Synthesis and in vitro anticancer activity of certain novel 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas as apoptosis-inducing agents

Article abstract of DOI:10.1080/14756366.2018.1547286

In connection with our research program on the development of novel anticancer candidates, herein we report the design and synthesis of novel series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. The target pyridins were evaluated for their in vitro anticancer activity against two cancer cell lines: non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Compound 5l emerged as the most active congener towards both A549 and HCT-116 cell lines with IC₅₀ values equal to 3.22 ± 0.2 and 2.71 ± 0.16 μM, respectively, which are comparable to those of Doxorubicin; 2.93 ± 0.28 and 3.10 ± 0.22, respectively. Furthermore, compound 5l stood out as the most potent pyridine derivative (mean % GI = 40), at US-NCI Developmental Therapeutic Program anticancer assay, with broad-spectrum antitumor activity against the most tested cancer cell lines from all subpanels. Compound 5l was able to provoke apoptosis in HCT-116 cells as evidenced by the decreased expression of the anti-apoptotic Bcl-2 protein, and the enhanced expression of the pro-apoptotic proteins levels; Bax, cytochrome C, p53, caspase-3 and caspase-9. Moreover, 5l disrupted the HCT-116 cell cycle via alteration of the Sub-G₁ phase and arresting the G₂-M stage. Also, 5l showed a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.99 to 15.76%.

Full text of DOI:10.1080/14756366.2018.1547286

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and in vitro anticancer activity of certain
novel 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas
as apoptosis-inducing agents
Wagdy M. Eldehna, Ghada S. Hassan, Sara T. Al-Rashood, Tarfah Al-Warhi,
Ahmed E. Altyar, Hamad M. Alkahtani, Abdulrahman A. Almehizia & Hatem
A. Abdel-Aziz
To cite this article: Wagdy M. Eldehna, Ghada S. Hassan, Sara T. Al-Rashood, Tarfah Al-
Warhi, Ahmed E. Altyar, Hamad M. Alkahtani, Abdulrahman A. Almehizia & Hatem A. Abdel-Aziz
(2019) Synthesis and inꢀvitro anticancer activity of certain novel 1-(2-methyl-6-arylpyridin-3-yl)-3-
phenylureas as apoptosis-inducing agents, Journal of Enzyme Inhibition and Medicinal Chemistry,
34:1, 322-332, DOI: 10.1080/14756366.2018.1547286
To link to this article: https://doi.org/10.1080/14756366.2018.1547286
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 322–332
https://doi.org/10.1080/14756366.2018.1547286
RESEARCH PAPER
Synthesis and in vitro anticancer activity of certain novel 1-(2-methyl-6-arylpyridin-
3-yl)-3-phenylureas as apoptosis-inducing agents
Wagdy M. Eldehnaa, Ghada S. Hassanb, Sara T. Al-Rashoodc, Tarfah Al-Warhid, Ahmed E. Altyare
Hamad M. Alkahtanic, Abdulrahman A. Almehiziac and Hatem A. Abdel-Azizf
,
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Department of Medicinal
c
Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, King
d
Saud University, Riyadh, Saudi Arabia; Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, Riyadh,
e
f
Saudi Arabia; Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; Department of Applied
Organic Chemistry, National Research Center, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 16 October 2018
Revised 6 November 2018
Accepted 8 November 2018
In connection with our research program on the development of novel anticancer candidates, herein we
report the design and synthesis of novel series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. The
target pyridins were evaluated for their in vitro anticancer activity against two cancer cell lines: non-small
cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Compound 5l emerged as the most
active congener towards both A549 and HCT-116 cell lines with IC50 values equal to 3.22 0.2 and
2.71 0.16 mM, respectively, which are comparable to those of Doxorubicin; 2.93 0.28 and 3.10 0.22,
respectively. Furthermore, compound 5l stood out as the most potent pyridine derivative (mean %
GI ¼ 40), at US-NCI Developmental Therapeutic Program anticancer assay, with broad-spectrum antitumor
activity against the most tested cancer cell lines from all subpanels. Compound 5l was able to provoke
apoptosis in HCT-116 cells as evidenced by the decreased expression of the anti-apoptotic Bcl-2 protein,
and the enhanced expression of the pro-apoptotic proteins levels; Bax, cytochrome C, p53, caspase-3 and
caspase-9. Moreover, 5l disrupted the HCT-116 cell cycle via alteration of the Sub-G1 phase and arresting
the G2-M stage. Also, 5l showed a significant increase in the percent of annexinV-FITC positive apoptotic
cells from 1.99 to 15.76%.
KEYWORDS
Anticancer agents;
apoptosis; cell cycle;
pyridine-urea; synthesis
Introduction
angiogenickinases VEGFR-1/3, FGFR1, PDGFRb, and Tie-2.
Regorafenib was approved by FDA, in September 2012, for the
treatment of metastatic colorectal cancer (mCRC)5. The anticancer
effect of Regorafenib is thought to be mediated by apoptosis
induction, in addition to its anti-angiogenic and anti-proliferative
Apoptosis, a self-automated cell death, represents the principal
pathway in tissue homeostasis and in animal development; in
addition, it is the main pathway for the clearance of aged or
defective cells in the body. Mainly, two major signaling pathways
for apoptotic cell death have been signified. The first one is the
extrinsic cytoplasmic pathway that is triggered via pro-apoptotic
ligands binding to the cell surface death receptor. Whereas, the
second is the intrinsic mitochondrial apoptotic pathway that
results from an intracellular cascade of events that are mainly pro-
duced by cellular stress, in which mitochondrial permeabilization
plays a crucial role. Both extrinsic and intrinsic pathways converge
onto the activation of effector caspases, resulting in apoptotic cell
death program. During cancer pathogenesis, apoptosis deregula-
R
effects6,7. Crizotinib (XalkoriV, Figure 1) is an orally active inhibitor
of multiple receptor tyrosine kinases, including anaplastic lymph-
oma kinase (ALK), Hepatocyte Growth Factor Receptor (HGFR, c-
Met), and Recepteur d’Origine Nantais (RON)8. Crizotinib was
approved for the treatment of adults with previously treated, ALK-
positive, advanced non-small cell lung cancer (NSCLC)9. Crizotinib
likely exerts its anticancer activity via multiple distinct mechanisms
such as apoptosis10.
Recently, our research group has explored the anticancer activ-
ity for novel series of 1–(2-methyl-6-(4-methoxy/3,4-dimethoxy-
phenyl)-pyridin-3-yl)-3-phenylureas11. All these derivatives were
evaluated for their growth inhibitory activity against the prolifer-
ation of breast cancer cell line (MCF-7), where they displayed
promising anti-proliferative activity. On the other hand, examin-
ation of their potential anti-angiogenic activity towards vascular
endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase
tion has been widely recognized as
a hallmark of cancer.
Accordingly, induction of apoptosis in tumor cells has stood out
as a successful tactic for combating different human malignancies,
in the current medical era1–3
.
On the other hand, non-fused pyridines have stood out as a
promising class of anticancer agents with efficient pro-apoptotic
R
V
activity. Regorafenib (Stivarga , Figure 1),
a pyridine-based
biphenyl urea derivative developed by Bayer4, inhibits unveiled their incompetence to inhibit VEGFR-2 significantly11.
CONTACT Sara T. Al-Rashood
salrashood@ksu.edu.sa
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451,
Saudi Arabia
Supplemental data for this article can be accessed here.
ß 2019 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
323
Figure 1. Structures of certain pyridine-based approved anticancer drugs, and the target pyridines 5a–l.
Based on the aforementioned findings and as a part of our 1-(6-(4-Fluorophenyl)-2-methylpyridin-3-yl)-3-(3-
(trifluoromethyl)phenyl)urea (5a)
ongoing quest towards developing potent anticancer agents12–20
,
herein we report the synthesis and biological evaluation of novel
series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. Ten
selected pyridines 5a, 5c–j and 5l were chosen to be in vitro eval-
uated for their antitumor activity at one dose (concentration
10ꢀ5 M) primary anticancer assay towards a panel including 85
cancer lines according to US-NCI protocol. In addition, all pyridines
5a–l were examined for their potential anti-proliferative activity
against non-small cell lung cancer A549 cell line and colon cancer
HCT-116 cell line. Furthermore, apoptosis induction potential of
the target pyridines was examined in HCT-116 cells, in order to
acquire more mechanistic insights and to verify and enlighten the
antitumor properties of the investigated pyridines.
White crystals (yield 70%), m.p. 223–225 ꢁC; IR (KBr, ꢀ cmꢀ1) 3393
(NH), 1731 (C¼O); H NMR (CDCl3-d) d ppm: 2.64 (s, 3H, CH3), 6.30
1
(s, 1H, NH, D2O exchangeable), 6.61 (s, 1H, NH, D2O exchange-
able), 7.15 (t, 2H, J ¼ 8.8 Hz, Ar-H), 7.38 (d, 1H, J ¼ 8.4 Hz, Ar-H),
7.46 (t, 1H, J ¼ 8.0 Hz, Ar-H), 7.60–7.65 (m, 2H, Ar-H), 7.71 (s, 1H,
Ar-H), 7.98 (dd, 2H, J ¼ 8.8 Hz, J ¼ 5.6 Hz, Ar-H), 8.06 (d, 1H,
J ¼ 8.4 Hz, Ar-H);13C NMR (DMSO-d6) d ppm: 21.34 (CH3), 115.36,
115.53, 117.69, 121.75, 128.04, 128.67, 130.06, 132.47, 135.00,
140.42, 148.02, 148.45, 152.62 (CO), 161.43, 163.38 (¼C-F); HRMS
(ESI) m/z calcd for [M þ H]þ (C20H16N3OF4): 390.12240,
found: 390.12286.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(6-(4-fluorophenyl)-2-
methylpyridin-3-yl)urea (5b)
White crystals (yield 65%), m.p. 235–237 ꢁC; IR (KBr, ꢀ cmꢀ1) 3390
Materials and methods
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.58 (s, 3H, CH3), 6.31
Chemistry
(s, 1H, NH, D2O exchangeable), 6.59 (s, 1H, NH, D2O exchange-
able), 7.17 (t, 2H, J ¼ 8.8 Hz, Ar-H), 7.59 (s, 1H, Ar-H), 7.63 (d, 1H,
J ¼ 8.4 Hz, Ar-H), 7.89 (s, 2H, Ar-H), 8.02–8.10 (m, 3H, Ar-H); 13 C
NMR (DMSO-d6) d ppm: 21.58 (CH3), 115.44, 115.61, 117.75,
128.09, 128.15, 128.66, 132.72, 135.10, 147.79, 148.34, 152.94
(C¼O), 161.49, 163.44 (¼C–F).
Melting points were measured with a Stuart melting point appar-
atus and were uncorrected. Infrared (IR) Spectra were recorded as
KBr disks using Schimadzu FT-IR 8400S spectrophotometer. 1H-
NMR and 13C-NMR experiments were carried out using Bruker
NMR spectrometer (400/100 MHz). Chemical shifts (dH) are
reported relative to TMS as the internal standard. All coupling
constant (J) values are given in hertz. Chemical shifts (dC) were
reported as follows: s, singlet; d, doublet; m, multiplet. High-reso-
lution mass spectra were recorded using a Bruker MicroTOF spec-
trometer (Bruker Daltonics, Bremen, Germany). All reagents and
solvents were dried and purified by the standard techniques.
Compounds 2-methyl-6-arylnicotinohydrazides 2a–c21–23 were pre-
viously prepared.
Ethyl 4-(3-(6-(4-fluorophenyl)-2-methylpyridin-3-
yl)ureido)benzoate (5c)
White crystals (yield 73%), m.p. 209–211 ꢁC; IR (KBr, ꢀ cmꢀ1) 3389
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 1.39 (t, 3H, J ¼ 7.2 Hz,
–OCH2CH3), 2.62 (s, 3H, –CH3), 4.37 (q, 2H, J ¼ 7.2 Hz, –OCH2CH3),
6.36 (s, 1H, NH, D2O exchangeable), 6.72 (s, 1H, NH, D2O
exchangeable), 7.14 (t, 2H, J ¼ 8.8 Hz, Ar-H), 7.49 (d, 2H, J ¼ 8.4 Hz,
Ar-H), 7.60 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.98–8.10 (m, 5H, Ar-H); 13 C
NMR (DMSO-d6) d ppm: 14.30 (CH3), 21.37 (CH3), 60.39 (CH2),
115.41, 115.58, 117.34, 117.75, 122.98, 128.08, 128.50, 130.51,
132.50, 135.04, 144.16, 147.93, 148.47, 152.35 (C¼O), 161.48,
163.43 (¼C–F), 165.48 (–COO–) HRMS (ESI) m/z calcd for [M þ H]þ
(C22H21N3O3F): 394.15615, found: 394.15628.
General procedures for preparation of the target pyridines 5a–l
A solution of hydrazides 2a–c (10 mmol) and sodium nitrite (1 g,
14 mmol) in hydrochloric acid was stirred for 1 h in an ice bath,
then stirring was continued for an additional 1 h at room tempera-
ture. The reaction mixture was poured over crushed ice. The
precipitated solid was filtered off and air-dried to yield 2-methyl-
6-arylnicotinoyl azides 3a–c, which were used in the next step
without further purification. Azides 3a–c were heated in refluxing
dry xylene for 1 h, then the appropriate aniline derivative was
added to this xylene solution. The reaction mixture was heated
under reflux temperature for 4 h. After cooling to room tempera-
ture, the formed precipitate was filtered, washed with ether and
recrystallized from ethanol to afford the target pyridines 5a–l.
1-(Benzo[d][1, 3]dioxol-5-yl)-3-(6-(4-fluorophenyl)-2-
methylpyridin-3-yl)urea (5d)
White crystals (yield 62%), m.p. 254–256 ꢁC; IR (KBr, ꢀ cmꢀ1) 3394
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.48 (s, 3H, CH3), 6.04
(s, 2H, CH2), 6.23 (s, 1H, NH, D2O exchangeable), 6.34 (s, 1H, NH,
D2O exchangeable), 6.84 (d, 1H, J ¼ 8.0 Hz, Ar-H), 6.97–7.02 (m, 2H,
Ar-H), 7.12 (t, 2H, J ¼ 8.4 Hz, Ar-H), 7.54–7.57 (m, 1H, Ar-H),
7.94–7.98 (m, 2H, Ar-H), 8.19 (d, 1H, J ¼ 8.0 Hz, Ar-H); 13C NMR
(DMSO-d6) d ppm: 21.36 (CH3), 100.82 (O–CH2–O), 108.20, 110.93,

324
W. M. ELDEHNA ET AL.
115.33, 115.50, 117.65, 127.99, 132.94, 133.89, 135.08, 142.16, 1-(4-Chlorophenyl)-3-(2-methyl-6-(thiophen-2-yl) pyridin-3-
yl)urea (5i)
147.27, 147.83, 152.66 (C¼O), 161.35, 163.30 (¼C–F); HRMS (ESI)
m/z
calcd
for
[M þ H]þ
(C20H17N3O3F):
366.12485,
White crystals (yield 71%), m.p. 234–236 ꢁC; IR (KBr, ꢀ cmꢀ1) 3398
found: 366.12405.
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.53 (s, 3H, CH3), 6.18
(s, 1H, NH, D2O exchangeable), 6.36 (s, 1H, NH, D2O exchange-
able), 7.06–7.14 (m, 1H, Ar-H), 7.35-7.40 (m, 5H, Ar-H), 7.52-7.54
(m, 2H, J ¼ 6.5 Hz, Ar-H), 7.99 (d, 1H, J ¼ 8.4 Hz, Ar-H); 13 C NMR
(DMSO-d6) d ppm: 21.08 (CH3), 116.48, 119.67, 123.88, 125.51,
127.13, 128.30, 128.72, 132.38, 138.53, 144.61, 145.51, 147.66,
152.46 (C¼O); HRMS (ESI) m/z calcd for [M-H]þ (C17H13N3OClS):
342.04733, found: 342.04752.
1-(6-(4-Chlorophenyl)-2-methylpyridin-3-yl)-3-(3-
(trifluoromethyl)phenyl)urea (5e)
White crystals (yield 68%), m.p. 241-242 ꢁC; IR (KBr, ꢀ cmꢀ1) 3378
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.48 (s, 3H, CH3), 6.25
(s, 1H, NH, D2O exchangeable), 6.36 (s, 1H, NH, D2O exchange-
able), 7.38 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.41 (d, 2H, J ¼ 8.8 Hz, Ar-H),
7.52-7.58 (m, 3H, Ar-H), 7.78 (s, 1H, Ar-H), 7.91 (d, 2H, J ¼ 8.4 Hz,
Ar-H), 8.24 (d, 1H, J ¼ 8.4 Hz, Ar-H); 13 C NMR (DMSO-d6) d ppm:
21.57 (CH3), 1117.91, 127.69, 128.41, 128.68, 133.05, 137.32,
147.75, 147.84, 152.82 (C¼O); HRMS (ESI) m/z calcd for [M-H]þ
(C20H14N3OClF3): 404.07830, found: 404.07779.
Ethyl 4-(3-(2-methyl-6-(thiophen-2-yl) pyridin-3-
yl)ureido)benzoate (5j)
White crystals (yield 69%), m.p. 203–204 ꢁC; IR (KBr, ꢀ cmꢀ1) 3393
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 1.39 (t, 3H, J ¼ 7.2 Hz,
1
–OCH2CH3), 2.55 (s, 3H, –CH3), 4.35 (q, 2H, J ¼ 7.2 Hz, –OCH2CH3),
6.53 (s, 1H, NH, D2O exchangeable), 6.98 (s, 1H, NH, D2O
exchangeable), 7.10 (t, 1H, J ¼ 4.4 Hz, Ar-H), 7.38 (d, 1H, J ¼ 5.2 Hz,
Ar-H), 7.46 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.52–7.55 (m, 2H, Ar-H),
7.99–8.02 (m, 3H, Ar-H); 13 C NMR (DMSO-d6) d ppm: 14.26 (CH3),
21.08 (CH3), 60.32 (O–CH2), 116.49, 117.28, 122.91, 123.97, 127.21,
128.25, 128.50, 130.45, 132.17, 144.12, 144.57, 145.73, 147.86,
152.26 (C¼O), 165.40 (–COO–); HRMS (ESI) m/z calcd for [M ꢀ H]þ
(C20H18N3O3S): 380.10744, found: 380.10764.
1-(6-(4-Chlorophenyl)-2-methylpyridin-3-yl)-3-(4-methoxyphenyl)
urea (5f)
White crystals (yield 55%), m.p. 264-265 ꢁC; IR (KBr, ꢀ cmꢀ1) 3392
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.41 (s, 3H, CH3), 3.86
(s, 3H, –OCH3), 6.27 (s, 1H, NH, D2O exchangeable), 6.33 (s, 1H,
NH, D2O exchangeable), 6.97 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.31 (d, 2H,
J ¼ 8.8 Hz, Ar-H), 7.41 (d, 2H, J ¼ 8.8 Hz, Ar-H), 7.57 (d, 1H,
J ¼ 8.0 Hz, Ar-H), 7.91 (d, 2H, J ¼ 8.4 Hz, Ar-H), 8.26 (d, 1H,
J ¼ 8.4 Hz, Ar-H); 13 C NMR (DMSO-d6) d ppm: 21.37 (CH3), 55.18
(OCH3), 114.08, 117.88, 119.92, 127.45, 127.57, 127.66, 128.61,
132.49, 132.87, 133.47, 137.30, 137.39, 147.13, 147.23, 147.69,
147.79, 152.67 (C¼O), 154.58 (¼C–OCH3); HRMS (ESI) m/z calcd for
[M ꢀ H]þ (C20H17N3O2Cl): 366.10148, found: 366.10152.
1-(Benzo[d][1, 3]dioxol-5-yl)-3-(2-methyl-6-(thiophen-2-
yl)pyridin-3-yl)urea (5k)
White crystals (yield 58%), m.p. 239–241 ꢁC; IR (KBr, ꢀ cmꢀ1) 3388
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.44 (s, 3H, CH3), 6.03
(s, 2H, –OCH2O–), 6.23 (s, 1H, NH, D2O exchangeable), 6.31 (s, 1H,
NH, D2O exchangeable), 6.77 (dd, 1H, J ¼ 2.0 Hz, J ¼ 8.0 Hz, Ar-H),
6.83 (d, 1H, J ¼ 8.0 Hz, Ar-H), 6.97 (d, 1H, J ¼ 2.0 Hz, Ar-H), 7.08 (dd,
1H, J ¼ 4.0 Hz, J ¼ 5.2 Hz, Ar-H), 7.35 (d, 1H, J ¼ 5.0 Hz, Ar-H), 7.53-
7.54 (m, 2H, Ar-H), 8.12 (d, 1H, J ¼ 8.4 Hz, Ar-H); 13 C NMR (DMSO-
d6) d ppm: 21.08 (CH3), 100.87 (O–CH2–O), 108.20, 110.96, 116.47,
123.74, 127.01, 127.92, 128.21, 132.66, 133.86, 142.18, 144.68,
145.20, 147.27, 152.61 (C¼O); HRMS (ESI) m/z calcd for [M ꢀ H]þ
(C18H14N3O3S): 352.07614, found: 352.07642.
1-(Benzo[d][1,3]dioxol-5-yl)-3-(6-(4-chlorophenyl)-2-
methylpyridin-3-yl)urea (5g)
White crystals (yield 63%), m.p. 271–273 ꢁC; IR (KBr, ꢀ cmꢀ1) 3388
1
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.47 (s, 3H, CH3), 6.04
(s, 2H, –OCH2O–), 6.28 (s, 1H, NH, D2O exchangeable), 6.38 (s, 1H,
NH, D2O exchangeable), 6.79–6.87 (m, 2H, Ar-H), 6.96 (d,
1H, J ¼ 2.1 Hz, Ar-H), 7.42 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.57 (d, 1H,
J ¼ 8.4 Hz, Ar-H), 7.91 (d, 2H, J ¼ 8.8 Hz, Ar-H), 8.22 (d, 1H,
J ¼ 8.4 Hz, Ar-H); 13 C NMR (DMSO-d6) d ppm: 21.36 (CH3), 100.84
(O–CH2–O), 108.21, 110.96, 117.88, 127.59, 128.62, 132.90, 133.32,
133.83, 137.36, 142.20, 147.28, 152.60 (C¼O); HRMS (ESI) m/z calcd
for [M-H]þ (C20H15N3O3Cl): 380.08074, found: 380.08115.
2-(3-(2-Methyl-6-(thiophen-2-yl)pyridin-3-
yl)ureido)benzenesulfonamide (5l)
White crystals (yield 60%), m.p. 265–266 ꢁC; IR (KBr, ꢀ cmꢀ1) 3369,
1
3207 (NH, NH2), 1733 (C¼O), 1330, 1157 (SO2); H NMR (DMSO-d6,
400 MHz) d ppm: 2.50 (s, 3H, CH3), 7.11 (t, 1H, H-4 of 2-thienyl,
J ¼ 4.0 Hz), 7.18 (t, 1H, H-4 of 2-(H2NO2S)-C6H4, J ¼ 7.6 Hz), 7.52-
7.56 (m, 2H, H-5 of 2-(H2NO2S)-C6H4, and H-5 of 2-thienyl), 7.60 (s,
2H, SO2NH2), 7.67 (d, 1H, H-3 of 2-thienyl, J ¼ 4.0 Hz), 7.71 (d, 1H,
H-5 pyridine, J ¼ 8.4 Hz), 7.82 (d, 1H, H-6 of 2-(H2NO2S)–C6H4,
J ¼ 7.6 Hz), 7.97 (d, 1H, H-3 of 2-(H2NO2S)-C6H4, J ¼ 8.0 Hz), 8.04 (d,
1-(4-Fluorophenyl)-3-(2-methyl-6-(thiophen-2-yl)pyridin-3-
yl)urea (5h)
White crystals (yield 60%), m.p. 217–219 ꢁC; IR (KBr, ꢀ cmꢀ1) 3393 1H, H-4 pyridine, J ¼ 8.4 Hz), 8.73 (s, 1H, 8.21 (s, 1H, NH, D2O
1
exchangeable), 9.15 (s, 1H, NH, D2O exchangeable).
(NH), 1733 (C¼O); H NMR (CDCl3-d) d ppm: 2.50 (s, 3H, CH3), 6.20
(s, 1H, NH, D2O exchangeable), 6.33 (s, 1H, NH, D2O exchange-
able), 7.07–7.13 (m, 3H, Ar-H), 7.35–7.39 (m, 3H, Ar-H), 7.54–7.56
Biological evaluation
(m, 2H, Ar-H), 8.05 (d, 1H, J ¼ 8.4 Hz, Ar-H); 13 C NMR (DMSO-d6) d
ppm: 21.09 (CH3), 115.30, 115.47, 116.47, 119.85, 119.91, 123.81,
127.07, 127.11, 128.16, 128.21, 132.55, 135.86, 144.65, 145.36,
147.51, 152.64 (C¼O); HRMS (ESI) m/z calcd for [M-H]þ
(C17H13N3OFS): 326.07688, found: 326.07718.
In vitro antitumor activity towards 60 cancer cell lines (NCI, USA)
The antitumor assay was performed according to the protocol of
the Drug Evaluation Branch, NCI, Bethesda24–26. A 48 h drug
exposure protocol was adopted, and sulforhodamine B (SRB)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
325
Scheme 1. Synthesis of target derivatives 5a–l; (i) Ethyl alcohol, NH2NH2ꢂH2O, reflux 3 h.; (ii) NaNO2, HCl, stirring 2 h.; (iii) Xylene, reflux 1 h.; (iv) Xylene, reflux 4 h.
assay27 was utilized to assess the cell growth and viability, as Cell cycle analysis
reported earlier17,28
.
HCT-116 cells were treated with pyridine 5l at its IC50 concentra-
tion (IC50 ¼ 2.71 lM) for 24 h, then cells were washed with ice-cold
phosphate-buffered saline (PBS). The treated cells were collected
by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with
PBS, re-suspended with 100 lg/mL RNase, stained with 40 lg/mL
PI, and analyzed by flow cytometry using FACS Calibur (Becton
Dickinson, BD, USA). The cell cycle distributions were calculated
In vitro anti-Proliferative activity towards A549 and HCT-116
cell lines
A549 (non-small cell lung cancer cell line) and HCT-116 (human
colon cancer cell line), were obtained from American Type using CellQuest software 5.1 (Becton Dickinson)33.
Culture Collection (Manassas, VA, USA). The cells were main-
tained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-
Aldrich, St. Louis, MO), and supplemented with 10% heat-inacti-
Annexin V-FITC apoptosis assay
Phosphatidylserine externalization was assayed using Annexin V-
vated FBS (Hyclone), 10 lg/mL of insulin (Manufacturer, Sigma,
St. Louis, MO, USA), and 1% penicillin-streptomycin. MTT assay29
was adopted to assess the in vitro antitumor activity of the
newly synthesized pyridines 5a–l according to the reported pro-
FITC/PI apoptosis detection kit (BD Biosciences, USA) according to
the manufacturer’s instructions, as reported earlier33,34
.
cedures30
,
using Doxorubicin as
a
standard treatment.
Results and discussion
Experimental conditions were tested using three replicates
(three wells of the 96-well plate per experimental condition) and
all experiments were carried out in triplicates. IC50 values were
calculated by the use of the equation for Boltzman sigmoidal
concentration–response curve using the nonlinear regression fit-
Chemistry
The method adopted for preparation of the target pyridines 5a–l is
depicted in Scheme 1. Firstly, esters 1a–c were hydrazinolyzed via
reaction with hydrazine hydrate in methanol under reflux tempera-
ting models by Graph Pad, Prism version 5 (GraphPad Software ture to furnish 2-methyl-6-arylnicotinohydrazides 2a–c in 75, 71 and
80% yields, respectively. Treatment of hydrazides 2a–c with sodium
nitrite in cold hydrochloric acid afforded 2-methyl-6-arylnicotinoyl
azides 3a–c, which subsequently subjected to Curtius rearrangement
upon heating in xylene to give the corresponding isocyanates deriva-
tives 4a–c. The target hybrids 5a–l was obtained by reaction of iso-
cyanates derivatives 4a–c with the appropriate aniline derivative in
xylene with 55–73% yield (Scheme 1).
Inc., La Jolla, CA).
ELISA immunoassay
The levels of the apoptotic markers Bax, cytochrome C, p53, cas-
pase-3 and caspase-9 as well as the anti-apoptotic protein Bcl-2
were evaluated using ELISA colorimetric kits per the manufac-
The structures of the newly prepared pyridines 5a–l were con-
firmed under the basis of spectral and elemental analyses which
turer’s instructions, as reported earlier31,32
.

326
W. M. ELDEHNA ET AL.
Table 1. Percentage growth inhibition (GI%) of in vitro subpanel tumor cell lines Table 2. Percentage growth inhibition (GI%) of in vitro subpanel tumor cell lines
at 10 lM concentration for pyridines 5a and 5c–f.
at 10 lM concentration for pyridines 5g–j and 5l.
Compounda
Compounda
Subpanel/Cell Line
5a
5c
5d
5e
5f
Subpanel/Cell Line
5g
5h
5i
5j
5l
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
25
11
50
33
52
44
53
17
31
–
18
24
15
34
60
–
–
13
–
–
–
23
–
22
21
20
12
15
41
10
–
–
–
–
–
18 Leukemia
12
13
14
14
28
25 Non-Small Cell Lung Cancer
–
–
–
19
–
–
–
40
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
–
24
21
18
–
22
32
–
–
–
–
–
–
–
38
–
–
–
–
11
–
–
–
–
–
–
–
–
34
–
29
–
–
–
17
25
27
–
–
–
–
–
10
15
22
29
24
18
32
–
–
–
–
16
–
–
39
–
14
22
26
12
18
–
–
–
–
–
–
–
–
–
–
–
24
–
12
20
18
–
–
–
–
–
–
–
–
–
–
–
–
13
19
–
22
–
–
–
15
16
23
–
20
–
15
–
–
29
–
–
–
–
–
–
–
28
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
23
19
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12
–
–
50
10
42
44
56
44
35
–
–
–
–
–
60
44
68
80
55
54
61
23
50
–
–
13
16
25
–
–
–
12
–
–
–
24
–
–
–
–
18
–
–
–
–
–
–
11
13
–
–
–
–
–
–
–
45
–
–
17
–
14
11
–
–
–
–
–
–
–
34
–
37
–
–
–
19
15
20
–
–
–
–
–
–
–
–
–
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
–
22
18
82
41
53
27
74
71
65
51
54
42
51
54
42
46
65
86
–
42
41
10
38
48
25
43
40
46
25
–
59
33
36
40
–
48
36
31
36
51
34
75
21
–
–
17
43
52
–
34
48
–
Colon Cancer
–
–
21
–
23
–
Colon Cancer
CNS Cancer
–
–
–
51
42
43
37
–
–
17
–
15
–
31
37
–
45
19
–
20
32
38
40
–
41
30
–
29
20
13
–
–
–
26
–
26
18
25
–
11
–
–
–
–
–
24
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
20
–
20
–
27
30
19
18
–
–
–
–
–
–
–
CNS Cancer
Melanoma
–
–
–
–
–
–
–
24
–
38
–
–
–
–
21
10
–
38
–
11
–
22
–
–
–
–
–
–
–
–
–
12
–
–
–
–
–
52
–
21
–
–
15
–
13
24
21 Melanoma
–
38
–
–
13
38
26
27
–
38
29
29
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Ovarian Cancer
Renal Cancer
–
–
–
–
–
–
–
–
–
21
–
–
–
–
–
Ovarian Cancer
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Renal Cancer
Prostate
–
–
–
–
–
–
–
–
–
–
–
–
15
55
11
30
28
–
Prostate
Breast Cancer
20
–
–
–
–
–
13
23 Breast Cancer
–
–
–
–
–
MDA-MB-231
HS 578T
BT-549
T-47D
MDA-MB-468
MDA-MB-231
HS 578T
BT-549
T-47D
MDA-MB-468
–
–
13
–
17
–
–
33
26
23
52
45
32
42
13
17
9
Sensitive cell lines no.
12
19 Sensitive cell lines no.
12
aOnly GI% higher than 10% are shown.
aOnly GI% higher than 10% are shown.
were in full agreement with the postulated structures
(Supplementary Material).
Therapeutic Program (www.dtp.nci.nih.gov). Ten pyridines 5a, 5c–j
and 5l were chosen to be in vitro evaluated for their antitumor
activity. The selected pyridines 5a, 5c–j and 5l were examined at
one dose (concentration 10ꢀ5 M) primary anticancer assay towards
a panel including 85 cancer lines. Nine different types of cancer
were tested in this assay: colon, ovarian, prostate, leukemia, mel-
anoma, CNS, renal, breast and lung cancers. A 48 h drug exposure
Biological evaluation
In vitro antitumor activity towards 60 cancer cell lines (NCI, USA)
The structures of all the newly synthesized pyridines 5a–l were
submitted to the National Cancer Institute (NCI) Developmental protocol was adopted, and sulforhodamine B (SRB) assay27 was

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
327
Figure 2. The most susceptible cancer cell lines towards the impact of target pyridines 5a and 5l according to the GI%.
over leukemia (CCRF-CEM, K-562, RPMI-8226 and SR), non-small
cell lung cancer (A549 and HOP-62), colon cancer (COLO205,
HT29, KM12 and SW-620), CNS (SF-295, SF-539 and U251), ovarian
(OVCAR-8 and prostate (PC-3) cell lines, Figure 2.
Furthermore, pyridine 5a was found to be the second most
active member (mean % GI ¼ 22) with broad spectrum activity
against 42 cell lines represent all subpanels. Compound 5a
exerted cytotoxic activity with GI more than 40% against leukemia
(K-562, RPMI-8226 and SR), non-small cell lung cancer (A549 and
NCI-H522), colon cancer (HCT-116, HCT-15 and HT29), melanoma
(M14 and UACC-62), ovarian (OVCAR-3), prostate (PC-3) and breast
(T-47D) cell lines (Figure 2).
Further investigation of results in Tables 1 and 2 unveiled that
all cell lines of the leukemia subpanel were sensitive to six tested
pyridines 5a, 5e, 5f, 5h, 5j and 5l with GI ranging from 10% to
91%. It is noteworthy that only non-small cell lung cancer A549
and NCI-H522 cells were sensitive to all the tested pyridines with
GI% range of 10–61% and 24–60%, respectively. Additionally, leu-
kemia SR (except 5i), leukemia HL-60 (except 5d) and colon can-
cer HT29 (except 5i) cell line were susceptible to nine tested
pyridines. The most susceptible cell lines towards the impact of
pyridines 5a and 5l are displayed in Figure 2.
Table 3. In vitro anti-proliferative activity of target pyridines 5a–l against A549
and HCT-116 cell lines.
H
N
H
N
R
O
Ar
N
5a-m
IC50 (mM)a
Compound
Ar
R
A549
HCT-116
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-F-C6H4
4-F-C6H4
4-F-C6H4
4-F-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
2-thienyl
2-thienyl
2-thienyl
2-thienyl
2-thienyl
–
3-CF3
3,5-(CF3)2
4-COOEt
3,4-Methylenedioxy
3-CF3
4-OCH3
3,4-Methylenedioxy
4-F
4-Cl
4-COOEt
3,4-Methylenedioxy
2-SO2NH2
–
6.83 0.42
24.05 1.78
9.61 1.03
12.48 0.85
11.87 0.92
7.90 0.54
6.72 0.38
10.64 0.86
8.73 0.71
8.04 0.59
19.17 2.05
3.22 0.25
2.93 0.28
5.49 0.30
16.03 1.52
NTb
10.37 0.84
7.05 0.72
12.61 1.08
NTb
8.25 0.84
NTb
5j
5k
5l
9.38 0.67
16.43 1.30
2.71 0.16
3.10 0.22
Dox.
aIC50 values are the mean SD of three separate experiments.
bNA: Not tested.
In vitro anti-proliferative activity against A549 and HCT-116
cell lines
utilized to assess the cell growth and viability. The results were
reported as mean-graph of the percentage growth of the treated
cells, and displayed as percentage growth inhibition (GI%) caused
by the test pyridines (Tables 1 and 2). Investigation of data in
Tables 1 and 2 revealed that the examined pyridines exhibited dis-
tinctive patterns of sensitivity and selectivity against the different
NCI cancer cell panels.
Inspecting the GI% values in Tables 1 and 2, highlighted that
compound 5l stood out as the most potent pyridine derivative
assayed in this study (mean % GI ¼ 40). Pyridine 5l possessed
broad spectrum antitumor activity against all tested cancer cell
lines from all subpanels with an exception to non-small cell lung
cancer (HOP-92 and NCI-H226), melanoma (MALME-3M), ovarian
cancer (OVCAR-5), renal cancer (A498) and breast cancer (HS 578T)
cell lines. In particular, 5l showed a potent growth inhibitory activ-
ity towards leukemia MOLT-4, non-small cell lung cancer NCI-
H460, colon cancer HCT-116 and HCT-15, melanoma LOX IMVI and
breast cancer MCF7 cell lines with inhibition % 80, 82, 74, 71, 86
All newly synthesized pyridines 5a–l were examined for their anti-
proliferative activity towards two cancer cell lines: non-small cell
lung cancer A549 cell line and colon cancer HCT-116 cell line. The
MTT colorimetric assay was adopted to assess the anti-proliferative
activity as described by Mosmann29. Doxorubicin was used as a
control in this assay. The results were expressed as median growth
inhibitory concentration (IC50) values that represent the com-
pound concentration required to produce a 50% inhibition of cell
growth after 48 h of incubation (Table 3).
The results of the MTT assay listed in Table 3 suggested that
the examined pyridines 5a–l exhibited excellent to moderate
growth inhibitory activity against the tested A549 and HCT-116
cancer cell lines. Also, HCT-116 cells were found to be more sensi-
tive to the impact of the tested compounds than A549 cells,
except compound 5j which is more effective towards A549 cells.
Interestingly, compound 5l emerged as the most active one
and 75, respectively. In addition, it displayed GI more than 50% towards both A549 and HCT-116 cell lines with IC50 values equal

328
W. M. ELDEHNA ET AL.
Table 4. Cytotoxicity of pyridines 5a–l towards non-tumorigenic human lung
fibroblast WI-38 cell line and their selectivity index (S. I.) towards lung A549
cancer cells.
Induction of apoptosis in colorectal cancer HCT-116 cells
To investigate the mechanism of antitumor activity of the target
pyridines and in continuation of our efforts to develop potent
pro-apoptotic anticancer agents35–39, the ability of sulfonamide 5l
to provoke apoptosis in HCT-116 cells was evaluated.
IC50 (mM)a
WI-38
S. I.
WI-38/A549
Compound
5a
5b
5c
5d
5e
5f
5g
5h
5i
93.55 5.28
151.37 8.12
122.61 10.17
107.28 7.03
130.44 9.22
115.86 9.61
63.48 5.08
142.60 8.38
129.31 11.95
107.29 7.02
138.74 10.40
67.05 3.82
13.7
6.3
12.8
8.6
11.0
14.7
9.4
13.4
14.8
13.3
7.2
Effects on mitochondrial apoptosis pathway proteins Bcl-2
and Bax
Bcl-2 and Bax are two discrete members of a gene family involved
in the regulation of cellular apoptosis known as BcL-2 family,
which finely tune the apoptotic switch on/off mechanism and
considered as an important gatekeeper to the apoptotic response.
While Bcl-2 protein is functionally characterized as an apoptosis-
suppressing factor, the Bax protein is more functionally character-
ized as an apoptosis-promoting factor. So, the intracellular Bax/
Bcl-2 ratio can profoundly influence the ability of a cell to respond
5j
5k
5l
17.6
aIC50 values are the mean SD of three separate experiments.
to an apoptotic signal40,41
.
Table 5. Impact of pyridine 5l on the expression levels of Bax and Bcl-2 in HCT-
116 cancer cells treated with the compound at its IC50 concentration.
In this study, treatment of HCT-116 cells with the IC50 of pyri-
dine 5l (IC50 ¼ 3.22 0.25 mM) resulted in a significant up-regula-
tion of the expression level of the pro-apoptotic Bax protein by 6-
fold compared to untreated control, with a concomitant signifi-
cant decrease in the expression level of the anti-apoptotic Bcl-2
protein by approximately 75% compared to control (Table 5).
These results revealed that pyridine 5l significantly boosted the
Bax/Bcl-2 ratio 25-fold in compared to control.
Bax
Bcl-2
Comp.
Pg/mL
ng/mL
Bax/Bcl-2 ratio
ꢃ
ꢃ
5l
Control
256.7
41.9
1.24
5.11
207
8.2
Data are represented as mean SD of three separate experiments.
ꢃ
Significantly different from control at p < .05.
Table 6. Impact of pyridine 5l on the expression levels of cytochrome C, p53,
active caspases-3 and -9, in HCT-116 cancer cells treated with the compound at
its IC50 concentration.
Effect on the level of cytochrome C
Cyt-c
Pg/mL
p53
Pg/mL
Caspase-9
ng/mL
Caspase-3
Pg/mL
The interplay between the pro-apoptotic Bax and anti-apoptotic
Bcl-2 proteins triggers the activated Bax to bind to the mitochon-
drial outer membrane which induces the opening of the mito-
chondrial voltage-dependent anion channel (VDAC), resulting in
the release of cytochrome C from mitochondria into cytosol where
it activates the caspase-dependent signaling and subsequent
apoptosis. Involvement of cytochrome C release from mitochon-
dria is an indicator of activation of the intrinsic apop-
totic pathway42.
Herein, we assessed the expression level of cytochrome C to
assure the adoption of the intrinsic pathway. As shown in Table 6,
the level of cytochrome C was induced significantly higher (12-
folds) in HCT-116 cells treated with pyridine 5l, compared to
Comp.
ꢃ
ꢃ
ꢃ
ꢃ
5l
Control
858
67
961.2
44.3
21.3
458.4
46.8
2.34
Data are mean SD of three separate experiments.
Significantly different from control at p < 0.05.
ꢃ
3.22 0.2 and 2.71 0.16 mM, respectively, which are comparable
to those of Doxorubicin: 2.93 0.28and 3.10 0.22, respectively.
Regarding activity against A549 cells, pyridines 5a, 5c, 5f, 5g,
5i and 5j displayed potent antitumor activity with IC50 values in
the range of 6.72–9.61 lM, whereas the remaining tested pyridines
exhibited moderate potency towards A549 cell line (IC50 range:
10.64–24.05 lM). On the other hand, investigation of the anti-pro- untreated control (Table 6).
liferative activity against HCT-116 cell line elucidated that 5a, 5e,
5 h and 5j had potent anti-proliferative activity with IC50 values
equal 5.49 0.30, 7.05 0.72, 8.25 0.84 and 9.38 0.67 lM,
respectively. Furthermore, pyridines 5b, 5d, 5f and 5k were mod-
erately active towards HCT-116 cells with IC50 values of
16.03 1.52, 10.37 0.84, 12.61 1.08 and 16.43 1.30 lM,
respectively.
Effect on the level of p53
One of the major apoptosis signaling pathways involves the p53
tumor suppressor. The ability of p53 to control apoptosis in
response to abnormal proliferative signals and stress is crucial for
its tumor suppression role. p53 tumor suppressor protein is a
nuclear transcription factor that regulates the expression of a
wide variety of genes involved in apoptosis. p53 is able to induce
Bax oligomerization and cytochrome
c
release from
In vitro cytotoxicity towards non-tumorigenic human WI-38 cells
The cytotoxic activity of all synthesized pyridines 5a–l were
assessed against non-tumorigenic human lung fibroblast WI-38
cell line to investigate their safety, using the MTT colorimetric
assay29. The results were expressed as IC50 values and the calcu-
lated selectivity index are presented in Table 4.
mitochondria43.
The effect of pyridine 5l on p53 expression in HCT-116 cells
was evaluated in this study. Results in Table 5 highlighted that
treatment of HCT-116 cells with pyridine 5l led to 21-fold
enhanced expression levels of p53, compared to control (Table 6).
The examined pyridines 5a–l displayed non-significant cyto-
toxic impact towards human lung fibroblast WI-38 cell line with
IC50 values spanning from 63.48 to 151.08 mM, thereby providing a
good safety profile as anticancer agents with selectivity index
range (6.3–17.6).
Effects on the levels of active caspase-3 and caspase-9
Caspases, cysteine-dependent aspartate-directed proteases, are
key factors in apoptotic cell death that have been shown to play
an important role in cleavage of vital structural and regulatory

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
329
ꢃ
Figure 3. Effect of compound 5l on the phases of cell cycle of HCT-116 cells. Significantly different from control at p < 0.05. (Two-way ANOVA test).
proteins important for cells survival, so activation of caspases is a considered significant remarks for pyridine 5l to induce apoptosis
hallmark for apoptosis induction44. The leading upstream caspases in HCT-116 cells.
are caspase-9 in the intrinsic pathway and caspase-8 in the extrin-
sic pathway, where both converge to caspase-3 which is the key
executioner of apoptosis45.
AnnexinV-FITC/propidium iodide analysis of apoptosis
Translocation of phosphatidylserine (PS) from the inner to the outer
membrane leaflet of the cell is an early apoptotic event, which could
In comparison with the untreated control, the expression levels
of active caspase-3 and caspases-9 in HCT-116 cells were 5.1- and
2.5-fold increased, respectively, in response to pyridine 5l treat-
ment with its IC50 concentration (Table 6).
be detected by fluorescein-labeled annexinV (annexinV-FITC), a Ca2þ
-
dependent phospholipid-binding protein with high affinity for PS.
Combined with propidium iodide PI (an indicator of cell integrity), a
measure of percentage cell population in early apoptosis can be
achieved. Cells displaying increased annexinV-FITC fluorescence with-
out a concurrent increase in PI fluorescence are considered to be in
early apoptosis, whereas an increase is seen in both fluorescence
channels, signifies a late apoptosis47.
Cell cycle analysis
Targeting the cell cycle of cancer cells has emerged as a promis-
ing approach for cancer therapy46. In the current study, pyridine
5l was examined for its effect on the cell cycle distribution in
HCT-116 cells (Figure 3). The results of the DNA flow cytometric
In this study, AnnexinV-FITC/PI dual staining assay was per-
assay showed that treatment of HCT-116 cells with pyridine 5l at formed to evaluate the effect of compound 5l on both early and
its IC50 concentration for 24 h resulted in a significant 7.3-fold late apoptosis percentages in HCT-116 cells (Figure 4, Table 7). As
increased percentage of HCT-116 cells at Sub-G1, with concurrent presented in Figure 4, the assay outcomes clearly indicate that the
significant reduction in the G2-M phase by approximately 2.2-fold. treatment of HCT-116 cells with 5l resulted in a significant
Both arrest of G2-M phase and alteration of the Sub-G1 phase are increase in the percentage of annexinV-FITC-positive apoptotic

330
W. M. ELDEHNA ET AL.
Figure 4. Effect of sulfonamide 5l on the percentage of annexin V-FITC-positive staining in HCT-116 cells. The experiments were done in triplicates. The four quadrants
identified as: LL: viable; LR: early apoptotic; UR: late apoptotic; UL: necrotic.
0.81% to 8.97%) that represent about eightfold total increase in
comparison with control.
Table 7. Distribution of apoptotic cells in the annexin V-FITC experiment.
Early Apoptosis
(Lower Right %)
Late Apoptosis
(Upper Right %)
Total
Comp.
(L.R % þ U.R %)
5l
6.79
1.18
8.97
0.81
15.76
1.99
Control
Disclosure statement
cells, including both the early and late apoptotic phases (LR; from
1.18% to 6.79%, and UR; from 0.81% to 8.97%), that represents
about eightfold total increase in comparison with control
(Table 7).
No potential conflict of interest was reported by the authors.
Funding
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for its
funding of this research through the Research Group Project no.
[RG-1439–65].
Conclusion
In summary, herein we report the synthesis of novel series of 1-(2-
methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. All the prepared pyr-
idins were evaluated for their in vitro anticancer activity against
two cancer cell lines: non-small cell lung cancer A549 cell line and
colon cancer HCT-116 cell line. Compound 5l was found to be the
most active congener towards both A549 and HCT-116 cell lines
with IC50 values equal to 3.22 0.2 and 2.71 0.16 mM, respect-
ively, which are comparable with those of Doxorubicin: 2.93 0.28
and 3.10 0.22, respectively. Furthermore, compound 5l stood out
as the most potent pyridine derivative (mean % GI ¼ 40), at US-
NCI Developmental Therapeutic Program anticancer assay, with
broad-spectrum antitumor activity against the most tested cancer
cell lines from all subpanels. The ability of sulfonamide 5l to pro-
voke apoptosis in HCT-116 cells was evaluated. Results revealed
that pyridine 5l significantly boosted the Bax/Bcl-2 ratio 25-fold
compared to control. Also, the expression levels of cytochrome C,
p53, active caspase-3 and caspases-9 in HCT-116 cells were 12-,
21-, 5.1- and 2.5-fold increased, respectively, in response to pyri-
dine 5l treatment. Furthermore, treatment of HCT-116 cells with
pyridine 5l at its IC50 concentration resulted in a significant 7.3-
fold increased percentage of HCT-116 cells at Sub-G1, with concur-
rent significant reduction in the G2-M phase by approximately
2.2-fold, in addition to a significant increase in the percentage of
annexinV-FITC-positive apoptotic cells, including both the early
and late apoptotic phases (LR; from 1.18% to 6.79%, and UR; from
ORCID
Ahmed E. Altyar
http://orcid.org/0000-0001-6210-3628
Abdulrahman A. Almehizia
8711-3873
http://orcid.org/0000-0001-
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