Novel 4-(piperazin-1-yl)quinolin-2(1H)-one bearing thiazoles with antiproliferative activity through VEGFR-2-TK inhibition

Article abstract of DOI:10.1016/j.bmc.2021.116168

A new series of 2-(4-(2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives were synthesized and evaluated for anticancer activity. All target compounds showed anticancer activity higher than that of their 2-oxo-4-piperazinyl-1,2-dihydroquinolin-2(1H)-one precursors. Multidose testing of target compounds was performed against breast cancer T-47D cell line. Five compounds showed higher cytotoxic activity than Staurosporine. The dihalogenated derivative showed the best cytotoxic activity with IC₅₀ 2.73 ± 0.16 μM. In addition, the VEGFR-2 inhibitory activity of all synthetic compounds was evaluated. Two compounds of 6-fluoro-4-(piperazin-1-yl)quinolin-2(1H)-ones showed inhibitory activity comparable to sorafenib with IC₅₀ 46.83 ± 2.4, 51.09 ± 2.6 and 51.41 ± 2.3 nM, respectively. The cell cycle analysis of two compounds namely, 2-(4-(6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide and N-(4-(4-chlorophenyl)thiazol-2-yl)-2-(4-(2-oxo-1-phenyl-1,2-dihydroquinolin-4-yl)piperazin-1-yl)acetamide revealed that the arrest of cell cycle occurred at S phase. In apoptosis assay, the same two compounds were able to induce significant levels of early and late apoptosis. In a similar manner to Sorafenib, docking of target compounds with VEGFR-2 protein 4ASD showed HB with Cys919 in hinge region of enzyme and HB with both Glu885 and Asp1046 in gate area. Using SwissADME, all target compounds were predicted to be highly absorbed from gastrointestinal tract with no BBB permeability. It is clear that the two compounds are promising antiproliferative candidates that require further optimization.

Full text of DOI:10.1016/j.bmc.2021.116168

Bioorg. Med. Chem. 40 (2021) 116168
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 4-(piperazin-1-yl)quinolin-2(1H)-one bearing thiazoles with
antiproliferative activity through VEGFR-2-TK inhibition
,
Abdelfattah Hassana, Mohamed Badrb, Heba A. Hassanc *, Dalia Abdelhamidc, Gamal El-
,d,*
Din A. Abuo-Rahmac
a Department of Medicinal Chemistry, Faculty of Pharmacy, South Valley University, Qena, Egypt
b Department of Biochemistry, Faculty of Pharmacy, Menoufia University, Menoufia, Egypt
c Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
d Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Deraya University, New Minia, Minia, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of 2-(4-(2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide de-
rivatives were synthesized and evaluated for anticancer activity. All target compounds showed anticancer ac-
tivity higher than that of their 2-oxo-4-piperazinyl-1,2-dihydroquinolin-2(1H)-one precursors. Multidose testing
of target compounds was performed against breast cancer T-47D cell line. Five compounds showed higher
cytotoxic activity than Staurosporine. The dihalogenated derivative showed the best cytotoxic activity with IC50
2.73 ± 0.16 µM. In addition, the VEGFR-2 inhibitory activity of all synthetic compounds was evaluated. Two
compounds of 6-fluoro-4-(piperazin-1-yl)quinolin-2(1H)-ones showed inhibitory activity comparable to sor-
afenib with IC50 46.83 ± 2.4, 51.09 ± 2.6 and 51.41 ± 2.3 nM, respectively. The cell cycle analysis of two
compounds namely, 2-(4-(6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)acet-
amide and N-(4-(4-chlorophenyl)thiazol-2-yl)-2-(4-(2-oxo-1-phenyl-1,2-dihydroquinolin-4-yl)piperazin-1-yl)
acetamide revealed that the arrest of cell cycle occurred at S phase. In apoptosis assay, the same two compounds
were able to induce significant levels of early and late apoptosis. In a similar manner to Sorafenib, docking of
target compounds with VEGFR-2 protein 4ASD showed HB with Cys919 in hinge region of enzyme and HB with
both Glu885 and Asp1046 in gate area. Using SwissADME, all target compounds were predicted to be highly
absorbed from gastrointestinal tract with no BBB permeability. It is clear that the two compounds are promising
antiproliferative candidates that require further optimization.
4-Piperazinylquinolin-2(1H)-one
4-Phenylthiazole
VEGFR-2 inhibitors
Breast cancer
Angiogenesis
1. Introduction
different vascular endothelial growth factor receptors including VEGFR-
1, VEGFR-2, and VEGFR-3. VEGFR-2 is the main tyrosine kinase receptor
One sixth of humankind deaths is due to cancer; cancer rates as the
second cause of death worldwide.1 Breast cancer is by far the most
prevalent cancer with about 7.8 million patients worldwide. In females
above 40 years, breast cancer has the highest mortality rate that is even
more than that of lung cancer.2 One of the markers of breast malignancy
is angiogenesis.3 Angiogenesis is a stratagem by which tumor expands its
own vasculature to guarantee sufficient import of oxygen and nutrients
and grow so quickly comparing to normal tissues. This process is
fundamental for both tumor growth and metastasis.4
through which Vascular endothelial growth factor VEGF exert its pro-
angiogenesis signaling.5 In addition to angiogenesis, activation of
VEGFR-2 triggers a cluster of other cellular events including protease
production, vascular permeability, platelet activating factor production,
cytoskeleton remodeling, cell survival, proliferation, and migration.6
The urgent demand of more selective antitumor agents provokes me-
dicinal chemists all over the world to design, develop and evaluate a
great number of TK inhibitor candidates. consequently, the majority of
FDA approved anticancer medications in last two decades are TK in-
hibitors with VEGFR-2 inhibitors group as a one of the most effective
weapons among all TK inhibitors arsenal.
Recently, cancer researches focused on drugs that inhibit signaling
pathways related to kinases as it is more specific than traditional
chemotherapy with fewer adverse effects. Amongst these kinases, the 3
In spite of a structural diversity of VEGFR-2 inhibitors, they all share
* Corresponding authors at: Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt (G.E.-D.A. Abuo-Rahma).
E-mail addresses: heba.hasan@mu.edu.eg (H.A. Hassan), gamal.aborahama@mu.edu.eg (G.E.A. Abuo-Rahma).
https://doi.org/10.1016/j.bmc.2021.116168
Received 31 January 2021; Received in revised form 2 April 2021; Accepted 19 April 2021
Available online 22 April 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
four conclusive pharmacophoric features namely HB heterocycle,
spacer, duo HBA/HBD domain and distal hydrophobic moiety (Fig. 3).7,8
A HB heterocycle is mainly an aromatic or non-aromatic, five or six-
membered mono or fused aza-heteroring like pyrazole, indole, thia-
zole, pyridine, quinoline, quinazoline, phthalazine, benzopyrazole,
pyrazolo[3,4-d]pyrimidine or benzoxazole.9–13 Albeit it sometimes can
be seven-membered heterocycle like benzodiazepine.14 Also, HB het-
erocycle sometimes can be oxa-heteroring as coumarin.3 HB heterocycle
occupies the hinge region of enzyme and interacts with hydrophobic
ATP-binding domain and is also capable of forming HB with Cys919 via
ring nitrogen atom. In addition, the presence of carboxamide or oxo
group on α-carbon to nitrogen as in Sorafenib and Sunitinib, respectively
adds an extra HB with Cys919 and enhance enzyme inhibition (Fig. 2).15
A Spacer is a 5-atom length moiety links between HB heterocycle and
duo HBD/HBA domain. This spacer is connected to HB heterocycle by
nitrogen or oxygen atom.10,13 Spacer is classically involved in phenyl or
heteroring but it can also be incorporated in aliphatic ring or
chain.8,12,16,17 The duo HBA/HBD domain can be urea, thiourea, car-
boxamide, dicarboxamide, sulfonamide, carbohydrazide, semi-
carbazone, thiosemicarbazone, N-sulfonylurea or even involved in
hetero-ring like thiopyrimidinone or pyrazole ring.8,10,12,17–20 It bonds
to both Glu885 and Asp1046 residues in the gate area of enzyme (Fig. 2).
Distal hydrophobic moiety occupies an allosteric site of enzyme and it
may be variable aliphatic, alicyclic, aromatic, and non-aromatic benzo
and heterocyclic groups with diverse electronic, lipophilic, and steric
properties.7,10,12,17,19
Fig. 2. 2D interactions of sorafenib with VEGFR-2 (PDB: 4ASD).22
All clinically used VEGFR-2 inhibitors satisfy this well-established
four pharmacophoric requirements for good enzyme inhibition. The
dual VEGFR/PDGFR inhibitor, Sunitinib and Nintedanib both contain
Fig. 3. Pharmacophoric requirements for VEGFR-2 inhibitory activity and
design of target compounds 8a-h.
indolin-2-one as a HB heterocycle and carboxamide as duo HBA/HBD
(Fig. 1). Moreover, both drugs have tertiary amine as distal hydrophobic
moiety, N,N-diethylaminoethyl in Sunitinib and 4-methylpiperazinyl-
methyl in Nintedanib that impacts positively in their pharmacoki-
netics.21 Both the dual VEGFR/PDGFR inhibitor, Sorafenib and its
fluoro-derivative, the multi-TK inhibitor, Regorafenib have a prototype
duo HBA/HBD urea group.22 Both the multi-TK inhibitor, Cabozantinib
and Lenvatinib have quinoline as HB heterocycle. The unique 2-cyclo-
propylmalondiamide drug, Cabozantinib exerts high VEGFR-2 inhibi-
tory activity at picogram range. Lenvatinib has
a characteristic
cyclopropyl ring as distal hydrophobic moiety that interacts with an
allosteric site of enzyme.23
Fig. 1. Some clinically used VEGFR-2 inhibitors.
2

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
2. Rationale and molecular design
sketched in (Scheme 1). The first step is the synthesis of the reported 4-
hydroxyquinolin-2(1H)-one derivatives 1a,b by heating a solution of
substituted aniline in diethyl malonate with 5 times by weight PPA at
160 ◦C for 2 hr.24 Classically, hydroxyl group of 4-hydroxyquinolin-2
(1H)-one was replaced directly with different aliphatic and aromatic
amines using neat condition in presence of hydrochloric acid or by
heating for long time in a high boiling point solvent like diphenyl ether
and DMSO.25–27 Unfortunately, this direct method did not work with
piperazine, so we had to replace the hydroxyl group with a good leaving
group -Cl to facilitate the nucleophilic substitution with piperazine. The
compound 3b was synthesized by heating compound 1b with phos-
phorus oxychloride at reflux for 2 h. In contrast to compound 1b, the
reaction of compound 1a with phosphorus oxychloride produced 2,4-
dichloro-6-fluoroquinoline compound 2.28 So, we had to hydrolyze the
2-chloro group back to hydroxyl group, this was carried out by heating
compound 2 in acetic acid at reflux overnight.24 4-Piperazinylquinolin-2
(1H)-ones 4a,b were synthesized by heating 3a,b respectively with 3
equivalents of piperazine in DMF at 80 ◦C.29,30 1H NMR of compounds
4a,b showed two characteristic signals at δH 3.2, 2.8 representing 2 sets
of piperazinyl eight protons in addition to presence of two sets of signals
of piperazinyl carbons at δC 46 and 53 in 13C NMR.
As aforementioned, there are four fundamental pharmacophoric re-
quirements for good VEGFR-2 inhibitory activity namely HB hetero-
cycle, spacer, duo HBA/HBD domain and distal hydrophobic moiety
Fig. 3.7,8 Through ring fusion strategy, the picolinamide ring of Sor-
afenib was replaced by quinoline-2(1H)-one in target compounds which
can enhance hydrophobic interaction with hinge region of enzyme.13
Furthermore, 2-oxo group in quinoline-2(1H)-one moiety of target
compounds can augment HB with Cys919 like 2-carboxamido group of
Sorafenib.13,15 The length of 5-atom spacer was conserved by replacing
oxyphenyl moiety of Sorafenib with more polar piperazinylmethyl
moiety that affords additional benefit on pharmacokinetics of target
compounds.16 Regarding to duo HBA/HBD, a bioisosteric modification
of urea of Sorafenib to N-(thiazol-2-yl)carboxamide moiety enhanced
HB with Glu885 and Asp1046 residues in the gate area of enzyme.12,16,23
Several 4-phenylthiazol-2-yl substituents were incorporated as distal
hydrophobic moiety to occupy an allosteric site instead of 3-trifluoro-
methyl-4-chlorophenyl group of Sorafenib in order to assess the effect
of different electron donating and withdrawing substituents on hydro-
phobic interactions with enzyme.7,10
All intermediates 4a,b and the target final compounds 8a-h under-
gone anticancer screening against sixty NCI cell line. In addition, cyto-
toxicity of target compounds against T-47D breast cancer cell line were
evaluated and IC50 was calculated and the VEGFR-2 inhibitory activity
was assessed for all compounds. For more investigation, the most active
compounds in both cytotoxicity and enzyme inhibition 8a and 8f were
selected for cell cycle analysis and apoptosis assay.
Compounds 7a-d were synthesized according to reported methods
(Scheme 2). Reaction of (us)substituted acetophenones with equivalent
amount of N-bromosuccinimide (NBS) in presence of p-toluene sulfonic
acid (p-TSA) under neat condition afforded phenacyl bromides 5a-d.31
4-phenylthiazole-2-amines 6a-d were synthesized by refluxing phenacyl
bromides 5a-d with thiourea in ethanol.32 acylation of compounds 6a-
d with bromoacetyl bromide in presence of K2CO3 yielded compounds
7a-d.33 Reaction of compounds 7-d with key intermediates 4a,b in DMF
in presence of K2CO3 afforded target final compounds 8a-h.33 1H NMR
of compound 8b, for example, showed two characteristic NH signals, the
first at δH 12.13 representing amide CONH and the second at δH 11.47
representing quinolinic NH. Moreover, singlet peak at δ 3.45 repre-
senting methylene group that linked amide and piperazinHe.
3. Result and discussion
3.1. Chemistry
The synthesis of key intermediates piperazinyl derivatives 4a,b was
3.2. Biology
3.2.1. NCI evaluation of in vitro antiproliferative activity
Anticancer activity of key intermediate compounds 4a,b and the
target compounds 8a-h were evaluated by National Cancer Institute
(NCI, Bethesda, ML, USA, http://www.dtp.nci.nih.gov.) at a single
concentration of 10 μM against a panel of sixty cancer cell lines of nine
different human tissues according to NCI protocol. The screening results
represent in the precent of growth inhibition of treated cells compared to
untreated ones, Table 1. Overall, the quinoline-2(1H)-one/thiazole hy-
brids compounds 8a-h showed higher cytotoxic activity than their 4-
piperazinylquinoline-2(1H)-one precursors 4a,b that reflects the of
positive impact of 4-phenylthiazole moiety on anticancer activity of
target compounds 8a-h. Compounds 4a,b showed weak cell growth
inhibition (GI) activity against most of the tested cell line. Most target
compounds showed good cytotoxic activity against leukemia cell lines.
Compound 8e exerted good activity against K-562 and MOLT-4 leuke-
mia cell line with GI 77.58% and 82.24%, respectively. For non-small
cell lung cancer, target compounds showed inhibition activity against
EKVX cell line and compound 8f is the best with GI 57.30%. Compound
8e exerted inhibition activity against colon HCT-15 cell line with GI
74.80%. In addition, compound 8e showed good activity against colon
HCT-15 cell line with GI 74.80%. Compound 8b exerted inhibition ac-
tivity against CNS SNB-75 cell line with GI 46.25%. Compound 8f
showed good inhibition against melanoma SK-MEL-5 cell line with GI
94.90%, and against ovarian cancer OVCAR-4 cell line with 80.34%.
Furthermore, 8f showed inhibition against renal cancer UO-31 cell line
with 70.52% and good inhibition against MDA-MB-468 cell line with GI
90.39%. Compound 8e exerted inhibition against prostate cancer PC-3
cell line with GI 67.53%. Generally, it is obvious that the screening re-
sults of target compounds 8a-h demonstrated specific activity against
Scheme 1. Synthesis of the intermediates 4a,b. Reagent and reaction condi-
tions: (a) PPA, 160 ◦C; (b) POCl3, reflux; (c) AcOH, reflux; (d) piperazine,
DMF, 80 ◦C.
3

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Compound
5a, 6a, 7a
5b, 6b, 7b
5c, 6c, 7c
5d, 6d, 7d
R3
H
Cl
Me
MeO
Compound
R1
H
H
H
H
R2
F
F
F
F
R3
H
Cl
Me
MeO
Compound
R1
Ph
Ph
Ph
Ph
R2
H
H
H
H
R3
H
Cl
Me
MeO
8a
8b
8c
8d
8e
8f
8g
8h
Scheme 2. Synthesis of target compounds 8a,h; Reagent and reaction conditions: (a) NBS, p-TSA, 60–80 ◦C; (b) thiourea, ethanol, reflux; (c) bromoacetyl bromide,
K2CO3, 0–5 ◦C; (d) 3a,b, K2CO3, DMF.
breast cancer cell lines.
3.2.4. VEGFR-2 inhibitory assay
To go deeper in molecular mechanism of the synthesized com-
pounds, their inhibitory activity on VEGFR-2 TK was investigated
(Table 4). All compounds showed their inhibitory activity at nanomole
range. Overall, intermediate compounds 4a,b showed inhibitory activ-
ity less than most of the final target hybrid compounds that confirms the
importance of 4-phenylthiazole moiety as a distal hydrophobic moiety
which occupies the allosteric hydrophobic pocket of kinase. Compounds
8a and 8d showed better inhibitory activity compared with sorafenib
with IC50 46.83 ± 2.4, 51.09 ± 2.6 and 51.41 ± 2.3 nM, respectively.
Also, compound 8f showed comparable activity to sorafenib with IC50 of
62.7 nM. On the other hand, 4a and 8c showed the least activity
amongst all synthesized series with 281.5 and 295.8 nM, respectively.
The results of both cytotoxicity against T-47D breast cancer cell lines
and inhibitory activity against VEGFR-2 TK demonstrates that com-
pounds 8a and 8f may be promising targeted anticancer agents.
Consequently, further studies like cell cycle analysis and apoptosis assay
for both compounds are necessary to be carried out.
3.2.2. Evaluation of in vitro cytotoxicity IC50 against breast cancer cell line
NCI in vitro anticancer screening for compounds 4a,b and 8a-h
demonstrates good antiproliferative activity against breast cancer cell
line especially T-47D cell line. Independently, the cytotoxic activity of
all synthesized compounds was evaluated against T-47D cell line using
MTT assay and the results are presented in (Table 2). All the target
compounds showed antiproliferative activity at micromole range.
Generally, quinoline-2(1H)-one/thiazole hybrid compounds 8a-h
showed prominent higher cytotoxic activity than their corresponding 4-
piperazinylquinoline-2(1H)-one precursors 4a,b that is in consistent
with the rationale of design and prevents the importance of introducing
4-phenylthiazole moiety in target compounds 8a-h. Intermediate com-
pounds 4a and 4b showed the least cytotoxic activity with IC50 37.70
and 92.1 µM, respectively. Five out of eight target compounds showed
higher cytotoxic activity than Staurosporine namely 8a, 8b, 8c, 8e and
8h. It is noteworthy that amongst all tested compounds, a dihalogenated
derivative 8b with (R2 = F and R3 = Cl) have the best cytotoxic activity
with IC50 2.73 µM. In addition, both compounds with unsubstituted 4-
phenylthiazole moiety 8a and 8e showed higher cytotoxic activity
than Staurosporine with IC50 7.20 and 10.30 µM, respectively. Also,
compound 8c with R3 = Me and 8h with R3 = OMe showed higher
cytotoxic activity than Staurosporine with IC50 8.41 and 8.11 µM,
respectively. Finally, Other three target compounds 8d, 8f and 8g
showed comparable cytotoxic activity to Staurosporine with IC50 of
16.90, 30.20 and 16.50 µM, respectively.
3.2.5. Cell cycle analysis and apoptosis assay
3.2.5.1. Cell cycle analysis. The cell cycle includes four phases G1, S, G2
and M. Firstly in G1 phase cell enlarges and DNA becomes ready for
duplication. In the second stage (S phase), the DNA starts to replicate
and chromatid duplicates; The third stage (G2 Phase), the new DNA is
repaired with continuous growth and finally in the fourth stage (M
phase) nuclear division occurs. The effect of both compounds 8a and 8f
on cell cycle development and induction of apoptosis in the T-47D cell
line was carried out. T-47D cell line was incubated with IC50 concen-
tration of compounds 8a and 8f for 24 h. Consequently, the cell line was
stained with PI/Annexin V and analyzed by flow cytometry using BD
FASCC alibur.28 The found results were presented in Table 5 and Fig. 4.
The results indicated that percentage of % Pre-G1 apoptosis induced by
8a and 8f on T47D were 43.05% and 25.12%, respectively. A high
percent of cell accumulation occurred in S phase in T-47D cell line
treated with compound 8a and 8f after 24 h incubation, that indicated
the arrest of cell cycle at S phase.
3.2.3. Evaluation of in vitro cytotoxicity IC50 against normal cell line
To assess the selectivity of target compounds 8a-h against cancer
cells over normal ones, the cytotoxicity of two selected compounds 8a
and 8f was evaluated against non-tumorigenic epithelial cell line, MCF
10A using Camptothecin as a reference Table 3. Two compounds 8a and
8f showed less toxicity against normal cell line MCF 10A comparing to
Camptothecin. Compound 8a was showed cytotoxic activity against
breast cancer cell line T-47D 5.5 times more than cytotoxic activity
against normal cell line MCF 10A while Compound 8f showed cytotoxic
activity against breast cancer cell line T-47D two-fold more than cyto-
toxic activity against normal cell line MCF 10A.
3.2.5.2. Apoptosis assay. After treatment of T-47D cell line with com-
pounds 8a and 8f, cell cycle analysis showed presence of pre-G1 peak
which indicates apoptosis. To ensure the ability of both compounds 8a
4

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Table 1
Cytotoxic activity represented in % GI for compounds 4a,b and 8a-h at a single concentration of 10 μM against a panel of sixty cancer cell lines.
Cell Line
4a
4b
8a
8b
8c
8d
8e
8f
8g
8h
Leukemia
CCRF-CEM
HL-60(TB)
K-562
–
–
–
–
–
–
–
–
–
–
–
33.30
38.30
44.81
52.80
38.30
29.11
47.04
40.59
42.38
53.25
51.11
47.49
29.81
23.50
30.08
46.70
40.93
34.71
60.38
62.85
77.58
82.24
53.84
48.67
49.34
73.75
75.77
71.01
63.29
43.18
14.79
19.88
49.33
32.18
NA
24.90
30.20
56.41
53.11
NA
–
–
MOLT-4
RPMI-8226
SR
13.31
–
–
21.19
–
11.21
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
–
–
–
44.42
43.34
–
29.71
12.55
28.79
21.30
18.08
25.94
43.68
42.13
–
39.74
11.70
21.63
18.88
21.36
22.32
29.51
24.94
–
43.81
54.54
33.55
NA
47.42
57.30
34.21
NA
12.01
26.51
–
13.81
17.32
29.72
–
11.09
22.85
19.67
35.21
12.37
–
18.69
35.73
12.63
15.90
30.42
56.17
–
14.62
HOP-62
–
–
–
–
–
–
–
–
–
HOP-92
16.98
10.93
25.44
–
NCI-H226
–
–
–
–
–
–
–
–
–
–
51.84
39.20
10.62
67.83
33.50
52.59
50.24
10.90
45.18
43.70
NCI-H23
16.62
12.64
12.51
21.61
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14.47
30.90
47.97
34.94
25.57
30.06
41.16
10.67
34.09
45.23
37.14
21.49
13.31
36.96
–
39.94
18.24
46.60
74.80
27.04
36.97
31.38
42.68
–
64.21
60.28
36.16
35.31
25.68
–
–
–
15.75
15.87
26.59
22.10
–
–
–
15.96
20.19
28.86
–
–
–
–
25.99
17.97
HT29
–
–
–
–
–
–
KM12
SW-620
34.58
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14.49
20.31
32.66
–
32.82
44.37
–
20.78
33.27
22.87
14.78
28.39
40.83
45.54
37.44
23.90
36.04
32.53
36.20
13.44
12.44
–
20.36
30.53
30.83
46.25
37.33
17.53
30.52
15.34
23.88
30.29
14.45
22.86
13.31
–
–
–
–
–
21.81
24.23
–
–
–
–
Melanoma
LOX IMVI
MALME-3M
M14
–
–
–
21.79
43.30
30.62
NA
23.89
43.48
27.46
NA
20.79
33.56
19.24
NA
50.55
30.27
26.60
NA
41.62
44.89
50.22
NA
12.15
15.03
–
–
–
–
–
–
–
–
–
16.11
NA
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
NA
–
NA
–
NA
NA
–
–
10.22
17.76
44.04
37.55
33.85
–
11.09
28.76
94.71
51.54
36.06
24.40
30.82
94.90
72.00
38.47
–
–
–
–
–
–
17.31
34.61
31.29
32.04
13.65
25.43
14.54
28.14
–
–
–
–
13.48
–
52.99
27.79
31.07
–
–
–
–
–
14.40
20.12
Ovarian Cancer
IGROV1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
16.09
50.75
16.85
39.10
–
23.98
41.91
11.94
52.56
–
45.64
–
32.54
27.49
76.71
–
30.17
37.28
29.25
34.48
32.85
80.34
–
40.61
45.08
28.60
20.22
21.46
17.12
70.36
–
OVCAR-3
–
–
–
–
–
–
–
–
–
–
–
–
OVCAR-4
30.65
–
25.13
–
OVCAR-5
OVCAR-8
23.23
38.36
11.03
17.24
20.48
10.36
15.47
–
NCI/ADR-RES
SK-OV-3
–
Renal Cancer
786–0
–
–
–
–
–
–
–
–
–
10.27
32.81
60.52
38.49
55.45
37.61
36.18
34.28
37.98
25.58
56.56
44.07
52.56
21.07
45.06
46.32
35.43
19.33
49.86
27.66
46.83
20.30
29.13
31.76
24.92
10.70
33.82
36.25
38.56
23.07
17.88
–
32.38
59.25
44.88
50.38
33.00
25.91
37.63
70.52
–
–
A498
26.00
27.87
18.61
15.06
–
12.96
19.00
16.09
–
ACHN
–
–
–
–
–
–
–
CAKI-1
RXF 393
SN12C
TK-10
17.35
–
13.19
13.14
–
–
16.04
–
27.93
–
UO-31
24.23
58.39
27.04
Prostate Cancer
PC-3
–
–
–
–
–
–
45.09
41.07
55.90
47.37
35.77
39.95
67.53
27.28
64.97
38.44
23.09
27.53
DU-145
–
–
Breast Cancer
MCF7
–
–
–
–
–
–
–
–
39.73
16.61
33.50
27.50
73.25
51.40
33.48
19.02
41.36
21.53
74.31
41.54
28.40
24.53
32.85
18.62
69.20
31.97
72.43
37.45
17.12
32.94
83.44
86.69
79.86
25.31
–
53.51
75.69
90.39
13.53
17.37
–
14.37
40.87
30.24
25.97
22.43
–
19.95
60.05
55.27
MDA-MB-231/ATCC
HS 578T
–
–
19.60
–
–
BT-549
–
T-47D
10.50
29.64
12.49
MDA-MB-468
–
NA: Not Available, (–) activity less than 10.00%.
5

A. Hassan et al.
Table 2
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Sorafenib into active site of enzyme. Investigation of different poses of
compound 8a and 8f revealed their interactions with Cys919, Glu885
and Asp1046 residues which is in consistent with that of Sorafenib
(Fig. 2). 2D style of compound 8a showed that quinoline-2(1H)-one
formed dual HB with Cys919 via both nitrogen and oxygen (3.5 Å and
2.52 Å, respectively) in competitive ATP-binding site of enzyme in
addition to hydrophobic interactions (Fig. 7). Furthermore, Compound
8a formed HB with Phe918 (2.87 Å) and Asp1046 (2.99 Å). Compound
8f showed HB with Cys919 via its oxygen atom (2.61 Å) in addition to pi-
H interaction of quinoline ring with both Gly922 and Leu840. Duo HBA/
ABD domain of compound 8f achieved its full job, exactly like Sorafenib,
in forming two HB with Glu885 (2.60 Å and 2.73 Å) and the third with
Asp1046 (3.26 Å) in the gate area of enzyme. Piperazine moiety of both
compounds 8a and 8f subrogated the central phenyl group of Sorafenib
to occupy the distance between hinge region and gate area of the
enzyme. Exactly like 3-trifluoromethyl-4-chlorophenyl of Sorafenib, 4-
phenylthiazole moiety of compound 8a and 8f occupied the hydro-
phobic allosteric site of enzyme that was built by the side chains of
Ile888, Leu892, Val898, Val899 and Cys1024 (Fig. 7).22
Cytotoxicity represented in IC50 µM for compounds 4a,
b and 8a-h against breast cancer cell lines (T-47D).
Compound
IC50 µM*
4a
37.70 ± 2.22
92.10 ± 5.43
7.20 ± 0.43
2.73 ± 0.16
8.41 ± 0.50
16.90 ± 1.34
10.30 ± 0.61
30.20 ± 1.78
16.50 ± 0.97
8.11 ± 0.48
11.70 ± 0.69
4b
8a
8b
8c
8d
8e
8f
8g
8h
Staurosporine
*
IC50 values are the mean ± SD of three separate
experiments.
Table 3
Cytotoxicity represented in IC50 µM for compounds 8a
and 8f against normal cell lines (MCF 10A).
3.4. In silico physicochemical and pharmacokinetic prediction
Compound
IC50 µM*
8a
39.40 ± 2.60
64.59 ± 4.20
24.77 ± 1.63
Away from efficacy and safety, many drug developments failed due
to inappropriate pharmacokinetics, so we utilize a free available Swis-
sADME website from the Swiss Institute of Bioinformatics (http://www.
swissadme.ch/index.php) to predict different physicochemical and
pharmacokinetic parameters of synthesized compounds.
8f
Camptothecin
Table 4
The Brain Or IntestinaL EstimateD permeation (BOILED-Egg)
method is a robust model that accurately predict both gastrointestinal
absorption and brain accessibility by calculating both the lipophilicity
(expressed in WLOGP) and polarity (expressed in TPSA) of selected
compounds Fig. 8. All target compounds 8a-h showed a high GI ab-
sorption while sorafenib showed low gastrointestinal absorption, this is
due to remarked decrease of lipophilicity (WLOGP 3.02–4.58) of syn-
thesized compounds comparing to sorafenib (WLOGP 6.32) in addition
to reasonable polarity of target compounds (TPSA 98.71–109.57 Å2)
(Table 7). Furthermore, target compounds 8a-h does not pass blood
brain barrier that confirming their good CNS safety profile.
Inhibitory activity against VEGFR-2 expressed in IC50
nM.
Compound
IC50 nM
4a
281.50 ± 12.0
111.90 ± 4.9
46.83 ± 2.4
169.70 ± 7.1
295.80 ± 13.2
51.09 ± 2.6
222.60 ± 9.8
62.70 ± 2.5
167.30 ± 6.4
174.50 ± 7.7
51.41 ± 2.3
4b
8a
8b
8c
8d
8e
8f
8g
In bioavailability radar Fig. 9, six physicochemical properties were
taken into account namely lipophilicity, size, polarity, solubility, flexi-
bility, and saturation. The molecular weights of all target compounds
are below 500 g/mol except N-phenyl derivatives 8e-h have molecular
weights above 500 g/mol, which represents the only Lipinski violation
in these target compounds. Otherwise, all target compounds satisfy
Lipinski rule with zero violation. All synthesized compounds have
higher Fraction Csp3 comparing to sorafenib as the aromatic spacer of
sorafenib was replaced by piperazine ring that positively impacted on
instauration parameter. All target compounds 8a-h have rotatable bonds
(6–8 bond) almost nearly that of sorafenib.34–36
8h
Sorafenib
*IC50 values are the mean ± SD of three separate
experiments.
Table 5
Cell cycle analysis results for compound 8a,f.
Phase
%G0-G1
%S
%G2/M
%Pre-G1
8a/T47D
8f/T47D
cont.T47D
41.31
45.69
52.59
52.39
49.36
34.62
6.30
4.95
12.79
43.05
25.12
2.61
4. Conclusion
and 8f to induce apoptosis, cells were stained with Annexin V/PI,
incubated for 24 h and analyzed. Analysis of early and late apoptosis
showed that compound 8a was able to induce significant levels of early
and late apoptosis percent 2.31% and 27.88%, respectively (Table 6,
Fig. 5 and Fig. 6) and necrosis percent was 12.86%. While compound 8f
induce early and late apoptosis percent 4.06% and 11.21%, respectively
and necrosis percent was 9.85% (Table 6).
A new series of 2-(4-(2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-
yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives were synthesized and
their structures were confirmed with spectral and elemental analyses.
NCI anticancer screening of key intermediates 4a,b and target com-
pounds 8a-h revealed higher anticancer activity of the later. Moreover,
NCI results demonstrated specific cytotoxic activity of target compounds
8a-h against breast cancer cell line. Consequently, further multidose
testing of compounds 4a,b and 8a-h was performed against breast
cancer T-47D cell line. Compounds 8a, 8b, 8c, 8e and 8h showed higher
cytotoxic activity than Staurosporine. The dihalogenated derivative 8b
showed the best cytotoxic activity with IC50 2.73 µM. In addition,
compounds 8a and 8d showed more inhibitory activity than sorafenib
with IC50 46.83 and 51.09 nM, respectively. The cell cycle analysis of
compounds 8a and 8f revealed that the arrest of cell cycle occurred at S
3.3. Molecular modeling studies
Using Molecular Operating Environment (MOE) software version
MOE 2014.0901 software, docking simulation of target compounds 8a-h
was performed with co-crystalized VEGFR-2 protein with Sorafenib
(PDB: 4ASD). Target compounds 8a-h showed similar fitness to
6

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Fig. 4. Cell cycle analysis results for compound 8a,f.
apoptosis; theses candidates require further in vivo, physicochemical and
Table 6
toxicity optimization.
Apoptosis induction analysis using Annexin V/PI for compounds 8a and 8f.
code
Apoptosis
Necrosis
5. Experimental
Total
Early
Late
5.1. Chemistry
8a/T47D
8f/T47D
cont.T47D
43.05
25.12
2.61
2.31
4.06
0.66
27.88
11.21
0.21
12.86
9.85
1.74
All starting materials were purchased and used without purification.
Follow up of reactions was performed by TLC (Kieselgel 60 F254pre-
coated plates, Merck, Darmstadt, Germany), the spots were detected by
exposure to UV lamp at 254 nm. Melting points were determined on an
electrothermal melting point apparatus (Stuart Scientific Co.) and were
uncorrected. NMR spectra were measured on a Bruker AV-400 spec-
trometer at Faculty of Pharmacy, Beni-Suef, Egypt. The 1H and 13C
chemical shifts are given relative to internal standard TMS = 0. Coupling
constants are stated in Hz. Mass Spectra were performed in Nawah
Scientific center, Cairo, Egypt, using APCI as ion source. Elemental an-
alyses for carbon, hydrogen, nitrogen, and sulfur were performed at The
Regional Center for Mycology and Biotechnology, Al-Azhar University,
phase. In apoptosis assay, both compounds 8a and 8f were able to
induce significant levels of early and late apoptosis. Exactly similar to
Sorafenib, docking of target compounds 8a-h with VEGFR-2 protein
4ASD showed HB with Cys919 in hinge region of enzyme and HB with
both Glu885 and Asp1046 in gate area. Using SwissADME, all target
compounds 8a-h were predicted to be highly absorbed from gastroin-
testinal tract with no BBB permeability. The present study introduces the
thiazolyl quinoline-2-one derivatives 8a and 8f as promising new anti-
proliferative candidates that target VEGFR-2 protein and induce
Fig. 5. Apoptosis induction analysis using Annexin V/PI for compounds 8a and 8f.
7

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Fig. 6. Apoptosis induction analysis using Annexin V/PI for compound 8a and 8f.
Cairo, Egypt. Methods of synthesis and instrumental data of compounds
5.1.1.2. 1-Phenyl-4-(piperazin-1-yl)quinolin-2(1H)-one 4b. White pow-
der; yield: 2.57 g (84%); mp: 203–204 ◦C (ethanol). 1H NMR (400 MHz,
DMSO‑d6) δ 7.83 (d, J = 7.3 Hz, 1H, Ar–H), 7.62 (t, J = 7.4 Hz, 2H,
Ar–H), 7.54 (t, J = 7.3 Hz, 1H, Ar–H), 7.40 (t, J = 7.2 Hz, 1H, Ar–H),
7.29 (d, J = 7.3 Hz, 2H, Ar–H), 7.25 (t, J = 7.4 Hz, 1H, Ar–H), 6.57 (d, J
1a,b, 2, 3a,b, 5a-d, 6a-d, and 7a-d were as reported.28
5.1.1. Synthesis of 4-(piperazin-1-yl)quinolin-2(1H)-one derivatives 4a,b
A mixture of 10 mmol compound 3a,b, 30 mmol (0.26 g) piperazine
was heated gradually in 10 mL DMF with continuous stirring over 2 h till
reach 80 ◦C then complete heating for another 6 hr. The solution was
evaporated under reduced pressure and the residue was triturated with
5% Na2CO3 solution, filtered, washed with water, and recrystallized
from the appropriate solvent.
=
8.4 Hz, 1H, Ar–H), 6.10 (s, 1H, quin-C3–H), 3.18 (s, 4H,
piperazinyl–H), 2.79 (s, 4H, piperazinyl–H); 13C NMR (101 MHz,
DMSO‑d6) δ 162.3, 159.4, 141.7, 138.4, 130.7, 130.4, 129.8, 128.9,
125.4, 121.9, 117.1, 116.4, 106.0, 53.3, 46.0; LC-MS: m/z calcd: 305.4,
found [M+H]+: 306.0. Anal. Calcd for C19H19N3O (305.38): C, 74.7; H,
6.3; N, 13.8. Found: C, 74.8; H, 6.4; N, 14.0.
5.1.1.1. 6-Fluoro-4-(piperazin-1-yl)quinolin-2(1H)-one 4a. White crys-
tals; yield: 2.10 g (85%); mp: 266–267 ◦C (ethanol). 1H NMR (400 MHz,
DMSO‑d6) δ 11.46 (s, 1H, quin-NH), 7.42–7.29 (m, 3H, quin-Ar–H), 5.91
(s, 1H, quin-C3–H), 2.95 (s, 8H, piperazinyl–H); 13C NMR (101 MHz,
DMSO‑d6) δ 163.4, 159.5, 158.4, 156.1, 136.2, 118.8, 117.2, 110.0,
106.9, 52.7, 45.5; TLC-MS: m/z calcd: 247.3, found [Mꢀ H]ꢀ : 246.20.
Anal. Calcd for C13H14FN3O (247.27): C, 63.15; H, 5.7; N, 17. Found: C,
63.2; H, 6.0; N, 16.8.
5.1.2. General synthesis of 2-(4-(2-oxo-1,2-dihydroquinolin-4-yl)
piperazin-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives 8a-h
Mixture of compound 4a,b (1 mmol), appropriate 2-bromo-N-(4-
phenylthiazol-2-yl)acetamide derivatives 7a-d (1.1–1.3 mmol) and
anhydrous K2CO3 (2 mmol, 276 mg) were stirred in 10 mL DMF over-
night. The solution was added to crashed ice, filtered, and recrystallized
from suitable solvent.
8

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Fig. 7. Docking of compounds 8a and 8f with VEGFR-2 (PDB:4ASD) illustrating 2D overlaid style (compound in green) with sorafenib (in red) and 3D overlaid style
(compound in blue) with sorafenib (in red).
5.1.2.1. 2-(4-(6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-
(4-phenylthiazol-2-yl)acetamide 8a. White crystals; yield: 370 mg (80%);
mp: 263–264 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ 11.47 (s, 1H, quin-
NH), 7.91 (d, J = 7.3 Hz, 2H, Ar–H), 7.66–7.64 (s, 1H, thiazole-Ar–H),
7.46–7.41 (m, 2H Ar–H), 7.39 (dd, J = 8.4, 2.7 Hz, 1H, Ar–H), 7.37–7.30
(m, 3H, quin-Ar–H), 5.97 (s, 1H, quin-C3–H), 3.45 (s, 2H, NHCOCH2),
3.10 (s, 4H, piperazinyl–H), 2.81 (s, 4H, piperazinyl–H); 13C NMR (101
MHz, DMSO‑d6) δ 169.2, 163.0, 158.7, 158.0, 156.0, 149.3, 136.4,
134.7, 129.2, 128.31 126.2, 119.0, 118.3, 117.1, 109.8, 108.6, 107.6,
60.4, 52.7, 51.6; TLC-MS: m/z calcd: 463.5, found [Mꢀ H]ꢀ : 462.5; Anal.
Calcd for C24H22FN5O2S (463.53): C, 62.2; H, 4.8; N, 15.1; S, 6.9. Found:
C, 62.4; H, 5.1; N, 15.0; S, 6.8.
Calcd for C24H21ClFN5O2S (497.97): C, 57.9; H, 4.25; N, 14.1; S, 6.4;
Found: C, 58.15; H, 4.4; N, 14.0; S, 6.2.
5.1.2.3. 2-(4-(6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-
(4-(p-tolyl)thiazol-2-yl)acetamide 8c. White powder; yield: 410 mg
(86%); mp: 192–194 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ 12.09 (s, 1H,
CONH), 11.47 (s, 1H, quin-NH), 7.80 (d, J = 8.1 Hz, 2H), 7.56 (s, 1H,
thiazole-Ar–H), 7.42–7.31 (m, 3H, quin-Ar–H), 7.24 (d, J = 8.0 Hz, 2H,
Ar–H), 5.97 (s, 1H, quin-C3–H), 3.45 (s, 2H, NHCOCH2), 3.10 (s, 4H,
piperazinyl–H), 2.81 (s, 4H, piperazinyl–H), 2.33 (s, 3H, Ar–CH3); 13
C
NMR (101 MHz, DMSO‑d6) δ 169.1, 163.0, 158.7, 157.8, 156.0, 149.4,
137.7, 136.4, 132.0, 129.8, 126.1, 119.0, 118.3, 117.2, 110.0, 107.7,
107.6, 60.4, 52.7, 51.6, 21.2; TLC-MS: m/z calcd: 477.6, found [Mꢀ H]ꢀ :
476.4; Anal. Calcd for C25H24FN5O2S (477.56): C, 62.9; H, 5.1; N, 14.7;
S, 6.7; Found: C, 63.2; H, 5.3; N, 14.6; S. 6.6.
5.1.2.2. N-(4-(4-chlorophenyl)thiazol-2-yl)-2-(4-(6-fluoro-2-oxo-1,2-
dihydroquinolin-4-yl)piperazin-1-yl)acetamide 8b. White powder; yield:
420 mg (85%); mp: 195–196 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ 12.13
(s, 1H, CONH), 11.47 (s, 1H, quin-NH), 7.93 (d, J = 8.6 Hz, 2H, Ar–H),
7.71 (s, 1H, thiazole-Ar–H), 7.49 (d, J = 8.6 Hz, 2H, Ar–H), 7.42–7.29
(m, 3H, quin-Ar–H), 5.97 (s, 1H, quin-C3–H), 3.45 (s, 2H, NHCOCH2),
3.10 (s, 4H, piperazinyl–H), 2.81 (s, 4H, piperazinyl–H); 13C NMR (101
MHz, DMSO‑d6) δ 169.2, 162.9, 158.6, 158.1, 156.0, 148.1, 136.5,
133.6, 132.8, 129.2, 127.9, 118.9, 118.4, 117.1, 110.0, 109.4, 107.8,
60.4, 52.7, 51.6. TLC-MS: m/z calcd: 498.0, found [Mꢀ H]ꢀ : 497.0; Anal.
5.1.2.4. 2-(4-(6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-
(4-(4-methoxyphenyl)thiazol-2-yl)acetamide 8d. White powder; yield:
400 mg (82%); mp: 190–191 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ 12.09
(s, 1H, CONH), 11.49 (s, 1H, quin-NH), 7.84 (d, J = 8.7 Hz, 2H), 7.46 (s,
1H, thiazole-Ar–H), 7.43–7.36 (m, 1H, quin-Ar–H), 7.33 (dd, J = 8.8,
5.2 Hz, 2H, Ar–H), 6.99 (d, J = 8.8 Hz, 2H, Ar–H), 5.98 (s, 1H, quin-
C3–H), 3.79 (s, 3H, Ar–OCH3), 3.44 (s, 2H, NHCOCH2), 3.09 (s, 4H,
9

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
Fig. 8. BOILED Egg plot for sorafenib, compounds 4a,b and 8a-h.
Table 7
Swiss ADME physicochemical and pharmacokinetic parameters of sorafenib, compounds 4a,b and 8a-h.
Compound
4a
4b
8a
8b
8c
8d
8e
8f
8g
8h
Sorafenib
M.W
247.27
0.31
1
305.37
0.21
2
463.53
0.21
6
497.97
0.21
6
477.55
0.24
6
493.55
0.24
7
521.63
0.17
7
556.08
0.17
7
535.66
0.19
7
551.66
0.19
8
464.82
0.10
9
Fraction Csp3
No of rotat.bonds
No. of HBA
No. of HBD
MR
3
2
5
5
5
6
4
4
4
5
7
2
1
2
2
2
2
1
1
1
1
3
75.89
48.13
1.56
High
Yes
0
100.9
37.27
2.46
High
Yes
0
135.73
109.57
3.2
140.74
109.57
3.75
High
No
140.69
109.57
3.55
High
No
142.22
118.8
3.17
High
No
160.75
98.71
4.08
High
No
165.76
98.71
4.6
165.71
98.71
4.39
High
No
167.24
107.94
3.88
High
No
112.48
92.35
4.11
Low
No
TPSA
Log P
GI absorption
BBB permeant
Lipin. violations
Bioavail. Score
High
No
High
No
0
0
0
0
1
1
1
1
0
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
Fig. 9. Bioavailability radar plot for compounds 8d,e and sorafenib.
piperazinyl–H), 2.81 (s, 4H, piperazinyl–H); 13C NMR (101 MHz,
DMSO‑d6) δ 169.1, 163.1, 159.4, 158.7, 157.8, 156.0, 149.2, 136.4,
127.5, 119.0, 118.7, 117.2, 117.1, 114.5, 110.0, 107.5, 106.6, 60.4,
55.6, 52.7, 51.6; TLC-MS: m/z calcd: 493.6, found [M+H]ꢀ : 493.9; Anal.
Calcd for C25H24FN5O3S (493.56): C, 60.8; H, 4.9; N, 14.2; S, 6.5. Found:
C, 61.0; H, 5.1; N, 13.95; S, 6.4.
5.1.2.5. 2-(4-(2-oxo-1-phenyl-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-
(4-phenylthiazol-2-yl)acetamide 8e. Yellow powder; yield: 420 mg
(80%); mp: 274–276 ◦C. 1H NMR (500 MHz, DMSO‑d6) δ 12.21 (s, 1H,
CONH), 7.92 (d, J = 9.4 Hz, 2H, Ar–H), 7.82 (d, J = 8.1 Hz, 1H, Ar–H),
7.67 (s, 1H, thiazole-Ar–H), 7.61 (t, J = 7.6 Hz, 2H, Ar–H), 7.54 (t, J =
7.4 Hz, 1H, Ar–H), 7.45 (t, J = 7.7 Hz, 2H, Ar–H), 7.39 (t, J = 8.5 Hz, 1H,
Ar–H), 7.34 (t, J = 7.4 Hz, 1H, Ar–H), 7.29 (d, J = 7.2 Hz, 2H, Ar–H),
7.24 (t, J = 7.6 Hz, 1H, Ar–H), 6.53 (d, J = 8.5 Hz, 1H, Ar–H), 6.11 (s,
10

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
1H, quin-C3–H), 3.49 (s, 2H, NHCOCH2), 3.19 (s, 4H, piperazinyl–H),
2.86 (s, 4H, piperazinyl–H); 13C NMR (126 MHz, DMSO‑d6) δ 169.1,
162.3, 158.8, 158.0, 149.3, 141.7, 138.3, 134.7, 130.8, 130.5, 129.8,
129.2, 129.0, 128.3, 126.2, 125.4, 122.1, 116.9, 116.5, 108.7, 106.4,
60.4, 52.7, 51.8; TLC-MS: m/z calcd: 521.6, found [M+H]ꢀ : 521.8; Anal.
Calcd for C30H27N5O2S (521.64): C, 69.1; H, 5.2; N, 13.4; S, 6.15. Found:
C, 69.3; H, 5.4; N, 13.2; S, 6.0.
5.2.2. Evaluation of in vitro cytotoxicity IC50 against breast cancer cell line
To inspect the effect of the synthesized compounds 4a,b and 8a-h on
breast cancer cells, MTT assay was performed against T-47D cell
line.37,38
5.2.3. Evaluation of in vitro cytotoxicity IC50 against normal cell line
The cytotoxicity of target compounds 8a and 8f on normal cell line
MCF 10A was performed using MTT assay.37,38
5.1.2.6. N-(4-(4-chlorophenyl)thiazol-2-yl)-2-(4-(2-oxo-1-phenyl-1,2-
dihydroquinolin-4-yl)piperazin-1-yl)acetamide 8f. White powder; yield:
470 mg (84%); mp: 265–267 ◦C. 1H NMR (500 MHz, DMSO‑d6) δ 12.22
(s, 1H, CONH), 7.94 (d, J = 8.6 Hz, 2H, Ar–H), 7.82 (d, J = 9.1 Hz, 1H,
Ar–H), 7.73 (s, 1H, thiazole-Ar–H), 7.61 (t, J = 7.6 Hz, 2H, Ar–H), 7.54
(d, J = 7.5 Hz, 1H, Ar–H), 7.51 (d, J = 8.6 Hz, 2H, Ar–H), 7.39 (t, J = 8.5
Hz, 1H, Ar–H), 7.29 (d, J = 7.2 Hz, 2H, Ar–H), 7.24 (t, J = 7.6 Hz, 1H,
Ar–H), 6.52 (d, J = 8.6 Hz, 1H, Ar–H), 6.11 (s, 1H, quin-C3–H), 3.49 (s,
2H, NHCOCH2), 3.19 (s, 4H, piperazinyl–H), 2.86 (s, 4H,
piperazinyl–H); 13C NMR (126 MHz, DMSO‑d6) δ 169.2, 162.3, 158.8,
158.2, 148.1, 141.7, 138.3, 133.6, 132.7, 130.8, 130.5, 129.8, 129.3,
129.0, 127.9, 125.4, 122.1, 116.9, 116.5, 109.4, 106.4, 60.4, 52.7, 51.8;
TLC-MS: m/z calcd: 556.1, found [Mꢀ H]ꢀ : 555.7; Anal. Calcd for
5.2.4. VEGFR-2 inhibitory assay
VEGFR-2 assay was performed for synthesized compounds 4a,b and
8a-h by a well-established reported method following the instructions of
(BPS VEGFR2(KDR) Kinase Assay Kit Catalog # 40325, bps bioscience,
San Diego, U.S.39
5.2.5. Cell cycle analysis and apoptotic assay
The effects of both compounds 8f and 8f on cell cycle development
and induction of apoptosis in the T-47D was performed using the
Annexin V-FITC Apoptosis Detection Kit (BioVision Research Products,
USA).40
C
30H26ClN5O2S (556.08): C, 64.8; H, 4.7; N, 12.6; S, 5.8. Found: C, 64.9;
5.3. Molecular modeling study
H, 4.8; N, 12.7; S, 5.9.
The molecular docking studies were achieved using Molecular
Operating Environment software version MOE 2014.0901. Firstly, the
co-crystalized VEGFR-2 protein with Sorafenib (PDB: 4ASD) was
downloaded from https://www.rcsb.org/. A library of target com-
pounds and Sorafenib were drawn and their energy was minimized by
Hamiltonian-Force Field-MMFF94x and the force field partial charges
for each molecule were calculated.16 3D protonation and correction was
performed and water removed. Docking was carried out by using Tri-
angle matcher placement and the rescoring function was London dG.
The docking methodology was validated by redocking of Sorafenib into
the active site of the enzyme and the results revealed an exact alignment
as the original one with the same interactions.
5.1.2.7. 2-(4-(2-oxo-1-phenyl-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-N-
(4-(p-tolyl)thiazol-2-yl)acetamide 8g. White crystals; yield: 500 mg
(84%); mp: 273–275 ◦C. 1H NMR (400 MHz, CDCl3) δ 10.28 (s, 1H,
CONH), 7.72 (d, J = 7.7 Hz, 1H, Ar–H), 7.67 (d, J = 7.4 Hz, 2H, Ar–H),
7.52 (d, J = 7.1 Hz, 2H, Ar–H), 7.45 (d, J = 6.7 Hz, 1H, Ar–H), 7.22 (d, J
= 6.9 Hz, 3H, Ar–H), 7.19–7.13 (m, 3H, Ar–H), 7.05 (s, 1H, thiazole-
Ar–H), 6.60 (d, J = 8.5 Hz, 1H, Ar–H), 6.23 (s, 1H, quin-C3–H), 3.34 (s,
2H, NHCOCH2), 3.26 (s, 4H, piperazinyl–H), 2.86 (s, 4H,
piperazinyl–H), 2.31 (s, 3H, Ar–CH3); 13C NMR (101 MHz, CDCl3) δ
168.1, 163.2, 158.4, 156.9, 150.3, 141.7, 138.0, 137.8, 131.6, 130.2,
129.5, 129.1, 128.8, 126.0, 124.6, 121.7, 117.2, 116.8, 107.4, 107.2,
61.3, 53.6, 51.6, 29.7; LC-MS: m/z calcd: 535.7, found [M+H]+: 536.0;
Anal. Calcd for C31H29N5O2S (535.67): C, 69.5; H, 5.5; N, 13.1; S, 6.0.
Found: C, 69.8; H, 5.7; N, 13.0; S, 5.9.
5.4. In silico physicochemical and pharmacokinetic prediction
5.1.2.8. N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-(4-(2-oxo-1-phenyl-1,2-
dihydroquinolin-4-yl)piperazin-1-yl)acetamide 8h. White crystals; yield:
450 mg (82%); mp: 276–277 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ 12.10
(s, 1H, CONH), 7.83 (t, J = 9.1 Hz, 3H, Ar–H), 7.61 (t, J = 7.1 Hz, 2H,
Ar–H), 7.54 (d, J = 7.2 Hz, 1H, Ar–H), 7.47 (s, 1H, thiazole-Ar–H), 7.39
(t, J = 7.6 Hz, 1H, Ar–H), 7.28 (d, J = 7.2 Hz, 2H, Ar–H), 7.24 (t, J = 7.5
Hz, 1H, Ar–H), 7.00 (d, J = 8.1 Hz, 2H, Ar–H), 6.53 (d, J = 8.2 Hz, 1H,
Ar–H), 6.10 (s, 1H, quin-C3–H), 3.79 (s, 3H, Ar–OCH3), 3.48 (s, 2H,
Physicochemical properties and pharmacokinetics prediction of
compounds 4a,b and 8a-h were performed using SwissADME which is
one of different free available services offered from the Swiss Institute of
Bioinformatics. BOILED Egg is a plot of TPSA versus WLOGP with the
white region represents the highest probability of gastrointestinal ab-
sorption, and the yolk region represents the highest probability to BBB
permeability. Lipophilicity is expressed in consensus log Po/w which is
calculated by SwissADME and it equals the arithmetic mean of the five
different log P values predicted by different freely available models
namely XLOGP3, MLOGP, SILICOS-IT, iLOGP in addition to their own
model WLOGP which also involved in BOILED Egg plot.41 Bioavail-
ability radar represents six physicochemical properties: lipophilicity
(-0.7 < XLOGP3 > +5.0), size (150 g/mol < MV > 500 g/mol), polarity
(20 Å2 <TPSA > 130 Å2), insolubility (0 < Log S (ESOL) > 6), insatu-
ration (0.25 < Fraction Csp3 > 1.0), and flexibility (0 < no. of rotatable
bonds > 9). The central pink hexagon represents the optimum range for
all six parameters. Lipinski filter is used to assess the drug-likeness of
synthesized compounds (MW ≤ 500, MLOGP ≤ 4.15, N or O ≤ 10, NH or
OH ≤ 5).42,43
NHCOCH2), 3.19 (s, 4H, piperazinyl–H), 2.87 (s, 4H, piperazinyl–H); 13
C
NMR (101 MHz, DMSO‑d6) δ 169.0, 162.3, 159.5, 158.8, 157.8, 149.2,
141.7, 138.3, 130.8, 130.4, 129.7, 129.0, 127.5, 125.4, 122.1, 117.0,
116.5, 114.6, 106.6, 106.3, 60.4, 55.6, 52.7, 51.8; LC-MS: m/z calcd:
551.7, found [M+H]+: 553.10; Anal. Calcd for C31H29N5O3S (551.67):
C, 67.5; H, 5.3; N, 12.7; S, 5.8. Found: C, 67.8; H, 5.5; N, 12.7; S, 5.6.
5.2. Biological evaluation
5.2.1. NCI antiproliferative assay
The methodology of the NCI procedures for initial anticancer
screening was illustrated on website (http://www.dtp.nci.nih.gov). The
protocol based on screening of anticancer candidates against sixty cell
lines panel derived from nine different human tumors. NCI-60 testing is
performed at a single concentration of 10ꢀ 5 M or 15
μg/ml in accor-
Declaration of Competing Interest
dance with the protocol of the Drug Evaluation Branch, National Cancer
Institute, Bethesda, USA.
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.
11

A. Hassan et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116168
18 Dawood DH, Nossier ES, Ali MM, Mahmoud AE. Synthesis and molecular docking
study of new pyrazole derivatives as potent anti-breast cancer agents targeting
VEGFR-2 kinase. Bioorg Chem. 2020;103916.
Acknowledgment
The authors are grateful to the National Cancer Institute (NCI),
Bethesda, MD for performing the anticancer screening for the target
compounds over their panel of cell lines. Authors would like to thank
Faculty of Pharmacy, Minia University, Minia and Faculty of Pharmacy,
South Valley University, Qena, Egypt
19 El-Adl K, Ibrahim MK, Khedr F, Abulkhair HS, Eissa IH. N-Substituted-4-
phenylphthalazin-1-amine-derived VEGFR-2 inhibitors: Design, synthesis, molecular
docking, and anticancer evaluation studies. Arch Pharm. 2020, e2000219.
20 Marzouk AA, Abdel-Aziz SA, Abdelrahman KS, et al. Design and synthesis of new 1,6-
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Appendix A. Supplementary material
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Supplementary data to this article can be found online at https://doi.
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