465
benzenesulfonamide or p-(2-aminoethyl)-benzenesulfo-
namide) were suspended in 15 mL anhydrous acetonitrile
and cooled to 2–5 °C in a salt-ice bath. Finely powdered
sulfonyl chloride (5 mmol) was added in small portions,
concomitantly with 5 mmol of pyridine or triethylamine,
maintaining the temperature under 10 °C. The reaction
mixture was then stirred at room temperature for 3–5 h
(t.l.c. control of the progress of the reaction), the solvent
evaporated in vacuo, and a small volume of water added.
The precipitated sulfonamides were filtered and recrys-
tallized from aqueous ethanol. Derivatives A11, B29,
C47 and D65 were prepared by reaction of the corre-
sponding amino-benzenesulfonamide derivatives with
2-sulfobenzoic acid cyclic anhydride in equimolar
amount, in anhydrous acetonitrile at reflux for 2 h. All
compounds have been extensively characterized and
analysed as mentioned above. Some typical spectral data
for a compound of each series are shown below.
1 255 (NO2), 1 150 and 1 171 (SO2sym), 1 328 (SO2as),
3 270 and 3 390 (NH and NH2); 1H-NMR (DMSO-d6), δ,
ppm: 3.15 (t, 2H, (CH2)); 3.71 (t, 2H, (CH2)); 6.86 (br s,
3H, NH2 + NH); 7.06 (m, AA’BB’, 4H, ArH from
phenylene-sulfonamido); 7.21 (m, AA’BB’, 4H, ArH
from nitro-phenylene). Anal C14H15N3O6S2 (C, H, N).
2.3 Pharmacology
2.3.1. Carbonic anhydrase inhibition
Initial rates of 4-nitrophenyl acetate (p-NPA) hydroly-
sis catalysed by different CA isozymes were monitored
spectrophotometrically, at 400 nm, with a Cary 3 instru-
ment interfaced with an IBM compatible PC [28]. Solu-
tions of substrate were prepared in anhydrous acetoni-
trile; the substrate concentrations varied between 2 × 10–2
and 1 × 10–6 M, working at 25 °C. A molar absorption
coefficient e of 18 400 M–1.cm–1 was used for the
4-nitrophenolate formed by hydrolysis, in the conditions
of the experiments (pH 7.40), as reported in the litera-
ture [28]. Non-enzymatic hydrolysis rates were always
subtracted from the observed rates. Duplicate experi-
ments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such
results. Stock solutions of inhibitor (1 mM) were pre-
pared in distilled-deionized water with 10–20% (v/v)
DMSO (which is not inhibitory at these concentra-
tions [18]) and dilutions up to 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were preincubated together for 10 min
at room temperature prior to assay, in order to allow for
the formation of the E-I complex. The IC50, representing
the molarity of inhibitor producing a 50% decrease of
enzyme catalysed p-NPA hydrolysis, has been determined
as described by Pocker and Stone [25, 28]. Enzyme
concentrations were 3.5 nM for CA II, 10.5 nM for CA I
and 35 nM for CA IV (this isozyme has a decreased
esterase activity [29] and higher concentrations had to be
used for the measurements).
2.2.1. 4-(4-Fluorobenzenesulfonylamido)-benzenesulfon-
amide A7
White crystals, m.p. 157–159 °C (from ethanol-water
3:1, v/v); IR (KBr), cm–1: 629, 710, 883, 1 165, 1 170,
1 290, 1 367, 1 425, 1 575, 3 075; 1H-NMR (DMSO-d6),
δ, ppm: 7.10–7.58 (m, AA’BB’,8H, ArH); 8.13 (br s, 2H,
SO2NH2); 8.46 (s, 1H, SO2NH); Anal C12H11FN2O4S2
(C, H, N).
2.2.2. 3-(2-Nitrobenzenesulfonylamido)-benzenesulfon-
amide B30
Pale yellow crystals, m.p. 201–204 °C, IR (KBr),
cm–1: 685, 717, 810, 943, 1 080 and 1 250 (NO2), 1 144
and 1 181 (SO2sym), 1 314 (SO2as), 3 275 and 3 400 (NH
and NH2); 1H-NMR (DMSO-d6), δ, ppm: 6.50 (br s, 3H,
NH2 + NH); 7.15–7.50 (m, 4H, ArH from 1,3-
phenylene); 7.33–7.78 (m, 4H, Ar H from ortho-
substituted phenyl). Anal C12H11N3O6S2 (C, H, N).
2.2.3. 4-(4-Nitrobenzenesulfonylamidomethyl)-benzene-
sulfonamide C50
Pale yellow crystals, m.p. 249–251 °C, IR (KBr),
cm–1: 661, 715, 762, 820, 884, 1 075 and 1 249 (NO2),
1 151 and 1 173 (SO2sym), 1 325 (SO2as), 3 270 and 3 390
2.4. Calculations
1
The atomic coordinates of the compounds listed in
table I were calculated using the program PC-
MODEL [30], using the MMX molecular mechanics
algorithm and the dihedral driver in an attempt to find a
global minimum. The geometry was further optimized
using the AM1 Hamiltonian and the program MOPAC
version 6 [31]. The first order polarizability was also
calculated at this stage.
(NH and NH2); H-NMR (DMSO-d6), δ, ppm: 4.90 (s,
2H, SO2NHCH2); 6.86 (br s, 3H, NH2 + NH); 7.05 (m,
AA’BB’, 4H, ArH from phenylene-sulfonamido); 7.23
(m, AA’BB’, 4H, ArH from nitro-phenylene). Anal
C13H13N3O6S2 (C, H, N).
2.2.4.
4-(4-Nitrobenzenesulfonylamidoethyl)-benzene-
sulfonamide D68
Pale yellow crystals, m.p. 237–240 °C, IR (KBr),
The optimized geometry was defined with the number-
ing of the common atoms from 1–17 as in figure 1 (D58,
cm–1: 690, 728, 779, 860, 898, 970, 1 045, 1 080 and