Synthesis and Biological Evaluation of Delavayin-C

The synthesis of a cyclic heptapeptide, delavayin-C, cyclo(gly-tyr-tyr-tyr-pro-val-pro) is described. The structure of this compound was established on the basis of analytical IR, 1H NMR and FAB mass spectral data. The antibacterial and antifungal activities of this peptide are also described.

the analysis of marketed formulations are well agreed with the label claim (Table 3). To study accuracy of the developed methods, recovery studies were carried out using standard addition method at four different levels for all the three brands and the % recovery was calculated ( Table 4). The results revealed no interference from the excipients. The developed methods are simple, precise and accurate. The statistical data proved that methods are reproducible and selective for the analysis of duloxetine HCl in its marketed formulations.
Cyclic peptides were found to exhibit various biological activities like antibacterial, antifungal, anthelmintic, insecticidal, antineoplastic, antitumor, antiinflammatory activities [1][2][3][4][5][6] . Keeping in view of the significant biological activities exhibited by various cyclic peptides, as a part of ongoing study, an attempt was made towards the synthesis of a cyclic heptapeptide, delavayin-C, cyclo(gly-tyrtyr-tyr-pro-val-pro), which was isolated from the roots of Stellaria delavayi and belongs the family Cariophyllaceae 7 . The synthesized compound was further subjected to antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Psuedomonas aeruginosa and antifungal activities against Candida albicans.
The synthesized compound has shown moderate antibacterial and antifungal activity comparable with the standard drug benzyl penicillin and standard antifungal agent fluconazole, respectively. Spectral interpretation and elemental analysis was done for the synthesized compound for structural elucidation.
In order to carry out the total synthesis of cyclic peptide, cyclo(gly-tyr-tyr-tyr-pro-val-pro), it was disconnected into three dipeptide units, Boc-glytyr-OMe 1, Boc-tyr-tyr-OMe 2, Boc-pro-val-OMe 3 and a single amino acid methyl ester hydrochloride unit, pro-OMe-HCl 4. The required dipeptides were prepared by coupling Boc amino acids with the respective amino acid ester hydrochlorides using DIPC, CHCl 3 and N-methyl morpholine according to Bondanszky 8 procedure with suitable modiÞ cations. The Boc-group of the dipeptide 2 was removed by using trifluoroacetic acid and the ester group of dipeptide 1 was removed by using LiOH. The deprotected units were then coupled to get a tetrapeptide Boc-gly-tyr-tyr-tyr-OMe 5. Similarly, the dipeptide 3 was coupled with single amino acid methyl ester hydrochloride unit, pro-OMe HCl 4 after appropriate deprotection to get a tripeptide Boc-pro-val-pro-OMe 6. The resulting tetrapeptide and tripeptide was then coupled together by using DIPC, NMM and CHCl 3 to get a linear heptapeptide Boc-gly-tyr-tyr-tyr-pro-val-pro-OMe 7. Finally cyclisation of this linear heptapeptide was carried out by p-nitrophenyl ester method. The intermediates and the final product were purified by recrystallisation from CHCl 3 . The retrosynthetic analysis of peptide is shown in the Scheme 1.
The newly synthesized compound was analyzed for C, H, N, and O by elemental analysis and structure was confirmed by IR, 1 H NMR and FAB mass spectral analysis. The characteristic IR absorption bands of -CO-NH-moiety was present in the cyclised product. The NMR spectrum of cyclised product clearly indicates the presence of all respective amino acid moieties. Furthermore, the mass spectrum of this cyclic heptapeptide showed a molecular ion peak at m/z 840, which corresponds to molecular formula C 44 H 53 O 10 N 7 .
The synthesized cyclic heptapeptide was screened in vitro for its antibacterial and antifungal activity by using disc diffusion method and tube dilution technique. The antibacterial activity was determined against four bacterial species (B. subtilis, S. aureus, E. coli and P. aeruginosa) and antifungal activity against Candida albican. In the disc diffusion method, the activity studies were carried out according to modiÞ ed Kirby-Bauer method 9 . Benzyl penicillin and fluconazole were used as standards against bacterial and fungal strains, respectively at a concentration of 50 µg/ml. Nutrient broth and Sabourds agar were used as a medium and dimethylformamide (DMF) was used as a solvent control for carrying out the activity. After preparation of the disc, allowed to stand for 24 h at 37 0 . The zone of inhibition, observed around the disks after incubation, was measured. The dipeptides 1 and 2 were used for the preparation of a tetrapeptide Boc-gly-tyr-tyr-tyr-OMe (5). The tripeptide Boc-pro-val-pro-OMe (6) was prepared by coupling a dipeptide Boc-pro-val-OMe (3) with pro-OMe HCl (4) unit. The resulting tetrapeptide and tripeptide were coupled by using DIPC and N-methyl morpholine (NMM) to obtain a linear heptapeptide Boc-gly-tyr-tyr-tyr-pro-val-pro-OMe (7). Cyclisation of this linear heptapeptide was carried out by using p-nitrophenyl ester method 10 . The ester group of the linear segment was removed with LiOH and the p-nitrophenyl ester group was introduced using the following procedure, The Boc-peptide carboxylic acid (1.5 mmol) was dissolved in CHCl 3 (15 ml) at 0 0 . Then p-nitrophenol was added (0.27 g, 2 mmol), and stirred for 12 h at room temperature. The reaction mixture was Þ ltered and the Þ ltrate was washed with NaHCO 3 solution (10%) until excess of p-nitrophenol was removed and Þ nally washed with 5% HCl (5 ml) to get Boc-peptide-pnp-ester.   Compound  21  15  11  10  18  Benzyl Penicillin  25  15  17  16  -Fluconazole  ----20  DMF  ------indicates no activity. Both test compounds and standard were tested at 50 µg/ml. signiÞ cant antibacterial activity against gram positive bacteria in comparison to the standard drug benzyl penicillin. It has also shown moderate antifungal activity in comparison with the standard drug ß uconazole.