Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis and biological evaluation of pyrimidine-benzimidazole conjugates

Article abstract of DOI:10.1016/j.ejmech.2018.01.072

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC₅₀ = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG₅₀ = 6.6 μM - 24.9 μM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20–23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Full text of DOI:10.1016/j.ejmech.2018.01.072

European Journal of Medicinal Chemistry 146 (2018) 687e708
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis
and biological evaluation of pyrimidine-benzimidazole conjugates
,
, d
Shadia A. Galal a *, Muhammad Khattab a, Samia A. Shouman b, Raghda Ramadan c
,
Omaima M. Kandil e, Omnia M. Kandil f, Ashraf Tabll g, Yasmine S. El Abd g,
Reem El-Shenawy g, Yasmin M. Attia b, Ahmed A. El-Rashedy a, Hoda I. El Diwani a
a Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National Research Centre, Cairo, 12622,
Egypt
b Department of Cancer Biology, National Cancer Institute, Cairo University, Egypt
c Radiobiology Unit, Belgian Nuclear Research Center (SCKꢀCEN), Mol, Belgium
d Department of Basic Medical Sciences, Physiology Group, Ghent University, Ghent, Belgium
e Department of Animal Reproduction & Artificial Insemination, Division, of Veterinary Research, National Research Centre, Cairo, 12622, Egypt
f Department of Parasitology, Animal Disease, Division, of Veterinary, National Research Centre, Cairo, 12622, Egypt
g Department of Microbial Biotechnology, Division of Genetic Engineering & Biotechnology, National Research Centre, 12622, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted
cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the
effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole con-
jugates were designed and twelve feasible derivatives were selected to be synthesized to investigate
their activity against Chk2 and subjected to study their antitumor activity alone and in combination with
the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ERþ) cell line (MCF-7).
The results indicated that the studied compounds inhibited Chk2 activity with high potency
(IC50 ¼ 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against
Received 11 July 2017
Received in revised form
20 January 2018
Accepted 22 January 2018
Available online 2 February 2018
Keywords:
2-Phenylbenzimidazoles
Pyrimidines
Chk2 inhibitors
Cell cycle
MCF-7 (IG50 ¼ 6.6
mM - 24.9 mM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of
the studied genotoxic drugs, whereas, compounds 9b and 20e23 antagonized their activity. Moreover,
the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combi-
nation of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more
than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed
on the basis of molecular modeling study using Molecular modeling Environment program (MOE).
© 2018 Elsevier Masson SAS. All rights reserved.
Genotoxic anticancer therapies
1. Introduction
drug resistance via development of chemo-resistance by cancer
cells, have emphasized the urgent need to solve their shortcomings
Cancer is one of the main causes of death around the world that
results from high rate of proliferation for abnormal cells [1,2]. Many
factors, as infectious agents, tobacco, chronic inflammation and UV
light, can lead to genomic instability which can stimulate cancerous
injuries [1,2]. Radiotherapy and chemotherapy are often used in
addition to surgery to decrease the probabilities cancer recurrence.
The generic mode of chemotherapies, genomic instability results
from targeting DNA in cancer treatment, the rapid emergence of
and find new strategy for cancer treatment [3,4]. The key compo-
nents of the DNA damage response pathway are two kinases named
as ataxia-telangiectasia mutated (ATM) and Rad3-related (ATR).
They subsequently phosphorylate several substrates essential for
DNA repair, cell cycle arrest, transcription, and apoptosis [5e9]. Cell
cycle checkpoints represent the restriction points between each
phase of the cell cycle whereby the entire process can be delayed/
stopped its progress to enable the proper sequential of the process
ensuring that each stage occurs in an imperative system or to allow
time for DNA repair [10e12]. Downstream phosphorylation targets
of ATM and ATR are the effectors serine/threonine checkpoint ki-
nases 1 and 2 (Chk1& Chk2) [13], which in turn phosphorylate
* Corresponding author.
E-mail addresses: sh12galal@hotmail.com, sh12galal@yahoo.com (S.A. Galal).
https://doi.org/10.1016/j.ejmech.2018.01.072
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
partially overlapping residues in other target proteins to induce cell
cycle arrest and facilitate DNA repair. Chk1 is activated by ATR
phosphorylation on Ser-317 and Ser-345 whereas Chk2 is activated
by ATM phosphorylation on Thr-68 [14]. Downstream targets of
Chk2 include Cdc25A and Cdc25C, which, on phosphorylation, go
through degradation and cytoplasmic relocalization, respectively,
and induce cell arrest at G1, S, and G2-M phases [15]. Another
important target of Chk2 is p53, the phosphorylation of which on
Ser20 regulates p53 transcriptional activation. Moreover, Chk2
phosphorylation of Hdmx Ser367, a negative regulator of p53, en-
hances its degradation [16,17] and endorses the accumulation of
p53 and transcriptional induction of p53 responsive genes. Chk2
also phosphorylates the transcription factor E2F-1 on Ser364,
thereby enhancing its stability and encourages apoptosis [18,19].
According to the current understanding of Chk2 function in
tumor cells, in both biological and genetic context, Chk2 plays an
important role in maintaining genomic integrity by acting as a
signal transducer of DNA damage. Chk2 is endogenously activated
in precancerous lesions with genomic instability and in cancer cells
grown in culture. Studies have shown that the activated Chk2 acts
as a survival factor for cancer cells. There are several rationales for
the development of Chk2 inhibitors. First, wide range of anticancer
drugs and ionizing radiation caused activation of Chk2 in tumor
cells which seriously limited their effect in these cells [20e26].
These shortcomings of current chemotherapeutic approaches can
be addressed by selective inhibition of Chk2. Second, inhibition of
Chk2 in normal cells leads to protection normal tissues during
chemotherapy or radiation therapy that increase the therapeutic
indices of DNA-targeted and ionizing radiation(IR) agents in these
cells. Therefore, Chk2 inhibitors would protect healthy tissues as
well as sensitize the tumor to chemotherapy. Therefore, inhibition
of Chk2 can address limitations of current cancer therapies and
rationalize the basis of using Chk2 inhibitors as chemotherapeutic
agents [27e30].
2. Results and discussion
2.1. Rational design
Although, 2-arylbenzimidazoles represent one of the most
potent selective class of Chk2 inhibitors and showed effective
radioprotection of human T-cells subjected to ionizing radiation
during radiotherapy [31e33], their antitumor effect is not reported
and so they cannot be used either as single agent in monotherapy
treatment or in combined chemotherapies where they do not
potentiate the antitumor effect of the genotoxic anticancer drugs
[42]. To address their shortage and following the same strategy
used in the formation of pyrazole-benzimidazole conjugates
[35,36] which succeeded in the synthesis of new candidates of 2-
arylbenzimidazoles with effective anticancer properties and
potentiated antitumor effect of genotoxic drugs [35,36] that formed
on the basis of hybridization two different bioactive molecules with
complementary pharmacophoric functions or with different
mechanisms of action often exhibited enhanced effects [43e45].
A small library 212 of pyrimidine-benzimidazole conjugates
were designed by replacing the lateral aryl group of 2-
arylbenzimidazoles (II) with pyrimidine moieties in formula I
(Fig. 2). Most of the designed pyrimidine-benzimidazole conjugates
of formula III (Fig. 2) revealed high binding affinities towards Chk2
receptor (PDB code: 2XBJ) [34] according to MOE program
(supplementary material: Table 2S). Twelve synthetically feasible
compounds were chosen among the best fifty candidates that
exhibiting the highest binding affinities into Chk2 for chemical
synthesis. Variation of the substituents permitted the deduction of
structure-activity relationship. Finally, their possible interactions
with Chk2 were investigated by docking using the MOE program
into the crystal structure of Chk2 in complex with the potent and
selective 2-(quinazolin-2-yl)phenol inhibitor (PDB code: 2XBJ) [34].
2-Arylbenzimidazole derivatives as 2-(4-(4-hydroxyphenylthio)
phenyl)-1H-benzo[d]imidazole-5-carboxamide (1) (Fig. 1A) are
defined as potent and selective inhibitors of Chk2 [31e33] and 4-
fluoro-2-(4-{[(3S,4R)-4-(2-hydroxypropan-2-yl)pyrrolidin-3-yl]
amino}-6,7-dimethoxyquinazolin-2-yl)phenol (XBJ) (Fig. 1B) were
described as a new series of potent and selective ATP-competitive
inhibitors of Chk2 that was generated on the basis of structure-
based design [34]. A series of pyrazole-benzimidazole conjugates
were developed by Galal et al. on the basis of structure based design
such as N-isopropyl-2-(4-(3-methyl-4-nitro-1-phenyl-1H-pyrazol-
2.2. Chemistry
The target pyrimidine-benzimidazole conjugates were synthe-
sized via straight forward synthetic routes as shown in Schemes
1e3. They were classified into three series according to the linker
(X) between 2-phenylbenzimidazoles and pyrimidines moieties
(Fig. 2). The desired pyrimidines derivatives 7a-g have been ach-
ieved
by
synthesis
of
6-aryl-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitriles (4a-f) that were prepared
following a previously described protocol [38,39]. The intermediate
5-yloxy)phenyl)-1H-benzo[d]imidazole-5-carboxamide
(2)
compounds,
dihydropyrimidin-2-ylthio)acetate (5) and ethyl 2-(5-cyano-4-(4-
chlorophenyl)-6-oxo-1,6-dihydropyrimidin-2-ylthio)acetate (6),
were obtained respectively by alkylation of compounds 4b and 4c
with ethyl bromoacetate in benzene and potassium carbonate.
Chloropyrimidine derivatives 7a-g were obtained through treat-
ment of hydroxypyrimidine derivatives 4a, 5, 4c, 4d, 4e, 4f and 6,
respectively, with phosphorus oxychloride (Scheme 1).
The first series of target compounds 9a-c and 10a-c with ether
linker (X ¼ O, Fig. 2) was obtained by reaction of 7a-c with 4-
hydroxybenzaldehyde in DMF, potassium hydroxide and copper
(I) iodide that yielded phenyl-pyrimidinyl ether derivatives 8a-c in
good yield. Subsequently, reaction of 3,4-diaminobenzoic with
compounds 8a-c in DMF and sodium thiosulphate afforded acid
derivatives of 2-arylbenzimidazoles (9a-c). Finally, coupling of 2-
arylbenzimidazoles 9a-c with ammonium carbonate in DMF and
CDI yielded amides of analogues of 2-arylbenzimidazoles 10a-c
(Scheme 2).
ethyl
2-(5-cyano-4-(4-florophenyl)-6-oxo-1,6-
(Fig. 1C) [35] and 2-(4-(4-((2-carbamoylhydrazono)methyl)-3-
methyl-1-phenyl-1H-pyrazol-5-yloxy)phenyl)-5-nitro-1H-benzo
[d]imidazole (3) [36] exhibiting high potency as Chk2 inhibitors
(Fig. 1D).
On the other hand, there are great numbers of antitumor active
agents possessing the pyrimidine nucleus [37e41] such as pyr-
imidinylmethyl benzimidazole derivatives (I) (Fig. 2) which
exhibited potent antitumor effect against panel of cancer cell lines
[38] and AZD6738 (Fig. 1E) is orally active anti-tumor single agent
acting as selective ATR inhibitor in phase I clinical trial and used in
cancer treatment as a monotherapy or in combination with chemo/
radiation therapy in patients with solid tumors [40,41].
The present study aimed to synthesize new candidates of 2-
arylbenzimidazoles, on the basis of molecular hybridization of 2-
arylbenzomidazoles and pyrimidines, guided by molecular dock-
ing studies, while assessing their biological activities not only as
Chk2 inhibitors and antitumor agents, but also in combination with
cisplatin or doxorubicin.
The second series of pyrimidine-benzimidazole conjugates

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
689
Fig. 1. A-E: Reported potent Chk2 inhibitors (AeD) and AZD6738, potent antitumor active agent with selective inhibition against ATR (E).
Fig. 2. Rational design of the target pyrimidine-benzimidazole conjugates via hybridization of effective antitumor active agents (I) and selective Chk2 inhibitors (II).

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
Scheme 1. Synthetic routes of chloropyrimidine derivatives 7a-g.
12e15 (Scheme 3) with amino linker (X ¼ NH; Fig. 2) were obtained
by reaction of chloropyrimidines 7d,e with 4-aminobenzoic acid to
form pyrimidinyl aminobenzoic acid derivatives 11a,b in the pres-
ence of DMF, potassium hydroxide and catalytic amount of copper
(I) iodide. Then, 3,4-diaminobenzoic acid was reacted with com-
pounds 11a,b in polyphosphoric acid to form acid derivatives of 2-
arylbenzimidazoles 12 and 13, respectively. Finally, coupling of 12
and 13 with ammonium carbonate in the presence of CDI and DMF
yielded the target amides of pyrimidine-benzimidazole conjugates
14 and 15, respectively (Scheme 3).
mercaptobenzoic acid to form phenyl pyrimidine thio-ether de-
rivatives 11e, 11c, 11d, and 11f, respectively, in the presence of
potassium hydroxide, catalytic amount of copper (I) iodide and
DMF. Thio-ether derivatives 11c-f were reacted with 3,4-
diaminobenzoic to afford pyrimidine-benzimidazole conjugates of
thioether linker 16e19 which followed by formation of the target
amides analogues 20e23 via coupling with ammonium carbonates
(Scheme 3).
The synthesized compounds were characterized by physico-
chemical and spectral means. The MS, 1HNMR, 13CNMR spectral
data and elemental analyses were found in accord with the
assigned molecular structures.
The third series of pyrimidine-benzimidazole conjugates 16e23
with thio-ether linker (Scheme 3) obtained via similar steps that
started by reaction of chloropyrimidines 7c, 7e, 7f and 7g with 4-

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
691
Scheme 2. Synthetic routes of pyrimidine-benzimidazole conjugates 9a-c and 10a-c.
2.3. Evaluation of the efficacy of target compounds as Chk2
inhibitors
20e23 in addition, to two reference compounds (as a positive
control) cisplatin and doxorubicin were tested on human breast
cancer cell line (MCF7) using sulphorhodamine-B (SRB) assay [46]
(Table 2). Compounds 9b, 10a-c, 14, 15 and 20e23 showed potential
The checkpoint Kinase Assay was used to demonstrate the
chemical inhibition of Chk2 function by compounds 9a-c, 10a-c, 14,
15 and 20e23. The IC50 values of synthesized compounds as Chk2
inhibitors in addition to 2-(4-(4-hydroxyphenylthio)phenyl)-1H-
benzo[d]imidazole-5-carboxamide (1) as lead compound have
been evaluated (Table 1). The activity of synthesized compounds
has not been examined against other kinases. The determination of
IC50 of studied compounds against Chk2 function profile, the newly
synthesized 2-arylbenzimidazoles are effective potent inhibitors
against Chk2 (Table 1) with IC50 in a range from 5.56 nM to
46.20 nM. Six candidates (9b, 23, 9a, 14, 9c and 20) out of twelve
synthesized 2-phenylbenzimidazoles were found to be more
potent than the lead compound 1 (IC50 ¼ 32.40 nM). Compounds 9b
and 23 are the most potent derivatives (IC50x 5.55 nM). Com-
pounds 10a, 10b, 21, 15 and 10c exhibited comparable inhibitory
effect with the lead compounds. In the first series with ether linker,
acid conjugates 9a-c are more potent than the amides analogues
10a-c. In the second series with amino linker, compound 14 is more
effective than compound 15. In the third series with thioether
linker, compound 22 (IC50 ¼ 46.20 3.44 nM) showed the least
potency among the studied compounds. The order of activity is as
follows: 23 ¼ 9b, 9a, 14, 9c, 20, 10a, 10b, 21, 15, 10c and finally 22
(Table 1).
cytotoxicity on MCF7. The compounds 14 (GI50 ¼ 6.6 0.047
mM),
M) showed
15 (GI50 ¼ 7.1 0.01
mM) and 23 (GI50 ¼ 6.2 0.07
m
higher cytotoxic effect than doxorubicin (GI50 ¼ 8.4 0.04) while, a
comparable toxicity to cisplatin was observed (GI50 ¼ 6.8 0.04).
On the other hand, compounds 9b, 10a-c and 20e22 exhibited less
potential cytotoxity than the two reference drugs, doxorubicin and
cisplatin with GI50 in range from (11.7 0.07 mM to 24.9 0.1 mM).
Preliminary structure activity relationships can be evaluated from
the obtained results as follow: compound 9b (GI50 ¼ 24.9 0.10) is
less cytotoxic than its amide analogue 10b (GI50 ¼ 14.6 0.06). For
compounds 10a (GI50 ¼ 20.2 0.02) and 10c (GI50 ¼ 11.7 0.07),
the presence of Cl in 10a negatively affected on the activity. For
amides of series 2, compounds 14 and 15 exhibited the highest
activity among the studied compounds. For compounds 20e23 of
series 3, compound 23 (GI50 ¼ 6.24 0.07) is the most active
compounds with obvious higher activity than its analogue 22
(GI50 ¼ 14.6 0.03) that revealed the importance of presence of
ethyl acetate group on thiol of pyrimidine moiety. For analogues 20
(GI50 ¼ 11.8 0.05), 21 (GI50 ¼ 15.0 0.03) and 22 exhibited com-
parable cytotoxic effect against MCF-7. Studying of SAR indicated
that acid conjugate 9b is less effective than its amide analogue 10b.
Ethyl thioacetate group is highly effective on the activity as
observed in compound 23. Compounds with amino linker as in
compounds 14 and 15 are more effective as cytotoxic agents than
that with ether or thio-ether linkers.
2.4. Cytotoxicity of the synthesized Chk2 inhibitors and their
combinations with doxorubicin and cisplatin
Resistance of the tumor to the used chemotherapy is a common
phenomenon that can result in rapid disease progression during or
The cytotoxicity of the studied compounds 9b, 10a-c, 14, 15 and

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
Scheme 3. Synthetic routes of pyrimidine-benzimidazole conjugates 12e23.
shortly after completion of treatment. One of the treatment mo-
dalities to overcome the impact of drug resistance is the concurrent
use of two or more chemotherapy agents with different mecha-
nisms of action and different modes of drug resistance. Therefore,
one of the main goals of the present study was to investigate the
combined effect of synthesized compounds 9b, 10a-c, 14, 15, and
20e23 with genotoxic drugs as cisplatin and doxorubicin. In this
study, combination effect was expressed as a potentiation index
(PI), which is the ratio of GI50 for the Chk2 inhibitor alone and GI50
for the Chk2 inhibitor in combination with anticancer drug
(Table 2). The studied compounds 10a-c, and 14, 15 potentiated the
cytotoxic effect of both doxorubicin and cisplatin (Table 2) with
dramatic decrease in their IG50. Compound 10b exhibited the best
potentiation effect with the highest PI whereas acid conjugate 9b
and compounds of series 3 with thio-ether linker 20e23 antago-
nize the cytotoxic effect of both drugs.
The foregoing results indicated that the synthesized compounds
are effective as Chk2 inhibitors as well as effective antitumor

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
693
Table 1
2.5. Cellular inhibitory effect of synthesized compounds against
Chk2 alone and in combination with genotoxic anticancer drug
Investigation of the activity of pyrimidine-benzimidazole conjugates 9a-9c, 10a-10c,
14, 15 and 20e23 as Chk2 inhibitors.
Compound#
IC50 (nM)
Compound#
IC50 (nM)
Several kinases in the DNA-repair pathway are activated after
cells are exposed to cisplatin. Among the two kinases involved in
checkpoint activation, namely ATM and ATR, cisplatin preferentially
activates ATR kinase [47,48], which phosphorylates p53 at serine-
15 to initiate activation of the p53 protein [49]. Also, cisplatin ac-
tivates Chk2, but the effect of cisplatin on Chk2 appears to be in-
dependent of ATM [48,50,51].
9a
9b
9c
10a
10b
10c
1
15.35 4.36
5.56 6.80
27.88 6.66
31.00 4.22
32.40 2.07
33.80 8.08
32.40 6.17
14
15
20
21
22
23
25.80 2.99
33.40 3.27
28.00 7.12
35.90 9.41
46.20 3.44
5.55 4.09
The chemical evaluation of the synthesized compounds proved
their potency as Chk2 inhibitors. It is important to determine their
cellular inhibitory effects against Chk2 in cancer cells alone and in
the combination with cisplatin. To address our target MCF-7 cells
were exposed to single agent treatment with IG50 of cisplatin, 9b,
1 ¼ Lead compound; 2-(4-(4-hydroxyphenylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxamide.
IC50: The concentration of synthesized compound that inhibits Chk2 by 50%.
IC50 values expressed in nM SD, all values are the mean of at four replicate
experiments.
10b, or their combination (IG50 of cisplatin þ0.1m M of studied
compound, 9b or 10b) (Fig. 3). Treated and untreated MCF-7 cells
(negative control) were harvested, and proteins were extracted,
separated on SDS-PAGE gel, transferred onto NC membrane and
then probed with phosphorylated anti-Chk2 antibody [52,53]. The
western blot reaction showed the immunogenic band recognized
phosphorylation (activation) of Chk2 at 64.8 kDa which was
observed in the untreated cancer cells (lane 4) and the cells treated
with cisplatin alone (lane 3) and in combination of compound 9b
(lane 2) (Fig. 3). A complete disappearance of the immunogenic
band of Chk2 phosphorylation reaction (activation process) in the
cases of treating cells with IG50 of compounds 9b (lane 1), 10b (lane
5) and the combination of 10b with cisplatin (lane 6), was observed.
These results suggest that the expression of Chk2 protein is induced
by cisplatin [49,50] and its combination with 9b whereas it was
completely inhibited in combination of cisplatin and compound
10b (Fig. 3). The specific blockade of Chk2 activity by compounds 9b
and 10b as well as combination of 10b with cisplatin in breast
cancer cells proved that both compounds both 9b and 10b alone
exhibited chemical inhibition of Chk2 activity but also the com-
bined treatment of 10b with cisplatin potentiates the kinase
inhibitory effect of compound 10b that suggest this combination
may be important therapy for resistant types of tumors. Interest-
ingly, combination of 9b and cisplatin induced expression of Chk2
which indicated that the combined treatment of 9b and cisplatin
antagonized the inhibitory effect of 9b which in accord with the
observed antagonism effect for antitumor activity on MCF-7 cells in
the potentiation assay.
Table 2
Cytotoxicity (GI50) of studied compounds 9b, 10a-c, 14, 15 and 20e23 alone and their
combination with genotoxic drugs as Doxorubicin and Cisplatin against MCF7 cells.
Compound GI50
(
m
M)
combination GI50
(
m
M)
PI
Doxorubicin Cisplatin
Doxorubicin Cisplatin
9b
24.90 0.10 >100
>100
<1
<1
10a
10b
10c
14
15
20
21
22
23
DOX
CIS
20.25 0.02 0.1 0.02
14.60 0.06 0.04 0.02
11.70 0.07 0.08 0.08
6.60 0.04
7.13 0.01
11.80 0.05 >100
15.00 0.03 >100
14.61 0.03 >100
06.24 0.07 >100
8.40 0.04
0.06 0.004 202.00
0.04 0.01
0.1 0.06
336.60
365.00
117.00
ND
142.00
<1
<1
<1
<1
365.00
146.25
132.00
78.80
<1
<1
<1
<1
0.05 0.041 ND
0.09 0.01
0.05 0.05
>100
>100
>100
>100
6.82 0.04
Each value is the mean SD of 5 independent experiments performed in triplicates;
the surviving fraction ¼ O.D. (treated cells)/O.D. (control cells). [48].
IG50: the concentration that inhibited growth by 50% relative to untreated cells.
PI: the ratio of the mean GI50 for the ChK2 inhibitor alone and the mean GI50 for the
ChK2 inhibitor in combination with a cytotoxic agent.
The statistical significance of the results was analyzed using one-way ANOVA fol-
lowed by Tukey multiple comparison test.
activity. The checkpoint kinase 2 (Chk2) is a critical enzyme
involved in the DNA damage-response pathway, which is activated
by phosphorylation prompting cellular response such as DNA
repair, cell-cycle regulation or apoptosis. Moreover, inhibition of
Chk2 is thought to sensitize p53-mutated or p53-deficient
cancerous cells leading to cell death [47]. Due to their multiple
pharmacological effects, the IC50 (Table 1) that measure the degree
of kinase inhibition of the synthesized compounds is different from
IG50 that is associated with cell death which may not confined by
only one pathway (Table 2). Their activities as Chk2 inhibitors result
from their binding to Chk2 receptor via interaction with its active
sites as reported in the literature data bases of 2-
arylbenzimidazoles class. So, we can deduce that there are
different modes of actions controlling their inhibitory effect against
Chk2 and their cytotoxicity on MCF-7 cells, which reflect the suc-
cess of the hybridization of pyrimidine and 2-arylbenzimidazole in
the production of molecules with complex modes of action that
impact our multiple targets simultaneously that are better in con-
trolling complex disease systems, less disposed to drug resistance
and being the standard of care in cancer treatment [43,45].
Fig. 3. Western Blot analysis showing reactivity of different samples on MCF-7 cells.
Lane M: Pre-stained molecular weight marker, Lane 1: IG50 of compound 9b (C9) Lane
2: IG50 of cisplatin þ0.1 M of compound 9b (Cis þ C9b), Lane 3: IG50 of cisplatin (Cis),
m
Lane 4: control, Lane 5: IG50 of compound 10b (C10b), Lane 6: IG50 of cisplatin þ0.1
m M
of compound 10b (Cis þ C10b).

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
2.6. Effect of synthesized Chk2 inhibitors on apoptosis
with cisplatin had the best apoptotic effect on the MCF-7 cells while
cells treated with compound 9b and cisplatin (Fig. 4D) showed the
worst anti-apoptotic effect compared to the untreated cells
(Fig. 4A). Regarding the MCF-7 cells treated with IG50 of cisplatin,
compounds 9b and 10b, the number of the living cells were
207 8.54 (Fig. 4B), 284 6.91 (Figs. 4C) and 232 18.69 cells
(Fig. 4E), respectively. These results can be summarized in Fig. 4G to
reflect the antitumor activity of compounds 9b and 10b as well as
The apoptotic effect was studied to further investigate anti-
tumor activity of synthesized compounds. The experiments
included untreated cells (control cells), treated MCF-7 cells with
GI50 of cisplatin, compound 9b or 10b alone or combination of GI50
of cisplatin with 0.1
mM of synthesized conjugates 9b or 10b
(Fig. 4AeG). As shown in Fig. 4F, combination of compound 10b
Fig. 4. A-G: Effect of synthesized Chk2 inhibitors on apoptosis of MCF7 cells. A, untreated cells (control). B, GI50 of cisplatin alone. C, GI50 of compound 9b. D, GI50 of cisplatin in
combination with 0.1 M of compound 9b. E, GI50 of compound 10b. F, GI50 of cisplatin in combination with 0.1 M of compound 10b. G, quantification of the apoptotic effect by
m for each of stained with propidium iodide by using confocal microscope (Zeiss LSM 710), the objective lens
m
m
calculating mean number of living cells in 5 point of dimension 1700
m
is 20X and magnification 200X.

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
695
Fig. 5. A-E: Effects of compound 10b on drug-induced cell cycle arrest in MCF-7 breast cancer cells. A, effects of cells treated with DMSO (control cells). B, effect of cells treated with
GI50 of doxorubicin alone. C, effects of cells treated with GI50 of compound 10b. D, effects of cells treated with GI50 of doxorubicin in combination with 0.1 M of compound 10b.
m
Histograms, cell cycle distribution were assessed by propidium iodide DNA staining as shown in Materials and Methods. E, quantification of the effects for total cells with studied
concentrations of compound 10b alone and in combination with doxorubicin-induced cell cycle arrest in MCF-7 cells at 48 h.

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
the potentiation effect of compound 10b to the cytotoxicity of
cisplatin in addition to the antagonism effect of compound 9b to
cisplatin.
refined using energy minimization with added hydrogens. Docking
calculations were carried out using standard default variables for
the MOE program [49].
The binding affinities were evaluated on the basis of the binding
free energy S-score and hydrogen bonds with their distance be-
tween the designed compounds and the amino acids in the re-
ceptor (Table 3).
All the synthesized compounds 9a-c, 10a-c, 14, 15 and 20e23
gave higher score than the Lead compound; 2-(4-(4-
hydroxyphenylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (1) which docked into Chk2 with S-score fitness in a
range from (ꢁ15.95 kcal/mol) to (ꢁ20.01 kcal/mol) (Table 3).
Compounds 9b, 9a, and 23 exhibited the highest score fitness
of ꢁ20.01, ꢁ18.73 and ꢁ18.07, respectively (Table 3).
2.7. Cell cycle analysis
Flow cytometry was used to check the activity of the synthe-
sized Chk2 inhibitors on the cell cycle phases. The experiments
were carried out on MCF-7 breast carcinoma cell line, including
untreated cells (control cells), treated cells with GI50 of compound
10b, treated MCF7 cells with GI50 of doxorubicin alone or GI50 of
doxorubicin in combination with 0.1 mM of compound 10b
(Fig. 5AeE). The analysis of control cells revealed that untreated
cells showed the expected pattern for continuously growing cells
and the existences of 75.09% of cells were in G1 phase, 17.24% in S-
phase and 7.67% in G2/M phase (Fig. 5A). While treating cells with
GI50 of doxorubicin, the number of the arrested cells in S-phase
increased (21.3%) and non-significant changes in the number of
cells in phases of G1 and G2/M by comparison to control cells
(Fig. 5B). Comparing the effect of compound 10b with untreated
control, compound 10b alone arrested MCF7 cells at G2/M by 11.19%
and almost no changes at G0/G1 phase (72.08%) or S phase(16.73%)
of the cell cycle (Fig. 5C). These results suggest that the G2-M phase
accumulation was caused by an increase in the duration of the G2-
M phase. The combination of doxorubicin with compound 10b
caused obvious change on the cell cycle distribution by comparison
to the effect of both doxorubicin or compound 10b with significant
arrest in S phase (43.8%) and a subsequent decrease in G1 to 56.6%
and complete absence of cells in G2-M (Fig. 5D). This result suggests
not only S phase accumulation but almost complete arrest of many
cells in the S phase. A summary of the cell cycle distribution found
in MCF-7 carcinoma cells treated with compounds 10b and com-
bination with doxorubicin with the studied concentrations
compared to control cells at 48 h (Fig. 5E).
The active-site of amino acids residues in Lead compound
formed one H-bond with GLU 308 and in the native ligand formed
three hydrogen bonds with amino acids residue GLU 308, GLU 351
and MET 304 beside two H-bonds with NO3 1516. The synthesized
compounds 9a-c, 10a-c, 14, 15 and 20e23 were bound into the
binding site through up to two hydrogen bonds, to a higher extent
with LYS 349, LYS 294, MET 304, NO3 1516 and GLY 232, and to a
lower extent with ASN 269 (C¼O), GLU 273, GLU 305 and ASP 368
amino acids (Table 3).
Compound 9b showed the highest binding free energies
(ꢁ20.0135 kcal/mol) among the studied compounds (Table 3). It
also bound into ChK2 kinase by two hydrogen bonds into the
binding site through its carboxylic group at position 5 with NH of
Met 304 and its terminal cyano group with NH of LYS 249 (Fig. 7).
The docking results for compound 9b are highly correlated to its
inhibitory activity against ChK2 kinase with highest activity among
the synthesized compounds (IC50 ¼ 5.56 6.80 nM).
Compound 10b exhibited S-score fitness into Chk2 kinase re-
ceptor (ꢁ16.01 kcal/mol) comparable to lead compound 1 as well as
they exhibited similar inhibitory activity against ChK2
(IC50 ¼ 32.40) (Table 1). Compound 10b also bound into the binding
site of the receptor by two hydrogen bonds with MET 304 and LYS
349 (Fig. 8AeB) (Table 3). Compound 10b and lead compound 1
revealed lower inhibitory activity than compound 9b (Table 1).
Compound 9a showed binding free energy (ꢁ18.73 kcal/mol)
higher than lead compound (ꢁ15.56 kcal/mol). Also, 9a showed
better interaction than lead compound 1 by forming 2 H-bonds
with the receptor binding site moieties MET 304 and LYS 249 and
carboxylate at position 5 and terminal cyano of 9a, respectively
(Fig. 9) compared to only 1 H-bond between the lead compound
and GLU308. The docking results for compound 9a is correlated to
its inhibitory activity against ChK2 kinase (IC50 ¼ 15.35 4.36 nM)
which is also higher than lead compound. The plausible binding
modes and the docking orientations for compounds 9a, 9b and 10b
superimposed in the binding site of Chk2 receptor that are sur-
rounded by a Gaussian contact surface are represented in Fig. 10.
2.8. Molecular modeling
The molecular modeling of the target compounds 9a-c, 10a-c,
14, 15 and 20e23 using MOE (molecular modeling environment)
program was studied to get deeper insight into the molecular bases
of the inhibitory potency and to find out the interaction between
the new synthesized pyrimidine-benzimidazole conjugates and
Chk2 receptor.
Molecular modeling calculations and local docking were done
using MOE [54] to evaluate the binding free energies of these in-
hibitors into the target to Chk2 kinase receptor (PDB: (2XBJ).
2.8.1. Validation of the docking performance and accuracy
To validate the docking accuracy of the program used, docking of
the native co-crystallized XBJ (4-fluoro-2-(4-{[(3S,4R)-4-(2-
hydroxypropan-2-yl)pyrrolidin-3-yl]amino}-6,7-
dimethoxyquinazolin-2-yl)phenol) ligand of Chk2 kinase (PDB:
(2XBJ) into the binding sites. The docked pose was docked super-
imposed on the native co-crystalized ligand on its binding sites
with a root mean square deviation (RMSD) of 1.8 Å, S-score
of ꢁ19.32 and bound by five hydrogen bonds (Fig. 6AeB).
2.8.3. Structure-activity relationship
Structure-activity relationship was deduced from the forgoing
results on the basis of molecular modeling by getting deeper insight
into the molecular bases of the inhibitory potency (Table 1) and by
correlating the interaction of pyrimidine-benzimidazole conjugates
9a-c, 10a-c, 14, 15, 20e23 and Chk2 receptor by the change in the
binding free energy according to change of substituents (Table 3).
Binding free energy score is a combination of different scoring
functions includes H-bonds, ionic interaction, hydrophobic inter-
action, metal ligation and the interaction between hydrophobic and
polar atoms. In the first series (X ¼ O), the binding free energies of
the acid derivatives, 9a-c, are higher than the amide analogues 10a-
c (Table 3) which in accord with the observed higher inhibitory
effect of the acid derivatives, 9a-c, than the amide analogues 10a-c
2.8.2. The binding affinities of the synthesized compounds into
ChK2 receptor
All synthesized compounds were docked into same groove of
the binding site of the native co-crystalized XBJ (4-fluoro-2-(4-
{[(3S,4R)-4-(2-hydroxypropan-2-yl)pyrrolidin-3-yl]amino}-6,7-
dimethoxyquinazolin-2-yl)phenol) ligand of Chk2 kinase into the
binding sites and for docking calculations, the protein structure
(PDB: (2XBJ) was first separated from the inhibitor molecule and

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
697
Fig. 6. A-B: Proposed binding mode for the interaction of the native co-crystallized ligand with Chk2 receptor (PDB: 2XBJ), five hydrogen bonds are formed with GLU 308, GLU 351,
NO3 1516, MET 304, NO3 1516 (violet dotted lines), (A: 2D and B: 3D). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
Table 3
Molecular Operating Environment (MOE) [54] flexible docking results for compounds 9a-c, 10a-c, 14, 15 and 20e23 docked by (MOE) 2008.10 into Chk2 kinase (PDB: (2XBJ) in
comparison to the co-crystallized XBJ ligand.
Compound
S- scorea (kcal/mol)
Hydrogen bonds between atoms of compounds and amino acids of receptor
Compounds
Atoms
receptor
Atoms
Type
Distance (Aꢀ)
Residues
XBJb
ꢁ19.32
H 4502
H 4512
N 4511
O 4539
N 4511
H 4964
O 5152
N 5153
O 5152
N 5211
O 5152
O 5152
N 2403
H 5117
O 5309
H 2418
N 2387
O 2415
H 2422
N 2383
H 2422
O 2409
O 2418
N 2386
N 2403
H 5013
N 4998
OE 1322
O 2049
O2 4562
N 1262
O2 4562
OE 1322
N 1262
NZ 2026
N 1262
NZ 2026
O1 4489
O1 4489
O2 2214
O 1267
NZ 2026
O1 2213
N 150
GLU 308
GLU 351
NO3 1516
MET 304
NO3 1516
GLU 308
MET 304
LYS 349
H-don
H-don
H-don
H-acc
H-acc
H-don
H-acc
H-acc
H-acc
H-acc
H-acc
H-don
H-acc
H-don
H-acc
H-don
H-acc
H-acc
H-don
H-acc
H-don
H-acc
H-acc
H-acc
H-acc
H-don
H-acc
2.03
2.23
2.86
2.79
2.86
1.19
1.49
2.79
1.51
3.00
1.92
1.92
2.55
1.59
2.88
2.57
2.85
3.13
1.90
2.52
1.55
2.79
2.88
2.99
2.60
2.25
2.98
1c
9a
ꢁ15.57
ꢁ18.73
9b
9c
ꢁ20.01
ꢁ16.43
MET 304
LYS 249
NO3 1516
NO3 1516
NO3 1516
MET 304
LYS 349
NO3 1516
GLY 232
ASN 269
GLU 273
GLY 232
GLU 305
LYS 349
ASP 368
GLY 232
NO3 1516
ALA 230
MET 304
10a
10b
ꢁ16.95
ꢁ16.01
10c
14
ꢁ16.22
ꢁ17.26
ND 338
OE 365
N 150
15
20
ꢁ16.42
ꢁ17.50
O 620
NZ 987
N 1122
N 150
O2 2214
O 298
21
22
ꢁ16.52
ꢁ15.95
23
s
N 1262
a
Binding free energy.
b
4-Fluoro-2-(4-{[(3S,4S)-4-(1-hydroxy-1-methylethyl)pyrrolidin-3-yl]amino}-6,7-dimethoxy-quinazolin-2-yl) phenol.
Lead compound: 2-(4-(4-hydroxyphenylthio)phenyl)-1H-benzo[d]imidazole-5-carboxamide (1).
c
(Table 1). For the acid candidates 9a-c: compound 9b is one of the
causes a great decrease in the activity of 22, which can be correlated
with the increase of binding free energy in case of substitution with
the methoxy group with respect to both methyl and chloro as
substituents in compounds 21 and 22, respectively.
most potent compound with the lowest IC50 ¼ 5.56 nM and the
highest binding free energy (S-score ¼ ꢁ20.01 kcal/mol, Table 3).
Decreasing of binding free energy by substitution with chlorine in
9c (S-score ¼ ꢁ16.43 kcal/mol) was observed and negatively
affected the inhibitory effect (IC50 ¼ 27.88 nM) compared to the
unsubstituted 9a (S-score ¼ ꢁ18.73 kcal/mol, IC50 ¼ 15.35 nM). For
the amide analogues 10a-c, comparable activities were detected
(IC50x32 nM) as well as comparable free energies (S-
scoresx ꢁ16 kcal/mol) were found. For amides of the second series
(compounds 14 and 15, X ¼ S), comparing the effect of the nitro
group in compound 14 to that of the methoxy group in compound
15, it can be deduced that the presence of the nitro group was more
favorable for activity (IC50 ¼ 25.80 nM) and led to enhancement of
both, the binding free energy (S-score ¼ ꢁ17.26 kcal/mol) and H-
bonding interaction with the receptor, (IC50 ¼ 33.40 nM, S-
score ¼ ꢁ16.42 kcal/mol). For the third series (20e23, X ¼ NH),
compound 23 exhibited the highest activity with a remarkable
decrease in the IC50 (5.55 nM) (Table 1) and an increase in the
binding free energy (S-score ¼ ꢁ18.07 kcal/mol, Table 3) with
respect to its compound 22, which exhibited the least potency
among the studied compounds (IC50 ¼ 46.20 nM, Table 1) and
lowest S-score that reflecting the importance of ethyl thioacetate as
substituent. This can be attributed to strong hydrophobic interac-
tion with the receptor and H-bonding interaction with its carbonyl
group which monopolizes the activity, no matters the compound
was an acid with the ether linker as 9b or an amide with amino
linker as 23, or the substituent on the phenyl ring was floro or
chloro. For analogues 20, 21 and 22, the OCH3 substitution was
favored as compound 20 was more active than 21 having CH3
substitution, or Cl in 22, which have negative effect on activity and
3. Conclusion
The studied compounds 9b, 10a-c, 14, 15 and 20e23 exhibited
potent inhibition effect against enzymatic activity of Chk2. The
inhibitory effect of compounds 9b and 10b was also evaluated by
western blot using anti-Chk2 which indicated that compounds 9b,
10b and combination of 10b with cisplatin blocked the enzymatic
activity of Chk2 which was proved by complete disappearance of
characteristic band of Chk2 at 64.8 KDa. Moreover, the studied
compounds 9b, 10a-c, 14, 15 and 20e23 exhibited remarkable
cytotoxicity against MCF-7. Compounds 10a-c, 14 and 15 potenti-
ated the activity of both cisplatin and doxorubicin whereas com-
pounds 9b and 20e23 antagonized their cytotoxicity. The
antitumor effect of compounds 9b and 10b was proved also by
studying their apoptotic effect. Flow cytometry indicated that
combination of compound 10b with doxorubicin blocked the cell
cycle to almost complete arrest in the S phase. The foregoing results
necessitated further elucidation of pyrimidine-benzimidazole
conjugates.
4. Material and methods
Microanalyses, spectral data of the compounds were performed
in the Microanalytical National Research Centre and Pharmaceu-
tical faculty, Cairo University, Egypt. The IR spectra (4000-
400 cmꢁ1) were recorded using KBr pellets in a Jasco FT/IR 300E

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
699
Fig. 7. A-B: Compound 9b exhibited good fitting with ChK2 receptor (pdb: 2XBJ) via hydrogen bonds and hydrophobic interaction. Two direct H-bonds with Met 304 and LYS 249
are formed. Green line represents the distance of the hydrogen bonds and violet dots represent the score percentage of the H-bonds interaction with the receptor (Fig. 7A). Docking
orientation of Compound 9b into ChK2 receptor (pdb: 2XBJ) receptor with a gaussian contact surface surrounding the van der Waals surface is drawn around the binding site
(Fig. 7B). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fourier transform infrared spectrophotometer on a Perkin Elmer
FT-IR 1650 spectrophotometer. Starting materials, reagents, and
solvents for reactions were reagent grade and used as purchased.
Chromatography solvents were HPLC grade and were used without
further purification. Thin layer chromatography (TLC) analysis was
performed using Merck silica gel 60 F-254 thin layer plates. Flash
column chromatography was carried out using columns prepacked
peak. The data is given as follows: chemical shift (d) in ppm, mul-
tiplicity (where applicable), coupling constants (J) in Hz (where
applicable), and integration (where applicable). The mass spectra
were recorded using a Finnigan mat SSQ 7000 (Thermo. Inst. Sys.
Inc., USA) spectrometer at 70 eV. The used petroleum ether had a
boiling temperature in the 60e80 ꢂC range.
with 40e63 mm silica or using standard chromatography tech-
4.1. Molecular docking
niques when performed on a large scale. NMR spectra were
recorded on a Bruker Advance 300 MHz spectrometer. The samples
were referenced to the appropriate internal non-deuterated solvent
The crystal structures of the Checkpoint kinase (PDB: 2XBJ) [34]
in complex with its co-crystalized native ligand: (XBJ): (4-fluoro-2-

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
Fig. 8. A-B: The interaction of compound 10b with Chk2 receptor (pdb: 2XBJ), 2 H-bonds are formed with MET 304 and LYS 349. Green line represents the distance of the hydrogen
bonds and violet dots represent the score percentage of the H-bonds interaction with the receptor (Fig. 8A). Docking orientation of Compound 10b into ChK2 receptor (pdb: 2XBJ)
receptor with a gaussian contact surface surrounding the van der Waals surface is drawn around the binding site (Fig. 8B). (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
ꢀ The constructed 3D structures of small library designed com-
(4-{[(3S,4R)-4-(2-hydroxypropan-2-yl)pyrrolidin-3-yl]amino}-6,7-
pounds about 200 derivatives were energetically minimized
through Force field MMFF94x Optimization using gradient of
0.0001 kcal/mol.
dimethoxyquinazolin-2-yl)phenol), as a potent and selective In-
hibitor of Checkpoint Kinase 2, was retrieved from the Protein Data
Bank, http://www.rcsb.org/pdb/home/home.do. The key amino
acids of the active site were identified using data in PDB sum,
http://www.ebi.ac.uk/pdbsum.
ꢀ Validation of the MOE software by re-docking of the native co-
crystalized on its binding site was done. The S-score
is: ꢁ19.32 kcal/mol and RMSD is 1.8 Å

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
701
Fig. 9. A-B: The interaction of compound 9a with Chk2 receptor (pdb: 2XBJ), 2 H-bonds are formed with MET 304 and LYS 349. Green line represents the distance of the hydrogen
bonds and violet dots represent the score percentage of the H-bonds interaction with the receptor (Fig. 7A). Docking orientation of Compound 9a into ChK2 receptor (pdb: 2XBJ)
receptor with a gaussian contact surface surrounding the van der Waals surface is drawn around the binding site (Fig. 7B). (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
ꢀ Docking calculations were carried out using standard default
variables for the MOE program and the tested compounds were
docked in the binding site of the native co-crystalized ligand.
ꢀ The Dock scoring in MOE software was done using London dG
scoring function, rotatable bonds were allowed; the best 10
poses were retained and analysed.
ꢀ The binding site of the human crystal structure of Chk2 is
composed of the following amino acids: Leu226, Gly227, Val234,
Ala247, Ile286, Leu301, Glu302, Leu303, Met304, Glu305,
Gly307, Glu308, Glu351, Asn352, and Leu354.
of compounds having simalr S-scores, hydrogen bonds between
the ligand and amino acids in Chk2 receptors were used in the
ranking of the compounds. Evaluation of the hydrogen bonds
was done by measuring the hydrogen bond length which
doesn't exceed 3 Å.
ꢀ Twelve compounds out of the best fifty were selected for
chemical synthesis in this study and biological evaluations were
performed as shown in the forgoing results.
ꢀ The mode of interaction of the native ligand 2XBJ receptors
within their crystal structures was used as a standard docked
model as well as for RMSD calculation.
ꢀ The binding free energy was used to rank the binding affinity of
the synthesize compounds to Chk2 receptor. In addition, in case

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S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
Fig. 10. Docking orientations and superimposition for Compound 9a "colored in blue", compound 9b "colored in red" along with ompound 10b "colored in yellow" into ChK2
receptor (pdb: 2XBJ) receptor. A gaussian contact surface surrounding the van der Waals surface is drown around the binding site. (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
4.2. Chemistry
crystals of compounds 7a, 7b, 7c-7f [38] and 7g.
4.2.1. Synthesis of compounds 5 and 6
4.2.2.1. Ethyl 2-(4-chloro-5-cyano-6-(4-fluorophenyl)pyrimidin-2-
ylthio)acetate (7b). Yield (50%), m.p. 143e145 ꢂC. 1H NMR
A
solution of 6-(4-fluorophenyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile (4b) or 6-(4-chlorophenyl)-
4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4c)
[39] (10 mmol) in benzene (50 mL) and K2CO3 (1.38g, 10 mmol) was
treated with ethyl bromoacetate (1.66g, 10 mmol) added dropwise.
The reaction mixtures were stirred for 1 h at room temperature
then were refluxed for 3 h. The solvent was evaporated under
reduced pressure. The solids obtained were filtered off and were
crystalized from benzene as yellow crystals.
(300 MHz, DMSO) d 1.10e1.24 (t, 3H, CH3), 4.07e4.20 (m, 4H, 2CH2),
7.37e7.40(m, 2H), 8.10e8.13(m, 2H, 2H-Ar). MS, m/z (%):351 (58%),
353 (19%) Mþ. Anal. Calcd for C15H11ClFN3O2S (FW: 351): C, 51.21; H,
3.15; Cl, 10.08; F, 5.40; N, 11.94; S, 9.11. Found: C, 51.09; H, 3.17; Cl,
10.22; N, 12.07; S, 9.21.
4.2.2.2. Ethyl 2-(4-chloro-6-(4-chlorophenyl)-5-cyanopyrimidin-2-
ylthio)acetate (7g). Yield (64%), m.p. 166e169 ꢂC.
d 1.05e1.13 (t,
3H, CH3), 4.09e4.20 (m, 4H, 2CH2), 7.54e7.57(d, J ¼ 8.22 Hz 2H),
7.98e9.01(d, J ¼ 8.22 Hz, 2H). MS, m/z (%): 368(19%), 366(60%) for
Mþ. Anal. Calcd for C15H11Cl2N3O2S (FW: 366): C, 48.93; H, 3.01; Cl,
19.26; N,11.41; S, 8.71. Found: C, 48.77; H, 3.24; Cl,19.00; N,11.39; S,
8.82.
4.2.1.1. Ethyl 2-(5-cyano-4-(4-florophenyl)-6-oxo-1,6-
dihydropyrimidin-2-ylthio)acetate
159e162 ꢂC. 1H NMR (300 MHz, DMSO)
3.99e4.19 (m, 4H, 2CH2), 7.39e7.49(m, 2H), 7.96e8.07(m, 2H, 2H-
Ar). 13C NMR (75 MHz, DMSO)
13.81, 32.93, 61.25, 93.00, 115.45,
(5). Yield,
(84%),
m.p.
d
1.03e1.12 (t, 3H, CH3),
d
115.57, 115.74, 115.81, 131.28, 131.34, 131.39, 130.77, 162.41, 165.12,
4.2.3. Synthesis of compounds 8a-c
165.96, 165.74, 167.91. MS, m/z (%): 333(Mþ, 28%), Anal. Calcd for
C
15H12FN3O2S (FW: 333): C, 54.05; H, 3.63; F, 5.70; N, 12.61; S, 9.62
4.2.3.1. Method A. A solution of 4-hydroxybenzaldehyde (1.22 g,
10 mmol), potassium hydroxide (2.24 g, 40 mmol), triphenylphos-
phine (0.524 g, 2 mmol) and cupper (I) iodide (0.19 g, 1 mmol) in
DMF (20 mL) was treated with compound 7a, 7b or 7c (10 mmol).
The reaction mixture was heated to 70 ꢂC and stirred under Argon
for 2 h. The reaction mixture was quenched with water and
extracted with ethyl acetate. The organic extracts were dried
(Na2SO4), concentrated, and purified by column chromatography
(0e25% EtOAc/n-hexane) to obtain yellow powders.
Found: C, 54.21; H, 3.44; N, 12.54; S, 9.77.
4.2.1.2. Ethyl 2-(4-(4-chlorophenyl)-5-cyano-6-oxo-1,6-
dihydropyrimidin-2-ylthio)acetate
(6). Yield,
(78%),
1.05e1.13 (t, 3H, CH3),
m.p.
178e180 ꢂC. 1H NMR (300 MHz, DMSO)
d
4.09e4.20 (m, 4H, 2CH2), 7.34e7.37 (d, J ¼ 8.22 Hz 2H), 7.48e7.51
(d, J ¼ 8.22 Hz, 2H). MS, m/z (%): 351 (15%), 349 (44%) for Mþ. Anal.
Calcd for C15H12ClN3O3S (FW: 349): C, 51.51; H, 3.46; Cl, 10.14; N,
12.01; S, 9.17. Found: C, 51.74; H, 3.55; Cl, 10.02; N, 12.22; S, 9.40.
4. 2. 3. 2. 4-(4-formylphenoxy)-6-phenyl-2-thioxo-1, 2-
4.2.2. Synthesis of compounds 7a-g
dihydropyrimidine-5-carbonitrile
(8a). Yield
(64%).
m.p.
Phosphorus oxychloride (20 mL) was added dropwise to com-
pounds 4a, 5, 4c, 4d, 4e, 4f or 6 (10 mmol) with stirring at r.t for 1 h
and then, stirred for 3 h at 50 οC. The reaction mixtures were left to
cool to room temperature and then were added to ice bath with
stirring. The yellow solids obtained were filtered off, washed with
petroleum ether and crystalized from dichloromethane as yellow
214e217 ꢂC. 1H NMR (300 MHz, DMSO) 7.17e7.20(d, J ¼ 8.70 Hz,
2H), 7.33e7.37(m, 1H), 7.46e7.49(m, 2H), 7.69e7.71(m, 2H),
7.88e7.91(d, J ¼ 8.70 Hz, 2H), 10.03(s, 1H, CHO), 12.33(br., 1H, D2O
exchangeable). MS, m/z (%): 333(Mþ, 24%). Anal. Calcd for
C18H11N3O2S (FW: 333): C, 64.85; H, 3.33; N, 12.60; S, 9.62. Found:
C, 64.69; H, 3.50; N, 12.44; S, 9.75.

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
703
4.2.3.3. Ethyl 2-(5-cyano-4-(4-fluorophenyl)-6-(4-formylphenoxy)
pyrimidin-2-ylthio) acetate (8b). Yield (64%), m.p. 153e155 ꢂC. 1H
21%). Anal. Calcd for C25H14ClN5O3S (FW: 499): C, 60.06; H, 2.82; Cl,
7.09; N, 14.01; S, 6.41. Found: C, 60.31; H, 2.75; Cl, 6.85; N, 14.31; S,
6.22.
NMR (300 MHz, DMSO)
7.47e7.53(m, 2H), 7.57e7.60(d, J ¼ 8.70 Hz, 2H), 8.05e8.11(m,
4H),10.07(s, 1H, CHO) . 13C NMR (75 MHz, DMSO)
13.81, 33.32,
d 1.02e1.07 (t, 3H), 3.92e3.99(m, 4H),
d
4.2.5. Synthesis of compounds 10a-c
61.14, 114.22,115.87, 116.17, 122.46, 130.96, 131.32, 131.53, 131.64,
134.31, 155.66, 167.63, 167.90, 168.77, 192.00. MS, m/z (%): 437(Mþ,
24%). Anal. Calcd for C22H16FN3O4S (FW: 437): C, 60.40; H, 3.69; F,
4.34; N, 9.61; S, 7.33. Found: C, 60.29; H, 3.65; N, 9.77; S, 7.42.
4.2.5.1. Method C. Compounds 9a-c (10 mmol) were dissolved in
DMF (20 mL), and 1,1-carbonyldiimidazole (0.16 g, 10 mmol) was
added. The mixture was stirred for 30 min at room temperature and
then cooled to 0 ꢂC. Ammonium carbonate (0.19 g, 20 mmol) was
added, and the reaction mixture was stirred for 24 h at room tem-
perature. The solvent was evaporated under reduced pressure, and
the residue quenched with water (30 mL) and extracted with ethyl
acetate washed with water (3 ꢃ 50 mL), and dried (MgSO4). The
organic extracts were then collected and concentrated. The residue
was purified by column chromatography (0e100% EtOAc/n-
hexane) and obtained as yellow powder.
4.2.3.4. 6-(4-Chlorophenyl)-4-(4-formylphenoxy)-2-thioxo-1,2-
dihydropyrimidine-5-carbonitrile
(8c). Yield
(64%),
m.p.
244e247 ꢂC. 1H NMR (300 MHz, DMSO) 7.33e7.37(d, J ¼ 8.52 Hz,
2H), 7.44e7.47(d, J ¼ 8.70 Hz, 2H), 7.89e7.91(d, J ¼ 8.52 Hz, 2H),
8.02e8.05(d, J ¼ 8.70 Hz, 2H), 10.07(s, 1H, CHO), 11.25(br., 1H, D2O
exchangeable). MS, m/z (%): 367(Mþ, 28%). Anal. Calcd for
C
18H10ClN3O2S (FW: 367): C, 58.78; H, 2.74; Cl, 9.64; N, 11.42; S,
8.72. Found: C, 58.55; H, 2.51; Cl, 9.44; N, 11.61; S, 8.97.
4.2.5.2. 2-(4-(5-cyano-6-phenyl-2-thioxo-1,2-dihydropyrimidin-4-
yloxy)phenyl)-1H-benzo[d]imidazole-5-carboxamide
(10a).
4.2.4. Synthesis of compounds 9-a-c
Yield (64%), m.p. 263e265 ꢂC. 1H NMR (300 MHz, DMSO)
4.2.4.1. Method B. Compounds 8a-c (10 mmol) were treated with a
solution of 3,4-diaminobenzoic acid (1.53g, 10 mmol) in DMF
(30 mL), followed by Na2S2O5 (2.85g, 15 mmol). The mixture was
heated to 90 ꢂC for 24 h. The reaction mixture was cooled to room
temperature then was quenched with water and the solid obtained
was filtered and purified by column chromatography (0e100%
EtOAc/n-hexane) to obtain yellow powders.
d
7.30e7.57(m, 5H), 7.66e7.69(m, 2H), 7.74e7.77(d, J ¼ 8.25 Hz, 1H),
7.96e7.99(d, J ¼ 8.25 Hz, 1H), 8.15 (s, 1H), 8.25e8.28 (d, J ¼ 8.70 Hz,
2H), 8.54 (br., 2H, D2O exchangeable), 11.42, (br., 1H, D2O
exchangeable). 13.01(br., 1H, D2O exchangeable). MS, m/z (%):465
(Mþ, 21%). Anal. Calcd for C25H16N6O2S (FW: 464): C, 64.64; H, 3.47;
N, 18.09; S, 6.90. Found: C, 64.68; H, 3.55; N, 17.94; S, 6.71.
4.2.5.3. 2-(4-(5-cyano-2-(2-ethoxy-2-oxoethylthio)-6-(4-
fluorophenyl)pyrimidin-4-yloxy) phenyl)-1H-benzo[d]imidazole-5-
carboxamide (10b). Yield (57%). m.p. 281e283 ꢂC. 1H NMR
4.2.4.2. 2-(4-(5-cyano-6-phenyl-2-thioxo-1,2-dihydropyrimidin-4-
yloxy)phenyl)-1H-benzo[d]imidazole-5-carboxylic
acid
(9a).
Yield (64%), m.p. 262e265 ꢂC. 1H NMR (300 MHz, DMSO)
(300 MHz, DMSO) d 0.98e1.22 (t, 3H, CH3), 3.99e4.21(m, 4H, 2CH2),
d
7.27e7.35(m, 3H), 7.50e7.53(d, J ¼ 8.70 Hz, 2H), 7.65e7.68(m, 2H),
7.38e7.47(m, 2H), 7.50e7.53(d, J ¼ 8.70 Hz, 2H), 7.69e7.72(d,
J ¼ 8.25 Hz, 1H), 7.88e7.91(d, J ¼ 7.2 Hz, 1H), 8.01e8.12(m, 2H), 8.19
(s, 1H), 8.32e8.35 (d, J ¼ 8.70 Hz, 2H), 8.69(br., 2H, D2O exchange-
able), 11.31(br., 1H, NH, D2O exchangeable). MS, m/z (%): 568(Mþ,
22%). Anal. Calcd for C29H21FN6O4S (FW: 568): C, 61.26; H, 3.72; F,
3.34; N, 14.78; S, 5.64. Found: C, 61.12; H, 3.57; N, 14.88; S, 5.80.
7.70e7.73(d, J ¼ 8.25 Hz, 1H), 7.88e7.91(d, J ¼ 7.2 Hz, 1H), 8.23 (s,
1H), 8.32e8.35 (d, J ¼ 8.70 Hz, 2H), 11.56 (br., 1H, D2O exchange-
able), 12.69 (br., 1H, D2O exchangeable), 13.07 (br., 1H, D2O
exchangeable). MS, m/z (%):465 (Mþ, 37%). Anal. Calcd for
C
25H15N5O3S (FW: 465): C, 64.51; H, 3.25; N, 15.05; S, 6.89. Found:
C, 64.32; H, 3.51; N, 15.21; S, 6.65.
4.2.5.4. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-thioxo-1,2-
dihydropyrimidin-4-yloxy)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (10c). Yield (61%). m.p. 278e280 ꢂC. 1H NMR
(300 MHz, DMSO) 7.21e7.24(d, J ¼ 8.70 Hz, 2H), 7.43e7.46 (d,
J ¼ 8.52 Hz, 2H), 7.63e7.66 (d, J ¼ 8.52 Hz, 2H), 7.78e7.81 (d,
J ¼ 8.20 Hz, 1H), 8.08e8.11 (d, J ¼ 8.20 Hz, 1H), 8.26 (s, 1H),
8.32e8.35 (d, J ¼ 8.70 Hz, 2H), 8.64 (br., 2H, D2O exchangeable),
11.68 (br., 1H, D2O exchangeable), 13.09 (br., 1H, D2O exchangeable).
MS, m/z (%):498 (Mþ, 27%), 500(Mþ, 8%). Anal. Calcd for
4.2.4.3. 2-(4-(5-cyano-2-(2-ethoxy-2-oxoethylthio)-6-(4-
fluorophenyl)pyrimidin-4-yloxy) phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (9b). Yield (67%), m.p. 271e273 ꢂC. 1H NMR
(300 MHz, DMSO) d 0.97e1.08 (t, 3H, CH3), 3.92e4.08(m, 4H, 2CH2),
7.37e7.48(m, 2H), 7.50e7.53(d, J ¼ 8.70 Hz, 2H), 7.70e7.73(d,
J ¼ 8.25 Hz, 1H), 7.88e7.91(d, J ¼ 8.25 Hz, 1H), 8.05e8.08(m, 2H),
8.23 (s, 1H), 8.32e8.35 (d, J ¼ 8.70 Hz, 2H), 10.56 (br., 1H, D2O
exchangeable), 11.57(br., 1H, D2O exchangeable). 13C NMR (75 MHz,
DMSO)
d
13.86, 33.35, 61.28, 110.29, 114.87, 115.87, 115.22, 116.29,
C25H15ClN6O2S (FW: 498): C, 60.18; H, 3.03; Cl, 7.11; N,16.84; S, 6.43.
120.87, 122.33, 128.11, 129.32, 130.96, 131.32, 131.52, 131.64, 134.31,
147.52, 148.22, 149.22, 150.22, 152.47, 156.66, 167.03, 167.89, 168.44.
MS, m/z (%): 569(Mþ, 38%). Anal. Calcd for C29H20FN5O5S (FW:
569): C, 61.15; H, 3.54; F, 3.34; N, 12.30; S, 5.63. Found: C, 61.01; H,
3.39; N, 12.56; S, 5.77.
Found: C, 60.02; H, 3.22; Cl, 7.04; N, 16.77; S, 6.56.
4.2.6. Synthesis of compounds 11a-f
4 . 2 . 6 .1. 4 - ( 5 - C ya n o - 6 - ( 4 - n i t ro p h e nyl ) - 2 - t h i o x o - 1, 2 -
dihydropyrimidin-4-ylamino)benzoic acid (11a). Prepared accord-
ing to method (A) by reaction of 4-chloro-6-(4-nitrophenyl)-2-
4.2.4.4. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-thioxo-1,2-
dihydropyrimidin-4-yloxy)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (9c). Yield (64%), m.p. 241e244 ꢂC. 1H NMR
(300 MHz, DMSO) 7.27e7.30(d, J ¼ 8.52 Hz, 2H), 7.40e7.43(d,
J ¼ 8.70 Hz, 2H), 7.55e7.58(d, J ¼ 8.52 Hz, 2H), 7.70e7.73(d,
J ¼ 8.25 Hz, 1H), 7.88e7.91(d, J ¼ 8.25 Hz, 1H), 8.06 (s, 1H),
8.12e8.13(d, J ¼ 8.70 Hz, 2H), 11.33 (br., 1H, D2O exchangeable),
13.07(br., 1H, D2O exchangeable). 13C NMR (75 MHz, DMSO)
thioxo-1,2-dihydropyrimidine-5-carbonitrile
(7d)
with
4-
aminobenzoic acid. Yield (57%). m.p. 259e262 ꢂC. 1H NMR
(300 MHz, DMSO) 4.52 (br., 1H, D2O exchangeable), 7.24e7.27(d,
J ¼ 8.70 Hz, 2H), 7.85e7.89(d, J ¼ 8.52 Hz, 2H), 8.09e8.11(d,
J ¼ 8.52 Hz, 2H), 8.32e8.35(d, J ¼ 8.70 Hz, 2H), 12.74 (br., 1H, D2O
exchangeable) 13.25 (br., 1H, D2O exchangeable). MS, m/z (%):
393(Mþ, 28%). Anal. Calcd for C18H11N5O4S (FW: 393): C, 54.96; H,
2.82; N, 17.80; S, 8.15. Found: C, 54.78; H, 2.97; N, 17.69; S, 8.27.
d
113.77, 115.44, 115.22, 116.98, 120.87, 124.21, 125.61, 129.11, 130.66,
131.96, 131.52, 131.64, 138.31, 146.31, 148.22, 149.22, 150.22, 153.00,
4.2.6.2. 4-(5-Cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
156.66, 164.03, 166.40, 167.89, 168.44, 179.44. MS, m/z (%):499 (Mþ,
dihydropyrimidin-4-ylamino)benzoic acid (11b). Prepared according

704
S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
to method (A) by reaction of 4-chloro-6-(4-methoxyphenyl)-2-
thioxo-1,2-dihydropyrimidine-5-carbonitrile(7e) with 4-
EtOAc/n-hexane).
aminobenzoic acid. Yield (42%). m.p. 271e274 ꢂC. 1H NMR
(300 MHz, DMSO) 3.83 (s, 3H), 5.22 (br., 1H, D2O exchangeable),
7.06e7.09 (d, J ¼ 8.64 Hz, 2H), 7.37e7.40(d, J ¼ 8.52 Hz, 2H),
7.59e7.61(d, J ¼ 8.52 Hz, 2H), 7.82e7.85(d, J ¼ 8.64 Hz, 2H), 12.44
(br., 1H, D2O exchangeable) 12.94 (br., 1H, D2O exchangeable). MS,
m/z (%): 378(Mþ, 28%). Anal. Calcd for C19H14N4O3S (FW: 378): C,
60.31; H, 3.73; N, 14.81; S, 8.47. Found: C, 60.48; H, 3.66; N, 14.94; S,
8.61.
4.2.7.2. 2-(4-(5-cyano-6-(4-nitrophenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylamino)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (12). Prepared by reaction of compound 11a with
3,4-diaminobenzoic acid according to method D. Yield (28%), m.p.
305e307 ꢂC. 1H NMR (300 MHz, DMSO)
d 5.65 (s., 1H, D2O
exchangeable), 7.17e7.19 (d, J ¼ 8.36 Hz, 2H), 7.64e7.66 (d,
J ¼ 8.12 Hz, 1H), 7.72e7.74(d, J ¼ 8.36 Hz, 2H), 7.82e7.84 (d,
J ¼ 8.12 Hz, 1H), 7.92e7.94 (d, J ¼ 8.36 Hz, 2H), 8.20 (s, 1H),
8.38e8.40(d, J ¼ 8.36 Hz, 2H), 12.04 (s., 1H, D2O exchangeable),
12.15 (s., 1H, D2O exchangeable), 12.75(br., 1H, D2O exchangeable).
MS, m/z (%):509 (Mþ, 45%). Anal. Calcd forC25H15N7O4S (FW: 509):
C, 58.93; H, 2.97; N, 19.24; S, 6.29. Found: C, 58.71; H, 2.68; N, 19.55;
S, 6.42.
4.2.6.3. 4-(5-Cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)benzoic acid (11c). Prepared according to
method (A) by reaction of 4-chloro-6-(4-methoxyphenyl)-2-
thioxo-1,2-dihydropyrimidine-5-carbonitrile
mercaptobenzoic acid. Yield (42%). m.p. 283e285 ꢂC. 1H NMR
(300 MHz, DMSO)
(7e)
with
4-
d
3.82 (s, 3H), 7.16e7.19 (d, J ¼ 8.64 Hz, 2H),
4.2.7.3. 2-(4-(5-cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylamino)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid(13). Prepared by reaction of compound 11b with
3,4-diaminobenzoic acid according to method D. Yield (25%), m.p.
7.57e7.60 (d, J ¼ 8.52 Hz, 2H), 7.68e7.71 (d, J ¼ 8.52 Hz, 2H),
8.02e8.05 (d, J ¼ 8.64 Hz, 2H), 12.74 (br., 1H, D2O exchangeable)
13.52 (br., 1H, D2O exchangeable). MS, m/z (%): 395(Mþ, 42%). Anal.
Calcd for C19H13N3O3S2 (FW: 395): C, 57.71; H, 3.31; N, 10.63; S,
16.22. Found: C, 57.65; H, 3.22; N, 10.71; S, 16.09.
287e289 ꢂC. 1H NMR (300 MHz, DMSO)
d3.81 (s, 3H), 5.24 (br., 1H,
D2O exchangeable), 7.05e7.08(d, J ¼ 8.35 Hz, 2H), 7.22e7.25(d,
J ¼ 8.25 Hz, 2H), 7.66e7.69(d, J ¼ 8.35 Hz, 2H), 7.88e7.91(m, 3H),
8.15e8.18(d, J ¼ 8.35 Hz, 1H), 8.23 (s, 1H), 11.43 (br., 1H, D2O
exchangeable), 12.55 (br., 1H, D2O exchangeable), 13.33(br., 1H, D2O
4.2.6.4. 4-(5-Cyano-2-thioxo-6-p-tolyl-1,2-dihydropyrimidin-4-
ylthio)benzoic acid (11d). Prepared according to method (A) by
reaction of 4-chloro-6-p-tolyl-2-thioxo-1,2-dihydropyrimidine-5-
carbonitrile (7f) with 4-mercaptobenzoic acid. Yield (51%), m.p.
exchangeable). 13C NMR (75 MHz, DMSO)
d 58.32, 115.17, 116.12,
116.54, 119.84, 120.87, 124.21, 125.61, 129.33, 129.98, 131.96, 131.46,
133.20, 139.01, 146.31, 147.27, 148.31, 150.75, 152.52, 159.24, 163.99,
165.24, 166.87, 181.07. MS, m/z (%):494 (Mþ, 45%). Anal. Calcd for-
252e254 ꢂC. 1H NMR (300 MHz, DMSO)
d 2.31 (s, CH3), 7.16e7.19 (d,
J ¼ 8.64 Hz, 2H), 7.27e7.30(d, J ¼ 8.33 Hz, 2H), 7.66e7.69 (d,
J ¼ 8.52 Hz, 2H), 8.08e8.11 (d, J ¼ 8.52 Hz, 2H), 12.79 (br., 1H, D2O
exchangeable) 13.10 (br., 1H, D2O exchangeable). MS, m/z (%):
379(Mþ, 40%). Anal. Calcd for C19H13N3O2S2 (FW: 379): C, 60.14; H,
3.45; N, 11.07; S, 16.90. Found: C, 60.02; H, 3.22; N, 11.31; S, 16.74.
C26H18N6O3S (FW: 494): C, 63.15; H, 3.67; N,16.99; S, 6.48. Found: C,
63.25; H, 3.70; N, 16.81; S, 6.66.
4.2.7.4. 2-(4-(5-cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (16). Prepared by reaction of compound 11c with
3,4-diaminobenzoic acid according to method D. Yield (27%), m.p.
4.2.6.5. 4-(6-(4-Chlorophenyl)-5-cyano-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)benzoic acid (11e). Prepared according to
method (A) by reaction of 4-chloro-6-(4-Chlorophenyl)-2-thioxo-
1,2-dihydropyrimidine-5-carbonitrile (7c) with 4-mercaptobenzoic
acid. Yield (62%), m.p. 274e277 οC. 1H NMR (300 MHz, DMSO)
301e303 ꢂC. 1H NMR (300 MHz, DMSO)
d3.81 (s, 3H), 7.01e7.04(d,
J ¼ 8.25 Hz, 2H), 7.46e7.49(m, 4H), 7.73e7.77(d, J ¼ 8.25 Hz, 2H),
7.85e7.88(d, J ¼ 8.35 Hz, 1H), 8.09e8.12(d, J ¼ 8.35 Hz, 1H), 8.23 (s,
1H), 11.61 (br., 1H, D2O exchangeable), 12.89 (br., 1H, D2O
exchangeable), 13.03(br., 1H, D2O exchangeable). MS, m/z (%): 511
(Mþ, 31%). Anal. Calcd forC26H17N5O3S2 (FW: 511): C, 61.04; H, 3.35;
N, 13.69; S, 12.54. Found: C, 61.22; H, 3.41; N, 13.48 S, 12.33.
d
7.35e7.38 (d, J ¼ 8.50 Hz, 2H), 7.48e7.51(d, J ¼ 8.50 Hz, 2H),
7.65e7.68(d, J ¼ 8.48 Hz, 2H), 8.08e8.11 (d, J ¼ 8.48 Hz, 2H), 12.55
(br., 1H, D2O exchangeable) 13.37 (br., 1H, D2O exchangeable). MS,
m/z (%):399 (Mþ, 28%), 401(Mþ, 9%) 28%). Anal. Calcd for
C
18H10ClN3O2S2 (FW: 398.99): C, 54.07; H, 2.52; Cl, 8.87; N, 10.51; S,
16.04. Found: C, 53.89; H, 2.34; Cl, 8.66; N, 10.64; S, 16.12.
4.2.7.5. 2-(4-(5-cyano-2-thioxo-6-p-tolyl-1,2-dihydropyrimidin-4-
ylthio)phenyl)-1H-benzo[d]imidazole-5-carboxylic
acid
(17).
4.2.6.6. 4-(6-(4-chlorophenyl)-5-cyano-2-(2-ethoxy-2-oxoethylthio)
pyrimidin-4-ylthio)benzoic acid (11f). Prepared according to
method (A) by reaction of 4-chloro-6-(4-Chlorophenyl)-2-(2-
ethoxy-2-oxoethylthio)-1,2-dihydropyrimidine-5-carbonitrile (7g)
with 4-mercaptobenzoic acid. Yield (54%), m.p. 264e267 ꢂC. 1H
Prepared by reaction of compound 11d with 3,4-diaminobenzoic
acid according to method D. Yield (27%), m.p. 265e267 ꢂC. 1H
NMR (300 MHz, DMSO)
d
2.33 (s, 3H), 7.12e7.15(d, J ¼ 8.26 Hz, 2H),
7.46e7.49(d, J ¼ 8.25 Hz, 2H), 7.68e7.71(d, J ¼ 8.36 Hz, 2H),
7.80e7.83(d, J ¼ 8.36 Hz, 2H), 7.91e7.94(d, J ¼ 8.35 Hz, 1H),
8.05e8.08(d, J ¼ 8.35 Hz, 1H), 8.21 (s, 1H), 11.61 (br., 1H, D2O
exchangeable), 12.78 (br.,1H, D2O exchangeable), 13.22 (br.,1H, D2O
exchangeable). MS, m/z (%): 495 (Mþ, 45%). Anal. Calcd for-
NMR (300 MHz, DMSO)
d 1.08e1.22 (t, 3H, CH3), 4.03e4.21(m, 4H,
2CH2), 2H), 7.55e7.759(m, 4H), 7.82e7.85(d, J ¼ 8.24 Hz, 2H),
8.01e8.04(d, J ¼ 8.52 Hz, 2H),12.50 (br., 1H, D2O exchangeable). MS,
m/z (%): 485 (Mþ, 31%), 487 (Mþ, 11%). Anal. Calcd for
C26H17N5O2S2 (FW: 495): C, 63.01; H, 3.46; N, 14.13; S, 12.94. Found:
C
22H16ClN3O4S2 (FW: 485): C, 54.37; H, 3.32; Cl, 7.30; N, 8.65; S,
C, 63.22; H, 3.59; N, 14.21; S, 13.09.
13.20. Found: C, 54.54; H, 3.49; Cl, 7.14; N, 8.57; S, 13.41.
4.2.7.6. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (18). Prepared by reaction of compound 11e with
3,4-diaminobenzoic acid according to method D. Yield (25%), m.p.
4.2.7. Synthesis of compounds 12, 13 and 16e19
4.2.7.1. Method D. Compounds 11a-f (10 mmol) were treated with
a solution of 3,4-diaminobenzoic acid (1.53g, 10 mmol) in poly-
phosphoric acid (20 mL). The mixture was heated to 150e180 ꢂC for
4 h. The reaction mixture was cooled to room temperature then was
quenched with water and neutralized with NH4OH the solid was
filtered and was purified by column chromatography (0e100%
322e324 ꢂC. 1H NMR (400 MHz, DMSO)
d
7.27e7.29(d, J ¼ 8.36 Hz,
2H), 7.55e7.57 (d, J ¼ 8.32 Hz, 2H), 7.63e7.65(d, J ¼ 8.36 Hz, 1H), 7.
74-7. 76 (d, J ¼ 8. 36 Hz, 2H), 7.91e7.93(d, J ¼ 8.36 Hz, 1H),
8.01e8.03(d, J ¼ 8.36 Hz, 1H), 8.22 (s, 1H), 12.04 (s., 1H, D2O

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
705
exchangeable), 12.16 (s., 1H, D2O exchangeable), 12.75 (br., 1H, D2O
exchangeable). 13C NMR (75 MHz, DMSO)
114.69, 114.99, 116.30,
4.2.8.4. 2-(4-(5-cyano-2-thioxo-6-p-tolyl-1,2-dihydropyrimidin-4-
ylthio)phenyl)-1H-benzo[d]imidazole-5-carboxamide (21).
d
117.50, 123.38, 124.22, 124.59, 125.16, 125.46, 129.26, 133.30, 135.43,
137.70, 140.45, 144.21, 154.95, 155.28, 161.73, 166.58, 167.77, 180.05.
MS, m/z (%): 515 (Mþ, 39%), 517 (Mþ, 14%). Anal. Calcd for
Prepared by reaction of compound 17 with ammonium carbonate
according to method C. Yield (27%), m.p. 299e302 ꢂC. 1H NMR
(300 MHz, DMSO)
d
2.33 (s, 3H), 7.12e7.13(d, J ¼ 8.25 Hz, 2H),
C
25H14ClN5O2S2 (FW: 515): C, 58.19; H, 2.73; Cl, 6.87; N, 13.57; S,
7.24e7.27(d, J ¼ 8.25 Hz, 2H), 7.46e7.49(d, J ¼ 8.70 Hz, 2H),
7.68e7.71(d, J ¼ 8.70 Hz, 2H), 7.91e7.94(d, J ¼ 8.25 Hz, 1H),
8.05e8.08(d, J ¼ 8.25 Hz, 1H), 8.21 (s, 1H), 8.61 (br., 2H, D2O
exchangeable), 12.78 (br.,1H, D2O exchangeable), 13.22 (br.,1H, D2O
12.43. Found: C, 58.31; H, 2.97; Cl, 6.56; N, 13.32; S, 12.54.
4.2.7.7. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-(2-ethoxy-2-
oxoethylthio) pyrimidin-4-ylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxylic acid (19). Prepared by reaction of compound 11f with
3,4-diaminobenzoic acid according to method D.
exchangeable). 13C NMR (75 MHz, DMSO)
d 21.59, 115.45,
116.3117.21, 122.34, 124.98, 127.49, 128.71, 128.99, 129.44, 130.10,
131.11, 134.58, 137.66, 140.21, 145.34, 152.13, 163.33, 164.03, 168.00,
180.27. MS, m/z (%): 494 (Mþ, 45%). Anal. Calcd forC26H18N6OS2
(FW: 494): C, 63.14; H, 3.67; N, 16.99; S, 12.97. Found: C, 63.14; H,
3.67; N, 16.99; S, 12.97.
1H NMR (300 MHz, DMSO)
d 1.18e1.29 (t, 3H, CH3), 4.02e4.18(m,
4H, 2CH2), 7.48e7.51(d, J ¼ 8.25 Hz, 2H), 7.62e7.65 (d, J ¼ 8.35 Hz,
2H), 7.72e7.75(d, J ¼ 8.35 Hz, 2H), 7.83e7.86 (d, J ¼ 8.25 Hz, 1H),
7.96e7.99 (d, J ¼ 8.35 Hz, 2H), 8.15e8.18 (d, J ¼ 8.22 Hz, 1H), 8.25 (s,
1H), 11.56 (br., 1H, D2O exchangeable), 12.75 (br., 1H, D2O
exchangeable). MS, m/z (%): 601(Mþ, 25%), 603(Mþ, 8%). Anal.
Calcd forC29H20ClN5O4S2 (FW: 601): C, 57.85; H, 3.35; Cl, 5.89; N,
11.63; S, 10.65. Found: C, 57.67; H, 3.29; Cl, 5.77; N, 11.42; S, 10.72.
4.2.8.5. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (22). Prepared by reaction of compound 18 with
ammonium carbonate according to method C. Yield (25%), m.p.
330e332 ꢂC. 1H NMR (300 MHz, DMSO)
d
7.22e7.25(d, J ¼ 8.70 Hz,
2H), 7.33e7.51(m, 4H), 7.65e7.68(d, J ¼ 8.70 Hz, 2H), 7.91e7.94(d,
J ¼ 8.25 Hz, 1H), 8.08e8.11(d, J ¼ 8.25 Hz, 1H), 8.21 (s, 1H), 8.65 (br.,
2H, D2O exchangeable),12.49 (br.,1H, D2O exchangeable),13.08 (br.,
4.2.8. Synthesis of compounds 14, 15 and 20e23
4.2.8.1. 2-(4-(5-cyano-6-(4-nitrophenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylamino)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (14). Prepared by the reaction of compound 12 with
ammonium carbonate according to method C. Yield (68%), m.p.
1H, D2O exchangeable). 13C NMR (75 MHz, DMSO)
d 115.22, 115.55,
116.54, 120.74, 122.20, 127.41, 127.87, 128.47, 129.82, 130.10, 131.87,
135.32, 135.55, 139.24, 145.90, 152.74, 163.04, 163.49, 168.11, 180.55.
MS, m/z (%): 514 (Mþ, 24% for Cl35.5), 516 (Mþ, 10% for Cl37.5). Anal.
Calcd for C25H15ClN6OS2 (FW: 514): C, 58.30; H, 2.94; Cl, 6.88; N,
16.32; S, 12.45. Found: C, 58.48; H, 2.77; Cl, 6.64; N, 16.45; S, 12.61.
291e293 ꢂC. 1H NMR (400 MHz, DMSO)
d 5.53e5.58 (br., 1H, D2O
exchangeable), 7.24e7.26(d, J ¼ 8.36 Hz, 2H), 7.76e7.78(d,
J ¼ 8.36 Hz, 1H), 7.94e7.96(d, J ¼ 8. 44 Hz, 2H), 7.97e7.99(m, 3H),
8.14 (s, 1H), 8.21e8.23(d, J ¼ 8.44 Hz, 2H), 8.51e8.55 (br., 2H, D2O
exchangeable), 12.17 (br., 1H, D2O exchangeable), 12.37 (br., 1H, D2O
exchangeable). MS, m/z (%):508 (Mþ, 24%). Anal. Calcd for-
4.2.8.6. 2-(4-(6-(4-chlorophenyl)-5-cyano-2-(2-ethoxy-2-
oxoethylthio) pyrimidin-4-ylthio) phenyl)-1H-benzo[d]imidazole-5-
carboxamide (23). Prepared by the reaction of compound 19 with
ammonium carbonate according to method C. Yield (28%), m.p.
C
25H16N8O3S (FW: 508): C, 59.05; H, 3.17; N, 22.04; S, 6.31. Found: C,
59.31; H, 3.25; N, 22.13; S, 6.19.
317e320 ꢂC. 1H NMR (300 MHz, DMSO)
d 1.21e1.29 (t, 3H, CH3),
4.2.8.2. 2-(4-(5-cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylamino)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (15). Prepared by reaction of compound 13 with
ammonium carbonate according to method C. Yield (67%), m.p.
4.04e4.18(m, 4H, 2CH2), 7.40e7.43(d, J ¼ 8.25 Hz, 2H), 7.52e7.55(d,
J ¼ 8.70 Hz, 2H), 7.67e7.70(d, J ¼ 8.25 Hz, 2H), 7.87e7.90(d,
J ¼ 8.20 Hz, 1H), 7.97e8.00(d, J ¼ 8.70 Hz, 2H), 8.12e8.15(d,
J ¼ 8.20 Hz, 1H), 8.23 (s, 1H), 8.55 (br., 2H, D2O exchangeable),
318e320 ꢂC. 1H NMR (400 MHz, DMSO)
d3.85 (s, 3H), 5.09 (br., 1H,
12.71(br., 1H, D2O exchangeable). 13C NMR (75 MHz, DMSO)
d14.12,
D2O exchangeable), 6.94e6.96 (d, J ¼ 8.24 Hz, 2H), 7.26e7.28(d,
J ¼ 8.36 Hz, 2H), 7.65e7.67(d, J ¼ 8.24 Hz, 2H), 7.72e7.74(d,
J ¼ 8.32 Hz, 1H), 7.88e7.90(d, J ¼ 8.36 Hz, 2H), 7.95e7.97 (d,
J ¼ 8.32 Hz, 1H), 8.13 (s, 1H), 8.61e8.65 (br., 2H, D2O exchangeable),
12.17 (br., 1H, D2O exchangeable), 12.37 (br., 1H, D2O exchangeable).
MS, m/z (%):493 (Mþ, 34%). Anal. Calcd forC26H19N7O2S (FW: 493):
C, 63.27; H, 3.88; N,19.87; S, 6.50. Found: C, 63.50; H, 3.77; N, 19.67;
S, 6.39.
35.46, 60.54, 100.44, 115.20, 116.87, 117.31, 122.34, 127.44, 127.65,
127.72, 128.78, 129.83, 129.99, 131.20, 131.68, 133.49, 134.09, 139.54,
145.63, 152.44, 164.47, 168.21, 169.87, 176.55, 192.21. MS, m/z (%):
600(Mþ, 39% for Cl35.5), 602(Mþ, 16% for Cl37.5). Anal. Calcd. for-
C29H21ClN6O3S2 (FW: 600): C, 57.95; H, 3.52; Cl, 5.90; N, 13.98; S,
10.67. Found: C, 57.79 H, 3.33; Cl, 5.87; N, 14.08; S, 10.81.
4.3. In vitro checkpoint Kinase Assay
4.2.8.3. 2-(4-(5-cyano-6-(4-methoxyphenyl)-2-thioxo-1,2-
dihydropyrimidin-4-ylthio)phenyl)-1H-benzo[d]imidazole-5-
carboxamide (20). Prepared by the reaction of compound 16 with
ammonium carbonate according to method C. Yield (60%), m.p,
The CycLex® Checkpoint Kinase Assay/Inhibitor Screening Kit
was used to measure the activities of checkpoint kinases by using a
phospho-Cdc25C (Ser216) monoclonal antibody to provide a spe-
cific and sensitive method. Chk2 positive control (CY-E1162-2) was
used. Assay conditions were based on published protocols with
minor modifications. The optical density (O.D.) of each well was
measured using a spectrophotometric plate reader at dual wave-
lengths of 450/492 nm using ELISA microplate reader. Then, assays
were repeated by using mixture of optimum concentration of
positive control that exhibited maximum activity of checkpoint
kinase 2 (0.01 unit/well) and specific concentration of each com-
325e327 ꢂC. 1H NMR (300 MHz, DMSO)
d3.81 (s, 3H), 7.03e7.06 (d,
J ¼ 8.35 Hz, 2H), 7.43e7.46(d, J ¼ 8.70 Hz, 2H), 7.60e7.63(d,
J ¼ 8.35 Hz, 2H), 7.76e7.79(d, J ¼ 8.70 Hz, 2H), 7.93e7.96(d,
J ¼ 8.20 Hz, 1H), 8.04e8.07(d, J ¼ 8.20 Hz, 1H), 8.23 (s, 1H), 8.69 (br.,
2H, D2O exchangeable), 12.47 (br., 1H, D2O exchangeable), 13.21(br.,
1H, D2O exchangeable). 13C NMR (75 MHz, DMSO)
d 55.99. 115.03,
115.92, 116.44, 121.17, 122.47, 123.66, 127.42, 127.89, 129.41, 130.11,
132.42, 134.55, 139.65, 144.89, 152.67, 160.99. 162.69, 163.41, 168.09,
180.13. MS, m/z (%): 510 (Mþ, 16%). Anal. Calcd for C26H18N6O2S2
(FW: 510): C, 61.16; H, 3.55; N, 16.46; S, 12.56. Found: C, 61.01; H,
3.42; N, 16.33; S, 12.72.
pound
D ¼ 0.050
A ¼ 2.5
m
mol/l,
B ¼ 1.5
m
mol/L,
C ¼ 0.1
mmol/L,
m
mol/L and E ¼ 0.025
m
mol/L. The IC50 (concentration
that produces 50% of enzyme inhibition) by synthesized com-
pounds as Chk2 inhibitors has been calculated as the mean value of

706
S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
at four replicate experiments. %inhibition¼ (1-(OD of the com-
pound at specific conc. e OD of negative control)÷ (OD of pos.
control - OD of negative control)) X100, (All data are represented in
supplementary material).
of 5 independent experiments performed in triplicates; the sur-
viving fraction ¼ O.D. (treated cells)/O.D. (control cells).
The statistical significance of the results was analyzed using
one-way ANOVA followed by Tukey multiple comparison tests.
4.4. Western blot
4.5.2. Combined effect of different concentrations of the tested
compounds and IG50 of cisplatin or doxorubicin on MCF7
MCF-7 breast carcinoma including untreated cells (control
cells), treated cells with GI50 of compound 8, 9, GI50 of cisplatin
alone or GI50 of cisplatin in combination with 0.1 mM of compound
The combination regimens were designed using GI50 of doxo-
rubicin and cisplatin with different concentrations of Chk2 in-
hibitors on MCF-7 cells. Different concentrations of the studied
compounds were chosen to test their combination with doxoru-
bicin and cisplatin for further experiments. MCF7 was plated in 96
well plates at concentration 3 ꢃ 103 cells/well and were left to
attach for 24 h before incubation with drugs. MCF7 cells were
treated with either GI50 of doxorubicin or cisplatin alone and with
different concentrations of the Chk2 inhibitors (0.05, 0.1, 0.15 and
8, 9. Cells were grown to ~70% confluency and reagents were added
as indicated concentrations. Cells were lysed in lysis buffer con-
taining Tris-HCl (20 mM), NaCl (0.5 M), Triton X-100(0.25%), EDTA
(1 mM), EGTA, (1 mM), glycophosphate (10 mM), NaF (10 mM),
Na3VO4 (300 lM), benzamidine (1 mM), PMSF (2 lM) and DTT
(1 mM). The protein concentration was determined by the BCA
protein assay (Thermo Scientific, Rockford, IL, USA). Proteins were
separated on SDS-PAGE, transferred on to nitrocellulose membrane
(NC). NC sheets were cut into 0.5 cm strips [52]. Followed by
blocking in 5% dry skimmed milk in TBS-T for 2 h on rocker platform
and then, following washing by TBS-T. The strips were probed with
phosphorylated anti-Chk2 antibody (Sigma-Aldrich) in 1:200
concentrations. Anti-mouse IgM peroxidase (Sigma-Aldrich)
diluted at 1: 1000 in TBS was added to NC strips for 1 h on a rocker
platform, The o-phenylene diamine tablet (Sigma-Aldrich) dis-
solved in 10 mL methanol and completed to 50 mL with TBS was
added to NC stripes for 30 min [53].
0.2 mmol) and incubated for 48 h. At the end of incubation, cells
were fixed with 20% trichloroacetic acid (TCA) and stained with
0.4% SRB dye as previously described [46].
4.6. Propidium iodide staining
Cells were plated in 60-mm dishes at 2 ꢃ 105 cells/mL RPMI. On
the following day, the cells were treated with GI50 of cisplatin,
compound 8 or 9 alone or combination of GI50 of cisplatin with
0.1 mM of synthesized conjugates 8 or 9 for 48 h, fixed with 2%
paraformaldehyde solution, and incubated with propidium iodide
buffer (0.1% sodium citrate, 0.1% Triton X-100, and 0.1 mg/mL pro-
pidium iodide). Subsequently, nuclei of the cells were observed
with confocal microscope (Zeiss LSM 710), the objective lens is 20X,
Immunoreactivity was detected using the ECL detection system
(Amersham Biosciences).
the cells were counted in 5 points of dimension 1700 m
m2 magni-
4.5. Cytotoxicity assay
fication 200X.
4.5.1. Cell lines
Breast carcinoma, estrogen receptor positive (ERþ) cell line
(MCF-7) was used in this study. It was obtained frozen in liquid
nitrogen (ꢁ180 ꢂC) from the American Type Culture Collection
(ATTC, USA). The tumor cell lines were maintained by serial sub
culturing at the National Cancer Institute, Cairo, Egypt. They were
cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 mg/mL streptomycin, and
3 mM glutamine and incubated in humidified incubator at 37 ꢂC
and 5% CO2. The cells were sub-cultured every three days. Cisplatin
and doxorubicin were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). The cytotoxicity of the synthesized compounds in
addition to two reference compounds (positive control) cisplatin
and doxorubicin were tested on MCF7 cancer cell lines, using
sulphorhodamine-B (SRB) method [46]. In brief; cells were seeded
at a density of 3 ꢃ 103 cells/well in 96-well microliter plates. They
were left to attach for 24 h before incubation with studied com-
pounds or drugs. Then, cells were treated with different concen-
4.7. Cell cycle analysis
Briefly cell cycle phase modifications were examined by flow on
propidium iodideestained cells using Cycle TEST™ PLUS DNA Re-
agent (BECTON DICKINSON, San Jose, California, U.S.A.) that was
used following the manufacturer's protocol, including that for
staining with propidium iodide for cell-cycle analysis on MCF-
7 cells that treated with DMSO or with either GI50 of doxorubicin
alone, and with 0.1 mM or with GI50 of studied compounds as pre-
viously described [46]. Cells were analyzed by flow cytometry to
quantify the numbers of apoptotic cells and the distribution of cells
throughout the cell cycle.
In conclusion, our synthesized compounds demonstrated strong
antitumor activity alone and in combination with other genotoxic
drugs as cisplatin and doxorubicin via inhibition of Chk2, induction
of apoptosis and cell cycle arrest.
trations of the Chk2 inhibitors (0.05, 0.1, 0.15 and 0.2
mmol). For
Acknowledgements
each concentration, three wells were used and incubation was
continued for 48 h. A negative control well-containing solvent as
DMSO its concentration does not exceed (1% v/v) was included. At
the end of incubation, cells were fixed with 20% trichloroacetic acid
(TCA), stained with 0.4% SRB dye. The optical density (O.D.) of each
well was measured spectrophotometrically at 570 nm using ELISA
microplate reader (TECAN sunrise™, Germany). The mean survival
fraction at each drug concentration was calculated as follows: O.D.
of the treated cells/O.D. of the control cells. The GI50 (concentration
that produces 50% of cell growth inhibition) value of each drug was
calculated using sigmoidal dose response curve fitting models
(Graph Pad Prizm software, version 5). Each value is the mean SD
We gratefully acknowledge the financial support of Science and
Technology Development Fund, Egypt (STDF) administered
through Basic and Applied Research Grants (Grant number ID: STDF
3130) to Dr. Shadia A. Galal and we deeply thank Prof. Dr. Maha A.
El-Demellawy for valuable comments and advises along the proj-
ect. Authors also thank the staff members of “Embryo and genetic
resources conservation bank” in National Research Centre for using
Confocal microscope (Zeiss LSM 710) in apoptotic analysis that was
administered by STDF grant number (STDF-CB, ID: 2339) and STDF-
RSDTG project of grant number ID: 6901 to Prof. Dr. Omaima M.
Kandil (Email: omaima_mk@yahoo.com).

S.A. Galal et al. / European Journal of Medicinal Chemistry 146 (2018) 687e708
707
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Appendix A. Supplementary data
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L. Huang, S. Crawford, C.R. Pandit, L. Karlsson, J.G. Breitenbucher, Checkpoint
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