V
R
SYNTHETIC COMMUNICATIONS
11
(
DMSO-d ) d (ppm): 7.17 (t, 1 H, Ar-H, J ¼ 8 Hz, J ¼ 1.2 Hz), 7.4 (dd, 2 H, Ar-H, J
6
o
m
o
¼
8 Hz, J ¼ 2 Hz), 7.47 (s, 1 H, CH¼), 7.51 (t, 2 H, Ar-H, J ¼ 8.4, 6.8 Hz), 7.58
m
o
(
t, 2 H, Ar-H, J ¼ 6.8, 7.6 Hz), 7.65 (t, 1 H, Ar-H, J ¼ 8.8 Hz, J ¼ 2 Hz), 7.72
o
o
m
(
d, 1 H, Ar-H, Jm ¼ 2 Hz), 7.92 (d, 1 H, Ar-H, J ¼ 7.6 Hz), 7.98 (dd, 1 H, Ar-H,
o
J ¼ 7.8 Hz, J ¼ 2 Hz), 8.78 (d, 1 H, Ar-H, J ¼ 7.6 Hz), 10.34 (br.s, 1 H, NHCO,
o
m
o
exchangeable), 12.16 (br.s, 1 H, NHCOPh, exchangeable), 13.48 (br.s, 1 H, OH,
þ.
þ.
þ.
exchangeable). MS (m/z): 457(M þ2, 4.8), 456 (M þ1, 11.8), 455 (M , 5.5), 439
(20.6), 438 (63), 437 (37.9), 436 (99.8), 403 (44), 401 (100), 105 (PhCO, 90.6), 104
(
24.7), 77 (Ph, 70.9); Anal. Calcd for: C H Cl N O (455.29): C, 60.68; H, 3.54; N,
2
3
16
2 2 4
6.15; Found: C, 60.43; H, 3.66; N, 6.41%
2
-(2-Benzamido-3-(4-methoxyphenyl) acrylamido) benzoic acid (2b)
ꢁ
ꢀ1
Yield 75%; white crystal; m.p. 250–252 C (EtOH/Dioxane); IR (cm ) ꢀ: 3428 (OH),
3
313, 3242 (NH), 3039, 3000 (CHaryl), 2924, 2931, 2836 (CHalkyl), 1687, 1670, 1650
1
(
3
CO), 1602, 1580, 1526(C¼C), 823 (d ), 695,757 (d ). H-NMR (DMSO-d ) d (ppm):
2
H
5H
6
.74 (s, 3 H, OCH ), 6.92 (d, 2 H, Ar-H, J ¼ 8.8 Hz), 7.12–7.15 (m, 1 H, Ar-H,
3
o
J ¼ 8.4,7.2 Hz), 7.51–7.64 (m, 7 H,Ar-H), 7.96 (s, 1 H, CH¼), 8.06 (d, 2 H, Ar-H,
J ¼ 8.4 Hz), 8.82 (d, 1 H, Ar-H, J ¼ 8.8 Hz)), 10.30 (br.s, 1 H, NHCO, exchangeable),
12.08 (br.s, 1 H, NHCOPh, exchangeable), 13.52 (br.s, 1 H, OH, exchangeable;
Anal.Calcd for: C H N O (398.4): C, 69.22; H, 4.84; N, 6.73; Found: C, 69.05; H,
2
4
20 2 5
4.49; N, 6.89%.
Biological evaluation
In vitro anticancer activity
Three human cancer cell lines, namely hepatocellular carcinoma (HePG-2), mammary
gland (MCF-7), and colorectal carcinoma (HCT-116) are used to determine in vitro the
anticancer activity of the synthesized compounds. The tested cell lines were supplied
[
30]
from the US National Cancer Institute. The reported standard procedure
was utilized
as follows. The tested cells were plated in 96-well Microplates; the total volume per well
ꢁ
was adjusted at 100 lL. Then, incubation of cells was performed at 37 C, 5% CO , 95%
2
air, and 100% relative humidity for 24 h before addition of synthesized compounds.
After 24 h, only two plates of each cell line were selected and fixed in situ with TCA, in
order to exemplify a measurement of the cell population for each cell line during drug
application. The title compounds and fluorouracil, the reference drug, were dissolved in
DMSO at 400-fold the desired final maximum test concentration and stored at freezing
point prior to use. During addition of drug, the frozen concentrate was dissolved and
diluted to twice the desired final maximum test concentration with gentamicin solution
(50 mg/mL). To reach the desired final drug concentrations, different tested compound
dilutions (100 mL) were added to the appropriate microtiter wells containing 100 mL of
medium. The tested compounds as well as 5-fluorouracil as reference drug were added.
ꢁ
Then, the plates were incubated for an additional 48 h at 37 C, 5% CO , 95% air, and
2
1
00% relative humidity. The assay was terminated by the addition of cold TCA for
ꢁ
adherent cells followed by incubation for 60 min at 4 C. The supernatant was removed,
and the plates were washed five times with excessive water and dried. A solution of