Anticancer activities, molecular docking and structure–activity relationship of novel synthesized 4H-chromene, and 5H-chromeno[2,3-d]pyrimidine candidates

Article abstract of DOI:10.1007/s00044-017-1961-3

In the present study, a series of 4H-chromene and 5H-chromeno[2,3-d]pyrimidine derivatives was synthesized and evaluated as potential cytotoxic agents. The cytotoxic activities of the target compounds were evaluated against four cancer cell lines MCF-7, H

Full text of DOI:10.1007/s00044-017-1961-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1961-3
ORIGINAL RESEARCH
Anticancer activities, molecular docking and structure–activity
relationship of novel synthesized 4H-chromene, and 5H-chromeno
[
2,3-d]pyrimidine candidates
1 ●
2 ●
3 ●
Ahmed H. Halawa Mahmoud M. Elaasser Ahmed M. El Kerdawy
1 ●
1 ●
1
Ahmed M. A. I. Abd El-Hady Hassein A. Emam Ahmed M. El-Agrody
Received: 9 January 2017 / Accepted: 14 June 2017
©
Springer Science+Business Media, LLC 2017
●
Abstract In the present study, a series of 4H-chromene and
H-chromeno[2,3-d]pyrimidine derivatives was synthesized
Keywords 4H-Chromene 5H-Chromeno[2,3-d]
●
●
●
●
5
pyrimidine Antitumor Lipophilicity SAR Molecular
docking
and evaluated as potential cytotoxic agents. The cytotoxic
activities of the target compounds were evaluated against
four cancer cell lines MCF-7, HCT-116, HepG-2, and A549
in comparison with vinblastine and colchicine as reference
drugs. We explored the structure–activity relationship of
4
H-chromenes with modification at the 2-,4- or 7-position,
Introduction
and fused pyrimidine ring at 2,3-position. Most of the
screened compounds showed marginal antitumor activity
against the different cell lines in comparison to the standard
drugs. The structure–activity relationship study revealed
that the antitumor activity of the synthesized compounds
was significantly affected by the lipophilicity of the sub-
stituent at the 2-,4- or 7-position for the 4H-chromenes, and
Chromene (Benzopyran) is a heterocyclic ring system in
which a benzene ring and a pyran ring are fused together.
Moreover, the function of substituted chromenes plays a
vital role in the synthetic approaches of promising com-
pounds in the field of medicinal chemistry including anti-
microbial (Vala et al. 2016; Bingi et al. 2015; Killander and
Sterner 2014; Chetan et al. 2012; Kathrotiya and Patel
2012), anti-inflammatory (Thomas and Zachariah 2013),
anti-proliferative (El-Agrody et al. 2016; Magedov et al.
2007), antioxidant (Singh et al. 2010; Vukovic et al. 2010),
herbicidal, analgesic and anticonvulsant (Bhat et al. 2008),
antitubercular (Nimesh et al. 2011), anticoagulant, estro-
genic antispasmolytic, estrogenic (Nareshkumar et al.
5
5
,8-position or fused pyrimidine ring at 2,3-positions for
H-chromeno[2,3-d]pyrimidines. Structure–activity rela-
tionship was elaborated with the help of molecular docking
studies. The structures of the synthesized compounds were
established on the basis of the spectral data, infrared, proton
nuclear magnetic resonance, 13-Carbon nuclear magnetic
resonance and mass spectroscopic data.
2009), TNF-α inhibitor (Cheng et al. 2003) effects and
activities, as well as inhibitor of diabetes-induced vascular
dysfunction (Birch et al. 1996). Such diverse biological and
pharmacological activities have made chromene derivatives
important for further development in organic synthesis and
medicinal studies (Thompson 2000; Nefzi et al. 1997).
Recently, 2-amino-4H-chromene derivatives are of great
interest for their antitumor activities (Kheirollahi et al.
2014; Saffari et al. 2014; Zhang et al. 2014; Olczak et al.
2013; Patil et al. 2013; Akbarzadeh et al. 2012; Rafinejad
et al. 2012; Sabry et al. 2011; Musa et al. 2010). In addition,
other 4H-chromene derivatives showed some biological and
pharmacological applications, such as, Crolibulin
*
Ahmed M. El-Agrody
elagrody_am@yahoo.com
1
2
3
Chemistry Department, Faculty of Science, Al-Azhar University,
Nasr City, 11884 Cairo, Egypt
The Regional Center for Mycology & Biotechnology (RCMP),
Al-Azhar University, Nasr City 11884 Cairo, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, 11562 Cairo, Egypt

Med Chem Res
Fig. 1 Structures of some
-amino-4H-chromenes with
2
diverse biological and
pharmacological activities
Scheme 1 Synthesis of
halogenated 7-hydroxy-4H-
chromene derivatives (4a, b)
Scheme 2 Synthetic protocol of
compounds (6, 8, 11, 12)
(
EPC2407) (A) which is currently in Phase I/II clinical trials
for the treatment of advanced solid tumors (Patil et al.
013). Meanwhile, pyranopyranone (B) that served as
In view of the above-mentioned findings, we intend to
synthesize novel series of 4H-chromenes and compounds
containing both 4H-chromene and pyrimidine moieties
(Schemes 1–4) to explore the synergistic effect that might
result from this combination on the antitumor activities. The
structure–activity relationship (SAR) of the 2-,4- or
7-position for 4H-chromene, 5,8-position or the fused
pyrimidine ring at 2,3-positions for 5H-chromeno[2,3-d]
pyrimidine was discussed and molecular docking was
studied.
2
precursor for the blood anticoagulant warfarin (Wiener
et al. 1962), and benzopyrane (C) has been known for its
anticancer therapeutic properties (Kemnitzer et al. 2005).
Furthermore, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-
2
6
carbonitrile (E) are well known as inhibitors of Bcl-2
protein and as apoptosis inducers (Doshi et al. 2006; Wang
et al. 2000), as shown in Fig. 1.
-oxoethyl)-4H-chromene-3-carboxylate (D) and 2-amino-
-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-
Among the different types of chromenes, our interest has
been focused on 2-amino-3-cyano-4H-chromene scaffolds,
which is one of the most active species that proved to be a
useful intermediate for the synthesis of 5H-chromeno[2,3-d]
pyrimidine derivatives. In addition, 4H-chromene and 5H-
chromeno[2,3-d]pyrimidine derivatives have been emerged
as promising and attractive scaffold in the development of
potent antitumor and antimicrobial agents (Fouda 2016;
Parthiban et al. 2015; El-Agrody et al. 2014; Kandeel et al.
Results and discussion
Chemistry
2-Amino-4-(4-chloro/bromophenyl)-7-hydroxy-4H-chromene-
3-carbonitrile (4a, b) were prepared via three-components
condensation of resorcinol (1) with a mixture of 4-
chlorobenzaldehyde (2a) or 4-bromobenzaldehyde (2b)
and malononitrile (3) in ethanolic piperidine solution under
reflux for 1 h as cited in Scheme 1.
2
013; Abd-El-Aziz et al. 2004).

Med Chem Res
Scheme 3 Synthesis of
4
H-chromene
derivatives (13–15)
Scheme 4 Synthetic protocol of
compounds (12, 15–18)
Acylation of compounds 4a and 4b with refluxed acetic
anhydride resulted in the isolation of the diacetyl derivatives
6a, b) with acylation of the hydroxyl group at 7-position
instead of the monoacetyl (5) or pyrimidine derivatives (7),
while condensation of compounds 4a, b with benzaldehyde
in refluxed ethanol under basic conditions afforded the
Schiff base products (8a, b) as shown in Scheme 2.
Hydrazinolysis of 8a, b in ethanol at room temperature
under stirring or at reflux returned to β-enaminonitrile 4
instead of the pyrimidine derivative (10) (Khafagy et al.
Reaction of the imidate 13a, b and 14a, b with dime-
thylamine in methanol at room temperature under stirring
for 1 h yielded the imidine 15a, b (m.p., mixed m.p.,
identical infrared (IR) and mass spectrum) with deacylation
of the acetoxy group at 7-position into hydroxyl group in
the case of imidate 13a, b Scheme 4. This imidine 15a, b
can also be obtained as described before from the reaction
of 4a, b with DMF–DPA (m.p., mixed m.p., identical IR
and MS spectrum) Scheme 3. In addition, treatment of the
imidate 13a, b and 14a, b with methylamine in methanol at
room temperature under stirring for 1 h gave the cyclo-
addition products 5-(4-chloro/bromophenyl)-4-imino-3-me-
thyl-4,5-dihydro-3H-chromeno[2,3-d]pyrimidin-8-ol (16a, b)
(m.p., mixed m.p., identical IR and MS spectrum) with
deacylation of the acetoxy group at 7-position into hydroxyl
group in the case of imidate 13a, b as shown in Scheme 4.
Hydrazinolysis of 13a, b and 14a, b afforded the cyclo-
addition products 3-amino-5-(4-chloro/bromophenyl)-4-im-
ino-4,5-dihydro-3H-chromeno[2,3-d]pyrimidin-8-ol (17a, b)
(m.p., mixed m.p., identical IR and MS spectrum) with
deacylation of the acetoxy group at 7-position into hydroxyl
group in the case of imidate 13a, b, while ammonolysis of
(
2
002). The formation of the β-enaminonitrile 4 can be
explained via elimination of benzaldehydehydrazone from
the addition product intermediate 9. Besides, reaction of
compounds 4a, b with urea in ethanol in the presence of
sodium ethoxide under reflux gave the cycloaddition pro-
ducts 4-amino-5-(4-chloro/bromophenyl)-8-hydroxy-1H-
chromeno[2,3-d]-pyrimidin-2(5 H)-one (11a, b), while
reaction of compounds 4a, b with formamide under reflux
gave the cycloaddition products 4-amino-5-(4-chloro/bro-
mophenyl)-5H-chromeno[2,3-d]pyrimi-din-8-ol (12a, b).
These results are depicted in Scheme 2.
Treatment of compounds 4a and 4b with triethyl
orthoformate in acetic anhydride under reflux afforded 7-
acetoxy-2-ethoxymethyleneamino-4-(4-chloro/bromophe-
nyl)-4H-chromene-3-carbonitrile (13a, b) with acylation
of the hydroxyl group at 7-position, while reaction of
compounds 4a, b with the neat triethyl orthoformate
13a, b and 14a, b with NH gas bubbled in methanol at
3
room temperature under stirring for 1 h gave the cycload-
dition products 4-amino-5-(4-chloro/bromophenyl)-5H-
chromeno[2,3-d]pyrimidin-8-ol (12a, b) (m.p., mixed m.p.,
identical IR and MS spectrum) with deacylation of the
acetoxy group at 7-position into hydroxyl group in the case
of imidate 13a, b. The aminopyrimidine derivatives (12a, b)
can be obtained as described before from the reaction of 4a,
b with formamide (m.p., mixed m.p., identical IR and MS
spectrum) (Scheme 2). Finally, interaction of the aminoi-
minopyrimidine derivative 17a, b with benzaldehyde in
ethanolic piperidine under reflux afforded the open chain
afforded
phenyl)-7-hydroxy-4H-chromene-3-carbonitrile (14a, b).
Furthermore, condensation of 4a, with N,N-
2-ethoxymethyleneamino-4-(4-chloro/bromo-
b
dimethylformamide–dineopentylacetal (DMF–DPA) in
benzene gave the 4-(4-chloro/bromophenyl)-2-dimethy-
laminomethyleneamino-7-hydroxy-4H-chromene-3-car-
bonitrile (15a, b) as shown in Scheme 3.

Med Chem Res
A549
Table 1 SAR of the aryl group,
-, 2, 3-positions and the
inhibitory concentration (IC50
in µg/ml) of target compounds
against the four human cancer
cell lines in comparison with
vinblastine and colchicine as
measured with the microculture
tetrazolium (MTT) method
a
Compound
Log P
IC50 (µg/ml)
2
,
MCF-7
HCT-116
HepG-2
b
b
b
c
4a
3.08 ± 0.50
3.26 ± 0.52
2.60 ± 0.67
2.77 ± 0.69
5.78 ± 0.62
5.95 ± 0.65
8.14 ± 0.69
8.31 ± 0.72
3.20 ± 0.68
3.37 ± 0.71
4.90 ± 0.63
5.08 ± 0.65
5.01 ± 0.63
5.18 ± 0.65
3.81 ± 0.64
3.98 ± 0.66
2.41 ± 0.75
2.58 ± 0.79
1.89 ± 0.75
2.07 ± 0.79
4.50 ± 0.78
4.67 ± 0.82
4.58 ± 0.66
0.92 ± 0.55
18.8 ± 0.3
5.4 ± 0.11
10.0 ± 0.1
0.99 ± 0.2
b
b
b
c
4
6
b
a
5.8 ± 0.16
12.2 ± 0.3
3.0 ± 0.11
4.76 ± 0.03
8.16 ± 0.06
3.13 ± 0.2
1.47 ± 0.03
45.8 ± 0.02
≥ 75
2.67 ± 0.18
12.4 ± 0.05
12.4 ± 0.02
6.22 ± 0.4
3.1 ± 0.23
≥ 75
38.1 ± 0.04
20.9 ± 0.11
12.4 ± 0.01
18.2 ± 0.01
≥ 75
6.13 ± 0.06
10.2 ± 0.07
2.24 ± 0.3
2.06 ± 0.07
64.2 ± 0.06
≥ 75
6b
8
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
≥ 75
≥ 75
16 ± 0.13
≥ 75
13.8 ± 0.01
≥ 75
6.55 ± 0.11
≥ 75
8.97 ± 0.2
≥ 75
7.81 ± 0.18
71.1 ± 0.2
3.39 ± 0.03
12.4 ± 0.3
38 ± 0.1
10.0 ± 0.23
≥ 75
3.99 ± 0.2
12.1 ± 0.3
1.77 ± 0.13
3.26 ± 0.12
31.6 ± 0.06
4.31 ± 0.16
1.5 ± 0.1
2.43 ± 0.4
6.12 ± 0.11
2.68 ± 0.2
5.21 ± 0.13
24.6 ± 0.2
2.89 ± 0.3
1.04 ± 0.06
1.38 ± 0.03
1.36 ± 0.6
0.96 ± 0.5
1.86 ± 0.4
1.64 ± 0.3
3.78 ± 0.01
21.3 ± 0.03
8.84 ± 0.3
6.99 ± 0.4
40.8 ± 0.2
6.4 ± 0.14
7.06 ± 0.13
5.96 ± 0.2
9.41 ± 0.4
4.1 ± 0.3
10.5 ± 0.5
2.04 ± 0.4
2.6 ± 0.08
42.8 ± 0.06
6.49 ± 0.2
2.95 ± 0.6
5.85 ± 0.03
6.15 ± 0.2
4.85 ± 0.14
2.54 ± 0.12
3.8 ± 0.18
6.1 ± 0.03
17.7 ± 0.03
1.81 ± 0.5
2.98 ± 0.15
1.1 ± 0.7
1.55 ± 0.03
1.59 ± 0.11
4.6 ± 0.01
10.6 ± 0.02
Vinblastine
Colchicine
a
IC50 values expressed in µg/ml as the mean values of triplicate wells from at least three experiments and are
reported as the mean ± standard error
b
El-Agrody et al. (2014) and
c
Fouda (2016)
products, 3-(benzylideneamino)-5-(4-chloro/bromophenyl)-
-imino-4,5-dihydro-3H-chromeno[2,3-d]pyrimidin-8-ol
vitro cytotoxic evaluation using cell viability assay was
performed at the Regional Center for Mycology & Bio-
technology (RCMP), Al-Azhar University using vinblastine
and colchicine as reference drugs. The results were
expressed as growth inhibitory concentration (IC ) values,
which represent the compound concentrations required to
produce a 50% inhibition of cell growth after 24 h of
incubation compared to untreated controls as shown in
Table 1 and Fig. 2.
4
(
18a, b). These results are depicted in Scheme 4.
The structure of compounds 6, 8, 11–18 was established
1
on the basis of IR, proton nuclear magnetic resonance ( H
5
0
1
3
NMR), 13-Carbon nuclear magnetic resonance ( C NMR)
and MS data.
Antitumor assays
The results from Table 1 explicated that some of the
synthesized compounds displayed excellent to modest or
fair growth inhibitory activity against the tested cell lines
MCF-7, HCT-116, HepG-2 and A549. Investigations of the
cytotoxic activity against MCF-7 indicated that compounds
The synthesized compounds 6, 8, 11–18 were evaluated for
their in vitro antitumor activity against four human cancer
cell lines: breast adenocarcinoma (MCF-7), human colon
carcinoma (HCT-116), hepatocellular carcinoma (HepG-2),
and lung carcinoma (A549) at various concentrations ran-
ging from 0 to 50 μg/ml and the cell viability was measured
by the microculture tetrazolium (MTT) assay as described
in the literature (Rahman et al. 2001; Mosmann 1983). In
1
8a, 16a, 8b, 14a, 18b, 17b, 4b, and 16b (IC50 = 2.54 ±
0.12, 2.95 ± 0.6, 3.1 ± 0.23, 3.39 ± 0.03, 3.8 ± 0.18, 4.85 ±
.14, 5.8 ± 0.16 and 5.85 ± 0.03 µg/ml, respectively) were
the most sensitive compared to the standard drugs
0

Med Chem Res
Fig. 2 IC50 values expressed in (µg/ml) of target compounds against the cell lines MCF-7, HCT-116, HepG-2 and A549
vinblastine (IC50 = 6.1 ± 0.03 µg/ml), colchicine (IC50
=
=
17a, 16b, 18b, a, 8b, a, 13a, 4b, 14a and 15b were the most
active analogs through this study (with IC50 = 0.96 ± 0.5,
0.99 ± 0.2, 1.04 ± 0.06, 1.36 ± 0.6, 1.38 ± 0.03, 1.64 ± 0.3,
1.86 ± 0.4, 2.06 ± 0.07, 2.24 ± 0.3, 2.43 ± 0.4, 2.67 ± 0.18,
2.68 ± 0.2 and 2.89 ± 0.3 µg/ml, respectively) compared to
vinblastine (IC50 = 3.78 ± 0.01 µg/ml) and colchicine (IC50
= 21.3 ± 0.03 µg/ml), while compounds 14b, 13b, 6a, 12a,
and 6b were more potent and efficacious (with IC50 = 5.21
± 0.13, 6.12 ± 0.11, 6.13 ± 0.06, 8.97 ± 0.2 and 10.2 ±
0.07 µg/ml, respectively) than colchicine. In addition, the
rest of compounds were moderately active or inactive for all
the cell lines MCF-7, HCT-116, HepG-2 and A549.
1
7.7 ± 0.03 µg/ml) and compounds 17a and 8a (IC50
6
.15 ± 0.2 and 6.22 ± 0.4 µg/ml) were almost equipotent as
vinblastine, while compounds 17a, 8a, 13a, 14b, 6a, b and
1
2a (IC50 = 6.15 ± 0.2, 6.22 ± 0.4, 7.81 ± 0.18, 12.4 ± 0.3,
1
2.4 ± 0.05, 12.4 ± 0.02 and 16 ± 0.13 µg/ml, respectively)
were found to be the most potent derivative against MCF-7
compared to colchicine (IC50 = 17.7 ± 0.03 µg/ml) and
compound 4a (IC50 = 18.8 ± 0.3 µg/ml) was almost equi-
potent as colchicine. Besides, compound 18b (IC50 = 2.04
±
1
0.4 µg/ml) possessed good cytotoxic activity against HCT-
16 compared to vinblastine (IC50 = 2.6 ± 0.08 µg/ml),
while compounds 18b, 17b, 4a, 16b, 15b, 14b, 16a, 14a,
7a, 13a, 18a, 4b, 8a, 12a, 8b, 6b, a and 15a (IC50 = 2.04
0.4, 4.1 ± 0.3, 5.4 ± 0.11, 5.96 ± 0.2, 6.4 ± 0.14, 6.99 ±
1
±
SAR studies
0
0
±
.4, 7.06 ± 0.13, 8.84 ± 0.3, 9.41 ± 0.4, 10.0 ± 0.23, 10.5 ±
.5, 12.2 ± 0.3, 12.4 ± 0.01, 13.8 ± 0.01, 18.2 ± 0.01, 20.9
0.11, 38.1 ± 0.04, and 40.8 ± 0.2 µg/ml, respectively)
The partition coefficient Log P (factor of the lipophilicity),
which is well known as an index of lipophilicity, is an
important physicochemical parameter that was measured by
ACD/Labs Log P calculated, ver.14.02 and is cited in
Table 1. The preliminary SAR study has focused on the
effect of the substituent at 2-, 4- or 7-position of the 4H-
chromene moiety and the substituent at 5-, 8-position or the
fused pyrimidine ring at 2,3-positions of the 5H-chromeno
[2,3-d]pyrimidine moiety, on the antitumor activities of the
synthesized compounds. Comparison of the cytotoxic
activities of the target compounds 4a, b and their analogs 6,
8, 11–18 against MCF-7, HCT-116, HepG-2, and A549, we
found that incorporating a pyrimidine nucleus at 2,3-posi-
tions with hydrophobic groups, 4-chlorophenyl or 4-
bromophenyl at 5-position, and more hydrophobic groups
(=NH-4, –N=CHPh-3; =NH-4, –NMe-3 and =NH-4,
–NH -3) for compounds 18a, 16a, 18b, 17b and 16b (IC
were found to be the most potent derivative overall the
tested compounds against HCT-116 compared to colchicine
(
IC50 = 42.8 ± 0.08 µg/ml). On the other hand, cytotoxicity
evaluation in HepG-2 cell line revealed that the compounds
17b, 8b, 16a, 18a, b, 14a, 16b, 17a, 4b, 8a, 14b, 13a and
15b (IC50 = 1.1 ± 0.7, 1.47 ± 0.03, 1.5 ± 0.1, 1.55 ± 0.03,
1.59 ± 0.11, 1.77 ± 0.13, 1.81 ± 0.5, 2.98 ± 0.15, 3.0 ±
0.11, 3.13 ± 0.2, 3.26 ± 0.12, 3.99 ± 0.2 and 4.31 ± 0.16
µg/ml, respectively) were more potent and efficacious than
vinblastine (IC50 = 4.6 ± 0.01 µg/ml), colchicine (IC50
0.6 ± 0.01 µg/ml) and compound 6a (IC50 = 4.76 ± 0.03
=
1
µg/ml) was almost equipotent as vinblastine, while com-
pounds 6a, 12a, 6b and 4a (with IC50 = 4.6 ± 0.01, 6.55 ±
0
.11, 8.16 ± 0.06 and 10.0 ± 0.1 µg/ml, respectively) dis-
2
50
played good cytotoxicity compared to colchicine. Con-
cerning activity against A549, compounds 17b, 4a, 16a,
= 2.54 ± 0.12, 2.95 ± 0.6, 3.8 ± 0.18, 4.85 ± 0.14 and 5.85
± 0.03 µg/ml, respectively), and a hydrophilic group

Med Chem Res
hydroxy group at 8-position has caused a remarkable
enhancement in the antitumor activity against MCF-7
compared to vinblastine and colchicine (IC50 = 6.1 ± 0.03
and 17.7 ± 0.03 µg/ml) with decreasing of the partition
coefficient Log P as shown in Table 1, while introduction of
hydrophobic groups (–N=CHPh and –N=CHOEt) at 2-
position with hydroxy group at 7-positions and 4-
bromophenyl or 4-chlorophenyl at 4-position for com-
pounds 8b and 14a (IC = 3.1 ± 0.23 and 3.39 ± 0.03 µg/ml)
coefficient Log P, and the 5H-chromeno[2,3-d]pyrimidine
derivatives showed superior in vitro antitumor activity than
the 4H-chromene derivatives.
Concerning activity against hepatocellular carcinoma
(HepG-2), compounds 17b, 8b, 16a, 18a, b, 14a, 16b, 17a,
4b, 8a, 14b, 13a and 15b with (IC50 = 1.1 ± 0.7, 1.47 ±
0.03, 1.5 ± 0.1, 1.55 ± 0.03, 1.59 ± 0.11, 1.77 ± 0.13, 1.81
± 0.5, 2.98 ± 0.15, 3.0 ± 0.11, 3.13 ± 0.2, 3.26 ± 0.12, 3.99
± 0.2, and 4.31 ± 0.16 µg/ml, respectively) were more
potent and efficacious than vinblastine (IC50 = 4.6 ± 0.01
µg/ml), colchicine (IC50 = 10.6 ± 0.02 µg/ml) and com-
pound 6a (IC50 = 4.76 ± 0.03 µg/ml) was almost equipotent
as vinblastine with decreasing partition coefficient Log P as
shown in Table 1, whiles compounds 6a, 12a, 6b and 4a
with (IC50 = 4.6 ± 0.01, 6.55 ± 0.11, 8.16 ± 0.06 and 10.0
± 0.1 µg/ml, respectively) displayed good cytotoxicity
compared to colchicines, implying that incorporating the
pyrimidine nucleus at 2,3-positions with hydrophobic
50
showed more activity than the standard drugs and com-
pound 17a (IC50 = 6.15 ± 0.2 µg/ml), with a pyrimidine
nucleus at 2,3-positions, hydrophobic groups (=NH-4,
–
NH -3) and 4-chlorophenyl at 5-position was almost
2
equipotent as vinblastine and active than colchicines, beside
introduction of hydrophobic groups (–N=CHPh, -N=
CHOEt and -NAc ) at 2-position with hydroxy or acetoxy
2
groups at 7-positions and 4-chlorophenyl or 4-bromophenyl
at 4-position for compounds 8a, 13a, 14b and 6a, b (IC50
=
6
0
.22 ± 0.4, 7.81 ± 0.18, 12.4 ± 0.3, 12.4 ± 0.05, and 12.4 ±
.02 µg/ml) or incorporating a pyrimidine nucleus at 2,3-
groups (=NH-4, -NH -3; =NH-4, –NMe-3 and =NH-4,
2
–N=CHPh-3) and a hydroxy group at 8-position showed
superior in vitro antitumor activity than the 4H-chromene
derivatives with hydrophobic groups (–N=CHPh-2,
–N=CHOEt-2, NH -2 and –N=CHNMe -2) and a hydroxy
positions with hydrophobic group (–NH -4) and 4-
2
chlorophenyl at 5-position with a hydroxy group at 8-
positions for compound 12a (IC50 = 16 ± 0.13 µg/ml)
represented the most active derivatives against MCF-7
compared to colchicine, suggesting that the pyrimidine
nucleus at 2,3-positions or 4H-chromene nucleus with cer-
tain hydrophobic or hydrophilic groups respectively, was
indispensable for the activities against breast adenocarci-
noma and 4-chloro-phenyl is favorable than 4-bromophenyl
at 5- or 4-position with decreasing of the partition coeffi-
cient Log P and the 5H-chromeno[2,3-d]pyrimidine deri-
vatives showed superior in vitro antitumor activity than the
2
2
or acetoxy groups at 7-positions against hepatocellular
carcinoma, and 4-bromophenyl is favorable than 4-
chlorophenyl at 5- or 4-position with decreasing of the
partition coefficient Log P.
Further investigation of the impact of substitution pattern
at the 2-,4-,7-position and 2-,3- 5-,8-positions on the pre-
pared chromenes and chromenopyrimidines activities
against lung carcinoma (A549) was then conducted.
Incorporating the pyrimidine nucleus at 2,3-positions with
4
H-chromene derivatives
hydrophobic groups (=NH-4, –NH -3; =NH-4, –NMe-3
2
Further investigation of the impact of the substitution at
and =NH-4, –N=CHPh-3) and a hydroxy group at 8-
position for compounds 17b, 16a, 17a, 16b, 18b, a with
(IC50 = 0.96 ± 0.5, 1.04 ± 0.06, 1.36 ± 0.6, 1.38 ± 0.03,
1.64 ± 0.3 and 1.86 ± 0.4 µg/ml, respectively) or introduc-
different positions, on the (SAR), the result from Table 1,
revealed that compound 18b (IC50 = 2.04 ± 0.4 µg/ml)
showed good activity against human colon carcinoma
(
HCT-116) compared to vinblastine and colchicine (IC50
=
tion of the hydrophobic group (NH -2, –N=CHPh-2,
2
2
4
6
0
0
0
.6 ± 0.08 and 42.8 ± 0.06 µg/ml), while compounds 17b,
–N=CHOEt-2 and –N=CHNMe -2) and hydroxy or acet-
oxy groups at 7-position for compounds 4a, 8b, a, 13a, 4b,
2
a, 16b, 15b, 14b, 16a, 14a, 17a, 13a, 18a, 4b, 8a, 12a, 8b,
b, a and 15a with (IC50 = , 4.1 ± 0.3, 5.4 ± 0.11, 5.96 ±
.2, 6.4 ± 0.14, 6.99 ± 0.4, 7.06 ± 0.13, 8.84 ± 0.3, 9.41 ±
.4, 10.0 ± 0.23, 10.5 ± 0.5, 12.2 ± 0.3, 12.4 ± 0.01, 13.8 ±
.01, 18.2 ± 0.01, 20.9 ± 0.11, 38.1 ± 0.04 and 40.8 ± 0.2
14a and 15b with (IC = 0.99 ± 0.2, 2.06 ± 0.07, 2.24 ± 0.3,
50
2.43 ± 0.4, 2.67 ± 0.18, 2.68 ± 0.2 and 2.89 ± 0.3 µg/ml,
respectively) showed increased in activity against A549
compared to vinblastine and colchicine (IC50 = 3.78 ±
0.01and 21.3 ± 0.03 µg/ml) with decreasing partition coef-
ficient Log P as shown in Table 1, suggesting that the
antitumor activity is significantly affected by the presence
of 5H-chromeno[2,3-d]pyrimidine with a hydroxy group at
8-position more than the 4H-chromene with hydrophobic
group at 2-position, a hydroxy or acetoxy groups at 7-
position and 4-bromophenyl is favorable than 4-
chlorophenyl at 5- or 4-position with decreasing partition
coefficient Log P.
µg/ml, respectively) were found to be the most potent
derivatives against HCT-116 compared to colchicines with
decreasing partition coefficient Log P as shown in Table 1,
suggesting that the antitumor activity is significantly
affected by the presence of the pyrimidine nucleus at 2,3-
positions with certain hydrophobic or hydrophilic groups;
some of the hydrophobic groups at 2-positions of the 4H-
chromene moiety and 4-bromophenyl are favorable than 4-
chlorophenyl at 5- or 4-position with decreasing partition

Med Chem Res
Fig. 3 Superimposition of the
co-crystallized (red) and the
docking pose (blue) of
colchicine in the tubulin active
site with RMSD of 0.942 Å
(
color figure online)
Finally, we discovered that the substitution pattern at
2
-,4- or 7-position of the 4H-chromene moiety and the
substituent at 5-,8-position or the fused pyrimidine ring at
,3-positionpositions of the 5H-chromeno[2,3-d]pyrimidine
2
moiety are crucial elements for the antitumor activity. The
incorporation of pyrimidine nucleus at 2,3-positions with
hydrophobic groups as (=NH-4, –N=CHPh-3; =NH-4,
–
NH -3 and =NH-4, –NMe-3) and a hydroxy group at 8-
2
position or (–N=CHPh2, –N=CHOEt-2, NH -2 and
2
–
N=CHNMe -2) and a hydroxy or acetoxy groups at 7-
2
position of the chromene nucleus are favorable and greatly
enhanced the antitumor activity than the other hydrophobic
groups and 4-bromophenyl is favorable than 4-chlorophenyl
at 5- or 4-position with decreasing of the partition coeffi-
cient Log P.
Fig. 4 2D Interaction diagram showing colchicine docking pose
interactions in the tubulin active site (distances in Å)
Molecular docking studies
Several crystal structures are available in the protein data
bank for tubulin, for this work we choose (PDB ID: 5EYP)
binds by its carbonyl moiety in ring A through hydrogen
bonding with the backbone NH of Val181A. The hydro-
phobic tricyclic structure of colchicine interacts through
hydrophobic interaction with the hydrophobic amino acids
in chain B lining the binding site (Leu242, Leu248, Ala250,
Leu255, Met259, Val315, ALa316, Ilu318 and Ilu378).
The ability of the synthesized compounds to interact with
the key amino acids in the active site rationalizes their good
activity as indicated by their docking pattern and docking
score compared to that of colchicine as shown in Table 2.
Table 2 shows that most of the newly synthesized
compounds are showing better docking score than that of
colchicine indicating that they have better tubulin inhibition
and so better antitumor activity as proven experimentally in
the cell line test.
(
Ahmad et al. 2016) which have tubulin co-crystallized with
colchicine as inhibitor.
First, the molecular docking setup was validated by
carrying out re-docking of colchicine near the tubulin active
site. The re-docking validation step reproduced the experi-
mental binding mode of the co-crystallized ligand quite
efficiently indicating the suitability of the used setup for the
intended docking study and this is indicated by the small
RMSD of 0.942 Å (<2 Å) between the docked pose and the
co-crystallized ligand (energy score (S) = −12.85 kcal/mol)
and by the ability of the docking poses to reproduce the key
interactions accomplished by the co-crystallized ligand with
the hot spots in the active site, Val181A Figs. 3 and 4.
As seen in Figs. 3 and 4, colchicine binds to β-tubulin at
its interface with α-tubulin, resulting in inhibition of tubulin
polymerization (Lu et al. 2012). The trimethoxyphenyl
group of colchicine (Ring A) is located in the β-tubulin
structure near the amino acid residue Cys241B. Colchicine
The docking study showed that the aryl rings in the
newly synthesized compounds overlap with ring A and ring
C of colchicine, and so share colchicine in their ability to
interact with the hydrophobic amino acids lining the

Med Chem Res
Table 2 Docking energy scores (S) in kcal/mol for the newly
synthesized compounds in tubulin Colchicine binding site
Compound
Energy score (S)
kcal/mol)
(
4
4
6
6
8
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
−12.01
−12.26
−13.77
−13.77
−13.87
−13.99
−12.63
−13.04
−12.58
−12.93
−12.70
−13.20
−12.45
−13.11
−12.71
−13.04
−13.10
−13.23
−13.13
−13.26
−14.29
−14.49
−12.85
b
a
b
a
b
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
Fig. 5 Superimposition of the co-crystallized colchicine ligand (red)
and the docking pose of compound 11a (blue) in the tubulin active site
showing the overlap of the aryl rings in the newly synthesized com-
pound with ring A and ring C of colchicine (color figure online)
against A549 than the standard drugs vinblastine and col-
chicines. From the SARs, we discovered that the incor-
poration of pyrimidine nucleus at 2,3-positions with
hydrophobic or hydrophilic group and some of hydrophobic
group at 2-position or hydrophilic group at 7-position of the
chromene nucleus is favorable, and greatly enhanced the
activity than the other hydrophobic groups and 4-
bromophenyl is favorable than 4-chlorophenyl at 5- or 4-
position with decreasing of the partition coefficient Log P.
Also, the compounds containing both 4H-chromenes and
pyrimidine moieties showed superior in vitro antitumor
activity than the 4H-chromene derivatives.
Colchicine
binding site (Leu242, Leu248, Ala250, Leu255, Met259,
Val315, ALa316, Ilu318, and Ilu378) as shown in
Figs. 5–7.
Conclusion
New series of 4H-chromenes 6, 8, 13–15 and 5H-chromeno
Experimental
[
2,3-d]pyrimidines 11, 12, 16–18 were synthesized and
evaluated for their cytotoxic activity against four cancer cell
lines, namely breast adenocarcinoma (MCF-7), human
colon carcinoma (HCT-116), hepatocellular carcinoma
All chemicals were purchased from Sigma-Aldrich Che-
mical Co. Melting points were determined with a Stuart
Scientific Co. Ltd apparatus and are uncorrected. IR spectra
were determined as KBr pellets on a Jasco FT/IR 460 plus
(
HepG-2) and lung carcinoma (A549). The results revealed
1
13
that compounds 18a, 16a, 8b, 14a, 18b, 17b, 4b, and 16b
displayed high growth inhibitory activity against MCF-7
cells, while compound 18b showed relatively potent anti-
tumor activity against HCT-116 as compared to vinblastine
and colchicine. Whereas, compounds 17b, 8b, 16a, 18a, b,
spectrophotometer. H NMR and C NMR spectra were
recorded using a Bruker High Performance Digital FT-
NMR Spectrometer Avance III 400 MHz spectrometer and
a Bruker AV 500 MHz spectrometer or Varian Gemini-
300BB at 300 MHz. The MS were measured on a Shimadzu
GC/MS-QP5 spectrometer. Elemental analyses were carried
out at the Regional Centre for Mycology & Biotechnology
(RCMP), Al-Azhar University, Cairo, Egypt and the results
were within ± 0.3%.
1
4a, 16b, 17a, 4b, 8a, 14b, 13a and 15b were more potent
and efficacious against HepG-2 than vinblastine and col-
chicines, besides compounds 17b, 4a, 16a, 17a, 16b, 18b,
a, 13a, 4b, 14a, and 15b exhibited good antitumor profile

Med Chem Res
Fig. 6 3D diagram of compound
1
1a showing its interaction with
the tubulin active site
2
-Amino-4-(4-chlorophenyl)-7-hydroxy-4H-chromene-3-
carbonitrile (4a)
Compound 4a was synthesized according to the literature
procedure (Makarem et al. 2008).
2
3
-Amino-4-(4-bromophenyl)-7-hydroxy-4H-chromene-
-carbonitrile (4b)
Compound 4b was synthesized according to the literature
procedure (Makarem et al. 2008).
Preparation of (6a, b)
A solution of 2-amino-4-(4-chloro/bromophenyl)-7-hydroxy-
4H-chromene-3-carbonitrile (4a, b) (0.01 mol) in acetic
anhydride (20 ml) was refluxed for ½, 3 or 6 h. The solvent
was removed under reduced pressure, and the solid obtained
was collected and washed with cooled methanol, filtered,
dried, and recrystallized from ethanol/benzene to afforded
Fig. 7 2D representation of compound 11a showing its interaction
with the tubulin active site
6
a, b. The physical and spectral data of compounds 6a, b
are as follows:
7-Acetoxy-4-(4-bromophenyl)-2-diacetylamino-4H-
7
-Acetoxy-4-(4-chlorophenyl)-2-diacetylamino-4H-
chromene-3-carbonitrile (6b)
chromene-3-carbonitrile (6a)
Colorless needles; m.p. 206–207 °C; yield 77%; IR (KBr)
−
1
Colorless crystals; m.p. 200–201 °C; yield 79%; IR (KBr)
υ (cm ): 3068, 3011, 2938 (CH stretching), 2223 (CN),
−
1
1
υ (cm ): 3061, 3018, 2946 (CH stretching), 2221 (CN),
1745, 1725 (CO); H NMR (DMSO-d , 400 MHz) δ =
7.65–6.55 (7 H, m, aromatic), 5.36 (1 H, s, H-4), 2.45 (6 H,
6
1
1
732, 1721 (CO); H NMR (DMSO-d , 400 MHz) δ =
6
7
.51–6.98 (7 H, m, aromatic), 5.36 (1 H, s, H-4), 2.44 (6 H,
s, 2COCH ), 2.26 (3 H, s, OCOCH ); MS m/z (%): 470
(M + 2, 7.85), 468 (M , 8.05) with a base peak at 187
3
3
+
+
s, 2COCH ), 2.25 (3 H, s, OCOCH ); MS m/z (%): 426
3
3
+
+
(
M + 2, 0.66), 424 (M , 2.00) with a base peak at 43
(100); anal. calcd for C H BrN O : C, 56.31; H, 3.65; N,
2
2
17
2 5
(
100); anal. calcd for C H ClN O : C, 62.20; H, 8.35; N,
5.97. Found: C, 56.06; H, 3.42; N, 5.73%.
2
2
17
2 5
6
.59. Found: C, 62.48; H, 8.61; N, 6.81%.

Med Chem Res
Synthesis of the Schiff base (8a, b)
2898, 2838, 2798 (CH stretching), 1747 (CO), 1627
1
(
C=N); H NMR (DMSO-d , 400 MHz) δ = 10.50 (1 H, bs,
6
Compounds 4a and 4b (0.01 mol), benzaldehyde (0.01
mol), piperidine (0.5 ml) in absolute ethanol (20 ml) was
refluxed for 2 h. The solid product which formed was fil-
tered, washed with cooled methanol, dried and recrys-
tallized from ethanol to give 8a, b. The physical and
spectral data of compounds 8a, b are as follows:
NH), 9.65 (1 H, bs, OH), 7.32–6.52 (7 H, m, aromatic), 6.50
+
(2 H, s, NH ), 5.04 (1 H, s, H-5); MS m/z (%): 343 (M + 2,
2
+
1.33), 341(M , 4.02) with a base peak at 230 (100); anal.
calcd for C H ClN O : C, 59.75; H, 3.54; N, 12.30.
1
7
12
3 3
Found: C, 60.00; H, 3.80; N, 12.56%.
4-Amino-5-(4-bromophenyl)-8-hydroxy-1H-chromeno
2
-Benzylideneamino-4-(4-chlorophenyl)-7-hydroxy-4H-
[2,3-d]pyrimidin-2(5 H)-one (11b)
chromene-3-carbonitrile (8a)
Brown powder; m.p. > 300 °C; yield 79%; IR (KBr) υ
−
1
Yellow needles; m.p. 217–218 °C; yield 83%; IR (KBr) υ
(cm ): 3452, 3382, 3331, 3211 (OH, NH & NH ), 3030,
2
−1
(
cm ): 3332, (OH), 3070, 3017, 2966 (CH stretching),
2888, 2835, 2781 (CH stretching), 1752 (CO), 1622
(C=N); H NMR (DMSO-d , 400 MHz) δ = 11.13 (1 H, bs,
1
1
2
183 (CN); H NMR (DMSO-d , 400 MHz) δ = 9.76 (1 H,
6
6
bs, OH), 9.11 (1 H, s, N=CH), 7.38–6.40 (12 H, m, aro-
NH), 9.87 (1 H, bs, OH), 7.46–6.80 (7 H, m, aromatic), 6.50
+
+
matic), 4.66 (1 H, s, H-4); MS m/z (%): 388 (M + 2, 0.61),
(2 H, s, NH ), 5.02 (1 H, s, H-5); 387 (M + 2, 14.65), 385
2
+
+
3
86 (M , 1.80) with a base peak at 187 (100); anal. calcd
(M , 14.77) with a base peak at 275 (100); anal. calcd for
for C H ClN O : C, 71.41; H, 3.91; N, 7.24. Found: C,
C H BrN O : C, 52.87; H, 3.13; N, 10.88. Found: C,
2
3
15
2
2
17 12
3 3
7
1.19; H, 3.68; N, 7.02%.
53.04; H, 3.34; N, 11.01%.
2
-Benzylideneamino-4-(4-bromophenyl)-7-hydroxy-4H-
Synthesis of the 4-aminopyrimidine derivatives (12a, b)
chromene-3-carbonitrile (8b)
Method (a) A mixture of compounds 4a, b (0.01 mol) and
formamide (0.02 mol) was stirred at reflux for 6 h. The
solvent was removed under vacuum. The solid obtained was
recrystallized from benzene to give 12a, b. The physical
and spectral data of compounds 12a, b are as follows:
Yellow needles; m.p. 269–270 °C; yield 80%; IR (KBr) υ
−1
(
2
cm ): 3382, (OH), 3055, 3007, 2796 (CH stretching),
1
202 (CN); H NMR (DMSO-d , 400 MHz) δ = 9.92 (1 H,
6
bs, OH), 9.12 (1 H, s, N=CH), 8.06–6.58 (12 H, m, aro-
+
matic), 5.10 (1 H, s, H-4); MS m/z (%): 432 (M + 2,
+
1
6.13), 430 (M , 16.92) with a base peak at 275 (100); anal.
4-Amino-5-(4-chlorophenyl)-5H-chromeno[2,3-d]pyrimi-
calcd for C H BrN O : C, 64.05; H, 3.51; N, 6.50.
Found: C, 63.85; H, 3.25; N, 6.29%.
din-8-ol (12a) Colorless crystals; m.p. 281–282 °C; yield
2
3
15
2 2
−1
71%; IR (KBr) υ (cm ): 3464, 3340, 3301 (OH & NH ),
2
1
3
097, 2819, 2762 (CH stretching), 1643 (C=N); H NMR
Synthesis of 1H-chromeno[2,3-d]pyrimidin-2(5H)-one
(DMSO-d , 400 MHz) δ = 9.72 (1 H, s, OH), 8.10 (1 H, s,
6
derivatives (11a, b)
H-2), 7.33–6.52 (7 H, m, aromatic), 6.80 (2 H, bs, NH ),
2
+
5
.18 (1 H, s, H-5); MS m/z (%): 327 (M + 2, 8.61), 325
+
A mixture of compounds 4a and 4b (0.01 mol) and urea
(M , 25.24) with a base peak at 276 (100); anal. calcd for
(
0.01 mol) with catalytic amount of sodium ethoxide in
ethanol (15 ml) was refluxed on water bath for 6–7 h
monitored by thin-layer chromatography (TLC)). After
C H ClN O : C, 62.68; H, 3.71; N, 12.90. Found: C,
62.91; H, 3.97; N, 13.16%.
1
7
12
3 2
(
completion of reaction, the reaction mixture was poured in
crushed ice (50 g) and neutralized by the diluted hydro-
chloric acid (1:1). The separated product was collected by
the filtration and washed with water. The crude product was
purified by crystallization from absolute ethanol to afforded
4-Amino-5-(4-bromophenyl)-5H-chromeno[2,3-d]pyrimi-
din-8-ol (12b) Colorless crystals; m.p. 292–293 °C; yield
−
1
70%; IR (KBr) υ (cm ): 3464, 3352, 3307 (OH & NH ),
2
1
3093, 2931, 2817 (CH stretching), 1648 (C=N); H NMR
(DMSO-d , 400 MHz) δ = 9.72 (1 H, s, OH), 8.10 (1 H, s,
6
1
1a,b. The physical and spectral data of compounds 11a, b
H-2), 7.46–6.52 (7 H, m, aromatic), 6.80 (2 H, bs, NH ),
2
+
are as follows:
5.17 (1 H, s, H-5); MS m/z (%): 371 (M + 2, 93.01), 369
+
(
M , 93.15) with a base peak at 104 (100); anal. calcd for
4
-Amino-5-(4-chlorophenyl)-8-hydroxy-1H-chromeno
C H BrN O : C, 55.15; H, 3.27; N, 11.35. Found: C,
17 12 3 2
[
2,3-d]pyrimidin-2(5 H)-one (11a)
54.89; H, 3.04; N, 11.16%.
Brown powder; m.p. > 300 °C; yield 80%; IR (KBr) υ
Method (b) A mixture of the imadate 13a, b or 14a, b
−1
(
cm ): 3448, 3367, 3339, 3218 (OH, NH and NH ), 3071,
(0.01 mol) and NH gas in methanol was stirred for 2 h, then
2
3

Med Chem Res
+
the mixture was left overnight. The solid product was col-
lected and crystallized from benzene to give 12a, b (m.p.,
mixed m.p., identical IR and MS spectrum).
354 (M , 4.38) with a base peak at 43 (100); anal. calcd for
C H ClN O : C, 64.32; H, 4.26; N, 7.90. Found: C,
64.22; H, 4.16; N, 7.80%.
1
9
15
2 3
2
-Ethoxymethyleneamino-4-(4-bromophenyl)-7-
Synthesis of the imidate 13a, b and 14a, b
hydroxy-4H-chromene-3-carbonitrile (14b)
A mixture of compounds 4a and 4b (0.01 mol) with triethyl
orthoformate (0.01 mol) and acetic anhydride (30 ml) or
without acetic anhydride was refluxed for 2 h. The solvent
was removed under reduced pressure and the resulting solid
was washed with methanol and recrystallized from benzene
to give 7-acetoxy-2-ethoxymethyleneamino-4-(4-chlor-
ophenyl)-4H-chromene-3-carbonitrile (13a) (Abd-El-Aziz
et al. 2004), 13b and 14a, b, respectively. The physical and
spectral data of compounds 13b and 14a, b are as follows:
Pale yellow needles; m.p. 189–190 °C; yield 77%; IR (KBr)
υ (cm ): IR (KBr) υ (cm ): 3371, (OH), 3071, 2974,
−
1
−1
1
2
925, 2858 (CH stretching), 2211 (CN); H NMR (DMSO-
d6, 400 MHz) δ = 9.83 (1 H, bs, OH), 8.66 (1 H, s, N=CH),
.88–6.53 (7 H, m, aromatic), 4.93 (1 H, s, H-4), 4.33 (2 H,
q, J = 7.1 Hz, O-CH CH ), 1.31 (3 H, t, J = 7.1 Hz, O-
7
2
3
1
3
CH CH ); C NMR (DMSO-d , 100 MHz) δ: 162.27 (C,
2
3
6
C-2), 158.28 (C, C-7), 157.99 (CH, N=CH), 149.35 (C, C-
8a), 130.47 (CH, C-5), 118.48 (C, CN), 113.69 (C, C-4a),
112.14 (CH, C-6), 103.30 (CH, C-8), 79.69 (C, C-3), 64.36
7
4
-Acetoxy-2-ethoxymethyleneamino-4-(4-bromophenyl)-
H-chromene-3-carbonitrile (13b)
(CH , O-CH CH ), 40.91 (CH, C-4), 14.33 (CH , O-
2 2 3 3
CH CH ), 144.42 (C, C-1′), 132.17 (CH, C-3′, C-5′),
2 3
1
30.62 (CH, C-2′, C-6′), 120.86 (C, C-4′); MS m/z (%): 400
+
+
Colorless needles; m.p. 173–174 °C; yield 86%; IR (KBr) υ
(M + 2, 10.01), 398 (M , 10.55) with a base peak at 187
−
1
(
(
(
cm ): 3070, 2985, 2866, 2800 (CH stretching), 2210
(100); anal. calcd for C H BrN O : C, 57.16; H, 3.79; N,
7.02. Found: C, 57.01; H, 3.65; N, 6.89%.
1
9
15
2 3
1
CN), 1767 (CO); H NMR (DMSO-d , 400 MHz) δ = 8.67
1 H, s, N=CH), 7.59–6.90 (7 H, m, aromatic), 5.10 (1 H, s,
6
H-4), 4.35 (2 H, q, J = 7.1 Hz, O–CH CH ), 2.26 (3 H, s,
Synthesis of the imidine 15a, b
2
3
1
3
OCOCH ), 1.33 (3 H, t, J = 7.1 Hz, O–CH CH ); C NMR
3
2
3
(
DMSO-d , 100 MHz) δ: 169.36 (C, COMe), 162.47 (C, C-
Method (a) A mixture of compounds 4a, b (0.01 mol) and
DMF–DPA (0.01 mol) in benzene (30 ml) was refluxed for
3 h. The solvent was removed under reduced pressure and
the resulting solid was recrystallized from benzene to give
the imidine 15a, b. The physical and spectral data of
compounds 15a, b are as follows:
6
2
8
1
), 158.20 (C, C-7), 150.50 (CH, N=CH), 148.91 (C, C-
a), 130.58 (CH, C-5), 119.52 (C, CN), 119.42 (C, C-4a),
18.19 (CH, C-6), 110.94 (CH, C-8), 79.53 (C, C-3), 64.50
(
CH , O-CH CH ), 41.00 (CH, C-4), 21.23 (CH , COMe),
2 2 3 3
1
3
4.31 (CH , O–CH CH ), 143.75 (C, C-1′), 132.33 (CH, C-
3 2 3
′, C-5′), 130.74 (CH, C-2′, C-6′), 121.21 (C, C-4′); MS m/z
+
+
(
%): 442 (M + 2, 24.27), 440 (M , 24.27) with a base
4-(4-Chlorophenyl)-2-dimethylaminomethyleneamino-7-
peak at 243 (100); anal. calcd for C H BrN O : C, 57.16;
H, 3.88; N, 6.35. Found: C, 57.41; H, 4.11; N, 6.62%.
hydroxy-4H-chromene-3-carbonitrile
crystals; m.p. 216–217 °C; yield 72%; IR (KBr) υ (cm ):
(15a) Colorless
2
1
17
2 4
−1
1
3464 (OH), 2931, 2896 (CH stretching), 2191 (CN); H
2
-Ethoxymethyleneamino-4-(4-chlorophenyl)-7-
NMR (DMSO-d , 400 MHz) δ = 9.72 (1 H, bs, OH), 8.41
6
hydroxy-4H-chromene-3-carbonitrile (14a)
(1 H, s, N=CH), 7.38–6.42 (7 H, m, aromatic), 4.67 (1 H, s,
+
H-4), 3.15, 3.00 (6 H, s, 2CH ); MS m/z (%): 355 (M + 2,
3
+
Pale yellow needles; m.p. 203–204 °C; yield 78%; IR (KBr)
4.71), 353 (M , 14.88) with a base peak at 326 (100); anal.
−
1
υ (cm ): 3396, (OH), 3076, 2985, 2869, 2804 (CH
calcd for C H ClN O : C, 64.50; H, 4.56; N, 11.88.
1
9
16
3 2
1
stretching), 2211 (CN); H NMR (DMSO-d , 400 MHz) δ
Found: 64.27; H, 4.30; N, 11.61%.
6
=
9.79 (1 H, bs, OH), 8.66 (1 H, s, N=CH), 7.43–6.42 (7 H,
m, aromatic), 5.18 (1 H, s, H-4), 4.33 (2 H, q, J = 7.1 Hz, O-
4-(4-Bromophenyl)-2-dimethylaminomethyleneamino-7-
1
3
CH CH ), 1.32 (3 H, t, J = 7.1 Hz, O-CH CH ); C NMR
hydroxy-4H-chromene-3-carbonitrile (15b) Colorless cry-
2
3
2
3
−1
(
DMSO-d , 100 MHz) δ: 162.31 (C, C-2), 158.30 (C, C-7),
stals; m.p. 250–250 °C; yield 71%; IR (KBr) υ (cm ):
6
1
1
1
57.98 (CH, N=CH), 149.36 (C, C-8a), 130.48 (CH, C-5),
18.48 (C, CN), 113.69 (C, C-4a), 112.23 (CH, C-6),
03.29 (CH, C-8), 79.74 (C, C-3), 64.35 (CH2,
3482, (OH), 3062, 2972, 2932, 2899 (CH stretching), 2191
1
(CN); H NMR (DMSO-d , 400 MHz) δ = 9.76 (1 H, bs,
6
OH), 8.40 (1 H, s, N=CH), 7.52–6.42 (7 H, m, aromatic),
1
3
O–CH CH ), 40.80 (CH, C-4), 14.34 (CH , O–CH CH ),
4.78 (1 H, s, H-4), 3.15, 3.00 (6 H, s, 2CH3); C NMR
2
3
3
2
3
1
1
44.03 (C, C-1′), 132.30 (C, C-4′), 130.26 (CH, C-2′, C-6′),
29.25 (CH, C-3′, C-5′); MS m/z (%): 356 (M + 2, 1.43),
(DMSO-d , 100 MHz) δ: 170.32 (C, C-2), 160.74 (C, C-7),
157.78 (CH, N=CH), 149.28 (C, C-8a), 130.30 (CH, C-5),
6
+

Med Chem Res
1
1
20.25 (C, CN), 113.20 (C, C-4a), 103.71 (CH, C-6),
02.78 (CH, C-8), 72.25 (C, C-3), 37.91(CH, C-4), 34.99
calcd for C H BrN O : C, 56.27; H, 3.67; N, 10.94.
Found: C, 56.50; H, 3.91; N, 11.18%.
1
8
14
3 2
[
CH ,N(CH ) ], 145.61 (C, C-1′), 131.99 (CH, C-3′, C-5′),
3 3 2
1
30.35 (CH, C-2′, C-6′), 120.78 (C, C-4′); MS m/z (%): 399
+
+
Synthesis of the 3-amino-4-iminopyrimidine derivatives
(
M + 2, 20.21), 397 (M , 20.40) with a base peak at 187
(
17a, b)
(
100); anal. calcd for C H BrN O : C, 57.30; H, 4.05; N,
1
9
16
3 2
1
0.55. Found: C, 57.58; H, 4.31; N, 10.81%.
Compound 17a, b were prepared from imadate 13a, b or
4a, b (0.01 mol) and methylamine (0.01 mol) according to
the procedure described for 15 (Method b) and recrys-
1
Method (b) A mixture of imadate 13a, b or 14a, b (0.01
mol) and dimethylamine in methanol (30 ml), was stirred
for 1 h and the mixture was left overnight. The solid product
was collected by filtration, washed with methanol and
recrystallized from ethanol/benzene to afford 15a, b (m.p.,
mixed m.p., identical IR and MS spectrum).
tallized from ethanol/benzene to afford 3-amino-5-
(4-chlorophenyl)-4-imino-4,5-dihydro-3H-chromeno[2,3-d]
pyrimidin-8-ol (17a) (Abd-El-Aziz et al. 2004) and 17b.
The physical and spectral data of compound 17b was as
follows:
Synthesis of the 3-methylpyrimidine derivatives (16a, b)
Compound 16a, b were prepared from imadate 13a, b or
3-amino-5-(4-bromophenyl)-4-imino-4,5-dihydro-3H-
chromeno[2,3-d]pyrimidin-8-ol (17b)
1
4a, b (0.01 mol) and methylamine in methanol (30 ml)
Colorless crystals; m.p. 234–235 °C; yield 81%; IR (KBr) υ
−1
−1
according to the procedure described for 15 (Method b) and
recrystallized from ethanol/benzene to afford 16a, b. The
physical and spectral data of compounds 16a, b are as
follows:
(cm ): IR (KBr) υ (cm ): 3633, 3454, 3201 (OH, NH &
1
NH ), 3001, 2931, 2800 (CH stretching), 1639 (C=N); H
2
NMR (DMSO-d , 400 MHz) δ = 9.71 (1 H, bs, OH), 8.07
6
(1 H, s, H-2), 7.45–6.50 (7 H, m, aromatic), 6.63 (1 H, s,
NH), 6.50 (2 H, s, NH ), 5.10 (1 H, s, H-5); MS m/z (%):
2
+
+
3
3
3
86 (M + 2, 25.39), 384 (M , 26.04) with a base peak at
5
-(4-Chlorophenyl)-4-imino-3-methyl-4,5-dihydro-3H-
68 (100); anal. calcd for C H BrN O : C, 53.00; H,
.40; N, 14.54. Found: C, 52.81; H, 3.20; N, 14.32%.
chromeno[2,3-d]pyrimidin-8-ol (16a)
17 13
4 2
Pale yellow crystals; m.p. 285–286 °C; yield 87%; IR (KBr)
−
1
υ (cm ): 3466, 3340 (OH & NH), 2939, 2898 (CH
1
Synthesis of the Schiff base (18a, b)
stretching), 1635 (C=N); H NMR (DMSO-d , 400 MHz)
6
δ = 9.70 (1 H, bs, OH), 8.11 (1 H, s, H-2), 7.33–6.47 (8 H,
A mixture of imino compounds 17a, b (0.01 mol) and
benzaldehyde (0.01 mol) in ethanol (30 ml) and piperidine
m, aromatic, NH), 5.10 (1 H, s, H-5), 3.27 (3 H, s, CH );
3
1
3
C NMR (DMSO-d , 100 MHz) δ: 157.63 (C,C-4), 157.24
6
(0.5 ml) was refluxed for 2 h. (TLC monitoring). The sol-
(
2
C, C-8), 151.83 (C, C-10a), 149.68 (C, C-9a), 144.67 (C-
), 129.66 (C, C-6), 114.51 (C, C-5a), 113.04 (C-7), 103.18
vent was removed under reduced pressure and the resulting
solid was recrystallized from ethanol/benzene to give the
open chain product 18a, b. The physical data of the com-
pounds 18a, b are as follows:
(
(
(
(
C-9), 98.14 (C-4a), 38.02 (CH, C-5), 35.81 (CH ), 131.75
3
C, C-1′), 130.47 (C, C-4′), 129.10 (CH, C-2′, C-6′), 127.47
+
CH, C-3′, C-5′); MS m/z (%): 341 (M + 2, 28.01), 339
+
M , 84.10) with a base peak at 187 (100); anal. calcd for
C H ClN O : C, 63.63; H, 4.15; N, 12.37. Found: C,
1
8
14
3 2
3
-(Benzylideneamino)-5-(4-chlorophenyl)-4-imino-4,5-
6
3.41; H, 3.95; N, 12.09%.
dihydro-3H-chromeno[2,3-d]pyrimidin-8-ol (18a)
5
-(4-Bromophenyl)-4-imino-3-methyl-4,5-dihydro-3H-
Yellow crystals; m.p. 296–297 °C; yield 87%; IR (KBr) υ
−1
−1
chromeno[2,3-d]pyrimidin-8-ol (16b)
(cm ): IR (KBr) υ (cm ): 3459, 3201 (OH & NH), 3011,
1
2
937 (CH stretching), 1647 (C=N); H NMR (DMSO-d ,
6
Pale yellow crystals; m.p. 271–273 °C; yield 85%; IR (KBr)
400 MHz) δ = 10.93 (1 H, s, NH), 9.74 (1 H, s, OH), 8.37
(1 H, s, N=CH), 8.16 (1 H, s, H-2), 7.71–6.61 (12 H, m,
−1
υ (cm ): 3469, 3348 (OH and NH), 3001, 2931, 2800 (CH
1
+
stretching), 1639 (C=N); H NMR (DMSO-d , 400 MHz)
aromatic), 5.99 (1 H, s, H-5); MS m/z (%): 430 (M + 2,
6
+
δ = 9.72 (1 H, bs, OH), 8.11 (1 H,s, H-2), 7.47–6.50 (8 H,
5.93), 428 (M , 13.40) with a base peak at 324 (100); anal.
m, aromatic, NH), 5.08 (1 H, s, H-5), 3.18 (3 H, s, CH );
MS m/z (%): 385 (M + 2, 98.55), 383 (M , 100); anal.
calcd for C H ClN O : C, 67.21; H, 4.00; N, 13.06.
Found: C, 67.45; H, 4.23; N, 13.24%.
3
24 17
4 2
+
+

Med Chem Res
3
-(Benzylideneamino)-5-(4-bromophenyl)-4-imino-4,5-
untreated cells. The relation between surviving cells and
drug concentration is plotted to get the survival curve of
each tumor cell line after treatment with the specified
compound. The 50% inhibitory concentration (IC ), the
concentration required to cause toxic effects in 50% of
intact cells, was estimated from graphic plots of the dose
response curve for each conc. using Graph pad Prism
software (San Diego, CA., USA) (Mosmann 1983).
dihydro-3H-chromeno[2,3-d]pyrimidin-8-ol (18b)
Yellow crystals; m.p. 311–312 °C; yield 84%; IR (KBr) υ
5
0
−1
(
cm ): 3413, 3197 (OH & NH), 3058, 2980, 2825 (CH
1
stretching), 1638 (C=N); H NMR (DMSO-d , 400 MHz)
δ = 10.96 (1 H, s, NH), 9.78 (1 H, s, OH), 8.37 (1 H, s,
N=CH), 8.17 (1 H, s, H-2), 7.71–6.60 (12 H, m, aromatic),
5
6
+
.98 (1 H, s, H-5); MS m/z (%): 474 (M + 2, 5.76), 472
+
(
M , 5.85) with a base peak at 368 (100); anal. calcd for
Molecular docking study
C H BrN O : C, 60.90; H, 3.62; N, 11.84. Found: C,
2
4
17
4 2
6
1.14; H, 3.85; N, 12.06%.
All the molecular modeling studies were carried out using
Molecular Operating Environment (MOE, 10.2008) soft-
ware. All minimizations were performed with MOE until an
Antitumor screening
−
1
−1
RMSD gradient of 0.05 kcal mol
Å
with MMFF94x
Cell culture
force field and the partial charges were automatically cal-
culated. The X-ray crystallographic structures of tubulin co-
crystallized with colchicine as inhibitor (PDB ID: 5EYP)
(Ahmed et al. 2016) were downloaded from the protein data
bank (http://www.rcsb.org/). The protein was prepared for
docking study by removal of chain F and keeping chains A
and B (α and β tubulin chains) as colchicine binding site is
at the interface between these two chains. Water molecules
and ligands that are not involved in the binding were also
removed. Then the protein was prepared using Protonate
3D protocol in MOE with default options. The co-
crystallized ligand was used to define the active site for
docking. Triangle Matcher placement method and London
dG scoring function were used for docking. Docking setup
was first validated by re-docking of the co-crystallized
ligand (Colchicine) in the vicinity of the active site of the
protein with energy score (S) = −12.85 kcal/mol and
RMSD of 0.942 Å. The validated setup was then used in
predicting the binding mode and the binding interactions of
the newly synthesized ligands at the active site of tubulin.
The tumor cell lines breast adenocarcinoma (MCF-7),
human colon carcinoma (HCT-116), hepatocellular carci-
noma (HepG-2) and lung carcinoma (A549) were obtained
from the American Type Culture Collection (ATCC,
Rockville, MD). The cells were grown on RPMI-1640
medium supplemented with 10% inactivated fetal calf
serum and 50 µg/ml gentamycin. The cells were maintained
at 37 °C in a humidified atmosphere with 5% CO and were
subculture two to three times a week.
2
Cytotoxicity evaluation using viability assay
The tumor cell lines were suspended in medium at con-
centration 5 × 104 cell/well in Corning 96-well tissue
®
culture plates and then incubated for 24 h. The tested
compounds with concentrations ranging from 0 to 50 μg/ml
were then added into 96-well plates (six replicates) to
achieve different conc. for each compound. Six vehicle
controls with media or 0.5% DMSO were run for each 96-
well plate as a control. After incubating for 24 h, the
numbers of viable cells were determined by the MTT test.
Briefly, the media was removed from the 96-well plates and
replaced with 100 μl of fresh culture RPMI 1640 medium
without phenol red then 10 µl of the 12 mM MTT stock
solution (5 mg of MTT in 1 ml of PBS) to each well
including the untreated controls. The 96-well plates were
Acknowledgements The authors deeply thank the RCMP, Al-Azhar
University for carrying out the antitumor study and Elemental
analyses.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
then incubated at 37 °C and 5% CO for 4 h. An 85-μl
2
aliquot of the media was removed from the wells, and 50 µl
of DMSO was added to each well and mixed thoroughly
with the pipette and incubated at 37 °C for 10 min. Then,
the optical density was measured at 590 nm with the
microplate reader (SunRise, TECAN, Inc, USA) to deter-
mine the number of viable cells and the percentage of
viability was calculated as [1 - (ODt/ODc)] × 100% where
ODt is the mean optical density of wells treated with the
tested sample and ODc is the mean optical density of
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