Orthogonal Protecting Groups in the Synthesis of Tryptophanyl-Hexahydropyrroloindoles

The synthesis of various polycyclic systems containing a C 3a - N i bond between a hexahydropyrrolo[2,3-b ]indole and an indole tryptophan is described here. A series of experiments was run to determine the best combination of five orthogonal protecting groups and the best reaction conditions for formation of said bond, which is a common feature among many recently discovered marine natural products.


Introduction
The tricyclic motif hexahydropyrrolo [2,3-b]indole (HPI) is present in many natural compounds with important bioactivities. [1]hese compounds all feature a substituent at the 3a-position of the HPI, such as a methyl group, in ( _ )-physostigmine; [2] a prenyl, in flustramines, [3] brevicompanines [4] and roquefortines; [5] and a newly discovered HPI linked by one aromatic carbon, in idiospermuline, [6] psychotridine [7] and quadrigemine. [8]Recently isolated natural compounds such as psychotrimine, [9] chaetomin and the chaetocochins [10] contain an unusual bond between the 3aposition of the HPI and the indole nitrogen of either a tryptamine or a tryptophan (Figure 1).Kapakahines are natural products with a bond between the C 4a of an α-carboline and the indole nitrogen of an N-Trp. [11]__________ [a]  Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain.

[b]
Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.

[d]
Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain.* albericio@irbbarcelona.org, mercedes.alvarez@irbbarcelona.orgSupporting information for this article is available on the WWW under http://www.eurjoc.org/or from the author.
To date, four total syntheses of psychotrimine have been reported. [12]Takayama et al. were the first to synthesize this compound, [12a] assembling the HPI motif from a phenylacetonitrile that contains an indoline at the appropriate α-nitrile position.In contrast, Newhouse and Baran [12b] prepared psychotrimine via simultaneous formation of the HPI and the N-C 3a bond.They later employed the same strategy to prepare kapakahines B and F [13] , and (+)-psychotetramine. [14] During the course of the present work, Rainier et al. published a study on N-C 3a bond formation via bromo-displacement of 3abromo-HPIC with the N-anion of indole. [15]The same group harnessed this chemistry to prepare kapakahines E and F, [16] and more recently, described a mechanism for the substitution. [17]mpound 1, which contains a bond between the C 3a of HPI and the N of an indole, could be used as a scaffold for the synthesis of many natural products and analogs.In the work reported here, 1 was synthesized via nucleophilic substitution of the bromine at position 3a of 3a-bromo-HPI with an N-indole anion (Figure 2).To ensure chemoselectivity during this chemistry, five orthogonal protecting groups were required.Studies to determine the best protecting groups and conditions for this bond formation were then performed and are described herein.

Figure 1.
Natural products containing a bond between the C 3a of an HPI, or the C 4a of an α-carboline, and the indole nitrogen of either a tryptamine or a tryptophan.Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)

Results and Discussion
First of all, various bromo analogs of 3a-bromo-1,2,3,3a,8,8ahexahydropyrrolo [2,3-b]indole-2-carboxylate (3a-Br-HPI; 3) were synthesized using two different procedures, which were subsequently compared for performance (Table 1).The first one follows the route described by Taniguchi and Hino, [18] based on cyclization of a protected Trp in acidic medium, followed by aniline protection and subsequent benzylic bromination of HPI-2carboxylate.The second procedure is an one-step brominationcyclization of a totally protected Trp using N-bromosuccinimide (NBS) and pyridinium p-toluenesulfonate (PPTS). [19]The resulting products 3 and their stereochemistries (endo/exo) are listed in Table 1.Although a three (two amino and one carboxylic protecting groups) orthogonal systems is desirable, the use of the same amino protecting groups (R 1 = R 2 ) for both amino groups were also studied (Table 1, Entries 2, 3, and 16) having in mind the different nucleophilicity of both amino functions.For the carboxylic protection, common esters such as methyl, t-butyl, and allyl were tested.On the other hand, for the amino function both alkoxycarbonyl, i.e., tert-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), 2,2,2-trichloroetoxycarbonyl (Troc), and metoxycarbonyl (Moc), and and sulfonyl, i.e., 2nitrobenzenesulfonyl (Nosyl) and SO 2 Ph were tested.Additionally, , protected amino acids ( R 2 and R 3 Table 1, Entries 14-16) were assayed with the idea of studying the size and/or the electronic properties of protecting groups.
The overall transformation of L-Trp-OMe (the starting material) into 3 was highly demanding, as illustrated by the yields, which ranged from poor to moderate.Method B, which is shorter, gave better yields for the same set of protecting groups (Table 1: Entries 7, 12 and 13) and has the important additional advantage of being amenable to use of various protecting groups for the α-amino group (R 2 ).Cyclization with H 3 PO 4 (Method A) gave better yields when methoxycarbonyl (R 2 =Moc) was used as N α -Trp protecting group compared to when trichloroethoxycarbonyl (R 2 =Troc) was used (see Table 1: Entries 12 and 7, respectively).
Despite various attempts in diverse conditions, we were unable to remove the Moc group from the N 1 of HPI-2-carboxylate. [21]12b,22] Compounds 3b and 3c possess two Boc groups at positions N 1 and N 8 which could be cleaved simultaneously; however, the amine of N 1 is more reactive than the aniline of N 8 , which enabled chemoselective acylation of N 1 as reported by Danishefsky et al.. [23] The 1 H-NMR signals corresponding to the protecting groups of R 2 -namely, the signals for the CH 2 of Cbz or Troc, and for the CH 3 of Moc-are broad or split, because the protons are diastereotopic.
The difference in stereochemistry of the products 3 obtained from each method is noteworthy.Comparison of the 1 H-NMR spectra of the products 3g obtained from Method A and from Method B revealed significant differences in the signals for the proton at position 2 (δ 4.67 vs. 3.98 ppm, respectively) and for the methyl ester (δ 3.21 vs. 3.74 ppm, respectively).Based on these data, the stereochemistry of the product from Method A was determined to be endo-3g, and that of the product from Method B, exo-3g (see Figure 3).The diamagnetic anisotropy of the phenyl ring shields the endo-methyl group (δ 3.21 ppm) and the exo-H2 (δ 3.98 ppm). [24]The same phenomenon occurred with the endo/exo 3l and 3m obtained with the appropriate method (see Supporting Information).
To obtain a more versatile intermediate during synthesis of 3n, 3o and 3p via Method B, protected Ala or Ile were used as N α -and O-protecting groups respectively.However, the synthesis of compound 6 required an indirect route (see Table 1, footnote d), because subjecting dipeptide 4, which was N α -Alloc-Ala protected, to the acidic conditions for cyclization furnished the dimer 5 (Figure 4).Formation of 5 could be explained based on electrophilic substitution between 4 and the indoline that had formed after its protonation.

exo-3g
d] exo [a] 3b, 3o and 3p were synthesized from 3c, 3a and 3c, respectively, after hydrolysis and subsequent esterification or coupling with the protected Ile (see Supporting Information).
[b] Ratio determined by HPLC. [20][c] Ratio determined by 1 H-NMR.Consequently, in the first step of the HPI formation in Method A, working with an N α -carbamate protecting group at this position, instead an amide bond, is rather important.
The second part of this work comprised formation of the bond between the C 3a of the HPI and the N i of the Trp.Several pairs of base and solvent were tested to generate the indole anion that would drive the substitution to give compound 1. [26] The best conditions found comprised NaH in DMF at 70 o C for 1.5 h.Every bromo derivative (3a-p) was tested with several protected Trp's.A distinguishing data point in the 13 C-NMR data for compounds 1 and 3 is the chemical shift of the quaternary C 3a , which is less shielded in 1 (δ 72.4 to 82.2 ppm) than in 3 (δ 53.7 to 67.9 ppm).The results of these substitutions are summarized in Table 2.
The best yields of 1 in the nucleophilic substitution were found using 3a, 3l, 3m and 3p (Table 2: Entries 1 and 7-10, respectively).Moderate yields were obtained for the substitutions with bromides 3c, 3d and 3g (Entries 3-6).However, very poor yields (< 10%, data not shown) were observed when bromides 3b, 3e, 3f, 3h-k and 3n were reacted with different protected versions of 7, which contains two additional protecting groups.Table 2. Nucleophilic substitution at the C3a of 3a-Br-HPI [a] See Table 1 for the protecting groups used in each compound 3.
[b] Percentage of each compound in the reaction crude (as measured by HPLC). [20].
[c] Yield of isolated compound.
Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com) The phthalamide (Phth) was introduced as R 4 because it is orthogonal to all protecting groups used in the first reactions (Table 2: Entries 1, 3, 4 and 10), as it eliminates all the N α acid protons in 7. The wide range of yields in the resulting substitution (from 21 to 41%) demonstrates the importance of the protecting groups in the starting bromide.The bromides 3l and 3m have the same protecting groups in both amino groups of HPI (R 1 = SO 2 Ph, R 2 = Moc).Interestingly, the yield was lower when the group at R 5 was tert-butyl ester (1g, Entry 7) compared to methyl ester (1h, Entry 8).Likewise, the yield was lower when R 3 was tert-butyl ester (1i, Entry 9) compared to methyl ester (1h, Entry 8).The same trend was observed for 1f (Entry 6) and 1e (Entry 5), although to a lesser extent: the tert-butyl in 1f is more sterically hindered than the methyl ester in 1e.The results obtained with a protected Ile-Trp dipeptide as nucleophile (Entries 7 and 9), and with a Br-HPI and a protected Ile (Entry 10), are interesting, as they can serve as the stepping stone to synthesis of peptides found in natural compounds.Additionally, owing to this Ile protection, 1j (R 3 = Ile-OAllyl, Entry 10) was obtained in higher yield than was 1c (R 3 = OMe, Entry 3), whose protecting groups are the same, except for at R 3 .
Reaction of bromide 3n and N α -Phth-Trp-OMe unexpectedly gave compound 8.The product was characterized by mono-and bidimensional NMR and by HRMS (see Supporting Information).Important spectroscopy data for compound 8 are the lack of Br, the α-proton of the Trp, and the fact that the two protons of the cyclopropane CH 2 (2d, J = 15.4Hz, at δ 3.43 and 3.91 ppm) only exhibit a geminal coupling constant.The significant difference in the chemical shift of the α-proton of the Ala in 3n (δ 5.02 ppm) and that of the Ala in 8 (δ 4.11 ppm) could be justified by the different electronic effects in each compound.One hypothetical mechanism for formation of 8 begins with deprotonation of the C 2 of the HPI, made possible by the basic conditions, followed by intramolecular bromine displacement and subsequent formation of cyclopropane, to afford intermediate B (see Figure 5).The high strain in B could drive opening of the aminal and subsequent cyclization, to give a more relaxed cyclohexane (see Figure 5).This is the first time that the authors of this paper have isolated a compound such as 8 after the nucleophilic substitution reaction with the aforementioned conditions.Recently, J.D. Rainier et al. have reported the behavior of 3c under basic conditions of KOtBu and have isolated a tetracycle-containing compound that resembles B. [27]

Conclusions
In conclusion, various analogs of 3a-bromo-1,2,3,4,4a,8,8ahexahydropyrrolo[2,3-b]indole-2-carboxylate (3), protected with different combinations of three orthogonal protecting groups, were prepared by two different routes.The routes were then compared for performance.Method A, based on sequential cyclization, protection and bromination, provided the thermodynamic endocompound; whereas Method B, based on one pot brominationcyclization of a fully protected Trp, afforded mainly the kinetic exo-bromide.The influence of the protecting groups on formation of the N-C 3a bond between the Trp and HPI to give compounds 1f, 1g, and 1i (containing five orthogonal protecting groups) and compounds 1a, 1d, 1e and 1j (containing four orthogonal protecting groups) also was evaluated.Some of these compounds contain a protected Ile as the R 4 to protect the α-amino Trp; the orthogonal protecting groups enable synthetic versatility for constructing more structurally complex molecules.The protecting groups in the bromides 3 determined the yields of compounds 1a, 1c, 1d and 1j, whose starting N α -Phth-Trp-OMe 7 is the same.Moreover, the importance of the carbamate protecting group at R 2 should be emphasized: unexpectedly, compound 5 was obtained from an attempted cyclization of 4 in acidic medium (using an Ala amide bond for protecting the N in 4) and compound 8 was obtained from an attempted nucleophilic substitution of the bromine at C 3a of 3n.

Experimental section
A solution of L-Trp-OMe•HCl (5.4 g, 21.2 mmol) and Et 3 N (2.9 mL, 21.2 mmol) in dry CH 2 Cl 2 (85 mL) was added to a solution of either di-tert-butyldicarbonate (5.5 g, 25.4 mmol) or an appropriate chloroformate (1.5 eq) in CH 2 Cl 2 (3 mL/mmol).The reaction mixture was stirred for 2 h at rt.The organic solution was washed with brine, and then dried over Na 2 SO 4 .The solvent was removed, and the crude was purified by flash chromatography (hexanes/EtOAc) to afford the desired product in 80 to 99% yield.
Syntheses of N  -protected-Trp-OtBu analogs (2f, 2h, 2k, 2m; R 1 = H) Hydrolysis of the methyl ester.N  -protected-Trp-methyl ester (9.9 mmol) was dissolved in 10:1 THF/H 2 O (168 mL) and 2M LiOH (15 mL, 30,0 mmol), and the solution was stirred at rt for 3 h.The solution was then diluted with water and subsequently brought to pH 5 by dropwise addition of 2N HCl.The aqueous solution was saturated with NaCl and the phases were separated.The aqueous layer was extracted with THF.The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo to give the carboxylic acid in quantitative yield.Formation of the tert-butyl ester.A mixture of N  -protected-Trp (8.4 mmol), BnEt 3 NCl (1.9 g, 8.4 mmol) and K 2 CO 3 (7.6 g, 54.7 mmol) in MeCN (25 mL) was vigorously stirred for 5 h.t-BuBr (9.9 mL, 88.4 mmol) was then added, and the solution was heated at 50 ºC for 2 h.The reaction mixture was treated with MeCN (13 mL), and then stirred for 24 h.The solvent was evaporated off and the resulting solid was dissolved in 2:1 EtOAc/H 2 O.The aqueous solution was extracted with EtOAc.The combined organic layers were washed with brine and dried over Na 2 SO 4 .The solvent was removed to give the tert-butyl N  -protected-Trp carboxylates 2f (84%), 2k (84%), 2h (55%) or 2m (58%).

indole-2-carboxylate (endo-3m).
To a solution of S6 (5.0 g, 12.0 mmol) in 10:1 THF/H 2 O (204 mL) was added 2M LiOH (12.0 mL, 24.0 mmol).The reaction mixture was stirred at reflux temperature for 2.5 h.The solution was diluted with water and subsequently brought to pH 5 by dropwise addition of 2N HCl.The aqueous solution was saturated with NaCl and extracted with THF.The combined organic layers were dried over Na 2 SO 4 , and then concentrated in vacuo to give the carboxylic acid in quantitative yield.Following the aforementioned method to generate a tert-butyl ester, S7 was obtained with 81% yield.endo-3m was obtained in 46% yield from S7 following the same method reported for endo-3g.

Method B: One-pot bromocyclization for exo-3a, exo-(3c-3n)
To a solution of PPTS (1.9 g, 7.4 mmol) and NBS (1.3 g, 7.4 mmol) was added N  -N i -protected-Trp alkyl ester (7.4 mmol) in anhydrous CH 2 Cl 2 (67 mL) under N 2 .The reaction mixture was stirred at rt for 4 h.The crude mixture was washed with 15% NaHCO 3 , 10% Na 2 S 2 O 4 and brine, and then dried over Na 2 SO 4 .The solvent was evaporated off, and the crude was purified by flash chromatography (see the following section for yields and solvent systems).

Synthesis of exo-3b
2M LiOH (1.5 mL, 3.0 mmol) was added to a solution of exo-3c (0.5 g, 1.0 mmol) in 10:1 THF/H 2 O (17 mL).The solution was stirred at reflux for 5 h.The solution was then diluted with water and subsequently brought to pH 5 by dropwise addition of 2N HCl.The aqueous layer was saturated with NaCl and extracted with THF.The combined organic layers were dried over Na 2 SO 4 and concentrated in vacuo to give the carboxylic acid in quantitative yield.
To a solution of the aforementioned acid (0.7 g, 1.5 mmol) in MeOH (10 mL) was added Cs 2 CO 3 (0.5 g, 1.6 mmol), and the solution was stirred for 30 min at rt.The reaction mixture was then concentrated, treated with dry DMF (5 mL) and AllylBr (0.25 mL, 3.0 mmol), and finally, stirred at rt for 4 h.The organic solution was diluted with EtOAc and washed with 5% NaHCO 3 and brine.The organic phase was dried over MgSO 4 , and then concentrated in vacuo.The resulting residue was purified by flash chromatography (hexanes/EtOAc, 80:20 to 70:30) to give exo-3b (0.2 g, 30%).

Synthesis of exo-3o and exo-3p exo-3a
or exo-3c were hydrolyzed following the aforementioned method for hydrolysis of a methyl ester of exo-3c.

Table 1 .
Synthesis of the bromo compounds 3a-p.
Reagents and solvents were purified according to Purification of Laboratory Chemicals (Armarego, W. and Chai, C.; Elsevier; 2003).Automatic flash chromatography was done in an Isco Combiflash medium pressure liquid chromatograph with Redisep silica gel columns (47-60 m).