Synthesis, antitumor activity of 2-amino-4H-benzo[h]chromene derivatives, and structure-activity relationships of the 3- and 4-positions

Article abstract of DOI:10.1007/s00044-013-0602-8

Several 2-amino-4H-benzo[h]chromenes (3a-i) and (5a-h) were obtained by reaction of 4-chloro-1-naphthol (1) with α-cyanocinnamonitrile (2a-i) or ethyl α-cyanocinnamate derivatives (4a-h), respectively. Structures of these compounds were established on the

Full text of DOI:10.1007/s00044-013-0602-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0602-8
ORIGINAL RESEARCH
Synthesis, antitumor activity of 2-amino-4H-benzo[h]chromene
derivatives, and structure–activity relationships of the 3- and
4-positions
•
Ahmed M. El-Agrody Ahmed M. Fouda
•
Essam Shawky A. E. H. Khattab
Received: 5 February 2013 / Accepted: 25 April 2013
Ó Springer Science+Business Media New York 2013
Abstract Several 2-amino-4H-benzo[h]chromenes (3a–i)
and (5a–h) were obtained by reaction of 4-chloro-1-naphthol
(1) with a-cyanocinnamonitrile (2a–i) or ethyl a-cyanocin-
namate derivatives (4a–h), respectively. Structures of these
compounds were established on the basis of spectral data. The
antitumor activity of the synthesized compounds was inves-
tigated in comparison with Vinblastine, Colchicine, and
Doxorubicin well-known anticancer drugs, using MTT col-
orimetric assay. Among them, the compounds 5e, 3c, 5f, b, d,
3d, 5c, a were the most active against MCF-7, 5a against
HCT-116 and 5a, 3e, a against HepG-2 as compared with the
standard drug Vinblastine, while the compounds 5e, 3c, 5f, b,
d, 3d, 5c, a, h, 3i, g, a, e were the most active against MCF-7,
5a, c, e, f, b, 3e, c, g, b, 5d, h, 3d, i, 5g against HCT-116, 5a,
3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g, 5h against HepG-2 as
compared with the standard drug Colchicine. The structure–
activity relationships of the 3- and 4-positions were discussed.
important biological activities. During the last decade, such
compounds had shown interesting pharmacological prop-
erties including antimicrobial (Kidwai et al., 2010; Alvey
et al., 2009; Kumar et al., 2009; Raj et al., 2009), antile-
ishmanial (Tanaka et al., 2007), anticancer (Sabry et al.,
2011; Rampa et al., 2005), antioxidant (Singh et al., 2010;
Vukovic et al., 2010), antiproliferative (Magedov et al.,
2007), antitumor (Mahmoodi et al., 2010; Endo et al.,
2010; Tseng et al., 2010) effects and activities, as well as
treatment of alzheimer’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).
They also exhibit hypolipidemic activity (Sashidhara
et al., 2011), DNA breaking activities and mutagenicity
(Hiramoto et al., 1997).
As a result of remarkable pharmacological efficiency of
4H-chromene and 4H-benzochromene derivatives and in
continuation of our program on the chemistry of 4H-pyran
derivatives (Al-Ghamdi et al., 2012; El-Agrody, 1994; El-
Agrody et al., 1997a, b, 2000, 2001, 2002, 2011, 2012a, b;
El-Agrody and Al-Ghamdi, 2011; Sabry et al., 2011; Abd-
El-Aziz et al., 2004, 2007; Eid et al., 2003; Khafagy et al.,
2002; Bedair et al., 2000, 2001; Sayed et al., 2000), we report
herein the synthesis of 4H-benzo[h]chromene derivatives
and the evaluation of their antitumor activities. The chemical
structures of the studied compounds and their structure–
activity relationships (SAR) are discussed in this study.
Keywords 4-Chloro-1-naphthol ꢀ
a-Cyanocinnamonitriles ꢀ Ethyl a-cyanocinnamates ꢀ
4H-Benzo[h]chromenes ꢀ Antitumor ꢀ SAR
Introduction
2-Amino-4H-chromenes and 2-amino-4H-benzochromenes
are an important class of heterocyclic compounds having
A. M. El-Agrody (&) ꢀ A. M. Fouda ꢀ E. S. A. E. H. Khattab
Department of Chemistry, Faculty of Science, King Khalid
University, P.O. Box Abha 9004, Abha 61413, Saudi Arabia
e-mail: elagrody_am@yahoo.com
Chemistry
A. M. El-Agrody ꢀ E. S. A. E. H. Khattab
Treatment of 4-chloro-1-naphthol (1) with a-cyano-4-sub-
stitutedcinnamonitriles (2a–g) or a-cyano-4-(4-piperdin-1-
Department of Chemistry, Faculty of Science, Al-Azhar
University, Nasr City 11884, Cairo, Egypt
123

Med Chem Res
ylphenyl/morpholinophenyl)cinnamonitrile (2h, i) in etha-
nolic piperidine under reflux afforded 2-amino-4-aryl-6-
compounds 3 and 5 gave also additional evidences for the
proposed structures.
chloro-4H-benzo[h]chromene-3-carbonitrile
(3a–i) (Scheme 1).
derivatives
In a similar manner, the reaction of 4-chloro-1-naphthol
(1) with ethyl a-cyano-4-substituted cinnamate (4a–g) or
ethyl a-cyano-4-(4-morpholinophenyl) cyanocinnamate
(4h) afforded ethyl 2-amino-4-aryl-6-chloro-4H-benzo[h]
chromene-3-carboxylate derivatives (5a–h) (Scheme 2).
The structures 3 and 5 were established on the basis of
IR, 1H NMR, 13C NMR, 13C NMR-DEPT, 13C NMR-APT,
MS data, and in conjunction with our previous work (Abd-
El-Aziz et al., 2004; El-Agrody, 1994; El-Agrody et al.,
1997a, b; Khafagy et al., 2002; Sayed et al., 2000; Sabry
et al., 2011). The IR spectra of 3a–i showed the appearance
of the a NH2 stretch at t 3471–3424, 3334–3326,
3222–3190 cm,-1 a CN stretch at t 2204–2192 cm,-1
while a NH2 stretch at t 3478–3380, 3325–3282 cm-1 and
Antitumor assays
Compounds 3a–i and 5a–h were evaluated for human
tumor cell growth inhibitory activity against three cell
lines: breast adenocarcinoma (MCF-7), lung carcinoma
(HCT-116), 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.
The inhibitory activity of the synthetic compounds
3a–i and 5a–h against the three cell lines MCF-7, HCT-116,
and HepG-2 are given in Table 1 and Figs. 1, 2, 3, 4,
5, and 6.
1
a CO stretch at t 1687–1669 cm-1 for 5a–h. The H and
13C NMR spectra of 3a–i and 5a–h revealed the presence
of 4H signals at
40.18–38.77 ppm (C-4). In compounds 5a–h the ester
d 5.28–4.79 (s, 1H, H-4) and
1
group gave H signals at 4.05–4.01 (q, J = 7.0–7.2 Hz,
Results and discussion
2H, CH2), 1.12–1.09 (t, J = 7.0–7.2 Hz, 3H, CH3) with the
corresponding signals in the 13C spectra at 58.86–58.63
(CH2) and 14.31–14.19 ppm (CH3), respectively. The 13C
NMR-DEPT spectra at 45°, 90°, and 135° and 13C NMR-
APT spectra of compounds 3 and 5 provided additional
evidence in support of the proposed structures. In addition,
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 pharma-
cological activities (Kidwai et al., 2010; Alvey et al., 2009;
Kumar et al., 2009; Raj et al., 2009; Tanaka et al., 2007;
Sabry et al., 2011; Rampa et al., 2005; Singh et al., 2010;
Vukovic et al., 2010; Magedov et al., 2007; Mahmoodi
et al., 2010; Endo et al., 2010; Tseng et al., 2010; Bruhl-
mann et al., 2001; Kesten et al., 1999; Lee et al., 2006; El-
Sayed and Ibrahim, 2010; Keri et al., 2010; Sashidhara
et al., 2011; Hiramoto et al., 1997).
1
the H NMR spectra for compounds 3 and 5 showed NH2
protons resonated at 7.34–7.14 (sharp singlet) and
8.00–7.82 (broad singlet lower field), respectively. This
deshielding is a result of replacement of CN group in 3 by
C=O group in 5 whose C=O anisotropy would deshield
these protons and in addition of the involvement of these
protons in hydrogen bonding with the C=O group. This
also, was supported by X-ray single crystal data (Al-Dies
et al., 2012; El-Agrody et al., 2012). The mass spectra of
In this study, seventeen compounds of 4H-benzo[h]
chromene derivatives were prepared. Structures of the
synthesized compounds were elucidated on the basis of IR,
Scheme 1 Synthesis
of 4H-benzo[h]chromene-3-
carbonitrile derivatives (3a–i)
9
a; Ar = C6H5
10
8
7
b; Ar = p-FC6H4
c; Ar = p-ClC6H4
d; Ar = p-BrC6H4
1
OH
O
2
3
NH2
CN
pip.
6a
EOH /
CN
Ar =
e:
-MeC H
p
A
6
f; Ar = p-MeOC64H4
r-CH=C
2a-i
+
reflux
Cl
6
4a
CN
4
5
g; Ar = p-NO2C6H4
Ar
h; Ar = p-piperdin-1-ylphenyl
i; Ar = p-morpholinophenyl
3a-i
Cl
1
Scheme 2 Synthesis of ethyl
4H-benzo[h]chromene-3-
carboxylate derivatives (5a–h)
9
a; Ar = C6H5
10
4a
8
OH
Cl
b; Ar = p-FC6H4
c; Ar = p-ClC6H4
d; Ar = p-BrC6H4
1
7
O
2
3
NH2
CO2Et
pip.
EOH /
CN
6a
A
r-CH=C
4a-h
=
+
e: Ar p-MeC6H4
reflux
Cl
CO2Et
6
f; Ar = p-MeOC6H4
4
5
g; Ar = p-NO2C6H4
1
Ar
5a-h
h; Ar = p-morpholinophenyl
123

Med Chem Res
Table 1 Effects of the treatment of MCF-7, HCT-116, and HepG-2 cells with various concentrations of the prepared compounds; cytotoxicity
(IC50) as measured with the MTT method
Compounds
Conc. (lg/ml)
MCF-7 Cell
viability %
IC50 (lg/ml)
HCT-116 Cell
viability %
IC50 (lg/ml)
HepG-2 Cell
viability %
IC50 (lg/ml)
Vinblastine
50
07.82
15.18
29.60
48.75
60.35
76.24
100.00
26.99
35.68
60.34
72.81
86.16
93.63
100.00
3.24
6.1 0.01
16.27
21.68
28.20
38.06
47.54
53.42
100.00
41.40
70.27
75.27
98.22
100.00
100.00
100.00
6.82
2.6 0.04
14.38
16.13
24.25
45.13
55.00
72.13
100.00
17.42
30.26
43.89
64.24
76.55
89.44
100.00
10.95
14.29
16.90
21.03
30.32
43.2
4.6 0.05
25
12.5
6.25
3.125
1.56
0
Colchicine
50
17.7 0.03
0.426 0.01
13.9 0.07
19.5 0.01
3.0 0.06
5.5 0.05
42.8 0.08
0.469 0.015
w
10.6 0.01
0.9 0.04
4.2 0.05
7.6 0.06
5.5 0.05
10.7 0.2
25
12.5
6.25
3.125
1.56
0
Doxorubicin
5 50
25
6.55
8.89
12.5
6.25
3.125
1.56
0
11.74
17.22
21.18
30.86
100.00
24.25
29.11
52.74
63.36
75.14
96.92
100.00
21.03
37.12
66.16
73.01
83.63
97.95
100.00
10.62
16.37
23.22
37.74
48.63
64.45
100.00
13.15
17.95
30.41
47.33
58.42
70.00
100.00
14.83
16.17
22.28
34.64
100.00
82.33
95.46
100.00
100.00
100.00
100.00
100.00
16.89
22.33
31.56
53.44
75.00
91.39
100.00
10.89
16.89
26.17
48.00
63.33
86.72
100.00
29.45
37.84
45.55
63.29
82.53
95.41
100.00
100.00
09.78
14.44
19.06
28.56
60.94
88.11
100.00
19.42
27.86
37.44
53.44
75.00
91.39
100.00
11.94
18.62
30.66
46.14
62.25
83.58
100.00
25.14
36.22
43.46
65.84
81.12
93.96
100.00
3a
3b
3c
3d
50
25
12.5
6.25
3.125
1.56
0
50
7.2 0.02
5.8 0.3
10.9 0.08
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
123

Med Chem Res
Table 1 continued
Compounds
Conc. (lg/ml)
MCF-7 Cell
viability %
IC50 (lg/ml)
HCT-116 Cell
viability %
IC50 (lg/ml)
HepG-2 Cell
viability %
IC50 (lg/ml)
3e
50
29.29
40.21
51.23
65.68
78.01
93.63
100.00
67.95
72.40
88.54
93.32
100.00
100.00
100.00
32.60
43.97
50.27
62.40
81.44
98.84
100.00
43.63
52.25
58.28
66.08
78.43
87.65
100.00
30.48
41.64
49.52
58.63
77.60
93.42
100.00
20.14
27.74
41.10
48.49
71.64
88.01
100.00
18.22
23.29
29.86
39.52
64.97
73.15
100.00
13.9 0.3
12.88
16.58
23.84
44.18
76.71
96.99
100.00
53.68
71.24
85.36
96.12
100.00
100.00
100.00
10.61
13.22
22.00
47.39
77.44
94.11
100.00
48.44
57.12
66.58
73.06
79.86
90.25
100.00
34.72
45.11
58.11
78.33
90.33
100.00
100.00
09.89
12.11
16.89
22.50
40.61
79.06
100.00
11.44
16.00
19.56
44.33
73.83
91.28
100.00
5.7 0.02
10.72
14.83
26.50
40.72
51.33
72.67
100.00
48.82
63.36
76.84
88.44
97.65
100.00
100.00
19.64
27.67
43.67
55.83
72.62
88.90
100.00
41.80
50.39
63.17
72.50
81.48
92.32
100.00
37.14
48.28
61.86
80.14
93.62
100.00
100.00
10.94
15.76
20.92
29.58
41.46
76.24
100.00
14.25
18.96
27.38
46.89
72.11
87.18
100.00
3.5 0.09
25
12.5
6.25
3.125
1.56
0
3f
50
w
w
48.0 0.4
9.2 0.03
26.1 0.02
23.4 0.06
2.8 0.03
5.9 0.013
25
12.5
6.25
3.125
1.56
0
3g
3h
3i
50
13.0 0.07
31.5 0.14
12.2 0.06
6.0 0.07
5.0 0.02
6.0 0.01
45.5 0.23
20.3 0.15
2.7 0.015
5.6 0.08
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
5a
50
25
12.5
6.25
3.125
1.56
0
5b
50
25
12.5
6.25
3.125
1.56
0
123

Med Chem Res
Table 1 continued
Compounds
Conc. (lg/ml)
MCF-7 Cell
viability %
IC50 (lg/ml)
HCT-116 Cell
viability %
IC50 (lg/ml)
HepG-2 Cell
viability %
IC50 (lg/ml)
5c
5d
5e
5f
50
17.53
20.75
27.74
44.93
78.49
94.11
100.00
12.88
17.40
23.15
38.84
69.59
90.62
100.00
07.60
10.48
13.84
25.62
34.18
69.73
100.00
07.88
10.07
14.32
36.03
60.34
82.60
100.00
41.10
70.21
85.14
95.48
98.22
100.00
100.00
10.27
13.22
32.33
55.27
87.26
99.11
100.00
5.8 0.4
12.06
15.11
18.56
36.61
49.56
82.22
100.00
10.78
13.66
20.67
57.72
85.28
96.00
100.00
12.47
14.11
20.00
40.82
59.93
88.84
100.00
12.12
14.38
21.78
40.68
68.49
92.19
100.00
33.72
60.50
68.78
78.78
92.17
100.00
100.00
11.71
15.41
34.79
79.04
93.56
100.00
100.00
3.1 0.06
11.61
15.17
26.50
53.83
75.83
95.11
100.00
12.54
16.93
27.42
56.06
82.95
94.12
100.00
14.12
18.48
27.96
42.14
61.37
87.28
100.00
11.91
17.24
26.85
46.44
67.12
86.96
100.00
27.88
53.42
69.14
80.86
89.12
97.08
100.00
14.22
21.18
37.14
77.48
90.32
97.86
100.00
7.1 0.2
25
12.5
6.25
3.125
1.56
0
50
5.1 0.01
2.4 0.02
4.5 0.013
42.8 0.28
7.7 0.03
7.6 0.03
4.7 0.036
5.2 0.028
34.8 0.35
10.4 0.13
7.6 0.09
5.0 0.04
5.7 0.08
28.3 0.16
10.5 0.41
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
50
25
12.5
6.25
3.125
1.56
0
5g
50
25
12.5
6.25
3.125
1.56
0
5h
50
25
12.5
6.25
3.125
1.56
0
W weak activity (IC50 [ 50 lg/ml)
IC50 values expressed in lg/ml as the mean values of triplicate wells from at least three experiments and are reported as the mean standard error
1H NMR, 13C NMR, 13C NMR-DEPT, 13C NMR-APT, and
MS data.
The results from Table 1 indicated that compounds 5e,
3c, 5f, b, d, 3d, 5c, a were the most active against MCF-7,
5a against HCT-116 and 5a, 3e, a against HepG-2 as
compared with the standard drug Vinblastine, while com-
pounds 5e, 3c, 5f, b, d, 3d, 5c, a, h, 3i, g, a, e were the most
active against MCF-7, 5a, c, e, f, b, 3e, c, g, b, 5d, h, 3d, i,
Compounds 3a–i and 5a–h were tested against three
tumer cell lines: MCF-7, HCT-116, and HepG-2. The cyto-
toxicity evaluation using viability assays and inhibitory
activities are given in Table 1 and Figs. 1, 2, 3, 4, 5, and 6.
123

Med Chem Res
Fig. 1 Evaluation of
cytotoxicity for compounds
(3a–i) against MCF-7cell line
Fig. 2 Evaluation of
cytotoxicity for compounds
(3a–i) against HCT-116 cell line
Fig. 3 Evaluation of
cytotoxicity for compounds
(3a–i) against HepG-2 cell line
123

Med Chem Res
Fig. 4 Evaluation of
cytotoxicity for compounds
(5a–h) against MCF-7 cell line
Fig. 5 Evaluation of
cytotoxicity for compounds
(5a–h) against HCT-116
cell line
Fig. 6 Evaluation of
cytotoxicity for compounds
(5a–h) against HepG-2 cell line
123

Med Chem Res
5g against HCT-116, 5a, 3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g,
5h against HepG-2 as compared with the standard drug
Colchicine and the remaining compounds exhibited near or
moderate to lower activities as compared with the standard
drugs Vinblastine, Colchicine, and Doxorubicin.
compound 5h (IC50 = 7.7 lg/ml)-exhibited good activity
and compound 5g (IC50 = 42.8 lg/ml) inactive.
In the case of HCT-116, investigation of (SAR) revealed
that compound 5a (IC50 = 2.7 lg/ml) has the most potent
activity against HCT-116 compared to the other compounds
3a–i, 5b–h and the standard drug Vinblastine (IC50 = 2.6 lg/
ml). This potency could be attributed to the presence of the
non-substituted phenyl group at the para-position of phenyl
ring at 4-position with the ester group at the 3-position, sug-
gesting that there might be a size limited pocket at the
4-position and the non-substituted phenyl (electron-donating
group) at the 4-position is preferred over the para-substituted
phenyl at the 4-position. In addition, compounds 5a, c, e, f, b,
3e, c, g, b, 5d, h, 3d, i, 5g (IC50 = 2.6–34.8 lg/ml) have the
most potent activity against HCT-116 compared to the other
compounds 3a, f and the standard drug Colchicine
(IC50 = 42.8 lg/ml). This potency could be attributed to the
presence of the non-substituted phenyl (electron-donating
group) at the 4-position and the substituted phenyl at the para-
position of phenyl ring at 4-position with the chloro (electron-
withdrawing), methyl, methoxy (electron-donating), fluoro
groups (electron-withdrawing) with the ester group at the
3-position for compounds 5a, c, e, f, b, the methyl (electron-
donating), chloro, nitro, fluoro groups (electron-withdrawing)
with the cyano group at the 3-position for compounds 3e, c, g,
b, the bromo (electron-withdrawing), morpholino groups
(electron-donating) with the ester group at the 3-position for
compounds 5d, h, the bromo (electron-withdrawing), mor-
pholino groups (electron-donating) with the cyano group at
the 3-position for compounds 3d, i and nitro group (electron-
withdrawing) with the ester group at the 3-position for com-
pound 5g, respectively, at the para-position of phenyl ring.
Furthermore, compounds 5a, 3e, a (IC50 = 2.8–4.2 lg/
ml) showed higher antitumor activities against HepG-2
than the standard drug Vinblastine (IC50 = 4.6 lg/ml).
This could be attributed to the presence of the non-
substituted phenyl group (electron-donating) at the
4-position with the ester group at the 3-position for com-
pound 5a and the substituted at the para-position of phenyl
ring at 4-position with the chloro (electron-withdrawing)
with the cyano group at the 3-position for compound 3e or
the non-substituted phenyl group (electron-donating) at the
4-position with the cyano group at the 3-position for
compound 3a, suggesting that there might be a size limited
pocket at the para-position of phenyl ring at 4-position and
the non-substituted phenyl (electron-donating group) at the
4-position is preferred over the substituted at the para-
position of phenyl ring at 4-position. In addition, com-
pounds 5a, 3e, a, 5e, 3c, 5d, c, f, 3b, 5g, 3g,5h
(IC50 = 2.8–10.5 lg/ml) showed higher antitumor activi-
ties against HepG-2 than the standard drug Colchicine
(IC50 = 10.6 lg/ml). This potency could be attributed to
the presence of the non-substituted phenyl (electron-
SAR studies
The antitumor activity (IC50) of compounds 3, 5 and its
analogs in the three cancer cell lines is summarized in
Table 1. By maintaining the 2-amino and 3-cyano groups
of 3a, the SAR studies at the 4-position was explored. The
non-substituted phenyl analog 3a was much less active
(IC50 = 13.9 lg/ml), confirming the importance of a sub-
stituent at the phenyl ring at the 4-position. Compounds 3c
and 3d have the higher potent antitumor activity
(IC50 = 3.0–5.5 lg/ml) against MCF-7 as compared to the
other compounds 3i, g, e, b, i, f (IC50 = 12.2–50.0 lg/ml)
and the standard drug Vinblastine (IC50 = 6.1 lg/ml). This
potency could be attributed to the presence of the chloro or
bromo atoms (electron-withdrawing groups) at the para-
position of phenyl ring at 4-position. Replacement of the
chloro or bromo atoms with other groups, resulted in
reduction of potency, suggesting that there might be a size
limited pocket at the para-position of phenyl ring at
4-position and an electron-withdrawing group is preferred
over an electron-donating group. In addition, compounds
3c, d, i, g, a, e have the higher potent antitumor activity
(IC50 = 3.0-13.9 lg/ml) against MCF-7, compared to
other compounds and the standard drug Colchicine
(IC50 = 17.7 lg/ml). This potency could be attributed to
the presence of the chloro, bromo, nitro (electron-with-
drawing), morpholino, and methyl (electron-donating)
groups at the para-position of phenyl ring at 4-position,
suggesting that there might be a size limited pocket at the
4-position of the phenyl ring and an electron-withdrawing
group is preferred over an electron-donating group.
Replacement of the electron-withdrawing group cyano
group by ester group at the 3-position for compound 5a and
its analogs improved the antitumor activities. Compounds
5e, f, b, d, c, a (IC50 = 2.4–6.0 lg/ml) have potent anti-
tumor activities against the MCF-7 than the other com-
pounds 5h, g (IC50 = 7.7–42.9 lg/ml) and the standard
drug Vinblastine (IC50 = 6.1 lg/ml). These data indicate
that the activities of compounds 5e, f, b, d, c, a are con-
siderably enhanced by the presence of the methyl, methoxy
(electron-donating), fluoro, bromo, chloro (electron-with-
drawing) at the para-position of phenyl ring at 4-position
or the phenyl ring at the 4-position, suggesting that there
might be a size limited pocket at the para-position of
phenyl ring at 4-position and an electron-donating group is
preferred over an electron-withdrawing group, while
123

Med Chem Res
donating group) at the 4-position, suggesting that there
might be a size limited pocket at the 4-position and the
non-substituted phenyl (electron-donating group) at the
4-position is preferred over the substituted at the para-
position of phenyl ring at 4-position.
obtained using attached proton test (APT), with this tech-
nique, the signals of CH and CH3 carbon atoms appears
normal (up) and the signal of CH2 and Cq environments
appears negative (down).The MS were measured on a Shi-
madzu GC/MS-QP5050A spectrometer. Elemental analyses
were performed on a Perkin-Elmer 240 microanalyser.
Finally, in the case of MCF-7, HCT-116, and HepG-2,
an investigation of SAR revealed that compounds 3a–i and
5a–h showed moderate to lower antitumor activities
against MCF-7, HCT-116, and HepG-2 as compared to the
standard drug Doxorubicin.
General procedure for the preparation of (3) and (5)
A solution of 4-chloro-1-naphthol (1) (0.01 mmol) in
EtOH (30 ml) and piperidine (0.5 ml) was treated with
a-cyanocinnamonitriles (2a–i) or ethyl a-cyanocinnamates
(4a–h) (0.01 mmol). The reaction mixture was heated
under reflux for 1–2 h. The solid product which formed
was collected by filtration, washed with MeOH, and re-
crystallized from ethanol or benzene. The physical and
spectral data of compounds (3) and (5) were as follows:
Conclusions
Our interest in the synthesis of 4H-benzo[h]chromene
derivatives is to focus on their antitumor activities as a part
of our recent research line that aims at the development of
new heterocyclic compounds as strong potent antitumor
agents (El-Agrody et al., 2011). Thus, in this paper we
revealed the synthesis of some 4H-benzo[h]chromene,
followed by antitumor evaluation for all of the synthesized
compounds. Seventeen compounds of 4H-benzo[h]-chro-
mene derivatives were prepared and their structures were
elucidated on the basis of IR, 1H NMR, 13C NMR, 13C NMR-
DEPT/APT, and MS data. Compounds 5e, 3c, 5f, b, d, 3d, 5c,
a were the most active against MCF-7, 5a against HCT-116
and 5a, 3e, a against HepG-2 as compared with the standard
drug Vinblastine, while compounds 5e, 3c, 5f, b, d, 3d, 5c, a,
h, 3i, g, a, e were the most active against MCF-7, 5a, c, e, f, b,
3e, c, g, b, 5d, h, 3d, i, 5g against HCT-116, 5a, 3e, a, 5e, 3c,
5d, c, f, 3b, 5g, 3g, 5h against HepG-2 as compared with the
standard drug Colchicine and the remaining compounds
exhibited near or moderate to lower activities as compared
with the standard drugs Vinblastine, Colchicine, and Doxo-
rubicin. A more extensive study is also warranted to deter-
mine additional antitumor parameters in order to give a
deeper insight to its structure–activity relationship and to
optimize the effectiveness of this series of molecules, which
can then be used in bigger scenarios such as drug design or
development of antitumor therapeutics.
2-Amino-6-chloro-4-phenyl- 4H-benzo[h]chromene-3-
carbonitrile (3a)
Colorless crystals from ethanol; yield 86 %; m.p.
220–221 °C; IR (KBr) t (cm-1): 3477, 3325, 3191 (NH2),
3082, 3017, 2855 (CH), 2199 (CN); H NMR (500 MHz)
1
(DMSO-d6) d: 8.36–7.26 (m, 10H, aromatic), 7.30 (s, 2H,
NH2, cancelled by D2O), 4.93 (s, 1H, H-4); 13C NMR
(125 MHz) (DMSO-d6) d: 159.91 (C-2), 145.11 (C-10b),
129.21 (C-9), 128.78 (C-6a), 127.66 (C-5), 127.62 (C-10a),
127.09 (C-8), 125.52 (C-6), 123.88 (C-7), 121.40 (C-10),
120.18 (C-4a), 118.56 (CN), 56.03 (C-3), 40.53 (C-4),
141.99, 128.57, 128.21, 125.86 (aromatic); MS m/z (%):
334 (M??2, 8.02), 332 (M?, 23.35) with a base peak at
256 (100); Anal. Calcd for C20H13ClN2O: C, 72.18; H,
3.94; N, 8.42. Found: C, 72.22; H, 3.97; N, 8.45 %.
2-Amino-6-chloro-4-(4-fluorophenyl)-4H-
benzo[h]chromene-3-carbonitrile (3b)
Pale yellow crystals from benzene; yield 86 %; m.p.
246–247 °C (Khafagy et al., 2002 m.p. 246 °C); IR (KBr)
t (cm-1): 3470, 3326, 3190 (NH2), 3092, 3037, 2983, 2865
(CH), 2197 (CN); 1H NMR (500 MHz) (DMSO-d6) d:
8.35–7.16 (m, 9H, aromatic), 7.31 (s, 2H, NH2, cancelled
by D2O), 4.97 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-
d6) d: 162.17 (C-2), 142.02 (C-10b), 129.57 (C-9), 129.29
(C-6a), 128.36 (C-5), 127.77 (C-10a), 125.84 (C-8), 123.93
(C-6), 123.83 (C-7), 121.46 (C-10), 120.11 (C-4a), 118.41
(CN), 55.94 (C-3), 40.07 (C-4), 160.23, 141.37, 129.64,
115.49 (aromatic); 13 C NMR-DEPT spectrum at 135° CH,
CH3 [positive (up)], CH2 [negative (down)], revealed the
following signals at d: 129.64 (aromatic :), 129.57 (C-9 :),
128.36 (C-5 :), 125.84 (C-8 :), 123.83 (C-7 :), 121.46
Experimental
Melting points were determined with a Stuart Scientific Co.
Ltd apparatus. IR spectra were determined as KBr pellets on
a Jasco FT/IR 460 plus spectrophotometer. 1H NMR and 13
C
NMR spectra were recorded using a Bruker AV 500 MHz
spectrometer. 13C NMR spectra were obtained using dis-
tortionless enhancement by polarization transfer (DEPT),
where the signals of CH and CH3 carbon atoms appear nor-
mal (up) and the signals of carbon atoms in CH2 environ-
ments appear negative (down). 13C NMR spectra were
123

Med Chem Res
(C-10 :), 40.07 (C-4 :), 115.49 (aromatic :). In the DEPT
spectrum at 90° only CH signals are positive (up) and
showed d: 129.64 (aromatic :), 129.57 (C-9 :), 128.36 (C-
5 :), 125.84 (C-8 :), 123.83 (C-7 :), 121.46 (C-10 :),
40.07 (C-4 :), 115.49 (aromatic :). In the DEPT spectrum
at 45° (CH, CH2, and CH3 positive)-revealed signals at d:
129.64 (aromatic :), 129.57 (C-9 :), 128.36 (C-5 :),
125.84 (C-8 :), 123.83 (C-7 :), 121.46 (C-10 :), 40.07 (C-
4 :), 115.49 (aromatic :); MS m/z (%): 352 (M??2, 5.14),
350 (M?, 17.09) with a base peak at 256 (100); Anal. Calcd
for C20H12ClFN2O: C, 68.48; H, 3.45; N, 7.99. Found: C,
68.13; H, 3.41; N, 7.95 %.
(s, 2H, NH2, cancelled by D2O), 4.88 (s, 1H, H-4), 2.27 (s,
3H, CH3); 13C NMR (125 MHz) (DMSO-d6) d: 159.80 (C-
2), 142.20 (C-10b), 129.32 (C-9), 129.18 (C-6a), 127.63 (C-
5), 127.54 (C-10a), 125.90 (C-8), 125.45 (C-6), 123.76 (C-
7), 121.39 (C-10), 120.20 (C-4a), 118.70 (CN), 56.16 (C-3),
40.17 (C-4), 20.54 (CH3), 141.90, 136.26, 128.16, 123.89
13
(aromatic);
C NMR-DEPT spectrum at 135o CH, CH3
[positive (up)], CH2 [negative (down)], revealed the fol-
lowing signals at d: 129.32 (C-9 :), 128.16 (aromatic :),
127.63 (C-5 :), 125.90 (C-8 :), 123.89 (aromatic :), 123.76
(C-7 :), 121.39 (C-10 :), 40.17 (C-4 :), 20.54 (CH3 :). In the
DEPT spectrum at 90° only CH signals are positive (up) and
showed d: 129.32 (C-9 :), 128.16 (aromatic :), 127.63 (C-5
:), 125.90 (C-8 :), 123.89 (aromatic :), 123.76 (C-7 :),
121.39 (C-10 :), 40.17 (C-4 :). In the DEPT spectrum at 45°
(CH, CH2 and CH3 positive) revealed signals at d: 129.32 (C-
9 :), 128.16 (aromatic :), 127.63 (C-5 :), 125.90 (C-8 :),
123.89 (aromatic :), 123.76 (C-7 :), 121.39 (C-10 :), 40.17
(C-4 :), 20.54 (CH3 :).13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the
following signals at d: 159.80 (C-2 ;), 142.20 (C-10b ;),
141.90 (aromatic ;), 136.26 (aromatic ;), 129.32 (C-9 :),
129.18 (C-6a ;), 128.16 (aromatic :), 127.63 (C-5 :), 127.54
(C-10a ;), 125.90 (C-8 :), 125.45 (C-6 ;), 123.89 (aromatic
:), 123.76 (C-7 :), 121.39 (C-10 :), 120.20 (C-4a ;), 118.70
(CN ;), 56.16 (C-3 ;), 40.17 (C-4 :), 20.54 (CH3 :); MS m/z
(%): 348 (M??2, 6.92), 346 (M?, 20.35) with a base peak at
252 (100); Anal. Calcd for C21H15ClN2O: C, 72.73; H, 4.36;
N, 8.08. Found: C, 72.76; H, 4.38; N, 8.10 %.
2-Amino-6-chloro-4-(4-chlorophenyl)-4H-
benzo[h]chromene-3-carbonitrile (3c)
Colorless needles from benzene; yield 88 %; m.p.
224–225 °C (Khafagy et al., 2002 m.p. 224 °C); IR (KBr)
t (cm-1): 3470, 3331, 3193 (NH2), 3095, 3047, 2986, 2867
(CH), 2194 (CN); 1H NMR (500 MHz) (DMSO-d6) d:
8.36–7.31 (m, 9H, aromatic), 7.34 (s, 2H, NH2, cancelled
by D2O), 4.98 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-
d6) d: 159.91 (C-2), 144.05 (C-10b), 129.53 (C-9), 128.76
(C-6a), 128.32 (C-5), 127.72 (C-10a), 125.72 (C-8), 123.88
(C-6), 123.76 (C-7), 121.43 (C-10), 120.03 (C-4a), 118.02
(CN), 55.64 (C-3), 40.05 (C-4), 142.04, 131.75, 129.30,
125.64 (aromatic); Anal. Calcd for C20H12Cl2N2O: C,
65.41; H, 3.29; N, 7.63. Found: C, 65.44; H, 3.32; N,
7.65 %.
2-Amino-6-chloro-4-(4-bromophenyl)-4H-
benzo[h]chromene-3-carbonitrile (3d)
2-Amino-6-chloro-4-(4-methoxyphenyl)-4H-
benzo[h]chromene-3-carbonitrile (3f)
Colorless needles from benzene; yield 90 %; m.p.
226–227 °C (Khafagy et al., 2002 m.p. 226 °C); IR (KBr) t
(cm-1): 3466, 3328, 3192 (NH2), 3093, 3037, 2986, 2863
(CH), 2193 (CN); 1H NMR (500 MHz) (DMSO-d6) d:
8.35–7.26 (m, 9H, aromatic), 7.34 (s, 2H, NH2, cancelled by
D2O), 4.97 (s, 1H, H-4); 13C NMR (125 MHz) (DMSO-d6)
d: 159.88 (C-2), 144.45 (C-10b), 129.28 (C-9), 128.33 (C-
6a), 127.73 (C-5), 125.72 (C-10a), 125.61 (C-8), 123.85 (C-
6), 123.78 (C-7), 121.41 (C-10), 120.26 (C-4a), 117.94 (CN),
55.53 (C-3), 40.03 (C-4), 142.01, 131.76, 129.87, 119.99
(aromatic); Anal. Calcd for C20H12BrClN2O: C, 58.35; H,
2.94; N, 6.80. Found: C, 58.31; H, 2.90; N, 6.77 %.
Pale yellow needles from benzene; yield 88 %; m.p.
233–234 °C; IR (KBr) t (cm-1): 3424, 3333, 3213 (NH2),
3076, 2953, 2834 (CH), 2193 (CN); H NMR (500 MHz)
1
(DMSO-d6) d: 8.35–6.90 (m, 9H, aromatic), 7.32 (s, 2H,
NH2, cancelled by D2O), 4.87 (s, 1H, H-4), 3.73 (s, 3H,
OCH3); 13C NMR (125 MHz) (DMSO-d6) d: 159.76 (C-2),
141.87 (C-10b), 129.19 (C-9), 128.75 (C-6a), 128.20 (C-5),
127.68 (C-10a), 125.97 (C-8), 125.46 (C-6), 123.93 (C-7),
121.43 (C-10), 120.24 (C-4a), 118.91 (CN), 56.35 (C-3),
55.00 (CH3), 40.08 (C-4), 158.30, 137.26, 128.16, 114.15
(aromatic); MS m/z (%): 364 (M??2, 4.31), 362 (M?,
18.7) with a base peak at 75 (100); Anal. Calcd for
C21H15ClN2O2: C, 69.52; H, 4.17; N, 7.72. Found: C,
69.56; H, 4.21; N, 7.75 %.
2-Amino-6-chloro-4-(4-methylphenyl)-4H-
benzo[h]chromene-3-carbonitrile (3e)
2-Amino-6-chloro-4-(4-nitrophenyl)-4H-
benzo[h]chromene-3-carbonitrile (3g)
Pale yellow needles from benzene; yield 90 %; m.p.
220–231 °C; IR (KBr) t (cm-1): 3450, 3334, 3222 (NH2),
3073, 3057, 2986, 2865 (CH), 2192 (CN); 1H NMR
(500 MHz) (DMSO-d6) d: 8.36–7.14 (m, 9H, aromatic), 7.27
Pale yellow needles from benzene; yield 88 %; m.p.
257–258 °C; IR (KBr) t (cm-1): 3448, 3326, 3197 (NH2),
123

Med Chem Res
1
3070, 2953, 2864 (CH), 2198 (CN); H NMR (500 MHz)
(DMSO-d6) d: 8.36–7.35 (m, 9H, aromatic), 7.44 (s, 2H,
NH2, cancelled by D2O), 5.18 (s, 1H, H-4); 13C NMR
(125 MHz) (DMSO-d6) d: 160.10 (C-2), 142.28 (C-10b),
129.49 (C-9), 129.03 (C-6a), 128.27 (C-5), 127.78 (C-10a),
125.86 (C-6), 125.64 (C-8), 123.86 (C-7), 121.51 (C-10),
signals are positive (up) and showed d: 128.19 (C-9 :),
128.13 (aromatic :), 127.69 (C-5 :), 125.36 (C-8 :),
123.82 (C-7 :), 121.40 (C-10 :), 40.18 (C-4 :), 115.92
(aromatic :). In the DEPT spectrum at 45° (CH, CH2, and
CH3 positive)-revealed signals at d: 128.19 (C-9 :), 128.13
(aromatic :), 127.69 (C-5 :), 125.36 (C-8 :), 123.82 (C-7
:), 121.40 (C-10 :), 49.39 (CH2 :), 40.18 (C-4 :), 25.25
(CH2 :), 23.82 (CH2 :), 115.92 (aromatic :). 13CNMR-
APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative
(down)], revealed the following signals at d: 159.74 (C-2
;), 150.65 (aromatic ;), 141.83 (C-10b ;), 135.01 (aro-
matic ;), 129.14 (C-6a ;), 128.19 (C-9 :), 128.13 (aromatic
:), 127.69 (C-5 :), 126.09 (C-10a ;), 125.36 (C-8 :),
123.95 (C-6 ;), 123.82 (C-7 :), 121.40 (C-10 :), 120.32
(C-4a ;), 119.25 (CN ;), 56.45 (C-3 ;), 49.39 (CH2 ;),
40.18 (C-4 :), 25.25 (CH2 ;), 23.82 (CH2 ;), 115.92
(aromatic :); MS m/z (%): 417 (M??2, 2.11), 415 (M?,
6.33) with a base peak at 55 (100); Anal. Calcd for
C25H22ClN3O: C, 72.19; H, 5.33; N, 10.10. Found: C,
72.22; H, 5.36; N, 10.14 %.
119.86 (C-4a), 117.26 (CN), 55.04 (C-3), 40.18 (C-4),
13
152.29, 146.61, 128.55, 124.14 (aromatic);
C NMR-
DEPT spectrum at 135o CH, CH3 [positive (up)], CH2
[negative (down)], revealed the following signals at d:
129.49 (C-9 :), 128.55 (aromatic :), 128.27 (C-5 :),
125.64 (C-8 :), 124.14 (aromatic :), 123.86 (C-7 :),
121.51 (C-10 :), 40.18 (C-4 :). In the DEPT spectrum at
90° only CH signals are positive (up) and showed d: 129.49
(C-9 :), 128.55 (aromatic :), 128.27 (C-5 :), 125.64 (C-8
:), 124.14 (aromatic :), 123.86 (C-7 :), 121.51 (C-10 :),
40.18 (C-4 :). In the DEPT spectrum at 45° (CH, CH2, and
CH3 positive) revealed signals at d: 129.49 (C-9 :), 128.55
(aromatic :), 128.27 (C-5 :), 125.64 (C-8 :), 124.14
(aromatic :), 123.86 (C-7 :), 121.51 (C-10 :), 40.18 (C-4
:).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2,
Cq [negative (down)], revealed the following signals at d:
160.10 (C-2 ;), 152.29 (aromatic ;), 146.61(aromatic ;),
142.28 (C-10b ;), 129.49 (C-9 :), 129.03 (C-6a ;), 128.55
(aromatic :), 128.27 (C-5 :), 127.78 (C-10a ;), 125.86 (C-
6 ;), 125.64 (C-8 :), 124.14 (aromatic :), 123.86 (C-7 :),
121.51 (C-10 :), 119.86 (C-4a ;), 117.26 (CN ;), 55.04 (C-
3 ;), 40.18 (C-4 :); MS m/z (%): 379 (M??2, 11.01), 377
(M?, 33.01) with a base peak at 72 (100); Anal. Calcd for
C20H12ClN3O3: C, 63.59; H, 3.20; N, 11.12. Found: C,
63.56; H, 3.27; N, 11.09 %.
2-Amino-6-chloro-4-(4-morpholinophenyl)-4H-
benzo[h]chromene-3-carbonitrile (3i)
Yellow needles from benzene; yield 82 %; m.p.
266–267 °C; IR (KBr) t (cm-1): 3451, 3327, 3208 (NH2),
3095, 3010, 2967, 2938, 2864, 2816 (CH), 2195 (CN); H
1
NMR (500 MHz) (DMSO-d6) d: 8.33–6.88 (m, 9H, aro-
matic), 7.18 (s, 2H, NH2, cancelled by D2O), 4.81 (s, 1H,
H-4), 3.72–3.70 (m, 4H, 2CH2), 3.08–3.06 (m, 4H, 2CH2);
13C NMR (125 MHz) (DMSO-d6) d: 159.74 (C-2), 141.83
(C-10b), 129.16 (C-6a), 128.22 (C-9), 127.69 (C-5), 126.06
(C-8), 125.38 (C-10a), 123.94 (C-6), 123.81 (C-7), 121.41
(C-10), 120.29 (C-4a), 119.11 (CN), 66.06 (CH2), 56.39
(C-3), 48.29 (CH2), 40.09 (C-4), 150.04, 135.81, 128.29,
115.27 (aromatic); 13 C NMR-DEPT spectrum at 135o CH,
CH3 [positive (up)], CH2 [negative (down)], revealed the
following signals at d: 128.29 (aromatic :), 128.22 (C-9 :),
127.69 (C-5 :), 126.06 (C-8 :), 123.81 (C-7 :), 121.41
(C-10 :), 66.06 (CH2 ;), 48.29 (CH2 ;), 40.09 (C-4 :),
115.27 (aromatic :). In the DEPT spectrum at 90° only CH
signals are positive (up) and showed d: 128.29 (aromatic
:), 128.22 (C-9 :), 127.69 (C-5 :), 126.06 (C-8 :), 123.81
(C-7 :), 121.41 (C-10 :), 40.09 (C-4 :), 115.27 (aromatic
:). In the DEPT spectrum at 45° (CH, CH2, and CH3
positive)-revealed signals at d: 128.29 (aromatic :), 128.22
(C-9 :), 127.69 (C-5 :), 126.06 (C-8 :), 123.81 (C-7 :),
121.41 (C-10 :), 66.06 (CH2 :), 48.29 (CH2 :), 40.09 (C-4
:), 115.27 (aromatic :).13CNMR-APT spectrum CH,CH3
[positive (up)], CH2, Cq [negative (down)], revealed the
following signals at d: 159.74 (C-2 ;), 150.04 (aromatic
;),141.83 (C-10b ;), 135.81 (aromatic ;),129.16 (C-6a ;),
128.29 (aromatic :), 128.22 (C-9 :), 127.69 (C-5 :),
2-Amino-6-chloro-4-(4-piperdin-1-ylphenyl)-4H-
benzo[h]chromene-3-carbonitrile (3h)
Yellow needles from benzene; yield 82 %; m.p.
252–253 °C; IR (KBr) t (cm-1): 3471, 3298, 3184 (NH2),
3075, 2993, 2938, 2864, 2813 (CH), 2204 (CN); H NMR
1
(500 MHz) (DMSO-d6) d: 8.32–6.86 (m, 9H, aromatic),
7.17 (s, 2H, NH2, cancelled by D2O), 4.79 (s, 1H, H-4),
3.09–3.07 (m, 4H, 2CH2), 1.60–1.57 (m, 4H, 2CH2), 1.51
(s, 2H, CH2); 13C NMR (125 MHz) (DMSO-d6) d: 159.74
(C-2), 141.83 (C-10b), 129.14 (C-6a), 128.19 (C-9), 127.69
(C-5), 126.09 (C-10a), 125.36 (C-8), 123.95 (C-6), 123.82
(C-7), 121.40 (C-10), 120.32 (C-4a), 119.25 (CN), 56.45
(C-3), 49.39 (CH2), 40.18 (C-4), 25.25 (CH2), 23.82 (CH2),
13
150.65, 135.01, 128.13, 115.92 (aromatic);
C NMR-
DEPT spectrum at 135o CH, CH3 [positive (up)], CH2
[negative (down)], revealed the following signals at d:
128.19 (C-9 :), 128.13 (aromatic :), 127.69 (C-5 :),
125.36 (C-8 :), 123.82 (C-7 :), 121.40 (C-10 :), 49.39
(CH2 ;), 40.18 (C-4 :), 25.25 (CH2 ;), 23.82 (CH2 ;),
115.92 (aromatic :). In the DEPT spectrum at 90° only CH
123

Med Chem Res
126.06 (C-8 :), 125.38 (C-10a ;), 123.94 (C-6 ;), 123.81
(C-7 :), 121.41 (C-10 :), 120.29 (C-4a ;), 119.11 (CN ;),
66.06 (CH2 ;), 56.39 (C-3 ;), 48.29 (CH2 ;), 40.09 (C-4 :),
115.27 (aromatic :); MS m/z (%): 419 (M??2, 17.98), 417
(M?, 55.84) with a base peak at 255 (100); Anal. Calcd for
C24H20ClN3O2: C, 68.98; H, 4.82; N, 10.06. Found: C,
68.95; H, 4.78; N, 10.05 %.
:).13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq
[negative (down)], revealed the following signals at d: 167.95
(CO ;), 161.60 (aromatic ;), 160.40 (C-2 ;), 159.68 (aromatic
;), 142.09 (C-10b ;), 129.17 (aromatic :), 129.10 (C-9 :),
128.27 (C-6a ;), 127.54 (C-5 :), 126.30 (C-8 :), 125.40 (C-
10a ;), 123.96 (C-6 ;), 123.77 (C-7 :), 121.46 (C-10 :),
121.33 (C-4a ;), 75.99 (C-3 ;), 58.68 (CH2 ;), 40.07 (C-4 :),
14.23 (CH3 :), 115.05 (aromatic :); Anal. Calcd for
C22H17ClFNO3: C, 66.42; H, 4.31; N, 3.52. Found: C, 66.46;
H, 4.35; N, 3.55 %.
Ethyl 2-amino-6-chloro-4-phenyl-4H-benzo[h]chromene-
3-carboxyalte (5a)
Colorless needles from ethanol; yield 78 %; m.p.
180–181 °C; IR (KBr) t (cm-1): 3393, 3285 (NH2), 3061,
3026, 2968, 2900 (CH), 1676 (CO); H NMR (500 MHz)
Ethyl 2-amino-6-chloro-4-(4-chlorophenyl)-4H-
benzo[h]chromene-3-carboxyalte (5c)
1
(DMSO-d6) d: 8.42-7.12 (m, 10H, aromatic), 7.86 (bs, 2H,
NH2), 5.06 (s, 1H, H-4), 4.01 (q, 2H, CH2, J = 7 Hz), 1.09
(t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6)
d: 168.02 (CO), 160.43 (C-2), 142.08 (C-10b), 129.03
(C-6a), 128.26 (C-9), 127.30 (C-5), 126.33 (C-10a), 126.18
(C-8), 123.94 (C-6), 123.73 (C-7), 121.56 (C-10), 121.42
(C-4a), 76.07 (C-3), 58.63 (CH2), 40.05 (C-4), 14.19
(CH3), 147.32, 127.91, 127.47, 125.30 (aromatic); 381
(M??2, 1.28), 379 (M?, 3.84) with a base peak at 314
(100); Anal. Calcd for C22H18ClNO3: C, 69.57; H, 4.78; N,
3.69. Found: C, 69.60; H, 4.81; N, 3.71 %.
Colorless crystals from ethanol; yield 80 %; m.p.
170–171 °C (Khafagy et al., 2002 m.p. 170 °C); IR (KBr)
t (cm-1): 3384, 3284 (NH2), 3075, 3061, 2989, 2975,
1
2927, 2904, 2889 (CH), 1669 (CO); H NMR (500 MHz)
(DMSO-d6) d: 8.44–7.32 (m, 9H, aromatic), 7.92 (bs, 2H,
NH2), 5.09 (s, 1H, H-4), 4.03 (q, 2H, CH2, J = 7 Hz), 1.11
(t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6)
d: 167.92 (CO), 160.44 (C-2), 142.12 (C-10b), 129.23 (C-
9), 129.02 (C-6a), 128.38 (C-8), 127.53 (C-5), 125.48 (C-
10a), 123.97 (C-6), 123.77 (C-7), 121.49 (C-10), 120.96
(C-4a), 75.74 (C-3), 58.74 (CH2), 40.09 (C-4), 14.24
(CH3), 146.34, 130.79, 129.16, 126.23 (aromatic); Anal.
Calcd for C22H17Cl2NO3: C, 63.78; H, 4.14; N, 3.38.
Found: C, 63.74; H, 4.11; N, 3.35 %.
Ethyl 2-amino-6-chloro-4-(4-fluorophenyl)-4H-
benzo[h]chromene-3-carboxyalte (5b)
Yellow needles from ethanol; yield 78 %; m.p. 184–185 °C
(Khafagy et al., 2002 m.p. 184 °C); IR (KBr) t (cm-1):
3384, 3290 (NH2), 3075, 3061, 2989, 2978, 2958, 2927, 2906,
2887 (CH), 1669 (CO); 1H NMR (500 MHz) (DMSO-d6) d:
8.42–7.06 (m, 9H, aromatic), 7.87 (bs, 2H, NH2), 5.09 (s, 1H,
H-4), 4.02 (q, 2H, CH2, J = 7 Hz), 1.10 (t, 3H, CH3,
J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6) d: 167.95
(CO), 160.40 (C-2), 142.09 (C-10b), 129.10 (C-9), 128.27 (C-
6a), 127.54(C-5), 126.30(C-8), 125.40(C-10a),123.96(C-6),
123.77 (C-7), 121.46 (C-10), 121.33 (C-4a), 75.99 (C-3),
58.68 (CH2), 40.07 (C-4), 14.23 (CH3), 161.60, 159.68,
129.17, 115.05(aromatic);13 C NMR-DEPTspectrumat135o
CH, CH3 [positive (up)], CH2 [negative (down)], revealed the
following signals at d: 129.17 (aromatic :), 129.10 (C-9 :),
127.54 (C-5 :), 126.30 (C-8 :), 123.77 (C-7 :), 121.46 (C-10
:), 58.68 (CH2 ;), 40.07 (C-4 :), 14.23 (CH3 :), 115.05
(aromatic :). In the DEPT spectrum at 90° only CH signals are
positive (up) and showed d: 129.17 (aromatic :), 129.10 (C-9
:), 127.54 (C-5 :), 126.30 (C-8 :), 123.77 (C-7 :), 121.46
(C-10 :), 40.07 (C-4 :), 115.05 (aromatic :). In the DEPT
spectrum at 45° (CH, CH2, and CH3 positive) revealed signals
at d: 129.17 (aromatic :), 129.10 (C-9 :), 127.54 (C-5 :),
126.30 (C-8 :), 123.77 (C-7 :), 121.46 (C-10 :), 58.68 (CH2
:), 40.07 (C-4 :), 14.23 (CH3 :), 115.05 (aromatic
Ethyl 2-amino-6-chloro-4-(4-bromophenyl)-4H-
benzo[h]chromene-3-carboxyalte (5d)
Colorless crystals from ethanol; yield 80 %; m.p.
162–163 °C (Khafagy et al., 2002 m.p. 162 °C); IR (KBr)
t (cm-1): 3385, 3282 (NH2), 3077, 3064, 2974, 2979,
1
2937, 2904, 2887 (CH), 1669 (CO); H NMR (500 MHz)
(DMSO-d6) d: 8.44-7.26 (m, 9H, aromatic), 7.93 (bs, 2H,
NH2), 5.08 (s, 1H, H-4), 4.04 (q, 2H, CH2, J = 7 Hz), 1.12
(t, 3H, CH3, J = 7 Hz); 13C NMR (500 MHz) (DMSO-d6)
d: 167.89 (CO), 160.41 (C-2), 142.10 (C-10b), 129.61 (C-
9), 129.40 (C-6a), 129.14 (C-5), 127.98 (C-8), 127.49 (C-
10a), 125.47 (C-6), 123.94 (C-7), 121.47 (C-10), 120.85
(C-4a), 75.66 (C-3), 58.73 (CH2), 40.08 (C-4), 14.23
(CH3), 146.74, 131.15, 129.78, 119.26 (aromatic); Anal.
Calcd for C22H17BrClNO3: C, 57.60; H, 3.74; N, 3.05.
Found: C, 57.65; H, 3.77; N, 3.10 %.
Ethyl 2-amino-6-chloro-4-(4-methylphenyl)-4H-
benzo[h]chromene-3-carboxyalte (5e)
Colorless crystals from ethanol; yield 79 %; m.p.
188–189 °C; IR (KBr) t (cm-1): 3478, 3315 (NH2), 3075,
3061, 2996, 2980, 2904, 2888 (CH), 1681 (CO); H NMR
1
123

Med Chem Res
(500 MHz) (DMSO-d6) d: 8.43–7.03 (m, 9H, aromatic), 7.85
(bs, 2H, NH2), 5.01 (s, 1H, H-4), 4.01 (q, 2H, CH2,
J = 7.2 Hz), 2.20 (s, 3H, CH3), 1.12 (t, 3H, CH3,
J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) d: 168.13
(CO), 160.42 (C-2), 142.08 (C-10b), 129.04 (C-9), 128.99
(C-6a), 127.86 (C-5), 127.00 (C-8), 125.32 (C-10a), 124.00
(C-6), 123.76 (C-7), 121.98 (C-10), 121.80 (C-4a), 76.25
(C-3), 58.69 (CH2), 40.00 (C-4), 20.49 (CH3), 14.25 (CH3),
144.46, 135.29, 128.85, 126.39 (aromatic); 13 C NMR-DEPT
spectrum at 135o CH, CH3 [positive (up)], CH2 [negative
(down)], revealed the following signals at d: 129.04 (C-9 :),
128.85 (aromatic :), 127.86 (C-5 :), 127.00 (C-8 :), 126.39
(aromatic :), 123.76 (C-7 :), 121.98 (C-10 :), 58.69 (CH2 ;),
40.00 (C-4 :), 20.49 (CH3 :), 14.25 (CH3 :). In the DEPT
spectrum at 90° only CH signals are positive (up) and showed
d: 129.04 (C-9 :), 128.85 (aromatic :), 127.86 (C-5 :),
127.00 (C-8 :), 126.39 (aromatic :), 123.76 (C-7 :), 121.98
(C-10 :), 40.00 (C-4 :). In the DEPT spectrum at 45° (CH,
CH2 and CH3 positive) revealed signals at d: 129.04 (C-9 :),
128.85 (aromatic :), 127.86 (C-5 :), 127.00 (C-8 :), 126.39
(aromatic :), 123.76 (C-7 :), 121.98 (C-10 :), 58.69 (CH2 :),
40.00 (C-4 :), 20.49 (CH3 :), 14.25 (CH3 :).13CNMR-APT
spectrum CH, CH3 [positive (up)], CH2, Cq [negative
(down)], revealed the following signals at d: 168.13 (CO ;),
160.42 (C-2 ;), 144.46 (aromatic ;), 142.08 (C-10b ;),
135.29 (aromatic ;), 129.04 (C-9 :), 128.99 (C-6a ;), 128.85
(aromatic :), 127.86 (C-5 :), 127.00 (C-8 :), 126.39 (aro-
matic :), 125.32 (C-10a ;), 124.00 (C-6 ;), 123.76 (C-7 :),
121.98 (C-10 :), 121.80 (C-4a ;), 76.25 (C-3 ;), 58.69 (CH2
;), 40.00 (C-4 :), 20.49 (CH3 :), 14.25 (CH3 :); MS m/z (%):
395 (M??2, 1.98), 393 (M?, 5.84) with a base peak at 255
(100); Anal. Calcd for C23H20ClNO3: C, 70.14; H, 5.12; N,
3.56. Found: C, 70.18; H, 5.16; N, 3.59 %.
(CH2 ;), 54.89 (CH3 :), 40.08 (C-4 :), 14.27 (CH3 :),
113.64 (aromatic :). In the DEPT spectrum at 90° only CH
signals are positive (up) and showed d: 128.85 (C-9 :),
128.29 (aromatic :), 127.88 (C-5 :), 127.47 (C-8 :),
123.76 (C-7 :), 121.42 (C-10 :), 40.08 (C-4 :), 113.64
(aromatic :). In the DEPT spectrum at 45° (CH, CH2 and
CH3 positive) revealed signals at d: 128.85 (C-9 :), 128.29
(aromatic :), 127.88 (C-5 :), 127.47 (C-8 :), 123.76 (C-7
:), 121.42 (C-10 :), 58.66 (CH2 :), 54.89 (CH3 :), 40.08
(C-4 :), 14.27 (CH3 :), 113.64 (aromatic :). 13CNMR-APT
spectrum CH, CH3 [positive (up)], CH2, Cq [negative
(down)], revealed the following signals at d: 168.08 (CO
;), 160.40 (C-2 ;), 157.62 (aromatic ;), 142.05 (C-10b ;),
157.62 (aromatic ;), 129.01 (C-6a ;), 128.85 (C-9 :),
128.29 (aromatic :), 127.88 (C-5 :), 127.47 (C-8 :),
127.47 (C-10a ;), 123.98 (C-6 ;), 123.76 (C-7 :), 121.42
(C-10 :), 121.42 (C-4a ;), 76.35 (C-3 ;), 58.66 (CH2 ;),
54.89 (CH3 :), 40.08 (C-4 :), 14.27 (CH3 :), 113.64
(aromatic :); MS m/z (%): 411 (M??2, 5.31), 409 (M?,
19.38) with a base peak at 75 (100); Anal. Calcd for
C23H20ClNO4: C, 67.40; H, 4.92; N, 3.42. Found: C, 67.38;
H, 4.89; N, 3.40 %.
Ethyl 2-amino-6-chloro-4-(4-nitrophenyl)-4H-
benzo[h]chromene-3-carboxyalte (5g)
Yellow crystals from ethanol; yield 78 %; m.p.
169–170 °C; IR (KBr) t (cm-1): 3467, 3317 (NH2), 3085,
3066, 2995, 2977, 2908, 2887 (CH), 1687 (CO); H NMR
1
(500 MHz) (DMSO-d6) d: 8.47–7.55 (m, 9H, aromatic),
8.00 (bs, 2H, NH2), 5.28 (s, 1H, H-4), 4.05 (q, 2H, CH2,
J = 7.2 Hz), 1.11 (t, 3H, CH3, J = 7.2 Hz); 13C NMR
(500 MHz) (DMSO-d6) d: 167.75 (CO), 160.51 (C-2),
142.30 (C-10b), 129.35 (C-6a), 128.95 (C-9), 128.49 (C-5),
127.65 (C-10a), 127.65 (C-8), 123.99 (C-6), 123.70 (C-7),
121.59 (C-10), 119.97 (C-4a), 75.16 (C-3), 58.86 (CH2),
Ethyl 2-amino-6-chloro-4-(4-methoxyphenyl)-4H-
benzo[h]chromene-3-carboxyalte (5f)
40.05 (C-4), 14.25 (CH3), 154.86, 146.00, 128.74, 126.13
C NMR-DEPT spectrum at 135o CH, CH3
13
Colorless crystals from ethanol; yield 80 %; m.p.
177–178 °C; IR (KBr) t (cm-1): 3468, 3325 (NH2), 3085,
3066, 2999, 2983, 2904, 2889 (CH), 1686 (CO); H NMR
(aromatic);
[positive (up)], CH2 [negative (down)], revealed the fol-
lowing signals at d: 128.95 (C-9 :), 128.74 (aromatic :),
128.49 (C-5 :), 127.65 (C-8 :), 126.13 (aromatic :),
123.70 (C-7 :), 121.59 (C-10 :), 58.86 (CH2 ;), 40.05 (C-4
:), 14.25 (CH3 :). In the DEPT spectrum at 90° only CH
signals are positive (up) and showed d: 128.95 (C-9 :),
128.74 (aromatic :), 128.49 (C-5 :), 127.65 (C-8 :),
126.13 (aromatic :), 123.70 (C-7 :), 121.59 (C-10 :),
40.05 (C-4 :). In the DEPT spectrum at 45° (CH, CH2 and
CH3 positive) revealed signals at d: 128.95 (C-9 :), 128.74
(aromatic :), 128.49 (C-5 :), 127.65 (C-8 :), 126.13
(aromatic :), 123.70 (C-7 :), 121.59 (C-10 :), 58.86 (CH2
:), 40.05 (C-4 :), 14.25 (CH3 :). 13CNMR-APT spectrum
CH, CH3 [positive (up)], CH2, Cq [negative (down)],
revealed the following signals at d: 167.75 (CO ;), 160.51
1
(500 MHz) (DMSO-d6) d: 8.40–6.81 (m, 9H, aromatic),
7.82 (bs, 2H, NH2), 5.01 (s, 1H, H-4), 4.02 (q, 2H, CH2,
J = 7.2 Hz), 3.68 (s, 3H, OCH3), 1.11 (t, 3H, CH3,
J = 7.2 Hz); 13C NMR (500 MHz) (DMSO-d6) d: 168.08
(CO), 160.40 (C-2), 142.05 (C-10b), 129.01 (C-6a), 128.85
(C-9), 127.88 (C-5), 127.47 (C-8), 127.47 (C-10a), 123.98
(C-6), 123.76 (C-7), 121.42 (C-10), 121.42 (C-4a), 76.35
(C-3), 58.66 (CH2), 54.89 (CH3), 40.08 (C-4), 14.27 (CH3),
13
157.62, 139.53, 128.29, 113.64 (aromatic);
C NMR-
DEPT spectrum at 135o CH, CH3 [positive (up)], CH2
[negative (down)], revealed the following signals at d:
128.85 (C-9 :), 128.29 (aromatic :), 127.88 (C-5 :),
127.47 (C-8 :), 123.76 (C-7 :), 121.42 (C-10 :), 58.66
123

Med Chem Res
(C-2 ;), 154.86 (aromatic ;), 146.00 (aromatic ;), 142.30
(C-10b ;), 129.35 (C-6a ;), 128.95 (C-9 :), 128.74 (aro-
matic :), 128.49 (C-5 :), 127.65 (C-10a ;), 127.65 (C-8 :),
126.13 (aromatic :), 123.99 (C-6 ;), 123.70 (C-7 :),
121.59 (C-10 :), 119.97 (C-4a ;), 75.16 (C-3 ;), 58.86
(CH2 ;), 40.05 (C-4 :), 14.25 (CH3 :); MS m/z (%): 426
(M??2, 18.32), 424 (M?, 57.87) with a base peak at 54
(100); Anal. Calcd for C22H17ClN2O5: C, 62.20; H, 4.03;
N, 6.59. Found: C, 62.24; H, 4.07; N, 6.62 %.
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
propagated in Dulbecco’s-modified Eagle’s medium
(DMEM) supplemented with 10 % heat-inactivated fetal
calf serum, 1 % L-glutamine, HEPES buffer and 50 lg/ml
gentamycin. All cells were maintained at 37 °C in a
humidified atmosphere with 5 % CO2 and were subcultures
two to three times a week.
Ethyl 2-amino-6-chloro-4-(4-morpholinophenyl)-4H-
benzo[h]chromene-3-carboxyalte (5h)
Yellow crystals from ethanol; yield 76 %; m.p. 199-200
oC; IR (KBr) t (cm-1): 3380, 3286 (NH2), 3087, 3068,
2995, 2978, 2928, 2906, 2887 (CH), 1683 (CO); H NMR
Cytotoxicity evaluation using viability assay
1
(500 MHz) (DMSO-d6) d: 8.40–6.80 (m, 9H, aromatic),
7.84 (bs, 2H, NH2), 5.08 (s, 1H, H-4), 4.01 (q, 2H, CH2,
J = 7.2 Hz), 3.98–3.67 (m, 4H, 2CH2), 3.36–3.00 (m, 4H,
2CH2), 1.10 (t, 3H, CH3, J = 7.2 Hz); 13C NMR
(500 MHz) (DMSO-d6) d: 160.41 (CO), 160.36 (C-2),
142.08 (C-10b), 129.17 (C-9), 128.06 (C-5), 127.87 (C-6a),
127.80 (C-10a), 126.48 (C-8), 125.39 (C-6), 123.79 (C-7),
121.47 (C-10), 121.37 (C-4a), 76.42 (C-3), 66.05 (CH2),
The in vitro cytotoxicity activity was studied against three
cell lines: MCF-7, HCT, and HepG-2 using the colori-
metric MTT assay (Mossman, 1983). The cells were
seeded in 96-well microtitre plate (Falcon, NJ, USA) at a
cell concentration of 1 9 104 cells per well in 100 ll of
growth medium. Fresh medium containing different con-
centrations of the test sample was added after 24 h of
seeding. Serial twofold dilutions of the metabolites were
added confluent cell monolayer. The microtiter plates
(polystyrene sterile tissue culture 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 concen-
tration of the test sample. Control 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 con-
centrations of sample were added, and the incubation was
continued for 48 h and viable cells yield was determined
by a colorimetric MTT method.
58.69 (CH2), 48.47 (CH2), 38.77 (C-4), 14.31 (CH3),
13
143.57, 138.20, 129.10, 114.90 (aromatic);
C NMR-
DEPT spectrum at 135o CH, CH3 [positive (up)], CH2
[negative (down)], revealed the following signals at d:
129.17 (C-9 :), 129.10 (aromatic :), 128.06 (C-5 :),
126.48 (C-8 :), 123.79 (C-7 :), 121.47 (C-10 :), 66.05
(CH2 ;), 58.69 (CH2 ;), 48.47 (CH2 ;), 38.77 (C-4 :),
14.31 (CH3 :), 114.90 (aromatic :). In the DEPT spectrum
at 90° only CH signals are positive (up) and showed d:
129.17 (C-9 :), 129.10 (aromatic :), 128.06 (C-5 :),
126.48 (C-8 :), 123.79 (C-7 :), 121.47 (C-10 :), 38.77 (C-
4 :), 114.90 (aromatic :). In the DEPT spectrum at 45°
(CH, CH2, and CH3 positive)-revealed signals at d: 129.17
(C-9 :), 129.10 (aromatic :), 128.06 (C-5 :), 126.48 (C-8
:), 123.79 (C-7 :), 121.47 (C-10 :), 66.05 (CH2 :), 58.69
(CH2 :), 48.47 (CH2 :), 38.77 (C-4 :), 14.31 (CH3 :),
114.90 (aromatic :). 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the
following signals at d:160.41 (CO ;) 160.36 (C-2 ;),
143.57 (aromatic ;), 142.08 (C-10b), 138.20 (aromatic ;),
129.17 (C-9 :), 129.10 (aromatic :), 128.06 (C-5 :),
127.87 (C-6a ;), 127.80 (C-10a ;), 126.48 (C-8 :), 125.39
(C-6 ;), 123.79 (C-7 :), 121.47 (C-10 :), 121.37 (C-4a ;),
76.42 (C-3 ;), 66.05 (CH2 ;), 58.69 (CH2 ;), 48.47 (CH2
;), 38.77 (C-4 :), 14.31 (CH3 :), 114.90 (aromatic :); MS
m/z (%): 466 (M??2, 22.28), 464 (M?, 69.83) with a base
peak at 101 (100); Anal. Calcd for C26H25ClN2O4: C,
67.17; H, 5.42; N, 6.03. Found: C, 67.13; H, 5.39; N,
6.06 %.
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. The cytotoxic effect of
each tested compound was calculated as [1 - (ODt/
ODc)] 9 100 % where ODt is the mean optical density of
wells treated with the tested compounds and ODc is the
mean optical density of untreated cells.
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
123

Med Chem Res
the Regional Center for Mycology & Biotechnology (RCMP),
Al-Azhar University for carrying out the antitumor study and Mr. Ali
El-Agrody AM, Abd El-Latif MS, El-Hady NA, Fakery AH, Bedair
AH (2001) Heteroaromatization with 4-hydroxycoumarin part II:
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1
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