Design and synthesis of tryptophan containing peptides as potential analgesic and anti-inflammatory agents

Article abstract of DOI:10.1002/psc.2431

A new series of smaller peptides with tryptophan at C-terminal and varying N-protected amino acids/peptides were designed, synthesized and characterized by analytical and spectroscopic techniques. Analgesic and anti-inflammatory properties of these peptides were carried out in vivo using tail-flick method and carrageenan-induced paw edema method, respectively, at different doses and different time intervals. Most of the peptides synthesized displayed enhanced activity, and particularly tetra and hexapeptides 29-31 were found to be even more potent than the reference standards used. Moreover, some peptides have exhibited promising activity even after 24h of administration, whereas the reference standards were active only up to 3h. Further, the compounds did not present any ulcerogenic liability.

Full text of DOI:10.1002/psc.2431

Research Article
Received: 10 February 2012
Revised: 10 May 2012
Accepted: 6 June 2012
Published online in Wiley Online Library: 9 July 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2431
Design and synthesis of tryptophan
containing peptides as potential analgesic
and anti-inflammatory agents
R. Suhas and D. Channe Gowda*
A new series of smaller peptides with tryptophan at C-terminal and varying N-protected amino acids/peptides were designed,
synthesized and characterized by analytical and spectroscopic techniques. Analgesic and anti-inflammatory properties of
these peptides were carried out in vivo using tail-flick method and carrageenan-induced paw edema method, respectively,
at different doses and different time intervals. Most of the peptides synthesized displayed enhanced activity, and particularly
tetra and hexapeptides 29–31 were found to be even more potent than the reference standards used. Moreover, some peptides
have exhibited promising activity even after 24 h of administration, whereas the reference standards were active only up to 3 h.
Further, the compounds did not present any ulcerogenic liability. Copyright © 2012 European Peptide Society and John
Wiley & Sons, Ltd.
Supporting Information may be found in the online version of this article.
Keywords: indole; synthesis; peptides; analgesics; anti-inflammatory agents
canine kidney renal tubular cells [21]. These agents do not act
by inhibiting lipid metabolic enzymes. They are not steroids,
Introduction
Inflammation is a reaction of a part of the body to injury or infec-
tion, characterized by swelling, heat, redness and pain. Non-
steroidal anti-inflammatory drugs are among the most widely
prescribed drugs worldwide [1]. Through their anti-inflammatory,
antipyretic and analgesic activities, they represent a choice of
treatment in various inflammatory diseases such as arthritis,
rheumatisms as well as to relieve the aches and pain of everyday
life [2]. However, their use is often limited by the side effects they
produce, particularly in the gastrointestinal tract and the kidney
[3]. Hence, the search for new therapeutic agents with high
degree of safety and freedom from normally associated gastrointes-
tinal toxic effects such as ulceration, hemorrhage and perforation
has been a priority of chemists and pharmacologists [4].
nor do they increase the circulating levels of endogenous gluco-
corticoids. Instead, they block recruitment of neutrophils into
inflammatory lesions and, when studied in vitro, inhibited T-cell
activation [19].
Led by the previous facts and coupled with our ongoing
project aimed at investigating new bioactive molecules derived
from amino acid/peptide conjugates [22–26], the present work
involves the synthesis of a series of smaller peptides with Trp at
the C-terminal as common entity and varying different protected
amino acids/peptides at the N-terminal (Figure 1).
Materials and Methods
The discovery of indomethacin [5] as a successful agent for
clinical treatment on anti-inflammatory disorders has led to the
exploration of indole moiety to obtain better anti-inflammatory
agents. In particular, tryptophan (Trp) is an essential amino acid
and as such is a constituent of most proteins; it also serves as a
biosynthetic precursor for a wide variety of indole-containing
metabolites, which are involved in a large and essential number
of physiological functions [6]. Furthermore, the indole nucleus
has been reported as a common dominator of psychotropism
and is of great value in the field of medicine and biochemistry
[7,8]. This nitrogen heterocycle and its derivatives have occupied
a unique place in chemistry because of its varied biodynamic
properties such as anti-inflammatory [9,10], anticancer [11,12],
antidepressant [13], antimicrobial [14,15], antidiabetic [16] and
antimalarial [17].
General
All the amino acids used except glycine were of L-configuration
unless otherwise mentioned. All amino acids, EDCI, HOBt and
TFA were purchased from Advanced Chem. Tech. (Louisville,
Kentucky, USA). IBCF and NMM were purchased from Sigma
Chemical Co. (St. Louis, MO). All solvents and reagents used for
the synthesis were of analytical grade. Silica gel (60–120mesh) for
column chromatography was purchased from Sisco Research
*
Correspondence to: D. Channe Gowda, Department of Studies in Chemistry,
University of Mysore, Manasagangotri, Mysore 570 006, India. E-mail:
dchannegowda@yahoo.co.in
Department of Studies in Chemistry, University of Mysore, Manasagangotri,
Mysore 570 006, India
Several researchers have shown that N-Fmoc amino acids exert
anti-inflammatory activity by recruitment of neutrophils into the
inflammatory site [18–20]. In addition, these compounds also
increased intracellular Ca2+ concentrations in Madin Darby
Abbreviations: EDCI, 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide ꢀ HCl;
IBCF, Isobutyl chloroformate; Z, Benzyloxycarbonyl.
J. Pept. Sci. 2012; 18: 535–540
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

R. SUHAS AND D. C. GOWDA
triturated with ether, filtered, washed with ether and dried to
obtain carboxyl free peptides as hygroscopic foam (20–31).
Pharmacology
For analgesic activity, healthy adult Swiss albino mice of either
sex weighing 25–30 g were used. For anti-inflammatory activity,
healthy adult wistar rats of either sex weighing between 75–125 g
were used. Ibuprofen and indomethacin served as reference
standards for analgesic and anti-inflammatory activities, respec-
tively. All the animals were maintained as per the norms of the
Committee for the Purpose of Control and Supervision of Experi-
ments on Animal (CPCSEA), and the study protocols were
approved by CPCSEA and Institutional Animal Ethics Committee
constituted for the purpose. The significant difference between
groups was statistically analyzed by one-way ANOVA followed by
Dunnett’s t-test.
Figure 1. Design of C-terminal indole moiety containing peptides.
Laboratories Pvt. Ltd., (Mumbai, India). The progress of the reac-
tion was monitored by TLC using silica gel coated on glass plates
with the solvent system comprising chloroform/methanol/acetic
acid in the ratio 95 : 5 : 3 (Rfa)/90 : 10 : 3 (Rbf ). 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 performed with
Lachrom-2000 (USA) Merck-Hitachi L7100 pump with a column
of RP18.250–4 mm and UV detector-UV-VISL 7400. 1H NMR spec-
tra were obtained on 400 MHz instrument using CDCl3, and 13C
NMR spectra were recorded in DMSO-d6 using Varian 100 MHz
instrument (Palo Alto, CA, USA). The chemical shifts are reported
as parts per million (d ppm) using TMS as an internal standard.
Electrospray ionization mass spectrometry was performed on a
Bruker electrospray mass spectrometer. Elemental analysis was
performed using Vario EL III Elementar (Germany) and the values
are within ꢁ0.4% of the calculated ones.
Analgesic activity
The analgesic activity of the synthesized compounds was deter-
mined by the standard tail immersion method in mouse [27,28].
Albino mice of either sex weighing 25–30 g were divided into
groups of five animals each. At intervals, the mouse was held
so that its tail was totally immersed in a bath at the temperature
of 55 ꢁ 5 ꢂC. The time until the typical reaction – a violent jerk of
the tail was recorded and noted as the basal reaction time. The
animals were administered orally with control (DMSO), test
compounds (20–31, 24a, 29a) and ibuprofen (standard) at a dose
of 100 mg/kg body weight. The compounds that showed potent
activity at 100 mg/kg body weight were treated with low dosage
such as 25 mg/kg and 50 mg/kg body weight. Time taken for tail
withdrawal response was recorded at 30 min, 1, 2, 3, 4 and 24 h
after administering the compounds. Increase in reaction time
(interval for withdrawal of tail by the animal) was considered
as an analgesic activity. The percentage analgesic activity was
calculated by using the formula,
Chemistry
General procedure for the coupling of Y-NH-Xaa-OH where Y = Fmoc,
Z, Boc; Xaa = Gly, Pro, Phe, Lys(Boc), His(Trt), Glu(OBut), GFG, GVGVP
and GFGFP with HClꢀ H-Trp-OMe (7)
ꢀ
ꢁ
To Y-NH-Xaa-OH (0.005 mol) and HOBt (0.765 g, 0.005 mol)
dissolved in DMF (10 ml/g of compound) and cooled to 0 ꢂC was
added NMM (0.55 ml, 0.005 mol). EDCI (0.956 g, 0.005mol) was
added under stirring while maintaining the temperature at 0ꢂC.
The reaction mixture was stirred for 10 min, and a pre-cooled solu-
tion of HCl ꢀ H-Trp-OMe (1.274 g, 0.005mol) and NMM (0.55 ml,
0.005mol) in DMF (13 ml) was added slowly. After 20 min, pH of
the solution was adjusted to 8 by the addition of NMM, and the
reaction mixture was stirred over night 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 30min. The precipitated peptide was taken into
CHCl3 and washed with 5% NaHCO3 solution (2ꢀ 30 ml), water
(2ꢀ 30ml), 0.1N cold HCl solution (2ꢀ 30 ml) and finally brine
solution (2ꢀ 30 ml). The CHCl3 layer was dried over anhydrous
Na2SO4, and the solvent was removed under reduced pressure.
The products (8–19) so obtained were recrystallized from ether/
petroleum ether.
T2 ꢃ T1
80 ꢃ T1
%Analgesic activity ¼
ꢀ 100
where T1 is reaction time before treatment, T2 is reaction time
after treatment and 80 is the cut-off time.
Potency of the tested compounds was calculated relative to
ibuprofen ‘reference standard’ treated group according to the
following equation.
ꢀ
ꢁ
%analgesic activity of test compound treated group
Potency ¼
%analgesic activity of ibuprofen treated group
Anti-inflammatory activity
The anti-inflammatory activity was evaluated using carrageenan-
induced rat hind paw edema method [29]. The animals fasted for
24 h were divided into control, standard and test groups each
consisting of five rats. The first group of rats was treated with
DMSO (control), second group was administered orally with a
dose of 100 mg/kg of indomethacin (standard), and test groups
(20–31, 24a, 29a) were treated with 100 mg/kg of test
compounds orally. The compounds that showed potent activity
at 100 mg/kg body weight were treated with low dosage such
as 25 mg/kg and 50 mg/kg body weight. After 30 min, the animals
were injected subcutaneously with 0.1 ml of freshly prepared
suspension of carrageenan solution (1% in 0.9% saline) into the
General procedure for the hydrolysis of methyl esters of 8–19
Each peptide (8–19) (0.003 mol) was hydrolysed in methanol
(10 ml/g of peptide) using cold solution of 1 N NaOH (15 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
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SYNTHESIS OF TRYPTOPHAN CONTAINING PEPTIDES
subplantar region of the right hind paw of rats. The volume of
hind paw was measured using vernier calipers both in control
and in animals treated with standard and test compounds at
30 min, 1, 2, 3, 4 and 24 h after carrageenan challenge. The initial
paw volume was measured within 30 s of the injection. The anti-
inflammatory activity was expressed as a percentage inhibition
of the inflammation in treated animals in comparison with the
control group [30].
ꢀ
ꢁ
Vt
%inhibition of edema ¼ 1 ꢃ
ꢀ 100
Vc
where Vc and Vt are the mean relative changes in the volume of
paw edema in the control and test-treated groups, respectively.
Potency of the tested compounds was calculated relative to
indomethacin ‘reference standard’ treated group according to the
following equation.
i. IBCF/ HOBt, NMM
ii. HCl/ dioxane
ꢀ
ꢁ
iii. IBCF, NMM
%edema inhibition of test compound-treated group
%edema inhibition of indomethacin-treated group
Protency ¼
iv. HCOONH4/ 10% Pd-C
Scheme 1. Schematic representation of the synthesis of Boc-GFG-OH,
Boc-GVGVP-OH and Boc-GFGFP-OH.
Ulcerogenic activity
Adult albino rats of either sex were fasted 24 h prior to the admin-
istration of compounds. Water was allowed ad libitum to the
animals. The compounds (which have shown promising anti-
inflammatory activity) and indomethacin were given orally and
the animals sacrificed 8 h after treatment. The stomach, duodenum
and jejunum were removed and examined with a hand lens for any
evidence of (i) shedding of epithelium (ii) red spots below skin and
bleeding and (iii) erosion or discrete ulceration with or without
perforation. The presence of any one of these was considered to
be an evidence of ulcerogenic activity [31].
Results and Discussion
Chemistry
The peptides Boc-GFG-OBzl, Boc-GVGVP-OBzl and Boc-GFGFP-OBzl
were synthesized by solution phase approach using inexpensive
IBCF/HOBt as coupling agent and NMM as base (Scheme 1). Trp
was converted to its methyl ester using SOCl2/MeOH, and its forma-
tion was confirmed by 1H NMR, which showed absence of ꢃCOOH
peak and the presence of a methyl ester peak at d 3.52. Further, this
compound was used for coupling with various N-protected amino
acids/peptides using EDCI/HOBt as coupling agent and NMM as
base (Scheme 2). All the products obtained presented characteristic
1H NMR signals for COOMe, aCH, NH and indole-NH at ~ d 3.50, 4.00,
8.00 and 10.90, respectively. The analytical and spectroscopic data
of the peptides (1–3/8–19) are provided in the supporting informa-
tion. Further, all the peptides synthesized were confirmed by mass
Scheme 2. Coupling of N-protected amino acids/peptides to 7 and
saponification where Y = Boc, Fmoc, Z; Xaa = Gly, Pro, Phe, Lys(Boc), His
(Trt), Glu(OBut), GFG, GFGFP, GVGVP. Include the reagents and conditions
here which is found below.
spectra and elemental analysis, which are in good agreement with
their structure.
Reagents and conditions: (a) SOCl2/MeOH, reflux, 4–5 h (b)
EDCI/HOBt/NMM, 0 ꢂC, overnight at rt (c) 1 N NaOH/MeOH, 0 ꢂC,
2 h at rt.
Comp. No.
Sequence
Comp. No.
Sequence
8/20
Fmoc-Gly-Trp-OMe/OH
Fmoc-Pro-Trp-OMe/OH
Fmoc-Phe-Trp-OMe/OH
Fmoc-Lys(Boc)-Trp-OMe/OH
Fmoc-His(Trt)-Trp-OMe/OH
Z-Gly-Trp-OMe/OH
14/26
15/27
16/28
17/29
18/30
19/31
Z-His(Trt)-Trp-OMe/OH
9/21
Boc-Gly-Trp-OMe/OH
10/22
11/23
12/24
13/25
Boc-Glu(OBut)-Trp-OMe/OH
Boc-Gly-Phe-Gly-Trp-OMe/OH
Boc-Gly-Phe-Gly-Phe-Pro-Trp-OMe/OH
Boc-Gly-Val-Gly-Val-Pro-Trp-OMe/OH
J. Pept. Sci. 2012; 18: 535–540 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

R. SUHAS AND D. C. GOWDA
Pharmacology
that basic amino acids have pronounced effects on the activity
[18], whereas the other Fmoc amino acids have shown moderate
results. In contrast, when an acidic amino acid, i.e. Glu(OBut) (28)
was present, it resulted in lower activity.
Analgesic and anti-inflammatory activities
Analgesic and anti-inflammatory activities of the synthesized
compounds were determined in vivo by tail-flick method in mice
and acute carrageenan-induced paw edema method in rats,
respectively, at a dose of 100 mg/kg body weight (Figures 2 and
3). The analgesic and anti-inflammatory properties were recorded
at successive time intervals of 30min, 1, 2, 3, 4 and 24 h and
compared with that of ibuprofen and indomethacin, respectively,
which were used as reference standards. When N-protected amino
acids/peptides alone (without Trp-OMe) were subjected to analge-
sic and anti-inflammatory activities, they showed ~10% and 20%
activity at 3-h interval, respectively. Hence, we intended to include
different amino acids/peptides at Xaa region of the designed
analogs (Figure 1). It was noted that the presence of methyl ester
at the C-terminal of the peptides did not present significant results.
Hence, methyl ester of the peptides was removed to obtain
corresponding free acid, which were used for the activity. From
the results, it was observed that the test compounds revealed
significant analgesic and anti-inflammatory properties.
In this context, we thought of replacing Fmoc by Z and Boc
groups, which are also important in peptide synthesis. It was
observed that there was a slight loss in the activity when Fmoc
was replaced by Z. This could be due to the presence of strong
aromatic fluorene ring in Fmoc when compared with the
presence of benzene in Z. Further decrease in the activity was
observed when Fmoc was replaced by aliphatic Boc. This observa-
tion was supported by His(Trt) (24> 26) and Gly (20> 25> 27)
containing peptides, i.e. Fmoc > Z > Boc. This fact revealed that
aromatic groups at the N-terminus play a major role. Further, to
obtain insight into the essentiality of protecting group at the N-
terminus, Boc of compound 29 was treated with TFA to obtain
29a, which resulted in decreased activity. This fact revealed that
substitution at the N-terminal is essential. Thus, structure–activity
relationships based on the observed data explain that the sub-
stitution at N-terminus has a significant role in developing the
pharmacological activities.
Among the N-Fmoc containing dipeptides such as Gly (20), Pro
(21), Phe (22), Lys(Boc) (23) and His(Trt) (24), compound 24 has
shown high activity, which could be due to aromaticity of His
as well as trityl group, acid–base character and H-binding affinity
of His. This is followed by Lys(Boc). This observation suggested
In this perspective, our next goal was to extend the length of
the peptide chain. It was noticed that increasing the peptide
chain length increased the pharmacological profile of the synthe-
sized peptides [23,24]. This fact is substantiated by the evidence
that the tetrapeptide GFGW (29) has exhibited 65% activity,
Figure 2. Diagramatic representation of the analgesic activity of the compounds.
Figure 3. Diagramatic representation of the anti-inflammatory activity of the compounds.
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SYNTHESIS OF TRYPTOPHAN CONTAINING PEPTIDES
whereas the hexapeptides have shown 71–73% activity. Further-
more, among the hexapeptides, GVGVPW (31) has demonstrated
better therapeutic activity over GFGFPW (30), although the latter
contains more hydrophobic Phe units. Subsequently, we focused
our attention on the importance of side-chain protecting groups.
When trityl group of compound 24 was removed, it resulted
in the decreased activity (24a). This highlighted the necessity
of the side-chain protecting group in exerting better pharma-
cological properties.
The activity of the most potent compounds was further
challenged through reduced doses such as 25 and 50-mg/kg body
weight, which is in accordance with the literature [6]. It was
observed that 100-mg/kg body weight is required to exhibit potent
activity (Figures 4 and 5). The synthesized peptides have exhibited
maximum reduction in the paw volume at 3-h interval and
decreased further (from 4 h). This implies that the peptides can
act against the inflammatory lesions produced and render protec-
tion up to 3 h from the time of administration. On the other hand,
an additional impetus to the present investigation is that some of
the synthesized peptides showed better results than the reference
standards even after 24 h of administration (Figures 6 and 7), i.e.
peptides have the ability to provide protection and act as better
pharmacological agents compared with the available compounds
in the market. A possible explanation for this observation would
Figure 6. Comparative diagramatic representation of analgesic activity
of the compounds at different time intervals.
Figure 7. Comparative diagramatic representation of anti-inflammatory
activity of the compounds at different time intervals.
be a better biocompatibility and bioavailability in the case of the
peptides and also amino acids [32,33].
Ulcerogenic activity
Considering the potent compounds of the series, they were
studied for ulcerogenic activity. The result clearly showed that the
compounds were less ulcerogenic and devoid of any evidences of
shedding of epithelium/red spots below skin/bleeding/erosion
and so on.
Figure 4. Diagramatic representation of the analgesic activity of the
potent compounds at different doses at 3-h interval.
Conclusion
In short, for the first time, we have developed a novel series of
peptides, which demonstrated significant pharmacological
properties. It was found that Fmoc group is essential for exerting
potent activity. Also, presence of bulky protecting groups at the
side-chain of the amino acids has a role in exhibiting the activity.
This study revealed that the activity is dose as well as time depen-
dent. The compounds showed highest activity at 3-h interval and
gradually decreased, which is in correlation with the standards
used. On the other hand, some peptides have exhibited promis-
ing activity even after 24 h of treatment, whereas the reference
standards were active only up to 3 h. Further, as the length of
the peptide chain increases, activity also increases. This article
also reports that peptides containing Z/Boc groups at the
Figure 5. Diagramatic representation of the anti-inflammatory activity of
the potent compounds at different doses at 3-h interval.
J. Pept. Sci. 2012; 18: 535–540 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

R. SUHAS AND D. C. GOWDA
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One of the authors, R. S., gratefully acknowledges Lady Tata
Memorial Trust, Mumbai for the award of Research Scholarship.
Also, the authors would like to thank Central Animal Facility,
University of Mysore, Mysore for extending the support to carry
out the pharmacology experiments.
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