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tmax./cmꢁ1 3450 (NH), 3040 (C–H aromatic), 1720 (C═O), 1590 (C═ H), and 8.5 (s, NH, D2O exchangeable). MS: m/z (% relative
C). 13C NMR (DMSO-d6): 187, 156, 146, 144, 139, 136, 133, 131, intensity)¼354 (Mþ, 100%).
128, 126, 118, 113, 53, 45. 1H NMR: d 2.3 (s, 1H, NH, D2O
exchangeable), 2.7–3.4 (m, 8H, piperazinyl protons), 6.7 (d, 1H,
3.3.3. 1-(4-(5-(3-Fluoropyridin-4-yl)-4,5-dihydro-1H-pyrazol-3-yl)
J¼ 15.0 Hz, CH═CH), and 6.9–7.8 (m, 8H, Ar-H, CH═). MS: m/z (%
relative intensity)¼311 (Mþ, 100%).
phenyl) piperazine (VIc)
Yield 70%, m.p. 135 ꢀC. Analysis calculated C18H20FN5; calcd.: % C
66.44; H, 6.20; N, 21.52; found: % C 66.49; H, 6.25; N, 21.548
IR: tmax./cmꢁ1 3390 (NH), 3020 (C–H aromatic), 1595 (C═ C). 13C
3.2.7. 3-(2-Hydroxy-5-nitrophenyl)-1-(4-(piperazin-1-yl)phenyl)prop-
2-en-1-one (Vg)
NMR (DMSO-d6): 152, 150, 147, 139, 138, 133, 130, 125, 124, 111,
Yield 65%, m.p. 185 ꢀC. Analysis calculated C19H19N3O4; calcd.: % C 54, 47, 45, 44. 1H NMR: d 2.3 (s, 1H, NH, D2O exchangeable),
64.58; H, 5.42; N, 11.89; found: % C, 64.50; H, 5.37; N, 11.83. IR: 2.7–3.2 (m, 8H, piperazinyl protons), 3.4 (dd, 1H, J ¼ 11.2, 5.0 Hz,
tmax./cmꢁ1 3400 (NH), 3320 (OH), 3010 (C–H aromatic), 1700 (C═ pyrazoline), 3.6 (dd, 1H, J¼ 11.6, 5.9 Hz, pyrazoline), 5.2 (dd, 1H,
O), 1590 (C═C). 13C NMR (DMSO-d6): 195, 163, 141, 140, 139, 131, J¼ 12.0, 5.7 Hz, pyrazoline), 7.0–7.9 (m, 7H, Ar-H), and 8.8 (s, NH,
129, 126, 125, 120, 119, 116, 112, 54, 45. H NMR: d 2.0 (s, 1H, NH, D2O exchangeable). MS: m/z (% relative intensity)¼325 (Mþ, 19%),
1
D2O exchangeable), 2.4–3.1 (m, 8H, piperazinyl protons), 6.6 (d, 229 (100%).
1H, J¼ 15.2 Hz, CH═), 7.3–7.9 (m, 8H, Ar-H, CH═), and 9.1 (s, 1H,
O-H). MS: m/z (% relative intensity)¼353 (Mþ, 10%), 290 (100%).
3.4. In vitro cytotoxicity
In vitro cytotoxicity was performed in NCI according to
3.2.8. 3-(Furan-2-yl)-1-(4-(piperazin-1-yl)phenyl)prop-2-en-1-one (Vh)
reported method61.
Yield 70%, m.p. 190 ꢀC. Analysis calculated C17H18N2O2; calcd.: % C
72.32; H, 6.43; N, 9.92; found: % C 72.37; H, 6.52; N, 9.90. IR: tmax.
/
cmꢁ1 3390 (NH), 3030 (C–H aromatic), 1700 (C═O), 1600 (C═C).
13C NMR (DMSO-d6): 188, 151, 140, 137, 130, 127, 126, 120, 114,
112, 110, 50, 45. 1H NMR: d 2.1 (s, 1H, NH, D2O exchangeable),
2.9–3.5 (m, 8H, piperazinyl protons), 6.7 (d, 1H, J¼ 15.5 Hz, CH═),
and 7.0–7.8 (m, 8H, Ar-H, CH═). MS: m/z (% relative intensity)¼282
(Mþ, 30%), 197 (100%).
3.5. VEGFR-2 inhibition assay
IC50s of Vd, Ve, Vf, VIa, VIb, and VIc compounds were evaluated
in vitro using colorimetric assay of human VEGFR-2 ELISA
R
(enzyme-linked immunosorbent assay) kits (HTScanV VEGF
Receptor 2 Kinase Assay Kit). It includes active VEGFR-2 kinase (a
biotinylated peptide substrate and a phospho-tyrosine antibody)
for detection of the phosphorylated form of the substrate peptide.
On a 96-well plate, a particular VEGFR-2 antibody was seeded and
100 mL of the normal solution or compound tested was applied,
incubated at room temperature for 2.5 h and washed.
Then, 100 mL of the prepared biotin antibody was added, incu-
bated for an additional 1 h at room temperature and washed.
Following, 100 mL of streptavidin solution was added at room tem-
perature, incubated for 45 min and then, 100 mL of TMB substrate
solution was applied and incubated at room temperature for
30 min. Finally, 50 mL stop solution was added and the absorption
was measured at 450 nm instantly. The standard curve, the X-axis
concentrations, and the Y-axis absorbance were drawn.
3.3. General method for the preparation of VIa–c
A mixture of the chalcone Va, Ve, and Vf (0.006 mol) and hydra-
zine hydrate (0.006 mol, 98%) in absolute ethanol (30 mL) was
heated for 12 h under reflux. The reaction was cooled, the formed
precipitate was filtered off and crystallised from ethanol to give
compounds VIa–c, respectively.
3.3.1. 2-(3-(4-(Piperazin-1-yl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)
benzonitrile (VIa)
Yield 64%, m.p. 165 ꢀC. Analysis calculated C20H21N5; calcd.: % C
72.48; H, 6.39; N, 21.13; found: % C 72.54; H, 6.42; N, 21.10. IR:
tmax./cmꢁ1 3320 (NH), 3030 (C–H aromatic), 2220 (CN), 1600 (C═
C). 13C NMR (DMSO-d6): 151, 146, 134, 133, 131, 129, 128, 127,
125, 115, 112, 111, 54, 47, 45, 42. 1H NMR: d 2.1 (s, 1H, NH, D2O
exchangeable), 2.4–2.9 (m, 8H, piperazinyl protons), 3.3 (dd, 1H,
J¼ 11.4, 5.1 Hz, pyrazoline), 3.4 (dd, 1H, J¼ 11.3, 6.1 Hz, pyrazo-
line), 5.0 (dd, 1H, J ¼ 11.0, 5.5 Hz, pyrazoline), 7.0–7.7 (m, 8H, Ar-H),
and 9.0 (s, NH, D2O exchangeable). MS: m/z (% relative
intensity)¼331 (Mþ, 14%), 77 (100%).
3.6. Cell cycle analysis
HCT-116 cells were seeded at concentrations of 1 ꢂ 105 cells per
well in a six-well plate, then incubated for 24 h. The cells were
treated for 24 h with vehicles (0.1% DMSO) or 10 mM of Vd or Ve
compounds. Using ice-cold, 70% ethanol at 4 ꢀC, cells were har-
vested and fixed for 12 h after that. Ethanol removal and cold PBS
washing of the cells were done. Then incubated in 0.5 mL of PBS
containing 1 mg/mL Ranse for 30 min at 37 ꢀC. In the dark, the
cells were stained with PI for 30 min. Flow cytometer was then
used to detect contents of DNA62.
3.3.2. 1-(4-(5-(4-Fluoro-3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-
3-yl)phenyl) piperazine (VIb)
Yield 70%, m.p. 195 ꢀC. Analysis calculated C20H23FN4O; calcd.: % C
67.78; H, 6.54; N, 15.81; found: % C 67.70; H, 6.59; N, 15.74. IR:
3.7. Annexin V-FITC apoptosis assay
tmax./cmꢁ1 3400 (NH), 3040 (C–H aromatic), 1590 (C═C). 13C NMR For this study, annexin V-FITC/PI apoptosis detection kit was used;
(DMSO-d6): 152, 150, 148, 140, 133, 130, 125, 120, 116, 112, 111, HCT-116 cells were stained with annexin V fluorescein isothiocyan-
56, 52, 50, 46, 43. 1H NMR: d 2.3 (s, 1H, NH, D2O exchangeable), ate (FITC) and PI counter-stained. 1 ꢂ 105 HCT-116 cells were 48 h
2.6–3.0 (m, 8H, piperazinyl protons), 3.2 (dd, 1H, J¼ 11.3, 6.1 Hz, incubated with compound Vd or Ve, trypsinised, washed with
pyrazoline), 3.3 (dd, 1H, J¼ 11.7, 5.9 Hz, pyrazoline), 3.8 (s, 3H, phosphate-buffered saline (PBS), stained in the dark at 37 ꢀC for
OCH3), 5.1 (dd, 1H, J¼ 11.9, 6.1 Hz, pyrazoline), 6.9–7.5 (m, 7H, Ar- 15 min. Then, analysed with a cytometer of FACS calibre flow63.