Total synthesis and pharmacological investigation of cordyheptapeptide A

Article abstract of DOI:10.3390/molecules22060682

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 6μg/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.

Full text of DOI:10.3390/molecules22060682

molecules
Article
Total Synthesis and Pharmacological Investigation of
Cordyheptapeptide A
Suresh Kumar 1,†, Rajiv Dahiya 2,*,†, Sukhbir Lal Khokra 1, Rita Mourya 3,
Suresh V. Chennupati 4 and Sandeep Maharaj 2
1
Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, Haryana, India;
sureshmpharma@rediffmail.com (S.K.); slkhokra@kuk.ac.in (S.L.K.)
Laboratory of Peptide Research and Development, School of Pharmacy, Faculty of Medical Sciences,
2
The University of the West Indies, St. Augustine, Trinidad & Tobago, West Indies;
Sandeep.Maharaj@sta.uwi.edu
School of Pharmacy, College of Medicine and Health Sciences, University of Gondar, P.O. Box 196,
3
Gondar 6200, Ethiopia; ritz_pharma@yahoo.co.in
Department of Pharmacy, College of Medical and Health Sciences, Wollega University, P.O. Box 395,
4
Nekemte, Ethiopia; sureshchennupati@rediffmail.com
*
†
Correspondence: Rajiv.Dahiya@sta.uwi.edu or drrajivdahiya@gmail.com; Tel.: +1868-493-5655
These authors contributed equally to this work.
Academic Editor: Nancy D. Turner
Received: 23 March 2017; Accepted: 18 April 2017; Published: 27 May 2017
Abstract: 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.
Keywords: cordyheptapeptide A; solution-phase peptide synthesis; coupling; cytotoxicity;
macrocyclization; biological activity; insect pathogenic fungus
1. 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 [
microorganisms such as fungi and bacteria, which produce a variety of natural products with
diverse bioactivities [ ]. Currently, microorganism-derived peptides (MdPs) are attracting the
attention of scientists [ ] because of the unique molecular structures and a wide array of
associated bioactivities like antimycobacterial and antimalarial activity [ ], antibacterial activity [ ],
1]. The previous literature is enriched with potential data indicating the ability of
2,3
4,5
6
,
7
8,9
cytotoxicity [10], and antiviral activity [11]. Cyclopeptides produced by microbes including
cycloaspeptides, halolitoralins [12], psychrophilins, talaromins, ustiloxins, and unguisins are associated
Molecules 2017, 22, 682; doi:10.3390/molecules22060682
www.mdpi.com/journal/molecules

Molecules 2017, 22, 682
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with many biological properties and some of the peptides from fungi have even gained entrance into
the pharmaceutical market. e.g., cyclosporins, ergopeptides, etc.
The structure of a homodectic heptapeptide, cordyheptapeptide A, previously isolated from the
insect pathogenic fungus Cordyceps sp. BCC 1788, was confirmed by spectroscopic techniques including
1
FT-IR, H/13C-NMR, distortionless enhancement of polarisation transfer (DEPT), high-resolution
electron ionization mass spectrometry (HREIMS) and the amino acid residues were established on
the basis of correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear
multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC)
spectroscopy. In terms of the biological response, natural cordyheptapeptide A exhibited antimalarial
activity against Plasmodium falciparum K1 and cytotoxicity to Vero cell lines with 50% inhibitory
concentration values of 5.35 and >56.88
µM, respectively [13]. Another peptide of the series,
cordyheptapeptide B, was isolated from a fungal strain Cordyceps sp. and the absolute configuration
of the cordyheptapeptide B was indicated by chromatographic analysis of the acid hydrolyzate.
Cordyheptapeptide B was found to exhibit cytotoxicity against several cell lines including KB, BC,
NCI-H187, and Vero cell lines with IC50 values in range of 0.66–3.1 µM [14]. In 2011, the total synthesis
of cordyheptapeptide B was reported for the first time utilizing a solution-phase technique [15].
Cordyheptapeptide B differs from cordyheptapeptide A by having an N-methyl-L-phenylalanine
residue instead of the N-methyl-L-tyrosine. Three new cyclic peptides, cordyheptapeptides C–E,
were isolated from the marine-derived fungus A. persicinum SCSIO 115 and their planar structures
were confirmed by extensive mass spectrometry (MS), 1D and 2D NMR spectroscopic analyses.
Cordyheptapeptides C and E displayed cytotoxicity against the SF-268, MCF-7, and NCI-H460 tumor
cell lines with IC50 values in the range of 2.5 to 12.1 µM [16].
Given the diverse pharmacological activities possessed by MdPs and other
cyclooligopeptides [17–19] and the continued efforts of our research group [15,20–45] for synthesizing
N-methylated and other cyclopolypeptides for quantitative yield in the laboratory, the present work
was directed toward the synthesis, characterization, and bioactivity screening of a phenylalanine-rich
cyclopolypeptide, cordyheptapeptide A for the antimicrobial and cytotoxic properties.
2. Results
2.1. Chemistry
The cycloheptapeptide molecule was split into three dipeptide units viz. Boc-L-Phe-N(Me)Gly-
OMe (
acid unit L-N(Me)Tyr-OMe
methyl esters was done by treatment with methyliodide and sodium hydride as per the
literature [46]. Dipeptide units ( ) 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
was coupled with dipeptide , deprotected at the amino terminus, to obtain the tetrapeptide
unit Boc-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OMe ( ). The carboxyl group of dipeptide was
deprotected by alkaline hydrolysis using lithium hydroxide (LiOH) and the deprotected peptide
was coupled with the amino acid unit , utilizing different carbodiimides to obtain the tripeptide
unit Boc-L-Leu-L-Ile-L-N(Me)Tyr-OMe ( ). After removal of the ester group of tripeptide
and the Boc-group of tetrapeptide , 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 ( ).
1
), Boc-L-Pro-D-N(Me)Phe-OMe (2), and Boc-L-Leu-L-Ile-OMe (3), and a single amino
·
HCl ( ). N-methylation of Boc-protected phenylalanine and tyrosine
4
1–3
·
·
·
1
2
5
3
4
6
6
5
,
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 (CF3COOH), 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

Molecules 2017, 22, 682
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(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, 1H-NMR spectroscopy, and elemental analysis. In addition, the mass spectra
and 13C-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.
O
N
O
O
O
O
N
O
N
O
N
N
NH
O
O
H
N
O
O
i, ii
iii
O
+
O
O
O
N
O
O
1
2
5
O
O
O
O
O
O
O
O
O
H2N+
Cl-
N
H
N
HN
O
HN
O
i, ii
iii
+
N
H
O
O
OH
O
3
4
6
HO
N
O
N
HN
i, ii
iii
O
5
6
+
OH
O
N
N
O
O
7
O
HN
O
O
i, iv
ii, v
O
HO
O
H
N
N
H
O
N
O
N
CH2
i = LiOH, THF:H2O (1:1), RT, 1h
ii = CF3COOH, CHCl3, RT, 1h
HN
O
N
iii = DIPC/EDC.HCl, TEA/NMM, HOBt,
DMF/THF, RT, 24h
O
NH
iv = DCC, CHCl3, pfp/pnp, RT, 12h
v = TEA/NMM/pyridine, 0 oC, 7d
N
O
O
8
Figure 1. Synthetic route of the N-methylated cyclopeptide, cordyheptapeptide A (8).
2.2. Pharmacological Activity Studies
The synthesized linear and cycloheptapeptide (
cytotoxicity study against the Dalton’s lymphoma ascites and Ehrlich’s ascites carcinoma cell lines at
concentrations of 62.5–3.91 g/mL using 5-fluorouracil (5-FU) as the reference compound. The
7, 8) were subjected to the short term in vitro
µ
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.

Molecules 2017, 22, 682
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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.
Table 1. Cytotoxic activity data for the linear and cycloheptapeptide (7, 8).
Dalton’s Lymphoma Ascites (DLA) Cells
Ehrlich’s Ascites Carcinoma (EAC) Cells
Conc.
(µg/mL)
Compd.
7
Live Cells
Counted
No. of
%
CTC50
Live Cells
Counted
No. of
Dead Cells
%
GI
CTC50
(µM)
Dead Cells GI a
(µM) b
62.50
31.25
15.63
7.81
0
38
27
17
12
4
38
31
23
16
10
0
0
0
0
0
38
38
28
25
16
100.0
71.05
44.74
34.21
10.53
100.0
81.58
60.53
42.11
28.95
–
0
8
17
21
24
0
28
20
11
7
100.0
71.43
39.29
25.00
14.29
100.0
85.71
60.71
35.71
21.43
–
11
21
25
34
0
19.9
10.6
–
22.6
14.6
–
3.91
4
8
62.50
31.25
15.63
7.81
28
24
17
10
6
0
0
0
0
7
4
15
22
27
38
38
38
38
38
0
11
18
22
28
28
28
28
28
0
3.91
Control
62.50
31.25
15.63
7.81
–
–
–
–
–
–
–
–
3.91
0
Std.
(5-FU)
62.50
31.25
15.63
7.81
100.0
100.0
73.68
65.79
42.11
28
28
17
9
100.0
100.0
60.71
32.14
17.86
0
0
10
13
22
37.4
11
19
23
b
90.6
3.91
5
a
% growth inhibition (GI) = 100
inhibiting 50% of growth.
−
[{(Celltotal – Celldead
)
×
100}/Celltotal]; CTC50 = cytotoxic concentration
Table 2. Antimicrobial evaluation data for the linear and cycloheptapeptide (7, 8).
Diameter of the Zone of Inhibition (mm)
Bacterial Strains
Fungal Strains
M. audo. A. niger
18(6)
Compound
B. sub.
S. aur.
P. aeru.
K. pneu.
C. alb.
T. menta.
7
8
10(50)
12(50)
–
–
10(25)
–
21(6)
25(6)
–
23(6)
26(6)
–
12(25)
15(25)
–
16(6)
20(6)
22(6)
9(25)
DMF †
DMSO ‡
Gatifloxacin
Griseofulvin
–
–
–
–
–
–
–
–
18(12.5) *
–
27(6)
–
23(6)
–
25(6)
–
20(6)
18(6)
20(12.5)
19(6)
B. sub.: Bacillus subtilis, S. aur.: Staphylococcus aureus, P. aeru.: Pseudomonas aeruginosa, K. pneu.: Klebsiella pneumoniae,
C. alb.: Candida albicans, M. audo.: Microsporum audouinii, A. niger: Aspergillus niger, T. menta.: Trichophyon
mentagrophytes; * Values in the bracket are the MIC values (
the antibacterial studies; ‡ dimethylsulfoxide—negative control for the antifungal studies.
µ
g/mL); † dimethylformamide—negative control for
3. 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

Molecules 2017, 22, 682
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absorption bands at 1744, 1268, 1393, 1381, and 939 cm−1 (C=Ostr, C–Ostr, ester and C–Hbend, CH3(rock)
tert-butyl group) in the IR spectra of . The formation of the cyclopeptide was further confirmed by the
,
8
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 1H-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 13C-NMR spectrum of
8, respectively. Furthermore,
the 1H-NMR and 13C-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 13C chemical
shift difference in the 13C chemical shifts between the C
∆δβγ = 32.0
N-methylated cyclopolypeptide
1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (FDAA) derivatized hydrolysis products of cyclopeptide
were 18.27 for the amino acid gly, 27.23 for L-leu, 28.90 for L-ile, 20.54 for L-tyr, 17.44 for L-phe, 22.36
β
and C
23.6 = 8.4) indicates a cis configuration for the proline residue in the synthesized
. The retention times (tR, min) of the observed peaks of the
γ signals of the proline residue
(
−
8
8
for D-phe, and 17.42 for L-pro, and were all in agreement with the values for standard amino acid
derivatives during the HPLC analysis. The appearance of the pseudomolecular ion peak (M + 1)+ at
m/z = 880 corresponding to the molecular formula C49H65N7O8 in the mass spectrum of 8, along with
other fragment ion peaks resulting from the cleavage at ‘Phe-N(Me)Tyr’, ‘Pro-N(Me)Gly’, ‘Ile-Leu’, and
‘Leu-N(Me)Phe’ amide bonds showed the exact sequence of the attachment of all of the seven amino
acid moieties in a chain (Figure 2). In addition, the presence of the immonium ion peaks at m/z 150
[N(Me)Tyr], 134 [N(Me)Phe], 120 [Phe], 86 [Leu/Ile], 70 [Pro], and 44 [N(Me)Gly] further confirmed
all the amino acid moieties in the cyclopeptide structure. Furthermore, the elemental analysis of
8
afforded the values with a tolerance of ±0.02 strictly in accordance with the molecular composition.
A (Cleavage at Ile-Leu)
D (Cleavage at Leu-N(Me)Phe)
O
O
NH
NH
OH
O
N
N
Me
Me
8
O
O
Me
N
N
NH
B (Cleavage at Phe-N(Me)Tyr)
O
O
C (Cleavage at Pro-N(Me)Gly)
NH2---Ile---CO---N(Me)Tyr---CO---Phe---CO---N(Me)Gly---CO---Pro---CO---N(Me)Phe---CO---Leu---CO+
A
B
263
410
481
578
739
852
114
291
438
509
606
767
880
NH2---Phe---CO---N(Me)Gly---CO---Pro---CO---N(Me)Phe---CO---Leu---CO---Ile---CO---N(Me)Tyr---CO+
191
288
449
562
675
852
148
219
316
477
590
703
880
C
D
NH2---Pro---CO---N(Me)Phe---CO---Leu---CO---Ile---CO---N(Me)Tyr---CO---Phe---CO---N(Me)Gly---CO+
231
344
457
634
781
852
98
259
372
485
662
809
880
NH2---Leu---CO---Ile---CO---N(Me)Tyr---CO---Phe---CO---N(Me)Gly---CO---Pro---CO---N(Me)Phe---CO+
199
376
523
594
691
852
114
227
404
551
622
719
880
Immonium ions: 150 (N(Me)Tyr), 134 (N(Me)Phe), 120 (Phe), 86 (Leu/Ile), 70 (Pro), 44 (N(Me)Gly)
Other fragments ions: 107, 93, 91, 77, 57, 43, 29, 17, 15
Figure 2. Mass fragmentation pattern for the newly synthesized N-methylated cyclopeptide,
cordyheptapeptide A (8).

Molecules 2017, 22, 682
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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
the DLA and EAC cell lines with CTC50 values of 10.6 and 14.6
standard drug, 5-fluorouracil (5-FU) (CTC50 values of 37.4 and 90.6
mechanism of the cytotoxic action of cycloheptapeptide 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 displayed more
bioactivity against the pathogenic microbes and cell lines when compared to its linear form . The
8
exhibited a good level of cytotoxic activity against
M, respectively, in comparison to the
M, respectively). The possible
µ
µ
8
8
7
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].
4. Materials and Methods
Amino acids, trifluoroacetic acid, pentafluorophenol (pfp), p-nitrophenol (pnp), pyridine
(C5H5N), N-methylmorpholine (NMM), triethylamine (TEA), Diisopropylcarbodiimide (DIPC),
Dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC HCl), and di-tert-butyl-pyrocarbonate (Boc2O) were purchased from Spectrochem Limited
·
(Mumbai, India). The IR spectra were recorded on a Shimadzu 8700 FTIR spectrophotometer
(Shimadzu, Japan) using a thin film supported on KBr pellets for the synthesized cyclic heptapeptide
and CHCl3 as the solvent for the intermediate semisolids. The 1H-NMR and 13C-NMR spectra were
recorded on a Bruker AC-NMR spectrometer (Brucker, Billerica, MA, USA) at 300 MHz using CDCl3
as the solvent and tetramethylsilane (TMS) as the internal standard. The mass spectrum was recorded
on a JMS-DX 303 Mass spectrometer (Jeol, Tokyo, Japan) operating at 70 eV using the fast atom
bombardment technique.
4.1. 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/separa◦ted
and washed with water, and purified from the mixture of CHCl3-petroleum ether (b.p. 60–80 C)
followed by cooling at 0 ◦C to obtain the title compounds.

Molecules 2017, 22, 682
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4.1.1. Tert-Butyloxycarbonyl-L-phenylalanyl-N(Me)glycyl-L-prolyl-D-N(Me)phenylalanine
Methyl Ester (5)
Semisolid mass; Yield 79%; [
α
]
=
−
52.3◦ (c = 0.25, CHCl3); Rf = 0.48 (CHCl3
MeOH—8:2); IR
·
D
(CHCl3): v 3136 (m, -NH str, amide), 2999, 2996 (m, -CH str, cyclic CH2, Pro), 2927, 2622, 2854–2851
(m, -CH str, asym and sym, CH2), 2870, 2866 (m, -CH str, sym, CH3), 1742 (s, -C=O str, ester), 1664–1659,
1642 (s, -C=O str, 3◦ and 2◦ amide), 1587, 1479 (m, skeletal bands, arom. rings), 1532 (m, -NH bend,
2◦ amide), 1391, 1376 (s, -CH bend, tert-Butyl group), 1270 (s, C-O str, ester), 933 (w, CH3 rocking,
tert-Butyl group), 726–722, 690, 687 (s, -CH bend, out-of-plane, arom. ring) cm−1; 1H-NMR (300 MHz,
CDCl3):
δ 7.52–7.48 (2H, tt, J = 6.8, 4.45 Hz, m-H’s, Phe-1), 7.15–7.12 (1H, t, J = 6.2 Hz, p-H, Phe-2),
7.05–7.01 (2H, tt, J = 6.75, 4.5 Hz, m-H’s, Phe-2), 6.93–6.90 (1H, t, J = 6.25 Hz, p-H, Phe-1), 6.86–6.84
(2H, dd, J = 8.8, 4.15 Hz, o-H’s, Phe-1), 6.75–6.73 (2H, dd, J = 8.75, 4.2 Hz, o-H’s, Phe-2), 6.51 (1H, br.
s, -NH, Phe-1), 5.39–5.36 (1H, t, J = 5.2 Hz,
4.45–4.42 (1H, t, J = 6.9 Hz, -H, Pro), 3.89 (2H, s,
3.54 (3H, s, OCH3), 3.17–3.11 (4H, m, -H’s, Phe-1 and Phe-2), 2.99 (3H, s, NCH3, Phe), 2.94 (3H, s,
NCH3, Gly), 2.69–2.64 (2H, q, -H’s, Pro), 1.96–1.89 (2H, m, -H’s, Pro), 1.52 (9H, s, tert-Butyl group)
α
-H, Phe-2), 4.87–4.83 (1H, q, J = 5.55 Hz,
α
-H, Phe-1),
α
α
-H, Gly), 3.59–3.56 (2H, t, J = 7.15 Hz,
δ-H’s, Pro),
β
β
γ
ppm; C33H44N4O7 (608): calcd. C 65.11, H 7.29, N 9.20; found C 65.10, H 7.32, N 9.22.
4.1.2. Tert-Butyloxycarbonyl-L-leucyl-L-isoleucyl-L-N(Me)tyrosine Methyl Ester (6)
Semisolid mass; Yield 83%; [
(CHCl3): v 3372 (m, -OH str, arom. ring), 3136, 3132 (m, -NH str, amide), 2929, 2854 (m, -CH str, asym
α
]
= +81.3◦ (c = 0.25, CHCl3); R = 0.71 (CHCl3
MeOH - 9:1); IR
·
D
f
and sym, CH2), 2967–2963, 2869, 2863 (m, -CH str, asym and sym, CH3), 1745 (s, -C=O str, ester), 1667,
◦
◦
1644–1641 (s, -C=O str, 3 and 2 amide), 1588, 1471 (m, skeletal bands, arom. ring), 1539, 1535 (m, -NH
bend, 2◦ amide), 1466 (m, -CH bend (scissoring), CH2), 1393, 1375 (s, -CH bend, tert-Butyl group),
1379, 1364 (s, -CH bend, iso-propyl group), 1274 (s, C-O str, ester), 933, 920 (w, CH3 rocking, tert-Butyl
and iso-propyl groups), 729, 685 (s, -CH bend, oop, arom. ring) cm−1; 1H-NMR (300 MHz, CDCl3):
δ
7.09 (1H, br. s, -NH, Ile), 6.87–6.83 (2H, dd, J = 8.6, 4.85 Hz, m-H’s, Tyr), 6.79–6.75 (2H, dd, J = 8.55,
5.25 Hz, o-H’s, Tyr), 6.06 (1H, br. s, -NH, Leu), 5.95 (1H, br. s, -OH, Tyr), 4.46–4.43 (1H, t, J = 5.8 Hz,
α
-H, Tyr), 4.37–4.34 (1H, t, J = 8.6 Hz,
OCH3), 3.11–3.09 (2H, d, J = 5.8 Hz, -H’s, Tyr), 3.04 (3H, s, NCH3), 2.06–1.95 (3H, m,
Ile), 1.65–1.59 (2H, m, -H’s, Ile), 1.58–1.53 (1H, m, -H’s, Leu), 1.50 (9H, s, tert-Butyl group), 1.04–1.02
(3H, d, J = 5.9 Hz, -H’s, Leu), 0.95–0.93 (3H, d, J = 7.75 Hz,
0-H’s, Ile), 1.01–0.99 (6H, d, J = 6.25 Hz,
α
-H, Ile), 4.22–4.18 (1H, q, J = 6.85 Hz,
α
-H, Leu), 3.57 (3H, s,
β
β-H’s, Leu and
γ
γ
γ
δ
δ-H’s, Ile) ppm; C28H45N3O7 (535): calcd. C 62.78, H 8.47, N 7.84; found C 62.75, H 8.49, N 7.85.
4.2. 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 CHCl3 (15 mL) and treated
with CF3COOH (2.28 g, 0.02 mol). The resulting solution was stirred at room temperature for 1 h,
and then washed with a saturated NaHCO3 solution (25 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by
the crystallization from CHCl3 and petroleum ether (b.p. 40–60 ◦C) to obtain the pure deprotected
compound 5a.
For deprotection at the carboxyl end, LiOH (0.36 g, 0.015 mol) was added to a solution of tripeptide
6
(5.35 g, 0.01 mol) in THF
for 1 h and then acidified to pH 3.5 with 1 M H2SO4. The aqueous layer was extracted with Et2O
(3 25 mL). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated
·
H2O (1:1, 36 mL) at 0 ◦C. The mixture was stirred at room temperature
×
under reduced pressure. The crude product was finally crystallized from methanol and ether to obtain
the pure deprotected compound 6a.

Molecules 2017, 22, 682
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4.2.1. L-Phenylalanyl-N(Me)glycyl-L-prolyl-D-N(Me)phenylalanine Methyl Ester (5a)
Semisolid mass; Yield 83%; [
α
]
= –84.1◦ (c = 0.25, CHCl3); R = 0.61 (CHCl3
·
MeOH - 8:2); IR
D
f
(CHCl3): v 3496, 3389 (w, -NH str, amine), 2997, 2995 (m, -CH str, cyclic CH2, Pro), 2926–2622, 2855–2849
(m, -CH str, asym and sym, CH2), 2868, 2865 (m, -CH str, sym, CH3), 1739 (s, -C=O str, ester), 1667–1662,
1643 (s, -C=O str, 3◦ and 2◦ amide), 1616 (m, -NH bend, amine), 1589, 1477 (m, skeletal bands, arom.
rings), 1534 (m, -NH bend, 2◦ amide), 1334 (m, -CN str, amine), 1269 (s, C-O str, ester), 725–722, 689,
685 (s, -CH bend, out-of-plane, arom. ring) cm−1; H-NMR (300 MHz, CDCl3):
δ 7.29–7.25 (2H, tt,
1
J = 6.75, 4.5 Hz, m-H’s, Phe-1), 7.17–7.14 (1H, t, J = 6.15 Hz, p-H, Phe-2), 7.06–7.02 (2H, tt, J = 6.8, 4.45
Hz, m-H’s, Phe-2), 6.97–6.94 (1H, t, J = 6.2 Hz, p-H, Phe-1), 6.77–6.69 (4H, m, o-H’s, Phe-1 and Phe-2),
5.38–5.35 (1H, t, J = 5.15 Hz,
α
-H, Phe-2), 4.46–4.43 (1H, t, J = 6.85 Hz,
-H, Gly), 3.61–3.58 (2H, t, J = 7.2 Hz, -H’s, Pro), 3.52 (3H, s, OCH3), 3.07–3.05
-H’s, Phe-2), 2.97 (3H, s, NCH3, Phe-2), 2.92 (3H, s, NCH3, Gly), 2.75–2.73 (2H,
d, J = 5.65 Hz, β-H’s, Phe-1), 2.68–2.63 (2H, q, β-H’s, Pro), 2.25 (2H, s, NH2, Phe-1), 1.95–1.89 (2H, m,
α-H, Pro), 3.98–3.93 (1H, m, α-H,
Phe-1), 3.82 (2H, s,
α
δ
(2H, d, J = 5.6 Hz,
β
γ-H’s, Pro) ppm; C28H36N4O5 (508): calcd. C 66.12, H 7.13, N 11.02; found C 66.10, H 7.15, N 11.05.
4.2.2. Tert-Butyloxycarbonyl-L-leucyl-L-isoleucyl-L-N(Me)tyrosine (6a)
Semisolid mass; Yield 79%; [
α
]
= +67.7◦ (c = 0.25, CHCl3); R = 0.82 (CHCl3
MeOH—9:1); IR
·
D
f
(CHCl3): v 3315–2530 (m/br, -OH str, -COOH), 3375 (m, -OH str, Tyr), 3133, 3129 (m, -NH str, amide),
2927, 2856 (m, -CH str, asym and sym, CH2), 2966–2962, 2868–2863 (m, -CH str, asym and sym, CH3),
1710 (m, -C=O str, -COOH), 1668, 1644–1639◦(s, -C=O str, 3◦ and 2◦ amide), 1589, 1469 (m, skeletal
bands, arom. ring), 1536–1532 (m, -NH bend, 2 amide), 1462 (m, -CH bend (scissoring), CH2), 1423 (m,
C-O-H bend, -COOH), 1392, 1377 (s, -CH bend, tert-Butyl group), 1378, 1362 (s, -CH bend, iso-propyl
group), 936, 919 (w, CH3 rocking, tert-Butyl and iso-propyl groups), 727, 683 (s, -CH bend, oop, arom.
ring) cm−1; 1H-NMR (300 MHz, CDCl3):
Ile), 6.82–6.78 (2H, dd, J = 8.55, 4.9 Hz, m-H’s, Tyr), 6.93–6.89 (2H, dd, J = 8.6, 5.3 Hz, o-H’s, Tyr), 6.02
(1H, br. s, -NH, Leu), 5.35–5.32 (1H, t, J = 8.55 Hz, -H, Ile), 4.52–4.49 (1H, t, J = 5.75 Hz, -H, Tyr),
4.19–4.15 (1H, q, J = 6.9 Hz, -H, Leu), 3.17 (3H, s, NCH3), 3.13–3.11 (2H, d, J = 5.75 Hz, -H’s, Tyr),
2.05–1.96 (3H, m, -H’s, Leu and Ile), 1.67–1.61 (2H, m, -H’s, Ile), 1.59–1.54 (1H, m, -H’s, Leu), 1.52
(9H, s, tert-Butyl group), 1.03–1.01 (3H, d, J = 5.85 Hz, -H’s,
0-H’s, Ile), 0.99–0.97 (6H, d, J = 6.3 Hz,
-H’s, Ile) ppm; C27H43N3O7 (521): calcd. C 62.17, H 8.31, N 8.06;
δ 8.19 (2H, br. s, -OH, Tyr and -COOH), 7.08 (1H, br. s, -NH,
α
α
α
β
β
γ
γ
γ
δ
Leu), 0.93–0.91 (3H, d, J = 7.8 Hz,
δ
found C 62.19, H 8.32, N 8.09.
4.3. Procedure for the Preparation of the Linear Heptapeptide Unit and Its Cyclized Form (7, 8)
The tetrapeptide methyl ester, L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OMe (5a, 5.08 g, 0.01 mol)
◦
was dissolved in 30 mL of THF, 0.021 mol of NMM was added at 0 C, and the resulting mixture
was stirred for 15 min. The Boc-protected tripeptide, Boc-L-Leu-L-Ile-L-N(Me)Tyr-OH (6a, 5.21 g,
0.01 mol) was dissolved in 30 mL of THF and DIPC/EDC HCl (1.26 g/1.92 g, 0.01 mol) and HOBt
·
(1.34 g, 0.01 mol) were added to the above mixture with stirring. The stirring was continued for
24 h, after which the reaction mixture was filtered and the filtrate was washed with 25 mL each of
the 5% NaHCO3 and saturated NaCl solutions. The organic layer was dried over the anhydrous
Na2SO4, filtered, and evaporated in vacuum. Finally, the recrystallization of the crude product
was carried out from a mixture of the CHCl3-petroleum ether (b.p. 40–60 ◦C) followed by cooling
◦
at 0 C to obtain the Boc-L-Leu-L-Ile-L-N(Me)Tyr-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OMe (
7) as
the pale-yellow semisolid mass. The linear heptapeptide unit (
7, 5.06 g, 0.005 mol) was then
deprotected at the carboxyl terminal using the lithium hydroxide (LiOH, 0.18 g, 0.0075 mol) to obtain
Boc-L-Leu-L-Ile-L-N(Me)Tyr-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-OH. To a solution of the deprotected
heptapeptide (4.99 g, 0.005 mol) in CHCl3 (50 mL), pentafluorophenol (pfp, 1.23 g, 0.0067 mol)
and DCC (1.06 g, 0.005 mol) were added followed by stirring at room temperature for 12 h. The
filtrate of the above reaction mixture was washed with 10% NaHCO3 (3
×
20 mL) and 5% HCl

Molecules 2017, 22, 682
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(2
×
20 mL) solutions to obtain the corresponding pentafluorophenyl ester Boc-L-Leu-L-Ile-L-N(Me)
The Boc-group of the resulting unit (4.66 g,
Tyr-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-Opfp.
0.004 mol) was removed using CF3COOH (0.91 g, 0.008 mol) to obtain the deprotected product,
L-Leu-L-Ile-L-N(Me)Tyr-L-Phe-N(Me)Gly-L-Pro-D-N(Me)Phe-Opfp, which was dissolved in CHCl3
(25 mL) and TEA/NMM/pyridine (2.8 mL/2.21 mL/1.61 mL, 0.021 mol) was added. Then,
the entire contents were kept at 0 ◦C for 7 days. Then the reaction mixture was washed
with 10% NaHCO3 (3
×
25 mL) and 5% HCl (2
×
25 mL) solutions. The organic layer
was dried over anhydrous Na2SO4 and the crude cyclized compound was recrystallized from
the CH2Cl2/n-hexane to obtain the cyclic product cyclo(L-leucyl-L-isoleucyl-L-N(Me)tyrosyl-L-
phenylalanyl-N(Me)glycyl-L-prolyl-D-N(Me)phenylalanyl) (8).
4.3.1. Tert-Butyloxycarbonyl-L-leucyl-L-isoleucyl-L-N(Me)tyrosyl-L-phenylalanyl-
N(Me)glycyl-L-prolyl-D-N(Me)phenylalanine Methyl Ester (7)
Semisolid mass; Yield 78%; [
α
]
=
−
103.2◦ (c = 0.25, MeOH); Rf = 0.73 (CHCl3
MeOH—9:1);
·
D
IR (CHCl3): v 3369 (m, -OH str, arom. ring), 3135–3129 (m, -NH str, amide), 2998–2994 (m, -CH str,
cyclic CH2, Pro), 2928, 2625, 2853–2849 (m, -CH str, asym and sym, CH2), 2968, 2962, 2872, 2866 (m,
-CH str, asym and sym, CH3), 1744 (s, -C=O str, ester), 1666–1659, 1643–1639 (s, -C=O str, 3◦ and 2◦
amide), 1589–1584, 1477–1473 (m, skeletal bands, arom. rings), 1537–1533 (m, -NH bend, 2◦ amide),
1393, 1381 (s, -CH bend, tert-Butyl group), 1375, 1364 (s, -CH bend, iso-propyl group), 1268 (s, C-O
str, ester), 939, 921 (w, CH3 rocking, tert-Butyl and iso-propyl groups), 728–723, 689, 685 (s, -CH bend,
out-of-plane, arom. rings) cm−1; 1H-NMR (300 MHz, CDCl3):
δ 9.22 (1H, br. s, -NH, Phe-1), 7.19–7.16
(2H, tt, J = 6.75, 4.45 Hz, m-H’s, Phe-1), 7.14–7.11 (1H, t, J = 6.15 Hz, p-H, Phe-2), 7.08 (1H, br. s, -NH,
Ile), 7.06–7.03 (2H, tt, J = 6.8, 4.5 Hz, m-H’s, Phe-2), 7.02–6.97 (2H, dd, J = 8.55, 4.9 Hz, m-H’s, Tyr),
6.92–6.89 (1H, t, J = 6.3 Hz, p-H, Phe-1), 6.85–6.83 (2H, dd, J = 8.75, 4.15 Hz, o-H’s, Phe-1), 6.74–6.69 (2H,
dd, J = 8.6, 5.25 Hz, o-H’s, Tyr), 6.75–6.73 (2H, dd, J = 8.8, 4.15 Hz, o-H’s, Phe-2), 6.05 (1H, br. s, -NH,
Leu), 5.97 (1H, br. s, -OH, Tyr), 5.38–5.35 (1H, t, J = 5.15 Hz,
-H, Phe-1), 4.45–4.42 (1H, t, J = 8.55 Hz, -H, Ile), 4.40–4.37 (1H, t, J = 6.85 Hz,
(1H, t, J = 5.75 Hz, -H, Tyr), 4.20–4.16 (1H, q, J = 6.9 Hz, -H, Leu), 3.91 (2H, s,
(2H, t, J = 7.2 Hz, -H’s, Pro), 3.56 (3H, s, OCH3), 3.11–3.07 (4H, m, -H’s, Phe-1 and Phe-2), 3.04 (3H, s,
NCH3, Tyr), 2.99 (3H, s, NCH3, Phe-2), 2.95 (3H, s, NCH3, Gly), 2.89–2.87 (2H, d, J = 5.75 Hz, -H’s,
Tyr), 2.70–2.65 (2H, q, -H’s, Pro), 2.05–1.93 (3H, m, -H’s, Leu and Ile), 1.91–1.86 (2H, m, -H’s, Pro),
1.67–1.61 (2H, m, -H’s, Ile), 1.58–1.53 (1H, m, -H’s, Leu), 1.51 (9H, s, tert-Butyl group), 1.07–1.05
(3H, d, J = 5.85 Hz, -H’s, Leu), 0.99–0.97 (3H, d, J = 7.8 Hz,
0-H’s, Ile), 1.03–1.01 (6H, d, J = 6.3 Hz,
-H’s, Ile) ppm; 13C-NMR (CDCl3):
= 175.3 (C=O, Tyr), 172.6 (C=O, Leu), 170.5, 169.5, 167.3 (3 C,
C=O, Phe-1, Ile and Phe-2), 166.9, 165.1 (2 C, C=O, Gly and Pro), 156.2 (p-C, Tyr), 154.5 (C=O, Boc),
139.2 ( -C, Phe-2), 136.6 ( -C, Phe-1), 130.8 (2 C, o-C’s, Tyr), 129.6 (2 C, o-C’s, Phe-2), 128.5 (2 C, m-C’s,
Tyr), 128.1 (2 C, o-C’s, Phe-1), 127.4 (2 C, m-C’s, Phe-1), 126.9 (2 C, m-C’s, Phe-2), 126.5 ( -C, Tyr), 126.1,
125.6 (2 C, -C’s, Phe-1 and Phe-2), 79.8 ( -C, Boc), 60.1, 57.7, 54.5 (3 C, -C’s, Tyr, Phe-2 and Pro), 52.9
(OCH3), 51.3, 48.2 (2 C, -C’s, Leu and Ile), 46.8, 45.3 (2 C, -C, Gly and Phe-1), 39.9 ( -C, Pro), 38.0,
37.2 (2 C, -C’s, Leu and Phe-1), 36.2 (NCH3, Gly), 34.4, 33.6, 32.3 (3 C, -C’s, Ile, Tyr and Phe-2), 31.5,
29.7 (2 C, NCH3, Phe-2 and Tyr), 29.2 ( -C, Pro), 28.3 (3 C, -C’s, Boc), 26.6, 25.2 (2 C, -C’s, Pro and
Ile), 24.4 (2 C, -C’s, Leu), 22.1 ( -C, Ile); C55H77N7O11 (1012): calcd. C
-C, Leu), 17.4 (γ0-C, Leu), 10.1 (
65.26, H 7.67, N 9.69; found C 65.25, H 7.65, N 9.72.
α
-H, Phe-2), 4.89–4.85 (1H, q, J = 5.6 Hz,
-H, Pro), 4.35–4.32
-H, Gly), 3.61–3.58
α
α
α
α
α
α
δ
β
β
β
β
γ
γ
γ
γ
δ
δ
δ
γ
γ
γ
γ
α
α
α
α
δ
β
β
β
β
γ
δ
γ
δ
4.3.2. Cyclo(L-leucyl-L-isoleucyl-L-N(Me)tyrosyl-L-phenylalanyl-N(Me)glycyl-L-prolyl-D-
N(Me)phenylalanyl) (8)
◦
◦
Pale yellow crystals; m.p. 178–180 C (179.6–179.8 C for natural cordyheptapeptide A [13]);
Yield 83% (NMM), 79% (C5H5N), 68% (TEA); [
68.6◦ (c = 0.56, CHCl3) (–68.5◦ for natural
cordyheptapeptide A [12]); Rf = 0.59 (CHCl3 MeOH—9:1); IR (KBr): v 3365 (m, -OH str, arom. ring),
3133, 3129–3126 (m, -NH str, amide), 2999–2993 (m, -CH str, cyclic CH2, Pro), 2929–2624, 2855, 2849
α
]
=
−
D
·

Molecules 2017, 22, 682
10 of 14
(m, -CH str, asym and sym, CH2), 2969–2963, 2874, 2867 (m, -CH str, asym and sym, CH3), 1668–1662,
1644, 1632 (s, -C=O str, 3◦ and 2◦ amide), 1588–1582, 1479–1474 (m, skeletal bands, arom. rings),
1539–1534 (m, -NH bend, 2◦ amide), 1376, 1364 (s, -CH bend, iso-propyl group), 923 (w, CH3 rocking,
iso-propyl group), 729–722, 686, 682 (s, -CH bend, out-of-plane, arom. rings) cm−1; 1H-NMR (300 MHz,
CDCl3):
δ 8.64 (1H, br. s, -NH, Phe), 8.23 (1H, br. s, -NH, Leu), 7.65 (2H, tt, J = 6.75, 4.5 Hz, m-H’s,
Phe-2), 7.44 (2H, tt, J = 6.8, 4.45 Hz, m-H’s, Phe-1), 7.23 (1H, t, J = 6.15 Hz, p-H, Phe-2), 7.01 (2H, dd,
J = 8.6, 4.85 Hz, m-H’s, Tyr), 6.85 (1H, t, J = 6.25 Hz, p-H, Phe-1), 6.71 (2H, dd, J = 8.8, 4.15 Hz, o-H’s,
Phe-1), 6.46 (2H, dd, J = 8.55, 5.3 Hz, o-H’s, Tyr), 6.37 (2H, dd, J = 8.8, 4.15 Hz, o-H’s, Phe-2), 5.95 (1H,
br. s, -OH, Tyr), 5.89 (1H, br. s, -NH, Ile), 5.59 (1H, t, J = 5.2 Hz,
5.37 (1H, q, J = 5.55 Hz, -H, Phe-1), 4.95 (1H, q, J = 6.85 Hz, -H, Leu), 4.46 (1H, t, J = 8.6 Hz,
Ile), 4.35 (1H, t, J = 6.85 Hz, -H, Pro), 3.64 (2H, t, J = 7.15 Hz, -H’s, Pro), 3.45 (1H, t, J = 5.8 Hz,
Tyr), 3.07 (3H, s, NCH3, Phe), 2.94 (3H, s, NCH3, Gly), 2.74 (2H, q,
2.46 (2H, d, J = 5.8 Hz, -H’s, Tyr), 2.13 (4H, m, -H’s, Phe-1 and Phe-2), 1.75 (2H, m,
(2H, m, -H’s, Ile), 1.47 (3H, m, -H’s, Leu and Ile), 1.25 (6H, d, J = 6.25 Hz, -H’s, Leu), 1.13 (3H, d,
J = 5.9 Hz, -H’s, Ile), 0.85 (1H, m,
0-H’s, Ile), 0.97 (3H, d, J = 7.75 Hz, -H’s, Leu) ppm; 13C-NMR
(CDCl3): = 174.4 (C=O, Leu), 172.5 (C=O, Pro), 170.7 (C=O, Ile), 170.4 (C=O, Tyr), 170.1, 168.7 (2 C,
C=O, Phe and NMePhe), 168.2 (C=O, Gly), 155.9 (p-C, Tyr), 139.4 ( -C, Phe-2), 137.3 ( -C, Phe-1), 133.1
-C, Tyr), 132.3 (2 C, m-C’s, Phe-1), 130.2 (2 C, o-C’s, Tyr), 129.8 (2 C, m-C’s, Tyr), 128.9 (2 C, o-C’s,
Phe-1), 128.1 (2 C, m-C’s, Phe-2), 127.2 (2 C, m-C’s, Phe-1), 122.9, 122.2 (2 C, -C’s, Phe-2 and Phe-1),
68.9, 58.6, 57.9, 54.8 (4 C, -C’s, Tyr, Ile, Pro and Phe-2), 50.6, 49.9, 47.3 (3 C, -C’s, Gly, Phe-1 and
Leu), 43.5 ( -C, Pro), 42.9, 42.3 (2 C, -C’s, Leu and Phe-1), 40.1 (NCH3, Tyr), 39.8, 36.9 (2 C, -C’s,
Tyr and Phe-2), 36.6 ( -C, Ile), 35.7 (NCH3, Phe), 32.0 ( -C, Pro), 30.3 (NCH3, Gly), 29.7, 24.9, 23.6
(3 C, -C’s, Leu, Ile and Pro), 22.0 (2 C, -C’s, Leu), 17.1 ( -C, Ile); MS (FAB, 70 eV):
0-C, Leu), 10.3 (
α-H, Phe-2), 5.44 (2H, s, α-H, Gly),
α
α
α
-H,
-H,
α
δ
α
β-H’s, Pro), 2.62 (3H, s, NCH3, Tyr),
β
β
γ
-H’s, Pro), 1.61
γ
β
δ
γ
δ
γ
δ
γ
γ
(γ
γ
α
α
δ
β
β
β
β
γ
δ
γ
δ
m/z (%) = 880 (100) [M + 1]+, 852 (13) [880-CO]+, 809 (77) [Pro-N(Me)Phe-Leu-Ile-N(Me)Tyr-Phe]+,
781 (29) [809-CO]+, 767 (49) [Ile-N(Me)Tyr-Phe-N(Me)Gly-Pro-N(Me)Phe]+, 739 (21) [767-CO]+, 719
(59) [Leu-Ile-N(Me)Tyr-Phe-N(Me)Gly-Pro]+, 703 (39) [Phe-N(Me)Gly-Pro-N(Me)Phe-Leu-Ile]+, 691
(13) [719-CO]+, 675 (26) [703-CO]+, 662 (52) [Pro-N(Me)Phe-Leu-Ile-N(Me)Tyr]+, 634 (32) [662-CO]+,
606 (69) [Ile-N(Me)Tyr-Phe-N(Me)Gly-Pro]+, 590 (78) [Phe-N(Me)Gly-Pro-N(Me)Phe-Leu]+, 578
(17) [606-CO]+, 562 (24) [590-CO]+, 551 (54) [Leu-Ile-N(Me)Tyr-Phe]+, 523 (25) [551-CO]+, 509
(44) [Ile-N(Me)Tyr-Phe-N(Me)Gly]+, 481 (22) [509-CO]+, 438 (52) [Ile-N(Me)Tyr-Phe]+, 410 (19)
[438-CO]+, 372 (44) [Pro-N(Me)Phe-Leu]+, 344 (25) [662-CO]+, 316 (66) [Phe-N(Me)Gly-Pro]+, 291
(41) [Ile-N(Me)Tyr]+, 288 (22) [316-CO]+, 263 (20) [291-CO]+, 259 (39) [Pro-N(Me)Phe]+, 231 (15)
[662-CO]+, 227 (37) [Leu-Ile]+, 219 (46) [Phe-N(Me)Gly]+, 199 (21) [227-CO]+, 191 (17) [219-CO]+,
150 (22) [N(Me)Tyr immonium ion, C9H12NO]+, 148 (16) [Phe]+, 134 (26) [N(Me)Phe immonium ion,
C9H12N]+, 120 (16) [Phe immonium ion, C8H10N]+, 114 (20) [Ile/Leu]+, 107 (27) [C7H7O]+, 98 (14)
[Pro]+, 93 (19) [C6H5O]+, 91 (23) [C7H7]+, 86 (29) [Leu/Ile immonium ion, C5H12N]+, 77 (20) [C6H5]+,
70 (24) [Pro immonium ion, C4H8N]+, 57 (18) [C4H9]+, 44 (9) [N(Me)Gly immonium ion, C2H6N]+, 43
(13) [C3H7]+, 29 (11) [C2H5]+, 17 (10) [OH]+, 15 (16) [CH3]+; C49H65N7O8 (879): calcd. C 66.87, H 7.44,
N 11.14; found C 61.85, H 7.47, N 11.15.
4.4. Preparation and Analysis of the Marfey Derivatives
About◦0.5 mg of the synthesized cycloheptapeptide
8 was hydrolyzed by heating in 1 mL of 6 M
HCl at 110 C for 24 h. After cooling, the solution was evaporated to dryness and redissolved in 50 µL
of water. Then 100
acid hydrolyzate solution (or to 50
of 20
L of the 1 M NaHCO3 solution, the mixture was incubated for 1 h at 40 ◦C. The reaction was
stopped by the addition of 10 L of 2 M HCl. Finally, the solvents were evaporated to dryness and
the residue was dissolved in 1 mL of MeOH. An aliquot of this solution (20 L for the cyclopeptide
and 10 L for the standards) was analyzed by HPLC (Phenomenex Luna C18, 4.6 250 mm, 5 m,
solvents: (A) water + 0.05% TFA, (B) MeCN, linear gradient: 0 min 35% B, 30 min 45% B, 1 mL min−1
µL of a 1% w/v solution of FDAA (Marfey’s reagent) in acetone was added to the
µL of a 50 mM solution of the respective amino acid). After addition
µ
µ
µ
8
µ
×
µ
,

Molecules 2017, 22, 682
11 of 14
25 ◦C). The retention times (min) of the FDAA amino acid derivatives used as the standards were as
follows: gly (18.24), L-ile (28.92), D-ile (33.32), L-tyr (20.51), D-tyr (33.08), L-leu (27.25), D-leu (25.05),
L-phe (17.41), D-phe (22.32), L-pro (17.45), and D-pro (18.39). Retention times (min) and relative peak
area (%) of the observed peaks of the FDAA derivatized hydrolysis products of cyclopeptide
8 were as
follows: gly (18.27, 2.39%), L-leu (27.23, 2.48%), L-ile (28.90, 3.67%), L-tyr (20.54, 5.24%), L-phe (17.44,
7.33%), D-phe (22.36, 6.26%), and L-pro (17.42, 7.82%).
4.5. Biological Evaluation Procedures
4.5.1. Cytotoxic Screening
The linear and cyclic heptapeptides (
study at 62.5–3.91 g/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 g/mL
were prepared in Dulbecco’s minimum essential medium and 0.1 mL of each diluted test compound
7, 8) were subjected to the short term in vitro cytotoxicity
µ
µ
was added to 0.1 mL of the DLA cells (1
×
106 cells/mL) and EAC cells (1
×
106 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 CTC50 values were determined by the
graphical extrapolation method. The controls were also tested at 62.5–3.91
lines. The results of the cytotoxicity studies are listed in Table 1.
µg/mL against both cell
4.5.2. 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.
The experimental details of the biological activity studies are described in our previously published
reports [51
N-methylated cycloheptapeptide
–
55]. As per the International Union of Pure and Applied Chemistry (IUPAC) rules, the
can be named as “3,15-Dibenzyl-9-(sec-butyl)-12-(4-hydroxybenzyl)
8
-6-isobutyl-2,11,17-trimethylperhydropyrrolo [1,2-a][1,4,7,10,13,16,19] heptaazacyclohenicosine-1,4,
7,10,13,16,19-heptaone”.

Molecules 2017, 22, 682
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5. 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.
Supplementary Materials: The following are available online. Figure S1: Mass spectrum for the N-methylated
cyclic heptapeptide, cordyheptapeptide A (8
), Figure S2: 13C-NMR spectrum for the N-methylated cyclic
heptapeptide, cordyheptapeptide A (8), Table S1: Various steric and lipophilicity parameters for the N-methylated
linear and cyclic heptapeptide (7, 8).
Acknowledgments: The authors are thankful to the Faculty of Pharmacy, Jamia Hamdard University, Delhi, India
for the spectral analysis. Also, the authors wish to thank the J.S.S. College of Pharmacy, Ooty, Tamilnadu, India
for the cytotoxic activity studies.
Author Contributions: S.K., S.L.K., and R.D. conceived and designed the experiments; S.K. performed
the experiments; S.L.K., S.M., R.M., S.K., and S.V.C. analyzed the data; S.M. and R.M. contributed
reagents/materials/analysis tools; S.K. and R.D. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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