Synthesis and in vitro cytotoxic activity of 2-amino-7-(dimethylamino)-4- [(trifluoromethyl)phenyl]-4H-chromenes

Article abstract of DOI:10.1155/2011/929673

Three 2-amino-4-(trifluoromethylphenyl)-3-cyano-7-(dimethylamino) -4H-chromene derivatives were synthesized and their cytotoxic activities were determined against six human tumor cell lines using MTT assay. Condensation of 3-(dimethylamino)phenol, trifluo

Full text of DOI:10.1155/2011/929673

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2011, 8(2), 598-602
http://www.e-journals.net
Synthesis and In Vitro Cytotoxic
Activity of 2-Amino-7-(dimethylamino)-4-
[(trifluoromethyl)phenyl]-4H-chromenes
M. MAHDAVI§, A. ASADIPOUR¶, S. RAJABALIAN¶, M. VOSOOGHI§,
L. FIROOZPOUR§, M. NAKHJIRI§, A. SHAFIEE§ and A. FOROUMADI§¶*
§Department of Medicinal Chemistry
Faculty of Pharmacy and Pharmaceutical Sciences Research Center
Tehran University of Medical Sciences, Tehran 14174, Iran
¶Kerman Neuroscience Research Center
Kerman University of Medical Sciences, Kerman, Iran
aforoumadi@yahoo.com
Received 13 July 2010; Accepted 4 September 2010
Abstract: Three 2-amino-4-(trifluoromethylphenyl)-3-cyano-7-(dimethylamino)
-4H-chromene derivatives were synthesized and their cytotoxic activities were
determined against six human tumor cell lines using MTT assay. Condensation
of 3-(dimethylamino)phenol, trifluoromethybenzaldehydes and malonitrile in
ethanol containing piperidine afforded corresponding chromenes (4a-c). The
structure of the synthesized compound was confirmed by 1H NMR, IR and Mass
spectral data. Among compounds tested, 3-trifluoromethyl analogue (3b) was the
most active against all human tumor cell lines (IC50=12-45 nM).
Keywords: Synthesis, 4H-chromenes, Cytotoxic activity.
Introduction
The correct balance between apoptosis and inhibition of apoptosis is important in preserving
tissue homeostasis and organ morphogenesis1. Apoptosis is one of the main types of
programmed cell death (PCD). This involves a series of biochemical events that lead to a
variety of morphological changes, including cell shrinkage, chromatin condensation, nuclear
and DNA fragmentation2. Since many cancer cells exhibit abnormal inhibition of apoptosis,
Scientifics are interested in the discovery and development of inducers of apoptosis as
potential anticancer agents3.

Synthesis and In Vitro Cytotoxic Activity
599
The discovery of compounds 1-3 (Figure 1) which belong to the 4-aryl-4H-chromenes
family has been reported and is shown to possess anti-cancer activity4. These compounds,
which are potent apoptosis inducers, were found to be highly active in the growth inhibition
MTT assay with IC50 values in low nanomolar ranges.
Herein, we would like to describe the synthesis of some 4-aryl-4H-chromenes having
substitution of trifluoromethyl moiety in 2-, 3- and 4-positions of the phenyl ring
(compounds 4a-c, Figure 1) as potential drugs effecting cancer. The cytotoxic activities of
synthesized compounds were determined against six cancer cell lines using growth
inhibition MTT assay.
OMe
O
O
MeO
Br
OMe
CN
CN
Me2N
O
NH2
Me2N
O
NH2
1
2
X
C
N
Me2N
O
NH2
3, X =Br, Cl, F
4a, X = 2-CF3; 4b, X = 3-CF3; 4c, X =4-CF3
Figure 1. Chemical structures of compounds 1-4
Experimental
All chemical reagents and solvents used in this study were purchased from Merck AG
(Darmstadt, Germany). Melting points were determined using Kofler hot stage apparatus
and are uncorrected. The IR spectra were obtained on a Shimadzu 470 spectrophotometer
(potassium bromide disks). NMR spectra were recorded using a Bruker 80 spectrometer
(Bruker Bioscience, Billerica, MA, USA) and chemical shifts are expressed as δ (ppm) with
tetramethylsilane as internal standard. MS spectra were measured with a Finnigan TSQ 70
Mass spectrophotometer at 70 eV. Elemental analyses were carried out on a HERAEUS
CHN-O rapid elemental analyzer (Heraeus GmbH, Hanau, Germany) for C, H and N and the
results are within 0.4% of the theoretical values. Merck silica gel 60 F254 plates were used
for analytical TLC.
Chemistry
2-Amino-7-(dimethylamino)-4-(trifluoromethylphenyl)-4H-chromene-3-carbonitriles (4a-c)
were synthesized by condensation of 3-(dimethylamino)phenol (5), corresponding
benzaldehyde (6a-c) and malonitrile (7) in ethanol in the presence of piperidine (Scheme 1)5.
1
The structure of synthesized compounds was characterized by IR, H NMR and Mass
spectral data.

600
A. FOROUMADI et al.
CF3
CHO
C
N
C
C
N
Piperidine, EtOH
350 C, 12h
+
CF3
+
N
N
O
NH2
N
OH
7
5
4a-c
6a-c
Scheme 1. Synthesis of 4-aryl-4H-chromenes 4a-c
2-Amino-7-(dimethylamino)-4-[(trifluoromethyl)phenyl]-4H-chromene-3-carbonitrile
(3a-c) (General procedure)
Piperidine (0.85 g, 10 mmol) was added to a mixture of 3-dimethylaminophenol 5 (0.68 g,
5 mmol), corresponding benzaldehyde 6a-c (5 mmol) and malonitrile 7 (0.03 g, 5 mmol) in
ethanol (20 mL). The reaction mixture was stirred at 35 °C for 12 h. After cooling, the
precipitated solid was filtered off, washed with cold ethanol and crystallized from the same
solvent.
2-Amino-7-(dimethylamino)-4-[2-(trifluoromethyl)phenyl]-4H-chromene-3-
carbonitrile (3a)
o
(M.W.: 359.35) Yield 45%; m.p.205-207 C; IR (KBr, cm-1): 3318, 3191 (NH2), 2197 (CN);
1HNMR (CDCl3, 80 MHz) δ: 7.80-7.10 (m, 4H, phenyl), 6.72 (d, 1H, J = 8.2 Hz, H5 chromene),
6.47 (dd, 1H, J = 8.2 and 2.5 Hz, H6 chromene), 6.30 (d, 1H, J = 2.5 Hz, H8 chromene), 5.14 (s, 1H,
H4 chromene), 4.60 (brs, 2H, NH2), 2.92 (s, 6H, NMe2); Ms (m/z, %): 359(M+,100), 341(25),
333(14), 240(87), 214(98), 197(95), 170(83), 134(87), 106(90), 77(91).
2-Amino-7-(dimethylamino)-4-[3-(trifluoromethyl)phenyl]-4H-chromene-3-
carbonitrile (3b)
(M.W.: 359.35) Yield 70%; m.p.199-200 oC; IR (KBr, cm-1): 3354, 3220 (NH2), 2197 (CN);
1HNMR (CDCl3, 80 MHz) δ: 7.56 - 7.32 (m, 4H, phenyl), 6.77 (d, 1H, J = 8.2 Hz, H5
chromene), 6.37 (dd, 1H, J = 8.2 and 2.5 Hz, H6 chromene), 6.26 (d, 1H, J = 2.5 Hz, H8
chromene), 4.72(s, 1H, H4 chromene), 4.64 (brs, 2H, NH2), 2.94 (s, 6H, NMe2); Ms (m/z, %):
359(M+,15), 214(100), 198(11).
2-Amino-7-(dimethylamino)-4-[4-(trifluoromethyl)phenyl]-4H-chromene-3-
carbonitrile (3c)
(M.W.: 359.35) Yield 36%; m.p.209-210 oC; IR (KBr, cm-1): 3329, 3196 (NH2), 2192 (CN);
1HNMR (CDCl3, 80 MHz) δ: 7.57 (d, 2H, J = 7.1 Hz, H3 and H5 phenyl), 7.32 (d, 2H, J =
7.1 Hz, H2 and H6 Phenyl), 6.76 (d, 1H, J = 8.5 Hz, H5 chromene), 6.41 (dd, 1H, J = 8.5 and
2.5 Hz, H6 chromene), 6.28 (d, 1H, J = 2.5 Hz, H8 chromene), 4.73
(s, 1H, H4 chromene),
4.62 (brs, 2H, NH2), 2.95 (s, 6H, NMe2); Ms (m/z, %): 359 (M+,10), 241(12), 214(100),
149(11), 105(11), 97(19), 84(95), 66(60).
Biological activity
Cell lines and cell cultures
The synthesized compounds were tested against six human cancer cell lines including KB
(nasopharyngeal epidermoid carcinoma), EJ (bladder carcinoma), MCF-7 (breast carcinoma),
1321N1 (astrocytoma), Saos-2 (osteosarcoma) and A 2780 CP (ovary carcinoma). The cell lines
were purchased from National Cell Bank of Iran (NCBI). The cells

Synthesis and In Vitro Cytotoxic Activity
601
were grown in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with
10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany), 100 µg/mL streptomycin and
100 u/mL penicillin, in a humidified air atmosphere at 37 °C with 5% CO2.
Cytotoxicity assay
The in vitro cytotoxic activity of each synthesized chromene derivatives 3a-c was
assessed in monolayer cultures using MTT colorimetric assay6,7. Briefly, each cell line
in log-phase of growth was harvested by tripsinization, resuspended in complete growth
medium to give a total cell count of 25×103 cells/ml. 100 micro liter (µL) of the cell
suspension was seeded into the wells of 96-well plates (Nunc, Denmark). The plates
were incubated in a humidified air atmosphere at 37 °C with 5% CO2 overnight. Then,
50 µL of the media containing various concentrations of the compound was added per
well in triplicate. The plates were incubated for further three days. The final
concentration of DMSO in the highest concentration of applied compound was 0.1%.
Vincristine was used as positive control for cytotoxicity while three wells containing
tumor cells cultured in 150 µL of complete medium were used as controls for cell
viability. After incubation, 30 µL of a 2.5 mg/mL solution of MTT (Sigma-Aldrich)8
was added to each well and the plates were incubated for another 1 h. The culture
medium was then replaced with 100 µL of DMSO and the absorbance of each well was
measured by using a microplate 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.
Results and Discussion
The compounds 3a-c was tested in vitro against a panel of six human tumor cell lines. The
percentage of growth was evaluated using MTT colorimetric assay versus controls not
treated with test agents. For each compound, 50% inhibitory concentration (IC50) was
determined and reported in Table 1.
Table 1. Cytotoxic activity (IC50, nM)a of compounds (4a–c) against different cell lines in
comparison with vincristine
CF3
C
N
Me2N
O
NH2
Cell line
MCF-7d 1321N1e
1280 28 305 77
Compound
KBb
175 7
EJc
Saos-2f
325 71
12 5
A 2780 CPg
235 49
12 2
4a (2-CF3)
550 95
30 4
145 35
4b (3-CF3) 12.5 0.7
4c (4-CF3) 113 23
45 5
910 42
8.90 3
15 1.5
195 11
5 1
315 21
2.60 0.50 0.18 0.03
140 84
Vincristine 0.10 0.01 0.24 0.08
a data represented in terms of mean SD. b KB: nasopharyngeal epidermoid carcinoma. c EJ: bladder
e
f
carcinoma. d MCF-7:breast carcinoma. 1321N1: astrocytoma. Saos-2:osteosarcoma. g A 2780 CP:
ovary carcinoma

602
A. FOROUMADI et al.
The data for vincristine was included for comparison. The obtained results revealed that
compounds 3a and 3c possessed poor activity (IC50 > 100 nM) against all cell lines in
contrast with 3b, which possess comparable activity with vincristine. These results
confirmed the reported activity of 3b as a potent inducer of apoptosis using cell-based
apoptosis assay9-11. In general, Table 1 shows that compound 3b was the most active
compound against all tested cell lines.
The results highlight the relationship between substitutions at 2, 3 or 4 positions of the
phenyl ring and cytotoxic activity. Among these positions, substitution at C3 position greatly
influences their potency and spectrum of cytotoxic activity.
Conclusion
In conclusion, we have explored the substitution of the 2, 3 and 4 positions of apoptosis inducing
4-aryl-4H-chromenes as potential anticancer agents. It was found that substitution of the
3-position resulted in a large increase of activity. Anti cancer effects of these compounds in
tumor cells indicated that 3b is a good candidate for further pharmacological studies to discover
effective chemotherapeutic for the treatment of human cancer diseases.
Acknowledgment
This work was supported by grants from the research council of Tehran University of
Medical Sciences and Iran National Science Foundation (INSF).
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