G.H. Al-Ansary et al.
Bioorganic Chemistry 107 (2021) 104640
50.63; H, 3.55; N, 17.35%.
and their viability was assessed using MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay (Stock No.
TOX-1, Sigma-Aldrich, Saint louis, Missouri, USA), according to the in-
structions provided by the manufacturer and as previously described
[28,29]. Optical density was measured at 570 nm. Represented data are
the mean of the three individual experiments.
4.1.2.7. 4-(3-(3,4-Dichlorophenyl)ureido)-N-(pyrimidin-2-yl)benzene-
sulfonamide (3g). Yellow powder, yield (79%), mp 253–255 ◦C; IR (KBr)
max/cmꢀ 1: 3421 (NH), 3352 (NH), 3209 (NH), 1691 (C O), 1324
–
ν
–
(SO2 Asym), 1157 (SO2 Sym); 1H NMR (400 MHz, DMSO‑d6): δppm = 6.55
(d, 2H, J = 8 Hz, CHAr), 7.00 (br s, 1H, pyrimidine-H5), 7.33 (d, 1H, J =
8 Hz, CHAr), 7.51 (d, 1H, J = 8 Hz, CHAr), 7.60 (d, 2H, J = 8 Hz, CHAr),
7.86 (s, 1H, CHAr), 8.48 (d, 2H, J = 4 Hz, pyrimidine-H4 and H6), 9.72
(br s, 2H, NH urea), 11.29 (br s, 1H, NH); 13C NMR (125 MHz,
DMSO‑d6): δppm = 112.6 (Pyrimidine-C5), 115.9, 117.4, 118.4, 119.4,
125.2, 129.5, 130.2, 131.5, 140.2, 144.0 (12 Ar-C), 152.6, 153.5, 157.7,
158.7 (3 Pyrimidine-C and CO); LC-ESI-Ms m/z: 436 [Mꢀ H]+; Anal.
Calcd. for C17H13Cl2N5O3S (437.01): C, 46.59; H, 2.99; N, 15.98%,
Found: C, 46.63; H, 3.06; N, 15.92%.
4.2.1.2. In vitro scratch-wound healing assay (HUVEC migration). The
wound-healing assay was performed using the CytoSelect™ 24-Well
Wound Healing Assay kit (Cell Biolabs, San Diego, CA, USA) accord-
ing to the manufacturer’s protocol. HUVEC cells (3 × 104) were seeded
in a 24-well chamber and cultured until a monolayer was formed. The
cells were treated with different concentrations of sorafenib or the target
compounds 3a-l (1 µM, 5 µM or 10 µM) or control (0.1% DMSO), in the
presence of 10 ng/mL VEGF, for 24 h and monitored for migration into
the wound field. Images of the scratches were taken immediately after
performing the scratch (0 h) and at 24 h. The area not covered by the
cells was quantified using Image J software.
4.1.2.8. 4-(3-(4-Chloro-3-trifluoromethyl-phenyl)ureido)-N-(pyrimidin-2-
yl)benzene-sulfonamide (3h). White powder, yield (84%), mp 269–270
◦C; IR (KBr)
ν
max/cmꢀ 1: 3367 (NH), 3340 (NH), 3102 (NH), 1724
(C O), 1318 (SO Asym), 1156 (SO2 Sym); 1H NMR (400 MHz,
4.2.1.3. Western blotting. HUVECs were cultured in 6-well plates at 10
x104 cells/well and cultured for 24 h. After 48 h, they were serum-
starved (0.1% serum) for 24 h to inhibit cell proliferation. Cells were
then pretreated with or without the target compounds 3a-l for 1 h,
followed by stimulation with 10 ng/mL of VEGF for 15 min (for VEGFR2
activation). Cells were washed twice with ice-cold PBS and lysed using
100 mL radioimmunoprecipitation assay (RIPA) lysis buffer with 1%
protease inhibitor and phosphatase inhibitor. The amount of protein
present in the cell lysate was assessed using the DC (Bio-Rad) protein
assay developed from Lowry’s protocol for protein assessment. Proteins
(15 mg) were separated by SDS-PAGE and then transferred to PVDF
membranes. Membranes were blocked with 3% fat-free dry milk in PBT
buffer and then probed for PTyr-1175-VEGFR2 (1:1000) or total
VEGFR2 (1:1000) and actin dilution. Protein bands were detected by
incubating with horse radish peroxidase-conjugated antibodies
(1:10,000) and visualized with enhanced chemiluminescence reagent
using Image Quant LAS4000 (GE Healthcare). Gel bands were stan-
dardized to total protein loading relative to actin by using a Gel-Pro
analyzer6.0 software [28].
–
–
2
DMSO‑d6): δppm = 7.04 (t, 1H, J = 4 Hz, pyrimidine-H5), 7.59–7.65 (m,
4H, CHAr), 7.90 (d, 2H, J = 8 Hz, CHAr), 8.11 (s, 1H, CHAr), 8.50 (d, 2H,
J = 4 Hz, pyrimidine-H4, pyrimidine-H6), 9.28 (s, 1H, NH urea), 9.29 (s,
1H, NH urea), 11.64 (br s, 1H, NH); 13C NMR (125 MHz, DMSO‑d6):
δppm = 112.6 (Pyrimidine-C5), 115.9, 116.2, 118.1, 123.8, 127.3, 129.4,
130.2, 132.5, 133.4, 139.3, 143.9 (12 × Ar-C, CF3), 152.5, 157.4, 158.7,
158.8 (CO, 3 × Pyrimidine-C); LC-ESI-Ms m/z: 470 [Mꢀ H]+; Anal.
Calcd. for C18H13ClF3N5O3S (471.04): C, 45.82; H, 2.78; N, 14.84%,
Found: C, 45.87; H, 2.81; N, 14.90%.
4.1.2.9. N-(4,6-Dimethylpyrimidin-2-yl)-4-(3-(4-nitrophenyl)thioureido)
benzenesulfona-mide (3j). Yellow powder, yield (74%), mp 256–257 ◦C;
IR (KBr)
ν
max/cmꢀ 1: 3427 (NH), 3352 (NH), 3208 (NH), 1531 (NO2
Asym), 1345 (NO2 Sym), 1324 (SO2 Asym), 1157 (SO2 Sym), 1082
1
–
–
(C S); H NMR (400 MHz, DMSO‑d ): δ
= 2.11 (s, 6H, 2 × CH3),
6
ppm
6.27 (s, 1H, pyrimidine-H5), 7.57 (d, 2H, J = 8 Hz, CHAr), 7.76 (d, 2H, J
= 8 Hz, CHAr), 7.87 (d, 2H, J = 8 Hz, CHAr), 8.13 (d, 2H, J = 8 Hz, CHAr),
10.70 (br s, 2H, NH urea), 11.55 (br s, 1H, NH); LC-ESI-Ms m/z: 457
[Mꢀ H]+; Anal. Calcd. for C19H18N6O4S2 (458.08): C, 49.77; H, 3.96; N,
18.33%, Found: C, 49.83; H, 4.01; N, 18.35%.
4.2.1.4. In vitro cytotoxicity assessment. Cells were seeded at a density of
2 × 104 for MCF-7, HCT-116, CaCo-2, HepG2 cells and at a density of 3
× 104 for RPE1 cells into 96 well plates and incubated to allow the
attachment for 24 h. After 24 h, the cells were treated with doxorubicin
as positive control or the synthesized compounds 3a-l at concentration
ranges (0.8–50 µM) for 48 h. After the incubation period, cells were
washed with PBS and their viability was assessed using IN VITRO
TOXICOLOGY ASSAY KIT, MTT BASED (Stock No. TOX-1, Sigma-
Aldrich, Saint louis, Missouri, USA), according to the instructions pro-
vided by the manufacturer. The plates were read at 570 nm. Represented
data are the mean of the three individual experiments.
4.1.2.10. 4-(3-(3,4-Dichlorophenyl)ureido)-N-(4,6-dimethylpyrimidin-2-
yl)benzene-sulfonamide (3l). White powder, yield (82%), mp 262–263
◦C; IR (KBr)
ν
max/cmꢀ 1: 3354 (NH), 3293 (NH), 3264 (NH), 1723
(C O), 1315 (SO Asym), 1144 (SO2 Sym); 1H NMR (400 MHz,
–
–
2
DMSO‑d6): δppm = 2.05 (s, 6H, 2 × CH3), 6.17 (s, 1H, pyrimidine-H5),
7.16 (d, 1H, J = 8 Hz, CHAr), 7.43–7.50 (m, 3H, CHAr), 7.74 (d, 2H, J = 8
Hz, CHAr), 7.95 (s, 1H, CHAr), 10.14 (s, 1H, NH urea), 10.45 (s, 1H, NH
urea), 11.55 (s, 1H, NH); 13C NMR (125 MHz, DMSO‑d6): δppm = 23.7 (2
CH3), 108.9 (Pyrimidine-C5), 111.9, 116.9, 119.1, 120.0, 128.6, 128.8,
129.3, 129.8, 131.2, 132.6 (12 Ar-C), 150.5, 164.0, 164.2, 166.1 (CO, 3
Pyrimidine-C); LC-ESI-Ms m/z: 464 [Mꢀ H]+; Anal. Calcd. for
4.2.2. Cellular mechanism of action
C
19H17Cl2N5O3S (465.04): C, 48.93; H, 3.67; N, 15.02%, Found: C,
48.97; H, 3.71; N, 15.07%.
4.2.2.1. Measurement of apoptosis of 3e using annexin-V-FITC apoptosis
detection kit. The BioVision annexin V-FITC apoptosis detection kit was
used for the apoptosis assay (Cat No.: K101; BioVision, Inc., Milpitas,
CA, USA) according to the manufacturer’s instructions [29]. Briefly,
HepG2 cells were treated with compound 3e at its IC50 concentration
4.2. Biological evaluation
4.2.1. Antiangiogenic studies
(21.3
μM) for 48 h. The cells were harvested by trypsinization and
washed twice with phosphate-buffered saline (PBS) and stained with 5
4.2.1.1. In vitro human umbilical vein endothelial cells (HUVECs) viability
assay. HUVECs were seeded (2 × 105 cells/mL) in 96-well plate,
allowed to grow for 24 h and then incubated with different dilutions of
sorafenib and target compounds 3a-l at a concentration range between
μ
L Annexin V-FITC and 5 μL PI for 10 min at room temperature in the
dark. Cells were analyzed using a FACS Calibur flow cytometer (BD
Biosciences, San Jose, CA) using CELLQUEST software (Becton Dick-
inson Immunocytometry Systems, San Jose, CA).
0.39 and 100 μM or control (0.1% DMSO), in the presence of 10 ng/mL
VEGF, for 24 h. After the incubation period, cells were washed with PBS
9