Novel benzimidazole-pyrimidine conjugates as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.011

As a continuation to our previous work in synthesizing antitumor benzimidazoles, a series of 2-((1H-benzo[d]imidazol-2-yl)methylthio)-4-(substituted)-6-phenylpyrimidine-5-carbonitriles was synthesized. Evaluation of the synthesized compounds for their in vitro cytotoxic activity against twelve cell lines namely, Cervical carcinoma (KB), Ovarial carcinoma (SK OV-3), CNS cancer (SF-268), Non small lung cancer (NCI H460), Colonadenocarcinoma (RKOP27), Leukaemia (HL60, U937, K562), Melanoma (G361, SK-MEL-28) and Neuroblastoma (GOTO, NB-1) revealed their marked potency when compared with known anticancer drugs.

Full text of DOI:10.1016/j.ejmech.2010.02.011

European Journal of Medicinal Chemistry 45 (2010) 2336–2344
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel benzimidazole–pyrimidine conjugates as potent antitumor agents
,
b
a
a
Heba T. Abdel-Mohsen a , Fatma A.F. Ragab , Mostafa M. Ramla , Hoda I. El Diwani
*
a Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries Research, National Research Centre, Cairo, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation to our previous work in synthesizing antitumor benzimidazoles, a series of 2-((1H-
benzo[d]imidazol-2-yl)methylthio)-4-(substituted)-6-phenylpyrimidine-5-carbonitriles was synthesized.
Evaluation of the synthesized compounds for their in vitro cytotoxic activity against twelve cell lines
namely, Cervical carcinoma (KB), Ovarial carcinoma (SK OV-3), CNS cancer (SF-268), Non small lung cancer
(NCI H460), Colonadenocarcinoma (RKOP27), Leukaemia (HL60, U937, K562), Melanoma (G361, SK-MEL-
28) and Neuroblastoma (GOTO, NB-1) revealed their marked potency when compared with known anti-
cancer drugs.
Received 25 October 2009
Received in revised form
27 January 2010
Accepted 2 February 2010
Available online 13 February 2010
Keywords:
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Benzimidazole
Pyrimidine
Antitumor activity
1. Introduction
the benzimidazoles I–VII (Fig. 1) by a basic moiety, which is
a methylthiopyrimidine, compound 3, (Fig. 2), on the antitumor
Benzimidazole moiety is a structural isostere of naturally
occurring nucleotides; hence, it has been extensively utilized as
a drug scaffold in the medicinal chemistry. Several promising
antitumor active agents were found to contain the benzimidazole
ring system. They were found to exert their antitumor activity by
acting mainly as topoisomerases inhibitors [1–3], alkylating agents
[4–6] and antiangiogenic agents [7–9]. In the last years, it has been
demonstrated that several bi- and ter-benzimidazole derivatives
act as topoisomerase I inhibitors. For example, Hoechst 33342 and
Hoechst 33258 bind to the minor groove of DNA, trapping the
activity. The pyrimidine ring carries a lipophilic phenyl group as
well as other polar groups as OH, Cl and CN. Investigation of the
effect of conversion of the 6-hydroxy group of compound 3 to
chlorine to form 4 and its subsequent replacement with various
amines including piperazine, as the reported cytotoxic activity of
aryl piperazines [16,17]; replacement of piperazine’s free NH of 15
by N-alkyl 16, 20, amide 17,18 and sulphonamides 21–23, as it is
known that arylsulphonamides posses a variety of mechanisms
for their antitumor action and building up an additional pyrimi-
dine nucleus in compounds 24 and 25 on the in vitro antitumor
activity was also conducted. We have discovered a new class of
potent antitumor benzimidazole-2-methylthiopyrimidines, as the
prepared compounds were found to have pronounced in vitro
activity against twelve cell lines, which will be subjected for
further investigations for their in vivo activity.
reversible complex derived from DNA and topoisomerase
I
producing a limited number of highly specific single strand DNA
breaks [10,11]. The benzimidazole derivatives I–VII (Fig. 1) were
previously synthesized by our laboratory group, where compounds
I (IC50 ¼ 4.2
m
M) and II (IC50 ¼ 8.29
mM) were found to have high
cytotoxic activity against breast cancer (MCF7) [12], compounds III
and IV possess significant inhibitory activity against Burkitt’s
lymphoma promotion [13] and V, VI (log GI50 ¼ ꢀ5.61) and VII have
high cytotoxic activity against non small lung cancer and also breast
cancer V (log GI50 ¼ ꢀ5.77), VI (log GI50 ¼ ꢀ5.36) [14,15].
2. Chemistry
A convergent synthesis was designed for the preparation of
compound 3. The first part of this synthesis involved the preparation
of 2-chloro-1H-benzo[d]imidazole 1 as previously reported [18]
(Scheme 1). The second part of this synthesis involved the prepa-
ration of 1,6-dihydro-2-mercapto-6-oxo-4-phenylpyrimidine-5-
carbonitrile 2 by ternary condensation of benzaldehyde with thio-
urea and ethylcyanoacetate according to the reported procedure
[19,20] (Scheme 2). Condensation of the 2 parts was then carried out
As a continuation to our previous work in synthesizing anti-
tumor benzimidazoles, we aimed in this manuscript to investigate
the effect of replacement of the methyl group in the 2-position of
* Corresponding author. Tel.: þ20 233371718; fax: þ20 233370931.
E-mail address: heldiwany@hotmail.com (H.T. Abdel-Mohsen).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.011

H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
2337
O2N
NH2
O2N
N
N
O
N
CH3
CH3
+
Cl
N
H
OH
NH2
S
N
H3C-CO-H2C-S
N
(a)
I
II
N
N
(IC50 = 4.2 µM)
(IC50 = 8.29 µM)
Cl
N
R
S
N
N
H
1
CH3
N
O
Reagents and conditions: (a) 4N HCl/4h reflux.
Scheme 1.
III- R=C6H5CO, (%EBV-EA = 12.3 (60))
IV- R= C6H4-OCH3, (%EBV-EA = 13.2 (60))
to obtain the required target compound 3, which was subsequently
halogenated by the reaction with POCl3 to yield compound 4. This
highly activated intermediate was then reacted with different
hydrazines, alkyl amines, aminoheterocycles, thioamides and amide
to obtain compounds 5–14 (Scheme 3). Compounds 15 and 16 were
obtained by nucleophilic substitution of the chlorine of 4 by piper-
azine and N-methylpiperazine respectively. Compound 15 was
further reacted with different acyl, alkyl and sulphonyl halides to
obtain compounds 17–23 (Scheme 2). An additional pyrimidine
nucleus was built up in compounds 24 and 25 by fusing 4 with
thiourea or urea respectively (Schemes 4 and 5).
CH3
N
HN
N
N
N
CH3
CH3
N
H
V, (Breast cancer, MCF7, GI50=1.82E-05)
(Breast cancer, T47D, GI50= 1.68E-06)
(Non small lung cancer, HOP92, GI50=1.49 E-05)
CH3
N
3. Pharmacology
O
N
N
N
3.1. Antitumor activity
CH3
CH3
N
H
All the newly synthesized compounds were investigated for
their in vitro antitumor activity on 12 cancer cell lines resembling
8 types of tumor using MTT assay according to Mosmann’s
VI, (Breast cancer, MCF7, GI50=2.87E-05)
(Breast cancer, T47D, GI50= 1.68E-06)
(Non small lung cancer, NCI-H522, GI50=1.25 E-06)
method [21]. Table 1 reported the IC50 (mM) values (concentration
required to achieve 50% inhibition of the tumor growth) of the
tested compounds and the standards. With respect to the in vitro
cytotoxic activity expressed in Table 1, almost all of the synthe-
sized compounds exhibited high antitumor activity when
compared with known anticancer agents. From the recorded IC50
N
CH3
N
H
N
H
OEt
O
N
S
S
O
H +
+
H2N
NH2
(a)
CN
VII, (Breast cancer, MCF7, GI50=1.82E-05)
(Breast cancer, T47D, GI50= 4.41E-06)
(Non small lung cancer, EKVX, GI50=1.49 E-05)
Fig. 1. Structures of previously synthesized antitumor benzimidazoles.
SH
SH
N
H
N
N
N
N
N
OH
O
N
H
S
CN
CN
CN
N
OH
2
Reagents and conditions: (a) EtOH/K2CO3/6h. reflux.
3
Fig. 2. Structure of compound 3.
Scheme 2.

2338
H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
SH
N
N
N
N
N
(a)
(c)
+
N
N
N
Cl
OH
S
CN
CN
H
CN
H
1
3
OH
NH
2
N
N
(b)
N
N
N
N
N
S
CN
S
N
H
4
H
Cl
5-14
R
Reagents and conditions: (a) THF/TEA/24h/rt. (b) POCl3/30min/rt. (c) RNH2/EtOH/TEA/2h rt/5h reflux.
No.
5
R
(%) Yield
81
NH2
6
82
H
N
7
8
9
CH3
CH2CH3
87
85
85
N
N
S
10
11
12
83
84
80
N
H
N
NH2
S
NH2
13
14
81
86
S
NH2
O
Scheme 3.
values in Table 1 it was observed that compound 3 was most
active against Non small lung cancer (NCI H460), Ovarial carci-
noma (SK OV-3) and against Leukaemia (HL60) but still less active
than the standards. Conversion of OH group of the pyrimidine ring
of 3 to Cl to obtain compound 4 does not seem to be of great
17 and the 4-nitrobenzoylpiperazino compound 18 were found to
be of the most active compounds against Melanoma (G 361), the
phenylacetylpiperazino compound 19 active against Leukaemia
(U937), the benzenesulfonylpiperazino compound 21 among
active the most active compounds against Cervical carcinoma (KB)
and Non small lung cancer (NCI H460), the tosyl piperazine
compound 22 one of the most active compounds against Colo-
nadenocarcinoma RKOP27 the 2-nitrobenzenesulfonylpiperazino
compound 23 was among active the most active compounds
against Neuroblastoma (NB-1). Building up additional pyr-
imidinethio or pyrimidinone ring fused to the pyrimidine ring of
compound 3 seems to increase the activity as compound 24 was
one of the most active compounds against Colonadenocarcinoma
(RKOP27) and 25 was one of the most active compounds against
Cervical carcinoma (KB).
interest as compound
4 was not among the most potent
compounds of the series. Replacement of Cl group of 4 by other
basic groups and moieties has a high effect on the activity e.g. the
hydrazino compound 5 was the most active against Cervical
carcinoma (KB) while the phenylhydrazino compound 6 was
found to be one of the most active compounds against Melanoma
(G 361) and Neuroblastoma (NB-1). The 4-aminopyridino
compound 9 and the 2-aminothiazolo compound 11 were found
to be the most active compounds against CNS cancer (SF-268)
while the 2-aminopyridino compound 10 was the most active one
against Leukaemia (U937) and Neuroblastoma (GOTO), the thio-
semicarbazido compound 12 was one of the most active
compounds against Neuroblastoma (GOTO), for the thiouryl
compound 13 was not highly active while the uryl compound 14
was the one of the most active compounds against Colonadeno-
carcinoma (RKOP27). Replacement of the Cl of the pyrimidino ring
by piperazine or substituted piperazine was found to have positive
effect on the activity as compound 15 was the most active against
Melanoma (SK-MEL-28), the N-methylpiperazino compound 16
was the most active among the series against Leukaemia (K562)
and Neuroblastoma (NB-1), the N-benzoylpiperazine compound
3.2. The median lethal dose LD50
The median lethal dose (dose required to kill half the members
of the tested population) of the tested compounds was determined
according to the procedure described by Lorke [22]. The geographic
mean of the least dose that killed mice and the highest dose that
did not kill mice was taken as the median lethal dose [23,24]. The
results of the LD50 in mg/kg of the synthesized compounds were
listed in Table 2.

H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
2339
N
N
N
(b)
N
N
N
N
N
H
S
CN
S
CN
H
N
4
16
Cl
N
CH3
(a)
N
N
(c)
N
N
N
N
N
H
N
H
S
CN
CN
S
N
N
17-23
15
N
N
R
H
Reagents and conditions: (a) Piperazine/EtOH /K2CO3/2h rt/8h reflux. (b) N-methylpiperazine/EtOH /TEA/ 1h rt. (c)
DMF/ NaH/30 min/ RCl/4h rt.
No.
17
R
O
(%)Yield
80
O
18
84
NO2
O
19
20
86
78
O
21
22
23
82
88
83
O2S
O2S
CH3
O2N
O2S
Scheme 4.
4. Conclusion
recorded on Bruker Vector 22 and Jasco FT/IR 300E Fourier trans-
former spectrophotometer. NMR spectra were recorded on Varian
GEMINI200 (200 MHz)andJEOLEX-270 and500 MHz spectrometers
using DMSO-d6 as a solvent. Mass spectrawere recorded on FINNGAN
MAT SSQ 7000. Elemental analysis was performed in the microana-
lytical laboratory of the National Research Center. The synthesized
compounds were named using ChemDraw Ultra software (v 10.0).
Successfully we have discovered a new class of 2-((1H-benzo[-
d]imidazol-2-yl)methylthio)-4-(substituted)-6-phenylpyrimidine-
5-carbonitriles of potent antitumor activity against 12 cell lines
namely, Cervical carcinoma (KB), Ovarial carcinoma (SK OV-3), CNS
cancer (SF-268), Non small lung cancer (NCI H460), Colonadeno-
carcinoma (RKOP27), Leukaemia (HL60, U937, K562), Melanoma
(G361, SK-MEL-28) and Neuroblastoma (GOTO, NB-1).
5.1.1. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-1,6-dihydro-6-
oxo-4-phenylpyrimidine-5-carbonitrile (3)
A solution of the thiouracil derivative 2 (42.17 mmol) in dry
tetrahydrofuran (60 mL) containing triethylamine (0.5 mL) was
stirred for 1 h. A solution of 1 (7 g, 42.17 mmol) in dry tetrahydro-
furan was then added portionwise and the reaction mixture was
stirred at room temperature for additional 24 h, poured onto
crushed ice with stirring. The precipitated product was filtered off
5. Experimental
5.1. Chemistry
Melting points are uncorrected and were recorded on a Gallen-
kamp thermometer melting point apparatus. IR spectra (KBr) were
N
N
(a)
N
NH
N
N
N
H
S
CN
N
H
24,25
S
N
NH .HCl
X
4
Cl
N
H
X=S,O
Reagents and conditions: (a) Thiourea or urea /fusion/250ºC/15min.
Scheme 5.

Table 1
IC50 M) values of compounds 3–25 against 12 cancer cell lines.
(
m
Compound no.
IC50
(mM)
Leukaemia
HL60
Melanoma
G361
Neuroblastoma
GOTO
KBa
SK OV-3b
SF-268c
NCI H460d
RKOP27e
U937
K562
SK-MEL-28
NB-1
3
4
6.20 ꢁ 10ꢀ3
3.7 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
2.9 ꢁ 10ꢀ3
2.7 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
2.7 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
20
2.5 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
2.6 ꢁ 10ꢀ3
6.09 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
4.9 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
4.46 ꢁ ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
6.57 ꢁ 10ꢀ3
3.96 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
20 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
3.9 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
1.92 ꢁ 10ꢀ02
2.63 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
3.80 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
4.63 ꢁ 10ꢀ3
1.13 ꢁ 10ꢀ02
14.7 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
1.32 ꢁ 10ꢀ02
4.38 ꢁ 10ꢀ3
10 ꢁ 10ꢀ3
3.43 ꢁ 10ꢀ3
1.25 ꢁ 10ꢀ02
14.7 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
7.70 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
7.81 ꢁ 10ꢀ3
8.62 ꢁ 10ꢀ3
2.68 ꢁ 10ꢀ3
6.41 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
3.96 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
5.1 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
4.80 ꢁ 10ꢀ3
1.13 ꢁ ꢁ 10ꢀ02
3.7 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
8.62 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
6.57 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
5.4 ꢁ 10ꢀ3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
4.54 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
2.80 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
2.84 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
9.6 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
5.3 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
4.80 ꢁ 10ꢀ3
4.80 ꢁ 10ꢀ3
2.6 ꢁ 10ꢀ3
20 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
2.63 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ33
3.9 ꢁ 10ꢀ3
3.7 ꢁ 10ꢀ3
1.25 ꢁ 10ꢀ02
4.38 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
3.80 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
2.63 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
5.40 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
7.81 ꢁ 10ꢀ3
4.80 ꢁ 10ꢀ3
2.6 ꢁ 10ꢀ3
7 ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
4.54 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
2.9 ꢁ 10ꢀ3
4 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
4.54 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
3.4 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
1.13 ꢁ 10ꢀ02
3 ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
3.96 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
9.6 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
2.70 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
7.81 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.63 ꢁ 10ꢀ3
1.60
20 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.68 ꢁ 10ꢀ3
5.2 ꢁ 10ꢀ3
2.55 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
6.20 ꢁ 10ꢀ3
4.1 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
2.80 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
6.90 ꢁ 10ꢀ3
2.80 ꢁ 10ꢀ3
5.81 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
2.6 ꢁ 10ꢀ3
2.87 ꢁ 10ꢀ3
7.81 ꢁ 10ꢀ3
3.00 ꢁ 10ꢀ3
3.50 ꢁ 10ꢀ3
2.84 ꢁ 10ꢀ3
2.80 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.9 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
4.23 ꢁ 10ꢀ3
4.23 ꢁ 10ꢀ3
4.23 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
3.1 ꢁ 10ꢀ3
4 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
6.41 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
1.32 ꢁ 10ꢀ02
2.9 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
6.09 ꢁ 10ꢀ3
2.5 ꢁ 10ꢀ3
3 ꢁ 10ꢀ3
8.62 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
2.77 ꢁ 10ꢀ3
4.54 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
3.50 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
8.62 ꢁ 10ꢀ3
4.38 ꢁ 10ꢀ3
9.6 ꢁ 10ꢀ3
2.4 ꢁ 10ꢀ3
4.54 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
6.75 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
3.28 ꢁ 10ꢀ3
1.13 ꢁ 10ꢀ02
4.6 ꢁ 10ꢀ3
7.35 ꢁ 10ꢀ3
2.5 ꢁ 10ꢀ3
22
23
24
25
3.28 ꢁ 10ꢀ3
4.44 ꢁ 10ꢀ3
7.30 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
4.46 ꢁ 10ꢀ3
22.7 ꢁ 10ꢀ3
3.70 ꢁ 10ꢀ3
3.00 ꢁ 10ꢀ3
2.84 ꢁ 10ꢀ3
1.13 ꢁ 10ꢀ02
14.7 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
14.7 ꢁ 10ꢀ3
5.60 ꢁ 10ꢀ3
2.50 ꢁ 10ꢀ3
2.70 ꢁ 10ꢀ3
3.37 ꢁ 10ꢀ3
3.20 ꢁ 10ꢀ3
11.9 ꢁ 10ꢀ3
8.9 ꢁ 10ꢀ3
Fluorouracil
Doxorubicin
Cytarabine
GemcitabineHCl
Capecitabine
Doxorubicin
Aldesleukin
Doxorubicin
4.16 ꢁ 10ꢀ3
7.68 ꢁ 10ꢀ3
2.13 ꢁ 10ꢀ3
4.33 ꢁ 10ꢀ3
1.13 ꢁ 10ꢀ3
4.45 ꢁ 10ꢀ3
6.12 ꢁ 10ꢀ3
6.66 ꢁ 10ꢀ3
3.45 ꢁ 10ꢀ3
4.73 ꢁ 10ꢀ3
5.15 ꢁ 10ꢀ3
IC50 values were estimated by logistic regression analysis. One-way ANOVA (P < 0.01) was used to test treatment difference in IC50. After significant factor by ANOVA, individual group differences were analyzed using Holm–
Sidak’s procedure for multiple comparisons versus control.
a
Cervical carcinoma.
Ovarial carcinoma.
CNS cancer.
Non small lung cancer.
b
c
d
e
Colonadenocarcinoma.

H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
2341
Table 2
D2O exchangeable), 7.25–7.30 (m, 2H, H5, H6 benzimidazole), 7.54–
7.62 (m, 5H, phenyl H), 7.83 (m, 2H, H4, H7 benzimidazole),12.60 (br,
1H, NH benzimidazole, D2O exchangeable); IR (KBr) 3422.2 (NH
benzimidazole), 3230.0 and 3152.7 (NH–NH2), 3077. (CH arom.), 2933
(CH aliph.), 2221.0 (CN), 1628.0 (C]N benzimidazole), 1549.7 and
1507.2 (C]N pyrimidine, C]C); MS (EI) m/z 373 (Mꢃþ,1.19%), m/z 375
(Mꢃþ þ 2, 6.9%), m/z 91 (100%). Anal. Calc. for (C19H15N7S): C, 61.10; H,
4.07; N, 26.26; S, 8.59. Found: C, 61.55; H, 4.32; N, 26.45; S, 8.67.
5.1.3.1.2. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(2-phenyl-
hydrazinyl)-6-phenyl pyrimidine-5-carbonitrile (6). Yield 82%; mp
The results of the LD50 in mg/kg of the synthesized compounds.
a
Compound no.
LD50 mg/kg
Compound no.
LD50a mg/kg
1
3
4
5
6
7
8
9
10
11
12
13
253.36 ꢄ 3
344.87 ꢄ 11
313.34 ꢄ 5
344.26 ꢄ 5
387.26 ꢄ 3
302.28 ꢄ 4
325.36 ꢄ 6
316.12 ꢄ 4
367.22 ꢄ 3
348.58 ꢄ 4
345 ꢄ 4
14
15
16
17
18
19
20
21
22
23
24
25
347 ꢄ 5
300 ꢄ 6
384 ꢄ 4
327.15 ꢄ 3
327.38 ꢄ 2
344.45 ꢄ 3
326.78 ꢄ 5
326.76 ꢄ 5
344.78 ꢄ 6
324.78 ꢄ 4
389 ꢄ 4
242–244 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.79 (s, 2H, CH2–S),
5.50 (br, 2H, NH–NH phenyl, D2O exchangeable), 7.13–7.17 (m, 2H,
H5, H6 benzimidazole), 7.25–7.28 (m, 2H, H200, H600 phenyl-
hydrazine), 7.45–7.53 (m, 3H, H300, H400, H500 phenylhydrazine),
7.62–7.64 (m, 3H, H30, H40, H50 phenyl), 7.85–7.90 (m, 2H, H20, H60
phenyl), 8.11–8.15 (m, 2H, H4, H7 benzimidazole), 12.75 (br, 1H, NH
benzimidazole, D2O exchangeable); IR (KBr) 3463.1 (NH benz-
imidazole), 3360.0 and 3263.0 (NH–NH phenyl), 3059.1 (CH arom.),
2970 (CH aliph.), 2219.1 (CN), 1611.1 (C]N benzimidazole), 1550.3
and 1511.1 (C]N pyrimidine, C]C); MS (EI) m/z 449 (Mꢃþ, 100%) m/
z 451 (Mꢃþ þ 2, 15%). Anal. Calc. for (C25H19N7S): C, 66.79; H, 4.27; N,
21.81; S, 7.13. Found: C, 66.50; H, 4.03; N, 21.56; S, 7.22.
327.48 ꢄ 5
346 ꢄ 3
Data expressed as means ꢄ SD for three independent experiments.
a
Dose required to kill half the members of the tested population.
and recrystallized from the appropriate solvent. Yield 73%; mp
153–155 ꢂC; 1H NMR (200 MHz, DMSO-d6)
4.77 (s, 2H, CH2–S),
d
7.27–7.31 (m, 2H, H5 and H6 benzimidazole), 7.52–7.64 (m, 5H,
phenyl H), 7.87–7.92 (m, 2H, H4 and H7 benzimidazole), 10.50 (br,
1H, OH enolic, D2O exchangeable), 11.65 (br, 1H, NH benzimidazole,
D2O exchangeable); IR (KBr) 3393.8 (NH benzimidazole), 3200.0–
2500.0 (br, enolic OH), 3079.0 (CH arom.), 2974.5 (CH aliph.), 2222.4
(CN), 1629.6 (C]N benzimidazole), 1602.7 and 1550.1 (C]N
pyrimidine, C]C); MS (EI) m/z 359 (Mꢃþ, 9.43%), m/z 361 (Mþ þ 2,
1.57%), m/z 57 (100%). Anal. Calc. for (C19H13N5OS): C, 63.49; H, 3.65;
N, 19.49; S, 8.92. Found: C, 63.16; H, 3.30; N, 19.85; S, 8.78.
5.1.3.1.3. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(methyl-
amino)-6-phenyl pyrimidine-5-carbonitrile (7). Yield 87%; mp 191–
193 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 2.87 (s, 3H, NH–CH3), 3.35
(s, 2H, CH2–S), 7.29–7.34 (m, 2H, C5H, C6H benzimidazole),
7.48–7.57 (m, 3H, C3aH, C4aH, C5aH), 7.65–7.70 (m, 2H, C2aH, C6aH),
7.73–7.76 (m, 2H, C4H, C7H benzimidazole), 10.97 (br, 1H, NH–CH3,
D2O exchangeable), 12.90 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3381.8 (NH benzimidazole), 3248.1
(NH–CH3), 3030. (CH arom.), 2923.5 (CH aliph.), 2199.1 (CN), 1612.4
(C]N benzimidazole), 1560.9 and 1585.2 (C]N pyrimidine, C]C);
MS (EI) m/z 372 (Mꢃþ, 11.5%), m/z 374 (Mꢃþ þ 2, 5.5%), m/z 239
(100%). Anal. Calc. for (C20H16N6S): C, 64.49; H, 4.34; N, 22.57; S,
8.61. Found: C, 64.72; H, 4.52; N, 22.85; S, 8.75.
5.1.2. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-chloro-6-
phenylpyrimidine-5-carbonitriles (4)
In an ice bath, phosphorous oxychloride (10 mL) was added
dropwise with gentle stirring to compound 3 (4 g, 11.14 mmol). The
reaction mixture was stirred at room temperature for 30 min, and
then refluxed on water bath at 70 ꢂC for 6 h. After cooling, the
reaction mixture was poured with continuous stirring onto crushed
ice. The formed solid was collected by vacuum filtration, washed
with ether (4.1 g, yield 97.61%) and recrystallized from methylene
5.1.3.1.4. 2-((1H-Benzo[d])imidazol-2-yl)methylthio)-4-(ethyl-
amino)-6-phenylpyrimidine-5-carbonitrile (8). Yield 85%; mp 173–
chloride; mp 160–162 ꢂC.
d
4.52 (s, 2H, CH2–S), 7.34–7.42 (m, 2H, H5
175 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 1.20 (t, 3H, CH2–CH3), 3.35
and H6 benzimidazole), 7.67–7.88 (m, 5H, phenyl H), 7.92–8.01
(m, 2H, H4 and H7 benzimidazole), 12.50 (br, 1H, NH benzimid-
azole, D2O exchangeable); MS (EI) m/z 375/373 (Mþ ꢀ 2, 69%/23% Cl
pattern); m/z 342 (Mþ ꢀ Cl, 11.37%), m/z 229 (100%). Anal. Calc. for
(C19H12ClN5S): C, 60.39; H, 3.21; N, 18.54; S, 8.49; Cl, 9.38. Found: C,
60.68; H, 3.25; N, 18.67; S, 8.71; Cl, 9.12.
(q, 2H, NH–CH2–CH3), 4.65 (s, 2H, CH2–S), 7.01–7.30 (m, 2H, C5H, C6H
benzimidazole), 7.35–7.60 (m, 5H, phenyl H), 7.74 (m, 2H, C4H, C7H
benzimidazole), 8.15 (br, 1H, NH–CH2CH3, D2O exchangeable), 12.90
(br, 1H, NH benzimidazole, D2O exchangeable); IR (KBr) 3392.6 (NH
benzimidazole), 3294.1 (NH–ethyl), 3056.8 (CH arom.), 2933.8 (CH
aliph.), 2208.7 (CN), 1625.0 (C]N benzimidazole), 1562.0 and 1545.1
(C]N pyrimidine, C]C); MS (EI) m/z 386 (Mꢃþ, 29%), m/z 388
(Mꢃþ þ 2, 8%), m/z 267 (100%). Anal. Calc. for (C21H18N6S): C, 65.25; H,
4.70; N, 21.75; S, 8.30. Found: C, 65.57; H, 4.78; N, 21.65; S, 8.41.
5.1.3.1.5. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(pyridin-4-ylamino)pyrimidine-5-carbonitrile (9). Yield 85%; mp
5.1.3. Preparation of 2-((1H-benzo[d]imidazol-2-yl)methylthio)-4-
(substituted)-6-phenyl pyrimidine-5-carbonitriles (5–14)
5.1.3.1. General method. To a well stirred solution of the appro-
priate amine (5.30 mmol) and triethylamine (0.5 mL) in absolute
ethanol (10 mL), equimolar amount of a solution of compound 4
(2 g, 5.30 mmol) in absolute ethanol (10 mL) was added portion-
wise. The reaction mixture was stirred for 2 h at room temperature
then heated under reflux for additional 5 h, the solvent was then
removed by distillation under reduced pressure and the remained
solid was washed with cold water and purified either by recrys-
tallization or by silica gel column chromatography.
Compounds 5 and 6 were prepared from the appropriate
hydrazine derivative using the same method but the reaction
mixture was left to stand overnight at room temperature and the
precipitated product was filtered off, washed with ether and
recrystallized from the appropriate solvent.
223–225 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.80 (s, 2H, CH2–S),
6.95 (d, J ¼ 7.2 Hz, 2H, H300, H500 pyridine), 7.12–7.29 (m, 2H, H5, H6
benzimidazole), 7.49–7.65 (m, 5H, phenyl H), 7.92–8.02 (m, 2H, H4,
H7 benzimidazole), 8.72 (br, 1H, NH–pyridine, D2O exchangeable),
9.22 (d, J ¼ 7.2 Hz, 2H, H200, H600 pyridine), 12.88 (br, 1H, NH benz-
imidazole, D2O exchangeable); IR (KBr) 3378.8 (NH benzimidazole),
3303.0 (NH–pyridine), 3073.6 (CH arom.), 2930 (CH aliph.), 2215.6
(CN), 1621.2 (C]N benzimidazole), 1551.5 and 1533.8 (C]N, C]C
groups); MS (EI) m/z 435 (Mꢃþ, 1.06%), m/z 437 (Mꢃþ þ 2, 0.25%), m/z
260 (100%). Anal. Calc. for (C24H17N7S): C, 66.18; H, 3.94; N, 22.51; S,
7.36. Found: C, 66.47; H, 4.00; N, 22.87; S, 7.47.
5.1.3.1.6. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(pyridin-2-ylamino)pyrimidine-5-carbonitrile (10). Yield 83%; mp
152–154 ꢂC; IR (KBr) 3384.5 (NH benzimidazole), 3194.3 (NH–
pyridine), 3059.8 (CH arom.), 2954.1 (CH aliph.), 2204.0 (CN),
1626.0 (C]N benzimidazole), 1567.4 and 1533.6 (C]N, C]C
5.1.3.1.1. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-hydra-
zinyl-6-phenylpyrimidine-5-carbonitrile (5). Yield 81%; mp 160–
162 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d
3.17 (br, 1H, NH hydrazine,
D2O exchangeable), 4.79 (s, 2H, CH2–S), 5.10 (br, 2H, NH2 hydrazine,

2342
H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
groups); m/z 432 (Mꢃþ ꢀ 3, 0.28%), m/z 55 (100%). Anal. Calc. for
(C24H17N7S): C, 66.18; H, 3.94; N, 22.51; S, 7.36. Found: C, 66.49; H,
4.09; N, 22.27; S, 7.39.
5.1.5. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(piperazin-1-yl)pyrimidine-5-carbonitrile (15)
A mixture of piperazine (6.81 g, 79.57 mmol) and anhydrous
potassium carbonate (1.82 g, 13.26 mmol) in absolute ethanol
(10 mL) was stirred for 30 min, a solution of compound 2 (5 g,
13.26 mmol) in absolute ethanol (15 mL) was then added portion-
wise with continuous stirring. The reaction mixture was stirred for
2 h at room temperature then heated under reflux for additional
8 h, after cooling it was filtered to remove insoluble material and the
filtrate was concentrated under vacuum then poured onto ice and
neutralized with dilute hydrochloric acid. Yield 44%; mp 182–184;
IR (KBr) 3421.9 (NH benzimidazole), 3300.0 (NH piperazine), 3058.5
(CH arom.), 2930.5 (CH aliph.), 2198.2 (CN), 1619.9 (C]N benz-
imidazole), 1584.1 and 1546.9 (C]N pyrimidine, C]C); MS (EI) m/z
427 (Mꢃþ, 0.8%), m/z 429 (Mꢃþ þ 2, 0.16%), m/z 350 (Mꢃþ ꢀ phenyl,
24.74%), 281 (100%). Anal. Calc. for (C23H21N7S): C, 64.61; H, 4.96; N,
22.93; S, 7.50. Found: C, 64.23; H, 4.86; N, 22.75; S, 7.56.
5.1.3.1.7. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(thiazol-2-ylamino)pyrimidine-5-carbonitrile (11). Yield 84%; mp
207–209 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.63 (s, 2H, CH2–S),
7.16–7.31 (m, 2H, H5, H6 benzimidazole), 7.41 (d, J ¼ 7.2 Hz, 1H,
H50thiazole), 7.50–7.55 (m, 5H, phenyl H), 7.95–8.04 (m, 3H, H4, H7
benzimidazole, H40 thiazole), 8.55 (br, 1H, NH–thiazole,
D2O exchangeable), 12.54 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3421.3 (NH benzimidazole), 3171.0 (NH
aminothiazole), 3062.0 (CH arom.), 2929.7 (CH aliph.), 2210.9 (CN),
1611.0 (C]N benzimidazole), 1590.0 and 1542.7 (C]N, C]C
groups); MS (EI) m/z 441 (Mꢃþ, 0.56%), m/z 443 (Mꢃþ þ 2, 0.23%), m/z
103 (100%). Anal. Calc. for (C22H15N7S2): C, 59.84; H, 3.43; N, 22.21;
S, 14.52. Found: C, 59.90; H, 3.56; N, 22.35; S, 14.62.
5.1.4. 1-(2-((1H-Benzo[d]imidazol-2-yl)methylthio)-5-cyano-6-
phenylpyrimidin-4-yl)thiosemicarbazide (12), thiourea or urea
(13,14)
5.1.6. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-
(methylpiperazin-1-yl)-6-phenylpyrimidine-5-carbonitrile (16)
A mixture of compound 2 (2 g, 5.3 mmol), 4-methylpiperazine
(0.53 g, 5.30 mmol) and triethylamine (0.5 mL) in absolute ethanol
(50 mL) was stirred for 1 h. The formed precipitate was filtered off,
washed with water and recrystallized from water/acetone. Yield
5.1.4.1. General method. To a well stirred solution of the appro-
priate thioamide or amide (5.30 mmol) and triethylamine (0.5 mL)
in absolute ethanol (15 mL), equimolar amount of a solution of 4
(2 g, 5.30 mmol) in absolute ethanol (10 mL) was added portion-
wise. The reaction mixture was stirred at room temperature for 2 h
then heated under reflux for additional 10 h, after cooling the
solvent was removed by distillation under reduced pressure and
the remained solid was washed with cold water and purified by
recrystallization from the appropriate solvent.
68%; mp 222–224 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 2.35 (s, 3H,
CH3), 2.55–2.59 (m, 4H, piperazine H), 3.98–4.04 (m, 4H, piperazine
H), 4.78 (s, 2H, CH2–S), 7.19–7.24 (m, 2H, H5, H6 benzimidazole),
7.55–7.66 (m, 5H, phenyl H), 7.84–7.92 (m, 2H, H4, H7 benzimid-
azole),12.52 (br,1H, NH benzimidazole, D2O exchangeable); IR (KBr)
3421.3 (NH benzimidazole), 3054.6 (CH arom.), 2946.5 (CH aliph.),
2209.9 (CN), 1621.9 (C]N benzimidazole), 1575.0 and 1526.8 (C]N
pyrimidine, C]C); MS (EI) m/z 441 (Mꢃþ, 9.65%), m/z 443 (Mꢃþ þ 2,
3.63%), m/z 310 (100%). Anal. Calc. for (C30H25N7OS): C, 67.77; H,
4.75; N, 18.44; S, 6.03. Found: C, 67.82; H, 4.87; N, 18.65; S, 6.15.
5.1.4.1.1. 1-(2-((1H-Benzo[d]imidazol-2-yl)methylthio)-5-cyano-
6-phenylpyrimidin-4-yl)thiosemicarbazide (12). Yield 80%; mp
129–132 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.50 (br, 1H, NH, D2O
exchangeable), 4.71 (s, 2H, CH2–S), 5.05 (br, 2H, NH2, D2O
exchangeable), 7.38–7.44 (m, 2H, H5, H6 benzimidazole), 7.53–7.62
(m, 5H, phenyl H), 7.68–7.72 (m, 2H, H4, H7 benzimidazole), 9.63
(br, 1H, NH, D2O exchangeable), 12.50 (br, 1H, NH benzimidazole,
D2O exchangeable); IR (KBr) 3420.3 (br, NH and NH2 groups),
3061.9 (CH arom.), 2929.4 (CH aliph.), 2219.1 (CN), 1620.0 (C]N
benzimidazole), 1583.0 and 1525.6 (C]N pyrimidine, C]C); MS
(EI) m/z 432 (Mꢃþ, 15%), m/z 434 (Mꢃþ þ 2, 6%), m/z 213 (100%). Anal.
Calc. for (C20H16N8S2): C, 55.53; H, 3.74; N, 25.91; S, 14.83. Found: C,
55.85; H, 3.56; N, 26.04; S, 14.78.
5.1.7. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(aryl or alkyl
piperazin-1-yl)-6-phenylpyrimidine-5-carbonitriles (17–23)
5.1.7.1. General method. To a solution of compound 13 (4.68 mmol)
in dimethylformamide (15 mL), sodium hydride (0.11 g, 4.68 mmol)
was added gradually under ice cooling. The reaction mixture was
stirred for 30 min, and then the appropriate halide (4.68 mmol)
was added portionwise. The whole mixture was warmed to room
temperature and stirred for 4 h, poured onto ice-water with
continuous stirring. The precipitated product was filtered off and
purified either by recrystallization or by silica gel column
chromatography.
5.1.4.1.2. 1-(2-((1H-Benzo[d]imidazol-2-yl)methylthio)-5-cyano-
6-phenylpyrimidin-4-yl)thiourea (13). Yield 81%; mp 118–120 ꢂC; 1H
NMR (200 MHz, DMSO-d6)
d 4.40 (br, 2H, NH2, D2O exchangeable),
4.90 (s, 2H, CH2–S), 7.38–7.44 (m, 2H, H5, H6 benzimidazole), 7.52–
7.60 (m, 3H, H30, H40, H50), 7.66–7.79 (m, 4H, H4, H7 benzimidazole,
H20, H60), 9.82 (br, 1H, NH, D2O exchangeable), 12.91 (br, 1H, NH
benzimidazole, D2O exchangeable); IR (KBr) 3396.8 (NH benzimid-
azole), 3286.1 and 3184.9 (NH, NH2), 3050 (CH arom.), 2921.6 (CH
aliph.), 2213.9 (CN),1607.6 (C]N benzimidazole),1563.9 and 1531.2
(C]N pyrimidine, C]C); MS (EI) m/z 417 (Mꢃþ, 2.32%), m/z 419
(Mꢃþ þ 2, 0.92), m/z 56 (100%). Anal. Calc. for (C20H15N7S2): C, 57.53;
H, 3.63; N, 23.49; S,15.36. Found: C, 57.75; H, 3.47; N, 23.64; S,15.21.
5.1.4.1.3. 1-(2-((1H-Benzo[d]imidazol-2-yl)methylthio)-5-cyano-
6-phenylpyrimidin-4-yl)urea (14). Yield 86%; mp 190–192 ꢂC; 1H
5.1.7.1.1. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-ben-
zoylpiperazin-1-yl)-6-phenylpyrimidine-5-carbonitrile
(17). Yield
80%; mp 164–166 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d
3.39–3.38
(m, 4H, piperazine H), 3.61–3.79 (m, 4H, piperazine H), 4.03 (s, 2H,
CH2–S), 7.11–7.43 (m, 6H, H30, H40, H50 phenyl, H300, H400, H500
benzoyl), 7.52–7.67 (m, 4H, H20, H60 phenyl, H200, H600 benzoyl), 7.84
(m, 2H, H5, H6 benzimidazole), 7.97 (m, 2H, H4, H7 benzimidazole),
9.95 (br, 1H, NH benzimidazole, D2O exchangeable); IR (KBr) 3423.5
(NH benzimidazole), 3057.9 (CH arom.), 2930.5 (CH aliph.), 2198.5
(CN), 1634.9 (C]O), 1625.0 (C]N benzimidazole), 1581.4 and
1547.6 (C]N pyrimidine, C]C); MS (EI) m/z 531 (Mꢃþ, 0.38%), m/z
104 (100%). Anal. Calc. for (C30H25N7OS): C, 67.77; H, 4.75; N, 18.44;
S, 6.03. Found: C, 67.82; H, 4.87; N, 18.65; S, 6.15.
NMR (200 MHz, DMSO-d6)
d 4.22 (br, 2H, NH2, D2O exchangeable),
4.80 (s, 2H, CH2–S), 7.10–7.25 (m, 2H, H5, H6 benzimidazole),
7.40–7.65 (m, 5H, phenyl H), 7.70–7.90 (m, 2H, H4, H7benzimidazole),
9.63 (br,1H, NH, D2O exchangeable),12.65 (br,1H, NH benzimidazole,
D2O exchangeable); IR (KBr) 3421.6 (br, NH and NH2 groups), 3059.8
(CH arom.), 2930.0 (CH aliph.), 2217.7 (CN), 1663.3 (C]O), 1627.6
(C]N benzimidazole), 1550.0 and 1533.3 (C]N pyrimidine, C]C);
MS (EI) m/z 399 (Mꢃþꢀ2,100%). Anal. Calc. for (C20H15N7OS): C, 59.83;
H, 3.77; N, 24.43; S, 7.99. Found: C, 59.48; H, 3.85; N, 24.52; S, 7.75.
5.1.7.1.2. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-(4-nitro-
benzoyl)piperazin-1-yl)-6-phenylpyrimidine-5-carbonitrile (18). Yield
84%; mp 127–129 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 3.12–3.85 (m,
8 H, piperazine H), 4.21 (s, 2H, CH2–S), 7.21–8.27 (m, 13H, arom. H),
12.30 (br, 1H, NH benzimidazole, D2O exchangeable); IR (KBr)
3410.49 (NH benzimidazole), 3060.0 (CH arom.), 2920.6 (CH aliph.),

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2343
2199.42 (CN), 1641.12 (C]O), 1610 (C]N benzimidazole), 1590.0
and 1545.67 (C]N pyrimidine, C]C), 1530.6 and 1373.2 (NO2); MS
(EI) m/z 576 (Mꢃþ, 0.52%), m/z 578 (Mꢃþ þ 2, 0.13%), m/z 150 (100%).
Anal. Calc. for (C30H24N8O3S): C, 62.48; H, 4.20; N, 19.43; S, 5.56.
Found: C, 61.77; H, 4.45; N, 19.65; S, 5.68.
(C29H24N8O4S2): C, 56.85; H, 3.96; N,18.29; S,10.47. Found: C, 56.95;
H, 3.65; N, 18.18; S, 10.38.
5.1.8. 7-((1H-Benzo[d]imidazol-2-yl)methylthio)-3,4-dihydro-4-
imino-5-phenylpyrimido[4,5-d]pyrimidin-2(1H)-(thione or one)
(24,25)
A mixture of compound 4 and thiourea or urea was fused
together at 250 ꢂC for 15 min. The formed mass was triturated with
ethanol and the remained solid was recrystallized from methylene
chloride to obtain 24 and 25 respectively.
5.1.7.1.3. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(4-(2-phenylacetyl)piperazin-1-yl)pyrimidine-5-carbonitrile (19).
Yield 86%; mp 162–164 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 3.09–
3.70 (m, 10H, piperazine H þ CO–CH2), 4.05 (s, 2H, CH2–S), 7.00–
8.13 (m, 14H, arom. H), 12.51 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3447.6 (NH benzimidazole), 3035.6
(CH arom.), 2923.8 (CH aliph.), 2229.9 (CN), 1676.3 (C]O), 1628.1
(C]N benzimidazole), 1596.1 and 1547.3 (C]N pyrimidine, C]C);
MS (EI) m/z 546 (Mꢃþ þ 1, 0.05%), m/z 105 (100%). Anal. Calc. for
(C31H27N7OS): C, 68.23; H, 5.00; N, 17.97; S, 5.88. Found: C, 68.52; H,
4.77; N, 17.76; S, 5.90.
5.1.8.1. 7-((1H-Benzo[d]imidazol-2-yl)methylthio)-3,4-dihydro-4-
imino-5-phenylpyrimido[4,5-d]pyrimidin-2(1H)-thione (24). Yield
71%; mp 210–212 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.75 (s, 2H,
CH2–S), 7.25–7.35 (m, 2H, H5, H6 benzimidazole), 7.55–7.70 (m, 5H,
phenyl H), 7.78–7.83 (m, 2H, H4, H7 benzimidazole), 8.22, 8.52, 8.60
(br, 3H, 3NH, D2O exchangeable), 12.50 (br, 1H, NH benzimidazole,
D2O exchangeable); IR (KBr) 3396.9 (NH benzimidazole), 3196.9
(br, NH groups), 3055.3 (CH arom.), 2922.3 (CH aliph.), 1624.1 (C]N
benzimidazole), 1598.0 and 1548.9 (C]N, C]C groups), 1240
(C]S); MS (EI) m/z 454 (Mꢃþ, 1.21%), m/z 84 (100%). Anal. Calc. for
(C20H15N7S2$HCl): C, 52.91; H, 3.56; N, 21.60; S, 14.13. Found: C,
52.75; H, 3.68; N, 21.23; S, 14.52.
5.1.7.1.4. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-(2-oxo-
2-phenylethyl)piperazin-1-yl)-6-phenylpyrimidine-5-carbonitrile
(20). Yield 78%; mp 120–122 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d
3.28–3.69 (m, 4H, piperazine H), 3.7–4.02 (m, 6H, CH2–CO,
piperazine H), 4.10 (m, 2H, CH2–S), 7.01–7.38 (m, 7H, H5, H6
benzimidazole, H30, H40, H50 phenyl, H300, H500 2-oxo-2-phenyl-
ethyl), 7.38–7.65 (m, 5H, H20, H60 phenyl, H200, H400, H600 2-oxo-2-
phenylethyl), 7.69–7.92 (m, 2H, H4, H7 benzimidazole), 9.50 (br, 1H,
NH benzimidazole, D2O exchangeable); IR (KBr) 3386.4 (NH benz-
imidazole), 3058.5 (CH arom.), 2928 (CH aliph.), 2199.4 (CN), 1656.5
(C]O), 1627.6 (C]N benzimidazole), 1587.1 and 1540.8 (C]N
pyrimidine, C]C); MS (EI) m/z 544 (Mꢃþ ꢀ 1, 0.77%), m/z 249.97
(100%). Anal. Calc. for (C31H27N7OS): C, 68.23; H, 5.00; N, 17.97; S,
5.88. Found: C, 68.45; H, 4.66; N, 17.85; S, 5.89.
5.1.8.2. 7-((1H-Benzo[d]imidazol-2-yl)methylthio)-3,4-dihydro-4-
imino-5-phenylpyrimido[4,5-d] pyrimidin-2(1H)-one (25). Yield 78%;
mp 177–179 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 4.56 (s, 2H, CH2–S),
7.21–7.28 (m, 2H, H5, H6 benzimidazole), 7.42–7.63 (m, 5H, phenyl
H), 7.82–7.95 (m, 2H, H4, H7 benzimidazole), 8.31, 8.56, 8.75 (br, 3H,
3NH, D2O exchangeable), 12.88 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3370.0 (NH benzimidazole), 3134.7 (br, NH
groups), 3053.0 (CH arom.), 2924.0 (CH aliph.), 1670.0 (C]O),
1626.6 (C]N benzimidazole), 1562.06 and 1520.0 (C]N, C]C
groups); MS (EI) m/z 437 (Mꢃþ, 0.48%), m/z 143 (100%). Anal. Calc.
for (C20H15N7OS$HCl): C, 54.85; H, 3.69; N, 22.39; S, 7.32. Found: C,
54.76; H, 3.23; N, 22.52; S, 7.12.
5.1.7.1.5. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(4-(phenylsulphonyl)piperazin-1-yl)pyrimidine-5-carbonitrile (21).
Yield 82%; mp 90–92 ꢂC; 1H NMR(200 MHz, DMSO-d6)
d 3.04 (m,
4H, piperazine H), 3.97 (m, 4H, piperazine H), 4.61 (s, 2H, CH2–S),
7.20–8.10 (m, 14H, arom. H), 13.14 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3394.1 (NH benzimidazole), 3055.2 (CH
arom.), 2950.8 (CH aliph.), 2199.4 (CN), 1615.1 (C]N benzimid-
azole), 1545.0 and 1520.0 (C]N pyrimidine, C]C), 1336.6 and
1145.0 (SO2); MS (EI) m/z 567 (Mꢃþ, 3.60%), m/z 569 (Mþ þ 2, 0.82%),
m/z 218 (100%). Anal. Calc. for (C29H25N7O2S2): C, 61.35; H, 4.45; N,
17.27; S, 11.29. Found: C, 61.53; H, 4.35; N, 17.53; S, 11.32.
5.2. Pharmacology
5.2.1. Antitumor activity
5.1.7.1.6. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-phenyl-6-
(4-tosylpiperazin-1-yl)pyrimidine-5-carbonitrile (22). Yield 88%; mp
5.2.1.1. In vitro cytotoxicity. The in vitro cytotoxicity of the synthe-
sized compounds against 12 different cancer cell lines was
performed with the MTT assay according to the Mosmann’s method
[21]. The MTT assay is based on the reduction of the soluble 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
into a blue–purple formazan product, mainly by mitochondrial
reductase activity inside living cells.
179–181 ꢂC; 1H NMR (200 MHz, DMSO-d6)
d 2.42 (s, 3H, p-CH3),
2.96–3.01 (m, 4H, H2, piperazine H), 3.86–3.97 (m, 6H, piperazine H,
CH2–S), 7.27–7.82 (m, 13H, arom. H), 12.84 (br, 1H, NH benzimid-
azole, D2O exchangeable); IR (KBr) 3421.7 (NH benzimidazole),
3033.0 (CH arom.), 2963.2 (CH aliph.), 2221.9 (CN), 1628.6 (C]N
benzimidazole), 1549.8 and 1507.5 (C]N pyrimidine, C]C), 1300.6
and 1157.7 (SO2); MS (EI) m/z 581 (Mꢃþ, 1.2%), m/z 583 (Mþ þ 2,
0.42%), m/z 91 (100%). Anal. Calc. for (C30H27N7O2S2): C, 61.94; H,
4.69; N, 16.86; S, 11.02. Found: C, 61.80; H, 4.57; N, 16.97; S, 11.10.
5.1.7.1.7. 2-((1H-Benzo[d]imidazol-2-yl)methylthio)-4-(4-(2-nitro-
benzensulphonyl)piperazin-1-yl)-6-phenylpyrimidine-5-carbonitrile
(23). Yield 83%; mp 117–119 ꢂC; 1H NMR (200 MHz, DMSO-d6)
The cells used in cytotoxicity assay were cultured in RPMI 1640
medium supplemented with 10% fetal calf serum. Cells suspended
´
in the medium (2Y 104/mL) were plated in 96-well culture plates
and incubated at 37 ꢂC in a 5% CO2 incubator. After 12 h, the test
´
sample (2 mL) was added to the cells (2Y 104) in 96-well plates and
cultured at 37 ꢂC for 3 days. The cultured cells were mixed with
20 mL of MTT solution and incubated for 4 h at 37 ꢂC. The super-
natant was carefully removed from each well and 100 mL of DMSO
was added to each well to dissolve the formazan crystals which
were formed by the cellular reduction of MTT. After mixing with
a mechanical plate mixer, the absorbance of each well was
measured by a microplate reader using a test wavelength of 570 nm.
The results were expressed as the IC50, which is the concentration of
the drugs inducing a 50% inhibition of cell growth of treated cells
when compared to the growth of control cells. Each experiment was
performed at least 3 times. There was a good reproducibility
between replicate wells with standard errors below 10%.
d
3.20–3.60 (m, 4H, piperazine H), 3.79–4.20 (m, 6H, 4 piperazine H,
CH2–S), 7.36–7.54 (m, 3H, H5 and H6 benzimidazole, H40), 7.79–7.93
(m, 5H, H20, H30, H50, H60 phenyl, H400 2-nitrobenzensulphonyl),
7.96–8.03 (m, 5H, H4 and H7 benzimidazole, H300, H500, H6002-
nitrobenzensulphonyl), 12.95 (br, 1H, NH benzimidazole, D2O
exchangeable); IR (KBr) 3396.7 (NH benzimidazole), 3035.4
(CH arom.), 2953.6 (CH aliph.), 2212.7 (CN), 1626.8 (C]N benz-
imidazole), 1583.0 and 1529.0 (C]N pyrimidine, C]C), 1512.2 and
1392.1 (NO2), 1330.0 and 1153.5 (SO2); MS (EI) m/z 535
(Mꢃþ ꢀ phenyl, 10.44%), m/z 174 (100%). Anal. Calc. for

2344
H.T. Abdel-Mohsen et al. / European Journal of Medicinal Chemistry 45 (2010) 2336–2344
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A. Greer, S. Hansel, W. Hurlburt, B. Jacobson, S. Krishnananthan, F.Y. Lee, A. Li,
T.-A. Lin, P. Liu, C. Ouellet, X. Sang, M.G. Saulnier, K. Stoffan, Y. Sun,
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The median lethal dose of the tested compounds was deter-
mined according to the procedure described by Lorke [22]. The
experiment was carried out on two phases; the first phase involved
three groups of three animals per each group. One dose was given
to each group intraperitoneally and the treated mice were moni-
tored for 24 h for mortality and general behaviors. The second
phase comprises 3–4 groups of one mouse per group were given
doses, based on the findings of phase 1, the mice were again
monitored for 24 h. The geographic mean of the least dose that
killed mice and the highest dose that did not kill mice was taken as
the median lethal dose [23,24].
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Acknowledgements
[13] M.M. Ramla, M.A. Omar, H. Tokuda, H.I. El-Diwani, Bioorg. Med. Chem. 15
(2007) 6489–6496.
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The authors express their deep thanks to ‘‘Sanofi-Aventis
Pharmaceutical Company’’ for evaluation of the synthesized
compounds for their in vitro antiviral and antitumor activities. We
are very grateful to the ‘‘US-Egypt Joint Science and Technology
Board Fund’’ administered through the USDA (BIO9-002-015).
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