Discovery of novel quinoline-based analogues of combretastatin A-4 as tubulin polymerisation inhibitors with apoptosis inducing activity and potent anticancer effect

Article abstract of DOI:10.1080/14756366.2021.1899168

A new series of quinoline derivatives of combretastatin A-4 have been designed, synthesised and demonstrated as tubulin polymerisation inhibitors. These novel compounds showed significant antiproliferative activities, among them, 12c exhibited the most potent inhibitory activity against different cancer cell lines (MCF-7, HL-60, HCT-116 and HeLa) with IC₅₀ ranging from 0.010 to 0.042 μM, and with selectivity profile against MCF-10A non-cancer cells. Further mechanistic studies suggest that 12c can inhibit tubulin polymerisation and cell migration, leading to G₂/M phase arrest. Besides, 12c induces apoptosis via a mitochondrial-dependant apoptosis pathway and caused reactive oxygen stress generation in MCF-7 cells. These results provide guidance for further rational development of potent tubulin polymerisation inhibitors for the treatment of cancer.Highlights A novel series of quinoline derivatives of combretastatin A-4 have been designed and synthesised. Compound 12c showed significant antiproliferative activities against different cancer cell lines. Compound 12c effectively inhibited tubulin polymerisation and competed with [³H] colchicine in binding to tubulin. Compound 12c arrested the cell cycle at G₂/M phase, effectively inducing apoptosis and inhibition of cell migration.

Full text of DOI:10.1080/14756366.2021.1899168

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of novel quinoline-based analogues
of combretastatin A-4 as tubulin polymerisation
inhibitors with apoptosis inducing activity and
potent anticancer effect
Tarek S. Ibrahim, Mohamed M. Hawwas, Azizah M. Malebari, Ehab S. Taher,
Abdelsattar M. Omar, Thikryat Neamatallah, Zakaria K. Abdel-Samii, Martin
K. Safo & Yaseen A. M. M. Elshaier
To cite this article: Tarek S. Ibrahim, Mohamed M. Hawwas, Azizah M. Malebari, Ehab
S. Taher, Abdelsattar M. Omar, Thikryat Neamatallah, Zakaria K. Abdel-Samii, Martin K.
Safo & Yaseen A. M. M. Elshaier (2021) Discovery of novel quinoline-based analogues of
combretastatin A-4 as tubulin polymerisation inhibitors with apoptosis inducing activity and potent
anticancer effect, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 802-818, DOI:
10.1080/14756366.2021.1899168
To link to this article: https://doi.org/10.1080/14756366.2021.1899168
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 802–818
https://doi.org/10.1080/14756366.2021.1899168
RESEARCH PAPER
Discovery of novel quinoline-based analogues of combretastatin A-4 as tubulin
polymerisation inhibitors with apoptosis inducing activity and potent
anticancer effect
Tarek S. Ibrahima,b , Mohamed M. Hawwasc, Azizah M. Malebaria , Ehab S. Taherc , Abdelsattar M. Omara,d
, Thikryat Neamatallahe, Zakaria K. Abdel-Samiib, Martin K. Safof and Yaseen A. M. M. Elshaierg
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; Department of
c
Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; Department of Pharmaceutical Organic Chemistry,
d
Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University,
e
f
Cairo, Egypt; Department of Pharmacology and toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; Institute for
Structural Biology, Drug Discovery and Development, Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth
g
University, Richmond, VA, USA; Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,
Menoufia, Egypt
ABSTRACT
ARTICLE HISTORY
Received 28 November 2020
Revised 27 January 2021
Accepted 27 February 2021
A new series of quinoline derivatives of combretastatin A-4 have been designed, synthesised and demon-
strated as tubulin polymerisation inhibitors. These novel compounds showed significant antiproliferative
activities, among them, 12c exhibited the most potent inhibitory activity against different cancer cell lines
(MCF-7, HL-60, HCT-116 and HeLa) with IC50 ranging from 0.010 to 0.042mM, and with selectivity profile
against MCF-10A non-cancer cells. Further mechanistic studies suggest that 12c can inhibit tubulin poly-
merisation and cell migration, leading to G2/M phase arrest. Besides, 12c induces apoptosis via a mito-
chondrial-dependant apoptosis pathway and caused reactive oxygen stress generation in MCF-7 cells.
These results provide guidance for further rational development of potent tubulin polymerisation inhibi-
tors for the treatment of cancer.
KEYWORDS
Combretastatin A-4;
quinoline; apoptosis; anti-
cancer; tubulin inhibitors
HIGHLIGHTS
ꢀ A novel series of quinoline derivatives of combretastatin A-4 have been designed and synthesised.
ꢀ Compound 12c showed significant antiproliferative activities against different cancer cell lines.
ꢀ Compound 12c effectively inhibited tubulin polymerisation and competed with [3H] colchicine in bind-
ing to tubulin.
ꢀ Compound 12c arrested the cell cycle at G2/M phase, effectively inducing apoptosis and inhibition of
cell migration.
GRAPHICAL ABSTRACT
isosteric replacment
of ring B with quinoline
CH3
OH
N
OCH3
B
- Antiproliferative effects
- Tubulin depolymerizing
- Colchicine site binding
- G2/M arrest and apoptosis
- ROS induction
OCH3
O
N
O
OCH3
OCH3
A
OCH3
OCH3
OCH3
H3CO
CA-4
12c
IC50 10-42 nM against different cancer cell lines
CONTACT Tarek S. Ibrahim
Abdulaziz University, Jeddah, 21589, Saudi Arabia; Martin K. Safo
tmabrahem@kau.edu.sa, tarekeldeeb1976@yahoo.com
msafo@vcu.edu
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King
Institute for Structural Biology, Drug Discovery and Development,
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23219, USA
ß 2021 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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
803
drug resistance18,19. Combretastatin A-4 (CA-4), (1, Figure 1) has
been reported as the most potent antimitotic agent of this family
against several tumour cells20. CA-4 was first isolated from the
bark of the willow tree Combretum caffrum from South Africa in
198921. CA-4 has a vascular disrupting activity against tumour cell
vasculature by preventing blood supply to solid tumour, resulting
in apoptosis22–24. Given its structural simplicity, CA-4 has been
studied as a lead pharmacophore for deciphering tubulin func-
tions and properties25. Phases II and III clinical studies are cur-
rently ongoing with tubulin-targeted drugs24,26. Structure activity
relationships (SAR) studies with CA-4 have revealed three import-
ant structural features (Figure 1). These include: (i) a 3,4,5-trime-
thoxy moiety on ring A that is essential for activity; (ii) a cis-
configuration of both aromatic rings that is essential for activity
(trans-orientation is inactive); (iii) the presence of small substituent
on ring B, e.g. methoxy group that is important for activity. The
cis-alkene configuration in CA-4 allows the aromatic rings to
assume optimal binding orientation for interactions with the col-
chicine binding site. Unfortunately, the cis configuration of CA-4
has a propensity for undergoing transformation to the inactive
trans configuration upon storage and during in vivo metabolism.
To overcome this, many structural modifications of CA-4 have
been undertaken where the cis double bond is replaced with het-
erocycles, either monocyclic, such as oxadiazole, isoxazole and
imidazole, resulting in compounds, such as 1, 2 and 3 respectively
(Figure 1)27–33 or fused heterocyclic, such as pyrazolopyridines34,
triazolopyridines35 and triazolothiadiazine derivatives36. These
compounds, like CA-4 showed pronounced activity against a panel
of cancer cell lines.
1. Introduction
Cancer is a disease of an uncontrolled growth and abnormal div-
ision of cells, which leads to death. In recent years, targeted anti-
neoplastic agents have become an effective treatment choice for
cancer1, with pharmaceutical companies focussing on targeted
therapies against different and special cancer types2. Tubulin poly-
merisation inhibitors represent one of the most well-known and
potential examples of such targeted cancer therapies3. Tubulin is
a globular protein that performs a substantial function in cell
mitosis. Microtubules (MTs), which represent the basic constituents
of eukaryotic cell, are cytoskeletons constructed by the association
of a- and b-tubulin heterodimers with a head and tail pattern to
form hollow cylindrical tubes (nearly 25 nm in diameter)4–7. MTs
play a crucial role in many fundamental cellular activities, such as
motility, cell formation, cell secretion, signalling, maintenance of
cell shape, regulation of intracellular transport and cell div-
ision8–10. Due to these multiple functions, microtubule system has
become an attractive target for cancer chemotherapy11,12
.
Disruption of MTs or tubulin dynamics exposes the cell to mitotic
arrest of the cell cycle at G2/M phase, and consequently induction
of cellular apoptosis13,14. Several microtubule-interfering agents
(MIAs) have been identified, e.g. paclitaxel, vincristine, and colchi-
cine that are obtained from the natural products, taxol, vinca and
colchicine, respectively. MIAs are known to bind to tubulin at spe-
cific binding sites that are classified as taxol, vinca and colchicine
sites to either enhance or inhibit tubulin polymerisation.15 For
example, microtubule stabilisers, e.g. paclitaxel stimulate micro-
tubule polymerisation16, while microtubule destabilizers, e.g. col-
chicine, and the vinca alkaloids vinblastine and vincristine inhibit
polymerisation of microtubules17.
Quinoline derivatives are popular for the treatment of mal-
aria37,38. Moreover, quinoline heterocyclic containing compounds
demonstrate potent anticancer activities with different modes of
Significant attention is now focussed on colchicine binding site
inhibitors due to their positive impact on ABC-transporter-mediated actions, including inhibition of proteasome, tyrosine kinases, and
(A)
(B)
Figure 1. (A) Chemical structure of CA-4 and its analogues; (B) tubulin targeting agents bearing quinoline moiety.

804
T. S. IBRAHIM ET AL.
Figure 2. Structure of CA-4 and our rationalised compounds.
tubulin polymerisation39–41. Previous studies have reported the (CH3)2SO appearing at d H 2.5 and the central resonance of the
CDCl3 “triplet” appearing at d C 77.0 and for (CD3)2SO “multiplet”
antiproliferative activity of CA-4, isoCA-4 or chalcone compounds
containing quinoline scaffold, either as ring A bioisoster, e.g. 4a23
and 4b42 or ring B bioisoster, e.g. 4c43–45 and 4d46. These com-
pounds demonstrate the potential of the quinoline ring as a tem-
plate for developing more promising tubulin polymerisation
inhibitors and antiproliferative agents.
appearing at d C 39.0 were used to reference 1H and 13C NMR
1
spectra, respectively. H NMR data are recorded as follows: chem-
ical shift (d) [multiplicity, coupling constant(s) J (Hz), relative inte-
gral] where multiplicity is defined as s ¼ singlet; d ¼ doublet;
t ¼ triplet; q ¼ quartette; and m ¼ multiplet or combinations of the
above. Elemental analyses were determined using Manual
Elemental Analyser Heraeus (Germany) and Automatic Elemental
Analyser CHN Model 2400 Perkin Elmer (Waltham, MA, USA) at
Microanalytical Centre, Faculty of Science, Cairo University, Egypt.
All the elemental analyses results corresponded to calculated val-
ues within experimental error. Progress of reactions was moni-
tored by thin-layer chromatography (TLC) using precoated TLC
sheets with Ultraviolet (UV) fluorescent silica gel (Merck 60F254),
and spots were visualised by iodine vapours or irradiation with UV
light (254 nm). All chemicals were purchased from Sigma-Aldrich
or Lancaster Synthesis Corporation (UK). Intermediates 6–8a–i
In this work, we optimised CA-4 into a series of novel hybrid
quinoline derivatives as potent tubulin inhibitor, which involves
introducing a rigid oxazolone or imidazolone between rings A and
B to maintain the cis configuration, as well as targeting the qui-
nolyl moiety (ring B), by varying the electronic substituents effect
while maintaining the 3,4,5-trimethoxyphenyl moiety as present in
ring A of CA-4 (Figure 2). Following, we synthesised several ana-
logues that constitute two classes of compounds: the oxazolones
(Compounds 12a–h) and the imidazolones (Compounds 13a–h).
The compounds have been screened for their antiproliferative
activities against a variety of cancer cell lines, as well as studied
for their mechanism of action. We expect the results to lead to
better understanding of the mechanistic mode of the compounds’
activity against tubulin and provide guidance for further develop-
ment of potent anticancer drugs.
were prepared according to reported procedure47,48
.
2.1.1. General procedure for preparation of oxazolones (12a–h)
A mixture of N-(3,4,5-trimethoxybenzoyl)glycine (0.30 g, 1.10 mmol)
and the appropriate aldehydes 8a–h (1.00 mmol) in acetic anhyd-
ride (1 ml) and fused sodium acetate (0.1 g, 1.2 mmol) was heated
on an oil bath at 80 ꢂC for 2 h. After cooling down at room tem-
perature the mixture was allowed to stand for 24 h at 0 ꢂC. The
precipitate was filtered off and washed three times with ice-
cooled ethanol (10 ml), and the product crystallised from ethanol.
2. Experimental section
2.1. Chemistry
Melting points were determined with a Gallenkamp (London, UK)
melting point apparatus and are uncorrected. IR spectra (KBr,
cmꢁ1) were recorded on Bruker Vector, 22FT-IR [Fourier Transform
Infra-red (FTIR), Germany] spectrometer. Unless otherwise speci-
fied, proton (1H) and carbon (13C) NMR spectra were recorded at
room temperature in base filtered CDCl3 on a spectrometer oper-
ating at 400 & 300 MHz for proton and 100 & 75 MHz for carbon
2.1.1.1. 4-[(2-Methoxyquinolin-3-yl)methylene]-2–(3,4,5-trimethoxy-
phenyl)oxazol-5(4H)-one (12a). Yellow solid, Yield (81%); m.p.
215–217 ꢂC. IR (KBr): t ¼ 1776 (C¼O), 1621 (C¼N), 1599 (C¼C)
1
cmꢁ1. H NMR (400 MHz, CDCl3) d: 9.48 (s, 1H, Ar-H), 7.86–7.81 (m,
nuclei. The signal due to residual CHCl3 appearing at d H 7.26 and 2H, Ar-H), 7.71–7.67 (m, 2H, Ar-H), 7.45–7.41 (m, 3H, Ar-H), 4.15 (s,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
805
3H, OCH3), 4.00 (s, 9H, 3 OCH3) ppm. 13C NMR (100 MHz, CDCl3) d: (70 eV): m/z (%): 450 (7.30) [Mþ]; Anal. Calcd for C24H22N2O7: C,
167.1, 163.9, 160.0, 153.5, 147.1, 143.1, 142.4, 134.6, 131.5, 128.9,
127.1, 125.2, 124.7, 123.6, 120.1, 118.5, 105.8, 61.1, 56.4, 54.1 ppm.
MS (70 eV): m/z (%): 420 (7.79) [Mþ]; Anal. Calcd for C23H20N2O6: C,
65.71; H, 4.80; N, 6.66. Found: C, 65.64; H, 4.74; N, 6.71
64.00; H, 4.92; N, 6.22. Found: C, 63.96; H, 4.96; N, 6.25.
2.1.1.7. 4-[(7-Isopropoxy-2-methoxyquinolin-3-yl)methylene]-
2–(3,4,5-trimethoxy phenyl)oxazol-5(4H)-one (12g). Yellow solid,
Yield (77%); m.p. 227–229 ꢂC. 1H NMR (400 MHz, CDCl3) d ppm:
9.41 (s, 1H, Ar-H), 7.74–7.67 (m, 2H, Ar-H), 7.41 (s, 2H, Ar-
H),7.16–7.00 (m, 2H, Ar-H), 4.87–4.76 (m, 1H, OCH-), 4.12 (s, 3H,
OCH3), 3.98 (s, 9H, 3 OCH3), 1.45 (d, J ¼ 4.0 Hz, 6H, 2 CH3) ppm.
13C NMR (100 MHz, CDCl3) d: 167.4, 163.1, 161.2, 160.7, 153.5,
149.5, 142.9, 141.9, 133.2, 130.3, 124.6, 120.4, 119.9, 118.2, 115.7,
107.9, 105.5, 70.3, 61.3, 56.4, 53.9, 22.0 ppm. MS (70 eV): m/z (%):
478 (9.50) [Mþ]; Anal. Calcd for C26H26N2O7: C, 65.26; H, 5.48; N,
5.85. Found: C, 65.21; H, 5.45; N, 5.91.
2.1.1.2. 4-[(2-Methoxy-6-methylquinolin-3-yl)methylene]-2–(3,4,5-
trimethoxyphenyl) oxazol-5(4H)-one (12b). Pale yellow solid, Yield
(79%); m.p. 218–220 ꢂC. 1H NMR (400 MHz, CDCl3) d: 9.36 (s, 1H,
Ar-H), 7.72 (t, J ¼ 4 Hz, 2H, Ar-H), 7.56 (s, 1H, Ar-H), 7.49 (d,
J ¼ 8.5 Hz, 1H, Ar-H), 7.41 (s, 2H, Ar-H), 4.11 (s, 3H, OCH3), 3.98 (d,
J ¼ 8.0 Hz, 9H, 3OCH3) and 2.51 (s, 3H, CH3) ppm. 13C NMR
(100 MHz, CDCl3) d: 167.2, 163.8, 159.7, 153.5, 145.7, 143.1, 141.8,
134.4, 134.3, 133.6, 127.8, 126.9, 125.1, 124.1, 120.3, 118.1, 110.0,
105.7, 61.1, 56.4, 53.9, 21.3 ppm. MS (70 eV): m/z (%): 434 (9.79)
[Mþ]; Anal. Calcd for C24H22N2O6: C, 66.35; H, 5.10; N, 6.45. Found:
C, 66.29; H, 5.04; N, 6.50.
2.1.1.8.
4-[(7-(Benzyloxy)-2-methoxyquinolin-3-yl)methylene]-
2–(3,4,5-trimethoxy phenyl)oxazol-5(4H)-one (12h). Pale yellow
solid, Yield (73%); m.p. 247–249 ꢂC. 1H NMR (400 MHz, CDCl3) d:
9.43 (s, 1H, Ar-H), 7.72 (t, J ¼ 8 Hz, 2H, Ar-H), 7.51–7.35 (m, 6H, Ar-
H), 7.28–7.26 (m, 2H, Ar-H), 7.14 (d, 1H, J ¼ 8.0 Hz, 1H, Ar-H), 5.21
(s, 2H, OCH2ꢁ), 4.12 (s, 3H, OCH3), 3.98 (s, 9H, 3 OCH3) ppm. 13C
NMR (100 MHz, CDCl3) d: 167.4, 163.3, 161.9, 160.8, 153.5, 149.4,
142.9, 141.9, 136.2, 133.5, 130.2, 128.7, 128.3, 127.7, 124.4, 120.4,
120.3, 117.5, 116.0, 107.5, 105.6, 70.3, 61.3, 56.4, 53.9 ppm. MS
(70 eV): m/z (%): 526 (7.30) [Mþ]; Anal. Calcd for C30H26N2O7: C,
68.43; H, 4.98; N, 5.35. Found: C, 68.39; H, 4.92; N, 5.41.
2.1.1.3. 4-[(2-Methoxy-7-methylquinolin-3-yl)methylene]-2–(3,4,5-
trimethoxyphenyl) oxazol-5(4H)-one (12c). Yellow solid, Yield
(84%); m.p. 233–235 ꢂC. 1H NMR (400 MHz, CDCl3) d: 9.50 (s, 1H,
Ar-H), 7.76–7.73 (m, 3H, Ar-H),7.45 (s, 2H, Ar-H), 7.30–7.28 (m, 1H,
Ar-H), 4.20 (s, 3H, OCH3), 4.00 (d, J ¼ 8.0 Hz, 9H, 3 OCH3), 2.58 (s,
3H, CH3) ppm. 13C NMR (100 MHz, CDCl3) d: 167.2, 163.8, 162.6,
160.2, 153.5, 143.1, 142.8, 142.5, 134.3, 128.7, 127.0, 126.3, 123.9,
123.1, 120.2, 117.6, 105.7, 61.1, 56.4, 54.6, 22.1 ppm. MS (70 eV): m/
z (%): 434 (6.90) [Mþ]; Anal. Calcd for C24H22N2O6: C, 66.35; H,
5.10; N, 6.45. Found: C, 66.32; H, 5.05; N, 6.47.
2.1.2. General procedure for preparation of (13a–h)
The appropriate oxazolones 12a–h (1 mmol) was stirred and
heated under reflux in ethanol (10 ml) containing ammonium
hydroxide (10 ml), and the reaction monitored by TLC. After com-
pletion of the reaction in 24 h, the solvent was concentrated and
cooled, and the precipitate was filtered off and crystallised
from ethanol.
2.1.1.4. 4-[(2-Methoxy-8-methylquinolin-3-yl)methylene]-2–(3,4,5-
trimethoxyphenyl) oxazol-5(4H)-one (12d). Yellow solid, Yield
(82%); m.p. 236–238 ꢂC. 1H NMR (400 MHz, CDCl3) d: 9.44 (s, 1H,
Ar-H), 7.73 (s, 1H, Ar-H), 7.64 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.52 (d,
J ¼ 6.40 Hz, 1H, Ar-H), 7.39 (s, 2H, Ar-H), 7.32–7.28 (m, 1H, Ar-H),
4.12 (s, 3H, OCH3), 3.98 (s, 9H, 3 OCH3), 2.68 (s, 3H, CH3) ppm. 13C
NMR (100 MHz, CDCl3) d: 167.2, 163.8, 159.1, 153.7, 146.0, 143.1,
142.7, 135.4, 134.3, 131.6, 126.7, 125.0, 124.3, 124.0, 120.2, 118.0,
105.6, 61.1, 56.3, 53.7, 17.5 ppm. MS (70 eV): m/z (%): 434 (9.50)
[Mþ]; Anal. Calcd for C24H22N2O6: C, 66.35; H, 5.10; N, 6.45. Found:
C, 66.30; H, 5.04; N, 6.51.
2.1.2.1. 5-[(2-Methoxyquinolin-3-yl)methylene]-2–(3,4,5-trimethoxy-
phenyl)-3,5-dihydro-4H-imidazol-4-one (13a). Yellow solid, Yield
(81%); m.p. 230–232 ꢂC. IR (KBr): t ¼ 3222 (NH), 1709 (C¼O), 1642
(C¼N), 1615, 1589 (C¼C) cmꢁ1
.
1H NMR (400 MHz, DMSO-d6) d:
12.22 (s, 1H, exch., NH), 9.69 (s, 1H, Ar-H), 8.03 (d, J ¼ 8.0 Hz, 1H,
Ar-H), 7.80–7.70 (m, 2H, Ar-H), 7.59 (s, 2H, Ar-H), 7.49 (t, J ¼ 8.0 Hz,
1H, Ar-H), 7.28 (s, 1H, Ar-H), 4.11 (s, 3H, OCH3), 3.94 (s, 6H, 2OCH3),
3.80 (s, 3H, OCH3) ppm. 13C NMR (100 MHz, DMSO-d6) d: 187.4,
165.0, 164.2, 161.3, 157.9, 153.5, 146.7, 140.9, 132.2, 128.9, 126.7,
125.2, 123.9, 118.4, 108.7, 104.2, 60.3, 56.3, 54.1 ppm. MS (70 eV):
m/z (%): 419 (5.40) [Mþ]; Anal. Calcd for C23H21N3O5: C, 65.86; H,
5.05; N, 10.02. Found: C, 65.81; H, 4.99; N, 10.09.
2.1.1.5. 4-[2,6-Dimethoxyquinolin-3-yl)methylene]-2–(3,4,5-trime-
thoxyphenyl)oxazol-5(4H)-one (12e). Yellow solid, Yieldꢁ(184%); m.p.
242–244 ꢂC. 1784 (C¼O), 1618 (C¼N), 1586 (C¼C) cm
.
1H NMR
(400 MHz, CDCl3) d: 9.33 (s, 1H, Ar-H), 7.73 (d, J ¼ 8.0 Hz, 2H, Ar-H),
7.42–7.32 (m, 3H, Ar-H), 7.12 (s, 1H, Ar-H), 4.10 (s, 3H, OCH3), 3.99
(s, 9H, 3 OCH3) 3.93 (s, 3H, OCH3) ppm. 13C NMR (100 MHz, CDCl3)
d: 167.1, 163.8, 158.9, 156.4, 153.5, 143.2, 143.0, 141.1, 134.5,
128.5, 125.7, 124.2, 123.0, 120.2, 118.4, 107.4, 105.9, 61.2, 56.5,
55.6, 53.8 ppm. MS (70 eV): m/z (%): 450 (8.40) [Mþ]; Anal. Calcd
for C24H22N2O7: C, 64.00; H, 4.92; N, 6.22. Found: C, 63.94; H, 4.87;
N, 6.28.
2.1.2.2. 5-[(2-Methoxy-6-methylquinolin-3-yl)methylene]-2–(3,4,5-
trimethoxyphenyl)-3,5-dihydro-4H-imidazol-4-one (13b). Yellow
1
solid, Yield (81%); m.p. 223–225 ꢂC. HNMR (400 MHz, DMSO-d6) d:
12.18 (s, 1H, exch., NH), 9.55 (s, 1H, Ar-H), 7.74–7.54 (m, 5H, Ar-H),
7.26–7.24 (d, J ¼ 6.6 Hz, 1H, Ar-H), 4.07 (s, 3H, OCH3), 3.93 (s, 6H,
OCH3), 3.80 (s, 3H, OCH3), 2.47 (s, 3H, CH3) ppm. 13C NMR
(100 MHz, DMSO-d6) d: 172.0, 162.2, 156.0, 153.7, 144.6, 142.6,
142.4, 141.2, 134.4, 133.4, 128.1, 126.8, 125.4, 123.2, 119.3, 116.2,
110.0, 106.2, 60.7, 56.8, 54.2, 21.3 ppm. MS (70 eV): m/z (%): 433
(5.60) [Mþ]; Anal. Calcd for C24H23N3O5: C, 66.50; H, 5.35; N, 9.69.
2.1.1.6. 4-[(2,7-Dimethoxyquinolin-3-yl)methylene]-2–(3,4,5-trime-
thoxyphenyl)oxazol-5(4H)-one (12f). Yellow solid, Yield (84%); m.p.
221–223 ꢂC. 1H NMR (400 MHz, CDCl3) d: 9.39 (s, 1H, Ar-H),
7.70–7.66 (m, 2H, Ar-H), 7.38 (s, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 7.04
(d, J ¼ 8.0 Hz, 1H, Ar-H), 4.11 (s, 3H, OCH3), 3.97 (s, 9H, 3OCH3) 3.95
(s, 3H, OCH3) ppm. 13C NMR (100 MHz, CDCl3) d: 167.4, 163.3,
162.8, 160.7, 153.5, 149.7, 142.8, 142.0, 133.3, 130.2, 124.4, 120.4,
120.1, 117.2, 115.9, 106.4, 105.5, 61.2, 56.2, 55.6, 53.9 ppm. MS Found: C, 66.41; H, 5.28; N, 9.75.

806
T. S. IBRAHIM ET AL.
2.1.2.3. 5-[(2-Methoxy-7-methylquinolin-3-yl)methylene]-2–(3,4,5- 2.1.2.8.
5-[(7-(Benzyloxy)-2-methoxyquinolin-3-yl)methylene]-
trimethoxyphenyl)-3,5-dihydro-4H-imidazol-4-one (13c). Yellow 2–(3,4,5-trimethoxy phenyl)-3,5-dihydro-4H-imidazol-4-one (13h).
Yellow solid, Yield (73%); m.p. 259–261 ꢂC. 1H NMR (400 MHz,
solid, Yield (84%); m.p. 236–238 ꢂC. IR (KBr): t ¼ 3215 (NH), 1711
(C¼O), 1639 (C¼N), 1590 (C¼C) cmꢁ1
.
1H NMR (400 MHz, DMSO-
DMSO-d6) d: 12.16 (s, 1H, exch., NH), 9.62 (s, 1H, Ar-H), 7.96–7.94
(d, J ¼ 8 Hz, 1H, Ar-H), 7.58–7.28 (m, 10H, ArH), 5.30 (s, 2H, OCH2-),
4.09 (s, 3H, OCH3), 3.94 (s, 6H, 2OCH3), 3.79 (s, 3H, OCH3) ppm. 13C
NMR (400 MHz, DMSO-d6) d: 163.9, 161.6, 158.5, 153.5, 148.8,
140.7, 140.3, 136.6, 130.2, 128.5, 128.0, 127.8, 118.9, 118.5, 117.8,
107.2, 105.9, 104.0, 69.7, 60.4, 56.3, 54.0 ppm. MS (70 eV): m/z (%):
525 (6.75) [Mþ]; Anal. Calcd for C30H27N3O6: C, 68.56; H, 5.18; N,
8.00. Found: C, 68.51; H, 5.13; N, 8.05.
d6) d: 12.17 (s, 1H, exch. NH), 9.63 (s, 1H, Ar-H), 7.92 (d, J ¼ 8.0 Hz,
1H, Ar-H), 7.58 (s, 3H, Ar-H), 7.32 (d, J ¼ 8 Hz, 1H, Ar-H), 7.27 (s, 1H,
Ar-H), 4.09 (s, 3H, OCH3), 3.94 (s, 6H, 2OCH3), 3.80 (s, 3H, OCH3),
2.53 (s, 3H, CH3) ppm. 13C NMR (100 MHz, DMSO-d6) d: 162.6,
162.5, 160.3, 153.9, 142.2, 141.5, 129.1, 127.1, 126.3, 123.6, 118.3,
115.8, 105.6, 60.8, 56.5, 54.2, 22.0 ppm. MS (70 eV): m/z (%): 433
(8.40) [Mþ]; Anal. Calcd for C24H23N3O5: C, 66.50; H, 5.35; N, 9.69.
Found: C, 66.44; H, 5.32; N, 9.72.
2.2. Biochemical evaluation of activity
2.1.2.4. 5-[(2-Methoxy-8-methylquinolin-3-yl)methylene]-2–(3,4,5-
trimethoxy phenyl)-3,5-dihydro-4H-imidazol-4-one (13d). Yellow
All biochemical assays were performed in triplicate on at least
three independent occasions for the determination of
mean values.
1
solid, Yield (82%); m.p. 233–235 ꢂC. H NMR (400 MHz, DMSO-d6) d:
12.16 (s, 1H, exch., NH), 9.59 (s, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 7.70
(d, J ¼ 8 Hz, 1H, Ar-H), 7.59–7.56 (m, 3H, Ar-H), 7.29 (s, 1H, Ar-H),
4.10 (s, 3H, OCH3), 3.95 (s, 6H, 2OCH3), 3.80 (s, 3H, OCH3), 2.49 (s,
3H, CH3) ppm. 13C NMR (100 MHz, DMSO-d6) d: 161.9, 158.2, 153.3,
142.2, 141.6, 141.5, 134.2, 131.3, 126.7, 124.8, 124.5, 118.6, 115.4,
105.3, 60.2, 56.1, 53.5, 17.1 ppm. MS (70 eV): m/z (%): 433 (7.12)
[Mþ]; Anal. Calcd for C24H23N3O5: C, 66.50; H, 5.35; N, 9.69. Found:
C, 66.47; H, 5.30; N, 9.73.
2.2.1. Cell culture
The four human tumour cell lines MCF-7, HCT-116, HL-60 and
HeLa used in this study were obtained from the VACSERA (Giza,
Egypt) cell culture unit that were originally acquired from ATCC
(Manassas, VA, USA). All the human tumour cell lines were cul-
tured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10%
foetal bovine serum, 2 mM L-glutamine and 100 mg/mL penicillin/
streptomycin. Cells were maintained at 37˚C in 5% CO2 in a
humidified incubator. All cells were sub-cultured 3 times/week by
trypsinisation using TrypLE Express (1X).
2.1.2.5. 5-[(2,6-Dimethoxyquinolin-3-yl)methylene]-2–(3,4,5-trime-
thoxyphenyl)-3,5-dihydro-4H-imidazol-4-one (13e). Yellow solid,
1
Yield (84%); m.p. 253–255 ꢂC. HNMR (400 MHz, DMSO-d6) d: 12.03
(s, 1H, exchangeable, NH), 9.50 (s, 1H, Ar-H), 7.83–7.77 (m, 1H, Ar-
H), 7.53 (s, 2H, Ar-H), 7.48 (s, 1H, Ar-H), 7.20–7.05 (m, 2H, Ar-H),
4.04 (s, 3H, OCH3), 3.90 (s, 9H, 3 OCH3), 3.79 (s, 3H, OCH3) ppm.
13C NMR (100 MHz, DMSO-d6) d: 164.6, 161.9, 157.1, 156.9, 154.1,
142.8, 141.9, 140.0, 128.4, 125.1, 124.1, 118.9, 110.0, 109.3, 108.0,
105.3, 60.7, 57.0, 56.2, 54.4 ppm. MS (70 eV): m/z (%): 449 (8.35)
[Mþ]; Anal. Calcd for C24H23N3O6: C, 64.16; H, 5.16; N, 9.35. Found:
C, 64.11; H, 5.12; N, 9.31.
2.2.2. Cell viability assay
The quinoline compounds were evaluated for antiproliferative
effect using the MTT viability assay of four cancer cell lines (MCF-
7, HCT-116, HL-60 and HeLa) and normal breast cells MCF-10A to
calculate the relative IC50 values for each compound. Cells were
seeded in triplicate in 96-well plates at a density of 10 ꢃ 103
cells/ml in a total volume of 200 ml per well. 0.1% of DMSO was
used as a vehicle control. Following, the cells were treated with
2 ml test compound (from stock solutions in ethanol) to furnish
the concentration range of study, 1 nM to 50 mM, and re-incubated
for a further 72 h. The culture medium was then removed, and
the cells washed with 100 mL phosphate buffered saline (PBS) and
50 mL MTT added, to reach a final concentration of 1 mg/mL. Cells
were incubated for 2 h in darkness at 37 ꢂC. Solubilisation was
begun through the addition of 200 ml DMSO, and the cells main-
tained at room temperature in darkness for 20 min to ensure thor-
ough colour diffusion before reading the absorbance. Plates were
incubated for 72 h at 37 ꢂC þ 5% CO2. The MTT (5 mg/mL in PBS)
was added and incubated for another 4 h, and the optical density
was detected with a microplate reader at 570 nm. Results were
expressed as percentage viability relative to vehicle control
(100%). Dose response curves were plotted and IC50 values (con-
centration of drug resulting in 50% reduction in cell survival) were
obtained using the commercial software package Prism (GraphPad
Software, Inc., La Jolla, CA, USA). All the experiments were
repeated in at least three independent experiments.
2.1.2.6. 5-[(2,7-Dimethoxyquinolin-3-yl)methylene]-2–(3,4,5-trime-
thoxyphenyl)-3,5-dihydro-4H-imidazol-4-one (13f). Yellow solid,
1
Yield (86%); m.p. 241–243 ꢂC. HNMR (400 MHz, DMSO-d6) d: 12.10
(s, 1H, exch., NH), 9.57 (s, 1H, Ar-H), 7.86 (d, J ¼ 8.0 Hz, 1H, Ar-H),
7.53 (s, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 7.15–7.08 (m, 2H, Ar-H), 4.07
(s, 3H, OCH3), 3.92 (s, 6H, 2OCH3), 3.79 (s, 3H, OCH3) ppm. 13C
NMR (100 MHz, DMSO-d6) d: 163.9, 162.6, 161. 5, 158.3, 153.3,
149.0, 140.7, 140.3, 129.9, 118.7, 118.2, 117.4, 106.3, 105.7, 104.1,
60.1, 56.3, 55.7, 54.1 ppm. MS (70 eV): m/z (%): 449 (5.65) [Mþ];
Anal. Calcd for C24H23N3O6: C, 64.16; H, 5.16; N, 9.35. Found: C,
64.13; H, 5.10; N, 9.29.
2.1.2.7.
5-((7-Isopropoxy-2-methoxyquinolin-3-yl)methylene)-
2–(3,4,5-trimethoxy phenyl)-3,5-dihydro-4H-imidazol-4-one (13g).
Yellow solid, Yield (77%); m.p. 238–240 ꢂC. 1HNMR (400 MHz,
DMSO-d6) d: 12.19 (s, 1H, exch.,NH), 9.61 (s, 1H, Ar-H), 7.92 (d,
J ¼ 8.0 Hz, 1H, Ar-H), 7.58 (s, 2H, Ar-H), 7.28 (s, 1H, Ar-H), 7.17 (s,
1H, Ar-H), 7.09–7.07 (d, J ¼ 8.0 Hz, 1H, Ar-H), 4.90–4.83 (m, 1H,
OCH), 4.09 (s, 3H, OCH3), 3.94 (s, 6H, 2OCH3), 3.79 (s, 3H, OCH3),
1.37 (s, 3H, CH3) 1.36 (s, 3H, CH3) ppm. 13C NMR (100 MHz, DMSO-
d6) d: 164.2, 161.3, 156.7, 154.2, 153.5, 142.0, 140.9, 139.7, 128.1,
124.8, 122.4, 118.6, 109.2, 108.5, 104.3, 69.8, 60.3, 56.3, 53.9,
21.7 ppm. MS (70 eV): m/z (%): 477 (9.25) [Mþ]; Anal. Calcd for
C26H27N3O6: C, 65.40; H, 5.70; N, 8.80. Found: C, 65.35; H, 5.66;
N, 8.86.
2.2.3. Tubulin polymerisation assay
The assembly of purified bovine tubulin was monitored using a
kit, BK006, purchased from Cytoskeleton Inc., (Denver, CO, USA).
The assay was carried out in accordance with the manufacturer’s

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
807
instructions using the standard assay conditions49. Briefly, purified cells), Annexin V positive and PI negative (Q3, early apoptosis),
(>99%) bovine brain tubulin (3 mg/mL) in a buffer consisting of Annexin V and PI positive (Q2, late apoptosis) and Annexin V
80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP and negative and PI positive (Q1, necrosis).
10% glycerol was incubated at 37 ꢂC in the presence of either
vehicle (2% (v/v) ddH2O), CA-4, or the quinoline compounds. Light
2.2.7. Evaluation of expression levels of anti-apoptotic proteins
bcl-2, pro-apoptotic proteins bax and caspase 9
is scattered proportionally to the concentration of polymerised
microtubules in the assay. Therefore, tubulin assembly was moni-
tored turbidimetrically at 340 nm in a Spectramax 340 PC spectro-
photometer (Molecular Devices, Sunnyvale, CA, USA). The
concentration that inhibits tubulin polymerisation by 50% (IC50)
was determined using area under the curve (AUC). The AUC of
the untreated controls were considered as 100% polymerisation.
The IC50 value for each compound was computed using GraphPad
Prism Software.
The level of the anti-apoptotic marker and apoptotic marker BAX
were assessed using Bcl-2 Elisa kit and human Bax ELISA Kit pur-
chased from Zymed laboratories, invitrogen and Cloud-Clone
Crop. (Katy, TX, USA), respectively, following the manufacturer’s
instructions. Briefly, Treated MCF-7 cell lysate with 250 nM of com-
pound 12c were prepared, and equal amount of cell lysates were
loaded and propped with specific antibodies. The samples were
measured and analysed at 450 nm in ROBONEK P2000 ELISA
reader52. In Vitro Caspase-9 Activation Assay was performed using
human active caspase-9 Invitrogen EIA kit according to the manu-
facturer’s instructions. Compound 12c at concentrations of 50 and
250 nM and CA-4 (50 nM) were prepared in dH2O up to a final vol-
ume of 50 mL/well followed by addition of 5 mL of active caspase-
9. Following, the cells were mixed and 50 mL of the Master Mix
was added to each well and allowed to react at 37 ꢂC for 1 h. The
fluorescence intensity of the test samples was recorded and ana-
lysed in a fluorescence plate reader at 400 nm excitation and
505 nm emissions. All experiments were conducted in triplicates.
2.2.4. Colchicine site competitive binding assay
The affinity of compounds 12c to colchicine binding site was
determined using Colchicine Site Competitive Assay kit
CytoDYNAMIX Screen15 (Cytoskeleton, Inc., Denver, CO, USA)
using the standard protocol of the manufacturer to determine Ki.
Biotin-labelled tubulin (0.5 mg) in 10 mL of reaction buffer was
mixed with [3H]colchicine (0.08 mM, PerkinElmer, Waltham, MA)
and the test compounds (positive control colchicine, negative con-
trol vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96-well plate
(final volume: 100 mL). After incubating for 2 h at 37 ꢂC with gentle
shaking, streptavidin-labelled yttrium SPA beads (80 mg in 20 mL
reaction buffer, PerkinElmer, Waltham, MA) were added to each
well and incubated for 30 min at 4 ꢂC. The plates were then read
2.2.8. Colony formation assay
MCF-7 cells (600 cells per well) were seeded in 6-well plates and
incubated for 24 h before being then treated with different doses
of the compound 12c (50 and 250 nM) for 14 days. Following, the
cells were washed with PBS twice and subsequently fixed with 4%
paraformaldehyde and stained with 0.05% crystal violet for
30 min. Finally, cells were visualised using an inverted microscope.
on
Microplate Reader) and the percentage of inhibition was
calculated50,51
a
scintillation counter (Packard Instrument, Topcount
.
2.2.5. Cell cycle analysis
MCF-7 cells were seeded at a density of 1 ꢃ 105 cells/well in 6-
well plates and treated with CA-4 (50 nM) and compound 12c (50
and 250 nM) for 24, 48 and 72 h. The cells were collected by tryp-
sinisation and centrifuged at 800ꢃg for 15 min. Cells were washed
twice with ice-cold PBS and fixed in ice-cold 70% ethanol over-
night at ꢁ20 ꢂC. Fixed cells were centrifuged at 800ꢃg for 15 min
and stained with 50 mg/mL of PI, containing 50 mg/mL of DNase-
free RNase A, at 37 ꢂC for 30 min. The DNA content of cells
(10,000 cells/experimental group) was analysed by flow cytometer
at 488 nm using a FACSCalibur flow cytometer (BD Biosciences,
San Jose, CA) and all data were recorded and analysed using the
CellQuest Software (Becton-Dickinson).
2.2.9. Wound healing assay
MCF-7 were grown in 6-well plates for 24 h, and scratches
were made using pipette tip and washed with PBS to remove
non-adherent cell debris. Subsequently, the cells were treated
with different concentrations of 12c for 24 h. The migrations
across the wound area were photographed under a phase con-
trast microscopy.
2.2.10. Measurement of mitochondrial depolarisation effect (Dwmt
and ROS levels in cells
Mitochondrial membrane potential (Dwmt) was measured by flow
cytometry with DiOC2(3) staining and additional labelling with an
annexin V conjugate. After treatment with compound 12c (50 and
)
2.2.6. Annexin V/PI apoptotic assay
Apoptotic cell death was detected by flow cytometry using 250 nM) and CA-4 (50 nM), cells were stained with DiOC2(3) dye
Annexin V and propidium iodide (PI). MCF-7 Cells were seeded in for 30 min in the incubator, then harvested and washed with PBS.
6 well plated at density of 1 ꢃ 105 cells/mL and treated with DiOC2(3)-stained cells were resuspended with 1X annexin binding
vehicle (0.1% (v/v) EtOH), positive control (CA-4) or compound buffer, followed by addition of annexin V conjugate and incubated
12c (50 and 250 nM) for 24, 48 and 72 h. Cells were then har- at 37 ꢂC for 15 min. The data from the flow cytometry were ana-
vested and prepared for flow cytometric analysis. Cells were lysed by Cell Quest software. Production of intracellular reactive
washed in 1X binding buffer (20X binding buffer: 0.1 M HEPES, pH oxygen species (ROS) was measured using 2,7–dichlorofluorescin
7.4; 1.4 M NaCl; 25 mM CaCl2 diluted in dH2O) and incubated in diacetate (H2-DCFDA) dye. MCF-7 cells were seeded and treated
the dark for 30 min on ice in Annexin V-containing binding buffer either with vehicle (0.1% DMSO) or with compound 12c (50 and
[1:100]. Cells were then washed once in binding buffer and then 250 nM) or CA-4 (50 nM) for 6, 12 and 24 h. H2O2 was used as a
re-suspended in PI-containing binding buffer [1:1000]. Samples positive control. The amount of ROS generated was estimated
were analysed immediately using the BD accuri flow cytometer after 2 h of selected compound treatment. The cells were collected
and prism software for analysis the data. Four populations are by centrifugation and washed twice with PBS. Cells were then
produced during the assay Annexin V and PI negative (Q4, healthy incubated with DCFDA dye (25 mM) in dark at 37 ꢂC for 1 h.

808
T. S. IBRAHIM ET AL.
Table 1. Antiproliferative activity of quinoline analogues against human cancer cell lines (IC50 [mM]).
IC50 value (mM)a
Compound number
X
R
HL-60
MCF-7
HCT-116
HeLa
12a
12b
12c
12d
12e
12f
12g
12h
13a
13b
13c
13d
13e
13f
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
NH
NH
–
H
6-CH3
7-CH3
8-CH3
6-OCH3
7-OCH3
7-OCH(CH3)2
7-OCH2Ph
H
6-CH3
7-CH3
3.309 0.022
0.197 0.041
0.019 0.059
0.268 0.093
0.068 0.034
0.352 0.021
1.822 0.640
4.660 0.260
1.561 0.055
0.240 0.012
0.661 0.026
0.096 0.006
0.272 0.050
0.210 0.098
5.114 0.410
4.931 0.260
0.076 0.004
1.712 0.040
0.132 0.013
0.010 0.003
0.154 0.071
0.056 0.027
0.052 0.0021
1.507 0.220
1.932 0.612
1.033 0.055
0.063 0.0011
0.223 0.056
0.137 0.023
0.042 0.0025
0.092 0.0062
5.562 0.133
1.695 0.821
0.019 0.004
2.012 0.080
0.184 0.007
0.022 0.001
0.191 0.001
0.031 0.001
0.066 0.005
7.880 0.300
1.563 0.720
8.21 0.0077
0.173 0.025
0.284 0.083
0.109 0.011
0.085 0.003
0.187 0.009
1.620 0.950
2.610 0.430
0.026 0.001
2.113 0.034
0.117 0.085
0.042 0.001
0.153 0.083
0.010 0.004
0.138 0.026
1.747 0.500
1.054 0.840
1.409 0.096
0.188 0.013
0.733 0.091
0.126 0.044
0.062 0.003
0.101 0.090
4.439 0.600
2.070 0.390
0.064 0.004
8-CH3
6-OCH3
7-OCH3
7-OCH(CH3)2
7-OCH2Ph
–
13g
13h
CA-4
aIC50 values are half maximal inhibitory concentrations required to block the growth stimulation of cells. Values represent the mean for three
experiments performed in triplicate.
Scheme 1. Synthetic route for preparation of aldehydes 8a–h. Reagents and conditions: (i) Ac2O, AcOH, 0 ꢂC,1h; (ii) DMF, POCl3, 70–90ꢂC, 18h; (iii) CH3ONa, MeOH,
40 ꢂC, 3–6h.
Fluorescence spectra (510 ꢁ 600 nm) were monitored using an form of either oxazolone or imidazolone between the two rings A
excitation wavelength of 488 nm53,54
.
and B, as well as isosterically replace ring B with quinolone.
Specifically, the rigidity of the molecules was increased by intro-
ducing 1,3-oxazol-5-ones and 1,3-imidazol-4-ones to the cis-ole-
finic bond of CA-4, which we anticipate would create a desirable
conformational and configurational restriction to prevent isomer-
isation of CA-4 into the inactive trans-isomer, as well as improve
on the anticancer activities of these compounds since chalcones
are well known for their anticancer properties55,56. The second
design step involves varying the electronic substituents effect on
the quinolyl moiety (ring B), while maintaining the natural active
compound 3,4,5-trimethoxyphenyl moiety, which we anticipate
will increase the potency of these compounds.
The syntheses of the proposed quinoline compounds 12a–h
(oxazolones) and 13a–h (imidazolones) (Table 1) are shown in
Schemes 1 and 2 and involve two core structural components: (i)
2-methoxyquinolyl-3-carbaldehyde nucleus 8a–h, and (ii) 3,4,5-tri-
methoxyphenyl moiety 11. A concise (three-step) synthesis was
3. Results and discussion
3.1. Design and chemistry
The natural product, Combretastatin A-4 (CA-4; Figure 1) exhibits
significant antiproliferative activities against several tumour cells
by binding to the colchicine site of tubulin to inhibit the protein
polymerisation20. However, the cis double bond of CA-4 has a pro-
pensity to isomerise into the inactive trans configuration, leading
to reduction in the molecule’s pharmacologic activity. Several
structural modifications of the CA-4 pharmacophore have subse-
quently been undertaken to overcome this disadvantage, e.g.
replacing the cis double bond with a heterocycle, oxadiazole, iso-
xazole and imidazole, resulting in compounds, such as 1, 2 and 3
respectively (Figure 1)27–32. In this work, we undertook a rational
design approach of introducing chalcone system (ring C) in the used for the synthesis of the first core structure, 2-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
809
Scheme 2. Synthetic route for preparation of Quinoline targets 12a–h and 13a–h. Reagents and conditions: (i) SOCl2,DCM, r.t; (ii) TEA, MeCN,H2O, r.t; (iii) AcONa,
AC2O, 80 ꢂC, 2h; (iv) NH4OH, EtOH, reflux, 18h.
Table 2. Inhibition of Tubulin Polymerisation and Colchicine Binding by quin-
oline compounds and CA-4.
3.2. Biological results and discussion
Tubulin assemblya
Colchicine bindingb
3.2.1. In vitro antiproliferative activities
All the synthesised compounds (with CA-4 as a positive reference)
were evaluated for their antiproliferative activities using MTT assay
5 mM drug with four different cancer cell lines – MCF-7 breast adenocarcin-
%
SD
Compound number
IC50 (mM)
1 mM drug
oma, HL-60 leukaemia, HCT-116 colorectal carcinoma, and HeLa
cervical adenocarcinoma. As shown in Table 1, most of the com-
pounds demonstrated moderate to highly potent antiproliferative
activities. In the oxazolone analogues (12a–h), compound 12a
without any substituent on the quinoline ring was the least active
when compared with quinoline ring substituted compounds. The
relative position of the substituent on the quinoline ring also
seemed to be critical for antiproliferative activity. Compound 12c
with methyl group at the 7-position ring displayed impressive
non-selective potency in nanomolar range against HL-60, MCF-7,
12a
12c
12e
12g
13c
13e
CA-4
13.98
1.21
2.26
8.23
20.29
1.48
2.17
nd
79
nd
nd
nd
nd
nd
87
nd
nd
nd
nd
2
1
86 0.9
97
2
aInhibition of tubulin polymerisation. Tubulin was at 10 mM.
bInhibition of [3H] colchicine binding. Tubulin and colchicine were at 1 and
5 mM concentrations, respectively.
methoxyquinoline-3-carbaldehyde derivatives 8a–h, as shown in HCT-116 and HeLa cell lines with IC50 of 0.019, 0.010, 0.022 and
Scheme 1. The synthesis was initiated with acetylation of the start- 0.042 mM, which compared to 0.076, 0.019. 0.026 and 0.064 mM for
ing aniline derivatives 5a–h using acetic anhydride and glacial CA-4, respectively. In contrast, both the 6-CH3 analog (12b) and 8-
acetic acid at 0 ꢂC. The produced amides 6a–h were subjected to CH3 analog (12d) were 3- to 15-fold less active than 12c. The
Vilsmeier–Haack reaction to give the corresponding quinoline-3- nature of the substituents on the quinoline ring of the oxazolone
aldehyde derivatives 7a–h. Addition of methoxy substituent to compounds was also found to significantly influence the biological
7a–h to give 8a–h was achieved through the use of sodium activity. For example, replacement of the methyl group in 12b
methoxide at 40 ꢂC in methanol47,48. The synthesis of the second and 12c with a stronger electron-releasing methoxy group yielded
core 3,4,5-trimethoxyphenyl moiety 11 started with acylation of compounds 12e and 12f, respectively, which resulted in better
the acid 9 under highly acidic condition using SOCl2 to give acyl antiproliferative activities. The methoxy-containing compound 12e
benzotriazole 10 (Scheme 2)57,58. Following, treatment of the acyl was 2.7- to 13-fold more active than the methyl-containing com-
benzotriazole 10 with glycine in aqueous acetonitrile gave the pound 12b with the four cancer cell lines (IC50 of 0.068, 0.056,
acyl glycine 11. Condensation of 11 with the appropriate quin- 0.031 and 0.010 mM in HL-60, MCF-7, HCT-116 and HeLa cancer
oline aldehydes 8a–h in the presence of acetic anhydride and cell lines, respectively). Compound 12f had a similar effect as 12c
catalytic amount of sodium acetate resulted in the formation of against MCF-7 and HCT-116 cells (0.052 and 0.066 mM, respect-
the oxazolones 12a–h. Aminolysis of 12a–h via condensation ively), but with reduction in activity against the other two cell
reaction with ammonia led to the formation of the imidazolones lines, HL-60 and HeLa (0.352 and 0.138 mM, respectively).
13a–h. It seems the nucleophilic ammonia attacks the carbonyl Introducing larger substituents at the quinoline ring as in 12g (7-
group of the oxazolone ring, followed by immediate intramolecu- tert-butyl) and 12h (7-benzyloxy) led to a dramatic decrease in
lar condensation and cyclisation to give the imidazolones 13a–h. activity compared to their corresponding analog 12f (7-methoxy).
In summary, two classes of compounds, 12a–h (oxazolones) and Summarily, adding smaller and/or polar groups to the quinoline
13a–h (imidazolones) were synthesised and used for further func- ring of the oxazolone resulted in significant improvement in the
tional and biological studies.
antiproliferative activity.

810
T. S. IBRAHIM ET AL.
Figure 3. (A) Effect of compound 12c on the cell cycle and apoptosis in MCF-7 cells. Cells were treated with either vehicle [0.1% ethanol (v/v)], CA-4 (50nM), 12c
(50nM and 250nM) for 24 h, 48 h and 72h. Cells were then fixed, stained with PI, and analysed by flow cytometry. Cell cycle analysis was performed on histograms of
gated counts per DNA area (FL2-A). The number of cells with (B) 4 N (G2/M), (C) 2 N(G0G1), and (D) <2 N (sub-G1) DNA content was determined with CellQuest soft-
ꢄ
ware. Values represent the mean SEM for three independent experiments. Statistical analysis was performed using two-way ANOVA ( p < 0.05;
ꢄꢄ
p < 0.01;
ꢄꢄꢄ
p < 0.001).
The imidazolones (Compound 13a–h) also resulted in impres- showed similar antiproliferative activities as the oxazolones (Table
sive antiproliferative activity with IC50 values ranging from 1), which could be due to similar electronic effects of the oxazo-
0.04–8.21 mM in all four cell lines. In general, the imidazolones lone and imidazolone rings. Like the oxazolone, lack of substituent

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
811
(A)
(B)
40
***
**
***
24 h
48 h
***
Vehicle
40
30
20
10
0
Vehicle
*
CA-4 (50 nM)
12c (50 nM)
12c (250 nM)
***
***
30
20
10
0
***
***
CA-4 (50 nM)
12c (50 nM)
12c (250 nM)
***
*
s
s
i
s
i
s
s
si
o
o
o
t
t
t
p
op
ap
o
op
p
a
ap
l
a
y
l
te
La
t
To
ar
E
72 h
***
***
***
***
**
40
***
Vehicle
CA-4 (50 nM)
12c (50 nM)
12c (250 nM)
30
20
10
0
s
s
s
i
i
i
s
o
s
os
t
op
t
to
p
po
op
p
a
a
ap
e
ly
al
t
t
To
r
La
Ea
Figure 4. (A) Effect of compound 12c at different time points on apoptosis in MCF-7 cells analysed by flow cytometry after double staining of the cells with Annexin-
V-FITC and PI. MCF-7 cells treated with 50 and 250 nM of compound 12c and 50nM of CA-4 for 24h, 48 h and 72 h and collected and processed for analysis. (B)
Quantitative analysis of apoptosis. Values represent the mean SEM for three independent experiments. Statistical analysis was performed using two-way ANOVA
ꢄ
ꢄꢄ
ꢄꢄꢄ
p < 0.001).
( p < 0.05 and p < 0.01;
on the quinolone ring as in compound 13a led to reduction in 13c and 8-CH3 13d led to potent activity in submicromolar range
activity, with IC50 values of more than 1 mM in all four cell lines,
similar to the results obtained with the oxazolone derivative 12a.
in all four cancer cell lines.
The position of the methoxy substituent on the quinoline het-
Methyl substitution on the quinoline ring, e.g. 6-CH3 13b, 7-CH3 erocycle also influenced the antiproliferative activity of the

812
T. S. IBRAHIM ET AL.
(A)
(A)
1.0
0.8
0.6
0.4
0.2
0.0
MCF-10A
MCF-7
100
50
0
e
)
l
c
M
i
-8
-6
-4
h
Ve
50 n
2
Log10 concentration (M)
12c (
(B)
(B)
MCF-10A
MCF-7
6
5
4
3
2
1
0
100
50
0
-8
-6
-4
e
cl
M)
i
n
h
Ve
Log10 concentration (M)
50
2
Figure 5. Dose response curve for (A) Compound 12c and (B) CA-4 on the prolif-
eration of breast cancer MCF-7 and normal breast MCF-10A cells. Cells were
grown in 96-well plates and treated with serial concentrations of compound 12c
or CA-4 for 72 h. Cell viability was expressed as percentage of vehicle control
[ethanol 1% (v/v)] treated cells and was measured by MTT assay (average of
three independent experiments).
12c (
(C)
40
30
20
10
0
compounds against the cancer cell lines. For example, the antipro-
liferative activity of 6-methoxy-substituted 13e was better than its
analog 7-methoxy-substituted 13f against MCF-7, HCT-116 and
HeLa cells with IC50 values of 0.042, 0.085 and 0.062 mM, which
compare to 0.092, 0.187 and 0.101 mM for 13f, respectively.
However, in HL-60, 13e exhibited less antiproliferative activity
with IC50 value of 0.272 mM. In
a similar trend as the
)
)
)
e
oxazolone derivatives, bulky substituents on the quinoline ring
13g (7-tert-butyl) and 13h (7-benzyloxy) resulted in drastic
decrease in activity in all four cancer cell lines with 14- to 125-fold
loss in potency compared to their corresponding 13f (7-methoxy
containing) compound.
In summary, both oxazolone and imidazolone compounds dis-
played potent antiproliferative effects, strengthening our hypoth-
esis that nitrogen-containing heterocycles, such as quinoline, are
beneficial surrogates for the ring B of CA-4. The different bio-
logical activities of the compounds are likely the result of differen-
ces in their mode of interaction with the colchicine binding site.
Due to its excellent antiproliferative activity, compound 12c was
studied in more details as described below.
M
M
M
cl
i
n
n
n
h
Ve
0
0
0
5
5
(
(5
4
(2
12c
12c
CA-
Figure 6. Effect of compound 12c on the expression (A) anti-apoptotic protein
Bcl2, (B) pro-apoptotic protein BAX and (C) Caspase 9 in MCF-7 cells.
at the colchicine binding site, resulting in inhibition of micro-
tubule polymerisation59,60. To confirm whether the quinoline com-
pounds similarly target the tubulin-microtubule system,
representative quinoline compounds, including four oxazolone
analogues (12a, 12c, 12e and 12g) and two imidazolone ana-
logues (13c and 13e), as well as the reference compound CA-4,
were evaluated for their antitubulin polymerisation activities and
the results presented in Table 2. The methyl and methoxy substi-
tuted oxazolone compounds 12c and 12e, respectively strongly
inhibited tubulin assembly with IC50 of 1.21 and 2.26 mM, respect-
3.2.2. In vitro inhibition of tubulin polymerisation and colchi-
cine binding
Trimethoxyphenyl (TMP) containing stilbenoid derived com-
pounds, such as colchicine, resveratrol and CA-4 bind to tubulin ively compared to that of CA-4 (IC50 of 2.17 mM), while the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
813
(A)
(B)
100
80
60
40
20
0
)
)
0
M
M
M)
50 n
50 n
50 n
2
(
(
4
-
A
12c
12c (
C
Figure 7. (A) Inhibition of colony formation in MCF-7 cells by 50 and 250 nM of compound 12c and 50nM of CA-4 for 48 h. (B) Quantitative analysis of colony forma-
tion. .
unsubstituted analogue 12a (IC50 of 13.98 mM) and tert-butyl ana- dependent manner. After 48 h, the percentage of G2/M phase
log 12g (IC50 of 8.23 mM) were 6- and 4-fold less active than CA-4. arrested cells were 28.4% and 38.3% at 50 nM and 250 nM,
The imidazolone compound 13e with IC50 of 1.48 mM also showed respectively compared to 9.2% of untreated cells (Figure 3(B)).
very potent tubulin polymerisation inhibition compared to CA-4. Moreover, there was an increase in the number of cells in G2/M
The methyl analogue 13c was inactive in the tubulin polymerisa- phase after 72 h (33.0% and 40.8% at 50 nM and 250 nM, respect-
tion assay (IC50 of 20.29 mM), and is 16-fold less active compared ively) with a concomitant decrease of cells in G0/G1 phase (40.3%
to its corresponding oxazolone derivative 12c, which is in agree- and 29.8% at 50 nM and 250 nM, respectively) compared to the
ments with the poor cell growth inhibitory activity of 13c com- control (57.3%). In a comparable finding, CA-4 (50 nM) also signifi-
pared to 12c.
cantly arrested G2/M phase at 24, 48 and 72 h (40.3%, 43.8% and
Compound 12c was also examined at two different concentra- 47.7%, respectively). Accordingly, a concomitant decrease of MCF-
tions (1 and 5 mM) for its ability to compete with colchicine for 7 cells was detected in G0 phase (Figure 3(C)). Furthermore, com-
binding to tubulin using
a
[3H] colchicine binding assay. pound 12c induced a gradual increase in apoptosis (16.2%, 23.4%
Compound 12c strongly inhibited colchicine binding to tubulin by and 32.7%) at 250 nM as the proportion of cells in the sub-G1
79% and 87% at 1 and 5 mM respectively, which compares with phase increased at 24, 48 and 72 h, respectively compared to
86% and 97% inhibition by CA-4, respectively. These results sug- untreated cells (1.5%) (Figure 3(D)). Similarly, 23.5%, 31.1% and
gest that compound 12c is involved in tubulin polymerisation 37.4% increase in apoptosis was observed for CA-4 at 24, 48 and
inhibition through the colchicine-binding site.
72 h, respectively. These findings are in agreement with previously
reported for a series of related quinoline analogues, which signifi-
cantly induced apoptosis and G2/M cycle arrest in MCF-7
cells23,42,46,64
.
3.2.3. Cell cycle analysis
Induction of cell cycle arrest at G2/M phase is strongly accompa-
nied with tubulin polymerisation inhibition. It is well established
that CA-4 arrests cell cycle at G2M phase61–63. To further gain
3.2.4. Cell apoptosis
insight into compound 12c potent antiproliferative activity, cell We investigated whether cell death induced by compound 12c
cycle analysis of MCF-7 cells was performed at two concentrations treatment was related to apoptosis using the Annexin-V/PI double
of 50 nM and 250 nM and at different time points of 0, 24, 48 and staining flow cytometric assay (Figure 4(A,B)). MCF-7 cells were
72 h. Figure 3(A) clearly demonstrates that 12c caused a signifi- treated with three different concentrations (0, 50 and 250 nM) of
cant arrest in G2/M phase and apoptosis in a dose- and time- compound 12c at different time points (24, 48 and 72 h).

814
T. S. IBRAHIM ET AL.
(A)
(B)
100
80
60
40
20
0
***
***
e
0
50
cl
i
25
h
Ve
Compound 12c
Concentration (nM)
Figure 8. (A) Inhibition of the migration of MCF-7 cells treated with compound 12c for 48h in the wound healing assay. (B) Quantitative analysis of wound closure
rate and was calculated as mean SEM for three independent experiments. Statistical analysis was performed using one-way ANOVA-Bonferroni post-hoc
ꢄꢄꢄ
test ( p < 0.001).
Compound 12c caused a significant accumulation of annexine-V than 50 mM in MCF-10A cells, which was significantly higher than
positive cells and induced both early and late apoptosis in a dose- the IC50 values of 19, 10, 22 and 42 nM in MCF-7, HL-60, HCT-116
and time-dependent manner compared to the untreated cells. As and HeLa cancer cell lines, respectively. Remarkably, 12c was
shown in Figure 4(B), when the cells were treated with 12c (0 and found to be less toxic in normal MCF-10A (IC50 >50 mM) when
250 nM) or CA-4 (50 nM) for 48 h, the percentage of Annexin V- compared to CA-4 (IC50 ¼ 6.1 mM) (Figure 5(B)), suggesting 12c to
staining positive cells significantly increased from 1% in untreated have better selective toxicity against cancer cells.
cells to 15%, 21% and 29% respectively. The percentage of early
and late apoptotic cells in the presence of 12c increased after
3.2.6. Expression of the apoptotic proteins in MCF-7 cell lines
The previous data clearly demonstrate that 12c is an effective
anti-mitotic quinoline compound in MCF-7 cell lines. Herein, the
effect of 12c on the expression of apoptosis pathway markers,
Bcl-2 anti-apoptotic protein and Bax pro-apoptotic protein was
investigated. MCF-7 cells treated with 12c at 250 nM for 48 h
decreased the expression level of the anti-apoptotic protein Bcl-2,
and correspondingly up-regulated the expression of the pro-apop-
totic protein Bax (Figure 6(A,B)).
72 h to 17.6% and 29.3% at 1 and 5 mM respectively when com-
pared to the untreated cells (2%). Based on the cell cycle arrest
and apoptosis findings (Figure 3(B–D)), it appears that compound
12c could efficiently induce apoptosis cell death in MCF-7 cells in
a dose- and time-dependent manner.
3.2.5. Assessment of toxicity to non-tumorigenic human cells
To assess the cytotoxicity and selectivity of 12c towards cancer
cells, normal epithelial breast MCF-10A cell viability study was car-
Activation of caspases initiates apoptosis, and in particular cas-
ried out. As shown in Figure 5(A), the IC50 value of 12c was more pase-9 is considered an important effector caspase responsible for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
815
(A)
(B)
40
30
20
10
0
Cont.
***
12c (50 nM)
12c (250 nM)
CA-4 (50 nM)
***
***
***
***
**
4
6
2
12
Time (h)
Figure 9. Assessment of mitochondrial membrane potential (Dwmt) after treatment of MCF-7 cells with 12c. Cells were treated with indicated concentration of com-
pound 12c for 6, 12 and 24h and then stained with fluorescent DiOC2(3) for analysis of mitochondrial potential. Cells were then analysed by flow cytometry as
described in the experimental section. Data are presented as mean SEM of three independent experiments. Statistical analysis was performed using two-way ANOVA
ꢄꢄ
p < 0.01;
ꢄꢄꢄ
p < 0.001).
(
programmed cell death apoptosis activated by CA-461,65,66. The 3.2.7. Inhibition of colony formation
amount of activated caspase-9 was examined in MCF-7 cells treated
with 12c. As observed from Figure 6(C), compound 12c at 50 and
250 nM produced about 8- and 16-fold increases in caspase-9 acti-
vation respectively when compared to 14-fold for CA-4 (50 nM).
This finding confirms that compound 12c like CA-4 enhanced the
rate of apoptosis in MCF-7 cell through caspase-9 activation.
Colony formation assay is one of the effective techniques for the
determination of long-term cell proliferation upon anticancer drug
exposure. The inhibitory potential of 12c on MCF-7 cells colony
formation is displayed in Figure 7. Compound 12c suppressed the
clonogenic formation potential of MCF-7 cells in a dose depend-
ent manner when compared to CA-4.

816
T. S. IBRAHIM ET AL.
above clearly suggests that compound 12c induced apoptosis via
the mitochondrial pathway.
50
40
30
20
10
0
Cont.
12c (50 nM)
12c (250 nM)
CA-4 (50 nM)
H2O2
***
***
4. Conclusion
***
In this study, we designed, synthesised and evaluated two classes
of novel quinoline compounds combretastatin A-4 derivatives as
potential inhibitors of tubulin polymerisation. Several other studies
have also reported derivatisation of the CA-4 pharmacophore with
varying success27–32. Unlike the previous compounds, we for the
first time introduced a chalcone system, including oxazolones and
imidazolones to the cis bond of CA-4 to give more rigidity to the
required active conformation. The chalcone system is well known
for its anticancer activities. Our design also kept the essential nat-
ural trimethoxyphenyl pharmacophore (found in CA-4), while vary-
ing the electronic substituents effect on the quinolyl moiety (ring
B) that were expected to enhance the potency of the compounds.
Most of the compounds showed significant and, in some instan-
ces, comparable antiproliferative activities against different cancer
cell lines as the previously studied combretastatin A-4 compound,
CA-4. One of the most promising compound 12c showed potent
anti-proliferative activities against HL-60, MCF-7, HCT-116 and
HeLA cancer cell lines with IC50 values of 0.019, 0.010, 0.022 and
0.042 mM, respectively, and simultaneously low cytotoxicity
towards MCF-10A non-cancer cells. The microtubule polymerisa-
tion inhibitory effect of 12c was confirmed with an in vitro tubulin
polymerisation and colchicine inhibition assays. Compound 12c
effectively block the G2/M phase at the cell cycle and induce MCF-
7 cell apoptosis together with significant change of Bax/Bcl
expression ratio indicating involvement of mitochondrial apoptosis
pathway. Further cellular mechanistic studies confirmed that 12c
inhibited MCF-7 cell migration and colony formation. In conclu-
sion, these results highlight our novel quinoline compounds and
particularly 12c as promising anti-tubulin agent for the treatment
of MCF-7 breast cancer cells. Moreover, the results point to a dir-
ection for rational development of potent tubulin polymerisation
inhibitors for the treatment of cancer.
***
***
Time (h)
Figure 10. Effect of compound 12c at different time points on ROS production
in MCF-7 cells. MCF-7 cells treated without and with 50 and 250 nM of com-
pound 12c and 50 nM of CA-4 and 100 mM H2O2 for 6 h, 12h and 24 h. Cells
were incubated with DCFDA (25 mM) dye for 1 h at 37 ꢂC in dark. Fluorescence
intensity per cell at 525 nm was calculated as mean SEM for three independent
experiments.
ANOVA ( p < 0.001).
Statistical
analysis
was
performed
using
two-way
ꢄꢄꢄ
3.2.8. Wound healing assay
Migration and motility of cancer cells are considered as critical fac-
tors in tumour progression and metastasis67,68. In order to investi-
gate the effect of compound 12c on the migration of MCF-7,
wound healing assay was performed. As illustrated in Figure
8(A,B), the untreated cells migrated to the scraped area while in
12c-treated wells, cell migration was significantly inhibited in a
dose-dependent manner. This significant difference in the wound
area confirms that 12c suppressed MCF-7 cell migration, an
important event in tumour metastasis.
3.2.9. Mitochondrial membrane potential
Mitochondria membrane potential plays a crucial role in the
propagation of apoptosis. Specifically, loss of mitochondrial mem-
brane potential Dwmt (MMP) is characteristic of early stage of
apoptosis69–71. To confirm whether compound 12c could decrease
the MMP of MCF-7 cancer cells, MMP was monitored by the fluor-
escence of the dye DiOC2(3). MCF-7 cells treated with 12c at 50
and 250 nM exhibited significant decrease in MMP in a dose- and
time-dependant manner (Figure 9(A)). This depletion in MMP was
associated with an increase of annexin-V positive early apoptotic
cells. Maximum decrease in MCF-7 MMP was detected after 24 h
treatment with 12c in which the percentage of apoptotic cells
increased from 1.1% to 24.9% and 31.3% at 50 and 250 nM,
respectively (Figure 9(B)). This indicates that compound 12c indu-
ces mitochondrial dysfunction in MCF-7, which eventually trig-
gered apoptotic cell death. These results are in agreement with
previously reported CA-4 analogues study that were shown to
Acknowledgements
The authors gratefully acknowledge DSR technical and finan-
cial support.
Disclosure statement
The authors declare no competing interests.
Funding
cause apoptosis through the mitochondrial pathway68,72,73
.
This project was funded by the Deanship of Scientific Research
(DSR), King Abdulaziz University, Jeddah, under grant number [RG-
18–166-41]. The authors, therefore, gratefully acknowledge DSR
technical and financial support.
3.2.10. Intracellular reactive oxygen species (ROS) production
The dissipation of mitochondrial potential is strongly associated
with mitochondrial production of reactive oxygen species
(ROS)71,73. The production of ROS after 12c treatment at 50 and
250 nM, as well as CA-4 (50 nM) with hydrogen peroxide H2O2 was
followed with 2,7–dichlorofluorescin diacetate (H2-DCFDA). As
shown in Figure 10, after 24 h of 12c treatment, the levels of ROS
in MCF-7 cells were 22.7 and 26.6% at 50 and 250 nM, respect-
ively. The level in untreated MCF-7 cells was 1.0%, while it
ORCID
Tarek S. Ibrahim
Azizah M. Malebari
http://orcid.org/0000-0002-3049-4617
http://orcid.org/0000-0003-4178-0341
Ehab S. Taher
http://orcid.org/0000-0002-1091-2885
increased only to 18.3% in CA-4-treated cells. This result along Abdelsattar M. Omar
with the significant loss of mitochondrial membrane potential Yaseen A. M. M. Elshaier
http://orcid.org/0000-0002-9825-3465
http://orcid.org/0000-0003-4332-6345

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