Design, synthesis and molecular modeling of new quinazolin-4(3H)-one based VEGFR-2 kinase inhibitors for potential anticancer evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.104695

Globally cancer is the second leading cause of death. So that this work is an attempt to develop new effective anti-cancer agents. In line with pharmacophoric features of VEGFR-2 kinase inhibitors, new nineteen quinazolin-4-one derivatives were designed,

Full text of DOI:10.1016/j.bioorg.2021.104695

Bioorganic Chemistry 109 (2021) 104695
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and molecular modeling of new quinazolin-4(3H)-one
based VEGFR-2 kinase inhibitors for potential anticancer evaluation
,
,
Abdallah E. Abdallaha *, Sally I. Eissab c, Maged Mohammed Saleh Al Warda,
Reda R. Mabrouka, Ahmed B.M. Mehanyd, Mohamed Ayman El-Zahabia
,
*
a Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11884, Egypt
c Faculty of Pharmacy Al-Maareffa University for Science and Technology, Riyadh, Saudi Arabia
d Zoology Department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Globally cancer is the second leading cause of death. So that this work is an attempt to develop new effective
anti-cancer agents. In line with pharmacophoric features of VEGFR-2 kinase inhibitors, new nineteen quinazolin-
4-one derivatives were designed, synthesized and biologically evaluated for their potential anticancer activity.
All target compounds were evaluated in vitro for VEGFR-2 tyrosine kinase inhibition. Then, nine compounds of
best results were further investigated by in vitro assay against three human cancer cell lines, namely HepG2, PC3
and MCF. N’-{2-](3-Ethyl-6-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio[acetyl}benzohydrazide (36) was found
to be the most potent candidate as it showed IC50 = 4.6 ± 0.06 µM against VEGFR-2 kinase. It also exhibited IC50
= 17.23 ± 1.5, 26.10 ± 2.2 and 30.85 ± 2.3 µg/mL against HepG2, PC3 and MCF, respectively. At the same time
it showed IC50 = 145.93 ± 1.1 µg/mL against the normal human lung fibroblasts cell line (WI-38), indicating
good selectivity index. Further investigation into HepG2 cell cycle showed the ability of compound 36 to induce
apoptosis and arrest cell growth at G2/M phase. Moreover, docking studies demonstrated the ability of com-
pound 36 to bind VEGFR-2 in a correct manner making three essential hydrogen bonds with the key residues
Glu885, Asp1046 and Cys919. In sum, this work suggests that compound 36 can serve as a lead for development
of effective anticancer agents targeting VEGFR-2.
Anticancer
Molecular docking
Quinazolin-4(3H)-one
VEGFR-2 kinase
1. Introduction
VEGFRs can be divided into three types VEGFR-1, VEGFR-2 and
VEGFR-3. VEGFR-2 is the most important and takes on a pivotal role in
Cancer is a worldwide major health problem. One of six death is
attributed to cancer. In U.S. 1,806,590 new cancer cases and 606,520
cancer deaths are the projected numbers to occur in 2020 [1]. So that
development of new anticancer agents is a global requirement to
improve the current situation.
angiogenesis process [6,7]. In comparison with normal vascular endo-
thelial cells, neo vascular tumor endothelial cells have extensive
expression of VEGFR-2 [8]. VEGFR-2 signaling pathway was shown in
many cancer types for example colorectal [9], breast [10], lung non-
small cell [11], urothelial [12] carcinoma. These facts drew a partic-
ular attention to inhibition of VEGFR-2 as a significant approach in
treatment of cancer. Examples for FDA approved anticancer drugs that
showed VEGFR-2 inhibition are pazopanib 1 [13], regorafenib 2 [14],
suntinib 3 [15], sorafenib 4 [16] and vatalanib 5 [17] and Tivozanib 6
[18] (Fig. 1).
To find an effective and safe anticancer agents, the characteristics of
cancer cells should be considered. Among which is secretion of growth
factors. One of which vascular endothelial growth factors (VEGFs)
which play a pivotal role in angiogenesis [2]. Angiogenesis is essential to
supply the cancer cells with oxygen and nutrients required for growth
[3]. It is also a potential cause for cancer progression and metastasis [3].
Inhibition of angiogenesis is reported as effective approach to cancer
treatment [4]. It can be achieved by vascular endothelial growth factor
receptors inhibition [5].
VEGFR-2 Kinase inhibitors can be classed into two main types [19].
Type I inhibitors e.g. sunitinib [18] occupy the region which originally
occupied by ATP adenine moiety. They identify the active conformation
of the receptor [20]. While type II kinase inhibitors e.g. sorafenib,
* Corresponding authors.
E-mail addresses: Abdulla_emara@azhar.edu.eg (A.E. Abdallah), Abelal_81@azhar.edu.eg (A.B.M. Mehany), malzahaby@azhar.edu.eg (M.A. El-Zahabi).
https://doi.org/10.1016/j.bioorg.2021.104695
Received 2 November 2020; Received in revised form 26 December 2020; Accepted 24 January 2021
Available online 8 February 2021
0045-2068/© 2021 Published by Elsevier Inc.

A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
regorafenib and tivozanib [18] stabilize the inactive conformation
(DFG-out) of the receptor which is formed by movement of the DFG
motif (Aspartate- Phenylalanine- Glycine motif). Type II inhibitors pri-
marily occupy an allosteric site which is a hydrophobic and appears
directly adjacent to the ATP binding domain. This allosteric site is
emerged only in the inactive conformation of the enzyme [21]. Type-II
kinase inhibitors are better than type-I inhibitors due to improved kinase
selectivity and slower off-rates [22].
1.1. Rationale of drug design.
The design was mainly based on the common essential pharmaco-
phoric features of type II VEGFR-2 kinase inhibitors as illustrated in
Fig. 2. Four features have been identified [26,27]; 1- flat hetero aromatic
moiety which occupies ATP binding domain and forms hydrogen bonds
with the key residues Cys919 and/or Glu917 [28,18]. 2-aryl linker
which occupies the area between ATP binding domain and DFG domain
[29]. 3- hydrogen bond acceptors and donors which forms hydrogen
bonds with the key residues Glu885 and Asp1046 in the DFG domain
[28]. 4- terminal hydrophobic moiety which binds via hydrophobic in-
teractions to the created hydrophobic allosteric pocket [28]. This pocket
is revealed when the phenylalanine residue of the DFG loop of the
enzyme flips out of its lipophilic pocket presenting DFG-out or inactive
conformation [30].
Quinazoline nucleus was used as a scaffold for development of potent
anticancer agents targeting VEGFR-2 [23,24]. One of quinazoline con-
taining compounds is the potent VEGFR-2 inhibitor AZD-2932 7 (Fig. 1)
[25].
Fig. 1. Chemical structures of some currently approved VEGFR-2 kinase inhibitors.
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Bioorganic Chemistry 109 (2021) 104695
Fig. 2. The common pharmacophoric features of type II VEGFR-2 tyrosine kinase inhibitors.
In the present work we used quinazoline as a scaffold to design
compounds consistent with the features of VEGFR-2 inhibitors as shown
in Fig. 3. Firstly, we selected substituted quinazoline-2,4-diones as
hetero aromatic moiety aiming to occupy ATP binding region and form
essential hydrogen bonds with Cys919 and/or Glu917. That is because
the quinazoline nucleus represents the hetero aromatic moiety in the
potent VEGFR-2 inhibitor AZD-2932 (Fig. 1). We made different sub-
stitutions at positions 3 (ethyl & phenyl), 6 (nitro & chloro) and 7 (nitro)
of quinazolin-4-one to find the most potent candidate and to establish
structure activity relationship. The essential hydrogen bonds can be
formed either via nitro group or chloro at positions 6 and 7 or via the
nitrogen or oxygen atoms of quinazolin4-one nucleus.
terminal hydrophobic groups to form hydrophobic interactions with the
allosteric pocket. Palin, 4-methoxy (electron donating) and 4-chloro
(electron withdrawing) phenyl were chosen to demonstrate the effect
of both substitution and electron density on the phenyl ring on the
biological effect.
2. Results and discussion
2.1. Chemistry
The strategy to obtain the target compounds was as follow; firstly,
four intermediate compounds (10a-c and 14) were prepared as illus-
trated in Scheme 1. The intermediates 10a-c were prepared by addition
of chloroacetyl chloride 9 drop wise to a solution of an appropriate
amine, namely aniline, 4-chloroaniline and 4-methoxyaniline [31].
Ethyl benzoate (12) was obtained by refluxing benzoic acid with cata-
lytic amount of conc. H2SO4 in absolute ethanol [32]. Refluxing com-
pound 12 with hydrazine hydrate in ethanol afforded the corresponding
Secondly, we selected amide which served as hydrogen bond
acceptor and donor in AZD-2932 (Fig. 1). The amidic group should form
essential hydrogen bonds with Glu885 and/or Asp1046 in the DFG
domain. In some designed compounds the amide was replaced with
oxohydrazide group as an effective hydrogen bond acceptor and donor.
Thirdly, substituted and unsubstituted phenyl rings were designed as
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A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
intermediate 14 [33].
Secondly, several salts of substituted mercaptoquinazolin-4-one 18a-f
were prepared as illustrated in Scheme 2. Refluxing a mixture of
substituted anthranilic acid namely 5-chloroanthranilic, 5-nitro-
anthranilic and 4-nitroanthranilic acid and an appropriate isothiocya-
nate namely ethyl isothiocyanate and phenyl isothiocyanate in absolute
ethanol in the presence of Et3N afforded the corresponding substituted
2-mercaptoquinazolin-4(3H)-one 17a-f. Treatment of compounds 17a-f
with equimolar amount of KOH afforded the corresponding salts 18a-f
.
Finally, refluxing the potassium salts of quinazolinone 18a-f with an
appropriate intermediate compound (10a-c and 14) gave the corre-
sponding final compounds 19–37 as illustrated in Scheme 2.
IR spectra of the final compounds show that intense bands at about
1535 and 1345 cmꢀ 1 were recoded specific to compounds containing
nitro group. Carbonyls of secondary amide were detected as strong
signals from about 1650 to 1701 cmꢀ 1. The intense amide II band
appeared from 1516 to 1554 cmꢀ 1. While carbonyls of quinazolinone
nucleus were detected from about 1630 to 1681 cmꢀ 1
.
1H NMR spectra of the final compounds show the appearance of
singlet signal of two protons integration at about 4.20 ppm which in-
dicates the presence of methylene group flanked between carbonyl
group and sulfur atom. With respect to 3-ethylquinazolinone de-
rivatives, two peaks are specific to them, the first is triplet of 3 protons
integration and appeared at about 1.33 ppm while the other detected at
about 4.14 ppm as quartet of 2 protons integration. Appearance of
singlet signal at about 3.72 ppm is characteristic of methoxy containing
derivatives. Presence of D2O exchangeable singlet peak of single proton
at about 10.40 ppm is attributed to NH of amide group. While spectra of
oxohydrazide containing derivatives revealed two exchangeable singlet
peaks at about 10.40 and 10.50 ppm.
Fig. 3. Model of the designed final compounds as VEGFR-2 kinase inhibitors
with the relevant pharmacophoric features.
hydrazide 13 [31].
Treatment of benzohydrazide 13 in DCM with chloro acetylchloride
in the presence of triethyl amine as a base under ice cooling gave the
Scheme 1. General procedures for synthesis of intermediates. i: DCM,Et3N, ice bath. ii: absolute ethanol, conc. H2SO4, reflux, 4 h. iii: NH2NH2, ethanol, reflux, 8 h.
iv: ClCOCH2Cl, DCM, Et3N, ice bath.
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A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
Scheme 2. General procedure for synthesis of final compounds. i: absolute ethanol, ET3N, reflux, 4 h. ii: alc. KOH, heating. iii: compounds 10a-c, KI, acetonitrile,
reflux, 2 h. iv: compound 14, KI, acetonitrile, reflux, 2 h.
2.2. Biological testing
potent than pazopanib which showed IC50 = 4.8 ± 0.07 µM.
Compounds 37, 28, 35 and 31 were weaker than pazopanib but close
to it. They showed IC50 = 4.9 ± 0.07, 5.4 ± 0.08, 6.6 ± 0.11 and 6.6 ±
0.13 µM, respectively.
2.2.1. In vitro VEGFR-2 tyrosine kinase inhibitory assay
All target compounds were evaluated for their VEGFR-2 tyrosine
kinase inhibitory activity. The concentrations that caused 50% enzyme
inhibition (IC50) were determined along with IC50 of pazopanib as a
reference drug.
Compounds 34, 25, 29, 32, 27, 30 and 33 should also be considered
as they showed potency exceeding half that of pazopanib.
The data indicate the particular significance of oxohydrazide con-
taining 6-nitroquinazolin-4(3H)-one derivatives especially compounds
36 and 37.
The results presented in Table 1 indicate that many compounds
exhibited potent VEGFR-2 tyrosine kinase inhibition with IC50 more or
less than that of pazopanib. Compound 36 was found to be the most
potent canditate as it demonstrated IC50 = 4.6 ± 0.06 µM. it was more
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A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
compounds 25, 32 and 29 demonstrated weak anticancer activity, no
compound was found to be very weak or inactive.
Table 1
IC50 of in vitro VEGFR-2 kinase inhibitory assay for all target compounds and
Pazopanib.
Serial
It is noticeable that compound 36 was the most potent candidate in
both VEGFR-2 inhibitory assay and antitumor assay.
Comp. no.
VEGFR-2 tyrosine kinase IC50 (µM)
1
19
13.7 ± 0.55
11.3 ± 0.24
10.7 ± 0.21
12.8 ± 0.45
11.1 ± 0.35
10.5 ± 0.22
7.6 ± 0.13
12.3 ± 0.38
9.3 ± 0.20
5.4 ± 0.08
8.5 ± 0.17
9.3 ± 0.19
6.6 ± 0.13
8.5 ± 0.18
9.3 ± 0.14
7.5 ± 0.12
6.6 ± 0.11
4.6 ± 0.06
4.9 ± 0.07
4.8 ± 0.07
2.2.3. Selective cytotoxicity test
2
20
3
21
One very important criterion of good anticancer agent is to be of
minimum or no side effect on normal cells. It can be achieved if the agent
has high degree of selectivity to cancer cell. So that safety of the target
compounds was further evaluated by in vitro cytotoxicity assay against
the normal human lung fibroblasts cell line (WI-38).
4
22
5
23
6
24
7
25
8
26
9
27
The results presented in Table 2 show that it is typical of all tested
compounds to have good degree of selectivity as their effect against
normal cell line was far weaker than that against cancer cell lines. In
particular, compound 36 which demonstrated selectivity indexes equal
8.47, 5.59 and 4.73 to HepG2, PC3 and MCF-7 cancer cell lines,
respectively.
10
11
12
13
14
15
16
17
18
19
20
28
29
30
31
32
33
34
The data presented in Tables 1 & 2 point to Compound 36 as the most
significant candidate. So that it was selected to further evaluation by two
more biological testing; effect on cell cycle progression and apoptosis
induction testing.
35
36
37
Pazopanib
2.2.4. Correlation of cytotoxicity with VEGFR-2 inhibition
2.2.2. In vitro antiproliferative activity assay
Results of VEGFR-2 inhibitory assay show the ability of our com-
pounds to inhibit VEGFR-2 kinase. To examine to what extent the
VEGFR-2 inhibition is the cause of antitumor effects of our compounds,
IC50 of VEGFR-2 inhibition were plotted against their corresponding
antiproliferative ones in simple linear regression setting [34]. The ob-
tained coefficients of determination (R2) imply the correlation between
the VEGFR-2 inhibition and the induced cytotoxicity. The R2 values for
HepG2, PC3, and MCF-7 were 0.4633, 0.4292, and 0.3775, respectively
(Fig. 4). Such R2 values indicate that the VEGFR-2 inhibition accounts to
a good extent for the cytotoxicity in the tested cell lines.
Nine Compounds of significant VEGFR-2 kinase inhibition were
selected for further in vitro antitumor investigation along with doxoru-
bicin as a reference drug. The assay was carried out against three human
tumor cell lines; hepatocellular carcinoma (HepG2), prostate cancer
(PC3), and mammary gland cancer (MCF-7). The concentrations caused
50% inhibition of cancer cell growth (IC50) were presented in Table 2.
The obtained results showed that six compounds are of strong to
moderate antiproliferative activity against the tested cell lines. The data
also demonstrated that HepG2 cell line is the most sensitive cell line to
the effect of the new derivatives, in particular compound 36.
Compound 36 was found to be the most potent derivative and had
demonstrated IC50 = 17.23 ± 1.5, 26.10 ± 2.2 and 30.85 ± 2.3 µg/mL
against HepG2, PC3 and MCF-7, respectively. Compound 34 was the
second most active compound with IC50 = 21.68 ± 1.9, 25.49 ± 2.0 and
33.70 ± 2.5 µg/mL against HepG2, PC3 and MCF-7, respectively.
Compounds 31, 28, 35 and 37 exhibited moderate activity. Despite of
2.2.5. Effect on cell cycle progression.
Cell cycle analysis was carried out for evaluation the effect of com-
pound 36 on HepG2 growth at different cell cycle phases. The obtained
results showed that compound 36 can arrest cell growth cycle at G2/M
phase. Fig. 5 illustrates the significant differences in the percent of cells
accumulation at G2/M and pre G1 phases between control HepG2 cells
and HepG2 cells treated with compound 36.
Table 2
2.2.6. Induction of apoptosis
IC50 of in vitro antiproliferative and cytotoxicity assay on tumor cell lines,
HePG2, PC3 and MCF-7 and on normal cell line WI-38 for the selected nine
compounds. a IC50 values are the mean ± S.D. of three separate experiments.b
IC50 (µg/mL): 1–10 (very strong), 11–25 (strong), 26–50 (moderate), 51–100
(weak).C ND: Not determined.
Induction of apoptosis in HepG2 cells by compound 36 was also
evaluated. The results illustrated in Fig. 6 show that the apoptosis per-
centage of control HepG2 is significantly increased from 2.37% to
18.33% upon treating with compound 36. On the other side, necrosis is
increased slightly from 1.17% to 2.44%. These data indicate that com-
pound 36 induces cancer cell apoptosis rather than necrosis.
Serial
Comp. no.
IC50 (µg/mL)a,b
HepG2
PC3
MCF-7
WI-38
7
25
54.43 ±
3.7
58.01 ±
3.9
62.82 ±
3.8
205.37 ±
1.7
2.3. In silico studies
10
11
13
14
16
17
18
19
21
28
36.27 ±
2.9
45.26 ±
3.0
46.33 ±
3.2
181.09 ±
1.6
2.3.1. Docking studies
To get insights on the binding pattern of target compounds into
VEGFR-2 binding site, molecular docking was performed using Molec-
ular Operating Environment (MOE) software. The crystal structure of
the enzyme complexed with pyrrolopyrimidine inhibitor was obtained
from protein data bank website (PDB code 3VHE). Validation of docking
process was carried out by redocking the ligand compound into the
active site of the receptor. The ligand was found to demonstrate the same
binding mode obtained by X-ray crystal structure with root mean square
deviation (RMSD) = 0.42 with free binding energy = -9.34 Kcal/mol.
Fig. 7 and Fig. 8 show that the ligand exhibited four hydrogen bonds;
one with Asp1046 and two with Glu885 via its urea moiety; the fourth
hydrogen bond was to Glu917 via NH of pyrimidine nucleus. Moreover,
it exhibited a further arene-H interaction with Leu840.
29
71.20 ±
4.1
82.55 ±
4.5
65.18 ±
3.9
167.44 ±
1.4
31
32.60 ±
2.5
34.09 ±
2.6
42.91 ±
3.1
119.75 ±
1.1
32
67.20 ±
3.9
89.35 ±
4.7
71.98 ±
4.1
246.12 ±
1.7
34
21.68 ±
1.9
25.49 ±
2.0
33.70 ±
2.5
105.33 ±
1.1
35
40.63 ±
3.2
37.16 ±
2.8
35.38 ±
2.6
154.37 ±
1.2
36
17.23 ±
1.5
26.10 ±
2.2
30.85 ±
2.3
145.93 ±
1.1
37
44.21 ±
3.5
48.83 ±
3.4
52.49 ±
3.6
161.76 ±
1.3
Doxorubicin
7.94 ± 0.6
8.87 ± 0.6
6.75 ± 0.4
NDc
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Bioorganic Chemistry 109 (2021) 104695
Fig. 4. Correlation of cytotoxicity with VEGFR-2 inhibition on three cell line models HepG2, PC3 and MCF-7.
Fig. 6. Effect of compound 36 on HepG2 cells apoptosis and necrosis compared
Fig. 5. Effect of compound 36 on percent accumulation of HePG2 cells at
with control HepG2 cells.
different phases of cell cycle compared with untreated HePG2 cells.
nitro group attached to quinazolin-4-one nucleus which was designed to
occupy ATP binding domain. Fig. 11 illustrates that the compound 36
and the ligand accommodated the pocket in the same manner which is
consistent with the design to a great extent. As the hetero aromatic
moiety of both occupied ATP binding domain. At the same time the urea
of the ligand and oxohydrazide group of compound 36 occupied the DFG
domain.
According to the binding pattern, our compounds can be divided into
two categories; the first showed a binding mode consistent with the
design and similar to the binding pattern of the ligand. This category
includes compounds 20, 21, 26, 27, 31, 33, 34 and 36.
Fig. 9 and Fig. 10 show that compound 36 revealed three hydrogen
bonds with the three essential amino acids in a correct manner consis-
tent with the design purpose. It made hydrogen bonds with Glu885 and
Asp1046 via its oxohydrazide group which was designed to occupy DFG
domain. The third hydrogen bond was formed between Cys919 and the
Fig. 12 and Fig. 13 show the correct binding pattern of compound 33.
As can be seen a hydrogen bond is formed with Glu885 via oxohydrazide
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Bioorganic Chemistry 109 (2021) 104695
Fig. 7. The binding pattern of the ligand to VEGFR-2 exhibited four essential hydrogen bonds with Glu885, Asp1046 and Glu917 as well as one arene-H interaction
with Leu840 (2D image).
Fig. 8. 3D image for binding of the pyrrolopyrimidine ligand with VEGFR-2, four essential hydrogen bonds were formed with Glu885, Asp1046 and Glu917 as well
as one arene-H interaction with Leu840.
moiety and another bond with Cys919 via nitro group attached to the
quinazoline nucleus in a manner consistent with the design. Further
hydrogen bond can be observed with Lys868. In the same manner Fig. 14
and Fig. 15 reveal the correct binding mode of compound 20 which
made two essential hydrogen bonds with Glu885 and Asp1046.
binding energies of 3-phenylquinazolin-4-one derivatives were found to
be comparable to that of 3-ethylquinazolin-4-one derivatives. That may
explain the comparable biological results.
It is noticeable that compound 36 is the single most efficient com-
pound. It is the only one who bound to the three essential amino acids.
Furthermore it was of the lowest binding energies (-7.42 Kcal/mol.).
These findings clearly explain the superiority of compound 36 in the
biological testing.
It is noticeable that the ethyl group at position 3 of quinazolin-4-one
can accommodate the pocket. So that 3-ethylquinazolin-4-one de-
rivatives showed the correct binding mode. While the phenyl group at
position 3 seems to be unable to accommodate the pocket so that all 3-
phenylquinazolin-4-one derivatives showed different binding patterns
which were not consistent with that of the ligand. Despite of that the
2.3.2. In silico ADMET analysis
ADMET studies were carried out for final compounds. Pazopanib was
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Bioorganic Chemistry 109 (2021) 104695
Fig. 9. 2D image shows the binding pattern of compound 36 to VEGFR-2. 3hydrogen bonds can be observed with the essential amino acid residues; Glu885 and
Asp1046 via oxohydrazide group; and with Cys919 via nitroquinazolinone moiety.
Fig. 10. 3D image for the binding of compound 36 to VEGFR-2 via 3 essential hydrogen bonds with Glu885, Asp1046 and Cys919.
used as reference compound. ADMET studies include many descriptors;
i) Blood brain barrier penetration. ii) Intestinal absorption which pre-
dicts human intestinal absorption (HIA) after oral administration. iii)
Aqueous solubility which predicts the solubility of each compound in
low. Accordingly, such compounds were expected to have no CNS side
effects. Compounds 25–30 were predicted to have high levels of BBB
penetration.
Compound 33 and 36 showed good levels of aqueous solubility. On
the other hand, the other candidates were expected to have low or very
low aqueous solubility.
◦
water at 25 C. iv) CYP2D6 binding which predicts cytochrome P450
2D6 enzyme inhibition. v) Plasma protein binding which predicts the
fraction of drug bound to plasma proteins in the blood [35]. Discovery
studio 4.0 was used to predict ADMET descriptors for all compounds.
The predicted descriptors are listed in Table 3.
Intestinal absorption is defined as a percentage absorbed compound
from the gut wall [36]. A well-absorbed compound is one that at least
90% absorbed into the bloodstream in humans [37]. According to the
ADMET studies, absorption levels of compounds 19–21, 25–28, 30, 31,
34, and 35 appeared in the good range, while compounds 22–24, 29,
ADMET - Blood Brain Barrier (BBB) penetration studies predicted
that BBB penetration levels of most of synthesized compounds are very
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Bioorganic Chemistry 109 (2021) 104695
Fig. 11. 3D image shows the overlay of compound 36 (blue colored molecule) and the ligand (green colored one). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 12. 2D binding pattern of compound 33, 3 hydrogen bonds were formed with Cys919, Glu885 and Lys868.
32, 33, and 36 showed moderate levels of intestinal absorption. In
contrary, compound 37 was predicted to have poor level of intestinal
absorption.
CYP2D6. Consequently, liver dysfunction side effect is not expected
upon administration of these compounds.
The plasma protein binding model predicts whether a compound is
likely to be highly bound (>= 90% bound) to carrier proteins in the
blood. All compounds were expected to bind plasma protein over than
more than 90% except compounds 21 and 36 (Fig. 16)
The cytochrome P450 2D6 (CYP2D6) model predicts CYP2D6
enzyme inhibition using 2D chemical structure as input. CYP2D6 is
involved in the metabolism of a wide range of drugs and hence its in-
hibition is expected to cause drug drug interactions. Therefore, CYP2D6
inhibition experiment is required as part of the regulatory procedures in
the drug discovery and development process [38]. The obtained results
predicted that all compounds have the advantage of non-inhibition of
2.4. Structure activity relationship (SAR)
On the basis of the obtained results SAR can be established to the
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Bioorganic Chemistry 109 (2021) 104695
Fig. 13. 3D image for binding pattern of compound 33, 3 hydrogen bonds were formed with Cys919, Glu885 and Lys868.
Fig. 14. 2D image for binding pattern of compound 20, it shows hydrogen bonds with the essential amino acid residues Glu 885 and Asp1046 as well as arene-H
interaction with Val848.
final compounds as follow
3- Chlorine atom at position 6 of quinazolin-4-one is more significant
than nitro group at position 7. Because it is likely that position 6 is
the correct position to orient the substituent to form hydrogen bond
with Cys919.
1- Nitro group at position 6 is far better than position 7 of quinazolin-4-
one. That may be explained by that the substituent at position 6 is
likely to be oriented towards the essential amino acid Cys919. Hence
the nitro group at position 6 is able to form hydrogen bond with
Cys919. While the substituent at position 7 is oriented away from
Cys919.
4- Ethyl group at position 3 of quinazolin-4-one is far better than phenyl
with regard to design of VEGFR-2 inhibitors type II.
5- N-oxohydrazide group is of a significant effect rather than amidic
group.
2- Nitro group at position 6 of quinazolin-4-one is better than chlorine
atom.
6- It was also found that the terminal plain phenyl has better activity
than substituted one.
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Bioorganic Chemistry 109 (2021) 104695
Fig. 15. 3D image for binding pattern of compound 20, it shows hydrogen bonds with the essential amino acid residues Glu 885 and Asp1046 as well as arene-H
interaction with Val848.
the data suggest that 3-ethyl-6-nitroquinazoline-4-one scaffold is of
Table 3
considerable importance and it should be considered for future devel-
: Predicted ADMET for the designed compounds and reference drugs.
opment of new potential VEGFR-2 kinase inhibitor agents. The results
Comp.
BBB
Solubility
level b
Absorption
level c
CYP2D6
PPB
also point to the relative importance of oxohydrazide moiety to be
incorporated in VEGFR-2 kinase inhibitors design. As it serves as
hydrogen bond acceptor and donor superior to amide group. Also on the
basis of the obtained results, structure activity relationship of the
designed compounds was clearly established. At the same time the
antiproliferative assay showed that sensitivity of hepatocellular carci-
noma to the designed compounds is more than prostate and breast
cancer. Moreover, the data of the present study indicate the considerable
importance of 36 to serve as a lead compound for further development
of potential anticancer agents targeting VEGFR-2 tyrosine kinase.
level
prediction d
prediction
a
e
19
4
4
4
4
4
4
1
1
2
1
1
1
4
4
4
3
4
4
4
4
2
2
2
2
1
2
2
2
2
1
1
1
2
2
3
2
2
3
2
1
0
0
0
1
1
1
0
0
0
0
1
0
0
1
1
0
0
1
2
1
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
TRUE
TRUE
FALSE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
FALSE
TRUE
TRUE
20
21
22
23
24
25
26
27
28
29
4. Materials and methods
30
31
4.1. Chemistry
32
33
34
All melting points were obtained by open capillary method on a
Gallen lamp Melting point apparatus and were uncorrected. Infrared
spectra were recorded on Pye Unicam SP 1000 IR spectrophotometer
35
36
37
(KBr discs) and were expressed in wave number (cmꢀ 1). 1H NMR and 13
C
Pazopanib
NMR spectra were recorded on a BRUKER 400 MHZ-NMR spectro-
photometer. TMS was used as internal standard in deuterated DMSO and
chemical shifts were measured in δppm. Mass spectra were recorded on
Varian MAT 311-A (70 e.v.). Progresses of the reaction was monitored
by TLC using TLC sheets precoated with UV fluorescent silica gel Merck
60 F254 plates and were visualized using UV lamp.
a
BBB level, blood brain barrier level, 0 = very high, 1 = high, 2 = medium, 3
= low, 4 = very low.
b
Solubility level, 1 = very low, 2 = low, 3 = good, 4 = optimal.
c
d
e
Absorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.
CYP2D6, cytochrome P2D6, TRUE = inhibitor, FALSE = non inhibitor.
PBB, plasma protein binding, FALSE means less than 90%, TRUE means
more than 90%
4.1.1. 4.1.1. General method of synthesis of compounds (17a-f
)
General method of synthesis of compounds (17a-f). To a solution of
anthranilic acid derivative namely, 4-nitroanthranilic acid, 5-chloroan-
thranilic acid and 5-nitroanthranilic acid (14.58 mmol) and Et3N (0.30
g, 0.42 mL, 2.92 mmol) in absolute ethanol (30 mL), an appropriate
isothiocyanate namely ethyl and phenyl isothiocyanate (16.04 mmol)
was added. The reaction mixture was heated under reflux for 5 h. Then
upon cooling to r.t., the obtained precipitate was filtered off and
recrystallized from ethanol to afford the corresponding substituted 2-
mercaptoquinazolin-4(3H)-one derivative [39,40].
3. Conclusion
In our effort to develop new effective anticancer agents, new nine-
teen quinazolinone derivatives were designed, synthesized and biolog-
ically evaluated. The design is mainly based on trisubstituted
quinazoline-4(3H)-one as a scaffold on which the pharmacophoric fea-
tures of VEGFR-2 kinase inhibitors is built. The data obtained from
VEGFR-2 tyrosine kinase inhibitory assay strongly support the signifi-
cance of VEGFR-2 kinase features illustrated in Fig. 2. At the same time
12

A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
Fig. 16. the expected ADMET profile for final compounds.
4.1.1.1. 4.1.1.1. 6-Chloro-3-ethyl-2-mercaptoquinazolin-4(3H)-one (17a)
the presence of a catalytic amount of KI. The reaction mixture was then
left to cool and diluted with cold water (50 mL) to give a precipitate. The
obtained precipitate was collected by filtration, washed with water,
dried and then crystallized from ethanol to afford the corresponding
final compound.
. 6-Chloro-3-ethyl-2-mercaptoquinazolin-4(3H)-one (17a). white solid
ꢀ 1
(yield 71.23%); m.p. ˃300 ◦C; IR (KBr, cm ): 3197 (NH), 3058 (C
H
–
1
–
aromatic), 2986 (C H aliphatic), 2550 (SH) and 1631 (CO amide); H
NMR (DMSO‑d6, 400 MHz) δ (ppm): 1.22 (t, J = 6.8 Hz, 3H, CH3), 4.41
(q, J = 6.8 Hz, 2H, CH2CH3), 7.35 (d, J = 8.4 Hz, 1H, Ar-H), 7.74 (dd, J
= 2.4 & 8.8 Hz, 1H, Ar-H), 7.83 (d, J = 2.4 Hz, 1H, Ar-H), 12.98 (s, 1H,
SH).
4.1.3.1. 2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]-N-
phenylacetamide. (19). 2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihydroquinazo-
line-2-yl)thio]-N-phenylacetamide. (19). yellow solid (yield 88.80%).
ꢀ 1
m.p. 190 ◦C; IR (KBr, cm ): 3286 (NH), 3082 (C H aromatic), 2993 &
–
4.1.1.2. 4.1.1.2. 3-Ethyl-2-mercapto-7-nitro-quinazolin-4(3H)-one (17e)
. 3-Ethyl-2-mercapto-7-nitro-quinazolin-4(3H)-one (17e). White solid
(yield 60.05%); m.p. 262–264 ◦C; IR (KBr, cmꢀ 1): 3116 (NH), 3095
–
2943 (C H aliphatic), 1693 (CO amide), 1651 (CO amide), 1550 (amide
II band) and 1535, 1342 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm):
1.33 (t, J = 7.0 Hz, 3H, CH3CH2), 4.14 (q, J = 7.0 Hz, 2H, CH3CH2), 4.27
(s, 2H, CH2CO), 7.06 (t, J = 7.4 Hz, 1H, Ar-H phenyl), 7.32 (dd, J = 7.4 &
8.0 Hz, 2H, Ar-H phenyl), 7.62 (d, J = 8.0 Hz, 2H, Ar-H phenyl), 8.13
(dd, J = 2.2 & 8.7 Hz, 1H, Ar-H quinazoline), 8.24 (d, J = 2.2 Hz, 1H, Ar-
H quinazoline), 8.27 (d, J = 8.7 Hz, 1H, Ar-H quinazoline), 10.45 (s, 1H,
D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 13.15,
37.36, 40.45, 119.69, 119.79, 121.20, 123.51, 124.01, 129.21, 129.28,
139.36, 147.31, 151.57, 159.46, 159.77, 166.10.
–
–
(C H aromatic), 2940, 2890 (C H aliphatic), 2600 (SH), 1627 (CO
amide) and 1534, 1338 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm):
1.24 (t, J = 7.2 Hz, 3H, CH3), 4.42 (q, J = 6.8 Hz, 2H, CH2CH3), 8.01 (dd,
J = 2.0 & 8.8 Hz, 1H, Ar-H), 8.11 (d, J = 2.0 Hz, 1H, Ar-H), 8.14 (d, J =
8.8 Hz, 1H, Ar-H), 13.16 (s, 1H, SH).
4.1.1.3. 4.1.1.3. 2-Mercapto-7-nitro-3-phenyl-quinazolin-4(3H)-one (17f)
. 2-Mercapto-7-nitro-3-phenyl-quinazolin-4(3H)-one (17f). Yellow
solid (yield 54.99%); m.p. 240–242 ◦C; IR (KBr, cmꢀ 1): 3269 (NH), 3090
–
(C H aromatic), 2600 (SH), 1658 (CO amide) and 1531, 1353 (NO2);
4.1.3.2. N-(4-Chlorophenyl)-2-[(3-ethyl-7-nitro-4-oxo-3,4-dihy-
1H NMR (DMSO‑d6, 400 MHz) δ (ppm): 7.30 (d, J = 6.8 Hz, 2H, Ar-H),
7.43 (t, J = 7.2 Hz, 1H, Ar-H), 7.50 (t, J = 7.2 Hz, 2H, Ar-H), 8.05 (dd, J
= 2.0 & 8.8 Hz, 1H, Ar-H), 8.17 (d, J = 8.8 Hz, 1H, Ar-H), 8.21 (d, J =
2.0 Hz, 1H, Ar-H), 13.30 (s, 1H, SH).
droquinazoline-2-yl)thio]-acetamide. (20). N-(4-Chlorophenyl)-2-[(3-eth
yl-7-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]-acetamide.
(20).
yellow solid (yield 92.20%). m.p. 215 C; IR (KBr, cmꢀ 1): 3344 (NH),
◦
–
–
3089 (C H aromatic), 2974 & 2916 (C H aliphatic), 1693 (CO amide)
and 1539, 1346 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm): 1.33 (t, J
= 7.0 Hz, 3H, CH3CH2), 4.14 (q, J = 7.0 Hz, 2H, CH3CH2), 4.27 (s, 2H,
CH2CO), 7.37 (d, J = 8.8 Hz, 2H, Ar-H phenyl), 7.65 (d, J = 8.8 Hz, 2H,
Ar-H phenyl), 8.13 (dd, J = 2.2 & 8.8 Hz, 1H, Ar-H quinazoline), 8.21 (d,
J = 2.2 Hz, 1H, Ar-H quinazoline), 8.27 (d, J = 8.7 Hz, 1H, Ar-H qui-
nazoline), 10.59 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6,
400 MHz) δ (ppm): 13.15, 29.45, 37.30, 119.68, 121.18, 123.54,
127.58, 129.21, 129.24, 130.05, 138.33, 147.30, 151.61, 159.41,
159.79, 166.35. Mass (m/z): 420 (M++2), 418 (M+), 380, 355 (100%,
base peak), 290, 247, 240, 231, 222, 148, 119, 76, 64 and 53.
4.1.2. 4.1.2. General procedure of synthesis of compounds (18a-f
)
General procedure of synthesis of compounds (18a-f) 2-Mercapto-
quinazolin-4(3H)-one derivative (17a-f) was added to alcoholic solution
of equimolar amount of potassium hydroxide. The reaction mixture was
heated with stirring until a sticky mass was obtained. Upon cooling to r.
t. and drying, the corresponding potassium salt was obtained in a
quantitative yield. All had melting points above 300 ◦C.
4.1.3. General procedure of synthesis of final compounds (19–37)
General procedure of synthesis of final compounds (19–37). A
mixture of an appropriate potassium salt of 2-mercaptoquinazolin-4
(3H)-one 18a-f (0.65 mmol) and an intermediate compound (3a-c and 7a,
b) (0.65 mmol) was heated under reflux for 2 h in acetonitrile (20 mL) in
4.1.3.3. 2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]-N-
(4-methoxyphenyl)acetamide. (21). 2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihy-
droquinazoline-2-yl)thio]-N-(4-methoxyphenyl)acetamide. (21). yellow
13

A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
solid (yield 87.50%). m.p. 185 ◦C; IR (KBr, cmꢀ 1): 3271 (NH), 3097 &
amide) and1550 (amide II band); 1H NMR (DMSO‑d6, 400 MHz) δ
(ppm): 1.32 (t, J = 7.04 Hz, 3H, CH3CH2), 4.12 (q, J = 7.0 Hz, 2H,
CH3CH2), 4.23 (s, 2H, CH2CO), 7.06 (t, J = 7.36 Hz, 1H, Ar-H phenyl),
7.32 (dd, J = 7.68 & 8.0 Hz, 2H, Ar-H phenyl), 7.47 (d, J = 8.72 Hz, 1H,
Ar-H quinazoline), 7.59 (d, J = 7.88 Hz, 2H, Ar-H phenyl), 7.79 (dd, J =
2.48 & 8.72 Hz, 1H, Ar-H quinazoline), 7.99 (d, J = 2.36 Hz, 1H, Ar-H
quinazoline), 10.40 (s, 1H, D2O exchangeable, NH). 13C NMR
(DMSO‑d6, 400 MHz) δ (ppm): 13.31, 37.19, 40.23, 119.68, 120.45,
123.98, 125.76, 128.41, 129.28, 130.42, 135.23, 139.37, 145.86,
157.22, 159.78, 166.10.
–
–
3059 (C H aromatic), 2993 & 2943 (C H aliphatic), 1685 (CO amide),
1651 (CO amide), 1550 (amide II band) and 1531, 1342 (NO2); 1H NMR
(DMSO‑d6, 400 MHz) δ (ppm): 1.33 (t, J = 7.08 Hz, 3H, CH3CH2), 3.72
(s, 3H, OCH3), 4.14 (q, J = 7.08 Hz, 2H, CH3CH2), 4.24 (s, 2H, CH2CO),
6.88 (d, J = 8.96 Hz, 2H, Ar-H phenyl), 7.52 (d, J = 8.96 Hz, 2H, Ar-H
phenyl), 8.14 (dd, J = 2.28 & 8.72 Hz, 1H, Ar-H quinazoline), 8.27 (d, J
= 2.20 Hz, 1H, Ar-H quinazoline), 8.28 (d, J = 8.84 Hz, 1H, Ar-H qui-
nazoline), 10.30 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6,
400 MHz) δ (ppm): 12.14, 13.16, 41.83, 55.64, 111.41, 114.41, 118.52,
119.86, 121.30, 129.26, 130.07, 139.85, 147.37, 151.65, 155.88,
159.84, 165.60.
4.1.3.8. 2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio]-N-
(4-chlorophenyl)acetamide. (26). 2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihy-
droquinazolin-2-yl)thio]-N-(4-chlorophenyl)acetamide. (26). white
solid (yield 91.45%). m.p. 205 ◦C; IR (KBr, cmꢀ 1): 3278 (NH), 3074 &
4.1.3.4. 2-[(7-Nitro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]-N-
phenylacetamide.
(22). 2-[(7-Nitro-4-oxo-3-phenyl-3,4-dihy-
–
–
droquinazoline-2-yl)thio]-N-phenylacetamide. (22). yellow solid (yield
3043 (C H aromatic), 2981 & 2920 (C H aliphatic), 1689 (CO amide),
ꢀ 1
1
85.50%). m.p. 213 ◦C; IR (KBr, cm ): 3290 (NH), 3082 & 3043 (C
1658 (CO amide) and 1546 (amide II band); H NMR (DMSO‑d6, 400
–
H
–
aromatic), 2993 (C H aliphatic), 1693 (CO amide), 1651 (CO amide),
1554 (amide II band) and 1535, 1342 (NO2); 1H NMR (DMSO‑d6, 400
MHz) δ (ppm): 4.13 (s, 2H, CH2CO), 7.06 (t, J = 7.4 Hz, 1H, Ar-H
phenyl), 7.32 (dd, J = 7.64 & 8.12 Hz, 2H, Ar-H phenyl), 7.54 (m, 2H,
Ar-H phenyl), 7.62 (m, 5H, Ar-H phenyl), 8.17 (dd, J = 2.24 & 8.72 Hz,
1H, Ar-H quinazoline), 8.29 (d, J = 8.68 Hz, 1H, Ar-H quinazoline), 8.34
(d, J = 2.04 Hz, 1H, Ar-H quinazoline), 10.39 (s, 1H, D2O exchangeable,
NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 37.87, 119.66, 119.95,
121.30, 124.03, 124.56, 129.29, 129.41, 129.66, 130.18, 130.79,
135.82, 139.33, 147.85, 151.78, 160.22, 160.58, 166.18.
MHz) δ (ppm): 1.31 (t, J = 7.0 Hz, 3H, CH3CH2), 4.12 (q, J = 7.0 Hz, 2H,
CH3CH2), 4.22 (s, 2H, CH2CO), 7.36 (d, J = 8.84 Hz, 2H, Ar-H phenyl),
7.44 (d, J = 8.72 Hz, 1H, Ar-H quinazoline), 7.63 (d, J = 8.84 Hz, 2H, Ar-
H phenyl), 7.79 (dd, J = 2.48 & 8.72 Hz, 1H, Ar-H quinazoline), 7.99 (d,
J = 2.44 Hz, 1H, Ar-H quinazoline), 10.55 (s, 1H, D2O exchangeable,
NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 13.32, 37.18, 40.24,
120.49, 121.19, 125.80, 127.51, 128.39, 129.21, 130.44, 135.28,
138.36, 145.86, 157.20, 159.77, 166.31. Mass (m/z): 411 (M++4), 409
(M++2), 407 (M+), 379, 345, 336, 332 (100%, base peak), 331, 316,
184, 269, 234, 211, 193, 181, 177, 162, 120, 107, 95, 73 and 47.
4.1.3.5. N-(4-Chlorophenyl)-2-[(7-nitro-4-oxo-3-phenyl-3,4-dihy-
4.1.3.9. 2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihydroquinazoline-2-yl)thio]-N-
droquinazoline-2-yl)thio]acetamide.
(23). N-(4-Chlorophenyl)-2-[(7-
(4-methoxyphenyl)acetamide.
(27). 2-[(6-Chloro-3-ethyl-4-oxo-3,4-
nitro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]acetamide.
dihydroquinazoline-2-yl)thio]-N-(4-methoxyphenyl)acetamide. (27).
(23). yellow solid (yield 80.42%). m.p. 245 C; IR (KBr, cmꢀ 1): 3294
white solid (yield 88.32%). m.p. 177 C; IR (KBr, cmꢀ 1): 3248 (NH),
◦
◦
–
–
–
–
(NH), 3059 & 3039 (C H aromatic), 2981 & 2920 (C H aliphatic),
1697 (CO amide), 1658 (CO amide), 1554 (amide II band) and 1527,
1346 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm): 4.12 (s, 2H,
CH2CO), 7.37 (d, J = 8.8 Hz, 2H, Ar-H phenyl), 7.53 (m, 2H, Ar-H
phenyl), 7.62 (m, 5H, Ar-H phenyl), 8.17 (dd, J = 2.16 & 8.72 Hz, 1H,
Ar-H quinazoline), 8.28 (d, J = 8.72 Hz, 1H, Ar-H quinazoline), 8.31 (d,
J = 2.04 Hz, 1H, Ar-H quinazoline), 10.53 (s, 1H, D2O exchangeable,
NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 37.82, 119.97, 121.17,
121.26, 124.56, 127.57, 129.21, 129.42, 129.65, 130.18, 130.80,
135.81, 138.30, 147.82, 151.78, 160.21, 160.52, 166.41. Mass (m/z):
468 (M++2), 466 (M+), 451, 439, 424, 415, 339, 283 (100%, base
peak), 261, 250, 231, 223, 88, 84 and 80.
3055 (C H aromatic), 2989 & 2935 (C H aliphatic), 1689 (CO amide),
1
1651 (CO amide) and 1554 (amide II band); H NMR (DMSO‑d6, 400
MHz) δ (ppm): 1.31 (t, J = 7.12 Hz, 3H, CH3CH2), 3.72 (s, 3H, OCH3),
4.12 (q, J = 7.08 Hz, 2H, CH3CH2), 4.20 (s, 2H, CH2CO), 6.90 (d, J =
8.96 Hz, 2H, Ar-H phenyl), 7.50 (m, 3H, Ar-H), 7.80 (dd, J = 2.48 &
8.72 Hz, 1H, Ar-H quinazoline), 8.00 (d, J = 2.44 Hz, 1H, Ar-H quina-
zoline), 10.25 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400
MHz) δ (ppm): 13.31, 37.08, 40.22, 55.62, 114.38, 120.45, 121.30,
125.77, 128.44, 130.41, 132.49, 135.25, 145.88, 155.86, 157.25,
159.80, 165.58.
4.1.3.10. 2-[(6-chloro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]-
N-phenylacetamide.
(28). 2-[(6-chloro-4-oxo-3-phenyl-3,4-dihy-
4.1.3.6. N-(4-Methoxyphenyl)-2-[(7-nitro-4-oxo-3-phenyl-3,4-dihy-
droquinazoline-2-yl)thio]acetamide. (24). N-(4-Methoxyphenyl)-2-[(7-
nitro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]acetamide.
(24). yellow solid (yield 83.57%). m.p. 210 ◦C; IR (KBr, cmꢀ 1): 3325 &
droquinazoline-2-yl)thio]-N-phenylacetamide. (28). white solid (yield
ꢀ 1
90.20%). m.p. 206 ◦C; IR (KBr, cm ): 3290 (NH), 3059 & 3039 (C
–
H
aromatic), 1701 (CO amide), 1651 (CO amide) and 1543 (amide II
band); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm): 4.09 (s, 2H, CH2CO), 7.04
(t, J = 7.40 Hz, 1H, Ar-H phenyl), 7.31 (t, J = 7.90 Hz, 2H, Ar-H phenyl),
7.51 (m, 2H, Ar-H phenyl), 7.58, (m, 2H, Ar-H phenyl), 7.60 (m, 3H, Ar-
H phenyl), 7.62 (d, J = 8.70 Hz, 1H, Ar-H quinazoline), 7.86, (dd, J =
8.70 & 2.50 Hz, 1H, Ar-H quinazoline), 8.01 (d, J = 2.40 Hz, 1H, Ar-H
quinazoline), 10.34 (s, 1H, D2O exchangeable, NH). 13C NMR
(DMSO‑d6, 400 MHz) δ (ppm): 37.81, 119.61, 121.37, 123.94, 126.00,
128.59, 129.27, 129.79, 130.09, 130.49, 130.62, 135.49, 136.04,
139.38, 146.34, 158.36, 160.18, 166.08.
–
3209 (NH), 3059 & 3032 (C H aromatic), 1650 (CO amide), 1631 (CO
amide), 1554 (amide II band) and 1530, 1323 (NO2); 1H NMR
(DMSO‑d6, 400 MHz) δ (ppm): 3.72 (s, 3H, OCH3), 4.10 (s, 2H, CH2CO),
6.87 (d, J = 9.00 Hz, 2H, Ar-H phenyl), 7.53 (m, 4H, Ar-H phenyl), 7.62
(m, 3H, Ar-H phenyl), 8.18 (dd, J = 2.24 & 8.72 Hz, 1H, Ar-H quina-
zoline), 8.29 (d, J = 8.68 Hz, 1H, Ar-H quinazoline), 8.35 (d, J = 2.12
Hz, 1H, Ar-H quinazoline), 10.24 (s, 1H, D2O exchangeable, NH). 13C
NMR (DMSO‑d6, 400 MHz) δ (ppm): 37.79, 55.63, 114.39, 119.94,
121.25, 121.32, 124.57, 129.41, 129.67, 130.17, 130.78, 132.47,
135.83, 147.88, 151.80, 155.87, 160.24, 160.60, 165.62.
4.1.3.11. 2-[(6-chloro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]-
N-(4-chlorophenyl)acetamide.
(29). 2-[(6-chloro-4-oxo-3-phenyl-3,4-
4.1.3.7. 2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio]-N-
dihydroquinazoline-2-yl)thio]-N-(4-chlorophenyl)acetamide.
(29).
white solid (yield 88.65%). m.p. 230 C; IR (KBr, cmꢀ 1): 3282 (NH),
◦
phenylacetamide.
(25). 2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihy-
–
–
droquinazolin-2-yl)thio]-N-phenylacetamide. (25). white solid (yield
3059 (C H aromatic), 2985 (C H aliphatic), 1697 (CO amide), 1654
(CO amide) and 1543 (amide II band); 1H NMR (DMSO‑d6, 400 MHz) δ
(ppm): 4.08 (s, 2H, CH2CO), 7.36 (d, J = 8.80 Hz, 2H, Ar-H phenyl), 7.51
ꢀ 1
91.15%). m.p. 180 ◦C; IR (KBr, cm ): 3340 (NH), 3066 & 3035 (C
–
H
–
aromatic), 2997 & 2947 (C H aliphatic), 1681 (CO amide), 1662 (CO
14

A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
(dd, J = 7.10, 2.50 Hz, 2H, Ar-H phenyl), 7.56 (t, J = 8.70 Hz, 1H, Ar-H
phenyl), 7.61 (m, 4H, Ar-H phenyl), 7.64 (d, J = 8.70 Hz, 1H, Ar-H
quinazoline), 7.86, (dd, J = 8.70 & 2.50 Hz, 1H, Ar-H quinazoline),
8.01 (d, J = 2.40 Hz, 1H, Ar-H quinazoline), 10.49 (s, 1H, D2O
exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 37.75,
121.15, 121.36, 126.00, 127.49, 128.55, 129.19, 129.78, 130.10,
130.51, 130.64, 135.49, 136.01, 138.33, 146.31, 158.29, 160.16,
166.33. Mass (m/z): 459 (M++4), 457 (M++2), 455 (M+), 449, 432,
431, 398, 386 (100%, base peak), 383, 340, 285, 279, 266, 263, 244,
222, 195, 193, 192, 130, 125, 101 and 71.
1H, D2O exchangeable, NH), 10.51 (s, 1H, D2O exchangeable, NH). 13
C
NMR (DMSO‑d6, 400 MHz) δ (ppm): 13.16, 34.46, 40.39, 119.86,
122.02, 123.45, 127.92, 128.93, 129.04, 132.35, 132.78, 147.45,
151.64, 158.93, 159.88, 165.94, 166.79.
4.1.3.16. N’-{2-[(6-Chloro-3-ethyl-4-oxo-3,4-dihydroquinazoline-2-yl)
thio]acetyl}benzohydrazide. (34). N’-{2-[(6-Chloro-3-ethyl-4-oxo-3,4-
dihydroquinazoline-2-yl)thio]acetyl}benzohydrazide. (34). brown solid
(yield 83.80%). m.p. 175 ◦C; IR (KBr, cmꢀ 1): 3194 (NH), 3070 & 3028
–
–
(C H aromatic), 2981 & 2935 (C H aliphatic), 1690 (CO amide), 1670
(CO amide) and 1546 (amide II band); 1H NMR (DMSO‑d6, 400 MHz) δ
(ppm): 1.31 (t, J = 7.00 Hz, 3H, CH3CH2), 4.11 (q, J = 7.00 Hz, 2H,
CH3CH2), 4.16 (s, 2H, CH2CO), 7.50 (d, J = 7.50 Hz, 2H, Ar-H phenyl),
7.58 (t, J = 7.50 Hz, 1H, Ar-H phenyl), 7.71 (d, J = 8.70 Hz, 1H, Ar-H
quinazoline), 7.87 (m, 3H, Ar-H), 8.02 (d, J = 2.40 Hz, 1H, Ar-H qui-
nazoline), 10.40 (s, 1H, D2O exchangeable, NH), 10.50 (s, 1H, D2O
exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 12.29,
13.35, 34.38, 120.48, 125.64, 127.94, 128.92, 129.04, 130.47, 132.32,
132.83, 135.13, 145.95, 156.74, 159.85, 165.86, 166.69.
4.1.3.12. 2-[(6-chloro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]-
N-(4-methoxyphenyl)acetamide. (30). 2-[(6-chloro-4-oxo-3-phenyl-3,4-
dihydroquinazoline-2-yl)thio]-N-(4-methoxyphenyl)acetamide. (30).
white solid (yield 83.35%). m.p. 200 C; IR (KBr, cmꢀ 1): 3302 (NH),
◦
–
–
3055 (C H aromatic), 2978 & 2916 (C H aliphatic), 1701 (CO amide),
1
1651 (CO amide) and 1540 (amide II band); H NMR (DMSO‑d6, 400
MHz) δ (ppm): 3.72 (s, 3H, OCH3), 4.06 (s, 2H, CH2CO), 6.87 (d, J =
9.00 Hz, 2H, Ar-H phenyl), 7.50 (m, 4H, Ar-H phenyl), 7.60 (m, 4H, Ar-
H), 7.86, (dd, J = 8.70 & 2.50 Hz, 1H, Ar-H quinazoline), 8.02 (d, J =
2.40 Hz, 1H, Ar-H quinazoline), 10.20 (s, 1H, D2O exchangeable, NH).
13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 37.72, 55.63, 114.37, 121.19,
121.36, 125.99, 128.60, 129.79, 130.08, 130.48, 130.61, 132.50,
135.49, 136.04, 146.36, 155.83, 158.38, 160.19, 165.53.
4.1.3.17. N’-{2-[(6-Chloro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)
thio]acetyl}benzohydrazide. (35). N’-{2-[(6-Chloro-4-oxo-3-phenyl-3,4-
dihydroquinazoline-2-yl)thio]acetyl}benzohydrazide. (35). White solid
(yield 78.20%). m.p. 200 ◦C; IR (KBr, cmꢀ 1): 3194 (NH), 3066 & 3032
–
–
(C H aromatic), 2978 (C H aliphatic), 1693 (CO amide) and 1546
1
4.1.3.13. 2-[(3-Ethyl-6-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]-N-
(amide II band); H NMR (DMSO‑d6, 400 MHz) δ (ppm): 4.03 (s, 2H,
phenylacetamide. (31). 2-[(3-Ethyl-6-nitro-4-oxo-3,4-dihydroquinazo-
CH2CO), 7.47 (m, 4H, Ar-H phenyl), 7.60 (m, 4H, Ar-H phenyl), 7.78 (d,
J = 8.70 Hz, 1H, Ar-H quinazoline), 7.85 (d, J = 7.40 Hz, 2H, Ar-H
phenyl), 7.91 (dd, J = 8.70 & 2.50 Hz, 1H, Ar-H quinazoline), 8.03 (d,
J = 2.40 Hz, 1H, Ar-H quinazoline), 10.40 (s, 1H, D2O exchangeable,
NH), 10.50 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400
MHz) δ (ppm): 34.92, 121.31, 125.86, 127.92, 128.93, 129.13, 129.79,
130.11, 130.57, 130.66, 132.32, 132.81, 135.40, 135.95, 146.39,
157.84, 160.25, 165.84, 166.68.
line-2-yl)thio]-N-phenylacetamide. (31). yellow solid (yield 88.15%).
ꢀ 1
m.p. 205 ◦C; IR (KBr, cm ): 3356 (NH), 3062 (C H aromatic), 2978 &
–
1
–
2939 (C H aliphatic), 1678 (CO amide) and 1539 & 1338 (NO2); H
NMR (DMSO‑d6, 400 MHz) δ (ppm): 1.34 (t, J = 7.00 Hz, 3H, CH3CH2),
4.16 (q, J = 6.90 Hz, 2H, CH3CH2), 4.30 (s, 2H, CH2CO), 7.07 (t, J =
7.30 Hz, 1H, Ar-H phenyl), 7.33 (dd, J = 7.80 & 7.30 Hz, 2H, Ar-H
phenyl), 7.61 (m, 3H, Ar-H), 8.51 (dd, J = 9.00 & 2.70 Hz, 1H, Ar-H
quinazoline), 8.76 (d, J = 2.60 Hz, 1H, Ar-H quinazoline), 10.45 (s,
1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm):
13.09, 37.42, 119.22, 119.71, 123.27, 124.02, 127.83, 129.27, 129.30,
139.35, 144.55, 150.92, 159.94, 161.36, 165.86.
4.1.3.18. N’-{2-[(3-Ethyl-6-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]
acetyl}benzohydrazide.
(36). N’-{2-[(3-Ethyl-6-nitro-4-oxo-3,4-dihy-
droquinazoline-2-yl)thio]acetyl}benzohydrazide. (36). brown solid
ꢀ 1
◦
–
H
(yield 83.50%). m.p. 165 C; IR (KBr, cm ): 3201 (NH), 3035 (C
–
4.1.3.14. 2-[(6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio]-
aromatic), 2989 & 2947 (C H aliphatic), 1693 (CO amide), 1516
(amide II band) and 1546, 1342 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ
(ppm): 1.34 (t, J = 7.00 Hz, 3H, CH3CH2), 4.16 (q, J = 6.80 Hz, 2H,
CH3CH2), 4.23 (s, 2H, CH2CO), 7.50 (dd, J = 7.50 & 7.20 Hz, 2H, Ar-H
phenyl), 7.58 (t, J = 7.20 Hz, 1H, Ar-H phenyl), 7.87 (m, 3H, Ar-H), 8.56
(dd, J = 9.00 & 2.60 Hz, 1H, Ar-H quinazoline), 8.78 (d, J = 2.60 Hz, 1H,
Ar-H quinazoline), 10.44 (s, 1H, D2O exchangeable, NH), 10.52 (s, 1H,
D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ (ppm): 13.03,
34.51, 40.48, 119.05, 123.07, 127.89, 128.46, 128.99, 129.02, 132.47,
132.61, 144.56, 150.92, 160.06, 160.79, 166.20, 166.73.
N-phenylacetamide.
(32). 2-[(6-nitro-4-oxo-3-phenyl-3,4-dihy-
droquinazoline-2-yl)thio]-N-phenylacetamide. (32). white solid (yield
ꢀ 1
88.60%). m.p. 230 ◦C; IR (KBr, cm ): 3352 (NH), 3093 & 3059 (C
–
H
aromatic), 1693 (CO amide), 1681 (CO amide) and 1539 & 1338 (NO2);
1H NMR (DMSO‑d6, 400 MHz) δ (ppm): 4.15 (s, 2H, CH2CO), 7.07 (t, J =
7.40 Hz, 1H, Ar-H phenyl), 7.52 (dd, J = 7.90 & 7.40 Hz, 2H, Ar-H
phenyl), 7.61 (m, 6H, Ar-H), 7.72 (d, J = 9.00 Hz, 1H, Ar-H phenyl),
7.87 (t, J = 8.80 Hz, 1H, Ar-H quinazoline), 8.59 (dd, J = 9.00 & 2.70
Hz, 1H, Ar-H quinazoline), 8.77 (d, J = 2.60 Hz, 1H, Ar-H quinazoline),
10.38 (s, 1H, D2O exchangeable, NH). 13C NMR (DMSO‑d6, 400 MHz) δ
(ppm): 39.41, 119.75, 120.12, 123.15, 124.14, 128.54, 129.09, 129.32,
129.58, 130.27, 130.62, 135.63, 139.04, 144.62, 145.34, 152.14,
161.06, 163.18.
4.1.3.19. N’-{2-[(6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)
thio]acetyl}benzohydrazide. (37). N’-{2-[(6-nitro-4-oxo-3-phenyl-3,4-
dihydroquinazoline-2-yl)thio]acetyl}benzohydrazide. (37). brown solid
(yield 84.00%). m.p. 190 ◦C; IR (KBr, cmꢀ 1): 3240 (NH), 3062 & 3035
4.1.3.15. N’-{2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihydroquinazoline-2-yl)thio]
(C H aromatic), 2889 (C H aliphatic), 1697 (CO amide), 1519 (amide
II band) and 1546, 1338 (NO2); 1H NMR (DMSO‑d6, 400 MHz) δ (ppm):
4.09 (s, 2H, CH2CO), 7.50 (m, 5H, Ar-H phenyl), 7.60 (m, 3H, Ar-H
phenyl), 7.86 (dd, J = 7.50 Hz, 2H, Ar-H phenyl), 7.93 (d, J = 9.00
Hz, 1H, Ar-H quinazoline), 8.63 (dd, J = 9.00 & 2.60 Hz, 1H, Ar-H
quinazoline), 8.79 (d, J = 2.50 Hz, 1H, Ar-H quinazoline), 10.38 (s,
–
–
acetyl}benzohydrazide.
(33). N’-{2-[(3-Ethyl-7-nitro-4-oxo-3,4-dihy-
droquinazoline-2-yl)thio]acetyl}benzohydrazide. (33). white solid
ꢀ 1
◦
–
H
(yield 87.30%). m.p. 235 C; IR (KBr, cm ): 3182 (NH), 3024 (C
–
aromatic), 2993 & 2943 (C H aliphatic), 1681 (CO amide), 1554
(amide II band) and 1535 & 1342 (NO2); 1H NMR (DMSO‑d6, 400 MHz)
δ (ppm): 1.33 (t, J = 6.68 Hz, 3H, CH3CH2), 4.14 (q, J = 6.84 Hz, 2H,
CH3CH2), 4.21 (s, 2H, CH2CO), 7.49 (dd, J = 7.32 & 7.04 Hz, 2H, Ar-H
phenyl), 7.57 (t, J = 6.88 Hz, 1H, Ar-H phenyl), 7.87 (d, J = 7.44 Hz, 2H,
Ar-H phenyl), 8.16 (d, J = 8.56 Hz, 1H, Ar-H quinazoline), 8.29 (d, J =
8.76 Hz, 1H, Ar-H quinazoline), 8.53 (s, 1H, Ar-H quinazoline), 10.48 (s,
1H, D2O exchangeable, NH), 10.48 (s, 1H, D2O exchangeable, NH). 13
C
NMR (DMSO‑d6, 400 MHz) δ (ppm): 35.14, 120.17, 123.18, 127.91,
128.54, 128.93, 129.39, 129.66, 130.24, 130.88, 132.34, 132.79,
135.66, 144.67, 151.51, 160.36, 162.05, 165.87, 166.51.
15

A.E. Abdallah et al.
Bioorganic Chemistry 109 (2021) 104695
4.2. Biological testing
Operating Environment (MOE 2010) software. The three-dimensional
structures and conformations of the enzymes were acquired from the
Protein Data Bank (PDB) website using VEGFR-2 complexed with pyr-
rolopyrimidine inhibitor (PDB code 3VHE). The ligand molecules were
constructed in MOE using the builder module and collected in a data-
base. The database was prepared by using the option “Protonate 3D” to
add hydrogens, calculate partial charges and minimize energy (using
Force Field MMFF94x). In addition, the protein structure was prepared
by deleting the repeated chains, water molecules and any surfactants,
hydrogens were also added to the atom of the receptor and the partial
charges were calculated. MOE was used to calculate the best score be-
tween the ligands and the enzymes’ binding sites. Scoring was deter-
mined using alpha HB as a scoring function. The resulted database
contained the score between the ligands’ conformers and the enzyme
binding sites in kcal/mol. To confirm the credibility of docking results,
self-docking was used to validate the adopted docking protocol in which
co-crystallized ligand were drawn in MOE, prepared as the targeted
compounds (hydrogens addition, partial charges calculation and energy
minimization), and then docked into the active site of the protein using
the same protocol. The top ranked pose exhibited Root mean square
deviation (RMSD) value of less than 0.42 Å from the experimental
crystal structure. This result indicated that the Molecular Operating
Environment (MOE) docking could reliably predict docking pose for the
studied compounds to an enzyme. It was reported that values less than
1.5 or 2 Å were a sign of a successful and reliable docking protocol.
4.2.1. In vitro VEGFR-2 tyrosine kinase inhibitory assay
IC50 of all compounds against VEGFR-2 kinase were determined with
enzyme linked immunosorbent assay (ELISA). Human VEGFR-2 ELISA
kit was used. VEGFR-2 specific antibody was seeded on a 96 well
microplate and 100 μL of solution of the standard or the tested com-
pound was added. All were then incubated for 2.5 h. at room tempera-
ture then washed. 100 L of prepared biotin antibody was then added
and incubated at room temperature for 1 h then washed. 100 L of
streptavidin solution was then added and incubated at room tempera-
ture for 45 min. then washed. 100 L of tetramethybenzidine (TMB)
Substrate solution was added then incubated at room temperature for
μ
μ
μ
30 min. Then 50 μL of the stop solution was added and immediately read
at 450 nm. The standard curve was hence drawn, concentration was on
the X-axis and the absorbance was on the Y-axis.
4.2.2. In vitro antiproliferative assay
Anti-proliferative activity screening was conducted against 3 human
cancer cell lines namely; HepG2, PC3 and MCF-7. The cell lines were
obtained from American Type Culture Collection (ATCC) via National
Cancer Institute (Cairo, Egypt). All chemicals used in the assay were of
high analytical grade and obtained from either Sigma-Alderich or Bio-
rad. The antitumor activity was measured quantitatively and the assay
was carried out according to MTT protocol [41]. This colorimetric assay
is based on the conversion of the yellow tetrazolium bromide (MTT) to a
purple formazan derivative by mitochondrial succinate dehydrogenase
in viable cells. The cells were cultured in RPMI-1640 medium with 10%
fetal bovine serum. Antibiotics added were 100 units/mL penicillin and
100 µg/mL streptomycin at 37C in a 5% CO2 incubator. The cells were
seeded in a 96-well plate at a density of 1.0x104 cells/well. at 37C for 48
h under 5% CO2. After incubation the cells were treated with different
concentration of compounds and incubated for 24 h. After 24 h of drug
treatment, 20 µL of MTT solution at 5 mg/ml was added and incubated
for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 µL is added into
each well to dissolve the purple formazan formed. The colorimetric
assay is measured and recorded at absorbance of 570 nm using a plate
reader (EXL 800, USA). The relative cell viability in percentage was
calculated as (A570 of treated samples/A570 of untreated sample) X
100.
4.3.2. In silico ADMET analysis
ADMET descriptors (absorption, distribution, metabolism, excretion
and toxicity) of the synthesized compounds were determined using
Discovery studio 4.0. At first, the CHARMM force field was applied then
the compounds were prepared and minimized according to the prepa-
ration of small molecule protocol. Then ADMET descriptors protocol
was applied to carry out these studies [44].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary material
4.2.3. Selective cytotoxicity assay
In vitro antiproliferative assay against the normal human cell line WI-
38 was carried out according to MTT protocol. The cell line was obtained
from ATCC via National Cancer Institute (Cairo, Egypt).
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104695.
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