Novel pyrazole-clubbed thiophene derivatives via Gewald synthesis as antibacterial and anti-inflammatory agents

Article abstract of DOI:10.1002/ardp.202000103

The aim of this study was to synthesize newer potent Schiff bases by condensing 2-amino-5-(2,4-dichlorophenyl)thiophene-3-carbonitrile and 1,3-disubstituted-1H-pyrazole-4-carbaldehydes, and to investigate their biological activity. The compounds were synthesized via Gewald synthesis and characterized by spectral data and elemental analyses. They were screened for their in vitro antibacterial and anti-inflammatory activities. The synthesized compounds were also evaluated for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv using the microplate Alamar Blue assay. Compounds 8b, 8c, 8f, 8g, 8k, 8n, and 8o showed promising antibacterial activity. The interactions between the substituted pyrazoles and bovine protein showed promising anti-inflammatory activity. The experimental results revealed compound 8a as a promising antitubercular agent. Hemolytic assays confirmed that the compounds are nontoxic, with percentage hemolysis ranging from 3.6 to 20.1, at a concentration of 1 mg/ml. The results suggest that the pyrazole ring and the substitution pattern on the heterocyclic moiety have an effect on the bioactivity.

Full text of DOI:10.1002/ardp.202000103

Received: 2 April 2020
Revised: 11 August 2020
Accepted: 13 August 2020
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DOI: 10.1002/ardp.202000103
F U L L P A P E R
Novel pyrazole‐clubbed thiophene derivatives via Gewald
synthesis as antibacterial and anti‐inflammatory agents
Soukhyarani G. Nayak | Boja Poojary
| Vinuta Kamat
Department of Chemistry, Mangalore
University, Mangaluru, Karnataka, India
Abstract
The aim of this study was to synthesize newer potent Schiff bases by condensing
2‐amino‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile and 1,3‐disubstituted‐1H‐
pyrazole‐4‐carbaldehydes, and to investigate their biological activity. The com-
pounds were synthesized via Gewald synthesis and characterized by spectral data
and elemental analyses. They were screened for their in vitro antibacterial and anti‐
inflammatory activities. The synthesized compounds were also evaluated for in vitro
antitubercular activity against Mycobacterium tuberculosis H37Rv using the micro-
plate Alamar Blue assay. Compounds 8b, 8c, 8f, 8g, 8k, 8n, and 8o showed promising
antibacterial activity. The interactions between the substituted pyrazoles and bovine
protein showed promising anti‐inflammatory activity. The experimental results
revealed compound 8a as a promising antitubercular agent. Hemolytic assays con-
firmed that the compounds are nontoxic, with percentage hemolysis ranging from
3.6 to 20.1, at a concentration of 1 mg/ml. The results suggest that the pyrazole ring
and the substitution pattern on the heterocyclic moiety have an effect on the
bioactivity.
Correspondence
Boja Poojary, Department of Chemistry,
Mangalore University, Mangalagangothri,
Mangaluru, Karnataka 574199, India.
Email: bojapoojary@gmail.com
K E Y W O R D S
anti‐inflammatory, antimicrobial, antitubercular, Gewald reaction, Schiff bases
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1
INTRODUCTION
their imine or azomethine (–C═N–) functional group. They are regularly
used as pigments, dyes, catalysts, and intermediates in organic synth-
Over recent decades, much effort has been dedicated to the devel-
opment of new antibacterial and anti‐inflammatory agents. Specifi-
cally, there is a significant amount of publications concerning the
synthesis and evaluation of Schiff base, thiophene, and pyrazole with
a potential bacteria inhibitory activity. Nevertheless, although a great
number of synthesized compounds were found to be potent as an-
tibacterial agents, only a portion of them revealed a safer pharma-
cological profile, compared with marketed antibacterial drugs.
A Schiff base is a privileged scaffold, which is found in a great
number of medicines and drug candidates including best‐selling drugs.
Schiff bases or imines are the product of the condensation of carbonyl
compounds (aldehydes or ketones) with primary amines. These are
important molecules that are extensively studied due to their broad
range of industrial and biomedical applications.[1] These are the fore-
most widely used organic compounds in medicinal chemistry due to
esis, and as polymer stabilizers.[2] Their importance is highly growing
due to their diverse pharmacological properties like antibacterial, an-
tifungal, antimalarial, anti‐inflammatory, and antiviral properties.[3–5]
These derivatives are also extensively studied due to their
antitubercular[6–10] activity. As this functionality has shown a relatively
well response as an antitubercular agent, it has become a topic of
interest in the field of research. Furthermore, as there are few anti-
tubercular drugs available and there is an ever‐increasing fear of drug
resistance, it is becoming more and more essential for the researcher to
synthesize some more drugs with higher potency and low toxicity. In
medicinal chemistry, thiophene derivatives have been familiar with
their therapeutic applications. There are a variety of reasons why
thiophene derivatives are of interest to the pharmaceutical chemists.
One reason is based on the concept of bioisosterism. Thiophene with
its three pairs of electrons aromaticity and being sterically similar to
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Arch Pharm. 2020;e2000103.
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© 2020 Deutsche Pharmazeutische Gesellschaft
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benzene derivatives may well exhibit similar activities. At the same
time, the presence of the heteroatom in thiophene may alter its me-
tabolic fate; thus, thiophene derivatives may have less toxic effects and
properties possessed by this important structural motif have attracted
the researchers for the development of new drug molecules. Literature
reports of some bioactive 1,3‐disubstituted‐1H‐pyrazole derivatives are
presented in Figure 1.
a
better therapeutic profile.[11] Pyrazoles have proven to be
a
remarkable scaffold for the synthesis of biologically active com-
pounds.[12–14] Their wide range of medical applications mainly includes
antimicrobial,[15–17] antifungal,[13,17,18] anti‐inflammatory,[12] analgesic,
and anxiolytic activities.[18,19] Some pyrazole derivatives have also
shown antitumor activities toward some cancer cell lines, including
leukemia (K562), HeLa cervix adenocarcinoma, and Fem‐x melano-
ma.[20] Consequently, in recent years, pyrazole‐based compounds have
attracted great interest. The various biological and pharmaceutical
Recently, there has been an increase in microbial infections
dramatically, because pathogens are becoming resistant to anti-
biotics, which has led to serious health hazards. The clinical limita-
tions of currently used antimicrobial drugs, such as drug resistance,
toxicity, and side effects, lead us for the development of novel, safer,
and more effective antimicrobial agents. In this context, considerable
attention has been focused on fused heterocycles. Taking into ac-
count the broad biological activities described for the Schiff base,
FIGURE 1 Literature reports of active 1,3‐disubstituted‐1H‐pyrazole derivatives[20,21]

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thiophene, and pyrazole derivatives including their therapeutic po-
tential, we planned to synthesize thiophene and pyrazole‐clubbed
Schiff bases, and to evaluate their potential bioactivity by measuring
the antimicrobial, anti‐inflammatory, and antitubercular activity.
into 2‐amino‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile 3 in the
presence of sulfur powder and sodium bicarbonate in tetra-
hydrofuran (THF) via Gewald synthesis.
1,3‐Disubstituted‐1H‐pyrazole‐4‐carbaldehydes 7a–p were
synthesized by Vilsmeier–Haack reaction in good yields. Finally,
preparation of 2‐{[(substituted phenyl‐1H‐pyrazol‐4‐yl)methylene]
amino}‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitriles 8a–p was
done by treating the intermediate 2‐amino‐5‐(2,4‐dichlorophenyl)
thiophene‐3‐carbonitrile 3 with 1,3‐disubstituted‐1H‐pyrazole‐4‐
carbaldehydes 7a–p in the presence of catalytic amount glacial
acetic acid in ethanol with a reasonably good yield. The formation of
substituted 2‐{[(phenyl‐1H‐pyrazol‐4‐yl)methylene]amino}‐5‐(2,4‐
dichlorophenyl)thiophene‐3‐carbonitriles 8a–p (Table 1) is evi-
denced by their elemental analysis and spectral data.
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
Our strategy to synthesize new heterocyclic Schiff bases involves the
use of 1,3‐disubstituted‐1H‐pyrazole‐4‐carbaldehydes 7a–p that ex-
hibit good pharmacological properties. The reaction sequence for the
synthesis of title compounds, 8a–p, is depicted in Scheme 1. Com-
mercially available 2,4‐dichloroacetophenone 1 upon reaction with
malononitrile, ammonium acetate in toluene, and a catalytic amount
of acetic acid under reflux condition afforded 2‐[1‐(2,4‐
dichlorophenyl)ethylidene]malononitrile 2. Then it was converted
The formation of target compounds 8a–p from corresponding
1,3‐disubstituted‐1H‐pyrazole‐4‐carbaldehydes was confirmed by
spectral data (infrared [IR], 1H nuclear magnetic resonance [NMR],
13C NMR, mass spectroscopy [MS]).
SCHEME 1 An outline for the synthesis. Reagents and conditions: (a) malononitrile, ammonium acetate, acetic acid, toluene, reflux: (b)
tetrahydrofuran, sulfur powder, sodium bicarbonate, water, room temperature (rt), 24 hr; (c) AcOH, MeOH, reflux, 4 hr; (d) DMF/POCl3, 0–5°C,
rt, 2 hr; (e) 7a–p, ethanol, catalytic amount of acetic acid

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TABLE 1 Physical data of the newly synthesized substituted 2‐{[(phenyl‐1H‐pyrazol‐4‐yl)methylene]amino}‐5‐(2,4‐dichlorophenyl)thiophene‐
3‐carbonitriles (8a–p)
Compound no.
Ar
R
Chemical formula
C27H16Cl2N4S
Molecular weight (g/mol)
499.41
Yield
73
76
71
75
73
77
77
73
74
71
70
72
73
75
72
74
Melting point (°C)
248–250
216–218
202–204
196–198
214–216
204–206
212–214
246–248
211–213
242–244
232–234
220–222
238–240
246–248
210–212
235–237
8a
8b
8c
8d
8e
8f
C6H5
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl‐C6H4
4‐F‐C6H4
4‐Br‐C6H4
3‐Br‐C6H4
4‐OCH3‐C6H4
4‐CH3‐C6H4
3‐Thienyl
C6H5
C27H15Cl3N4S
533.86
C27H15Cl2FN4S
C27H15Cl2BrN4S
C27H15Cl2BrN4S
C28H18Cl2N4OS
C28H18Cl2N4S
517.40
578.31
578.31
529.44
8g
8h
8i
513.44
C
25H14Cl2N4S2
505.44
C27H15Cl3N4S
C27H14Cl4N4S
C27H14Cl3FN4S
C27H14Cl3BrN4S
C27H14Cl3BrN4S
C28H17Cl3N4OS
C28H17Cl3N4S
C25H13Cl3N4S2
533.86
8j
4‐Cl‐C6H4
4‐F‐C6H4
4‐Br‐C6H4
3‐Br‐C6H4
4‐OCH3‐C6H4
4‐CH3‐C6H4
3‐Thienyl
568.30
8k
8l
551.85
612.75
8m
8n
8o
8p
612.75
563.88
547.88
539.88
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2.2
Biological activity
standard drug (ciprofloxacin). However, in the case of Gram‐positive
bacterial strains (S. aureus and E. faecalis), the synthesized compounds
showed a moderate activity. From the screening results, it could be
seen that the majority of the tested compounds displayed a moderate‐
to‐good antibacterial activity (MIC values of 1.56–12.5 µg/ml) as
compared with the standard drug (ciprofloxacin). The antibacterial
screening of compounds 8a–p revealed that halogen‐, methoxy‐, and
methyl‐substituted derivatives are highly active.
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2.2.1
Antibacterial activity
It is well known that pyrazole derivatives and also Schiff bases have
unveiled a promising antimicrobial activity. Therefore, antibacterial
potency of newly synthesized derivatives (8a–p) was evaluated by
determining their antibacterial activity using the broth dilution
method.[22] Two Gram‐positive bacteria, Staphylococcus aureus and
Enterococcus faecalis, and two Gram‐negative bacteria, Escherichia coli
and Pseudomonas aeruginosa, were selected to investigate the anti-
microbial activity. Ciprofloxacin was used as a reference drug. The
minimum inhibitory concentration (MIC) was defined as the lowest
concentration without visible growth. The results of the preliminary
antibacterial activity of compounds 8a–p are presented in Table 2. The
results obtained revealed that almost all the newly synthesized com-
pounds showed excellent antibacterial activity against Gram‐negative
bacterial strains (E. coli and P. aeruginosa) as compared with the
The antibacterial bioscreening data given in Table 2 displayed
that most of the Schiff bases were potent. Among all the synthesized
compounds, 8b and 8j, with a 4‐chlorophenyl ring; 8c and 8k, bonding
to 4‐fluorophenyl ring; 8f and 8n, possessing 4‐methoxyphenyl ring;
and 8g and 8o, bearing 4‐methylphenyl ring, derivatives showed a
good activity as compared with the standard drug (ciprofloxacin).
Compound 8b (Ar = 4‐Cl‐C6H4 and R = 4‐H) showed an MIC va-
lue of 1.56 µg/ml against E. faecalis (Gram‐positive) and P. aeruginosa
(Gram‐negative). But derivative 8b (Ar = 4‐Cl‐C6H4 and R = 4‐H) did
not show a good activity against S. aureus and E. coli. One more

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TABLE 2 Antibacterial activity of synthesized compounds, 8a–p, by the broth dilution method (incubation time 24 hr at 37°C)
Antibacterial activity data of the target compounds (8a–p) in terms of minimum inhibitory concentration in µg/ml (MIC) (incubation time 24 hr
at 37°C)
Gram‐positive
Gram‐negative
Escherichia coli
3.125
12.5
Entry
8a
8b
8c
Ar
R
Staphylococcus aureus
Enterococcus faecalis
Pseudomonas aeruginosa
C6H5
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐H
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
4‐Cl
6.25
12.5
1.56
3.125
12.5
1.56
50
3.125
1.56
12.5
25
1.56
1.56
1.56
25
4‐Cl‐C6H4
4‐F‐C6H4
4‐Br‐C6H4
3‐Br‐C6H4
4‐OCH3‐C6H4
4‐CH3‐C6H4
3‐Thienyl
C6H4
12.5
8d
8e
8f
1.56
1.56
12.5
25
3.125
1.56
3.125
12.5
1.56
25
8g
8h
8i
1.56
50
25
50
12.5
25
3.125
12.5
1.56
3.125
3.125
1.56
3.125
25
6.25
25
8j
4‐Cl‐C6H4
4‐F‐C6H4
4‐Br‐C6H4
3‐Br‐C6H4
4‐OCH3‐C6H4
4‐CH3‐C6H4
3‐Thienyl
Ciprofloxacin
1.56
3.125
1.56
3.125
1.56
3.125
1.56
25
8k
8l
3.125
12.5
3.125
12.5
1.56
50
3.125
1.56
8m
8n
8o
8p
Std
3.125
1.56
3.125
25
6.25
6.25
3.125
6.25
Note: Significance of bold face at lower value of concentration the derivative are active.
Abbreviation: MIC, minimum inhibitory concentration.
chloro‐substituted derivative, 8j (Ar = 4‐Cl‐C6H4 and R = 4‐Cl), was
found to be highly active against E. coli at a lower concentration
(1.56 mg/ml). When chloro is replaced by fluoro compound, 8c
(Ar = 4‐F‐C6H4 and R = 4‐H) showed a good MIC value of 1.56 µg/ml
against two bacterial strains, namely S. aureus and P. aeruginosa, but
failed to show activity against E. faecalis and E. coli bacterial strains.
However, compound 8k (Ar = 4‐F‐C6H4 and R = 4‐Cl) showed a good
activity against E. faecalis and P. aeruginosa. Halogen derivatives
showed good activity results, which are summarized in Table 2.
Schiff bases possessing 4‐methoxyphenyl ring were highly active.
Among these, compound 8f (Ar = 4‐OMe‐C6H4 and R = 4‐H) showed
an activity at a lower concentration (1.56 mg/ml) against S. aureus and
E. coli, and it was least active against E. faecalis and P. aeruginosa. In a
similar way, compound 8f (Ar = 4‐OMe‐C6H4 and R = 4‐Cl) was active
against E. faecalis and E. coli. Methyl‐substituted derivatives also
showed good activity results. Compounds 8g (Ar = 4‐Me‐C6H4 and
R = 4‐H) and 8o (Ar = 4‐Me‐C6H4 and R = 4‐Cl) were found to be
active against S. aureus, E. coli, and P. aeruginosa bacterial strains.
Remaining derivatives were moderately active against the tested
bacterial strains as compared with the standard drug (ciprofloxacin).
However, compounds 8a–p were less active against Gram‐positive
bacteria (S. aureus and E. faecalis) and active against Gram‐negative
bacteria (E. coli and P. aeruginosa) as compared with the standard drug
(ciprofloxacin). The results obtained are presented in Table 2.
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2.2.2
Anti‐inflammatory activity
Inflammation is a biological response of the body to the harmful
pathogens, and alteration of neighboring protein tissue is one of the
causes of inflammatory condition. There are complications in using
animal models in in vivo research, such as lack of groundwork, lack of
maintenance, lack of animal risk management, and ethical disputes
for their use. Inflammation is associated with pain, and it involves an
increase in protein denaturation. In vivo denaturation of proteins
leads to the synthesis of autoantigens in some arthritic diseases,[23]
and therefore the compounds that inhibit denaturation are desirable
for anti‐inflammatory discovery. The worthwhile condition for anti‐
inflammatory drug discovery is to stop the protein denaturation by
the formation of complexes. Nonsteroidal anti‐inflammatory drugs
(NSAIDs) are commonly used for the management of inflammatory
conditions. Heat‐induced protein denaturation prevention by syn-
thesized compounds was analyzed, using diclofenac sodium as a
standard drug. One of the properties noticed in several NSAIDs is to

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TABLE 3 Anti‐inflammatory activity results of substituted 2‐
{[(phenyl‐1H‐pyrazol‐4‐yl)methylene]amino}‐5‐(2,4‐dichlorophenyl)-
thiophene‐3‐carbonitrile derivatives 8a–p
20–100 mg/ml and are summarized in Table 3. A graphical re-
presentation of anti‐inflammatory results of synthesized compounds
is presented in Figure 2. From the results, it was found that most of
the compounds show an excellent anti‐inflammatory activity with
IC50 values close to the standard drug. Electron‐withdrawing
halogen‐substituted compounds 8b (Ar = 4‐Cl‐C6H4 and R = 4‐H), 8d
(Ar = 4‐Br‐C6H4 and R = 4‐H), 8e (Ar = 3‐Br‐C6H4 and R = 4‐H), 8i
(Ar = C6H5 and R = 4‐Cl), and 8m (Ar = 3‐Br‐C6H4 and R = 4‐Cl)
showed a noticeable anti‐inflammatory activity with IC50 values of
34.1, 34.3, 46.3, 40.3, and 49 μg/ml, respectively, when comparing
with the standard drug. Electron‐withdrawing functional groups in
the para position of the phenyl group enhance the anti‐inflammatory
activity. In the same way, electron‐donating –H in 8a (Ar = 4‐H‐C6H4
and R = 4‐H), –OCH3 in 8f (Ar = 4‐OCH3‐C6H4 and R = 4‐H), and ‐3‐
thiophene in 8h (Ar = 3‐thienyl and R = 4‐H) and 8p (Ar = 3‐thienyl
and R = 4‐Cl) groups of phenyl ring also showed a more potent in-
hibitory action by obtaining IC50 values of 40.3, 41, 40.9, and
42.8 μg/ml, respectively, when compared with the IC50 value, 31.4, of
diclofenac sodium (control). Compounds 8g and 8o showed the least
activity with IC50 values of 104.5 and 90 μg/ml, respectively, and
remaining compounds (8c, 8j, 8k, 8l, and 8n) showed a moderate
activity against the protein denaturation of bovine albumin. Overall,
the compound 8b showed a remarkable anti‐inflammatory activity,
and remaining compounds (8a, 8d, 8e, 8f, 8h, 8i, 8m, and 8p) having
electron‐withdrawing and ‐donating substitutions at para position of
phenyl ring showed a good anti‐inflammatory activity. By observing
these results, we conclude that the activity result enhances on the
basis of the substitution on the phenyl ring.
Inhibition of protein denaturation (%)
Compounds
Conc. (μg/ml) 20
40
60
80
100
93.2
86
IC50 (µg/ml)
31.4
Standard
8a
35.4
58.1
43.1
50.5
32.8
50.5
33
71.2
65.8
69.9
47.5
67.3
55
82.6
74.1
79
27.2
34.1
13.9
33.1
26
40.3
8b
8c
87
34.1
69
82.3
92.6
86
60.9
8d
8e
82.1
68
34.3
46.3
8f
28.3
2.9
46.5
18.8
42.8
42.9
38.3
28.3
28.8
32.5
34.1
22.8
42.5
57.1
25.2
54.6
59.5
42.6
34.6
32.1
41.6
42.5
31.5
59.6
69.8
34.6
71.2
78.2
50.1
51.3
65.1
68.9
56.9
48.6
68.9
88.3
48.9
83.9
87.1
70.9
78.6
78.2
89.3
76.5
59.3
79.9
41.0
8g
104.5
40.9
8h
8i
34.3
28.6
19.7
19.6
16.2
25.3
24.3
15.3
30.2
40.3
8j
65.4
8k
66.1
8l
64.8
8m
8n
8o
8p
49.0
58.5
90.0
42.8
Note: Significance of bold face at lower value of concentration the
derivative are active.
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Antitubercular activity
alleviate heat‐exposed albumin denaturation at a physiological pH
range of 6.2–6.5.[24] However, these drugs have several adverse side
effects, especially gastric irritation, leading to the formation of gastric
ulcers. Therefore, the search for natural sources and phytochemicals
with anti‐inflammatory activity has greatly increased in recent years.
As a part of the investigation on the mechanism of the anti‐
inflammatory activity, the ability of the synthesized derivatives,
8a–p, to inhibit protein denaturation was studied using diclofenac
sodium as a standard drug. IC50 values showed the concentration‐
dependent inhibition of protein denaturation in the range of
2.2.3
The antitubercular properties of all prepared compounds were as-
sessed against Mycobacterium tuberculosis at different concentrations
ranging from 0.2 to 100 µg/ml using streptomycin, ciprofloxacin, and
pyrazinamide as standard drugs. The results of in vitro antitubercular
activity of the synthesized compounds against M. tuberculosis H37Rv
are listed in Table 4. Few compounds were found active against M.
tuberculosis, as shown in Figure 3. M. tuberculosis is unique due to
thick and waxy surrounding cell walls, owing to which effective
FIGURE 2 A graphical representation of in
vitro protein denaturation of Schiff base
analogs 8a–p with the standard drug

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TABLE 4 Antitubercular activity of compounds by the microplate Alamar Blue assay method against Mycobacterium tuberculosis
Compound no.
Ar
R
100 µg/ml
50 µg/ml
25 µg/ml
12.5 µg/ml
6.25 µg/ml
3.125 µg/ml
1.6 µg/ml
0.8 µg/ml
8b
8j
4‐Cl‐C6H4
4‐Cl‐C6H4
4‐F‐C6H4
4‐Br‐C6H4
4‐OCH3‐C6H4
4‐CH3‐C6H4
3‐Thienyl
C6H5
4‐H
4‐Cl
4‐Cl
R
R
R
R
S
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
8k
8l
8n
8o
8p
8a
4‐Cl
4‐Cl
4‐Cl
4‐H
S
R
S
Abbreviations: R, resistant; S, sensitive.
antitubercular drug should be reasonably lipophilic to penetrate the
bacterial cell wall.[25] The MIC was defined as the lowest drug con-
centration that prevented the color change from blue to pink. The
lowest concentration where the fragment shows a blue color is the
MIC value. In Table 4, we have highlighted the compounds in terms of
sensitivity and resistivity based on their activeness. The term re-
sistance indicated that the compound is not active at a particular
concentration. The term sensitive refers to the color change and that
the compound is active at a particular concentration.
were resistant at the lower concentration. Further studies for the
higher concentration for the compound can be done.
|
2.2.4
Hemolytic assay
The safety of the products is one of the major questions to address
during the drug development process. Many of the biologically active
compounds may pose toxicity issues in terms of hemolysis, char-
acterized by the rupture of red blood cells (erythrocytes) to release
hemoglobin. The free hemoglobin in plasma may cause damage to
Among the tested compounds, the compound that contains only
the phenyl group on the aryl part showed inhibition at 50 µg/ml
concentration, which means the compound is sensitive; in other
words, in the next concentration, it changed color from blue to pink.
Among the tested compounds that contain methyl and chlorine
substitution on the aryl part also showed the color change from blue
to pink at a concentration of 100 µg/ml. The remaining compounds
TABLE 5 The toxicity assay of the compounds in terms of
percentage hemolysis at a concentration of 1 mg/ml
Compound
% Hemolysis
4.81
8a
8b
8c
8d
8e
8f
6.07
5.35
6.02
8.98
20.1
8g
8h
8i
10.64
3.6
5.04
8j
8.25
8k
8l
12.54
6.91
8m
8n
8o
8p
13.1
6
5.25
18.26
Note: Significance of bold face at lower value of concentration the
FIGURE 3 The antitubercular activity of the synthesized
compounds
derivative are active.

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NAYAK ET AL.
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vital organs, such as heart, kidney, and liver.[26,27] Hence, it is es-
sential to investigate the hemolytic activity of the biologically active
compounds. One of the simplest, inexpensive, and quick methods for
preliminary toxicity evaluation is the hemolytic assay, as it can be
evident from several reports that the results obtained by this method
complement those obtained by the cytotoxicity assay carried out
against normal cell lines.[28,29] To test the preliminary toxicity, the
compounds were treated with human erythrocytes (red blood cells)
for hemolysis; the results are presented in Table 5. Most of the
compounds were nontoxic, so they were found to be safe even at
higher concentrations.
4.1.2
General synthetic procedure for 2‐[1‐(2,4‐
|
dichlorophenyl)ethylidene]malononitrile (2)[30]
Malononitrile (1.1 mmol), ammonium acetate (0.1 mmol), and acetic
acid (0.2 mmol) were added to the solution of 1‐(2,4‐dichlorophenyl)
ethan‐1‐one (1; 1 mmol) in dry toluene. The reaction mixture was
maintained under reflux condition for 24–28 hr using a Dean–Stark
water separator. Completion of the reaction was monitored by TLC.
Upon cooling the reaction mixture, the solid product was separated.
The crude product was recrystallized from ethyl acetate and toluene
mixture to afford the title compound in a 75% yield.
|
|
3
CONCLUSION
4.1.3
General synthetic procedure for 2‐amino‐5‐
(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (3)[30]
A library of new pyrazole‐clubbed thiophene derivatives was syn-
thesized and evaluated for their antibacterial, anti‐inflammatory, and
antitubercular studies. Compounds 8a, 8b, 8d, 8e, 8f, 8g, 8h, 8i, 8k,
8m, 8n, 8o, and 8p showed high potency toward both antimicrobial
and anti‐inflammatory activitites when compared with standard
drugs ciprofloxacin and diclofenac sodium, respectively. Compound
8a exhibited a potent activity against M. tuberculosis strain. These
results suggested that it is a new and potential route in the discovery
of drug against antibacterial, anti‐inflammatory, and antitubercular
activities.
A mixture of 2‐[1‐(2,4‐dichlorophenyl)ethylidene]malononitrile (2;
1 mmol) and sulfur powder (1.3 mmol) was suspended in THF (25 ml)
and warmed to an internal temperature of 35°C. A solution of sodium
bicarbonate (1.4 mmol) in 5 ml of water was added over a period of
1 hr. The mixture was stirred for 24 hr. After completion of the re-
action (TLC), the reaction mass was cooled and quenched to ice‐cold
water then filtered and washed with water. The crude product was
recrystallized from toluene and ethyl acetate mixture to afford
compound 3 in a 75% yield.
|
|
4
EXPERIMENTAL
4.1.4
General procedure for synthesis of 1‐(aryl)‐2‐
[1‐(aryl)ethylidene]hydrazines 6a–p[30]
|
4.1
4.1.1
Chemistry
To a solution of substituted acetophenones 4a–h (5 g, 19 mmol) in
methanol (40 ml), substituted phenyl hydrazine 5a, 5b (2.1 g,
19 mmol) was added, followed by glacial acetic acid (15 ml), at room
temperature. The resulting reaction mixture was heated to reflux for
4 hr. After the reaction solvent was evaporated under reduced
pressure, and the resulting mass was stirred for 15 min with ice
water (25 ml) at 10°C. The precipitated solid was filtered washed
with ice water (20 ml), followed by n‐pentane (20 ml) and dried
under vacuum to afford 1‐(1‐(substituted phenyl)ethylidene)‐2‐
phenylhydrazines 6a–p.
|
General
All the required chemicals and reagents for this study were obtained
from Merck, S.D. Fine, and Sigma‐Aldrich. The reagents and solvents
purchased were of analytical grade and were used without purifica-
tion. The melting points of the synthesized compounds were de-
termined in open glass capillaries and were uncorrected. FT(ATR)‐IR
absorption spectra were recorded on Thermo Nicolet, Avatar 370 in
range 4,000–400 cm−1 1H NMR spectra and 13C NMR spectra were
.
recorded on Bruker Avance III, 500 MHz, and chemical shift values
were expressed in δ ppm. Mass spectra were recorded on Waters
Synapt G2 high detection mass spectrometer and were uncorrected.
Elemental analysis was carried out using CHNS Elementar Vario EL
III. All reactions were monitored by thin‐layer chromatography (TLC)
analyses and were performed on silica plates; spots were visualized
either by UV light or on exposure to iodine vapors. Hexane/ethyl
acetate (9:1) was adopted as a solvent system. Compounds were
prepared according to reported procedures.
|
4.1.5
General procedure for the synthesis of
1,3‐diaryl‐1H‐pyrazole‐4‐carbaldehydes 7a–p[30]
Phosphorus oxychloride (6.2 ml, 66 mmol) was added to N,N‐
dimethylformamide (DMF; 26.5 ml, 339 mmol) at −5°C to 0°C drop-
wise over a period of 10 min and stirred at the same temperature for
30 min, followed by the addition of 6a–p (6.6 g, 22 mmol) dissolved in
DMF (10 ml) dropwise at −5°C over a period of 15 min. The reaction
mixture was allowed to attain room temperature and stirred for
1.5 hr. After completion (TLC), the reaction mixture was slowly added
to ice‐cold water and basified with saturated NaHCO3 solution to
The original spectra of the investigated compounds are pro-
vided as Supporting Information Data. Their InChI codes, together
with the biological activity data, are also provided as Supporting
Information Data.

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NAYAK ET AL.
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adjust the pH at 7–8. The mixture was then stirred for 1 hr at room
temperature and the precipitated solid was filtered, washed with
water, and dried under vacuum to get the crude compound (7a–p).
The crude compound was washed with methanol, filtered, and dried
under vacuum.
(100 MHz, DMSO, δ ppm): 82.8, 113.0, 115.3, 119.9, 123.1, 125.5,
126.2, 127.4, 128.3, 129.3, 130.3, 130.9, 132.0, 132.1, 133.6, 135.0,
135.7, 139.7, 141.7, 150.4, 160, 160.3; EI–MS: m/z [M+], 516,
[M+2+H], 518; anal. calcd. for C27H15Cl2FN4S (517.40) C, 62.68; H,
2.92; N, 10.83. Found: C, 62.67; H, 2.93; N, 10.84%.
2‐({[3‐(4‐Bromophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]methylene}amino)‐
5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8d)
|
4.1.6
General procedure for 2‐{[(aryl‐1H‐pyrazol‐4‐
yl)methylene]amino}‐5‐(2,4‐dichlorophenyl)-
thiophene‐3‐carbonitrile derivatives 8a–p
Yield: 75%; m.p.: 196–198°C; IR (KBr, νmax in cm−1): 3,354 (C–H),
2,215 (C≡N), 1,605 (C═N), 1,435 (N═N), 1,257 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30 (1H, s, Ar‐H), 7.41–7.55 (6H, m, Ar‐H),
7.65–7.70 (4H, m, Ar‐H), 7.78 (2H, d, J = 8 Hz), 8.44 (1H, s, Py‐H), 8.46
(1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113.0, 115.3,
119.9, 123.1, 125.5, 126.2, 127.4, 128.3, 129.3, 130.3, 130.9, 132.0,
132.1, 133.6, 135, 135.7, 139.7, 141.7, 150.4, 160, 160.3; EI–MS: m/z
[M+], 576, [M+2+H], 578; anal. calcd. for C27H15Cl2BrN4S (578.31) C,
56.08; H, 2.61; N, 9.69. Found: C, 56.07; H, 2.62; N, 9.69%.
2‐Amino‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile 3 (1 mmol)
and 1,3‐disubstituted‐1H‐pyrazole‐4‐carbaldehydes 8a–p (1 mmol)
were dissolved in 20 ml of warm ethanol containing a catalytic
amount of glacial acetic acid. The reaction mixture was refluxed for
5–7 hr. The major part of the product was precipitated when the
reaction mixture was hot. The solid formed on cooling was filtered,
washed with hot ethanol, and dried under vacuum to give respective
Schiff bases 8a–p. The crude material was purified with ethyl acetate
recrystallization.
2‐({[3‐(3‐Bromophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]methylene}amino)‐
5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8e)
Yield: 73%; m.p.: 214–216°C; IR (KBr, νmax in cm−1): 3,354 (C–H),
2,215 (C≡N), 1,605 (C═N), 1,435 (N═N), 1,257 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30 (1H, s, Ar‐H), 7.41–7.55 (6H, m, Ar‐H),
7.65–7.70 (4H, m, Ar‐H), 7.78 (2H, d, J = 8 Hz), 8.44 (1H, s, Py‐H), 8.46
(1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113.0, 115.3,
119.9, 122.2, 125.5, 126.2, 127.4, 128.3, 129.3, 130.3, 130.9, 132.0,
132.1, 133.6, 135, 135.7, 139.7, 141.7, 150.4, 160, 160.3; EI–MS: m/z
[M+], 576, [M+2+H], 578; anal. calcd. for C27H15Cl2BrN4S (578.31) C,
56.08; H, 2.61; N, 9.69. Found: C, 56.09; H, 2.62; N, 9.68%.
5‐(2,4‐Dichlorophenyl)‐2‐{[(1,3‐diphenyl‐1H‐pyrazol‐4‐yl)methylene]-
amino}thiophene‐3‐carbonitrile (8a)
Yield: 73%; m.p.: 248–250°C; IR (KBr, νmax in cm−1): 3,143 (C–H),
2,411 (C≡N), 1,623 (C═N), 1,456 (N═N), 1,312 (C═C); 1H (500 MHz,
dimethyl sulfoxide [DMSO]‐d6, δ ppm): 7.30 (1H, s, Ar‐H), 7.41–7.54
(7H, m, Ar‐H), 7.65–7.70 (6H, m, Ar‐H), 8.44 (1H, s, Py‐H), 8.46 (1H, s,
–N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113.0, 115.3, 119.9,
125.5, 126.2, 127.4, 127.5, 128.7, 129.2, 129.3, 130.3, 130.9, 133,
133.6, 135, 135.7, 139.7, 141.7, 150.4, 160.0, 160.3; electron ioni-
zation mass spectroscopy (EI–MS): m/z [M+], 498, [M+2+H]+, 500;
anal. calcd. for C27H16Cl2N4S (499.41) C, 64.94; H, 3.23; N, 11.22.
Found: C, 64.96; H, 3.25; N, 11.23%.
5‐(2,4‐Dichlorophenyl)‐2‐({[3‐(4‐methoxyphenyl)‐1‐phenyl‐1H‐
pyrazol‐4‐yl]methylene}amino)thiophene‐3‐carbonitrile (8f)
Yield: 77%; m.p.: 204–206°C; IR (KBr, νmax in cm−1): 2,224 (C≡N),
1,594 (C═N), 1,484 (C═C), 754 (C–Cl); 1H (500 MHz, DMSO‐d6, δ
ppm): 3.83 (3H, s, –OCH3), 7.06–7.09 (2H, m, Ar‐H), 7.43 (1H, t, Ar‐
H), 7.54–7.60 (5H, m, Ar‐H), 7.84 (1H, d, J = 2 Hz), 7.96–7.98 (2H, m,
Ar‐H), 8.03 (2H, d, J = 8.75 Hz), 8.71 (1H, s, Py‐H), 9.25 (1H, s,
–N═CH); 13C (100 MHz, DMSO, δ ppm): 55.7, 105.1, 114.4, 114.5,
114.8, 118.8, 119.6, 121.7, 124.3, 128.0, 128.2, 129.8, 130.2, 130.9,
132, 132.8, 133.4, 133.8, 134.9, 137.2, 139.1, 153.6, 156.0, 160.4,
165.1; EI–MS: m/z [M+], 528.00, [M+2+H]+, 530.00; anal. calcd. for
C28H18Cl2N4OS (529.44) C, 63.52; H, 3.43; N, 10.58. Found: C, 63.53;
H, 3.44; N, 10.58%.
2‐({[3‐(4‐Chlorophenyl)‐1‐phenyl‐1H‐pyrazol‐4‐yl]methylene}amino)‐
5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8b)
Yield: 76%; m.p.: 216–218°C; IR (KBr, νmax in cm−1): 2,229 (C≡N),
1,593 (C═N), 754 (C–Cl); 1H (500 MHz, DMSO‐d6, δ ppm): 7.45 (1H,
s, Ar‐H), 7.54–7.60 (7H, m, Ar‐H), 7.84 (1H, d, Ar‐H, J = 1.5 Hz), 8.02
(2H, d, Ar‐H, J = 8 Hz), 8.12 (2H, d, Ar‐H, J = 8.5 Hz), 8.75 (1H, s, Py‐
H), 9.29 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 84.23, 105.5,
114.7, 119.1, 119.7, 121.9, 128.2, 129.0, 129.8, 130.2, 130.9, 131.3,
131.9, 133.4, 133.8, 133.9, 134.2, 134.9, 137.1, 139.0, 152.1, 155.6,
164.8; EI–MS: m/z [M+], 532, [M+2+H]+, 534, [M+4+H]+, 536; anal.
calcd. for C27H15Cl3N4S (533.86) C, 60.75; H, 2.83; N, 10.49. Found:
C, 60.74; H, 2.85; N, 10.48%.
5‐(2,4‐Dichlorophenyl)‐2‐({[1‐phenyl‐3‐(p‐tolyl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)thiophene‐3‐carbonitrile (8g)
Yield: 77%; m.p.: 212–214°C; IR (KBr, νmax in cm−1): 3,117 (C–H),
2,262 (C≡N), 1,631 (C═N), 1,497 (N═N), 1,320 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 2.34 (3H, s, –CH3), 7.15 (2H, d, J = 8 Hz), 7.30 (1H,
s, Ar‐H), 7.41–7.70 (10H, m, Ar‐H), 8.44 (1H, s, Py‐H), 9.32 (1H, s,
–N═CH); 13C (100 MHz, DMSO, δ ppm): 21.3, 82.8, 113.0, 114.8,
115.3, 119.9, 125.3, 125.5, 126.2, 127.4, 128.5, 129.5, 130.3, 130.9,
133.6, 135, 135.7, 139.7, 141.7, 150.4, 160.0, 160.3; EI–MS: m/z
5‐(2,4‐Dichlorophenyl)‐2‐({[3‐(4‐fluorophenyl)‐1‐phenyl‐1H‐pyrazol‐
4‐yl]methylene}amino)thiophene‐3‐carbonitrile (8c)
Yield: 71%; m.p.: 202–204°C; IR (KBr, νmax in cm−1): 3,122 (C–H),
2,383 (C≡N), 1,659 (C═N), 1,429 (N═N), 1,343 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30–7.52 (7H, m, Ar‐H), 7.65–7.70 (4H, m, Ar‐H),
7.86 (2H, d, J = 8 Hz), 8.44 (1H, s, Py‐H), 9.32 (1H, s, –N═CH); 13C

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NAYAK ET AL.
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[M+], 512, [M+2+H]+, 514; anal. calcd. for C28H18Cl2N4S (513.44) C,
65.50; H, 3.53; N, 10.91. Found: C, 65.51; H, 3.53; N, 10.92%.
(551.85) C, 58.77; H, 2.56; N, 10.15. Found: C, 58.77; H, 2.58;
N, 10.16%.
5‐(2,4‐Dichlorophenyl)‐2‐({[1‐phenyl‐3‐(thiophen‐3‐yl)‐1H‐pyrazol‐
4‐yl]methylene}amino)thiophene‐3‐carbonitrile (8h)
2‐({[3‐(4‐Bromophenyl)‐1‐(4‐chlorophenyl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8l)
Yield: 72%; m.p.: 220–222°C; IR (KBr, νmax in cm−1): 3,162 (C–H),
2,347 (C≡N), 1,601 (C═N), 1,403 (N═N), 1,320 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30–7.41 (4H, m, Ar‐H), 7.55 (4H, d, J = 8 Hz),
7.65–7.70 (2H, m, Ar‐H), 7.78 (2H, d, J = 8 Hz), 8.44 (1H, s, Py‐H), 8.46
(1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113, 115.3, 116,
119.8, 123.1, 125.5, 127.4, 128.3, 129.4, 130.3, 132, 132.1, 133.6, 135,
135.7, 137.8, 141.7, 150.4, 160.0, 160.3; EI–MS: m/z [M+], 610,
[M+2+H]+, 612, [M+4+H]+, 614; anal. calcd. for C27H14Cl3BrN4S
(612.75) C, 52.92; H, 2.30; N, 9.14. Found: C, 52.93; H, 2.31; N, 9.15%.
Yield: 73%; m.p.: 246–248°C; IR (KBr, νmax in cm−1): 3,182 (C–H),
2,462 (C≡N), 1,679 (C═N), 1,497 (N═N), 1,300 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30 (1H, s, Ar‐H), 7.38–7.52 (5H, m, Ar‐H),
7.65–7.70 (4H, m, Ar‐H), 7.89 (1H, d, J = 8 Hz), 7.98 (1H, s, Ar‐H), 8.46
(1H, s, Py‐H), 8.47 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm):
82.8, 114.0, 115.3, 119.9, 121.7, 125.5, 126.2, 127.4, 128.2, 128.3,
129.3, 130.3, 130.9, 133.3, 133.6, 135.0, 135.7, 139.7, 141.7, 142.3,
160.0, 160.3; EI–MS: m/z [M+], 504, [M+2+H]+, 506; anal. calcd. for
C25H14Cl2N4S2 (505.44) C, 59.41; H, 2.79; N, 10.09. Found: C, 59.42;
H, 2.78; N, 10.09%.
2‐({[3‐(3‐Bromophenyl)‐1‐(4‐chlorophenyl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐
2‐({[1‐(4‐Chlorophenyl)‐3‐phenyl‐1H‐pyrazol‐4‐yl]methylene}amino)‐
5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8i)
carbonitrile (8m)
Yield: 74%; m.p.: 211–213°C; IR (KBr, νmax in cm−1): 3,119 (C–H),
2,222 (C≡N), 1,591 (C═N), 1,482 (N═N); 1H (500 MHz, DMSO‐d6, δ
ppm): 7.16–7.18 (1H, m, Ar‐H), 7.56–7.71 (7H, m, Ar‐H), 7.84 (1H, d,
J = 1.5 Hz), 7.97–8.00 (3H, m, Ar‐H), 8.45 (1H, d, J = 3.5 Hz), 8.84 (1H,
s, Py‐H), 9.29 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 105.7,
114.8, 118.7, 121.1, 121.2, 122.1, 128.2, 128.5, 129.6, 129.8, 130.2,
131.9, 132.3, 132.5, 133.4, 133.7, 133.8, 133.9, 134.7, 134.9, 137.1,
137.2, 137.6, 147.7, 155.2, 164.9; EI–MS: m/z [M+], 532, [M+2+H]+,
534, [M+4+H]+, 536; anal. calcd. for C27H15Cl3N4S (533.86) C, 60.75;
H, 2.83; N, 10.49. Found: C, 60.76; H, 2.84; N, 10.48%.
Yield: 73%; m.p.: 238–240°C; IR (KBr, νmax in cm−1): 3,287 (C–H),
2,242 (C≡N), 1,622 (C═N), 1,441 (N═N), 1,241 (C═C), 731 (C–Cl); 1H
(500 MHz, DMSO‐d6, δ ppm): 7.30–7.45 (6H, m, Ar‐H), 7.55 (2H, d,
J = 8 Hz), 7.65–7.78 (4H, m, Ar‐H), 8.44 (1H, s, Py‐H), 8.46 (1H, s,
–N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113, 115.3, 116, 119.8,
123.1, 125.5, 127.4, 128.3, 129.4, 130.3, 132, 132.1, 133.6, 135, 135.7,
137.8, 141.7, 150.4, 160.0, 160.3; EI–MS: m/z [M+], 610, [M+2+H]+,
612, [M+4+H]+, 614; anal. calcd. for C27H14Cl3BrN4S (612.75) C,
52.92; H, 2.30; N, 9.14. Found: C, 52.93; H, 2.31; N, 9.15%.
2‐({[1‐(4‐Chlorophenyl)‐3‐(4‐methoxyphenyl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐
2‐({[1,3‐Bis(4‐chlorophenyl)‐1H‐pyrazol‐4‐yl]methylene}amino)‐5‐
(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8j)
carbonitrile (8n)
Yield: 71%; m.p.: 242–244°C; IR (KBr, νmax in cm−1): 3,182 (C–H),
2,124 (C≡N), 1,582 (C═N), 1,404 (N═N), 1,217 (C═C), 723 (C–Cl); 1H
(500 MHz, DMSO‐d6, δ ppm): 7.30 (1H, s, Ar‐H), 7.35–7.41 (3H, m,
Ar‐H), 7.52–7.55 (4H, m, Ar‐H), 7.65–7.70 (2H, m, Ar‐H), 7.98 (2H, d,
J = 8 Hz), 8.44 (1H, s, Py‐H), 8.46 (1H, s, –N═CH); 13C (100 MHz,
DMSO, δ ppm): 82.8, 113, 115.3, 119.8, 125.5, 127.4, 128.9, 129.3,
129.4, 130.3, 130.9, 131.1, 131.8, 133.6, 134.3, 135, 135.7, 137.8,
141.7, 150.4, 160.0, 160.3; EI–MS: m/z [M+], 566, [M+2+H]+, 568,
[M+4+H], 570; anal. calcd. for C27H14Cl4N4S (568.30) C, 57.06; H,
2.48; N, 9.86. Found: C, 57.07; H, 2.49; N, 9.85%.
Yield: 75%; m.p.: 246–248°C; IR (KBr, νmax in cm−1): 3,387 (C–H), 2,222
(C≡N), 1,602 (C═N), 1,444 (N═N), 1,249 (C═C); 1H (500 MHz, DMSO‐
d6, δ ppm): 3.81 (3H, s, –OCH3), 7.03 (2H, d, J = 8 Hz), 7.30–7.41 (4H, m,
Ar‐H), 7.55 (4H, d, J = 8 Hz), 7.65–7.70 (2H, m, Ar‐H), 8.44 (1H, s, Py‐H),
8.46 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 55.8, 82.8, 113,
114.8, 115.3, 119.8, 125.3, 125.5, 127.4, 128.5, 129.4, 130.3, 130.9,
131.8, 133.6, 135, 135.7, 137.8, 141.7, 150.4, 160.0, 160.3; EI–MS: m/z
[M+], 562, [M+2+H]+, 564, [M+4+H]+, 566; anal. calcd. for
C28H17Cl3N4OS (562.02) C, 59.64; H, 3.04; N, 9.94. Found: C, 59.65; H,
3.05; N, 9.95%.
2‐({[1‐(4‐Chlorophenyl)‐3‐(4‐fluorophenyl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐
2‐({[1‐(4‐Chlorophenyl)‐3‐(p‐tolyl)‐1H‐pyrazol‐4‐yl]methylene}-
amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐carbonitrile (8o)
carbonitrile (8k)
Yield: 72%; m.p.: 210–212°C; IR (KBr, νmax in cm−1): 2,224 (C≡N),
1,594 (C═N), 1,455 (N═N), 770 (C–Cl); 1H (500 MHz, DMSO‐d6, δ
ppm): 2.47 (3H, m, –CH3), 7.36 (2H, d, Ar‐H, J = 8 Hz), 7.54–7.61 (3H,
m, Ar‐H), 7.65 (2H, d, Ar‐H, J = 8.5 Hz), 7.85 (1H, d, Ar‐H, J = 2 Hz),
7.88 (2H, d, Ar‐H, J = 8 Hz), 8.09 (2H, d, Ar‐H, J = 9 Hz), 8.69 (1H, s,
Py‐H), 9.30 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 23.37,
114.5, 114.5, 114.6, 115.3, 115.5, 118.5, 119.1, 121.3, 122.6, 124.1,
124.3, 130.1, 130.6, 130.9, 132.1, 132.3, 135.7, 137.9, 155.6, 160.5,
160.6, 185.07; EI–MS: m/z [M+], 546, [M+2+H]+, 548, [M+4+H]+,
Yield: 70%; m.p.: 232–234°C; IR (KBr, νmax in cm−1): 3,429 (C–H),
2,212 (C≡N), 1,504 (C═N), 1,440 (N═N); 1H (500 MHz, DMSO‐d6, δ
ppm): 7.30–7.41 (6H, m, Ar‐H), 7.55 (2H, d, J = 8 Hz), 7.65–7.70 (2H,
m, Ar‐H), 7.86 (2H, d, J = 8 Hz), 8.44 (1H, s, Py‐H), 8.46 (1H, s,
–N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8, 113, 115.3, 116, 119.8,
125.5, 127.4, 128.6, 129.4, 130.3, 130.6, 130.9, 131.8, 133.6, 135,
135.7, 137.8, 141.7, 150.4, 160.0, 160.3; EI–MS: m/z [M+], 550, [M
+2+H]+, 552, [M+4+H]+, 554; anal. calcd. for C27H14Cl3FN4S

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550; anal. calcd. for C28H17Cl3N4S (547.89) C, 61.38; H, 3.13; N,
10.23. Found: C, 61.37; H, 3.14; N, 10.23%.
second tube to make 10 dilutions. The serial dilution was repeated up to
10 dilutions for each vehicle. From the maintained stock cultures of the
required microorganisms, 5 ml was taken and added to 2 ml of BHI broth.
In each serially diluted tube, 200 ml of the above culture suspension was
added. The last tube contained only the media and the culture suspen-
sion, that is, the negative control. The tubes were kept for incubation for
24 hr at 37°C in a bacteriological incubator and observed for turbidity.
2‐({[1‐(4‐Chlorophenyl)‐3‐(thiophen‐3‐yl)‐1H‐pyrazol‐4‐yl]-
methylene}amino)‐5‐(2,4‐dichlorophenyl)thiophene‐3‐
carbonitrile (8p)
Yield: 74%; m.p.: 235–237°C; IR (KBr, νmax in cm−1): 3,427 (C–H),
2,400 (C≡N), 1,496 (C═N), 1,438 (N═N), 1,408 (C═C); 1H (500 MHz,
DMSO‐d6, δ ppm): 7.30–7.41 (5H, m, Ar‐H), 7.55 (2H, d, Ar‐H,
J = 8 Hz), 7.65–7.70 (2H, m, Ar‐H), 7.89–7.98 (2H, m, Ar‐H), 8.47 (1H,
s, Py‐H), 9.32 (1H, s, –N═CH); 13C (100 MHz, DMSO, δ ppm): 82.8,
114, 115.3, 119.8, 121.7, 125.5, 127.4, 128.2, 128.3, 129.2, 129.4,
130.3, 130.9, 131.8, 133.3, 133.6, 135, 135.7, 137.8, 141.7, 142.3,
160, 160.3; EI–MS: m/z [M+], 538, [M+2+H]+, 540, [M+4+H]+, 542;
anal. calcd. for C25H13Cl3N4S2 (539.89) C, 55.62; H, 2.43; N, 10.38.
Found: C, 55.63; H, 2.42; N, 10.39%.
|
4.2.2
Anti‐inflammatory activity
The anti‐inflammatory activity of the compounds was studied by
using inhibition of albumin denaturation technique, which was stu-
died according to Govindappa et al.[31] and Sakat et al.[32] The reac-
tion mixture consisted of test compounds and 1% aqueous solution of
bovine albumin fraction; the pH of the reaction mixture was adjusted
using a small amount of 1 N HCl. The sample extracts were incubated
at 37°C for 20 min and then heated to 51°C for 20 min. After cooling
the samples, the turbidity was measured at 660 nm (UV–Visible
spectrophotometer). The experiment was performed in triplicate.
Diclofenac sodium was used as the standard compound.
|
4.2
Biological studies
|
4.2.1
Antibacterial assay
The anti‐inflammatory activity of the compounds was estimated
on the basis of the percentage of inhibition of albumin denaturation
as the following equation:
The susceptibility of the test organisms to synthetic compounds was
assessed using broth dilution assay, as MIC. Triplicates were per-
formed for each of the standard strains.
Control absorbance − Sample absorbance
% Inhibition =
× 100.
Control absorbance
Culture media
Brain Heart Infusion (BHI) broth test organisms. Four microorgan-
isms were selected for the study: S. aureus Microbial Type Culture
Collection MTCC 12598, E. faecalis MTCC 35550, E. coli MTCC 443,
and P. aeruginosa MTCC 25668. All microorganisms were previously
subcultured in appropriate media and under gaseous conditions to
confirm their purity at 35°C for 48 hr before testing of the vehicles.
|
4.2.3
Antitubercular activity
The antitubercular activity of compounds was assessed against M. tu-
berculosis (ATTC 2729415) using the microplate Alamar Blue assay.[33]
The methodology is nontoxic, which uses a thermally stable reagent and
shows a good correlation with proportional and BACTEC radiometric
methods. Briefly, 200 µl of sterile deionized water was added to all outer
perimeter wells of sterile 96‐well plates (Falcon 3072; Becton Dickinson,
Lincoln Park, NJ) to minimize evaporation of the medium in the test wells
during incubation. The 96 plates received 100 µl of the Middlebrook 7H9
Broth (Difco Laboratories, Detroit, MI), and a serial dilution of the com-
pounds was performed directly on the plate. The final drug concentra-
tions tested were 100–0.2 µg/ml. Plates were covered and sealed with a
parafilm and incubated at 37°C for 5 days. After this time, 25 µl of a
freshly prepared 1:1 mixture of Alamar Blue (AccuMed International,
Westlake, OH) reagent and 10% Tween‐80 was added to the plate and
incubated for 24 hr. Blue color in the well indicated no bacterial growth
and pink color indicated growth. MIC was defined as the lowest drug
concentration, which prevented a color change from blue to pink.
Standard values for the anti‐Tb test that was performed are as
follows:
Inoculum preparation
The growth method or the log phase method was performed as follows.
At least three to five well‐isolated colonies of the same morphological
type were selected from an agar culture plate. The top of each colony
was scooped with a loop, and they were transferred into a tube con-
taining 4–5 ml of BHI broth. The broth culture was incubated at 35°C for
2–6 hr until it achieved the turbidity of the 0.5 McFarland standard. The
turbidity of actively growing broth culture was adjusted with the broth to
obtain a final turbidity, optically comparable to that of the 0.5 McFarland
standard, which was done visually by comparing the inoculum tube and
the standard against a white card with contrasting black lines.
Broth dilution method
A total of 10 tubes were taken and nine dilutions of the vehicle were
done with BHI for MIC. In the initial tube, only 200 ml of the vehicle was
added. For further dilutions, 200 ml of BHI broth was added to the next
nine tubes separately. In the second tube, 200 ml of the vehicle was
added which already contained 200 ml of BHI broth. This was considered
as 10 dilutions. From the 10 diluted tubes, 200 ml was transferred to the
Pyrazinamide (P): 3.125 µg/ml
Ciprofloxacin (C): 3.125 µg/ml
Streptomycin (S): 6.25 µg/ml

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|
4.2.4
Hemolytic assay
[3] C. M. da Silva, D. L. da Silva, L. V. Modolo, R. B. Alves, M. A. de
Resende, C. V. B. Martins, A. de Fatima, J. Adv. Res. 2011, 2, 1.
[4] P. Panneerselvam, M. G. Priya, N. R. Kumar, G. Saravanan, Indian
J. Pharm. Sci. 2009, 71, 428.
The hemolytic assay was carried out as per the literature report.[34]
The human blood was collected in a vacutainer containing EDTA
(2 mg/ml). The resulting suspension was centrifuged at 800g for a
period of 10 min to separate buffy coat and plasma. Successively, the
settled erythrocytes were suspended in saline to acquire a suspension
of 5% erythrocytes after washing three times with normal saline
(0.9%). The cells were later incubated in the presence of test com-
pounds (1 mg/ml or 1,000 µg/ml) for 2 hr at 37°C. After incubation,
the suspensions were subjected to centrifugation at 800g for 10 min,
followed by measurement of the absorbance of the supernatant so-
lutions in a UV spectrophotometer at 540 nm. The 2% Triton X‐100
(Sigma‐Aldrich, MO) served as a positive control. The percentage of
hemolysis is expressed as a percentage of Triton X‐100‐induced
hemolysis and is calculated by the following formula:
[5] M. A. Neelakantan, M. Esakkiammal, S. S. Mariappan, J. Dharmaraja, T.
Jeyakumar, Indian J. Pharm. Sci. 2010, 72, 216.
[6] G. More, S. Bootwala, S. Shenoy, J. Mascarenhas, K. Arun, Orient.
J. Chem. 2018, 34, 800.
[7] T. AbouI‐Fad, F. A. H. Mohammed, E. A. S. Hassan, Arch. Pharm. Res.
2003, 26, 778.
[8] M. J. Hearn, M. H. Cynamon, M. F. Chen, R. Coppins, J. Davis, H. J. O.
Kang, A. Noble, B. Tu‐Sekine, M. S. Terrot, D. Trombino, M. Thai, E. R.
Webster, R. Wilson, Eur. J. Med. Chem. 2009, 44, 4169.
[9] K. K. Sivakumar, A. Rajasekaran, J. Pharm. BioAllied Sci. 2013, 5,
126.
[10] P. K. Das, R. Sahu, P. Satapathy, D. Dash, B. Garnaik, Asian J. Chem.
2018, 30, 556.
[11] D. Gramec, L. P. Masicc, M. S. Dolenc, Chem. Res. Toxicol. 2014, 27,
1344.
[12] A. A. Bekhit, H. M. A. Ashour, Y. S. A. Ghany, A. E. A. Bekhit, A. Baraka,
Eur. J. Med. Chem. 2008, 43, 456.
(Absorbance of the sample) − (Absorbance of blank)
% Hemolysis =
[13] W. J. Si, X. Wang, M. Chen, M. Q. Wang, A. M. Lua, C. L. Yang, New
J. Chem. 2013, 00, 1.
Highest absorbace of positive control
[14] Y. S. Hassan, Molecules 2013, 18, 2683.
× 100.
[15] S. Guniz Kuçukguzel, S. Rollas, H. Erdeniz, M. Kiraz, A. Cevdet Ekinci,
A. Vidin, Eur. J. Med. Chem. 2000, 35, 761.
ACKNOWLEDGMENTS
[16] K. Karrouchi, S. Radi, Y. Ramli, J. Taoufik, Y. N. Mabkhot, F. A. Al‐
aizari, M. Ansar, Molecules 2018, 23, 134.
We are thankful to DST‐PURSE, Mangalore University, and SAIF
Punjab for providing elemental and spectral analysis of newly syn-
thesized compounds. We are grateful to Dr. Prabhakar Kore Basic
Science Research Centre, KLE Academy of Higher Education and
Research, Belgaum, and Maratha Mandal's NGH Institute of Dental
Sciences and Research Centre for their help in carrying out the
biological studies.
[17] A. M. Farag, A. S. Mayhoub, S. E. Barakat, A. H. Bayomi, Bioorg. Med.
Chem. 2008, 16, 4569.
[18] N. E. A. El‐Gamel, T. A. Farghaly, Spectrochim. Acta, Part A 2013, 115,
469.
[19] A. L. Iglesias, V. Miranda‐Soto, D. A. Pompa‐Monroy, J. G. Martinez‐
Ortiz, G. C. Diaz‐Trujillo, l. J. Villarreal‐Gomez, Indian J. Pharm. Sci.
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[20] I. A. M. Radini, Molecules 2018, 23, 2092.
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CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
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Nkehchungag, J. E. Iputo, J. Med. Plants Res. 2010, 4, 789.
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Boja Poojary
http://orcid.org/0000-0002-4977-3501
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SUPPORTING INFORMATION
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How to cite this article: Nayak SG, Poojary B, Kamat V. Novel
pyrazole‐clubbed thiophene derivatives via Gewald synthesis
as antibacterial and anti‐inflammatory agents. Arch Pharm.
2020;e2000103. https://doi.org/10.1002/ardp.202000103
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