Synthesis, characterization and biological evaluation of some new indomethacin analogs with a colon tumor cell growth inhibitory activity

Article abstract of DOI:10.1007/s00044-017-1932-8

Focusing particularly on colorectal cancer, and suggesting research strategies that may help to accelerate the future clinical application of indomethacin for the treatment of cancer, the molecular structures of indomethacin was used as starting scaffold to design novel amide analogs, and the effects of those analogs on the proliferation of human cancer cells were evaluated against three colon cancer cell lines, namely, HCT-116, CACO-2, and HT-29. Compared to indomethacin, the new derivatives displayed significantly increased activities. Interestingly two of the indomethacin analogs 7a and 8c displayed high growth inhibitory activity in nano-molar to micro-molar range against all three human colon cancer cell lines with IC50 values ranging from 0.055 to 4.0 μg/ml compared to 0.7–5.45 μg/ml for 5-fluorouracil (5-FU). Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 7a and 8c on the HT-29 and HCT-116 cell lines respectively were studied. The results indicated that compounds 7a and 8c arrested the cell cycle at G1/S and G0/G1 phase in HT-29 and HCT-116 cells respectively and might induced apoptosis via caspase-3 dependent pathway.

Full text of DOI:10.1007/s00044-017-1932-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1932-8
ORIGINAL RESEARCH
Synthesis, characterization and biological evaluation of some new
indomethacin analogs with a colon tumor cell growth inhibitory
activity
Sally I. Eissa1
Received: 18 January 2017 / Accepted: 22 May 2017
© Springer Science+Business Media New York 2017
Abstract Focusing particularly on colorectal cancer, and
suggesting research strategies that may help to accelerate
the future clinical application of indomethacin for the
treatment of cancer, the molecular structures of indo-
methacin was used as starting scaffold to design novel
amide analogs, and the effects of those analogs on the
proliferation of human cancer cells were evaluated against
three colon cancer cell lines, namely, HCT-116, CACO-2,
and HT-29. Compared to indomethacin, the new derivatives
displayed significantly increased activities. Interestingly
two of the indomethacin analogs 7a and 8c displayed high
growth inhibitory activity in nano-molar to micro-molar
range against all three human colon cancer cell lines with
IC50 values ranging from 0.055 to 4.0 µg/ml compared to
0.7–5.45 µg/ml for 5-fluorouracil (5-FU). Moreover, the
potential mechanisms of the cytotoxic activity of the pro-
mising compounds 7a and 8c on the HT-29 and HCT-116
cell lines respectively were studied. The results indicated
that compounds 7a and 8c arrested the cell cycle at G1/S
and G0/G1 phase in HT-29 and HCT-116 cells respectively
and might induced apoptosis via caspase-3 dependent
pathway.
Introduction
Cancer colon is one of the most common malignant tumors
affecting humans, and represents the third leading cause of
cancer death among adults (Sinha and Richa 2009). The
disease sometimes begins as a benign adenomatous polyp,
which develops into an advanced adenoma with high-grade
dysplasia and then progresses to an invasive cancer (Con-
nell et al. 2014). Apoptosis is the programmed cell death
which maintains the healthy survival/death balance in nor-
mal cells. Disruption of apoptotic pathway has been
established as a prominent hallmark of several cancers
(Cotter 2009). Evidences are increasingly available to sup-
port the hypothesis that failure of apoptosis may be an
important factor in the evolution of colorectal cancer and its
poor response to chemotherapy and radiation (Watson
2004). Deeper understanding of the molecular mechanisms
of apoptosis and its defective status opens the gate for a new
class of targeted therapeutics (Hassan et al. 2014). There is a
growing body of evidence suggests that the non-steroidal
anti-inflammatory drug indomethacin has a potent antic-
olorectal cancer activity as well as chemopreventive effect
from in vitro and in vivo models of colorectal cancer by a
variety of reported methods including cyclooxygenase
inhibition and peroxisome proliferator-activated receptor c
activation (Pollard and Luckert 1983; Blitzer and Huang
1983; Heneghan 1985; Michael et al. 2002; Kumar et al.
2013; Chan 2002; Hawcraft et al. 2002; Zhu et al. 1999a;
Blidner et al. 2015). In recent years, it is widely reported
that, inhibiting cell proliferation and inducing cell apoptosis
is another mechanism by which indomethacin and other non
steroidal anti-inflammatory drugs inhibit tumor. The fact
that the anti-proliferative effects of indomethacin in a
variety of transformed cells that do not express cyclo-
oxygenase (COX)-enzyme would argue against the
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●
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Keywords Synthesis Indomethacin Anticancer activity
●
●
Colon cancer Apoptosis Cell cycle arrest
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1932-8) contains supplementary material,
which is available to authorized users.
* Sally I. Eissa
Sarazizo2005@yahoo.com
1
Pharmaceutical Chemistry Department, Faculty of Pharmacy
(Girls), Al-Azhar University, Nasr City, Cairo, Egypt

Med Chem Res
relevance of COX inhibition as a target in colorectal epi-
thelial cells (Groot et al. 2007, 2005; Rigas and Shiff 1999;
Rigas and Kashfi 2005; Hanif et al.1996; Zhu et al. 1999a,
b; Schror 2011; Elder et al. 1997; Kralj et al. 2001; Piazza
et al. 1997, 1995; Wu and Xu 2001; Jones et al. 1999;
Tinsley et al. 2010). This study is an attempt to improve the
indomethacin anticancer potency through chemical mod-
ification to attain an active antitumor agent with potentiated
activity and selectivity toward colon cancerous cells, hence
indomethacin was used as a lead compound for the synth-
esis of a series of new derivatives through systematic
modification of carboxylic acid moiety. The newly syn-
thesized derivatives were evaluated as antiproliferative
agents against colon cancer cells,namely, HCT-116,
CACO-2, and HT-29. For the most potent compounds, the
effect on normal cell cycle profile and apoptosis were per-
formed to analyze the possible mechanism underlying their
antiproliferative effect.
was poured over crushed ice, few drops of HCl was added
and the separated solid product was filtered, dried and
recrystallized from ethanol to give:
Ethyl 4-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido)benzoate
(2a) Beige
needles
(EtOH): Yield 67%; m.p. 120–122 °C. IR (KBr) νmax:3292
(NH), 3060 (CH–Ar), 2908 (CH-aliphatic), 1713 (CO,
ESTER),1668 (br, CO) cm−1 1H NMR (DMSO, 300
.
MHz,) δ: 1.24 (t, 3H, CH2CH3), 2.25 (s, 3H, CH3); 3.74
(s, 2H, CH2); 3.80 (s, 3H, OCH3); 4.28 (m, 2H, CH2CH3),
6.58–7.92(m, 11H, ArH); 10.61 (s, 1H, NH exchangeable
with D2O).13C NMR (DMSO, 75 MHz,);δ 13.89 (CH3 pyrole),
14.65 (COOCH2–CH3), 31.62(NHCOCH2), 55.95 (OCH3),
60.85(COO-CH2-CH3),102.49(CH, C5 indole), 111.70 (C,
C3 indole), 114.37 (CH, C7 indole), 115.00 (CH, C8 indole),
116.94 (C, C4 indole), 118.92 (C, C9 indole), 124.00 (CH, C2b
Ph-COO), 124.63 (CH, C6b Ph-COO), 129.50 (C, C4b Ph-
COO), 130.63 (CH, C3b Ph-COO), 130.77 (CH, C5b
Ph-COO), 131.33 (CH, C2a Cl-benzoyl), 131.61 (CH, C6a
Cl-benzoyl), 134.64 (C, C1a Cl-benzoyl), 135.92 (C, C2
indole), 138.03 (CH, C3a Cl-benzoyl), 138.07 (CH, C5a
Cl-benzoyl) 144.03 (C, C1b Ph-COO), 156.02 (C,C4a
Cl-benzoyl),165.02 (C, carboxyl), 156.97 (C,C6 indole),
168.15 (C, carbonyl), 169.63 (C, amide). EI MS m/z: (%):
504 ([M+2]+, 3.04), 504 ([M+]+, 13.31), 41.01 (100).
Anal. calcd. for C28H25ClN2O5: C, 66.60; H, 4.99; N, 5.55;
found: C, 66.82; H, 5.06; N, 5.64.
Experimental
Chemistry
All chemicals and reagents used in the current study were of
analytical grade. Melting points (uncorrected) were deter-
mined on open capillary tubes using Griffin melting point
1
apparatus. All the H (nuclear magnetic resonance) NMR
and 13C NMR spectra were performed on Varian Gemini
300 MHz, 75 MHz spectrophotometer, respectively, using
tetramethylsilane as internal standard. Chemical shift values
(δ) are given using parts per million scale (ppm) at the
Armed Forces Laboratories. The infrared (IR) spectra were
recorded using Bruker ATR/FTIR Spectrophotometer at the
Armed Forces Laboratories. Mass spectra were made on a
DI-150 Unit of Shimadzu GC/MS-QP 5050A at the
Regional Centre for Mycology and Biotechnology, AL-
Azhar University. Analytical thin-layer chromatography was
performed on precoated silica gel plates 60-F-254 (Merck;
0.25 mm). Elemental analyses (C, H, N) were performed by
Micro Analytical Center, The Regional Center for Mycology
and Biotechnology, Al-Azher University, and they were
within 0.4% of the theoretical values.
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido) benzoic acid (2b) Dark yellow(EtOH):
Yield 78%; m.p. 110–112 °C. IR (KBr) νmax:3380 (br,
carboxylic OH), 3214 (NH), 3080 (CH- Ar), 2943 (CH-
1
aliphatic), 1677 (Br, CO) cm−1. H NMR (DMSO, 300
MHz,) δ: 2.27 (s, 3H, CH3); 3.71 (s, 2H, CH2); 3.85 (s, 3H,
OCH3); 6.68–8.60 (m, 11H, ArH); 11.21(s, 1H, NH
exchangeable with D2O); 13.42(s, 1H, COOH exchange-
able with D2O). 13C NMR (d6-DMSO, 75 MHz,) δ 13.67
(CH3 pyrole), 33.81 (NHCOCH2), 55.82 (OCH3), 101.71
(CH, C5 indole), 112.08 (C, C 3 indole), 112.89 (CH, C7 indole),
115.30 (CH, C8 indole), 119.92 (C, C4 indole), 123.15 (C, C9
indole), 129.54 (CH, C2b Ph-COOH), 130.93 (CH, C6b Ph-
COOH), 130.94 (C, C4b Ph-COOH), 131.63 (CH, C3b
Ph-COOH), 131.77 (CH, C5b Ph-COOH), 134.54 (CH, C2a
Cl-benzoyl), 134.56 (CH, C6a Cl-benzoyl), 136.62 (C, C1a
Cl-benzoyl), 138.10 (C, C2 indole), 141.13 (CH, C3a Cl-
benzoyl), 138.07 (CH, C5a Cl-benzoyl) 144.03 (C, C1b Ph-
COOH), 154.02 (C, C4aCl-benzoyl),165.09 (C, carboxyl),
168.45 (C,C6 indole),169.40 (C, carbonyl), 169.58 (C,
amide).
General procedure for synthesis of 2-(2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl) N-
substituted acetamide derivatives) (2a–e)
A mixture of equimolar amounts (0.01 mol, 3.7 g) of
2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetyl chloride (1) and appropriate amine (0.01 mol) in
dimethylformamide solvent (DMF) (10 mL) containing few
drops of pyridine was refluxed for 2 h. The reaction mixture
EI MS m/z: (%): 476 ([M], 9.02), 477 ([M+]+, 2.9), 139
(100). Anal. calcd. for C26H21ClN2O5: C, 65.48;H, 4.44; N,
5.87; found: C, 65.69; H, 4.49; N, 5.96.

Med Chem Res
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-1-(piperazin-1-yl)ethanone (2c) Red needles (EtOH):
Yield 69%; m.p. >300 °C. IR (KBr) νmax: 3382 (NH),3063
3.77 (s, 3H, OCH3); 6.68–7.70 (m, 12H, ArH); 9.28(s, 1H,
NH exchangeable with D2O). 13C NMR (DMSO, 75 MHz,)
δ
11.64 (CH3 pyrazole), 13.93 (CH3 pyrole), 32.05
1
(CH–Ar), 2926 (CH-aliphatic), 1677 (Br, CO) cm−1. H
(NHCOCH2), 36.48 (NCH3 pyrazole), 55.88 (OCH3), 102.22
(CH, C5 indole), 108.04 (C=CCH3 pyrazole),111.87 (C, C3
indole), 114.67 (CH, C7 indole), 115.00 (CH, C8 indole), 123.86
(C, C4 indole), 129.49 (CH, C2b benzene), 129.53 (CH, C6b
benzene), 130.26 (CH, C4b benzene), 131.2 (CH, C3b ben-
zene), 131.63 (CH, C5b benzene), 131.62 (C, C1a Cl-ben-
zoyl), 134.69 (CH, C5a Cl-benzoyl), 134.14 (CH, C3a Cl-
benzoyl), 135.49 (C, C1b benzene), 135.66 (CH, C2a indole),
135.49 (CH, C6a Cl-benzoyl), 138.01 (C=CCH3 pyrazole),
152.81 (C, C4a Cl-benzoyl), 156.02 (C, C6 indole), 162.23
(C, CO pyrazole), 168.34 (C, carbonyl), 169.68 (C, amide). EI
MS m/z (%): 542 ([M]+, 0.77), 105, (100%). Anal. calcd.
for C30H27ClN4O4; C, 66.36; H, 5.01; N, 10.32; found: C,
66.52; H, 5.08; N, 10.47.
NMR (DMSO, 300 MHz,) δ: 2.19 (s, 3H, CH3); 2.96 (m,
4H, 2(CH2), piperazine); 3.05 (m, 4H, 2(CH2) piperazine);;
3.73 (s, 2H, CH2); 3.85 (s, 3H, OCH3) 6.69–7.76 (m, 7H,
ArH); 12.24(s, 1H, NH exchangeable with D2O) 13C NMR
(DMSO, 75 MHz) δ 13.82 (CH3 pyrole), 29.81 (NHCOCH2),
42.00 (CH, piprazine), 43.70 (CH, piprazine), 55.86 (OCH3),
101.71 (CH, C5 indole), 111.48 (C, C3 indole), 114.40 (CH, C7
indole), 115.30 (CH, C8 indole), 119.51 (C, C4 indole), 123.15
(C, C9 indole), 130.71 (CH, C2a Cl-benzoyl), 134.67 (CH,
C6a Cl-benzoyl), 138.01 (C, C1a Cl-benzoyl), 138.01 (C, C2
indole), 141.17 (CH, C3a Cl-benzoyl), 138.22 (CH, C5a Cl-
benzoyl) 154.02 (C,C4aCl-benzoyl), 155.45 (C,C6 indole),
169.40 (C, carbonyl), 168.25 (C, amide). EI MS m/z: (%):
426 ([M+1]+, 1.93), 42.2, (100) Anal. calcd. for
C23H24ClN3O3: C, 64.86;H, 5.68; N, 9.87; found: C, 65.03;
H, 5.65; N, 10.04.
N-(2-aminoethyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl)acetamide (3)
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(naphthalen-2-yl)acetamide (2d) Dark yellow nee-
dles (EtOH): Yield 69%; m.p. 280–282 °C. IR (KBr)
νmax:3208 (NH), 3004 (CH–Ar), 2793 (CH-aliphatic),
1675 (Br, CO) cm−1. 1H NMR (DMSO, 300 MHz,) δ: 2.35
(s, 3H, CH3); 3.76 (s, 2H, CH2); 3.91 (s, 3H, OCH3);
6.79–8.08 (m, 14H, ArH); 10.11(s, 1H, NH exchangeable
A mixture of equimolar amounts of compound (1) (0.01
mol, 3.7 g) and ethylenediamine (0.01 mol) in DMF (10
mL) containing few drops of pyridine was refluxed for 2 h.
The reaction mixture was poured over crushed ice, few
drops of HCl was added and the separated solid product was
filtered, dried, and recrystallized from ethanol. Dark yellow
needles (EtOH), 84%; m.p. 260–262 °C. IR (KBr)
νmax:3281 (Br, NH), 3069 (CH–Ar), 2926 (CH-aliphatic),
with D2O). 13C NMR (DMSO, 75 MHz)
δ 13.93
(CH3 pyrole), 32.05 (NHCOCH2), 55.90 (OCH3), 102.24
(CH, C5 indole), 111.86 (C, C3 indole), 114.72 (CH, C7 indole),
115.11 (CH, C8 indole), 122.46 (C, C4 indole), 123.07 (C,
1
1677(Br, CO) cm−1. H NMR (DMSO, 300 MHz)δ: 2.08
(s, 3H, CH3); 2.91 (m, 2H, CH2-CH2-NH2); 3.31 (s, 2H,
CH2); 3.41 (m, 2H, CH2-CH2-NH2); 3.84 (s, 3H, OCH3);
6.67–7.96 (m, 7H, ArH); 8.07, 8.96 (2s, 2NH exchangeable
with D2O). 13C NMR (DMSO, 75 MHz) δ 13.77 (CH3
pyrole), 31.59 (NHCOCH2), 37.62 (CH2CH2), 39.08
(CH2CH2)), 55.87 (OCH3), 102.26 (CH, C5 indole), 111.69
(C, C 3 indole), 114.56 (CH, C7 indole), 114.96 (CH, C8 indole),
129.45 (C, C4 indole), 130.72 (C, C9 indole), 131.28 (CH, C2a
Cl-benzoyl), 131.59 (CH, C6a Cl-benzoyl), 134.69 (C, C1a
Cl-benzoyl), 135.64 (C, C2 indole), 137.96 (CH, C3a Cl-
benzoyl), 138.22 (CH, C5a Cl-benzoyl) 154.02 (C, C4aCl-
benzoyl), 155.99 (C, C6 indole), 168.28 (C, carbonyl), 170.10
(C, amide). EI MS m/z (%): 399 ([M]+, 3.43), 105, (100).
Anal. calcd. for C21H22ClN3O3: C, 63.08; H, 5.55; N,
10.51; found: C, 63.21; H, 5.62; N, 10.76.
C9 indole), 125.88 (CH, C2b naphthalene), 126.01 (CH, C11b
naphthalene), 126.24 (C, C3b naphthalene), 126.49 (CH,
C4b naphthalene), 128.34 (CH, C5b naphthalene), 129.49
(CH, C6b naphthalene), 130.78 (CH, C7b naphthalene),
131.77 (CH, C8b naphthalene), 131.62 (C, C9b naphtha-
lene), 133.39 (CH, C10b naphthalene), 131.62 (CH, C2a
Cl-benzoyl), 133.39 (CH, C6a Cl-benzoyl), 134.14 (C, C1a
Cl-benzoyl), 134.70 (C, C2 indole), 135.92 (CH, C3a Cl-
benzoyl), 138.03 (CH, C5a Cl-benzoyl) 156.02 (C,C4a
Cl-benzoyl), 156.09 (C, C6 indole),168.34 (C, carbonyl),
169.68 (C, amide). EI MS m/z: (%): 482 ([M]+, 0.97), 104,
(100). Anal. calcd. for C29H23ClN2O3; C, 72.12; H, 4.80; N,
5.80; found: C, 72.31; H, 4.87; N, 5.89.
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyra-
zol-4-yl)acetamide (2e) Yellowish brown needles (EtOH):
Yield 73%; m.p. 110–112 °C. IR (KBr) νmax: 3267 (NH),
General procedure for synthesis of (E)-2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(2-
((4-substituted-benzylidene)amino)ethyl)acetamide (4a–c)
3018 (CH–Ar), 2926 (CH-aliphatic), 1660 (Br, CO) cm−1
.
1H NMR (DMSO, 300 MHz): δ 2.05 (s, 3H, CH3-pyrazole);
A mixture of equimolar amounts (0.01 mol, 3.7 g) of com-
2.26 (s, 3H, CH3); 3.02 (s, 3H, N-CH3); 3.70 (s, 2H,CH2)
pound (3) and appropriate aldehyde (0.01 mol) in glacial

Med Chem Res
acetic acid (10 mL) was refluxed for 2 h. The reaction
mixture was poured over crushed ice, and the separated
solid product was filtered, dried and recrystallized from
ethanol to give:
(E)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)-N-(2-((4-fluorobenzylidene)amino)ethyl)acetamide
(4c) A dark yellow needle, Yield: 66%; m.p. 229–231 °C.
IR (KBr) νmax: 3281 (NH), 3080 (CH–Ar), 2967 (CH-
1
aliphatic), 1668 (Br, CO) cm−1. H NMR (DMSO, 300
(E)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)-N-(2-((4-methoxybenzylidene)amino)ethyl)acetamide
(4a) Beige needles (EtOH): Yield 65%; m.p. 230–232 °C.
IR (KBr) νmax: 3281 (NH), 3075 (CH–Ar), 2928 (CH-
MHz) δ: 2.18 (s, 3H, CH3); 2.70 (m, 2H, CH2-CH2-N=Ph);
3.12 (m, 2H, CH2-CH2-N=Ph); 3.30 (s, 2H, CH2); 3.75 (s,
3H,OCH3); 6.99–8.01 (m, 12H, ArH+NH). 13C NMR
(DMSO, 75 MHz) δ 12.05 (CH3 pyrole), 25.33 (NHCOCH2),
28.79 (CH2CH2), 30.22 (CH2CH2)), 55.81 (OCH3), 100.84
(CH, C5 indole), 104.67 (C, C 3 indole), 109.87 (CH, C7 indole),
110.00 (CH, C3b benzylidene), 111.35 (CH, C5b benzylidene),
113.25 (CH, C8 indole), 129.20 (C, C4 indole), 129.23 (C,
C9 indole), 130.04 (CH, C2b benzylidene), 130.04 (CH, C6b
benzylidene), 130.34 (C, C1b benzylidene),130.57 (CH, C2a
Cl-benzoyl), 131.59 (CH, C6a Cl-benzoyl), 132.55 (C, C1a
Cl-benzoyl), 132.89 (C, C2 indole), 134.52 (CH, C3a Cl-
benzoyl), 138.25 (CH, C5aCl-benzoyl), 144.26 (C, C4a
Cl-benzoyl), 153.47 (C, C4b benzylidene) 155.98 (C,C6 indole),
166.92 (CH, N=CH), 171.03 (C, carbonyl), 186.89 (C,
amide). EI MS m/z (%): 507 ([M+2]+, 1.61%), 505 [M]+,
(2.08), 40.18, (100). Anal. calcd. for C28H25ClFN3O3: C,
66.47; H, 4.98; N, 8.30; found: C, 66.63; H, 5.06; N, 8.45.
1
aliphatic), 1660 (Br, CO) cm−1. H NMR (DMSO, 300
MHz) δ: 2.18 (s, 3H, CH3); 3.07 (m, 2H, CH2-CH2-N=Ph);
3.32 (m, 2H, CH2-CH2-N=Ph); 3.76 (s, 2H,CH2CO); 3.82,
3.86 (2s, 6H, 2OCH3); 6.90–8.69 (m, 11H, ArH); 9.84 (s,
1H, NH exchangeable with D2O); 13C NMR (DMSO, 75
MHz) δ 13.78 (CH3 pyrole), 31.56 (NHCOCH2), 43.83
(CH2CH2), 45.74 (CH2CH2)), 55.87 (OCH3), 55.91
(OCH3), 102.30 (CH, C5 indole), 111.72 (C, C indole),
3
114.64 (CH, C7 indole), 114.6 (CH, C8 indole), 114.6 (CH, C3b
benzylidene), 114.6 (CH, C5b benzylidene), 126.99 (C, C4 indole),
129.45 (C, C9 indole), 130.42 (CH, C2b benzylidene), 130.70
(CH, C6b benzylidene), 131.29 (C, C1b benzylidene), 131.58 (CH,
C2a Cl-benzoyl), 132.59 (CH, C6a Cl-benzoyl), 134.69 (C,
C1a Cl-benzoyl), 135.62 (C, C2 indole), 137.96 (CH, C3a Cl-
benzoyl), 138.22 (CH, C5a Cl-benzoyl) 154.02 (C, C4aCl-
benzoyl), 155.99 (C, C6 indole), 162.11 (CH, N=CH),168.28
(C, carbonyl), 170.04 (C, C4b benzylidene), 170.13 (C,amide).
EI MS m/z (%): 518 ([M]+, 0.86%), 287, (100%). Anal.
calcd. for C29H28ClN3O4: C, 67.24; H, 5.45; N, 8.11; found:
C,67.39; H, 5.52; N, 8.24.
General procedure for synthesis of 2-(1-(4-chlorobenzoyl)-
5-methoxy-2-methyl-1H-indol-3-yl)-N-(4-(N-(5-substituted-
3-yl)sulfamoyl)phenyl)acetamide derivatives (5a–c)
A mixture of equimolar amounts (0.01 mol, 3.7 g) of
2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetyl chloride (1) and appropriate sulpha drug (0.01 mol)
in DMF (10 mL) containing few drops of pyridine was
refluxed for 4 h. The reaction mixture was poured over
crushed ice, few drops of HCl was added and the separated
solid product was filtered, dried and recrystallized from
ethanol to give:
(E)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)-N-(2-((4-chlorobenzylidene)amino)ethyl)acetamide
(4b) Light brown needles (EtOH): Yield 71%; m.p.
235–237 °C. IR (KBr) νmax: 3280 (NH),3077 (CH–Ar),
2927 (CH-aliphatic), 1666 (Br, CO) cm−1 1H NMR
.
(DMSO, 300 MHz) δ: 2.16 (s, 3H, CH3); 2.72 (m, 2H, CH2-
CH2-N=Ph); 2.88 (m, 2H, CH2-CH2-N=Ph); 3.06 (s, 2H,
CH2); 3.74 (s, 3H,OCH3); 6.67–8.20 (m, 12H, ArH+NH).
13C NMR (DMSO, 75 MHz) δ 13.74 (CH3 pyrole), 31.59
(NHCOCH2), 36.22 (CH2CH2), 39.07 (CH2CH2)), 55.84
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(4-sulfamoylphenyl)acetamide (5a) Dark yellow
needles (EtOH), Yield 83%; m.p. 80–82 °C. IR (KBr)
νmax: 3286 (Br NH), 3050(CH–Ar), 2988 (CH-aliphatic),
(OCH3), 102.23 (CH, C5 indole), 111.67 (C, C indole),
3
114.50 (CH, C7 indole), 114.6 (CH, C3b benzylidene), 114.6
(CH, C5b benzylidene), 115.01 (CH, C8 indole), 129.37(C, C4
indole), 129.45 (C, C9 indole), 129.54 (CH, C2b benzylidene),
1
1671 (Br, CO) cm−1. H NMR (DMSO, 300 MHz) δ: 2.16
(s, 3H, CH3); 3.70 (s, 2H, CH2); 3.782 (s, 3H, OCH3);
6.69–8.73 (m, 14H, Ar H+NH & NH2). 13C NMR (DMSO,
75 MHz) δ 13.67 (CH3 pyrole), 34.63 (NHCOCH2), 55.84
(OCH3), 102.02 (CH, C5 indole), 111.74 (C, C3 indole),
111.89 (CH, C7 indole), 113.27 (CH, C8 indole), 113.88 (C, C4
indole), 115.05 (C, C9 indole), 129.19 (CH, C2b Ph), 129.51
(CH, C6b Ph), 130.18 (C, C4b Ph), 130.64 (CH, C3b Ph),
130.65 (CH, C5b Ph), 130.97 (CH, C2a Cl-benzoyl), 131.61
(CH, C6a Cl-benzoyl), 134.55 (C, C1a Cl-benzoyl), 134.60
(C, C2 indole), 135.60 (CH, C3a Cl-benzoyl), 135.80 (CH,
129.47 (CH, C6b benzylidene), 131.29 (C, C1b
benzyli-
dene),131.27 (CH, C2a Cl-benzoyl), 131.59 (CH, C6a Cl-
benzoyl), 133.05 (C, C1a Cl-benzoyl), 134.67 (C, C2 indole),
135.66 (C, C4b benzylidene), 137.96 (CH, C3a Cl-benzoyl),
138.22 (CH, C5a Cl-benzoyl) 139.02 (C, C4aCl-benzoyl),
155.98 (C, C6 indole), 160.11 (CH, N=CH), 168.28 (C,
carbonyl), 170.13 (C, amide). EI MS m/z (%): 521 ([M]+,
0.52), 54, (100). Anal. calcd. for C28H25Cl2N3O3: C, 64.37;
H, 4.82; N, 8.04; found: C, 64.52; H, 4.89; N, 8.13.

Med Chem Res
C5a Cl-benzoyl) 138.10 (C, C1b Ph),156.01 (C, C4aCl-ben-
zoyl), 168.31 (C, C6 indole), 170.96 (C, carbonyl), 172.5 (C,
amide). EI MS m/z (%): 511 ([M]+, 0.78), 137, (100). Anal.
calcd. for C25H22ClN3O5S: C, 58.65; H, 4.33; N, 8.21;
found: C, 58.79; H, 4.39; N, 8.34.
70–72 °C. IR (KBr) νmax: 3280 (Br, NH), 3041 (CH–Ar),
2987 (CH-aliphatic), 1699 (Br, CO) cm−1 1H NMR
.
(DMSO, 300 MHz): δ 2.17 (s, 3H, CH3); 2.48 (s, 3H,
CH3-isoxazol); 3.65 (s, 2H, CH2); 3.75 (s, 3H, OCH3);
6.69–7.96 (m, 13H, 12Ar H+NH); 8.43 (s, NH exchange-
able with D2O). 13C NMR (DMSO, 75 MHz) δ 13.67 (CH3
pyrole), 14.58 (CH3 methylisoxazole), 34.64 (NHCOCH2), 55.83
(OCH3), 102.00 (CH, C5 indole), 111.75 (CH, C2- isoxazole),
111.89 (C, C 3 indole), 113.88 (CH, C7 indole), 115.05 (CH,
N-(4-(N-acetylsulfamoyl)phenyl)-2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl)acetamide (5b) Brown
needle (EtOH): Yield 74%; m.p. 100–102 °C. IR (KBr)
νmax: 3280 (Br NH), 3046 (CH–Ar), 2988 (CH-aliphatic),
1671 (Br, CO) cm−1. 1H NMR (DMSO, 300 MHz): 2.17 (s,
3H, CH3); δ 2.43 (s, 3H, COCH3); 3.71; (s, 2H, CH2); 3.85
(s, 3H, OCH3); 6.76–8.70 (m, 13H, Ar H+2NH). 13C NMR
(DMSO, 75 MHz) δ; 13.67 (CH3 pyrole), 29.82 (COCH3),
34.66 (NHCOCH2), 55.84 (OCH3), 102.02 (CH, C5 indole),
111.75 (C, C 3 indole), 111.89 (CH, C7 indole), 113.27 (CH,
C8 indole), 129.51 (C, C4 indole), 130.62 (C, C9 indole), 130.64
(CH, C2b Ph), 130.97 (CH, C6b Ph), 131.18 (C, C4b Ph),
131.56 (CH, C3b Ph), 131.60 (CH, C5b Ph), 131.62 (CH,
C2a Cl-benzoyl), 134.55 (CH, C6a Cl-benzoyl), 134.61 (C,
C1a Cl-benzoyl), 135.60 (C, C2 indole), 135.81 (CH, C3a Cl-
benzoyl), 138.04 (CH, C5a Cl-benzoyl) 138.09 (C, C1b Ph),
150.99 (C, C1- isoxazole) 156.00 (C, C4aCl-benzoyl), 168.31
(C, C6 indole), 168.31 (C, C3-isooxazole), 170.96 (C, carbonyl),
172.50 (C, amide). EI MS m/z (%): 592 ([M]+, 5.88), 137,
(100). Anal.calcd. for C29H25ClN4O6: C, 58.73; H, 4.25; N,
9.45; found: C, 58.91; H, 4.32; N, 9.62.
C8 indole), 113.88 (C, C4 indole), 115.05 (C, C9 indole), 129.19
(CH, C2b Ph), 130.56 (CH, C6b Ph), 130.18 (C, C4b Ph),
130.65 (CH, C3b Ph), 130.87 (CH, C5b Ph), 130.97 (CH,
C2a Cl-benzoyl), 131.65 (CH, C6a Cl-benzoyl), 134.53 (C,
C1a Cl-benzoyl), 134.61 (C, C2 indole), 135.90 (CH, C3a Cl-
benzoyl), 135.81 (CH, C5a Cl-benzoyl) 138.09 (C, C1b Ph),
155.99 (C, C4aCl-benzoyl), 168.31 (C, C6 indole), 170.96 (C,
carbonyl), 172.5 (C, amide). EI MS m/z (%): 554 ([M]+,
4.07), 280, (100). Anal.calcd. for C27H24ClN3O6S: C,
58.53; H, 4.37; N, 7.85; found: C, 58.70; H, 4.41; N, 7.67.
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamide
(5e) Dark yellow needles (EtOH), Yield 59%; m.p. 80–82
°C. IR (KBr) νmax: 3287 (Br, NH), 3080 (CH–Ar), 2927
1
(CH-aliphatic), 1687 (Br, CO) cm−1. H NMR (DMSO,
300 MHz): δ 2.14 (s, 3H, CH3); 3.65 (s, 2H, CH2); 3.75 (s,
3H,OCH3); 6.69–7.96 (m, 15H, Ar H+NH); 12.39 (s, 1H,
NH exchangeable with D2O). 13C NMR (DMSO, 75 MHz)
δ 13.64 (CH3 pyrole), 34.55 (NHCOCH2), 55.85 (OCH3),
102.14 (CH, C5 indole), 111.75 (CH, C5- diazine), 111.89 (C,
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)acetamide
(5c) Dark yellow needles (EtOH), Yield 79%; m.p.
150–152 °C. IR (KBr) νmax: 3283 (Br NH),3102 (CH–Ar),
2988 (CH-aliphatic), 1699 (Br, CO) cm−1 1H NMR
.
C3 indole), 113.89 (CH, C7 indole), 115.01 (CH, C8 indole),
(DMSO, 300 MHz): δ 2.26 (s, 3H, CH3); 3.65 (s, 2H, CH2);
3.75 (s, 3H, OCH3); 6.69–8.76 (m, 15H, Ar H+2NH). 13C
NMR (DMSO, 75 MHz) δ 13.67 (CH3 pyrole), 34.70
(NHCOCH2), 55.85 (OCH3), 102.01 (CH, C5 indole), 102.15
(CH, C4-thiazole), 111.75 (C, C 3 indole), 111.89 (CH, C7
indole), 113.27 (CH, C8 indole), 113.88 (C, C4 indole), 115.02
(C, C9 indole), 129.52 (CH, C2b Ph), 130.63 (CH, C6b Ph),
130.97 (C, C4b Ph), 131.18 (CH, C3b Ph), 131.60 (CH, C5b
Ph), 134.56 (CH, C2a Cl-benzoyl), 134.61 (CH, C6a Cl-
benzoyl), 135.60 (C, C1a Cl-benzoyl), 135.81 (C, C2 indole),
138.04 (CH, C3a Cl-benzoyl), 138.09 (CH, C5a Cl-benzoyl)
138.90 (C, C1b Ph), 155.99 (CH, C5-thiazole) 156.99 (C,
C4aCl-benzoyl), 168.31 (C, C6 indole), 170.02 (C, C2-thiazole),
170.96 (C, carbonyl), 172.50 (C, amide). EI MS m/z (%):
594 ([M1]+, 8.47), 69, (100). Anal. calcd. for
C28H23ClN4O5S2: C, 56.51; H, 3.90; N, 9.41; found: C,
56.59; H, 3.87; N, 9.52.
127.26 (C, C4 indole), 129.52 (C, C9 indole), 130.62 (CH, C2b
Ph), 131.59 (CH, C6b Ph), 131.61 (C, C4b Ph), 134.60 (CH,
C3b Ph), 135.60 (CH, C5b Ph), 135.80 (CH, C2a Cl-ben-
zoyl), 138.09 (CH, C6a Cl-benzoyl), 134.61 (C, C1a Cl-
benzoyl), 135.60 (C, C2 indole), 135.81 (CH, C3a Cl-ben-
zoyl), 138.04 (CH, C5a Cl-benzoyl) 138.09 (C, C1b Ph),
143.38 (CH, C4- diazine), 145.33 (CH, C6- diazine), 156.00
(C,C4aCl-benzoyl), 168.31 (C, C6 indole), 168.31 (CH,
C2-diazine), 170.96 (C, carbonyl), 172.50 (C, amide). EI MS
m/z (%): 589 ([M]+, 0.89), 264 (100). Anal. calcd. for
C29H24ClN5O5S: C, 59.03; H, 4.10; N, 11.87; found: C,
59.34; H, 4.19; N, 11.98.
2-((1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)methyl)-4H-benzo[d][1,3]oxazin-4-one (6)
A
mixture of 2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-
2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)-N-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl)acet-
amide (5d) Dark brown needles (EtOH), Yield 61%; m.p.
methyl-1H-indol-3-yl)acetamido)benzoic acid (2) (14 g,
0.03 mol) and acetic anhydride (30 g, 0.3 mol) was heated
under reflux for 4 h. The solvent was removed under

Med Chem Res
reduced pressure. The residue was triturated with petroleum
ether 40–60. The separated solid was collected by filtration,
washed with petroleum ether 40–60, dried and crystallized
from ethanol to give a yellowish needle (EtOH), Yield 75
%; m.p.168–170 °C. IR (KBr) νmax: 3057 (CH–Ar), 2983,
benzoyl), 135.60 (C, C2 indole), 135.29(CH, C3a Cl-ben-
zoyl), 135.58 (CH, C5a Cl-benzoyl), 135.90 (CH, C4c- Ph),
138.90 (CH, C1c Ph) 139.23 (C, C1b Ph), 156.00 (C,
C4aCl-benzoyl), 168.31 (C, C6 indole), 166.90 (C, carbonyl),
169.77 (C, amide), 172.50 (C, amide). EI MS m/z (%):
631 [M+1]+, 630 [M]+,, 155, (100). Anal. calcd. for
C32H27ClN4O6S: C, 60.90; H, 4.31; N, 8.88; found: C,
61.03; H, 4.35; N, 9.02.
2830 (CH-aliphatic), 1772 (CO-lactone),1670 (CO) cm−1
.
1H NMR (DMSO, 300 MHz): δ 2.29 (s, 3H, CH3); 3.71 (s,
3H, OCH3); 4.13 (s, 2H, CH2); 6.69–8.08 (m, 11H, ArH).
13C NMR (DMSO, 75 MHz) δ 13.80 (CH3 pyrole), 29.95
(NHCOCH2), 55.82 (OCH3), 102.32 (CH, C5 indole), 111.89
(C, C3 indole), 115.08 (CH, C7 indole), 117.03 (CH, C8 indole),
126.91 (C, C4 indole), 128.36 (C, C9 indole), 129.02 (CH, C2a
Cl-benzoyl), 129.11 (CH, C6a Cl-benzoyl), 129.51 (C, C1a
Cl-benzoyl), 129.54 (CH, benoxazine), 130.93 (CH,
benoxazine), 130.94 (CH, benoxazine), 130.73 (CH, C3a
Cl-benzoyl), 130.96 (CH, C5a Cl-benzoyl), 131.96 (C, C2
indole), 132.05 (CH, benoxazine), 134.51 (C, benoxazine),
136.39 (C, benoxazine), 146.39 (C, C4aCl-benzoyl),156.03
(C, carboxyl), 159.63 (C, C6 indole), 160.40 (C, carbonyl),
164,20 (C, N=C-OCO) 169.58 (C, amide). EI MS m/z (%):
458, [M], (35.62), 460, [M+2], (11.6), 139, (100). Anal.
calcd. for C26H19ClN2O4: C, 68.05; H, 4.17; N, 6.10; found:
C, 68.23; H, 4.21; N, 6.19.
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(4-(N-pyrimidin-2-ylsulfamoyl)phenyl)
benzamide (7b) Brown needles (EtOH); Yield 65%; m.p.
151–153 °C; IR (KBr) νmax: 3216 (Br, NH), 3085
1
(CH–Ar), 2932 (CH-aliphatic), 1679 (Br, CO) cm−1: H
NMR (DMSO, 300 MHz) δ: 2.38 (s, 3H, CH3), 3.72 (s, 2H,
CH2); 4.08 (s, 3H, OCH3); 6.61–8.62 (m, 18H, ArH); 10.5,
11.14 (2s, 2H, NH cancelled with D2O). 13C NMR (DMSO,
75 MHz) δ: 11.60 (CH3 pyrole), 34.51(NHCOCH2), 55.83
(OCH3), 100.14 (CH, C5 indole), 111.53 (C, C3 indole),
111.45 (CH, C5- diazine), 117.03 (CH, C7 indole), 119.01 (CH,
C8 indole), 129.26 (C, C4 indole), 129.49 (C, C9 indole), 129.61
(CH, C2c- Ph), 129.61 (CH, C6c- Ph), 130.09 (CH, C2b Ph),
130.94 (CH, C6b- Ph), 131.50 (C, C4b Ph), 131.59 (CH, C3b
Ph), 131.72 (CH, C5b Ph), 131.75 (CH, C3c- Ph), 131.75
(CH, C3c- Ph), 134.59 (CH, C2a Cl-benzoyl), 138.09 (CH,
General procedure for synthesis of 2-(2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetamido)-N-(4-(N-(4-substituted)sulfamoyl)phenyl)
benzamide (7a–c)
C6a Cl-benzoyl), 134.61 (C, C1a Cl-benzoyl), 135.60 (C, C2
indole), 135.29 (CH, C3a Cl-benzoyl), 135.58 (CH, C5a Cl-
benzoyl), 135.90 (CH, C4c- Ph), 138.90(CH, C1c- Ph)
139.23 (C,C1b Ph), 149.38 (CH, C4- diazine), 149.33 (CH,
C6- diazine), 156.00 (C, C4aCl-benzoyl), 168.31 (CH, C2-
diazine), 168.31 (C,C6 indole), 166.81 (C, carbonyl), 169.70
(C, amide), 171.50 (C, amide). EI MS m/z (%): 708 [M]+,
174, (100). Anal. calcd. for C36H29ClN6O6S: C, 60.97; H,
4.12; N, 11.85; found: C, 61.42; H, 4.17; N, 12.02.
A mixture of equimolar amounts (0.01 mol, 4.5 g) of of
benzoxazinone derivative (6) and appropriate sulpha drug
(0.01 mol) in DMF (10 mL) containing few drops of pyr-
idine was refluxed for 4 h. The reaction mixture was poured
over crushed ice, few drops of HCl was added and the
separated solid product was filtered, dried and recrystallized
from ethanol to give:
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(4-(N-(4-methylisoxazol-3-yl)sulfa-
moyl)phenyl)benzamide (7c) Brown needles (EtOH);
Yield 64%; m.p. 140–142 °C; IR (KBr) νmax: 3230 (Br,
NH), 3085 (CH–Ar.), 2932(CH-aliphatic), 1678 (Br, CO)
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(4-sulfamoylphenyl)benzamide (7a)
Beige needles (EtOH); Yield 68%; m.p. 100–102 °C; IR
(KBr) νmax: 3349 (Br, NH), 3075 (CH–Ar), 2931, 2833
1
cm−1: H NMR (DMSO, 300 MHz): δ 2.29, 2.38 (2s, 6H,
(CH-aliphatic.),1679 (Br, CO) cm−1
:
1H NMR (DMSO,
2CH3), 3.70 (s, 2H, CH2); 3.77 (s, 3H, OCH3), 6.62–8.64
(m, 16H, ArH); 10.74, 11.14 (2s, 2H, NH cancelled with
D2O); 13C NMR (DMSO, 75 MHz) δ: 11.64 (CH3 pyrole),
13.76 (CH3 methyl-isoxazole), 34.55 (NHCOCH2), 55.71
(OCH3), 100.12 (CH, C5 indole), 103.47 (CH, C2- isoxazole),
110.21 (C, C3 indole), 111.53 (CH, C7 indole), 116.07 (CH, C8
indole), 119.78 (C, C4 indole), 122.67 (C, C9 indole), 129.11
(CH, C2c- Ph), 129.20 (CH, C6c- Ph), 130.40 (CH, C2b Ph),
130.50 (CH, C6b- Ph), 130.75 (C, C4b Ph), 131.57 (CH, C3b
Ph), 131.72 (CH, C5b Ph), 131.75 (CH, C3c- Ph), 131.75
(CH, C3c- Ph), 134.59 (CH, C2a Cl-benzoyl), 138.09
(CH, C6a Cl-benzoyl), 134.61 (C, C1a Cl-benzoyl), 135.60
300 MHz) δ:2.30 (s, 3H, CH3); 3.74 (s, 2H, CH2); 3.87 (s,
3H,OCH3); 6.70–8.63 (m, 17H, ArH+NH2); 11.16 (s, 1H,
NH cancelled with D2O). 13C NMR (DMSO, 75 MHz) δ:
11.64 (CH3 pyrole), 34.55 (NHCOCH2), 55.85 (OCH3),
100.14 (CH, C5 indole), 111.54 (C, C3 indole), 116.03 (CH, C7
indole), 119.01 (CH, C8 indole), 129.26 (C, C4 indole), 129.49
(C, C9 indole), 129.61 (CH, C2c- Ph), 129.61 (CH, C6c- Ph),
130.07 (CH, C2b Ph), 130.74 (CH, C6b- Ph), 131.50 (C, C4b
Ph), 131.59 (CH, C3b Ph), 131.72 (CH, C5b Ph), 131.75
(CH, C3c- Ph), 131.75 (CH, C3c- Ph), 134.59 (CH, C2a Cl-
benzoyl), 138.09 (CH, C6a Cl-benzoyl), 134.61 (C, C1a Cl-

Med Chem Res
(C, C2 indole), 135.29 (CH, C3a Cl-benzoyl), 135.58 (CH,
C5a Cl-benzoyl), 135.90 (CH, C4c- Ph), 138.90 (CH,
cancelled with D2O); 13C NMR (DMSO, 75 MHz)δ: 11.85
(CH3 pyrole), 34.55 (NHCOCH2), 55.70 (OCH3), 103.43
(CH, C5 indole), 116.04 (C, C 3 indole), 119.41 (CH, C7 indole),
121.41 (CH, C8 indole), 122.35 (C, C4 indole), 127.56 (C, C9
indole), 128.94 (CH, C2c- Ph), 129.09 (CH, C6c- Ph), 129.20
(CH, C2b Ph), 129.22 (CH, C6b- Ph), 129.50 (C, C4b Ph),
129.69 (CH, C3b Ph), 129.75 (CH, C5b Ph), 130.07 (CH,
C3c- Ph),130.12 (CH, C3c- Ph), 130.75 (CH, C2a Cl-ben-
zoyl), 131.50 (CH, C6a Cl-benzoyl), 131.56 (C, C1a Cl-
benzoyl), 131.58 (C, C2 indole), 133.79 (CH, C3a Cl-ben-
zoyl), 137.58 (CH, C5a Cl-benzoyl), 138.30 (CH, C4c- Ph),
138.41 (CH, C1c- Ph) 141.61 (C, C1b Ph), 160.18 (C, C4aCl-
benzoyl), 164.96 (C, C6 indole), 166.89 (C, carbonyl),
169.77 (C, amide), 171.05 (C, amide). EI MS m/z (%):
588 [M+2], 586 [M]+, 139, (100). Anal. calcd. for
C32H25Cl2N3O4: C, 65.54; H, 4.30; N, 7.16; found: C,
60.56; H, 4.34; N, 7.33.
C1c- Ph) 139.23 (C, C1b Ph), 141.60 (C, C1- isoxazole) 153.61
(C, C4aCl-benzoyl), 164.31 (C,C6 indole), 165.35 (C,
C3-isooxazole), 166.89 (C, carbonyl), 169.76 (C, amide),
171.02 (C, amide). EI MS m/z (%): 711 [M]+, 137, (100).
Anal. calcd. C36H30ClN5O7S: C, 60.71; H, 4.25; N, 9.83;
found: C, 60.89; H, 4.34; N, 9.79.
General procedure for synthesis of 2-(2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetamido)-N-(4-substituted phenyl)benzamides (8a–c)
A mixture of equimolar amounts (0.01 mol, 4.5 g) of ben-
zoxazinone derivative (6) and appropriate aromatic amines
(0.01 mol) in DMF (10 mL) containing few drops of pyr-
idine was refluxed for 2 h. The reaction mixture was poured
over crushed ice, few drops of HCl was added and the
separated solid product was filtered, dried and recrystallized
from ethanol to give.
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(2,6-dimethylphenyl)benzamide (8c)
Brown needles (EtOH); Yield 70%; m.p. 114–116 °C; IR
(KBr) νmax: 3372 (Br, NH), 3071 (CH-arom.), 2830 (CH-
1
aliph.), 1679 (Br, CO) cm−1; H NMR (DMSO, 300 MHz)
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(4-fluorophenyl)benzamide (8a) Yel-
low needles (EtOH); Yield 74%; m.p. 60–62 °C; IR (KBr)
νmax: 3377 (Br, NH), 3080 (CH-arom.), 2936 (CH-aliph.),
δ: 2.17, 2.27, 2.38 (3s, 9H, 3CH3), 3.77 (s, 3H, OCH3), 3.96
(s, 2H, CH2), 6.61–8.14 (m, 14H, Ar–H), 8.68, 12.50 (2s,
2H, NH cancelled with D2O). 13C NMR (DMSO, 75 MHz)
δ: 11.54 (CH3- Ph), 19.23 (CH3 -Ph), 19.50 (CH3 pyrole),
34.45 (NHCOCH2), 55.75 (OCH3), 102.13 (CH, C5 indole),
112.53 (C, C 3 indole), 116.73 (CH, C7 indole), 119.00 (CH,
1
1679 (Br, CO) cm−1. H NMR (DMSO, 300 MHz) δ: 2.34
(s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.94 (s, 2H,CH2),
6.64–8.63 (m, 15H, Ar–H), 9.44, 11.18 (2s, 2H, NH can-
celled with D2O): 13C NMR (DMSO, 75 MHz) δ:11.40
(CH3 pyrole), 34.51 (NHCOCH2), 55.75 (OCH3), 101.14
(CH, C5 indole), 111.64 (C, C 3 indole), 115.03 (CH, C7 indole),
118.05 (CH, C8 indole), 129.36 (C, C4 indole), 129.60 (C, C9
indole), 129.61 (CH, C2c- Ph), 129.63 (CH, C6c- Ph), 130.07
(CH, C2b Ph), 130.84 (CH, C6b- Ph), 131.44 (C, C4b Ph),
131.59 (CH, C3b Ph), 131.62 (CH, C5b Ph), 131.75 (CH,
C3c- Ph),131.79 (CH, C3c- Ph), 134.59 (CH, C2a Cl-ben-
zoyl), 138.09 (CH, C6a Cl-benzoyl), 134.51 (C, C1a Cl-
benzoyl), 135.60 (C, C2 indole), 135.29 (CH, C3a Cl-ben-
zoyl), 135.55 (CH, C5a Cl-benzoyl), 135.93 (CH, C4c- Ph),
138.67 (CH, C1c- Ph) 139.23 (C,C1b Ph), 156.03 (C,C4aCl-
benzoyl), 166.60 (C, carbonyl), 168.43 (C, C6 indole), 169.00
(C, amide), 171.40 (C, amide). EI MS m/z (%): 570 [M]+,,
339 (100). Anal. calcd. for C32H25ClFN3O4: C, 67.43; H,
4.42; N, 7.37; found: C, 67.75; H, 4.51; N, 7.52.
C8 indole), 129.23 (C, C4 indole), 129.48 (C, C9 indole), 129.67
(CH, C2c- Ph), 129.69 (CH, C6c- Ph), 130.00 (CH, C2b Ph),
131.14 (CH, C6b- Ph), 131.50 (C, C4b Ph), 131.60 (CH, C3b
Ph), 131.72 (CH, C5b Ph), 131.75 (CH, C3c- Ph), 131.79
(CH, C3c- Ph), 133.19 (CH, C2a Cl-benzoyl), 133.89 (CH,
C6a Cl-benzoyl), 134.51 (C, C1a Cl-benzoyl), 135.60 (C, C2
indole), 135.20 (CH, C3a Cl-benzoyl), 135.50 (CH, C5a Cl-
benzoyl), 135.90 (CH, C4c- Ph), 137.80 (CH, C1c- Ph)
139.23 (C,C1b Ph), 156.23 (C,C4aCl-benzoyl), 168.01 (C,C6
indole), 165.90 (C, carbonyl), 169.07 (C, amide), 172.00 (C,
amide). EI MS m/z (%): 580 [M]+, (0.66), 139, (100). Anal.
calcd. for C34H30ClN3O4: C, 70.40; H, 5.21; N, 7.24; found:
C, 70.55; H, 5.27; N, 7.41.
In vitro anticancer screening of the synthesized
compounds against human colon cell lines HCT-116,
HT-29, and CACO-2
2-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamido)-N-(4-chlorophenyl)benzamide (8b) Yel-
lowish brown needles (EtOH); Yield 75%; m.p. 120–122 °C;
IR (KBr) νmax: 3238 (Br, NH), 3065 (CH-arom.), 2887
Cell culture
1
(CH-aliph.), 1669 (Br, CO) cm−1, H NMR (DMSO, 300
Cancer cells from different colon cancer cell lines HCT-
116, HT-29 or Caco-2 were originally purchased from
American type Cell Culture collection (ATCC, Manassas,
MHz): δ 2.27 (s, 3H, CH3), 3.66 (s, 3H, OCH3), 3.76 (s, 2H,
CH2), 6.61–8.28 (m, 15H, ArH); 10.33, 11.14 (2s, 2H, NH

Med Chem Res
USA) and were maintained in the tissue culture lab of the
Egyptian company for vaccines and sera (Vacsera, Giza.
Egypt). Cells were then transferred to our lab and were
grown on Roswell Park Memorial Institute medium
(RPMI 1640) supplemented with 1% of 100 mg/ mL
streptomycin, 100 units/mL penicillin and 10% of heat-
inactivated fetal bovine serum in a humidified, 5% (v/v)
CO2 atmosphere at 37 °C (Huang et al. 2013; Sheen et al.
2003).
Mountain View, CA) and the FlowJo software (Ashland,
OR). For each measurement, at least 10,000 cells were
counted.
Apoptotic analysis
HT-29 and HCT-116 cells were seeded in 24-well tissue
culture plates. After 24 h, the medium was changed and 7c
and 8c at IC50 concentrations were added. After treatment
for 48 h, cells floating in the medium were collected. The
adherent cells were detached with 0.05% trypsin. Then
culture medium containing 10% FBS (and floating cells)
was added to inactivate trypsin. When gentle pipetting was
completed, the cells were centrifuged for 5 min at 1500 g.
The supernatant was removed and cells were stained with
annexin V-fluorescein isothiocyanate and propidium iodide
(PI) according to the manufacturer’s instructions. Untreated
cells were used as control for double staining. Immediately
after staining the cells were analyzed by a FACScan flow
cytometer. For each measurement, at least 20,000 cells were
counted.
Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT)
Exponentially growing cells from different cancer cell lines
were trypsinized, counted, and seeded at the appropriate
densities (2000-1000 cells/0.33 cm2 well) into 96-well
microtiter plates. Cells then were incubated in a humidi-
fied atmosphere at 37 ˚C for 24 h. Then, cells were exposed
to different concentrations of compounds (0.1, 10, 100,
1000 µg/ml) for 72 h. Then the viability of treated cells were
determined using MTT technique as follow. Media were
removed; cells were incubated with 200 μl of 5%
MTT solution/well (Sigma Aldrich, MO) and were allowed
to metabolize the dye into a colored-insoluble formazan
crystals for 2 h. The remaining MTT solution were dis-
carded from the wells and the formazan crystals were dis-
solved in 200 µl/well acidified isopropanol for 30 min,
covered with aluminum foil and with continuous shaking
using a MaxQ 2000 plate shaker (Thermo Fisher Scientific
Inc, MI) at room temperature. Absorbance were measured at
570 nm using a Stat FaxR 4200 plate reader (Awareness
Technology, Inc., FL). The cell viability were expressed as
percentage of control and the concentration that induces
50% of maximum inhibition of cell proliferation (IC50)
were determined using Graph Pad Prism version 5 software
(Graph Pad software Inc, CA).
Caspase activity assay
Caspase activities were assayed using caspase enzyme
Assay kits (Geno Technology). In brief, HT-29 and HCT-
116 cells were treated with IC50 concentration of 7a and 8c
respectively for 24 or 48 h, collected by trypsinization,
washed once with PBS, and cell pellets resuspended in 350
ml lysis buffer. The cells were lysed by freeze and thaw five
times. The lysates were centrifuged at 12,000 rpm for 30
min at 4 °C. Supernatants were collected and used for
measuring caspase-3, and -9, activities by Enzyme-linked
Immunosorbent Assay (ELISA) cell-based assay according
to the manufacturer’s instructions. Caspase-3, and caspase-9
activities were detected using the specific caspases fluoro-
genic substrate, DEVD peptide conjugated to 7-amino-4-
trifluoromethyl coumarin (AFC). Samples were read
on a microplate reader at 405 nm (ThermoLabsystems,
Chantily, Va.)
Cell cycle analysis
The cell cycle profile was assayed by flow cytometry after
staining with PI/RNase. HT-29 and HCT-116 cells were
seeded in 24-well tissue culture plates. On the second day,
the medium was changed, and cells were treated with 7a
and 8c, at IC50 concentrations (7a 0.1 μg/ml, 8c 0.4 μg/
ml). Cells were incubated for 48 h before harvesting. The
cells were fixed gently with 80% ethanol before being
placed in a freezer for 2 h. They were then treated with
0.25% triton X-100 for 5 min in an ice bath. The cells
were resuspended in 30 μl of phosphate buffered saline
(PBS) containing 40 μg/ml propidium iodide and 0.1 mg/
ml RNase. Cells were incubated in a dark room for
20 min at room temperature before cell cycle analysis
with a FACScan flow cytometer (Becton Dickinson,
CDK2/A enzyme assay
ELISA was used for profiling evaluation of protein kinase
targets. The microtiter plate provided in this kit has been
pre-coated with an antibody specific to CDK-2A. Samples
are then added to the appropriate microtiter plate wells with
a biotin-conjugated antibody specific to CDK2. Next,
Avidin conjugated to Horseradish peroxidase (HRP) is
added to each microplate well and incubated. Then the
TMB substrate solution is added, only those wells that
contain CDK2, biotin-conjugated antibody, and enzyme-
conjugated Avidin will exhibit a change in color. The

Med Chem Res
enzyme-substrate reaction is terminated by the addition of
1N sulfuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 10 nm.
The concentration of CDK2 in the samples is then deter-
mined by blotting the O.D. of the samples to the standard
curve.
generate the diamide derivatives 2-(2-(1-(4-chlorobenzoyl)-
5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(4-(N-(4-
substituted)sulfamoyl)phenyl)benzamide derivatives (7a–c)
and
2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido)-N-(4-substituted phenyl)benzamides
(8a–c) where the benzoxazinone derivative (6) underwent
nucleophilic ring opening via the reaction with different
aromatic amines or appropriate sulfa drug in dry pyridine,
Scheme 2.
All the synthesized compounds were crystallized using
appropriate solvents and characterized by H1, C13, NMR as
well as by MS and elemental analysis.
Results and discussion
Chemistry
The synthesis of the target compound is outlined in Schemes 1
and 2. Indomethacin was used as starting scaffolds to design
our targeted new monoamide and diamide analogs containing
various substituent with different electronic environment
which might be beneficial to their biological activity. Indo-
methacin acid chloride (1) which was prepared from indo-
methacin via reported process (Eissa et al. 2013 and Khalifa
et al. 2012) was used as key intermediate for successful
transformation of a carboxylate moiety of the parent com-
pound into, amide or sulphonamide functions, that were
known to contribute to the enhancement of the antitumor
activity, via a single chemical derivatization method (amida-
tion) (Kalgutkar et al. 2000). The reaction between the key
intermediate (1) and different aromatic amine containing
compounds afforded the 2-(2-(1-(4-chlorobenzoyl)-5-meth-
oxy-2-methyl-1H-indol-3-yl) N-substituted acetamide analo-
gues (2a–e). Different sulfonamide derivatives 2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(4-(N-
Biological activity
In vitro cytotoxic activity
Based on the widly reported colo-rectal cancer activity of
indomethacin, the cytotoxic activity of the newly synthe-
sized analoges was evaluated using (MTT) in vitro assay
(Huang et al. 2013) against three human colon cancer cell
lines: HCT-116, CACO-2, and HT-29, compared to the
parent compound (indomethacin) and (5-FU) as a reference
standard (cell lines selection was encouraged by the
reported antiproliferative activity of indomethacin against
colon cancer cell lines). The concentrations that cause 50%
inhibition of cancer cell growth against various cell lines are
expressed as IC50 values and are summarized in Table 1.
Analysis of the data in Table 1 showed that, all the tested
cell lines were susceptible to the influence of the tested
compounds. With the exception of compounds 2a, 4a, b,
5a–e, all the tested compounds exhibited stronger cytotoxic
activities than that of parent compound for all three human
cancer cell lines.
Interestingly, three of our tested compounds 7a, 8a, and
8c were found to exhibit excellent anti-proliferation activ-
ities for almost all three human cell lines. Surprisingly,
compound 7a showed 99 fold higher cytotoxic activities of
5-FU against CACO-2 cell line with IC50 = 0.055 µg/ml,
7.5 fold higher cytotoxic activities of 5-FU against HT-29
with IC50 = 0.1 µg/ml, nearly half of cytotoxic activity of
5-FU with IC50 = 4.0 µg/ml. The diamide derivative 8a had
potent growth inhibitory activity for all the tested cell lines
with the IC50 values smaller than 1 µg/ml (IC50 = 0.78,
0.505, 0.13 μg/ml compared to 1.8, 0.75, 5.45 μg/ml for 5-
FU against HCT-116, HT-29, and CACO-2 cell lines
respectively.
(5-substituted-3-yl)sulfamoyl)phenyl)acetamide
(5a–e)
were prepared to investigate the effect of replacement of the
amide function in (2a–e) derivatives with a different sul-
fonamide moites. On the other hand, acid chloride key
intermediate (1) was allowed to react with the correspond-
ing ethylenediamine to generate the N-(2-aminoethyl)-2-(1-
(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acet-
amide intermediate (3), which subsequently reacted via
condensation with different appropriate aromatic aldehydes
in glacial acetic acid to afford the respective, arylidene
derivatives,
(E)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl)-N-(2-((4-substituted-benzylidene)
amino)ethyl)acetamide (4a–c), Scheme 1.
In order to evaluate the effect of substituents variation
(linker group variation) on the cytotoxic activity of the
synthesized derivatives, benzoxazin-4-one derivative,
named as, 2-((1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-yl)methyl)-4H-benzo[d][1,3]oxazin-4-one (6)
was obtained in reasonable yield via refluxing,the amide
analog, 2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido) benzoic acid (2b) in acetic anhydride
(Noolvi et al. 2011; Al-ObaidA et al. 2009). The latter
compound (6) reacted in the similar design strategy to
At the same time compound 8c of the same series
exhibited high cytotoxic activity against HCT-116 and
CACO-2 with (IC50 = 0.4, 4.7 μg/ml compared to 1.8,
5.45 μg/ml for 5-FU respectively, Table 1.

Med Chem Res
Scheme 1 General procedure
for preparation of target
compounds 2a–e, 3, 4a–c and
5a–e
Cell cycle analysis
in (Figs. 1, 2, 3) in comparison with control cells, exposure
of HT-29 cells to 7a (0.1 μg/ml) for 24 h increase in the
percentage of cells at the pre-G phase (pre-G1 apoptosis)
and induced cell cycle arrest at G1/S phase, manifested by
an increase of the cell number in the G1/S phase with a
concomitant reduction in the percentage of cells at G2/M
phase compared to control, Meanwhile, exposure of HCT-
116 cells to 8c for 24 h markedly increase in the percentage
of cells at the pre-G phase (pre-G1 apoptosis) and induced
cell cycle arrest at G0/G1 phase manifested by an increase
of the cell number in the G0/G1 phase with a concomitant
reduction in the number of cells in S phase compared to the
control. These results indicate that, compound 7a and 8c
arrested cells in G1/S and G0/G1 phase respectively, with
subsequent induction of apoptosis.
In response to DNA damage, cells arrest primarily at the
G1/S and G2/M boundaries. G1/S arrest blocks entry of
damaged DNA into the S phase, and G2/M arrest prohibits
entry of damaged DNA into mitosis (Sheen et al. 2003). To
analyze the possible mechanism underlying the anti-
proliferative effect exerted by our new compounds, HT-29
and HCT-116 cell cycle progression was assessed following
treatment with compounds 7a and 8c (compounds that had
the marked highest antiproliferative activity (IC50 0.1 and
0.4 µg/ml) on the human colorectal adenocarcinoma HT-29
and HCT-116 cell line respectively for 24 using propidium
iodide flow cytometry analysis. The apoptosis inducing
activity of 7a and 8c was also characterized by flow cyto-
metric analysis of the DNA profile in HT-29 and HCT-116
cells respectively (Table 2, Figs. 1, 2, 3).
Caspase-3 activity (key executor of apoptosis)
The cell cycle distribution was monitored by flow cyto-
metry analysis after propidium iodide staining of the cel-
lular DNA (Huang et al. 2013; Sheen et al. 2003). As seen
Study of apoptosis has considered the basis for novel tar-
geted therapies that can bring death in malignant cells

Med Chem Res
Scheme 2 General procedure
for preparation of target
compounds 6, 7a–c and 8a–c
Table 1 Cytotoxic activity of the newly synthesized compounds
against human colon cell lines (HCT-116, HT-29, and CACO-2). IC50
values are mean of three separate experiments S.D
Table 2 HCT-116 and HT-29 cell cycle distribution of compounds 6a
and 7c
Comp.no.
Cell cycle distribution (%)
Compound
HCT-116
HT-29
CACO-2
Sample
code
Used conc. %G0-G1 %S %G2-M %Apoptosis
μg/ml
IC50 μg/mL
(μM)
IC50 μg/mL
(μM)
IC50 μg/mL
(μM)
7a/HT-29
0.1
0.4
61.31
78.6
24.62 0.42
13.62
6.58
2a
2c
2d
2e
3
43.6 0.503
13.5 0.136
155 3.603
2.9 0.1
77.6 1.20
15.5 0.351
22 2.51
8c/HCT-
116
14.82 0.0
8
0.251
1.0 0.386
13.5 0.251
4.5 0.152
26 1.525
160 3.601
9.5 0.152
21.5 0.152
27 2.516
14.5 0.152
69 3.511
91 3.511
0.1 0.511
5.5 0.288
5.52 0.3
Cont.HT-29
0
0
71.18
68.7
23.45 4.85
23.49 7.33
0.52
0.48
32.5 0.461
1.0 0.2
18
1
Cont.
HCT116
1.0 0.163
1.0 6.082
616 2.645
4a
4b
4c
5a
5b
5c
5d
5e
7a
7b
7c
8a
8b
8c
120 2.081
347 7.505
10.5 0.208
46 3.055
102 5.866
76 4.509
43.6 0.416
51 3.055
Bold values are significantly different from control at p<0.05
14
51
1
2
(Hassan et al. 2014). Consequently, the activation of
apoptosis is emerging as a key approach to treat colorectal
tumor. The unique characteristics of apoptosis is the acti-
vation various enzymes that are cysteine protease families
called caspases (Sheen et al. 2003). Caspase-3 and caspase-
9 are two key proteins of the caspase family, which are
highly conserved in multicellular organisms and function as
central regulators of apoptosis (Alnemri et al. 1996; Kim
et al. 2000; Creagh and Martin 2001; D’Amelio 2010;
Janicke et al. 1998) Clarifying the role of caspases in newly
synthesized indomethacin analogue-induced colon cancer
cell death is important for understanding molecular
mechanisms of the antineoplastic effect of those analogue.
To elucidate a pathway leading to new derivatives induced
cell death we determined the ability of compounds 7a and
8c to activate caspase-3/9 in colon cancer cell lines HT-29
53.7 3.156
52.5 1.60
36 3.511
72.4 1.793
0.055 0.285
2.9 0.152
2.66 0.02
0.13 0005
8.7 0.251
4.7 0.152
30.2 0.461
5.45 0.25
4
0.215
5.7 0.3
5.1 0.251
0.78 0.011
446.2 5.047
0.4 0.219
0.505 0.221
10.9 0.152
28.8 0.757
53.7 0.173
0.75 0.045
Indomethacin 50.11 0.548
5-FU 1.8 0.2

Med Chem Res
Fig. 1 Percentage of each cell
cycle phase with various
treatments or with control
Fig. 2 a, a1 Effects of 7a on HT-29 cell cycle profile at its IC50
compared to untreated cell using flow cytometry data for 24 h. a
Untreated HT-29 cell a1 HT-29 cell treated with 8C. b, b1 7a Induced
apoptosis in human HT-29 cell as assayed by annexin V staining
compared to control cell using flow cytometryfor 24 h., b control cell,
b1 Representative scatter Plots of PI (y-axis) vs. annexin V (x-axis)
showing the effects of 7a on HT-29 cell apoptosis
and HCT-116. Compared with the control, we tested the
increase in caspase-3 and -9 activities in HT-29 and HCT-
116 cells in response to 7a and 8c treatment at its IC50, the
level of active caspase-3/9 was measured in ng/g protein
using caspase apoptosis assay kits (ELISA-based assay) for
24 and 48 h. As shown in Table 3, Fig. 4, the activities of
caspase-3 and caspase-9 were increased following the
treatment of HT-29 and HCT-116 cells with 7a and 8c

Med Chem Res
Fig. 3 a, a1 Effects of 8c on HCT-116 cell cycle profile at its IC50
compared to untreated cell using flow cytometry data. a Untreated
HCT-116 cell a1 HCT-116 cell treated with 8c. b, b1 8c Induced
apoptosis in human HCT-116 cell as assayed by annexin V staining
compared to control cell using flow cytometry, b control cell, b1
Representative scatter Plots of PI (y-axis) vs. annexin V (x-axis)
showing the effects of 8c on HCT-116 cell apoptosis. Data are mean
SEM. * significantly different from control at p < 0.05
Table 3 7a and 8c induced increases in caspase-3/9 activity in human HT-29 and HCT-116 cells treated with IC50 of compound 7a and 8c for 24
and 48 h respectively measured by ELISA-based assay
Cell line
Comp 24 h
Control 24 h Ratio of activation 48 h
Control 48 h Ratio of activation
Caspase-3 conc. ng/ml HT-29
7a
0.3119 0.03849
0.1925 0.02293
8 fold
0.5055 0.04884 10.5 fold
HCT-116 8c
8.5 fold
7.3 fold
3 fold
0.2952 0.03277
9 fold
Caspase-9 conc. ng/ml HT-29
7a
19.74
13.44
2.707
4.188
28.33
25.66
2.064
3.169
13.7 fold
8 fold
HCT-116 8c
respectively. In details, treatment of HT-29 cells with 7a for
24 and 48 h caused a significant increase in caspase-3 level
by about 8 and 10.5 folds respectively, compared to control,
while treatment of HCT-116 cells with 8a for 24 and 48 h
caused a significant increase in caspase-3 level by about 8.5
and 9 folds respectively, compared to control. At the same
time treatment of HT-29 cells with 7a for 24 and 48 h
caused a significant increase in caspase-9 level by about 8
and 10.5 folds respectively, compared to control, while
treatment of HCT-116 cells with 8a for 24 and 48 h caused
a significant increase in caspase-9 level by about 8.5 and 9
folds respectively, compared to control. These results sug-
gest that 7a and 8c may induce apoptosis in of HT-29 and
HCT-116 cells via a caspase-dependent pathway.

Med Chem Res
Fig. 4 Effect of 7a and 8c on
Caspase-3/9 activity in HT-29
and HCT-116 cells for 24 and
48 h respectively measured by
ELISA-based assay. The
Ca3
0.6
0.5
0.4
0.3
0.2
0.1
0
Series1
experiment was done in
triplicate. Data are mean SEM.
*Significantly different from
control at p < 0.05
Ca9
30
25
20
15
10
5
Series1
0
Fig. 5 The effect of 7a and 8c
(expressed as % inhibitionon) on
CDK2 expression in HT-29 7
HCT-116 cells respectivelly,
compared to control cells using
using radioisotope ELISA assay.
The experiment was done in
triplicate. Data are mean SEM.
*Significantly different from
control at p < 0.05
c:
concentrations using ELISA assay (Bertoli et al. 2013 and
Ma et al. 2008). The profiling data in Fig. 5 showed that
CDK-2A activities of HT-29 were inhibited by 55 and 79%
after 24 and 48 respectivelly compared to control cell due to
the effect of 7a. On the other hand, 8c showed potent inhi-
bition of enzyme at its IC50 concentration, the CDK-2A
activities were inhibited by 93 and 90% respectively com-
pared to control. In summary, experiments might have
proved that 7a and 8c might inhibits cell proliferation, alters
cell cycle and arrests G1/S and G1phase, respectively, pos-
sibly through down-regulating CDK-2A (Fig. 5).
Cyclin-dependent kinases (CDKs) are a family of protein
kinases that are participating in the regulation of the cell
cycle and promote cell proliferation. Different types of
cyclins and CDKs play their roles at various stages of the cell
cycle (Woo et al. 1998) and conidered the main regulatory
point to start cell cycle is in G1 phase. During G1 phase,
growth-dependent CDK, and specifically, CDK-2A activity
promotes DNA replication and initiates G1-to-S phase tran-
sition and considered the rate-limiting step for progression of
G1 phase (Pan et al. 2002). Activation of CDK2 and CDK4
can yield many cell cycle related proteins, thus prompting
progression from G1 phase to S phase (Wolter et al. 2001).
To verify if the G1/S phase arrest caused by compound 7a
and G0/G1 phase arrest caused by compound 8c is mediated
through CDK-2A inhibition, the kinase inhibitory effect of
7a and 8c was evaluated against CDK-2A at IC50
Conclusion
In this study, the molecular structure of indomethacin was
used as starting scaffolds to design novel analogs and their

Med Chem Res
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effects on the proliferation of three human colon cancer cell
lines were evaluated compared to indomethacin and 5FU as
a reference. The results indicated that 12 compounds, out of
21, 2c, 2d, 2e, 3, 4a, 4c, 7a, 7b, 7c, 8a, 8b, and 8c exhibited
cytotoxic activities stronger than that of parent compound
(indomethacin) for all three human cancer lines. Interest-
ingly, three of our tested compounds 7a, 8a, and 8c were
found to exhibit excellent anti-proliferation activities for
almost all three human cell lines. The most potent com-
pound 7a, showed 99 and, 7.5, fold higher cytotoxic
activities against CACO-2 and HT-29 cell lines respectively
than 5-FU, while the diamide derivative 8C had potent
growth inhibitory activity for all the tested with the IC50
values smaller than 1 µg/ml. Furthermore, we observed the
combined effect on cell apoptosis, cell cycle, caspase, and
CDK-2A assays to elucidate the possible mechanism of 7a
and 8c effects on colorectal cancer cells. The data indicated
that 7a and 8c inhibited tumor proliferation and inducing
cell cycle arrest in the G1/S and G0/G1 respectively might
be through potent inhibition of CDK-2A and induced cancer
cell apoptosis, propably, via caspase-3/9 dependent path-
way. Previous findings have shown that the most active
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interests.
Huang KB, Chen ZF, Liu YC, Li ZQ, Wei JH, Wang M, Zhang GH,
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