Microwave-assisted synthesis and in-vitro anti-tumor activity of 1,3,4-triaryl-5-N-arylpyrazole-carboxamides

Article abstract of DOI:10.1016/j.ejmech.2010.02.026

Regioselective 1,3-dipolar cycloaddition of nitrilimines with 5-arylidene-2-arylimino-4-thiazolidinones and with 2-(4-arylidene)thiazolo[3,2-a]benzimidazol-3(2H)-ones afforded the corresponding 1,3,4-triaryl-5-N-arylpyrazole-carboxamides and pyrazolylbenzimidazoles. All reactions were carried out under conventional thermal heating and/or microwave irradiation. Both the pyrazole-5-carboxamides and pyrazolylbenzimidazoles were examined for their in-vitro anti-tumor activities against two tumor cell lines, Hep-2 and colon CaCo-2. Most of the obtained compounds exhibited significant activity against CaCo-2 and Hep-2 cell lines.

Full text of DOI:10.1016/j.ejmech.2010.02.026

European Journal of Medicinal Chemistry 45 (2010) 2427e2432
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave-assisted synthesis and in-vitro anti-tumor activity of
1,3,4-triaryl-5-N-arylpyrazole-carboxamides
,
Hatem A. Abdel-Aziz a, Heba S.A. El-Zahabi b, Kamal M. Dawood c
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
c Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Regioselective 1,3-dipolar cycloaddition of nitrilimines with 5-arylidene-2-arylimino-4-thiazolidinones
and with 2-(4-arylidene)thiazolo[3,2-a]benzimidazol-3(2H)-ones afforded the corresponding 1,3,4-tri-
aryl-5-N-arylpyrazole-carboxamides and pyrazolylbenzimidazoles. All reactions were carried out under
conventional thermal heating and/or microwave irradiation. Both the pyrazole-5-carboxamides and
pyrazolylbenzimidazoles were examined for their in-vitro anti-tumor activities against two tumor cell
lines, Hep-2 and colon CaCo-2. Most of the obtained compounds exhibited significant activity against
CaCo-2 and Hep-2 cell lines.
Received 26 June 2009
Received in revised form
7 February 2010
Accepted 10 February 2010
Available online 16 February 2010
Keywords:
Pyrazoles
Ó 2010 Elsevier Masson SAS. All rights reserved.
Benzimidazoles
Thiazolidinones
Microwave irradiation
Anti-tumor activity
1. Introduction
Moreover, many pyrazole derivatives especially that bearing aryl
and/or carboxamide group(s) are important for the creation
Pyrazole derivatives have been attracting a great deal of interest
due to their various pharmaceutical applications [1]. Numerous
synthetic pyrazoles were used as molecular scaffolds in several
drugs such as metamizol [2], difenamizole [3], lonazolac [4], phe-
nidone [5], and mepirizole [6].
of promising new anti-tumor agents [13e16]. For example, some
N-arylpyrazoles have remarkable activities as tumor necrosis
inhibitors [17]. Recently, pyrazole-3-carboxamide derivatives
showed significant activity toward Ehrlich Ascites carcinoma tumor
cell [18]. It is also reported that some 3-arylpyrazoles suppressed
A549 lung cancer cell growth [19].
The combination of pharmacophores may provide a synergistic
effect to improve the activity and reducing the risk of side effects.
On the other hand, CDK (cyclin-dependent kinases) inhibitors have
been intensively pursued as potential anti-cancer agents [20].
Recently, a series of 3-(benzimidazol-2-yl)pyrazoles showed potent
CDK1 inhibitory activities and inhibited in-vitro cellular prolifera-
tion in HeLa, HCT116, and A375 human tumor cell lines [21]. These
results encouraged us to synthesize functionally pyrazole
combined with benzimidazole moiety.
Based on the previous findings and in continuation of our
interest in the synthesis of bioactive heterocycles [22e30]
including a promising anti-tumor agents [28e30], the aim of the
present study is to investigate the regioselective synthesis of some
arylpyrazoles that are bearing carboxamide function or benzimid-
azole moiety to evaluate their in-vitro anti-tumor activities against
two tumor cell lines Hep-2 and CaCo-2.
Additionally, aryl pyrazoles have been reported to possess
several biological activities including non-nucleoside HIV-1 reverse
transcriptase inhibitory activity [7] and their anti-inflammatory
activity such as the N-arylpyrazole derivative; Celecoxib, a well-
known COX-2 inhibitor [8]. On the other hand, the pyrazole-1-car-
boxamide derivative MPCA was reported as antinociceptive agent
[9]. Furthermore, the N-arylpyrazole-3-carboxamide; Rimonabant
(SR 141716A) acted as CB1 receptor antagonist useful for the treat-
ment of obesity [10] and as effective therapeutic agents for the
treatment of hypercholesterolemia and prevention of coronary
heart disease [11]. In addition, N-arylpyrazole-5-carboxamides
DPC423, Razaxaban and their analogs were reported as highly
potent FXa inhibitor agents for treatment of thromboembolic
diseases [12].
* Corresponding author. Tel.: þ202 35676602; fax: þ202 35676501.
E-mail address: dr_dawood@yahoo.com (K.M. Dawood).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.026

2428
H.A. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 45 (2010) 2427e2432
2. Results and discussion
were failed. Interestingly, compound 8a was previously published
by Shawali and his co-workers [42] through completely
a
2.1. Chemistry
different route and our product 8a is identical with the reported
one.
5-Arylidene-2-imino-4-thiazolidinones 3a, 3c and 3d have been
synthesized by the conventional heating of 2-imino-4-thiazolidi-
nones 2 with the appropriate aldehyde under basic or acidic
conditions [31,32]. Recently, compound 3a was prepared by a one-
pot three component solvent-free reaction of N,N-diphenylthiourea
1a with chloroacetic acid and benzaldehyde under microwave
irradiation [33]. In this study, we synthesized compounds 3aed
according to the latter reported procedure and their Z configuration
was assigned as shown in Scheme 1 [34e38]. The structure of the
new derivative 3b was confirmed from its elemental and spectral
analyses.
Cycloaddition reaction of 5-arylidene-2-imino-4-thiazolidi-
nones 3aed with the appropriate hydrazonoyl chloride 4a or 4b
was attempted. At first, the reaction between 5-benzylidene-2-
imino-4-thiazolidinone (3a) with an equimolar quantity of the
hydrazonoyl chloride 4a and triethy amine was conducted under
conventional thermal heating in dry toluene at reflux until the
starting substrates were completely consumed resulting in the
formation of a single product as examined by TLC. The same
reaction between 3a and an equimolar quantity of 4a, in the
presence of equivalent amount of triethy amine, was repeated
under microwave irradiation condition at 130 ꢀC and 250 W for
15 min to a product identical to that obtained under thermal
heating in higher yield. The elemental analysis and mass spec-
trum (Mþ 415 m/e) of the reaction product were compatible with
the molecular formula C28H21N3O but not with the expected
spiropyrazoline cycloadduct structure 6a or 9a having the
molecular formula C35H26N4OS. The 1H NMR spectrum of the
obtained compound showed no signals due to the aliphatic pyr-
1,3-Dipolar cycloaddition reaction of 5-arylidene-2-imino-4-
thiazolidinones 3bed with the appropriate hydrazonoyl chloride
4a or 4b was similarly performed in the presence of triethylamine
in refluxing toluene or solvent-free, 130 ꢀC at 250 W for
mw irra-
diation to give in each case a fine needle product. In all cases,
phenylisothiocyanate or 4-tolylisothiocyanate were lost from the
intermediates 9bed to give the products 6bed. It is noteworthy to
mention here that compounds 8cee have been hitherto unreported
(Scheme 2).
As illustrated in Scheme 2, the formation of the pyrazoles 8aee
is assumed to occur via the regioselective 1,3-dipolar cycloaddition
of the nitrilimine 5a, b (which generated in situ from the hydra-
zonoyl chlorides 4a, b) to the exocyclic-arylidene double bond of
thiazolidinones 3a-d to give the non-isolable spiropyrazoline
derivatives 6aee followed by aromatization of the pyrazoline rings
accompanied with ring-closure of the thiazolidinone moieties via
1,3-hydrogen shift to give the thiourea derivatives 7aee which loss
aryl isothiocyanate to furnish the N-arylcarboxamido pyrazoles
8aee.
Microwave irradiation has also been applied for a rapid and
efficient synthesis of 2-arylidenethiazolo[3,2-a]benzimidazol-3
(2H)-one 13a, b from 2-mercapto-1H-benzimidazole (12) using
the same procedure for synthesis of compounds 3aee
(Scheme 3).
Then, the reaction of nitrilimine 5a with 2-arylidenethiazolo
[3,2-a]benzimidazol-3(2H)-one 13a,
b was performed under
similar reaction conditions, used for synthesis of compounds of
8aee, and afforded in each case only one isolable product as
examined by TLC. Spectroscopic analyses (IR, MS, 1H and 13C NMR)
confirmed the regioselective 1,3-dipolar cycloaddition of the
nitrilimine 5a to the exocyclic double bond of 13a, b to afford the
corresponding pyrazolylbenzimidazole derivatives 16a, b but not
17a, b as shown in Scheme 3. Moreover, the structure of the iso-
lated products was supported by the alternate synthesis of an
authentic sample of compound 16a. Thus the reaction of 1,3,4-
triphenyl-1H-pyrazole-5-carbonyl chloride (18) with 2-mercapto-
1H-benzimidazole (12) in pyridine gave a product identical in all
aspects with that obtained from the reaction of the nitrilimine 5a
azoline-4-CH proton (typically
line-5-CH proton (typically
d
4.7e5.1) [39,40] or the pyrazo-
5.6) [41] which excluded the
d
possible spiropyrazoline structures 6 and 9, respectively. The 13C
NMR spectrum of the product was free of any aliphatic CH. In
addition, NH function was confirmed in both 1H NMR and IR
spectra of the reaction product. From the foregoing results it can
be concluded that the spiropyrazoline cycloadduct structures 6a
or 9a are non-isolable intermediates and readily loss phenyl-
isothiocyanate molecule to give a product has either the structure
8a or 11a as outlined in Scheme 2. It would be of great support to
get an unequivocal evidence for one of these two possible
isomeric structures 8a or 11a by carrying out a single crystal X-ray
analysis of the reaction product, however all attempts to get such
crystals for 8a or any of its further derivatives for X-ray diffraction
with
(Z)-2-benzylidenethiazolo[3,2-a]benzimidazol-3(2H)-one
(12a) (Scheme 3).
2.2. Anti-tumor activity
The in-vitro anti-tumor activity of the tested compounds was
evaluated in the National Institute of Cancer, Cairo, Egypt.
Compounds 8aee and 16a, b were tested for their in-vitro anti-
tumor activity against colon and laryngeal carcinoma cell lines,
CaCo-2 and Hep-2, respectively. Doxorubicin was used as a refer-
Ar
O
S
N
(a)
Ar
Ar
N
H
N
H
N
S
3a-d
ence and showed IC50 ¼ 1.0 and 0.4
mg/ml against CaCo-2 and Hep-
Ar
Ar1
1a,b
2 cell lines, respectively. Most of the tested compounds showed
effects against both cancer cell lines as shown in Table 1.
Compounds 8b (p-chlorophenyl at C4) and 8e (p-tolyl at C4)
showed remarkable activity against Hep-2 with IC50 ¼ 0.74
Ar
O
N
(b)
(c)
and 0.81 mg/ml, respectively. On the other hand, compound 8d
N
S
2a,b
(p-chlorophenyl at C3) revealed a significant activity against CaCo-2
cell line. Compounds 16a and 16b did not exert a pronounced anti-
tumor effect against both of the two cells (Table 1). The effect of 8b
and 8e on laryngeal carcinoma Hep-2 is depicted in Fig. 1A and B,
respectively, while the effect of 8d on colon carcinoma CaCo-2 is
shown in Fig. 1C.
Ar
1,2: a, Ar = C6H5
b, Ar = 4-CH3C6H4
Scheme 1. Reagents and conditions: (a) ClCH2COOH, Ar1CHO, solvent free, MWI, 50 W
(110 ꢀC), 20 min, 70e82%; (b) ClCH2COOH, AcONa, EtOH, reflux 6e8 h, 50e58%;
(c) EtOH/piperidine or, AcOH/AcONa, reflux 3 h, 55e63%.

H.A. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 45 (2010) 2427e2432
2429
Cl
H
N
Ar2
N
4a,b
Ph
Ph
Ar
O
Ar
O
Ar2
N
Ar2
N
N
Ph
N
N
- ArNCS
N
N
S
S
Ar
N
Ar
Ar1
Ar1
Ar
O
N
H
H
10a-e
Ph
Ar2
N
5a,b
N
9a-e
N
S
O
Ar
HN
(a)
Ar
Ar1
Ar2
3a-d
Ar
O
N
Ph
Ar1
N
N
N
Ph
11a-e
N
N
S
6-11 Ar
Ar1
Ph
Ar2
Ph
Ar
Ar1
H
6a-e
Ar2
a
b
c
d
e
Ph
Ph
4-ClC6H4 Ph
4-MeC6H4
Ph
4-MeC6H4 Ph
4-MeC6H4
Ph
4-ClC6H4
4-ClC6H4 Ph
H
S
O
Ar1
Ar2
N
Ph
Ar
N
N
N
- ArNCS
O
N
Ar
Ar1
N
NH
Ar
Ph
8a-e
Ar2
7a-e
Scheme 2. Reagents and conditions: (a) toluene, reflux 8e11 h, 72e80% (for thermal heating) or solvent-free, 130 ꢀC at 250 watt for 15 min (for microwave irradiation).
In conclusion, we described a facile one-pot approach to 1,3,4,5-
tetra-substituted pyrazoles 8a-e and 14a, b via (2Z,5Z)-5-arylidene-
2-arylimino-4-thiazolidinones 3aed and 2-(4-arylidene)thiazolo
[3,2-a]benzimidazol-3(2H)-one 11a, b, respectively, under thermal
as well as microwave irradiation. The synergistic action was not
achieved by the designed hybrids between pyrazole and benzimid-
azole moieties in compounds 14a, b while the most potent anti-
tumor compounds were the pyrazolecarboxamide derivatives 8aee.
Ph
N
O
N
O
Ar
Ph
N
(b)
(a)
N
N
N
H
SH
S
S
Ar
N
H
N
12
13a,b
14a,b
H
S
Ar
Ph
H
S
N
Ar
Ar
Ph
Ph
NCS
N
N
H
N
N
N
N
N
N
Ph
N
Ph
N
Ph
O
O
16a,b
O
17a,b
15a,b
(c)
16a,
Ar = Ph
Ar
16
Ph
Ph
N
Ph
a
b
Cl
SH
+
N
N
H
4-ClC6H4
N
Ph
18
O
12
Scheme 3. (a) ClCH2COOH, Ar1CHO, solvent free, MW, 50 W (110 ꢀC), 20 min, 85e88%; (b) 4a toluene, reflux 8e11 h, 72e80%; (c) pyridine, r.t., stirring 3 h, 37%.

2430
H.A. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 45 (2010) 2427e2432
Table 1
Hydrazonoyl chlorides 4a, b, [43] 1,3,4-triphenyl-1H-pyrazole-5-
carbonyl chloride (16) [44] and 2-mercapto-1H-benzimidazole (12)
[45] were prepared following the methods reported in the litera-
ture. 1,3-Diarylthioureas 1a, b, and chloroacetic acid were used as
obtained commercially.
IC50 of tested compounds for their in-vitro anti-tumor activity against colon cancer
cell CaCo-2 and laryngeal carcinoma cell Hep-2.
Compounds
IC50 (mg/mL)
CaCo-2
Hep-2
8a
8b
8c
8d
1.68
1.74
2.21
0.67
1.54
1.48
3.29
1.0
1.50
0.74
1.21
1.21
0.81
7.18
1.54
0.40
3.1.2. Synthesis of (2Z,5Z)-5-arylidene-2-arylimino-
4-thiazolidinones 3aed under microwave irradiation
A one-pot three component mixture of the appropriate N,N-
diarylthiourea 1a, b (10 mmol), chloroacetic acid (1.0 g, 11 mmol)
and the appropriate aldehyde (10 mmol) were mixed in a process
vial. The vial was capped properly, and thereafter the mixture was
heated under microwave irradiating conditions at 110 ꢀC and 50
watt for 20 min. Then left to cool and the mixture was then treated
with ethanol (10 mL). The resulting residue was recrystallized from
EtOH to afford the corresponding thiazolidine derivatives 3aed in
70e82% yield.
8e
16a
16b
Doxorubicin
3. Experimental
3.1. Chemistry
3.1.1. General
Melting points were measured with a Gallenkamp apparatus
and are uncorrected. IR spectra were recorded on Shimadzu FT-IR
8101 PC infrared spectrophotometer. NMR spectra were deter-
mined in DMSO-d6 at 300 MHz (1H NMR) and at 75 MHz (13C NMR)
on a Varian Mercury VX 300 NMR spectrometer using TMS as an
internal standard. Mass spectra were measured on a GCMS-QP1000
EX spectrometer at 70 eV. Elemental analyses were carried out at
the Microanalytical center of Cairo University. Microwave experi-
3.1.3. Synthesis of thiazolidinones 3aed under conventional
thermal heating
3.1.3.1. General procedure for the preparation of compounds 2a,
b. The appropriate N,N-diarylthiourea 1a,
b (10 mmol) and
chloroacetic acid (0.95 g, 10 mmol), were refluxed for 6e8 h in
absolute ethanol (50 mL) in the presence of sodium acetate (0.82 g,
10 mmol). Then left to cool and the mixture was then triturated
with water (15 mL). The resulting residue was recrystallized from
EtOH to afford the corresponding thiazolidine derivatives 2a, b. The
physical constants (mp and spectra) of 2a, b were identical with the
reported ones [25e27].
ments were carried out using
microwave apparatus (300 W with ChemDriverÔ Software).
a CEM Discover LabmateÔ
A
B
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
conc. g/mL
conc. g/mL
C
4.0
3.6
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
conc. g/mL
Fig. 1. (A) Effect of 8b on laryngeal carcinoma Hep-2. (B) Effect of 8e on laryngeal carcinoma Hep-2. (C) Effect of 8d on colon carcinoma CaCo-2. Cell proliferation was determined by
measuring the absorbance at 570 nm (SRB method). Data were represented in three individual experiments, performed in three replicates. Data were expressed as the mean
absorbance value ꢁ S.E. Significant difference of absorbance of treated cells compared to control value at p < 0.05.

H.A. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 45 (2010) 2427e2432
2431
3.1.3.2. General procedure for the preparation of compounds
3aed. A mixture of the appropriate thiazolidinone 2a, b (10 mmol)
and the appropriate aldehyde (10 mmol) in glacial acetic acid
(30 mL) in the presence of sodium acetate (0.82 g, 10 mmol) was
refluxed for 3 h. After cooling, the resulting residue was purified by
recrystallization from ethanol to afford the corresponding benzy-
lidines 3aed in 55e63% yields. Using of ethanol (30 mL) and
piperidine (0.3 mL) gave the same yields. The overall yield of
compounds 3aed by conventional thermal heating method is
28e37%. The physical constants (mp and spectra) of the known
derivatives 3a, 3c and 3d were identical with the reported ones
[25e27]. The data of compound 3b is as following.
3.1.5.3. 1,3,4-Triphenyl-N-(4-tolyl)-1H-pyrazole-5-carboxamide
(8c). White needles in 80%; mp 240e242 ꢀC; IR (KBr) nmax/cmꢂ1
3278 (NH), 1646 (C]O), 1605 (C]N); 1H NMR (DMSO-d6)
2.23 (s,
d
3H, CH3), 7.09 (d, 2H, ArH, J ¼ 8.34 Hz), 7.31e7.56 (m,15H, ArH), 7.70
(d, 2H, ArH, J ¼ 8.13 Hz), 10.72 (br. s, 1H, NH); 13C NMR (DMSO-d6)
d
20.4, 119.6, 120.2, 122.7, 127.5, 127.9, 128.1, 128.4, 128.5, 129.2,
129.4, 129.5, 131.2, 132.2, 133.5, 135.5, 137.9, 139.3, 149, 158.8; MS
m/z (%) 429 (Mþ, 100%), 323 (94.5%), 293 (5.1%),190 (10.3), 165 (8.9),
106 (14.5), 77 (29.2). Anal. Calcd for C29H23N3O: C, 81.09; H, 5.40; N,
9.78. Found: C, 79.92; H, 5.51; N, 9.72.
3.1.5.4. 3-(4-Chlorophenyl)-1,4-diphenyl-N-(4-tolyl)-1H-pyrazole-5-
carboxamide (8d). White fibers in 72%; mp 255e257 ꢀC; IR (KBr)
nmax/cmꢂ1 3247 (NH), 1649 (C]O), 1600 (C]N), 1530; 1H NMR
3.1.3.2.1. (2Z,5Z)-5-(4-Chlorobenzylidene)-3-phenyl-2-(phenyl-
imino)thiazolidin-4-one (3b). Yellow needles in 78% yield, mp
200e205 ꢀC; IR (KBr) nmax/cmꢂ1 1715 (C]O), 1646 (C]N); 1H NMR
(DMSO-d6)
7.31e7.56 (m, 14H, ArH), 7.69 (d, 2H, ArH, J ¼ 8.22 Hz), 10.72 (br. s,
1H, NH, D2O-exchangeable); 13C NMR (DMSO-d6)
20.4, 119.7,
d
2.23 (s, 3H, CH3), 7.08 (d, 2H, ArH, J ¼ 8.31 Hz),
(DMSO-d6)
d (ppm), 6.94e7.00 (m, 2 H, AreH), 7.18e7.20 (m, 1 H,
AreH), 7.37e7.58 (m, 11 H, AreH), 7.82 (s, 1H, CH]C); MS m/z (%)
390 (Mþ, 4.3), 194 (52.5), 168 (42.3), 133 (15.8), 89 (46.6), 77 (100).
Anal. Calcd for C22H15ClN2OS: C, 67.60; H, 3.87; N, 7.17; S, 8.20.
Found: C, 67.42; H, 3.95; N, 7.11; S, 8.39.
d
120.3, 122.8, 127.7, 128.1, 128.6, 129.2, 129.4, 129.5, 129.8, 130.9,
131.1, 132.9, 133.6, 135.4, 138.1, 139.2, 147.8, 158.7; MS m/z (%) 463
(Mþ, 100), 386 (5.3), 357 (84.6), 321 (23.5%), 293 (23.2%), 226 (8.3),
190 (31.9), 106 (88.6), 77 (85.1). Anal. Calcd for C29H22ClN3O: C,
75.07; H, 4.78; N, 9.06. Found: C, 75.20; H, 4.79; N, 9.12.
3.1.4. Microwave-assisted synthesis of pyrazoles 8aee
A solvent-free mixture of the appropriate arylidine derivative
3aed (2 mmol), hydrazonoyl chloride 4a, b (2 mmol) and trie-
thylamine (0.5 mL) were mixed in a process vial. The vial was
capped properly, and thereafter the mixture was heated under
microwave irradiating conditions at 130 ꢀC and 250 W for 15 min
then left to cool. The solid mixture was then triturated with
methanol (5 mL) and product was collected by filtration, washed
with water followed by methanol and dried. Recrystallization from
ethanol afforded the corresponding pyrazole derivatives 8aef,
respectively, in 72e80% yields.
3.1.5.5. 4-(4-Chlorophenyl)-1,3-diphenyl-N-(4-tolyl)-1H-pyrazole-5-
carboxamide (8e). White needles in 79%; mp 280e282 ꢀC; IR (KBr)
nmax/cmꢂ1 3279 (NH), 1644 (C]O), 1603 (C]N); 1H NMR (DMSO-d6)
d
2.21 (s, 3H, CH3), 6.98e7.44 (m, 14H, ArH), 7.58 (d, 2H, ArH,
J ¼ 8.03 Hz), 10.42 (br. s, 1H, NH, D2O-exchangeable); MS m/z (%) 463
(Mþ, 76.3), 386 (3.1), 357 (38.6), 321 (34.5%), 293 (16.2%), 226 (12.1),
190(49.7),123(42.5),106(91.8), 77(100). Anal. CalcdforC29H22ClN3O:
C, 75.07; H, 4.78; N, 9.06. Found: C, 75.24; H, 4.75; N, 9.01.
3.1.6. 2-Arylidenethiazolo[3,2-a]benzimidazol-3(2H)-one 13a,
b [46]
These compounds were synthesized using the same method
(microwave irradiation) that described for synthesis of compounds
3aed to give 13a and 13b in 85 and 88% yields, respectively.
3.1.5. General procedure for synthesis of 8aee under conventional
thermal heating
To a stirred solution of the appropriate hydrazonoyl chloride 4a,
b (2 mmol) and the arylidine derivative 3aed (2 mmol) in dry
toluene (30 mL), triethylamine (0.5 mL) was added and the mixture
was refluxed for 7e11 h, then left to cool to room temperature. The
precipitated triethylamine hydrochloride was then filtered off, and
the filtrate was evaporated under reduced pressure. The residue
was triturated with methanol and the solid product, so formed in
each case, was collected by filtration, washed with methanol and
dried. Recrystallization from ethanol afforded the corresponding
pyrazole derivatives 8aef, respectively, in 58e70% yields.
3.1.7. Synthesis of the pyrazole derivatives 16a, b
This reaction was carried out using the same procedure
(conventional thermal heating) that described for the synthesis of
compounds 8aee using the benzylidine derivatives 13a, b instead
of 3aed. Recrystallization from EtOH/DMF afforded the corre-
sponding pyrazoles 16a, b in 72 and 68% yields, respectively.
3.1.7.1. 1-[(1,3,4-triphenyl-1H-pyrazol-5-yl)carbonyl]-2,3-dihydro-
1H-benzimidazole-2-thione (16a). Reaction time 15 h, white nee-
dles in 72%; mp 242e244 ꢀC; IR (KBr) nmax/cmꢂ1 3367 (NH), 1670
3.1.5.1. N,1,3,4-tetraphenyl-1H-pyrazole-5-carboxamide (8a) [42].
White needles in 75% yield; mp 242e243 ꢀC (Lit mp 240e242 ꢀC);
IR (KBr) nmax/cmꢂ1 3313 (NH), 1643 (C]O), 1597 (C]N); 1H NMR
(C]O), 1580 (C]N), 1511 (C ¼ S); 1H NMR (DMSO-d6)
d 7.09e7.79
(DMSO-d6)
d
7.05e7.71 (m, 21H, ArH), 10.76 (br. s, 1H, NH, D2O-
119.1, 119.8, 122.8, 124.4,
(m, 16H, ArH), 7.80e7.89 (m, 2H, ArH), 8.04 (d, 1H, ArH, J ¼ 7.50 Hz),
exchangeable); 13C NMR (DMSO-d6)
d
9.59 (br. s, 1H, NH, D2O-exchangeable); 13C NMR (DMSO-d6)
d 119.7,
124.5, 127.7, 127.9, 128.0, 128.3, 128.6, 128.6, 128.9, 129.2, 130.1,
131.2, 131.9, 132.5, 137.8, 138.0, 139.1, 149.0, 158.9, 187.1; MS m/z (%)
415 (Mþ, 89.5%), 323 (100%), 293 (10.6%), 218 (10.2), 190 (23.7), 165
(17.5), 77 (42.2).
120.4, 122.2, 123.1, 124.2, 126.2, 127.3, 128.1, 128.7, 129.2, 129.4,
129.6, 131.1, 132.2, 137.7, 139.3, 143.2, 147.1, 148.9, 156.7, 158.9; MS
m/z (%) 472 (Mþ, 4.6), 412 (5.1), 395 (4.3), 295 (19.2), 275 (74.4), 193
(11.9), 160 (17.7), 147 (50.8), 118 (13.8), 43 (100). Anal. Calcd for
C29H20N4OS: C, 73.71; H, 4.27; N, 11.86; S, 6.79. Found: C, 73.55; H,
4.16; N, 11.73; S, 6.90.
3.1.5.2. 4-(4-Chlorophenyl)-N,1,3-triphenyl-1H-pyrazole-5-carbox-
amide (8b) [42]. White needles in 72%; mp 235e236 ꢀC (Lit mp
234 ꢀC); IR (KBr) nmax/cmꢂ1 3288 (NH),1645 (C]O),1589 (C]N); 1H
3.1.7.2. 1-[(3-(4-Chlorophenyl)-1,4-diphenyl-1H-pyrazol-5-yl)]carbonyl-
2,3-dihydro-1H-benzimidazole-2-thione (16b). Reaction time 40 h,
white powder in 66%; mp 214e216 ꢀC; IR (KBr) nmax/cmꢂ1 3367
(NH), 1673 (C]O), 1580 (C]N), 1492 (C]S); 1H NMR (DMSO-d6)
NMR (DMSO-d6)
7.71 (d, 2H, ArH, J ¼ 8.01 Hz), 10.85 (br. s, 1H, NH, D2O-exchange-
able); 13C NMR (DMSO-d6)
119.1, 119.7, 122.8, 124.4, 124.5, 127.9,
d 7.07e7.13 (m, 2H, ArH), 7.27e7.69 (m, 15, ArH),
d
128.1,128.3, 128.5,128.6,128.9,129.4,130.1,131.2,131.9,132.3,137.8,
137.9, 139.1, 149.1, 158.9, 187.2; MS m/z (%) 450 (Mþ þ 1, 31.3), 449
(Mþ, 85), 357 (100), 321 (23.0), 293 (5.6),190 (5.7),123 (3.4), 77 (7.2).
d 7.12e7.66 (m, 14H, ArH), 7.80e7.84 (m, 2H, ArH), 8.04 (d, 2H, ArH,
J ¼ 7.71 Hz), 9.77 (br. s, 1H, NH, D2O-exchangeable); 13C NMR
(DMSO-d6) d 119.7, 122.2, 122.6, 123, 124.2, 126.2, 126.4, 127.9, 128.4,

2432
H.A. Abdel-Aziz et al. / European Journal of Medicinal Chemistry 45 (2010) 2427e2432
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S. Kshirsagar, B. Mishra, V. Takale, S. Gupta, P. Pandya, P. Kapadnis, M. Solanki,
H. Patel, P. Mitra, M.R. Jain, P.R. Patel, Bioorg. Med. Chem. Lett. 19 (2009)
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128.7, 129.4, 129.9, 131.1, 131.8, 132.3, 133, 137.7, 139.2, 143.5, 147.1,
156.7, 158.8; MS m/z (%) 507 (Mþ, 100), 295 (22.2), 275 (68.9), 192
(14.8), 160 (19.3). Anal. Calcd for C29H19ClN4OS: C, 68.70; H, 3.78; N,
11.05; S, 6.32. Found: C, 68.73; H, 3.65; N, 10.93; S, 6.24.
3.1.8. Alternate synthesis of 16a
(b) B.K. Srivastava, R. Soni, J.Z. Patel, S. Jha, S.A. Shedage, N. Gandhi, K.V.V.
M. Sairam, V. Pawar, N. Sadhwani, P. Mitra, M.R. Jain, P.R. Patel, Bioorg. Med.
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To a cold solution of 1,3,4-triphenyl-1H-pyrazole-5-carbonyl
chloride (18) [44] (0.36 g, 1 mmol) in pyridine (20 ml), 2-mercapto-
1H-benzimidazole (12) (0.15 g, 1 mmol) was added portion-wise
while stirring over a period of 30 min. After complete addition, the
reaction mixture was stirred for further 3 h at room temperature
and then the reaction mixture was poured onto an ice-water
mixture with stirring. The precipitated solid was collected by
filtration washed with dilute hydrochloric acid followed by cold
water, then dried and finally recrystallized from EtOH/DMF to
afford compound 16a as white powder in 37% yield.
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3.2. Anti-tumor activity
The potential cytotoxicity of the tested compounds was evalu-
ated using the method of Skehan et al. [47]. Cells were plated in
96-multiwell plate (104 cells/well) for 24 h before treatment with
the prepared compounds to allow the attachment of cells to the
wall of the plate. The tested compounds were dissolved in dime-
thylsulfoxide (DMSO) and diluted 1000-fold in the assay. Concen-
trations 1, 2.5, 5 and 10 mg/ml of the tested compounds were added
to the cell monolayer. The monolayer cells were incubated with the
compounds for 48 h at 37 ꢀC, in atmosphere of 5% CO2. After 48 h,
the cells were fixed, washed and stained with Sulfo-Rhodamine-B
stain (SRB). Excess stain was washed with acetic acid. The attached
stain was recovered with TriseEDTA buffer. Cell survival and drug
activity were determined by measuring color intensity using an
ELISA reader. Data are representative of three individual experi-
ments, performed in three replicates for each individual dose and
measured by SRB assay. Control values did not exhibit significant
changes compared to the DMSO vehicle. The IC50 was determined
by using a program Graph-Pad PRISM version 3. Statistical signifi-
cance was determined by using one-way analysis of variance
(ANOVA) test. P value more than 0.05 was considered insignificant.
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Acknowledgement
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One of the authors (KMD) is deeply indebted to the Alexander-
von-Humboldt (AvH) Foundation for donating him a CEM Discover
LabmateÔ microwave apparatus.
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