Synthesis of elastin based peptides conjugated to benzisoxazole as a new class of potent antimicrobials - A novel approach to enhance biocompatibility

Article abstract of DOI:10.1016/j.ejmech.2010.12.005

The peptides of elastin sequences chosen for the present study included tetrapeptides, pentapeptides and tricosapeptides (30 amino acids), synthesized by classical solution phase method and conjugated to [3-(4-piperidyl)-6-fluoro- 1,2-benzisoxazole]. The

Full text of DOI:10.1016/j.ejmech.2010.12.005

European Journal of Medicinal Chemistry 46 (2011) 704e711
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of elastin based peptides conjugated to benzisoxazole as a new class
of potent antimicrobials e A novel approach to enhance biocompatibility
*
R. Suhas, S. Chandrashekar, D. Channe Gowda
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570 006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The peptides of elastin sequences chosen for the present study included tetrapeptides, pentapeptides
and tricosapeptides (30 amino acids), synthesized by classical solution phase method and conjugated to
[3-(4-piperidyl)-6-fluoro-1,2-benzisoxazole]. The structures of the compounds were confirmed by
physical and spectroscopic techniques followed by the antimicrobial evaluation by both agar well
diffusion and microdilution methods. Here we wish to report the effect of conjugation of these moieties
which enabled us to identify a novel set of peptides conjugated to heterocycle which have exhibited
more potent antimicrobial activity than the conventional drugs used. Further, conjugates of tricosamers
Received 7 October 2010
Received in revised form
23 November 2010
Accepted 7 December 2010
Available online 14 December 2010
Keywords:
34 and 35 were able to inhibit the growth of fungal species at 3e5 mg/mL which is nearly 5 fold more
potent than the reference drug.
Elastin sequences
Benzisoxazole
Conjugates
Ó 2010 Elsevier Masson SAS. All rights reserved.
Potent antimicrobials
Hydrophobicity
Biocompatibility
1. Introduction
2-benzisoxazoles inhibit acetyl cholinesterase, making them suitable
candidates for the palliative treatment of Alzheimer’s disease [7]. In
view of the broad spectrum of the biological activity exhibited by
these benzisoxazoles, the present work was aimed to incorporate [3-
(4-piperidyl)-6-fluoro-1,2-benzisoxazole] which consists of benzi-
soxazole, piperidineand also fluoro groupwhich wouldmake them to
be explored as better antimicrobials.
Elastin protein-based polymers have their origin in repeating
sequences of the mammalian elastic protein, elastin [8,9]. The most
prominent repeating sequence occurs in bovine elastin; it can be
represented as (Val1-Pro2-Gly3-Val4-Gly5)n where n is eleven
without a single substitution. Another repeat first found in porcine
elastin is (Val1-Pro2-Gly3-Gly4)n but this repeat has not been found
to occur with n greater than 2 without substitution. The next most
common recurring sequence in mammalian elastin is a poly-
hexapeptide (Ala1-Pro2-Gly3-Val4-Gly5-Val6)n where, with but
a couple of isomorphous hydrophobic residue replacements such as
Val by Ile or Leu, n is 8 in man [10]. The monomers, oligomers, and
high polymers of these repeats have been synthesized and con-
formationally characterized [11]. These polymers have a number of
medical and non-medical applications [12,13].
Antibiotics are among the most prescribed drugs in the world
today, and since their development and commercialization, have
saved countless millions of lives. The ideal antimicrobial agents are
selective only in targeting the microorganism but not host cells.
Resistance to antimicrobial agents is now recognized as a major
global public health problem. Although there are new classes of
compounds that are now frequently used to treat microbial infec-
tions, the frequency of deeply invasive microbial agents has
increased 10 fold during the past decade. With the emergence of
new microbial strain resistant to many currently available antibiotic
treatments, there is increasing interest in the discovery of novel
antimicrobial agents [1,2].
Many reports portray interesting biological activities of 1,2-ben-
zisoxazoles and its derivatives. These have significant pharmacolog-
ical and biological activities such as anticonvulsant [3], antipsychotic
[4], anticancer [5] presented affinity for serotonergic and dopami-
nergic receptors [6]. In particular, 3-(N-benzylpiperidinylethyl)-1,
Abbreviations: Boc, t-butoxycarbonyl; EDCI, 1-(3-dimethylaminopropyl)
-3-ethyl-carbodiimide HCl; HOBt, 1-hydroxybenzotriazole; IBCF, isobutyl chlor-
oformate; NMM, N-methyl morpholine; TFA, trifluoroacetic acid.
Earlier reports have shown that conjugation of different amino
acids/peptides to various biologically active scaffolds has fetched
the remarkable results which are very promising and even enthu-
siastic ([14e17] and several unpublished works). Moreover, the
* Corresponding author. Tel.: þ91 9449834348 (mobile), 0821 2419664 (office).
E-mail address: dchannegowda@yahoo.co.in (D.C. Gowda).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.005

R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
705
Table 1
1H NMR data of the final protected peptides (2e8).
Entry
1H NMR data (CDCl3,
d ppm)
Boc-G1G2A3P4-OBzl (2)
Boc ¼ 1.40 (9H, s); eNH ¼ 8.01e8.15 (3H, m); Gly1 ¼ 3.87 (2H, s, eaCH); Gly2 ¼ 4.14 (2H, s, eaCH); Ala3 ¼ 1.34
(1H, d, ebCH3), 4.62 (1H, m, eaCH); Pro4 ¼ 1.97e2.10 (2H, m, egCH2), 2.01e2.18 (2H, m, ebCH2), 3.64, 3.79 (2H, m, edCH2),
4.60 (1H, m, eaCH); OBzl ¼ 5.07 (2H, m, eCH2), 7.10e7.34 (5H, m, Ar-H)
Boc-G1G2I3P4-OBzl (3)
Boc-G1G2F3P4-OBzl (4)
Boc-G1V2G3V4P5-OBzl (5)
Boc-G1F2G3F4P5-OBzl (6)
Boc ¼ 1.47 (9H, s); eNH ¼ 8.14e8.26 (3H, m); Gly1 ¼ 3.91 (2H, s, eaCH); Gly2 ¼ 4.62 (2H, s, eaCH);
Ile3 ¼ 0.96e0.97 (6H, m, e(CH3)2), 1.43 (2H, m, egCH2), 2.18 (1H, m, ebCH), 4.63 (1H, m, eaCH); Pro4 ¼ 1.97e2.18 (2H, m, egCH2),
2.01e2.17 (2H, m, ebCH2), 3.50, 3.91 (2H, m, edCH2), 4.61 (1H, m, eaCH); OBzl ¼ 5.10 (2H, m, eCH2), 7.15e7.42 (5H, m, Ar-H)
Boc ¼ 1.41 (9H, s); eNH ¼ 8.51e8.53 (3H, m); Gly1 ¼ 3.87 (2H, s, eaCH); Gly2 ¼ 4.18 (2H, s, eaCH);
Phe3 ¼ 3.67, 3.73 (2H, d, ebCH2), 4.80 (1H, t, eaCH); 7.21e7.32 (5H, m, ArH); Pro4 ¼ 2.00e2.05 (2H, m, egCH2), 2.14,
2.17 (2H, m, ebCH2), 3.70e3.82 (2H, m, edCH2), 4.80 (1H, m, eaCH); OBzl ¼ 5.12 (2H, m, eCH2), 7.21e7.32 (5H, m, Ar-H)
Boc ¼ 1.43 (9H, s); eNH ¼ 8.34e8.42 (4H, m); Gly1 ¼ 3.83 (2H, s, eaCH); Val2 ¼ 0.98 (6H, d, e(CH3)2), 2.70 (1H, m, ebCH),
4.75 (1H, s, eaCH); Gly3 ¼ 4.13 (2H, s, eaCH); Val4 ¼ 0.98 (6H, d, e(CH3)2), 2.69 (1H, m, ebCH),
4.78 (1H, s, eaCH); Pro5 ¼ 2.09e3.64 (6H, m, eCH2), 4.59 (1H, m, eaCH); OBzl ¼ 5.05 (2H, m, eCH2), 7.17e7.36 (5H, m, Ar-H)
Boc ¼ 1.41 (9H, s); eNH ¼ 8.04e8.12 (4H, m); Gly1 ¼ 3.87 (2H, s, eaCH); Phe2 ¼ 3.53, 3.57 (2H, m, ebCH2), 4.79 (1H, s, eaCH),
7.21e7.32 (5H, m, ArH); Gly3 ¼ 4.12 (2H, s, eaCH); Phe4 ¼ 3.47, 3.61 (2H, m, ebCH2), 4.81 (1H, s, eaCH), 7.21e7.32 (5H, m, ArH);
Pro5 ¼ 1.80e3.64 (6H, m, eCH2), 4.55 (1H, m, eaCH); OBzl ¼ 5.08 (2H, m, eCH2), 7.21e7.32 (5H, m, Ar-H)
Boc-GE(OcHx)GFP GVGVP GVGVP Boc ¼ 1.42 (9H, s); eNH ¼ 8.29e9.30 (24H, m); Gly ¼ 4.09, 4.94 (24H, s, eaCH); Val ¼ 0.98, 1.00 (36H, m, (eCH3)2),
GVGVP GFGFP GFGFP-OBzl (7)
2.46, 2.63 (6H, m, ebCH), 4.44 (6H, m, eaCH); Glu ¼ 1.11e1.24 (10H, m, eCH2 of cyclohexyl ring), 2.05, 2.19 (4H, m, ebgCH2),
3.69 (1H, m, eCH of cyclohexyl ring), 4.75 (1H, m, eaCH); Phe ¼ 3.54, 3.70 (10H, m, ebCH2), 4.20, 4.65 (5H, m, eaCH),
7.00e7.63 (25H, m, ArH); Pro ¼ 2.00, 3.52, 3.66 (36H, m, eCH2), 4.76 (6H, m, eaCH); OBzl ¼ 5.10 (2H, m, eCH2),
7.00e7.63 (5H, m, Ar-H)
Boc-GE(OcHx)GFP GVGVP GVGFP Boc ¼ 1.40 (9H, s); eNH ¼ 8.38e9.34 (24H, m); Gly ¼ 4.08, 4.90 (24H, s, eaCH); Val ¼ 0.96, 1.02 (36H, m, (eCH3)2), 2.42,
GFGFP GVGVP GVGFP-OBzl (8) 2.60 (6H, m, ebCH), 4.39 (6H, m, eaCH); Glu ¼ 1.10e1.22 (10H, m, eCH2 of cyclohexyl ring), 2.07, 2.14 (4H, m, eb,gCH2),
3.65 (1H, m, eCH of cyclohexyl ring), 4.69 (1H, m, eaCH); Phe ¼ 3.55, 3.74 (10H, m, ebCH2), 4.19, 4.57 (5H, m, eaCH),
7.19e7.69 (25H, m, ArH); Pro ¼ 2.03, 3.54, 3.70 (36H, m, eCH2), 4.72 (6H, m, eaCH); OBzl ¼ 5.13 (2H, m, eCH2),
7.19e7.69 (5H, m, Ar-H)
amino acid/peptide based drugs have low toxicity, ample bio-
availability and permeability, modest potency and good metabolic
and pharmacokinetic properties [18].
Armed with such valuable information and to shed some more light
on the importance of conjugation, the overarching goal of this study is
to get some insight into the hydrophobic elastin based peptide
conjugates of benzisoxazoles with improved biocompatibility.
strains of human pathogens of both gram positive bacteria namely
Klebsiella pneumoniae and Coagulase positive staphylococcus and
gram negative organisms like Escherichia coli and Xanthomonas
oryzae and antifungal studies against Aspergillus niger, Aspergillus
flavus and Fusarium oxysporum. The results obtained as zone of
inhibition (mm) and minimum inhibitory concentration (mg/mL)
are presented in Table 3 and Table 4 respectively. Amoxicillin and
bavistin served as standard drugs for antibacterial and antifungal
studies respectively.
2. Results and discussion
It is unambiguous from the results obtained that all the
heterocycle conjugated amino acids/peptides have shown
enhanced activity than either heterocycle or free peptides which
Previous work has shown that it is possible to improve the
activities and/or reduce the toxicities of naturally occurring peptide
antibiotics by the modification of primary and/or secondary
structures [19]. Using the new ideas as departure points, we have
developed a new class of biocompatible elastin based peptides
conjugated to biolabile benzisoxazole derivative. Hence, the
present study included the provision for these two entities as
a platform to investigate the effect of conjugation intricacies upon
antimicrobial activity.
are inactive or weakly active (>50 mg/mL).
Interest was generated from the results of the earlier studies
revealed by aromaticity and hydrophobicity [14] and hence initially
simple aromatic amino acids such as Phe, Trp and Tyr were selected
for conjugation. A more important gain would be that the Phe
coupled heterocycle (26) has shown improved activity which is
nearly one and half times more active than the standard drug used
against all the species tested. But to our surprise other two amino
acid conjugates 27 and 28 have shown moderate activity in spite of
being aromatic and also having indole group in Trp and phenolic
group in Tyr. Hence, the effect of substitution of Phe by other
aromatic amino acids is not salutatory.
In view of this, developing novel antimicrobial agents became
an area of great interest encompassing the concept of hydropho-
bicity and biocompatibility of elastin based peptide sequences with
varied chain length. Next we focused our attention on tetrapeptide
elastin sequences with varying hydrophobicity. Among the analogs
tested GGAP (29), GGIP (30) and GGFP (31), the latter has exerted
activity to its fullest i.e., GGFP which comprises of Phe has shown
activity which is almost two times more active than the standard.
But the remaining conjugates of tetrapeptide have exposed activity
not as much as GGFP which could be attributed to the hydropho-
bicity dependency. Thus the order of activity among tetrapeptide
conjugates is GGFP > GGIP > GGAP.
2.1. Chemistry
2.1.1. Peptide synthesis and characterization
Peptides were synthesized by classical solution phase method
employing Boc chemistry. 1H NMR data of the final protected
peptides (2e8) are given in Table 1. The C terminal protected benzyl
ester of these peptides was removed and then conjugated to ben-
zisoxazole derivative 1 using EDCI/HOBt as coupling agent and
NMM as base. The yields of the compounds obtained were found to
be >84% and were characterized by TLC, M.P., 1H NMR and mass
spectroscopic analyses. The physical and analytical data of the
conjugated compounds are provided in Table 2. The 1H NMR and
mass data were found to be in good agreement with the structures
assigned.
2.2. Biological studies
In the light of the above findings, our subsequent goal was to opt
pentapeptide elastin sequences GVGVP and GFGFP. The conjugates
of these two peptides have revealed the activity which is more than
The potentiality of the synthesized compounds as antimicro-
bials was appraised for their antibacterial studies against different

706
R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
Table 2
Physical and analytical data of the conjugated compounds (16e25).
Entry Rf value Yield (%) M.P.ꢀC
Theoretical Actual MS 1H NMR data (CDCl3,
d ppm)
mol. wt.
values (Mþ)
467.5
16
17
0.86
0.72
92.0
96.0
Gummy
108
468
Boc ¼ 1.44 (9H, s); Phe ¼ 3.64e3.73 (2H, d, ebCH2), 4.94 (1H, t, eaCH), 7.10e7.36 (5H, m, ArH),
8.11 (1H, s, eNH); Heterocycle ¼ 1.89 (4H, m, eCH2), 2.92 (1H, m, eCH), 3.60e3.61 (4H, m, eCH2),
7.10e7.36 (3H, m, ArH)
507
506.5
Boc ¼ 1.43 (9H, s); Trp ¼ 3.58e3.66 (2H, d, ebCH2), 4.95 (1H, t, eaCH), 7.04 (1H, s, eCH of indole),
7.11e7.49 (4H, m, ArH), 10.32 (1H, s, eNH of indole), 8.08 (1H, s, eNH);
Heterocycle ¼ 1.91e1.93 (4H, m, eCH2), 2.94 (1H, m, eCH), 3.66e3.67 (4H, m, eCH2),
7.11e7.49 (3H, m, ArH)
18
19
0.71
0.70
90.9
90.5
112
643
603
642.5
602.6
Boc ¼ 1.44 (9H, s); Tyr(2,6-Cl2-Bzl) ¼ 3.20e3.52 (2H, d, ebCH2), 4.88 (1H, t, eaCH),
5.24 (2H, s, eCH2 of side chain protecting group), 6.94e7.66 (7H, m, ArH), 8.00 (1H, s, eNH);
Heterocycle ¼ 1.89 (4H, m, eCH2), 2.98 (1H, m, eCH), 3.66 (4H, m, eCH2), 6.94e7.66 (3H, m, ArH)
Boc ¼ 1.40 (9H, s); eNH ¼ 8.08e8.15 (3H, s); Gly1 ¼ 3.87 (2H, s, eaCH); Gly2 ¼ 4.14 (2H, s, eaCH);
Ala3 ¼ 1.34 (3H, d, ebCH3), 4.62 (1H, m, eaCH); Pro4 ¼ 1.97e2.10 (2H, m, egCH2),
2.11e2.18 (2H, m, ebCH2), 3.64,3.79 (2H, m, edCH2), 4.60 (1H, m, eaCH);
82e85
Heterocycle ¼ 1.88,1.90 (4H, m, eCH2), 2.88 (1H, m, eCH), 3.60 (4H, m, eCH2), 6.98e7.49 (3H, m, ArH)
Boc ¼ 1.40 (9H, s); eNH ¼ 8.18e8.23 (3H, m); Gly1 ¼ 3.87 (2H, s, eaCH); Gly2 ¼ 4.14 (2H, s, eaCH);
Ile3 ¼ 0.84e1.06 (6H, m, e(CH3)2), 1.48 (2H, m, egCH2), 2.13 (1H, m, ebCH), 4.62 (1H, m, eaCH);
Pro4 ¼ 1.97e2.10 (2H, m, egCH2), 2.01e2.18 (2H, m, ebCH2), 3.64,3.79 (2H, m, edCH2),
4.60 (1H, m, eaCH); Heterocycle ¼ 1.88,1.90 (4H, m, eCH2), 2.88 (1H, m, eCH), 3.60 (4H, m, eCH2),
6.98e7.49 (3H, m, ArH)
20
21
22
23
24
0.75
0.74
0.64
0.60
0.72
86.0
88.1
89.1
88.5
84.3
70e72
80e82
645
679
644.7
678.7
729.4
825.8
3130.5
Boc ¼ 1.44 (9H, s); eNH ¼ 8.51e8.53 (3H, s); Gly1 ¼ 3.84 (2H, s, eaCH); Gly2 ¼ 4.13 (2H, s, eaCH);
Phe3 ¼ 3.62, 3.71 (2H, d, ebCH2), 4.96 (1H, t, eaCH); 6.99e7.26 (5H, m, ArH);
Pro4 ¼ 2.00e2.05 (2H, m, egCH2), 2.14, 2.17 (2H, m, ebCH2), 3.70e3.82 (2H, m, edCH2),
4.80 (1H, m, eaCH); Heterocycle ¼ 1.90e1.97 (4H, m, eCH2), 2.94 (1H, m, eCH), 3.57,
3.58 (4H, m, eCH2), 6.99e7.26 (3H, m, ArH)
119e121 730
125e127 826
178e181 3125
Boc ¼ 1.41 (9H, s); eNH ¼ 8.16e8.24 (4H, s); Gly1 ¼ 3.82 (2H, s, eaCH); Val2 ¼ 0.98 (6H, m, eCH3)2),
2.61 (1H, m, ebCH), 4.71 (1H, s, eaCH); Gly3 ¼ 4.13 (2H, s, eaCH); Val4 ¼ 0.97 (6H, m, eCH3)2),
2.66 (1H, m, ebCH), 4.92 (1H, s, eaCH); Pro5 ¼ 2.11e3.64 (6H, m, eCH2), 4.52 (1H, m, eaCH);
Heterocycle ¼ 1.98e1.99 (4H, m, eCH2), 2.89 (1H, m, eCH), 3.68, 3.70 (4H, m, eCH2),
7.11e7.64 (3H, m, ArH)
Boc ¼ 1.41 (9H, s); eNH ¼ 8.38e8.49 (4H, s); Gly1 ¼ 3.82 (2H, s, eaCH); Phe2 ¼ 3.51, 3.60 (2H, m, ebCH2),
4.71 (1H, s, eaCH), 7.11e7.64 (5H, m, ArH); Gly3 ¼ 4.13 (2H, s, eaCH); Phe4 ¼ 3.44, 3.63 (2H, m, ebCH2),
4.92 (1H, s, eaCH), 7.11e7.64 (5H, m, ArH); Pro5 ¼ 2.11e3.64 (6H, m, eCH2), 4.52 (1H, m, eaCH);
Heterocycle ¼ 1.98e1.99 (4H, m, eCH2), 2.89 (1H, m, eCH), 3.68, 3.70 (4H, m, eCH2),
7.11e7.64 (3H, m, ArH)
Boc ¼ 1.45 (9H, s); eNH ¼ 8.05e8.13 (24H, m); Gly ¼ 4.04, 4.80 (24H, m, eaCH); Val ¼ 1.06,
1.09 (36H, m, (eCH3)2), 2.45, 2.59 (6H, m, ebCH), 4.40 (6H, m, eaCH);
Glu ¼ 1.17e1.30 (10H, m, eCH2 of cyclohexyl ring), 2.09, 2.14 (4H, m, eb,gCH2),
3.73 (1H, m, eCH of cyclohexyl ring), 4.71 (1H, m, eaCH); Phe ¼ 3.51, 3.73 (10H, m, ebCH2),
4.19, 4.67 (5H, m, eaCH), 7.16e7.58 (25H, m, ArH); Pro ¼ 2.04, 3.62, 3.68 (36H, m, eCH2),
4.80 (6H, m, eaCH); Heterocycle ¼ 1.93, 1.96 (4H, m, eCH2), 2.98 (1H, m, eCH),
3.60, 3.67 (4H, m, eCH2), 7.16e7.58 (3H, m, ArH)
25
0.70
84.6
150e153 3125
3130.5
Boc ¼ 1.42 (9H, s); eNH ¼ 8.01e8.11 (24H, m); Gly ¼ 4.06, 4.85 (24H, m, eaCH); Val ¼ 1.04,
1.07 (36H, m, (eCH3)2), 2.44, 2.60 (6H, m, ebCH), 4.43 (6H, m, eaCH); Glu ¼ 1.11e1.24
(10H, m, eCH2 of cyclohexyl ring), 2.10, 2.15 (4H, m, eb,gCH2), 3.70 (1H, m, eCH of cyclohexyl ring),
4.69 (1H, m, eaCH); Phe ¼ 3.54, 3.75 (10H, m, ebCH2), 4.20, 4.61 (5H, m, eaCH), 7.19e7.69
(25H, m, ArH); Pro ¼ 2.00, 3.58, 3.71 (36H, m, eCH2), 4.78 (6H, m, eaCH); Heterocycle ¼ 1.95, 1.97
(4H, m, eCH2), 2.96 (1H, m, eCH), 3.62, 3.68 (4H, m, eCH2), 7.19e7.69 (3H, m, ArH)
Table 3
Inhibitory zone (diameter) mm of the synthesized conjugates (26e35) against tested bacterial and fungal strains by agar well diffusion method.
Entry
Antibacterial activity
Antifungal activity
Zone of inhibitiona (mm) ꢁ SD (n ¼ 3)
E. coli
X. oryzae
K. pneumoniae
C. positive staphylococcus
A. niger
A. flavus
F. oxysporum
26
27
28
29
30
31
32
33
14 ꢁ 0.57
08 ꢁ 0.40
07 ꢁ 0.35
18 ꢁ 0.50
20 ꢁ 0.32
22 ꢁ 0.36
23 ꢁ 0.45
25 ꢁ 0.28
15 ꢁ 0.35
12 ꢁ 0.36
03 ꢁ 0.20
11 ꢁ 0.26
e
11 ꢁ 1.00
06 ꢁ 0.36
05 ꢁ 0.12
13 ꢁ 0.51
15 ꢁ 0.43
16 ꢁ 0.43
17 ꢁ 0.21
20 ꢁ 0.43
18 ꢁ 0.25
14 ꢁ 0.05
04 ꢁ 0.05
08 ꢁ 0.11
e
10 ꢁ 0.50
05 ꢁ 0.55
04 ꢁ 0.50
12 ꢁ 0.60
14 ꢁ 0.55
15 ꢁ 0.41
17 ꢁ 0.15
18 ꢁ 0.25
17 ꢁ 0.30
11 ꢁ 0.20
03 ꢁ 0.10
08 ꢁ 0.15
e
09 ꢁ 0.76
04 ꢁ 0.50
04 ꢁ 0.25
11 ꢁ 0.40
12 ꢁ 0.04
14 ꢁ 0.55
15 ꢁ 0.40
18 ꢁ 0.07
20 ꢁ 0.61
16 ꢁ 0.25
03 ꢁ 0.15
07 ꢁ 0.47
e
16 ꢁ 0.61
11 ꢁ 0.70
10 ꢁ 0.56
19 ꢁ 0.45
22 ꢁ 0.55
25 ꢁ 0.40
27 ꢁ 0.20
29 ꢁ 0.17
36 ꢁ 0.15
34 ꢁ 0.20
03 ꢁ 0.20
e
11 ꢁ 0.35
04 ꢁ 0.05
03 ꢁ 0.55
13 ꢁ 0.15
14 ꢁ 0.15
16 ꢁ 0.26
17 ꢁ 0.32
21 ꢁ 0.35
32 ꢁ 0.51
28 ꢁ 0.30
02 ꢁ 0.21
e
14 ꢁ 0.15
07 ꢁ 0.47
06 ꢁ 0.47
16 ꢁ 0.24
18 ꢁ 0.47
20 ꢁ 0.60
21 ꢁ 0.37
23 ꢁ 0.45
31 ꢁ 0.34
28 ꢁ 0.25
04 ꢁ 0.13
e
34
35
Heterocycle
Amoxicillin
Bavistin
12 ꢁ 0.20
07 ꢁ 0.15
10 ꢁ 0.25
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases.

R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
707
Table 4
Minimum inhibitory concentration (MIC) in
mg/mL of the synthesized conjugates (26e35) against tested bacterial and fungal strains by microdilution method.
Entry
Antibacterial activity
Minimum inhibitory concentration (MIC) in
Antifungal activity
m
g/mLa
E. coli
X. oryzae
K. pneumoniae
C. positive staphylococcus
A. niger
A. flavus
F. oxysporum
26
27
28
29
30
31
32
33
20
38
40
17
15
14
11
08
10
12
>50
>50
24
e
15
26
29
13
12
11
10
09
09
10
>50
>50
18
e
16
31
34
14
12
11
08
06
09
10
>50
>50
20
e
18
36
39
16
14
13
10
08
11
13
>50
>50
23
e
18
32
34
15
13
11
08
06
03
04
>50
>50
e
22
41
43
18
16
15
09
08
03
04
>50
>50
e
21
39
41
19
17
16
10
08
04
05
>50
>50
e
34
35
Heterocycle
Peptides
Amoxicillin
Bavistin
22
26
25
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases.
two fold the conventional drugs used. Inspection of the results
further revealed that, irrespective of the same chain length of the
peptides presence of two more hydrophobic Phe units in 33 has
caused a slight increase in the activity compared to 32 which
comprises of two Val moieties which is relatively less hydrophobic.
It is also attractive to speculate the observations that the presence of
single Phe residue in 26 has shown enhanced activity (∼1.5 times)
while the retaining of only one Phe unit and increasing the peptide
chain length has caused further increase in the activity (∼two times)
which is a case of GGFP (31). Also the other two analogs of tetrapeptide
conjugates GGIP and GGAP have shown increased activity compared to
Phe alone (26). On the other hand, when two Phe groups were intro-
duced (greater hydrophobicity) as well as the peptide chain length
increased, GFGFP (33) which resulted in the activity more than two
times the standard drug used so also the case of GVGVP conjugate (32).
Hence, it can be inferred that as the length of the peptide chain as well
as the hydrophobicity increases the activity also increases. In short,
based on the length of the peptide chain and hydrophobicity, the order
of activity of the heterocyclic conjugated peptides is found to be
GFGFP > GVGVP > GGFP > GGIP > GGAP > Phe>Trp > Tyr.
by tricosamers 7 and 8 having improved hydrophobicity and also
a polar species, Glu in the chain. Tricosamers prepared elsewhere in
order to study the hydrophobicity-induced pKa shifts were used as
such. Theoutcome of the conjugationof thesetricosamers, 34and35
has yielded the antibacterial activity which is almost two times
greater than the standard drug. Though it is twice active, the activity
is less when compared to the GFGFP conjugate (33). This could be
due to the long peptide chain length in spite of being a more
hydrophobic entity and that would have resulted in the difficult
passage of the molecule across the cell membranes of bacteria.
Contrary to conventional wisdom, antifungal activity of the
tricosamers conjugated heterocycle has fetched a different set of
results. Serendipitously, we succeeded in identifying highly potent
antifungal agents of tricosamer conjugates which were able to
inhibit the growth of fungal species at 3e5
mg/mL which is five
times greater than the antibiotic used. Thus, both conjugates of
tricosamers might better qualify as highly potent antifungal agents.
Further insight into the results revealed that, among the two
conjugates of tricosamers, the former 34 has put forth a slight
enhancement over the latter (35). This may probably be due to
variations of the positions of Phe and Val units in the tricosamers
and also the distance as well as the charges present which would
Upon getting the interesting results, our quest for a more reliable
and suitable elastin based sequence for conjugation was portrayed
Gly
Gly
X
Pro
Gly
Pro
Gly
Z
Z
Boc
Boc
H
OH
H
OBzl
OBzl
OBzl
OBzl
Boc
Boc
H
OBzl
OH
iii
i
OBzl
ii
Boc
Boc
OBzl Boc
OBzl Boc
H
ii
iii
ii
OH
H
OBzl
OBzl
OH
Boc
Boc
OH
iii
iv
i
ii
Boc
OH
H
OBzl
Boc
Boc
Boc
OBzl
OH
H
iii
iv
Boc
Boc
OBzl
OH
iii
OBzl
iv
OH
Scheme 2. Schematic representation of the synthesis of pentapeptides Boc-GZGZ0P-
OH where Z ¼ Z0 ¼ Val for GVGVP; Z ¼ Z0 ¼ Phe for GFGFP; Z ¼ Val and Z0 ¼ Phe for
GVGFP; Z ¼ Glu(OcHx) and Z0 ¼ Phe for GE(OcHx)GFP by (3 þ 2) fragment coupling
method. i. IBCF/HOBt, NMM, ii. HCl/dioxane, iii. IBCF, NMM, iv. HCOONH4/10%Pd-C or
1N NaOH/MeOH.
Scheme 1. Schematic representation of the synthesis of tetrapeptides Boc-GGXP-OH
where X ¼ Ala for GGAP; Ile for GGIP and Phe for GGFP by stepwise approach. i. IBCF/
HOBt, NMM, ii. HCl/dioxane, iii. IBCF, NMM, iv. HCOONH4/10%Pd-C

708
R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
GE(OcHx)GFP
GVGVP
Boc
GVGVP
Boc
GVGFP
OBzl
GFGFP
GVGFP
H OBzl
OH
H
OH
i
i
Boc
OBzl
OBzl
OBzl
Boc
H
OBzl
OBzl
OBzl
OcHx
Boc
OH
ii
ii
Boc
H
OH
OcHx
Boc
i
i
Boc
H
OcHx
Boc
ii
iii
OH
OBzl
OBzl
OcHx
Boc
i
OcHx
Boc
iii
OH
Scheme 3. Schematic representation of the synthesis of tricosapeptides by [(5 þ 5 þ 5) þ (5 þ 5 þ 5)] fragment coupling method. i. EDCI/HOBt, NMM, ii. TFA, iii. 1N NaOH/MeOH.
F
F
HCl
+
HN
Boc
Xaa
Boc Xaa
COOH
CO
N
O
O
N
N
16-25
1
b
F
F
c
TFA .
NH2
Ybb
Boc
Ybb
CO
N
CO
N
O
O
N
N
26-35
Scheme 4. Schematic representation of the coupling of amino acids/peptides to heterocycle 1. Reagents and conditions: (a) EDCI/HOBt, NMM, 0 ꢀC, overnight at rt (b) Polymer
supported HCOOꢂNHþ3/10% PdeC (c) TFA, 40 min,rt. Xaa ¼ Phe, Trp, Tyr(2,6-Cl2-Bzl), GGAP, GGIP, GGFP, GVGVP, GFGFP, GE(OcHx)GFP GVGVP GVGVP GVGVP GFGFP GFGFP, GE
(OcHx)GFP GVGVP GVGFP GFGFP GVGVP GVGFP. Ybb ¼ Phe, Trp, Tyr, GGAP, GGIP, GGFP, GVGVP, GFGFP, GEGFP GVGVP GVGVP GVGVP GFGFP GFGFP, GEGFP GVGVP GVGFP GFGFP
GVGVP GVGFP.
facilitate to acquire better conformation of the molecule to trespass
microbial environment.
Finally our stress on the existing study is to ponder over the
consequences that may occur out of conjugation. In order to further
refine this theme interest, we conducted an experiment in which
equal amount of peptide and heterocycle were just mixed in
technique (>50 mg/mL). Our findings, thus, represent a break
through in the longstanding impasse on the effect of conjugation.
This observation provided the fact that the highly potent activity of
these analogs is not due to the additive effect irrespective of the
rank of hydrophobicity and length of the peptide chain and only it
is because of conjugative effect which would provide additional
interaction with the microbial cell membranes.
required volume of DMSO to get the concentration of 50 mg/mL and
performed the assay. The results revealed that there was only very
less or no inhibition of the growth of microorganisms by this
In summary, the present study involved the development of
several conjugates of elastin peptides and benzisoxazole moiety
Fig. 1. Diagramatic representation of antibacterial activity of the synthesized conju-
Fig. 2. Diagramatic representation of antibacterial activity of the synthesized conju-
gates by agar well diffusion method.
gates by microdilution method.

R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
709
Kentucky, USA). IBCF and NMM were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). All solvents and reagents used for
the synthesis were of analytical grade. All the chemicals and
reagents used for antimicrobial studies were of bacteriological grade
unless otherwise indicated. Nutrient broth and nutrient agar were
purchased from Hi-media chemicals (Mumbai, India). Silica gel
(60e120 mesh) for column chromatography was purchased from
Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). The pathogens
used for the microbial studies were obtained from a local hospital.
The progress of the reaction was monitored by TLC using silica gel
coated on glass plates with the solvent system comprising chloro-
form/methanol/acetic acid in the ratio 95:5:3 throughout the study
and the compounds on TLC plates were detected by iodine vapors.
Melting points were determined on a Thomas Hoover melting point
apparatus and are uncorrected. All the HPLC analyses were per-
formed with Lachrom-2000 Merck-Hitachi L7100 pump with
RP18.250e4 mm column and UV detector-UV-VISL 7400. 1H NMR
spectra were obtained on VARIAN 400 MHz instrument using CDCl3
Fig. 3. Diagramatic representation of antifungal activity of the synthesized conjugates
by agar well diffusion method.
and the chemical shifts are reported as parts per million (d ppm)
using TMS as an internal standard. Mass spectra were obtained on
LCMSD-Trap-XCT instrument.
4.2. Synthesis
The [3-(4-piperidyl)-6-fluoro-1,2-benzisoxazole]HCl
1 was
prepared as previously reported [20]. The peptides were
synthesized by classical solution phase method using Boc chem-
istry. The Boc group used for temporary Na protection was
removed with 4 N HCl in dioxane or TFA. The C terminus carboxyl
group was protected by the benzyl ester and its removal was
effected by hydrogenolysis using HCOONH4 as hydrogen donor
and 10% Pd on carbon as catalyst [21] or hydrolysed using 1 N
NaOH/MeOH. The hydroxyl group of Tyr was protected by 2,6-Cl2-
Bzl and g-carboxyl group of Glu was protected by cyclohexyl ester
Fig. 4. Diagramatic representation of antifungal activity of the synthesized conjugates
and removed by treatment with polymer supported HCOOꢂNH3þ,
10% PdeC [22]. All the coupling reactions for peptide synthesis
were achieved with IBCF. The procedure followed for the
synthesis of tetrapeptides, pentapeptides and tricosamers are
outlined in the Scheme 1e3 respectively. The protected peptides
were purified by column chromatography over silica gel and
characterized by physical and analytical techniques. The purity of
free peptides was checked by HPLC and found to have purity
>95% in each case.
by microdilution method.
having high potency as antimicrobials. Results of variation of
different amino acid residues in different positions and also the
chain length will be the subject of future reports.
3. Conclusion
Several amino acids/elastin based peptides were conjugated to
benzisoxazole derivative and some representatives of this series
were identified as novel and highly potent antimicrobial agents. Our
studies revealed that conjugation plays a paramount role in inhibit-
ing the growth of microorganisms tested. All the peptide conjugates
4.2.1. Synthesis of tetrapeptides
The tetrapeptide Boc-GGXP-OH [where X ¼ Ala (9), Ile (10) and
Phe (11)] was synthesized by a stepwise approach as shown in
Scheme 1. In this stepwise approach, Boc-X-Pro-OBzl (X ¼ Ala, Ile
and Phe) was synthesized by the mixed anhydride method in the
presence of HOBt, deblocked, and coupled with Boc-Gly-OH to
obtain Boc-Gly-X-Pro-OBzl. This was further deblocked, and
coupled with Boc-Gly-OH to obtain Boc-Gly-Gly-X-Pro-OBzl. This
was then hydrogenolysed to the corresponding free acid using
HCOONH4/10% PdeC.
have shownimprovedantimicrobialactivity(6e19
to their precursors tested alone (>50 g/mL) where as amino acids
conjugates have shown moderate activity (15e43 g/mL). Further,
conjugates of tricosamers have revealed extraordinary activity
(3e5 g/mL)against all thefungalspeciestestedwhichwerefoundto
mg/mL)compared
m
m
m
be almost five times more potent than the reference drug used. Thus,
these analogs could be regarded as most promising antimicrobial
agents and developed as lead.
4.2.2. Synthesis of pentapeptides
The pentapeptide Boc-GZGZ0P-OH [where Z ¼ Z0 ¼ Val for
GVGVP; Z ¼ Z0 ¼ Phe for GFGFP; Z ¼ Val and Z0 ¼ Phe for GVGFP;
Z ¼ Glu(OcHx) and Z0 ¼ Phe for GE(OcHx)GFP] was synthesized by
(3 þ 2) coupling strategy, the tripeptide Boc-Gly-Z-Gly-OH and the
dipeptide HCl$NH2-Z0-Pro-OBzl were synthesized separately and
coupled to obtain the pentamer as shown in Scheme 2. In this
approach, Boc-Z0P-OBzl was synthesized by the mixed anhydride
method in the presence of HOBt, deblocked to obtain HCl$NH2-Z0P-
OBzl. On the other hand, Boc-Z-Gly-OBzl was synthesized by the
4. Experimental
4.1. Materials
All the amino acids used except glycine were of L-configuration
unless otherwise mentioned. All Boc-amino acids, EDCI, HOBt and
TFA were purchased from Advanced Chem. Tech. (Louisville,

710
R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
mixed anhydride method, deblocked and coupled to Boc-Gly-OH to
obtain Boc-Gly-Z-Gly-OBzl, hydrogenolysed using HCOONH4/10%
PdeC or hydrolysed using 1 N NaOH/MeOH to get Boc-Gly-Z-Gly-
OH. Now the tripeptide and dipeptide fragments were coupled by
the mixed anhydride method to obtain Boc-Gly-Z-Gly-Z0-Pro-OBzl.
These were then treated with HCOONH4/10% PdeC or 1 N NaOH/
MeOH to get corresponding free acid.
under stirring while maintaining the temperature at 0 ꢀC. The
reaction mixture was stirred for an additional 10 min and pre-
cooled solution of [3-(4-piperidinyl)-6-fluoro-1,2-benzisoxazole]
HCl (1.285 g, 0.005 mol) and NMM (0.55 mL, 0.005 mol) in DMF
(13 mL) was added slowly (Scheme 4). After 20 min, pH of the
solution was adjusted to 8 by the addition of NMM and the reaction
mixture was stirred overnight at room temperature. DMF was
removed under reduced pressure and the residue was poured into
about 200 mL ice-cold 90% saturated KHCO3 solution and stirred for
30 min. The precipitated product was taken into CHCl3 and washed
with 5% NaHCO3 solution (2 ꢃ 20 mL), water (2 ꢃ 20 mL), 0.1 N cold
HCl solution (2 ꢃ 20 mL) and finally brine solution (2 ꢃ 20 mL). The
CHCl3 layer was dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The products so obtained were
recrystallized from ether/petroleum ether to get white colored
desired conjugates (16e25).
4.2.3. Synthesis of tricosamers
The tricosapeptides Boc-GE(OcHx)GFP GVGVP GVGVP GVGVP
GFGFP GFGFP-OH (14) and Boc-GE(OcHx)GFP GVGVP GVGFP GFGFP
GVGVP GVGFP-OH (15) were synthesized by the [(5 þ 5 þ 5) þ
(5 þ 5 þ 5)] fragment coupling strategy in solution phase as shown
in Scheme 3. The pentamers required for this purpose were
synthesized with appropriate side chain protection as described
above. Among the tricosamers 14 and 15, synthesis of 15 has been
given below and the synthesis of 14 was followed in a similar way.
In this approach, Boc-GVGVP-GVGFP-OBzl was synthesized using
EDCI/HOBt, deblocked, and coupled to Boc-GFGFP-OH to obtain
Boc-GFGFP-GVGVP-GVGFP-OBzl. This fragment was deblocked
further to get TFA.GFGFP-GVGVP-GVGFP-OBzl. In an another appr
oach, Boc-GVGVP-GVGFP-OBzl was synthesized using EDCI/HOBt,
deblocked, and coupled to Boc-GE(OcHx)GFP-OH to get Boc-GE
(OcHx)GFP-GVGVP-GVGFP-OBzl. This fragment was hydrolysed
using 1 N NaOH/MeOH to get Boc-GE(OcHx)GFP-GVGVP-GVGFP-
OH. Now the 15mer fragments were coupled using EDCI/HOBt to
get Boc-GE(OcHx)GFP GVGVP GVGFP GFGFP GVGVP GVGFP-OBzl,
hydrolysed using 1 N NaOH/MeOH to get Boc-GE(OcHx)GFP GVGVP
GVGFP GFGFP GVGVP GVGFP-OH.
4.2.7. Deprotection of 2,6-Cl2-Bzl of Tyr and OcHx of Glu of the
conjugates 18, 24 and 25
To a solution of the conjugates 18, 24 and 25 (0.001 mol) in
methanol (10 mL/g of compound), 10% PdeC (100 mg) and polymer
supported formate (1 g) were added and the mixture was stirred at
room temperature for 8 h. After completion of the reaction moni-
tored by TLC, catalyst and the polymer were filtered, washed with
methanol. The solvent was evaporated under reduced pressure and
the product was taken into CHCl3, washed with saturated NaCl and
the solvent was dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure and triturated with ether and
dried to get side chain deprotected conjugates.
4.2.4. General procedure for the hydrogenolysis of benzyl esters of
peptides
4.2.8. General procedure for the deblocking of Boc-Ybb-Heterocycle
Each conjugate (16e25) (0.001 mol) was deblocked with TFA
(10 mL/g of compound) for 40 min. The excess solvent was removed
under reduced pressure, triturated with ether, filtered, washed
with ether and dried under vacuum (yield 100%) to obtain
TFA$NH2-Ybb-Heterocycle (26e35) and these were used for the
antibacterial and antifungal studies.
Each peptide 2e6 (0.006 mol) was hydrogenolysed in methanol
(10 mL/g of peptide) using ammonium formate (2.0 equiv.) and 10%
PdeC (0.1 g/g of peptide) for 30 min at room temperature. The
completion of the reaction was monitored by TLC. The catalyst was
filtered and washed with methanol. The combined washings and
filtrate were evaporated and the residue taken into CHCl3, washed
with water and dried over anhydrous Na2SO4. The solvent was
removed under pressure and triturated with ether, filtered, washed
with ether and dried to obtain debenzylated peptides (9e13).
4.3. Biology
4.3.1. Antibacterial activity
4.2.5. General procedure for the hydrolysis of benzyl esters of
tricosapeptides
In vitro antibacterial activity was evaluated against human
pathogens of both gram positive organisms namely C. positive
staphylococcus and K. pneumoniae and gram negative organisms
namely X. oryzae and E. coli by agar well diffusion method (Fig. 1) as
well as microdilution method (Fig. 2).
Each tricosamer 7, 8 (0.006 mol) was hydrolysed in methanol
(10 mL/g of peptide) using cold solution of 1 N NaOH (30 mL) for
2 h. The completion of the reaction was monitored by TLC, solvent
was evaporated, cooled, neutralized with cold 1 N HCl, extracted
with CHCl3, washed with 1 N HCl followed by water and dried over
anhydrous Na2SO4. The solvent was removed under pressure and
triturated with ether, filtered, washed with ether and dried to
obtain debenzylated peptides (14, 15).
The C terminal free peptides so obtained were characterized by
Rf values and M.P. (ꢀC) and the data are as follows: (9) Rf 0.47; M.P.
67e69 (Lit. 66) [23] (10) Rf 0.54; M.P. 99 (Lit. 99) [23] (11) Rf 0.44;
M.P. 106e109 (Lit. 105) [23] (12) Rf 0.45; M.P. 125 (Lit. 127) [24] (13)
Rf 0.46; M.P. 117 (Lit. 118) [24] (14) Rf 0.48; M.P. 200e203 (15) Rf
0.46; M.P. 166e169.
4.3.1.1. Agar well diffusion method. The microorganisms were
inoculated in to the sterilized nutrient broth and maintained at
37 ꢀC for 24 h. On the day of testing, bacteria were subcultured
separately into 25 mL of sterilized nutrient broth. Inoculated sub-
cultured broths were kept at room temperature for the growth of
inoculums. Each test compounds (26e35) and standard drug
(amoxicillin) of 10 mg was dissolved in 10 mL of DMSO to get
a concentration of 1 mg/mL and further diluted to get a final
concentration of 50 mg/mL. About 15e20 mL of molten nutrient
agar was poured into each of the sterile plates. With the help of
cork borer of 6 mm diameter, the cups were punched and scooped
out of the set agar and the plates were inoculated with the
suspension of particular organism by spread plate technique. The
cups of inoculated plates were then filled with 0.1 mL of the test
solution, amoxicillin solution and DMSO (negative control). The
plates were allowed to stay for 24 h at 37 ꢀC and zone of inhibition
(mm) was then measured.
4.2.6. General procedure for the coupling of benzisoxazole
derivative 1 with Boc-Xaa-OH where Xaa ¼ Phe, Trp, Tyr(2,6-Cl2-
Bzl) and peptides 9e15
To Boc-Xaa-OH (0.005 mol) and HOBt (0.765 g, 0.005 mol) dis-
solved in DMF (10 mL/g of peptide) and cooled to 0 ꢀC was added
NMM (0.55 mL, 0.005 mol). EDCI (0.956 g, 0.005 mol) was added

R. Suhas et al. / European Journal of Medicinal Chemistry 46 (2011) 704e711
711
4.3.1.2. Microdilution method. All the microorganisms were grown
Acknowledgements
in Muller-Hinton broth. After cultivation for 16e18 h at 37 ꢀC, the
bacteria were harvested and their density was determined by
measuring O.D at A600. MIC of the compounds was determined by
agar dilution method. Suspension of each microorganism was
prepared to contain approximately (1 ꢃ 104e2 ꢃ 104 CFU/mL) and
applied to the plates with serially diluted compounds (dissolved in
DMSO) to be tested and also reference drug and incubated at 37 ꢀC
overnight. The minimum inhibitory concentration was considered
to be the lowest concentration that completely inhibited the
growth of microorganisms on the plates. Zone of inhibition (mm)
was measured after 24 h and MIC values were determined.
One of the authors RS gratefully acknowledges Lady Tata Memo-
rial Trust, Mumbai for the award of Junior Research Scholarship.
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species namely A. niger, A. flavus and F. oxysporum by agar well
diffusion method (Fig. 3) as well as microdilution method (Fig. 4).
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incubated for one day to obtain the inoculums. Each test
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