Synthesis and structure-activity relationships of novel benzene sulfonamides with potent binding affinity for bovine carbonic anhydrase II

Article abstract of DOI:10.1016/j.bmcl.2005.08.113

This manuscript reports the identification of a novel series of mono- and bis- benzene sulfonamides with potent binding affinity for bovine carbonic anhydrase II (bCAII). These compounds exhibited nanomolar equilibrium dissociation constants with K_i<

Full text of DOI:10.1016/j.bmcl.2005.08.113

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5429–5433
Synthesis and structure–activity relationships of novel
benzene sulfonamides with potent binding affinity
for bovine carbonic anhydrase II
Sally-Ann Poulsen,* Laurent F. Bornaghi and Peter C. Healy
Chemical Biology Group, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan Campus,
Brisbane 4111, Australia
Received 5 August 2005; revised 30 August 2005; accepted 30 August 2005
Available online 6 October 2005
Abstract—This manuscript reports the identification of a novel series of mono- and bis- benzene sulfonamides with potent binding
affinity for bovine carbonic anhydrase II (bCAII). These compounds exhibited nanomolar equilibrium dissociation constants with
KiÕs ranging from 4.7 to 9.3 nM. All compounds were ester derivatives of the weak affinity bCAII inhibitor, 4-carboxybenzenesulf-
onamide. Structure–activity relationships for this novel series of compounds are discussed.
ꢀ 2005 Elsevier Ltd. All rights reserved.
S
S
The carbonic anhydrase (CA) family of Zn(II) metalloen-
zymes (EC 4.2.1.1) catalyzes the reversible hydration of
CO2 to HCO3À. This regulatory reaction underpins many
physiological processes associated with pH control, ion
transport, and fluid secretion.1,2 Classically, an aromatic
or heteroaromatic sulfonamide moiety (ArSO2NH2)
was the primary recognition element necessary for small
molecules to bind the active site of CA.1,2 Coordination
of the nitrogen atom of the ionized sulfonamide anion
(ArSO2NHÀ) to the active site Zn(II) of CA facilitates this
protein–small molecule interaction.1,2 The inhibition of
CAs has been exploited clinically for several decades for
the treatment of a variety of conditions including glauco-
ma, epilepsy, and gastric ulcers.1,2 More recently, CA
inhibition has been implicated as playing an important
role in cancer tumor progression.3,4 Clinically used CA
inhibitors include acetazolamide (AAZ), methazolamide
(MZA), ethoxazolamide (EZA), dichlorophenamide
(DCP), brinzolamide (BRZ) and dorzolamide (DZA).
Indisulam (IND) is in Phase II clinical trials as an antican-
cer agent to treat solid tumors.5
MeCOHN
SO2NH2
MeCON
Me
SO2NH2
N
N
N N
AAZ
MZA
Cl
Cl
SO2NH2
N
S
SO2NH2
EtO
SO2NH2
DCP
EZA
NHEt
NHEt
SO2NH2
SO2NH2
S
S
S
O
MeO(CH2)3
S
Me
O
O
O
BRZ
DZA
O
O
S
SO2NH2
In 1958, Beasley and colleagues reported that 4-carboxy-
benzenesulfonamide (1), containing the well-known
primary recognition motif for CA enzyme binding,
NH
NH
Cl
IND
Keywords: Carbonic anhydrase; Sulfonamides.
*
was minimally more active (4-fold) than the then lead
compound sulfanilamide (2) for the inhibition of CA
Corresponding author. Tel.: +61 7 3735 7825; fax: +61 7 3735
7656; e-mail: s.poulsen@griffith.edu.au
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.113

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S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5429–5433
SO2NH2
SO2NH2
SO2NH2
HO
O
H2N
O
O
1
2
3
in vitro.6 A remarkable increase in activity was, howev-
er, observed for simple aliphatic esters of 1. In this study
compound 3, the allyl ester of 1, possessed 900-fold
greater in vitro activity than that of sulfanilamide 2,
while the propyl ester analogue of 1 was 500-fold greater
in activity. It is now extensively documented that signif-
icant enhancement of CA activity (especially for the
most abundant CA isoform CAII) can be achieved
through coupling the primary recognition aromatic sul-
fonamide motif with secondary binding elements, the so-
called ÔtailÕ approach.1,7,8 Even so, since this early study
of Beasley no further literature analysis of the CA activ-
ity of 3 or CA structure–activity study based on 3 has
been reported.
mides have shown improved CAII activity compared
to the corresponding mono-sulfonamide analogues.6,10
As the bis-sulfonamides of this study were symmetrical,
the effect of the second sulfonamide moiety could be
determined by synthesis and evaluation of compound
6, which lacks the second sulfonamide group of 4. Com-
pounds 7 and 8 were prepared to delineate the effect on
bCAII affinity of a small, polar hydroxyl group (in place
of the bulky, largely hydrophobic substituents of 4–6)
on the allyl functionality. The pentenyl analogue 9 is a
two-methylene chain extension of 3. Compound 9 was
prepared to investigate the effect on bCAII affinity of
increasing chain length between the aromatic sulfon-
amide motif and the terminal alkene moiety. This man-
uscript discusses synthesis and preliminary structure–
activity relationships for this family of novel ester-linked
benzene sulfonamides.
The dual functionality of 3, possessing both an aromatic
sulfonamide and a terminal alkene group, made it an
ideal candidate building block for our ongoing investi-
gation into the development of the cross metathesis
reaction for dynamic combinatorial chemistry, with
bCAII as the protein target.9 The benzene sulfonamide
motif gives reliable bCAII affinity, while the alkene per-
mits the evaluation of cross metathesis chemistry to gen-
erate libraries for screening against bCAII.
The synthesis of the lead compound 3 was carried out by
the acid catalyzed esterification of 1 with allyl alcohol.11
Compound 3 afforded crystalline material suitable for
X-ray crystallography analysis.12–14 An ORTEP-315 rep-
resentation of 3 is shown in Figure 1. The molecular
structure is essentially planar with the exception of the
peripheral NH2 and CH2 groups (O4-C8-C9-
C10 = À140.4(6)ꢁ, N1-S1-C1-C2 = 104.6(2)ꢁ). The mole-
cules are linked through strong intermolecular N-HÁ Á ÁO
hydrogen bonds between the amide and sulfonyl oxygen
O1 and the carbonyl oxygen O3, respectively. The syn-
thesis of 4 and 5 proceeded by the 1,3-dicyclohexylcar-
bodiimide (DCC) mediated esterification of 1 with
0.5 equiv of trans-2-butene-1,4-diol and cis-2-butene-
1,4-diol, respectively.11 Reaction of 1 in the presence
of an excess of each of these diols generated the mono-
The symmetrical trans bis sulfonamide 4 and cis bis-sul-
fonamide 5 are the two possible homodimer reaction
products from cross metathesis of 3. These homodimers
will form the background bCAII binding affinity re-
sponse in libraries generated from cross metathesis of
3 with other alkene containing building blocks. An accu-
rate determination of the bCAII binding affinity of
authentic 4 and 5 was therefore of interest. Early reports
of CAII activity of unrelated symmetrical bis-sulfona-
SO2NH2
SO2NH2
O
O
O
O
O
O
O
O
NH2SO2
NH2SO2
5
4
SO2NH2
O
O
O
O
6
SO2NH2
SO2NH2
SO2NH2
O
O
O
HO
O
O
O
HO
7
8
9

S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5429–5433
5431
(Ki = 9.3 nM) had similar affinity to 3. Compound 6,
which lacks the second sulfonamide moiety of 4, had
identical bCAII affinity to 4 (Ki = 4.7 nM). This result
demonstrates that the second sulfonamide moiety is
clearly not important for bCAII affinity. Compounds 7
and 8, with a hydroxyl group in place of the second aro-
matic sulfonamide moiety of 4 and 5, exhibited minimal
change in affinity when compared to 4 and 5, respectively
(Ki of 7 = 5.3 nM, Ki of 8 = 9.1 nM). The trans isomers 4
and 7 were each ꢀ2-fold higher in affinity than their cis
counterparts 5 and 8, as well as the unsubstituted 3. Col-
lectively compounds 4, 5, 7, and 8 demonstrate that (i)
the enzyme is very tolerant to functionality on the allyl
substituent of 3 (size, polarity, and hydrophobicity)
and (ii) the enzyme exhibits a preference for trans stereo-
chemistry about the substituted alkene. The pentenyl es-
ter analogue 9, with two additional methylenes between
the ester linkage and terminal alkene when compared
to 3, had a Ki of 7.7 nM, similar to that for 3. This result
also confirms that bCAII exhibits tolerance for the steric
nature of the secondary binding elements.
Figure 1. ORTEP-3 plot for 3.15
Table 1. bCAII enzyme binding assay results expressed as Ki in nM
SO2NH2
RO
O
Compound
R
BCAII
Kia (R2)
This study has demonstrated that simple allyl ester
derivatives of 4-carboxybenzenesulfonamide have pro-
duced compounds with high affinity for bCAII. A termi-
nal substituent on the allyl alkene bond revealed both a
tolerance from bCAII for the properties of this function-
ality (steric, hydrophobic, and polar) and also a subtle
steric preference for trans stereochemistry over cis
stereochemistry.
1
3
4
5
6
7
8
9
H
151 (0.97)
8.6 (0.97)
CH2CH@CH2
trans-CH2CH@CHCH2OCOPhSO2NH2 4.9 (0.97)
cis-CH2CH@CHCH2OCOPhSO2NH2
trans-CH2CH@CHCH2OCOPh
trans-CH2CH@CHCH2OH
cis-CH2CH@CHCH2OH
9.3 (0.98)
4.7 (0.96)
5.3 (0.97)
9.1 (0.95)
7.7 (0.96)
CH2CH2CH2CH@CH2
a bCAII binding data utilizing competitive displacement of DNSA
from bCAII, experiments performed in triplicate. Kd of DNSA was
0.3 lM.16
Acknowledgments
The authors thank the Australian Research Council
(Grant Nos. F00103312 and DP0343419) and Griffith
University for research funding.
esters 7 and 8, respectively. Esterification of trans-2-bu-
tene-1,4-diol with 1 equiv of benzoyl chloride followed
by 1 equiv of 1 produced 6 in two steps. Compound 9
was synthesized by the DCC mediated esterification of
1 with 1-pentene-5-ol. All compounds were extensively
characterized.11
References and notes
1. Scozzafava, A.; Supuran, C. T. Curr. Med. Chem. Immun.
Endocr. Metab. Agents 2001, 1, 61.
2. Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T.
J. Enzyme Inhib. Med. Chem. 2004, 19, 199.
3. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925.
4. Pastorekova, S.; Casini, A.; Scozzafava, A.; Vullo, D.;
Pastorek, J.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2004, 14, 869.
5. Supuran, C. T. Expert Opin. Investig. Drugs 2003, 12, 283.
6. Beasley, Y. M.; Overell, B. G.; Petrow, V.; Stephenson, O.
J. Pharm. Pharmacol. 1958, 10, 696.
7. Sigal, G. B.; Whitesides, G. M. Bioorg. Med. Chem. Lett.
1996, 6, 559.
8. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42,
2641.
9. For a review of Dynamic Combinatorial Chemistry,
see Lehn, J.-M. Chem. Eur. J. 1999, 5, 2455.
10. Supuran, C. T.; Scozzafava, A. J. Enzyme Inhib. 2000, 15,
597.
11. Preparation of allyl-4-(aminosulfonyl)benzoate (3). To a
solution of 1 (2.0 g, 9.94 mmol) in allyl alcohol (80 mL)
at 0 ꢁC was added H2SO4 (4 drops). The reaction was
Parent compound 1, lead compound 3, and the families
of novel compounds 4–9 were each assayed for bCAII
enzyme binding by a fluorescent competitive binding as-
say, results of which are presented in Table 1.16 The
fluorescence-based assay relies on the competition for
the active site of bCAII between the ligand 5-(dimeth-
ylamino)-1-naphthalenesulfonamide (DNSA) and the
test compounds.1,7,17 Upon excitation at 290 nm (an
absorption minimum for DNSA) fluorescence is detect-
ed at 460 nm (from the bCAII–DNSA complex). The
equilibrium dissociation constant (Kd) of DNSA was
measured as 0.3 lM.16
The parent compound 1 was a relatively weak inhibitor
of bCAII, with an equilibrium dissociation constant
(Ki) of 151 nM. The simple derivatization of the carbox-
ylic acid of 1 to generate esters 4–9 yielded compounds
with high affinity for bCAII. The allyl ester 3 had a Ki
of 8.6 nM, 17-fold > 1. The bis-sulfonamide trans isomer
4 had higher bCAII affinity again (Ki = 4.9 nM, 31-
fold > 1, 1.8-fold > 3), while the cis isomer
5

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S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5429–5433
stirred at 70 ꢁC for 48 h, then cooled to room temper-
(m, 2H, OCH2CH@), 4.80 (br s, 1H, OH), 4.09–4.08 (m,
2H, @CHCH2OH); 13C NMR (100 MHz, CDCl3, ppm):
d 165.3 (CO), 148.8 (OCH2CH@), 136.1, 133.0, 130.6,
126.7, 124.1 (ArCH,@CHCH2OH), 61.9 (OCH2CH@),
57.9 (@CHCH2OH); ESI-MS: m/z [MÀH]À 269.9. Anal.
Calcd for C11H13NO5S: C, 48.70; H, 4.83; N, 5.16.
Found: C, 48.73; H, 4.85; N, 5.18.
1-Pentenyl-4-(aminosulfonyl)benzoate (9). Preparation
from 4-penten-1-ol. (Yield 55%). 1H NMR (200 MHz,
CDCl3, ppm): d 8.12–8.09 (m, 2H, ArH), 7.94–7.91 (m,
2H, ArH), 7.52 (br s, 2H, NH2), 5.83 (tt, 1H, J = 17.2,
10.4, 6.4 Hz, @CH), 5.00 (ddt, 1H, J = 17.2, 10.4 Hz,
@CH2), 4.28 (t, 2H, J = 13.2 Hz, OCH2), 2.15 (m, 2H,
OCH2CH2CH2), 1.79 (m, 2H, OCH2CH2CH2); 13C
NMR (100 MHz, CDCl3, ppm): d 165.5 (CO), 148.7
(@CH), 138.4, 133.2, 130.6, 126.7 (ArCH), 116.1
(@CH2), 65.3 (OCH2), 30.3 (OCH2CH2CH2), 27.9
(OCH2CH2CH2), ESI-MS: m/z [MÀH]À 268.1. Anal.
Calcd for C12H15NO4S: C, 53.52; H, 5.61; N, 5.20.
Found: C, 53.53; H, 5.36; N, 5.07.
Preparation of (2E)-4-(benzoyloxy)but-2-enyl-4-(amin-
osulfonyl)benzoate (6). To a solution of (2E)-butene-1,4-
diol (1 g, 11.3 mmol) in CH2Cl2/pyridine (4:1, v/v, 20 mL)
was added benzoyl chloride (397 mg, 2.8 mmol) dropwise
over 2 h. The solution was stirred at room temperature
for 24 h and then concentrated. The residue was redis-
solved in CH2Cl2 (25 mL) and washed with 1 M HCl
solution (2 · 20 mL), saturated NaHCO3 (1 · 20 mL),
and saturated brine (1 · 20 mL). The organic phase was
dried over MgSO4 and evaporated to afford the mono-
benzoate intermediate (1.7 g, 78%), which was used in the
next step without further purification. To a mixture of
the mono-benzoate in DMF (10 mL) were added 1 (2.3 g,
11.3 mmol), DCC (2.33 g, 11.3 mmol), and DMAP
(50 mg, 0.4 mmol). The solution was stirred at room
temperature for 6 h. The reaction mixture was filtered
through Celite and the filtrate was concentrated to give a
clear oil. Purification by solid-phase extraction on
normal-phase silica sorbent (eluted with DCM/methanol
20:1, v/v) to generate 6 as a white solid (198 mg) in 9 %
yield over two steps. 1H NMR (200 MHz, CDCl3, ppm):
d 8.12–8.09 (m, 2H, ArH), 7.95–7.991 (m, 4H, ArH),
7.65–7.61 (m, 1H, ArH), 7.44 (br s, 2H, NH2), 7.51–7.47
(m, 2H, ArH), 5.93–5.91 (m, 2H, @CH), 5.00–4.96 (m,
4H, CH2); 13C NMR (100 MHz, CDCl3, ppm): d 165.9
(CO),163.2 (CO), 148.8 (OCH2CH@CH), 148.7
(OCH2CH@CH), 134.1, 133.1, 130.6, 129.8, 129.3,
128.7, 126.7 (ArCH), 61.7 (OCH2CH@), 61.2
(OCH2CH@): ESI-MS: m/z [MÀH]À 374.0. Anal. Calcd
for C18H17NO6S: C, 57.59; H, 4.56; N, 3.73. Found: C,
57.93; H, 4.79; N, 3.78.
ature and neutralized by addition of solid NaHCO3. The
crude reaction mixture was concentrated and the residue
was dissolved in DCM (25 mL). The organic phase was
washed with H2O (2 · 25 mL), dried over MgSO4,
filtered, and the solvent removed. The crude material
was recrystallized from MeOH to yield compound 3
1
(690 mg, 29% yield). H NMR (200 MHz, CDCl3, ppm):
d 8.15–8.11 (m, 2H, ArH), 7.95–7.92 (m, 2H, ArH), 7.54
(br s, 2H, NH2), 6.07–5.98 (m, 1H, @CH), 5.33 (ddt,
2H, J = 16.6, 10.4 Hz, @CH2), 4.81 (dt, 2H, J = 5.2, 1.6
Hz, CH2); 13C NMR (100 MHz, CDCl3, ppm): d 165.1
(CO), 148.8 (@CH), 133.0, 132.9, 130.6, 126.8 (ArCH),
118.9 (@CH2), 66.3 (CH2); ESI MS: m/z [MÀH]À 240.3;
HRMS (ESI). Calculated for m/z [MÀH]ÀC10
H10N1O4SÀ: 240.0336. Found: 240.0341; Anal. Calcd
for C10H11N1O4S: C, 49.78; H, 4.60; N, 5.81. Found: C,
49.82; H, 4.66; N, 5.67; mp: 106 ꢁC (in agreement with
literature6).
Preparation of (2E)-but-2-en-1,4-diyl-bis[(aminosulfo-
nyl)benzoate] (4) as a representative of substituted allyl
ester-benzene sulfonamides 4, 5, 7–9. To a mixture of 1
(0.2 g, 1 mmol) and (2E)-butene-1,4-diol (43 mg, 0.5
mmol) in DMF (10 mL) were added DCC (206 mg,
1 mmol) and DMAP (5 mg, 0.041 mmol). The reaction
mixture was stirred at room temperature for 4 h. The
reaction mixture was filtered through Celite and the
filtrate was concentrated under high vacuum to give a
clear oil. The crude material was purified by solid-phase
extraction on normal-phase silica sorbent (eluted with
DCM/methanol 20:1, v/v) to give a white solid. Recrys-
tallization from MeOH yielded 4 (168 mg, 74% yield) as
1
small white needles. H NMR (200 MHz, CDCl3, ppm):
d 8.13–8.10 (m, 4H, ArH), 7.94–7.92 (m, 4H, ArH), 7.53
(br s, 4H, NH2), 5.94–5.92 (m, 2H, @CH), 5.01–5.00 (m,
4H, CH2); 13C NMR (100 MHz, CDCl3, ppm): d 165.3
(CO), 148.8 (@CH), 132.9, 130.6, 128.9, 126.8 (ArCH),
61.8 (CH2); ESI-MS: m/z[MÀH]À 453.0; Anal. Calcd for
C18H18N2O8S2: C, 47.57; H, 3.99; N, 6.16. Found: C,
47.58; H, 4.03; N, 6.06.
(2Z)-But-2-en-1,4-diyl-bis[(aminosulfonyl)benzoate]
(5).
Preparation from (2Z)-butene-1,4-diol. (Yield 21%). 1H
NMR (200 MHz, CDCl3, ppm): d 8.13-8.10 (m, 4H,
ArH), 7.94–7.91 (m, 4H, ArH), 7.53 (br s, 4H, NH2),
5.94–5.92 (m, 2H, @CH), 5.01–5.00 (m, 4H, CH2); 13C
NMR (100 MHz, CDCl3, ppm): d 165.3 (CO), 148.8
(@CH), 132.9, 130.6, 128.9, 126.8 (ArCH), 61.8 (CH2);
ESI-MS: m/z [M-H]À 452.9. Anal. Calcd for
C18H18N2O8S2: C, 47.57; H, 3.99; N, 6.16. Found: C,
47.41; H, 4.02; N, 6.11.
(2E)-4-Hydroxybut-2-enyl-4-(aminosulfonyl)benzoate (7).
Preparation from (2E)-butene-1,4-diol. (Yield 26%). 1H
NMR (200 MHz, CDCl3, ppm): d 8.11–8.08 (m, 2H,
ArH), 7.94–7.91 (m, 2H, ArH), 7.49 (br s, 2H, NH2),
5.74 (dtt, 1H, J = 11.2, 5.6, 1.2 Hz, @CHCH2OH), 5.61
(dtt, 1H, J = 11.2, 6.4, 1.6 Hz, OCH2CH@), 4.89–4.87
(m, 2H, OCH2CH@), 4.79 (br s, 1H, OH), 4.09–4.08 (m,
2H, @CHCH2OH); 13C NMR (100 MHz, CDCl3, ppm):
d 165.3 (CO), 148.8 (OCH2CH@CH), 136.1, 133.0, 130.6,
126.7, 124.1 (ArCH, @CHCH2OH), 61.9 (OCH2CH@),
57.9 (@CHCH2OH); ESI-MS: m/z [MÀH]À 269.9. Anal.
Calcd for C11H13NO5S: C, 48.70; H, 4.83; N, 5.16.
Found: C, 48.67; H, 4.86; N, 4.97.
(2Z)-4-Hydroxybut-2-enyl-4-(aminosulfonyl)benzoate (8).
Preparation from (2Z)-butene-1,4-diol. (Yield 32%). 1H
NMR (200 MHz, CDCl3, ppm): d 8.12-8.09 (m, 2H,
ArH), 7.94–7.91 (m, 2H, ArH), 7.53 (br s, 2H, NH2),
5.74 (dtt, 1H, J = 11.2, 6.0, 1.6 Hz, @CHCH2OH), 5.61
(dtt, 1H, J = 11.2, 4.8, 1.2 Hz, OCH2CH@), 4.89–4.87
12. Crystal data for 3 were obtained with a Rigaku AFC7R
˚
diffractometer, Mo Ka radiation (k = 0.71073 A), graphite
monochromator, C10H11NO4S, monoclinic, space group
P21/a, cell dimensions a = 16.180(2), b = 5.162(1), c =
3
13.495(2) A, b = 98.14(1), V = 1115.7(3) A , Dcalc = 1.44 g cmÀ3
,
˚
˚
Z = 4, F(000) = 504, and l = 0.288 mmÀ1. Data were
collected at 295(2) K using x À 2h scans in the range
h = 2.80–25.0ꢁ. A total of 2236 reflections were collected,
1961, were unique (Rint = 0.0192). The structure was refined
by full-matrix least squares on F2. The non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were con-
˚
strained as riding atoms with C–H = 0.95 A, N–
˚
H = 0.85 A. Uiso (H) values were set to 1.2 Ueq of the parent
atom. Atom coordinates, bond lengths, angles, and thermal
parameters have been deposited at the Cambridge Crystal-
lographic Data Centre: (CCDC 278809)..
13. TeXsan for Windows V1.06, 2001; Molecular Structure
Corporation: The Woodlands, TX, 2001.

S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5429–5433
5433
14. Sheldrick, G. M. SHELX-97 Program for Crystal Structure
Determination, University of Go¨ttingen: Go¨ttingen, 1997.
15. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
16. Procedure for bCAII enzyme binding assay. Compounds 1,
3–9 were assessed for their ability to inhibit the binding of
DNSA to bCAII (CAII from bovine erythrocytes, Sigma–
Aldrich, catalog No. C2522, lot number 044K6064).
Enzyme assays were carried out in 96-well microtiter plates
(Nunc F96) in an assay volume of 200 lL. Each assay
contained bCAII (180 nM); DNSA (3 lM, equals 10 times
the Kd value), incubation buffer (phosphate buffer, pH 7.2),
and test compound (at 15 concentrations, triplicate deter-
minations) in DMSO. The final DMSO concentration in the
assay was 1%, this concentration of DMSO did not decrease
control binding. The assay was incubated for 4 h at 25 ꢁC.
Fluorescence measurements were carried out on a Varian
Cary-Eclipse spectrophotometer in fluorescence mode
using a multiwell plate reader at 25 ꢁC (excitation wave-
length of 290 nM, emission wavelength of 460 nM). Known
compounds (AAZ and EZA) were used to characterize this
assay procedure. Data were fitted to a sigmoidal dose–
response equation using nonlinear regression analysis
(GraphPad Prism V4, San Diego, CA, USA). The measure-
ment of the Kd of DNSA was determined by titrating bCAII
(180 nM in pH 7.2 phosphate buffer) with DNSA (100–
3500 nM) and monitoring the fluorescence as described
above. Data were fitted to an equilibrium one-site binding
model using nonlinear regression analysis. The Kd of DNSA
was calculated as 0.3 lM and is comparable with the
literature.2
17. Chen, R. F.; Kernohan, J. C. J. Biol. Chem. 1967, 242,
583.