Synthesis, characterization and antimicrobial activity of novel ethyl 1-(N-substituted)-5-phenyl-1H-pyrazole-4-carboxylate derivatives

Article abstract of DOI:10.1007/s00044-011-9796-9

In the present study, a novel series of Pyrazole derivatives (3a-m) were synthesized by condensing ethyl-3-(dimethylamino)-2-(phenylcarbonyl)prop-2- enoate with different aromatic and aliphatic hydrazines. These newly synthesized compounds were characteri

Full text of DOI:10.1007/s00044-011-9796-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2702–2708
DOI 10.1007/s00044-011-9796-9
ORIGINAL RESEARCH
Synthesis, characterization and antimicrobial activity of novel
ethyl 1-(N-substituted)-5-phenyl-1H-pyrazole-4-carboxylate
derivatives
•
B. Chandrakantha Arun M. Isloor Prakash Shetty
•
•
•
Shrikrishna Isloor Shridhar Malladi Hoong Kun Fun
•
Received: 30 January 2011 / Accepted: 10 September 2011 / Published online: 22 September 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In the present study, a novel series of Pyrazole
derivatives (3a–m) were synthesized by condensing ethyl-
3-(dimethylamino)-2-(phenylcarbonyl)prop-2-enoate with
different aromatic and aliphatic hydrazines. These newly
synthesized compounds were characterized by NMR, mass
spectral, IR spectral studies as well as by C, H, and N
analyses. All the newly synthesized compounds were
screened for their antibacterial properties against Staphy-
lococcus aureus, Bacillus subtilis, Escherichia coli, and
Pseudomonas aeruginosa. Among the screened samples,
3c, 3f, 3k, and 3l have showed excellent antibacterial
activity against all the tested bacterial strains as compared
to the standard drug Ceftriaxone. Few of the compounds
were found to be biologically potent. Molecular structure
of compound 3i was confirmed by single crystal X-ray
analysis.
Keywords Pyrazole Á Antibacterial study Á MIC
Introduction
Pyrazoles and their derivatives play an important role in
medicinal chemistry research. Several derivatives of pyr-
azole are of pharmaceutical interest due to their analgesic
power (Hall et al., 2008; Isloor et al., 2000). Among them,
derivatives of 5-isopyrazolone and pyrazolidine-3,5-dione
(Deng et al., 2008) are worth noting due to their clinical
interest. Several, substituted pyrazolines are reported to
possess moderate antibacterial and antifungal activities
(Soliman et al., 2001; Isloor et al., 2009) Besides, a number
of nitrofurylpyrazoline derivatives were found to possess
antibacterial activity (Ragavan et al., 2010; Premsai Rai
et al., 2009; Holla et al., 2000). Pyrazole molecules also
exhibits anticancer, anti-inflammatory (Sunil et al., 2009),
antidepressant, anticonvulsant (Isloor et al., 2000), and anti
HIV properties (Kelekci et al., 2007). The incorporation of
aryl system into the pyrazole ring enhances the biological
activities to a great extent. The presence of different sub-
stituents, both on the pyrazole ring and on the phenyl ring,
can severely modify the biological properties of such
molecules (El-Sabbagh et al., 2009; Bernard et al., 1985).
During the past few years, considerable evidence has been
accumulated to demonstrate the efficacy of pyrazole
derivatives. Prompted by these observations and in con-
tinuation of our search on biologically active heterocycles
(Kalluraya et al., 2004; Chandrakantha et al., 2010;
Isloor et al., 2010), we describe in this article the syn-
thesis of novel pyrazole derivatives carrying aryl ring
system, by condensing ethyl-3-(dimethylamino)-2-(phenyl-
carbonyl)prop-2-enoate with different aromatic/aliphatic
hydrazines.
B. Chandrakantha
Department of Chemistry, Manipal Institute of Technology,
Manipal University, Manipal, India
A. M. Isloor (&) Á S. Malladi
Department of Chemistry, Organic Chemistry Division, National
Institute of Technology, Surathkal, Karnataka 575 025, India
e-mail: isloor@yahoo.com
P. Shetty
Department of Printing, Manipal Institute of Technology,
Manipal University, Manipal, India
S. Isloor
Department of Microbiology, Veterinary College, Veterinary
University, Hebbal, Bangalore, India
H. K. Fun
X-ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, 11800 USM, Penang, Malaysia
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Med Chem Res (2012) 21:2702–2708
Results and discussion
2703
values for all the compounds and C, H, and N analyses are
given in the experimental part. Also, the single crystal
X-ray analysis of 3j further confirmed the structure of the
synthesized compounds. Figure 1 shows crystal structure
of compound 3i. The detailed spectral study has been given
under the experimental and characterization part.
Synthesis and characterization
The reaction between ethylbenzoylacetate (1) and N,N
dimethylformamide dimethyl acetal for 18 h under reflux
condition and further on distilling excess of acetal under
reduced pressure, yielded yellow colored ethyl-3-(dimeth-
ylamino)-2-(phenylcarbonyl)prop-2-enoate (2). Further
reaction between ethyl-3-(dimethylamino)-2-(phenylcar-
bonyl) prop-2-enoate (2) with different series of aromatic/
aliphatic hydrazines under reflux, yielded whitish-yellow-
ish crystalline solid (3) in reasonable yield. Synthetic route
has been presented in Scheme 1.
All the compounds were screened for their possible
antibacterial activity against S. aureus, B. subtilis, E. coli,
and P. aeruginosa. Compounds 3c, 3f, 3k, and 3l have
shown excellent antibacterial activity against all the tested
bacterial strains as compared with the standard drug Cef-
triaxone, which was active at 3.125, 1.6125, 1.6125, and
1.6125 lg/ml against S. aureus, B. subtilis, E. coli, and
P. aeruginosa strains, respectively. Interestingly all these
active compounds are halogen-substituted derivatives,
which is responsible for the enhanced activity of the
compounds.
Formation of the ethyl-1-(N-substituted)-5-phenyl-
1H-pyrazole-4-carboxylate derivatives was confirmed by
1
recording their IR, H-NMR, 13C-NMR, mass spectra, and
by single crystal X-ray analysis. IR spectrum of compound
3a showed absorption at 3500 and 2990 cm-1, which is
due to the aromatic stretching. Absorption band at
1600 cm-1 is due to C=N of pyrazole ring, a band at
1509 cm-1 is due to C=C, absorptions at 1230 cm-1 and at
1700 cm-1 are due to the C–O, C=O stretch of ester,
Experimental
Chemistry
1
respectively. The H-NMR of spectrum of 3a showed a
All the chemicals were procured from Aldrich Co, India
and were used without any further purification. Reactions
were monitored, and purity of the products was checked by
TLC which was performed on MERCK 60F-254 silica gel
plates. Melting points were determined using BUCHI
Melting point B-545 instrument. The IR spectra (in KBr
pellets) were recorded on NICOLET 6700FT-IR spectro-
broad singlet near d 13.3 for carboxylic group in the phenyl
ring and a singlet at d 8.23, which is due to the pyrazole
–CH; a doublet observed in the region of d 7.88–7.86 is due
to the two aromatic protons. Similarly, another multiplet
observed in the range of d 7.43–7.28 is due to the seven
protons of the aromatic ring, while a multiplet and triplet at
the regions d 4.13–4.08 and d 1.12–1.089 correspond to
–CH2 and –CH3 of ethyl ester functional group, respec-
tively. The mass spectrum of compound 3a showed the
molecular ion peak at m/z 337, which is in agreement with
the molecular formula C19H16N2O4. Similarly, the spectral
1
photometer. H-NMR spectra were recorded on BRUKER
(400 MHz) spectrometer in DMSO-d6 solvent. Mass
spectra were recorded on LC–MS-Agilent 1100 series with
MSD (Ion trap) using 0.1% aqueous TFA in acetonitrile
system on C18-BDS column for 10 min duration. The
Scheme 1 Synthetic route for
the pyrazole derivatives.
R = Phenyl, 4-carboxyphenyl,
2-carboxyphenyl, 4-CF3-phenyl,
4-tert-butylphenyl,
O
O
O
O
N
N,N-DMF Acetal
Reflux, 18 hrs
O
O
4-fluorophenyl, 2-bromophenyl,
Quinolinyl, 4-tolyl, tertbutyl,
Methyl, 2,4-dicholorophenyl,
Piperidyl, and Cyclohexyl
2
1
R-NH-NH2
EtOH, 80°C
O
O
N
N
R
3
123

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Med Chem Res (2012) 21:2702–2708
Fig. 1 X-ray crystal structure
of compound (3i)
elemental analysis was performed using THERMO Fin-
ningan FLASH EA 1112 CHN analyzer. Single crystal
X-ray analyses were performed using Bruker APEX2
instrument. Column chromatography was performed on
silica gel (60–120 mesh) supplied by Acme Chemical
Co.(India) for compound purification.
absolute ethanol (10 vol) for 2 h, and the excess of solvent
was evaporated under reduced pressure. The residue was
washed with 1.5 N HCl, and the solid separated was fil-
tered and dried under vacuum. The solid obtained was
purified by column chromatography using silica gel 60–120
mesh size and petroleum ether:ethyl acetate as eluent to
afford different N-substituted-5-phenyl-1H-pyrazole-4-
ethyl carboxylate as white and pale yellow crystalline
solid. (3a–3m) in 65–85% yield.
General procedure for the preparation of ethyl-3-
(dimethylamino)-2-(phenylcarbonyl)prop-2-enoate (2)
A mixture of ethylbenzoylacetate (1) (10 g, 0.0520 mol)
and N,N dimethyl formamide dimethyl acetal (30.9 g,
0.26 mol) was heated to reflux for 18 h. The excess of
acetal was distilled off under reduced pressure, and the
residue was purified by column chromatography using
60–120 silica gel mesh size using chloroform and methanol
as an eluent to give yellow solid.(2) (11 g, 85%) with
melting point 65–70°C.
4-[4-(Ethoxycarbonyl)-5-phenyl-1H-pyrazol-1-yl] benzoic
acid (3a)
(TLC, Pet-ether/EtOAc, 1:1, Rf = 0.2), Yield 75%; white
solid.; m.p. 105–110°C; 1H-NMR (DMSO-d6, 24°C): d
13.13 (bs, 1H, –COOH group), 8.23 (s, 1H, Pyrazole –CH),
7.88–7.86 (d, 2H, J = 8.56 Hz, Ar–H), 7.43–7.28 (m, 7H,
Ar–H), 4.13–4.08 (q, 2H), 1.12–1.08 (t, 3H, J = 7.12 Hz).
13C-NMR (DMSO-d6) 165.15, 162.95, 145.33, 142.07,
139.00, 131.67, 130.44, 129.49, 128.78, 128.34, 127.98,
126.01, 113.62., 59.78, 13.83. MS: m/z = 337.3 (M?);
Method: A-0.1% HCOOH, B-MEOH, Coloumn: Atlantis
dC18 (50 9 4.6 mm) 5 mm. Flow rate 1.0 ml/min. IR
(KBr) cm-1: 3500, 2990 (Ar–H), C=N (1600-stretch
of Pyrazole ring), C=C (1509), C–O (1230), C=O
General procedure for preparation of different substituted
pyrazole derivatives (3a–n)
To a solution of ethyl-3-(dimethylamino)-2-(phenylcar-
bonyl) prop-2-enoate (2) (1.0 eq) in different series of
aromatic/aliphatic hydrazines (1.1 eq) were refluxed with
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Med Chem Res (2012) 21:2702–2708
2705
Ethyl 1-(4-fluorophenyl)-5-phenyl-1H-pyrazole-4-
carboxylate (3e)
(1700-stretch of ester); Anal. Calcd. (Found) for C19H16
N2O4: C, 67.85 (67.66); H, 4.79 (4.88); N, 8.33 (8.56).
(TLC, Pet-ether/EtOAc, 9:1, Rf = 0.3) Yield 76%; white
1
solid; m.p. 140–150°C; H-NMR (DMSO-d6): d 8.17 (s,
4-[3-(Ethoxycarbonyl)-5-phenyl-1H-pyrazol-1-yl]benzoic
acid (3b)
1H, pyrazole –CH), 7.38–7.32 (m, 3H, Ar–H), 7.29–7.27
(m, 4H, Ar–H), 7.27–7.18 (m, 2H, Ar–H), 4.12–4.07 (q,
2H), 1.11–1.08 (t, 3H, J = 7.08 Hz). MS: m/z = 311.3
(M?) Method: A- 0.1%TFA, B-MEOH, Column: XBridge
C18 (50 9 4.6 mm) 3.5 mm. Flow rate 2.0 ml/min IR
(KBr) cm -1: 3570, 2865 (Ar–H), C=N (1690-stretch of
Pyrazole ring), C=C (1575), C–O (1445), C=O (1670-
stretch of ester). Anal. Calcd. (Found) for C18H15FN2O2: C,
69.67 (69.71); H, 4.87 (4.87); N, 9.03 (9.03).
(TLC, Pet-ether/EtOAc, 1:1, Rf = 0.40), Yield 82%; white
1
solid.; m.p. 90–100°C; H-NMR (DMSO-d6): d 13.17 (bs,
1H, –COOH), 8.21 (s, 1H, pyrazole –CH), 7.88–7.86
(d, 1H, J = 7.44 Hz, Ar–H), 7.78 (s, 1H, Ar–H), 7.47–
7.29 (m, 7H, Ar–H), 4.13–4.07 (q, 2H), 1.15–1.08 (t,
3H, J = 7.04 Hz).13C-NMR (DMSO-d6) 166.15, 161.95,
145.33, 142.07, 139.00, 131.67, 130.44, 129.49, 129.32,
129.20, 128.78, 128.34, 127.98, 126.01, 113.62., 59.68,
13.93. MS: m/z = 337.3 (M?); Method: A-0.1% HCOOH,
B-MEOH, Column: Atlantis dC18 (50 9 4.6 mm) 5 mm.
Flow rate 1.0 ml/min. IR (KBr) cm-1: 3453, 2996 (Ar–H),
C=N (1634-stretch of Pyrazole ring), C=C (1560), C–O
(1300), C=O (1642-stretch of ester); MS: m/z = 337.3
(M?); Anal. Calcd.(Found) for C19H16N2O4: C, 67.85
(67.80); H, 4.79 (4.82); N, 8.33 (8.42).
Ethyl 1-(2-bromophenyl)-5-phenyl-1H-pyrazole-4-
carboxylate (3f)
(TLC, Pet-ether/EtOAc, 9:1, Rf = 0.3) Yield 80%; white
1
solid.; m.p. 80–90°C; H-NMR (DMSO-d6): d 8.2(s, 1H,
Pyrazole –CH), 7.68–7.66 (d, 1H, J = 7.9 Hz, Ar–H),
7.60–7.58 (d, 1H, J = 7.76 Hz, Ar–H), 7.45–7.41 (m, 1H,
Ar–H), 7.38–7.28 (m, 6H, Ar–H), 4.13–4.08(q, 2H),
1.12–1.09 (t, 3H, J = 7.08 Hz). 13C-NMR (DMSO-d6)
162.04, 146.74, 141.84, 137.94, 133.01, 131.59, 130.77,
130.13, 129.13, 128.39, 127.82, 127.61, 121.59, 112.55,
59.67, 13.95. MS: m/z = 371.3 (M?) Method: A- 0.1%TFA,
B-MEOH, Column: XBridge C18 (50 9 4.6 mm) 3.5 mm.
Flow rate 2.0 ml/min; IR (KBr) cm-1: 3550, 2855(Ar–H),
C=N (1680-stretch of Pyrazole ring), C=C (1585), C–O
(1485), C=O (1680-stretch of ester); Anal. Calcd. (Found)
for C18H15BrN2O2: C, 58.24 (58.55); H, 4.07 (4.15); N, 7.55
(7.62).
Ethyl 5-phenyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-
4-carboxylate (3c)
(TLC, Pet-ether/EtOAc, 8:2, Rf = 0.5), Yield 78%; white
solid; m.p. 98–110°C; 1H-NMR (DMSO-d6): d 8.25 (s, 1H,
Pyrazole -CH), 7.75–7.73 (d, 2H, J = 8.48 Hz, Ar–H),
7.44–7.39 (m, 5H, Ar–H), 7.36-7.31 (t, 2H, J = 11.6 Hz,
Ar–H), 4.13–4.08 (q, 2H), 1.12–1.08 (t, 3H, J = 7.08 Hz)
MS: m/z = 361.3 (M?) Method: A- 0.1%TFA, B-MEOH,
Column: XBridge C18 (50 9 4.6 mm) 3.5 mm. Flow rate
2.0 ml/min. IR (KBr) cm-1: 3553, 2896 (Ar–H), C=N
(1658-strecth of Pyrazole ring), C=C (1567), C–O (1412),
C=O (1650-stretch of ester);; Anal. Calcd. (Found) for
C19H15F3N2O2: C, 63.33 (63.33); H, 4.20 (4.24); N, 7.77
(7.72).
Ethyl 5-phenyl-1-quinolin-2-yl-1H-pyrazole-4-carboxylate
(3g)
(TLC, Pet-ether/EtOAc, 7:3, Rf = 0.4) Yield 82%; pale
1
yellow solid.; m.p. 150–160°C; H-NMR (DMSO-d6): d
Ethyl 1-(4-tert-butylphenyl)-5-phenyl-1H-pyrazole-4-
carboxylate (3d)
8.52–8.50 (d, 1H, J = 8.76 Hz, quinoline H), 8.2 (s, 1H,
Pyrazole –CH), 8.01–7.99 (d, 1H, J = 8.08 Hz, Ar–H),
7.78–7.73 (d, 1H, J = 8.7 Hz, Ar–H), 7.70–7.68 (t, 1H,
J = 7.08 Hz, Ar–H), 7.62–7.58 (t, 1H, J = 7.96 Hz,
Ar–H), 7.47–7.45 (d, 1H, J = 8.36 Hz), 7.33–7.30 (m, 5H,
Ar–H) 4.15–4.09 (q, 2H), 1.14–1.05 (t, 3H, J = 7.12 Hz).
13C-NMR (DMSO-d6) 161.91, 150.06, 145.98, 145.25,
142.36, 139.31, 130.63, 130.20, 129.45, 128.60, 128.15,
127.88, 127.40, 127.28, 126.87, 117.12, 114.33, 59.77,
13.90. MS: m/z = 344.3 (M?) Method: A- 0.1%TFA,
B-MEOH, Column: XBridge C18 (50 9 4.6 mm) 3.5 mm.
Flow rate 2.0 ml/min. IR (KBr) cm-1: 3530, 2835 (Ar–H),
C=N (1630-stretch of Pyrazole ring), C=C (1535), C–O
(1455), C=O (1680-stretch of ester); Anal. Calcd. (Found)
(TLC, Pet-ether/EtOAc, 8:2, Rf = 0.5) Yield 85%; white
1
solid; m.p. 125–130°C; H NMR (DMSO-d6): d 8.15 (s,
1H, Pyrazole –CH), 7.38–7.32 (m, 5H, Ar–H), 7.29–7.28
(d, 2H, J = 7.64 Hz, Ar–H), 7.14–7.12 (d, 2H,
J = 8.52 Hz, Ar–H), 4.11–4.06 (q, 2H), 1.22(s, 9H, tert-
buty) 1.10–1.07 (t, 3H, J = 7.08 Hz) MS: m/z = 349.4
(M?) Method: A- 0.1%TFA, B-MEOH, Column: XBridge
C18 (50 9 4.6 mm) 3.5 mm. Flow rate 2.0 ml/min. IR
(KBr) cm-1: 3560, 2885 (Ar–H), C=N (1670-stretch of
Pyrazole ring), C=C (1585), C–O (1415), C=O (1660-
stretch of ester); Anal. Calcd.(Found) for C22H24N2O2: C,
75.83 (75.63); H, 6.94 (7.00); N, 8.04 (7.95).
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Med Chem Res (2012) 21:2702–2708
Ethyl 1-(2,4-dichlorophenyl)-5-phenyl-1H-pyrazole-4-
carboxylate (3k)
for C21H17N3O2: C 73.45 (73.45), H 4.99 (4.97), N
12.24(12.20).
(TLC, Pet-ether/EtOAc, 7:3, Rf = 0.5) Yield 84%; pale
1
yellow solid.; m.p. 125–135°C; H-NMR (DMSO-d6): d
Ethyl 1-(4-methylphenyl)-5-phenyl-1H-pyrazole-4-
carboxylate (3h)
8.22 (s, 1H, pyrazole -H), 8.21–7.4 (m, 1H, Ar–H), 7.4–7.17
(m, 7H, Ar–H), 4.14 (q, 2H), 1.13–1.09 (t, J = 7.08 Hz, 3H).
13C-NMR (DMSO-d6) 161.04, 144.74, 142.84, 136.94,
134.01, 132.59, 131.77, 131.13, 128.13, 129.39, 128.82,
126.61, 123.59, 114.55, 59.77, 13.92. MS: m/z = 362.2
(M?) Method: A- 0.1% HCOOH, B-MEOH, Column:
Atlantis dC18 (50 9 4.6 mm) 5 mm. Flow rate 1.0 ml/min;
IR (KBr) cm-1: 3620, 2825 (Ar–H), C=N (1620-stretch of
Pyrazole ring), C=C (1525), C–O (1425), C=O (1620-stretch
of ester); Anal. Calcd. (Found) for C18H14Cl2N2O2: C, 59.85
(59.80); H, 3.91(3.97); N, 7.76 (7.72).
(TLC, Pet-ether/EtOAc, 8:2, Rf = 0.3) Yield 80%; pale
yellow solid; m.p. 105–115°C; 1H-NMR (DMSO-d6): d
8.15 (s, 1H, pyrazole-CH), 7.37–7.31 (m, 3H, Ar–H),
7.27–7.25 (t, 2H, J = 7.68 Hz, Ar–H), 7.14–7.12 (d, 1H,
J = 8.321 Hz, Ar–H), 7.09–7.07 (d, 2H, J = 8.40 Hz,
Ar–H), 4.11–4.06 (q, 2H), 2.26 (s, 1H, –CH3 1.12–1.09 (t,
3H, J = 7.08 Hz). 13C-NMR (DMSO-d6)162.04, 145.12,
141.55, 137.78, 136.45, 130.40, 129.33, 128.96, 128.65,
127.84, 125.41, 113.17, 59.56, 20.51, 13.94. MS:
m/z = 307.4 (M?) Method: A- 0.1% HCOOH, B-MEOH,
Column: Atlantis dC18 (50 9 4.6 mm) 5 mm. Flow rate
1.0 ml/min IR (KBr) cm -1: 3670, 2855 (Ar–H), C=N
(1690-stretch of Pyrazole ring), C=C (1545), C–O (1475),
C=O (1640-stretch of ester); Anal. Calcd. (Found) for
C19H18N2O2: C, 74.49 (74.60); H, 5.92 (6.00); N, 9.14
(9.23).
Ethyl 5-phenyl-1-piperidin-4-yl-1H-pyrazole-4-carboxylate
(3l)
(TLC, chloroform/methanol, 8:2, Rf = 0.35) Yield 81%;
1
pale yellow solid; m.p. 60–70°C; H-NMR (DMSO-d6): d
8.01 (s, 1H, pyrazole H), 7.52–7.49 (m, 3H, Ar–H),
7.41–7.37 (m, 2H, Ar–H), 4.13–4.06 (q, 2H), 3.92 (m, 1H),
3.1–2.9 (m, 3H), 2.8–2.49 (m, 1H), 2.01–1.96 (m, 2H),
1.67–1.64 (m, 1H), 1.33–1.30 (m, 1H), 1.10–1.06
(t, J = 7.08 Hz, 3H). 13C-NMR (DMSO-d6) 162.05,
145.11, 140.43, 129.89, 129.23, 128.62, 127.64, 111.86,
59.24, 54.82, 50.10, 24.44, 13.88. MS: m/z = 300.4 (M?)
Method: A- 0.1% HCOOH, B-MEOH, Column: Atlantis
dC18 (50 9 4.6 mm) 5 mm. Flow rate 1.0 ml/min; IR
(KBr) cm-1: 3620 (Ar–H), C=N (1650-stretch of Pyrazole
ring), C=C (1555), C–O (1455), C=O (1650-stretch of
ester), Anal. Calcd. (Found) for C17H21N3O2: C, 68.20
(68.20); H, 7.07 (7.01); N, 14.04 (14.01).
Ethyl 1-tert-butyl-5-phenyl-1H-pyrazole-4-carboxylate (3i)
(TLC, Pet-ether/EtOAc, 8:2, Rf = 0.2) Yield 70%; pale
yellow solid; m.p. 69–75°C; Molecular structure of the
compound has been presented in Fig. 1. 1H-NMR (DMSO-
d6): d 7.88 (s, 1H, Ar–H), 7.46–7.40 (m, 3H, Ar–H),
7.34–7.31 (m, 2H, Ar–H), 3.93 (q, 2H), 1.35 (s, 9H, tert
butyl), 0.94–0.90 (t, J = 7.12 Hz, 3H). MS: m/z = 273.3
(M?) Method: A- 0.1% HCOOH, B-MEOH, Column:
Atlantis dC18 (50 9 4.6 mm) 5 mm. Flow rate 1.0 ml/min
IR (KBr) cm -1: 3630, 2835 (Ar–H), C=N (1630-stretch of
Pyrazole ring), C=C (1535), C–O (1435), C=O (1630-
stretch of ester). Anal. Calcd.(Found) for C16H20N2O2: C,
70.56 (70.65); H, 7.40 (7.35); N, 10.29 (10.33).
Ethyl 1-cyclohexyl-5-phenyl-1H-pyrazole-4-carboxylate
(3m)
(TLC, Pet-ether/EtOAc, 7:3, Rf = 0.35) Yield 78%; pale
1
yellow solid; m.p. 85–90°C; H-NMR (DMSO-d6): d 8.12
Ethyl 1-methyl-5-phenyl-1H-pyrazole-4-carboxylate (3j)
(s, 1H, pyrazole H), 7.65–7.25 (m, 5H, Ar–H), 4.13–4.06
(q, 2H), 3.3–3.2 (m, 1H), 3.1–2.5 (m, 2H), 2.45–2.20 (m,
4H), 2.2–1.99 (m, 4H), 1.12–1.07 (t, J = 7.08 Hz, 3H).
13C-NMR (DMSO-d6) 161.05, 144.11, 142.43, 128.89,
128.23, 128.62, 127.84, 11.96, 59.24, 54.82, 54.60, 50.10,
24.44, 13.88. MS: m/z = 299.4 (M?) Method: A- 0.1%TFA,
B-MEOH, Column: XBridge C18 (50 9 4.6 mm) 3.5 mm.
Flow rate 2.0 ml/min IR (KBr) cm-1: 3630 (Ar–H), C=N
(1630-stretch of Pyrazole ring), C=C (1535), C–O (1435),
C=O (1630-stretch of ester), Anal. Calcd. (Found) for
C18H22N2O2: C, 72.46 (72.50); H, 7.43 (7.39); N. 9.39
(9.39).
(TLC, Pet-ether/EtOAc, 9:1, Rf = 0.23) Yield 78%; pale
yellow solid. m.p. 70–75°C. 1H-NMR (DMSO-d6): d
8.33(s, 1H, Pyrazole H), 7.47–7.33 (m, 5H, Ar–H),
4.12–4.06 (q, 2H), 2.16 (s, 3H,-CH3), 1.10–1.06
(t, J = 7.08 Hz, 3H). MS: m/z = 230.3 (M?) Method:
A- 0.1% HCOOH, B-MEOH, Column: Atlantis dC18
(50 9 4.6 mm) 5 mm. Flow rate 1.0 ml/min IR (KBr)
cm-1: 3640, 2825 (Ar–H), C=N (1690-stretch of Pyra-
zole ring), C=C (1565), C–O (1435), C=O (1700-stretch
of ester); Anal. Calcd.(Found) for C13H14N2O2: C, 67.81
(67.81); H, 6.13 (6.11); N, 12.17 (12.12).
123

Med Chem Res (2012) 21:2702–2708
2707
Antimicrobial studies
containing six tubes. Then, 1.9 ml of trypticase soya broth
was added in the first tube in each row and 1 ml in the
remaining tubes. Now, 100 ll of filtered anti microbial
suspension was added to the first tube in each row and after
mixing the content, 1 ml was serially transferred from these
tubes to the second tube in each of the rows. The contents in
the second tube of each of the rows were mixed and trans-
ferred to the third tube in each of the rows. This serial dilution
was repeated till the sixth tube in each of the rows. This
provided anti-microbial concentrations of 50, 25, 12.5, 6.25,
3.125, and 1.6125 lg/ml in the first to sixth tube, respec-
tively, in each row. Finally, 1 ml of 105 cfu/ml of S. aureus,
B. subtilis, E. coli, and P. aerogenosa suspension were added
to the first, second, third, and fourth rows of tubes, respec-
tively. Along with the test samples and Ceftriaxone (stan-
dard), the inoculums control (without antimicrobial
compound) and broth control (without antimicrobial com-
pound and inoculum) were maintained. All the test samples
and control tubes were then incubated for 16 h at 37°C.
All the newly synthesized compounds were screened for
their antibacterial activity. For this, S. aureus, B. subtilis,
E. coli, and P. aeruginosa microorganisms were employed.
Antimicrobial study was assessed by Minimum Inhibitory
Concentration (MIC) by serial dilution method (Mackie and
McCartney 1989) Several colonies of S. aureus, B. subtilis,
E. coli, and P. aeruginosa were picked off a fresh isolation
plate and inoculated in corresponding tubes containing 5 ml
of trypticase soya broth. The broth was incubated for 6 h at
37°C until there was visible growth. Mc Farland No.5 stan-
dard was prepared by adding 0.05 ml of 1% w/v BaCl2.2H2O
in Phosphate Buffered saline (PBS) to 9.95 ml of 1% v/v
H2SO4 in PBS. The growths of all the four cultures were
adjusted to Mc Farland No.5 turbidity standard using sterile
PBS. This gives a 108 cfu/ml suspension. The working
inoculums of aforementioned four different microorganisms
containing 105 cfu/ml suspension was prepared by diluting
the 108 cfu/ml suspension, 103 times in trypticase soya broth.
Interpretation
Preparation of anti-microbial suspension (50 lg/ml)
After incubation, the tubes showing no visible growth were
considered to be representing the MIC. The details of results
are furnished in Table 1. Inoculums control showed visible
growth, whereas the broth control showed no growth.
Dissolved 0.5 mg of each compound in 10 ml of trypticase
soya broth to get 50 lg/ml. This suspension was filter
sterilized in syringe filters.
Preparation of dilutions
Conclusions
In all, for each of the 14 anti-microbial compounds and
standard antimicrobial, i.e., Ceftriaxone, 24 tubes of 5-ml
capacity were arranged in four rows with each row
A novel series of ethyl-1-(N-substituted) 5-phenyl-1H-
pyrazole-4 carboxylate derivative were synthesized and
Table 1 Antibacterial data for the newly synthesized pyrazole derivatives in MIC (lg/ml)
Comp.
S. aureus
B. subtilis
E. coli
P. aeruginosa
3a
3b
3c
3d
3e
3f
50.00
Growth in all concentrations Growth in all concentrations 25.00
Growth in all concentrations Growth in all concentrations Growth in all concentrations Growth in all concentrations
3.125 1.6125 1.6125 1.6125
Growth in all concentrations 12.50 Growth in all concentrations 12.50
6.250
3.125
3.125
3.125
1.6125
3.125
1.6125
1.6125
6.250
1.6125
1.6125
3.125
3g
3h
3i
Growth in all concentrations Growth in all concentrations Growth in all concentrations Growth in all concentrations
Growth in all concentrations Growth in all concentrations Growth in all concentrations Growth in all concentrations
Growth in all concentrations Growth in all concentrations Growth in all concentrations Growth in all concentrations
3j
3k
3l
1.6125
3.125
6.250
1.6125
3.125
1.6125
1.6125
12.50
3.250
1.6125
6.250
3m
6.250
Ceftriaxone (Standard) 3.125
1.6125
1.6125
1.6125
Inoculum
Growth in all concentrations Growth in all concentrations Growth in all concentrations Growth in all concentrations
No growth No growth No growth No growth
Broth control
123

2708
Med Chem Res (2012) 21:2702–2708
characterized by IR, 1H-NMR and mass spectrometry stud-
ies. Molecular structure 3i was also confirmed by single
crystal X-ray analysis. All the synthesized compounds were
screened for their antibacterial activity by serial dilution
(MIC) method. As regards the relationships between the
structure of the heterocyclic scaffold and the detected anti-
bacterial properties, it showed varied biological activity.
Among the screened samples, 3c, 3f, 3k, and 3l have shown
excellent antibacterial activity against all the tested bacterial
strains as compared with the standard drug Ceftriaxone,
which was active at 3.125, 1.6125, 1.6125, and 1.6125 lg/ml
against S. aureus, B.subtilis, E.coli, and P.aeruginosa
strains, respectively. Interestingly all these active com-
pounds are halogen-substituted derivatives, which is respon-
sible for the enhanced activity. Compound 3e, which is
fluorine substituted compound, has shown significant activ-
ity. Compounds 3g and 3l, which have quinolin and piperidin
substituents, respectively, also have shown significant anti-
bacterial activity against all bacterial strains. All the
remaining compounds have exhibited poor antibacterial
activity. In our recently published study (Vijesh et al., 2011),
pyrazole incorporated imidazoles were shown to have sig-
nificant antibacterial activity. In conclusion, pyrazole moiety
is one of the active components present in all the synthesized
molecules, which has shown significant activity in the
presence of halogen substituent.
El-Sabbagh OI, Baraka MM, Ibrahim SM, Pannecouque C, Andrei G,
Snoeck R, Balzarin J, Rashad AA (2009) Synthesis and antiviral
activity of new pyrazole and thiazole derivatives. Eur J Med
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Acknowledgment AMI is thankful to Board of Research in Nuclear
Sciences, Department of Atomic Energy, Government of India for the
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Premsai Rai N, Narayanaswamy VK, Shashikanth S (2009) Synthesis,
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