 1969 and broadly applied by our research group.499,500,501 Starting from known diketone 125, a transannular reductive amination took place affording compound 126 in 57% yield (Scheme 57 ). In this step, reaction with benzylamine yielded an imine intermediate, which was reduced following a 1,4-conjugated - like mechanism upon the addition of a hydride source in a one-pot fashion. This intramolecular attack is possible due to the presence of a ?-homoconjugated system, similar to that of 25. 499 Geigy A. G. Deri vat i ve s of 2 - o xa - ad aman ta n e. GB 1 1236 09 . 500 Gagn eu x, A. R.; M eier, R. Tetrahedron Lett. 1969, 10, 1365 - 1368 . 501 Duqu e, M. D.; Camp s, P.; Pro fire, L.; Mon ta n er, S.; V?z q u ez , S.; Sured a, F. X.; Mallol, J.; L ?p ez - Q u ero l , M.; Naes en s, L.; D e Cl ercq , E.; Pra th alin gam, S. R.; Ke lly, J. M . Bioorg. Med. Chem. 2009, 17 , 3198 - 3 206 . 188 CHAPTER 3: sEH inhibition Scheme 57. Formation of benzylamine 126 from diketone 125 and subsequent precipitation as the hydrochloride salt. The proton shifts have been avoided for the sake of clarity. With benzylamine 126 in hand, hydrogenolysis of the benzyl group led to hemiaminal 120 in quantitative yield, isolated as its hydrochloride salt (Scheme 58). Scheme 58. Deprotection of the benzyl group by catalytic hydrogenation at atmospheric pressure. 1.1.1.2 Preparation of (4-oxahexacyclo[5.4.1.0 2,6.03,10.05,9.08,11 ]dodec -3-yl)amine, 121: The synthesis of oxahexacyclo derivative 121 was based on the work of Govender and co-workers, and started by using the so-called CooNson?s dione 127.502 Analogously to the latter intermediate, 127 was subjected to a reductive amination with benzylamine. In this procedure, azeotropic distillation with toluene led the dehydration of the reaction intermediate to afford imine 128, which was then reduced with sodium borohydride. Subsequent catalytic hydrogenation provided deprotected amine 121 in 30% overall yield (Scheme 59). 502 Onaj o le, O. K.; Coovad ia, Y.; Kru ger, H. G.; Magu ire, G. E. M.; Pillay , M.; Goven d er, T. Eur. J. Med. Chem. 2012, 54, 1 - 9 . Results & Discussion 189 Scheme 59. Preparation of oxahexacyclo amine 121 starting from commercially available diketone 127. In accordance to what was described in chapter 1 (see page 66), the formation of aza- cage compound 130 was reported by Marchand and co-workers from the selective reduction of the imine with sodium cyanoborohydride instead of the ketone in compound 128.503 The resulting amine attacked the carbonyl group providing a hydroxyl -aza-bridged compound (Scheme 60). To avoid this, sodium borohydride is needed. Thus, the ketone is first reduced, releasing a hydroxyl function ready to add to the imine moiety, and finally affording the desired ?bird-cage? compound 129. Scheme 60. Selective reduction of keto-imine 128. 1.1.1.3 Synthesis of (tricyclo[3.3.0.0 3,7 ]oct -1-yl)amines 122 and 123: Amines 122 and 123 were prepared following a long and tedious synthetic pathway inspired from the previous work of the group. The key step of the route revolves around an intramolecular enolate homocoupling of diesters 131 and 132, which can lead to the desired amines after classical synthetic transformations (Fig. 85). Diester 132 was taken from previously prepared samples by former group members. 503 March an d , A. P.; Arn ey , B. E. Jr.; Da ve, P. R.; Sat y an ar ay an a, N. J. Org. Chem. 1988 , 53, 26 44 - 26 47 . 190 CHAPTER 3: sEH inhibition Fig. 85. Planned synthesis of amine 122 and 123 from diester 131 and 132, respectively. For the preparation of diester 131, the first step was the synthesis of diketone 133 through a Weiss-Cook condensation in a multigram scale. Following the procedure reported in Organic Syntheses , 504 two equivalents of 1,3-dimethyl-acetondicarboxylate were condensed with glyoxal in the presence of sodium hydroxide as a base (Scheme 61). After neutralization of disodium salt 134, dienol-tetraester 135 was obtained in 55% yield. The reaction mechanism is analogous to that from scheme 3 in chapter 1, and consisted in a double aldol condensation prior to a double Michael addition. Scheme 61. Weiss-Cook condensation of 1,3-dimethyl-acetondicarboxylate and glyoxal . Without further purification, tetraester 135 was treated with acidic aqueous solution for the hydrolysis of the ester group and concomitant decarboxylation of the resulting ?- keto carboxylic acids, giving diketone 133 in 71% yield (Scheme 62 ). Scheme 62. ?-decarboxylation of tetraester 135 to yield dione 133 after acid hydrolysis. The synthesis continued with the reaction of diketone 133 with sodium cyanide and sulphuric acid.505 In this step, the slow addition of 40% H 2SO4 solution to sodium cyanide in aqueous media supplied in situ- formed cyanide ions that added to the carbonyl groups of 133, giving a stereoisomeric mixture of bis-cyanohydrins 136 (Scheme 63). 504 Bertz , S. H.; Coo k, J. M.; Gawi sh , A.; Wei ss, U. Org. Synth. Coll. Vol. VII 1990, 50 - 56 . 505 Camp s, P.; Igles ias, C.; Rod r? gu ez, M. L.; Gran ch a, M. D.; Gregori, M. E.; Loz an o , R.; Mira n d a, M. A.; Figu ered o , M.; Lin ar es, A. Chem. Ber. 1988, 121 , 647 - 654 . Results & Discussion 191 Scheme 63. Double addition of cyanide to diketone 133 for obtaining a mixture of bis- cyanohydrins 136. Bis -cyanohydrins 136 were transformed into vinyl cyanides in the presence of thionyl chloride and pyridine under reflux. Under these conditions, dehydration of 136 led to a regioisomeric mixture of dinitriles 137 and 138 in 44% yield (Scheme 64). The reaction begins with the addition of the oxygen a tom of the hydroxyl group to SOCl2 and later elimination of a chloride ion. The base subtracts the ?-proton to form the vinyl system, eliminating sulphur dioxide and eventually hydrochloric acid, which is trapped by the base. Scheme 64. Dehydration of bis-cyanohydrins 136 with thionyl chloride in the presence of pyridine. Following the procedure, the syn - and anti- mixture of both vinyl cyanides 137 and 138 was submitted to a very slow catalytic hydrogenation due to the steric encumbrance around the double bonds. After 3 days at 27 atm of hydrogen, a complex stereoisomer ic mixture of saturated nitriles 139 was obtained in a ratio of 2:1.3:1; (1?,3?,5?,7?), (1?,3?,5?,7?) and (1?,3?,5?,7?) respectively, in 87% yield (Scheme 65 ). 192 CHAPTER 3: sEH inhibition Scheme 65. Formation of the stereoisomeric mixture 139 after hydrogenation of 137 and 138. The next step was the hydrolysis of the nitrile groups with potassium hydroxide and water, followed by a Fischer esterification with anhydrous methanol and conc. H 2SO4, providing a mixture of dimethyl esters 141 in 62% yield (2 steps) (Scheme 66).506 Scheme 66. Hydrol ysis of dinitriles 139 and esterification of 140 to 141. The next step can be consider as the most important tran sformation of the whole synthetic route, consisting of the intramolecular homocoupling of enolates to build the tricyclic system (Fig. 86). Fig. 86. Intramolecular cyclization of 141. Many different procedures have been reported since the first dimerization of enolates was described in 1935, where Ivanoff et al. published the generation of the enolate of a carboxylic acid with a Grignard reagent for the posterior treatment with bromine to give the desired dimer.507 Later on, similar methods for the homo- and heterocoupling of enolates appeared, all of them with common features: use of a strong base for the ?- deprotonation, such as lithium N -(tert-butyl)cyclohexanamide or lithim diisopropylamide 506 M?nd ez, N. M.S. The sis , Univ ersity of Barc elon a, 1997 . 507 Ivan o ff, D.; Spa sso ff , A. Bull. Soc. Chim. Fr. 1935, 2, 76 - 78. Results & Discussion 193 (LDA), and an oxidant agent, such as copper(II) bromide or chloride, iron(III) chloride, titanium(IV) chloride or iodine.508,509,510,511,512,513,514 Despite these alternatives, we decided to apply the method already employed by the group.515,506 The tricyclic system of 131 was thus formed starting from the stereoisomeric mixture of diesters 141 by an oxidative homocoupling with iod ine as oxidant and LDA as base (formed in situ from diisopropylamine and n-butyllithium) in 36% yield (Scheme 67). The actual mechanism of this reaction is still unclear, albeit different studies have been performed to understand it.516 It is believed that the treatment of diesters 141 with LDA generates enolates A that can undergo two different reaction pathways: i) reaction with iodine by a single electron transfer (SET) to give diradical B (path a), which can collapse to directly provide the ring-closed diester 131; ii) formation of iodide C by an ionic mechanism (path b), which can be displaced in an intramolecular SN2-type reaction. The steric hindrance of the dianions plays an important role in determining the reaction pathway. 508 Rath ke, M. W.; Lin d ert, A. J. Am. Chem. Soc. 1971, 93, 4605 - 460 6. 509 Ito, Y.; Kon o ike, T.; Sa egu sa, T. J. Am. Chem. Soc. 1975, 97, 2912 - 2914. 510 Fraz ier, R. H. Jr.; Harlo w, R. L. J. Org. Chem. 1980, 45, 54 08 - 5411. 511 Kise, N.; Tokio ka , K.; Aoy ama, Y.; Mat su mu ra , Y. J. Org. Chem. 1995, 60, 1100 - 11 01. 512 Paqu e tt e, L. A.; Bzo wej, E. I.; Bran an , B. M.; Sta n to n , K. J. J. Org. Chem. 1995, 60, 7277 - 72 83. 513 Bara n , P. S.; DeMart in o , M. P. Angew. Chem. Int. Ed. Engl. 2006, 45, 7083 - 70 86. 514 DeMartin o , M. P.; Chen , K.; Baran , P. S. J. Am. Chem. Soc. 2008, 130, 115 46 - 1156 0. 515 Camp s, P.; Lu q u e, F. J. ; Oro zc o , M.; P?rez, F.; V?zq u ez, S. Tetrahedron Lett. 1996, 37, 8605 - 86 08 . 516 Renau d , P.; Fox , M. A. J. Org. Chem. 1988, 53, 374 5 - 3 752. 194 CHAPTER 3: sEH inhibition Scheme 67. Intramolecular oxidative homocoupling of diester 141. At this point, the syntheses of amines 122 and 123 were concurrently run. With the goal of achieving these monoamines from diesters 131 and 132, we needed to differentiate the ester functions in order to selectively eliminate one of them (Scheme 68). For this purpose, diesters 131 and 132 were hydrolysed under basic conditions to furnish the corresponding carboxylic acids 142 and 143 in quantitative yields. Afterwards, the treatment of the aforementioned with neat acetic anhydride produced anhydrides 144 and 145 in 72% and quantitative yield, respectively. Then, the anhydride unit was opened in the presence of sodium methoxide and methanol to give hemiesters 146 and 147 in low yields due to the presence of adventitious water from the solvent. In both cases, diacids 142 and 143 were recovered. Results & Discussion 195 Scheme 68. Functional group differentiation of 131 and 132. Next, hemiesters 146 and 147 were converted to the corresponding monoesters 148 and 149 through a Barton radical decarboxylation. 517,518 The procedure consists in the protodecarboxylation of a carboxylic acid 150 in the presence of a suitable hydrogen bond donor (H -donor) to furnish the corresponding hydrocarbon 151. Fundamental to the Barton decarboxylation protocol is the alkyl thiohydroxamic ester 152, also known as Barton ester, which is readily prepared from the reaction of an activated carboxylic acid and the sodium salt of 1-hydroxypyridine -2(1H)-thione.519,520 By means of applying light and heat, the Barton ester undergoes rapid homolytic decomposition yielding the corresponding alkyl acyloxy radical 153, which is subjected to decarboxylation. The remaining alkyl radical 154 intercepts a proton from the H -donor, whereupon 151 is recovered (Scheme 69). Scheme 69. Mechanism of reaction of the Barton decarboxylation . 517 Bart o n , D. H. R.; Crich , D.; Mo th erwel l, W. B. J. Chem. Soc. Chem. Commun. 1983, 939 - 94 1 . 518 Bart o n , D. H. R.; Herv?, Y.; Po tier, P.; Thier ry , J. J. Chem. Soc. Chem. Commun. 1984, 1298 - 1299. 519 Bart o n , D. H. R.; Crich , D. Moth erwel l, W. B. Tetrahedron 1985 , 41, 3901 - 39 24. 520 Bart o n , D. H. R.; Samad i, M. Tethahedron 1992, 48, 7083 - 7 090. 196 CHAPTER 3: sEH inhibition Former protocols of the Barton decarboxylation applied by our group entailed the use of 22?-dithiobis(pyridine- N -oxide) as precursor of the Barton ester and n-tributylphosphine for its pre-activation.172 As H -donor, t-butylthiol was the preferred proton source, despite the fact that its use implied that the reaction should be set up in a special laboratory for highly smelly and toxic reactions. On the other hand, the utility of chloroform as both solvent and H -donor in Barton decarboxylation has been recently demonstrated. 521,522 Taking advantage of this finding, we performed the reaction under t-butylthiol-free conditions within chloroform (Scheme 70). Pleasingly, hemiesters 146 and 147 were easily transformed to their corresponding monoesters 148 and 149 (86% yield for 149). Because of the high volatility of 148, the solvents were distilled off after the work up to give the monoester along with reaction by- products. The crude was used in next step without any purification. Scheme 70. Barton decarboxylation of hemiesters 39 and 40 with chloroform as H -donor. The decarboxylation was followed by a basic hydrolysis of monoesters 148 and 149 to monoacids 155 and 156 in 73% and 61 % yield (2 steps), respectively (Scheme 71). Scheme 71. Basic hydrolysis of monoesters 148 and 149. The last step for the preparation of monoamines 122 and 123 involved a Curtius rearrangement, which led to the one-step functional interconversion of a carboxylic acid to a primary amine with concomitant loss of one carbon atom through an acyl azide intermediate. In this transformation, the thermal decomposition of carboxylic azides produces an isocyanate, which can be hydrolysed to yield the corresponding amine. In our case Ze applied the 220 dec. 174 150-152 182 206-207 175 211-213 183 257-259 176 152-154 Except for compounds 157, 181 and 183 that did not alter or reduce somewhat the value with respect to the standards, the new scaffolds provided compounds with lower melting points. This observation is in accordance to the overall increase in the water solubility from table 20. 1.4 Lipophilic ligand efficiency: a new metric for drug discovery The strong relationship between solubility and lipophilicity is often discussed in the literature, and unsurprisingly as clog P increases, solubility on average decreases.84 Compounds may be less water soluble if i) they are lipophilic and/or ii) they form cohesive, stable crystalline lattices. Indeed, Banerjee et al. suggested an empirical equation that relates solubility, melting point and log P [log P = 6.5 ? 0.89(log S) ? 0.015mp]. 542 Hence, these three characteristics are interrelated. In consequence, fine-tuning of lipophilicity is one of the most applied strategies for improving water solubility, and eventually reIining the ?drug- 542 Ban erjee, S. Environ. Sci. Technol. 1980, 14, 1227 - 12 29. Results & Discussion 217 liNeness.? 27,536 In Iact this has been our approach Zith the modulation oI adamantane?s structure (scaffold-hopping), thus affecting log P, as it will be discussed later on. In the last few years, novel metrics diIIerent Irom the traditional /ipinsNi?s rule oI Iive have appeared, and are employed to expedite the optimization process by combining s everal of these factors into guidelines for medicinal chemists.543,544,545 All of them are designed to deIine the ?drug-liNe? space Among them, ligand efficiency (LE) is one of the most important and commonly employed metrics in drug discovery, first published by Kuntz in 1999 and redefined by Hopkins in 2004 .546,547 LE quantifies the average contribution of a heavy atom to the binding (Equation 2). As the name suggests, it measures the efficiency of the ligand in the potency. /( ?*  number oI heav\ atoms or LE = pIC 50 (or pKi) / number of heavy atoms Equation 2 Another major metric is the lipophilic ligand efficiency (LLE or LipE), which i s an estimate of the specificity of a molecule in binding to the target relative to lipophilicity. In other words, LipE normalizes potency relative to log P.548 It is calculated by applying a relative simple equation 3. LipE = ? log (Ki or IC50) ? log D (or clog P) Equation 3 It reasserts the principle of minimal hydrophobicity, which states that ?Zithout convincing evidence to the contrary, drugs should be made as hydrophilic as possible Zithout loss oI eIIicac\?549 Increasing LipE is likely to be associated with better selectivity, efficient polar atom ligand-protein contacts, and specific hydrophobic binding.83 Based on the properties of an average oral drug, with a clog P of ~2.5?3.0 and potency in the range of ~1?10 nM, an ideal LipE value for an optimized drug candidate is ~5?7 units or greater. Negative LipE values are clearly unfavourable. Many oral drugs occupy optimal combined ligand efficiency space for their specific targets, showing the importance of balancing biological activity, size, and lipophilicity in lead optimization. In order to assess the changes on the clog P, and hence on the LipE, as a result of our scaffold-hopping strategy, we first calculated the clog P using Bio-Loom ? software, and then we applied equation 3. The results are summarized in table 22. 543 Mean wel l, N. A . Chem. Res. Toxicol. 2011, 24, 1420 - 145 6. 544 Abad - Za p at ero , C.; Champ n e ss, E. J.; S egall, M. D. Future Med. Chem. 2014, 6, 577 - 593. 545 Hop kin s, A. L.; Keser? , G. M.; Lee so n , P. D.; Rees, D. C.; Rey n o ld s, C. H. Nat. Rev. Drug Discov. 2014, 13, 105 - 1 21. 546 Kuntz , I. D.; Chen , K.; Sh ar p , K. A.; Kollman , P. A. Proc. Natl. Acad. Sci. USA 1999, 96, 9997 - 1000 2. 547 Hopkin s, A. L.; Gro o m, C. R.; Alex, A. Drug Discov. Today 2004 , 9, 430 - 431. 548 Free man - Coo k, K. D. ; Hoff ma n , R. L.; Joh n so n , T. W. Future Med. Chem. 2013, 5, 113 - 115. 549 ,ansch, C.; ??r?roth, :. W.; Leo, A. J. Pharm. Sci. 1987, 76, 6 63 - 68 7. 218 CHAPTER 3: sEH inhibition Table 22. Values of clog P and LipE for the new sEH inhibitors. Ureas 108 and 109 were used as standards. Comp. LHS clog P LipE Comp. LHS clog P LipE 108 4.04 4.07 177 2.93 4.92 109 5.08 3.31 178 3.96 5.11 170 2.71 5.58 179 5.36 3.02 171 3.23 4.44 180 5.19 2.80 172 3.76 4.33 157 2.59 4.84 173 5.26 2.50 181 3.55 4.04 174 4.27 3.40 182 5.48 3.62 175 2.14 5.18 183 4.21 3.38 Results & Discussion 219 176 1.67 5.83 Some interesting aspects could be deduced from the evaluation of the clog P values: ? The clog P varied with every different scaffold, with values ranging from 1.67 to 5.48. Since adamantyl standards 108 and 109 showed clog P values of 4.04 and 5.08, respectively, most part of the new compounds lowered the lipophilicity, whilst others increased moderately the clog P. ? Compounds bearing an oxygen atom 170-176 displayed a significant reduction in the clog P, except for compounds 173 and 174 (clog P of 5.26 and 4.27, respectively), which contain bulkier hydrophobic substituents. Outstanding are ureas 170, 175 and 176 (clog P of 2.71, 2.14 and 1.67, respectively) with a clog P value < 3 an d 1 to 2 log units less than adamantyl ureas 108 and 109 (clog P of 4.04 and 5.08, respectively). ? For both 2-oxaadamantan -1-yl and tricyclo[3.3.0 .03,7 ]oct -1-yl families, the lipophilicity was enhanced as the number of atoms increased, as expected. ? Among the ring-contracted scaffolds, compounds 177 and 178 (clog P of 2.93 and 3.96, respectively) lowered the clog P significantly compared with 1-adamantyl-urea 108. ? Surprisingly, enlarged pentacyclic ureas 157 and 181 (clog P of 2.59 and 3.55, respectively) reduced lipophilicity around 0.5-2.5 log units when compared to standards 108 and 109 (clog P of 4.04 and 5.08, respectively). ? Finally, scaffolds featuring a phenyl ring 182 and 183 (clog P of 5.48 and 4.21, respectively) displayed a larger clog P than the adamantyl ureas 108 and 109 because of their expanded hydrocarbon skeleton. Besides, the insertion of a heteroatom in 183 reduced the lipophilicity compared with its analogue 182. Evaluation of the LipE values led us to the identification of three different situations: ? Enhanced LipE with a reduction of both IC50 and clog P values: this is case of compounds 170 and 178 (LipE of 5.58 and 5.11, respectively). ? Enhanced LipE because of a remarkable reduction in the lipophilicity: here are included compounds 171 (LipE of 4.44), 172 (LipE of 4.33), 175 (LipE of 5.18), 176 (LipE of 5.83), 177 (LipE of 4.92), 157 (LipE of 4.84) and 181 (LipE of 4.04). ? Reduced LipE due to loss in the inhibitory activity and to an increase in the lipophilicity: this behaviour is showed in compounds 173 (LipE of 2.50), 174 (LipE of 3.40), 179 (LipE of 3.02), 180 (LipE of 2.80) and 183 (LipE of 3.38). 220 CHAPTER 3: sEH inhibition A special case is urea 182 (LipE of 3.62), with a LipE in between both standards 108 and 109 (LipE of 4.07 and 3.31, respectively), which despite of the dramatic gain in the lipophilicity (clog P of 5.48), is more efficient than the adamantyl analogues in the binding, as it is reflected in its IC50 (0.80 nM). This last observation, along with the former statements, showed that the adamantane ring is not the most efficient bulky hydrophobic group as LHS of sEH inhibitors, and that a variety of scaffolds may replace it with properties that are more appropriate. 2. New ureas with selected scaffolds In order to confirm the ability of the new scaffolds to surrogate the adamantane in sEH inhibitors, we aimed to build a second batch of compounds with the more promising scaffolds. The selection of the polycycles were in accordance to the overall performance in the inhibition assay, solubility measurement and LipE calculation, but also to the ease on the synthesis of the scaffold. Figure 91 resumes the selected scaffolds. Fig. 9 1. Selection of scaffolds from the previous series of compounds. We applied the scaffolds from compounds 170, 176 and 178 to the synthesis of further ureas with the aim of proving the efficiency of these structures as lipophilic groups in the design of sEH inhibitors. In this case, we selected the RHS of the known inhibitors t-AUCB and APAU. Both compounds display potent activities against sEH (IC 50 values of 1.3 nM and 7 nM, respectively) and improved physicochemical properties (Fig. 92).484,483 Results & Discussion 221 Fig. 9 2. New approach for the synthesis of new ureas. 2.1 Synthesis of new ureas with selected scaffolds For the preparation of the next ureas, we could not apply the synthetic approach used for the previous batch, since no required isocyanate was commercially available. In the exploration of the different strategies to build ureas groups, two procedures stood out: i) formation of an intermediate isocyanate with triphosgene from the first amine, and subsequent attack of the second amine, or ii) use of the coupling agent carbonyldiimidazole (CDI) (Scheme 83). Both routes implied the use of a transfer reagent, which functionalized one of the amine counterparts in order to react with the other one.550 Scheme 83. Two alternatives to form a urea functionality. To test the feasibility of both protocols in our scaffolds, we applied both conditions for the preparation of the N -(1-acetylpiperidin-4-yl)-N ?-(oxaadamantan -1-yl)urea 184, analogue to APAU. We decided to make first the corresponding isocyanate 185 from the treatment of (2-oxaadamantan -1-yl)amine 120 with triphosgene, a source of the highly toxic 550 Pdiy a, K. J.; Gavad e, S. ; Kard il e, B.; Ti war i, M. ; Bajar e, S.; M an e, M.; Gawar e, V.; Vargh es e , S.; Harel, D.; Kurh ad e, S. Org. Lett. 2012, 14 , 2814 - 2817. 222 CHAPTER 3: sEH inhibition phosgene.551,552,553 Triphosgene has the advantage of being safer than phosgene when working with the substance since it is a solid crystal, as opposed to phosgene that is a gas. Its name came from the idea that triphosgene contains three molecules of phosgene, which are released upon reaction. Thus, the reaction mechanism starts with the attack of the nucleophilic amine to the triphosgene carbonyl, liberating one molecule of phosgene and the carbamate intermediate 186. This intermediate eliminates another molecule of phosgene to give the desired isocyanate 185 in 86% yield (Scheme 84 ). The two in situ- formed molecules of phosgene can react with the starting amine 120. Hence, only a third of the equivalents of triphosgene is enough for the reaction to be efficient. Scheme 84. Formation of an isocyanate via the reaction of an amine with triphosgene. Next, isocyanate 185 was reacted with 1-acetyl-4-aminopiperidine under the same conditions used for the preparation of the former trifluorophenyl ureas. In this manner, urea 184 was produced in 52% yield after pur ification by column chromatography (Scheme 85). Scheme 85. Reaction of oxaadamantyl isocyanate 185 with a commercially available amine. For the second alternative, we proceeded to the synthesis of the corresponding imidazole carboxamide 187 by reacting CDI with 1-acetyl-4-aminopiperidine in 1,2- dichloroethane (Scheme 86).554 The titled compound 187 was obtained in 75% yield, and fully characterized. 551 Sciu to , A. M.; Hurt, H. H. Inhal. Toxicol. 2004, 16, 565 - 58 0. 552 Ichika wa, Y.; Mat su ka wa, Y.; Nish iy ama, T.; I sob e, M. Eur. J. Org. Chem. 2004 , 2004, 58 6 - 591. 553 Davis, M. C.; Dah l, J. E. P.; Car lso n , R. M. K. Synth. Commun. 2008, 38, 115 3 - 1 158. 554 Rawlin g, T.; McDon agh , A. M. ; Tat ta m, B.; Mu rra y , M. Tetrahedron 2012, 68, 6065 - 607 0. Results & Discussion 223 Scheme 86. Preparation of the intermediate 187 through an addition-elimination reaction mechanism. Then, imidazole carboxamide 187 was subjected to the attack of (2-oxaadamantan -1- yl)amine 120 to afford the desired urea 184 in 53% yield, comparable to the previous procedure with triphosgene (Scheme 87). Scheme 87. Synthesis of final urea 184 following the protocol with CDI as the transfer reagent. With these two syntheses, we concluded that both alternatives were comparable in terms of yield. Nevertheless, the procedure with CDI as transfer reagent needed harsher conditions (long reaction times while heating). Accordingly, we applied the triphosgene route, whenever possible, for the preparation of the following ureas. Analogously, oxaahexacyclo derivative 188 was built by means of the preparation of its corresponding isocyanate 189 in quantitative yield from amine 121, and subsequent transformation to urea 188 with the addition of 1-acetyl-4-aminopiperidine in 26% yield (Scheme 88). 224 CHAPTER 3: sEH inhibition Scheme 88. Synthetic sequence for the preparation of urea 188. In the case of the analogues of the sEH inhibitor t-AUCB, the corresponding amine 190 was not commercially available. So, we followed the reported methodologies by Hwang et al. for the construction of the RHS of the molecule. 483,555 The synthetic pathway consisted in the nucleophilic aromatic substitution of fluorobenzonitrile by trans-4- aminocyclohexanol, an d consecutive hydrolysis of nitrile 191 to carboxylic acid 190 under basic conditions in 44% overall yield (Scheme 89 ). The success of the nucleophilic aromatic substitution was due to the presence of an EWG in para-position (nitrile). Scheme 89. Nucleophilic aromatic substitution and basic hydrolysis for the preparation of intermediate amine 190 as its hydrochloride salt. 555 Hwan g, S. H.; Wecksl er, A. T.; Zhan g, G.; Moriss eau , C.; Ngu y en , L. V; Fu, S. H.; Hammo ck, B. D. Bioorg. Med. Chem. Lett. 2013, 23, 37 32 - 37 37. Results & Discussion 225 Once intermediate amine 190 was prepared in sufficient amounts, ureas 192 and 193 were obtained in 24 and 50% yield, respectively, from the corresponding isocyanates 185 and 189 (Scheme 90). Scheme 90. Attack of the primary amine of 190 to each one of the isocyanates to provide final ureas 192 and 193. Special cases were the ring-contracted analogues with general structure V I, the last scaffold to prepare (Fig. 93). Fig. 9 3. Bisnoradamantane ureas to prepare. For the synthesis of these particular ureas, we encountered two handicaps: 1. We envisioned that the corresponding isocyanate 194 should be volatile under the reaction conditions and posterior work-up procedure (Fig. 94). This volatility may be due to the small molecular weight (177.12 Da) and to the lack of strong intermolecular interactions. 226 CHAPTER 3: sEH inhibition Fig. 9 4. We hypothesized the volatility of isocyanate 194. 2. Because of the low-yielding synthetic route to construct the bisnoradamantane scaffold, we had only enough amounts of starting amine 123 to prepare a single derivative. All in all, Ze pursued the s\nthesis oI A3AU?s derivative 195 implementing the procedure that entailed CDI as transfer reagent. In this sense, amine 123 was reacted with the imidazole carboxamide 187 in the presence of triethylamine to furnish final urea 195 in 77% yield (Scheme 91 ). Scheme 91. Synthesis of the last derivative of the series. 2.2 Pharmacological evaluation of new ureas with the selected scaffolds The five new ureas were tested as sEH inhibitors under our o ptimized screening assay (Fig. 95). Results & Discussion 227 Fig. 9 5. New ureas to test as sEH inhibitors. We performed the inhibitory screening assay under the previous conditions for the determination of the IC50 values, using the commercially available t-AUCB as standard (Table 23). 228 CHAPTER 3: sEH inhibition Table 23. Inhibition of the sEH by the new ureas. t-AUCB was used as standard.483,484 Comp. LHS RHS IC50 ? SE (nM) t-AUCB 1.87 ? 0.14 (reported 1.3 nM) APAU reported 7 nM 184 19.8 ? 6.2 188 50.8 ? 11.9 192 13.4 ? 4.0 193 40.6 ? 8.9 195 8.41 ? 0.59 According to the above data, the following conclusions may be drawn for this second batch of ureas: ? Any new scaffold improved the inhibitory activity of the standard t-AUCB under the assay conditions. Results & Discussion 229 ? Among the three different scaffolds tested, bisnoradamantane 195 (IC50 of 8.41 nM) was the most potent inhibitor compared with other structures. ? Regarding the oxa -derivatives, oxaadamantyl ureas 184 and 192 (IC50 of 19.8 and 13.4 nM, respectively) displayed lower IC50 values than oxahexacyclic ureas 188 and 193 (IC50 of 50.8 and 40.6 nM, respectively). ? Between the two different RHSs assayed (when a direct comparison was possible), APAU derivatives showed lower inhibitory potency than t-AUCB derivatives; compare 184 with 192 (IC50 of 19.8 and 13.4 nM, respectively) and 188 and 193 (IC50 of 50.8 and 40.6 nM, respectively). Although showing higher IC50 values than the adamantyl standard, the potent activity of the selected scaffolds as sEH inhibitors underpinned their ability to surrogate the adamantane nucleus in this kind of bioactive molecules. Taking into account the excellent potency of the ring -contracted APAU derivative 195 (IC50 of 8.41 nM), and that the inhibitors bearing the RHS from t-AUCB displayed higher activities, it seems logical that the missing piece of this puzzle is the corresponding ring- contracted t-AUCB derivative 196 (Fig. 96). Unfortunately, there was not enough time during the present thesis to prepare this promising compound. Fig. 9 6. Urea to prepare to combine the best findings from table 23. 2.3 Water solubility, melting points and LipE To further assess the new sEH inhibitors, we aimed to determine some physicochemical properties as in the case of the former series of compounds. Thus, we measured the water solubility, determined the melting points, and calculated the clog P and the LipE in order to compare these structures with the adamantyl standards (Table 24). Worth to mention is that, in the case of compounds 184, 188 and 195, which lack a chromophore group and therefore showed lower absorbance in the HPLC measurement , a mathematical adjustment was required in order to extrapolate the va lues to the calibration curve (see Materials & Methods). 230 CHAPTER 3: sEH inhibition Table 24. Water solubility, melting points, clog P and LipE of the new ureas. t-AUCB was used as standard.483 mp: melting point. ND: not determined. Comp. LHS RHS S ? SD (?g/mL) mp ( ? C) clog P LipE t-AUCB reported > 200 250-255 4.84 3.89 APAU reported > 200 205-206 0.77 7.38 184 232.9 ? 12.1 172-173 -0.37 8.07 188 258.1 ? 17.4 164-166 -1.41 8.70 192 72.1 ? 2.9 255-257 3.70 4.17 193 138.6 ? 3.5 254-256 2.67 4.72 195 295.9 ? 26.6 165-167 0.69 7.39 The data shown in table 24 revealed interesting features: ? Although we could not afford to determine the water solubility of the standards under our procedure, all the prepared ureas displayed good solubility values, better suited than the previous ones for a possible pharmaceutical. Results & Discussion 231 ? Compound 195 showed the highest water solubility (295.9 ?g/mL), whereas tAUCB derivatives 192 and 193 exhibited the worst results (72.1 and 138.6 ?g/mL, respectively). This is in accordance with t he high melting points for these two ureas. Presumably, the extraordinary energy lattices of the crystal structure of these compounds affects significantly their water solubility. ? clog P values extremely decreased when 1-acetylpiperidine was placed as the RHS (compounds 184, 188 and 195), to a level that they were no longer considerable as drug candidates (-1.41 to 0.69). Because of this, LipE values were remarkably high for these type of structures. ? t-AUCB derivatives 192 and 193 shoZed a more ?drug-liNe? proIile with acceptable water solubilities, optimum clog P values, and appropriate LipE. Compiling the data obtained so far, we have identified some scaffolds as excellent surrogates of the adamantane nucleus in sEH inhibitors, a few of them with remarkable physicochemical properties. From this point, the next step was the evaluation of these new sEH inhibitors in in vitro studies, so as to assess their ability to inhibit the sEH enzyme in a cellular level. Because of the close relationship of the sEH in the development of the endoplasmic reticulum (ER) stress (vide infra ), we aimed to test a few of our more promising compounds in ER stress studies. Consequently, we selected three compounds that displayed a perfect balance between inhibitory activity and clog P (expressed in the LipE) and that entailed easy synthetic routes for the preparation of the scaffolds. The latter point was envisioning that these structures would progress throughout the drug discovery process. The selected molecules are disclosed in figure 97. Fig. 9 7. Selected compounds for in vitro studies. 3. ER stress amelioration with selected sEH i nhibitors. In vitro studies The ER is a membranous network that functions in the synthesis, folding and maturation of newly secretory and membrane proteins, and is highly responsive to nutrients and energy status of the cell. When certain pathological stress conditions disrupt ER homeostasis, such as nutrient excess or deficiency, stress, inflammation, hypoxia or infection, the folding capacity of the ER is exceeded and consequently misfolded proteins 232 CHAPTER 3: sEH inhibition accumulate and lead to the so-called ER stress.556 For instance, obesity is a chronic stimulus for ER stress in peripheral tissues, and is a core mechanism involved in triggering insulin resistance and T2DM.557 In addition, obese individuals exhibit increased expression of multiple markers of ER stress.558 On the other hand, ER stress also provides several links with the emergence of inflammatory processes, mainly by activation of both c-Jun amino- terminal kinase (JNK) and inhibitor of ?? kinase (IKK), both related to the development of insulin resistance.559,560 Taken together, the effector arms of ER stress and the associated stress responses are tightly linked to inflammatory pathways at many levels that are crucial for insulin action and metabolic homeostasis. Thus, understanding the role of ER stress may be of therapeutic potential for the treatment of metabolic diseases, specially obesity and T2DM.301,561 In addition, ER stress is a key piece to the pathogenesis of other diseases such as neurodegenerative disorders and cancer. 440 In response to the accumulation of unfolded proteins in the ER, a sequential process takes place; the rate of general translation initiation is attenuated, the expression of ER resident protein chaperones and protein foldases is induced, the ER compartment proliferates, and ER-associated degradation (ERAD) is activated to eliminate irreparable misfolded proteins.556,562 If these survival mechanisms fail to facilitate ER homeostasis, ER- stress triggers apoptotic processes.563,564 The adaptive mechanisms to mitigate ER stress and to restore homeostasis are collectively termed as the unfolded protein response (UPR), which consists of a signal transduction system linking the ER lumen with the cytoplasm and nucleus.565 UPR signalling consists of three branches initiated by the ER transmembrane proteins PKR-like ER-regulated kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6).559,566,567 These sensor proteins respond to changes in protein folding status in the ER and convey information to activate distinct and sometimes overlapping pathways, leading to diverse developmental and metabolic processes. PERK phosphorylates the ?-subunit of eukaryotic translation initiation factor 2 (eIF2) at Ser51, leading to rapid and transient attenuation of protein 556 Schr? d er, M. ; Kau f man , R. J. Annu. Rev. Biochem. 2005, 74 , 739 - 7 89 . 557 Ozcan , U.; Cao, Q.; Yil maz , E.; Lee, A. H. ; Iwak o sh i, N. N.; Ozd elen , E.; Tun cman , G. ; G?r g? n , C.; Gli mch er, L. H.; Hota misli gil, G. S. Science 2004, 306, 457 - 46 1 . 558 Sharma, N. K. ; Das, S. K.; Mo n d al, A. K.; Hack n ey , O. G.; Ch u , W. S.; Kern , P. A.; Raso u li, N .; Spen cer, H . J.; Yao - Bor en ga s ser, A.; Elb ein , S. C. J. Clin. Endocrinol. Metab. 2008, 93, 4532 - 45 41 . 559 Humm asti, S. ; Hota misl igil, G. S. Circ. Res. 2010, 107, 5 79 - 5 91. 560 Hota misligi l, G. S. Int. J. Obs. 2008, 32, S52 - S 54. 561 Eizirik , D . L.; Card o zo , A . K.; Cno p , M . Endocr. Rev. 2008, 29 , 42 - 61 . 562 Wu, J.; Kau fman , R. J. Cell Death Differ. 2006, 13, 374 - 38 4. 563 Zin szn er, H.; Kuro d a, M.; Wan g, X.; Bat ch var o va, N.; Ligh tf o o t, R. T.; Re mo tt i, H.; Ste ven s, J. L.; Ron , D . Genes Dev. 1998, 12, 9 82 - 995 . 564 Nish ito h , H.; Matsu za wa, A.; Tob iu me, K.; Saegu sa, K.; Tak ed a, K.; In ou e, K.; Hori, S.; Kak izuka , A.; Ich ijo , H. Genes Dev. 2002, 16, 13 45 - 1355 . 565 Pluq u et, O.; Pou rtier, A.; Abb ad ie, C. Am. J. Physiol. Cell Physiol. 2015 , 308, C41 5 - C425. 566 Ron , D.; Walter, P. Nat. Rev. Mol. Cell Biol. 2007 , 8, 519 - 52 9 . 567 Hota misligi l, G. S. Cell 2010 , 140, 900 - 917. Results & Discussion 233 synthesis to decrease protein influx into the ER lumen .568,569 eIF2? promotes the activating transcription factor 4 (ATF4), which induces genes involved in ER function, ER-induced apoptosis, and an inhibitory feedback to prevent hyperactivation of the UPR, among other effects.570,571 IRE1 activation leads to the unconventional splicing of X-box -binding protein 1 (XBP1) mRNA, leading to the synthesis of a nuclear XBP1 form that induces the transcription of genes encoding ER chaperones and ERAD.572,573,574 The third canonical branch includes ATF6, which, upon ER stress, traffics to the Golgi apparatus, where it is cleaved to liberate a fragment that translocates to the nucleus to induce genes encoding ER chaperones and ERAD functions, independently or synergistically with XBP1 and ATF4.575,576 The master initiator of the UPR is the immunoglobulin-heavy-chain-binding protein or BiP, which is a negative effector of the UPR, known as well as 78 kDa glucose-regulated protein (GRP78). In physiological states, this chaperone is known to be expressed constitutively and binds to the luminal domains of PERK, IRE1, and ATF6 in their inactive forms.577 Under conditions that promote accumulation of misfolded proteins, GRP78 is overexpressed and preferentially binds to hydrophobic regions of unfolded/misfolded proteins that accumulate in the ER lumen.578 Consequently, BiP is sequestered and dissociates from the ER stress sensors to allow their signalling.579,580,581 As stated before, chronic or severe ER stress activates the UPR leading to apoptosis. Persistent activation of PERK-eIF2?-ATF4 and ATF6 pathways culminates in the induction of the CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP or GADD153), which up-regulates apoptosis-related genes.563,582,583 568 Shi, Y.; Vat te m, K. M.; Soo d , R.; An, J. ; Lia n g, J.; Stra m m, L. ; Wek, R. C. Mol. Cell. Biol. 1998 , 18, 7 499 - 7509 . 569 Novoa, I.; Zh an g, Y.; Zen g, H.; Jun greis, R. ; Hard in g, H. P.; Ron , D. EMBO J. 2003 , 22, 1180 - 1187 . 570 Hard in g, H. P.; Y. Zh an g, H. ; Z en g, I. ; Novo a, P. D. ; L u, M. ; Calfon , N. ; Sad ri, C. ; Yun , B. ; Po p ko , R. ; Pau le s Sto jd l, D. F .; Bell, J. C.; Hett ma n n , T.; Leid en , J. M.; Ron , D. Mol. Cell. 2003, 11, 619 - 633 . 571 Hiramat su , N.; Me s sah , C.; Ha n , J.; LaVail, M. M.; Kau fman , R. J.; Lin , J. H. Mol. Biol. Cell 2014 , 25, 1411 - 1420. 572 Sidrau ski, C.; Walter, P. Cell 1997, 90, 1031 - 10 39 . 573 Yosh id a, H.; Matsu i, T. ; Yama mo to , A.; Okad a, T.; Mori, K. Cell 2001, 107, 88 1 - 8 91. 574 Calfo n , M. ; Zeng, H.; Uran o , F.; Till, J. H.; Hub b ar d , S. R.; Hardin g, H. P.; Clar k, S. G.; Ron , D. Nature 2002, 415, 92 - 96 . 575 Ye, J. ; Ra wso n , R. B. ; Ko mu ro , R.; Chen , X.; Dav ?, U. P.; P ry we s, R.; Bro wn , M. S. ; G old st ein , J. L. Mol. Cell 2000, 6, 13 55 - 1364 . 576 Okad a, T.; Yosh id a, H.; Akaz awa, R.; Negi sh i, M.; Mori, K. Biochem. J. 2002, 366, 585 - 594. 577 Bert o lot ti, A.; Zh an g, Y.; Hen d ersh o t, L. M.; Hard in g, H. P.; Ron , D. Nat. Cell Biol . 2000, 2, 326 - 3 32. 578 San d erso n , T.; Ga llaway , M.; Kumar , R. Int. J. Mol. Sci. 2015 , 16, 7133 - 7 142. 579 Shen , J.; Chen , X.; Hen d ersh o t, L.; Pry wes, R. Dev. Cell 2002 , 3, 99 - 111. 580 Liu , C. Y.; Wo n g, H. N.; Sch au erte, J. A. ; Kau fman , R. J. J. Biol. Chem . 2002, 277, 183 46 - 1 8356. 581 Kimat a, Y.; Kimat a, Y. I. ; Shim i zu , Y.; Abe, H.; Farca san u , I. C.; Tak eu ch i, M.; Ros e, M. D.; Koh n o , K. Mol. Biol. Cell 2003 , 14, 2559 - 25 69 . 582 Ma , Y.; Bre w er , J . W.; Di eh l , J . A .; Hen d ersh o t , L . M . J. Mol. Biol. 2002, 318 , 1351 - 136 5 . 583 Rutk o wski , D . T, .; Arn o ld , S . M.; Mill er , C . N.; Wu , J.; Li , J.; Gun n iso n , K . M.; Mori , K.; Sad ighi Akh a , A . A.; Rad en , D.; Kau f man , R . J . PLoS Biol. 2006, 4, e374. 234 CHAPTER 3: sEH inhibition Figure 98 summarizes what has been discussed so far. Fig. 98. UPR activation in response to the ER stress. The machinery concerning ER stress is thus intricate and complex. Although s till unknown to some extent, different proteins have been identified to play a trivial role in the development of the ER stress. Among them, activating transcription factor 3 (ATF3) is an ER stress marker whose expression is increased in such conditions. It is related to insulin signalling and lipogenesis.584 Over the multiple studies that have been performed in relation to the ER stress, different ER stress-inducers have been identified. Among them, the saturated fatty acid palmitate,585,586,587 followed by thapsigargin (by depletion of Ca2+ from the ER), tunicamycin (by blockade of protein glycosylation in the ER)588 and borrelidin.589 On the other hand, several substances have emerged as restorers of ER stress, such as unsaturated fatty acids 584 Koh , I.; Lim, J. H.; Jo e, M. K.; Kim, W. H.; Jun g, M. H.; Yoon , J. B.; Son g, J. FEBS J. 2010, 277, 2304 - 2317. 585 Guo , W.; Won g , S.; Xie , W.; L ei , T.; Lu o , Z . Am. J. Physiol. Endocrinol. Metab. 2007, 293, E576 - E 586 . 586 Gwiaz d a, K. S.; Yan g, T. L.; Lin , Y.; Joh n so n , J . D. Am. J. Phisiol. Endocrinol. Metab. 2009, 296 , E690 - 701 . 587 Wei , Y .; Wan g , D.; Gen til e , C . L.; Paglias so tt i , M . J . Mol. Cell. Biochem. 2009, 331, 31 - 4 0. 588 Sriva sta va, R. K. ; Sol lot t, S. J.; Kh an , L.; Han sfo rd , R .; Laka tt a , E. G.; Lon go, D. L. Mol. Cell Biol. 1999, 19, 5659 - 5674 . 589 Sidh u , A.; Miller, J. R.; Trip at h i, A.; Garsh o tt , D. M.; Brown ell, A. L.; Chiego , D. J. ; Are van g, C. - J. ; Zeng, Q .; Jack so n , L. C.; Bech l er, S. A .; Callagh an , M. U.; Yoo, G. H. ; Seth i, S.; Lin , H. - S.; Cal lagh an , J. H.; Tamay o - Ca stillo, G.; Sher man , D. H.; Kau fman , R. J. ; Frib l ey , A. M. ACS Med. Chem. Lett. 2015, ASAP . DOI: 10.1021 /acs med ch eml ett .5b 0 0133. Results & Discussion 235 (oleate and linolate),590,591 the PPAR? agonist fenofibrate,592 and the chemical chaperon 4- phenyl butyric acid (PBA),593 and tauroursodeoxy cholic acid (TUDCA).594 In 2013, sEH was identified as a key regulator of the ER stress.595 Epoxy fatty acids have proven to be upstream modulators of ER stress pathways.596,597 In consequence, attenuation or inhibition of sEH leads to an amelioration of the ER stress, therefore reasserting its potential as a therapeutic target for obesity-related comorbidities. Taken together these studies and due to the wide expert ise of the research group of Dr. Manuel V?zquez Carrera in the study of the ER stress in skeletal muscle and in animal models of insulin resistance,590,598 we envisioned that our sEH inhibitors would be able to attenuate the ER stress when produced by a stimuli. For our studies, human hepatocytes, from the cell line Huh -7,599 were chosen because of their availability and ease to maintain for cell cultures, coupled with the fact that sEH is widely expressed in liver , as seen in the introductory part of this chapter. Cells were treated with palmitate prior to conjugation of this fatty acid with BSA , as previously described by the group.590 Based on our experience in ER stress and the reported studies, we proceeded to run the in vitro assays in a single final concentration of each inhibitor (170, 176 and 192) and palmitate (1 ?M/L and 0.5 mM /L, respectively) during 48 hours. After the treatment, samples were recovered for RNA and total protein extraction. We used again urea 109 as standard for comparison. Worth to note is the fact that adamantyl urea 109 has not been tested in human cell cultures prior to our studies. To assess the resolution of ER stress with our sEH inhib itors, we examined their effects on the expression of the ER stress markers Bip/Grp78 , C hop and Atf3 . Levels of mRNA of the three genes were determined by Real Time-PCR with GAPDH as housekeeping gene. Human Huh -7 hepatocytes exposed to 0.5 mmol/L palmitate showed an increase in Bip/Grp78 , C hop and Atf3 mRNA levels compared with cells exposed only to BSA (Fig. 99). Interestingly, co-incubation of cells with palmitate (0.5 mmol/L ) and each one of the inhibitors (1 ?mol/L ) considerably blocked the effects of the saturated fatty acid, with a 590 Salvad ? , L.; Coll, T. ; G? mez - Foix, A. M.; Sal mer? n , E.; Bar ro so , E.; Palomer, X. ; V?z q u e z - Carrera , M. Diabetologia 2013, 56, 1372 - 1 382. 591 Maru y ama, H.; Tak ah ashi, M.; Sekimot o , T. ; Shimad a, T. ; Yo ko su ka , O. Lipids Health Dis. 2014 , 13, 1 - 8. 592 Rah man , S. M.; Qad ri, I.; Jan s sen , R. C.; Fried man , J. E. J. Lipid Res. 2009, 50, 21 93 - 2202. 593 Xiao , C.; Giacca, A. L ewi s, L. F. Diabetes 2011, 60, 918 - 924. 594 Ozcan , U.; Yil maz , E.; Ozcan , L.; Furu h ashi, M.; V aillan cou rt, E.; Smith , R. O.; G?r g? n , C. Z.; Hota mislig il , G. S. Science 2006, 313, 1137 - 1140. 595 Bett aieb, A .; Naga ta , N.; Abo u b ech ar a, D.; Cha h ed , S. ; Mor iss eau , C.; Ham mo ck, B. D.; H aj , F. G. J. Biol. Chem. 2013, 288, 14 189 - 141 9 9. 596 L?pez - Vicar io, C.; Alcar az - Qu iles, J.; Garc?a - Alon so , V.; Riu s, B.; H wan g, S. H.; Tito s, E.; Lop at egi, A. ; Hammo ck, B. D. ; Arro y o , V.; Cl?ria , J. Proc. Natl. Acad. Sci. USA 2015, 112, 536 - 54 1. 597 Inceo glu, B.; Bett ai eb , A.; Tri n d ad e da Silva, C. A.; Le e, K. S. S.; Haj , F. G.; Ham mo ck, B. D. Proc. Natl. Acad. Sci. U SA 2015, 112, 908 2 - 908 7. 598 Salvad ? , L.; Palomer, X. ; Barro so , E.; V?z q u ez - Carr era , M. Trends Endocrinol. Metab. 2015, 26 , 438 - 4 4 8. 599 Huh - 7 cell li n e we b site. htt p : //h u h 7.com/ (acc e ss ed on 18 th Septe mb er 20 15). 236 CHAPTER 3: sEH inhibition significant reduction of the mRNA levels for the three markers. This trend was more remarkable for Atf3 (Fig. 99C). A B C Fig. 9 9. Assessment by quantitative real-time RT-PCR of mRNA abundance of Bip/Grp78 (A), C hop (B) and Atf3 (C). Huh -7 hepatocytes were incubated for 48 h in the absence (Control, CT) or in the presence of 0.5 mmol/L palmitate (PAL ) and the compounds tested. Data are presented as the mean ? SD (n = 6 per group). ***: P < 0.001 vs control. # : P < 0.05 , # # : P < 0.01 and ## # : P < 0.001 vs palmitate-exposed cells . In agreement with the changes in C hop mRNA levels, CHOP protein levels were only increased in cells exposed to palmitate (Fig. 100). Results & Discussion 237 A B Fig. 10 0. Levels of protein of CHOP (A) and their quantification (B). Huh -7 hepatocytes were incubated for 48 h in the absence (Control, CT) or in the presence of 0.5 mmol/L palmita te (PAL) and the compounds tested. Data are presented as the mean ? S D (n = 3 per group). ***: P < 0.001 vs control. # # : P < 0.01 and ## # : P < 0.001 vs palmitate-exposed cells. The reduction of the mRNA and protein levels of BiP, CHOP and ATF3 markers resulting from the sEH inhibition from our three tested compounds prompted the following conclusions: ? In general, sEH inhibition at a low concentr ation provided a remarkable protection against palmitate-induced ER stress and reduced the levels of theses markers to those present in control cells. ? Except for compound 192, new inhibitors 170 and 176 led to an extremely significant reduction of the ER stress comparable with the adamantyl standard 109. ? Addition of compound 192, although providing less benefit than the other tested compounds, resulted in a very significant recovery of the ER stress. ? Only in mRNA levels, compound 170 showed a better behaviour preventing palmitate-induced ER stress than the adamantane analogue 109. ? The positive outcomes of these in vitro studies showed that the four sEH inhibitors are able to cross cell membrane and are not cytotoxic. As a whole, the findings of this study show that sEH inhibition contributes to the prevention of palmitate-induced ER stress, and that our scaffolds compare well with the adamantane nucleus. 238 CHAPTER 3: sEH inhibition To our delight, these experiments with sEH inhibitors in Huh -7 cell line are, to date and to the best of our knowledge, the first in vitro studies concerning sEH and ER stress performed under this new protocol. Conclusi ons Conclusions 241 This chapter involves the synthesis of 20 new ureas with 15 different adamantane-like polycycles through a scaffold-hopping approach; none of them had been previously described in the literature. The new structures have been thoroughly characterized by spectroscopic and analytical means. None of the scaffolds had been previously applied in the design and synthesis of sEH inhibitors. The pharmacological evaluation was carried out, and entailed the optimization of the assay conditions, followed by the determination of the IC50 values of each new compound. Furthermore, an unexplored method for the water solubility measurement was applied, and the values of solubility were used for the assessment of each scaffold. Finally, the lipophilic ligand efficiency of each new sEH inhibitor was calculated from the IC 50 and clog P data. As the main conclusions of this chapter, in chronological order: 1. All the 15 polycyclic scaffolds were potent sEH inhibitors, with IC 50 values ranging from 0.80 to 50.8 nM. 2. Three different structures were identified with higher inhibitory activities than the adamantanyl standards 108 and 109 (IC50 of 7.74 and 4.04 nM, respectively) (Fig. 101). Fig. 101 . Most potent compounds found in this chapter. 3. According to the results obtained from water solubility and LipE, and taking into account the ease in the synthesis of each structure, three scaffolds were selected for the preparation of optimized sEH inhibitors (Fig. 102 ). Fig. 102 . Selected scaffolds as adamantane surrogates for the second batch of compounds. 4. Five new ureas were prepared following described methods, in order to test their i n vi tro potency against human sEH. Albeit showing higher IC 50 values than the standard t -AUCB, all of them were potent sEH inhibitors (from 8.41 to 50.8 nM). 242 CHAPTER 3: sEH inhibition 5. After the determination of their water solubility and LipE, these scaffolds have proven to be efficient surrogates of the adamantane ring for this specific target, with more appropriate physicochemical properties than the adamantane group. 6. I n vi tro cellular studies have demonstrated their ability to cross cell membrane and to attenuate ER stress induced by palmitate, without promoting cell death. These experiments with the human Huh -7 cell line are, to date, the first ever reported that link sEH and ER stress under this protocol. This work has been focused on investigating structure-activity relationships of adamantane-like derivatives as human sEH inhibi tors, which provided an important base for developing valuable compounds that are not only highly potent inhibitors but show improved physicochemical properties. As a general conclusion of this dissertation, we have demonstrated that the adamantane nucleus is not necessarily the perfect lipophilic bullet, and that a fine-tuning is required in order to optimize the binding mode of the molecule for each specific target as well as to adjust its physicochemical properties. Exploration around adamantane-like scaffolds for the discovery of improved drug candidates is still an underexploited research area. MATERIALS & METHODS General Methods 245 General Methods Melting points were determined in open capillary tubes with a MFB 59510M Gallenkamp or a B?chi B ? 540 melting point apparatuses. 300 MHz 1H NMR spectra, 400 MHz 1H /100.6 MHz 13C NMR spectra, and 500MHz 1H /125.7 MHz 13C NMR spectra were recorded on Varian Gemini 300, Varian Mercury 400, and Varian Inova 500 spectrometers, respectively. The chemical shifts are reported in ppm (? scale) relative to internal tetramethylsilane, or to solvent peak, and coupling constants are reported in Hertz (Hz). The used abbreviations were: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; or combinations thereof. IR spectra were run on FTIR Perkin-Elmer Spectrum RX I spectrophotometer using potassium bromide (KBr) pellets or attenuated total reflectance (ATR) technique. Absorption values are expressed as wavenumbers (cm -i); only significant absorption bands are given. The GC/MS analyses were carried out in an inert Agil ent Technologies 5975 gas chromatograph equipped with a DB-5MS (30 m x 25 mm) capillary column with a stationary phase of phenylmethylsilicon (5% diphenyl -9% dimethylpolysoloxane), using the following conditions: initial temperature of 50 ?C ( 1 min), with a gradient of 15 ?C/min up to 300 ?C, and a temperature in the source of 230 ?C. solvent decay of 3 minutes and pressure of 7.5 psi. The electron impact (70 eV) or chemical ionization (CH 4) techniques were used. Only significant ions are given, those with higher relative ratio, except for the ions with higher m/z values. The accurate mass anal\ses Zere carried out at Unitat d?(spectrometria de Masses dels Centres Cient?fics i Tecnol?gics de la Universitat de Barcelona (CCiTUB), Faculty of Chemistry, using a LC/MSD -TOF spectrophotometer. The elemental analyses were carried out in a Flash 1112 series Thermofinnigan elemental microanalyzator (A5) to determine C, H, and N, and in a tiroprocessor Methrom 808 to determine Cl, at the Servei de Microan?lisi of IIQAB (CSIC) of Barcelona. To concentrate solvents in vacuo a B?chi GKR-50 rotavapor was used. Column chromatography was perfomed on either silica gel 60 ? (35-70 mesh, SDS), or on aluminium oxide, neutral, 60 ? (50 -200 ?m, Brockmann I). Thin-layer chromatography was performed with aluminium?backed sheets with either silica gel 60 F254 or aluminum oxide 60 ?, and spots were vi sualized with UV light, 1% aqueous solution of KMnO4 and/or iodine. All new compounds that were subjected to a pharmacological evalu ation, possessed a purit\ ?  as evidenced b\ their anal\tical data iI possible 246 Materials & Methods Solvent purification was carried out following the procedures described in: Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals , 4th Edition, Butterworth- Heinemann: Oxford, 1996. The NMR data and the synthetic procedure of all the compounds synthesized for the first time in our laboratory are included in the current manuscript. Regarding the compounds described previously in the literature, the synthetic procedure is described along with the reference corresponding with their preparation. A complete characterization of all the new compounds synthesized in this thesis, was carried out including 1H and 13C, IR, elemental analysis or accurate mass and GC/MS. All 1H and 13C NMR signlas were assigned by reason of homocorrelation 1H / 1 H (COSY and NOESY) and heterocorrelation 1H / 13C (HSQC) experiments. C hapter 1 CHAPTER 1 249 Preparation of 5,6,8,9 -tetrahydro -5,9 -propanebenzocycloheptane -7,11 -dione, 27 and 7,11 -epoxi -6,7,8,9 -tetrahydro -5,9 -propane-5H-benzocycloheptane -7,11 -diol, 28 168 a) Tetraester?s Iormation To a solution of o-phthaldialdehyde (9.80 g, 73.1 mmol) and dimethyl 1,3- acetonedicarboxylate (25.9 g, 149 mmol) in m ethanol (200 mL) were added 15 drops of diethylamine, and the solution was heated at reflux for 1.5 hours. Then 12 drops more of diethylamine were added and the mixture was cooled to 4 ?C overnight. The resulting precipitated was filtered in vacuo and washed with cold methanol (25 mL) to give the intermediate tetraester as white crystals (22.2 g, 68% yield). b) Preparation of the diketone 27 and its hydrate 28: To a round bottom flask containing the intermediate tetraester prepared in the last step (22.2 g, 49.8 mmol), glacial acetic acid (125 mL) and conc. HC l (35 mL) were added and the mixture was heated at reflux for 12 hours. The acids were then removed under vacuo and the resulting solid was digested with diethyl ether (150 mL) for 15 minutes and cooled to 4 ?C overnight. Filtration in vacuo gave a mixture of diketone 27 and hydrate 28 with a 1:3 ratio as a white solid (10.5 g). Dehydration of the mixture by a Dean -Stark system with toluene (250 mL) as the solvent gave the pure diketone 27 (9.8 g, 93% yield), whose spectroscopic data matched with those previously published. Preparation of 5,6,8,9 -tetrahydro -5,9 -propanebenzocycloheptane -11-ene-7-one, 25 165 A suspension of sodium hydride, 60% dispersion in mineral oil (1.00 g, 25.17 mmol) in anh. DMSO (50 mL) was heated to 75 ?C for 45 minutes under N2 atmosphere. After the reaction mixture was tempered, a solution of triphenylmethylphophonium iodide (10.55 g, 25.17 mmol) in anh. DMSO (60 mL) was added and the resulting yellow solution was stirred at room temperature for 20 minutes. Then a suspension of diketone 27 (4.31 g, 20.14 mmol) in anh. DMSO (50 mL) was added and the obtained solution was heated to 75 ?C overnight. The resulting black solution was allowed to cool to room temperature and 250 Materials & Methods then poured into water (150 mL). Hexane (50 mL) was added and the phases were separated. The aqueous phase was extracted with further hexane (2 x 50 mL) and the combined organic phases were washed with brine (50 mL), dried over anh. Na2SO4, filtered and concentrated in vacuo to give a white solid (3.93 g). Purification by packing the solid with silica gel and extracting with 10% petrol eum ether/diethyl ether mixture gave 25 (3.34 g, 84% yield) as a white solid. The spectroscopic data were identical to those previously published. Preparation of 2-chloro-N-(9 -hydroxy -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)acetamide , 34 To a solution of enone 25 (2.09 g, 9.86 mmol) in DCM (15 mL) was added chloroacetonitrile (0.62 mL, 9.86 mmol) and the mixture was cooled to 0 -5 ?C with an ice bath. Then conc. H 2SO4 (0.79 mL, 14.82 mmol) was added dropwise without reaching a temperature greater than 10 ?C. After the addition, the reaction mixture was allowed to reach room temperature and stirred overnight. Then to the resulting solution was added ice (25 g) and the mixture was stirred at room temperature for few minutes. DCM (30 mL) was added, the phases were separated and the aqueous phase was extracted with further DCM (2 x 30 mL). The combin ed organic phases were dried over anh. Na2SO4, filtered and evaporated in vacuo to give a white solid (2.68 g). Purification by column chromatography (Al2O3, 0-5% m ethanol/DCM) gave 34 (1.46 g, 49% yield) as a white solid. Analytical and spectroscopic data of compound 34: Melting point: 184 ? 185 ?C. IR (ATR) ?: 3300 ? 2800 (3315, 3247, 3217, 3065, 3021, 2925, 2851), 1911, 1664, 1560, 1494, 1441, 1413, 1361, 1300, 1224, 1202, 1151, 1107, 1037, 911, 757, 668, 571, 512, 471 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.78 [d, J = 12.8 Hz, 2 H, 10(13) -H b], 1.97 [m , 2 H, 10(13) - H a ], 2.04 [d, J = 12.8 Hz, 2 H, 6(12) - H b], 2.12 (s, 2 H, 8 -H 2), 2.19 [ m, 2 H , 6(12)- H a ], 3.21 [t, J = 6.4 Hz, 2 H, 5(11) -H], 3.93 (s, 2 H, C H 2Cl), 6.36 (s, 1 H, NH), 7.08 [m, 2 H, 1(4)-H], 7.13 [m, 2 H, 2(3)-H]. 13C-NMR (100.6 MHz, CDCl 3) ?: 38.2 [CH 2, C6(12)], 39.9 [CH, C5(11)], 42.4 [CH 2, C10(13)], 42.8 (CH 2, CH 2Cl), 47.9 (CH 2, C8), 57.3 (C, C7), 70.7 (C, C9), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 144.9 [C, C4a(C11a)], 164.6 (C, CO). CHAPTER 1 251 MS (EI), m /z (%); significant ions: 307 (10), 305 (M? + , 30), 270 [(M -35Cl) + , 12], 212 (100), 197 (17), 194 (22), 179 (26), 169 (13), 155 (58), 142 (25), 129 (32), 115 (26), 91 (7), 77 (9), 65 (2), 55 (4), 49 (4). HRMS -ESI+ m /z [ M +H ] + calcd for [C 17H 20ClNO2+ H ] + : 306.1255, found: 306.1249. Preparation of 9-amino-5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-ol hydrochloride , 46 ?HCl To a solution of chloroacetamide 37 (710 mg, 2.32 mmol) in abs. ethanol (40 mL) were added thiourea (0.212 g, 2.79 mmol) and glacial acetic acid (1.4 mL) and the mixture was heated at reflux overnight. The resulting suspension was then tempered to room temperature, water (20 mL) was added and the pH adjusted to ~12 with 5 N NaOH solution. DCM (20 mL) was added, the phases were separated and the aqueous phase was extracted with further DCM (2 x 20 mL). The combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give 46 as a white solid. Its hydrochloride was obtained by adding an excess of Et 2O?HCl to a solution of the amine in DCM, followed by filtration of the white precipitated (602 mg, quantitative yield). The analytical sample was obtained by crystallization with methanol/d iethyl ether. Analytical and spectroscopic data of compound 46?HCl: Melting point: > 315 ?C (dec.). IR (ATR) ?: 3273, 2902, 2872, 2638, 2569, 2088, 1633, 1531, 1493, 1441, 1355, 1314, 1272, 1106, 1094, 1015, 972, 902, 764, 640, 580 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.75 [d, J = 12.5 Hz, 2 H, 6(12 )-H b], 1.81 [d, J = 13 .0 Hz, 2 H, 10(13 )- H b], 1.89 (s, 2 H, 8 -H 2), 1.98 [ m, 2 H, 6(12 )- H a ], 2.05 [ m, 2 H, 10(13 )- H a ], 3.30 [ tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 7.13 (m, 4 H, Ar-H). 13C-NMR (125.7 MHz, CD 3OD) ?: 39.0 [CH 2, C10(13)], 40.7 [CH, C5(11)], 42.8 [CH 2, C6(12)], 47.9 (CH 2, C8), 57.5 (C, C7), 70.9 (C, C9), 128.3 [CH, C2(3)], 129.3 [CH, C1(4)], 145.7 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 229 (M ? + , 100), 214 (10), 196 (C15H 16? + , 10), 187 (37), 186 (16), 172 (14), 171 (11), 170 (23), 168 (18), 158 (11), 157 (20), 156 (29), 155 (12), 144 (49), 143 (47), 129 (28), 128 (32), 115 (26), 110 (33), 96 (29). 252 Materials & Methods Elemental analysis: Calculated for C15H 20ClNO: C 67.79% H 7.58% Cl13.34% N 5.27% Calculated for C15H 20ClNO?0.20H 2O: C 66.87% H 7.63% Cl 13.16% N 5.20% Found: C 66.87% H 7.71% Cl 13.08% N 4.98% Preparation of 9-(d imethylamino) -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-ol hydrochloride, 50?HCl To a solution of amine 46 (250 mg, 1.09 mmol) in methanol (8 mL) were added formaldehyde (0.25 mL, 37% wt in aqueous solution, 3.32 mmol), glacial acetic acid (0.23 mL) and sodium cyanoborohydride (206.2 mg, 3.12 mmol) and the mixture was stirred at room temperature for 7 hours. Then further NaBH 3CN (206.2 mg, 3.12 mmol) and formaldehyde (0.25 mL, 37% wt in aqueous solution, 3.32 mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was then evaporated to dryness in vacuo and the resulting residue partitioned between water (5 mL) and EtOAc (5 mL). The pH was adjusted to ~12 with 5 N NaOH solution and the phases were separated. The aqueous phase was extracted with further EtOAc (2 x 5 mL) and the combined organic layers were dried over anh. Na2SO4, filtered and concentrated in vacuo. The residue was taken in DCM and the amine 50 was precipitated as its hydrochloride (104.9 mg, 33% yield) by adding an excess of Et 2O?HCl. The analytical sample was ob tained by crystallization with methanol/d iethyl ether. Analytical and spectroscopic data of compound 50?HCl: Melting point: 241-242 ?C. IR (ATR) ?: 3424, 3224, 2956, 2855, 2533, 2429, 1665, 1487, 1451, 1412, 1361, 1348, 1310, 1211, 1183, 1141, 1094, 1078, 990, 978, 969, 908, 769, 702, 621, 591 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.74 [d, J = 14.0 Hz, 2 H, 6(12 )-H b], 1.96-2.02 [ m, 6 H, 10(13)-H b, 8-H 2, 6(12)-H a ], 2.14 [ m, 2 H, 10(13 )-H a ], 2.83 (s, 6 H, N -CH 3), 3.38 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 7.14 (s, 4 H, Ar -H). 13C-NMR (125.7 MHz, CD 3OD) ?: 34.1 [CH 2, C10(13)], 37.5 (CH 3, N-CH 3), 40.5 [CH, CHAPTER 1 253 C5(11)], 42.6 [CH 2, C6(12)], 45.5 (CH 2, C8), 69.0 (C, C7), 71.6 (C, C9), 128.3 [CH, C2(3)], 129.3 [CH, C1(4)], 145.6 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 257 (M?+ , 100), 242 (16), 240 (20), 215 (43), 214 (14), 198 (14), 187 (54), 184 (19), 172 (18), 155 (16), 138 (18), 129 (19), 128 (22), 127 (12), 124 (23), 115 (19), 85 (17). Elemental analysis: Calculated for C17H 24ClNO: C 69.49% H 8.23% Cl 12.07% N 4.77% Calculated for C17H 24ClNO?0.50H 2O: C 67.42% H 8.32% Cl 11.71% N 4.63% Found: C 67.70% H 8.51% Cl 11.80% N 4.44% Preparation of 9-bromo-5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine, 47 To a solution of amine 46 (700 mg, 3.06 mmol) in toluene (23mL) and few drops of DCM was added thionyl bromide (7 mL, 90.05 mmol). The resulting orange solution was stirred at room temperature for 1.5 hours. The reaction mixture was then concentrated to dryness in vacuo. Toluene (50 mL) was added and the resulting solution concentrated in vacuo. The procedure was repeated five times more until it was obtained an orange solid (1.33 g). The crude was partitioned between DCM (15 mL) and saturated aqueous NaHCO 3 solution (15 mL) and the phases were separated. The aqueous layer was extracted with further DCM (2 x 15 mL) and the combined organic layers were dried over anh. Na2SO4, filtered and concentrated in vacuo to give the amine 47 (709.5 mg, 79% yield) as a brown solid. The product was used in next steps without further purification or characterization. 254 Materials & Methods Preparation of 5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 -dimethanobenzo[9]annulen -7- amine hydrochloride , 23 ?HCl To a solution of amine 47 (700 mg, 2.54 mmol) in dry and deoxygenated toluene (14.2 mL) under N2 atmosphere were added tri-n-but\ltin h\dride (12 m/  mmol and 22?- azobisisobutyronitrile (AIBN) (62.5 mg, 0.38 mmol). The resulting solution was heated to 95 ?C for 1 hour. After addition of further AIBN (62.5 mg, 0.38 mmol) the solution was kept to 95 ?C for 90 minutes. The reaction mixture was then cooled to room temperature and concentrated to dryness in vacuo. The residue was partitioned between DCM (15 mL) and 2 N HCl solution (15 mL). The phases were separated and the organic layer was extracted wit h further 2 N HCl solution (2 x 10 mL). The combined aqueous phases were basified to pH ~12 with 10 N NaOH solution and then extracted with DCM (3 x 15 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow oil (236 mg). The residue was taken in DCM and the amine 23 was precipitated as its hydrochloride (72.9 mg, 12% yield) by adding an excess of Et 2O?HCl. Analytical and spectroscopic data of compound 23?HCl: Melting point: > 330 ?C (dec.). IR (ATR) ?: 3426, 2987, 2903, 2856, 2633, 2545, 2159, 2050, 1604, 1506, 1493, 1446, 1371, 1312, 1273, 1220, 1119, 1092, 1059, 1006, 954, 752, 611 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.79 [dm, J = 14.0 Hz, 2 H, 10(13) -H b], 1.91 [d, J = 13.0 Hz, 2 H, 6(12) - H b], 1.97 (s, 2 H, 8 -H 2), 2.01 [m, 2 H, 10(13) -H a ], 2.13 [ m, 2 H, 6(12) - H a ], 2.45 [m, 1 H, 9 -H], 3.17 [tt, J = 6.0 Hz, -? = 2.0 Hz, 2 H, 5(11) -H], 7.09 (broad s, 4 H, Ar - H). 13C-NMR (125.7 MHz, CD 3OD) ?: 32.2 (CH, C9), 34.7 [CH 2, C10(13)], 39.9 [CH 2, C6(12)], 40.5 (CH 2, C8), 41.8 [CH, C5(11)], 54.0 (C, C7), 127.9 [CH, C2(3)], 129.3 [CH, C1(4)], 146.8 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 213 (M? + , 90), 198 (20), 172 (15), 171 (91), 170 (30), 157 (21), 156 (100), 155 (15), 144 (28), 143 (21), 141 (31), 130 (19), 129 (27), 128 (34), 115 (34), 94 (26), 77 (11), 57 (12). Elemental analysis: Calculated for C15H 20ClN: C 72.13% H 8.07% Cl 14.19% N 5.61% CHAPTER 1 255 Calculated for C15H 20ClN?0.60H 2O: C 69.14% H 8.20% C l 13.60% N 5.38% Found: C 68.85% H 7.91% Cl 13.68% N 5.417% Preparation of N,N -d imethyl -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo [9]annulen -7-amine hydrochloride, 24 ?HCl To a solution of amine 23 (140 mg, 0.55 mmol) in methanol (4 mL) were added formaldehyde (0.13 mL, 37% wt in aqueous solution, 1.71 mmol), glacial acetic acid (0.12 mL) and sodium cyanoborohydride (107 mg, 1.62 mmol) and the mixture was stirred at room temperature for 8 hours. Then further NaBH 3CN (107 mg, 1.62 mmol) and formaldehyde (0.13 mL, 37% wt in aqueous solution, 1.71 mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was then evaporated to dryness in vacuo and the resulting residue partitioned between water (5 mL) and DCM (5 mL). The pH was adjusted to ~12 with 10 N NaOH solution and the phases were separated. The aqueous phase was extracted with further DCM (2 x 5 mL) and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo. An excess of Et 2O?HCl was added to a solution of the amine 24 in DCM to form its hydrochloride, followed by evaporation to dryness in vacuo (101.4 mg, 69 yield). Analytical and spectroscopic data of compound 24?HCl: Melting point: 253 - 255 ?C. IR (ATR) ?: 3414, 3014, 2930, 2904, 2850, 2599, 2473, 2151, 1635, 1491, 1447, 1416, 1377, 1309, 1287, 1272, 1220, 1188, 1156, 1089, 1046, 1011, 957, 902, 756, 631, 608 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.78 [broad d, J = 14.0 Hz, 2 H, 6(12) -H b], 2.00 -2.09 [m, 6 H, 6(12) -H a, 8-H, 10(13) -H b], 2.19 [dm, J = 12.5 Hz, J ? = 6.5 Hz, 2 H, 10(13) -H a ], 2.53 [m, 1 H, 9 -H], 2.80 [s, 6 H, N -CH 3], 3.26 [broad t, J = 6.5 Hz, 2 H, 5(11) -H], 7.10 (m, 4 H, Ar - H). 13C-NMR (125.7 MHz, CD 3OD) ?: 32.8 (CH, C9), 34.6 [CH 2, C6(12)], 35.0 [CH 2, C10(13)], 37.0 (CH 3, N-CH 3), 37.8 (CH 2, C8), 41.8 [CH, C5(11)], 65.6 (C, C7), 128.0 [CH, C2(3)], 129.2 [CH, C1(4)], 146.7 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 241 (M?+ , 84), 226 (21), 200 (18), 199 (100), 198 (33), 184 (39), 172 (21), 171 (16), 170 (15), 155 (30), 141 (43), 129 (30), 128 (35), 122 (20), 115 256 Materials & Methods (33), 108 (21), 85 (43), 84 (14), 71 (16), 70 (21), 58 (18). Elemental analysis: Calculated for C17H 24ClN: C 73.49% H 8.71% Cl 12.76% N 5.04% Calculated for C17H 24ClN?1.3H 2O?0.15HCl: C 66.57% H 8.79% Cl 13.29% N 4.57% Found: C 66.51% H 8.52% Cl 13.08% N 4.60% Preparation of 2-chloro-N-(9 -chloro-5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)acetamide , 4 0 A solution of chloroacetamide 37 (500 mg, 1.64 mmol) in thionyl chloride (20.5 mL) was heated at reflux for 1 hour. The reaction mixture was then tempered to room temperature and evaporated to dryness in vacuo. Toluene (25 mL) was added and the resulting solution concentrated in vacuo. The procedure was repeated twice more to give a yellow oil (592mg). Purification by column chromatography (Al2O3, 0-10% methanol/DCM) gave 40 (297 g, 56% yield) as a white solid. The analytical sample was obtained by crystallization with methanol/diethyl ether. Analytical and spectroscopic data of compound 40: Melting point: 195 - 197 ?C. IR (ATR) ?: 3292, 3074, 2947, 2910, 2856, 1654, 1552, 1492, 1450, 1426, 1359, 1331, 1307, 1281, 1251, 1206, 1153, 1091, 1044, 998, 941, 892, 803, 756, 734, 692, 676 cm-1. 1H -NMR (400 MHz, CDCl3) ?: 2.05 [d, J = 13.2 Hz, 2 H, 6(12) - H b], 2.18 [d, J = 13.2 Hz, 2 H, 10(13) - H b], 2.27 [m, 2 H, 6(12) -H a ], 2.40 [m, 2 H, 10(13) -H a ], 2.55 [s, 2 H, 8 -H], 3.28 [tt, J = 6.8 Hz, J ? = 1.6 Hz, 2 H, 5(11) -H], 3.94 [s, 2 H, 14 -H], 6.35(s, 1 H, NH), 7.07 [ m, 2 H, 2(3) -H], 7.13 [m, 2 H, 1(4) -H]. 13C-NMR (100.6 MHz, CDCl 3) ?: 37.8 [CH 2, C6(12)], 41.0 [CH, C5(11)], 42.8(CH 2, C14), 44.3 [CH 2, C10(13)], 49.6 (CH 2, C8), 56.7 (C, C9), 68.7 (C, C7), 127.0 [CH, C2(3)], 128.2 [CH, C1(4)], 144.4 [C, C4a(C11a)], 164 .6 (C, C14). MS (EI), m /z (%); significant ions: 327 [M ? + (37Cl2), 5], 325 [M ? + (35Cl37Cl), 20], 323 [M ? + (35Cl2), 32], 290 [(C 17H 1937ClNO)?+ , 13], 288 [(C 17H 1935ClNO)? + , 34], 233 (14), 232 (42), 231 CHAPTER 1 257 (40), 230 [(C 15H 1535Cl)?+ , 100], 207 (21), 196 (19), 195 [(C 15H 15)? + , 88), 194 (23), 181 (28), 179 (24), 175 (23), 165 (24), 155 (36), 153 (24), 141 (41), 129 (24), 128 (31), 115 (33), 77 (15). HRMS -ESI+ m /z [ M +H ] + calcd for [C 17H 19Cl2NO+H] + : 324.0916, found: 324.0913. Preparation of 9-chloro-5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine hydrochloride, 42?HCl To a solution of chloroacetamide 40 (350 mg, 1.08 mmol) in abs. ethanol (23 mL) were added thiourea (98mg, 1.29 mmol) and glacial acetic acid (0.75 mL) and the mixture was heated at reflux overnight. The resulting suspension was then tempered to room temperature, water (10 mL) was added and the pH adjusted to ~12 with 5 N NaOH solution. DCM (10 mL) was added, the phases were separated and the aqueous phase was extracted with further DCM (2 x 10 mL). The combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give 42 as a yellow solid. An excess of Et2O?HCl was added to a solution of the amine in DCM to form its hydrochloride, followed by filtration of the white precipitated (230.3 mg, 73 yield). The analytical sample was obtained by crystallization with methanol/diethyl ether. Analytical and spectroscopic data of compound 42?HCl: Melting point: > 315 ?C (dec.). IR (ATR) ?: 2906, 2850, 2673, 2559, 2176, 1601, 1509, 1494, 1452, 1361, 1309, 1297, 1213, 1170, 1090, 1070, 1036, 1007, 967, 945, 877, 804, 756 cm-1. 1H -RMN (500 MHz, CD 3OD) ?: 1.87 [d, J = 13 Hz, 2 H, 10(13) -H b], 2.10 -2.17 [complex signal, 4 H, 10(13)-H a, 6(12)-H b], 2.35 (s, 2 H, 8 -H 2), 2.41 [m, 2 H, 6(12) - H a ], 3.33 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 7.12 -7.17 (complex signal, 4 H, Ar -H). 13C-RMN (125.7 MHz, CD 3OD) ?: 38.3 [CH 2, C10(13)], 41.8 [CH, C5(11)], 45.0 [CH 2, C6(12)], 50.0 (CH 2, C8), 56.9 (C, C7), 68.4 (C, C9), 128.5 (CH ) and 129.4 (CH ) [C1(4) and C2(3)] , 145.1 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 248 [(M+H) ? + , 12], 214 (52), 213 [(M -Cl+H) ? + , 100], 212 (62), 158 (16), 157 (27), 156 (21), 155 (19), 152 (10), 129 (12), 128 (14), 115 (15). HRMS -ESI+ m /z [ M +H ] + calcd for [C 15 H 18ClNO+H] + : 248.1021, found: 248.1202. 258 Materials & Methods Elemental analysis: Calculated for C15H 19Cl2N: C 63.39% H 6.74% N 4.93% Calculated for C15H 19Cl2N?0.50H 2O: C 61.44% H 6.87% N 4.78% Found: C 61.37% H 6.61% N 4.78% Preparation of N,N -d imethyl -9-chloro-5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine hydrochloride , 49 ?HCl To a solution of amine 42 (200 mg, 0.80 mmol) in methanol (6 mL) were added 37% formaldehyde solution (0.19 mL, 2.48 mmol), glacial acetic acid (0.17 mL) and sodium cyanoborohydride (155 mg, 2.35 mmol) and the mixture was stirred at room temperature for 6 hours. Then further NaBH 3CN (155 mg, 2.35 mmol) and 37% formaldehyde solution (0.19 mL, 2.48 mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was then evaporated to dryness in vacuo and the resulting residue partitioned between water (5 mL) and EtOAc (5 mL). The pH was adjusted to ~12 with 5 N NaOH solution and the phases were separated. The aqueous phase was extracted with further EtOAc (2 x 5 mL) and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo. Purification by column chromatography (Al2O3, 0-2% m ethanol/DCM) gave 49 (75.2 g, 57% column yield) as a white solid. The purified product was taken in DCM and the amine 49 was precipitated as its hydrochloride (44.4 mg, 19.6% overall yield) by adding an excess of Et 2O?HCl. Analytical and spectroscopic data of compound 49?HCl: Melting point: 265 - 270 ?C. IR (ATR) ?: 3484, 3415, 2937, 2867, 2553, 2441, 1481, 1467, 1449, 1367, 1316, 1252, 1218, 1177, 1158, 1143, 1091, 1048, 1012, 975, 936, 900, 822, 802, 783, 764, 737, 632 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 2.02 [d, J = 12.5 Hz, 2 H, 6(12) -H b], 2.13 [d, J = 13.5 Hz, 2 H, 10(13) -H b], 2.23 [m, 2 H, 6(12) -H a ], 2.44 [m, 2 H, 8 -H, 10(13) -H a ], 2.47 (s, 2 H, 8 -H 2), 2.85 [s, 6 H, N(C H 3)2 ], 3.41 [t, J = 7 Hz, 2 H, 5(11) -H], 7.16 (s, 4 H, Ar -H). 13C-NMR (125.7 MHz, CD 3OD) ?: 33.6 [CH 2, C6(12)], 37.7 [CH 3, N(CH 3)2], 41.6 [CH, C5(11)], 44.9 [CH 2, C10(13)], 47.2 (CH 2, C8), 68.2 (C, C7), 69.2 (C, C9), 128.6 (CH ) and CHAPTER 1 259 129.4 (CH ) [C1(4) and C2(3)] , 145.0 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 275 (M ? + , 18), 240 [ (M-Cl)?+ , 100] , 233 (11), 184 (17). HRMS -ESI+ m /z [ M +H ] + calcd for [C 17H 23ClN+H] + : 276.1514, found: 276.1514. Elemental analysis: Calculated for C17H 23Cl2N: C 65.39% H 7.42% Cl 22.70% N 4.49% Calculated for C17H 23Cl2N?0.80MeOH: C 63.27% H 7.81% Cl 20.98% N 4.16% Found: C 63.12% H 7.54% Cl 20.74% N 4.16% Preparation of 9-f luoro -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine hydrochloride , 48 ?HCl A solution of amine 46 (400 mg, 1.75 mmol) in DCM (6 mL) was cooled to -30 ?C with a dry ice in acetone bath. Then (diethylamino)sulfur trifluoride (DAST) (0.97 mL, 6.98 mmol) was added and the reaction mixtu re was stirred with the dry ice in acetone bath overnight. To the resulting solution was added water (10 mL) and the pH adjusted to ~12 with a 1 N NaOH solution. The phases were separated and the aqueous phase was extracted with further DCM (2 x 8 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to about 5 mL. An excess of Et 2O?HCl was added and the amine 48?HCl was recovered by filtration in vacuo (205.5 mg, 44% yield) as a white solid. Analytical and spectroscopic data of compound 48?HCl: Melting point: 280 ?C (dec.). IR (ATR) ?: 3280, 2934, 2854, 2701, 2649, 2580, 1626, 1542, 1493, 1453, 1367, 1326, 1259, 1218, 1094, 998, 900, 860, 757, 708, 594, 577 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.85 [broad d, J = 15 Hz, 2 H, 6(12) -H b], 1.89 [broad d, J = 10 Hz, 2 H, 10(13) -H b], 2.10 -2.15 [m , 4 H, 6(12) -H a, 8-H 2], 2.19 [m, 2 H, 10(13) -H a ], 3.39 [m, 2 H, 5(11)-H], 7.15 (s, 4 H, Ar -H). 13C-NMR (125.7 MHz, CD 3OD) ?: 38.7 [CH 2, s, C6(12)], 40.2 [CH, d, 3J C - F = 12.8 Hz, C5(11)], 40.6 [ CH 2, d, 2 J C - F = 20.5 Hz, C10(13)], 45.9 (CH 2, d, 2J C - F = 20.5 Hz, C8), 58.2 (C, 260 Materials & Methods d, 3J C - F = 10.7 Hz, C7), 94.5 (C, d, 1J C - F = 179.1 Hz, C9), 128.5 [CH, s, C2(3)], 129.4 [CH, s, C1(4)], 145.2 [C, s, C4a(C11a)]. MS (EI), m /z (%); significant ions: 231 (M? + , 100), 216 (17), 211 (11), 196 (20), 190 (12), 189 (73), 188 (12), 170 (34), 169 (18), 168 (56), 159 (13), 156 (44), 155 (13), 144 (18), 141 (14), 129 (17), 128 (24), 115 (26), 112 (25). HRMS -ESI+ m /z [ M +H ] + calcd for [C 15 H 18FN+H] + : 232.1496, found: 232.1496. Elemental analysis: Calculated for C15H 19ClFN: C 67.28% H 7.15% Cl 13.24% N 5.23% Calculated for C15H 19ClFN?0.75H 2O: C 64.05% H 7.35% Cl 12.60% N 4.98% Found: C 63.61% H 7.32% Cl 13.11% N 4.75% Preparation of 9-f luoro -N,N -dimethyl -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine hydrochloride , 51 ?HCl To a solution of amine 48 (228 mg, 0.99 mmol) in methanol (8 mL) were added formaldehyde (0.23 mL, 37% wt in aqueous solution, 3.01 mmol), glacial acetic acid (0.21 mL) and sodium cyanoborohydride (189.2 mg, 2.86 mmol) and the mixture was stirred at room temperature for 6 hours. Then further NaBH 3CN (189.2 mg, 2.86 mmol) and formaldehyde (0.23 mL, 37% wt in aqueous solution, 3.01 mmol) were added and the solution was stirred at room temperature overnight. The reaction mixture was then evaporated to dryness in vacuo and the resulting residue partitioned between water (5 mL) and EtOAc (5 mL). The pH was adjusted to ~12 with 5 N NaOH solution and the phases were separated. The aqueous phase was extracted with further EtOAc (2 x 10 mL) and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to about 5 mL. An excess of Et 2O?HCl was added and the solvents were removed under vacuo to give the amine 51?HCl (67 mg, 23% yield) . Analytical and spectroscopic data of compound 51?HCl: Melting point: 249 - 251 ?C. IR (ATR) ?: 3398, 3034, 2936, 2860, 2511, 2415, 1470, 1368, 1341, 1322, 1220, 1186, 1136, CHAPTER 1 261 1088, 1057, 983, 968, 929, 903, 882, 757, 656, 582 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.89 [d, J = 12.5 Hz, 2 H, 6(12) -H b], 1.99 [d, J = 12 Hz, 2 H, 10(13) -H b], 2.19 -2.24 [ m, 6 H, 6(12) -H a, 8-H 2, 10(13)-H a ], 2.85 (s, 6 H, N -CH 3), 3.47 [m, 2 H, 5(11) -H], 7.17 (s, 4 H, Ar -H). 13C-NMR (125.7 MHz, CD 3OD) ?: 33.9 [CH 2, s, C6(12)], 37.8 (CH 3, s, N-CH 3), 39.9 [CH, d, 3JC-F = 12.8 Hz, C5(11)], 40.6 [CH 2, d, 2J C - F = 20.5 Hz, C10(13)], 43.3 (CH 2, d, 2 J C - F = 21.6 Hz, C8), 69.5 (C, d, 3 J C - F = 10.7 Hz, C7), 94.8 (C, d, 1J C - F = 179.1 Hz, C9), 128.3 [CH, s, C2(3)], 129.3 [CH, s, C1(4)], 145.1 [C, s, C4a(C11a)]. MS (EI), m /z (%); si gnificant ions: 259 (M? + , 100), 258 (18), 244 (13), 217 (55), 216 (13), 184 (23), 173 (12), 159 (10), 141 (11), 140 (12), 128 (12), 115 (12), 85 (10). Elemental analysis: Calculated for C17H 23ClFN: C 69.02% H 7.84% Cl 11.98% N 4.73% Calculated for C17H 23ClFN?0.30H 2O?0.05HCl : C 67.38% H 7.87% Cl 12.28% N 4.62% Found: C 67.32% H 7.91% Cl 12.20% N 4.67% Preparation of 9-methoxy -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-ol, 53 165 To a solution of enone 25 (500 mg, 2.36 mmol) in methanol (10 mL) was added p- toluenesulfonic acid monohydrate (112 mg, 0.59 mmol) and the reaction mixture was stirred at room temperature overnight. To the resulting solution was then concentrated in vacuo and the obtained residue partitioned between DCM (10 mL) and saturated aqueous NaHCO 3 solution (10 mL). The phases were separated and the aqueous one was extracted with further DCM (2 x 10 mL). The combined organic layers were dried over anh. Na2SO4, filtered and evaporated in vacuo to give a yellow gum (541.3 mg). Purification by column chromatography (Al2O3, DCM/m ethanol mixture) gave 53 (367.5 mg, 64% yield) as a white solid. Analytical and spectroscopic data of compound 53: Melting point: 97 - 98 ?C. 262 Materials & Methods IR (ATR) ?: 3359, 2930, 2851, 1723, 1495, 1444, 1356, 1296, 1239, 1193, 1094, 1061, 981, 900, 846, 758, 663 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.74 [dm, J = 12.0 Hz, 2 H, 10(13) -H b], 1.78 [s, 2 H, 8 -H 2], 1.81 [d, J = 13.0 Hz, 2 H, 6(12) -H b], 1.90 [ m, 4 H, 6(12) -H a, 10(13)-H a ], 3.23 [tt, J = 6.4 Hz, -? = 2.0 Hz, 2 H, 5(11) -H], 3.24 (s, 3 H, O CH 3), 7.07-7.15 (m, 4 H, Ar -H). 13C-NMR (100.6 MHz, CDCl 3) ?: 37.8 [CH 2, C10(13)], 39.6 [CH, C5(11)], 42.7 [CH 2, C6(12)], 48.4 (CH 3, OCH 3), 48.9 (CH 2, C8), 72.5 (C, C9), 76.5 (C, C7), 126.7 [CH, C2(3)], 128.1 [CH, C1(4)], 145.1 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 244 (M? + , 100), 213 (42), 201 (17), 172 (22), 171 (30), 159 (38), 158 (72), 157 (28), 155 (48), 154 (16), 153 (16), 144 (17), 143 (22), 141 (23), 132 (12), 130 (12), 129 (63), 128 (52), 127 (19), 125 (40), 115 (48), 111 (25), 107 (12), 91 (13). HRMS -ESI+ m /z [ M +H ] + calcd for [C 16H 19O+H; fragment] + : 227.1431, found: 227.1430. Preparation of 2-chloro-N-(9 -methoxy -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[ 9]annulen -7-yl)acetamide , 54 To a solution of alcohol 53 (545 mg, 2.23 mmol) in DCM (5 mL) was added chloroacetonitrile (0,140 mL, 2.23 mmol) and the mixture was cooled to 0 -5 ?C with an ice bath. Then conc. H 2SO4 (0.180 mL, 3.38 mmol) was added dropwise without reaching a temperature greater than 10 ?C. After the addition, the reaction mixture was stirred at room temperature overnight. Then the resulting solution was poured into ice (10 g) and DCM (10 mL) was added. The phases were separated and the aqueous phase was extracted with further DCM (2 x 10 mL). The combined organic phases were dried over anh. Na2SO4, filtered and evaporated in vacuo to give a white gum (570 mg). Column chromatography (Al2O3, Hexane/EtOAc mixture) gave in order of elution 54 (222 mg, 31% yield), 34 (210 CHAPTER 1 263 mg, 37% column yield) and 53 (91 mg, 16% column yield) a s white solids. The product was used in next steps without further purification or characterization. Preparation of 9-methoxy -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-amine, 52 To a solution of chloroacetamide 54 (136.8 mg, 0.43 mmol) in abs. ethanol (9 mL) were added thiourea (39.2 mg, 0.52 mmol) and glacial acetic acid (0.3 mL) and the mixture was heated at reflux overnight. The reaction mixture was then tempered to room temperature and concentrated in vacuo. The crude was partitioned between water (10 mL) and DCM (10 mL) and the aqueous phase was acidified to pH ~2 with 2 N HCl solution. The phases were separated, the pH adjusted to ~12 with 2 N NaOH solution and the aqueous phase was then extracted with DCM (3 x 10 mL). The combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give 52 (62 mg, 59% yield) as a white solid. Analytical and spectroscopic data of compound 52: Melting point: 71 ? 73 ?C. IR (ATR) ?: 3500?2850 (3507, 3389, 3329, 3307, 3263, 3126, 3019, 2936, 2852), 1651, 1605, 1490, 1441, 1360, 1210, 1113, 1059, 1047, 1010, 969, 947, 893, 847, 754, 666, 630, 584 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.61-1.66 [ m, 4 H, 6(12) -H b, 8-H 2], 1.77 [ m, 2 H, 6(12) -H a ], 1.83-1.90 [ m, 4 H, 10(13)-H 2], 3.19 [broad t, J = 6.0 Hz, 2 H, 5(11) -H], 3.23 (s, 3 H, OC H 3), 7.05-7.13 (m, 4 H, Ar -H). 13C-NMR (100.6 MHz, CDCl 3) ?: 37.8 [CH 2, C10(13)], 40.2 [CH, C5(11)], 43.3 [CH 2, C6(12)], 48.2 (CH 3, OCH 3), 49.5 (CH 2, C8), 52.5 (C, C7), 75.4 (C, C9), 126.6 [CH, C2(3)], 128.0 [CH, C1(4)], 145.4 [C, C4a(C11a)]. MS (EI), m /z (%); significant ions: 243 (M? + , 100), 228 (17), 212 (27), 201 (13), 200 (14), 171 (20), 170 (18), 156 (23), 155 (14), 144 (20), 129 (15), 128 (19), 124 (28), 115 (18), 110 (41). HRMS -ESI+ m /z [ M +H ] + calcd for [C 16H 21O+H] + : 244.1696, found: 244.1704. C hapter 2 Benzoho moad am antane scaffolds CHAPTER 2 269 Preparation of 5,6,8,9 -tetrahydro -5,9 -propanebenzocycloheptane -7,11 -diene, 29 165 A suspension of sodium hydride, 60% dispersion in mineral oil (0. 78 g, 19.64 mmol) in anh. DMSO (50 mL) was heated to 75 ?C for 45 minutes. After the reaction mixture was tempered, a solution of triphenylmethylphophonium iodide (8.2 g, 19.64 mmol) in anh. DMSO (70 mL) was added and the resulting yellow solution was stirred at room temperature for 20 minutes. Then a suspension of diketone 27 (2 g, 9.35 mmol) in anh. DMSO (20 mL) was added and the obtained solution was heated to 75 ?C overnight. The resulting black solution was allowed to cool to room temperature and then poured into water (150 mL). Hexane (100 mL) was added and the phases were separated. The aqueous phase was extracted with further hexane (3 x 50 mL) and the combined organic phases were washed with brine (50 mL), dried over anh. Na2SO4, filtered and concentrated in vacuo to give an orange gum (2.32 g). Purification by packing the solid with silica gel and extracting with 100% petrol eum ether gave 29 (1.56 g, 80% yield) as a white solid. The spectroscopic data were identical to those previously published. Preparation of N-(6,7,8,9,10,11 -hexahy dro -9-methyl -5,7:9,11 -dimethano -5H- benzocyclononen -7-yl)chloroacetamide , 55 98 A solution of diene 29 (5.02 g, 23.9 mmol) in chloroacetonitrile (6.05 mL, 95.6 mmol) and glacial acetic acid (15 mL) was cooled to 0-5 ?C with an ice bath. Then conc. H 2SO4 (7.6 mL, 143.4 mmol) was added dropwise without reaching a temperature greater than 10 ?C. After the addition, the reaction mixture was stirred at room temperature overnight. Then the resulting solution was poured into ice (100 g) and DCM (40 mL) was added. The phases were separated and the aqueous phase was extracted with further DCM (2 x 40 mL). The combined organic phases were dried over anh. Na2SO4, filtered and evaporated in vacuo to give 55 (5.86 g, 81% yield) as a white solid, whose spectroscopic data were those corresponding to the previously published. 270 Materials & Methods Preparation of 6,7,8,9,10,11 -hexahydro -9-methyl -5,7:9,11 -dimethano -5H- benzocyclononen -7-amine, 19 98 To a solution of chloroacetamide 55 (5.86 g, 19.4 mmol) in abs. ethanol (150 mL) were added thiourea (1.76 g, 23.2 mmol) and glacial acetic acid (14 mL) and the mixture was heated at reflux overnight. The reaction mixture w as then tempered to room temperature and concentrated in v acuo . The crude was partitioned between water (200 mL) and EtOAc (100 mL) and the aqueous phase was acidified to pH ~2 with 1N HCl solution. The phases were separated, the pH adjusted to ~12 with 5N NaOH solution and the aqueous phase was then extracted with EtOAc (4 x 100 mL). The combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give 19 (3.03 g, 69% yield) as a white solid. The spectroscopic data were identical to those previously published. Preparation of (2 R) -tert-butyl 2 -[ (9 -hydroxy -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethan obenzo[9]annulen -7-yl)carbamoyl] pyrrolidine -1-carboxylate , 71 To a solution of amine 46 (500 mg, 2.18 mmol) in DMF (10 mL) were added Boc-D- proline (445.8 mg, 2.07 mmol), HATU (328.4 mg, 2.18 mmol) and triethylamine (0.6 mL, 4.36 mmol) and the resulting solution was stirred at room temperature overnight. The reaction mixture was then diluted with EtOAc/ benzene (2:1, 25 mL) and washed with 0.5 N HCl aqueous solution (2 x 25 mL), brine (25 mL), saturated NaHCO 3 aqueous solution (2 x 25 mL) and brine (25 mL). The organic phase was separated, dried over anh. Na 2SO4, filtered and concentrated in vacuo to give an orange oil (765 mg). Column chromatography (Al2O3, hexane/ ethyl acetate mixture) gave 12 (438 mg, 50% yield) as a white solid. The analytical sample was obtained by crystallization from EtOAc/p entane. Analytical and spectroscopic data of compound 71: Melting point: 98 ? 103 ? C. IR (ATR) ?: 3408, 3327, 2975, 2927, 1677, 1538, 1451, 1400, 1366, 1300, 1247, 1163, 1119, CHAPTER 2 271 1088, 1045, 978, 922, 862, 758 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.45 [ s, 9 H, OC(CH 3)3], 1.73 (m, 2 H, 17 -H), 1.76 [d, J = 13.0 Hz, 2 H, 10(13) -H b ], 1.83 (broad signal, 1 H, 1 6-H a), 1.93-2.31 [complex signal, 9 H, 10(13)-H a, 6(12)-H 2, 8-H, 16-H b], 3.17 [t, J = 6.5 Hz, 2 H, 5(11) -H], 3.37 (m, 2 H, 1 8-H), 4. 21 (m, 1 H, 1 5-H), 5.81 (broad s, 1 H, OH), 6.94 (broad s, 1 H, NH), 7.06 [m, 4 H, 1(4) -H], 7.10 [m, 2 H, 2(3) -H]. 13C-NMR (125.7 MHz, CDCl 3) ? (mixture of two rotamers): 23.7 (CH 2) and 24.1 (CH 2) (two rotamers, C17), 28.4 [CH 3, OC(CH 3)3], 31.1 (broad CH 2, C16), 38.4 [CH 2, C6(12)], 40.0 [CH, C5(11)], 42.4 [CH 2, C10(13)], 47.1 (CH 2, C18), 48.2 (CH 2, C8), 56.6 (C, C7), 61.2 (CH) and 61.9 (CH) (C15, two rotamers), 70.8 (C, C9), 80.4 (C, C20), 126.6 [CH, C2(3)], 128.1 [CH, C1(4)], 145.2 [d, C, C4a(C11a)], 154.7 (C) and 156.1 (C) (NCO, two rotamers), 170.9 (C) and 171.6 (C) (CO, two rotamers). HRMS -ESI+ m /z [ M +H ] + calcd for [C 25H 34N2O4+H ] + : 427.2591, found: 427.2593. Preparation of (2 R) -N-(9 -hydroxy -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide , 72 A solution of Boc-protected pyrrolidine 71 (438.5 mg, 1.03 mmol) in DCM (5 mL) and 85% o-phosphoric acid (1.05 mL, 15.4 mmol) was stirred at room temperature for 3 hours. To the reaction mixture was then added water (8 mL) and the pH was adjusted to ~12 with 5 N NaOH solution. The phases were separated, the aqueous phase was extracted with further DCM (2 x 8 mL) and the combined organic phases were dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic layers gave 72 (290.8 mg, 87% yield) as a white solid. Analytical and spectroscopic data of compound 72: Melting point: 175 ? 180 ? C. IR (ATR) ?: 3441, 3368, 3262, 2945, 2863, 1639, 1521, 1507, 1475, 1434, 1401, 1378, 1361, 1336, 1300, 1230, 1194, 1115, 1050, 887, 846, 799, 767, 731, 698, 552 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.66 (complex signal , 2 H, 17 -H 2), 1.78 [d, J = 13 Hz, 2 H, 272 Materials & Methods 10(13)-H b], 1.85 (m, 1H, 16 -H a), 1.90-1.99 [complex signal, 4 H, 6(12) -H b, 10(13)-H a ], 2.0 4- 2.11 (complex signal, 4 H, 8 -H, 16 -H b), 2.15-2.25 [complex signal, 2 H, 6(12) -H a ], 2.83 (dt, J = 10.5 Hz, J ?= 6.5 Hz, 1 H, 18 -H a), 2.97 (dt, J = 10.5 Hz, J ?= 6.5 Hz, 1 H, 18 -H b), 3.17 [ broad t, J = 6.5 Hz, 2 H, 5(11) -H], 3.59 (dd, J = 9 Hz, J ? = 5.5 Hz, 1 H, 15-H), 7.06 [m, 2 H, 1(4) - H], 7.08 [m, 2 H, 2(3) -H], 7.60 (broad s, 1 H, NH). 13C-NMR (125.7 MHz, CDCl 3) ?: 26.1 (CH 2, C17), 30.8 (CH 2, C16), 38.5 (CH 2) and 38.6 (CH 2) (C6 and C12), 40.1 [CH, C5(11)], 42.4 [CH 2, C10(13)], 47.2 (CH 2, C18), 48.1 (CH 2, C8), 55.9 (C, C7), 61.0 (CH, C15), 70.7 (C, C9), 126.6 [CH, C2(3)], 128.1 [CH, C1(4)], 145.3 [C, C4a(C11a)], 174.1 (C, CO). Elemental analysis: Calculated for C20 H 26N2O2: C 73.59% H 8.03% N 8.58% Calculated for C20 H 26N2O2?0.15DCM: C 71.36% H 7.82% N 8.26% Found: C 71.48% H 7.78% N 8.07% Preparation of (2 R) -1-ethyl -N-(9 -hydroxy -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide , 56 A solution of pyrrolidine 72 (170 mg, 0.52 mmol) in DMF (3 mL) was cooled to 5 ? C with an ice bath. Then KI (8.64 mg, 0.052 mmol) and triethylamine (0.278 mL, 2.08 mmol) were added, followed by the dropwise addition of ethyl bromide (0.04 mL, 0.55 mmol). The reaction mixture was stirred at room temperature in an ice -water bath overnight. The resulting suspension was then filtered and the solids were washed with EtOAc (10 mL). The combined filtrates were washed with saturated aqueous NaHCO 3 solution (10 mL) and brine (10 mL). The organic phase was separated, dried over anh. Na2SO4, filtered and concentrated in vacuo to give 56 (133 mg, 72% yield) as a white solid. The analytical sample was obtained by crystallization from EtOAc/p entane. Analytical and spectroscopic data of compound 56: Melting point: 150 ? 152 ? C. IR (ATR) ?: 3388, 2931, 2871, 1647, 1511, 1440, 1384, 1358, 1335, 1304, 1234, 1194, 1117, CHAPTER 2 273 1088, 1060, 761, 647 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.04 (t, J = 7.5 Hz, 3 H, NCH 2CH 3), 1.59-1.82 (complex signal, 3 H, 16 -H a, 17-H ), 1.77 [d, J = 12.5 Hz, 2 H, 10(13) -H b], 1.92 [d, J = 13 Hz, 2 H, 6(12) - H b], 1.97 [ dd, J = 12.5 Hz, J ?  Hz, 2 H, 10(13) -H a ], 2.02 (broad s, 1 H, OH), 2.11 (m, 1 H, 16 -H b), 2.14 (s, 2 H, 8-H), 2.17-2.30 [complex signal, 3 H, 6(12) -H a, 19-H a ], 2.45 (m, 1 H, 18-H a), 2.58 (m, 1 H, 18 -H b), 3.85 (dd, J = 10 .0 Hz, J ? = 4.5 Hz, 1 H, 15 -H), 3.12 (m, 1 H, 19-H b), 3.18 [dt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 7.07 [m, 2 H, 1(4) -H], 7.11 [m, 2 H, 2(3)-H], 7.45 (broad s, 1 H, NH). 13C-NMR (125.7 MHz, CDCl 3) ?: 14.8 (CH 3, NCH 2CH 3), 24.1 (CH 2, C17), 30.7 (CH 2, C16), 38.6 (CH 2) and 38.7 (CH 2) (C6 and C12), 40.1 [CH, C5(11)], 42.4 [CH 2, C10(13)], 48.1 (CH 2, C8), 49.7 (CH 2, C18), 53.8 (CH 2, NCH 2CH 3), 55.8 (C, C7), 67.8 (CH, C15), 70.7 (C, C9), 126.6 [CH, C2(3)], 128.1 [CH, C1(4)], 145.3 [d, C, C4a(C11a)], 174.3 (C, CO). Elemental analysis: Calculated for C22 H 30N2O2: C 74.54% H 8.53% N 7.90% Calculated for C22 H 30N2O2?0.1EtOAc: C 74.06% H 8. 55% N 7.71% Found: C 73.93% H 8.64% N 7.71% Preparation of (2 R) -tert-butyl -2-[(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)carbamoyl]pyrrolidine -1-carboxylate, 73 Boc-D-proline (336 mg, 1.56 mmol), HOBt (316 mg, 2.34 mmol), EDC (363 mg, 2.34 mmol) and triethylamine (0.48 mL, 3.43 mmol) were added to a solution of amine 19 (388 mg, 1.71 mmol) in EtOAc (20 mL) and the mixture was stirred at room temperature overnight. To the resulting suspension was then added water (15 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic layers gave 73 (556 mg, 84% yield) as a white solid, that was washed with pentane for obtaining an analytical sample. 274 Materials & Methods Analytical and spectroscopic data of compound 73: Melting point: 168 ? 169 ?C. IR (ATR) ?: 3296, 2917, 1706, 1684, 1657, 1560, 1450, 1385, 1363, 1287, 1238, 1179, 1161, 1123, 1089, 1048, 1009, 982, 920, 756, 673, 574 cm-1. 1H -NMR (400 MHz, CDCl 3) ? (two rotamers): 0.91 (s, 3 H, C9 -CH 3), 1.46 [s, 9 H, OC(CH 3)3], 1.53 [broad d, J = 13.5 Hz, 2 H, 10(13) -H b], 1.65 [broad dd, J = 13.5 Hz, J ? = 6.5 Hz, 2 H, 10(13) -H a ], 1.75 -1.88 (complex signal, 4 H, 8 -H, 17 -H), 2.01 [broad d, J = 13 Hz, 2 H, 6(12) -H b], 2 .13 [m, 2 H, 6(12) -H a ], 2.3 (very broad signal, 1 H, 16 -H b), 3.06 [t, J = 6 Hz, 2 H, 5(11) -H], 3.39 (broad m, 2 H, 18 -H), 4.11 (very broad signal, 1 H, 15 -H), 6.77 (broad s, 1 H, NH), 7.05 [m, 4 H, Ar -H]. 13C-NMR (100.6 MHz, CDCl 3) ? (two rotamers): 24.2 (broad CH 2, C17), 28.4 [CH 3, C(CH 3)3], 31.1 (broad CH 2, C16), 32.2 (CH 3, C9-CH 3), 33.6 (C, C9), 38.9 and 39.0 [CH 2, C6(12)], 41.0 and 41.1 [CH, C5(11)], 41.11 and 41.15 [CH 2, C10(13)], 47.0 (CH 2, C8), 47.2 (broad signal, CH 2, C18), 54.1 (C, C7), 60.6 and 61.9 (CH, C15), 126.2 [CH, C2(3)], 127.9 [CH, C1(4)], 146.2 [broad C, C4a(C11a)], 170.8 (very broad C, CO). MS-DIP (EI), m /z (%); significant ions: 424 (M ? + , <1), 211 (C16H 19+ , 14), 171 (22), 170 (14), 155 (19), 140 (8), 115 (22), 114 (100), 70 (60), 57 (22). HRMS -ESI+ m /z [ M +H ] + calcd for [C 26H 36N2O3+H + ]: 425.2799, found: 425.2803. Preparation of (2 R) -N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide , 74 A solution of Boc-protected pyrrolidine 73 (556.1 mg, 1.31 mmol) in DCM (6 mL) and 85% o-phosphoric acid (1.33 mL, 19.6 mmol) was stirred at room temperature for 18 hours. To the reaction mixture was then added water (15 mL) and the aqueous phase was basified until pH ~12 with 5 N NaOH solution. The phases were separated and the aqueous phase was extracted with further DCM (2 x 10 mL). The combined organic layers were dried over anh. Na2SO4, filtered and evaporated in vacuo to give 74 (419.5 mg, quantitative CHAPTER 2 275 yield) as a white solid. The analytical sample was obtained by crystallization from EtOAc/pentane. Analytical and spectroscopic data of compound 74: Melting point: 116 ? 119 ?C. IR (ATR) ?: 3289, 3257, 3017, 2920, 2897, 2864, 2839, 1646, 1515, 1492, 1467, 1450, 1359, 1345, 1307, 1245, 1208, 1153, 1135, 1110, 1090, 1046, 1006, 948, 914, 877, 856, 800, 754, 700, 613, 644, 558, 488 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 0.91 (s, 3 H, C9 -CH 3), 1.53 [d, J = 13.0 Hz, 2 H, 10(13) -H b], 1.66 [complex si gnal, 4 H, 17 -H, 10(13) -H a ], 1.86 (complex signal, 3 H, 8 -H, 16 -H a), 1.99 [dq, J = 13.0 Hz, J ? = 1.5 Hz, 2 H, 6(12) -H b], 2.07 (m, 1 H, 16 -H b), 2.17 [m, 2 H, 6(12) -H a ], 2.84 (dt, J = 10.5 Hz, J ?= 6.5 Hz, 1 H, 18 -H a), 2.97 (dt, J = 10.5 Hz, J ?= 6.5 Hz, 1 H, 18 -H b), 3.06 [tq, J = 6.5 Hz, J ?= 1.5 Hz, 2 H, 5(11) -H], 3.60 (dd, J = 9.0 Hz, J ?= 5.5 Hz, 1 H, 15 -H), 7.02-7.08 [complex signal, 4 H, Ar -H], 7.50 (broad s, 1 H, NH). 13C-NMR (125.7 MHz, CDCl 3) ?: 26.1 (CH 2, C17), 30.8 (CH 2, C16), 32.2 (CH 3, C9-CH 3), 33.5 (C, C9), 38.9 (CH 2) and 39.0 (CH 2) (C6 and C12), 41.0 [CH, C5(11)], 41.1 (CH 2) and 41.2 (CH 2) (C10 and C13), 47.0 (CH 2, C8), 47.2 (CH 2, C18), 53.3 (C, C7), 61.1 (CH, C15), 126.2 [CH, C2(3)], 127.9 [CH, C1(4)], 146.3 [d, C, C4a(C11a)], 173.9 (C, CO). MS (CI), m /z (%); significant ions: 324 (M ? + , <1), 211 (C 16H 19?+ , 1), 155 (4), 70 (100). Elemental analysis: Calculated for C21H 28N2O: C 77.74% H 8.70% N 8.63% Calculated for C21H 28N2O?0.2EtOAc?0.15Pentane: C 76.75% H 8.97% N 7.94% Found: C 76.78% H 8.95% N 7.89% Preparation of (2 R) -1-ethyl -N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide , 57 ?tartrate A solution of pyrrolidine 74 (504 mg, 1.55 mmol) in DMF (10 mL) was cooled to 5 ?C with an ice bath. Then KI (26 mg, 0.16 mmol) and triethylamine (0.87 mL, 6.2 mmol) were 276 Materials & Methods added, followed by the dropwise addition of ethyl bromide (0.12 mL, 1.63 mmol). The reaction mixture was stirred at room temperature in an ice -water bath overnight. To the resulting solution was added EtOAc (5 mL) and water (20 mL). The phases were separated and the aqueous layer was extracted with further EtOAc (2 x 15 mL). The combined o rganic phases were washed with saturated aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4, filtered and concentrated in vacuo to give a clear oil (446 mg). Column chromatography (Al2O3, hexane/EtOAc mixtures) gave 57 (255 mg, 44% yield) as a white solid. A solution of L-(+) -tartaric acid (108.8 mg, 0.72 mmol) in methanol (2 mL) was added to 57 directly. The solvents were removed under vacuo to give 57 as its tartrate salt. The analytical sample was obtained by crystallization from methanol/diethyl ether. Analytical and spectroscopic data of compound 57?tartrate: Melting point: 177-179 ?C. IR (ATR) ?: 3426, 3250, 3023, 2915, 1694, 1667, 1573, 1453, 1361, 1306, 1243, 1217, 1116, 1080, 948, 893, 800, 761, 666 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 0.94 (s, 3 H, C9 -CH 3), 1.27 (t, J = 7.5 Hz, 3 H, NCH 2CH 3), 1.50 [d, J = 13.5 Hz, 2 H, 10(13) -H b], 1.69 [dd, J = 13.0 Hz, J ? = 6.0 Hz, 2 H, 10(13) -H a ], 1.80 (s, 2 H , 8-H 2), 1.93-1.99 (complex signal, 3 H, 16 -H a, 17-H), 2.11 [m, 2 H, 6(12) -H a ], 2.17 [d, J = 13 Hz, 2 H, 6(12) -H b], 2.46 (m, 1 H, 16 -H b), 3.08 [tt, J = 6.5 Hz, J ?= 2 Hz, 2 H, 5(11) -H], 3.13 (m, 1 H, 18 -H a), 3.19 (q, J = 7.5 Hz, 2 H, NC H 2CH 3), 3.66 (m, 1 H, 18 -H b), 3.96 (dd, J = 9 Hz, J ?= 6.5 Hz, 1 H, 15 -H), 4.41 (s, 2 H, tartrate -CH ), 7.02-7.065 [complex signal, 4 H, Ar-H]. 13C-NMR (125.7 MHz, CD 3OD) ?: 11.5 (CH 3, NCH 2CH 3), 24.0 (CH 2, C17), 31.2 (CH 2, C16), 32.8 (CH 3, C9-CH 3), 34.5 (C, C9), 39.5 (CH 2) and 39.6 (CH 2) (C6 and C12), 42.4 [CH and CH 2, C5(11), C10(13)], 48.0 (CH 2, C8), 51.3 (CH 2, NCH 2CH 3), 55.6 (CH 2, C18), 56.1 (C, C7), 68.6 (CH, C15), 74.1 (CH, tartrate -CH), 127.5 [CH, C2(3)], 129.0 [CH, C1(4)], 147.4 [C, C4a(C11a)], 168.6 (C, CO), 176.6 (C, tartrate-CO). MS (CI), m /z (%); significant ions: 393 (M+41, 7), 381 (M+29, 18), 354 (27), 353 (M+1, 100), 211 (12), 98 (26). Elemental analysis: Calculated for C27H 38N2O7: C 64.52% H 7.62% N 5.57% Calculated for C27H 38N2O7?1H 2O: C 62.29% H 7.74% N 5.38% Found: C 62.50% H 7.50% N 5.13% CHAPTER 2 277 Preparation of 4 -amino-3,5 -dichloro -N-(9 -hydroxy -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)benzamide, 58 4-Amino-3,5-dichlorobenzoic acid (220.9 mg, 1.07 mmol), 1-hydroxybenzotriazole (HOBt) (216.7 mg, 1.6 mmol), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (248.0 mg, 1.6 mmol) and triethylamine (0.327 mL, 2.2 mmol) were added to a solution of amine 46 (270 mg, 1.18 mmol) in EtOAc (15 mL) and DMF (1 mL) and the reaction mixture was stirred at room temperature for 21 hours. To the resulting suspension was then added water (20 mL) and the phases were separated. The organic phase was washed with 5% aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic phases gave 58 (422 mg, 95% yield) as a white solid. The analytical sample was obtained by crystallization from DCM. Analytical and spectroscopic data of compound 58: Melting point: 236 ? 237 ?C. IR (ATR) ?: 3362, 2923, 2853, 2160, 1976, 1643, 1606, 1547, 1519, 1492, 1452, 1321, 1299, 1269, 1230, 1201, 1101, 1020, 864, 748, 570 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.58 (broad s, 1 H, OH), 1.81 [d, J = 12.5 Hz, 2 H, 10(13) - H b], 2.00 [m, 2 H, 10(13) -H a ], 2.12 [d, J = 13 Hz, 2 H, 6(12) -H b], 2.19 (s, 2 H, 8 -H), 2.27 [ddd, J = 13.0 Hz, J ? = 6.5 Hz, J ?? = 1.0 Hz, 2 H, 6(12) -H a ], 3.23 [tt, J = 6.5 Hz, J ?= 1.5 Hz, 2 H, 5 (11)-H], 4.73 (broad s, 2 H, Ar -NH 2), 5.72 (broad s, 1 H, CON H ), 7.09 [m, 2 H, 1(4) - H], 7.13 [m, 2 H, 3(2) -H], 7.54 [s, 2 H, 16(20)]. 13C-NMR (125.7 MHz, CDCl 3) ?: 38.6 [CH 2, C6(12)], 40.0 [CH, C5(11)], 42.5 [CH 2, C10(13)], 48.4 (CH 2, C8), 57.4 (C, C7), 70.9 (C, C9), 118.9 [C, C17(19)], 125.4 (C, C15), 126.7 [C, C16(20)], 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 142.6 (C, C18), 145.1 [d, C, C4a(C11a)], 164.2 (C, CO). MS (EI), m /z (%); significant ions: 418 (21), 416 (M ?+ , 32), 212 (43), 207 (36), 205 (46), 194 (16), 190 (63), 188 (100), 179 (15), 160 (15), 155 (33), 154 (29), 142 (16), 141 (17), 129 (19), 128 (20), 124 (16), 115 (16). Elemental analysis: Calculated for C22 H 22Cl2N2O2: C 63.32% H 5.31% N 6.71% 278 Materials & Methods Found: C 63.32% H 5.35% N 6.52% Preparation of 4 -amino-3,5 -dichloro -N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)benzamide , 59 4-Amino-3,5-dichlorobenzoic acid (165 mg, 0.80 mmol), 1-hydroxybenzotriazole (HOBt) (162 mg, 1.2 mmol), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (186 mg, 1.2 mmol) and triethylamine (0.25 mL, 1.76 mmol) were added to a solution of amine 19 (200 mg, 0.88 mmol) in ethylacetate (10 mL) and DMF (1 mL) and the reaction mixture was stirred at room temperature for 22 hours. To the resulting suspension was then added water (20 mL) and the phases were separated. The organic phase was washed with 5% aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4 and filtered. Evaporation in v acuo of the combined organic phases gave an orange solid (320 mg). Column chromatography (Al2O3, 100% DCM) gave 59 (176.7 mg, 53% yield) as a white solid. The analytical sample was obtained washing the solid with n -pentane. Analytical and spectroscopic data of compound 59: Melting point: 173 ? 175 ?C. IR (ATR) ?: 3460, 3352, 2920, 2852, 1627, 1610, 1532, 1484, 1452, 1322, 1306, 1270, 1232, 1126, 1091, 1049, 1006, 947, 908, 880, 837, 789, 757, 720, 695, 663, 581 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 0.95 (s, 3 H, C9 -CH 3), 1.58 [dt, J = 13.5 Hz, J ? = 1.5 Hz, 2 H, 10(13) -H b], 1.69 [ddm, J = 13.5 Hz, J ? = 6.5 Hz, 2 H, 10(13) -H a ], 1.93 (s, 2 H, 8 -H), 2.15 [dt, J = 13.0 Hz, J ? = 1.5 Hz, 2 H, 6(12) -H b], 2.25 [ddm, J = 13.0 Hz, J ? = 6.5 Hz, 2 H, 6(12) - H a ], 3.11 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 4.71 (broad s, 2 H, NH 2), 5.68 (broad s, 1 H, NH), 7.04 -7.11 [complex signal, 4 H, 1(4) -H, 2(3) -H], 7.54 [s, 2 H, 16(20)]. 13C-NMR (125.7 MHz, CDCl 3) ?: 32.2 (CH 3, C9-CH 3), 33.7 (C, C9), 39.1 [CH 2, C6(12)], 41.0 [CH, C5(11)], 41.1 [CH 2, C10(13)], 47.3 (CH 2, C8), 55.0 (C, C7), 118.8 [C, C17(19)], 125.7 (C, C15), 126.3 [CH, C2(3)], 12 6.6 [CH, C16(20)], 128.0 [CH, C1(4)], 142.4 (C, C18), 146.1 [d, C, C4a(C11a)], 164.1 (C, CO). MS (EI), m /z (%); significant ions: 416 (25), 415 (11), 414 (M ?+ , 38), 211 (20), 210 (100), 195 (26), 190 (48), 188 (74), 182 (19), 181 (19), 160 (12), 156 (14), 155 (78), 154 (30), 143 (13), 142 (14), 141 (18), 129 (18), 128 (19), 124 (15), 115 (14). CHAPTER 2 279 Elemental analysis: Calculated for C23 H 24Cl2N2O2: C 66.51% H 5.82% N 6.74% Calculated for C23 H 24Cl2N2O2?0.1C5H 12: C 66.79% H 6.01 % N 6.63% Found: C 66.82% H 6.04% N 6.54% Preparation of N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)cyclohexanecarboxamide , 60 Cyclohexanecarboxylic acid (104.9 mg, 0.82 mmol), HOBt (166.1 mg, 1.23 mmol), EDC (190.5 mg, 1.23 mmol) and triethylamine (0.251 mL, 1.80 mmol) were added to a solution of amine 19 (204.8 mg, 0.90 mmol) in EtOAc (15 mL) and the reaction mixture was stirred at room temperature overnight. To the resulting suspension was then added water (15 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4 and filtered. Evaporation in v acuo of the combined organic layers gave 60 (265 mg, 96% yield) as a white solid. The analytical sample was obtained by crystallization with EtOAc/pentane. Analytical and spectroscopic data of compound 60: Melting point: 197-198 ?C. IR (ATR) ?: 3299, 3060, 2917, 2850, 1645, 1545, 1490, 1447, 1381, 1361, 1335, 1316, 1261, 1214, 1091, 1047, 1005, 960, 942, 894, 753, 675, 665 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 0.91 (s, 3 H, 14 -H), 1.15 -1.27 [complex signal, 4 H, 18-H, 17(19)-H a ], 1.37 [m, 2 H, 16(20) -H a ], 1.53 [broad d, J = 13.5 Hz, 2 H, 10(13) -H b], 1.65 [m, 2 H, 10(13) -H a ], 1.72 -1.83 [m, complex signal, 4 H, 16(20) -H b, 17(19)-H b], 1.85 (s, 2 H, 8 -H), 1.93 (tt, J = 11.5 Hz, J ?= 3.5 Hz, 1 H, 15 -H]), 1.97 [dt, J = 13 Hz, J ?= 1.5 Hz, 2 H, 6(12) -H b], 2.16 [ddm, J = 13.0 Hz, J ? = 6.5 Hz, 2 H, 6(12) -H a ], 3.06 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11)-H], 5.14 (broad s, 1 H, NH), 7.02 -7.08 [complex signal, 4 H, Ar -H]. 13C-NMR (125.7 MHz, CDCl 3) ?: 25.7 [CH 2, C17(19)], 25 .8 (CH 2, C18), 29.8 [CH 2, C16(20)], 32.2 (CH 3, C14), 33.6 (C, C9), 39.1 [CH 2, C6(12)], 41.0 [CH, C5(11)], 41.1 [CH 2, C10(13)], 46.4 (CH, C16), 47.2 (CH 2, C8), 54.0 (C, C7), 126.2 [CH, C2(3)], 127.9 [CH, C1(4)], 146.2 [d, C, C4a(C11a)], 175.3 (C, CO). 280 Materials & Methods MS (EI), m /z (%); significant ions: 338 (18), 337 (M ? + , 68), 282 (49), 212 (22), 211 (92), 210 (61), 169 (18), 156 (19), 155 (100), 154 (20), 143 (34), 141 (30), 129 (27), 128 (23), 83 (17), 55 (22). Elemental analysis: Calculated for C23 H 31NO: C 81.85% H 9.26% N 4.15% Found: C 81.82% H 9.34% N 4.14% Preparation of N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)thiourea , 76 a) N -[(9 -methyl-6,7,8,9,10,11-hexahydro -5,9:7,11-dimethano-5H-benzocyclononen-7- yl)carbamothioyl]benzamide formation: Benzoyl isothiocyanate (0.327 mL, 2.43 mmol) was added to a solution of amine 19 (500 mg, 2.21 mmol) in CHCl 3 (20 mL) and the reaction mixture was stirred at room temperature overnight. The resulting solution was concentrated in vacuo to give a brown gum (1.11 g). b) N -[(9 -methyl-6,7,8,9,10,11-hexahydro -5,9:7,11-dimethano-5H -benzocyclononen-7- yl)thiourea formation: K2CO3 (1.97 g, 14.26 mmol) and water (10 mL) were added to a solution of the aforementioned benzamide (1.11 g, aprox. 2.85 mmol) in methanol (22 mL) and THF (10 mL), and the mixture was stirred at room temperature overnight. The resulting suspension was filtered and the solids were washed with methanol (15 mL) to give 76 (330.3 mg, 52% overall yield) as a white solid. Analytical and spectroscopic data of compound 76: Melting point: 197-199 ?C. IR (ATR) ?: 3481, 3209, 3135, 3097, 3012, 2916, 2840, 1618, 1529, 1491, 1450, 1359, 1302, 1238, 1209, 1134, 1090, 1048, 1022, 941, 815, 799, 758, 719, 708, 643, 611 cm-1. 1H -RMN (400 MHz, DMSO -d6) ?: 0.88 (s, 3 H, 14 -H), 1.36 [broad d, J = 13.0 Hz, 2 H, 10(13)-H b], 1.61 [ddd, J = 13.0 Hz, J ? = 6.0 Hz, J ?? = 1.2 Hz, 2 H, 10(13) -H a ], 1.77 (s, 2 H, 8 - CHAPTER 2 281 H 2), 2.06 [ddd, J = 12.0 Hz, J ? = 5.6 Hz, J ?? = 2.0 Hz, 2 H, 6(12) -H a ], 3.04 [m, 2 H, 5(11) -H], 6.76 (broad s, 2 H, NH 2), 7.02-7.09 (complex signal, 4 H, Ar -H), 7.31 (b road s, 1 H, NH). 13C-RMN (100.6 MHz, DMSO -d6) ?: 32.1 (CH 3, C14), 33.2 [CH 2, C10(13)], 38.2 [CH, C5(11)], 41.0 [CH 2, C6(12)], 47.5 (CH 2, C8), 50.1 (C, C7), 55.0 (C, C9), 126.1 [CH, C2(3)], 127.7 [CH, C1(4)], 146.2 [d, C, C4a(C11a)], 181.2 (C, CS). MS-DIP (EI), m /z (%); significant ions: 287 (15), 286 (M ? + , 66), 285 (66), 212 (10), 211 [(C 16H 19) + , 50], 210 (14), 169 (15), 156 (17), 155 (100), 154 (12), 153 (12), 143 (37), 141 (33), 129 (29), 128 (24), 117 (11), 115 (24). HRMS -ESI+ m /z [ M +H ] + calcd for [C 17H 22N2S+H + ] : 287.1576, found: 287.1578. Preparation of 5,5 -dimethyl -2-[(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)amino]thiazol -4(5 H) -one, 61 Thiourea 76 (300 mg, 1.05 mmol), ethyl 2-bromoisobutyrate (0.29 mL, 1.95 mmol), DIPEA (0.36 mL) and abs. ethanol (3.5 mL) were placed into a microwave vial. The white suspension was heated to 130 ?C, 250 psi and 250 W for 3 hours. The solvents were then removed under vacuo and the resulting solid was partitioned between DCM (10 mL) and 0.5 N HCl solution (10 mL) and the phases were separated. The aqueous phase was extracted with further DCM (3 x 10 mL), and the combined organic phases were dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic layers gave a white solid (315 mg). Column chromatography (Al2O3, DCM/methanol mixtures) gave 61 (47 mg, 13% yield) as a white solid. The analytical sample was obtained washing the solid with pentane. Analytical and spectroscopic data of compound 61: Melting point: 294 ? 299 ?C. IR (ATR) ?: 3223, 2906, 1673, 1586, 1540, 1505, 1458, 1376, 1361, 1293, 1281, 1255, 1235, 1182, 1128, 1091, 1033, 1005, 951, 756, 651, 633, 602, 534 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 0.93 (s, 3 H, C -CH 3), 1.53 [d, J = 13.5 Hz, 2 H, 10(13) -H b], 1.62 [s, 6 H, C(C H 3)2 ], 1.67 [ddm, J = 12.5 Hz, J ? = 6.5 Hz, 2 H, 10(13) -H a ], 1.95 (s, 2 H, 8 - H), 2.17 [d, J = 12.5 Hz, 2 H, 6(12) -H b], 2.29 [dd, J = 12.5 Hz, J ? = 6.5 Hz, 2 H, 6(12) -H a ], 282 Materials & Methods 3.09 [t, J = 6.5 Hz, 2 H, 5(11) -H], 5.75 (broad s, 1 H, NH), 7.04 [m, H, 1(4) -H], 7.08 [m, H, 2(3)-H]. 13C-NMR (125.7 MHz, CDCl 3) ?: 27.9 [CH 3, C(CH 3)2], 32.0 (CH 3, C-CH 3), 33.9 (C, C9), 38.9 [CH 2, C6(12)], 40.8 [CH 2, C10(13)], 40.9 [CH, C5(11)], 47.1 (CH 2, C8), 59.4 (C, C7), 61.0 [C, C(CH 3)2 ], 126.5 [CH, C2(3)], 128.0 [CH, C1(4)], 145.7 [d, C, C4a(C11a)], 176.2 (C, CN), 193.8 (C, CO). MS (EI), m /z (%); significant ions: 355 (24), 354 (M ? + , 100), 285 (34), 211 [(C 16H 19) + , 35], 210 (11), 169 (14), 156 (15), 155 (100), 154 (10), 143 (35), 141 (29), 129 (27), 128 (21), 117 (11), 115 (18). Elemental analysis: Calculated for C21H 26N2OS: C 71.15% H 7.39% N 7.90% S 9.04% Calculated for C21H 26N2OS?0.75CH 3OH ?0.1DCM: C 67.81% H 7.60% N 7.24% S 8.28% Found: C 67.98% H 7.22% N 7.29% S 7.88% Preparation of N-(9 -hydroxy -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)piperidine -1-carboxamide , 62 1-Piperidinecarbonyl chloride (0.162 mL, 1.30 mmol) and triethylamine (0.328 mL, 2.36 mmol) were added to a solution of amine 46 (270 mg, 1.18 mmol) in DCM (10 mL) and the reaction mixture was stirred at room temperature o vernight. Saturated aqueous NaHCO 3 solution (15 mL) was added and the phases were separated. The aqueous phase was extracted with further DCM (2 x 15 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow solid (472 mg). Column chromatography (Al2O3, DCM/m ethanol mixture s) gave 62 (288 mg, 64% overall yield) as a white solid, that was washed with pentane for obtaining an analytical sample. Analytical and spectroscopic data of compound 62: Melting point: 188 ? 190 ? C. IR (ATR) ?: 3419, 2934, 2899, 2852, 1635, 1502, 1444, 1414, 1375, 1356, 1334, 1291, 1249, 1201, 1161, 1148, 1111, 1067, 1028, 992, 969, 907, 848, 762, 728, 625, 570, 544 cm-1. 1H -RMN (500 MHz, CDCl 3) ?: 1.48-1.59 [complex signal, 6 H, 16(18) -H 2, 17-H 2], 1.76 [d, J CHAPTER 2 283 = 13.0 Hz, 2 H, 10(13) -H b], 1.90 -2.02 [complex signal, 4 H, 6(12) -H b, 10(13)-H a ], 2.09 (s, 2 H, 8 -H 2), 2.17 [ddd, J = 13.0 Hz, J ?  Hz, J ?? 1 Hz, 2 H, 6(12) -H a ], 3.16 [tt, J = 6.5 Hz, J? 1 Hz, 2 H, 5(1 1)-H], 3.23 [m, 4 H, 15(19) -H 2], 7.05 [m, 2 H, 1(4) -H], 7.09 [m, 2 H, 3(2)-H]. 13C-RMN (125.7 MHz, CDCl 3) ?: 24.3 (CH 2, C17), 25.5 [CH 2, C16(18)], 39.4 [CH 2, C6(12)], 40.1 [CH, C5(11)], 42.4 [CH 2, C10(13)], 44.9 [CH 2, C15(19)], 49.1 (CH 2, C8), 56.3 (C, C7), 71.0 (C, C9), 126.5 [CH, C2(3)], 128.0 [CH, C1(4)], 145.4 [d, C, C4a(C11a)], 156.4 (C, CO). MS-DIP (EI), m /z (%); significant ions: 341 (11), 340 (M?+ , 48), 255 (41), 212 (9), 157 (13), 155 (22), 144 (13), 141 (13), 129 (35), 128 (33), 127 (43), 115 (21), 112 (20), 86 (19), 85 (70), 84 (100), 69 (20), 57 (10), 56 (14). Elemental analysis: Calculated for C21H 28N2O2: C 74.08% H 8.29% N 8.23% Calculated for C21H 28N2O2?0.45CH 3OH ?0.2C5H 12: C 73.01% H 8.7 9% N 7.59% Found: C 73.17% H 8.43% N 7.23% Preparation of N-(9 -methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)piperidine -1-carboxamide , 63 1-Piperidinecarbonyl chloride (0.247 mL, 1.98 mmol) and triethylamine (0.367 mL, 2.64 mmol) were added to a solution of amine 19 (300 mg, 1.32 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature overnight. To the resulting mixture was added saturated aqueous NaHCO 3 solution (15 mL) and the phases were separated. The aqueous phase was extracted with further DCM (2 x 15 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow solid (419 mg). Column chromatography (Al2O3, DCM/ methanol mixture s) gave 63 (265 mg, 59% yield) as a white solid. The analytical sample was obtained washing with pentane. Analytical and spectroscopic data of compound 63: Melting point: 170 ? 171 ? C. IR (ATR) ?: 3361, 2914, 2850, 1617, 1525, 1479, 1439, 1404, 1360, 1343, 1275, 1261, 1228, 284 Materials & Methods 1211, 1168, 1022, 978, 949, 852, 758, 576 cm-1. 1H -RMN (500 MHz, CDCl 3) ?: 0.91 (s, 3 H, C9 -CH 3), 1.49-1.59 [complex signal, 8 H, 10(13)-H b, 16(18)-H 2, 17-H 2], 1.65 [ddm, J = 13. 5 Hz, J ? = 6.5 Hz, 2 H, 10(13) -H a ], 1.84 (s, 2 H, 8 -H), 2.00 [d, J = 13.0 Hz, 2 H, 6(12) -H b], 2.16 [ddm, J = 13.0 Hz, J ? = 6.5 Hz, 2 H, 6(12) - H a ], 3.06 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 3.22 -3.25 [complex signal, 4 H, 15(19) - H], 4.25 (s, 1 H, NH ), 7.03 [m, 2 H, 1(4) -H], 7.06 [m, 2 H, 3(2) -H]. 13C-RMN (125.7 MHz, CDCl 3) ?: 24.4 (CH 2, C17), 25.6 [CH 2, C16(18)], 32.3 (CH 3, C9- CH 3), 33.7 (C, C9), 39.8 [CH 2, C6(12)], 41.2 [CH, C5(11)], 41.3 [CH 2, C10(13)], 44.9 [CH 2, C15(19)], 48.0 (CH 2, C8), 53.6 (C, C7), 126.1 [CH, C2(3)], 127.9 [CH, C1(4)], 146.4 [d, C, C4a(C11a)], 156.6 (C, CO). MS-DIP (EI), m /z (%); significant ions: 254 (18), 253 [ (M-C5H 11N)? + , 100] , 238 (25), 195 (10), 182 (14), 155 (32), 143 (13), 141 (15), 129 (17), 128 (15), 115 (15). Elemental analysis: Calculated for C22 H 30N2O: C 78.06% H 8.93% N 8.28% Found: C 78.05% H 9.11% N 8.06% Preparation of N-(9 -fluoro -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)piperidine -1-carboxamide , 64 1-Piperidinecarbonyl chloride (0.1 mL, 0.80 mmol) and triethylamine (0.213 mL, 1.54 mmol) were added to a solution of amine 48 (177.5 mg, 0.79 mmol) in DCM (9 mL) and the reaction mixture was stirred at room temperature overnight. To the resulting mixture was added saturated aqueous NaHCO 3 solution (10 mL) and the phases were separated. The aqueous phase was extracted with further DCM (2 x 10 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow gum (246.3 mg). Column chromatography (Al2O3, DCM/methanol mixtures) gave 64 (68 mg, 26% yield) as a white solid. The analytical sample was obtained b y crystallization with EtOAc/pentane. Analytical and spectroscopic data of compound 64: Melting point: 178 ? 179 ?C. CHAPTER 2 285 IR (ATR) ?: 3285, 2932, 2856, 1614, 1538, 1470, 1442, 1359, 1278, 1259, 1230, 1088, 1024, 1005, 968, 915, 865, 851, 804, 756, 648, 605, 570 cm-1. 1H -RMN (500 MHz, CDCl 3) ?: 1.50-1.60 [complex signal, 6 H, 16(18) -H 2, 17-H 2], 1.91 [d, J = 12.5 Hz, 2 H, 10(13) -H b ], 2.03 [d, J = 13.5 Hz, 2 H, 6(12) -H b], 2.12 -2.22 [complex signal, 4 H, 6(12) -H a, 10(13)-H a ], 2.25 (d, J C - F = 6.5 Hz, 2 H, 8 -H 2), 3.20-3.27 [complex signal, 6 H, 5(11)-H, 15(19) -H 2], 4.32 (s, 1 H, NH), 7.07 [m, 2 H, 1(4) -H], 7.11 [m, 2 H, 3(2) -H]. 13C-RMN (125.7 MHz, CDCl 3) ?: 24.4 (CH 2, C17), 25.6 [CH 2, C16(18)], 39.3 [CH 2, C6(12)], 39.6 [CH, d, 3 J C - F = 13.3 Hz, C5(11)], 40.1 [CH 2, d, 2J C - F = 20.1 Hz, C10(13)], 44.9 [CH 2, C15(19)], 46.8 (CH 2, d, 2J C - F = 17.8 Hz, C8), 51.1 (C, d, 3J C - F = 11.4 Hz, C7), 95.4 (C, d, 1J C - F = 173.2 Hz, C9), 126.8 [CH, C2(3)], 128.1 [CH, C1(4)], 144.9 [d, C, C4a(C11a)], 156.3 (C, CO). MS (EI), m /z (%); significant ions: 258 (17), 257 [(C 16H 16FNO)+ , 100], 215 (12), 170 (10), 159 (13), 155 (10), 141 (9), 129 (13), 128 (12), 115 (15). Elemental analysis: Calculated for C21H 27FN2O: C 73.65% H 7.95% N 8.18% Calculated for C21H 27FN2O?0.1EtOAc: C 73.17% H 7.98% N 7.97% Found: C 73.35% H 8.06% N 7.93% Preparation of N-(9 -methoxy -5,6,8,9,10,11 -hexahydro -7H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)piperidine -1-carboxamide , 65 1-Piperidinecarbonyl chloride (0.04 mL, 0.33 mmol) and triethylamine (0.09 mL, 0.62 mmol) were added to a solution of amine 252(76 mg, 0.31 mmol) in DCM (3.5 mL) and the reaction mixture was stirred at room temperature ove rnight. To the resulting mixture was added saturated aqueous NaHCO 3 solution (5 mL) and the phases were separated. The aqueous phase was extracted with further DCM (2 x 5 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow gum (201.4 mg). Column chromatography (Al2O3, DCM/ methanol mixture s) gave 65 (42 mg, 38% yield) as a white solid. Analytical and spectroscopic data of compound 65: 286 Materials & Methods Melting point: 172 ? 173 ? C. IR (ATR) ?: 3293, 2926, 2850, 2820, 1616, 1540, 1491, 1441, 1409, 1383, 1360, 1344, 1265, 1256, 1231, 1151, 1080, 1064, 1045, 1025, 994, 984, 969, 914, 848, 756, 638, 602 cm-1. 1H -RMN (500 MHz, CDCl 3) ?: 1.47-1.57 [complex signal, 6 H, 16(18) -H 2, 17-H 2], 1 .43 [d, J = 13.0 Hz, 2 H, 10(13) -H b], 1.96 [m, 2 H, 10(13) -H a ], 2.08 (s, 2 H, 8 -H 2), 2.07 [d, J = 13.5 Hz, 2 H, 6(12) -H b], 2.14 [m, 2 H, 6(12) -H a ], 3.18 [tt, J = 6.5 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 3.23-3.27 [complex signal, 7 H, 15(19) -H, C9 -OCH 3 ], 4.31 (s, 1 H, NH), 7.05 [m, 2 H, 1(4) - H], 7.09 [m, 2 H, 3(2) -H]. 13C-RMN (125.7 MHz, CDCl 3) ?: 24.4 (CH 2, C17), 25.5 [CH 2, C16(18)], 38.3 [CH 2, C6(12)], 39.6 [CH 2, C10(13)], 39.9 [CH, C5(11)], 44.9 [CH 2, C15(19)], 45.4 (CH 2, C8), 48.2 (CH 3, OCH 3), 56.1 (C, C7), 74.8 (C, C9), 126.5 [CH, C2(3)], 128.0 [CH, C1(4)], 145.5 [d, C, C4a(C11a)], 156.5 (C, CO). MS (EI), m /z (%); significant ions: 270 (18), 269 [ (C17H 19NO2) + , 100] , 238 (36), 226 (12), 195 (15), 182 (20), 171 (22), 159 (32), 158 (91), 155 (35), 153 (15), 150 (17), 141 (20), 129 (26), 128 (30), 127 (13), 115 (32). Elemental analysis: Calculated for C22 H 30N2O2: C 74.54% H 8. 53% N 7.90% Calculated for C22 H 30N2O2?0.1CH 3OH : C 74.21% H 8. 57% N 7.83% Found: C 74.11% H 8.54% N 7.67% Preparation of (2 R) -tert-butyl -2-[(9 -fluoro -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)carbamo yl]pyrrolidine -1-carboxylate, 78 Boc-D-proline (55 mg, 0.26 mmol), HOBt (52 mg, 0.38 mmol), EDC (59 mg, 0.38 mmol) and triethylamine (0.14 mL, 1.02 mmol) were added to a solution of amine 48?HCl (75 mg, 0.28 mmol) in EtOAc (4 mL) and the mixture was stirred at room temperature CHAPTER 2 287 overnight. To the resulting suspension was then added water (5 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO 3 solution (5 mL) and brine (5 mL), dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic layers gave 78 (110 mg, 92% yield) as a beige solid. The product was used in next step without further purification or characterization. Preparation of (2 R) -N-(9 -fluoro -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide, 79 A solution of Boc-protected pyrrolidine 78 (110 mg, 1.26 mmol) in DCM (2 mL) and 85% o-phosphoric acid (0.26 mL, 3.90 mmol) was stirred at room temperature for 4 hours. To the reaction mixture was then added water (5 mL) and the aqueous phase was basified until pH ~12 with 5 N NaOH solution. The phases were separated and the aqueous phase was extracted with further DCM (2 x 5 mL). The combined organic layers were dried over anh. Na2SO4, filtered and evaporated in vacuo to give 79 (82 mg, quantitative yield) as a maroon gum. The product was used in next step without further characterization or purification. Preparation of (2 R) -1-ethyl -N-(9 -fluoro -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl)pyrrolidine -2-carboxamide , 77 ?tartrate A solution of pyrrolidine 79 (82 mg, 0.25 mmol) in DMF (1.5 mL) was cooled to 5 ?C with an ice bath. Then KI (4 mg, 0.03 mmol) and triethylamine (0.14 mL, 1.00 mmol) were added, followed by the dropwise addition of a solution of ethyl bromide (29 mg, 0.26 mmol) in DMF (0.5 mL). The reaction mixture was stirred at room temperature in an ice - water bath overnight. To the resulting solution was added EtOAc (5 mL) and water (5 mL). The phases were separated and the aqueous layer was ext racted with further EtOAc (2 x 5 mL). The combined organic phases were washed with saturated aqueous NaHCO 3 solution 288 Materials & Methods (5 mL) and brine (5 mL), dried over anh. Na2SO4, filtered and concentrated in vacuo to give an orange oil (74 mg). Column chromatography (SiO2, DCM/methanol mixtures) gave 77 (33 mg, 35% yield) as a white solid. A solution of L-(+) -tartaric acid (11 mg, 0.07 mmol) in methanol (1 mL) was added to 77 directly. The solvents were removed under vacuo to give 77 as its tartrate salt. Analytical and spectroscopic data of compound 77?tartrate: Melting point: 94-96 ?C. IR (ATR) ?: 3500-2800 (3432, 3381, 3285, 3063, 2923, 2856), 2553, 2363, 2182, 1962, 1721, 1667, 1563, 1441, 1395, 1358, 1341, 1301, 1235, 1118, 1074, 997, 863, 757, 663 cm- 1. 1H -RMN (500 MHz, CD 3OD) ?: 1.28 (t, J = 7.5 Hz, 3 H, NCH 2CH 3), 1.84 [d, J = 11.5 Hz, 2 H, 10(13)-H b], 1.99 (complex signal, 2 H, 16 -H a, 17-H a), 2.08 [d, J = 13 Hz, 2 H, 6(12) -H b], 2.16 [complex signal, 6 H, 6(12) -H a, 10(13)-H a, 17-H a ], 2.24 (d, J = 6.5 Hz, 2 H, 8 -H), 2.50 (m, 1 H, 16 -H b], 3.17 (complex signal, 3 H, NC H 2CH 3, 18-H a), 3.27 [m, 2 H , 5(11)-H], 3.68 (m, 1 H, 18 -H b), 3.95 (dd, J = 8.5 Hz, J ?  Hz, 1 H, 15 -H), 4.45 (s, 2 H, tartrate -CH ), 7.11 [complex signal, 4 H, Ar -H] . 13C-RMN (125.7 MHz, CD 3OD) ?: 11.4 (CH 3, NCH 2CH 3), 24.0 (CH 2, C17), 31.1 (CH 2, C16), 39.1 [CH 2, d, 4JC-F = 7.4 Hz, C6(12)], 40.9 [CH, d, 3JC-F = 12.9 Hz, C5(11)], 41.2 [CH 2, d, 2JC-F = 20.1 Hz, C10(13)], 46.6 (CH 2, d, 2JC-F = 18.9 Hz, C8), 51.4 (CH 2, NCH 2CH 3), 55.6 (CH 2, C18), 59.3 (C, d, 3JC-F = 11.2 Hz, C7), 68.6 (CH, C15), 73.8 (CH, tartrate -CH), 94.6 (C, d, 1JC-F = 177.6 Hz, C9), 128.1 [CH, C2(3)], 129.2[CH, C1(4)], 146.1 [C, C4a(C11a)], 168.4 (C, CO), 176.9 (C, tartrate-CO). MS (DEPEI), m /z (%); significant ions: 99 (27), 98 (100), 97 (26), 96 (11), 76 (13), 71 (10), 70 (34), 69 (14), 68 (11). HRMS -ESI+ m /z [ M +H ] + calcd for [C 22H 29FN2O+H + ]: 357.2337, found: 357.2338. Hexacycl ic scaffolds CHAPTER 2 291 Preparation of dimethyl pentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8,9 -dicarboxylate, 82391 a) Formation of 9,10-dihydrofulvalene, 81: In a 2 L reactor equipped with a mechanic stirrer, thermometer, gas inlet and pressure- equalizing dropping funnel was prepared a suspension of 95% dry sodium hydride (50 g, 2.08 mol) in anh. THF (1 L). The stirred suspens ion was cooled to ~0 ?C with an ice bath and then freshly distilled cyclopentadiene (137 g, 2.08 mol) was added dropwise over 70 minutes avoiding excess foaming. After the addition, the cooling bath was removed and the resulting dark red colour suspension was stirred at room temperature for one hour. Aferwards, copper(I)-bromide-dimethyl sulphide complex (750 mg) was added and the mixture was cooled to -78 ?C with a dry ice in acetone bath. A solution of iodine (265 g, 1.04 mol) in anh. THF (400 mL) was added dropwise over 2 hours without re aching a temperature greater than -40 ?C. The dark suspension was stirred at low temperature for 15 minutes. b) Diels-Alder reaction of 81 with dimethyl acetylenedicarboxylate: Dimethyl acetylenedicarboxylate (165 g, 1.16 mol) was added rapidly dropwise over 10 minutes to the previous slurry. The mixture was kept at low temperature for 30 minutes and then allowed to reach room temperature with the stirring maintained for 4 hours. The reaction mi[ture Zas the Iiltered through a Celite? pad and the solid Zas Zashed repeatedly with THF (1 L). The combined filtrates were concentrated under vacuo at a temperature not above 40 ?C with the obtaining of a dark oil, which was dissolved in diethyl ether (750 mL) and stirred at room temperature for 15 minutes. The mixture was again Iiltered through Celite? pad and evaporated under vacuo without exceeding 40 ?C to give a mixture of diesters 82 and 83, which was stored at 0 ?C until next step. c) Selective hydrolysis and isolation of 82: 292 Materials & Methods In a 2 L reactor equipped with mechanical stirrer, thermometer, gas outlet and pressure-equalizing dropping funnel was dissolved the concentrated from part b in methanol (1 L) and the solution was cooled to -5 ?C by means of an ice/ NaCl bath. Then a precooled (0 ?C) solution of potassium hydroxide (110 g, 85%, 1.67 mol) in water (200 mL) was added dropwise at such a rate as to keep the reaction temperature below 10 ?C. The reaction mixture was stirred at 0 ?C for 2 hours and at room temperature for an additional 2 hours prior to the addition of glacial acetic acid (50 mL), followed by powder sodium carbonate until pH ~ The resulting suspension Zas then Iiltered through Celite? pad. The filtrate was concentrated at 40 ?C under vacuo to about 500 mL, the residue was diluted with water (1 L) and extracted with petroleum ether (6 x 300 mL). The combined organics were washed with 10% sodium thiosulfate solution (2 x 200 mL), dried over anh. Na2SO4, filtered and concentrated under reduced pressure at 40 ?C to give the diester 82 as an orange solid (20.0 g, 7% overall yield), whose spectroscopic data were in agreement with those described in the bibliography. Preparation of pent acyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8,9 -dicarboxylic acid, 88391 To a solution of pentacyclic diester 82 (20.0 g, 73.4 mmol) in methanol (75 mL) was added a solution of potassium hydroxide (20 g , 85%, 0.30 mol ) in water (75 mL). The reaction mixture was heated at reflux for 1 hour. The methanol was removed from the resulting brown solution under vacuo, water (145 mL) was added and the solution was heated for further 5 hours. Thereafter, activated charcoal (6 g) was added and the black suspension Zas stirred at room temperature overnight )iltration through Celite? pad Zas followed by cooling the filtrate with an ice bath. Conc. HCl was added dropwise until p H 1 with the formation of a white solid, which was filtered under vacuo. The solid was dissolved in EtOAc (1 L), dried over anh. Na2SO4, filtered and concentrated under reduced pressure to give the diacid 88 as a white solid. In order to recover further amounts of desired product, the aqueous layer was extracted with EtOAc (3 x 50 mL) and the combined organics were dried over anh. Na2SO4, filtered and concentrated under vacuo to give a total amount of 14.5 g (81% yield) of diacid 88. The spectroscopic data were identical to those previously published. CHAPTER 2 293 Preparation of 3-azahexacyclo[7 . 6. 0. 01, 5.0 5, 12.0 6, 10.0 11,15 ]pentad eca-7,1 3-diene -2,4 -di one, 8990 Pentacyclic diacid 88 (3.60 g, 14.8 mmol) and urea (4.44 g, 74.0 mmol) were placed in a round-bottom flask equipped with a magnetic stirrer, gas outlet and Liebig condenser apparatus. The mixture wa s heated slowly to fusion (150 ?C) and once melted, the resulting paste was heated at 220 ?C for 30 minutes. Then the reaction mixture was tempered, water (120 mL) was added and the suspension was extracted with DCM (7 x 50 mL). The combined organic phases were washed with brine (100 mL), dried over anh. Na2SO4 and filtered. Evaporation under vacuo gave the imide 89 as a white solid (2.85 g, 86% yield). The spectroscopic data matched with those described in the bibliography. Preparation of 3 -azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadeca -7,13 -diene, 9090 To a suspension of pentacyclic imide 89 (2.85 g, 12.6 mmol) in anh. toluene (90 mL) was added dropwise Red-Al? (17.9 mL, 63.4 mmol) under nitrogen, keeping the temperature lower than 35 ?C and with some gas releasing. The reaction mixture was heated at reflux for 48 hours, monitoring the starting material conversion by TLC. Then the solution was tempered to room temperature and quenched with 30 wt. % aqueous KOH solution (45 mL). The phases were separated and the aqueous layer was extracted with DCM (3 x 25 mL). The combined organic phases were dried over anh. Na2SO4, filtered and evaporated under vacuo to give a brown oil. In order to purify the crude, this was dissolved in DCM (20 mL) and extracted with 1 N HCl solution (2 x 15 mL). The pH of the combined aqueous phases was adjusted to 12 with 5 N NaOH solution and extracted with DCM (3 x 20 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under vacuo to give the amine 90 as a brownish oil (2.28 g, 92% yield), its spectroscopic data and those previously published were identical. 294 Materials & Methods Preparation of 3 -azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadecane hydrochloride, 95?HCl90 A suspension of amine 90 (231 mg, 1.17 mmol) and 5 wt. % palladium on carbon (50% in water, 37 mg , 16% of the weight of 90) in abs. ethanol (37 mL) was stirred at room temperature and atmospheric pressure under hydrogen for 4 hours. The suspension was then filtered and the solids were washed with EtOH (15 mL). An excess of HCl/Et 2O was added and the solvents were removed under vacuo to give the reduced amine 95?HCl (239 mg, 86% yield) as white solid. The s pectroscopic data matched with those described in the bibliography. Preparation of tert-butyl ( R) -2-( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ] -pentadecane -3- carbonyl )pyrrolidine -1-carboxylate, 96 To a solution of amine 95 (239 mg, 1.01 mmol) in ethylacetate (15 mL) were added Boc-D-Proline (197 mg, 0.92 mmol), HOBt (188 mg, 1.38 mmol), EDC (214 mg, 1.38 mmol) and triethylamine (0.560 mL, 4.05 mmol) and the reaction mixture was stirred at room temperature overnight. To the resulting suspension was then added water (15 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO 3 solution (15 mL) and brine (15 mL), dried over anh. Na2SO4 and filtered. The organic layer was concentrated under vacuo to give 96 along with reaction by-products (360 mg, quantitative yield) as a clear oil. The product was used in the next step without further purification or characterization. CHAPTER 2 295 Preparation of 3-[ (2 R) -prolyl] -3-azahexacyclo[ 7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadecane , 97 A solution of Boc-protected pyrrolidine 96 (385 mg, 0.97 mmol) in DCM (5 mL) and 85 wt. % o-phosphoric acid (1.00 mL, 14.5 mmol) was stirred at room temperature for 6 hours. To the reaction mixture was then added water (10 mL) and the aqueous phase was basified until pH ~12 with 5 N NaOH solution. The phases were separated and the aqueous phase was extracted with further DCM (2 x 10 mL). The combined organic layers were dried over anh. Na2SO4, filtered and evaporated in vacuo to give a brown oil (202 mg). The crude was purified by column chromatography (Al2O3, DCM/m ethanol mixture). The appropriate fractions were combined and concentrated under reduced pressure to give 97 (104 mg, 36% yield) as a white solid that was used in the next step without further purification or characterization. Preparation of 3-[ (2 R) -N-ethylprolyl] -3- azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadecane , 98 ?tartrate A solution of pyrrolidine 97 (98 mg, 0.33 mmol) in DMF (2 mL) was cooled to 5 ?C with an ice bath. Then potassium iodide (5.5 mg, 10% mmol ) and triethylamine (0.180 mL, 1.32 mmol) were added, followed by the slowly addition of ethyl bromide (37.6 mg, 0.35 mmol). The reaction mixture was stirred at room temperature in an ice -water bath overnight. Then the resulting precipitated was filtered off and washed with EtOAc (5 mL). The filtrate was washed with saturated aqueous NaHCO 3 solution (5 mL) and brine (5 mL), dried over anh. Na2SO4, filtered and concentrated in vacuo to give clear oil (90 mg). A solution of tartaric acid (41 mg, 0.28 mmol) in methanol (1 mL) was added to the crude directly. The solvents were removed under vacuo to give 98 as tartrate salt (106 mg, 68% yield). Analytical and spectroscopic data of compound 98?tartrate: Melting point: 184 - 185 ?C. 296 Materials & Methods IR (ATR) ?: 3450-2800 (3453, 3038, 2931, 2868), 1712, 1632, 1463, 1424, 1355, 1227, 1201, 1118, 1074, 1027, 893, 836, 678 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.31 (t, J = 7 Hz, 3 H, C H 3CH 2), 1.48-1.63 (complex signal, 8 H, 7 -H 2, 8-H 2, 13-H 2, 14-H 2), 2.03-2.14 (complex signal , 2 H, ?-H a ?-H a), 2.16-2.26 (complex signal, 5 H, 6 -H, 9 -H, 12 -H, 15 -H, ?-H b), 2.49 (m, 2 H, 10 -H, 11 -H), 2.66 (m, 1 H, ?-H b), 3.20 (m, 1 H, ?-H a), 3.28 (complex signal, 4 H, 2 -H 2 or 4-H 2, CH 3CH 2), 3.51 (m, 2 H, 4 -H 2 or 2-H 2), 3.78 (m, 1 H, ?-H b), 4.41 (s, 2.2 H, tartrate -CH ), 4.53 (m, 1 H, 2?-H) . 13C-NMR (125.7 MHz, CD 3OD) ?: 11.4 (CH 3, CH 3CH 2), 22.3 (CH 2), 22.4 (CH 2), 22.6 (CH 2), and 22.7 (CH 2), (C7, C8, C13 and C14), 24.0 (CH 2, C4?  2 (CH 2, C3?  42.6 (CH 2, C2 or C4), 43.1 (CH 2, C4 or C2), 50.6 (CH, C10 or C11), 51.2 (CH, C11 or C10), 51.5 (CH 2, CH 3CH 2), 55.5 (CH 2, C5?  55.8 (CH), 55.9 (CH), 56.3 (CH) and 56.5 (CH) (C6, C9, C12 and C15), 56.4 [d, CH, C12(15) or C6(9)], 58.8 (C, C1 or C5], 60.8 (C, C5 or C1], 67.6 (CH, C2 ?  1 (CH, tartra te-CH), 167.3 (C, CO), 176.6 (C, tartrate -CO). MS (CI), m/z (%); significant ions: 327 [(M ? + )+1, 100], 325 (41), 258 (14), 230 (19), 98 (68). Elemental analysis: Calculated for C21H 30N2O?1C4H 6O6: C 63.01% H 7.61% N 5.88% Calculated for C21H 30N2O?1.25C4 H 6O6: C 60.75% H 7.35% N 5.45% Found: C 61.03% H 7.51% N 5.19% Preparation of ( 4-amino-3,5 -dichloro phenyl) ( 3- azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ] pentadeca -7,13 -dien -3-yl)methanone, 99 To a solution of amine 90 (400 mg, 2.03 mmol) in EtOAc (30 mL) and DMF (2 mL) were added 4-amino-3,5-dichlorobenzoic acid (380 mg, 1.85 mmol), 1-hydroxybenzotriazole (HOBt ) (375 mg, 2.78 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (430 mg, 2.78 mmol) and triethylamine (0.560 mL, 4.07 mmol) and the reaction mixture was stirred at room temperature overnight. To the resulting suspension was then added water (20 mL) and the phases were separated. The organic phase was washed with saturated aqueous NaHCO 3 solution (20 mL) and brine (20 mL), dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the organic phase gave 677 mg of a yellow solid. The crude CHAPTER 2 297 was purified by column chromatography (Al2O3, DCM/m ethanol mixture). The appropriate fractions were combined and concentrated under vacuo to give compound 99 as a white solid (332 mg, 47% yield). Analytical and spectroscopic data of compound 99: Melting point: 236 - 238 ?C. IR (ATR) ?: 3449, 3306, 3250, 3204, 2954, 2867, 2150, 1597, 1538, 1501, 1456, 1410, 1384, 1342, 1316, 1295, 1244, 1225, 1192, 1054, 1002, 943, 896, 875, 791, 745, 733, 686, 667, 643 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 2.61 [m, 2 H, 10(11) -H], 2.85 [ broad s, 2 H, 6( 12)-H or 9(15)- H], 2.94 [ broad s, 2 H, 9(15)-H or 6( 12)-H], 3.13 (broad s, 2 H, 2-H or 4 -H), 3.33 ( broad s, 2 H, 4 -H or 2 -H), 5.91 [ m, 2 H, 7( 13)-H or 8(14)-H], 6.03 [ m, 2 H, 8(14)-H or 7( 13)-H], 7.19 (s, 2 H, Ar -H). 13C-NMR (125.7 MHz, CDCl 3) ?: 45.5 (CH 2, C2 or C4), 49.9 (CH 2, C4 or C2), 61.8 [CH, C6(12), C9(15)], 62.8 [CH, C10(11)], 68.8 (C, C1 or C5), 71.0 (C, C5 or C1), 118.7 (C, Ar - Cmeta), 126.7 (C, Cipso), 127.1 (CH, Ar -Cortho), 132.9 [CH, C7( 13) or 8(14)], 133.9 [CH, C 8(14) or C7(13)], 141.3 (C , Ar-Cpara), 166.8 (C, CO). MS (EI), m/z (%); significant ions: 388 [(C 21H 1837Cl2N2O)?+ , 2], 386 [(C 21H 1837Cl35ClN2O)?+ , 12], 384 [(C 21H 1835Cl2N2O)? + , 18], 192 [(C 7H 437Cl2NO) + , 10], , 190 [(C 7H 437Cl35ClNO) + , 63], 188 [ (C7H 435Cl2NO) + , 100] , 180 (12), 160 (12), 124 (16). HRMS -ESI+ m/z [ M +H ] + calcd for [C 21H 18Cl2N2O+H] + : 385.0869, found: 385.0875. Preparation of ( 4-amino-3,5 -dichloro phenyl) ( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ] pentadecan -3-yl ) methanone, 100 To a solution of amide 99 (200 mg, 0.78 mmol) in abs. ethanol (24 mL) was added 5 wt. % palladium on carbon (50% in water, 24 mg, 8% of the weight of 99) and the suspension was hydrogenated at room temperature and atmospheric pressure for 5 hours. The reaction mixture was then filtered , the solids were washed with EtOH (15 mL) and the solvent was removed under vacuo to give a yellow solid (207 mg). Column chromatography 298 Materials & Methods (Al2O3, DCM/m ethanol mixture) gave the desired amide 100 (180 mg, 89% yield) as white solid. Analytical and spectroscopic data of compound 100: Melting point: 243 - 244 ?C. IR (ATR) ?: 3455, 3283, 3238, 3186, 2931, 2865, 1631, 1597, 1545, 1498, 1456, 1425, 1332, 1307, 1269, 1232, 1216, 1118, 1070, 1035, 944, 895, 789, 769, 745, 693, 647, 629 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.43-1.60 [complex signal, 8 H, 7( 13)-H 2, 8(14)-H 2], 2.04 [ broad s, 2 H, 6( 12)-H or 9(15)-H ], 2.12 [ broad s, 2 H, 9(15)-H or 6( 12)-H] , 2.41 [ m, 2 H, 10(11)-H], 3.2 9 (s, 2 H, 2 -H 2 or 4-H 2), 3.53 (s, 2 H, 4 -H 2 or 2-H 2), 7.40 (s, 2 H, Ar -H). 13C-NMR (125.7 MHz, CDCl 3) ?: 21.5 [CH 2, C7(13) or C8(14)], 21.8 [CH 2, C8(14) or C7(13)], 40.8 (CH 2, C2 or C4), 45.5 (CH 2, C4 or C2), 49.6 [CH, C10(11)], 54.8 [ broad CH, C6( 12) and C9(15)], 57.9 (C, C1 or C5), 59.6 (C, C5 or C1), 118.8 (C, Ar-Cmeta), 126.9 (C, Cipso), 127.2 (CH, Ar -Cortho), 141.4 (C, Ar-Cpara), 170.0 (C, CO). MS (EI), m/z (%); significant ions: 392 [(C 21H 2237Cl2N2O)?+ , 8], 390 [(C 21H 2237Cl35ClN2O)?+ , 42], 388 [(C 21H 2235Cl2N2O)? + , 63], 200 (18), 192 [(C 7H 437Cl2NO) + , 11], 190 [(C 7H 437Cl35ClNO) + , 64], 188 [(C 7H 435Cl2NO) + , 100], , 184 (33), 169 (13), 160 (12), 124 (13). Elemental analysis: Calculated for C21H 22Cl2N2O: C 64.79% H 5.70% Cl 18.21% N 7.20 % Calculated for C21H 22Cl2N2O?0.50H 2O: C 63.32% H 5.82% Cl 17.80% N 7.03% Found: C 63.23% H 5.71 % Cl 17.82% N 6.77% Preparation of ( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ] pentadeca -7,13 -dien -3- yl)(cyclohexyl)methanone , 101 To a solution of amine 90 (400 mg, 2.03 mmol) in ethylacetate (30 mL) were added cyclohexanecarboxylic acid (237 mg, 1.85 mmol), HOBt (375 mg, 2.78 mmol), EDC (430 mg, 2.78 mmol) and triethylamine (0.570 mL, 4.07 mmol) and the reaction mixture was stirred at room temperature overnight. To the resulting suspension was then added water (20 mL) and the phases were separated. The organic phase was washed with saturated CHAPTER 2 299 aqueous NaHCO 3 solution (20 mL) and brine (20 mL), dried over anh. Na2SO4 and filtered. Evaporation in vacuo of the combined organic layers gave an orange oil (627 mg). The crude was purified by column chromatography (Al2O3, DCM/m ethanol mixture). The appropriate fractions were combined and concentrated under vacuo to give the desired amide 101 as a white solid (260 mg, 42% yield). The analytical sample was obtained by crystallization from tert-butanol. Analytical and spectroscopic data of compound 101: Melting point: 111 - 113 ?C. IR (ATR) ?: 2961, 2930, 2853, 1630, 1443, 1425, 1347, 1305, 1223, 1194, 1138, 1067, 998, 897, 878, 830, 780, 749, 734, 696, 659, 646 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.17-1.24 [complex signal, 3 H, ?-H ax  ?(? -H ax ], 1.43 [complex signal, 2 H, 2?(? -H ax ], 1.60 -1.66 [complex signal, 3 H, 2?(? -H eq ?-H eq], 1.75 -1.78 [complex signal, 2 H, ?(? -H eq], 2.13 (tt, J = 12.0 Hz, J ? = 3.5 Hz, 1 H, 1?-H), 2.67 [m, 2 H, 10(11)-H], 2.98 [complex signal, 4 H, 6(12) -H, 9(15) -H], 3.18 (s, 2 H, 2 -H 2 or 4-H 2), 3.19 (s, 2 H, 4 -H 2 or 2-H 2), 6.00 [d dd, J = 6 Hz, J ?= 3 Hz, J ??= 1.5 Hz, 2 H, 7(13) -H or 8(14) -H], 6.04 [ddd, J = 6 Hz, J ?= 3 Hz, J ??= 1 Hz, 2 H, 8(14) -H or 7(13) -H]. 13C-NMR (125.7 MHz, CDCl 3) ?: 25.8 (CH 2, C4?  2 [CH 2, C3?(? @ 2 [CH 2, C2?(? @ 42.5 (CH, C1 ?   (CH 2, C2 or C4), 46.7 (CH 2, C4 or C2), 62.0 [CH, C6(12) and C9(15)], 62.8 [CH, C10(11)], 69.1 (C, C1 or C5), 70.8 (C, C5 or C1), 132.8 [CH, C7(13) or 8(14)], 134.1 [CH, C8(14) or C7(13)], 174.4 (C, CO). MS (EI), m/z (%); significant ions: 308 (20), 307 (M ? + , 83), 252 (16), 242 (25), 198 (17), 197 (100), 196 (C14H 14N?+ , 39), 182 (19), 181 (15), 180 (41), 179 (16), 168 (24), 167 (25), 166 (15), 165 (38), 156 (21), 153 (23), 152 (27), 132 (64), 131 (100), 130 (64), 128 (17), 118 (19), 117 (19), 115 (23), 91 (18), 83 [(C 6H 11) + , 85], 77 (15), 55 (72). HRMS -ESI+ m/z [ M +H ] + calcd for [C 21H 25NO+H] + : 308.2009, found: 308.2003. Preparation of ( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ] pentadecane -3- yl)(cyclohexyl)methanone , 102 300 Materials & Methods To a solution of amide 101 (118 mg, 0.40 mmol) in abs. ethanol (13 mL) was added 5 wt. % palladium on carbon (50% in water, 12.6 mg , 10% of the weight of 101) and the suspension was hydrogenated at room temperature and atmospheric pressure for 5 hours. The reaction mixture was then filtered, the solids were washed with EtOH (15 mL) and the solvents were removed under vacuo to give the amide 102 (94 mg, 78% yield) as white solid. The analytical sample was obtained by crystallization from DCM/diethyl ether . Analytical and spectroscopic data of compound 102: Melting point: 134 - 135 ?C. IR (ATR) ?: 2932, 2851, 1621, 1463, 1426, 1357, 1286, 1202, 1120, 1104, 1036, 1013, 969, 925, 886, 860, 825, 768, 732, 702, 657, 627 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.25 [ complex signal, 3 H, ?-H ax  ?(? -H ax ], 1.48 -1.57 [complex signal, 10 H, 7(13) -H 2, 8(14)-H 2, 17(21)-H ax ], 1.68 (m, 1 H, ?-H eq), 1.75-1.82 [complex signal, 4 H, 2?(? -H eq ?(? -H eq ], 2.08 (broad signal, 4 H, 6(12) -H, 9(15) -H], 2.36 (tt, J = 12 Hz, J ?= 3.5 Hz, 1 H, 1?-H), 2.41 [m, 2 H, 10(11) -H] , 3.29 [s, 4 H, 2(4) -H 2 ]. 13C-NMR (125.7 MHz, CDCl 3) ?: 21.5 [CH 2, C7(13) or C8(14)], 21.8 [CH 2, C8(14) or C7(13)], 25.8 (CH 2, C4?  2 [CH 2, C3?(? @ 20 [CH 2, C2?(? @ 0 (CH 2, C2 or C4), 42.0 (CH 2, C4 or C2), 42.9 (CH, C1 ?   [CH, C10(11)], 55.0 [CH, C6(12) or C9(15)], 55.1 [CH, C9(15) or C6(12)], 57.5 (C, C1 or C5], 59.3 (C, C5 or C1], 175.1 (C, CO). MS (EI), m/z (%); significant ions: 312 (23), 311 (M ? + , 100), 270 (14), 257 (19), 256 (99), 243 (12), 228 [( C15 H 18NO) + 13], 202 (12), 201 (30), 200 (13), 184 (29), 169 (11), 129 (15), 128 (15), 91 (16), 83 [(C 6H 11) + , 24], 55 (25). Elemental analysis: Calculated for C21H 29NO: C 80.98% H 9.39% N 4.50% Found: C 80.76% H 9.61% N 4.33% Preparation of ( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadeca -7,13 -diene -3- yl ) ( piperidin -1-yl)methanone , 103 To a solution of amine 90 (400 mg, 2.03 mmol) in DCM (18 mL) were added 1- piperidinecarbonyl chloride (0.26 mL, 2.13 mmol) and triethylamine (0.56 mL, 4.06 mmol) CHAPTER 2 301 and the mixture was stirred at room temperature overnight. To the resulting mixture was added saturated aqueous NaHCO 3 solution (20 mL) and the phases were separated. The aqueous phase was extracted with further DCM (2 x 10 mL), and the combined organic phases were dried over anh. Na2SO4, filtered and concentrated in vacuo to give a yellow oil (719 mg). Column chromatography (Al2O3, DCM/Methanol mixture) gave 103 (443 mg, 71% yield) as a clear oil. Several attempts to crystallize this product met with failure. The product was used in the next step without further purification or characterization. MS (EI), m/z (%); significant ions: 308 (M? + , 46), 196 [(C 14H 14N) + , 17], 165 (14), 130 (15), 112 [(C 6H 10NO) + , 100] , 84 [(C 5 H 10N) + , 17], 69 (41). Preparation of ( 3-azahexacyclo[7.6.0.0 1,5 .0 5,12 .0 6,10 .0 11,15 ]pentadeca -3-yl )(piperidin -1- yl)methanone , 104 A suspension of urea 103 (235 mg, 0.76 mmol) and 5 wt. % palladium on carbon (50% in water, 24 mg, 10% of the weight of 103) in abs. ethanol (25 mL) was stirred at room temperature and atmospheric pressure under hydrogen for 3 hours. The suspension was then filtered and the solids were washed with EtOH (1 0 mL). The solvents were removed under vacuo to give the reduced urea 104 (171 mg, 72% yield) as white solid. Analytical and spectroscopic data of compound 104: Melting point: 124 - 126 ?C. IR (ATR) ?: 3457, 3291, 2936, 2867, 2839, 1615, 1538, 1461, 1415, 1370, 1332, 1304, 1252, 1226, 1202, 1159, 1124, 1110, 1027, 991, 915, 882, 851, 767, 722, 632 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.47-1.58 [complex signal, 14 H, 7(8 ,13,14)-H 2, ?(?)-H 2, ?- H 2], 2.05 [ m, 4 H, 6(9 ,12,15)-H ], 2.38 [m , 2 H, 10(11)-H], 3.19 [m, 8 H, 2(4)-H 2, 2?(?)-H 2]. 13C-NMR (125.7 MHz, CDCl 3) ?: 21.7 [CH 2, C7(8, 13, 14)], 24.8 ( CH 2, C?), 25.9 [CH 2, C?(?)], 43.3 [CH 2, C2(4)] , 47.8 [CH 2, C16(20)], 49.6 [CH, C10(11)], 54.8 [CH, C6(9, 12, 15)], 58.8 [ C, C1(5)] , 163.3 (C, CO). MS (EI), m/z (%); significant ions: 312 (M ? + , 100), 201 (15), 200 [ (C14H 18N) + , 82] , 184 (33), 129 (36), 112 [(C 6H 10NO) + , 54] , 91 (16), 84 [(C 5H 10N) + , 59] , 69 (28). Elemental analysis: 302 Materials & Methods Calculated for C20 H 28N2O: C 76.88% H 9.03% N 8.97% Found: C 76.60% H 9.21% N 8.74% C hapter 3 Chemist r y CHAPTER 3 307 Preparation of N-benzyl(2 -oxaadaman tan-1-yl)amine hydrochloride, 126 ?HCl500 In a three-necked round bottom flask equipped with a magnetic stirrer and gas inlet, benzylamine (4.36 mL, 40.3 mmol) was added to a solution of diketone 125 (6 g, 39.4 mmol) in anh. THF (120 mL). The reaction mixture was heated at reflux for 0.5 hours. Afterwards the solution was cooled in an ice bath. In a separate flask, lithium aluminium hydride (3.16 g, 78.9 mmol) was added in small portions to anh. Et2O (80 mL). Then the cooled solution prepared before was added dropwise under vigorous stirring. After the addition, the suspension was heated to 40 ? C for 6 hours. The reaction mixture was tempered to room temperature and filtered through a Celite? pad and the filtrate was evaporated under vacuo. The residue was partitioned between DCM (50 mL) and water (50 mL) and the phases were separated. The aqueous layer was extracted with further DCM (3 x 50 mL). The combined organic phases were washed with brine (100 mL), dried over anh. Na2SO4, filtered and concentrated under vacuo. To a solution of the residue in acetone (120 mL) was added an excess of conc. HCl. The formed precipitated was filtered under vacuo to give 126?HCl (6.25 g, 57% yield) as a white solid. The spectroscopic data was identical to those previously described. Preparation of (2 -oxaadama ntan-1-yl)amine hydrochloride, 120 ?HCl501 A suspension of benzilamine 126 (720 mg, 2.58 mmol) and 5% wt. palladium on carbon (55 mg) in abs. ethanol (80 mL) was hydrogenated at room temperature and atmospheric pressure until no further hydrogen consumption was observed. The suspension was then filtered and the solid washed with methanol (30 mL). An excess of Et2O?HCl was added and the solvent was evaporated under vacuo to give the amine 120?HCl (466 mg, 96% yield) as a w hite solid. The spectroscopic data matched with those previously described. 308 Materials & Methods Preparation of N-benzyl(4 -oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodec -3-yl)amine , 129 502 In a three-necked round bottom flask equipped with a magnetic stirrer and gas inlet, CooNson?s diNetone (5 g, 28.7 mmol) was dissolved in anh. THF (50 mL) an d the solution was cooled to 5 ? C with an ice bath while stirring for 30 minutes under nitrogen. The resulting white solid (hydroxylamine) was filtered under vacuo and washed with cold THF (20 mL). The solid (11.25 g) was suspended in toluene (125 mL) and dehydrated under Dean-Stark conditions for one hour. The reaction mixture was tempered to room temperature and concentrated under vacuo to give the imine (6.95 g) as a yellow oil. Anh. THF (150 mL), anh. methanol (30 mL) and sodium borohydride (1.63 g, 43.1 mmol) were added and the suspension was stirred at room temperature for 24 hours. The reaction mixture was then evaporated under vacuo and the residue was partitioned between water (100 mL) and DCM (100 mL). The phases were separated and the aqueous layer was extracted with further DCM (3 x 50mL). The combined organic phases were dried over anh. Na2SO4, filtered and concentrated under vacuo to give a yellow solid (6.93 g). Column chromatography (SiO2, Hexanes/Ethyl acetate mixtures) gave 129 (2.67 g, 35% yield) as a white solid, whose spectroscopic data matched with those previously published. Preparation of 4-oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodec -3-yl)amine hydrochloride, 121?HCl502 A suspension of benzilamine 129 (2.67 mg, 10.0 mmol) and 10 wt. % palladium on carbon (1.33 g) in methanol (100 mL) was hydrogenated at room temperature and atmospheric pressure until no further hydrogen consumption was observed. The suspension was then filtered and the solid washed with methanol (40 mL). An excess of Et2O?HCl was added an d the solvent was evaporated under vacuo to give the amine 121?HCl (1.79 g, 85% yield) as a white solid. The spectroscopic data matched with those previously described. CHAPTER 3 309 Preparation of tetramethyl (1 ?,4?,5?,8?) -3,7 -hydroxy -bicyclo[3.3.0]octa -2,6 -diene - 2,4,6,8 -tetracarboxylate , 135 504 a) Formation of the disodium enolate salt of tetramethyl (1?,4?,5?,8?)-3,7-hydroxy - bicyclo[3.3.0]octa -2,6-diene-2,4,6,8-tetracarboxylate, 134: In 2 L round-bottom flask equipped with a pressure-equalizing dropping funnel, condenser apparatus, high temperature thermometer and stir bar a solution of sodium hydroxide (64 g, 1.64 mol) in methanol (1.15 L) was cooled to 10 ?C with an ice bath. Then dimethyl 1,3-acetondicarboxylate (273 g, 1.57 mol) was added dropwise over 1 hour. The mixture was heated at reflux, whereupon 40 wt. % glyoxal solution in water (128 g, 0.89 mol) was added over 15-20 minutes, keeping the inner temperature at 65 ?C. Once added, the reaction mixture was stirred at room temperature overnight. The resulting solid was filtered under vacuo, washed with cold methanol and dried under reduced pressure to give 134 as a disodium salt (339 g). b) Synthesis of tetramethyl (1?,4?,5?,8?)-3,7-hydroxy -bicyclo[3.3.0]octa -2,6-diene- 2,4,6,8-tetracarboxylate, 135: In a 5 L Erlenmeyer flasks equipped with a large magnetic stirrer the disodium salt 134 (339 g, 0.82 mol) was partitioned between water (1.9 L) and DCM (1.2 L). The two-phase mixture was vigo rously stirred as 5 N HCl (400 mL) was added. The layers were separated and the aqueous phase was extracted with further DCM (2 x 500 mL). The combined organic layers were washed with brine (2 x 250 mL), dried over anh. Na2SO4, filtered and evaporated under vacuo, keeping the water bath temperature below 40 ?C. The resulting waxy oil was crystallized from 2:1 hexane/EtOAc affording 135 (179 g, 55% overall yield) as a white solid, which was used in next step without further charac terization. Preparation of cis-bicyclo[3.3.0]octane -3,7 -dione, 133 504 A 2 L round-bottom flask equipped with three condenser apparatus and pumice stone as a stirrer was charged with the tetraester 135 (179 g, 0.48 mol), glacial acetic acid (90 mL) and 1 M HCl solution (800 mL). The reaction mixture was heated at reflux for 2.5 hours, 310 Materials & Methods with vigorous foaming formation. Afterwards, the mixture was tempered to room temperature, the pumice stone decanted and the solution extracted with DCM (4 x 400 mL). The combined organic phases were evaporated under vacuo, keeping the water bath temperature below 40 ?C, until most of the acetic acid was removed. The residue was dissolved in further DCM (400 mL) and the solution washed with saturated aqueous NaHCO 3 solution (3 x 300 mL). The organic layer was dried over anh. Na2SO4, filtered and concentrated cautiously to give de diketone 133 (48 g, 71% yield) as a white solid, whose spectroscopic data were in accord to those described in the bibliography. Preparation of the stereoisomeric mixture of cis-3,7 -dihydroxybicyclo[3.3.0]octa -3,7 - dicarbonitrile, 136 505 In a 2 L round-bottom flask equipped with a pressure-equalizing dropping funnel, low temperature thermometer, magnetic stirrer and 10 N NaOH solution trap, a solution of diketone 133 (42 g, 0.3 mol) in water (315 mL) was prepared. To that, sodium cyanide (91 g, 1.86 mol) was added and the solution was cooled to 10-15 ?C with an ice/NaCl bath. Then 40% H 2SO4 (380 mL, 7.06 mol) was added dropwise while keeping the inner temperature < 15 ?C, with the formation of a thick slurry. After the addition, the ice/NaC l bath was removed and the reaction mixture was stirred at room temperature for 3 hours. Diethyl ether (300 mL) was added and the phases were separated. The aqueous layer was extracted with further Et 2O (5 x 300 mL) and the combined organic layers were was hed with water (2 x 150 mL), dried over Na 2SO4 and filtered. Evaporation under vacuo gave the stereoisomeric mixture 136 (31 g, 53% yield) as a brown oil. The spectroscopic data matched with those previously described. Preparation of the mixture of cis-bicyclo[3.3.0]octa -2,7 -diene -3,7 -dicarbonitrile, 137 , and cis-bicyclo[3.3.0]octa -2,6 -diene -3,7 -dicarbonitrile, 138505 In a round-bottom flask equipped with a pressure-equalizing dropping funnel, condenser apparatus and stir bar, thionyl chloride (40 mL) was added dropwise over 3 hours to a solution of bis-cyanohydrines 136 (31 g, 0.16 mol) in pyridine (200 mL). The CHAPTER 3 311 black solution was heated at reflux for 5 hours. The mixture was tempered overnight and 2 N HCl solution (20 0 mL) was carefully poured into reaching acid pH. The black precipitated was filtered under vacuo and washed diluted HCl solution and water (300 mL approx.). The solid was extracted with hot DCM (7 x 100 mL) and the combined organic extracts were washed with water (2 x 150 mL), dried over anh. Na 2SO4, filtered and concentrated under vacuo to give a solid (7.9 g). In order to recover further product, the filtrate was concentrated until a third of its volume and DCM (300 mL) was added. The immiscible mixtur e was filtered through a Celite? pad and the phases Zere separated The aqueous phase was extracted with further DCM (200 mL) and the combined organic layers were dried over anh. Na2SO4, filtered and evaporated under vacuo to give an oil (12 g). Crystallization of the obtained product from EtOAc/hexane afforded the mixture of unsaturated dinitriles 137 and 138 (11.2 g, 44% yield) as a yellow solid. The spectroscopic data were identical to those described in the bibliography. Preparation of the stereoisomer ic mixture of cis-bicyclo[3.3 .0]octane -3,7 -dicarbonitrile, 139505 A suspension of the unsaturated dinitriles 137 and 139 (11.14 g, 70 mmol) and 10% wt. palladium on carbon (2.15 g) in methanol (200 mL) was stirred under hydrogen atmosphere (27 atm) at room temperature until no further hydrogen consumption was observed. The suspension was then filtered and the solid washed with methanol (50 mL). The solvent was evaporated under vacuo and crystallization of the residue from EtOAc gave the stereoisomeric mixture of saturated nitriles 139 (9.9 g, 87% yield) as a yellow solid in a ratio of 2:1.3:1; (1?,3?,5?,7?), (1?,3?,5?,7?) and (1?,3?,5?,7?) respectively. The spectroscopic data matched with those previously described. Preparation of the stereoisomeric mixture of dimethyl cis-bicyclo[3.3.0]octane -3,7 - d icarboxylate, 141 506 a) Synthesis of the stereoisomeric mixture of cis-bicyclo[3.3.0]octane -3,7-dicarboxylic acid, 140: 312 Materials & Methods A solution of the saturated dinitriles 139 (7.1 g, 44.2 mmol) in 40% (p/v) KOH in methanol (53 mL) was heated at reflux for 3 hours. Water (155 mL) was added and the reaction mixture was heated at reflux for further 3 hours. The solution was cooled with an ice bath and conc. HCl was added until acid pH. The resulting solution was concentrated under vacuo and the residue extracted with hot Et 2O (5 x 100 mL). The combined organic extracts were dried over anh. Na2SO4, filtered and evaporated under vacuo to give the diacid mixture 140 (6.3 g, 72% yield) as a yellow solid which was used without any further purification or characterization. b) Synthesis of the stereoisomeric mixture of dimethyl cis-bicyclo[3.3.0]octane -3,7- dicarboxylate, 141: To a solution of diacid mixture 140 (6.45 g, 32.6 mmol) in anh. methanol (130 mL) was added conc. H 2SO4 (13 mL) and the reaction mixture was heated at reflux overnight. Then the resulting solution was tempered to room temperature and anh. K2CO3 was added until reaching basic pH (~ 12). The solid was filtrated and the filtrate was concentrated under vacuo. The residue was dissolved in DCM (100 mL) and dried over anh. Na2SO4 and filtered. Evaporation under vacuo afforded the diester mixture 141 (6.3 g, 86% yield) as a brown oil, whose spectroscopic data belong to those previously described. Any further purification or characterization was carried out. Preparation of dimethyl tricyclo[3.3.0.0 3,7 ]octane -1,5 -dicarboxylate, 131 505 In a three-necked round bottom flask equipped with a pressure-equalizing dropping funnel, low temperature thermometer and gas inlet, a solution of anh. diisopropylamine (10.8 mL, 77.0 mmol) in anh. THF (110 mL) was cooled to -78 ? C by means of a dry ice in CHAPTER 3 313 acetone bath. 2.5 M n-butyllithium in hexanes (25.6 mL, 64.2 mmol) was added dropwise and the resulting clear solution was stirred at ~0 ? C with an ice bath for 1 hour. Afterwards the reaction mixture was cooled down to -10 ? C and a solution of the diester mixture 141 (6.3 g, 27.9 mmol) in anh. THF (82 mL) was added dropwise. The resulting beige suspension was stirred at -10 ? C for 1 hour and at -78 ? C for a further hour. Next, a solution of iodine (7.1 g, 27.9 mmol) in anh. THF (185 mL) was added dropwise over 2 hours. After the addition, the resulting deep-red solution was stirred at 0 ? C for 1 hour and then at room temperature overnight. The reaction mixture was acidified with 5 N HCl solution until acid pH (~ 25 mL) and the phases were separated. The aqueous layer was extracted with Et20 (5 x 50 mL) and the combined organic phases were washed with 10% NaS 2O3 solution (3 x 50 mL) and brine (2 x 50 mL), dried over anh. Na2SO4 and filtered. Concentration under vacuo gave a brown oil (6.6 g). The crude was purified by column chromatography (SiO2, Hexane/ EtOAc mixture) and evaporation in vacuo of the appropriate fractions provided the desired tricyclic diester 131 (2.23 g, 36% yield) as yellow solid. The spectroscopic data were identical to those described in the bibliography. Preparation of tricyclo[3.3.0.0 3,7 ] octane-1,5 -dicarboxylic acid, 141 515 A suspension of diester 131 (2.23 g, 9.9 mmol) in 40% (p/v) KOH in methanol (15 mL) was heated at reflux for 3 hours. Then water (15 mL) was added and the resulting brown solution was heated at reflux fo r further 3 hours. Cooling to ~ 0 ? C with an ice bath was followed by the addition of conc. HCl until acid pH, and the resulting suspension was concentrated under vacuo. The residue was extracted with hot Et 2O (7 x 30 mL) and the combined organic extracts were dried over anh. Na2SO4, filtered and concentrated under vacuo to give 142 (1.90 g, 97% yield) as a brown solid, whose spectroscopic data matched with those described in the bibliography. Preparation of 3 -oxatetracyclo[5.2.1.1 5,8 .0 1,5 ]undeca -2,4 -dione, 144 172 314 Materials & Methods Diacid 142 (1.9 g, 9.7 mmol) was placed in a round bottom flask equipped with a condenser apparatus and a stir bar. Acetic anhydride (55 mL) was added and the solution was heated at reflux for 90 minutes. The reaction was tempered to room temperature prior the evaporation under vacuo to give a brown solid. Purification by sublimation at 105 ? C and 0.5 Torr provided the desired anhydride 144 (1.24 g, 72% yield) as a yellow solid. Its spectroscopic data were identical to those described. Preparation of 5 -( methoxycarbonyl)tricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid, 146 172 In a round bottom flask equipped with a condenser apparatus, CaCl2 tube and magnetic stirrer was prepared a solution of anhydride 144 (1.24 g, 7.0 mmol) in anh. methanol (100 mL). To that, sodium methoxide (1.88 g, 34.8 mmol) was added and the suspension was heated at reflux overnight. Thereupon the resulting solution was tempered to room temperature and the solvents were removed under vacuo. The residue was partitioned between water (25 mL) and EtOAc (25 mL) and the layers were separated. The aqueous phase was adjusted with conc. HCl to pH ~ 1 and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under reduced pressure to give an orange oil (1.36 g) as a mixture of the desired hemiester 146 and the diacid 142. Purification by column chromatography (SiO2, hexane/EtOAc mixture) gave in order of elution the hemiester 146 (747 mg, 51% yield) and the diacid 142 (601 mg, 44% yield). The spectroscopic data were identical to those previously published. Preparation of tricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid, 155 172 a) Synthesis of the methyl tricyclo[3.3.0.0 3,7]octane -1-carboxylate, 148: CHAPTER 3 315 In a three-necked round bottom flask equipped with a magnetic stirrer and gas inlet, 22?-dithiobis(pyridine-N -oxide) (198 mg, 0.78 mmol) was added to a solution of hemiester 148 (129 mg, 0.61 mmol) in anhydrous and deoxygenated CHCl 3 (5 mL). The resulting solution was wrapped in aluminium foil and cooled to 0 ? C by means of an ice/NaCl bath. Then tributylphosphine (0.22 mL, 0.86 mmol) was added and the yellow solution stirred at room temperature for 2 hours. Next the aluminium foil removed and the reaction mixture stirred at room temperature under irradiation of a 100 W lamp for 3 hours. Afterwards the reaction mixture was washed with saturated aqueous NaHCO 3 solution (3 x 10 mL), 6 N HCl soluti on (3 x 10 mL), water (2 x 10 mL) and brine (2 x 10 mL). The organic layer was dried over anh. Na2SO4 and filtered. The solvents were distilled off to give the monoester 148 along with reaction by-products (aprox. 420 mg). The product was used in next step without any further purification or characterization due to its high volatility. b) Synthesis of the tricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid, 155: A suspension of monoester 148 (aprox. 420 mg, 2.56 mmol) in 10% (p/v) KOH in methanol (3.2 mL) was heated at reflux for 3 hours. Water (3.2 mL) was added and the reaction mixture was heated at reflux for further 3 hours. The solution was tempered to room temperature and then concentrated under vacuo. The residue was partitioned between water (10 mL) and EtOAc (10 mL). The phases were separated and the aqueous layer was washed with further EtOAc (10 mL) followed by acidification with 2 N HCl solution until acid pH and extraction with EtO Ac (3 x 10 mL). The combined organic layers were dried over anh. Na2SO4, filtered and evaporated under vacuo to give a grey solid (93 mg). Purification by column chromatography (SiO2, Hexane/ EtOAc mixture) afforded the desired monoacid 155 (68 mg, 73% yiel d) as a white solid, whose spectroscopic data were in accord to those previously described. Preparation of (tricyclo[3.3.0.0 3,7 ]octa -1-yl)amine hydrochloride, 122 ?HCl172 In a round bottom flask equipped with a condenser apparatus and magnetic stirrer a solution of acid 155 (71 mg, 0.47 mmol) in toluene (1.4 mL) was prepared, to which was 316 Materials & Methods added diphenylphosphoryl azide (0.150 mL, 0.69 mmol) followed by triethylamine (0.09 mL, 1.34 mmol). The solution was heated at reflux for 3 hours, whereupon the reaction mixture was tempered to room temperature and washed with cold 6 N HCl solution (8 x 5 mL). Further 6 N HCl solution (2.3 mL) was added and the biphasic system heated at re flux for 24 hours. Then the reaction mixture was tempered to room temperature and the phases were separated. The aqueous layer was washed with Et2O (3 x 5 mL), the pH was adjusted to ~ 12 with 10 N NaOH solution and extracted with Et 2O (4 x 5 mL). The combined organic phases were dried over anh. Na2SO4 and filtered. To the combined extracts an excess of Et2O?HCl was added and the solvent was removed under vacuo keeping the bath temperature below 40 ? C, to give the amine 122?HCl (23 mg, 31% yield). The spectroscopic data matched with those described in the literature. Preparation of 3,7 -dimethyltricyclo[3.3.0.0 3,7 ] octane-1,5 -dicarboxylic acid, 143 600 A suspension of diester 132 (2 g, 7.9 mmol) in 40% (p/v) KOH in methanol (15 mL) was heated at reflux for 3 hours. Then water (15 mL) was added and the resulting brown solution was heated at reflux fo r further 3 hours. Cooling to ~0 ? C with an ice bath was followed by the addition of conc. HCl until acid p H, and the resulting suspension was concentrated under vacuo. The residue was extracted with hot Et 2O (5 x 30 mL) and the combined organic extracts were dried over anh. Na2SO4, filtered and concentrated under vacuo to give 143 (1.77 g, quantitative yield) as a yellow solid, whose spectroscopic data matched with those described in the bibliography. Preparation of 7,8 -dimethyl -3-oxatetracyclo[5.2.1.1 5,8 .0 1,5 ]undeca -2,4 -dione, 145 524 Diacid 143 (2.6 g, 11.4 mmol) was placed in a round bottom flask equipped with a condenser apparatus and a stir bar. Acetic anhydride (70 mL) was added and the solution 600 Camp s, P.; Fon t - Bard ia, M.; P ?rez, F.; Solan s, X.; V?z q u ez, S . Angew. Chem. Int. Ed. Engl. 1995 , 34, 912 - 9 14 . CHAPTER 3 317 was heated at reflux for 90 minutes. The reaction was tempered to room temperature prior the evaporation under vacuo to give the desired anhydride 145 (2.29 g, 97% yield) as a yellow solid, which was used in next step without any further purification or characterization. Its spectroscopic data were identical to those described. Preparation of 3,7 -dimethyl -5-(methoxycarbonyl)tricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid, 147 164 In a round bottom flask equipped with a condenser apparatus, CaCl2 tube and magnetic stirrer was prepared a solution of anhydride 145 (2.29 g, 11.1 mmol) in anh. methanol (160 mL). To that, sodium methoxide (3.00 g, 55.6 mmol) was added and the solution was heated at reflux for 20 hours. Thereupon the resulting suspension was tempered to room temperature and the solvents were removed under vacuo. The residue was partitioned between water (100 mL) and EtOAc (50 mL) and the layers were separated. The aqueous phase was adjusted with conc. HCl to pH ~ 1 and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under reduced pressure to give an orange oil (2.52 g) as a mixture of the desired hemiester 147 and the diacid 143. Purification by column chromatography (SiO2, Hexane/EtOAc mixture) gave in order of elution a mixture of 147 and 143 in a 1:1 ratio (920 mg, 37% yield), the hemiester 147 (293 mg, 11% yield) and the diacid 143 (1.22 g, 48% yield). The spectroscopic data were identical to those previously published. Preparation of methyl 3,7 -dimethyltricyclo[3.3.0.0 3,7 ]octane -1-carboxylate, 149 164 In a three-necked round bottom flask equipped with a magnetic stirrer and gas inlet, 22?-dithiobis(pyridine-N -oxide) (271 mg, 1.08 mmol) was added to a solution of hemiester 147 (200 mg, 0.84 mmol) in anhydrous and deoxygenated CHCl 3 (10 mL). The resulting solution was wrapped in aluminium foil and cooled to 0 ? C by means of an ice/NaCl bath. 318 Materials & Methods Then tributylphosphine (0.29 mL, 1.18 mmol) was added and the yellow solution stirred at room temperature for 2 hours. Next the aluminium foil removed and the reaction mixture stirred at room temperature under irradiation of a 60 W lamp for 2 hours. Afterwards the reaction mixture was washed with saturated aqueous NaHCO 3 solution (3 x 10 mL), 6 N HCl solution (3 x 1 0 mL), water (2 x 10 mL) and brine (2 x 10 mL). The organic layer was dried over anh. Na2SO4, filtered and concentrated under vacuo to give a yellow oil. The crude was purified by column chromatography (SiO2, Hexane/ EtOAc mixture), evaporation under vacuo of the appropriate fractions provided the monoester 149 (138 mg, 86% yield). The spectroscopic data were in accord to those described in the bibliography. Preparation of 3,7 -dimethyltricyclo[3.3.0.0 3,7 ]octane -1-carboxylic acid, 156 164 A solution of monoester 149 (454 mg, 2.34 mmol) in 10% (p/v) KOH in methanol (5 mL) was heated at reflux for 3 hours. Water (5 mL) was added and the reaction mixture was heated at reflux for further 3 hours. The solution was tempered to room temperature and then concentrated under vacuo. The residue was partitioned between water (15 mL) and EtOAc (15 mL). The phases were separated and the aqueous layer was washed with further EtOAc (2 x 15 mL) followed by acidification with 5 N HCl solution until acid pH and extraction with EtOAc (4 x 15 mL). The combined organic layers were washed with brine (3 x 15 mL), dried over anh. Na 2SO4, filtered and evaporated under vacuo to afforded the desired monoacid 156 (256 mg, 61% yield) as a yellow solid, whose spectroscopic data were in accord to those previously described. Preparation of 3,7 -dimethyl(tricyclo[3.3.0.0 3,7 ]octa -1-yl )amine hydrochloride, 123 ?HCl95 In a round bottom flask equipped with a condenser apparatus and magnetic stirrer a solution of acid 36 (221 mg, 1.23 mmol) in toluene (3.7 mL) was prepared, to which was added diphenylphosphoryl azide (0.390 mL, 1.80 mmol) followed by triethylamine (0.230 mL, 1.64 mmol). The solution was heated at reflux for 3 hours, whereupon the reaction CHAPTER 3 319 mixture was tempe red to room temperature and washed with cold 6 N HCl solution (10 x 5 mL). Further 6 N HCl solution (6 mL) was added and the biphasic system heated at reflux for 24 hours. Then the reaction mixture was tempered to room temperature and the phases were separated. The aqueous layer was washed with Et2O (3 x 5 mL), the pH was adjusted to ~ 12 with 10 N NaOH solution and extracted with Et 2O (2 x 10 mL) and EtOAc (2 x 10 mL). The combined organic phases were dried over anh. Na2SO4 and filtered. To the combined extracts an excess of Et2O?HCl was added and the solvent was removed under vacuo to give the amine 123?HCl (230 mg, quantitative yield). The spectroscopic data matched with those described in the literature. Preparation of 1-adamantanecarbonyl azide , 164601 a) Formation of 1-adamantanecarbonyl chloride, 163: In a round-bottom flasks equipped with a stir bar, condenser apparatus and CaCl2 tube, a solution of 1-adamantanecarboxylic acid (100 mg, 0.55 mmol) in thionyl chloride (2 mL) was heated at reflux for 1.5 hours. The reaction mixture was then tempered to ro om temperature and concentrated in vacuo. The residue was dissolved in toluene (5 mL) and the solvent removed under reduced pressure. The procedure was repeated twice to obtain 163 (108 mg, 98% yield) as a yellow solid. The product was used in next step wi thout any further purification or characterization. IR (ATR) ?: 1786 (CO band) cm-1. b) Preparation of 1-adamantanecarbonyl azide, 164: A solution of acyl chloride 163 (108 mg, 0.55 mmol) in acetone (3.6 mL) was added dropwise to a solution of sodium azide (131 mg, 2.02 mmol) in water (1.9 mL) prior cooling at 0 ? C with an ice/NaCl bath. The resulting suspension was stirred at room temperature for 4 hours. Water (5 mL) and Et2O (5 mL) were added, the phases separated and the aqueous layer was extracted with further Et 2O (2 x 5 mL). The combined organic layers were washed with saturated aqueous NaHCO 3 solution (2 x 10 mL) and water (2 x 10 mL), 601 Oliver, J. E.; Sto kes, J. B. J. Med Chem. 1970, 13, 779 - 780 . 320 Materials & Methods dried over anh. Na2SO4, filtered and evaporated under vacuo to give the desired acyl azide 164 (102 mg, 91% yield) as a yellow solid. The product was used in next step without further purification. IR (ATR) ?: 1702 (CO band), 2136 and 2905 (azide band) cm-1. Preparation of 1-(1 -adamantyl) -3-(2,3,4 -trifluorophenyl)urea , 166525 a) Formation of 1-isocyanatoadamantane, 165: In a round-bottom flasks equipped with a stir bar and condenser apparatus anh. toluene (17 mL) was heated at reflux under nitrogen. Then a solution of 1- adamantanecarbonyl azide 164 (102 mg, 0.50 mmol) in anh. toluene (3.5 mL) was added dropwise and the reaction heated at reflux overnight. The reaction completion was monitored by IR. IR (NaCl) ?: 2248 (isocyanate band) cm-1. b) Preparation of 1-(1-adamantyl)-3-(2,3,4-trifluorophenyl)urea, 166: 2,3,4-trifluoroaniline (74 mg, 0.51 mmol) was added to anh. THF (10 mL) and cooled to -78 ? C by means of a dry ice in acetone bath. To that 2.5 M n-butyllithium in hexanes (0.21 mL, 0.51 mmol) was added dropwise. The reaction mixture was removed from the dry ice in acetone bath and tempered to 0 ? C with an ice bath. Afterwards the solution of the isocyanate in anh. toluene from the previous step already tempered to room temperature was added gradually and the mixture stirred at room temperature overnight. Methanol (2 mL) was then added to quench any unreacted butyllithium. The solvents were evaporated under vacuo to give an impure mixture of the urea 166 plus reaction by-products (334 mg). Any further purification or characterization was fulfilled. IR (ATR) ?: 1509 (CO band) cm-1. CHAPTER 3 321 Preparation of pentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -die ne-8,9 -dicarboxylic anhydride, 160602 Pentacyclic diacid 88 (14.5 g, 59.4 mmol) was placed in a round-bottom flask equipped with a magnetic stirrer and condenser apparatus and acetic anhydride (370 mL, 5.47 mol) was added. The suspension was heated at reflux for 1 hour. After that, the resulting solution was tempered to room temperature and then concentrated under vacuo. The crude was purified by sublimation at 100 ? C and 0.7 Torr giving pure anhydride 160 (11.82 g, 88% yield) as a white solid. The spectroscopic data matched with those described in the bibliography. Preparation of 9 -methoxycarbonilpentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8- carboxylic acid, 161 164 In a round-bottom flask equipped with a magnetic stirrer, condenser apparatus and CaCl2 tube was prepared a solution of pentacyclic anhydride 160 (1.98 g, 8.76 mmol) in anh. MeOH (130 mL). Sodium methoxide (2.36 g, 43.8 mmol) was added and the reaction mixture was heated at reflux for 1 4 hours. Thereupon the resulting solution was tempered to room temperature and the solvents were removed under vacuo. The residue was partitioned between water (100 mL) and EtOAc (50 mL) and the layers were separated. The aqueous phase was adjusted with conc. H Cl to pH ~ 1 and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under reduced pressure to give a white solid (2.17 g) as a mixture of the desired hemiester 161 and the diacid 88. Purification by column chromatography (SiO2, Hexane/EtOAc mixture) gave in order of elution the hemiester 161 (1.37 g, 60% yield), a mixture of 161 and 88 in a 1:1 ratio (450 mg, 21% yield), and the diacid 88 (310 mg, 14% yield). The spectroscopic data were identical to those previously published. 602 Camp s, P.; Pu jo l, X.; Ro ssi, R. A.; V?z q u ez, S. Synthesis 1999 , 5, 854 - 858 . 322 Materials & Methods Preparation of methyl pentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]do deca -5,11 -diene -8-carboxylate, 162164 In a three-necked round bottom flask equipped with a magnetic stirrer and gas inlet, 22?-dithiobis(pyridine-N -oxide) (1.49 g, 5.93 mmol) was added to a solution of hemiester 161 (1.19 g, 4.63 mmol) in anhydrous and deoxygenated CHCl 3 (43 mL). The resulting solution was wrapped in aluminium foil and cooled to 0 ? C by means of an ice/NaCl b ath. Then tributylphosphine (1.61 mL, 6.48 mmol) was added and the yellow solution stirred at room temperature for 2 hours. Next the aluminium foil removed and the reaction mixture stirred at room temperature under irradiation of a 60 W lamp for 2 hours. Afterwards the reaction mixture was washed with saturated aqueous NaHCO 3 solution (3 x 20 mL), 6 N HCl solution (3 x 20 mL), water (2 x 20 mL) and brine (2 x 20 mL). The organic layer was dried over anh. Na2SO4, filtered and concentrated under vacuo to give a yellow oil (1.50 g). The crude was purified by column chromatography (SiO2, Hexane/ EtOAc mixture), evaporation under vacuo of the appropriate fractions provided the monoester 162 (858 mg, 59% yield). The spectroscopic data matched with those described in the bibliography. Preparation of pentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8-carboxylic acid, 159 164 A solution of sodium hydroxide (1.05 g) in water (4 mL) was added to a solution of pentacyclic monoester 162 (858 mg, 4.00 mmol) in methanol (75 mL). The reaction mixture was heated at reflux for 1 hour. The solution was tempered to room temperature and the methanol evaporated under vacuo. Water (15 mL) was added and the solution was heated for further 5 hours. Thereafter, the reaction mixture was tempered to room temperature, activated charcoal (200 mg) was added and the black suspension was stirred at room temperature overnight. The suspension was filtered through Celite? pad and the filtrate washed with EtOAc (3 x 30 mL). The aqueus layer was cooled to ~ 0 ? C with an ice bath and conc. HCl was added until pH ~ 1. The resulting white precipitated was filtered CHAPTER 3 323 under vacuo and washed with water (10 mL). The solid was left in the vacuum dryer until constant weight (103 mg). In order to recover further product, the aqueous phase was extracted with EtOAc (3 x 30 mL) and the combined organics were dried over anh. Na2SO4, filtered and concentrated under vacuo to give a total amount of 254 mg (31% yield) of monoacid 159. The spectroscopic data were identical to those previously published. Preparation of pentacyclo[6.4.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8-carbonyl azide , 169 a) Formation of pentacyclo[6.4.0 2,10.03,7.04,9]dodeca -5,11-diene-8-carbonyl chloride, 168: In a round-bottom flasks equipped with a stir bar, condenser apparatus and CaCl2 tube, a solution of pentacyclic carboxylic acid 159 (100 mg, 0.50 mmol) in thionyl chloride (2 mL) was heated at reflux for 1 hour. The reaction mixture was then tempered to room temperature and concentrated in vacuo. The residue was dissolved in toluene (5 mL) and the solvent removed under reduced pressure. The procedure was repeated twice to obtain 168 (108 mg, quantitative yield) as a beige solid. The product was used in next step without any further purification or characterization. IR (ATR) ?: 1778 (CO band) cm-1. b) Preparation of pentacyclo[6.4.0 2,10.03,7.04,9 ]dodeca -5,11-diene-8-carbonyl azide, 169: A solution of acyl chloride 168 (108 mg, 0.50 mmol) in acetone (3.4 mL) was added dropwise to a solution of sodium azide (127 mg, 1.95 mmol) in water (1.8 mL) prior cooling at 0 ? C with an ice/NaCl bath. The resulting suspension was stirred at room temperature for 4 hours. Water (5 mL) and Et2O (10 mL) were added, the phases separated and the aqueous layer was extracted with further Et 2O (2 x 5 mL). The com bined organic layers were washed with saturated aqueous NaHCO 3 solution (2 x 10 mL) and water (2 x 10 mL), dried over anh. Na2SO4, filtered and evaporated under vacuo to give the desired acyl azide 169 (98 mg, 88% yield) as a beige solid. Any further purification or characterization was carried out. IR (ATR) ?: 1697 (CO band), 2129 and 2964 (azide band) cm-1. 324 Materials & Methods Preparation of 1 -( pentacyclo[6.4.0.0 2,10 .0 3,7 .0 4,9 ]dodeca -5,11 -diene -8-yl ) -3-(2,3,4 - trifluorophenyl)urea, 157 a) Formation of 8-isocyanatopentacyclo[6.4.0 2,10.03,7.04,9]dodeca -5,11-diene, 158: In a round-bottom flasks equipped with a stir bar and condenser apparatus anh. toluene (15 mL) was heated at reflux und er nitrogen. Then a solution of pentacyclic azide 169 (98 mg, 0.43 mmol) in anh. toluene (3 mL) was added dropwise and the reaction heated at reflux overnight. The reaction completion was monitored by IR. IR (NaCl) ?: 2258 (NCO band) cm-1. b) Preparation of 1-(pentacyclo[6.4.0.0 2,10.03,7.04,9 ]dodeca -5,11-diene-8-yl)-3-(2,3,4- trifluorophenyl)urea, 157: 2,3,4-trifluoroaniline (64 mg, 0.43 mmol) was added to anh. THF (8 mL) and cooled to -78 ? C by means of a dry ice in acetone bath. To that 2.23 M n-butyllithium in hexanes (0.19 mL, 0.43 mmol) was added dropwise. The reaction mixture was removed from the dry ice in acetone bath and tempered to 0 ? C with an ice bath. Afterwards the solution of the isocyanate in anh. toluene from the previous step already tempered to room temperature was added gradually and the mixture s tirred at room temperature for 20 hour. The reaction completion was controlled by 1H -NMR, and once there was any further progression, methanol (2 mL) was then added to quench any unreacted butyllithium. The solvents were evaporated under vacuo to give a brown solid (173 mg, IR (ATR) ?: 1509 (CO band) cm-1). Purification by column chromatography (SiO2, Hexane/EtOAc mixture) gave the desired urea 157 (37 mg, 25% yield). The analytical sample was obtained by crystallization from methanol/d iethyl ether. Analytical and spectroscopic data of compound 157: Melting point: 217 - 219 ? C. CHAPTER 3 325 IR (ATR) ?: 3350, 2964, 2522, 2158, 1977, 1656, 1639, 1562, 1511, 1471, 1339, 1290, 1246, 1186, 1167, 1101, 1074, 1032, 1012, 999, 975, 882, 805, 767, 741, 725, 689, 661, 620 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.57 (s, 1 H, 9 -H), 2.60 [d, J = 2.4 Hz, 2 H, 2(3)], 2.94 [broad s, 2 H, 4(10) -H], 3.60 [broad s, 2 H, 1(7) -H], 4.85 (broad s, 1 H, 1 -NH ), 5.93 [ddd, J = 6 Hz, J ? = 3 Hz, J ?? = 0.8 Hz, 2 H, 6(12) -H], 6.08 [ddd, J = 6 Hz, J ? = 3 Hz, J ?? = 0.8 Hz, 2 H, 5(11) - H], 6.38 (broad s, 1 H, 3 -NH ), 6.90 (m, 1 H, ?-H), 7.78 (m, 1 H, ?-H). 13C-NMR (100.6 MHz, CDCl 3) ?: 58.5 (CH, C9), 60.2 [CH, C2(3) and C4(10)], 61.3 [CH, C1(7)], 73.7 (C, C8), 111.5 (CH, dd, 2J C - F = 17.5 Hz, 3J C - F = 3.8 Hz, C5 ?  110 (CH, broad t, 3J C - F = 5.4 Hz, C6 ?  12 (C, dd, 2J C - F = 8 Hz, 3J C - F = 3.3 Hz Ar -C1?  11 [CH, C6(12)], 133.9 [CH, C 5(11)], 139.8 (C, dd, 1J C - F = 250 Hz, 2J C - F = 15 Hz, Ar -C3?  120 (C, dd, 1J C - F = 245 Hz, 2J C - F = 15.5 Hz, Ar -C4?  1 (C, dd, 1J C - F = 245 Hz, 2J C - F = 10 Hz, Ar -C2?  1 (C, CO). MS (DIP), m /z (%); significant ions: 344 (M? + , 68), 279 (66), 171 (31), 170 [(C 12H 12N) + , 70], 169 (15), 168 (26), 156 (26), 155 (31), 154 (73), 153 (60), 152 (24), 143 (15), 132 (25), 130 (25), 129 (16), 128 (36), 127 (16), 119 (23), 115 (26), 106 (100), 93 (25), 91 (19), 80 (15), 78 (22), 77 (28), 65 (16). HRMS -ESI+ m /z [ M +H ] + calcd for [C 19H 15F3N2O+H] + : 345.1209, found: 345.12105. General method for the synthesis of urea compounds 108 -109 and 170 -183 In a round-bottom flask equipped with a stir bar under nitrogen atmosphere 1.2 eq. of the appropriate amine hydrochloride was added to anh. DCM (~ 110 mM). To this suspension 1.0 eq. of 2,3,4-trifluorophenyl isocyanate followed by 7 eq. of triethylamine was added. The reaction mixture was stirred at room temperature overnight. Then the solvent was removed under vacuo and the resulting crude was purified as specified in each compound (vide infra ). 326 Materials & Methods Preparation of 1-(1 -adamantyl) -3-(2,3,4 -trifluorophenyl)urea , 1 08497 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 108 (280 mg, quantitative yield). The analytical sample was obtained by crystallization from methanol. The spectroscopic data coincide with those described in the bibliography. Preparation of 1 -(2 -adamantyl) -3-(2,3,4 -trifluorophenyl)urea, 109 497 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 109 (270 mg, 94% yield). The analytical sample was obtained by crystallization from EtOAc/p entane. The spectroscopic data were identical to those previously published. Preparation of 1 -(2 -oxa adamant -1-yl) -3-(2,3,4 -trifluorophenyl)urea, 170 Purification by column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and evaporation in vacuo of the appropriate fractions gave the urea 170 (163 mg, 94% yield) as a white solid. Analytical and spectroscopic data of compound 170: Melting point: 196 - 198 ? C. IR (ATR) ?: 3300-2800 (3293, 3232, 3127, 2933, 2857), 1702, 1640, 1621, 1563, 1509, 1489, 1471, 1446, 1373, 1349, 1340, 1317, 1294, 1257, 1239, 1227, 1200, 1165, 1117, 1099, 1080, 1020, 996, 976, 963, 932, 912, 884, 840, 805, 788, 757, 683, 653 cm-1. CHAPTER 3 327 1H -NMR (500 MHz, CD3OD) ?: 1.68 [dm, J = 12.5 Hz, 2 H, 4(10) -H a ], 1.89 [complex signal, 4 H, 6-H 2, 8(9)-H a ], 2.00 [dm, J = 12.5 Hz, 2 H, 4(10) -H b], 2.26 [broad signal, 2 H, 5(7) -H], 2.32 [d, J = 12.5 Hz, 8(9) -H b], 4.23 (broad s, 1 H, 3 -H), 7.03 (m, 1 H, ?-H), 7.75 (m, 1 H, ?-H). 13C-NMR (125.7 MHz, CD3OD) ?: 29.6 [CH, C5(7)], 35.6 [CH 2, C4(10)] , 35.8 (CH 2, C6), 40.9 [CH 2, C8(9)], 72.5 (CH, C3), 82.2 (C, C1), 112.3 (CH, dd, 2J C - F = 18 Hz, 3J C - F = 4 Hz, C5?  11 (CH, C6 ?  12 (C, d, 2 J C - F = 8.8 Hz, Ar -C1?  110 (C, dt, 1J C - F = 246 Hz, 2J C - F = 15 Hz, Ar -C3?  1 (C, dd, 1 J C - F = 244 Hz, 2J C - F = 11 Hz, Ar -C4?  1 (C, dd, 1J C - F = 244 Hz, 2J C - F = 9 Hz, Ar -C2?  10 (C, CO). MS (DIP), m /z (%); significant ions: 328 (6), 327 (5), 179 (11), 172 (18), 149 (97), 148 (100), 146 (36), 121 (12), 120 (10), 118 (13), 111 (11), 95 (17), 94 (26), 93 (11), 79 (20), 68 (18). Elemental analysis: Calculated for C16H 17F3N2O2: C 58.89% H 5.25% F 17.47% N 8.58% Found: C 59.00% H 5.60 % F 17.22% N 8.57% Preparation of 1 -(3 -methyl -2-oxa adamant -1-yl) -3-(2,3,4 -trifluorophenyl)urea, 171 Column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and concentration in vacuo of the appropriate fractions gave the urea 171 (156 mg, 93% yield) as a white solid. The analytical sample was obtained by crystallization from methanol/d iethyl ether. Analytical and spectroscopic data of compound 171: Melting point: 195 - 197 ? C. IR (ATR) ?: 3300-2800 (3270, 3227, 3128, 2976, 2927, 2856), 1701, 1641, 1622, 1564, 1509, 1492, 1471, 1373, 1341, 1322, 1301, 1286, 1256, 1228, 1213, 1200, 1171, 1136, 1106, 1090, 1072, 1034, 1006, 991, 972, 959, 921, 899, 885, 804, 788, 755, 682, 670, 652 cm-1. 1H -NMR (500 MHz, CD3OD) ?: 1.19 (s, 3 H, 3C -CH 3), 1.63 [dm, J = 12.5 Hz, 2 H, 4(10) - H a ], 1.73 [dm, J = 12.5 Hz, 2 H, 4(10) -H b], 1.75 -1.82 (complex signal, 2 H, 6 -H 2), 1.83 [dm, J = 12.5 Hz, 2 H, 8(9) -H a ], 2.15 [dm, J = 12.5 Hz, 2 H, 8(9) -H b], 2.29 [m, 2 H, 5(7)-H], 7.03 (m, 1 H, ?-H), 7.80 (m, 1 H, ?-H). 328 Materials & Methods 13C-NMR (125.7 MHz, CD3OD) ?: 29.2 (CH 3, C3-CH 3), 30.1 [CH, C5(7)], 34.9 (CH 2, C6), 40.3 [CH 2, C8(9)], 41.5 [CH 2, C4(10)], 75.1 (C, C3), 83.3 (C, C1), 112.4 (CH, dd, 2J C - F = 17.8 Hz, 3J C - F = 4 Hz, C5 ?  11 (CH, C6 ?  12 (C, C1?  110 (C, dt, 1J C - F = 248 Hz, 2J C - F = 15 Hz, Ar -C3?  1 (C, dd, 1J C - F = 245.2 Hz, 2 J C - F = 9.5 Hz, Ar -C4?  1 (C, dd, 1 J C - F = 242.6 Hz, 2J C - F = 8.9 Hz, Ar -C2?  1 (C, CO). MS (DIP), m /z (% ); significant ions: 342 (7), 172 (13), 150 (14), 149 (100), 148 (80), 147 (25), 109 (10), 108 (14), 107 (11), 95 (10), 93 (25). Elemental analysis: Calculated for C17H 19F3N2O2: C 59.99% H 5.63 % F 16.75% N 8.23 % Found: C 59.91% H 5.90 % F 16.52% N 8.22 % Preparation of 1 -(3 -ethyl -2-oxa adamant -1-yl) -3-(2,3,4 -trifluorophenyl)urea, 172 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 172 (116 mg, 96% yield) as a white solid. Analytical and spectroscopic data of compound 172: Melting point: 165 - 166 ? C. IR (ATR) ?: 3300-2800 (3288, 3238, 3128, 2970, 2927, 2850), 1702, 1641, 1622, 1563, 1509, 1471, 1371, 1341, 1322, 1301, 1254, 1227, 1209, 1172, 1091, 1010, 996, 965, 939, 921, 896, 803, 788, 755, 669, 653 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 0.92 (t, J = 7.5 Hz, 3 H, 3C-CH 2CH 3), 1.50 (broad q, J = 7.5 Hz, 2 H, 3C -CH 2CH 3), 1.60 [broad d, J = 12 Hz, 2 H, 4(10) -H a ], 1.69 [broad d, J = 12 Hz, 2 H, 4(10) -H b], 1.80 (complex signal, 2 H, 6 -H 2), 1.86 [broad d, J = 13 Hz, 2 H, 8(9) -H a ], 2.13 [broad d, J = 13 Hz, 2 H, 8(9) -H b], 2.31 [broad s, 2 H, 5(7) -H], 7.02 (m, 1 H, ?-H), 7.80 (m, 1 H, ?-H). 13C-NMR (125.7 MHz, CD3OD) ?: 7.4 (CH 3, 3C-CH 2CH 3), 30.0 [CH, C5(7)], 35.3 (CH 2, C6), 35.7 (CH 2, 3C-CH 2CH 3), 39.0 [CH 2, C4(10)], 40.6 [CH 2, C8(9)], 77.3 (C, C3), 83.2 (C, C1), 112.4 (CH, dd, 2 J C - F = 18 Hz, 3J C - F = 4 Hz, C5 ?  11 (CH, C6 ?  12 (C, 2J C - F = 8 Hz, C1 ?  110 (C, dt, 1J C - F = 248.5 Hz, 2J C - F = 15 Hz, Ar -C3?  1 (C, dd, 1J C - F = 245.5 Hz, CHAPTER 3 329 2J C - F = 10.7 Hz, Ar -C4?  1 (C, dd, 1J C - F = 243.5 Hz, 2J C - F = 8.9 Hz, Ar-C2?  1 (C, CO). MS (DIP), m /z (%); significant ions: 354 (M?+ , 5), 148 (14), 147 [(C 6H 4F3N)? + , 100], 94 (10), 93 (10). Elemental analysis: Calculated for C18 H 21F3N2O2: C 61.01% H 5.97 % F 16.08% N 7.91 % Found: C 60.97% H 6.06 % F 16.23% N 7.84% Preparation of 1 -(3 -cycloh ex yl -2-oxa adamant -1-yl) -3-(2,3,4 -trifluorophenyl)urea, 173 Purification by column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and evaporation in vacuo of the appropriate fractions gave the urea 173 (70 mg, 94% yield) as a white solid. The analytical sample was obtained by crystallization from methanol/d iethyl ether. Analytical and spectroscopic data of compound 173: Melting point: 193 - 195 ? C. IR (ATR) ?: 3300-2800 (3309, 3227, 3107, 2925, 2855), 1681, 1622, 1537, 1513, 1470, 1326, 1300, 1256, 1234, 1211, 1084, 1061, 1014, 994, 975, 892, 853, 825, 809, 763, 702, 678, 655 cm-1. 1H -NMR (500 MHz, CD Cl3) ?: 0.98-1.06 [dq, J = 12.5 Hz, J ?= 3.0 Hz, 2 H, 12(16) -H ax ], 1.10 - 1.16 (tq, J = 13.0 Hz, J ? = 3.5 Hz, 1 H, 14 -H ax ), 1.21-1.29 [tq, J = 13 Hz, J ? = 3.5 Hz, 2 H, 13(15)-H ax ], 1.40 (tt, J = 12 Hz, J ? = 3 Hz, 1 H, 11 -H), 1.62-1.72 [complex signal, 8 H, 4(10) - H 2, 8(9)-H a, 6-H 2], 1.75 -1.84 [complex signal, 5 H, 12(16) -H eq, 13(15)-H eq and 14-H eq], 2.09 [d, J = 14 Hz, 2 H, 8(9) -H b ], 2.35 [broad s, 2 H, 5(7) -H], 5.03 (broad s, 1 H, 1 -NH ), 6.91 (m, 1 H, ?-H), 7.88 (m, 1 H, ?-H), 8.58 (broad s, 1 H, 3 -NH ). 13C-NMR (125.7 MHz, CD Cl3) ?: 26.4 [CH 2, C12(16)], 26.6 [CH 2, C14 and C13(15)], 28.4 [CH, C5(7)], 34.5 (CH 2, C6), 35.8 [CH 2, C4(10)], 40.0 [CH 2, C8(9)], 48.3 (CH, C11), 78.9 (C, C3), 82.1 (C, C1), 111.4 (CH, d, 2J C - F = 18 Hz, C5 ?  11 (CH, C6 ?  12 (C, C1?  139.7 (C, dt, 1J C - F = 248.8 Hz, 2J C - F = 15 Hz, Ar -C3?  12 (C, d, 1J C - F = 247.7 Hz, Ar -C4?  330 Materials & Methods 146.6 (C, d, 1J C - F = 244 Hz, Ar -C2?  1 (C, CO). MS (DIP), m /z (%); significant ions: 408 (M? + , 5), 178 (38), 176 (21), 173 (19), 152 (23), 148 (11), 147 [(C 6 H 4F3N)? + , 100], 135 (16), 94 (19), 83 [(C 6H 11) + , 15], 55 (16). Elemental analysis: Calculated for C22 H 27F3N2O2: C 64.69% H 6.66% N 6.86 % Calculated for C22 H 27F3N2O2?0.50MeOH : C 63.26% H 6.91 % N 6.56% Found: C 63.44% H 7.17% N 6.63 % Preparation of 1 -(3 -phenyl -2-oxa adamant -1-yl) -3-(2,3,4 -trifluorophenyl)urea, 174 Column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and concentration in vacuo of the appropriate fractions gave the urea 174 (31 mg, 70% yield) as a white solid. Analytical and spectroscopic data of compound 174: Melting point: 150 - 152 ? C. IR (ATR) ?: 3300-2800 (3312, 3238, 3118, 2922, 2856), 1697, 1621, 1555, 1514, 1470, 1324, 1262, 1235, 1208, 1179, 1094, 1079, 1017, 993, 976, 945, 897, 803, 751, 696, 669, 653 cm- 1. 1H -NMR (500 MHz, CDCl 3) ?: 1.84 [d, J = 12.5 Hz, 2 H, 8(9) -H a ], 1.88 -1.96 (complex signal, 2 H, 6 -H 2), 1.98 [d, J = 11 Hz, 2 H, 4(10) -H a ], 2.05 [d, J = 12.5 Hz, 2 H, 4(10) -H b], 2.24 [d, J = 13 Hz, 2 H, 8(9) -H b], 2.48 [m, 2 H, 5(7) -H], 5.29 (broad s, 1 H, 1 -NH ), 6.89 (m, 1 H, ?- H), 7.28 (t, J = 7.5 Hz, 1 H, Ar ?-H para), 7.37 (t, J = 7.5 Hz, 2 H, Ar ?-H meta ), 7.45 (d, J = 7 Hz, 2 H, Ar ?-H ortho ), 7.88 (m, 1 H, ?-H), 8.51 (broad s, 1 H, 3 -NH ). 13C-NMR (125.7 MHz, CDCl 3) ?: 28.7 [CH, C5(7)], 33.8 (CH 2, C6), 39.6 [CH 2, C8(9)], 40.6 [CH 2, C4(10)], 77.4 (C, C3), 82.5 (C, C1), 111.3 (CH, dd, 2J C - F = 17.6 Hz, 3J C - F = 3.9 Hz, C5?  11 (CH, t, 3 J C - F = 5.5 Hz, C6 ?  12 (CH, Ar ?-Cortho ), 124.7 (C, dd, 2 J C - F = 8 Hz, 3J C - F = 3.3 Hz, Ar -C1?  127.2 (CH, Ar ?-Cpara), 128.3 (CH, Ar ?-Cmeta ), 139.6 (C, dt, 1J C - F = 249.5 Hz, 2J C - F = 15 Hz, Ar -C3?  120 (C, ddd, 1J C - F = 245.5 Hz, 2J C - F = 10.7 Hz, 3 J C - F = 2.4 Hz, Ar -C4?  CHAPTER 3 331 146.4 (C, dd, 1 J C - F = 244 Hz, 2J C - F = 10 Hz, Ar -C2?  1 (C, Ar?-Cipso), 154.5 (C, CO). MS (DIP), m /z (%); significant ions: 402 (M? + , 13), 255 (19), 184 (15), 172 (25), 171 (15), 155 (22), 147 (100), 145 (15), 142 (27), 129 (16), 120 (16), 118 (26), 110 (17), 105 (26), 91 (17), 77 (23). Elemental analysis: Calculated for C22 H 21F3N2O3: C 65.66% H 5.26% N 6.96% Calculated for C22 H 21F3N2O3?1.0H 2O: C 62.85% H 5.51% N 6. 66% Found: C 62.79% H 5.45% N 6.69 % Preparation of 1 -(2 -oxaadamant -5-yl) -3-(2,3,4 -trifluorophenyl)urea, 175 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 175 (24 mg, 83% yield) as a white solid. Analytical and spectroscopic data of compound 175: Melting point: 211 - 213 ? C. IR (ATR) ?: 3344, 2937, 2856, 2478, 2362, 1681, 1621, 1556, 1504, 1473, 1434, 1402, 1361, 1320, 1288, 1236, 1183, 1146, 1107, 1076, 1035, 1020, 1005, 992, 948, 921, 890, 810, 793, 775, 761, 724, 686, 656, 636 cm-1. 1H -NMR (400 MHz, CD 3OD) ?: 1.66 [dm, J = 12.8 Hz, 2 H, 8(10) -H a ], 1.98 [dm, J = 12.4 Hz, 2 H, 8(10) -H b], 2.06 [d, J = 12 Hz, 2 H, 4(9) -H a ], 2.14 -2.21 [complex signal, 4 H, 4(9)-H a, 6-H 2], 2.25 (m, 1 H, 7 -H), 4.17 [ broad s, 2 H, 1(3) -H], 7.01 (m, 1 H, ?-H), 7.68 (m, 1 H, ?- H). 13C-NMR (100.6 MHz, CD 3OD) ?: 28.8 (CH, C7), 36.0 [CH 2, C8(10)], 41.2 [CH 2, C6], 41.8 [CH 2, C4(9)], 50.6 (C, C5), 70.9 [CH, C1(3)], 112.2 (CH, dd, 2J C - F = 17.8 Hz, 3J C - F = 3.7 Hz, C5 ?  11 (CH, C6 ?  12 (C, d, 2J C - F = 6.5 Hz, Ar -C1?  110 (C, d, 1J C - F = 247 Hz, Ar - C3?  1 (C, d, 1J C - F ~ 245 Hz, Ar -C4?  1 (C, d, 1J C - F = 242 Hz, Ar -C2?  12 (C, CO). MS (DIP), m /z (%); significant ions: 173 [(C 7H 2F3NO)? + , 4], 149 (100), 148 (62), 147 (16), 137 [(C 9H 13O) + , 8], 93 (49). 332 Materials & Methods Elemental analysis: Calculated for C16H 17F3N2O2: C 58.89% H 5.25% N 8.58% Calculated for C16H 17F3N2O2?0.50H 2O: C 57.31% H 5.41 % N 8.35% Found: C 57.46% H 5.70% N 8.22% Preparation of 1-(4 -oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodecan -3-yl) -3-(2,3,4 - trifluorophenyl)urea , 176 Purification by column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and evaporation in vacuo of the appropriate fractions gave the urea 176 (374 mg, 94% yield) as a white solid. Analytical and spectroscopic data of compound 176: Melting point: 152 - 154 ? C. IR (ATR) ?: 3329, 3192, 3118, 2975, 1701, 1641, 1621, 1559, 1509, 1470, 1342, 1321, 1287, 1253, 1205, 1183, 1166, 1127, 1071, 1020, 996, 951, 933, 911, 867, 839, 798, 757, 708, 688, 655, 635, 593 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.60 (dt, J = 10.8 Hz, J ? = 1.6 Hz, 1 H, 12 -H a), 1.95 (dt, J = 10.8 Hz, J ? = 1.6 Hz, 1 H, 12 -H b), 2.50 (broad t, J = 5.2 Hz, 1 H, 7 -H), 2.57 (t, J = 4.8 Hz, 1 H, 1 -H), 2.66 (m, 8 H, 2 -H), 2.75 -2.84 (complex signal, 3 H, 2 -H, 10 -H, 11 -H), 2.88 -3.01 (complex signal, 2 H, 6 -H, 9 -H), 4.83 (t, J = 5.2 Hz, 1 H, 5 -H), 5.94 (broad s, 1 H, 1 -NH ), 6.90 (m, 1 H, ?-H), 7.82 (m, 1 H, ?-H), 8.06 (broad s, 1 H, 3 -NH ). 13C-NMR (100.6 MHz, CDCl 3) ?: 41.4 (CH, C8), 41.6 (CH, C11), 43.2 (CH, C1), 43.5 (CH 2, C12), 44.6 (CH, C9), 44.9 (CH, C7), 46.3 (CH, C10), 54.8 (CH, C6), 57.0 (CH, C2), 84.1 (CH, C5), 103.1 (C, C3), 111.3 (CH, dd, 2 J C - F = 17.7 Hz, 3J C - F = 3.9 Hz, C5 ?  115.2 (CH, m, C6?  124.5 (C, dd, 2J C - F = 8 Hz, 3J C - F = 3.5 Hz, C1 ?  1 (C, ddd, 1J C - F = 252 Hz, 2J C - F = 16.4 Hz, 2J C - F = 13.7 Hz, C3 ?  12 (C, ddd, 1J C - F = 251 Hz, 2J C - F = 11.7 Hz, 3 J C - F = 3.2 Hz, C4 ?  146.6 (C, ddd, 1J C - F = 245.8 Hz, 2 J C - F = 10 Hz, 3J C - F = 2.7 Hz, C2 ?  1 (C, CO). MS (DIP), m /z (%); significant ions: 348 (M? + , 18), 173 [(C 7H 2F3NO)? + , 17], 159 [(C 11H 11O) + , CHAPTER 3 333 35], 147 (100), 146 (13), 131 (24), 91 (15). Elemental analysis: Calculated for C18 H 15F3N2O2: C 62.07% H 4.34% N 8.04% Found: C 62.04% H 4.43% N 7.98% Preparation of 1 -( tricyclo [3.3.0.0 3,7 ]oct -1-yl ) -3-(2,3,4 -trifluorophenyl)urea, 177 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 177 (24 mg, 66% yield) as a white solid. Analytical and spectroscopic data of compound 177: Melting point: 185 - 186 ? C. IR (ATR) ?: 3331, 3105, 2970, 2943, 2894, 2159, 1656, 1640, 1620, 1563, 1510, 1467, 1318, 1288, 1244, 1204, 1171, 1107, 1076, 1065, 1017, 979, 823, 800, 764, 723, 710, 690, 668, 645, 620 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.51 [d, J = 8.8 Hz, 2 H, 4(6) -H a ], 1.74 [complex signal, 6 H, 4(6)-H a, 2(8)-H 2], 2.34 [broad s, 2 H, 3(7) -H], 2.42 (m, 1 H, 5 -H), 5.42 (broad s, 1 H, 1 -NH ), 6.67 (broad s, 1 H, 3 -NH ), 6.90 (m, 1 H, ?-H), 7.82 (m, 1 H, ?-H). 13C-NMR (100.6 MHz, CDCl 3) ?: 32.8 [CH, C3(7)], 43.0 (CH, C5), 46.6 [CH 2, C4(6)], 51.1 [CH 2, C2(8)], 61.6 (C, C1), 111.4 (CH, dd, 2J C - F = 17.7 Hz, 3J C - F = 3.7 Hz, C5 ?  111 (CH, t, 3J C - F = 5.6 Hz, C6 ?  12 (C, dd, 2 J C - F = 8 Hz, 3J C - F = 3.5 Hz, C1 ?  1 (C, dt, 1J C - F = 245 Hz, 2J C - F = 15 Hz, C3 ?  122 (C, dd, 1 J C - F = 225 Hz, 2J C - F = 12 Hz, C4 ?  11 (C, dd, 1J C - F = 246 Hz, 2J C - F = 10 Hz, Ar -C2?  1 (C, CO). MS (DIP), m /z (%); significant ions: 296 (M? + , 34), 268 (14), 267 (24), 254 (22), 147 [(C 6H 4F3N)? + , 77], 146 (23), 119 (14), 95 (29), 94 (100), 82 (29), 81 (62), 80 (20), 79 (18). Elemental analysis: 334 Materials & Methods Calculated for C15H 15F3N2O: C 60.81% H 5. 10% N 9.45 % Found: C 60.87% H 5.34% N 9.19 % Preparation of 1 -( 3,7 -dimethyl(tricyclo[3.3.0.0 3,7 ]oct -1-yl ) ) -3-(2,3,4 -trifluorophenyl)urea, 178 Purification by column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and evaporation in vacuo of the appropriate fractions gave the urea 178 (50 mg, 47% yield) as a white solid. Analytical and spectroscopic data of compound 178: Melting point: 174 - 175 ? C. IR (ATR) ?: 3335, 2957, 2930, 2882, 2158, 2005, 1686, 1656, 1637, 1621, 1565, 1509, 1471, 1308, 1289, 1242, 1204, 1165, 1154, 1118, 1081, 1064, 1020, 1009, 964, 946, 816, 796, 719, 694, 678, 657 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.15 [s, 6 H, 3(7) -CH 3], 1.39 [dd, J = 8.4 Hz, J ? = 3.6 Hz, 2 H, 4(6) -H a ], 1.64 [dd, J = 7.6 Hz, J ?= 3.6 Hz, 2 H, 2(8) -H a ], 1.76 [dd, J = 8.4 Hz, J ? = 3.0 Hz, 2 H, 4(6) -H b], 1.82 [d, J = 7.6 Hz, 2 H, 2(8) -H b], 2.38 (t, J = 3.0 Hz, 1 H, 5 -H), 5.47 (broad s, 1 H, 1 -NH ), 6.75 (broad s, 1 H, 3 -NH ), 6.89 (m, 1 H, ?-H), 7.80 (m, 1 H, ?-H). 13C-NMR (100.6 MHz, CDCl 3) ?: 16.5 [CH 3, C3(7)-CH 3], 44.8 (CH, C5), 46.2 [C, C3(7)], 53.1 [CH 2, C4(6)], 57.5 [CH 2, C2(8)], 61.8 (C, C1), 111.4 (CH, dd, 2J C - F = 17.7 Hz, 3J C - F = 3.8 Hz, C5 ?  110 (CH, t, 3J C - F = 5 Hz, C6 ?  12 (C, dd, 2J C - F = 8 Hz, 3J C - F = 3.4 Hz, C1 ?  1 (C, ddd, 1J C - F = 249 Hz, 2J C - F = 16.3 Hz, 2J C - F = 13.7 Hz, C3 ?  121 (C, ddd, 1J C - F = 244 Hz, 2J C - F = 11.9 Hz, 3J C - F = 3.2 Hz, C4 ?  11 (C, ddd, 1J C - F = 245 Hz, 2J C - F = 10 Hz, 3J C - F = 2.8 Hz, C2 ?  154.6 (C, CO). MS (EI), m /z (%) ; significant ions: 324 (M? + , 8), 268 (15), 148 (44), 147 [(C 6 H 4F3N)?+ , 100], 146 (23), 136 (17), 134 (84), 122 (89), 121 (54), 120 (40), 119 (81), 110 (20), 109 (51), 108 (50), 107 (28), 106 (16), 105 (21), 96 (17), 95 (70), 94 (54), 93 (52), 92 (18), 91 (44), 81 (17), 80 (18), 79 (37), 77 (33), 67 (24), 55 (16), 41 (23). CHAPTER 3 335 Elemental analysis: Calculated for C17H 19F3N2O: C 62.95% H 5.90% N 8.64 % Found: C 63.12% H 6.17% N 8.48 % Preparation of 1 -[ ( 3,7 -dimethyl(tricyclo[3.3.0.0 3,7 ]oct -1-yl ) methyl] -3-(2,3,4 - trifluorophenyl)urea, 179 Column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and concentration in vacuo of the appropriate fractions gave the urea 179 (82 mg, 97% yield) as a white solid. Analytical and spectroscopic data of compound 179: Melting point: 133 - 134 ? C. IR (ATR) ?: 3323, 2952, 2881, 1637, 1621, 1570, 1510, 1474, 1292, 1244, 1177, 1045, 1001, 985, 809, 794, 755, 682, 653 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 1.17 [s, 6 H, C3(7) -CH 3], 1.32 -1.38 [complex signal, 4 H, 4(6)-H a and 2(8)-H a ], 1.42 [d, J = 7.5 Hz, 2 H, 2(8) -H b], 1.59 [dd, J = 8 Hz, J ? = 3 Hz, 2 H, 4(6)-H b], 2.10 (t, J = 3 Hz, 1 H, 5 -H), 3.42 (s, 2 H, C H 2-N), 7.01 (m, 1 H, ?-H), 7.75 (m, 1 H, ?-H). 13C-NMR (125.7 MHz, CD3OD) ?: 17.1 [CH 3, C3(7)-CH 3], 42.7 (CH, C5), 43.8 [CH 2, CH 2- N], 48.5 [C, C3(7)], 52.3 (C, C1), 54.9 [CH 2, C4(6)], 57.2 [CH 2, C2(8)], 112.3 (CH, dd, 2J C - F = 17.7 Hz, 3 J C - F = 3.9 Hz, C5 ?  11 (CH, C6 ?  120 (C, d, 2J C - F = 6 Hz, C1 ?  11 (C, dt, 1J C - F = 247 Hz, 2 J C - F = 15 Hz, C3 ?  1 (C, dd, 1 J C - F = 246 Hz, 2J C - F = 12 Hz, C4 ?  1 (C, dd, 1J C - F = 243 Hz, 2J C - F = 9 Hz, C2 ?  1 (C, CO). MS (DIP), m /z (%); significant ions: 338 (M?+ , 1), 149 [(C 11H 17) + , 43], 148 (86), 147 [(C 6H 4F3N)? + , 42], 136 (19), 135 [(C 10H 15) + , 100], 119 (18), 107 (56), 106 (15), 105 (15), 93 (42), 91 (28), 79 (16), 77 (16). Elemental analysis: Calculated for C18 H 21F3N2O: C 63.89% H 6.26% N 8.28 % 336 Materials & Methods Found: C 63.83% H 6.52% N 8.26% Preparation of 1 (?) -[ (tricyclo[ 3.3.1 .0 3,7 ]non -3-yl) ethyl] -3-(2,3,4 -trifluorophenyl)urea, 180 The crude was purified by column chromatography (SiO2, hexane/ EtOAc mixture). Evaporation in vacuo of the appropriate fractions gave the urea 180 (53 mg, 63% yield) as a white solid. The analytical sample was obtained by crystallization from EtOAc/p entane. Analytical and spectroscopic data of the 180: Melting point: 148 - 150 ? C. IR (ATR) ?: 3302, 2929, 2861, 1717, 1637, 1620, 1561, 1509, 1474, 1291, 1242, 1190, 1173, 1138, 1077, 1030, 1015, 964, 797, 754, 682, 653 cm-1. 1H -NMR (500 MHz, CDCl 3) ? (mixture of rotamers): 1.11 (d, J = 6.5 Hz) and 1.13 (d, J = 6.5 Hz) (3 H, 2 CHC H 3), 1.20-1.70 [complex signal, 10 H, 2(4) -H 2, 6(8)-H 2, 9-H 2], 2.04 (t, J = 6.5 Hz) and 2.16 (t, J = 6.5 Hz) (1 H, 7 -H), 2.18 (broad s) and 2.22 (broad s) [2 H, 1(5) -H], 3.97 (m, 1 H, C H CH 3), 5.00 (s) and 6.65 (s) (1 H, 1 -NH ), 6.90 (m, 1 H, ?-H), 7.15 (m, 1 H, 3 -NH ), 7.74 (m) and 7.81 (m) (1 H, ?-H). 13C-NMR (125.7 MHz, CDCl 3) ? (mixture of rotamers): 17.4 and 17.9 (CH 3, CH CH 3), 35.1 and 35.4 (CH 2, C9), 36.9 and 37.1 (CH, C1 or C5), 37.4 and 37.5 (CH, C5 or C1), 41.1 (CH, C7), 43.6 and 43.7 (CH 2, C6 or C8), 44.0 and 44.1 (CH 2, C8 or C6), 44.5 and 44.8 (CH 2, C2 or C4), 46.1 and 46.2 (CH 2, C4 or C2), 51.4 and 52.4 (CH, CHCH 3), 52.9 and 53.3 (C, C3), 111.5 and 112.9 (CH, dt, 2J C - F = 17.7 Hz, 3J C - F = 4.3 Hz, C5 ?  11 and 125.2 (CH, broad s, C6 ?  121-125.2 (C, several peaks, C1?  1-153.0 (C, several peaks, C2? C3? and C4?  11 and 154.6 (C, CO). MS (DIP), m /z (%); significant ions: 338 (M? + , 1), 146 (100). Elemental analysis: Calculated for C18 H 21F3N2O: C 63.89% H 6.26% N 8.28 % Calculated for C18 H 21F3N2O ?0.15EtOAc: C 63.55% H 6.36% N 7.97 % Found: C 63.26% H 6.59% N 8.26 % CHAPTER 3 337 Preparation of 1 -(pentacyclo[6.4.0.0 2,10 .0 3,7 .0 4,9 ]dodec -8-yl) -3-(2,3,4 -trifluorophenyl)urea, 181 Purification by column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and evaporation in vacuo of the appropriate fractions gave the urea 181 (14 mg, 60% yield) as a white solid. Analytical and spectroscopic data of compound 181: Melting point: > 220 ? C (dec.). IR (ATR) ?: 3388, 3260, 2958, 2932, 2866, 1645, 1546, 1504, 1481, 1324, 1267, 1248, 1231, 1188, 1168, 1059, 1044, 991, 786, 719, 691, 670, 631 cm-1. 1H -NMR (500 MHz, CDCl 3) ?: 1.12 (t, J = 2.5 Hz, 1 H, 9 -H), 1.49 -1.67 [complex signal, 8 H, 5(11) -H 2 and 6(12)-H 2], 2.19 [broad s, 2 H, 4(10) -H], 2.28 [broad s, 2 H, 2(3) -H], 2.71 [broad s, 2 H, 1(7) -H], 4.92 (broad s, 1 H, N H -Al), 6.47 (broad s, 1 H, N H -Ar), 6.90 (m, 1 H, ?-H), 7.83 (m, 1 H, ?-H). 13C-NMR (125.7 MHz, CDCl 3) ?: 21.5 [CH 2, C6(12)], 24.4 [CH 2, C5(11)], 46.9 [CH, C2(3)], 52.8 [CH, C4(10)], 53.7 [CH, C1(7)], 55.6 (CH, C9), 65.6 (C, C8), 111.5 (CH, dd, 2J C - F = 17.7 Hz, 3J C - F = 3.9 Hz, C5 ?  11 (CH, C6 ?  12 (C, dd, 2J C - F = 8.0 Hz, 3J C - F = 3.3 Hz Ar - C1?  1 (C, d, 1J C - F = 247.7 Hz, Ar -C3?  1 (C, d, 1J C - F = 245.2 Hz, Ar -C4?  11 (C, d, 1J C - F = 249.0 Hz, Ar -C2?  12 (C, CO). MS (DIP), m /z (%); si gnificant ions: 348 (M? + , 31), 320 (30), 202 [(C 13H 16NO) + , 12], 147 [(C 6H 4F3N)? + , 100], 146 (19), 119 (16), 91 (11). HRMS -ESI+ m /z [ M +H ] + calcd for [C 19H 19F3N2O+H] + : 349.1522, found: 349.152 338 Materials & Methods Preparation of 1 -( 9-methyl -6,7,8,9,10,11 -hexahydro -5H-5,9:7,11 - dimethanobenzo[9]annulen -7-yl ) -3-(2,3,4 -trifluorophenyl)urea, 182 For the preparation of compound 182, the starting amine was used as a free base, hence less equivalents of triethylamine (4 eq.) were needed following the above general procedure. Column chromatography (SiO2, hexane/ EtOAc mixture) of the crude and concentration in vacuo of the appropriate fractions gave the urea 182 (38 mg, 13% yield) as a white solid. Analytical and spectroscopic data of compound 182: Melting point: 206 - 207 ? C. IR (ATR) ?: 3331, 2903, 2839, 1654, 1556, 1510, 1473, 1361, 1344, 1290, 1237, 1174, 1101, 1038, 1019, 1004, 800, 756, 690, 669, 625 cm-1. 1H -NMR (500 MHz, CD 3OD) ?: 0.94 (s, 3 H, C H 3), 1.50 [d, J = 13.5 Hz, 2 H, 10(13) -H b], 1.69 [m, 2 H, 10(13) -H a ], 1.77 (s, 2 H, 8 -H), 2.10 [m, 2 H, 6(12) -H a ], 2.15 [d, J = 13 Hz, 2 H, 6(12)-H b], 3.08 [tt, J = 6 Hz, J ? = 1.5 Hz, 2 H, 5(11) -H], 6.98 (m, 1 H, ?-H), 7.04 [s, 4 H, 1(2,3,4)-H], 7.66 (m, 1 H, ?-H). 13C-NMR (125.7 MHz, CD3OD) ?: 32.9 (CH 3, CH 3), 34.6 (C, C9), 40.6 [CH 2, C6(12)], 42.5 [CH, C5(11)], 42.5 [CH 2, C10(13)], 44.9 [CH 2, C16(20)], 49.0 (CH 2, C8), 54.5 (C, C7), 112.2 (CH, dd, 2 J C - F = 17.8 Hz, 3 J C - F = 3.9 Hz, C5 ?  11 (CH, C6 ?  120 (C, dd, 2 J C - F = 8.0 Hz, 3J C - F = 2.4 Hz Ar -C1?  12 [CH, C2(3)], 129.0 [CH, C1(4)], 141.0 (C, dt, 1J C - F = 247.8 Hz, 2 J C - F = 14.9 Hz, Ar -C3?  1 (C, dd, 1J C - F = 245.7 Hz, 2J C - F = 12.8 Hz, Ar -C4?  1 (C, dd, 1J C - F = 242.6 Hz, 2 J C - F = 10.3 Hz, Ar -C2?  1 [C, C4a(C11a)], 156.1 (C, CO). MS (DIP), m /z (%); significant ions: 400 (M? + , <1), 253 (19), 228 (14), 211 [(C 16H 19) + , 16], 172 (23), 155 (54), 149 (56), 148 (100), 147 (52), 143 (22), 141 (20), 129 (21), 128 (18), 115 (16). Elemental analysis: Calculated for C23 H 23F3N2O: C 68.99% H 5.79% N 7.00 % Found: C 68.94% H 5.92% N 6.71 % CHAPTER 3 339 Preparation of 1 -( 5-methyl -1,2,3,5,6,7 -hexahydro -1,5:3,7 -dimethano -4-benzoxonin -3-yl) - 3-(2,3,4 -trifluorophenyl)urea, 183 For the preparation of compound 183, the starting amine was used as a free base, hence less equivalents of triethylamine (4 eq.) were needed following the above general procedure. Any purification the urea was needed. The desired urea 183 was obtained as a white solid (205 mg, 54% yield) as a white solid. Analytical and spectroscopic data of compound 183: Melting point: 257 - 259 ? C. IR (ATR) ?: 3295, 3241, 3118, 2916, 2173, 1693, 1620, 1564, 1510, 1493, 1468, 1462, 1356, 1345, 1320, 1302, 1286, 1273, 1254, 1229, 1210, 1181, 1167, 1111, 1091, 1074, 1049, 1035, 1008, 999, 958, 906, 820, 812, 763, 646 cm-1. 1H -NMR (400 MHz, DMSO-d6) ?: 1.18 (s, 3 H, CH 3), 1.56 [d, J = 13.6 Hz, 2 H, 6(13) -H b], 1.84 [m, 2 H, 6(13) -H a ], 1.97 [d, J = 13.2 Hz, 2 H, 2(12) -H b], 2.20 [m, 2 H, 2(12) -H a ], 3.16 [t, J = 5.5 Hz, 2 H, 1(7) -H], 4.06 (s, 1 H, N H -Al), 7.14 (complex signal, 5 H, 15 -H, ?-H), 7.84 (m, 1 H, ?-H), 8.52 (broad s, 1 H, N H -Ar). 13C-NMR (100.6 MHz, DMSO-d6) ?: 31.1 (CH 3, CH 3), 37.4 [CH 2, C2(12)], 38.1 [CH 2, C6(13)], 38.2 [CH, C1(7)], 73.4 (C, C5), 82.7 (C, C3), 111.6 (CH, dd, 2J C - F = 17.2 Hz, 3J C - F = 3.5 Hz, C5 ?  11 (CH, broad s, C6 ?  120 (C, dd, 2J C - F = 7.8 Hz, 3J C - F = 3.0 Hz Ar -C1?  126.5 [CH, C9(10)], 128.2 [CH, C8(11)], 139.0 (C, dd, 1 J C - F = 246 Hz, 2 J C - F = 15 Hz, Ar -C3?  141.0 (C, dd, 1 J C - F = 248 Hz, 2J C - F = 12 Hz, Ar -C4?  1 (C, dd, 1J C - F = 241 Hz, 2J C - F = 11 Hz, Ar-C2?  145.5 [C, C7a(C11a)], 152.3 (C, CO). MS (DEPEI), m /z (%); significant ions: 402 (M? + , 48), 171 (13), 170 (34), 169 (21), 157 (20), 156 (18), 155 (53), 154 (14), 153 (11), 148 (18), 147 [(C 6H 4F3N)? + , 100], 146 (53), 145 (15), 143 (25), 142 (21), 141 (23), 131 (12), 130 (15), 129 (65), 128 (46), 127 (22), 116 (12), 115 (55), 91 (17), 84 (19), 83 (28), 71 (15), 70 (16), 69 (21). Elemental analysis: Calculated for C22 H 21F3N2O2: C 65.66% H 5.26% N 6.96 % Calculated for C22 H 21F3N2O2?0.1H 2O: C 65.37% H 5.29% N 6.93% 340 Materials & Methods Found: C 65.18% H 5.31% N 6.73 % HRMS -ESI+ m /z [ M +H ] + calcd for [C 22H 21F3N2O2+ H ] + : 403.1633, found: 403.1631. Preparation of (2 -oxaadamant -1-yl)isocyanate, 185 In a three-necked round-bottom flask equipped with a stir bar, low temperature thermometer and gas inlet, triphosgene (392 mg, 1.32 mmol) was added in a single portion to a solution of amine hydrochloride 120?HCl (500 mg, 2.63 mmol) in DCM (35 mL) and saturated NaHCO 3 solution (15 mL). The biphasic mixture was stirred vigorously at 4 ?C for 30 minutes. Afterwards, the phases were separated and the organic layer was washed with brine (20 mL), dried over anh. Na2SO4 and filtered. Evaporation under vacuo provided 185 (408 mg, 86% yield), that was used in next step without further purification. IR (ATR) ?: 2235 (isocyanate band) cm-1. Preparation of 1-(1 -acetylpiperidin -4-yl) -3-(oxaadamant -1-yl)urea, 184 Under anhydrous conditions, a solution of isocyanate 185 (323 mg, 1.80 mmol) in anh. DCM (20 mL) was added to a solution of 1-acetyl-4-aminopiperidine (308 mg, 2.16 mmol) in anh. DCM (10 mL), followed by TEA (0.50 mL, 3.61 mmol). The reaction mixture was s tirred at room temperature overnight. The solution was then concentrated under vacuo to give an orange gum (720 mg). Purification by column chromatography (SiO2, DCM/methanol mixture) gave the titled compound 184 (300 mg, 52% yield) as a white solid. The analytical sample was obtained by washing with pentane. Analytical and spectroscopic data of compound 184: Melting point: 172 - 173 ? C. IR (ATR) ?: 3322, 2920, 2850, 2188, 2153, 2000, 1637, 1549, 1428, 1369, 1313, 1264, 1234, 1192, 1139, 1090, 1046, 995, 959, 879, 816, 773, 731 cm-1. CHAPTER 3 341 1H -NMR (400 MHz, CDCl 3) ?: 1.28-1.41 (complex signal, 2 H, 3 -H ax and 5-H ax ), 1.58-1.96 [complex signal, 9 H, ?-H a ?-H a ?-H 2 10?-H 2 ?-H 2, and 3-H eq or 5-H eq], 1.98 -2.22 (complex signal, 6 H, COC H 3 ?-H b ?-H b and 5-H eq or 3-H eq), 2.27 [broad s, 2 H, ?(? - H], 2.86 (dt, J = 11.2 Hz, J ? = 2.8 Hz, 1 H, 6 -H ax or 2-H ax ), 3.18 (dt, J = 10.8 Hz, J ? = 2.8 Hz, 1 H, 2 -H ax or 6-H ax ), 3.70 (dm, J = 14 Hz, 1 H, 2 -H eq or 6-H eq), 3.87 (m, 1 H, 4 -H), 4.27 (broad s, 1 H, ?-H), 4.35 (broad d, J = 14.0 Hz, 1 H, 6 -H eq or 2-H eq), 4.75 (s, 1 H, 3 -NH ), 6.06 (d, J = 8 Hz, 1 -NH ). 13C-NMR (100.6 MHz, CDCl 3) ?: 21.4 (CH 3, CH 3CO), 27.9 [CH, C5 ?(? @ 21 (CH 2, C3 or C5), 33.2 (CH 2, C5 or C3), 34.6 (broad CH 2, C4? C6? and C10?  00 (CH 2, C8? or C9?  02 (CH 2, C9? or C8?  0 (CH 2, C2 or C6), 45.1 (CH 2, C6 or C2), 46.5 (CH, C4), 71.0 (CH, C3 ?  0 (C, C1?  1 (C, NH CONH), 168 .9 (C, CH 3CO). MS (DIP), m /z (%); significant ions: 321 (M? + , 34), 197 (32), 179 [(C 10H 13NO2)? + , 34], 154 (100), 153 (18), 137 [(C 9H 13O) + , 33], 136 (32), 126 (15), 125 (51), 122 (21), 111 (17), 96 (41), 95 (18), 94 (45), 84 (19), 83 (37), 82 (54), 79 (22), 67 (20), 57 (23), 56 (32), 55 (15). Elemental analysis: Calculated for C17H 27N3O3: C 63.53% H 8.47% N 13.07% Calculated for C17H 27N3O3?0.25H 2O: C 62.65% H 8.50% N 12. 89% Found: C 62.64% H 8.61 % N 12.74% Preparation of N-(1 -acetylpiperidin -4-yl) -1H-imidazole -1-carboxamide, 187 11?-carbonyldiimidazole (400 mg, 2.46 mmol) was suspended in anh. 1,2- dichloroethane (15 mL) under nitrogen. Then 1-acetyl-4-aminopiperidine (250 mg, 1.76 mmol) was added and the reaction mixture was heated to 50 ?C for 21 hours. With an external ice bath, the mixture was cooled down for 30 minutes. The resulting solid was collected by filtration under vacuo and washed with 1,2-DCE (20 mL) yielding the titled product 187 (312 mg, 75% yield) as a white solid. Analytical and spectroscopic data of compound 187: Melting point: 191 - 193 ? C. IR (ATR) ?: 3216, 3118, 3038, 2918, 2342, 2074, 1709, 1613, 1542, 1479, 1463, 1441, 1369, 342 Materials & Methods 1358, 1320, 1281, 1272, 1233, 1195, 1137, 1111, 1090, 1068, 1053, 1001, 984, 974, 916, 902, 859, 799, 748, 652 cm-1. 1H -NMR (400 MHz, CDCl3) ?: 1.37 (complex signal, 2 H, 3 -H ax , 5-H ax ), 1.99 (dm, J = 12.8 Hz, J ?= 4 Hz, 1 H) and 2.21 (dm, J = 12.8 Hz, J ?= 4 Hz, 1 H) (?-H eq and ?-H eq), 2.09 (s, 3 H, COC H 3), 2.69 (ddd, J = 13 Hz, J ?= 2.6 Hz, 1 H) and 3.21 (ddd, J = 13 Hz, J ? = 2.6 H z, 1 H) (2?-H ax and ?-H ax ), 3.86 (dm, J = 13.6 Hz, 1 H) and 4.67 (dm, J = 13.6 Hz, 1 H) (2?-H eq and ?-H eq), 4.10 (m, 1 H, ?-H), 7.06 (dd, J = 1.6 Hz, J ?= 0.8 Hz, 1 H, 4 -H), 7.29 (broad d, J = 7.6 Hz, 1 H, N H ), 7.60 (dd, J = 1.6 Hz, J ? =1.2 Hz, 1 H, 5 -H), 8.29 (dd, J = 1.2 Hz, J ? = 0.8 Hz, 1 H, 2 -H). 13C-NMR (100.6 MHz, CDCl 3) ?: 21.5 (CH 3, COCH 3), 31.4 and 33.1 (CH 2, C3? and C5?  40.9 and 45.6 (CH 2, C2? and C6?  2 (CH, C4 ?  112 (CH, C5), 130.3 (CH, C4), 136.2 (CH, C2), 148.5 ( C, NH CNH), 169.2 (C, COCH 3). MS (DIP), m /z (%); significant ions: 169 (10), 168 (100), 153 (19), 126 (53), 125 (31), 85 (19), 84 (42), 83 (20), 82 (23), 81 (21), 68 (98), 57 (40), 56 (56), 55 (16). HRMS -ESI+ m /z [ M +H ] + calcd for [C 11H 16N4O2+H ] + : 237.1346, found: 237.1345. Preparation of 1-(1 -acetylpiperidin -4-yl) -3-(oxaadamantan -1-yl)urea, 184 In a round bottom flask equipped with a condenser apparatus and magnetic stirrer a solution of amine hydrochloride 120?HCl (18 mg, 0.09 mmol) in chloroform (2 mL) was prepared, to which was added 187 (45 mg, 0.19 mmol) followed by triethylamine (10 mg, 0.10 mmol). The solution was heated to 50 ? C for 16 hours, whereupon the reaction mixture was tempered to room temperature and evaporated under vacuo to dryness (78 mg). Purification by column chromatography (SiO2, DCM/methanol mixture) afforded the desired product 184 (16 mg, 53% yield) as a white solid. CHAPTER 3 343 Preparation of 4-oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodec -3-yl)isocyanate , 189 In a three-necked round-bottom flask equipped with a stir bar, low temperature thermometer and gas inlet, triphosgene (98 mg, 0.33 mmol) was added in a single portion to a solution of amine hydrochloride 121?HCl (50 mg, 0.24 mmol) in DCM (3.6 mL) and saturated NaHCO 3 solution (2.3 mL). The biphasic mixture was stirred vigorously at 4 ?C for 30 minutes. Afterwards, the phases were separated and the organic layer was washed with brine (10 mL), dried over anh. Na2SO4 and filtered. Evaporation under vacuo provided the isocyanate 189 (47 mg, quantitative yield). Any purification or characterization was fulfilled and the product was used in next step. IR (ATR) ?: 2256 (NCO band) cm-1. Preparation of 1-(1 -acetylpiperidin -4-yl) -3-(4 -oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodec -3- yl)urea , 188 Under anhydrous conditions, a solution of isocyanate 189 (365 mg, 1.81 mmol) in anh. DCM (20 mL) was added to a solution of 1-acetyl-4-aminopiperidine (309 mg, 2.18 mmol) in anh. DCM (10 mL), followed by TEA (0.51 mL, 3.63 mmol). The reaction mixture was stirred at room temperature overnight. The solution was th en concentrated under vacuo to give a beige solid (736 mg). Purification by column chromatography (SiO2, DCM/methanol mixture) gave the titled compound 188 (159 mg, 26% yield) as a white. The analytical sample was obtained by crystallization with ethyl acetate. Analytical and spectroscopic data of compound 188: Melting point: 164 - 166 ? C. IR (ATR) ?: 3501, 3455, 3272, 2948, 2857, 2180, 1985, 1650, 1543, 1425, 1357, 1310, 1300, 1266, 1223, 1147, 1136, 1095, 1061, 1006, 973, 912, 870, 779, 731, 659 cm-1. 344 Materials & Methods 1H -NMR (400 MHz, CDCl 3) ?: 1.32 [complex signal, 2 H, 3(5) -H ax ], 1.57 (broad d, J = 10.4 Hz, 1 H, 12?-H a), 1.90 (complex signal, 2 H, 12?-H b, 3-H eq or 5-H eq), 2.01 (complex signal, 1 H, 3 -H eq or 5-H eq), 2.07 (s, 3 H, COC H 3), 2.46 (tt, J = 5.2 Hz, J ? = 4.4 Hz, 1 H, ?-H), 2.51 (t, J = 4.4 Hz, 1 H, 1?-H), 2.62 (m, 1 H, ?-H), 2.72 (complex signal, 3 H, 2?-H, 10?-H, 11?- H), 2.83 (complex signal, 3 H, ?-H, ?-H, 2 -H ax or 6-H ax ), 3.17 (m, 1 H, 6 -H ax or 2-H ax ), 3.70 (dm, J = 13.6 Hz, 1 H, 2 -H eq or 6-H eq), 3.86 (m, 1 H, 4 -H), 4.36 (dm, J = 13.6 Hz, 1 H, 6 - H eq or 2-H eq), 4.71 (t, J = 5.2 Hz, 1 H, ?-H), 5.32 (s, 1 H, 3 -NH ), 5.69 (dd, J = 10 Hz, J ? = 8 Hz, 1 H, 1 -NH ). 13C-NMR (100.6 MHz, CDCl 3) ?: 21.4 (CH 3, COCH 3), 32.0 (CH 2, C3 or C5), 33.0 (CH 2, C5 or C3), 40.3 (CH 2, C2 or C6), 41.4 (CH, C8 ?  1 (CH, C2 ?  1 (CH, C1 ?   (CH 2, C12?   (CH, C9 ?   (CH, C7 ?  1 (CH 2, C6 or C2), 45.7 (CH, C10 ?   (CH, C4), 54.6 (CH, C6 ?   (CH, C11 ?   (CH, C5 ?  101 (C, C3?  1 (C, NH CONH), 168.8 (C, COCH 3). MS (DIP), m /z (%); significant ions: 343 (M? + , 16), 325 (62), 260 (42), 201 (15), 177 (12), 176 (82), 168 (15), 160 (18), 159 [(C 11H 11O) + , 100], 158 (29), 146 (10), 132 (13), 131 (53), 139 (20), 129 (26), 128 (15), 126 (15), 125 (33), 124 (89), 116 (16), 115 (19), 94 (17), 91 (37), 84 (25), 83 (83), 82 (59), 81 (19), 80 (18), 56 (29), 55 (16). HRMS -ESI+ m /z [ M +H ] + calcd for [C 19H 25N3O3+H ] + : 344.1969, found: 344.1972. Preparation of trans-4-(4 -amino-cyclohexyloxy) -benzonitrile, 191555 In a three-necked round bottom flask equipped with a condenser apparatus, magnetic stirrer and gas inlet a solution of trans-4-aminocyclohexanol (3 g, 26.0 mmol) in anh . DMF (130 mL) was cooled to 0 ? C with an ice/NaCl bath. Sodium hydride, 60% dispersion in mineral oil (1.15 g, 28.7 mmol) was added and the suspension was stirred for 1 hour while cooling. 4-fluorobenzonitrile (3.9 g, 32.6 mmol) was added and the reaction mixture was heated to 60 ? C for 2 hours and stirred at room temperature overnight. The resulting suspension was diluted with EtOAc (150 mL) and washed with water (2 x 100 mL) and brine (100 mL). The organic layer was extracted with 1N HCl solution (2 x 50 mL) and the combined aqueous phases were basified with 10N NaOH solution until pH ~ 12 and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under vacuo to give a total amount of 2.59 g (46% yield) of nitrile 191. The spectroscopic data were identical to those previously published. CHAPTER 3 345 Preparation of trans-4-(4 -amino-cyclohexyloxy) -benzoic acid, 190?HCl 6 N NaOH solution (30 mL) was added to a solution of nitrile 191 (2.58 g, 11.9 mmol) in ethanol (100 mL) and the reaction mixture was heated to 80 ? C for 18 hours. The solution was then tempered to room temperature and evaporated under vacuo. Water (100 mL) was added and the solution was acidified with conc. HCl at 0 ? C until acidic pH. The resulting precipitate was filtered under vacuo to give the desired product 190?HCl (3.07 g, 95% yield) as a white solid, whose spectroscopic were in good agreement with those previously published. Preparation of trans-4-[4 -(3 -oxaadamant -1-yl -ureido) -cyclohexyloxy] -benzoic acid , 192 A solution of isocyanate 185 (400 mg, 2.23 mmol) in anh. DCM (25 mL) was added to a solution of amine hydrochloride 190?HCl (728 mg, 2.68 mmol) in anh. DCM (12 mL), followed by TEA (1.24 mL, 8.94 mmol) under nitrogen. The reaction mixture was stirred at room temperature overnight. Water (50 mL) was then added and the phases were separated. The organic layer was extracted with further water (2 x 50 mL) and the pH of the combined aqueous phases was adjusted to pH ~2 with 5N HCl solution, prior extraction with DCM (3 x 50 mL). The combined organic layers were dried over anh. Na2SO4, filtered and concentrated under vacuo yielding 192 (220 mg, 24% yield) as a white solid. The analytical sample was obtained by crystallization with methanol/diethyl ether. Analytical and spectroscopic data of compound 192: Melting point: 255 - 275 ? C. IR (ATR) ?: 3364, 3267, 3198, 3061, 2922, 2559, 2348, 2187, 2068, 2011, 1977, 1672, 1601, 1552, 1443, 1369, 1347, 1320, 1231, 1196, 1172, 1110, 1091, 1049, 1027, 989, 959, 863, 346 Materials & Methods 828, 774, 698, 640 cm-1. 1H -NMR (400 MHz, CD 3OD) ?: 1.37 [m, 2 H, ?(? -H ax ], 1.55 [m, 2 H, 2?(? -H ax ], 1.65 [dm, J = 12.4 Hz, 2 H, ??(10?? -H a ], 1.78 [dm, J = 11.6 Hz, 2 H, ??(?? -H a ], 1.86 (complex signal, 2 H, ??-H 2), 1.94 [m, 2 H, ??(10?? -H b], 2.01 [m, 2 H, ?(? -H eq], 2.10 [m, 2 H, 2?(? -H eq], 2.22 [broad s, 2 H, ??(?? -H], 2.2 8 [dm, J = 15.5 Hz, 2 H, ??(?? -H b], 3.57 (m, 1 H, ?-H), 4.18 (broad s, 1 H, ??-H), 4.40 (m, 1 H, 1?-H), 6.92 [d, J = 8.8 Hz, 2 H, 3(5) -H], 7.93 [d, J = 8.8 Hz, 2 H, 2(6) -H]. 13C-NMR (100.6 MHz, CD 3OD) ?: 29.6 [CH, C5 ??(?? @ 10 [CH 2, C2?(? @ 31.4 [CH 2, C3?(? @  [CH 2, C4??(10?? @  (CH 2, C6??  10 [CH 2, C8??(?? @  (CH, C4 ?  2 (CH, C3 ??   (CH, C1 ?  1 (C, C1??  11 [CH, C3(5)], 126.8 (C, C1), 132.6 [CH, C2(4)], 159.0 (C, NH CONH), 162.4 (C, C4 and CO2H). MS (DIP), m /z (%); significant ions: 414 (M? + , 0.2), 179 [(C 10H 13NO2)?+, 27], 138 [(C 9H 14O) + , (C7H 5O3) + , 100], 122 (29), 121 (39), 111 (21), 108 (10), 98 (99), 96 (30), 94 (45), 82 (18), 81 (97), 79 (41), 67 (19), 65 (15), 56 (42), 55 (16). Elemental analysis: Calculated for C23 H 30N2O5: C 66.65% H 7.30% N 6.76% Calculated for C23 H 30N2O5?0.1H 2O: C 66.36% H 7.31% N 6.73% Found: C 66.13% H 7.32% N 6.64% Preparation of trans-4-[4 -[ 3-(4 -oxahexacyclo[5.4.1.0 2,6 .0 3,10 .0 5,9 .0 8,11 ]dodec -3- yl )ureido]cyclohexyloxy] -benzoic acid , 193 A solution of isocyanate 189 (387 mg, 1.93 mmol) in anh. DCM (20 mL) was added to a solution of amine hydrochloride 190?HCl (627 mg, 1.93 mmol) in anh. DCM (10 mL), followed by TEA (1.07 mL, 7.70 mmol) under nitrogen. The reaction mixture was stirred at room temperature overnight. Water (80 mL) was then added and the phases were separated. The organic layer was extracted with further water (2 x 80 mL) and the pH of CHAPTER 3 347 the combined aqueous phases was adjusted to pH ~ 2 with 5N HCl solution, prior extraction with DCM (3 x 80 mL). The combine d organic layers were dried over anh. Na2SO4, filtered and concentrated under vacuo to give a white solid (556 mg). Purification by column chromatography (SiO2, DCM/methanol mixture) yielded 193 (418 mg, 50% yield) as a white solid. Analytical and spectroscopic data of compound 193: Melting point: 254 - 256 ? C. IR (ATR) ?: 3358, 3267, 2939, 2856, 2473, 2369, 2216, 2159, 1669, 1600, 1558, 1487, 1446, 1305, 1242, 1168, 1108, 1033, 992, 910, 847, 776, 694 cm-1. 1H -NMR (500 MHz, DMSO -d6) ?: 1.27 [complex signal, 2 H, ?(? -H ax ], 1.46 [complex signal, 4 H, 12 -H a 2?(? -H ax ], 1.86 [complex signal, 4 H, 12 -H b ?(? -H eq ], 2.01 [complex signal, 2 H, 2?(? -H eq], 2.37 (complex signal, 2 H, 1??-H and ??-H), 2.53 (m, 1 H, 2??-H), 2.62 (m, 1 H  ??-H), 2.70 (complex signal, 2 H, ??-H and ??-H), 2.84 (m, 1 H, 11??-H), 2.90 (t, J = 7.5 Hz, 1 H, 10??-H), 3.42 (m, 1 H, ?-H), 4.43 (m, 1 H, 1?-H), 4.54 (t, J = 5.5 Hz, 1 H, ??-H), 5.84 (d, J = 7.5 Hz, 1 H, 1 -NH ), 6.69 (s, 1 H, 3 -NH ), 7.00 [m, 2 H, 3(5)-H], 7.85 [m, 2 H, 2(6) -H]. 13C-NMR (125.7 MHz, DMSO -d6) ?: 29.6 [CH 2, C2?(? @ 01 [CH 2, C3?(? @ 0 (CH, C2??  10 (CH, C8 ??  2 (CH, C1 ??  0 (CH 2, C12??   (CH, C9 ??  2 (CH, C7 ??  44.6 (CH, C10 ??   (CH, C4 ?   (CH, C6??   (CH, C11 ??   (CH, C1 ?  1 (CH, C5 ??  102 (C, C2??  110 [CH, C3(5)], 122.9 (C, C1), 131.3 [CH, C2(6)], 155.9 (C, C4), 161.0 (C, NH CONH), 167.0 (C, CO2H). MS (EI), m /z (%); significant ions: 200 (28), 158 (13), 135 (43), 132 (18), 131 (95), 130 (53), 129 (41), 128 (21), 122 (18), 119 (19), 117 (17), 116 (28), 115 (36), 104 (19), 94 (16), 92 (30), 91 (100), 81 (22), 79 (24), 78 (64), 77 (42), 70 (16), 66 (26), 65 (38), 64 (15), 63 (17). H RMS-ESI+ m /z [ M +H ] + calcd for [C 25H 28N2O5+H ] + : 437.2076, found: 437.2071. H RMS-ESI m /z [ M -H] + calcd for [C 25H 28N2O5 H ] + : 435.1920, found: 435.1905. Elemental analysis: Calculated for C25 H 28N2O5: C 68.79% H 6.47% N 6.42% Calculated for C25H 28N2O5?0.1CH 3OH ?0.2DCM: C 66.54% H 6.36% N 6.13% Found: C 66.70% H 6.55% N 5.73% 348 Materials & Methods Preparation of 1-(1 -acetylpiperidin -4-yl) -3-(3 ,7 -dimethyl(tricyclo[3.3.0.0 3,7 ]octa -1-yl)urea, 195 In a round bottom flask equipped with a condenser apparatus and magnetic stirrer a solution of amine hydrochloride 123?HCl (68 mg, 0.36 mmol) in chloroform (5 mL) was prepared, to which was added 187 (172 mg, 0.73 mmol) followed by triethylamine (0.06 mL, 0.40 mmol). The solution was heated to 50 ? C for 25 hours, whereupon the reaction mixture was tempered to room temperature and evaporated under vacuo to dryness (384 mg). Purification by column chromatography (SiO2, DCM/methanol mixture) afforded the desired product 195 (90 mg, 77% yield) as a white solid. Analytical and spectroscopic data of compound 195: Melting point: 165 - 167 ? C. IR (ATR) ?: 3359, 3244, 2947, 2878, 2170, 2034, 1960, 1613, 1556, 1477, 1443, 1371, 1318, 1264, 1227, 1151, 1096, 1033, 978, 717, 639 cm-1. 1H -NMR (400 MHz, CDCl 3) ?: 1.11 [s, 6 H, ?(? -CH 3], 1.22 [complex signal, 2 H, 2(6) -H a ], 1.34 [dd, J = 8.2 Hz, J ?= 3.4 Hz, 2 H, ?(? -H a ], 1.54 [dd, J = 7.4 Hz, J ?= 3.4 Hz, 2 H, 2?(? - H a ], 1.70 [dd, J = 8.2 Hz, J ?= 2.6 Hz, 2 H, ?(? -H b], 1.75 [d, J = 7.6 Hz, 2 H, 2?(? -H b], 1.90 and 2.03 (complex signal, 2 H, 3 -H ax and 5-H ax ), 2.07 (s, 3 H, COC H 3), 2.27 (t, J = 2.6 Hz, 1 H, ?-H), 2.75 (dt, J = 14.0 Hz, J ? = 2.8 Hz, 1 H, 2 -H ax or 6-H ax ), 3.14 (dt, J = 11.2 Hz, J ? = 2.8 Hz, 2 H, 6 -H ax or 2-H ax ), 3.73 (broad d, J = 13 Hz, 2 H, 6 -H eq or 2-H eq), 3.83 (m. 1 H, 4 - H), 4.42 (broad d, J = 13 Hz, 2 H, 2 -H eq or 6-H eq), 4.79 (d, J = 7.6 Hz, 1 H, 1 -NH ), 5.18 (broad s, 1 H, 3 -NH ). 13C-NMR (100.6 MHz, CDCl 3) ?: 319 (M? + , 1), 277 [(C 16H 27N3O)? + , 58], 263 (20), 177 [(C 11H 15NO)?+ , 61], 169 [(C 8 H 13N2O2) + , 25], 151 (18), 150 [(C 10H 16N) + , 22], 148 (25), 143 [(C 6H 13N3O)? + , 100], 136 (26), 135 [(C 10H 15) + , 43], 134 (31), 127 (16), 126 (23), 125 (17), 122 (86), 121 (29), 119 (25), 110 (22), 109 (86), 108 (48), 96 (18), 95 (62), 94 (29), 93 (16), 91 (15), 84 (31), 83 (16), 82 (33), 56 (25), 55 (17). MS (DIP), m /z (%); significant ions: 278 (10), 277 (58), 263 (20), 178 (10), 169 (25), 151 (18), 150 (22), 148 (25), 143 (100), 136 (26), 135 (43), 134 (31), 127 (16), 126 (23), 125 (17), 123 (11), 122 (86), 121 (29), 119 (25), 110 (22), 109 (86), 108 (48), 96 (18), 95 (62), 94 (29), CHAPTER 3 349 93 (16), 91 (15), 84 (31), 83 (16), 82 (33), 80 (11), 79 (12), 77 (11), 67 (11), 57 (13), 56 (25), 55 (17). Elemental analysis: Calculated for C18 H 29N3O2: C 67.68% H 9.15% N 13.15% Calculated for C18 H 29N3O2?1.0H 2O: C 64.07% H 9.26 % N 12.45% Found: C 64.00% H 9.31% N 12.40% IC50 det e rminatio n CHAPTER 3 353 IC50 assay determination a) Buffer prep arati on : The buffer utilized was b i s -tris with an ionic strength of 25 mM, supplemented with 0.1 mg BSA fraction V per mL of buffer and prepared with water filtered through a MilliQ reagent water system (Millipore Corp., Bedford, MA, USA). The pH was adjusted to 7.0 with 1 N HCl solution. b) Substrate prep arati on : (3-Phenyl-oxiranyl) -acetic acid cyano-(6-methoxy -naphthalen-2-yl)-methyl ester (PHOME , Cayman Chemical, item number 10009134) was employed as a fluorescent substrate at a final concentration of 5 ?M. As the total volume of the assay is 200 ?L in all the wells, a 200 ?M-solution of PHOME in DMSO was prepared pri or to the assay performance. c) E nz ym e dil uti on : Beforehand to assaying, human recombinant sEH, purchased by Cayman Chemical, was diluted with cold assay buffer (588 ?L) and kept on ice during the assay, no longer than 4 hours. d) Preparati on o f i nhi bi tors : Each compound was dissolved in DMSO so as to prepare an 800 ?M-stock solution. Serial dilutions in DMSO were followed in order to have a range of concentrations for the compound testing (from 0.05 to 20 nM). In that the final concentration of the assay is 40x diluted, the compounds were prepared 40 times more concentrated (from 2 to 800 nM). Five different concentrations were used for each compound. e) A ssay pe rform anc e : For every determination, the 96-well black microplate (Greiner Bio-one, item n? 655900) was designed so to have at least a duplicate of the background and a triplicate of each concentration of inhibitor, as well as a triplicate of 100% activity . Table 25 discloses the pipetting summary for performing the assay. 354 Materials & Methods Table 25. Reagents of the inhibitory assay and addition order. Well Assay Buffer Solvent (DMSO) Inhibitor sEH Assay Buffer + Substrate Background 90 ?L 5 ?L - - 100 ?L + 5 ?L Positive control 85 ?L 5 ?L - 5 ?L 100 ?L + 5 ?L Inhibitor 85 ?L - 5 ?L 5 ?L 100 ?L + 5 ?L Addition order 1 2 3 4 5 As shown in Table 25, a pre-mixture of the assay buffer and the substrate was prepared for its final addition with a multichannel pipette. After the addition of the substrate, the microplate was carefully shaken for 10 seconds and incubated for 5 minutes at room temperature. Enzyme activity was measured by means of the fluorescence appearance of the hydrolysed product 6-methoxy -2-naphthaldehyde over 10 min with a FLUOstar OPTIMA microplate reader (BMG) (excitation wavelength: 337 nm; emission wavelength: 460 nm). f) Pharm aco l ogi c al resul ts : Results were obtained by regression analysis from at least three data points in a linear region of the curve. IC50 values are average of minimum three independent replicates. Results are given as means ? standard error . Water solubility CHAPTER 3 357 Apparent or semi-equilibrium solubility measurements a) C ali brati on c urve of a stand ard , 109: Compound 109 was used as a standard for calculating the linear regression of concentration v s area. In this manner, the calibration curve was obtained from six standard stock solutions with UV detection at 225 and 254 nm ( r = 0.99) (Table 26). Table 26. Area values for six standard stock solutions at 225 and 254 nm. Concentration Area ?g/mL ?mol/mL 225 nm 254 nm 100 0.3042 6056764 965689 50 0.1521 2966801 560875 10 0.0304 503418 125001 5 0.0152 200315 70518 1 0.0030 34244 10530 0,1 0.0003 30309 3147 The measurement was performed by HPLC (Waters), with an Akady ODS-C18 column (250 mm x 4.6 mm, 5 ?m) at room temperature. Ten microliters of the samples were injected into the column. The conditions were the following: flow rate of 0.55 mL/min with an isocratic elution for 25 min (0.1% formic acid in acetonitrile/0.1% formic acid in water, 75:25 (v/v) ). b) Sam pl e p reparati on for sol ubi l i ty measurem ents : Solubility samples were prepared according to the method described by Tsai et al. 539 Each compound (~1 mg) was added into sodium phosphate buffer (0.1 M sodium phosphate, pH 7.4, 1 m L) to create a suspension, which was equilibrated for 24 h at room temperature. The suspension was then centrifuged (8,000 rpm, 5 min, RT) using centrifuge. The supernatant Zas transIerred to neZ )alcon? tube and Zas diluted 10 times with methanol to precipitate the salts. The solution was centrifuged (8,000 rpm, 5 min, RT) by centrifuge and the supernatant was filtered through PTFE membrane filters (0.22 ?m) to a vial. The samples were kept at -20 ?C before being analysed by HPLC, if necessary. c) Sol ubi l i ty determi nati on : Two different conditions were applied depending on the compound for testing: 358 Materials & Methods ? Conditions A: flow rate of 0.55 mL/min with an isocratic elution for 25 min ( 0.1% formic acid in acetonitrile/0.1% formic acid in water, 75:25 (v/v) ). ? Conditions B: flow rate of 0.60 mL/min with an isocratic elution for 15 min ( 0.1% formic acid in acetonitrile/0.1% formic acid in water, 92:8 (v/v) ). Compounds 170-183 and 192-193 were measured applying conditions A, whereas compounds 184, 188 and 195 were tested using conditions B, either at 225 nm or at 254 nm. d) C orrecti on facto r cal c ulati on : The absorbance of a 1 mM solution of 184, 188 and 195 in methanol was measured by HPL C, as well as of a 1 mM solution of the standard 109 in methanol. The correction factors were determined for each compound (Table 27). Table 27. Correction factor applied for the determination of the solubility of compounds 184, 188 and 195. Comp. Correction factor 184 2.28 188 2.81 195 3.27 e) Results : Apparent solubility results are average of minimum two independent replicates in ?g/ mL ? standard deviation. In vitro studies CHAPTER 3 361 In vitro cell cultures a) C el l c ulture : Human Huh -7 were maintained in a humid atmosphere of 5% CO 2 at 37 ?C in high glucose (25 mmol/L Dulbecco?s modiIied (agle?s medium (D0(0 supplemented with 10% heat -inactivated fetal bovine serum (FBS), 1% of penicillin/streptomycin (10. 000 units/mL of penicillin and 10.000 ?g/mL of streptomycin) and 1% of amphotericin B (250 ?g/mL). b) C el l treatm ent : Huh -7 cells were serum-starved overnight pri or to treatment. Lipid-containing media were prepared by conjugation of palmitic acid with fatty acid -free BSA, as previously described.575 Cells were pre-treated with the inhibitors (final concentration 1 ? mol/L ) for 1 hour before treatment with palmitate (final concentration 0.5 mmol/L ) and inhibitors (final concentration 1 ? mol/L ). For each condition, at least 3 replicates were performed. Following 48 hours of incubation, RNA or protein were extracted as described below. c) I m m unoblo tti ng : To obtain total protein, hepatocytes were homogenized in RIPA lysis buffer (Sigma) with 5 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L sodium orthovanadate and 5.4 ?g/mL aprotinin at 4 ?C for 30 min. The homogenate was centrifuged at 10.000 g for 20 min at 4 ?C. Protein concentration was measured by the Bradford method. Total proteins (30 ?g) were separated by SDS -PAGE on 10% separation gels and transferred to Immun-Blot? polyvinylidene difluoride membranes (BioRad). Western blot analysis was performed using antibodies against BiP, CHOP and ATF3 (Cell Signalling Technology or Santa Cruz Biotechnology). Detection was achieved exposing to Hyperfilm -ECL films, and analysed using an imaging system (Alpha Innotech, CA) to obtain densitometric values. The equal loading of proteins was assessed by detection of GAPDH . The size of proteins detected was estimated using protein molecular-mass standards (BioRad). d) Real - T im e PC R : Total RNA in hepatocytes was harvested by TRIsure (Bioline) according to the manuIacturer?s instructions The e[tracted 51A Zas dissolved in 51ase-free water and concentrations of total RNA were quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific). First-stranded cDNA was synthesized from 0.5 ?g total RNA (Fermentas Life Science). Primer Express Software (Applied Biosystems, Foster City, CA, USA) was used to design the primers examined with SYBR Green I (Table 28). The PCR reaction contained 10 ng of reverse-transcribed RNA, 2X I4? 6