Synthesis and antimicrobial activity of novel thiazole substituted pyrazole derivatives

Article abstract of DOI:10.1002/jhet.1513

A series of new 1-[4-(2,3,4-substituted-phenyl) thiazol-2-yl]-3-(2,3,4- substituted-phenyl)-1H-pyrazole-4-carbaldehyde (4a-m), 4-[4-(4-substituted- phenyl) thiazol-2-yl]-3-(4-substituted-phenyl)-1-phenyl-1H-pyrazole (7a-i), 4-[4-(4-substituted phenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine (10a-g) have been synthesized by using Vilsmeier Haack formylation and Hantzsch reaction in high yield. All the synthesized compounds were tested qualitative (Zone of inhibition) and quantitative antimicrobial activities (MIC). Most of the synthesized compounds showed potent antimicrobial activity against gram positive and gram negative bacteria as well as fungi species.

Full text of DOI:10.1002/jhet.1513

May 2013
Synthesis and Antimicrobial Activity of Novel Thiazole Substituted
Pyrazole Derivatives
519
b
a
a
*
Nitin D. Gaikwad, Sachin V. Patil, and Vivek D. Bobade
aDepartment of Chemistry, H.P.T Arts and R.Y.K. Science College, Nashik 422005, India
bDepartment of Chemistry, K.T.H.M. College, Nashik 422005, India
*
E-mail: v_bobade31@rediffmail.comReceived May 22, 2011
DOI 10.1002/jhet.1513
Published online in Wiley Online Library (wileyonlinelibrary.com).
A series of new 1-[4-(2,3,4-substituted-phenyl) thiazol-2-yl]-3-(2,3,4-substituted-phenyl)-1H-
pyrazole-4-carbaldehyde (4a–m), 4-[4-(4-substituted-phenyl) thiazol-2-yl]-3-(4-substituted-phenyl)-
1-phenyl-1H-pyrazole (7a–i), 4-[4-(4-substituted phenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine (10a–g)
have been synthesized by using Vilsmeier Haack formylation and Hantzsch reaction in high yield.
All the synthesized compounds were tested qualitative (Zone of inhibition) and quantitative antimicrobial
activities (MIC). Most of the synthesized compounds showed potent antimicrobial activity against gram
positive and gram negative bacteria as well as fungi species.
J. Heterocyclic Chem., 50, 519 (2013).
INTRODUCTION
agents, we report herein the synthesis of some new
thiazole substituted pyrazole derivatives.
Pyrazole and thiazole rings are privileged scaffolds for
the generation of target compounds for drug discovery.
The structural diversity and biological importance of
pyrazoles [1–18] and thiazoles [19–34] have made them
attractive targets for synthesis. Pyrazole and thiazole rings
present in the same molecule could be convenient models
for investigation of their biological activity. Literature
revealed many syntheses of such thiazolyl-pyrazoles, which
include condensation of thiocarbamoyl pyrozoles with
phenacyl bromides or condensation of hydrazino thiazoles
with a-cyanoacetophenones that showed anti-inflammatory
activity [35,36]. Alternatively, condensation of hydrazino
thiazoles with arylidine malononitriles also gave the
thiazolyl-pyrazoles [37]. Another approach uses condensa-
tion of 4-(chloroacetyl)-antipyrine with thiourea [38].
Three different series of thiazole substituted pyrazole
derivatives (4a–m, 7a–i, and 10a–g) were synthesized
and screened for their antimicrobial activity.
CHEMISTRY
Considering the importance of pyrazole and thiazole
derivatives, it was thought worthwhile to synthesize thiazole
substituted pyrazole derivatives. In the present work, we
report the synthesis of 1-[4-(substituted-phenyl) thiazol-
2-yl]-3-(substituted-phenyl)-1H-pyrazole-4-carbaldehyde
4a–m, 4-[4-(substituted-phenyl) thiazol-2-yl]-3-(substituted-
phenyl)-1-phenyl-1H-pyrazole 7a–i, 4-[4-(substituted
phenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine 10a–g
using Vilsmeier Haack formylation and Hantzsch
reaction.
• In continuation of our effort to synthesize such com-
pounds, that may be potent and less toxic antimicrobial
© 2013 HeteroCorporation

520
N. D. Gaikwad, S. V. Patil, and V. D. Bobade
Vol 50
Scheme 1. Synthetic pathway for the formation of compounds 4a–m.
Scheme 2. Synthetic pathway for the formation of compounds 7a–i.
Scheme 3. Synthetic pathway for the formation of compounds 10a–g.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Synthesis and Antimicrobial Activity of Novel Thiazole Substituted Pyrazole Derivatives
521
Compounds 4a–m, were synthesized (Scheme 1) from the
substituted acetophenone 1a–m, which were converted
into their corresponding thiosemicarbazones 2a–m. The
thiosemicarbazones 2a–m upon Hantzsch reaction with
phenacyl bromide gave thiazoles 3a–m. The thiazoles 3a–m
upon Vilsmeier Haack formylation gave 1-[4-(substituted-
phenyl) thiazol-2-yl]-3-(substituted-phenyl)-1H-pyrazole-4-
carbaldehyde (4a–m). Compounds 7a–i, were synthesized
(Scheme 2) from the key intermediate nitrile pyrazoles 5a–i,
which in turn were prepared by the known literature method
[18]. The reaction of nitrile pyrazole 5a–i with hydrogen
sulfide gas in pyridine and catalytic amount of triethyl amine
gave corresponding thioamides 6a–i. These thioamides 6a–i
upon Hantzsch reaction with phenacyl bromide gave 4-[4-
(substituted-phenyl) thiazol-2-yl]-3-(substituted-phenyl)-
1-phenyl-1H-pyrazole 7a–i. Compounds 10a–g were
synthesized as delineated in Scheme 3. Reaction of phenyl
hydrazine, triethyl orthoformate, and malonyl nitrile gave
3-amino-4-cyno pyrazole 8a–g [39]. Compounds 8a–g
were converted into corresponding thioamides 9a–g as in
the case of nitrile pyrazole 5a–i. These thioamides 9a–g
upon Hantzsch reaction with phenacyl bromide gave 4-[4-
(substituted phenyl) thiazol-2-yl]-1-phenyl-1H-pyrazol-3-
amine 10a–g.
BIOLOGICAL RESULTS AND DISCUSSION
All the synthesized compounds were tested for
antibacterial activity against bacteria Bacillus subtilis
(2250), Staphylococcus aureus (2079), Escherichia coli
(2109), and Pseudomonas aeruginosa (2036) and antifun-
gal activity against two fungi Candida albicans (3471)
and Aspergillus niger (545). To evaluate the activity of
the synthesized compounds, the zone of inhibition
(128 mg/mL in dimethyl sulfoxide (DMSO)) and minimum
inhibitory concentrations (MICs; at 128, 64, 32, 16, 8, 4, 2,
and 1 ug/mL in DMSO) were determined using agar diffu-
sion method [40–42]. Known antibiotic Chloramphenicol
Table 1
Antimicrobial screening of synthesized compounds 4a–10g (zone diameter of growth inhibition in mm).
Microorganisms
Staphylococcus
aureus
Escherichia
coli
Bacillus
subtilis
Pseudomonas
aeruginosa
Aspergillus
niger
Candida.
albicans
Compoundsa
4a
22.1
-
20.25
-
-
-
4b
-
17.62
-
18.53
18.23
17.56
4c
-
18.84
-
20.4
-
-
4d
18.22
20.4
-
-
-
-
4e
24.41
-
16.7
-
-
19.09
4f
-
-
12.51
-
-
15.02
4g
-
12.79
-
16.44
-
-
4h
4i
4j
4k
4l
4m
7a
7b
7c
7d
7e
7f
7g
7h
-
-
-
14.22
-
-
-
-
-
-
18.29
-
14.36
24.14
-
-
-
-
-
13.63
-
-
-
13.18
-
-
17.86
18.42
-
17.58
-
-
21.12
-
-
17.2
-
12.51
13.48
-
19.35
18.02
20.08
15.74
17.44
-
19.38
-
-
-
24.68
19.03
27.13
-
11.81
14.3
-
19.32
15.21
17.33
17.52
-
18.64
-
18.94
18.81
-
-
-
18.32
26.68
-
-
18.20
18.36
-
19.94
14.38
18.68
-
-
7i
10a
10b
10c
10d
10e
21.58
19.48
22.25
-
-
-
-
-
22.38
-
14.38
-
-
-
-
-
-
-
-
-
-
15.24
-
-
15.74
15.38
-
16.32
12.81
-
11.25
12.50
-
17.54
15.84
-
10f
10g
-
-
-
21.54
-
NA
23.87
-
-
-
15.38
NA
29.24
13.32
20.32
NA
Nystatin
Chloramphenicol
NA
31.4
NA
27.55
22.03
NA
aChloromphenicol (128mg/disk) and aNystatin (128mg/disk) were used as reference; synthesized compounds (128 mg/disk); NA, not applicable; -, inactive.
Journal of Heterocyclic Chemistry
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N. D. Gaikwad, S. V. Patil, and V. D. Bobade
Vol 50
(the reference for antibacterial drugs) and Nystatin (the
reference for antifungal drug) were used for comparison.
The zone of inhibition and MIC against micro organisms
tested is reported in Tables 1 and 2, respectively. As shown
in Table 1 most of the compounds were active against gram
positive and gram negative bacteria as well as both the fungi
species.
Careful analysis of the MICs in Table 2 provides some
lead molecules with good antibacterial and antifungal
activity. Of the compounds 4a–10g tested, compounds with
electron-withdrawing F, Cl, Br, CF3, and NO2 at the phenyl
ring expressed a moderate to good activity against most of
the tested pathogens, they inhibited the Gram-negative and
Gram-positive pathogens equally. Compounds 4a to 10g
required about 32–128 mg/mL against Gram-positive and
Gram-negative bacteria as well as both the fungi species,
whereas 4e required 32 mg/mL, 4j, 7a, 7e and 7g
required 64mg/mL, and 4a, 4d, 7b, 7f, 7i, 10a, 10b required
128 mg/mL against S. aureus. Also compounds 4b, 4c, 4d,
4g, 4i, 4l, 4m, 7b, 7e, 7h, 10c, and 10d registered their
MIC at 128 mg/mL against E. coli. However, the CF3 substit-
uent did not enhance the activity. Introduction of the F, Cl,
Br, and NO2 substituent on the phenyl ring showed an
improvement in its activity. Compounds 7b, 7i and 4a, 4e,
4f, 4h, 7d, 7e, 10b, 10f, 10g showed MIC at 64 and
128 mg/mL, respectively against B. subtilis (they are four
and eightfold less potent than Chloramphenicol). Com-
pounds 4b, 4c, 4g, 4l, 7a, 7b, 7g, and 7h registered MIC at
128 mg/mL against P. aeruginusa.
Table 2 also describes the MIC of synthesized com-
pounds for their antifungal activity. The compound 4a with
unsubstituted phenyl ring did not exhibit activity even at
128 mg/mL. However, the introduction of F, Cl, Br, and
NO2 substituents exhibited moderate to good activity
against C. albicans and A. niger, whereas, except the 4a,
4c, 4d, 4g, 4i, 7h, and 10f, all other compounds showed
Table 2
Antimicrobial screening of synthesized compounds 4a–10g MIC in mg/mL.
Microorganisms
Staphylococcus
aureus
Escherichia
coli
Bacillus
subtilis
Pseudomonas
aeruginosa
Aspergillus
niger
Candida
albicans
Compoundsa
4a
4b
4c
4d
4e
4f
4g
128
-
-
128
32
-
-
128
-
-
-
128
128
-
-
-
32
-
-
-
-
64
-
128
128
128
-
128
128
-
-
-
64
128
-
-
-
-
-
-
128
128
4h
4i
-
-
-
128
-
-
-
128
-
-
-
128
4j
4k
4l
4m
7a
7b
7c
7d
7e
7f
7g
7h
7i
10a
10b
10c
64
-
-
-
-
-
-
-
-
-
-
-
64
64
128
128
-
32
64
64
32
64
-
-
128
-
128
128
-
128
128
-
128
128
-
128
-
-
128
-
-
128
-
-
64
128
64
-
32
128
64
64
32
32
32
-
64
-
128
128
-
-
-
128
64
-
128
128
128
-
-
-
-
-
-
-
-
128
128
-
-
-
-
-
-
-
-
64
-
128
-
-
-
128
128
NA
16
-
128
-
-
128
128
-
-
-
64
128
-
128
128
-
128
16
NA
128
128
-
-
-
10d
10e
10f
10g
-
-
-
Nystatin
Chloramphenicol
NA
16
NA
16
NA
16
16
NA
aChloromphenicol (mg/mL) and aNystatin (mg/mL) were used as reference; NA, not applicable; -, inactive.
Journal of Heterocyclic Chemistry
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Synthesis and Antimicrobial Activity of Novel Thiazole Substituted Pyrazole Derivatives
523
addition of POCl3, the reaction mixture was further stirred at
0ꢀC for 1 h. To this well stirred and cooled reaction mixture, a
solution of 3a–m in anhydrous DMF was added dropwise
during 1 h; after complete addition, reaction mixture was
heated at 65–70ꢀC for 2 h. The reaction mixture was poured
into crushed ice and left overnight in a refrigerator, during
which the product separates out as solid mass. The product was
filtered, washed with Na2CO3 (5%, 30 mL), water, and
recrystallized from DMF-ethanol mixture. The yields of 4a–m
for this step are reported along with the spectral data given in
the succeeding text.
moderate to good activity against A. niger and C. albican.
Of the halo-substituted compounds 4b, 7a, 7e, 7f, and 7g
registered a good activity against A. niger at 32 mg/mL, also
compounds 7d, 7e, and 10a recorded moderate activity at
64 mg/mL, which is fourfold lower than standard Nystatin.
In addition, compounds 7b and 7f registered the MIC at
32 mg/mL against C. albicans. Introduction of the halo
substituent on the phenyl ring moderately inhibited the
growth of A. niger and C. albican.
General procedure for the synthesis of 3-(substituted phenyl)-1-
phenyl-1H-pyrazole-4-carbothioamide (6a–i). Carbonitrile 5a–i
(1 mol) and triethylamine (1 mL) were taken in pyridine (25 mL),
and excess of hydrogen sulfide gas was passed through this solution
for 2h. The reaction mixture was poured in ice cold water, the
product separated out was filtered, washed with water and
recrystallized from ethanol.
General procedure for the synthesis of 4-[4-(substituted-
phenyl) thiazol-2-yl]-3-(4-substituted-phenyl)-1-phenyl-1H-
pyrazole (7a–i). A mixture of 6a–i (1 mol) and substituted
phenacyl bromide (1 mol) in absolute ethanol (25 mL) was
refluxed for 1 h. On completion of reaction, the reaction mixture
was cooled to room temperature, the product thus separated was
filtered, washed with ethanol, dried, and recrystallized from
ethanol: DMF.
General procedure for the synthesis of 3-amino-1-phenyl-
1H- pyrazole-4-carbonitrile (8). A mixture of malononitrile
(1.5 mol) and triethylorthoformate (1 mol) was added slowly to
the solution of phenyl hydrazine (1 mol) in absolute ethanol
with stirring. After complete addition, the solution was heated
to gentle boiling for 2 h. The solution was kept overnight in a
refrigerator. The product was filtered, washed with water, and
recrystallized from hot water.
General procedure for the synthesis of 3-amino-1-phenyl-
1H-pyrazole-4-carbothioamide (9). Carbonitrile (1 mol) 8 and
triethylamine (1 mL) were dissolved in pyridine (25 mL), and
excess of hydrogen sulfide gas was passed through this solution
for 2 h. The reaction mixture was poured in ice cold water to
obtain a solid product that was filtered, washed with water, and
recrystallized from ethanol.
CONCLUSION
In conclusion, a series of new compounds 4a–m, 7a–i,
and 10a–g were synthesized. The pharmacological studies
were undertaken to evaluate the effect of substituents and
position of thiazole on pyrazole ring for their antimicrobial
activities. Most of the synthesized compounds exhibited
moderate to good activity towards Gram-positive and
Gram-negative bacteria as well as both the fungi species.
The enhancement in antibacterial and antifungal activity
can be attributed to the presence of pharmacologically
active F, Cl, Br, and NO2 groups irrespective of their position
in the molecule.
EXPERIMENTAL
Melting points were determined in capillary tubes in silicon oil
bath using a Veego melting point apparatus and are uncorrected.
1H (300 MHz) NMR and 13C (75 MHz) NMR spectra were
recorded on Varian mercury XL-300. Chemical shifts are reported
from internal tetramethylsilane standard and are given in d units.
The solvent for NMR spectra was CDCl3 and DMSO-d6. Infrared
spectra were taken on Shimadzu FTIR-408 in KBr. The mass
spectra were recorded on Shimadzu GC-MS QP 2010A mass
spectrometer with an ionization potential of 70 eV. Elemental
analysis was performed on a Hosli CH-analyzer.
General procedure for the synthesis of (E)-1-[1-(substituted
phenyl) ethylidene] thiosemicarbazide (2a–m). A mixture of
substituted acetophenone 1a–m (1 mol), thiosemicarbazide
(1 mol), and acetic acid (1 mL) in ethanol (20 mL) was refluxed
for 30 min. After the completion of the reaction, as monitored
on TLC, the reaction mixture was cooled at room temperature.
The product was filtered, washed with water, dried, and
recrystallized from ethanol.
General procedure for the synthesis of 4-[4-(substituted
phenyl) thiazol-2-yl)-1-phenyl-1H-pyrazol-3-amine (10a–g). A
mixture of 3-amino-1-phenyl-1H-pyrazole-4-carbothioamide
9
(1mol) and substituted phenacyl bromide (1 mol) in absolute
ethanol (25 mL) was refluxed for 1 h. After completion of
reaction, as monitored on TLC, reaction mixture was cooled to
room temperature. The product thus separated was filtered,
washed with ethanol, dried, and recrystallized in ethanol.
General procedure for the synthesis of (E)-2-[4-( substituted
phenyl) thiazol-2-yl] -1-[(1-(substituted phenyl) ethylidene]
hydrazine (3a–m). A mixture of (E)-1-[1-(4-substituted phenyl)
ethylidene] thiosemicarbazide 2a–m (1 mol) and substituted
phenacyl bromide (1mol) in absolute ethanol (25 mL) was
refluxed for 1 h, on completion of reaction, reaction mixture
was cooled to room temperature. The product separated out was
filtered, washed with ethanol, dried, and recrystallized from
appropriate solvent.
1-[4-(4-Bromophenyl)thiazol-2-yl]-3-phenyl-1H-pyrazole-4-
carbaldehyde (4a). Yield: (74%); mp: 205–207ꢀC; IR (KBr,
cmÀ1) 1689 (CHO); 1H NMR (CDCl3): d 7.48–7.55 (m, 5H,
Ar–H); 7.80 (d, J = 8.5 Hz, 2H); 7.66 (d, J = 8.5 Hz, 2H); 10.11
(s, 1H, CHO); 7.30 (s, 1H, pyrazole); 9.10 (s, 1H, thiazole);
13C NMR (CDCl3): d 104; 110; 121; 123; 128; 129; 132; 137;
139; 142; 148; 152; 158; 182; Anal. Calcd for C19H12BrN3OS:
C, 55.62; H, 2.95; N, 10.24; Found: C, 55.27; H, 3.08; N,
10.32; ms: m/z 408.99 (M+), 409.99 (M + 1), 410.99.
General procedure for the synthesis of 1-[4-(substituted-phenyl)
thiazol-2-yl]-3-(substituted-phenyl)-1H-pyrazole-4-carbaldehyde
(4a–m). To a well stirred and cooled (0ꢀC) DMF solution,
POCl3 was added drop wise during 1 h. After complete
3-[3,5-Bis(trifluoromethyl)phenyl]-1-[4-(3-fluoro-4-ethoxyphenyl)
thiazol-2-yl]-1H-pyrazole-4-carbaldehyde (4b). Yield: (63%);
mp: 214–216ꢀC; IR (KBr, cmÀ1) 1690 (CHO); 1H NMR
(CDCl3): d 8.54 (s, 2H); 7.98 (s, 1H); 10.11 (s, 1H, CHO); 7.30
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N. D. Gaikwad, S. V. Patil, and V. D. Bobade
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(s, 1H, pyrazole); 9.10 (s, 1H, thiazole); 7.68 (d, J = 8.5 Hz, 1H);
7.64 (d, J = 8.5Hz, 1H); 7.05 (d, J = 8.5 Hz, 1H); 3.96 (s, 3H);
Anal. Calcd for C22H12F7N3O2S: C, 51.27; H, 2.35; N, 8.15;
Found: C, 51.53; H, 2.45; N, 8.72; ms: m/z 515.05 (M+), 516.06
(M+ 1), 517.05.
3-(4-Bromophenyl)-1-[4-(4-nitrophenyl) thiazol-2-yl]-1H-
pyrazole-4-carbaldehyde (4c). Yield: (65%); mp: >300ꢀC; IR
(KBr, cmÀ1) 1685 (CHO); 1H NMR (CDCl3): d 7.81 (d,
J = 8.5 Hz, 2H); 7.61 (d, J = 8.5Hz, 2H); 10.14 (s, 1H, CHO);
7.43 (S, 1H, pyrazole); 9.115 (s, 1H, thiazole); 8.22 (d, J = 8.5 Hz,
2H); 8.38 (d, J = 8.5 Hz, 2H); 13C NMR: d 104; 110; 121;123;
128; 129; 132; 137; 139; 142; 148; 152; 158; 182 (CHO); Anal.
Calcd for C19H11BrN4O3S: C, 50.12; H, 2.44; N, 12.31; Found:
C, 50.12; H, 2.44; N, 12.3; ms: m/z: 455.97[M+], 453.97[M + 1],
456.97.
C19H11Br2N3OS: C, 46.65; H, 2.27; N, 8.59; Found: C, 46.51; H,
2.39; N, 8.62; ms: m/z 488.90 (M+), 489.89 (M + 1), 490.85.
3-[3,5-Bis(trifluoromethyl)phenyl]-1-[4-(4-bromophenyl)thiazol-
2-yl]-1H-pyrazole-4-carbaldehyde (4j). Yield: (66%); mp: 278–
280ꢀC; IR (KBr, cmÀ1) 1690 (CHO); 1H NMR (CDCl3): d 8.53
(s, 2H); 7.98 (s, 1H); 10.11 (s, 1H, CHO); 7.42 (s, 1H,
pyrazole); 9.10 (s, 1H, thiazole); 7.80 (d, J = 8.5 Hz, 2H); 7.59
(d, J = 8.5 Hz, 2H); 13C NMR: d 104; 110; 123; 124; 129; 131;
132; 133; 137; 142; 158; 152; 182; Anal. Calcd for
C21H10BrF6N3OS: C, 46.17; H, 1.85; N, 7.69; Found: C,
46.38; H, 1.77; N, 7.58; ms: m/z 544.96 [M+], 545.96 [M + 1].
3-[3,5-Bis(trifluoromethyl)phenyl]-1-[4-(4-chlorophenyl)thiazol-
2-yl]-1H-pyrazole-4-carbaldehyde (4k). Yield: (77%); mp:
1
281–283ꢀC; IR (KBr, cmÀ1) 1685 (CHO); H NMR (CDCl3):
d 8.53 (s, 2H); 7.98 (s, 1H); 10.11(s, 1H, CHO); 7.41 (s, 1H,
pyrazole); 9.10 (s, 1H, thiazole); 7.86 (d, J = 8.5 Hz, 2H); 7.44
(d, J = 8.5 Hz, 2H); 13C NMR (CDCl3): d 104; 110; 123; 124;
128; 129; 131; 133; 134; 137; 142; 152; 158; 182; Anal. Calcd
for C21H10ClF6N3OS: C, 50.26; H, 2.01; N, 8.37; Found: C,
50.44; H, 2.08; N, 8.45; ms: m/z 501.01 [M+], 502.02 [M + 1],
503.01.
3-(4-Bromophenyl)-1-[4-(4-chlorophenyl)thiazol-2-yl]-1H-
pyrazole-4-carbaldehyde (4l). Yield: (68%); mp: 244–246ꢀC; IR
(KBr, cmÀ1) 1682 (CHO); 1H NMR (CDCl3): d 7.81 (d,
J = 8.5Hz, 2H); 7.66 (d, J = 8.5 Hz, 2H); 10.09 (s, 1H, CHO);
7.60 (s, 1H, pyrazole); 9.12 (s, 1H, thiazole); 7.88 (d, J = 8.5 Hz,
2H); 7.46 (d, J = 8.5 Hz, 2H); Anal. Calcd for C19H11BrClN3OS:
C, 51.31; H, 2.49; N, 9.45; Found: C, 51.18; H, 2.85; N,
9.72; ms: m/z 444.95 [M+], 446.09 [M + 1].
3-(4-Chlorophenyl)-1-[4-(4-nitrophenyl) thiazol-2-yl]-1H-
pyrazole-4-carbaldehyde (4m). Yield: (64%); mp: 210–212ꢀC; IR
(KBr, cmÀ1) 1688 (CHO); 1H NMR (CDCl3): d 7.87 (d, J = 8.5 Hz,
2H); 7.50 (d, J = 8.5 Hz, 2H); 10.09 (s, 1H, CHO); 7.60 (s, 1H,
pyrazole); 9.06 (s, 1H, thiazole); 8.09 (d, J = 8.5 Hz, 2H); 8.33 (d,
J = 8.5Hz, 2H); 13C NMR (CDCl3): d 104; 110; 121; 128; 129;
131; 134; 137; 139; 142; 148; 152; 158; 182; Anal. Calcd for
C19H11ClN4O3S: C, 55.55; H, 2.70; N, 13.64; Found: C, 55.58;
H, 2.75; N, 13.60; ms: m/z 410.02 [M+], 411.03 [M + 1], 412.02.
3-(4-Fluorophenyl)-4-[4-(4-nitrophenyl)thiazol-2-yl]-1-phenyl-
1H-pyrazole (7a). Yield: (65%); mp: 232–234ꢀC; 1H NMR
(CDCl3): d 7.82 (d, J = 8.5Hz, 2H); 7.59 (d, J = 8.5Hz, 2H); 8.43
(d, J = 8.5Hz, 2H); 7.92 (d, J = 8.5 Hz, 2H); 7.50 (m, 5H); 8.10
(s, 1H, thiazole); 7.71 (s, 1H, pyrazole); 13C NMR (CDCl3):
d 106; 110; 116; 120; 126; 128; 129; 131; 139; 145; 148; 152;
154; 162; Anal. Calcd for C24H15FN4O2S: C, 65.15; H, 3.42; N,
12.66; Found: C, 65.31; H, 3.72; N, 12.38; ms: m/z 442.09 (M+),
443.09 (M + 1), 444.09.
4-[4-(4-Chlorophenyl)thiazol-2-yl]-3-(4-fluorophenyl)-1-phenyl-
1H-pyrazole (7b). Yield: (66%); mp: 190–192ꢀC; 1H NMR
(CDCl3): d 7.85 (d, J = 8 Hz, 2H); 7.44 (d, J = 8 Hz, 2H); 7.82
(d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H); 7.18 (m, 2H), 7.54
(m, 3H); 9.25(s, 1H, thiazole); 7.75 (1H, s, pyrazole); 13C NMR
(CDCl3): d 106; 110; 116; 120; 126; 128; 129; 131; 134; 139;
145; 152; 154; 163; Anal. Calcd for C24H15ClFN3S: C, 66.74; H,
3.50; N, 9.73; Found: C, 66.66; H, 3.57; N, 9.62; ms: m/z 431.07
[M+], 432.07 [M+ 1], 433.06, 434.07.
3-[3,5-Bis(trifluoromethyl)phenyl]-1-[4-(3-nitrophenyl)thiazol-
2-yl]-1H-pyrazole-4-carbaldehyde (4d). Yield: (63%); mp:
1
235–237ꢀC; IR (KBr, cmÀ1) 1690 (CHO); H NMR (CDCl3): d
8.549 (s, 2H); 8.018 (s, 1H); 10.14 (s, 1H, CHO); 7.60 (s, 1H,
pyrazole); 9.162 (s, 1H, thiazole); 8.81 (s, 1H); 8.27 (d,
J = 8.5 Hz, 1H); 8.24 (dd, J = 8.5 Hz, 1H); 7.67 (dd, J = 8.5 Hz and
J = 2 Hz, 1H); Anal. Calcd for C21H10F6N4O3S: C, 49.23; H, 1.97;
N, 10.93; Found: C, 49.32; H, 1.91; N, 10.98; ms: m/z 512.04
[M+], 513.04 [M+ 1], 514.
1-[4-(3-Fluoro-4-methoxyphenyl) thiazol-2-yl]-3-(4-nitrophenyl)-
1H-pyrazole-4-carbaldehyde (4e). Yield: (68%); mp: 240–
1
242ꢀC; IR (KBr, cmÀ1) 1690 ( CHO); H NMR (CDCl3): d 8.34
(d, J = 8.5 Hz, 2H); 8.22 (d, J = 8.5 Hz, 2H,); 10.06 (s, 1H, CHO);
8.02 (s, 1H, pyrazole); 9.54 (s, 1H, thiazole); 7.82 (d, J = 8 Hz,
1H); 7.23 (d, J = 8 Hz, 1H); 7.78 (d, 1H); 3.89 (s, 3H); Anal.
Calcd for C20H13FN4O4S: C, 43.57; H, 2.38; N, 10.16; Found: C,
43.41; H, 2.46; N, 10.28; ms: m/z 550.97 [M+], 551.97 [M + 1].
1-[4-(4-Bromophenyl) thiazol-2-yl]-3-(4-nitrophenyl)-1H-
pyrazole-4-carbaldehyde (4f). Yield: (66%); mp: >300ꢀC; IR
(KBr, cmÀ1): 1685 (CHO); 1H NMR (CDCl3): d 8.16 (d,
J = 8.5 Hz, 2H); 8.33 (d, J = 8.5Hz, 2H); 10.15 (s, 1H, CHO);
7.52 (s, 1H, pyrazole); 9.15 (s, 1H, thiazole); 7.78 (d, J = 8.5 Hz,
2H); 7.62 (d, J = 8.5 Hz, 2H); Anal. Calcd for C19H11BrN4O3S: C,
50.12; H, 2.44; N, 12.31; Found: C, 49.96; H, 2.78; N, 12.45; ms:
m/z 512.04 (M+), 513.04 (M + 1), 514.03.
1-[4-{3,5-Bis(trifluoromethyl)phenyl} thiazol-2-yl]-3-(4-
nitrophenyl)-1H-pyrazole-4-carbaldehyde (4g). Yield: (72%);
mp: 224–226ꢀC; IR (KBr, cmÀ1) 1687 (CHO); 1H NMR
(CDCl3): d 8.16 (d, J = 8.5 Hz, 2H); 8.33 (d, J = 8.5 Hz, 2H);
10.15 (s, 1H, CHO); 7.52 (s, 1H, pyrazole); 9.15 (s, 1H, thiazole);
7.87 (s, 1H); 8.66 (s, 2H); Anal. Calcd for C21H10F6N4O3S: C,
49.23; H, 1.97; N, 10.93; Found: C, 49.32; H, 1.89; N, 10.83; ms:
m/z 512.04 [M+], 513.04 [M + 1], 514.03.
3-[3,5-Bis(trifluoromethyl) phenyl]-1-[4-{3,5-zis(trifluoromethyl)
phenyl} thiazol-2-yl]-1H-pyrazole-4-carbaldehyde (4h). Yield:
1
(66%); mp: 222–224ꢀC; IR (KBr, cmÀ1) 1685 (CHO); H NMR
(CDCl3): d 8.54 (s, 2H); 7.96 (s, 1Hs); 10.11 (s, 1H, CHO); 7.43
(s, 1H, pyrazole); 9.10 (s, 1H, thiazole); 8.61 (s, 2H); 7.77 (s, 1H);
13C NMR (CDCl3): d 104; 110; 123; 124 (CF3); 129; 131; 133;
137; 142; 152; 158; 182; Anal. Calcd for C23H9F12N3OS: C,
45.78; H, 1.50; N, 6.96; Found: C, 45.63; H, 1.67; N, 6.84; ms:
m/z 603.03 (M+), 604.03 (M + 1), 605.02.
3-(4-Bromophenyl)-1- [4-(4-bromophenyl)thiazol-2-yl]-1H-
pyrazole-4-carbaldehyde (4i). Yield: (62%); mp: >300ꢀC; IR
(KBr, cmÀ1) 1690 (CHO); 1H NMR (CDCl3): d 7.80 (d, 4H,
J = 8.5 Hz); 7.53–7.59 (d, J = 8.5 Hz, 4H); 10.11 (s, 1H, CHO);
7.42 (s, 1H, pyrazole); 9.10 (s, 1H, thiazole); Anal. Calcd for
3-(4-Fluorophenyl)-1-phenyl-4- (4-phenylthiazol-2-yl)-1H-
pyrazole (7c). Yield: (64%); mp: 185–187ꢀC; 1H NMR (CDCl3):
d 7.82 (d, J = 8.5 Hz, 2H); 7.59 (d, J = 8.5 Hz, 2H); 7.97 (d,
J = 8.5Hz, 2H); 7.55 (d, J = 8.5 Hz, 5H); 7.5 (m, 5H,); 8.38 (s,
1H, thiazole); 7.74 (s, 1H, pyrazole); 13C NMR (CDCl3): d 106;
110; 116; 120; 126; 127; 128; 129; 131; 133; 139; 145; 152; 154;
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2013
Synthesis and Antimicrobial Activity of Novel Thiazole Substituted Pyrazole Derivatives
525
162; Anal. Calcd for C24H16FN3S: C, 72.52; H, 4.06; N, 10.57;
Found: C, 72.48; H, 4.13; N, 10.63; ms: m/z 397.10 [M+], 398.1
[M+ 1], 399.2, 400.1.
4-[4-(4-Chlorophenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine
(10b). Yield: (67%); mp: 202–204ꢀC; IR (KBr, cmÀ1) 3487, 3336
(NH2); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 7.62 (d, J=8.5Hz,
2H); 7.85 (d, J= 8.5 Hz, 2H); 7.86 (s, 1H, thiazole); 7.47 (s, 1H,
pyrazole); 5.71 (bs, 2H, NH2); 13C NMR: d 110; 120; 126; 128;
129; 131; 134; 139; 150; 152; 154; Anal. Calcd for C18H13ClN4S:
C, 61.27; H, 3.71; N, 15.88; Found: C, 61.69; H, 3.62; N, 15.57;
ms: m/z 351.9 [M+], 352.7 [M + 1], 353.8 [M + 2], 350.6 [M-1],
337.6, 324.8, 296.8, 254.7, 215.5, 174.4, 146.4, 112.3, 78.7.
4-[4-(4-Bromophenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine
(10c). Yield: (70%); mp: 192–194ꢀC; IR (KBr, cmÀ1) 3480, 3335
(NH2 ); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 7.55 (d, J = 8.5 Hz,
2H); 7.90 (d, J = 8.5 Hz, 2H); 7.89 (s, 1H, thiazole); 7.47 (s, 1H,
pyrazole); 5.69 (bs, 2H, NH2); 13C NMR (CDCl3): d 110; 120;
123; 126; 128; 129; 132; 139; 150; 152; 154; Anal. Calcd for
C18H13BrN4S: C, 54.42; H, 3.30; N, 14.10; Found: C, 54.89; H,
3.77; N, 14.59; ms: m/z 395.8 [M+], 396.7 [M + 1], 397.5, 366.7,
360.9, 324.7, 282.8, 268.8, 255.8, 148.5, 78.7.
4-[4-(4-Bromophenyl)thiazol-2-yl-3-(4-fluorophenyl)-1-phenyl-
1H-pyrazole (7d). Yield: (67%); mp: 208–210ꢀC; 1H NMR
(CDCl3): d 7.83 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H);
7.79 (d, J = 8.5Hz, 2H); 7.56 (d, J = 8.5 Hz, 2H); 7.17 (m, 2H);
7.5 (m, 3H); 7.85 (s, 1H, thiazole); 7.49 (s, 1H, pyrazole); 13C
NMR (CDCl3): d 106; 110; 116; 120; 123; 126; 128; 129;
131; 132; 139; 145; 152; 154; 162; Anal. Calcd for
C24H15BFrN3S: C, 60.51; H, 3.17; N, 8.82; Found: C, 60.57;
H, 3.23; N, 8.83; ms: m/z 477.01 [M+], 478.0 [M+ 1], 479,
480, 477, 476, 398, 205.
3-(4-Fluorophenyl)-4-[4-(4-fluorophenyl)thiazol-2-yl]-1-phenyl-
1H-pyrazole (7e). Yield: (60%); mp: 193–195ꢀC; 1H NMR
(CDCl3): d 7.92 (d, J = 8.5 Hz, 2H); 7.84 (d, J = 8.5 Hz, 2H); 7.89
(d, J = 8.5 Hz, 2H); 7.77 (d, J = 8.5 Hz, 2H); 7.15 (m, 2H); 7.5 (m,
3H); 8.10 (s, 1H, thiazole); 7.74 (s, 1H, pyrazole); 13C NMR
(CDCl3): d 106; 110; 116; 120; 126; 128; 129; 131; 139; 145;
152; 154; 162; Anal. Calcd for C24H15F2N3S: C, 69.38; H, 3.64;
N, 10.11; Found: C, 69.81; H, 3.27; N, 10.74; ms: m/z 415.1
(M+), 416.1 (M+ 1), 417.1, 418.1.
3-(4-Fluorophenyl)-1-phenyl-4-(4-p-tolylthiazol-2-yl)-1H-
pyrazole (7f). Yield: (75%); mp: 212–214ꢀC; 1H NMR (CDCl3): d
7.92 (d, J = 8.5 Hz, 2H); 7.84 (d, J = 8.5 Hz, 2H); 7.63 (d, J = 8.5 Hz,
2H); 7.53 (d, J = 8.5 Hz, 2H); 7.46 (m, 5H); 8.37 (s, 1H, thiazole);
7.74 (s, 1H, pyrazole); Anal. Calcd for C25H18FN3S: C, 72.97; H,
4.41; N, 10.21; Found: C, 72.22; H, 4.71; N, 10.36; ms: m/z
411.12 (M+), 412.12 (M + 1), 413.12.
3-(4-Bromophenyl)-1-phenyl-4-(4-p-tolylthiazol-2-yl)-1H-
pyrazole (7g). Yield: (63%); mp: 223–225ꢀC; 1H NMR (CDCl3):
d 7.97 (d, J = 8 Hz, 2H); 7.74 (d, J = 8.5 Hz, 2H); 7.63 (d,
J = 8.5 Hz, 2H), 7.53 (d, J = 8.5Hz, 2H), 7.46 (m, 5H), 8.37 (s,
1H, thiazole); 7.74 (s, 1H, pyrazole); Anal. Calcd for
C25H18BrN3S: C, 63.56; H, 3.84; N, 8.90; Found: C, 63.57; H,
3.94; N, 8.83; ms: m/z 471.9 [M+], 472.04 [M+ 1], 420.9, 348.8,
304.8, 255.9, 194.6, 148.6, 101.7.
3-(4-Bromophenyl)-4-[4-(4-nitrophenyl)thiazol-2-yl]-1-phenyl-
1H-pyrazole (7h). Yield: (67%); mp: >300ꢀC; 1H NMR (CDCl3):
d 8.12 (d, J = 8.5 Hz, 2H); 7.73 (d, J = 8.5 Hz, 2H); 8.43 (d,
J = 8.5 Hz, 2H); 7.92 (d, J = 8.5 Hz, 2H); 7.50 (m, 5H); 8.10 (s,
1H, thiazole); 7.71 (s, 1H, pyrazole); Anal. Calcd for
C24H15BrN4O2S: C, 57.27; H, 3.00; N, 11.13; Found: C, 57.35;
H, 3.07; N, 11.18; ms: m/z 501.0 [M+], 502.9 [M+ 1], 503.9,
504.9, 476.1, 433.1, 318.9, 255.8, 176.7, 120.5, 88.0.
3-(4-Bromophenyl)-1-phenyl-4-(4-phenylthiazol-2-yl)-1H-
pyrazole (7i). Yield: (73%); mp: 215–217ꢀC; 1H NMR (CDCl3): d
7.98 (d, J = 8.50Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H); 7.97 (d, J = 8 Hz,
2H); 7.55 (d, J = 8.5 Hz, 5H); 7.5 (m, 5H); 8.38 (s, 1H, thiazole);
7.74 (s, 1H, pyrazole); Anal. Calcd for C24H16BrN3S: C, 62.89;
H, 3.52; N, 9.17; Found: C, 62.74; H, 3.58; N, 9.25; ms: m/z
458.8 [M+], 459.9 [M+ 1], 457.0, 413.0, 323.6, 284.3, 238.6,
197.5, 88.5.
1-Phenyl-4-(4-p-tolylthiazol-2-yl)-1H-pyrazol-3-amiÀn1e
Compound (10d). Yield: (65%); mp: 188–190ꢀC; IR (KBr, cm
)
3476, 3338 (NH2 ); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 7.53
(d, J = 8.5 Hz, 2H); 7.63 (d, J = 8.5 Hz, 2H); 7.85 (s, 1H, thiazole);
7.46 (s, 1H, pyrazole); 5.67(bs, 2H, NH2); Anal. Calcd for,
C19H16N4S: C, 68.65; H, 4.85; N, 16.85; Found: C, 68.37; H,
4.78; N, 16.25; ms: m/z 332.1 [M+], 333.2 [M + 1], 331.2, 318.8,
270.9, 256.8, 191.6, 129.5.78.6, 15.0.
1-Phenyl-4-(4-phenylthiazol-2-yl)-1H-pyrazol-3-amine
(10e). Yield: (68%); mp: 186–188ꢀC; IR (KBr, cmÀ1) 3480, 3330
1
(NH2 ); H NMR (CDCl3): d 7.48–7.70 (m, 10H); 7.89 (s, 1H,
thiazole);7.44 (s, 1H, pyrazole); 5.65 (bs, 2H, NH2); 13C NMR
(CDCl3): d 94; 110; 120; 126; 127; 128; 129; 133; 139; 150;
152; 154; Anal. Calcd for C18H14N4S: C, 67.90; H, 4.43; N,
17.60; Found: C, 67.57; H, 4.72; N, 17.24; ms: m/z 317.8[M+],
318.7[M+ 1], 319.1, 303.9, 255.9, 241.8, 22.6, 176.6, 132.6,
114.5.
4-[4-(4-Nitrophenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine
(10f). Yield: (71%); mp: 220–222ꢀC; IR (KBr, cmÀ1) 3484, 3340
(NH2 ); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 7.97 (d, J = 8.5 Hz,
2H); 8.32 (d, J = 8.5 Hz, 2H); 7.87 (s, 1H, thiazole); 7.45 (s, 1H,
pyrazole); 5.69 (bs, 2H, NH2); 13C NMR (CDCl3): d 94; 110;
120; 121; 126; 128; 129; 139; 148; 150; 152; 154; Anal. Calcd for
C18H13N5O2S: C, 59.49; H, 3.61; N, 19.27; Found: C, 59.89; H,
3.73; N, 19.57; ms: m/z 362.8 [M+], 363.8 [M + 1], 361.6, 317.8,
255.9, 148.5, 78.7.
4-[4-(3-Nitrophenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine
(10 g). Yield: (66%); mp: 218–220ꢀC; IR (KBr, cmÀ1) 3478, 3335
(NH2 ); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 8.81 (s, 1H,); 8.27
(d, J = 8.5 Hz, 1H); 8.24 (dd, J = 8.5 and 2 Hz, 1H); 7.67 (dd,
J = 8.5 and 2 Hz, 1H); 7.84 (s, 1H, thiazole); 7.45 (s, 1H,
pyrazole); 5.71(bs, 2H, NH2); Anal. Calcd for C18H13N5O2S: C,
59.49; H, 3.61; N, 19.27; Found: C, 59.89; H, 3.73; N, 19.57;
ms: m/z 362.7 [M+], 363.8 [M + 1], 361.7, 338.8, 317.8, 296.7,
255.5, 148.6, 78.6.
4-[4-(4-Fluorophenyl)thiazol-2-yl]-1-phenyl-1H-pyrazol-3-amine
(10a). Yield: (71%); mp: 197–199ꢀC; IR (KBr, cmÀ1) 3476, 3335
(NH2); 1H NMR (CDCl3): d 7.48–7.63 (m, 5H); 7.55 (d, J = 8.5 Hz,
2H); 7.90 (d, J = 8.5 Hz, 2H); 7.85 (s, 1H, thiazole); 7.45 (s, 1H,
pyrazole); 5.71 (bs, 2H, NH2); 13C NMR (CDCl3): d 94; 110;
116; 120; 126; 127; 128; 129; 133; 139; 150; 152; 154; 162;
Anal. Calcd for C18H13FN4S: C, 64.27; H, 3.90; N, 16.66; Found:
C, 64.89; H, 3.52; N, 16.17; ms: m/z 336.1 [M+], 337.1 [M+ 1],
338.0, 322.9, 274.9, 241.8, 195.6, 152.6, 133.5, 78.7.
Antimicrobial activity.
For antibacterial activity, solid
medium used for the study was Muller–Hinton agar (MHA; Hi
media) of the following composition, beef infusion 300 g/L, casein
acid hydrolysate 17.5g/L, starch 1.5 g/mL, agar–agar 17g/L, and
sterilized distilled water 1000mL adjusted to pH7.4. soyabean
casein digest agar (SCDA; casein enzymatic hydrolysate 17.0g/L,
papain digest of soyabean 3.0 g/L, NaCL 5.0g/L, dipotassium
phosphate 2.5 g/L, and distilled water 1000 mL adjusted to
pH7.3), was used for biological assays.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

526
N. D. Gaikwad, S. V. Patil, and V. D. Bobade
Vol 50
well isolated colonies of the same morphological type selected
from an agar plate culture. The cultures were further diluted 10-
fold to obtain an inoculums size of 1.2 Â 107 CFU/mL. Stock
solutions of 4 mg/mL of each compound were prepared in
For antifungal activity, solid medium used for the study was
potato dextrose agar (Hi media) of the following composition:
potato 250 g, dextrose 10 g, agar–agar 20 g, and sterile distilled
water 100 mL adjusted to pH 7.3.
Test microorganisms. All the synthesized compounds were
screened for their in vitro antibacterial activity against the
standard strains B. subtilis (2250), S. aureus (2079), E. coli
(2109), and P. aeruginosa (2036) and for their antifungal
activity against C. albicans (3471) and A. niger (545). All the
strains were obtained from microbial type culture collection
(MTCC) at the NCIM, Pune, India.
Primary screening. The antibacterial activity of all the newly
synthesized compounds was carried out by the agar-well diffusion
assay technique [40,41]. Bacterial cultures (24-h-old) of all test
microorganisms were used as inoculums, which were adjusted to
0.5 McFarland standards, that is, 1.5 Â 108 CFU/mL. The stock
solutions of all test compounds (128 mg/mL) were prepared by
dissolving 128 mg of the test compound in DMSO (1 mL).
Chloramphenicol and DMSO were used as positive and negative
controls, respectively.
Twenty milliliter of molten and cooled MHA and 320 mL of
each test bacterial culture were mixed (separate flasks were used
for each bacterial culture) and poured in sterilized and labeled
Petri plates. The wells of 6 mm were punched in the solidified
Petri plates, aseptically. Fifty microliters from stock solutions of
all compounds as well as controls was added to each well of
labeled Petri plates and incubated at 35ꢀC for 24 h. The diameter
of the zone of growth inhibition around each well was measured
after incubation by using vernier caliper.
DMSO and were appropriately diluted to obtain
a final
concentration of 128, 64, 32, 16, 8, 4, 2, and 1 mg/mL. Standard
antibiotic Chloramphenicol was also diluted to obtain a final
concentration in the same manner. Three hundred and twenty
microliters of each dilution was added to 20 mL molten and
cooled MHA (separate flasks were taken for each dilution).
After thorough mixing, the medium was poured in sterilized
Petri plates. The test bacterial cultures were spotted in a
predefined pattern by ascetically transferring 5 mL of each
bacterial culture on the surface of solidified agar plates and
incubated at 35ꢀC for 24 h.
For antifungal activity, the MICs of synthesized compounds
4a–10 g were determined in the range of concentrations from
128 to 1 mg/mL. The standardized micro broth dilution methods
were used according to the guidelines of Clinical and Laboratory
Standards Institute (formerly National Committee for Clinical and
Laboratory Standards) [42]. Table 2 summarizes the minimum
concentration of each derivative necessary to completely inhibit
(MIC90) the growth of two standardized opportunistic pathogenic
fungi including C. albicans and A. Niger.
Acknowledgments. We are grateful to the UGC, New Delhi and
BCUD, University of Pune, India for financial assistance. One of
the authors (NDG) is sincerely thankful to Principal, K.T.H.M.
College, Nashik for providing laboratory facilities.
For the antifungal activity, sliced potatoes were taken with
500 mL of distilled water in a pan and boiled for half an hour till
a spoon when placed on a slice can pierce into it. Filtered it while
hot and broth was again taken in a pan with rest of the distilled wa-
ter. Dextrose dissolved in distilled water and weighed agar was
added to the broth and heated it to boil. The medium thus obtained
was sterilized in pressure cooker for 30 min. Sterilized medium
(15 mL each) was pipette out into flat Petri plates. When it solidi-
fied, 15 mL of warm seeded agar was applied over it. The seeded
agar was made by cooling the medium to 40ꢀC and then adding
spore suspension to seeded medium. The spores were obtained
from 10 days culture of C. albicans and A. niger species. The final
inoculums size was adjusted to 1 Â 106 spore mLÀ1. Nystatin and
DMSO were used as positive and negative controls, respectively.
Before the solidification of agar, the plate was tilted to ensure that
coverage should be even. These Petri plates were then put into the
refrigerator upside down to prevent condensation of moisture.
Concentration, 128 mg/mL, of the synthesized compounds were
prepared by dissolving the required quantity of compounds in
DMSO, sterilized Whatmann filter paper number 541 disks were
prepared by cutting 6 mm diameter with cork borer and were spread
individually with needle and planted upon the chilled seeded
medium. The culture plates were then incubated for 24 to 72h at
37ꢀC, and inhibition zone around each disk was measured from
the center of the disks. The diameter of growth inhibition zone
was calculated by vernier caliper.
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