Novel coumarin-6-sulfonamides as apoptotic anti-proliferative agents: synthesis, in vitro biological evaluation, and QSAR studies

Article abstract of DOI:10.1080/14756366.2018.1477137

Herein, we report the synthesis of different novel sets of coumarin-6-sulfonamide derivatives bearing different functionalities (4a, b, 8a–d, 11a–d, 13a, b, and 15a–c), and in vitro evaluation of their growth inhibitory activity towards the proliferation of three cancer cell lines; HepG2 (hepatocellular carcinoma), MCF-7 (breast cancer), and Caco-2 (colon cancer). HepG2 cells were the most sensitive cells to the influence of the target coumarins. Compounds 13a and 15a emerged as the most active members against HepG2 cells (IC₅₀ = 3.48 ± 0.28 and 5.03 ± 0.39 μM, respectively). Compounds 13a and 15a were able to induce apoptosis in HepG2 cells, as assured by the upregulation of the Bax and downregulation of the Bcl-2, besides boosting caspase-3 levels. Besides, compound 13a induced a significant increase in the percentage of cells at Pre-G1 by 6.4-folds, with concurrent significant arrest in the G2-M phase by 5.4-folds compared to control. Also, 13a displayed significant increase in the percentage of annexin V-FITC positive apoptotic cells from 1.75–13.76%. Moreover, QSAR models were established to explore the structural requirements controlling the anti-proliferative activities.

Full text of DOI:10.1080/14756366.2018.1477137

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Novel coumarin-6-sulfonamides as apoptotic anti-
proliferative agents: synthesis, in vitro biological
evaluation, and QSAR studies
Ahmed Sabt, Omaima M. Abdelhafez, Radwan S. El-Haggar, Hassan M. F.
Madkour, Wagdy M. Eldehna, Ezz El-Din A. M. El-Khrisy, Mohamed A. Abdel-
Rahman & Laila. A. Rashed
To cite this article: Ahmed Sabt, Omaima M. Abdelhafez, Radwan S. El-Haggar, Hassan M. F.
Madkour, Wagdy M. Eldehna, Ezz El-Din A. M. El-Khrisy, Mohamed A. Abdel-Rahman & Laila. A.
Rashed (2018) Novel coumarin-6-sulfonamides as apoptotic anti-proliferative agents: synthesis,
inꢀvitro biological evaluation, and QSAR studies, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 1095-1107, DOI: 10.1080/14756366.2018.1477137
To link to this article: https://doi.org/10.1080/14756366.2018.1477137
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1095–1107
https://doi.org/10.1080/14756366.2018.1477137
RESEARCH PAPER
Novel coumarin-6-sulfonamides as apoptotic anti-proliferative agents: synthesis,
in vitro biological evaluation, and QSAR studies
Ahmed Sabta, Omaima M. Abdelhafeza, Radwan S. El-Haggarb, Hassan M. F. Madkourc, Wagdy M. Eldehnad,
Ezz El-Din A. M. El-Khrisya, Mohamed A. Abdel-Rahmane and Laila. A. Rashedf
b
aChemistry of Natural Compounds Department, National Research Centre, Dokki, Egypt; Pharmaceutical Chemistry Department, Faculty of
c
d
Pharmacy, Helwan University, Cairo, Egypt; Chemistry Department, Faculty of Science, Ain-shams University, Cairo, Egypt; Department of
e
Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt; Department of Biochemistry, Faculty of Medicine, Cairo University,
f
Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 22 March 2018
Revised 10 May 2018
Accepted 10 May 2018
Herein, we report the synthesis of different novel sets of coumarin-6-sulfonamide derivatives bearing differ-
ent functionalities (4a, b, 8a–d, 11a–d, 13a, b, and 15a–c), and in vitro evaluation of their growth inhibi-
tory activity towards the proliferation of three cancer cell lines; HepG2 (hepatocellular carcinoma), MCF-7
(breast cancer), and Caco-2 (colon cancer). HepG2 cells were the most sensitive cells to the influence of
the target coumarins. Compounds 13a and 15a emerged as the most active members against HepG2 cells
(IC50 ¼ 3.48 0.28 and 5.03 0.39 mM, respectively). Compounds 13a and 15a were able to induce apop-
tosis in HepG2 cells, as assured by the upregulation of the Bax and downregulation of the Bcl-2, besides
boosting caspase-3 levels. Besides, compound 13a induced a significant increase in the percentage of cells
at Pre-G1 by 6.4-folds, with concurrent significant arrest in the G2-M phase by 5.4-folds compared to con-
trol. Also, 13a displayed significant increase in the percentage of annexin V-FITC positive apoptotic cells
from 1.75–13.76%. Moreover, QSAR models were established to explore the structural requirements con-
trolling the anti-proliferative activities.
KEYWORDS
Anticancer; Apoptosis;
Coumarin-6-sulfonamides;
2D-QSAR; Synthesis
Introduction
inhibition12–14 (as G8935, Figure 1), steroid sulfatase inhibition15–17
(as Irosustat, Figure 1), CDC25 phosphatases inhibition18–20 (as
SV37, Figure 1), tubulin polymerization inhibition21–23, aromatase
Cancer is a very complex, widespread and lethal disease that
affects approximately 14 million people every year. In 2018, the
American Cancer Society predict that the new diagnosed cancer
cases will reach to 1,735,350 and cancer death 595,690 cancer
deaths in the United States1. Accordingly, the discovery of new
anti-cancer agents with promising bioactivity and high therapeutic
index is still an urgent need and a major challenge for researchers.
It is well established that natural and synthetic coumarin deriv-
atives have attracted a great deal of interest due to their variety
of biological and pharmacological properties, such as their anti-
inflammatory2, antibacterial3, antiviral4, and anti-cancer activities5.
In the current medical era, much attention has been extensively
paid to modify and update coumarin-based drug leads from the
point of view of drug design and medicinal chemistry to fulfill
inhibition24,25, COX-2 inhibition26, and apoptosis induction27–31
.
On the other hand, sulfonamide derivatives constitute another
important class of organic compounds that displayed interesting
biological activities including anti-carbonic anhydrase and anti-
cancer activities32,33. It is noteworthy to mention that there is a
continuing interest in the synthesis of coumarin sulfonamide deriv-
atives as potent anti-cancer agents34–37. SLC-0111, Figure 1, an
ureido benzenesulfonamide derivative, is currently in phase I/II
clinical trials as anti-cancer drug. SLC-0111 II is characterized by its
selectivity towards inhibition of the transmembrane isoforms hCA
IX/XII (over the cytosolic isoforms hCA I/II). SLC-0111 was also able
to block human breast cancer invasion, delay tumor growth and
diminish the cancer stem cell population in vivo38–40
.
more effective and safe anti-cancer agents6–8
.
KCN1, Figure 1, a novel synthetic sulfonamide, is a HIF pathway
inhibitor with in vitro and in vivo anti-pancreatic cancer activities
and preclinical pharmacology. KCN1 specifically inhibited HIF
reporter gene activity in several glioma cell lines at the nanomolar
level. Also, KCN1 effectively inhibited the growth of subcutaneous
malignant glioma tumor xenografts with low side effects on the
host41,42. Moreover, Indisulam (N-(-3-chloro-7-indolyl)-1,4-benzene-
The promising biological profile of coumarins as anti-cancer
agents, and their easy synthetic modifications paved the way for
design and synthesis of various coumarin-based derivatives with
diverse activities against cancer. In this context, many coumarin-
based molecules were widely reported to possess anti-cancer
activity through binding to different targets and diverse pharma-
cological mechanisms, to name just a few, selective estrogen
receptor modulation9–11 (as CHF4227, Figure 1), MEK1 disulfonamide), Figure 1, is an orally active sulfonamide anti-tumor
CONTACT Wagdy M. Eldehna
wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1096
A. SABT ET AL.
Figure 1. Structure of some reported coumarins and sulfonamides with effective anti-cancer activities.
agent that possesses anti-cancer properties through down-regula- (2 mmol) in absolute ethanol (25 ml) was refluxed for 12 h. The
obtained solid was filtered, dried, and recrystallized from ethanol
to give compounds 4a, b.
tion of various cell-cycle checkpoint molecules, thereby blocking
the phosphorylation of retinoblastoma protein and inducing p53
and p21. Pre-clinical and clinical studies have established synergy
of Indisulam with nucleoside analogs as well as topoisomerase
inhibitors43–47
.
2-Oxo-N-(4-sulfamoylphenyl)-2H-chromene-6-sulfonamide (4a)
Brown crystals, mp 266–268 ꢀC, yield (43%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 6.47 (d, H, J ¼ 9.5 Hz, H3 of coumarin), 6.94 (d,
2H, J ¼ 8.2 Hz, Ar), 7.31 (d, 1H, J ¼ 8.5 Hz, H8 of coumarin), 7.61 (d,
2H, J ¼ 8.4 Hz, Ar), 7.71–7.71 (m, 3H, NH2 and H7 of coumarin),
7.95 (d, 1H, J ¼ 10 Hz, H4 of coumarin), 8.11 (s, 1H, H5 of couma-
rin), 10.95 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6); d ppm:
116.32(2C), 116.90, 118.40, 118.84, 125.96, 125.97, 127.87 (2C),
Based on the aforementioned findings, herein we report the
synthesis of novel sets of coumarin sulfonamide derivatives (4a, b,
8a–d, 11a–d, 13a, b and 15a–c) and their in vitro growth inhibi-
tory activity towards the proliferation of three cancer cell lines;
HepG2 (hepatocellular carcinoma), MCF-7 (breast cancer), and
Caco-2 (colon cancer). Additionally, the synthesized coumarin sul-
fonamides were further examined regarding their potential apop-
totic induction and their effects on cell cycle progression in the 129.81 (2C), 137.55, 144.94 (C4 of coumarin), 153.88 (C9 of couma-
rin), 160.40 (C¼O of coumarin); MS m/z [%]: 380 [9.21], 92 [100].
Hep-G2 cancer cells to acquire a perception for the mechanism of
their anti-cancer activity.
Analysis for C15H12N2O6S2 (380), Calcd.: % C, 47.36; H, 3.18; N, 7.36;
O, 25.24; S, 16.86. Found: % C, 47.28; H, 3.22; N, 7.42; O, 25.18;
S, 16.78.
Materials and methods
Chemistry
2-Oxo-N-(4-(N-(pyridin-2-yl)sulfamoyl)phenyl)-2H-chromene-6-
Melting points were determined on Electrothermal IA 9000 appar- sulfonamide (4b)
atus and were uncorrected. Elemental microanalyses were per-
White crystals, mp 237–239 ꢀC, yield (40%). 1H NMR (400 MHz,
formed on Elementar, Vario EL, at the Micro-analytical Laboratory,
National Research Centre, Dokki, Cairo. The 1H NMR and 13C NMR
spectra were recorded with a BrukerAvance 400 MHz spectrometer
at Turku University, Finland and JEOL ACA 500 NMR spectrometer,
at the National Research Centre, Dokki, Cairo, Egypt. The mass
spectra were performed on Mass Spectrometer Finnigan MAT SSQ-
7000 and GCMS-QP 1000EX Shimadzu Gas Chromatography MS
Spectrometer at Faculty of Science, Cairo University, Egypt. The
reactions were followed by TLC (silica gel, aluminum sheets 60
F254, Merck) using chloroform/methanol (9.5:0.5 v/v) as eluent.
DMSO-d6) d ppm: 6.62 (d, 1H, J ¼ 9.6 Hz, H3 of coumarin), 6.83
(t, 1H, J ¼ 12.4 Hz, H3 of pyridine), 7.08 (d, 1H, J ¼ 8.8 Hz, H5 of
pyridine), 7.24 (d, 2H, J ¼ 8.8 Hz, Ar), 7.55 (d, 1H, J ¼ 8.8 Hz, H8 of
coumarin), 7.67 (t, 1H, J ¼ 8.8 Hz, H4 of pyridine), 7.75 (d, 2H,
J ¼ 8.8 Hz, Ar), 7.94–7.96 (m, 2H, H2 of pyridine and H7 of couma-
rin), 8.15 (d, 1H, J ¼ 10 Hz, H4 of coumarin), 8.28 (s, 1H, H5 of cou-
marin), 11.05 (s, 1H, NH, D2O exchangeable), 11.92 (s, 1H, NH, D2O
exchangeable); 13 C NMR (100 MHz, DMSO); d ppm: 114.23 (C5 of
pyridine), 118.42 (2C), 118.99 (2C), 119.49, 128.29, 128.66, 128.55,
129.88, 129.96, 135.38, 137.18, 140.90, 141.01, 141.29, (Ar, pyridine
and coumarin), 143.76 (C4 of coumarin), 153.57 (C¼N of pyridine),
156.51 (C9 of coumarin), 159.54 (C¼O of coumarin); Analysis for
C20H15N3O6S2 (457), Calcd.: % C, 52.51; H, 3.30; N, 9.19; O, 20.98; S,
14.02. Found: % C, 52.58; H, 3.40; N, 9.22; O, 21.01; S, 14.09.
Synthesis of coumarin-6-sulfonyl chloride 2
Compound 2-oxo-2H-chromene-6-sulfonyl chloride 2 was obtained
from a reaction of coumarin with chlorosulfonic acid48.
Synthesis of N-(4-acetylphenyl)-2-oxo-2H-chromene-6-
sulfonamide (6)
Synthesis of coumarin-6-sulfonamide derivatives 4a,b
A solution of 2-oxo-2H-chromene-6-sulfonyl chloride 2 (0.5 g,
Compound
6
was prepared according to the literatures
2 mmol) and the appropriate sulfanilamide 3a or sulfapyridine 3b procedures49.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1097
Synthesis of schiff base coumarin sulfonamides 8a-d
2-Oxo-N-(4–(1-(2-tosylhydrazono)ethyl)phenyl)-2H-chromene-6-
sulfonamide (8d)
To a mixture of compound 6 (0.25 g, 0.729 mmol) and the appro-
priate phenylhydrazine 7a, 2,4-dinitrophenylhydrazine 7b, 2,4,6-tri-
chlorophenylhydrazine 7c or p-toluenesulfonyl hydrazide 7d
(0.911 mmol) in absolute ethanol (30 ml), three drops of glacial
acetic acid was added and the reaction mixture was refluxed for
8 h. After cooling, the precipitate was filtered, washed with water,
purified via crystallization from ethanol to give compounds 8a–d,
respectively.
White crystals, mp 163–165 ꢀC, yield (76%). 1H NMR (400 MHz,
DMSO-d6) d ppm: 2.03 (s, 3H, CH3), 2.31 (s, 3H, CH3 - tosyl), 6.60 (d,
1H, J ¼ 9.6 Hz, H3 of coumarin), 7.11 (d, 2H, J ¼ 8.4 Hz, Ar), 7.39 (d,
2H, J ¼ 8.4 Hz, Ar-tosyl), 7.50–7.56 (m, 3H, Ar, and H8 of coumarin),
7.78 (d, 2H, J ¼ 8.4 Hz, Ar-tosyl), 7.91 (dd, 1H, J ¼ 2 and 2 Hz, H7 of
coumarin), 8.17 (d, 1H, J ¼ 9.6 Hz, H4 of coumarin), 8.18 (s, 1H, H5 of
coumarin), 10.44 (s, 1H, NH), 10.56 (s, 1H, NH); 13 C NMR (100 MHz,
DMSO); d ppm: 14.47 (CH3), 21.47 (CH3), 118.30, 118.45, 118.53,
119.42, 119.75, 125,77, 127.50, 128.04 (2C), 128.18, 129.91, 129.97
(2C), 133.45, 135.70, 136.57, 138.90, 143.79, 143.83 (C4 of coumarin),
152.92 (C¼N), 156.37 (C9 of coumarin), 159.56 (C¼O of coumarin);
Analysis for C24H21N3O6S2 (511), Calcd.: % C, 56.35; H, 4.14; N, 8.21; O,
2-Oxo-N-(4–(1-(2-phenylhydrazono)ethyl)phenyl)-2H-chromene-6-
sulfonamide (8a)
Brown crystals, mp 178–180 ꢀC, yield (66%). 1H NMR (500 MHz, 18.77; S, 12.54. Found: % C, 56.40; H, 4.08; N, 8.16; O, 18.71; S, 12.50.
DMSO-d6) d ppm: 2.19 (s, 3H, CH3), 6.47 (d, 1H, J ¼ 12 Hz, H3 of
coumarin), 6.87–7.83 (m, 11H, NH, Ar, and H8 of coumarin), 7.95
2–(1-(4–(2-Oxo-2H-chromene-6-sulfonamido)phenyl)ethylidene)
(dd, 1H, J ¼ 6 and 10.5 Hz, H7 of coumarin), 8.04 (d, 1H, J ¼ 2.4 Hz,
hydrazine carbothioamide (9)
H4 of coumarin), 8.23 (s, 1H, H5 of coumarin), 11.09 (s, 1H, NH);
13C NMR (125 MHz, DMSO); d ppm: 13.10 (CH3), 113.21 (2C), 113.25,
A mixture of N-(4-acetylphenyl)-2-oxo-2H-chromene-6-sulfonamide
118.59, 119.25, 120.47, 122.66, 126.50, 128.18, 128.29, 129.32 (2C),
6 (0.5 g, 1.45 mmol), and thiosemicarbazide (1.45 mmol) in abso-
129.40, 130.33 (2C), 132.62, 135.93, 135.95, 143.81 (C4 of couma- lute ethanol (20 ml) and catalytic amount of acetic acid was
refluxed for 6 h. The obtained solid was filtered, dried, and washed
rin), 154.54 (C¼N), 156.31 (C9 of coumarin), 160.02 (C¼O of cou-
marin); HRMS (ESI): m/z calculated for C23H19N3O4S [M þ H]þ,
with hot ethanol to afford compound 9.
Light yellow crystals, mp 253–254 ꢀC, yield (34%). 1H NMR
434.1169; found, 434.1163.
(400 MHz, DMSO-d6) d ppm: 2.18 (s, 3H, CH3), 6.58 (d, 1H, J ¼ 4.4 Hz,
H3 of coumarin), 7.25 (d, 2H, J ¼ 6.8 Hz, Ar), 7.55 (d, 1H, J ¼ 2.4 Hz,
H8 of coumarin), 7.79–7.84 (m, 4H, NH2, Ar), 7.94 (dd, 1H, J ¼ 6 and
N-(4–(1-(2–(2,4-dinitrophenyl)hydrazono)ethyl)phenyl)-2-oxo-2H-
1.6 Hz, H7 of coumarin), 8.16 (d, 1H, J ¼ 10 Hz, H4 of coumarin),
chromene-6-sulfonamide (8b)
8.29 (d, 1H, J ¼ 2.4 Hz, H5 of coumarin), 10.13 (s, 1H, NH), 10.60 (s,
Red crystals, mp 269–270 ꢀC, yield (60%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 2.36 (s, 3H, CH3), 5.72 (s, 1H, NH), 6.61 (d, 1H,
J ¼ 6.50 Hz, H3 of coumarin), 7.21(d, 2H, J ¼ 11 Hz, Ar), 7.57–7.85
(m, 3H, Ar, and H8 of coumarin), 7.96 (dd, 1H, J ¼ 2.5 and 2.5 Hz,
1H, NH); 13 C NMR (100 MHz, DMSO-d6); d ppm: 14.17 (CH3), 113.57,
118.30, 118.34, 119.43 (2C), 119.69, 128.14 (2C), 130.01, 133.82,
135.86, 138.72, 143.82 (C4 of coumarin), 147.53 (C¼N), 156.36 (C9
of coumarin), 159.54 (C¼O of coumarin), 179.22 (C ¼ S), Analysis for
H7 of coumarin), 8.03 (d, 1H, J ¼ 12 Hz, H4 of coumarin), 8.19 (d, C18H16N4O4S2 (416), Calcd.: % C, 51.91; H, 3.87; N, 13.45; O, 15.37; S,
15.40. Found: %C, 51.79; H, 3.82; N, 13.31; O, 15.42; S, 15.47.
1H, J ¼ 12 Hz, Ar), 8.29 (s, 1H, H5 of coumarin), 8.36 (d, 1H,
J ¼ 12 Hz, NO2-CH, Ar), 8.87 (s, 1H, H3 of 2,4-(NO2)2C6H3), 11.06 (s,
H, NH); 13 C NMR (125 MHz, DMSO-d6); d ppm: 13.83 (CH3), 118.26,
General procedures for preparation of target compounds (11a–d
and 13a, b)
118.29, 118.38, 118.42, 118.60, 119.41, 119.50, 120.01, 127.26,
128.18, 128.29, 130.00, 130.05, 130.31, 132.60, 135.88, 138.33,
142.38, 143.86 (C4 of coumarin), 154.37 (C¼N), 156.34 (C9 of cou-
A mixture of thiosemicarbazone 9 (10 mmol) and the appropriate
marin), 159.56 (C¼O of coumarin); Analysis for C23H17N5O8S (523), a-halocarbonyl compounds 10a–d, phenacyl bromide 12a or cou-
marin-3-acetylbromide 12 b (10 mmol) in dioxane (25 ml) contain-
ing catalytic amount of triethylamine was heated under reflux for
8 h and then cooled. The solution was poured onto water-ice and
concentrated hydrochloric acid. The solid produced was collected
by filtration and crystallized from ethanol to furnish compounds
11a–d and 13a, b, respectively.
Calcd.: % C, 52.77; H, 3.27; N, 13.38; O, 24.45; S, 6.13. Found: % C,
52.84; H, 3.20; N, 13.32; O, 24.40; S, 6.18.
2-Oxo-N-(4–(1-(2–(2,4,6-trichlorophenyl)hydrazono)ethyl)phenyl)-
2H-chromene-6-sulfonamide (8c)
White crystals, mp 259–260 ꢀC, yield (58%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 2.21 (s, 3H, CH3), 6.55 (d, 1H, J ¼ 12 Hz, H3 of
coumarin), 7.11 (d, 2H, J ¼ 10.5 Hz, Ar), 7.54–7.60 (m, 3H, Ar, and
H8 of coumarin), 7.64 (s, 2H, H3 and H5 of 2,4,6-(Cl)3C6H2), 7.92
(dd, 1H, J ¼ 2 and 2 Hz, H7 of coumarin), 8.04 (s, 1H, NH), 8.16 (d,
1H, J ¼ 12.5 Hz, H4 of coumarin), 8.22 (s, 1H, H5 of coumarin),
10.51 (s, 1H, NH); 13 C NMR (125 MHz, DMSO-d6); d ppm: 12.98
(CH3), 118.23, 118.29, 118.36, 119.39, 120.21 (2C), 126.78 (2C),
128.06, 128.17, 128.86, 129.14 (2C), 130.03, 134.92, 135.86, 137.80,
139.14, 143.85 (C4 of coumarin), 145.64 (C¼N), 156.33 (C9 of cou-
marin), 159.54 (C¼O of coumarin); Analysis for C23H16Cl3N3O4S
(534), Calcd.: % C, 51.46; H, 3.00; Cl, 19.81; N, 7.83; O, 11.92; S,
5.97. Found: % C, 51.49; H, 2.96; Cl, 19.85; N, 7.85 O, 11.87; S, 5.92.
N-(4–(1-(2–(4-methylthiazol-2-yl)hydrazono)ethyl)phenyl)-2-oxo-
2H-chromene-6-sulfonamide (11a)
Brown crystals, mp 257–259 ꢀC, yield (78%). 1H NMR (500 MHz,
DMSO-d6) d ¼ 2.15 (s, 3H, CH3), 2.45 (s, 3H, CH3-thiazole), 5.99
(s, 1H, CH thiazole), 6.57 (d, 1H, J ¼ 9.6 Hz, H3 of coumarin), 7.19
(d, 2H, J ¼ 8.6 Hz, Ar), 7.52 (d, 1H, J ¼ 8.6 Hz, H8 of coumarin), 7.80
(d, 2H, J ¼ 8.6 Hz, Ar), 7.91 (dd, 1H, J ¼ 9.5 and 2 Hz, H7 of couma-
rin), 8.12 (d, 1H, J ¼ 10 Hz, H4 of coumarin), 8.27 (s, 1H, H5 of
coumarin), 10.55 (s, 1H, NH, D2O exchangeable), 11.01 (s, 1H, NH,
D2O exchangeable); 13 C NMR (125 MHz, DMSO); d ppm: 13.83
(CH3), 14.55 (CH3-thiazole), 118.26, 118.42, 118.60, 119.41, 119.50,
120.01, 127.26, 128.18, 130.31, 132.60, 135.88, 138.33, 142.38

1098
A. SABT ET AL.
(Ar, C5-thiazole and coumarin), 143.86 (C4 of coumarin), 154.33 Calcd.: % C, 58.73; H, 4.22; N, 14.68; O, 11.18; S, 11.20. Found: % C,
(C¼N), 154.37 (C4-thiazole), 156.34 (C9 of coumarin), 159.56 (C¼O 58.82; H, 4.14; N, 14.77; O, 11.19; S, 11.16.
of coumarin), 168.66 (S-C¼N): Analysis for C21H18N4O4S2 (454),
Calcd.: % C, 55.49; H, 3.99; N, 12.33; O, 14.08; S, 14.11. Found: % C,
55.40; H, 3.96; N, 12.36; O, 14.05; S, 14.14.
2-Oxo-N-(4–(1-(2–(4-phenylthiazol-2-yl)hydrazono)ethyl)phenyl)-
2H-chromene-6-sulfonamide (13a)
Yellow crystals, mp 170–171 ꢀC, yield (80%). 1H NMR (500 MHz,
Ethyl 4-methyl-2–(2-(1–(4-(2-oxo-2H-chromene-6-
DMSO-d6) d ppm: 2.19 (3H, s, CH3), 6.56 (d, 1H, J ¼ 5.75 Hz, H3 of
sulfonamido)phenyl) ethylidene)hydrazinyl)thiazole-5-
coumarin), 7.16 (d, 2H, J ¼ 8.8 Hz, Ar), 7.27–7.36 (m, 5H, Ar), 7.54
carboxylate (11b)
(d, 1H, J ¼ 8.65 Hz, H8 of coumarin), 7.61 (d, 2H, J ¼ 8.4 Hz, Ar), 7.94
Yellow crystals, mp 180–182 ꢀC, yield (53%). 1H NMR (500 MHz,
(dd, 1H, J ¼ 2.4 and 8 Hz, H7 of coumarin), 8.15(d, 1H, J ¼ 8 Hz, H4
of coumarin), 8.20 (s, 1H-thiazole), 8.29 (d, 1H, J ¼ 2.5 Hz, H5 of
coumarin), 10.59 (s, 1H, NH), 10.99 (s, 1H, NH); 13C NMR (125 MHz,
DMSO-d6); d ppm: 14.29 (CH3), 104.44 (C4-thiazole), 118.29, 118.58,
119.43, 120.11, 125.96, 128.20, 128.29, 129.06 (2C), 130.05, 130.33,
132.61, 134.20, 135.21, 135.61, 135.86, 138.26, 142.35 (Ar, and cou-
marin), 143.86 (C4 of coumarin), 146.27 (C5-thiazole), 156.36
(C¼N), 156.50 (C9 of coumarin), 159.56 (C¼O of coumarin), 170.21
(S-C¼N);. MS m/z [%]: 516 [93], 132 [100]; Analysis for
C26H20N4O4S2 (516), Calcd.: % C, 60.45; H, 3.90; N, 10.85; O, 12.39;
S, 12.41 Found: % C, 60.39; H, 3.88; N, 10.87; O, 12.33; S, 12.36.
DMSO-d6) d ppm: 1.21 (t, 3H, J ¼ 11.48 Hz, CH3), 2.19 (s, 3H, CH3),
2.41 (s, 3H, CH3-thiazole), 4.14 (q, 2H, J ¼ 6.65 Hz, OCH2), 6.56 (d,
1H, J ¼ 9.2 Hz, H3 of coumarin), 7.14 (d, 2H, J ¼ 8.4 Hz, Ar), 7.53 (d,
1H, J ¼ 8.6 Hz, H8 of coumarin), 7.63 (d, 2H, J ¼ 8.4 Hz, Ar), 7.91
(dd, 1H, J ¼ 9.6 and 5 Hz, H7 of coumarin), 8.14 (d, 1H, J ¼ 9.6 Hz,
H4 of coumarin), 8.22 (s, 1H, H5 of coumarin), 10.67 (s, 1H, NH),
11.04 (s, 1H, NH); 13 C NMR (125 MHz, DMSO-d6); d ppm: 14.62
(CH3), 14.87 (CH3), 26.93 (CH3-ester), 60.69 (OCH2), 112.87, 118.34,
118.67, 119.48, 120.01, 127.51, 128.25, 130.13 (2C), 130.37, 135.91
(Ar, C5-thiazole and coumarin), 143.87 (C4 of coumarin), 152.41
(C¼N), 156.41 (C4-thiazole), 156.53 (C9 of coumarin), 159.48 (C¼O
of coumarin), 169.66 (S-C¼N), 185.45 (C¼O); HRMS (ESI): m/z calcu-
lated for C24H22N4O4S2 [M þ H]þ, 527.1054; found, 527.1052.
2-Oxo-N-(4–(1-(2–(4-(2-oxo-2H-chromen-3-yl)thiazol-2-
yl)hydrazono)ethyl)phenyl)-2H-chromene-6-sulfonamide (13b)
Yellow crystals, mp 201–202 ꢀC, yield (63%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 2.16 (s, 3H, CH3), 6.58 (d, 1H, J ¼ 9.3 Hz, H3 of
coumarin), 7.20 (d, 2H, J ¼ 8.1 Hz, Ar), 7.34–8.80 (m, 7H, 8H couma-
rin and Ar), 7.97 (dd, 1H, J ¼ 8.6 and 9.5 Hz, H7 of coumarin), 8.11
(s, 1H, H4 of coumarin), 8.15 (d, 1H, J ¼ 9.5 Hz, H4 of coumarin),
8.28 (d, 1H, J ¼ 8.6 Hz, H5 of coumarin), 8.52 (s, 1H-thiazole), 10.60
(s, 1H, NH); 10.99 (s, 1H, NH); 13 C NMR (125 MHz, DMSO-d6); d
ppm: 14.32 (CH3), 111.46 (C4-thiazole), 116.36, 118.30, 118.43,
118.58 (2C), 119.50, 120.05, 125.20, 127.17, 128.29, 129.20 (2C),
129.98, 130.33 (2C), 132.61, 134.03, 135.61, 135.84, 142.35, 143.80
(C4 of coumarin), 152.75 (C¼N), 156.36 (C5-thiazole), 156.50 (2C9
of coumarin), 159.51 (2C¼O of coumarin), 169.67 (S-C¼N); Analysis
for C29H20N4O6S2 (584), Calcd.: % C, 59.58; H, 3.45; N, 9.58; O,
16.42; S, 10.97. Found: % C, 59.52; H, 3.48; N, 9.56; O, 16.36;
S, 10.94.
N-(4–(1-(2–(5-((4-chlorophenyl)diazenyl)-4-methylthiazol-2-
yl)hydrazono)ethyl) phenyl)-2-oxo-2H-chromene-6-
sulfonamide (11c)
Red crystals, mp 250–252 ꢀC, yield (88%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 2.41 (s, 3H, CH3), 2.57(s, 3H, CH3-thiazole), 6.62
(d, 1H, J ¼ 12 Hz, H3 of coumarin), 7.21 (d, 2H, J ¼ 10.5 Hz, Ar),
7.35–7.39 (m, 4H, Ar), 7.59 (d, 1H, J ¼ 8.6 Hz, H8 of coumarin), 7.85
(d, 2H, J ¼ 8.8 Hz, Ar), 7.97 (dd, 1H, J ¼ 3 and 3 Hz, H7 of coumarin),
8.19 (d, 1H, J ¼ 12 Hz, H4 of coumarin); 8.30 (s, 1H, H5 of couma-
rin), 10.65 (br, 2H, 2NH,); 13 C NMR (125 MHz, DMSO) d ppm: 14.55
(CH3), 15.50 (CH3), 118.35, 118.38, 118.64, 119.47 (2C), 119.98,
123.52, 124.53, 128.22, 128.54, 129.64 (2C), 129.99, 130.04, 130.31,
133.07, 135.92, 139.30, 140.32 (Ar, C5-thiazole and coumarin),
143.84 (C4 of coumarin), 147.89 (C4-thiazole), 151.37 (C¼N), 156.42
(C9 of coumarin), 159.54 (C¼O of coumarin), 171.80 (S-C¼N);
Analysis for C27H21ClN6O4S2 (592), Calcd.: % C, 54.68; H, 3.57; Cl,
5.98; N, 14.17; O, 10.79; S, 10.81. Found: % C, 54.61; H, 3.52; N,
14.20; O, 10.71; S, 10.70.
General procedures for synthesis of thiazolidinones (15a–c)
In 50 ml round-bottom flask, thiosemicarbazone 9 (10 mmol) was
dissolved in 15 ml acetic acid, followed by addition of anhydrous
sodium acetate (30 mmol) under magnetic stirring and warming.
After 20 min, bromoacetic acid 14a, 2-bromopropanoic acid 14b
or maleic anhydride 14c (15 mmol) was added and the reaction
mixture was maintained under reflux for 8 h. After cooling, the
precipitate was filtered, washed with water, dried, and recrystal-
lized from ethanol to give compounds 15a–c, respectively.
N-(4–(1-(2–(4-methyl-5-(p-tolyldiazenyl)thiazol-
2yl)hydrazono)ethyl)phenyl)-2-oxo-2H-chromene-6-
sulfonamide (11d)
Red crystals, mp 258–259 ꢀC, yield (83%). 1H NMR (500 MHz,
DMSO-d6) d ppm: 2.25 (s, 3H, CH3), 2.41 (s, 3H, CH3), 2.56 (s, 3H,
CH3-thiazole), 6.62 (d, 1H, J ¼ 12 Hz, H3 of coumarin), 7.13 (d, 2H,
J ¼ 10 Hz, Ar), 7.20–7.25 (m, 4H, Ar), 7.59 (d, 1H, J ¼ 11 Hz, H8 of
coumarin), 7.85 (d, 2H, J ¼ 10.5 Hz, Ar), 7.97 (dd, 1H, J ¼ 3 and 3 Hz,
H7 of coumarin), 8.19 (d, 1H, J ¼ 12 Hz, H4 of coumarin), 8.29 (s,
1H, H5 of coumarin), 10.47 (s, 1H, NH), 10.77 (s, 1H, NH); 13 C NMR
(125 MHz, DMSO); d ppm: 15.48 (CH3), 16.94 (CH3), 20.85 (CH3),
114.68, 118.39, 118.60, 119.47 (2C), 128.23, 128.28, 128.52, 129.99,
2-Oxo-N-(4–(1-(2–(5-oxo-4,5-dihydrothiazol-2-
yl)hydrazono)ethyl)phenyl)-2H-chromene-6-sulfonamide (15a)
Yellow crystals, mp 191–192 ꢀC, yield (64%). 1H NMR (400 MHz,
CDCl3) d ppm: 2.23 (s, 3H, CH3), 3.8 (s, 2H, CH2, thiazolidinone),
6.62 (d, 1H, J ¼ 10.8 Hz, H3 of coumarin), 7.25 (d, 2H, J ¼ 12 Hz, Ar),
130.04, 130.20, 130.33, 131.65, 133.26, 135.63, 135.83, 137.96, 7.55 (d, 1H, J ¼ 8.4 Hz, H8 of coumarin); 7.85 (d, 2H, J ¼ 8.4 Hz, Ar),
140.10, 141.61, 142.35 (Ar, thiazole and coumarin), 143.83 (C4 of 7.95 (dd, 1H, J ¼ 2.4 and 2 Hz, H7 of coumarin), 8.16 (d, 1H,
coumarin), 151.15 (C¼N), 156.44 (C9 of coumarin), 159.54 (C¼O J ¼ 10 Hz, H4 of coumarin), 8.29 (s, 1H, H5 of coumarin), 10.66
of coumarin), 171.79 (S-C¼N); Analysis for C28H24N6O4S2 (572), (s, 1H, NH), 10.97 (s, 1H, NH); 13 C NMR (100 MHz, CDCl3);

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1099
d ppm: 15.33 (CH3), 56.30 (CH2), 118.39, 118.64 (2C), 119.72, compounds as well as doxorubicin as the reference compound.
127.93, 128.26, 129.99 (2C), 130.30, 132.66, 135.87, 142.35, 143.79 Following 72 h of treatment, the cells were fixed with 10% tri-
(C4 of coumarin), 152.56 (C¼N), 156.50 (C9 of coumarin), 159.48 chloroacetic acid for 1 h at 4 ꢀC. Wells were stained for 10 min at
room temperature with 0.4% SRB dissolved in 1% acetic acid. The
(C¼O of coumarin), 166.33 (S-C¼N), 196.90 (C¼O); Analysis for
plates were air dried for 24 h, and the dye was solubilized with
Tris–HCl for 5 min on a shaker at 1600 rpm. The optical density
(OD) of each well was measured spectrophotometrically at 564 nm
with an ELISA microplate reader (ChroMate-4300, FL, USA). The
IC50 values were calculated according to the equation for
Boltzmann sigmoidal concentrationeresponse curve using the non-
linear regression models (GraphPad, Prism Version 5). The results
reported are means of at least three separate experiments.
Significant differences were analyzed by one-way ANOVA wherein
the differences were considered to be significant at p < .05.
C20H16N4O5S2 (456), Calcd.: % C, 52.62; H, 3.53; N, 12.27; O, 17.52;
S, 14.05. Found: % C, 52.58; H, 3.50; N, 12.19; O, 17.50; S, 14.00.
N-(4–(1-(2–(4-methyl-5-oxo-4,5-dihydrothiazol-2-
yl)hydrazono)ethyl)phenyl)-2-oxo-2H-chromene-6-
sulfonamide (15b)
Brown crystals, mp 180–182 ꢀC, yield (50%). 1H NMR (400 MHz,
CDCl3) d ppm: 1.59 (d, 3H, J ¼ 9 Hz, CH3 thiazolidinone), 2.27 (s, 3H,
CH3), 3.96 (q, 1H, J ¼ 9 Hz CH thiazolidinone), 5.23 (s, 1H, NH), 6.44
(d, 1H, J ¼ 10 Hz, H3 of coumarin), 7.05 (d, 2H, J ¼ 10.5 Hz, Ar), 7.30
(d, 1H, J ¼ 10.5 Hz, H8 of coumarin), 7.61 (d, 1H, J ¼ 12 Hz, H4 of
coumarin), 7.69 (d, 2H, J ¼ 10.5 Hz, Ar), 7.84 (dd, 1H, J ¼ 5.5 and
3 Hz, H7 of coumarin), 7.95 (s, 1H, H5 of coumarin), 11.02 (s, 1H,
NH); 13 C NMR (100 MHz, CDCl3); d ppm: 13.70 (CH3), 18.17 (CH3),
41.47 (CH of thiazolidinone), 117.10, 117.49, 117.88, 119.85 (2C),
126.66, 127.03 (2C), 129.05, 134.25, 136.40, 140.69, 141.30 (C4 of
coumarin), 148.51 (C¼N), 155.60 (C9 of coumarin); 158.13 (C¼O of
coumarin), 172.21 (S-C¼N), 180.55 (C¼O); Analysis for
C21H18N4O5S2 (470), Calcd.: % C, 53.61; H, 3.86; N, 11.91; O, 17.00;
S, 13.63. Found: % C, 53.55; H, 3.83; N, 11.87; O, 16.98; S, 13.60.
ELISA immunoassay
The levels of the apoptotic markers (Bax, caspase-3) and the anti-
apoptotic marker (Bcl-2) were examined using ELISA colorimetric
kits per the manufacturer’s protocol and referring to reported
instructions51,52
.
Cell cycle analysis
The HepG2 cells were treated with compound 13a for 24 h (at IC50
concentration), then cells were washed twice with ice-cold phos-
phate-buffered saline (PBS). Subsequently, the treated cells were
collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol,
washed with PBS, re-suspended with 0.1 mg/mL RNase, stained
with 40 mg/mL PI, and analyzed by flow cytometry using FACS
Calibur (Becton Dickinson, BD). The cell cycle distributions were
calculated using CellQuest software (Becton Dickinson)53.
2–(4-Oxo-2–(2-(1–(4-(2-oxo-2H-chromene-6-sulfonamido)
phenyl)ethylidene) hydrazinyl)-4,5-dihydrothiazol-5-yl)acetic
acid (15c)
White crystals, mp 193–194 ꢀC, yield (40%). 1H NMR (400 MHz,
CDCl3) d ppm: 2.43 (s, 3H, CH3), 2.86 (t, 1H, J ¼ 6.4 Hz CH thiazolidi-
none), 3.04 (d, 2H, J ¼ 12.8 Hz CH2), 6.58 (d, 1H, J ¼ 7.6 Hz, H3 of
coumarin), 7.22 (d, 2H, J ¼ 6.4 Hz, Ar); 7.53 (d, 1H, J ¼ 7.2 Hz, H8 of
coumarin), 7.82 (d, 2H, J ¼ 6.4 Hz, Ar); 7.95 (dd, 1H, J ¼6.8 and 2 Hz,
H7 of coumarin), 8.16 (d, 1H, J ¼ 8 Hz, H4 of coumarin), 8.27 (s, 1H,
H5 of coumarin), 10.99 (s, 1H, NH), 11.14 (s, 1H, NH), 13.21 (s, 1H,
COOH); 13 C NMR (100 MHz, CDCl3); d ppm: 16.36 (CH3), 38.04
(CH2), 42.56 (CH of thiazolidinone), 118.42, 118.59 (2C), 119.51,
128.29, 129.99 (2C), 130.32 (2C), 132.62, 135.63, 142.37, 143.37 (C4
of coumarin), 152.24 (C¼N), 156.56 (C9 of coumarin), 159.51 (C¼O
of coumarin), 165.86 (S-C¼N), 172.48 (C¼O of COOH), 196.92
(C¼O); Analysis for C22H18N4O7S2 (514), Calcd.: % C, 51.35; H, 3.53;
N, 10.89; O, 21.77; S, 12.46. Found: % C, 51.30; H, 3.51; N, 10.86; O,
21.74; S, 12.44.
Annexin V–FITC apoptosis assay
Phosphatidylserine externalization was measured using Annexin V-
FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA) accord-
ing to the manufacturer's instructions, as reported earlier53. HepG2
cells were treated with 13a at defined concentrations for 24 h.
Results and discussion
Chemistry
The synthetic routes employed to prepare the new target deriva-
tives are depicted in Schemes 1–4. The key intermediate 2-oxo-2H-
chromene-6-sulfonyl chloride 2 was obtained from a reaction of
coumarin 1 with chlorosulfonic acid, which subsequently reacted
with sulfanilamide 3a and sulfapyridine 3 b in refluxing ethanol to
furnish the corresponding target compounds 4a, b, respectively
(Scheme 1).
In Scheme 2, synthesis of N-(4-acetylphenyl)-2-oxo-2H-chro-
mene-6-sulfonamide 6 was achieved via stirring compound 2 with
4-aminoacetophenone 5 in dichloromethane at room temperature.
The later was refluxed with phenylhydrazine 7a, 2,4-dinitrophenyl-
hydrazine 7b, 2,4,6-trichlorophenylhydrazine 7c and p-toluenesul-
fonyl hydrazide 7d in absolute ethanol to afford target
coumarins 8a–d.
Biological evaluation
In vitro anti-proliferative activity
HepG2 liver cancer, MCF-7 breast cancer and Caco-2 cancer cell
lines were obtained from the National Cancer Institute (Cairo,
Egypt). Caco-2 cells were grown in DMEM while HepG2 and MCF-7
were grown in RPMI-1640. Media were supplemented with 10%
heat-inactivated FBS, 50 units/mL of penicillin and 50 g/mL of
streptomycin and maintained at 37 ꢀC in a humidified atmosphere
containing 5% CO2. The cells were maintained as a “monolayer
culture” by serial subculturing. Cytotoxicity was determined using
the sulforhodamine B (SRB) method as previously described by
Refluxing of compound 6 with thiosemacarbazide in ethanol
Skehan et al.50 Exponentially growing cells were collected using and catalytic amount of acetic acid yielded the key intermediate 9,
0.25% trypsin-EDTA and seeded in 96-well plates at 1000–2000 which utilized for preparation of diverse derivatives. Treatment of
cells/well in supplemented DMEM medium. After 24 h, cells were intermediate 9 with the appropriate a-halocarbonyl compounds
incubated for 72 h with various concentrations of the tested 10a–d in refluxing dioxane furnished the corresponding thiazole

1100
A. SABT ET AL.
Scheme 1. Synthesis of target compounds 4a, b; Reagents and conditions: (i) Heating 100ꢀC, 4 h; (ii) EtOH/reflux 12 h.
Scheme 2. Synthesis of target compounds 8a–d; Reagents and conditions: (i) Pyridine/stirring, rt 24h; (ii) EtOH/AcOH (catalytic)/reflux 8 h.
derivatives 11a–d. Additionally, refluxing of 9 with phenacyl brom-
ide 12a and coumarin-3-acetylbromide 12b in dioxane afforded the
corresponding thiazoles 13a, b, respectively (Scheme 3). Moreover,
treatment of intermediate 9 with each of bromoacetic acid 14a, 2-
bromopropanoic acid 14b and maleic anhydride 14c in acetic acid
in the presence of anhydrous sodium acetate afforded the target
thiazolidinone derivatives 15a–c, respectively (Scheme 4).
Postulated structures of the newly synthesized coumarin sulfo-
namides were in full agreement with their spectral and elemental
analyses data (Supplementary material).
From the displayed results, it was obvious that several of the
prepared coumarin sulfonamides possess excellent to modest
growth inhibitory activity towards the tested cancer cell lines.
Examinations of the anti-proliferative activity towards HepG2 cells
revealed that it is the most sensitive cell line to the influence of the
target coumarin sulfonamide derivatives. Compounds 13a and 15a
emerged as the most active coumarins against HepG2 cells through
this study with IC50 values of 3.48 0.28 and 5.03 0.39 mM, respect-
ively. They displayed 1.5- and 1.1-fold increased potency than doxo-
rubicin (IC50 ¼ 5.43 0.24 mM). Besides, compounds 4b and 15c with
IC50 values of 8.08 0.51 and 7.57 0.66 mM, respectively, displayed
excellent activity in comparison to doxorubicin. Also, compounds
8b and 13b (IC50 ¼ 11.84 1.34 and 11.80 1.16 mM, respectively)
exhibited good anti-proliferative activity. Moreover, compounds 6,
8a, 8c, and 15b had moderate activity with IC50 values ranging
from 25.07 2.08 to 29.80 2.21mM.
Concerning activity against MCF-7 cells, compounds 15a and
15b were the most active members that displayed potent anti-
proliferative activity with IC50 values of 10.95 0.96 and
10.62 1.35 mM, respectively, in comparison to the standard drug
doxorubicin (IC50 ¼ 3.18 0.32 mM). Moreover, compounds 8a, 11a,
and 15c were moderately active against MCF-7 cells with IC50 val-
ues: 14.30 1.18, 13.86 1.19 and 16.32 1.48 mM, respectively. On
the other hand, anti-proliferative activity evaluation in Caco-2 cells
showed that compound 8a possessed the best growth inhibitory
Biological evaluation
In vitro anti-proliferative activity
All the newly synthesized target coumarin sulfonamides were eval-
uated for their in vitro anti-proliferative activity against three human
tumor cancer cell lines, HepG2 hepatocellular carcinoma, MCF-7
breast cancer, and Caco-2 colon cancer, using SRB colorimetric assay
as described by Skehan et al.50 Doxorubicin was involved in the
experiments as a reference cytotoxic compound. The results were
expressed as growth inhibitory concentration (IC50) values which
represent the compound concentrations required to produce a 50%
inhibition of cell growth after 72h of incubation compared to
untreated controls. The results were summarized in Table 1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1101
Scheme 3. Synthesis of target compounds 11a–d and 13a, b; Reagents and conditions: (i) EtOH/AcOH(catalytic)/reflux 6h (ii) Dioxane/TEA (catalytic)/reflux 8h.
Scheme 4. Synthesis of target compounds 15a–c; Reagents and conditions: (i) AcOH/anhydrous AcONa/reflux 8h.
activity (IC50 ¼ 8.53 0.72), with twofold decreased activity than safety of the newly synthesized coumarin-6-sulfonamides towards
doxorubicin (IC50 ¼ 4.10 1.37 mM). In addition, compound 11a the normal cells. The results were expressed as IC50 values, and
showed good anti-proliferative activity against Caco-2 cancer cell selectivity index was calculated (Table 2). The tested coumarin-6-
line (IC50 ¼ 10.12 0.90). Whilst, both compounds 8d and 11d dis- sulfonamides (13a and 15a) showed non-significant cytotoxic
played moderate activity against Caco-2 cancer cell line with IC50 action with IC50 values of 73.20 3.47 and 55.92 0.39 mM, respect-
values: 16.02 1.32 and 16.06 1.28, respectively. These results ively, with good selectivity index range 21 and 11, thereby provid-
suggest that diverse functionalities could be incorporated into the ing a high safety profile as anticancer agents.
coumarin sulfonamide scaffold to obtain promising anti-prolifera-
tive activity against different types of cancer cells.
Apoptosis induction in HepG2 cells
Apoptosis induction in cancer cells represents one of the most
In vitro cytotoxicity towards human normal WI-38 cells
successful strategies for the development of cancer therapy54–56
.
The cell growth inhibitory activity of the most potent compounds As displayed above, coumarins 13a and 15a emerged as the most
13a and 15a was examined towards non-tumorigenic human nor- active ones towards HepG2 liver cancer cells. Accordingly, we
mal lung fibroblast cell line (WI-38) to investigate the potential investigated the ability of compounds 13a and 15a to provoke

1102
A. SABT ET AL.
Table 1. In vitro anti-proliferative activity of the target coumarin sulfonamide
derivatives against HepG2, MCF-7 and Caco-2 cancer cell lines.
Table 3. Effect of compounds 13a and 15a on the active caspase-
3 level, and the expression levels of Bcl-2 and Bax in HepG2 can-
cer cells treated with each compound at its IC50 concentration.
IC50 (mM)a
Caspase-3
Bax
Bcl-2
Compound
HepG2
MCF7
Caco-2
Compound (ng/mg protein) (Pg/mg protein) (ng/mg protein)
0.3021 (6.6)a
0.2625 (5.7)a
0.0457
453.3 (16.5)a
394.3 (14.3)a
27.52
1.51 (0.21)a
2.73 (0.39)a
7.07
4a
4b
6
8a
8b
8c
8d
9
35.48 2.93
8.08 0.51
25.62 1.36
26.99 2.01
11.84 1.34
29.80 2.21
89.91 7.63
163.03 10.22
>200
54.31 3.87
72.50 6.33
>200
3.48 0.28
11.80 1.16
5.03 0.39
25.07 2.08
7.57 0.66
5.43 0.24
50.73 4.61
22.95 2.04
>200
14.30 1.18
126.08 10.87
>200
192.69 15.33
66.88 6.07
81.54 6.89
8.53 0.72
89.59 7.26
76.93 6.59
16.02 1.32
>200
10.12 0.90
145.46 8.02
108.41 11.36
16.06 1.28
83.43 7.04
96.64 5.37
158.38 9.39
174.91 12.30
46.06 3.17
4.10 1.37
13a
15a
Control
aNumbers given between parentheses are the numbers of folds
of control.
>200
>200
HepG2 cells were treated with 13a at concentration equals to its
IC50. Figure 2 showed that exposure of HepG2 cells to compound
13a induced a significant increase in the percentage of cells at
Pre-G1 by 6.4-folds, with concurrent significant arrest in the G2-M
phase by 5.4-folds compared to control. Alteration of the Pre-G1
phase and arrest of G2-M phase were significant remarks for com-
pound 13a to induce apoptosis in HepG2 cells.
11a
11b
11c
11d
13a
13b
15a
15b
15c
Doxorubicin
13.86 1.19
63.42 5.69
34.73 2.94
>200
83.23 6.85
>200
10.95 0.96
10.62 1.35
16.32 1.48
3.18 0.32
Annexin V-FITC apoptosis assay
aIC50 values are the mean SE of three separate experiments.
The apoptotic effect of coumarin 13a was further assured by
Annexin V-FITC/PI (AV/PI) dual staining assay to investigate the
occurrence of phosphatidylserine externalization (Figure 3). Flow
cytometric analysis revealed that HepG2 cells treated with 13a
showed a significant increase in the percent of annexin V-FITC
positive apoptotic cells (UR þ LR) from 1.75% to 13.76% which
comprises about 7.9 folds compared to control.
Table 2. In vitro cytotoxic activity against normal WI-38 cells, and selectivity
index of the most active coumarins.
IC50 (mM)
Compound
WI-38
HepG2
Selectivity index
13a
15a
73.20 3.47
55.92 0.39
3.48 0.28
5.03 0.39
21
11
2D QSAR study
apoptosis in HepG2 cells to define the principle mechanism for
their anti-proliferative activity.
Development of QSAR models. With the aim to assess the struc-
tural basis for the anti-proliferative activity of the newly pre-
pared coumarin sulfonamides (4a, b, 6, 8a–d, 9, 11a–d, 13a, b
and 15a–c), 2D-QSAR analysis was carried out. This analysis was
run by means of the DS 4.0 software (Discovery Studio 4.0,
Accelrys, Co. Ltd)57. A set of the newly synthesized compounds
(4a, b, 6, 8a–d, 9, 11a–d, 13a, b and 15a–c) was used as train-
ing set with their measured pIC50 against HepG2 and Caco-2
for QSAR modeling, compounds 6, 8d, 15a and 15c were
chosen as statistical outliers while building model for HepG2
whereas, compounds 6, 9, and 13a were chosen for the Caco-
2 model.
“Calculate Molecular Properties” module was used for calculat-
ing the molecular properties. Genetic function approximation
(GFA) protocol was applied in order to choose the best descrip-
tors that characterize the activity. Multiple linear regression (MLR)
protocol was then employed to search for optimal QSAR models
with the best statistical validation measures and capable of corre-
lating bioactivity variation across the used training set collection.
QSAR model was validated employing leave one-out cross-valid-
ation by setting the folds to a number much larger than the num-
ber of samples, r2 (squared correlation coefficient value) and r2
prediction (predictive squared correlation coefficient value), resid-
uals between the predicted and experimental activity of the test
set and training set (Table 4).
Effects on mitochondrial apoptosis pathway proteins Bcl-2
and Bax
The Bcl-2 proteins family is mainly responsible for synchronizing
the mitochondrial apoptotic pathway, and classified into two
major classes: anti-apoptotic proteins such as Bcl-2 protein and
the counteracting pro-apoptotic proteins including Bax protein. In
this study, we examined the impact of coumarins 13a and 15a
on the level of the anti-apoptotic Bcl2 and the level of the pro-
apoptotic Bax (Table 3). As shown in Table 3, compound 13a
induced the protein expression of Bax with 16.5 folds of the con-
trol while 14.3 folds were recorded for compound 15a. On the
other hand, treatment of HepG2 cells with compounds 13a and
15a significantly reduced the expression levels of the anti-apop-
totic protein Bcl-2 by 21.4 and 38.6%, respectively, compared to
the control.
Effects on the levels of active caspase-3 (key executor
of apoptosis)
As a key executioner protease, caspase-3 is activated by upstream
initiator caspases as caspase-9. Herein, treatment of HepG2 cells
with coumarins 13a and 15a resulted in a significant elevation in
the level of active caspase-3 by about 6.6 and 5.7 folds, respect-
ively, compared to control (Table 3).
QSAR study results
Equation (1) Represents the best performing QSAR model for the
activity against HepG2;
Cell-cycle analysis
The impact of compound 13a on cell cycle progression was exam-
ined in HepG2 cancer cell line after 24 h of treatment (Figure 2).
This impact was illustrated by DNA flow cytometric analysis where
ꢁlogIC50 ¼ ꢁ0:7926 – 1:598CHI3C þ 0:2775DipoleY
(1)
ꢁ3:102e ꢁ 002JursPNSA3 þ 0:04829ShadowYZ

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1103
Figure 2. Effect of compound 13a on the phases of cell cycle of HepG2 cells.
Figure 3. Effect of compound 13a on the percentage of annexin V-FITC-positive staining in HepG2 cells. The experiments were done in triplicates. The four quadrants
ꢂ
identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic. Significantly different from control at p < .05.
Table 4. Experimental activities of the synthesized derivatives against the predicted activity according to Equations (1) and (2).
Caco-2
HepG2
Experimental activity
(pIC50
Predicted activity
Experimental activity
(pIC50
Predicted activity
Compound
)
(pIC50
)
Residual
)
(pIC50
)
Residual
4a
4b
6
ꢁ2.2849
ꢁ1.8253
—
ꢁ2.2003
ꢁ2.0638
—
ꢁ0.0845
0.2385
—
ꢁ1.5500
ꢁ0.9074
—
ꢁ1.5052
ꢁ0.9211
—
ꢁ0.0447
0.0137
—
8a
8b
8c
8d
ꢁ0.9309
ꢁ1.9523
ꢁ1.8861
ꢁ1.2047
—
ꢁ0.8882
ꢁ1.9601
ꢁ1.8261
ꢁ1.1348
—
ꢁ0.0427
0.0078
ꢁ0.0600
ꢁ0.0699
—
ꢁ1.4312
ꢁ1.0734
ꢁ1.4742
—
ꢁ2.2123
ꢁ2.3118
ꢁ1.7349
ꢁ1.8603
ꢁ2.3222
ꢁ0.5416
ꢁ1.0719
—
ꢁ1.5247
ꢁ1.0444
ꢁ1.6672
—
ꢁ2.1558
ꢁ2.1523
ꢁ1.9201
ꢁ1.9921
ꢁ2.2134
ꢁ0.6399
ꢁ1.0082
—
0.0935
ꢁ0.0290
0.1930
—
ꢁ0.0565
ꢁ0.1595
0.1852
0.1317
ꢁ0.1088
0.0983
ꢁ0.0637
—
9
11a
11b
11c
11d
13a
13b
15a
15b
15c
—
—
—
ꢁ2.1627
ꢁ2.0351
ꢁ1.2058
ꢁ1.9213
ꢁ1.9852
ꢁ2.1997
ꢁ2.2428
ꢁ1.6633
ꢁ2.1391
ꢁ2.0812
ꢁ1.5517
ꢁ1.8689
ꢁ1.7932
ꢁ2.1829
ꢁ2.1671
ꢁ1.6425
ꢁ0.0237
0.0462
0.3459
ꢁ0.0524
ꢁ0.1920
ꢁ0.0168
ꢁ0.0757
ꢁ0.0208
ꢁ1.3992
ꢁ1.1459
ꢁ0.2533
—
—
—
Equation (2) Represents the best performing QSAR model for versus the predicted bioactivity values –log IC50 for the training
the activity against Caco-2;
set compounds as shown in Figures 4 and 5.
The two MLR models exhibited good correlation coefficient r2
of 0.940 and 0.896, r2 (adj) ¼ 0.910 and 0.865, respectively, r2
(pred) ¼ 0.878 and 0.789, Least-Squared error ¼ 0.0168 and 0.0173
respectively, where r2 (adj) is r2 adjusted for the number of terms
in the model; r2 (pred) is the prediction r2, equivalent to q2 from a
ꢁlogIC50 ¼ ꢁ6:6095 ꢁ 3:7896IACMean þ 0:11713DipoleX
ꢁ 0:1615ShadowYlength
(2)
According to the former equations these QSAR models were
represented graphically by scattering plots of the experimental leave-1-out cross-validation58.

1104
A. SABT ET AL.
Figure 4. Predicted versus experimental pIC50 of the tested compounds against HepG2 according to Equation (1) (r2 ¼ 0.940).
Figure 5. Predicted versus experimental pIC50 of the tested compounds against Caco-2 according to Equation (2) (r2 ¼ 0.896).
Equations (1) and (2) for HepG2 and Caco-2 suggested that anti-proliferative activity towards HepG2 was also affected by the
the anti-proliferative activity of the prepared compounds was posi- high and negative value of Shadow_YZfrac while for Caco-2
tively affected by Dipole, which is a 3D electronic descriptors affected by Shadow_Ylength. Shadow indices are a set of geomet-
describing the strength and orientation behavior of a molecule ric descriptors that characterize the shape of the molecules. They
in an electrostatic field. Both the magnitude and the components are calculated by projecting the model surface on three mutually
(X, Y, Z) of the dipole moment are calculated (Debyes). It is esti- perpendicular planes: xy, yz, and xz. These descriptors depend not
mated by utilizing partial atomic charges and atomic coordinates. only on conformation, but also on the orientation of the model. In
Partial atomic charges are computed using Gasteiger if not order to calculate them, the models are first rotated to align the
present. Dipole properties have been correlated to long range principal moments of inertia with the x-, y-, and z-axes.
ligand-receptor recognition and subsequent binding. The Shadow_YZ is area of the molecular shadow in the yz plane while

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1105
Table 5. External validation for the established QSAR models.
Caco-2
HepG2
Experimental activity
(pIC50
Predicted activity
(pIC50
Experimental activity
(pIC50
Predicted activity
(pIC50
Compound
)
)
Residual
)
)
Residual
6
8d
9
13a
15a
15c
ꢁ1.9114
ꢁ1.8158
ꢁ0.0956
ꢁ1.4086
ꢁ1.9538
—
—
–0.7016
–0.8791
ꢁ0.9815
ꢁ1.9191
—
—
–0.8816
–1.1608
ꢁ0.4271
ꢁ0.0347
—
—
0.1801
0.2817
—
—
—
ꢁ2.3424
ꢁ1.0052
—
ꢁ1.7150
ꢁ1.7283
—
ꢁ0.6275
0.7231
—
—
—
—
Shadow_Ylength is indicator for length of molecule in the y doxorubicin (IC50 ¼ 4.10 1.37 mM). Compounds 13a and 15a were
dimension59.
able to induce apoptosis in HepG2 cells, as assured by the up-
Equation (1) also showed that the anti-proliferative activity was regulation of the Bax and down-regulation of the Bcl-2, besides
boosting caspase-3 levels. Besides, compound 13a induced a sig-
nificant increase in the percentage of cells at Pre-G1 by 6.4-folds,
with concurrent significant arrest in the G2-M phase by 5.4-folds
compared to control. Also, 13a displayed significant increase in
the percentage of annexin V-FITC positive apoptotic cells from
1.75% to 13.76%. Moreover, QSAR models were established to
explore the structural requirements controlling the anti-prolifera-
tive activities.
also affected by jurs descriptors. Jurs descriptors are a group of
geometric descriptors that combine both shape and electronic
information which may characterize the molecules60. In particular,
Jurs_PNSA_3, which contributes inversely to the activity, repre-
sents the atomic charge weighted negative surface area and calcu-
lated by Sum of the product of solvent-accessible surface
area ꢃ partial charge for all negatively charged atoms. This indi-
cates increasing this value in a molecule could increase reuptake
inhibition activity because it is negatively correlated with the activ-
ity. Equations (1) and (2) are also affected by topological descrip-
tors which are a special class of descriptors that do not rely on a
three-dimensional model. All calculations are derived from the
two-dimensional topology of the molecule. As CHI_3_C which is
connectivity indices while IAC_Mean indicate Graph-Theoretical
InfoContent descriptors.
Disclosure statement
No potential conflict of interest was reported by the authors.
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