Syntheses, characterization and antimicrobial evaluation of some 1, 3, 5-trisubustituted pyrazole derivatives

Article abstract of DOI:10.1155/2010/947215

A series of 1, 3, 5-trisubustituted pyrazole derivatives were synthesized and screened for antimicrobial activity. The compounds (2j-o) were evaluated against two gram-positive and two gram-negative bacteria and one fungus, at concentrations of 10 μg/mL a

Full text of DOI:10.1155/2010/947215

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2010, 7(4), 1190-1195
http://www.e-journals.net
Syntheses, Characterization and
Antimicrobial Evaluation of Some 1, 3, 5-
Trisubustituted Pyrazole Derivatives
VERTIKA GAUTAM*, VINEY CHAWLA,
PANKAJ K. SONAR and SHAILENDRA K. SARAF
Faculty of Pharmacy, Northern India Engineering College,
Lucknow-227105, Uttar Pradesh, India.
dirpharmniec@gmail.com
Received 6 October 2009; Revised 17 December 2009; Accepted 10 February 2010
Abstract: A series of 1, 3, 5-trisubustituted pyrazole derivatives were
synthesized and screened for antimicrobial activity. The compounds (2j-o) were
evaluated against two gram-positive and two gram-negative bacteria and one
fungus, at concentrations of 10 µg/mL and 50 µg/mL. The compounds were
founds to be inactive against P. aeruginosa and A. niger but exhibited moderate
activity against B. subtilis, E. coli and S. aureus. It can be concluded that the
newly synthesized compounds possess promising antimicrobial activity.
Keywords: Pyrazoles, Antimicrobial activity, Diketo compounds, FTIR.
Introduction
In the past few decades, the development of resistant microbes has been greatly accelerated
by several concurrent trends. These have worked to increase the number of infections and
thus expand the need for antimicrobials1.
In this situation, enlargement of innovative drugs with dual activity against bacterial and
fungal pathogens is required to over come this problem2. Pyrazoles are important nitrogen-
containing five-membered heterocyclic compounds possessing some important
pharmacological activities like antitumour3, immunosuppressive4, antibacterial5, antitubercular
anti-inflammatory6, anticancer7, antidiabetic8 and antidepressant properties9. Some novel
pyrazolo[3,4-d] pyrimidine derivatives have been reported for antimicrobial potential against
E. coli, S. aureus, P. aerunginosa, B. subtilis, C. albicans and A. fumigatus10. A series of 3, 4-
diarylpyrazoles was synthesized and evaluated for the ability to selectively inhibit
cyclooxygenase-2 (COX-2)11. Pyrazole-4-carboxylic acid derivatives were found to possess in
vivo hypoglycemic activity12. Some unsymmetrical N-endocyclic and N-exocyclic derivatives
from benzoylation of 3- and 5- aminopyrazole were shown to have anticonvulsant activity13.

1191
V. GAUTAM et al.
Halogenated substituents can alter not only the course of the reaction but also the
biological activities. Thus, introduction of chlorine atoms provide more lipophilic and
pharmacologically significant compounds as compared to their non-halogenated analogues.
The present research was intended to synthesize some novel1, 3, 5-trisubstituted chloro-
derivatives of pyrazole and evaluate them for antibacterial and antifungal activities.
Experimental
All the chemicals were of synthetic grade and procured from S. D. Fine-Chem. Limited;
Mumbai and Sigma-Aldrich Chemie GmbH, Germany. Melting points were determined
in open capillary tubes and are uncorrected. Thin Layer Chromatography was performed
on precoated silica plates using chloroform: methanol (9:1). The spots were observed
under ultraviolet lamp with short and long wavelength and also in an iodine chamber.
The IR spectra of the compounds were recorded (in KBr) on a Shimadzu 8400 FTIR
spectrophotometer and mass spectra on a Jeol/ SX-102/ DA-600 FAB mass
spectrophotometer at SAIF, C.D.R.I., Lucknow.
The antimicrobial activity of the prepared compounds was determined by agar diffusion
method in concentrations of 10 and 50 µg/mL in 10% DMSO against the microbial strains,
MTCC 441 (Bacillus subtilis), MTCC 1430 (Staphylococcus aureus), MTCC 424 (Pseudomonas
aeruginosa), MTCC 1573 (Escherichia coli) and MTCC 2546 (Aspergillus niger) respectively.
Bacterial and fungal strains were procured from IMTECH, Chandigarh, India.
Syntheses of diketo derivatives 1(a-c)
The sodium ethoxide was synthesized by routine method using dry sodium (0.5 mol) and
refluxing it with absolute ethanol (0.5 mol). 2 mol of pure dry ethyl acetate and 0.5 mol of
substituted acetophenone were added in the synthesized sodium ethoxide (0.5 mol) to get
corresponding sodium salt of diketo compounds (1a-c) after 2 h stirring on ice bath.
O
O
O
CH3
H3C
O
CH3
+
-
Na OEt
+
Ethanol, Reflux
R
OEt
R
R= CH3, C2H5, Cl
1(a-c)
Scheme 1
1-p-Tolylbutane-1, 3-dione (1a)
Yield: 67.77%, melting range 164-168 °C. IR (KBr pellet) (Ar) C-H str, 2974 cm-1; C=O str,
1612 cm-1; (Aliphatic) C-H str, 1417 cm-1. FAB-MS m/z: 176 (M+ peak).
1-(4-Ethylphenyl) butane-1, 3-dione (1b)
Yield: 71.36%, melting range 128-132 °C. IR (KBr pellet) C=O str, 1741.40 cm-1;
(Aliphatic) C-H str, 1411 cm-1. FAB-MS m/z: 191 (M+1 peak).
1-(4-Chlorophenyl) butane-1, 3-dione (1c)
Yield: 77.77%, melting range 112-116 °C. IR (KBr pellet) (Ar) C-H str, 3415 cm-1; C=O str,
1691.81 cm-1; (Ar) C-Cl str, 1098.83 cm-1. FAB-MS m/z: 196 (M+ peak).
Synthesis of p-nitro aryl derivatives of pyrazole (2a-c)
0.01 Mol of p-nitro phenyl hydrazine and a drop of aqueous 15% hydrochloric acid were
slowly added to a solution of 0.01 mol of previously prepared diketo compounds (1a-c) in
ethanol.

Syntheses, Characterization and Antimicrobial Evaluation
1192
CH3
O
O
NHNH2
N
Ethanol, HCl
Reflux
+
CH3
N
R
O2N
R
(1a-c)
(2a-c)
NO2
HCl
CH3
Sn
R= CH3, C2H5, Cl
N
R1= CH3Cl, C2H5Cl.
N
R
(2d-f)
CH3
NaNO2
NH2
CH3
NaN3
N
N
Ethanol, R1Cl
Reflux
N
N
R
HCl
R
N3
NHR1
(2j-o)
(2g-i)
Scheme 2
After refluxing for 2 h and further evaporation of solvent, the residue was recrystallized
from industrial spirit affording the pyrazoles (2a-c).
3-Methyl-1-(4-nitrophenyl)-5-p-tolyl-1H-pyrazole (2a)
Yield: 53.94%, melting range 140-143 °C. IR (KBr pellet) N-H str, 3523.70 cm-1; Ring str,
1336.51 cm-1; (Ar) N=O str 1521.73 cm-1; (Ali) C-H str, 1456.16 cm-1. FAB-MS m/z: 293
(M+ peak).
5-(4-Ethylphenyl)-3-methyl-1-(4-nitrophenyl)-1H-pyrazole (2b)
Yield: 65.34%, melting range 114-118 °C. IR (KBr pellet) N-H str, 3423.76 cm-1; Ring str,
1347.09 cm-1; (Ar) N=O str 1521.73 cm-1; (Methyl) C-H str, 1697.24 cm-1. FAB-MS m/z:
307 (M+ peak).
5-(4-Chlorophenyl)-3-methyl-1-(4-nitrophenyl)-1H-pyrazole (2c)
Yield: 75.87%, melting range 102-105 °C. IR (KBr pellet) N-H str, 3423.76 cm-1; Ring str,
1347.09 cm-1; (Ar) N=O str 1521.73 cm-1; (Ar) C-Cl str, 1097.42. cm-1. FAB-MS m/z: 314
(M+1 peak).

1193
V. GAUTAM et al.
Reduction of nitro group into amino group (2d-f)
A solution of 0.01 mol of the synthesized p-nitroaryl derivatives of pyrazole (2a-c) and 0.15 mol
of granulated tin in concentrated hydrochloric acid (30 mL) were placed in a round-
bottomed flask, equipped with a reflux condenser and the shaken steadily to ensure thorough
mixing. Finally, the mixture was heated on a boiling water bath for 1 h, cooled to room
temperature and a solution of 9 g of sodium hydroxide in 15 mL of water was gradually added.
After cooling, the reduced product separated out and was extracted using ether.
4-(3-Methyl-5-p-tolyl-1H-pyrazol-1-yl) aniline (2d)
Yield: 56.67%, melting range 96-98 °C. IR (KBr pellet) (Htr) C-H str, 3076.25 cm-1 ; ( Htr)
N-H str, 3207.40 cm-1; Ring str, 1431.08 cm-1; C-N str (-NH2),1317.29 cm-1; (Methyl) C-H
str, 2916.17 cm-1. FAB-MS m/z: 264 (M+1 peak).
4-(5-(4-Ethylphenyl)-3-methyl-1H-pyrazol-1-yl) aniline (2e)
Yield: 63.06%, melting range 108-110 °C. IR (KBr pellet) (Htr) C-H str, 3076.25 cm-1; (Ar)
C-H str, 2916.17 cm-1; C-N str (-NH2), 1232.43 cm-1; (Htr) N-H str, 3207.40 cm-1. FAB-MS
m/z: 277 (M+ peak).
4-(5-(4-Chlorophenyl)-3-methyl-1H-pyrazol-1-yl) aniline (2f)
Yield: 64.32%, melting range 143-145 ºC. IR (KBr pellet) (Htr) C-H str, 2923.89 cm-1; (Htr)
N-H str, 3442.70 cm-1; Ring str, 1396.36 cm-1; C-N str (-NH2), 1306.72 cm-1; (Ar) C-Cl str,
1097.42 cm-1. FAB-MS m/z: 284 (M+1 peak).
Synthesis of azide derivatives of pyrazole (2g-i)
0.01 Mole of synthesized amine derivatives of pyrazole (2d-f) were added in a 20% v/v solution
of sulfuric acid and the suspension was cooled to 0-5 ºC in an ice-salt bath. A solution of
sodium nitrite (0.16 mol) in 30 mL of water was added drop-wise over a period of 15 minutes.
A thick precipitate of the sparingly soluble diazonium salt separated out from the initially clear
solution. A solution of sodium azide (0.17 mol) in 40 mL of water was added with rapid stirring,
which was continued for 1 h. The solid was filtered, washed with water and dried.
1-(4-Azidophenyl)-3-methyl-5-p-tolyl-1H-pyrazole (2g)
Yield: 61.56%, melting range 100-104 ºC. IR (KBr pellet) (Htr) C-H str, 2879.52 cm-1; (Htr)
N-H str, 3357.54 cm-1; C-N str (-N3), 1306.72 cm-1. FAB-MS m/z: 289 (M+ peak).
1-(4-Azidophenyl)-5-(4-ethylphenyl)-3-methyl-1H-pyrazole (2h)
Yield: 57.98%, melting range 180-185 ºC. IR (KBr pellet) (Htr) N-H str, 3357.64 cm-1; Ring
str, 1305.72 cm-1; C-N str (-N3), 1305.08 cm-1. FAB-MS m/z: 303 (M+ peak).
1-(4-Azidophenyl)-5-(4-chlorophenyl)-3-methyl-1H-pyrazole (2i)
Yield: 53.23%, melting range 110-115 ºC. IR (KBr pellet) (Htr) C-H str, 2923.89 cm-1; (Htr)
N-H str, 3442.70 cm-1; C-N str (-N3), 1306.72 cm-1; (Ar) C-Cl str, 1095.49 cm-1. FAB-MS
m/z: 309 (M+ peak).
Syntheses of N-substituted aniline derivatives of pyrazole (2j-o)
Equimolar quantities of the synthesized azide derivatives of pyrazole (2g-i) and methyl
chloride were refluxed in ethanol for 3 h. The reaction mixture was concentrated in a rotary
evaporator, filtered and dried in a desiccator.
N-Methyl-4-(3-methyl-5-p-tolyl-1H-pyrazol-1-yl) aniline (2j)
Yield: 47.78%, melting range 146-148 ºC. IR (KBr pellet) (Htr) N-H str, 3475.40 cm , 3417.63 cm-1;
-1
(Htr) C-H str, 3024.18 cm-1; C-N str (-NHR), 1348.13 cm-1. FAB-MS m/z: 277 (M+ peak).

Syntheses, Characterization and Antimicrobial Evaluation
1194
4-(5-(4-Ethylphenyl)-3-methyl-1H-pyrazol-1-yl)-N-methylaniline (2k)
Yield: 32.45%, melting range 86-88 ºC. IR (KBr pellet) (Htr) N-H str, 3321.10 cm-1, 3415.70 cm-1;
C-N str (-NHR), 1288.36 cm-1; Ring str, 1595.02 cm-1. FAB-MS m/z: 291 (M+ peak).
4-(5-(4-Chlorophenyl)-3-methyl-1H-pyrazol-1-yl)-N-methylaniline (2l)
Yield: 38.65%, melting range 98-102 ºC. IR (KBr pellet) (Htr) N-H str, 3438.84 cm-1; C-N
str (-NHR), 1255.57 cm-1; Ar C-Cl str, 1074.19 cm-1. FAB-MS m/z: 298 (M+1 peak).
N-Ethyl-4-(3-methyl-5-p-tolyl-1H-pyrazol-1-yl) aniline (2m)
Yield: 28.33%, melting range 114-116 ºC. IR (KBr pellet) (Htr) N-H str, 3489.40 cm-1; (Htr)
C-H str, 3076.25 cm-1; C-N str (-NHR), 1317.29 cm-1. FAB-MS m/z: 291 (M+ peak).
N-Ethyl-4-(5-(4-ethylphenyl)-3-methyl-1H-pyrazol-1-yl) aniline (2n)
Yield: 34.89%, melting range 163-165 ºC. IR (KBr pellet) (Htr) N-H str, 3305.76 cm-1; (Htr)
C-H str, 3026.25 cm-1; C-N str (-NHR), 1317.29 cm-1. FAB-MS m/z: 305 (M+ peak).
4-(5-(4-Chlorophenyl)-3-methyl-1H-pyrazol-1-yl)-N-ethylaniline (2o)
Yield: 33.07%, melting range 118-122 ºC. IR (KBr pellet) (Htr) N-H str, 3442.70 cm-1; (Htr)
C-H str, 2923.65 cm-1; C-N str (-NHR), 1305.72 cm-1; (Ar) C-Cl str, 1097.42 cm-1. FAB-MS
m/z: 311 (M+ peak).
Anti-microbial activity
The test organisms were inoculated in nutrient broth and were incubated for 48 hours. A
definite volume of this suspension was mixed with nutrient agar (cooled to 40 ºC) and
poured into petri plates to obtain a uniform thickness. Wells were prepared in the agar and
filled with equal volume of respective solution of synthesized compounds (2j-o). After a
period of pre-incubation diffusion, the plates were incubated in the specified conditions 14, 15
Zones of inhibition were measured with the help of zone reader and are reported in Table 1.
.
Table 1. Results of Antimicrobial activity of the tested compounds
Compounds
Concentration,
µg /mL
Comparision of diameters of zones of inhibition, mm
Gram +ve Bacteria Gram –ve Bacteria Fungus
B. subtils S. aureus P. aeruginosa E. coli A. niger
50
10
50
10
50
10
50
10
50
10
50
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
-
-
-
-
-
-
-
-
-
-
+
++
-
++
-
++
-
++
-
-
-
-
-
-
-
-
-
-
-
+
2j
2k
2l
2m
2n
2o
+
-
+
+
-
+
++
-
++
10
10
10
10% v/v
-
+++
+
-
+++
+
-
+++
+
-
+++
+
-
+
+++
-
Norfloxacin
Fluconazole
Control (DMSO)
-
-
-
-
Highly active = +++ (zone of inhibition > 12 mm), moderately active = ++ (zone of inhibition > 9-12 mm),
slightly active = + (zone of inhibition > 7-9 mm), Inactive = - (zone of inhibition < 7 mm).

1195
V. GAUTAM et al.
Results and Discussion
All compounds proposed in the scheme were synthesized using the pyrazole ring formed by
the substrate diketo and p-nitro phenyl hydrazine and further derivatising by reduction and
final substitution by alkyl halides.
The synthesized compounds were subjected to physicochemical and spectral
characterization. Single spot in TLC established the purity of the compounds. IR and mass
spectra confirmed the synthesis of intermediates and final compounds (2j-o). Antimicrobial
activity of the final derivatives was determined by agar diffusion method and was found to
be concentration dependent. None of the compounds was found to be effective against
P. aeruginosa and A. niger. The compounds showed moderate activity at the concentration
of 50 µg/mL, against B. subtilis, S. aureus and E. coli
Conclusion
The study establishes that the 1, 3, 5-trisubstituted pyrazole derivatives exhibit antimicrobial
activity. The activity of the derivatives is more pronounced for Gram-negative
microorganisms than for the Gram-positive ones. However, the derivatives are almost
inactive against the fungus.
The study thus provides a lead for the syntheses and evaluation of more pyrazole
derivatives for antimicrobial activity as the same could lead to the discovery of some
promising agents.
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