One pot synthesis of thiazolo[2,3-b]dihydropyrimidinone possessing pyrazole moiety and evaluation of their anti-inflammatory and antimicrobial activities

Article abstract of DOI:10.1007/s00044-017-2058-8

A series of pyrazole integrated thiazolo[2,3-b]dihydropyrimidinone derivatives were synthesized as dual anti-inflammatory and antimicrobial agents. Among the compounds studied, 3-fluoro-4-methylphenyl analogues (3a, 3e, and 3i) are considered to be promising leads for novel anti-inflammatory agents compared with the standard drug. The superior antimicrobial property of the compounds 3a, 3b, and 3d indicates that 3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazole substitution is a favourable site for high activity. Molecular docking studies were carried out in order to predict the hypothetical binding mode of these compounds to the COX-2 isoenzyme. The results of the present study suggest that 1,3-diaryl pyrazole substitution on thiazolo[2,3-b]dihydropyrimidinone derivatives might potentially constitute a novel class of anti-inflammatory agents with antimicrobial property and could be an interesting approach for the design of new selective COX-2 inhibitory agents.

Full text of DOI:10.1007/s00044-017-2058-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2058-8
ORIGINAL RESEARCH
One pot synthesis of thiazolo[2,3-b]dihydropyrimidinone
possessing pyrazole moiety and evaluation of their anti-
inflammatory and antimicrobial activities
Shivapura Viveka1 Dinesha1 Gundibasappa Karikannar Nagaraja1
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Prasanna Shama2 Guru Basavarajaswamy3 K. Poornachandra Rao4
Marikunte Yanjarappa Sreenivasa4
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Received: 24 September 2015 / Accepted: 30 August 2017
© Springer Science+Business Media, LLC 2017
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Abstract A series of pyrazole integrated thiazolo[2,3-b]
dihydropyrimidinone derivatives were synthesized as dual
anti-inflammatory and antimicrobial agents. Among the
compounds studied, 3-fluoro-4-methylphenyl analogues
(3a, 3e, and 3i) are considered to be promising leads for
novel anti-inflammatory agents compared with the standard
drug. The superior antimicrobial property of the compounds
3a, 3b, and 3d indicates that 3-(3,4-dichlorophenyl)-1-
phenyl-1H-pyrazole substitution is a favourable site for high
activity. Molecular docking studies were carried out in
order to predict the hypothetical binding mode of these
compounds to the COX-2 isoenzyme. The results of the
present study suggest that 1,3-diaryl pyrazole substitution
on thiazolo[2,3-b]dihydropyrimidinone derivatives might
potentially constitute a novel class of anti-inflammatory
agents with antimicrobial property and could be an inter-
esting approach for the design of new selective COX-2
inhibitory agents.
Keywords Pyrazole Thiazolo[2 3-b]
●
●
dihydropyrimidinone Anti-inflammatory Antimicrobial
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are com-
monly prescribed for the treatment of acute and chronic
inflammation, pain, and fever (Allavena et al. 2008; Sorbera
et al. 2001; Palomer et al. 2002). Most currently used
NSAIDs have limitations for therapeutic use since they
cause gastrointestinal and renal side effects, which are
inseparable from their pharmacological activities (Smith
et al. 2000; Dogne et al. 2005). In addition, inflammation is
known not only as a symptom of a great deal of common
diseases, but also as an early phase of some life-threatening
diseases like cancer, heart vascular diseases, Alzheimer’s
dementia, etc (Dillingham et al. 2002; Dekhane et al. 2011;
Selkoe 2001). Therefore, investigations for the development
of safer and more effective anti-inflammatory agents are of
great interest for medicinal researchers. The rapidly
increasing incidence of antimicrobial resistance also repre-
sents a serious problem worldwide. Therefore, the devel-
opment of new and different antimicrobial drugs is a very
important objective and many research programmes are
directed towards the design of new antimicrobial agents (Li
and Ma 2015; Beekmann et al. 2005; Chua et al. 2008). This
situation highlights the need to identify lead molecules from
newer classes of compounds, in order to develop novel and
effective NSAIDs and antimicrobial agents in the future.
Among various classes of chemical moieties, pyrazole
and its derivatives represent important building blocks in
drug research (Kumar et al. 2015, 2013a). In addition, they
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2058-8) contains supplementary
material, which is available to authorized users.
* Gundibasappa Karikannar Nagaraja
nagarajagk@gmail.com
1
Department of Studies in Chemistry, Mangalore University,
Mangalagangotri 574199, Karnataka, India
2
Department of Pharmacology, N.G.S.M. Institute of
Pharmaceutical Sciences, Paneer Deralakatte, Mangalore 574160,
Karnataka, India
3
Acharya & B.M. Reddy College of Pharmacy, Soldevanahalli,
Chikkabanavara Post, Bangalore 560 090, Karnataka, India
4
Department of Microbiology, Manasagangothri, Mysore
University, Mysore 570006, Karnataka, India

Med Chem Res
are of interest in their own right due to their pharmacolo-
gical properties. The changes in their structure offer a high
degree of diversity, which is useful for the development of
new therapeutic agents (Fustero et al. 2011, 2009). Anti-
pyrine is the first pyrazoline derivative used as an anti-
inflammatory agent. However, its use is restricted due to its
GI side effect (Sivakumar et al. 2014). The literature survey
reveals that many pyrazole and pyrazoline derivatives have
been used for clinical application as NSAIDs, i.e., Phe-
nylbutazone, Celecoxib, and Deracoxib (Fig. 1) (Capone
et al. 2003; Kumar et al. 2013b). Furthermore, much
attention was given to pyrazoles as antimicrobial agents
after the discovery of the natural pyrazole C-glycosde
pyrazofurin (Fig. 2), which demonstrated a broad spectrum
of antimicrobial activity (Comber et al. 1991). The dis-
covery of a dual anti-inflammatory-antimicrobial agent with
potential activity and fewer adverse effects is a priority of
the current global research, and consequently has occupied
our prime interest in recent years (Sivakumar et al. 2014,
Keche et al. 2012; Li and Zhao 2014; Bekhit et al. 2010).
Pyrimidines have a long distinguished history as
important constituents of nucleic acids to their current use in
Fig. 1 Structures of some known pyrazole NSAIDs
Fig. 2 Structures of antibiotic pyrazofurin and structures of some reported thiazolo pyrimidinone (a) and pyrazole integrated thiazolo pyr-
imidinone derivatives (b and c) with anti-inflammatory and antimicrobial activity

Med Chem Res
chemotherapy for AIDS (Nagarapu et al. 2013). One pos-
sible reason for their activity is the presence of the pyr-
imidine base in thymine, cytosine, and uracil, which are
essential building blocks of nucleic acids, DNA, and RNA
(Sondhi et al. 2005). In addition, thiazolopyrimidines are
pharmacological scaffolds that represent a wide range of
biological activities such as antimicrobial, anti-inflamma-
tory, analgesic, antioxidant, insecticides, antidepressant,
antiviral, antitumour, calcium channel blocker, etc
(Mohamed et al. 2010a, b; Abu-Hashem et al. 2011; Fatima
et al. 2012; Khalifa et al. 2015). The reported significance of
such synthons (A, Fig. 2) generated interest in the designing
and synthesis of new thiazolo[3,2-a]pyrimidine-6-carbox-
ylate derivatives (Ashok et al. 2007).
However, only a few approaches can be found in lit-
erature to synthesize these compounds. The major limita-
tions of these reported methods are long reaction time, more
solvent, and less yield due to multistep reaction condition.
Multi-component reactions (MCRs) have emerged as an
efficient and powerful tool in modern synthetic organic
chemistry in which three or more different starting materials
react to give a final product in a one pot procedure (Kiyani
and Ghorbani 2015; Viveka et al. 2012, 2015a, d, 2014).
Some of the reported pyrazole integrated thiazolo pyr-
imidinone derivatives (B and C) (Fig. 2) showed pro-
nounced anti-inflammatory and antimicrobial activities
(Bekhit et al. 2003). Prompted by these observations and in
continuation of our research work related to pyrazole
chemistry (Viveka et al. 2012, 2015a, b, c, d), we hereby
report the synthesis of a new series of pyrazole embodied
thiazolopyrimidinone in a single framework. All the com-
pounds were synthesized via MCR approach and their anti-
inflammatory and antimicrobial activities was evaluated.
compounds has been reported in our previous publication
(Viveka et al. 2012, 2015d). The structures of these com-
pounds were established on the basis of elemental analysis,
1
IR, H NMR, 13C NMR, and mass spectral studies. The
synthetic strategy to prepare the targeted compounds 3(a–p)
is illustrated in Scheme 1.
The FTIR spectrum of pyrazoline analogue 3e showed
absorption band at 1737 cm−1 for carbonyl stretching. Also,
the appearance of the absorption band at 1682 and 1591
cm−1 due to C=N, C=C stretching further supported the
1
proposed structure. The H NMR spectrum of 3e, appearing
as a triplet and quartet at δ 1.10 and 4.02 ppm, clearly con-
firmed the presence of the ethyl carboxylate group. In addi-
tion, two CH3 protons appearing as singlets at δ 2.17 and
2.38 ppm, supported the proposed structure. A singlet at δ
5.97 ppm confirmed the presence of proton in the pyrimidine
ring. Pyrazole H-5 proton appeared as a singlet at δ 8.79 ppm
and all the other aromatic protons resonated as a complex
multiplet in the region of δ 6.99–8.02 ppm. Additional evi-
dence was obtained by its 13C NMR spectrum, where two
characteristic signals appeared at δ 14.39 and δ 60.64 ppm
corresponding to the OCH2CH3 carbons of the proposed
structure. The carbons of two other methyl groups appeared
at δ 14.10 and 22.81 ppm and peaked at δ 54.98 ppm cor-
responding to the CH of the pyrimidine ring. Furthermore,
the 13C NMR spectrum showed two characteristic peaks at δ
165.3 and 164.5 ppm, which was attributed to the ester and
cyclic carbonyl group of the target compound. The absence
of C=S prominent peak at δ 174.5 ppm in the 13C NMR
spectrum gave strong evidence to the formation of thiazolo
[2,3-b]dihydropyrimidinone derivative 3e. Also, the dis-
appearance of the NH absorption peak (3100–3400 cm−1) in
the IR spectrum and the absence of the two singlets at higher
δ values corresponding to the –NH of 1(a–d) clearly estab-
lished the conversion of 1(a–d) to 3(a–p). The mass spectra
of all the final derivatives showed comparable molecular ion
peak with respect to the molecular formula. Similarly, all the
target compounds were also well-characterized by FTIR, 1H
NMR, 13C NMR, and mass spectral studies. The physical
data of the target compounds is given in Table 1.
Results and discussion
Chemistry
The key intermediates dihydropyrimidinones (DHPMs) 1
(a–d) were obtained by the Biginelli reaction involving
substituted benzaldehyde, ethyl acetoacetate, and thiourea
in the presence of HCl catalyst according to the procedure
reported in the literature (Patel and Patel 2013). The ver-
satile Vilsmeier reaction was employed to synthesize a
series of substituted 4-formyl pyrazole 2(a–d) in good
yields from the corresponding phenylhydrazone, according
to our previous reports (Viveka et al. 2012, 2015a, b, d).
These highly activated intermediates, 1(a–d) and 2(a–d),
were then reacted with monochloroacetic acid, anhydrous
sodium acetate in acetic acid-acetic anhydride medium,
which resulted in the formation of the target compounds 3
(a–p). The synthesis and characterization of such
Pharmacological screening
Anti-inflammatory activity
The anti-inflammatory screening of the synthesized com-
pounds was carried out using the carrageenan-induced paw
edema method of inflammation in rats at successive inter-
vals of 1, 2, and 4 h compared with that of the standard drug
Indomethacin. The results of the anti-inflammatory analysis
are reported in Table 2. The results are expressed as mean
SEM. The percentage inhibition for the synthesized com-
pounds as well as the standard drug was determined. The

Med Chem Res
Scheme 1 Synthetic pathway for the synthesis of the targeted compounds 3(a–p)
Table 1 Characterization data of compounds 3(a–p)
Compound 3(a–p)
R
R1 R2 Yield (%) Melting point Mol.formula (Mol. wt)
Elemental analysis (%) calculated
(found)
C
H
N
3a
3b
3c
3d
3e
3f
3F-4-CH3
Cl Cl 91
127–130
98–101
C33H25Cl2FN4O3S (647.54) 61.21 (61.18) 3.89 (3.92) 8.65 (8.66)
C34H28Cl2N4O5S (675.58) 60.45 (60.49) 4.18 (4.19) 8.29 (8.26)
2,5-(OCH3)2 Cl Cl 78
H
Cl Cl 67
Cl Cl 88
135–137
112–114
166–167
105–107
134–136
167–169
90–93
C32H24Cl2N4O3S (615.52) 62.44 (62.41) 3.93 (3.95) 9.10 (9.06)
4-OCH3
3F-4-CH3
2,5-(OCH3)2
H
C33H26Cl2N4O4S (645.55) 61.40 (61.34) 4.06 (4.01) 8.68 (8.70)
F
F
72
74
81
73
59
72
70
49
66
75
86
76
C33H25F3N4O3S (614.63)
C34H28F2N4O5S (642.67)
C32H24F2N4O3S (582.61)
C33H26F2N4O4S (612.64)
64.69 (64.72) 4.10 (4.06) 9.12 (9.06)
63.54 (63.49) 4.39 (4.41) 8.72 (8.69)
65.97 (66.01) 4.15 (4.10) 9.62 (9.59)
64.70 (64.78) 4.28 (4.23) 9.15 (9.14)
F
F
3g
3h
3i
F
F
4-OCH3
3F-4-CH3
F
F
Cl
H
H
H
H
H
H
H
H
C33H26ClFN4O3S (613.10) 64.65 (64.65) 4.27 (4.31) 9.14 (9.11)
3j
2,5-(OCH3)2 Cl
103–105
118–120
182–185
134–136
99–101
C33H29 ClN4O5S (641.13)
C32H25ClN4O3S (581.08)
63.69 (63.68) 4.56 (4.63) 8.74 (8.79)
66.14 (66.18) 4.34 (4.33) 9.64 (9.69)
64.86 (64.85) 4.45 (4.40) 9.17 (9.16)
66.43 (66.41) 4.39 (4.40) 9.39 (9.40)
65.37 (65.40) 4.68 (4.70) 8.97 (8.99)
68.07 (68.01) 4.46 (4.48) 9.92 (9.89)
66.65 (66.63) 4.58 (4.60) 9.42 (9.40)
3k
3l
H
Cl
Cl
F
4-OCH3
3F-4-CH3
2,5-(OCH3)2
H
C33H27ClN4O4S (611.10)
C33H26F2N4O3S (596.64)
C34H29FN4O5S (624.68)
3m
3n
3o
3p
F
F
182–185
163–165
C32H25FN4O3S (564.62)
4-OCH3
F
C33H27FN4O4S (594.65)

Med Chem Res
tested compounds exhibited moderate anti-inflammatory
within 2 h; the activity increased and reached peak level at
4 h and declined after 4 h.
Antimicrobial activity
The synthesized compounds 3(a–p) were also evaluated for
their in vitro antibacterial and antifungal activities against
Gram-positive organism (Staphylococcus aureus), Gram-
negative organisms (Klebsiella pneumoniae, Pseudomonas
aerogenosa, and Escherichia coli), and fungi (Aspergillus
flavus and Fusarium verticillioides), which was compared
with that of the standard drugs Streptomycin and Nystain.
The zone of inhibition was determined by the agar well
diffusion method. The results of the tested compounds are
listed in Table 3. The minimum inhibitory concentration
(MIC) for the active compounds showing maximum activity
was determined by the agar dilution method (Table 4).
As observed in the antibacterial activity, compounds 3a,
3b, and 3d show good activity for all the tested species at
MICs 3.12–25 μg/mL that contained electron withdrawing
3,4-dichloro phenyl groups on the pyrazole ring. The com-
pound 3a exhibited maximum activity among all the com-
pounds with MIC values of 3.12 μg/mL (E. coli, S. aureus),
12.5 μg/mL (K. pneumoniae), and 25 μg/mL (P. aerogenosa).
Similarly, 3b also showed appreciable result against E. coli
and K. pneumoniae (MICs 3.12 μg/mL) and was significantly
active against S.aureus (6.25 μg/mL) and P. aerogenosa
(12.5 μg/mL). The compound 3d was found to be active
against all bacterial strains with MICs 6.25–25 μg/mL. From
The activity data depicts the anti-inflammatory potential
of the synthesized compounds, and majority of the analo-
gues were found to be active. From the anti-inflammatory
activity result analysis, it was observed that the compounds
3a, 3b, 3e, 3f, 3i, 3m, and 3n showed good activity with
67.61 to 85.33% inhibition of the edema. The compounds
3a (85.33), 3e (81.32), and 3i (80.75) showed potent anti-
inflammatory activity compared with the other test com-
pounds and are comparable with the standard Indomethacin
(86.76) (Table 2). This emphasizes the presence of the
3F–4CH3– substituted phenyl ring on the 5th position of the
3-oxothiazolopyrimidine nucleus in this pyrazole series.
The presence of electron donating methoxy groups at 2, 5
position on the same phenyl ring [3b (75.61), 3f (79.56), 3j
(63.86), and 3n (67.61)] showed superior activity over the
compounds containing p-methoxy electron donating sub-
stituted analogues [3d (65.11), 3h (67.14), 3l (63.68), and
3p (55.92)]. The phenyl ring without any substituent on
pyrimidine 3c, 3g, 3k, and 3o did not show considerable
influence on the activity (Fig. 3). It was also demonstrated
from the data that there was increase in the activity profile
when the dihalogen groups present in the phenyl ring
attached to the 3rd position of the pyrazole moiety.
Table 2 In vivo anti-
Test sample
1h
2h
4h
inflammatory effect of the
compounds against carrageenan-
induced rat paw model
Swelling
% Inhibition Swelling
% Inhibition Swelling
% Inhibition
3a
3b
3c
3d
3e
3f
1.11 0.05* 63.90
1.22 0.34* 60.36
1.42 0.04* 53.99
1.42 0.06* 63.36
0.70 0.05* 85.33
1.24 0.06* 67.87
0.74 0.04* 80.74
1.17 0.04* 75.61
1.97 0.06* 59.06
1.75 0.09
43.41
2.12 0.06
45.15
1.68 0.11
65.11
1.24 0.07* 59.90
1.29 0.06* 58.14
1.58 0.10* 48.90
1.49 0.11* 61.39
1.54 0.06* 60.10
0.90 0.11* 81.32
0.98 0.07* 79.56
3g
3h
3i
2.32 0.11
2.01 0.05
40.07
48.15
2.22 0.29
1.58 0.08
53.89
67.14
1.47 0.04
52.30
1.40 0.09* 54.76
1.91 0.09* 38.30
1.58 0.10* 59.07
2.42 0.08* 37.60
0.92 0.13* 80.75
3j
1.74 0.16* 63.86
3k
3l
1.57 0.16
1.72 0.09
1.83 0.10
49.28
44.40
40.74
2.10 0.08
1.91 0.09
2.13 0.12
45.70
50.61
45.07
2.74 0.07
1.75 0.08
43.15
63.68
3m
3n
3o
3p
1.50 0.07* 68.89
1.56 0.08* 67.61
1.44 0.11* 53.36
1.40 0.16* 63.87
1.98 0.23
2.45 0.08
35.92
20.87
3.07 0.08
2.76 0.04
20.87
28.64
3.44 0.04
2.12 0.23
28.64
55.92
Indomethacin 0.92 0.18* 70.20
Control 3.10 0.09
0.76 0.15* 80.30
3.88 0.10
0.62 0.15* 86.96
4.83 0.11
–
–
–
ANOVA analysis followed by Dunnett’s-t-test, (n = 6) significance levels
*P < 0.05, **P < 0.01, ***P < 0.001 as compared with the respective control
Bold valus are anti-inflammatory activity result analysis of compounds 3a, 3b, 3e, 3f, 3i, 3m, and 3n showed
good activity with 67.61 to 85.33% inhibition of the edema

Med Chem Res
Fig. 3 SAR analysis of
synthesized compounds 3(a–p)
Table 3 Antibacterial activity
of compounds 3(a–p)
Test sample
Antibacterial activity
E. coli S. aureus
Antifungal activity
P. aerogenosa K. pneumonia A.flavus F.verticelloides
3a
17.9 1.9 15.9 1.4 15.9 1.9
17.5 1.6 15.2 1.3 14.4 1.8
11.2 1.1 11.9 1.4 10.6 0.4
16.3 1.4 13.7 1.2 15.2 3.3
12.2 1.5 12.4 1.1 11.1 0.9
15.4 1.3
16.2 2.0
12.9 0.9
14.4 0.9
10.4 2.4
10.3 1.2
8.1 1.4
9.4 1.3
10.6 1.3
12.4 1.6
5.1 2.0
7.0 1.2
9.6 1.2
12.8 1.2
9.1 2.4
11.1 1.4
16.6 1.6
–
12.2 1.5 11.4 1.6
11.6 1.3 6.0 0.8
8.6 4.8 9.8 1.6
11.2 1.5 4.4 2.1
3b
3c
3d
3e
8.2 1.4
9.4 2.1
10.6 1.6
8.3 2.4
3f
9.9 1.3
7.1 1.1
9.3 1.6
9.5 1.6
9.5 1.7
5.3 1.2
9.1 2.0
11.7 1.2
10.6 5.3
7.2 0.6
3g
10.3 1.2 9.5 1.2
3h
6.8 1.8
6.0 2.2
5.8 1.2
5.9 1.6
4.7 1.4
7.4 2.8
6.5 1.8
5.2 1.2
6.2 1.2
–
6.1 1.2
4.8 2.1
4.9 1.2
4.4 1.8
5.9 1.6
12.3 1.2
6.6 1.2
4.1 1.2
5.4 1.2
–
3i
11.2 1.2 12.2 2.4
3j
11.6 5.6 8.2 1.4
7.7 1.6 5.2 1.3
11.6 1.8
10.4 1.1
3k
3l
10.9 1.4 11.5 2.6 10.1 1.4
8.1 1.0 9.9 0.9 5.4 2.4
10.1 1.1 11.4 0.9 11.6 2.6
3m
3n
3o
7.2 1.2
7.4 0.8
9.6 1.4
11.4 1.2
3p
10.0 1.6 11.2 1.2
Streptomycin
Nystain
18.5 1.8 16.2 2.2 19.2 1.4
–
–
–
17.8 1.4 18.4 1.6
Mean of the triplicate values standard deviation, sample concentration
Bold valus are anti-inflammatory activity result analysis of compounds 3a, 3b, 3e, 3f, 3i, 3m, and 3n showed
good activity with 67.61 to 85.33% inhibition of the edema
the inhibition results (Table 3), it was observed that com-
pounds with 3,4-dichlorophenyl at the 3-position of the pyr-
azole showed enhanced antibacterial activity compared with
3,4-difluoro, 4-chloro and 4-fluorophenyl substituents. Sig-
nificant enhancement was also observed in the antibacterial
activity with the introduction of electron donating methoxy
(3b, 3d, 3f, 3h, 3j, 3l, 3n, and 3p) and methyl (3a, 3e, 3i, and
3m) substituted on the 5th position of the 3-
oxothiazolopyrimidine observed against E. coli, S. aureus,
and K. pneumoniae bacterial species. As a result, the intro-
duction of 2,5-dimethoxyphenyl groups on the pyrimidine
ring are promising candidates for antibacterial research in this
series of compounds (Fig. 3). The results of the antifungal
activity revealed that the compounds 3a, 3b, 3d, 3g, and 3m
showed average antifungal activity against both the tested
species. It was also observed that there was no significant
change in the antifungal activity with the substitution on the
phenyl ring attached to the pyrimidine. However, the dihalo
substituted phenyl ring on the pyrazole ring was compara-
tively more effective than the 4-halo substituted phenyl group
and none of the compounds was superior to the reference
drug.

Med Chem Res
Molecular docking study
COX-2 via three hydrogen bonds and three π-cation inter-
action. The fluorine atom of the phenyl ring, the sulphur
atom of the thiazolidinone ring, and the nitrogen atom of the
pyrimidine ring form hydrogen bonds (F···H–N: 2.26 Å),
(S···H–O: 2.23 Å), (N···H–N: 2.45 Å) with the amino
hydrogen atom of the ARG 120 residue. The difluor-
ophenyl, pyrazole, thiazolidinone rings forms π-cation
interaction with the amino cation ARG 120 and ARG
513. In general, the molecular docking results were in
agreement with the pharmacological data in terms of eva-
luation of the binding energy and binding mode.
To understand the rationale behind the type of interactions
between the compounds and the enzymes, molecular
docking study was performed using AutoDock tools 1.5.6
(ADT 4.2). The synthesized compounds (3a–p) were
docked successfully into the active sites of the COX-2
enzyme. The molecular docking studies revealed that the
compounds 3a, 3e, 3f, and 3i were more selective towards
the COX-2 enzyme. The calculated binding energy of the
docked compounds towards COX-2 was found to be
between −129.88 and −540.47 kcal/mol (Table 5). The
binding mode of the most active inhibitors 3a and 3e to the
COX-2 protein and the interactions are shown in Fig. 4. On
observing the dock results, it can be ascertained that inter-
action with amino acid ARG 120 was common for the
active compounds. The compound 3a is nicely bonded to
COX-2 via two hydrogen bonds and two π-cation interac-
tion. The nitrogen atom of the pyrimidine ring of the
compound 3a forms hydrogen bonds (N···H–N: 2.21 Å)
with the amino hydrogen atom of ARG 120, and the oxygen
atom of the ester group of the compound 3a forms hydrogen
bonds (O···H–N: 2.08 Å) with the amino hydrogen atom of
the LYS 83 residue. The dichlorophenyl, thiazolidinone
rings forms π-cation interaction with the amino cation ARG
513 and ARG 120. Compound 3e is satisfactorily bonded to
Conclusion
In conclusion, in an attempt to develop a new class of anti-
inflammatory and antimicrobial agents, a novel thiazolo[2,3-
b]dihydropyrimidinone possessing pyrazole moiety has been
synthesized. The target compounds are achieved by
employing the Knoevenagel reaction approach. All the
compounds were prepared using the convenient one pot
MCR sequence with less reaction time and good yield. To
the best of our knowledge, the anti-inflammatory activity
and the antimicrobial data of the above mentioned deriva-
tives has been firstly reported by our group. The results of
the in vivo anti-inflammatory activity assays demonstrated
that the compounds 3a, 3b, 3e, 3f, 3i, 3m, and 3n showed
promising anti-inflammatory activity, and among these 3a,
3e, and 3i were found to be comparable with the standard
Indomethacin. Again, the presence of the 3-fluoro-4-
methylphenyl ring on the 3-oxothiazolopyrimidine nucleus
is a favourable site for high activity, followed by 2,5-
dimethoxyphenyl substituent and p-methoxyphenyl sub-
stituent. The structure activity relationships (SARs) revealed
that the 3,4-dichlorophenyl substituted pyrazole analogues
(3a, 3b, and 3d) were identified as compounds with potent
antibacterial activity and showed moderate antifungal
property. Also, the molecular docking studies revealed that
thiazolo[2,3-b]dihydropyrimidinone possessing pyrazole
analogues exhibited good interaction with the active site of
the docked receptor cyclooxygenase-2. The results of the
present study suggest that pyrazole–thiazolopyrimidine
hybrid can be an interesting approach for the design of new
selective COX-2 inhibitory agents.
Table 4 Minimum inhibitory concentrations (MICs) of the selected
compounds
Test sample
Minimum inhibitory concentrations (MICs) in μg/mL
E. coli S. aureus P. aerogenosa K. pneunoniae
3a
3b
3d
3.12
3.12
6.25
3.12
6.25
25
12.5
12.5
6.25
3.8
6.25
3.12
6.25
2.8
Streptomycin 2.8
2.5
Table 5 Binding energy of compounds (3a, 3e, 3f, and 3i) after
docking in to COX-2 active site
Compound C-Docker
energy
H-bond
distance (Å)
Hydrogen bonding
residues
3a
3e
3f
3i
−540.474
−315.736
−412.813
−129.883
2.08713
2.21156
2.4950
LYS 83
ARG 120
2.4542
ARG 120
Experimental section
Materials and methods
2.23841
2.26208
2.48284
2.30603
1.82602
2.13139
ARG 120
ARG 513
All the reagents were purchased from commercial suppliers
Sigma-Aldrich, Spectrochem India and used without further
purification. Melting points were determined in an open
ARG 120

Med Chem Res
Fig. 4 Docking figures of 3a and 3e in COX-2 protein cavity
capillary tube and were uncorrected. The progress of each
reaction was monitored by ascending thin layer chromato-
graphy (TLC) on silica gel G (Merck 1.05570.0001),
visualized by UV light. The IR spectra (in KBr pellets) were
recorded on a Shimadzu-FTIR spectrometer and the wave
(C–O), 1097 (C=S), 1024 (C–F). 1H NMR (DMSO-d6, 400
MHz, δ ppm): 1.08 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.24 (s,
3H, pyrimidine-CH3), 2.62 (s, 3H, Ar–CH3), 3.96 (q, 2H, J
= 5.6 Hz, –CH2–CH3), 5.35 (s, 1H, pyrimidine-CH),
7.06–7.54 (m, 3H, Ar–H), 9.67 (s, 1H, pyrimidine-NH),
10.54 (s, 1H, pyrimidine-NH); 13C NMR (DMSO-d6, 100
MHz, δ ppm): 14.23 (–CH2–CH3), 14.75, 17.48 (CH3),
54.68 (pyrimidine-CH), 60.72 (–CH2–CH3), 102.34,
113.75, 122.04, 123.54, 130.53, 141.82, 159.52, 161.52,
165.41 (ester C=O), 174.53 (C=S); LCMS (m/z): 309.13
[M+H]. Anal. Calcd. for C15H17FN2O2S: C, 58.42; H,
5.56; N, 9.08. Found: C, 58.46; H, 5.53; N, 9.06.
1
numbers were given in cm−1. The H NMR and 13C NMR
spectra were recorded (CDCl3/DMSO-d6 mixture) on a
Bruker AMX-400 NMR spectrometer with 5 mm PABBO
BB-1H TUBES with TMS as internal standard. Mass
spectra were recorded in Agilent Technology LC-mass
spectrometer. Elemental analyses were carried out using
VARIO EL-III (Elementar Analysensysteme GmBH).
Procedure for the preparation of ethyl 4-(substitutedphenyl)-
6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxy-
late 1(a–d)
Ethyl 4-(2,5-dimethoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (1b) Yield: 56%; m.
p. 203–205 °C; FT-IR (KBr) νmax (cm−1): 3286, 3110
(N–H), 2951 (Ar C–H), 1726 (C=O), 1566 (C=C), 1284
(C–O), 1137 (C=S); 1H NMR (DMSO-d6, 400 MHz, δ
ppm): 1.07 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.37 (s, 3H,
pyrimidine-CH3), 3.75 (s, 6H, Ar–OCH3 × 2), 3.98 (q, 2H,
J = 5.6 Hz, –CH2–CH3), 5.42 (s, 1H, pyrimidine-CH),
7.18–7.77 (m, 3H, Ar–H), 9.79 (s, 1H, pyrimidine-NH),
10.29 (s, 1H, pyrimidine-NH); 13C NMR (DMSO-d6, 100
MHz, δ ppm): 14.31 (–CH2–CH3), 17.85 (CH3), 54.13
(pyrimidine-CH), 55.12, 56.59 (–OCH3 × 2), 60.84 (–
CH2–CH3), 102.48, 112.74, 113.68, 115.91, 122.18,
148.52, 152.32, 160.26, 165.94 (ester C=O), 174.64
(C=S); LCMS (m/z): 337.01 [M+H]. Anal. Calcd. for
C16H20N2O4S: C, 57.12; H, 5.99; N, 8.33. Found: C, 57.16;
H, 5.93; N, 8.36.
A mixture of simple or substituted benzaldehyde (0.05
mol), ethylacetoacetate (0.05 mol), thiourea (0.05 mol) and
a few drops of HCl as catalyst was refluxed in ethanol (30
mL) for 1–4 h and the progress of the reaction was mon-
itored by TLC. After completion of reaction, the resulting
mixture was cooled and poured onto crushed ice. The solid
was collected by filtration, washed several times with
water to remove unreacted thiourea, and then dried.
The product was purified by recrystallization from
methanol.
Ethyl 4-(3-fluoro-4-methylphenyl)-6-methyl-2-thioxo-1,2,3,
4-tetrahydropyrimidine-5-carboxylate (1a) Yield: 88%; m.
p. 175–177 °C; FT-IR (KBr) νmax (cm−1): 3255, 3132
(N–H), 2986 (Ar C–H), 1738 (C=O), 1579 (C=C), 1279

Med Chem Res
Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimi-
dine-5-carboxylate (1c) Yield: 66%; m.p. 178–180 °C;
FT-IR (KBr) νmax (cm−1): 3236, 3097 (N–H), 2918 (Ar
C–H), 1724 (C=O), 1564 (C=C), 1205 (C–O), 1089
128.43, 129.10, 130.24, 130.61, 131.21, 131.77, 132.34,
137.15, 138.88, 149.98, 184.87 (aldehyde C=O); LCMS
(m/z): 317.12 [M+H] (35Cl2), 319.14 [M+H+2] (35Cl37Cl),
321.24 [M+H+4] (37Cl2); Anal. Calcd. for C16H10Cl2N2O:
C, 60.59; H, 3.18; N, 8.83. Found: C, 60.56; H, 3.13; N,
8.86.
1
(C=S); H NMR (DMSO-d6, 400 MHz, δ ppm): 1.04 (t,
3H, J = 7.2 Hz, –CH2–CH3), 2.26 (s, 3H, pyrimidine-CH3),
3.95 (q, 2H, J = 7.2 Hz, –CH2–CH3), 5.14 (s, 1H, pyr-
imidine-CH), 7.18–7.33 (m, 5H, Ar–H), 9.62(s, 1H, pyr-
imidine-NH), 10.30 (s, 1H, pyrimidine-NH); 13C NMR
(DMSO-d6, 100 MHz, δ ppm): 14.44 (–CH2–CH3), 17.57
(CH3), 54.45 (pyrimidine-CH), 60.02 (–CH2–CH3), 101.16,
126.81, 128.09, 128.97, 143.93, 145.45, 165.56 (ester
C=O), 174.69 (C=S); LCMS (m/z): 277.12 [M+H]. Anal.
Calcd. for C14H16N2O2S: C, 60.85; H, 5.84; N, 10.14.
Found: C, 60.86; H, 5.83; N, 10.16.
3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazole-4-carbalde-
hyde (2b) Yield: 76%; m.p. 95–98 °C; FT-IR (KBr) νmax
(cm−1): 3056 (Ar C–H), 1675 (C=O), 1578 (C=N), 1453
(C=C), 1086 (C–F); 1H NMR (DMSO-d6, 400 MHz, δ
ppm): 7.44–9.37 (m, 9H, Ar–CH), 9.96 (s, 1H, CHO); 13C
NMR (DMSO-d6, 100 MHz, δ ppm): 117.99, 119.71,
122.58, 126.04, 128.33, 129.18, 130.19, 136.83, 138.88,
148.48, 149.19, 150.43, 150.91, 151.65, 184.87 (aldehyde
C=O); LCMS (m/z): 285.2 [M+H]; Anal. Calcd. for
C16H10F2N2O: C, 67.60; H, 3.55; N, 9.85. Found: C, 67.62;
H, 3.53; N, 9.86.
Ethyl 4-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tet-
rahydropyrimidine-5-carboxylate (1d) Yield: 78%; m.p.
150–152 °C; FT-IR (KBr) νmax (cm−1): 3236, 3143 (N–H),
2922 (Ar C–H), 1763 (C=O), 1523 (C=C), 1273 (C–O),
3-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(2c) Yield: 82%; m.p. 140–142 °C; FT-IR (KBr) νmax
(cm−1): 3022 (Ar C–H), 1698 (C=O), 1554 (C=N), 1496
(C=C), 789 (C–Cl); 1H NMR (DMSO-d6, 400 MHz, δ
1
1174 (C=S); H NMR (DMSO-d6, 400 MHz, δ ppm): 1.04
(t, 3H, J = 6.4 Hz, –CH2–CH3), 2.26 (s, 3H, pyrimidine-
CH3), 3.59 (s, 3H, Ar–OCH3), 3.96 (q, 2H, J = 6.4 Hz, –
CH2–CH3), 5.14 (s, 1H, pyrimidine-CH), 6.69–6.99 (m, 4H,
Ar–H), 9.63 (s, 1H, pyrimidine-NH), 10.35 (s, 1H, pyr-
imidine-NH); 13C NMR (DMSO-d6, 100 MHz, δ ppm):
14.42 (–CH2–CH3), 17.60 (CH3), 53.86 (pyrimidine-CH),
53.94 (–OCH3), 60.08 (–CH2–CH3), 100.74, 128.74,
129.00, 132.70, 142.82, 145.80, 165.42 (ester C=O),
174.70 (C=S); LCMS (m/z): 307.12 [M+H]. Anal. Calcd.
for C15H18N2O3S: C, 58.80; H, 5.92; N, 9.14. Found: C,
58.86; H, 5.93; N, 9.16.
ppm): 7.14–8.37 (m, 10H, Ar–CH), 9.65 (s, 1H, CHO); 13
C
NMR (DMSO-d6, 100 MHz, δ ppm): 106.54, 120.32,
126.54, 128.53, 129.56, 130.04, 130.89, 131.43, 134.77,
139.78, 150.54 (Ar–C), 187.58 (aldehyde C=O); LCMS
(m/z): 283.12 [M+H] (35Cl), 285.14 [M+H+2] (37Cl);
Anal. Calcd. for C16H11ClN2O: C, 67.97; H, 3.92; N, 9.91.
Found: C, 67.96; H, 3.93; N, 9.86.
3-(4-Fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(2d) Yield: 64%; m.p. 110–112 °C; FT-IR (KBr) νmax
(cm−1): 3011 (Ar C–H), 1694 (C=O), 1574 (C=N), 1463
(C=C), 1081 (C–F); 1H NMR (DMSO-d6, 400 MHz, δ
General procedure for the synthesis of 4-Formylpyrazole 2
(a–d)
ppm): 7.16–8.16 (m, 10H, Ar–CH), 9.58 (s, 1H, CHO); 13
C
NMR (DMSO-d6, 100 MHz, δ ppm): 106.28, 116.59,
120.70, 126.54, 127.93, 129.08, 129.96, 130.08, 139.74,
150.50, 160.78 (Ar–C), 187.61 (aldehyde C=O); LCMS
(m/z): 267.34 [M+H]; Anal. Calcd. for C16H11FN2O: C,
72.17; H, 4.16; N, 10.52. Found: C, 72.21; H, 4.19; N,
10.56.
(Substituted phenyl) ethanone phenylhydrazone (0.01 mol)
was added to the mixture of Vilsmeier-Haack reagent
(prepared by drop-wise addition of 1.2 mL POCl3 in ice
cooled 5 mL DMF) and refluxed for 6 h. The reaction
mixture, after cooling, was poured onto crushed ice, fol-
lowed by neutralization using sodium bicarbonate. The
solid product thus obtained was collected by filtration,
washed with water and recrystallized from ethanol (Viveka
et al. 2015a, b, d).
Procedure for the synthesis of ethyl 2-((3-(substitutedphenyl)
-1-phenyl-1H-pyrazol-4-yl)methylene)-5-(substituted phenyl)-
7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-
carboxylate 3(a–p)
3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbalde-
hyde (2a) Yield: 83%; m.p. 150–152 °C; FT-IR (KBr)
νmax (cm−1): 3041 (Ar C–H), 1678 (C=O), 1571 (C=N),
A solution of ethyl 6-methyl-4-(substituted phenyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate 1 (10 mmol), 3-
(substitutedphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 2
(10 mmol), monochloroacetic acid (15 mmol) and anhydrous
1
1489 (C=C), 813 (C–Cl); H NMR (DMSO-d6, 400 MHz,
δ ppm): 7.42–7.83 (m, 9H, Ar–CH), 9.97 (s, 1H, CHO). 13
C
NMR (DMSO-d6, 100 MHz, δ ppm): 119.79, 122.75,

Med Chem Res
sodium acetate (2 g) in glacial acetic acid (25 mL)-acetic
anhydride (20 mL) mixture was refluxed for 2–6 h and
monitored by TLC. The mixture was cooled to room tem-
perature and then poured onto crushed ice with stirring. The
precipitated solid was collected by filtration, washed with
cold water and recrystallized from methanol (Viveka et al.
2012, 2015d).
thiazolo[3,2-a]pyrimidine-6-carboxylate (3c) Yield: 67%;
m.p. 135–137 °C; FT-IR (KBr) νmax (cm−1): 2994 (Ar
C–H), 1736 (C=O), 1680 (C=N), 1589 (C=C), 1232
1
(C–O), 759 (C–Cl); H NMR (CDCl3, 400 MHz, δ ppm):
1.16 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.35 (s, 3H,
pyrimidine-CH3), 4.09 (q, 2H, J = 5.6 Hz, –CH2–CH3),
6.18 (s, 1H, pyrimidine-CH), 7.26–7.78 (m, 14H, Ar–H),
8.19 (s, 1H, pyrazole CH); 13C NMR (CDCl3, 100 MHz, δ
ppm): 14.21 (–CH2–CH3), 22.32 (CH3), 55.73 (pyrimidine-
CH), 61.69 (–CH2–CH3), 107.56, 120.12, 121.49, 122.31,
124.67, 125.13, 126.42, 127.23, 127.41, 128.42, 129.03,
129.92, 130.02, 130.81, 133.45, 134.32, 139.71, 142.52,
143.52, 150.31, 154.57, 160.41, 164.52 (cyclic C=O),
165.51(ester C=O); LCMS (m/z): 615.15 [M+H] (35Cl2),
617.15 [M+H+2] (35Cl37Cl), 619.15 [M+H+4] (37Cl2);
Anal. Calcd. for C32H24Cl2N4O3S: C, 62.44; H, 3.93; N,
9.10. Found: C, 62.41; H, 3.95; N, 9.06.
Ethyl 2-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(3-fluoro-4-methylphenyl)-7-methyl-3-oxo-
3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate
(3a) Yield: 91%; m.p. 127–130 °C; FT-IR (KBr) νmax
(cm−1): 2966 (Ar C–H), 1726 (C=O), 1672 (C=N), 1587
(C=C), 1210 (C–O), 1031 (C–F) 761 (C-Cl); 1H NMR
(CDCl3, 400 MHz, δ ppm): 1.19 (t, 3H, J = 5.6 Hz, –CH2–
CH3), 2.21 (s, 3H, pyrimidine-CH3), 2.51 (s, 3H, Ar–CH3),
4.10 (q, 2H, J = 5.6 Hz, –CH2–CH3), 6.13 (s, 1H, pyr-
imidine-CH), 7.02–7.77 (m, 12H, Ar–H), 8.18 (s, 1H,
pyrazole CH); 13C NMR (CDCl3, 100 MHz, δ ppm): 14.10
(–CH2–CH3), 14.22, 22.70 (CH3), 55.11 (pyrimidine-CH),
60.53 (–CH2–CH3), 109.33, 113.92, 116.26, 119.24,
119.57, 123.46, 127.16, 127.84, 128.95, 129.14, 129.42,
129.74, 129.91, 130.11, 130.20, 132.33, 135.20, 139.10,
151.94, 153.58, 155.17, 159.74, 164.73 (cyclic C=O),
165.54 (ester C=O); LCMS (m/z): 647.24 [M+H] (35Cl2),
649.24 [M+H+2] (35Cl37Cl), 650.24 [M+H+4] (37Cl2);
Anal. Calcd. for C33H25Cl2FN4O3S: C, 61.21; H, 3.89; N,
8.65. Found: C, 61.18; H, 3.92; N, 8.66.
Ethyl 2-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(4-methoxyphenyl)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3d)
Yield: 88%; m.p. 112–114 °C; FT-IR (KBr) νmax (cm−1):
2989 (Ar C–H), 1742 (C=O), 1652 (C=N), 1567 (C=C),
1
1224 (C–O), 767 (C-Cl); H NMR (CDCl3, 400 MHz, δ
ppm): 1.21 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.49 (s, 3H,
pyrimidine-CH3), 3.75 (s, 3H, Ar–OCH3), 3.99 (q, 2H, J =
5.6 Hz, –CH2–CH3), 6.13 (s, 1H, pyrimidine-CH), 6.98–7.89
(m, 13H, Ar–H), 8.21 (s, 1H, pyrazole CH); 13C NMR
(CDCl3, 100 MHz, δ ppm): 14.34 (–CH2–CH3), 21.86
(CH3), 54.83 (pyrimidine-CH), 55.62 (–OCH3), 60.86 (–
CH2–CH3), 107.53, 112.53, 120.91, 121.54, 122.76, 125.43,
126.34, 127.40, 128.81, 129.54, 130.41, 130.89, 132.50,
133.50, 133.72, 135.71, 139.72, 142.51, 150.12, 154.56,
158.71, 162.71, 164.71 (cyclic C=O), 165.79(ester C=O);
LCMS (m/z): 645.12 [M+H] (35Cl2), 647.12 [M+H+2]
(35Cl37Cl), 649.12 [M+H+4] (37Cl2); Anal. Calcd. for
C33H26Cl2N4O4S: C, 61.40; H, 4.06; N, 8.68. Found: C,
61.34; H, 4.01; N, 8.70.
Ethyl 2-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(2,5-dimethoxyphenyl)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3b)
Yield: 78%; m.p. 98–101 °C; FT-IR (KBr) νmax (cm−1):
2978 (Ar C–H), 1723 (C=O), 1653 (C=N), 1565 (C=C),
1
1226 (C–O), 786 (C–Cl); H NMR (CDCl3, 400 MHz, δ
ppm): 1.23 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.45 (s, 3H,
pyrimidine-CH3), 3.69 (s, 6H, Ar–OCH3 × 2), 4.01 (q, 2H,
J = 5.6 Hz, –CH2–CH3), 6.20 (s, 1H, pyrimidine-CH),
6.88–7.79 (m, 12H, Ar–H), 8.21 (s, 1H, pyrazole CH); 13C
NMR (CDCl3, 100 MHz, δ ppm): 14.21 (–CH2–CH3),
22.69 (CH3), 53.79 (pyrimidine-CH), 55.79, 56.25
(–OCH3 × 2), 60.24 (–CH2–CH3), 108.21, 112.43, 114.13,
115.17, 116.15, 119.16, 120.23, 121.32, 127.02, 127.73,
128.32, 128.89, 129.32, 129.71, 130.26, 131.18, 133.20,
133.39, 139.72, 142.54, 152.81, 154.08, 159.74, 161.17,
164.51 (cyclic C=O), 165.21(ester C=O); LCMS (m/z):
675.21 [M+H] (35Cl2), 677.22 [M+H+2] (35Cl37Cl),
Ethyl 2-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(3-fluoro-4-methylphenyl)-7-methyl-3-oxo-3,
5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate
(3e) Yield: 72%; m.p. 166–167 °C; FT-IR (KBr) νmax
(cm−1): 2969 (Ar C–H), 1737 (C=O), 1682 (C=N), 1591
1
(C=C), 1224 (C–O), 1055 (C–F); H NMR (CDCl3, 400
MHz, δ ppm): 1.10 (t, 3H, J = 7.2 Hz, –CH2–CH3), 2.17 (s,
3H, pyrimidine-CH3), 2.38 (s, 3H, Ar–CH3), 4.02 (q, 2H, J
= 4.0 Hz, –CH2–CH3), 5.97 (s, 1H, pyrimidine-CH),
6.99–8.02 (m, 12H, Ar–H), 8.79 (s, 1H, pyrazole CH); 13C
NMR (CDCl3, 100 MHz, δ ppm): 14.10 (–CH2–CH3),
14.39, 22.81 (CH3), 54.98 (pyrimidine-CH), 60.64
(–CH2–CH3), 108.86, 114.70, 116.26, 119.54, 119.73,
123.04, 123.45, 125.48, 125.62, 127.25, 127.94, 128.00,
679.21
[M+H+4]
(37Cl2);
Anal.
Calcd.
for
C34H28Cl2N4O5S: C, 60.45; H, 4.18; N, 8.29. Found: C,
60.49; H, 4.19; N, 8.26.
Ethyl 2-((3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-

Med Chem Res
129.78, 130.48, 130.84, 131.43, 131.55, 133.26, 133.39,
138.96, 139.46, 139.52, 152.22, 152.45, 155.01, 160.23,
162.19, 164.51 (cyclic C=O), 165.34 (ester C=O); LCMS
(m/z): 615.29 [M+H]; Anal. Calcd. for C33H25F3N4O3S: C,
64.69; H, 4.10; N, 9.12. Found: C, 64.72; H, 4.06; N, 9.06.
6.18–7.92 (m, 13H, Ar–H), 8.33 (s, 1H, pyrazole CH); 13C
NMR (CDCl3, 100 MHz, δ ppm): 14.18 (–CH2–CH3),
22.37 (CH3), 54.74 (pyrimidine-CH), 55.96 (–OCH3),
61.57 (–CH2–CH3), 107.51, 113.72, 119.98, 121.38,
122.89, 125.12, 126.38, 128.12, 128.37, 129.19, 130.14,
131.01, 132.45, 133.18, 133.99, 135.17, 137.52, 142.71,
150.15, 153.16, 157.98, 161.91, 163.97 (cyclic C=O),
164.99 (ester C=O); LCMS (m/z): 613.16 [M+H]; Anal.
Calcd. for C33H26F2N4O4S: C, 64.70; H, 4.28; N, 9.15.
Found: C, 64.78; H, 4.23; N, 9.14.
Ethyl 2-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(2,5-dimethoxyphenyl)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3f)
Yield: 74%; m.p. 105–107 °C; FT-IR (KBr) νmax (cm−1):
2972 (Ar C–H), 1726 (C=O), 1676 (C=N), 1587 (C=C),
1
1249 (C–O), 1031 (C–F); H NMR (CDCl3, 400 MHz, δ
ppm): 1.22 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.43 (s, 3H,
pyrimidine-CH3), 3.75 (s, 6H, Ar–OCH3 × 2), 4.09 (q, 2H,
J = 5.6 Hz, –CH2–CH3), 6.24 (s, 1H, pyrimidine-CH),
6.76–7.77 (m, 12H, Ar–H), 8.19 (s, 1H, pyrazole CH); 13C
NMR (CDCl3, 100 MHz, δ ppm): 14.17 (–CH2–CH3),
22.81 (CH3), 53.92 (pyrimidine-CH), 55.67, 56.14
(–OCH3 × 2), 60.35 (–CH2–CH3), 107.42, 112.62, 114.49,
115.85, 116.03, 116.37, 117.12, 119.15, 119.50, 122.79,
125.30, 127.02, 127.63, 128.22, 128.42, 129.03, 129.72,
130.67, 139.17, 151.67, 152.36, 153.13, 153.74, 155.07,
162.27, 164.63 (cyclic C=O), 165.83 (ester C=O); LCMS
(m/z): 643.17 [M+H]; Anal. Calcd. for C34H28F2N4O5S: C,
63.54; H, 4.39; N, 8.72. Found: C, 63.49; H, 4.41; N, 8.69.
Ethyl 2-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)me-
thylene)-5-(3-fluoro-4-methylphenyl)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3i)
Yield: 59%; m.p. 90–93 °C; FT-IR (KBr) νmax (cm−1):
2967 (Ar C–H), 1716 (C=O), 1684 (C=N), 1572 (C=C),
1
1234 (C–O), 1041 (C–F), 758 (C-Cl); H NMR (CDCl3,
400 MHz, δ ppm): 1.29 (t, 3H, J = 5.6 Hz, –CH2–CH3),
2.16 (s, 3H, pyrimidine-CH3), 2.48 (s, 3H, Ar–CH3), 4.18
(q, 2H, J = 5.6 Hz, –CH2–CH3), 5.98 (s, 1H, pyrimidine-
CH), 6.92–8.01 (m, 13H, Ar–H), 8.24 (s, 1H, pyrazole CH);
13C NMR (CDCl3, 100 MHz, δ ppm): 14.18 (–CH2–CH3),
14.51, 22.18 (CH3), 56.13 (pyrimidine-CH), 61.28 (–
CH2–CH3), 107.13, 113.78, 120.13, 121.52, 122.34,
122.78, 123.34, 126.57, 128.34, 129.54, 130.02, 130.84,
131.59, 132.76, 134.76, 138.76, 141.84, 142.54, 150.57,
154.79, 160.09, 161.2, 164.12 (cyclic C=O), 165.76 (ester
C=O); LCMS (m/z): 613.12 [M+H] (35Cl), 615.12 [M+H
+2] (37Cl); Anal. Calcd. for C33H26ClFN4O3S: C, 64.65; H,
4.27; N, 9.14. Found: C, 64.65; H, 4.31; N, 9.11.
Ethyl 2-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thia-
zolo[3,2-a]pyrimidine-6-carboxylate (3g) Yield: 81%; m.
p. 134–136 °C; FT-IR (KBr) νmax (cm−1): 2978 (Ar C–H),
1734 (C=O), 1684 (C=N), 1569 (C=C), 1218 (C–O), 1054
1
(C–F); H NMR (CDCl3, 400 MHz, δ ppm): 1.22 (t, 3H, J
= 5.6 Hz, –CH2–CH3), 2.41 (s, 3H, pyrimidine-CH3), 4.15
(q, 2H, J = 5.6 Hz, –CH2–CH3), 6.07 (s, 1H, pyrimidine-
CH), 7.51–7.98 (m, 14H, Ar–H), 8.23 (s, 1H, pyrazole CH);
13C NMR (CDCl3, 100 MHz, δ ppm): 14.23 (–H2–CH3),
22.31 (CH3), 54.82 (pyrimidine-CH), 61.06 (–CH2–CH3),
108.16, 119.98, 120.92, 121.86, 123.75, 125.22, 125.69,
126.54, 127.11, 128.31, 129.01, 129.36, 130.11, 130.99,
133.45, 134.89, 137.34, 142.17, 144.02, 150.17, 154.57,
160.38, 164.18 (cyclic C=O), 165.85 (ester C=O); LCMS
(m/z): 583.16 [M+H]; Anal. Calcd. for C32H24F2N4O3S: C,
65.97; H, 4.15; N, 9.62. Found: C, 66.01; H, 4.10; N, 9.59.
Ethyl
2-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(2,5-dimethoxyphenyl)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3j)
Yield: 72%; m.p. 103–105 °C; FT-IR (KBr) νmax (cm−1):
2978 (Ar C–H), 1724 (C=O), 1677 (C=N), 1598 (C=C),
1215 (C–O), 756 (C–Cl); 1H NMR (DMSO-d6, 400 MHz, δ
ppm): 1.10 (t, 3H, J = 7.2 Hz, –CH2–CH3), 2.26 (s, 3H,
pyrimidine-CH3), 3.61 (s, 6H, Ar–OCH3 × 2), 3.90 (q, 2H,
J = 6.8 Hz, –CH2–CH3), 6.02 (s, 1H, pyrimidine-CH),
6.63–8.00 (m, 13H, Ar–H), 8.75 (s, 1H, pyrazole CH); 13C
NMR (DMSO-d6, 100 MHz, δ ppm): 14.36 (–CH2–CH3),
22.91 (CH3), 53.93 (pyrimidine-CH), 55.89, 56.68
(–OCH3 × 2), 60.36 (–CH2–CH3), 107.39, 113.84, 114.52,
115.98, 117.43, 119.75, 119.78, 119.88, 122.13, 128.02,
128.65, 128.88, 129.01, 129.44, 130.05, 130.17, 130.61,
130.77, 134.33, 139.16, 151.06, 152.99, 153.98, 159.68,
161.120, 164.51 (cyclic C=O), 165.21(ester C=O); LCMS
(m/z): 641.12 [M+H] (35Cl), 643.12 [M+H+2] (37Cl);
Anal. Calcd. for C34H29ClN4O5S: C, 63.69; H, 4.56; N,
8.74. Found: C, 63.68; H, 4.63; N, 8.79.
Ethyl 2-((3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-5-(4-methoxyphenyl)-7-methyl-3-oxo-3,5-dihy-
dro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate
(3h)
Yield: 73%; m.p. 167–169 °C; FT-IR (KBr) νmax (cm−1):
2984 (Ar C–H), 1734 (C=O), 1661 (C=N), 1558 (C=C),
1
1218 (C–O), 1045 (C–F); H NMR (CDCl3, 400 MHz, δ
ppm): 1.34 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.56 (s, 3H,
pyrimidine-CH3), 3.46 (s, 3H, Ar–OCH3), 4.13 (q, 2H, J =
5.6 Hz, –CH2–CH3), 6.18 (s, 1H, pyrimidine-CH),

Med Chem Res
Ethyl 2-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)me-
thylene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thiazolo
[3,2-a]pyrimidine-6-carboxylate (3k) Yield: 70%; m.p.
118–120 °C; FT-IR (KBr) νmax (cm−1): 2942 (Ar C–H),
1734 (C=O), 1682 (C=N), 1591 (C=C), 1234 (C–O), 768
(C–Cl); 1H NMR (CDCl3, 400 MHz, δ ppm): 1.31 (t, 3H, J
= 5.6 Hz, –CH2–CH3), 2.46 (s, 3H, pyrimidine-CH3), 4.18
(q, 2H, J = 5.6 Hz, –CH2–CH3), 6.11 (s, 1H, pyrimidine-
CH), 7.03–8.02 (m, 15H, Ar–H), 8.22 (s, 1H, pyrazole CH);
13C NMR (CDCl3, 100 MHz, δ ppm): 14.22 (–CH2–CH3),
23.04 (CH3), 54.98 (pyrimidine-CH), 61.30 (–CH2–CH3),
106.37, 119.97, 121.43, 122.34, 126.38, 127.07, 127.89,
128.12, 128.94, 129.34, 129.88, 130.40, 131.34, 134.78,
139.86, 142.50, 143.86, 150.29, 155.73, 161.78, 164.57
(cyclic C=O), 165.39 (ester C=O); LCMS (m/z): 581.13
[M+H] (35Cl), 583.13 [M+H+2] (37Cl); Anal. Calcd. for
C32H25ClN4O3S: C, 66.14; H, 4.34; N, 9.64. Found: C,
66.18; H, 4.33; N, 9.69.
154.86, 161.19, 163.28, 165.24 (cyclic C=O), 166.15 (ester
C=O); LCMS (m/z): 597.23 [M+H]; Anal. Calcd. for
C33H26F2N4O3S: C, 66.43; H, 4.39; N, 9.39. Found: C,
66.41; H, 4.40; N, 9.40.
Ethyl 5-(2,5-dimethoxyphenyl)-2-((3-(4-fluorophenyl)-1-
phenyl-1H-pyrazol-4-yl)methylene)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3n)
Yield: 75%; m.p. 99–101 °C; FT-IR (KBr) νmax (cm−1):
2973 (Ar C–H), 1759 (C=O), 1665 (C=N), 1563 (C=C),
1
1230 (C–O), 1054 (C–F); H NMR (DMSO-d6, 400 MHz,
δ ppm): 1.19 (t, 3H, J = 7.2 Hz, –CH2–CH3), 2.38 (s, 3H,
pyrimidine-CH3), 3.14 (s, 6H, Ar–OCH3 × 2), 3.95 (q, 2H,
J = 6.8 Hz, –CH2–CH3), 6.12 (s, 1H, pyrimidine-CH),
6.89–8.03 (m, 13H, Ar–H), 8.69 (s, 1H, pyrazole CH); 13C
NMR (DMSO-d6, 100 MHz, δ ppm): 14.14 (–CH2–CH3),
22.84 (CH3), 53.98 (pyrimidine-CH), 54.92, 56.33
(–OCH3 × 2), 60.14 (–CH2–CH3), 106.89, 112.16, 114.34,
115.78, 117.58, 118.87, 119.34, 119.78, 121.86, 127.56,
128.79, 128.96, 129.05, 129.86, 130.05, 130.18, 130.68,
131.24, 135.12, 139.84, 151.12, 152.96, 153.73, 161.12,
162.34, 164.86 (cyclic C=O), 165.86 (ester C=O); LCMS
(m/z): 625.22 [M+H]; Anal. Calcd. for C34H29FN4O5S: C,
65.37; H, 4.68; N, 8.97. Found: C, 65.40; H, 4.70; N, 8.99.
Ethyl 2-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)me-
thylene)-5-(4-methoxyphenyl)-7-methyl-3-oxo-3,5-dihydro-
2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3l) Yield: 49%;
m.p. 182–185 °C; FT-IR (KBr) νmax (cm−1): 2981 (Ar C–H),
1734 (C=O), 1678 (C=N), 1541 (C=C), 1234 (C–O), 763
(C–Cl); 1H NMR (CDCl3, 400 MHz, δ ppm): 1.17 (t, 3H, J =
5.6 Hz, –CH2–CH3), 2.51 (s, 3H, pyrimidine-CH3), 3.85 (s,
3H, Ar–OCH3), 4.09 (q, 2H, J = 4.4 Hz, –CH2–CH3), 6.13 (s,
1H, pyrimidine-CH), 6.82–7.83 (m, 14H, Ar–H), 8.18 (s, 1H,
pyrazole CH); 13C NMR (CDCl3, 100 MHz, δ ppm): 14.16
(–CH2–CH3), 21.72 (CH3), 53.93 (pyrimidine-CH), 56.12
(–OCH3), 61.50 (–CH2–CH3), 106.68, 113.76, 120.14,
121.36, 122.81, 126.54, 128.10, 128.96, 129.14, 129.89,
130.10, 131.84, 134.50, 135.56, 139.70, 141.68, 150.32,
154.27, 158.75, 160.72, 164.52 (cyclic C=O), 165.78 (ester
C=O); LCMS (m/z): 611.28 [M+H] (35Cl), 613.27 [M+H
+2] (37Cl); Anal. Calcd. for C33H27ClN4O4S: C, 64.86; H,
4.45; N, 9.17. Found: C, 64.85; H, 4.40; N, 9.16.
Ethyl 2-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)-7-methyl-3-oxo-5-phenyl-3,5-dihydro-2H-thia-
zolo[3,2-a]pyrimidine-6-carboxylate (3o) Yield: 86%; m.
p. 182–185 °C; FT-IR (KBr) νmax (cm−1): 2964 (Ar C–H),
1721 (C=O), 1674 (C=N), 1576 (C=C), 1231 (C–O), 1048
1
(C–F); H NMR (CDCl3, 400 MHz, δ ppm): 1.36 (t, 3H,
J = 5.6 Hz, –CH2–CH3), 2.54 (s, 3H, pyrimidine-CH3),
4.38 (q, 2H, J = 5.6 Hz, –CH2–CH3), 6.74 (s, 1H, pyr-
imidine-CH), 7.02–8.04 (m, 15H, Ar–H), 8.31 (s, 1H,
pyrazole CH); 13C NMR (CDCl3, 100 MHz, δ ppm): 14.24
(–CH2–CH3), 23.86 (CH3), 54.50 (pyrimidine-CH), 61.98
(–CH2–CH3), 106.57, 119.68, 121.54, 122.64, 126.75,
127.79, 127.92, 128.23, 128.98, 129.53, 130.14, 130.96,
131.76, 134.56, 139.85, 142.16, 143.74, 150.85, 155.26,
161.58, 164.19 (cyclic C=O), 165.50 (ester C=O); LCMS
(m/z): 565.26 [M+H]; Anal. Calcd. for C32H25FN4O3S: C,
68.07; H, 4.46; N, 9.92. Found: C, 68.01; H, 4.48; N, 9.89.
Ethyl 5-(3-fluoro-4-methylphenyl)-2-((3-(4-fluorophenyl)-
1-phenyl-1H-pyrazol-4-yl)methylene)-7-methyl-3-oxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate
(3m) Yield: 66%; m.p. 134–136 °C; FT-IR (KBr) νmax
(cm−1): 2976 (Ar C–H), 1731 (C=O), 1654 (C=N), 1582
1
(C=C), 1238 (C–O), 1041 (C–F); H NMR (CDCl3, 400
Ethyl 2-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)me-
thylene)-5-(4-methoxyphenyl)-7-methyl-3-oxo-3,5-dihy-
dro-2H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3p) Yield:
76%; m.p. 163–165 °C; FT-IR (KBr) νmax (cm−1): 2975 (Ar
C–H), 1728 (C=O), 1673 (C=N), 1586 (C=C), 1232 (C–O),
MHz, δ ppm): 1.22 (t, 3H, J = 5.6 Hz, –CH2–CH3), 2.05 (s,
3H, pyrimidine-CH3), 2.55 (s, 3H, Ar–CH3), 4.37 (q, 2H, J
= 5.6 Hz, –CH2–CH3), 5.69 (s, 1H, pyrimidine-CH),
6.87–8.05 (m, 13H, Ar–H), 8.16 (s, 1H, pyrazole CH); 13C
NMR (CDCl3, 100 MHz, δ ppm): 14.36 (–CH2–CH3),
14.84, 22.92 (CH3), 56.54 (pyrimidine-CH), 61.10
(–CH2–CH3), 107.28, 114.10, 120.84, 121.78, 122.57,
122.98, 124.01, 126.76, 128.34, 129.18, 130.13, 130.94,
131.68, 132.84, 134.86, 138.88, 141.42, 143.22, 150.17,
1
1038 (C–F); H NMR (CDCl3, 400 MHz, δ ppm): 1.23 (t,
3H, J = 5.6 Hz, –CH2–CH3), 2.68 (s, 3H, pyrimidine-CH3),
3.68 (s, 3H, Ar–OCH3), 4.12 (q, 2H, J = 4.4 Hz, –
CH2–CH3), 6.25 (s, 1H, pyrimidine-CH), 6.94–7.95 (m, 14H,
Ar–H), 8.23 (s, 1H, pyrazole CH); 13C NMR (CDCl3, 100

Med Chem Res
MHz, δ ppm): 14.36 (–CH2–CH3), 21.58 (CH3), 53.86
(pyrimidine-CH), 56.05 (–OCH3), 61.87 (–CH2–CH3),
106.14, 112.86, 119.86, 121.24, 122.68, 126.53, 128.94,
129.10, 129.75, 130.57, 131.48, 134.82, 135.32, 139.17,
141.35, 150.18, 154.57, 158.25, 160.68, 162.18, 164.58
(cyclic C=O), 164.64 (ester C=O); LCMS (m/z): 595.23 [M
+H]; Anal. Calcd. for C33H27FN4O4S: C, 66.65; H, 4.58; N,
9.42. Found: C, 66.63; H, 4.60; N, 9.40.
Antifungal activity
The newly synthesized compounds were screened for their
antifungal activity against Aspergillus flavus and Fusarium
verticillioides in DMSO by serial plate dilution method
(James et al. 1970; Fenlon and Cynamon 1986). Sabour-
aud’s agar media were prepared by dissolving peptone (1 g),
D-glucose (4 g) and agar (2 g) in distilled water (100 mL)
and the pH was adjusted to 5.7. Normal saline was used to
make a suspension of the spores of the fungal strain for
lawning. A loopful of a particular fungal strain was trans-
ferred to 3 mL of saline to get a suspension of the corre-
sponding species. Agar media of 20 mL was poured into
each petri dish. An excess of the suspension was decanted,
and the plates were dried by placing them in an incubator at
37 °C for 1 h. Using an agar punch, wells were made on
these seeded agar plates, and 10–50 mg/mL of the test
compounds in DMSO was added into each well labelling.
Similarly, a control was also prepared using the solvent
DMSO. The petri dishes were prepared in triplicate and
maintained at 37 °C for 3–4 days. Antifungal activity was
determined by measuring the inhibition zone. The obtained
data was compared with the standard Nystain.
Pharmacology
Anti-inflammatory activity
The newly synthesized compounds were evaluated for their
anti-inflammatory activity against carrageenan-induced rat
paw edema assay model (Winter et al. 1962). Male Wistar
rats (200–250 g) were fasted with free access to water at
least 12 h prior to the experiments and divided randomly
into different groups (control, standard, and test groups) of
five rats each. The first group of rats was treated with 1 mL
of 1% gum acacia suspension orally (control), the second
group was administered a dose of 20 mg/kg of the Indo-
methacin (standard) and the third group was treated with 50
mg/kg suspension of the test compounds. After 1 h, the
animals were injected with 0.1 mL of 1% carrageenan in
normal saline subcutaneously to the sub-planar region of the
right hind paw. The paw volume was measured after 1, 2,
and 4 h using the Plethysmometer.
Molecular docking study
Molecular docking study was performed using AutoDock
tools 1.5.6 (ADT 4.2). The 3D structures of the compounds
in pdb form were prepared using CS ChemDraw Ultra 12.0.
The 3D crystal structure of COX-2 was downloaded from
the Protein Data Bank (PDB ID: 1CX2). Hetero atoms and
water molecules were removed and hydrogen was added to
the protein for correct calculation of the partial atomic
charges. The grid box was set at 64, 64, and 64 Å (x, y, and
z) with centre x = 25.0, y = 23.0, and z = 19.0 for 1CX2.
pdb. The Lamarckin genetic algorithm (LCA) was applied
to search for conformers that possessed the lowest binding
energy. The results of the molecular docking analysis were
obtained as estimated free energy of binding in kcal/mol.
Antimicrobial activity
Antibacterial activity
Newly synthesized pyrazoles were investigated for their
antibacterial activity against Gram-positive organism (Sta-
phylococcus aureus) and Gram-negative organisms (Kleb-
siella pneumoniae, Pseudomonas aerogenosa, and
Escherichia coli) by the disc diffusion method (Viveka et al.
2015a; Nandakumar et al. 2010). The discs 6.25 mm in
diameter were punched from Whatman No.1 filter paper.
Batches of 100 discs were dispensed in each screw-capped
bottle and sterilized by dry heat at 140 °C for an hour. The
test compounds were prepared with different concentration
using dimethylformamide (DMF). One millilitre containing
100 times the amount of chemical was added to each bottle,
which contained 100 discs, which were then placed in tri-
plicate in a nutrient agar medium separately seeded with
fresh bacteria. The incubation was carried out at 37 °C for 24
h. Solvent and growth controls were kept, and the zones of
inhibition were noted, and minimum inhibitory concentration
(MIC) for the active compounds was determined. The results
were compared with the standard Streptomycin.
Acknowledgements The authors gratefully acknowledge the UGC,
SAP, and DST-PURSE for the financial assistance. We are grateful to
IISC Bangalore, Mysore University and USIC Mangalore University
for providing the spectral analysis. We gratefully acknowledge Pra-
sanna Shama, N.G.S.M. Institute of Pharmaceutical Sciences, Paneer
Deralakatte, Mangalore, for anti-inflammatory activity and Guru
Basavarajaswamy from Acharya & B.M. Reddy College of Pharmacy
for molecular docking study. Also, thankful to Poornachandra Rao K
and Sreenivasa MY from the Department of Microbiology Manasa-
gangothri, Mysore University, Mysore, for antimicrobial study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.

Med Chem Res
micro technique for antimicrobial susceptibility testing. Appl
Microbiol 20:46–53
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