Microwave-assisted synthesis and biological evaluation of pyrazole-4-carbonitriles as antimicrobial agents

Article abstract of DOI:10.1002/jhet.4003

An efficient microwave-assisted method of synthesis of pyrazole-4-carbonitriles has been developed. Condensation of pyrazole-4-carbaldehydes with hydroxylamine hydrochloride followed by reaction of the resulting oximes with the Vilsmeier-Haack reagent pre-formed from phthaloyl dichloride and dimethylformamide under microwave irradiation afforded the corresponding pyrazole-4-carbonitriles in 73percent to 91percent yield. The operational simplicity, avoidance of toxic reagents such as POCl₃, shorter reaction time, higher yield compared to the classical version, easy work up, and the use of the by-product in the regeneration of phthaloyl dichloride are the advantages of this methodology. All the target compounds were tested for antimicrobial activity against Gram-positive bacteria Bacillus cereus and Staphylococcus aureus; Gram-negative bacteria Escherichia coli and Yersinia enterocolitica, and the fungal species Candida albicans.

Full text of DOI:10.1002/jhet.4003

Received: 3 February 2020
DOI: 10.1002/jhet.4003
Revised: 2 April 2020
Accepted: 9 April 2020
A R T I C L E
Microwave-assisted synthesis and biological evaluation of
pyrazole-4-carbonitriles as antimicrobial agents
1
1
2
1
Sumit Sood | Poonam Kumari | Ajar Nath Yadav | Anil Kumar
|
1
Karan Singh
1
Department of Chemistry, Akal College
Abstract
of Basic Sciences, Eternal University, Baru
Sahib, India
An efficient microwave-assisted method of synthesis of pyrazole-4-carbonitriles
has been developed. Condensation of pyrazole-4-carbaldehydes with hydroxyl-
amine hydrochloride followed by reaction of the resulting oximes with the
Vilsmeier-Haack reagent pre-formed from phthaloyl dichloride and
dimethylformamide under microwave irradiation afforded the corresponding
pyrazole-4-carbonitriles in 73% to 91% yield. The operational simplicity, avoid-
2
Department of Biotechnology, Dr. KSG
Akal College of Agriculture, Eternal
University, Baru Sahib, India
Correspondence
Karan Singh, Department of Chemistry,
Akal College of Basic Sciences, Eternal
University, Baru Sahib, Sirmaur-173101,
Himachal Pradesh, India.
ance of toxic reagents such as POCl , shorter reaction time, higher yield com-
3
pared to the classical version, easy work up, and the use of the by-product in
the regeneration of phthaloyl dichloride are the advantages of this methodol-
ogy. All the target compounds were tested for antimicrobial activity against
Gram-positive bacteria Bacillus cereus and Staphylococcus aureus; Gram-
negative bacteria Escherichia coli and Yersinia enterocolitica, and the fungal
species Candida albicans.
Email: karansinghji@rediffmail.com,
karan.sagwal@yahoo.com
1
| INTRODUCTION
Microwave-assisted organic synthesis is an eco-
friendly and efficient method for the synthesis of organic
The organic compounds possessing nitrile ( C N) func-
tional group have been used as vital building blocks for
the synthesis of a variety of medicinally as well as biologi-
cally important nitrogen containing heterocyclic com-
compounds. Obviating the use of large quantity of sol-
vents and reagents, lesser reaction time, operational sim-
plicity, safety, less environmental pollution, and higher
chemical yields are the advantages of microwave method
[1,2]
[7]
pounds.
The multi-substituted pyrazoles containing
which makes it superior to the conventional method.
nitrile ( C N) functionality have been well known
throughout pharmaceuticals, agrochemicals, and related
The Vilsmeier-Haack reagent generated in situ from
tertiary amide (eg, DMF or NMP) and acid chloride/bis-
(trichloromethyl)carbonate (BTC, triphosgene) has
emerged as a versatile reagent responsible for OH-
[3,4]
drug candidates.
Some of the multi-substituted
cyanopyrazole based bioactive compounds are Fipronil,
Pyriprole, Pyrafluprole and Ethiprole (insecticides),
Zaleplon (sedative-hypnotic), and Lorlatinib (ALK/ROS I
[
8,9]
activation of oximes.
However, most of the Vilsmeier-
Haack reagents reported in the literature are not suitable
for organic synthesis due to hazardous nature of the
reagents or by-products. The Vilsmeier-Haack reagent
(OPC-VH) prepared by Kimura and his co-workers using
o-phthaloyl dichloride and dimethylformamide is
obtained in pure form, obviating the use of toxic reagents
and the by-product is reused in the preparation of
[5]
inhibitors).
In addition to the biological activities,
nitrile functional group can be converted into various
synthetic derivatives such as acids, ketones, imines, alde-
hydes, amines, amides, and even tetrazoles, thus provid-
[
5,6]
ing a facile access to structurally diversified pyrazoles.
[
10–13]
Sumit Sood and Poonam Kumari contributed equally to this study.
phthaloyl dichloride (Scheme 1).
J Heterocyclic Chem. 2020;1–9.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC
1

2
SOOD ET AL.
Although, OPC-VH reagent mediated convenient syn-
thesis of benzothiazolyl 4-cyanopyrazole hybrids using
yield. With the same objective, we were wondering
whether this OPC-VH mediated Vilsmeier-Haack reac-
tion could be further optimized owing to microwave acti-
vation. It is well documented in the literature that
microwave can increase the rate of reactions and overall
[14]
thermal condition has been already explored,
the OH-
activation of aldoximes using OPC-VH under microwave
condition still have the scope for further investigation.
Taking into account of molecular hybridization approach
and owing the importance of OPC-VH reagent for OH-
activation of aldoximes, the syntheses of 1H-pyrazole-
yield of product. As
a
blank experiment, the
4-formylpyrazole 3a was reacted with hydroxylamine
hydrochloride and followed by treatment with OPC-VH
ꢀ
4
-carbonitriles using microwave irradiation have been
(1.5 equiv.) at 40 C in acetonitrile for 0.5 hour afforded
executed.
the desired nitrile product 4a in 63% yield (entry
1
, Table 1). The reaction was also attempted with o-
ꢀ
phthaloyl dichloride (1.5 equiv.) at 40 C in acetonitrile
2
| RESULTS AND DISCUSSION
for 0.5 hour but no product 4a was detected (entry
2, Table 1). Microwave irradiation using Anton Paar
Recently, the conversion of 4-formylpyrazoles to their
corresponding 4-cyanopyrazoles using hydroxylamine
hydrochloride followed by treatment with OPC-VH
Monowave-300 reactor (Gaas, Austria), operating at a fre-
quency of 2.455 GHz with continuous irradiation power
of 0 to 850 W was next studied, varying the solvent, tem-
perature, and the amount of OPC-VH.
[14]
reagent has been reported in the literature.
A series of
experiments with different solvents were executed to
determine the most appropriate solvent and had
established the best condition which was used for further
exploration, preparing a series of compounds having a
number of substituents. Under the optimal reaction con-
ditions, yields were uniformly good and were not particu-
larly substituent-dependent.
These results encouraged us to probe the microwave-
assisted synthesis of some 1H-pyrazole-4-carbonitriles
The use of microwave proved to be efficient, allowing
the formation of desired product 4a in 68% yield within
5 minutes, leading to a 5% increase of the yield at the same
temperature (entry 5 vs 1 Table 1). Raising the reaction
time from 2 to 10 minutes for DMF as solvent resulted a
decrease of the yield of the nitrile product 4a from 86% to
75% (entries 9-11, Table 1). In DMF, repeating the experi-
[14]
ꢀ
ꢀ
ment at 60 C and 80 C resulted in a significant decrease of
the yield (71% and 68%, respectively, for entries 12 and
13, Table 1). Whereas the conventional thermal heating of
4
using Vilsmeier-Haack reagent (OPC-VH) (Scheme 2).
ꢀ
The 4-formylpyrazoles 3 required for the present
aldoxime at 40 C with OPC-VH (1.5 equiv.) in DMF for
study were synthesized by the condensation of aryl
methyl ketones 1 with phenyl hydrazine using acidic con-
dition followed by cyclization of hydrazones using OPC-
0.5 hour afforded the desired nitrile product 4a in 69% yield
(entry 8, Table 1). To check the requirement of OPC-VH
reagent, one more experiment was performed without
ꢀ
ꢀ
VH reagent in DMF at 60 C and according to the litera-
using OPC-VH reagent in DMF at 40 C for 20 minutes, no
[15]
ture procedure as outlined in Scheme 2.
reaction was observed (entry 14, Table 1). The best condi-
tion appeared thus to be the use of 1.5 equivalents of OPC-
VH in DMF (entry 9, Table 1).
Consequently, the reaction condition of entry 9 of
Table 1 was used for further exploration of the preparing
nitrile compounds 4, having different aryl substituents.
The results are summarized in Table 2. Under the opti-
mal reaction condition, the yields of isolated nitrile
Having these 4-formylpyrazoles in hand, the current
study was initiated with 3a as a model substrate.
[14]
According to the previous report,
the treatment of
aldoximes, obtained from 4-formylpyrazoles by treating
with hydroxylamine hydrochloride, with OPC-VH
ꢀ
reagent in acetonitrile at 40 C within 0.5 hour gave the
corresponding pyrazole-4-carbonitriles in 84% to 92%
SCHEME 1 Synthesis of
Vilsmeier-Haack reagent (OPC-VH
reagent)
SCHEME 2 Synthesis of 1H-
pyrazole-4-carbonitriles 4 using OPC-
VH reagent

SOOD ET AL.
3
TABLE 1
OPC-VH mediated Vilsmeier-Haack
transformation
Optimization of
ꢀ
a
Entry OPC-VH (equiv.) Solvent
Temp ( C) Reaction time Yield (%)
b
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1.5
0.0
1.0
1.2
1.5
1.5
2.0
1.5
1.5
1.5
1.5
1.5
1.5
0.0
Acetonitrile 40
Acetonitrile 40
Acetonitrile 40
Acetonitrile 40
Acetonitrile 40
0.5 hours
63
c
0.5 hours
NR
10 minutes
10 minutes
5 minutes
10 minutes
10 minutes
0.5 hours
52
60
68
61
Toluene
Toluene
DMF
40
40
40
40
40
40
60
80
40
58
b
69
DMF
2 minutes
5 minutes
10 minutes
2 minutes
2 minutes
20 minutes
86
78
0
1
2
3
4
DMF
DMF
75
DMF
71
DMF
68
DMF
NR
Note: Reaction conditions: Oxime (1 equiv), OPC-VH, Solvent (5 mL), μW, power, temperature.
a
Yields of isolated products.
The reaction was performed at 40 C without any μW activation.
b
ꢀ
c
ꢀ
Oxime (1 equiv), o-phthaloyl dichloride (1.5 equiv.), acetonitrile, 40 C, 0.5 hours.
on condensation with phenacyl bromides 7 according the
well-known Hantzsch-thiazole synthesis gave the
corresponding hydrazones 8. The microwave-assisted cycli-
zation of hydrazones 8 using OPC-VH reagent in DMF
at 60 C within 10 minutes afforded the corresponding
1-(thiazol-2-yl)-1H-pyrazole-4-carbaldehydes 9. The
TABLE 2
transformation
Scope of the microwave-assisted OPC-VH
a
b
Entry
Product
Ar
Yield (%)
1
2
3
4
5
6
4a
4b
4c
4d
4e
4f
C
6
H
5
86
82
77
83
73
82
80
ꢀ
4-ClC
4-CH
3-Br-4-FC
4-OCH
4-BrC
2-ClC
6
H
4
1
-(thiazol-2-yl)-1H-pyrazole-4-carbaldehydes 9 were
3
C
6
H
4
further subjected to the optimal reaction condition for 4a to
obtain 1-(thiazol-2-yl)-1H-pyrazole-4-carbonitriles 10 in
excellent yields (85%-91%) and the results are summarized
in Table 3.
6
H
3
3 6 4
C H
6
H
4
7
4g
6 4
H
In order to compare the effect of microwave on the
a
Reaction conditions: Oxime (1 equiv.), OPC-VH (1.5 equiv.), DMF
reaction yield, the earlier reported 1-(benzo[d]thiazol-
ꢀ
[14]
(
5 mL), μW, 850 W, 40 C.
Yields of isolated products.
2-yl)-3-phenyl-1H-pyrazole-4-carbonitrile 12
was fur-
b
ther studied. A mixture of 1-(benzo[d]thiazol-2-yl)-3-phe-
nyl-1H-pyrazole-4-carbaldehyde 11 and hydroxylamine
hydrochloride in ethanol was refluxed for 6 hours and
cooled to room temperature. The separated solid was fil-
tered and dissolved in DMF. The resulting mixture was
subjected to the optimized microwave condition afforded
1-(benzo[d]thiazol-2-yl)-3-phenyl-1H-pyrazole-4-car-
bonitrile 12 in 91% yield as compared to 59% yield
obtained in thermal heating method (Scheme 4).
compounds were uniformly good (73%-86%) and were
not particularly substituents dependent.
The investigation study was not restricted to pyrazole-
4
-carbonitriles 4. Indeed, we extended the scope and
generality of this approach and synthesized one
more series of 1-(thiazol-2-yl)-1H-pyrazole-4-carbonitriles
1
0a-g (Scheme 3).
The IR spectrum of 1-(thiazol-2-yl)-1H-pyrazole-4-car-
bonitrile 10a showed IR band at 2231 cm− (C N
1
The phenacyl bromides 7 needed for the investigation
−
−
1
studies were prepared by the bromination of the
corresponding propiophenone in acetic acid according to
the literature procedure.
stretch) and the absence of (CO stretch) at 1679 cm
1
along with C-H stretching bands at 2776 and 2861 cm
[15]
1
Treatment of aryl methyl
suggested the formation of nitrile 10a. H NMR spectrum
ketones 5 with thiosemicarbazides in refluxing methanol
using acidic condition afforded thiosemicarbazones 6 which
(400 MHz, CDCl ) showed the absence of one-proton sin-
glet at δ 10.02 which was appeared in the NMR spectrum
3

4
SOOD ET AL.
SCHEME 3 Synthesis of
1
-(thiazol-2-yl)-1H-pyrazole-
-carbonitriles 10a-g using
4
microwave-assisted OPC-VH reagent
TABLE 3
pyrazole-4-carbonitriles 10a-g
Yields of a series of 1H-
afford intermediate B. Then chloride anion abstracts the
proton from intermediate B which loses a molecule of
HCl and DMF molecule resulted into the formation of
0
a
Entry
Product
10a
R
R
Yield (%)
1
2
3
4
5
6
7
C
6
H
5
C
C
C
C
6
6
6
6
H
H
H
H
5
5
5
5
87
91
85
90
85
87
90
4-cyanopyrazoles 4 and 10 (Scheme 5).
10b
4-BrC
4-CH
4-OCH
6 4
H
10c
3
C
6
H
4
2.1 | Antimicrobial activities
10d
3
C
6
H
4
10e
C
6
H
5
3-ClC
3-ClC
3-ClC
6
6
6
H
4
H
4
H
4
The minimum inhibitory concentrations (MICs) of 4a-g
and 10a-g were evaluated against Gram-positive (Bacillus
cereus MTCC1272 and Staphylococcus aureus MTCC737)
and Gram-negative (Escherichia coli MTCC118 and
Yersinia enterocolitica MTCC861) bacteria, and the fungal
species Candida albicans MTCC227 by the broth macro-
10f
4-BrC
4-CH
6 4
H
10g
3 6 4
C H
a
Yields of isolated products.
[
16–19]
of corresponding pyrazole-4-carbaldehyde 9a and was
assigned to CHO group. The appearance of a singlet at
δ 8.83 was ascribed to pyrazole-H. The two 2-proton dou-
blet of doublets were appeared at δ 8.07 (J = 8.0 and
dilution method.
All strains used were procured
from Microbial Type Culture Collection (MTCC), Insti-
tute of Microbial Technology (IMTECH), Chandigarh.
Remarkable bacterial growth inhibition was observed
with pyrazole-4-carbonitriles 4 and 10 as shown in
Table 4 as compared to the one of standard drug ampicil-
lin. Compounds 4a-g, 10a, 10b, 10d, 10e, and 10f
exhibited MIC of 78.1 μg/mL while compound 10c and
10g exhibited MIC of 39.06 μg/mL against the Gram
(−ve) bacterial strain E. coli. The MIC value of these com-
pounds was found comparable to the reference standard
ampicillin. The compounds 4a, 4c, 4e, and 4f were hav-
ing MIC of 39.06 μg/mL against the Gram (−ve) bacterial
strains Y. enterocolitica. The compound 10g was found an
excellent compound to be active against all Gram (−ve)
and Gram (+ve) bacterial strains E. coli, Y. enterocolitica,
B. cereus, and S. aureus. Regarding the antifungal evalua-
tion, the compounds 4c, 10a, 10d, 10e, and 10f showed
better antifungal activity against C. albicans with MIC
15.62 to 39.06 μg/mL as compared to reference standard
Miconazole.
1
.8 Hz) and δ 7.67 (J = 7.2 and 1.4 Hz) and a five protons
complex multiplet in the region of 7.46 to 7.54 were
assigned to the aromatic protons. The rest one-proton
triplet appeared at δ 7.41 (J = 7.2 Hz) was also assigned
to the aromatic proton. A three-proton sharp singlet
appeared at δ 2.61 was assigned to thiazole-CH . The
downfield signal in the
3
13
C
NMR spectrum of
4
-cyanopyrazole 10a appeared at δ 113.1 further con-
firmed the presence of nitrile group. The resonance found
in the aliphatic region at δ 12.8 was the main characteris-
tics and assigned to thiazole-CH The formation of com-
pound 10a was further supported by its ESI-MS spectrum
which exhibited its molecular ion peak as the base peak
3.
+
of the spectrum at m/z 343.1 [M + H] .
The structures of all novel 1H-pyrazole-4-carbonitriles
1
4
and 10 were determined by spectroscopic (IR, H NMR,
C NMR, and ESI-MS) methods (Appendix S1) and
1
3
purity was further determined by its elemental analysis.
We believe that the OPC-VH reagent formed from
phthaloyl dichloride and DMF reacting with the hydroxy
group of aldoxime leading to the formation of intermedi-
ate A which subsequently loses a molecule of HCl to
As evident by structure-activity relationship, the
tested compounds 10c and 10g having methyl group
resulted in superior antibacterial compounds against
E. coli while the compounds 4a, 4c, 4e, and 4f showed
comparable antibacterial activity against Y. enterocolitica

SOOD ET AL.
5
SCHEME 4 Synthesis of
1
-(benzo[d]thiazol-2-yl)-3-phenyl-1H-
pyrazole-4-carbonitrile 12:
Comparison between conventional
heating and microwave-assisted
methods
SCHEME 5 Possible reaction
scheme for the synthesis of
4
-cyanopyrazoles 4 and 10 from the
corresponding aldoximes with OPC-
VH reagent
TABLE 4
Minimum inhibitory concentrations (MICs) of test compounds 4 and 10 against fungal and bacterial isolates
MIC (μg/mL)
Gram-negative bacteria
Gram-positive bacteria
Fungi
Test compounds
Escherichia coli
Yersinia enterocolitica
Bacillus cereus
156.25
156.25
78.1
Staphylococcus aureus
Candida albicans
4
4
4
4
4
4
4
1
1
1
1
1
1
1
a
78.1
78.1
78.1
78.1
78.1
78.1
78.1
78.1
78.1
39.06
78.1
78.1
78.1
39.06
78.1
-
39.06
78.1
156.25
156.25
78.1
78.1
78.1
39.06
78.1
78.1
78.1
78.1
39.06
78.1
78.1
39.06
19.53
19.53
78.1
-
b
c
39.06
78.1
d
156.25
156.25
78.1
78.1
e
39.06
39.06
78.1
78.1
f
78.1
g
78.1
78.1
0a
0b
0c
0d
0e
0f
0g
156.25
156.25
156.25
156.25
156.25
156.25
78.1
78.1
156.25
156.25
156.25
78.1
78.1
156.25
78.1
78.1
78.1
156.25
39.06
39.06
-
78.1
78.1
a
Ampicillin
39.06
-
78.1
b
Miconazole
-
78.1
a
Standard antibacterial agent.
Standard antifungal agent.
b
as compared to ampicillin. The compound 10g against
B. cereus and 4c-g and 10d-g against S. aureus resulted
comparable activities as compared to standard drug. All
the synthesized compounds, specially 10e and 10f have
resulted in superior antifungal compounds against
C. albicans as compared to Miconazole. But there is still a
need to carry out further modifications in pyrazole-
4-carbonitriles structure in order to detect more effica-
cious antimicrobial lead molecules. Moreover, cytotoxic-
ity evaluation on test microorganisms and mammalian
cell lines is also necessary to establish correlation
between antimicrobial potential and therapeutic safety of

6
SOOD ET AL.
the pyrazole-4-carbonitriles under investigation. Follow-
up studies with most promising and non-cytotoxic deriva-
tives are important to elucidate their possible molecular
mechanism of action.
a frequency of 2.455 GHz with continuous irradiation
power of 0 to 850 W. The reactions were carried out in a
G-4 borosilicate glass vial sealed with Teflon septum and
placed in a microwave cavity. Initially, microwave of
required power was used, and temperature was being
ramped from room temperature to the desired tempera-
ture. Once this temperature was attained, the process vial
was held at this temperature for a required time. The
reactions were continuously stirred. Temperature was
measured by an IR sensor. After the experiments, a
cooling-jet cooled the reaction vessel to ambient
temperature.
3
| CONCLUSIONS
In summary, a convenient microwave-assisted Vilsmeier-
Haack reagent (OPC-VH) mediated synthesis of pyrazole-
4
-carbonitriles has been reported. This method can be
useful for the chemists having interest in developing
potential pyrazole based drugs due to the benefits such as
operational simplicity, avoiding the use of toxic reagents,
easy work up, and reuse of the by-product in the prepara-
tion of phthaloyl dichloride. Some of the synthesized
compounds showed significant antibacterial and antifun-
gal activities as evident from their MIC. Notably, 1H-
pyrazole-4-carbonitriles can be further used as versatile
building blocks for the synthesis of various useful hetero-
cyclic compounds that may be of great interest in devel-
oping the novel pyrazole-based drugs.
A
mixture of pyrazole-4-carbaldehydes
3
or
9
(0.938 mmol) and hydroxylamine hydrochloride
(0.091 g, 1.311 mmol) in ethanol (5 mL) was refluxed for
6 hours. The reaction mixture was cooled to room tem-
perature. The separated solid was filtered and dried. A
mixture of separated solid in DMF (2 mL) in a microwave
vial was added OPC-VH reagent (0.18 g, 1.408 mmol) at
ꢀ
0 C and stirred for 1 minute. The reaction mixture was
placed in the Anton Paar Monowave-300 reactor and was
ꢀ
ꢀ
irradiated at 40 C for 2 minutes. The reaction mixture
was cooled to 0 C and poured into ice-cooled water. The
solid separated on cooling was filtered, washed with
water and dried under vacuum. The crude compound
was crystallized from ethanol to get pure compound
4 or 10.
4
| MATERIALS AND METHODS
Melting points were determined in open glass capillaries
on a Fisher-Johns melting point apparatus and are
uncorrected. H NMR (400 MHz) was recorded at room
1
temperature in CDCl as solvent and tetramethylsilane
4.1.1 | 1,3-Diphenyl-1H-pyrazole-
4-carbonitrile 4a
3
(TMS) as an internal standard (δ = 0 ppm), and the
values were reported in the following order: chemical
shift (δ = 0 ppm), multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet), coupling constant
ꢀ
ꢀ
[20]
White solid; 86% yield; m.p. 121 C to 124 C (Lit.
ꢀ
ꢀ
−1
1
125 C-127 C). IR (υ cm , KBr): 2223 (C N str).
H
13
(
J in Hz), and integration. C NMR (100 MHz) spectra
NMR (400 MHz, CDCl ): δ 8.37 (s, 1H, Pyrazole-H), 8.08
3
were recorded in CDCl solutions with TMS as internal
(d, J = 7.2 Hz, 2H, ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH),
3
standard. IR spectra were taken as KBr pellets on a
Perkin Elmer RXI FT-IR spectrometer. The mass spectra
were recorded on a JOEL JMS 600 mass spectrometer at
an ionising potential of 70 eV (EI) electron impact
ionisation mode using the direct inlet system. All the
reactions were monitored by thin-layer chromatography
on pre-coated silica gel 60 F254 (mesh); spots were vis-
ualised under UV light at 254 nm. Elemental analyses
were obtained using a Carlo Erba EA1108 microanalyser.
7.39 to 7.55 (m, 6H, ArH). ESI-MS (m/z): 246.1 [M
+
+ H] . Anal. Calcd for C H N : C, 78.35; H, 4.52%.
16
11 3
Found: C, 78.31; H, 4.47%.
4.1.2 | 3-(4-Chlorophenyl)-1-phenyl-1H-
pyrazole-4-carbonitrile 4b
ꢀ
ꢀ
[20]
Yellow solid; 82% yield; m.p. 130 C to 132 C (Lit.
1
ꢀ
ꢀ
−1
1
38 C-140 C). IR (υ cm , KBr): 2225 (C N str). H NMR
(
400 MHz, CDCl ): δ 8.35 (s, 1H, Pyrazole-H), 8.03 (d,
3
4
4
.1 | General procedure of pyrazole-
-carbonitriles (4a-g, 10a-g)
J = 8.8 Hz, 2H, ArH), 7.73 (d, J = 8.8 Hz, 2H, ArH), 7.52
(t, J = 7.2 Hz, 2H, ArH), 7.46 (d, J = 8.8 Hz, 2H, ArH),
7
.42 (t, J = 8.8 Hz, 1H, ArH). ESI-MS (m/z): 280.16 [M
35
+
37
+
All compounds 4 and 10 were synthesized using Anton
+ H, Cl] , 282.14 [M + H, Cl] . Anal. Calcd for
Paar Monowave-300 reactor (Gaas, Austria), operating at
C H ClN : C, 68.70; H, 3.60%. Found: C, 68.63; H, 3.56%.
16
10
3

SOOD ET AL.
7
7
9
+
81
+
4
.1.3 | 1-Phenyl-3-(4-methylphenyl)-1H-
+ H Br] , 326.34 [M + H, Br] . Anal. Calcd for
pyrazole-4-carbonitrile 4c
C H BrN : C, 59.28; H, 3.11%. Found: C, 59.21; H, 3.15%.
16
10
3
ꢀ
ꢀ
[20]
Light brown solid; 77% yield; m.p. 107 C to 111 C (Lit.
1
ꢀ
ꢀ
−1
1
02 C-104 C). IR (υ cm , KBr): 2222 (C N str).
H
4.1.7 | 3-(2-Chlorophenyl)-1-phenyl-1H-
pyrazole-4-carbonitrile 4g
NMR (400 MHz, CDCl ): δ 8.35 (s, 1H, Pyrazole-H), 7.97
3
(d, J = 7.6 Hz, 2H, ArH), 7.74 (d, J = 7.6 Hz, 2H, ArH),
ꢀ
ꢀ
−1
7
.51 (t, J = 7.6 Hz, 2H, ArH), 7.39 (t, J = 7.2 Hz, 1H,
Yellow solid; 80% yield; m.p. 111 C to 113 C. IR (υ cm ,
1
ArH), 7.29 (d, J = 8.0 Hz, 2H, ArH), 2.41 (s, 3H, ArCH ).
KBr): 2231 (C N str). H NMR (400 MHz, CDCl ): δ 8.39 (s,
3
3
+
ESI-MS (m/z): 260.25 [M + H] . Anal. Calcd for
1H, Pyrazole-H), 7.73 (d, J = 8.8 Hz, 2H, ArH), 7.51 to 7.59
13
C H N : C, 78.74; H, 5.05%. Found: C, 78.69; H, 5.01%.
(m, 4H, ArH), 7.36 to 7.44 (m, 3H, ArH). C NMR (100 MHz,
17
13 3
CDCl ): δ 149.2, 138.3, 132.8, 131.7, 131.1, 130.3, 129.9, 129.4,
3
1
28.9, 128.7, 125.9, 118.9, 113.2 (C N), 92.1. ESI-MS (m/z):
35
+
37
+
4
1
.1.4 | 3-(3-Bromo-4-fluorophenyl)-
-phenyl-1H-pyrazole-4-carbonitrile 4d
280.11 [M + H Cl] , 282.12 [M + H, Cl] . Anal. Calcd for
C H ClN : C, 68.70; H, 3.60%. Found: C, 68.66; H, 3.64%.
16
10
3
ꢀ
ꢀ
−1
Yellow solid; 83% yield; m.p. 168 C to 171 C. IR (υ cm ,
1
KBr): 2223 (C N str). H NMR (400 MHz, CDCl ): δ 8.37
4.1.8 | 1-(5-Methyl-4-phenylthiazol-2-yl)-
3-phenyl-1H-pyrazole-4-carbonitrile 10a
3
(
s, 1H, Pyrazole-H), 8.30 (dd, J = 6.8 and 2.4 Hz, 1H,
ArH), 8.03 (ddd, J = 8.8, 4.8 and 2.4 Hz, 1H, ArH), 7.74
d, J = 8.0 Hz, 2H, ArH), 7.54 (t, J = 8.0 Hz, 2H, ArH),
.43 (t, J = 7.6 Hz, 1H, ArH), 7.24 (t, J = 8.0 Hz, 1H,
ꢀ
ꢀ
−1
(
7
Yellow solid; 87% yield; m.p. 184 C to 186 C. IR (υ cm ,
1
KBr): 2231 (C N str). H NMR (400 MHz, CDCl ): δ 8.83 (s,
3
13
ArH). C NMR (100 MHz, CDCl ): δ 163.3, 149.4, 138.1,
1H, Pyrazole-H), 8.07 (dd, J = 8.0 and 1.8 Hz, 2H, ArH), 7.67
3
1
33.2, 132.8, 129.4, 128.9, 128.4, 125.9, 118.9, 117.8, 113.2
(dd, J = 7.2 and 1.4 Hz, 2H, ArH), 7.46 to 7.54 (m, 5H, ArH),
79
+
13
(C N), 110.1, 92.3. ESI-MS (m/z): 342.13 [M + H, Br] ,
7.41 (t, J = 1.2 Hz, 1H, ArH), 2.61 (s, 3H, thiazole-CH3).
C
81
+
3
44.13 [M + H, Br] . Anal. Calcd for C H BrFN : C,
NMR (100 MHz, CDCl ): δ 157.3, 156.4, 142.1, 139.1, 138.2,
16
9
3
3
5
6.16; H, 2.65%. Found: C, 56.10; H, 2.71%.
132.2, 130.8, 128.4, 128.2, 127.4, 113.1 (C N), 91.6, 12.8
+
(
CH ). ESI-MS (m/z): 343.1 [M + H] . Anal. Calcd for
3
C H N S: C, 70.15; H, 4.12%. Found: C, 70.06; H, 4.08%.
20 14 4
4
.1.5 | 3-(4-Methoxyphenyl)-1-phenyl-1H-
pyrazole-4-carbonitrile 4e
4
.1.9 | 3-(4-Bromophenyl)-1-(5-methyl-
ꢀ
ꢀ
−1
Brown solid; 73% yield; m.p. 118 C to 121 C. IR (υ cm ,
4-phenylthiazol-2-yl)-1H-pyrazole-
4-carbonitrile 10b
1
KBr): 2225 (C N str). H NMR (400 MHz, CDCl ): δ 8.34
s, 1H, Pyrazole-H), 8.03 (d, J = 8.0 Hz, 2H, ArH), 7.74 (d,
J = 8.4 Hz, 2H, ArH), 7.51 (t, J = 8.0 Hz, 2H, ArH), 7.40
t, J = 7.2 Hz, 1H, ArH), 7.01 (d, J = 8.0 Hz, 2H, ArH),
.80 (s, 3H, ArOCH3). C NMR (100 MHz, CDCl ): δ
59.3, 149.7, 138.3, 132.8, 128.9, 128.1, 125.9, 122.8, 118.8,
13.8, 112.9 (C N), 92.3, 51.3 (OCH ). ESI-MS (m/z):
76.29 [M + H] . Anal. Calcd for C H N O: C,
3
(
ꢀ
ꢀ
Light yellow solid; 91% yield; m.p. 203 C to 205 C. IR (υ
cm , KBr): 2228 (C N str). H NMR (400 MHz, CDCl ): δ
8.96 (s, 1H, Pyrazole-H), 7.80 (d, J = 8.4 Hz, 2H, ArH), 7.67
(d, J = 7.3 Hz, 2H, ArH), 7.63 (d, J = 8.4 Hz, 2H, ArH), 7.47
(t, J = 7.7 Hz, 2H, ArH), 7.40 (t, J = 7.3 Hz, 1H, ArH), 2.60
(s, 3H, thiazole-CH3). C NMR (100 MHz, CDCl ): δ 154.8,
153.3, 148.6, 133.9, 133.4, 132.2, 128.6, 128.5, 128.4, 128.2,
−1
1
(
3
13
3
1
1
2
7
3
3
+
13
1
7
13
3
3
4.17; H, 4.76%. Found: C, 74.12; H, 4.72%.
1
27.0, 124.5, 113.4 (C N), 92.6, 12.8 (CH ). ESI-MS (m/z):
3
79
+
81
+
421.3 [M + H Br] , 423.1 [M + H, Br] . Anal. Calcd for
4
.1.6 | 3-(4-Bromophenyl)-1-phenyl-1H-
C H BrN S: C, 57.02; H, 3.11%. Found: 56.95; H, 3.07%.
20 13 4
pyrazole-4-carbonitrile 4f
ꢀ
ꢀ
[20]
Yellow solid; 82% yield; m.p. 133 C to 135 C (Lit.
1
4.1.10 | 1-(5-Methyl-4-phenylthiazol-
2-yl)-3-(4-methylphenyl)-1H-pyrazole-
4-carbonitrile 10c
ꢀ
ꢀ
−1
1
32 C-134 C). IR (υ cm , KBr): 2225 (C N str). H NMR
400 MHz, CDCl ): δ 8.36 (s, 1H, Pyrazole-H), 7.96 (d,
(
3
J = 8.8 Hz, 2H, ArH), 7.73 (d, J = 8.0 Hz, 2H, ArH), 7.62
d, J = 8.4 Hz, 2H, ArH), 7.53 (t, J = 8.0 Hz, 2H, ArH).
.42 (t, J = 7.2 Hz, 1H, ArH). ESI-MS (m/z): 324.34 [M
ꢀ
ꢀ
(
7
Light yellow solid; 85% yield; m.p. 187 C to 188 C. IR (υ
−1
1
cm , KBr): 2231 (C N str). H NMR (400 MHz, CDCl ):
3

8
SOOD ET AL.
δ 8.80 (s, 1H, Pyrazole-H), 7.95 (d, J = 8.2 Hz, 2H, ArH),
CDCl ): δ 155.2, 153.4, 148.6, 134.0, 133.4, 132.7, 132.2,
3
7
.66 (d, J = 7.0 Hz, 2H, ArH), 7.47 (t, J = 8.4 Hz, 2H,
131.8, 130.5, 129.5, 128.5, 128.4, 128.1, 126.9, 124.2, 122.6,
ArH), 7.39 (s, 1H, ArH), 7.30 (d, J = 8.0 Hz, 2H, ArH),
114.3 (C N), 12.4 (CH ). ESI-MS (m/z): 454.9 [M + H,
3
13
79 35
81 35
79 37
2
.59 (s, 3H, thiazole-CH ), 2.42 (s, 3H, ArCH ). C NMR
Br Cl], 456.9 [M + H, Br Cl and Br Cl], 458.9 [M
3
3
81
37
(
100 MHz, CDCl ): δ 155.1, 154.5, 148.4, 140.3, 134.3,
+ H,
Br Cl]. Anal. Calcd for C H BrClN S: C,
20 12 4
3
1
33.2, 129.7, 128.5, 128.4, 128.1, 126.8, 126.8, 113.7
52.71; H, 2.65%. Found: C, 52.65; H, 2.61%.
(
[
C N), 92.5, 21.5 (CH ), 12.8 (CH ). ESI-MS (m/z): 357.1
M + H] . Anal. Calcd for C H N S: C, 70.76; H, 4.52%.
21 16 4
3
3
+
Found: C, 70.70; H, 4.48%.
4.1.14 | 1-(4-[3-Chlorophenyl]-
5
-methylthiazol-2-yl)-3-(4-methylphenyl)-
1H-pyrazole-4-carbonitrile 10g
4
1
.1.11 | 3-(4-Methoxyphenyl)-
-(5-methyl-4-phenylthiazol-2-yl)-1H-
ꢀ
ꢀ
Light yellow solid; 90% yield; m.p. 228 C to 230 C. IR (υ
−1
1
pyrazole-4-carbonitrile 10d
cm , KBr): 2230 (C N str). H NMR (400 MHz, DMSO-
d6): δ 9.55 (s, 1H, Pyrazole-H), 7.84 (d, J = 8.0 Hz, 2H,
ArH), 7.81 (s, 1H, ArH), 7.71 (d, J = 7.6 Hz, 1H, ArH),
7.54 (t, J = 8.0 Hz, 1H, ArH), 7.49 (d, J = 8.0 Hz, 1H,
ArH), 7.40 (d, J = 8.0 Hz, 2H, ArH), 2.62 (s, 3H, thiazole-
ꢀ
ꢀ
Light yellow solid; 90% yield; m.p. 209 C to 210 C. IR (υ
cm , KBr): 2229 (C N str). H NMR (400 MHz, CDCl ):
δ 8.79 (s, 1H, Pyrazole-H), 8.02 (d, J = 8.8 Hz, 2H, ArH),
−
1
1
3
13
7
.66 (d, J = 7.2 Hz, 2H, ArH), 7.47 (t, J = 7.8 Hz, 2H,
ArH), 7.39 (t, J = 7.3 Hz, 1H, ArH), 7.02 (d, J = 8.8 Hz,
H, ArH), 3.87 (s, 3H, ArOCH ), 2.60 (s, 3H, thiazole-
CH ), 2.39 (s, 3H, ArCH ). C NMR (100 MHz, CDCl ): δ
3
3
3
155.2, 154.6, 146.9, 140.4, 135.7, 134.5, 133.2, 129.8, 129.7,
129.0, 128.5, 128.1, 127.7, 126.9, 126.7, 113.6 (C N), 92.7,
21.5 (CH ), 12.8 (CH ). ESI-MS (m/z): 391.1 [M + H,
2
3
13
CH3). C NMR (100 MHz, CDCl ): δ 161.1, 155.1, 148.4,
3
3
3
35
+
37
+
1
34.0, 133.1, 128.5, 128.4, 128.1, 126.7, 122.2, 114.3, 113.8
Cl] , 393.3 [M + H,
Cl] . Anal. Calcd for
(
3
6
C N), 92.2, 55.4 (OCH ), 12.1 (CH ). ESI-MS (m/z):
C H ClN S: C, 64.53; H, 3.87%. Found: C,
64.58; H, 3.91%.
3
3
21 15
4
+
73.2 [M + H] . Anal. Calcd for C H N OS: C,
21
16 4
7.72; H, 4.33%. Found: C, 67.62; H, 4.29%.
4
.2 | Biological assay
4
5
4
.1.12 | 1-(4-[3-Chlorophenyl]-
-methylthiazol-2-yl)-3-phenyl-1H-pyrazole-
-carbonitrile 10e
4.2.1 | Test microorganisms
Bacterial cultures (Bacillus cereus MTCC1272, Staphylo-
coccus aureus MTCC737, Escherichia coli MTCC118, and
Yersinia enterocolitica MTCC861) and fungus Candida
albicans MTCC227 were obtained from Microbial Type
Culture Collection, IMTECH, Chandigarh (India). Cul-
tures were regularly maintained in the laboratory using
standard media and incubation conditions.
ꢀ
ꢀ
−1
Yellow solid; 85% yield; m.p. 178 C to 179 C. IR (υ cm ,
1
KBr): 2234 (C N str). H NMR (400 MHz, CDCl ): δ 8.83
s, 1H, Pyrazole-H), 7.38 to 7.85 (m 9H, ArH), 2.61 (s, 3H,
thiazole-CH3). C NMR (100 MHz, CDCl ): δ 155.2,
54.5, 146.9, 135.7, 134.5, 133.3, 130.2, 129.8, 129.5, 129.0,
28.5, 128.2, 127.8, 126.9, 126.4, 113.5 (C N), 92.9, 12.8
CH ). ESI-MS (m/z): 377.2 [M + H, Cl] , 379.3 [M
H, Cl] . Anal. Calcd for C H ClN S: C, 63.74; H,
20 13 4
3
(
13
3
1
1
(
+
35
+
3
37
+
3
.48%. Found: C, 63.67; H, 3.45%.
4.2.2 | Antimicrobial activity
Pyrazole-4-carbonitriles 4 and 10 were evaluated for their
4
1
1
.1.13 | 3-(4-Bromophenyl)-
-(4-[3-chlorophenyl]-5-methylthiazol-2-yl)-
H-pyrazole-4-carbonitrile 10f
antimicrobial activities by broth macrodilution method
[
16]
[17]
as described previously by NCCLS,
To et al,
with slight modifica-
Modiya
[
18]
[19]
and Patel,
and Berghaus et al
tions. Briefly, to prepare the stock solution, 10 mg of the
test compounds were dissolved in DMSO (4 mL) and sim-
ilarly, the standard drugs ampicillin and miconazole were
dissolved in appropriate quantity of sterile distilled water
(2.5 mg/mL). The bacterial and fungal inocula were made
in saline solution (0.85% NaCl) and cell counts were
ꢀ
ꢀ
Light yellow solid; 87% yield; m.p. 167 C to 168 C. IR (υ
−
1
1
cm , KBr): 2233 (C N str). H NMR (400 MHz, DMSO-
d6): δ 10.02 (s, 1H, Pyrazole-H), 7.92 (d, J = 8.4 Hz, 2H,
ArH), 7.82 (d, J = 8.4 Hz, 2H, ArH), 7.77 (s, 1H, ArH),
7
.74 (d, J = 8.4 Hz, 2H, ArH), 7.44 (t, J = 8.4 Hz, 1H,
13
7
ArH), 2.61 (s, 3H, thiazole-CH3). C NMR (100 MHz,
adjusted to 5 × 10 cfu/mL by using 0.5 McFarland

SOOD ET AL.
9
standard. MICs of test compounds and test drugs were
determined in sterile test tubes containing 2.0 mL of
nutrient broth or Mueller-Hinton broth (for bacteria) and
Sabouraud dextrose broth (for Candida). Two millilitre
stock solution of test compounds and standard antimicro-
bial agents were added to first tube of their respective
series and diluted serially to obtain different concentra-
tions. The resultant diluted concentrations of test com-
pounds and standard antimicrobial agents were 2500,
[6] O. Prakash, R. Pundeer, P. Ranjan, K. Pannu, Y. Dhingra,
K. Aneja, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem.
2
009, 48B, 563.
7] D. Swarnkar, R. Ameta, R. Vyas, Int. J. Pharm. Sci. Drug Res.
014, 6, 200.
8] W. Su, Y. Zhang, J. Li, P. Li, Org. Prep. Proced. Int. 2008,
0, 543.
[
[
2
4
[9] Y. Chen, D. Cantillo, C. O. Kappe, Eur. J. Org. Chem. 2019,
2019, 2163.
10] Y. Kimura, D. Matsuura, T. Hanawa, Y. Kobayashi, Tetrahe-
dron Lett. 2012, 53, 1116.
11] R. Bala, P. Kumari, S. Sood, V. Kumar, N. Singh, K. Singh,
J. Heterocyclic Chem. 2018, 55, 2507.
12] R. Bala, P. Kumari, S. Sood, H. Phougat, A. Kumar, K. Singh,
J. Heterocyclic Chem. 2019, 56, 1787.
[
[
[
[
1
250, 625, 312.5, 156.25, 78.1, 39.06, 19.53, 9.76 μg/mL.
Broth tubes were inoculated with 10 μL of microbial sus-
5
pension so that final counts were 5 × 10 cfu/mL. Tubes
containing medium or medium plus DMSO, and tubes
without microbial inoculum served as growth and steril-
ity controls, respectively. Broth tubes were incubated at
13] Y. Kimura, D. Matsuura, Int. J. Org. Chem. 2013, 3, 1.
[14] R. Bala, P. Kumari, S. Sood, A. Kumar, K. Singh, Org. Prep.
Proced. Int. 2019, 51, 547.
ꢀ ꢀ
3
5 C to 37 C for 24 hours and examined for the presence
[
15] P. Kumari, S. Sood, A. Kumar, K. Singh, J. Heterocyclic Chem.
020, 57, 796.
or absence of growth. The minimum concentration of test
compounds and standard antimicrobials that resulted in
complete growth inhibition was recorded as MIC.
2
[
16] NCCLS (National Committee for Clin Lab Standards). Ref-
erence method for broth dilution antifungal susceptibility
testing for Yeast Proposed standard. Document M27-P.
National Committee for Clin Lab Standards, Villanova,
PA. 1992.
ACKNOWLEDGEMENTS
The authors are grateful to Prof. H.S. Dhaliwal, Vice-
Chancellor, Eternal University, Baru Sahib for providing
facilities and financial assistance. The authors would like
to acknowledge GVK Biosciences Pvt. Ltd. Hyderabad
and Regional Sophisticated Instrumentation Centre, Pun-
jab University, Chandigarh, for recording the analytical
data during the study.
[17] W. K. To, A. W. Fothergill, M. G. Rinaldi, J. Clin. Microbiol.
1995, 33, 2660.
[
[
18] P. R. Modiya, C. N. Patel, Org. Med. Chem. Lett. 2012, 2, 29.
19] L. J. Berghaus, S. Giguère, K. Guldbech, E. Warner, U. Ugorji,
R. D. Berghaus, J. Clin. Microbiol. 2015, 53, 314.
[20] E. M. Sharshira, R. I. El Sokkary, N. Morsy, Z. Naturforsch. B
2018, 73, 389.
ORCID
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
Karan Singh https://orcid.org/0000-0001-8956-5558
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[