New carboxamide derivatives bearing benzenesulphonamide as a selective COX-II inhibitor: Design, synthesis and structure-activity relationship

Article abstract of DOI:10.1371/journal.pone.0183807

Sixteen new carboxamide derivatives bearing substituted benzenesulphonamide moiety (7a-p) were synthesized by boric acid mediated amidation of appropriate benzenesulphonamide with 2-amino-4-picoline and tested for anti-inflammatory activity. One compound 7c showed more potent anti-inflammatory activity than celecoxib at 3 h in carrageenan-induced rat paw edema bioassay. Compounds 7g and 7k also showed good anti-inflammatory activity comparable to celecoxib. Compound 7c appeared selectivity index (COX-2/COX-1) better than celecoxib. Compound 7k appeared selectivity index (COX-2/COX-1) a little higher than the half of celecoxib while compound 7g is non-selective for COX-2. The LD₅₀ of compounds 7c, 7g and 7k were comparable to celecoxib.

Full text of DOI:10.1371/journal.pone.0183807

RESEARCH ARTICLE
New carboxamide derivatives bearing
benzenesulphonamide as a selective COX-II
inhibitor: Design, synthesis and structure-
activity relationship
David Izuchukwu Ugwu1,2*, Uchechukwu Chris Okoro1, Hilal Ahmad2
1 Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka, Nigeria, 2 Department of
Chemistry, Indian Institute of Technology, Kanpur, India
* izuchukwu.ugwu@unn.edu.ng
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Abstract
Sixteen new carboxamide derivatives bearing substituted benzenesulphonamide moiety
(7a-p) were synthesized by boric acid mediated amidation of appropriate benzenesulphona-
mide with 2-amino-4-picoline and tested for anti-inflammatory activity. One compound 7c
showed more potent anti-inflammatory activity than celecoxib at 3 h in carrageenan-induced
rat paw edema bioassay. Compounds 7g and 7k also showed good anti-inflammatory activ-
ity comparable to celecoxib. Compound 7c appeared selectivity index (COX-2/COX-1) bet-
ter than celecoxib. Compound 7k appeared selectivity index (COX-2/COX-1) a little higher
than the half of celecoxib while compound 7g is non-selective for COX-2. The LD50 of com-
pounds 7c, 7g and 7k were comparable to celecoxib.
OPEN ACCESS
Citation: Ugwu DI, Okoro UC, Ahmad H (2017)
New carboxamide derivatives bearing
benzenesulphonamide as a selective COX-II
inhibitor: Design, synthesis and structure-activity
relationship. PLoS ONE 12(9): e0183807. https://
doi.org/10.1371/journal.pone.0183807
Editor: Luis Eduardo M Quintas, Universidade
Federal do Rio de Janeiro, BRAZIL
Received: April 12, 2017
Introduction
Accepted: August 13, 2017
Published: September 18, 2017
The initial stage of transformation of arachidonic acid to prostanoids are catalysed by cycloox-
ygenases (COXs). It exists as three distinct isozymes; cyclooxygenase-1 (COX-I), cyclooxygen-
ase-2 (COX-II) and cyclooxygenase-3 (COX-III). Selective COX-II inhibitors are a class of
potential anti-inflammatory, analgesic and antipyretic drugs with reduced gastrointestinal
(GI) side effects compared to non-selective inhibitors [1–3]. Cyclooxygenase (COX)-2 is one
of the rate-limiting enzyme in the production of prostaglandin from arachidonic acid. Non-
steroidal anti-inflammatory drugs (NSAIDs) are the competitive inhibitors of cyclooxygenase
(COX), the enzyme which mediates the bioconversion of arachidonic acid to inflammatory
prostaglandins (PGs). The inhibition of COX-2 gives rise to the anti-inflammatory activity of
NSAIDs whereas the undesired side effects arise from inhibition of COX-1 activity. Thus, it
was thought that more selective COX-2 inhibitors would have reduced side effects [3]. Based
upon a number of selective COX-2 inhibitors (rofecoxib, celecoxib, valdecoxib etc.) were
developed as safer NSAIDs with improved gastric safety profile [4]. However, the recent mar-
ket removal of some COXIBs such as rofecoxib due to its adverse cardiovascular side effects
clearly encourages researchers to explore and evaluate alternative templates with COX-2
Copyright: © 2017 Ugwu et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: The data are
contained in the manuscript and supporting
document.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors declare that
there is no competing interests.
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Synthesis of new anti-inflammatory agent comparable to celecoxib
inhibitory activity [3]. Recognition of new avenues for selective COX-2 inhibitors in cancer
chemotherapy and neurological diseases such as Parkinson and Alzheimer’s diseases still
continues to attract investigations on the development of COX-2 inhibitors [5]. COX-2 is
induced by stimuli such as mitogens, cytokines, growth factors and tumor promoters, and has
been elucidated to be up-regulated not only at the sites of inflammation but also in various
cancer tissues such as colon, stomach, breast, lung, head and neck including oral cavity [6].
The biosynthesis of prostanoids, which include the prostaglandins (PGs) and thromboxanes,
occurs in three steps: (a) the mobilization of a fatty acid substrate, typically arachidonic acid
(AA), from membrane phospholipids through the action of a phospholipase A2; (b) biotrans-
formation of AA by cyclooxygenase in a bifunctional action which leads to the generation of
unstable PGG2 by the cyclooxygenase reaction, and its immediate conversion into PGH2 by
the same enzyme in a peroxidase reaction; (c) the conversion of PGH2 to specific prostanoids
through the action of synthases and specific isomerases [6]. The successful inhibition of COX-
2 will arrest the synthesis of prostaglandin which has been implicated in varieties of physiologi-
cal and pathophysiological conditions, including inflammation [7].
Sulphonamides have been the centre of drug structures as they are quite stable and well tol-
erated in human beings [8]. Sulphonamides constitute an important class of chemotherapeutic
agents with applications ranging from their traditional antibacterial agent [8] to anticancer [9],
antimalarial [10], anticonvulsant [11], antiretroviral [12], antidiabetic [13], anti-insomnia
[14], anti-inflammatory [15], diuretics [16] and antileukemic [17] agents to mention but a few.
Carboxamides are also ubiquitous functionality in drug molecules as pharmacophore [18].
Carboxamides are present in drug molecules used in the blockage of cholesterol synthesis [19],
treatment of hypertension and angina [20], blockade of angiotensin-II receptors [21], inhibi-
tion of angiotensin converting enzyme [22], treatment of HIV [23], and management of heart
disease [24] to mention but a few.
We therefore exploited the synergistic biological properties arising from the successful
incorporation of carboxamides in substituted benzenesulphonamides in this report.
Experimental
Instrumentation
All reactions requiring inert atmosphere were carried out under nitrogen atmosphere. Drying
of solvents was achieved using molecular sieve for 48 h. All reagents were purchased from
commercial suppliers, Aldrich, Merck, Fluka, Avra, SD fine and Alfa Aesar. Thin layer chro-
matography was carried out using silica plates purchased from Avra. The plates were visualized
under UV light (popular India). FT-IR spectroscopy of the compounds were run in PerkinEl-
mer Spectrum version 10.03.06 and the bands presented in wavenumber. 1H NMR and 13C
NMR spectroscopy were run in DMSOd6 and CD3OD, unless otherwise stated on either Jeol
500 MHz or 400 MHz. The chemical shifts were reported in part per million with reference to
tetramethylsilane. Mass spectroscopy were carried out using micro TOF electrospray time of
flight (ESI-TOF) mass spectrometer, sodium formate was used as the calibrant. All experi-
ments were carried out at Prof. Sandeep Verma’s Laboratory, department of Chemistry, Indian
Institute of Technology, Kanpur. Melting points were determined using digital melting point
apparatus and were uncorrected.
General procedure for the synthesis of substituted benzenesulphonamoyl alkanamides
(3a-p). Sodium carbonate (Na2CO3, 1.590 g, 15 mmol) was added to a solution of amino
acids (2a-h, 12.5 mmol) in water (15 mL) with continuous stirring until all the solutes had dis-
solved. The solution was cooled to -5˚C and the appropriate benzenesulphonyl chloride (1a-c,
15 mmol) was added in four portions over a period of 1 h. The slurry was further stirred at
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Synthesis of new anti-inflammatory agent comparable to celecoxib
room temperature for about 4 h. The progress of the reaction was monitored using TLC
(MeOH/DCM, 1:9). Upon completion, the mixture was acidified using 20% aqueous hydro-
chloric acid to pH 2. The crystals was filtered via suction and washed with pH 2.2 buffer. The
pure products (3a-p) were dried over self-indicating fused silica gel in a desiccator.
General procedure for the synthesis of N-benzoyl derivatives of benzenesulphonamides
(5a-f and 5i-n). Appropriate benzenesulphonamides (3a-f, and 3i-n, 1.0 mmol) was dis-
solved in NaOH (10%, 10 mL) in a 50 mL round bottom flask. Benzoyl chloride (8, 1.1 mmol,
0.2 mL) was transferred into the solution of appropriate benzenesulphonamide and stirred at
room temperature. The reaction progress was monitored by TLC (3% MeOH/CH2Cl2) to the
disappearance of the benzenesulphonamide spot. Upon completion, the solution was trans-
ferred into a beaker containing crushed ice and then acidified to pH of 3 with concentrated
hydrochloric acid. The solid was collected via suction filtration and transferred into a beaker
containing CCl4 (10 mL) and covered with watch glass boiled for 10 min. the mixture was
allowed to cool slightly and then filtered. The products (5a-f and 5i-n) obtained were washed
with 10–20 mL of CCl4 and dried over fused self-indicating silica gel in a dessicator.
Boric acid catalysed direct amidation of unactivated carboxylic acid and 2-amino-4-pic-
oline. To a suspension of N-benzoyl-substituted-benzenesulphonamides (5a-f and 5i-n, 1.0
mmol) in dry toluene (40 mL) equipped with Dean-Stark apparatus for azeotropic removal of
water, was added 4-picoline (1.0 mmol) and boric acid (0.1 mmol) at room temperature and
then refluxed for 6 h. On completion (as monitored by TLC), reaction mixture was precipi-
tated in to amides by adding about 40 mL n-hexane. The carboxamides were obtained via suc-
tion filtration, washed with n-hexane and dried over fused silica gel or concentrated using
rotary evaporator and dried over vacuum in the case of oily products.
N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phenylformamido]acet-
amide (7a)
Appearance: brownish oil; Yield (0.4471 g, 98.5%), FTIR (KBr, cm-1): 3420 (NH), 3064
(C-H aromatic), 2976 (C-H aliphatic), 1701, 1669 (C = O), 1631 (C = N), 1601, 1494, 1454
(C = C), 1334, 1310 (2S = O), 1166, 1123 (SO2N), 1091, 1015 (C-N). 1H NMR (DMSO-d6, 400
MHz) δ: 8.34–8.31 (m, 1H, ArH), 8.24–8.22 (m, 1H, ArH), 8.14–8.09 (m, 2H, ArH), 7.91
(d, J = 6.88 Hz, 2H, ArH), 7.74 (d, J = 8.72 Hz, 2H, ArH), 7.66 (d, J = 5.96 Hz, 2H, ArH), 7.55
(t, J = 7.76 Hz, 1H, ArH), 7.44 (t, J = 7.32 Hz, 2H, ArH), 7.24–7.01 (m, 2H, ArH), 6.43 (s, 2H,
ArH), 3.24 (s, 2H, CH2), 2.17 (s, 3H, CH3). 13C NMR (DMSO-d6, 400 MHz) δ: 173.9, 168.3
(C = O), 157.7, 151.8, 149.5, 147.2, 142.0, 137.7, 129.8, 129.4, 129.0, 128.6, 128.3, 126.8, 124.5,
114.1, 110.4 (fifteen aromatic carbons), 58.9, 21.6 (two aliphatic carbons). HRMS (ESI-TOF,
m/z): 454.0958 (M+), calculated, 454.0947.
(2S)-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phenylformamido]-
3-phenyl propanamide (7b)
Appearance: pale brown solids; Yield (0.5304 g, 97.5%),mp, 156.10–156.80˚C, FTIR (KBr,
cm-1): 3383 (NH), 3034 (C-H aromatic), 2964 (C-H aliphatic), 1773, 1661 (C = O), 1624
(C = N), 1602, 1575, 1446, 1412 (C = C), 1525 (NO2), 1353, 1304 (2S = O), 1199, 1168 (SO2N),
1093, 1044, 1013 (C-N). 1H NMR (DMSO-d6, 400 MHz) δ: 8.36–8.30 (m, 3H, ArH), 8.16 (d,
J = 8.72 Hz, 1H, ArH), 8.02–7.98 (m, 3H, ArH), 7.90 (d, J = 7.32 Hz, 1H, ArH), 7.71 (d,
J = 5.96 Hz, 2H, ArH), 7.58 (t, J = 7.56 Hz, 1H, ArH), 7.45 (t, J = 7.10 Hz, 2H, ArH), 7.14–7.08
(m, 2H, ArH), 6.72 (s, 1H, NH), 6.46–6.43 (m, 2H, ArH), 3.89–3.85 (m, 1H, CH), 2.96 (dd,
J = 4.60, 4.56 Hz, 1H, CHa of CH2), 2.71 (dd, J = 9.60, 10.08 Hz, 1H, CHb of CH2), 2.15 (s, 3H,
CH3). 13C NMR (DMSO-d6, 400 MHz) δ: 170.7, 167.9 (C = O), 157.4 (C = N), 152.0, 149.9,
149.9, 147.0, 146.2, 142.0, 133.3, 129.8, 129.1, 128.9, 128.7, 128.2, 124.9, 124.7, 124.6, 114.2,
110.4 (seventeen aromatic carbons), 48.9, 44.5, 21.5 (three aliphatic carbons). HRMS (ESI-
TOF, m/z): 562.0107 (M+NH4), calculated, 562.0110.
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Synthesis of new anti-inflammatory agent comparable to celecoxib
(2S)-3-(1H-indol-2-yl)-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phe-
nylformamido]propanamide (7c)
Appearance: yellowish-brown solids; Yield (0.5799 g, 99.4%), mp, 100.50–100.80˚C, FTIR
(KBr, cm-1): 3415, 3365 (NH), 3087 (C-H aromatic), 2891 (C-H aliphatic), 1702, 1669 (C = O),
1623 (C = N), 1601, 1491 (C = C), 1528 (NO2), 1350, 1311 (2S = O), 1163, 1121 (SO2N), 1093
(C-N). 1H NMR (DMSO-d6, 500 MHz) δ: 10.6664 (s, 1H, NH of indole), 8.58 (s, 1H, ArH),
7.91 (d, J = 6.90 Hz, 1H, ArH), 7.81 (d, J = 8.60 Hz, 2H, ArH), 7.75–7.70 (m, 1H, ArH), 7.58 (t,
J = 7.45 Hz, 1H, ArH), 7.48–7.45 (m, 3H, ArH), 7.25–7.20 (m, 1H, ArH), 7.12 (t, J = 7.40 Hz,
1H, ArH), 7.05 (d, J = 8.00 Hz, 1H, ArH), 6.99–6.98 (m, 1H, ArH), 6.88–6.82 (m, 2H, ArH),
6.32 (d, J = 5.15 Hz, 1H, ArH), 6.26 (s, 1H, ArH), 5.97 (s, 1H, NH), 3.88 (t, J = 4.28 Hz, 1H,
CH-C = O), 3.04 (dd, J = 4.60, 4.00 Hz, 1H, CHa of CH2), 2.78 (dd, J = 10.30, 10.30 Hz, 1H,
CHa, CH2), 2.11 (s, 3H, CH3). 13C NMR (DMSO-d6, 500 MHz) δ: 173.5, 167.9 (C = O), 159.7
(C = N), 148.9, 148.7, 146.5, 136.5, 133.4, 129.8, 129.4, 129.1, 128.7, 127.4, 126.9, 125.9, 124.8,
123.8, 121.2, 118.8, 118.2, 113.9, 111.7, 109.3, 108.9 (twenty one aromatic carbons), 57.2, 28.4,
21.2 (three aliphatic carbons). HRMS (ESI-TOF, m/z): 583.1236 (M+), 583.1526.
(2S)-4-Methyl-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phenylfor-
mamido]pentanamide (7d)
Appearance: yellowish oil; Yield (0.4989 g, 97.8%), FTIR (KBr, cm-1): 3333 (NH), 3106
(C-H aromatic), 2957, 2871 (C-H aliphatic), 1691, 1671 (C = O), 1619 (C = N), 1603, 1583,
1492, 1452 (C = C), 1530 (NO2), 1316, 1301 (2S = O), 1172, 1148 (SO2N), 1093, 1071, 1025
(C-N). 1H NMR (DMSO-d6, 500 MHz) δ: 8.36–8.24 (m, 2H, ArH), 8.12–8.05 (m, 2H, ArH),
7.97 (d, J = 8.72 Hz, 2H, ArH), 7.90 (d, J = 7.76 Hz, 1H, ArH), 7.66 (m, 1H, ArH), 7.55 (t,
J = 7.32 Hz, 1H, ArH), 7.43 (t, J = 7.56 Hz, 1H, ArH), 7.20–7.06 (m, 2H, ArH), 3.67 (t, J = 7.32
Hz, 1H, CH-C = O), 2.17 (s, 3H, CH3), 1.92–1.87 (m, 1H, CH), 1.63–1.55 (m, 1H, CHa of
CH2), 1.41–1.37 (m, 1H, CHb of CH2). 13C NMR (DMSO-d6, 400 MHz) δ: 174.4, 168.2, 156.9,
152.7, 149.8, 147.3, 140.8, 129.8, 129.4, 128.9, 128.7, 125.8, 124.6, 114.2, 110.8 (thirteen aro-
matic carbons), 55.4, 41.5, 24.5, 23.2, 21.5 (five aliphatic carbons). HRMS (ESI-TOF, m/z):
511.2299 (M+H), calculated, 510.1573.
(2S)-3-Methyl-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phenylfor-
mamido]pentanamide (7e)
Appearance: pale yellow solids; Yield (0.5009 g, 98.2%), 110.90–111.40˚C, FTIR (KBr,
cm-1): 3422 (NH), 3071 (C-H aromatic), 2962 (C-H aliphatic), 1673, 1662 (C = O), 1618
(C = N), 1601, 1496, 1470 (C = C), 1521 (NO2), 1332, 1311 (2S = O), 1169, 1136 (SO2N), 1093,
1051, 1016 (C-N). 1H NMR (DMSO-d6, 500 MHz) δ: 8.34 (d, J = 9.15 Hz, 2H, ArH), 7.98 (d,
J = 8.60 Hz, 2H, ArH), 7.69 (d, J = 5.15 Hz, 1H, ArH), 7.15 (m, 5H, ArH), 6.30 (d, J = 5.15 Hz,
1H, ArH), 6.23 (s, 1H, ArH), 5.91 (s, 1H, NH), 3.58 (d, J = 5.70 Hz, 1H, CH-C = O), 2.10 (s,
3H, CH3), 1.69–1.65 (m, 1H, CH), 1.36–1.30 (m, 1H, CHa of CH2), 1.11–1.03 (m, 1H, CHb of
CH2), 0.82–0.73 (m, 6H, 2CH3). 13C NMR (DMSO-d6, 500 MHz) δ: 172.6, 165.4 (C = O),
159.9, 158.5, 149.9, 148.4, 147.2, 146.8, 146.2, 129.4, 128.7, 125.9, 124.8, 113.9, 108.9 (thirteen
aromatic carbons), 61.1, 37.4, 24.9, 21.1, 15.9, 11.5 (six aliphatic carbons). HRMS (ESI-TOF,
m/z): 510.1582 (M+), calculated, 510.1583.
(2S)-3-Methyl-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-1-phenylfor-
mamido]butanamide (7f)
Appearance: yellow solid; Yield (0.4869 g, 98.2%), 143.30–143.90˚C, FTIR (KBr, cm-1):
3418 (NH), 2964 (C-H aliphatic), 1672, 1663 (C = O), 1620 (C = N), 1604, 1495 (C = C), 1520
(NO2), 1356, 1311 (2S = O), 1169, 1140 (SO2N), 1093, 1047, 1016 (C-N). 1H NMR (DMSO-d6,
400 MHz) δ: 8.31 (d, J = 8.72 Hz, 2H, ArH), 7.78 (d, J = 9.16 Hz, 2H, ArH), 7.89 (d, J = 7.32
Hz, 1H, ArH), 7.67 (d, J = 5.46 Hz, 1H, ArH), 7.56 (t, J = 7.56 Hz, 1H, ArH), 7.44 (t, J = 7.56
Hz, 1H, ArH), 7.27–7.009 (m, 2H, ArH), 6.31 (d, J = 5.52 Hz, 1H, ArH), 6.26 (s, 1H, ArH),
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Synthesis of new anti-inflammatory agent comparable to celecoxib
6.0654 (s, 1H, NH), 3.51 (d, J = 5.96 Hz, 1H, CH-C = O), 2.09 (s, 3H, CH3), 1.96–1.89 (m, 1H.
CH), 0.80–0.75 (m, 6H, 2CH3). 13C NMR (DMSO-d6, 500 MHz) δ: 172.8, 168.1 (C = O), 159.4,
149.8, 149.1, 147.3, 145.9, 129.8, 129.4, 129.1, 128.7, 125.8, 124.7, 113.9, 109.2 (thirteen aro-
matic carbons), 62.3, 30.9, 21.2, 19.6, 18.3 (five aliphatic carbons). HRMS (ESI-TOF, m/z):
497.0419 (M+H), calculated, 497.0422.
(2S)-4-Hydroxy-N-(4-methylpyridin-2-yl)-1-(4-nitrobenzenesulfonyl)pyrrolidine-
2-carboxamide (7g)
Appearance: crystalline yellow; Yield (0.4061 g, 100%), 100.20–100.60˚C, FTIR (KBr, cm-1):
3414 (OH), 3323 (NH), 3107 (C-H aromatic), 2953 (C-H aliphatic), 1670 (C = O), 1635
(C = N), 1606, 1491 (C = C), 1530 (NO2), 1352, 1308 (2S = O), 1199, 1161 (SO2N), 1093, 1010
(C-N, C-O). 1H NMR (DMSO-d6, 400 MHz) δ: 8.35 (d, J = 9.16 Hz, 2H, ArH), 8.02 (d, J = 8.72
Hz, 2H, ArH), 7.69 (d, J = 5.52 Hz, 1H, ArH), 6.28 (d, J = 5.48 Hz, 1H, ArH), 6.22 (s, 1H,
ArH), 5.82 (s, 1H, NH), 4.16 (s, 1H, OH), 4.09 (t, J = 7.80 Hz, 1H, CH-C = O), 3.19 (d, J = 5.45
Hz, 2H, CH2N), 2.08 (s, 3H, CH3), 2.03–1.98 (m, 1H, CH-OH), 1.93–1.86 (m, 2H, CH2). 13
C
NMR (DMSO-d6, 400 MHz) δ: 173.8 (C = O), 160.1, 150.3, 148.2, 147.2, 143.6, 129.5, 124.8,
113.9, 108.8 (nine aromatic carbons), 69.0, 60.6, 57.1, 21.1 (four aliphatic carbons). HRMS
(ESI-TOF, m/z): 407.1023 (M+H), calculated, 407.1027.
(2S)-N-(4-methylpyridin-2-yl)-1-(4-nitrobenzenesulfonyl)pyrrolidine-2-carboxamide
(7h)
Appearance: off-yellow crystals; Yield (0.3900 g, 99.9%), 156.70–157.20˚C, FTIR (KBr,
cm-1): 3265 (NH), 3009 (C-H aromatic), 2981, 2893 (C-H aliphatic), 1678 (C = O), 1641
(C = N), 1602, 1492, 1449 (C = C), 1320, 1306 (2S = O), 1183, 1163 (SO2N), 1088, 1045 (C-N).
1H NMR (DMSO-d6, 400 MHz) δ: 8.35 (d, J = 9.16 Hz, 2H, ArH), 8.05 (d, J = 9.16 Hz, 2H,
ArH), 7.68 (d, J = 5.48 Hz, 1H, ArH), 6.34 (d, J = 5.48 Hz, 1H, ArH), 6.31 (s, 1H, ArH), 4.15
(dd, J = 3.20, 3.68 Hz, 1H, CH-C = O), 3.37–3.34 (m, 1H, CHa of CH2N), 3.24–3.18 (m, 1H,
CHb of CH2N), 2.12 (s, 3H, CH3), 1.96–1.91 (m, 1H, CH of CH2), 1.86–1.75 (m, 2H, CH2),
1.66–1.59 (m, 1H, CH of CH2). 13C NMR (DMSO-d6, 400 MHz) δ: 174.1 (C = O), 159.1, 150.3,
149.7, 144.9, 143.9, 129.4, 125.1, 113.9, 109.5 (nine aromatic carbons), 61.5, 48.9, 31.0, 24.7,
21.3 (five aliphatic carbons). HRMS (ESI-TOF, m/z): 389.0603 (M-H), calculated, 389.0604.
2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-(4-methylpyridin-2-yl)acet-
amide (7i)
Appearance: white crystals; Yield (0.4201 g, 99.3%), mp, 137.10–137.60˚C, FTIR (KBr,
cm-1): 3311 (NH), 3039 (C-H aromatic), 2091 (C-H aliphatic), 1703, 1664 (C = O), 1644
(C = N), 1602, 1579, 1496, 1426 (C = C), 1392, 1329 (2S = O), 1185, 1162 (SO2N), 1093, 1018
(C-N). 1H NMR (DMSO-d6, 500 MHz) δ: 7.91 (d, J = 8.60 Hz, 3H, ArH), 7.72 (d, J = 5.75 Hz,
1H, ArH), 7.64 (d, J = 8.00 Hz, 1H, ArH), 7.57 (t, J = 7.45 Hz, 1H, ArH), 7.45 (t, J = 7.73 Hz,
2H, ArH), 7.33–7.27 (m, 2H, ArH), 6.32 (d, J = 5.15 Hz, 1H, ArH), 6.28 (s, 1H, ArH), 6.15 (s,
1H, NH), 3.46 (s, 2H, CH2), 2.32 (s, 3H, CH3-Ar), 2.11 (s, 3H, CH3-Py). 13C NMR (DMSO-d6,
500 MHz) δ: 171.3, 168.2 (C = O), 159.7 (C = N), 148.8, 146.3, 143.1, 138.3, 133.2, 131.7, 129.9,
129.8, 129.0, 128.7, 127.1, 113.9, 109.1 (thirteen aromatic carbons), 44.8, 21.5, 21.2 (three ali-
phatic carbons). HRMS (ESI-TOF, m/z): 423.0782 (M+), calculated, 423.0783.
(2S)-2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-(4-methylpyridin-2-yl)-
3-phenyl propanamide (7j)
Appearance: off-white solids; Yield (0.5009 g, 97.6%), mp, 91.50–92.30˚C, FTIR (KBr,
cm-1): 3267 (NH), 3062 (C-H aromatic), 1700, 1679 (C = O), 1641 (C = N), 1599, 1494, 1452
(C = C), 1383, 1301 (2S = O), 1174, 1157 (SO2N), 1091, 1025 (C-N). 1H NMR (DMSO-d6, 500
MHz) δ: 7.92 (d, J = 7.45 Hz, 3H, ArH), 7.71 (d, J = 5.20 Hz, 2H, ArH), 7.56 (t, J = 7.45 Hz, 2H,
ArH), 7.45 (t, J = 7.43 Hz, 4H, ArH), 7.22–7.08 (m, 5H, ArH), 6.35 (d, J = 5.15 Hz, 1H, ArH),
6.33 (s, 1H, ArH), 5.47 (s, 1H, NH), 3.77 (t, J = 5.75 Hz, 1H, CH-C = O), 2.90 (dd, J = 5.75,
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Synthesis of new anti-inflammatory agent comparable to celecoxib
5.75 Hz, 1H, CHa of CH2), 2.72 (dd, J = 8.05, 8.55 Hz, 1H, CHb of CH2), 2.26 (s, 3H, CH3-Ar),
2.12 (s, 3H, CH3-Py). 13C NMR (DMSO-d6, 500 MHz) δ: 173.7, 168.3 (C = O), 159.2 (C = N),
149.6, 145.1, 142.7, 138.7, 137.7, 133.1, 131.9, 129.7, 129.4, 128.9, 128.7, 128.6, 126.9, 125.9,
113.9, 109.5 (seventeen aromatic carbons), 58.4, 38.6, 21.4, 21.2 (four aliphatic carbons).
HRMS (ESI-TOF, m/z): 512.0759 (M-H), calculated, 512.0762.
(2S)-3-(1H-indol-2-yl)-2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-
(4-methylpyridin-2-yl)propanamide (7k)
Appearance: creamy solids; Yield (0.5502 g, 99.6%), mp, 114.40–114.70˚C, FTIR (KBr,
cm-1): 3421, 3264 (2NH), 3020 (C-H aromatic), 1692, 1684 (C = O), 1641 (C = N), 1599, 1494,
1454 (C = C), 1326, 1299 (2S = O), 1183, 1148 (SO2N), 1067, 1025 (C-N). 1H NMR (DMSO-
d6, 500 MHz) δ: 10.73 (s, 1H, NH of indole), 8.01 (s, 1H, ArH), 7.91 (d, J = 8.60 Hz, 1H, ArH),
7.71 (d, J = 5.20 Hz, 1H, ArH), 7.58 (t, J = 7.45 Hz, 1H, ArH), 7.44 (m, 3H, ArH), 7.21 (m, 3H,
ArH), 7.12 (t, J = 8.60 Hz, 3H, ArH), 7.01–6.99 (m, 2H, ArH), 6.88 (t, J = 8.60 Hz, 1H, ArH),
6.32 (d, J = 5.15 Hz, 1H, ArH), 6.27 (s, 1H, ArH), 3.81 (t, J = 4.30 Hz, 1H, CH-C = O), 3.01
(dd, J = 6.30, 6.30 Hz, 1H, CHa of CH2), 2.81 (dd, J = 8.00, 7.40 Hz, CHb of CH2), 2.27 (m, 3H,
CH3-Ar), 2.11 (s, 3H, CH3-Py). 13C NMR (DMSO-d6, 500 MHz) δ: 173.3, 167.7 (C = O), 159.6
(C = N), 148.8, 146.3, 142.7, 133.3, 129.8, 129.7, 129.4, 129.1, 128.7, 127.5, 126.8, 125.9, 124.4,
121.3, 118.8, 118.4, 113.9, 111.9, 109.5, 109.0 (twenty one aromatic carbons), 57.2, 28.8, 21.6,
21.5 (four aliphatic carbons). HRMS (ESI-TOF, m/z): 553.1335 (M+H), calculated, 553.1339.
(2S)-4-Methyl-2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-(4-methylpyri-
din-2-yl) pentanamide (7l)
Appearance: yellowish oil; Yield (0.4689 g, 97.8%), FTIR (KBr, cm-1): 3196 (NH), 3063
(C-H aromatic), 2959 (C-H aliphatic), 1692, 1675 (C = O), 1642 (C = N), 1600, 1493, 1451
(C = C), 1383, 1316 (2S = O), 1161 (SO2N), 1093, 1070, 1025 (C-N). 1H NMR (DMSO-d6, 500
MHz) δ: 7.93–7.91 (m, 3H, ArH), 7.69 (d, J = 5.52Hz, 1H, ArH), 7.61 (d, J = 8.24 Hz, 1H,
ArH), 7.57–7.53 (m, 1H, ArH), 7.46–7.42 (m, 3H, ArH), 7.28–7.26 (d, J = 7.80 Hz, 1H, ArH),
7.21–7.17 (t, J = 7.34 Hz, 1H, ArH), 7.12–7.09 (t, J = 7.10 Hz, 1H, ArH), 6.37 (s, 2H, ArH),
3.59–3.58 (t, J = 6.88 Hz, 1H, CH-C = O), 2.31–2.24 (m, 3H, CH3-Ar), 2.12 (s, 3H, CH3-Py),
1.59–1.52 (m, 1H, CH), 1.34 (t, J = 6.88 Hz, 2H, CH2), 0.79–0.65 (m, 6H, 2CH3). 13C NMR
(DMSO-d6, 500 MHz) δ: 174.7, 168.4 (C = O), 158.8, 150.3, 144.1, 142.9, 138.8, 133.1, 131.9,
129.8, 129.4, 128.9, 128.7, 127.1, 125.8, 113.9, 109.8 (fifteen aromatic carbons), 54.9, 24.4, 23.1,
21.7, 21.3, 15.9 (six aliphatic carbons). HRMS (ESI-TOF, m/z): 480.1989 (M+H), calculated,
480.1992.
(2S)-3-Methyl-2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-(4-methylpyri-
din-2-yl) pentanamide (7m)
Appearance: off-white solids; Yield (0.4709 g, 98.3%), mp, 91.20–91.80˚C, FTIR (KBr,
cm-1): 3268 (NH), 3012 (C-H aromatic), 2965 (C-H aliphatic), 1692, 1679 (C = O), 1641
(C = N), 1598, 1496 (C = C), 1383, 1301 (2S = O), 1161, 1123 (SO2N), 1093, 1024 (C-N). 1H
NMR (DMSO-d6, 500 MHz) δ: 7.92 (d, J = 6.88 Hz, 4H, ArH), 7.68 (d, J = 5.96 Hz, 2H, ArH),
7.61 (d, J = 8.24 Hz, 2H, ArH), 7.54 (t, J = 7.34 Hz, 2H, ArH), 7.43 (t, J = 7.56 Hz, 4H, ArH),
7.24 (d, J- 8.24 Hz, 2H, ArH), 6.82 (s, 1H, ArH), 6.36 (d, J = 5.52 Hz, 3H, ArH), 3.45 (d,
J = 5.52 Hz, 1H, CH-C = O), 2.29–2.23 (m, 3H, CH3-Ar), 2.12 (s, 3H, CH3-Py), 1.66–1.60 (m,
1H, CH), 1.37–1.31 (m, 1H, CHa of CH2), 1.09–1.02 (m, 1H, CHa of CH2), 0.77–0.67 (m, 6H,
2CH3). 13C NMR (DMSO-d6, 500 MHz) δ: 173.7, 168.6 (C = O), 158.8, 150.4, 143.9, 142.8,
138.8, 133.0, 132.1, 129.7, 128.9, 127.2, 125.8, 113.9, 109.8 (thirteen aromatic carbons), 61.1,
37.7, 24.4, 21.4, 21.3, 16.0, 11.6 (seven aliphatic carbons). HRMS (ESI-TOF, m/z): 479.1888
(M+), calculated, 479.1879.
(2S)-3-Methyl-2-[N-(4-methylbenzenesulfonyl)-1-phenylformamido]-N-(4-methylpyri-
din-2-yl) butanamide (7n)
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Appearance: yellowish oil; Yield (0.4554 g, 97.9%), FTIR (KBr, cm-1): 3322 (NH), 3064
(C-H aromatic), 2965 (C-H aliphatic), 1691, 1673 (C = O), 1623 (C = N), 1601, 1493, 1451
(C = C), 1386, 1316 (2S = O), 1161 (SO2N), 1093, 1071, 1025 (C-N). 1H NMR (DMSO-d6, 400
MHz) δ: 7.92–7.90 (m, 2H, ArH), 7.70 (d, J = 8.24 Hz, 1H, ArH), 7.61 (d, J = 8.24 Hz, 1H,
ArH), 7.55 (t, J = 7.32 Hz, 1H, ArH), 7.44 (t, J = 7.80 Hz, 3H, ArH), 7.32–7.26 (m, 2H, ArH),
6.35 (d, J = 5.52 Hz, 1H, ArH), 6.32 (s, 1H, ArH), 3.42 (d, J = 5.52 Hz, 1H, CH-C = O), 2.31–
2.25 (m, 3H, CH3-Ar), 2.12 (s, 3H, CH3-Py), 1.92–1.87 (m, 1H, CH), 0.79–0.74 (m, 6H, 2CH3).
13C NMR (DMSO-d6, 500 MHz) δ: 173.4, 168.3 (C = O), 159.1 (C = N), 149.7, 144.9, 142.8,
138.8, 133.2, 131.8, 129.8, 129.0, 127.1, 126.9, 113.9, 109.5 (thirteen aromatic carbons), 62.1,
30.9, 21.4, 21.2, 19.6, 18.4 (six aliphatic carbons). HRMS (ESI-TOF, m/z): 466.1392 (M+H),
calculated, 466.1393.
(2S)-4-Hydroxy-1-(4-methylbenzenesulfonyl)-N-(4-methylpyridin-2-yl)pyrrolidine-
2-carboxamide (7o)
Appearance: brownish oil; Yield (0.3754 g, 100%), FTIR (KBr, cm-1): 3421 (OH), 3362
(NH), 3028 (C-H aromatic), 2967, 2891 (C-H aliphatic), 1682 (C = O), 1612 (C = N), 1601,
1573, 1455 (C = C), 1342, 1312 (2S = O), 1176, 1121 (SO2N), 1092, 1073 (C-N). 1H NMR
(DMSO-d6, 400 MHz) δ: 7.70 (d, J = 5.48 Hz, 1H, ArH), 7.63 (d, J = 8.24 Hz, 2H, ArH), 7.35
(d, J = 7.80 Hz, 2H, ArH), 6.34 (d, J = 5.52 Hz, 1H, ArH), 6.30 (s, 1H, ArH), 6.18 (s, 1H, NH),
4.17 (s, 1H, OH), 4.00 (t, J = 7.80 Hz, 1H, CH-C = O), 3.43–3.39 (m, 1H, CH-OH), 3.03 (d,
J = 5.25 Hz, 2H, CH2N), 2.34 (s, 3H, CH3-Ar), 2.11 (s, 3H, CH3-Py), 1.91–1.85 (m, 2H, CH2).
13C NMR (DMSO-d6, 500 MHz) δ: 173.9 (C = O), 159.2 (C = N), 149.5, 145.4, 143.7, 135.0,
130.1, 129.4, 128.7, 127.9, 114.0, 109.3 (eleven aromatic carbons), 68.9, 60.3, 56.8, 21.5, 21.2
(five aliphatic carbons). HRMS (ESI-TOF, m/z): 376.1333 (M+H), calculated, 376.1336.
(2S)-1-(4-Methylbenzenesulfonyl)-N-(4-methylpyridin-2-yl)pyrrolidine-2-carboxamide
(7p)
Appearance: yellowish oil; Yield (0.3509 g, 97.7%), FTIR (KBr, cm-1): 3401 (NH), 3001
(C-H aromatic) 2980 (C-H aliphatic), 1673 (C = O), 1640 (C = N), 1598, 1494, 1451, 1402
(C = C), 1338, 1305 (2S = O), 1202, 1156 (SO2N), 1093, 1015 (C-N). 1H NMR (DMSO-d6, 400
MHz) δ: 7.71–7.67 (m, 3H, ArH), 7.39–7.37 (m, 2H, ArH), 6.33 (d, J = 5.15 Hz, 2H, ArH), 6.28
(s, 1H, ArH), 6.06 (s, 1H, NH), 4.44 (dd, J = 5.15, 4.00 Hz, 1H, CH-C = O), 4.20 (dd, J = 4.00,
4.00 Hz, 1H, CHa of CH2N), 4.03–4.01 (m, 1H, CHb of CH2N), 3.54–3.50 (m, 1H, CH), 2.36–
2.32 (m, 3H, CH3-Ar), 2.11 (s, 3H, CH3-Py), 1.92–1.72 (m, 2H, CH2), 1.52–1.49 (m, 1H, CH).
13C NMR (DMSO-d6, 400 MHz) δ: 173.9, 159.6 (C = O), 148.9, 146.1, 135.7, 130.4, 130.3,
129.4, 128.7, 127.7, 113.9, 109.1 (nine aromatic carbons), 61.2, 59.5, 48.9, 30.9, 21.5, 21.2 (six
aliphatic carbons). HRMS (m/z): 359.1057 (M+), calculated, 359.1059.
Ethics statement on animal use. The animal use and care was approved by University of
Nigeria, Nsukka animal ethics committee in confirmation to international standards for the
project number PG/PhD/14/69629. The animal house of the Department of Biochemistry was
used for the experiment. The project was designed to use humane endpoints for death of ani-
mals. During the entire experimental session, the mice were well nursed to reduce all kinds of
stress. All efforts were made to minimize the suffering of the mice by strictly adhering with the
regulation of animal protection committee.
In vitro anti-inflammatory activities determination. Male albino rats weighing 300 g
where purchased from the Department of Zoology and Environmental Biology, University of
Nigeria, Nsukka and kept at room temperature in a light controlled animal house. They were
fasted with free access to water at least 12 h prior to the experiments. The tested compounds
were prepared as suspension in vehicle (0.5% methylcellulose) and celecoxib was used as a
standard drug. The positive control received celecoxib while the negative control received only
the vehicle. Edema was produced by injecting 0.2 mL of a solution of 1% carrageenan in the
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Synthesis of new anti-inflammatory agent comparable to celecoxib
hind paw. The rats were injected intraperitoneally with 1 mL suspension in 0.5% methylcellu-
lose of the tested compounds and reference drug. Paw volume was measured by water dis-
placement with a plethysmometer (UGO BASILE) before, 30 min, 1 h, 2 h and 3 h after
treatment. The percentage was calculated by the following equation [25].
Anti-inflammatory activity (%) = (1-D/C)-100, where D represents the difference in paw
volume before and after drug administration to the rats and C represents the difference of vol-
ume in the control groups. The approval for the use of animal was obtained from the Univer-
sity of Nigeria committee on experimental animal use. No sign of death was observed during
the experiment.
Determination of LD50 for the active anti-inflammatory compounds. Male mice were
divided into various groups and test compounds were administered in various doses intraperi-
toneally. Following treatments, the animals were observed for up to 6 h continuously and were
then kept under observation for 72 h. All behavioral changes and deaths during the observa-
tion periods were recorded. The percentage of death at each dose level was then calculated,
converted to probits and the LD50 (μM/kg) values were calculated [26]. To observe the health
status of the mice, they were monitored 4 times a day. Humane endpoints were used when the
animal shows sign of weight loss, weakness accompanied by inability to get food, complete
anorexia and convulsion for 24 h. For the purpose of ameliorating the suffering of the dying
mice, CO2 euthanasia was applied. All the dead mice were disposed in bio-safety containers in
accordance with local standard protocols. The mortality rate in each group was calculated
according to the formula:
Mortality rateð%Þ ¼ ðthe number of dead mice=the number of mice in the groupÞ Â 100:
In vitro cyclooxygenase inhibitory assay. The in vitro ability of test compounds and cele-
coxib to inhibit the COX-1 and COX-2 isozymes was carried out using Cayman colorimetric
COX (ovine) inhibitor screening assay kit supplied by Cayman chemicals, USA. The calcula-
tions were performed as per the kit guidelines [27].
Results and discussion
Chemistry
Considering the ubiquitous pharmacological activities of sulphonamides and carboxamides as
found in the literature and the need for anti-inflammatory agents with reduced side effects, we
undertook the design and synthesis of some novel hybrids which possess advantages of the
three pharmacophores of sulphonamide, carboxamide and 2-picoline in single molecular
backbone. Our design strategy employed the use of boric acid in the condensation of 2-pico-
line with substituted benzenesulphonamides derived from L-amino acids. The use of boric
acid was aimed to achieve the successful formation of carboxamide from un-activated carbox-
ylic acid end of the L-amino acid in the presence of sulphonamide functionality. During the
course of this study, we synthesized and characterized hybrid compounds containing sulpho-
namide, carboxamide and 2-picoline motifs. We took into consideration the problems of oral
bioavailability and transportation of drug molecules in the design and as such the pharmacoki-
netics prediction of the designed molecules were evaluated prior to synthesis to eliminate com-
pounds with associated oral bioavailability and transport problems.
The reaction of substituted benzenesulphonyl chloride (1a-b) with various amino acids
gave the substituted benzenesulphonamoyl alkanamides (3a-p). The base promoted reaction
of 3a-f and 3i-n with benzoyl chloride under nitrogen at room temperature afforded the N-
benzoylated derivatives (5a-f and 5i-n). Further reaction of compounds 3g-h, 3o-p, 5a-f and
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Fig 1. Synthesis protocol. A representation of the stepwise reactions leading to the desired compounds 7a-p.
https://doi.org/10.1371/journal.pone.0183807.g001
5i-n with 2-amino-4-picoline in the presence of catalytic amount of boric acid gave the new
carboxamides 7a-p in excellent yield (Figs 1–4).
In the FTIR of the substituted benzenesulphonamoyl alkanamides (3a-p), the bands
between 3297–3253 cm-1 was due to NH group; the band between 1753–1705 cm-1 were
assigned to the C = O of the carboxylic acid. In compounds 3a-h, the N-O of NO2 appeared
between 1532–1403 which were absent in the p-toluene derivatives 3i-p. In the 1H NMR, the
diagnostic NH peak appeared as a triplet in compounds 3a and 3i around 8.44–8.41 ppm; a
doublet in compounds 3b-3f and 3j-3n around 8.69–8.42 ppm and disappeared in compounds
3g-h and 3o-p. In the 13C NMR, the diagnostic carbonyl appeared between 173.4–170.7 ppm.
Additionally, all the carbons were accounted for.
In the benzoyl derivatives (5a-f and 5i-n), the FTIR showed additional C = O band between
1691–1685 cm-1. The disappearance of the NH bands in the benzoyl derivatives further indi-
cate successful coupling of the benzoyl chloride with substituted benzenesulphonamoyl alka-
namides. In the 1H NMR, the disappearance of the NH peak is diagnostic. In addition, the
benzoyl protons were accounted for in the aromatic region.
The FTIR spectra of the p-nitro derivatives 7a-h (Fig 2) showed diagnostic bands at 3422–
3265, 1773–1670, 1671–1661, 1641–1618 and 1535–1521 cm-1 corresponding to NH of carbox-
amide, C = O of carboxamide, C = O of benzoyl group, C = N of pyridine and NO2 of p-nitro-
benzene respectively. The 1H NMR showed peaks between 7.75–7.73 and 2.17–2.16 ppm are
diagnostic of pyridine ring and methyl of pyridine respectively. The 13C NMR of the com-
pounds at 159.7–157.6, 152.0–148.9 and 21.5–21.2 ppm corresponds to C = N, C-N and methyl
carbon of pyridine.
The p-toluenebenzenesulphonamide derivatives (7i-p, Fig 3) showed similar pattern of
absorption in the FTIR, 1H NMR, 13C NMR and HRMS as recorded in compounds 7a-h
worthy of mention in the FTIR is the absorptions between 3401–3196 cm-1 due to NH
stretch, 1692–1673 cm-1 due to C = O stretch and 1641–1612 cm-1 due to C = N of picoline.
We observed a reduction in the bands of the C = O in the derivatives which is attributable
to successful coupling. These bands are indicative of successful coupling of the 4-aminopi-
coline with the carboxylic end of the 4-methylbenzenesulphonamides. In the 1H NMR
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Fig 2. Description of p-nitrobenzenesulphonamide derivatives 7a-f. A structural representation of the p-nitrobenzenesulphonamide
derivatives 7a-f and the intermediates 3a-f and 5a-f.
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Fig 3. Description of p-toluenesulphonamide derivatives 7i-n. A structural representation of the p-toluenesulphonamide derivatives 7i-n
and the intermediates 3i-n and 5i-n.
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Fig 4. Description of L-proline derived sulphonamides 7g-h and 7o-p. A structural representation of the L-
proline derivatives 7g, 7h, 7o and 7p and the intermediates 3g-h and 3o-p.
https://doi.org/10.1371/journal.pone.0183807.g004
(S1–S57 Figs), the peaks between δ7.71–7.69 (d, 1H); δ6.35–6.32 (d, 1H) and δ6.82–6.27
(s, 1H) were assigned to the three hydrogen of picoline in all the derivatives. In the 13
C
NMR spectra (S1–S57 Figs) of the 4-methylbenzenesulphonamide derivatives 7i-p, the
appearance of the azomethine peak between 159.6–158.8 ppm indicated the successful for-
mation of the target molecules. The spectral analysis above in addition to the molecular ion
peaks showed that there was successful coupling of the amino group of 4-aminopicooline
with the carboxylic acid of the 4-methylbenzenesulphonamoyl ethanamides.
The yield suggests that compounds 7i-p had better yield than compounds 7a-h, this
suggests that the presence of an electron donating group at the para position in the
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Fig 5. Graphical abstract.
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benzenesulphonamoyl group favours the reaction more than an electron withdrawing nitro
group. Nonetheless, all the derivatives had excellent yields ranging from 97.5–100 in the
para nitro derivatives and 97.6–100 in the para methyl derivatives. The 3D structures of the
most active derivatives 7c and 7k in comparison with celecoxib are shown in the graphical
abstract (Fig 5).
Biological evaluations
Anti-inflammatory activity. The anti-inflammatory activity was performed to get per-
centage inhibition of carrageenan induced rat paw edema for each of the tested compound
after 0.5, 1, 2 and 3 h and to compare it with reference drug celecoxib, however, the significant
decrease in the activity of all compounds except 7c, 7g, 7k and celecoxib after 2 h was noticed
(Table 1). Only compounds 7c, 7g and 7k (% inhibition = 88.89, 69.12 and 61.58% respec-
tively) showed anti-inflammatory activities comparable with celecoxib (% inhibition = 82.60%)
[28]. The L-tryptophan and L-4hydroxyproline derivatives were the most active derivatives.
This result suggests that derivatives with having atoms capable of forming hydrogen bond had
better anti-inflammatory activity. The substitution of the methyl group in p-toluenesulphonyl
derivative 7k with a nitro group (atom capable of hydrogen bonding) led to a better anti-
inflammatory activity. The presence of nitro group at the para position seem to increase the
anti-inflammatory activities as shown in Table 1; compounds 7a-h having the para nitro group
showed better anti-inflammatory activities than the corresponding methyl derivatives (7i-p).
In addition, LD50 of compound 7c, 7g and 7k (LD50 = 8.3, 9.9 and 9.2 μM/kg respectively)
related to celecoxib (LD50 = 9.8 μM/kg).
In vitro COX inhibitory activity. Percentage inhibition of COX-1 and COX-2 and selec-
tivity (COX-2/COX-1) of test compounds at concentration of 2.0 μM were illustrated in
Table 1. Compounds 7g, 7h, 7l, 7m and 7n are non-selective for COX-2 inhibitors whereas
compound 7c showed better selectivity towards COX-2. Its COX-2/COX-1 selectivity is 35.
Compound 7k showed good selectivity towards COX-2. Its COX-2/COX-1 selectivity is 18.67,
more than half of selectivity of celecoxib (COX-2/COX-1 = 30.5). This results indicate that the
mechanism of action of test compounds could be COX-2 inhibition but we discovered that
compound 7g and 7k did not show comparable selectivity in-spite of their comparable anti-
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Table 1. In vitro anti-inflammatory activity.
S/N
7a
0.5 h (%)
24.62
1 h (%)
18.82
2 h (%)
14.24
3 h (%)
11.85
LD50 (μM/kg)
12.9
14.2
8.3
COX-1 (%)
COX-2 (%)
49
S I
14
8
3.5
7b
32.31
49.23
52.31
26.15
24.62
46.92
27.69
24.62
41.54
42.12
26.15
13.85
21.54
21.54
29.15
56.92
17.06
62.35
17.65
25.88
17.65
53.53
17.06
23.53
40.59
56.12
19.88
12.35
16.47
11.23
28.24
63.53
17.03
75.75
10.54
19.38
14.84
58.84
16.59
16.78
39.84
70.89
18.28
8.59
12.96
88.89
10.15
16.05
1.85
52
6.5
7c
2
70
35
7d
12.6
13.1
15.8
9.9
10
4
20
2.00
4.25
3.6
7e
17
7f
15
18
30
3
54
7g
61.58
16.05
12.47
36.42
69.12
16.67
8.58
20
1.11
0.43
6.00
3.00
18.67
0.95
1.47
1.50
3.00
2.5
7h
10.7
18.2
17.6
9.2
13
7i
18
7j
10
3
30
7k
56
7l
12.5
11.9
20.1
10.1
9.9
20
17
32
6
19
7m
7n
25
10.56
8.78
3.09
48
7o
5.56
18
7p
30.34
64.84
33.26
82.60
8
20
Celecoxib
9.8
2
61
30.5
https://doi.org/10.1371/journal.pone.0183807.t001
inflammatory activity with celecoxib. The implication therefore is that the mechanism of test
compounds needs further investigations.
Assessment of oral bioavailability property. Lipinski’s rule of five (ro5) alongside topo-
logical polar surface area (TPSA) properties were used to assess the oral bioavailability poten-
tial of the newly synthesized 4-picoline derivatives. Topological polar surface area is frequently
used in drug design as surrogate property for cell permeability with a rule of thumb that a mol-
ecule with TPSA of less than 140 Å2 would be able to permeate the cell. It has also been used as
a surrogate for penetrating the blood-brain-barrier (BBB) if the TPSA is ꢀ 90 Å2. The results
showed that all the new compounds can permeate cells but only compounds 7i-p can permeate
blood-brain-barriers and hence can be used in treating brain inflammation.
Lipinski’s ro5, derived from 90th percentile of drug candidates that reach phase II clinical
trials, to be drug-like, a drug candidate should have lipophilicity (logP) ꢀ 5, number of hydro-
gen bond acceptor (HBA) ꢀ 10, molecular weight (MW) ꢀ 500 and number of hydrogen
bond donor (HBD) ꢀ 5. This rule claims that violation of more than one property will disqual-
ify a drug candidate based on associated bioavailability problem. The results (Table 2) showed
that all the synthesized compounds are drug-like considering ro5 since none of the com-
pounds violated more than one property. Verber et al [29] reported that the number of rotat-
able bond (nRB) influences bioavailability in rats and recommended NRB ꢀ 10 for good oral
bioavailability property. Compounds 7b-7e were disqualified because they had NRB of 11
which was greater than the recommended value. Other derivatives reported had good oral bio-
availability with respect to number of rotatable bond. Additionally, the number of acid was
zero which was a good requirement for blood-brain-barriers.
Conclusion
In conclusion, 3-(1H-indol-2-yl)-N-(4-methylpyridin-2-yl)-2-[N-(4-nitrobenzenesulfonyl)-
1-phenyl formamido]propanamide, 3-(1H-indol-2-yl)-2-[N-(4-methylbenzenesulfonyl)-
1-phenylformamido]-N-(4-methylpyridin-2-yl)propanamide and 4-Hydroxy-N-(4-methyl-
pyridin-2-yl)-1-(4-nitrobenzene sulfonyl)pyrrolidine-2-carboxamide 7c, 7k and 7g were
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Table 2. Physicochemical properties for drug-likeness.
S/N
7a
7b
7c
7d
7e
7f
MW
454
544
583
509
509
496
406
390
423
513
552
479
479
465
375
359
logP
HBA
7
HBD
0
TPSA (Å2)
135.4
nRB
9
nAc
0
NV
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0.319
2.094
1.701
2.28
7
0
135.4
135.4
135.4
135.4
135.4
118.33
118.33
92.26
92.26
92.26
92.26
92.26
92.26
75.19
75.19
11
11
11
11
10
6
0
8
0
0
7
0
0
2.069
1.50
7
0
0
7
0
0
7g
7h
7i
-0.95
0.077
0.705
2.48
7
0
0
6
0
6
0
7
0
8
0
7j
7
0
10
10
10
10
9
0
7k
7l
2.087
2.666
2.455
1.886
0.463
-0.57
8
0
0
7
0
0
7m
7n
7o
7p
7
0
0
7
0
0
6
0
5
0
7
0
5
0
MW = molecular weight; HBA = hydrogen bond acceptor; HBD = hydrogen bond donor; TPSA = topological polar surface area; nRB = number of rotatable
bond; nAc = number of acid and NV = number of violations
https://doi.org/10.1371/journal.pone.0183807.t002
found to have excellent anti-inflammatory activity with percentage inhibition of carrageenan
induced rat paw edema of 88.89, 69.12 and 61.58% respectively comparable with that of cele-
coxib (% inhibition = 82.60%) after 3 h of administration. Compound 7c appeared COX-2/
COX-1 selectivity higher than celecoxib while compound 7k appeared COX-2/COX-1 selec-
tivity a little higher than half of celecoxib. Compound 7g however is non-selective for COX-
2. These finding suggests further investigation to the mechanism of action of the test com-
pounds. On a broad basis, compounds 7c, g and k seemed to be a promising anti-inflamma-
tory agents.
Supporting information
S1 Fig. C-13 NMR spectrum of 7a.
(TIF)
S2 Fig. 1H NMR spectrum of 7b.
(TIF)
S3 Fig. 1H NMR spectrum of 7b (expansion).
(TIF)
S4 Fig. C-13 NMR spectrum of 7b.
(TIF)
S5 Fig. 13C NMR spectrum of 7b (expansion).
(TIF)
S6 Fig. 1H NMR spectrum of 7c.
(TIF)
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Synthesis of new anti-inflammatory agent comparable to celecoxib
S7 Fig. 1H NMR spectrum of 7c (expansion).
(TIF)
S8 Fig. 1H NMR spectrum of 7c (expansion).
(TIF)
S9 Fig. 13C NMR spectrum of 7c.
(TIF)
S10 Fig. 13C NMR spectrum of 7c (expansion).
(TIF)
S11 Fig. 1H NMR spectrum of 7d.
(TIF)
S12 Fig. 1H NMR spectrum of 7d (expansion).
(TIF)
S13 Fig. 1H NMR spectrum of 7d (expansion).
(TIF)
S14 Fig. 13C NMR spectrum of 7d (expansion).
(TIF)
S15 Fig. 1H NMR spectrum of 7e.
(TIF)
S16 Fig. 1H NMR spectrum of 7e (expansion).
(TIF)
S17 Fig. 13C NMR spectrum of 7e.
(TIF)
S18 Fig. 1H NMR spectrum of 7f.
(TIF)
S19 Fig. 1H NMR spectrum of 7f (expansion).
(TIF)
S20 Fig. 13C NMR spectrum of 7f.
(TIF)
S21 Fig. 1H NMR spectrum of 7g.
(TIF)
S22 Fig. 1H NMR spectrum of 7g (expansion).
(TIF)
S23 Fig. 13C NMR spectrum of 7g.
(TIF)
S24 Fig. 1H NMR spectrum of 7h.
(TIF)
S25 Fig. 1H NMR spectrum of 7h (expansion).
(TIF)
S26 Fig. 1H NMR spectrum of 7h (expansion).
(TIF)
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Synthesis of new anti-inflammatory agent comparable to celecoxib
S27 Fig. 13C NMR spectrum of 7d.
(TIF)
S28 Fig. 1H NMR spectrum of 7i.
(TIF)
S29 Fig. 1H NMR spectrum of 7i (expansion).
(TIF)
S30 Fig. 13C NMR spectrum of 7i.
(TIF)
S31 Fig. 1H NMR spectrum of 7j.
(TIF)
S32 Fig. 1H NMR spectrum of 7j (expansion).
(TIF)
S33 Fig. 1H NMR spectrum of 7j (expansion).
(TIF)
S34 Fig. 13C NMR spectrum of 7j.
(TIF)
S35 Fig. 1H NMR spectrum of 7k.
(TIF)
S36 Fig. 1H NMR spectrum of 7k (expansion).
(TIF)
S37 Fig. 1H NMR spectrum of 7k (expansion).
(TIF)
S38 Fig. 13C NMR spectrum of 7k.
(TIF)
S39 Fig. 13C NMR spectrum of 7k.
(TIF)
S40 Fig. 1H NMR spectrum of 7l.
(TIF)
S41 Fig. 1H NMR spectrum of 7l (expansion).
(TIF)
S42 Fig. 1H NMR spectrum of 7l (expansion).
(TIF)
S43 Fig. 13C NMR spectrum of 7l.
(TIF)
S44 Fig. 1H NMR spectrum of 7m.
(TIF)
S45 Fig. 1H NMR spectrum of 7m (expansion).
(TIF)
S46 Fig. 1H NMR spectrum of 7m (expansion).
(TIF)
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Synthesis of new anti-inflammatory agent comparable to celecoxib
S47 Fig. 13C NMR spectrum of 7m.
(TIF)
S48 Fig. 1H NMR spectrum of 7n.
(TIF)
S49 Fig. 1H NMR spectrum of 7n.
(TIF)
S50 Fig. 13C NMR spectrum of 7n.
(TIF)
S51 Fig. 1H NMR spectrum of 7o.
(TIF)
S52 Fig. 13C NMR spectrum of 7o.
(TIF)
S53 Fig. 1H NMR spectrum of 7p.
(TIF)
S54 Fig. 1H NMR spectrum of 7p (expansion).
(TIF)
S55 Fig. 1H NMR spectrum of 7p (expansion).
(TIF)
S56 Fig. 1H NMR spectrum of 7p (expansion).
(TIF)
S57 Fig. 13C NMR spectrum of 7p.
(TIF)
Acknowledgments
Authors wish to thank the Department of Chemistry, Indian Institute of Technology, Kanpur
for providing the facilities for the synthesis. We also acknowledge the help received from the
Department of Biology and Bioengineering, Indian Institute of Technology, Kanpur during
the in silico studies. We acknowledge the Department of Biochemistry, University of Nigeria,
Nsukka for the in vivo anti-inflammatory screening.
Author Contributions
Conceptualization: David Izuchukwu Ugwu, Uchechukwu Chris Okoro.
Data curation: David Izuchukwu Ugwu, Hilal Ahmad.
Formal analysis: David Izuchukwu Ugwu, Uchechukwu Chris Okoro, Hilal Ahmad.
Funding acquisition: David Izuchukwu Ugwu.
Investigation: David Izuchukwu Ugwu, Uchechukwu Chris Okoro, Hilal Ahmad.
Methodology: David Izuchukwu Ugwu, Uchechukwu Chris Okoro.
Project administration: Uchechukwu Chris Okoro.
Resources: David Izuchukwu Ugwu, Hilal Ahmad.
PLOS ONE | https://doi.org/10.1371/journal.pone.0183807 September 18, 2017
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Synthesis of new anti-inflammatory agent comparable to celecoxib
Software: David Izuchukwu Ugwu.
Supervision: Uchechukwu Chris Okoro.
Validation: David Izuchukwu Ugwu, Uchechukwu Chris Okoro.
Visualization: David Izuchukwu Ugwu, Uchechukwu Chris Okoro, Hilal Ahmad.
Writing – original draft: David Izuchukwu Ugwu.
Writing – review & editing: David Izuchukwu Ugwu, Uchechukwu Chris Okoro, Hilal
Ahmad.
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