Toward the synthesis and biological evaluation of hirsutide

Article abstract of DOI:10.1007/s00706-008-0052-z

The present investigation deals with the synthesis of a N-methylated cyclotetrapeptide, hirsutide (2), by coupling of the dipeptide units Boc-l-phenylalanyl-l-N-methylphenylalanine-OH and l-valyl-l-N- methylphenylalanine-OMe followed by cyclization of the

Full text of DOI:10.1007/s00706-008-0052-z

Monatsh Chem (2009) 140:121–127
DOI 10.1007/s00706-008-0052-z
ORIGINAL PAPER
Toward the synthesis and biological evaluation of hirsutide
Rajiv Dahiya Æ Monika Maheshwari Æ
Anil Kumar
Received: 24 July 2008 / Accepted: 30 July 2008 / Published online: 14 October 2008
Ó Springer-Verlag 2008
Abstract The present investigation deals with the
synthesis of a N-methylated cyclotetrapeptide, hirsutide
(2), by coupling of the dipeptide units Boc-L-phenylalanyl-
L-N-methylphenylalanine-OH and L-valyl-L-N-methylphe-
nylalanine-OMe followed by cyclization of the linear
tetrapeptide fragment. The chemical structure was estab-
lished on the basis of analytical as well as spectroscopic
data. The newly synthesized cyclic peptide was subjected
to pharmacological screening and found to be highly potent
against the gram-negative bacteria Pseudomonas aerugin-
osa and Klebsiella pneumoniae at 6 lg cm-3. In addition,
potent antihelmintic activity against the earthworms
Megascoplex konkanensis and Pontoscotex corethruses at 1
and 2 mg cm-3, and potent cytotoxic activity against
Dalton’s lymphoma ascites and Ehrlich’s ascites carci-
noma cell lines with IC50 values of 14 and 22 lM were also
observed. Studies revealed that the pentafluorophenyl ester
method employing a catalytic amount of N-methylmor-
pholine proved to be better for cyclization of the linear
tetrapeptide unit.
Introduction
Literature is enriched with several findings suggesting the
vital role of natural products in pharmaceutical research as
biomedically useful agents or as lead compounds for drug
development. Among these, cyclopeptides and related
congeners having unique structures and wide pharmaco-
logical profiles have emerged as an important class of
organic compounds, which may prove better candidates to
overcome the problem of resistance towards conventional
agents [1–3]. Fungi-derived natural cyclic peptides exhibit
a variety of bioactivities, such as cytotoxic activity [4, 5],
antimalarial activity [6], insecticidal activity [7],
antibacterial activity [8], antimycotic activity [9], anti-
dinoflagellate activity [10], antimycobacterial activity [11],
and inhibitory activity toward the spore germination of
fungi [12]. Hirsutide (2), a cyclotetrapeptide, has been
isolated from the spider-derived entomopathogenic fungus
Hirsutella sp. using semipreparative HPLC under isocratic
conditions [13] and is unique in having two N-methylated
phenylalanine units in its structure. Having only minute
quantities of this cyclopeptide obtained from natural
resources has restricted scientists from investigating its
biological profile in detail. Further, the widespread increase
of resistance against conventional antibiotics encourages
the development of novel bioactive congeners with unex-
ploited mechanisms of action.
Keywords Natural products Á Hirsutide Á Peptides Á
Cyclizations Á Total synthesis Á Biological activity
Hence, keeping in view the biological potential of
extracts of Hirsutella sp. and to obtain a bioactive peptide
in good yield, the present work was aimed at the first total
synthesis of the natural peptide hirsutide (2) using the
solution-phase technique in a simple and economical
manner. The study also includes testing of the peptide for
its antibacterial, antifungal, antihelmintic, and cytotoxic
effects.
R. Dahiya (&) Á M. Maheshwari Á A. Kumar
Department of Pharmaceutical Chemistry,
Rajiv Academy for Pharmacy, PO Chattikara,
Mathura 281 001, Uttar Pradesh, India
e-mail: rajivdahiya02@yahoo.com;
rajivdahiya77@rediffmail.com
123

122
R. Dahiya et al.
Results and discussion
NMM/TEA/pyridine to obtain cyclo(L-Phe-L-N-(Me)Phe-L-
Val-L-N-(Me)Phe)—hirsutide (2) (Scheme 1).
Chemistry
The structures of the newly synthesized cyclic peptide as
well as the linear tetrapeptide were confirmed by FTIR, 1H-
NMR, 13C-NMR as well as elemental analysis. In addition,
a mass spectra was recorded for the cyclotetrapeptide.
The synthesis of hirsutide (2) was accomplished with
good yield utilizing three different carbodiimides. The pfp
ester was more suitable than the pnp ester for cyclization of
the linear tetrapeptide unit possibly due to the low reac-
tivity of the pnp ester. Four signals between 5.16 and
5.75 ppm in the proton spectrum of 2 suggested a peptidic
structure for the compound, with these signals being
attributable to the alpha protons of four amino acid units.
The structure of the newly synthesized cyclic tetrapeptide
was found to be identical with natural hirsutide (2) as
indicated by two broad singlets at 9.58 and 9.54 ppm
corresponding to the imino protons of the phenylalanine
For the synthesis of hirsutide (2), the cyclic tetrapeptide
molecule was disconnected into the two dipeptide units
Boc-L-Phe-L-N-(Me)Phe-OMe
and
Boc-L-Val-L-N-
(Me)Phe-OMe. The required amino acid methyl ester
hydrochloride L-Phe-OMe.HCl and Boc-protected amino
acids viz. Boc-L-Phe-OH and Boc-L-Val-OH were prepared
according to previously reported procedures [14–17]. The
free NH2 group of L-Phe-OMe.HCl was protected by
introduction of the Boc-group to get Boc-L-Phe-OMe.
N-methylation of the Boc-protected phenylalanine methyl
ester was achieved by treatment with methyl iodide and
sodium hydride [18] to yield Boc-L-N-(Me)Phe-OMe. The
Boc-group of the resulting unit was then removed using
trifluoroacetic acid (TFA) to give L-N-(Me)Phe-OMe. The
required dipeptide units were prepared by coupling of
N-methyl-phenylalanine methyl ester with Boc-L-Phe-OH
and Boc-L-Val-OH employing dicyclohexylcarbodiimide/
N,N-diisopropylcarbodiimide/1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (DCC/DIPC/EDC.HCl)
as the coupling agents and TEA as base [19–28]. Ester
group of the dipeptide Boc-L-Phe-L-N-(Me)Phe-OMe was
removed by alkaline hydrolysis with LiOH, and the
deprotected peptide was coupled with the dipeptide Boc-L-
Val-L-N-(Me)Phe-OMe after deprotection at the amino
terminal, to get the linear tetrapeptide unit Boc-L-Phe-L-N-
(Me)Phe-L-Val-L-N-(Me)Phe-OMe (1). The methyl ester
group of the linear peptide fragment was then replaced by
the p-nitrophenyl/pentafluorophenyl (pnp/pfp) ester group.
The Boc-group of the resulting compound was removed
using TFA, and the deprotected linear tetrapeptide frag-
ment was now cyclized by keeping the whole contents at
0 °C for 7 days in the presence of a catalytic amount of
1
and valine moieties in the H NMR. The proton spectrum
also showed signals for two N-methyl groups at 2.91 and
2.87 ppm, which are attached to the remaining two phen-
ylalanine units. This fact was further supported by the
presence of four signals due to carbonyl groups between
169.91 and 174.53 ppm in the 13C NMR spectrum of 2.
The presence of two NH-protons and two N-methyl singlets
in the proton spectrum of the tetrapeptide suggested a
cyclic structure, which also is in accordance with the
molecular formula of C34H40N4O4, derived from the
FABMS. Presence of (M ? 1)? ion peak at m/z 569.70 in
mass spectrum of 2 [(M ? 1)? at m/z 569.31 for natural
hirsutide], along with other fragment ion peaks resulting
from cleavage at ‘Phe–N(Me)Phe’ and ‘N(Me)Phe–Val’
amide bond levels, showed the exact sequence of attach-
ment of all the four amino acid moieties in a chain. Further,
presence of immonium ion peaks at m/z 134.20
(N(Me)Phe), 120.20 (Phe) and 72.10 (Val) further
Scheme 1
O
H
O
N
N
O
N
H
O
H
N
a-d
N
O
N
O
N
O
O
O
N
H
O
O
1
2
a = LiOH, THF:H2O (1:1), RT, 1 h;
b = DCC, pfp/pnp, RT, 12 h;
d = NMM/TEA/C5H5N, CHCl3, 7 days, 0 oC.
c = TFA, CHCl3, RT, 1 h;
123

Toward the synthesis and biological evaluation of hirsutide
123
confirmed presence of these amino acid moieties in the
cyclopeptide structure. Moreover, appearance of peaks at
m/z 422.60, 394.50, 261.40 and 233.30 indicated that the
fragmentation also occurs at ‘Val–N(Me)Phe’ and
‘N(Me)Phe–Phe’ amide bond levels to some extent. In
addition, elemental analysis of 2 afforded values (with
6 lg cm-3. Furthermore, the synthesized peptide (2)
showed moderate to good antifungal activity against the
dermatophyte M. audouinii and the plant pathogenic fun-
gus Ganoderma species, in comparison to the standard
drug griseofulvin. However, 2 displayed only a moderate
level of activity against the pathogenic fungi C. albicans
and A. niger.
tolerance of
0.03) strictly in accordance with the
molecular composition (Fig. 1).
Analysis of pharmacological activity data revealed that
the linear tetrapeptide 1 displayed less bioactivity against
pathogenic microbes, earthworms and cell lines when
compared to its cyclic form 2. This is because cyclization
of peptides reduces the degree of freedom for each con-
stituent within the ring and thus substantially leads to
reduced flexibility, increased potency, and selectivity of
cyclic peptides. Further, the inherent flexibility of linear
peptides leads to different conformations that can bind to
more than one receptor, resulting in undesirable adverse
effects. On passing toxicity tests, the synthesized cyclo-
tetrapeptide 2 may prove a good candidate for clinical
studies and can be a novel cytotoxic, antihelmintic, and
antibacterial drug of the future.
Analysis of cytotoxic activity data suggested that the
cyclotetrapeptide 2 exhibited a high level of cytotoxic
activity against DLA and EAC cell lines with IC50 values
of 14 and 22 lM (7.81 and 12.57 lg cm-3) in comparison
to the standard drug—5-FU (IC50 values—37 and 91 lM)
and the linear tetrapeptide 1 [IC50 values—21 and 25 lM
(14.78 and 17.39 lg cm-3)]. Antihelmintic activity data
revealed that 2 showed higher activity against M. kon-
kanensis and P. corethruses at 1 and 2 mg cm-3, in
comparison to the standard drugs, albendazole and
mebendazole. Antimicrobial activity data indicated that 2
exhibited a higher antibacterial activity than the reference
drug, gatifloxacin, against the pathogenic gram-negative
bacteria P. aeruginosa and K. pneumoniae at the
In conclusion, the pentafluorophenyl ester was proved to
be better for the activation of the acid functionality of the
linear tetrapeptide unit. NMM was found to be a good base
for intramolecular cyclization of the linear peptide frag-
ment. DCC was found to be a yield-effective coupling
agent giving a highly insoluble by-product dicyclohexyl-
urea (DCU), in comparison to EDC.HCl and DIPC.
A significant level of pharmacological potential was
observed for the synthesized cyclotetrapeptide against
gram-negative bacteria, earthworms, and DLA/EAC cell
lines. Gram-positive bacteria and Candida/Aspergillus
species were found to be the least sensitive to the cyclic
peptide.
IV
I
H
N
O
N
O
N
O
N
O
II
H
III
2
H2N--Phe--CO--N(Me)Phe--CO--NH--Val--CO--N(Me)Phe--CO+
I
281.4
309.4
380.5
408.5
569.7
541.7
148.2
HN(Me)Phe--CO--NH--Phe--CO--N(Me)Phe--CO--NH--Val--CO+
Experimental
II
569.7
134.2
162.2
281.4
309.4
442.6
470.6
Melting point was determined by open capillary method
and is corrected. L-Amino acids, di-tert-butylpyrocarbonate
(Boc2O), DCC, TFA, TEA, NMM, and pyridine were
purchased from SpectroChem Pvt. Ltd., Mumbai, India.
EDC.HCl, DIPC, methyl iodide, sodium hydride, p-nitro-
phenol, and pentafluorophenol were procured from
RANKEM RFCL Ltd., New Delhi, India. IR spectra were
recorded on Shimadzu 8700 FTIR spectrophotometer
(Shimadzu, Japan) using a thin film supported on KBr or
541.7
H2N--Val--CO--N(Me)Phe--CO--NH--Phe--CO--N(Me)Phe--CO+
III
380.5
408.5
233.3
261.4
569.7
541.7
100.2
1
CHCl3 as solvent. H NMR and 13C NMR spectra were
HN(Me)Phe--CO--NH--Val--CO--N(Me)Phe--CO--NH--Phe--CO+
IV
recorded on a Bruker AC NMR spectrometer (300 MHz)
(Brucker, USA) using CDCl3 as solvent and TMS as
internal standard. Mass spectra were recorded on a JMS-
DX 303 Mass spectrometer (Jeol, Tokyo, Japan) operating
at 70 eV using fast atom bombardment technique.
134.2
162.2
394.5
422.6
569.7
541.7
233.3
261.4
Fig. 1 Fragmentation pattern for hirsutide (2) in the mass spectrum
123

124
R. Dahiya et al.
Elemental analyses of all compounds were performed on
Vario EL III elemental analyzer (Elementar, Germany) and
found to be in good agreement with the calculated values.
Optical rotation was measured on automatic polarimeter
(Optics Tech, Ghaziabad, India) in a 2-dm tube at 25 °C
using sodium D-light. Purity of all compounds was
checked by TLC on precoated silica gel G plates (Kieselgel
0.25 mm, 60G F254, Merck, Germany) utilizing CHCl3/
MeOH (9/1) and CHCl3/AcOH/H2O (3/2/5) as developing
solvents.
174.42 (C=O, Val), 172.03 (C=O, Phe-1), 153.51 (C=O,
boc), 138.20, 135.28, 133.02 (3C, C-c, Phe-3, Phe-2 and
Phe-1), 130.41, 129.53 (4C, C-m, Phe-1 and Phe-2),
130.13, 129.48, 128.29 (6C, C-o, Phe-1, Phe-2 and Phe-
3), 128.04, 127.72 (2C, C-p, Phe-1 and Phe-2), 127.21,
126.04 (3C, C-m and C-p, Phe-3), 79.63 (C-a, boc),
61.11, 59.23 (2C, C-a, Phe-2 and Phe-3), 53.80 (OCH3),
51.42, 48.14 (2C, C-a, Phe-1 and Val), 38.03, 35.33,
33.72 (3C, C-b, Phe-1, Phe-2 and Phe-3), 32.20 (NCH3,
Phe-1), 29.63 (NCH3, Phe-3), 28.79 (C-b, Val), 29.02
(3C, C-b, boc), 18.58 (2C, C-c, Val) ppm.
Synthesis of linear tetrapeptide unit: Boc-L-Phe-L-N-
(Me)Phe-L-Val-L-N-(Me)Phe-OMe (1, C40H52N4O7)
Synthesis of hirsutide: cyclo (L-Phe-L-N-(Me)Phe-L-Val-
L-N-(Me)Phe) (2, C34H40N4O4)
A
solution of 2.92 g of L-Val-L-N-(Me)Phe-OMe
(10 mmol) in 25 cm3 of DMF was neutralized with
2.21 cm3 of NMM (21 mmol) at 0 °C, and the resulting
mixture was stirred for 15 min; 4.27 g of Boc-L-Phe-L-N-
(Me)Phe-OH (10 mmol) was dissolved in 25 cm3 of DMF
and the resulting solution with 2.1 g of DCC [(10 mmol)/
1.92 g of EDC.HCl (10 mmol)/1.26 g of DIPC
(10 mmol)] was added to the above mixture. Stirring was
first done for 1 h at 0–5 °C and then further for 24 h at
RT. After the completion of the reaction, the reaction
mixture was diluted with an equal amount of water. The
precipitated solid was filtered, washed with water, and
recrystallized from a mixture of chloroform and petro-
leum ether followed by cooling at 0 °C to get 5.9 g (84%)
1 as a semisolid mass. Rf 0.75 (CHCl3:AcOH:H2O/3:2:5);
To synthesize cyclopeptide 2, 3.5 g of linear tetrapeptide
unit 1 (5 mmol) was deprotected at the carboxyl end using
0.18 g of LiOH (7.5 mmol) to get Boc-L-Phe-L-N-
(Me)Phe-L-Val-L-N-(Me)Phe-OH. Now, 3.43 g of depro-
tected tetrapeptide unit (5 mmol) was dissolved in 50 cm3
of CHCl3 at 0 °C. To this solution, 0.94 g/1.23 g of pnp/
pfp (6.7 mmol) and 1.06 g of DCC (5 mmol) were added,
and stirring was done at RT for 12 h. The reaction mixture
was filtered, and the filtrate was washed with 10%
NaHCO3 solution (3 9 15 cm3) and finally washed with
5% HCl (2 9 10 cm3) to get the corresponding p-nitro-
phenyl/pentafluorophenyl ester Boc-L-Phe-L-N-(Me)Phe-L-
Val-L-N-(Me)Phe-Opnp/Boc-L-Phe-L-N-(Me)Phe-L-Val-L-
N-(Me)Phe-Opfp. To 3.23 g/3.41 g of this compound
(4 mmol) dissolved in 35 cm3 of CHCl3, 0.91 g of TFA
(8 mmol) was added, stirred at RT for 1 h, and washed
with two proportions each 25 cm3 of 10% NaHCO3
solution. The organic layer was dried over anhydrous
Na2SO4 to get L-Phe-L-N-(Me)Phe-L-Val-L-N-(Me)Phe-
Opnp/L-Phe-L-N-(Me)Phe-L-Val-L-N-(Me)Phe-Opfp which
was dissolved in 25 cm3 of CHCl3 and 2.8 cm3/2.21 cm3/
1.61 cm3 of TEA/NMM/pyridine (21 mmol) was added.
Then, whole contents were kept at 0 °C for 7 days. The
reaction mixture was washed with 10% NaHCO3
(3 9 25 cm3) and 5% HCl (3 9 15 cm3) solutions. The
organic layer was dried over anhydrous Na2SO4,
and the crude cyclized product was crystallized from
CHCl3/n-hexane to get 2.3 g (81%, NMM), 1.95 g
(67%, C5H5N), 1.5 g (53%, TEA) of pure 2 as white
solid. M.p.: 202 °C; Rf 0.61 (CHCl3:AcOH:H2O/3:2:5);
[a]D –192.2 9 10-1 deg cm2 g-1 (Ref. [13]: [a]D for nat-
ural hirsutide: –192.0 9 10-1 deg cm2 g-1) (c, 0.2 in
20
[a]D = -81.6 9 10-1 deg cm2 g-1 (c, 0.9 in DMF); IR
ꢀ
(CHCl3): m = 3,222–3,217, 3,112 (N–H str, amide),
2,968, 2,928–2,922 (C–H str, asym, CH3 and CH2),
2,877–2,873, 2,847 (C–H str, sym, CH3 and CH2), 1746
(C=O str, ester), 1,668–1,663, 1,639 (C=O str, amide),
1,589–1,585, 1,478–1,473 (skeletal bands, rings), 1,540–
1,536 (N–H def, amide), 1,387, 1,368 (C–H def, butyl-t),
1,380, 1,364 (C–H bend, propyl-i), 1,269 (C–O str, ester),
715–705, 698–685 (C–H bend, out-of-plane, rings) cm-1
;
1H NMR (300 MHz, CDCl3): d = 9.05 (brs, 1H, NH,
Val), 7.50 (tt, 2H, J = 7.15, 4.35 Hz, H-m, Phe-1), 7.26
(tt, 2H, J = 7.2, 4.4 Hz, H-m, Phe-2), 7.14 (t, 1H,
J = 6.15 Hz, H-p, Phe-3), 7.08–7.02 (m, 3H, H-m, Phe-3
and H-p, Phe-2), 6.89 (t, 1H, J = 6.2 Hz, H-p, Phe-1),
6.85 (dd, 2H, J = 8.8, 4.15 Hz, H-o, Phe-1), 6.77–6.72
(m, 4H, H-o, Phe-2 and Phe-3), 6.42 (brs, 1H, NH, Phe),
4.74–4.69 (m, 1H, H-a, Val), 4.65–4.59 (m, 1H, H-a, Phe-
1), 4.40 (t, 1H, J = 4.85 Hz, H-a, Phe-2), 4.27 (t, 1H,
J = 4.9 Hz, H-a, Phe-3), 3.58 (3H, s, OCH3), 3.14–3.06
(m, 6H, H-b, Phe-1, Phe-2 and Phe-3), 3.07 (s, 3H,
NCH3, Phe-1), 3.02 (s, 3H, NCH3, Phe-3), 2.09–2.03 (m,
1H, H-b, Val), 1.54 (9H, s, butyl-t), 1.02 (6H, d,
J = 4.55 Hz, H-c, Val) ppm; 13C NMR (125 MHz,
CDCl3): d = 175.82 (C=O, ester), 175.34 (C=O, Phe-2),
ꢀ
CH2Cl2); IR (KBr): m = 3,225–3,216, 3,110 (N–H str,
amide), 2,966, 2,927–2,922 (C–H str, asym, CH3 and CH2),
2,875–2,869, 2,845 (C–H str, sym, CH3 and CH2), 1,668–
1,662, 1,642–1,638 (C=O str, amide), 1,588–1,582, 1,476–
1,472 (skeletal bands, rings), 1,539–1,532 (N–H def,
amide), 1,378, 1,365 (C–H bend, propyl-i), 717–706, 697–
123

Toward the synthesis and biological evaluation of hirsutide
125
Val), 5.35–5.31 (m, 1H, H-a, Phe-1), 5.25–5.21 (m, 1H,
H-a, Phe-2), 5.20–5.16 (m, 1H, H-a, Phe-3), 2.91 (s, 3H,
NCH3, Phe-1), 2.87 (s, 3H, NCH3, Phe-3), 2.69–2.58 (m,
6H, H-b, Phe-1, Phe-2 and Phe-3), 1.65–1.58 (m, 1H, H-b,
Val), 1.15 (6H, d, J = 4.6 Hz, H-c, Val); 13C NMR
(125 MHz, CDCl3): d = 174.53 (C=O, Phe-1), 174.14
(C=O, Val), 172.20 (C=O, Phe-3), 169.91 (C=O, Phe-2),
139.02, 137.68 (3C, C-c, Phe-2, Phe-3 and Phe-1), 131.18
(2C, C-o, Phe-1), 130.59, 129.76 (4C, C-m, Phe-2 and
Phe-3), 129.23 (2C, C-m, Phe-1), 128.74, 128.21 (4C, C-o,
Phe-2 and Phe-3), 127.90 (2C, C-p, Phe-2 and Phe-3),
127.21 (C-p, Phe-1), 63.83, 60.22 (2C, C-a, Phe-2 and Phe-
3), 57.50, 55.87 (2C, C-a, Val and Phe-1), 45.13, 43.64,
40.18 (3C, C-b, Phe-1, Phe-2 and Phe-3), 34.59 (NCH3,
Phe-1), 32.36 (NCH3, Phe-3), 31.66 (C-b, Val), 18.73 (2C,
C-c, Val) ppm; FABMS (70 eV): m/z = 569.70
[(M ? 1)?, 100], 541.70 [(569.70–CO)?, 23], 470.60
[(N(Me)Phe-Phe-N(Me)Phe)?, 41], 442.60 [(470.60–CO)?,
11], 422.60 [(N(Me)Phe-Val-N(Me)Phe)?, 16], 408.50
[(Phe-N(Me)Phe-Val)?/(Val-N(Me)Phe-Phe)?, 29], 394.50
[(422.60–CO)?, 37], 380.50 [(408.50–CO)?, 63], 309.40
Table 1 Antibacterial activity data
Compd Zone of inhibition/mm
Bacterial strains
C. pyogenes S. aureus P. aeruginosa K. pneumoniae
1
8 (25)
10 (25)
–
9 (13)
11 (13)
–
19 (6)
28 (6)
–
21 (6)
26 (6)
–
2
Control
Std1
20 (13)
28 (6)
24 (6)
25 (6)
Values in brackets are MIC values (lg cm-3
Std1 Gatifloxacin
)
Table 2 Antifungal activity data
Compd
Zone of inhibition/mm
Fungal strains
C. albicans M. audouinii A. niger Ganoderma
sp.
1
8 (13)
9 (13)
–
10 (6)
14 (6)
–
8 (25)
11 (25)
–
16 (6)
20 (6)
–
[(N(Me)Phe-Phe)?/(N(Me)Phe-Phe)?,
19],
281.40
2
[(309.40–CO)?, 58], 261.40 [(N(Me)Phe-Val)?/(Val-
N(Me)Phe)?, 11], 233.30 [(261.40–CO)?, 17], 162.20
[(N(Me)Phe)?, 69], 148.20 [(Phe)?, 13], 134.20
[(C9H12N)?/(162.20–CO)?, 34], 120.20 [(C8H10N)?, 8],
100.20 [(Val)?, 11], 91.10 [(C7H7)?, 17], 72.10
[(C4H10N)?, 21], 65.10 [(C5H5)?, 14], 43.10 [(C3H7)?, 6],
42.10 [(C3H6)?, 4], 15.10 [(CH3)?, 3].
Control
Std2
20 (6)
17 (6)
18 (13)
22 (6)
Values in brackets are MIC values (lg cm-3
Std2 Griseofulvin
)
686 (C–H bend, oop, rings) cm-1; H NMR (300 MHz,
CDCl3): d = 9.58 (brs, 1H, NH, Phe), 9.45 (brs, 1H, NH,
Val), 7.27–7.22 (m, 4H, H-m, Phe-2 and Phe-3), 7.15 (tt,
2H, J = 7.15, 4.35 Hz, H-m, Phe-1), 7.08–7.02 (m, 2H, H-
p, Phe-2 and Phe-3), 6.98 (t, 1H, J = 6.15 Hz, H-p, Phe-1),
6.87 (dd, 2H, J = 8.75, 4.15 Hz, H-o, Phe-1), 6.75–6.69
(m, 4H, H-o, Phe-2 and Phe-3), 5.75–5.70 (m, 1H, H-a,
1
Pharmacological activities
The newly synthesized linear as well as cyclic tetrapep-
tides 1 and 2 were screened for antimicrobial activity [29]
Table 3 Antihelmintic activity data
Compd
Conc./mg
Earthworm species
M. konkanensis
Mpt/min
P. corethruses
Eudrilus sp.
Mdt/min
Mpt/min
Mdt/min
Mpt/min
Mdt/min
1
100
200
100
200
100
200
100
200
–
10.28 ( 0.36)
06.44 ( 0.50)
07.44 ( 0.43)
04.09 ( 0.30)
11.25 ( 0.18)
06.40 ( 0.37)
13.85 ( 0.64)
08.21 ( 0.48)
–
19.54 ( 0.23)
13.27 ( 0.22)
14.52 ( 0.21)
10.41 ( 0.17)
19.44 ( 0.40)
13.12 ( 0.83)
22.90 ( 0.53)
15.11 ( 0.28)
–
12.46 ( 0.16)
07.50 ( 0.20)
09.28 ( 0.33)
05.34 ( 0.61)
13.20 ( 0.54)
08.60 ( 0.50)
17.85 ( 0.48)
12.44 ( 0.21)
–
23.09 ( 0.47)
17.13 ( 0.36)
19.04 ( 0.28)
12.33 ( 0.43)
24.22 ( 0.11)
18.04 ( 0.19)
29.64 ( 0.73)
21.05 ( 0.39)
–
15.22 ( 0.51)
10.29 ( 0.41)
12.40 ( 0.50)
09.42 ( 0.31)
12.32 ( 0.33)
07.37 ( 0.28)
13.54 ( 0.45)
10.18 ( 0.77)
–
25.38 ( 0.23)
18.12 ( 0.49)
23.45 ( 0.31)
16.55 ( 0.66)
23.35 ( 0.42)
16.11 ( 0.21)
24.05 ( 0.62)
18.52 ( 0.30)
–
2
Std3
Std4
Control
All data are given as mean SD (n = 3)
Mpt Mean paralyzing time, Mdt mean death time, Std3 albendazole, Std4 mebendazole
123

126
R. Dahiya et al.
Table 4 Cytotoxic activity data
Compd
Conc./lg cm-3
DLA cells
Dead cells
EAC cells
Dead cells
%GI
IC50
%GI
IC50
1
62.50
31.25
15.63
07.81
03.91
62.50
31.25
15.63
07.81
03.91
62.50
31.25
15.63
07.81
03.91
62.50
31.25
15.63
07.81
03.91
38
31
20
11
07
38
34
25
19
12
00
00
00
00
00
38
38
28
25
16
100.00
81.58
52.63
28.95
18.42
100.00
89.47
65.79
50.00
31.58
–
28
22
13
03
01
28
24
16
06
03
00
00
00
00
00
28
28
17
09
05
100.00
78.57
46.43
10.71
03.57
100.00
92.86
68.42
21.43
10.71
–
21
25
2
14
22
Control
–
–
–
–
–
–
–
–
–
–
% Growth inhibition
(%GI) = 100 – [{(Celltotal
Std5
100.00
100.00
73.68
65.79
42.11
100.00
100.00
60.71
32.14
17.86
–
Celldead) 9 100}/Celltotal], IC50
cytotoxic concentration
inhibiting 50% of percentage
growth (in lM)
37
91
Std5 5-fluorouracil (5-FU)
against the four bacterial strains Corynebacterium
pyogenes (MUMC 73), Staphylococcus aureus (MUMC
377), Pseudomonas aeruginosa (MUMC 266) and Kleb-
siella pneumoniae (MUMC 95) and four fungal strains
Microsporum audouinii (MUMC 545), Candida albicans
(MUMC 29), and Aspergillus niger (MUMC 77) and
Ganoderma sp. (MUMC 218) at 25–6 lg cm-3 using
gatifloxacin and griseofulvin as standard drugs. MIC val-
ues of the test compound were determined by tube dilution
technique using sterile DMF. The petri plates inoculated
with bacterial/fungal cultures were incubated at 37 °C for
18 and 48 h. The average diameters of the zones of inhi-
bition (in mm) of the test compound were calculated for
triplicate sets and compared with that produced by the
standard drugs.
3.91 lg cm-3 using 5-fluorouracil (5-FU) as reference
compound. Activity was assessed by determining the per-
centage inhibition of DLA and EAC cells. IC50 values were
determined by the graphical extrapolation method.
Detailed procedures of pharmacological activity studies
are mentioned in our previously published reports [32]. The
results of biological activity studies are compiled in
Tables 1, 2, 3 and 4.
Acknowledgments We are grateful to USIC, DU, Delhi, India, and
RSIC, IIT, Delhi, India, for spectral analysis and CPCRI, Kasaragod,
Kerala, India, for providing earthworms for testing the antihelmintic
activity. Also, great thanks to JSS College of Pharmacy, Ooty, India,
for cytotoxic activity studies.
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