Utilization of 2-Chloronicotinonitrile in the Syntheses of Novel Fused Bicyclic and Polynuclear Heterocycles of Anticipated Antitumor Activity

Article abstract of DOI:10.1002/jhet.1631

Some of synthetic bicyclic and polynuclear heterocyclic compounds based on pyridine scaffold were tested as in vitro antitumor agents, and these compounds found to exhibit interesting activities. Fused heterocycles containing sulfur such as thienopyridine

Full text of DOI:10.1002/jhet.1631

May 2016
Utilization of 2-Chloronicotinonitrile in the Syntheses of Novel Fused
Bicyclic and Polynuclear Heterocycles of Anticipated Antitumor Activity
953
a
b
b
a
*
Wafaa S. Hamama, Mohamed A. Waly, Ibrahim I. El-Hawary, and Hanafi H. Zoorob
aChemistry Department, Faculty of Science, Mansoura University, Mansoura ET-35516, Egypt
bChemistry Department, Faculty of Science, Mansoura University, Damietta, Egypt
*
E-mail: wshamama@yahoo.com
Received October 16, 2011
DOI 10.1002/jhet.1631
Published online 21 January 2016 in Wiley Online Library (wileyonlinelibrary.com).
CH3 NH
NH CH3
CH3
N
CH3
O
S
O
N
N
NH
R
H3C
N
N
S
S
S
N
CH3
N
H3C
H3C
N
11
5, R= COOEt
6, R= H
7
O
12
Some of synthetic bicyclic and polynuclear heterocyclic compounds based on pyridine scaffold were
tested as in vitro antitumor agents, and these compounds found to exhibit interesting activities. Fused hetero-
cycles containing sulfur such as thienopyridine, pyridothiazine, and some related fused heterocycles as new
ring systems were achieved. Bicyclic thienopyridine 6 and pyrido[3,2-e][1,3]thiazin-4(3H)-one 7 exhibited
good antitumor activities compared with 5-fluorouracil.
J. Heterocyclic Chem., 53, 953 (2016).
INTRODUCTION
2,3-dihydrothieno[2,3-b]pyridine-2-carboxylate (5), with
carefully working up at ꢀ20ꢁC. Attempting for working
up at 0ꢁC causes the hydrolysis and decarboxylation of
Several pyridine ring systems “binary and fused” have
attracted the attention of several research groups because
of their potential biological activities as anti-inflammatory
[1–4], anticancer [5–9], and antimicrobial agents [6]. Re-
cently, we have reported a modified synthetic method for
the synthesis of 2-chloro-4,6-dimethylnicotinonitrile (1)
through reaction of acetylacetone with malononitrile. Com-
pound 1 was used as a starting material for the syntheses of
fused or binary heterocycles containing nitrogen [10]. It
was reported that the fused 5,7-dimethyl-[1,2,4]triazolo
[4,3-a]pyridine-8-carbonitrile (2) have higher affinity than
5-fluorouracil [10]. In this work, our aim is to obtain novel
biologically active heterocyclic compounds [11] through
building up different fused bicyclic and polynuclear
heterocyclic ring systems containing sulfur on the basis
of 2-chloropyridine. The reaction of compound 1 with thio-
urea in refluxing ethanol afforded 2-mercapto-4,6-
dimethylnicotinonitrile (3) [12] (Fig. 1).
the b-ketoester derivative
5
to give the 4,6-
dimethylthieno[2,3-b]pyridin-3(2H)-one (6). Compound 6
was obtained in this route through hydrolysis and decar-
boxylation of compound 5 in a mixture of acetic acid and
hydrochloric acid. Another route for the preparation of
compound 6 was achieved by direct alkylation of com-
pound 3 with ethyl chloroacetate and cyclization in the
presence of sodium hydride as a base in a one-pot synthesis
(Scheme 1). The 1H NMR spectrum of 6 revealed the pres-
ence of singlet signal at d 3.8 ppm corresponding to meth-
ylene protons of thiophene ring.
The bicyclic fused six-membered ring system was
achieved through Schmidt rearrangement for compound
6. Refluxing of 6 with sodium azide in DMF in the pres-
ence of few drops of H2SO4 afforded 5,7-dimethyl-2H-pyr-
ido[3,2-e][1,3]thiazin-4(3H)-one (7) (Scheme 1). The IR
spectrum of 7 showed absorption band at 1684 cmꢀ1
1
corresponding to amide carbonyl group, whereas its H
RESULTS AND DISCUSSION
NMR spectrum revealed the presence of singlet signal at
d 3.33 ppm because of methylene protons of thiazine ring
and a signal at d 13.8 ppm because of NH proton.
Some newly fused polynuclear heterocycle-incorporated
thienopyridine moieties of anticipated antitumor activity were
further synthesized. Thus, refluxing of compound 3 with 2-
(1H-benzo[d]imidazol-2-yl)-4-chloro-3-oxobutanenitrile (8)
[13,14] in DMF containing sodium carbonate afforded
benzo[d]imidazol-thieno[2,3-b:4,5-b0]dipyridine derivative
The synthetic procedures adopted to obtain the target
compounds were depicted in Schemes 1–3. Synthesis of
five fused bicyclic ring systems was achieved via the alkyl-
ation of pyridinethiol derivative 3 with ethyl chloroacetate
in the presence of sodium carbonate to afford the acetate
ester derivative 4 (which is consistent with the published
data [12]). Refluxing of a solution of acetate ester 4 with
sodium hydride in THF gave ethyl 4,6-dimethyl-3-oxo-
© 2016 HeteroCorporation

954
W. S. Hamama, M. A. Waly, I. I. El-Hawary, and H. H. Zoorob
Vol 53
CH3
CH3
CH3
10. Compound 10 was obtained through intramolecular
cyclization of the intermediate 9 (Scheme 2). The struc-
ture 10 was elucidated by IR spectrum that showed absorp-
tion bands at 1660cmꢀ1 because of carbonyl group and
bands at 3392, 3327, and 3290cmꢀ1 because of NH2 and
CN
CN
Cl
CN
SH
H3C
N
N
N
H3C
N
H3C
N
1
2
3
1
NH groups, respectively, whereas its H NMR spectrum
showed signals at d 4.2 ppm because of NH2 protons and
Figure 1. Structures of different heterocycles containing pyridine moiety.
Scheme 1. Synthesis of thieno[2,3-b]pyridinone 5 and pyrido[3,2-e][1,3]thiazin-4(3H)-one 7.
CH3
DMF/Na2CO3
CH3
O
1) THF/NaH
2) HCl, 0oC
CN
S
COOEt
COOEt
S
H3C
N
COOEt
H3C
N
CH3
4
5
Cl
CN
SH
AcOH/HCl
COOEt
CH3
CH3
O
S
H3C
N
O
S
Cl
3
NaN3
NH
H2SO4
1) THF/NaH
2) HCl, 0oC
H3C
N
H3C
N
6
7
Scheme 2. Synthesis of benzo[d]imidazol-thieno[2,3-b:4,5-b0]dipyridine derivative 10.
CH3
NH2
CN
CH3
CN
SH
O
N
DMF
N
+
H3C
N
S
Cl
Na2CO3
N
H
H3C
N
O
HN
CN
9
3
8
CH3
N
O
NH2
N
H3C
N
S
HN
10
Scheme 3. Reactions of compound 1 with different nucleophiles.
N
HOOC
CH3
NH CH3
CN
O
SH
N
N
H
HO
TEA
H3C
N
S
N
CH3
N
COOH
11
13
1
NH2
S
CH3
CH3 NH
CN
N
N
H
N
NH2
N
CH3
H2N
O
H3C
N
N
H
H3C
N
N
S
NC
O
14
12
CH3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2016
Novel Heterocycles Derived from 2-Chloronicotinonitrile
955
Table 1
multiplet signals at d 7.1–7.5ppm indicating the presence of
aromatic protons.
In vitro cytotoxicity of pyridine derivatives using Ehrlich ascites
cell assay.
In addition, refluxing of compound 1 with 1H-benzo[d]
imidazole-2-thiol in DMF catalyzed by triethylamine
afforded the corresponding benzo[4,5]imidazo[2,1-b]pyr-
ido[3,2-e][1,3]thiazine 11 in 66% yield (Scheme 3). Its
1H NMR spectrum showed multiplet signals at d 7.2–
7.9 ppm because of aromatic protons and signals at d
10.6 ppm because of imino proton.
Also, 5-imino-2,4-dimethyl-9H-pyrido[3,2-e]thiazolo[3,4-a]
pyrimidin-9-one (12) was obtained by refluxing of 1 with
2-aminothiazol-4(5H)-one in the presence of a few drops of
triethylamine (Scheme 3). The IR spectrum of 12 showed
absorption band at 1663 cmꢀ1 as a sharp peak indicated to
the presence of lactam group and a band at 3434 cmꢀ1
because of imino group. The 1H NMR spectrum of
12 showed signals at d 3.2 ppm corresponding to methylene
protons of thiazole moiety.
On the other hand, compound 1 was allowed to react
with salicylic acid in DMF in the presence of sodium car-
bonate to give 2-(3-cyano-4,6-dimethylpyridin-2-yloxy)
benzoic acid 13 (Scheme 3). The structure 13 was eluci-
dated by its correct elemental analysis and spectral data.
Its IR spectrum showed absorption band at 2213 cmꢀ1
indicated the presence of cyano group, at 3436 cmꢀ1
corresponding to hydroxyl group, and at 1680 cmꢀ1 for
carbonyl group. Its 1H NMR spectrum showed two singlet
signals at d 2.39 and 2.54 ppm because of two methyl
groups in pyridine ring and multiplet signals at d 7.1–
7.5 ppm because of aromatic protons.
% Dead
Compound no.
ED100 mL
ED50 mL
ED25 mL
5-FU
1
3
4
5
95.2
46.0
61.7
53.2
54.0
88.0
81.2
56.0
73.3
50.3
62.2
60.0
62.0
28.9
31.3
27.1
28.2
49.0
47.5
30.0
41.0
29.8
28.1
29.0
38.0
20.0
17.2
20.0
21.6
28.0
26.9
16.0
23.0
13.4
12.4
13.3
6
7
10
11
12
13
14
ED100, ED50, and ED25 are the effective doses at 25, 50, and 100 mL, re-
spectively, of the compounds used. The dead % refers to the % of the dead
tumor cells, and 5-FU is 5-fluorouracil as a well-known cytotoxic agent.
By comparing the cytotoxicity results in Table 1, the
following structure–activity relationships were drawn:
(i) fused sulfur heterocyclic ring systems increase the
activity of the tested compounds and (ii) the presence
of basic skeleton is necessary for the broad spectrum
of antitumor activity toward Ehrlich ascites carcinoma
cells.
Modification of produced fused bicyclic and polynu-
clear heterocycle compounds is potential for further
development as anticancer agents. With these preliminary
screening results, compounds 6 and 7 showed significant
activity in certain cancer cells and have been targeted for
further studies. Additional research, including mode of
action studies, is planned to accurately establish relative
activity for structure–activity relationships and rational
design.
Moreover, refluxing of 1 with ethylenediamine in aceto-
nitrile containing sodium carbonate afforded dimmeric
product 14 (Scheme 3). Its IR spectrum showed absorption
band as a sharp peak at 3311 cmꢀ1 because of (NH) group,
1
besides the presence of a sharp band at 2203 cmꢀ
corresponding to two symmetrical cyano groups. More-
over, its H NMR spectrum showed signals at d 5.4 ppm
1
because of two NH protons and at d 6.3 ppm because of
pyridyl proton.
CONCLUSION
The present investigation offers rapid and effective
novel procedures for the syntheses of a new class of fused
and ring junction pyridines. It involves reactions of 2-
mercapto-4,6-dimethylnicotinonitrile (3) with ethyl chlor-
oacetate at different conditions and a-halo ketone to
afford thieno[2,3-b]pyridinone, pyrido[3,2-e][1,3]thia-
zin-4(3H)-one, and benzo[d]imidazol-thieno[2,3-b:4,5-b0]
dipyridine ring systems. Moreover, reactions of 2-chloro-
nicotinonitrile (1) toward different nucleophiles were in-
vestigated. The newly prepared ring systems seem to be
interesting for biological studies. It is worth mentioning
that fused sulfur heterocyclic ring systems were crucial
for the antitumor activity as in the case of compounds
6 and 7.
BIOLOGICAL ACTIVITY
Antitumor activity. Effect of drugs on the viability of
Ehrlich ascites carcinoma cells in vitro.
To examine
whether these substances have a direct cytotoxic effect on
Ehrlich ascites cell (EAC) viability, the percentage of
viable cells was estimated by the trypan blue [15]
exclusion test. Eleven fused bicyclic and polynuclear
heterocycles were tested for cytotoxicity against EACs
in vitro. Results for the ED100, ED50, and ED25 values of
the active compounds are summarized in Table 1. The
data showed clearly that compounds 6 and 7 exhibited
more potent activities, whereas the other compounds
showed moderate activities compared with 5-fluorouracil.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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W. S. Hamama, M. A. Waly, I. I. El-Hawary, and H. H. Zoorob
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5,7-Dimethyl-2H-pyrido[3,2-e][1,3]thiazin-4(3H)-one (7).
A
EXPERIMENTAL
mixture of 6 (1.79 g, 0.01 mol) and sodium azide (1.0 g) in DMF
(30 mL) in the presence of a few drops of conc. H2SO4 was
refluxed with stirring for 5 h. After cooling, the reaction mixture
was poured onto ice water (50 mL). The precipitated solid was
filtered off, dried, and recrystallized from methanol to give
compound 7; (1.0 g, 51.5%), mp 280–282ꢁC. IR (KBr) υ
Melting points (uncorrected) (Electronic Melting Point Appa-
ratus, Great Britain, London) were determined on a Gallenkamp
melting point apparatus. The IR spectra were recorded on a Jasco
4100 FTIR spectrophotometer (Microanalytical Unit, Mansoura
University, Mansoura, Egypt) in KBr discs (υmax in cmꢀ1). The
1H NMR and 13C NMR spectra were recorded on a Bruker
Avance 300 spectrometer operating at 300 and 75 MHz by using
CDCl3 and DMSO-d6 as solvents, respectively. Chemical shifts
were expressed in d-scale downfield as part per million (ppm)
against TMS as an internal standard. Elemental analyses (C, H, N,
and S) were recorded on Perkin-Elmer 2400 elemental analyzer.
The completion of reaction and the purity of the compounds
were controlled by TLC on silica gel-precoated aluminum sheets
(Type 60 F254, Merck, Darmstadt, Germany), and spots were
exposed to iodine vapor. Ethyl 2-(3-cyano-4,6-dimethylpyridin-
2-ylthio)acetate (4) [12] and 2-(1H-benzo[d]imidazol-2-yl)-4-
chloro-3-oxobutanenitrile (8) [13,14] were prepared according
to the previously reported methods.
1
(cmꢀ1), 1684 (CO), 3450 (NH); H NMR (CDCl3): d/ppm, 2.41
(s, 3H, CH3), 2.50 (s, 3H, CH3), 3.33 (s, 2H, CH2), 6.34 (s, 1H),
13.8 (s, 1H, exchangeable NH). Anal. Calcd for C9H10N2OS
(194.25): C, 55.65; H, 5.19; N, 14.42; S, 16.51%. Found: C,
55.54; H, 5.24; N, 14.54; S, 16.42%.
2-Amino-3-(1H-benzo[d]imidazol-2-yl)-7,9-dimethylthieno
[2,3-b:4,5-b0]dipyridin-4(3H)-one (10).
A mixture of 3
(1.64 g, 0.01 mol), 2-(1H-benzo[d]imidazol-2-yl)-4-chloro-3-
oxobutanenitrile (8) (2.2 g, 0.01 mol), and sodium carbonate
(1.05 g, 0.01 mol) in DMF (50 mL) was refluxed with stirring
for 4 h. The reaction mixture was then poured onto ice–cold
water (50 mL), and the formed solid product was filtered off,
dried, and recrystallized from DMF to give compound 10 (2.5 g,
69.44%), mp 131–133ꢁC. IR (KBr) υ (cmꢀ1), 1660 (CO); 3392,
Ethyl 2-(3-cyano-4,6-dimethylpyridin-2-ylthio)acetate (4).
A
1
3327 (NH2), 3290 (NH); H NMR (CDCl3): d/ppm, 2.31 (s, 3H,
mixture of 3 (3.28 g, 0.02 mol), ethyl chloroacetate (2.44 g,
0.02 mol), and sodium carbonate (2.1 g, 0.02 mol) in DMF (50 mL)
was refluxed with stirring for 4 h. After cooling, it was poured onto
ice–cold water, and the solid formed was collected and recrystallized
from methanol to give compound 4 (4.4 g, 88% yield), mp 90–92ꢁC,
Lit. [12]; IR (KBr) υ (cmꢀ1), 2217 (CN), 1735 (CO, ester).
CH3), 2.4 (s, 3H, CH3), 6.1 (s, 1H, CH-CO), 6.3 (s, 1H,
pyridyl), 7.5 (m, 4H, phenyl). Anal. Calcd for C19H15N5OS
(361.42): C, 63.14; H, 4.18; N, 19.38; O, 4.43; S, 8.87%.
Found: C, 63.22; H, 4.07; N, 19.51; O, 4.34; S, 8.81%.
2,4-Dimethyl-5H-benzo[4,5]imidazo[2,1-b]pyrido[3,2-e][1,3]
thiazin-5-imine (11).
A mixture of 1 (3.2 g, 20mmol) and
Ethyl 4,6-dimethyl-3-oxo-2,3-dihydrothieno[2,3-b]pyridine-
1H-benzo[d]imidazole-2-thiol (3g, 20mmol) in DMF (30 mL)
containing few drops of triethylamine was refluxed for 6 h.
After cooling, the reaction mixture was poured into ice–water,
and the formed precipitate was filtered off, dried, and
recrystallized from chloroform to give compound 11 (66%), mp
2-carboxylate (5).
A solution of 4 (2.5 g, 0.01mol) in THF
(20 mL) added to sodium hydride (0.8 g, 60%, 0.02 mol) under
nitrogen in THF (40 mL) was refluxed for 3 h. The reaction
mixture was cooled in ice bath at ꢀ20ꢁC and then ethanol
(10 mL) was added followed by cold water (50mL) and acidified
with dilute HCl. The formed solid product was collected and
recrystallized from methanol to give compound 5 (1.83 g, 73%),
mp 183–140ꢁC; IR (KBr) υ (cmꢀ1), 1701, 1666 (b-keto ester);
1H NMR (CDCl3): d/ppm, 1.31 (t, 3H, CH3, J = 7.1 Hz), 2.42 (s,
3H, CH3), 2.71 (s, 3H, CH3), 4.22 (q, 2H, CH2, J = 7.1 Hz), 6.70
(s, 1H, C-2), 7.01 (s, H, C-5); 13C NMR (CDCl3): d, 197.54,
163.58, 162.45, 159.31, 144.20, 123.92, 118.18, 61.44, 60.37,
20.34, 19.75, 13.95. Anal. Calcd for C12H13NO3S (251.3):
C, 57.35; H, 5.12; N, 5.57; S, 12.76%. Found: C, 57.24; H, 5.26;
N, 5.66; S, 12.83%.
1
74–76ꢁC; IR (KBr) υ (cmꢀ1), 2203 (CN); 3377 (NH); H NMR
(CDCl3): d/ppm, 2.4 (s, 3H, CH3-C═N), 2.5 (s, 3H, CH3-C═C),
5.0 (s, H, NH), 6.7 (s, H, pyridyl), 7.5 (m, 4H, phenyl).
Anal. Calcd for C15H12N4S (280.35): C, 64.26; H, 4.31; N,
19.98; S, 11.44%. Found: C, 64.31; H, 4.37; N, 19.85; S, 11.41%.
5-Imino-2,4-dimethyl-9H-pyrido[3,2-e]thiazolo[3,4-a]pyrimidin-
9-one (12).
A mixture of 1 (3.2 g, 20 mmol) and 2-
aminothiazol-4(5H)-one (2.3 g, 20 mmol) treated with sodium
carbonate (0.01 mol) in DMF (40 mL) was refluxed for 4 h.
After cooling, the reaction mixture was poured into ice–water,
and the formed precipitate was filtered off, dried, and
recrystallized from hexane to give compound 12 (72%), mp
123–125ꢁC; IR (KBr) υ (cmꢀ1), 1663 (CO); 3434 (NH); 1H
NMR (CDCl3): d/ppm, 2.3 (s, 3H, CH3-C═N), 2.4 (s, 3H, CH3-
C═C), 3.2 (s, H, S-CH), 4.9 (s, H, NH), 6.3 (s, H, pyridyl).
Anal. Calcd for C11H10N4OS (246.29): C, 53.64; H, 4.09; N,
22.75; O, 6.50; S, 13.02%. Found: C, 53.48; H, 4.29; N, 22.93;
O, 6.98; S, 13.28%.
4,6-Dimethylthieno[2,3-b]pyridine-3(2H)-one (6).
Method A. A solution of 3 (1.64 g, 0.01 mol) in THF (20 mL)
added to sodium hydride (0.8 g, 60%, 0.02 mol) under nitrogen
in THF (40 mL) was refluxed for 3 h. The reaction mixture was
cooled in ice bath at 0ꢁC and then ethanol (10 mL) was added
followed by cold water (50 mL) and acidified with dilute HCl.
The formed solid product was collected and recrystallized
from methanol to give compound 6; (85%), mp 198–200ꢁC;
IR (KBr) υ (cmꢀ1), 1681 (CO); 1H NMR (CDCl3): d/ppm,
2.56 (s, 3H, CH3), 2.59 (s, 3H, CH3), 3.80 (s, 2H, CH2),
6.71 (1H, s, C-5). Anal. Calcd for C9H9NOS (179.24): C,
60.31; H, 5.06; N, 7.81; S, 17.89%. Found: C, 60.38; H, 5.18;
N, 7.95; S, 17.71%.
Method B. A solution of b-ketoester 5 (1.25 g, 0.005 mol) in
acetic acid (20 mL) and concentrated HCl (10 mL) was refluxed for
4 h. After cooling, the mixture was poured onto cold water, and
solid formed was filtered off and recrystallized from methanol to
give the same pure product 6 (TLC control, mp), (0.7 g, 77.7%).
2-(3-Cyano-4,6-dimethylpyridin-2-yloxy)benzoic acid (13).
A
mixture of 1 (3.2 g, 20 mmol) and salicylic acid (2.8 g, 20 mmol)
treated with sodium carbonate (0.01 mol) in DMF (40 mL) was
refluxed for 5 h. After cooling, the reaction mixture was poured
into ice–water, and the precipitated solid product was filtered off,
dried, and recrystallized from ethanol to give compound 13
(72%), mp 123–125ꢁC; IR (KBr) υ (cmꢀ1), 3436 (OH), 2223
1
(CN), 1680 (CO); H NMR (CDCl3): d/ppm, 2.39 (s, 3H, CH3-
C═N), 2.54 (s, 3H, CH3-C═C), 6.3 (s, H, pyridyl), 7.1–7.5 (m,
4H, Ar-H); 13C NMR (CDCl3): d/ppm, 169.24, 166.14, 161.86,
Journal of Heterocyclic Chemistry
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Novel Heterocycles Derived from 2-Chloronicotinonitrile
957
155.27, 154.18, 133.34, 132.53, 123.40, 121.21, 120.84, 117.91,
117.00, 91.71, 25.46, 17.65. Anal. Calcd for C15H12N2O3
(268.27): C, 67.16; H, 4.51; N, 10.44; O, 17.89%. Found: C,
67.36; H, 4.63; N, 10.36; O, 17.73%.
were discarded, and the entire procedure was repeated for
three times.
2,20-(Ethane-1,2-diylbis(azanediyl))bis(4,6-dimethylnicoti-
nonitrile) (14). A mixture of 1 (1.64 g, 10mmol) and ethane-1,2-
diamine (1 mL) in acetonitrile (20 mL) containing Na2CO3 (1 g) was
refluxed for 3 h. After cooling, the reaction mixture was poured into
ice–water, and the precipitated solid product was filtered off, dried,
and recrystallized from ethanol to give compound 14 (71%), mp
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1
83–85ꢁC; IR (KBr) υ (cmꢀ1), 2203 (CN); 3311 (NH); H NMR
(CDCl3): d/ppm, 2.33 (s, 6H, 2 ꢂ CH3-C═N), 2.38 (s, 6H,
2 ꢂ CH3-C═C), 3.5 (t, 4H, 2 ꢂ CH2-NH, J =11Hz), 5.4 (s, 2H,
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Antitumor activity. Different concentrations of the tested
compounds were prepared (ED100, ED50, and ED25 1 g/mL
DMSO). The amount of DMSO was adjusted to give a final
concentration of 0.1%. Ascites fluid obtained was aseptically
aspirated from the peritoneal cavity of the donor animal
(National Cancer Institute, Cairo, Egypt), which contains
Ehrlich cell. The cells were grown partially floating and attach
in AQ4, a suspension culture (RPMI 1660 medium, Sigma
Chemical, St. Louis), supplemented with 10% fetal bovine
serum (GIBCO, UK). They were maintained at 37ꢁC in
humidified atmosphere with 5% CO2 for 2 h. Viability of the
cell obtained and used in control experiments (DMSO only
without drug) exceeded 95% as determined by microscopic
examination by using a hemocytometer and trypan blue stain
(stain only the dead cells). Below this percentage, the cells
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet