Total Synthesis and Pharmacological Investigation of Cordyheptapeptide A

The present investigation reports the synthesis of a phenylalanine-rich N-methylated cyclopeptide, cordyheptapeptide A (8), previously isolated from the insect pathogenic fungus Cordyceps sp. BCC 1788, accomplished through the coupling of N-methylated tetrapeptide and tripeptide fragments followed by cyclization of the linear heptapeptide unit. Structure elucidation of the newly synthesized cyclopolypeptide was performed by means of FT-IR, 1H-NMR, 13C-NMR, and fast atom bombardment mass spectrometry (FABMS), and screened for its antibacterial, antidermatophytic, and cytotoxic potential. According to the antimicrobial activity results, the newly synthesized N-Methylated cyclopeptide exhibited potent antibacterial activity against Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumoniae and antifungal activity against dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at a concentration of 6 μg/mL, in comparison to the reference drugs, gatifloxacin and griseofulvin. In addition, cyclopolypeptide 8 displayed suitable levels of cytotoxicity against Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) cell lines.


Introduction
Nature is an attractive source of new therapeutic compounds. In drug discovery, microorganisms play a tremendous role especially by producing a number of candidates that are effective against microbes [1]. The previous literature is enriched with potential data indicating the ability of microorganisms such as fungi and bacteria, which produce a variety of natural products with diverse bioactivities [2,3]. Currently, microorganism-derived peptides (MdPs) are attracting the attention of scientists [4,5] because of the unique molecular structures and a wide array of associated bioactivities like antimycobacterial and antimalarial activity [6,7], antibacterial activity [8,9], cytotoxicity [10], and antiviral activity [11]. Cyclopeptides produced by microbes including cycloaspeptides, halolitoralins [12], psychrophilins, talaromins, ustiloxins, and unguisins are associated

Chemistry
The cycloheptapeptide molecule was split into three dipeptide units viz. Boc-L-Phe-N(Me)Gly-OMe (1), Boc-L-Pro-D-N(Me)Phe-OMe (2), and Boc-L-Leu-L-Ile-OMe (3), and a single amino acid unit L-N(Me)Tyr-OMe·HCl (4). N-methylation of Boc-protected phenylalanine and tyrosine methyl esters was done by treatment with methyliodide and sodium hydride as per the literature [46]. Dipeptide units (1-3) were prepared by the coupling of Boc-amino acids such as Boc-L-Phe, Boc-L-Pro, and Boc-L-Leu with the corresponding amino acid methyl ester hydrochlorides such as N(Me)Gly-OMe·HCl, D-N(Me)Phe-OMe·HCl, and L-Ile-OMe·HCl by following the modified Bodanzsky method [47]. After deprotection at the carboxy terminus, dipeptide 1 was coupled with dipeptide 2, deprotected at the amino terminus, to obtain the tetrapeptide unit Boc-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OMe (5). The carboxyl group of dipeptide 3 was deprotected by alkaline hydrolysis using lithium hydroxide (LiOH) and the deprotected peptide was coupled with the amino acid unit 4, utilizing different carbodiimides to obtain the tripeptide unit Boc-L-Leu-L-Ile-L-N(Me)Tyr-OMe (6). After removal of the ester group of tripeptide 6 and the Boc-group of tetrapeptide 5, the deprotected units (5a, 6a) were coupled to obtain the linear heptapeptide unit Boc-L-Leu-L-Ile-L-N(Me)Tyr-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OMe (7). The methyl ester group of the linear peptide fragment was replaced by the pentafluorophenyl (pfp)/p-nitrophenyl (pnp) ester group. The Boc-group of the resulting compound was removed using trifluoroacetic acid (CF 3 COOH), and the deprotected linear fragment was now cyclized by keeping the entire contents at 0 • C for 7 days in the presence of catalytic amounts of triethylamine (TEA) or N-methylmorpholine (NMM) or pyridine to get the cyclic product 8. The structures of the newly synthesized cycloheptapeptide as well as that of the intermediate tri/tetra/heptapeptides were confirmed by FT-IR, 1 H-NMR spectroscopy, and elemental analysis. In addition, the mass spectra and 13 C-NMR spectroscopy were recorded for the linear and cyclic heptapeptide only. The synthetic pathway for the newly synthesized N-methylated heptacyclopeptide is shown in Figure 1. In addition, the mass spectra and 13 C-NMR spectroscopy were recorded for the linear and cyclic heptapeptide only. The synthetic pathway for the newly synthesized N-methylated heptacyclopeptide is shown in Figure 1.

Pharmacological Activity Studies
The synthesized linear and cycloheptapeptide (7,8) were subjected to the short term in vitro cytotoxicity study against the Dalton's lymphoma ascites and Ehrlich's ascites carcinoma cell lines at concentrations of 62.5-3.91 μg/mL using 5-fluorouracil (5-FU) as the reference compound. The activity was determined by measuring the inhibition (%) of the cell lines [48]. The CTC50 values were determined by the graphical extrapolation method. The results of the cytotoxic activity studies are presented in Table 1.

Pharmacological Activity Studies
The synthesized linear and cycloheptapeptide (7,8) were subjected to the short term in vitro cytotoxicity study against the Dalton's lymphoma ascites and Ehrlich's ascites carcinoma cell lines at concentrations of 62.5-3.91 µg/mL using 5-fluorouracil (5-FU) as the reference compound. The activity was determined by measuring the inhibition (%) of the cell lines [48]. The CTC 50 values were determined by the graphical extrapolation method. The results of the cytotoxic activity studies are presented in Table 1.
Moreover, the linear and heptacyclopeptide (7,8) were further evaluated for their antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae, dermatophytes Microsporum audouinii, Trichophyton mentagrophytes, diamorphic fungi Candida albicans, and other fungal strains, including Aspergillus niger at concentrations of 50-6.25 µg/mL by using the modified Kirby-Bauer disc diffusion method [49]. The minimum inhibitory concentration (MIC) values of the test compounds were determined by the tube dilution technique. Gatifloxacin and griseofulvin were used as the reference drugs and DMF/DMSO were used as the control. The results of the antibacterial and antifungal studies are presented in Table 2.

Discussion
The synthesis of the N-methylated cyclopolypeptide 8 was accomplished with 83% yield, and the N-methylmorpholine proved to be an effective base for the cyclization of the linear heptapeptide unit. The cyclization of the linear peptide fragment was supported by the disappearance of the absorption bands at 1744, 1268, 1393, 1381, and 939 cm −1 (C=O str , C-O str , ester and C-H bend , CH 3(rock) , tert-butyl group) in the IR spectra of 8. The formation of the cyclopeptide was further confirmed by the disappearance of the singlets at 3.56 and 1.51 ppm corresponding to the three protons of the methyl ester group and the nine protons of the tert-butyl group of the Boc in the 1 H-NMR spectrum and the disappearance of the singlets at 154.5 and 79.8, and 52.9 and 28.3 ppm corresponding to the carbon atoms of the ester and tert-butyl groups in the 13 C-NMR spectrum of 8, respectively. Furthermore, the 1 H-NMR and 13 C-NMR spectra of the synthesized cyclic heptapeptide showed the characteristic peaks confirming the presence of all of the 65 protons and 49 carbon atoms. A large 13 C chemical shift difference in the 13     Comparison of the antibacterial activity data suggested that the cycloheptapeptide 8 possessed potent bioactivity against the Gram-negative bacteria K. pneumonia and P. aeruginosa, in addition to a satisfactory level of activity against the Cutaneous fungi M. audouinii and T. mentagrophytes with the MIC values of 6 µg/mL when compared to the reference drugs, gatifloxacin and griseofulvin. No significant level of bioactivity was observed against the pathogenic fungi C. albicans and A. niger and the Gram-positive bacteria B. subtilis and S. aureus, in comparison to the standard drugs. Moreover, the newly synthesized N-methylated cyclopeptide 8 exhibited a good level of cytotoxic activity against the DLA and EAC cell lines with CTC 50 values of 10.6 and 14.6 µM, respectively, in comparison to the standard drug, 5-fluorouracil (5-FU) (CTC 50 values of 37.4 and 90.6 µM, respectively). The possible mechanism of the cytotoxic action of cycloheptapeptide 8 might be through apoptosis via induction of the early cell death, nuclear fragmentation, and the internucleosomal DNA scission. In addition, the analysis of the pharmacological activity data revealed that cycloheptapeptide 8 displayed more bioactivity against the pathogenic microbes and cell lines when compared to its linear form 7. The enhanced activity is due to the reduction in the degree of freedom for each constituent within the ring by the cyclization of the peptides. Usually, cyclic peptides show better biological activity compared to their linear counterparts due to the conformational rigidity, which decreases the entropy term of the Gibbs free energy, therefore allowing the enhanced binding toward target molecules, or receptor selectivity. Another advantage from the cyclic structure is the resistance to hydrolysis by the exopeptidases due to the lack of both amino and carboxyl termini. Furthermore, the cyclic peptides can be resistant even to the endopeptidases, as the structure is less flexible than the linear peptides [50]. Cordyheptapeptide A differs from the cordyheptapeptide B in one amino acid moiety i.e., N-methyl-L-tyrosine in place of the N-methyl-L-phenylalanine residue. This structural change results in the enhanced bioactivity against the Gram-negative bacteria and variation in the cytotoxic properties against the DLA and EAC cell lines [14].

General Method for the Synthesis of Linear N-methylated Tri/Tetrapeptide Units (5, 6)
The N-methyl amino acid methyl ester hydrochloride or dipeptide methyl ester (0.01 mol) was dissolved in DMF (25 mL). To this, TEA (0.021 mol) was added at 0 • C and the reaction mixture was stirred for 15 min. The Boc-dipeptide (0.01 mol) in the DMF (25 mL), DIPC (1.26 g, 0.01 mol), and HOBt (1.34 g, 0.01 mol) were added with stirring. The stirring was first done for 1 h at 0-5 • C and then for a further 24 h at room temperature (RT). After the completion of the reaction, the reaction mixture was diluted with an equal amount of water. The precipitated solid/semisolid was then filtered/separated and washed with water, and purified from the mixture of CHCl 3 -petroleum ether (b.p. 60-80 • C) followed by cooling at 0 • C to obtain the title compounds.

Deprotection of the Tetrapeptide Unit (5) at the Amino End and Tripeptide Unit (6) at the Carboxyl End
The Boc-protected tetrapeptide 5 (6.08 g, 0.01 mol) was dissolved in CHCl 3 (15 mL) and treated with CF 3 COOH (2.28 g, 0.02 mol). The resulting solution was stirred at room temperature for 1 h, and then washed with a saturated NaHCO 3 solution (25 mL). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by the crystallization from CHCl 3 and petroleum ether (b.p. 40-60 • C) to obtain the pure deprotected compound 5a.

Cytotoxic Screening
The linear and cyclic heptapeptides (7,8) were subjected to the short term in vitro cytotoxicity study at 62.5-3.91 µg/mL against the DLA and EAC cell lines using 5-fluorouracil (5-FU) as the reference compound. The different dilutions of both compounds ranging from 62.5-3.91 µg/mL were prepared in Dulbecco's minimum essential medium and 0.1 mL of each diluted test compound was added to 0.1 mL of the DLA cells (1 × 10 6 cells/mL) and EAC cells (1 × 10 6 cells/mL). The resulting suspensions were incubated at 37 • C for 3 h followed by performing the tryphan blue dye test and the calculation of the growth inhibition (%). The CTC 50 values were determined by the graphical extrapolation method. The controls were also tested at 62.5-3.91 µg/mL against both cell lines. The results of the cytotoxicity studies are listed in Table 1.

Antimicrobial Screening
The newly synthesized linear and cyclic heptapeptides (7,8) were evaluated for their antibacterial and antifungal potential against two Gram-positive bacteria, B. subtilis and S. aureus, two Gram-negative bacteria, P. aeruginosa and K. pneumoniae, and the diamorphic fungal strain C. albicans and three other fungal strains, including A. niger and two Cutaneous fungal strains M. audouinii and T. mentagrophytes at the concentrations of 50-6.25 µg/mL. The MIC values of the test compounds were determined by the tube dilution technique. Both the linear and cyclic heptapeptides were dissolved separately to prepare a stock solution of 1 mg/mL using the DMF or DMSO. The stock solution was aseptically transferred and suitably diluted with the sterile broth medium to contain seven different concentrations of each test compound ranging from 200-3.1 µg/mL in the different test tubes. The inoculation of all the tubes was carried out with one of the test microbes. The process was repeated with different test bacteria/fungi and the different samples. The tubes inoculated with the bacterial cultures were incubated at 37 • C for 18 h and the fungal cultures were incubated at 37 • C for 48 h. Finally, the presence/absence of growth of the bacteria/fungi was observed. From these results, the MIC of each test compound was determined against each test bacterium/fungus. Gatifloxacin and griseofulvin were used as the reference drugs with the pure solvents (DMF and DMSO) as the negative controls for the antibacterial and antifungal studies, respectively. The Petri plates inoculated with the bacterial cultures were incubated at 37 • C for 18 h and those inoculated with the fungal cultures were incubated at 37 • C for 48 h. The diameters of the inhibition zones (in mm) were measured and the average diameters for the test samples were calculated in triplicate. The diameters obtained for the test samples were compared with that produced by the standard drug. The results of the antimicrobial studies are presented in Table 2.

Conclusions
The first successful synthesis of a N-methylated cyclopeptide, cordyheptapeptide A (8), was accomplished in the present study, with reasonable yield via the coupling reactions utilizing different carbodiimides. The EDC·HCl/TEA coupling method proved to be yield-effective, in comparison to the method utilizing the DIPC/TEA or NMM, providing 9-10% extra yield. The pentafluorophenyl ester was shown to be better than the p-nitrophenyl ester, for the activation of the acid functionality of the linear heptapeptide unit. The NMM was found to be a good base for the intramolecular cyclization of the linear peptide fragment in comparison to the TEA or pyridine. The synthesized heptacyclopeptide displayed potent antibacterial activity especially against the Gram-negative bacteria, and effectiveness against the pathogenic dermatophtytes and the significant level of cytotoxicity. The Gram-negative bacteria were found to be more sensitive than the Gram-positive bacteria towards the newly synthesized peptide. On passing the toxicity tests, the N-methylated cyclopeptide 8 may prove to be a good candidate for clinical studies and can be a new antibacterial, anti-dermatophyte, and the anticancer drug of the future.