RECENT ADVANCES IN LAMELLARIN ALKALOIDS : ISOLATION , SYNTHESIS AND ACTIVITY

a: Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, 08028-Barcelona, Spain; b: CIBER-BBN Networking Centre on Bioengineering, B iomaterials and Nanomedicine, E-08028 Barcelona, Spain; c: Department of Organic Chemistry, University of B arcelona, 08028-Barcelona, Spain; d: Laboratory of Organic Chemistry, Faculty of Phar m cy, University of Barcelona, 08028-Barcelona, Spain


Introduction:
Lamellarins are a large family of marine alkaloids characterized by their unusual structures and important activities.From a structural perspective, two groups of lamellarins can be found.Members of the larger of the two groups possess a pentacyclic system of 6-oxobenzo[b]pyrano [3,4-b]pyrrolo[2,1-a] with a substituted phenyl ring at position 14.Pentacyclic lamellarins may be saturated (Table 1) or unsaturated (Table 2) between positions 8 and 9.
Several reviews on lamellarins have recently been published [16].Herein is covered work related to their isolation, synthesis and activity that was published between 2004 and December 2007.

Synthesis
Lamellarins are rather complex structural targets.Several approaches to their synthesis can be found in the literature.These fall into two main synthetic categories: (a) pyrrole formation as the cornerstone of the synthesis; and (b) transformation of a pre-existing pyrrole derivative through cross-coupling reactions.

a) Pyrrole ring formation
A highly efficient synthesis of Lam-K and Lam-L was described by Ruchirawat et al. [17].The pyrrole ring was constructed via Michael addition followed by a ring-closing reaction of benzyldihydroisoquinoline derivatives with ethoxycarbonyl-β-nitrostyrenes (Figure 2).Formation of the pyrrole ring produces the dihydropyrrolo[2,1-a]isoquinoline in which all the phenol groups were protected as benzyl-ethers.
Deprotection by hydrogenolysis followed by base-mediated lactonization gave the natural products.The same methodology was used to prepare several natural saturated and unsaturated lamellarins, as well as various analogs [18].The same authors reported an elegant preparation of the lamellarin skeleton using a slightly different pyrrole ring formation from benzyldihydroisoquinoline and a phenacyl bromide [19].They used polymersupported reagents to simplify the work-up and obviate column chromatography.The pyrrole ring was constructed in one pot by quaternization of the isoquinoline followed by an aldol-type condensation.Several lamellarins and derivatives were obtained by solid-phase synthesis (SPS) on an appropriate solid support and under different cleavage conditions [20].The lamellarin skeleton was synthesized on solid phase through formation of the pentacyclic system from an open chain dihydroisoquinolinium salt by an intramolecular [3+2] cycloaddition [21].The use of different Lewis acids as cleavage-deprotection reagents in SPS has been exploited for introducing diversity to produce analogs for screening (Figure 4).A biomimetic synthesis of lamellarin and lukianol skeleton was developed by Steglich et al. [22].It is based on formation of 3,4-diarylpyrrole-2,5-dicarboxylic acid from aryl pyruvic acids and 2arylethylamines.The method has been used for the synthesis of ningalin B, Lam-G, Lam-K, lukianol A and a lukianol-lamellarin hybrid (Figure5).

Fig. (7). Synthesis of lamellarin α 20-sulfate
A similar procedure employing the appropiate boronic acids for the cross-coupling reaction has been used for the total synthesis of Lam-D, Lam-L, and Lam-N [27].
The 1,2-diaryl-substituted pyrrolo[2,1-a]isoquinoline skeleton of lamellarins has been obtained by a new route via 1,5-dipolar electrocyclization of azomethine ylides (Figure 8) [28].The reagents comprised the stilbenic amides 2 available from the acids obtained by condensation of substituted benzaldehydes with phenylacetic acid.Cyclization of the amide 2 using the Bischler-Napieralski procedure afforded 3,4dihydroisoquinolines 3. Subsequent reaction of the isoquinolines with ethyl bromoacetate gave the quaternary salts, which upon treatment with triethylamine in dry ethanol afforded the pyrrole derivatives.
Removal of the allylic protecting group with Pd-C and TsOH resulted in simultaneous formation of the pentacyclic lamellarin skeleton and lactonization.This elegant method was not used for the preparation of a natural product; it was only applied to the construction of the lamellarin skeleton.

Fig. (9). Synthesis of pyrroloisoquinoline core structures of lamellarins b) Transformation of a pyrrole derivative through cross-coupling reactions
Lam-Q and Lam-O have been synthesized on Merrifield resin with N-protected 3,4-dibromopyrrole-2carboxylate as scaffold (Figure 10) [30].The process comprises incorporation of a substituted pyrrole ring onto a p-alkoxy iodo phenyl resin through a Negishi cross-coupling reaction, followed by Suzuki crosscoupling to introduce the second substituted phenyl ring, and finally, N-alkylation.A Lewis acid was used for the final cleavage.The beauty of this strategy is that diversity can be introduced at each step, including the final cleavage (by using the appropriate Lewis acid).

N Br
Br

Activity and mechanism of action:
Lamellarins and their derivatives are multi-drug resistance (MDR) reversal agents.As some of them are highly cytotoxic, they have been tested against various cancer cell lines.The results are summarized in Table 3. Lam-D, Lam-K and Lam-M are among the most cytotoxic molecules in the series.The best studied member is Lam-D, which is highly cytotoxic to a wide range of tumor cell lines, particularly human prostate cancer cells [8] and leukemia cells [37].Several molecular targets have been described for Lam-D and other lamellarins.

Molecular structure-activity determinants
Lamellarins with a C8-C9 double bond are generally more cytotoxic than their corresponding saturated analogs.This is very clear in the case of Lam-D, which is considerably more cytotoxic than its synthetic saturated analog, Lam-501 (Figure 14), which has no effect on topoisomerase I.As such, Bailly et al.
postulated that the planarity of the pentacyclic structure is important for cytotoxicity [38].These results agree with those of a molecular dynamics study performed by Iwao et al. [37] to establish molecular interactions for the complex of Lam-D and the enzyme topoisomerase I.These researchers proposed that the guanidinium group of Arg 364 maintains a close relationship with the lactone ring of the molecule.Moreover, they observed that direct hydrogen bonding interactions between the 3-OH oxygen and the Glu 356 carboxylate oxygen, and between the 11-OH oxygen and the side chain amide nitrogen of Asn 722 , were maintained throughout the entire simulation.
A library of open lactone analogs of Lam-D [34] was recently synthesized (Table 5).The 45 members of the library all feature a methyl 8-hydroxy-9-methoxypyrrolo[2,1-a]isoquinoline-3-carboxylate scaffold, which differs from Lam-D primarly in that it lacks a lactone ring.The absence of the pyranone ring affords flexibility to the derivatives, and more importantly, greater solubility.Two series of compounds were prepared: derivatives of the 1-aryl-scaffold and of the 1,2-diaryl-scaffold.Members of both series feature either a single or double bond between C5 and C6 (which correspond to the C8 and C9 in lamellarins).The compounds from each series differ in their respective numbers and positions of the OH/OMe substituents on the aryl rings, and in the presence of functional groups such as NO 2 , NMe 2 , OCF 3 and heterocycles instead of aryl rings.All the compounds were tested for cytotoxicity against a panel of three human tumor cell lines: A-549 lung carcinoma, HT-29 colon carcinoma and MDA-MB-231 breast adenocarcinoma.The most active compounds are shown in Table 5. Structurally simplified analogs of Lam-D, in which the lactone ring was removed, and, in the case of derivatives 11 and 13, an additional aryl group was removed, all had lower activity than Lam-D.These data reveal that the complete structure is crucial for biological activity, despite being less soluble in biological media than simpler molecules.In a general overview, oxidized derivatives showed greater activity than the corresponding reduced analogs [34].These data reveal that the complete structure is crucial for biological activity, despite being less soluble in biological media than simpler molecules.In a general overview, oxidized derivatives showed greater activity than the corresponding reduced analogs.
This fact can probably be attributed to the greater hydrogen bonding capacity of these analogs-namely, with active sites, as has been described for Lam-D [37].The donor effect of the methoxy-substituents may explain why 12c and 14b were quite active despite not being able to act as hydrogen bond donors.
Compounds 14g, 14a, 14h and Lam-D have identical substituents on their scaffolds and at position 1 of their aryl rings.For these compounds, the greater the substitution of the aryl ring at position 2 of the scaffold, the lower the activity.The simplified analog 14a maintained 63% of activity of Lam-D in HT29 cells, and 14g, which has a hydroxy group at C4" (the same position as C-3 in Lam-D) was nearly as active.
The open lactone compound 14h may undergo lactonization in physiological conditions.Therefore, 14h merits further study as a pharmacodynamic improvement on Lam-D, a validated lead compound.

Topoisomerases, the initial biomolecular engines of cell growth
Topoisomerases, nuclear enzymes than can change the topology of DNA [40,41], are amongst the most promising targets for inhibiting cellular proliferation.DNA topoisomerases are crucial in cellular replication; hence, they are especially attractive targets for cancer therapy.Interaction of a drug with a DNA topoisomerase can produce a stable, cytotoxic complex that inhibits post-cleavage DNA religation processes [42].Indeed, this mode of action has been reported as a novel mechanism for many anticancer drugs [41].15).Structure of (+)-camptothecin P glycoprotein, the most common protein efflux pump in cells, also highly favours the activity of Lam-D in detrimental of recognized substrates like camptothecin.Investigations into the mechanism of action of Lam-D revealed that it is not sensitive to MDR-mediated drug efflux by P glycoprotein without active transporters to carry it out of the cell cytoplasm.

Inhibition of topoisomerase I by
Although the pro-apoptotic effects of Lam-D could be understood as the final consequence of its stabilization of topoisomerase complexes, experiments [44] have revealed that it has other cellular targets.
It has also been suggested that Lam-D induces apoptosis of leukemia cells by disrupting the mitochondrial transmembrane potential (MTP).
Using reliable real-time flow cytometry techniques and swelling of mitochondria isolated from leukemia cells, Bailly et al. showed that Lam-D directly induces MPT.Furthermore, they discovered that mitochondria are required to mediate Lam-D-induced nuclear apoptosis in a cell-free system [44].
In summary, Lam-D is rich in pharmacological potential which should be exploited for the development of treatments against chemoresistant cancer cells.

Docking of LAM-D with topoisomerase I
Computational techniques have been used to elucidate the structural basis and the mode of interaction of the covalent complex formed by Lam-D, topoisomerase I and DNA [37,38]  featuring Lam-D superimposed in the active site [35] Bailly et al. removed topotecan from the original structure to obtain a template on which to model the drugfree covalent complexes.The main difference between the two approaches [37,38] remains in the exocyclic phenyl ring of Lam-D, which is rotated 180° relative to the conformation reported in a previous very similar proposal, such that the methoxy group at C13 is close to the 6-amino group of adenosine in the major groove.
Finally, to support the latter refined [37] model of the cleavable Top1-DNA complex stabilized by Lam-D, a quantitative estimation of the contribution to the free energy of binding of the crucial 20-OH group was obtained through a set of precise, thermodynamic-integration free-energy simulations.
The inhibition of topoisomerase functionality alone probably does not result in cell death, but when the Lam-D stabilized ternary complex encounters a replication fork, the single DNA strand break is converted into a double DNA strand break which kills the cell.

Lam-D: taking control of mitochondria
Mitochondria [46,47] are subcellular organelles evolved from bacterial symbiosis and therefore contain their own genome.Cancer cells have unlimited replicative potential; are resistant to cell death stimuli; exhibit several mitochondrial disorders (e.g.dysfunction, and genetic instability with alterations such as mutations, deletions or translocations of the mitochondrial DNA [mtDNA]); and are highly glycolytic.The rapid and continuous growth of tumor cells is highly energy-dependent, and cancer cells often develop drug resistance, consequently becoming unaffected by pro-apoptotic signals.The dependency of cancer cells on glycolysis for ATP synthesis indicates that the mitochondrial engineering of the respiratory chain might be inefficient.The significance of mitochondria in mediating apoptosis has led to an interest in exploiting radio-and chemo-therapeutic agents to trigger cancer cell death.To date, direct and specific targeting of mitochondria to obtain a persistent antitumor response has not been achieved, but there have been several encouraging cases in which some level of activity was reached.The vast majority of conventional anticancer drugs indirectly exploit mitochondria to exert cytotoxicity via multiple activation pathways that implicate p53 or death receptors.mtDNA metabolism can also be targeted by topoisomerase inhibitors.Type I and type II topoisomerases have been identified in mitochondria, and have been shown to be inhibited there by known topoisomerase inhibitors.
Lam-D induces early disruption of the inner MPT through induction of pore opening [44].This is considered as a predominant mechanism for mediating the release of pro-apoptotic molecules such as cytochrome c to the cytoplasm.Hence, agents that permeabilize cancer cell mitochondria may eliminate the resistance of these cells to apoptosis.Early studies have revealed that MPT pore opening precedes the proteolytic activation of caspase-3 in Lam-D mediated apoptosis.Furthermore, a greater gain in cell depolarization was observed in tumor cells (P388 leukemia, A549 lung cancer and MCF-7 breast cancer) rather than in non-tumor ones (NIH3T3 fibroblasts and H9C2 cardiomyocytes).The direct targeting of mitochondria by Lam-D is highly advantageous over classical anticancer drugs.Lam-D may be effective for treating cancers in which signal transducing systems are interrupted (e.g.those implying mutations of p53).

Focus on HIV integrase
Current HIV treatments comprise reverse transcriptase inhibition, which prevents single-stranded viral RNA genome form being translated into double stranded DNA, and protease inhibition, which blocks the production of mature infectious virions.Whereas drugs that target these two viral enzymes have been in use for more than ten years, inhibitors of the third HIV enzyme, integrase (IN), have yet to be developed.
Integrase is a viral protein of 32 kDa responsible for the insertion of newly reverse-transcribed doublestranded viral DNA into the host genome [48] The mechanisms by which small molecule inhibitors of recombinant HIV-1 IN act are unknown.Important structural motifs identified to date for HIV-integrase inhibitors are 1,2-and 1,4-diphenols, which can be oxidated to the corresponding quinones [49,50].
Ridley et al. [50] reported that the sulfate group is critical for the anti-HIV-1 integrase activity of Lam-α 20-sulfate, because Lam-α showed no inhibition of HIV-1 integrase at concentrations up to 1.6 µM.HIV-1 integrase has been demonstrated to be a DNA manipulating enzyme and is a rarely exploited target.The low cytotoxicity of the sulfate compounds is interesting in the context of antiviral agents.Indeed, during a screening program aimed at identifying inhibitors of HIV-1 integrase.Reddy et al. [9] discovered that Lamα 20-sulfate [25] strongly inhibited both terminal cleavage of integrase and strand transfer in vitro.
However, they reported that the disulfate analog Lam-α 13,20-disulfate is less selective than Lam-α 20sulfate, as they observed it to inhibit molluscum contagiosum virus (MCV) topoisomerase at roughly the same concentration as that used in the HIV-1 integrase assay.

Fig. ( 16 ).
Fig. (16).Crystallographic model of topotecan-DNA-topoisomerase (Protein Data Bank entry 1k4t) . An IN inhibitor could offer improvements in selectivity, despite the fact that the enzyme is only briefly active in the replication cycle of the virus.Integration of viral DNA into host cell chromosomal DNA to form a provirus is an essential step in the viral life cycle.IN is an ideal target for drug design because it does not have any known cellular homologs in mammals, and therefore, the reactions that it catalyzes are unique.Moreover, IN is required for viral replication and mutations in key residues.During the past 15 years many IN inhibitors have been identified, some of which are highly selective against IN and block viral replication.IN inhibitors fall into two major classes: catechol-containing hydroxylated aromatics and diketoacid-containing aromatics.