Indole based peptidomimetics as anti-inflammatory and anti-hyperalgesic agents: Dual inhibition of 5-LOX and COX-2 enzymes

Article abstract of DOI:10.1016/j.ejmech.2015.04.044

The indoles bearing a tosyl group at N-1 and a dipeptide substituent at C-3 were screened for anti-inflammatory and anti-hyperalgesic activities. Some of the compounds made significant reduction in the dextran induced swelling and capsaicin induced pain in the albino mice. About 95% reversal in capsaicin induced pain occurred in the presence of 5 mg kg^-1of compound 7b, 7d and 7h while diclofenac showed 90% reversal when its 10 mg kg^-1 dose was used. In order to examine the mode of action of these compounds; COX-1, COX-2 and 5-LOX enzyme immunoassays were performed. The IC₅₀ of compound 7b for COX-2 and 5-LOX were in the nM range: 5-LOX, IC₅₀ Combining double low line 2.0 nM; COX-2, IC₅₀ Combining double low line 6.3 nM, selectivity for COX-2 over COX-1 was 351. The interactions of the compounds with COX-2 and 5-LOX were supported by the physical parameters including K_i, K_a and δ G. The most potent compounds 7b, 7d and 7h showed no toxicity to the animals and were identified as the promising leads for anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2015.04.044

Accepted Manuscript
Indole Based Peptidomimetics as Anti-inflammatory and Anti-hyperalgesic Agents:
Dual Inhibition of 5-LOX and COX-2 Enzymes
Palwinder Singh, Parteek Prasher, Parvirti Dhillon, Rajbir Bhatti
PII:
S0223-5234(15)30007-6
10.1016/j.ejmech.2015.04.044
EJMECH 7859
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2015
Revised Date: 10 April 2015
Accepted Date: 20 April 2015
Please cite this article as: P. Singh, P. Prasher, P. Dhillon, R. Bhatti, Indole Based Peptidomimetics
as Anti-inflammatory and Anti-hyperalgesic Agents: Dual Inhibition of 5-LOX and COX-2 Enzymes,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.044.
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ACCEPTED MANUSCRIPT
Indole Based Peptidomimetics as Anti-inflammatory
and Anti-hyperalgesic Agents: Dual Inhibition of 5-
LOX and COX-2 Enzymes
,a
a
b
b
Palwinder Singh,* Parteek Prasher, Parvirti Dhillon, Rajbir Bhatti
a
UGC Sponsored Centre for Advanced Studies, Department of Chemistry, Guru Nanak Dev
b
University, Amritsar – 143005, India; Department of Pharmaceutical Sciences, Guru Nanak
Dev University, Amritsar – 143005, India
Graphical Abstract
Presence of both Gly and Trp residues at C-3 substituent of N-tosylated indole increased the anti-
inflammatory activity of the molecules.
1

ACCEPTED MANUSCRIPT
Indole Based Peptidomimetics as Anti-inflammatory
and Anti-hyperalgesic Agents: Dual Inhibition of 5-
LOX and COX-2 Enzymes
,a
a
b
b
Palwinder Singh,* Parteek Prasher, Parvirti Dhillon, Rajbir Bhatti
a
UGC Sponsored Centre for Advanced Studies, Department of Chemistry, Guru Nanak Dev
University, Amritsar – 143005, India
b
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar – 143005, India
KEYWORDS. Peptidomimetics; Anti-inflammatory; Anti-hyperalgesic; COX-2, 5-LOX
inhibitors.
ABSTRACT. The indoles bearing a tosyl group at N-1 and a dipeptide substituent at C-3 were
screened for anti-inflammatory and anti-hyperalgesic activities. Some of the compounds made
significant reduction in the dextran induced swelling and capsaicin induced pain in the albino
-1
mice. About 95% reversal in capsaicin induced pain occurred in the presence of 5 mg Kg of
-1
compound 7b, 7d and 7h while diclofenac showed 90% reversal when its 10 mg Kg dose was
used. In order to examine the mode of action of these compounds; COX-1, COX-2 and 5-LOX
enzyme immunoassays were performed. The IC of compound 7b for COX-2 and 5-LOX were
5
0
in the nM range: 5-LOX, IC = 2.0 nM; COX-2, IC = 6.3 nM, selectivity for COX-2 over
5
0
50
COX-1 was 351. The interactions of the compounds with COX-2 and 5-LOX were supported by
2

ACCEPTED MANUSCRIPT
the physical parameters including K , K and ∆G. The most potent compounds 7b, 7d and 7h
i
a
showed no toxicity to the animals and were identified as the promising leads for anti-
inflammatory drugs.
3

ACCEPTED MANUSCRIPT
INTRODUCTION
The discovery of arachidonic acid metabolism [1] helped in understanding the root cause for
several inflammatory diseases [2]. The arachidonic acid cascade catalyzed by the enzymes
lipoxygenases [3] and cyclooxygenases [4] for the biosynthesis of leukotrienes [5] and
prostaglandins [6], respectively is responsible for the chronic inflammation in humans. The
consequent ailments are expressed in the form of asthma [7], atherosclerosis [8], irritable bowel
syndrome [9], rheumatoid arthritis [10] and cancer [11]. Hence, the continuous threat of the
severe inflammation problems is making the medicinal research to focus on exploiting new
treatment strategies. Targeting the induced isoform of cyclooxygenase, viz. cyclooxygenase-2
(
COX-2), the anti-inflammatory medication [12] made a long journey from SAIDs’ and NSAIDs’
to COXIBS [13]. Likewise, the consistent efforts have made zileuton [14], MK885 and MK0591
15] (Chart 1) to reach to the market for the treatment of inflammatory diseases through the
[
inhibition of LOX enzymes. Irrespective of the development of a number of new chemical
entities targeting COX-2 and LOX enzymes, the progress of these compounds towards clinical
stage is hampered by their severe cardiovascular side effects [16]. Rofecoxib and valdecoxib
were even withdrawn from the market due to these limitations [17].
Recently, we reported cyclic amine / amino acid appended indoles as highly effective 5-LOX
inhibitors [18, 19]. Compounds 4 and 5 (Chart 1) have shown appreciable IC for 5-LOX and
5
0
also exhibited significant anti-inflammatory activities. Compound 5 was also found to inhibit
COX-2 enzymatic activity with IC 9 – 13 μM. The advantage of developing the new drugs by
5
0
the modification of the existing ones [20] and the benefits of the practice of polypharmacology
21] made us to refine the structures of compounds 4 and 5 so that their anti-inflammatory
[
activity gets improved. Since the activity of compounds 5, carrying one amino acid at C-3
4

ACCEPTED MANUSCRIPT
substituent, was improved over that of compounds 4; it was planned to extend the amino acid
chain at the C-3 substituent of compound 5 and thereby compounds 6 and 7 (Chart 1) were
designed. Care was taken that LogP and total polar surface area (TPSA) of the compounds are
within the range of Lipinski values when amino acids were chosen for the extension of C-3
substituent in compounds 6.
<
Chart 1>
2
. Results and Discussion
.1. Selection of the compounds
2
Amongst the previous set of compounds [18], those having Gly and Trp residues as a part of
their C-3 substituent showed appreciable inhibition of 5-LOX. It was envisaged that the
combination of these amino acids in one molecule may increase its anti-inflammatory potency.
Hence, introducing Gly – Trp dipeptide at the C-3 substituent and the possible stereoisomers led
to the design of compounds 6a – d. In order to increase the variation, His and Glu based
dipeptide was also introduced at C-3 position of indole, thus compounds 6e – h were
synthesized. Compounds 7, the respective acids of compounds 6, were also included for the
biological screening (Chart 2).
<
Chart 2>
Compounds 6a – h and 7a – h were docked in the active site of 5-LOX and COX-2 (Table S1 –
S3). Also, the LogP and TPSA of these compounds were calculated [22] (Table S4) which were
found to be within the limits of Lipinski values. It was noticed that the isomers of compounds 6
and 7 with S- configuration at the C position of amino acid/s exhibited better interactions
α
(docking score) when docked in the active site of 5-LOX and COX-2 enzymes (Table S1 – S3).
Hence, compounds 6b, 6d, 6h and their corresponding acids 7b, 7d, 7h were selected as the
5

ACCEPTED MANUSCRIPT
potential candidates for the present studies. For making the comparison, compounds 6a, 6c, 6e,
6
2
i and 7a, 7c, 7e, 7i were also synthesized and included in the biological assays.
.2. Synthesis
Oxalyl chloride was slowly added to the ice cooled solution of indole (8, Scheme 1) in dry ether.
The yellow colored solid (9) was filtered and dissolved in dry acetonitrile (ACN) followed by the
addition of K CO The appropriate L-amino acid ester hydrochloride, after neutralization with
2
3.
triethylamine, was added to the ACN solution of compound 9. All additions to the reaction
o
mixture were made at 0 C. C-3 Substituted indoles 10 – 12 (Scheme 1) were purified through
column chromatography using ethyl acetate – hexane as the eluent.
<
Scheme 1>
After washing with dry hexane, NaH (1 mmol) was suspended in dry ACN. Compound 10 (1
mmol) and p-toluenesulphonyl chloride (1.2 mmol) were added to the NaH suspension in ACN
o
keeping the reaction temperature 0 – 5 C. On usual work up of the reaction mixture, compound
1
3 was isolated. Using the same reaction conditions, tosyl group was introduced at N-1 of
compounds 11 and 12 and respective compounds 14 and 15 were procured (Scheme 2). Further
treatment of compounds 13 – 15 with 1N NaOH in acetone – H O (1:1) provided compound 16 –
2
1
8 (Scheme 2).
<
Scheme 2>
In order to introduce second amino acid; the solution of compound 16, triethyl amine and
o
ethyl chloroformate in dry DCM at 0 C was treated with neutralized solution of glycine methyl
ester hydrochloride. The coupling reaction was completed in 25 – 30 min and compound 6i was
isolated (Scheme 3). Similarly, compound 6d, 6b and 6h were synthesized from compound 16,
1
7 and 18, respectively.
6

ACCEPTED MANUSCRIPT
<
Scheme 3>
Compounds 6 were converted into corresponding compounds 7 by hydrolyzing the ester
component with 1N NaOH (Scheme 4). The optical purity of compounds 7 was checked by
®
recording their LC-MS using chirobiotic T 10 µm chiral HPLC column. Synthesis of
compounds 6a, 6c, 6e and their corresponding acids 7 is given in the supplementary data.
<
Scheme 4>
3
3
. Biological studies
.1. Anti-inflammatory and anti-hyperalgesic activity
The anti-inflammatory and anti-hyperalgesic activities of the compounds were checked on
Swiss albino mice (25-35 g) of either sex [23, 24]. The animals were divided into different
groups of five each. Group I, the control, comprised of animals treated with vehicle, and group II
-1
was treated with standard reference drug – diclofenac (10 mg kg ). All the test compounds were
-1
administered at two doses of 5 and 10 mg Kg . Compound 7i was given to groups III, IV; 7d to
groups V, VI; 7b to groups VII, VIII and groups IX, X were given 7h at both doses,
intraperitoneally. Dextran was administered at a dose of 1% by subplantar injection 30 min after
the respective treatments and paw thickness was measured using vernier calipers after 1, 2, 3, 4,
6
and 24 h of dextran injection.
Anti-inflammatory activity of 7b and 7h was observed to be most significant in comparison to
-1
the control. For 7b, the dose of 5 mg Kg produced 47% reduction in the paw thickness (w.r.t.
-1
control) which further improved to 49% on increasing the dosage to 10 mg Kg at 4h (Figure 1).
-1
Similar to the effect shown by compound 7b, 5 mg Kg dose of compound 7h caused 45%
-1
reduction in the paw thickness after 4h which was improved to 48% when 10 mg Kg dose was
used. The effect of both 7b and 7h was comparable to that of diclofenac (42%).
7

ACCEPTED MANUSCRIPT
<
Figure 1>
For studying the anti-hyperalgesic effect of the compounds, 20 µL of capsaicin was injected
into the plantar surface of the right hind paw. Animals were observed individually for 10 min
after capsaicin administration and the number of paw lickings and twitchings was recorded as an
indicator of hyperalgesia. Animals were divided into 10 groups of 5 each. All treatments were
administered intraperitoneally 30 min before capsaicin injection. Group I was the control group
in which the animals were injected vehicle; in group II, the animals were injected diclofenac at a
-1
dose of 10 mg Kg . Group III and IV animals were injected compound 7i at doses of 5 and 10
-1
mg Kg , respectively; group V and VI animals were injected compound 7d at doses of 5 and 10
-1
mg Kg , respectively; group VII and VIII animals were given compound 7b at doses of 5 and 10
-1
mg Kg , respectively and group IX and X animals were injected compound 7h at doses of 5 and
-1
1
0 mg Kg , respectively.
Compounds 7b, 7d, 7h and 7i were found to have a significant anti-hyperalgesic effect at dose
-1
-1
of 5 mg Kg and a further increase in dose (10 mg Kg ) of compound 7i, 7b and 7h did not
result in corresponding increase in anti-hyperalgesic effect whereas compound 7d showed an
-1
appreciable increase in the anti-hyperalgesic effect at dose 10 mg Kg (Figure 2). The number of
paw lickings was remarkably reduced by 96% (w.r.t. control) on treatment with compound 7b. A
similar effect was observed in the presence of compound 7h (95%) and 7i (90%). Unlike other
compounds, amplification in the analgesic effect when increasing the dose was observed for
-1
compound 7d which made 88% reduction in paw lickings at 5 mg Kg and 96% reduction at 10
-1
mg Kg . Interestingly, all the compounds showed slightly better but not statistically significant
anti-hyperalgesic response as compared to diclofenac (90%).
<
Figure 2>
8

ACCEPTED MANUSCRIPT
3
.2. Toxicity
Studies for checking acute toxicity of the most active compound 7b were conducted as per
OECD guidelines- 423 (OECD, 2001) [25]. Three groups of three animals in each group were
administered compound 7b at 5 mg, 50 mg and 300 mg dose. The animals were observed
continuously for the first 4 h for any signs of toxicity. Thereafter, the animals were observed at
regular intervals for the first 24h followed by once daily for the next 14 days. After 14 days, the
histological studies on the liver, kidney and heart did not show any signs of behavioural
abnormality. The histological studies also did not show any gross structural variations as
compared to the control (Figure S113).
4
. Mechanistic investigation for the mode of action of the compounds
Prostaglandins and leukotrienes, the metabolites of arachidonic acid, generated by the
cyclooxygenase (COX) and lipooxygenase (LOX), respectively are amongst the major culprits in
mediating pain and inflammation. With due support from the results of the docking studies, the
compounds which showed best anti-inflammatory and anti-hyperalgesic activities were checked
for the inhibition of COX and LOX enzymes.
4
.1. 5-LOX inhibitory activities
-4
-8
Compounds 6 and 7 at 10 M – 10 M concentrations were examined for the inhibition of
5
-LOX enzymatic activity [26]. Similar to the previous observations [18]; compounds 7, the
corresponding acids of compounds 6, exhibited better IC for 5-LOX. Compounds 7b and 7d
5
0
having Gly-Trp dipeptide exhibited IC 0.002 μM and 0.004 μM, respectively against 5-LOX
5
0
which was considerably improved with comparison to compounds 16 and 17 [18]. Compound
h with His-Glu dipeptide has IC50 0.08 µM which was also improved over the parent
7
compound 18, the single amino acid homologue of 7h having His as a part of its C-3
9

ACCEPTED MANUSCRIPT
substituent. Compound 7i, containing Gly-Gly dipeptide showed IC 0.005 µM – better than
5
0
that of compound 16 (Table 1). Hence, supporting the design of the molecules, the
combination of those amino acids which were part of the previous active compounds (5/16,
1
7), into a single molecule resulted in considerable increase in the 5-LOX inhibitory activity.
Additionally, compound 7h having His-Glu combination showed appreciable enzyme
inhibition activity. Overall, we observed that 5-LOX inhibitory activities of compounds 6 and
7
were comparable/better than the commercially available drugs zileuton and MK0591.
Remarkably, compounds 6a, 6c, 6e, 7a, 7c and 7e, which exhibited poor docking score for 5-
LOX, were found to be less potent than their corresponding enantiomer/diastereomer for the
inhibition of 5-LOX enzymatic activity.
<
Table 1>
4
.2. COX-1/2 inhibitory activities
Compounds 6a-e, 6h, 6i, and 7a-e, 7h, 7i were screened for COX-1 and COX-2 inhibition at
-4
-8
five different molar concentrations (10 M – 10 M) [27]. As evidenced by the IC and
5
0
selectivity index data, these compounds displayed higher efficacy towards the inhibition of
COX-2 enzyme over COX-1 enzyme (Table 1). Compound 7b, with IC 0.0063 µM for COX-
5
0
2
and 2.21 µM for COX-1 and selectivity index 351 (for COX-2 over COX-1), was found to be
the most active. Compound 7d with IC 0.099 µM against COX-2 and a selectivity index 440
5
0
was another potent and selective COX-2 inhibitor. Compound 7h also displayed appreciable
IC 0.54 µM for COX-2 enzyme but its selectivity over COX-1 was poor. The IC of
5
0
50
compounds 7b and 7d against COX-2 was much better than that of commercially available
drugs wogonin [28], indomethacin [29], celecoxib [30], SC-558 and diclofenac [31].
1
0

ACCEPTED MANUSCRIPT
Intriguingly, these compounds were identified with highest anti-inflammatory and anti-
hyperalgesic activities amongst the compounds investigated here. In this enzyme
immunoassay, compound 6b exhibited IC 0.047 for COX-2 with selectivity index 210. The
5
0
enzyme inhibitory activities of compounds 6a, 6c, 6e, 7a, 7c and 7e were found to be less than
the other isomeric compounds.
Therefore, it seems that 5-LOX and COX-2 are the cellular targets of these compounds and
inhibition of their enzymatic activity imparts anti-inflammatory features to the compounds.
The interactions of the compounds with these enzymes were also supported by the other
physical experiments.
4
.3. Kinetic Studies: UV-vis and isothermal calorimetric experiments.
In order to support the results of enzyme immunoassays and for checking the extent of
interactions between the compounds and COX-2/5-LOX enzymes, the kinetic and
thermodynamics of the enzyme – ligand interactions were investigated.
4
.3.1 K and K of compounds 6 and 7 for 5-LOX and COX-2.
a i
Ka (association constant) quantifies the strength of the enzyme inhibitor complex
E + I
EI) while K is the concentration of the inhibitor which is required to decrease the
(
i
maximum rate of reaction by half. Smaller the K , lesser is the concentration of inhibitor required
i
to decrease the enzymatic activity. For non-competitive or uncompetitive kinetics, K equals IC
i
50
whereas for competitive kinetics, K is different from IC . Hence, it is a relevant parameter for
i
50
not only determining the effectiveness of the compound (inhibitor) against the enzyme but also
hints towards the mode of inhibition of the enzyme. K and K of compounds 6 and 7 for 5-LOX
a
i
and COX-2 were calculated from the UV spectral data with the aid of Benesi Hildebrand
equation 1 [32].
1
1

ACCEPTED MANUSCRIPT
1
/(A - A ) = 1/(A -A ) + 1/K (A -A )[L]
(1)
f
obs
f
fc
a
f
fc
A = absorbance of free host, A = absorbance observed, A = absorbance at saturation
f
obs
fc
The K (inhibitor concentration required to produce half maximum inhibition) was calculated
i
with equation 2.
K = IC /(1+[L]/K )
(2), K = dissociation constant
d
i
50
d
Using K , ∆G (Gibbs free energy) for the enzyme-compound complex was also calculated
a
from the equation 3.
∆G = -2.303 RT log Ka
(3), K = association constant
a
<
Table 2>
It is evident from the data given in table 2 that the compounds 7b and 7d, with best IC for 5-
5
0
-1
-1
6
-1
LOX, are showing ∆G -37.20 and -36.02 KJ mol K , respectively; K 3.36 x 10 M and 2.06 x
a
6
-1
1
0 M , respectively and K 0.042 and 0.064 μM, respectively against 5-LOX. Compounds 6i
i
and 7h also exhibited appreciable free energy change while interacting with the enzyme, having
-1
-1
5
-1
5
-1
respective ∆G -30.33 and -32.22 KJ mol K ; K 2.08 x 10 M and 1.99 x 10 M and K 0.097
a
i
and 0.536 μM for 5-LOX. The K and K values were also calculated for COX-2 enzyme.
a
i
-1
-1
6
-1
Compound 7d was having ∆G -37.15 KJ mol K ; K and K 3.25 x 10 M and 0.005 μM,
a
i
respectively while the same parameters for compound 7b and 7h were ∆G -31.48, -33.60 KJ
-1
-1
5
-1
5
-1
mol K ; K and K 3.29 x 10 M , 0.109 μM and 7.76 x 10 M , 0.012 μM (Table 2). A
a
i
comparison of the IC50 and K values indicated that these compounds exhibit competitive
i
inhibition of the enzymes which was also verified by the Lineweaver-Burk plots.
4
.3.2. Isothermal calorimetric data for free energy change (∆G) during interaction of
compounds 6 and 7 with the enzymes.
1
2

ACCEPTED MANUSCRIPT
Regarded as a method of choice for characterizing the thermodynamics and stoichiometry of
the molecular interactions with high sensitivity, the isothermal calorimetry (ITC) experiments
were performed. The binding constant (K), standard molar enthalpy (ΔH), standard molar
entropy (ΔS) and stoichiometry (N) of binding of compounds 7b, 7d, 7h and 7i with 5-LOX and
COX-2 were determined. The compound solution was titrated into the sample cell (containing
the enzyme) using a 250 µL rotating stirrer syringe set at 500 rpm. The reference cell contained
HEPES buffer. Each experiment consisted of 19 consecutive injections of 2 µL of 10 µM of the
compound to the enzyme contained in the sample cell after regular time intervals of 120 s to
guarantee the equilibrium in each titration point. Control experiments were performed for
comparison. The total heat Q produced or absorbed in the active cell volume V determined at
0
th
fractional saturation Θ after the i injection is given by equation 4
Q = n ΘM ΔHV
(4)
t
0
Where, M is the total concentration of the macromolecule
t
n is the total number of binding sites in the macromolecule and
ΔH is the molar heat of ligand binding
th
The enthalpy change for the i injection ΔH(i) for an injection volume dVi is defined by
equation 5.
The various parameters determined from ITC experiments are given in table 3 and table 4
Figure 3, Figure S112). Remarkably, the isothermal calorimetric data was quite in agreement
ΔH(i) = Q(i) + dV /V [Q(i) – Q(i-1)/2] – Q(i-1)
(5)
i
0
(
with the physical constants calculated from the UV-vis spectral data. It was evident from all the
four experiments that 5-LOX and COX-2 have one binding site for the test compound.
<
<
Table 3>
Table 4>
1
3

ACCEPTED MANUSCRIPT
<
Figure 3>
4
.3.3. Competitive/non-competitive interaction of compounds 7 with 5-LOX and COX-2
In addition to the information obtained from the comparison of K and IC , the double
i
50
reciprocal plots were also used for further confirmation about competitive/non-competitive
inhibition of the enzymes by compounds 7. The rate of change in absorbance on incremental
addition of the substrate (arachidonic acid) to a fixed concentration of the enzyme during the
progress of the reaction was plotted in the form of a double reciprocal plot (green trail).
Similarly, the change in absorbance of the enzyme – inhibitor solution on incremental addition of
arachidonic acid was recorded. The red and blue trails in figure 4 correspond to the solutions
containing 10 μM and 20 μM of the inhibitor, respectively. The point where the three trails meet
on extrapolation indicates the nature of inhibition: competitive or non competitive. Compounds
7
b, 7d, 7i and 7h displayed competitive inhibition of both 5-LOX and COX-2 (Figure 4).
Figure 4>
<
4
.4. Further evidences in support of mode of action of the compounds
Experiments were designed to see the effect of compound 7 on misoprostol and substance P
stimulated reversal of paw lickings. Misoprostol is a synthetic prostaglandin E analogue while
substance P is known to stimulate lipooxygenase and increase the production of LTB [33] and
4
also substance P has been reported to stimulate PGE production by increasing the expression of
2
COX-2 in human colonic epithelial cells [34]. Each of the seven groups having five animals were
given all the treatments intraperitoneally 30 min before the capsaicin injection. Group I was
control wherein the animals were injected normal saline. In group II and III animals were
-1
injected compound 7b and 7h, respectively at a dose of 5 mg Kg ; in group IV and V, animals
were injected with misoprostol (200 µg) 30 min before compound 7b and 7h, respectively (5 mg
1
4

ACCEPTED MANUSCRIPT
-1
Kg ) followed by capsaicin after 30 min. In group VI and VII, animals were injected with
-1
substance-P (10 µg) 30 min before compound 7b and 7h, respectively (5 mg Kg ) followed by
capsaicin after 30 min.
The treatment with compound 7b and 7h was found to produce a marked decrease in the
number of paw lickings after capsaicin injection. Pretreatment of the animals with misoprostol
increased the number of paw lickings slightly but not significantly. However, pretreatment with
substance P was found to reverse the antihyperalgesic effect of compounds 7b and 7h completely
(Figure 5). Therefore, these findings corroborate to the fact that compounds 7b and 7h decrease
the hyperalgesic effect of capsaicin by inhibiting COX-2 and 5-LOX and not by COX pathway
alone as misoprostol could not reverse the antinociceptive effect of compounds 7b and 7h.
<
Figure 5>
5
. Docking of compounds 7b in the active site of 5-LOX and COX-2. In order to support the
experimental results, interactions of compound 7b in the active site of 5-LOX and COX-2 were
investigated with the help of molecular docking. Compound 7b sits in the active site of COX-2
(Figure 6A, B) and interacts with Ser516 (2.14Å, 2.20Å) and Tyr371 (2.50 Å) amino acid
residues through the carbonyl oxygen of amide group and carboxylic group, respectively. When
docked in the active site of 5-LOX, compound 7b exhibited H-bond interactions with Phe177
(2.61 Å, 3.57 Å) and Asn554 (1.10 Å) (Figure 7A, B).
<
<
Figure 6>
Figure 7>
6
. Conclusions
We have performed a stepwise modification of the compounds for developing highly
efficacious anti-inflammatory agents. Improved over the previous set of compounds, presence of
1
5

ACCEPTED MANUSCRIPT
a dipeptide unit in the C-3 substituent of N-tosylated indole and a careful selection of the
stereoisomers enabled us to identify compounds with significant anti-inflammatory and anti-
hyperalgesic activities. Significant reversal in the dextran induced swelling and capsaicin
induced lickings in the hind paw of the mice were observed in the presence of compounds 7b
and 7h. Remarkably, the analgesic effectiveness of these compounds was as good as shown by
diclofenac. IC of compound 7b for COX-2 and 5-LOX was 2 nM and 6.3 nM respectively and
5
0
these results indicated that COX-2 and 5-LOX might be the cellular targets of 7b. Similar results
were shown by compound 7d. The physical parameters including K , K and ∆G of compounds
a
i
7
b, 7d and 7h for both COX-2 and 5-LOX enzymes were compromising for the possible hit-to-
lead development. Other general observations during this work are supporting the fact that
stereochemistry of the molecule play critical role during its interaction with the cellular target.
Moreover the compounds with free carboxyl group at the end of C-3 substituent (7) were more
potent than their corresponding esters 6.
7
7
. EXPERIMENTAL PROCEDURES
.1. Chemistry
All reactions were performed in oven-dried glassware with magnetic stirring. Diethyl ether
was distilled over activated anhydrous calcium chloride and further dried by passing sodium
wire. Acetonitrile was dried by refluxing over anhydrous P O , then over anhydrous K CO and
2
5
2
3
stored over activated 4 Å molecular sieves. The reactions were monitored by thin-layer
chromatography using silica gel GF254; visualization of the developed chromatogram was
performed by UV and staining with iodine. Column chromatography was performed with silica
1
13
gel of 100-200 mesh using hexane and ethyl acetate as eluents. H and C NMR spectra were
recorded at 500 MHz and 125 MHz NMR spectrometer using CDCl and DMSO as solvents and
3
1
6

ACCEPTED MANUSCRIPT
1
TMS as internal standard. Data for H NMR spectra are reported as chemical shift (δ ppm),
multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, dd = double doublet, br s = broad
1
3
singlet) and coupling constant (J in Hz). Data for C and DEPT-135 NMR spectra are given in
terms of chemical shift; +ve signals correspond to CH and CH carbons, -ve signals correspond
3
to CH carbons while absent means a quaternary carbon. Infrared spectra were examined in KBr
2
pallets using SP 300 PYE UNI CAM Infrared Spectrophotometer. Mass spectra were acquired
using Bruker micrOTOF-Q-II mass spectrometer in +ve ESI mode.
General method of preparation of compounds 10-12:
Oxalyl chloride was slowly added to the ice cooled solution of indole (8) in dry ether. The
yellow colored solid (9) was filtered and dissolved in dry ACN followed by the addition of
K CO L-Amino acid ester hydrochloride was neutralized with K CO and added to the ACN
2
3.
2
3
o
solution of compound 9. All the additions were done at 0 C. C-3 Substituted indoles 10-12 were
purified through column chromatography using ethyl acetate -hexane as eluent.
General method of preparation of compounds 13-18:
Compounds 10-12 (1 mmol) and NaH (1.2 mmol) (after washing with dry hexane) were taken
o
in dry ACN. Keeping the reaction temperature 0 – 5 C, p-toluene sulphonyl chloride (1.2 mmol)
was added and the reaction mixture was stirred for 30 min. After filtration, the filtrate was
concentrated under vacuum and purified by column chromatography to procure compounds 13-
1
5 (Scheme 2). Compounds 13-15 were further treated with 1N NaOH in acetone – H O (1:1) to
2
get compound 16 – 18. All the compounds were characterized with NMR, IR, mass spectral data.
General method of preparation of compounds 6:
o
Solution of compound 16 and triethylamine in dry DCM was stirred at 0 C. After 2-3 min of
stirring, ethylchloroformate was added to the reaction mass. Eventually, the neutralized solution
1
7

ACCEPTED MANUSCRIPT
of glycine methyl ester hydrochloride (neutralized with 1.5 equivalent solution of triethylamine)
was added to the reaction mass. The coupling reaction was completed in 25 – 30 min to provide
the product in 50-60% yield (Scheme 3). It should be taken into consideration that all the
o
additions to the reaction mixture were done at 0 C. The crude product was purified through
column chromatography using ethyl acetate-hexane as eluent to get compound 6i. Similarly,
compound 6d was synthesized from 16. Through the same reaction procedure as depicted in
scheme 5, compounds 6b and 6h were procured
General method of preparation of compounds 7:
Compounds 6 were dissolved in acetone-water (2:1). 1N NaOH was eventually added to this
solution. The hydrolysis reaction was completed in 15-20 min to yield compounds 7 (Scheme 4).
The crude product was purified through column chromatography using ethyl acetate-hexane as
eluent. The optical purity of compounds 7 was checked by recording their LC-MS using
®
chirobiotic T 10 µm chiral HPLC column. All the compounds were characterized with NMR
and high resolution mass spectra (Supporting information).
[2-(1H-Indol-3-yl)-2-oxo-acetylamino]-acetic acid methyl ester (10). Indole (1 mmol) was
dissolved in dry ether (10 ml) and allowed to stir in an ice bath for a while till a clear solution is
obtained. Afterwards, dropwise addition of oxalyl chloride (1.2 mmol) was done to this solution
which immediately gives a yellow solid through an exothermic reaction. This yellow solid was
filtered under vacuum and dissolved in dry ACN (25 ml) followed by the addition of K CO (1.5
2
3
mmol). Eventually, the neutralized solution of glycine methyl ester hydrochloride (1.2 mmol)
neutralized with 1.5 equivalents of triethyl amine) was added to the reaction mass. The progress
(
of the reaction was monitored with TLC. Reaction completes in 15-20 min. The reaction mass
was washed with a minimum amount of water and extracted with ethyl acetate. Finally, the ethyl
1
8

ACCEPTED MANUSCRIPT
acetate was distilled off to leave behind a crude product which was washed with absolute ethanol
o
-1
to get the product. Yield 95%. White solid. Mp 167 C. IR νmax (KBr, cm ): 1681 (C=O), 1750
1
(
C=O), 2857 (C-H), 3487 (NH). H NMR (500 MHz, CDCl ) : 3.83 (s, 3H, OCH ), 4.20 (d,
3
3
J=1.5 Hz, 2H, CH ), 7.39-7.43 (m, 2H, ArH), 7.82-8.01 (m, 2H, ArH), 8.35 (s, 1H, ArH), 8.78
2
13
(
s, 1H, NH). C NMR (normal/DEPT- 135) (125 MHz, CDCl ) : 41.09 (CH , -ve), 52.74
3
2
(
OCH ), 122.79 (CH), 125.17 (CH), 125.98 (CH), 128.04 (C), 130.27 (CH), 134.43 (C), 138.48
3
(
CH), 161.30 (C=O), 169.30 (C=O), 180.63 (C=O). ESI-MS (HRMS) calcd for C H N O Na
1
3
12
2
4
+
2
3
83.0689. Found m/z 283.0670 [M+H] .
-(1H-Indol-3-yl)-2-[2-(1H-indol-3-yl)-2-oxo-acetylamino]-propionic acid methyl ester (11).
Indole (1 mmol) was dissolved in dry ether (10 ml) and allowed to stir in an ice bath for a while
till a clear solution is obtained. Afterwards, dropwise addition of oxalyl chloride (1.2 mmol) is
done to this solution which immediately gives a yellow solid through an exothermic reaction.
This yellow solid was filtered under vacuum and dissolved in dry ACN (25 ml) followed by the
addition of K CO (1.5 mmol). Eventually, the neutralized solution of L-tryptophan methyl ester
2
3
hydrochloride (1.5 mmol) (neutralized with 1.5 equivalents of triethyl amine) was added to the
reaction mass. The progress of the reaction was monitored with TLC. Reaction completes in 15-
2
0 min. The reaction mass was washed with a minimum amount of water and extracted with
ethyl acetate. Finally, the ethyl acetate was distilled off to leave behind a crude product which
was further refined through column chromatography using ethyl acetate-hexane as eluents to get
o
-1
the product. Yield 89%. Grey solid. Mp 146 C. IR νmax (KBr, cm ): 1681 (C=O), 1759 (C=O),
1
2
1
987 (CH), 3307 (NH). H NMR (500 MHz, CDCl ) : 3.44-3.47 (dd, J = 7.5 Hz, J = 1.5 Hz,
3
H, CH ), 3.58-3.63 (dd, J = 9.0 Hz, J = 1.0 Hz, 1H, CH ), 3.88 (s, 3H, OCH ), 4.51 (m, 1H,
2
2
3
CH), 6.70 (d, J=3.5 Hz, 1H, ArH), 7.16-7.28 (m, 5H, ArH), 7.32-7.50 (m, 4H, ArH), 8.160 (s,
1
9

ACCEPTED MANUSCRIPT
1
3
1
5
H, NH). C NMR (normal/DEPT- 135) (125 MHz, CDCl ) : 26.06 (CH , -ve), 51.41 (CH),
3
2
4.75 (OCH ), 117.61 (CH), 117.84 (CH), 120.45 (CH), 121.51 (CH), 123.23 (CH), 123.53
3
(CH), 124.74 (CH), 126.10 (C), 126.11 (CH), 126.75 (C), 129.98 (CH), 130.72 (C), 134.63 (C),
1
34.73 (C), 136.53 (C), 145.40 (C), 160.72 (C=O), 161.41 (C=O), 169.56 (C=O). ESI-MS
+
(
HRMS) calcd for C H N O 390.1448. Found m/z 390.1444 [M+H] .
2
2
19
3
4
3
-(3H-Imidazol-4-yl)-2-[2-(1H-indol-3-yl)-2-oxo-acetylamino]-propionic acid methyl ester
(12). Indole (1 mmol) was dissolved in dry ether (10 ml) and allowed to stir in an ice bath for a
while till a clear solution is obtained. The yellow solid obtained after dropwise addition of oxalyl
chloride (1.2 mmol) to this solution was filtered under vacuum and dissolved in dry ACN (25
ml) followed by the addition of K CO (1.5 mmol). Eventually, the neutralized solution of L-
2
3
histidine methyl ester hydrochloride (1.34 mmol) (neutralized with 1.5 equivalents of triethyl
amine) was added to the reaction mass. The progress of the reaction was monitored with TLC.
Reaction completes in 15-20 min. The reaction mass was washed with a minimum amount of
water and extracted with ethyl acetate. Finally, the ethyl acetate was distilled off to leave behind
a crude product which was further refined through column chromatography using ethyl acetate-
o
-1
hexane as eluents to get the product. Yield 90%. Yellow solid. Mp 118 C. IR ν
(KBr, cm ):
max
1
1
688 (C=O), 1750 (C=O), 2857 (C-H), 3537 (NH). H NMR (500 MHz, DMSO-d ) : 3.33 (d,
6
J= 6.5 Hz, 2H, CH ), 3.77 (s, 3H, OCH ), 4.46 (m, 1H, CH), 6.82 (s, 1H, ArH), 6.83-7.28 (m,
2
3
3
H, ArH), 7.28-7.33 (m, 2H, ArH), 7.33-7.39 (m, 2H, ArH, NH), 7.58 (s, 1H, NH), 9.10 (s, 1H,
1
3
NH); C NMR (normal/DEPT- 135) (125 MHz, DMSO- d ) : 25.56 (CH , -ve), 51.51 (CH),
6
2
5
3.51 (OCH ), 109.89 (CH), 113.53 (CH), 118.57 (CH), 122.02 (CH), 123.88 (CH), 125.10
3
(CH), 127.12 (C), 127.15 (CH), 127.39 (CH), 130.65 (CH), 130.98 (C), 134.49 (C), 134.52
2
0

ACCEPTED MANUSCRIPT
(
CH), 169.07 (C=O), 174.51 (C=O), 179.89 (C=O); ESI-MS (HRMS) calcd for C H N O
1
7
16
4
4
+
3
41.1244. Found m/z 341.1241 [M+H] .
{2-Oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-acetic acid methyl ester (13).
NaH (1.2 mmol) was washed with dry hexane (5 ml) and suspended in a solution of dry ACN
o
(
20 ml). Compound 10 (1 mmol) was added to this suspension and allowed to stir at 0 C for 2-3
min until the whole reactant gets dissolved. Eventually, the p-toluyl sulphonylchloride (1.2
mmol), recrystallized over warm hexane, was added to the reaction mass. The progress of the
reaction was monitored with TLC. On completion, the reaction was quenched by adding a
minimum amount of ice cold water. The crude product was extracted from aqueous layer by
ethyl acetate. The organic layer was dried over Na SO and concentrated under vacuum. The
2
4
crude residue was purified with column chromatography using ethyl acetate-hexane as eluents to
o
-1
procure the product. Yield 68%. Yellow solid. Mp 180 C. IR ν
(KBr, cm ): 1130 (S=O),
max
1
1
661 (C=O), 1757 (C=O), 2957 (C-H), 3407 (NH). H NMR (500 MHz, CDCl ) : 2.38 (s, 3H,
3
CH ), 3.83 (s, 3H, OCH ), 4.20 (d, J = 5.5 Hz, 2H, CH ), 7.28-7.43 (4H, ArH), 7.82-7.84 (m, 1H,
3
3
2
1
3
ArH), 7.89-8.01 (m, 2H, ArH), 8.35-8.36 (m, 2H, ArH), 9.39 (s, 1H, NH). C NMR
(
normal/DEPT- 135) (125 MHz, CDCl ) : 21.66 (CH ), 41.06 (CH -ve), 52.62 (OCH ), 113.27
3
3
2 ,
3
(CH), 115.93 (C), 122.81 (CH), 125.17 (CH), 125.98 (CH), 127.38 (CH), 128.11 (C), 130.30
(CH), 134.23 (C), 134.43 (C), 138.48 (CH), 146.09 (C), 161.27 (C=O), 169.34 (C=O), 180.74
+
(
C=O). ESI-MS (HRMS) calcd for C H N O S 437.0778. Found m/z 437.0855 [M+Na] .
2
0
18
2
6
3
-(1H-Indol-3-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-propionic
acid methyl ester (14). NaH (1.2 mmol) was washed with dry hexane (5 ml) and suspended in
dry ACN (20 ml). Compound 11 (1mmol) was added to this suspension and allowed to stir at 0
o
C for 2-3 min until the whole reactant gets dissolved. Eventually, p-toluyl sulphonylchloride
2
1

ACCEPTED MANUSCRIPT
(1.2 mmol), recrystallized from warm hexane, was added to the reaction mass. The progress of
the reaction was monitored with TLC. On completion, the reaction was quenched by adding a
minimum amount of ice cold water. The crude product was extracted from aqueous layer by
ethyl acetate. After drying over anhydrous Na SO , the organic layer was concentrated under
2
4
vacuum. The crude residue was purified through column chromatography by using ethyl acetate-
o
hexane as eluents to procure the product. Yield 79 %. Brown solid. Mp 131 C. IR νmax (KBr,
-1
1
cm ): 1135 (S=O), 1651 (C=O), 1741 (C=O), 2927 (CH), 3411 (NH). H NMR (500 MHz,
CDCl ) : 2.36 (s, 3H, CH ), 3.44-3.49 (dd, J = 15.0 Hz, J = 7.5 Hz, 1H, CH ), 3.59-3.63 (dd, J =
3
3
2
1
5.0 Hz, J=2.7 Hz, 1H, CH ), 3.88 (s, 3H, OCH ), 4.51 (m, 1H, CH), 6.70 (d, J = 3 Hz, 1H,
2 3
ArH), 7.16-7.18 (m, 2H, ArH), 7.22-7.28 (m, 5H, ArH), 7.32-7.35 (m, 3H, ArH), 7.41 (d, J = 10
Hz, 1H, ArH), 7.49 (d, J = 10 Hz, 1H, ArH), 7.56 (m, 1H, NH), 7.77 (d, J = 10 Hz, 1H, ArH),
1
3
7
.97 (m, 1H, NH). C NMR (normal/DEPT- 135) (125 MHz, CDCl ) : 21.47 (CH ), 26.11
3
3
(
CH , -ve), 54.09 (CH), 54.11 (CH), 105.46 (C), 109.56 (CH), 110.93 (C), 111.92 (CH), 113.19
2
(C), 113.43 (CH), 115.46 (C), 117.63 (CH), 117.72 (C), 120.45 (CH), 121.51 (CH), 123.23(CH),
1
1
23.53 (CH), 124.71 (CH), 124.74 (CH), 126.10 (C), 126.16 (CH), 126.75 (CH), 129.98 (CH),
30.84 (C), 134.63 (C), 134.76 (C), 136.56 (C), 145.41 (C), 169.61 (C=O). ESI-MS (HRMS)
+
calcd for C H N O S 566.1356. Found m/z 566.1366 [M+Na] .
2
9
25
3
6
3
-(3H-Imidazol-4-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
propionic acid methyl ester (15) NaH (1.2 mmol) was washed with dry hexane (5 ml) and
suspended in a dry ACN (20 ml). Compound 12 (1 mmol) was added to this suspension and
o
allowed to stir at 0 C for 2-3 min until the whole reactant gets dissolved. Eventually, p-toluyl
sulphonylchloride (1.2 mmol), recrystallized from warm hexane, was added to the reaction mass.
The progress of the reaction was monitored with TLC. On completion, the reaction was
2
2

ACCEPTED MANUSCRIPT
quenched by adding a minimum amount of ice cold water. The crude product was extracted from
aqueous layer by ethyl acetate. After drying over anhydrous Na SO , the organic layer was
2
4
concentrated under vacuum. The crude product was purified through column chromatography by
o
using ethyl acetate-hexane as eluents to procure the product. Yield 85%. Grey solid. Mp 121 C.
-1
1
IR νmax (KBr, cm ): 1087.19 (S=0), 1628 (C=O), 1744 (C=O), 2925 (CH), 3401 (N-H). H
1
NMR (300 MHz, DMSO-d ) : H NMR (500 MHz, DMSO d ): 2.29 (s, 3H, CH ), 3.33-3.35
6
6
3
(
d, J = 7.0 Hz, 2H, CH ), 3.72 (s, 3H, OCH ), 4.49 (t, J = 7.0 Hz, 1H, CH), 6.82 (d, J= 5 Hz, 1H,
2 3
ArH), 7.23 (t, J = 5 Hz, 1H, ArH), 7.31 (s, 1H, ArH), 7.35 (t, J = 10 Hz, 3H, ArH), 7.54 (s, 1H,
ArH), 7.59 (d, J = 10 Hz, 1H, ArH), 7.77 (d, J = 5 Hz, 1H, ArH), 7.84 (d, J = 10 Hz, 1H, ArH),
1
3
7
.92 (d, J = 10 Hz, 1H, ArH), 9.10 (s, 1H, NH). C NMR (normal/DEPT- 135) (125 MHz,
CDCl ) : 21.46 (CH ), 25.51 (CH , -ve), 51.49 (CH), 53.51 (OCH ), 109.88 (CH), 113.53 (CH),
3
3
2
3
1
1
18.57 (CH), 122.07 (CH), 123.89 (CH), 125.10 (CH), 127.12 (C), 127.15 (CH), 127.38 (CH),
27.39 (C), 130.68 (CH), 130.93 (C), 134.52 (CH), 134.56 (C), 134.63 (C), 145.92 (C), 169.02
(
C=O), 174.65 (C=O), 179.82 (C=O). ESI-MS (HRMS) calcd for C H N O S 495.1323. Found
24 22 4 6
+
m/z 495.1333 [M+H] .
2-Oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-acetic acid (16). Compound 13
1 mmol) was dissolved in 10 ml acetone - water (2:1) and allowed to stir at room temperature.
{
(
Eventually 1N solution of NaOH was added to the reaction mass. The progress of reaction was
monitored with TLC. The reaction completes in 15-20 min. On completion, the reaction mass
was heated to evaporate acetone. On dropwise addition of 0.1N HCl to the aqueous part, the
solid product precipitates out which was filtered and purified through column chromatography
o
-
using ethyl acetate - hexane as eluents. Yield 80%. Brown solid. Mp 140 C. IR νmax (KBr, cm
1
1
)
: 1120 (S=O), 1630 (C=O), 1764 (C=O), 3412 (NH); H NMR (500 MHz, CDCl ) : 2.38 (s,
3
2
3

ACCEPTED MANUSCRIPT
3
H, CH ), 4.20 (d, J= 4.5 Hz. 2H, CH ), 7.13-7.15 (d, J = 10 Hz, 2H, ArH), 7.26-7.28 (t, J = 5
3
2
Hz, 2H, ArH), 7.50 (m, 2H, ArH), 7.54 (m, 1H, ArH), 8.25 (m, 1H, ArH), 8.76 (m, 1H, ArH),
1
3
8
.95 (s, 1H, NH), 12.35 (s, 1H, OH). C NMR (normal/DEPT- 135) (125 MHz, CDCl ) : 21.98
3
(
CH ), 41.07 (CH -ve), 113.30 (CH), 115.79 (C), 122.81 (CH), 125.17 (CH), 125.74 (CH),
3 2
1
1
4
3
27.38 (CH), 128.11 (CH), 134.24 (C), 134.43 (C), 138.48 (CH), 146.09 (C), 161.27 (C=O),
69.35 (C=O), 180.66 (C=O). ESI-MS (HRMS) calcd for C H16N O S 423.0621. Found m/z
1
9
2
6
+
23.0641 [M+Na] .
-(1H-Indol-3-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-propionic
acid (17). Compound 14 (1 mmol) was taken in 10 ml acetone - water (2:1) to which 1N NaOH
was added. On completion of the reaction (TLC), the reaction mass was heated to evaporate
acetone. On dropwise addition of 0.1N HCl to the aqueous part, the solid product precipitates out
which was vacuum filtered and purified through column chromatography using ethyl acetate -
o
-1
hexane as eluents. Yield 69%. Cream solid. Mp 110 C. IR ν
(KBr, cm ): 1110 (S=O), 1632
max
1
(
C=O), 1735 (C=O), 3421 (NH). H NMR (500 MHz, DMSO-d ) : 2.30 (s, 3H, CH ), 3.25 (d, J
6
3
=
5 Hz, 2H, CH ), 4.17 (m, 1H, CH), 6.82 (d, J = 5 Hz, 1H, ArH), 7.01 (t, J = 5 Hz, 1H, ArH),
2
7
3
.10 (t, J = 5 Hz, 1H, ArH), 7.24 (t, J = 5 Hz, 1H, ArH), 7.32-7.39 (m, 3H, ArH), 7.56-7.60 (m,
H, ArH), 7.78 (d, J = 5 Hz, 1H, ArH), 7.85 (d, J = 10 Hz, 2H, ArH), 7.93 (d, J = 10 Hz, 1H,
1
3
ArH), 8.19 (s, 2H, NH), 11.058 (s, 1H, OH). C NMR (normal/DEPT- 135) (125 MHz, DMSO-
d ) : 21.44 (CH ), 26.61 (CH , -ve), 53.05 (CH), 107.07 (C), 109.85 (CH), 111.98 (CH), 112.29
6
3
2
(C), 113.53 (CH), 114.60 (C), 116.90 (C), 118.66 (CH), 119.09 (CH), 119.20 (C), 121.65 (CH),
1
1
22.05 (CH), 123.88 (CH), 125.08 (CH), 125.41 (CH), 127.15 (CH), 127.38 (CH), 127.47 (C),
30.67 (CH), 130.93 (C), 134.57 (C), 134.66 (C), 136.76 (C), 145.92 (C), 171.35 (C=O). ESI-
+
MS (HRMS) calcd for C H N O S 552.1200. Found m/z 552.1243 [M+Na] .
2
8
23
3
6
2
4

ACCEPTED MANUSCRIPT
3
-(3H-Imidazol-4-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
propionic acid (18). Compound 15 (1 mmol) was dissolved in 10 ml acetone - water (2:1) and
allowed to stir at room temperature. Eventually 1N NaOH was added to the reaction mass. The
progress of the reaction was monitored with TLC. On completion, the reaction mass was heated
to evaporate acetone. On dropwise addition of 0.1N HCl to the aqueous part, the solid product
precipitates out which was vacuum filtered and purified by column chromatography using ethyl
o
-1
acetate - hexane as eluents. Yield 65%. Yellow solid. Mp 135 C. IR ν
(KBr, cm ): 1065
max
1
(
S=O), 1630 (C=O), 1735 (C=O), 3395 (NH). H NMR (500 MHz, DMSO-d ) : 2.29 (s, 3H,
6
CH ), 3.28 (d, J = 6.0 Hz, 2H, CH ), 4.12 (t, J = 5.5 Hz, 1H, CH), 7.00 (t, J= 5 Hz, 1H, ArH),
3
2
7
.09-7.12 (m, 3H, ArH), 7.14 (s, 1H, ArH), 7.24 (d, J = 5 Hz, 1H, ArH), 7.37 (d, J = 10 Hz, 1H,
ArH), 7.50 (d, J = 10 Hz, 2H, ArH), 7.57 (d, J = 5 Hz, 1H, ArH), 7.59 (d, J = 10 Hz, 1H, ArH),
1
3
8
.30-8.33 (b, 2H, NH), 11.08 (s, 1H, OH). C NMR (normal/DEPT- 135) (125 MHz, DMSO-d )
6

: 21.24 (CH ), 26.54 (CH , -ve), 53.04 (CH), 107.08 (C), 111.97 (CH), 116.64 (C), 118.71
3
2
(CH), 119.05 (CH), 121.59 (CH), 125.46 (CH), 125.96 (CH), 127.49 (C), 128.61 (CH), 136.73
(C), 138.36 (C), 145.77 (C=O), 158.64 (C=O), 158.95 (C=O), 171.27 (C=O). ESI-MS (HRMS)
+
calcd for C H N O S 481.1176. Found m/z 481.1171 [M+Na] .
2
3
20
4
6
(2-{2-Oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]- acetylamino}-acetylamino)-acetic acid
methyl ester (6i). Compound 16 (1 mmol) was dissolved in dry DCM (25 ml) and stirred for 2-3
min to make a clear solution. Triethylamine and ethylchloroformate were added to this solution.
Eventually, neutralized solution of glycine methyl ester hydrochloride in triethylamine was
o
added to the reaction mass at 0 C. The progress of the reaction was monitored with TLC. On
completion, the reaction mass was neutralized with 0.1N HCl and washed repeatedly with 1M
NaHCO till the effervescence persists. Eventually, the crude organic product was extracted with
3
2
5

ACCEPTED MANUSCRIPT
ethyl acetate. Further purification of the crude material was done through column
o
chromatography using ethyl acetate-hexane as eluent. Yield 68%. Brown solid. Mp 171 C. IR
-1
1
νmax (KBr, cm ): 1135 (S=O) 1666 (C=O), 1717 (C=O), 2950 (C-H), 3477 (NH). H NMR (500
MHz, CHCl + TFA) : 2.29 (s, 3H, CH ), 3.34 (d, J = 7.0 Hz, 2H, CH ), 3.72 (s, 3H, OCH ),
3
3
2
3
4
.49 (t, J = 7.0 Hz, 2H, CH ), 6.82 (s, 1H, ArH), 7.31-7.36 (m, 4H, ArH), 7.58-7.60 (m, 1H,
2
ArH), 7.54 (s, 1H, ArH), 7.77-7.78 (m, 1H, ArH), 7.83-7.85 (m, 1H, ArH), 7.91-7.93 (m, 1H,
ArH), 9.10 (s, 1H, NH). (normal/DEPT- 135) (125 MHz, CHCl + TFA) : 21.40 (CH ), 25.78
3
3
(
(
(
CH , -ve), 29.66 (CH , -ve), 51.78 (OCH ), 109.82 (CH), 113.53 (CH), 114.55 (CH), 116.85
2 2 3
CH), 122.03 (CH), 123.86 (CH), 125.06 (CH), 127.14 (CH), 127.35 (CH), 130.93 (CH), 130.65
CH), 134.57 (CH), 134.66 (CH), 145.90 (CH), 171.23 (C=O), 173.70 (C=O). ESI-MS (HRMS)
+
calcd for C H N O S 471.1100. Found m/z 471.1103 [M+H] .
2
2
21
3
7
(2-{2-Oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino)-acetic acid (7i). Compound
6
i was dissolved in acetone - water (2:1) and stirred for 2-3 min to make a homogeneous
solution. Eventually, 1N NaOH was added to the reaction mass and allowed to stir at room
temperature. The progress of the reaction was monitored through TLC. The reaction mass was
heated to evaporate acetone. On dropwise addition of 0.1N HCl to the reaction mass, the crude
product precipitated out which was filtered under vaccum and washed repeatedly with diethyl
o
-1
ether to procure pure product. Yield 59%. Brown solid. Mp 191 C. IR νmax (KBr, cm ): 1099
1
(
S=O), 1610 (C=O), 1717 (C=O), 2885 (C-H), 3402 (NH); H NMR (500 MHz, DMSO-d +
6
TFA) : 2.29 (s, 3H, CH ), 3.28 (d, J = 6.0 Hz, 2H, CH ), 4.12 (d, J = 5.5 Hz, 2H, CH ), 6.99-
3
2
2
7
.07 (m, 2H, ArH), 7.09-7.14 (m, 3H, ArH), 7.24 (d, J = 5 Hz, 1H, ArH), 7.36-7.58 (m, 3H,
1
3
ArH), 8.30-8.33 (b, 2H, ArH), 11.08 (s, 1H, COOH); C NMR (normal/DEPT- 135) (125MHz,
DMSO-d + TFA) : 21.54(CH ), 41.07 (CH , -ve), 43.20 (CH , -ve), 112.28 (C), 113.94 (CH),
6
3
2
2
2
6

ACCEPTED MANUSCRIPT
1
1
20.33 (CH), 122.79 (CH), 123.85 (CH), 125.16 (CH), 127.84 (CH), 128.00 (C), 129.98 (C),
0
30.26 (CH), 134.98 (C), 143.99 (C=O), 145.23 (C=O), 167.75 (C=O), 169.81 (C=O). [α] + 22
D
+
(
c 1, MeOH), ESI-MS (HRMS) calcd for C H N O S 457.0944. Found m/z 457.0955 [M+H] .
2
1
19
3
7
3
-(1H-indol-2-yl)-2-(2-{2-oxo-2-[1-toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
acetylamino)-propionic acid methyl ester (6b). Compound 17 was dissolved in dry DCM and
stirred for 2-3 min to make a clear solution. Triethylamine and ethylchloroformate were added to
this solution. Eventually, neutralized solution of glycine methyl ester hydrochloride in
o
triethylamine was added to the reaction mass at 0 C. The progress of the reaction was monitored
with TLC. On completion, the reaction mass was neutralized with 0.1N HCl and washed
repeatedly with 1M NaHCO till the effervescence persists. Eventually, the crude product was
3
extracted with ethyl acetate. Further purification of the crude material was done through column
o
chromatography using ethyl acetate-hexane as eluent. Yield 78 %. Yellow solid. Mp 181 C. IR
-1
1
νmax (KBr, cm ): 1139 (S=O), 1695 (C=O), 1721 (C=O), 2999 (CH), 3415 (N-H). H NMR (500
MHz DMSO-d + TFA) : 2.33 (s, 3H, CH ), 3.26 (t (dd), J = 5.5 Hz, 2H, CH ), 3.70 (s, 3H,
6
3
2
OCH ), 4.04 (d, J = 6.0 Hz, 1H, CH), 4.15 (t, J = 5.5 Hz, 1H, CH), 6.99-7.98 (m, 10H, ArH),
3
8
.00-8.26 (m, 4H, ArH), 9.11 (s, 1H, NH), 9.31 (t, J = 5Hz, 1H, NH), 11.04 (s, 1H, NH).
(
normal/DEPT- 135) (125 MHz, DMSO-d + TFA) : 21.45 (CH ), 26.57 (-ve, CH ), 41.12 (-ve,
6
3
2
CH ), 52.35 (CH), 53.05 (OCH ), 107.04 (C), 111.96 (CH), 112.09 (C), 113.61 (CH), 116.14
2
3
(
(
(
(
[
C), 116.67 (C), 118.63 (CH), 118.97 (C), 119.06 (CH), 121.62 (CH), 122.62 (CH), 125.39
CH), 125.75 (CH), 126.66 (CH), 127.45 (C), 127.76 (C), 127.83 (CH), 129.06 (C), 131.11
CH), 133.57 (C), 133.93 (C), 136.76 (C), 137.69 (CH), 162.85 (C=O), 171.01 (C=O), 171.34
C=O), 182.78 (C=O). ESI-MS (HRMS) calcd for C H N O S 600.1678 Found m/z 600.1680
3
1
28
4
7
+
M+H] .
2
7

ACCEPTED MANUSCRIPT
3
-(1H-indol-2-yl)-2-(2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
acetylamino)-propionic acid (7b). Compound 6b was dissolved in acetone-water (2:1) and
stirred for 2-3 min to make a homogeneous solution. Eventually, 1N NaOH was added to the
reaction mass and allowed to stir at room temperature. After completion of the reaction (TLC),
the reaction mixture was heated to evaporate acetone. On dropwise addition of 0.1N HCl to the
aqueous part, the crude product precipitated out which was filtered under vacuum and washed
o
repeatedly with diethyl ether to procure the product. Yield 65%. Cream solid. Mp 190 C. IR
-1
1
νmax (KBr, cm ): 1111 (S=O); 1602 (C=O), 1730 (C=O), 3420 (NH). H NMR (500 MHz,
DMSO-d + TFA) : 2.28 (s, 3H, CH ), 2.64 (s, 2H, CH ), 3.30 (d, J = 6.5 Hz, 2H, CH ), 4.11 (t,
6
3
2
2
J = 6.0 Hz, 1H, CH), 6.80-6.98 (m, 1H, ArH), 7.00-7.38 (m, 4H, ArH), 7.56-7.69 (m, 6H, ArH),
.70-7.92 (m, 4H, ArH, NH), 8.31 (m, 2H, NH), 11.12 (s, 1H, COOH) (normal/DEPT- 135)
7
;
(
125MHz, DMSO-d + TFA) : 21.34 (CH ), 26.57 (CH , -ve), 40.08 (CH , -ve), 53.16 (CH),
6
3
2
2
1
07.17 (C), 109.82 (C), 111.95 (C), 113.50 (CH), 113.77 (C), 116.13 (C), 118.49 (C), 118.64
(
CH), 119.00 (CH), 120.85(C), 121.54(CH), 122.02(CH), 123.86(CH), 125.06(CH), 125.40
CH), 127.11 (CH), 127.29 (CH), 127.50 (C), 129.08 (C), 130.61 (CH), 130.92 (C), 131.99 (C),
(
1
34.54 (C), 134.62 (C), 136.76 (C), 145.93 (C), 159.61 (C=O), 169.40 (C=O), 171.28 (C=O).
0
[
α] + 21 (c 1, MeOH), ESI-MS (HRMS) calcd for C H N O S 587.1522. Found m/z
D
30 26
4
7
+
5
2
87.1520 [M+H] .
-(3-(3H-imidazol-4-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
propionylamino)-pentanedioic acid dimethyl ester (6h). Compound 18 was dissolved in dry
DCM and stirred for 2-3 min to make a clear solution. Triethylamine and ethylchloroformate
were added to the above solution. Eventually, neutralized solution of L-glutamic dimethyl ester
o
hydrochloride in triethylamine was added to the reaction mass at 0 C. The progress of the
2
8

ACCEPTED MANUSCRIPT
reaction was monitored with TLC. On completion, the reaction mass was neutralized with 0.1N
HCl and washed repeatedly with 1M solution of NaHCO till the effervescence persists.
3
Eventually, the crude product was extracted with ethyl acetate and purified through column
o
chromatography using ethyl acetate-hexane as eluent. Yield 72%. Yellow solid. Mp 141 C. IR
-1
1
νmax (KBr, cm ): 1080 (S=O), 1632 (C=O), 1699 (C=O), 2906 (C-H), 3434 (NH). H NMR (500
MHz, DMSO-d ) : 2.08-2.13 (m, 2H, CH ), 2.28 (s, 3H, CH ), 2.48-2.62 (m, 4H, 2xCH ), 3.33
6
2
3
2
(
d, J = 6.0 Hz, 1H, CH), 3.60 (s, 3H, OCH ), 3.73 (s, 3H, OCH ), 4.08 (m, 1H, CH), 6.82 (s, 1H,
3 3
ArH), 7.00-7.10 (m, 1H, ArH), 7.21-7.35 (m, 3H, ArH), 7.38-7.58 (m, 3H, ArH), 7.83-7.94 (m,
1
3
3
H, ArH), 8.45 (s, 1H, NH), 8.76 (s, 1H, NH). C NMR (normal/DEPT- 135) (125MHz,
DMSO-d ) : 21.39 (CH ), 25.58 (CH , -ve), 26.50 (CH , -ve), 29.32 (CH , -ve), 51.94 (OCH ),
6
3
2
2
2
3
5
1
1
1
3.06 (CH), 53.20 (OCH ), 107.12 (C), 109.84 (CH), 111.94 (CH), 113.52 (CH), 114.81 (C),
3
17.12 (C), 118.75 (CH), 119.00 (CH), 121.52 (CH), 122.03 (CH), 123.86 (CH), 125.0 (CH),
25.49 (CH), 127.12 (CH), 127.33 (CH), 127.53 (C), 130.63 (CH), 130.93 (C), 134.57 (C),
34.64 (C), 136.73 (C), 145.88 (C), 169.98 (C=O), 171.22 (C=O), 172.63 (C=O). ESI-MS
+
(
HRMS) calcd for C H N O S 637.1843. Found m/z 637.1856 [M+H] .
3
0
31
5
9
2
-(3-(3H-imidazol-4-yl)-2-{2-oxo-2-[1-(toluene-4-sulfonyl)-1H-indol-3-yl]-acetylamino}-
propionylamino)-pentanedioic acid (7h) Compound 6h was dissolved in acetone-water (2:1)
and stirred for 2-3 min to make a homogeneous solution. Eventually, 1N NaOH was added to the
reaction mass and allowed to stir at room temperature. The progress of the reaction was
monitored through TLC. On completion, the reaction mass was heated to evaporate acetone. On
dropwise addition of 0.1N HCl to the aqueous part, the crude product precipitated out which was
filtered under vaccum and washed repeatedly with diethyl ether to procure the product. Yield
o
-1
8
5%. Orange solid. Mp 175 C. IR ν
(KBr, cm ): 1099 (S=O), 1611 (C=O), 1797 (C=O),
max
2
9