Studies on the synthesis, in vitro antitumor activity of 4H-benzo[h]chromene, 7H-benzo[h]chromene[2,3-d]pyrimidine derivatives and structure-Activity relationships of the 2-,3- and 2,3-positions

Article abstract of DOI:10.1007/s00044-013-0904-x

Some 4H-benzo[h]chromene and 7H-benzo[h]chromeno[2,3-d]pyrimidine derivatives were prepared as potential cytotoxic agents. The in vitro cytotoxic activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard

Full text of DOI:10.1007/s00044-013-0904-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0904-x
ORIGINAL RESEARCH
Studies on the synthesis, in vitro antitumor activity of 4H-
benzo[h]chromene, 7H-benzo[h]chromene[2,3-d]pyrimidine
derivatives and structure–activity relationships of the 2-,3-
and 2,3-positions
•
Ahmed M. El-Agrody Ahmed M. Fouda
•
Al-Anood M. Al-Dies
Received: 1 November 2013 / Accepted: 21 December 2013
Ó Springer Science+Business Media New York 2014
Abstract Some 4H-benzo[h]chromene and 7H-benzo[h]
chromeno[2,3-d]pyrimidine derivatives were prepared as
potential cytotoxic agents. The in vitro cytotoxic activity of
the synthesized compounds was investigated in comparison
with the well-known anticancer standard drugs Vinblastine,
Colchicine, and Doxorubicin using MTT colorimetric
assay. It was found that compounds 23, 15, 20, and 21
showed the highest anticancer activity against the three
tumor cell lines MCF-7, HCT, and HepG-2, compared with
Vinblastine and Colchicine, while compound 23 was the
most active against HepG-2 as compared with Doxorubi-
cin. We explored the SAR of 4H-benzo[h]chromenes with
modification at the 2-,3- positions and 7H-benzo[h]chro-
meno[2,3-d]pyrimidine at 2,3-positions. The structure–
activity relationship (SAR) study revealed that the antitu-
mor activity on 4H-benzo[h]chromene and 7H-benzo[h]
chromeno[2,3-d]pyrimidine derivatives were significantly
affected by the lipophilicity (hydrophobic or hydrophilic),
of the substituent at 2-,3- and 2,3-positions. Structures of
these compounds were established on the basis of spectral
Keywords 4-Methoxy-1-naphthol ꢀ
a-Cyanocinnamonitriles ꢀ 4H-Benzo[h]chromenes ꢀ
7H-Benzo[h]chromeno[2,3-d]pyrimidines ꢀ Antitumor ꢀ
SAR
Introduction
2-Amino-4H-chromenes and their derivatives occupy an
important place in the realm of natural and synthetic organic
chemistry because of their biological and pharmacological
activities such as antimicrobial (Kidwai et al., 2010; Alvey
et al., 2009), antioxidant (Singh et al., 2010; Vukovic et al.,
2010), antitumor (Sabry et al., 2011; Kemnitzer et al., 2008.
Kemnitzer et al., 2007; Mahmoodi et al., 2010; Endo et al.,
2010; Tseng et al., 2010), vascular disrupting (Kasibhatla
et al., 2004), antileishmanial (Tanaka et al., 2007), anti-
cancer (Vosooghi et al., 2010; Gourdeau et al., 2004;
Kemnitzer et al., 2004), antiproliferative (Magedov et al.,
2007), effects and activities, as well as treatment of alzhei-
mer’s disease (Bruhlmann et al., 2001), and schizophrenia
disorder (Kesten et al., 1999). Fused chromene ring systems
have blood platelet antiaggregating (Lee et al., 2006) and
analgesic activities (El-Sayed and Ibrahim, 2010; Keri et al.,
2010). In addition, polyfunctionalized 4H-chromenes con-
stitute a structural unit of a number of natural products and
because of the inherent reactivity of the inbuilt pyran ring are
versatile synthons (Cingolant and Pigini, 1969). They also
exhibit hypolipidemic activity (Sashidhara et al., 2011),
DNA breaking activities, and mutagenicity (Hiramoto et al.,
1997).
1
data, IR, H NMR, 13C NMR, 13C NMR-DEPT, and MS
data.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0904-x) contains supplementary
material, which is available to authorized users.
A. M. El-Agrody
Chemistry Department, Faculty of Science, Al-Azhar University,
Nasr City, Cairo 11884, Egypt
These findings stimulated our interest in the syntheses
of 4H-benzo[h]chromene and 7H-benzo[h]chromeno[2,3-
d]pyrimidine derivatives and in continuation of our pro-
gram on the chemistry of 4H-pyran derivatives (Al-Ghamdi
A. M. El-Agrody (&) ꢀ A. M. Fouda ꢀ A.-A. M. Al-Dies
Chemistry Department, Faculty of Science, King Khalid
University, P.O. Box 9004, Abha 61413, Saudi Arabia
e-mail: elagrody_am@yahoo.com
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Med Chem Res
Scheme 1 Synthesis of 4H-
benzo[h] chromene derivatives
(7, 8)
9
6
OH
10
8
7
1
O
O
NH2
a,b,c or d
10a
10b
e,f,g or h
NH2
CN
2
3
a
b
c
h
f
6a
g
e
MeO
CO2Et
d
MeO
4a
1
OMe
4
5
8
Ar
Ar = p-ClC6H4
a = ArCHO (2) / CH2(CN)2 (3) EtOH / pip. 1 hr reflux
Ar
7
b = ArCHO (2) / CH2(CN)2 (3) EtOH / pip. MW / 400 W / 2 min.
c = ArCH=C(CN)2 (4) / EtOH / pip. 1 hr reflux
d = ArCH=C(CN)2 (4) / EtOH / pip. MW / 400 W / 2 min.
e = ArCHO (2) / CNCH2 CO2 Et (5) EtOH / pip. 2 hr reflux
f = ArCHO (2) / CNCH2 CO2 Et (5) EtOH / pip. MW / 400 W / 2 min.
g = ArCH=C(CN)(CO2 Et) (6) / EtOH / pip. 2 hr reflux
h = ArCH=C(CN)(CO2 Et) (6) / EtOH / pip. MW / 400 W / 2 min.
Scheme 2 Synthesis
2-Benzylideneamino-4-(4-
chlorophenyl)-6-methoxy-4H-
benzo[h] chromene-3-
carbonitrile (9)
O
NH2
CN
O
N=CHPh
CN
PhCHO
MeO
MeO
Ar
7
9
Ar
NH2NH2.H2O or
PhNHNH2
O
N
Ph
H
O
NNHR
CHPh
N
MeO
NHR
7
N
-PhCH=NNHR
NH
11
Ar
H
MeO
CN
Ar = p-ClC6H4
10
Ar
et al., 2012; El-Agrody et al., 2011, 2012, 2013; El-Agrody
and Al-Ghamdi, 2011; Sabry et al., 2011; Abd-El-Aziz
et al., 2004, 2007), we report herein the synthesis of
4H-benzo[h]chromene and 7H-benzo[h]chromeno[2,3-d]pyrim-
idine derivatives and the evaluation of their antitumor
activities. The chemical structures of the studied compounds
and their structure–activity relationships (SAR) at 2-,3- and
2,3-positions are discussed in this work.
maximum power of Microwave irradiation was optimized
by carrying out the same reaction at different watt powers.
Microwave radiations at 400 W were chosen as the optimum
power, as the highest yield was obtained at this power.
Condensation of 7 with benzaldehyde in the ethanolic
piperidine solution under reflux gave 2-benzylideneamino-
4-(4-chlorophenyl)-6-methoxy-4H-benzo[h]chromene-3-
carbonitrile (9). When 9 was treated with hydrazine hydrate
or phenyl hydrazine in ethanol at room temperature or
under reflux, the addition product 10 was formed (R = H
or Ph, respectively). From the intermediate 10, benzalde-
hyde hydrazone or benzaldehyde phenyl hydrazone was
eliminated to give b-enaminonitrile 7 (Khafagy et al.,
2002) rather than the pyrimidine derivative 11. These
results are depicted in (Scheme 2).
Chemistry
Treatment of 4-methoxy-1-naphthol (1) with a mixture
of 4-chlorobenzaldehyde (2) and malononitrile (3) or
a-cyano-4-chlorocinnamonitrile (4) in ethanolic piperidine
solution under reflux for 1 h or under Microwave irradia-
tion conditions for 2 min at 140 °C gave 2-amino-4-(4-
chlorophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbo-
nitrile (7), while reaction of 1 with 2 and ethyl cyanoace-
tate (5) or ethyl a-cyano-4-chlorocinnamate (6) afforded
ethyl 2-amino-4-(4-chlorophenyl)-6-methoxy-4H-benzo[h]
chromene-3-carboxylate (8), respectively, (Scheme 1). The
reactions were controlled using TLC technique. The
Treatment of 7 with acetic anhydride under reflux for
1/2 or 6 h gave the same compound, 2-diacetylamino-4-(4-
chlorophenyl)-6-methoxy-4H-benzo[h]chromene-3-car-
bonitrile (12), while reaction of 7 with triethyl orthoformate
in acetic anhydride or dimethylformamide-dipentylacetal
(DMF-DPA) in benzene under reflux for 2 h afforded 4-(4-
chlorophenyl)-2-ethoxymethyleneamino-6-methoxy-4H-
123

Med Chem Res
O
N=CHOEt
CN
O
N(Ac)2
CN
O
NH2
CN
Ac2O / 1/2 or
6 hr reflux
HC(OEt)3
Ac2O
MeO
MeO
MeO
reflux
13
Ar
Ar
12
Ar
7
Me2NH / MeOH
stirring for 1 hr
NH3 gas / MeOH
stirring for 1 hr
DMF-DPA / benzene reflux
O
N=CHNMe2
O
N=CHNH2
CN
MeO
CN
MeO
14
Ar
Ar
15
Ar = p-ClC6H4
Scheme 3 Synthetic protocol of compounds (12–15)
O
N=CHNMe2
DMF-DPA
O
N=CHOEt
CO2Et
O
NH2
HC(OEt)3
MeO
CO2Et
benzene
reflux
Ac2O
reflux
MeO
MeO
CO2Et
18
Ar
Ar
16
8
Ar
H2O
Ar = p-ClC6H4
O
H
O
NHCH
OEt
O
NHCHO
-EtOH
MeO
CO2Et
MeO
CO2Et
Ar
Ar
17
Scheme 4 Synthetic protocol of compounds (17, 18)
benzo[h]chromene-3-carbonitrile (13) and 4-(4-chloro-
phenyl)-2-dimethylaminomethyleneamino-6-methoxy-
4H-benzo[h]chromene-3-carbonitrile (14), respectively,
(Scheme 3). Interaction of the imidate 13 with dimethyl-
amine in methanol at room temperature under stirring for
1 h yielded the imidine 14, which can be obtained as
described before from the reaction of 7 and DMF-DPA
(m.p., mixed m.p. and identical IR and MS spectrum),
while reaction of 13 with NH3 gas bubbled in methanol at
room temperature for 1 h yielded the open chain product
2-aminomethyleneamino-4-(4-chlorophenyl)-6-methoxy-
4H-benzo[h]chromene-3-carbonitrile 15 (Scheme 3). The
reactions were controlled using TLC technique.
benzo[h]chromene-3-carboxylate (18). The formation of 17
can be rationalized through the initial product of addition of
H2O to ethoxymethyleneamino group (–N=CHOEt) of 16,
which lose ethanol to give 2-formamido derivatives 17. These
results are depicted in (Scheme 4).
Compounds 7 and 13 were subjected for further reac-
tions to produce fused heterotetracyclic systems incorpo-
rating pyrimidine nucleus at 2,3-positions in addition to
4H-chromene moiety. Thus, condensation of 7 with formic
acid under reflux gave 7-(4-chlorophenyl)-5-methoxy-
7H,9H-benzo[h]chromeno[2,3-d]pyrimidin-8-one (19), while
reaction of 7 with formamide under reflux was unsuccessful,
the 8-amino-7-(4-chlorophenyl)-5-methoxy-7H-benzo[h]chro-
meno[2,3-d]pyrimidine (20) was not formed. Compound 20
can be prepared by cyclization of 15 in ethanolic piperidine
solution under reflux (Khafagy et al., 2002). These results
are depicted in (Scheme 5). The reactions were controlled
using TLC technique.
In a similar manner, reaction of 8 with triethyl orthofor-
mate gave the ethyl 4-(4-chlorophenyl)-2-formamido-6-
methoxy-4H-benzo[h]chromene-3-carboxylate (17), instead
of ethyl 4-(4-chlorophenyl)-2-ethoxymethyleneamino-6-
methoxy-4H-benzo[h]chromene-3-carboxylate (16), while
condensation of 8 with DMF-DPA give the ethyl 4-(4-chlo-
rophenyl)-2-dimethylaminomethyleneamino-6-methoxy-4H-
Reaction of the imidate 13 with methylamine in ethanol
at room temperature under stirring for 1 h gave the open
123

Med Chem Res
2
Scheme 5 Synthetic protocol
of compounds (19, 20)
1
3
4
1
O
O
N
1a
1b
2
N
O
NH2
CN
10
HCONH2
reflux
a
c
b
d
HCO2H
reflux
h
4a
5
g
a
b
N
f
e
d
c
NH
MeO
6a
MeO
3
9
8
MeO
7
6
NH2
Ar
20
O
Ar
Ar
19
7
EtOH / pip. reflux
Ar = p -ClC6 H4
O
N=CHNH2
MeO
CN
15 Ar
O
N=CHOEt
O
N
NH2NH2.H2O
N
EtOH stirring
for 1hr
MeO
13
CN
MeO
NH2
NH
Ar
Ar
MeNH2
23
EtOH
stirring for 1h
PHCHO / EtOH
pip. reflux
O
N=CHNHMe
CN
O
N
N
O
N
MeO
N
MeO
N=CHPh
MeO
Me
21
Ar
NH
24
Ar
NH
Ar
22
Ar = p -ClC6H4
Scheme 6 Synthetic protocol of compounds (21, 23, 24)
chain product 2-methylaminomethyleneamino-4-(4-chlo-
rophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile
(21), instead of cycloaddition product 7-(4-chlorophenyl)-
5-methoxy-8-imino-9-methyl-7H-benzo[h]chromeno[2,3-
d]pyrimidine (22), while reaction of 13 with hydrazine
hydrate afforded the cycloaddition product 9-amine-7-(4-
chlorophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno
[2,3-d]pyrimidine (23). Condensation of the imino com-
pound 23 with benzaldehyde in ethanolic piperidine solu-
tion under reflux gave the open chain product 9-benzy-
lideneamino-7-(4-chlorophenyl)-5-methoxy-8-imino-7H-
benzo[h]chromeno[2,3-d]pyrimidine (24). These results are
depicted in (Scheme 6). The reactions were controlled
using TLC technique.
carcinoma (HCT), and hepatocellular carcinoma (HepG-2).
The measurements of cell growth and the viabilities were
determined as described in the literature (Rahman et al.,
2001). In vitro cytotoxicity evaluation using viability assay
was performed at the Regional Center for Mycology &
Biotechnology (RCMP), Al-Azhar University using Vin-
blastine, Colchicine, and Doxorubicin as standard drugs. All
the standard drugs and all new synthesized compounds
(controls) were tested at the same concentrations, to see the
effectiveness action for these new synthesized compounds in
comparing with control drugs. The inhibitory activity of the
synthetic compounds 7–9, 12–15, 17–21, 23, and 24 against
the three cell lines MCF-7, HCT, and HepG-2 is given in
Table 3 which included in supporting information (Supple-
mentary material 27) and Fig. 1.
The structures of 9, 12–15, 17–21, 23, and 24 were
established on the basis of IR, 1H NMR, 13C NMR, and MS
data.
Results and discussion
Antitumor assays
4H-Benzo[h]chromene derivatives were selected for this
study as their families are well known to contain active
compounds with a wide range of biological and pharmaco-
logical activities (Kidwai et al., 2010; Alvey et al., 2009;
Compounds 7–9, 12–15, 17–21, 23, and 24 were evaluated
for human tumor cell growth inhibitory activity against three
different cell lines: breast adenocarcinoma (MCF-7), lung
123

Med Chem Res
120
100
80
60
40
20
0
Fig. 1 IC50 values expressed in
lM of 4H-benzo[h]chromene
and 7H-benzo[h]chromeno[2,3-
d]pyrimidine derivatives against
MCF-7, HCT, and HepG-2
tumor cells
MCF-7
HCT
HEPG-2
Compound
Vosooghi et al., 2010; Singh et al., 2010; Vukovic et al.,
2010; Sabry et al., 2011; Kemnitzer et al., 2008; Mahmoodi
et al., 2010; Endo et al., 2010; Tseng et al., 2010; El-Sayed
and Ibrahim 2010; Keri et al., 2010; Sashidhara et al., 2011).
In the present study, several of 4H-benzo[h]chromenes
and 7H-benzo[h]chromeno[2,3-d]pyrimidines were pre-
pared. Structures of the synthesized compounds were elu-
cidated on the basis of IR, 1H NMR, 13C NMR, 13C NMR-
DEPT, and MS data. Compounds 7–9, 12–15, 17–21, 23, and
24 were tested against three different tumor cell lines: MCF-
7, HCT, and HepG-2. The cytotoxicity evaluation using
viability assays and inhibitory activities is given in Supple-
mentary Table 3 and Fig. 1. The results from Supplementary
Table 3 indicated that compounds 23, 15, and 20 were the
most active against MCF-7, compounds 21 and 15 were the
most active against HCT and compounds 23, 15, 20, 21, and
8 were the most active against HepG-2 as compared with the
standard drug Vinblastine, while compounds 23, 15, 20, 21,
9, 13, 24, 8, and 19 were the most active against MCF-7,
compounds 21, 15, 20, 23, 13, 8, 19, 12, 9, 17, 18, 7, and 24
were the most active against HCT, and compounds 23, 15,
20, 21, 8, 9, 19, and 13 were the most active against HepG-2
as compared with the standard drug Colchicine. In addition,
compound 23 was the most active against HepG-2 as com-
pared with the standard drug Doxorubicin and the remaining
compounds exhibited moderate to lower activities as com-
pared with the standard drugs Vinblastine, Colchicine, and
Doxorubicin.
explored. The SAR studies of 7 and its analogs revealed that
compound 15 have the highest significant potent antitumor
activity (IC50 = 4.6 lM) against MCF-7 as shown in
Table 1, compounds 21, 9, 13 showed antitumor activities
(IC50 = 9.4–12.7 lM) near to the standard drug Vinblastine
(IC50 = 7.5 lM), while compounds 15, 21, 9, and 13 have
the highest significant potent antitumor activities (IC50
=
4.6–12.7 lM) against MCF-7, Table 1 as compared to the
standard drug Colchicine (IC50 = 44.3 lM), and the other
compounds 12 and 14 (IC50 = 101.8–108.4 lM) are con-
sidered to be inactive. These data indicate that the activity of
compounds 15, 21, 9, and 13 was considerably attributed to
the presence of the hydrophobic groups (–N=CHNH2-2,
–N=CHNHMe-2, –N=CHPh-2 and –N=CHOEt-2) in 4H-
benzo[h]chromene moiety at position C-2 and the hydro-
phobic groups is preferred at position C-2. Blocking of the
(–NH2-2) group with other hydrophobic groups such as
(–NAc2-2 and N=CHNMe2-2) in compounds 12 and 14
resulted in reduction of potency. Replacing the cyano-3 with
ester-3 group (hydrophobic group) resulted in strong
improvement of potency for compound 8 (IC50 = 21.5 lM)
as compared to the compound 7 (IC50 = 106.9 lM), while
blocking of the (–NH2-2) group for compound 8 with
hydrophilic groups (–NHCHO-2) or hydrophobic group
(–N=CHNMe2-2) resulted in reduction of potency of the
compounds 17 and 18 (IC50 = 48.1–51.9 lM), suggesting
that the small hydrophobic group (–NH2-2) is preferred over
the other groups and the hydrophilic group (ester-3) is pre-
ferred over an cyano-3 group. Incorporating a pyrimidine
nucleus at 2, 3-positions with hydrophobic groups (=NH-8,
–NH2-9) for compound 23 and (–NH2-8) for compound 20
SAR studies
The cytotoxicity activity (IC50) of compounds 7, 8, and their
analogs against the three different cancer cell lines is sum-
marized in Supplementary Table 3. The SAR studies at the
2-,3- and 2,3-positions and the relationship between lipo-
philicity (hydrophilic or hydrophobic groups) and SAR were
resulted in strong improvement of potency (IC50 =
3.0–7.7 lg/ml) as compared to the standard drugs Vinblas-
tine (IC50 = 7.5 lM) and Colchicine (IC50 = 44.3 lM)
with the highest significant potent antitumor activity as
shown in Table 1 and compound 24 (=NH-8, –N=CHPh-9)
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Med Chem Res
Table 1 Positive and negative controls and the effectiveness of the
test compounds against MCF-7, HCT, and HEPG-2
Table 2 Positive and negative controls and the effectiveness of the
test compounds against HCT, HEPG-2
Control/compound IC50 (lM) Cell line F ratio
p value
Compound
IC50 (lM)
Cell line
Vinblastine
7.5a
3.0b
4.6c
44.3a
3.0b
4.6c
7.7d
9.4e
10.9f
12.7g
16.5h
21.5i
29j
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
HCT
44.113 0.000 (HS)
Vinblastine
21
3.2
HCT
HCT
23
3.0
15
T test
3.873
0.018 (S)
5.7
Colchicine
1,060.418 0.000 (HS)
p value
Vinblastine
15
23
HEPG-2
HEPG-2
15
3.3
20
T test
45.088
0.000 (HS)
1.7
21
p value
Doxorubicin
23
9
HEPG-2
HEPG-2
13
1.7
24
T test
0.000
1.000 (NS)
8
p value
19
Positive control (active compounds) and negative control (standard
drugs)
Colchicine
107.2a
3.0b
8,196.746 0.000 (HS)
21
HCT
S significant, HS highest significant, NS not significant
15
3.6c
HCT
suggesting that an hydrophobic group is preferred over an
hydrophilic group and the pyrimidine moiety is preferred at
2,3-positions.
20
5.7d
HCT
23
6.2ed
14.1f
17.8g
19.7h
21.3f
21.3i
51.9j
59.3k
63.0l
80.7m
26.5a
1.7b
3.3c
7.2d
7.4e
9.3f
12.0g
17.9h
24.0i
25.4j
HCT
13
HCT
In the case of HCT, investigation of SAR revealed that
compound 21 (IC50 = 3.0 lM) has the most potent with
significant activity against HCT as compared to compound 7
and its analogs and the standard drug Vinblastine
(IC50 = 3.2 lM) observed in Table 2, while compound 15
showed antitumor activity (IC50 = 3.6 lM) near to the
standard drug Vinblastine. This potency could be attributed
to the presence of the hydrophobic groups (–N=CHNHMe-2
and –N=CHNH2-2) at the 2-position, while the blocking of
the (–NH2-2) group with other groups such as (–N=CHOEt-
2, –NAc2-2 and –N=CHPh-2) with cyano-3 group in com-
pounds 13, 12, and 9 (IC50 = 14.1–21.3 lM) resulted in a
reduction of potency or with the hydrophobic group
(–N=CHNHMe-2) or hydrophilic group (–NHCHO-2) with
ester-3 group in compounds 17 and 18 (IC50 = 51.9–
59.3 lM) resulted in more reduction of potency, suggesting
that the hydrophobic groups (–N=CHNHMe-2 and –N=
CHNH2-2) at position C-2 are preferred over the other
groups. Incorporating a pyrimidine nucleus at 2, 3-positions
with hydrophobic groups (–NH2-8) for 20 and (=NH-8,
–NH2-9) for 23 resulted in strong improvement of potency
(IC50 = 5.7–6.2 lM) as compared to the standard drug
Vinblastine (IC50 = 3.2 lM), while the presence of the
8
HCT
19
HCT
12
HCT
9
HCT
17
HCT
18
HCT
7
HCT
24
HCT
Colchicine
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
HEPG-2
872.398 0.000 (HS)
23
15
20
21
8
9
19
24
13
Positive control (active compounds) and negative control (standard
drugs). Same letters in column do not differ statistically (ANOVA),
medium evaluated with LSD test (a = 0.05)
HS highest significant
hydrophilic group (–C=O-8) for compound 19 (IC50
=
19.7 lM) resulted in a reduction of potency and more
reduction of potency (IC50 = 80.7 lM) for compound 24
(–N=CHPh-9), suggesting that an hydrophobic group is
preferred over an hydrophilic group and the pyrimidine
moiety is preferred at 2, 3-positions. In addition, compounds
21, 15, 13, 8, 12, 9, 17, 18, and 7 (IC50 = 3.0–63.0 lM),
showed moderated activity (IC50 = 16.5 M) as compared to
the standard drug Vinblastine and was active as compared to
the standard drug Colchicine, while the presence of hydro-
philic group (–C=O-8) for compound 19 (IC50 = 29.0 lM)
resulted in a little reduction of potency with the highest
significant potent antitumor activity as shown in Table 1,
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Med Chem Res
respectively, have the most potent highest significant activity
against HCT as compared to the standard drug Colchicine
(IC50 = 107.2 lM) as shown in Table 1, while the pyrimi-
dine compounds 20, 23, and 19 (IC50 = 5.7–19.7 lM) resul-
ted in strong improvement of potency with the highest
significant activity as shown in Table 1 and compound 24 has
less potency with the highest significant as shown in Table 1
(IC50 = 80.7 lM) than the standard drug Colchicine.
Furthermore, compounds 15 (IC50 = 3.3 lM) showed
the highest significant antitumor activity, Table 2 against
HepG-2 as compared to compound 7 (IC50 = 78.5 lM) and
activity as shown in Table 2, this potency due to the
incorporating a pyrimidine nucleus at 2,3-positions with
the hydrophobic groups (=NH-8, –NH2-9) for compound
23, suggesting that hydrophobic group is preferred over the
hydrophilic group and the pyrimidine moiety is preferred at
2,3-positions. In addition, compounds 15, 20, 21, and 8
showed activities (IC50 = 3.3–9.3 lM) near to the standard
drug Doxorubicin and the other compounds showed more
reduction of potency (IC50 = 12.0–110.8 lM).
its analogs and the standard drug Vinblastine (IC50
=
Conclusions
5.7 lM), while compounds 21 and 8 showed antitumor
activities (IC50 = 7.4–9.3 lM) against HepG-2 near to the
standarddrugVinblastine. These dataindicatethattheactivity
of compounds 15, 21, and 8 was considerably attributed to
the presence of the hydrophobic groups (–N=CHNH2-2,
–N=CHNHMe-2 and –NH2-2) at the 2-position, suggesting
that the hydrophobic groups (–N=CHNHMe-2 and –N=
CHNH2-2) at position C-2 are preferred over the other groups,
while the blocking of the (–NH2-2) group with other hydro-
phobic groups such as (–N=CHPh-2, –N=CHOEt-2 and
In conclusions, several compounds of 4H-benzo[h]chrom-
enes and fused 4H-benzo[h]chromenes were prepared.
Compounds 23, 15, and 20 were the most active against
MCF-7, compounds 21 and 15 were the most active against
HCT and compounds 23, 15, 20, 21, and 8 were the most
active against HepG-2 as compared with the standard drug
Vinblastine, while compounds 23, 15, 20, 21, 9, 13, 24, 8,
and 19 were the most active against MCF-7, compounds
21, 15, 20, 23, 13, 8, 19, 12, 9, 17, 7, 18, and 24 against
HCT, compounds 23, 15, 20, 21, 8, 9, 19, and 13 were the
most active against HepG-2 as compared with the standard
drug Colchicine, and compound 23 was the most active
against HepG-2 as compared with the standard drug
Doxorubicin. In addition, we have explored the SAR study
of 4H-benzo[h]chromene and 7H-benzo[h]chromeno[2,3-
d]pyrimidine compounds as antitumor agents via modifi-
cation at the 2-,3- and 2,3-positions, and it was found that
there might be a size limited pocket at positions C-2, C-3,
the hydrophobic group is preferred over the hydrophilic
group, and the 7H-benzo[h]chromeno[2,3-d]pyrimidine
moiety is preferred over the 4H-benzo[h]chromene moiety.
Further investigations are essential to gain deeper insight
into structure–activity aspects and to predict the optimal
structural parameters, which could be beneficial in devel-
opment of antitumor therapeutics.
–NAc2-2) with cyano-3 in compounds 9, 13, and 12 (IC50
=
12.0–27.8 lM) resulted in a reduction of potency, and the
other compounds 18, 17, and 14 showed more reduction of
potency (IC50 = 37.3–110.8 lM). In addition, compounds
15, 21, 8, 9, and 13 (IC50 = 3.3–25.4 lM)showedthe highest
significant antitumor activities against HepG-2 as compared
to compound 7 (IC50 = 78.5 lM) as shown in Table 1 and its
analogs and the standard drug Colchicine (IC50 = 26.5 lM).
This is due to the presence of the hydrophobic groups
(–N=CHNH2-2, –N=CHNHMe-2, –NH2-2, –N=CHPh-2 and
–N=CHOEt-2), while compounds 12 and 18 showed a little
reduction of potency (IC50 = 27.8–37.3 lM)and compounds
17 and 14 are inactive (IC50 = 84.7–110.8 lM). Incorporat-
ing a pyrimidine nucleus at 2,3-positions with the hydropho-
bic groups (=NH-8, –NH2-9) for compound 23 and (–NH2-8)
for 20 resulted in strong improvement of potency with the
highest significant activities (IC50 = 1.7–7.2 lM), Tables 1,
2
as compared to the standard drugs Vinblastine
(IC50 = 5.7 lM) and Colchicine (IC50 = 26.5 lM), while
the other compounds 19 and 24 showed moderate activities
(IC50 = 17.9–24.0 lM) as compared to the standard drug
Vinblastineandcompound19and24 (IC50 = 17.9–24.0 lM)
showed the highest significant activities less than the standard
drug Colchicine (IC50 = 26.5 lM) as shown in Table 1,
suggesting that the hydrophobic group is preferred over an the
hydrophilic group and the pyrimidine moiety is preferred at
2,3-positions.
Experimental
Melting points were determined with a Stuart Scientific Co.
Ltd apparatus. IR spectra were determined as KBr pellets
1
on a Jasco FT/IR 460 plus spectrophotometer. H NMR
and 13C NMR spectra were recorded on a BRUKER AV
500/600 MHz spectrometer using tetramethylsilane (TMS)
as an internal reference and results are expressed as d
(ppm) values. 13C NMR spectra were obtained using dis-
tortionless enhancement by polarization transfer (DEPT),
with this technique, the signals of CH and CH3 carbon
atoms appear normal (up) and the signal of carbon atoms in
CH2 environments appears negative (down). The
Finally, in the case of HepG-2, an investigation of SAR
revealed that compound 23 (IC50 = 1.7 lM) has the same
potent activity against HepG-2 as compared to the standard
drug Doxorubicin (IC50 = 1.7 lM) with not significant
123

Med Chem Res
Microwave apparatus used is Milestone Sr1, Microsynth.
The MS was measured on a Shimadzu GC/MS-QP5050A
spectrometer. Elemental analyses were performed on a
Perkin-Elmer 240 microanalyser.
washed with cold ethanol, filtered, dried, and recrystallized
from ethanol to afford 12 as yellow powder; m.p.
162–163 °C; yield 73 %; IR (cm-1) in (KBr) t: 3,003,
2,942, 2,875 (CH), 2,219 (CN), 1,736 (CO); 1H NMR
(500 MHz, CDCl3) d (ppm): 8.25–6.22 (m, 9H, aromatic),
5.15 (s, 1H, H-4), 3.86 (s, 3H, OCH3), 2.58 (s, 6H,
2COCH3); 13C NMR (125 MHz, CDCl3) d (ppm): 170.7
(CO), 170.4 (CO), 153.7 (C-2), 152.7 (C-6), 138.0 (C-10b),
129.3 (C-10a), 128.0 (C-9), 126.6 (C-6a), 125.7 (C-8),
124.2 (C-7), 122.4 (C-10), 120.6 (C-4a), 115.2 (CN), 102.0
(C-5), 91.8 (C-3), 55.8 (CH3), 43.7 (C-4), 25.7 (CH3), 25.2
(CH3), 140.2, 134.3, 129.7, 126.7 (aromatic); MS m/z (%):
448 (M??2, 9.01), 446 (M?, 27.49) with a base peak at 75
(100); Anal. Calcd. for C25H19ClN2O4: C, 67.19; H, 4.29;
N, 6.27. Found: C, 67.21; H, 4.39; N, 6.28 %.
2-Amino-4-(4-chlorophenyl)-6-methoxy-4H-
benzo[h]chromene-3-carbonitrile (7)
Prepared as previously described (Al-Sehemi et al., 2012;
El-Agrody et al., 2014).
Ethyl 2-amino-4-(4-chlorophenyl)-6-methoxy-4H-
benzo[h]chromene-3-carboxyalte (8)
Prepared as previously described (Al-Sehemi et al., 2012;
El-Agrody et al., 2014).
General procedure for the preparation of 13 and 17
2-Benzylideneamino-4-(4-chlorophenyl)-6-methoxy-
4H-benzo[h]chromene-3-carbonitrile (9)
A mixture of 7 or ethyl 2-amino-4-(4-chlorophenyl)-6-
methoxy-4H-benzo[h]chromene-3-carboxylate (8) (0.01 mol)
with triethyl orthoformate (0.01 mol) and acetic anhydride
(30 ml) was refluxed for 2 h. The solvent was removed under
reduced pressure and the resulting solid was washed with
methanol and recrystallized from proper solvent to give 13
and 17. The physical data of the compounds 13 and 17 are as
follows:
A mixture of 2-amino-4-(4-chlorophenyl)-6-methoxy-4H-
benzo[h]chromene-3-carbonitrile (7) (0.01 mol) with benz-
aldehyde (0.01 mol) and piperidine (0.5 ml) in ethanol
(20 ml) was refluxed for 2 h. The solid product which
formed was filtered, washed with cold methanol, dried, and
recrystallized from ethanol to afford 9 as yellow needles;
m.p. 235–236 °C; yield 70 %; IR (cm-1) in (KBr) t: 3,071,
1
3,029, 2,962, 2,937, 2,853 (CH), 2,211 (CN); H NMR
4-(4-Chlorophenyl)-2-ethoxymethyleneamino-6-methoxy-
(600 MHz, CDCl3) d (ppm): 9.40 (s, 1H, N=CH) 8.52–6.53
(m, 14H, aromatic), 5.34 (s, 1H, H-4), 3.84 (s, 3H, OCH3);
13C NMR (150 MHz, CDCl3) d (ppm): 163.7 (N=CH),
160.7 (C-2), 151.9 (C-6), 136.9 (C-10b), 127.7 (C-10a),
126.7, (C-9), 124.7 (C-8), 124.0 (C-6a), 121.6 (C-7), 121.6
(C-10), 120.3 (C-4a), 116.1 (CN), 103.1 (C-5), 87.0 (C-3),
55.8 (CH3), 42.2 (C-4), 139.6, 134.8, 133.5, 132.8, 130.1,
129.3, 129.1, 177.8 (aromatic); MS m/z (%): 452 (M??2,
12.99), 450 (M?, 26.98) with a base peak at 340; Anal.
Calcd. for C28H19ClN2O2: C, 74.58; H, 4.25; N, 6.21.
Found: C, 74.60; H, 4.34; N, 6.30 %.
4H-benzo[h]chromene-3-carbonitrile (13)
Yellow needles from benzene; m.p. 192–193 °C; yield
78 %; IR (cm-1) in (KBr) t: 3,079, 3,054, 2,984, 2,958,
2,941, 2,900, 2,857 (CH), 2,210 (CN); 1H NMR (500 MHz,
CDCl3) d (ppm): 8.51 (s, 1H, N=CH), 8.19–6.11 (m, 9H,
aromatic), 4.84 (s, 1H, H-4), 4.40 (q, 2H, CH2, J = 7.25 Hz),
3.76 (s, 3H, OCH3), 1.34 (t, 3H, CH3, J = 7.25 Hz); 13C
NMR (125 MHz, CDCl3) d (ppm): 159.3 (N=CH), 157.3
(C-2), 152.7 (C-6), 137.8 (C-10b), 129.2 (C-9), 127.5 (C-8),
125.7 (C-10a), 124.5 (C-6a), 122.4 (C-7), 120.5 (C-10),
118.2 (C-4a), 115.3 (CN), 102.6 (C-5), 80.5 (C-3), 64.3
(CH2), 55.7 (CH3), 43.3 (C-4), 14.0 (CH3), 141.8, 133.6,
129.7, 126.4 (aromatic); 13C NMR-DEPT 135° CH, CH3 (:),
CH2 (;) (125 MHz, CDCl3) d (ppm): 159.3 (N=CH :), 129.7
(aromatic :), 129.2 (C-9 :), 127.5 (C-8 :), 126.4 (aromatic
:), 122.4 (C-7 :), 120.5 (C-10 :), 102.6 (C-5 :), 64.3 (CH2 ;),
55.7 (CH3 :), 43.3 (C-4 :), 14.0 (CH3 :); 13C NMR-DEPT
90° CH (:) (125 MHz, CDCl3) d (ppm): 159.3 (N=CH :),
129.7 (aromatic :), 129.2 (C-9 :), 127.5 (C-8 :), 126.4
(aromatic :) 122.4 (C-7 :), 120.5 (C-10 :), 102.6 (C-5 :),
43.3 (C-4 :); 13C NMR-DEPT 45° CH, CH2, CH3 (:)
(125 MHz, CDCl3) d (ppm): 159.3 (N=CH :), 129.7 (aro-
matic :), 129.2 (C-9 :), 127.5 (C-8 :), 126.4 (aromatic :)
122.4 (C-7 :), 120.5 (C-10 :), 102.6 (C-5 :), 64.3 (CH2 :),
Reaction of 9 with hydrazine derivatives
A mixture of 7 (0.01 mol) and hydrazine hydrate or phenyl
hydrazine (0.01 mol) in EtOH was stirred at room tem-
perature or refluxed for 2 h to give 7 (m.p. and mixed m.p.
218–219 °C) yield (80 %).
2-Diacetylamino-4-(4-chlorophenyl)-6-methoxy-4H-
benzo[h]chromene-3-carbonitrile (12)
A solution of 7 (0.01 mol) in acetic anhydride (20 ml) was
refluxed for 1/2 or 6 h. The solvent was removed under
reduced pressure and the resulting solid was collected and
123

Med Chem Res
55.7 (CH3 :), 43.3 (C-4 :), 14.0 (CH3 :); MS m/z (%): 420
(M??2, 11.95), 418 (M?, 34.05) with a base peak at 251
(100); Anal. Calcd. for C24H19ClN2O3: C, 68.82; H, 4.57; N,
6.69. Found: C, 68.85; H, 4.60; N, 6.71 %.
(N=CH), 153.7 (C-2), 151.9 (C-6), 138.0 (C-10b), 129.4
(C-9), 127.1 (C-8), 125.5 (C-10a), 124.6 (C-6a), 122.3
(C-7), 120.6 (C-10), 120.5 (C-4a), 116.0 (CN), 103.0 (C-5),
73.6 (C-3), 55.6 (CH3), 43.3 (C-4), 41.2 (CH3), 143.0,
133.0, 129.0, 126.0 (aromatic); 13C NMR-DEPT 135° CH,
CH3 (:), CH2 (;) (125 MHz, CDCl3) d (ppm): 159.6
(N=CH :), 129.4 (C-9 :), 129.0 (aromatic :), 127.2 (C-8
:), 126.0 (aromatic :) 122.3 (C-7 :), 120.6 (C-10 :), 103.0
(C-5 :), 55.6 (CH3 :), 43.3 (C-4 :), 41.2 (CH3 :); 13C
NMR-DEPT 90° CH (:) (125 MHz, CDCl3) d (ppm):
159.6 (N=CH :), 129.4 (C-9 :), 129.0 (aromatic :), 127.1
(C-8 :), 126.0 (aromatic :), 122.3 (C-7 :), 120.6 (C-10 :),
103.0 (C-5 :), 43.3 (C-4 :); 13C NMR-DEPT 45° CH, CH2,
CH3 (:) (125 MHz, CDCl3) d (ppm): 159.6 (N=CH :),
129.4 (C-9 :), 129.0 (aromatic :), 127.1 (C-8 :), 126.0
(aromatic :), 122.3 (C-7 :), 120.6 (C-10 :), 103.0 (C-5 :),
55.6 (CH3 :), 43.3 (C-4 :), 41.2 (CH3 :); MS m/z (%): 419
(M??2, 30.12), 417 (M?, 88.76) with a base peak at 251
(100); Anal. Calcd. for C24H20ClN3O2: C, 68.98; H, 4.82;
N, 10.06. Found: C, 69.00; H, 4.85; N, 10.09 %.
Ethyl 4-(4-chlorophenyl)-2-formamido-6-methoxy-4H-
benzo[h]chromene-3-carboxylate (17)
Brown needles from benzene; m.p. 239–240 °C; yield
50 %; IR (cm-1) in (KBr) t: 3,282 (NH or OH), 3,069,
3,007, 2,945, 2,838 (CH), 1,722 (CO), 1,675(CO); 1H
NMR (600 MHz, DMSO-d6) d (ppm): 11.38 (s, 1H, CHO),
11.00 (bs, 1H, NH),8.52–6.30 (m, 9H, aromatic), 5.05 (s,
1H, H-4), 4.17 (q, 2H, CH2, J = 7.2 Hz), 3.89 (s, 3H,
OCH3), 1.26 (t, 3H, CH3, J = 7.2 Hz); 13C NMR
(150 MHz, DMSO-d6) d (ppm): 169.0 (CO), 167.3 (CO),
159.4 (C-2), 153.0 (C-6), 137.52 (C-10b), 132.6 (C-10a),
127.7 (C-9), 125.1 (C-6a), 126.3 (C-8), 122.4 (C-7), 121.8
(C-10), 120.2 (C-4a), 102.7 (C-5), 85.9 (C-3), 60.9 (CH2),
55.7 (CH3), 40.8 (C-4), 14.2 (CH3), 144.9, 136.7, 129.4,
128.7 (aromatic); 13C NMR-DEPT 135° CH, CH3 (:), CH2
(;) (125 MHz, CDCl3) d (ppm): 129.4 (aromatic :), 128.7
(aromatic :), 127.7 (C-9 :), 126.3 (C-8 :), 122.4 (C-7 :),
121.8 (C-10 :), 102.7 (C-5 :), 60.9 (CH2 ;), 55.7 (CH3 :),
40.8 (C-4 :), 14.2 (CH3 :); 13C NMR-DEPT 90° CH (:)
(125 MHz, CDCl3) d (ppm): 129.4 (aromatic :), 128.7
(aromatic :), 127.7 (C-9 :), 126.3 (C-8 :), 122.4 (C-7 :),
121.8 (C-10 :), 102.7 (C-5 :), 40.8 (C-4 :); 13C NMR-
DEPT 45° CH, CH2, CH3 (:) (125 MHz, CDCl3) d (ppm):
129.4 (aromatic :), 128.7 (aromatic :), 127.7 (C-9 :),
126.3 (C-8 :), 122.4 (C-7 :), 121.8 (C-10 :), 102.7 (C-5 :),
60.9 (CH2 :), 55.7 (CH3 :), 40.8 (C-4 :), 14.2 (CH3 :); MS
m/z (%): 439 (M??2, 3.58), 437 (M?, 11.78) with a base
peak at 298; Anal. Calcd. for C24H20ClNO5: C, C, 65.83;
H, 4.60; N, 3.20. Found: C, 65.79; H, 4.67; N, 3.26 %.
Ethyl 4-(4-chlorophenyl)-2-
dimethylaminomethyleneamino-6-methoxy-4H-
benzo[h] chromene-3-carboxylate (18)
Brown crystals from benzene; m.p. 185–186 °C; yield
67 %; IR (cm-1) in KBr) t: 3,085, 3,044, 3,004, 2,969,
2,929, 2,890, 2,808 (CH), 1,706 (CO); 1H NMR (500 MHz,
CDCl3) d (ppm): 8.18 (s, 1H, N=CH), 8.25–6.44 (m, 9H,
aromatic), 5.23 (s, 1H, H-4), 4.17 (q, 2H, CH2,
J = 7.5 Hz), 3.90 (s, 3H, OCH3), 3.22, 3.17 (2 s, 6H,
2CH3), 1.28 (t, 3H, CH3, J = 7.5 Hz); 13C NMR
(125 MHz, CDCl3) d (ppm): 167.9 (CO), 160.7 (N=CH),
155.6 (C-2), 151.9 (C-6), 138.5 (C-10b), 128.6 (C-10a),
128.4 (C-9), 128.2 (C-6a), 125.6 (C-8), 122.2 (C-7), 120.9
(C-10), 119.5 (C-4a), 103.3 (C-5), 91.4 (C-3), 59.7 (CH2),
55.7 (CH3), 43.4 (C-4), 40.8 (CH3), 34.6 (CH3), 14.4
(CH3), 145.9, 131.9, 129.5, 126.7(aromatic); 13C NMR-
DEPT 135° CH, CH3 (:), CH2 (;) (125 MHz, CDCl3) d
(ppm): 160.7 (N=CH :), 129.5 (aromatic :), 128.4 (C-9 :),
126.7 (aromatic :), 125.6 (C-8 :), 122.2 (C-7 :), 120.9
(C-10 :), 103.3 (C-5 :), 59.7 (CH2 ;), 55.7 (CH3 :), 43.4
(C-4 :), 40.8 (CH3 :), 34.9 (CH3 :), 14.4 (CH3 :); 13C
NMR-DEPT 90° CH (:) (125 MHz, CDCl3) d (ppm):
160.7 (N=CH :), 129.5 (aromatic :), 128.4 (C-9 :), 126.7
(aromatic :), 125.6 (C-8 :), 122.2 (C-7 :), 120.9 (C-10 :),
103.3 (C-5 :), 43.4 (C-4 :); 13C NMR-DEPT 45° CH, CH2,
CH3 (:) (125 MHz, CDCl3) d (ppm): 160.7 (N=CH :),
129.5 (aromatic :), 128.4 (C-9 :), 126.7 (aromatic :),
125.6 (C-8 :), 122.2 (C-7 :), 120.9 (C-10 :), 103.3 (C-5 :),
59.7 (CH2 :), 55.7 (CH3 :), 43.4 (C-4 :), 40.8 (CH3 :),
34.9 (CH3 :), 14.4 (CH3 :), MS m/z (%): 466 (M??2,
20.31), 464 (M?, 9.64) with a base peak at 75 (100); Anal.
General procedure for the preparation of 14 and 18
A mixture of 7 or 8 (0.01 mol) with DMF-DPA (0.01 mol)
and 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 14 and 18. The phys-
ical data of the compounds 14 and 18 are as follows:
4-(4-Chlorophenyl)-2-dimethylaminomethyleneamino-6-
methoxy-4H-benzo[h]chromene-3-carbonitrile (14)
Colorless crystals from benzene; m.p. 200–201 °C; yield
65 %; IR (cm-1) in (KBr) t: 3,079, 2,981, 2,968, 2,926,
1
2,813 (CH), 2,196 (CN); H NMR (500 MHz, CDCl3) d
(ppm): 8.38 (s, 1H, N=CH), 8.22–6.10 (m, 9H, aromatic),
4.71 (s, 1H, H-4), 3.81 (s, 3H, OCH3), 3.21, 3.15 (2 s, 6H,
2CH3); 13C NMR (125 MHz, CDCl3) d (ppm): 159.6
123

Med Chem Res
Calcd. for C26H25ClN2O4: C, 67.17; H, 5.42; N, 6.03.
Found: C, 67.10; H, 5.37; N, 6.00 %.
C23H18ClN3O2: C, 68.40; H, 4.49; N, 10.40. Found: C,
68.36; H, 4.46; N, 10.36 %.
7-(4-Chlorophenyl)-5-methoxy-7H,9H-
General procedure for the preparation of 14, 15 and 21
benzo[h]chromeno[2,3-d]pyrimidin-8-one (19)
A mixture of imidate 13 (0.01 mol), dimethylamine, and
NH3 gas or methylamine in methanol or ethanol (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 proper solvent to afford
14, 15, and 21. The physical data of the compounds 14, 15,
and 21 are as follows:
A mixture of 7 (0.01 mol) and formic acid (30 ml) was
refluxed for 3–5 h. The solvent was removed under
reduced pressure and the resulting solid was recrystallized
from ethanol to give 19 as yellow crystals from ethanol;
m.p. 196–197 °C; yield 63 %; IR (cm-1) in (KBr) t: 3,503
(NH), 3,071, 3,013, 2,958, 2,939, 2,892 (CH), 1,762 (CO);
1H NMR (500 MHz, CDCl3) d (ppm): 8.30–6.18 (m, 10H,
aromatic, H-10), 8.02 (bs, 1H, NH, canceled by D2O), 4.71
(s, 1H, H-7), 3.91 (s, 3H, OCH3); 13C NMR (125 MHz,
CDCl3) d (ppm): 160.0 (CO), 159.8 (C-11a), 153.4 (C-5),
153.1 (C-10), 135.1 (C-1b), 128.9 (C-1a), 128.1 (C-4a),
127.2 (C-2), 124.3 (C-3), 122.4 (C-7), 122.1 (C-1), 120.9
(C-6a), 113.8 (C-6), 101.7 (C-7a), 55.9 (CH3), 41.2 (C-7),
140.0, 135.0, 129.8, 126.1 (aromatic); MS m/z (%): 392
(M??2, 38.49), 390 (M?, 12.20) with a base peak at 67
(100); Anal. Calcd. for C22H15ClN2O3: C, 67.61; H, 3.87;
N, 7.17. Found: C, 67.57; H, 3.78; N, 7.06 %.
4-(4-Chlorophenyl)-2-dimethylaminomethyleneamino-6-
methoxy-4H-benzo[h]chromene-3-carbonitrile (14)
Described before.
2-Aminomethyleneamino-4-(4-chlorophenyl)-6-methoxy-
4H-benzo[h]chromene-3-carbonitrile (15)
Colorless needles from ethanol; m.p. 235–236 °C; yield
91 %; IR (cm-1) in (KBr) t: 3,444, 3,367, 3,182 (NH2),
3,012, 2,954, 2,939 (CH), 2,204 (CN); 1H NMR (600 MHz,
DMSO-d6) d (ppm): 8.63–6.52 (m, 9H, aromatic), 8.26 (s,
1H, N=CH), 7.77 (s, 2H, NH2), 5.02 (s, 1H, H-4), 3.82 (s,
3H, OCH3); 13C NMR (150 MHz, DMSO-d6) d (ppm):
161.7 (N=CH), 155.0 (C-2), 151.3 (C-6), 137.2 (C-10b),
128.3 (C-10a), 127.4 (C-9), 126.3(C-6a), 124.5 (C-8),
124.0 (C-7), 121.7 (C-10), 120.8 (C-4a), 115.6 (CN), 103.3
(C-5), 72.1 (C-3), 55.7 (CH3), 39.9 (C-4), 141.1, 132.5,
129.7, 128.0 (aromatic); MS m/z (%): 391(M??2, 20.31),
389 (M?, 16.64) with a base peak at 279; Anal. Calcd. for
C22H16ClN3O2: C, 67.78; H, 4.14; N, 10.78. Found: C,
67.81; H, 4.17; N, 10.80 %.
8-Amino-7-(4-chlorophenyl)-5-methoxy-7H-
benzo[h]chromeno[2,3-d]pyrimidine (20)
A mixture of 2-aminomethyleneamino-4-(4-chlorophenyl)-
6-methoxy-4H-benzo[h]chromene-3-carbonitrile (15) (0.01 mol)
in ethanol (30 ml) and piperidine (0.5 ml) was heated under
reflux for 2 h. The solid product formed was collected by
filtration, washed with methanol, and recrystallized from
ethanol to give 15 as colorless needles; m.p. 250–251 °C;
yield 68 %; IR (cm-1) in (KBr) t: 3,470, 3,414, 3,294
(NH2), 3,006, 2,994, 2,868 (CH); 1H NMR (600 MHz,
DMSO-d6) d (ppm): 8.27-6.60 (m, 9H, aromatic), 8.18 (s,
1H, H-10), 6.90 (s, 2H, NH2), 5.38 (s, 1H, H-7), 3.90 (s, 3H,
OCH3); 13C NMR (150 MHz, DMSO-d6) d (ppm): 162.7
(C-8), 162.3 (C-11a), 156.7 (C-10), 151.4 (C-5), 143.0
(C-1b), 127.5 (C-1a), 126.3 (C-2), 124.5 (C-4a), 124.2 (C-3),
121.7 (C-7), 120.7 (C-1), 118.7 (C-6a), 103.2 (C-6), 95.2
(C-7a), 55.8 (CH3), 38.2 (C-7), 137.7, 131.6, 129.8, 128.6
(aromatic); Anal. Calcd. for C22H16ClN3O2: C, 67.78; H,
4.14; N, 10.78. Found: C, 67.80; H, 4.17; N, 10.80 %.
2-Methylaminomethyleneamino-4-(4-chlorophenyl)-6-
methoxy-4H-benzo[h]chromene-3-carbonitrile (21)
Pale yellow crystals from ethanol/benzene; m.p.
230–231 °C; yield 66 %; IR (cm-1) in (KBr) t: 3,302
(NH), 3,074, 3,004, 2,954, 2,895, 2,819 (CH), 2,202 (CN);
1H NMR (500 MHz, CDCl3) d (ppm): 8.66 (s, 1H, N=CH),
8.22–6.52 (m, 9H, aromatic), 5.80 (s, 1H, NH), 4.89 (s, 1H,
H-4), 3.81 (s, 3H, OCH3), 3.12 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3) d (ppm): 159.4 (N=CH), 156.8 (C-2),
151.1 (C-6), 136.8 (C-10b), 129.4 (C-9), 127.3 (C-8), 124.4
(C-10a), 123.7 (C-6a), 121.5 (C-7), 120.6 (C-10), 117.6
(C-4a), 115.5 (CN), 103.3 (C-5), 85.0 (C-3), 56.0 (CH3),
40.7 (C-4), 30.3 (CH3), 141.7, 131.7, 129.4, 126.2 (aro-
matic); MS0 m/z (%): 405 (M??2, 10.19), 403 (M?,
31.01) with a base peak at 75 (100); Anal. Calcd. for
9-Amine-7-(4-chlorophenyl)-5-methoxy-8-imino-7H-
benzo[h]chromeno[2,3-d]pyrimidine (23)
A mixture of imidate 13 (0.01 mol) and hydrazine hydrate
(0.01 mol) in ethanol (30 ml) was stirred at room temper-
ature for 1 h. The solid product was collected by filtration,
washed with methanol, and recrystallized from benzene to
give 23 as colorless crystals; m.p. 233–234 °C; yield 75 %;
123

Med Chem Res
IR (cm-1) in (KBr) t: 3,329, 3,272 (NH and NH2), 3,062,
3,011, 2,953, 2,862, 2,828 (CH); 1H NMR (500 MHz,
DMSO-d6) d (ppm): 8.21-6.63 (m, 9H, aromatic), 8.16 (s,
1H, H-10), 6.66 (bs, 1H, NH, canceled by D2O), 5.72 (bs,
2H, NH2, canceled by D2O), 5.33 (s, 1H, H-7), 3.85 (s, 3H,
containing different concentrations of the test sample was
added after 24 h of seeding. Serial twofold dilutions of the
metabolites were added confluent cell monolayer. The
microtitre plates were incubated at 37 °C in a humidified
incubator with 5 % CO2 for a period of 48 h. Three wells
were used for each concentration of the test sample. Con-
trol cells were incubated without the test sample and with
or without DMSO. The little percentage of DMSO present
in the wells (maximal 0.1 %) was found not to affect the
experiment. After incubation of the cells for 24 h at 37 °C,
various concentrations of sample were added, and the
incubation was continued for 48 h and viable cells yield
was determined by a colorimetric MTT method.
13
OCH3); C NMR (125 MHz, DMSO-d6) d (ppm): 155.9
(C-8), 151.4 (C-11a), 150.7 (C-5), 137.5 (C-10), 131.3
(C-1b), 127.3 (C-1a), 126.2 (C-2), 125.1 (C-4a), 124.4
(C-3), 121.6 (C-4), 120.7 (C-1), 117.9 (C-6a), 103.5 (C-6),
97.8 (C-7a), 55.7 (CH3), 40.0 (C-7), 143.6, 129.8, 128.4,
124.0 (aromatic); MS m/z (%): 406 (M??2, 5.9), 404 (M?,
17.52) with a base peak at 76; Anal. Calcd. for
C22H17ClN4O2: C, 65.27; H, 4.23; N, 13.84. Found: C,
65.30; H, 4.30; N, 13.90 %.
In brief, after the end of the incubation period, crystal
violet solution (1 %) was added to each well for 30 min.
The stain was removed and the plates were rinsed using tap
water until all excess stain is removed. Glacial acetic acid
was then added to all wells and mixed thoroughly, and the
plates were read on ELISA reader, using a test wavelength
of 490 nm. Treated samples were compared with the
control in the absence of the tested samples. All experi-
ments were carried out in triplicate and all the new syn-
thesized compounds are pure samples with purity between
95 and 100 %. The cytotoxic effect of each tested com-
pound was calculated.
9-Benzylideneamino-7-(4-chlorophenyl)-5-methoxy-8-
imino-7H-benzo[h]chromeno[2,3-d]pyrimidine (24)
A mixture of imino compound 23 (0.01 mol) and benzal-
dehyde (0.01 mol) in ethanol (30 ml) and piperidine
(0.5 ml) was refluxed for 2 h. The solvent was removed
under reduced pressure and the resulting solid was re-
crystallized from benzene to give the open chain product
24 as colorless powder; m.p. 260–261 °C; yield 98 %; IR
(cm-1) in (KBr) t: 3,214 (NH), 3,065, 3,010, 2,971, 2,860
(CH stretching), 1,629 (C=N); MS m/z (%): 494 (M??2,
2.07), 492 (M?, 5.29) with a base peak at 385 (100); Anal.
Calcd. for C29H21ClN4O2: C, 70.66; H, 4.29; N, 11.37.
Found: C, 70.70; H, 4.33; N, 11.42 %.
Statistical analysis
All statistical calculations were done using computer pro-
grams, Microsoft excel version 10, SPSS (statistical package
for the social science version 20.00) statistical program at
0.05, 0.01, and 0.001 level of probability (Snedecor and
Cochran, 1982). Comparisons of inhibiting tumor growth
between treatment groups or the control were done using
Student’s T test, One-way ANOVA, and Post hoc-LSD tests
(the least significant difference) measurement.
Antitumor screening
Cell culture
MCF-7, HCT, and HepG-2 cells were grown on RPMI-1640
medium supplemented with 10 % inactivated fetal calf
serum and 50 lg/ml gentamycin. Vero cells were propa-
gated in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10 % heat-inactivated fetal calf serum,
1 % L-glutamine, HEPES buffer, and 105 lM gentamycin.
All cells were maintained at 37 °C in a humidified atmo-
sphere with 5 % CO2 and were subcultures two to three times
a week.
Acknowledgments This research was supported by a program to
support research and researchers at King Khalid University, Abha,
Saudi Arabia and No. KKU-SCI-11-028. The authors deeply thank
the Regional Center for Mycology & Biotechnology (RCMP),
Al-Azhar University for carrying out the antitumor study, and Mr. Ali
1
Y. A. Alshahrani for making the H NMR and 13C NMR spectra.
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