Month 2014
Synthesis and Cytotoxic Activity of Some Novel Dihyrobenzo[h]pyrano[3,2-c]
chromene Derivatives
8.31 (d, J = 7.7 Hz, 1H, H10); Anal. Calcd for C25H18N2O5:
C, 70.42; H, 4.25; N, 6.57. Found: C, 70.66; H, 4.48; N, 6.83.
3-Amino-12-oxo-1-(3,4,5-trimethoxyphenyl)-1,12-dihydrobenzo
3-Amino-1-(2-fluorophenyl)-12-oxo-1,12-dihydrobenzo[h]
pyrano[3,2-c]chromene-2-carbonitrile (4p). Yield 86%, mp
248–250ꢀC. IR (KBr, υ, cmꢁ1): 3403, 3325 (NH2), 2200 (CN),
1
[h]pyrano[3,2-c]chromene-2-carbonitrile (4j). Yield 75%,
1713 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 4.77 (s, 1H,
mp 293–295ꢀC. IR (KBr, υ, cmꢁ1): 3448, 3310 (NH2), 2188
0
0
0
CH, H1), 7.14–7.19 (m, 2H, H3 , H5 ), 7.30–7.40 (m, 2H, H4 ,
1
0
(CN), 1721 (CO); H NMR (500MHz, DMSO-d6) d ppm: 3.64
H6 ), 7.48 (s, 2H, NH2), 7.72–7.78 (m, 2H, H8, H9), 7.90
(d, J = 8.7 Hz, 1H, H5), 7.99 (d, J = 8.7 Hz, 1H, H6), 8.09
(d, J = 8.0 Hz, 1H, H7), 8.30 (d, J = 8.0 Hz, 1H, H10); 13CNMR
(125 MHz, DMSO-d6) d ppm: 31.3, 56.4, 102.19, 108.3, 115.4,
115.6, 117.8, 119.0, 121.4, 121.8, 124.5, 124.6, 127.8, 128.2,
129.2, 129.8, 129.9, 130.3, 134.6, 149.5, 154.7, 158.3, 159.3,
160.3 (JC–F = 231.2 Hz); Anal. Calcd for C23H13FN2O3: C, 71.87;
(s, 3H, OCH3), 3.73 (s, 6H, OCH3), 4.48 (s, 1H, CH, H1), 6.57
0
0
(s, 2H, H2 , H6 ), 7.41 (s, 2H, NH2), 7.73–7.77 (m, 2H, H8, H9),
7.89 (d, J = 8.7 Hz, 1H, H5), 7.97 (d, J = 8.7 Hz, 1H, H6), 8.08
(d, J = 8.0Hz, 1H, H7), 8.31 (d, J = 8.0 Hz, 1H, H10); Anal. Calcd
for C26H20N2O6: C, 68.42; H, 4.42; N, 6.14. Found: C, 68.22; H,
4.13; N, 5.93.
H, 3.41; N, 7.29. Found: C, 71.59; H, 3.18; N, 7.44.
3-Amino-1-(2,4-dimethlphenyl)-12-oxo-1,12-dihydrobenzo
3-Amino-1-(3-fluorophenyl)-12-oxo-1,12-dihydrobenzo[h]
[h]pyrano[3,2-c]chromene-2-carbonitrile (4k). Yield 74%, mp
251–253ꢀC. IR (KBr, υ, cmꢁ1): 3411, 3321 (NH2), 2197 (CN),
pyrano[3,2-c]chromene-2-carbonitrile (4q). Yield 79%, mp
270–272ꢀC. IR (KBr, υ, cmꢁ1): 3415, 3326 (NH2), 2199 (CN),
1
1735 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 2.21 (s, 3H,
1
1712 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 4.56 (s, 1H,
CH3), 2.47 (s, 3H, CH3), 4.73 (s, 1H, CH, H1), 6.91–6.98 (m, 3H,
0
CH, H1), 7.09 (dd, J = 5.6, 2.5 Hz, 1H, H2 ), 7.10-7.18 (m, 2H,
0
0
0
H3 , H5 , H6 ), 7.35 (s, 2H, NH2), 7.73–7.76 (m, 2H, H8, H9), 7.91
(d, J = 8.7 Hz, 1H, H5), 7.99 (d, J = 8.7 Hz, 1H, H6), 8.09 (d,
J = 8.0 Hz, 1H, H7), 8.29 (d, J = 8.0 Hz, 1H, H10); Anal. Calcd for
C25H18N2O3: C, 76.13; H, 4.60; N, 7.10. Found: C, 76.30; H,
0
0
0
H4 , H6 ), 7.35–7.39 (m, 1H, H5 ), 7.48 (s, 2H, NH2), 7.72–7.76
(m, 2H, H8, H9), 7.89 (d, J = 8.7 Hz, 1H, H5), 7.97 (d, J = 8.7 Hz,
1H, H6), 8.08 (d, J = 8.0 Hz, 1H, H7), 8.29 (d, J = 8.0 Hz, 1H,
H10); Anal. Calcd for C23H13FN2O3: C, 71.87; H, 3.41; N, 7.29.
4.35; N, 7.34.
Found: C, 71.54; H, 3.56; N, 6.98.
3-Amino-1-(2-chlorophenyl)-12-oxo-1,12-dihydrobenzo[h]
3-Amino-1-(4-fluorophenyl)-12-oxo-1,12-dihydrobenzo[h]
pyrano[3,2-c]chromene-2-carbonitrile (4l). Yield 77%, mp 260–
262ꢀC. IR (KBr, υ, cmꢁ1): 3407, 3328 (NH2), 2196 (CN), 1713
(CO); 1H NMR (500 MHz, DMSO-d6) d ppm: 5.06 (s, 1H,
pyrano[3,2-c]chromene-2-carbonitrile (4r). Yield 90%, mp
248–250ꢀC. IR (KBr, υ, cmꢁ1): 3422, 3320 (NH2), 2194 (CN),
1
1711 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 4.51 (s, 1H,
0
0
0
CH, H1), 7.27–7.28 (m, 2H, H4 , H6 ), 7.34-7.35 (m, 1H, H5 ),
0
0
0
0
CH, H1), 7.31–7.34 (m, 2H, H2 , H3 , H5 , H6 ), 7.45 (s, 2H, NH2),
7.73–7.77 (m, 2H, H8, H9), 7.94 (d, J = 8.7 Hz, 1H, H5), 7.98
(d, J = 8.7 Hz, 1H, H6), 8.05 (d, J = 8.0 Hz, 1H, H7), 8.26
(d, J = 8.0 Hz, 1H, H10); Anal. Calcd for C23H13FN2O3: C, 71.87;
H, 3.41; N, 7.29. Found: C, 72.08; H, 3.62; N, 7.50.
0
7.42–7.44 (m, 1H, H3 ), 7.46 (s, 2H, NH2), 7.71–7.77 (m, 2H, H8,
H9), 7.89 (d, J = 8.7 Hz, 1H, H5), 7.98 (d, J = 8.7 Hz, 1H, H6),
8.08 (d, J = 7.8 Hz, 1H, H7), 8.28 (d, J = 7.8 Hz, 1H, H10);
13C NMR (125MHz, DMSO-d6) d ppm: 34.3, 56.5, 102.4, 108.3,
117.9, 118.8, 121.4, 121.8, 124.5, 127.6, 127.8, 128.2, 128.8,
129.2, 129.5, 130.7, 132.4, 134.6, 140.2, 149.5, 154.8, 158.1,
159.2; Anal. Calcd for C23H13ClN2O3: C, 68.92; H, 3.27; N, 6.99.
Biology. Reagents and chemicals. Roswell Park Memorial
Institute (RPMI) 1640, fetal bovine serum (FBS) and phosphate-
buffered saline (PBS) were purchased from Biosera (Ringmer,
Found: C, 69.14; H, 2.98; N, 7.22.
3-Amino-1-(3-chlorophenyl)-12-oxo-1,12-dihydrobenzo[h]
UK).
3-(4,5-Dimethylthiazol-2-yl)-2,5
diphenyltetrazolium
bromide (MTT) was obtained from Sigma (Saint Louis, MO,
USA), and penicillin⁄streptomycin was purchased from
Invitrogen (San Diego, CA, USA). Doxorubicin and dimethyl
sulfoxide were obtained from EBEWE Pharma (Unterach,
Austria) and Merck (Darmstadt, Germany), respectively.
pyrano[3,2-c]chromene-2-carbonitrile (4m). Yield 73%, mp
259–261ꢀC. IR (KBr, υ, cmꢁ1): 3384, 3320 (NH2), 2207
1
(CN), 1699 (CO); H NMR (500MHz, DMSO-d6) d ppm: 4.54
0
0
0
0
(s, 1H, CH, H1), 7.28–7.38 (m, 4H, H2 , H4 , H5 , H6 ), 7.50
(s, 2H, NH2), 7.71–7.77 (m, 2H, H8, H9), 7.87 (d, J = 8.7 Hz, 1H,
H5), 7.96 (d, J = 8.7 Hz, 1H, H6), 8.07 (d, J = 8.0 Hz, 1H, H7),
8.28 (d, J = 8.0Hz, 1H, H10); Anal. Calcd for C23H13ClN2O3:
Cell lines and cell culture. HL-60 (Human promyelocytic
leukemia cells) and MOLT4 (Human acute lymphoblastic
leukemia cell line) 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,
100 units⁄mL penicillin-G, and 100 mg⁄mL streptomycin. Cells were
grown in monolayer cultures, except for Raji cells, which were
C, 68.92; H, 3.27; N, 6.99. Found: C, 68.72; H, 3.05; N, 6.78.
3-Amino-1-(4-chlorophenyl)-12-oxo-1,12-dihydrobenzo[h]
pyrano[3,2-c]chromene-2-carbonitrile (4n). Yield 81%, mp
244–246ꢀC. IR (KBr, υ, cmꢁ1): 3385, 3305 (NH2), 2197 (CN),
1
1710 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 4.53 (s, 1H,
grown in suspension, at 37ꢀC in humidified air containing 5% CO2.
Cytotoxicity assay. Cell viability following exposure to
0
0
CH, H1), 7.34 (d, J = 7.5 Hz, 2H, H2 , H6 ), 7.37 (d, J = 7.5 Hz,
0
0
2H, H3 , H5 ), 7.48 (s, 2H, NH2), 7.72–7.76 (m, 2H, H8, H9), 7.89
(d, J = 8.7Hz, 1H, H5), 7.97 (d, J = 8.7 Hz, 1H, H6), 8.07
(d, J = 8.0Hz, 1H, H7), 8.30 (d, J = 8.0 Hz, 1H, H10); Anal. Calcd
for C23H13ClN2O3: C, 68.92; H, 3.27; N, 6.99. Found: C, 68.64;
synthetic compounds was estimated using the MTT reduction
assay [20,21]. HL-60 and MOLT4 cells were plated in 96-well
microplates at a density of 5 ꢃ 104 cells⁄mL (100 mL⁄well).
Control wells contained no drugs, and blank wells contained
only growth medium for background correction. After overnight
incubation at 37ꢀC, half of the growth medium was removed,
and 50 mL of medium supplemented with different
concentrations of synthetic compounds dissolved in DMSO was
added in triplicate. Plates were centrifuged before this
procedure. Maximum concentration of DMSO in the wells was
0.5%. Cells were further incubated for 72 h. At the end of the
incubation time, the medium was removed, and MTT was
added to each well at a final concentration of 0.5 mg⁄mL, and
H, 3.50; N, 7.31.
3-Amino-1-(2,3-dichlorophenyl)-12-oxo-1,12-dihydrobenzo
[h]pyrano[3,2-c]chromene-2-carbonitrile (4o). Yield 70%, mp
256–259ꢀC. IR (KBr, υ, cmꢁ1): 3432, 3311 (NH2), 2195 (CN),
1
1709 (CO); H NMR (500 MHz, DMSO-d6) d ppm: 5.11 (s, 1H,
0
0
0
CH, H1), 7.31–7.37 (m, 3H, H4 , H5 , H6 ), 7.53 (s, 2H, NH2),
7.73–7.77 (m, 2H, H8, H9), 7.90 (d, J = 8.7 Hz, 1H, H5), 7.99
(d, J = 8.7Hz, 1H, H6), 8.09 (d, J = 8.0 Hz, 1H, H7), 8.30
(d, J = 8.0Hz, 1H, H10); Anal. Calcd for C23H12Cl2N2O3:
C, 63.47; H, 2.78; N, 6.44. Found: C, 63.19; H, 3.02; N, 6.71.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet