N-Substituted-4-phenylphthalazin-1-amine-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking, and anticancer evaluation studies

Article abstract of DOI:10.1002/ardp.202000219

In accordance with the significant impetus of the discovery of potent vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors, herein, we report the design, synthesis, and anticancer evaluation of 12 new N-substituted-4-phenylphthalazin-1-amine

Full text of DOI:10.1002/ardp.202000219

Received: 26 June 2020
Revised: 4 September 2020
Accepted: 24 October 2020
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DOI: 10.1002/ardp.202000219
F U L L P A P E R
N‐Substituted‐4‐phenylphthalazin‐1‐amine‐derived VEGFR‐
2 inhibitors: Design, synthesis, molecular docking, and
anticancer evaluation studies
Khaled El‐Adl1,2
| Mohamed‐Kamal Ibrahim1 | Fathalla Khedr1
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Hamada S. Abulkhair3,4
| Ibrahim H. Eissa1
1Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University, Cairo,
Egypt
Abstract
In accordance with the significant impetus of the discovery of potent vascular en-
dothelial growth factor receptor 2 (VEGFR‐2) inhibitors, herein, we report the de-
sign, synthesis, and anticancer evaluation of 12 new N‐substituted‐4‐
phenylphthalazin‐1‐amine derivatives against HepG2, HCT‐116, and MCF‐7 cells
as VEGFR‐2 inhibitors. The results of the cytotoxicity investigation indicated that
HCT‐116 and MCF‐7 were the most sensitive cell lines to the influence of the newly
synthesized derivatives. In particular, compound 7a was found to be the most po-
tent derivative among all the tested compounds against the three cancer cell lines,
HepG2, HCT116, and MCF‐7, with IC50 = 13.67 1.2, 5.48 0.4, and 7.34 0.6 µM,
respectively, which is nearly equipotent to that of sorafenib (IC50 = 9.18 0.6,
5.47 0.3, and 7.26 0.3 µM, respectively). All synthesized derivatives, 4a,b−8a−c,
were evaluated for their inhibitory activities against VEGFR‐2. The tested com-
pounds displayed high to low inhibitory activity, with IC50 values ranging from
0.14 0.02 to 9.54 0.85 µM. Among them, compound 7a was found to be the most
potent derivative that inhibited VEGFR‐2 at an IC50 value of 0.14 0.02 µM, which
is nearly 72% of that of the sorafenib IC50 value (0.10 0.02 µM). Compounds
7b, 8c, 8b, and 8a exhibited very good activity with IC50 values of 0.18 0.02,
0.21 0.03, 0.24 0.02, and 0.35 0.04 µM, respectively. Molecular modeling stu-
dies were carried out for all compounds against the VEGFR‐2 active site. The data
obtained from biological testing highly correlated with that obtained from molecular
modeling studies. However, these modifications led to new phthalazine derivatives
with higher VEGFR‐2 inhibitory activities than vatalanib and which are nearly
equipotent to sorafenib.
2Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
3Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy, Al‐Azhar
University, Cairo, Egypt
4Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Horus University, New
Damietta, Egypt
Correspondence
Khaled El‐Adl, Ibrahim H. Eissa, and Fathalla
Khedr, Pharmaceutical Medicinal Chemistry
and Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University, Nasr
City, Cairo 11884, Egypt.
Email: eladlkhaled74@azhar.edu.eg, khaled.
eladl@hu.edu.eg, eladlkhaled74@yahoo.com,
(K. E.‐A.), ibrahimeissa@azhar.edu.eg (I. H. E.),
and fkhedr2020@gmail.com (F. K.)
K E Y W O R D S
4‐phenylphthalazin‐1‐amine, anticancer agents, molecular docking, VEGFR‐2 inhibitors
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Arch Pharm. 2020;e2000219.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000219

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1
INTRODUCTION
discovery of several leading phthalazines with different cellular and
enzymatic targets. For example, AMG 900 I (Figure 1) was synthe-
sized by Amgen as an orally bioavailable, potent, and highly selective
pan‐Aurora kinase inhibitor that is active against taxane‐resistant
tumor cell lines.[6] AMG 900 I was found to be active against an
AZD1152‐resistant HCT116 variant cell line that harbors an Aurora‐
B mutation (W221L).[7] Thereafter, Cee et al.[13] have discovered two
selective and orally bioavailable pyridinyl‐pyrimidine phthalazines
Aurora kinase inhibitors. Vatalanib (PTK787) II[14] (Figure 1) inhibits
both VEGFR‐1 and VEGFR‐2 with IC50 values of 380 and 20 nM,
respectively. Vatalanib is well absorbed orally and shows an in vivo
antitumor activity against a panel of human tumor xenograft models;
however, vatalanib is currently in phase III clinical trials for the
treatment of colorectal cancer.[15,16] In addition, many anili-
nophthalazines have been reported as potent inhibitors of VEGFR‐2,
such as AAC789 III and IM‐023911 IV with IC50 = 20 and 48 nM,
respectively (Figure 1).[8‐11,17,18]
Extensive studies have been reported concerning the synthesis of
several phthalazine derivatives[1‐7] as promising anticancer agents
and potent vascular endothelial growth factor receptor 2 (VEGFR‐2)
inhibitors.[8‐11] Phthalazin‐1,4‐diones have been reported as potent
type II IMP dehydrogenase inhibitors and as effective anti-
proliferative agents against different human and murine tumor cells,
particularly against hepatocellular carcinoma.[3] Moreover, 1,4‐
disubstituted phthalazines have attracted considerable attention as
promising and effective anticancer agents.[3,5] In addition, many
triazolo[3,4‐a]phthalazine derivatives were reported to exhibit pro-
mising antitumor activities against MGC‐803, EC‐9706, HeLa, and
MCF‐7 human cancer cell lines.[12]
During the last two decades, there was a growing interest in the
synthesis of several phthalazines as promising drug candidates for
the treatment of cancer. The latter research efforts have led to the
FIGURE 1 Structures of the lead anticancer phthalazine derivatives I−IV, sorafenib, and the designed target 4‐phenylphthalazinon‐1‐amine
derivatives 4−9a–c

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In this context, the 1‐substituted‐4‐phenylphthalazine scaffold,
was reported to be substantially upregulated in HepG2 cells in a
dose‐dependent manner with the stimulation of the hepatocyte
growth factor, which is involved in cell proliferation, invasion, and
angiogenesis of hepatocellular carcinoma.[30‐32] Blockade of VEGFR‐
2 signaling revealed a marked inhibition on both the growth and
metastasis of HCC.[32,33] Also, VEGFR‐2 was found to be crucial for
cell survival, which regulates endothelial differentiation in both the
breast cancer cells (MCF‐7)[32,33] and human colorectal carcinoma
(HCT‐116).[34,35] The overexpression of VEGFR‐2 receptors in breast
cancer cells has been documented as a contributor in resistance of
such cancer type to the chemotherapeutic effect of tamoxifen.[36]
VEGFR‐2 degraders were documented to impair the in vitro en-
dothelial differentiation and to promote angiogenesis, suggesting a
VEGFR‐2‐mediated mechanism of antiproliferative activity in human
colorectal carcinoma.[34]
bearing different 4‐substituted anilines, in particular, emerged as an
interesting scaffold for designing VEGFR inhibitors (Figure 1). The
abovementioned facts have aggravated us to design novel
N‐substituted‐4‐phenylphthalazin‐1‐amine derivatives in an attempt
to obtain more potent anticancer agents. Sorafenib (Nexavar®, V)
(Figure 1) is a potent VEGFR‐2 inhibitor, which has been approved as
an antiangiogenic drug.[19,20]
The common pharmacophoric features shared by various
VEGFR‐2 inhibitors, for example, vatalanib and sorafenib, revealed
four main features, as shown in Figure 1[21‐23]: (i) The core structure
of most inhibitors that occupied the catalytic adenosine triphosphate
(ATP)‐binding domain consists of a flat heteroaromatic ring system
that contains at least one N atom. (ii) A central aryl ring (hydrophobic
spacer) is present, occupying the linker region between the ATP‐
binding domain and the DFG domain of the enzyme.[24] (iii) Also,
there exists a hydrophilic linker containing a functional group (e.g.,
amino or urea) acting as pharmacophore that possesses both H‐bond
donor and acceptor to bind with Glu883 and/or Asp1044, the two
crucial residues in the DFG (Asp−Phe−Gly) motif, which is an es-
sential tripeptide sequence in the active kinase domain. The NH
group of the linker moiety usually forms one hydrogen bond with
Glu883, whereas the C═O group forms another hydrogen bond with
Asp1044. (iv) The hydrophobic interactions are usually achieved
when a terminal hydrophobic moiety of the inhibitors occupies the
newly formed allosteric hydrophobic pocket, which is exposed when
the DFG loop phenylalanine residue flips out of its lipophilic pock-
et.[25] Furthermore, the X‐ray analysis of the VEGFR‐2 receptor
confirmed the presence of adequate space available for various
substituents around the terminal heteroaromatic ring.[26,27]
In continuation of our efforts to obtain new anticancer
agents,[37‐43] the goal of the present work was the synthesis of new
agents with the same essential pharmacophoric features of the re-
ported and clinically used VEGFR‐2 inhibitors (e.g., vatalanib and
sorafenib). The main core of our molecular design rationale com-
prised bioisosteric modification strategies of VEGFR‐2 inhibitors at
the four different pharmacophoric positions (Figure 1).
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2
RESULTS AND DISCUSSION
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2.1
Rationale and structure‐based design
N‐Substituted‐4‐phenylphthalazin‐1‐amine derivatives have the es-
sential pharmacophoric features of VEGFR‐2 inhibitors[22,39‐44]
(Figure 1), which include the following: First, there exists a six‐
membered heteroaromatic ring, phthalazine, as a central aryl moiety,
substituted with phenyl ring, as a hydrophobic portion, forming the
4‐phenylphthalazine scaffold. Second, the target phenyl group at
VEGFR‐2 is the earliest recognized marker for the development
of endothelial cells. VEGFR‐2 has been documented as a regulator of
tumor angiogenesis; therefore, several VEGFR‐2 inhibitor molecules
have been developed as effective anticancer agents.[28,29] VEGFR‐2
FIGURE 2 Superimposition of compound 7a and vatalanib inside the binding pocket of vascular endothelial growth factor receptor 2 (1YWN)

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FIGURE 3 Superimposition of compound 7a and sorafenib inside the binding pocket of vascular endothelial growth factor receptor
2 (1YWN)
position‐4 is used to replace pyridine and N‐methylpicolinamide
moieties of vatalanib and sorafenib (Figures 2 and 3), respectively. N
at position‐2 of phthalazine nucleus acts as an H‐bond acceptor
(HBA), forming an H‐bond with the essential amino acid
residue Asp1044. The third strategy is using NH linker (as NH of
vatalanib) which acts as H‐bond donor (HBD), forming an H‐bond
with the essential amino acid residue Glu883. Fourth, the hydro-
phobic substituted phenyl tail of the reported ligands vatalanib and
sorafenib (Figures 2 and 3) is replaced by other one substituted with
distal hydrophobic moieties linked through amide (NHCO), urea
(NHCONH), thiourea (NHCSNH), and/or amide (CONH) linkers as in
6, 7a,b, 8a−c, and/or 9a−c derivatives, respectively.
these amide, urea, and/or thiourea linkers were used to resemble the
urea linker of sorafenib to obtain both linkers of vatalanib and sor-
afenib in the same compound to increase H‐bonding interactions
with the active site of VEGFR‐2 and consequently the anticancer
activities. These linkers increased the length of the structures to
enable the distal moieties to occupy new hydrophobic grooves
formed by Arg1025, His1024, Ile1023, Cys1022, Lys1021, Arg1020,
Leu1017, Ile890, His889, and Ile886 (Figures 2 and 3).
Additionally, the substitution pattern was selected to ensure
different electronic and lipophilic environments that could influence
the activity of the target compounds. These modifications
were performed to carry out further elaboration of the phthalazine
scaffolds and to explore a valuable structure–activity relationship
(SAR). The designed target phthalazine derivatives were synthesized
and evaluated as potential VEGFR‐2 inhibitors and antitumor
The used substituted hydrophobic phenyl tail occupied the hy-
drophobic pocket formed by Asp1044, Cys1043, Ile1042, Val897,
His892, Gly891, Ile890, Leu887, Ile886, and Glu883. Furthermore,
_
(2)
(3)
(4a b)
(1)
SCHEME 1 The synthetic route for the preparation of the target compounds 4a,b

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agents against three human tumor cell lines, namely, hepatocellular
carcinoma (HepG2), breast cancer (Michigan Cancer Foundation‐7
[MCF‐7]), and human colorectal carcinoma‐116 (HCT‐116).
the reported procedure.[45] The produced amino derivative 5 was al-
lowed to react with benzoic acid, the appropriate isocyanates, namely,
phenyl and cyclohexyl isocyanates, and/or the appropriate iso-
thiocyanates, namely, propyl, butyl, and cyclohexyl isothiocyanates, to
afford the corresponding amide derivative 6, urea 7a,b, and/or thiourea
8a–c derivatives, respectively (Scheme 2). Finally, the acid derivative 4b
was reacted with the appropriate amine, namely, aniline, 4‐chloroaniline,
and/or 4‐methylaniline, following the mixed anhydride method to give
the corresponding acid amide derivatives 9a−c, respectively (Scheme 3).
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2.2
Chemistry
The synthetic strategy for the preparation of the target compounds (4−9)
is depicted in Schemes 1‐3. The synthesis was initiated by cyclo-
condensation of 2‐benzoylbenzoic acid (1) with hydrazine hydrate to
afford the corresponding 4‐phenylphthalazin‐1(2H)‐one (2), which un-
derwent chlorination by reaction with phosphorous oxychloride to afford
1‐chloro‐4‐phenylphthalazine (3).[3,4] The chloro derivative (3) was re-
fluxed with the appropriate 4‐substituted aniline, namely, 4‐nitroaniline
and/or 4‐aminobenzoic acid, to afford the corresponding amino deriva-
tives 4a,b, respectively (Scheme 1). However, the 4‐nitro derivative 4a
was reduced to the corresponding 4‐amino derivative 5 by heating with
SnCl2 as a reducing agent in the presence of hydrochloric acid following
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2.3
In vitro antiproliferative activity
The antiproliferative activity of the newly synthesized phthalazine deri-
vatives 4a,b−9a−c was examined against three human tumor cell lines,
namely, hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT‐
116), and breast cancer (MCF‐7), using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐
diphenyltetrazolium bromide (MTT) colorimetric assay as described by
SCHEME 2 The synthetic route for the preparation of the target compounds 5−8a−c

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(4b)
(9a c)
SCHEME 3 The synthetic route for preparation of the target compounds 9a−c
Mosmann.[46‐48] Sorafenib was used as a reference cytotoxic drug. The
results were expressed as growth inhibitory concentration (IC50) values,
which represent the compound concentrations required to produce a
50% inhibition of cell growth after 72 h of incubation, calculated from the
concentration−inhibition response curve and summarized in Table 1.
From the obtained results, it was explicated that most of the prepared
compounds displayed an excellent to low growth inhibitory activity
against the tested cancer cell lines. Investigations of the cytotoxic activity
indicated that HCT‐116 and MCF‐7 were the most sensitive cell lines to
the influence of the new derivatives, respectively. In particular, com-
pound 7a was found to be the most potent derivative over all the tested
compounds against the three HepG2, HCT116, and MCF‐7 cancer cell
lines with IC50 = 13.67 1.2, 5.48 0.4, and 7.34 0.6 µM, respectively,
which is nearly equipotent to that of sorafenib (IC50 = 9.18 0.6,
5.47 0.3, and 7.26 0.3 µM, respectively).
With respect to the HepG2 hepatocellular carcinoma cell line,
compounds 7b and 8c displayed very good anticancer activities with
IC50 = 19.69 1.8 and 29.46 2.6 µM, respectively. Compounds 8b,
8a, 9c, and 9b, with IC50 = 41.45 3.2, 45.24 3.3, 49.40 3.6, and
52.60 3.7 µM, respectively, displayed good cytotoxicity. Com-
pounds 6 and 9a, with IC50 = 79.41 4.8 and 82.16 4.8 µM, re-
spectively, exhibited moderate cytotoxicity. However, compounds
4a, 4b, and 5 with IC50 > 100 µM displayed the lowest cytotoxicity.
Cytotoxicity evaluation against colorectal carcinoma (HCT‐116)
cell line revealed that compounds 7b and 8c displayed very good
anticancer activities with IC50 = 9.38 0.9 and 17.38 1.5 µM, re-
spectively. Compounds 8a, 8b, 9c, and 9b, with IC50 = 38.23 2.5,
39.49 2.6, 43.90 3.0, and 46.28 3.1 µM, respectively, exhibited
good cytotoxicity. Compounds 6 and 9a, with IC50 = 73.46 4.2 and
77.06 4.5 µM, displayed moderate cytotoxicity. However, com-
pounds 4a, 4b, and 5, with IC50 ranging from 93.93 4.9 to
97.43 5.3 µM, displayed the lowest cytotoxicity.
TABLE 1 In vitro cytotoxic activities of the newly synthesized
compounds against HepG2, HCT‐116, and MCF‐7 cell lines and
VEGFR‐2 kinase assay
IC50 (µM)a
Compound
HepG2
HCT116
MCF‐7
VEGFR‐2
4a
>100
97.43 5.3
95.67 5.3
93.93 4.9
73.46 4.2
5.48 0.4
9.38 0.9
38.23 2.5
39.49 2.6
17.38 1.5
77.06 4.5
46.28 3.1
43.90 3.0
5.47 0.3
99.09 5.1
96.97 5.1
96.43 5.1
86.55 4.9
7.34 0.6
8.41 0.8
49.06 3.2
44.83 3.0
14.16 1.3
88.16 5.0
55.35 3.6
54.21 3.5
7.26 0.3
9.54 0.85
8.99 0.74
6.76 0.62
3.02 0.07
0.14 0.02
0.18 0.02
0.35 0.04
0.24 0.02
0.21 0.03
3.68 0.36
1.99 0.06
1.56 0.05
0.10 0.02
4b
>100
With respect to the MCF‐7 cell line, compounds 7b and 8c
displayed very good anticancer activities with IC50 = 8.41 0.8
and 14.16 1.3 µM, respectively. Compounds 8b, 8a, 9c,
and 9b, with IC50 = 44.83 3.0, 49.06 3.2, 54.21 3.5, and
55.35 3.6 µM, respectively, exhibited good cytotoxicity. Com-
pounds 6 and 9a, with IC50 = 86.55 4.9 and 88.16 5.0 µM,
displayed moderate cytotoxicity. However, compounds 4a, 4b,
and 5, with IC50 ranging from 96.43 5.1 to 99.09 5.1 µM, dis-
played the lowest cytotoxicity.
5
>100
6
79.41 4.8
13.67 1.2
19.69 1.8
45.24 3.3
41.45 3.2
29.46 2.6
82.16 4.8
52.60 3.7
49.40 3.6
9.18 0.6
7a
7b
8a
8b
8c
9a
9b
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2.4
In vitro VEGFR‐2 kinase assay
9c
Sorafenib
All the synthesized derivatives, 4a,b−8a−c, were evaluated for
their inhibitory activities against VEGFR‐2 by using an
aIC50 values are the mean SD of three separate experiments.

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antiphosphotyrosine antibody with the Alpha Screen system (Perki-
nElmer). The results were reported as a 50% inhibition concentration
value (IC50), which is calculated from the concentration−inhibition
response curve and summarized in Table 2. Sorafenib was used as a
positive control in this assay. The tested compounds displayed high
to low inhibitory activity with IC50 values ranging from 0.14 0.02 to
9.54 0.85 µM (Figure 4). Among them, compound 7a was found to
be the most potent derivative that inhibited VEGFR‐2 at an IC50
value of 0.14 0.02 µM, which is nearly 72% of that of sorafenib IC50
value (0.10 0.02 µM). Compounds 7b, 8c, 8b, and 8a exhibited a
very good activity with IC50 values of 0.18 0.02, 0.21 0.03,
0.24 0.02, and 0.35 0.04 µM, respectively. Also, compounds 9c
and 9b possessed good VEGFR‐2 inhibition with IC50 values of
1.56 0.05 and 1.99 0.06 µM, respectively. Finally, compounds 4a,
4b, 5, 6, and 9a displayed the lowest VEGFR‐2 inhibition with IC50
values ranging from 3.02 0.07 to 9.54 0.85 µM, respectively.
FIGURE 4 IC50(µM) of the synthesized compounds and sorafenib
as vascular endothelial growth factor receptor 2 inhibitors
hydrophobic grooves formed by Arg1025, His1024, Ile1023,
Cys1022, Lys1021, Arg1020, Leu1017, Ile890, His889, and Ile886.
From the structure of the synthesized derivatives and the data
shown in Table 1, we can divide these tested compounds into three
groups. The first group contains amino and (thio)urea linkers as in
compounds 7a,b, and 8a–c. Generally, in this group, the urea linker
derivatives 7a,b exhibited higher anticancer activities than that
containing thiourea linkers 8c, 8b, and 8a, respectively. The hydro-
phobic electron‐donating (+inductive [+I]) aliphatic cyclohexyl moiety
in compound 7a showed higher anticancer activities than the hy-
drophobic electron‐withdrawing (−I) phenyl one, 7b. The more hy-
drophobic electron‐donating (+I) aliphatic cyclohexyl moiety
connected to thiourea linker in compound 8c displayed higher an-
ticancer activities than the butyl 8b and propyl 8a against the
HepG2, HCT116, and MCF‐7 cell lines, except for the propyl deri-
vative 8a, which exhibited a higher anticancer activity than the butyl
one, 8b, against MCF‐7 cell lines.
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2.5
Structure–activity relationship
The preliminary SAR study focused on the effect of hydrophobic and
electronic nature of the substituents used in this study. Also, it fo-
cused on the effect of the type, length, and the number of linkers
used and the distal moieties. The data obtained revealed that the
tested compounds displayed different levels of anticancer activity
and possessed a distinctive pattern of selectivity against the HCT116
cell lines. Generally, the 4‐phenylphthalazine scaffold, bearing dif-
ferent 4‐substituted anilines, was connected with the hydrophobic
distal moieties through amide, urea, and/or thiourea linkers con-
taining HBA−HBD. Lipophilicity and electronic nature of the distal
moieties exhibited an important role in VEGFR‐2 inhibition and,
consequently, the anticancer activities.
In molecular docking studies, the presence of the amino and urea
linkers, of both vatalanib and sorafenib in the same molecule,
like 7a,b, imparts higher VEGFR‐2 binding affinity, and consequently
higher anticancer activity, compared with either vatalanib (with only
NH linker) or sorafenib (with only urea linker). This higher affinity of
such derivative may be attributed to the increased length of the
molecular structures so as to enable the distal moieties to occupy the
The second group, 6 and 9a−c, contains shorter amide CONH
and NHCO linkers, respectively. Generally, the 4‐substituted phenyl
distal moieties, as in compounds 9c and 9b, exhibited higher activ-
ities than the unsubstituted ones, 6 and 9a, respectively, against the
HepG2, HCT116, and MCF‐7 cell lines. In this group, the arrange-
ment of CO and NH atoms is very important for the activity.
In compound 6, the NHCO linker showed higher activities than the
TABLE 2 The calculated free energy of binding (ΔG in kcal/mol) for the ligands
Compound
ΔG (kcal/mol )
−56.38
RMSD (Å)
0.98
Compound
ΔG (kcal /mol )
−88.86
RMSD (Å)
1.15
4a
4b
5
8b
−59.88
1.05
8c
−90.66
0.83
−61.67
0.95
9a
−76.50
0.92
6
−77.27
1.02
9b
−81.74
0.96
7a
7b
8a
−99.06
1.19
9c
−82.44
1.16
−94.99
1.04
Vatalanib
Sorafenib
−77.51
1.02
−87.24
1.12
−95.36
1.06
Abbreviation: RMSD, root‐mean‐square deviation.

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CONH linker in compound 9c, where the two compounds have the
same structure but only differ in the arrangement of the amide lin-
kers. The presence of hydrophobic electron‐donating (+I) methyl
group 9c showed higher activities than that substituted with hy-
drophobic electron‐withdrawing (−I) chloro group 9b.
bounded by a membrane‐binding domain that serves as an entry
channel for the substrate to the active site (Figure 5). The obtained
results of the free energy of binding (ΔG) explained that most of
these compounds had a good binding affinity toward the re-
ceptor and the computed values reflected the overall trend (Table 1).
The proposed binding mode of sorafenib revealed an affinity
value of −95.66 kcal/mol and exhibited four H‐bonds. The urea linker
formed one H‐bond with the key amino acid Glu883 (2.01 Å) through
its NH group and one H‐bond with Asp1044 (2.09 Å) through its
carbonyl group. The N‐methylpicolinamide moiety was stabilized by
the formation of two H‐bonds with Cys917, where the pyridine N
atom formed one H‐bond with the NH of Cys917 (2.51 Å), whereas
its NH group formed one H‐bond with the carbonyl of Cys917
(1.95 Å). The N‐methylpicolinamide moiety occupied the hydrophobic
groove formed by Leu1033, Gly920, Lys918, Cys917, Phe916,
Glu915, Leu838, and Ala864. Moreover, the central phenyl ring oc-
cupied the hydrophobic pocket formed by Cys1043, Leu1033,
Val914, Val897, Lys866, and Val865. Furthermore, the hydrophobic
3‐trifluoromethyl‐4‐chlorophenyl moiety attached to the urea linker
occupied the hydrophobic pocket formed by Asp1044, Ile1042,
His1024, Leu1017, His892, Gly891, Ile890, Leu887, and Glu883
(Figure 6).
The third group is the one with no distal moieties, 4a, 4b, and 5
derivatives. This group generally contains only the hydrophobic phenyl
tail substituted at position‐4 with nitro, carboxylic, and/or amino groups,
respectively. The hydrophilic electron‐donating amino group
5
(−I and +mesomeric effect [+M]) exhibited higher activities than the
hydrophilic electron‐withdrawing carboxylic group 4b (−I and −M) and
hydrophobic electron‐withdrawing nitro group 4a (−I and −M) against the
HCT116, MCF‐7, and HepG2 cell lines, respectively. The obtained data
were correlated with the Hansch equation for a linear relationship.
The data obtained from VEGFR‐2 inhibition assay concluded
that the presence of the hydrophobic distal moieties connected to
the urea linkers 7a and/or 7b exhibited higher VEGFR‐2 inhibition
activities than that attached to thiourea linkers 8c, 8b, and/or 8a,
respectively. The longer urea linkers 7a and 7b and thiourea linkers
8c, 8b, and 8a displayed higher activities than CONH 9c, 9b, NHCO
6, and CONH 9a, respectively. Finally, compounds having distal
moieties exhibited higher activities than that containing only hy-
drophobic phenyl tails, 5, 4b, and 4c, respectively.
The proposed binding mode of vatalanib revealed an affinity
value of −77.51 kcal/mol and exhibited three H‐bonds. The NH linker
formed one H‐bond with the key amino acid Glu883 (1.73 Å) through
its carbonyl group. The N‐2 of phthalazine nucleus formed one
H‐bond with the essential amino acid residue Asp1044 (2.74 Å). The
pyridine N atom also formed one H‐bond with the NH of Cys917
(2.39 Å). The pyridine moiety occupied the hydrophobic groove
formed by Leu1033, Cys917, Phe916, Glu915, Ala864, and Leu838.
Moreover, the central phthalazine scaffold occupied the hydrophobic
pocket formed by Asp1044, Cys1043, Glu915, Val914, Val897,
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2.6
Docking studies
All modeling experiments in the present work were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
the Brookhaven Protein Data Bank (PDB ID 1YWN).[49]
All studied ligands have a similar position and orientation inside
the recognized binding site of VEGFR‐2, which reveals a large space
FIGURE 5 Superimposition of some docked compounds inside the binding pocket of 1YWN

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FIGURE 6 The predicted binding mode of sorafenib with 1WYN. H‐bonded atoms are indicated by dotted lines
Lys866, Val865, and Ala864. Furthermore, the hydrophobic
4‐chlorophenyl moiety attached to the urea linker occupied the
hydrophobic pocket formed by Asp1044, Ile1042, Leu1017, His892,
Gly891, Ile890, Leu887, Ile886, and Glu883 (Figure 7).
These findings encourage us to use different linkers resembling
sorafenib and vatalanib in the same compound, hoping to obtain
potent VEGFR‐2 inhibitors.
As planned, the proposed binding mode of compound 7a is vir-
tually the same as that of sorafenib and vatalanib, which revealed an
affinity value of −99.06 kcal/mol and formed four H‐bonds. The NH
linker formed one H‐bond with the key amino acid Glu883 (1.60 Å)
The urea and NH linkers played an important role in the binding
affinities toward the VEGFR‐2 enzyme, where it was responsible for
the higher binding affinities of sorafenib and vatalanib, respectively.
FIGURE 7 The predicted binding mode of vatalanib with 1WYN. H‐bonded atoms are indicated by dotted lines

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EL‐ADL ET AL.
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through its carbonyl group, whereas the N‐2 of phthalazine nucleus
formed one H‐bond with the essential amino acid residue Asp1044
(2.95 Å). Moreover, the urea linker formed two H‐bonds with Asp1044
(2.88 Å) and His1024 (2.91 Å). The 4‐phenyl group occupied the hydro-
phobic groove formed by Leu1033, Cys917, Phe916, Glu915, and
Ala864. Moreover, the central phthalazine scaffold occupied the hydro-
phobic pocket formed by Asp1044, Cys1043, Glu915, Val914, Val897,
Lys866, Val865, and Ala864. The hydrophobic phenyl tail occupied the
hydrophobic pocket formed by Asp1044, Cys1043, Val897, His892,
Gly891, Ile890, Leu887, Ile886, and Glu883. Furthermore, the distal
cyclohexyl moiety occupied the hydrophobic groove formed by Arg1025,
His1024, Ile1023, Cys1022, Lys1021, Arg1020, Leu1017, Ile890, His889,
and Ile886 (Figure 8). These interactions may explain the highest antic-
ancer activity of compound 7a.
(Figure 9). These interactions of compound 7b may explain its high
anticancer activity.
The proposed binding mode of compound 8c is virtually the same as
that of 7a and 7b, which revealed an affinity value of −90.66 kcal/mol and
three H‐bonds. The NH linker formed one H‐bond with the key amino
acid Glu883 (1.70 Å) through its carbonyl group, whereas the N‐2 of
phthalazine nucleus formed one H‐bond with the essential amino acid
residue Asp1044 (2.58 Å). Moreover, the thiourea linker formed one
H‐bond with Asp1044 (2.75 Å). The 4‐phenyl group occupied the hy-
drophobic groove formed by Leu1033, Cys917, Phe916, Glu915, and
Ala864. Moreover, the central phthalazine scaffold occupied the hydro-
phobic pocket formed by Asp1044, Cys1043, Glu915, Val914, Val897,
Lys866, Val865, and Ala864. The hydrophobic phenyl tail occupied the
hydrophobic pocket formed by Asp1044, Cys1043, Val897, His892,
Gly891, Ile890, Leu887, Ile886, and Glut883. Furthermore, the distal
cyclohexyl moiety occupied the hydrophobic groove formed by His1024,
Cys1022, Lys1021, Arg1020, Leu1017, Ile890, His889, and Ile886
(Figure 10). These interactions of compound 8c may explain its high
anticancer activity.
The proposed binding mode of compound 7b is virtually the
same as that of 7a, which revealed an affinity value of −94.99 kcal/
mol and displayed four H‐bonds. The NH linker formed one
H‐bond with the key amino acid Glu883 (2.63 Å) through its car-
bonyl group, whereas the N‐2 of phthalazine nucleus formed one
H‐bond with the essential amino acid residue Asp1044 (2.96 Å).
Moreover, the urea linker formed two H‐bonds with Asp1044
(2.90 Å) and His1024 (2.89 Å). The 4‐phenyl group occupied the
hydrophobic pocket formed by Leu1033, Cys917, Phe916,
Glu915, and Ala864. Moreover, the central phthalazine scaffold
occupied the hydrophobic pocket formed by Asp1044, Cys1043,
Glu915, Val914, Val897, Lys866, Val865, and Ala864. The hy-
drophobic phenyl tail occupied the hydrophobic pocket formed by
Asp1044, Cys1043, Val897, His892, Gly891, Ile890, Leu887,
Ile886, and Glu883. Furthermore, the distal phenyl ring occupied
the hydrophobic groove formed by Arg1025, His1024, Ile1023,
Cys1022, Lys1021, Arg1020, Leu1017, Ile890, His889, and Ile886
From the obtained docking results (Table 2), we concluded that the
NH linker occupied the same groove occupied by NH and urea linkers of
vatalanib and sorafenib, and played the same role, which is essential for
higher affinity toward VEGFR‐2 enzyme. The distal hydrophobic moieties
increased hydrophobic interactions and, consequently, affinities
toward VEGFR‐2 enzyme. The phthalazine enables the new compounds
to form a new H‐bond through its N atom at position‐2 with the amino
acid Asp1044. The use of urea, thiourea, and/or amide linkers leads to
elongations of the structures, which plays an important role in their
VEGFR‐2 inhibitory activities. The hydrophobic distal moieties and lin-
kers formed hydrophobic and hydrogen bonding interactions, which in-
creased the affinity toward the VEGFR‐2 enzyme. However, these
FIGURE 8 The predicted binding mode of 7a with 1WYN

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FIGURE 9 The predicted binding mode of 7b with 1WYN
FIGURE 10 The predicted binding mode of 8c with 1WYN
modifications result in obtaining new phthalazine derivatives with higher
VEGFR‐2 inhibitory activities than vatalanib to be nearly equipotent to
sorafenib.
hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT‐116),
and breast cancer (MCF‐7), as VEGFR‐2 inhibitors. All the tested
compounds showed variable anticancer activities. The molecular
modeling was performed to investigate the binding mode of the
proposed compounds with the VEGFR‐2 active site. The data ob-
tained from biological testing highly correlated with that obtained
from molecular modeling studies. Investigations of the cytotoxic
activity indicated that HCT‐116 and MCF‐7 were the most sensitive
cell lines to the influence of the new derivatives, respectively.
In particular, compound 7a was found to be the most potent
|
3
CONCLUSION
In summary, 12 new N‐substituted‐4‐phenylphthalazin‐1‐amine de-
rivatives have been designed, synthesized, and evaluated for their
anticancer activities against three human tumor cell lines,

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derivative over all the tested compounds against the HepG2,
HCT116, and MCF‐7 cancer cell lines with IC50 = 13.67 1.2,
5.48 0.4, and 7.34 0.6 µM, respectively, which is nearly equipo-
tent to that of sorafenib (IC50 = 9.18 0.6, 5.47 0.3, and
7.26 0.3 µM, respectively). All synthesized derivatives 4a,b−8a−c
were evaluated for their inhibitory activities against VEGFR‐2. The
tested compounds displayed a high to low inhibitory activity with
IC50 values ranging from 0.14 0.02 to 9.54 0.85 µM. Among them,
compound 7a was found to be the most potent derivative that in-
hibited VEGFR‐2 at an IC50 value of 0.14 0.02 µM, which is nearly
72% of that of the sorafenib IC50 value (0.10 0.02 µM). Compounds
7b, 8c, 8b, and 8a exhibited a very good activity with IC50 values of
0.18 0.02, 0.21 0.03, 0.24 0.02, and 0.35 0.04 µM, respec-
tively. Also, compounds 9c and 9b possessed good VEGFR‐2 inhibi-
tion with IC50 values of 1.56 0.05 and 1.99 0.06 µM, respectively.
From the obtained docking results, it was concluded that the NH
linker occupied the same groove occupied by NH and urea linkers of
vatalanib and sorafenib, respectively, and played the same role,
which is essential for higher affinity toward the VEGFR‐2 enzyme.
The distal hydrophobic moieties increased hydrophobic interactions
and, consequently, affinities toward the VEGFR‐2 enzyme. The
phthalazine enables the new compounds to form a new H‐bond
through its N atom at position‐2 with the amino acid Asp1044. The
use of urea, thiourea, and/or amide linkers leads to elongations of the
structures and enables the hydrophobic distal moieties to occupy a
new hydrophobic groove, which increases the affinity toward the
VEGFR‐2 enzyme. However, these modifications result in obtaining
new phthalazine derivatives with higher VEGFR‐2 binding affinities
than vatalanib to be nearly equipotent to sorafenib.
Inlet part to Single Quadropole mass analyzer in Thermo Scientific
GCMS model ISQ LT using Thermo X‐Calibur software at the
Regional Center for Mycology and Biotechnology, Al‐Azhar
University. Elemental analyses (C, H, N) were performed on a CHN
analyzer at Regional Center for Mycology and Biotechnology, Al‐
Azhar University. All compounds were within 0.4 of the theoretical
values. The reactions were monitored by thin‐layer chromatography
(TLC) using TLC sheets precoated with UV fluorescent silica gel
Merck 60 F254 plates and were visualized using a UV lamp and
different solvents as mobile phases.
4‐Phenylphthalazin‐1(2H)‐one (2) and 1‐chloro‐4‐phenylph
thalazine (3) were obtained according to the reported
procedures.[3,4]
The original spectra of the investigated compounds, together
with their InChI codes and some biological activity data, are provided
as Supporting Information.
|
4.1.2
General procedure for the synthesis of
compounds 4a,b
Equimolar quantities of 1‐chloro‐4‐phenylphthalazine (3) (2.40 g,
0.01 mol) and the appropriate p‐substituted aniline, namely, 4‐
nitroaniline and/or 4‐aminobenzoic acid (0.01 mol), in acetonitrile
(20 ml) were heated under reflux for 6 h. The mixture was left to cool
and the separated solid was filtered and crystallized from ethanol to
give the target compounds 4a,b, respectively.
N‐(4‐Nitrophenyl)‐4‐phenylphthalazin‐1‐amine (4a)
Yield, 84%; m.p. 180–182°C; IRνmax (cm−1): 3,418 (N–H), 3,073 (C–H
aromatic), 1,513 (C═N), and 1,334 (NO2); 1H NMR (400 MHz,
dimethyl sulfoxide [DMSO]‐d6): 7.69–7.71 (m, 3H, H‐3, H‐4, H‐5 of
C6H5), 7.77 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 6.22), 8.05 (d, 1H, H‐5
of phthalazine, J = 8.20), 8.14 (d, 1H, H‐6 of phthalazine, J = 7.36),
8.18 (d, 2H, H‐2, H‐6 of C6H5, J = 6.12), 8.29 (d, 1H, H‐7 of phtha-
lazine, J = 7.76), 8.33 (d, 2H, H‐2, H‐6 of NH–C6H4, J = 6.22), 9.01
(d, 1H, H8 of phthalazine, J = 8.28), and 10.33 (s, 1H, NH, D2O ex-
changeable); Anal. calcd. for C20H14N4O2 (342.11): C, 70.17; H, 4.12;
N, 16.37. Found: C, 69.89; H, 4.34; N, 16.53.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All melting points were determined by the open capillary method on
a Gallenkamp melting point apparatus at the Faculty of Pharmacy
Al‐Azhar University and were uncorrected. The infrared spectra
were recorded on a pyeUnicam SP 1000 IR spectrophotometer at
the Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al‐Azhar
University, using the potassium bromide disc technique. The proton
magnetic resonance 1H NMR spectra were recorded on a Jeol 400
MHZ‐NMR spectrophotometer at Microanalytical Center, Faculty of
Pharmacy, Ain Shams University, and a Jeol 400 MHZ‐NMR spec-
trophotometer at Microanalytical Unit, Faculty of Pharmacy, Cairo
University. The 13C NMR spectra were recorded on a Bruker 100
MHZ‐NMR spectrophotometer at Microanalytical Unit, Faculty of
Pharmacy, Cairo University. Tetramethylsilane was used as an in-
ternal standard and chemical shifts were measured in the δ scale
(ppm). The mass spectra were performed on Direct Probe Controller
4‐[(4‐Phenylphthalazin‐1‐yl)amino]benzoic acid (4b)
Yield, 82%; m.p. 245–247°C; IRνmax (cm−1): 3,363 (NH), 3,039
(C–H aromatic), 1,786 (C═O acid), and 1,541 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 7.68–7.72 (m, 3H, H‐3, H‐4, H‐5 of C6H5),
7.74 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 8.00), 7.97–8.02 (m, 5H,
H‐2, H‐6 of C6H5, H‐2, H‐6 of NH–C6H4 and H‐5 of phthalazine),
8.13 (t, 1H, H‐6 of phthalazine, J = 8.60), 8.25 (t, 1H, H‐7 of
phthalazine, J = 8.60), 9.12 (d, 1H, H‐8 of phthalazine, J = 7.8 Hz),
and 10.93 (s, 1H, OH, D2O exchangeable); MS (m/z): 342 (M++1,
11.12%), 341 (M+, 56.49%), and 340 (M+−1, 100%, base peak);
Anal. calcd. for C21H15N3O2 (341.12): C, 73.89; H, 4.43; N, 12.31.
Found: C, 74.15; H, 4.62; N, 12.58.

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Synthesis of N1‐(4‐phenylphthalazin‐1‐yl)-
(10.98%); Anal. calcd. for C27H20N4O (416.16): C, 77.87; H, 4.84; N,
13.45. Found: C, 78.21; H, 4.97; N, 13.62.
|
4.1.3
benzene‐1,4‐diamine (5)
A
mixture of N‐(4‐nitrophenyl)‐4‐phenylphthalazin‐1‐amine (4a)
|
General procedure for the synthesis of
compounds 7a,b
(3.42 g, 0.01 mol) and SnCl2 (19.0 g, 0. 1 mol) was heated under reflux
in ethanol (100 ml) for 4 h in the presence of a catalytic amount of
HCl (0.02 M). After cooling to room temperature, the reaction mix-
ture was neutralized with aqueous NaOH. The ethanol was evapo-
rated and the aqueous solution was continuously extracted with
ether. The ether solution was dried over Na2SO4, concentrated, and
the precipitated solid was washed with water, filtered, dried, and
crystallized from ethanol to give the corresponding target com-
pound (5). Yield, 62%; m.p. 217–219°C; IRνmax (cm−1): 3,291 (NH2),
3,167 (NH), 3,014 (C–H aromatic), and 1,506 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 4.93 (s, 2H, NH2, D2O exchangeable), 6.63
(d, 2H, H‐3, H‐5 of NH–C6H4, J = 8.80), 7.56–7.61 (m, 5H, H‐3, H‐4,
H‐5 of C6H5 and H‐2, H‐6 of NH–C6H4), 7.62 (d, 2H, H‐2,H‐6 of
C6H5, J = 9.60), 7.81 (d, 2H, H‐6, H‐7 of phthalazine, J = 7.40), 7.92 (d,
1H, H‐5 of phthalazine, J = 7.40), 8.59 (d, 1H, H‐8 of phthalazine,
J = 9.60), and 8.95 (s, 1H, NH, D2O exchangeable); MS (m/z): 313
(M++1, 13.43%), 314 (M+, 72.98%), 311 (M+−1, 100%, base peak),
and 77 (6.07%); Anal. calcd. for C20H16N4 (312.14): C, 76.90; H, 5.16;
N, 17.94. Found: C, 76.72; H, 5.33; N, 18.12.
4.1.5
A mixture of the N1‐(4‐phenylphthalazin‐1‐yl)benzene‐1,4‐diamine 5
(0.31 g, 0.001 mol) and the appropriate isocyanate, namely, cyclo-
hexylisocyanate and/or phenylisocyanate (0.001 mol), was refluxed
in absolute ethanol (25 ml) for 3 h. The solution was cooled and the
obtained solid was filtered and recrystallized from ethanol to pro-
duce the corresponding urea derivatives 7a,b, respectively.
1‐Cyclohexyl‐3‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}urea (7a)
Yield, 83%; m.p. 262–264°C; IRνmax (cm−1): 3,207 (3NH), 3,010 (C–H
aromatic), 2,931 (C–H aliphatic), and 1,656 (C═O amide); 1H NMR
(400 MHz, DMSO‐d6): 1.06–1.19 (m, 2H, C‐4 of cyclohexyl),
1.32–1.58 (m, 4H, C‐3 and C‐5 of cyclohexyl), 1.64–1.82 (m, 4H, C‐2
and C‐6 of cyclohexyl), 3.74–3.78 (m, 1H, C‐1 of cyclohexyl), 6.03
(s, 1H, NH‐cyclohexyl, D2O exchangeable), 7.39 (d, 2H, H‐3, H‐5 of
NH–C6H4, J = 8.00), 7.56–7.70 (m, 5H, H‐3, H‐5 of C6H4 and H‐2, H‐6
of NH–C6H4), 7.78 (m, 5H, H‐2, H‐6 of C6H5, H‐5, H‐6 of phthhala-
zine, NH–C═O, D2O exchangeable), 8.05 (d, 1H, H‐5 of phthalazine,
J = 7.40), 8.27 (d, 1H, H‐7 of phthalazine, J = 8.80), 8.64 (d, 1H, H‐8 of
phthalazine, J = 8.20), and 9.14 (s, 1H, NH–C6H4, D2O exchangeable);
Anal. calcd. for C27H27N5O (437.22): C, 74.12; H, 6.22; N, 16.01.
Found: C, 74.38; H, 6.39; N, 16.34.
|
4.1.4
Synthesis of N‐{4‐[(4‐phenylphthalazin‐1‐yl)-
amino]phenyl}benzamide (6)
A mixture of benzoic acid (0.19 g, 0.0016 mol) and triethanola-
mine (TEA) (0.18 g, 0.0018 mol) in methylene chloride (20 ml) was
put in an ice−salt bath while stirring. Next, a solution of ethyl
chloroformate (0.19 g, 0.0018 mol) in methylene chloride (10 ml) was
added to the reaction mixture dropwise for over 20 min and left for
1 h while stirring at −10°C to −5°C. Then, N1‐(4‐phenylphthalazin‐1‐
yl)benzene‐1,4‐diamine (5) (0.46 g, 0.15 mmol) was added to the re-
action mixture portionwise. The reaction mixture was stirred for
3 h at ambient temperature. The formed precipitate was filtered and
washed with water and ethanol to afford the target compound (6).
Yield, 75%; m.p. 247–249°C; IRνmax (cm−1): 3,207 (2NH), 3,010 (C–H
aromatic), and 1,656 (C═O amide); 1H NMR (400 MHz, DMSO‐d6):
7.52 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 7.40), 7.59 (d, 1H, H‐4 of
C═O–C6H5, J = 6.80), 7.64–7.68 (m, 4H, H‐2, H‐6 of NH–C6H4 and
H‐3, H‐5 of C═O–C6H4), 7.69–7.72 (m, 3H, H‐3, H‐4, H‐5 of C6H5),
7.95 (t, 1H, H‐5 of phthalazine, J = 7.20), 8.01 (dd, 4H, H‐2, H‐6 of
C6H5, H‐2, H‐6 of C═O–C6H4, J = 8.40), 8.16 (t, 1H, H‐6 of phtha-
lazine, J = 7.00), 8.23 (t, 1H, H‐7 of phthalazine, J = 7.00), 9.27 (s, 1H,
NH, D2O exchangeable), and 11.70 (s, 1H, NH–C═O, D2O ex-
changeable); 13C NMR (100 MHz, DMSO‐d6): 119, 121.68, 121.83
(2C), 125.88, 126.01, 126.57, 127.67, 128.24 (2C), 128.50, 128.89
(2C), 129.37 (2C), 130.26 (2C), 130.83, 132.17, 134.82, 135.19,
135.98, 138.72, 152.35, 152.80, 154.08 and 166.11; MS (m/z): 417
(M+1, 27.43%), 416 (M+, 100% base peak), 415 (M+−1, 90%), and 77
1‐Phenyl‐3‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}urea (7b)
Yield, 85%; m.p. 253–255°C; IRνmax (cm−1): 3,489, 3,252, 3,260
(3NH), 3,042 (C–H aromatic), 1,681 (C═O amide), and 1,545
(C═N); 1H NMR (400 MHz, DMSO‐d6): 6.97 (t, 1H, H‐4 of NH‐C6H5,
J = 8.40), 7.26 (dd, 2H, H‐3, H‐5 of NH‐C6H5, J = 8.80), 7.47 (m, 4H,
H‐2, H‐6 of NH–C6H5 and H‐3, H‐5 of NH–C6H4), 7.53–7.59 (m, 3H,
H‐3, H‐4, H‐5 of C6H5), 7.65 (d, 2H, H‐2, H‐6 of NH–C6H4, J = 7.60),
7.85–7.94 (m, 4H, H‐2, H‐6 of C6H5 and H‐6, H‐7 of phthalazine),
7.99 (d, 1H, H‐5 of phthalazine, J = 8.60), 8.64–8.67 (m, 3H, H‐8 of
phthalazine, NH–C═O–NH, D2O exchangeable), and 9.20 (s, 1H, NH,
D2O exchangeable); MS (m/z): 431 (M+, 2.25), 430 (M++1, 1.13), and
337 (100%, base peak); Anal. calcd. for C27H21N5O (431.17): C,
75.16; H, 4.91; N, 16.23. Found: C, 75.34; H, 5.12; N, 16.45.
|
4.1.6
General procedure for the synthesis of
compounds 8a–c
A mixture of the N1‐(4‐phenylphthalazin‐1‐yl)benzene‐1,4‐diamine 5
(0.31 g, 0.001 mol) and the appropriate isothiocyanate, namely, pro-
pyl isothiocyanate, butyl isothiocyanate, and/or cyclohexyl iso-
thiocyanate (0.001 mol), was refluxed in absolute ethanol (25 ml) for
3 h. The solution was cooled and the formed solid was filtered and

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General procedure for the synthesis of
recrystallized from ethanol to obtain the corresponding thiourea
4.1.7
derivatives 8a−c, respectively.
compounds 9a–c
1‐{4‐[(4‐Phenylphthalazin‐1‐yl)amino]phenyl}‐3‐propylthiourea (8a)
Yield, 83%; m.p. 202–204°C; IRνmax (cm−1): 3,194 (3NH), 3,095 (C–H
aromatic), 2,993 (C–H aliphatic), and 1,634 (C═O amide); 1H NMR
(400 MHz, DMSO‐d6): 0.87 (t, 3H, CH3, J = 8), 1.52 (q, 2H, CH2CH3,
J = 8.00), 3.44 (t, 2H, CH2N, J = 8.00), 7.36 (d, 2H, H‐3, H‐5 of
NH–C6H4, J = 8.40), 7.52–7.56 (m, 3H, H‐3, H‐4, H‐5 of C6H5),
7.65–7.68 (m, 3H, H‐2, H‐6 of NH‐C6H4 and NH–CH2, D2O ex-
changeable), 7.87–7.94 (m, 4H, H‐2, H‐6 of C6H5 and H‐6, H‐7 of
phthalazine), 7.99 (d, 1H, H‐5 of phthalazine, J = 9.00), 8.65 (d, 1H,
H‐8 of phthalazine, J = 10.80), 9.29 (s, 1H, NH–C═S, D2O ex-
changeable), and 9.40 (s, 1H, NH–C6H4, D2O exchangeable); 13C
NMR (100 MHz, DMSO‐d6): 11.86, 22.35, 46.12, 118.90 (2C),
121.66, 123.12, 124.49, 126.20, 128.89, 129.01, 130.13 (2C), 132.05
(2C), 132.63 (2C), 133.94, 137.24 (2C), 137.78, 152.29, 153.77, and
180.91; Anal. calcd. for C24H23N5S (413.17): C, 69.71; H, 5.61; N,
16.94. Found: C, 69.47; H, 5.87; N, 17.12.
A mixture of 4‐[(4‐phenylphthalazin‐1‐yl)amino]benzoic acid (4b)
(0.50 g, 1.48 mmol) and TEA (0.18 g, 1.78 mmol) in methylene chlor-
ide (20 ml) was put in an ice−salt bath with stirring. Next, a solution
of ethyl chloroformate (0.19 g, 1.77 mmol) in methylene chloride
(10 ml) was added to reaction mixture dropwise for over 20 min and
left for 1 h while stirring at −10 to −5°C. Then, the appropriate
amine, namely, aniline, 4‐chloroaniline, and/or 4‐methylaniline
(1.48 mmol), was added to the reaction mixture portionwise.
The reaction mixture was stirred for 3 h at ambient temperature.
The formed precipitate was filtered and washed with water and
ethanol to afford the corresponding compounds 9a−c, respectively.
N‐Phenyl‐4‐[(4‐phenylphthalazin‐1‐yl)amino]benzamide (9a)
Yield, 83%; m.p. 253–255°C; IRνmax (cm−1): 3,365 (2NH), 3,095 (C–H
aromatic), and 1,671 (C═O amide); 1H NMR (400 MHz, DMSO‐d6): 7.15
(t, 1H, H‐4 of NH–C6H5, J = 10.80), 7.35 (t, 2H, H3, H‐5 of NH–C6H5,
J = 10.80), 7.69–7.72 (m, 3H, H‐3, H‐4, H‐5 of C6H5), 7.75 (d, 2H, H‐3, H‐5
of NH–C6H4, J = 8.00), 7.82 (d, 2H, H‐2, H‐6 of NH–C6H5, J = 10.40), 7.99
(d, 2H, H‐2, H‐6 of C6H5, J = 7.20), 8.04 (t, 1H, H‐5 of phthalazine,
J = 6.40), 8.13 (d, 2H, H‐2, H‐6 of NH–C6H4, J = 10.00), 8.18 (t, 1H, H‐6 of
phthalazine, J = 7.60), 8.31 (t, 1H, H‐7 of phthalazine, J = 7.60), 9.08
(d, 1H, H‐8 of phthalazine, J = 8.40), 10.33 (s, 1H, NH–C6H4, exchange-
able with D2O), and 10.93 (s, 1H, C═O–NH, exchangeable with D2O);
Anal. calcd. for C27H20N4O (416.16): C, 77.87; H, 4.84; N, 13.45. Found:
C, 78.11; H, 4.97; N, 13.69.
1‐Butyl‐3‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}thiourea (8b)
Yield, 85%; m.p. 216–218°C; IRνmax (cm−1): 3,177 (3NH), 3,050 (C–H
aromatic), 2,978 (C–H aliphatic), 1,675 (C═O amide); 1H NMR
(400 MHz, DMSO‐d6): 0.89 (t, 3H, CH3), 1.28 (m, 2H, –CH2–CH3),
1.50 (m, 1H, CH2CH2CH3), 3.48 (t, 2H, CH2CH2CH2CH3), 7.35 (d, 2H,
H‐3, H‐5 of NH–C6H4, J = 9.60), 7.53–7.60 (m, 4H, H‐3, H‐4, H‐5 of
C6H5 and NH‐CH2, D2O exchangeable), 7.65 (d, 4H, H‐2,H‐6 of NH‐
C6H4,J = 8.80), 7.86–7.95 (m, 4H, H‐2, H‐6 of C6H5 and H‐6, H‐7 of
phthalazine), 8.02 (d, 1H, H‐5 of phthalazine, J = 7.20), 8.65 (d, 1H,
H‐8 of phthalazine, J = 9.20), 9.30 (s, 1H, NH–C═S, D2O exchange-
able), 9.38 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz,
DMSO‐d6): 14.23, 20.10, 31.20, 44.08, 118.90, 121.66 (2C), 123.12,
124.43 (2C), 126.20 (2C), 128.89 (2C), 129.01, 130.13 (2C), 132.04,
132.63, 133.94, 137.25, 137.76, 152.28, 153.76, and 180.85 (C═S);
Anal. calcd. for C25H25N5S (427.18): C, 70.23; H, 5.89; N, 16.38.
Found: C, 70.05; H, 5.97; N, 16.59.
N‐(4‐Chlorophenyl)‐4‐[(4‐phenylphthalazin‐1‐yl)amino]benzamide (9b)
Yield, 81%; m.p. 287–289°C; IRνmax (cm−1): 3,143 (2NH), 3,040 (C–H
aromatic), and 1,667 (C═O amide); 1H NMR (400 MHz, DMSO‐d6):
7.32 (d, 2H, H‐3, H‐5 of 4‐Cl–C6H4, J = 8.00), 7.49 (d, 2H, H3, H‐5 of
C6H5–NH, J = 10.40), 7.57–7.64 (m, 3H, H‐3, H‐4, H‐5 of C6H5), 7.71
(d, 2H, H‐4, H‐6 of 4‐Cl–C6H4, J = 6.00), 7.95–8.04 (m, 3H, H‐2, H‐6
of C6H5 and H‐5 of phthalazine), 8.08 (dd, 2H, H‐2, H‐6 of NH–C6H4,
J = 8.40), 8.16 (d, 1H, H‐6 of phthalazine, J = 10.80), 8.27 (d, 1H, H‐7
of phthalazine, J = 10.80), 8.71 (d, 1H, H‐8 of phthalazine, J = 8.40),
9.09 (s, 1H, NH–C6H4, D2O exchangeable), and 9.88 (s, 1H, C═O‐NH,
D2O exchangeable); Anal. calcd. for C27H19ClN4O (450.12): C, 71.92;
H, 4.25; N, 12.43. Found: C, 71.69; H, 4.38; N, 12.08.
1‐Cyclohexyl‐3‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}-
thiourea (8c)
Yield, 81%; m.p. 230–232°C; IRνmax (cm−1): 3,143 (3NH), 3,040
(C–H aromatic), 2,965 (C–H aliphatic), and 1,667 (C═O
amide); 1H NMR (400 MHz, DMSO‐d6): 1.01–1.08 (m, 2H, C‐4 of
cyclohexyl), 1.23–1.58 (m, 4H, C‐3 and C‐5 of cyclohexyl),
1.66–1.82 (m, 4H, C‐2 and C‐6 of cyclohexyl), 4.11–4.16 (m, 1H,
C‐1 of cyclohexyl), 7.42 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 8.00),
7.50–7.57 (m, 4H, H‐3, H‐4, H‐5 of C6H5, NH‐cyclohexyl, D2O
exchangeable), 7.67 (d, 2H, H‐2, H‐6 of NH–C6H4, J = 8.00),
7.90–7.98 (m, 4H, H‐2, H‐6 of C6H5 and H‐6, H‐7 of phthalazine),
8.03 (d, 1H, H‐5 of phthalazine, J = 10.00), 8.12–8.14 (d, 2H, H‐6,
H‐7 of phthalazine), 8.67 (d, 1H, H‐8 of phthalazine, J = 8.00),
9.28 (s, 1H, NH–C═S, D2O exchangeable), and 9.58 (s, 1H, NH‐
C6H4, D2O exchangeable); Anal. calcd. for C27H27N5S (453.20): C,
71.49; H, 6.00; N, 15.44. Found: C, 71.68; H, 6.13; N, 15.51.
4‐[(4‐Phenylphthalazin‐1‐yl)amino]‐N‐(p‐tolyl)benzamide (9c)
Yield, 85%; m.p. 262–264°C; IRνmax (cm−1): 3,320 (2NH), 3,065 (C–H
aromatic), 2,965 (C–H aliphatic), 1,660 (C═O amide); 1H NMR
(400 MHz, DMSO‐d6): 2.29 (s, 3H, CH3), 7.15 (d, 2H, H‐3, H‐5 of
4‐CH3–C6H4, J = 10.80), 7.55–7.63 (m, 3H, H‐3, H‐4, H‐5 of C6H5),
7.68–7.701 (m, 4H, H‐2, H‐6 of C6H4–CH3 and H‐3, H‐5 of
NH–C6H4), 7.90–8.09 (m, 5H, H‐2, H‐6 of C6H5, H‐2, H‐6 of
NH–C6H4 and H‐5 of phthalazine), 8.17 (d, 2H, H‐6, H‐7 of phtha-
lazine, J = 10.00), 8.72 (d, 1H, H‐8 of phthalazine, J = 7.60), 9.62
(s, 1H, NH–C6H4, D2O exchangeable) and 10.07 (s, 1H, C═O–NH,
D2O exchangeable); MS (m/z): 363 (M+, 3.5%), 256 (71.6%), 82 (base

EL‐ADL ET AL.
15 of 16
|
|
beak, 100%), and 76 (17.84%); Anal. calcd. for C28H22N4O (430.18):
C, 78.12; H, 5.15; N, 13.01. Found: C, 78.40; H, 4.98; N, 13.23.
4.4
In vitro VEGFR‐2 kinase assay
The kinase activity of VEGFR‐2 was measured by using an antipho-
sphotyrosine antibody with the Alpha Screen system (PerkinElmer) ac-
cording to the manufacturer's instructions.[50] Enzyme reactions were
performed in 50 mM Tris‐HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01%
Tween‐20, and 2 mM dithiothreitol, containing 10 μM ATP, 0.1 μg/ml
biotinylated poly‐GluTyr (4:1), and 0.1 nM VEGFR‐2 (Millipore). Before
catalytic initiation with ATP, the tested compounds at final concentra-
tions ranging from 0 to 300 μg/ml and enzyme were incubated for 5 min
at room temperature. The reactions were quenched by the addition of
25 μl of 100 mM EDTA, 10 μg/ml Alpha Screen streptavidin donor beads,
and 10 μg/ml acceptor beads in 62.5 mM 4‐(2‐hydroxyethyl)‐1‐
piperazineethanesulfonic acid, pH 7.4, 250 mM NaCl, and 0.1% bovine
serum albumin. The plate was incubated in the dark overnight and then
read by ELISA Reader (PerkinElmer). Wells containing the substrate and
the enzyme without compounds were used as the reaction control. Wells
containing biotinylated poly‐GluTyr (4:1) and enzyme without ATP
were used as the basal control. The percent inhibition was calculated by
the comparison of compounds treated with control incubations. The
concentration of the test compound causing 50% inhibition (IC50) was
calculated from the concentration–inhibition response curve (triplicate
determinations) and the data were compared with sorafenib (Sigma‐
Aldrich) as a standard VEGFR‐2 inhibitor.
|
4.2
Docking studies
In the present work, all the target compounds were subjected to
docking study to explore their binding mode toward the VEGFR‐2
enzyme. All modeling experiments were performed using molsoft
program, which provides a unique set of tools for the modeling of
protein/ligand interactions. It predicts how small flexible molecule
such as substrates or drug candidates bind to a protein of known
three‐dimensional structure represented by grid interaction poten-
tials (http://www.molsoft.com/icm_pro.html). Each experiment used
the biological target VEGFR‐2 downloaded from the Brookhaven
Protein Data Bank (http://www.rcsb.org/pdb/explore/explore.do?
structureId=1YWN). To qualify the docking results in terms of ac-
curacy of the predicted binding conformations in comparison with
the experimental procedure, the reported VEGFR‐2 inhibitor drugs
vatalanib and sorafenib were used as reference ligands.
|
4.3
In vitro cytotoxic activity
The cytotoxicity assays were performed at Pharmacology and Toxicol-
ogy Department, Faculty of Pharmacy, Al‐Azhar University, Cairo, Egypt.
Cancer cells from different cancer cell lines, hepatocellular carcinoma
(HepG2), colorectal carcinoma (HCT‐116), and breast cancer (MCF‐7),
were purchased from American Type Cell Culture Collection and grown
on the appropriate growth medium, Roswell Park Memorial Institute
medium‐1640, supplemented with 100 mg/ml of streptomycin, 100
units/ml of penicillin, and 10% of heat‐inactivated fetal bovine serum, in a
humidified, 5% (v/v) CO2 atmosphere at 37°C. Cytotoxicity assay was
carried out by using 3‐[4,5‐dimethylthiazole‐2‐yl]‐2,5‐diphenyltetrazolium
bromide (MTT).
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
ORCID
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
Hamada S. Abulkhair
http://orcid.org/0000-0001-6479-4573
http://orcid.org/0000-0002-6955-2263
Ibrahim H. Eissa
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How to cite this article: El‐Adl K, Ibrahim M‐K, Khedr F,
Abulkhair HS, Eissa IH. N‐substituted‐4‐phenylphthalazin‐1‐
amine‐derived VEGFR‐2 inhibitors: Design, synthesis, molecular
docking, and anticancer evaluation studies. Arch Pharm.
2020;e2000219. https://doi.org/10.1002/ardp.202000219
[29] G. Bold, K.‐H. Altmann, J. Frei, M. Lang, P. W. Manley, P. Traxler,
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P. Furet, F. Hofmann, G. Martiny‐Baron, J. Mestan, J. Rösel,
M. Sills, D. Stover, F. Acemoglu, E. Boss, R. Emmenegger,
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