Comparison of sulfamate and sulfamide groups for the inhibition of carbonic anhydrase-II by using topiramate as a structural platform

Article abstract of DOI:10.1021/jm040124c

This paper examines the relative effectiveness of sulfamate and sulfamide groups for the inhibition of carbonic anhydrase-II (CA-II). Topiramate (1) and its sulfamide analogue 4, and 4,5-cyclic sulfate 6 and its sulfamide analogue 5, were compared for inhibition of human CA-II. A colorimetric assay, based on the pH shift that accompanies hydration of carbon dioxide, and an esterase assay were used. For these bioisosteric pairs, 1/4 and 6/5, the sulfamate compound was markedly more potent than its sulfamide counterpart. A similar, large difference in potency was also observed for the sulfamate/sulfamide pairs 14/15 and 16/17. These results indicate that the sulfamide moiety is not particularly suitable for obtaining potent carbonic anhydrase inhibition. A discussion of this structure-activity relationship with respect to the interactions of 1 and 6 with CA-II from published X-ray data is presented. A metabolic acidosis study was performed in rats with 1, 4, 6, and 2, and the results are discussed with respect to the degree of inhibition of CA-II in vivo.

Full text of DOI:10.1021/jm040124c

J. Med. Chem. 2005, 48, 1941-1947
1941
Comparison of Sulfamate and Sulfamide Groups for the Inhibition of Carbonic
Anhydrase-II by Using Topiramate as a Structural Platform†
Bruce E. Maryanoff,* David F. McComsey, Michael J. Costanzo, Coralie Hochman,
Virginia Smith-Swintosky, and Richard P. Shank
Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, Spring House, Pennsylvania 19477-0776
Received June 30, 2004
This paper examines the relative effectiveness of sulfamate and sulfamide groups for the
inhibition of carbonic anhydrase-II (CA-II). Topiramate (1) and its sulfamide analogue 4, and
4,5-cyclic sulfate 6 and its sulfamide analogue 5, were compared for inhibition of human CA-
II. A colorimetric assay, based on the pH shift that accompanies hydration of carbon dioxide,
and an esterase assay were used. For these bioisosteric pairs, 1/4 and 6/5, the sulfamate
compound was markedly more potent than its sulfamide counterpart. A similar, large difference
in potency was also observed for the sulfamate/sulfamide pairs 14/15 and 16/17. These results
indicate that the sulfamide moiety is not particularly suitable for obtaining potent carbonic
anhydrase inhibition. A discussion of this structure-activity relationship with respect to the
interactions of 1 and 6 with CA-II from published X-ray data is presented. A metabolic acidosis
study was performed in rats with 1, 4, 6, and 2, and the results are discussed with respect to
the degree of inhibition of CA-II in vivo.
Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc-contain-
ing enzyme that catalyzes a reversible reaction involving
the hydration of carbon dioxide and the dehydration of
-
bicarbonate: CO2 + 2H2O a HCO3 + H3O+.1-4 This
enzymatic reaction is essential to many physiological
anion-exchange processes,5 and CA inhibitors are effec-
tive drugs for the treatment of glaucoma by reducing
aqueous humor formation to lower intraocular pres-
sure.3,6,7 Of the three CA families, designated R, â, and
γ, the mammalian R-CA family encompasses 14 geneti-
cally distinct isozymes, CA-I through CA-XIV, with
different tissue and intracellular distribution.4 CA-II,
the most widely studied form, which is found in many
different organs and cell types, has very high catalytic
activity for CO2 hydration. Numerous potent inhibitors
of CA-II, generally having a key primary sulfonamide
functionality, R-SO2NH2, have been reported.3,8,9 The
sulfonamide in these compounds coordinates with the
tetrahedral ZnII atom in the enzyme active site, which
is also coordinated to the imidazole groups of three
histidine residues (His-94, His-96, and His-119 in hu-
man CA-II), thereby displacing the water/hydroxide
ligand that is intimately involved in CO2 hydration.9,10
Our interest in carbonic anhydrase enzymes derives
from working with topiramate (1),11 a novel sugar
sulfamate anticonvulsant that is marketed worldwide
for the treatment of epilepsy and migraine.12 Following
our discovery of topiramate,11b,c we were prompted to
investigate its potential to inhibit CA because of the
presence of a sulfamate functionality (R-OSO2NH2).
This effort entailed the use of CA isozymes derived from
different tissues and the determination of CO2
hydration.11a,b,13,14 In a CO2 hydration bioassay involving
enzymes from rat blood (CA-I + CA-II) or rat myelin
(CA-II), we found that topiramate is a moderate inhibi-
tor with IC50 values of 8.9 or 1.4 µM, respectively.11a,16
Also, topiramate is reported to be a moderate inhibitor
of purified human erythrocyte CA-II, with a Ki value of
5 µM, which is 125 times less potent than the value
obtained with the reference sulfonamide acetazolamide
(2).13,17 With respect to other CA isozymes, topiramate
is generally a moderate inhibitor, with IC50 or Ki values
usually well above 100 nM.18 However, recent publica-
tions have reported low-nanomolar inhibition of human
recombinant CA-II (Ki ) 5 nM) by topiramate via an
esterase assay (rather than a CO2-hydration assay),
twice as potent as acetazolamide (Ki ) 12 nM).19 The
study by Casini et al.19a also brought to light some
sulfamide derivatives that are structurally related to
topiramate, and they found that close analogue 3 is
essentially devoid of CA-II inhibition in the esterase
assay (Ki > 100 µM). By contrast, other sulfamides were
found to be potent CA-II inhibitors,19a,20 and the study
by Abbate et al.20 also suggests that the sulfamide group
should be favorable for binding within the CA active site
cleft. Because 319a differs from topiramate by having a
* To whom correspondence should be addressed. E-mail:
bmaryano@prdus.jnj.com. Phone: 215-628-5530 Fax: 215-628-4985.
† This paper is dedicated to the memory of Dr. Paul A. J. Janssen,
one of the most prolific pharmaceutical researchers of all time.
10.1021/jm040124c CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/13/2004

1942 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Maryanoff et al.
Scheme 1
Scheme 2
carbonyl group installed, not just by changing a sulfa-
mate to a sulfamide, we thought that it would be
important to determine the CA-II inhibition for the
direct sulfamide analogue of topiramate, i.e., 4.11a
Herein, we report the evaluation of sulfamide 4, as well
as its 4,5-cyclic sulfate congener, 5, which is the direct
sulfamide analogue of 6, a very potent sulfamate-based
CA inhibitor.11a Our results for sulfamate and sulfamide
bioisosteric pairs indicate that the sulfamide moiety is
not particularly suitable for obtaining potent carbonic
anhydrase inhibition.
can be sensitive to nucleophilic ring opening, especially
at higher temperatures such as 100 °C. Because of the
cyclic sulfate group, the azide displacement step was
carried out at a lower temperature (23 °C) than that
used in the synthesis of 4. Compounds 14-17 were
prepared in a straightforward manner (Supporting
Information).
Results and Discussion
Synthesis. Sulfamide 4, reported earlier,11a was
synthesized by an improved method, as outlined in
Scheme 1. Commercially available diacetone fructose
(DAF) was treated with triflic anhydride to give triflate
7, which was immediately converted to azide 8 in near
quantitative yield. Catalytic hydrogenation cleanly af-
forded the desired amine 9 in a superior yield (ca. 80%
from DAF) compared with the previously reported
method (ca. 50%).11a Reaction of amine 9 with sulfamide
in refluxing 1,4-dioxane gave target 4 in 75% yield,
which was far superior to the 20% yield obtained in the
original synthesis that employed sulfamoyl chloride and
sodium hydride. Sulfamide 5 was synthesized in good
overall yield from alcohol 1011a in a similar manner, via
11-13 (Scheme 2). It is remarkable that the sulfamide
reaction could be effected with high efficiency despite
the presence of the cyclic sulfate functionality, which
Enzyme Inhibition. Compounds 1, 2, 4, 5, and 6
were tested for inhibition of purified human CA-II
obtained from erythrocytes (Sigma Chemical Co.) by
using an assay involving the native action of the
enzyme, namely, the hydration of CO2, via a pH-shift
measurement (Table 1). Sulfamates 1 and 6 inhibited
CA-II with Ki values of 0.50 and 0.012 µM, respectively,
which compare with a Ki value of 0.0033 µM for
acetazolamide (2), which is an expected level of inhibi-
tion for this reference compound and serves to validate
the enzyme assay (positive control). By contrast, sulfa-
mides 4 and 5 exhibited comparatively weak CA-II
inhibition, with Ki values of 650 and 20 µM, respectively.
From these data, the sulfamide-based bioisosteres, 4
and 5, are approximately 1000 times less potent than
their sulfamate counterparts, 1 and 6.

Sulfamates vs. Sulfamides for CA-II Inhibition
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1943
Table 1. Carbonic Anhydrase Inhibition Dataa
cmpd
pH-shift CA-II, Ki (µM)b
esterase CA-II, Ki (µM)b
1
2
0.50 (0.39-0.64)
0.0033 (0.0026-0.0043)
650 (390-1070)
20 (12-35)
0.43 (0.39-0.49)
0.018 (0.016-0.020)
340 (240-490)
4
5
25 (22-29)
6
0.012 (0.011-0.015)
9.6 (5.3-17.2)
0.038 (0.030-0.047)
7.7 (6.0-9.7)
0.75 (0.64-0.88)
L-1c
L-6d
0.91 (0.59-1.4)
a Inhibition of human CA-II, as described in the Experimental
Section. b The 95% confidence intervals are given in parentheses.
See the Experimental Section for details on the method. IC50
values for 1, 2, 4, 5, and 6 in the pH-shift assay: 1.5, 0.011, 1950,
c
d
60, and 0.036 µM. L enantiomer of 1. L enantiomer of 6.
With the above confirmation of our earlier results
with 1 as an inhibitor of CA-II, we became curious about
the much more potent CA-II inhibition reported for the
esterase method.19 In our hands, the esterase assay for
topiramate (1) and acetazolamide (2) afforded Ki values
of 0.43 and 0.018 µM, respectively (Table 1), instead of
0.005 and 0.012 µM, as reported by Supuran and co-
workers.19 The Ki values in the esterase assay for
sulfamate 6 and sulfamides 4 and 5, were 0.038, 340,
and 25 µM, respectively. It is not clear to us what factors
are behind the observed discrepancy between the Ki
values for 1 and 2 measured by each group, although
the source of CA-II is different (purified native vs.
recombinant21). Fairly consistent results were obtained
between the two assay modes (Table 1), except for
acetazolamide (2), which had a pH-shift Ki value of
0.0033 and an esterase Ki value of 0.018 µM. Our results
for the L enantiomer of 1 and the L enantiomer of 6 in
the paired assays were also consistent (Table 1).
Sulfamates and Sulfamides as Inhibitors of CA-
II. Although aryl and heteroaryl sulfonamides are
archetypal structures for inhibitors of carbonic anhy-
drases, sulfamate and sulfamide derivatives can also
give rise to potent inhibitors.11a,e,19-21 In an X-ray
crystallographic study of human CA-II complexed with
sulfamic acid and sulfamide, the parent core structures
for the sulfamate and sulfamide derivatives, respec-
tively, the expected ZnII-nitrogen binding is reflected,
as are some new interactions resulting from the het-
eroatoms (O or N).20 These new interactions were
suggested to offer opportunities for the design of new
inhibitors. However, because sulfamic acid and sulfa-
mide themselves are relatively weak inhibitors of CA-
II (esterase assay Ki values of 97 and 82 µM, respec-
tively),19a,21 the relevance of those new interactions to
CA inhibitor design might be questioned. Consequently,
it would be worthwhile to compare sulfamate and
sulfamide molecules that are strict bioisosteres and that
represent meaningfully potent inhibitors of CA. Given
that the cyclic sulfate topiramate analogue 6 is a very
potent inhibitor of human CA-II with a Ki value of 12
nM (Table 1), it provides a suitable benchmark com-
pound for the sulfamate species. However, its sulfamide
isostere, 5, has remarkably weaker human CA-II inhibi-
tory activity (Ki ) 20 µM; 1000-fold less potent). A
similar dichotomy is observed between the human CA-
II inhibition of topiramate (1) (Ki ) 0.50 µM) and that
of its sulfamide analogue, 4 (Ki ) 650 µM; ca. 1000-fold
less potent). This pattern for the sulfamate/sulfamide
pairs also applies to the CA-II inhibition results from
the esterase-based assay (Table 1).
Figure 1. Schematic representation of interactions of 6 within
the active site of human CA-II.
Since these substantial potency differences emanate
from sulfamate/sulfamide bioisosteric pairs, it would
appear that the sulfamide moiety is not especially
competent for obtaining potent inhibition of CA-II.
Considering the importance of this observation, we
sought to probe this issue further with some additional
sulfamate/sulfamide pairs. Thus, we synthesized and
examined the pairs 14/15 and 16/17, which have some
structural distinction from the other pairs studied. In
this manner, we could gain support for the generality
of this effect. CA-II inhibition IC50 values for the pairs
14/15 and 16/17 were determined in the pH-shift assay.
Sulfamate 14 and sulfamide 15 had IC50 values of 0.13
and 71 µM, respectively, and sulfamate 16 and sulfa-
mide 17 had IC50 values of 0.28 and 129 µM, respec-
tively. Again, in the case where sulfamate compounds
are reasonably potent inhibitors of CA-II, the corre-
sponding sulfamide compounds are considerably less
potent, by a large factor of approximately 500.22
Binding Interactions of Sulfamates 1 and 6 with
CA-II. We have described the X-ray structure of sulfa-
mate 6 complexed with human CA-II,11e the collected
interactions for which are depicted schematically in
Figure 1. Casini et al. have reported on the X-ray
structure of topiramate (1) complexed with human CA-
II,19a the interactions for which are depicted in Figure
3
2. The pyranose rings of 1 and 6 adopt the S0 skew
conformation (Figures 1 and 2), which we have cor-
related with potent anticonvulsant activity.11a,e The key
interactions between each inhibitor molecule and the
CA active site are very similar for these two complexes
and dependent on this 3S0 skew conformation for a good
steric fit (Figure 3). However, the two ligands manage
to dock into the active site of CA-II in a different
structural context (Figure 3). The sulfamate nitrogen
of both 1 and 6 is coordinated to ZnII and hydrogen
bonded to the hydroxyl of Thr199 in basically the same
manner. Likewise, the binding modes of both 1 and 6
have the sulfamate oxygen hydrogen bonding to the
R-amino group of Thr199 and the C2 oxygen of the 2,3-

1944 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Maryanoff et al.
The bridging sulfamate oxygen (CH2-O-S) does not
play a meaningful role in the binding of 1 and 6 with
CA-II.11e,19a By contrast, in the complex of sulfamic acid
with CA-II there are important interactions of the
bridging sulfamate oxygen.20 It would appear that the
anionic properties of the oxygen in the sulfamic acid case
govern interactions within that enzyme complex and
thereby distinguish it from the sulfamate ester cases.
In the absence of interactions of the bridging sulfamate
oxygen of 1 and 6, how might one explain the poor
CA-II inhibitory activity of sulfamide bioisosteres 4 and
5, respectively? Perhaps, the bioisosteric CH2NHSO2
group has an altered conformation in the ligand such
that the terminal NH2 (or NH-) group cannot complex
effectively with the Zn atom and still retain appropriate
hydrophobic and hydrogen-bonding interactions for the
rest of the molecule. Another contributing factor could
be the difference in pKa between the sulfamate and
sulfamide groups. For sulfamates 1 and 6, we deter-
mined pKa values of 8.66 and 8.51, respectively, whereas
for sulfamide 4 we determined a pKa value of 10.7.
Because 4 is much less acidic, it will have a much lower
population of the anionic form that is required for
binding to ZnII in the active site of CA-II.9,10,23
Figure 2. Schematic representation of interactions of topi-
ramate (1) within the active site of human CA-II.
CA Inhibition and Topiramate’s Antiepileptic
Action. Given the topic under discussion in this paper,
it would be instructive to consider the relevance of CA
inhibition to the antiepileptic activity of topiramate.
While attempting to elucidate the mechanisms of action
for topiramate, we suggested that in some forms of
epilepsy the inhibition of CA-II or CA-IV may contribute
to the pharmacological activity.11d However, a cross-
tolerance study with acetazolamide and topiramate
indicated that inhibition of CA-II and CA-IV is not a
major factor for topiramate’s anticonvulsant activity in
mice.11c Also, when we conducted pharmacological stud-
ies in rats with sulfamate 6, which is much more potent
than topiramate as a CA-II inhibitor,11a we observed
frank diuresis and alkalinization of the urine, which is
characteristic of CA inhibition.24 Such diuresis has not
been observed with topiramate in preclinical experi-
ments or in clinical practice. However, there have been
clinical reports of metabolic acidosis, which presumably
emanates from inhibition of CA-II and possibly other
CA isozymes.25 To follow up on this point, we investi-
gated the effects of topiramate (1), 6, acetazolamide (2,
positive control), and 4 (negative control) on the pH
values of arterial blood in rats. Since inhibition of CA-
II is likely to result in metabolic acidosis, with a dose-
dependent relationship, the blood pH readout should
reflect the degree of CA-II inhibition in vivo.26 With a
normal pH for rat blood of 7.40-7.43, the threshold for
metabolic acidosis would be a pH of less than 7.40.
acetonide ring hydrogen bonding to the side chain amino
group of Gln92. In addition, various hydrogen-bonding
interactions occur between both inhibitors and key
residues of CA-II: Asn62, Gln92, and Thr200. However,
the binding modes of 1 and 6 differ in that the molecules
are rotated by ca. 180°, relative to each other, about the
sugar anomeric C-CH2 bond, i.e., the 2,3-ring of one
ligand has exchanged position with the 4,5-ring of the
other ligand. Hence, the 4,5-cyclic sulfate of 6 is directed
toward the solvent and forms hydrogen bonds with two
water molecules and the hydroxyl of Thr200. In addi-
tion, the C2 oxygen of the 2,3-acetonide and the pyra-
nose ring oxygen of 6 form hydrogen bonds with the
amide side chain of Asn62. By contrast, for 1 the 2,3-
acetonide is directed toward the solvent and hydrogen
bonded to a water molecule and Gln92. In addition, the
C5 oxygen of the 4,5-acetonide forms a hydrogen bond
with the amide side chain of Asn62. The difference in
binding modes for 1 and 6 can be rationalized by the
structural differences between each ligand. Perhaps, it
is energetically more favorable for the more polar 4,5-
cyclic sulfate of 6 to be solvated (e.g., by water). This
viewpoint is consistent with the fact that the 4,5-cyclic
sulfate has three hydrogen bonding interactions (Figure
1) and three associated water molecules, whereas the
colocalized 2,3-acetonide of 1 has only two hydrogen-
bonding interactions and one associated water molecule
(Figure 2). The remarkable distinction in binding modes
for 1 and 6 underscores the flexibility that is available
in the design of active-site-directed inhibitors of CA-II.
However, it is important to note that the active site of
CA-II does not accommodate the L isomers11a of 1 and
6 nearly as well as the D isomers, which is clearly
reflective of specific steric requirements (Table 1). In
the pH-shift assay, the L enantiomer of 111a was ca. 20
times less potent than D isomer 1, and the L enantiomer
of 611a was ca. 80 times less potent than D isomer 6.
The weaker affinities of the L isomers of 1 and 6 relative
to the D isomers is consistent with our structural
analysis.
We orally dosed adult male rats with 100 mg/kg of
topiramate, 6, acetazolamide, or 4, sampled their arte-
rial blood 2 h later, and measured the pH. Acetazola-
mide (CA-II Ki ) 11 nM) gave a pH of 7.26 ( 0.06,
reflecting frank acidosis, whereas 4 (Ki ) 650,000 nM)
gave a pH of 7.40 ( 0.03, reflecting a lack of activity
(essentially the same as the value for vehicle, pH ) 7.42
( 0.07). Compound 6 (Ki ) 12 nM) was very potent in
inducing acidosis (pH ) 7.25 ( 0.07), whereas topira-
mate (Ki ) 500 nM) exhibited weak acidosis activity (pH

Sulfamates vs. Sulfamides for CA-II Inhibition
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1945
Figure 3. Stereoview of 1 (green) and 6 (white) complexed with human CA-II, from the relevant X-ray crystal structures.
1-[(Aminosulfonyl)amino]-1-deoxy-2,3-O-(isopropyl-
idene)-4,5-O-sulfonyl-â-d-fructopyranose (5).27 A solution
of 2,3-O-(isopropylidene)-4,5-O-sulfonyl-â-D-fructopyranose
(10)11a and pyridine (6 equiv) in dichloromethane was treated
at -20 °C with triflic anhydride (3 equiv), and the reaction
was stirred for 2 h. The crude triflate was purified by flash
column chromatography to give oily 11, which was dissolved
in DMF and treated with sodium azide (1.1 equiv) at 23 °C
for 24 h. Crude azide 12 in ethanol was hydrogenated in the
presence of Pd/C to give amine 13, which was reacted with
sulfamide (4 equiv) in refluxing 1,4-dioxane for 30 min to
produce crude 5. Purification by flash column chromatography
furnished pure sulfamide 5. (MS M - 1: 337. 1H NMR: C, H,
N.)
) 7.37 ( 0.04). Our data indicate that the degree of
blood acidification, in terms of pH, is reasonably con-
sistent with the level of CA-II inhibition for the com-
pounds of interest. It is clear that topiramate causes
limited metabolic acidosis compared to the more potent
CA-II inhibitors. Because topiramate does not manifest
strong inhibition of human CA-II in vitro and induces
just mild acidosis in rats in vivo, we suggest that other,
CNS-based mechanisms are probably the dominant
factors in topiramate’s antiepileptic action.11d
Conclusion
We examined some sulfamate/sulfamide bioisosteric
pairs to determine the relative effectiveness of the
sulfamate and sulfamide groups for inhibiting human
CA-II. Topiramate (1) and its sulfamide analogue 4, and
4,5-cyclic sulfate 6 and its sulfamide analogue 5, were
compared, and a dramatic difference in potency was
observed. This finding also applied to the sulfamate/
sulfamide pairs 14/15 and 16/17. Given our results for
these sulfamate/sulfamide pairs, it would appear that
the sulfamide moiety is not particularly suitable for
obtaining potent carbonic anhydrase inhibition. Our
study of metabolic acidosis in rats with topiramate (1),
6, acetazolamide (2), and 4, which reflects on the
inhibition of CA-II in vivo, indicates that topiramate
causes limited metabolic acidosis compared to the more
potent CA-II inhibitors, 6 and 2. Thus, we suggest that
other, CNS-based mechanisms are probably the prin-
cipal source of topiramate’s antiepileptic action.
Carbonic Anhydrase Inhibition. pH-Shift Assay. Puri-
fied human CA-II, obtained from erythrocytes, was purchased
from Sigma Chemical Co. Inhibition of CA-II was determined
in a “pH-shift” assay performed according to the following
procedure.28 The experiments involved ca. 0.5 mg of CA-II
dissolved in 20 mL of water and then diluted 10-fold to 20-
fold with water (amount of CA-II per sample ) 0.1-0.2 µg).
The decrease in pH resulting from CO2 and H2O being
-
converted to H+ and HCO3 was measured by the change in
the color of the dye bromthymol blue. The reaction was
performed at 0-2 °C under conditions that allowed the
catalyzed reaction to reach an endpoint within 15-25 s and
the uncatalyzed reaction to reach an endpoint in 90-120 s. A
10 mM aqueous HEPES solution was buffered to pH 7.7 at 23
°C with Tris containing bromthymol blue (50 mg/L) and
dithiothreitol (160 mg/L). For the catalyzed reaction, the
contents of each sample included 0.5 mL of the buffered dye
solution, 0.1 mL of CA-II solution, 0.1 mL of test compound
solution (0.1 mL of water for catalyzed control samples), and
0.3 mL of fresh seltzer water. The temperature of the solutions
(final volume ) 1.0 mL) was maintained at 0-2 °C. For
uncatalyzed control samples, 0.1 mL of water was substituted
for the 0.1 mL of enzyme solution. The seltzer water was added
last to initiate the timed reaction. Each sample was gently
mixed, placed in ice water, and gently mixed every 15-30 s
until the solution turned yellow, at which point the time was
recorded. Each compound was tested in triplicate at each
specified concentration. Test compounds were dissolved in
dimethyl sulfoxide at 100 mM and then diluted in water to a
concentration 10 times the final concentration in the test tube.
In this assay, the concentration of the substrate (CO2) de-
creases from ca. 18 to 14 mM and, on average, is about twice
the concentration of the Km for CO2. By use of the Cheng-
Experimental Section27
1-[(Aminosulfonyl)amino]-1-deoxy-2,3:4,5-bis-O-(iso-
propylidene)-â-D-fructopyranose (4).27 Diacetone fructose
(DAF) and pyridine (6 equiv) in dichloromethane were treated
with triflic anhydride (3 equiv) at -20 °C, and the mixture
was stirred for 2 h. The crude triflate was purified by column
chromatography to give oily 7, which was dissolved in di-
methylformamide (DMF) and treated with sodium azide (1.1
equiv) at 55 °C for 6 h. Crude azide 8 in ethanol was
hydrogenated in the presence of Pd/C, and acid/base workup
gave amine 9. Reaction of 9 with sulfamide (2 equiv) in
refluxing 1,4-dioxane for 2 h produced crude 4, which was
purified by flash column chromatography to give pure sulfa-
Prusoff equation,29 the Ki value determined is approximately
1
/
of the IC50 value. Concentration-inhibition curves were
3
1
mide 4. (MS M - 1: 337. H NMR: C, H, N.)
generated for each compound (5-10 concentrations) in 4-12

1946 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Maryanoff et al.
(10) Lindskog, S. Structure and Mechanism of Carbonic Anhydrase.
Pharmacol. Ther. 1997, 74, 1-20.
independent experiments. The IC50 values were obtained using
the PRISM (GraphPad) curve-fitting program in the competi-
tive inhibition mode.
(11) (a) Maryanoff, B. E.; Costanzo, M. J.; Nortey, S. O.; Greco, M.
N.; Shank, R. P.; Schupsky, J. J.; Ortegon, M. E.; Vaught, J. L.
Structure-Activity Studies on Anticonvulsant Sugar Sulfamates
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Derivatives. J. Med. Chem. 1998, 41, 1315-1343. (b) Maryanoff,
B. E.; Nortey, S. O.; Gardocki, J. F.; Shank, R. P.; Dodgson, S.
P. Anticonvulsant O-Alkyl Sulfamates. 2, 3: 4, 5-Bis-O-(1-
methylethylidene)-â-D-fructopyranose Sulfamate and Related
Compounds. J. Med. Chem. 1987, 30, 880-887. (c) Shank, R.
P.; Gardocki, J. F.; Vaught, J. L.; Davis, C. B.; Schupsky, J. J.;
Raffa, R. B.; Dodgson, S. J.; Nortey, S. O.; Maryanoff, B. E.
Topiramate: Preclinical Evaluation of a Structurally Novel
Anticonvulsant. Epilepsia 1994, 35, 450-460. (d) Shank, R. P.;
Gardocki, J. F.; Streeter, A. J.; Maryanoff, B. E. An Overview of
the Preclinical Aspects of Topiramate: Pharmacology, Pharma-
cokinetics, and Mechanism of Action. Epilepsia 2000, 41 (Suppl.
1), S3-S9. (e) Recacha, R.; Costanzo, M. J.; Maryanoff, B. E.;
Chattopadhyay, D. Crystal Structure of Human Carbonic An-
hydrase II Complexed with an Anticonvulsant Sugar Sulpha-
mate. Biochem. J. 2002, 361, 437-441.
Carbonic Anhydrase Inhibition. Esterase Assay. This
method was performed with purified enzyme (see above) and
is based on procedures described previously.30,31 Stock solutions
of the substrate, 4-nitrophenyl acetate (4-NPA), were made
up in 95% ethanol at 4 mM. In most experiments, the reaction
medium was buffered using HEPES (10 mM) adjusted to a
pH of 7.7 with Tris. Preliminary experiments showed that
similar catalytic activity was obtained by using Tris Cl- or
Na+ phosphate as buffering agents. The reaction medium was
comprised as follows: 70 µL of buffer, 10 µL of test compound
(see above), 10 µL of 4-NPA, and 10 µL of enzyme. Water was
substituted for test compound or enzyme as appropriate to
establish the control catalyzed and uncatalyzed reaction rates.
In each experiment, compounds were tested in quadruplicate
by using 96-well plates at 23 °C. The reaction was initiated
by adding enzyme, and the reaction course was monitored at
400 nm. Data for each sample was recorded at each minute
from 0 to 20 min. Because the concentration of substrate (0.4
mM) was at least 25-fold lower than the Km (>10 mM), the
IC50 values obtained were essentially the same as the Ki
values.29,30 The data were analyzed as described above for the
pH-shift assay.
(12) (a) Maryanoff, B. E.; Margul, B. L. Topiramate. Drugs Future
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Rat Metabolic Acidosis Assay. Male Sprague-Dawley
rats (250-300 g) were fasted overnight and dosed orally with
vehicle (0.5% methylcellulose) or 100 mg/kg of topiramate (1),
acetazolamide (2), 6, or 4. Arterial blood samples were collected
2 h postdosing. Blood chemistries, including pH, were mea-
sured by using an iSTAT portable clinical analyzer. The data
in the text represent the mean ( SD for each treatment group,
with eight rats per group (N ) 8).
Acknowledgment. We thank Dr. Brett Tounge for
technical assistance with computer modeling and Dr.
Michael Parker for preparing 16 and 17.
(13) Dodgson, S. J.; Shank, R. P.; Maryanoff, B. E. Topiramate as
an Inhibitor of Carbonic Anhydrase Isozymes. Epilepsia 2000,
41 (Suppl. 1), S35-S39.
Supporting Information Available: General procedures,
synthetic details and analytical data for 4 and 5, detailed
synthetic procedures, and analytical data for 14-17. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(14) (a) A procedure that we used for CA inhibition relies on a color
change of a pH-sensitive dye, such as bromthymol blue.11a,15
Hydrogen ions generated by the consumption of CO2 cause a
decrease in pH with a concomitant change in color. Reactions
were performed at 0-2 °C under conditions that led to an end
point for the catalyzed reaction in 10-20 s (the endpoint for the
uncatalyzed reaction occurred in 90-110 s). (b) Another proce-
dure that we used for CA inhibition relies on a mass spectro-
metric technique for CO2 analysis.13,15 Reactions were generally
performed at 25 °C.
(15) Forster, R. E. Methods for Measurement of Carbonic Anhydrase
Activity. In The Carbonic Anhydrases: Cellular Physiology and
Molecular Genetics; Dodgson, S. J., Tashian, R. E., Gros, G.,
Carter, N. D., Eds.; New York: Plenum Press: 1991; pp 79-98.
(16) The corresponding IC50 values for acetazolamide (2) were 0.037
or 0.024 µM, respectively.
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