Toward the synthesis and improved biopotential of an n-methylated analog of a proline-rich cyclic tetrapeptide from marine bacteria

Article abstract of DOI:10.3390/md16090305

An N-methylated analog of a marine bacteria-derived natural proline-rich tetracyclopeptide was synthesized by coupling the deprotected dipeptide fragments Boc-L-prolyl-L-N-methylleucine-OH and L-prolyl-L-N-methylphenylalanine-OMe. A coupling reaction was accomplished utilizing N,N⁰-Dicyclohexylcarbodidimde (DCC) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC·HCl) as coupling agents and Triethylamine (TEA) or N-methylmorpholine (NMM) as the base in the presence of the racemization suppressing agent. This was followed by the cyclization of the linear tetrapeptide fragment under alkaline conditions. The structure of the synthesized cyclooligopeptide was confirmed using quantitative elemental analysis, FTIR (Fourier-transform infrared spectroscopy),¹H NMR (Nuclear magnetic resonance spectroscopy),¹³C NMR, and mass spectrometry. From the bioactivity results, it was clear that the newly synthesized proline-rich tetracyclopeptide exhibited better anthelmintic potential against Megascoplex konkanensis, Pontoscotex corethruses, and Eudrilus eugeniae at a concentration of 2 mg/mL as well as improved antifungal activity against pathogenic dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at a concentration of 6 μg/mL, as compared to non-methylated tetracyclopeptide. Moreover, N-methylated tetracyclopeptide displayed significant activity against pathogenic Candida albicans.

Full text of DOI:10.3390/md16090305

marine drugs
Article
Toward the Synthesis and Improved Biopotential of
an N-methylated Analog of a Proline-Rich Cyclic
Tetrapeptide from Marine Bacteria
1
, ,† ID
2, ,† ID
2
3
Rajiv Dahiya *
, Suresh Kumar *
, Sukhbir Lal Khokra , Sheeba Varghese Gupta ,
3
4
ID
5
1
Vijaykumar B. Sutariya , Deepak Bhatia
Sandeep Maharaj 1
, Ajay Sharma , Shamjeet Singh and
1
Laboratory of Peptide Research and Development, School of Pharmacy, Faculty of Medical Sciences,
The University of the West Indies, Saint Augustine, Trinidad and Tobago; Shamjeet.Singh@sta.uwi.edu (S.S.);
Sandeep.Maharaj@sta.uwi.edu (S.M.)
2
3
4
5
Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, Haryana, India;
slkhokra@kuk.ac.in
Department of Pharmaceutical Sciences, USF College of Pharmacy, University of South Florida,
Tampa, FL 33612-4749, USA; svarghes@health.usf.edu (S.V.G.); vsutariy@health.usf.edu (V.B.S.)
Department of Pharmacogenomics, ICPH Fairfax Bernard J. Dunn School of Pharmacy,
Shenandoah University, Fairfax, VA 22031, USA; dbhatia@su.edu
Department of Pharmacognosy, Amity Institute of Pharmacy, Amity University,
Gwalior 474020, Madhya Pradesh, India; asharma5@gwa.amity.edu
*
Correspondence: Rajiv.Dahiya@sta.uwi.edu or drrajivdahiya@gmail.com (R.D.);
sureshmpharma@rediffmail.com (S.K.); Tel.: +1-868-493-5655 (R.D.)
†
These two authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 21 June 2018; Accepted: 27 August 2018; Published: 30 August 2018
Abstract: An N-methylated analog of a marine bacteria-derived natural proline-rich tetracyclopeptide
was synthesized by coupling the deprotected dipeptide fragments Boc-L-prolyl-L-N-methylleucine-OH
and L-prolyl-L-N-methylphenylalanine-OMe. A coupling reaction was accomplished utilizing
0
N,N -Dicyclohexylcarbodidimde (DCC) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
(
EDC HCl) as coupling agents and Triethylamine (TEA) or N-methylmorpholine (NMM) as the base in
·
the presence of the racemization suppressing agent. This was followed by the cyclization of the linear
tetrapeptide fragment under alkaline conditions. The structure of the synthesized cyclooligopeptide
was confirmed using quantitative elemental analysis, FTIR (Fourier-transform infrared spectroscopy),
1
13
H NMR (Nuclear magnetic resonance spectroscopy), C NMR, and mass spectrometry. From the
bioactivity results, it was clear that the newly synthesized proline-rich tetracyclopeptide exhibited
better anthelmintic potential against Megascoplex konkanensis, Pontoscotex corethruses, and Eudrilus
eugeniae at a concentration of 2 mg/mL as well as improved antifungal activity against pathogenic
dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at a concentration of 6 µg/mL,
as compared to non-methylated tetracyclopeptide. Moreover, N-methylated tetracyclopeptide
displayed significant activity against pathogenic Candida albicans.
Keywords: N-methylation; tetracyclopeptide; Pseudomonas sp.; macrocyclization; marine sponge;
Pseudoalteromonas sp.; solution-phase peptide synthesis; pharmacological activity
1
. Introduction
The marine environment is distinguished by unique groups of organisms which are a source
of extremely interesting structures with a wide array of biological activities [1]. The enormous
Mar. Drugs 2018, 16, 305; doi:10.3390/md16090305
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2018, 16, 305
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biodiversity of marine habitats is mirrored by the molecular diversity of secondary metabolites
found to be associated with marine sponges, bacteria, fungi, mollusks, etc. Among these metabolites,
cyclopeptides have emerged as a unique group of bioactive analogs with interesting pharmacological
and biochemical properties including cytotoxicity [
2–
4], insecticidal activity [5], antimicrobial
activity [ ], antitubercular activity [ ], anti-inflammatory activity [9
6
,
7
8
], Human Immunodeficiency
Virus (HIV)-inhibitory activity [10], chymotrypsin-inhibitory activity [11], antimalarial activity [12],
etc. Among cyclooligopeptides, N-methylated peptides have attracted the attention of researchers
and scientists in terms of their unique structures and diverse pharmacological activities [13,14].
Evolutionarily speaking, nature has employed the N-methylation of peptides as an ingenious technique
to modulate biological function [15]. The N-methylation of amino acids and their derivatives can
be carried out with dimethyl sulfate in the presence of sodium hydride and a catalytic amount
of water [16], by utilizing 2-chlorotrityl chloride resin/nosyl group and diazomethane [17], by the
reductive amination, and via other novel methods [18]. In recent years, through the advancement in
synthetic approaches, the potential of N-methylation has begun to be revealed in terms of modulating
the biological activity, selectivity, and pharmacokinetic properties of peptides, but also in delivering
novel drugs [19,20]. Many studies have shown the N-methylation of the cyclic peptides to offer
a number of advantages, including the improvement of the therapeutic efficacy of peptides by
fine-tuning their selectivity for a receptor, enhancing the hydrophobicity by reducing the number
of hydrogen-bond donors and by preventing the formation of intermolecular and intramolecular
hydrogen bonds, which in turn improves the oral bioavailability [21].
Keeping in view the utilization of N-methylation in modulating the biological properties of
peptides and, further, in continuation of the efforts of our research group in the synthesis of
bioactive cyclic peptides [14,22–25], the present study was directed toward the first total synthesis
of an N-methylated analog of a natural tetracyclopeptide. The non-methylated natural peptide was
previously isolated from the marine bacteria Pseudomonas sp. and Pseudoalteromonas sp., associated
with the seaweed Diginea sp. and the sponge Halisarca ectofibrosa [26]. Further, the newly synthesized
N-methylated peptide was subjected to antibacterial, antifungal, antidermatophytic, and anthelmintic
screening and its bioproperties were compared with those of the non-methylated analog.
2
. Results
2
.1. Chemistry
The solution-phase technique of peptide synthesis was employed to prepare an N-methylated
analog of a natural tetracyclopeptide. In the present study, p-nitrophenol (pnp) was used for the
esterification of a linear tetrapeptide chain and compared with pentafluorophenol (pfp) during the
synthesis of tetracyclopeptide 4, affording it in a 74–89% yield, utilizing N-methylmorpholine and
pyridine as bases. There is a literature report indicating the synthesis of non-methylated natural cyclic
tetrapeptide employing the solution-phase technique [27], an N-methylated analog of which was
selected for synthesis in the present investigation. In this study, the peptide units were prepared by the
Bodanszky and Bodanszky method with certain modifications [28]. Prior to coupling, the amino groups
of L-amino acids were protected using Di-tert-butyl dicarbonate (Boc O) and the deprotection of amino
2
groups was achieved using trifluoroacetic acid (TFA). Similarly, the carboxyl groups of L-amino acids
were protected by an esterification reaction with Methanol in the presence of Chlorotrimethylsilane
and the deprotection of carboxyl groups was realized by alkaline hydrolysis with lithium hydroxide
(
LiOH). Racemization was avoided by the utilization of 1-Hydroxybenzotriazole (HOBt) in all
coupling reactions.
In order to carry out the synthesis of N-methylated tetracyclopeptide, Boc-amino acid viz.
Boc-L-Pro-OH was coupled with the corresponding N-methylated amino acid methyl esters such
as L-N-(Me)Leu-OMe and L-N-(Me)Phe-OMe. The required amino acid methyl ester hydrochloride,
such as L-Leu-OMe
·
HCl, L-Phe-OMe HCl, and Boc-protected amino acid viz. Boc-L-Pro-OH, were
·

Mar. Drugs 2018, 16, 305
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prepared according to previously reported procedures in the literature [29]. The free NH groups of
2
L-Leu-OMe
·
HCl and L-Phe-OMe HCl were protected by the introduction of Boc-groups producing
·
Boc-L-Leu-OMe and Boc-L-Phe-OMe, respectively. The N-methylation of Boc-protected leucine and
phenylalanine methyl ester was achieved by treatment with methyl iodide and sodium hydride [30],
producing Boc-L-N-(Me)Leu-OMe and Boc-L-N-(Me)Phe-OMe. The Boc-groups of the resulting
units were removed using TFA producing L-N-(Me)Leu-OMe and L-N-(Me)Phe-OMe, respectively.
The required dipeptide units Boc-L-Pro-L-N-(Me)Leu-OMe (
) were prepared by the coupling of N-methylated leucine and phenylalanine methyl esters
with Boc-L-Pro-OH, employing DCC/EDC HCl as the coupling agent and TEA as the base.
1) and Boc-L-Pro-L-N-(Me)Phe-OMe
(2
·
The ester group of the dipeptide unit Boc-L-Pro-L-N-(Me)Leu-OMe was removed by alkaline
hydrolysis with LiOH and the deprotected peptide was coupled with another dipeptide unit
Boc-L-Pro-L-N-(Me)Phe-OMe after deprotection at the amino terminal, producing the linear tetrapeptide
unit Boc-L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-OMe (3). The methyl ester group of the linear peptide
fragment was replaced by a p-nitrophenyl (pnp)/pentafluorophenyl (pfp) ester group. The Boc-group
of the resulting compound was removed using TFA and the deprotected linear fragment was then
◦
cyclized by maintaining the entire content at 0 C for 7 days in the presence of catalytic amounts of
NMM or pyridine, producing cyclo(L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe) (4). The structure of the
newly synthesized tetracyclopeptide as well as that of the intermediate di- and tetrapeptides were
1
13
confirmed by FT-IR, H/ C NMR spectroscopy, and elemental analysis. In addition, mass spectrum
was recorded for the N-methylated cyclic product 4. The synthetic pathway for the newly synthesized
tetracyclopeptide is given in Figure 1.
O
O
O
O
O
N
O
O
O
O
O
a
N
N
+
HO
HN
1
+
O
O
O
O
O
O
O
a
O
N
N
+
HO
O
HN
N
O
2
O
b, c
a
N
N
O
O
N
O
O
O
O
O
O
b, d
c, e
O
N
N
N
N
N
O
4
3
a: DCC/EDC.HCl, TEA/NMM, HOBt, DCM/THF, RT, 24 h; b: LiOH, THF:H O (1:1), RT, 1 h;
2
o
c: TFA, CHCl , RT, 1 h; d: DIPC, pnp/pfp, CHCl , RT, 12 h; e: NMM/C H N, CHCl , 7 days, 0 C
3
3
5
5
3
Figure 1. Synthetic route for N-methylated proline-rich tetracyclopeptide 4.
. Boc-L-Pro-L-N-(Me)Phe-OMe;
L-N-(Me)Leu-L-Pro-L-N-(Me)Phe).
1
. Boc-L-Pro-L-N-(Me)Leu-OMe;
2
3. Boc-L-Pro-L-N-(Me)Leu-L-Pro-L-N- (Me)Phe-OMe; and 4. cyclo(L-Pro-

Mar. Drugs 2018, 16, 305
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2
.2. Pharmacological Activity
The anthelmintic screening of linear and cyclic tetracyclopeptides (
3,
4) was performed against
three earthworm species (Megascoplex konkanensis, Pontoscotex corethruses, and Eudrilus eugeniae) at
a concentration of 2 mg/mL using the modified Garg’s method [31]. The bioactivity results are
presented in Table 1. Further, antibacterial and antifungal evaluations of peptides 3, 4 were conducted
against four Gram-positive and Gram-negative bacteria—Bacillus subtilis and Staphylococcus aureus,
Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively; dermatophytes Microsporum audouinii
and Trichophyton mentagrophytes; and diamorphic fungi Candida albicans and Aspergillus niger using the
modified Kirby-Bauer disk diffusion method [32]. The antimicrobial activity results are compiled in
Table 2.
Table 1. Anthelmintic activity data for the proline-rich linear and cyclic tetracyclopeptides 3, 4.
Earthworm Species
P. cor.
Compound Ә
M. kon.
E. eug.
Mean
Paralyzing
Time (min)
Mean
Paralyzing
Time (min)
Mean
Paralyzing
Time (min)
Mean Death
Time (min)
Mean Death
Time (min)
Mean Death
Time (min)
‡
‡
III *
IV *
3
4
14.06 ± 0.36
10.25 ± 0.22
13.43 ± 0.22
08.12 ± 0.41
-
22.09 ± 0.27
18.22 ± 0.14
21.13 ± 0.49
16.45 ± 0.26
-
18.55 ± 0.19
12.36 ± 0.37
17.08 ± 0.16
09.55 ± 0.29
-
29.44 ± 0.29
21.25 ± 0.14
27.16 ± 0.22
20.05 ± 0.44
-
14.15 ± 0.26
12.34 ± 0.41
12.57 ± 0.33
09.50 ± 0.24
-
24.32 ± 0.36
21.04 ± 0.21
22.08 ± 0.25
18.05 ± 0.39
-
#
Control
Mebendazole
13.58 ± 0.38
22.59 ± 0.29
17.58 ± 0.40
29.56 ± 0.15
13.50 ± 0.44
24.09 ± 0.49
‡
M. kon.: Megascoplex konkanensis; P. cor.: Pontoscotex corethruses; E. eug.: Eudrilus eugeniae. Data are given as
mean ± S.D. (n = 3). * Bioactivity data for III, IV (non-methylated compounds) were obtained under the same
experimental conditions as those used for methylated derivatives and compared with the results of a previously
#
Ә
published report [27]. Tween 80 (0.5%) in distilled water. Compounds were tested at a concentration of 2 mg/mL.
Table 2. Antimicrobial activity data for proline-rich linear and cyclic tetracyclopeptides 3, 4.
Diameter of Zone of Inhibition (mm)
Compound Ә
Bacterial Strains
Fungal Strains
B. sub.
S. aur.
P. aer.
K. pne.
C. alb.
M. aud.
A. nig.
T. men.
12 (25) †
16 (25)
16 (12.5)
21 (12.5)
-
III *
IV *
3
-
-
-
-
-
-
17 (25)
19 (25)
18 (12.5)
23 (12.5)
-
26 (6)
-
9 (12.5)
13 (12.5)
18 (6)
24 (6)
-
17 (6)
25 (6)
19 (6)
27 (6)
-
-
-
-
-
-
-
18 (6)
26 (6)
22 (6)
28 (6)
-
10 (12.5)
12 (12.5)
-
27 (6)
-
4
Control #
Gatifloxacin
Griseofulvin
-
18 (12.5) †
-
23(6)
-
-
-
-
20 (6)
18 (6)
20 (12.5)
19 (6)
B. sub.: Bacillus subtilis; S. aur.: Staphylococcus aureus; P. aer.: Pseudomonas aeruginosa; K. pne.: Klebsiella pneumoniae;
C. alb.: Candida albicans; M. aud.: Microsporum audouinii; A. nig.: Aspergillus niger; T. men.: Trichophyton mentagrophytes.
*
Bioactivity data for III, IV (non-methylated compounds) were obtained under the same experimental conditions
as those used for methylated derivatives and compared with the results of a previously published report [27].
†
#
Values in brackets are minimum inhibitory concentration (MIC) values (
µ
g/mL). Dimethylformamide/Dimethyl
Ә
sulfoxide (DMF/DMSO), Compounds were tested at a concentration in the range of 6–25 µg/mL.
3
. Discussion
The synthesis of an N-methylated cyclooligopeptide (
4) was accomplished with an 89% yield,
and NMM proved to be an effective base for the cyclization of the linear tetrapeptide segment in
comparison to TEA and pyridine. Cyclization was supported by the disappearance of absorption bands
−
1
at 1742, 1273 and 1395, 1373 cm due to C=Ostr, C–Ostr, ester, and C–Hdef, tert-butyl groups in the IR
spectra of compound . The formation of the cyclopeptide was further confirmed by the disappearance
of singlets at 3.54 and 1.52 ppm, corresponding to three protons of the methyl ester group and nine
4

Mar. Drugs 2018, 16, 305
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1
protons of the tert-butyl group of Boc in the H NMR spectrum, in addition to the disappearance of the
singlets at 155.4, 80.4 and 53.0, 29.6 ppm, corresponding to carbon atoms of tert-butyl and ester groups
in the 13C NMR spectrum of compound
4. Furthermore, the H NMR and C NMR spectra of the
1
13
synthesized N-methylated tetracyclopeptide showed characteristic peaks confirming the presence of
+
all 38 protons and 27 carbon atoms. The pseudomolecular ion peak (M + 1) appearing at m/z = 483
corresponded to the molecular formula C H N O in the mass spectrum of 4, along with other
27
38
4
4
fragment ion peaks resulting from the cleavage at ‘N(Me)Leu-Pro’, ‘Pro-N(Me)Phe’, ‘N(Me)Phe-Pro’,
and ‘Pro-N(Me)Leu’ amide bonds (Figure 2). In addition, the presence of the immonium ion peaks at
m/z = 134 [N(Me)Phe], 100 [N(Me)Leu], and 70 [Pro] further confirmed all of the amino acid moieties in
the cyclopeptide structure. Furthermore, the elemental analysis of tetracyclopeptide
4 afforded values
with a strict tolerance of ±0.03, in accordance with the molecular composition.
C (Cleavage at N(Me)Phe-Pro)
O
N
N
D (Cleavage at Pro-N(Me)Leu)
O
4
O
N
N
B (Cleavage at Pro-N(Me)Phe)
O
A (Cleavage at N(Me)Leu-Pro)
+
A
B
C
D
NH(Me)Leu---CO---Pro---CO---N(Me)Phe---CO---Pro---CO
128
225
386
483
1
97
358
455
+
NHPro---CO---N(Me)Leu---CO---Pro---CO---N(Me)Phe---CO
9
8
322
483
2
94
455
+
NH(Me)Phe---CO---Pro---CO---N(Me)Leu---CO---Pro---CO
1
62
259
483
1
34
231
455
+
NHPro---CO---N(Me)Phe---CO---Pro---CO---N(Me)Leu---CO
9
8
356
483
328
455
Immonium ions: 134 [N(Me)Phe], 100 [N(Me)Leu], 70 [Pro]
Figure 2. Fragmentation pattern for N-methylated proline-rich tetracyclopeptide 4 at diverse amide
bond levels.
In the present study, we observed that the proline-rich tetracyclopeptide
4 exhibits better
anthelmintic activity than the standard drug Mebendazole at a concentration of 2 mg/mL against
all three of the tested earthworm species. Comparison of antimicrobial activity data suggested that
N-methylated tetracyclopeptide
T. mentagrophytes and pathogenic fungi C. albicans with minimum inhibitory concentration (MIC) values
of 6 g/mL when compared to the reference drug Griseofulvin. Moreover, moderate activity was seen
4 possesses potent bioactivity against dermatophytes M. audouinii,
µ

Mar. Drugs 2018, 16, 305
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against Gram-negative bacteria P. aeruginosa and K. pneumonia for the newly synthesized N-methylated
tetracyclopeptide, in comparison to the standard drug Gatifloxacin. However, compound displayed
4
little activity against Gram-positive bacteria S. aureus but no significant activity against B. subtitis
nor A. niger. In addition, the analysis of the pharmacological activity data revealed that proline-rich
N-methylated tetracyclopeptide
earthworms than its linear form,
4
displayed higher bioactivity against pathogenic microbes and
, which is due to the fact that the cyclization of the peptides
3
reduces the degree of freedom for each constituent within the ring and thus substantially leads
to the reduced flexibility, as well as increased potency and selectivity of the cyclic peptide [33].
Further, comparison of biological activity data of compound
IV, previously synthesized by our research group [27], suggested that proline-rich N-methylated
tetracyclopeptide displayed improved pharmacological activity against pathogenic dermatophytes,
4 with its non-N-methylated analog
4
earthworms, and Gram-negative bacteria than its non-N-methylated analog IV, which is due to the
fact that N-methylation is the simplest chemical modification occurring in peptides and proteins to
improve the potency. In particular, better improvement was observed in the antifungal activity of
compound 4 against pathogenic C. albicans in comparison to its non-N-methylated analog IV.
Detailed investigation of the structures and biological potential of naturally occurring
cyclooligopeptides suggested that there are various natural cyclic peptides in the literature rich
in proline units which have shown biological potential [4]. In most instances, the arrangement of two
proline units was found to be alternate to each other, as was also observed in the cyclopeptide chosen
for the present study. Besides this, the same type of arrangement of two proline units has been found
in the natural/synthetic cyclopeptide ‘Hymenamide E’, which has already shown potent anthelmintic
and antifungal effects [29]. This arrangement of two proline units in a cyclic structure in an alternate
pattern appears to be responsible for the biological potential of the tetracyclopeptide.
The possible mechanism of action for antimicrobial activity of N-methylated proline-rich
tetracyclopeptides may involve the active transport inside the bacterial cell where peptides can bind
and inactivate the specific targets such as the bacterial ribosome and inhibit protein synthesis, similar to
other proline-rich antimicrobial peptides [34]. Antifungal activity may be attributed to the inhibition of
chitin synthesis, which is a cell wall component essential to the maintenance of the structural integrity
of the fungus; the inhibition of 1–3
β glucan synthase, a multiunit membrane-integrated enzyme which
is critical to cell wall integrity; and the ability to traverse the energized membrane and interact with an
intracellular target or incite the intracellular induction of reactive oxygen species (ROS), which are
toxic to the fungi, as reported for the established anti-fungal peptides [35].
Dermatophytes such as Microsporum and Trichophyton are the prevailing causes of fungal infection
of the skin, hair, and nails due to their ability to utilize keratin. They are also responsible for athlete’s
foot. In temperate regions, foot ringworm (athlete’s foot) accounts for 75% of all tinea diseases.
These dermatophytes grow in the non-living tissues of hair, nails, and skin, in the region above the
layers where keratin is deposited, and cause a complex of diseases known clinically as tinea (ringworm)
in humans and other vertebrates [36,37]. The zoophilic fungi, such as M. audouinii, more commonly
affect children and tend to evoke a more acute inflammatory response than do the anthropophilic
fungi. Besides this, T. mentagrophytes is among the most common dermatophytes responsible for
infections [38]. Candida albicans and other Candida species can cause cutaneous infections at many sites
on the body, especially those that are moist, such as folds of flesh and armpits. Candida can cause a
different and serious disease if the cells enter and spread within the body [37]. Further, K. pneumoniae
is an opportunistic pathogen that can cause severe hospital-acquired infections such as septicaemia,
pneumonia, urinary tract infection, and soft tissue infection. It is frequently found in the flora of the
mouth, skin, and intestines [39]. Another Gram-negative bacterium, P. aeruginosa, has emerged as an
important pathogen which causes between 10% and 20% of infections in most hospitals. Pseudomonas
infection is especially prevalent among patients with burn wounds, cystic fibrosis, acute leukemia,
organ transplants, and intravenous-drug addiction [40].

Mar. Drugs 2018, 16, 305
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As per the literature, and despite its apparent simplicity, Caenorhabditis elegans has developed into
an important model for biomedical research, particularly in the functional characterization of novel
drug targets. Models of the nematode worm C. elegans can be used to advance the understanding of
the molecular mechanisms of drug action and disease pathogenesis. Several features of C. elegans—as it
is easy to culture; undergoes rapid reproduction with a short generation time, enabling the large-scale
production of animals; has a small size; is transparent, which enables the use of fluorescent markers to
study biological processes in vivo; and exhibits cellular complexity—make it a powerful tool for the
pharmaceutical industry [41]. Using mutant strains, RNA interference (RNAi), and passive optical
techniques, C. elegans can be used as an early model to evaluate mechanisms and pathways that
formerly would have required more expensive and time-consuming methods. Further, spectroscopic
techniques for C. elegans are also valuable preliminary tools for finding novel developmental disruptors,
anthelminthic agents, neurotransmitter agonists/antagonists, and proteasome regulators [42].
4
. Materials and Methods
The melting point was determined by the open capillary method and is uncorrected.
The FTIR spectra for all of the synthesized compounds were recorded using an FTIR-8400S Fourier
1
13
transform spectrophotometer (Shimadzu, Kyoto, Japan). The H NMR and C NMR spectra of the
tetracyclopeptide and intermediate linear peptide units were recorded on a Bruker AC 300 spectrometer
at 300 MHz (Brucker, IL, USA). The mass spectra of N-methylated tetracyclopeptide were recorded on a
JMS-DX 303 spectrometer (Jeol, Tokyo, Japan). The elemental analysis was performed on a Vario EL
III elemental analyzer (Elementar Vario EL III, Hanau, Germany). The optical rotation was measured
on an Optics Technology automatic polarimeter (Optics Technology, Delhi, India). The purity of the
synthesized tetracyclopeptide and the intermediate linear peptide units was checked by Thin Layer
Chromatography (TLC) on precoated silica gel G plates (Kieselgel 0.25 mm, 60G F254, Merck, Germany).
4
.1. Procedure for the Preparation of N-Methylated Dipeptide Units (1, 2)
The N-methylated-L-amino acid methyl ester (L-N-(Me)Leu-OMe (1.59 g, 0.01 mol)) was dissolved
◦
in Dichloromethane (DCM, 30 mL). To this, TEA (2.8 mL, 0.021 mol) was added at 0 C and the reaction
mixture was stirred for 15 min. Boc-L-Pro-OH (2.15 g, 0.01 mol) was dissolved in DCM (30 mL) followed
by the addition of DCC (2.12 g, 0.01 mol) and HOBt (1.34 g, 0.01 mol). The resulting mixture was added
to the above solution with constant shaking and stirring was continued for 24 h. This reaction was
repeated using EDC
was filtered and the residue was washed with DCM (25 mL) and added to the filtrate. The filtrate was
washed with 5% NaHCO and saturated NaCl solutions. The organic layer was dried over anhydrous
·
HCl (1.92 g, 0.01 mol) as the coupling agent instead of DCC. The reaction mixture
3
Na SO , filtered, and evaporated in vacuum. The crude product was recrystallized from a mixture
2
4
◦
◦
of chloroform and petroleum ether (Boiling point (b.p.) 40–60 C) followed by cooling at 0 C to
obtain the title compound Boc-L-Pro-L-N-(Me)Leu-OMe ( ). Similarly, the N-methylated dipeptide
1
unit Boc-L-Pro-L-N-(Me)Phe-OMe (2) was prepared by coupling L-N-(Me)Phe-OMe (1.93 g, 0.01 mol)
and Boc-L-Pro-OH (2.15 g, 0.01 mol) under similar conditions.
4
.1.1. tert-Butyloxycarbonyl-L-Prolyl-L-N-Methyl-L-Leucine Methyl Ester (1)
◦
Semisolid mass; yield 79%; [
α
] =
D
−
32.1 (c = 0.25, MeOH); R = 0.62 (CHCl
·
MeOH—8:2); IR
f
3
(
(
(
CHCl ): v = 2995–2988 (C–Hstr), 2966, 2927 (C–Hstr), 2796 (C–Hstr N-Me), 1744 (C=Ostr), 1677, 1638
C=Ostr), 1398, 1373 (C–Hdef), 1388, 1367 (C–Hdef), 1269 (C–Ostr) cm ; H NMR (CDCl ):
3
−
1 1
δ
= 4.52
3
t, J = 6.1 Hz,1H, H-
H, H-δ, Pro), 2.99 (s, 3H, NCH ), 2.57 (q, J = 5.65 Hz, 2H, H-β, Pro), 1.96–1.88 (m, 2H, H-γ, Pro), 1.49
α, Leu), 4.10 (t, J = 7.15 Hz, 1H, H-α, Pro), 3.65 (s, 3H, OCH ), 3.25 (t, J = 7.2 Hz,
3
2
3
(
(
5
s, 9H, tert-Butyl), 1.45–1.34 (m, 3H, H-
CDCl3): = 176.4 (C=O, Pro), 170.3 (C=O, Leu), 159.8 (C=O, Boc), 79.4 (C-
5.9 (C- , Leu), 54.2 (OCH ), 46.7 (C- , Pro), 40.7 (C- , Leu), 35.3 (NCH ), 30.4 (C-
β
and H-
γ
, Leu), 0.94 (d, 6H, J = 6.35 Hz, H-
, tert-Butyl), 60.7 (C-
, Pro), 29.1 (3C, C-β,
δ
, Leu); 13C NMR
, Pro),
δ
α
α
α
δ
β
β
3
3

Mar. Drugs 2018, 16, 305
8 of 14
tert-Butyl), 26.6, 24.1 (2C, C-
γ
, Leu and Pro), 21.8 (2C, C-
δ
, Leu); C H N O (356): calcd. C 60.65,
18
32
2
5
H 9.05, N 7.86; found C 60.66, H 9.02, N 7.88.
4
.1.2. tert-Butyloxycarbonyl-L-Prolyl-L-N-Methyl-Phenylalanine Methyl Ester (2)
◦
Semisolid mass; yield 81%; [
α
] = +44.9 (c = 0.25, MeOH); R = 0.48 (CHCl
·
MeOH—8:2); IR
D
f
3
(
1
CHCl ): v = 2996-2987 (C–Hstr), 2965, 2929, 2877 (C–Hstr), 2802 (C–Hstr N-Me), 1741 (C=Ostr), 1672,
3
−
1 1
636 (C=Ostr), 1544, 1415 (C=C), 1389, 1377 (C–Hdef), 1273 (C–Ostr), 725, 686 (C–Hdef) cm ; H
= 7.15 (t, J = 5.65 Hz, 1H, H-p, Phe), 7.05–6.97 (m, 2H, H-m, Phe), 6.76 (dd, J = 5.8 Hz,
.15 Hz, 2H, H-o, Phe), 5.16 (t, J = 5.65 Hz, 1H, H- , Phe), 4.11 (t, J = 6.45 Hz, 1H, H- , Pro), 3.54 (s, 3H,
OCH ), 3.21 (t, J = 7.15 Hz, 2H, H- , Pro), 3.09 (d, J = 4.85 Hz, 2H, H- , Phe), 3.05 (s, 3H, NCH ), 2.57
, Pro), 1.92-1.86 (m, 2H, H-
, Pro), 1.46 (s, 9H, tert-Butyl); 13C NMR (CDCl3):
= 175.4, 170.1 (2C, C=O, Pro and Phe), 159.7 (C=O, Boc), 138.8 (C- , Phe), 130.2 (2C, C-o, Phe), 127.6
2C, C-m, Phe), 126.2 (C-p, Phe), 80.3 (C- , tert-Butyl), 62.7, 59.3 (2C, C- , Pro and Phe), 53.8 (OCH3),
6.9 (C- , Pro), 34.9, 32.7 (2C, C- , Phe and Pro), 32.1 (NCH ), 29.5 (3C, C- , tert-Butyl), 24.0 (C- , Pro);
C H N O (390): calcd. C 64.60, H 7.74, N 7.17; found C 64.59, H 7.71, N 7.19.
NMR (CDCl3):
δ
7
α
α
δ
β
3
3
(
δ
q, J = 5.45 Hz, 2H, H-
β
γ
γ
(
4
α
α
δ
β
β
γ
3
21
30
2
5
4
.2. Procedure for the Synthesis of Linear Tetrapeptide Segments (3)
To the solution of the dipeptide methyl ester, L-Pro-L-N-(Me)Phe-OMe (2.90 g, 0.01 mol) in
◦
tetrahydrofuran (THF, 25 mL), NMM (2.23 mL, 0.021 mol) was added at 0 C, and the reaction mixture
was stirred for 25 min. To a solution of Boc-L-Pro-L-N-(Me)Leu-OH (3.42 g, 0.01 mol) in THF (25 mL),
DCC (2.12 g, 0.01 mol) and HOBt (1.34 g, 0.01 mol) were added with stirring and the resulting mixture
was added to the above reaction mixture. The stirring of the resulting mixture was continued for 24 h
at RT. This reaction was repeated using EDC
DCC. The reaction mixture was filtered and the residue was washed with THF (25 mL) and added
to the filtrate. The filtrate was washed with 5% NaHCO and saturated NaCl solutions. The organic
·
HCl (1.92 g, 0.01 mol) as a coupling agent in place of
3
layer was dried over anhydrous Na SO , filtered, and evaporated in vacuum. The crude product was
2
4
◦
recrystallized from a mixture of chloroform and petroleum ether (b.p. 40–60 C) followed by cooling
at 0 C to obtain the title compound Boc-L-Pro-L-N-(Me) Leu-L-Pro-L-N-(Me)Phe-OMe (3).
◦
tert-Butyloxycarbonyl-L-Prolyl-L-N-Methyl-L-Leucyl-L-Prolyl-L-N-Methyl-Phenylalanine
Methyl Ester (3)
◦
Semisolid mass; yield 83%; [
α
]D = –53.6 (c = 0.25, MeOH); R = 0.82 (CHCl
·
MeOH-7:3); IR
f
3
(
(
(
(
(
CHCl ): v 2997-2986 (C–Hstr), 2962, 2925–2921, 2875 (C–Hstr), 2801–2794 (C–Hstr N-Me), 1742
3
C=Ostr), 1676, 1645–1640 (C=Ostr), 1544, 1418 (C=C), 1395, 1373 (C–Hdef), 1386, 1362 (C–Hdef), 1273
−
1 1
C–Ostr), 729, 684 (C–Hdef) cm ; H NMR (CDCl ):
δ
= 7.19 (t, J = 5.65 Hz, 1H, H-p, Phe), 7.02–6.95
m, 2H, H-m, Phe), 6.72 (dd, J = 5.75 Hz, 7.1 Hz, 2H, H-o, Phe), 5.38 (t, J = 5.55 Hz, 1H, H- , Phe), 4.45
t, J = 7.15 Hz, 1H, H- , Pro-2), 4.36 (t, J = 6.15 Hz, 1H, H- , Leu), 3.95 (t, J = 7.15 Hz, 1H, H- , Pro-1),
.68 (t, J = 7.2 Hz, 2H, H- , Pro-2), 3.54 (s, 3H, OCH ), 3.24 (t, J = 7.15 Hz, 2H, H- , Pro-1), 3.09 (d,
, Pro-2),
, Pro-2 and Pro-1), 1.94–1.83 (m, 3H, H- and
, Leu); 13C NMR (CDCl3):
= 175.2, 169.3
, Phe),
, Leu), 57.5
, Pro-1 and Pro-2), 36.6
, Pro-2), 29.6 (3C,
, Leu); C H N O
3
α
α
α
α
3
δ
δ
3
J = 4.75 Hz, 2H, H-
2
H-
β
, Phe), 3.06 (s, 3H, NCH ), 2.98 (s, 3H, NCH ), 2.71 (q, J = 5.45 Hz, 2H, H-
β
3
3
.59 (q, J = 5.7 Hz, 2H, H-
β
, Pro-1), 1.98–1.89 (m, 4H, H-
γ
β
γ
, Leu), 1.52 (s, 9H, tert-Butyl), 0.99 (d, J = 6.35 Hz, 6H, H-
δ
δ
(
1
(
(
2C, C=O, Pro-2 and Leu), 167.8, 164.4 (2C, C=O, Phe and Pro-1), 155.4 (C=O, Boc), 139.4 (C-
29.8 (2C, C-o, Phe), 127.9 (2C, C-m, Phe), 126.1 (C-p, Phe), 80.4 (C- , tert-Butyl), 59.2 (C-
, Phe), 53.0 (OCH ), 46.2, 43.6 (2C, C-
γ
α
α
C-
C-
α
, Pro-2), 55.7(C-
, Leu), 33.9 (NCH ), 33.0, 32.3 (2C, C-
α
, Pro-1), 53.9 (C-
α
δ
3
β
β
, Phe and Pro-1), 31.7 (NCH ), 30.8 (C-
β
3
3
C-
β
, tert-Butyl), 27.2 (C-
γ
, Pro-1), 25.5 (2C, C-
δ
, Leu), 23.9 (C-
γ
, Pro-2), 22.2 (C-γ
33
50
4
7
(614): calcd. C 64.47, H 8.20, N 9.11; found C 64.45, H 8.19, N 9.14.

Mar. Drugs 2018, 16, 305
9 of 14
4
.3. Procedure for the Synthesis of N-Methylated Tetracyclopeptide (4)
The linear tetrapeptide unit, Boc-L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-OMe (
3, 3.07 g,
0.005 mol) was deprotected at the carboxyl terminal using LiOH (0.18 g, 0.0075 mol) to
obtain Boc-L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-OH (3a). To a solution of the deprotected
tetrapeptide 3a (3.00 g, 0.005 mol) in CHCl (50 mL), p-nitrophenol (pnp, 0.94 g, 0.0067 mol)
3
0
and N,N -Diisopropylcarbodiimide (DIPC) (0.63 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%
NaHCO (3
×
25 mL) and 5% HCl (2
×
25 mL) solutions to obtain the corresponding p-nitrophenyl
3
ester. This reaction was repeated using pentafluorophenol (pfp, 1.23 g, 0.0067 mol) in place of
p-nitrophenol, producing pentafluorophenyl ester. The Boc-group of the resulting units Boc-L-Pro-L-N-
(
Me)Leu-L-Pro-L-N-(Me)Phe-Opnp (2.89 g, 0.004 mol)/Boc-L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-Opfp
3.07 g, 0.004 mol) was removed using TFA (0.91 g, 0.008 mol) to obtain the deprotected
products L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-Opnp/L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe-Opfp,
which were dissolved in CHCl3 (20 mL), followed by the addition of TEA/NMM/C H N
(
5
5
◦
(
2.8 mL/2.21 mL/1.61 mL, 0.021 mol). Then, all contents were maintained at 0 C for 7 days. The reaction
mixtures were washed with 10% NaHCO (3 20 mL) and 5% HCl (2 20 mL) solutions. The organic
crude cyclized compound was recrystallized using
×
×
3
layer was dried over anhydrous Na SO and
the
2
4
DCM and n-hexane to obtain the pure cyclic product cyclo (L-Pro-L-N-(Me)Leu-L-Pro-L-N-(Me)Phe) (4).
Cyclo (L-Prolyl-L-N-Methyl-L-Leucyl-L-Prolyl-L-N-Methyl-Phenylalanyl) (4)
◦
◦
67.4
Pale white solid; m.p. 189–190 C (d); yield 89% (NMM), 78% (pyridine), 74% (TEA); [
α
] =
D
−
(
2
(
c = 0.1, MeOH); R = 0.59 (CHCl3
·
MeOH
−
9:1); IR (KBr): v = 2999–2989 (C–Hstr), 2969, 2925, 2919,
f
879 (C–Hstr), 2805-2797 (C–Hstr N-Me), 1674, 1643, 1636-1632 (C=Ostr), 1545, 1411 (C=C), 1382, 1366
−
1 1
C–Hdef), 726, 688 (C–Hdef) cm ; H NMR (CDCl ): δ = 7.38, 7.27 (dd, J=5.55 Hz, 7.15 Hz, 2H, H-m,
3
Phe), 7.02 (t, J = 5.6 Hz, 1H, H-p, Phe), 6.73, 6.64 (dd, J = 5.8 Hz, 7.15 Hz, 2H, H-o, Phe), 5.47 (t, J = 6.1 Hz,
1
1
H, H-
H, H-
α
, Leu), 4.97 (t, J = 5.6 Hz, 1H, H-
, Pro-1), 3.63 (t, J = 7.2 Hz, 2H, H-
α
, Phe), 4.48 (t, J = 7.15 Hz, 1H, H-
α
, Pro-2), 4.37 (t, J = 7.2 Hz,
, Pro-2), 3.02 (s, 3H,
, Pro-2), 2.33 (q, J = 5.65 Hz, 2H, H- , Pro-1),
, Pro-2 and Pro-1), 1.39 (t, J = 6.25 Hz, 2H,
, Leu); 13C NMR (CDCl3):
= 173.5,
, Phe), 130.6 (2C, C-m,
α
δ
, Pro-1), 3.48 (t, J = 7.15 Hz, 2H, H-δ
NCH ), 2.68 (s, 3H, NCH ), 2.41 (q, J = 5.5 Hz, 2H, H-
β
β
3
3
2
.14 (d, J = 4.7 Hz, 2H, H-
H-
71.2 (2C, C=O, Leu and Pro-2), 170.4, 168.8 (2C, C=O, Phe and Pro-1), 137.2 (C-
Phe), 127.9 (2C, C-o, Phe), 126.5 (C-p, Phe), 58.8 (C- , Phe), 55.5 (C- , Pro-2), 54.2 (C-
Leu), 48.3, 47.1 (2C, C- , Pro-1 and Pro-2), 39.2 (C-
C- , Leu and Pro-1), 31.5 (C-
β
, Phe), 1.88–1.62 (m, 4H, H-
γ
β
, Leu), 0.97 (d, J = 6.3 Hz, 6H, H-
δ
, Leu), 0.77–0.62 (m, 1H, H-
γ
δ
1
γ
α
α
α
, Pro-1), 51.8 (C-
α
,
δ
β
, Phe), 37.4 (NCH ), 35.7 (NCH ), 34.1, 32.4 (2C,
3
3
β
β
, Pro-2), 28.5 (C-
γ
, Leu), 24.6 (2C, C-
δ
, Leu), 22.9 (C-
γ
, Pro-1), 21.8 (C-
γ
+
,
+
+
Pro-2); FABMS: m/z = 483 [(M + H) , 100], 455 [(483–CO) , 15], 386 [(HN(Me)Leu-Pro-N(Me)Phe) ,
+
+
+
+
5
3
4
6], 358 [(386–CO) , 22], 356 [Pro-N(Me)Phe-Pro) , 42], 328 [(356–CO) , 18], 322 [(Pro-N(Me)Leu-Pro) ,
9], 294 [(322–CO) , 19], 259 [(HN(Me)Phe-Pro) , 66], 231 [(259–CO) , 19], 225 [(HN(Me)Leu-Pro) ,
7], 197 [(225–CO) , 27], 162 [(HN(Me)Phe) , 29], 134 [N(Me)Phe immonium ion (C H N) , 16], 128
+
+
+
+
+
+
+
9
12
+
+
+
+
[
(HN(Me)Leu) , 32], 100 [N(Me)Leu immonium ion (C H N) , 14], 98 [(Pro) , 28], 91 [(C H ) , 9], 77
6 14 7 7
+
+
+
+
+
[(C H ) , 10], 70 [Pro immonium ion (C H N) , 21]; 57 [(C H ) , 8], 43 [(C H ) , 11], 15 [(CH ) , 19];
6
5
4
8
4
9
3
7
3
C H N O (482): calcd. C 67.20, H 7.94, N 11.61; found C 67.19, H 7.95, N 11.63.
27
38
4
4
4
.4. Pharmacological Studies
4
.4.1. Anthelmintic Screening
The anthelmintic activity studies for the newly synthesized linear and cyclic N-methylated
tetrapeptides ( ) were carried out against the three different species of the earthworms M. konkanensis,
3, 4
P. corethruses, and E. eugeniae at a concentration of 2 mg/mL. Suspensions of the samples were prepared
by triturating the synthesized compounds (100 mg) with Tween 80 (0.5%) and distilled water and
the resulting mixtures were stirred using a mechanical stirrer for 30 min. The suspensions were

Mar. Drugs 2018, 16, 305
10 of 14
diluted to contain 0.2% w/v of the test samples. Suspensions of the reference drug, Mebendazole, were
prepared with the same concentration in a similar way. Five earthworms per earthworm species under
investigation were evaluated in triplicate. Earthworms of similar size (2 inches in length) were placed
in Petri plates having a 4-inch diameter, containing 50 mL of the suspension of the test sample and
the reference drug at room temperature (RT). Another set of five earthworms was maintained as a
control in 50 mL of suspension of the distilled water and Tween 80 (0.5%). The paralysis and death
times were noted and their mean was calculated for the triplicate sets. The death time was ascertained
◦
by placing the earthworms in warm water (50 C), which stimulated movement if the worm was alive.
The results of the anthelmintic studies are compiled in Table 1.
4
.4.2. Antibacterial Screening
The newly synthesized linear and cyclic N-methylated proline-rich tetrapeptides (3, 4) were
evaluated for their antibacterial potential against two Gram-positive bacteria (B. subtilis and S. aureus)
and two Gram-negative bacteria (P. aeruginosa and K. pneumoniae) at concentrations ranging from
6.25–50 µg/mL. The minimum inhibitory concentration (MIC) values of test compounds were
determined by the Tube Dilution Technique. Both the linear and cyclic tetrapeptides were dissolved
separately to prepare a stock solution of 1 mg/mL using dimethylformamide (DMF). 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 3.1–200 µg/mL in different test
tubes. The tubes were inoculated with one loopful of overnight growth culture of the test bacteria.
The process was repeated with the different test bacteria and the different samples. The tubes inoculated
◦
with bacterial cultures were incubated at 37 C for 18 h and the presence/absence of growth of the
bacteria was observed. From these results, the MIC of each test compound was determined against
each test bacterium. A possible spore suspension was prepared in sterile distilled water from 5-day-old
culture of the test bacteria growing on nutrient broth media. About 20 mL of the growth medium
was transferred into the sterilized Petri plates and inoculated with 1.5 mL of the spore suspension
4
(spore concentration ~6
×
10 spores/mL). Filter paper disks having a 6-mm diameter and a 1-mm
◦
thickness were sterilized by autoclaving at 121 C (15 psi) for 15 min. Each Petri plate was divided into
five equal portions along the diameter to place one disc. Three discs of the test sample were placed on
three portions together with one disc with the reference drug (Gatifloxacin) and a disk impregnated
with the solvent (DMF) as the negative control. The Petri plates inoculated with bacterial cultures were
◦
incubated at 37 C for 18 h. Diameters of the zones of inhibition (ZOI in mm) were measured and the
average diameters of 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 antibacterial
studies are presented in Table 2.
4
.4.3. Antifungal Screening
The serial plate dilution method was used for the evaluation of antifungal activity against 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 concentrations ranging from 6.25–50 g/mL for
the newly synthesized linear and cyclic tetrapeptides ( ). The MIC values of the test compounds
µ
3, 4
were determined by employing the same technique as that used for the antibacterial studies using
dimethyl sulfoxide (DMSO) instead of DMF, and the tubes inoculated with fungal cultures were
◦
incubated at 37 C for 48 h. After incubation, the presence/absence of the fungal growth was observed
and MIC of the test compounds was determined against each test fungus. A spore suspension in
normal saline (0.91% w/v of NaCl) was prepared from the culture of the test fungi on Sabouraud’s
broth media. After transferring the growth medium, the Petri plates were inoculated with the spore
suspension. After drying, wells were made using an agar punch and the test samples, reference
drug (Griseofulvin), and negative control (DMSO) were placed in the labeled wells in each Petri plate.
◦
The Petri plates inoculated with the fungal cultures were incubated at 37 C for 48 h. Antifungal activity

Mar. Drugs 2018, 16, 305
11 of 14
was determined by measuring the diameter of the inhibition zone for the triplicate sets. The activity of
each compound was compared with the reference standard. The results of the antifungal studies are
given in Table 2.
The experimental details of the biological activity studies are described in our earlier reports [43–52].
Further, in order to describe the intermolecular forces of drug receptor interaction as well as the
transport and distribution of drugs in a quantitative manner, various steric and the lipophilicity
parameters need to be calculated for the synthesized linear and cyclic tetracyclopeptides (
As per International Union of Pure and Applied Chemistry (IUPAC) rules, tetracyclopeptide
can be named as 6-Benzyl-14-isobutyl-7,15-dimethylperhydrodipyrrolo[1,2-a:1,2-g] [ 10
tetraazacyclododecine-5,8,13,16-tetraone.
3, 4).
4
1,4,7, ]
5
. Conclusions
An efficient strategy was developed toward the first total synthesis of an N-methylated
analog of tetracyclopeptide ( ) utilizing carbodiimide chemistry. The EDC HCl/NMM coupling
method provided 9–10% additional yield in comparison to the methods utilizing EDC HCl/TEA
4
·
·
and DCC/TEA or NMM. The p-nitrophenyl ester was shown to be better for the activation of
the acid functionality of the linear tetrapeptide unit in comparison to pentafluorophenyl ester.
The NMM was found to be a better base for the intramolecular cyclization of the linear peptide
segment in comparison to pyridine. Like other proline-containing synthetic cyclooligopeptides,
the synthesized N-methylated tetracyclopeptide displayed potent anthelmintic activity against M.
konkanensis, P. corethruses, and E. eugeniae as well as effectiveness against pathogenic M. audouinii,
T. mentagrophytes, and C. albicans. Further, Gram-negative bacteria were found to be more sensitive
than Gram-positive bacteria towards the newly synthesized tetracyclopeptide at the concentrations
tested. The improved pharmacological activity results of the N-methylated analog further proved
the potential of N-methylation in modulating biological activity and selectivity. On passing toxicity
tests, tetracyclopeptide 4 may prove to be a good candidate for clinical studies and perhaps eventual
consideration as a new anthelmintic and antifungal drug.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/
1
13
9
/305/s1, Figures S1–S3: H NMR, C NMR and mass spectrum for newly synthesized N-methylated
proline-rich tetracyclopeptide, Table S1: Various steric and the lipophilicity parameters for the linear and
cyclic tetracyclopeptides.
Author Contributions: All authors were involved uniformly in all aspects of the work conducted for this paper.
Acknowledgments: The authors are thankful to the Faculty of Pharmacy, Jamia Hamdard University, Jamia
Hamdard, Delhi, India for spectral analysis. Also, sincere thanks to the C.P.C.R.I., Kasaragod, Kerala, India for
providing the earthworms for the testing of anthelmintic activity.
Conflicts of Interest: The authors declare no conflict of interest.
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