2-Amino-3-cyano-4-(5-arylisoxazol-3-yl)-4H-chromenes: Synthesis and in vitro cytotoxic activity

Article abstract of DOI:10.1002/ardp.201100345

A new series of 4-aryl-4H-chromenes bearing a 5-arylisoxazol-3-yl moiety at the C-4 position were prepared as potential anticancer agents. The in vitro cytotoxic activity of the synthesized compounds was investigated against a panel of tumor cell lines in

Full text of DOI:10.1002/ardp.201100345

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
1
Full Paper
2-Amino-3-cyano-4-(5-arylisoxazol-3-yl)-4H-chromenes:
Synthesis and In Vitro Cytotoxic Activity
Tahmineh Akbarzadeh1, Ali Rafinejad1, Javad Malekian Mollaghasem1, Maliheh Safavi1,2
,
Asal Fallah-Tafti1, Mahboobeh Pordeli2, Sussan Kabudanian Ardestani2, Abbas Shafiee1,
and Alireza Foroumadi1
1 Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development Research
Center, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
A new series of 4-aryl-4H-chromenes bearing a 5-arylisoxazol-3-yl moiety at the C-4 position were prepared
as potential anticancer agents. The in vitro cytotoxic activity of the synthesized compounds was
investigated against a panel of tumor cell lines including MCF-7 (breast cancer), KB (nasopharyngeal
epidermoid carcinoma), Hep-G2 (liver carcinoma), MDA-MB-231 (breast cancer), and SKNMC (human
neuroblastoma) using the MTT colorimetric assay. Doxorubicin, a well-known anticancer drug, was used
as positive standard drug. Among the synthesized compounds, the 5-(3-methylphenyl)isoxazol-3-yl analog
(7j) showed the most potent cytotoxic activity against all five human tumor cell lines.
Keywords: 4-(5-Arylisoxazol-3-yl)-4H-chromenes / Cancer / Cytotoxic activity / Synthesis
Received: October 2, 2011; Revised: November 16, 2011; Accepted: November 25, 2011
DOI 10.1002/ardp.201100345
Introduction
apoptosis, or programmed cell death, is one of the best
understood and most studied cell death pathways.
Excessive inhibition of the normal apoptosis pathway
due to cellular changes results in tumor growth, metasta-
sis, and resistance to chemotherapeutic agents [2, 4–6].
Therefore, targeting the apoptosis pathway to find new
therapeutic agents for neoplastic diseases represents
an opportunity to selectively kill malignant cells while
reducing systemic toxicity.
Cancer has been known as the leading cause of death in
economically developed countries and the second leading
cause of death in developing countries for several years.
Increasing incidence of cancer in economically developing
countries may be a result of population aging and growth
as well as adoption of cancer-associated lifestyle choices includ-
ing smoking, physical inactivity, and fast food diets [1].
The disease is characterized by the uncontrolled growth of
abnormal cells which are self-sufficient in growth signals,
insensitive to antigrowth signals, sustained angiogenesis,
metastasis, and evasion of apoptosis [2].
Many 4-aryl-4H-chromenes (A and B) have been reported to be
potent apoptosis inducers (Fig. 1) [7, 8]. The proposed mechan-
ism for these series of compounds is that they were found to be
tubulin destabilizers, binding at or close to the binding site of
colchicine. They have also shown activity in drug-resistant
cancer cell lines including the paclitaxel-resistant and multi-
drug resistant tumor cells [9, 10]. In addition, diverse groups of
compounds bearing the isoxazole ring have shown cytotoxic
effects [11]. In continuation of our efforts to design or identify
new scaffolds as cytotoxic agents [8, 12, 13], herein, we decided
to introduce the isoxazole ring to the 4 position of a chromene-
based structure (Fig. 1). Thus, we report the synthesis and
evaluation of selected 2-amino-3-cyano-7-dimethylamino-4-(5-
arylisoxazol-3-yl)-4H-chromenes (7a–o) as inhibitors of cell
growth.
Cancer therapy is based on killing cancer cells selectively
without harming the normal cells. A series of regulated
events is involved in cell death of both malignant and
non-malignant cells [3]. Several different mechanisms
of cell death, including apoptosis, necrosis, mitotic
catastrophe, and autophagy have been proposed. Of these,
Correspondence: Alireza Foroumadi, Drug Design & Development
Research Center, Tehran University of Medical Sciences, Tehran, Iran.
E-mail: aforoumadi@yahoo.com
Fax: þ98 21 66461178
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T. Akbarzadeh et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Figure 1. Potent apoptosis inducer chromene-
based structures (A and B), and synthesized
4-(5-arylisoxazol-3-yl)-4H-chromenes (7a–o).
Results and discussion
SKNMC (human neuroblastoma). The percentage of growth
inhibitory activity was evaluated using the MTT colorimetric
assay in comparison with doxorubicin as standard drug. For
each compound, the 50% inhibitory concentration (IC50) was
determined and is reported in Table 1.
Chemistry
The synthetic pathways for the synthesis of key intermediates
4a–o and target compounds 2-amino-7-dimethylamino-4-(5-
arylisoxazol-3-yl)-4H-chromene-3-carbonitrile 7a–o are out-
lined in Scheme 1 and Scheme 2, respectively.
In general, compounds 7c (R ¼ 4-F), 7j (R ¼ 3-CH3), and 7k
(R ¼ 4-CH3) displayed good activity against all tested cell
lines with IC50 values of 6.5 ꢀ 1.4 to 12.3 ꢀ 0.5 mM; these
compounds were also active against MDA-MB-231, SKNMC,
and KB cell lines, while other compounds were inactive or
moderately active. Generally, the MCF-7 and Hep-G2 cell lines
were more sensitive to all the tested compounds compared to
the other cell lines, in which compounds 7c, 7j, and 7k
exhibited higher cytotoxic activity on the mentioned cell
lines.
Ethyl 2,4-dioxo-4-arylbutanoate derivatives 1a–o were
reacted with hydroxylamine hydrochloride to give corre-
sponding ethyl 5-arylisoxazole-3-carboxylates 2a–o, which
reacted with sodium borohydride and converted to 5-aryli-
soxazol-3-ylmethanol derivatives 3a–o. Oxidation of the alco-
hols 3a–o by using MnO2 afforded the desired 5-arylisoxazole-
3-carboxaldehydes 4a–o (Scheme 1) [14]. One-pot three-com-
ponent condensation of the 5-arylisoxazole-3-carboxalde-
hydes 4a–o, malonitrile 5, and 3-(dimethylamino)phenol 6
in the presence of piperidine in EtOH afforded target com-
pounds 7a–o (Scheme 2) [15, 16].
The study of structure activity relationship of these series of
4-aryl-4H-chromenes revealed that 3-methylphenyl substituted
analog 7j was the most potent compound of these series
against all the tested cell lines. This compound showed an
IC50 value of 6.7 ꢀ 2.6 mM in inhibiting the growth of MCF-7.
In comparison, 4-methylphenyl substituted analog 7k demon-
strated decrease in inhibitory potency when compared to 7j
in all tested cell lines. However, in the 4-methoxyphenyl
analogue (compound 7m) changing the position of methoxy
In vitro cytotoxic activity
The synthesized compounds 7a–o were tested against a panel
of five human tumor cell lines including MCF-7 (breast
cancer), KB (nasopharyngeal epidermoid carcinoma), Hep-
G2 (liver carcinoma), MDA-MB-231 (breast cancer), and
Scheme 1. Synthesis of key intermediates 5-
arylisoxazole-3-carboxaldehydes 4a–o.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
4-(5-Arylisoxazol-3-yl)-4H-chromenes with Cytotoxic Activity
3
Scheme 2. One-pot synthesis of 4-(5-aryl-
isoxazol-3-yl)-4H-chromenes 7a–o.
group (R ¼ 3-OCH3) has led to loss of activity. On the other
hand, 3,4-dimethoxyphenyl substitution in compound 7n
showed increased activity compared to compound 7l. In
addition, 3,4,5-tri-substituted analog 7o was less active than
7n, suggesting that bulky substitutions may be less preferred
for cytotoxic activity in these compounds. Comparison of
compound 7h with compounds 7j and 7l showed that electron
withdrawing groups on the 3 position can deteriorate the
cytotoxic activity. Comparison of IC50 values of unsubstituted
compound 7a with other compounds reveals that this com-
pound has considerable activity against all tested cell lines.
In halo-substituted analogues, compound 7c (R ¼ 4-F) was
the most potent compound, while movement of the fluorine
atom to position 2 of the phenyl ring in compound 7b
led to decreased activity in all tested cell lines. Other halo-
and nitro-substituted compounds showed moderate to week
cytotoxic activity and the influence of the position of the
halogen atom or nitro group on the cytotoxic activity was
variable.
This study provides insights for further optimization of the
4-aryl-4H-chromene scaffold for generating novel anticancer
agents.
Table 1. Cytotoxic activity (IC50 in mM) of compounds 7a–o against different cancer cell lines
Compounds
R
MCF7
Hep-G2
MDA-MB-231
KB
SKNMC
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
H
2-F
4-F
2-Cl
4-Cl
4-Br
10.9 ꢀ 0.4
15.9 ꢀ 1
8.8 ꢀ 1.1
11.8 ꢀ 1.6
10.6 ꢀ 4.7
12.0 ꢀ 1.4
13.3 ꢀ 3.6
>30
22.6 ꢀ 2.6
6.7 ꢀ 2.6
9.8 ꢀ 0.9
16.1 ꢀ 1.3
>30
11.5 ꢀ 0.4
13.2 ꢀ 0.9
9.5 ꢀ 0.9
10 ꢀ 1.3
19.3 ꢀ 5.4
>30
17.5 ꢀ 1.3
22.3 ꢀ 2.3
11.9 ꢀ 0.8
>30
16.5 ꢀ 1.5
20.5 ꢀ 4.9
14.3 ꢀ 3.8
13.8 ꢀ 1.6
15.6 ꢀ 2.0
12.0 ꢀ 5.0
>30
>30
>30
8.7 ꢀ 0.9
12.3 ꢀ 0.5
23.6 ꢀ 1.7
>30
17.5 ꢀ 12.1
>30
>30
15.2 ꢀ 0.3
10.4 ꢀ 1.6
16.2 ꢀ 3.8
26.7 ꢀ 4.6
16.1 ꢀ 4.5
6.5 ꢀ 1.4
10.9 ꢀ 0.1
16.3 ꢀ 3.2
>30
>30
15 ꢀ 0.1
14.7 ꢀ 0.7
2,4-Cl
3-NO2
4-NO2
3-CH3
4-CH3
3-OCH3
4-OCH3
3,4-OCH3
3,4,5-OCH3
–
>30
>30
>30
>30
>30
>30
12.0 ꢀ 4.1
12.1 ꢀ 2.6
>30
11.5 ꢀ 0.6
11.6 ꢀ 4.9
>30
>30
20.1 ꢀ 6.5
>30
12.2 ꢀ 0.7
15.1 ꢀ 0.9
18.5 ꢀ 1.2
1.4 ꢀ 0.4
12.7 ꢀ 1.0
18.5 ꢀ 7.5
0.5 ꢀ 0.1
12.6 ꢀ 0.6
>30
0.93 ꢀ 0.4
>30
4.3 ꢀ 0.2
>30
1.4 ꢀ 0.3
Doxorubicin
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
vacuum and recrystallized from dichloromethane to give
compounds 3a–o.
Experimental
Chemistry
(5-Phenylisoxazol-3-yl)methanol 3a
All starting materials, reagents, and solvents were purchased
from Merck AG (Germany). The purity of the synthesized com-
pounds was confirmed by thin layer chromatography (TLC) using
various solvents of different polarities. Merck silica gel 60 F254
plates were applied for analytical TLC. Melting points were
determined on a Kofler hot stage apparatus (Vienna, Austria)
and are uncorrected. 1H-NMR spectra were recorded using a
Bruker 500 spectrometer (Bruker, Rheinstetten, Germany), and
chemical shifts are expressed as d (ppm) with tetramethylsilane
(TMS) as internal standard. The IR spectra were obtained on
a Shimadzu 470 (Shimadzu, Tokyo, Japan) spectrophotometer
(potassium bromide disks). The mass spectra were run on a
FinniganTSQ-70 spectrometer (Finnigan, USA) at 70 eV.
Elemental analyses were carried out on a CHNO rapid elemental
analyzer (Heraeus GmbH, Hanau, Germany) for C, H, and N, and
the results are within ꢀ0.4% of the theoretical values.
Yield: 80%; m.p.: 91–928C; IR (KBr, cmꢂ1) ymax: 3322 (OH), 3055
(C–H aromatic), 2951 (C–H, aliphatic); 1H-NMR (CDCl3) d: 7.81–
7.76 (m, 2H, arom.), 7.53–7.44 (m, 3H, arom.), 6.59 (s, 1H,
isoxazole), 4.83 (s, 2H, CH2); MS (m/z, %): 175 [Mþ] (100), 145
(15), 105 (20), 77 (10). Anal. calcd. for C10H9NO2: C, 68.56;
H, 5.18; N, 8.00. Found: C, 68.42; H, 5.28; N, 8.17.
(5-(2,4-Dichlorophenyl)isoxazol-3-yl)methanol 3g
Yield: 84%; m.p.: 78–798C; IR (KBr, cmꢂ1) ymax: 3220 (OH), 2923 (C–H,
aliphatic), 1H-NMR (CDCl3) d: 7.89 (d, J ¼ 8.4 Hz, 1H, H6phenyl), 7.54
(d, J ¼ 1.6 Hz, 1H, H3phenyl), 7.39 (dd, J ¼ 8.4 and 1.6 Hz, 1H,
H5phenyl), 7.01 (s, 1H, isoxazole), 4.85 (s, 2H, CH2), 1.45 (t, 3H,
CH3); MS (m/z, %): 245 [Mþþ2] (17), 243 [Mþ] (30), 242 (53), 214
(8), 212 (12), 175 (68), 173 (100). Anal. calcd. for C10H7Cl2NO2:
C, 49.21; H, 2.89; N, 5.74. Found: C, 49.27; H, 3.04; N, 5.82.
General procedure for preparation of 5-substituted phenyl
isoxazole-3-carboxylate 2a–o
General procedure for preparation of 5-substituted phenyl
isoxazole-3-carboxaldehyde 4a–o
A mixture of hydroxylamine hydrochloride (20 g, 0.23 mole) and
the appropriate ethyl acylpyruvate (0.080 mole) in ethanol
(200 mL) was heated under reflux for 3 h. The mixture was
partially concentrated under vacuum, diluted with water
(200 mL), and extracted with diethyl ether (3 mL ꢁ 100 mL).
The organic extracts were washed with brine, then with 1 N
sodium hydroxide solution (50 mL), and finally with brine.
Evaporation of the solvent and distillation afforded the isoxazole
esters 2a–o.
A mixture of 5-substituted phenyl isoxazole-3-methanoles 3a–o
(0.02 mole) and MnO2 (0.288 mole) in chloroform (100 mL) was
stirred at room temperature for 24 h. The mixture was then
filtered through a pad of celite and the filtrate was concentrated
under reduced pressure. The product was crystallized from
methanol/water to afford the corresponding aldehydes 4a–o.
5-Phenylisoxazole-3-carbaldehyde 4a
Yield: 64%; m.p.: 59–618C; IR (KBr, cmꢂ1) ymax: 3056 (C–H aro-
1
matic), 1714 (C O); H-NMR (CDCl ) d: 10.21 (s, 1H, C–H aldehyde),
–
–
3
7.85–7.80 (m, 2H, arom.), 7.53–7.48 (m, 3H, arom.), 6.91 (s, 1H,
isoxazole); MS (m/z, %): 173 [Mþ] (100), 171 (100), 106 (15), 77 (90),
63 (23). Anal. calcd. for C10H7NO2: C, 69.36; H, 4.07; N, 8.09. Found:
C, 69.25; H, 4.16; N, 8.22.
Ethyl 5-phenyl isoxazole-3-carboxylate 2a
Yield: 50%; m.p.: 52–538C; IR (KBr, cmꢂ1) ymax: 3063 (C–H aro-
1
matic), 2924 (C–H, aliphatic), 1732 (C O); H-NMR (CDCl ) d: 7.84–
–
–
3
7.80 (m, 2H, arom.), 7.53–7.47 (m, 3H, arom.), 6.94 (s, 1H,
isoxazole), 4.48 (q, 2H, CH2), 1.45 (t, 3H, CH3); Anal. calcd. for
C12H11NO3: C, 66.35; H, 5.10; N, 6.45. Found: C, 66.42; H, 5.28;
N, 6.12.
5-(2,4-Dichlorophenyl)isoxazole-3-carbaldehyde 4g
Yield: 52%; m.p.: 109–1108C; IR (KBr, cmꢂ1) ymax: 3101 (C–H aro-
1
matic), 1716 (C O); H-NMR (CDCl ) d: 10.22 (s, 1H, C–H aldehyde),
–
–
3
7.94 (d, J ¼ 8.8 Hz, 1H, H6phenyl), 7.57 (d, J ¼ 2.0 Hz, 1H,
H3phenyl), 7.42 (dd, J ¼ 8.8 and 2.0 Hz, 1H, H5phenyl), 7.32
(s, 1H, isoxazole); MS (m/z, %): 241 [Mþ] (23), 240 (44), 175 (63),
173 (100), 144 (24), 109 (18), 68 (8). Anal. calcd. for C10H5Cl2NO2:
C, 49.62; H, 2.08; N, 5.79. Found: C, 49.55; H, 2.18; N, 5.82.
Ethyl 5-(2,4-dichlorophenyl)isoxazole-3-carboxylate 2g
Yield: 57%; m.p.: 97–988C; IR (KBr, cmꢂ1) ymax: 3103 (C–H aro-
1
matic), 2986 (C–H, aliphatic), 1726 (C O); H-NMR (CDCl ) d: 7.93
–
–
3
(d, J ¼ 8.8 Hz, 1H, H6phenyl), 7.56 (d, J ¼ 1.6 Hz, 1H, H3phenyl),
7.42 (dd, J ¼ 8.8 and 1.6 Hz, 1H, H5phenyl), 7.34 (s, 1H, isoxazole),
4.49 (q, 2H, CH2), 1.45 (t, 3H, CH3); MS (m/z, %): 285 [Mþ] (5), 240
(11), 239 (17), 212 (30), 175 (64), 173 (100). Anal. calcd. for
C12H9Cl2NO3: C, 50.38; H, 3.17; N, 4.90. Found: C, 50.27;
H, 3.04; N, 4.72.
General procedure for preparation of 2-amino-3-cyano-7-
dimethylamino-4-(5-arylisoxazol-3-yl)-4H-chromenes 7a–o
Piperidine (10 mmol) was added to a mixture of the appropriate
aldehyde 4a–o (5 mmol), malonitrile (10.5 mmol), and 3-(dime-
thylamino)phenol (11.5 mmol) in ethanol (20 mL). The reaction
mixture was stirred at 358C for 12 h. After cooling, the precipi-
tated solid was filtered, washed with cold ethanol, and crystal-
lized from ethanol.
General procedure for preparation of 5-substituted phenyl
isoxazole-3-methanoles 3a–o
To an ice cooled and stirred solution of the isoxazole ester (2a–
o) (10 g, 0.048 mole) in dry ethanol (100 mL) was added por-
tion-wise sodium borohydride (4 g, 0.11 mole). The resulting
solution was stirred at room temperature for 3 h, carefully
acidified with 1 N hydrochloric acid, and concentrated
under vacuum. The aqueous solution was extracted with
diethyl ether (3 mL ꢁ 100 mL) and concentrated under
2-Amino-3-cyano-7-dimethylamino-4-(5-phenylisoxazol-3-
yl)-4H-chromene 7a
Yield: 30%; m.p.: 214–2168C; IR (KBr, cmꢂ1) ymax: 3428 and 3315
(NH2), 2921 (C–H, aliphatic), 2197 (CN); H-NMR (CDCl3) d: 7.73–
1
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
4-(5-Arylisoxazol-3-yl)-4H-chromenes with Cytotoxic Activity
5
7.70 (m, 2H, arom.), 7.43–7.39 (m, 3H, arom.), 7.03 (d, J ¼ 8.4 Hz,
1H, H5chromene), 6.48 (dd, J ¼ 2.4 and 8.4 Hz, 1H, H6
chromene), 6.32 (s, 1H, isoxazole), 6.29 (d, J ¼ 2.4 Hz, 1H,
H8chromene), 4.98 (s, 1H, H4chromene), 4.71 (s, 2H, NH2),
2.94 (s, 6H, 2 ꢁ CH3); MS (m/z, %): 359 [Mþþ1] (5), 358 [Mþ]
(27), 214 (100), 198 (17), 105 (28), 77 (38), 51 (18). Anal. calcd.
for C21H18N4O2: C, 70.38; H, 5.06; N, 15.63. Found: C, 70.71;
H, 5.38; N, 15.95.
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-bromophenyl)-
isoxazol-3-yl)-4H-chromene 7f
Yield: 40%; m.p.: 202–2048C; IR (KBr, cmꢂ1) ymax: 3457 and 3287
(NH2), 2920 (C–H, aliphatic), 2179 (CN); H-NMR (CDCl3) d: 7.61–
1
7.50 (m, 4H, arom.), 7.02 (d, J ¼ 9.6 Hz, 1H, H5chromene), 6.48
(dd, J ¼ 4.1 and 9.6 Hz, 1H, H6chromene), 6.32 (s, 1H, isoxazole),
6.29 (d, J ¼ 4.1 Hz, 1H, H8chromene), 4.98 (s, 1H, H4chromene),
4.71 (s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3); MS (m/z, %): 438 [Mþþ2]
(47), 436 [Mþ] (47), 356 (9), 281 (10), 214 (100), 198 (72), 185 (65),
155 (34). Anal. calcd. for C21H17BrN4O2: C, 57.68; H, 3.92; N, 12.81.
Found: C, 57.95; H, 4.08; N, 12.95.
2-Amino-3-cyano-7-dimethylamino-4-(5-(2-fluorophenyl)-
isoxazol-3-yl)-4H-chromene 7b
Yield: 40%; m.p.: 212–2148C; IR (KBr, cmꢂ1) ymax: 3430 and 3315
(NH2), 2921 (C–H, aliphatic), 2197 (CN); H-NMR (CDCl3) d: 7.96–
1
2-Amino-3-cyano-7-dimethylamino-4-(5-(2,4-
7.90 (m, 1H, arom.), 7.42–7.35 (m, 1H, arom.), 7.27–7.20 (m, 1H,
arom.), 7.21–7.11 (m, 1H, arom.), 7.01 (d, J ¼ 8.4 Hz, 1H,
H5chromene), 6.50 (d, J ¼ 2.4 Hz, H8chromene), 6.47 (dd,
J ¼ 8.4 and 2.4 Hz, 1H, H6chromene), 6.30 (d, 1H, isoxazole),
5.01 (s, 1H, H4chromene), 4.72 (s, 2H, NH2), 2.94 (s, 6H,
2 ꢁ CH3); MS (m/z, %): 376 [Mþ] (10), 336 (4), 213 (87), 186 (14),
123 (100), 95 (34), 75 (16). Anal. calcd. for C21H17FN4O2: C, 67.01;
H, 4.55; N, 14.89. Found: C, 67.39; H, 4.26; N, 15.11.
dichlorophenyl)isoxazol-3-yl)-4H-chromene 7g
Yield: 43%; m.p.: 188–1908C; IR (KBr, cmꢂ1) ymax: 3420 and 3314 (NH2),
3109 (C–H aromatic), 2898 (C–H, aliphatic), 2198 (CN); 1H-NMR (CDCl3)
d: 77.89 (d, J ¼ 8.8 Hz, 1H, arom.), 7.49 (d, J ¼ 2.0 Hz, 1H, arom.),
7.36 (dd, J ¼ 2.0 and 8.8 Hz, 1H, arom.), 7.01 (d, J ¼ 8.8 Hz, 1H,
H5chromene), 6.74 (s, 1H, isoxazole), 6.48 (dd, J ¼ 2.4 and 8.8 Hz,
1H, H6chromene), 6.29 (d, J ¼ 2.4 Hz, 1H, H8chromene), 5.01 (s, 1H,
H4chromene), 4.71 (s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3); Anal. calcd. for
C21H16Cl2N4O2: C, 59.03; H, 3.77; N, 13.11. Found: C, 59.37; H, 4.02;
N, 13.45.
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-fluorophenyl)-
isoxazol-3-yl)-4H-chromene 7c
Yield: 45%; m.p.: 200–2028C; IR (KBr, cmꢂ1) ymax: 3460 and 3288
(NH2), 2920 (C–H, aliphatic), 2179 (CN); 1H-NMR (CDCl3) d: 7.73–
7.67 (m, 2H, arom.), 7.11 (t, 2H, arom.), 7.02 (d, J ¼ 8.0 Hz, 1H,
H5chromene), 6.48 (dd, J ¼ 2.8 and 8.0 Hz, 1H, H6chromene), 6.29
(d, J ¼ 2.8 Hz, 1H, H8chromene), 6.27 (s, 1H, isoxazole), 4.98 (s, 1H,
H4chromene), 4.71 (s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3); MS (m/z, %): 376
[Mþ] (5), 318 (17), 213 (90), 186 (22), 136 (35), 123 (100), 95 (68), 75 (30).
Anal. calcd. for C21H17FN4O2: C, 67.01; H, 4.55; N, 14.89. Found:
C, 66.88; H, 4.23; N, 14.89.
2-Amino-3-cyano-7-dimethylamino-4-(5-(3-nitrophenyl)-
isoxazol-3-yl)-4H-chromene 7h
Yield: 38%; m.p.: 178–1808C; IR (KBr, cmꢂ1) ymax: 3466 and 3345
(NH2), 2922 (C–H, aliphatic), 2188 (CN), 1527 and 1348 (NO2); H-
1
NMR (CDCl3) d: 8.54–8.51 (m, 1H, arom.), 8.28–8.24 (m, 1H, arom.),
8.07 (d, 1H, arom.), 7.64 (t, 1H, arom.), 7.01 (d, J ¼ 8.8 Hz, 1H,
H5chromene), 6.49 (dd, J ¼ 2.0 and 8.8 Hz, 1H, H6chromene), 6.48
(s, 1H, isoxazole), 6.29 (d, J ¼ 2.0 Hz, 1H, H8chromene), 5.02 (s, 1H,
H4chromene), 4.78 (s, 2H, NH2), 2.96 (s, 6H, 2 ꢁ CH3). Anal. calcd.
for C21H17N5O4: C, 62.53; H, 4.25; N, 17.36. Found: C, 62.88; H, 4.39;
N, 17.12.
2-Amino-3-cyano-7-dimethylamino-4-(5-(2-chlorophenyl)-
isoxazol-3-yl)-4H-chromene 7d
Yield: 35%; m.p.: 202–2048C; IR (KBr, cmꢂ1) ymax: 3424 and 3316
(NH2), 2921 (C–H, aliphatic), 2198 (CN); 1H-NMR (CDCl3) d: 7.97–
7.91 (m, 1H, arom.), 7.49–7.42 (m, 1H, arom.), 7.41–7.31 (m, 2H,
arom.), 7.02 (d, J ¼ 8.8 Hz, 1H, H5chromene), 6.74 (s, 1H, isoxa-
zole), 6.49 (dd, J ¼ 2.4 and 8.8 Hz, 1H, H6chromene), 6.29
(d, J ¼ 2.4 Hz, 1H, H8chromene), 5.01 (s, 1H, H4chromene), 4.65
(s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3); MS (m/z, %): 394 [Mþþ2] (5), 392
[Mþ] (15), 362 (100), 214 (76), 198 (16), 139 (18), 57 (35). Anal. calcd.
for C21H17ClN4O2: C, 64.21; H, 4.36; N, 14.26. Found: C, 64.21;
H, 4.67; N, 14.12.
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-nitrophenyl)-
isoxazol-3-yl)-4H-chromene 7i
Yield: 45%; m.p.: 208–2108C; IR (KBr, cmꢂ1) ymax: 3453 and 3287
(NH2), 2921 (C–H, aliphatic), 2179 (CN), 1519 and 1336 (NO2); 1H-NMR
(CDCl3) d: 8.30 (d, J ¼ 8.8 Hz, 2H, arom.), 7.91 (d, J ¼ 8.0 Hz, 2H,
arom.), 6.98 (d, J ¼ 8.0 Hz, 1H, H5chromene), 6.54 (s, 1H, isoxazole),
6.48 (dd, 1–H, H6chromene), 6.30 (bs, 1H, H8chromene), 5.65 (s, 1H,
H4chromene), 4.95 (s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3); MS (m/z, %): 403
[Mþ] (26), 264 (6), 214 (100), 198 (17), 150 (47), 104 (18), 76 (20), 57 (17).
Anal. calcd. for C21H17N5O4: C, 62.53; H, 4.25; N, 17.36. Found:
C, 62.35; H, 4.11; N, 17.09.
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-chlorophenyl)-
2-Amino-3-cyano-7-dimethylamino-4-(5-(3-methylphenyl)-
isoxazol-3-yl)-4H-chromene 7e
Yield: 37%; m.p.: 199–2018C; IR (KBr, cmꢂ1) ymax: 3456 and 3285
(NH2), 2921 (C–H, aliphatic), 2181 (CN); 1H-NMR (CDCl3) d: 7.65
(d, J ¼ 8.0 Hz, 2H, arom.), 7.39 (d, J ¼ 8.0 Hz, 2H, arom.), 7.02
(d, J ¼ 8.8 Hz, 1H, H5chromene), 6.48 (dd, J ¼ 2.8 and 8.8 Hz,
1H, H6chromene), 6.31 (s, 1H, isoxazole), 6.29 (d, J ¼ 2.8 Hz, 1H,
H8chromene), 5.01 (s, 1H, H4chromene), 4.65 (s, 2H, NH2), 2.94
(s, 6H, 2 ꢁ CH3); MS (m/z, %): 394 [Mþþ2] (8), 392 [Mþ] (23),
362 (53), 214 (100), 198 (19), 139 (52), 111 (34). Anal. calcd. for
C21H17ClN4O2: C, 64.21; H, 4.36; N, 14.26. Found: C, 64.04; H, 4.09;
N, 14.52.
isoxazol-3-yl)-4H-chromene 7j
Yield: 25%; m.p.: 181–1838C; IR (KBr, cmꢂ1) ymax: 3432 and 3320 (NH2),
2920 (C–H, aliphatic), 2196 (CN); 1H-NMR (CDCl3) d: 7.58–7.48 (m, 2H,
arom.), 7.35–7.27 (m, 2H, arom.), 7.23–7.19 (m, 1H, H5chromene), 7.03
(d, 1H, H6chromene), 6.52–6.45 (m,1H, H8chromene), 6.30 (s, 1H,
isoxazole), 6.28 (s, 1H, H8chromene), 4.97 (s, 1H, H4chromene), 4.72
(s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3), 2.37 (s, 3H, CH3); MS (m/z, %): 372
[Mþ] (29), 362 (20), 214 (100), 198 (17), 71 (57), 57 (93). Anal. calcd. for
C22H20N4O2: C, 70.95; H, 5.41; N, 15.04. Found: C, 71.22; H, 5.65;
N, 14.87.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
T. Akbarzadeh et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-methylphenyl)-
Biological activity
Cell lines and cell culture
isoxazol-3-yl)-4H-chromene 7k
Yield: 27%; m.p.: 182–1848C; IR (KBr, cmꢂ1) ymax: 3461 and 3285
(NH2), 2922 (C–H, aliphatic), 2179 (CN); 1H-NMR (CDCl3) d: 7.60
(d, J ¼ 8.0 Hz, 2H, arom.), 7.21 (d, J ¼ 8.0 Hz, 2H, arom.), 7.03
(d, J ¼ 8.8 Hz, 1H, H5chromene), 6.48 (dd, 1H, H6chromene),
6.29 (d, 1H, H8chromene), 6.26 (s, 1H, isoxazole), 4.97 (s, 1H,
H4chromene), 4.74 (s, 2H, NH2), 2.94 (s, 6H, 2 ꢁ CH3), 2.37
(s, 3H, CH3); MS (m/z, %): 372 [Mþ] (31), 343 (1), 280 (2), 214
(100), 198 (17), 170 (5), 119 (16), 91 (16). Anal. calcd. for
C22H20N4O2: C, 70.95; H, 5.41; N, 15.04. Found: C, 70.76;
H, 5.22; N, 15.36.
The synthesized compounds were tested against different
human cancer cell lines including MCF-7 (breast cancer), KB
(nasopharyngeal epidermoid carcinoma), Hep-G2 (liver carci-
noma), MDA-MB-231 (breast cancer), and SKNMC (human neuro-
blastoma). The cell lines were purchased from the National Cell
Bank of Iran (NCBI). The cells were grown in Dulbecco’s modified
Eagle medium (DMEM, Sigma–Aldrich) supplemented with 10%
heat-inactivated fetal calf serum (Biochrom, Berlin, Germany),
100 mg/mL streptomycin, and 100 U/mL penicillin, in a humidi-
fied air atmosphere at 378C with 5% CO2.
2-Amino-3-cyano-7-dimethylamino-4-(5-(3-
methoxyphenyl)isoxazol-3-yl)-4H-chromene 7l
Cytotoxicity assay
The in vitro cytotoxic activity of each synthesized chromene deriva-
tive 7a–o was assessed in monolayer cultures using MTT colori-
metric assay [17]. Briefly, each cell line in log-phase of growth was
harvested by trypsinization, resuspended in complete growth
medium to give a total cell count of 5 ꢁ 104 cells/mL. Hundred
microliters of the cell suspension was seeded into the wells
of 96-well plates (Nunc, Denmark). The plates were incubated
overnight in a humidified air atmosphere at 378C with 5% CO2.
Then, 50 mL of the media containing various concentrations
of the compound was added per well in triplicate. The plates
were incubated for further 3 days. The final concentration
of DMSO in the highest concentration of the applied compounds
was 0.1%. Doxorubicin was used as positive control for cyto-
toxicity while three wells containing tumor cells cultured
in 150 mL of complete medium were used as controls for
cell viability. After incubation, 30 mL of a 2.5 mg/mL solution
of MTT (Sigma–Aldrich) was added to each well and the
plates were incubated for another 1 h. The culture medium
was then replaced with 100 mL of DMSO and the absorbance
of each well was measured by using a micro-plate reader at
570 nm. Each set of experiments was independently performed
three times. For each compound, the concentration causing
50% cell growth inhibition (IC50) compared with the control
was calculated from concentration–response curves by
regression analysis.
Yield: 30%; m.p.: 216–2188C; IR (KBr, cmꢂ1) ymax: 3452 and 3277
(NH2), 2923 (C–H, aliphatic), 2177 (CN); H-NMR (CDCl3) d: 7.37–
1
7.23 (m, 3H, arom.), 7.03 (d, J ¼ 8.8 Hz, 1H, H5chromene), 6.96–
6.91 (m, 1H, arom.), 6.48 (dd, J ¼ 2.8 and 8.8 Hz, H6chromene),
6.31 (s, 1H, isoxazole), 6.29 (d, J ¼ 2.8 Hz, 1H, H8chromene), 5.01
(s, 1H, H4chromene), 4.73 (s, 2H, NH2), 3.84 (s, 3H, OCH3), 2.94
(s, 6H, 2 ꢁ CH3); Anal. calcd. for C22H20N4O3: C, 68.03; H, 5.19;
N, 14.42. Found: C, 68.37; H, 4.89; N, 14.13.
2-Amino-3-cyano-7-dimethylamino-4-(5-(4-
methoxyphenyl)isoxazol-3-yl)-4H-chromene 7m
Yield: 30%; m.p.: 206–2088C; IR (KBr, cmꢂ1) ymax: 3464 and 3288 (NH2),
2927 (C–H, aliphatic), 2178 (CN); 1H-NMR (CDCl3) d: 7.65 (dd, J ¼ 1.6
and 6.8 Hz, 2H, arom.), 7.03 (d, J ¼ 8.8 Hz, 1H, H5chromene), 6.92
(dd, J ¼ 1.6 and 6.8 Hz, 2H, arom.), 6.47 (dd, J ¼ 2.4 and 8.4 Hz,
H6chromene), 6.29 (d, J ¼ 2.4 Hz, 1H, H8chromene), 6.19 (s, 1H, iso-
xazole), 4.95 (s, 1H, H4chromene), 4.70 (s, 2H, NH2), 3.84 (s, 3H, OCH3),
2.94 (s, 6H, 2 ꢁ CH3); Anal. calcd. for C22H20N4O3: C, 68.03; H, 5.19;
N, 14.42. Found: C, 67.88; H, 5.47; N, 14.69.
2-Amino-3-cyano-7-dimethylamino-4-(5-(3,4-
dimethoxyphenyl)isoxazol-3-yl)-4H-chromene 7n
Yield: 28%; m.p.: 192–1948C; IR (KBr, cmꢂ1) ymax: 3441 and 3352
(NH2), 2923 (C–H, aliphatic), 2187 (CN); 1H-NMR (CDCl3) d: 7.29
(dd, J ¼ 2.4 and 8.8 Hz, 1H, arom.), 7.22 (s, 1H, arom.), 7.03
(d, J ¼ 8.4 Hz, 1H, H5chromene), 6.89 (d, J ¼ 8.8 Hz, 2H, arom.),
6.48 (dd, J ¼ 2.4 and 8.4 Hz, H6chromene), 6.29 (d, J ¼ 2.4 Hz, 1H,
H8chromene), 6.21 (s, 1H, isoxazole), 4.97 (s, 1H, H4chromene),
4.70 (s, 2H, NH2), 3.92 (d, J ¼ 4.4 Hz, 6H, 2 ꢁ OCH3), 2.94
(s, 6H, 2 ꢁ CH3); MS (m/z, %): 418 [Mþ] (20), 354 (20), 280 (10),
214 (100), 189 (34), 165 (72), 150 (18), 79 (20). Anal. calcd. for
C23H22N4O4: C, 66.02; H, 5.30; N, 13.39. Found: C, 66.07; H, 5.05;
N, 13.12.
This research was supported by a grant from Tehran University of Medical
Sciences.
The authors have declared no conflict of interest.
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