38 Y. Akbaba et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 35–42
for 15 h. After the solvent was evaporated, the residue was N-(5,6-dimethoxy-2,3-dihydro-1H-inden-2-yl)methanesulfona-
chromatographed on silica gel (30 g) column with 30% EtOAc– mide (32)
hexane. Sulfonamide 28 was synthesized as white solid (0.53 g,
1
78% yield; mp.: 162–164 ꢂC. H-NMR (400 MHz, CDCl3) ꢀ 6.72
80% yield).
(s, 2H, Ar-H), 4.89 (bd, 1H, NH, J ¼ 8.4 Hz), 4.31–4.22 (m, 1H,
CH–N), 3.83 (s, 6H, 2OCH3), 3.24 (A part of AB, dd, 2H of CH2,
J ¼ 7.2 and J ¼ 15.5 Hz), 2.98 (s, 3H, CH3), 2.83 (B part of AB,
N-(5,6-dimethoxyindane-1-yl)methanesulfonamide (28)
dd, 2H of CH2, J ¼ 5.8 and J ¼ 15.5 Hz). 13C-NMR (100 MHz,
1
Melting point (m.p.): 144–146 ꢂC. H-NMR (400 MHz, CDCl3)
CDCl3) ꢀ 148.7 (2C), 131.8 (2C), 108.1 (2CH), 56.3 (2OCH3),
55.3 (CH–N), 41.7 (CH3) and 41.0 (2CH2). IR (CH2Cl2): 3249,
2958, 2930, 2840, 1605, 1560, 1504, 1443, 1404, 1311, 1219,
1188, 1169, 1152, 1096 cmꢁ1. Anal. Calcd for (C12H17NO4S): C
53.12, H 6.32, N 5.16 and S 11.82; Found C 53.10, H 6.31, N 5.18
and S 11.85.
ꢀ 6.90 (s, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 4.93–4.88 (m, 1H, CH-
N), 4.66 (bd, 1H, NH, J ¼ 9.2 Hz), 3.85 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 3.03 (s, 3H, CH3), 2.94 (A part of AB, ddd, 1H, CH2,
J ¼ 3.9, J ¼ 8.3 and J ¼ 15.5 Hz), 2.81–2.73 (B part of AB, m, 1H,
CH2), 2.61 (dddd, 1H, CH2, J ¼ 4.0, J ¼ 7.8, J ¼ 12.1 and
J ¼ 16.8 Hz), 1.96–1.87 (m, 1H, CH2). 13C-NMR (100 MHz,
CDCl3) ꢀ 149.9 (C), 148.9 (C), 134.9 (C), 133.5 (C), 107.7 (CH),
107.2 (CH), 59.4 (CH–N), 56.4 (OCH3), 56.2 (OCH3), 42.1
(CH3), 35.5 (CH2), 30.1 (CH2). IR (CH2Cl2, cmꢁ1): 3284, 3002,
2935, 2857, 2834, 1608, 1504, 1465, 1412, 1309, 1264, 1221,
1188, 1148, 1097. Anal. Calcd for (C12H17NO4S): C 53.12, H
6.32, N 5.16 and S 11.82; Found C 53.08, H 6.34, N 5.12 and S
11.80.
N-(Indane-2-yl)methanesulfonamide (33)
80% yield; m.p.: 134–136 ꢂC. 1H-NMR (400 MHz, CDCl3)
ꢀ 7.23–7.17 (m, 4H, Ar-H), 4.64 (ddd, 1H, CH–N, J ¼ 5.5,
J ¼ 7.7 and J ¼ 13.0 Hz), 4.70 (bd, 1H, NH, J ¼ 7.7 Hz), 4.31 (m,
1H, CH–N), 3.30 (A part of AB, dd, 2H, 2Ha of CH2, J ¼ 7.1 and
J ¼ 15.7 Hz), 3.01 (s, 3H, CH3) 2.91 (B part of AB, dd, 2H, 2Hb
of CH2, J ¼ 5.9 and J ¼ 15.7 Hz). 13C-NMR (100 MHz, CDCl3)
ꢀ 140.15 (2C), 127.3 (2CH), 124.95 (2CH), 55.1 (CH–N), 41.81
(2CH2) and 41.02 (CH3). IR (CH2Cl2): 3230, 3064, 2964, 2930,
2901, 1459, 1436, 1306, 1228, 1135, 1103, 1018 cmꢁ1. Anal.
Calcd for (C10H13NO2S): C 56.85, H 6.20, N 6.63 and S 15.18;
Found C 56.88, H 6.23, N 6.60 and S 15.21.
N-(Indane-1-yl)methanesulfonamide (29)
Yield 82%; m.p.: 93–95 ꢂC. 1H-NMR (400 MHz, CDCl3)
ꢀ 7.37–7.19 (m, 4H, Ar-H), 5.06 (bd, 1H, NH, J ¼ 9.0 Hz),
4.89–4.83 (m, 1H, CH–N), 3.00–2.93 (m, 4H, 1H of CH2 and
CH3), 2.84–2.76 (m, 1H of CH2), 2.55 (dddd, 1H of CH2,
J ¼ 3.6, J ¼ 7.8, J ¼ 11.3 and J ¼ 15.8 Hz), 1.89 (dddd, 1H of
CH2, J ¼ 3.6, J ¼ 8.1, J ¼ 12.8 and J ¼ 15.8 Hz). 13C-NMR
(100 MHz, CDCl3) ꢀ 143.0 (C), 142.4 (C), 128.5 (CH), 127.1
(CH), 125.1 (CH), 124.5 (CH), 59.0 (CH-N), 41.9 (CH3), 35.1
(CH2), 30.2 (CH2). Anal. Calcd for (C10H13NO2S): C 56.85, H
6.20, N 6.63 and S 15.18; Found C 56.82, H 6.18, N 6.66
and S 15.21.
Purification of hCA I and hCA II by affinity
chromatography
CA isoenzymes were purified affinity chromatography using a
column packed with Sepharose 4B-L-Tyrosine sulphanilamide
resin via a simple one-step method using Sepharose-4B-L
tyrosine-sulphanilamide affinity gel chromatography; described
previously27,28
.
N-(5-metoksi-1,2,3,4-tetrahydronaphthalene-1-yl)methanesulfo-
namide (30)
Hydratase and esterase activity assays
Yield 83%; m.p.: 142–144 ꢂC. 1H-NMR (400 MHz, CDCl3) ꢀ 7.18
(dd, 1H, Ar-H, J ¼ 7.7 and J ¼ 8.1 Hz), 7.02 (d, 1H, Ar-H,
J ¼ 7.7 Hz), 6.75 (d, 1H, Ar-H, J ¼ 8.1 Hz), 4.64 (ddd, 1H, CH–N,
J ¼ 5.5, J ¼ 7.7 and J ¼ 13.0 Hz), 4.50 (d, 1H, NH, J ¼ 7.7 Hz), 3.82
(s, 3H, OCH3), 3.07 (s, 3H, CH3), 2.72 (A part of AB, dt, 1H, Ha of
CH2, J ¼ 6.1 and J ¼ 18.0 Hz), 2.57 (B part of AB, dt, 1H, Hb of
CH2, J ¼ 7.1 and J ¼ 18.0 Hz), 2.07–1.79 (m, 4H, 2CH2). 13C-
NMR (100 MHz, CDCl3) ꢀ 157.4 (C), 137.0 (C), 127.0 (CH), 126.9
(C), 120.8 (CH), 109.0 (CH), 55.6 (OCH3), 52.5 (CH-N), 42.4
(CH3), 31.1 (CH2), 22.9 (CH2) and 18.8 (CH2). IR (CH2Cl2): 3647,
3280, 3008, 2935, 2862, 1587, 1470, 1438, 1408, 1319, 1252, 1149,
1101, 1078 cmꢁ1. Anal. Calcd for (C12H17NO3S): C 56.45, H 6.71,
N 5.49 and S 12.56; Found C 56.42, H 6.72, N 5.49 and S 12.57.
The effect of novel sulfonamides 28–33 on HCA isozyme activity
was determined colorimetrically using CO2-hydration method of
Wilbur and Anderson29 described previously9. Esterase activity
was determined according to the method described by Verpoorte
et al30.
Protein determination
The yield of protein during the purification steps was determined
spectrophotometrically at 595 nm according to the Bradford
method31, explained previously using bovine serum albumin as
the standard protein32.
SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis (SDS-PAGE) by the
method of Laemmli33 was performed for determination of
isoenzymes purity as described previously34.
N-(1,2,3,4-tetrahydronaphthalene -2-yl)methanesulfonamide (31)
85% yield; m.p.: 126–128 ꢂC. 1H-NMR (400 MHz, CDCl3)
ꢀ 7.16–7.05 (m, 4H, Ar-H), 4.52 (d, 1H, NH, J ¼ 7.5 Hz), 3.87–
3.81 (m, 1H, CH–N), 3.17 (A part of AB, dd, 1H, Ha of CH2,
J ¼ 4.7 and J ¼ 16.2 Hz), 3.01 (s, 3H, CH3) 2.92 (t, 2H, CH2,
J ¼ 6.6 Hz), 2.77 (B part of AB, dd, 1H, Hb of CH2, J ¼ 8.2 and
J ¼ 16.2 Hz), 2.18–2.11 (m, 1H, CH2), 1.90–1.81 (m, 1H, CH2).
13C-NMR (100 MHz, CDCl3) ꢀ 135.2 (C), 133.5 (C), 129.6 (CH),
129.1 (CH), 126.7 (CH), 126.4 (CH), 50.0 (CH-N), 42.2 (CH3),
37.2 (CH2), 30.3 (CH2) and 27.2 (CH2). IR (CH2Cl2): 3647, 3272,
Results and discussion
Chemistry
Reduction of ketones 4–6 with NaBH4 in MeOH at 0–25 ꢂC gave
corresponding alcohols 7,18 819 and 920. Mitsunobu reactions of
sec-alcohols with HN3 have been given in the literature
clearly35,36. By applying this method to compounds 7–9, we
failed to synthesize azides 10–12. In the literature, the synthesis of
benzylazides from benzylalcohols has been managed with DPPA
3020, 2928, 2852, 1494, 1439, 1316, 1150, 1113, 1072 cmꢁ1
.
Anal. Calcd for (C11H15NO2S): C 58.64, H 6.71, N 6.22 and S
14.23; Found C 58.63, H 6.71, N 6.28 and S 14.25.