Synthesis, anti-inflammatory, antimicrobial potential and molecular docking studies of 4,5-disubstituted-1,2,4-triazole thioacetate derivatives

Article abstract of DOI:10.2174/1570180815666180810122226

Background: In the present study synthesis and biological assessment of nine new ethyl [(4,5-disubstituted-4H-1,2,4-triazol-3-yl)sulfanyl]acetate derivatives 2(a-i) is performed. Methods: The title compounds were characterized by their analytical and spec

Full text of DOI:10.2174/1570180815666180810122226

734
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Letters in Drug Design & Discovery, 2019, 16, 734-745
RESEARCH ARTICLE
ISSN: 1570-1808
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Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular
Docking Studies of 4,5-Disubstituted-1,2,4-Triazole Thioacetate Derivatives
BENTHAM
S
CIENCE
Muhammad Nouman Arif a,c, Humaira Nadeema,*, Rehan Zafar Parachab, Arif-ullah Khand,
Muhammad Imrana and Fawad Alid,e
aDepartment of Pharmaceutical Chemistry, Riphah Institute of Pharmaceutical Sciences, Riphah International Universi-
ty, Islamabad, Pakistan; bResearch Center for Modeling and Simulation, National University of Science and Technolo-
gy, Islamabad, Pakistan; cDepartment of Pharmaceutical Chemistry, Margalla College of Pharmacy, Margalla Institute
d
of Health Sciences, Rawalpindi, Pakistan; Department of Basic Medical Sciences, Riphah Institute of Pharmaceutical
Sciences, Riphah International University, Islamabad, Pakistan; eDepartment of Pharmacy, Kohat University of Science
and Technology, Kohat, Pakistan
Abstract: Background: In the present study synthesis and biological assessment of nine new ethyl
[(4,5-disubstituted- 4H-1,2,4-triazol-3-yl)sulfanyl]acetate derivatives 2(a-i) is performed.
Methods: The title compounds were characterized by their analytical and spectral data. All the syn-
thesized compounds were screened for their in vivo anti-inflammatory activity using carrageenan-
induced rat paw oedema method and in vitro antimicrobial activity. All the compounds exhibited
good anti-inflammatory activity; especially compound 2h produced the maximum effect i.e., 62.5 %
A R T I C L E H I S T O R Y
comparable to that of standard, diclofenac. The antimicrobial screening results indicated that some
of the newly synthesized compounds showed good antibacterial activity, especially against Esche-
richia coli.
Received: April 17, 2018
Revised: July 17, 2018
Accepted: August 03, 2018
Results: All the synthesized thioacetate derivatives of triazoles were also studied for their interac-
tions with the enzymes COX-I and COX-II, two important targets of inflammation pathway,
DOI:
through docking analysis. All the compounds showed good binding affinities with both the enzymes
10.2174/1570180815666180810122226
with a maximum value of -8.1 for 2e kcal/mol against COX-I.
Conclusion: Docking analysis predicted that our compounds reduce inflammation nonselectively
by inhibiting both COX-I and COX-II of inflammatory pathway just like other nonselactive
NSAIDS.
Keywords: 1,2,4-triazole thioacetate derivatives, biological evaluation, molecular docking, COX inhibitor, in vivo, in vitro,
nonslactive NSAIDS
1. INTRODUCTION
series of pathological changes coupled with local vascular
reaction and cellular response of the living tissue to an injury
insufficient to kill the tissue. A large number of non-steroidal
anti-inflammatory drugs (NSAIDs) are available clinically to
take care of inflammatory disorders. Long-term use of
NSAIDs results in gastrointestinal ulceration and bleeding
and platelet dysfunction [2]. Moreover, there are evidence to
suggest that free –COOH present in NSAIDS is responsible
for GI irritation [3]. So, there is a need to develop new com-
pounds with better anti-inflammatory potential and lesser
side effects. Among the nitrogen-containing heterocycles,
the mercapto-substituted 1,2,4-triazole ring systems have
been well investigated and a large number of biological ac-
tivities have been reported for many of their derivatives [4].
There is enough literature available highlighting the im-
portance of 1,2,4-triazole nucleus in the development of
novel compounds with antiphlogistic [5-7], antitumor [8, 9],
Despite the accessibility to wide array of compounds,
there is a substantial number of diseases for which either
there is no offered drug therapy or existing drugs are unable
to cure the disease completely. So, there is always a liberty
for the progress by developing new drugs with a wide thera-
peutic window and fewer side effects [1]. Amid many doable
strategies for improving the therapeutic effectiveness of nu-
merous drugs, one of the most imperative is the synthesis of
new derivatives with tailored structure. Inflammation is a
*Address correspondence to this author at the Department of Pharmaceutical
Chemistry, Riphah Institute of Pharmaceutical Sciences, Riphah Interna-
tional University St. 41, G-7/4, 7th Avenue, Islamabad, Pakistan; UAN:
+92-51-111-510-511; Tel: +92 (51) 289 1835-8, +92 (51) 810 8793-5;
Fax: +92(51)2890690; Mobile: 03235010850;
E-mail: humaira.nadeem@riphah.edu.pk
1875-628X/19 $58.00+.00
©2019 Bentham Science Publishers

Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular Docking Studies
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7 735
antifungal [10, 11], antitubercular action [12-14], analgesic
[15], anticonvulsant [16, 17] and antibacterial action [18-22].
Triazole containing ring system is present in many therapeu-
tically interesting drug candidates. Many of them are ap-
proved drugs like alprazolam [23] or vibunazole [24]. Letro-
zole, vorozole, and anastrozole are used for cancer as non-
steroidal drugs [25]. Triazoles are also used for the synthesis
of very effective antifungal agents such as fluconazole,
voriconazole, and itraconazole [26, 27]. Furthermore, the
literature survey showed that 1,2,4-triazole-3-acetate bearing
aryl groups or heteroaryl residues [28] have shown promi-
nent anti-inflammatory activity.
THR-530
VAL-523
PHE-525
ASN-529
GLN-526
ASP-331
PHE-330
-522
LEU-328
LYS-329
Provoked by these annotations and in the persistence of our
hunt for better therapeutic agents, the present study was designed to
synthesize ethyl [(4,5-disubstituted-4H-1,2,4-triazol-3-yl) sul
fanyl]acetate derivatives and screening them for anti-
inflammatory and antimicrobial potential. These newly syn-
thesized compounds have the distinctive feature of having
esterified hydroxyalkyl chain to minimize the effects of free
carboxylic group. Further to investigate the mechanism,
these compounds were docked against both COX-1 and
COX-2, the major targets involved in the inflammation
pathway.
U-333
ASN
ARG-78
Fig. (2). Binding interactions of compound 2a with COX-II.
Picture from Discovery studio visualizer showing Ethyl {[4-(4-
chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetate
binding interactions with the binding site of COX-II.
LEU-92
IIE-314
SER-90
ARG-89
SER
MET-441
MEN-491
2. EXPERIMENTAL
2.1. Chemistry
GLY-440
LEU-86
THR-87
VAL-88
Solvents and chemicals from Merck and Sigma Aldrich
were used without further purification. All the compounds
synthesized were purified by recrystallization in suitable
solvent. Elemental analyses (C, H, N) were in agreement
with the proposed structures and within 0.5% of the theo-
retical values. Monitoring of the reactions to check the purity
of the final products was done using thin layer chromatog-
raphy with silica gel-precoated aluminum sheets (60 F254;
Merck Schuchardt, Darmstadt, Germany) and visualization
with ultraviolet light at 365 nm and 254 nm. Characterization
of synthesized compounds was done by spectrophotometric
LEU-328
AL-85
PRO-55
MET-82
HIS-59
LEU-8
LEU-
PHE-6
LEU-62
LEU-81
THR-58
ARG-52
PRO-53
ARG-78
LEU-61
LEU-51
VAL-72
Fig. (3). Binding interactions of compound 2e with COX-I. Picture
from Discovery studio visualizer showing Ethyl {[4-cyclohexyl-
5-(4-methylphenyl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetate binding
interactions with the binding site of COX-I.
PHE-439
SER-56
MET-441
HIS-59
PHE-60
PRO-55
LEU
GLY-440
ARG-89
THR-58
-61
analysis i.e., FT-IR (Thermoscientific NICOLET IS10 Spec-
trophotometer), 1H NMR and 13C NMR spectroscopy
(Bruker AM-300 spectrophotometer) using DMSO as sol-
vent (Figs. 1-6).
ASN-
VAL-05
AR
VAL-88
2.1.1. Synthesis of 4,5-disubstituted-4H-1,2,4-triazole-3-
thiols 1(a-i)
ASN-49
ARG-48
Equimolar mixture of carboxylic acid hydrazide and
isothiocynate (0.03 mol) was refluxed with stirring for 6-8
hours. After completion, the reaction mixture was cooled and
the corresponding thiosemicarbazide was separated and dis-
solved in 4N NaOH solution. The solution was refluxed for
4-5 hours and after completion, it was filtered and the filter-
ate acidified to pH of 4-5. The solid separated was filtered
and recrystallized from ethanol.
LEU-51
EU-81
LEU-84
Fig. (1). Binding interactions of compound 2a with COX-I. Pic-
ture from Discovery studio visualizer showing Ethyl {[4-(4-
chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetate
binding interactions with the binding site of COX-I.

736 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7
Arif et al.
ASN-72
ASN-73
GLU-333
VAL-57
LEU-328
ILE-60
ASP-331
LYS-329
HIS-63
PHE-330
LEU-61
IE-64
GLU-315
ASN-529
ILE-81
PHE-67
THR-530
Fig. (6). Binding interactions of compound 2g with COX-II.
Picture from Discovery studio visualizer showing Ethyl [(4-
cyclohexyl-5-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetate binding
interactions with the binding site of COX-II.
Fig. (4). Binding interactions of compound 2e with COX-II.
Picture from Discovery studio visualizer showing Ethyl {[4-
cyclohexyl-5-(4-methylphenyl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetate
binding interactions with the binding site of COX-II.
SER-54
PRO-55
2.1.1.3. 4-(4-fluorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-
3-thiol (1c)
LEU-62
THR-58
Yield 74.2%, M.p.: 267-269°C, Rf 0.78 (ethyl acetate :
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 7.52-
PRO-53
8.56 (m, 6H, ArH), 8.65 (d, 2H, ArH), 11.60 (bs, 1H,
NH/SH); IR cm-1: 3212 (NH), 1588 (C=N), 1397 (C=C),
1289 (C=S).
2.1.1.4. 4-hexyl-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-
3-thiol (1d)
ARG-52
Yield 74.4%, M.p.: 191-192°C, Rf 0.86 (ethyl acetate:
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm:0.85 (t,
VAL-85
LEU-51
3H, CH3), 1.55-1.71 (m, 6H, hexyl CH2), 1.85 (m, 2H, hexyl
CH2, 3.78 (s, 9H, OCH3), 4.16 (t, 2H, NH-CH2), 6.98 (s, 2H,
ArH), 11.54 (bs, 1H, NH/SH); IR cm-1: 3219 (NH), 1602
(C=N), 1421 (C=C), 1296 (C=S).
LEU-81
VAL-88
2.1.1.5. 4-cyclohexyl-5-(4-methylphenyl)-4H-1,2,4-triazole-
3-thiol (1e)
Fig. (5). Binding interactions of compound 2g with COX-I.
Picture from Discovery studio visualizer showing Ethyl [(4-
cyclohexyl-5-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetate binding
interactions with the binding site of COX-I.
Yield 70.1%, M.p.: 178-180°C, Rf 0.88 (ethyl acetate:
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 1.28-
1.75 (m, 10H, cyclohexyl CH2), 2.31 (s, 3H, Ar-CH3), 4.45
(m, 1H, cyclohexyl CH), 7.31-7.40 (m, 4H, ArH), 11.63 (bs,
1H, NH/SH); IR cm-1: 3221 (NH), 1581 (C=N), 1409 (C=C),
1279 (C=S).
2.1.1.1. 4-(4-chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-
3-thiol (1a)
Yield 71.7%, M.p.: 240-241°C, Rf 0.85 (ethyl acetate:
2.1.1.6.
thiol (1f)
4-hexyl-5-(4-methylphenyl)-4H-1,2,4-triazole-3-
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 7.52-
8.01 (m, 6H, ArH), 8.65 (d, 2H, ArH), 11.65 (bs, 1H,
NH/SH); IR cm-1: 3171 (NH), 1559 (C=N), 1289 (C=S).
Yield 66.34%, M.p.: 90-91°C, Rf 0.83 (ethyl acetate:
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 0.86 (t,
2.1.1.2.
thiol (1b)
4-ethyl-5-(4-methylphenyl)-4H-1,2,4-triazole-3-
3H, CH3), 1.52-1.70 (m, 6H, hexyl CH2), 1.79 (m, 2H, hexyl
CH2), 2.31 (s, 3H, Ar-CH3), 4.14 (t, 2H, NH-CH2), 7.36-7.42
(m, 4H, ArH), 11.65 (bs, 1H, NH/SH); IR cm-1 : 3239 (NH),
1596 (C=N), 1265 (C=S).
Yield 69.7%, M.p.: 147-148°C, Rf 0.83 (ethyl acetate:
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 1.32 (t,
J = 7.0 Hz, 3H, CH3), 2.3 (s, 3H, Ar-CH3), 4.15 (q, J = 7.1
Hz, 2H, CH2), 7.34-7.48 (m, 4H, ArH), 11.75 (bs, 1H,
NH/SH); IR cm-1: 3199 (NH), 1579 (C=N), 1404 (C=C),
1277 (C=S).
2.1.1.7. 4-cyclohexyl-5-phenyl-4H-1,2,4-triazole-3-thiol (1g)
Yield 70.3%, M.p.: 198-200°C, Rf 0.77 (ethyl acetate:
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 1.28-

Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular Docking Studies
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7 737
1.81 (m, 10H, cyclohexyl CH2), 4.38 (m, 1H, cyclohexyl
CH), 7.32-7.38 (m, 5H, ArH), 11.69 (bs, 1H, NH/SH); IR
cm-1 : 3207 (NH), 1583 (C=N), 1418 (C=C), 1294 (C=S).
2.1.2.3. Ethyl {[4-(4-fluorophenyl)-5-(pyridin-4-yl)-4H-
1,2,4-triazol-3-yl]sulfanyl}acetate (2c)
Yield: 76.7%, M.p.: 138-140°C. Analysis for C17H15FN4
O2S, Calculated: C 56.97%, H 4.22, N 15.63%. Found C
2.1.1.8. 5-benzyl-4-cyclohexyl-4H-1,2,4-triazole-3-thiol (1h)
1
56.91%, H 4.17, N 15.64%. H NMR (DMSO-d6) δ (ppm):
Yield 68.1%, M.p.: 156-157°C, Rf 0.79 (ethyl acetate:
1.3 (t, 3H, J = 7.2 Hz, CH3), 4.11 (s, 2H, SCH2.), 4.20 (q,
2H, J = 7.4 Hz,CH2), 7.23-7.35 (m, 6H, ArH), 8.50 (d, 2H,
ArH); 13C NMR (DMSO-d6) δ (ppm): 173.1 (C=O), 156.9
(triazole-C5), 144.1 (triazole-C3), Ar C (pyridine) [125.1
(C1), 125.7 (C2 and C6), 154.9 (C3 and C5)] Ar C [117.2 (C1),
123.8 (C2 and C6), 118.3 (C3 and C5), 163.3 (C4)], 61.6
(OCH2), 32.0 (SCH2), 14.2 (CH3). IR cm-1: 1729 (C=O es-
ter), 1157 (C-O) 1598 (C=N).
1
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 1.28-
1.81 (m, 10H, cyclohexyl CH2), 3.72 (s, 2H, CH2), 4.38 (m,
1H, cyclohexyl CH), 7.32-7.40 (m, 5H, ArH), 11.83 (bs, 1H,
NH/SH); IR cm-1: 3193 (NH), 1611 (C=N), 1414 (C=C),
1288 (C=S).
2.1.1.9. 4-hexyl-5-phenyl-4H-1,2,4-triazole-3-thiol (1i)
Yield 71.7%, M.p.: 109-110°C, Rf 0.91 (ethyl acetate:
1
2.1.2.4.
Ethyl{[4-hexyl-5-(3,4,5-trimethoxyphenyl)-4H-
petroleum ether 2:1); H NMR (DMSO-d6) δ (ppm): 0.80 (t,
3H, J = 6.5Hz, CH3), 1.20-1.69 (m, 6H, hexyl CH2), 1.81 (m,
2H, hexyl CH2), 4.10 (t, 2H, N-CH2), 7.42-7.54 (m, 5H,
ArH), 11.70 (bs, 1H, NH/SH); IR cm-1 : 3251 (NH), 1589
(C=N), 1414 (C=C), 1275 (C=S).
1,2,4-triazole-3-yl]sulfanyl}acetate (2d)
Yield: 67.7%, M.p.: 117-118°C. Analysis for C21H31N3
O5S (437.55) Calculated: C 57.64%, H 7.14, N 9.6%; Found
C 57.60%, H 7.13, N 9.6%. 1H NMR (DMSO-d6) δ (ppm):
0.86 (t, 3H, J = 7.1 Hz, CH3), 1.16 (t, J = 7.2 Hz, 3H, CH3.),
1.28-1.32 (m, 6H, hexylCH2), 1.91 (m, 2H, CH2), 3.61 (s,
6H, OCH3), 3.73 (s, 2H, SCH2), 3.85 (s, 3H, OCH3), 3.91 (t,
2H, J = 7.0 Hz, NCH2), 4.15 (q, 2H, J = 7.2Hz, OCH2), 6.9
(s, 2H, ArH); 13C NMR (DMSO-d6) δ (ppm): 175.9 (C=O),
155.3 (triazole-C5), 144.8 (triazole-C3), Ar C [118.8 (C1),
129.9 (C2 and C6), 118.6 (C3 and C5), 161.4 (C4)], ArOCH3
[58.4 (C3-C5), 61.8 (C1)], 32.1 (SCH2), 64.1 (OCH2), Hexyl
C [ 49.3 (C1), 27.43, 26.8, 31.6, 23.7, 14.6], 14.27 (CH3);
FTIR cm-1: 1723 (C=O ester), 1077 (C-O) 1587 (C=N).
2.1.2. Formats: Synthesis of Ethyl [(4,5-disubstituted-4H-
1,2,4-triazol-3-yl)sulfanyl]acetate Derivatives 2(a-i)
To a solution of 4,5-disubstitutedl-4H-1,2,4-triazole-3-
thiol derivatives (0.003 mol) in absolute ethanol (20 ml),
0.003 moles of KOH were added and refluxed for 30
minutes. After cooling 0.003 moles of ethyl chloroacetate
were added and reaction mixture was refluxed further for 8-
10 hours. The progress of reaction was monitored by TLC
(Petroleum ether: Ethyl acetate 1:2). After completion of
reaction, the solvent was removed under reduced pressure to
give the solid product. The crude product was recrystallized
from ethanol: water (1:1).
2.1.2.5. Ethyl {[4-cyclohexyl-5-(4-methylphenyl)-4H-1,2,4-
triazol-3-yl]sulfanyl}acetate (2e)
Yield: 71.8%, M.p.: 128-129°C. Analysis for C19H25N3
O2S (359.49) Calculated: C 63.48%, H 7.01, N 11.69%;
Found C 63.49%, H 7.01, N 11.63%.1H NMR (DMSO-d6) δ
(ppm): 1.23 (t, 3H, J = 7.0 Hz, CH3), 1.28-1.75 (m, 10H,
cyclohexyl CH2), 2.38 (s, 3H, ArCH3), 3.67 (s, 2H, SCH2),
4.04 (q, 2H, J = 7.1 Hz, CH2), 4.13 (m, 1H, cyclohexyl CH),
7.35-7.54 (m, 4H, ArH); 13C NMR (DMSO-d6) δ (ppm):
176.0 (C=O), 155.2 (triazole-C5), 144.5 (triazole-C3), Ar C
[118.7 (C1), 129.5 (C2 and C6), 118.6 (C3 and C5), 161.6
(C4)], 64.1 (OCH2), Cyclohexyl C [ 58.4 (C1), 31.4, 27.4,
26.9 ], 23.4 (ArCH3), 32.1 (SCH2), 14.2 (CH3). FTIR cm-1:
1730 (C=O ester), 1064 (C-O) 1653 (C=N).
2.1.2.1. Ethyl {[4-(4-chlorophenyl)-5-(pyridin-4-yl)-4H-
1,2,4-triazol-3-yl]sulfanyl}acetate (2a)
Yield: 72.6%, M.p.: 141-142°C. Analysis for C17H15ClN4
O2S (374.84) Calculated: C 54.47%, H 4.03, N 14.95%;
Found C 54.51%, H 4.01, N 14.89%. 1H NMR (DMSO-d6) δ
(ppm): 1.19 (t, 3H, J = 7.1 Hz, CH3), 4.01 (s, 2H, SCH2),
4.13 (q, 2H, J = 7.2 Hz, OCH2), 7.73-7.92 (m, 6H, ArH),
8.67 (d, 2H, ArH); 13C NMR (DMSO-d6) δ (ppm): 171.8
(C=O), 152.8 (triazole-C5), 145.3 (triazole-C3), Ar C (pyri-
dine) [128.6 (C1), 122.3 (C2 and C6), 151.5 (C3 and C5)] Ar
C [116.4 (C1), 128.4 (C2 and C6), 117.1 (C3 and C5), 161.4
(C4)], 63.8 (OCH2), 32.1(SCH2), 14.2 (CH3). FTIR cm-1:
1708 (C=O ester), 1609 (C=N), 1199 (C-O).
2.1.2.6.
Ethyl
{[4-hexyl-5-(4-methylphenyl)-4H-1,2,4-
triazole-3-yl]sulfanyl}acetate (2f)
2.1.2.2. Ethyl {[4-ethyl-5-(4-methylphenyl)-4H-1,2,4-triazol-3-
yl]sulfanyl}acetate (2b)
Yield: 70.1%, M.p.: 130-131°C. Analysis for C19H27N3
O2S: (361.50) Calculated: C 66.13%, H 7.535%, N 11.62%;
Found C 66.09%, H 7.48, N 11.60%. 1H NMR (DMSO-d6) δ
(ppm): 0.87 (t, 3H, J= 7.2 Hz, hexyl CH3), 1.19 (t, 3H, J =
7.3 Hz, CH3), 1.29-1.34 (m, 6H, hexyl CH2), 1.83 (m, 2H,
hexyl CH2), 2.26 (s, 3H, ArCH3), 3.77 (s, 2H, SCH2), 4.13
(q, 2H, J = 7.4 Hz, OCH2), 4.18 (t, 2H, J= 7.2 Hz, NCH2),
7.71-7.82 (m, 4H, ArH); 13C NMR (DMSO-d6) δ (ppm):
175.5 (C=O), 155.8 (triazole-C5), 144.4 (triazole-C3), Ar C
[118.7 (C1), 129.7 (C2 and C6), 118.6 (C3 and C5), 161.4
(C4)], Ar C [118.8 (C1), 129.7 (C2 and C6), 118.5 (C3 and
C5), 161.4 (C4)], 63.4 (OCH2), Hexyl C [ 49.3 (C1), 27.4,
26.8, 31.6, 23.7, 14.6] 23.41 (Ar-CH3), 32.1 (SCH2), 14.27
(CH3) IR cm-1: 1731 (C=O ester), 1086 (C-O) 1597 (C=N).
Yield: 70.5%, M.p.: 125-126°C. Analysis for C15H19N3
O2S (305.40) Calculated: C 58.99%, H 6.27, N 13.76%;
Found C 58.78%, H 6.13, N 13.57% 1H NMR (DMSO-d6) δ
(ppm): 1.17 (t, 3H, J = 7.0 Hz, CH3), 1.81 (t, 3H, J = 6.9 Hz,
CH3.), 2.39 (s, 3H, ArCH3), 3.68 (s, 2H, SCH2), 4.01 (q, 2H,
J = 7.1 Hz, CH2), 4.11 (q, 2H, J = 7.0 Hz, N-CH2), 7.34-7.42
(m, 4H, ArH); 13C NMR (DMSO-d6) δ (ppm): 175.8 (C=O),
155.8 (triazole-C5), 144.3 (triazole-C3), Ar C [118.7 (C1),
129.6 (C2 and C6), 118.3 (C3 and C5), 161.4 (C4)], 64.1
(OCH2), 46.7 (N-CH2), 32.1(SCH2), 23.4 (Ar-CH3), 15.8
(CH3), 14.2 (CH3). FTIR cm-1: 1713 (C=O ester), 1170 (C-
O) 1611 (C=N).

738 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7
Arif et al.
2.1.2.7. Ethyl [(4-cyclohexyl-5-phenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetate (2g)
intraperitoneally in 0.5% aqueous carboxymethyl cellulose
suspension. After 30 min of drug administration, car
rageenan (0.1 ml, 1% w/v solution in normal saline) as phlo-
gistic agent was injected into the plantar surface of the right
hind paw of every animal. The volume of the right hind paw
was determined before phlogistic agent injection and then
after 1 hour, 2 hours, 3 hours and 4 hours. The mean differ-
ence in the volume of the right hind paw of rats was com-
pared with control and standard. Percent inhibition was cal-
culated by;
Yield: 72.1%, M.p.: 136-137°C. Analysis for
C18H23N3O2S (345.46) Calculated: C 62.58%, H 6.71, N
1
12.16%: Found C 62.55%, H 6.69, N 12.13%; H NMR
(DMSO-d6) δ (ppm): 1.15 (t, 3H, J = 7.0 Hz, CH3), 1.37-1.52
(m, 6H, cyclohexyl H), 1.96 (m, 4H, cyclohexyl H), 3.95 (s,
2H, SCH2), 4.01 (q, 2H, J = 7.2 Hz, OCH2), 4.65 (m, 1H,
CH-N), 7.77-7.90 (m, 5H, ArH); 13C NMR (DMSO-d6) δ
(ppm): 175.6 (C=O), 155.8 (triazole-C5), 144.3 (triazole-C3),
Ar C [118.7 (C1), 129.76 (C2 and C6), 118.6 (C3 and C5),
161.4 (C4)], 64.2 (OCH2), cyclohexyl C [ 58.43 (C1), 31.45,
27.43, 26.98], 14.27 (CH3); FTIR cm-1: 1738 (C=O ester),
1083 (C-O), 1643 (C= N).
% inhibition = (VcꢀVt / Vc) x100
Where Vc is the mean relative change in paw oedema
volume in control group and Vt is mean relative change in
paw edema volume in test group [29].
2.1.2.8. Ethyl [(5-benzyl-4-cyclohexyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetate (2h)
2.2.2. Antimicrobial Assay
The antibacterial and antifungal activity of synthesized
triazole esters 2(a-i) was evaluated by agar well diffusion
method [30, 31]. Three bacterial strains namely Escherichia
coli (ATCC-25922), Pseudomonas aeruginosa (ATCC
10145), Staphylococcus aureus (ATCC 6538) and two fun-
gal strains, Candida glabrata (ATCC 62934) and Candida
albicans (ATCC 60387) were used. Ciprofloxacin was taken
as positive control for antibacterial activity and Nystatin was
used as positive control for antifungal activity. DMSO was
taken as a negative control in both antibacterial and antifun-
gal assays. Nutrient agar/ Sabouraud dextrose agar and nutri-
ent broth were sterilized in the autoclave at 121°C for 15
minutes. Sterile agar plates were prepared by pouring 25mL
sterile nutrient agar into each plate and kept in an incubator
at 37°C for 24 hours. The fresh bacterial/fungal strains were
prepared in sterile nutrient broth and incubated at 37°C for
24hrs. Lawn of each bacterial/fungal stain was made on the
agar plates and wells were made using sterile borer (6mm).
With the help of micropipette 50µg/ml of the synthesized
compounds, ciprofloxacin and DMSO were poured into the
individual well. The concentration of test compounds and
positive control was 10mg/ml and 100µg/ml respectively.
Finally the plates were incubated for 24 hours at 37°C in
case of bacteria and at 28°C for four days in antifungal activ-
ity. The zones of inhibition were measured accordingly.
Yield: 69.8%, M.p.: 136-137°C. Analysis for C19H25N3
O2S (359.49) Calculated: C 63.48%, H 7.01, N 11.69%
1
Found: C 63.44%, H 7.02, N 11.67%. H NMR (DMSO-d6)
δ (ppm): 1.07 (t, 3H, J = 7.4 Hz, CH3), 1.36-1.41 (m, 10H,
cyclohexyl H.), 2.63 (s, 2H, ArCH2), 3.86 (s, 2H, SCH2),
4.01 (q, 2H, J = 7.5 Hz, OCH2), 4.10 (m, 1H, CH-N), 7.2-7.3
(m, 5H, ArH); 13C NMR (DMSO-d6) δ (ppm): 175.8 (C=O),
155.8 (triazole-C5), 144.3 (triazole-C3), Ar C [141.7 (C1),
129.7 (C2 and C6), 128.6 (C3 and C5), 129.4 (C4)], 64.1
(OCH2), cyclohexyl C [58.43 (C1), 31.45, 27.43, 26.98],
32.47 (ArCH2), 32.2 (SCH2), 14.27 (CH3). FTIR cm-1: 1741
(C=O ester), 1146 (C-O) 1619 (C=N).
2.1.2.9. Ethyl [(4-hexyl-5-phenyl-4H-1,2,4-triazol-3-yl)
sulfanyl]acetate (2i)
Yield: 73.3%, M.p.: 106-107°C. Analysis for C18H25N3
O2S, (347.48) Calculated: C 62.22%, H 7.25, N 12.09%;
1
Found: C 62.19%, H 7.24, N 12.05%. H NMR (DMSO-d6)
δ (ppm): 0.86 (t, 3H, J = 7.1 Hz, hexyl CH3), 1.23 (t, 3H, J =
7.2 Hz, CH3), 1.25-1.39 (m, 6H, hexyl CH2), 1.83 (m, 2H,
hexyl CH2), 3.85 (s, 2H, SCH2), 4.13 (q, 2H, J = 7.1 Hz,
NCH2), 4.22 (t, 2H, J= 7.2 Hz, OCH2), 7.47-7.71 (m, 5H,
ArH); 13C NMR (DMSO-d6) δ (ppm): 175.8 (C=O), 155.6
(triazole-C5), 144.3 (triazole-C3), Ar C [118.7 (C1), 129.7
(C2 and C6), 118.6 (C3 and C5), 161.4 (C4)], 63.5 (OCH2),
Hexyl C [ 49.37 (C1), 27.4, 26.8, 31.6, 23.7, 14.6], 32.1
(SCH2), 14.27 (CH3). FTIR cm-1: 1738 (C=O ester), 1122
(C-O) 1586 (C=N).
2.3. Docking Studies
ChemSketch 12.0 was used for drawing nine different
ligands. Different derivatives of main nucleus ethyl (4,5-
disubstituted-4H-1,2,4-triazol-3-ylsulfanyl)acetate were de-
signed by substituting different groups at 4 and 5 positions
of triazole. Uniprot (www.uniprot.org) [32] was used for
obtaining the sequences of COX-1 (M1-L599: (ID No.
P23219)). Three dimensional (3D) homologous sequences of
structures present in PDB were obtained by submitting se-
quences to NCBI given protein specific iteration (PSI)-
BLAST [33].Structure having PDB ID: 1CQE (The X-ray
crystal structure of the membrane protein prostaglandin H2
synthase-1 resolved at 3.1 Å) [34] with 93% sequence
similarity with human COX-1. This three-dimensional
structure was used as a model/template for COX-1 ho-
mology modelling. Structural sequencing between the
COX-I and its template was done by a web server expresso.
2.2. Pharmacological Screening
2.2.1. In vivo Anti-inflammatory Activity
The antiꢀinflammatory potential was assessed using car-
rageenanꢀinduced rat paw oedema method. Sprague Dawley
rats (140-180g) were divided into eleven groups each group
having 5 animals. Animals were starved overnight with wa-
ter ad libitum prior to the day of the experiment. Group 1
taken as negative control group & group 11 was taken as
positive control which received diclofenac sodium 10 mg/kg,
group 2 to 10 received synthesized compounds 2a to 2i
30mg/kg respectively. The initial volume of right hind paw
of Sprague Dawley rat was determined using plethysometer
without administration of drug. The drug was administered

Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular Docking Studies
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7 739
H
S
H
N
H
N
R'
N
S
R
N
NH2
+
R
N
H
R'
O
O
N
4N NaOH
N
1) KOH
N
O
N
R
SH
N
R
2) ClCH2COOC2H5
S
1(a-i)
R'
N
O
R'
2(a-i)
R
R'
0
4-C5H4N
4-ClC6H4
C2H5
1a, 2a
4-CH3C6H4
4-C5H4N
1b, 2b
1c, 2c
4-FC6H4
C6H13
3,4,5-OCH3C6H2
4-CH3C6H4
1d, 2d
1e, 2e
C6H11
4-CH3C6H4
C6H5
1f, 2f
C6H13
C6H11
1g, 2g
C6H5CH2
C6H5
C6H11
C6H13
1h, 2h
1i, 2i
Scheme 1. Synthesis of 4,5-disubstituted-1,2,4-triazole-3-thioacetate derivatives
(http://www.tcoffee.org/Projects/expresso/) [35]. Homology
modeling of COX-1 was done by the use of MODELLER.
Hundred models were prepared which were divided into dif-
ferent groups depending upon RMSD among the residues in
their structures by the use of NMRCLUST. Typical models
were obtained from each group on the basis of their potential
energies and scores given by MODELLER, ERRAT, Rama-
chandran plot, Qmean, PROCHECK and PROSA. Similarly,
the structure of COX-2 with PDB ID: 5F1A (The Crystal
Structure of Salicylate Bound to Human Cyclooxygenase-2
resolved at 2.38 Å) [36] was used for additional analyzation.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The purpose of the present work was the synthesis,
spectral analysis and screening of antimicrobial and
anti-inflammatory activities of a series of Ethyl [(4,5-
disubstituted-4H-1,2,4-triazol-3-yl)sulfanyl]acetate deriva-
tives (2a-i). The target compounds were synthesized accord-
ing to steps outlined in Scheme 1. In the first step different
carboxylic acid hydrazides were condensed with respective
isothiocyanates to yield thiosemicarbazides which were cy-
clized in alkaline media to the corresponding 4, 5-
disubstituted-4H-1, 2, 4-triazole-3-thiols 1(a-i). The triazoles
were finally reacted with KOH and ethyl chloroacetate to
yield the target thioacetates 2(a-i). Physical data of target
triazole derivatives is presented in Table 1.
The re-docking procedure is used to validate docking
protocols [37].
For validation of docking protocols, we used the COX-II
crystallographic structure with a PDB ID: 5F1A, docking
protocols were performed into the active site of COX-II and
pose were compared with the original crystallographic struc-
ture. Re-docked ligand binds to pCOX-II with similar bind-
ing mode and interaction compared to the original crystallo-
graphic structure.
All the synthesized compounds are stable solids and dis-
solved in DMSO at room temperature. Each compound
yielded a single spot in a different solvent system establish-
ing the purity of synthesized compounds. The triazoles (1a-i)
are known compounds and characterized by comparing their
melting points and spectral data with the reported values
[40]. FTIR data showed strong peak of C=N in each com-
pound. The strong peak of C=S was also observed in each
case indicating the presence of thione form as the predomi-
nant form. In the 1H NMR spectra of triazoles downfield
signal of tautomeric proton NH/SH was observed confirming
the formation of products.
Protein-Ligand docking was carried out using Auto
dockvina [38], Open Babel GUI [39], Discovery Studio 4.1
Visualizer and Pymol were utilized for preparing and con-
version of protein molecules by removing the ligand and
water. The results obtained were shown in the form of bind-
ing affinity.

740 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7
Arif et al.
The final compounds 2(a-i) were characterized by FTIR,
1H NMR, 13C NMR and elemental analysis data. FTIR
spectral data of ester derivatives 2(a-i) showed a strong peak
of C=O (ester) in each compound in the range 1708-1738
cm-1. Strong peak of C=N was also observed in each com-
pound in the range 1609-1619 cm -1. Also appearance of C-
O peak indicated the formation of ester derivatives. 1H NMR
data confirmed the formation of ethyl [(4,5-disubstituted-4H-
1,2,4-triazol-3-yl)sulfanyl]acetate derivatives. Triplet of
CH3 protons was observed at 1.3 ppm in each compound.
The quartet of CH2 protons of ester moiety appeared at
higher chemical shift in the range of 4.01 ppm to 4.13 ppm
which was deshielded due to the electronegative effect of
adjacent oxygen atom. Another evidence for the formation of
sulfanyl acetate derivatives was the appearance of singlet of
S-CH2 in all the newly synthesized compounds in the range
of 3.68 ppm to 4.13 ppm due to adjacent electronegative
sulphur. In the 13C NMR spectra the most downfield peak of
ester carbonyl confirmed the formation of ester derivatives.
The two carbons of triazole moiety resonated in the range
144.1-155.8 ppm. Also, methylene protons of ester moiety
attached to oxygen were observed at 61.1-64.1 ppm. Ele-
mental analysis data further confirmed the formation of thio-
acetate derivatives of triazoles.
induced paw edema of 62.5% at 30 mg/kg dose. The com-
pound 2h produced inhibition quite comparable with that of
standard (Diclofenac), which was 46.6 % inhibition after 1
hour, 56.25 % inhibition after 2 hours, 57.8% inhibition after
3 hours and 62.5% inhibition after 4 hours while standard
drug produced 26.49% inhibition after 1 hour, 39.50% inhi-
bition after 2 hours, 57.37% inhibition after 3 hours and
74.07% after 4 hours. Compounds 2a, 2b, 2c and 2e also
showed good activities i.e., 51.23%, 50.0%, 56.23% and
56.25% respectively after 4 hours. Compound 2g and 2f
were moderately active while 2d and 2i showed weak anti-
inflammatory activity. All the compounds were also docked
against COX-I and COX-II to find their potential interactions
with these two targets of inflammation pathway. All the
compounds showed good binding affinities with both COX-I
and COX-II especially with COX-I. Compound 2h having
maximum anti-inflammatory activity also exhibited high
binding affinity of -6.5 and -7.7 kcal/mol with COX-II and
COX-I respectively. Compound 2c showed maximum bind-
ing affinity with COX-II i.e. -6.7 kcal/mol while with com-
pound 2g has highest binding affinity of -8.0 with COX-I.
The structure-activity relationship of the tested triazole
deriva¬tives 2(a-i) revealed that hexyl-substituted tria-
zoles 2e and 2h were the most active compounds with
maximum inhibition of 56.25 and 62.50% respectively.
Also 4-halosubstituted triazoles 2a and 2c exhibited good
activities while cyclohexyl containing triazoles 2d, 2f and
2i showed weak activity. In general, it can be inferred
from the results that hexyl substitution is the most benefi-
cial in terms of anti-inflammatory activity of triazole thio-
acetate derivatives.
3.2. Pharmacology
3.2.1. In vivo Anti-inflammatory Activity
The acute anti-inflammatory activity of synthesized tar-
get compounds 2(a-i) was determined in vivo using the car-
rageenan-induced paw edema method in rats. The com-
pounds were tested at dose level of 30 mg/kg. The volume of
right hind paw of rats was measured before carrageenen in-
jection and then after 1, 2, 3 and 4 hours. The mean differ-
ence in the volume of the right hind paw of rats was com-
pared with control and standard. The results for the anti-
inflammatory activity of compounds (at 30 mg/kg) and the
potent anti-inflammatory drug indomethacin (at 10 mg/kg)
are listed in Table 2. The best activity was shown by com-
pounds 2h which produced strong inhibition of carrageenan-
3.2.2. Antimicrobial Assay
All newly synthesized ester derivatives were also ex-
plored for their antibacterial and antifungal potential. Agar
well diffusion method was used to determine the antimicro-
bial activity of the synthesized compounds. Staphylococus
aureus (ATCC 6538), Escherichia coli (ATCC-25922), and
Pseudomonas aeruginosa (ATCC 10145), were selected
bacterial stains while antifungal stains were Candida albicans
Table 1. Physical data of compounds 2(a-i).
Color
Molecular
Formula
MW Calculated
(gm/mol)
M.P
(°C)
Physical
State
%
Yield
Rf
Value
Comp
2a
2b
2c
2d
2e
2f
C17H15ClN4O2S
C15H19N3O2S
C17H15FN4O2S
C21H31N3O5S
C19H25N3O2S
C19H27N3O5S
C18H23N3O2S
C19H25N3O2S
C18H25N3O2S
374.84
305.40
358.39
437.55
359.49
361.50
345.46
359.49
347.48
141
125
138
117
128
130
136
125
106
Powder
Powder
White
White
White
White
White
White
White
White
White
72.6
70.1
76.7
67.4
71.8
69.6
72.1
69.8
73.3
0.52
0.38
0.41
0.28
0.33
0.55
0.25
0.28
0.43
Powder
Crystalline Powder
Crystalline Powder
Crystalline Powder
Powder
2g
2h
2i
Powder
Crystalline powder

Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular Docking Studies
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7 741
Table 2. Anti-inflammatory activity of synthesized compounds 2(a-i).
Mean Paw Volume SEM (ml) and % Inhibition
Time after Carrageenan Injection
2 hr
Normal Paw
Volume
Compounds
0 hr
1 hr
3 hr
4 hr
Control
0.251 0.008
0.212 0.013
0.381 0.006
0.33 0.015
0.402 0.015
0.413 0.009
0.441 0.011
0.413 0.013
Standard
0.323 0.021
(26.49%)
0.310 0.011
(39.50%)
0.293 0.019
(57.37%)
0.254 0.016
(74.07%)
2a
2b
2c
2d
2e
2f
0.203 0.017
0.31 0.005
0.222 0.011
0.26 0.012
0.28 0.013
0.30 0.006
0.201 0.011
0.29 0.018
0.25 0.008
0.35 0.0037
0.44 0.009
0.34 0.03
0.327 0.005
(17.88%)
0.304 0.004
(37.65%)
0.292 0.006
(53.16%)
0.282 0.005
(51.23%)
0.44 0.011
(13.3%)
0.41 0.013
(37.5%)
0.40 0.008
(52.63%)
0.39 0.007
(50.0%)
0.326 0.029
(31.12%)
0.321 0.028
(38.89%)
0.313 0.03
(52.10%)
0.293 0.026
(56.17%)
0.39 0.008
0.41 0.016
0.45 0.015
0.38 0.027
0.39 0.014
0.40 0.011
0.38 0.011
(20.0%)
0.38 0.007
(25.0%)
0.39 0.013
(31.58%)
0.38 0.009
(20.0%)
0.39 0.011
(26.67%)
0.37 0.009
(43.75%)
0.37 0.012
(52.63%)
0.35 0.008
(56.25%)
0.46 0.011
(-6.67%)
0.44 0.009
(12.5%)
0.43 0.01
(31.57%)
0.39 0.0011
(43.75%)
2g
2h
2i
0.344 0.018
(5.29%)
0.353 0.021
(6.17%)
0.322 0.011
(36.31%)
0.301 0.017
(38.27%)
0.37 0.009
(46.6%)
0.36 0.007
(56.25%)
0.37 0.012
(57.8%)
0.35 0.015
(62.5%)
0.40 0.01
(0.0%)
0.39 0.008
(12.5%)
0.39 0.012
(26.3%)
0.38 0.009
(18.75%)
Table 3. Antimicrobial activity of synthesized compounds 2(a-i).
Antimicrobial Activity of Synthesized Compounds (Zone of Inhibition mm)
Bacterial Stains
Fungal Stains
Compounds
Escherichia
coli
Pseudomonas
aeruginosa
Staphylococcus
aureus
Candida
glabrata
Candida
albicans
2a
16.2
18.4
17.4
16.6
15.2
15.3
16.3
13.1
14.2
23.7
------
7.2
7.9
-------
-------
-------
-------
-------
-------
-------
-------
-------
23.9
7.7
8.4
6.8
----
7.1
2b
2c
8.3
11.3
----
2d
7.6
7.9
2e
8.0
9.4
---
2f
8.1
7.9
---
2g
2h
8.3
----
---
7.5
7.3
11.2
9.6
2i
7.7
8.2
Ciprofloxacin
Nystatin
24.0
-------
-------
23.2
-------
24.1
-------

742 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7
Arif et al.
Table 4. Binding affinities of compounds 2 (a-i) with COX-I
and COX-II.
(ATCC 60387) and Candida glabrata (ATCC 62934).
Ciprofloxacin was used as a reference for antibacterial assay
whereas; Nystatin was used as a reference for antifungal assay.
Binding Affinity
All the synthesized ester derivatives showed moderate to
good antibacterial potential against gram negative bacterial
species and didn’t show any remarkable potential against
gram positive species. The results of antimicrobial activity
are given in Table 3. Compounds 2b, 2d, 2f and 2i having
alkyl group at 4 position showed good activity against Esch-
erichia coli with the zone of inhibition (mm) 18.4, 16.6, 15.3
and 14.2 respectively. Compound 2b with ethyl group at 4
position exhibited highest activity, substituting position 4
with higher alkanes like hexanes showed a slight decrease in
activity. Substitution of phenyl or substituted phenyl at posi-
tion 4 also showed good activity against Escherichia coli.
All the compounds exhibited weak activity against Pseud-
monas aeruginosa. Presence of benzyl ring at position 5 in
compound 2h showed decreased activity against both Esche-
richia coli and Pseudmonas aeruginosa with zone of inhibi-
tion 13.1 mm and 7.5 mm respectively. None of the com-
pounds showed activity against Staphylococcus aureus.
Against fungal stains all the compounds 2(a-i) showed very
unpredictable results. By substitution at 4 and 5 position no
general trend has been shown regarding antifungal activity.
Compounds 2a, 2c, 2h and 2i showed weak potential against
both Candida glabarata and Candida albicans. Compound
2e and 2f only showed mild activity against Candida gla-
barata whereas compound 2d showed slight potential against
Candida albicans. Rest of the compounds did not show any
potential against both fungal stains.
Protein Target
Ligand
(kcal/mol)
2a
-7.5
-6.9
-7.7
-6.0
-7.9
-6.4
-7.6
-7.7
-6.3
-7.5
2b
2c
2d
2e
COX-II
2f
2g
2h
2i
Diclofenac
Binding Affinity
(kca/mol)
Protein Target
Ligand
2a
-7.9
-7.2
-7.9
-6.9
-8.1
-7.1
-8.0
-7.7
-7.0
-7.6
2b
2c
2d
2e
COX-I
3.3. Docking Analysis
2f
Computational ligand-target binding approach was em-
ployed in analyzing the complexes of synthesized com-
pounds with target proteins (COX-I and COX-II) in order to
interpret the structural basis of target protein specificity.
Compounds were docked into the active site of COX-1 sur-
rounding by Val85, Thr 58, Arg 89, Val 85, Asn 49, Phe 68,
Ser 56, Met 441, Phe 439, Leu 84, Gly 440, His 59, Phe 60
and with COX-2 residues Phe 330, Asp 331, Lys 329, Leu
328, Arg 89, Asn 49, Thr 53, Pro 52, Gly 323, Val 85,
Arg322, Val523, His 58, Glu 493. Leu534. Results of dock-
ing of compounds 2 (a-i) in the form of binding affinities
(kcal/mol) are given in Table 4. Docking results showed that
all the compounds 2 (a-i) bind to both COX-I and COX-II at
their active site successfully. Compound 2e showed the max-
imum binding affinity against COX-II, found to be -
7.9Kcal/mol. While compound 2e also showed the maximum
binding affinity against COX-I, which was -8.1Kcal/mol.
The binding affinities of newly synthesized ligands against
COX-II and COX-I were better than Diclofenac, which are -
7.5 and -7.6 Kcal/mol respectively. In general, the triazole
derivatives show higher binding affinity for COX-I as com-
pared to COX-II. However, the overall anti-inflammatory
effect of our synthesized compounds is supposed to be due to
nonselective inhibition of both COX-I and COX-II.
2g
2h
2i
Diclofenac
as compared to diclofenac. Diclofenac is relatively a simple
molecule and interacts with the COX-II mainly by pi-alkyl
interactions involving residues mainly leucine and valine.
Moreover, the carbonyl carbon of diclofenac showed con-
ventional hydrogen bonding with residues like tyrosine. On
the other hand, newly synthesized triazoles derivatives are
relatively complex structures, thereby enhancing the binding
interactions like pi-sulfur interactions, hydrophobic interac-
tions and hydrogen bonding, van der Waals interactions with
different amino acid residues such as cystine, proline and
asparagine respectively compared to diclofenac as shown in
Fig. (7).
Similarly with diclofenac interact with the COX-I mainly
by pi-anion interaction and hydrogen bonding with glutamic
acid and asparagine respectively. Whereas triazole deriva-
tives showed hydrogen bonding, pi-pi interaction and pi-
sigma interaction with various amino acids like leucine, his-
tidine and lysine as shown in the Fig. (8).
The newly synthesizes derivatives interact with COX-II
and COX-I at the binding site in a slightly different manner

Synthesis, Anti-inflammatory, Antimicrobial Potential and Molecular Docking Studies
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7 743
ILE
B:274
PRO
B:156
VAL
ASP
CYS
B:291
O
TYR
B:157
HIS
B:47
B:136
B:214
O
PRO
B:35
N
GLY
TYR
B:409
B:135
N
S
N
N
COH
MET
B:48
THR
B:603
B:212
GLN
A:327
ASP
B:158
CYS
SER
LYS
B:36
B:49
LEU
B:211
HIS
B:294
B:207
TRP
A:323
GLU
A:322
ASN
ASN
B:34
B:222
GLN
B:203
GLN
B:289
Interactions
van der Waals
Interactions
Pi-Sulfur
Alkyl
van der Waals
Alkyl
Conventional Hydrogen Bond
Carbon Hydrogen Bond
Conventional Hydrogen Bond
Pi-Pi Stacked
Pi-Alkyl
Fig. (7). Comparison of binding modes of Diclofenac (left hand side) and proposed compound 2e (right hand side) with COX-II. (The color
version of the figure is available in the electronic copy of the article).
THR
PHE
B:212
GLN
A:210
A:289
LYS
COH
ASP
B:215
HIS
LYS
GLU
A:602
B:213
B:214
GLN
A:211
A:236
B:203
O
VAL
A:291
THR
O
N
A:237
THR
A:212
N
O
N
O
COH
ASN
N
B:603
A:222
S
LEU
B:294
VAL
ASN
B:291
B:222
GLU
A:290
ILE
A:274
HIS
B:207
Interactions
van der Waals
Pi-Pi Stacked
Pi-Pi T-shaped
Alkyl
Conventional Hydrogen Bond
Pi-Cation
Interactions
van der Waals
Conventional Hydrogen Bond
Pi-Anion
Pi-Sigma
Pi-Alkyl
Pi-Sulfur
Fig. (8). Comparison of binding modes of Diclofenac (left hand side) and proposed compound 2e (right hand side) with COX-I. (The color
version of the figure is available in the electronic copy of the article).
pounds showed good binding affinities with both COX-I and
COX-II explaining their nonselective mode of inhibition.
CONCLUSION
This study reports the synthesis of nine thioacetae deriva-
tives of substituted 1,2, 4 triazoles. In the first step, the 5-
substituted-1,2,4 triazole-3-thiols 1 a-i were synthesized
from corresponding hydrazides which were treated with
ethyl chloroacetate to get the corresponding triazol-3-
yl(sulfanyl)acetate derivatives 2 a-i. All the newly synthe-
sized acetate derivatives were screened for their anti-
inflammatory, antibacterial and antifungal potential. The best
activity was shown by compound 2h which produced strong
inhibition of carrageenan-induced paw edema of 62.5% at 30
mg/kg dose. 2g and 2f also exhibited moderate anti-
inflammatory effect. None of the derivatives showed promi-
nent antibacterial or antifungal potential. Further the inhibi-
tory potential of thioacetates was further investigated by
docking analysis against COX-I and COX-II enzymes of
inflammation pathway using autodock vina. All the com-
ETHICS
APPROVAL
AND
CONSENT
TO
PARTICIPATE
Ethical approval for this study was obtained from Re-
search Ethics Committee-Riphah Institute of Pharmaceutical
Sciences, Pakistan.
HUMAN AND ANIMAL RIGHTS
No humans were used in this study. All animal proce-
dures were conducted in accordance with the animals ethical
guidelines by National Institute of Health (NIH) have been
followed.
CONSENT FOR PUBLICATION
Not applicable.

744 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 7
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AVAILABILITY OF DATA AND MATERIALSꢀ
The data supporting the findings of the article is available at
https://drive.google.com/drive/folders/1F51nPVu_FHGozYygELO
uvH9oArke1eNs
[15]
[16]
FUNDINGꢀ
None.ꢀ
[17]
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
[18]
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ACKNOWLEDGEMENTS
Declared none.
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