Synthesis, molecular modeling and biological evaluations of novel pyrrolidine derivatives as potential cyclooxygenase-2 (COX-2) inhibitors

Article abstract of DOI:10.1007/s13738-020-02151-2

Abstract: Toward the development of selective cyclooxygenase-2 inhibitors, a series of pyrrolidine derivatives is described. All the compounds containing sulfonamide, ester, nitrile, acid, amide and urea functionalities were computationally screened, and

Full text of DOI:10.1007/s13738-020-02151-2

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Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02151-2
ORIGINAL PAPER
Synthesis, molecular modeling and biological evaluations of novel
pyrrolidine derivatives as potential cyclooxygenase‑2 (COX‑2)
inhibitors
Kamlesh H. Chavan1 · Nathrao Ankushrao Kedar1,2 · Ashish M. Kanhed3 · Vishal Kumar Agrahari4 ·
Anshuman Sinha4,5
Received: 27 July 2020 / Accepted: 26 December 2020
© Iranian Chemical Society 2021
Abstract
Toward the development of selective cyclooxygenase-2 inhibitors, a series of pyrrolidine derivatives is described. All the
compounds containing sulfonamide, ester, nitrile, acid, amide and urea functionalities were computationally screened, and
binding afnity scores for all synthesized compounds with cyclooxygenase-1 and cyclooxygenase -2 were compared. The
computational observations showed three top-ranked compounds (8b, 8d and 10a) having selectively more afnity for
cyclooxygenase -2. These were selected for pharmacological evaluation using carrageenan-induced rat paw edema model.
Compound 8b showed maximum activity (54.83%) which was closer to standard drug indometacin (57.48%). The safety
parameter of the potent compound (8b) was assessed using aspirin induced gastric ulceration animal model.
Graphic abstract
Keywords Pyrrolidine derivatives · Cyclooxygenase-2 · Docking study · Carrageenan-induced rat paw edema model ·
Gastric ulceration animal model
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are an
important therapeutic agent widely used for treatment of
a variety of infammatory conditions over the Globe. The
* Nathrao Ankushrao Kedar
dnakedar07@gmail.com
Extended author information available on the last page of the article
Vol.:(0123456789)
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NSAIDS exerts their pharmacological actions by inhibition
of the enzymes COX-1 and COX-2 which resulted in the
decreased level of the prostaglandins (PGs) and thrombox-
anes (TXs) [1]. The enzyme COX-1 involved in the produc-
tion of PGs, functioning as homeostatic in most of the tis-
sues, and serves as housekeeping enzyme. However, COX-2
produced as a response toward the infammatory stimuli
upon increased levels of PGs. The classical NSAIDs are
associated with the undesired side efects such as gastro-
intestinal ulcers, bleeding, and platelet dysfunction, due to
the simultaneous inhibition of the COX-1 enzyme [2–4].
The use of the selective COX-2 inhibitor (coxib’s) reduced
toxicity which are associated with classical NSAIDs [5]. The
coxibs improved the overall anti-infammatory potential;
however, these are associated with risk of adverse cardio-
vascular outcomes [6–9]. In this direction, to overcome the
adverse efects of existing COX-2 inhibitors, the develop-
ment of selective COX-2 inhibitor with improved COX-1/
COX-2 selectivity index with an improved safety profle is
still an urgent need.
N-methyl-(2S,4R)- trans -4-hydroxy-l–proline exhibits the
anti-infammatory potential through the inhibition of infam-
matory cytokine TNF-α [15]. Ugwu et al. proline bearing
bezothiothiazole motifs showed anti-infammatory activity
by selective COX-2 inhibitions [16]. Due to its usefulness
in medicinal, organic chemistry and abundant in natural
products, the number of publications increased in the recent
years; however, the 4-amino-proline bearing, amide, sulfona-
mide and urea remain unexplored. In our interest to fnd
out the new selective COX-2 inhibitor, we have designed
the novel compound by modifying trans-4-amino-proline
core. We reported the synthesis of the new library of trans-
4-amino-proline analogs with the aim of identifying the
binding afnity and selectivity toward the COX-2 enzyme.
The newly synthesized derivatives were docked in the active
site of human COX-1 and COX-2. The highly ranked hits
from modeling studies were evaluated for in vivo anti-
infammatory potential using carrageenan-induced rat paw
edema model. Furthermore, compounds accessed for ulcer
genic potential.
Pyrrolidine derivatives are recognized as an important
class of heterocycles which exhibits the range of biological
activities. Pyrrolidine derivatives are abundant in nature,
the alkaloid nicotine, hygrine contains this core, and the
other examples of the pyrrolidine natural products include
cocaine, allosecurinine, stemofoline, quinocarcin, coccinine,
etc. Moreover, the amino acid proline, 4-hydroxy-proline,
contains the pyrrolidine ring. Pyrrolidine rings containing
compounds have reached in the market and many under
clinical investigations. Also, pyrrolidine and its deriva-
tives serve as catalyst in many important organic transfor-
mations. The plethora of the literature was documented
regarding its biological signifcance as antibacterial [10],
anticancer [11], antidiabetic [12], etc., including the anti-
infammatory activity [13, 14]. Pedro Everson et al. reported
Result and discussion
Chemistry
Synthesis of designed derivatives of (2S,4S)-ethyl 4-amino-
pyrrolidine-2-carboxylate/acid (6) bearing amide (7, 8)/sul-
fonamide (11, 12) and urea (9, 10) functionality is achieved
as per the reaction sequence depicted in Schemes 1 and 2.
The key intermediate (2S,4S)-ethyl 4-aminopyrrolidine-
2-carboxylate (6) was synthesized from commercially avail-
able (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (1) as
per the reaction sequence depicted in Scheme 1.
MsO
HO
HO
HO
O
O
O
O
O
O
O
b
a
c
N
R
N
H
OH
N
H
N
R
2
1
3
4
R=
N3
H2N
O
O
Ph
OPh
e
N
R
N
O
O
R
6
5
OBn
Scheme 1 Synthesis of (2S,4S)-ethyl 4-aminopyrrolidine-2-car-
boxylate derivative (6). Reagents and conditions: a ethanol, SOCl2,
0–5 °C, 15 min, refux, 8 h; b R-Br, triethylamine, rt, 4 h; c meth-
ane sulfonyl chloride, pyridine, DCM, 25–30 °C, 3 h; d NaN3, DMF,
80 °C, 3 h; e PPh3, THF, 25–30 °C, 4 h
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O
O
H
O
R1
N
(S)
O
O
c
O
(S)
H2N
d
a
OH
O
HN
O
O
N
HN
HN
R2
N
R
N
N
R
HN
R2
R
R
6
O
7
9
10
e
b
H
N
O
S
O
H
N
H
O
O
O
R1
O
N
f
R3
S
R3
O
O
OH
N
R
N
R
OH
O
N
R
8
11
12
Scheme 2 Synthesis of designed amide (7, 8)/Sulfonamide (11,
12)/urea (9, 10) derivatives. Reagents and conditions: a RCOOH,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridin-
ium 3-oxide hexafuorophosphate (HATU), N, N-diisopropylethyl-
amine (DIPEA), N, N-dimethylformamide (DMF), 25–30 °C, 4 h; b
ethanol, 2 M LiOH, 25–30 °C, 2 h; c RNCO, triethylamine, DCM,
25–30 °C, 4 h; d ethanol, 2 M LiOH, 25–30 °C, 2 h; e RSO2Cl,
triethylamine, DCM, 0 °C to 25–30 °C, 4 h; f ethanol, 2 M LiOH,
25–30 °C, 2 h
(2S,4R)-4-Hydroxypyrrolidine-2-carboxylic acid (1) is
converted into corresponding ethyl ester (2) upon refuxing
in ethanol in the presence of thionyl chloride. Intermedi-
ate 2 on reaction with aryl bromide in the presence of tri-
ethylamine in DCM at 25–30 °C to obtain intermediate 3.
The (2S,4S)-ethyl 4-hydroxypyrrolidine-2-carboxylate (3)
is converted into mesyl derivative 4, which upon treatment
with sodium azide gives the azide analogs 5, intermediate
5 reduced under staudinger condition gives (2S,4S)-ethyl
4-aminopyrrolidine-2-carboxylate (6). The synthesis of
designed analogous of (2S,4S)-ethyl 4-aminopyrrolidine-
2-carboxylate/acid (6) bearing amide (7, 8)/sulfonamide
(11, 12) and urea (9, 10) was achieved as per the reaction
sequence outlined in Scheme 2. The intermediate (2S,4S)-
ethyl 4-aminopyrrolidine-2-carboxylate (6) on reaction
with appropriate carboxylic acid/isocyanate/sulfonyl chlo-
ride gives corresponding amide (7)/sulfonamide (11)/urea
analogues (9) of (2S,4S)-ethyl 4-aminopyrrolidine-2-car-
boxylate which are hydrolyzed in the presence of lithium
hydroxide to corresponding carboxylic acid derivatives (8,
10, 12). The structures of the newly synthesized compounds
were in full agreement with their NMR and mass spectral
data. The purity of the newly synthesized compounds was
checked by using HPLC analysis.
respectively, and exists as homodimers. These two isoforms
show more than 60% similarity in their sequence. COX sub-
unit is mainly divided into three domains, viz. epidermal
growth factor domain, membrane binding domain and the
catalytic domain which covers both the cyclooxygenase and
peroxidase active sites and is considered as a major part of
the protein with residue count 73–116. In the active site of
COX-2, valine is present at position 523, whereas isoleucine
is present at the same position in COX-1. This major difer-
ence in COX-1 and COX-2 diferentiates the receptor active
binding sites among these isoforms. In COX-2, the small
Val523 provides space to interact with the hydrophobic
side pocket, which is in fact sterically hindered by Ile523 in
COX-1. In this report, the binding afnity of novel pyrroli-
dine derivatives was studied by performing docking studies
with the active site of COX2 and COX1 receptors and the
ligand receptor interactions were identifed. For this study,
the PDB structures were selected from protein data bank
with PDB Code: 2OYE (COX-1) and 6 COX (COX-2). The
docking scores with COX-1 and COX-2 for all synthesized
derivatives are mentioned in Table 1.
On the basis of binding score, compound 8b and 10a
were found to be having more afnity and selectivity toward
COX-2 receptor, whereas compounds 8b, 8d, and 10a
showed poor afnity toward COX-1 receptor. Other than
these compounds, majorities of compounds exhibited almost
similar afnity toward both the receptors under study. To
analyze the COX-2 selective ligands under study, the com-
parative interactions of compound 8b and 10a with COX-2
and COX-1 receptor active sites are discussed here. Com-
pound 8b exhibited maximum afnity toward the COX-2
active site in comparison with COX-1. The 3,4-dichloroben-
zyloxy group of compound 8b was observed to be inter-
acting in the hydrophobic pocket of aromatic amino acids
Computational study
COX-1 and COX-2 are the two recognized isoforms of
cyclooxygenase enzyme. COX-1 constructively is involved
in various physiological functions, while the COX-2 is pro-
duced in cells by endotoxins and cytokines in response to
infammation. COX-2 is mainly responsible for amplifed
levels of prostaglandins during infammation. The COX-1
and COX-2 consist of 576 and 581 amino acid sequence,
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Table 1 The docking scores
the compound showed only pi–pi interaction with TYR-355
and no salt bridges were observed between any polar amino
acid of protein with either pyrrolidine or carboxylate ion
of the ligand. Further the phenoxybenzyl group was found
to be facing the solvent front, and thus, the compound was
found to be having more interactions with COX-2 receptors
in comparison with COX-1 (Fig. 1a, b).
Compound Docking score
for all synthesized compounds
(8a–12b) for COX-1 and COX-2
COX2
COX1
8a
−1.864 NI
8b
−9.485 −6.486
−6.294 −1.208
−7.882 −4.912
−6.816 NI
−6.409 −4.759
−8.008 NI
−3.192 −3.622
−2.192 −7.374
−4.912 NI
−1.134 −1.534
−1.748 −3.538
−3.045 −2.992
−5.944 −6.397
−4.645 −5.543
−4.504 −7.916
8c
8d
Similarly, in compound 10a, the phenoxy group showed
very promising pi–pi interaction with TRP-387. The amine
groups of urea stabilized the ligand receptor complex by
forming two strong hydrogen bonds with TYR-355, whereas
no interactions were observed between compound 10a and
COX-1 receptor (Fig. 2).
9a
9b
10a
10b
10c
10d
10e
10f
10 g
10h
12a
12b
Compound 8b at COX-2 showed highest binding afnity
promisingly because of pi–pi interaction and hydrophobic
interactions of scafold along with the salt bridge formed
by the carboxylate anion and pyrrolidine of same scafold,
whereas the least active compound 10f did not show salt
NI no interaction observed
TYR-385, TRP-387, Phe-518 along with a nonpolar hydro-
phobic VAL-349, whereas the benzamido ring exhibited
pi–pi interaction with TYR-355 and provided very good
stability of the ligand receptor complex. The benzyl group of
4-phenoxybenzyl present in scafold expressed very promis-
ing pi-cation interaction with LYS-83, while the phenoxy
ring imparted stability to the complex by showing pi–pi
interaction with TYR-115. Additionally, the carboxylate
anion of the pyrrolidine ring was found to provide stability
of the ligand receptor complex by generating a salt bridge
with the ARG-120. At physiological pH, the quaternized
N of pyrrolidine ring system stabilized the ligand receptor
complex by forming a salt bridge interaction with GLU-
524. Considering same molecule with the COX-1 active site,
Fig. 2 Interaction of compound 10a with COX2 receptor active site;
no interaction of compound 10a was observed with COX1
Fig. 1 a Interaction of compound 8b with COX2 receptor active site; b interaction of compound 8b with COX1 receptor active site
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