Synthesis and cytotoxic activity of novel benzopyrano [3,2-c]chromene-6,8- dione derivatives

Article abstract of DOI:10.1007/s00044-010-9340-3

4-Hydroxycoumarins constitute the structural nucleus of many natural products, drugs, and pesticides. Promising biological properties in new families of synthetic coumarins were recently reported. Therefore, efficient synthesis of new benzopyrano[3,2-c]ch

Full text of DOI:10.1007/s00044-010-9340-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:466–474
DOI 10.1007/s00044-010-9340-3
ORIGINAL RESEARCH
Synthesis and cytotoxic activity of novel benzopyrano
[3,2-c]chromene-6,8-dione derivatives
•
Abbas Shafiee Radineh Motamedi Omidreza Firuzi
•
•
•
Savis Meili Ahmad Reza Mehdipour Ramin Miri
•
Received: 6 August 2009 / Accepted: 3 March 2010 / Published online: 23 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract 4-Hydroxycoumarins constitute the structural
nucleus of many natural products, drugs, and pesticides.
Promising biological properties in new families of
synthetic coumarins were recently reported. Therefore,
efficient synthesis of new benzopyrano[3,2-c]chromene-
6,8-dione was undertaken and the structures of 15 com-
pounds were confirmed by their IR, Mass, 1H-NMR, and C,
H, N analysis. Then, the cytotoxic activities of these
compounds were assessed on four different human cancer
cell lines (Raji, HeLa, LS180, and MCF-7). The results
showed that these compounds had weak-to-moderate anti-
tumoral activities and their IC50 ranged from 49 to more
than 100 lM. Among the compounds 9,10-dihydro-7-(3-
methoxyphenyl)-7H,11H-benzopyrano[3,2-c]chromene-6,8-
dione [4k] demonstrated the highest activity. Furthermore,
conformational analysis revealed that ortho substituents
were clearly different from meta and para substituents.
Keywords 4-Hydroxycoumarin Á Benzopyran Á
Chromene Á Cytotoxicity
Introduction
4-Hydroxycoumarins constitute the structural nucleus of
many natural products, drugs and pesticides (Feuer, 1974;
Obaseki and Porter, 1982). They are the key intermediates
for various widely used oral anticoagulants and rodenticides
(Hermodson et al., 1971). Similarly, several derivatives of
pyran or fused pyran ring systems have been shown to
possess different types of biological activities (Bedair et al.,
2000) such as tyrosinase inhibitory (Kang et al., 2009) and
may lay a role against Alzheimer disease (Jin et al., 2002).
Various other activities exhibited by pyran derivatives
include antimicrobial (Georgiadis et al., 1992), antiprolif-
erative (Kasaplar et al., 2009), antifungal, antidepressant
(Ermili et al., 1979) and selective MAO-B inhibitory (Matos
et al., 2009) activities. They have also been developed as
photosensitive drugs (Farag and El-Tayeb, 2010), potent and
selective human dopamine D4 antagonists (Kesten et al.,
1999), potential HIV inhibitors (Mahajan et al., 2009) or as
antibiotic agents such as novebiocin (Crow et al., 1999).
Moreover, promising biological properties have been
reported for new families of (semi)synthetic coumarins
(Stanchev et al., 2008; Su et al., 2009). One family, repre-
sented by 2,4-diaryl-4H,5H-pyrano[3,2-c] benzopyran-5-
ones, exhibits strong antiproliferative activities in MCF-7
breast carcinoma cells by a mechanism that remains to be
determined (Colotta et al., 1990). The second group, rep-
resented by 1-benzopyrano[3,4-b][1,4] benzothiazin-6-
ones, display interesting antioxidant and estrogenic like
effects in HepG2 and MCF-7 cells, respectively (Fig. 1)
(Colotta et al., 1990, 1991a, b).
A. Shafiee
Department of Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
A. Shafiee
Pharmaceutical Sciences Research Center, Tehran University
of Medical Sciences, Tehran 14174, Iran
R. Motamedi
Department of Chemistry, Payame Noor University,
Delijan, Iran
O. Firuzi Á S. Meili Á A. R. Mehdipour Á R. Miri (&)
Medicinal & Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, P.O. Box 3288-71345,
Shiraz, Iran
e-mail: mirir@sums.ac.ir
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Med Chem Res (2011) 20:466–474
467
Fig. 1 Two novel families of
synthetic coumarins
R
R
O
O
HN
O
S
R
O
O
2,4-Diaryl-4H, 5H-pyrano[3,2-c] benzopyran-5-ones
1-Benzopyrano[3,4-b][1,4]benzothiazine-6-ones
Thus, it is evident from various reports that extensive
efforts have been focused on 4-hydroxycoumarin and pyran
derivatives. In the present paper, we report the efficient
synthesis of new benzopyrano[3,2-c]chromene-6,8-diones
(4a–4o) by a simple one-pot method. The cytotoxicity of
synthesized compounds was investigated on different
human cultured cancer cell lines, including HeLa (human
cervical adenocarsinoma), MCF-7 (human breast adeno-
carcinoma), Raji (human B lymphoma), and LS180
(human colorectal adenocarcinoma) cells.
A plausible mechanism for the formation of compounds
4a–o is shown in Scheme 2. It is reasonable to assume that
in the first step, aldol condensation between arylaldehydes
and 1,3-cyclohexadione provides compound 5. In the sec-
ond step, Michael addition of 4-hydroxycoumarin to
compound 5 leads to the formation of the intermediates 6,
which undergo cyclization followed by dehydration to give
the desired compounds 4 (Scheme 2). The structures of
1
compounds 4 were determined from their IR, Mass, H-
NMR, and C, H, N analysis.
Results and discussion
Cytotoxic activity
Chemistry
The cytotoxic activity of synthesized compounds was eval-
uated in four human cancer cell lines and the data are shown
in Table 2. IC16 and IC50 values were calculated for each
compound. The highest cytotoxic effect was seen on Raji cell
line, while the lowest activity was observed in LS180, since
only three compounds had IC50s lower than 100 lM in this
cell line. Among the tested compounds, 9,10-dihydro-7-(3-
methoxyphenyl)-7H,11H-benzopyrano
Due to the enol lactone structure of 4-hydroxycoumarin,
3-C shows nucleophilicity. Thus, a number of 4-hydroxy-
coumarin derivatives, namely, pyrano[3,2-c]benzopyrans
can be synthesized through the Michael addition of 4-hy-
droxycoumarins to a,b unsaturated carbonyl compounds
utilizing this nucleophilicity (Trivedi et al., 2001; Sagar
et al., 2005). Recently, some other methods have been
described that utilize nucleophilicity for asymmetric syn-
thesis of 4-hydroxycoumarin derivatives (Robinson et al.,
1996; Cravottoo et al., 2001; Halland et al., 2003). In our
investigation, the reaction of 4-hydroxyl coumarin 3, 1,3
cyclohexadione 1, and arylaldehydes 2 in different solvents
(ethanol, acetic acid, acetonitrile, and benzene) to provided
7-aryl-9,10-dihydro-7H,11H-benzopyrano[3,2-c]chromene-
6,8-diones 4a–o (Scheme 1). The highest yield was found
in boiling acetic acid (65–78%) (Table 1).
[3,2-c]chromene-6,8-dione [4k] was the most potent one
with IC50 values smaller than 100 lM in all cell lines (IC50
range 44–99 lM).
The presence of the methoxy group, especially in the meta
position, appeared to confer the highest cytotoxic activity,
sine compound 4k was the only compound, which had an
IC50 of lower than 100 lM in all cell lines. On the other hand,
the weakest activities were observed for compounds 4m–
o with a methyl group. These compounds had IC50 values of
higher than 100 lM in all cell lines except for Raji cells. Due
10
Scheme 1 General synthetic
pathway for the preparation of
benzopyrano[3,2-c]chromene-
6,8-dione derivatives
11
9
11a
12
8
7a
O
O
CHO
OH
1
4
12a
1a
14
2
7
13
6a
+
15
+
3
R
O
O
6
O
O
4a
O
O
18
5
16
17
R
2
1
3
4
123

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Med Chem Res (2011) 20:466–474
Table 1 Physical properties for compounds 4a–4o
O
O
O
CH
R
O
R
Mp (°C)
Yield (%)
Rf
4a
4b
4c
4d
4e
4f
2-nitro
222–223
218–220
195–197
238–239
265–264
170–171
294–295
247–248
244–245
245–246
188–189
196–197
268–269
218–219
216–215
65
70
75
78
60
71
65
65
65
78
60
64
68
73
74
0.47
0.45
0.47
0.43
0.39
0.38
0.41
0.38
0.40
0.45
0.43
0.46
0.41
0.41
0.39
3-nitro
4-nitro
2-bromo
3-bromo
4-bromo
2-chloro
3-chloro
4-chloro
2-methoxy
3-methoxy
4-methoxy
2-methyl
3-methyl
4-methyl
4g
4h
4i
4j
4k
4l
4m
4n
4o
Scheme 2 Possible mechanism
of formation of 4 in the reaction
pathway
OH
O
CHO
O
O
+
+
CH
O
O
O
R
1
3
2
R
5
HO
O
O
O
O
O
O
O
OH
O
H
CH
CH
CH
Ar
Ar
Ar
O
O
O
O
4
6
7
to low solubility, three compounds (two containing chlorine
and one containing bromine) were tested at the maximum
concentrations of 25 and 50 lM.
Structures of six compounds are represented in Fig. 2. As is
seen, the cyclohexenone ring exhibited a semi-boat con-
former and the aryl group at C7 position positioned at the
axial coordinate. Moreover, the aryl ring made a dihedral
angle with main structure which is the main difference
between different derivatives occurred (Table 3). The
derivatives containing ortho substituent show a complete
antiplanar position with respect to main four-ring structure,
Three-dimensional structure
The optimized 3D structures of molecules were obtained
by Ab initio (RHF) calculations at the level of STO-3G.
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Med Chem Res (2011) 20:466–474
469
Table 2 Cytotoxic activity of compounds assessed by the MTT reduction assay
Compound HeLa cells
LS180 cells
MCF-7 cells
Raji cells
ICa16 (lM)
ICa50 (lM)
IC16 (lM)
IC50 (lM)
IC16 (lM)
IC50 (lM)
IC16 (lM)
IC50 (lM)
4a
4b
4c
4d
4e
4f
18.1 8.6
26.2 5.2
[100
[100
[100
[100
28.0 11.8
23.1 2.1
26.0 7.9
[100
59.5 22.8
80.3 7.8
61.7 5.6
[100
14.9 1.4
55.8 37.9
47.7 21.4
[100
54.8 9.2
[100
63.1 2.5
[100
60.9 19.2
32.6 8.6
[100
[100
75.7 16.8
[100
[100
[100
[100
[100
21.9 5.0
[100
61.6 11.0
[100
35.1 0.2
[100
83.8 0.4
[100
[ 50
15.7 1.3
[ 50
49.3 4.7
11.3 4.7
22.4 5.1
[25
49.3 25.5
82.4 55.6
[25
4g
4h
4i
[25
[25
[25
[25
[25
[25
[50
[50
[50
[50
[50
[50
23.9 1.3
59.0 3.5
30.3 14.8
13.1 2.3
33.1 14.6
26.4 1.4
26.7 15.9
8.5 2.8
[50
17.9 1.2
37.6 13.2
25.3 4.9
48.7 1.2
42.9 3.8
41.7 26.1
38.8 13.4
55.8 4.8
74.3 26.3
89.9 3.9
97.9 4.5
[100
[100
[100
54.7 26.2
[100
[100
[100
4j
52.1 19.9
48.9 3.2
[ 100
[100
[100
[100
4k
4l
99.7 10.0
[100
22.2 5.0
35.9 9.2
30.9 10.3
42.8 6.2
49.2 9.6
58.6 6.1
76.3 7.6
[100
49.2 11.6
[100
4m
4n
4o
53.8 0.6
47.0 9.9
74.2 14.0
[100
79.7 13.7
72.7 22.6
78.9 10.2
[100
[100
[100
[100
[100
[100
Doxorubicin 0.015 0.001 0.053 0.007 0.030 0.013 0.130 0.021 0.051 0.006 0.211 0.024 0.012 0.001 0.039 0.004
Values represent the mean SD of at least three different experiments. The maximum concentration of the compound tested for cytotoxicity
was 100 lM, except for 4e, 4g, and 4h, which, due to lower solubility, were tested at the maximum concentrations of 50, 25, and 50 lM,
respectively
a
IC16 and IC50 values are defined as the concentration of the compound necessary to inhibit cell growth by 16 and 50%, respectively
while other derivatives (containing para and meta substit-
uents) do not posses a complete antiplanar and show a
variation from this position. As seen in Table 3, the
derivatives containing ortho substituent have significant
difference in dihydral angles with para and meta substit-
uents (except methyl substituents).
spectrometer, and chemical shifts are expressed as d (ppm)
with tetramethylsilane as internal standard. The mass
spectra were run on a Finnigan TSQ-70 spectrometer at
70 eV. Merck silica gel 60 F254 plates were used for
analytical TLC; column chromatography was performed on
Merck silica gel (70–230 mesh). Yields were calculated for
purified products and were not optimized.
Conclusion
General procedure for the synthesis of 7-aryl-9,
10-dihydro-7H,11H-benzopyrano[3,2-c]chromene-6,
8-dione derivatives
In search of novel antitumoral compounds, a set of ben-
zopyrano[3,2-c]chromene-6,8-dione derivatives were
designed and synthesized by a simple one-pot method. The
cytotoxic activity of these compounds was evaluated in
vitro on four different cancer cell lines. The results of
cytotoxicity study show that these compounds have weak-
to-moderate in vitro anti-cancer effect.
4-hydroxyl coumarin 3 (0.162 g, 1 mmol), 1,3 cyclohex-
adione 1 (0.112 g, 1 mmol) and arylaldehydes (1 mmol),
were refluxed in acetic acid (50 ml) for 5 h. The mixture
was cooled to room temperature and the solid was filtered,
washed with cold ethyl acetate, dried, and recrystallized
from ethyl acetate to give pure product 4 (Table 1).
Experimental protocols
9,10-Dihydro-7-(2-nitrophenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4a)
Chemistry
IR (KBr) m: 3078 (C–H aromatic), 2950 (C–H aliphatic),
1731 (C=O), 1526, 1357 (NO2) cm-1
.
Chemicals and all solvents used in this study were pur-
chased from Merck AG and Aldrich Chemicals. Melting
points were determined on a Kofler hot stage apparatus and
are uncorrected. The IR spectra were obtained on a Shi-
matdzu 470 spectrophotometer (potassium bromide disks).
1H-NMR spectra were measured using a Bruker FT-500
1H NMR (CDCl3) d: 2.05–2.14 (m, 2H, H10), 2.40–2.42
(m, 2H, H11), 2.72–2.79 (m, 1H, H9), 2.83–2.89 (m, 1H,
H9), 5.75 (s, 1H, H7), 7.31 (t, 1H, J = 7.5 Hz, H16), 7.34
(d, 1H, J = 8.0 Hz, H4), 7.38 (t, 1H, J = 8.0 Hz H2), 7.47
(t, 1H, J = 7.5 Hz, H17), 7.54 (d, 1H, J = 7.5 Hz, H18),
123

470
Med Chem Res (2011) 20:466–474
Fig. 2 The optimized three-
dimensional structural
representation of six selected
compounds
7.59 (t, 1H, J = 8.0 Hz, H3), 7.78 (d, 1H, J = 7.5 Hz,
H15), 7.91 (d, 1H, J = 8.0 Hz H1).
MS: m/z (%), 389 (M?, 33), 342 (40), 267 (100), 149
(23), 122 (57), 83 (31), 55 (91).
H9), 5.09 (s, 1H, H7), 7.36 (d, 1H, J = 8.0 Hz, H4), 7.41 (t,
1H, J = 8.0 Hz, H2), 7.48 (t, 1H, J = 8.0 Hz, H17), 7.62 (t,
1H, J = 8.0 Hz, H3), 7.94 (m, 2H, H18, H1), 8.05–8.08 (m,
2H, H14, H16).
Anal. Calcd for C22H15NO6: C, 67.86; H, 3.88; N, 3.60.
Found: C, 67.63; H, 4.15; N, 3.89.
MS: m/z (%), 390 (M??1, 29), 389 (M?, 8), 373 (86),
343(16), 267 (100).
Anal. Calcd for C22H15NO6: C, 67.86; H, 3.88; N, 3.60.
Found: C, 68.15; H, 3.65; N, 3.85.
9,10-Dihydro-7-(3-nitrophenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4b)
9,10-Dihydro-7-(4-nitrophenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4c)
IR (KBr) m: 3100 (C–H aromatic), 2934 (C–H aliphatic),
1731 (C=O), 1526, 1362 (NO2) cm-1
.
1H NMR (CDCl3) d: 2.08–2.21 (m, 2H, H10), 2.40–2.50
(m, 2H, H11), 2.77–2.85 (m, 1H, H9), 2.92–2.99 (m, 1H,
IR (KBr) m: 3068 (C–H aromatic), 2914 (C–H aliphatic),
1721 (C=O), 1526, 1362 (NO2) cm-1
.
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Med Chem Res (2011) 20:466–474
471
7-(3-Bromophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4e)
Table 3 Geometrical features of the optimized structures of syn-
thesized compounds
a
b
c
Compound
A1 (°)
A2 (°)
A3 (°)
IR (KBr) m: 3050 (C–H aromatic), 2950 (C–H aliphatic),
1731 (C=O) cm-1
4a
4b
4c
4d
4e
4f
-117.79
-111.19
-110.45
-114.02
-109.37
-109.31
-115.58
-109.74
-109.73
-112.97
-108.46
-108.74
-107.71
-108.62
-108.62
60.08
55.52
58.75
60.58
59.46
58.95
60.88
59.78
58.88
59.94
59.50
59.40
58.94
58.93
59.14
37.89
34.45
34.13
35.75
33.81
33.77
36.66
33.91
33.84
35.83
33.55
33.57
33.06
33.51
33.52
.
1H NMR (CDCl3) d: 2.18–2.09 (m, 2H, H10), 2.50–2.30
(m, 2H, H11), 2.74–2.80 (m, 1H, H9), 2.88–2.93 (m, 1H,
H9), 4.95 (s, 1H, H7), 7.15 (t, 1H, J = 8.0 Hz, H17), 7.29
(dt, 1H, J = 8.0, 1.0 Hz,H18), 7.34 (d, 1H, J = 8.0 Hz,
H4), 7.38 (t, 1H, J = 8.0 Hz, H2),7.39 (t, 1H, J =
1.0 Hz,H14), 7.41 (dt, 1H, J = 8.0, 1.0 Hz, H16), 7.58 (td,
1H, J = 8.0, 1.0 Hz H3), 7.90 (dd, 1H, J = 8.0, 1.0 Hz,
H1).
4g
4h
4i
MS : m/z (%), 424 (M?? 2, 10), 422 (M?, 10), 343 (50),
4j
4k
4l
267 (41), 249 (100), 122 (23), 93 (10).
Anal. Calcd for C22H15BrO4: C, 62.43; H, 3.57. Found:
C, 62.68; H, 3.82.
4m
4n
4o
a
7-(4-Bromophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4f)
Dihedral angle of cyclohexanone ring (C7a–C8–C9–C10
)
b
c
Dihedral angle between phenyl and pyran rings (C6a–C7–C13–C14
)
)
Dihedral angle between phenyl and pyran rings (C12a–C6a–C7–C13
IR (KBr) m: 3080 (C–H aromatic), 2939 (C–H aliphatic),
1716 (C=O) cm-1
.
1H NMR (CDCl3) d: 2.07–2.18 (m, 2H, H10), 2.42–2.46
(m, 2H, H11), 2.78–2.80 (m, 1H, H9), 2.85–2.90 (m, 1H,
H9), 4.94 (s, 1H, H7), 7.25 (d, 2H, J = 8.5 Hz, H14, H18),
7.34 (dd, 1H, J = 8.0, 1.0 Hz, H4), 7.36 (td, 1H, J = 8.0,
1.0 Hz, H2), 7.37 (d, 2H, J = 8.5 Hz, H15, H17),7.59 (td,
1H, J = 8.0, 1.5 Hz,H3), 7.88 (dd, 1H, J = 8.0, 1.5 Hz,
H1)
MS: m/z (%), 424 (M?? 2, 18), 422 (M?, 16), 342 (40),
267 (100), 154 (25), 122 (18), 93 (38), 74 (42).
Anal. Calcd for C22H15BrO4: C, 62.43; H, 3.57. Found:
C, 62.67; H, 3.81.
1H NMR (CDCl3) d: 2.08–2.21 (m, 2H, H10), 2.44–2.47
(m, 2H, H11), 2.79–2.84 (m, 1H, H9), 2.89–2.90 (m, 1H,
H9), 5.08 (s, 1H, H7), 7.36 (dd, 1H, J = 8.0, 1.0 Hz, H4),
7.40 (td, 1H, J = 8.0, 1.0 Hz, H2), 7.57 (d, 2H,
J = 8.5 Hz, H14, H18), 7.62 (td, 1H, J = 8.0, 1.5 Hz, H3),
7.91 (dd, 1H, J = 8.0, 1.5 Hz, H1), 8.13 (d, 2H,
J = 8.5 Hz, H15, H17).
MS : m/z (%), 389 (M?, 11), 372 (47), 342 (22), 267
(100), 149 (17), 122 (13), 83 (23), 55 (40).
Anal. Calcd for C22H15NO6: C, 67.86; H, 3.88; N, 3.60.
Found: C, 67.69; H, 3.70; N, 3.82.
7-(2-Chlorophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4g)
7-(2-Bromophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4d)
IR (KBr) m: 3062 (C–H aromatic), 2955 (C–H aliphatic),
1726 (C=O) cm-1
.
IR (KBr) m: 3052 (C–H aromatic), 2919 (C–H aliphatic),
1H NMR (CDCl3) d: 1.93 (m, 2H, H10), 2.29–2.33 (m,
2H, H11), 2.82–2.85 (m, 2H, H9), 5.06 (s, 1H, H7), 7.16
(t, 1H, J = 7.6 Hz, H16), 7.24 (t, 1H, J = 7.6 Hz, H17),
7.29 (d, 1H, J = 7.6, H18), 7.34 (d, 1H, J = 8.0 Hz, H4),
7.46 (t, 1H, J = 8.0 Hz, H2), 7.50 (d, 1H, J = 7.6 Hz,
H15), 7.7 (td, 1H, J = 8.0, 1.0 Hz, H3), 7.98 (dd, 1H,
J = 8.0, 1.0 Hz, H1).
1726 (C=O) cm-1
.
1H NMR (CDCl3) d: 2.05–2.157 (m, 2H, H10), 2.36–2.45
(m, 2H, H11), 2.72–2.78 (m, 1H, H9), 2.82–2.88 (m, 1H, H9),
5.25 (s, 1H, H7), 7.06 (t, 1H, J = 7.5 Hz, H16), 7.24 (t, 1H,
J = 7.5 Hz, H17), 7.33 (d, 1H, J = 8.0 Hz, H4), 7.35 (t, 1H,
J = 8.0 Hz, H2),7.49-7.46 (m, 2H, H15, H18), 7.57 (td, 1H,
J = 8.0, 1.0 Hz, H3), 7.90 (dd, 1H, J = 8.0, 1.0 Hz, H1).
MS : m/z (%), 424 (M?? 2, 10), 422 (M?, 10), 343
(100), 267 (22).
MS: m/z (%), 380 (M?? 2, 3), 378 (M?, 10), 344 (100),
343 (55), 268 (25).
Anal. Calcd for C22H15ClO4: C, 69.75; H, 3.99. Found:
C, 69.46; H, 3.70.
Anal. Calcd for C22H15BrO4: C, 62.43; H, 3.57. Found:
C, 62.71; H, 3.38.
123

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Med Chem Res (2011) 20:466–474
7-(3-Chlorophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4h)
9,10-Dihydro-7-(3-methoxyphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4k)
IR (KBr) m: 3075 (C–H aromatic), 2965 (C–H aliphatic),
IR (KBr) m: 3080 (C–H aromatic), 2939 (C–H aliphatic),
1726 (C=O) cm-1
.
1736 (C=O) cm-1
.
1H NMR (CDCl3)d: 2.09–2.17 (m, 2H, H10), 2.38–2.50
(m, 2H, H11), 2.74–2.80 (m, 1H, H9), 2.88–2.92 (m, 1H,
H9), 4.96 (s, 1H, H7), 7.15 (dt, 1H, J = 7.5, 1.5 Hz, H18),
7.20 (t, 1H, J = 7.5 Hz, H17), 7.26 (t, 1H, J = 1.5, H14),
7.33 (dd, 1H, J = 8.0, 1.0 Hz,H4), 7.36–7.39 (m, 2H, H16,
H2), 7.59 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.89 (dd, 1H,
J = 8.0, 1.5 Hz, H1).
1H NMR (CDCl3) d: 2.08–2.16 (m, 2H, H10), 2.41–2.49
(m, 2H, H11), 2.73–2.79 (m, 1H, H9), 2.85–2.90 (m, 1H,
H9), 3.78(s, 3H, CH3), 4.99 (s, 1H, H7), 6.72 (dd, 1H,
J = 8.0, 1.5 Hz, H18), 6.94 (t, 1H, J = 1.5 Hz, H14), 6.99
(dd, 1H, J = 8.0, 1.5 Hz H16), 7.17 (t, 1H, J = 8.0 Hz,
H17), 7.32 (d, 1H, J = 8.0 Hz,H4), 7.36 (t, 1H, J = 8.0 Hz
H2), 7.57 (td, 1H, J = 8.0, 1.0 Hz, H3), 7.88 (dd, 1H,
J = 8.0, 1.0 Hz, H1).
MS: m/z (%), 380 (M?? 2, 3.5), 378 (M?, 10), 344
(100), 268 (38), 111 (10).
MS: m/z (%), 374 (M?, 70), 343(65), 267 (100), 93 (90),
76 (95).
Anal. Calcd for C22H15ClO4 : C, 69.75; H, 3.99. Found:
C, 69.47; H, 4.28.
Anal. Calcd for C23H18O5: C, 73.79; H, 4.05. Found: C,
73.95; H, 3.82.
7-(4-Chlorophenyl)-9,10-dihydro-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4i)
9,10-Dihydro-7-(4-methoxyphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4l)
IR (KBr) m: 3100 (C–H aromatic), 2939 (C–H aliphatic),
IR (KBr) m: 3080 (C–H aromatic), 2939 (C–H aliphatic),
1716 (C=O) cm-1
.
1726 (C=O) cm-1
.
1H NMR (CDCl3) d: 2.05–2.16 (m, 2H, H10), 2.42-2.46
(m, 2H, H11), 2.74–2.80 (m, 1H, H9), 2.85–2.91 (m, 1H,
H9), 4.96 (s, 1H, H7), 7.23 (d, 2H, J = 8.5 Hz, H14, H18),
7.33 (d, 2H, J = 8.5 Hz, H15, H17), 7.34–7.39 (m, 2H, H2,
H4),7.61 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.88 (dd, 1H,
J = 8.0, 1.5 Hz, H1).
1H NMR (CDCl3) d: 2.07–2.16 (m, 2H, H10), 2.41–2.46
(m, 2H, H11), 2.73–2.79 (m, 1H, H9), 2.85–2.90 (m, 1H,
H9), 3.74 (s, 3H, CH3), 4.94 (s, 1H, H7), 6.79 (d, 2H,
J = 9.0 Hz, H15, H17), 7.30 (d, 2H, J = 9.0 Hz, H14, H18)
7.32 (dd, 1H, J = 8.0, 1.0 Hz,H4), 7.36 (td, 1H, J = 8.0,
1.0 Hz, H2), 7.57 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.87 (dd,
1H, J = 8.0, 1.5 Hz, H1).
MS: m/z (%), 380 (M??2, 8), 378 (M?, 25), 344 (15),
327(85), 266(80), 216(100), 111(65), 54(95).
Anal. Calcd for C22H15ClO4 : C, 69.75; H, 3.99. Found:
C, 69.97; H, 3.76.
MS: m/z (%), 374 (M?, 18), 343 (10), 300 (10), 267
(100).
Anal. Calcd for C23H18O5: C, 73.79; H, 4.05. Found: C,
73.48; H, 3.83.
9,10-Dihydro-7-(2-methoxyphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4j)
9,10-Dihydro-7-(2-methylphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4m)
IR (KBr) m: 3067 (C–H aromatic), 2945 (C–H aliphatic),
1721 (C=O) cm-1
.
IR (KBr) m: 3078 (C–H aromatic), 2965 (C–H aliphatic),
1H NMR (CDCl3) d: 2.02–2.13 (m, 2H, H10), 2.34–2.43
(m, 2H, H11), 2.70–2.78 (m, 2H, H9), 3.70 (s, 3H, CH3),
5.06 (s, 1H, H7), 6.79 (dd, 1H, J = 8.0, 1.0 Hz, H15), 6.94
(td, 1H, J = 8.0, 1.0 Hz, H17), 7.17 (td, 1H, J = 8.0,
1.5 Hz H16), 7.32 (dd, 1H, J = 8.0, 1.0 Hz, H4), 7.35 (td,
1H, J = 8.0, 1.0 Hz H2), 7.52 (dd, 1H, J = 8.0, 1.5 Hz,
H18), 7.55 (td, 1H, J = 8.0, 1.5 Hz, H3, 7.94 (dd, 1H,
J = 8.0, 1.5 Hz, H1).
1731 (C=O) cm-1
.
1H NMR (CDCl3) d: 2.10–2.13 (m, 2H, H10), 2.40–2.42
(m, 2H, H11), 2.73–2.81 (m, 1H, H9), 2.86–2.90 (m, 1H,
H9), 2.87 (s, 3H, CH3), 5.10 (s, 1H, H7), 6.96–6.97 (m, 1H,
H15), 7.01–7.04 (m, 2H, H16, H17), 7.11–7.13 (m, 1H, H18),
7.32 (dd, 1H, J = 8.0,1.0 Hz, H4), 7.36 (td, 1H, J = 8.0,
1.0 Hz, H2), 7.56 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.90 (dd,
1H, J = 8.0, 1.5 Hz, H1).
MS: m/z (%), 374 (M?, 55), 344 (30), 319 (18), 268
(100), 122 (25), 56 (12).
MS: m/z (%), 358 (M?, 10), 343 (5), 268 (100), 225 (5),
197 (22).
Anal. Calcd for C23H18O5: C, 73.79; H, 4.05. Found: C,
73.98; H, 4.33.
Anal. Calcd for C23H18O4: C, 77.08; H, 5.06. Found: C,
77.31; H, 5.28.
123

Med Chem Res (2011) 20:466–474
473
9,10-Dihydro-7-(3-methylphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4n)
and LS180 (human colorectal adenocarcinoma) cells were
obtained from the National Cell Bank of Iran, Pasteur
Institute, Tehran, Iran. All cell lines were maintained in
RPMI 1640 supplemented with 10% FBS, and 100 units/ml
penicillin-G and 100 lg/ml streptomycin. Cells were grown
in monolayer cultures at 37°C in humidified air containing
5% CO2.
IR (KBr) m: 3100 (C–H aromatic), 2955 (C–H aliphatic),
1726 (C=O) cm-1
.
1H NMR (CDCl3)(400 MHz) d: 1.90–1.97 (m, 1H, H10),
2.01–2.04 (m, 1H, H10), 2.06 (s, 3H, CH3), 2.30–2.38 (m,
2H, H11), 2.51–2.81 (m, 1H, H9), 2.83–2.94 (m, 1H, H9),
4.68 (s, 1H, H7), 6.95 (dd, 1H, J = 9.0, 1.0 Hz, H18),
7.04–7.12 (m, 3H, H14, H16, H17), 7.45 (dd, 1H,
J = 8.0,1.0 Hz, H4), 7.47 (td, 1H, J = 8.0, 1.0 Hz, H2),
7.70 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.95 (dd, 1H, J = 8.0,
1.5 Hz, H1).
Cytotoxicity assay
Cell viability following exposure to synthetic compounds
was estimated by using the MTT reduction assay (Mehdi-
pour et al., 2007; Jabbar et al., 1989; Miri et al., 2004).
MCF-7 and Raji cells were plated in 96-well microplates at
a density of 5 9 104 cells/ml (100 ll per well). LS180 and
HeLa cells were plated at densities of 1 9 105 and
2.5 9 104 cells/ml, respectively. Control wells contained
no drugs, and blank wells contained only growth medium
for background correction. After overnight incubation at
37°C, half of the growth medium was removed and 50 ll
of medium supplemented with different concentrations of
synthetic compounds dissolved in DMSO were added in
quadruplicate. Plates with Raji cells were centrifuged
before this procedure. Maximum concentration of DMSO
in the wells was 0.5%. Cells were further incubated for
72°h, except for HeLa cells, which were incubated for
96 h. At the end of the incubation time, the medium was
removed and MTT was added to each well at a final con-
centration of 0.5 mg/ml and plates were incubated for
another 4 h at 37°C. Then formazan crystals were solubi-
lized in 200 ll DMSO. The optical density was measured
at 570 nm with background correction at 655 nm using a
Bio-Rad microplate reader (Model 680). The percentage of
viability compared to control wells was calculated for each
concentration of the compound and IC16 and IC50 values
(Tallarida and Murray, 1986) were calculated with the
software CurveExpert version 1.34 for Windows. Each
experiment was repeated at least three times and data
presented as mean SD.
MS: m/z (%), 358 (M?, 8), 327 (100), 267 (20), 225
(20), 214 (58), 200 (60), 112 (45), 85 (40).
Anal. Calcd for C23H18O4: C, 77.08; H, 5.06. Found: C,
76.85; H, 4.87.
9,10-Dihydro-7-(4-methylphenyl)-7H,11H-
benzopyrano[3,2-c]chromene-6,8-dione (4o)
IR (KBr) m: 3068 (C–H aromatic), 2955 (C–H aliphatic),
1731 (C=O) cm-1
.
1H NMR (CDCl3) d: 2.05–2.18 (m, 2H, H10), 2.22 (s,
3H, CH3), 2.49–2.37 (m, 2H, H11), 2.73–2.79 (m, 1H, H9),
2.85–2.90 (m, 1H, H9), 4.96 (s, 1H, H7), 7.06 (d, 2H,
J = 8.0 Hz, H15, H17), 7.27(d, 2H, J = 8.0 Hz, H14, H18),
7.34 (dd, 1H, J = 8.0,1.0 Hz, H4),7.37(td, 1H, J = 8.0,
1.0 Hz, H2), 7.56 (td, 1H, J = 8.0, 1.5 Hz, H3), 7.88 (dd,
1H, J = 8.0, 1.5 Hz, H1).
MS: m/z (%), 358 (M?, 20), 344 (65), 267 (100), 225 (5).
Anal. Calcd for C23H18O4: C, 77.08; H, 5.06. Found: C,
77.33; H, 4.87.
Cytotoxicity section
Reagents and chemicals
RPMI 1640, fetal bovine serum (FBS), trypsin, and phos-
phate buffered saline (PBS) were purchased from Biosera
(Ringmer, UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) was obtained from Sigma
(St. Louis, MO, USA) and penicillin/streptomycin were
purchased from Invitrogen (San Diego, CA, USA). Doxo-
rubicin and dimethyl sulphoxide were obtained from EB-
EWE Pharma (Unterach, Austria) and Merck (Darmstadt,
Germany), respectively.
QSAR studies
Chemical structure of the molecules was built by Hyper-
chem software (Version 7, Hypercube Inc., http://www.
hyper.com, USA) for the structural chemistry. Gaussian98
was operated to optimize the molecular structure (Frisch
et al., 1998). The structures were optimized by ab initio
calculations at the level of RHF/STO-3G. No molecular
symmetry constraint was applied; rather full optimization
of all bond lengths and angles was carried out. The root
mean square of kcal mol-1 was used to end geometry
optimization. Then, the molecules were reloaded to
Cell lines and cell culture
HeLa (human cervical adenocarsinoma), MCF-7 (human
breast adenocarcinoma), Raji (Human Burkitt’s lymphoma),
123

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Med Chem Res (2011) 20:466–474
Halland N, Velgoard T, Jørgensen KA (2003) Direct asymmetric
Michael reactions of cyclic 1,3-dicarbonyl compounds and
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Hermodson MA, Barker WM, Link KP (1971) Studies on the
4-hydroxycoumarins. Synthesis of the metabolites and some
other derivatives of warfarin. J Med Chem 14:167–169
Jabbar SA, Twentyman PR, Watson JV (1989) The MTT assay
underestimates the growth inhibitory effects of interferons. Br J
Cancer 60:523–528
Hyperchem and optimized structures were achieved (Miri
et al., 2006).
Acknowledgments This research was supported by grants from the
research council of Tehran University of Medical Sciences, TWAS-IC
(The Academy of Sciences for the Developing World- Iran Chapter)
and INSF (Iran National Science Foundation).
Jin LW, Hua DH, Shie FS, Maezawa I, Sopher B, Martin GM (2002)
Novel tricyclic pyrone compounds prevent intracellular APP
C99-induced cell death. J Mol Neurosci 19:57–61
Kang SS, Kim HJ, Jin C, Lee YS (2009) Synthesis of tyrosinase
inhibitory (4-oxo-4H-pyran-2-yl)acrylic acid ester derivatives.
Bioorg Med Chem Lett 19:188–191
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