Design, synthesis and biological evaluation of a novel series of 1,3,4-oxadiazole bearing N-methyl-4-(trifluoromethyl)phenyl pyrazole moiety as cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2012.03.056

On account of the reported anticancer activity of pyrazoles and oxadiazoles, we have designed and synthesized a novel combinatorial library of S-substituted-1,3,4-oxadiazole bearing N-methyl-4-(trifluoromethyl)phenyl pyrazole moiety and tested for in-vitro cytotoxic activity by MTT assay. Amongst the tested compounds, the compound 5e was the most promising anticancer agent with IC₅₀ value of 15.54 μM in MCF-7 cells, compared to Doxorubicin as standard drug. The newly synthesized compounds were characterized by NOE, IR, ¹H NMR, ¹³C NMR and LC-MS analysis.

Full text of DOI:10.1016/j.ejmech.2012.03.056

European Journal of Medicinal Chemistry 53 (2012) 203e210
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of a novel series of 1,3,4-oxadiazole
bearing N-methyl-4-(trifluoromethyl)phenyl pyrazole moiety as cytotoxic agents
,
,
c
d
Pushpan Puthiyapurayil a b, Boja Poojarya , Chandrashekhar Chikkanna , Sunil Kumar Buridipad
*
a Department of Chemistry, Mangalore University, Mangalagangotri 574199, Karnataka, India
b Syngene International Ltd, Biocon Park, Bangalore 560099, Karnataka, India
c Department of Medicinal Chemistry, SDM College, Ujire, D.K. 574342, Karnataka, India
d Department of Pharmacy College, NET Pharmacy College, Raichur 584103, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
On account of the reported anticancer activity of pyrazoles and oxadiazoles, we have designed and
synthesized a novel combinatorial library of S-substituted-1,3,4-oxadiazole bearing N-methyl-4-(tri-
fluoromethyl)phenyl pyrazole moiety and tested for in-vitro cytotoxic activity by MTT assay. Amongst the
tested compounds, the compound 5e was the most promising anticancer agent with IC50 value of
Received 26 February 2012
Received in revised form
28 March 2012
Accepted 29 March 2012
Available online 6 April 2012
15.54
were characterized by NOE, IR, 1H NMR, 13C NMR and LCeMS analysis.
Ó 2012 Elsevier Masson SAS. All rights reserved.
mM in MCF-7 cells, compared to Doxorubicin as standard drug. The newly synthesized compounds
Keywords:
N-Methyl-4(trifluoromethyl)phenyl
Pyrazole
Cytotoxic activity
MTT assay
Doxorubicin
Nuclear overhauser effect
1. Introduction
antimicrobial [4], antiviral [5], fungicidal [6], antineoplastic [7],
anticancer [8] and inhibition of tyrosinase [9]. 5-Substituted-1,3,4-
oxadiazole-2-thiones posses CNS depresent [10] and pesticidal
property [11,12]. Moreover it is considered that the presence of
toxophoric eN]CeOe linkage is responsible for their potent bio-
logical activity. Further, 1,3,4-oxadiazole heterocycles are very good
bioisosters of amide and ester functionalities with substantial
improvement in biological activity by participating in hydrogen
bonding interactions with different receptors [13,14].
Over the years much attention was paid to pyrazole as
a potential antimicrobial agent after the discovery of the natural
pyrazole C-glycoside, pyrazofurin which demonstrated a broad
spectrum of antimicrobial activity [15], it has also been reported
that pyrazofurin has been evaluated against several cell lines.
Consequently, it has only been used clinically as an anticancer
agent. Many pyrazole derivatives have long been well known for
their biological activities such as antiviral/antitumor [16e18],
antibacterial [19], anti-inflammatory [20], analgesic [21], fungi-
static and anti-hyperglycaemic activity [22,23]. It has been well
established that fluorinated in particular CF3 substituted hetero-
cycles have got significant place in modern medicinal chemistry
[24]. Their biological studies clearly indicate that the presence of
trifluoromethyl group gives useful biological activity and are the
Currently cancer is one of the causes of death and it is likely
become the most common disease in the near future. The incidence
and mortality of cancer patients have become one of the important
issues discussed worldwide. Unfortunately, development of resis-
tance to chemotherapeutic agents is a common obstacle in the
treatment of different types of cancer [1,2]. Several important drugs
including 5-Flurouracil (5FU), Doxorubicin, Celecoxib, Mitoxan-
trone, Hydrazine carbathioamide and Indomethacin with different
structures and mechanisms of antitumor activities fail to end these
problems completely. Due to the several side effects, drug resis-
tance and failure of antitumor drugs to exert their effects in certain
cases of cancer, looking for new chemotherapeutic agents with
synthesis or natural origin is one of the hot topics in cancer
research laboratories.
1,3,4-Oxadiazoles are an important class of heterocyclic
compounds [3] with a broad range of biological activities such as
* Corresponding author. Tel.: þ91 824 2287262; fax: þ91 824 2287367.
E-mail addresses: pppushpan@rediffmail.com (P. Puthiyapurayil), bojapoojary@
gmail.com (B. Poojary).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.056

204
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
subject of considerable growing interest [25]. The increased
activity is attributed to the presence of fluorine atoms (highly
electronegative) in the molecules which increases the lip-
ophilicity and affects the partitioning of molecules into
membranes and facilitates hydrophobic interactions of the
molecules with specific binding sites on either receptor or
enzymes. In this paper we therefore are interested in exploring
the biological activities of such molecules through structural
modifications. However to our knowledge very few reports have
been reported in the literature to the synthesis of 1,3,4-oxadiazole
containing N-methyl pyrazole moiety. In the course of our studies
directed towards the synthesis of 1,3,4-oxadiazole bearing N-
methyl-4-(trifluoromethyl)phenyl pyrazole moiety, to our
surprise, when compound 1 was cyclized using methyl hydrazine
sulphate we were ended up with two isomers (2 & 6), wherein
the spectral and homo nuclear NOE experiment together
confirmed the structures of compound 2 & 6. The compounds (2 &
6) were hydrazinolyzed using hydrazine hydrate in methanol at
100 ꢀC to get aryl hydrazides (3 & 7). 1H NMR spectrum displayed
two signals at
d 9.41, 4.41 ppm which correlated with eNHe and
eNH2 protons respectively. The compounds (3 & 7) on reaction
with carbon disulphide in methanolic potassium hydroxide under
reflux condition yielded corresponding 5-substituted-1,3,4-
oxadiazole-2-(3H)-thione (4 & 8). These compounds exist in
keto-enol form as shown in Scheme 3. The method for structural
determination was similar; first, its molecular formula was
determined to be C13H9F3N4OS, based on elemental analysis and
detection of its molecular ion peak in mass spectrum (m/z 327,
Mþ). On its 1H NMR spectrum, the signal at
d 14.77 ppm, was
corresponding to the eNH proton and 13C NMR showing a signal at
176.79 ppm corresponding to C]S. The compounds (4 & 8) then
treated with different substituted aryl halides using potassium
carbonate in dimethyl formamide at 80 ꢀC to afford the final target
compounds (5aeh & 9aef). The formation of S-alkylated products
were confirmed by the absence of C]S characteristic peak at
176.79 ppm in 13C NMR spectrum. Further the structures of final
target compounds were confirmed by IR, 1H NMR and LCeMS
analysis. The chemical profiles of the compounds are as shown
in Table 1.
compound
2 was the major isomer (74% by LCeMS) and
compound 6 was the minor isomer (24% by LCeMS). Both the
isomers were isolated by flash column chromatography and
characterized by homo nuclear NOE experiment.
On the basis of these observations, we thought of designing and
synthesizing a new class of heterocycles, wherein potent 1,3,4-
oxadiazole moiety is linked to biologically active N-methyl-4-(tri-
fluoromethyl)phenyl pyrazole moiety at C-5 position and different
S-substituted aryl groups at C-2 position on the basis of combina-
torial synthesis, which is the current trend being practiced in most
of the drug discoveries.
3. Pharmacology
2. Results and discussion
3.1. In-vitro cytotoxic evaluation and time course MTT assay
2.1. Chemistry
In-vitro cytotoxicity of the synthesized compounds was
assessed by standard MTT bioassay in different cancer cells at
24 h of drug administration. In EAC cells, the compounds carrying
2-substituted phenyl-5-{5-[(4-trifluoromethyl)phenyl]-1-methyl-
1H-pyrazol-3-yl}-1,3,4-oxadiazole (5aeh) were found to be more
effective when compared with the compounds carrying 2-
substituted phenyl-5-{3-[(4-trifluoromethyl)phenyl]-1-methyl-
1H-pyrazol-5-yl}-1,3,4-oxadiazole (9aef). To assess the efficacy
and to select the promising compounds human cancer cells,
breast cancer cell line (MCF-7) and alveolar adenocarcinoma cell
line (A549) were used. Human breast cancer lines MCF-7 and
alveolar adenocarcinoma cell lines (A549), procured from
National centre for cell sciences (NCCS), Pune, India, were
The synthetic methods for target compounds 5 and 9 were
illustrated in Scheme 1. For the preparation of intermediates 2 & 6,
the starting compound 4-trifluoromethyl acetophenone was
treated with sodium methoxide and dimethyl oxalate in methanol
at RT to yield compound (1) by the reported procedure [26], which
upon cyclization using N-methyl hydrazine sulphate in glacial
acetic acid at 105 ꢀC yielded the corresponding N-methyl-4-(tri-
fluoromethyl)phenyl pyrazole carboxylic acid methyl esters (2 &
6). LCeMS analysis of the crude sample shows 74% major isomer at
RT ¼ 3.11 min and 25% of minor isomer at RT ¼ 3.49 min. The
derivatives 2 and 6 were characterized by spectral studies using IR,
1H NMR and NOE. For the major isomer (2) IR absorption peak at
1712 cmꢁ1 corresponding to ester keto group, 1H NMR showing
cultured in MEM medium supplemented with 10% FBS, 1%
L
glutamine and 50 g/mL gentamicin sulphate in a CO2 incubator
m
two distinct doublets at
to aromatic protons of the phenyl ring,
to pyrazole ring proton and two singlet’s at
corresponding to O-methyl and N-methyl protons respectively.
Similarly for the minor isomer (6), IR absorption peak at 1724 cmꢁ1
corresponding to ester keto group. 1H NMR showing two doublets
d
7.76 ppm and
d
7.57 ppm corresponding
6.91 ppm corresponding
in a humidified atmosphere of 5% CO2 and 95% air. The EAC cells
were maintained for 12e14 days in the peritoneal cavity of Swiss
albino mice. The tumour cell cultures were started from mouse
Ehrlich Ascites with at least one passage in vitro prior to use.
In-vitro cytotoxicity was determined using a standard MTT
assay [27] with protocol appropriate for the individual test
system. In brief, exponentially growing cells were plated in 96-
d
d
3.97, 3.95 ppm were
at
phenyl ring, a singlet at
proton and two singlet’s at
d
7.91 and 7.67 ppm corresponds to aromatic protons of the
7.17 ppm corresponding to pyrazole ring
4.24, 3.92 ppm corresponding to N-
d
well plates (104 cells/well in 100
mL of medium) and incubated
d
for 24 h for attachment. Test compounds were prepared prior to
the experiment by dissolving in 0.1% DMSO and diluted with
medium. The cells were then exposed to different concentrations
methyl and O-methyl protons respectively. Furthermore two
isomers were characterized by NOE experiment (Scheme 2). Upon
irradiation of the aromatic proton signals at
d
7.57 ppm of (2), there
6.91
of the drugs (1e100 mM) in the volume of 100 mL/well. Cells in the
control wells received the same volume of medium containing
0.1% DMSO. After 24 h, the medium was removed and cell
were enhancements in the signal at 7.75 ppm (lower field),
d
d
and 3.96 ppm. Upon irradiation of N-methyl proton at
there was enhancement in the aromatic-Hb signal at
d
3.96 ppm,
7.56 ppm.
d
cultures were incubated with 100
4 h at 37 ꢀC, The formazan produced by the viable cells was
solubilised by addition of 100 L DMSO. The suspension was
mL MTT reagent (1 mg/mL) for
Whereas the lower filed aromatic-Ha signal remained nearly
unaffected. Thus the signal at 7.56 ppm has to be attributed to the
d
m
aromatic-Hb due to the special closeness of Hb to the N-methyl
protons. Similarly for the other isomer (6), upon irradiation of the
placed on micro-vibrator for 5 min and absorbance was recorded
at 540 nm by the ELISA reader. The experiment was performed in
triplicate. The percentage cytotoxicity was calculated using the
formula.
aromatic-Hb signal at
d
7.67 ppm, there was no enhancement in
4.24 ppm and vice versa. Overall
the signal of N-methyl proton at
d

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
205
O
F
F
F
a
O-Na+
O
O
F
O
1
F
F
b
O
N
O
O
O
N
N
N
c
O
F
F
6
2
F
F
F
F
c
NH2
NH
O
NH
NH2
N
N
N
N
F
F
7
3
F
d
F
F
d
F
F
F
SH
N
SH
N
N
N
N
O
O
N
N
N
F
8
4
F
F
F
F
e
e
S
S
N
N
R
R
N
N
N
N
O
O
N
N
F
F
9
5
F
F
F
Scheme 1. Reagents and conditions: (a) Dimethyl oxalate, NaOMe, MeOH, RT; (b) Methyl hydrazine hydrate, Gla. AcOH, 105 ꢀC; (c) N2H4.H2O, MeOH, 100 ꢀC; (d) CS2, KOH, MeOH,
Reflux; (e) ReX, K2CO3, DMF, 80 ꢀC.
3.2. Biological results
% Cytotoxicity ¼ ðControl abs ꢁ Blank absÞ
ꢁ ðTest abs ꢁ Blank absÞ=
ꢂ ðControl abs ꢁ Blank absÞ ꢂ 100
In MCF-7 cells, IC50 values for the compounds carrying 2-
substituted
phenyl-5-{5-[(4-trifluoromethyl)phenyl]-1-methyl-
1H-pyrazol-3-yl}-1,3,4-oxadiazoles (5aeh) and 2-substituted
phenyl-5-{3-[(4-trifluoromethyl)phenyl]-1-methyl-1H-pyrazol-5-
yl}-1,3,4-oxadiazoles (9aef) were in the range of 15.54e32.24 and
For MTT time course study, MCF-7 cells (5 ꢂ 103 cells/well
seeded in 96-well plates were exposed to different concentrations
of test compounds (1e50 mM). The percentage cytotoxicity and IC50
values were determined at 24, 48 and 72 h of drug incubation. The
results were tabulated in Tables 2and 3.
27.10e60.22
IC50 values for (5aeh and 9aef) were found to be in the range of
29.21e54.28 and 39.80e84.22 M respectively. In MCF-7 cells, the
mM respectively (Table 2). Similarly in A549 cells, the
m

206
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
Table 2
O
N
O
NOE
NOE
In-vitro cytotoxic activity of compounds (4, 5aeh, 8, 9aef).
O
NOE
NOE
O
Hc
Hb
Hc
Hb
Compounds
IC50
EAC
(m
M)a
N
MCF-7
A549
NTb
N
Ha
Ha
F
NOE
N
4
5a
5b
5c
5d
5e
5f
5g
5h
8
9a
9b
9c
9d
9e
9f
68.25 ꢃ 0.022
28.18 ꢃ 0.002
25.18 ꢃ 0.011
36.84 ꢃ 0.0039
46.24 ꢃ 0.012
20.54 ꢃ 0.009
29.22 ꢃ 0.03
>100
26.22 ꢃ 0.011
75.34 ꢃ 0.002
38.50 ꢃ 0.011
35.10 ꢃ 0.022
56.52 ꢃ 0.0022
27.25 ꢃ 0.005
39.54 ꢃ 0.0022
>100
>100
F
26.18 ꢃ 0.023
23.12 ꢃ 0.0039
32.24 ꢃ 0.011
27.22 ꢃ 0.044
15.54 ꢃ 0.012
25.22 ꢃ 0.022
>100
38.01 ꢃ 0.012
48.12 ꢃ 0.005
54.28 ꢃ 0.017
44.18 ꢃ 0.012
29.21 ꢃ 0.022
39.02 ꢃ 0.009
NTb
Hb
Hb
F
F
F
NOE
F
Ha
Ha
6
2
NOE
NOE
Scheme 2. NOE of two isomers of N-methylpyrazoles.
18.22 ꢃ 0.022
48.22 ꢃ 0.009
>100
NTb
46.20 ꢃ 0.023
33.30 ꢃ 0.0039
60.22 ꢃ 0.011
27.10 ꢃ 0.022
45.40 ꢃ 0.011
>100
72.22 ꢃ 0.011
68.12 ꢃ 0.005
84.20 ꢃ 0.011
39.80 ꢃ 0.0039
84.22 ꢃ 0.005
NTb
H
SH
N
N
S
N
N
N
N
N
O
O
Doxorubicinc
5.22 ꢃ 0.03
2.82 ꢃ 0.0176
3.28 ꢃ 0.18
a
Each experiment was independently performed three times and expressed as
N
mean ꢃ SD.
b
c
F
NT: not tested.
Doxorubicin was used as reference drug.
F
F
4'
4
F
F
F
Scheme 3. Keto-enol form of 1,3,4-oxadiazole.
15.54
mM. We have also observed that, compounds from the
first series (5aeh) were more potent than the second series
(9aef). The enhanced activity of compounds 5a, 5b, 5d, 5e, 5f,
5h and 9d was mainly attributed to the presence of biologically
active groups like 2,6-difluorobenzylthio, 2-(2-fluorophenoxy)
ethylthio, 3,4-dichlorobenzylthio, 4-(trifluoromethyl)benzylthio, 4-
fluorophenethylthio and 4-chloro-2-fluorobenzylthio groups at C-2
position of the 1,3,4-oxadiazole ring. Except compound 5d, all the
active molecules (5a, 5b, 5e, 5f, 5h and 9d) have fluoro substitution
on the phenyl ring at C-2 position. These results further revealed
that, the enhanced cytotoxic activity was mainly attributed to the
presence of biologically active N-methyl-4-(trifluoromethyl)phenyl
pyrazole moiety at C-5 position and fluoro substituted phenyl
groups at C-2 position of the 1,3,4-oxadiazole ring system.
In the MTT time course study, the selected compounds (5a, 5b,
5e, 5f and 5h) showed dose dependent and time dependent
activities. Experiments revealed that (Table 3), there was substan-
tial increase in cytotoxicity in MCF-7 cells with increasing exposure
to drug concentration. The IC50 values of the promising compounds
at 48 h and 72 h were significantly reduced in comparison with 24 h
values. The most promising compound (5e) causes 1.75 and 2.96
times reduction in IC50 values at 48 h and 72 h.
compounds 4, 5g, 8 and 9f did not show 50% inhibition even at
a concentration of 100 M, hence they were not evaluated in A549
m
cells. Present study revealed that among the human cancer cell
lines tested, MCF-7 cells are more sensitive to all the tested
compounds than A549 cells. Many anticancer drugs are effective
against MCF-7 and A549 cells, by causing apoptosis through the
expression of caspase-3, generating reactive oxygen species (ROS)
and damaging DNA [28]. Chemotherapeutic agents such as doxo-
rubicin and mitoxantrone cause cytotoxicity by generating ROS
[29]. Hence like the cytotoxic drugs, the synthesized compounds
may act as effective anticancer drug by similar mechanism. The
results of the in-vitro cytotoxicity revealed that, the compounds 4
and 8 having a free mercapto group showed poor cytotoxic activity,
so various aryl halides were considered for the synthesis of the title
compounds to draw the structure activity relationship (SAR)
studies in relation to cytotoxic activity. Results from the Table 2
revealed that, compounds 5a, 5b, 5d, 5f, 5h and 9d showed
moderate to good cytotoxic activity in MCF-7 cells. Compound 5e
was the most promising cytotoxic agent with IC50 value of
Table 1
Characterization data of 2,5-disubstituted-1,3,4-oxadiazoles (5aeh, 9aef).
Compounds
R
Mol. formula
Mol. weight
m.p. (ꢀC)
Yield (%)
5a
5b
5c
5d
5e
5f
5g
5h
9a
9b
9c
9d
9e
9f
2,6-F2eC6H3eCH2e
2-FeC6H4eOeCH2eCH2e
2,3-Cl2eC6H3eCH2e
3,4-Cl2eC6H3eCH2e
4eCF2eC6H4eCH2e
4-FeC6H4eCH2eCH2e
3-NO2eC6H4eCH2e
4-Cle2-FeC6H3eCH2e
2,6-F2eC6H3eCH2e
2-FeC6H4eOeCH2eCH2e
2,3-Cl2eC6H3eCH2e
4-CF2eC6H4eCH2e
C20H13F5N4OS
C21H16F4N4O2S
C20H13Cl2F3N4OS
C20H13Cl2F3N4OS
C21H14F6N4OS
C21H16F4N4OS
C20H14F3N5O3S
C20H13ClF4N4OS
C20H13F5N4OS
C21H16F4N4O2S
C20H13Cl2F3N4OS
C21H14F6N4OS
C21H16F4N4OS
C20H14F3N5O3S
452.42
464.43
485.31
485.31
484.42
448.44
461.42
468.85
452.42
464.43
485.31
484.42
448.44
461.42
141.3e142.8
100.2e101.6
151.4e152.8
143.2e144.5
151.2e152.5
119.2e120.1
113.3e114.7
114.1e115.3
121.2e122.4
156.2e157.6
170.2e171.7
150.2e151.6
170.2e171.3
136.2e137.3
94
92
83
78
91
87
89
79
73
87
88
79
84
94
4-FeC6H4eCH2eCH2e
3-NO2eC6H4eCH2e

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
207
Table 3
5.1.2. Synthesis of 1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-
pyrazole-3-carboxylate acid methyl ester (2) and 1-methyl-3-[4-
(trifluoromethyl)phenyl]-1H-pyrazole-5-carboxylate acid methyl
ester (6)
In-vitro cytotoxic activity of compounds (5a, 5b, 5e, 5f and 5h) in MCF-7 cells at
different time points of drug exposure by MTT assay.
Compounds
IC50 (m
M)a
To a mixture of compound (1) (11.3 g, 38.1 mmol) and methyl
hydrazine sulphate (8.25 g, 57.2 mmol) glacial acetic acid (152 mL)
was added under N2 atmosphere and heated at 105 ꢀC for 1 h. The
completion of the reaction was confirmed by TLC. Upon completion
of the reaction, the solvent was removed in vacuum. Residue
obtained was diluted with water (100 mL) and extracted in ethyl
acetate (3 ꢂ 150 mL). The combined extract was washed with 10%
sodium bicarbonate solution, water followed by brine and dried
over anhydrous sodium sulphate. The solvent removed under
reduced pressure and the crude product was purified by flash
column chromatography (230e400 size mesh) using 18e19% ethyl
acetate in pet ether to afford the minor isomer (compound 6) and
28e29% ethyl acetate in pet ether to afford the major isomer
(compound 2).
24 h
48 h
72 h
5a
5b
5e
5f
26.18 ꢃ 0.023
23.12 ꢃ 0.0039
15.54 ꢃ 0.012
25.22 ꢃ 0.022
18.22 ꢃ 0.022
20.52 ꢃ 0.011
19.18 ꢃ 0.009
8.86 ꢃ 0.0012
21.12 ꢃ 0.012
12.20 ꢃ 0.012
16.22 ꢃ 0.0022
14.25 ꢃ 0.003
5.25 ꢃ 0.0011
18.10 ꢃ 0.005
9.80 ꢃ 0.0069
5h
a
Each experiment was independently performed three times and expressed as
mean ꢃ SD.
4. Conclusion
In conclusion, this work demonstrates the synthesis of a novel
combinatorial library of
oxadiazoles and their biological evaluation of cytotoxic activity. In
case of cytotoxic study by MTT assay, the results revealed that, the
compound 5e was the most promising cytotoxic agent with IC50
a series of 2,5-disubstituted-1,3,4-
5.1.2.1. 1-Methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carb-
oxylate acid methyl ester (2). Yield (6.4 g, 63%), off white solid,
m.p. ¼ 108.5e109.9 ꢀC. Found: C, 55.23; H, 4.15; N, 10.05. Calcd. for
value of 15.54 mM in MCF-7 cells. Upon exposure to drug concen-
tration, compound 5e even causes 1.75 and 2.96 times reduction in
IC50 values at 48 h and 72 h respectively. These results are therefore
conclusive in showing that, 1,3,4-oxadiazole bearing N-methyl-4-
(trifluoromethyl)phenyl pyrazole moiety make them certain lead
molecules for further optimization in the development of novel
anticancer agent.
C13H11F3N2O2: C, 54.93; H, 3.90; N, 9.86. IR (KBr) n
/cmꢁ1: 3132,
3081 (AreH), 3023, 2954 (CeH), 1712 (C]O), 1618 (C]N), 1474
(C]C). 1H NMR (400 MHz, CDCl3):
d
7.76 (d, J ¼ 8.1 Hz, 2H), 7.57 (d,
J ¼ 8.0 Hz, 2H), 6.91 (s, 1H), 3.97 (s, 3H), 3.95 (s, 3H). MS (MM-
ES þ APCI) 285.2 (M þ H)þ.
5.1.2.2. 1-Methyl-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-carb-
oxylate acid methyl ester (6). Yield (1.6 g, 14%), off white solid,
m.p. ¼ 73.3e78.3 ꢀC. Found: C, 55.30; H, 4.18; N, 10.10. Calcd. for
5. Experimental protocols
5.1. General
C13H11F3N2O2: C, 54.93; H, 3.90; N, 9.86. IR (KBr) n
/cmꢁ1: 3128
(AreH), 3015, 2961 (CeH),1724 (C]O), 1618 (C]N),1513 (C]C). 1H
NMR (400 MHz, CDCl3):
1H NMR and 13C NMR spectra were recorded on 400 MHz and
100 MHz Bruker spectrometer, respectively, and elemental analysis
was performed on a Thermo Finnigan FLASH EA 1112 CHN analyzer.
Melting points were recorded (uncorrected) on Buchi melting B-
545 instrument. Infrared spectra were recorded on a Thermo
scientific Nicolet 6700 FT-IR spectrometer. Coupling constants were
reported whenever it was necessary in hertz (Hz). The mass spectra
were recorded on Agilent LC/MSD SL 1100 instrument. Reactions
were carried out in an oven dried three necked round bottomed
flask. Yields in table refer to isolated yields of compounds with
purity >94e99% and single impurity <3% as determined by LCeMS
analysis. All the reagents and solvents used were of analytical
grade, and were used as supplied, unless otherwise stated. TLC was
performed on aluminium plates coated with silica gel (Merck) for
monitoring the reactions.
d
7.91 (d, J ¼ 8.0 Hz, 2H), 7.67 (d, J ¼ 8.2 Hz,
2H), 7.17 (s, 1H), 4.24 (s, 3H), 3.92 (s, 3H). MS (MM-ES þ APCI) 285.2
(M þ H)þ.
5.1.3. General procedure for the synthesis of 1-methyl-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazole-3-carbohydrazide (3) and 1-
methyl-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-
carbohydrazide (7)
To a stirred solution of compound (2 or 6) (1 mmol) in methanol
(2 mL) hydrazine hydrate (2 mmol) was added at RT under N2
atmosphere. The reaction mass was allowed to heat at 100 ꢀC for
2 h. After the completion of the reaction as monitored by TLC,
solvent was removed in vacuum. The residue obtained was diluted
with ethyl acetate, washed with water, brine and dried over
anhydrous sodium sulphate. The solvent removed under reduced
pressure and the crude product was recrystallised from methanol
to afford the pure product (compounds 3 & 7).
5.1.1. Synthesis of sodium; (Z)-3-methoxy carbonyl-3-oxo-1-(4-
trifluoromethyl)phenyl propen-1-olate (1)
To a stirred solution of 4-trifluoromethyl acetophenone (15 g,
79.7 mmol) in anhydrous methanol (15 mL), cooled at 0 ꢀC was
added a solution of (0.5 M) sodium methoxide (8.6 g, 435 mL,
159 mmol). Reaction mass allowed to stir at RT for 1 h. A solution of
dimethyl oxalate (18.8 g, 159 mmol) in anhydrous methanol
(20 mL) was added drop wise and allowed to stir at RT for 24 h. The
progress of the reaction was monitored by TLC. Upon completion of
the reaction, the precipitate obtained was isolated and washed with
cooled methanol followed by diethyl ether and dried in vacuum to
obtain the pure product as sodium salt.
5.1.3.1. 1-Methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carb-
ohydrazide (3). Yield (90%), off white solid, m.p. ¼ 142.1e144.8 ꢀC.
Found: C, 51.03; H, 4.15; N, 19.91. Calcd. for C12H11F3N4O: C, 50.71;
H, 3.90; N, 19.71. IR (KBr)
(eNH2), 3059 (AreH), 2935 (CeH), 1598 (C]O), 1537 (C]C). 1H
NMR (400 MHz, DMSO-d6):
n
/cmꢁ1: 3315 (eNHe), 3265, 3204
d
9.44 (s, 1H), 7.88 (d, J ¼ 8.3 Hz, 2H),
7.82 (d, J ¼ 8.2 Hz, 2H), 6.90 (s, 1H), 4.4 (s, 2H), 3.92 (s, 3H). MS
(MM-ES þ APCI) 285.2 (M þ H)þ
5.1.3.2. 1-Methyl-3-[4-(trifluoromethyl)phenyl]-1H-pyrazole-5-carb-
ohydrazide (7). Yield (91%), off white solid, m.p. ¼ 165.2e167.1 ꢀC.
Found: C, 51.06; H, 4.16; N, 19.95. Calcd. for C12H11F3N4O: C, 50.71;
Yellow colour semi solid. Yield: 11.3 g (48%). 1H NMR (400 MHz,
DMSO-d6):
d
7.94 (d, J ¼ 8.0 Hz, 2H), 7.88 (d, J ¼ 8.0 Hz,1.3H), 7.74 (d,
J ¼ 8.2 Hz, 2H), 7.66 (d, J ¼ 8.1 Hz, 1.3H), 6.26 (s, 1H), 5.30 (s, 0.7H),
H, 3.90; N,19.71. IR (KBr)
n
/cmꢁ1: 3277 (eNHe), 3025 (AreH), 2995,
3.65 (s, 3H), 3.57 (s, 2H). MS (MM-ES þ APCI) 273.0 (M ꢁ H)þ.
2921 (CeH), 1673 (C]O), 1617 (C]N), 1521 (C]C). 1H NMR

208
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
(400 MHz, DMSO-d6):
d
9.88 (s, 1H), 7.96 (d, J ¼ 8.1 Hz, 2H), 7.80 (d,
113.0, 112.4, 112.1, 107.4, 39.0, 24.8. MS (MM-ES þ APCI) 453.0
J ¼ 8.2 Hz, 2H), 7.31 (s, 1H), 4.57 (s, 2H), 4.13 (s, 3H). MS (MM-
(M þ H)þ.
ES þ APCI) 285.2 (M þ H)þ.
5.1.4.2. 2-[2-(2-Fluorophenoxy)ethylthio]-5-{5-[4-(trifluoromethyl)
phenyl]-1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5b). Off
white solid, found: C, 54.19; H, 3.32; N, 11.94. Calcd. for
C21H16F4N4O2S: C, 54.31; H, 3.47; N, 12.06. IR (KBr)
/cmꢁ1: 3126,
3071 (AreH), 2952 (CeH), 1611 (C]N), 1482 (C]C), 1064 (CeF).
1H NMR (400 MHz, DMSO-d6):
5.1.3.3. General procedure for the synthesis of 5-{-5-[4-(tri-
fluoromethyl)phenyl]-1-methyl-1H-pyrazole-3-yl}-1,3,4-oxadiazole-
2-thione (4) and 5-{3-[4-(trifluoromethyl)-phenyl]-1-methyl-1H-
pyrazole-5-yl}-1,3,4-oxadiazole-2-thione (8). To a stirred solution
of compound (3 or 7) (1 mmol) in anhydrous methanol (5 mL),
potassium hydroxide (1.5 mmol) was added followed by carbon
disulphide (1.5 mmol) at 0 ꢀC under N2 atmosphere. Reaction mass
heated at 65 ꢀC for overnight period. Starting material consump-
tion was monitored by TLC, upon completion of the reaction,
solvent was removed in vacuum. Residue obtained was diluted
with water and extracted in ethyl acetate. Organic phase was
washed with water, brine solution and dried over anhydrous
sodium sulphate. The solvent removed under reduced pressure
and the crude product was purified by flash column chromatog-
raphy (60e120 size mesh) using 35e36% ethyl acetate in pet ether
to afford the pure product (compound 4 & 8).
n
d
7.92 (d, J ¼ 8.9 Hz, 2H), 7.89 (d,
J ¼ 8.9 Hz, 2H), 7.25e7.18 (m, 2H), 7.16 (s, 1H), 7.13e7.09 (m, 1H),
6.97e6.93 (m, 1H), 4.44 (t, J ¼ 6.0 Hz, 2H), 4.00 (s, 3H), 3.74 (t,
J ¼ 6.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6):
d 163.0, 160.7,
152.8, 150.4, 145.8, 143.5, 135.2, 132.8, 129.5, 125.8, 124.8, 121.6,
116.0, 106.9, 67.0, 38.9, 31.4. MS (MM-ES
þ
APCI) 465.2
(M þ H)þ.
5.1.4.3. 2-(2,3-Dichlorobenzylthio)-5-{5-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5c). Off white solid,
found: C, 49.85; H, 3.03; N, 11.90. Calcd. for C20H13Cl2F3N4OS: C,
49.50; H, 2.70; N, 11.54. IR (KBr)
(CeH), 1612 (C]N), 1471 (C]C), 481 (CeCl). 1H NMR (400 MHz,
CDCl3):
7.79 (d, J ¼ 8.1 Hz, 2H), 7.60e7.57 (m, 3H), 7.44 (dd, J ¼ 8.0,
1.4 Hz, 1H), 7.19 (t, J ¼ 7.8 Hz, 1H), 6.97 (s, 1H), 4.60 (s, 2H), 4.00 (s,
3H). 13C NMR (100 MHz, CDCl3):
163.6, 161.1, 144.0, 136.2, 135.9,
n
/cmꢁ1: 3108, 3010 (AreH), 2959
5.1.3.4. 5-{-5-[4-(Trifluoromethyl)phenyl]-1-methyl-1H-pyrazole-3-
yl}-1,3,4-oxadiazole-2-thione (4). Yield (91%), off white solid,
m.p. ¼ 226.4e227.8 ꢀC. Found: C, 48.05; H, 3.00; N, 17.32. Calcd. for
d
d
C13H9F3N4OS: C, 47.85; H, 2.78; N, 17.17. IR (KBr)
n
/cmꢁ1: 3113
133.4,132.6,131.4,131.0,130.2,129.6,129.1,127.3,125.9,121.8,106.8,
(AreH), 2947 (CeH), 2605 (eSH), 1622 (C]N), 1440 (C]C), 1318
(eNeN]C). 1H NMR (400 MHz, DMSO-d6):
14.77 (br, s, 1H), 7.91
(d, J ¼ 8.6 Hz, 2H), 7.87 (d, J ¼ 8.6 Hz, 2H), 7.14 (s, 1H), 3.99 (s, 3H).
38.2, 35.1. MS (MM-ES þ APCI) 486.3 (M þ H)þ.
d
5.1.4.4. 2-(3,4-Dichlorobenzylthio)-5-{5-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5d). Off white solid,
found: C, 49.73; H, 2.93; N, 11.80. Calcd. for C20H13Cl2F3N4OS: C,
13C NMR (100 MHz, DMSO-d6):
d 176.7, 153.2, 148.2, 135.6, 128.4,
127.7, 125.7, 122.8, 106.6, 39.0. MS (MM-ES þ APCI) 325.0 (M þ H)þ
49.50; H, 2.70; N, 11.54. IR (KBr)
1612 (C]N), 1464 (C]C), 478 (CeCl). 1H NMR (400 MHz, CDCl3):
7.79 (d, J ¼ 8.2 Hz, 2H), 7.60 (d, J ¼ 7.5 Hz, 2H), 7.58 (s, 1H), 7.41 (d,
J ¼ 8.2 Hz, 1H), 7.34 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.97 (s, 1H), 4.46 (s, 2H),
4.00 (s, 3H). 13C NMR (100 MHz, CDCl3):
163.1, 161.1, 144.0, 136.2,
n
/cmꢁ1: 3073 (AreH), 2945 (CeH),
5.1.3.5. 5-{3-[4-(Trifluoromethyl)-phenyl]-1-methyl-1H-pyrazole-5-
yl}-1,3,4-oxadiazole-2-thione (8). Yield (80%), off white solid,
m.p. ¼ 260.1e261.8 ꢀC. Found: C, 48.07; H, 2.90; N, 17.31. Calcd. for
d
C13H9F3N4OS: C, 47.85; H, 2.78; N, 17.17. IR (KBr)
n
/cmꢁ1: 3123
d
(AreH), 2939 (CeH), 2756 (eSH), 1617 (C]N), 1478 (C]C), 1322
135.9, 132.6, 132.2, 131.5, 131.0, 129.1, 128.4, 125.9, 125.4, 121.8,
(eNeN]C). 1H NMR (400 MHz, DMSO-d6):
d
15.03 (br, s, 1H), 8.13
106.8, 38.2, 35.3. MS (MM-ES þ APCI) 486.3 (M þ H)þ.
(d, J ¼ 8.1 Hz, 2H), 7.81 (d, J ¼ 8.3 Hz, 2H), 7.65 (s, 1H), 4.16 (s, 3H).
MS (MM-ES þ APCI) 325.0 (M þ H)þ.
5.1.4.5. 2-[4-(Trifluoromethyl)benzylthio]-5-{5-[4-(trifluoromethyl)
phenyl]-1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole
white solid, found: C, 51.87; H, 2.76; N, 11.41. Calcd. for
C21H14F6N4OS: C, 52.07; H, 2.91; N, 11.57. IR (KBr)
/cmꢁ1: 3126,
3064 (AreH), 2951 (CeH), 1610 (C]N), 1479 (C]C), 1064 (CeF).
1H NMR (400 MHz, DMSO-d6):
7.92 (d, J ¼ 8.7 Hz, 2H), 7.89 (d,
J ¼ 8.7 Hz, 2H), 7.72 (s, 4H), 7.18 (s, 1H), 4.66 (s, 2H), 4.01 (s, 3H).
13C NMR (100 MHz, CDCl3):
163.2, 161.1, 144.0, 139.7, 136.2,
(5e). Off
5.1.4. General procedure for the synthesis of 2,5-disubstituted-1,3,4-
oxadiazoles (5aeh, 9aef)
n
To a stirred solution of 5-substituted-1,3,4-oxadiazole-2-thione
(0.91 mmol) in anhydrous dimethyl formamide (9 mL), anhydrous
potassium carbonate (1.37 mmol) was added at RT and allowed to
stir for 10 min. Substituted aryl halides (0.91 mmol) was added to
the above and allowed to heat at 80 ꢀC for 1 h. The progress of the
reaction was monitored by TLC. After the completion of the reac-
tion, the solvent was removed in vacuum. Residue obtained was
diluted with water, extracted in ethyl acetate (2 ꢂ 50 mL).
Combined organic extract was washed with water, brine solution
and dried over anhydrous sodium sulphate. Solvent removed under
reduced pressure and the crude product was recrystallised from
ethyl acetate/pet ether to afford the pure title compounds
(compounds 5aeh & 9aef).
d
d
132.6, 131.5, 131.1, 130.4, 129.9, 129.4, 129.0, 125.9, 125.6, 122.0,
121.8, 106.8, 38.2, 35.9. MS (MM-ES þ APCI) 485.0 (M þ H)þ.
5.1.4.6. 2-(4-Fluorophenethylthio)-5-{5-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5f). Pale brown solid,
found: C, 56.47; H, 3.85; N,12.69. Calcd. for C21H16F4N4OS: C, 56.25;
H, 3.60; N, 12.49. IR (KBr)
1610 (C]N), 1512 (C]C), 1069 (CeF). 1H NMR (400 MHz, CDCl3):
7.80 (d, J ¼ 8.0 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 2H), 7.27e7.22 (m, 2H),
n
/cmꢁ1: 3128, 3080 (AreH), 2947 (CeH),
d
7.03 (d, J ¼ 8.5 Hz, 2H), 6.99 (s, 1H), 4.01 (s, 3H), 3.54 (t, J ¼ 7.2 Hz,
5.1.4.1. 2-(2,6-Difluorobenzylthio)-5-{5-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5a). Off white solid,
found: C, 53.32; H, 3.05; N,12.63. Calcd. for C20H13F5N4OS: C, 53.10;
2H), 3.16 (t, J ¼ 7.7 Hz, 2H). 13C NMR (100 MHz, CDCl3):
d 163.3,
160.1, 144.0, 136.3, 134.7, 132.7, 131.5, 131.0, 130.2, 129.1, 125.9,
121.8, 115.5, 115.2, 106.8, 38.2, 34.6, 33.7. MS (MM-ES þ APCI) 449.0
(M þ H)þ.
H, 2.90; N, 12.38. IR (KBr)
1621 (C]C), 1603 (C]N), 1468 (C]C), 1069 (CeF). 1H NMR
(400 MHz, DMSO-d6):
7.92 (d, J ¼ 9.0 Hz, 2H), 7.89 (d, J ¼ 9.1 Hz,
2H), 7.47e7.40 (m, 1H), 7.17 (s, 1H), 7.12e7.10 (m, 2H), 4.54 (s, 2H),
4.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
162.2, 162.1, 161.8,
159.8, 144.0, 135.4, 133.2, 131.3, 130.0, 129.6, 126.2, 125.8, 123.1,
n
/cmꢁ1: 3070 (AreH), 2954, 2926 (CeH),
d
5.1.4.7. 2-(3-Nitrobenzylthio)-5-{5-[4-(trifluoromethyl)phenyl]-1-
methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole (5g). Off white solid,
found: C, 52.24; H, 3.28; N, 15.41. Calcd. for C20H14F3N5O3S: C,
d
52.06; H, 3.06; N, 15.18. IR (KBr) n
/cmꢁ1: 3146, 3058 (AreH),

P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
209
2947, 2867 (CeH), 1610 (C]N), 1526 (NO2, anti), 1484 (C]C),
1328 (NO2, sym) 1065 (CeF). 1H NMR (400 MHz, CDCl3):
8.36 (s,
4.36 (s, 3H), 3.56 (t, J ¼ 7.1 Hz, 2H), 3.19 (t, J ¼ 7.8 Hz, 2H). 13C NMR
d
(100 MHz, CDCl3): d 164.5, 163.0, 160.5, 158.1, 149.4, 135.5, 134.4,
1H), 8.18 (dd, J ¼ 8.2, 1.6 Hz, 1H), 7.88 (d, J ¼ 7.7 Hz, 1H), 7.79 (d,
J ¼ 8.1 Hz, 2H), 7.60 (d, J ¼ 8.2 Hz, 2H), 7.55 (t, J ¼ 7.9 Hz, 1H), 6.97
(s, 1H), 4.60 (s, 2H), 4.00 (s, 3H). 13C NMR (100 MHz, CDCl3):
130.1, 129.8, 128.3, 125.7, 115.5, 105.6, 39.9, 33.8. MS (MM-
ES þ APCI) 449.0 (M þ H)þ.
d
162.9, 161.3, 148.3, 144.1, 138.0, 136.2, 135.3, 132.6, 131.5, 129.7,
5.1.4.14. 2-(3-Nitrobenzylthio)-5-{3-[4-(trifluoromethyl)phenyl]-1-
methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9f). Off white solid,
found: C, 52.34; H, 3.32; N, 15.44. Calcd. for C20H14F3N5O3S: C,
129.1, 126.0, 124.1, 123.0, 106.9, 38.3, 35.6. MS (MM-ES þ APCI)
462.0 (M þ H)þ.
52.06; H, 3.06; N, 15.18. IR (KBr)
(CeH), 1618 (C]N), 1528 (NO2, anti), 1468 (C]C), 1322 (NO2,
sym) 1060 (CeF). 1H NMR (400 MHz, CDCl3):
8.38 (s, 1H), 8.20
n
/cmꢁ1: 3084 (AreH), 2938, 2872
5.1.4.8. 2-(4-Chloro-2-fluorobenzylthio)-5-{5-[4-(trifluoromethyl)
phenyl]-1-methyl-1H-pyrazol-3-yl}-1,3,4-oxadiazole
white solid, found: C, 51.41; H, 2.97; N, 12.14. Calcd. for
C20H13ClF4N4OS: C, 51.24; H, 2.79; N, 11.95. IR (KBr)
/cmꢁ1: 3130,
3074 (AreH), 2951 (CeH), 1610 (C]N), 1481 (C]C), 1070 (CeF), 485
(CeCl). 1H NMR (400 MHz, CDCl3):
(5h). Off
d
(dd, J ¼ 8.2, 1.3 Hz, 1H), 7.94 (d, J ¼ 8.1 Hz, 2H), 7.89 (d, J ¼ 7.6 Hz,
n
1H), 7.69 (d, J ¼ 8.2 Hz, 2H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.13 (s, 1H),
4.61 (s, 2H), 4.34 (s, 3H). 13C NMR (100 MHz, CDCl3):
d 163.2,
d
7.79 (d, J ¼ 8.1 Hz, 2H), 7.60 (d,
158.5, 149.5, 148.4, 137.7, 135.4, 130.2, 129.7, 128.1, 125.8, 125.6,
123.9, 123.1, 122.2, 105.7, 39.9, 35.6. MS (MM-ES þ APCI) 462.0
(M þ H)þ.
J ¼ 8.0 Hz, 2H), 7.5 (t, J ¼ 8.0 Hz, 1H), 7.13e7.09 (m, 2H), 6.97 (s, 1H),
4.51 (s, 2H), 4.00 (s, 3H). MS (MM-ES þ APCI) 469 (M þ H)þ.
5.1.4.9. 2-(2,6-Difluorobenzylthio)-5-{3-[4-(trifluoromethyl)phenyl]-
1-methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9a). Off white solid,
found: C, 53.32; H, 3.11; N, 12.57. Calcd. for C20H13F5N4OS: C, 53.10;
Acknowledgements
The Author PP thanks Dr. Ashis Baran Mandal, Dr. Santosh Kul-
karni and Dr. John Kallikat, Syngene international ltd. for helpful
discussions. The Authors also grateful to Dr. Chadrashekar, Dept. of
Pharmacology, HSK College, Bagalkote, India for providing the
facilities to determine the anticancer activity.
H, 2.90; N, 12.38. IR (KBr)
(C]C), 1591 (C]N), 1471 (C]C), 1065 (CeF). 1H NMR (400 MHz,
CDCl3):
7.94 (d, J ¼ 8.1 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz, 2H), 7.33e7.28
(m, 1H), 7.15 (s, 1H), 6.97 (t, J ¼ 8.0 Hz, 2H), 4.60 (s, 2H), 4.36 (s, 3H).
13C NMR (100 MHz, CDCl3):
163.4, 162.9, 159.6, 158.4, 149.4, 135.5,
n
/cmꢁ1: 3159 (AreH), 2953 (CeH), 1620
d
d
130.3, 129.7, 128.3, 125.8, 125.7, 125.6, 122.2, 111.9, 105.7, 39.9, 24.2.
Appendix A. Supplementary material
MS (MM-ES þ APCI) 453.0 (M þ H)þ.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.03.056.
5.1.4.10. 2-[2-(2-Fluorophenoxy)ethylthio]-5-{3-[4-(trifluoromethyl)
phenyl]-1-methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9b). Off white
solid, found: C, 54.16; H, 3.35; N, 11.91. Calcd. for C21H16F4N4O2S: C,
References
54.31; H, 3.47; N, 12.06. IR (KBr)
2876 (CeH), 1615 (C]N), 1504 (CeO), 1463 (C]C), 1059 (CeF). 1H
NMR (400 MHz, CDCl3):
n
/cmꢁ1: 3164, 3074 (AreH), 2943,
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d
7.94 (d, J ¼ 8.0 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz,
2H), 7.15 (s, 1H), 7.12e7.01 (m, 3H), 6.97e6.94 (m, 1H), 4.51 (t,
J ¼ 6.0 Hz, 2H), 4.35 (s, 3H), 3.76 (t, J ¼ 5.9 Hz, 2H). MS (MM-
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5.1.4.11. 2-(2,3-Dichlorobenzylthio)-5-{3-[4-(trifluoromethyl)phen-
yl]-1-methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9c). Off white
solid, found: C, 49.73; H, 2.92; N, 11.77. Calcd. for
C20H13Cl2F3N4OS: C, 49.50; H, 2.70; N, 11.54. IR (KBr)
3124, 3065 (AreH), 2950 (CeH), 1616 (C]N), 1469 (C]C), 474
(CeCl). 1H NMR (400 MHz, DMSO-d6):
n :
/cmꢁ1
d
8.11 (d, J ¼ 8.1 Hz, 2H),
7.81 (d, J ¼ 8.2 Hz, 2H), 7.65e7.62 (m, 2H), 7.61 (s, 1H), 7.40 (t,
J ¼ 7.7 Hz, 1H), 4.72 (s, 2H), 4.22 (s, 3H). MS (MM-ES þ APCI) 486.0
(Mþ1H)þ.
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5.1.4.12. 2-[4-(Trifluoromethyl)benzylthio]-5-{3-[4-(trifluoromethyl)
phenyl]-1-methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9d). Off white
solid, found: C, 52.29; H, 3.14; N, 11.77. Calcd. for C21H14F6N4OS:
C, 52.07; H, 2.91; N, 11.57. IR (KBr)
1617 (C]N), 1471 (C]C), 1059 (CeF). 1H NMR (400 MHz,
CDCl3):
n
/cmꢁ1: 2954, 2927 (CeH),
d
7.93 (d, J ¼ 8.3 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz, 2H), 7.63 (s,
4H), 7.11 (s, 1H), 4.58 (s, 2H), 4.34 (s, 3H). MS (MM-ES þ APCI)
485.0 (M þ H)þ.
5.1.4.13. 2-(4-Fluorophenethylthio)-5-{3-[4-(trifluoromethyl)phen-
yl]-1-methyl-1H-pyrazol-5-yl}-1,3,4-oxadiazole (9e). Off white
solid, found: C, 56.59; H, 3.95; N, 12.81. Calcd. for C21H16F4N4OS:
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C, 56.25; H, 3.60; N, 12.49. IR (KBr)
2928 (CeH), 1615 (C]N), 1508 (C]C), 1061 (CeF). 1H NMR
(400 MHz, CDCl3):
7.95 (d, J ¼ 8.2 Hz, 2H), 7.70 (d, J ¼ 8.3 Hz, 2H),
7.25 (dd, J ¼ 8.4, 3.0 Hz, 2H), 7.14 (s, 1H), 7.05 (t, J ¼ 8.6 Hz, 2H),
n
/cmꢁ1: 3142, 3052 (AreH),
d

210
P. Puthiyapurayil et al. / European Journal of Medicinal Chemistry 53 (2012) 203e210
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