Phthalazine-based VEGFR-2 inhibitors: Rationale, design, synthesis, in silico, ADMET profile, docking, and anticancer evaluations

Article abstract of DOI:10.1002/ardp.202100201

In the designed compounds, a new linker was inserted in the form of fragments with verified VEGFR-2 inhibitory potential, including an α,β-unsaturated ketonic fragment, pyrazole, and pyrimidine. Also, new distal hydrophobic moieties were attached to these

Full text of DOI:10.1002/ardp.202100201

Received: 27 May 2021
Revised: 28 July 2021
Accepted: 30 July 2021
|
|
DOI: 10.1002/ardp.202100201
F U L L P A P E R
Phthalazine‐based VEGFR‐2 inhibitors: Rationale, design,
synthesis, in silico, ADMET profile, docking, and anticancer
evaluations
Fathalla Khedr1 | Mohamed‐Kamal Ibrahim1 | Ibrahim H. Eissa1
|
Hamada S. Abulkhair3,4
| Khaled El‐Adl1,2
1Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University,
Nasr City, Cairo, Egypt
Abstract
In the designed compounds, a new linker was inserted in the form of fragments with
verified VEGFR‐2 inhibitory potential, including an α,β‐unsaturated ketonic frag-
ment, pyrazole, and pyrimidine. Also, new distal hydrophobic moieties were attached
to these linkers that are expected to increase the hydrophobic interaction with
VEGFR‐2 and, consequently, the affinity. These structural optimizations have led us
to identify the novel dihydropyrazole derivative 6e as a promising hit molecule. All
the new derivatives were evaluated to assess their anticancer activity against three
human cancer cell lines, including HepG2, HCT‐116, and MCF‐7. The results of the
in vitro anticancer evaluation study revealed the moderate to excellent cytotoxicity
of 6c, 6e, 6g, and 7b, with IC50 values in the low micromolar range. The inhibitory
activity of VEGFR‐2 was investigated for 16 of the designed compounds. The en-
zyme assay results of the new compounds were compared with those of sorafenib as
a reference VEGFR‐2 inhibitor. The obtained results demonstrated that our deri-
vatives are potent VEGFR‐2 inhibitors. The most potent derivatives 6c, 6e, 6g, and 7b
showed IC50 values in the range of 0.11–0.22 µM. Molecular docking and pharma-
cokinetic studies were also conducted to rationalize the VEGFR‐2 inhibitory activity
and to evaluate the ability of the most potent derivatives to be developed as good
drug candidates.
2Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
3Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy, Al‐Azhar
University, Nasr City, Cairo, Egypt
4Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Horus University–Egypt,
New Damietta, Egypt
Correspondence
Khaled El‐Adl and Ibrahim H. Eissa,
Pharmaceutical Medicinal Chemistry and Drug
Design Department, Faculty of Pharmacy
(Boys), Al‐Azhar University, Nasr City 11884,
Cairo, Egypt.
Email: eladlkhaled74@yahoo.com,
eladlkhaled74@azhar.edu.eg,
khaled.eladl@hu.edu.eg and
ibrahimeissa@azhar.edu.eg
Hamada S. Abulkhair, Pharmaceutical Organic
Chemistry Department, Faculty of Pharmacy,
Al‐Azhar University, Nasr City 11884, Cairo,
Egypt.
Email: hamadaorganic@azhar.edu.eg
K E Y W O R D S
anticancer, dihydropyrazole, molecular docking, phthalazines, pyrimidinone, VEGFR‐2
inhibitors
pathological angiogenesis mechanisms, such as cancer.[1] Over-
expression of this receptor has been observed in a number of solid
|
1
INTRODUCTION
Vascular endothelial growth factors (VEGF) are transmembrane
proteins that trigger angiogenesis through VEGF receptor (VEGFR)
signaling.[1] To date, three types of VEGF tyrosine kinase receptors
are known: VEGFR‐1, VEGFR‐2, and VEGFR‐3. All of them display a
high affinity toward VEGF. However, VEGFR‐2 is the only receptor
that transmits the angiogenic signals.[2] The VEGFR‐2 receptor has
been documented to play major roles in both physiological and many
cancers[3] and hematological malignancies.[4] Consequently, VEGFR‐2
is an ideal target for the development of novel chemotherapeutic
agents.[3] Tyrosine kinase inhibitors are grouped into two types: type
I and type II.[5] Unlike type I, type II inhibitors interact with the
adenosine triphosphate (ATP) binding site as well as the allosteric
hydrophobic site and display high selectivity.[6,7] The architectural
design of the VEGFR‐2 active site comprises two pockets: the front
|
Arch. Pharm. 2021;e2100201.
wileyonlinelibrary.com/journal/ardp © 2021 Deutsche Pharmazeutische Gesellschaft
1 of 18
https://doi.org/10.1002/ardp.202100201

2 of 18
KHEDR ET AL.
|
and the back. The front pocket has two associated key residues,
Glu917 and Cys919, while the back hydrophobic pocket has another
two key residues: Glu885 and Asp1046.[8] The pharmacophoric
features shared by all of the reported VEGFR‐2 inhibitors showed the
presence of four main features as shown in Figure 1:[9,10] (i) the core
structure, which occupies the catalytic ATP‐binding domain and
consists of a flat aromatic ring; (ii) the central hydrophobic spacer,
which occupies the linker region between the ATP‐binding domain
and the DFG domain; (iii) a hydrophilic linker, for example, amino or
urea, to act as both a H‐bond donor and acceptor with the above‐
mentioned two key amino acid residues, Glu885 and Asp1046; and
(iv) a terminal hydrophobic moiety to occupy the allosteric hydro-
phobic pocket.[11]
binding site.[26,27] Moreover, the α,β‐unsaturated ketonic fragment is
also present in a number of synthetic derivatives with potent VEGFR‐
2 inhibitory activity.[28,29]
|
1.1
Rationale and aim of the work
Over the last few years, our research group members were inter-
ested in the construction and evaluation of heterocyclic molecules
with expected biological activity, particularly those with anticancer
activity.[30–36] The objective of this study is to develop a novel
inhibitor that has the potential to interact with the VEGFR‐2
protein. Accordingly, the above‐mentioned facts have encouraged
us to design novel N‐substituted‐4‐phenylphthalazin‐1‐amine de-
rivatives linked with fragments of verified VEGFR‐2 inhibitory
potentials, including an α, β‐unsaturated ketonic fragment,[28,29]
pyrazole,[23,24] and pyrimidine, to investigate the anticancer and
VEGFR‐2 inhibitory potential of the designed compounds. All the
designed compounds retained the essential pharmacophoric fea-
tures of the reported and clinically used VEGFR‐2 inhibitors
(Figure 2), with two additional bioisosteric modifications: (i)
insertion of a new bioisosteric linker with that reported in the most
recent molecular docking and molecular dynamic simulation
studies.[13] The new linker was inserted in the form of pharma-
cophoric fragments with reported anticancer potential including an
α,β‐unsaturated ketonic fragment,[28,29] dihydropyrazole,[23,24]
and pyrimidine[25,26] and (ii) attachment of new distal bioisosteric
hydrophobic moieties with that of AMG 900 (IV) that are expected
to increase the hydrophobic interaction with VEGFR‐2 and con-
sequently the affinity. The substitution patterns of the new distal
bioisosteric hydrophobic moieties were selected to ensure differ-
ent electronic and lipophilic environments that could influence the
Over the last two decades, a number of VEGFR‐2 inhibitors have
demonstrated success as targeted anticancer therapeutics. Among
these, sorafenib (I), the 1,4‐disubstituted phthalazine derivatives,
vatalanib (II), AAC789 (III), AMG 900 (IV), and IM‐023911 (V) re-
vealed potent inhibitory activity against VEGFR‐2 in nanomolar levels
[12,13]
of IC50
.
Sorafenib is an FDA‐approved antiangiogenic drug that
shares the above pharmacophoric features.[14]
The phthalazine ring is a vital pharmacophoric scaffold present in
the core structures of many anticancer molecules[11,15–19] with po-
tent activity against hepatocellular carcinoma,[18] colon cancer,[11]
and breast cancer,[19] and as degraders of VEGFR‐2.[15,20] Many
phthalazine derivatives were reported to have VEGFR‐2 and EGFR
kinase inhibitory activities, with IC50 values in the nanomolar range;
however, compounds were more selective and better inhibitors of
VEGFR‐2 compared with EGFR.[21,22] Other pharmacophoric het-
erocycles have been implanted in the structures of recently reported
anticancer agents with a VEGFR‐2 inhibitory effect, for example,
pyrazole[23,24] and pyrimidine.[25,26] The latter were reported to have
spatial configurations that enable both to interact with the VEGFR‐2
FIGURE 1 Structures and pharmacophoric features of the reported VEGFR‐2 inhibitors

KHEDR ET AL.
3 of 18
|
FIGURE 2 Rational design of the target 4‐phenylphthalazinon‐1‐amine derivatives 4–7
activity of the target compounds. These modifications were per-
formed to carry out further elaboration of the phthalazine scaf-
folds and to explore the potentially useful structure–activity
relationship (SAR).
conduct the in vitro anticancer evaluation study of the newly
designed phthalazine derivatives.
|
The designed target derivatives were synthesized and evaluated
as potential VEGFR‐2 inhibitors and anti‐tumor agents against three
human tumor cell lines, namely, hepatocellular carcinoma (HepG2),
human colorectal carcinoma‐116 (HCT‐116), and breast cancer (Mi-
chigan Cancer Foundation‐7 [MCF‐7]).
2
RESULTS AND DISCUSSION
|
2.1
Structure‐based design
N‐Substituted‐4‐phenylphthalazin‐1‐amine derivatives possess the
essential pharmacophoric features of VEGFR‐2 inhibitors[8–10,44,45]
(Figure 1). The 4‐phenylphthalazine scaffold represents the central
aryl moiety. The phenyl group at position‐4 replaces the pyridine and
N‐methylpicolinamide moieties of vatalanib and sorafenib (Figures 3
and 4), respectively. The N‐2 atoms of the phthalazine nucleus act as
a H‐bond acceptor (HBA), forming a H bond with the essential amino
acid residues Asp1046. The NH linker resembles that of vatalanib,
which acts as H‐bond donor (HBD), forming a H bond with the es-
sential amino acid residues Glu885. Moreover, the new
linkers, prop‐2‐en‐1‐one, dihydropyrazole, and/or pyrimidin‐2(1H)‐
one at position‐4 of the hydrophobic phenyl tails, resemble the
pyridin‐2‐yl‐oxy fragment of compound IV. These linkers joined to
different substituted distal hydrophobic phenyl moieties as in the
new derivatives 5a–g, 6a–g, and 7a–c. The 4‐phenyl group occupied the
In a dose‐dependent manner, VEGFR‐2 was reported to be
considerably upregulated in HepG2 cells with the stimulation of the
hepatocyte growth factor, which is involved in the cell proliferation
of hepatocellular carcinoma (HCC).[37,38] Blockade of VEGFR‐2 sig-
naling markedly inhibited the growth and metastasis of HCC.[38,39]
Also, VEGFR‐2 is a crucial regulator of endothelial differentiation in
breast cancer cells (MCF‐7)[40,41] and human colorectal carcinoma
(HCT‐116).[42,43] Overexpression of VEGFR‐2 receptors in breast
cancer cells has been documented as a contributor to the resistance
of such cancer types to the curative effect of tamoxifen.[40]
Degraders of VEGFR‐2 were verified to impair the in vitro endothelial
differentiation and to promote angiogenesis, suggesting a VEGFR‐2‐
mediated mechanism of antiproliferative activity in human colorectal
carcinoma.[42] Consequently, we selected these three cancer cells to

4 of 18
KHEDR ET AL.
|
FIGURE 3 Superimposition of compound 6e (S) and vatalanib inside the binding pocket of VEGFR‐2 (4ASD)
FIGURE 4 Superimposition of compound 6e (S) and sorafenib inside the binding pocket of VEGFR‐2 (4ASD)
|
hydrophobic ATP‐binding groove formed by Leu1035, Cys919,
Phe918, Glu917, Val848, and Leu840. This ATP‐binding groove was
occupied by pyridine and N‐methylpicolinamide moieties of vatalanib
and sorafenib (Figures 3 and 4), respectively. Moreover, the central
phthalazine scaffold occupied the linker hydrophobic pocket formed
by Asp1046, Cys1045, Thr916, Lys868, and Val848. The hydro-
phobic phenyl tail occupied the allosteric hydrophobic pocket formed
by Asp1046, Cys1045, His1026, and Glu885. Furthermore, the distal
moiety occupied the new hydrophobic groove formed by His1026,
Ile1025, Cys1024, Arg1022, Leu1019, Ile892, His891, and Ile888.
2.2
Docking studies
All modeling experiments in the present work were performed using
Molsoft software. The VEGFR‐2 experimental structure was down-
loaded from the Protein Data Bank (PDB ID 4ASD; https://www.rcsb.
org/structure/4asd).[46] All studied ligands have similar positions and
orientations inside the recognized binding site of VEGFR‐2. This active
site reveals a large space bound 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) showed that

KHEDR ET AL.
5 of 18
|
FIGURE 5 Superimposition of some docked compounds inside the binding pocket of 4ASD
TABLE 1 Calculated free energy of binding (ΔG in kcal/mol) for
the ligands
protein. It was found that the root‐mean‐square deviation value
between the re‐docked conformer and the co‐crystallized conformer
of sorafenib was 0.3 Å, which confirms the validity of the docking
protocol (Figure 6).
Compound
ΔG (kcal/mol)
−72.15
−90.97
−85.12
−91.92
−84.02
−94.65
−90.35
−96.12
−96.86
−94.99
Compound
ΔG (kcal/mol)
−99.88
4
6c
The proposed binding mode of sorafenib revealed an
affinity value of −100.55 kcal/mol and four H bonds. The N‐
methylpicolinamide moiety was stabilized by the formation of
two H bonds with the essential amino acid Cys919, where the
pyridine N atom formed one H bond with the NH of Cys919
(2.51 Å), while its NH group formed one H bond with the carbonyl
of Cys919 (2.10 Å). The urea linker formed one H bond with the
key amino acid Glu885 (1.77 Å) through its NH group and one H
bond with Asp1046 (1.50 Å) through its carbonyl group. The
N‐methylpicolinamide moiety occupied the hydrophobic
ATP‐binding pocket formed by Leu1035, Lys920, Cys919,
Phe918, Glu917, Val848, and Leu840. Moreover, the central
phenyl ring occupied the linker hydrophobic pocket formed by
Cys1045, Leu1035, Thr916, Lys868, and Val848. Furthermore,
the hydrophobic 3‐trifluromethyl‐4‐chlorophenyl moiety at-
tached to the urea linker occupied the allosteric hydrophobic
pocket formed by Asp1046, Cys1045, His1026, Ile892, Ile888,
and Glu885 (Figure 7).
5a
5b
5c
5d
5e
5f
5g
6a
6b
6d
−92.07
6e
−101.98
−99.18
6f
6g
−101.17
−95.16
7a
7b
−100.67
−99.49
7c
Vatalanib
Sorafenib
−87.11
−100.55
Since the compounds of the dihydropyrazole series, for ex-
ample, 6e, bear a chiral center, it has both (S)‐ and (R)‐enantiomers.
The (S)‐enantiomer showed higher affinities than that of the
(R)‐enantiomer.
FIGURE 6 Superimposition of the co‐crystallized ligand (purple)
and the re‐docked conformer (turquoise)
As planned, the proposed binding mode of the (S)‐enantiomer of
compound 6e is almost the same as that of sorafenib, which revealed
an affinity value of −101.98 kcal/mol and formed four H bonds. The
NH linker formed one H bond with the key amino acid Glu885
(1.59 Å) through its carbonyl group, while the two N atoms of the
most of these compounds had good binding affinity toward the receptor
and the computed values reflected the overall trend (Table 1).
To validate the docking process, the co‐crystallized ligand (sor-
afenib) was re‐docked against the isolated pocket of the target

6 of 18
KHEDR ET AL.
|
FIGURE 7 Predicted binding mode for sorafenib with VEGFR‐2. H‐bonded atoms are indicated by dotted lines
FIGURE 8 Predicted binding mode for 6e (S) with 4ASD
phthalazine nucleus formed two H bonds with the essential amino
acid residue Asp1046 (2.22, 2.97 Å). Moreover, the NH of the dihy-
dropyrazole linker formed a H bond with Ile1025 (2.22 Å). The
4‐phenyl group occupied the hydrophobic ATP‐binding groove
formed by Leu1035, Cys919, Phe918, Glu917, Val848, and Leu840.
Moreover, the central phthalazine scaffold occupied the linker hydro-
phobic pocket formed by Asp1046, Cys1045, Thr916, Lys868, and
Val848. The hydrophobic phenyl tail occupied the allosteric hydro-
phobic pocket formed by Asp1046, Cys1045, His1026, and Glu885.
Furthermore, the distal 4‐methylphenyl moiety occupied the new hy-
drophobic groove formed by His1026, Ile1025, Cys1024, Arg1022,
Leu1019, Ile892, His891, and Ile888 (Figure 8). These interactions may
explain the highest anticancer activity of compound 6e.
As planned, the proposed binding mode of the (R)‐enantiomer of
compound 6e is almost the same as that of the (S)‐enantiomer, but
revealed an affinity value of −99.93 kcal/mol and formed only 3 H
bonds. The NH linker formed one H bond with the key amino acid
Glu885 (1.71 Å) through its carbonyl group, while the two N atoms of

KHEDR ET AL.
7 of 18
|
the phthalazine nucleus formed two H bonds with the essential
amino acid residue Asp1046 (2.53, 2.97 Å) (Figure 9).
corresponding chalcone derivatives 5a–g following the reported
procedure[47,48,50] (Scheme 1). The produced chalcone derivatives
5a–g underwent binucleophilic cyclocondensation reactions with
hydrazine hydrate and/or urea[51,52] to yield the corresponding
From the obtained docking results (Table 1), we concluded that
the NH linker occupied the same groove as that occupied by NH and
urea linkers of vatalanib and sorafenib, respectively. It played the
same role that is essential for higher affinity toward the VEGFR‐2
enzyme. The distal hydrophobic moieties induced an increase in hy-
drophobic interactions and consequently affinities toward the
VEGFR‐2 enzyme. Phthalazine enables the new compounds to form
new H bonds through its N atoms with the amino acid Asp1046. The
use of pyrazoline and/or pyrimidin‐2(1H)‐one linkers leads to elon-
gations of the structures and enables the hydrophobic distal moieties
to form new hydrophobic interactions. Compounds of the dihy-
dropyrazole series, for example, 6e, bear a chiral center, so it has both
(S)‐ and (R)‐enantiomers. The (S)‐enantiomer showed higher affinities
than that of the (R) one, which may be attributed to the formation of
an extra H bond with Ile1025.
pyrazoline 6a–g and/or pyrimidin‐2(1H)‐one 7a–c, respectively
(Scheme 2).
|
2.4
In vitro cytotoxic activity
As outlined before, VEGFR‐2 receptors have been reported to act as
regulators of endothelial differentiation in both breast cancer cells
(MCF‐7)[40,41] and human colorectal carcinoma (HCT‐116).[42,43] In
addition, blockade of VEGFR‐2 signaling revealed a marked inhibition
of both the growth and metastasis of hepatocellular carcinoma.[38,39]
Consequently, the antiproliferative activity of the newly synthesized
phthalazine derivatives was examined against these three human cell
lines using the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium
bromide (MTT) colorimetric assay as described by Mosmann.[53]
Sorafenib and doxorubicin were used as reference cytotoxic drugs.
|
2.3
Chemistry
The results were described as growth‐inhibitory concentration (IC50
)
The adopted synthetic strategies for the preparation of target com-
pounds (4–7) are shown in Schemes 1 and 2. The synthesis was
initiated by cyclocondensation of 2‐benzoylbenzoic acid (1) with
hydrazine hydrate to afford the corresponding 4‐phenylphthalazin‐
1(2H)‐one[11,47,48] (2), which underwent chlorination by reaction
with phosphorous oxychloride[11,33,49] to afford 1‐chloro‐4‐
phenylphthalazine (3). The chloro derivative 3 was heated under re-
flux with the 4‐aminoacetophenone to afford the corresponding
acetyl derivative 4. On the contrary, the acetyl derivative 4 was
condensed with the appropriate benzaldehyde to yield the
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 2. From the obtained results, it was found
that most of the prepared compounds showed excellent to low
growth‐inhibitory activity against the tested cancer cell lines. In-
vestigations of the cytotoxic activity indicated that HCT‐116 was the
most sensitive cell line to the influence of the new derivatives. In
particular, compound 6e was found to be the most potent derivative
among all the tested compounds against the three HepG2, HCT116,
FIGURE 9 Predicted binding mode for 6e (R) with 4ASD

8 of 18
KHEDR ET AL.
|
SCHEME 1 Synthetic route for the preparation of the target compounds 4 and 5a–g
SCHEME 2 Synthetic route for the preparation of the target compounds 6a–g and 7a–c

KHEDR ET AL.
9 of 18
|
TABLE 2 In vitro cytotoxic activities of the newly synthesized
compounds against HepG2, HCT‐116, and MCF‐7 cell lines and the
VEGFR‐2 kinase assay
42.47 2.9, 43.90 3.0, and 47.12 3.3 µM, respectively. Chalcone
derivatives 5b, 5c, and 5e showed moderate cytotoxicity, with
IC50 = 61.35 3.5, 63.82 3.6, and 66.16 3.8 µM, respectively. The
α,β‐unsaturated carbonyl derivatives 5a, 5d, 5g, 6a, 6b, and the pyr-
imidinone 7a, with IC50 ranging from 75.87 4.3 to 91.75 5.3 µM,
displayed the lowest cytotoxicity. On the contrary, ketonic com-
pound 4 showed no activity, with IC50 > 100 µM.
IC50 (µM)a
Compound
HepG2
HCT116
MCF‐7
VEGFR‐2
NT
4
>100
>100
>100
5a
84.29 4.9
59.27 4.0
69.56 4.3
90.45 5.2
70.55 4.4
56.98 3.8
>100
78.50 4.5
61.35 3.5
63.82 3.6
81.25 4.7
66.16 3.8
47.12 3.3
91.75 5.3
75.87 4.3
79.18 4.6
26.08 1.9
43.90 3.0
10.12 1.0
42.47 2.9
12.91 1.3
78.81 4.3
22.37 1.8
31.28 2.2
5.47 0.3
8.07 0.8
92.16 5.5
67.06 4.1
71.45 4.3
89.26 5.2
74.80 4.6
64.79 3.9
>100
0.82 0.08
0.63 0.07
0.63 0.07
0.87 0.08
0.65 0.06
0.44 0.02
NT
With respect to the MCF‐7 cell line, the dihydropyrazole deri-
5b
vative 6g displayed very good anticancer activities, with IC50
=
5c
18.49 1.6 µM. Compounds 7b, 6c, and 7c displayed good cytotoxi-
city, with IC50 = 25.16 2.0, 27.10 2.2, and 39.86 2.8 µM, re-
spectively. Compounds 5b, 5c, 5e, 5f, 6a, 6d, 6f, and 7a showed
moderate cytotoxic activity, with IC50 ranging from 51.68 3.4 to
74.80 4.6 µM. Compounds 5a, 5d, and 6b displayed the lowest cy-
totoxicity, with an IC50 range of 89.26–94.85 µM. On the contrary,
compounds 4 and 5g showed no activity, with IC50 > 100 µM.
5d
5e
5f
5g
6a
86.01 4.9
87.31 5.0
23.92 2.3
49.40 3.6
11.23 1.1
48.56 3.5
15.80 1.4
88.08 4.9
18.15 1.7
37.52 2.9
9.18 0.6
7.94 0.6
60.70 3.9
94.85 5.8
27.10 2.2
54.21 3.5
13.92 1.2
51.68 3.4
18.49 1.6
61.99 3.9
25.16 2.0
39.86 2.8
7.26 0.3
6.75 0.4
0.65 0.06
0.84 0.08
0.22 0.02
0.44 0.02
0.11 0.02
0.40 0.03
0.13 0.02
0.67 0.07
0.17 0.02
0.28 0.03
0.10 0.02
NT
6b
6c
|
2.5
In vitro VEGFR‐2 kinase assay
6d
Sixteen of the newly synthesized derivatives 5a–f, 6a–g, and 7a–c were
evaluated for their inhibitory activities against VEGFR‐2 using an
anti‐phosphotyrosine antibody with the AlphaScreen system (Perki-
nElmer).[54] The results were reported as a 50% inhibition con-
centration value (IC50) calculated from the concentration–inhibition
response curve. The results of the VEGFR‐2 enzyme assay are
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 0.11 0.02 to 0.87 0.08 µM.
Among these, compound 6e was found to be the most potent deri-
vative that inhibited VEGFR‐2 at an IC50 value of 0.11 0.02 µM,
which is nearly equipotent to the IC50 value of sorafenib
(0.10 0.02 µM). Compounds 6g, 7b, and 6c showed very good ac-
tivity, with IC50 values of 0.13 0.02, 0.17 0.02, and
0.22 0.02 µM, respectively. Also, compounds 7c, 6f, 5f, and 6d
showed good VEGFR‐2 inhibition with IC50 values of 0.28 0.03,
0.40 0.03, 0.44 0.02, and 0.44 0.02 µM, respectively. Moreover,
compounds 5b, 5c, 5e, 6a, and 7a possessed good VEGFR‐2 inhibition,
with IC50 values of 0.63 0.07, 0.63 0.07, 0.65 0.06, 0.65 0.06,
and 0.67 0.07 µM, respectively. Finally, compounds 5a, 5d, and 6b
displayed the lowest VEGFR‐2 inhibition, with IC50 values ranging
from 0.82 0.08 to 0.87 0.08 µM, respectively.
6e
6f
6g
7a
7b
7c
Sorafenib
Doxorubicin
Abbreviation: NT, not tested.
aIC50 values are the mean SD of three separate experiments.
and MCF‐7 cancer cell lines, with IC50 = 11.23 1.1, 10.12 1.0, and
13.92 1.2 µM, respectively, compared to sorafenib (IC50 = 9.18
0.6, 5.47 0.3, and 7.26 0.3 µM, respectively) and doxorubicin
(IC50 = 7.94 0.6, 8.07 0.8, and 6.75 0.4 µM, respectively).
With respect to the HepG2 hepatocellular carcinoma cell line,
compounds 6g, 7b, and 6c displayed very good anticancer activities,
with IC50 = 15.80 1.4, 18.15 1.7, and 23.92 2.3 µM, respectively.
Compounds 7c, 6f, and 6d, with IC50 = 37.52 2.9, 48.56 3.5, and
49.40 3.6 µM, respectively, displayed good cytotoxicity. Com-
pounds 5b, 5c, 5e, and 5f, with IC50 ranging from 56.98 3.8 to
70.55 4.4 µM, showed moderate cytotoxicity. Compounds 5a, 5d,
6a, 6b, and 7a, with IC50 ranging from 84.29 4.9 to 90.45 5.2 µM,
displayed the lowest cytotoxicity. On the contrary, compounds 4 and
5g, with IC50 > 100 µM, showed no activity.
|
2.6
SAR
The preliminary SAR study has focused on the effect of the hydro-
phobic and electronic nature of the substituents used in this study.
Also, it focused on the effect of the type, length, and 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
On cytotoxicity evaluation against the colorectal carcinoma
(HCT‐116) cell line, it was found that compounds 6g and 7b displayed
very good anticancer activities with IC50 = 12.91 1.3 and
22.37 1.8 µM, respectively. Compounds 6c, 7c, 6f, 6d, and 5f dis-
played good cytotoxicity effects, with IC50 = 26.08 1.9, 31.28 2.2,

10 of 18
KHEDR ET AL.
|
different 4‐substituted anilines, was joined to the hydrophobic distal
moieties through the new linkers prop‐2‐en‐1‐one, pyrazoline, and/
or pyrimidin‐2(1H)‐one. Lipophilicity and the electronic nature of the
distal moieties played an important role in VEGFR‐2 inhibition and
consequently the anticancer activities.
higher anticancer activities than the more hydrophobic electron‐
withdrawing (−I) 2,6‐dichloro 6d and the unsubstituted one 6a
against the three cancer cell lines. Finally, the hydrophobic
electron‐withdrawing (−I) 2‐chloro one 6b displayed the lowest
activities against the three cancer cell lines.
In molecular docking studies, generally, the presence of the new
pyrazoline linkers as compounds 6a–g imparts higher VEGFR‐2 bind-
ing affinity and consequently higher anticancer activity compared
with pyrimidin‐2(1H)‐one as compounds 7a–c and prop‐2‐en‐1‐one
linkers as compounds 5a–g, respectively.
The third group has the new pyrimidin‐2(1H)‐one linkers 7a–c. In
this group, the hydrophobic electron‐donating (+I) 4‐methyl group 7b
showed higher anticancer activities than the hydrophobic electron‐
donating (+I) 4‐methoxy 7c and the unsubstituted one 7a against the
three cancer cell lines.
The data obtained from biological testing matched very well
with those obtained from molecular modeling studies. The pyrazo-
line derivatives 6a–g showed higher anticancer activities against the
three HepG2, HCT116, and MCF‐7 cell lines than pyrimidin‐2(1H)‐
|
2.7
ADMET profiling study
one derivatives 7a–c and prop‐2‐en‐1‐one derivatives 5a–g
,
The in silico investigational study of the four most active com-
pounds (6e,c,g and 7b) was conducted to evaluate their physico-
chemical properties following Lipinski's rule.[55] Lipinski stated that
the absorption is more likely to be good if the compound follows
three or more of the following rules: (a) HB donors ≤5; (b) HB ac-
ceptors ≤10; (c) molecular weight <500; and (d) log P < 5. The newly
designed compounds 6e,c,g and 7b violated only one rule (log P).
Determination of the ADMET profiles was performed using the
protocol of the pkCSM descriptor algorithm.[56] The results of the
ADMET profiles of the most efficient new compounds as VEGFR‐2
inhibitors are shown in Table 3. From the tabulated data, we can
conclude that these derivatives have the advantage of better solu-
bility in water than sorafenib. Also, the intestinal absorption of new
phthalazines in humans is better than that of doxorubicin. This ad-
vantage may be attributed to the higher lipophilicity of the new
ligands. Drugs with reasonable lipophilicity can pass more easily
through biological membranes,[57] and consequently, may show
good bioavailability following oral administration. As shown in
Table 3, doxorubicin, sorafenib and our new derivatives shared the
advantage of no Ames toxicity which indicate non‐mutagenic po-
tentials of these compounds.[58] Similarly, they also showed no
predicted hERG I inhibitory activity, which indicates their safety for
the electrical activity of the heart in humans.[59] Additionally, our
designed phthalazines revealed lower Minnow toxicity values, re-
vealing the lower hazard and risk of the new compounds in the
aquatic environment.[60]
respectively.
From the structure of the synthesized derivatives and the data
shown in Table 2, we can divide these tested compounds into three
groups. The first group contains the new prop‐2‐en‐1‐one linkers
as in compounds 5a–g. Generally, in this group, the substituents at
position‐2 of the distal moieties showed higher activities than that
at position‐4 against the three cancer cell lines. The hydrophobic
electron‐donating (+ inductive [+I]) 2‐methoxy group in compound
5f showed higher anticancer activities than the hydrophobic
electron‐withdrawing (−I) 2‐chloro one 5b against the three cancer
cell lines. The hydrophobic electron‐donating (+I) 2‐methoxy group
in compound 5f showed higher anticancer activities than the hy-
drophobic electron‐donating (+I) 4‐methoxy one in compound 5g
against the three cancer cell lines. The hydrophobic electron‐
withdrawing (−I) 2‐chloro group in compound 5b showed higher
anticancer activities than the hydrophobic electron‐withdrawing
(−I) 4‐chloro 5c and the more hydrophobic electron‐withdrawing
(−I) 2,6‐dichloro one 5d, respectively, against the three cancer cell
lines. The hydrophobic electron‐withdrawing (−I) 4‐chloro group
5c showed higher anticancer activities than the hydrophobic
electron‐donating (+I) 4‐methyl 5e and the electron‐donating (+I) 4‐
methoxy one 5d, respectively, against the three cancer cell lines.
The unsubstituted 5a displayed higher anticancer activities than the
hydrophobic electron‐withdrawing (−I) 2,6‐dichloro 5d one against
HepG2, HCT116, but lower activity against MCF‐7 cell lines. All the
derivatives showed higher activities than the hydrophobic
electron‐donating (+I) 4‐methoxy one 5g against the three cancer
cell lines.
|
3
CONCLUSION
The second group 6a–g contains the new pyrazoline linkers.
Generally, the 4‐substituted phenyl distal moieties as in com-
pounds 6e, 6g, and 6c showed higher activities than the
2‐substituted 6f and unsubstituted one 6a, respectively, against
the three HepG2, HCT116, and MCF‐7 cell lines. In this group, the
hydrophobic electron‐donating (+I) 4‐methyl group 6e showed
higher anticancer activities than the hydrophobic electron‐
donating (+I) 4‐methoxy 6g and the hydrophobic electron‐
withdrawing (−I) 4‐chloro one 6c against the three cancer cell lines.
The hydrophobic electron‐donating (+I) 2‐methoxy 6f showed
In this study, we describe the design and synthesis of 18 new
N‐substituted‐4‐phenylphthalazin‐1‐amine derivatives. In the de-
signed compounds, new linkers in the form of fragments with verified
VEGFR‐2 inhibitory potential, including an α,β‐unsaturated ketonic
fragment, pyrazole, and pyrimidine, were introduced at position‐4 of
the phenyl tail. Also, new distal hydrophobic moieties were attached
to these linkers, which are expected to increase the hydrophobic
interaction with the VEGFR‐2 enzyme. These structural optimizations
have led to the identification of the novel dihydropyrazole derivative

KHEDR ET AL.
11 of 18
|
TABLE 3 ADMET profile of the four most active derivatives and the reference VEGFR‐2 inhibitor and anticancer agents
Parameter
6e
6c
6g
7b
Sorafenib
Doxorubicin
Molecular properties
Molecular weight
Log P
475.983
455.565
471.564
481.559
464.831
543.525
7.1325
6.78752
6.4877
6.76612
5.5497
0.0013
Rotatable bonds
Acceptors
5
5
6
5
5
5
5
5
6
5
4
12
Donors
2
2
2
2
3
6
Surface area
208.050
204.111
209.225
213.265
185.111
222.081
Absorption
Water solubility
−4.119
1.164
88.347
−2.735
Yes
−4.136
1.174
89.805
−2.735
Yes
−3.649
1.233
90.168
−2.735
Yes
−2.967
0.735
92.497
−2.735
Yes
−4.888
0.613
84.731
−2.776
Yes
−2.915
0.457
62.372
−2.735
Yes
Caco2 permeability
Intestinal abs. (human)
Skin permeability
P‐glycoprotein substrate
P‐glycoprotein I inhibitor
P‐glycoprotein II inhibitor
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Distribution
VDss (human)
−1.063
−1.073
−1.185
−0.984
−0.233
1.647
0.215
Fraction unbound
(human)
0.171
0.172
0.205
0.291
0.058
BBB permeability
CNS permeability
0.51
0.523
−0.086
−1.554
−1.272
−1.366
−1.682
−1.995
−1.379
−4.307
−1.243
−1.284
Metabolism
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP3A4 inhibitor
No
No
Yes
No
Yes
No
No
No
Yes
No
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Excretion
Total clearance
Renal OCT2 substrate
0.072
No
0.218
No
0.255
No
0.376
No
−0.212
0.987
No
No
Toxicity
Ames toxicity
No
No
No
No
No
No
Max. tolerated dose
(human)
0.388
0.388
0.369
0.433
0.677
0.081
hERG I inhibitor
hERG II inhibitor
Oral rat acute
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
3.103
3.115
3.103
2.741
2.595
2.408
toxicity (LD50
)
Oral rat chronic toxicity
(LOAEL)
2.262
2.215
2.22
1.613
1.054
3.339
Hepatotoxicity
Yes
Yes
Yes
Yes
Yes
Yes
Skin sensitization
No
No
No
No
No
No
Tetrahymena pyriformis
0.285
0.285
0.285
0.285
0.349
0.285
toxicity
Minnow toxicity
−0.225
−0.007
−0.664
0.411
−0.421
4.412
Abbreviations: BBB, blood–brain barrier; CNS, central nervous system.

12 of 18
KHEDR ET AL.
|
6e as the most promising hit molecule. The designed compounds
were evaluated for their anticancer activities against three human
tumor cell lines: hepatocellular carcinoma (HepG2), colorectal carci-
noma (HCT‐116), and breast cancer (MCF‐7). In addition, the VEGFR‐
2 inhibitory potential was investigated for 16 of the new derivatives.
All the tested compounds showed mild to good anticancer activities
against the selected cancer cells. Also, a molecular docking study was
performed to investigate the proposed binding mode of the new
compounds with the VEGFR‐2 active site. The data obtained from
biological testing were highly correlated with those obtained from
the docking study. Investigations of the cytotoxic activity indicated
that HCT‐116 was the most sensitive cell line that had an influence
on the new derivatives. In particular, compound 6e was found to be
the most potent derivative among all the tested compounds against
the three cancer cell lines, HepG2, HCT116, and MCF‐7, with
IC50 = 11.23 1.1, 10.12 1.0, and 13.92 1.2 µM, respectively, in
comparison to sorafenib (IC50 = 9.18 0.6, 5.47 0.3, and
7.26 0.3 µM, respectively). With respect to the HepG2 hepatocel-
lular carcinoma cell line, compounds 6g, 7b, and 6c displayed very
good anticancer activities, with IC50 = 15.80 1.4, 18.15 1.7, and
23.92 2.3 µM, respectively. Cytotoxicity evaluation against the
colorectal carcinoma (HCT‐116) cell line showed that compounds
Supporting Information) were recorded on a Bruker 100 MHz NMR
spectrophotometer at the Microanalytical Unit, Faculty of Pharmacy,
Cairo University. Tetramethylsilane was used as an internal standard,
and chemical shifts were measured in the δ scale (ppm). The mass
spectra were recorded on the Direct Probe Controller Inlet part to a
single quadropole mass analyzer in theThermo Scientific GCMS model
ISQ LT using Thermo Xcalibur software at the Regional Center for
Mycology and Biotechnology, Al‐Azhar University. Elemental analyses
(C, H, N) were performed on a CHN analyzer at the Regional Center for
Mycology and Biotechnology, Al‐Azhar University. All compounds
were within 0.4 of the theoretical values. The reactions were mon-
itored by thin‐layer chromatography (TLC) using TLC sheets precoated
with UV fluorescent silica gel Merck 60 F254 plates and were visua-
lized using a UV lamp and different solvents as mobile phases.
4‐Phenylphthalazin‐1(2H)‐one (2) and 1‐chloro‐4‐phenylphthalazine
(3) were obtained according to the reported procedures.[11]
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as the Supporting
Information.
|
4.1.2
Synthesis of 1‐{4‐[(4‐phenylphthalazin‐1‐
6g and 7b displayed very good anticancer activities, with IC50
=
yl)amino]phenyl}ethan‐1‐one (4)
12.91 1.3 and 22.37 1.8 µM, respectively. With respect to the
MCF‐7 cell line, compound 6g displayed very good anticancer ac-
tivities, with IC50 = 18.49 1.6 µM. The results of the VEGFR‐2
enzyme assay of the tested compounds revealed high to low in-
hibitory activity, with IC50 values ranging from 0.11 0.02 to
0.87 0.08 µM. Among these, compound 6e was found to be the
most potent derivative that inhibited VEGFR‐2 at an IC50 value of
0.11 0.02 µM, which is almost equipotent to the IC50 value
of sorafenib (0.10 0.02 µM). Compounds 6g, 7b, and 6c showed
very good activity, with IC50 values of 0.13 0.02, 0.17 0.02, and
0.22 0.02 µM, respectively.
Equimolar quantities of 1‐chloro‐4‐phenylphthalazine (3) (2.40 g,
0.01 mol) and p‐aminoacetophenone (1.35 g, 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 compound 4.
Yield, 88%; m.p. 255–257°C; IRνmax (cm−1): 3252 (NH), 3045
(C–H aromatic), 2965 (C–H aliphatic), 1664 (C═O), 1564 (C═N); 1H
NMR (400 MHz, dimethyl sulfoxide [DMSO]‐d6): 2.57 (s, 3H, COCH3),
7.62–7.67 (m, 3H, H‐3, H‐4, H‐5 of C6H5), 7.71 (d, 2H, H‐3, H‐5 of
NH–C6H4, J = 7.40), 7.97–8.03 (m, 4H, H‐2, H‐6 of C6H5 and H‐2,
H‐6 of NH–C6H4), 8.10–8.12 (m, 3H, H‐5, H‐6, H‐7 of phthalazine),
8.83 (d, 1H, H‐8 of phthalazine, J = 7.8 Hz), and 10.01 (s, 1H, NH,
D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6): 27.08, 121.58
(2C), 122.31, 125.11, 127.55 (3C), 128.73, 129.39 (2C), 129.92 (2C),
130.57, 131.23, 133.26, 135.14, 135.47, 143.35, 153.26, 153.63,
197.20; MS (m/z): 341 (M++2, 1.55%), 340 (M++1, 13.59%), 339 (M+,
62.53%), 338 (M+−1, 100% base peak), 324 (15.15%), 165 (11.25%),
and 77 (39%). Anal. calcd. for C22H17N3O (339.14): C, 77.86; H, 5.05;
N, 12.38. Found: C, 77.58; H, 5.23; N, 12.60.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All melting points were determined using the open capillary method on
a Gallenkamp melting point apparatus at the Faculty of Pharmacy, Al‐
Azhar University, and were uncorrected. Infrared spectra were re-
corded on a pyeUnicam SP 1000 IR spectrophotometer at Pharma-
ceutical Analytical Unit, Faculty of Pharmacy, Al‐Azhar University,
using the potassium bromide disc technique. Proton magnetic
resonance 1H nuclear magnetic resonance (NMR) spectra were re-
corded on a JEOL 400 MHz NMR spectrophotometer at the Micro-
analytical Center, Faculty of Pharmacy, Ain Shams University, and a
JEOL 400 MHz NMR spectrophotometer at the Microanalytical Unit,
Faculty of Pharmacy, Cairo University. 13C NMR spectra (see the
|
4.1.3
General procedure for the synthesis of
compounds 5a–g
To
a mixture of the acetyl derivative (4) (0.40 g, 0.001 mol)
and the appropriate aromatic aldehyde, namely, benzaldehyde,
2‐chlorobenzaldehyde, 4‐chlorobenzaldehyde, 2,6‐dichlorobenzaldehyde,
4‐methylbenzaldehyde, 2‐methoxybenzaldehyde, and/or 4‐methoxybe‐
nzaldehyde (0.001 mol) in ethyl alcohol (10 ml), 5% alcoholic NaOH

KHEDR ET AL.
13 of 18
|
(10 ml) was added dropwise over 15 min. The reaction mixture was re-
fluxed for 3 h, then cooled and the formed precipitate was filtered, air‐
dried, and then recrystallized from ethanol to give the corresponding
chalcones 5a–g, respectively, as trans (E)‐isomers.
calcd. for C29H20ClN3O (461.13): C, 75.40; H, 4.36; N, 9.10.
Found: C, 75.62; H, 4.49; N, 9.17.
(E)‐3‐(2,6‐Dichlorophenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (5d)
(E)‐3‐Phenyl‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}prop‐2‐
en‐1‐one (5a)
Yield, 88%; m.p. 190–1922°C; IRνmax (cm−1): 3180 (NH), 3010 (C–H
aromatic), 2962 (C–H aliphatic), 1660 (C═O), 1610 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 7.42 (t, 1H, H‐4 of 2,6‐di‐Cl–C6H3, J = 8.00),
7.59 (d, 2H, H‐3, H‐5 of 2,6‐di‐Cl–C6H3, J = 8.80), 7.69 (m, 3H, H‐3,
H‐4, H‐5 of C6H5), 7.73 (d, 1H, α‐C═H), 7.77 (d, 2H, H‐3, H‐5 of
NH–C6H4, J = 6.40), 7.84 (d, 1H, β‐C═H), 8.02 (d, 1H, H‐2 of C6H5,
J = 7.60), 8.15–8.19 (m, 5H, H‐6 of C6H5, H‐2, H‐6 of NH–C6H4 and
H‐6, H‐7 of phthalazine), 8.26 (d, 1H, H‐5 of phthalazine, J = 7.60),
9.34 (d, 2H, H‐8 of phthalazine, J = 8.40), 11.29 (s, 1H, NH, D2O
exchangeable). Anal. calcd. for C29H19Cl2N3O (495.09): C, 70.17;
H, 3.86; N, 8.47. Found: C, 70.34; H, 3.91; N, 8.63.
Yield, 83%; m.p. 160–162°C; IRνmax (cm−1): 3168 (NH), 3058 (C–H
aromatic), 2976 (C–H aliphatic), 1666 (C═O) and 1598 (C═N); 1H
NMR (400 MHz, DMSO‐d6): 7.29 (d, 1H, H‐4 of CH═CH–C6H5,
J = 8.40), 7.47 (d, 2H, H‐3, H‐5 of C6H5, J = 8.00), 7.59–7.61 (m,
3H, H‐3, H‐4, H‐5 of C6H5), 7.71 (d, 2H, H‐3, H‐5 of NH–C6H4,
J = 9.60), 7.77 (d, 1H, α‐C═H), 7.89–7.91 (m, 4H, H‐2, H‐6 of
CH═CH–C6H5 and H‐2, H‐6 of C6H5), 7.96 (d, 1H, β‐C═H), 8.03 (t,
1H, H‐5 of C6H5, J = 10.00), 8.21–8.29 (m, 4H, H‐2, H‐6 of
NH–C6H4, and H‐6, H‐7 of phthalazine), 8.82 (d, 1H, H‐8 of
phthalazine, J = 10.40) and 9.95 (s, 1H, NH, D2O exchangeable);
13C NMR (100 MHz, DMSO‐d6): 119.39, 119.69 (2C), 122.63,
123.38, 126.32 (2C), 126.39 (2C), 127.49 (2C), 128.94, 129.23,
129.38, 129.45, 130.21 (2C), 130.24, 130.86, 131.33, 132.36,
133.00, 135.37, 136.98, 143.42, 146.18, 152.10, 154.96, 188.11.
Anal. calcd. for C29H21N3O (427.17): C, 81.48; H, 4.95; N, 9.83.
Found: C, 81.62; H, 5.13; N, 9.97.
(E)‐1‐{4‐[(4‐Phenylphthalazin‐1‐yl)amino]phenyl}‐3‐(p‐tolyl)prop‐2‐
en‐1‐one (5e)
Yield, 87%; m.p. 195–197°C; IRνmax (cm−1): 3175 (NH), 3092 (C–H
aromatic), 2959 (C–H aliphatic), 1667 (C═O), 1597 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 2.36 (s, 3H, 4‐CH3), 7.27 (d, 2H, H‐3, H‐5 of
4‐CH3–C6H4, J = 7.20), 7.58–7.60 (m, 3H, H‐3, H‐4, H‐5 of C6H5),
7.69 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 7.20), 7.73 (d, 1H, α‐C═H),
7.77 (d, 2H, H‐2, H‐6 of 4‐CH3–C6H4, J = 7.20), 7.92 (d, 2H, H‐2, H‐6
of C6H5, J = 7.20), 7.98 (d, 1H, β‐C═H), 8.05 (d, 1H, H‐5 of C6H5,
J = 7.60), 8.22 (m, 4H, H‐2, H‐6 of NH–C6H4 and H‐6, H‐7 of
phthalazine), 8.75–8.73 (d, 1H, H‐8 of phthalazine, J = 10.80) and
9.78 (s, 1H, NH, D2O exchangeable). Anal. calcd. for C30H23N3O
(441.18): C, 81.61; H, 5.25; N, 9.52. Found: C, 81.43; H, 5.37; N, 9.76.
(E)‐3‐(2‐Chlorophenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (5b)
Yield, 82%; m.p. 172–174°C; IRνmax (cm−1): 3452 (NH), 3084 (C–H
aromatic), 1606 (C═O), 1517 (C═N); 1H NMR (400 MHz, DMSO‐d6):
7.46 (d, 2H, H‐4, H‐5 of 2‐Cl–C6H4, J = 8.00), 7.57 (d, 1H, α‐C═H),
7.63 (3H, H‐3, H‐3, H‐5 of C6H5), 7.72 (d, 2H, H‐3, H‐5 of
NH–C6H4, J = 6.80), 7.95 (d, 1H, β‐C═H), 8.04 (m, 3H, H‐3 of
2‐Cl–C6H4 and H‐2, H‐6 of C6H5) 8.13 (d, 1H, H‐5 of C6H5,
J = 8.00), 8.27–8.34 (m, 5H, H‐6 of 2‐Cl–C6H4, H‐2, H‐6 of
NH–C6H4 and H‐6, H‐7 of phthalazine), 9.08 (d, 1H, H‐8 of
phthalazine, J = 9.60), 10.52 (s, 1H, NH, D2O exchangeable); MS
(m/z): 464 (M++3, 6.63%), 463 (M++2, 30.63%), 462 (M++1, 51.73%),
461 (M+, 89.69), 460 (M+−1, 100% base peak) and 424 (37.68%).
Anal. calcd. for C29H20ClN3O (461.13): C, 75.40; H, 4.36; N, 9.10.
Found: C, 75.57; H, 4.43; N, 9.28.
(E)‐3‐(2‐Methoxyphenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (5f)
Yield, 82%; m.p. 203–205°C; IRνmax (cm−1): 3428 (NH), 3051 (C–H
aromatic), 2933 (C–H aliphatic), 1602 (C═O), 1507 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 3.92 (s, 3H, 2‐O–CH3), 7.05 (t, 1H, H‐3 of
2‐O–CH3–C6H5), 7.13 (d, 1H, H‐4 of 2‐O–CH3–C6H5), 7.44 (d, 1H,
α‐C═H), 7.59–7.64 (m, 3H, H‐3, H‐4, H‐5 of C6H5), 7.71 (d, 2H, H‐3,
H‐5 of NH–C6H4, J = 9.20), 7.93 (m, 4H, H‐5, H‐6 of 2‐O–CH3–C6H5
and H‐2, H‐6 of C6H5), 8.05 (d, 1H, β‐C═H), 8.09 (d, 1H, H‐5 of C6H5,
J = 8.40), 8.21 (m, 4H, H‐2, H‐6 of NH–C6H4 and H‐6, H‐7 of
phthalazine), 8.72 (d, 2H, H‐8 of phthalazine, J = 8.60), 9.74 (s, 1H,
NH, D2O exchangeable). Anal. calcd. for C30H23N3O2 (457.18): C,
78.75; H, 5.07; N, 9.18. Found: C, 78.98; H, 5.23; N, 9.32.
(E)‐3‐(4‐Chlorophenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (5c)
Yield, 81%; m.p. 182–184°C; IRνmax (cm−1): 3339 (NH), 3057 (C–H
aromatic), 1613 (C═O), 1530 (C═N); 1H NMR (400 MHz, DMSO‐
d6): 7.53 (d, 2H, H‐4, H‐5 of 4‐Cl–C6H4, J = 7.60), 7.59–7.62 (m,
3H, H‐3, H‐4, H‐5 of C6H5), 7.70 (d, 2H, H‐3, H‐5 of NH–C6H4,
J = 6.00), 7.75 (d, 1H, α‐C═H), 7.94–7.96 (m, 4H, H‐2, H‐6 of
4‐Cl–C6H4 and H‐2, H‐6 of C6H5), 7.99 (d, 1H, β‐C═H), 8.05
(d, 1H, H‐5 of C6H5, J = 7.00), 8.23 (m, 4H, H‐2, H‐6 of NH–C6H4
and H‐6, H‐7 of phthalazine), 8.70 (d, 1H, H‐8 of phthalazine,
J = 9.60), 9.74 (s, 1H, NH, D2O exchangeable); MS (m/z): 464
(M++3, 8.29%), 463 (M++2, 32.37%), 462 (M++1, 54.98%), 461
(M+, 85.56), 460 (M+−1, 100% base peak), 424 (32.78%). Anal.
(E)‐3‐(4‐Methoxyphenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (5g)
Yield, 84%; m.p. 212–214°C; IRνmax (cm−1): 3334 (NH), 3058 (C–H
aromatic), 2946 (C–H aliphatic), 1610 (C═O), 1539 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 3.83 (s, 3H, 4‐O–CH3), 7.02 (d, 2H, H‐3, H‐5
of 4‐O–CH3–C6H4, J = 8.00), 7.58–7.61 (m, 3H, H‐3, H‐4, H‐5 of
C6H5), 7.69 (d, 2H, H‐3, H‐5 of NH–C6H4, J = 8.60), 7.73 (d, 1H,
α‐C═H), 7.84–7.92 (m, 2H, H‐2, H‐6 of 4‐O–CH3–C6H4 and H‐2, H‐6

14 of 18
KHEDR ET AL.
|
of C6H5), 7.95 (d, 1H, β‐C═H), 8.04 (d, 1H, H‐5 of C6H5, J = 7.40),
8.21 (m, 4H, H‐2, H‐6 of NH–C6H4 and H‐6, H‐7 of phthalazine), 8.72
(d, 2H, H‐8 of phthalazine, J = 8.80), 9.73 (s, 1H, NH, D2O ex-
changeable). Anal. calcd. for C30H23N3O2 (457.18): C, 78.75; H, 5.07;
N, 9.18. Found: C, 78.91; H, 5.29; N, 9.41.
of pyrazole, J = 10.40), 4.83 (dd, 1H, H‐4 of pyrazole, J = 10.40),
7.40–7.43 (m, 4H, H‐3, H‐5 of 4‐Cl–C6H4 and H‐2, H‐6 of NH–C6H4),
7.48 (s, 1H, NH of pyrazole, D2O exchangeable), 7.55–7.61 (m, 3H, H‐3,
H‐4, H‐5 of C6H5), 7.67–7.71 (m, 4H, H‐3, H‐5 of C6H4 and H‐2, H‐6 of
4‐Cl–C6H4), 7.89 (d, 2H, H‐2 of C6H5, J = 7.40), 7.94 (d, 1H, H‐6 of
C6H5, J = 7.40), 8.03 (m, 3H, H‐5, H‐6, H‐7 of phthalazine), 8.68 (d, 1H,
H‐8 of phthalazine, J = 8.80), 9.41 (s, 1H, NH–C6H4, D2O exchangeable);
MS (m/z): 478 (M++3, 2.90%), 477 (M++2, 7.04%), 476 (M++1, 10.32%),
475 (M+, 30.95), 447 (73.66), 446 (100% base peak), 77 (14.31%). Anal.
calcd. for C29H22ClN5 (475.16): C, 73.18; H, 4.66; N, 14.71. Found: C,
73.34; H, 4.78; N, 14.85.
|
4.1.4
General procedure for the synthesis of
compounds 6a–g
A mixture of the appropriate chalcone 5a–g (0.005 mol) and hydrazine
hydrate (2.5 ml, 0.005 mol, 98%) in absolute ethanol (25 ml) was he-
ated under reflux for 6 h. After cooling, the separated material
was filtered, air‐dried, and recrystallized from ethanol to yield the
corresponding pyrazole derivatives 6a–g, respectively, as racemic
mixtures (R,S).
(R,S)‐N‐{4‐[3‐(2,6‐Dichlorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]phenyl}‐4‐phenylphthalazin‐1‐amine (6d)
Yield, 78%; m.p. 135–137°C; IRνmax (cm−1): 3180 (NH), 3010 (C‐H
aromatic), 2962 (C–H aliphatic and 1610 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 3.21 (dd, 1H, H‐4 of pyrazole, J = 10.800), 3.47 (dd, 1H,
H‐5 of pyrazole, J = 10.80), 5.60 (dd, 1H, H‐4 of pyrazole, J = 10.80),
7.34 (t, 1H, H‐4 of 2,6‐di‐Cl–C6H3, J = 9.60), 7.46 (s, 1H, NH– of
pyrazole, D2O exchangeable), 7.48 (d, 2H, H‐2, H‐6 of NH–C6H4,
J = 6.80), 7.54–7.61 (m, 3H, H‐3, H‐3, H‐5 of C6H5), 7.65 (d, 2H, H‐3,
H‐5 of 2,6‐di‐Cl–C6H3, J = 9.60), 7.67 (d, 2H, H‐2, H‐6 of NH–C6H4,
J = 10.40), 7.89 (d, 2H, H‐2, H‐6 of C6H5, J = 9.20), 8.03 (m, 3H, H‐5,
H‐6, H‐7 of phthalazine), 8.68 (d,2H, H‐8 of phthalazine, J = 9.20),
9.41 (s, 1H, NH–C6H4, D2O exchangeable); MS (m/z): 513.61 (M++4,
5.84%), 512.81 (M++3, 9.72%), 511 (M++2, 11.78%), 510 (M++1,
18.16%), 509 (M+, 14.67), 507 (24.12%), 506 (44.79%), 334 (65.31%),
77 (100% base peak). Anal. calcd. for C29H21Cl2N5 (509.12): C, 68.24;
H, 4.15; N, 13.72. Found: C, 68.01; H, 4.23; N, 13.18.
(R,S)‐4‐Phenyl‐N‐[4‐(3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)phenyl]‐
phthalazin‐1‐amine (6a)
Yield, 73%; m.p. 135–137°C; IRνmax (cm−1): 3165 (NH), 3042 (C–H
aromatic), 2954 (C–H aliphatic), 1598 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 2.85 (dd, 1H, H‐4 of pyrazole, J = 11.60), 3.12 (dd, 1H, H‐5
of pyrazole, J = 11.60), 5.16 (t, 1H, H‐4 of pyrazole, J = 11.60), 7.15–7.17
(m, 4H, H‐3, H‐5 of pyrazole‐C6H5 and H‐2, H‐6 of NH–C6H4),
7.42–7.47 (m, 3H, H‐2, H‐3, H‐5 of C6H5), 7.58–7.68 (m, 3H, H‐3, H‐5
of NH–C6H5 and H‐4 of pyrazole‐C6H5), 7.92–8.03 (m, 5H, H‐3, H‐5
of pyrazole‐C6H5, H‐5, H‐6, H‐7 of phthalazine), 8.71 (d, 1H, H‐8 of
phthalazine, J = 8.60), 9.43 (s, 1H, NH‐pyrazole, D2O exchangeable),
9.74 (s, 1H, NH–C6H4, D2O exchangeable). Anal. calcd. for C29H23N5
(441.20): C, 78.89; H, 5.25; N, 15.86. Found: C, 79.01; H, 5.34; N, 15.41.
(R,S)‐4‐Phenyl‐N‐{4‐[3‐(p‐tolyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
(R,S)‐N‐{4‐[3‐(2‐Chlorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]phenyl}phthalazin‐1‐amine (6e)
yl]phenyl}‐4‐phenylphthalazin‐1‐amine (6b)
Yield, 70%; m.p. 155–157°C; IRνmax (cm−1): 3175 (NH), 3092 (C–H
aromatic), 2959 (C–H aliphatic), 1597 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 2.29 (s, 3H, CH3), 2.80 (dd, 1H, H‐4 of pyrazole, J = 10.20),
3.41 (dd, 1H, H‐4 of pyrazole, J = 10.20), 4.78 (t, 1H, H‐5 of pyrazole,
J = 10.20), 7.15 (d, 2H, H‐3, H‐5 of 4‐CH3–C6H4, J = 7.60), 7.28 (d, 2H,
H‐2, H‐6 of NH–C6H4, J = 7.92), 7.57–7.61 (m, 3H, H‐3, H‐4, H‐5 of
C6H5), 7.64–7.79 (m, 4H, H‐3, H‐5 of NH–C6H4 and H‐2, H‐6 of
4‐CH3–C6H4), 7.89 (d, 2H, H‐2, H‐6 of C6H5, J = 8.00), 8.03–8.05
(m, 3H, H‐5, H‐6, H‐7 of phthalazine), 8.23 (s, 1H, NH–C6H4, D2O
exchangeable), 8.68 (d, 1H, H‐8 of phthalazine, J = 8.24), 9.40 (s, 1H, NH
of pyrazole, D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6): 21.15,
41.42, 63.83, 119.83, 120.95, 121.37, 123.27, 125.49, 126.34, 126.45,
127.00, 127.73, 128.08, 128.95, 129.14, 129.27, 129.38, 129.81,
129.87, 130.16, 130.21, 132.22, 132.40, 132.56, 132.80, 133.03,
136.92, 143.55, 184.34, 185.75. Anal. calcd. for C30H25N5 (455.21):
C, 79.10; H, 5.53; N, 15.37. Found: C, 78.91; H, 5.68; N, 15.70.
Yield, 75%; m.p. 140–142°C; IRνmax (cm−1): 3185 (NH), 3084 (C–H
aromatic), 2994 (C–H aliphatic), 1606 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 2.80 (dd, 1H, H‐4 of pyrazole, J = 10.40), 3.49 (dd, 1H, H‐5
of pyrazole, J = 10.40), 4.83 (dd, 1H, H‐4 of pyrazole, J = 10.40),
7.40–7.45 (m, 4H, H‐4, H‐5 of 2‐Cl–C6H4 and H‐2, H‐6 of NH–C6H4),
7.48 (s, 1H, NH of pyrazole, D2O exchangeable), 7.55–7.60 (m, 3H, H‐3,
H‐4, H‐5 of C6H5), 7.64–7.69 (m, 4H, H‐3, H‐5 of NH–C6H4 and H‐3,
H‐6 of 2‐Cl–C6H4), 7.88 (d, 2H, H‐2 of C6H5, J = 7.30), 7.93 (d, 2H, H‐6
of C6H5, J = 7.30), 8.01 (t, 3H, H‐5, H‐6, H‐7 of phthalazine, J = 8.20),
8.69 (d, 1H, H‐8 of phthalazine, J = 7.60), 9.43 (s, 1H, NH–C6H4, D2O
exchangeable); MS (m/z): 477 (M++2, 10.47%), 476 (M++1, 50.96%), 475
(M+, 30.28), 355 (31.68%), 260 (38.04%), 97 (71.49%), 55 (100% base
peak). Anal. calcd. for C29H22ClN5 (475.16): C, 73.18; H, 4.66; N, 14.71.
Found: C, 73.02; H, 4.53; N, 14.97.
(R,S)‐N‐{4‐[3‐(4‐Chlorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]phenyl}‐4‐phenylphthalazin‐1‐amine (6c)
Yield, 76%; m.p. 146–148°C; IRνmax (cm−1): 3182 (NH), 3105 (C–H
aromatic), 2965 (C–H aliphatic), 1599 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 2.82 (dd, 1H, H‐4 of pyrazole, J = 10.40), 3.49 (dd, 1H, H‐5
(R,S)‐N‐{4‐[3‐(2‐Methoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]phenyl}‐4‐phenylphthalazin‐1‐amine (6f)
Yield, 75%; m.p. 162–164°C; IRνmax (cm−1): 3163 (NH), 3031 (C–H
aromatic), 2955 (C–H aliphatic), 1659 (C═O), 1601 (C═N); 1H NMR

KHEDR ET AL.
15 of 18
|
(400 MHz, DMSO‐d6): 2.71 (dd, 1H, H‐4 of pyrazole, J = 10.00),
3.42–3.49 (dd, 1H, H‐4 of pyrazole, J = 10.00), 3.84 (s, 3H, 2‐O–CH3),
5.01 (t, 1H, H‐5 of pyrazole, J = 10.00), 6.92 (t, 1H, H‐6 of
2‐OCH3–C6H4, J = 8.00), 7.01 (t, 1H, H‐3 of 2‐OCH3–C6H4, J = 8.00),
7.27 (d, 2H, H‐2 and H‐6 of NH–C6H4, J = 8.20), 7.40 (s, 1H,
NH–C6H4, D2O exchangeable), 7.57–7.60 (m, 3H, H‐3, H‐4, H‐5 of
C6H5), 7.67–7.71 (m, 4H, H‐3, H‐5 of NH–C6H4, H‐4, H‐5 of
2‐O–CH3–C6H4), 7.88–7.94 (d, 2H, H‐2, H‐6 of C6H5, J = 7.20),
8.02–8.07 (m, 3H, H‐5, H‐6, H‐7 of phthalazine), 8.68 (d, 1H, H‐8 of
phthalazine, J = 8.00), 9.39 (s, 1H, NH of pyrazole, D2O exchange-
able). Anal. calcd. for C30H25N5O (471.21): C, 76.41; H, 5.34;
N, 14.85. Found: C, 76.59; H, 5.38; N, 14.73.
(467.17): C, 77.07; H, 4.53; N, 14.98. Found: C, 77.17; H, 4.33;
N, 15.16.
4‐(4‐Methylphenyl)‐6‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}‐
pyrimidin‐2(1H)‐one (7b)
Yield, 44%; m.p. 160–162°C; IRνmax (cm−1): 3152 (2NH), 3052 (C–H
aromatic), 1681 (C═O amide); 1H NMR (400 MHz, DMSO‐d6): 2.51 (s,
3H, CH3), 7.55–7.62 (m, 5H, CH of pyrimidinone, H‐3, H‐5 of
4‐CH3–C6H4, H‐3, H‐5 of NH–C6H4), 7.69 (d, 3H, H‐3, H‐4, H‐5 of
C6H5), 7.92 (d, 2H, H‐2, H‐6 of 4‐CH3–C6H4, J = 8.08), 7.98–8.05 (m,
5H, H‐2, H‐6 of C6H5, H‐2, H‐6 of NH–C6H4, H‐5 of phthalazine),
8.16 (d, 2H, H‐6, H‐7 of phthalazine, J = 8.60), 8.70 (d, 1H, H‐8 of
phthalazine, J = 8.28), 9.68 (s, 1H, NH–C6H4, D2O exchangeable),
12.11 (s, 1H, NH‐pyrimidinone, D2O exchangeable); 13C NMR
(100 MHz, DMSO‐d6): 21.51, 116.34, 117.74, 119.83 (2), 121.71,
122.10 (2), 123.74 (2), 124.59, 129.60 (2), 130.04, 130.41 (2), 130.71,
131.77 (2), 132.12 (2), 132.88 (2), 138.26, 141.12, 147.11 (2), 150.98
(2), 164.85, 166.21. Anal. calcd. for C31H23N5O (481.19): C, 77.32; H,
4.81; N, 14.54. Found: C, 77.61; H, 4.75; N, 14.89.
(R,S)‐3‐(4‐Methoxyphenyl)‐1‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]‐
phenyl}prop‐2‐en‐1‐one (6g)
Yield, 75%; m.p. 175–177°C; IRνmax (cm−1): 3175 (NH), 3028 (C–H
aromatic), 2956 (C–H aliphatic), 1602 (C═N); 1H NMR (400 MHz,
DMSO‐d6): 2.80 (dd, 1H, H‐4 of pyrazole, J = 10.68), 3.75 (dd, 1H,
H‐5 of pyrazole, J = 10.68), 3.88 (s, 3H, 4‐O–CH3), 4.77 (t, 1H, H‐4 of
pyrazole, J = 10.68), 6.91 (d, 2H, H‐3, H‐5 of 4‐O–CH3–C6H4,
J = 8.68), 7.31 (d, 2H, H‐2, H‐6 of NH–C6H4, J = 10.00), 7.57–7.61 (m,
3H, H‐3, H‐4, H‐5 of C6H5), 7.64–7.68 (m, 4H, H‐3, H‐5 of NH–C6H4,
H‐2, H‐6 of 4‐OCH3–C6H4), 7.89–7.93 (dd, 2H, H‐2, H‐6 of C6H5,
J = 8.20), 8.02–8.04 (m, 3H, H‐5, H‐6, H‐7 of phthalazine), 8.22 (s, 1H,
NH‐pyrazole, D2O exchangeable), 8.68 (d, 1H, H‐8 of phthalazine,
J = 8.28), 9.40 (s, 1H, NH–C6H4, D2O exchangeable); 13C NMR
(100 MHz, DMSO‐d6): 41.42, 55.54, 63.60, 114.23, 114.62, 119.70,
119.84, 120.94, 123.28, 125.77, 126.25, 126.33, 126.45, 126.90,
127.77, 128.06, 128.22, 128.78, 128.95, 129.26, 129.92, 130.15,
130.21, 132.40, 132.78, 133.02, 136.93, 163.46, 184.36, 184.79.
Anal. calcd. for C30H25N5O (471.21): C, 76.41; H, 5.34; N, 14.85.
Found: C, 76.32; H, 5.49; N, 15.08.
4‐(4‐Methoxyphenyl)‐6‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}‐
pyrimidin‐2(1H)‐one (7c)
Yield, 40%; m.p. 171–173°C; IRνmax (cm−1): 3139 (NH), 3094 (CH
aromatic), 2938 (CH aliphatic), 1688 (C═O), 1516 (C═N); 1H NMR
(400 MHz, DMSO‐d6): 3.87 (s, 3H, 4‐OCH3), 7.12 (d, 2H, H3, H‐5 of
4‐OCH3–C6H4, J = 8.72), 7.47 (s, 1H, pyrimidinone), 7.59–7.61 (m,
4H, H‐3, H‐5 of NH–C6H4, J = 6.52, H‐6, H‐7 of phthalazine), 7.70 (d,
2H, H‐3, H‐5 of C6H5), 7.93 (m, 1H, H‐4 of C6H5), 7.99 (d, 1H, H‐6 of
C6H5, J = 7.24), 8.09 (d, 1H, H‐2 of C6H5, J = 8.08), 8.17–8.22 (m, 5H,
H2, H‐6 of 4‐OCH3–C6H4, H‐2, H‐6 of NH–C6H4, H‐8 of phthala-
zine), 8.72 (d, 1H, H‐5 of phthalazine, J = 7.36), 9.63 (s, 1H, NH–C6H4,
D2O exchangeable), 11.81 (s, 1H, NH‐pyrimidinone, D2O exchange-
able); 13C NMR (100 MHz, DMSO‐d6): 55.18, 120.09, 122.40, 127.27
(2), 127.32 (2), 127.48 (2), 127.56 (2), 128.57 (2), 128.84 (2), 130.18,
130.34 (2), 130.40 (2), 131.22, 139.43, 140.45, 140.97, 151.35,
152.95 (2), 166.19, 166.40, 166.79, 168.99; MS (m/z): 497 (M+,
1.74%), 495 (6.33%), 493 (5.68%), 457 (61.77%), 449 (58.24), 312
(25.53%), 311 (100% base peak). Anal. calcd. for C31H23N5O2
(497.19): C, 74.83; H, 4.66; N, 14.08. Found: C, 74.98; H, 4.60;
N, 14.27.
|
4.1.5
General procedure for the synthesis of
compounds 7a–c
A mixture of the appropriate chalcone 5a,d,g (0.005 mol) and urea
(0.50 g, 0.005 mol) in ethanol (20 ml) was refluxed for 8 h in the
presence of conc. HCl (5 ml). The reaction mixture was concentrated
to half its volume, cooled, and neutralized with NH4OH solution. The
precipitated solid was filtered, washed with water, air‐dried, and re-
crystallized from ethanol to yield the corresponding pyrimidine de-
rivatives 7a–c, respectively.
|
4.2
Docking studies
In the present work, all the target compounds were subjected to a
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 molecules
such as substrates or drug candidates bind to a protein of a known
3D structure represented by grid interaction potentials (http://www.
molsoft.com/icm_pro.html). Each experiment used the biological
target VEGFR‐2 downloaded from the Brookhaven Protein Data
4‐Phenyl‐6‐{4‐[(4‐phenylphthalazin‐1‐yl)amino]phenyl}pyrimidin‐
2(1H)‐one (7a)
Yield, 52%; m.p. 155–157°C; IRνmax (cm−1): 3212 (2NH), 3060 (C–H
aromatic), 1659 (C═O amide); 13C NMR (100 MHz, DMSO‐d6):
112.20, 121.02, 121.14, 122.71, 123.77, 124.02, 124.92, 124.98,
125.48, 125.82, 126.51, 126.57, 129.13, 129.16, 131.51, 131.79,
132.57 (2), 133.77, 133.88, 138.68, 141.71, 150.16, 150.18, 157.89,
158.02, 159.12, 159.16, 168.51, 174.28. Anal. calcd. for C30H21N5O

16 of 18
KHEDR ET AL.
|
Bank
(http://www.rcsb.org/pdb/explore/explore.do?structureId=
streptavidin donor beads, and 10 μg/ml acceptor beads in 62.5 mM
HEPES pH 7.4, 250 mM NaCl, and 0.1% bovine serum albumin. The
plate was incubated in the dark overnight and then read using an
ELISA reader (PerkinElmer). Wells containing the substrate and the
enzyme without compounds were used as reaction controls. Wells
containing biotinylated poly‐GluTyr (4:1) and enzymes without ATP
were used as basal controls. Percent inhibition was calculated by
comparison of compounds subjected to control incubations. The
concentration of the test compound that induced 50% inhibition
(IC50) was calculated from the concentration–inhibition response
curve (triplicate determinations) and the data were compared with
sorafenib (Sigma‐Aldrich) as the standard VEGFR‐2 inhibitor.
3B8Q). To qualify the docking results in terms of accuracy of the
predicted binding conformations in comparison with the experi-
mental 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 the Pharmacology &
Toxicology Department, Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt. Cancer cells from different cancer cell lines hepato-
cellular carcinoma (HepG2), colorectal carcinoma (HCT‐116), and
breast cancer (MCF‐7) were purchased from the American Type
Cell Culture Collection (ATCC) and grown on the appropriate
growth medium Roswell Park Memorial Institute medium (RPMI‐
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 MTT.[53]
ACKNOWLEDGMENTS
The authors extend their appreciation and gratitude to Dr. Fatma M.
I. A. Shoman, MD, in Clinical Pathology, Blood bank specialist, Blood
bank directorate manager, Ministry of Health, Cairo, Egypt, for help
with the pharmacological part.
CONFLICTS OF INTERESTS
Exponentially growing cells from different cancer cell lines were
trypsinized, counted and seeded at the appropriate densities
(2000–1000 cells/0.33 cm2 well) into 96‐well microtiter plates.
Cells were then incubated in a humidified atmosphere at 37°C for
24 h. Then, cells were exposed to different concentrations of
compounds (0.1, 10, 100, and 1000 µM) for 72 h. After that, the
viability of the treated cells was determined using the MTT tech-
nique as follows: Cells were incubated with 200 μl of 5% MTT so-
lution/well (Sigma‐Aldrich) and were allowed to metabolize the dye
into colored‐insoluble formazan crystals for 2 h. The formazan
crystals were dissolved in 200 µl/well acidified isopropanol for
30 min, covered with an aluminum foil, and subjected to continuous
shaking using a MaxQ 2000 plate shaker (Thermo Fisher Scientific
Inc.) at room temperature. Absorbance was measured at 570 nm
using a Stat FaxR 4200 plate reader (Awareness Technology, Inc.).
The cell viability was expressed as a percentage of control, and the
concentration that induces 50% of maximum inhibition of cell pro-
liferation (IC50) was determined using GraphPad Prism version 5
software (GraphPad Software Inc.).
The authors declare that there are no conflicts of interests.
ORCID
Ibrahim H. Eissa
http://orcid.org/0000-0002-6955-2263
http://orcid.org/0000-0001-6479-4573
Hamada S. Abulkhair
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
REFERENCES
[1] M. Shibuya, Genes Cancer 2011, 2, 1097. https://doi.org/10.1177/
1947601911423031
[2] J. Li, N. Zhou, K. Luo, W. Zhang, X. Li, C. Wu, J. Bao, Int. J. Mol. Sci.
2014, 15, 15994. https://doi.org/10.3390/ijms150915994
[3] N. R. Smith, D. Baker, N. H. James, K. Ratcliffe, M. Jenkins,
S. E. Ashton, G. Sproat, R. Swann, N. Gray, A. Ryan,
J. M. Jurgensmeier, C. Womack, Clin. Cancer Res. 2010, 16, 3548.
https://doi.org/10.1158/1078‐0432.CCR‐09‐2797
[4] T. Padró, R. Bieker, S. Ruiz, M. Steins, S. Retzlaff, H. Bürger,
T. Büchner, T. Kessler, F. Herrera, J. Kienast, C. Müller‐Tidow,
H. Serve, W. Berdel, R. Mesters, Leukemia 2002, 16, 1302. https://
doi.org/10.1038/sj.leu.2402534
[5] K.‐C. Hsu, T.‐Y. Sung, C.‐T. Lin, Y.‐Y. Chiu, J. T.‐A. Hsu, H.‐C. Hung,
C.‐M. Sun, I. Barve, W.‐L. Chen, W.‐C. Huang, C.‐T. Huang, C.‐H.
Chen, J.‐M. Yang, Sci. Rep. 2015, 5, 10938. https://doi.org/10.
1038/srep10938
[6] O. Fedorov, S. Müller, S. Knapp, Nat. Chem. Biol. 2010, 6, 166.
https://doi.org/10.1038/nchembio.297
|
4.4
In vitro VEGFR‐2 kinase assay
[7] T. Honda, H. Nagahara, H. Mogi, M. Ban, H. Aono, Bioorg. Med. Chem.
Lett. 2011, 21, 1782. https://doi.org/10.1016/j.bmcl.2011.01.063
[8] M. A. Zeidan, A. S. Mostafa, R. M. Gomaa, L. A. Abou‐Zeid, M. El‐
Mesery, M. A.‐A. El‐Sayed, K. B. Selim, Eur. J. Med. Chem. 2019,
168, 315. https://doi.org/10.1016/j.ejmech.2019.02.050
[9] A.‐G. A. El‐Helby, H. Sakr, I. H. Eissa, H. Abulkhair, A. A. Al‐
Karmalawy, K. El‐Adl, Arch. Pharm. (Weinheim) 2019, 352, 1900113.
https://doi.org/10.1002/ardp.201900113
The kinase activity of VEGFR‐2 was measured using an anti‐
phosphotyrosine antibody with the AlphaScreen system according to
the manufacturer's instructions.[54] Enzyme reactions were per-
formed in 50 mM Tris‐HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01%
Tween‐20, and 2 mM DTT, containing 10 μM ATP, 0.1 μg/ml bioti-
nylated poly‐GluTyr (4:1), and 0.1 nM of VEGFR‐2 (Millipore). Before
catalytic initiation with ATP, the tested compounds at final con-
centrations 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 AlphaScreen
[10] J. Dietrich, C. Hulme, L. H. Hurley, Bioorg. Med. Chem. 2010, 18,
5738. https://doi.org/10.1016/j.bmc.2010.05.063
[11] K. El‐Adl, M. K. Ibrahim, F. Khedr, H. S. Abulkhair, I. H. Eissa, Arch.
Pharm. (Weinheim) 2020, 354, e202000219. https://doi.org/10.
1002/ardp.202000219

KHEDR ET AL.
17 of 18
|
[12] M. Payton, H.‐K. Cheung, M. S. S. Ninniri, C. Marinaccio,
W. C. Wayne, K. Hanestad, J. D. Crispino, G. Juan, A. Coxon, Mol.
Cancer Ther. 2018, 17, 2575. https://doi.org/10.1158/1535‐7163.
MCT‐18‐0186
[13] S. Rampogu, A. Baek, A. Zeb, K. W. Lee, BMC Cancer 2018, 18, 264.
https://doi.org/10.1186/s12885‐018‐4050‐1
[14] S. M. Mennen, M. L. Mak‐Jurkauskas, M. M. Bio, L. S. Hollis,
K. A. Nadeau, A. M. Clausen, K. B. Hansen, Org. Process Res. Dev.
2015, 19, 884. https://doi.org/10.1021/acs.oprd.5b00135
[15] A.‐G. A. El‐Helby, R. R. A. Ayyad, H. Sakr, K. El‐Adl, M. M. Ali,
F. Khedr, Arch. Pharm. (Weinheim) 2017, 350, 1700240. https://doi.
org/10.1002/ardp.201700240
[34] K. El‐Adl, A. A. El‐Helby, R. R. Ayyad, H. A. Mahdy, M. M. Khalifa,
H. A. Elnagar, A. B. M. Mehany, A. M. Metwaly, M. A. Elhendawy,
M. M. Radwan, M. A. ElSohly, I. H. Eissa, Bioorg. Med. Chem. 2020,
29, 115872. https://doi.org/10.1016/j.bmc.2020.115872
[35] A. M. Sayed, F. A. Taher, M. R. K. Abdel‐Samad, M. S. A. El‐Gaby,
K. El‐Adl, N. M. Saleh, Bioorg. Chem. 2021, 108, 104669. https://
doi.org/10.1016/j.bioorg.2021.104669
[36] N. E. A. Abd El‐Sattar, K. El‐Adl, M. A. El‐Hashash, S. A. Salama,
M. M. Elhady, Bioorg. Chem. 2021, 115, 105186. https://doi.org/10.
1016/j.bioorg.2021.105186
[37] M. Li, X. Yu, W. Li, T. Liu, G. Deng, W. Liu, H. Liu, F. Gao, Oncotarget
2018, 9, 152. https://doi.org/10.18632/oncotarget.22077
[38] H. Wang, B. Rao, J. Lou, J. Li, Z. Liu, A. Li, G. Cui, Z. Ren, Z. Yu, Front.
Cell Dev. Biol. 2020, 8, 55. https://doi.org/10.3389/fcell.2020.
00055
[16] K. El‐Adl, A.‐G. A. El‐Helby, R. R. A. Ayyad, H. Sakr, M. M. Ali,
F. Khedr, Anticancer Agents Med. Chem. 2018, 18, 1184. https://doi.
org/10.2174/1871520618666180412123833
[17] H. S. Abulkhair, A. Turky, A. Ghiaty, H. E. A. Ahmed, A. H. Bayoumi,
Bioorg. Chem. 2020, 100, 103899. https://doi.org/10.1016/j.bioorg.
2020.103899
[39] Y. Zhang, X. Gao, Y. Zhu, D. Kadel, H. Sun, J. Chen, Q. Luo, H. Sun,
L. Yang, J. Yang, Y. Sheng, Y. Zheng, K. Zhu, Q. Dong, L. Qin, J. Exp.
Clin. Cancer Res. 2018, 37, 93. https://doi.org/10.1186/s13046‐
018‐0750‐2
[40] R. Aesoy, B. C. Sanchez, J. H. Norum, R. Lewensohn, K. Viktorsson,
B. Linderholm, Mol. Cancer Res. 2008, 6, 1630. https://doi.org/10.
1158/1541‐7786.MCR‐07‐2172
[41] S. Svensson, K. Jirström, L. Rydén, G. Roos, S. Emdin,
M. C. Ostrowski, G. Landberg, Oncogene 2005, 24, 4370. https://
doi.org/10.1038/sj.onc.1208626
[18] A. Turky, A. H. Bayoumi, A. Ghiaty, A. S. El‐Azab, A. A.‐M. Abdel‐
Aziz, H. S. Abulkhair, Bioorg. Chem. 2020, 101, 104019. https://doi.
org/10.1016/j.bioorg.2020.104019
[19] H. G. Ezzat, A. H. Bayoumi, F. F. Sherbiny, A. M. El‐Morsy,
A. Ghiaty, M. Alswah, H. S. Abulkhair, Mol. Divers. 2021, 25, 291.
https://doi.org/10.1007/s11030‐020‐10070‐w
[20] S. Elmeligie, A. M. Aboul‐Magd, D. S. Lasheen, T. M. Ibrahim,
T. M. Abdelghany, S. M. Khojah, K. A. M. Abouzid, J. Enzyme Inhib.
Med. Chem. 2019, 34, 1347. https://doi.org/10.1080/14756366.
2019.1642883
[42] Z. Liu, L. Qi, Y. Li, X. Zhao, B. Sun, BMC Cancer 2017, 17, 593.
https://doi.org/10.1186/s12885‐017‐3578‐9
[43] M. Zhong, N. Li, X. Qiu, Y. Ye, H. Chen, J. Hua, P. Yin, G. Zhuang, Int.
J. Biol. Sci. 2020, 16, 272. https://doi.org/10.7150/ijbs.37906
[44] R. N. Eskander, K. S. Tewari, Gynecol. Oncol. 2014, 132, 496.
https://doi.org/10.1016/j.ygyno.2013.11.029
[21] S. Zaib, I. Khan, Bioorg. Chem. 2020, 105, 104425. https://doi.org/
10.1016/j.bioorg.2020.104425
[22] S. Elmeligie, A. M. Aboul‐Magd, D. S. Lasheen, T. M. Ibrahim,
T. M. Abdelghany, S. M. Khojah, K. A. M. Abouzid., J. Enzyme Inhib.
Med. Chem. 2019, 34(1), 1347. https://doi.org/10.1080/14756366.
2019.1642883.y
[45] V. A. Machado, D. Peixoto, R. Costa, H. J. C. Froufe, R. C. Calhelha,
R. M. V. Abreu, I. C. F. R. Ferreira, R. Soares, M.‐J. R. P. Queiroz,
Bioorg. Med. Chem. 2015, 23, 6497. https://doi.org/10.1016/j.bmc.
2015.08.010
[23] P. Ravula, H. B. Vamaraju, M. Paturi, J. N. G. N. Sharath Chandra,
Arch. Pharm. (Weinheim) 2018, 351, 1700234. https://doi.org/10.
1002/ardp.201700234
[46] M. McTigue, B. W. Murray, J. H. Chen, Y. Deng, J., Solowiej,
R.S., Kania, Proc Natl. Acad. Sci. U.S.A. 2012, 109, 18281. https://
doi.org/10.1073/pnas.1207759109
[24] N. M. Saleh, M. G. El‐Gazzar, H. M. Aly, R. A. Othman, Front. Chem.
2020, 7, 917. https://doi.org/10.3389/fchem.2019.00917
[25] W. Sun, S. Hu, S. Fang, H. Yan, Bioorg. Chem. 2018, 78, 393. https://
doi.org/10.1016/j.bioorg.2018.04.005
[47] A.‐G. A. El‐Helby, R. R. A. Ayyad, M. F. Zayed, H. S. Abulkhair,
H. Elkady, K. El‐Adl, Arch. Pharm. (Weinheim) 2019, 352, 1800387.
https://doi.org/10.1002/ardp.201800387
[26] J. Li, W. Gu, X. Bi, H. Li, C. Liao, C. Liu, W. Huang, H. Qian, Bioorg.
Med. Chem. 2017, 25, 6674. https://doi.org/10.1016/j.bmc.2017.
11.010
[48] M. H. El‐Shershaby, K. M. El‐Gamal, A. H. Bayoumi, K. El‐Adl,
M. Alswah, H. E. A. Ahmed, A. A. Al‐Karmalamy, H. S. Abulkhair, New
J. Chem. 2021. 45, 13986. https://doi.org/10.1039/D1NJ02838C
[49] A. A. El‐Helby, H. Sakr, R. R. A. Ayyad, K. El‐Adl, M. M. Ali, F. Khedr,
Anticancer Agents. Med. Chem. 2018, 18(8), 1184. https://doi.org/
10.2174/1871520618666180412123833
[27] Y. Oguro, N. Miyamoto, K. Okada, T. Takagi, H. Iwata, Y. Awazu,
H. Miki, A. Hori, K. Kamiyama, S. Imamura, Bioorg. Med. Chem. 2010,
18, 7260. https://doi.org/10.1016/j.bmc.2010.08.017
[28] D. Jandial, C. Blair, S. Zhang, L. Krill, Y.‐B. Zhang, X. Zi, Curr. Cancer Drug
Targets 2014, 14, 181. https://doi.org/10.2174/15680096146661
40122160515
[50] M. K. Ibrahim, K. El‐Adl, A. A. Al‐Karmalawy, Bull. Fac. Pharm.
(Cairo Univ.) 2015, 53, 101. https://doi.org/10.1016/j.bfopcu.
2015.05.001
[29] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chem. Rev.
2017, 117, 7762. https://doi.org/10.1021/acs.chemrev.7b00020
[30] K. El‐Adl, H. Sakr, S. S. A. El‐Hddad, A. A. El‐Helby, M. Nasser,
H. S. Abulkhair, Arch. Pharm. (Weinheim) 2021, 354, e2000491.
https://doi.org/10.1002/ardp.202000491
[51] K. M. El‐Gamal, M. S. Hagrs, H. S. Abulkhair, Bull. Fac. Pharm.
(Cairo Univ.) 2016, 54, 263. https://doi.org/10.1016/j.bfopcu.
2016.08.002
[52] M. H. Hannoun, M. Hagras, A. Kotb, A.‐A. M. M. El‐Attar,
H. S. Abulkhair, Bioorg. Chem. 2020, 94, 103364. https://doi.org/
10.1016/j.bioorg.2019.103364
[31] K. El‐Adl, H. Sakr, M. Nasser, M. Alswah, F. M. A. Shoman, Arch.
Pharm. (Weinheim) 2020, 353, e2000079. https://doi.org/10.1002/
ardp.202000079
[53] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
[54] J. Kendrew, C. Eberlein, B. Hedberg, K. McDaid, N. R. Smith,
H. M. Weir, S. R. Wedge, D. C. Blakey, I. Foltz, J. Zhou, J. S. Kang,
S. T. Barry, Mol. Cancer Ther. 2011, 10, 770. https://doi.org/10.
1158/1535‐7163.MCT‐10‐0876
[32] K. El‐Adl, A.‐G. A. El‐Helby, H. Sakr, S. S. A. El‐Hddad, Arch.
Pharm. 2020, 353, e2000068. https://doi.org/10.1002/ardp.
202000068
[33] K. El‐Adl, A. A. El‐Helby, H. Sakr, R. R. Ayyad, H. A. Mahdy,
M. Nasser, H. S. Abulkhair, S. El‐Hddad, Arch. Pharm. 2020, 354,
e2000279. https://doi.org/10.1002/ardp.202000279
[55] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug
Deliv. Rev. 1997, 23, 3. https://doi.org/10.1016/S0169‐409X(96)
00423‐1

18 of 18
KHEDR ET AL.
|
[56] D. E. V. Pires, T. L. Blundell, D. B. Ascher, J. Med. Chem. 2015, 58,
4066. https://doi.org/10.1021/acs.jmedchem.5b00104
[57] A. Beig, R. Agbaria, A. Dahan, PLOS One 2013, 8, e68237. https://
doi.org/10.1371/journal.pone.0068237
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[58] A. Hebert, M. Bishop, D. Bhattacharyya, K. Gleason, S. Torosian,
Appl. Nanosci. 2015, 5, 763. https://doi.org/10.1007/s13204‐014‐
0373‐7
[59] S. Roy, M. K. Mathew, J. Biol. Chem. 2018, 293, 4289. https://doi.
org/10.1074/jbc.RA117.000432
How to cite this article: F. Khedr, M.‐K. Ibrahim, I. H. Eissa,
H. S. Abulkhair, K. El‐Adl, Arch. Pharm. 2021, e2100201.
https://doi.org/10.1002/ardp.202100201
[60] X. Wu, Q. Zhang, J. Hu, Environ. Res. 2016, 27, 147. https://doi.org/
10.1080/1062936X.2015.1137353