Synthesis of benzo[: D] thiazole-hydrazone analogues: Molecular docking and SAR studies of potential H+/K+ ATPase inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1039/c7md00111h

A series of new benzo[d]thiazole-hydrazones were synthesized and characterized by analytical and spectroscopic techniques. All the compounds were screened for their in vitro inhibition of H⁺/K⁺ ATPase and anti-inflammatory effects. The results revealed that compounds 6-8, 13-15, 18-20, 22, 23 and 27-30 displayed excellent inhibitory activity against H⁺/K⁺ ATPase, and their IC₅₀ values were lower than those of the standard compound omeprazole. Compounds 2-5, 9-12, 28 and 30 exhibited better anti-inflammatory activity in comparison to the standard compound indomethacin. Studies of the structure-activity relationship (SAR) showed that electron-donating groups (OH and OCH₃) favored inhibitory activity against H⁺/K⁺ ATPase, whereas electron-withdrawing groups (F, Cl, Br and NO₂) favored anti-inflammatory activity, and derivatives with both electron-donating (OH and OCH₃) and electron-withdrawing (Br) groups (16-18) displayed reasonable activity, whereas aliphatic analogues (24-26) exhibited less activity and heterocyclic analogues (27-30) displayed moderate activity in both biological studies. Molecular docking studies were performed for all the synthesized compounds, among which compounds 19 and 20 exhibited the highest docking scores for inhibitory activity against H⁺/K⁺ ATPase, whereas compounds 10 and 12 displayed the highest docking scores for anti-inflammatory activity.

Full text of DOI:10.1039/c7md00111h

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RESEARCH ARTICLE
Synthesis of benzo[d]thiazole-hydrazone
analogues: molecular docking and SAR studies of
potential H+/K+ ATPase inhibitors and anti-
inflammatory agents†‡
Cite this: DOI: 10.1039/c7md00111h
Shi-Meng Wang,a Gao-Feng Zha,a K. P. Rakesh,
a N. Darshini,b T. Shubhavathi,b
*
c
b
a
*
*
H. K. Vivek, N. Mallesha and Hua-Li Qin
A series of new benzo[d]thiazole-hydrazones were synthesized and characterized by analytical and
spectroscopic techniques. All the compounds were screened for their in vitro inhibition of H+/K+ ATPase
and anti-inflammatory effects. The results revealed that compounds 6–8, 13–15, 18–20, 22, 23 and 27–30
displayed excellent inhibitory activity against H+/K+ ATPase, and their IC50 values were lower than those of
the standard compound omeprazole. Compounds 2–5, 9–12, 28 and 30 exhibited better anti-inflammatory
activity in comparison to the standard compound indomethacin. Studies of the structure–activity relation-
ship (SAR) showed that electron-donating groups (OH and OCH3) favored inhibitory activity against H+/K+
ATPase, whereas electron-withdrawing groups (F, Cl, Br and NO2) favored anti-inflammatory activity, and
derivatives with both electron-donating (OH and OCH3) and electron-withdrawing (Br) groups (16–18)
displayed reasonable activity, whereas aliphatic analogues (24–26) exhibited less activity and heterocyclic
analogues (27–30) displayed moderate activity in both biological studies. Molecular docking studies were
performed for all the synthesized compounds, among which compounds 19 and 20 exhibited the highest
docking scores for inhibitory activity against H+/K+ ATPase, whereas compounds 10 and 12 displayed the
highest docking scores for anti-inflammatory activity.
Received 2nd March 2017,
Accepted 4th April 2017
DOI: 10.1039/c7md00111h
rsc.li/medchemcomm
gastric acid secretion.2 As a result, H+/K+ ATPase could be a
therapeutic target for various acid-related diseases. Although
1. Introduction
Gastric and duodenal ulcers are common diseases of the gas-
trointestinal tract with high clinical incidence rates, which
may result in severe superior gastrointestinal bleeding. They
may be caused by an imbalance between aggressive and de-
fensive forces in the stomach and duodenum. The reduction
of acid secretion has been proven to be a useful means for
promoting the healing of ulcers.1 H+/K+ ATPase catalyzes the
terminal step in gastric acid secretion, and the inhibition of
H+/K+ ATPase can provide an intrinsically greater reduction in
a number of H+/K+ ATPase inhibitors are available on the
market, there is always a need for new agents with even better
efficacy and safety profiles.
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
most commonly used anti-inflammatory agents. They exhibit
their anti-inflammatory activity by inhibiting the production
of prostaglandin, which is a potent mediator of inflamma-
tion. The pharmacological target of NSAIDs is cyclooxygenase
(COX), which catalyses the first committed step in arachi-
donic acid metabolism.3 The key cyclooxygenase enzyme ex-
ists in two isoforms, namely, the constitutive form COX-1
and the inducible form COX-2. COX-1 is present in most tis-
sues, whereas COX-2 is expressed during inflammation.4
However, traditional NSAIDs that inhibit both cyclooxygenase
enzymes may cause gastrointestinal side effects and selective
COX-2 inhibitors such as coxibs have been reported to cause
significant cardiovascular side effects.5 In order to overcome
the above drawbacks, it is of great significance to develop
novel compounds with improved profiles.
a Department of Pharmaceutical Engineering, School of Chemistry, Chemical
Engineering and Life Science, Wuhan University of Technology, 205 Luoshi Road,
Wuhan 430073, PR China. E-mail: rakeshasg@gmail.com, qinhuali@whut.edu.cn;
Fax: +86 27 87749300
b SRI RAM CHEM, R & D Centre, Plot No. 31, JCK Industrial Park, Belagola
Industrial Area, Mysore 570016, Karnataka, India.
E-mail: research@sriramchem.com; Tel: +91 821 4255588
c Department of Biotechnology, Sri Jayachamarajendra College of Engineering,
Mysuru 570006, Karnataka, India
† The authors declare no competing interests.
Benzo[d]thiazoles are bicyclic ring systems that are wide-
spread and abundant in natural products. They have
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7md00111h
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attracted increasing attention because of their diverse biolog-
ical activities such as antimicrobial,6 antimalarial,7 anticon-
vulsant,8 antioxidant,9 anti-inflammatory,10 antitumor,11 an-
algesic,12 antitubercular,13 antidiabetic14 activities, and so
on. Hydrazone Schiff bases are compounds that contain an
azomethine group (–CHN–) in their structure, which is gen-
erally synthesized by the condensation of hydrazine deriva-
tives with active carbonyl groups. Different derivatives of
benzothiazole substituted at the 2 position have diverse ther-
apeutic applications15 and anticancer effects.16 Hence, on the
basis of the above observations and our ongoing research
into the chemistry and bioactivity of novel heterocyclic
compounds,2,17–20 we discuss our attempt to synthesize a
new series of benzo[d]thiazole Schiff bases as potential H+/K+
ATPase inhibitors and anti-inflammatory agents in this pa-
per. Some of the reported hydrazones with potent inhibitory
activity against H+/K+ ATPase and anti-inflammatory activity
are shown in Fig. 1.2,17,21–24
amine (A) was synthesized according to the method reported
in the literature.25,26 To a suspension of compound A in eth-
ylene glycol, an excess amount of hydrazine hydrate and a
catalytic amount of concentrated HCl were added. The mix-
ture was refluxed for 3–4 h to obtain compound B. Com-
pound B was continually reacted with various aldehydes in
the presence of a catalytic amount of glacial acetic acid to af-
ford hydrazones (1–30). All the derivatives were obtained in
high yields by simple manipulation. The structures of all the
newly synthesized compounds, including intermediates, were
1
confirmed by IR, H NMR, 13C NMR and mass spectral analy-
sis (see ESI‡). The formation of hydrazones was confirmed by
the presence of an absorption band at 1612–1630 cm−1 for
the imine group, i.e., –NCH–, in the IR spectra and a sin-
glet peak at δ = 7.80–8.02 ppm in the 1H NMR spectra. The
chemical structures were confirmed by spectroscopic
techniques.
2.2 Biology
2. Results and discussion
2.2.1 H+/K+ ATPase inhibition activity. All the synthesized
compounds were screened for their in vitro inhibitory activity
against H+/K+ ATPase, and the results are tabulated in
Table 1. Omeprazole was used as a standard compound.
2.1 Chemistry
The preferred compounds were synthesized according to the
steps illustrated in Scheme 1. 7-Methylbenzoijd]thiazol-2-
Fig. 1 Structures of representative hydrazones with potential inhibitory activity against H+/K+ ATPase and anti-inflammatory activity.
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Scheme 1 Synthesis of the title compounds (1–30).
Among the derivatives, compound 1, which had no substit-
2.2.2 Anti-inflammatory activity. All the synthesized com-
pounds were investigated for their in vitro anti-inflammatory
activity using a known literature procedure on human eryth-
rocytes.28 A significant number of compounds were identified
as displaying excellent to moderate inhibitory activity in com-
parison to the standard drug indomethacin. The IC50 values
determined for the compounds showed that more than 50%
of the compounds had higher inhibitory concentrations
(Table 1). Compounds 2, 3, 4, 5, 9, 10, 11, 12, 28 and 30
displayed excellent activity, with IC50 values of 28, 24, 32, 20,
18, 12, 16, 10, 28 and 30 μg mL−1, respectively, which were
much lower than that of the standard drug indomethacin
(IC50 = 40 μg mL−1).
In general, the presence of electron-withdrawing groups
(2–5 and 9–12) favored anti-inflammatory activity,17 and the
presence of two electron-withdrawing groups (9–12) made the
molecules highly active. The presence of electron-donating
groups in combination with electron-withdrawing groups de-
creased the activity (16–18). Derivatives with electron-
donating groups (OH and OCH3) on the phenyl ring and the
aliphatic analogues 24–26 displayed the least activity, and
among those with heterocyclic moieties 28 and 29 exhibited
good anti-inflammatory activity.
2.2.3 Docking studies. PLA2 is the first upstream enzyme
in the inflammatory cascade, followed by COX-2 or LOXs,
which are activated during tissue damage or inflammation.29
The current therapy for inflammation comprises non-
steroidal anti-inflammatory drugs (NSAIDs), owing to their
inherent capability to inhibit the first downstream enzymes,
namely, cyclooxygenases (COXs), which are the key enzymes
involved in arachidonic acid metabolism. However, the long-
term use of NSAIDs may give rise to marked gastrointestinal
(GI) irritation and ulceration owing to their unwanted inhibi-
tion of COX-1. Hence, we aimed to assess the potency of our
ligands against both main enzymes, which are COX-2 and
H+/K+ ATPase during the process of inflammation and GI
uents on the phenyl ring, exhibited activity with IC50 = 84
μg mL−1 but was thus less potent than omeprazole (IC50
=
46 μg mL−1). It can be inferred that compounds without
substituents on the phenyl ring display less activity. There-
fore, the effects of substituents on the phenyl ring were fur-
ther investigated. Benzo[d]thiazoles with different electron-
donating (OH and OCH3) or electron-withdrawing (Cl, NO2,
F and Br) groups on the phenyl ring and those in which
the phenyl ring was replaced with an aliphatic or heterocy-
clic substituent were synthesized and their antiulcer and
anti-inflammatory activities were also evaluated. It was
found that when there were hydroxyl and/or methoxy
groups substituted on the phenyl ring, the corresponding
derivatives exhibited highly potent inhibitory activity against
H+/K+ ATPase.2,27 Compounds 6, 7, 8, 13, 14, 15, 19, 20, 27,
28 and 30 displayed excellent inhibitory activity against H+/
K+ ATPase, with IC50 values of 26, 30, 34, 14, 20, 26, 12, 8,
38, 30 and 28 μg mL−1, respectively, which were much
lower than that of the standard compound omeprazole
(IC50 = 46 μg mL−1). The inhibitory activity increased to-
gether with an increase in the number of hydroxyl and
methoxy groups. When three hydroxyl (20) or three methoxy
(19) groups were present on the phenyl ring, the derivatives
exhibited the most potent activity, with IC50 values of
8 and 12 μg mL−1, respectively, and the trends in activity
were 1OH or OCH3 < 2OH or OCH3 < 3OH or OCH3. Fur-
thermore, we were also interested in designing the mole-
cule by replacing the aromatic aldehydes with aliphatic or
heterocyclic aldehydes (23 and 27–30) and testing the prod-
ucts for their inhibitory activity against H+/K+ ATPase. Ana-
logues derived from furan (27), thiophene (28) and indole
(30) displayed good activity against H+/K+ ATPase. Deriva-
tives with electron-withdrawing groups (Cl, NO2, F and Br)
on the phenyl ring and aliphatic analogues (24–26)
exhibited least antiulcer activity.2
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Table 1 Activities against H+/K+ ATPase and anti-inflammatory activities of the synthesized compounds (1–30)
Sl. no.
01
Structure
Antiulcer activitya (IC50 μg mL−1
)
Anti-inflammatory activitya (IC50 μg mL−1
)
84.1 1.02
80.14 1.02
02
03
04
05
06
07
08
09
10
11
68.17 1.23
72.17 0.58
66.10 1.09
60.11 0.46
26.33 0.47
30.44 0.74
34.17 1.14
68.94 0.68
80.63 1.84
70.56 0.47
28.74 1.25
24.07 1.07
32.15 0.36
20.07 0.47
60.01 1.07
72.69 1.62
54.31 1.39
18.17 0.74
12.74 0.43
16.14 0.45
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Table 1 (continued)
Sl. no.
12
Structure
Antiulcer activitya (IC50 μg mL−1
)
Anti-inflammatory activitya (IC50 μg mL−1
)
66.14 0.40
10.12 0.34
13
14
15
16
17
18
19
20
21
14.12 0.77
20.37 0.46
26.19 0.33
48.06 0.88
54.38 0.66
42.07 0.74
12.77 0.17
8.99 0.78
56.38 0.75
58.17 0.62
68.10 0.64
56.18 1.34
52.38 0.95
58.64 0.74
56.38 0.74
74.58 0.52
68.56 0.73
60.89 0.78
22
42.86 0.23
66.19 0.17
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Table 1 (continued)
Sl. no.
23
Structure
Antiulcer activitya (IC50 μg mL−1
)
Anti-inflammatory activitya (IC50 μg mL−1
)
40.18 0.85
54.19 0.49
24
82.19 1.59
90.74 0.45
25
26
80.17 0.99
72.15 0.88
78.46 0.74
82.16 0.77
27
38.26 0.47
50.14 0.17
28
29
30.44 0.18
42.56 0.87
28.74 0.17
46.18 0.48
30
28.14 0.49
30.18 0.74
Omeprazole
46.14 0.15
—
Indomethacin
—
40.04 0.45
a
The values are the means of three determinations, the ranges of which were <5% of the mean in all cases.
irritation. The active site of COX-2 was identified by creat-
ing a grid of 20 Å × 20 Å × 20 Å in x, y and z coordi-
nates around diclofenac bound to COX-2. Indomethacin
interacted with Arg120, Tyr387 and Ser530 (Fig. 3). Cur-
rent anti-inflammatory drugs such as NSAIDs are closely
related to gastric ulcers,30 as anti-inflammatory drugs
cause gastric ulcers owing to the presence of free carbox-
ylic groups.31 Hence, we docked the 30 newly synthesized
ligands with H+/K+ ATPase to assess their antiulcer
activity.
was chosen as
a reference ligand to calculate RMSD
values for the newly synthesized ligands. Among the 30 li-
gands that were docked with COX-2, the most potent li-
gands 12 and 10 gave D-scores of −8.90 and −8.69, respec-
tively (Table 2). The docked configuration of ligand 12
displayed interactions with Arg120, Tyr385 and Tyr387 via
π–π stacking (Fig. 2). These hydrophobic interactions indi-
cated that ligand 12 binds deep in the core of the active
site where the reference ligand binds. However, ligand 10
Molecular docking studies of H+/K+ ATPase have provided a
homology model of H+/K+-ATPase; hence, H+/K+ ATPase was
used to dock the ligands.32 Ligands 20 and 19 exhibited good
binding affinity, with G-scores of −5.67 and −5.64, respectively.
Ligand 20 displayed strong interactions with Asp897 and Glu899
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(Fig. 4), whereas ligand 19 exhibited a favourable energy con-
figuration towards Glu905 (Fig. 5).
potent inhibitory activity against H+/K+ ATPase than the stan-
dard drug. Compounds 2–5 and 9–12, which had Cl, NO2, F
and/or Br groups on the phenyl ring (electron-withdrawing),
demonstrated excellent anti-inflammatory activity. Com-
ADME studies of these ligands (except for ligands 21 and
22) showed key characteristics of drugs, as they followed
Lipinski's rule of a molecular weight below 500 Da, fewer
than five hydrogen bond donors and fewer than ten accep-
tors. The value of QP log Po/w (octanol/water partition coeffi-
cient) for all the ligands was less than 5.33 All the ligands sat-
isfied the criteria for the values of the octanol/gas (QP log
Poct), water/gas (QP logPw), and brain/blood (QP logBB) parti-
tion coefficients, skin permeability (QP log Kp) and aqueous
solubility (QP log S), which can predict the properties of li-
gands within the permissible ranges (Table 3).
pounds 27–30, which contained
a heterocyclic moiety,
exhibited good inhibitory activity against H+/K+ ATPase as
well as good anti-inflammatory activity. Compounds 24, 25
and 26 displayed less potent anti-H+/K+ ATPase and anti-
inflammatory activities. Molecular docking studies were
performed for all the synthesized compounds, among which
compounds 19 and 20 gave the highest docking scores for in-
hibitory activity against H+/K+ ATPase, whereas compounds
10 and 12 gave the highest docking scores for anti-
inflammatory activity.
3. Conclusions
4. Experimental
4.1 General
In the present study, novel H+/K+ ATPase-inhibiting and anti-
inflammatory hydrazones were synthesized by conjugating
benzo[d]thiazoles with different aldehydes. Our studies re-
vealed that compounds 6–8, 13–15, 18–20, 22 and 23, which
had CH3, OH and/or OCH3 groups on the phenyl ring
(electron-donating) or a heterocyclic moiety, exhibited more
All chemicals and reagents were obtained from Merck (India)
and Avra Synthesis (India) and were used without further pu-
rification. Melting points were determined with a Superfit
melting point apparatus (India) and were uncorrected. FT-IR
Table 2 Docking scores of all synthesized compounds against COX-2 and H+/K+ ATPase
Protein
COX-2
H+/K+ ATPase
Glide
Glide
Glide
ligand
Glide
ligand
RMSD
Docking ligand
efficiency Glide
Glide RMSD
Docking ligand
efficiency Glide
Glide
Ligand
OPLS-2005 score
efficiency Sa
Gscore lipo
OPLS-2005 score
efficiency Sa
Gscore lipo
M-1
M-2
M-3
M-4
M-5
M-6
M-7
M-8
0.005
0.033
0.046
0.038
0.028
0.021
0.037
0.023
0.022
0.002
0.023
0.001
0.015
0.008
0.011
0.030
0.006
0.001
0.014
0.016
0.009
0.017
0.042
0.016
0.002
0.037
0.017
0.004
0.004
0.023
−7.19
−8.33
−8.40
−8.30
−8.54
−7.56
−7.36
−7.86
−8.63
−8.69
−8.65
−8.90
−7.73
−7.43
−7.86
−7.96
−7.56
−7.83
−7.35
−7.38
−7.48
−7.44
−7.89
−7.05
−7.27
−7.12
−8.13
−8.38
−8.04
−8.16
−8.89
—
−0.34
−0.46
−0.42
−0.44
−0.43
−0.34
−0.32
−0.36
−0.43
−0.46
−0.46
−0.42
−0.37
−0.34
−0.37
−0.40
−0.36
−0.31
−0.33
−0.35
−0.44
−0.34
−0.38
−0.47
−0.32
−0.36
−0.45
−0.42
−0.45
−0.41
−0.68
—
−0.95
−1.21
−1.14
−1.17
−1.16
−0.96
−0.91
−1.00
−1.17
−1.22
−1.22
−1.17
−1.02
−0.95
−1.03
−1.08
−0.99
−0.92
−0.94
−0.97
−1.13
−0.95
−1.04
−1.16
−0.90
−0.97
−1.18
−1.14
−1.17
−1.11
−1.14
—
−8.53 −4.08 0.046
−4.94
−5.11
−5.03
−5.16
−5.22
−5.53
−5.50
−5.36
−5.07
−5.03
−5.06
−5.14
−5.60
−5.57
−5.55
−5.25
−5.23
−5.30
−5.64
−5.67
−5.23
−5.26
−5.31
−4.95
−5.03
−5.04
−5.31
−5.43
−5.29
−5.51
—
−0.25
−0.30
−0.25
−0.18
−0.24
−0.25
−0.26
−0.26
−0.24
−0.23
−0.25
−0.27
−0.25
−0.29
−0.29
−0.24
−0.29
−0.27
−0.27
−0.28
−0.19
−0.25
−0.21
−0.28
−0.20
−0.19
−0.21
−0.27
−0.26
−0.26
—
−0.67
−0.77
−0.68
−0.56
−0.67
−0.70
−0.72
−0.70
−0.67
−0.64
−0.69
−0.72
−0.71
−0.78
−0.78
−0.67
−0.76
−0.72
−0.74
−0.77
−0.57
−0.69
−0.62
−0.72
−0.59
−0.58
−0.62
−0.74
−0.72
−0.72
—
−5.25
−5.12
−5.61
−5.51
−5.65
−5.94
−5.93
−5.80
−5.72
−5.25
−5.47
−5.61
−5.69
−5.64
−5.96
−5.66
−5.64
−5.41
−5.76
−6.10
−5.76
−5.55
−5.49
−5.37
−5.86
−5.39
−5.40
−5.87
−5.69
−5.92
—
−2.93
−2.53
−2.78
−3.12
−2.33
−3.04
−2.86
−2.61
−3.04
−2.29
−2.73
−2.56
−2.49
−2.74
−2.97
−3.02
−2.76
−2.63
−2.43
−3.01
−3.61
−2.76
−2.59
−2.14
−2.74
−3.18
−2.43
−2.64
−2.72
−3.23
—
−8.74
−8.70
−8.76
−9.01
−7.97
−7.77
−8.28
−9.05
−9.09
−8.72
−9.33
−8.17
−7.84
−8.27
−8.07
−8.25
−8.66
−7.57
−8.02
−7.48
−8.96
−8.01
−7.06
−8.60
−8.45
−8.54
−8.82
−8.45
−8.62
−7.91
—
−4.33 0.009
−4.82 0.032
−4.29 0.036
−4.82 0.024
−4.46 0.021
−3.91 0.013
−4.01 0.015
−4.11 0.016
−4.12 0.014
−4.03 0.021
−4.50 0.038
−4.03 0.046
−4.19 0.023
−3.97 0.002
−4.15 0.023
−5.03 0.004
−4.39 0.03
−3.87 0.042
−4.28 0.022
−3.69 0.022
−4.04 0.006
−4.13 0.013
−3.36 0.017
−4.37 0.001
−4.50 0.048
−3.87 0.008
−4.23 0.004
−4.04 0.048
−4.76 0.001
M-9
M-10
M-11
M-12
M-13
M-14
M-15
M-16
M-17
M-18
M-19
M-20
M-21
M-22
M-23
M-24
M-25
M-26
M-27
M-28
M-29
M-30
Indomethacin 0.002
Omeprazole
−4.13
—
—
—
0.001
−6.31
−0.26
−0.62
−6.49
−2.29
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Fig. 2 (A) Molecular interaction of COX-2 enzyme with ligand 12. (B) Electrostatic surface representation of the protein depicting the best docking
configuration for ligand 12.
spectroscopy was performed with a Jasco spectrometer (Ja-
pan) using Nujol media. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded with an Agilent Technolo-
gies (USA) spectrometer using CDCl3 as the solvent. High-
resolution mass spectroscopic analysis was performed using
a Bruker MicroTOF QII mass spectrometer in positive mode.
The progress of the reaction was monitored by TLC using sil-
ica gel coated on glass plates with a solvent system compris-
ing chloroform/methanol/acetic acid in the ratio of 95 : 5 : 3
(Rf), and the compounds on the TLC plates were detected un-
der UV light.
4.1.1 Synthesis of 2-hydrazinyl-7-methylbenzoijd]thiazole
(B). Concentrated hydrochloric acid (10 mL) was added
dropwise to a suspension of hydrazine hydrate (0.06 mol) in
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Fig. 3 (A) Molecular interaction of COX-2 enzyme with ligand 10. (B) Electrostatic surface representation of the protein depicting the best docking
configuration for ligand 10.
ethylene glycol (40 mL) at 5–6 °C, and then compound
(A) (0.02 mol) was added. The mixture was refluxed for
4.1.2 General procedure for the synthesis of hydrazones
(1–30). Compound B (1 mmol) was dissolved in ethanol (10
mL g−1 compound) and treated with the appropriate aldehyde
(1 mmol) in the presence of 5–7 drops (0.3–0.5 mL) of glacial
acetic acid. The reaction mixture was refluxed for 8–10 h, and
the completion of the reaction was monitored by TLC. After
the completion of the reaction, the solvent was removed
2–3
h and cooled to room temperature. The reaction
mixture was filtered and the resulting precipitate was
washed with distilled water. Then, the resulting crude
product was crystallized from ethanol to obtain the prod-
uct (B).
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Fig. 4 (A) Molecular interaction of H+/K+ ATPase enzyme with ligand 20. (B) Electrostatic surface representation of the protein depicting the best
docking configuration for ligand 20.
under reduced pressure and the mixture was cooled by
adding ice-cold water. The resulting precipitate was filtered,
washed with water and recrystallized from ethanol to obtain
the desired benzo[d]thiazole-hydrazone (1–30).
4.1.3 2-Hydrazinyl-7-methylbenzoijd]thiazole [B]. Yield
78.9%, light yellow solid, Rf = 0.34, m.p. 208–210 °C, IR KBr
(cm−1): 1920, 3310; 1H NMR (CDCl3) δ ppm: 2.54 (s, 3H,
CH3), 4.82 (s, 2H, NH2), 7.01 (t, J = 8.0 Hz, 1H, Ar–H), 7.11 (d,
J = 7.2 Hz, 1H, Ar–H), 7.46 (d, J = 8.0 Hz, 1H, Ar–H), 11.20 (s,
1H, NH); 13C NMR (CDCl3) δ ppm: 18.2, 118.5, 121.6, 126.6,
128.7, 130.4, 151.1, 173.4; HRMS m/z [M + 1]: calcd for
C8H9N3S: 180.0517; found: 180.0526.
4.1.4 2-(2-Benzylidenehydrazinyl)-7-methylbenzoijd]thiazole
[1]. Yield 91.2%, light yellow solid, Rf = 0.58, m.p. 170–172
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Fig. 5 (A) Molecular interaction of H+/K+ ATPase enzyme with ligand 19. (B) Electrostatic surface representation of the protein depicting the best
docking configuration for ligand 19.
1
°C, IR KBr (cm−1): 1611, 3314; H NMR (CDCl3) δ ppm: 2.54
(s, 3H, CH3), 7.08 (t, J = 7.6 Hz, 1H, Ar–H), 7.16 (d, J = 7.2 Hz,
1H, Ar–H), 7.34–7.41 (m, 3H, Ar–H), 7.52 (d, J = 7.6 Hz, 1H, Ar–
H), 7.63–7.66 (m, 2H, Ar–H), 7.86 (s, 1H, –NCH), 9.63 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 18.0, 118.8, 122.2, 125.1, 126.9,
127.0, 127.7, 128.6, 129.7, 134.0, 143.9, 148.2, 167.8; HRMS
m/z [M + 1]: calcd for C15H13N3S: 268.0830; found: 268.0834.
4.1.5 2-(2-(4-Chlorobenzylidene)hydrazinyl)-7-methylbenzo-
ijd]thiazole [2]. Yield 83.9%, white solid, Rf = 0.44, m.p. 188–
189 °C, IR KBr (cm−1): 1611, 3348; 1H NMR (CDCl3) δ ppm: 2.52
(s, 3H, CH3), 7.05 (t, J = 7.2 Hz, 1H, Ar–H), 7.17 (d, J = 6.2 Hz,
1H, Ar–H), 7.50 (d, J = 7.8 Hz, 1H, Ar–H), 7.60–7.62 (m, 2H, Ar–
H), 7.77–7.79 (m, 2H, Ar–H), 7.89 (s, 1H, –NCH), 10.8 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 18.7, 117.9, 123.4, 125.7, 127.3,
128.4, 129.7, 130.1, 133.5, 137.9, 142.7, 148.0, 167.2; HRMS m/z
[M + 1]: calcd for C15H12ClN3S: 302.0440; found: 302.0428.
4.1.6 7-Methyl-2-(2-(4-nitrobenzylidene)hydrazinyl)benzo-
ijd]thiazole [3]. Yield 88.5%, yellow solid, Rf = 0.49, m.p. 201–
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Table 3 Computer-aided ADME screening of the synthesized compounds
Ligand
Mol wt
a*
b*
c*
d*
e*
f*
g*
h*
i*
j*
k*
l*
M-1
M-2
M-3
M-4
M-5
M-6
M-7
M-8
267.35
301.79
312.35
285.34
346.24
283.35
297.37
281.38
336.24
357.34
303.33
425.14
299.35
327.40
313.37
362.24
376.27
392.27
357.43
315.35
359.45
389.47
268.34
247.36
219.30
275.41
256.32
272.38
255.34
305.40
31.20
32.52
32.99
31.49
32.87
31.07
33.12
33.07
33.70
34.41
31.83
34.37
30.98
35.13
33.04
32.70
35.07
34.58
37.58
30.85
42.05
43.35
30.41
28.26
24.49
31.62
29.52
29.66
29.93
35.44
8.36
8.12
9.54
8.14
8.13
10.46
8.65
8.06
7.98
10.46
8.03
7.96
12.61
8.88
10.75
10.23
8.46
10.48
7.99
14.65
8.34
9.93
9.83
6.74
6.89
6.32
6.77
4.85
7.84
7.19
3.61
4.11
2.82
3.85
4.18
2.88
3.63
3.92
4.42
2.39
3.97
4.69
2.12
3.84
3.04
3.46
4.21
3.68
4.54
1.37
5.79
5.25
2.61
3.22
2.48
3.99
3.99
4.31
3.83
4.70
−4.38
−5.11
−4.52
−4.74
−5.23
−4.12
−4.63
−4.95
−5.59
−4.45
−5.05
−5.94
−3.83
−4.89
−4.35
−4.89
−5.54
−5.07
−5.63
−3.54
−6.85
−6.35
−3.77
−4.16
−3.38
−5.06
−4.59
−4.65
−4.63
−5.81
−6.09
−6.01
−6.04
−5.97
−6.04
−5.97
−6.00
−6.01
−5.85
−5.78
−5.88
−5.97
−5.87
−5.96
−5.94
−5.92
−6.01
−5.88
−5.81
−5.76
−7.26
−7.22
−5.90
−5.03
−4.80
−5.59
−5.84
−5.48
−5.85
−6.29
4111
4111
389
4111
4111
1245
3262
4110
3465
114
3277
4139
367.69
4132
1489
1451
3261
2114
4086
158
4087
3985
1762
3021
2537
2552
5072
3526
3385
2249
0.03
0.20
3803
9389
282
6881
10 000
1046
2805
3803
10 000
79
8283
10 000
265
3825
1203
3104
7295
4529
3692
110
3693
3600
1443
2668
2210
2223
4688
5481
3028
1928
−0.58 0.15
−0.75 0.27
−2.69 0.11
−0.71 0.19
−0.75 0.29
−1.64 −0.01
−0.88 0.17
−0.77 0.31
−1.02 0.37
−3.77 0.04
−1.01 0.23
−0.87 0.41
−2.69 −0.17
−0.75 0.18
−1.53 0.01
−1.65 0.11
−1.03 0.32
−1.36 0.13
−0.87 0.51
−3.43 −0.33
−0.03 1.00
−0.01 0.65
−1.43 −0.16
−1.51 0.09
−1.76 −0.15
−1.37 0.31
−0.60 0.34
−1.02 0.37
−0.97 0.34
−1.09 0.72
100
100
89.85
100
100
100
100
100
100
77.73
100
100
85.26
100
100
100
100
100
100
74.35
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
−1.15
0.14
0.21
−0.57
−0.16
0.02
M-9
0.23
M-10
M-11
M-12
M-13
M-14
M-15
M-16
M-17
M-18
M-19
M-20
M-21
M-22
M-23
M-24
M-25
M-26
M-27
M-28
M-29
M-30
−1.75
0.12
0.37
−1.21
−0.11
−0.59
−0.35
0.01
−0.25
−0.12
−1.67
−0.07
−0.22
−0.36
−0.17
−0.17
−0.48
0.19
0.08
0.00
−0.27
95% range 130.0/725.0 13.0/70.0 4.0/45.0 −2.0/6.5 −6.5/0.5 Concern if <25 poor,
−3.0/1.2 <25 poor,
>500 good
—
−1.5/1.5 >80% >4
for drugs >500 good
below −5
is high
a*: QP polarizability (Å3); b*: QP log P for water/gas; c*: QP log P for octanol/water; d*: QP log S for aqueous solubility; e*: HERG K+ channel
blockage, log IC50; f*: apparent Caco-2 permeability (nm s−1); g*: QP log BB for brain/blood; h*: apparent MDCK permeability (nm s−1); i*: QP
log Kp for skin permeability; j*: QP log K hsa serum protein binding; k*: percentage human oral absorption; l*: Lipinski's rule of 5 violations.
202 °C, IR KBr (cm−1): 1610, 3337; 1H NMR (CDCl3) δ ppm:
2.56 (s, 3H, CH3), 7.07 (t, J = 6.8 Hz, 1H, Ar–H), 7.14 (d, J =
6.0 Hz, 2H, Ar–H), 7.26 (d, J = 7.0 Hz, 1H, Ar–H), 7.48 (d, J =
5.8 Hz, 1H, Ar–H), 7.52 (s, 1H, Ar–H), 7.65 (d, J = 6.2 Hz, 1H,
Ar–H), 8.02 (s, 1H, –NCH), 11.10 (s, 1H, NH); 13C NMR
(CDCl3) δ ppm: 17.1, 118.7, 122.6, 124.8, 125.1, 127.1, 128.3,
129.6, 133.3, 142.8, 148.5, 152.3, 168.3; HRMS m/z [M + 1]:
calcd for C15H12N4O2S: 313.0681; found: 313.0652.
4.1.7 2-(2-(4-Fluorobenzylidene)hydrazinyl)-7-methylbenzo-
ijd]thiazole [4]. Yield 92.1%, off-white solid, Rf = 0.47, m.p.
177–178 °C, IR KBr (cm−1): 1631, 3354; 1H NMR (CDCl3) δ
ppm: 2.49 (s, 3H, CH3), 6.79–6.81 (m, 2H, Ar–H), 7.01 (t, J =
7.0 Hz, 1H, Ar–H), 7.12 (d, J = 8.0 Hz, 1H, Ar–H), 7.41 (d, J =
7.2 Hz, 1H, Ar–H), 7.62–7.64 (m, 2H, Ar–H), 7.81 (s, 1H, –N
CH), 11.04 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 17.8,
115.1, 118.3, 123.0, 126.1, 127.4, 128.3, 129.9, 131.0, 144.0,
148.6, 160.7, 168.3; HRMS m/z [M + 1]: calcd for C15H12FN3S:
286.3393; found: 286.3367.
6.8 Hz, 1H, Ar–H), 7.65 (m, 2H, Ar–H), 7.87 (s, 1H, –NCH),
11.10 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 18.1, 119.4,
123.7, 125.3, 127.4, 128.0, 129.6, 130.1, 131.0, 132.4, 142.7,
147.6, 167.2; HRMS m/z [M + 1]: calcd for C15H12BrN3S:
346.1449; found: 346.1452.
4.1.9 4-((2-(7-Methylbenzoijd]thiazol-2-yl)hydrazono)-
methyl)phenol [6]. Yield 85.6%, cream solid, Rf = 0.41, m.p.
166–167 °C, IR KBr (cm−1): 1617, 3325, 3541; 1H NMR (CDCl3)
δ ppm: 2.36 (s, 3H, CH3), 6.89–6.93 (m, 2H, Ar–H), 7.01–7.08
(m, 3H, Ar–H), 7.19–7.22 (m, 1H, Ar–H), 7.27–7.31 (m, 1H,
Ar–H), 7.36 (s, 1H, –NCH), 8.31 (s, 1H, OH), 10.8 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 17.4, 116.8, 118.1, 119.6,
122.4, 123.0, 125.0, 127.7, 130.8, 131.3, 152.8, 158.3, 166.8;
HRMS m/z [M + 1]: calcd for C15H13N3OS: 284.0813; found:
284.0869.
4.1.10 2-(2-(4-Methoxybenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [7]. Yield 88.9%, white solid, Rf
=
0.53, m.p. 157–159 °C, IR KBr (cm−1): 1624, 3328; 1H
NMR (CDCl3) δ ppm: 2.53 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 6.90 (d, J = 8.4 Hz, 2H, Ar–H), 7.07 (t, J = 7.6 Hz,
1H, Ar–H), 7.15 (d, J = 6.8 Hz, 1H, Ar–H), 7.52 (d, J = 7.6
Hz, 1H, Ar–H), 7.57 (d, J = 8.8 Hz, 2H, Ar–H), 7.82 (s,
1H, –NCH), 11.0 (s, 1H, NH); 13C NMR (CDCl3) δ ppm:
4.1.8 2-(2-(4-Bromobenzylidene)hydrazinyl)-7-methylbenzo-
ijd]thiazole [5]. Yield 88.41%, light yellow solid, Rf = 0.56, m.
1
p. 191–193 °C, IR KBr (cm−1): 1606, 3354; H NMR (CDCl3) δ
ppm: 2.54 (s, 3H, CH3), 7.08 (t, J = 7.0 Hz, 1H, Ar–H), 7.16 (d,
J = 7.8 Hz, 1H, Ar–H), 7.38–7.40 (m, 2H, Ar–H), 7.53 (d, J =
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18.1, 55.3, 114.2, 118.8, 121.9, 126.9, 127.0, 127.4, 128.4,
129.8, 144.0, 148.6, 161.0, 168.3; HRMS m/z [M + 1]: calcd
for C16H15N3OS: 298.2398; found: 298.2364.
166.9; HRMS m/z [M + 1]: calcd for C15H11Br2N3S: 425.1409;
found: 425.1454.
4.1.16 4-((2-(7-Methylbenzoijd]thiazol-2-yl)hydrazono)-
methyl)benzene-1,3-diol [13]. Yield 88.1%, off-white solid, Rf
= 0.29, m.p. 188–189 °C, IR KBr (cm−1): 1601, 3314, 3510,
3547; 1H NMR (CDCl3) δ ppm: 2.48 (s, 3H, CH3), 6.92–6.93
(m, 1H, Ar–H), 7.03 (t, J = 8.0 Hz, 1H, Ar–H), 7.12 (d, J = 7.8
Hz, 1H, Ar–H), 7.42 (d, J = 6.8 Hz, 1H, Ar–H), 7.55–7.57 (m,
2H, Ar–H), 7.80 (s, 1H, –NCH), 8.90 (s, 2H, OH), 9.90 (s,
1H, NH); 13C NMR (CDCl3) δ ppm: 18.9, 104.8, 109.3, 111.5,
118.7, 123.2, 125.9, 127.3, 131.4, 135.2, 144.7, 148.3, 160.8,
162.3, 167.9; HRMS m/z [M + 1]: calcd for C15H13N3O2S:
300.0762; found: 300.0715.
4.1.17 2-(2-(2,4-Dimethoxybenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [14]. Yield 85.6%, off-white solid, Rf =
0.45, m.p. 169–170 °C, IR KBr (cm−1): 1612, 3549; 1H NMR
(CDCl3) δ ppm: 2.51 (s, 3H, CH3), 3.85 (s, 6H, 2OCH3), 6.66–
6.68 (m, 2H, Ar–H), 7.05 (t, J = 7.8 Hz, 1H, Ar–H), 7.10 (t, J =
6.8 Hz, 1H, Ar–H), 7.47 (d, J = 8.8 Hz, 1H, Ar–H), 7.62–7.63
(m, 1H, Ar–H), 7.89 (s, 1H, –NCH), 11.04 (s, 1H, NH); 13C
NMR (CDCl3) δ ppm: 18.7, 54.9, 55.4, 102.7, 106.9, 109.4,
118.7, 122.4, 125.9, 128.0, 131.4, 133.7, 142.8, 147.3, 160.4,
163.4, 167.9; HRMS m/z [M + 1]: calcd for C17H17N3O2S:
328.1041; found: 328.1047.
4.1.18 2-Methoxy-5-((2-(7-methylbenzoijd]thiazol-2-yl)-
hydrazono)methyl)phenol [15]. Yield 91.7%, off-white solid,
Rf = 0.39, m.p. 177–179 °C, IR KBr (cm−1): 1621, 3314, 3542;
1H NMR (CDCl3) δ ppm: 2.53 (s, 3H, CH3), 3.96 (s, 1H, OH),
3.98 (s, 3H, OCH3), 6.92 (d, J = 8.0 Hz, 1H, Ar–H), 7.04–7.09
(m, 2H, Ar–H), 7.14 (d, J = 7.2 Hz, 1H, Ar–H), 7.34 (d, J = 1.2
Hz, 1H, Ar–H), 7.47 (d, J = 7.6 Hz, 1H, Ar–H), 7.83 (s, 1H, –N
CH), 9.82 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 17.5, 55.6,
108.5, 114.4, 118.2, 122.1, 126.2, 127.2, 128.7, 130.8, 137.3,
142.9, 145.0, 146.4, 149.7, 166.1; HRMS m/z [M + 1]: calcd for
C16H15N3O2S: 314.1119; found: 314.1187.
4.1.11 7-Methyl-2-(2-(4-methylbenzylidene)hydrazinyl)-
benzoijd]thiazole [8]. Yield 87.8%, brown solid, Rf = 0.51, m.
p. 152–154 °C, IR KBr (cm−1): 1618, 3310; 1H NMR (CDCl3)
δ ppm: 2.20 (s, 3H, CH3), 2.51 (s, 3H, CH3), 6.90–6.92 (m,
2H, Ar–H), 7.02 (d, J = 7.8 Hz, 1H, Ar–H), 7.11 (d, J = 4.8
Hz, 1H, Ar–H), 7.42 (d, J = 7.8 Hz, 1H, Ar–H), 7.68–7.70 (d,
J = 8.0 Hz, 2H, Ar–H), 7.80 (s, 1H, –NCH), 10.7 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 19.2, 22.8, 119.3, 123.7,
125.0, 126.8, 127.1, 129.3, 130.4, 132.4, 139.4, 144.3, 148.7,
167.3; HRMS m/z [M + 1]: calcd for C16H15N3S: 282.1020;
found: 282.1031.
4.1.12 2-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [9]. Yield 86.9%, off-white solid, Rf =
0.48, m.p. 183–185 °C, IR KBr (cm−1): 1616, 3351; 1H NMR
(CDCl3) δ ppm: 2.53 (s, 3H, CH3), 7.05 (t, J = 7.2 Hz, 1H, Ar–
H), 7.12 (d, J = 7.2 Hz, 1H, Ar–H), 7.24–7.27 (m, 1H, Ar–H),
7.37 (d, J = 2.0 Hz, 1H, Ar–H), 7.47 (d, J = 8.0 Hz, 1H, Ar–H),
7.97 (d, J = 8.8 Hz, 1H, Ar–H), 8.20 (s, 1H, –NCH), 9.82 (s,
1H, NH); 13C NMR (CDCl3) δ ppm: 17.9, 118.8, 122.4, 125.4,
127.2, 127.4, 127.7, 129.5, 130.2, 134.0, 135.6, 139.3, 147.8,
150.1, 167.6; HRMS m/z [M + 1]: calcd for C15H11Cl2N3S:
336.0051; found: 336.0074.
4.1.13 2-(2-(2,4-Dinitrobenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [10]. Yield 79.6%, yellow solid, Rf =
0.52, m.p. 170–171 °C, IR KBr (cm−1): 1620, 3310; 1H NMR
(CDCl3) δ ppm: 2.54 (s, 3H, CH3), 6.90–6.91 (m, 2H, Ar–H),
7.07 (t, J = 7.8 Hz, 1H, Ar–H), 7.13 (t, J = 5.8 Hz, 1H, Ar–H),
7.41 (d, J = 7.8 Hz, 1H, Ar–H), 7.65–7.67 (m, 1H, Ar–H), 7.70–
7.72 (m, 1H, Ar–H), 8.01 (s, 1H, –NCH), 10.8 (s, 1H, NH);
13C NMR (CDCl3) δ ppm: 18.7, 118.2, 121.0, 123.4, 125.9,
127.0, 129.8, 130.7, 132.5, 135.4, 142.6, 148.6, 149.2, 151.7,
168.1; HRMS m/z [M + 1]: calcd for C15H11N5O4S: 358.0823;
found: 358.0819.
4.1.14 2-(2-(2,4-Difluorobenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [11]. Yield 88.9%, light yellow solid,
Rf = 0.40, m.p. 184–186 °C, IR KBr (cm−1): 1601, 3322; 1H
NMR (CDCl3) δ ppm: 2.50 (s, 3H, CH3), 6.75–6.80 (m, 1H, Ar–
H), 6.89–6.94 (m, 1H, Ar–H), 7.02 (t, J = 7.2 Hz, 1H, Ar–H),
7.11 (d, J = 7.2 Hz, 1H, Ar–H), 7.45 (d, J = 7.2 Hz, 1H, Ar–H),
7.92–7.98 (m, 1H, Ar–H), 8.06 (s, 1H, –NCH), 9.11 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 17.8, 103.9, 112.0, 118.5,
122.3, 127.1, 127.7, 127.8, 129.4, 136.3, 142.6, 147.8, 159.7,
162.3, 167.6; HRMS m/z [M + 1]: calcd for C15H11F2N3S:
304.0642; found: 304.0666.
4.1.19 2-Bromo-4-((2-(7-methylbenzoijd]thiazol-2-yl)-
hydrazono)methyl)phenol [16]. Yield 82.6%, light yellow
solid, Rf = 0.40, m.p. 158–159 °C, IR KBr (cm−1): 1622, 3360,
1
3548; H NMR (CDCl3) δ ppm: 2.49 (s, 3H, CH3), 6.95 (d, J =
8.0 Hz, 1H, Ar–H), 7.06–7.09 (m, 2H, Ar–H), 7.40 (d, J = 6.8
Hz, 1H, Ar–H), 7.62–7.68 (m, 2H, Ar–H), 7.90 (s, 1H, –N
CH), 9.10 (s, 1H, OH), 11.6 (s, 1H, NH); 13C NMR (CDCl3) δ
ppm: 19.0, 113.4, 117.0, 118.3, 122.8, 124.8, 127.6, 128.3,
129.4, 130.3, 132.4, 144.1, 148.3, 158.3, 167.8; HRMS m/z [M +
1]: calcd for C15H12BrN3OS: 362.9864; found: 362.9860.
4.1.20 2-(2-(3-Bromo-4-methoxybenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [17]. Yield 86.4%, brown solid, Rf =
0.52, m.p. 188–189 °C, IR KBr (cm−1): 1610, 3345; 1H NMR
(CDCl3) δ ppm: 2.53 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.90–
6.92 (m, 1H, Ar–H), 7.02 (t, J = 6.8 Hz, 1H, Ar–H), 7.14 (d, J =
8.0 Hz, 1H, Ar–H), 7.40 (d, J = 7.0 Hz, 1H, Ar–H), 7.69–7.71
(m, 2H, Ar–H), 7.84 (s, 1H, –NCH), 10.9 (s, 1H, NH); 13C
NMR (CDCl3) δ ppm: 19.4, 54.6, 111.4, 112.7, 118.3, 123.0,
125.6, 126.8, 128.2, 128.9, 129.7, 131.6, 143.8, 148.7, 160.4,
167.9; HRMS m/z [M + 1]: calcd for C16H14BrN3OS: 376.2790;
found: 376.2751.
4.1.15 2-(2-(2,4-Dibromobenzylidene)hydrazinyl)-7-
methylbenzoijd]thiazole [12]. Yield 83.6%, brown solid, Rf =
0.55, m.p. 167–169 °C, IR KBr (cm−1): 1612, 3314; 1H NMR
(CDCl3) δ ppm: 2.52 (s, 3H, CH3), 6.82–6.84 (m, 1H, Ar–H),
7.06 (t, J = 6.8 Hz, 1H, Ar–H), 7.16 (d, J = 5.8 Hz, 1H, Ar–H),
7.47 (d, J = 7.0 Hz, 1H, Ar–H), 7.60–7.61 (m, 1H, Ar–H), 7.70–
7.71 (m, 1H, Ar–H), 7.89 (s, 1H, –NCH), 9.28 (s, 1H, NH);
13C NMR (CDCl3) δ ppm: 18.6, 118.1, 122.4, 123.7, 124.3,
125.8, 127.6, 129.4, 131.0, 133.7, 134.6, 136.7, 142.1, 147.9,
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4.1.21 2-Bromo-6-methoxy-4-((2-(7-methylbenzoijd]thiazol-
2-yl)hydrazono)methyl)phenol [18]. Yield 80.4%, brown solid,
Rf = 0.40, m.p. 201–203 °C, IR KBr (cm−1): 1623, 3345, 3560;
1H NMR (CDCl3) δ ppm: 2.51 (s, 3H, CH3), 3.80 (s, 3H,
OCH3), 6.87–6.88 (m, 1H, Ar–H), 7.03 (t, J = 5.8 Hz, 1H, Ar–
H), 7.11 (d, J = 7.2 Hz, 1H, Ar–H), 7.42 (d, J = 7.8 Hz, 1H, Ar–
H), 7.72–7.73 (m, 2H, Ar–H), 7.88 (s, 1H, –NCH), 8.95 (s,
1H, OH), 11.4 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 18.9,
55.7, 111.7, 115.3, 119.0, 122.7, 123.6, 125.7, 127.3, 129.4,
132.7, 142.0, 144.3, 148.6, 151.7, 167.8; HRMS m/z [M + 1]:
calcd for C16H14BrN3O2S: 392.0024; found: 392.0324.
4.1.22 7-Methyl-2-(2-(3,4,5-trimethoxybenzylidene)-
hydrazinyl)benzoijd]thiazole [19]. Yield 78.6%, off-white solid,
Rf = 0.46, m.p. 208–209 °C, IR KBr (cm−1): 1607, 3540; 1H
NMR (CDCl3) δ ppm: 2.53 (s, 3H, CH3), 3.80 (s, 9H, 3OCH3),
7.03 (t, J = 7.8 Hz, 1H, Ar–H), 7.11–7.15 (m, 3H, Ar–H), 7.46
(d, J = 6.8 Hz, 1H, Ar–H), 7.79 (s, 1H, –NCH), 11.2 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 19.2, 55.7, 59.7, 104.7, 118.4,
122.7, 125.3, 127.8, 128.9, 132.4, 140.9, 143.7, 148.3, 151.7,
167.8; HRMS m/z [M + 1]: calcd for C18H19N3O3S: 358.1181;
found: 358.1189.
(d, J = 7.2 Hz, 1H, Ar–H), 7.50 (d, J = 7.6 Hz, 1H, Ar–H), 7.54
(d, J = 6.0 Hz, 2H, Ar–H), 7.83 (s, 1H, –NCH), 8.61 (d, J =
6.0 Hz, 2H, Ar–H), 11.7 (s, 1H, NH); 13C NMR (CDCl3) δ ppm:
17.8, 118.9, 120.7, 122.7, 125.4, 127.1, 131.4, 141.0, 141.4,
148.7, 150.2, 169.4; HRMS m/z [M + 1]: calcd for C14H12N4S:
269.0783; found: 269.0756.
4.1.27 7-Methyl-2-(2-(3-methylbutylidene)hydrazinyl)benzo-
ijd]thiazole [24]. Yield 82.6%, light yellow solid, Rf = 0.44, m.
1
p. 170–171 °C, IR KBr (cm−1): 1612, 3334; H NMR (CDCl3) δ
ppm: 0.97 (d, J = 6.8 Hz, 6H, (CH3)3), 1.85–1.91 (m, 1H, CH),
2.20 (t, J = 5.6 Hz, 2H, CH2), 2.56 (s, 3H, CH3), 7.03 (t, J = 6.8 Hz,
1H, Ar–H), 7.12 (d, J = 7.2 Hz, 1H, Ar–H), 7.20 (t, J = 5.6 Hz,
1H, Ar–H), 7.47 (d, 1H, –NCH), 11.1 (s, 1H, NH); 13C NMR
(CDCl3) δ ppm: 18.0, 23.3, 26.7, 40.9, 118.6, 121.8, 123.7,
125.7, 126.7, 131.4, 146.9, 169.4; HRMS m/z [M + 1]: calcd for
C13H17N3S: 248.1143; found: 248.1160.
4.1.28 7-Methyl-2-(2-propylidenehydrazinyl)benzoijd]-
thiazole [25]. Yield 86.4%, off-white solid, Rf = 0.49, m.p.
142–143 °C, IR KBr (cm−1): 1604, 3318; 1H NMR (CDCl3) δ
ppm: 1.11 (t, J = 7.0 Hz, 3H, CH3), 2.29–2.34 (m, 2H, CH2),
2.50 (s, 3H, CH3), 7.03 (t, J = 6.8 Hz, 1H, Ar–H), 7.12 (d, J =
7.2 Hz, 1H, Ar–H), 7.23 (t, J = 5.6 Hz, 1H, Ar–H), 7.47 (s, 1H,
–NCH), 11.12 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 10.7,
18.3, 25.7, 118.6, 121.4, 126.6, 127.1, 129.7, 149.2, 150.6,
167.3; HRMS m/z [M + 1]: calcd for C11H13N3S: 219.0830;
found: 219.0877.
4.1.29 2-(2-Heptylidenehydrazinyl)-7-methylbenzoijd]-
thiazole [26]. Yield 80.4%, off-white solid, Rf = 0.56, m.p.
150–152 °C, IR KBr (cm−1): 1610, 3345; 1H NMR (CDCl3) δ
ppm: 0.91 (t, J = 8.6 Hz, 3H, CH3), 1.20–1.35 (m, 8H, (CH2)4),
1.67–1.69 (m, 2H, CH2), 2.53 (s, 3H, CH3), 7.10 (t, J = 7.8 Hz,
1H, Ar–H), 7.15 (d, J = 8.2 Hz, 1H, Ar–H), 7.44 (d, J = 6.8 Hz,
1H, Ar–H), 7.91 (s, 1H, –NCH), 10.89 (s, 1H, NH); 13C NMR
(CDCl3) δ ppm: 15.2, 19.2, 20.7, 23.4, 26.7, 29.3, 31.0, 119.1,
123.5, 125.6, 127.3, 132.5, 144.0, 148.6, 168.9; HRMS m/z [M +
1]: calcd for C15H21N3S: 276.1490; found: 276.1453.
4.1.23 7-Methyl-2-(2-(3,4,5-trihydroxybenzylidene)-
hydrazinyl)benzoijd]thiazole [20]. Yield 77.6%, off-white solid,
Rf = 0.32, m.p. 212–214 °C, IR KBr (cm−1): 1627, 3349, 3540,
1
3556; H NMR (CDCl3) δ ppm: 2.51 (s, 3H, CH3), 5.28 (s, 2H,
OH), 6.84–6.86 (m, 2H, Ar–H), 7.06 (t, J = 8.0 Hz, 1H, Ar–H),
7.14 (s, J = 6.0 Hz, 1H, Ar–H), 7.41 (d, J = 7.2 Hz, 1H, Ar–H),
7.89 (s, 1H, –NCH), 9.20 (s, 1H, OH), 11.3 (s, 1H, NH); 13C
NMR (CDCl3) δ ppm: 18.6, 108.7, 119.1, 122.8, 125.7, 127.6,
129.6, 133.1, 142.6, 144.7, 148.1, 148.7, 168.9; HRMS m/z [M +
1]: calcd for C15H13N3O3S: 316.0711; found: 316.0726.
4.1.24 7-Methyl-2-(2-(4-phenoxybenzylidene)hydrazinyl)-
benzoijd]thiazole [21]. Yield 90.7%, light yellow solid, Rf
=
0.56, m.p. 193–195 °C, IR KBr (cm−1): 1604, 3360; 1H NMR
(CDCl3) δ ppm: 2.45 (s, 3H, CH3), 7.08–7.14 (m, 6H, Ar–H),
7.46–7.50 (m, 4H, Ar–H), 7.72–7.76 (m, 2H, Ar–H), 7.91 (s,
1H, –NCH), 10.81 (s, 1H, NH); 13C NMR (CDCl3) δ ppm:
19.2, 117.2, 118.4, 119.6, 121.4, 123.4, 125.9, 126.4, 128.1,
128.9, 130.4, 131.4, 143.8, 148.9, 156.4, 160.7, 168.7; HRMS
m/z [M + 1]: calcd for C21H17N3OS: 360.1126; found: 360.1195.
4.1.25 2-(2-(4-Benzyloxy-3-methoxybenzylidene)hydrazinyl)-
7-methylbenzoijd]thiazole [22]. Yield 82.4%, off-white solid, Rf
4.1.30
2-(2-(Furan-2-ylmethylene)hydrazinyl)-7-
methylbenzoijd]thiazole [27]. Yield 91.2%, cream solid, Rf =
0.51, m.p. 164–166 °C, IR KBr (cm−1): 1619, 3365; 1H NMR
(CDCl3) δ ppm: 2.52 (s, 3H, CH3), 6.65–6.72 (m, 2H, Ar–H),
7.02 (t, J = 6.8 Hz, 1H, Ar–H), 7.14 (d, J = 7.8 Hz, 1H, Ar–H),
7.41 (d, J = 7.0 Hz, 1H, Ar–H), 7.71–7.72 (m, 1H, Ar–H), 7.86
(s, 1H, –NCH), 10.99 (s, 1H, NH); 13C NMR (CDCl3) δ ppm:
18.6, 112.7, 118.5, 119.3, 123.0, 125.1, 127.3, 132.4, 141.7,
144.5, 148.7, 150.1, 168.7; HRMS m/z [M + 1]: calcd for
C13H11N3OS: 258.0656; found: 258.0621.
1
= 0.53, m.p. 162–163 °C, IR KBr (cm−1): 1620, 3360; H NMR
(CDCl3) δ ppm: 2.50 (s, 3H, CH3), 3.96 (s, 3H, OCH3), 5.19 (s,
2H, CH2), 6.87 (d, J = 8.4 Hz, 1H, Ar–H), 7.04 (t, J = 8.0 Hz,
2H, Ar–H), 7.12 (d, J = 7.2 Hz, 1H, Ar–H), 7.29–7.39 (m, 4H,
Ar–H), 7.44 (t, J = 7.2 Hz, 3H, Ar–H), 7.87 (s, 1H, –NCH),
9.69 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 18.0, 21.5, 55.9,
70.9, 108.9, 113.4, 118.8, 121.3, 122.1, 126.9, 127.2, 127.5,
127.9, 128.4, 128.5, 131.5, 136.7, 144.9, 149.9, 168.3, 177.0;
HRMS m/z [M + 1]: calcd for C23H21N3O2S: 404.1354; found:
404.1359.
4.1.31 7-Methyl-2-(2-(thiophen-2-ylmethylene)hydrazinyl)-
benzoijd]thiazole [28]. Yield 85.6%, off-white solid, Rf = 0.49,
1
m.p. 187–190 °C, IR KBr (cm−1): 1601, 3214; H NMR (CDCl3)
δ ppm: 2.46 (s, 3H, CH3), 7.06 (t, J = 7.0 Hz, 1H, Ar–H),
7.12–7.15 (m, 2H, Ar–H), 7.38 (d, J = 6.2 Hz, 1H, Ar–H),
7.68–7.70 (m, 2H, Ar–H), 7.89 (s, 1H, –NCH), 9.89 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 19.2, 118.3, 123.7, 125.1,
126.7, 127.9, 128.6, 129.7, 133.5, 137.9, 142.8, 148.6, 167.3;
HRMS m/z [M + 1]: calcd for C13H11N3S2: 274.0428; found:
274.0454.
4.1.26 7-Methyl-2-(2-(pyridin-4-ylmethylene)hydrazinyl)-
benzoijd]thiazole [23]. Yield 90.8%, off-white solid, Rf = 0.42,
1
m.p. 151–152 °C, IR KBr (cm−1): 1619, 3344; H NMR (CDCl3)
δ ppm: 2.53 (s, 3H, CH3), 7.09 (t, J = 7.6 Hz, 1H, Ar–H), 7.16
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4.1.32 2-(2-(1H-Pyrrol-2-ylmethylene)hydrazinyl)-7-
methylbenzoijd]thiazole [29]. Yield 81.6%, brown solid, Rf =
0.42, m.p. 210–211 °C, IR KBr (cm−1): 1612, 3345, 3348; 1H
NMR (CDCl3) δ ppm: 2.53 (s, 3H, CH3), 6.60–6.72 (m, 3H, Ar–
H), 7.04 (t, J = 6.8 Hz, 1H, Ar–H), 7.15 (t, J = 7.0 Hz, 1H, Ar–
H), 7.44 (d, J = 9.2 Hz, 1H, Ar–H), 7.90 (s, 1H, –NCH), 9.89
(s, 1H, NH), 11.20 (s, 1H, NH); 13C NMR (CDCl3) δ ppm: 19.1,
110.7, 118.6, 119.4, 122.7, 124.6, 125.9, 127.6, 131.4, 133.5,
140.8, 148.6, 169.7; HRMS m/z [M + 1]: calcd for C13H12N4S:
257.0816; found: 257.0841.
4.1.33 2-(2-(1H-Indol-2-ylmethylene)hydrazinyl)-7-
methylbenzoijd]thiazole [30]. Yield 82.6%, light yellow solid,
Rf = 0.45, m.p. 196–198 °C, IR KBr (cm−1): 1610, 3214, 3342;
1H NMR (CDCl3) δ ppm: 2.49 (s, 3H, CH3), 6.86–6.92 (m, 3H,
Ar–H), 7.07 (t, J = 8.0 Hz, 1H, Ar–H), 7.17 (t, J = 5.8 Hz, 1H,
Ar–H), 7.41 (d, J = 6.8 Hz, 1H, Ar–H), 7.61–7.65 (m, 2H, Ar–
H), 7.81 (s, 1H, –NCH), 10.81 (s, 1H, NH), 11.26 (s, 1H,
NH); 13C NMR (CDCl3) δ ppm: 18.4, 101.4, 112.7, 118.7,
119.6, 120.7, 121.4, 122.9, 123.5, 126.7, 127.3, 128.4, 131.6,
133.4, 141.6, 148.9, 168.9; HRMS m/z [M + 1]: calcd for
C17H14N4S: 307.3849; found: 307.3856.
Inorganic phosphorus estimation. Inorganic phosphorus
was estimated according to the Fiske Subbarow method.35 Al-
iquots of a working standard solution (40 μg mL−1) were
added to 8 fresh and dry test tubes in volumes of 0 to 1 mL,
and 5 μL and 10 μL of the enzyme sample were added to the
7th and 8th test tubes, respectively. The volumes of all solu-
tions were made up to 8.6 mL using 10% TCA. Ammonium
molybdate (1 mL) and ANSA reagent (0.4 mL) were added to
all test tubes. All test tubes were allowed to stand for 10 min
at RT. Then the color was developed and the λmax value at
660 nm was recorded.
5.2 ATPase activity
ATPase activity was determined using a reported method.36
The basal activity of Mg2+-dependent ATPase was calculated
in 1 mL reaction mixture comprising 2 mmol L−1 ATP and 50
mmol L−1 Tris-HCl buffer (pH = 7.5). The activities of K+-stim-
−
ulated and HCO3 -stimulated ATPase were tested in basal me-
dium. The ATPase reaction was started by the addition of the
substrate (ATP), carried out at 37 °C for 10–15 min and then
terminated by the addition of 1 mL cold 20% TCA. The
amount of inorganic phosphate liberated from ATP was esti-
mated using the literature method.36
The activity of the enzyme sample in the presence of ATP
was 0.066 μmol.
The activity of the enzyme sample in the absence of ATP
was 0.042 μmol.
5. Biological activity
5.1 Gastric H+/K+ ATPase activity
Isolation of parietal cells from sheep stomach. The fundic
portion of a sheep stomach was collected soon after sacrifice
and rinsed with Krebs Ringer buffer (250 mM sucrose, 2 mM
MgCl2, 1 mM EGTA and 2 mM Hepes-Tris of a pH of 7.4).
The upper layer was pinned with the help of needles on a dis-
section table. Mucosal scrapings were suspended in 10 vol-
umes of Krebs Ringer buffer (pH = 7.4) containing sucrose
(250 mM) and homogenized by 20 strokes of a mortar-driven
Teflon pestle homogenizer. The tissues were discarded and
the filtrate was subjected to subcellular fractionation. The
pellets obtained were dissolved in 2 mL sucrose-EGTA buffer
and used as an enzyme sample.
Protein estimation. The protein content was calculated
using the Lowry method,34 and bovine serum albumin was
used as a standard (0–75 μg). Eight clean and dry test tubes
were taken and aliquots of various concentrations of the syn-
thesized derivatives were prepared. To the 7th and 8th test
tubes were added unknown samples (5 μL and 10 μL of cells
isolated from the sheep stomach), for which the protein con-
tent was to be determined. In every test tube, the solution
was made up to 1 mL by the addition of distilled water,
followed by the addition of 5 mL Lowry's reagent. All the test
tubes were incubated at RT for 8–10 min, and then 0.5 mL
Folin–Ciocalteu reagent was added and the tubes were again
incubated at RT for 30 min. The absorbance of each solution
was recorded at 670 nm against a blank solution. A graph
was plotted using the concentration of protein on the X-axis
and OD on the Y-axis. From the standard graph obtained, the
concentration of protein in the unknown sample was calcu-
lated, and it was found to contain 21 mg protein per 8 g tis-
sue homogenate.
100% activity of the enzyme was 0.066 μmol − 0.042 μmol
= 0.024 μmol.
5.3 Anti-inflammatory activity
Human erythrocyte suspension. Human blood was col-
lected in a heparinized vacutainer from a healthy volunteer
who had not taken any NSAIDs for 2 weeks before the experi-
ment. The collected healthy human blood was washed with
0.9% saline and centrifuged for 10 minutes at 3000 rpm. The
packed cells were washed with 0.9% saline, and a 40% (v/v)
suspension was prepared with isotonic phosphate buffer of
154 mM NaCl in 10 mM sodium phosphate buffer at a pH of
7.4 and used as a stock erythrocyte (RBC) suspension.
Hypotonic solution-induced haemolysis. The activity of
the synthesized compounds was tested according to
a
reported method.28 The tested samples, which consisted of
0.5 mL stock erythrocyte (RBC) suspension, 5 mL hypotonic
solution (50 mM NaCl in 10 mM sodium phosphate-buffered
saline at a pH of 7.4) and samples of different concentrations
(20, 40, 60, 80 and 100 μg mL−1), were prepared. The blank
control consisted of 0.5 mL mixed RBC suspension and 5 mL
hypotonic buffered solution alone. The mixtures were incu-
bated for 10 minutes at room temperature and centrifuged
for 10 minutes at 3000 rpm, and the supernatant was mea-
sured spectrophotometrically at 540 nm. The percentage inhi-
bition of haemolysis was calculated according to the follow-
ing formula:
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where:
A1 = absorbance of hypotonic buffered solution alone; and
A2
= absorbance of test/standard sample in hypotonic
solution.
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5.4 Molecular docking and ADME predictions
The coordinates of COX-2 and H+/K+ ATPase were obtained
from the Brookhaven Protein Data Bank, and their PDB IDs
are 1PXX and 2ZXE, respectively. The ligands were drawn
using Maestro 2D sketcher, and energy minimization was
performed with OPLS 2005. Protein structures were obtained
by retrieval from the Maestro platform (Schrödinger, Inc.).
Protein structures were corrected using the Schrödinger
Prime software module to correct the missing loops in the
protein. Water molecules in COX-2 and H+/K+ ATPase were re-
moved beyond 5 Å from the respective heteroatom. Water
molecules that were thought to be important in aiding the
interaction with the receptor were optimized using Protein
Preparation Wizard. Necessary bonds, bond orders, hybridi-
zation, explicit hydrogens and charges were assigned auto-
matically. The OPLS 2005 force field was applied to the pro-
tein to restrain minimization, and an RMSD of 0.30 Å was set
to converge heavy atoms during preprocessing of the protein
before docking was started. Using extra-precision (XP)
docking and scoring, each compound was docked into a re-
ceptor grid of dimensions of 20 Å × 20 Å × 20 Å, and the
docking calculations were judged on the basis of the Glide
score, ADME results and Glide energy. The prediction pro-
gram QikProp was used to calculate the ADME properties of
all the ligands, and molecular visualization was performed in
the Maestro workspace.37
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Acknowledgements
We are grateful to Wuhan University of Technology, Wuhan,
China for financial support. The authors are also thankful to
the management of SRI RAM CHEM (India) for their continu-
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