Discovery of pyridine- sulfonamide hybrids as a new scaffold for the development of potential VEGFR-2 inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2021.104842

New sulfonamide derivatives have been synthesized and tested as antitumor agents. All newly synthesized compounds were tested in vitro against 60 lines of human cancer cells. Compound VIIb shows broad-spectrum activity with a mean inhibition value of 91.67% against all cell lines. It exhibited potent anticancer activity with GI50 values of 1.06–8.92 μM relative to most of the tested cancer cell lines. Compound VIIb has been tested for enzyme inhibition activity toward vascular endothelial growth factor receptor 2, where VEGFR-2 was potently inhibited at a lower IC50 value of 3.6 μM, compared with sorafenib (IC50 = 4.8 μM). Hybrid VIIb was also able to induce cell cycle disturbance and apoptosis in Renal UO-31 cells, as shown by DNA flow cytometry and Annexin V-FITC/PI assays. It has also revealed lower Bcl-2 protein expression anti-apoptotic levels and higher BAX, p53, and caspases 3 expression levels.

Full text of DOI:10.1016/j.bioorg.2021.104842

Bioorganic Chemistry 111 (2021) 104842
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of pyridine- sulfonamide hybrids as a new scaffold for the
development of potential VEGFR-2 inhibitors and apoptosis inducers
a,
b,
*
a
Marwa F. Ahmed
, Eman Y. Santali
a
Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
New sulfonamide derivatives have been synthesized and tested as antitumor agents. All newly synthesized
compounds were tested in vitro against 60 lines of human cancer cells. Compound VIIb shows broad-spectrum
activity with a mean inhibition value of 91.67% against all cell lines. It exhibited potent anticancer activity with
Sulfonamides
Antitumor
Vascular endothelial growth factor receptor
GI50 values of 1.06–8.92
enzyme inhibition activity toward vascular endothelial growth factor receptor 2, where VEGFR-2 was potently
inhibited at a lower IC50 value of 3.6 M). Hybrid VIIb was also able
μ
M relative to most of the tested cancer cell lines. Compound VIIb has been tested for
Cell cycle
Apoptosis
μ
M, compared with sorafenib (IC50 = 4.8 μ
to induce cell cycle disturbance and apoptosis in Renal UO-31 cells, as shown by DNA flow cytometry and
Annexin V-FITC/PI assays. It has also revealed lower Bcl-2 protein expression anti-apoptotic levels and higher
BAX, p53, and caspases 3 expression levels.
1
. Introduction
Vascular endothelial growth factor receptors (VEGFRs) have been
Analysis of structure–activity relationship and typical Pharmaco-
phoric characteristics shared with sorafenib, as well as various inhibitors
of VEGFR-2 have shown that the majority of VEGFR-2 inhibitors have
four major characteristics [15–17] as seen in Fig. 1: (A) The main
structure of most inhibitors is a flat heteroaromatic ring system
comprising at least one N-atom, (B) a central aryl ring (hydrophobic
spacer) [18], (C) a linker comprising a functional group acting as a
pharmacophore (e.g., amino or urea) containing both an H-bond
acceptor and a donor; (D) Inhibitor terminal hydrophobic moiety. Thus,
hydrophobic interactions in this allosteric binding region are commonly
achieved [19]. In addition, an analysis of X-ray structure in several
VEGFR 2-linked inhibitors confirmed that there is adequate space
around the heteroaromatic ring for different substitutes [20,21].
Sulfonamides constitute a major class of therapeutic agents consid-
ered to display a wide range of biological activities such as antitumor
[22–27], diuretic [28], antibacterial [29] and hypoglycemic activity
[30]. Several sulfonamides have been tested for their antitumor activity,
and there is a wide range of pathways for their action against cancer,
such as cell cycle arrest [22] and VEGFR-2 inhibitor [31–34]. In addi-
tion, several chalcones [35–37], pyrazole [38,39], isoxazole [40–42],
and pyridine [43–46] moieties were reported as VEGFR-2 inhibitors.
identified for the development of novel anticancer agents as an excellent
remedial target [1]. VEGF is a powerful mitogen specific to vascular
endothelial cells [2] which increases the vessel’s permeability and en-
hances cell formation, proliferation, and differentiation [3]. The path-
ogenesis of many diseases, including cancer and rheumatoid arthritis, is
affected by angiogenesis [4–6]. VEGFR-2 is a class V tyrosine kinase
(
RTK) receptor triggered by the specific binding of the VEGFR-2 extra-
cellular regulatory domain in both endothelial and multiple tumor cells.
When stimulated, VEGFR-2 undergoes autophosphorylation, which
activates signaling pathways leading to proliferation of endothelial cells
as well as tumor angiogenesis to encourage development of the tumor
[
7]. Several VEGFR-2 small molecule inhibitors found the anti-
angiogenic potential agents for the treatment of a variety of cancers by
start competing for the VEGFR-2 intracellular kinase binding site with
adenosine triphosphate (ATP) [8]. Sorafenib [9], Sunitinib and pazo-
panib [10] have been licensed by The Food and Drug Administration to
treat severe renal cell carcinoma [10,11]. Sorafenib is a powerful in-
hibitor of VEGFR-2 and has been approved as an anti-angiogenic drug
[
12–14].
In particular, a molecular hybridization strategy involving a
*
Corresponding author at: Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia; Department
of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt
E-mail addresses: marwa.farg@tu.edu.sa, marwa_farag@pharm.helwan.edu.eg (M.F. Ahmed).
https://doi.org/10.1016/j.bioorg.2021.104842
Received 23 December 2020; Received in revised form 10 February 2021; Accepted 16 March 2021
Available online 22 March 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
combination of two or more groups with specific biological character-
istics was applied, depending on ligand-based drug design [47], In order
to acquire new molecules with promising antitumor activity, the mo-
lecular hybridization of sulfonamides and other successful anti-tumor
molecules was carried out. Our study focused on the synthesis of new
compounds with similar essential pharmacophoric characteristics re-
ported and currently used as inhibitors of VEGFR-2 (e.g., sorafenib). Our
molecular design argument included bioisosteric alteration strategies in
four different positions for VEGFR-2 inhibitors (Fig. 1). First, we adopted
a bioisosteric replacement of the pyridine ring of the reported ligand
sorafenib by pyrazole (compounds V a-d) and/or isoxazole (compounds
VI a-d) or use pyridine ring (compounds VII a-d) or dihydropyridine ring
2. Results and discussion
2.1. Chemistry
The synthetic pathways used in this study for the preparation of new
hybrid chalcones derivatives are represented in (Scheme 1). Claisen –
Schmidt condensation of sulphonamide derivative III with different ar-
omatic aldehyde namely, 2- Flurobenzaldehyde, 3- Flurobenzaldehyde,
and m-Tolualdehyde in ethanolic sodium hydroxide solution obtained
the chalcones IVa-c in good yields. The corresponding pyrazoline de-
rivatives Va-c (Scheme 2) is afforded after the treatment of chalcone
derivatives IV a-c with hydrazine hydrate in ethanol.
(
compounds VIII a-d). The second strategy is to use phenyl moiety to
Further cyclo condensation reaction was performed with hydroxyl-
amine from chalcone derivatives IV b,c to obtain the corresponding
isoxazole analogs VI a,b. (Scheme 2).
replace the central aryl ring of the lead structure. The third strategy is to
use HBA-HBD functional group containing sulfonamide linkers that
possess H-bond acceptors and/or donors. Also, we use tolyl moiety
instead of the hydrophobic substituted phenyl tail of the lead structure.
In the light of previous studies and in an effort to discover new potent
antagonists of VEGFR-2 as anticancer agents, novel hybrid compounds
including sulfonamide-chalcones, pyrazole, isoxazole and/or pyridine
backbone have been designed to test their cytotoxic efficacy. All new
synthesized compounds were sent to the National Cancer Institute (NCI)
to test their anti-cancer efficacy against 60 lines of human cancer cells.
In addition, the inhibitory activity of the most active derivative has been
tested against VEGFR-2.
Reaction of chalcones with malononitrile in the presence of ammo-
nium acetate afford pyridine derivatives VIIa-c (Scheme 3). While cyclo
condensation of chalcone derivatives with ethyl cyanoacetate gives de-
rivatives VIIIa-c (Scheme 3).
2
2
.2. Biological activity
.2.1. Preliminary single (10 mM) dose screening and SAR discussion:
All final synthesized compounds tested at a concentration (10 M)
μ
against different human tumor cell lines, derived from nine clinically
isolated types of cancer for their in vitro anticancer activity (Figs. 2 and
3
). In this research study, we have five series (IV, V, VI, VII, VIII) each
series contain sulfonamide moiety binding to chalcone, pyrazole,
Fig. 1. Design strategy of the new synthesized compounds.
2

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Scheme 1. Synthesis of compounds IV a-c. Reagents and conditions: (i) Pyridine, reflux, 2 h; (ii) Aromatic aldehydes, alcoholic NaOH (40%), stirring 5 h.
isoxazole, pyridine, and /or dihydropyridine moiety, respectively.
Data analysis shows that first series IV a-c with chalcone moiety
demonstrate good sensitivity against Colon cancer (HCT-116), Mela-
noma (UACC-62), Breast cancer (MCF7). Compound IVa bearing ortho
Fluro group at phenyl moiety show inhibition % (28.97, 35.64, 45.15)
against the three cell lines respectively. Compound IVb with meta Fluro
moiety show inhibition % (78.21, 28.88, 56.27) on the same cell lines.
On the other hand, compound IVc with meta methyl moiety show in-
hibition % (37.86, 30.38, 41.34) respectively. Second series V a-c which
contains pyrazole moiety show moderate selectivity against Renal can-
cer (CAKI-1). Compound Va with ortho Fluro group displayed cell
growth inhibition for Renal cancer CAKI-1(35.63%), UO-31 (31.09%)
and Melanoma UACC-62 (25.37%). Vb with meta Fluro moiety display
cell growth inhibition for Renal cancer CAKI-1 (26.9%), and Melanoma
UACC-62 (30.55%), while Vc show cell growth inhibition for Renal
cancer CAKI-1 (23.36%).
(57.29, 43.38, 49.77, 44.28, 58.73, 42.59, 62.68, 67.15) respectively
against the previous cell lines. On the other hand, compounds VIIc with
meta methyl group show inhibition % (41.78, 40.64, 47.11, 36.61,
38.77, 46.31, 59.21 and 57.72) on the same cell lines. On the other hand,
compound VII b bearing meta Fluro moiety shows broad spectrum ac-
tivity against all cell lines it shows mean inhibition value 91.67%.
furthermore, it shows inhibition percent more than 100% on Leukemia
(HL-60(TB)), (RPMI-8226), Non-small cell lung cancer (HOP-92, NCI-
H522), Colon cancer (HCT-116, HCT-15 HT29, SW-620), CNS cancer
(SNB-75), Melanoma (M14, SK-MEL-28, UACC-62), Ovarian cancer
(OVCAR-3, OVCAR-4, IGROV1), Breast cancer (T-47D, MDA-MB-468)
and Renal cancer (ACHN, RXF 393, TK-10, UO-31).
Series VIII a-c with dihydropyridine moiety has very good inhibitory
activity against Leukemia (RPMI-8226), Colon cancer (HCT-116), Renal
cancer (CAKI-1) and Breast cancer (MCF7). Compound VIIIa with ortho
Fluro group show inhibition % (41.18, 78.32, 44.65, 74.95) respec-
tively, while compound VIIIb with meta Fluro moiety show inhibition %
(80.63, 91.39, 39.77, 83.14) on the same cell line. also compound VIIIb
displays cell growth inhibition for Leukemia K-562 (45.10%), Leukemia
SR (45.10%), Colon cancer HCT-29 (45.36%) and Breast cancer T-47D
(58.05%). Compound VIIIc with meta methyl moiety show cell growth
inhibition for Leukemia K-562 (53.27%), Leukemia RPMI-8226
(82.39%), Colon cancer HCT-116, HCT-15 and HT29 (88.31%,
52.91%, 60.8%), Renal cancer CAKI-1 (35.48%) and Breast cancer
MCF7 (81.29%).
Series VI a,b with isoxazole moiety show good sensitivity toward
lung cancer HOP-92 and Renal cancer (CAKI-1). Compound VIa with
meta Fluro group show inhibition % (27.33 and 37.17) while compound
VIb with meta methyl group display inhibition % (32.34 and 31.34)
against the two cell lines respectively. Furthermore, compound VIa
show also cell growth inhibition for Non-small cell lung cancer A549/
ATCC (40.36%), CNS cancer U251 (37.77%) and Breast cancer T-47D
(
36.48%).
Series VII a-c with pyridine moiety was the most active series it
shows potent cytotoxic activity against Leukemia (CCRF-CEM), (K-562),
Non-small cell lung cancer (HOP-92), CNS cancer (SNB-75), Ovarian
cancer (IGROV1, OVCAR-4), Renal cancer (CAKI-1) and Breast cancer
2.2.2. In vitro full NCI panel five dose assay
Compound VIIb was chosen for further study at 5 different minimum
(
T-47D). Compound VIIa with ortho Fluro moiety displays inhibition %
concentrations (0.01, 0.1, 1, 10 and 100 M) against the full 60-cell
μ
3

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Scheme 2. Synthesis of compounds V a-c and VI a,b. Reagents and conditions: (i) Hydrazine hydrate, absolute ethanol, reflux 10 h.; (ii) Hydroxylamine, alcoholic
NaOH (10%), reflux 18 h.
panel (Table 1 and Fig. 4), based on the promising results obtained from
single dose testing. For hybrid VIIb, in each of the cancer cell lines
evaluated, three response parameters (GI50, TGI and LC50) have been
evaluated. GI50 values reflect the inhibitory effect level for growth,
while TGI represents cytostatic impact. Additionally, the parameter
LC50 is the cytotoxicity parameter for the hybrid being tested. In
addition, a mean midpoint graph (MG-MID) was measured at the GI50
parameter level, providing an average potency parameter for the com-
pound VIIb (Table 1). Compound VIIb has exhibited great anti-
proliferative activity with the GI50 values ranging from 1.06 to 8.92
2.2.3.1. DNA fragmentation assay. A main feature of apoptosis is the
DNA fragmentation which is used as an apoptosis marker and for the
identification of apoptotic cells using the DNA laddering assay [48]. The
gel electrophoresis DNA ladder assay was performed to further delineate
the apoptotic activation process by compound VIIb. The electrophoresis
of the agarose gel of the compound VIIb-treated DNA displayed a
ladder-like fragmentation pattern (Fig. 5). These results suggest that
IIIa-induced cancer growth inhibition exists by inducing apoptosis.
2.2.3.2. Cell cycle assay. Cell cycle analysis was conducted to provide
an insight into the cytotoxic nature of compound VIIb. As seen in
Table 3, Figs. 6 and 7, the percentage of cell population in the G2/M
phase increased to 49.68 percent in the compound VIIb treated group,
while in the untreated control group it was 17.22 percent, indicating
that the compound VIIb triggered a significant G2/M phase arrest.
μ
M against most of the cancer cell lines tested.
2
.2.3. In vitro VEGFR-2 kinase assay
The most active cytotoxic compound VIIb has been chosen to eval-
uate its inhibitory activity against VEGFR-2. Results have been reported
as a 50 percent concentration inhibition value (IC50). Sorafenib was
used in this assay as a positive control (Table 2). VIIb showed high
2
.2.3.3. Apoptosis assay. Apoptosis induction in cancer cells is the
inhibitory activity at IC50 value of 3.62
sorafenib (4.58 M).
μM relative to the IC50 value of
principal mechanism by which certain cytotoxic agents destroy tumor
cells [49]. Cellular changes occurring during apoptosis are the
μ
4

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Scheme 3. Synthesis of compounds VII a-c and VIIIa-c. Reagents and conditions: (i) Malononitrile, anhydrous ammonium acetate, ethanol, reflux 12 h.; (ii) Ethyl
cyanoacetate, anhydrous ammonium acetate, butanol, reflux 10 h.
Fig. 2. The growth inhibition % of compounds IVa-c, Va-c, VIa,b, VIIa,c, VIIIa-c over the most sensitive cell lines.
translocation of phosphatidylserine from the inside to the outside
through the plasma membrane. Annexin-V, which links to phosphati-
dylserine, is used on the plasma membrane’s outer side. as a sensitive
probe for PS [50]. The annexin-V/PI staining methodology has been
conducted. As shown in Figs. 8, and 9 and Table 4 Treatment of UO-31
cells with GI50 concentration (1.32 M) of compound VIIb for 24 h
μ
resulted in an increase in the early apoptosis ratio from 1.26% of the
control sample to 7.14% and a substantial increase in the late apoptosis
cell ratio from 0.25% to 9.08%. These findings suggest that compound
VIIb can induce the apoptotic mechanism of programmed cell death.
5

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 3. Single dose screening of compound VIIb as GI% of cancer cells.
.2.3.4. BAX, p35 and BCL-2 activity. The tumor suppressor gene, p53,
2
compound showed similar position and orientation inside the VEGFR-2
binding sites. The calculated ΔG (free energy binding) of compound
VIIb and the reference drug against VEGFR-2 is summarized in Table 6.
The binding mode of compound VIIb demonstrated a strong fitting to
VEGFR-2 active site as sorafenib with a docking score of ꢀ 27.09 kcal/
mol, which was higher than that of sorafenib. The sulfonamide (phar-
macophore) group formed two hydrogen bonds with Glu883 and
Asp1044. The phenyl (spacer) group formed five hydrophobic in-
teractions with Cys1043, Val914, Val89, Lys886, and Phe1045. The 2-
amino-4-(3-fluorophenyl)nicotinonitrile moiety formed six hydropho-
bic interactions with Phe1045, Val846, Leu1033, Ala864 and Leu838.
The terminal (hydrophobic tail) also formed two hydrophobic bonds
with Ile886 (Figs. 15–17).
is a crucial regulator of the central cell cycle that has an important role
in both cell arrest and cell death [51,52]. P53 may have been inactivated
by tumor cells due to sequencing mutations or by disabling those main
components of the p53 transcriptional signaling pathway upstream or
downstream [53]. P53 can also act in a transcription-independent
′
manner because of its ability to inhibit Bcl-2 s antiapoptotic activity or
to stimulate Bax’s apoptotic potential [54]. Study results showed that
treatment with VIIb raised the expression levels of p53 in UO-31 cells by
8
.9 folds compared with untreated control. Results also show significant
higher BAX protein expression level and significant lower Bcl2 level
when compared to untreated cells (Table 5).
2
.2.3.5. Caspases-3. Caspases considered to be the real apoptosis
workers are triggered at the early stages of apoptosis resulting in the
cleavage of main cellular components necessary for normal cellular
function [55]. Compared to untreated control, active caspase-3 expres-
sion level was 7.58 folds higher in PC3 cells, in response to VIIb treat-
3. Conclusion
In conclusion, a series of sulfonamide hybrids were designed, syn-
thesized, and biologically evaluated via NCI protocol for their anticancer
activity. Among the synthesized compounds, VIIb exhibited the most
powerful cytotoxic activity in the five-dose assay of the NCI against all
ment with concentration 1.32 M (Fig. 10).
μ
tested cell lines with a powerful 1.06–8.92
μ
M GI50 subpanel range.
2
.3. Molecular docking
In this work, molecular docking studies have been conducted for
Moreover, the most active compound VIIb has been tested for inhibitory
activity against VEGFR-2. Interestingly, the result of an inhibition of
VEGFR-2 was compatible with the cytotoxicity results. In addition,
compound VIIb arrested the cell cycle in phase G2/M and induced
apoptosis in UO-31 cells by upregulating BAX, Caspase 3 and P53 and
downregulating the expression of Bcl-2. These results indicate that
compound VIIb can be applied for further studies as a lead in the design
of new anticancer compounds.
compound VIIb and sorafenib as a reference drug. This investigation
was carried out in order to gain further insight into the binding modes of
compound VIIb to the VEGFR-2 binding site (PDB ID: 2OH4). The
binding free energies (ΔG) have been reported in Table 6. The key
binding site reported for VEGFR-2 consists of Glu883, Asp1044 and
Cys1043 [56,57].
The co-crystallized ligand (sorafenib) was re-docked against the
isolated pocket of the target protein to validate the docking process. The
RMSD value between the redocked conformer and the cocrystallized
conformer of sorafenib was found to be 1.09 which confirms the validity
of the docking protocol (Fig. 11).
4
. Experimental
4
.1. Chemistry
Digital melting point appliances Electrothermal Stuart 5MP3 is used
The proposed binding mode of sorafenib (Figs. 12–14) showed an
affinity value of-26,16 kcal/mol. Significant interactions with residues
at the active site of VEGFR-2 have been demonstrated. The urea (phar-
macophore) group formed three hydrogen bonds with Glu883 and
Asp1044. In addition, the phenyl group (spacer) formed four hydro-
phobic interactions with Cys1043, Val914, Lys866 and Val897. The
pyridine moiety (hetero-aromatic ring) formed three hydrophobic in-
teractions with Leu1033, Ala864 and Cys917, while nitrogen atom of the
pyridine moiety and the carbonyl group of acetamide moiety formed
two hydrogen bonds with Cys917. The terminal phenyl group (hydro-
phobic tail) also formed two hydrophobic bonds with Cys1022 and
Ile886.
to measure melting points. Elemental analyses have been performed in
the microanalytical units of Cairo University, Egypt. IR spectra have
been measured at FT. IR spectrophotometer-Nexus 670-Spectrophotom-
eter Nicolet, USA, and Perkin Elmer-9712. The Bruker NMR spectrom-
1
13
eter (400/100 MHz) was used to record H NMR and C NMR spectra.
Finnigan Mat SSQ 7000 EI 70 ev (Thermo Inst. Sys. Inc. USA) measured
mass spectrums. Compound III was prepared according to reported
procedure [58]
4
.1.1. Synthesis of compounds IVa-c
Compound III (0.01 mol) and aromatic aldehydes (0.01 mol) in 30 ml
ethanol and sodium hydroxide solution (5 ml, 40%) were stirred for 7 h.
The findings of the docking experiments revealed that the target
6

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
exchangeable). 13C NMR (DMSOd
) δ 23, 114, 116, 121, 122, 125, 126,
Table 1
6
Results of five-dose NCI-USA screening for compound VII b in vitro.
127, 128, 129, 130, 132, 136, 137, 143, 146, 159, 191.
Panel
Subpanel
GI50
mM)
MG-
MID
TGI
LC50
(mM
(
(mM)
4.1.1.2. N-(4-(3-(3-fluorophenyl)acryloyl)phenyl)-4-methyl-
◦
ꢀ 1
benzenesulfonamide (IVb). Yield 65%, mp. 145–147 C. IR (KBr, cm ):
3240 (NH), 3100 (CH-arom.), 1695 (CO). MS: m/z = 395; Anal. For
Leukemia
CCRF-CEM
HL-60(TB)
K-562
2.26
1.86
2.63
3.20
2.21
2.70
3.11
2.17
4.25
1.06
1.86
2.48
3.74
3.53
1.69
2.18
1.72
1.69
1.88
3.40
3.10
2.00
2.97
3.33
1.56
3.15
2.75
1.82
1.61
2.01
2.02
1.96
1.96
1.77
1.93
1.52
1.35
1.63
1.77
1.73
2.43
2.92
8.92
1.71
3.70
1.58
1.60
1.44
2.20
1.66
1.32
2.19
2.33
2.32
1.95
2.47
30.8
6.07
>100
30.2
8.92
10.5
15.7
6.32
17.2
2.37
4.21
7.96
15.5
12.6
4.91
4.48
3.28
3.23
4.85
12.3
10.2
4.40
12.8
14.5
3.02
11.6
9.62
3.66
3.25
4.38
4.46
4.22
4.08
3.23
4.24
3.44
2.75
3.12
3.67
3.46
7.82
10.8
23.3
3.12
16.2
2.92
3.16
2.79
7.11
3.17
2.59
7.57
5.45
10.0
4.60
>100
>100
>100
>100
>100
>100
>100
29.2
47.3
5.31
DNS
44.3
41.1
53.7
55.4
9.21
6.24
6.16
>100
>100
60.1
9.71
70.3
40.5
5.83
35.5
33.2
7.35
6.57
9.53
9.88
9.07
8.48
5.90
9.33
7.78
5.61
6.01
7.62
6.93
>100
97.6
56.9
5.69
41.4
5.40
6.25
5.41
34.6
6.06
5.09
46.0
17.7
68.3
30.9
22 3
C H18FNO S; Calcd. C, 66.82; H, 4.59; N, 3.54; Found: C, 66.77; H,
MOLT-4
RPMI-8226
SR
1
4
.63; N, 3.50. H NMR (DMSOd
6
): δ 2.28 (3H, s, CH
3
), 7.17–7.82 (13H,
), 10.02 (1H, s, NH, D
) δ 25, 112, 113, 114, 119, 125, 126,
128, 129, 131, 132, 135, 136, 138, 139, 142, 160, 190.
–
H, CH
–
), 7.97 (1H, d, J = 15.5 Hz, CH
–
–
2
O
m, Ar
1
3
Non-small cell
lung cancer
A549/ATCC
EKVX
2.65
exchangeable). C NMR (DMSOd
6
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
4
.1.1.3. 4-methyl-N-(4-(3-m-tolylacryloyl)phenyl)benzenesulfonamide
(IVc). Yield 70%, mp. 120–122 ◦C. IR (KBr, cm ): 3235 (NH), 3030
ꢀ 1
(CH-arom.), 1690 (CO). MS: m/z = 391; Anal. For C23
0.57; H, 5.41; N, 3.58; Found: C, 70.50; H, 5.38; N, 3.54. H NMR
DMSOd ): δ 2.27 (3H, s, CH ), 2.35 (3H, s, CH
3
H21NO S; Calcd. C,
1
7
(
6
3
3
), 6.87–7.90 (13H, m,
Colon cancer
2.28
–
H, CH
–
), 8.08 (1H, d, J = 15.0 Hz, CH
–
–
), 10.12 (1H, s, NH, D
) δ 21, 23, 114, 120, 121, 125, 126,
28, 129, 131, 132, 134, 135, 136, 137, 138, 140, 143, 185.
2
O
Ar
1
3
exchangeable). C NMR (DMSOd
6
1
KM12
4
.1.2. Synthesis of compounds Va-c
SW-620
SF-268
CNS cancer
Melanoma
2.75
1.62
A mixture of IV a-c (0.002 mmol) and hydrazine hydrate (99%,
SF-295
0.002 mmol was refluxed in 30 ml ethanol for 10 h then cooled, filtered,
and crystallized from ethanol.
SF-539
SNB-19
U251
LOX IMVI
MALME-3 M
M14
4.1.2.1. N-(4-(5-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-
◦
4-methylbenzenesulfonamide (Va). Yield 68%, mp. 174–176 C. IR (KBr,
ꢀ 1
cm ): 3250 (NH), 3230 (NH), 3105 (CH-arom.). MS: m/z = 409; Anal.
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786–0
3 2
For C22H20FN O S; Calcd. C, 64.53; H, 4.92; N, 10.26; Found: C, 64.48;
1
6 3
H, 4.96; N, 10.34. H NMR (DMSOd ): δ 2.33 (3H, s, CH ), 3.16 (1H, dd,
J = 12.3, 6.1 Hz, pyrazoline), 3.81 (1H, dd, J = 11.5, 5.8 Hz, pyrazo-
line), 4.59 (1H, dd, J = 11.7, 6.1 Hz, pyrazoline), 6.95–7.68 (12H, m, Ar-
13
2 6
H), 9.26, 9.85 (2H, 2 s, 2NH, D O exchangeable). C NMR (DMSOd ) δ
Ovarian cancer
2.96
2
0, 45, 46, 110, 113, 115, 121, 123, 124, 125, 126, 127, 128, 129, 136,
137, 140, 155.
4
4
.1.2.2. N-(4-(5-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-
◦
-methylbenzenesulfonamide (Vb). Yield 74%, mp. 164–166 C. IR (KBr,
ꢀ 1
Renal cancer
1.90
cm ): 3255 (NH), 3220 (NH), 3112 (CH-arom.). MS: m/z = 409; Anal.
A498
For C22
3 2
H20FN O S; Calcd. C, 64.53; H, 4.92; N, 10.26; Found: C, 64.59;
ACHN
1
H, 4.86; N, 10.32. H NMR (DMSOd
6 3
): δ 2.37 (3H, s, CH ), 3.23 (1H, dd,
CAKI-1
J = 12.3, 6.1 Hz, pyrazoline), 3.97 (1H, dd, J = 11.5, 5.8 Hz, pyrazo-
RXF 393
SN12C
line), 4.94 (1H, dd, J = 11.7, 6.1 Hz, pyrazoline), 7.12–7.72 (12H, m, Ar-
2 6
H), 9.29, 10.35 (2H, 2 s, 2NH, D ) δ
O exchangeable). 13C NMR (DMSOd
TK-10
UO-31
2
1, 44, 50, 108, 112, 113, 122, 123, 125, 130, 132, 136, 137, 138, 140,
Prostate cancer
Breast cancer
PC-3
2.26
1.85
1
45, 150, 164.
DU-145
MCF7
MDA-MB-
4.1.2.3. 4-methyl-N-(4-(5-m-tolyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)
2
31/ATCC
◦
ꢀ 1
benzenesulfonamide (Vc). Yield 80%, mp. 112–115 C. IR (KBr, cm ):
HS 578 T
BT-549
2.28
1.35
1.48
1.72
7.48
2.78
3.66
3.76
>100
5.75
9.05
8.19
3
243 (NH), 3230 (NH), 3107(CH-arom.). MS: m/z = 405; Anal. For
23 23 3 2
C H N O S; Calcd. C, 68.12; H, 5.72; N, 10.36; Found: C, 68.19; H,
T-47D
1
MDA-MB-468
5.67; N, 10.30. H NMR (DMSOd
6
): δ 2.33 (3H, s, CH
), 3.26 (1H, dd, J = 12.3, 6.1 Hz, pyrazoline), 3.78 (1H, dd, J = 11.5,
.8 Hz, pyrazoline), 4.68 (1H, dd, J = 11.7, 6.1 Hz, pyrazoline),
.83–7.68 (12H, m, Ar-H), 9.16, 9.48 (2H, 2 s, 2NH, D O exchangeable).
) δ 22, 24, 44, 54, 115, 124, 125, 126, 127, 128, 129,
3
), 2.36 (3H, s,
CH
3
DNS: Data not shown.
5
6
2
The reaction was left overnight at room temperature and poured onto
crushed ice, filtered and crystallized from ethanol.
13
C NMR (DMSOd
6
1
30, 131, 132, 136, 138, 141, 143, 146
4
.1.1.1. N-(4-(3-(2-fluorophenyl)acryloyl)phenyl)-4-methyl-
4
.1.3. Synthesis of compounds VIa-b
◦
ꢀ 1
benzenesulfonamide (IVa). Yield 72%, mp. 137–139 C. IR (KBr, cm ):
3
A mixture of chalcones IV b,c (0.004 mol) and hydroxylamine hy-
230 (NH), 3050 (CH-arom.), 1690 (CO). MS: m/z = 395; Anal. For
18FNO S; Calcd. C, 66.82; H, 4.59; N, 3.54; Found: C, 66.79; H,
.50; N, 3.62. H NMR (DMSOd
drochloride (0.004 mol) in ethanolic sodium hydroxide solution (15 ml,
0%) has been refluxed for 18 h concentrated, and neutralized with HCl.
Isolated product filtered and crystallized with ethanol.
C
22
H
3
1
1
4
6
): δ 2.32 (3H, s, CH
), 8.11 (1H, d, J = 15.3 Hz, CH
3
), 7.17–8.92 (13H,
–
–
), 10.15 (1H, s, NH, D
–
–
H, CH
2
O
m, Ar
7

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 4. Five-dose assay graph of compound VIIb for the NCI cancer cell lines of nine panels.
Table 2
IC50 for compound VIIb as VEGFR-2 inhibitor.
Comp.
IC50 M
μ
VIIb
3.62 ± 0.04
4.85 ± 0.05
Sorafenib
4
.1.3.1. N-(4-(5-(3-fluorophenyl)isoxazol-3-yl)phenyl)-4-methyl-
◦
ꢀ 1
benzenesulfonamide (VIa). Yield 69%, mp. 136–138 C. IR (KBr, cm ):
3
225 (NH), 3110 (CH-arom.). MS: m/z = 408; Anal. For C22
Calcd. C, 64.69; H, 4.20; N, 6.86; Found: C, 64.64; H, 4.25; N, 6.80. H
NMR (DMSOd ): δ 2.35 (3H, s, CH
12H, m, Ar-H), 10.55 (1H, s, NH, D
DMSOd ) δ 20, 98, 114, 115, 116, 118, 121, 124, 125, 126, 128, 129,
36, 137, 138, 162, 164, 170.
2 3
H17FN O S;
1
6
3
), 6.68 (1H, s, isoxazole), 7.17–7.89
(
(
2
O exchangeable). 13C NMR
6
1
4
.1.3.2. 4-methyl-N-(4-(5-(m-tolyl)isoxazol-3-yl)phenyl)benzenesulfona-
◦
ꢀ 1
mide (VIb). Yield 69%, mp. 120–122 C. IR (KBr, cm ): 3225 (NH),
3
110 (CH-arom.). MS: m/z = 404; Anal. For C23
8.30; H, 4.98; N, 6.93; Found: C, 68.37; H, 4.94; N, 6.90. H NMR
): δ 2.27 (3H, s, CH ), 2.34 (3H, s, CH ), 6.67 (1H, s, isoxazole),
.34–7.88 (12H, m, Ar-H), 10.02 (1H, s, NH, D
O exchangeable). 13
) δ 21, 22, 100, 112, 118, 122, 126, 128, 129, 130, 131,
33, 134, 135, 136, 137, 138, 165, 172.
20 2 3
H N O S; Calcd. C,
1
6
(
DMSOd
6
3
3
Fig. 5. DNA fragmentation of UO-31 cells after incubation with VIIb.
7
2
C
NMR (DMSOd
6
ethanol for 12 h. Then cooled, poured onto crushed ice, filter and
crystallized from ethanol.
1
4
.1.4. Synthesis of compounds VIIa-c
4
4
.1.4.1. N-(4-(6-amino-5-cyano-4-(2-fluorophenyl)pyridin-2-yl)phenyl)-
-methylbenzenesulfonamide (VIIa). Yield 65%, mp. 199–201 ◦C. IR
A mixture of chalcone IV a-c (0.002 mol), malononitrile (0.002 mol),
and anhydrous ammonium acetate (0.016 mol) was refluxed in 25 ml
8

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Table 3
C
26
H
22
N
4
O
2
S; Calcd. C, 68.70; H, 4.88; N, 12.33; Found: C, 68.64; H,
1
Effect of compound VIIb on cell cycle progression in UO-31 cells.
4.83; N, 12.30. H NMR (DMSOd
): δ 2.18 (3H, s, CH
H), 9.42 (2H, s, NH, D O exchangeable),
O exchangeable). C NMR (DMSOd ) δ 21, 23, 86,
12, 113, 117, 124, 125, 126, 127, 128, 129, 130, 131, 135, 136, 138,
139, 142, 149, 156, 161.
3
), 2.32 (3H, s,
6
CH
9.93 (1H, s, NH, D
3
), 7.22–8.15 (13H, m, Ar
–
2
%
G0-
%S
%G2-
M
%Pre-
G1
Comment
1
3
G1
2
6
1
VIIb/ UO-
26.29
24.03
32.47
49.68
17.22
17.75
1.92
Cell growth
3
1
arrest@G2/M
Cont. (UO-
1)
50.31
3
4
.1.5. Synthesis of compounds VIIIa-c
A mixture of chalcone IV a-c (0.002 mol), ethyl cyanoacetate (0.002
mol), and anhydrous ammonium acetate (0.016 mol) was refluxed in 25
ml butanol for 10 h. Then cooled, filter and crystallized from ethanol.
4
.1.5.1. N-(4-(5-cyano-4-(2-fluorophenyl)-6-oxo-1,6-dihydropyridin-2-
yl)phenyl)-4-methylbenzenesulfonamide
(VIIIa). Yield
74%,
mp.
16–118 ◦C. IR (KBr, cm ): 3242 (NH), 3233(NH), 3105 (CH-arom.),
ꢀ 1
1
1
3
2
695 (CO). MS: m/z = 459; Anal. For C25
.95; N, 9.15; Found: C, 65.30; H, 3.91; N, 9.17. H NMR (DMSOd
.33 (3H, s, CH
), 6.76 (1H, s, dihydropyridine), 6.93–7.72 (12H, m,
H), 9.12, 10.23 (2H, 2 s, 2NH, D
DMSOd ) δ 24, 104, 109, 113, 114, 115, 120, 121, 124, 125, 126, 127,
28, 130, 132, 134, 135, 158, 160, 164, 170.
3 3
H18FN O S; Calcd. C, 65.35; H,
1
6
): δ
3
1
3
Ar
–
2
O exchangeable). C NMR
(
6
1
4
.1.5.2. N-(4-(5-cyano-4-(3-fluorophenyl)-6-oxo-1,6-dihydropyridin-2-
yl)phenyl)-4-methylbenzenesulfonamide
128–130 ◦C. IR (KBr, cm ): 3240 (NH), 3238(NH), 3107(CH-arom.),
(VIIIb). Yield
61%,
mp.
ꢀ 1
Fig. 6. The apoptotic influence of compound VIIb on the distribution of the cell
cycle of UO-31 cell line.
1696 (CO). MS: m/z = 459; Anal. For C25
H, 3.95; N, 9.15; Found: C, 65.32; H, 3.90; N, 9.19. H NMR (DMSOd
.25 (3H, s, CH
), 6.44 (1H, s, dihydropyridine), 6.93–7.71 (12H, m,
Ar H), 9.06, 10.19 (2H, 2 s, 2NH, D
DMSOd ) δ 21, 104, 110, 111, 112, 113, 116, 119, 123, 127, 128, 129,
32, 133, 134, 135, 137, 157, 158, 159, 164.
3 3
H18FN O S; Calcd. C, 65.35;
1
6
): δ
(
KBr, cm 1): 3250–3232 (NH, NH
ꢀ
2
), 3100 (CH-arom.). MS: m/z = 458;
2
3
1
3
–
2
O exchangeable). C NMR
Anal. For C25
H
19FN
5.54; H, 4.14; N, 12.27. H NMR (DMSOd
.35–8.28 (13H, m, Ar H), 9.57 (2H, s, NH, D
O exchangeable). 13C NMR (DMSOd
6
4 2
O S; Calcd. C, 65.49; H, 4.18; N, 12.22; Found: C,
1
(
6
6
7
6
): δ 2.37 (3H, s, CH
O exchangeable), 10.65
) δ 25, 85, 110, 111,
3
),
–
1
2
(
1H, s, NH, D
2
1
1
14, 122, 123, 124, 126, 129, 130, 132, 133, 134, 135, 136, 154, 155,
59, 161.
4
4
.1.4.2. N-(4-(6-amino-5-cyano-4-(3-fluorophenyl)pyridin-2-yl)phenyl)-
◦
-methylbenzenesulfonamide (VIIb). Yield 71%, mp. 178–180 C. IR
ꢀ 1
(
KBr, cm ): 3248–3235 (NH, NH ), 3110 (CH-arom.). MS: m/z = 458;
2
Anal. For C25
H
19FN
5.53; H, 4.22; N, 12.18. H NMR (DMSOd
.24–8.26 (13H, m, Ar H), 9.34 (2H, s, NH, D
O exchangeable). 13C NMR (DMSOd
6
4 2
O S; Calcd. C, 65.49; H, 4.18; N, 12.22; Found: C,
1
6
7
6
): δ 2.30 (3H, s, CH
O exchangeable), 10.35
) δ 20, 90, 113, 114,
3
),
–
2
(
1H, s, NH, D
2
1
1
15, 116, 117, 123, 125, 127, 128, 129, 130, 135, 136, 137, 141, 151,
54, 160, 162.
4
.1.4.3. N-(4-(6-amino-5-cyano-4-(m-tolyl)pyridin-2-yl)phenyl)-4-meth-
◦
ꢀ 1
ylbenzenesulfonamide(VIIc). Yield 65%, mp. 193–195 C. IR (KBr, cm ):
251, 3225 (NH, NH
), 3110 (CH-arom.). MS: m/z = 454; Anal. For
3
2
Fig. 8. Apoptosis and necrosis impact of compound VIIb.
Fig. 7. Impact of compound VIIb on the cell cycle process in UO-31 cells.
9

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 9. The effect of compound 4b on binding to annexin-V.
1
H, 4.65; N, 9.22; Found: C, 68.59; H, 4.60; N, 9.26. H NMR (DMSOd
6
): δ
Table 4
2
.24 (3H, s, CH
3
), 2.30 (3H, s, CH
3
), 6.53 (1H, s, dihydropyridine),
Percentage of apoptotic and necrotic cells in UO-31 treated and control cells.
6
.92–7.72 (12H, m, Ar
–
2
H), 9.21, 10.65 (2H, 2 s, 2NH, D O exchange-
Apoptosis
Total
Necrosis
13
able). C NMR (DMSOd
6
) δ 20, 23, 100, 114, 115, 116, 117, 119, 122,
Early
Late
123, 124, 125, 126, 127, 128, 132, 136, 137, 138, 153, 159, 163.
VIIb/ UO-31
17.75
1.92
7.14
1.26
9.08
0.25
1.53
0.41
Cont. (UO-31)
4
4
.2. Biology
.2.1. Cancer cell line screening at the NCI
Table 5
The NCI-USA anticancer screening was performed following the
Impact of compound VIIb on p53, BAX and Bcl-2 protein expression levels in UO-
standard protocol, using the SRB protein assay [59–62].
3
1 cells.
Comp.
BAX conc.pg
FLD
P53 conc.
FLD
Bcl-2 Conc. pg
FLD
4
.2.2. In vitro VEGFR-2 kinase assay
VIIb
380.66
51.47
7.4
1
471.27
52.49
8.97
1
2.14
5.7
0.38
1
Human VEGFR-2 ELISA (Enzyme-Linked Immunosorbent Assay) kit
Cont.
was used to determine inhibitory activity against VEGFR-2. A specific
VEGFR-2 antibody was seeded on a 96-well plate, and 100 L of the
standard solution or the compound examined was applied, both incu-
bated at room temperature for 2.5 h. Washed, adding 100 L of the
prepared biotin antibody, then incubated for an hour at room temper-
ature. Washed, 100 L of streptavidin solution was applied at room
temperature, then incubated for 45 min. Washed again, added, and
μ
4
.1.5.3. N-(4-(5-cyano-6-oxo-4-(m-tolyl)-1,6-dihydropyridin-2-yl)
μ
phenyl)-4-methylbenzenesulfonamide
(VIIIc). Yield
70%,
mp.
12–215 ◦C. IR (KBr, cm ): 3235 (NH), 3232(NH), 3108(CH-arom.),
ꢀ 1
2
1
μ
693 (CO). MS: m/z = 455; Anal. For C26
21 3 3
H N O S; Calcd. C, 68.55;
Fig. 10. Caspase-3 activity in UO-31 cells after incubation with VIIb compared with untreated (control) cells.
Table 6
Docking of binding free energies of compound VIIb and reference drug (sorafenib) against VEGFR-2.
Comp.
Score
rmsd_refine
E_conf
E_place
E_score1
E_refine
E_score2
VIIb
ꢀ 27.09
ꢀ 26.16
1.51
1.68
6.82
ꢀ 75.73
ꢀ 66.51
ꢀ 22.07
ꢀ 20.46
ꢀ 7.73
ꢀ 27.09
ꢀ 26.16
Sorafenib
ꢀ 12.09
ꢀ 17.62
Score: lower scores are more favorable, rmsd_refine; the root mean square deviation of the pose from the docking pose compared to the co-crystal ligand position,
E_conf; free binding energy of the conformer, E_place; free binding energy from the placement stage, E_score 1; free binding energy from the first rescoring stage,
E_refine; free binding energy from the refinement stage, E_score 2; free binding energy from the second rescoring stage.
1
0

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 11. Superimpose of the co-crystallized pose (green) and the docking pose (maroon). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 12. 3D of the predicted sorafenib binding mode with VEGFR-2 active site.
Fig. 13. 2D of Sorafenib’s predicted binding mode with VEGFR-2 active site.
1
1

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 14. Surface mapping for sorafenib in the predicted binding mode with the VEGFR-2 active site.
Fig. 15. 3D of Compound VIIb’s predicted binding mode with VEGFR-2 active sites.
incubated 100
perature. 50
μ
L of TMB Substrate reagent for 30 min. at room tem-
4.2.5. Apoptosis assay
μ
L of the stop solution was applied and immediately read at
Apoptosis was measured by staining over Renal cancer cells UO-31
cells with Annexin V–fluorescein isothiocyanate (FITC) and propidium
iodide (PI) counterstaining. Briefly, 1 x105 cell/well were exposed to
4
50 nm.
4
.2.3. DNA fragmentation analysis
(0.1% DMSO) or GI50 concentration (1.32 M) of compound VIIb for 24
μ
The fragmentation of genomic DNA was assessed using the agarose
h. The cells were obtained by trypsinization and were washed with
phosphate buffered saline (PBS) and stained with Annexin V–FITC and
PI in 1 × binding buffer (BD Biosciences, San Jose, CA, USA) for fifteen
minutes at room temperature. Analysis was performed using flow cy-
tometer [65].
gel electrophoresis of DNA isolates obtained by the salting-out method
63]. UO-31cells were cultured in the presence or absence of compound
[
VIIb for this reason. Cells were then exposed to DNA extraction after
counting and washing. In the TE buffer, the DNA samples were resus-
pended carefully. The sample was placed onto 1,5 percent TAE agarose
gel; the laddering of DNA was visualized by ethidium bromide staining
on a UV transilluminator.
4.2.6. BAX, p35 and BCL-2 activity
These assays were performed as previously recorded [66,67].
4
.2.4. Cell cycle assay
4.2.7. Caspases-3
Flow cytometric analysis was conducted to assess the UO-31 cell
The caspase-3 activity was evaluated using DRG Caspase-3 (human)
ELISA (EIA-4860) kit (DRG International Inc., USA), according to the
manufacturer’s instructions.
cycle distributions after 24 h GI50 concentration (1.32 M) of compound
μ
VIIb was treated and the non - treated cells were used as controls. The
cells were fixed with ethanol and processed in compliance with the in-
structions [64].
4.3. Molecular docking
The VEGFR-2 crystal structure has been downloaded from the
1
2

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Fig. 16. 2D of the predicted compound VIIb mode binding with VEGFR-2 active sites.
Fig. 17. Surface mapping of the predicted compound VIIb binding mode with the active VEGFR-2 sites.
Protein Data Bank, http://www.rcsb.org/pdb (PDB ID: 2OH4, resolu-
tion: 2.05 Å). The molecular operating environment (MOE) has been
used for docking analysis [68]. In these tests, the free energies and
binding modes of the molecules measured against the target proteins
were determined. At first the water molecules were separated from the
crystal structures of the target proteins, keeping only one chain, which is
necessary for binding. Co-crystallized ligands (sorafenib) were used as a
reference ligand. The protein structure was then protonized and the
hydrogen atoms were hidden. The energy was then minimized, and the
binding pocket was defined.
running the docking process for only the co-crystal ligand. The low
RMSD value between docked and crystal conformations suggests a valid
performance [68]. The docking process was conducted using the stan-
dard protocol. In each case, 10 docked structures were generated using a
genetic algorithm. The output of MOE software was further analyzed
and visualized using Discovery Studio 4.0 software.
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.
The structure of the studied compound and the co-crystallized ligand
were drawn using ChemBioDraw Ultra 14.0 and stored as SDF formats.
Then the saved files were opened using MOE software and the 3D
structures were protonated. Next the energy of the molecules has been
reduced. Validation process was conducted for the target receptor by
1
3

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
Acknowledgement
[24] C.T. Supuran, A. Scozzafava, Carbonic anhydrase inhibitors and their therapeutic
potential, Exp. Opin. Ther. Patents 10 (2000) 575–600.
[
25] C.T. Supuran, A. Scozzafava, Carbonic anhydrase inhibitors, Curr. Med. Chem.
The authors are thankful to the National Cancer Institute (NCI), USA,
for performing the anticancer screening tests.
Immunol. Endocrinol. Metab. Agents 1 (2001) 61–97.
[26] K.L. Lobb, P.A. Hipskind, J.A. Aikins, E. Alvarez, Y. Cheung, E.L. Considine, A. De
Dios, G.L. Durst, R. Ferritto, C.S. Grossman, D.D. Giera, B.A. Hollister, Z. Huangn,P.
W. Iversen, K.L. Law, L.I. Tiechao, B. Ho-Shen Lin Lopez, J.E. Lopez, L.M.M.
Cabrejas, Denis J. McCann, V. Molero, J.E. Reilly, M.E. Richett, C. Shih, B. Teicher,
J.H. Wikel, W.T. White, M.M. Mader, Acyl sulfonamide anti-proliferatives: Benzene
substituent structureꢀ activity relationships for a novel class of antitumor agents,
J. Med. Chem. 47 (2004) 5367–5380.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104842.
[
27] J.C. Medina, D. Roche, B. Shan, R.M. Learned, W.P. Frankmoelle, D.L. Clark,
T. Rosen, J.C. Jaen, Novel halogenated sulfonamides inhibit the growth of
multidrug resistant MCF-7/ADR cancer cells, Bioorg. Med. Chem. Lett. 9 (1999)
References
1
843–1846.
[
28] T.H. Maren, Annu, Relations between structure and biological activity of
sulfonamides, Rev. Pharmacol. Toxicol. 16 (1976) 309–327.
[
[
[
1] H. Takahashi, M. Shibuya, The vascular endothelial growth factor (VEGF)/VEGF
receptor system and its role under physiological and pathological conditions, Clin.
Sci. 109 (2005) 227–241.
[
[
29] J. Drew, Drug discovery: a historical perspective, Science 287 (2000) 1960–1964.
30] A.E. Boyd, Sulfonylurea receptors, ion channels, and fruit flies, Diabetes 37 (1988)
2] H.F. Dvorak, L.F. Brown, M. Detmar, A.M. Dvorak, Vascular permeability factor/
vascular endothelial growth factor, microvascular hyperpermeability, and
angiogenesis, Am. J. Pathol. 146 (1995) 1029–1039.
8
47–850.
[
[
[
[
31] M.M. Ghorab, M.S. Alsaid, A.M. Soliman, F.A. Ragab, VEGFR-2 inhibitors and
apoptosis inducers: synthesis and molecular design of new benzo [g] quinazolin
bearing benzenesulfonamide moiety, J. Enzyme Inhib. Med. Chem. 32 (1) (2017)
3] N. Ferrara, H.P. Gerber, J. Le Couter, The biology of VEGF and its receptors, Nat.
Med. 9 (2003) 669–676.
8
93–907.
[
[
4] J. Folkman, Y. Shing, Angiogenesis, J. Biol. Chem. 267 (1992) 10931–10934.
5] M. Klagsbrun, P.A. D’Amore, Regulators of angiogenesis, Annu. Rev. Physiol. 53
32] A. Desroches-Castan, D. Qu ´e lard, M. Demeunynck, J.F. Constant, C. Dong,
M. Keramidas, J.L. Coll, C. Barette, L. Lafanech `e re, J.J. Feige, A new chemical
inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling
pathway upstream of Ras, Oncotarget 6 (7) (2015) 5382.
(
1991) 217–239.
[
[
6] J. Folkman, Tumors are angiogenesis dependent? J. Natl. Cancer Inst. 82 (1990)
–6.
4
33] M.M. Ghorab, M.S. Alsaid, Y.M. Nissan, A.E. Ashour, A.A. Al-Mishari, A. Kumar, S.
F. Ahmed, Novel sulfonamide derivatives carrying a biologically active 3, 4-
dimethoxyphenyl moiety as VEGFR-2 inhibitors, Chem. Pharm. Bull. 64 (12)
7] L.M. Strawn, G. McMahon, H. App, R. Schreck, W.R. Ku-chler, M.P. Longhi, T.
H. Hui, C. Tang, A. Levitzki, A. Gazit, I. Chen, G. Keri, L. Orfi, W. Risau, I. Flamme,
A. Ullrich, K.P. Hirth, L.K. Shawver, Flk-1 as a target for tumor growth inhibition,
Cancer Res. 56 (1996) 3540–3545.
(
2016) 1747–1754.
34] M.I. Shahin, D.A. Abou El Ella, N.S. Ismail, K.A. Abouzid, Design, synthesis and
biological evaluation of type-II VEGFR-2 inhibitors based on quinoxaline scaffold,
Bioorg. Chem. 56 (2014) 16–26.
[
[
8] S. Baka, A.R. Clamp, G.C. Jayson, A review of the latest clinical compounds to
inhibit VEGF in pathological angiogenesis, Expert Opin. Ther. Targets 10 (2006)
8
67–876.
[
[
35] M. Shibuya, Vascular endothelial growth factor Receptor-2: its unique signaling
and specific ligand, VEGF-E, Cancer Sci. 94 (9) (2003) 751–756.
9] T. Ahman, T. Eisen, Kinase inhibition with BAY 43–9006 in renal cell carcinoma,
Clin. Cancer Res. 10 (18) (2004) 6388S–6392S.
36] Y.S. Sun, Cancer Center, South China Institute of Botany, Chinese Academy of
Sciences assignee. Use of 20,40 dihydroxy-60-methoxy-30,50 dimethylchalcone for
preparing anticancer medicine, Patent CN1454895 A. (2003).
[
[
10] K. Podar, L.P. Catley, Y. Tai, R. Shringarpure, P. Carvalho, T. Hayashi, R. Burger, R.
L. Schlossman, P.G. Richardson, L.N. Pandite, R. Kumar, T. Hideshima,
D. Chauhan, K.C. Anderson, GW654652, the pan-inhibitor of VEGF receptors,
blocks the growth and migration of multiple myeloma cells in the bone marrow
microenvironment, Blood 103 (2004) 3474–3479.
[
[
[
37] S.U.F. Rizvi, H.L. Siddiqui, M. Nisar, N. Khan, I. Khan, Discovery and molecular
docking of quinolyl-thienyl chalcones as anti-angiogenic agents targetingmVEGFR-
2
tyrosine kinase, Bioorg. Med. Chem. Lett. 22 (2012) 942–944.
11] A. Morabito, E. De Maio, M. Di Maio, N. Normanno, F. Perrone, Tyrosine kinase
inhibitors of vascular endothelial growth factor receptors in clinical trials: current
status and future directions, Oncologist 11 (2006) 753–764.
38] N. Mainolfi, R. Karki, F. Liu, K. Anderson, Evolution of a new class of VEGFR-2
inhibitors from scaffold morphing and redesign, ACS Med. Chem. Lett. 7 (2016)
3
63–367.
[
[
12] P. Wu, T.E. Nielsen, M.H. Clausen, FDA-approved small-molecule kinase inhibitors,
Trends Pharmacol. Sci. 36 (2015) 422–439.
39] N. Miyamoto, N. Sakai, T. Hirayama, K. Miwa, Y. Oguro, H. Oki, K. Okada,
T. Takagi, H. Iwata, Y. Awazu, S. Yamasaki, Discovery of N-[5-({2-
13] S. Wilhelm, C. Carter, M. Lynch, T. Lowinger, J. Dumas, R.A. Smith, B. Schwartz,
R. Simantov, S. Kelley, Discovery and development of sorafenib: a multikinase
inhibitor for treating cancer, Nat. Rev. Drug Discov. 5 (2006) 835–844.
14] A. Pircher, W. Hilbe, I. Heidegger, J. Drevs, A. Tichelli, M. Medinger, Biomarkers in
tumor angiogenesis and anti-angiogenic therapy, Int. J. Mol. Sci. 12 (2011)
[
(cyclopropylcarbonyl) amino] imidazo [1, 2-b] pyridazin-6-yl} oxy)-2-
methylphenyl]-1, 3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly
potent VEGFR2 kinase inhibitor, Bioorg. Med. Chem. 21 (2013) 2333–2345.
40] N. Mainolfi, R. Karki, F. Liu, K. Anderson, Evolution of a new class of VEGFR-2
inhibitors from scaffold morphing and redesign, ACS Med. Chem. Lett. 7 (4) (2016)
[
[
[
[
[
7
077–7099.
3
63–367.
15] Q.Q. Xie, H.Z. Xie, J.X. Ren, L.L. Li, S.Y. Yang, Pharmacophore modeling studies of
type I and type II kinase inhibitors of Tie2, J. Mol. Graph. Model. 27 (2009)
[
[
41] L. Huang, Z. Huang, Z. Bai, R. Xie, L. Sun, K. Lin, Development and strategies of
VEGFR-2/KDR inhibitors, Future Med. Chem. 4 (14) (2012) 1839–1852.
42] R. Tumma, S. Siliveri, H.B. Vamaraju, M.R. Bommineni, Synthesis, biological
evaluation and molecular docking studies of isoxazole synchronized quinazolinone
derivatives, J. Pharm. Res. 11 (2017) 895–902.
7
51–758.
16] K. Lee, K.W. Jeong, Y. Lee, J.Y. Song, M.S. Kim, G.S. Lee, Y. Kim, Pharmacophore
modeling and virtual screening studies for new VEGFR-2 kinase inhibitors, Eur. J.
Med. Chem. 45 (2010) 5420–5427.
[
43] W. Gu, Y. Dai, H. Qiang, W. Shi, C. Liao, F. Zhao, W. Huang, H. Qian, Discovery of
novel 2-substituted-4-(2-fluorophenoxy) pyridine derivatives possessing
pyrazolone and triazole moieties as dual c-Met/VEGFR-2 receptor tyrosine kinase
inhibitors, Bioorg. Chem. 72 (2017) 116–122.
17] R.N. Eskander, K.S. Tewari, Gynecol, Incorporation of anti-angiogenesis therapy in
the management of advanced ovarian carcinoma—mechanistics, review of phase
III randomized clinical trials, and regulatory implications, Oncol. 132 (2014)
4
96–505.
[
[
[
44] A. Balupuri, P.K. Balasubramanian, S.J. Cho, 3D-QSAR study of tetrahydro-3H-
imidazo [4, 5-c] pyridine derivatives as VEGFR-2 kinase inhibitors using various
charge schemes, Arch. Pharm. 38 (8) (2015) 1434–1442.
[
[
18] V.A. Machado, D. Peixoto, R. Costa, H.J. Froufe, R.C. Calhelha, R.M. Abreu, I.
C. Ferreira, R. Soares, M.J.R. Queiroz, Synthesis, antiangiogenesis evaluation and
molecular docking studies of 1-aryl-3-[(thieno [3, 2-b] pyridin-7-ylthio) phenyl]
ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase
inhibitors, Bioorg. Med. Chem. 23 (2015) 26497–26509.
45] A.S. Kiselyov, M. Semenova, V.V. Semenov, D. Milligan, Inhibitors of VEGF
receptors-1 and-2 based on the 2-((pyridin-4-yl) ethyl) pyridine template, Bioorg.
Med. Chem. Lett. 16 (7) (2006) 1913–1919.
19] J. Dietrich, C. Hulme, L.H. Hurley, The design, synthesis, and evaluation of 8
hybrid DFG-out allosteric kinase inhibitors: a structural analysis of the binding
interactions of Gleevec®, Nexavar®, and BIRB-796, Bioorg. Med. Chem. 18 (2010)
46] Y. Oguro, D.R. Cary, N. Miyamoto, M. Tawada, H. Iwata, H. Miki, A. Hori,
S. Imamura, Design, synthesis, and evaluation of novel VEGFR2 kinase inhibitors:
discovery of [1, 2, 4] triazolo [1, 5-a] pyridine derivatives with slow dissociation
kinetics, Bioorg. Med. Chem. 21 (15) (2013) 4714–4729.
5
738–5748.
[
[
20] A. Garofalo, L. Goossens, P. Six, A. Lemoine, S. Ravez, A. Farce, P. Depreux, Impact
of aryloxy-linked quinazolines: a novel series of selective VEGFR-2 receptor
tyrosine kinase inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 2106–2112.
21] M.A. Aziz, R.A. Serya, D.S. Lasheen, A.K. Abdel-Aziz, A. Esmat, A.M. Mansour, A.N.
B. Singab, K.A. Abouzid, Discovery of potent VEGFR-2 inhibitors based on
furopyrimidine and thienopyrimidne scaffolds as cancer targeting agents, Sci. Rep.
[
[
[
47] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E. J. Barreiro, C.A.M. Fraga,
Curr. Med. Chem. 14 (2007) 1829–1852.
48] A.H. Wyllie, Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation, Nature 284 (5756) (1980) 555–556.
49] H.M. Alkahtani, M.M. Alanazi, F.S. Aleanizy, F.Y. Alqahtani, A. Alhoshani, F.
E. Alanazi, A.A. Almehizia, A.N. Abdalla, M.G. Alanazi, A.S. El-Azab, A.A. Abdel-
Aziz, Synthesis, anticancer, apoptosis-inducing activities and EGFR and VEGFR2
assay mechanistic studies of 5, 5-diphenylimidazolidine-2, 4-dione derivatives:
Molecular docking studies, Saudi Pharm. J. 27 (5) (2019) 682–693.
50] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, A novel assay for
apoptosis flow cytometric detection of phosphatidylserine expression on early
apoptotic cells using fluorescein labelled annexin V, J. Immunol. Methods 184
6
(2016) 24460.
[
[
22] C.T. Supuran, A. Casini, A. Scozzafava, A. Mastrolorenzo, Sulfonamides and
sulfonylated derivatives as anticancer agents, Curr. Cancer Drug Targets 2 (2002)
5
5–75.
[
23] T. Owa, T. Nagasu, Novel sulphonamide derivatives for the treatment of cancer,
Exp. Opin. Ther. Patents 10 (2000) 1725–1740.
(
1995) 39–51.
1
4

M.F. Ahmed and E.Y. Santali
Bioorganic Chemistry 111 (2021) 104842
[
[
[
51] B. Vogelstein, D. Lane, A.J. Levine, Surfing the p53 network, Nature 408 (6810)
anticancer drug screen using a diverse panel of cultured human tumor cell lines,
J. Natl. Cancer Inst. 83 (1991) 757–766.
(
2000) 307–310.
52] J.J. Fuster, S.M. Sanz-Gonz ´a lez, U.M. Moll, V. Andr ´e s, Classic and novel roles of
p53: prospects for anticancer therapy, Trends Mol. Med. 13 (5) (2007) 192–199.
53] J.J. Leu, P. Dumont, M. Hafey, M.E. Murphy, D.L. George, Mitochondrial p53
activates Bak and causes disruption of a Bak–Mcl1 complex, Nat. Cell Biol. 6 (5)
[61] B.A. Teicher, (Ed.), Anticancer drug development guide: preclinical screening,
clinical trials, and approval. Springer Science & Business Media, 2013.
[62] M.R. Boyd, K.D. Paull, Some practical considerations and applications of the
National Cancer Institute in vitro anticancer drug discovery screen, Drug Dev. Res.
34 (1995) 91–109.
(
2004) 443–450.
[
[
54] J.E. Chipuk, T. Kuwana, L. Bouchier-Hayes, N.M. Droin, D.D. Newmeyer,
M. Schuler, D.R. Green, Direct activation of Bax by p53 mediates mitochondrial
membrane permeabilization and apoptosis, Science 303 (5660) (2004) 1010–1014.
55] D. Chauhan, T. Hideshima, S. Rosen, J.C. Reed, S. Kharbanda, K.C. Anderson, Apaf-
[63] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting
DNA from human nucleated cells, Nucleic Acids Res. 16 (1988) 1215.
[64] A. Sabt, O.M. Abdelhafez, R.S. El-Haggar, H.M. Madkour W.M., Eldehna, E.E.D.A.
El-Khrisy, M.A. Abdel-Rahman, L.A. Rashed, L.A., Novel coumarin- 6-sulfonamides
as apoptotic anti-proliferative agents: Synthesis, in vitro biological evaluation, and
QSAR studies. J. Enzyme Inhib. Med. Chem. 33 (2018) 1095–1107.
[65] K.K. Lo, T.K. Lee, J.S. Lau, W.L. Poon, S.H. Cheng, Luminescent biological probes
derived from ruthenium (II) estradiol polypyridine complexes, Inorg. Chem. 47
(2008) 200–208.
1
/cytochrome c-independent and Smac-dependent inductionof apoptosis in
multiple myeloma (MM) cells, J. Biol. Chem. 276 (27) (2001) 24453–24456.
56] Y. Liu, N.S. Gray, Rational design of inhibitors that bind to inactive kinase
conformations, Nat. Chem. Biol. 2 (7) (2006) 358–364.
[
[
57] J. Dietrich, C. Hulme, L.H. Hurley, The design, synthesis, and evaluation of 8
hybrid DFG-out allosteric kinase inhibitors: A structural analysis of the binding
interactions of Gleevec®, Nexavar®, and BIRB-796, Bioorg. Med. Chem. 18 (15)
[66] E.F. Abdelhaleem, M.K. Abdelhameid, A.E. Kassab, M.M. Kandeel, Design and
synthesis of thienopyrimidine urea derivatives with potential cytotoxic and
proapoptotic activity against breast cancer cell line MCF-7, Eur. J. Med. Chem. 143
(2018) 1807–1825.
(
2010) 5738–5748.
[
58] M.M. Ghorab, F.A. Ragab, H.I. Heiba, M.S. Elsayed, W.M. Ghorab, Design,
synthesis and molecular modeling study of certain 4-Methylbenzenesulfonamides
with CDK2 inhibitory activity as anticancer and radio-sensitizing agents, Bioorg.
Chem. 80 (2018) 276–287.
[67] M.F. Ahmed, E.Y. Santali, E.M.M. El-Deen, I.A. Naguib, R. El-Haggar, Development
of pyridazine derivatives as potential EGFR inhibitors and apoptosis inducers:
design, synthesis, anticancer evaluation, and molecular modeling studies, Bioorg.
Chem. 106 (2021), 104473.
[68] K.M. El-Gamal, A.M. El-Morsy, A.M. Saad, I.H. Eissa, M. Alswah, Synthesis,
docking, QSAR, ADMET and antimicrobial evaluation of new quinoline-3-
carbonitrile derivatives as potential DNA-gyrase inhibitors, J. Mol. Struct. 1166
(2018) 15–33.
[
[
59] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren,
H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for
anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107–1112.
60] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, Feasibility of a high-flux
1
5