Urea derivatives of piperazine doped with pyrazole-4-carboxylic acids: Synthesis and antimicrobial evaluation

Article abstract of DOI:10.1002/jhet.4325

A series of novel N-cycloalkyl/aryl-4-(1,3-diphenyl-1H-pyrazole-4-carbonyl)piperazine-1-carboxamides 9a-g has been synthesized for biological interest by simple base catalyzed reaction of various N-cycloalkyl/aryl isocyanates with (1,3-diphenyl-1H-pyrazol

Full text of DOI:10.1002/jhet.4325

Received: 7 April 2021
DOI: 10.1002/jhet.4325
Revised: 24 May 2021
Accepted: 12 June 2021
A R T I C L E
Urea derivatives of piperazine doped with pyrazole-
4-carboxylic acids: Synthesis and antimicrobial evaluation
Harshita Phougat1,2 | Vandana Devi3 | Sanjay Rai2 | T. Shreedhar Reddy2
Karan Singh1,4
|
1Department of Chemistry, Akal College
Abstract
of Basic Sciences, Eternal University, Baru
Sahib, India
A series of novel N-cycloalkyl/aryl-4-(1,3-diphenyl-1H-pyrazole-4-carbonyl)
2Medicinal Chemistry Division, GVK
Biosciences Pvt. Ltd., Hyderabad, India
3Department of Chemistry, National
Institute of Technology, Kurukshetra,
India
piperazine-1-carboxamides 9a-g has been synthesized for biological interest by
simple base catalyzed reaction of various N-cycloalkyl/aryl isocyanates with
(1,3-diphenyl-1H-pyrazol-4-yl)(piperazin-1-yl)methanone hydrochloride 8. The
compound 8 was synthesized in excellent yield by the reaction of 1,3-diphenyl-
1H-pyrazole-4-carbonyl chloride with boc-piperazine followed by deprotection
of boc group with dioxane-HCl. All the novel intermediates and urea deriva-
tives have been screened for antimicrobial activity. The compound 9a was
found an excellent compound to be active against all Gram (Àve) and Gram
(+ve) bacterial strains E. coli, Y. enterocolitica, B. cereus, and S. aureus. It also
showed comparable antifungal activity against C. albicans with MIC 78.1 μg/ml
as compared to reference standard miconazole.
4Department of Chemistry, Indira Gandhi
University Meerpur, Rewari, India
Correspondence
Karan Singh, Department of Chemistry,
Indira Gandhi University, Rewari - Lokri
Rd, Meerpur, Rewari, Haryana 122502,
India.
Email: karansinghji@rediffmail.com;
karan.sagwal@yahoo.com; karansingh.
chem@igu.ac.in
1 | INTRODUCTION
Furthermore, among nitrogen containing heterocy-
clic compounds, piperazine derivatives have gained
The pyrazole structural motif has been well recognized
presently as a “privileged core” in the medicinal chemis-
try due to the presence of this heterocycle in numerous
therapeutic agents exhibiting a broad range of pharma-
ceutical activities [1–3]. A literature survey revealed that
many heterocyclic derivatives having pyrazole ring sys-
tems were reported to have a wide range of biological
activities [4–13]. Many pyrazole ring containing drugs
have been commercialized successfully such as Celebrex,
Viagra, Lonazolac, Fipronil, and Acomplia [14–16] which
are enlisted among the most selling drugs albeit few have
been retrieved from the market owing to side effects.
Interestingly, some of the pyrazole derivatives have been
also employed as agrochemicals like fungicides, herbi-
cides, and insecticides for the crops protection [17]. How-
ever, the amide class of compounds derived from
3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic
acid have been commercialized as fungicides [17].
much importance in the medicinal field and were
originally used in veterinary medicines [18]. In addi-
tion, the urea-based compounds also exhibited broad
spectrum of biological activities. Recently, due to
unique binding mode and kinase inhibition profile,
the urea-based derivatives act as kinase inhibitors and
novel therapeutic agents in cancer treatment [19,20].
Due to much importance in pharmaceuticals and agro
fields, the development of new chemical entities
(NCE's) containing pyrazole moiety has attracted the
attention of researchers over the past several decades
[21–27].
Owing to the findings regarding the urea and pipera-
zine based compounds and continuation of our research
work aimed at the synthesis of a variety of heterocyclic
systems for biological and pharmacological evaluations
[28–33], we report in the present work an efficient syn-
thesis and antimicrobial evaluation of N-cycloalkyl/aryl-
J Heterocyclic Chem. 2021;1–8.
wileyonlinelibrary.com/journal/jhet
© 2021 Wiley Periodicals LLC.
1

2
PHOUGAT ET AL.
4-(1,3-diphenyl-1H-pyrazole-4-carbonyl)piperazine-1-carbox
amide derivatives.
various cycloalkyl/aryl isocyanates in tetrahydrofuran
using triethylamine as a base as shown in Scheme 1.
The physical characteristics of the title compounds
9a–g have been represented in Table 1.
2 | RESULTS AND DISCUSSION
The structures of the novel urea derivatives 9a–g were
characterized by various spectroscopic techniques such
1
The synthesis of urea derivatives 9 of piperazine doped
with pyrazole-4-carboxylic acid 5 has been accomplished
and depicted in Scheme 1. By following the literature pro-
cedure [34], commercially available acetophenone on
condensation with phenyl hydrazine in ethanol con-
taining a catalytic amount of glacial acetic acid at room
temperature provided acetophenone phenyl hydrazone 3.
The cyclisation of hydrazone 3 using Vilsmeier-Haack
reagent in dimethylformamide at 80ꢀC within 2 h yielded
the corresponding 1H-pyrazole-4-carbaldehyde 4 in good
yield [30]. Aldehyde 4 thus obtained was oxidized to key
derivative, 1H-pyrazole-4-carboxylic acid 5 with KMnO4
in basic medium with good yield [35]. The pyrazole-
4-carboxylic acid 5 was further converted into acid chlo-
ride 6 using thionyl chloride in dichloromethane. The
acid chloride 6 on treating with boc-piperazine gave
the corresponding amide 7. The deprotection of the boc
group from amide 7 with HCl in dioxane resulted into
the formation of desired key intermediate 8 in good yield.
Finally, the urea derivatives 9a–g were achieved in good
to excellent yields via the reaction of intermediate 8 with
as IR, H NMR, 13C NMR, MS, and elemental analysis
(Supporting information). The IR spectrum of compound
9a displayed strong absorption band at 1615.39, 1600.55
due to C═O stretching of amide bonds and a medium
absorption band at 1230.80 appeared due to C─N
stretching indicating the formation of titled urea deriva-
1
tive 9a. In the H NMR spectrum of the compound 9a, a
one-proton sharp singlet appeared at δ 8.67 is assigned to
C5-H of the pyrazole ring. The one-proton doublet
(J = 2.4 Hz) appeared at δ 6.49 and a one-proton multi-
plet in the region δ 2.44–2.55 which are assigned to pro-
ton of –NH (exchangeable in D2O) and N─CH,
respectively. 13C NMR spectra gave signals at 165.35 and
157.32 ppm which are assigned to amide ─C═O and urea
─C═O, respectively. The appearance of the molecular ion
peak at m/z 416.10 in the mass spectra has given further
evidence for the characterization of the synthesized com-
pound 9a. The spectral data of titled compounds 9a–g are
presented in the experimental part.
A plausible mechanism for the synthesis of (1H-
pyrazole-4-carbonyl)piperazine-1-carboxamide
SCHEME 1 Synthesis of (1H-pyrazole-4-carbonyl)piperazine-1-carboxamide derivatives 9

PHOUGAT ET AL.
3
derivatives has been given in Scheme 2. The resonance
structure of the isocyanate group indicates that the elec-
tron density is smaller in the carbon atom, average in
nitrogen, and larger in oxygen. The reactions occur
through addition of piperazine N─H to C═N bond. An
active hydrogen atom-containing piperazine attacks the
electrophilic carbon atom of isocyanate and active hydro-
gen is added to the nitrogen atom.
bacterial strain Y. enterocolitica. The MIC value of these
compounds was found comparable to the reference stan-
dard ampicillin. The compounds 7, 9a, 9b, 9c, 9d, and 9e
were having MIC of 78.01 μg/ml against the Gram (+ve)
bacterial strains S. aureus. The compound 9a was found
an excellent compound to be active against all Gram
(Àve) and Gram (+ve) bacterial strains E. coli,
Y. enterocolitica, B. cereus, and S. aureus. Regarding the
antifungal evaluation, the compounds 9a, 9b, 9c, 9d, and
9g showed comparable antifungal activity against
C. albicans with MIC 78.1 μg/ml as compared to refer-
ence standard miconazole. All other compounds dis-
played lower activity against all bacterial and fungal
isolates.
But there is still a need to carry out further modifica-
tions in pyrazole containing piperazine-1-carboxamides
structure in order to detect more efficacious antimicrobial
lead molecules. Moreover, cytotoxicity evaluation on test
microorganisms and mammalian cell lines is also neces-
sary to establish a correlation between antimicrobial
potential and therapeutic safety of these pyrazole deriva-
tives under investigation. Follow-up studies with the
most promising and non-cytotoxic derivatives are impor-
tant to elucidate their possible molecular mechanism of
action.
2.1 | Antimicrobial activities
The Minimum inhibitory concentrations (MICs) of the
intermediates and the title compounds 7, 8 (free base),
and 9a–g were examined against Bacillus cereus, Staphy-
lococcus aureus, Escherichia coli, and Yersinia enter-
ocolitica bacterial strains, and against Aspergillus flavus,
Aspergillus niger, Candida albicans fungal strains by
broth macrodilution method [30] using standard drugs
ampicillin and miconazole and the results have been
given Table 2. As shown in Table 2, the compounds 7, 9a,
9b, and 9c exhibited equipotent activity to the reference
standard ampicillin against the Gram (Àve) bacterial
strain E. coli evidenced by minimum inhibitory concen-
tration of 78.1 μg/ml. While compounds 9a and 9c
exhibited MIC of 39.06 μg/ml against the Gram (Àve)
3 | MATERIALS AND METHODS
TABLE 1 Physical data of titled urea derivatives 9a–g
Unless otherwise noted, reagents and solvents were used
as received from commercial suppliers. Melting points
were determined in open glass capillaries on a Fisher-
Johns melting point apparatus and are uncorrected. Pro-
ton nuclear magnetic resonance spectra were obtained on
a Bruker AVANCE 400 spectrometer at 400 MHz, and
Agilent 300 MHz. Tetramethylsilane was used as an
internal standard for proton spectra. Coupling constants
(J) are reported in Hz; those for common alkyl groups
were in the expected range and are not separately
Entry
R
Product
Yield %
86
Mp
1
2
3
4
5
6
7
Cyclopropyl
Cyclopentyl
Cyclohexyl
4-OCH3C6H4
3-F-4-CH3C6H3
3-F-4-ClC6H3
4-CH3C6H3
9a
9b
9c
9d
9e
9f
105–107ꢀC
118–120ꢀC
148–150ꢀC
171–173ꢀC
198–200ꢀC
200–202ꢀC
154–156ꢀC
72
70
68
77
66
9g
79
SCHEME 2 A plausible mechanism of the
Et3N mediated urea formation from piperazine
hydrochloride and isocyanates

4
PHOUGAT ET AL.
TABLE 2 Minimum inhibitory concentrations (MICs) of test compounds 7, 8 (free base), 9a–g against fungal and bacterial isolates
MIC (μg/ml)
Gram-negative bacteria
Gram-positive bacteria
Fungi
Test compds
E. coli
78.1
Y. Enterocolitica
B. cereus
78.1
78.1
39.06
78.1
78.1
78.1
78.1
78.1
78.1
39.06
-
S. aureus
78.1
A. flavus
78.1
A. niger
78.1
C. albicans
156.25
156.25
78.1
7
78.1
78.1
39.06
78.1
39.06
78.1
78.1
78.1
156.25
39.06
-
8 (free base)
156.25
78.1
156.25
78.1
156.25
78.1
156.25
78.1
9a
9b
78.1
78.1
78.1
78.1
78.1
9c
78.1
78.1
78.1
78.1
78.1
9d
156.25
156.25
156.25
156.25
78.1
78.1
156.25
78.1
156.25
78.1
78.1
9e
78.1
156.25
156.25
78.1
9f
156.25
156.25
78.1
156.25
156.25
78.1
156.25
156.25
78.1
9g
Ampicillina
Miconazoleb
-
-
-
39.06
39.06
78.1
aStandard antibacterial agent.
bStandard antifungal agent.
marked. 13C NMR (100 MHz) spectra were recorded in
CDCl3 solutions with tetramethylsilane as internal stan-
dard. Thin-layer chromatography was performed using
Merck TLC silica-gel 6OF254 plates. Visualization of TLC
plates was performed using UV light (254 nm). The mass
spectra were obtained on a Waters Quattro micro
API/Waters SQD2/Waters Quattro Premier Spectrometer
using electrospray ionization and atmospheric-pressure
chemical ionization. Elemental analyses were obtained
using a Carlo Erba EA1108 microanalyser.
completed, the reaction mixture was diluted with H2O
(50 ml) and extracted with dichloromethane (2 Â 50 ml).
Combined organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel (100–200 mesh)
column chromatography (5%–10% EtOAc in pet ether as
eluent) to afford 7a (19 g, 65%) as a yellow solid.
1H NMR (CDCl3, 400 MHz): δ 8.16 (s, 1H, Pyrazole-
H), 7.70–7.77 (m, 4H, ArH), 7.34–7.51 (m, 6H, ArH),
3.65–3.76 (m, 2H, Piperazine-H), 3.36–3.44 (m, 2H,
Piperazine-H), 3.07–3.17 (m, 2H, Piperazine-H), 2.85–
2.89 (m, 2H, Piperazine-H), 1.42 (s, 9H, ─C[CH3]3); MS
(ESI): m/z 433.10 [M + H+].
3.1 | Preparation of tert-butyl
4-(1,3-diphenyl-1H-pyrazole-4-carbonyl)
piperazine-1-carboxylate (7a)
3.2 | Preparation of (1,3-diphenyl-1H-
pyrazol-4-yl)(piperazin-1-yl)methanone
hydrochloride (8)
To a round bottom flask containing 1,3-diphenyl-1H-
pyrazole-4-carboxylic acid 5a (22 g, 83.33 mmol), SOCl2
(60.4 ml, 833.3 mmol) was added drop-wise at 0ꢀC and
allowed the reaction mixture to reflux for 1 h. The reac-
tion progress was monitored by thin-layer chromatogra-
phy. When the reaction was completed, SOCl2 was
removed under vacuum using rotaevaporator. The crude
compound was further treated with dichloromethane
To a stirred solution of 7a (10 g, 23.14 mmol) in
1,4-dioxane (100 ml) at 0ꢀC was added 4 N HCl in 1,4--
dioxane (57.8 ml). Then the reaction mass was stirred at
room temperature for 16 h. The reaction progress was
monitored by thin-layer chromatography. When the reac-
tion was completed, the reaction mass was concentrated
under reduced pressure and dried to afford 8 (7 g, crude)
as an off-white solid which was used for the next step
directly without further purification.
(150 ml) and
a solution of tert-butyl piperazine-
1-carboxylate (20 g, 70.92 mmol) in CH2Cl2 (200 ml)
along with DIPEA (30.19 ml, 212.7 mmol) was added at
0ꢀC. The resulting reaction mass was stirred at room tem-
perature for 1 h. The reaction progress was monitored by
thin-layer chromatography. When the reaction was
The formation of compound 8 was confirmed by mass
spectra analysis. MS (ESI): m/z 333 [M + H+].

PHOUGAT ET AL.
5
3.3 | General procedure for synthesis of
N-cycloalkyl/aryl-4-(1,3-diphenyl-1H-
pyrazole-4-carbonyl)piperazine-
1-carboxamides 9
J = 6.9 Hz, 2H, ArH), 7.34–7.57 (m, 5H, ArH), 6.27 (d,
J = 6.6 Hz, 1H, ArH), 5.60 (d, J = 7.2 Hz, 1H, NH [D2O
exchangeable]), 3.78–3.89 (m, 2H, Piperazine-H), 3.52–
3.64 (m, 2H, Piperazine-H), 3.14–3.23 (m, 2H, Piperazine-
H), 2.90–3.04 (m, 2H, Piperazine-H), 1.23–1.76 (m, 9H,
CH, 4 Â CH2); 13C NMR (CDCl3, 100 MHz): δ 164.60
(C═O), 156.92 (C═O), 149.98 (C═N), 139.20, 131.91,
129.45, 128.79, 128.70, 128.48, 127.38, 127.10, 119.12,
116.34, 52.47, 46.71, 42.94, 33.42, 23.49; MS (ESI): m/z
444.42 [M + H+]. Anal. Calcd for C26H29N5O2: C,
70.41; H, 6.59%. Found: C, 70.33; H, 6.55%.
To a stirred solution of 8 (0.08 g, 0.217 mmol) in dry tet-
rahydrofuran (3 ml) was added triethylamine (0.043 ml,
0.434 mmol) and isocyanatocyclopropane (18 mg,
0.217 mmol) at 0ꢀC. The resulting reaction mass was
stirred at room temperature for 1 h. The reaction progress
was monitored by thin-layer chromatography. When the
reaction was completed, the reaction mixture was diluted
with H2O (5 ml) and extracted with dichloromethane
(2 Â 5 ml). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pres-
sure. The crude product was purified by column chroma-
tography (0%–2% MeOH in CH2Cl2 as eluent) using 100–
200 mesh silica. The pure fraction was dried under
reduced pressure to afford compound 9a. The other com-
pounds 9b–g were prepared by following the same
procedure.
3.6 | N-Cyclohexyl-4-(1,3-diphenyl-1H-
pyrazole-4-carbonyl)piperazine-
1-carboxamide 9c
Off white solid, yield 51 mg (70%); m.p. 148–150ꢀC. IR (υ
cmÀ1, KBr): 1618.80, 1600.51 (C═O str), 1232.30 (C─N
str). 1H NMR (DMSO-d6, 300 MHz): δ 8.65 (s, 1H,
Pyrazole-H), 7.90 (d, J = 7.8 Hz, 2H, ArH), 7.71 (dd,
J = 8.4 and 1.5 Hz, 2H, ArH), 7.33–7.55 (m, 5H, ArH),
5.91 (d, J = 7.5 Hz, 1H, ArH), 5.41 (d, J = 7.5 Hz, 1H, NH
[D2O exchangeable]), 3.27–3.47 (m, 6H, Piperazine-H),
3.16–3.19 (m, 2H, Piperazine-H), 3.09–3.13 (m, 1H, CH),
1.46–1.81 (m, 4H, 2 Â CH2), 0.98–1.30 (m, 6H, 3 x CH2);
13C NMR (CDCl3, 100 MHz): δ 164.59 (C═O), 156.44
(C═O), 149.99 (C═N), 139.21, 131.93, 129.45, 128.79,
128.69, 128.49, 127.39, 127.09, 119.13, 116.35, 49.44,
46.68, 42.98, 33.77, 25.47, 24.91; MS (ESI): m/z 458.43 [M
+ H+]. Anal. Calcd for C27H31N5O2: C, 70.87; H, 6.83%.
Found: C, 70.77; H, 6.89%.
3.4 | N-Cyclopropyl-4-(1,3-diphenyl-1H-
pyrazole-4-carbonyl)piperazine-
1-carboxamide 9a
Off white solid, yield 85.7 mg (86%); m.p. 105–107ꢀC. IR
(υ cmÀ1, KBr): 1615.39, 1600.55 (C═O str), 1230.80 (C─N
str). 1H NMR (DMSO-d6, 300 MHz): δ 8.67 (s, 1H,
Pyrazole-H), 7.90 (d, J = 7.8 Hz, 2H, ArH), 7.67 (d,
J = 6.9 Hz, 2H, ArH), 7.41–7.60 (m, 6H, ArH), 6.49 (d,
J = 2.4 Hz, 1H, NH [D2O exchangeable]), 3.56–3.63 (m,
2H, Piperazine-H), 3.18–3.36 (m, 4H, Piperazine-H),
2.88–2.95 (m, 2H, Piperazine-H), 2.44–2.55 (m, 1H,
N─CH), 0.55–0.57 (m, 2H, CH2), 0.35–0.36 (m, 2H, CH2);
13C NMR (CDCl3, 100 MHz): δ 164.62 (C═O), 158.08
(C═O), 150.03 (C═N), 139.23, 131.94, 129.49, 128.85,
128.72, 128.53, 127.43, 127.15, 119.17, 116.34, 46.73,
42.97, 23.37, 6.55; MS (ESI): m/z 416.10 [M + H]+. Anal.
Calcd for C24H25N5O2: C, 69.38; H, 6.06%. Found: C,
69.47; H, 5.99%.
3.7 | 4-(1,3-Diphenyl-1H-pyrazole-
4-carbonyl)-N-(4-methoxyphenyl)
piperazine-1-carboxamide 9d
Off white solid, yield 47 mg (68%); m.p. 171–173ꢀC. IR (υ
cmÀ1, KBr): 1634.80, 1619.40 (C═O str), 1223.30 (C─N
str). 1H NMR (DMSO-d6, 400 MHz): δ 8.82 (s, 1 H,
Pyrazole-H), 8.40 (s, 1 H, NH [D2O exchangeable]), 7.94
(dd, J = 8.8, 0.8 Hz, 2H, ArH), 7.71 (dd, J = 8.4, 1.2 Hz,
2H, ArH), 7.56 (t, J = 8.4 Hz, 2H, ArH), 7.49 (t,
J = 8.4 Hz, 2H, ArH), 7.34–7.44 (m, 2H, ArH), 7.30 (d,
J = 7.6 Hz, 2H, ArH), 6.81 d, J = 7.6 Hz, 2H, ArH), 3.64–
3.69 (m, 5H, ─OCH3 and Piperazine-H), 3.42–3.52 (m,
2H, Piperazine-H), 3.26–3.31 (m, 2H, Piperazine-H),
3.06–3.17 (m, 2H, Piperazine-H); 13C NMR (CDCl3,
100 MHz): δ 164.66 (C═O), 155.88 (C═O), 155.28, 149.99
(C═N), 139.19, 131.92, 131.51, 129.49, 128.88, 128.75,
128.45, 127.40, 127.16, 122.70, 119.14, 116.23, 113.99,
3.5 | N-Cyclopentyl-4-(1,3-diphenyl-1H-
pyrazole-4-carbonyl)piperazine-
1-carboxamide 9b
Off white solid, yield 54 mg (72%); m.p. 118–120ꢀC. IR (υ
cmÀ1, KBr): 1618.04, 1603.24 (C═O str), 1240.79 (C─N
str). 1H NMR (DMSO-d6, 300 MHz): δ 8.79 (s, 1H,
Pyrazole-H), 7.94 (d, J = 8.1 Hz, 2H, ArH), 7.69 (d,

6
PHOUGAT ET AL.
55.34 (OCH3), 46.75, 43.32; MS (ESI): m/z 482.21 [M
+ H+]. Anal. Calcd for C28H27N5O3: C, 69.84; H, 5.65%.
Found: C, 69.77; H, 5.69%.
3.10 | 4-(1,3-Diphenyl-1H-pyrazole-
4-carbonyl)-N-(p-tolyl)piperazine-
1-carboxamide 9g
Off white solid, yield 56 mg (79%); m.p. 154–156ꢀC. IR (υ
cmÀ1, KBr): 1638.87, 1622.16 (C═O str), 1244.92 (C─N
str). 1H NMR (DMSO-d6, 400 MHz): δ 8.82 (s, 1H,
Pyrazole-H), 8.46 (s, 1H, NH [D2O exchangeable]), 7.95
(dd, J = 8.4, 0.8 Hz, 2H, ArH), 7.71 (dd, J = 8.4, 1.2 Hz,
2H, ArH), 7.56 (t, J = 8.4 Hz, 2H, ArH), 7.49 (t,
J = 8.4 Hz, 2H, ArH), 7.36–7.44 (m, 2H, ArH), 7.29 (d,
J = 8.4 Hz, 2 H, ArH), 7.02 (d, J = 8.0 Hz, 2H, ArH),
3.62–3.72 (m, 2H, Piperazine-H), 3.42–3.48 (m, 2H,
Piperazine-H), 3.23–3.29 (m, 2H, Piperazine-H), 3.07–
3.18 (m, 2H, Piperazine-H), 2.21 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz): δ 164.68 (C═O), 154.99 (C═O), 150.01
(C═N), 139.20, 135.95, 132.88, 131.93, 129.49, 129.23,
128.90, 128.76, 128.48, 127.42, 127.17, 120.52, 119.15,
116.23, 46.76, 43.21, 20.65 (CH3); MS (ESI): m/z 466.24
[M + H+]. Anal. Calcd for C28H27N5O2: C, 72.24; H,
5.85%. Found: C, 72.15; H, 5.89%.
3.8 | 4-(1,3-Diphenyl-1H-pyrazole-
4-carbonyl)-N-(3-fluoro-4-methylphenyl)
piperazine-1-carboxamide 9e
Off white solid, yield 53 mg (77%); yield; m.p. 198–200ꢀC.
IR (υ cmÀ1, KBr): 1651.76, 1615.05 (C═O str), 1222.65
1
(C─N str). H NMR (DMSO-d6, 400 MHz): δ 8.82 (s, 1H,
Pyrazole-H), 8.52 (s, 1H, NH [D2O exchangeable]), 7.95
(d, J = 7.6 Hz, 2H, ArH), 7.72 (d, J = 8.8 Hz, 2H, ArH),
7.56 (t, J = 8.4 Hz, 2H, ArH), 7.49 (t, J = 7.6 Hz, 2H,
ArH), 7.34–7.45 (m, 2H, ArH), 7.30 (dd, J = 7.2, 2.4 Hz,
1 H, ArH), 7.18–7.25 (m, 1H, ArH), 6.98 (t, J = 9.2 Hz,
1H, ArH), 3.66–3.70 (m, 2H, Piperazine-H), 3.46–3.70 (m,
2H, Piperazine-H), 3.24–3.30 (m, 2H, Piperazine-H),
3.07–3.16 (m, 2H, Piperazine-H), 2.17 (s, 3H, CH3); 13C
NMR (DMSO-d6, 100 MHz): δ 164.03 (C═O), 157.68 ([d,
1JCF = 243 Hz]), 155.38 (C═O), 149.75 (C═N), 139.55
(C─N), 136.68 (C─NH), 132.47, 130.10, 129.52, 129.25 (d,
2
2JCF = 19 Hz), 127.51 (d, JCF = 17 Hz), 124.13, 123.96,
3.11 | Biological assay
123.18, 119.18, 119.06, 115.03, 114.80, 47.13, 43.93, 14.85
(CH3); MS (ESI): m/z 484.36 [M + H+]. Anal. Calcd for
C28H26FN5O2: C, 69.55; H, 5.42%. Found: C,
69.47; H, 5.39%.
3.11.1 | Test microorganisms
Bacterial cultures (Bacillus cereus MTCC1272, Staphylo-
coccus aureus MTCC737, Escherichia coli MTCC118,
Yersinia enterocolitica MTCC861) and fungus (Aspergillus
flavus MTCC2798, Aspergillus niger MTCC872, 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.
3.9 | N-(4-Chloro-3-fluorophenyl)-
4-(1,3-diphenyl-1H-pyrazole-4-carbonyl)
piperazine-1-carboxamide 9f
Off white solid, yield 43.5 mg (66%); m.p. 200–202ꢀC. IR
(υ cmÀ1, KBr): 1677.03, 1644.79 (C═O str), 1230.74 (C─N
str). 1H NMR (DMSO-d6, 400 MHz): δ 8.81 (s, 1H,
Pyrazole-H), 8.75 (s, 1H, NH [D2O exchangeable]), 7.95
(dd, J = 8.8, 1.2 Hz, 2H, ArH), 7.69–7.72 (m, 3H, ArH),
7.56 (t, J = 8.4 Hz, 2H, ArH), 7.49 (t, J = 8.4 Hz, 2H,
ArH), 7.34–7.44 (m, 3H, ArH), 7.28 (t, J = 9.2 Hz, 1H,
ArH), 3.68–3.72 (m, 2H, Piperazine-H), 3.47–3.53 (m, 2H,
Piperazine-H), 3.20–3.29 (m, 2H, Piperazine-H), 3.12–
3.19 (m, 2H, Piperazine-H); 13C NMR (DMSO-d6,
100 MHz): δ 163.55 (C═O), 154.51 (C═O), 153.50 (d,
1JCF = 238.9 Hz), 149.29 (C═N), 139.05 (C─N), 137.64
3.11.2 | Antimicrobial activity
The title compounds 7, 8 (free base), and 9a–g were eval-
uated for their antimicrobial activities by broth macro-
dilution method as described previously by Sood et al.
[30]. Briefly, to prepare the stock solution, 10 mg of the
test compounds were dissolved in DMSO (4 ml) and simi-
larly, 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
adjusted to 5 Â 107cfu/ml by using 0.5 McFarland stan-
dard. MICs of test compounds and test drugs were deter-
mined in sterile test tubes containing 2.0 ml of nutrient
broth or Mueller-Hinton broth (for bacteria) and
(C─NH),
131.97,
129.62,
129.08,
128.77
(d,
2JCF = 18.3 Hz), 127.04 (d, JCF = 18.2 Hz), 120.67,
119.52, 119.46, 118.73, 118.56, 116.53, 116.33, 46.57,
43.39, 14.85 (CH3); MS (ESI): m/z 504.31 [M + H+].
Anal. Calcd for C27H23ClFN5O2: C, 64.35; H, 4.60%.
Found: C, 64.42; H, 4.53%.
2

PHOUGAT ET AL.
7
Sabouraud dextrose broth (for fungus). Two milliliter
stock solution of test compounds and standard antimicro-
bial agents were added to the 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,
1250, 625, 312.5, 156.25, 78.1, 39.06, 19.53,
9.76 μg/ml. Broth tubes were inoculated with 10 μl of
microbial suspension so that final counts were
5 Â 105cfu/ml. Tubes containing medium or medium
plus DMSO, and tubes without microbial inoculum
served as growth and sterility controls, respectively. Broth
tubes were incubated at 35–37ꢀC for 24 h and examined
for the presence or absence of growth. The minimum
concentration of test compounds and standard antimicro-
bials that resulted in complete growth inhibition was
recorded as MIC.
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4 | CONCLUSIONS
In summary, a convenient synthesis and antimicrobial screen-
ing results of a series of novel N-cycloalkyl/aryl-4-(1,
3-diphenyl-1H-pyrazole-4-carbonyl)piperazine-1-carboxamides
has been reported. This synthetic strategy can be useful for
the organic as well as medicinal chemists having interest in
developing potential pyrazole based drugs due to its opera-
tional simplicity and easy work up in each step. Some of the
synthesized compounds showed significant antibacterial and
antifungal activities as evident from their minimum inhibi-
tory concentrations (MIC). Notably, (1,3-diphenyl-1H-pyrazol-
4-yl)(piperazin-1-yl)methanone hydrochloride can be further
used as versatile building block for the synthesis of various
useful organic compounds that may be of great interest in
developing the novel piperazine doped pyrazole-based drugs.
ACKNOWLEDGMENTS
The authors are grateful to Hon'ble Vice-Chancellor,
Eternal University, Baru Sahib for providing facilities and
financial assistance. The authors would like to acknowl-
edge GVK Biosciences Pvt. Ltd. Hyderabad and Regional
Sophisticated Instrumentation Centre, Punjab University,
Chandigarh, for recording the analytical data during the
study.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are avail-
able in the supplementary material of this article.
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ORCID
Karan Singh https://orcid.org/0000-0001-8956-5558

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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: H. Phougat, V. Devi,
S. Rai, T. S. Reddy, K. Singh, J Heterocyclic Chem
2021, 1. https://doi.org/10.1002/jhet.4325