Synthesis and antimicrobial activities of novel 1,5-diaryl pyrazoles

Article abstract of DOI:10.1016/j.ejmech.2009.12.042

Novel 1,5-diaryl pyrazole derivatives viz. 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-carboxamides (2a-e, 3, 3a-f), 2-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-yl)thiazoles (6a-c,7, 8, 9a-c, 10, 11) were synthesized by varying the active part (amide group) of pyrazole, characterized using IR, ¹H NMR, mass spectral data and screened for their antibacterial activity against Escherichia coli (ATTC-25922), Staphylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATTC-27853), Klebsiella pneumonia. Similarly all these compounds were screened for their antifungal activity against Aspergillus flavus (NCIM No. 524), Aspergillus fumigates (NCIM No. 902), Penicillium marneffei and Trichophyton mentagrophytes. Interestingly all the synthesized compounds exhibited good antibacterial (except 2a-c, 8, 9a) and antifungal activity (except 2a-c, 6a, 8, 9a).

Full text of DOI:10.1016/j.ejmech.2009.12.042

European Journal of Medicinal Chemistry 45 (2010) 1173–1180
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activities of novel 1,5-diaryl pyrazoles
,
,
c
R. Venkat Ragavan a b, V. Vijayakumar a , N. Suchetha Kumari
*
a Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, India
b Syngene International Pvt. Ltd., Biocon Park, Bangalore 560 099, India
c Department of Biochemistry, K.S. Hegde Medical Academy, Deralakatte 574 162, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 1,5-diaryl pyrazole derivatives viz. 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-carbox-
amides (2a–e, 3, 3a–f), 2-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-yl)thiazoles (6a–c,7, 8,
9a–c, 10, 11) were synthesized by varying the active part (amide group) of pyrazole, characterized using
IR, 1H NMR, mass spectral data and screened for their antibacterial activity against Escherichia coli (ATTC-
25922), Staphylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATTC-27853), Klebsiella pneu-
monia. Similarly all these compounds were screened for their antifungal activity against Aspergillus flavus
(NCIM No. 524), Aspergillus fumigates (NCIM No. 902), Penicillium marneffei and Trichophyton menta-
grophytes. Interestingly all the synthesized compounds exhibited good antibacterial (except 2a–c, 8, 9a)
and antifungal activity (except 2a–c, 6a, 8, 9a).
Received 26 July 2009
Received in revised form
6 December 2009
Accepted 18 December 2009
Available online 28 December 2009
Keywords:
1,5-Diaryl pyrazoles
5-(4-Chlorophenyl)-1-(4-fluorophenyl)-
1H-pyrazole-3-carboxamides
2-(5-(4-Chlorophenyl)-1-(4-fluorophenyl)-
1H-pyrazole-3-yl)thiazoles
4-Piperidine amide pharmacophore
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antibacterial activity
Antifungal activity
1. Introduction
biological activity, we are interested to have 4-fluoro or 4-chloro
substitution in the aryls of 1,5-diaryl pyrazoles. As part of our on
Antibacterial and antifungal activities of the azoles are most
widely studied and some of them are in clinical practice as anti-
microbial agents. However, the azole resistant strains led to
develop a new antimicrobial compounds. In particular pyrazole
derivatives are extensively studied and used as antimicrobial
agents [1–21]. Pyrazole is an important class of heterocyclic
compound and many pyrazole derivatives are reported to have the
broad spectrum of biological activities, such as anti-inflammatory
[21–23], antifungal [24], herbicidal [25,26], antitumour, cytotoxic,
molecular modelling [27–29], and antiviral [30,31] activities.
Pyrazole derivatives also acting as antiangiogenic agents [32], A3
adenosine receptor antagonists [33], neuropeptide YY5 receptor
antagonists [34], kinase inhibitor for treatment of type 2 diabetes,
hyperlipidemia, obesity [35], and thrombopiotinmimetics [36].
Recently urea derivatives of pyrazoles have been reported as potent
inhibitors of p38 kinase [37]. Since the high electronegativity of
halogens (particularly chlorine and fluorine) in the aromatic part of
the drug molecules plays an important role in enhancing their
going research aiming the synthesis of new antimicrobial
compounds [38], here in we report the synthesis of novel pyrazole
derivatives and their microbial activities.
2. Chemistry
The b-diketo ester was synthesized by the available procedure in
literature [39], then condensed with 4-flurophenyl hydrazine in
ethanol to get pyrazole carboxylic acid ethyl ester (1) which upon
hydrolysis by lithium hydroxide (Scheme 1) afford 1,5-diaryl pyr-
azole-3-carboxylic acid (1a). The acid chloride of 1a was synthe-
sized using oxalyl chloride, and then coupled with various amines
to get 2a–b, 2d, and 2e. 2c was obtained by the hydrolysis of 2b
using lithium hydroxide (Scheme 2).
The de-protection of t-butyl oxy carbonyl group by TFA in 2d
under acidic conditions yield key scaffold 3 which, was converted
into its amide derivatives (3a–b) by acylation in the presence of
triethylamine as base in dichloromethane solvent. The reaction of
various isocyanides with 3 in THF, yielded the urea derivatives
3c–e. The 3f was obtained by the alkylation of 3 in the presence of
potassium carbonate using propargyl bromide (Scheme 3).
* Corresponding author.
E-mail address: kvpsvijayakumar@gmail.com (V. Vijayakumar).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.042

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R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
O
NHNH2
O
O
OC2H5
F
LiHMDS
O
EtOH/ CH3COOH
THF/ 0ºC
Cl
Cl
Diethyl oxalate
80ºC
F
F
Li(OH)2/ THF
N
N
OC2H5
O
OH
N
N
H2O/ MeOH
RT
O
Cl
Cl
1
1a
Scheme 1. Synthetic protocol for compound 1a.
Compound 5 was obtained in two steps from 1a. In the first step
carboxylic acid of 1a was converted into corresponding amide (4)
by ammonia, and then refluxed with Lawesson’s reagent in THF/
Toluene to get thioamide (5) in second step, which under the
3. Pharmacology
3.1. Antibacterial activity
refluxing condition with various
expected thiazole derivatives (Scheme 4).
a
-bromo ketones afford the
We have investigated the newly synthesized pyrazoles for their
antibacterial activity against Escherichia coli (ATTC-25922), Staph-
ylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATTC-
27853), and Klebsiella pneumonia (recultured) bacterial strains by
the disc diffusion method [40–42]. The discs with 6.25 mm diam-
eter 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 an hour. The test compounds were prepared
with different concentrations using Dimethylformamide (DMF).
One millilitre containing 100 times the amount of chemical in each
disc was added to each bottle, which contains 100 discs. The discs of
Ester group of 6b was converted into the corresponding
hydrazide (7) by refluxing it with hydrazine hydrate in ethanol.
Compound 7 was cyclized using 1,10-carbonyl diimidazole and
triethylamine in THF to get 8. Urea and thiourea derivatives of 9
were prepared by reaction of 9 with different isocyanides or isothio
cyanides in THF (Scheme 5).
Ketone of 2e was reduced into 10 using sodium borohydride in
methanol which upon reaction with methane sulfonyl chloride in
DCM (Scheme 6) affords 11.
F
F
1. (COCl)2 / DCM
DMF (cat.) / RT
N
N
R
N
OH
N
O
2. RNH2 / DCM
O
TEA / O°C
Cl
Cl
1a
2
H
N
O
N
2d ,
2e ,
R=
R=
N
2a
2b
,
R=
O
N
Cl
COOC2H5
O
S
N
,
R=
R=
N
H
N
COOH
S
2c
,
N
H
N
Scheme 2. Synthetic protocol to synthesis derivatives of 2.

R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
1175
F
F
Boc
NH
N
N
N
N
N
TFA / DCM / RT
N
N
O
O
2d
3
Cl
Cl
DCM / TEA
°
0 C / Acid Chloride
K2CO3 / DMF / RT
THF/ 0°C
Isocyanide
Br
F
R
N
F
R
N
N
N
N
F
R
O
N
N
N
N
Cl
N
N
N
O
O
Cl
Cl
O
O
, R=
3c
, R=
, R=
3a
3b
N
H
CN
O
O
, R=
3f
O
3d , R=
3e , R=
N
H
N
H
Scheme 3. Synthetic protocol to synthesis various derivatives of 3.
each concentration were placed in triplicate in a nutrient agar
medium separately seeded with fresh bacteria. The incubation 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. Results of these studies are given in Table 1, and
compared with the standard ciprofloxacin.
inhibition zone. The results of these studies are given in Table 2 and
compared with the standard Ciclopiroxolamine.
4. Results and discussion
All the synthesized compounds were characterized through IR,
1H NMR, LCMS techniques and screened for their antimicrobial
activities. Interestingly all the synthesized compounds (except 2a–
c, 8, 9a) exhibited good antibacterial activity against S. aureus, E. coli
and moderate activity against P. aeruginosa, K. pneumonia (2e, 3, 3b,
10 and 11 were found to be more active against K. pneumonia). The
structure–activity relationship (SAR) studies revealed that the
aromatic amide linkage in 2a–c, thiazolo oxadiazolone group in 8
and thiourea group in 9a, are responsible for their lower activity.
SAR studies further revealed that, the presence of aliphatic amide
linkage is important for their bacterial activity and the results of 2e,
10, 11 also confirm the same. 2e, 10, 11 were active against all the
bacteria tested, which impressed us to pursue further studies on
these derivatives.
Similarly all these synthesized compounds were exhibiting
pronounced activity against P. marneffei and T. mentagrophytes
(except 2a–c, 6a, 8, 9a). 2e, 3, 3a–c, 10, 11 were more active against
A. fumigates whereas 3a, 3d–f, 6b, 7, 9b–c were moderately active.
2a–c, 6a, 6c, 8 and 9 were not active against A. fumigates. 2e, 10 and
11 were most active against A. flavus, whereas 3, 3b–c, 3e, 6b, 9c
shown moderate activity, and all the other compounds found to be
less active. From SAR studies it is clear that compounds with
aromatic amide linkage are not active against any of the fungi
3.2. Antifungal activity
Newly synthesized pyrazoles were screened for their antifungal
activity against Aspergillus flavus (NCIM No. 524), Aspergillus fumi-
gates (NCIM No. 902), Penicillium marneffei (recultured) and Tri-
chophyton mentagrophytes (recultured) in DMSO by serial plate
dilution method [38–40]. Sabouraud’s agar media were prepared
by dissolving peptone (1 g),
D-glucose (4 g) and agar (2 g) in
distilled water (100 ml) and the pH was adjusted to 5.7. Normal
saline was used to make a suspension of spores of fungal strain for
lawning. A loopful of particular fungal strain was transferred to
3 ml of saline to get a suspension of corresponding species. Agar
media of 20 ml was poured into each Petri dish. An excess of
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/ml of the test
compounds in DMSO was added into each well labelling. A control
was also prepared for plates in the same way using solvent DMSO.
The Petri dishes were prepared in triplicate and maintained at 37 ꢀC
for 3–4 days. Antifungal activity was determined by measuring the

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R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
F
F
N
N
OH
N
NH2
N
Lawesson reagent
THF / Toluene / 85 C
HOBT / EDC.HCl
DCM / TEA/ 0°C
°
O
O
NH3 (g)
4
Cl
Cl
1a
F
F
O
Br
N
R1
R1
N
N
N
NH2
N
R2
EtOH / 90°C
S
S
R2
Cl
5
Cl
6
6a , R1 =
R2 = H
,
6b ,
R1 =
R2 = COOC2H5
,
Cl
6c ,
R1 =
,
R2 = H
CN
Scheme 4. Synthetic protocol to synthesis various derivatives of 6.
tested. Thiazolo oxadiazolone group in 8 and thiourea group in 9a
are responsible for their lower antifungal activity. The pronounced
activity of 2e, 10, 11 with 4-piperdine amide linkage indicates the
importance of 4-piperidine amide pharmacophore for antifungal
activity. It is essential to carry out further studies on 2e, 10 and 11 to
develop them as drug molecules.
5. Conclusion
A group of novel 1,5-diaryl pyrazoles was synthesized by
altering the active part (amide linkage) and tested for their bio-
logical activities. Compounds having the aliphatic amide linkage
are shown pronounced activity; particularly compounds with
F
F
N
N
N
N
N
N
º
NH2NH2 / EtOH / 85 C
S
S
COOC2H5
CONHNH2
7
6b
Cl
Cl
CDI / THF /
TEA / RT
THF / Isocyanide
(or) Isothiocyanide / O C
F
º
F
N
N
N
N
N
N
S
N
H
NH
S
N
Cl
O
R
N
9
8
H
O
Cl
O
S
O
O
O
O
,
R =
9a
N
9b, R =
, R =
9c
N
H
N
H
H
CF3
Scheme 5. Synthetic protocol to synthesis various derivatives of 9.

R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
1177
F
O
F
OH
CH3SO2Cl / TEA /
DCM / RT
N
N
N
NaBH / MeOH / RT
4
°
N
N
N
O
O
Cl
10
2e
Cl
F
OSO2CH3
N
N
N
O
11
Cl
Scheme 6. Synthetic protocol to synthesis compound 11.
4-piperidine amide linkage are more active against all the bacteria
and fungi tested, where as the compounds bearing aromatic amide
linkage are not active. The thiazolo oxadiazolo group and thiourea
group retarded the biological activity. The results of our present
study conferred that the aliphatic amide pharmacophore is
important for antimicrobial activities of studied pyrazoles; specif-
ically the presence of 4-piperidine moiety enhances the activities.
Further, derivatization of the active scaffolds 2e, 10, 11 and their in
vitro and in vivo screening also required.
approximately 0.03 M solutions in d6-DMSO at 300 MHz or
400 MHz with TMS as internal reference. IR spectra were recorded
using NICOLET 6700 FT-IR (Thermo scientific). LCMS were obtained
using Agilent 1200 series LC and Micromass zQ spectrometer.
Column chromatography was performed using a Silica gel (230–
400 mesh). 1H NMR, IR, LCMS and melting point were recorded for
all final compounds.
6.2. Synthesis of 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid ethyl ester (1)
6. Experimental
The mixture of
b-diketo ester (1 mmol) and 4-flurophenyl
6.1. General
hydrazine (1 mmol) in ethanol (100 ml) and acetic acid (10 ml)
was refluxed overnight. Reaction mixture (RM) was concentrated
to remove ethanol, then neutralized with Na2CO3 and the
obtained solid was filtered, dried and purified by recrystallisation
using ethanol. Off-white solid, 78% yield, m.p. 120.6–121.7 ꢀC, 1H
NMR (400 MHz, d6-DMSO):
7.10 (s, 1H). MS calcd. for C22H20ClFN2O2: 398.86. Found: 399.8
(Mþ).
All reagents were purchased from Aldrich and used as received.
LiHMDS solutions were kept under nitrogen atmosphere after
opening. All chemistry was performed under a nitrogen atmo-
sphere using standard techniques. Melting points were determined
by Buchi B-545 apparatus. All the NMR spectra were measured
using either Bruker AMX 400 or Bruker DPX 300 instrument with
5 mm PABBO BB-1H tubes. 1H NMR spectra measured for
d
¼ 13.0 (br s, 1H), 7.45–7.25 (m, 8H),
Table 1
Table 2
Antibacterial activities of the newly synthesized compounds (zone of inhibition in
mm, MIC in mg/ml given in parenthesis).
Antifungal activities of the newly synthesized compounds (zone of inhibition in mm,
MIC in mg/ml given in parenthesis).
Compound no.
S. aureus
E. coli
P. aeruginosa
Klebsiella sp.
Compound no.
Trichophton
Penicillium
AS flavous
A. fumigates
2a
2b
2c
2e
3
3a
3b
3c
3d
3e
3f
6a
6b
6c
7
8
<10(100)
<10(100)
<10(100)
24(12.5)
<10(100)
18(12.5)
20(12.5)
18(12.5)
21(12.5)
20(12.5)
18(12.5)
<10(100)
16(12.5)
19(12.5)
20(12.5)
<10(100)
<10(100)
19(12.5)
21(12.5)
23(12.5)
26(12.5)
23(6.25)
<10(100)
<10(100)
<10(100)
27(12.5)
<10(100)
18(12.5)
20(12.5)
19(12.5)
19(12.5)
19(12.5)
18(50)
21(12.5)
18(12.5)
20(12.5)
18(12.5)
<10(100)
<10(100)
20(12.5)
22(12.5)
24(12.5)
28(12.5)
32(6.25)
<10(100)
<10(100)
<10(100)
18(12.5)
19(50)
<10(100)
17(100)
<10(100)
<10(100)
19(100)
<10(100)
16(100)
19(100)
19(50)
19(100)
<10(100)
<10(100)
<10(100)
<10(100)
29(100)
21(50)
<10(100)
<10(100)
<10(100)
25(12.5)
19(12.5)
21(100)
19(12.5)
17(50)
19(100)
19(50)
17(50)
17(100)
19(50)
17(100)
19(50)
<10(100)
<10(100)
19(50)
21(100)
21(12.5)
23(12.5)
24(6.25)
2a
2b
2c
2e
3
3a
3b
3c
3d
3e
3f
6a
6b
6c
7
8
<10(100)
<10(100)
<10(100)
24(50)
<10(100)
<10(100)
<10(100)
29(12.5)
<10(100)
19(12.5)
21(12.5)
19(12.5)
23(12.5)
24(12.5)
18(100)
<10(100)
19(12.5)
22(12.5)
20(12.5)
<10(100)
<10(100)
22(12.5)
21(12.5)
24(12.5)
29(12.5)
23(6.25)
<10(100)
<10(100)
<10(100)
18(50)
<10(100)
<10(100)
<10(100)
26(12.5)
23(12.5)
28(100)
22(12.5)
18(12.5)
24(100)
16(50)
19(50)
<10(100)
18(50)
<10(100)
19(50)
<10(100)
<10(100)
25(100)
23(100)
27(12.5)
25(12.5)
26(6.25)
19(50)
25(50)
20(12.5)
20(12.5)
24(12.5)
21(12.5)
22(12.5)
<10(100)
19(100)
20(12.5)
22(12.5)
19(12.5)
<10(100)
<10(100)
17(12.5)
19(12.5)
26(12.5)
28(12.5)
27(3.125)
<10(100)
24(100)
17(50)
<10(100)
19(100)
<10(100)
<10(100)
22(100)
<10(100)
<10(100)
<10(100)
<10(100)
<10(100)
21(100)
29(50)
9a
9b
9c
10
9a
9b
9c
10
11
11
31(100)
27(3.125)
Ciprofloxacin
28(6.25)
Ciclopiroxolamine

1178
R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
6.3. Synthesis of 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid (1a)
(br s, 2H), 3.4 (br s, 4H). IR (cmꢂ1): 3126.2, 3001.0, 2928.5, 2977.8,
1699.3, 1621.8. MS calcd. for C23H20ClFN4O: 422.282. Found: 429.1
(M-tert-Bu).
To the solution of pyrazole carboxylic acid ethyl ester 1 (1 mmol)
in THF (50 ml), methanol (15 ml), water (20 ml), and lithium
hydroxide (1.2 mmol) were added at room temperature (RT) and
stirred overnight. RM was concentrated to remove volatiles, the
resulting residue was dissolved in water (50 ml) and ethyl acetate
(100 ml), acidified using 1 N HCl to the pH w5 and then extracted
with ethyl acetate (2 ꢁ 100 ml), the combined organic layer was
washed with water (50 ml), brine solution (20 ml), dried over
Na2SO4 and then evaporated to dryness. Off-white solid, 92% yield,
6.4.5. 1-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazine-1-carboxylic acid tert-butyl ester (2e)
Whitesolid, 76%yield, m.p.163.2–164.5 ꢀC,1HNMR(400 MHz, d6-
DMSO):
d
¼ 7.47–7.39 (m, 4H), 7.34–7.28 (m, 4H), 7.03 (s,1H), 4.24 (br
s, 2H), 3.29 (br s, 2H), 2.50 (br s, 4H). IR (cmꢂ1): 3047.2, 2921.3,1710.2,
1623.2. MS calcd. for C22H18ClFN2O2: 396.84. Found: 397.8 (Mþ).
6.5. Synthesis of [5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carbonyl]-piperazin-1-yl-methanone (3)
m.p. 172.2–173.1 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 7.4–7.25 (m,
8H), 7.16 (s, 1H), 4.33 (q, J ¼ 7.08 Hz, 2H), 1.30 (t, J ¼ 7.08 Hz, 3H). MS
calcd. for C22H20ClFN2O2: 398.86. Found: 345.8 (Mþ).
To the solution of 2d (1 mmol) in DCM (20 ml), TFA (5 ml) was
added at 0 ꢀC and stirred at RT. After completion of the reaction, RM
was diluted with water (20 ml), and made alkaline using NaHCO3,
and then extracted with DCM (2 ꢁ100 ml), the combined organic
layer was washed with water (50 ml), brine solution (20 ml), dried
over Na2SO4, crude was directly taken for the next step. Yellow
solid, 83% yield, m.p.143.8–144.7 ꢀC, 1H NMR (400 MHz, d6-DMSO):
6.4. General procedure for the synthesis derivatives of 5-(4-
chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-carboxamide (2)
To the solution of 1 (1 mmol) in DCM (20 ml), oxalyl chloride
(1.5 mmol) was added at 0 ꢀC, after 5 min catalytic amount of DMF
(three drops) was added and stirred at RT. After completion of the
reaction, RM was concentrated to remove excess of oxalyl chloride
under nitrogen atmosphere to get the neat acid chloride. To the
solution of acid chloride in fresh DCM (25 ml), triethylamine
(2 mmol) was added at 0 ꢀC, followed by amine (1.1 mmol) at 0 ꢀC
and allowed to stand. After completion of the reaction, RM was
diluted with water (50 ml), extracted with DCM (2 ꢁ 100 ml), and
then the combined organic layer was washed with water (50 ml),
brine solution (20 ml), dried over Na2SO4 and then evaporated to
dryness and washed with diethyl ether.
d
¼ 7.44 (d, J ¼ 8.48 Hz, 2H), 7.43–7.35 (m, 2H), 7.32–7.26 (m, 4H),
6.94 (s, 1H), 3.82 (br, 2H), 3.57 (br s, 2H), 3.32 (br s, 2H), 2.71 (br,
2H). IR (cmꢂ1): 3147.3, 2921.4, 1689.7, 1536.9. MS calcd. for
C20H18ClFN4O: 384.84. Found: 385.1 (Mꢂ).
6.6. General procedure to synthesize compounds 3a–b
Triethylamine (2 mmol) was added to the solution of 3 (1 mmol)
in DCM (20 ml), then acid chloride (1.2 mmol) was added at 0 ꢀC and
stirred at RT. After completion of the reaction, RM was diluted with
water (20 ml), made alkaline using NaHCO3, and extracted with DCM
(2 ꢁ100 ml), the combined organic layer was washed with water
(50 ml), brine solution (20 ml), dried over Na2SO4, and then evapo-
rated to dryness, the resulting solid was washed with diethyl ether.
6.4.1. 5-(4-Chlorophenyl)-N-(6-chloropyridin-3-yl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamide (2a)
Off-white solid, 77% yield, m.p. 182.2–183.4 ꢀC, 1H NMR
(400 MHz, d6-DMSO):
d
¼ 10.69 (s, 1H), 8.85 (s, 1H), 8.36 (d,
J ¼ 8.90 Hz,1H), 7.52–7.44 (m, 5H), 7.37–7.29 (m, 4H), 7.22 (s, 1H). IR
(cmꢂ1): 3295.7, 3116.8, 1685.6, 1586.3, 1522.1. MS calcd. for
C21H13Cl2FN4O: 427.26. Found: 425.2 (Mꢂ).
6.6.1. 1-{4-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazin-1-yl}-hexan-1-one (3a)
White solid, 71% yield, m.p. 94.4–95.6 ꢀC, 1H NMR (400 MHz, d6-
DMSO):
d
¼ 7.46–7.26 (m, 4H), 6.99 (s, 1H), 4.07 (br s, 2H), 3.65 (br s,
6.4.2. Ethyl 2-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-4-methyl-thiazole-5-carboxylate (2b)
Yellow solid, 92% yield, m.p. 185.5–186.4 ꢀC, 1H NMR (400 MHz,
2H), 3.56 (br s, 4H), 2.33 (m, 4H), 1.44 (m, 4H), 0.83 (m, 5H). IR
(cmꢂ1): 3514.2, 3210.8, 2821.6, 1618.0, 1507.1. MS calcd. for
C26H28Cl2FN4O2: 482.98. Found: 483.0 (Mþ).
d6-DMSO):
d
¼ 12.68 (s, 1H), 7.50–7.46 (m, 5H), 7.40–7.30 (m, 4H),
4.27 (q, J ¼ 7.20 Hz, 2H), 2.59 (s, 3H), 1.30 (t, J ¼ 7.2 Hz, 3H). IR
(cmꢂ1) 3380.7, 2984.9, 2361.4, 1709.1, 1681.8, 1508.3. MS calcd. for
C23H18ClFN4O3S: 484.93. Found: 485.9 (Mþ).
6.6.2. [5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-yl]-
(4-cyclopropanecarbonyl-piperazin-1-yl)-methanone (3b)
White solid, 67% yield, m.p. 179.7–180.4 ꢀC, 1H NMR (400 MHz,
d6-DMSO):
d
¼ 7.46–7.38 (m, 4H), 7.33–7.26 (m, 4H), 7.00 (s, 1H),
6.4.3. Synthesis of 2-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-4-methyl-thiazole-5-carboxylic acid (2c)
To the solution of 2b (1 mmol) in methanol:THF:water (1:2:1),
lithium hydroxide (1.5 mmol) was added and stirred at RT. After
completion of the reaction, RM was concentrated to remove vola-
tiles and then the residue was extracted with ethyl acetate
(2 ꢁ 50 ml), washed with water (20 ml), brine solution (10 ml),
dried over Na2SO4 and then evaporated to dryness. White solid, 87%
4.05 (br s, 1H), 3.93 (br s, 1H), 3.75 (br s, 3H), 3.36 (br s, 1H), 3.32 (br
s, 2H), 1.97 (br s, 1H), 1.77 (m, 1H), 1.08–1.02 (m, 4H). IR (cmꢂ1):
3018.5, 2886.9, 1608.4, 1512.6. MS calcd. for C24H22ClFN4O2: 452.91.
Found: 453.1 (Mþ).
6.7. General procedure to synthesize compounds 3c–e
To the solution of 3 (1 mmol) in THF (20 ml), isocyanate
(1.2 mmol) was added at 0 ꢀC, and stirred at RT for 30 min. After
completion of the reaction, RM was concentrated and the obtained
solid washed with diethyl ether.
yield, m.p. 299.1–299.7 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 12.8
(br, 1H), 7.50–7.46 (m, 4H), 7.40 (s, 1H), 7.37–7.30 (m, 4H), 2.57 (s,
1H). IR (cmꢂ1): 3291.0, 2927.5, 2819.3, 1635.2, 1514.4. MS calcd. for
C21H14ClFN4O3S: 456.88. Found: 457.0 (Mþ).
6.7.1. 4-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazine-1-carboxylic acid ethylamide (3c)
6.4.4. 4-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazine-1-carboxylic acid tert-butyl ester (2d)
Off-white solid, 45% yield, m.p. 83.1–84.2 ꢀC, 1H NMR (400 MHz,
White solid, 74% yield, m.p. 222.7–223.4 ꢀC, 1H NMR (400 MHz,
d6-DMSO):
d
¼ 7.47–7.38 (m, 4H), 7.34–7.27 (m, 3H), 6.99 (s, 1H),
d6-DMSO):
d
¼ 7.43–7.28 (m, 8H), 6.97 (s, 1H), 3.94 (br s, 2H), 3.65
6.54 (br s, 1H), 3.93 (br s, 2H), 3.62 (br s, 2H), 3.32 (br s, 2H), 3.05 (q,

R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
1179
J ¼ 5.2 Hz, 2H), 1.01 (t, J ¼ 7.2 Hz, 3H). IR (cmꢂ1): 3344.4, 2971.3,
6.11.1. 4-(4-Chlorophenyl)-2-[5-(4-chlorophenyl)-1-(4-
fluorophenyl)-1H-pyrazol-3-yl]-thiazole (6a)
2930.5, 1616.0, 1546.5. MS calcd. for C23H23ClFN5O2: 455.91. Found:
456.8 (Mþ).
Purified by crystallisation using ethanol. Yellow solid, 75% yield,
m.p. 186.5–187.4 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 8.01 (d,
6.7.2. 4-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazine-1-carboxylic acid (4-cyanophenyl)-amide (3d)
Pale yellow solid, 77% yield, m.p. 212.5–213.0 ꢀC, 1H NMR
J ¼ 8.12 Hz, 2H), 7.89 (s, 1H), 7.47 (d, J ¼ 8.00 Hz, 2H), 7.40 (d,
J ¼ 7.64 Hz, 4H), 7.32 (d, J ¼ 8.08 Hz, 2H), 7.24 (s, 1H), 7.20 (d,
J ¼ 8.64 Hz, 2H). IR (cmꢂ1): 3129.7, 3073.9, 1596.8, 1507.8. MS calcd.
for C23H23ClFN5O2: 466.36. Found: 466.2 (Mþ).
(400 MHz, d6-DMSO):
d
¼ 9.07 (s, 1H), 7.67–7.64 (m, 4H), 7.46–7.39
(m, 4H), 7.33–7.27 (m, 4H), 7.00 (s, 1H), 4.03 (br s, 2H), 3.71 (br s,
2H), 3.56 (br s, 4H). IR (cmꢂ1): 3314.3, 3064.6, 2215.5, 1672.9,1597.1.
MS calcd. for C28H22ClFN6O2: 528.97. Found: 527.0 (M).
6.11.2. 2-[5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-yl]-
4-methyl-thiazole-5-carboxylic acid ethyl ester (6b)
Purified by crystallisation using ethanol. White solid, 81% yield,
6.7.3. 4-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carbonyl]-piperazine-1-carboxylic acid allylamide (3e)
m.p. 138.3–140.0 ꢀC, 1H NMR (300 MHz, d6-DMSO):
d
¼ 7.35–7.30
(m, 4H), 7.27 (s, 1H), 7.23 (s, 1H), 7.18 (d, J ¼ 8.55 Hz, 2H), 7.09
(t, J ¼ 8.37 Hz, 2H), 4.37 (q, J ¼ 7.11 Hz, 2H), 2.83 (s, 1H), 1.39
(t, J ¼ 7.14 Hz, 3H). IR (cmꢂ1): 2923.8, 2853.6, 1707.0, 1506.4. MS
calcd. for C22H17ClFN3O2S: 441.91. Found: 442.2(Mþ).
White solid, 67% yield, m.p. 147.9–149.1 ꢀC, 1H NMR (400 MHz,
d6-DMSO):
d
¼ 7.41–7.26 (m, 8H), 6.98 (s, 1H), 6.75 (br s, 1H), 5.83–
5.75 (m, 1H), 5.11–4.98 (m, 2H), 3.92 (br s, 2H), 3.62 (br s, 4H), 3.38
(br s, 4H). IR (cmꢂ1): 3380.4, 3240.4, 1983.0, 1652.6, 1505.9. MS
calcd. for C25H24ClFN4O2: 466.94. Found: 468.1 (Mþ).
6.11.3. 4-{2-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazole-
3-yl]-thiazol-4-yl}-benzonitrile (6c)
6.8. General procedure to synthesize compound [5-(4-
chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-yl]-(4-prop-2ynyl-
piperazin-1-yl)-methanone (3f)
Purified by crystallisation in ethanol. Yellow solid, 76% yield,
m.p. 206.9–207.8 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 8.23 (d,
J ¼ 8.28 Hz, 2H), 8.10 (s, 1H), 7.83 (d, J ¼ 8.28 Hz, 2H), 7.44–7.40 (m,
4H), 7.32 (d, J ¼ 8.52 Hz, 2H), 7.25–7.20 (m, 3H). IR (cmꢂ1): 3116.8,
3075.0, 2233.1, 1604.5, 1523.2. MS calcd. for C25H14ClFN4S: 456.92.
Found: 457.2 (Mþ).
To the solution of 3 (1 mmol) in DMF (10 ml) and K2CO3
(1.2 mmol), propargyl bromide (1.2 mmol) were added at 0 ꢀC and
stirred at RT overnight. After completion of the reaction, RM was
filtered through celite bed and concentrated to get the compound.
Light brown liquid, 65% yield, 1H NMR (400 MHz, d6-DMSO):
6.12. Synthesis of 2-[5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazol-3-yl]-4-methyl-thiazole-5-carboxlic acid hydrazide (7)
d
¼ 7.33–7.24 (m, 4H), 7.17–7.14 (m, 2H), 7.10–7.05 (m, 2H), 6.92 (s,
1H), 4.15 (br s, 2H), 3.88 (br s, 2H), 3.37 (d, J ¼ 2.32 Hz, 2H), 2.68–
2.65 (m, 4H), 2.28 (s, 1H). IR (cmꢂ1) 3325.6, 2921.1, 1627.0. MS calcd.
for C23H20ClFN4O: 422.28. Found: 423.1 (Mþ).
The mixture of 6a (1 mmol) and hydrazine hydrate (10 ml) in
ethanol (50 ml) was refluxed overnight. RM was concentrated and
the resulting solid was filtered, washed with diethyl ether. White
solid, 69% yield, m.p. 197.0–199.0 ꢀC, 1H NMR (400 MHz, d6-DMSO):
6.9. Synthesis of 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid amide (4)
d
¼ 9.57 (br s, 1H), 7.47–7.40 (m, 4H), 7.33–7.30 (m, 4H), 7.22 (s, 1H).
IR (cmꢂ1): 3292.6, 2923.0, 1596.8, 1508.9. MS calcd. for
C20H15ClFN5OS: 427.88. Found: 427.9 (M).
To the solution of
1 (1 mmol), HOBT (1 mmol), EDC.HCl
(1.5 mmol), TEA (4 mmol) in DCM (40 ml), ammonia gas was purged
at 0 ꢀC for 10 min then stirred at RT overnight. RM was diluted with
water (20 ml), and then extracted with DCM (2 ꢁ100 ml), and the
combined organic layer was washed with water (50 ml), brine
solution (20 ml), dried over Na2SO4 and then concentrated under
reduced pressure. Crude was directly taken for the next step. 65%
yield. Off-white solid, m.p. 83.1–84.2 ꢀC, 1H NMR (300 MHz, d6-
6.13. Synthesis of 5-{2-[5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazol-3-yl]-4-methyl-thiazol-5-yl}-3H-[1,3,4]oxadiazole-2-one (8)
To the solution of 7 (1 mmol), TEA (2 mmol) in THF (25 ml), CDI
(1.2 mmol) was added at 0 ꢀC and stirred at RT overnight. RM was
diluted with water (50 ml), extracted with ethyl acetate
(2 ꢁ100 ml), the combined organic layer was washed with water
(50 ml), brine solution (20 ml), dried over Na2SO4 and then evap-
orated to dryness. Crude was recrystallized using ethanol. 62%
Yield. Off-white solid, m.p. 279.5–281.5 ꢀC, 1H NMR (400 MHz, d6-
DMSO):
d
¼ 7.72 (s, 1H), 7.45–7.37 (m, 4H), 7.33–7.24 (m, H), 7.01 (s,
1H). MS calcd. for C23H20ClFN4O: 315.72. Found: 316.9 (Mþ).
6.10. Synthesis of 5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carbothioic acid amide (5)
DMSO):
d
¼ 12.51 (br s, 1H), 7.48–7.43 (m, 4H), 7.36–7.32 (m, 4H),
7.28 (s, 1H), 2.67 (s, 3H). IR (cmꢂ1): 3131.4, 2804.7, 1806.0, 1598.7.
MS calcd. for C21H13ClFN5O2S: 453.88. Found: 453.8 (Mþ).
The mixture of 4 and Lawesson’s reagent in THF (20 ml) and
toluene (40 ml) was refluxed overnight. RM was diluted with water
(50 ml) and ethyl acetate (100 ml), washed with 10% NaHCO3 solu-
tion, and the organic layer was washed with water (50 ml), brine
solution (20 ml), dried over Na2SO4 and then evaporated to dryness.
Crude was purified by column chromatography (30% EtoAC in Pet
ether). 78% Yield, m.p. 83.1–84.2 ꢀC (after purification the compound
wasused directly forfurtherstepswithout spectralcharacterization).
6.14. General procedure to synthesize compounds 9a–c
To the solution of 7 (1 mmol) in THF (20 ml), the corresponding
isocyanate (or) isothiocyanate (1.2 mmol) was added at 0 ꢀC and
stirred at RT. After the completion of the reaction, the RM was
concentrated; the residue was recrystallized using ethanol.
6.14.1. N-(N0-{2-[5-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazol-3-yl]-4-methyl-thiazole-5-carbonyl}-
6.11. General synthesis of compounds 6a–c
hydrazinocarbothionyl)-benzamide (9a)
The mixture of 5 (1 mmol) and
ethanol (50 ml) was refluxed overnight. RM was concentrated to
get the crude.
a
-halo ketone (1 mmol) in
Purified by crystallisation using ethanol. White solid, 78% yield,
m.p. 230.0–231.6 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 12.37 (s,
1H), 11.82 (s, 1H), 11.05 (s, 1H), 7.97 (d, J ¼ 7.4 Hz, 2H), 7.65 (d,

1180
R.V. Ragavan et al. / European Journal of Medicinal Chemistry 45 (2010) 1173–1180
J ¼ 7.48 Hz, 1H), 7.55–7.51 (m, 2H), 7.48–7.43 (m, 4H), 7.35–7.31 (m,
4H), 7.28 (s, 1H). IR (cmꢂ1): 3278.1, 3056.5, 1653.1, 1507.6. MS calcd.
for C28H20ClFN6O2S2: 591.08. Found: 589.0 (Mꢂ).
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d
¼ 10.85
(s, 1H), 10.48 (s, 1H), 8.11 (m, 3H), 7.93–7.86 (m, 3H), 7.48–7.35 (m,
4H), 7.34–7.28 (m, 5H), 2.69 (s, 3H). IR (cmꢂ1): 3173.3, 2999.6,
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600.0 (M).
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allylamide (9c)
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Purified by crystallisation using ethanol. White solid, 63% yield,
m.p. 204.0–205.5 ꢀC, 1H NMR (400 MHz, d6-DMSO):
d
¼ 9.93 (s, 1H),
8.01 (s, 1H), 7.48–7.31 (m, 8H), 7.26 (s, 1H), 6.68 (br s, 1H), 5.82–5.77
(m, 1H), 5.18–5.02 (m, 2H), 3.67 (br s, 2H), 2.70 (s, 3H). IR (cmꢂ1):
3062.1, 2903.3, 1617.7, 1542.6. MS calcd. for C24H19ClFN5O2S:
495.96. Found: 496.8 (Mþ).
6.15. Synthesis of [5-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
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To the solution of 2e (1 mmol) in methanol (20 ml), sodium
borohydride (1.2 mmol) was added in portions over the period of
5 min, and then stirred at RT for 2 h. After completion of reaction,
RM was concentrated to remove methanol, the residue was dis-
solved in water (50 ml) and then extracted with ethyl acetate
(2 ꢁ100 ml), the combined organic layer was washed with water
(50 ml), brine solution (20 ml), dried over Na2SO4 and then evapo-
rated to dryness. Crude was directly taken for the next step without
purification. White solid, 84% yield, m.p. 243.3–244.8 ꢀC, 1H NMR
(400 MHz, d6-DMSO):
d
¼ 7.44 (d, J ¼ 8.44 Hz, 12H), 7.39–7.34 (m,
3H), 7.32–7.26 (m, 4H), 6.93 (s, 1H), 4.78 (d, J ¼ 4.16 Hz, 1H), 4.22 (br
s, 1H), 4.06 (br s, 1H), 3.75 (br s, 1H), 3.48 (br s, 1H), 3.21 (br s, 1H),
1.77 (br s, 2H), 1.36 (br s, 2H). IR (cmꢂ1) 3331.2, 2949.6, 1626.3,
1514.2. MS calcd. for C22H20ClFN2O2: 398.86. Found: 399.8 (Mþ).
6.16. Synthesis of methanesulfonic acid 1-[5-(4-chlorophenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carbonyl]-piperidine-4-yl ester (11)
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TEA (2 mmol) was added to the solution of 10 (1 mmol) in DCM
(10 ml), then methane sulfonyl chloride (1.2 mmol) was added at
0 ꢀC. After completion of the reaction, RM was diluted with water
(20 ml) and extracted with DCM (2 ꢁ 50 ml), the combined organic
layer was washed with water (20 ml), brine solution (10 ml), dried
over Na2SO4 and then concentrated. The crude was purified by
column (30% ethyl acetate in pet ether). Light brown gummy liquid.
Off-white, 71% yield, m.p. 132.2–135.0 ꢀC, 1H NMR (400 MHz, d6-
DMSO):
d
¼ 7.45–7.36 (m, 4H), 7.32–7.26 (m, 4H), 6.97 (s, 1H), 4.94
(br s, 1H), 4.24 (br s, 1H), 3.98 (br s, 1H), 3.75 (br s, 1H), 3.48 (br s,
1H), 3.22 (s, 3H), 2.01 (br s, 2H), 1.73 (br s, 2H). IR (cmꢂ1): 3133.2,
3058.5, 2926.0, 1642.2, 1601.5. MS calcd. for C23H22ClFN2O4S:
476.95. Found: 477.8 (Mþ).
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Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2009.12.042.