Biological evaluation and in silico molecular docking study of a new series of thiazol-2-yl-hydrazone conglomerates

Article abstract of DOI:10.1007/s11164-018-3261-z

A new series of hybridized thiazol-2-yl-hydrazone derivatives having diverse substituents were designed, synthesized, and screened for their anti-inflammatory property by a carrageenan-induced paw edema method. The compounds 11a, 11b, 11c, 11d, 11e, 11g, 11m and 11p revealed significant inhibition when compared to Diclofenac sodium. Subsequently, two highly potent compounds (11d and 11e) were evaluated for their cytotoxic effect on the tumor cell line. The binding interactions of thiazol-2-yl-hydrazones with the cyclooxygenase-2 (COX-2) protein (PDB: 3LN1) displayed effective interactions with Arg-120, Tyr-385 and Tyr-355 amino acids, the main criteria of the COX-2 inhibitor. In addition, all the compounds showed moderate to good in vitro antibacterial activity. Most active benzyloxy derivatives were also tested to understand the radical scavenging efficacy by the 2,2-diphenyl-1-picrylhydrazyl method. Graphical Abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-018-3261-z

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3261-z
Biological evaluation and in silico molecular docking
study of a new series of thiazol-2-yl-hydrazone
conglomerates
Mahima Bhat1 P. M. Gurubasavaraja Swamy2 Boja Poojary1
•
•
•
B. C. Revanasiddappa3 M. Vijay Kumar3 Vasantha Kumar4
•
•
Received: 27 March 2017 / Accepted: 8 January 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract A new series of hybridized thiazol-2-yl-hydrazone derivatives having diverse
substituents were designed, synthesized, and screened for their anti-inflammatory
property by a carrageenan-induced paw edema method. The compounds 11a, 11b, 11c,
11d, 11e, 11g, 11m and 11p revealed significant inhibition when compared to
Diclofenac sodium. Subsequently, two highly potent compounds (11d and 11e) were
evaluated for their cytotoxic effect on the tumor cell line. The binding interactions of
thiazol-2-yl-hydrazones with the cyclooxygenase-2 (COX-2) protein (PDB: 3LN1)
displayed effective interactions with Arg-120, Tyr-385 and Tyr-355 amino acids, the
main criteria of the COX-2 inhibitor. In addition, all the compounds showed moderate to
good in vitro antibacterial activity. Most active benzyloxy derivatives were also tested to
understand the radical scavenging efficacy by the 2,2-diphenyl-1-picrylhydrazyl method.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3261-z) contains supplementary material, which is available to authorized users.
&
Boja Poojary
bojapoojary@gmail.com
P. M. Gurubasavaraja Swamy
gurubasavaraj@acharya.ac.in
B. C. Revanasiddappa
evergreen_revan@rediffmail.com
Vasantha Kumar
vasantha.kumar1886@gmail.com
1
2
3
4
Department of Chemistry, Mangalore University, Mangalagangothri, Mangaluru,
Karnataka 574199, India
Medicinal Chemistry Research Laboratory, Acharya and B. M. Reddy College of Pharmacy,
Bangalore, Karnataka 560090, India
Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences Nitte
University, Mangaluru, Karnataka, India
Department of Chemistry, SDM College (Autonomus), Ujire, Karnataka 574240, India
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M. Bhat et al.
Graphical Abstract
Keywords Benzyloxy Á Paw edema Á Anti-inflammatory Á Antibacterial Á Anti-
oxidant Á COX-2
Introduction
In recent years, heterocycles have fascinated researchers as a vital scaffold of
natural products. Of note, incorporation of small heterocyclic moieties especially a
five-membered sulfur- and nitrogen-containing thiazole ring is an enthralling area of
research in enhancing the potency of the drug candidate [1]. The literature has
promulgated that 2,4-disubstituted thiazoles have prevalent biomedicinal utility and
are also effective as anti-inflammatory [2–5], anticandida [6], antimicrobial,
antibacterial, antibiotic, antimycobacterial, anti-oxidant, anticancer, antitumor,
antiviral [7], and anti-Alzheimer [8] agents. On the other hand, thiazole acts as a
potent inhibitor of H?/K?-ATPase [28], MAO-B [9], histone acetyltransferase [10],
procaspase-3 kinase [11], and Trypanosoma cruzi [12] (Fig. 1).
Moreover, the benzyloxy group paves the way to develop novel molecules with
their germaneness in the field of medicinal chemistry as an important pharma-
cophore. Pharmacological acceptance of the benzyloxy group has been materialized
as
a potential anticancer and anti-oxidant [13], anti-breast cancer [14],
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Biological evaluation and in silico molecular docking study…
Fig. 1 Commercially available drugs which are incorporated with 2,4-disubstituted thiazoles and
synthetic strategy of the series
antileishmanial [15], antiproliferative [16], antiprotozoal [17], antitubercular [18],
and anticonvulsant [19] agents. They also act as inhibitors of Plasmodium
falciparum [20] and monoamine oxidase B [21]. Additionally, morpholine and
piperidine derivatives are known to possess varied medicinal applications [22–28].
In recent decades, the treatment of inflammatory disorders has become one of the
major challenges for the scientists. However, non-steroidal anti-inflammatory drugs
(NSAIDs) are the paramount therapeutics for these and act by inhibiting the COXs
enzymes which are responsible for the prostaglandin biosynthesis. To date, three
isoforms of COX enzymes have been identified as COX-1, COX-2 and COX-3 [29].
Interestingly, COX-2 is the responsible isoenzyme for the formation of
prostaglandin from arachidonic acid which has been the chief reason of inflamma-
tion. On the other hand, COX-1 is mainly involved in maintaining the physiological
action of the tissues [30]. Hence, COX-2 has been recognised as the appropriate
target for the discovery of anti-inflammatory drugs. Moreover, NSAIDs existing
from the past few decades have major side effects due to the lack of inhibitory
specificity for the COX-2 enzyme [31]. Thus, there is a need for the development of
the new NSAIDs having specificity with minimum toxicity.
Nevertheless, multi-drug resistance acquired by the microbes for the drugs,
which are being extensively used for the microbial infection, creates an incessant
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M. Bhat et al.
demand for the development of new drugs with high efficacy and less toxicity [32].
Additionally, the usage of multi-drugs for the treatment of an inflammatory
disorder, concomitant with the microbial infection, may cause severe side effects in
the patients with liver and kidney disorders [33]. The utility of monotherapy along
with the multi-targeted drug is a major endeavor in curing inflammatory conditions
and with advantages over multi-drug therapy [34, 35].
Furthermore, the role of anti-oxidants in alleviating the inflammation by
scavenging the free radicals is never neglected and numerous anti-inflammatory
agents have been reported to act by the same mechanism [36]. Hence, the active
benzyloxy derivatives, which showed good anti-inflammatory activity, were
evaluated to check their potency as radical scavengers.
All the aforementioned literature encouraged us to design and synthesize a series
of simple, bioactive novel hybrids with diverse thiazol-2-yl hydrazones. Moreover,
anti-inflammatory activity of various benzyloxy hydrazones was compared with the
potency of (5-bromothiophen-2-yl)methylene), (4-(piperidin-1-yl)benzylidene), and
(4-morpholino benzylidene) moieties. The mechanism of binding mode and
interaction of molecules with the COX-2 [PDB ID: 3LN1] has been scrutinized
to understand the effectiveness of the molecules as anti-inflammatory agents. As we
are searching for multi-targeted drugs, molecules were also expected to have
antimicrobial and anti-oxidant activities.
Experimental
Materials and methods
Laboratory-grade chemicals and standard techniques were utilized to undertake the
designed reactions. Melting points of the aimed compounds were determined via the
open capillary method. The compounds synthesized were confirmed and characterized
with the aid of thin-layer chromatography (TLC), AT-IR, 1H NMR and 13C NMR data
with the help of a Bruker DRX-300 (400 MHz NMR) and Bruker DRX-75 (100 MHz
NMR) spectrometer in (DMSO)-d6, respectively, using tetramethylsilane (TMS; an
internal standard) and LC mass spectra by a Shimadzu LCMS 2010 spectrometer.
1-(4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene)thiosemicarbazide (9b)
Creamy solid; Yield: 82%; FT IR (ATR, mmax, cm-1): 3381 and 3356 (N–H2), 3251
(N–H), 3104 (Ar–H), 2923, 2871 (C–H), 1615 (C=N), 1579 (C=C), 1247 (C–N),
1089 (C=S); 1H NMR (400 MHz, DMSO-d6, d ppm): 3.80 (s, 3H, –OCH3), 5.12 (s,
2H, –OCH2), 7.02 (d, 2H, J = 8.0 Hz, C5–H of 3-OCH3-C6H3O), 7.12 (dd, 1H,
J = 1.6 and 8.0 Hz, C6–H of 3–OCH3–C6H3O), 7.46 (dd, 1H, J = 2.4 and 8.8 Hz,
C5–H of 2,4-Cl2-C7H3), 7.52 (d, 1H, J = 1.6 Hz, C3–H of 3-OCH3-C6H3O), 7.57
(d, 1H, J = 8.0 Hz, C6–H of 2,4-Cl2-C7H3), 7.65 (d, 1H, J = 2.0 Hz, C3–H of 2,4-
Cl2-C7H3), 7.94 (s, 1H, =C–H), 7.99 (bs, 1H, NH2), 8.14 (bs, 1H, NH2), 11.30 (s,
1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 178.1, 150.1, 149.6, 142.8,
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Biological evaluation and in silico molecular docking study…
134.0, 134.1, 133.9, 131.8, 129.3, 128.3, 128.0, 122.4, 113.7, 109.6, 67.4, 56.3;
ESI–MS: 383.98 (M ? H)?, 385.94 [(M ? H)??2], 388.03 [(M ? H)??4].
1-(4-(2-Fluorobenzyloxy)-3-methoxybenzylidene)thiosemicarbazide (9c)
Creamy solid; Yield: 81%; FT IR (ATR, mmax, cm-1): 3452 and 3332 (N–H2), 3249
(N–H), 3120 (Ar–H), 2947, 2827 (C–H), 1609 (C=N), 1543 (C=C), 1292 (C–N), 1169
1
(C–F), 1110 (C=S); H NMR (400 MHz, DMSO-d6, d ppm): 3.94 (s, 3H, –OCH3),
5.26 (s, 2H, –OCH2), 7.09 (d, 1H, J = 8.0 Hz, C5–H of 3-OCH3-C6H3O), 7.15 (dd,
1H, J = 1.6 and 8.8 Hz, C6–H of 3-OCH3-C6H3O), 7.24 (d, 1H, J = 2.0 Hz, C2–H of
3-OCH3-C6H3O), 7.37–7.41 (m, 1H, C3–H of 2- F-C7H6), 7.50–7.53 (m, 1H, C5–H of
2- F-C7H6), 7.54–7.57 (m, 1H, C6–H of 2- F-C7H6), 7.61–7.65 (m, 1H, C4–H of 2-
F-C7H6), 7.92 (s, 1H, =C–H), 7.98 (bs, 1H, NH2), 8.19 (bs, 1H, NH2), 11.29 (s, 1H, N–
H); 13C NMR (100 MHz, DMSO-d6, d ppm): 178.1, 159.63 (d, C-2, JC,F = 245.1 Hz),
149.6–132.2 (Ar–C), 132.1 (d, C-6, JC,F = 3.8 Hz), 130.6 (d, C-5, JC,F = 8.3 Hz),
125.1 (d, C-1, JC,F = 3.0 Hz), 124.2 (d, C-4, JC,F = 14.4 Hz), 115.5 (d, C-3,
JC,F = 21.3 Hz), 129.3, 113.7, 109.6, 67.4; ESI–MS: 334.09 (M ? H)?.
1-((5-Bromothiophen-2-yl)methylene)thiosemicarbazide (9d)
Creamy yellow solid; Yield: 86%; FT IR (ATR, mmax, cm-1): 3426 and 3273 (N–
H2), 3156 (N–H), 3028 (Ar–H), 2999, 2966 (C–H), 1607 (C=N), 1591 (C=C), 1279
1
(C–N), 1090 (C=S), 559 (C–Br); H NMR (400 MHz, DMSO-d6, d ppm): 7.23 (d,
1H, J = 4.0 Hz, thiophene-H), 7.27 (d, 1H, J = 4.0 Hz, thiophenyl-H), 7.63 (bs,
1H, NH2), 8.14 (s, 1H, =C–H), 8.21 (bs, 1H, NH2), 11.49(s, 1H, N–H); 13C NMR
(100 MHz, DMSO-d6, d ppm): 177.6, 140.6, 136.6, 131.2, 131.09, 114.5; ESI–MS:
263.92 (M ? H)?, 266.19 [(M ? H)??2].
1-(4-(Piperidin-1-yl)benzylidene)thiosemicarbazide (9e)
Greenish-yellow solid; Yield: 86%; FT IR (ATR, mmax, cm-1): 3452 and 3332 (N–
H2), 3249 (N–H), 3120 (Ar–H), 2947, 2827 (C–H), 1609 (C=N), 1110 (C=S), 1534
(C=C), 1061 (C–N); 1H NMR (400 MHz, DMSO-d6, d ppm): 1.56 (s, 6H, piperidyl-
0
H), 3.25 (s, 4H, piperidyl-H), 6.90 (d, 2H, J = 9.2 Hz, C2 and C2 of C6H4), 7.58 (d,
0
2H, J = 8.8 Hz, C3 and C3 of C6H4), 7.78 (bs, 1H, NH2), 7.94 (s, 1H, =C–H), 8.01
(bs, 1H, NH2), 11.21(s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 177.2,
152.3, 143.1, 128.5, 123.2, 114.5, 48.4, 24.9, 23.9; ESI–MS: 263.10 (M ? H)?.
1-(4-Morpholinobenzylidene)thiosemicarbazide (9f)
Orange-red crystals; Yield: 80%; FT IR (ATR, mmax, cm-1): 3456 and 3314 (N–H2),
3296 (N–H), 3068 (Ar–H), 2966, 2875 (C–H), 1615 (C=N), 1560 (C=C), 1110 (C–
1
O–C), 1101 (C–N); H NMR (400 MHz, DMSO-d6, d ppm): 3.09-3.11 (m, 4H,
morpholine-CH2), 3.57–3.69 (m, 4H, morpholine-CH2), 6.84 (d, 2H, J = 9.2 Hz, C2
0
0
and C2 of C6H4), 7.56 (d, 2H, J = 8.8 Hz, C3 and C3 of C6H4), 7.75 (bs, 1H, NH2),
7.90 (s, 1H, =C–H), 8.00 (bs, 1H, NH2), 11.19 (s, 1H, N–H); 13C NMR (100 MHz,
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M. Bhat et al.
DMSO-d6, d ppm): 177.6, 153.0, 142.9, 129.1, 114.1, 66.5, 48.3, 25.0; ESI–MS:
264.92 (M ? H)?.
General procedure for the synthesis of 11a–r is in the supplementary data.
1-(4-(2,4-Dichlorobenzyloxy)benzylidene)-2-(4-p-tolylthiazol-2-yl)hydrazine (11a)
Creamy solid; Yield: 86%; MP: 178–180 °C; FT IR (ATR, mmax, cm-1): 3448 (N–H),
3067 (Ar–H), 2920, 2727 (C–H), 1620 (C=N), 1566 (C=C), 1099 (C–N); H NMR
1
(400 MHz, DMSO-d6, d ppm): 2.46 (s, 3H, –CH3), 5.16 (s, 2H, -OCH2), 7.07 (d, 2H,
0
J = 8.8 Hz, C3 and C3 –H of C6H4O), 7.17–7.19 (m, 3H, thiazolyl-H, C3 and C3 –H of
0
4-CH3-C6H4), 7.45–7.47 (dd, 1H, J = 2.0 and 8.0 Hz, C5–H of 2,4-Cl2-C7H3),
0
0
7.57–7.61 (m, 3H, C6–H of C7H5Cl2 and C2 C2 of 4-CH3-C6H4), 7.67 (d, 1H,
0
J = 2.0 Hz, C3–H of 2,4-Cl2-C7H3), 7.70 (d, 2H, J = 8.0 Hz, C2 and C2 –H of
C6H4O), 7.97 (s, 1H, =C–H), 11.91 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d
ppm): 168.7, 159.4, 141.8, 133.8, 132.8, 102.9, 66.9, 21.2; ESI–MS of C24H19N3Cl2-
OS: 467.96 (M ? H)?, 469.95 [(M ? H)??2], 471.95 [(M ? H)??4].
1-(4-(2,4-Dichlorobenzyloxy)benzylidene)-2-(4-(2,4-dichlorophenyl)thiazol-2-
yl)hydrazine (11c)
Creamy solid; Yield: 88%; MP: 172–174 °C; FT IR (ATR, mmax, cm-1): 3250 (N–
H), 3073 (Ar–H), 2926, 2866 (C–H), 1699 (C=N), 1570 (C=C), 1096 (C–N); H
1
NMR (400 MHz, DMSO-d6, d ppm): 5.15 (s, 2H, -OCH2), 7.06 (d, 2H, J = 8.8 Hz,
0
C3 and C3 –H of C6H4O), 7.34 (s, 1H, thiazolyl-H), 7.43–7.48 (m, 2H, C3–H and
C5–H of 2,4-Cl2-C7H3), 7.58–7.65 (m, 5H, Ar–H), 7.85 (d, 1H, J = 8.8 Hz, C6–H
of 2,4-Cl2-C6H3), 8.04 (s, 1H, =C–H), 12.04 (s, 1H, N–H); 13C NMR (100 MHz,
DMSO-d6, d ppm): 167.9, 159.6, 142.6, 134.0, 133.7, 133.1, 132.7, 132.2, 131.8,
130.1, 129.4, 128.5, 128.1, 128.0, 127.9, 115.6, 109.4, 66.9; ESI–MS of
C23H15N3Cl4OS:
521.98
(M ? H)?,
523.89
[(M ? H)??2],
525.94
[(M ? H)??4], 527.79 [(M ? H)? ?6], 530.01 [(M ? H)??8].
1-(4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene)-2-(4-p-tolylthiazol-2-
yl)hydrazine (11d)
Creamy solid; Yield: 90%; MP: 98–100 °C; FT IR (ATR, mmax, cm-1): 3172 (N–H),
3080 (Ar–H), 2923, 2854 (C–H), 1623 (C=N), 1565 (C=C), 1097 (C–N); H NMR
1
(400 MHz, DMSO-d6, d ppm): 2.28 (s, 3H, -CH3), 4.31 (s, 1H, –OCH3), 5.12 (s, 2H, -
OCH2), 7.03 (d, 1H, J = 8.4 Hz, C5–H of 3-OCH3–C6H3O), 7.12 (dd, 1H, J = 2.0 and
8.0 Hz, C6–H of 3-OCH3-C6H3O), 7.19 (s, 1H, thiazolyl-H), 7.46 (dd, 1H, J = 2.0 and
8.0 Hz, C5–H of 2,4-Cl2-C7H3), 7.53 (d, 1H, J = 2.0 Hz, C2–H of 3-OCH3-C6H3O),
0
7.55-7.59 (m, 3H, C6–H of 2,4-Cl2-C7H3 and C3,C3 of 4-CH3-C6H4), 7.66 (s, 1H, C3–
0
H of 2,4-Cl2-C7H3), 7.69 (d, 2H, J = 8.4 Hz, C2,C2 of 4-CH3-C6H4), 7.91 (s, 1H, =C–
H), 12.01 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 168.7, 159.4, 141.8,
133.8, 132.8, 102.9, 66.9, 21.2; ESI–MS of C24H19N3Cl2OS: 497.96 (M ? H)?,
499.96 [(M ? H)??2], 501.96 [(M ? H)??4].
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Biological evaluation and in silico molecular docking study…
1-(4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene)-2-(4-(4-bromophenyl)thiazol-
2-yl) hydrazine (11e)
Creamy solid; Yield: 86%; MP: 166–168 °C; FT IR (ATR, mmax, cm-1): 3178 (N–
H), 3077 (Ar–H), 2924, 2854 (C–H), 1624 (C=N), 1566 (C=C), 1099 (C–N); H
1
NMR (400 MHz, DMSO-d6, d ppm): 4.33 (s, 1H, -OCH3), 5.13 (s, 2H, -OCH2),
7.06 (d, 1H, J = 8.8 Hz, C5–H of 3-OCH3-C6H3O), 7.14 (dd, 1H, J = 1.6 and
8.4 Hz, C6–H of 3-OCH3-C6H3O), 7.27 (d, 1H, J = 1.6 Hz, C2–H of 3-OCH3-
C6H3O), 7.35 (s, 1H, thiazolyl-H), 7.46 (dd, 1H, J = 1.6 and 8.4 Hz, C5–H of 2,4-
0
Cl2-C7H3), 7.55–7.59 (m, 3H, C6–H of 2,4-Cl2-C7H3 and C2, C2 –H of 4-Br-C6H4),
0
7.65 (d, 1H, J = 1.6 Hz, C3–H of 2,4-Cl2-C7H3), 7.77 (d, 2H, J = 8.8 Hz, C3,C3 –H
of p-Br-C6H4), 7.95 (s, 1H, =C–H), 12.24 (s, 1H, N–H); 13C NMR (100 MHz,
DMSO-d6, d ppm): 168.9, 114.3, 109.5, 104.8, 67.5, 56.0; ESI–MS of C24H18N3-
BrCl2O2S: 561.84 (M ? H)?, 563.84 [(M ? H)??2], 565.83 [(M ? H)??4],
566.83 [(M ? H)??6].
1-(4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene)-2-(4-(2,4-
dichlorophenyl)thiazol-2-yl) hydrazine (11f)
Creamy solid; Yield: 89%; MP: 176–178 °C; FT IR (ATR, mmax, cm-1): 3180 (N–
H), 3081 (Ar–H), 2926, 2857 (C–H), 1625 (C=N), 1568 (C=C), 1089 (C–N); H
1
NMR (400 MHz, DMSO-d6, d ppm): 3.81 (s, 1H, –OCH3), 5.14 (s, 2H, -OCH2),
7.07 (d, 1H, J = 8.4 Hz, C5–H of 3-OCH3-C6H3O), 7.16 (dd, 1H, J = 2.0 and
8.0 Hz, C6–H of 3-OCH3-C6H3O), 7.29 (d, 1H, J = 1.6 Hz, C2–H of 3-OCH3-
C6H3O), 7.35 (s, 1H, thiazolyl-H), 7.45–7.49 (m, 2H, C5–H of 2,4-Cl2-C7H3 and
0
C5 –H of C6H3), 7.59 (d, 2H, J = 8.4 Hz, C6–H of 2,4-Cl2-C7H3), 7.65 (d, 2H,
0
J = 2.4 Hz, C3–H of 2,4-Cl2-C7H3 and C3 –H of C6H3), 7.87 (d, 1H, J = 8.0 Hz,
0
C6 –H of 2,4-Cl2-C7H3), 7.99 (s, 1H, =C–H), 12.23 (s, 1H, N–H); 13C NMR
(100 MHz, DMSO-d6, d ppm): 167.9, 149.9, 149.2–120.5, 114.2, 109.6, 109.4, 67.5,
56.0; ESI–MS of C24H17N3Cl4O2S: 551.85 (M ? H)?, 553.93 [(M ? H)??2],
555.79 [(M ? H)??4], 557.83 [(M ? H)??6], 559.90 [(M ? H)??8].
1-(4-(2-Fluorobenzyloxy)-3-methoxybenzylidene)-2-(4-p-tolylthiazol-2-yl)hydrazine
(11g)
Creamy solid; Yield: 84%; MP: 138–140 °C; FT IR (ATR, mmax, cm-1): 3421 (N–H),
3071 (Ar–H), 2928, 2862 (C–H), 1620 (C=N), 1512 (C=C), 1099(C–N), 759 (C-F); 1H
NMR (400 MHz, DMSO-d6, d ppm): 2.39 (s, 1H, -CH3), 3.95 (s, 1H, -OCH3), 5.27 (s,
2H, -OCH2), 6.65 (s, 1H, C2–H of 3-OCH3-C6H3O), 6.95 (d, 2H, J = 8.4 Hz, C3–H
0
and C3 –H of 4-CH3-C6H4), 7.07 (d, 1H, J = 8.4 Hz, C5–H of 3-OCH3-C6H3O),
7.11–7.17 (m, 3H, C3–H and C5–H of 2-F-C7H6 and C6–H of 3-OCH3-C6H3O), 7.25(s,
1H, thiazolyl-H), 7.27–7.31 (m, 1H, C6–H of 2-F-C7H6), 7.48–7.51 (m, 1H, C4–H of
0
2-F-C7H6), 7.59 (d, 2H, J = 8.0 Hz, C2–H and C2 –H of 4-CH3-C6H4), 8.17 (s, 1H,
=C–H), 11.77 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 167.9, 160.8 (d,
C-2 JC,F = 238 Hz), 149.6–132.1 (Ar–C), 131.2 (d, C-6, JC,F = 10.0 Hz), 130.8 (d,
C-5 JC,F = 10.0 Hz), 124.9 (d, C-1 JC,F = 10.0 Hz), 124.0 (d, C-4 JC,F = 20.0 Hz),
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115.7 (d, C-3 JC,F = 20.0), 130.0, 128.0, 127.7, 120.4, 114.0, 109.5, 109.2, 64.5, 56.0;
ESI–MS of C25H22N3FO2S: 448.04 (M ? H)?.
1-(4-(2-Fluorobenzyloxy)-3-methoxybenzylidene)-2-(4-(4-bromophenyl)thiazol-2-
yl) hydrazine (11h)
Creamy solid; Yield: 86%; MP: 156–158 °C; FT IR (ATR, mmax, cm-1): 3231 (N–
H), 3047 (Ar–H), 2968, 22941 (C–H), 1620 (C = N), 1595 (C=C), 1099 (C–N), 758
(C-F); 1H NMR (400 MHz, DMSO-d6, d ppm): 3.97 (s, 1H, -OCH3), 5.29 (s, 2H, -
OCH2), 7.10 (d, 1H, J = 8.0 Hz, C5–H of 3-OCH3-C6H3O), 7.17 (dd, 1H, J = 1.6
and 8.4 Hz, C6–H of 3-OCH3-C6H3O), 7.20–7.25 (m, 2H, C3–H of 2-F-C7H6 and
C5–H of 2-F-C7H6), 7.28 (d, 1H, J = 2.0 Hz, C2–H of 3-OCH3-C6H3O), 7.35 (s,
1H, thiazolyl-H), 7.37–7.41 (m, 1H, C6–H of 2-F-C7H6), 7.51–7.54 (m, 1H, C4–H of
0
2-F-C7H6), 7.59 (d, 2H, J = 8.0 Hz, C2, C2 –H of 4-Br-C6H4), 7.79 (d, 2H,
0
J = 8.4 Hz, C3, C3 –H of 4-Br-C6H4), 8.01 (s, 1H, =C–H), 11.86 (s, 1H, N–H); 13C
NMR (100 MHz, DMSO-d6, d ppm): 169.2, 161.4 (d, C-2 JC,F = 240 Hz,),
149.6–132.1 (Ar–C), 131.2 (d, C-6, JC,F = 10.0 Hz), 130.8 (d, C-5 JC,F = 10.0 -
Hz), 125.0 (d, C-1 JC,F = 3.0 Hz), 124.0 (d, C-4 JC,F = 21.0 Hz), 115.8 (d, C-3
JC,F = 21.1), 130.0, 128.0, 127.7, 120.4, 114.0, 109.5, 109.2, 64.5, 56.0; ESI–MS of
C24H19N3BrFO2S: 512.05 (M ? H)?, 514.12 [(M ? H)??2].
1-(4-(2-Fluorobenzyloxy)-3-methoxybenzylidene)-2-(4-(2,4-dichlorophenyl)thiazol-
2-yl) hydrazine (11i)
Creamy solid; Yield: 84%; MP: 197–199 °C; FT IR (ATR, mmax, cm-1): 3343 (N–
H), 3070 (Ar–H), 2941, 2882 (C–H), 1626 (C=N), 1576 (C=C), 1033 (C–N), 1138
1
(C-F), 760 (C–Cl); H NMR (400 MHz, DMSO-d6, d ppm): 3.78 (s, 1H, -OCH3),
5.12 (s, 2H, -OCH2), 7.11 (d, 1H, J = 8.8 Hz, C5–H of 3-OCH3-C6H3O), 7.16 (dd,
1H, J = 1.6 and 8.8 Hz, C6–H of 3-OCH3-C6H3O), 7.19–7.24 (m, 2H, C3–H of 2-F-
C7H6 and C5–H of 2-F-C7H6), 7.27 (s, 1H, C2–H of 3-OCH3-C6H3O), 7.33 (s, 1H,
thiazolyl-H), 7.36–7.40 (m, 1H, C6–H of 2-F-C7H6), 7.46 (dd, 1H, J = 2.0 and
8.4 Hz, C5–H of 2,4-Cl2-C6H3), 7.50–7.52 (m, 1H, C4–H of 2-F-C7H6), 7.65 (d, 1H,
J = 2.4 Hz, C3–H of 2,4-Cl2-C6H3), 7.85 (d, 1H, J = 8.8 Hz, C6–H of 2,4-Cl2-
C6H3), 8.02 (s, 1H, =C–H), 11.79 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d
ppm): 167.9, 160.9 (d, C-2, JC,F = 245.1 Hz), 149.5–132.2 (Ar–C), 131.3 (d, C-6,
JC,F = 3.8 Hz), 130.9 (d, C-5, JC,F = 8.3 Hz), 125.0 (d, C-1, JC,F = 3.0 Hz), 124.2
(d, C-4, JC,F = 14.4 Hz), 115.8 (d, C-3, JC,F = 21.3 Hz), 130.1, 128.2, 127.9,
120.6, 114.1, 109.6, 109.4, 64.7, 56.0; C24H18N3Cl2FO2S: 501.93 (M ? H)?,
503.93 [(M ? H)??2], 505.93 [(M ? H)??4].
1-((5-Bromothiophen-2-yl)methylene)-2-(4-4-tolylthiazol-2-yl)hydrazine (11j)
Creamy solid; Yield: 90%; MP: 180–181 °C; FT IR (ATR, mmax, cm-1): 3227 (N–
H), 3053 (Ar–H), 2963, 2932 (C–H), 1574 (C=C), 1052 (C–N), 557 (C–Br); H
1
NMR (400 MHz, DMSO-d6, d ppm): 3.04 (s, 3H, -CH3), 7.18 (d, 1H, J = 4.0 Hz,
C3–H of 5-Br-thiophene ring), 7.20 (d, 1H, J = 3.6 Hz, C4–H of 5-Br- thiophene
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Biological evaluation and in silico molecular docking study…
0
ring), 7.35 (s, 1H, thiazolyl-H), 7.58 (d, 2H, J = 8.4 Hz, C3 and C3 –H of 4-CH3-
0
C6H4), 7.77 (d, 2H, J = 8.0 Hz, C2 and C2 –H of 4-CH3-C6H4), 8.04 (s, 1H, =C–H),
12.16 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 167.74, 140.9, 135.4,
131.1, 130.5, 129.0, 127.0, 120.2, 113.1, 104.3; C15H12N3BrS2: 377.92 (M ? H)?,
379.89 [(M ? H)??2].
2-(4-(4-Bromophenyl)thiazol-2-yl)-1-((5-bromothiophen-2-yl)methylene)hydrazine
(11k)
Greenish crystalline solid; Yield: 90%; MP: 184–186 °C; FT IR (ATR, mmax, cm-1):
3261 (N–H), 3086 (Ar–H), 3016, 2955 (C–H), 1692 (C = N), 1595 (C=C), 1092
(C–N), 554 (C–Br); 1H NMR (400 MHz, DMSO-d6, d ppm): 7.23 (d, 1H,
J = 4.0 Hz, C3–H of 5-Br- thiophene ring), 7.20 (d, 1H, J = 3.6 Hz, C4–H of 5-Br-
0
thiophene ring), 7.40 (s, 1H, thiazolyl-H), 7.60 (d, 2H, J = 8.8 Hz, C3 and C3 –H of
0
4-Br-C6H4), 7.80 (d, 2H, J = 8.4 Hz, C2 and C2 –H of 4-Br-C6H4), 8.14 (s, 1H, =C–
H), 12.23 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 167.8, 141.0,
135.9, 131.5, 131.2, 129.5, 127.5, 120.5, 113.3, 104.8; C14H9N3Br2S2: 441.91
(M ? H)?, 443.94 [(M ? H)??2], 445.89 [(M ? H)??4].
1-((5-Bromothiophen-2-yl)methylene)-2-(4-(2,4-dichlorophenyl)thiazol-2-
yl)hydrazine (11l)
Greanish solid; Yield: 89%; MP: 138–140 °C; FT IR (ATR, mmax, cm-1): 3379 (N–
H), 3186 (Ar–H), 3065, 2953 (C–H), 1626 (C = N), 1578 (C=C), 1074 (C–N), 797
(C–Cl), 559 (C–Br); 1H NMR (400 MHz, DMSO-d6, d ppm): 7.22 (d, 1H,
J = 3.6 Hz, C3–H of 5-Br- thiophene ring), 7.25 (d, 1H, J = 3.6 Hz, C4–H of 5-Br-
thiophene ring), 7.39 (s, 1H, thiazolyl-H), 7.50 (dd, 1H, J = 2.4 and 8.4 Hz, C5–H
of 2,4-Cl2-C6H3), 7.66 (d, 1H, J = 2.0 Hz, C3–H of 2,4-Cl2-C6H3), 7.87 (d, 1H,
J = 8.4 Hz, C6–H of 2,4-Cl2-C6H3), 8.15 (s, 1H, =C–H), 12.19 (s, 1H, N–H); 13C
NMR (100 MHz, DMSO-d6, d ppm): 168.0, 141.2, 140.1, 131.6, 131.4, 129.7,
127.8, 120.5, 113.5, 104.9; C14H9N3Br2S2: 431.94 (M ? H)?, 433.87
[(M ? H)??2], 435.91 [(M ? H)??4], 437.96 [(M ? H)??6].
1-(4-(Piperidin-1-yl)benzylidene)-2-(4-4-tolylthiazol-2-yl)hydrazine (11m)
Brown solid; Yield: 75%; MP: 168–170 °C; FT IR (ATR, mmax, cm-1): 3343 (N–H),
3064 (Ar–H), 2953, 2874 (C–H), 1619 (C = N), 1564 (C=C), 1067 (C–N); 1H NMR
(400 MHz, DMSO-d6, d ppm): 1.60–1.72 (m, 6H, pipiridine-CH2), 2.28 (s, 3H, -
0
CH3), 3.29–3.40 (m, 4H, pipiridine-CH2), 6.98 (d, 2H, J = 8.4 Hz, C3, C3 –H of
0
C6H4), 7.15 (d, 2H, J = 8.8 Hz, C3, C3 –H of 4-CH3-C6H4), 7.29 (d, 2H,
0
J = 8.4 Hz, C2, C2 –H of C6H4), 7.30 (s, 1H, thiazolyl-H), 7.57 (d, 2H,
0
J = 8.4 Hz, C2, C2 –H of 4-CH3-C6H4), 7.98 (s, 1H, =C–H), 11.71 (s, 1H, N–H);
13C NMR (100 MHz, DMSO-d6, d ppm): 168.2, 149.1, 133.6, 131.0, 127.4, 127.3,
120.1, 104.2, 24.3, 24.2; C22H24N4S: 377.14 (M ? H)?.
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M. Bhat et al.
1-(4-(Piperidin-1-yl)benzylidene)-2-(4-(4-bromophenyl)thiazol-2-yl)hydrazine
(11n)
Brown solid; Yield: 78%; MP: 206–208 °C; FT IR (ATR, mmax, cm-1): 3351 (N–H),
3069 (Ar–H), 2955, 2875 (C–H), 1622 (C = N), 1567 (C=C), 1070 (C–N), 557 (C–
1
Br); H NMR (400 MHz, DMSO-d6, d ppm): 1.62–1.74 (m, 6H, pipiridine-CH2),
0
3.31–3.40 (m, 4H, pipiridine-CH2), 7.18 (d, 2H, J = 8.4 Hz, C3, C3 –H of C6H4),
0
7.30 (d, 2H, J = 8.0 Hz, C2, C2 –H of C6H4), 7.31 (s, 1H, thiazolyl-H), 7.63 (d, 2H,
0
0
J = 8.8 Hz, C2 and C2 –H of 4-Br-C6H4), 7.80 (d, 2H, J = 8.4 Hz, C3 and C3 –H of
4-Br-C6H4) 8.00 (s, 1H, =C–H), 11.89 (s, 1H, N–H); 13C NMR (100 MHz, DMSO-
d6, d ppm): 168.4, 149.3, 133.8, 131.5, 131.2, 127.6, 127.5, 120.5, 104.4, 24.4, 24.3;
C21H21N4BrS: 440.97 (M ? H)?, 442.96 [(M ? H)??2].
1-(4-(Piperidin-1-yl)benzylidene)-2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazine
(11o)
Brown solid; Yield: 80%; MP: 180–182 °C; FT IR (ATR, mmax, cm-1): 3332 (N–H),
3071 (Ar–H), 2973, 2884 (C–H), 1622 (C = N), 1569 (C=C), 1082 (C–N), 793 (C–
1
Cl); H NMR (400 MHz, DMSO-d6, d ppm): 1.63–1.73 (m, 6H, pipiridine-CH2),
0
3.30–3.39 (m, 4H, pipiridine-CH2), 7.18 (d, 2H, J = 8.4 Hz, C3, C3 –H of C6H4),
0
7.30 (d, 2H, J = 8.8 Hz, C2, C2 –H of C6H4), 7.32 (s, 1H, thiazolyl-H), 7.46 (dd,
1H, J = 2.0 and 8.4 Hz, C5–H of 2,4-Cl2-C6H3), 7.64 (d, 1H, J = 2.4 Hz, C3–H of
C6H4), 7.86 (d, 1H, J = 8.4 Hz, C6–H of 2,4-Cl2-C6H3), 8.00 (s, 1H, =C–H), 11.74
(s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 168.4, 149.4, 133.7, 131.3,
127.5, 127.3, 120.3, 104.3, 24.3, 24.1; C21H20N4Cl2S: 431.12 (M ? H)?, 433.23
[(M ? H)??2], 435.18 [(M ? H)??4].
1-(4-Morpholinobenzylidene)-2-(4-4-tolylthiazol-2-yl)hydrazine (11p)
Brown solid; Yield: 76%; MP: 226–228 °C; FT IR (ATR, mmax, cm-1): 3298 (N–H),
3070 (Ar–H), 2965, 2874 (C–H), 1612 (C = N), 1559 (C=C), 1109 (C–O–C), 1099
(C–N); 1H NMR (400 MHz, DMSO-d6, d ppm): 2.27 (s, 3H, -CH3), 3.13–3.15 (m, 4H,
morpholine-CH2), 3.69–3.73 (m, 4H, morpholine-CH2), 6.96 (d, 2H, J = 8.0 Hz, C3,
0
C3 –H of C6H4), 7.16 (d, 2H, J = 8.8 Hz, C3, C3 –H of 4-CH3-C6H4), 7.31 (d, 2H,
0
0
J = 8.4 Hz, C2, C2 –H of C6H4), 7.33 (s, 1H, thiazolyl-H), 7.57 (d, 2H, J = 8.4 Hz, C2
0
and C2 –H of 4-CH3-C6H4), 7.97 (s, 1H, =C–H), 11.91 (s, 1H, N–H); 13C NMR
(100 MHz, DMSO-d6, d ppm): 167.9, 149.7, 142.3, 132.6, 132.5, 131.8, 130.0, 127.5,
125.1, 109.0, 66.1, 47.8; C21H22N4OS: 378.95 (M ? H)?.
1-(4-Morpholinobenzylidene)-2-(4-(4-bromophenyl)thiazol-2-yl)hydrazine (11q)
Brown solid; Yield: 77%; MP: 258–260 °C; FT IR (ATR, mmax, cm-1): 3303 (N–H),
3075 (Ar–H), 2981, 2879 (C–H), 1626 (C = N), 1565 (C=C), 1124 (C–O–C), 1099
1
(C–N), 558 (C–Br); H NMR (400 MHz, DMSO-d6, d ppm): 3.17–3.19 (m, 4H,
morpholine-CH2), 3.74–3.77 (m, 4H, morpholine-CH2), 7.19 (d, 2H, J = 8.0 Hz,
0
C3, C3 –H of C6H4), 7.32 (d, 2H, J = 8.0 Hz, C2 and C2 –H of C6H4), 7.34 (s, 1H,
0
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Biological evaluation and in silico molecular docking study…
0
thiazolyl-H), 7.63 (d, 2H, J = 8.4 Hz, C3 and C3 –H of 4-Br-C6H4), 7.81 (d, 2H,
0
J = 8.8 Hz, C2 and C2 –H of 4-Br-C6H4), 8.01 (s, 1H, =C–H), 12.19 (s, 1H, N–H);
13C NMR (100 MHz, DMSO-d6, d ppm): 168.2, 152.3, 142.5, 133.1, 132.8, 132.5,
132.1, 130.4, 127.9, 125.5, 109.4, 66.5, 48.4; C20H19N4BrOS: 443.13 (M ? H)?,
445.21 [(M ? H)??2].
1-(4-Morpholinobenzylidene)-2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazine (11r)
Brown solid; Yield: 80%; MP: 248–250 °C; FT IR (ATR, mmax, cm-1): 3312 (N–H),
3080 (Ar–H), 2982, 2881 (C–H), 1619 (C=N), 1564 (C=C), 1112 (C–O–C), 1084
1
(C–N), 757 (C–Cl); H NMR (400 MHz, DMSO-d6, d ppm): 3.15–3.18 (m, 4H,
morpholine-CH2), 3.71–3.73 (m, 4H, morpholine-CH2), 6.97 (d, 2H, J = 8.8 Hz, C3
0
0
and C3 –H of C6H4), 7.34 (s, 1H, thiazolyl-H), 7.46–7.51 (m, 3H, C2 and C2 –H of
C6H4 and C5–H of 2,4-Cl2-C6H3), 7.66 (d, 1H, J = 2.4 Hz, C3–H of 2,4-Cl2-C6H3),
7.88 (d, 1H, J = 8.8 Hz, C6–H of 2,4-Cl2-C6H3), 7.92 (s, 1H, =C–H), 11.92 (s, 1H,
N–H); 13C NMR (100 MHz, DMSO-d6, d ppm): 168.0, 152.2, 142.5, 132.8, 132.7,
132.6, 132.0, 130.2, 127.9, 125.3, 115.0, 109.2, 66.4, 48.1; C20H18N4Cl2OS: 433.13
(M ? H)?, 435.14 [(M ? H)??2], 437.23 [(M ? H)??4].
Anti-inflammatory activity
In vivo anti-inflammatory activity of the synthesized compounds (11a–r) was
studied by fabricating the inflammatory reaction with the help of irritants in the
form of paw edema in Wistar albino rats weighing 150–250 g. Carrageenan-induced
paw edema is the most frequently used experimental method [37, 38]. Carrageenan
is a sulfated polysaccharide obtained from seaweed (Rhodophyceae) causing the
release of histamine, 5-HT, bradykinin and prostaglandins which produce inflam-
mation and edema.
The weighed Wistar albino rats were numbered and a mark was made on the left
hind paw just beyond tibiotarsal junction so that every time the paw is dipped in the
mercury column up to the fixed mark to ensure constant paw volume. After 1 h,
0.1 mL of 1% carrageenan suspension in isosaline solution was injected into the
sub-plantar tissue of the right hind paw. The linear paw circumference was
measured at an hourly interval for 5 h. The initial paw volume of each rat was noted
by the mercury displacement method. The animals were divided into different
groups, each containing 6 rats. The first group of rats was treated with carrageenan
(control), the second group was administered with a dose of 20 mg/kg of Diclofenac
(standard), and the suspension of test compounds (20 mg/kg) was injected into the
remaining groups (the dose of the test compounds was fixed by conducting acute
toxicity studies as per OECD 425 guidelines and there were no signs of toxicity at
the given dose level). The paw volume of both legs was noted for 1 h, 2 h, and after
3 h carrageenan challenge. The mean paw edema value obtained for the test group is
compared with its mean value of the control group.
Anti-inflammatory activity was measured as the percentage reduction in edema
level when the drug was present, relative to control. Percentage inhibition was
calculated by using the relationship,
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M. Bhat et al.
% inhibition ¼ f1 À Vt=Vcg  100
where Vt is the edema volume in the drug-treated group and Vc is the edema volume
in the control group.
Cytotoxicity
The test compounds were studied for short-term in vitro cytotoxicity using Dalton’s
lymphoma ascites cells (DLA) [39]. The tumor cells aspirated from the peritoneal
cavity of tumor mice were washed twice with PBS or normal saline. Cell viability
was determined by the trypan blue exclusion method. A viable cell suspension
(1 9 106 cells in 0.1 mL) was added with various concentrations of the test
compounds prepared (10, 20, 50, 100 and 200 lg/mL) and the volume was made up
to 1 mL using phosphate-buffered saline (PBS). The control tube was with the cell
suspension. These assay mixtures were incubated for 3 h at 37 °C. Further, the cell
suspension was mixed with 0.1 mL of 1% trypan blue and kept for 2–3 min and
loaded on a hemocytometer. Dead cells take up the blue colour of trypan blue while
live cells do not take up the dye. The numbers of stained and unstained cells were
counted separately using this equation:
No: of dead cells
% Cytotoxicity ¼
 100
No: of live cells + No: of dead cells
Anti-oxidant activity
A free radical scavenging activity of the title compounds were tested as per the
literature [36]. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) was procured from
Sigma Aldrich. A 1-mL solution of 0.135 mM DPPH in methanol was prepared and
was mixed with 1 mL of varying concentrations (0, 100, 200, 300, 400 and 500 lg/
mL) of the test solutions. The reaction mixture prepared was vortexed properly and
left in the dark for 30 min at room temperature. The purple color of DPPH generally
vanishes when it undergoes reaction with an anti-oxidant present in the medium and
hence the absorbance. The absorbance of the mixture was measured using ascorbic
acid as the reference standard at 517 nm. Anti-oxidant property of the test
compounds is calculated using the following formula:
% DPPH radical scavenging activity
ðAbsorbance of control À Absorbance of sampleÞ
¼
 100
Absorbance of control
The anti-oxidant property of the test compound was demonstrated by IC50 values
(50% inhibitory concentration) depending upon the percentage of DPPH radicals
utilized in the reaction. Lower IC50 values indicate the highest anti-oxidant activity.
Imax (maximum percentage inhibition) values for the compounds were also cal-
culate using ‘GraphPad Software’ [40].
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Biological evaluation and in silico molecular docking study…
Antibacterial activity
The antibacterial activity of the novel compounds was determined using the
literature [41]. Solutions were created by dissolving the test compounds in dimethyl
sulfoxide (DMSO) and diluted with distilled water to get the concentration of 75 lg/
mL under aseptic conditions. Standard drugs, Ciprofloxacin and Streptomycin, were
procured from HIMEDIA were used to compare the activity of the synthesized
molecules at 75 lg/mL. Nutrient Agar Media was prepared by dissolving 38 g of
Mueller–Hinton agar with 1000 mL of distilled water and was sterilized by
autoclaving at 121 °C for 20 min with 15 psi pressure. The prepared media were
cooled at 45 °C with stirring and bacterial culture was inoculated onto the media
and poured into Petri plates under aseptic condition. The plates were kept for
solidification for 1 h. Then, 6-mm-diameter bores were made at equal distances
using a sterile borer and were filled with the test solution. A zone of inhibition was
measured after incubating the plates at 37 °C for 24 h.
Molecular docking
The binding interactions of the synthesized molecules with the COX-2 protein were
assessed with the aid of molecular docking. The docking study was executed by means
of a C-Dock suite. The crystallographic structure of the COX-2 protein with PDB ID:
3LN1 was collected from the RSCB protein Data Bank (www.rscb.org). The protein
obtained was pre-processed by deleting all the chains of the protein except Chain-A,
and heteroatoms and water molecules were also being deleted. With the help of the
Chemistry tool, hydrogen atoms were added to the protein. Using the receptor–ligand
interaction tool, protein was prepared for docking. A binding site in the protein was
identified and utilized for docking, leaving the remaining part of the protein. Ligands
drawn in Chemsketch were uploaded on Discovery studio 3.5 and were prepared with
the help of the Small molecule tool. Docking has been carried out to understand the
receptor–ligand interactions in producing the desired activity.
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M. Bhat et al.
Results and discussion
Chemistry
In the current exploratory study, target compounds were synthesized, as depicted in
Schemes 1, 2, 3, and 4. The condensation of thiosemicarbazide with 4-(Benzy-
loxy)benzaldehydes (3a–c), 5-bromothiophene-2-carbaldehyde (4), 4-(piperidin-1-
yl)benzaldehyde (7a) and 4-morpholinobenzaldehyde (7b) afforded the respective
thiosemicarbazones (9a–f) as reported in the literature [42]. Compounds (3a–c)
have been synthesized according to the procedure given by Bhattarai et al. [43]. The
nucleophilic substitution of fluorine in the compound (6) with cyclic amines resulted
in the formation of (7a) and (7b), respectively [44]. Thiosemicarbazones (9a–f)
were made to react with respective phenacyl bromides and chlorides to get the
desired molecules (11a–r) [2]. Completion of the reaction was monitored using
TLC, recrystallization of the product was carried out using ethanol to afford the pure
product. Formation of the new compounds was confirmed by spectral techniques
like Attenuated Total Reflection–Fourier Transform Infrared (ATR–FTIR), Proton
and 13Carbon Nuclear Magnetic Resonance (1H and 13C NMR), and Mass (MS).
Physical parameters of the novel derivatives have been provided in the spectral
section.
Scheme 1 Synthesis of 4-(benzyloxy)benzaldehydes. Reagents and condition: (i) K2CO3, DMF, reflux,
6 h
Scheme 2 Synthesis of compounds (7a–b). Reagents: (i) K2CO3, Acetone, Reflux for 8 h
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Biological evaluation and in silico molecular docking study…
Scheme 3 Synthesis of substituted thiosemicarbazones (9a–f). Reagents: (i) EtOH, AcOH, 6 h
Scheme 4 Synthesis of the target compounds (11a–r). Reagents: EtOH, Reflux for 8 h
The formation of intermediates, thiosemicarbazones (9a–f) was supported by the
IR (ATR) spectral data. The characteristic bands for NH2 and C=S supported the
formation of the products. For the prototype compound (9a), two characteristic
absorption bands at 3379 and 3354 cm-1 were discerned for NH2, and the
occurrence of two strong bands near 1618 and 1086 cm-1 affirmed the presence of
1
C=N and C=S groups, respectively. As in the case of IR, H NMR also gave
evidence for the thiosemicarbazone formation (9a) by a typical singlet for an
exocycli C=C–H at d 7.97 ppm. In addition to this, a doublet with J = 9.2 Hz was
identified at d 7.72 ppm for a proton on C6 of the 2,4-Cl2-C7H5 ring. A doublet at d
7.63 ppm (J = 2.0 Hz) was identified for a C3–H of 2,4-Cl2-C7H5 due to meta-
coupling. A doublet of doublet was observed at d 7.43 ppm (J = 2.4 and 8.4 Hz) for
C5–H of the 2,4-Cl2-C7H5 ring. The spectrum also showed two doublets at d 7.02
and 7.58 ppm with J = 8.8 and 8.0 Hz, respectively, for the four protons of the
benzylidine ring. A singlet for –benzyl (OCH2) protons was assigned at d 5.14 ppm.
In the spectrum, two broad singlets were observed due to tautomerization of free N–
H2 at d 7.99 and 8.14 ppm, respectively. The N–H group has given a singlet at d
11.31 ppm. Characteristic peaks at d 178.2 ppm (C=S), d 159.8 ppm (–O–C=C–), d
142.5 ppm (–C=N), d 66.9 ppm (–O–CH2) in the 13C NMR spectrum ratified the
product formation. Mass spectrum confirmed the product (9a) by giving isotopic
peaks at 354.1 (M ? H)?, 355.94 [(M ? H)??2] and 358.02 [(M ? H)??4],
respectively.
The formation of target compounds 1-(4-(arylbenzylidene)-2-(arylthiazol-2-
yl)hydrazine (11a–r) was also favored by their IR data. The disappearance of the
bands for NH2 and C=S near 3300 and 1080 cm-1 and the appearance of a band for
C–S–C near 660 cm-1 evidenced the product. The compound 11b (Fig. 4,
Supplementary data) displayed stretching vibrations for -NH at 3290 cm-1
,
3088 cm-1(Ar–H), 2933 and 2879 cm-1 (–C–H), 1625 cm-1 (C=N), 1512 cm-1
(C=C) and 663 cm-1 (C–S–C), respectively. Further, the 1H NMR spectrum of 11b
(Fig. 11, Supplementary data) evidenced a characteristic singlet at d 7.37 ppm for
123

M. Bhat et al.
thiazolyl-H. Similarly, two more singlets at d 5.19 and 8.01 ppm were due to –O–
CH2 and =C–H protons, respectively. Along with this, the spectrum showed a
doublet of doublet for the proton of C5 of the 2,4-Cl2-C7H5 ring at d 7.49 ppm
(J = 2.0 and 8.4 Hz). A doublet was observed for C3–H due to meta-coupling at d
7.70 with J = 2.0 Hz. Two more doublets at d 7.80 ppm (J = 8.8 Hz) and
7.60 ppm (J = 8.8 Hz), respectively, were observed for 4 protons which are ortho
and meta to the carbon-bearing bromine atom. A multiplet was predicted for two
benzylidine protons and one benzyloxy protons in the range d 7.63–7.65 ppm. A
0
doublet was exhibited by C4–H and C4 –H protons of the benzylidine moiety at d
7.10 ppm with J value 8.8 Hz. In the 13C NMR of 11b (Fig. 28, Supplementary
data), purging of a signal for C=S and the emergence of a peak for -C-S- justified the
thiazol-2-yl-hydrazone formation. Further, a signal observed at d 66.4 ppm was
manifested for O-CH2 carbon. The 2nd and 5th carbon atoms of the thiazole ring
resonated at d 168.4 ppm and 104.3 ppm, respectively. Peaks at d 159.1 ppm and
149.2 ppm correspond to C4 of benzylidine as well as C4 of the thiazole ring. An
exocyclic –C=N carbon was found at d 141.4 ppm and C1 of the 2,4-Cl2-C7H5 ring
was resonated at d 133.8 ppm. All the remaining aromatic carbons resonated in the
region at d 133.6, 133.3, 131.5, 131.4, 128.9, 127.9, 127.6, 127.5, 120.5 and
1
115.2 ppm, correspondingly. Chemical shift values of H and 13C NMR observed
for the remaining compounds are summarised in the ‘‘Experimental’’ section. The
ESI–MS spectral data of the same has assisted in confirming the new product
formation, which showed a (M ? 1)? peak at the m/z value of 531.9, along with
isotopic peaks at m/z values of 534.03, 536.14 and 537.9 for [(M ? 1)? ? 2],
[(M ? 1)? ? 4] and [(M ? 1)? ? 6], respectively, which is in accordance with its
molecular formula weight.
Biological activity
Anti-inflammatory activity and structural activity relationship (SAR)
To facilitate the prospective anti-inflammatory agent, the synthesized novel
molecules (11a–r) were screened for their in vivo activity by opting carrageenan-
induced paw edema method [37] in rats and the results are systematized in Table 1.
The results obtained are distinctly showing that the compounds are modest to
comparable activity to that of the Diclofenac sodium (standard). The compounds
11a, 11b, 11c, 11d, 11e and 11g showed significant activity when compared to the
standard (53.2%). The derivatives 11c, 11d and 11e exhibited modest activity in 3 h
duration after administration of the test compound and they started to act on the
inflammation from the first hour. The compound encompassed by the (4-(2,4-
dichlorobenzyloxy)-3-methoxybenzylidene) part in combination with (4-bro-
mophenyl)thiazol-2-yl)hydrazine (11e) evolved as a potent moiety among the
series, with 56% inhibition in 3 h duration and 58% inhibition was observed during
the 2nd hour. The compound containing (4-(2,4-dichlorobenzyloxy)-3-methoxy-
benzylidene) which is substituted with 4-methylphenyl on the thiazole unit also
exhibited 54% activity after 2 h of administration and remained active even after the
123

Biological evaluation and in silico molecular docking study…
123

M. Bhat et al.
3rd hour. However, the compound with -2,4-Cl2-C6H3 substitution on thiazol-2-yl-
hydrazone is inactive. Compounds 11a, 11b, 11c and 11g displayed average 53%
inhibition, remained effective up to the 3rd hour and the compound 11a starts acting
after the first hour of an injection. In the series, the compound bearing the (4-(2-
fluorobenzyloxy)-3-methoxybenzylidene) moiety which is amalgamated with the
(4-methylphenyl-thiazol-2-yl)hydrazine part is less active during the first hour, and
activity increased consequently with time. In view of the result, (4-(2-fluoroben-
zyloxy)-3-methoxybenzylidene) derivatives 11h and 11i appeared to be moderately
active with 38 and 35% inhibition, respectively. Molecules with the 5-bromoth-
iophene part were found to be less active (23–28%) compared to the benzyloxy
derivatives. Similarly, piperidine- and morpholine-bearing molecules were also
having less activity except for 11m and 11p, which is having the 4-methlphenyl
component at the thiazole portion, increasing the activity (53 and 40% inhibition,
correspondingly). However, piperidine and morpholine part did not play any vital
role in increasing the inhibitory action. It is clear that the molecules with
benzyloxybenzylidine were found to be the most active compounds in the series.
Moreover, 2,4-dichlorobenzyloxy nucleus substituted with 3-methoxybenzylidine
snippet displayed significant activity (Fig. 2).
The results of the anti-inflammatory screening highlighted the facts about the
relationship between the structure and the potency of the molecules (11a–r), which
could assist us to select the propitious pharmacophore in this series. Interestingly,
the results reflected the dependence of the molecular efficacy on the structural
variations, which means that variations in the structure have altered the potency of
the molecule. Fascinatingly, 4-(2,4-dichlorobenzyloxy)benzylidene derivatives
produced effective anti-inflammatory activity. Further, substitution of the methoxy
group on the 3rd position of the phenyl ring of the 4-(2,4-dichlorobenzyloxy)ben-
zylidene enhanced the activity. However, the same with the 2,4-dichlorophenyl
substituent on the thiazol-2-yl moiety substantially reduced the potency to 28.4%.
However, replacement of 2,4-dichlorobenzyloxy by a 2-fluoro substituent decreased
the anti-inflammatory property except for the compound with 4-methylphenylth-
iazol-2-yl, but the activity of 4-bromophenylthiazol-2-yl and 2,4-dichlorophenylth-
iazol-2-yl derivatives reduced to 37.9 and 34.5%, respectively. Notably, when the
benzyloxy group was replaced by the 5-bromothiophene nucleus, a substantial
decrease in the activity of all derivatives was observed. Further, replacement of the
benzyloxy group by the 1-phenylpiperidine and 4-phenylmorpholine moieties
significantly reduced the anti-inflammatory activity, but the derivatives with the
4-methylphenylthiazol-2-ly substituent did not reduce the activity.
In conclusion, benzyloxy-bezylidene was proved to be the key moiety to display
the activity, which becomes further improved upon substitution with the –OMe
group at the 3rd position of the benzylidene unit of the benzyloxy-benzylidine.
Additionally, keen observation on the activity profile shows that the 4-Me
substitution on the aryl moiety of thiazol-2-yl is also an essential facture to exhibit
the activity.
123

Biological evaluation and in silico molecular docking study…
Fig. 2 A Effect of derivatives 11a–r on swelling. B Percentage inhibition of inflammation using novel
derivatives 11a–r
123

M. Bhat et al.
Cytotoxicity
The compounds which are potent anti-inflammatory agents, 11d and 11e, were
evaluated for their cytotoxicity against DLA (Dalton’s lymphoma ascites) tumor
cells at various concentrations (10, 20, 50, 100 and 200 lg/mL) using the trypan
blue dye method. The cytotoxic effect of the tested compounds on the cell line is
represented in Table 2. Both the tested compounds exhibited lower cytotoxicity on
the tumor cells. Interestingly, the results reflect that the novel compounds 11d and
11e are nontoxic to the cells and showed only 30% of cell death at the highest
concentration used (200 lg/mL).
Anti-oxidant property
The novel compounds bearing the benzyloxy moiety which showed significant anti-
inflammatory activity were investigated for their anti-oxidant property by the DPPH
scavenging method according to the Ref. [45]. IC50 and Imax values of the test
compounds are represented in Table 3. From the results, it is noticeable that the
compounds are less active when compared with the standard ascorbic acid, but
compounds 11g, 11h and 11c showed moderate activity. However, the maximum
inhibition of 90.5% was observed for compound 11c, and the compounds 11g and
11e exhibited 78 and 76% inhibition, respectively. Interestingly, the derivatives
which possess fluoro-substituents on the benzyloxy part were found to have better
free radical scavenging activity when compared with the derivatives having chloro-
Table 2 Cytotoxicity of potent compounds 11d and 11e against the DAL tumor cell line
Compound
% Cell death
200 (lg/mL)
100 (lg/mL)
50 (lg/mL)
20 (lg/mL)
10 (lg/mL)
11d
11e
26
30
16
18
5
8
2
6
0
0
Table 3 Anti-oxidant activity
(IC50) and Imax values of the
active compounds 11a–i
Compounds
IC50 (lg/mL)
Imax (%)
11a
309.33
273.75
175.93
267.52
237.04
209.77
110.00
146.96
206.75
18.68
69.4
74.9
90.5
69.6
76.4
65.4
78.1
65.3
75.4
–
11b
11c
11d
11e
11f
11g
11h
IC50 concentration at which
50% inhibition was observed;
Imax maximum % inhibition
11i
Ascorbic acid
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Biological evaluation and in silico molecular docking study…
substitution. Further, the structural adaptation on the thiazole unit may enhance the
anti-oxidant activity.
Antibacterial activity
Eighteen molecules of the series were investigated for their in vitro antibacterial
activity using the agar well diffusion method [41]. The zone of inhibition was
determined at a concentration of 75 lg/mL against Staphylococcus aureus and
Bacillus subtilis (Gram-positive) and Pseudomonas aeruginosa and Escherichia coli
(Gram-negative) bacterial strains using Ciprofloxacin and Streptomycin as
standards. Inhibitions obtained for the test compounds are tabulated in the Table 4.
From the result, it is clear that almost all the compounds showed moderate activity
against the tested bacterial strains. The compounds 11a, 11d, 11e and 11r showed
modest activity against the Gram-negative as well as the Gram-positive bacteria.
Benzyloxy derivatives as well as 5-bromothiphene derivatives showed nearly
similar activity. Except for 11r, all the other molecules with piperidine and
morpholine were less potent when compared to benzyloxy derivatives.
Table 4 Zone of inhibition of target molecules 11a–r in mm
Compounds
Pseudomonas
aeruginosa
Escherichia coli
Bacillus subtilis
Staphy-
lococcus
aureus
11a
11.10
9.57
11.21
08.31
12.15
19.71
11.14
10.24
9.24
11.53
14.16
15.35
14.61
12.09
8.22
11.44
15.12
9.41
11b
11c
9.06
11d
13.07
13.23
13.20
10.27
9.85
9.46
11e
11.09
11.15
12.11
11.50
13.36
9.24
11f
11g
9.31
11h
11.76
11.55
11.04
12.14
12.24
9.37
13.72
15.41
12.63
14.42
13.25
10.34
11.57
12.78
10.74
13.45
19.50
20.54
24.04
11i
10.56
11.12
12.16
10.36
10.33
9.43
11j
11k
12.25
10.16
7.56
11l
11m
11n
10.47
11.57
9.46
7.68
11o
10.34
11.58
10.30
13.43
22.20
24.18
8.47
11p
12.45
8.87
11q
10.16
10.81
21.10
23.14
11r
9.75
Ciprofloxacin
Streptomycin
23.31
23.21
Mean of triplicate values
123

M. Bhat et al.
In silico molecular docking
An in silico molecular docking analysis has been undertaken to investigate the
significant binding interactions of the target molecules with the binding pockets of
the COX-2 protein [PDB ID: 3LN1], which is the principal requirement for
producing the biological effect. The docking results obtained from the binding
interaction of the molecules (11a–r) with the protein are set out in Table 5. The
cDock energy of the compounds varied from - 48.1 to 12.8 kcal/mol.
In this series, most of the compounds (11a, 11b, 11f, 11i, 11j, 11k, 11l and 11r)
were found to bind actively with the Arg-120 of the COX-2 enzyme, which is the
imperative amino acid among COX isoenzymes. The importance of Arg-120 is
highly augmented for their involvement in increasing the specificity of NSAIDs
among COX enzymes [46]. According to the literature on bovine COX, to have
improved COX inhibitory activity there should be an interaction between Arg-120
and the fatty acids of the substrate via the guanidinium group. A x-end of Arg-120
is situated in a hydrophobic area having a Ser-530 mantle in a scenario such that
carbon-13 of the substrate has been positioned below Tyr-385 to have an
appropriate COX reaction, and the Tyr-355 with phenolic side chain is sited close
to the mouth of the conduit facing Arg-120 [47]. Additionally, the biochemical
study of a complex of arachidonic acid (AA) and eicosapentaenoic acid (EPA) with
COX-2 demonstrated that the flexibility of the Leu-531 side chain is the responsible
feature in influencing the binding mode of AA and EPA with the protein, which in
turn enhances the volume of the COX-2 channel for oxygenation of the wide array
of fatty acids and esters. This analysis also demonstrated the importance of the
interaction of the Agr-120 side chain with AA and EPA at the carboxylate end of the
substrate in conquering the conformation flexibility. This scrutiny also confirmed
that the side chain of Arg-120 has a conformational disparity in its arrangement
around the substrates in COX-1 in contrast to COX-2 [48].
Furthermore, the literature clearly evidences the significance of Arg-120 in
catalyzing the COX reactions. In addition, Arg-120 has also played an active role in
the inhibitory effect of many of the commercially available drugs such as the
inhibitor flurbiprofen binds to COX-2 through ion–pair interaction to the residues of
Arg-120 via the carboxylate moiety [49]. Further, discernment of the structure of a
complex of Diclofenac with murine COX-2 revealed that the drug has bonded to the
protein with the carboxylate unit in an overturned configuration which concluded
the interaction of Tyr-385 and Ser-530 through the hydrogen bond, in turn expelling
the salt bridge formation with Arg-120. The excavation also advocated the
importance of Ser-530 interaction of nimesulide in the inhibitory process [50].
The mechanism of action of novel compounds which showed significant in vivo
activity (11a, 11b, 11c, 11d, 11f and 11m) was predicted via the molecular docking
study. In compound 11a (Fig. 23, supplementary data), an aryl ring exhibited pi–pi
interaction and S of the thiazole ring and N–H of the molecule formed a hydrogen
˚
bond (2.37605 and 2.44656 A) with Arg-120 and Val-523, respectively. The
derivative 11b (Fig. 24, supplementary data) interacted with Arg-120 by forming a
hydrogen bond with S of thiazole ring and double-bonded N with a bond distance of
˚
2.46517 and 2.42724 A, correspondingly. Two aryl rings of the compound also
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Biological evaluation and in silico molecular docking study…
123

M. Bhat et al.
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Biological evaluation and in silico molecular docking study…
communicated with Arg-120 and Lys-83 through pi-stacking. Similarly, the two
aryl ring and a thiazole ring of the compound 11c (Fig. 25, supplementary data),
appeared to form a pi interaction with Arg-120, Arg-513 and Lys-83 with binding
energy - 19.58 kcal/mol. Double-bonded nitrogen and thiazole S formed the
hydrogen bond with Tyr-355. However, in the compound 11d (Fig. 26, supple-
mentary data), S of thiazole and chlorine present in the second position of
benzyloxy part formed the hydrogen bonds with Tyr-355 and Lys-83 respectively.
In unison, the compound established pi bonding with Lys-83.
According to Duggan et al., substitution of Tyr-355 by a surrogate phenylalanine
countermanded the inhibitory role of naproxen. It necessitates the participation of
Tyr-355 in inhibiting the COX-2 enzyme by thiazole-based molecules [51].
Fascinatingly, two aryl rings of the molecule 11e (Fig. 27, supplementary data)
exhibited two pi interactions with Lys-83 and Arg-120. Ring S formed a hydrogen
bond with Tyr-355 amino acid. Intriguingly, Loll et al. [52] explained that Tyr-355
and Arg-120 are placed along the mouth of the channel and the catalytic receptacle
in the COX. The interaction between these two residues indicates the conforma-
tional flexibility of the synthesized molecule.
It is evident that the variation in the docking energy and in vivo results may be
attributed to the lack of structural flexibility of novel molecules. However,
molecular docking interactions obviously indicated that the novel thiazole
molecules have successfully communicated with the active site of the target protein.
Conclusions
In the present work, much attention has been given to the development of novel,
small COX-2 inhibitors with significant anti-inflammatory, antimicrobial and anti-
oxidant activities. From the data, it is evident that the molecules 11a, 11b, 11c, 11d,
11e and 11g are found to be significant inhibitors for inflammatory disorders among
the series. However, the compounds 11d and 11e emerged as the most potent among
them. The in silico binding interaction of the novel compounds with the COX-2
protein (PDB: 3LN1) revealed the vital binding interaction of the ligand with Arg-
120, Tyr-385, Tyr-355 and Ser-530 amino acids, a necessary factor to act as a
significant COX-2 inhibitor. Results acquired from the antibacterial screening
legitimized the role of novel compounds in curing the infection caused by the
intruders during the inflammatory disorder. Anti-oxidant activity results depicted
the moderate effectiveness of the molecules in scavenging the free radicals
generated in the medium.
In conclusion, the benzyloxy benzyledine is the key moiety in producing the
good anti-inflammatory, antimicrobial, and anti-oxidant properties of the new
thiazol-2-yl-hydrazones. Along with this, 4-methylphenyl substituent on the thiazol-
2-yl unit and 3-OMe on the benzyloxybenzylidene played a prominent role in
enhancing the activity. Further, the linkers (H–N–N=C–) S of the thiazole and aryl
rings were helpful in producing the effective binding interactions with the amino
acids of the protein. However, there is further scope to modify the molecular
123

M. Bhat et al.
structure to enhance the microbiological and anti-oxidant properties by varying the
substituent at the aryl part of the thiazol-2-yl moiety.
Acknowledgements The authors are obliged to Mangalore University for providing the facility and also
grateful to Mysore University and SAIF Cochin for providing spectral data. The authors are also grateful
to Vaishali M. Rai (Department of Microbiology, Mangalore University, Mangaluru), for the needful
help.
Compliance with ethical standards
Conflict of interest All the authors declare that, they have no Conflict of Interest.
Ethical approval This article contains the studies performed on animals (mice). The appropriate care
and use of animals were performed according to the international, national, and institutional guidelines.
The study on mice was in accordance with the ethical standards of the institutional and national research
committee. This study does not include any human participants. Ethical No. SCSCP/IAEC/08/2016-2017.
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