Carbonic Anhydrase Inhibitors
A R T I C L E S
layer was washed with water and dried over anhydrous Na2SO4. After
solvent evaporation, the solid obtained was recrystallized with CHCl3/
hexane. Yield: 0.42 g (28%). 1H NMR (300 MHz, CDCl3): δ 1.28 (t,
6H, J ) 7.1 Hz), 3.51 (s, 2H), 3.61 (s, 4H), 4.22 (q, 4H, J ) 7.1 Hz),
7.21 (bs, 1H), 7.39 (m, 1H), 7.80 (d, 1H, J ) 7.5 Hz), 7.99 (m, 1H),
8.24 (s, 1H).
added to the residue, and the product was extracted with ethyl acetate
and washed with 4% citric acid and 4% NaHCO3 solution and finally
with brine. The pure product was obtained by silica gel column
chromatography with 3-5% MeOH in CHCl3 (Rf 0.2 in 8% MeOH in
CHCl3). Yield: 0.45 g (48%). 1H NMR (400 MHz, CDCl3): δ 1.24 (t,
6H, J ) 14 Hz), 2.93-2.96 (m, 2H), 3.42-3.46 (m, 2H), 3.54 (s, 4H),
4.13-4.17 (m, 4H), 5.27 (br s, 2H), 7.90 (distorted doublet, 2H, J )
8 Hz), 8.01 (distorted doublet, 2H, J ) 8 Hz), 8.25 (br s, 1H).
The above ester (0.21 g, 0.51 mmol) was dissolved in MeOH/CH2-
Cl2 (1:1, 12 mL), and solid LiOH (65 mg, 1.54 mmol) was added. The
reaction mixture was stirred at room temperature for 12 h. The pH of
the solution was lowered to 3 by adding 2 N HCl when a precipitate
appeared. The precipitate was filtered and washed with methanol/
dichloromethane. Drying under vacuum afforded the acid as a white
BR22. The reaction between the acid 6 (0.2 g, 0.55 mmol) and
p-aminobenzenesulfonamide HCl salt (0.112 g, 0.55 mmol) was carried
out in the presence of benzotriazol-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate (BOP) (0.25 g, 0.56 mmol) and Et3N
(0.2 mL, 1.4 mmol) in CHCl3/dimethylformamide (DMF) at room
temperature. The reaction mixture was stirred at room temperature for
8 h. The reaction was quenched with brine, and the solvent was removed
under reduced pressure. Water was added to the residue, and the product
was extracted with ethyl acetate, then washed successively with 4%
citric acid, 4% NaHCO3 solution, and brine. The crude product was
purified by silica gel column chromatography with 8% MeOH in CHCl3
(Rf 0.4). Yield: 0.285 (95%). 1H NMR (300, CDCl3-CD3OD-D2O):
δ 1.25-1.32 (m, 6H), 3.00 (t, 2H, J ) 6.5 Hz), 3.52 (s, 2H), 3.59 (s,
4H), 3.69 (t, 2H, J ) 6.5 Hz), 4.16-4.24 (m, 4H), 7.33 (d, 2H, J )
8.0 Hz), 7.39 (t, 1H, J ) 8.0 Hz), 7.47 (d, 1H, J ) 8.0 Hz), 7.58 (d,
1H, J ) 8.0 Hz), 7.85 (d, 2H, J ) 8.0 Hz), 8.03 (s, 1H). 13C NMR
(125 MHz, CDCl3-CD3OD): δ 14.44, 35.45, 40.91, 56.69, 60.43,
61.65, 116.86, 122.51, 123.75, 127.17, 129.66, 129.92, 135.63, 138.33,
140.82, 144.51, 167.58, 170.29, 171.73.
1
solid. Yield: 0.14 g (77%). H NMR (400 MHz, D2O): δ 2.92-2.96
(m, 2H), 3.42-3.46 (m, 2H), 3.49 (s, 4H), 7.86 (distorted doublet, 2H,
J ) 8 Hz), 7.97 (distorted doublet, 2H, J ) 8 Hz).
The acid (66 mg, 0.18 mmol) was dissolved in MeOH (10 mL),
and CuCl2‚2H2O (32 mg, 0.18 mmol) was added. Immediate color
change was observed. No precipitation was obtained upon continuation
of stirring for six more hours at room temperature. The solvent was
removed, and the residue was dried under vacuum. It was redissolved
in ethanol and precipitated with CH2Cl2. The precipitate was filtered
and washed with ethanol/CH2Cl2 and dried under vacuum, affording a
blue solid (66 mg). Yield: 81%. Anal. Calcd for C13H15CuN3O7S‚
H2O: C, 35.58; H, 3.90; N, 9.57. Found: C, 35.23; H, 3.78; N, 9.88.
Isothermal Titration Calorimetry. ITC experiments were per-
formed on a Microcal MCS calorimeter, and the data were analyzed
as described by Wiseman and colleagues.21 The calorimeter was
calibrated by known heat pulses as described in the MCS-ITC manual.
During titration, the reference cell was filled with a 0.03% azide solution
in water. Prior to titration, both carbonic anhydrase and inhibitor solu-
tions were thoroughly degassed. The sample cell was filled either with
1.8 mL (effective volume ) 1.36 mL) of buffer (for control) or with
an appropriately diluted enzyme. The contents of the sample cell were
titrated with increasing aliquots (first aliquot 1 µL, and subsequent
aliquots 4 µL each) of inhibitors. During the titration, the reaction mix-
ture was constantly stirred at 400 rpm. The enzyme concentration was
adjusted by 2% (as recommended by the manufacturer) to include a
dilution effect of the enzyme solution, which occurs following a buffer
rinse.
All calorimetric titration data were presented after subtracting the
background signal, deduced from the amplitude of heat pulses at the
end of the titration. The raw experimental data were presented as the
amount of heat produced per second following each injection of
inhibitor into the enzyme solution (minus the blank) as a function of
time. The amount of heat produced per injection was calculated by
integration of the area under individual peaks by the Origin software.
Final data are presented as the amount of heat produced per injection
versus the molar ratio of inhibitor to enzyme. Whereas the binding
isotherm for the CAII-benzenesulfonamide complex was analyzed
according to the “one-site” binding model, that for the CAII-BR30
complex was analyzed by the “two-site” binding model, as described
by Wiseman and colleagues.21
The above ester (0.184 g, 0.337 mmol) was dissolved in methanol
(MeOH)/CH2Cl2 (6:4 mL), and solid LiOH (50 mg, 1.20 mmol) was
added and stirred for 6 h at room temperature. The pH of the solution
was lowered to 3 by adding 2 N HCl when a precipitate appeared. The
precipitate was filtered and washed with methanol/dichloromethane.
Drying under vacuum afforded the acid as a white solid. Yield: 120
1
mg (73%). H NMR (300 MHz, D2O): δ 2.95 (t, 2H, J ) 6.6 Hz),
3.30 (s, 4H), 3.47 (s, 2H), 3.62 (t, 2H, J ) 6.6 Hz), 7.35-7.40 (m,
3H), 7.43 (t, 1H, J ) 8.0 Hz), 7.56 (d, 1H, J ) 8.0 Hz), 7.70-7.75
(m, 3H).
The above acid (110 mg, 0.223 mmol) was dissolved in MeOH (8
mL), and solid CuCl2‚2H2O (38 mg, 0.223 mmol) was added. The
mixture was stirred at room temperature for 6 h. The solvent was
removed, and the residue was dried under vacuum. It was redissolved
in ethanol and precipitated with CH2Cl2. The precipitate was filtered
and washed with ethanol/CH2Cl2 and dried under vacuum, affording a
blue solid. Yield: 110 mg (86%). Anal. Calcd for C21H22CuN4O8S‚
H2O: C, 44.09; H, 4.23; N, 9.79. Found: C, 44.19; H, 4.56; N, 10.03.
BR30.
To a solution of 4-carboxybenzenesulfonamide (0.46 g, 2.28 mmol)
and Et3N (1.7 mL, 12.21 mmol) in CHCl3/DMF (1:1, 20 mL) was added
a solution of amine 728 (0.7 g, 2.28 mmol) in DMF (10 mL) followed
by the addition of BOP (1.01 g, 2.29 mmol). The reaction mixture was
stirred for 12 h at room temperature. The reaction was quenched with
brine. The solvent was removed under reduced pressure. Water was
Crystallography. CAI and CAII were purified as described.14,29
Crystals of recombinant CAII were grown by the hanging drop
method: 5 µL of protein solution (10 mg/mL protein, 1 mM methyl
mercuric acetate, 50 mM Tris-sulfate, pH 8.0) and 5 µL of precipitant
solution (2.5 M (NH4)2SO4, 50 mM Tris-sulfate, pH 7.7) were mixed
and suspended over a reservoir containing 1 mL of precipitant solution
at 4 °C. Crystals formed within 3 days. Single crystals were transferred
to fresh sitting drops containing inhibitor (1.4-2.0 mM inhibitor from
10 mM stock in dimethyl sulfoxide/acetonitrile), 50 mM Tris-sulfate,
pH 7.7, and 2.05 M (NH4)2SO4 over a well of 1-mL precipitant solution.
After 2 days, crystals were transferred to a drop containing 30% glycerol
(24) Håkansson, K.; Wehnert, A.; Liljas, A. Acta Crystallogr. 1994, D50, 93-
100.
(25) (a) Eriksson, E. A.; Jones, T. A.; Liljas, A. In The Zinc Enzymes; Bertini,
I.; Luchinat, C.; Maret, W.; Zeppezauer, M., Eds.; Birkha¨user Boston:
Boston, MA, 1986; pp 317-328. (b) Sumalan, S. L.; Casanova, J.; Alzuet,
G.; Borra´s, J.; Castin˜eiras, A.; Supuran, C. T. J. Inorg. Biochem. 1996, 62,
31-39.
(26) Conroy, C. W.; Maren, T. H. Mol. Pharmacol. 1995, 48, 486-491.
(27) Burks, E.; Koshti, N.; Jacobs, H.; Gopalan, A. Synlett 1998, 1285-1287.
(28) del Olmo, E.; Macho, A.; Alves, M.; Lo´pez, J. L.; el Banoua, F.; Mun˜oz,
E.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2002, 12, 2621-2626.
(29) Banerjee, A. L.; Swanson, M.; Mallik, S.; Srivastava, D. K. Protein
Expression Purif. 2004, 37, 450-454.
9
J. AM. CHEM. SOC. VOL. 128, NO. 9, 2006 3017