New Polyfunctional Phragmalin Limonoids from Neobeguea mahafalensis.

Neobeguea mahafalensis is used as a medicinal plant in Madagascar. A decoction of the stem bark of this species is reported to treat back pain. Recently, it was reported that a decoction of the root bark, containing two novel phragmalin limonoids as identified active constituents, exhibited an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents.From the dichloromethane extract of the root barks of N. mahafalensis, nine phragmalin limonoids were isolated, of which eight were novel compounds. The structures were established mainly by extensive use of 2D NMR spectroscopic techniques and high-resolution mass spectrometry. One of the new compounds named dodoguin displayed sleep-inducing activity in Swiss albino mice. The amount of 3-10 mg/kg of this compound induced sleep 18-22 min after its administration with a duration of 16-18 min.

Semisynthesis of libiguin A and its analogues by trans-lactonization of phragmalin.

Libiguins are limonoids with highly potent sexual activity enhancing effects, originally isolated from the Madagascarian Meliaceae species Neobeguea mahafalensis, where they exist in only minute quantities. Their low natural abundance has hampered mapping of their biological effects. Here we describe an approach to the semisynthesis of libiguin A and its close analogues 1-3 starting from phragmalin, which is a limonoid present in high amounts in a commercially cultivated Meliaceae species, Chukrasia tabularis, allowing the preparation of libiguins in appreciable quantities.

Libiguins A and B: novel phragmalin limonoids isolated from Neobeguea mahafalensis causing profound enhancement of sexual activity.

In a screening programme directed towards the discovery of drugs that could enhance sexual activity, we found that a decoction of the root bark of Neobeguea mahafalensis displayed an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents. Bioassay-guided fractionation led to the isolation of two pharmacoactive constituents, which turned out to be novel 1,8,9-orthoacetate phragmalin limonoids that we named libiguins A and B, each with a C-16/30 δ-lactone ring. Chemical structures were established by the interpretation of their 1D and 2D NMR data. In vivo pharmacological tests showed that starting with a treatment from 0.004-0.4 mg/kg/day for three consecutive days, over a 3-h sampling period, these limonoids induced a long-lasting augmentation of frequency and sustainment of mounting behaviour in male rodents, with an effect lasting for up to 11 days post-treatment. Libiguin A proved to be markedly more potent than libiguin B. This report is the first of limonoids having such an effect, and the findings could lead to novel therapies for the treatment of sexual dysfunction. Moreover, the results can serve as an opening to elucidate the central physiological control of mating behaviour, which is still not well mapped out.

Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B-NS3 proteases.

A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B-NS3 proteases of the four subtypes of dengue virus, DEN-1-4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2 µM for the DEN-1-4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.

A new protolimonoid from Capuronianthus mahafalensis.

From stem barks of Capuronianthus mahafalensis (Meliaceae) endemic to Madagascar, a new protolimonoid named capulin containing a four membered ring in its side chain was isolated by repeated silica gel column chromatography. Its structure was determined by 1D and 2D NMR spectroscopy and high-resolution MS. To the best of our knowledge, this is the first time that a four-membered ring occurs in the side chain of protolimonoids.

Oligomerization of indole derivatives with incorporation of thiols.

Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl)-1-(2-mercaptoethylthio)ethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl) ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid) of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.

Design and synthesis of a library of tertiary amides: evaluation as mimetics of the melanocortins' active core.

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl(4). Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K(i)s reached 400 nM on MC(1), 2 microM on MC(3) and 1 microM on MC(4) and MC(5) receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.

N-alkylated dipeptide amides and related structures as imitations of the melanocortins' active core.

Thirty-three low molecular mass structures combining both peptide and peptoid features were prepared and tested on human melanocortin receptors MC1,3-5R. Most of them displayed low micromolar activity with preference for diamines, guanidino and 2-naphthyl derivatives compared to monoacetylated, amino and 3-indolyl counterparts. Some contained L- or D-histidine residues, but the change did not influence affinity. QSAR modelling yielded excellent models for the MC3-5 receptors explaining R2Y=0.89-0.91 and predicting Q2=0.77-0.80 of the affinity variations. One compound displayed MC1R selectivity (13-fold and more). An NMR study of showed that it exists as a mixture of four rotamers at its tertiary amide bonds. Comparisons with earlier data for melanocortin core tetrapeptide analogues indicate that the novel peptide-peptoids interact with the melanocortin receptors in a different way.

New substituted piperazines as ligands for melanocortin receptors. Correlation to the X-ray structure of "THIQ".

A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule agonist "THIQ" was synthesized and characterized structurally and pharmacologically. First, several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermediate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e., preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving (R,R)-23c and (S,S)-23c, respectively. Melanocortin MC(1,3-5) receptor binding studies showed that all tested piperazine derivatives were active. Several compounds showed clear selectivity for MC4R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-23c, displayed a biphasic curve in displacement of [125I]NDP-MSH on MC4R [K(i)high = 1 nM and K(i)low = 260 nM]. This biphasic competition curve was similarly biphasic to the competition curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate salt revealed two closely related conformations, which resemble the shape of the letter "Y", where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-23c, allowed us to suggest a possible "bioactive" conformation of (R,R)-23c that is similar to the crystal conformation of THIQ.