molecules
Article
Crystallography, Molecular Modeling, and COX-2 Inhibition
Studies on Indolizine Derivatives
Katharigatta N. Venugopala 1,2, * , Sandeep Chandrashekharappa 3,4, * , Christophe Tratrat 1 , Pran Kishore Deb 5 ,
Rahul D. Nagdeve 6 , Susanta K. Nayak 6 , Mohamed A. Morsy 1,7 , Pobitra Borah 8 , Fawzi M. Mahomoodally 9 ,
Raghu Prasad Mailavaram 10 , Mahesh Attimarad 1 , Bandar E. Aldhubiab 1 , Nagaraja Sreeharsha 1,11 ,
Anroop B. Nair 1 , Osama I. Alwassil 12 , Michelyne Haroun 1 , Viresh Mohanlall 2 , Pottathil Shinu 13 ,
Rashmi Venugopala 14 , Mahmoud Kandeel 15,16 , Belakatte P. Nandeshwarappa 17 and Yasmine F. Ibrahim 7






Citation: Venugopala, K.N.;
Chandrashekharappa, S.; Tratrat, C.;
Deb, P.K.; Nagdeve, R.D.; Nayak, S.K.;
Morsy, M.A.; Borah, P.; Mahomoodally,
F.M.; Mailavaram, R.P.; et al.
Crystallography, Molecular Modeling,
and COX-2 Inhibition Studies on
Indolizine Derivatives. Molecules
2021, 26, 3550. https://doi.org/
10.3390/molecules26123550
Academic Editor: Ritesh Raju
Received: 26 April 2021
Accepted: 8 June 2021
Published: 10 June 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 3550. https://doi.org/10.3390/molecules26123550
1 Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; ctratrat@kfu.edu.sa (C.T.); momorsy@kfu.edu.sa (M.A.M.);
mattimarad@kfu.edu.sa (M.A.); baldhubiab@kfu.edu.sa (B.E.A.); sharsha@kfu.edu.sa (N.S.);
anair@kfu.edu.sa (A.B.N.); mharoun@kfu.edu.sa (M.H.)
2 Department of Biotechnology and Food Technology, Durban University of Technology,
Durban 4001, South Africa; vireshm@dut.ac.za
3 Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER-R)
Raebareli, Lucknow UP 226002, India
4 Institute for Stem Cell Science and Regenerative Medicine, NCBS, TIFR, GKVK, Bellary Road,
Bangalore 560065, India
5 Faculty of Pharmacy, Philadelphia University, Amman 19392, Jordan; prankishore1@gmail.com
6 Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur 440010, Maharashtra, India;
rahulnagdeve3@gmail.com (R.D.N.); sknayak@chm.vnit.ac.in (S.K.N.)
7 Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt;
yasmine.ibrahim@mu.edu.eg
8 Pratiksha Institute of Pharmaceutical Sciences, Chandrapur Road, Panikhaiti, Guwahati 781026, Assam, India;
pobitrab.phe15@itbhu.ac.in
9 Department of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius,
Réduit 80835, Mauritius; f.mahomoodally@uom.ac.mu
10 Department of Pharmaceutical Chemistry, Shri Vishnu College of Pharmacy, Vishnupur,
Bhimavaram 534202, India; raghumrp@svcp.edu.in
11 Department of Pharmaceutics, Vidya Siri College of Pharmacy, Off Sarjapura Road, Bangalore 560035, India
12 Department of Pharmaceutical Sciences, College of Pharmacy, King Saud bin Abdulaziz University for
Health Sciences, Riyadh 11481, Saudi Arabia; wassilo@ksau-hs.edu.sa
13 Department of Biomedical Sciences, College of Clinical Pharmacy, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; spottathail@kfu.edu.sa
14 Department of Public Health Medicine, Howard College Campus, University of KwaZulu-Natal,
Durban 4001, South Africa; rashmivenugopala@gmail.com
15 Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; mkandeel@kfu.edu.sa
16 Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt
17 Department of Studies in Chemistry, Shivagangotri, Davangere University, Davangere,
Karnataka 577007, India; belakatte@davangereuniversity.ac.in
* Correspondence: kvenugopala@kfu.edu.sa (K.N.V.); c.sandeep@niperraebareli.edu.in (S.C.);
Tel.: +966-1358-98842 (K.N.V.); +91-94486-39413 (S.C.)
Abstract: The cyclooxygenase-2 (COX-2) enzyme is an important target for drug discovery and
development of novel anti-inflammatory agents. Selective COX-2 inhibitors have the advantage of
reduced side-effects, which result from COX-1 inhibition that is usually observed with nonselective
COX inhibitors. In this study, the design and synthesis of a new series of 7-methoxy indolizines
as bioisostere indomethacin analogues (5a–e) were carried out and evaluated for COX-2 enzyme
inhibition. All the compounds showed activity in micromolar ranges, and the compound diethyl 3-(4-
cyanobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5a) emerged as a promising COX-2 inhibitor
with an IC50 of 5.84 µM, as compared to indomethacin (IC50 = 6.84 µM). The molecular modeling
study of indolizines indicated that hydrophobic interactions were the major contribution to COX-2 in-
hibition. The title compound diethyl 3-(4-bromobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5c)
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 3550 2 of 20

was subjected for single-crystal X-ray studies, Hirshfeld surface analysis, and energy framework cal-
culations. The X-ray diffraction analysis showed that the molecule (5c) crystallizes in the monoclinic
crystal system with space group P 21 /n with a = 12.0497(6)Å, b = 17.8324(10)Å, c = 19.6052(11)Å,
α = 90.000◦ , β = 100.372(1)◦ , γ = 90.000◦ , and V = 4143.8(4)Å3 . In addition, with the help of Crystal
Explorer software program using the B3LYP/6-31G(d, p) basis set, the theoretical calculation of the
interaction and graphical representation of energy value was measured in the form of the energy
framework in terms of coulombic, dispersion, and total energy.

Keywords: indolizine derivatives; molecular modeling; COX-2 inhibition; crystal structure; Hirshfeld
surface analysis; energy framework

1. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly pre-
scribed drugs for the treatment of inflammatory conditions worldwide [1]. NSAIDs ex-
hibit their anti-inflammatory activity via inhibition of cyclooxygenase (COX), an enzyme
involved in the biosynthesis of prostaglandin G2 (PGG2). By inhibiting the formation
of PGG2, the pathway, which would ultimately lead to the inflammatory response, is
blocked [2,3]. The COX enzyme has two important isoforms, namely, COX-1 and COX-2.
The COX-1 isoform represents the constitutive type that is normally expressed in various
regions of the body, such as the kidney and the gastrointestinal tract (GIT), where it is
responsible for maintaining certain physiological functions including protection of the
gastric mucosa [4–7]. On the other hand, the COX-2 isoform represents the inducible type
that is expressed in response to various inflammatory stimuli and certain substances such
as mitogens and cytokines that are produced during injuries. Nonselective NSAIDs that
inhibit both COX-1 and COX-2 can have severe undesirable side-effects due to inhibition
of the customarily expressed isoform, COX-1. For example, GIT ulceration is a commonly
observed adverse effect associated with the use of nonselective NSAIDs [8,9]. Thus, in
order to develop anti-inflammatory agents with reduced side-effects, compounds with
high selectivity for inhibiting the COX-2 isoform over the COX-1 isoform are required.
Synthetic indolizine derivatives have been shown to interact with a wide range of
drug targets such as calcium channels [10], histamine receptors [11], and phospholipase
A2 [12]. In addition, they have shown numerous pharmacological properties [13] such
as analgesic [14], COX-2-inhibitory [15–18], anticancer [19,20], antidiabetic [21], antihis-
taminic [11], antileishmanic [22], antimicrobial [23], antimutagenic [24], antioxidant [25],
antiviral [26], larvicidal [27,28], herbicidal [29], antitubercular [30–37], and alpha-7 nico-
tinic acetylcholine receptor (α-7 nAChR)- [38], N-meningitidis-, N-acetylneuraminic acid
synthese (NmeNANAS)-inhibitory activities [39].
In persistence of our interest in pharmacologically active heterocyclic compounds [40–48],
polymorphism studies [49–52], and the discovery of anti-inflammatory agents [53–59], in
the present investigation, we synthesized a range of 7-methoxy indolizine derivatives
as indomethacin analogues (Figure 1 and Scheme 1) to evaluate their potential as anti-
inflammatory agents. The title compounds (5a–e) were evaluated for their pharmacological
activity against the COX-2 enzyme in order to study the influence of ethyl ester at the 1-
and 2-positions, ethyl at the 2-position, and the diverse substituent on the benzoyl ring at
the 3-positions of the indolizine core on the biological action.
 Molecules 2021, 26, 3550 3 of 20
s 2021, 26, x FOR PEER REVIEW 3 of 20
Molecules 2021, 26, x FOR PEER REVIEW 3 of 20

Figure 1. TheFigure 1. The

proposed proposed
7-methoxy 7-methoxy
indolizine indolizine
analogues and analogues andavailable
commercially commercially available
nonselective nonselective(COX)
cyclooxygenase
cyclooxygenaseFigure
(COX)1.inhibition
The proposed
inhibitor 7-methoxyfor
(indomethacin) indolizine analogues action.
COX-2 inhibition and commercially available nonselective
inhibitor (indomethacin) for COX-2 action.
cyclooxygenase (COX) inhibitor (indomethacin) for COX-2 inhibition action.

Scheme
heme 1. Synthetic 1. Synthetic
1.
outline for theoutline
Synthetic for
outline forthethe
construction construction of diethyl
ofconstruction 7-methoxy-3-(3-substituedbenzoyl)indolizine-1,2-dicarboxylates
of diethyl 7-methoxy-3-(3-substituedbenzoyl)indolizine-1,2-dicarbox-
diethyl 7-methoxy-3-(3-substituedbenzoyl)indolizine-1,2-dicarbox-
ates (5a–d) [35] and ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-1-carboxylates (5e). (5e). (5e).
ylates
(5a–d) (5a–d)
[35] [35]
and and
ethyl ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-1-carboxylates
3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-1-carboxylates

2. Results and2. Results

Results and
2.Discussion and Discussion
Discussion
Chemistry
2.1. Chemistry
2.1. Chemistry
The design of the target compounds (5a–e) was mainly based on the close chemical
The design of the target compounds (5a–e) was mainly based on the close chemical
structural relationship with the commercially available available NSAIDs
NSAIDs indomethacin
indomethacin (Figure (Figure 1).
1).
structural relationship with the commercially available NSAIDs indomethacin (Figure 1).
The synthesis of the target compounds is depicted in Scheme 1. The intermediates (3a–e)
The synthesis of the target compounds is depicted in Scheme 1. The intermediates (3a–e)
were obtained by stirring a mixture of 4-methoxy pyridine and para- and meta-substituted
were obtainedphenacyl
by stirring a mixture
bromides
bromides in of 4-methoxy
inacetone
acetone medium
medium pyridine
atat5 h5 and
h andand
were para-
werethen and
then meta-substituted
further reacted
further withwith
reacted diethyl but-
diethyl
phenacyl bromides in
2-ynedioate acetone
in the medium
existence at
of 5 h and
potassium were then
carbonate further
in reacted
dimethylformamide
but-2-ynedioate in the existence of potassium carbonate in dimethylformamide solvent with diethyl
solvent medium
but-2-ynedioatefor in
30 the
medium min.forexistence
The resulting
30 min. of potassium
The title compounds
resulting carbonate
title were in
compounds dimethylformamide
purified
werebypurified
column by solvent
chromatography
column using
chromatog-
medium for 30 min.using
mixture
raphy The resulting
of ethyl acetate
mixture oftitle
and
ethylcompounds
hexane
acetate and were
as an purified
eluent,
hexane asand by column
an the purity
eluent, chromatog-
andofthe thepurity
compounds was
of the com-
more than
raphy using mixture
pounds of
was 99%
ethyl
morewith
acetate
thana satisfactory
andwith
99% hexane yield as(69% to 77%).
an eluent,
a satisfactory yield The
and(69% physicochemical
the topurity
77%). of The property of the
thephysicochemical
com-
pounds was moretargetthan
property compound
of99% ethylacompound
with
the target 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-1-carboxylate
satisfactory yield
ethyl (69% to 77%). The physicochemical
3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine- (5e)
property of theis presented
target compound
1-carboxylate in Table 1. The chemical structure of this compound
ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-
(5e) is presented (5e)
in Table 1. The chemical structure of this compound (5e) was confirmed with
spectroscopic techniques such 1 H-NMR and 13 C-NMR, and LC–MS. The Fourier-
1-carboxylatewas(5e)confirmed
is presented with inspectroscopic
Table 1. as
TheFT-IR,
chemical
techniques structure
such as FT-IR,of this compound
1H-NMR and 13(5e)
C-NMR, and
was confirmed transform
LC–MS. infrared
with spectroscopic (FT-IR)
The Fourier-transform spectroscopy
techniques infrared
such revealed
(FT-IR)
as FT-IR, benzoyl
spectroscopy
1H-NMR and and ester
revealed
13C-NMR, carbonyl
benzoyl
and groups
and esterat
1699 and 1668 − 1
cmat ,1699 respectively
carbonyl
LC–MS. The Fourier-transformgroups
1 H-NMRinfrared
1668(spectra
and(FT-IR) cm are available(spectra
−1, respectively
spectroscopy as Electronic
revealed Supplementary
are available
benzoyl and ester Mate-
as Electronic
rials). The
Supplementary spectra revealed the methoxy group at 3.86 ppm, ester peak triplet ap-
carbonyl groups at 1699 andMaterials).
1668 cm−1The 1 H-NMR spectra
, respectively (spectrarevealed the methoxy
are available group
as Electronic at 3.86 ppm,
13 C-NMR spec-
pearance
ester of 1.34
peak triplet ppm, and ester group quartet appearance of 4.30 ppm. The
Supplementary Materials). Theappearance
1H-NMR spectra of 1.34revealed
ppm, and theester
methoxygroup quartet
group appearance
at 3.86 ppm, of 4.30
tra revealed
ppm. The the appearance
13C-NMR spectra of benzoyl
revealed theand ester carbonyl
appearance of groupsand
benzoyl at 186.00
ester and 166.07
carbonyl ppm,
ester peak triplet appearance
respectively. of 1.34 ppm,
The molecular and ester
ion peaks of thisgroup
compoundquartet (5e)appearance 4.30 groups
were in goodofagreement with
at 186.00
ppm. The 13C-NMR and 166.07
spectra revealed ppm, respectively.
thecompounds
appearancediethyl The molecular
of benzoyl andion esterpeaks of thisgroups
carbonyl compound (5e)
its molecular mass. Title 3-(4-cyanobenzoyl)-7-methoxyindolizine-
at 186.00 andwere
166.07in good
ppm,agreement
1,2-dicarboxylate respectively. withThe
(5a), diethyl
its molecular
molecularmass. Title compounds
ion peaks of this compound diethyl 3-(4-cyanoben-
3-(4-fluorobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5e)
zoyl)-7-methoxyindolizine-1,2-dicarboxylate (5a), diethyl 3-(4-fluorobenzoyl)-7-methoxy-
were in good agreement
(5b), diethylwith its molecular mass. Title compounds diethyl 3-(4-cyanoben-
3-(4-bromobenzoyl)-7-methoxyindolizine-1,2-dicarboxylate (5c), and diethyl
indolizine-1,2-dicarboxylate (5b), diethyl 3-(4-bromobenzoyl)-7-methoxyindolizine-1,2-
zoyl)-7-methoxyindolizine-1,2-dicarboxylate (5a), diethyl 3-(4-fluorobenzoyl)-7-methoxy-
indolizine-1,2-dicarboxylate (5b), diethyl 3-(4-bromobenzoyl)-7-methoxyindolizine-1,2-
Molecules 2021, 26, x FOR PEER REVIEW 4 of 20

Molecules 2021, 26, 3550 4 of 20

dicarboxylate (5c), and diethyl 7-methoxy-3-(3-methoxybenzoyl)indolizine-1,2-dicarbox-

Molecules 2021, 26, x FOR PEER REVIEW 4 of 20
ylate (5d) were resynthesized [35], and their physicochemical properties are presented in
7-methoxy-3-(3-methoxybenzoyl)indolizine-1,2-dicarboxylate (5d) were resynthesized
Table 1. The proposed general reaction mechanism for the target compound (5e) is illus- [35],
and their physicochemical properties are presented in Table 1. The proposed general
trated in Figure 2.
reaction mechanism
dicarboxylate fordiethyl
(5c), and the target compound (5e) is illustrated in Figure 2.
7-methoxy-3-(3-methoxybenzoyl)indolizine-1,2-dicarbox-
ylate (5d)ofwere
Table 1. Physicochemical properties resynthesized
the target [35],ethyl
compound and their physicochemical properties are presented in
3-(4-bromobenzoyl)-2-ethyl-7-methoxy-indolizine-1-
Table 1. Physicochemical
carboxylate (5e). properties
Table 1. of
Thethe target
proposed compound
general ethyl
reaction 3-(4-bromobenzoyl)-2-ethyl-7-methoxy-indolizine-1-
mechanism for the target compound (5e) is illus-
carboxylate (5e). trated in Figure 2.

Table 1. Physicochemical properties of the target compound ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxy-indolizine-1-

carboxylate (5e).

Compound
Compound Mol Formulae(Mol
Mol Formulae (Mol Mass)
Mass) R R R1R1 Yield(%)
Yield (%)a,b a,b m.p.(◦(°
m.p. C)C) cLogPc c
cLogP
5a CC 23H20N2O6 (420) 4-CN COOC 2H5 72 171 3.4454
5a 23 H20 N2 O6 (420) 4-CN COOC 2 H5 72 171 3.4454
Compound
5b MolCFormulae
22H20FNO(Mol 6 (413)Mass) R4-F R1
COOC 2H5 Yield (%)77
a,b m.p. (°C)147 cLogP c 4.0199
5b C22 H20 FNO6 (413) 4-F COOC2 H5 77 147 4.0199
5c 5a C H N
C22H20BrNO6 (473)
23 20 2 O 6 (420) 4-CN
4-Br COOC
COOC 2H5 2H5 72 73 171 134 3.4454 4.7399
5c C22 H BrNO 6 (473) 4-Br COOC H 73 147 142 4.0199 4.7399
134
5d 5b CC232220
HH2320NO
FNO 6 (413)
7 (425)
4-F
3-OCH 3
COOC
COOC 22H55
2H5
77 75 4.0294
5d 5c CH
C23 22H NO
20 BrNO
7 6 (473)
(425) 3-OCH4-Br3 COOC
COOC22H H55 73 75 134 142 4.7399 4.0294
5e C21H23 20BrNO4 (430) 4-Br C2H5 69 108 6.2773
5d C23H
C21was
H 23NO7 (425)
BrNO 3-OCH 3 COOC H25H5final yield 75 142 108 4.0294 6.2773
5e target
a The compound 4 (430)
20confirmed 4-Br
by physical and spectral data.Cb2The 69
obtained after purification by column
5e C21H20BrNO4 (430) 4-Br C2H5 69 108 6.2773
chromatography. c cLogP
a The target compound was calculated
was confirmed using
by physical andChemBioDraw Ultra
spectral data. b The final16.0v.
yield obtained after purification by column chromatography.
a The target compound was confirmed by physical and spectral data. b The final yield obtained after purification by column
c cLogP was calculated using ChemBioDraw Ultra 16.0v.
chromatography. c cLogP was calculated using ChemBioDraw Ultra 16.0v.

Figure 2. Plausible reaction mechanism for the construction of ethyl 3-(4-bromobenzoyl)-2-ethyl-7-

Figure 2. Plausible reaction mechanism for the construction of ethyl 3-(4-bromobenzoyl)-2-ethyl-7-
Figure 2. Plausible reaction mechanism
methoxyindolizine-1-carboxylate for the construction of ethyl 3-(4-bromobenzoyl)-2-ethyl-7-
(5e) [35,60].
methoxyindolizine-1-carboxylate (5e) [35,60].
methoxyindolizine-1-carboxylate (5e) [35,60].
The construction of the final indolizine compound (5e) was achieved via 1,3-dipolar
The construction
cycloaddition of the final indolizine
of intermediate compound (5e) was achieved via 1,3-dipolar
The construction of the pyridinium salt 3e,compound
final indolizine generating ylide 3e(1)achieved
(5e) was using a base.
viaThis
1,3-dipolar
cycloaddition
ylide carbanion of intermediate pyridinium salt 3e, generating ylidebond
3e(1)ofusing a base.
cycloaddition ofsubsequently
intermediateattacks the electron-deficient
pyridinium acetylene
salt 3e, generating triple
ylide 3e(1) using reac-
a base. This
This
tant ylide carbanion
(4). This triple bondsubsequently attacks
anion then attacks thethe electron-deficient
carbocation of compound acetylene triple bond of
3e(1), yielding
ylide carbanion subsequently attacks the electron-deficient acetylene triple bond of reac-
reactant
compound (4).3e(2).
This triple bond
This loss anion then
of hydrogen attacksoxidation
through the carbocation of compound
leads to the of in-yielding
construction3e(1),
tant (4). This triple bond anion then attacks the carbocation of compound 3e(1), yielding
compound 3e(2).5e This
dolizine nucleus loss
(Figure 2).of hydrogen through oxidation leads to the construction of
compound 3e(2). This
indolizine nucleus loss of hydrogen
5e (Figure 2). through oxidation leads to the construction of in-
dolizine nucleus 5e (Figure 2).
Molecules 2021, 26, 3550 5 of 20

2.2. Crystallography
The crystallographic details of 5c are listed in Table 2. The crystal structure of 5c
crystallized in the monoclinic space group P 21 /n with eight molecules in the unit cell. The
lattice parameters were a = 12.0497(6)Å, b = 17.8324(10)Å, c = 19.6052(11)Å, α = 90.000◦ ,
β = 100.372(1)◦ , γ = 90.000◦ , and V = 4143.8(4)Å3 . The asymmetric unit of 5c crystal
structure showed two molecules which preferred the intramolecular C–H···O interactions
shown as a dotted line and 50% thermal ellipsoidal probability of non-hydrogen atoms
(Table 3 and Figure 3). There were eight molecules in the unit cell of 5c, where inter-
molecular weak hydrogen bonds C2A–H2A···O6B and C4B–H4B1···O4A were the major
interactions, along with C–H···π interactions that stabilized the molecular assembly, as
shown in Figures 4 and 5 (Table 3).

Table 2. The crystallographic refinement parameters of 5c (diethyl 3-(4-bromobenzoyl)-7-methoxy-

indolizine-1,2-dicarboxylate).

DATA 5c
Formula C22 H20 BrNO6
Formula weight 474.30
CCDC 2,045,116
Temperature (K) 173(2)
Wavelength (Å) 0.71073
Crystal system Monoclinic
Space group P 21 /n
a (Å) 12.0479(6)
b (Å) 17.8324(10)
c (Å) 19.6052(11)
α (◦ ) 90.000
β (◦ ) 100.372(1)
γ (◦ ) 90.000
V (Å3 ) 4143.8(4)
Z’, Z 2, 8
Density (g·cm−3 ) 1.52
µ (mm−1 ) 2.023
F (000) 1936.0
θ (min, max) 1.6, 28.3
hmin , max , kmin , max , lmin , max . −16, 15; −23, 23; −26, 26
No. of refl. 10,282
No of unique ref./Obs. ref. 10,282/6833
No. parameters 576
Rall , Robs 0.079, 0.041
wRall , wRobs 0.100, 0.088
∆ρmin , max (eÅ−3 ) −0.473, 0.393
G.O.O.F. 1.024

Table 3. Intra- and intermolecular interactions of 5c.

D–X···A D–X (Å) X···A (Å) D···A (Å) <D–X···A (◦ )

C21B–H21B···O1B i 0.95 2.55 3.109(3) 118
C1A–H1A···O6A i 0.95 2.27 2.851(3) 119
C1A–H1A···O6A i 0.95 2.26 2.855(3) 120
C2A–H2A···O6B ii 0.95 2.52 3.466(3) 174
C4B–H4B1···O4A iii 0.98 2.50 3.442(3) 162
C13B–H13C···π iv 0.98(24) 2.92 3.735(3) 140
C13B–H13C···π* v 0.98(24) 2.86 3.785(3) 156
Symmetry codes: (i) 1 + x, y, z; (ii) 3/2 − x, 1/2 + y, 1/2 − z; (iii) −1/2 + x, 1/2 − y, 1/2 + z; (iv) x, y, z; (v) x, y, z;
Note: −π and π* are the centroids of the (N1A–C6A–C7A–C11A–C15A) and (N1A–C1A–C2A–C3A–C5A–C6A)
aromatic rings, respectively.
es 2021, 26, x FOR PEER REVIEW 6 of 20

C13B–H13C···π* v 0.98(24) 2.86 3.785(3) 156

Symmetry codes: (i) 1 + x, y, z; (ii) 3/2 − x, 1/2 + y, 1/2 − z; (iii) −1/2 + x, 1/2 − y, 1/2 + z; (iv) x, y, z; (v)
C13B–H13C···π* v 0.98(24) 2.86 3.785(3) 156
x, y, z; Note: −π and π* are the centroids of the (N1A–C6A–C7A–C11A–C15A) and (N1A–C1A–
Molecules 2021, 26, 3550
Symmetry codes: (i) 1 + x, y, z; (ii) 3/2 − x, 1/2 + y, 1/2 − z; (iii) −1/2 + x, 1/2 − y, 1/2 + z; (iv) x, y, z; (v) 6 of 20
C2A–C3A–C5A–C6A) aromatic rings, respectively.
x, y, z; Note: −π and π* are the centroids of the (N1A–C6A–C7A–C11A–C15A) and (N1A–C1A–
C2A–C3A–C5A–C6A) aromatic rings, respectively.

Figure
Figure 3. The ORTEP3. The ORTEP
of 5c at 50%ofellipsoidal
5c at 50% ellipsoidal
probabilityprobability and atomoflabeling
and atom labeling of non-hydrogen
non-hydrogen atoms.
atoms. The intramolecular
Figure
The3.intramolecular
The ORTEP ofC–H· 5c at
··O50% ellipsoidal
interactions are probability
shown as anddotted
green atom lines.
labeling of non-hydrogen atoms.
C–H···O interactions are shown as green dotted lines.
The intramolecular C–H···O interactions are shown as green dotted lines.

Figure 4. Crystal packing of 5c prefers the weak C–H···O interactions.

Figure 4. Crystal packing

Figure 4. of 5c prefers
Crystal theofweak
packing C–H·
5c prefers ··Oweak
the interactions.
C–H···O interactions.
 Molecules
x FOR PEER REVIEW2021, 26, 3550 7 of 20 7 of 20

Figure 5. TheFigure
C–H···π contacts
5. The C–H···πexist in 5c
contacts crystal
exist in 5c structure in its in
crystal structure molecular assembly.
its molecular assembly.

2.3. Hirshfeld Surface

2.3.Analysis
Hirshfeld Surface Analysis
The Hirshfeld
The Hirshfeld surfaces surfacesstructure
of the crystal of the crystal structure
5c were 5c were investigated
investigated to illustrate to illustrate
the the
nature of intermolecular interactions and visualization of intermolecular close contacts in
nature of intermolecular interactions and visualization of intermolecular close contacts in
its crystal structure using Crystal Explorer 17.5 [60], which mapped over de , dnorm , shape
its crystal structure using
index, andCrystal
curvedness,Explorer 17.5in[60],
as shown which
Figure 6. Themapped
contribution over de, dnorm, intermolecular
of individual shape
index, and curvedness, as shown
interactions on the inHirshfeld
Figure 6.surface
The contribution
can be definedofbyindividual
color codes.intermolecu-
On the dnorm surface,
lar interactions on the
theredHirshfeld
color shows surface can be
the shorter definedcontacts
molecular by color andcodes.
the blueOn theondthe
color normdsur-
norm surface
face, the red colorarea
showsrepresents the longer
the shorter molecularcontacts
molecular contacts. The
andwhitethe color
blue on the don
color normthesurface
dnormindicates
the contact
surface area represents around
the longer the van der
molecular Waals radii.
contacts. In the dcolor
The white norm surfaces,
on the dthe red color shows
norm surface
the hydrogen bonding H···O contacts, whereas the blue surface area represents the H···H
indicates the contact around the van der Waals radii. In the dnorm surfaces, the red color
contacts (Figure 6a). The de surface features appear as a relatively flat green region where
shows the hydrogen thebonding H···O contacts,
contact distances whereas
are similar (Figurethe
6b).blueThesurface
adjacentarea represents
highlighted the yellow
red and
H···H contacts (Figure 6a).onThe
regions the d e surface
shape indexfeatures appear
surface also showas thea strong
relatively flat green
hydrogen bonding region
interactions
where the contact distances
present in thearemolecule
similar (Figure 6b).whereas
(Figure 6c), The adjacent
the bluehighlighted
curved and yellowred and yel- on the
regions
low regions on thecurvedness
shape index surfaces shows
surface also H···H interactions
the show the strong(Figure
hydrogen 6d). The 2D fingerprint
bonding interac- plots [61]
tions present in the molecule (Figure 6c), whereas the blue curved and yellow regions on in the
show the sharp spike, which represents the intermolecular interactions present
molecule (Figure 7a). Again, it shows that C–H···O interactions were predominant (23.2%)
the curvedness surfaces shows the H···H interactions (Figure 6d). The 2D fingerprint plots
after the H···H contacts, which led to the highest contribution of 35.8% in comparison to
[61] show the sharpother
spike, which represents
interactions, the intermolecular
suggesting that weak C–H···O hydrogen interactions
bonding present
plays aninessential
the role
molecule (Figure 7a). Again,
in its crystalitpacking.
shows that C–H···O interactions
The percentage contributions were predominant
of other intermolecular (23.2%)
interactions
were as follows: C ··· H/H ···
after the H···H contacts, which led to the highest contribution of 35.8% in comparison Hto(12.2%),
in this crystal structure C (18.4%), H ··· Br/Br ···
···O/O···C (2.5%),
other interactions,Csuggesting C···C (2.1%),
that weak C–H··N ·O···hydrogen
H/H···N (1.7%),bonding etc. (Figure
plays7b).an essential
role in its crystal packing. The percentage contributions of other intermolecular interac-
tions in this crystal structure were as follows: C···H/H···C (18.4%), H···Br/Br···H (12.2%),
C···O/O···C (2.5%), C···C (2.1%), N···H/H···N (1.7%), etc. (Figure 7b).
Molecules 2021, 26, 3550 8 of 20
Molecules 2021, 26, x FOR PEER REVIEW 8 of 20
Molecules 2021, 26, x FOR PEER REVIEW 8 of 20

Figure6.6.Hirshfeld
Figure Hirshfeldsurfaces
surfacesofof5c5cmapped
mappedwith
with(a)(a)dnorm
dnorm(b)
(b)ddee (c)
(c) shape
shape index,
index, and
and(d)
(d)curvedness.
curvedness.
Figure 6. Hirshfeld surfaces of 5c mapped with (a) dnorm (b) de (c) shape index, and (d) curvedness.

Figure 7. Cont.
 Molecules 2021, 26, 3550 9 of 20
cules 2021, 26, x FOR 2021,
Molecules PEER26,REVIEW
x FOR PEER REVIEW 9 of 20 9 of 20

Figure 7. (a) The 2D fingerprint

Figure 7.
7. (a)(a)
The plots
2D
The of the compound
fingerprint
2D fingerprintplots 5c
of the
plots with
ofcompounda percentage
the compound 5c with 5cof interaction.
awith
percentage (b)interaction.
of
a percentage Theof interaction.
(b) The
short contact contributions
short contact derived from
contributions H· ··
H, O· ··
H/H·
derivedderived ·
·O,
from H·from C·
··H, O···H/H· · ·
C, Br···
H/H· ·
· Br, O· ·
· C/C· ··O,
(b) The short contact contributions H····
·H/H·
H, O ··O, C···H/H·
···H/H ···O,··C
C,···Br···H/H·
H/H ···C,··Br, O·
Br··· ··C/C·
H/H ·····Br,
O,
and C···C contacts.
and The
C···C···values mentioned
contacts. in mentioned
the pie chart inare inpie
percentage form.
O···C/C O, and CThe
···C values
contacts. The values the
mentioned chartinare
theinpie
percentage
chart are in form.
percentage form.

2.4. Energy Framework Calculation

2.4. Energy Framework Calculation
Furthermore,Furthermore,
the Crystal Explorer 17.5Explorer
Crystal
the Crystal software17.5
Explorer wassoftware
17.5 used to wasevaluate
usedthe interaction
to evaluate the interaction
energies for energies
the crystal
energies structure 5c. Energy frameworks have a strong and
for the crystal structure 5c. Energy frameworks have a strong and remarkable
for the crystal structure 5c. Energy frameworks have a remarkable
strong and remarkable
way of imagining
way the
of supramolecular
imagining
way of imagining the existence ofexistence
the supramolecular
supramolecular molecular
existenceofof crystal
molecularstructures.
molecular crystal The inter- The
crystalstructures.
structures. Theinterac-
inter-
action energies between
tion energies
action the
energies molecules
between
betweenthe are obtained
themolecules
moleculesare using monomer
areobtained
obtainedusing wave
usingmonomer functions
monomerwave at the
wavefunctions
functions atat the
the
B3LYP/6-31GB3LYP/6-31G
(d, p) level. [62].
B3LYP/6-31G (d,p)
(d, p)As prescribed,
level.
level. [62]. As
[62]. Asthe tube sizethe
prescribed,
prescribed, used
the in size
tube
tube all
sizethe energy
used
used allframe-
in all
in the energy
the energy frame-
frame-
works was 80 (scalewas
works
works factor),
was 80 and factor),
80 (scale
(scale the cutoff
factor), andfor
and the the
the energy
cutoff
cutoff for threshold
forthe
theenergy
energy value was value
threshold
threshold setvalue
to was
zero.
wassetset
to zero. In
to zero.
In the 3D topological
the 3D images,
topological the diameter
images, the of the
diameter tubeof cylinder
the tube reflects
cylinder the interaction
reflects
In the 3D topological images, the diameter of the tube cylinder reflects the interaction en- the en-
interaction energy
in theinmolecular
ergy in the molecular
ergy thepacking packing
molecular for the for thefor
corresponding
corresponding
packing interaction.
interaction.
the corresponding The The molecules
molecules
interaction. Thepresent presentpresent
molecules within
the
within the 3.8 Å 3.8 Å
circle circle
within within
1 × 1 ×1 1× 1
unit × 1
cell unit cell
dimensions dimensions
were were
selected selected
for this
within the 3.8 Å circle within 1 × 1 × 1 unit cell dimensions were selected for this calcula- for this
calcula- calculation
(Figure
tion (Figure 8a).
tion 8a). 8a).
(Figure

Figure 8. (a) Selected

Figure 8.molecules for molecules
(a) Selected 5c presentfor
within 3.8 Å and cylindrical
5c present Å andtube formation for the
cylindrical
within 3.8 Å cylindrical tube formation for the
coulombic energy as red
coulombic tubes (b), for dispersion energy as green tubes (c), and tubesenergy
total andastotal
blueenergy as blue
coulombic energy
energy as
as red
red tubes
tubes (b),
(b), for
for dispersion energy as
dispersion energy as green
green tubes (c), and
(c), total energy as blue
tubes (d). tubes (d).
tubes (d).
Energies between molecular pairs
Energies are expressed as cylinders asthat connect that
molecular
Energies between
between molecular
molecular pairs
pairs are
are expressed cylinders
expressed as cylinders connect molecular
that connect molecular
pair centroids with a cylindrical radius proportional to the energy
pair centroids with a cylindrical radius proportional to theinteraction
the energy magnitude.
energy interaction
interaction magnitude.
magnitude.
The energy framework
The energywas outlinedwas
framework as red cylinders
outlined forcylinders
as red Eelec, green
forcylinders forcylinders
Eelec, green Edis, and for Edis, and
Molecules 2021, 26, 3550 10 of 20

The energy framework was outlined as red cylinders for Eelec , green cylinders for Edis , and
blue cylinders for Etot , as shown in (Figure 8b–d), and the relative strength of molecular
packing was expressed in various directions by these tubes. The supramolecular nature
of the crystal structure was, thus, visualized by energy structures in a special way. The
calculated energy values are listed in Table 4 for electrostatic, polarization, dispersion,
and total interaction energy, which suggests that 5c crystal structure preferred dispersion
energy over others.

Table 4. Interaction energies as obtained from the Crystal Explorer 17.5 (in kJ/mol) for the 5c compound.

Color N Symop R E_ele E_pol E_dis E_rep E_tot

1 - 4.55 −22.8 −5.1 −98.5 37.6 −84.7
1 - 14.58 −0.5 0 −0.4 0 −0.8
1 - 11.1 4.2 −5.5 −31.3 16.2 −14.4
1 - 14.41 0.2 0 −0.3 0 −0.1
1 - 13.98 0 0 −1.3 0 −1.3
1 - 17.79 0.2 0 −0.2 0 0
1 - 11.16 1 −4.4 −22.6 8.9 −15
1 - 16.73 0.1 0 −0.1 0 0
1 - 18.83 0.3 0 −0.2 0 0.2
1 - 12.4 0.8 0 −0.8 0 0.1
1 −x + 1/2, y + 1/2, −z + 1/2 11.15 −16.2 −1.2 −22.8 49.6 2.5
1 x + 1/2, −y + 1/2, z + 1/2 12.57 −0.6 0 −0.6 0 −1.2
1 −x, −y, −z 18.25 0.3 0 −0.1 0 0.2
2 −x + 1/2, y + 1/2, −z + 1/2 12.35 −0.3 −0.1 −2.1 0 −2.3
1 x + 1/2, −y + 1/2, z + 1/2 10.75 −2.2 −0.3 −3.1 0 −5.2
1 x, y, z 12.05 −11.3 −2.6 −26.1 14.3 −25.2
1 −x, −y, −z 23.43 0.1 0 0 0 0.1
1 x, y, z 17.83 −0.1 0 −0.2 0 −0.3
1 −x, −y, −z 16.07 −0.3 0 −0.1 0 −0.4
1 x + 1/2, −y + 1/2, z + 1/2 22.54 0 0 0 0 0

2.5. Pharmacology
The COX-2-inhibitory activity of the target compounds (5a–5e) is presented in Table 5.
As can be seen from Table 5, all the indolizines displayed interesting inhibitory activity
against COX-2 similar to the commercially available drug indomethacin. Compound 5a
with a 4-cyanobenzoyl group attached to the 3-position of the indolizine scaffold, having
two ethyl carboxylate groups attached to the 1- and 2-position of the scaffold, emerged as
the most promising compound with the highest COX-2-inhibitory activity (IC50 = 5.84 µM).
Replacement of the electron-withdrawing nitrile group with halogens such as fluorine and
bromine atoms at the 4-position of the benzoyl ring exhibited detrimental COX-2 inhibitory
activity for compounds 5b and 5c with IC50 values 6.73 µM and 6.99 µM, respectively.
It is remarkable to note that title compound 5e with only one ethyl ester moiety at the
first position of the indolizine pharmacophore exhibited a further reduction in activity
(IC50 = 7.38 µM) as compared to the structurally similar compound 5c (IC50 = 6.99 µM).
The compound 5d, substituted with a methoxy functional group at the 3-position
of the benzoyl ring, displayed the least inhibitory activity (IC50 = 8.49 µM). In general,
the availability of the electron-withdrawing functional groups at the para position of the
benzoyl ring was found to be favorable for COX-2-inhibitory activity as compared to the
presence of the electron-donating functional groups at the meta position. Previously these
derivatives demonstrated excellent safety profiles [35]; thus, they could be considered as
lead molecules for further improvement of novel potential COX-2 inhibitors.
Molecules 2021, 26, 3550 11 of 20
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Molecules 2021, 26, x FOR PEER REVIEW 11 of 20
Molecules 2021, 26, x FOR PEER REVIEW 11 of 20
Molecules 2021, 26, x FOR PEER REVIEW 11 of 20
Table 5. In vitro inhibitory study of cyclooxygenase-2 (COX-2) on diethyl 7-methoxy-3-(3-substitued-
Molecules 2021, 26, x FOR PEER REVIEW 11 of 20
Table 5. In vitro inhibitory study of cyclooxygenase-2
benzoyl)-indolizine-1,2-dicarboxylates (5a–d) and ethyl(COX-2) on diethyl 7-methoxy-3-(3-substit-
3-(4-bromobenzoyl)-2-ethyl-7-methoxy-
Table 5. In vitro inhibitory study of cyclooxygenase-2
ued-benzoyl)-indolizine-1,2-dicarboxylates (5a–d) and(COX-2) on diethyl 7-methoxy-3-(3-substit-
ethyl 3-(4-bromobenzoyl)-2-ethyl-7-meth-
indolizine-1-carboxylate (5e).study of cyclooxygenase-2
Table 5. In vitro inhibitory
ued-benzoyl)-indolizine-1,2-dicarboxylates (5a–d) and (COX-2) on diethyl 7-methoxy-3-(3-substit-
ethyl 3-(4-bromobenzoyl)-2-ethyl-7-meth-
oxy-indolizine-1-carboxylate
Table (5e). of cyclooxygenase-2
5. In vitro inhibitory study (COX-2) on diethyl 7-methoxy-3-(3-substit-
ued-benzoyl)-indolizine-1,2-dicarboxylates
oxy-indolizine-1-carboxylate
Table 5. In (5a–d) and ethyl 3-(4-bromobenzoyl)-2-ethyl-7-meth-
(5e). of cyclooxygenase-2 (COX-2) on diethyl 7-methoxy-3-(3-substit-
vitro inhibitory study
ued-benzoyl)-indolizine-1,2-dicarboxylates
Compound
oxy-indolizine-1-carboxylate (5e). Compound (5a–d)Structure
Compound and ethyl 3-(4-bromobenzoyl)-2-ethyl-7-meth-
ICIC * *(µM)
50 50
Compound
ued-benzoyl)-indolizine-1,2-dicarboxylates (5a–d)Structure (µM)
and ethyl 3-(4-bromobenzoyl)-2-ethyl-7-meth-
oxy-indolizine-1-carboxylate
Compound (5e). Compound Structure IC50 * (µM)
oxy-indolizine-1-carboxylate
Compound (5e). Compound Structure IC50 * (µM)
Compound Compound Structure IC50 * (µM)
Compound Compound Structure IC50 * (µM)

5a
5a 5.84
5.84 ±± 0.03
0.03 a a
5a 5.84 ± 0.03 a
5a 5.84 ± 0.03 a
5a 5.84 ± 0.03 a
5a 5.84 ± 0.03 a

5b 6.73 ± 0.03 a,b

5b
5b 6.73
6.73 a,b a,b
± 0.03
± 0.03
5b 6.73 ± 0.03 a,b
5b 6.73 ± 0.03 a,b
5b 6.73 ± 0.03 a,b

5c 6.99 ± 0.03 b
5c 6.99 ± 0.03 b
5c
5c 6.99
6.99 b b
± 0.03
± 0.03
5c 6.99 ± 0.03 b
5c 6.99 ± 0.03 b

5d 8.49 ± 0.03, b,c

5d 8.49 ± 0.03, b,c
5d 8.49 ± 0.03, b,c
5d
5d 8.49
8.49 b,c b,c
± 0.03,
± 0.03,
5d 8.49 ± 0.03, b,c

5e 7.38 ± 0.03 c,d

5e 7.38 ± 0.03 c,d
5e 7.38 ± 0.03 c,d
5e 7.38 ± 0.03 c,d
5e
5e 7.38
7.38 c,d c,d
± 0.03
± 0.03

Indomethacin 6.84 ± 0.03 b,d

Indomethacin 6.84 ± 0.03 b,d
Indomethacin 6.84 ± 0.03 b,d
Indomethacin 6.84 ± 0.03 b,d
Indomethacin 6.84 ± 0.03 b,d
Molecules 2021, 26, 3550 12 of 20

5e 7.38 ± 0.03 c,d

Table 5. Cont.

Compound Compound Structure IC50 * (µM)

Indomethacin
Indomethacin 6.84
6.84 b,d b,d
± 0.03
± 0.03

Molecules 2021, 26, x FOR PEER REVIEW 12 of 20

Celecoxib
Celecoxib 0.05
0.05 ± ±0.03 a a
0.03

* IC50 value is defined as the concentration of test and standard substances required to produce
* IC50 value is defined as the concentration of test and standard substances required to produce 50% inhibition of
50% inhibition
human recombinant of COX-2
humanenzyme
recombinant
by means COX-2
of threeenzyme by means
determinations usingofthe
three determinations
enzyme-linked immuneusing the
sorbent
enzyme-linked
assay immune sorbent
kit. a–d Title compounds assay
not sharing a letter Title
kit. a–dvary compounds
significantly (p <not sharing a letter vary signifi-
0.05).
cantly (p < 0.05).
2.6. Computational Studies
The compound
Molecular docking5d, studies
substituted with a methoxy
are considered functionalin
an invaluable group
silicoatapproach
the 3-position of
to cor-
the benzoyl
relate ring,structure–activity
the in vitro displayed the least inhibitory (SAR)
relationship activityof(IC 50 = 8.49 µM). In general, the
chemical compounds [63]. To
gain insight into the inhibitory activity of indolizines (5a–e),the
availability of the electron-withdrawing functional groups at wepara position of
investigated the key
their ben-
zoyl ring was found to be favorable for COX-2-inhibitory activity as compared
interactions with the COX-2 receptor through a computational approach. The docking to the pres-
ence of
study theconducted
was electron-donating functional
with Accelrys groupsStudio
Discovery at the meta position.
Client Previously
4.0 software. these de-
The docking
interaction energies and the residue interactions of indolizines 5a–e and indomethacin areas
rivatives demonstrated excellent safety profiles [35]; thus, they could be considered
lead molecules
reported in Tablefor
6. further
All the improvement of novel potential
compounds demonstrated COX-2
favorable inhibitors.
docking energy ranging
from −38.22 to −53.29 kcal/mol, indicating that they have a good binding affinity with the
2.6. Computational
COX-2 receptor, as Studies
demonstrated from their biological activities.
The predicted
Molecular docking
docking poses
studies ofconsidered
are indolizinesan 5a–e and indomethacin
invaluable are depicted
in silico approach in
to corre-
Figure
late the9.inAll thestructure–activity
vitro compounds adopted a similar
relationship (SAR)conformation
of chemicaltocompounds
that of indomethacin,
[63]. To gain
where
insightthe indolizine
into ring is activity
the inhibitory taken into
of aindolizines
sandwich between
(5a–e), we theinvestigated
amino-acid their
residues
key Ala527,
interac-
Val523 and the
tions with Val349,
COX-2 Leu352. Thethrough
receptor benzoyla ring is orientedapproach.
computational toward the residues
The dockingTyr385
studyandwas
Trp387, while the methoxy group is located in the deep region of
conducted with Accelrys Discovery Studio Client 4.0 software. The docking interaction the receptor. As can
be observed
energies andfrom the binding
the residue poses, indolizines
interactions of indolizines 5a–eand
5a, 5b, demonstrated
and5dindomethacin favorable
are reported
hydrogen
in Table 6. bonding
All theinteraction
compounds between Arg120 with
demonstrated the ester
favorable groupenergy
docking at the 1-position
ranging from of
the indolizine
−38.22 to −53.29scaffold, while
kcal/mol, indomethacin
indicating showed
that they have hydrogen bonding
a good binding and ionic
affinity bonding
with the COX-
interactions
2 receptor, asbetween the samefrom
demonstrated residue
theirArg120 and its
biological carboxylic acid group. This indicated
activities.
that the ionic interaction with Arg120 is not a requirement for maintaining the potency
Table 6. Docking results ofofindolizines
the compounds.
(5a–e) andFurthermore, the indolizines
indomethacin against 5c and(COX-2)
cyclooxygense-2 5e containing
receptor a(PDB
bromine
4COX). atom
at the para position of the benzoyl ring showed no hydrogen bonding involvement with
the residue Arg120. Therefore, the major contribution to the COX-2 activity of our com-
pounds principally involves hydrophobic interactions with the indolizine ring and with
the substituents at positions 2 and 3 of indolizine. It can be noted that only the bromine
substituent on the benzoyl ring (5c and 5e) demonstrated hydrophobic interactions with
residues Leu384 and Met522, while pi–pi interactions were observed for all indolizines
with the exception of fluoro indolizine 5b and indomethacin. Therefore, the substituent
in the benzoyl ring has a minor contribution to the activity of indolizine. However, it has
been demonstrated that the benzoyl ring on indomethacin is important to bioactivity since
CDocker of N-benzoyl by N-benzylResidues
the replacement led to a reduction
Interactionin COX-2 inhibition [64].
Entry R1 R2 Interaction pi-alkyl Alkyl-alkyl
H-bonding pi–pi
Energy pi-halogen Alkyl-halogen
Trp387 Val349, Leu352, Val116, Val349,
5a CO2Et 4-CN 39.51 Arg120
Gly526 Ala527, Val523, Tyr355 Leu531, Leu534
 tions with the COX-2 receptor through a computational approach. The docking study was
conducted with Accelrys Discovery Studio Client 4.0 software. The docking interaction
energies and the residue interactions of indolizines 5a–e and indomethacin are reported
in Table 6. All the compounds demonstrated favorable docking energy ranging from
Molecules
Molecules 2021,
2021, 26,
26, x3550
FOR PEER REVIEW 13
13 of
of 20
20
−38.22 to −53.29 kcal/mol, indicating that they have a good binding affinity with the COX-
2 receptor, as demonstrated from their biological activities.

Table 6. Docking results of indolizines (5a–e) and indomethacin against cyclooxygense-2 (COX-2) receptor (PDB 4COX).
Table 6. Docking results of indolizines (5a–e) and indomethacin Tyr385
against cyclooxygense-2 (COX-2) receptor
Val349, Leu352, (PDB 4COX).
Val116, Leu359,
5d CO2Et 3-OCH3 53.29 Arg120 Trp387
Ala527, Val523, Tyr355 Leu531
Gly526
Val349, Leu352, Val116, Val349,
Ala527, Val523, Leu359, Leu 531,
5e Et 4-Br 48.54 - Trp387
Tyr385, Trp387, Leu534, Leu384,
Phe381 Met522
Val349, Ala527,
Val349, Leu352,
Indomethacin 55.36 Arg120 (ionic) Leu531, Leu384,
Ala527, Val523, Trp387
CDocker Residues Interaction Met522
CDocker Residues Interaction
Entry
Entry R1 R1 R 2R2 Interaction
Interaction H-bonding pi-alkyl
pi-alkyl Alkyl-alkyl
Alkyl-alkyl
The predicted docking poses
H-Bonding pi–pi
of indolizines
pi–pi 5a–e and indomethacin are depicted in
Energy
Energy pi-halogen
pi-halogen Alkyl-halogen
Alkyl-halogen
Figure 9. All the compounds adopted a similar conformation to that of indomethacin,
Trp387 Val349, Leu352, Val116, Val349,
5a CO2Et 4-CN where39.51 the indolizineArg120
ring is taken into a sandwichVal349,
Trp387
Leu352,
between the amino-acid
Val116, residues
Val349,
5a CO2 Et 4-CN 39.51 Arg120 Gly526 Ala527, Val523, Ala527,Tyr355
Val523, Leu531, Leu534
Ala527, Val523 and Val349, Leu352. The benzoyl ring is oriented toward
Gly526 the Leu534
Leu531, residues
Tyr355
Val349, Leu352, Val116, Leu359,
5b CO2Et 4-F Tyr385 and Trp387,Arg120
38.22 while the methoxy group is located in the deep region of the receptor.
As can be observed from the binding poses, Ala527, Val523,
5a,Tyr355
Val349,
indolizines Leu352, Leu531
5b, and 5d demonstrated
Val116, Leu359,fa-
5b CO2 Et 4-F 38.22 Arg120 Ala527,
Val349, Val523,
Leu352, Val116, Val349,
vorable hydrogen bonding interaction between Arg120 with Tyr355
the ester groupLeu531
at the 1-po-
sition of the indolizine scaffold, while Ala527,
indomethacin Val523,
showed hydrogen Leu359,
bonding Leuand531,
ionic
5c CO2Et 4-Br 46.69 - Tyr385 Val349, Leu352, Val116, Val349,
bonding interactions between the same residue Tyr385,Arg120 Trp387, Leu534,
and its carboxylic Leu384,This
acid group.
Ala527, Val523, Leu359, Leu 531,
5c CO2 Et 4-Br indicated46.69 -
that the ionic interaction withTyr385 Phe381
Arg120 is not a requirement Met522
Tyr385, Trp387, forLeu534,
maintaining
Leu384,the
potency of the compounds. Furthermore, the indolizinesPhe381 5c and 5e containing a bromine
Met522
atom at the para position of the benzoyl ring showed no hydrogen bonding involvement
Tyr385 Val349, Leu352,
3- with the residue Arg120. Therefore, the major contribution to the COX-2 Val116, Leu359,
activity of our
5d CO2 Et 53.29 Arg120 Trp387 Ala527, Val523,
OCH3 compounds principally involves hydrophobic interactions with the indolizine Leu531ring and
Gly526 Tyr355
with the substituents at positions 2 and 3 of indolizine. It can
Val349, be noted that
Leu352, onlyVal349,
Val116, the bro-
mine substituent on the benzoyl ring (5c and 5e) demonstrated hydrophobic
Ala527, Val523, Leu359,interactions
Leu 531,
5e Et 4-Br 48.54 - Trp387
with residues Leu384 and Met522, while pi–pi interactions
Tyr385,were observed
Trp387, for allLeu384,
Leu534, indoliz-
Phe381
ines with the exception of fluoro indolizine 5b and indomethacin. Therefore,Met522
the substit-
uent in the benzoyl ring has a minor contribution to Val349,
the activity of indolizine.
Leu352, Val349,However,
Ala527,
Arg120
Indomethacin it has been demonstrated that the benzoyl ring on indomethacin
55.36 is important
Ala527, Val523, to bioactivity
Leu531, Leu384,
(ionic)
since the replacement of N-benzoyl by N-benzyl led to aTrp387 reduction in COX-2 Met522
inhibition
[64].

Indolizine 5a Indolizine 5b
Figure 9. Cont.
Molecules 2021, 26, 3550 14 of 20
Molecules 2021, 26, x FOR PEER REVIEW 14 of 20

Indolizine 5c Indolizine 5d

Indolizine 5e Indomethacin
Figure
Figure 9.9. Predicted
Predicted docking
dockingpose
poseof
ofindolizines
indolizines(5a–e)
(5a–e)and
andindomethacin
indomethacin(salmon-filled
(salmon-filledspheres)
spheres)ininthe
thecyclooxygense-2
cyclooxygense-
COX-2
2 COX-2 domain (PDB 4COX). Hydrogen bonding and pi–pi interactions are represented in greenviolet
domain (PDB 4COX). Hydrogen bonding and pi–pi interactions are represented in green and dotteddotted
and violet lines,
respectively.
lines, respectively.

The
The molecular
molecular modeling
modeling studystudy provided
provided insight
insight into
into the
the structural
structural requirement
requirement of of
indolizines
indolizines for COX-2-inhibitory activity. Moreover, indolizines 5a–e are more likely
for COX-2-inhibitory activity. Moreover, indolizines 5a–e are more likely to
to be
be
selective COX-2inhibitors.
selective COX-2 inhibitors.ItIthas
has been
been validated
validated thatthat
COX-1 COX-1 and COX-2
and COX-2 selectivity
selectivity is mainlyis
mainly dueionic
due to the to theinteraction
ionic interaction with residue
with residue Arg120Arg120 since
since the the corresponding
corresponding ester andester and
amide
amide of indomethacin
of indomethacin derivatives
derivatives presented
presented good selectivity
good selectivity in favor
in favor of COX-2
of COX-2 inhibition
inhibition [64].
[64].
3. Materials and Methods
3. Materials
3.1. Chemistryand Methods
3.1. Chemistry
All the commercially offered chemicals and solvents were purchased from Sigma-
AldrichAll Co. (St. Louis, MO,
the commercially USA).chemicals
offered All the chemical reactions
and solvents were
were performed
purchased fromin hot-air-
Sigma-
dried glassware
Aldrich in theMO,
Co. (St. Louis, presence
USA).ofAll
a nitrogen atmosphere
the chemical reactionsconsuming dry solvents.
were performed A
in hot-air-
Shimadzu FT-IR spectrophotometer (Columbia, MD, USA) was used to record
dried glassware in the1presence13of a nitrogen atmosphere consuming dry solvents. A Shi- the FT-IR
spectra.FT-IR
madzu Furthermore, H- and C-NMR
spectrophotometer spectraMD,
(Columbia, wereUSA)
documented
was usedat ambient temperature
to record the FT-IR
on Bruker AVANCE III 400 MHz instruments (San Jose, CA, USA) using
spectra. Furthermore, H- and C-NMR spectra were documented at ambient temperature
1 13 CDCl3 and
DMSO-d
on Bruker 6 as solvents.III An
AVANCE 400Agilent 1200 series (San
MHz instruments instrument (Santa
Jose, CA, USA)Clara,
usingCA,
CDClUSA) in
3 and
conjunction with a 6140 single-quadrupole mass spectrometer using positive and negative
DMSO-d6 as solvents. An Agilent 1200 series instrument (Santa Clara, CA, USA) in con-
ESI mode with a mass selective detector (MSD) range of 100–2000, as well as 0.1% aqueous
junction with a 6140 single-quadrupole mass spectrometer using positive and negative
trifluoroacetic acid in an acetonitrile system on the C18-BDS column, was used to record
ESI mode with a mass selective detector (MSD) range of 100–2000, as well as 0.1% aqueous
liquid chromatography–mass spectrometry (LC–MS) spectra. Then, an elemental analysis
trifluoroacetic acid in an acetonitrile system on the C18-BDS column, was used to record
was carried out using the analyzer FLASH EA 1112 CHN (Thermo Finnigan LLC, New
liquid chromatography–mass spectrometry (LC–MS) spectra. Then, an elemental analysis
York, NY, USA). A single-crystal X-ray diffraction study was performed using a Bruker
was carried out using the analyzer FLASH EA 1112 CHN (Thermo Finnigan LLC, New
Molecules 2021, 26, 3550 15 of 20

KAPPA APEX II DUO diffractometer (Madison, WI, USA) equipped with a charge-coupled
device (CCD) detector; monochromated Mo Kα radiation (λ = 0.71073 Å) was used. Data
collection was carried out using an Oxford Cryostream cooling system featuring the Bruker
Apex II software (Madison, WI, USA) at 173(2) K [16].

3.2. General Synthetic Procedure for 4-methoxy-1-(2-(substituted phenyl)-2-oxoethyl)pyridinium

Bromides (3a–e)
To a solution of 4-methoxypyridine (1) (0.0091 mol, 1 g) in dry acetone solvent (10 mL),
substituted-phenacylbromide (0.0091 mol, 2.03 g) was added and agitated at room temper-
ature for 5 h. The completion of the reaction was observed on thin-layer chromatography
(TLC). The product obtained was separated, filtered, and desiccated under vacuum to yield
92–99% 1-(2-(substituted phenyl)2-oxoethyl)-4-methoxypyridinium bromides.

3.3. Synthetic Procedure for the Synthesis of Ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-

1-carboxylate (5e)
To a stirred solution of 1-(2-(4-bromophenyl)-2-oxoethyl)-4-methoxypyridinium bro-
mide (3e) (0.0026 mol, 1 g), in dry dimethylformamide, ethyl pent-2-ynoate (4) (0.0025 mol,
0.512 g) and K2 CO3 (0.0051 mol, 0.713 g) were added. It was stirred at room temperature
for 30 min. The completion of the reaction was monitored on TLC. After completion of the
reaction, the solvent was evaporated under reduced pressure and diluted with ethyl acetate.
The organic layer was washed with water, brine, and dried with sodium sulfate. The crude
compound was purified by column chromatography to afford a 69% yield of compound
5e. The physicochemical characteristics are tabulated in Table 1. Appearance: light-yellow
crystalline compound. FT-IR (KBR neat cm−1 ): 1699, 1668, 1639, 1602. 1 H-NMR (400 MHz
CDCl3 ) δ = 9.18 (d, J = 7.2 Hz, 1H), 7.69–7.60 (m, 3H), 7.10–7.05 (m, 2H), 6.57–6.54 (m, 1H),
4.30 (q, J = 7.2 Hz, 2H), 3.86 (s, 3H), 2.57 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H), 0.91 (t,
J = 7.2 Hz, 3H). 13 C-NMR (100 MHz CDCl3 ) δ = 186.00, 166.07, 165.01, 163.56, 159.37, 144.79,
142.77, 137.58, 137.55, 130.95, 130.86, 129.61, 121.61, 121.10, 115.64, 115.42, 108.20, 102.82,
97.57, 59.64, 55.57, 20.06, 15.99, 14.41. Analysis calculated for C21 H20 BrNO4 : C, 58.62; H,
4.68; N, 3.26; found: C, 58.69: H, 4.52: N, 3.24. The spectra are available as Electronic
Supplementary Materials.

3.4. Crystallography
Single-crystal X-ray diffraction data of 5c were collected on a Bruker KAPPA APEX II
DUO diffractometer using graphite-monochromated Mo-Kα radiation (χ = 0.71073 Å). Data
collection was carried out at 173(2) K. Oxford Cryostream was used to control temperature
(Oxford Cryostat). The cell refinement and data reduction for 5c were performed using the
program SAINT [65], and the absorption correction was performed using SADABS [65].
The crystal structure of 5c was solved by direct methods using SHELXS-18 [66] and
refined by the full-matrix least-squares method based on F2 using SHELXL-2018 [66]. The
program WinGx [67] was used to prepare molecular graphic images. All non-hydrogen
atoms were refined anisotropically, and all hydrogen atoms were placed in idealized
positions and refined in riding models with Uiso assigned 1.2 or 1.5 times Ueq of the parent
atoms [67]. The C–H bond distances was constrained to 0.95 Å for aromatic hydrogen and
1.00 Å for methyl hydrogen. The crystallographic parameters are listed in Table 2.

3.5. In Vitro COX-2 Inhibition Assay

The title compounds 5a–e were tested for in vitro human recombinant COX-2 enzyme
inhibition activity following our previously reported protocol [68].

3.6. Computational Studies

The computational study for test compounds 5a–e was conducted with Accelrys
Discovery Studio Client 4.0 (Waltham, MA, USA) using the indomethacin crystal structure
PDB 4COX following our previously reported protocol [68].
Molecules 2021, 26, 3550 16 of 20

4. Conclusions
In this study, a series of diethyl 7-methoxy-3-(3-substituted benzoyl)indolizine-1,2-
dicarboxylate derivatives (5a–d) and ethyl 3-(4-bromobenzoyl)-2-ethyl-7-methoxyindolizine-
1-carboxylate (5e) were synthesized and evaluated for the inhibition of COX-2 enzyme
activity. All the compounds were demonstrated to be active inhibitors of COX-2, with
the most active compound (5a) having an IC50 value comparable to that of indomethacin,
a marketed COX inhibitor. Computational studies were conducted to analyze the key
interactions of these compounds with the amino-acid residues of the COX-2 receptor.
Hydrophobic interactions were observed to be mainly responsible for the inhibitory COX-
2 activity of indolizines. The compound 5c was crystallized in a monoclinic crystal system
with space group P 21 /n. The molecule was observed to have both intra- and intermolecu-
lar hydrogen bonds and exhibited C–H···π interactions for stability. In order to understand
and visualize the contribution of different intermolecular interactions, Hirshfeld surface
analysis with 2D fingerprint plots was carried out to provide insight into the stability of
the crystal structure. In terms of electrostatic, dispersion, and total energy, the systematic
and theoretical energy was calculated using the software program Crystal Explorer, which
further provided 3D topological images. Indolizines 5a–e could be considered as lead
compounds for developing novel COX-2 inhibitors.

Supplementary Materials: The following are available online: Figure S1. FT-IR of FT-IR of di-
ethyl 3-(4-cyano benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5a); Figure S2. 1 H-NMR of di-
ethyl 3-(4-cyano benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5a); Figure S3. 13 C-NMR of di-
ethyl 3-(4-cyano benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5a); Figure S4. FT-IR of diethyl
3-(4-fluoro benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5b); Figure S5. 1 H-NMR of diethyl
3-(4-fluoro benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5b); Figure S6. 13 C-NMR of diethyl
3-(4-fluoro benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5b); Figure S7. FT-IR of diethyl 3-
(4-bromobenzoyl)7-methoxyindolizine-1,2-dicarboxylate (5c); Figure S8. 1 H-NMR of diethyl 3-(4-
bromobenzoyl)7-methoxyindolizine-1,2-dicarboxylate (5c); Figure S9. 13 C-NMR of diethyl 3-(4-
bromobenzoyl)7-methoxyindolizine-1,2-dicarboxylate (5c); Figure S10. FT-IR of diethyl 7-methoxy-3-
(3-methoxybenzoyl)indolizine-1,2-dicarboxylate (5d); Figure S11. 1 H-NMR of diethyl 7-methoxy-3-(3-
methoxy benzoyl)indolizine-1,2-dicarboxylate (5d); Figure S12. 13 C-NMR of diethyl 7-methoxy-3-(3-
methoxy benzoyl)indolizine-1,2-dicarboxylate (5d); Figure S13. FT-IR of ethyl 3-(4-bromobenzoyl)-
2-ethyl-7-methoxyindolizine-1-carboxylate (5e); Figure S14. 1 H-NMR of ethyl 3-(4-bromobenzoyl)-
2-ethyl-7-methoxyindolizine-1-carboxylate (5e); Figure S15. 13 C-NMR of ethyl 3-(4-bromobenzoyl)-
2-ethyl-7-methoxyindolizine-1-carboxylate (5e); Figure S16. checkCIF/PLATON report of check-
CIF/PLATON report of diethyl-3-(4-bromo benzoyl)7-methoxyindolizine-1,2-dicarboxylate (5c).
Author Contributions: Conceptualization, K.N.V. and C.T.; methodology, K.N.V., R.D.N., S.K.N.,
P.B., C.T., S.C., M.A., M.H., V.M., R.V., M.K., B.P.N. and P.S.; software, K.N.V., P.K.D., S.K.N. and
C.T.; validation, K.N.V., P.K.D., R.D.N., C.T., S.C. and N.S.; formal analysis, K.N.V., F.M.M., C.T.,
S.C. and M.H.; investigation, K.N.V., R.D.N., S.C., R.P.M. and A.B.N.; resources, K.N.V., P.K.D., S.C.,
S.K.N., R.P.M., M.A.M., B.E.A., M.A., N.S., O.I.A., R.V., M.K., B.P.N. and Y.F.I.; data curation, K.N.V.
and M.A.M.; writing—original draft preparation, K.N.V., P.K.D., S.K.N., P.B., R.D.N., F.M.M., C.T.,
S.C., R.P.M., M.A.M., B.E.A., M.A., A.B.N., N.S., O.I.A., M.H., V.M., P.S., R.V., M.K., B.P.N. and Y.F.I.;
writing—review and editing, K.N.V., S.C., P.K.D., S.K.N., P.B., F.M.M., C.T., M.A.M., B.E.A., A.B.N.,
O.I.A., V.M., P.S., R.V., M.K., B.P.N. and Y.F.I.; visualization, K.N.V. and C.T.; supervision, K.N.V. and
S.K.N.; project administration, K.N.V.; funding acquisition, K.N.V. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at King Faisal University,
Al-Ahsa, Saudi Arabia (Nasher Track Grant No. 186333).
Data Availability Statement: Crystal data of compound 5c were deposited in the Cambridge Crys-
tallographic Data Center (CCDC) (https://www.ccdc.cam.ac.uk/, accessed on 18 September 2020)
with number 2045116.
Molecules 2021, 26, 3550 17 of 20

Acknowledgments: The authors acknowledge the Deanship of Scientific Research at King Faisal
University, Kingdom of Saudi Arabia, for their financial support under Nasher Track (Grant No.
186333) and their encouragement. The authors thank Hong Su, Center for Supramolecular Chemistry
Research, Department of Chemistry, University of Cape Town, Rondebosch 7701, for single-crystal
X-ray data collection. R.D.N and S.K.N. thank UGC-New Delhi for providing financial assistance
under UGC-JRF (JUNE18-136885) through a fellowship and the Department of Chemistry, VNIT,
Nagpur for research facilities and infrastructure.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.

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