JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
4-(3-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ben-
zenesulfonamide (13)
contained SDS (0.1%), respectively. Activities of CA I and II isoen-
zymes were determined according to the esterase method by
Verpoorte et al.26. The increase in absorbance of reaction medium
was spectrophotometrically recorded at 348 nm (UV–VIS
Spectrophotometer, Shimadzu, UVmini-1240, Kyoto, Japan). Also,
the quantity of protein was determined at 595 nm according to
Bradford method27. Bovine serum albumin was used as standard
protein. The IC50 values were obtained from activity (%) versus
compounds plots. For calculation of Ki values, three different con-
centrations were used. The Lineweaver–Burk curves were drawn
and calculations were realised28.
Mp 202–204 ꢀC. 3.03 g (87%). 1H NMR (400 MHz, DMSO-d6, ppm)
d ¼ 7.82 (dd, 2H, J ¼ 8.8, 5.5 Hz), 7.57 (d, 2H, J ¼ 9.1 Hz), 7.34–7.22
(m, 7H), 7.05 (d, 2H, J ¼ 9.1 Hz), 6.99 (s, 2H, NH2), 5.62 (dd, 1H,
J ¼ 12.1, 5.1 Hz), 3.95 (dd, 1H, J ¼ 17.9, 12.1 Hz), 3.17 (dd, 1H,
J ¼ 17.9, 5.1 Hz); 13C NMR (100 MHz, DMSO-d6, ppm) d ¼ 163.4 (d,
1J ¼ 247 Hz), 149.5, 146.6, 142.3, 133.8, 129.8, 129.1 (d, 4J ¼ 3 Hz),
129.0 (d, 3J ¼ 9 Hz), 128.3, 127.8, 126.4, 116.4 (d, 2J ¼ 21 Hz), 112.7,
63.2,
43.8;
HRMS
(ESI-MS):
calcd.
for
C21H19FN3O2S
[M þ H]þ 396.1177; found 396.1166.
Results and discussion
4-(3-(4-Bromophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ben-
zenesulfonamide (14)
The compounds designed were successfully synthesized. The
chemical structures of the compounds were confirmed by 1H
NMR, 13C NMR, and HRMS spectra. The CA inhibition effects of the
compounds were evaluated towards hCA I and hCA II isoenzymes.
The inhibition values are presented in Table 1. As shown in
Table 1, IC50 values were in the range of 402.9–554.8 nM towards
hCA I, while they were in the range of 458.6–620.4 nM towards
hCA II. The IC50 values of the reference compound AZA towards
hCA I and hCA II were 985.8 nM and 489.4 nM, respectively. All
compounds had lower IC50 value than AZA toward hCA I, while
the compounds 11, 14, and 16 had lower IC50 value than AZA
towards hCA II. According to IC50 values of the compounds, chlor-
ine-bearing compound 12 and bromine-bearing compound 14
were the most effective compounds towards hCA I while it was
hydroxy derivative 16 towards hCA II.
When Ki values of the compounds were considered, Ki
values of the compounds were in the range of
316.7 9.6–533.1 187.8 nM towards hCA I, while Ki values were
412.5 115.4–624.6 168.2 nM towards hCA II. The Ki values of ref-
erence compound AZA were 278.8 44.3 nM and 293.4 46.4 nM
towards hCA I and hCA II, respectively. When Ki values of the com-
pounds were considered, 12 with chlorine, 14 with bromine, and
15 with nitro substituents, which have very close Ki values, are the
leader compounds of series towards hCA I, while 14 was the
leader compound of the series towards hCA II because of the low-
est Ki values. All compounds were less effective than AZA on both
hCA I and II isoenzymes, since Ki value of the compound had
higher values than AZA’s.
Mp 190–192 ꢀC. 2.86 g (90%). 1H NMR (400 MHz, DMSO-d6, ppm)
d ¼ 7.71 (d, 2H, J ¼ 8.8 Hz), 7.62 (d, 2H, J ¼ 8.8 Hz), 7.57 (d, 2H,
J ¼ 9.0 Hz), 7.34–7.31 (m, 2H), 7.25–7.22 (m, 3H), 7.06 (d, 2H,
J ¼ 9.0 Hz), 7.01 (s, 2H, NH2), 5.63 (dd, 1H, J ¼ 12.2, 5.3 Hz), 3.94
(dd, 1H, J ¼ 17.8, 12.2 Hz), 3.16 (dd, 1H, J ¼ 17.8, 5.3 Hz); 13C NMR
(100 MHz, DMSO-d6, ppm) d ¼ 149.3, 146.4, 142.2, 134.0, 132.4,
131.7, 129.8, 128.7, 128.4, 127.8, 126.4, 123.1, 112.8, 63.2, 43.4;
HRMS (ESI-MS): calcd. for C21H19BrN3O2S [M þ H]þ 456.0376; found
456.0364.
4-(3-(4-Nitrophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benze-
nesulfonamide (15)
Mp 216–218 ꢀC. 2.74 g (82%). 1H NMR (400 MHz, DMSO-d6, ppm)
d ¼ 8.25 (d, 2H, J ¼ 8.6 Hz), 7.99 (d, 2H, J ¼ 8.6 Hz), 7.61 (d, 2H,
J ¼ 8.8 Hz), 7.35–7.32 (m, 2H), 7.26–7.24 (m, 3H), 7.14 (d, 2H,
J ¼ 8.8 Hz), 7.06 (s, 2H, NH2), 5.74 (dd, 1H, J ¼ 12.4, 5.3 Hz), 4.01
(dd, 1H, J ¼ 17.8, 12.4 Hz), 3.24 (dd, 1H, J ¼ 17.8, 5.3 Hz); 13C NMR
(100 MHz, DMSO-d6, ppm) d ¼ 148.3, 147.7, 145.8, 141.9, 138.8,
134.9, 129.9, 128.5, 127.8, 127.5, 126.4, 124.7, 113.4, 63.7, 43.1;
HRMS (ESI-MS): calcd. for C21H19N4O4S [M þ H]þ 423.1122; found
423.1120.
4-(3-(4-Hydroxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (16)
Mp 184–186 ꢀC. 0.77 g (22%). 1H NMR (400 MHz, DMSO-d6, ppm)
d ¼ 9.86 (s, 1H, OH), 7.61 (d, 2H, J ¼ 8.8 Hz), 7.54 (d, 2H, J ¼ 8.8 Hz),
7.33–7.30 (m, 2H), 7.24–7.21 (m, 3H), 6.99 (d, 2H, J ¼ 9.1 Hz), 6.97
(s, 2H, NH2), 6.81 (d, 2H, J ¼ 8.8 Hz), 5.54 (dd, 1H, J ¼ 11.7, 5.1 Hz),
3.89 (dd, 1H, J ¼ 17.6, 11.7 Hz), 3.10 (dd, 1H, J ¼ 17.6, 5.1 Hz); 13C
NMR (100 MHz, DMSO-d6, ppm) d ¼ 159.5, 150.6, 146.8, 142.5,
133.1, 129.8, 128.5, 128.2, 127.8, 126.4, 123.4, 116.2, 112.3, 62.7,
43.9; HRMS (ESI-MS): calcd. for C21H20N3O3S [M þ H]þ 394.1220;
found 394.1206.
Any substitution rather than hydrogen at the para position of
phenyl ring decreased Ki value of the substituted compound
toward hCA I by comparing with 9, which is non-substituted
derivative. This means that substitution at the para position of
phenyl ring was a useful modification to increase the effect of
compound toward hCA I. Exception was 11, which is a methoxy-
Table 1. Human CA isoenzymes (hCA I and II) inhibition value of the com-
pounds (9–16) by the esterase method with 4-nitrophenyl acetate as substrate.
Biological activity
IC50 (nM)
r2
hCA II
554.8 0.9759 586.3 0.9723 506.3 133.5 624.6 168.2
Ki (nM)
Compounds hCA I
r2
hCA I
hCA II
Carbonic anhydrase enzyme assay
9
The purification of cytosolic CA isoenzymes (CA I and II) were pre-
viously described with a simple one-step method by a Sepharose-
4B-L tyrosine-sulfanilamide affinity chromatography23. The protein
quantity in the column effluents was determined spectrophoto-
metrically at 280 nm. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was applied with a Bio-Rad Mini Gel
system (Mini-PROTEAN Tetra System, Guangdong, China) after
purification of both CA isoenzymes24,25. Briefly, it was performed
in acrylamide for the running (10%) and the stacking gel (3%)
10
11
12
13
14
15
16
AZA
491.8 0.9816 620.4 0.9565 476.5 81.1 561.5 133.9
483.9 0.9743 486.3 0.9688 533.1 187.8 469.0 63.0
402.9 0.9646 559.3 0.9814 377.6 113.8 520.6 131.8
493.2 0.9709 536.0 0.9737 462.2 133.5 587.3 188.5
404.8 0.9817 470.5 0.9602 316.7 9.6
416.5 0.9815 534.3 0.9631 325.8 29.4
528.2 0.9710 458.6 0.9772 472.0 57.5
985.8 0.9811 489.4 0.9972 278.8 44.3
412.5 115.4
430.3 87.3
591.9 134.5
293.4 46.4
Acetazolamide (AZA) was used as a standard inhibitor for all hCA isoenzymes.
The results were expressed as nanomolar (nM).