Synthesis of new pyrazole derivatives via multicomponent reaction and evaluation of their antimicrobial and antioxidant activities

Article abstract of DOI:10.1007/s00706-015-1428-5

Abstract This work describes a facile synthesis and characterization of a new series of pyrazole containing pyrimidine, 1,4-dihydropyridine, and imidazole derivatives using substituted 4-formylpyrazole as a key intermediate via multicomponent reaction seq

Full text of DOI:10.1007/s00706-015-1428-5

Monatsh Chem
DOI 10.1007/s00706-015-1428-5
ORIGINAL PAPER
Synthesis of new pyrazole derivatives via multicomponent
reaction and evaluation of their antimicrobial and antioxidant
activities
•
•
•
Shivapura Viveka Dinesha Leelavathi Narayana Madhu
Gundibasappa Karikannar Nagaraja
Received: 25 August 2014 / Accepted: 19 January 2015
Ó Springer-Verlag Wien 2015
Abstract This work describes a facile synthesis and
characterization of a new series of pyrazole containing
pyrimidine, 1,4-dihydropyridine, and imidazole derivatives
using substituted 4-formylpyrazole as a key intermediate
via multicomponent reaction sequence. The structures of
these unknown compounds were elucidated by IR, 1H
NMR, 13C NMR, LC–MS, and elemental analysis. The
synthesized products were screened for their in vitro anti-
microbial and antioxidant properties. Among the tested
3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazol-4-yl, incorpo-
rated acetyl dihydropyrimidine compounds exhibited
promising antimicrobial activity and DPPH radical scav-
enging activity with levels of inhibition at 89.4 and 83.3 %,
respectively.
Keywords Pyrazole ꢀ Pyrimidine ꢀ Dihydropyridine ꢀ
Imidazole ꢀ Antimicrobial ꢀ Antioxidant
Introduction
Antimicrobial agents are considered one of the most sig-
nificant milestones in modern medicines. Infections from
resistant bacteria are now relatively usual, and some
pathogens have become resistant to multiple types or
classes of antibiotics. However, the incidence of systemic
microbial infections has increased relentlessly due to
increase of immune compromised hosts [1]. Alongside, the
side effects of some antibiotics can sometimes prove to be
more difficult to tackle than the ailment they are mean to
cure. In order to handle this situation, the continued
development of new efficient antibiotic agents is more
crucial than ever [2]. Much more could be achieved by
better and more widespread application of these measures,
and there are many promising opportunities for innovation
in this area [3]. Many researchers have shown that anti-
oxidants of natural or synthetic origin could have a great
importance as therapeutic agents [4]. There are increasing
experimental and clinical evidences showing the involve-
ment of oxidative stress induced by reactive oxygen and
nitrogen species in a variety of disorders and various other
diseases are caused because of their violent reactivity.
Antioxidants can minimize or inhibit the oxidative damage
byinterrupting the free radical formation or terminating the
chain reaction [5]. Moreover, the applications of antioxi-
dants such as food preservatives and skin-protective source
in cosmetics have also attracted much interest [6].
Graphical abstract
S. Viveka ꢀ Dinesha ꢀ G. K. Nagaraja (&)
Department of Studies in Chemistry, Mangalore University,
Mangalagangotri 574199, Karnataka, India
e-mail: nagarajagk@gmail.com
Pyrazole and halosubstituted 1,3-diarylpyrazoles [7] are
considered to be an important chemical synthon of various
physiological significance and pharmaceutical utility [8–
L. N. Madhu
Post Graduation Department of Biochemistry, St. Aloysius
College, Mangalore 575003, India
123

S. Viveka et al.
11]. Antimicrobial and antioxidant-like biological activities
have been claimed for compounds comprising pyrazole
ring [12–14]. Moreover, pyrazole functionality makes the
core structure of medicinally important molecules such as
lonazolac, celecoxib, and deracoxib [15]. The incorpora-
tion of the aryl system into the pyrazole can severely
modify the biological properties of such molecules [16].
Dihydropyrimidinones (DHPMs) and their derivatives
have attracted considerable interest because of their phar-
macological and therapeutic properties such as
antibacterial, antiviral, antitumour, and anti-inflammatory
[17]. Hantzsch 4-aryl-1,4-dihydropyridines are biologically
and medicinally important class of compounds due to their
high potency and selectivity of action as calcium channel
blockers [18]. In addition, this class of compounds have
been broadly studied for different pharmacological prop-
erties such as antimicrobial, antioxidant, anti-
inflammatory, and antiulcer [19, 20]. Pyrazole tagged to
1,4-dihydropyridines (DHPs) are found to be potent anti-
bacterial and antioxidant agents [21]. Similar to DHPMs
and DHPs, imidazole and its derivatives also possess a
broad spectrum of pharmacological activities such as
antiinflammatory, analgesic, antitubercular, and antimi-
crobial [22, 23]. The literature survey revealed that
DHPMs, DHPs, and imidazoles are other important phar-
Scheme 1
macodynamic
heterocyclic
nuclei, which
when
are 4-formylpyrazoles 2a, 2b, which were synthesized by
the Vilsmeier-Haack reaction from the corresponding
hydrazones [27]. In the present study, 4-formylpyrazoles
were converted into dihydropyrimidinone derivatives 3a–
3f by one-pot three-component Biginelli reaction. A series
of pyrazoles bearing 1,4-dihydropyridine in the 4-position
was constructed by the classical Hantzsch condensation
4a–4f reaction. The 2,4,5-trisubstituted imidazoles 5a, 5b
were obtained in excellent yields by refluxing key aldehyde
with 1,2-diketone and ammonium acetate in acetic acid for
6–7 h via Debus reaction [28]. The structures of all the
compounds were confirmed by their characterization data
(Table 1).
incorporated in different heterocyclic templates have been
reported to possess potent antimicrobial as well as anti-
oxidant activity [20, 24, 25]. 2,4,5-Triarylimidazole
compounds also have gained remarkable importance due to
their widespread biological activities [26].
Based on these precedents, we sought to construct
hybrid molecular architectures by combining pyrazole with
biologically active DHPMs, DHPs, and imidazole phar-
mocophores through a multicomponent reaction sequences.
We envisioned that the resultant conjugates by virtue of the
presence of critical structural features might serve as a
prototype for new drugs that could be used in antimicrobial
and antioxidant research. In continuation of our research
work related to pyrazole, imidazole chemistry, and anti-
oxidant activity [27–29], we report here the synthesis,
in vitro antimicrobial, and antioxidant activities of new
pyrazole tagged multi-functionalized dihydropyrimidinon-
es (DHPMs), dihydropyridines (DHPs), and imidazoles.
Antimicrobial activity
All the synthesized compounds were screened for their
antimicrobial activity by disc diffusion method. The com-
pounds were evaluated for their antibacterial activities
against Staphylococcus aureus MTCC-7443, Escherichia
coli MTCC-443, Pseudomonas Aeruginosa MTCC-424,
and Klebsiella pneumonia MTCC-139, whereas antifungal
activities were tested against Aspergillus niger MTCC-281
and Aspergillus flavus MTCC-871. The activities and the
minimum inhibitory concentration (MIC) of the com-
pounds are presented in Table 2. Streptomycin and
itraconozole were used as standards for antibacterial and
Results and discussion
Chemistry
Synthetic strategies for the investigation of new antimi-
crobial and antioxidant derivatives are illustrated in
Scheme 1. The key intermediates used for the present study
123

Synthesis of new pyrazole derivatives
Table 1 Characterization data of compounds 3a–3f, 4a–4f, and 5a, 5b
Comp.
R
R1
Yield/%
M.p./°C
3a
3b
3c
3d
3e
3f
Cl
Cl
Cl
F
OC2H5
OCH3
CH3
90
88
77
89
80
72
74
79
64
72
65
62
86
78
147–149
132–136
187–192
169–171
184–186
167–169
112–115
123–126
118–121
119–123
129–131
135–137
210–212
197–199
OC2H5
OCH3
CH3
F
F
4a
4b
4c
4d
4e
4f
Cl
Cl
Cl
F
OC2H5
OCH3
CH3
OC2H5
OCH3
CH3
F
F
5a
5b
Cl
F
–
–
Table 2 Antimicrobial activity of compounds (3a–f), (4a–f) and 5a, 5b
Comp.
MIC/lg cm-3 (zone of inhibition/mm)
Antibacterial activity
Antifungal activity
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
Aeruginosa
Klebsiella
pneumonia
Aspergillus
niger
Aspergillus
Flavus
3a
3b
3c
3d
3e
3f
25 (13)
12.5 (15)
6.25 (18)
12.5 (12)
25 (12)
12.5 (16)
12.5 (14)
50 (9)
25 (14)
12.5 (15)
6.25(18)
12.5 (14)
12.5 (13)
6.25 (16)
12.5 (14)
25 (11)
50 (11)
25 (10)
12.5 (15)
50 (8)
NP
50 (9)
25 (11)
12.5(12)
12.5 (11)
25 (11)
6.25(19)
25 (12)
100 (4)
NP
25 (12)
12.5 (14)
12.5 (14)
12.5 (13)
25 (11)
6.25 (19)
25 (10)
25 (9)
NP
12.5 (10)
12.5 (15)
25 (11)
25 (10)
12.5 (12)
12.5 (14)
12.5 (13)
50 (8)
25 (10)
6.25(19)
NP
50 (10)
6.25 (17)
25 (11)
NP
4a
4b
4c
4d
4e
4f
50 (10)
25 (11)
12.5 (13)
50 (7)
50 (9)
NP
NP
12.5(12)
12.5(15)
NP
50 (8)
12.5 (14)
50 (7)
NP
25 (11)
12.5 (15)
NP
25(10)
100 (8)
12.5(10)
25(9)
5a
5b
NP
25(12)
NP
NP
50 (10)
25(11)
100 (5)
6.25 (18)
–
NP
100 (7)
–
Streptomycin 6.25 (20)
Itraconozole
6.25 (17)
–
6.25 (19)
–
–
–
6.25 (19)
6.25 (18)
NP indicates bacterial strains are resistant to the compounds [100 lg cm-3
antifungal drugs for comparison. It is more attractive to
speculate on the observation that the result of the antimi-
crobial activity of the various compounds 3a–3f, 4a–4f, 5a,
and 5b displayed variable inhibitory effects on the growth
of the bacteria depending on the nature of the substituents
with the pyrazole.
The antibacterial screening results of compounds 3a–3f
revealed that acetyl-substituted pyrimidinone compounds
3c and 3f showed broad spectrum of antimicrobial activity
against E. coli, P. Aeruginosa and K. pneumonia compa-
rable with the standard streptomycin. With the introduction
of ethoxy 3a and 3d and methoxy 3b and 3e groups in
123

S. Viveka et al.
place of acetyl substituent, gradual decrease in the activity
against the tested bacterium was noticed. In classifying
antibacterial activity of 3a–3f, generally showed that 1,3-
diarylpyrazole incorporated acetyl substituted pyrimidi-
none emerged as potent molecules. In the pyrazole-
substituted 1,4-dihydropyridine series 4a–4f, 4a, 4d, and 4e
showed moderate activity against all the bacterial strains,
while they were less inhibited by compounds 4c and 4f,
which contained acetyl substitution on the dihydropyridine.
The anti-bacterial data of compounds 5a and 5b revealed
that this series of compounds are less active against all the
tested bacterial strains.
series 4a–4f, acetyl-substituted 4c and 4f have exhibited
maximum radical scavenging activity compared to ester
(4a, 4b, 4d, and 4e)-substituted compounds. This may be
due to the rapid keto-enol tautomerism attributed by these
compounds. By comparing the antioxidant results, it is
observed that there is a gradual decrease in the activity of
acetyl (–CO–CH3) substituent 3c, 3f, 4c, 4f followed by
methoxy (–CO–OCH3), 3b, 3e, 4b, 4e and ethoxy (–CO–
OC2H5) ester substituents 3a, 3d, 4a, 4d. This result
implies that modulation of the basic structure through ring
substituents and/or additional functionalization decreases
the antioxidant activity. Compounds with imidazole sub-
stituent 5a and 5b exhibited poor activity than 3a–3f and
4a–4f.
All the series of compounds displayed moderate to good
activity against the various fungal pathogens. Majority of
the compounds 3a–3f showed appreciable antifungal
activity results against A. niger and A. Flavus. Compound
3f showed excellent antifungal activity comparable with
the standard itraconozole against A. niger. However, other
compounds 3a–3e showed a good antifungal profile against
both fungal strains. In the series of 4a–4f, 4e showed some
degree of inhibition for the fungal strains. The compounds
4a, 4b, and 4d against all the tested organisms, showed less
activity. In contrast, compounds with acetyl substituted
1,4-dihydropyridine 4c and 4d have not shown significant
antifungal activity against both the fungal strains. The
results of 5a and 5b support the facts that the imidazole
ring with pyrazole does not contribute to the antifungal
efficacy.
Reducing power assay
Reducing power is associated with antioxidant activity and
may serve as a significant reflection to it [30]. Compounds
with reducing power indicate that they are electron donors
and can reduce the oxidized intermediates of lipid perox-
idation processes so that they can act as primary and
secondary antioxidants. The substances, which have
reduction potential, react with potassium ferricyanide to
form potassium ferrocyanide, which then reacts with ferric
chloride to form ferrous complex that has an absorption
maximum at 700 nm. Increased absorbance of the reaction
mixture indicates the increased reducing power [31]. The
results are summarized in Table 3.
Reducing power assay is expressed in effective con-
centration (mg/cm3) equivalent of 0.5 absorbance
glutathione. Among the tested compounds, compounds 3a,
3b, 3e, and 4a showed good reducing power capacity while
compounds 3c, 3d, 4c, 4f, and 5b exhibited moderate
reducing power capacity in comparison with the standard
glutathione.
Antioxidant activity
DPPH radical scavenging assay
In the present study, the antioxidant potential of synthe-
sized compounds was studied using the DPPH radical
scavenging technique spectrophotometrically. Radical
scavenging activities of all compounds were determined by
the interacting ability of compounds with stable free radical
DPPH. The degree of discolouration indicated the scav-
enging potential of the antioxidant compounds or extracts
in terms of hydrogen-donating ability.
Conclusions
In conclusion, we have synthesized a new series and highly
functionalized substituted pyrazole structure by one-pot
reaction under simple reaction conditions that resulted in
high yields with high compatibility. The structures of the
derivatives were confirmed by various spectral studies such
The results of the in vitro antioxidant activity are sum-
marized in Table 3. Anti-oxidant activity for all the series
of synthesized compounds showed promising DPPH free
radical scavenging ability. The obtained result gives an
idea that the substituents appear to be an important factor
in their antioxidant results. In the series of newly synthe-
sized compounds, promising antioxidant activity was
shown by dihydropyrimidinones (DHPMs) series 3a–3f.
The potential antioxidant activity emerged from the acidic
proton in the pyrimidinone ring and other groups as a
substituent. Compounds 3c (89.41 %) and 3f (83.34 %)
exhibited excellent DPPH radical scavenging activity as
compared to glutathione (89.09 %). In the dihydropyridine
1
as IR, H NMR, 13C NMR, and LC–MS followed by ele-
mental analysis studies, and screened for their
antimicrobial and antioxidant activities. The compounds 3c
and 3f showed potent antimicrobial and antioxidant agents.
From these studies, we have concluded that pyrimidinone-
incorporated halo-substituted 1,3-diarylpyrazole is a better
molecule from a biological activity point of view and these
molecules can be designed as potential drugs with a
structural modification.
123

Synthesis of new pyrazole derivatives
Table 3 Antioxidant activity of compounds 3a–3f, 4a–4f, and 5a, 5b
Compounds
% DPPH scavenging
Reducing power assay
3a
61.36 9.64
69.54 4.35
89.41 1.58
57.26 6.42
61.36 9.64
83.34 2.50
42.09 1.42
46.81 0.64
67.26 6.42
28.30 1.12
36.81 0.64
60.94 3.14
29.09 1.42
25.34 2.23
89.09 1.09
0.88 0.01
1.75 0.19
7.07 1.66
2.16 0.25
1.12 0.07
11.32 0.45
1.49 0.11
25.00 2.78
2.15 0.19
11.90 0.01
22.72 4.67
4.94 3.14
14.28 2.56
8.33 1.98
0.595 0.01
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
Glutathione
3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbalde-
Experimental
hyde (2a, C16H10Cl2N2O)
ꢀ
Yield 87 %; m.p.: 150–152 °C; IR (KBr): m = 3,041 (Ar
All the reagents and solvents, purchased from commer-
cial suppliers Sigma-Aldrich, Spectrochem India, were
used without further purification. The solvents were all
dried and distilled before use. Melting points were
determined in an open capillary tube. The progress of
each reaction was monitored by ascending thin layer
C–H), 1,678 (C=O), 1,571 (C=N), 1,489 (C=C), 813 (C–
Cl) cm-1; 1H NMR (400 MHz, DMSO-d6): d = 7.42–7.83
(m, 9H, Ar–CH), 9.97 (s, 1H, CHO) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 119.79, 122.75, 128.43,
129.10, 130.24, 130.61, 131.21, 131.77, 132.34, 137.15,
138.88, 149.98 (Ar–C), 184.87 (aldehyde C=O) ppm; LC–
MS: m/z = 317 (M?), 319 (M??2).
chromatography (TLC) on silica gel
G
(Merck
1.05570.0001) and visualized by UV light. The IR
spectra (in KBr pellets) were recorded on a Shimadzu-
1
FTIR spectrometer. The H NMR spectra were recorded
3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazole-4-carbalde-
hyde (2b, C16H10F2N2O)
(CDCl3/DMSO-d6 mixture) on a Bruker AMX 400 NMR
spectrometer with 5 mm PABBO BB-1H TUBES with
TMS as internal standard. LCMS was obtained using
Agilent 1200 series LC and Micromass zQ spectrometer.
Mass spectra of some compounds were recorded on a
Jeol SX-102 (FAB) mass spectrometer. Elemental anal-
yses were carried out using VARIO EL-III (Elementar
Analysensysteme GmBH).
ꢀ
Yield 75 %; m.p.: 95–98 °C; IR (KBr): m = 3,056 (Ar C–
H), 1,675 (C=O), 1,578 (C=N), 1,453 (C = C), 1,086 (C–
F) cm-1; H NMR (400 MHz, DMSO-d6): d = 7.44–9.37
1
(m, 9H, Ar–CH), 9.96 (s, 1H, CHO) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 117.99, 119.71, 122.58,
126.04, 128.33, 129.18, 130.19, 136.83, 138.88, 148.48,
149.19, 150.43, 150.91, 151.65 (Ar–C), 184.87 (aldehyde
C=O) ppm; LC–MS: m/z = 285 (M??1).
General procedure for the preparation of 4-
General procedure for the synthesis of pyrazole
formylpyrazoles 2a, 2b
substituted pyrimidine derivatives 3a–3f
(3,4-Dihalophenyl)ethanone phenylhydrazone (0.01 mol)
was added to a mixture of the Vilsmeier-Haack reagent
(prepared by dropwise addition of 1.2 cm3 POCl3 to
10 cm3 ice-cooled DMF) and refluxed for 6 h. The reaction
mixture was poured into crushed ice followed by neutral-
ization using sodium bicarbonate. Crude product was
isolated and crystallized from methanol.
A mixture of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-
4-carbaldehyde 2 (2.0 mol), ethylacetoacetate (methylace-
toacetate/acetylacetone) (2.2 mol), urea (3.0 mol), and
0.5 cm3 HCl in ethanol medium was heated to reflux for
6 h. Completion of the reaction was monitored by TLC.
The resulting solution was cooled to room temperature and
poured into cold water with vigorous stirring. The resulting
123

S. Viveka et al.
2.14 (s, 3H, CH3), 3.86 (q, 2H, CH2, J = 7.2 Hz), 5.16 (s,
1H, CH), 7.26–7.87 (m, 9H, Ar–H), 8.46 (s, 1H, NH), 9.11
(s, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): d = 14.56
(ester CH3), 17.24 (–CH3), 47.37 (pyrimidine CH), 59.52
(ester CH2), 101.90, 118.69, 123.58, 125.95, 126.90,
127.78, 129.74, 130.07, 130.98, 131.84, 132.86, 134.96,
138.87, 146.58, 148.63 (Ar–C), 153.05 (pyrimidine ring
C=O), 162.52 (ester C=O) ppm; LC–MS: m/z = 438 (M?).
solid was filtered under suction, washed with 50 % ethanol,
and recrystallized from hot ethanol [38].
Ethyl 4-[3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-
yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylate (3a, C23H20Cl2N4O3)
ꢀ
IR (KBr): m = 3,392 (NH), 2,960 (Ar C–H), 1,670 (C=O),
1
1,602 (C=N), 1,579 (C=C), 840 (C–Cl) cm-1; H NMR
(400 MHz, DMSO-d6): d = 0.85 (t, 3H, CH3, J = 7.2 Hz),
2.24 (s, 3H, CH3), 3.78 (q, 2H, CH2, J = 7.2 Hz), 5.34 (s,
1H, CH), 7.30–7.95 (m, 9H, Ar–H), 8.39 (s, 1H, NH), 9.15
(s, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): d = 14.18
(ester CH3), 18.49 (–CH3), 46.43 (pyrimidine CH), 60.24
(ester CH2), 100.90, 119.16, 124.57, 126.87, 126.94,
127.61, 129.42, 130.27, 130.62, 132.48, 132.83, 133.07,
139.53, 146.53, 148.52 (Ar–C), 153.01 (pyrimidine ring
C=O), 165.33 (ester C=O) ppm; LC–MS: m/z = 471 (M?),
473 (M??2), 475 (M??4).
Methyl 4-[3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-
yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylate (3e, C22H18F2N4O3)
ꢀ
IR (KBr): m = 3,406 (NH), 2,956 (Ar C–H), 1,705 (C=O),
1
1,606 (C=N), 1,581 (C=C), 1,101 (C–F) cm-1; H NMR
(400 MHz, DMSO-d6): d = 2.36 (s, 3H, CH3), 3.62 (s, 3H,
OCH3), 5.47 (s, 1H, CH), 7.22–7.81 (m, 9H, Ar–H), 8.01 (s,
1H, NH), 9.06 (s, 1H, NH) ppm; 13C NMR (100 MHz,
CDCl3): d = 46.35 (ester CH3), 18.44 (–CH3), 48.27 (pyrim-
idine CH), 100.75, 117.46, 119.11, 124.45, 124.56, 124.66,
126.70, 126.87, 129.40, 130.10, 139.54, 146.82, 148.92,
149.15, 151.64(Ar–C), 153.30(pyrimidineringC=O), 165.79
(ester C=O) ppm; LC–MS: m/z = 426 (M??2).
Methyl 4-[3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-
yl]-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylate (3b, C22H18Cl2N4O3)
ꢀ
IR (KBr): m = 3,374 (NH), 2,994 (Ar C–H), 1,668 (C=O),
1
1,598 (C=N), 1,545 (C=C), 864 (C–Cl) cm-1; H NMR
5-Acetyl-4-[3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-
yl]-6-methyl-3,4-dihydropyrimidin-2(1H)-one
(3f, C22H18F2N4O2)
(400 MHz, DMSO-d6): d = 1.68 (s, 3H, CH3), 3.32 (s, 3H,
OCH3), 5.47 (s, 1H, CH), 7.48–7.97 (m, 9H, Ar–H), 8.43
(s, 1H, NH), 9.25 (s, 1H, NH) ppm; 13C NMR (100 MHz,
CDCl3): d = 46.35 (ester CH3), 18.44 (–CH3), 48.27
(pyrimidine CH), 101.90, 117.23, 125.71, 126.53, 126.89,
128.52, 129.52, 130.26, 131.04, 132.68, 132.87, 134.12,
138.48, 145.68, 147.52 (Ar–C), 153.50 (pyrimidine ring
C=O), 164.76 (ester C=O) ppm; LC–MS: m/z = 457 (M?),
459 (M??2), 461 (M??4).
ꢀ
IR (KBr): m = 3,367 (NH), 2,974 (Ar C–H), 1,648 (C=O),
1
1,587 (C=N), 1,532 (C=C), 1,045 (C–F) cm-1; H NMR
(400 MHz, DMSO-d6): d = 1.63 (s, 3H, CH3), 2.54 (s, 3H, –
CO–CH3), 5.44 (s, 1H, CH), 7.51–7.97 (m, 9H, Ar–H), 8.39
(s, 1H, NH), 9.18 (s, 1H, NH) ppm; 13C NMR (100 MHz,
CDCl3): d = 27.34 (CO–CH3), 18.24 (–CH3), 48.02 (pyrim-
idine CH), 104.23, 114.58, 123.89, 125.08, 126.68, 128.92,
129.31, 130.52, 131.10, 131.88, 132.26, 135.34, 138.92,
145.37, 147.54 (Ar–C), 158.92 (pyrimidine ring C=O),
178.64 (ester C=O) ppm; LC–MS: m/z = 408 (M?).
5-Acetyl-4-[3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-
4-yl]-6-methyl-3,4-dihydropyrimidin-2(1H)-one
(3c, C22H18Cl2N4O2)
ꢀ
IR (KBr): m = 3,400 (NH), 3,012 (Ar C–H), 1,657 (C=O),
1
1,606 (C=N), 1,577 (C = C), 862 (C–Cl) cm-1; H NMR
General procedure for the synthesis of pyrazole
substituted 1,4-dihydropyridine derivatives 4a–4f
(400 MHz, DMSO-d6): d = 1.71 (s, 3H, CH3), 2.56 (s, 3H,
–CO–CH3), 5.43 (s, 1H, CH), 7.49–7.93 (m, 9H, Ar–H),
8.58 (s, 1H, NH), 9.24 (s, 1H, NH) ppm; 13C NMR
(100 MHz, CDCl3): d = 28.35 (–CO–CH3), 17.46 (–CH3),
47.26 (pyrimidine CH), 102.90, 116.25, 124.79, 125.89,
126.98, 129.02, 129.94, 130.22, 131.08, 132.12, 132.68,
135.45, 139.01, 145.66, 147.54 (Ar–C), 154.34 (pyrimidine
ring C=O), 181.72 (ester C=O) ppm; LC–MS: m/z = 441
(M?), 445 (M??4).
3-(3,4-Dihalophenyl)-1-phenyl-1H-pyrazole-4-carbalde-
hyde 2 (1.0 mol), ethylacetoacetate (methylacetoacetate/
acetyl acetone) (2.0 mol), and ammonium acetate
(1.12 mol) in 20 cm3 ethanol were refluxed for 8 h in an
water bath. After the completion of the reaction, reaction
mixture was concentrated and poured into crushed ice. The
precipitated product was filtered, washed with water, and
recrystallized from hot ethanol.
Ethyl 4-[3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-
6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxyl-
ate (3d, C23H20F2N4O3)
Diethyl 4-[3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-
yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4a, C28H27Cl2N3O4)
ꢀ
IR (KBr): m = 3,392 (NH), 2,978 (Ar C–H), 1,662 (C=O),
1
1,602 (C=N), 1,542 (C=C), 1,032 (C–F) cm-1; H NMR
ꢀ
IR (KBr): m = 3,261 (NH), 3,109 (Ar C–H), 1,666 (C=O),
1,597 (C=N), 1,508 (C=C), 1,230 (C–O), 837 (C–Cl) cm-1
;
(400 MHz, DMSO-d6): d = 0.93 (t, 3H, CH3, J = 7.2 Hz),
123

Synthesis of new pyrazole derivatives
1H NMR (400 MHz, DMSO-d6): d = 0.93 (t, 6H, 2 CH3,
J = 7.2 Hz), 2.24 (s, 6H, 2 CH3), 3.66 (q, 2H, CH2,
J = 7.2 Hz), 3.87 (q, 2H, CH2, J = 7.2 Hz), 5.10 (s, 1H,
CH), 7.27-8.19 (m, 9H, Ar–H), 8.84 (s, 1H, pyridine-NH)
ppm; 13C NMR (100 MHz, CDCl3): d = 14.25 (ester
CH3), 19.52 (–CH3), 29.81 (pyridine CH), 59.79 (ester
CH2), 104.34, 118.89, 126.34, 127.59, 128.24, 129.05,
129.28, 129.85, 130.61, 131.84, 131.33, 134.98, 139.86,
143.39, 148.56 (Ar–C), 167.32 (ester C=O) ppm; LC–MS:
m/z = 540 (M?), 542 (M??2), 544 (M??4).
145.12, 148.76 (Ar–C), 169.54 (ester C=O) ppm; LC–MS:
m/z = 507 (M?).
Dimethyl 4-[3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4e, C26H23F2N3O4)
ꢀ
IR (KBr): m = 3,269 (NH), 3,035 (Ar C–H), 1,651 (C=O),
1
1,564 (C=N), 1,502 (C=C), 1,120 (C–F) cm-1; H NMR
(400 MHz, CDCl3): d = 2.31 (s, 6H, 2 CH3), 3.40 (s, 6H, 2
OCH3), 5.21 (s, 1H, CH), 7.21–7.83 (m, 9H, Ar–H), 5.59
(s, 1H, pyridine-NH) ppm; 13C NMR (100 MHz, CDCl3):
d = 50.69 (ester CH3), 19.42 (–CH3), 29.49 (pyridine CH),
104.36, 116.66, 116.83, 117.52, 117.70, 118.92, 124.76,
126.32, 127.53, 129.04, 131.98, 139.86, 143.63, 148.61,
151.08 (Ar–C), 167.65 (ester C=O) ppm; LC–MS: m/
z = 480 (M??1), 481 (M??2).
Dimethyl 4-[3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4b, C26H23Cl2N3O4)
ꢀ
IR (KBr): m = 3,267 (NH), 3,039 (Ar C–H), 1,647 (C=O),
1,558 (C=N), 1,500 (C=C), 1,240 (C–O), 837 (C–Cl) cm-1
;
1H NMR (400 MHz, DMSO-d6): d = 3.21 (s, 6H, 2 CH3),
1.84 (s, 6H, 2 CH3), 4.10 (s, 1H, CH), 7.46–8.03 (m, 9H,
Ar–H), 8.67 (s, 1H, pyridine-NH) ppm; 13C NMR
(100 MHz, CDCl3): d = 51.65 (ester CH3), 18.53
(–CH3), 30.02 (pyridine CH), 102.36, 118.52, 125.15,
126.89, 128.59, 129.06, 129.54, 129.89, 131.02, 132.33,
135.44, 138.68, 142.58, 146.51, 148.55 (Ar–C), 170.25
(ester C=O) ppm; LC–MS: m/z = 512 (M?), 514 (M??2),
516 (M??4).
4-[3-(3,4-Difluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-diethanone
(4f, C26H23F2N3O2)
ꢀ
IR (KBr): m = 3,276 (NH), 3,012 (Ar C–H), 1,656 (C=O),
1
1,568 (C=N), 1,530 (C=C), 1,021 (C–F) cm-1; H NMR
(400 MHz, DMSO-d6): d = 1.34 (s, 6H, 2 CH3), 2.79 (s,
6H, 2 –CO–CH3), 5.96 (s, 1H, CH), 7.62–7.97 (m, 9H, Ar–
H), 8.34 (s, 1H, pyridine-NH) ppm; 13C NMR (100 MHz,
CDCl3): d = 46.35 (–CO–CH3), 17.08 (–CH3), 31.02
(pyridine CH), 100.68, 116.56, 123.16, 125.98, 127.98,
128.16, 129.54, 130.58, 131.68, 132.51, 134.96, 138.55,
141.78, 144.98, 147.78 (Ar–C), 176.18 (C=O) ppm; LC–
MS: m/z = 447 (M?).
4-[3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,6-
dimethyl-1,4-dihydropyridine-3,5-diethanone
(4c, C26H23Cl2N3O2)
ꢀ
IR (KBr): m = 3,271 (NH), 2,998 (Ar C–H), 1,665 (C=O),
1,565 (C=N), 1,530 (C=C), 1,252 (C–O), 840 (C–Cl) cm-1
;
1H NMR (400 MHz, DMSO-d6): d = 1.75 (s, 6H, 2 CH3),
2.68 (s, 6H, 2 CH3), 5.34 (s, 1H, CH), 7.52–7.93 (m, 9H,
Ar–H), 8.72 (s, 1H, pyridine-NH) ppm; 13C NMR
(100 MHz, CDCl3): d = 48.15 (–CO–CH3), 16.32
(–CH3), 30.72 (pyridine CH), 101.31, 117.52, 124.13,
125.99, 127.95, 128.12, 129.12, 130.01, 131.23, 132.35,
134.88, 138.67, 142.57, 145.33, 148.38 (Ar–C), 177.25
(C=O) ppm; LC–MS: m/z = 480 (M?), 482 (M??2), 484
(M??4).
General procedure for the synthesis
of 2,4,5-trisubstituted imidazoles 5a, 5b
A mixture of 3-(3,4-dihalophenyl)-1-phenyl-1H-pyrazole-
4-carbaldehyde 2 (0.01 mol), benzil (0.01 mol), and
ammonium acetate (0.05 mol) in 30 cm3 acetic acid was
refluxed for 6–7 h at 120 °C. After completion of the
reaction, the reaction mixture was allowed to cool and
filtered to remove any precipitate. The filtrate was added to
the ice cold water and the precipitated product was col-
lected by filtration. The crude product was recrystallized
using ethanol-DMF mixture.
Diethyl 4-[3-(3,4-difluorophenyl)-1-phenyl-1H-pyrazol-
4-yl]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(4d, C28H27F2N3O4)
ꢀ
IR (KBr): m = 3,278 (NH), 3,005 (Ar C–H), 1,661 (C=O),
3-(3,4-Dichlorophenyl)-4-(4,5-diphenyl-1H-imidazol-2-yl)-
1-phenyl-1H-pyrazole (5a, C30H20Cl2N4)
1,578 (C=N), 1,512 (C=C), 1,241 (C–O), 1,040 (C–F)
cm-1; 1H NMR (400 MHz, DMSO-d6): d = 1.53 (t, 6H, 2
CH3, J = 7.3 Hz), 2.58 (s, 6H, 2 CH3), 3.56 (q, 2H, CH2,
J = 7.2 Hz), 3.74 (q, 2H, CH2, J = 7.2 Hz), 5.17 (s, 1H,
CH), 7.34–8.04 (m, 9H, Ar–H), 8.78 (s, 1H, pyridine-NH)
ppm; 13C NMR (100 MHz, CDCl3): d = 14.13 (ester
CH3), 18.98 (–CH3), 30.08 (pyridine CH), 58.74 (ester
CH2), 104.52, 117.84, 126.53, 127.64, 128.62, 129.09,
129.32, 129.79, 131.52, 132.38, 134.99, 139.89, 143.82,
IR (KBr): mꢀ = 3,414 (NH-str), 2,962 (Ar C–H), 1,654
(C=N), 1,562 (C=C), 802 (C–Cl) cm-1
;
1H NMR
(400 MHz, DMSO-d6): d = 7.19–9.05 (m, 19H, Ar–H),
12.53 (s, 1H, imidazole-NH) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 113.55, 118.99, 127.53, 128.66, 129.16,
129.91, 130.23, 130.64, 131.09, 131.20, 131.21, 133.63,
139.39, 139.67, 147.67 (Ar–C) ppm; LC–MS: m/z = 507
(M?), 509 (M??2), 511 (M??4).
123

S. Viveka et al.
3-(3,4-Difluorophenyl)-4-(4,5-diphenyl-1H-imidazol-2-yl)-
measuring the inhibition zone. The results studies were
compared with the standard itraconozole.
1-phenyl-1H-pyrazole (5b, C30H20F2N4)
ꢀ
IR (KBr): m = 3,271 (NH-str), 2,939 (Ar C–H), 1,602
(C=N), 1,508 (C=C), 1,087 (C–F) cm-1 1H NMR
;
DPPH radical scavenging assay
(400 MHz, DMSO-d6): d = 7.21–9.05 (m, 19H, Ar–H),
12.12 (s, 1H, imidazole-NH) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 111.55, 117.22, 127.51, 128.66, 128.52,
129.18, 130.15, 130.78, 131.06, 131.89, 131.54, 133.28,
139.58, 139.62, 142.62 (Ar–C) ppm; LC–MS: m/z = 474
(M??1).
The DPPH assay was based on the reported method [36].
Briefly, 1 mM solution of DPPH in ethanol was prepared,
and this solution (1 cm3) was added to sample solutions
1 mg/cm3 of DMSO. The mixture was shaken vigorously
and allowed to stand at room temperature for 20 min. Then
the absorbance was measured at 517 nm in a spectropho-
tometer. The lower absorbance of the reaction mixture
indicated higher free radical scavenging activity. The
capability to scavenge the DPPH radical was calculated
using the following equation:
Antibacterial activity
The newly synthesized pyrazoles were investigated for
their antibacterial activity against Staphylococcus aureus
MTCC-7,443, Escherichia coli MTCC-443, Pseudomonas
Aeruginosa MTCC-424, and Klebsiella pneumonia MTCC-
139 bacterial strains by the disc diffusion method [32, 33].
The discs 6.25 mm in diameter were punched from
Whatman No.1 filter paper. Batches of 100 discs were
dispensed to each screw-capped bottle and sterilized by dry
heat at 140 °C for 1 h. The test compounds were prepared
with different concentrations using dimethylformamide
(DMF). One cm3 containing 100 times the amount of
chemical was added to each bottle, which contained 100
discs, which were then placed in triplicate in a nutrient agar
medium separately seeded with fresh bacteria. The incu-
bation was carried out at 37 °C for 24 h. Solvent and
growth controls were kept, and the zones of inhibition and
minimum inhibitory concentrations (MIC) were noted. The
results were compared with the standard streptomycin.
DPPH scavenging effect ð%Þ ¼ ðA0 ꢁ A1Þ =A0 ꢂ 100;
where A0 is the absorbance of the control reaction and A1 is
the absorbance in the presence of the samples or standards.
Each sample was assayed at 1 mg/cm3 and all experiments
were carried out in triplicate.
Reducing power assay
The reducing powers of the synthesized compounds were
determined according to the reported method [37]. Dif-
ferent concentrations of the samples (100–1,000 lg/cm3) in
1 cm3 DMSO were mixed with 2.5 cm3 phosphate buffer
(0.2 M, pH = 6.6) and 2.5 cm3 potassium ferricyanide
(1 % solution). The mixture was incubated at 50 °C for
20 min. after which 2.5 cm3 10 % trichloroacetic acid was
added to the mixture and centrifuged for 10 min. The upper
layer of the solution (2.5 cm3) was mixed with 2.5 cm3
distilled water and 0.5 cm3 FeCl3 (0.1 %), and then the
absorbance at 700 nm was measured using a spectropho-
tometer. The higher absorbance of the reaction mixture
indicated greater reducing power. All experiments were
carried out in triplicate and the reducing power assay was
represented by effective concentration (mg/cm3) equivalent
to 0.5 absorbance glutathione.
Antifungal activity
Newly synthesized pyrazoles were screened for their anti-
fungal activity against Aspergillus niger and Aspergillus
Flavus in DMSO by serial plate dilution method [34, 35].
Sabouraud’s agar media were prepared by dissolving 1 g
peptone, 4 g D-glucose, and 2 g agar in 100 cm3 distilled
water and the pH was adjusted to 5.7. Normal saline was
used to make a suspension of the spores of the fungal strain
for lawning. A loopful of a particular fungal strain was
transferred to 3 cm3 of saline to get a suspension of the
corresponding species. Agar media of 20 cm3 was poured
into each Petri dish. An excess of the suspension was
decanted, and the plates were dried by placing them in an
incubator at 37 °C for 1 h. Using an agar punch, wells were
made on these seeded agar plates, and 10–50 mg/cm3 of
the test compounds in DMSO was added into each well
labeling. A control was also prepared for the plates in the
same way using the solvent DMSO. The Petri dishes were
prepared in triplicate and maintained at 37 °C for
3–4 days. Antifungal activity was determined by
Acknowledgments The authors gratefully acknowledge the Uni-
versity Grants Commission (UGC) and Promotion of University
Research and Scientific Excellence (PURSE) for the financial assis-
tance. They are grateful to IISC Bangalore and USIC Mangalore
University for providing the spectral analysis.
References
1. Hitchock CA (1993) Biochem Soc Trans 21:1039
2. Amir M, Javed SA, Hassan MZ (2012) Med Chem Res 21:1261
3. Walsh C (2000) Nature 406:775
4. Laguerre M, Lecomte J, Villeneuve P (2007) Prog Lipid Res
46:244
123

Synthesis of new pyrazole derivatives
5. Aruoma OI (1998) J Am Oil Chem Soc 75:199
6. Winkler C, Frick B, Schroecksnadel K, Schennach H, Fuchs D
(2006) Food Chem Toxicol 44:2003
7. Malladi S, Isloor AM, Peethambar SK, Fun HK (2013) Med
Chem Res 22:2654
8. Thumar NJ, Patel MP (2011) Med Chem Res 21:1751
9. Zelenin KN, Bezhan IP, Pastushenkov LV, Gromova EG, Les-
iovskaja EE, Chakchir BA, Melnikova LF (1999)
Arzneimittelforsch 49:843
10. Hall A, Billinton A, Brown SH, Clayton NM, Chowdhury A,
Gerald MP, Goldsmith GP, Hayhow TG, Hurst DN, Kilford IR,
Naylor A, Passingham B (2008) Bioorg Med Chem Lett 18:3392
11. Rashad AE, Hegab MI, Abdel-Megeid RE, Micky JA, Abdel-
Megeid FME (2008) Bioorg Med Chem 16:7102
12. Kumar KA, Jayaroopa P (2013) Int J PharmTech Res 5:1473
13. Chauhan A, Sharma PK, Kaushik N (2011) Int J ChemTech Res
3:11
21. Zhou K, Wang X, Zhao Y, Cao Y, Fu Q, Zhang S (2011) Med
Chem Res 20:1325
22. Achar KCS, Hosamani KM, Seetharamareddy HR (2010) Eur J
Med Chem 45:2048
23. Vijesh AM, Isloor AM, Telkar S, Peethambar SK, Rai S, Isloor N
(2011) Eur J Med Chem 46:3531
24. Sharma V, Chitranshi N, Agarwal AK (2014) Int J Med Chem
2014:202784
25. Tirzitis A, Tirzite D, Hyvonen Z (2001) Czech J Food Sci 19:81
26. Kalidhar U, Kaur A (2011) Res J Pharm Biol Chem Sci 2:1116
27. Zarghi A, Arfaei S, Ghodsi R (2012) Med Chem Res 21:1803
28. Viveka S, Dinesha, Laxmeshwar SS, Nagaraja GK (2012) Mol-
bank 3:M776
29. Viveka S, Prabhuswamy M, Dinesha, Lokanath NK, Nagaraja
GK (2014) Mol Cryst Liq Cryst 588:83
30. Dinesha, Viveka S, Naik P, Nagaraja GK (2014) Med Chem Res
23:4189
14. Jamwal A, Javed A, Bhardwaj V (2013) J Pharm BioSci 3:114
15. Kumar P, Chandak N, Kaushik P, Sharma C, Kaushik D, Aneja
KR, Sharma PK (2012) Med Chem Res 21:3396
16. El-Sabbagh OI, Baraka MM, Ibrahim SM, Pannecouque C, An-
drei G, Snoeck R, Balzarin J, Rashad AA (2009) Eur J Med Chem
44:3746
31. Oktay M, Gulcin I, Kufrevioglu OI (2003) LWT Food Sci
Technol 36:263
32. Samshuddin S, Narayana B, Sarojini BK, Madhu LN (2013) Med
Chem Res 22:3002
33. Nandakumar A, Thirumurugan P, Perumal PT, Vembu P, Pon-
nuswamy MN, Ramesh P (2010) Bioorg Med Chem Lett 20:4252
34. James D, Lowry M, Jaqua MJ, Selepak ST (1970) Appl Microbiol
20:46
17. Sadanadam YS, Shetty MM, Diwan PV (1992) Eur J Med Chem
27:87
18. Baranda AB, Alonso RM, Jimenez RM, Weinmann W (2006)
Forensic Sci Int 156:23
35. Fenlon CH, Cynamon MH (1986) Antimicrob Agents Chemother
29:386
19. Klegeris A, Liutkevicius E, Mikalauskiene G, Duburs G, McGeer
PL, Klusa V (2002) Eur J Pharmacol 441:203
20. Swarnalatha G, Prasanthi G, Sirisha N, Chetty CM (2011) Int J
ChemTech Res 3:75
36. Arthington-Skaggs BA, Motley M, Warnock DW, Morrison CJ
(2000) J Clin Microbiol 38:2254
37. Blois MS (1958) Nature 181:1199
38. Oyaizu M (1986) Japan J Nutr 44:307
123