Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents

Article abstract of DOI:10.1080/14756366.2017.1279155

Treatment of patients with triple-negative breast cancer (TNBC) is challenging due to the absence of well- defined molecular targets and the heterogeneity of such disease. In our endeavor to develop potent isatin-based anti-proliferative agents, we utiliz

Full text of DOI:10.1080/14756366.2017.1279155

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and in vitro anti-proliferative activity of
some novel isatins conjugated with quinazoline/
phthalazine hydrazines against triple-negative
breast cancer MDA-MB-231 cells as apoptosis-
inducing agents
Wagdy M. Eldehna, Hadia Almahli, Ghada H. Al-Ansary, Hazem A. Ghabbour,
Mohamed H. Aly, Omnia E. Ismael, Abdullah Al-Dhfyan & Hatem A. Abdel-
Aziz
To cite this article: Wagdy M. Eldehna, Hadia Almahli, Ghada H. Al-Ansary, Hazem A.
Ghabbour, Mohamed H. Aly, Omnia E. Ismael, Abdullah Al-Dhfyan & Hatem A. Abdel-Aziz (2017)
Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/
phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-
inducing agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 600-613, DOI:
10.1080/14756366.2017.1279155
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1279155
© 2017 The Author(s). Published by Informa
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Group.
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Date: 13 February 2017, At: 15:21

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 600–613
http://dx.doi.org/10.1080/14756366.2017.1279155
RESEARCH ARTICLE
Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated
with quinazoline/phthalazine hydrazines against triple-negative breast cancer
MDA-MB-231 cells as apoptosis-inducing agents
c
d
e,f
Wagdy M. Eldehnaa, Hadia Almahlia,b , Ghada H. Al-Ansary , Hazem A. Ghabbour †, Mohamed H. Aly ,
ꢀ
ꢀ
Omnia E. Ismaelg, Abdullah Al-Dhfyanh and Hatem A. Abdel-Azizi
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt; Department of Chemistry,
c
Faculty of Pharmacy, University of Oxford, Oxford, UK; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University,
d
e
Cairo, Abbassia, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department
f
of Pharmacology and Toxicology, Faculty of Pharmacy, British University in Egypt, Cairo, Egypt; Department of Biology, The American University
in Cairo, New Cairo, Egypt; Department of Biochemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt; Stem Cell &
Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, Riyadh, Saudi Arabia; Department of
g
h
i
Applied Organic Chemistry, National Research Center, Dokki, Giza, Egypt
ABSTRACT
ARTICLE HISTORY
Received 10 August 2016
Revised 23 December 2016
Accepted 1 January 2017
Treatment of patients with triple-negative breast cancer (TNBC) is challenging due to the absence of
well- defined molecular targets and the heterogeneity of such disease. In our endeavor to develop potent
isatin-based anti-proliferative agents, we utilized the hybrid-pharmacophore approach to synthesize three
series of novel isatin-based hybrids 5a–h, 10a–h and 13a–c, with the prime goal of developing potent
anti-proliferative agents toward TNBC MDA-MB-231 cell line. In particular, compounds 5e and 10g were
the most active hybrids against MDA-MB-231 cells (IC50 ¼ 12.35 0.12 and 12.00 0.13 lM), with 2.37- and
2.44-fold increased activity than 5-fluorouracil (5-FU) (IC50 ¼ 29.38 1.24 lM). Compounds 5e and 10g
induced the intrinsic apoptotic mitochondrial pathway in MDA-MB-231; evidenced by the reduced expres-
sion of the anti-apoptotic protein Bcl-2, the enhanced expression of the pro-apoptotic protein Bax and the
up-regulated active caspase-9 and caspase-3 levels. Furthermore, 10g showed significant increase in the
percent of annexin V-FITC positive apoptotic cells from 3.88 to 31.21% (8.4 folds compared to control).
KEYWORDS
Isatin; quinazoline;
phthalazine; synthesis;
triple-negative breast
cancer
Introduction
patients largely receive systemic chemotherapy that puts them
under higher risk of suffering the devastating side effects.
This urgent necessity motivates medicinal chemists to search for
alternative treatments targeting apoptotic machinery as a novel
approach for TNBC therapy.
Heading the list of the critical health problems for females, breast
cancer continues to be the major life-threatening health issue.
According to the recent records, an estimated 1.7 million women
are expected to be diagnosed with breast cancer by 2020 with a
make-up of 26% increase from current recorded levels1,2. Despite
the huge and continuous efforts of the scientific community, no
tangible improvements were recorded in the last decade. With the
development of resistance toward the available anticancer drugs,
the mission becomes even more challenging and the efficacious
Targeting critical regulators of apoptosis with the goal of induc-
ing apoptosis in cancer cells still stands as an attractive and
successful strategy to drug discovery and development of new
anti-cancer agents7–11. On April 11, 2016, the U.S. food and drug
R
administration (FDA) approved Venetoclax (VenclextaV) for the
therapy remains questionable3,4
.
treatment of patients with chronic lymphocytic leukemia (CLL)
whose tumors have a specific genetic alteration. Venclexta is the
first FDA-approved drug that targets the BcL-2 protein, an anti-
apoptotic regulatory protein12.
Molecular hybridization is a contemporary concept in drug
design and development gaining momentum worldwide. There
are two main ways that can be explored to design and construct
affordable and efficient hybrid molecules. The first method merges
haptophoric moieties of different drugs and the second technique
combines two or more entire drugs together using a linker
Targeting breast cancer with tailored drugs depends on the
fact of immunohistochemical expression of estrogen receptors
(ERs), progesterone receptors (PRs) and human epidermal growth
factor receptors-2 (HER-2). Being devoid of the three aforemen-
tioned targetable proteins, triple-negative breast cancer (TNBC)
stands even as an unbeatable challenge5. Besides their well-known
resistance to the current chemotherapy, TNBC is an aggressive
phenotype of cancer with high recurrence risk, poor prognosis,
reduced survival and distant metastasis that it may reach the
lungs and even the central nervous system (CNS)5,6. Accordingly, chain13,14. This strategy affords novel molecule hybrids with
CONTACT Wagdy M. Eldehna
wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh,
Egypt
ꢀ
These authors contributed equally to this work.
†Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
Supplemental data for this article can be accessed here.
ß 2017 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, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
601
O
N
N
N
O
HO
O
N
NH
O
NH
F
O
N
H
HO
HN
S
N
N
O
O
O
O
N
H
N
H
N
H
O
Raltitrexed III
Sunitinib I
Nintedanib II
N
O
F
F
NH
O
N
O
O
HN
Cl
HN
N
Cl
N
O
O
HN
O
N
N
N
F
O
N
N
O
Olaparib VI
Gefitinib V
Figure 1. Structures of some isatin-, quinazoline- and phthalazine-based I–VI approved anticancer drugs.
Afatinib IV
improved affinity and efficacy when compared to the parent MB-231 cell line. Figure 2 displays some of these derivatives with
drugs, modified selectivity profile, different and/or dual modes of their IC50 values or growth inhibition (GI) percentage against
action and reduced undesired side effects. Thus, hybrid molecules MDA-MB-231 cells.
In light of the above findings and as a part of our ongoing
emerged as magic bullets that trigger two or more cytocidal
pharmacological mechanisms of action acting in synergy to inhibit
effort to develop potent isatin-based anti-proliferative agents39–45
,
cancer tumor growth15–17
.
we utilized the hybrid pharmacophore approach to design and
synthesize three different series of novel isatin-based hybrids
5a–h, 10a–h and 13a–c (Figure 2), via merging the pharmaco-
phoric elements of isatin and phthalazine or quinazoline in a sin-
gle chemical framework through a hydrazine linker, with the
prime goal of developing potent anti-proliferative agents against
the TNBC MDA-MB-231 cell line. The three synthesized series were
in vitro evaluated for their potential anti-proliferative activity
against TNBC MDA-MB-231 cell line. Compounds 5e and 10g were
further estimated for their apoptosis induction potential in MDA-
MB-231 cells, to gain mechanistic insights into the anti-prolifera-
tive activity of the prepared hybrids. Furthermore, the activities
against A549 alveolar carcinoma, Caco-2 colon cancer, LoVo
human colorectal carcinoma and HepG2 hepatocellular carcinoma
were in vitro examined.
Pertaining to its wide presence endogenously in both human
and other mammalian tissues and fluids besides its prevalence in
many naturally occurring compounds, notably alkaloids, fungal
metabolites and marine natural products, isatin (1H-indole-2,3-
dione) as a privileged scaffold is endowed with excellent anti-
cancer profile18. Thus, medicinal chemists embarked on exploring
diverse isatin derivatives comprehending their potential to create
novel bioactive compounds. By 2006, the FDA approval of
R
Sunitinib (SutentV) I (Figure 1) for the treatment of advanced renal
carcinoma and gastrointestinal stromal tumors furnished the path
for the creation of new isatin derivatives as drug targets19.
R
Nintedanib (OfevV) II (Figure 1), an orally available triple angioki-
nase inhibitor, received its first global approval in the US in
October 2014 for the treatment of idiopathic pulmonary fibrosis
(IPF)20. By 2015, Nintedanib II has been approved by the European
medicines agency (EMA) in Europe, as a second-line treatment in
combination with docetaxel of locally advanced, metastatic or
locally recurrent non-small cell lung cancer of adenocarcinoma20.
On the other hand, quinazolines constitute a leading class of
heterocycles that displayed interesting biological activities, chiefly
Materials and methods
Chemistry
Melting points were measured with a Stuart melting point appar-
atus (Bibby Scientific Limited, Staffordshire, UK) and were uncor-
rected. Infrared spectra were recorded as potassium bromide discs
on Schimadzu FT-IR 8400S spectrophotometer (Shimadzu Scientific
Instruments, Kyoto, Japan) and expressed in wave number (cmꢁ1).
R
R
anticancer activity. Raltitrexed (TomudexV) III, Afatinib (GilotrifV) IV
R
and Gefitinib (IressaV) V (Figure 1) are examples for the clinically
approved quinazoline-based anticancer drugs21. Besides, phthala-
zine nucleus has emerged as a promising and attractive one in
The NMR spectra were recorded on Varian Gemini-300BB 300 MHz
the development of novel anticancer agents22–24
.
Olaparib FT-NMR spectrometers (Varian Inc., Palo Alto, CA). H and 13C spec-
1
R
(LynparzaV) VI (Figure 1) is an oral small molecule phthalazine- tra were run at 300 and 75 MHz, respectively, in deuterated
based poly ADP-ribose polymerase (PARP) inhibitor being devel- dimethyl sulfoxide (DMSO-d6). Chemical shifts (dH) are reported
oped for the treatment of solid tumors. In December 2014, relative to tetramethylsilane (TMS) as internal standard. All cou-
Olaparib was approved in the EU and USA for the treatment of pling constant (J) values are given in hertz. Chemical shifts (dC) are
BRCA-mutated ovarian cancer25.
reported relative to DMSO-d6 as internal standards. The abbrevia-
tions used are as follows: s, singlet; d, doublet; m, multiplet.
Surveying the literature revealed that different isatin26–30
,
2-phenylquinazoline31–35 and 4-arylphthalazine23,36–38 derivatives Elemental analyses were carried out at the Regional Center for
were developed with significant activity against the TNBC MDA- Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.

602
W. M. ELDEHNA ET AL.
Ph
N
O2N
O
N
O
N
HN
N
S
S
N
S
O
S
OH
N
N
N
O
N
Br
Br
O
O
O
O
N
N
N
N
H
H
H
N
VII IC
50 (µM) = 0.3
VIII IC50 (µM) = 2.52
IX GI (%)
= 70.36
X IC50 (µM) = 19.15
F
NH
H
N
O
HN
N
O
N
O
Cl
O
N
Cl
N
N
N
Cl
O
N
N
N
H
IXV IC
50 (µM) = 4.21
XI IC50 (µM) = 9.91
XII IC50 (µM) = 3.1
XIII IC50 (µM) = 4.8
O
O
O
O
N
N
N
H
O
N
N
NH
N
O
N
O
N
N
N
O
XV IC
XVI IC50 (µM) = 8.5
50 (µM) = 4.1
XVII IC50 (µM) = 1.65
HN
R1
R1
O
HN
N
R1
N
O
O
HN
N
N
N
N
N
HN
NH
N
N
N
R
R
5a-h
13a-c
R
10a-h
Figure 2. Structures of some reported isatins, quinazolines and phthalazines VII–XVII with anti-proliferative activity against the triple-negative breast cancer MDA-
MB-231 cells, and structures of the target hybrids 5a–h, 10a–h and 13a–c.
Reaction courses and product mixtures were routinely monitored General procedure for preparation of 4-chloroquinazolines 2a,b
by thin layer chromatography (TLC) on silica gel precoated F254
Merck plates (Merck Group, Darmstadt, Germany). Unless other-
wise noted, all solvents and reagents were commercially available
and used without further purification.
To a cooled stirred mixture of phosphorus oxychloride (30 mL) and
N,N-dimethylaniline (1 mL), 2-Substituted-quinazolin-4(3H)-ones
1a,b (10 mmol) were added portion-wise. After refluxing for 6 h,
the reaction mixture was cooled then poured onto ice-water and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
603
alkalinized with 2 N NaOH. The aqueous solution was extracted NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d: 112.68,
with methylene chloride. The combined organic layer was dried 119.95, 120.10, 121.67, 123.47, 126.75, 127.50, 128.49, 128.68,
over anhydrous Na2SO4 then filtered, and the solvent was 129.72, 134.29, 135.51, 136.11, 140.41, 151.58, 156.02, 157.90,
removed under reduced pressure. The obtained solid was crystal- 163.08 (C ¼ O); Anal. Calcd. for C22H13Cl2N5O: C, 60.85; H, 3.02; N,
lized from isopropanol to afford compounds 2a,b46.
16.13; Found C, 60.97; H, 2.98; N, 16.05.
5-Bromo-3–(2–(2–(4-chlorophenyl)quinazolin-4-yl)hydrazono)indo-
lin-2-one (5d). Orange powder (yield 77%), m.p. >300 ꢂC; IR
(KBr, m cmꢁ1): 3245 (NH) and 1679 (C ¼ O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 6.94 (d, 1H, Ar–H, J ¼ 8.1 Hz), 7.54 (dd, 1H, Ar–H,
J ¼ 2.1, 8.1 Hz), 7.61 (d, 2H, H-3 and H-5 of 4-Cl-C6H4, J ¼ 8.7 Hz),
7.69–7.79 (m 2H, Ar–H), 7.87 (s, 1H, Ar–H), 7.98 (d, 2H, Ar–H,
J ¼ 3.9 Hz), 8.56 (d, 2H, H-2 and H-6 of 4-Cl-C6H4, J ¼ 8.7 Hz), 11.49
(s, 1H, NH, D2O exchangeable), 13.88 (s, 1H, NH, D2O exchange-
able); Anal. Calcd. for C22H13BrClN5O: C, 55.20; H, 2.74; N, 14.63;
Found C, 55.33; H, 2.71; N, 14.54.
General procedure for preparation of 4-hydrazinyl-2-substituted
quinazoline 3a,b
To a magnetically stirred solution of 4-chloroquinazolines 2a,b
(5 mmol) in ethanol (15 mL), hydrazine hydrate 99% (2.5 mL,
50 mmol) was added. The stirring was continued at refluxing tem-
perature for 4 h. Upon cooling, the obtained precipitate was fil-
tered, washed with methanol and water, dried and recrystallized
from ethanol to furnish compounds 3a,b.
2–(2,6-Dichlorophenyl)-4-hydrazinylquinazoline (3b). White crys-
3–(2–(2–(2,6-Dichlorophenyl)quinazolin-4-yl)hydrazono)indolin-2-
tals (yield 86%), m.p. 237–239 ꢂC; IR (KBr, m cmꢁ1): 3295 (NH, NH2)
one (5e). Orange powder (yield 65%), m.p. > 300 ꢂC; IR (KBr, m
1
cmꢁ1; H NMR (DSMO-d6) d ppm: 4.77 (s, 2H, NH2, D2O exchange-
1
cmꢁ1): 3170 (NH) and 1683 (C ¼ O); H NMR (300 MHz, DMSO-d6) d
able), 7.51–7.54 (m, 3H, Ar–H), 7.69–7.87 (m, 3H, Ar–H), 8.21 (d, 1H,
Ar–H, J ¼ 8.1 Hz), 9.77 (s, 1H, NH, D2O exchangeable); Anal. Calcd.
for C14H10Cl2N4:C, 55.10; H, 3.30; N, 18.36; Found C, 54.89; H, 3.33;
N, 18.41.
ppm: 6.88 (d, 1H, Ar–H, J ¼ 7.5 Hz), 7.08 (t, 1H, Ar–H, J ¼ 7.5 Hz),
7.31 (t, 1H, Ar–H, J ¼ 7.5 Hz), 7.58–7.84 (m, 5H, Ar–H), 8.01 (s, 1H,
Ar–H), 8.42 (d, 2H, Ar–H, J ¼ 7.5 Hz), 10.62, 11.38 (s, 1H, NH, D2O
exchangeable), 12.11, 13.93 (s, 1H, NH, D2O exchangeable); Anal.
Calcd. for C22H13Cl2N5O: C, 60.85; H, 3.02; N, 16.13; Found 60.97; H,
2.99; N, 16.03.
General procedure for preparation of the target
isatin-quinazoline hybrids 5a–h
3–(2–(2–(2,6-Dichlorophenyl)quinazolin-4-yl)hydrazono)-5-fluoroin-
dolin-2-one (5f). Orange powder (yield 70%), m.p. > 300 ꢂC; IR (KBr,
1
m cmꢁ1): 3185 (NH) and 1675 (C ¼ O); H NMR (300 MHz, DMSO-d6)
To a stirred solution of hydrazine derivatives 3a,b (1 mmol), in
absolute ethyl alcohol, the appropriate isatin 4a–d (1 mmol) and
catalytic amount of glacial acetic acid were added. The mixture
was heated under reflux for 0.5 h, filtered while hot. The obtained
solid was washed with ethanol collected and crystallized from an
ethanol/DMF mixture to obtain hybrids 5a–h.
3–(2–(2–(4-Chlorophenyl)quinazolin-4-yl)hydrazono)indolin-2-one
(5a). Orange powder (yield 75%), m.p. 291–292 ꢂC; IR (KBr, m cmꢁ1):
3210 (NH) and 1675 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm:
6.98 (d, 1H, Ar–H, J ¼ 7.8 Hz), 7.14 (t, 1H, Ar–H, J ¼ 7.5 Hz), 7.38 (t, 1H,
Ar–H, J ¼ 7.8 Hz), 7.61 (d, 2H, H-3 and H-5 of 4-Cl-C6H4, J ¼ 8.4 Hz),
7.69–7.76 (m, 3H, Ar–H), 7.98–8.00 (m, 2H, Ar–H), 8.54 (d, 2H, H-2
and H-6 of 4-Cl-C6H4, J ¼ 8.7 Hz), 11.38 (s, 1H, NH, D2O exchange-
able), 13.89 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz,
DMSO-d6) d: 111.23, 112.70, 120.00, 120.62, 122.63, 123.37,
127.46, 128.49, 128.66, 129.72, 131.19, 134.11, 135.48, 136.24,
141.83, 151.51, 156.15, 157.99, 163.29 (C ¼ O); Anal. Calcd. for
C22H14ClN5O: C, 66.09; H, 3.53; N, 17.52; Found C, 66.21; H, 3.49; N,
17.45.
d ppm: 6.85 (dd, 1H, Ar–H, J ¼ 4.5, 8.7 Hz), 7.20 (t, 1H, Ar–H,
J ¼ 8.7 Hz), 7.55–7.87 (m, 6H, Ar–H), 8.03 (s, 1H, Ar–H), 8.45 (d, 1H,
Ar–H, J ¼ 8.4 Hz), 10.65, 11.39 (s, 1H, NH, D2O exchangeable),
12.25, 13.91 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz,
DMSO-d6) d: 112.37, 117.99, 121.07, 123.75, 127.34, 128.35, 129.42,
132.76, 134.26, 135.28, 136.92, 138.10, 139.76, 146.37, 151.24,
155.92, 156.80, 158.99, 159.42, 159.95, 163.40, 165.44 (C ¼ O); Anal.
Calcd. for C22H12Cl2FN5O: C, 58.43; H, 2.67; N, 15.49; Found C,
58.29; H, 2.71; N, 15.58.
5-Chloro-3–(2–(2–(2,6-dichlorophenyl)quinazolin-4-yl)hydrazono)in-
dolin-2-one (5g). Orange powder (yield 75%), m.p. >300 ꢂC; IR (KBr,
1
m cmꢁ1): 3203 (NH) and 1688 (C ¼ O); H NMR (300 MHz, DMSO-d6)
d ppm: 6.89 (d, 1H, Ar–H, J ¼ 8.4 Hz), 7.37 (t, 1H, Ar–H, J ¼ 8.7 Hz),
7.58 (d, 1H, Ar–H, J ¼ 8.4 Hz), 7.72–7.88 (m, 5H, Ar–H), 8.01 (s, 1H,
Ar–H), 8.39 (d, 1H, Ar–H, J ¼ 8.4 Hz), 10.75, 11.48 (s, 1H, NH, D2O
exchangeable), 12.37, 13.85 (s, 1H, NH, D2O exchangeable); Anal.
Calcd. for C22H12Cl3N5O: C, 56.37; H, 2.58; N, 14.94; Found C, 56.51;
H, 2.53; N, 14.82.
3–(2–(2–(4-Chlorophenyl)quinazolin-4-yl)hydrazono)-5-fluoroindo-
5-Bromo-3–(2–(2–(2,6-dichlorophenyl)quinazolin-4-yl)hydrazono)in-
lin-2-one (5b). Orange powder (yield 77%), m.p. >300 ꢂC; IR (KBr,
dolin-2-one (5h). Orange powder (yield 72%), m.p. >300 ꢂC; IR (KBr,
1
m cmꢁ1): 3190 (NH) and 1690 (C ¼ O); H NMR (300 MHz, DMSO-d6)
1
m cmꢁ1): 3305 (NH) and 1670 (C ¼ O); H NMR (300 MHz, DMSO-d6)
d ppm: 6.95 (dd, 1H, Ar–H, J ¼ 4.2, 8.7 Hz), 7.19 (t, 1H, Ar–H,
J ¼ 8.7 Hz), 7.59–7.63 (m, 3H, Ar–H), 7.71–7.77 (m, 2H, Ar–H), 7.96
(d, 2H, Ar–H, J ¼ 7.8 Hz), 8.53 (d, 2H, H-2 and H-6 of 4-Cl-C6H4,
J ¼ 8.4 Hz), 11.39 (s, 1H, NH, D2O exchangeable), 13.86 (s, 1H, NH,
D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d: 107.93, 112.72,
116.75, 121.22, 127.43, 128.54, 129.78, 133.67, 134.29, 135.54,
136.16, 137.95, 138.07, 151.61, 156.13, 157.95, 163.44, 167.34
(C ¼ O); Anal. Calcd. for C22H13ClFN5O: C, 63.24; H, 3.14; N, 16.76;
Found C, 63.39; H, 3.11; N, 16.68.
d ppm: 6.84 (d, 1H, Ar–H, J ¼ 8.7 Hz), 7.49–8.02 (m, 7H, Ar–H), 8.36
(d, 1H, Ar–H, J ¼ 8.4 Hz), 8.53 (s, 1H, Ar–H), 10.76, 11.48 (s, 1H, NH,
D2O exchangeable), 12.15, 13.85 (s, 1H, NH, D2O exchangeable);
Anal. Calcd. for C22H12BrCl2N5O: C, 51.49; H, 2.36; N, 13.65; Found
C, 51.30; H, 2.39; N, 13.71.
1-Chloro-4-phenylphthalazines 8a,b
Compounds 8a,b were prepared according to the literature
procedure47,48
.
5-Chloro-3–(2–(2–(4-chlorophenyl)quinazolin-4-yl)hydrazono)indo-
lin-2-one (5c). Orange powder (yield 70%), m.p. > 300 ꢂC; IR (KBr, m
1
cmꢁ1): 3285 (NH) and 1672 (C ¼ O); H NMR (300 MHz, DMSO-d6) d
General procedure for preparation of 1-hydrazinyl-4-phenylphtha-
lazines 9a,b
ppm: 6.98 (d, 1H, Ar–H, J ¼ 8.4 Hz), 7.41 (d, 1H, Ar–H, J ¼ 8.4 Hz),
7.61 (d, 2H, H-3 and H-5 of 4-Cl-C6H4, J ¼ 8.4 Hz), 7.69–7.77 (m 3H,
Ar–H), 7.97 (d, 2H, Ar, J ¼ 7.8 Hz), 8.53 (d, 2H, H-2 and H-6 of 4-Cl-
C6H4, J ¼ 8.7 Hz), 11.49 (s, 1H, NH, D2O exchangeable), 13.86 (s, 1H,
Compounds 9a,b were prepared according to the literature
procedure49
.

604
W. M. ELDEHNA ET AL.
General procedure for preparation of the target isatin- (s, 1H, NH, D2O exchangeable); Anal. Calcd. for C22H14ClN5O: C,
66.09; H, 3.53; N, 17.52; Found C, 65.89; H, 3.58; N, 17.61.
3–(2–(4–(4-Chlorophenyl)phthalazin-1-yl)hydrazono)-5-fluoroindo-
lin-2-one (10f). Orange powder (yield 73%), m.p. >300 ꢂC; IR (KBr, m
phthalazine hybrids (10a–h)
Hydrazino phthalazines 9a,b (1 mmol) and catalytic amount of gla-
cial acetic acid were added to a stirred solution of indoline-
2,3-dione 4a–d (1 mmol) in refluxing absolute ethyl alcohol, then
the reaction mixture was refluxed for 1 h. The precipitated solid
was filtered while hot, dried and recrystallized from ethanol/DMF
mixture to afford the target hybrids 10a–h.
1
cmꢁ1): 3247 (NH) and 1670 (C ¼ O); H NMR (300 MHz, DMSO-d6) d
ppm: 6.86 (dd, 1H, H-6 isatin, J ¼ 4.2, 8.1 Hz), 7.13 (t, 1H, H-7 isatin,
J ¼ 8.1 Hz), 7.64–7.66 (m, 4H, H-2, H-3, H-5 & H-6 of 4-Cl-C6H5), 7.70
(d, 1H, H-8 phthalazine, J ¼ 8.1 Hz), 7.92 (t, 1H, H-6 phthalazine,
J ¼ 7.8 Hz), 8.01 (t, 1H, H-7 phthalazine, J ¼ 7.8 Hz), 8.20 (dd, 1H, H-
4 isatin, J ¼ 3.0, 8.4 Hz), 8.66 (d, 1H, H-5 phthalazine, J ¼ 7.8 Hz),
10.58 (s, 1H, NH, D2O exchangeable), 13.05 (s, 1H, NH, D2O
exchangeable); Anal. Calcd. for C22H13ClFN5O: C, 63.24; H, 3.14; N,
16.76; Found C, 63.08; H, 3.09; N, 16.82.
3–(2–(4-Phenylphthalazin-1-yl)hydrazono)indolin-2-one
(10a).
Orange powder (yield 65%), m.p. > 300 ꢂC; IR (KBr, m cmꢁ1): 3183
(NH) and 1677 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm: 6.87
(d, 1H, H-7 isatin, J ¼ 7.8 Hz), 7.05 (t, 1H, H-5 isatin, J ¼ 7.5 Hz), 7.29
(t, 1H, H-6 isatin, J ¼ 7.5 Hz), 7.56–7.63 (m, 5H, H-2, H-3, H-4, H-5 &
H-6 of C6H5), 7.68 (d, 1H, H-8 phthalazine, J ¼ 8.1 Hz), 7.89 (t, 1H,
H-6 phthalazine, J ¼ 7.8 Hz), 7.96 (t, 1H, H-7 phthalazine, J ¼ 7.8 Hz),
8.48 (d, 1H, H-4 isatin, J ¼ 7.8 Hz), 8.68 (d, 1H, H-5 phthalazine,
J ¼ 7.8 Hz), 10.56 (s, 1H, NH, D2O exchangeable), 12.83 (s, 1H, NH,
D2O exchangeable); Anal. Calcd. for C22H15N5O: C, 72.32; H, 4.14;
N, 19.17; Found C, 72.44; H, 4.11; N, 19.12.
5-Chloro-3–(2–(4–(4-chlorophenyl)phthalazin-1-yl)hydrazono)indo-
lin-2-one (10g). Orange powder (yield 70%), m.p. >300 ꢂC; IR (KBr,
1
m cmꢁ1): 3195 (NH) and 1673 (C ¼ O); H NMR (300 MHz, DMSO-d6)
d ppm: 6.90 (d, 1H, H-6 isatin, J ¼ 8.1 Hz), 7.35 (d, 1H, H-7 isatin,
J ¼ 8.4 Hz), 7.66–7.69 (m, 4H, H-2, H-3, H-5 & H-6 of 4-Cl-C6H5), 7.70
(d, 1H, H-8 phthalazine, J ¼ 8.1 Hz), 7.92 (t, 1H, H-6 phthalazine,
J ¼ 8.1 Hz), 8.01 (t, 1H, H-7 phthalazine, J ¼ 7.8 Hz), 8.43 (s, 1H, H-4
isatin), 8.61 (d, 1H, H-5 phthalazine, J ¼ 7.8 Hz), 10.69 (s, 1H, NH,
D2O exchangeable), 13.12 (s, 1H, NH, D2O exchangeable); 13C NMR
(75 MHz, DMSO-d6) d: 111.44, 118.91, 125.29, 126.05, 126.35,
126.87, 128.75, 130.13, 131.32, 133.40, 133.84, 134.07, 141.45,
142.72, 148.30, 151.91, 165.62; Anal. Calcd. for C22H13Cl2N5O: C,
60.85; H, 3.02; N, 16.13; Found C, 61.03; H, 2.97; N, 16.06.
5-Fluoro-3–(2–(4-phenylphthalazin-1-yl)hydrazono)indolin-2-one
(10b). Orange powder (yield 60%), m.p. >300 ꢂC; IR (KBr, m cmꢁ1):
3234 (NH) and 1677 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm:
6.86 (dd, 1H, H-6 isatin, J ¼ 4.2, 8.7 Hz), 7.14 (t, 1H, H-7 isatin,
J ¼ 8.4 Hz), 7.57–7.64 (m, 5H, H-2, H-3, H-4, H-5 & H-6 of C6H5),
7.71 (d, 1H, H-8 phthalazine, J ¼ 7.8 Hz), 7.91 (t, 1H, H-6 phthala-
zine, J ¼ 7.8 Hz), 7.96 (t, 1H, H-7 phthalazine, J ¼ 7.8 Hz), 8.20 (dd,
1H, H-4 isatin, J ¼ 3.0, 8.4 Hz), 8.68 (d, 1H, H-5 phthalazine,
J ¼ 7.5 Hz), 10.58 (s, 1H, NH, D2O exchangeable), 13.04 (s, 1H, NH,
D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d: 109.52, 113.88,
118.19, 123.64, 126.08, 126.62, 127.00, 128.54, 129.37, 132.69,
133.78, 134.55, 139.06, 143.32, 149.31, 151.86, 155.86, 158.98,
165.87, 171.92; Anal. Calcd. for C22H14FN5O: C, 68.92; H, 3.68; N,
18.27; Found C, 68.77; H, 3.73; N, 18.33.
5-Chloro-3–(2–(4-phenylphthalazin-1-yl)hydrazono)indolin-2-one
(10c). Orange powder (yield 72%), m.p. >300 ꢂC; IR (KBr, m cmꢁ1):
3195 (NH) and 1679 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm:
6.89 (d, 1H, H-6 isatin, J ¼ 8.4 Hz), 7.34 (d, 1H, H-7 isatin, J ¼ 8.4 Hz),
7.57–7.64 (m, 5H, H-2, H-3, H-4, H-5 & H-6 of C6H5), 7.72 (d, 1H,
H-8 phthalazine, J ¼ 8.1 Hz), 7.92 (t, 1H, H-6 phthalazine, J ¼ 8.1 Hz),
8.02 (t, 1H, H-7 phthalazine, J ¼ 8.1 Hz), 8.45 (s, 1H, H-4 isatin), 8.62
(d, 1H, H-5 phthalazine, J ¼ 8.1 Hz), 10.68 (s, 1H, NH, D2O
exchangeable), 13.03 (s, 1H, NH, D2O exchangeable); Anal. Calcd.
for C22H14ClN5O: C, 66.09; H, 3.53; N, 17.52; Found C, 66.21; H,
3.49; N, 17.42.
5-Bromo-3–(2–(4–(4-chlorophenyl)phthalazin-1-yl)hydrazono)indo-
lin-2-one (10h). Yellow powder (yield 75%), m.p. >300 ꢂC; IR (KBr, m
1
cmꢁ1): 3210 (NH) and 1678 (C ¼ O); H NMR (300 MHz, DMSO-d6) d
ppm: 6.85 (d, 1H, H-6 isatin, J ¼ 8.1 Hz), 7.47 (d, 1H, H-7 isatin,
J ¼ 8.4 Hz), 7.66–7.69 (m, 4H, H-2, H-3, H-5 & H-6 of 4-Cl-C6H5), 7.71
(d, 1H, H-8 phthalazine, J ¼ 7.8 Hz), 7.92 (t, 1H, H-6 phthalazine,
J ¼ 8.1 Hz), 8.01 (t, 1H, H-7 phthalazine, J ¼ 7.8 Hz), 8.58–8.61 (m,
2H, H-4 isatin and H-5 phthalazine), 10.74 (s, 1H, NH, D2O
exchangeable), 13.17 (s, 1H, NH, D2O exchangeable); 13C NMR
(75 MHz, DMSO-d6) d: 111.83, 113.01, 119.40, 123.42, 125.07,
126.06, 126.89, 128.66, 129.11, 131.32, 132.69, 133.39, 133.85,
134.07, 141.80, 142.60, 148.30, 151.89, 165.49; Anal. Calcd. for
C22H13BrClN5O: C, 55.20; H, 2.74; N, 14.63; Found C, 55.36; H, 2.77;
N, 14.55.
N-Benzylindoline-2,3-diones 12a,b
Compounds 12a,b were prepared according to the literature
procedure45.
5-Bromo-3–(2–(4-phenylphthalazin-1-yl)hydrazono)indolin-2-one
(10d). Orange powder (yield 75%), m.p. >300 ꢂC; IR (KBr, m cmꢁ1):
3315 (NH) and 1672 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm: General procedure for preparation of the target hybrids (13a-c)
6.85 (d, 1H, H-6 isatin, J ¼ 8.4 Hz), 7.47 (d, 1H, H-7 isatin, J ¼ 8.1 Hz), Following the same procedures described for preparation of the
7.57–7.64 (m, 5H, H-2, H-3, H-4, H-5 & H-6 of C6H5), 7.72 (d, 1H, H- hybrids 5a–h, using N-benzylindoline-2,3-diones 7a,b instead of
8 phthalazine, J ¼ 8.4 Hz), 7.92 (t, 1H, H-6 phthalazine, J ¼ 8.1 Hz), indoline-2,3-dione 4a–d.
1-Benzyl-3–(2–(2–(4-chlorophenyl)quinazolin-4-yl)hydrazono)indo-
8.01 (t, 1H, H-7 phthalazine, J ¼ 8.1 Hz), 8.59–8.62 (m, 2H, H-4 isatin
lin-2-one (13a). Orange powder (yield 65%), m.p. 291–293 ꢂC; IR
and H-5 phthalazine), 10.70 (s, 1H, NH, D2O exchangeable), 13.06
(s, 1H, NH, D2O exchangeable); Anal. Calcd. for C22H14BrN5O: C,
59.47; H, 3.18; N, 15.76; Found C, 59.61; H, 3.14; N, 15.68.
(KBr, m cmꢁ1): 1681 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm:
5.00, 5.06 (s, 2H, –CH2–), 6.99 (d, 1H, Ar–H, J ¼ 7.8 Hz), 7.16 (t, 1H,
3–(2–(4–(4-Chlorophenyl)phthalazin-1-yl)hydrazono)indolin-2-one
Ar–H, J ¼ 7.5 Hz), 7.27–7.46 (m, 6H, Ar–H), 7.59–7.98 (m, 6H, Ar),
(10e). Yellow powder (yield 70%), m.p. >300 ꢂC; IR (KBr, m cmꢁ1): 8.10 (d, 1H, Ar–H, J ¼ 7.5 Hz), 8.47–8.57 (m, 2H, Ar–H), 11.42, 13.78
3218 (NH) and 1676 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm: (s, 1H, NH, D2O exchangeable); Anal. Calcd. for C29H20ClN5O: C,
6.87 (d, 1H, H-7 isatin, J ¼ 7.8 Hz), 7.05 (t, 1H, H-5 isatin, J ¼ 7.5 Hz), 71.09; H, 4.11; N, 14.29; Found C, 70.91; H, 4.16; N, 14.21.
7.29 (t, 1H, H-6 isatin, J ¼ 7.5 Hz), 7.60–7.65 (m, 4H, H-2, H-3, H-5 &
1-Benzyl-3–(2–(2–(2,6-dichlorophenyl)quinazolin-4-yl)hydrazono)in-
H-6 of 4-Cl-C6H5), 7.67 (d, 1H, H-8 phthalazine, J ¼ 7.8 Hz), 7.89 (t, dolin-2-one (13b). Orange powder (yield 70%), m.p. >300 ꢂC; IR
1H, H-6 phthalazine, J ¼ 7.8 Hz), 7.96 (t, 1H, H-7 phthalazine, (KBr, m cmꢁ1): 1674 (C ¼ O); 1H NMR (300 MHz, DMSO-d6) d ppm:
J ¼ 7.8 Hz), 8.47 (d, 1H, H-4 isatin, J ¼ 7.2 Hz), 8.68 (d, 1H, H-5 4.98, 5.07 (s, 2H, –CH2–), 6.98 (d, 1H, Ar–H, J ¼ 7.5 Hz), 7.10 (t, 1H,
phthalazine, J ¼ 7.5 Hz), 10.63 (s, 1H, NH, D2O exchangeable), 13.04 Ar–H, J ¼ 7.8 Hz), 7.25–7.37 (m, 5H, Ar–H), 7.42 (t, 1H, Ar–H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
605
J ¼ 7.8 Hz), 7.61 (d, 1H, Ar–H, J ¼ 7.8 Hz), 7.68–7.89 (m, 4H, Ar–H), overnight. The medium was replaced with fresh one containing
8.03 (d, 1H, Ar–H, J ¼ 8.4 Hz), 8.51–8.53 (m, 2H, Ar–H), 12.28, 13.87
the desired concentrations of the test compounds.
(s, 1H, NH, D2O exchangeable); Anal. Calcd. for C29H19Cl2N5O: C,
66.42; H, 3.65; N, 13.36; Found C, 66.57; H, 3.69; N, 13.28.
Anti-proliferative activity of the prepared hybrids against MDA-
MB-231 cells was evaluated by the cell GI assay using 5-
Fluorouracil (5-FU) as a standard treatment. This assay was con-
ducted by the use of WST-1 reagent for determination of IC50 for
each compound and the results are given in Table 1. After 48 h of
incubation with the synthesized agents in the treatment groups or
the vehicle (1% DMSO) in the control group, 10 lL of the WST-1
reagent were added to each well and the plates were re-incubated
for 4 h at 37 ꢂC. The amount of formazan was quantified using
ELISA reader at 450 nm. Moreover, cytotoxicity of the compounds
and the reference drug, doxorubicin in LoVo, HepG2 and A549
cells was assessed against control group at the end of exposure
using the sulforhodamine B (SRB) assay. Experimental conditions
were tested using three replicates (three wells of the 96-well plate
per experimental condition) and all experiments were performed
in triplicates. IC50 was calculated according to the equation for
Boltzman sigmoidal concentration–response curve using the non-
linear regression fitting models by Graph Pad, Prism version 5
(GraphPad Software Inc., La Jolla, CA).
3–(2–(2–(4-Chlorophenyl)quinazolin-4-yl)hydrazono)-1–(4-fluoro-
benzyl) indolin-2-one (13c). Orange powder (yield 68%),
m.p. > 300 ꢂC; IR (KBr, m cmꢁ1): 1677 (C ¼ O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 4.99, 5.05 (s, 2H–CH2–), 7.02 (d, 1H, Ar–H,
J ¼ 7.2 Hz), 7.12–7.19 (m, 4H, Ar–H), 7.30 (t, 1H, Ar–H, J ¼ 7.5 Hz),
7.42–7.81 (m, 6H, Ar–H), 7.98 (s, 1H, Ar–H), 8.18 (d, 1H, Ar–H,
J ¼ 8.1 Hz), 8.48–8.56 (m, 2H, Ar–H), 11.67, 13.79 (s, 1H, NH, D2O
exchangeable); Anal. Calcd. for C29H19ClFN5O: C, 68.57; H, 3.77; N,
13.79; Found C, 68.72; H, 3.81; N, 13.73.
Biological evaluation
In vitro anti-proliferative activity
Human breast cancer (MDA-MB-231) cells, LoVo human colorectal
carcinoma cells, human hepatoma (HepG2) cells and human non-
small cell lung cancer (A549) cells were obtained from American
Type Culture Collection (ATCC). Cells were maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, St.
Louis, MO), supplemented with 10% fetal bovine serum (Lonza
Group, Basel, Switzerland), 100 IU/mL penicillin, 100 mg/mL
streptomycin and 2 mmol/L L-glutamine (Sigma). Cells were
ELISA immunoassay
Effects of treatment of MDA-MB-231 cells with the most promising
seeded into 96-well plates at 0.4 ꢃ 104/well and incubated compounds 5e and 10g on the cells’ apoptotic machinery were
Table 1. In vitro anti-proliferative activity of the newly synthesized hybrids against MDA-MB-231 cell line.
HN
R1
O
R1
N
HN
N
HN
R1
O
O
N
N
N
N
N
HN
NH
N
N
N
R
R
R
5a-h
10a-h
13a-c
IC50 (lM)a
MDA-MB-231
Compound
R
R1
5a
5b
5c
5d
5e
5f
5g
5h
10a
10b
10c
10d
10e
10f
10g
10h
13a
13b
13c
5-FU
4-Cl
4-Cl
4-Cl
4-Cl
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
H
H
H
H
Cl
Cl
Cl
Cl
4-Cl
2,6-Cl2
4-Cl
H
F
Cl
Br
H
F
Cl
Br
H
F
Cl
Br
H
F
13.18 0.36
15.15 0.15
17.93 0.11
19.68 0.26
12.35 0.12
12.95 0.31
14.59 0.23
17.31 0.55
13.96 0.10
15.62 0.14
17.63 0.37
21.39 0.13
12.86 0.12
12.94 0.23
12.00 0.13
15.21 0.15
26.21 2.07
48.72 2.59
34.17 2.42
29.38 1.24
Cl
Br
H
H
F
aIC50 values are the mean SD of three separate experiments.

606
W. M. ELDEHNA ET AL.
further investigated. The levels of the apoptotic markers caspase-9, incubation period of 30 min at 4 ꢂC. Cells were then rinsed in bind-
ing buffer and re-suspended in 150 lL of binding buffer with the
addition of 1 lL of DAPI (1 lg/lL in PBS) (Invitrogen, Life
Technologies, Darmstadt, Germany). Cells were then analyzed
using the flow cytometer BD FACS Canto II (BD Biosciences San
Jose, CA) and the results were interpreted with FlowJo7.6.4 soft-
ware (Tree Star, FlowJo LLC, Ashland, OR).
caspase-3 and Bax as well as the anti-apoptotic marker Bcl-2 were
assessed using ELISA colorimetric kits (Neogen, Lexington, KY) as
per the manufacturer’s instructions.
MDA-MB-231 cells were cultured as a monolayer in T-25 flasks
and were seeded to attain 30% confluency prior to treatment.
Cells were then treated separately with compounds 5e and 10g at
their IC50 concentrations (12.35 and 12 lM, respectively) for 48 h.
At the end of treatment, cells were collected via trypsinization and
centrifuged at 10,000 rpm. The pellet was then rinsed with phos-
phate buffered saline (PBS) and lysed in radio immunoprecipita-
tion assay (RIPA) lysis buffer at 4 ꢂC for 45 min, then centrifuged at
14,000 rpm for 20 min to remove the cellular debris. Lysates were
then collected and stored at ꢁ80 ꢂC for later protein determin-
ation using Pierce BCA Protein Assay Kit (Pierce Biotechnology,
Rockford, IL) according to manufacturer’s recommendations.
The cell lysate was diluted 10 times, and 100 lL (50 mg pro-
tein) was added to the wells of four separate microtiter plates
for the four ELISA kits that were pre-coated with primary anti-
bodies specific to caspase-9, caspase-3, Bax and Bcl-2 proteins,
respectively. A secondary biotin-linked antibody specific to the
protein captured by the primary antibody was further added to
bind the captured protein, forming a “sandwich” of specific anti-
bodies around the desired protein in the cell lysate. The strep-
tavidin-horseradish peroxidase (HRP) complex was then used to
bind the biotin-linked secondary antibody through its streptavi-
din portion. The HRP domain reacted with the added TMB sub-
strate, forming a colored product that was measured at 450 nm
by a plate reader (ChroMate-4300, Awareness Technology, Inc.,
Palm City, FL) after the reaction was terminated by the addition
of stop solution.
Statistical analysis
Data are presented as means SD. Individual groups were com-
pared using the two-tailed independent student’s t-test. Multiple
group comparisons were carried out using one-way analysis of
variance (ANOVA) followed by the Tukey–Kramer test for post-hoc
analysis. Statistical significance was accepted at a level of p < 0.05.
All statistical analyses were performed using GraphPad InStat soft-
ware, version 3.05 (GraphPad Software, Inc., La Jolla, CA). Graphs
were sketched using GraphPad Prism software, version 5.00
(GraphPad Software, Inc., La Jolla, CA).
ADME profiling
Absorption, distribution, metabolism and excretion (ADME)
profiling for the prepared hybrids was carried out using
Discovery Studio 2.5 (Accelrys, San Diego, CA). All the tested
hybrids were drawn as a small library then prepared using lig-
and protocol to find the suitable orientation in 3D. ADME
profiling was predicted for the designed library using ADME
descriptors protocol.
Results and discussion
Chemistry
Annexin V-FITC apoptosis
The synthetic strategies employed to prepare the new target
hybrids are depicted in Schemes 1–3. In Scheme 1, 4-chloro-2-phe-
nylquinazolines 2a,b were obtained (in 75 and 72% yield, respect-
ively), by chlorination of 2-phenylquinazolin-4(3H)-ones 1a,b in
excess refluxing phosphorous oxychloride. Next, 4-chloro-2-phenyl-
quinazolines 2a,b were refluxed with hydrazine hydrate in ethanol
to furnish the hydrazine derivatives 3a,b (in 81 and 86% yield,
respectively), which were condensed with different isatins in
Apoptotic cells were further analyzed by Annexin V-FITC/DAPI
assay (Cayman Chemical, Ann Arbor, MI). Briefly, MDA-MB-231 cells
were cultured to a monolayer then treated with compound 10g at
the IC50 concentration (12 lM) as described earlier. Cells were then
harvested via trypsinization, and rinsed twice in PBS followed by
binding buffer. Moreover, cells were re-suspended in 100 lL of
binding buffer with the addition of 1 lL of FITC-Annexin V (Becton
Dickinson BD PharmingenTM, Heidelberg, Germany) followed by an refluxed ethanol in the presence of a catalytic amount of glacial
HN
R1
R1
O
HN
NH2
N
Cl
N
O
HN
N
N
4a-d
O
HN
N
O
NH
i
ii
iii
N
N
R
R
R
N
R
5a-h
1a,b
3a,b
2a,b
5
R1
R
1 2 3 a
, &
b
; R= 4-Cl, ; R= 2,6-Cl2
:
a
b
c
d
e
f
4-Cl
4-Cl
4-Cl
4-Cl
2,6-Cl2
2,6-Cl2
H
F
Cl
Br
H
F
g
h
2,6-Cl2 Cl
Br
2,6-Cl2
Scheme 1. Reagents and conditions: i, POCl3/N,N-dimethylaniline/reflux 6 h; ii, NH2NH2.H2O/EtOH/reflux 4 h; iii, EtOH/AcOH (catalytic)/reflux 0.5 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
607
NH2
O
Cl
HN
O
i
ii
iii
NH
N
N
N
N
N
OH
O
R
R
R
R
7a,b
6a,b
8a,b
9a,b
6, 7, 8& 9 a
b
: ; R=H, ; R=Cl
R1
HN
O
iv
4a-d
O
HN
R1
O
10
R
R1
H
F
Cl
Br
H
F
Cl
Br
N
HN
a
b
c
d
e
f
H
H
H
H
Cl
Cl
Cl
Cl
N
N
g
h
R
10a-h
Scheme 2. Reagents and conditions: i, NH2NH2.H2SO4/NaOH/reflux 1 h; ii, POCl3/N,N-dimethylaniline/reflux 6 h; iii, NH2NH2.H2O/EtOH/reflux 7 h. iv, EtOH/AcOH (cata-
lytic)/reflux 1 h.
R1
R1
N
O
O
Br
O
11a,b
i
ii
O
O
N
NH
N
N
H
N
4
R1
12a,b
, b; R
N
R
11 & 12: a; R
= H
= F
13a-c
R1
13
R
a
b
c
4-Cl
2,6-Cl2
4-Cl
H
H
F
Scheme 3. Reagents and conditions: i, DMF/K2CO3/reflux 3 h; ii, Compounds 4a,b/EtOH/AcOH (catalytic)/reflux 0.5 h.
acetic to afford the isatin-quinazoline hybrids 5a–h (in 65–77%
yield).
For the synthesis of isatin-phthalazine hybrids 10a–h,
2-benzoylbenzoic acids 6a,b were cyclocondensed with hydrazine
IR spectra of the latter hybrids displayed absorption bands due sulfate in the presence of sodium hydroxide to afford phthalazi-
to the NH groups in the region 3170–3305 cmꢁ1, in addition to a none 7a,b. Next, chlorination of compounds 7a,b was carried out
carbonyl band in the region 1672–1690 cmꢁ1. The 1H NMR via refluxing with excess phosphorus oxychloride to furnish
spectra of compounds 5a–h confirmed the presence of two 1-chloro-4-phenylphthalazines 8a,b (in 65 and 72% yield, respect-
D2O-exchangeable singlet signals attributable to NH protons of ively). The chloro intermediates 8a,b were reacted with hydrazine
the hydrazine function ( ¼ N–NH–) and the isatin in the range d hydrate in refluxing ethanol to provide the 1-hydrazinyl-4-phenyl-
10.62–11.49 and 12.11–13.93 ppm, respectively.
phthalazines 9a,b (in 84 and 80% yield, respectively), which upon

608
W. M. ELDEHNA ET AL.
reaction with different isatins afforded the target hybrids 10a–h atom (more bulky and lipophilic than chlorine) decreased the anti-
proliferative activity (IC50 ¼ 19.68 0.26 and 17.31 0.55 lM,
(in 60–75% yield) (Scheme 2).
respectively). Thus, the order of activity of the halogenated hybrids
in such series, were smoothly decreased in the order of
F > Cl > Br, hinting that increasing the lipophilicity and the size of
the substituents of isatin moiety at the 5-position is not affirmative
for the activity of the isatin-quinazoline hybrids regardless of the
type of substitution of the 2-phenyl group of the quinazoline
moiety.
Regarding the isatin-phthalazine hybrids 10a–h, the above-
mentioned relationship could be, also, proposed for the hybrids
10a–d with unsubstituted 4-phenyl group in the phthalazine moi-
ety. While, substitution of hybrids 10e–h (containing 4–(4-chloro-
phenyl) group) with F or Cl at 5-position of isatin moiety has no
significant effect, introduction of Br led to a decreased activity
toward the MDA-MB-231 cell line.
Further examination of the effect of the substitution pattern of
the phenyl group on the prepared hybrids was then conducted.
Regarding the isatin-quinazoline hybrids 5a–h, the increased IC50
values of members 5a–d with incorporated 4-chlorophenyl group
(13.18 0.36, 15.15 0.15, 17.93 0.11 and 19.68 0.26 lM, respect-
ively) than that of their corresponding analogs 5e–h with 2,6-
dichlorophenyl group (12.35 0.12, 12.95 0.31, 14.59 0.23 and
17.31 0.55 lM, respectively) indicated that 2,6-dichloro substitu-
tion is more beneficial for activity rather than 4-chloro substitu-
tion, which could be attributed to the non-coplanarity or the
increased lipophilicity. On the other hand, substitution of the 4-
phenyl group in the isatin-phthalazine hybrids was found to be
advantageous to the activity rather than unsubstitution, that is evi-
denced by the decreased IC50 values of compounds 10e–f
(12.86 0.12, 12.94 0.23, 12.00 0.13 and 15.21 0.15 lM, respect-
ively) than those of their corresponding analogs 10a–d
IR spectra of 10a–h revealed the presence of an NH stretching
band about 3200 cmꢁ1. Their 1H NMR spectra showed two D2O-
exchangeable singlet signals from the hydrazine function
( ¼ N–NH–) and the isatin protons in the range d 10.56–10.74 and
12.83–13.17 ppm.
Finally, isatin was refluxed with benzyl bromide or 4-fuoroben-
zyl bromide in dry DMF and anhydrous K2CO3 to furnish N-substi-
tuted isatins 12a,b, respectively (in 88 and 82% yield,
respectively). The reaction of derivatives 12a,b with the appropri-
ate 4-hydrazinyl-2-phenylquinazoline derivative 3 in ethanol in the
presence of a catalytic amount of glacial acetic acid afforded the
target hybrids 13a–c (Scheme 3).
The structures of hybrids 13a–c were confirmed under the
basis of spectral and elemental analyses which were in full agree-
ment with the proposed structures.
Biological evaluation
In vitro anti-proliferative activity against TNBC MDA-MB-231
cell line
Anti-proliferative activity of the newly prepared hybrids 5a–h,
10a-h and 13a–c was evaluated against MDA-MB-231 TNBC cell
line using the WST-1 assay as described by Ngamwongsatit
et al.50. Being a well-known broad-spectrum anticancer drug and a
first line therapy for many tumors management, 5-FU was chosen
as
a
positive control51,52. The anti-proliferative activity was
expressed as the half maximal inhibitory concentration (IC50) val-
ues (Table 1).
From the obtained results, it is obvious that most of the
tested hybrids have excellent to moderate growth inhibitory (13.96 0.10, 15.62 0.14 17.63 0.37 and 21.39 0.13 lM,
activity toward the tested MDA-MB-231 cancer cell line. In par- respectively).
Finally, investigation of the effect of N-benzylation of isatin
moiety showed that this was not an advantageous approach for
the anti-proliferative activity against MDA-MB-231 cell line, where
the N-benzyl hybrids 13a–c displayed decreased potency
(26.21 2.07, 48.72 2.59 and 34.17 2.42 lM, respectively) than
their unsubstituted counterparts 5a and 5e (IC50 ¼ 13.18 0.36
and 12.35 0.12 lM, respectively).
In conclusion, we can deduce that unsubstitution of isatin moi-
ety at C-5 or N-1 positions, disubstitution of the 2-phenyl group of
quinazoline moiety and substitution of the 4-phenyl group of
phthalazine moiety are crucial elements for the anti-proliferative
activity against MDA-MB-231.
ticular, compounds 5e and 10g were the most active members
against MDA-MB-231 cells and
(IC50 ¼ 12.35 0.12
12.00 0.13 lM, respectively), with 2.37- and 2.44-fold increased
activity than the reference drug, 5-FU (IC50 ¼ 29.38 1.24 lM).
Besides, compounds 5a–d, 5f–h, 10a–f, 10h and 13a displayed
good anti-proliferative activity with IC50 values ranging from
12.86 0.12 to 26.21 2.07 lM. Whilst, hybrids 13a and 13b
were moderately active with IC50 values of 48.72 2.59 and
34.17 2.42 lM, respectively.
Structure activity relationship SAR
By scrutinizing the aforementioned biological data, one can snugly
reveal a clear structure activity relationship. First, we investigated
the impact of the substitution at the 5-position of the isatin moi-
ety. Regarding the isatin-quinazoline hybrids 5a–h, incorporation
of unsubstituted isatin resulted in compounds 5a and 5e with
moderate activity against the MDA-MB-231 cell line
(IC50 ¼ 13.18 0.36 and 12.35 0.12 lM, respectively). As it has an
electronic and size properties similar to those of hydrogen, fluor-
ine is introduced as a classical bioisostere to the hydrogen atom.
Compounds 5b and 5f bearing fluorine substituent, displayed
slight decrease in the anti-proliferative activity (IC50 ¼ 15.15 0.15
and 12.95 0.31 lM, respectively) compared to the unsubstituted
analogs, suggesting that halogens incorporation may not be
advantageous for the activity. Furthermore, introduction of more
bulky and lipophilic chlorine atom; compounds 5c and 5g, caused
decrease of activity against MDA-MB-231 cells (IC50 ¼ 17.9 0.11
Effects on mitochondrial apoptosis pathway proteins Bax and
Bcl-2
Induction of apoptosis in cancer cells is one of the successful strat-
egies for the development of cancer therapy53–55. Therefore, we
investigated the potential pro-apoptotic effects of our isatin-quina-
zoline and isatin-phthalazine hybrids attempting to explore the
underlying mechanism for their anti-proliferative activity.
As indicated by the cytotoxicity results, compounds 5e and
10g were found to be the most active agents against MDA-MB-
231 breast cancer cells. Thus, we investigated the potential pro-
apoptotic effects of these promising agents in an attempt to
define the principle mechanism for their anti-proliferative activity.
The Bcl-2 family of proteins is responsible for synchronizing the
mitochondrial apoptotic pathway56. These proteins are classified
into two groups: anti-apoptotic proteins such as Bcl-2 protein and
and 14.59 0.23 lM, respectively). Also, incorporation of bromine the counteracting pro-apoptotic proteins including Bax protein57.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
609
Figure 3. Effect of compounds 5e and 10g on the protein levels of A) Bax; B) Bcl-2 in MDA-MB-231 cells treated with the compounds at their IC50 concentrations
ꢀ
ꢀꢀ
against control (1% DMSO). Data are mean SD (n ¼ 3). The experiment was done in triplicates. Significantly different from control at p < 0.05. Significantly differ-
ꢀꢀꢀ
ent from control at p < 0.01.
Significantly different from control at p < 0.001.
Table 2. Effect of compounds 5e and 10g on active caspase-9 and -3 levels, and the expression levels of Bcl-2 and Bax in MDA-MB-231 cancer cells treated with the
compounds at their IC50 concentrations.
Compound
Bcl-2 ng/mg protein
Bax ng/mg protein
Bax/Bcl-2 ratio
Caspase-9 nmol/mg protein
Caspase-3 nmol/mg protein
Control (1% DMSO)
5e
10g
12.31 0.97
8.76 0.42b
5.90 0.53c
6.92 0.51
9.05 0.77a
12.47 1.08c
0.56
1.033
2.11
3.07 0.22
6.98 0.73c
8.27 0.139c
2.56 0.17
6.12 0.54c
9.48 0.73c
Data are mean SD of three separate experiments.
aSignificantly different from control (1% DMSO) at p < 0.05.
bSignificantly different from control (1% DMSO) at p < 0.01.
cSignificantly different from control (1% DMSO) at p < 0.001.
It has been previously indicated that the overexpression of the caspases such as caspase-960. Therefore, the elevated Bax/Bcl-2
anti-apoptotic Bcl-2 protein is a contributing factor to tumorigen- ratios obtained with these treatments triggered the investigation
esis in a multitude of cancers including breast cancer58. In this of the protein expression levels of active caspases-9 and -3.
In this study, treatment of MDA-MB-231 cells with compounds
5e and 10g resulted in a significant elevation of active caspase-9
protein levels by around 2.3 and 2.7 folds, respectively, compared
study, exposure of MDA-MB-231 breast cancer cells to compounds
5e and 10g for 48 h resulted in a significant increase in the
expression of the pro-apoptotic protein Bax by ꢄ130 and 180%,
respectively, compared to the control (Figure 3(A), Table 2). On to control (Figure 4(A), Table 2). Moreover, MDA-MB-231 cells
the other hand, treatment of MDA-MB-231 breast cancer cells with upon treatment with compounds 5e and 10g, exhibited a signifi-
compounds 5e and 10g significantly reduced the expression levels cant upregulation of active caspase-3 protein levels by ꢄ2.4 and
of the anti-apoptotic protein Bcl-2 by ꢄ29 and 52%, respectively, 3.7 folds, respectively, compared to control (Figure 4(B), Table 2).
It is worth noting that upon comparing the relative expression
levels of caspases with the relative elevation patterns of Bax/Bcl-2
ratio observed in both treatments, it was found that the magni-
tude of the Bax/Bcl-2 ratio elevation is proportional to that of cas-
pase-9 and that of caspase-3 up-regulation. This observation
strengthens the correlation between the increased Bax/Bcl-2 ratio
and the activation of proteolytic caspases that act as key players
in the execution of apoptosis, which might be responsible for the
compared to the control (Figure 3(B), Table 2). A rather more
important parameter is the ratio between Bax (apoptosis inducer)
and Bcl-2 (apoptosis suppressor) that gave more real insight to
the apoptotic activity of the compounds as it is considered a key
indicator of therapeutic response to chemotherapy. Analyzing the
results discloses that compound 5e increased the Bax/Bcl-2 ratio
two folds compared to the control, while compound 10g even
boosted it four folds in comparison to the control. The ability of
the two compounds to down regulate Bcl-2 levels while boosting anti-proliferative activity of the tested compounds. Moreover, com-
Bax levels further supports their effectiveness as apoptosis pound 10g exhibited a more potent activity than compound 5e in
inducers.
terms of both elevating Bax/Bcl-2 ratio and further activating the
caspases cascade at a relatively lower IC50. This finding directed
further investigation of the apoptotic effect of this compound on
MDA-MB-231 cells.
Effects on the level of active caspase-9 and caspase-3
The down-regulation of the anti-apoptotic Bcl-2 results in
increased levels of free pro-apoptotic Bax which then accumulates
at the inner mitochondrial membrane forming channels thus alter- Annexin V–FITC apoptosis assay
ing membrane permeability. Apoptotic factors then leak into the Externalization of the phospholipid phosphatidylserine (PS) at the
cytoplasm resulting in the activation of caspases cascade59
.
cell membrane is one of the hallmarks of cells going into apop-
Caspases are cysteine-containing aspartic acid-specific proteases, tosis61. Having a high Ca2þ-dependent binding affinity to nega-
which exist as zymogens in the cytoplasm and play a pivotal role tively charged phospholipid surfaces. Annexin A5 (AnxV A5), a 36-
in the execution of cell death upon activation60. Caspase-3 is the kDa human protein, is a suitable candidate for apoptosis imaging.
key executioner protease that is activated by upstream initiator Besides, Anx V-based flow cytometry analysis is a useful tool to

610
W. M. ELDEHNA ET AL.
Figure 4. Effect of compounds 5e and 10g on the protein levels of A) active caspase-9; B) active caspase-3 in MDA-MB-231 cells treated with the compounds at their
ꢀꢀꢀ
IC50 concentrations against control (1% DMSO). Data are mean SD (n ¼ 3). The experiment was done in triplicates.
Significantly different from control at p < 0.001.
Figure 5. Effect of compounds 10g on the percentage of Annexin V-FITC-positive staining in MDA-MB-231 cells versus control (1% DMSO). Data are mean SD (n ¼ 3).
The experiments were done in triplicates. The four quadrants identified as: Normal, viable; Apo, early apoptotic; LATE APO, late apoptotic; NEC, necrotic.
ꢀꢀꢀ
Significantly different from control at p < 0.001 (student’s t-test).
comprehend whether cell death is due to physiological apoptosis (an initiator caspase) together with the downstream caspase-3 (the
or nonspecific necrosis61.
hallmark key executor) belonging to the intrinsic apoptotic path-
Further evaluation of the apoptotic effect of the most potent
compound 10g was carried out using Anx V-FITC/DAPI dual stain-
ing assay (Figure 5). MDA-MB-231 cells treated with compound
10g showed a significant increase in the percent of Anx V-FITC
positive apoptotic cells (apoptotic and late apoptotic) from 3.88 to
31.21% which comprises about 8.4-folds (p < 0.001) compared to
control (Figure 5). Flow cytometric analysis of the differential bind-
ing of the cells to Anx V-FITC displayed a significant increase in
the proportion of early apoptotic cells. This observation is in line
way. This strongly suggests as well that a cascade of proteinases’
activation has been triggered as a consequence of an increase in
Bax/Bcl-2 ratio, eventually leading to apoptosis. These findings
clearly imply that compound 10g has potent pro-apoptotic activity
that unravels the mechanism of its anti-proliferative activity.
In conclusion, the reduced expression of the anti-apoptotic
protein Bcl-2 in addition to the enhanced expression of the pro-
apoptotic protein Bax as well as the up-regulated active caspase-9
and caspase-3 levels together with a harmonized increase in the
with the simultaneous up-regulation of the upstream caspase-9 Bax/Bcl-2 ratio, suggests that the cytotoxic effect of compounds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
611
5e and 10g might be attributed, at least in part, to the induction A549 with IC50 values of 28.73 1.59 and 18.42 1.52 lM, respect-
ively. Besides, most compounds possessed good to moderate
activity against LoVo cell line with IC50 values ranged from
13.09 0.33 to 48.62 2.21 lM. Regrettably, among the tested can-
cer cell lines, the colon Caco-2 and hepatocellular HepG2 were not
susceptible cell lines to almost all hybrids influence.
of the intrinsic apoptotic mitochondrial pathway.
In vitro anti-proliferative activity against four different cell lines
The in vitro anti-proliferative activity of the prepared hybrids 5a–h,
10a–h and 13a–c was further examined against a panel of cell
lines, namely A549 alveolar carcinoma, Caco-2 colon cancer, LoVo
human colorectal carcinoma and HepG2 hepatocellular carcinoma
using SRB colorimetric assay as described by Skehan et al.62, to
carry out further elaboration for their anti-proliferative activity. The
results were expressed as IC50 values and listed in Table 3.
ADME study The ADME of the prepared hybrids 5a–h, 10a–h and
13a–c was predicted through a theoretical kinetic study per-
formed by Discovery Studio software (Table 4). To evaluate the lip-
ophilicity and polar surface area, AlogP98 and PSA_2D descriptors
were calculated. Besides, absorption, solubility and CYP2D inhib-
ition levels were predicted. All compounds were expected to have
very low-to-low aqueous solubility. Meanwhile, most of the exam-
ined derivatives seemed to possess good to poor absorption lev-
els, and predicted to be CYP2D inhibitors except compounds 10a,
10e–h and 13b which are expected not to inhibit CYP2D.
The presented data revealed that some hybrids displayed mod-
erate to fair anti-proliferative activity toward A549 cell line. In par-
ticular, compounds 5d and 10d showed the better activity against
Table 3. In vitro anti-proliferative activities of the newly synthesized hybrids
against A549, Caco-2, LoVo and HepG2 cell lines.
IC50 (lM)a
Conclusion
Compound
A549
Caco-2
NAb
LoVo
HepG2
NAb
In an effort to develop potent anti-proliferative agents, the
molecular hybridization approach was adopted to design and
synthesize new different series of isatin-quinazoline hybrids
5a–h, isatin-phthalazine hybrids 10a–h and N-benzylisatin-quina-
zoline hybrids 13a–c. The in vitro anti-proliferative activity of the
newly synthesized hybrids was evaluated against the TNBC MDA-
MB-231 breast cancer. Compounds 5e and 10g displayed the
highest potency toward MDA-MB-231 with IC50 values of
12.35 0.12 and 12.00 0.13 lM, respectively. Subsequently, com-
pounds 5e and 10g were further estimated for their apoptosis
induction potential. Both compounds 5e and 10g proved to
induce apoptosis, which was assured by the reduced expression
of the anti-apoptotic protein Bcl-2 in addition to the enhanced
expression of the pro-apoptotic protein Bax as well as the up-
regulated active caspase-9 and caspase-3 levels together with a
harmonized increase in the Bax/Bcl-2 ratio. Furthermore, com-
pound 10g showed a significant increase in the percent of
annexin V-FITC positive apoptotic cells from 3.88 to 31.21%
which comprises about 8.4 folds compared to control. Finally the
5a
5b
5c
5d
5e
5f
5g
5h
10a
10b
10c
10d
10e
10f
10g
10h
13a
13b
13c
Dox.
97.04 4.31
NAb
15.71 1.52
17.58 2.18
36.59 3.05
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
3.01 0.17
NAb
NAb
NAb
28.73 1.59
NAb
NAb
14.48 0.78
23.77 1.75
13.86 0.96
39.00 2.13
13.83 0.66
38.92 2.73
15.38 0.83
25.46 2.07
14.29 1.32
13.09 0.33
14.40 1.92
16.91 1.36
48.62 2.21
NAb
NAb
37.51 2.08
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
18.42 1.52
91.57 5.36
56.71 2.93
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
5.23 0.49
NAb
0.82 0.05
2.74 0.11
aIC50 values are the mean SD of three separate experiments.
bNA: Compounds having IC50 value >100 lM.
Table 4. Computer aided ADME study for the prepared hybrids.
Compound
AlogP98a
PSA_2Db
Solubilityc
Solubility leveld
Absorption levele
CYP2D6f
CYP2D6 probability
5a
5b
5c
5d
5e
5f
5g
5h
10a
10b
10c
10d
10e
10f
10g
10h
13a
13b
13c
4.911
5.117
5.576
5.660
5.576
5.781
6.240
6.324
4.306
4.511
4.970
5.054
4.970
5.176
5.635
5.719
6.701
7.365
6.906
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
76.766
67.308
67.308
67.308
ꢁ6.987
ꢁ7.346
ꢁ7.757
ꢁ7.834
ꢁ7.784
ꢁ8.138
ꢁ8.549
ꢁ8.626
ꢁ6.276
ꢁ6.638
ꢁ7.050
ꢁ7.127
ꢁ7.035
ꢁ7.390
ꢁ7.802
ꢁ7.879
ꢁ8.103
ꢁ8.777
ꢁ8.316
1
1
1
1
1
0
0
0
1
1
1
1
1
1
1
1
0
0
0
0
0
1
1
1
1
2
2
0
0
0
0
0
0
1
1
2
3
2
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
1
0
1
0.534
0.544
0.613
0.574
0.524
0.683
0.673
0.584
0.455
0.613
0.514
0.514
0.356
0.316
0.386
0.316
0.544
0.366
0.544
aLipophilicity descriptor.
bPolar surface area.
cSolubility parameter. (0:ꢁ2 ¼ optimal, ꢁ2:ꢁ4 ¼ good, ꢁ4:ꢁ6 ¼ low, ꢁ6:ꢁ8 ¼ very low).
dSolubility level (0 ¼ extremely low, 1 ¼ very low but possible, 2 ¼ low, 3 ¼ good, 4 ¼ optimal).
eAbsorption level (0 ¼ good, 1 ¼ moderate, 2 ¼ low, 3 ¼ very low).
fCYP2D inhibition (0 ¼ non inhibitor, 1 ¼ inhibitor).

612
W. M. ELDEHNA ET AL.
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a
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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-1436–038.
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Disclosure statement
The authors declare no conflict of interest.
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Funding
The Deanship of Scientific Research at King Saud University
funded this research through the Research Group Project no. RG-
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