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digital apparatus. Absorption spectra were recorded on
a
141.02, 139.18, 136.88, 129.74, 128.59, 127.59, 103.05, 63.34, 60.38,
Shimadzu UV-1800 spectrophotometer. CA esterase activity was 56.63, 39.68, 21.50. Anal. calcd. for: C20H23N3O3S: C, 62.32; H, 6.01;
determined according to Verpoorte et al.22
N, 10.90; Found: C, 62.31; H, 6.02; N, 10.89.
2.2. General methods for the synthesis of chalcones (1–5)
2.2.5. 3-(4-Aminophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (9)
An aqueous solution of NaOH (60%, 10 ml) was added into the
ethanol (6 ml) solution of substituted carbaldehyde (20.0 mmol)
and a suitable acetophenone (20.0 mmol). The mixture was stirred
for a day at room temperature and it was then poured on ice-
water. The mixture was neutralised using 6 M hydrochloric acid.
The yellow precipitate obtained was filtered and crystallized from
IR (ATR), ꢀ/cmꢀ1: 3443, 3304, 1576, 1357. 1H-NMR (DMSO-d6),
(d:ppm): 3.06 (1H, dd, J ¼ 2.8 and 2.8 Hz), 3.70 (3H, s), 3.71 (3H, s),
3.82 (1H, dd, J ¼ 7.2 and 6 Hz), 5.73 (1H, s), 5.81 (1H, dd, J ¼ 2.8
and 2.4 Hz), 6.57 (2H, d, J ¼ 8.8 Hz), 6.77 (1H, d, J ¼ 2), 6.85 (1H, d,
J ¼ 8.4), 7.53 (2H, d, J ¼ 8.8 Hz). 13 C-NMR (DMSO-d6), (d:ppm):
175.51, 156.48, 151.87, 149.08, 148.16, 136.04, 130.61, 129.17,
114.78, 113.73, 112.29, 110.21, 65.92, 55.99, 55.93, 42.93. Anal.
calcd. for: C18H20N4O2S: C, 60.65; H, 5.66; N, 15.72; Found: C, 60.64;
H, 5.63; N, 15.72.
ethanol-water.
(E)-1-(4-aminophenyl)-3-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one (1), (E)-1-(4-bromophenyl)-3-(3,4,5-trimethoxyphe-
nyl)prop-2-en-1-one (2), (E)-1-p-tolyl-3-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (3), (E)-1-(4-aminophenyl)-3-(3,4-dimethoxyphenyl)
prop-2-en-1-one (4), (E)-3-(4-(dimethylamino)phenyl)-1-(4-hydroxy-
phenyl)prop-2-en-1-one (5) were synthesised according to the lit-
erature,23 respectively.
2.2.6. 5-(4-(Dimethylamino)phenyl)-3-(4-hydroxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (10)
IR (ATR), ꢀ/cmꢀ1: 3430, 3260, 1580, 1346. 1H-NMR (DMSO-d6),
(d:ppm): 2.99 (6H, s), 3.06 (1H, dd, J ¼ 2.8 and 2.0 Hz), 3.72 (1H, dd,
J ¼ 10.8 and 11.2 Hz), 5.78 (1H, dd, J ¼ 2.4 and 2.4 Hz), 6.65 (2H, d,
J ¼ 8.8 Hz), 6.83 (2H, d, J ¼ 8.8 Hz), 6.95 (2H, d, J ¼ 8.4 Hz), 7.71 (2H,
d, J ¼ 8.4 Hz), 7.82 (br, –NH2), 11.20 (br, –OH). 13 C-NMR (DMSO-d6),
(d:ppm): 175.75, 160.32, 156.50, 151.91, 130.31, 129.21, 127.15,
119.01, 113.95, 63.81, 42.05, 41.78. Anal. calcd. for: C18H20N4OS: C,
63.50; H, 5.92; N, 16.46; Found: C, 63.54; H, 5.88; N, 16.44.
2.2.1. General methods for the synthesis of pyrazoles (6–10)
A mixture of (0.007 mol) chalcone and (0.014 mol) thiosemicarba-
zide were refluxed in ethanol (15 ml) while stirring vigorously.
After complete dissolution of the reactants,
a solution of
(0.014 mol) of KOH in ethanol (15 ml) was added dropwise. The
solution was refluxed for another 18 h, allowed to warm at room
temperature and then stirred for 4 h. The crude product was refri-
gerated overnight. The precipitate formed was filtered off and
crystallized from ethanol twice yielding yellow crystals.
2.3. Biological activity
2.3.1. Inhibition studies of carbonic anhydrase I and II isoforms
Enzyme activity was determined spectrophotometrically by follow-
ing the change in absorbance at 348 nm of 4-nitrophenylacetate
to 4-nitrophenolate over a period of 3 min at 25 ꢁC.21 The enzym-
atic reaction contained 1.4 ml 0.05 M Tris-SO4 buffer (pH 7.4), 1 ml
3 mM 4-nitrophenylacetate, 0.5 ml H2O and 0.1 ml enzyme solu-
tion, in a total volume of 3.0 ml.24 Inhibitory effects of compounds
1–10 were compared with acetazolamide (AZA). Different inhibitor
concentrations were used and all compounds were tested in tripli-
cate at each concentration used. Control cuvette activity was
acknowledged as 100% in the absence of inhibitor. An Activity% –
[Inhibitor] graph was drawn for each inhibitor.25 The curve-fitting
algorithm allowed for obtaining the IC50 values, working at the
lowest concentration of substrate of 0.15 mM, from which Ki val-
ues were calculated.20,21 The catalytic activity of these enzymes
was calculated from Lineweaver-Burk plots, as reported previously,
and represent the mean from at least three different determina-
tions. The hCA I and II isoenzymes used here were purified from
human blood as previously described.21
2.2.2.
3-(4-Aminophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (6)
IR (ATR), ꢀ/cmꢀ1: 3439, 3331, 1591, 1342. 1H-NMR (DMSO-d6),
(d:ppm): 3.10 (1H, dd, J ¼ 3.2 and 3.2 Hz), 3.62 (3H, s), 3.70 (6H, s),
3.86 (1H, dd, J ¼ 13.6 and 10.8 Hz), 5.80 (1H, dd, J ¼ 3.2 and
2.8 Hz), 6.40 (2H, s), 6.56 (2H, d, J ¼ 8.8 Hz), 7.53 (2H, d, J ¼ 8.4 Hz).
13 C-NMR (DMSO-d6), (d:ppm): 175.80, 156.52, 153.35, 151.91,
139.31, 136.82, 129.21, 118.08, 113.71, 103.07, 62.93, 60.38, 56.59,
42.95. Anal. calcd. for: C19H22N4O3S: C, 59.05; H, 5.74; N, 14.50;
Found: C, 59.07; H, 5.71; N, 14.48.
2.2.3. 3-(4-Bromophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (7)
IR (ATR), ꢀ/cmꢀ1: 3439, 3263, 1587, 1334. 1H-NMR (DMSO-d6),
(d:ppm): 3.21 (1H, dd, J ¼ 3.6 and 3.2 Hz), 3.64 (3H, s), 3.71 (6H, s),
3.90 (1H, dd, J ¼ 12.6 and 12.0 Hz), 5.89 (1H, dd, J ¼ 2.8 and
2.8 Hz), 6.43 (2H, s), 7.65 (2H, d, J ¼ 8.4 Hz), 7.82 (2H, d, J ¼ 8.4 Hz).
13 C-NMR (DMSO-d6), (d:ppm): 176.20, 154.52, 153.80, 139.64,
136.90, 132.07, 130.42, 129.54, 124.46, 104.37, 63.57, 59.99, 55.98,
42.75. Anal. calcd. for: C19H20BrN3O3S: C, 50.67; H, 4.48; N, 9.33;
Found: C, 50.65; H, 4.47; N, 9.29.
2.4. Computational section
2.4.1. Ligand and protein preparation
As a crucial step to meet minimum requirements for further com-
putational calculations, all the studied ligands and target proteins
2.2.4.
3-p-Tolyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyra-
were prepared. LigPrep tool26 interfaced with Maestro module of
zole-1-carbothioamide (8)
26
IR (ATR), ꢀ/cmꢀ1: 3433, 3263, 1580, 1338. 1H-NMR (DMSO-d6),
(d:ppm): 2.35 (3H, s), 3.20 (1H, dd, J ¼ 3.6 and 3.2 Hz), 3.64 (3H, s),
3.71 (6H, s), 3.92 (1H, dd, J ¼ 12.0 and 11.0 Hz), 5.88 (1H, dd,
€
Schrodinger suite was used for the ligand preparation. The 3 D
structures including all possible tautomers and ionisation states at
pH 7.0 2.0 of all the ligands 1–10 and the reference compound
J ¼ 3.2 and 2.8 Hz), 6.43 (2H, s), 7.27 (2H, d, J ¼ 4 Hz), 7.76 (2H, d, AZA were generated and geometrically minimised using opti-
J ¼ 4 Hz). 13 C-NMR (DMSO-d6), (d:ppm): 176.76, 155.73, 153.40, mised potential liquid simulations (OPLS3) force field.27