Synthesis and antitumor evaluation of some new 1,3,4-oxadiazole-based heterocycles

Article abstract of DOI:10.1016/j.ejmech.2011.12.013

The synthetic strategies and characterization of some novel 1,3,4-oxadiazole derivatives carrying different pharmacophores and heterocyclic rings that are relevant to potential antitumor and cytotoxic activities are described. The antitumor activities of

Full text of DOI:10.1016/j.ejmech.2011.12.013

European Journal of Medicinal Chemistry 48 (2012) 192e199
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor evaluation of some new 1,3,4-oxadiazole-based
heterocycles
,
b
,
a
a
c
Samir Bondock a , Shymaa Adel , Hassan A. Etman , Farid A. Badria
*
a Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
b Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
c Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, ET-35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthetic strategies and characterization of some novel 1,3,4-oxadiazole derivatives carrying
different pharmacophores and heterocyclic rings that are relevant to potential antitumor and cytotoxic
activities are described. The antitumor activities of the newly synthesized compounds were evaluated
according to the protocol of the National Cancer Institute (NCI) in-vitro disease-oriented human cells
screening panel assay. The results revealed that five compounds, namely 2, 7a, 11a, 12b, and 17; displayed
promising in-vitro antitumor activity in the 4-cell lines assay. Incorporating a thiazole ring to 1,3,4-
oxadiazole skeleton resulted in better antitumor activities than those displayed by the pyrazole and
thiophene ring systems. Transformation of 1,3,4-oxadiazole 2 to N-(6-amino-7H-pyrazolo[5,1-c][1,2,4]
triazol-3-yl)benzamide (15) diminished the antitumor activity.
Received 17 October 2011
Received in revised form
2 December 2011
Accepted 7 December 2011
Available online 13 December 2011
Keywords:
1,3,4-Oxadiazole
Pyrazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
Thiazole
Thiophene
Antitumor activity
1. Introduction
Cancer treatment has been a major endeavor of research and
development in academia and pharmaceutical industry for the last
1,3,4-Oxadiazoles are an important class of heterocyclic
compounds with a wide range of biological activities such as anti-
viral [1], antimicrobial [2], antineoplastic [3], fungicidal [4], anti-
cancer [5e8], inhibition of tyrosinase [9] and cathepsin K [10]. They
are also useful intermediates in organic synthesis [11] and widely
employed as electron transporting and hole-blocking materials [12].
Further, 1,3,4-oxadiazole heterocycles are very good bioisosteres of
amides and esters, which can contribute substantially in increasing
pharmacological activity by participating in hydrogen bonding
interactions with the receptors [13]. 2,4-Disubstituted 1,3,4-
oxadiazoles have also attracted significant interest because of their
applications in organic light-emitting diodes, photoluminescence,
polymers, and material science [14,15]. Inview of the great medicinal
significance and material applications a number of synthetic routes
have been developed for 1,3,4-oxadiazole. The majority of them are
based on the cyclization of the diacylhydrazides or acylth-
iosemicarbazides and the oxidation of acylhydrazones [16] with
a variety of reagents such as thionyl chloride, phosphorus oxy-
chloride, or sulfuric acid, usually under harsh reaction conditions.
many years as it is one of the leading causes of death [17]. Many of
the available anticancer agents exhibit undesirable side effects such
as reduced bioavailability, toxicity and drug-resistance [18e22].
Therefore, the search for novel and selective anticancer agents is
urgently required due to problems associated with currently
available anticancer drugs.
Encouraged by the afore-mentioned findings and in a continua-
tion of an ongoing program aiming at finding new structural leads
with potential chemotherapeutic activities [23e30]; it was ratio-
nalized to synthesize some novel 1,3,4-oxadiazoles-substituted
heterocycles that would produce anticancer activity. The proposed
candidates were supported with avariety of pharmacophoric groups
which would impart various electronic and lipophilic properties.
Synthesis of 1,3,4-oxadiazoles with an amide group was rationalized
on the fact that many antitumor antibiotics such as bleomycin, and
pyrazofurin incorporate in their structures amidic group [31].
2. Results and discussion
2.1. Chemistry
* Corresponding author. Department of Chemistry, Faculty of Science, Mansoura
University, El-gomhuria street, ET-35516 Mansoura, Egypt. Tel.: þ20 502369267;
fax: þ20 502246781.
The suggested synthetic plans to obtain the target compounds
are shown in Schemes 1 and 2. The versatile hitherto unreported
E-mail address: Bondock@mans.edu.eg (S. Bondock).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.013

S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
193
N
N
N
N
CN
S
CN
S
PhOCHN
O
S
PhOCHN
O
H
N
O
ArCHO
Ph
N
H
N
H
CN
Ph
N
AcONa/AcOH
Ph
N
O
O
O
1
4
Ar
5
5
Ar
HgO / EtOH
/ -H2S
ClCOCH2Cl
a
b
1,3-diphenylpyrazol-4-yl
antipyrinyl
N
N
N
N
N
N
CN
Ph-N=C=S
KOH / DMF
CN
CN
S
HCl
PhOCHN
O
PhOCHN
PhOCHN
PhOCHN
O
O
SK
PhHN
2
PhHN
9A
3
I
e
M
N
RCOCH2Cl
N
CN
SMe
O
N
N
N
N
PhHN
10
CN
S
CN
PhOCHN
PhOCHN
O
O
R
SH
PhHN
6
PhHN
9B
O
RNHNH2
EtOH
H2N
N
RCOCH2Cl
TEA/EtOH
N
N
PhOCHN
O
N
N
N
H2N
R
CN
N
N
PhHN
COR
PhOCHN
O
11
11
PhOCHN
R
O
S
S
N
Ph
R
PhHN
a
H
7-8
R
b
Ph
a
b
Me
8
Ph
7
Scheme 1. Synthetic pathway to compounds 2e11.
starting material, N-(5-(cyanomethyl)-1,3,4-oxadiazol-2-yl)benza-
mide (2) could be easily obtained via cyclodesulfurization of
4-benzoyl-1-cyanoacetylthiosemicarbazide (1) [32] upon boiling in
ethanolic mercuric oxide solution.
appearance of a cyano absorption band around 2199 cmꢀ1 in their
IR spectra, and the presence of a characteristic singlet signal was
due to the thiazoline H-5 proton at 5.15 ppm in their 1H NMR
spectra.
The reactivity of hydrogen atoms at C-5 is the most outstanding
The appearance of the cyano function in the IR spectra of the
reaction products supports structures 7a,b and ruled out the
possibility of the other structures 8a,b. Formation of compounds
7a,b was assumed to proceed via the initial alkylation of 3 to give
the non-isolable thioether intermediate 6 followed by in situ het-
erocyclization through nucleophilic addition of the secondary
amino group to the carbonyl group and elimination of water
molecule (Scheme 1).
Treatment of the intermediate 3 with cold HCl afforded the
thiocarbamoyl derivative 9, which can exist in two tautomeric
thione-thiol forms (9A and 9B). The thiol form 9B was verified by its
1H NMR spectrum which displayed a downfield singlet signal at
14.12 ppm due to SH proton, besides the other expected signals. The
chemical property of 5-cyanomethyl-1,3,4-oxadiazole
2 which
undergoes the characteristic condensation and electrophilic substi-
tution reactions.
To show the synthetic potentiality of compound 2, the reactivity
2 with isothiocyantes was examined. Thus, treatment of
of
compound 2 with phenyl isothiocyanate in potassium hydroxide
solution afforded the non-isolable intermediate 3, which was con-
verted in situ to thiazolidin-5-one 4 upon treatment with chlor-
oacetyl chloride. The structure of the latter product was established
on the basis of elemental analysis, spectral data, and chemical
transformation. The ClaiseneSchmidt condensation of thiazolidin-
5-one 4 with heterocyclic aldehydes, namely, 1,3-diphenyl-1H-
pyrazole-4-carbaldehyde and/or 4-formylantipyrine, in glacial ace-
tic acid containing anhydrous sodium acetate as a basic catalyst
furnished the arylidene derivatives 5a,b. The structure of latter
products were confirmed based on elemental analysis and spectral
data (see experimental).
reaction of the latter product with a variety of
a-halocarbonyl
compounds, as a key step for the synthesis of polysubstituted
thiophene derivatives was examined. Thus, compound 9 reacted
with each of chloroacetone and phenacyl chloride in refluxing
ethanol containing a catalytic amount of triethylamine, to afford
a single product, in each case, identified as the aminothiophene
derivatives 8a,b based on the elemental analysis and spectral data
of the isolated products (see experimental).
The non-isolable potassium salt 3 was exploited to synthesis
some new thiazoline derivatives. Thus, treatment of 3 with
-chloroketones namely, chloroacetone and phenacyl chloride at
a
room temperature afforded a single product, in each case, that was
identified as 2,3-dihydrothiazolines 7a,b on the basis of its spectral
data. The structures of the latter products were confirmed by the
The plausible mechanism for the formation of the amino-
thiophenes 8a,b is attributed to the initial alkylation of 9B with
a-chloroketones to form the non-isolable thioether 6 which in situ

194
S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
H2N
N
N
N
N
ArCHO
NH2NH2
-H2
CN
N
NH
PhOCHN
PhOCHN
PhOCHN
O
EtOH / TEA
O
13
Ar
Ar
12
12-13
Ar
C6H4-4-OCH3
1,3-diphenylpyrazol-4-yl
a
b
N
N
N
N
CN
NH2NH2
CHO
PhOCHN
N
N
N
NH2
NH2
15
14
OH
2
N
N
N
N
dil HCl
PhOCHN
PhOCHN
PhOCHN
O
O
EtOH / piperidine
O
O
O
HN
16
17
NO
OH
N
N
N
N
N
N
dil HCl
PhOCHN
PhOCHN
O
O
EtOH / piperidine
O
O
O
HN
18
19
OH
NO
HN
O
N
N
N
N
O
O
dil HCl
PhOCHN
O
O
EtOH / piperidine
N
N
21
20
Scheme 2. Synthetic pathway to compounds 12e21.
underwent intramolecular cyclization via the nucleophilic addition
of the active methylene group to the cyano function and tauto-
merization according to the ThorpeeZiegler reaction [33].
As an extension of our study, the behavior of 2 towards salicy-
laldehyde and nitrosonaphthols as a convenient synthetic route to
some new coumarin and naphtho[1,2-b]oxazines incorporating
1,3,4-oxadiazole moiety was also investigated. Thus, condensation
of 2 with salicylaldehyde in ethanolic piperidine solution furnished
coumarin derivative 17 (Scheme 2).
In a similar manner, treatment of 2 with either 1-nitroso-2-
naphthol or 2-nitroso-1-naphthol in boiling ethanolic piperidine
solution afforded a single product, in each case, identified as N-(5-
(3-oxo-3H-naphtho[2,1-b] [1,4]oxazin-2-yl)-1,3,4-oxadiazol-2-yl)
benzamide (19) and N-(5-(2-oxo-2H-naphtho[1,2-b] [1,4]oxazin-3-
yl)-1,3,4-oxadiazol-2-yl)benzamide (21), respectively.
The plausible mechanism for the formation of compounds 17,19,
and 21 may be attributed to the initial Knoevenagel condensation
of the active methylene nitrile of 2 with each of the carbonyl group
of salicyaldehyde and nitroso group of nitrosonaphthols followed
by an intramolecular 1,6-dipolar cyclization via the addition of the
phenolic OH group to the cyano function to give the intermediates
16, 18, and 20 those underwent acid catalyzed hydrolysis to afford
the target compounds.
As an extension of our investigation, treatment of 3 with methyl
iodide afforded the ketene N,S-acetal 10. The novel ketene N,S-
acetal 10 represents the versatile precursors for the synthesis of
aminopyrazoles. Thus, treatment of the ketene N,S-acetal 10 with
each of hydrazine hydrate and phenyl hydrazine in boiling ethanol
afforded, in each case, a single product, that was identified as 3-
aminopyrazole derivatives 11a,b based on elemental analysis and
spectral data (see experimental).
Pyrazolyleoxadiazoles have recently received considerable
attention because of their synthetic and pharmaceutical importance
[34e37]. In the present work we explore the synthetic potentialities
of 2 to obtain some novel pyrazolyloxadiazole derivatives. Thus, the
Knoevenagel condensation of 2 with each of p-anisaldehyde and 1,3-
diphenylpyrazole-4-carbaldehyde in refluxing ethanol containing
a catalytic amount of triethylamine furnished the arylidene deriv-
atives 12a,b. Treatment of 12a,b with hydrazine hydrate in ethanol,
under reflux, furnished the pyrazolyloxadiazole derivatives 13a,b.
Formation of compounds 13a,b is supposed to proceed through
the Michael addition of hydrazine hydrate to
a
,b
-unsaturated
3. Pharmacology
nitrile 12 and in situ intramolecular 1,5-dipolar cyclization via the
addition of amino group to a cyano function to give dihydropyr-
azole which underwent auto-oxidation to give the target pyrazoles.
On the other hand, the solvent-free reaction of 2 with an excess
of hydrazine hydrate furnished N-(6-amino-7H-pyrazolo[5,1-c]
[1,2,4]triazol-3-yl)benzamide (15) in a reasonable chemical yield.
It is worthwhile to mention that transformation of 1,3,4-
oxadiazole ring to 1,2,4-triazole ring by hydrazine hydrate is in
the line with reported results by Padmavathi and coworkers [38].
3.1. Cytotoxicity and antitumor evaluation
Out of the newly synthesized compounds, seventeen analogs
were selected to be evaluated for their in-vitro anticancer effect via
the standard MTT method [39e41], against a panel of four human
tumor cell lines namely; heptacelluar carcinoma HepG2, lung
fibroblasts WI 38, kidney of a normal adult African green monkey
VERO, and breast cancer MCF-7. The cell lines were obtained from

S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
195
ATCC via the Holding company for biological products and vaccines
3.2. Bleomycin-dependent DNA damage
(VACSERA), Cairo, Egypt. 5-Fluorouracil (5-Fu) was used as a stan-
dard anticancer drug for comparison. The results of cytotoxic
activity is reported in Table 1.
The bleomycins are a family of glycopeptide antibiotics that are
used routinely as antitumor agents. The bleomycin assay has been
adopted for assessing the pro-oxidant effects of food antioxidants.
The antitumor antibiotic bleomycin binds iron ions and DNA. The
bleomycineiron complex degrades DNA that, upon heating with
thiobarbituric acid (TBA), yields a pink chromogen. Upon the
addition of suitable reducing agents antioxidants compete with
DNA and diminish chromogen formation [42].
To show the mechanism of action of our two potent compounds
2 and 7a, their protective activity against DNA damage induced by
the bleomycineiron complex were examined. The results in Table 2
show that compounds 2 and 7a have high protection against DNA
damage induced by the bleomycineiron complex, thus diminishing
chromogen formation between the damaged DNA and TBA.
MTT assay is a standard colorimetric assay for measuring
cell growth. It is used to determine cytotoxicity of potential
medicinal agents and other toxic materials. In brief, yellow MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is
reduced to purple formazan by mitochondrial dehydrogenases of
living cells. A suitable solvent is added to dissolve the insoluble
purple formazan product into a colored solution. The absorbance of
this colored solution can be quantified by measuring at a certain
wavelength. When the amount of purple formazan produced by
cells treated with an agent is compared with that produced by
unreacted control cells, the effectiveness of the agent in causing
death of cells can deduced, through the production of a dos-
eeresponse curve.
The obtained results revealed that five of the tested compounds
namely; 2, 7a, 11a, 12b, 17 exhibited variable degrees of inhibitory
activity towards the four tested human tumor cell lines. As for
activity against hepatocellular carcinoma HepG2, the highest
cytotoxic activity was displayed by compounds 2 and 7a which
3.3. Structure activity relationship
By comparing the experimental cytotoxicity of the compounds
reported in this study to their structures, the following structure
activity relationships (SAR) were postulated.
showed the percentage viability IC50 at 21.2 and 12.4
mg/ml,
respectively. Remarkable inhibitory activity was also demonstrated
by compounds 12b and 17.
The lung fibroblasts WI 38 cell line showed highest sensitivity
towards the tested compounds, as its growth was found to be
initiated by three compounds. The best activity was demonstrated
by compounds 2, 7a, and 12b which have IC50 at 24.4, 17.3, and
ꢁ The presence of a basic skeleton 1,3,4-oxadiazole is necessary
for the broad spectrum of cytotoxic activity towards different
cell lines (HepG2, WI 38, VERO and MCF-7).
ꢁ Introducing a thiazolidin-5-one ring in position 2 to 1,3,4-
oxadiazole reduced the activity towards HepG2 and dimin-
ished the activity towards WI38.
ꢁ Cyclization of thioamide 9 to 2,3-dihydrothiazole derivative 7a
enhances the antitumor activity against all cell lines.
ꢁ Conversion of ketene N,S-acetal 10 to 3-aminopyrazole deriv-
ative 11a increases the cytotoxic activity.
ꢁ Transformation of 1,3,4-oxadiazole ring system to pyrazolo
[5,1-c][1,2,4]triazole reduces the antitumor activity.
ꢁ Introducing of naphtho[2,1-b][1,4]oxazine in position 2 to
1,3,4-oxadiazole diminishes the activity against all cell lines.
24.1
mg/ml, respectively. The remaining compounds exhibited less
inhibitory activity with percentage inhibition range of 51e100
mg/ml.
On the other hand, Kidney carcinoma VERO was proved to be the
least sensitive among the cell lines tested as it was affected by the
only three test compounds. However, an outstanding growth
inhibition was shown by compound 7a with IC50 15.8 mg/ml. The
remaining two active compounds, namely 2 and 17 showed
moderate activity against the same cell line with IC50 30.3 and
34.5 mg/ml.
Further interpretation of the results revealed that compounds 2,
7a, 11a, 12b, 17 showed moderate anticancer activity against breast
4. Conclusion
cancer MCF-7 with percentage inhibition range of 26e50 mg/ml.
Compound 5a showed no cytotoxicity against all four cell lines.
The objective of the present study was to synthesize and
investigate the anticancer activity of some novel 1,3,4-oxadiazoles
with the hope of discovering new structure leads serving as anti-
cancer agents. The results of the anticancer screening revealed that
five compounds were found to exhibit variable degrees of anti-
cancer activities against the four used cell lines. Compounds 2 and
7a showed a considerable broad spectrum of anticancer activity
against the four tested human tumor cell lines. In particular
compound 7a proved to the most active member in this study with
special effectiveness against the human HepG2, VERO, and WI-38.
Compound 2 was found to possess high activity against HepG2
and moderate activity against MCF-7. In addition, compounds 2 and
7a showed high protection against DNA damage induced by the
Table 1
Cytotoxicity (IC50) of tested compounds on different cell lines.
Comp. no.
IC50 (m
g/ml)a
HepG2
WI-38
VERO
MCF-7
2
4
5a
5b
7a
8a
8b
9
10
11a
11b
12a
12b
15
17
19
21
5-Fu
21.2
51.2
250.2
145.6
12.4
70.2
90.7
100.2
125.4
33.7
98.7
69.5
38.1
65.6
35.6
189.8
111.4
8.6
24.4
52.4
340.5
175.0
17.3
84.6
89.6
84.6
98.7
38.4
90.5
71.2
24.1
65.0
35.8
180.7
90.9
3.2
30.3
50.3
256.6
169.8
15.8
76.4
100.6
82.4
132.4
36.3
98.8
95.6
39.6
69.8
34.5
189.2
95.4
6.5
39.2
69.2
410.6
166.8
25.8
75.5
100.5
77.5
107.3
38.3
99.1
49.9
37.4
66.8
32.6
221.8
96.9
2.3
Table 2
Results of the bleomycin-dependent DNA damage assay of
compounds 2 and 7a.
Sample
Absorbancea
2
7a
0.006 ꢂ 0.01
0.004 ꢂ 0.24
0.0038 ꢂ 0.32
Ascorbic acid
a
All experiments were performed three times. The data
are expressed as the meanꢀstandard error of the mean
(S.E.M.).
a
IC50 (mg/ml): 1e10 (very strong), 11e25 (strong), 26e50 (moderate), 51e100
(weak), 100e200 (very weak), Above 200 (non-cytotoxic).

196
S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
bleomycineiron complex, thus diminishing chromogen formation
between the damaged DNA and TBA.
reaction mixture was heated for 6 h, where upon the solid product
partially crystallized out. The reaction mixture was left to cool and
the separated solid product was filtered off, washed with water,
dried, and recrystallized from ethanol to give compounds 5a,b.
5. Experimental
All melting points were measured on a Gallenkamp electro-
thermal melting point apparatus. IR spectra were recorded for KBr
disc on a Mattson 5000 FTIR spectrophotometer. NMR spectra were
measured in CDCl3 or DMSO-d6 as solvent at 300 MHz (1H NMR) and
at 75 MHz (13C NMR) on a Varian MercuryVX300 NMR spectrometer
using TMS as an internal standard and chemical shifts are expressed
as dppm. Mass spectra were determined on GCMS-QP1000 EX spec-
trometer and JEOL JMS600 spectrometer at 70 eV. Elemental anal-
yses were carried out in the Microanalytical Unit of the Faculty of
Science, CairoUniversity. 4-Benzoyl-1-cyanoacetylthiosemicarbazide
(1) was prepared according to literature procedure [32].
5.3.1. N-(5-(Cyano-4e-((1,3-diphenyl-1H-pyrazol-4-yl)methylene-
5-oxo-3-phenylthiazolidin-2-ylidene)methyl)-1,3,4-oxadiazol-2-yl)
benzamide (5a)
Yellow powder; yield 69%; m.p. 120e121 ꢃC. IR (KBr):
n
/cmꢀ1 ¼3259 (NH), 2195 (CN),1700 (thiazolidin-5-one),1671 (CO),
1602 (C]C), 1550 (C]N). 1H NMR (DMSO-d6): dppm ¼ 7.45e8.02
(m, 20H, AreH), 9.32 (s, 1H, pyrazole-H-5), 9.99 (s, 1H, CH]), 11.95
(s, 1H, NH). 13C NMR (DMSO-d6): dppm ¼ 90.2, 100.8, 115.6, 119.8,
122.7,128.3,128.8,129.1, 129.2,129.7,130.3,131.8,135.4,139.2,147.9,
150.1, 153.2, 164.8, 171.2, 185.2. MS (EI, 70 ev) m/z (%) ¼ 633 (Mþ,
6.8), 327 (2.3), 269 (2.5), 247 (100), 246 (70.2), 244 (17.8), 230 (3.2),
219 (7.9), 218 (21.9), 190 (3.9), 170 (7.4), 120 (8.6), 97 (13.4), 82 (9.2),
73 (77.9). Anal. Calcd. for C36H23N7O3S (633.68): C, 68.23; H, 3.66; N,
15.47%; Found: C, 68.18; H, 3.64; N, 15.41%.
5.1. Synthesis of N-(5-(cyanomethyl)-1,3,4-oxadiazol-2-yl)
benzamide (2)
A
mixture of 4-benzoyl-1-cyanoacetylthiosemicarbazide
1
5.3.2. N-(5-(Cyano(-4e-((1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)methylene)-5-oxo-3-phenylthiazolidin-2-
ylidene)methyl)-1,3,4-oxadiazol-2-yl)benzamide (5b)
(2.62 g, 0.01 mol) and excess yellow mercuric oxide (3.25 g,
0.015 mol) in ethanol (30 mL) was refluxed for 4 h. The reaction
mixture was allowed to cool to room temperature (to allow the
sedimentation of the black mercuric sulfide), was filtered and the
mercuric sulfide was washed with hot ethanol. The filtrate and
alcoholic washing were combined, treated with water until
a permanent turbidity existed, and allowed to stand overnight. The
product was separated, filtered off, dried, and crystallized from
ethanol to give compound 2.
Orange powder; yield 62%; m.p. 184e185 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3403 (NH), 2208 (CN), 1697 (thiazolidin-5-one), 1674
(CO), 1590 (C]C). 1H NMR (DMSO-d6): dppm ¼ 2.36 (s, 3H, CH3),
3.32 (s, 3H, CH3), 7.25e7.46 (m, 15 H, AreH), 9.89 (s, 1H, CH]),
12.02 (s, 1H, NH). MS (EI, 70 ev) m/z (%) ¼ 601 (Mþ, 10), 510 (0.1),
451 (0.2), 401 (0.9), 339.8 (2.6), 297.7 (0.6), 268 (34.4), 256 (2.6),
236 (19.3), 212 (1.5), 194 (6.2), 188 (11.4), 150 (4.6), 135 (19.6), 105
(7.5), 102.8 (27.7), 95 (100.0), 88 (37.6), 76 (75.4), 69 (11.0), 60 (6.6).
Anal. Calcd. for C32H23N7O4S (601.63): C, 63.88; H, 3.85; N, 16.30%;
Found: C, 63.93; H, 3.89; N, 16.37%.
Pale yellow powder; Yield 85%; mp 210e211 ꢃC; IR (KBr) ymax
/
cmꢀ1 ¼ 3320 (NH), 2263 (CN), 1695 (CO), 1631 (C]N). 1H NMR
(DMSO-d6): dppm ¼ 4.68 (s, 2H, CH2CN), 7.55e8.05 (m, 5H, AreH),
12.15 (s, 1H, NH). 13C NMR (DMSO-d6): dppm ¼ 16.1, 115.0, 128.8,
129.9, 132.7, 133.5, 155.9, 159.2, 165.6. MS (EI, 70 ev) m/z (%): 228
(Mþ, 16.5), 227 (25.5), 159 (0.3), 137 (2.2), 122 (8.2), 95 (100), 89
(5.4), 77 (4.9), 76 (33.3), 65 (1.2). Anal. Calcd. for C11H8N4O2
(228.21): C, 57.89; H, 3.53; N, 24.55%; Found: C, 57.92; H, 3.57; N,
24.58%.
5.4. Synthesis of 2,3-dihydrothiazoline derivates 7a,b
To a stirred solution of potassium hydroxide (60 mg, 1 mmol) in
dimethylformamide (15 mL), compound 2 (0.23 g, 1 mmol) was
added. After stirring for 30 min, phenyl isothiocyanate (0.135 g,
1 mmol) was added to the resulting mixture. The stirring was
5.2. Synthesis of N-(5-(cyano(5-oxo-3-phenylthiazolidin-2-yl)
continued for a further 6 h, then the appropriate a-chloroketones
methyl)-1,3,4-oxadiazol-2-yl)benzamide (4)
namely chloroacetone or phenacyl chloride (1 mmol) was added
and the reaction mixture was further stirred overnight then diluted
with cold water (5 mL). The solid product was filtered off, washed
with water, dried, and finally recrystallized from DMF/EtOH to
afford the corresponding 2,3-dihydrothiazoline derivatives 7a,b.
A mixture of compound 2 (2.28 g, 0.01 mol), KOH (0.56 g,
0.01 mol), and phenyl isothiocyanate (1.35 g, 0.01 mol) was stirred
in DMF (20 ml) at 0e5 ꢃC for 10 h. Simultaneously chloroacetyl
chloride (1.13 g, 0.01 mol) was added dropwise, and stirring was
continued for 4 h. The reaction mixture was poured onto crushed
ice. The formed precipitate was filtered off, dried, and recrystallized
from ethanol to afford compound 4.
5.4.1. N-(5-(Cyano(4-methyl-3-phenylthiazol-2(3H)-ylidene)
methyl)-1,3,4-oxadiazol-2-yl)benzamide (7a)
Yellow powder; yield 81%; m.p. 175e176 ꢃC. IR (KBr):
Pale yellow crystals; yield 86%; m.p. 140e141 ꢃC. IR (KBr):
n
/cmꢀ1 ¼3418 (NH), 2199 (CN),1685 (CO),1658 (C]C),1600 (C]N).
n
/cmꢀ1 ¼ 3448 (NH), 2221 (CN),1736 (thiazolidin-5-one),1666 (CO),
1H NMR (CDCl3): dppm ¼ 1.95 (s, 3H, CH3), 5.15 (s, 1H, CH, thiazole),
7.27e8.11 (m, 10H, AreH), 10.02 (s, 1H, NH). MS (EI, 70 ev) m/z
(%) ¼ 401 (Mþ, 7.9), 376 (6.1), 346 (2.9), 334 (11.4), 308 (0.6), 287
(7.1), 264 (2.1), 256 (15.0), 239.5 (5.3), 229.6 (40.9), 214 (11.0), 183
(5.9),152 (3.8),135(12.0),105 (3.0), 95 (100.0), 76 (51.0), 73 (15.3), 65
(2.5). Anal. Calcd. for C21H15N5O2S (401.44): C, 62.83; H, 3.77; N,
17.45%; Found: C, 62.91; H, 3.73; N, 17.42%.
1631 (C]C), 1596 (C]N). 1H NMR (DMSO-d6): dppm ¼ 4.24 (s, 2H,
SCH2), 7.24e8.01 (m, 10H, AreH), 12.01 (s, 1H, NH). MS (EI, 70 ev)
m/z (%) ¼ 403 (Mþ, 7.0), 340 (10.1), 297 (8.8), 226 (5.3), 193 (14.0),
149 (97.4), 105 (100), 77 (63.6). Anal. Calcd. for C20H13N5O3S
(403.41): C, 59.55; H, 3.25; N, 17.36%; Found: C, 59.53; H, 3.21; N,
17.32%.
5.3. Reaction of thiazolidin-5-one derivative 4 with aromatic
aldehydes
5.4.2. N-(5-(Cyano(3,4-diphenylthiazol-2(3H)-ylidene)methyl)-
1,3,4-oxadiazol-2-yl)benzamide (7b)
Orange powder; yield 85%; m.p. 224e225 ꢃC. IR (KBr):
To a solution of compound 4 (0.41 g, 0.001 mol) and anhydrous
sodium acetate (0.12 g, 0.0015 mol) in glacial acetic acid (10 mL),
was added an appropriate aromatic aldehydes (0.001 mol). The
n
/cmꢀ1 ¼3405 (NH), 2198 (CN),1689 (CO),1645 (C]C),1600 (C]N).
1H NMR (CDCl3): dppm ¼ 5.16 (s, 1H, CH, thiazole), 7.19e8.13 (m,15H,
AreH), 10.26 (s, 1H, NH). MS (EI, 70 ev) m/z (%) ¼ 463 (Mþ, 17.0), 376

S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
197
(6.1), 346 (2.9), 334 (11.4), 308 (0.6), 287 (7.1), 264 (2.1), 256 (15.0),
239.5 (5.3), 229.6 (40.9), 214 (11.0),183 (5.9),152 (3.8),135 (12.0),105
(3.0), 95 (100.0), 76 (51.0), 73 (15.3), 65 (2.5). Anal. Calcd. for
C26H17N5O2S (463.51): C, 67.37; H, 3.70; N,15.11%; Found: C, 67.41; H,
3.71; N, 15.22%.
added. After stirring for 30 min, phenyl isothiocyanate (0.135 g,
1 mmol) was added to the resulting mixture. The stirring was
continued for a further 6 h, then methyl iodide (0.141 g, 1 mmol)
was added and the reaction mixture was further stirred 4 h then
diluted with cold water (5 mL). The solid product was filtered off,
washed with water, dried, and finally recrystallized from ethanol to
afford compound 10.
5.5. Synthesis of the thioacetanilide derivative 9
Yellow crystals; yield 90%; m.p. 200e201 ꢃC. IR (KBr):
To a stirred solution of potassium hydroxide (0.11 g, 2 mmol) in
dimethylformamide (20 mL), compound 2 (0.46 g, 2 mmol) was
added. After stirring for 30 min, phenyl isothiocyanate (0.27 g,
2 mmol) was added to the resulting mixture. Stirring was continued
for 6 h, then poured over crushed ice containing hydrochloric acid.
The solid product that formed was filtered off, washed with water,
dried, and finally recrystallized from ethanol to afford compound 9.
Yellow crystals; yield 83%; m.p. 160e161 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3430 (NH), 3218 (NHePh), 2206 (CN), 1704 (CO), 1630
(C]C), 1580 (C]N). 1H NMR (DMSO-d6): dppm ¼ 2.31 (s, 3H, SCH3),
7.23e7.99 (m, 10H, AreH), 10.46 (s, 1H, NH), 11.94 (s, 1H, NH, ami-
dic). MS (EI, 70 ev) m/z (%) ¼ 377 (Mþ, 56.7), 377 (3.5), 364 (30.0),
340 (48.4), 326 (18.0), 305 (8.0), 283 (25.0), 265 (4.9), 256 (10.5),
242 (31.6), 231 (16.0), 227 (25.8), 197 (5.0), 195 (30.0), 170 (14.9),
160 (3.6), 153 (30.5), 126 (8.6), 99 (100.0), 92 (3.9), 85 (17.9), 73
(38.0), 63 (2.5). Anal. Calcd. for C19H15N5O2S (377.42): C, 60.46; H,
4.01; N, 18.56%; Found: C, 60.41; H, 4.07; N, 18.53%.
n
/cmꢀ1 ¼ 3477 (NH), 3334 (NHePh), 2196 (CN), 1700 (CO), 1621
(C]C), 1590 (C]N). 1H NMR (DMSO-d6): dppm ¼ 7.09e8.02 (m, 10H,
AreH), 9.98 (s, 1H, NHPh), 12.17 (s, 1H, NHCO), 14.12 (s, 1H, SH). MS
(EI, 70 ev) m/z (%) ¼ 363 (Mþ, 10.0), 327 (1.0), 299 (1.5), 256 (10.0),
239.8 (4.4), 216 (6.0), 186 (12.8), 167 (4.3), 147 (21.6), 135 (16.0), 122
(6.3), 103 (44.4), 95 (100), 88 (13.8), 76 (72.5), 63 (5.9), 57 (29.0), 55
(16.6). Anal. Calcd. for C18H13N5O2S (363.39): C, 59.49; H, 3.61; N,
19.27%; Found: C, 59.46; H, 3.59; N, 19.19%.
5.8. Reaction of N-(5-(1-cyano-2-(methylthio)-2-(phenylamino)
vinyl)-1,3,4-oxadiazol-2-yl)benzamide (10) with hydrazines
A mixture of an equimolar amounts of 10 (0.38 g, 0.001 mol) and
hydrazine derivatives namely; hydrazine hydrate (0.05 g,
0.001 mol) or phenyl hydrazine (0.11 g, 0.001 mol) in ethanol
(20 mL) was refluxed for 4 h. The reaction mixture was left to cool
then poured onto ice-cold water (10 mL), filtered off, washed with
water, dried, and recrystallized from ethanol to afford compounds
11a and 11b, respectively.
5.6. Reaction of thioacetanilide derivative 9 with a-haloketones
To a solution of 9 (0.73 g, 2 mmol) in ethanol (20 mL), the
appropriate -chloroketone namely; chloroacetone or phenacyl
a
chloride (2 mmol) was added followed by few drops of triethyl-
amine. The mixture was refluxed for 2 h, then allowed to cool to
room temperature. The formed solid was filtered off, washed with
ethanol, and recrystallized from EtOH/DMF to afford the corre-
sponding thiophene derivatives 8a,b.
5.8.1. N-(5-(3-amino-5-(phenylamino)-1H-pyrazol-4-yl)-1,3,4-
oxadiazol-2-yl)benzamide (11a)
Colorless powder; yield 76%; m.p. 184e185 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3448e3375 (NH2), 3343, 3220 (2NH), 1675 (CO), 1600
(C]N). 1H NMR (DMSO-d6): dppm ¼ 6.93 (bs, 2H, NH2), 7.32e7.83
(m, 10H, AreH), 8.96 (s, 1H, NHepyrazole), 10.32 (s, 1H, NH), 11.53
(s, 1H, NH, amidic). 13C NMR (DMSO-d6): dppm ¼ 88.9, 115.9, 116.3,
118.9, 121.9, 125.9, 129.2, 130.6, 142.8, 144.4, 150.5, 151.2, 166.2,
170.6. MS (EI, 70 ev) m/z (%) ¼ 361 (Mþ, 10.5), 303 (1.68), 258 (2.13),
243 (45.7), 174 (100.0), 160 (80.7), 104 (82.4), 77 (74.9). Anal. Calcd.
for C18H15N7O2 (361.36): C, 59.83; H, 4.18; N, 27.13%; Found: C,
59.86; H, 4.22; N, 27.18%.
5.6.1. N-(5-(5-acetyl-4-amino-2-(phenylamino)thiophen-3-yl)
1,3,4-oxadiazol-2-yl)benzamide (8a)
Yellow powder; yield 67%; m.p. 138e139 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3417e3334 (NH2), 3207, 3185 (2NH), 1697 (CO), 1658
(CONh), 1580 (C]C). 1H NMR (CDCl3) dppm ¼ 2.25 (s, 3H, CH3), 6.47
(s, 2H, NH2), 6.99e7.56 (m, 10H, AreH), 9.81 (bs, 1H, NH), 11.96 (s,
1H, NH amidic). MS (EI, 70 ev) m/z (%) ¼ 419 (Mþ, 1.7), 344 (1.5), 285
(1.7), 253 (100), 225 (2.2), 208 (4.3), 181 (12.6), 117 (8.3), 105 (6.5),
99 (14.2), 91 (11.9), 84 (18.9), 73 (33.0), 69 (15.5). Anal. Calcd. for
C21H17N5O3S (419.46): C, 60.13; H, 4.09; N, 16.70%; Found: C, 60.18;
H, 4.12; N, 16.67%.
5.8.2. N-(5-(3-amino-1-phenyl-5-(phenylamino)-1H-pyrazol-4-
yl)-1,3,4-oxadiazol-2-yl)benzamide (11b)
Colorless powder; yield 69%; m.p. 287e288 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3444e3385 (NH2), 3203 (NH), 1704 (CO), 1600 (C]N). 1H
NMR (CDCl3): dppm ¼ 6.25 (bs, 2H, NH2), 7.27e7.45 (m, 15H, AreH),
9.07 (s,1H, NH). MS (EI, 70 ev) m/z (%) ¼ 437 (Mþ, 4.2), 347 (1.6), 333
(34.7), 319 (5.9), 294 (0.7), 286 (11.7), 263 (5.4), 255 (39.0), 229
(39.1), 224 (10.4), 221 (2.3), 197 (9.9), 188 (15.6), 169 (7.6), 129 (1.5),
103 (80.0), 100 (20.5), 95 (100.0), 87 (4.7), 76 (66.2), 65 (4.4). Anal.
Calcd. for C24H19N7O2 (437.45): C, 65.89; H, 4.38; N, 22.41%; Found:
C, 65.92; H, 4.42; N, 22.49%.
5.6.2. N-(5-(4-amino-5-benzoyl-2-(phenylamino)thiophen-3-yl)
1,3,4-oxadiazol-2-yl)benzamide (8b)
Pale yellow powder; yield 74%; m.p. 270e271 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3463e3411 (NH2), 3353, 3278 (2NH), 1706 (COPh), 1660
(CONH), 1600 (C]C). 1H NMR (DMSO-d6) dppm ¼ 7.15 (bs, 2H, NH2),
7.42e8.05 (m,15H, AreH), 9.68 (s,1H, NH),12.15 (s,1H, NH, amidic).
13C NMR (DMSO-d6): dppm ¼ 93.6, 121.8, 125.9, 127.5, 128.8, 129.0,
129.2, 130.3, 131.0, 132.8, 133.5, 140.3, 141.7, 155.2, 156.3, 157.2,
159.2, 165.2, 185.3. MS (EI, 70 ev) m/z (%) ¼ 481 (Mþ, 4.0), 377 (1.7),
335 (1.5), 319 (4.3), 215 (0.9), 160 (1.4), 105 (100), 77 (95.9). Anal.
Calcd. for C26H19N5O3S (481.53): C, 64.85; H, 3.98; N, 14.54%;
Found: C, 64.92; H, 4.03; N, 14.51%.
5.9. Reaction of compound 2 with aromatic aldehydes
A few drops of triethylamine were added to an ethanolic solu-
tion (25 mL) of compound 2 (0.23 g, 1 mmol) and each of p-ani-
saldehyde (0.14 g, 1 mmol) and 1,3-diphenyl-1H-pyrazole-4-
carbaldehyde (0.25 g, 1 mmol), and the reaction mixture was
refluxed for 6 h. The solvent was evaporated under reduced pres-
sure, and the residue was treated with ethanol. The solid product
was filtered off, washed with ethanol, dried, and purified by
recrystallization from ethanol to afford compounds 12a,b.
5.7. Synthesis of (N-(5-1-cyano-2-(methylthio)-2-(phenylamino)
vinyl)-1,3,4-oxadiazol-2-yl)benzamide (10)
To a stirred solution of potassium hydroxide (60 mg, 1 mmol) in
dimethylformamide (15 mL), compound 2 (0.23 g, 1 mmol) was

198
S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
5.9.1. N-(5-(1-cyano-2-(4-methoxyphenyl)vinyl)-1,3,4-oxadiazol-
2-yl)benzamide (12a)
(CO), 1615 (C]N). 1H NMR (DMSO-d6) dppm ¼ 2.61 (s, 2H, CH2), 6.86
(s, 2H, NH2), 7.21e7.87 (m, 5H, AreH), 12.13 (s,1H, amidic NH). Anal.
Calcd. for C11H10N6O (242.24): C, 54.54; H, 4.16; N, 34.69%; Found:
C, 54.47; H, 4.08; N, 34.57%.
Yellow crystals; yield 74%; m.p. 270e271 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3405 (NH), 2206 (CN), 1695 (CO), 1622 (C]C), 1582 (C]
N). 1H NMR (CDCl3) dppm ¼ 3.92 (s, 3H, OCH3), 7.29e7.82 (m, 5H,
AreH), 8.23 (s, 1H, CH]), 8.42 (d, J ¼ 6.8 Hz, 2H, AreH), 8.53 (d,
J ¼ 6.8 Hz, 2H, AreH), 11.13 (s, 1H, exchangeable with D2O, NH,
amidic). MS (EI, 70 ev) m/z (%) ¼ 346 (Mþ, 2.7), 345 (Mþ ꢀ 1, 22.0),
341 (10.9), 340 (4.4), 310 (2.6), 309 (10.9), 283 (1.0), 269 (4.3), 231
(2.4), 228 (18.2), 203 (15.2), 198 (1.2), 186 (22.9), 170 (3.4), 146 (2.6),
122 (2.3), 103 (8.0), 95 (100.0), 82 (5.9), 76 (36.9), 64 (2.3). Anal.
Calcd. for C19H14N4O3 (346.34): C, 65.89; H, 4.07; N, 16.18%; Found:
C, 65.82; H, 4.04; N, 16.18%.
5.12. Synthesis of coumarin 17 and naphthoxazines 19 and 21
derivatives
To a solution of compound 2 (0.23 g, 1 mmol) in ethanol
(25 mL) containing a few drops of piperidine, either salicylalde-
hyde (0.12 g, 1 mmol), 1-nitroso-2-naphthol (0.173 g, 1 mmol), or
2-nitroso-1-naphthol (0.173 g, 1 mmol) was added. The reaction
mixture, in each case, was heated under reflux for 4 h. The solid
products formed upon pouring onto an ice-water mixture con-
taining few drops of hydrochloric acid was collected by filtration,
dried, and recrystallized from ethanol to afford compounds
16e18.
5.9.2. N-(5-(1-cyano-2-(1,3-diphenyl-1H-pyrazol-4-yl)vinyl)-1,3,4-
oxadiazol-2-yl)benzamide (12b)
Yellow powder; yield 81%; m.p. 307e308 ꢃC. IR (KBr):
n
/cmꢀ1 ¼3422 (NH), 2205 (CN),1697 (CO),1626 (C]C),1589 (C]N).
1H NMR (DMSO-d6) dppm ¼ 7.29e8.02 (m, 15H, AreH), 8.41 (s, 1H,
CH]), 9.25 (s, 1H, pyrazoleeH5), 12.31 (s, br., 1H, NH). MS (EI, 70 ev)
m/z (%) ¼ 458 (Mþ, 5.5), 425 (1.3), 382 (1.6), 353 (1.0), 340 (12.2), 295
(1.8), 268 (1.1), 248 (8.4), 227 (24.4),188 (1.5),158 (1.5),122 (1.8),115
(5.5), 95 (100.0), 88 (6.8), 76 (36.5), 65 (1.9). Anal. Calcd. for
C27H18N6O2 (458.47): C, 70.73; H, 3.96; N,18.33%; Found: C, 70.78; H,
4.02; N, 18.39%.
5.12.1. N-(5-(2-oxo-2H-chromen-3-yl)-1,3,4-oxadiazol-2-yl)
benzamide (17)
Orange Crystals; yield 72%; m.p. 185e186 ꢃC. IR (KBr):
n
/cmꢀ1 ¼3345 (NH),1735 (CO),1671 (CO),1605 (C]C),1543 (C]N).
1H NMR (DMSO-d6) dppm ¼ 7.13e7.76 (m, 9H, AreH), 8.43 (s, 1H,
coumarin-H4),10.68 (s,1H, NH, amidic). MS (EI, 70 ev) m/z (%) ¼ 332
(Mþ ꢀ 1, 3.9),186 (10.8),130 (7.7),105 (38.8), 51 (100). Anal. Calcd. for
C18H11N3O4 (333.3): C, 64.86; H, 3.33; N, 12.61%; Found: C, 64.95; H,
3.35; N, 12.68%.
5.10. Synthesis of pyrazolyleoxadiazoles 13a,b
Hydrazine hydrate (80%, 0.1 mL, 1 mmol) was added to an
equimolar amounts of solution of arylidene derivatives 12aeb
(1 mmol) in 20 mL of ethanol. The reaction mixture was heated
under reflux for 4 h, then left to cool. The solid product obtained
was filtered off, washed with ethanol, dried, and recrystallized from
ethanol to afford compounds 13a,b.
5.12.2. N-(5-(3-oxo-3H-naphtho[2,1-b][1,4]oxazin-2-yl)-1,3,4-
oxadiazol-2-yl)benzamide (19)
Violet crystals; yield 85%; m.p. 215e216 ꢃC. IR (KBr):
n
/cmꢀ1 ¼3336 (NH),1730 (CO),1668 (CO),1602 (C]C),1583 (C]N).
1H NMR (DMSO-d6) dppm ¼ 7.27e8.36 (m, 11H, AreH), 11.22 (s, 1H,
NH, amidic). 13C NMR (DMSO-d6): dppm ¼ 117.5, 118.2, 120.8, 121.6,
123.3,125.5,126.2,127.6,128.3,128.7,129.3,131.0,131.2,133.9,150.4,
155.0,158.4,164.2,165.4. MS (EI, 70 ev) m/z (%) ¼ 384 (Mþ, 20.0), 322
(40.1), 305 (20.3), 277 (20.2), 196 (20.0), 170 (100.0), 154 (40), 133
(40.3), 105 (12.7), 83 (20.1), 69 (40.1). Anal. Calcd. for C21H12N4O4
(384.34): C, 65.62; H, 3.15; N, 14.58%; Found: C, 65.67; H, 3.21; N,
14.66%.
5.10.1. N-(5-(3-amino-5-(4-methoxyphenyl)-1H-pyrazol-4-yl)-
1,3,4-oxadiazol-2-yl)benzamide (13a)
Colorless powder; yield 76%; m.p. 212e213 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3407 (NH), 3276e3196 (NH2), 3141 (NH), 1686 (CO), 1598
(C]N). 1H NMR (DMSO-d6) dppm ¼ 3.91 (s, 3H, OCH3), 6.65 (s, 2H,
NH2), 7.26e8.17 (m, 9H, AreH), 8.47 (s, 1H, pyrazoleeNH), 11.12
(s, 1H, amidic NH). Anal. Calcd. for C19H16N6O3 (376.37): C, 60.63; H,
4.28; N, 22.33%; Found: C, 60.58; H, 4.18; N, 22.28%.
5.12.3. N-(5-(2-oxo-2H-naphtho[1,2-b][1,4]oxazin-3-yl)-1,3,4-
oxadiazol-2-yl)benzamide (21)
Violet crystals; yield 78%; m.p. 302e303 ꢃC. IR (KBr):
5.10.2. N-(5-(5-amino-10,30-diphenyl-10H,2H-3,40-bipyrazol-4-yl)-
1,3,4-oxadiazol-2-yl)benzamide (13b)
n
/cmꢀ1 ¼3305 (NH),1729 (CO),1674 (CO),1609 (C]C),1589 (C]N).
1H NMR (DMSO-d6) dppm ¼ 7.14e8.14 (m, 11H, AreH), 11.17 (s, 1H,
NH, amidic). Anal. Calcd. for C21H12N4O4 (384.34): C, 65.62; H, 3.15;
N, 14.58%; Found: C, 65.70; H, 3.12; N, 14.51%.
Colorless powder; yield 71%; m.p. 220e221 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3423e3395 (NH2), 3345 (NH), 3141 (NH), 1674 (CO), 1598
(C]N). 1H NMR (DMSO-d6) dppm ¼ 6.15 (s, 2H, NH2), 7.28e8.35 (m,
15H, AreH), 8.68 (s, 1H, pyrazoleeH5), 10.89 (s, 1H, pyrazoleeNH),
11.19 (s, 1H, amidic NH). MS (EI, 70 ev) m/z (%) ¼ 488 (Mþ, 44.2), 388
(5.6), 360 (2.1), 322 (5.8), 246 (82.5), 216 (4.7),116 (18.6), 77 (100.0).
Anal. Calcd. for C27H20N8O2 (488.5): C, 66.38; H, 4.13; N, 22.94%;
Found: C, 66.32; H, 4.08; N, 22.87%.
6. Cytotoxicity and antitumor evaluation
6.1. Materials and methods
The reagents RPMI-1640 medium (Sigma Co., St. Louis, USA)
Fetal Bovine serum (GIBCO, UK) and the cell lines HepG2, WI38,
VERO, MCF-7 which obtained from ATCC were used.
5.11. Synthesis of N-(6-amino-7H-pyrazolo[5,1-c][1,2,4]triazol-3-
yl)benzamide (15)
6.1.1. Procedure
A mixture of compound 2 (0.23 g, 1 mmol) and hydrazine
hydrate (80%, 0.2 mL, 2 mmol) was heated in an oil bath at 160 ꢃC
for 1 h, then left to cool. The obtained solid product was triturated
with ethanol (10 mL), filtered off, washed with ethanol, dried, and
recrystallized from DMF to afford compound 15.
The stock samples were diluted with RPMI-1640 medium to
desired concentrations ranging from 10 to 1000 mg/mL. The final
concentration of dimethylsulfoxide (DMSO) in each sample did not
exceed 1% v/v. The cytotoxic activity of the compounds was tested
against human hepatocellular liver carcinoma cell line (HepG2),
human lung fibroblast cell line (WI 38), human caucasian breast
adeno-carcinoma cell line (MCF-7), and normal adult African green
Colorless powder; yield 65%; m.p. 263e264 ꢃC. IR (KBr):
n
/cmꢀ1 ¼ 3386e3304 (NH2), 3165 (NH), 2986 (CH, aliphatic), 1675

S. Bondock et al. / European Journal of Medicinal Chemistry 48 (2012) 192e199
199
monkey kidney cell line (VERO). The % viability of cell was exam-
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Appendix. Supplementary material
Supplementary material associated with this article can be
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