Design, synthesis and QSAR studies of 2-amino benzo[d]thiazolyl substituted pyrazol-5-ones: novel class of promising antibacterial agents

Article abstract of DOI:10.1007/s00044-017-1898-6

Abstract: Novel analogs of 3-(4-substituted benzylideneamino)-N-(6-substituted-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5a–s) were designed and synthesized by reacting 3-amino-N-(6-substituted-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (4a–d) with p-substituted benzaldehydes. The Infrared Spectroscopy, ¹H-Nuclear Magnetic Resonance, ¹³C-Nuclear Magnetic Resonance, and High Resolution Mass Spectra spectral data confirmed the structures of all the novel synthesized compounds. Among the series tested, two compounds 5k and 5o displayed promising antibacterial activity especially against Staphylococcus aureus (MIC = 3.14 and 1.57 μg/mL) and Bacillus subtilis (MIC = 3.12 and 1.84 μg/mL) respectively. Further, these title compounds were also assessed for their cytotoxic activity (IC₅₀) against mammalian Vero cell line using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide assay, indicating that the compounds exhibit antibacterial activity at non-cytotoxic concentrations. Field based three-dimensional quantitative structure–activity relationships were also discussed based on the antimicrobial screening data. Graphical Abstract: Synthesis, spectral studies, antibacterial evaluation and QSAR studies of nineteen novel Schiff bases of pyrazol-5-one derivatives derived from 2-aminobenzothiazole nucleus are described.

Full text of DOI:10.1007/s00044-017-1898-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1898-6
ORIGINAL RESEARCH
Design, synthesis and QSAR studies of 2-amino benzo[d]thiazolyl
substituted pyrazol-5-ones: novel class of promising antibacterial
agents
Mahesh B. Palkar1,2 Aniket Patil1 Girish A. Hampannavar2
●
●
●
Mahamadhanif S. Shaikh2 Harun M. Patel2 Ashish M. Kanhed3
●
●
●
Mange Ram Yadav3 Rajshekhar V. Karpoormath2
●
Received: 6 January 2015 / Accepted: 13 April 2017
© Springer Science+Business Media New York 2017
Abstract Novel analogs of 3-(4-substituted benzylidenea-
mino)-N-(6-substituted-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-
5-oxo-pyrazole-1-carboxamide (5a–s) were designed and
synthesized by reacting 3-amino-N-(6-substituted-1,3-benzo
[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(4a–d) with p-substituted benzaldehydes. The Infrared
Spectroscopy, 1H-Nuclear Magnetic Resonance, 13C-
Nuclear Magnetic Resonance, and High Resolution Mass
Spectra spectral data confirmed the structures of all the
novel synthesized compounds. Among the series tested, two
compounds 5k and 5o displayed promising antibacterial
activity especially against Staphylococcus aureus (MIC =
3.14 and 1.57 µg/mL) and Bacillus subtilis (MIC = 3.12 and
1.84 µg/mL) respectively. Further, these title compounds
were also assessed for their cytotoxic activity (IC50) against
mammalian Vero cell line using 3-(4,5-dimethylthiazo-2-
yl)-2,5-diphenyl-tetrazolium bromide assay, indicating that
the compounds exhibit antibacterial activity at non-cytotoxic
concentrations. Field based three-dimensional quantitative
structure–activity relationships were also discussed based on the
antimicrobial screening data.
Graphical Abstract Synthesis, spectral studies, anti-
bacterial evaluation and QSAR studies of nineteen novel
Schiff bases of pyrazol-5-one derivatives derived from 2-
aminobenzothiazole nucleus are described.
●
●
Keywords Benzo[d]thiazole Pyrazol-5-one Antibacterial
●
●
activity Cytotoxicity 3D-QSAR
Introduction
The infectious diseases caused by pathogenic microorgan-
isms are a major cause of morbidity and mortality for the
human kind. According to the recent reports from World
Health Organization (Cohen 2000), approximately 57 mil-
lion deaths occur per annum worldwide. A matter of grave
concern is a rapid development of resistance to che-
motherapeutic agents or antibiotics by multidrug-resistant
strains of Gram-positive (Streptococcus, vancomycin-
resistant Enterococcus, and methicillin-resistant Staphylo-
coccus aureus) and Gram-negative pathogens (Escherichia,
Salmonella, and certain Pseudomonas strains). Thus there is
a precipitously growing global crisis in the clinical man-
agement of severe infectious disease, which demands for
the discovery and development of novel antimicrobial leads
with high efficacy (Coleman 2004; Abbanat et al. 2003).
* Mahesh B. Palkar
palkarmahesh4u@rediffmail.com
* Rajshekhar V. Karpoormath
Karpoormath@ukzn.ac.za
rvk2006@gmail.com
1
Department of Pharmaceutical Chemistry, K.L.E.University’s
College of Pharmacy, VidyanagarHubballi 580031 Karnataka,
India
2
Department of Pharmaceutical Chemistry, College of Health
Sciences, University of KwaZulu-Natal, Westville Campus,
Durban 4000, South Africa
3
Department of Pharmacy, Faculty of Technology and Engineering,
MS University of Baroda, Kala BhavanVadodara 390001 Gujarat,
India

Med Chem Res
Fig. 1 Literature reported structures of benzo[d]thiazole, pyrazole and Schiff base derivatives. Design of 2-aminobenzo[d]thiazolyl substituted
pyrazol-5-one Schiff base analogs using molecular hybridization approach
Heterocyclic skeletons serve as ideal scaffolds on which
pharmacophores can be appended to yield potential drug
candidates (Thompson and Ellman 1996). From literature it
is known that the molecular manipulation of 2-amino benzo
[d]thiazole moiety, one of the versatile scaffold in the drug
discovery, resulted in compounds that exhibited wide spec-
trum of biological activities such as anticancer, antimicrobial,
anti-inflammatory, analgesic, antitubercular, and muscle
relaxants (Axford et al. 2013; Kitagawa et al. 2007; Telvekar
et al. 2012; Cho et al. 2008; Manjula et al. 2009; Dogruer
et al. 1998). The structural variations along with their asso-
ciated biological activities of 2-aminobenzo[d]thiazole moi-
ety are depicted in Fig. 1. Similarly, nitrogen containing five
membered heterocyclic scaffolds such as pyrazole and pyr-
azolone moieties serves as a privileged scaffolds for the
generation of drug-like libraries in medicinal chemistry as
illustrated in Fig. 1. The pyrazolone class of compounds,
containing N–N bond display a wide range of biological
activities like anti-inflammatory, analgesic, antitubercular,
antimicrobial, anticancer, and anti-human immune-deficiency
virus (Ronkin et al. 2010; Tanitame et al. 2004; Castagnolo
et al. 2008; El-Hawash et al. 2006; Markovic et al. 2011;
Makhija et al. 2004). Much attention has been given to
pyrazoles as therapeutic agents after the discovery of pyr-
azofurin (pyrazole C-glycoside), which demonstrated potent
antimicrobial and anticancer activity (Genin et al. 2000).
Recently, Sun et al. reported the synthesis of series of N′-(4-
methoxy)benzoyl-3-(4-bromophenyl)-1H-pyrazole-5-carbo-
hydrazide analogs containing double amide bridge, which
displayed potent antibacterial activity (Sun et al. 2013).
Figure 1 also highlights the molecular manipulation of Schiff
bases (also known as azomethine) and their resultant biolo-
gical activities. Schiff bases have been found to possess a
broad range of biological activities, which includes anti-
fungal, antibacterial, antimalarial, antiproliferative, anti-
inflammatory, antiviral, and antipyretic properties (Rathelot
et al. 1995; Sriram et al. 2006; Makawana et al. 2013; da
Silva et al. 2011; Sztanke et al. 2013).

Med Chem Res
Scheme 1 Synthesis of 4-(6-
substituted-1,3-benzo[d]thiazol-
2-yl)semicarbazide (3a–d).
Reagents and conditions: (i)
CH3COOH, KSCN, Br2, NH3,
0 °C, stir, 10–12 h; (ii) Ethyl
chloroformate, anhydrous
K2CO3, acetone, stir, 50 °C,
reflux, 9–15 h; (iii)
O
H2C CH3
O
O
NH2
NH
N
S
N
S
N
(i)
(ii)
(iii)
NH2
R
NH
NH
NH2
S
R
R
R
2a-d
3a-d
1a-d
R = F
R = Cl
R = Br
3a: R = F
3b: R = Cl
3c: R = Br
1a: R = F
1b: R = Cl
1c: R = Br
2a: R = F
2b: R = Cl
2c: R = Br
2d: R = NO2
R = NO2
3d: R = NO2
1d: R = NO2
NH2NH2·H2O (99%), methanol,
reflux, 4–6 h
Scheme 2 Synthesis of novel
series of 3-(4-
substitutedbenzylideneamino)-
N-(6-substituted-1,3-benzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-
pyrazole-1-carboxamide (5a–s)
derivatives. Reagents and
conditions: (iv) ethyl-2-
cyanoacetate, DMF, reflux,
8–12 h; (v) p-substituted
benzaldehydes, toluene
(anhydrous), CH3COOH, Dean
stark, reflux, 13–20 h
Over the years, molecular hybridization-based drug
design approach (Viegas-Jr et al. 2007) has been exploited
by many researchers in order to develop new hybrid che-
mical entities as promising drug candidates. It is known that
more efficacious antibacterial agents with synergistic anti-
microbial activity can be designed by joining two or more
biologically active pharmacophores or heterocyclic systems
in a single molecular framework. Based on the above
mentioned facts, we envisaged to synthesize novel hybrid
structure having 6-substituted-2-aminobenzo[d]thiazole and
substituted pyrazol-5-one Schiff bases linked with an amide
bond (See Fig. 1). In continuation of our search for biolo-
gically active hybrid molecules (Hampannavar et al. 2016;
Palkar et al. 2015; Shaikh et al. 2014; Aragade et al. 2013),
we herein report the design, synthesis, and spectral studies
of a series of 19 new Schiff bases of pyrazol-5-one deri-
vatives derived from benzo[d]thiazole nucleus (5a–s).
These hybrid compounds were evaluated for their pre-
liminary in vitro antibacterial and cytotoxicity studies fol-
lowed by field-based three-dimensional quantitative
structure–activity relationships (3D-QSAR) studies on their
biological results.
achieved through versatile and efficient synthetic route as
outlined in Schemes 1 and 2. Various starting materials i.e.,
6-substituted-2-aminobenzo[d] thiazoles (1a–d) were pre-
pared by reacting p-substituted aniline and potassium thio-
cyanate in glacial acetic acid at 0 °C by following earlier
reported methods (Brewster and Dains 1936; Palkar et al.
2010). The subsequent intermediates i.e., ethyl(6-sub-
stituted-1,3-benzo[d]thiazol-2-yl)carbamates (2a–d) were
prepared by nucleophilic substitution reaction of com-
pounds 1a–d in the presence of ethyl chloroformate and
anhydrous K2CO3 in acetone. Subsequently, compounds
2a–d were reacted with hydrazine hydrate (99%) in
methanol to afford different carbohydrazide derivatives
(3a–d) as depicted in Scheme 1. Compounds 3a–d were
further reacted with ethyl 2-cyanoacetate in glacial acetic
acid and dimethyl formamide (DMF) under reflux to obtain
various
3-amino-N-(6-substituted-1,3-benzo[d]thiazol-2-
yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide derivatives
(4a–d) through a cyclo-condensation process (Parashar
et al. 2012; Mruthyunjayaswamy and Basavarajaiah 2009).
These compounds contain free amino (NH2) group at 3rd
position of the pyrazol-5-one ring, and which can be
appreciably labile to participate in the Schiff reaction.
Condensation of compounds 4a–d with appropriately p-
substituted benzaldehydes in toluene (anhydrous) and cat-
alytic amounts of glacial acetic acid resulted in a series of
Schiff bases (5a–s) with moderate to good yields (see
Scheme 2). This reaction was carried out using Dean stark
apparatus in order to remove the in situ water formed, thus
Chemistry
The synthesis of a novel series of 3-(4-substitutedben-
zylideneamino)-N-(6-substituted-1,3-benzo[d]thiazol-2-yl)-4,
5-dihydro-5-oxo-pyrazole-1-carboxamide
(5a–s)
was

Med Chem Res
favoring the formation of Schiff bases (da Silva et al. 2011).
It was observed that the crystalline appearance of Schiff
bases (5a–s) were of different shades of yellow color, which
could be attributed to the presence of chromophoric group
(CH=N) in their molecular structure.
Promega Cell Titer 96, a non-radioactive cell proliferation
assay method (Mosmann 1983).
QSAR studies
In the present study nineteen derivatives (5a–s) and their
corresponding MIC values in nanomolar (nM) scale were
used for field-based QSAR generation. The field-based
QSAR models are based on CoMFA (Cramer 1988) and
CoMSIA (Glide 2013; Klebe and Abraha 1999). CoMFA
field-based models are constructed by calculating the value
of fields, such as the electrostatic field, on a rectangular grid
that encompasses the molecules in the training set. The grid
locations are the independent variables that are used in a
partial-least-squares (PLS) fitting procedure to produce a
relationship between the values of the fields and the activity
of the training set molecules.
The MIC values were converted to 1/log MIC in nM
terms and were used as dependent variables in QSAR study.
The 3D structures of the molecules were built using ‘build’
module within Schrödinger. All the structures were mini-
mized using MacroModel by OPLS_2005 with Powell-
Reeves conjugate gradient convergence criterion of 0.05
kcal/mol force field at a maximum of 3500 iteration. An 8
and 20 Å cut-off was applied for both van der Waals
interactions and electrostatic energies, respectively. One of
the fundamental assumptions wherein 3D-QSAR studies are
based is that a geometric similarity should exist between the
structures. So, taking compound 5o as bioactive con-
formation all the compounds are aligned on the backbone of
compound 5o. The 19 compounds were divided into train-
ing set (14 compounds) and test set (5 compounds) for the
generations of QSAR model.
The steric and electrostatic potential fields were calcu-
lated at each lattice intersection of a regularly spaced grid of
1.0 Å. The grid box dimensions were determined auto-
matically in such a way that the region boundaries were
extended beyond 3 Å in each direction from co-ordinates of
each molecule. The van der Waals potentials and Cou-
lombic terms, which represent steric and electrostatic fields,
respectively, were calculated using five Gaussian fields for
the QSAR model field (Gaussian 2009). Cross-validated
PLS regressions method was performed to generate QSAR
equation. The QSAR equation for antibacterial potential
against S. aureus was evaluated by cross validated corre-
lation coefficient (r2cv), standard deviation (s), F-test (F)
and correlation coefficient (r2).
Material and methods
In vitro antibacterial activity
The antibacterial activity of the synthesized compounds (5a–
s) was tested against two Gram positive bacteria namely S.
aureus (ATCC 25923), B. subtilis (ATCC 6633) and two
Gram negative bacteria namely, E. coli (ATCC 25922), P.
aeruginosa (ATCC 27853) using MH medium (Mueller-
Hinton medium: casein hydrolysate 17.5 g, soluble starch
1.5 g, beef extract 1000 mL). The minimum inhibitory
concentrations (MICs) of the test compounds were deter-
mined by a colorimetric method using the dye 3-(4,5-dime-
thylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). A
stock solution of the synthesized compound (100 µg/mL)
in DMSO was prepared and graded quantities of the test
compounds were incorporated in specified quantity of
sterilized liquid MH medium. A specified quantity of the
medium containing the compound was poured into micro-
titration plates. The bacterial inocula were prepared by
suspending 24 h old bacterial colonies from Muller Hinton
Agar media in 0.85% saline. The inocula were adjusted to
0.5 McFarland Standard (1.5 × 108 CFU/mL) and applied
to microtitration plates with serially diluted compounds in
DMSO to be tested and incubated at 37 °C for 24 h. After
the MICs were visually determined on each of the micro-
titration plates, 50 µL of phosphate buffered saline (0.01
mol/L, pH 7.4, Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g,
NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL) containing
2 mg of MTT/mL was added to each well. Incubation was
continued at room temperature for 4–5 h. The content of
each well was removed, and 100 µL of isopropanol con-
taining 5% 1 mol/L HCl was added to extract the dye. After
12 h of incubation at room temperature, the optical density
was measured with a microplate reader at 550 nm (CLSI
guidelines 2015; Stevens and Olsen 1993; McFarland
1907).
Cytotoxic activity
The cellular conversion of MTT into a formazan product
was used to evaluate cytotoxic activity (IC50) of a novel
series of title compounds (5a–s) against mammalian Vero
cell line upto concentrations of 62.5 µg/mL. After 72 h of
exposure, cell viability was assessed on the basis of cellular
conversion of MTT into a formazan product using the
Results and discussion
The synthesis of 2-amino benzo[d]thiazolyl substituted
pyrazol-5-ones was achieved through an efficient and

Med Chem Res
versatile synthetic route as illustrated in Scheme 1. Struc-
tures of key intermediates 4a–d and their respective Schiff
bases (5a–s) were confirmed based on their physicochem-
corresponding signals for aromatic and heteroaromatic
carbons resonated at around δ 148.9–105.3 ppm, while the
methoxyl carbons appeared at around δ 58.3–55.7 ppm. In
addition, the formation of title compounds was confirmed
by recording their mass spectra (HRMS), which were in full
agreement with their molecular weights. Electron impact
mass spectra (EIMS) displayed an accurate molecular ion
peaks at m/z 382.2, 412.9, 425.3, 398.1, 428.3, 441.5,
433.6, 471.2, 408.6, 438.1, and 424.4 for compounds 5a–c,
5f–i, 5l, 5o, 5p, and 5s, respectively.
All the newly synthesized compounds (5a–s) were fur-
ther evaluated for their in vitro antibacterial activity against
two Gram positive bacteria; Staphylococcus aureus (ATCC
25923), Bacillus subtilis (ATCC 6633) and two Gram
negative bacteira; Escherichia coli (ATCC 25922), Pseu-
domonas aeruginosa (ATCC 27853), which could be causal
agents of some serious infections in humans. The anti-
bacterial activity was carried out in MH medium. Ampi-
cillin and Gatifloxacin were used as a reference standard.
The MIC values of in vitro antibacterial screening of the test
compounds are summarized in Table 1. All the reported
compounds exhibited moderate to significant in vitro
activity against the tested bacterial strains compared to
reference drugs. A systematic analysis of the data as
depicted in Table 1 revealed that some of the synthesized
compounds exhibited enhanced antibacterial activity, espe-
cially against Gram positive S. aureus (ATCC 25923) and
B. subtilis (ATCC 6633).
From this series, three compounds (5k, 5n, and 5o) exhib-
ited excellent antibacterial activity for Gram positive bacteria,
while compounds 5e, 5f, 5h, 5j, 5q, and 5s showed moderate
to good antibacterial activity against the tested organisms.
Compound 5o displayed two fold higher activity than the
standard drugs against S. aureus with MIC 1.57 µg/mL,
whereas almost comparable activity was observed for com-
pounds 5k and 5n with MIC value of 3.14 µg/mL with respect
to that of positive control Ampicillin (MIC = 3.12 µg/mL) and
Gatifloxacin (MIC = 3.12 µg/mL).
In the case of B. subtilis, compound 5o (MIC = 1.84 µg/mL)
exhibited significant antibacterial activity, whereas com-
pounds 5a, 5e, 5f, 5j, 5k, 5q, and 5s showed moderate to
good antibacterial activity when compared to reference
drugs. Besides this, compounds 5k (MIC = 4.48 µg/mL)
and 5o (MIC = 5.26 µg/mL) displayed moderate to good
antibacterial activity against Gram negative E. coli, when
compared to standard Ampicillin (MIC = 3.12 µg/mL) and
Gatifloxacin (MIC = 0.39 µg/mL). Further, moderate activ-
ity against P. aureginosa was observed for compound 5k
(MIC = 6.25 µg/mL). However, all other compounds in this
series exhibited little or poor activity against Gram negative
bacteria. From the activity data an interesting pattern
between the compounds and antibacterial activity against
S. aureus was observed. In general, a close inspection of the
1
ical and spectral (FTIR, H-NMR (nuclear magnetic reso-
nance), 13C-NMR, and High Resolution Mass Spectra
(HRMS)) analysis. All the synthesized compounds were in
agreement with their respective analytical data, which are
summarized in experimental section. In general, the Infrared
Spectroscopy (IR) spectra of compounds 4a–d showed the
absorption bands ranging from 3268 to 3233 cm−1 for N–H,
1715–1699 cm−1 for C=O (amide), 1601–1586 cm−1 for
C=N as well as 748–726 cm−1 for C–S–C groups, respec-
tively. Further, the formation of 3-amino-pyrazol-5-one
nucleus was confirmed by the appearance of distinct char-
acteristic bands at 3477–3434 and 1680–1658 cm−1
assigned to the stretching vibration of the NH2 and C=O
(pyrazol-5-one ring) groups. This was further substantiated
1
from H-NMR spectra of compounds 4a–d, which exhib-
ited the prominent singlet signals around δ 5.35–5.16 and
3.60–3.34 ppm accounting for amino (NH2) and methylene
(CH2) protons indicating the pyrazol-5-one ring formation
from their respective carbohydrazides (3a–d) by simple
cyclo-condensation process. This observation is consistent
with similar reported compounds (Parashar et al. 2012;
Mruthyunjayaswamy and Basavarajaiah 2009). Further the
IR spectra of Schiff base (5a–s) showed disappearance of
the characteristic bands at 3477–3434 cm−1 of amino group
and appearance of prominent bands at around 1711–1672
and 1670–1642 cm−1 accounted for carbonyl (C=O) groups
of amide and pyrazol-5-one, respectively. The IR spectra of
these compounds also showed moderately strong bands at
around 3304–3190, 1616–1585, and 759–729 cm−1 con-
firming the presence of N–H, C=N and C–S–C groups,
respectively. The 1H-NMR spectra of these compounds
revealed the most informative and attributable signals as
singlets at around δ 10.48–9.97 and 9.06–8.65 ppm for –NH
(amide) and –CH=N– (imine) protons indicating the
formation of Schiff bases. A characteristic singlet signal
due to the CH2 protons of pyrazol-5-one ring appeared at δ
3.56–3.19 ppm. Further the 1H-NMR spectra of the title
compounds showed prominent signals for the benzo[d]
thiazole protons between δ 8.14 and 7.51 ppm and the
aromatic protons appeared between δ 7.60 and 6.95 ppm,
1
respectively. The H-NMR spectra of the compounds 5b,
5c, 5g, 5l, 5p, and 5s also exhibited peaks resonating around
δ 5.54, 3.88–3.80 and 2.92–2.79 ppm confirming the
presence of OH, OCH3 and N(CH3)2 protons in their
respective structures. In 13C-NMR spectrum the character-
istic signals appeared at around
δ
174.9–171.1,
168.3–165.3, 163.6–160.2, and 39.4–33.6 ppm, strongly
suggesting the presence of C=O (of amide), C=O (of
pyrazol-5-one), –CH=N (of imine) and CH2 (of pyrazol-5-
one) groups, respectively. It was also observed that the

Med Chem Res
Table 1 The in vitro antibacterial and cytotoxic activity of the newly synthesized compounds (5a–s)
Code Compounds
Minimum inhibitory concentrations (MIC)a
Cytotoxicity IC50
(µM)b
Gram positive
Gram negative
S. aureus B. subtilis E. coli
P. aeruginosa
5a
10.38
08.46
12.48
13.24
129.7
5b
25.43
14.40
10.52
15.76
161.9
5c
16.57
22.10
>50.00
>50.00
143.7
5d
13.04
16.25
>50.00
>50.00
127.2
5e
08.06
06.50
10.26
12.62
145.5
5f
04.60
07.50
12.68
10.24
114.9

Med Chem Res
Table 1 continued
Code Compounds
Minimum inhibitory concentrations (MIC)a
Cytotoxicity IC50
(µM)b
Gram positive
Gram negative
S. aureus B. subtilis E. coli
P. aeruginosa
5g
18.77
16.68
16.24
18.16
128.1
5h
08.06
10.78
21.66
>50.00
168.1
5i
12.54
18.43
>50.00
>50.00
178.9
5j
05.56
06.25
13.12
12.60
120.3
5k
03.14
03.12
04.48
06.25
163.4

Med Chem Res
Table 1 continued
Code Compounds
Minimum inhibitory concentrations (MIC)a
Cytotoxicity IC50
(µM)b
Gram positive
Gram negative
S. aureus B. subtilis E. coli
P. aeruginosa
5l
25.43
23.66
>50.00
>50.00
118.3
5m
16.57
13.79
>50.00
>50.00
134.8
5n
03.14
03.50
09.40
12.25
218.4
5o
01.57
01.84
05.26
06.27
151.3
5p
17.27
10.38
15.25
18.50
149.7

Med Chem Res
Table 1 continued
Code Compounds
Minimum inhibitory concentrations (MIC)a
Cytotoxicity IC50
(µM)b
Gram positive
Gram negative
S. aureus B. subtilis E. coli
P. aeruginosa
5q
06.34
06.34
10.48
12.50
192.2
5r
16.57
20.99
26.88
>50.00
174.5
5s
09.64
10.26
15.22
12.54
249.7
Ampicillin
Gatifloxacin
a
03.12
03.12
01.56
01.56
03.12
0.39
03.12
0.78
>430.0
>575.0
MIC values are determined by broth dilution method (two fold dilution) and are expressed in μg/ml
b Mammalian toxicity testing using VERO cell lines and is expressed as IC50, is the concentration of compound, which is reduced by 50% of the
optical density of treated cells with respect to untreated cells using the MTT assay
screening results indicated that the compounds with halo or
nitro (–NO2) group at position C-6 of benzo[d]thiazole
nucleus has exerted significant influence on the antibacterial
armament, whereas p-substitution on the aromatic ring had
little or no influence on the antibacterial activity. From this
novel series, compounds 5k and 5o have emerged as pro-
mising antibacterial leads especially against the Gram
positive bacterial organisms.
The title compounds 5a–s were further examined for
their toxicity (IC50) profile on mammalian Vero cell line up
to 62.5 µg/mL concentrations. After 72 h of exposure, cell
viability was assessed on the basis of cellular conversion of
MTT into a formazan product. Promega Cell Titer 96, a
non-radioactive cell proliferation assay method was
employed and their results are summarized in Table 1. The
IC50 values ranging from 114.9 to 249.7 µM was observed

Med Chem Res
Table 2 The MIC (experimental) and pMIC (predicted) along with
residual activity of the training and test set compounds against S.
aureus
for the 19 derivatives tested indicating no significant
activity against mammalian Vero cell line at concentrations
<100 µM. Compounds 5o–s containing nitro (NO2) group
at position C-6 of benzo[d]thiazole ring showed less cyto-
toxicity with IC50 ranging from 174.5 to 249.7 μM when
compared to that of fluoro, chloro, and bromo-substituted
analogs. With the promising activity profile along with
increased cyto-friendly properties exhibited, the synthesized
compounds could be attractive lead molecules in develop-
ing safe and potential antimicrobial agents.
Compound Experimental Activity Predicted Activity Residual
5a
5b
5ct
5dt
5et
5f
0.1016
0.1405
0.1219
0.1115
0.0906
0.0662
0.1273
0.0906
0.1098
0.0745
0.0496
0.1405
0.1219
0.0496
0.0195
0.1237
0.0802
0.1219
0.0984
0.1020
0.1401
0.1223
0.1121
0.0912
0.0667
0.1278
0.0900
0.1091
0.0752
0.0490
0.1412
0.1224
0.0489
0.0188
0.1246
0.0809
0.1224
0.0993
−0.0004
0.0004
−0.0004
−0.0006
−0.0006
−0.0005
−0.0005
0.0006
Based on the promising antibacterial activity profile (low
MICs) and the effect of various substituents on the motifs
(benzo[d]thiazole/pyrazol-5-one) of the core skeleton and
their subsequent consequence on the activity, encouraged us
to develop a suitable 3D-QSAR model to further validate
the observed results. The predictivity of the generated 3D-
QSAR model was analyzed by test set predictions. Con-
sidering the flexibility of the molecules, the predictive
qualities of the generated 3D-QSAR model was found to be
satisfactory. The generated 3D-QSAR model displayed
acceptable statistics, which was based on the parameters
like R2, Q2, SD, and RMSE, as well as on the high value of
Pearson-R. The predicted and observed activities of the
training and test set compounds are summarized in Table 2
and represented in Fig. 2. The training set correlation was
characterized by PLS factors (R2 = 0.874, SD = 0.009, F =
34.8, P = 3.13e−005). The test set correlation was char-
acterized by PLS factors (Q2 = 0.621, RMSE = 0.01,
Pearson R = 0.929) of the field-based 3D-QSAR. The
relative contributions of the steric, electrostatic, hydro-
phobic, and hydrogen bond interaction fields were found to
be 67.49, 4.27, 13.58, and 14.64%, respectively, reflecting
the relative importance of steric, electrostatic, hydrophobic,
and the hydrogen bonding interactions in the antibacterial
mechanism.
5g
5h
5i
0.0007
5j
−0.0007
0.0006
5k
5l
−0.0007
−0.0005
0.0007
5m
5n
5o
5pt
5q
5r
0.0007
−0.0009
−0.0007
−0.0005
−0.0009
5st
t test set
The 3D-QSAR steric and electrostatic contours are
represented in Fig. 3a, b. The steric interaction was depicted
by green and yellow contours, while electrostatic interaction
was denoted by blue and red contours. For steric interac-
tions, the green contour map represents sterically favorable
region, while the yellow contour map represents the unfa-
vorable region. A yellow contour around the 4th position
phenyl ring of compound 5o suggesting that groups with
low steric tolerance are required at this position to increase
the activity (See Fig. 3a). This could be a possible reason,
why compounds 5b, 5c, 5d, 5g, 5i, 5l, 5m, 5p, and 5r have
poor antibacterial activity. It was also observed that the
presence of bulky group at 3rd position of the phenyl ring
could increase the biological activity. In the case of com-
pound 5o, green contour near the 6th position of the benz[d]
thiazole ring indicated that bulky substituents were pre-
ferred in this region (See Fig. 3a). Similarly, in compounds
5a, 5e, 5f, 5h, 5j, 5k, 5n, 5q, and 5s containing fluoro,
Fig. 2 Correlation graph between MIC (experimental) and pMIC
(predicted) of the training and test set compounds along with residual

Med Chem Res
Fig. 3 Showing the field based
3D-QSAR visualization for a
Compound 5o: effect for steric
interaction; b Compound 5n:
effect for electrostatic
interaction; c Compound 5h;
effect for hydrophobic; d
Compound 5n: effect for
hydrogen bond donor interaction
chloro, bromo, and nitro groups extended towards the large
green contour, which might be a favorable region for
bulkier groups. This also explains that these compounds
displayed high binding affinity.
Conclusion
In this communication, we report the synthesis, spectral
studies, and biological evaluation of a novel series of 3-(4-
substitutedbenzylideneamino)-N-(6-substituted-1,3-benzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5a–s) derivatives. It was observed from the in vitro anti-
bacterial screening of the title compounds (5a–s) that
compounds 5k, 5n, and 5o displayed excellent antibacterial
activity against Gram positive bacteria. Compounds 5k and
5o also exhibited moderate to good antibacterial activity
against Gram negative organisms. The cytotoxic activity
(IC50) against mammalian Vero cell line (MTT assay)
indicated that the title compounds were non-cytotoxic and
no significant cytotoxicity was observed at concentrations
<100 µM. Hence, the molecular hybridization strategy
chosen for the current study to combine pharmacophoric
key features from benzo[d]thiazole and pyrazol-5-one using
amide linkage was successful. Developed 3D - QSAR
results are in close agreement with the observed
structure–activity relationship. Statistically significant 3D -
QSAR model was obtained with good results in terms of q2
and r2 values. The QSAR model established the most sig-
nificant correlation of steric and hydrogen bond acceptor
fields with the biological activities for the title compounds,
while the influence of the steric, electrostatic, hydrophobic,
and hydrogen-bond acceptor fields on their activities was
clarified by analyzing the field based contour maps. Overall,
the results described in this study provide a fruitful insight
into the different field contributions around the molecules,
and their influence on the antibacterial activity. In conclu-
sion, compound 5o was found to be the most promising lead
and could be further exploited for developing novel
potential and safe antibacterial agents.
The electrostatic contour plot is presented in Fig. 3b. A
blue contour favors the electropositive substituent for
high binding affinity, while the red contour indicates the
electronegative substituents are favorable for high
binding affinity with the protein. For compounds 5c
and 5h the decrease in activity could be attributed to the
increase in electron density on the aromatic ring due to the
presence of N,N-dimethyl group at 4th position. Thus,
explaining the reduced biological activity for some
compounds.
The hydrophobic interactions as illustrated in Fig. 3c are
represented by pink (favorable) and magenta (unfavorable)
contours for biological activity. The presence of small
hydrophobic groups occupying the pink contours enhances
the activity, while larger hydrophobic groups extending up
to magenta region could result in decrease of biological
activity. This explains the biological activity of some
compounds 5c and 5h. Similarly hydrogen bond interac-
tions are represented in Fig. 3d. Magenta contours represent
regions favoring hydrogen bond acceptors on the ligands
while red contours indicate regions, where hydrogen bond
acceptor substituents on ligands are unfavorable for the
activity. From the contour maps, it was clear that presence
of hydrogen bond acceptor substituents like carbonyl and/or
nitro groups at 4th position on the phenyl ring might
enhance the biological activity. This indicates the
increase in activity for compounds (5e, 5j, and, 5n)
having nitro group at 4th position of the phenyl ring.
However for compounds 5k and 5o such observation was
not noticed.

Med Chem Res
Experimental
6-Fluoro-2-amino-benzo[d]thiazole (1a)
All research chemicals were purchased from Sigma-Aldrich
(St. Louis, Missouri, USA) or Lancaster Co. (Ward Hill,
MA, USA) and used as such for the reactions. The reference
drugs (Ampicillin and Gatifloxacin) used in this work
was received as a gift sample from Fourrtis (India) Lab.
Pvt. Ltd., Chennai (Tamilnadu, India). Solvents except
laboratory reagent grade were dried and purified accor-
ding to the literature when necessary. The progress of
reactions and purity of compounds were monitored by
thin-layer chromatography (TLC) on pre-coated silica
gel plates procured from E. Merck and Co. (Darmstadt,
Germany) using methanol (3%) in dichloromethane as
mobile phase and iodine vapors as visualizing agent.
Melting points of synthesized compounds were deter-
mined in Thermonik (Mumbai, India) melting point appa-
ratus and are uncorrected. IR spectra were recorded on
ThermoNicolet IR200 FT-IR Spectrometer (Madison, WI,
White crystals, yield 77.3%, mp 183–185 °C; FTIR (KBr,
cm−1): 3374 (N–H), 1598 (C=N); 1H NMR (400 MHz,
DMSO-d6, δ, ppm): 7.64–7.62 (d, 1H, J = 8.5 Hz, H-7 of
benzo[d]thiazole), 7.41–7.38 (d, 1H, J = 8.2, H-4 of benzo
[d]thiazole), 7.22–7.19 (d, 1H, J = 8.7 Hz, H-5 of benzo[d]
thiazole), 6.38 (s, 2H, NH2, D2O exchangeable); EIMS m/z:
149.3 (M+).
6-Chloro-2-amino-benzo[d]thiazole (1b)
Colorless crystals, yield 68.5%, mp 203–205 °C; FTIR
1
(KBr, cm−1): 3403 (N–H), 1585 (C=N); H NMR (400
MHz, DMSO-d6, δ, ppm): 7.60 (s, 1H, H-7 of benzo[d]
thiazole), 7.35–7.33 (d, 2H, J = 8.3 Hz, H-4 and H-5 of
benzo[d]thiazole), 6.44 (s, 2H, NH2, D2O exchangeable);
EIMS m/z: 165.2 (M+).
1
USA) by using KBr pellets. The H NMR and 13C NMR
6-Bromo-2-amino-benzo[d]thiazole (1c)
were recorded on Bruker AVANCE 400 (Bruker, Rhein-
stetten/Karlsruhe, Germany) using appropriate solvent.
Chemical shifts are reported in δ ppm units with respect to
TMS as internal standard. Mass spectra and HRMS were
recorded on Autospec Mass Spectrometer under the electron
impact at 70 eV. The synthesized title compounds were
purified by using flash chromatography SP-01 (Biotage,
Sweden).
Yellow solid, yield: 72.3%, mp 217–219 °C; FTIR (KBr,
cm−1): 3410 (N–H), 1589 (C=N); 1H NMR (400 MHz,
DMSO-d6, δ, ppm): 7.72 (s, 1H, H-7 of benzo[d]thiazole),
7.46–7.42 (d, 2H, J = 8.5 Hz, H-4 and H-5 of benzo[d]
thiazole), 6.52 (s, 2H, NH2, D2O exchangeable); EIMS m/z:
210.1 (M+).
6-Nitro-2-amino-benzo[d]thiazole (1d)
General procedure for synthesis of 6-substituted-2-
amino-1,3-benzo[d]thiazole (1a–d)
Yellow crystalline solid, yield 70.7%, mp 196–198 °C;
1
FTIR (KBr, cm−1): 3416 (N–H), 1591 (C=N); H NMR
(400 MHz, DMSO-d6, δ, ppm): 7.79 (s, 1H, H-7 of benzo
[d]thiazole), 7.40–7.36 (d, 2H, J = 8.6 Hz, H-4 and H-5 of
benzo[d]thiazole), 6.65 (s, 2H, NH2, D2O exchangeable);
EIMS m/z: 176.3 (M+).
An appropriately p-substituted aniline (0.1 mol) and potas-
sium thiocyanate (KCNS, 9.718 g, 0.1 mol) were dissolved
in 100 mL of glacial acetic acid (AcOH), cooled in
ice–salt mixture and stirred mechanically, while a solution
of bromine (15.980 g, 0.1 mol) in AcOH (20 mL)
was slowly added drop by drop (Palkar et al. 2010).
External cooling was applied throughout the process to keep
the temperature below 10 °C and the stirring was continued
for 30 min. After all of the bromine had been added, the
solution was stirred for 2 h below room temperature and at
room temperature for 10 h. It was then allowed to stand
overnight during which the precipitate of imino-benzo[d]
thiazole hydrobromide salt thus separated out was filtered,
washed with acetic acid, dried, dissolved in hot water and
basified to pH 11.0 with ammonia solution (NH4OH) and
the resulting precipitate was filtered, washed with water and
dried to get the desired products 2-amino-6-substituted
benzo[d]thiazole (1a–d). The progress of the reaction was
monitored by TLC using acetone (20%) in toluene solvent
system.
General procedure for synthesis of ethyl(6-substituted-
1,3-benzo[d]thiazol-2-yl) carbamate (2a–d)
To a constantly stirred solution of 6-substituted-2-amino-
1,3-benzo[d]thiazole (one of 1a–d, 0.03 mol) in anhydrous
acetone (50 mL), anhydrous potassium carbonate (6.22 g,
0.045 mol) was added and stirring was further continued at
50 °C for 1.5 h (Parashar et al. 2012). Ethyl chloroformate
(3.256 g, 0.03 mol) was added in a dropwise manner at 50 °
C with constant stirring and then the reaction mixture was
refluxed for 9–15 h. The progress of the reaction was
monitored by TLC. The reaction mixture was allowed attain
the room temperature. The mixture was then poured into the
water and extracted successively with 25 mL of dichloro
methane (DCM, four times). To the combined DCM frac-
tion, anhydrous Na2SO4 was added, filtered and the filtrate

Med Chem Res
was evaporated in vaccuo to yield the desired compounds
2a–d.
was filtered, washed with n-hexane, dried under vacuum to
afford the respective compounds 3a–d.
Ethyl(6-fluoro-1,3-benzo[d]thiazol-2-yl)carbamate (2a)
N-(6-fluorobenzo[d]thiazol-2-yl)hydrazinecarboxamide
(3a)
Off while solid, yield 61.2%, mp 198–200 °C; FTIR (KBr,
1
cm−1): 3273 (N–H), 1690 (C=O), 1591 (C=N); H NMR
White pluffy crystals, yield 66.3%, mp 229–232 °C; FTIR
(KBr, cm−1): 3397 (N–H), 1666 (C=O), 1590 (C=N), 812
(C–F), 738 (C–S–C); H NMR (400 MHz, DMSO-d6, δ,
ppm): 8.68 (s, 1H, NHC=O), 7.88 (s, 1H, H-7 of benzo[d]
thiazole), 7.69–7.64 (d, 2H, J = 8.6 Hz, H-4 and H-5 of
benzo[d]thiazole), 7.02 (s, 1H, C=ONH), 4.22 (m, 2H,
NH2); EIMS m/z: 226.1 (M+).
(400 MHz, DMSO-d6, δ, ppm): 8.49 (s, 1H, –NH),
7.77–7.75 (d, 1H, J = 8.6 Hz, H-7 of benzo[d]thiazole),
7.39–7.36 (d, 1H, J = 8.3 Hz, H-4 of benzo[d]thiazole),
7.24–7.20 (d, 1H, J = 8.8 Hz, H-5 of benzo[d]thiazole),
3.25 (s, 2H, –CH2), 1.31 (t, 3H, –CH3); EIMS m/z: 225.3
(M+ + 1), 224.1 (M+).
1
Ethyl(6-chloro-1,3-benzo[d]thiazol-2-yl)carbamate (2b)
N-(6-chlorobenzo[d]thiazol-2-yl)hydrazinecarboxamide
(3b)
While solid, yield 58.7%, mp 215–218 °C; FTIR (KBr,
1
cm−1): 3292 (N–H), 1705 (C=O), 1601 (C=N); H NMR
Colorless crystals, yield 73.4%, mp 242–244 °C; FTIR
(KBr, cm−1): 3368 (N–H), 1674 (C=O), 1602 (C=N), 831
(C–Cl), 755 (C–S–C); H NMR (400 MHz, DMSO-d6, δ,
ppm): 8.95 (s, 1H, NHC=O), 7.92 (s, 1H, H-7 of benzo[d]
thiazole), 7.56–7.52 (d, 2H, J = 8.5 Hz, H-4 and H-5 of
benzo[d]thiazole), 6.72 (s, 1H, C=ONH), 4.25 (m, 2H,
NH2); EIMS m/z: 244.2 (M+ + 2), 242.3 (M+).
(400 MHz, DMSO-d6, δ, ppm): 8.55 (s, 1H, –NH), 7.63 (s,
1H, H-7 of benzo[d]thiazole), 7.46–7.42 (d, 2H, J = 8.2 Hz,
H-4 and H-5 of benzo[d]thiazole), 3.16 (q, 2H, –CH2), 1.21
(t, 3H, –CH3); EIMS m/z: 242.5 (M+ + 2), 240.3 (M+).
1
Ethyl(6-bromo-1,3-benzo[d]thiazol-2-yl)carbamate (2c)
Pale yellowish brown solid, yield 67.1%, mp 195–197 °C;
FTIR (KBr, cm−1): 3302 (N–H), 1706 (C=O), 1580
(C=N); H NMR (400 MHz, DMSO-d6, δ, ppm): 8.42 (s,
N-(6-bromobenzo[d]thiazol-2-yl)hydrazinecarboxamide
(3c)
1
1H, –NH), 7.81 (s, 1H, H-7 of benzo[d]thiazole), 7.45–7.40
(d, 2H, J = 8.4 Hz, H-4 and H-5 of benzo[d]thiazole), 3.06
(q, 2H, –CH2), 1.29 (t, 3H, –CH3); EIMS m/z: 284.7 (M+ +
2), 282.5 (M+).
Beige colored solid, yield 52.9%, mp 216–218 °C; FTIR
(KBr, cm−1): 3402 (N–H), 1657 (C=O), 1595 (C=N), 850
(C–Br), 743 (C–S–C); H NMR (400 MHz, DMSO-d6, δ,
ppm): 9.12 (s, 1H, NHC=O), 8.03 (s, 1H, H-7 of benzo[d]
thiazole), 7.57–7.54 (d, 2H, J = 8.4 Hz, H-4 and H-5 of
benzo[d]thiazole), 7.08 (s, 1H, C=ONH), 4.21 (m, 2H,
NH2); EIMS m/z: 287.4 (M+).
1
Ethyl(6-nitro-1,3-benzo[d]thiazol-2-yl)carbamate (2d)
Light yellow solid, yield 78.2%, mp 218–220 °C; FTIR
1
(KBr, cm−1): 3313 (N–H), 1711 (C=O); 1593 (C=N); H
N-(6-nitrobenzo[d]thiazol-2-yl)hydrazinecarboxamide (3d)
NMR (400 MHz, DMSO-d6, δ, ppm): 8.89 (s, 1H, –NH),
7.94 (s, 1H, H-7 of benzo[d]thiazole), 7.64–7.61 (d, 2H, J
= 8.7 Hz, H-4 and H-5 of benzo[d]thiazole), 3.11 (q, 2H,
–CH2), 1.35 (t, 3H, –CH3); EIMS m/z: 267.4 (M+).
Light yellow solid, yield 57.3%, mp 175–178 °C; FTIR
(KBr, cm−1): 3414 (N–H), 1660 (C=O), 1609 (C=N), 736
(C–S–C). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.79 (s,
1H, NHC=O), 7.98 (s, 1H, H-7 of benzo[d]thiazole),
7.76–7.73 (d, 2H, J = 8.5 Hz, H-4 and H-5 of benzo[d]
thiazole), 6.47 (s, 1H, C=ONH), 4.13 (m, 2H, NH2); EIMS
m/z: 269.1 (M+).
General procedure for synthesis of N-(6-substituted
benzo[d]thiazol-2-yl)hydrazine carboxamide (3a–d)
To a warm solution of ethyl(6-substituted-1,3-benzo[d]
thiazol-2-yl)carbamate (one of 2a–d, 0.005 mol) in anhy-
drous methanol (25 mL), hydrazine hydrate (99%, 0.005
mol) was added in a dropwise manner with continuous
stirring. The reaction mixture was then refluxed for 4–6 h
and allowed to attain room temperature. The TLC mon-
itored the completion of the reaction. The reaction mixture
was then cooled in ice-cold condition; the separated solid
General procedure for synthesis of 3-amino-N-(6-
substituted-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-
pyrazole-1-carboxamide (4a–d)
To a solution of N-(6-substituted benzo[d]thiazol-2-yl)
hydrazinecarboxamide (one of 3a–d, 0.003 mol) in DMF
(20 mL), ethyl 2-cyanoacetate (0.339 g, 0.003 mol) was

Med Chem Res
added in the presence of catalytic amount of glacial acetic
acid (0.3 mL) and the reaction mixture was refluxed in an
anhydrous condition for 8–12 h (Parashar et al. 2012). The
progress of the reaction was monitored by the TLC. The
reaction mixture was allowed to attain the room tempera-
ture. The mixture was then poured into the ice-cold water.
The resulting solid products (4a–d) were filtered, dried, and
recrystallized from methanol.
7.75–7.71 (d, 2H, J = 8.5 Hz, H-4 and H-5 of benzo[d]
thiazole), 5.28 (s, 2H, NH2), 3.52 (s, 2H, –CH2 of pyrazol-
5-one). EIMS m/z: 320.5 (M+).
General procedure for synthesis of3-(4-
substitutedbenzylideneamino)-N-(6-substituted-1,3-
benzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-
carboxamide (5a–s)
3-amino-N-(6-fluoro-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-
5-oxo-pyrazole-1-carboxamide (4a)
To a constantly stirred solution of compound (one of 4a–d,
0.001 mol) in anhydrous toluene (50 mL), an appropriately
p-substituted benzaldehydes (0.0012 mol) and glacial acetic
acid (0.3 mL) were slowly added. The reaction mixture was
then heated at reflux temperature for about 13–20 h, until no
water appears (monitored with a Dean stark apparatus). The
solvent was removed in vacuo and the resultant residue was
washed with n-hexane and recrystallized from appropriate
solvent to afford the title compounds (5a–s).
White crystalline solid, yield 51.4%, mp 210–212 °C; FTIR
(KBr, cm−1): 3434, 3387 (NH2), 3256 (N–H), 1703 (C=O
of amide), 1680 (C=O of pyrazol-5-one), 1586 (C=N), 796
1
(C–F), 741 (C–S–C); H NMR (400 MHz, DMSO-d6, δ,
ppm): 10.13 (br, s, 1H, NHC=O), 7.93 (s, 1H, H-7 of benzo
[d]thiazole), 7.65–7.62 (d, 2H, J = 7.7 Hz, H-4 and H-5 of
benzo[d] thiazole), 5.16 (s, 2H, NH2), 3.6 (s, 2H, -CH2 of
pyrazol-5-one); EIMS m/z: 293.3 (M+).
3-(Benzylideneamino)-N-(6-fluorobenzo[d]thiazol-2-yl)-
4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5a)
3-Amino-N-(6-chloro-1,3-benzo[d]thiazol-2-yl)-4,5-
dihydro-5-oxo-pyrazole-1-carboxamide (4b)
Yield 68.1%, mp 185–187 °C; FTIR (KBr, cm−1): 3190
(N–H), 3056 (Ar–H), 1689 (C=O of amide), 1657 (C=O of
pyrazol-5-one), 1599 (C=N), 752 (C–S–C);1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.48 (brs, 1H, NH), 9.03 (s, 1H,
CH=N of imine), 8.00 (s, 1H, H-7 of benzo[d]thiazole),
7.89–7.86 (d, 2H, J = 8.6 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.56–7.54 (d, 1H, J = 8.4, Ar–H), 7.45–7.43 (d,
1H, J = 8.2 Hz, Ar–H), 7.34–7.31 (m, 3H, Ar–H), 3.19 (s,
2H, –CH2 of pyrazol-5-one). 13C NMR (101 MHz, DMSO-
d6, δ, ppm): 171.7 (C=O of amide), 167.5 (C=O of pyr-
azol-5-one), 162.8 (CH=N of imine), 156.3, 147.5, 130.5,
128.8, 127.9, 127.7, 126, 121.6, 120.8, 114.3, 106.9, 36.2
(CH2 of pyrazol-5-one). EIMS m/z (relative intensity):
382.2 (M+ + 1, 23%), 292.2 (100%), 230.1 (12%), 196.6
(34%), 157.0 (22%), 125.2 (38%), 91.8 (17%). HRMS (EI):
for C18H12FN5O2S [M+ + H], calcd. 382.0694 m/z; found,
382.0635 m/z.
Colorless solid, yield 47.5%, mp 199–202 °C; FTIR (KBr,
cm−1): 3458, 3332 (NH2), 3241 (N–H), 1708 (C=O of
amide), 1673 (C=O of pyrazol-5-one), 1594 (C=N), 815
1
(C–Cl), 748 (C–S–C); H NMR (400 MHz, DMSO-d6, δ,
ppm): 10.21 (br, s, 1H, NHC=O), 8.07 (s, 1H, H-7 of benzo
[d]thiazole), 7.79–7.74 (d, 2H, J = 8.2 Hz, H-4 and H-5 of
benzo[d]thiazole), 5.22 (s, 2H, NH2), 3.47 (s, 2H, –CH2 of
pyrazol-5-one); EIMS m/z: 309.4 (M+).
3-Amino-N-(6-bromo-1,3-benzo[d]thiazol-2-yl)-4,5-
dihydro-5-oxo-pyrazole-1-carboxamide (4c)
Yellowish-orange solid, yield 53.0%, mp 189–191 °C;
FTIR (KBr, cm−1): 3477, 3356 (NH2), 3268 (N–H), 1699
(C=O of amide), 1667 (C=O of pyrazol-5-one), 1601
1
(C=N), 879 (C–Br), 739 (C–S–C); H NMR (400 MHz,
DMSO-d6, δ, ppm): 9.88 (br, s, 1H, NHC=O), 8.12 (s, 1H,
H-7of benzo[d]thiazole), 7.83–7.80 (d, 2H, J = 8.6 Hz, H-4
and H-5 of benzo[d] thiazole), 5.35 (s, 2H, NH2), 3.34 (s,
2H, –CH2 of pyrazol-5-one); EIMS m/z: 354.2 (M+).
3-(4-Methoxybenzylideneamino)-N-(6-fluorobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5b)
Yield 55.6%, mp 194–196 °C; FTIR (KBr, cm−1): 3220
(N–H), 3033 (Ar–H), 1688 (C=O of amide), 1665 (C=O of
pyrazol-5-one), 1602 (C=N), 747 (C–S–C); 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.42 (brs, 1H, NH), 9.01 (s, 1H,
CH=N of imine),8.14 (s, 1H, H-7 of benzo[d]thiazole),
7.72–7.69 (d, 2H, J = 8.3 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.41–7.39 (d, 2H, J = 8.2 Hz, Ar–H), 7.18–7.16
(d, 2H, J = 8.8 Hz, Ar–H), 3.86 (s, 3H, OCH3), 3.25 (s, 2H,
–CH2 of pyrazol-5-one). 13C NMR (101 MHz, DMSO-d6,
3-Amino-N-(6-nitro-1,3-benzo[d]thiazol-2-yl)-4,5-dihydro-
5-oxo-pyrazole-1-carboxamide (4d)
Cream-yellow solid, yield 55.6%, mp 235–237 °C; FTIR
(KBr, cm−1): 3472, 3351 (NH2), 3233 (N–H), 1715 (C=O
of amide), 1658 (C=O of pyrazol-5-one), 1598 (C=N), 726
1
(C–S–C); H NMR (400 MHz, DMSO-d6, δ, ppm): 10.05
(br, s, 1H, NHC=O), 8.09 (s, 1H, H-7 of benzo[d]thiazole),

Med Chem Res
δ, ppm): 171.1 (C=O of amide), 167.4 (C=O of pyrazol-5-
one), 163.6 (CH=N of imine), 157.4, 147.9, 132.2, 129,
126.6, 124.1, 121.5, 120.5, 113.8, 105.3, 57.5 (OCH3), 35.9
(CH2 of pyrazol-5-one). EIMS m/z (relative intensity):
412.9 (M+ + 1, 16%), 411.1 (35%), 381.1 (21%), 292.1
(100%), 230.0 (15%), 195.3 (42%), 158.1 (20%), 124.9
(29%), 91 (33%). HRMS (EI): for C19H14FN5O3S [M+ +
H], calcd. 413.0086 m/z; found, 413.0111 m/z.
MHz, DMSO-d6, δ, ppm): 10.25 (brs, 1H, NH), 8.89 (s, 1H,
CH=N of imine), 8.01 (s, 1H, H-7 of benzo[d]thiazole),
7.82–7.79 (d, 2H, J = 8.2 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.42–7.40 (d, 2H, J = 8.5 Hz, Ar–H), 7.21–7.18
(d, 2H, J = 8.6 Hz, Ar–H), 3.42 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm):172.6 (C=O
of amide), 168.1 (C=O of pyrazol-5-one), 163.1 (CH=N of
imine), 156.3, 147.3, 131.2, 129.3, 127.1, 123.4, 121.9,
120.9, 114.4, 105.5, 35.8 (CH2 of pyrazol-5-one).
3-(4-(N,N-dimethylamino)benzylideneamino)-N-(6-
fluorobenzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-
carboxamide (5c)
3-(Benzylideneamino)-N-(6-chlorobenzo[d]thiazol-2-yl)-
4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5f)
Yield 46.3%, mp 158–160 °C; FTIR (KBr, cm−1): 3297
(N–H), 3064 (Ar–H), 1693 (C=O of amide), 1661 (C=O of
pyrazol-5-one), 1610 (C=N), 746 (C–S–C); 1H NMR (400
MHz, DMSO-d6, δ, ppm): 9.97 (brs, 1H, NH), 8.9 (s, 1H,
CH=N of imine), 8.08 (s, 1H, H-7 of benzo[d]thiazole),
7.68–7.65 (d, 2H, J = 8.7 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.30–7.27 (d, 2H, J = 8.2 Hz, Ar–H), 7.15–7.12
(d, 2H, J = 8.3 Hz, Ar–H), 3.31 (s, 2H, –CH2 of pyrazol-5-
one), 2.92 (s, 6H, N(CH3)2). 13C NMR (101 MHz, DMSO-
d6, δ, ppm): 172 (C=O of amide), 168.2 (C=O of pyrazol-
5-one), 163.2 (CH=N of imine), 156.9, 148, 132.7, 129.5,
127.2, 123.2, 121.2, 120.3, 114.2, 106.8, 44.6 (–N(CH3)2),
36.3 (CH2 of pyrazol-5-one). EIMS m/z (relative intensity):
425.3 (M+ + 1, 41%), 379.1 (28%), 293.1 (100%), 230.2
(60%), 195.2 (21%), 157.5 (38%), 124.9 (14%), 91.3
(32%). HRMS (EI): for C20H17FN6O2S [M+ + H], calcd.
425.0011 m/z; found, 425.0614 m/z.
Yield 64.2%, mp 237–239 °C; FTIR (KBr, cm−1): 3279
(N–H), 3044 (Ar–H), 1696 (C=O of amide), 1654 (C=O of
pyrazol-5-one), 1601 (C=N), 733 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.31 (brs, 1H, NH), 8.72 (s, 1H,
CH=N of imine), 7.97 (s, 1H, H-7 of benzo[d]thiazole),
7.66–7.61 (d, 2H, J = 8.7 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.40–7.37 (d, 2H, J = 8.5 Hz, Ar–H), 7.14–7.11
(m, 3H, Ar–H), 3.33 (s, 2H, –CH2 of pyrazol-5-one). 13C
NMR (101 MHz, DMSO-d6, δ, ppm): 174.9 (C=O of
amide), 168.2 (C=O of pyrazol-5-one), 163.1 (CH=N of
imine), 155.8, 147.4, 131, 128.2, 127.5, 126.6, 123.9,
120.4, 115.1, 107.3, 37.1 (CH2 of pyrazol-5-one). EIMS
m/z (relative intensity): 398.1 (M+ + 1, 26%), 321.6 (13%),
308.4 (100%), 229.8 (62%), 165.4 (37%), 124.4 (19%),
91.4 (27%). HRMS (EI): for C18H12ClN5O2S [M+ + H],
calcd. 398.0405 m/z; found, 398.0389 m/z.
3-(4-Chlorobenzylideneamino)-N-(6-fluorobenzo[d]thiazol-
2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5d)
3-(4-Methoxybenzylideneamino)-N-(6-chlorobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5g)
Yield 69.4%, mp 191–194 °C; FTIR (KBr, cm−1): 3252
(N–H), 3080 (Ar–H), 1684 (C=O of amide), 1670 (C=O of
pyrazol-5-one), 1596 (C=N), 729 (C–S–C); 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.09 (brs, 1H, NH), 9.06 (s, 1H,
CH=N of imine), 8.09 (s, 1H, H-7 of benzo[d]thiazole),
7.84–7.82 (d, 2H, J = 8.5 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.38–7.34 (d, 2H, J = 8.7 Hz, Ar–H), 7.08–7.01
(d, 2H, J = 8.5 Hz, Ar–H), 3.50 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 172.3
(C=O of amide), 167.8 (C=O of pyrazol-5-one), 162.9
(CH=N of imine), 157.2, 147.8, 130.9, 128.9, 126.9, 124.3,
122.3, 120.7, 113.4, 107.1, 36.1 (CH2 of pyrazol-5-one).
Yield 50.7%, mp 192–194 °C; FTIR (KBr, cm−1): 3289
(N–H), 3054 (Ar–H), 1672 (C=O of amide), 1645 (C=O of
pyrazol-5-one), 1593 (C=N), 759 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.42 (brs, 1H, NH), 9.06 (s, 1H,
CH=N of imine), 7.95 (s, 1H, H-7 of benzo[d]thiazole),
7.65–7.62 (d, 2H, J = 8.4 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.33–7.30 (d, 2H, J = 8.7 Hz, Ar–H), 7.12–7.08
(d, 2H, J = 8.9 Hz, Ar–H), 3.82 (s, 3H, OCH3), 3.27 (s, 2H,
–CH2 of pyrazol-5-one). 13C NMR (101 MHz, DMSO-d6,
δ, ppm): 174.1 (C=O of amide), 168.3 (C=O of pyrazol-5-
one), 161.8 (CH=N of imine), 155.2, 148.3, 131.8, 127.8,
127.4, 125.4, 123.4, 121.3, 120.9, 115.2, 108.8, 58.3
(OCH3), 37.5 (CH2 of pyrazol-5-one). EIMS m/z (relative
intensity): 428.3 (M+ + 1, 33%), 323.2 (18%), 307.9
(100%), 230.4 (20%), 165.1 (53%), 126.0 (44%), 91.6
(21%). HRMS (EI): for C19H14ClN5O3S [M+ + H], calcd.
428.0510 m/z; found, 428.0532 m/z.
3-(4-Nitrobenzylideneamino)-N-(6-fluorobenzo[d]thiazol-
2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5e)
Yield 60.7%, mp 202–206 °C; FTIR (KBr, cm−1): 3302
(N–H), 3069 (Ar–H), 1689 (C=O of amide), 1652 (C=O of
pyrazol-5-one), 1599 (C=N), 736 (C–S–C). 1H NMR (400

Med Chem Res
3-(4-(N,N-dimethylamino)benzylideneamino)-N-(6-
chlorobenzo[d]thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-
carboxamide (5h)
thiazole), 7.49–7.45 (d, 2H, J = 8.3 Hz, Ar–H), 7.21–7.17
(d, 2H, J = 8.8 Hz, Ar–H), 3.20 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 172.2
(C=O of amide), 168.2 (C=O of pyrazol-5-one), 161.9
(CH=N of imine), 154.4, 148.1, 132.6, 132.3, 130.1, 128.3,
126.5, 123.9, 122.6, 121.1, 114.5, 109.9, 35.6 (CH2 of
pyrazol-5-one).
Yield 43.5%, mp 226–228 °C; FTIR (KBr, cm−1): 3260
(N–H), 3061 (Ar–H), 1685 (C=O of amide), 1649 (C=O of
pyrazol-5-one), 1597 (C=N), 738 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.40 (brs, 1H, NH), 8.94 (s, 1H,
CH=N of imine), 8.01 (s, 1H, H-7 of benzo[d]thiazole),
7.60–7.57 (d, 2H, J = 8.3 Hz,H-4 and H-5 of benzo[d]
thiazole), 7.41–7.39 (d, 2H, J = 8.6 Hz, Ar–H), 7.27–7.24
(d, 2H, J = 8.2 Hz, Ar–H), 3.34 (s, 2H, –CH2 of pyrazol-5-
one), 2.79 (s, 6H, N(CH3)2). 13C NMR (101 MHz, DMSO-
d6, δ, ppm): 172.7 (C=O of amide), 167.4 (C=O of pyr-
azol-5-one), 162.3 (CH=N of imine), 153.1, 147.6, 130.9,
128.8, 127.5, 125.6, 123.2, 121.6, 120.8, 115.4, 107.8, 42.5
(–N(CH3)2), 35.9 (CH2 of pyrazol-5-one). EIMS m/z
(relative intensity): 441.5 (M+ + 1, 19%), 395.2 (32%),
308.1 (100%), 229.7 (72%), 127.2 (24%), 91.1 (30%).
HRMS (EI): for C20H17ClN6O2S [M+ + H], calcd.
441.0925 m/z; found, 441.0815 m/z.
3-(Benzylideneamino)-N-(6-bromobenzo[d]thiazol-2-yl)-
4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5k)
Yield 61.6%, mp 207–209 °C; FTIR (KBr, cm−1): 3304
(N–H), 3082 (Ar–H), 1706 (C=O of amide), 1651 (C=O of
pyrazol-5-one), 1609 (C=N), 759 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.38 (brs, 1H, NH), 8.75 (s, 1H,
CH=N of imine), 8.14 (s, 1H, H-7 of benzo[d]thiazole),
7.72–7.68 (d, 2H, J = 8.4 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.40–7.35 (m, 3H, Ar–H), 7.13–7.10 (d, 2H,
J = 8.7 Hz, Ar–H), 3.44 (s, 2H, –CH2 of pyrazol-5-one).
13C NMR (101 MHz, DMSO-d6, δ, ppm): 172.7 (C=O of
amide), 167.4 (C=O of pyrazol-5-one), 162.7 (CH=N of
imine), 153.5, 147.4, 130.8, 128.9, 127.6, 125.8,
123.3, 121.9, 120.4, 115.8, 108.2, 35.9 (CH2 of pyrazol-5-
one).
3-(4-Chlorobenzylideneamino)-N-(6-chlorobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5i)
3-(4-Methoxybenzylideneamino)-N-(6-bromobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5l)
Yield 58.2%, mp 180–182 °C; FTIR (KBr, cm−1): 3272
(N–H), 3038 (Ar–H), 1697 (C=O of amide), 1653 (C=O of
pyrazol-5-one), 1616 (C=N), 751 (C–S–C). 1H NMR
(400 MHz, DMSO-d6, δ, ppm): 10.18 (brs, 1H, NH), 8.65
(s, 1H, CH=N of imine), 8.11 (s, 1H, H-7 of benzo[d]
thiazole), 7.69–7.66 (d, 2H, J = 8.5 Hz, H-4 and H-5 of
benzo[d]thiazole), 7.56–7.54 (d, 2H, J = 8.6 Hz, Ar–H),
7.15–7.12 (d, 2H, J = 8.4 Hz, Ar–H), 3.24 (s, 2H, –CH2 of
pyrazol-5-one). 13C NMR (101 MHz, DMSO-d6, δ,
ppm): 173.2 (C=O of amide), 167.7 (C=O of pyrazol-5-
one), 161.5 (CH=N of imine), 153.4, 148.9, 135.2, 133.3,
130.9, 128.9, 127.6, 125.2, 122.9, 121.1, 120.1, 114.9, 33.6
(CH2 of pyrazol-5-one). EIMS m/z (relative intensity):
433.6 (M+ + 1, 40%), 395.1 (21%), 308.5 (100%), 230.2
(62%), 123.2 (24%), 91.2 (35%). HRMS (EI): for
C18H11Cl2N5O2S [M+ + H], calcd. 433.5010 m/z; found,
433.5025 m/z.
Yield 59.3%, mp 232–235 °C; FTIR (KBr, cm−1): 3281
(N–H), 3047 (Ar–H), 1678 (C=O of amide), 1646 (C=O of
pyrazol-5-one), 1585 (C=N), 735 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.26 (brs, 1H, NH), 8.93 (s, 1H,
CH=N of imine), 7.91 (s, 1H, H-7 of benzo[d]thiazole),
7.57–7.54 (d, 2H, J = 8.6 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.22–7.19 (d, 2H, J = 8.5 Hz, Ar–H), 6.98–6.95
(d, 2H, J = 8.3 Hz, Ar–H), 3.80 (s, 3H, OCH3), 3.43 (s, 2H,
–CH2 of pyrazol-5-one). 13C NMR (101 MHz, DMSO-d6,
δ, ppm): 174.7 (C=O of amide), 167.3 (C=O of pyrazol-5-
one), 160.2 (CH=N of imine), 154.2, 147.4, 134.9, 131.2,
127.9, 125.1, 119.8, 114.2, 107.7, 55.4 (OCH3), 39.4 (CH2
of pyrazol-5-one). EIMS m/z (relative intensity): 473.2 (M+
+ 2, 14%), 471.2 (M+, 19%), 394.3 (27%), 352.4 (100%),
257.0 (51%), 167.2 (12%), 124.5 (43%), 91.5 (15%).
HRMS (EI): for C19H14BrN5O3S [M+], calcd. 471.0710
m/z; found, 471.0635 m/z.
3-(4-Nitrobenzylideneamino)-N-(6-chlorobenzo[d]thiazol-
2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5j)
Yield 56.8%, mp 213–216 °C; FTIR (KBr, cm−1): 3264
(N–H), 3026 (Ar–H), 1690 (C=O of amide), 1642 (C=O of
pyrazol-5-one), 1586 (C=N), 740 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.32 (brs, 1H, NH), 8.68 (s, 1H,
CH=N of imine), 8.07 (s, 1H, H-7 of benzo[d]thiazole),
7.87–7.84 (d, 2H, J = 8.3 Hz, H-4 and H-5 of benzo[d]
3-(4-Chlorobenzylideneamino)-N-(6-bromobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5m)
Yield 63.4%, mp 209–211°C; FTIR (KBr, cm−1): 3291
(N–H), 3079 (Ar–H), 1685 (C=O of amide), 1652 (C=O of

Med Chem Res
pyrazol-5-one), 1600 (C=N), 748 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm):10.35 (brs, 1H, NH), 8.71 (s, 1H,
CH=N of imine), 8.1 (s, 1H, H-7 of benzo[d]thiazole),
7.79–7.76 (d, 2H, J = 8.6 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.44–7.40 (d, 2H, J = 8.6 Hz, Ar–H), 7.17–7.13
(d, 2H, J = 8.5 Hz, Ar–H), 3.42 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 174.6
(C=O of amide), 168.6 (C=O of pyrazol-5-one), 162.3
(CH=N of imine), 155.4, 148.7, 131.2, 128.7, 127.2, 125.6,
123.7, 121.2, 115.2, 107.7, 36.2 (CH2 of pyrazol-5-one).
HRMS (EI): for C18H11BrClN5O2S [M+], calcd. 475.7120
m/z; found, 475.7365 m/z.
3-(4-Methoxybenzylideneamino)-N-(6-nitrobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5p)
Yield 53.2%, mp 212–214 °C; FTIR (KBr, cm−1): 3299
(N–H), 3036 (Ar–H), 1691 (C=O of amide), 1658 (C=O of
pyrazol-5-one), 1596 (C=N), 758 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.37 (brs, 1H, NH), 8.84 (s, 1H,
CH=N of imine), 7.90 (s, 1H, H-7 of benzo[d]thiazole),
7.71–7.68 (d, 2H, J = 8.7 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.34–7.32 (d, 2H, J = 8.4 Hz, Ar–H), 6.98–6.96
(d, 2H, J = 8.7 Hz, Ar–H), 3.86 (s, 3H, OCH3), 3.35 (s, 2H,
–CH2 of pyrazol-5-one). 13C NMR (101 MHz, DMSO-d6,
δ, ppm): 173.8 (C=O of amide), 166.7 (C=O of pyrazol-5-
one), 161.4 (CH=N of imine), 154.9, 145.5, 133.6, 132.2,
128.2, 126.3, 125.8, 120.5, 114.9, 105.3, 55.7 (OCH3), 36.2
(CH2 of pyrazol-5-one). EIMS m/z (relative intensity):
438.1 (M+, 18%), 361.4 (15%), 319.5 (100%), 251.4
(31%), 180.6 (54%), 124.8 (23%), 91.6 (22%). HRMS (EI):
for C19H14N6O5S [M+], calcd. 438.0675 m/z; found,
438.0727 m/z.
3-(4-Nitrobenzylideneamino)-N-(6-bromobenzo[d]thiazol-
2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide (5n)
Yield 60.2%, mp 218–220 °C; FTIR (KBr, cm−1): 3287
(N–H), 3058 (Ar–H), 1677 (C=O of amide), 1644 (C=O of
pyrazol-5-one), 1598 (C=N), 732 (C–S–C).1H NMR (400
MHz, DMSO-d6, δ, ppm):10.22 (brs, 1H, NH), 8.95 (s, 1H,
CH=N of imine), 8.03 (s, 1H, H-7 of benzo[d]thiazole),
7.83–7.80 (d, 2H, J = 8.3 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.43–7.41 (d, 2H, J = 8.4 Hz, Ar–H), 7.22–7.19
(d, 2H, J = 8.6 Hz, Ar–H), 3.39 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 174.4
(C=O of amide), 167.9 (C=O of pyrazol-5-one), 163.1
(CH=N of imine), 154.2, 147.1, 131.3, 128.2, 127.4, 125.4,
123.3, 120.3, 114.5, 106.6, 37.5 (CH2 of pyrazol-5-one).
HRMS (EI): for C18H11BrN6O4S [M+], calcd. 486.2090
m/z; found, 486.2174 m/z.
3-(4-Chlorobenzylideneamino)-4,5-dihydro-N-(6-
nitrobenzo[d]thiazol-2-yl)-5-oxo-pyrazole-1-carboxamide
(5q)
Yield 63.8%, mp 206–209 °C; FTIR (KBr, cm−1): 3292
(N–H), 3042 (Ar–H), 1681 (C=O of amide), 1650 (C=O of
pyrazol-5-one), 1602 (C=N), 754 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm):10.32 (brs, 1H, NH), 8.98 (s, 1H,
CH=N of imine), 7.93 (s, 1H, H-7 of benzo[d]thiazole),
7.63–7.60 (d, 2H, J = 8.3 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.340–7.36 (d, 2H, J = 8.5 Hz, Ar–H), 7.12–7.08
(d, 2H, J = 8.3 Hz, Ar–H), 3.37 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 172.1
(C=O of amide), 167.3 (C=O of pyrazol-5-one), 162.8
(CH=N of imine), 153.8, 147.9, 131.3, 128.4, 127.2, 124.9,
123.2, 120.8, 119.7, 115.4, 35.1 (CH2 of pyrazol-5-one).
HRMS (EI): for C18H11ClN6O4S [M+], calcd. 443.8340
m/z; found, 443.8408 m/z.
3-(Benzylideneamino)-N-(6-nitrobenzo[d]thiazol-2-yl)-4,5-
dihydro-5-oxo-pyrazole-1-carboxamide (5o)
Yield 65.5%, mp 179–182 °C; FTIR (KBr, cm−1): 3294
(N–H), 3043 (Ar–H), 1702 (C=O of amide), 1660 (C=O of
pyrazol-5-one), 1603 (C=N), 746 (C–S–C). 1H NMR
(400 MHz, DMSO-d6, δ, ppm): 10.20 (brs, 1H, NH), 9.04
(s, 1H, CH=N of imine), 8.02 (s, 1H, H-7 of benzo[d]
thiazole), 7.62–7.59 (d, 2H, J = 8.2 Hz, H-4 and H-5 of
benzo[d]thiazole), 7.41–7.37 (m, 3H, Ar–H), 7.09–7.07
(d, 2H, J = 8.7 Hz, Ar–H), 3.44 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 171.6
(C=O of amide), 166.9 (C=O of pyrazol-5-one), 160.7
(CH=N of imine), 155.8, 146.9, 132.3, 131.5, 128.4, 127.6,
125.7, 122.5, 120.1, 114.4, 106, 35.1 (CH2 of pyrazol-
5-one). EIMS m/z (relative intensity): 408.6 (M+, 25%),
360.8 (13%), 319.4 (100%), 258.2 (28%), 181.3
(16%), 125.2 (47%), 91.7 (34%). HRMS (EI): for
C18H12N6O4S [M+], calcd. 408.0544 m/z; found, 408.
0618 m/z.
3-(4-Nitrobenzylideneamino)-4,5-dihydro-N-(6-nitrobenzo
[d]thiazol-2-yl)-5-oxo-pyrazole-1-carboxamide (5r)
Yield 66.3%, mp 198–200 °C; FTIR (KBr, cm−1): 3295
(N–H), 3033 (Ar–H), 1683 (C=O of amide), 1652 (C=O of
pyrazol-5-one), 1597 (C=N), 740 (C–S–C). 1H NMR (400
MHz, DMSO-d6, δ, ppm): 10.36 (brs, 1H, NH), 8.95 (s, 1H,
CH=N of imine), 7.97 (s, 1H, H-7 of benzo[d]thiazole),
7.62–7.59 (d, 2H, J = 8.4 Hz, H-4 and H-5 of benzo[d]
thiazole), 7.41–7.38 (d, 2H, J = 8.4 Hz, Ar–H), 7.19–7.16
(d, 2H, J = 8.2 Hz, Ar–H), 3.39 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 174.4

Med Chem Res
Axford LC, Agarwal PK, Anderson KH, Andrau LN, Barker S,
Bennett JM, Blair M, Collins I, Atherall J, Czaplewski LG,
Davies DT (2013) Design, synthesis and biological evaluation of
∝-substituted isonipecotic acid benzo[d]thiazole analogues as
potent bacterial type II topoisomerase inhibitors. Bioorg Med
Chem Lett 23:6598–6603
Brewster RQ, Dains FB (1936) Studies on the thiazoles obtained by
direct thiocyanogenation. J Am Chem Soc 58:1364–1366
Castagnolo D, De Logu A, Radi M, Bechi B, Magnani FMM,
Supino S, Meleddu R, Chisu L, Botta M (2008) Synthesis,
biological evaluation and SAR study of novel pyrazole analogues
as inhibitors of M. tuberculosis. Bioorg Med Chem
16:8587–8591
(C=O of amide), 168.3 (C=O of pyrazol-5-one), 163.7
(CH=N of imine), 155.5, 147.3, 132.2, 128.6, 127.4, 125.2,
123.3, 120.8, 115.3, 104.8, 35.7 (CH2 of pyrazol-5-one).
HRMS (EI): for C18H11N7O6S [M+], calcd. 453.3509 m/z;
found, 453.4125 m/z.
3-(4-Hydroxybenzylideneamino)-N-(6-nitrobenzo[d]
thiazol-2-yl)-4,5-dihydro-5-oxo-pyrazole-1-carboxamide
(5s)
Yield 55.6%, mp 218–220 °C; FTIR (KBr, cm−1): 3440 (br,
O–H), 3263 (N–H), 3075 (Ar–H), 1711 (C=O of amide),
1662 (C=O of pyrazol-5-one), 1608 (C=N), 747 (C–S–C).
1H NMR (400 MHz, DMSO-d6, δ, ppm): 10.35 (brs, 1H,
NH), 9.02 (s, 1H, CH=N of imine), 7.98 (s, 1H, H-7 of
benzo[d]thiazole), 7.64–7.60 (d, 2H, J = 8.5 Hz, H-4 and
H-5 of benzo[d]thiazole), 7.46–7.43 (d, 2H, J = 8.4 Hz,
Ar–H), 7.13–7.11 (d, 2H, J = 8.3 Hz, Ar–H), 5.54 (s, 1H,
OH, D2O exchangeable), 3.56 (s, 2H, –CH2 of pyrazol-5-
one). 13C NMR (101 MHz, DMSO-d6, δ, ppm): 174.8
(C=O of amide), 165.3 (C=O of pyrazol-5-one), 160.3
(CH=N of imine), 159.2, 155.1, 147.9, 132.5, 131.2, 129.2,
126.8, 125.1, 123.7, 121.4, 115.1, 106.6, 35.7 (CH2 of
pyrazol-5-one). EIMS m/z (relative intensity): 424.4 (M+,
27%), 408.2 (47%), 363.6 (15%), 319.5 (100%), 249.1
(12%), 180.3 (30%), 125.2 (25%), 91.6 (41%). HRMS (EI):
for C18H12N6O5S [M+], calcd. 424.0600 m/z; found,
424.0613 m/z.
Cho Y, Ioerger TR, Sacchettini JC (2008) Discovery of novel nitro-
benzothiazole inhibitors for Mycobacterium tuberculosis ATP
phosphoribosyltransferase (HisG) through virtual screening. J
Med Chem 51:5984–5992
CLSI. (2015) Methods for Dilution Antimicrobial Susceptibility Tests
for Bacteria That Grow Aerobically; Approved Standard—10th
Edition. CLSI document M07-A10. Wayne, PA: Clinical and
Laboratory Standards Institute
Cohen ML (2000) Changing patterns of infectious disease. Nature
406:762–767
Coleman K (2004) Recent advances in the treatment of gram-positive
infections. Drug Discov Today Ther Strateg 1:455–460
Cramer RD, Patterson DE, Bunce JD (1988) Comparative molecular
field analysis (CoMFA). 1. Effect of shape on binding of steroids
to carrier proteins. J Am Chem Soc 110:5959–5967
da Silva CM, da Silva DL, Modolo LV, Alves RB, de Resende MA,
Martins CVB, Fatima A (2011) Schiff bases: a short review of
their antimicrobial activities. J Adv Res 2:1–8
Dogruer DS, Unlu S, Sahin MF, Yesilada E (1998) Antinociceptive
and anti inflammatory activity of some new (2-benzoxazolon-3-yl
and 2-benzothiazolon-3-yl)acetic acid derivatives. Il Farmaco
53:80–84
El-Hawash SA, Badawey SA, El-Ashmawey IM (2006) Non-
steroidalanti-inflammatory agents-part
2
antiinflammatory,
Acknowledgements The authors are thankful to KLE University’s
College of Pharmacy, Hubballi (India) and College of Health Sciences,
University of KwaZulu-Natal (UKZN), Durban (South Africa), for
providing necessary facilities to carry out this research work. We
sincerely express our gratitude to Dr.L.V.G.Nargund, Director, Nar-
gund College of Pharmacy, Bangalore (India), for his scientific
encouragement. Authors also express special thanks to Sapco
Laboratories Pvt. Ltd., Chennai (India) for providing gift samples of
Ampicillin and Gatifloxacin. Authors are grateful to NMR center,
School of Chemistry and Physics, UKZN (South Africa) and The
Director, SAIF, Punjab University (India) for providing spectral ana-
lytical data.
analgesic and antipyretic activity of some substituted 3-pyrazolin-
5-ones and 1,2,4,5,6,7-3H-hexahydroindazol-3-ones. Eur J Med
Chem 41:155–165
Gaussian 09 (2009) Revision B.01. Gaussian, Inc, Wallingford CT
Genin MJ, Allwine DA, Anderson DJ, Barbachyn MR, Emmert DE
(2000) Substituent effects on the antibacterial activity of nitrogen
−carbon-linked (Azolylphenyl) oxazolidinones with expanded
activity against the fastidious Gram-negative organisms H.
influenzae and M. catarrhalis. J Med Chem 43:953–970
Glide (2013) Version 5.9 Schrödinger. LLC, New York, NY
Hampannavar GA, Karpoormath R, Palkar MB, Shaikh MS, Chan-
drasekaran B (2016) Dehydrozingerone inspired styryl hydrazine
thiazole hybrids as promising class of anti-mycobacterial agents:
design, synthesis and in vitro characterization. ACS Med Chem
Lett 7:686–691
Compliance with ethical standards
Kitagawa H, Ozawa T, Takahata S, Iida M (2007) Phenylimidazole
derivatives as new inhibitors of bacterial enoyl-ACP reductase
FabK. Bioorg Med Chem Lett 17:4982–4986
Conflict of interest The authors declare that they have no competing
interests.
Klebe G, Abraham U (1999) Comparative molecular similarity index
analysis (CoMSIA) to study hydrogen-bonding properties and to
score combinatorial libraries. J Comput Aided Mol Des 13
(1999):1–10
Makawana JA, Sun J, Zhu HL (2013) Schiff’s base derivatives bearing
nitroimidazole moiety: new class of antibacterial, anticancer
agents and potential EGFR tyrosine kinase inhibitors. Bioorg
Med Chem Lett 23:6264–6268
References
Abbanat D, Macielag M, Bush K (2003) Novel antibacterial agents for
the treatment of serious gram-positive infections. Expert Opin
Investig Drugs 12:379–399
Aragade P, Palkar MB, Ronad PM, Satyanarayana D (2013) Cou-
marinylpyrazole derivatives of INH: promising antimycobacterial
agents. Med Chem Res 22:2279–2283
Makhija MT, Kasliwal RT, Kulkarni VM, Neamati N (2004) De novo
design and synthesis of HIV-1 integrase inhibitors. Bioorg Med
Chem 12:2317–2333

Med Chem Res
Manjula SN, Noolvi MN, Parihar VK, Reddy SAM, Ramani V, Gadad
AK (2009) Synthesis and antitumor activity of optically active
thiourea and their 2-amino benzothiazole derivatives: novel class
of anticancer agents. Eur J Med Chem 44:2923–2929
Markovic V, Eric S, Stanojkovic T, Gligorijevic N, Aranđelovic S,
Todorovic N (2011) Antiproliferative activity and QSAR studies
of a series of new 4-aminomethylidene derivatives of some
pyrazol-5-ones. Bioorg Med Chem Lett 21:4416–4421
McFarland J (1907) Thenephelometer: an instrument for estimating the
number of bacteria in suspensions used for calculating the
opsonic index and for vaccines. J Am Med Assoc 14:1176–1178
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
evaluation of in vitro antimalarial activity. Eur J Med Chem
30:503–508
Ronkin SM, Badia M, Bellon S, GrillotAL, Gross CH, Grossman TH,
Stamos, Trudeau M, Charifson PS, Wei Y (2010) Discovery of
pyrazolthiazoles as novel and potent inhibitors of bacterial gyr-
ase. Bioorg Med Chem Lett 20:2828–2831
Shaikh MS, Palkar MB, Patel HM, Rane RA, Alwan WS, Shaikh MM,
Shaikh IM, Hampannavar GA, Karpoormath R (2014) Design
and synthesis of novel carbazolo-thiazoles by molecular hybri-
dization approach as anti-mycobacterial agents. RSC Adv
4:62308–62320
Sriram D, Yogeeswari P, Myneedu NS, Saraswat V (2006) Abacavir
prodrugs: microwave-assisted synthesis and their evaluation of
anti-HIV activities. Bioorg Med Chem Lett 16:2127–2129
Stevens MG, Olsen SC (1993) Comparative analysis of using MTT
and XTT in colorimetric assays for quantitating bovine neutrophil
bactericidal activity. J Immunol Methods 157:225–231
Sun J, Lv PC, Yin Y, Yuan RJ, Ma J, Zhu HL (2013) Synthesis,
structure and antibacterial activity of potent DNA gyrase inhibi-
Mruthyunjayaswamy BHM, Basavarajaiah SM (2009) Synthesis and
antimicrobial activity of novel ethyl-5-(ethoxycarbonyl)-4-
methylthiazol-2-yl-carbamate compounds. Indian
J
Chem
48B:1274–1278
Palkar MB, Noolvi MN, Sankangoud R, Maddi V, Gadad AK, Nar-
gund LVG (2010) Synthesis and antibacterial activity of a novel
series of 2,3-diaryl substituted imidazo(2,1-b) benzo[d]thiazole-
derivatives. Archiv der Pharmazie Chem Life Sci 343:353–359
Palkar MB, Jalalpure SS, Rane RA, Patel HM, Shaikh MS, Ham-
pannavar GA, Alwan WS, Bolakatti GS, Karpoormath R (2015)
Novel series of coumarinyl substituted-thiazolidin-2,4-dione
analogs as anticancer agents: design, synthesis, spectral studies
and cytotoxicity evaluation. Anticancer Agents Med Chem 15
(8):970–979
tors:
N-9-Benzoyl-3-(4-bromophenyl)1H-pyrazole-5-carbohy-
drazide derivatives. PLoS One 8:69751
Sztanke K, Maziarka A, Sztanke M (2013) An insight into synthetic
Schiff bases revealing antiproliferative activities in vitro. Bioorg
Med Chem 21:3648–3666
Tanitame A, Oyamada Y, Ofuji K, Fujimoto M, Iwai N (2004)
Synthesis and antibacterial activity of a novel series of potent
DNA gyrase inhibitors: pyrazole derivatives. J Med Chem
47:3693–3696
Parashar B, Sharma VK, Jain A, Bharadwaj S (2012) Synthesis and
pharmacological properties of some novel pyrazolidine and pyr-
azole derivatives. Med Chem Res 21:1692–1699
Patel RV, Patel PK, Kumari P, Rajani DP, Chikhalia KH (2012)
Synthesis of benzimidazolyl-1,3,4-oxadiazol-2ylthio-N-phenyl
(benzothiazolyl)acetamides as antibacterial, antifungal and anti-
tuberculosis agents. Eur J Med Chem 53:41–51
Telvekar VN, Bairwa VK, Satardekar K, Bellubi A (2012) Novel
2-(2-(4-aryloxy benzylidene) hydrazinyl)benzothiazole deriva-
tives as anti-tubercular agents. Bioorg Med Chem Lett
22:649–652
Thompson LA, Ellman JA (1996) Synthesis and applications of small
molecule libraries. Chem Rev 96:555–600
Viegas-Jr C, Danuello C, da Silva BV, Barreiro EJ, Fraga CA (2007)
Molecular hybridization: a useful tool in the design of new drug
prototypes. Curr Med Chem 14:1829–1852
Rathelot P, Vanelle P, Gasquet M, Delmas F, Crozet MP, Timon DP
(1995) Synthesis of novel functionalized 5-nitroisoquinolines and