Design, synthesis, and biological evaluation of novel carbohydrate-based sulfamates as carbonic anhydrase inhibitors

Article abstract of DOI:10.1021/jm101525j

Carbonic anhydrases (CAs) IX and XII are enzymes with newly validated potential for the development of personalized, first-in-class cancer chemotherapies. Here we present the design and synthesis of novel carbohydrate-based CA inhibitors, several of which were very efficient inhibitors (K_i<10 nM) with good selectivity for cancer-associated CA isozymes over off-target CA isozymes. All inhibitors comprised a carbohydrate core with one hydroxyl group derivatized as a sulfamate. Five different carbohydrates were chosen to present a selection of molecular shapes with subtle stereochemical differences to the CA enzymes active site. Variable modifications of the remaining sugar hydroxyl groups were incorporated to provide an incremental coverage of chemical property parameters that are associated with biopharmaceutical performance. All sulfamate inhibitors displayed ligand efficiencies that are consistent with those reported for good drug lead candidates.

Full text of DOI:10.1021/jm101525j

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Novel
Carbohydrate-Based Sulfamates as Carbonic Anhydrase Inhibitors
Marie Lopez,† Jonathan Trajkovic,† Laurent F. Bornaghi,† Alessio Innocenti,‡ Daniela Vullo,‡
Claudiu T. Supuran,*,‡ and Sally-Ann Poulsen*,†
†Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland 4111, Australia
‡Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Universitꢀa degli Studi di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy
S Supporting Information
b
ABSTRACT: Carbonic anhydrases (CAs) IX and XII are enzymes
with newly validated potential for the development of personalized,
first-in-class cancer chemotherapies. Here we present the design and
synthesis of novel carbohydrate-based CA inhibitors, several of
which were very efficient inhibitors (Ki<10 nM) with good selec-
tivity for cancer-associated CA isozymes over off-target CA iso-
zymes. All inhibitors comprised a carbohydrate core with one
hydroxyl group derivatized as a sulfamate. Five different carbohy-
drates were chosen to present a selection of molecular shapes with
subtle stereochemical differences to the CA enzymes active site.
Variable modifications of the remaining sugar hydroxyl groups were
incorporated to provide an incremental coverage of chemical
property parameters that are associated with biopharmaceutical
performance. All sulfamate inhibitors displayed ligand efficiencies
that are consistent with those reported for good drug lead
candidates.
Scheme 1. Endogenous Reaction Catalyzed by Carbonic
Anhydrases
’ INTRODUCTION
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc metalloen-
zymes that catalyze the reversible hydration of carbon dioxide
(CO2) to generate bicarbonate anion (HCO3-) and a proton
(Hþ) (Scheme 1).1,2 This equilibrium contributes to a range of
physiological functions that involve the production, transport, or
consumption of CO2, Hþ, and HCO3-.1 CA isozymes IX and
XII are transmembrane enzymes with an extracellularly oriented
active site. They are predominantly expressed in poorly vascular-
ized hypoxic tumors and have minimal expression in healthy
cells.3 Tumor cells experience elevated metabolism, and thus
increased acid production, compared to healthy cells.3-6 If this
acid load is allowed to accumulate, then intracellular pH (pHi)
would fall and disrupt critical biological functions. Recent
evidence has demonstrated that cell-generated CO2, in addition
to lactic acid, is predominantly responsible for the removal of
excess acid from tumor cells.4 CO2 is freely membrane permeable
and rapidly exits metabolically active tumor cells via passive
diffusion.5 The role of CAs IX and XII is to catalyze the hydration
of tumor generated CO2 to form HCO3- and Hþ (Scheme 1).
The net effect is to trap Hþ extracellularly, lowering extracellular
pH (pHe) while maintaining normal pHi.3-5 This process favors
tumor growth and invasion, allowing hypoxic tumor cells to
survive and proliferate.4,7
The importance of modulating pH in the hypoxic tumor
microenvironment underpins a strong case to develop well
designed small-molecule inhibitors of CAs IX and XII for
application as chemical probes. These compounds are needed
to appraise the potential of cancer-associated CAs as a possible
mode for therapeutic intervention in cancer.1,8 The extracellular
active site location of CAs IX and XII together with their
expression in hypoxic cancer cells (overexpression) compared
to healthy cells (minimal expression) improves the odds for the
development of tumor selective CA inhibitors.
The implied target for small molecule drug design of CA
inhibitors is the CA active site Zn2þ cation, and both sulfonamide
(-SO2NH2) and, less frequently, sulfamate (-OSO2NH2) zinc
binding groups feature in the structures of many small molecule
CA inhibitors.1,9 In the past few years our group has established a
Received: November 28, 2010
Published: February 11, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
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novel approach to targeting the inhibition of CAs IX and XII
using carbohydrate-based small molecules.10-15 Druglike carbo-
hydrate-based molecules are now viewed as a vast but relatively
untapped source of potential new therapeutics.16,17 We have
demonstrated that through appending sugar moieties to the
Ar-SO2NH2 zinc binding pharmacophore of classical CA inhibi-
tors we were able to deliver potent inhibitors for CAs IX and XII
in vitro.18 More recently we reported a new class of compound
that incorporates a sulfonamide moiety directly attached to the
anomeric center of a mono- or disaccharide, i.e., [sugar]-
SO2NH2.19 The value of these anomeric sulfonamides was
3-fold. First, they were novel chemical entities. Second, they
lacked the classical aromatic component of classical CA inhibi-
tors, and third, they allowed us to tune the physicochemical
properties of the inhibitor toward selective inhibition of CAs IX
and XII owing to the glycoconjugates compromised ability to
diffuse across cell membranes. The CA inhibition activity of these
compounds was, however, only moderate with Kis in the micro-
molar range. In the present contribution we extend our focus on
modulating structure-property relationships (SPRs) of CA
inhibitors while delivering novel compounds with good CA
inhibition activity and isozyme selectivity. Specifically we present
the design, synthesis, and biological evaluation of carbohydrate-
based sulfamates of the motif [sugar]-OSO2NH2.
Figure 1. Structure of topiramate, a fructose sulfamate CA inhibitor.
stick models of sulfamates 7, 10, 12, and 13 (Figure 2b). All
compounds 7-18 preserve the sulfamate zinc binding group that
is critical for good CA inhibition.
Chemical Synthesis. The carbohydrate sulfamate com-
pounds 7-18 of this study were derived from commercially
available monosaccharides D-glucose, D-galactose, D-mannose,
and methyl R-glucopyranoside and the disaccharide maltose.
The synthetic route to the monosaccharide sulfamates is pre-
sented in Scheme 2. The regioselective protection of the
6-position of the monosaccharides was achieved by reaction with
trityl chloride in pyridine to introduce a trityl group, shown for
D-glucose in Scheme 2.26 The remaining secondary hydroxyl
groups were then acylated with acetic anhydride, propionic
anhydride, or butyric anhydride to give 1, 2, or 3, respectively.
A mixture of R- and β-anomers was obtained with D-glucose, D-
galactose, and D-mannose, from which the β-anomer was either
separated or carried through the detritylation step as a mixture of
anomers, then separated. The R/β ratio was dependent both on
the glycosyl residue and on the nature of the protecting group.
The trityl ether protecting groups were removed with NaI/
TMSCl to unmask the C-6 primary hydroxyl groups, providing
precursor compounds 4-6. The yield for detritylation using this
method is low compared to more traditional conditions; how-
ever, these reagents avoid acyl group migration and the asso-
ciated mixture of products.27,28 Selective sulfamoylation of the
now free C-6 hydroxyl groups of 4-6 with sulfamoyl chloride
(generated in situ from formic acid and chlorosulfony-
lisocyanate) gave target sulfamates 7-9.29 Lastly, the O-acetate
groups of the carbohydrate moiety of the sulfamates were
removed using Zemplen’s conditions30 to liberate the fully
deprotected sugar analogue 14 as a mixture of anomers. Sulfa-
mates 10, 11, 13, 15, 16, and 18 were prepared similarly to
Scheme 2.
’ RESULTS AND DISCUSSION
Compound
Design. Topiramate
(2,3:4,5-bis-O-(l-
methylethy1idene)-6-D-fructopyranosyl sulfamate, TPM) is a
billion dollar drug that is marketed worldwide for the treatment
of epilepsy and migraine.20 TPM is a fructose sulfamate and is an
excellent inhibitor of CAs in vitro.21 The chemical structure of
TPM has two isopropylidene groups masking four secondary
hydroxyl groups, with the primary hydroxyl group sulfamoylated
(Figure 1).
The sulfamate compounds 7-18 of this study were derived
from the monosaccharides D-glucose, D-galactose, D-mannose,
and methyl R-glucopyranoside and the disaccharide maltose
(Figure 2a). The sulfamate group is on the C-6 (or C-60 for
maltose) primary hydroxyl group. Compounds 13 and 18 are
methyl glycosides, while the remaining hydroxyls of compounds
are either acylated (7-13) or fully deprotected to provide free
sugars (14-18).
In addition to inspiration from the impressive pharmaceutical
pedigree of the carbohydrate-sulfamate drug TPM, the design
principle toward the target molecules 7-18 was to develop a
family of compounds that provided incremental coverage of
chemical property space. The application of esters as a prodrug
strategy to mask polar hydroxyl groups is prevalent across
medicinal chemistry and chemical biology.22 Such prodrugs take
advantage of hydrolysis by nonspecific esterases to unmask the
hydroxyl groups in vivo, the prodrugs often survive the GI tract
and may be absorbed into the bloodstream where they are
hydrolyzed by plasma enzymes.23 Esterification of the hydroxyl
groups of carbohydrates has been used to modulate the mem-
brane permeability and downstream activity of carbohydrate-
based molecules.24,25 In this study we explore the acetyl, pro-
pionyl, and butryl acyl groups; each is expected to be substrates of
esterases. The five different sugar types of this study were chosen
to present a selection of molecular shapes with subtle stereo-
chemical differences to the CA enzyme active site and contribute
to building valuable SAR. This is demonstrated by an overlay of
The synthesis of the target maltose sulfamates 12 and 17
followed an analogous strategy to the monosaccharides; how-
ever, several additional steps were needed to accommodate
selectivity in the presence of the two primary alcohols of the
disaccharide (Scheme 3). First the 40,60-O-benzylidene acetal of
maltose is prepared, and the remaining six hydroxyl groups were
acetylated. Removal of the benzylidene acetal with aqueous
acetic acid provides a diol precursor that is manipulated similarly
to the procedure outlined for the monosaccharides. All target
sulfamates 7-18 were spectroscopically characterized using 1D
and 2D NMR (1H, 13C, gCOSY, gHSQC). The characterization
data were consistent with the target structures, with a character-
istic broad singlet for the sulfamate protons (O-SO2NH2) for all
compounds observed at δ 7.40-7.60 ppm in DMSO-d6.
Carbonic Anhydrase Inhibition. The enzyme inhibition
data for sulfamates 7-18 as well as the sulfamate drug TPM
were determined by assaying the CA catalyzed hydration of CO2
(Table 1).31 Enzyme inhibition data for the physiologically
dominant CAs I and II and tumor-associated CAs IX and XII
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ARTICLE
Figure 2. (a) Target carbohydrate sulfamates. (b) Overlay of stick models of sulfamates 7, 10, 12, and 13. Common scaffold is colored by atom type
(red, gray, blue and yellow). Variable acetate groups are colored black.
was the only potent CA II inhibitor of the novel sulfamates
(Ki = 11.3 nM). The remaining monosaccharide sulfamates were
moderately active at this off-target enzyme, with Ki ranging from
66 to 307 nM. Given that the sole structural difference between
compound 11 and compounds 7 and 10 is the stereochemistry of
one position on the monosaccharide moiety, this is intriguing
SAR that may assist to identify further opportunities in CA
inhibitor design. As for CA I, the maltose derivatives 12 (Ki = 754
nM) and 17 (Ki = 513 nM) were the weakest inhibitors at CA II,
while the three different acyl groups of 7-9 also had little impact
on SAR at CA II.
Scheme 2. Synthetic Route to Target Monosaccharide Sul-
famates, Shown for D-Glucose Derivatives 7-9 and 14a
Cancer-Associated CA Isozymes IX and XII. Sulfamates 11,
13, 14, and 18 were good CA IX inhibitors with inhibition
constants clustered below 10 nM (Ki = 7.9-9.0 nM) with very
good selectivity over the off-target CA I (124- to 674-fold) and
up to 14-fold selectivity against CA II. The latter is very good and
contrasts with the behavior of TPM, which is nonselective for CA
II. At hCA IX compounds 7-10, 15, and 16 had Kis ranging from
53 to 86 nM similar to TPM (Ki of 58 nM). These compounds
generally had good selectivity over CA I (up to 112-fold) but
were nonselective over CA II. At CA XII sulfamates were
generally more effective inhibitors than for other isozymes, with
Kis for compounds 13-16 ranging from 7.3 to 9.5 nM, com-
pared to TPM with a Ki of 3.8 nM. Compounds 10 and 14-16
had >10-fold selectivity over CA II and >70-fold selectivity over
CA I. With the exception of 14, these compounds were also
selective over CA IX (5.6- to 8.2-fold), a property that is likely to
prove valuable for applications as chemical probes. Compounds
7-9 and 18 were moderate CA XII inhibitors with Kis ranging
from 85 to 96 nM. These compounds had good selectivity over
CA I but were nonselective compared to CA II inhibition.
Comparison with Anomeric Sulfonamides. Recently we
reported the CA inhibition properties of S-glycosyl primary
sulfonamides.19,32 These were a new class of compound wherein
a primary sulfonamide moiety was directly bonded to a ster-
ochemically rich carbohydrate scaffold, notably without an
intervening aromatic moiety. The enzyme inhibition data and
isozyme selectivity ratios for glucose, galactose, and maltose
anomeric sulfonamides 19-24 at CAs I, II, IX, and XII are
provided in Table 2. When the anomeric sulfonamides 19-24
are compared with their corresponding sulfamates 7, 14, 10, 15,
12, and 17, it is readily apparent that the sulfamates are far
superior CA inhibitors at CAs II, IX, and XII while both groups of
compounds are weak CA I inhibitors. As well, the anomeric
a Reagents and conditions: (a) Ph3CCl, pyridine, 80 °C, 1.5 h; (b) R2O,
H2O, 0 °C, 1 h, yield over two steps 37-59%; (c) TMSCl, NaI, MeCN,
0 °C, then Na2S2O3, 19-57%; (d) HCO2H, ClSO2NCO, 0 °C to room
temp, 30 min; (e) ClSO2NH2 from (d), DMA, 0 °C to room temp, 1 h,
yield over two steps 26-70%; (f) compound 7, MeONa, MeOH, room
temp, 1 h, 71%.
were obtained. Isozyme selectivity ratios for 7-18 and TPM are
also provided (Table 1).
Off-Target CA Isozyme I. The sulfamate compounds were
micromolar inhibitors of the off-target CA I isozyme, with Kis
ranging from 0.86 to 12.6 μM. Inhibition at CA I is weaker than
for the other CA isozymes. The TPM inhibition profile behaves
similarly, also showing poorer inhibition of CA I (Ki = 250 nM)
than for the other CA isozymes. The structural variability
provided by the three different acyl groups had no remarkable
impact on structure-activity relationships (SAR) at CA I with
the Ki values for acetyl 7 (Ki = 4.6 μM), propionyl 8 (Ki = 3.4
μM), and butryl 9 (Ki = 5.6 μM) glucose sulfamates being
similar. Notable, however, was that the free sugar glucose
analogue 14 was a better CA I inhibitor (Ki = 1.2 μM) than
the acylated analogues 7-9. The disaccharide maltose deriva-
tives 12 (Ki = 12.6 μM) and 17 (Ki = 8.75 μM) were weaker
inhibitors than the monosaccharides.
Off-Target CA Isozyme II. CA II is a much more efficient
enzyme than CA I, and inhibition of this isozyme is largely
responsible for side effects associated with clinically used CA
inhibitors.1 TPM is a very potent CA II inhibitor with a Ki of
5.0 nM. Compound 11, the acetylated mannose derivative,
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ARTICLE
Scheme 3. Synthetic Route To Target Disaccharide Sulfamates 12 and 17 Starting from Commercially Available Maltosea
a Reagents and conditions: (a) R,R0-dimethoxytoluene, p-TSA, DMF, 40 °C, 40 mbar; (b) Ac2O, NaOAc, reflux, 0.5 h, yield over two steps 72%
(β-anomer); (c) 80% AcOHaq, room temp, 30 min, 46%; (d) Ph3CCl, pyridine, 80 °C, 1.5 h; (e) Ac2O, H2O, 0 °C, 1 h, yield over two steps 81%;
(f) TMSCl, NaI, MeCN, 0 °C, then Na2S2O3, 60%; (g) HCO2H, ClSO2NCO, 0 °C to room temp, 30 min; (h) ClSO2NH2 from (g), DMA, 0 °C to
room temp, 1 h, yield over two steps 50%; (i) MeONa, MeOH, room temp, 1 h, 37%.
Table 1. Inhibition and Isozyme Selectivity Ratio Data of hCA Isozymes I, II, IX, and XII with Sulfamates 7-18, and the
Carbohydrate Sulfamate Drug TPM
Ki (nM)a
selectivity ratiod
II/IX
compd
CA Ib
CA IIb
CA IXc
CA XIIc
I/IX
I/XII
II/XII
TPMe
7
250
5.0
58
3.8
85
4.3
65.8
0.1
4.2
1.4
1.3
1.4
1.5
1.2
7.9
9.5
1.5
1.97
1.0
14.3
1.3
4610
3360
5640
860
307
105
114
106
11.3
754
66
73
63.2
43.6
65.6
11.5
124.4
20.0
673.8
137.2
72.6
112.5
17.6
132.2
54.2
3.6
8
77
96
35.0
1.1
9
86
87
64.8
1.3
10
11
12
13
14
15
16
17
18
75
9.5
8.4
145
7.6
7.3
7.6
9.5
138
93
90.5
11.2
1.4
970
7.8
631
8.4
8.6
62
115.5
96.9
12600
5660
1180
4500
5960
8750
1190
5.2
744.7
161.6
592.1
627.4
63.4
8.7
82
11.2
12.2
10.9
3.7
93
104
513
129
53
497
9.0
12.8
1.4
a Errors in the range of (5-10% of the reported value, from three determinations. b Human (cloned) isozymes. c Catalytic domain of human (cloned)
isozymes. d These are Ki ratios and are indicative of isozyme selectivity in vitro. e The values for TPM are consistent with those of previous reports.18
sulfonamides showed no isozyme selectivity, while the reverse is
true for the C-6 sulfamates, some of which display excellent
selectivity for the cancer-associated CAs. Using protein X-ray
crystallography, we demonstrated that the shape of the anomeric
sulfonamide ligands resulted in less than optimal CA active site
interactions.19 The design of the new compounds of this study addres-
ses this limitation and has provided better performing compounds.
Structure-Property Relationships (SPR). The incorpora-
tion of variable acyl protecting groups onto the carbohydrate
core of glucose derivatives 7-9, 13, 14, and 18 has encapsulated
a range of log P and topological polar surface area (TPSA) values
(Table 3). Manipulating these physicochemical parameters is a
useful way to refine druggability, and notably this coverage of
chemical property space was achieved with retention of the
underlying glucose sulfamate core.33,34 Values for TPSA, calcu-
lated log P (cLogP), molecular weight (MW), and ligand effi-
ciency (LE) for TPM and all glucose sulfamates are provided in
Table 3. TPSA is a descriptor for characterizing lipid membrane
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ARTICLE
Table 2. Inhibition and Isozyme Selectivity Ratio Data of
hCA Isozymes I, II, IX, and XII with Anomeric Sulfonamides
19-2419
by esterase activity to give a more polar molecule (compound
14) that as a consequence selectively targets the extracellular
active site of cancer-associated CAs.
LE represents the relationship between a compound's potency
and its MW and is now emerging as an important metric to guide
drug discovery campaigns. The LE value is indicative of the
prospects for druggability properties such as bioavailability,
stability, and solubility and permits the comparison of the relative
quality of different leads.37 Perola recently reported an analysis of
60 lead/drug pairs.38 The findings from this analysis revealed a
number of useful threshold values to guide medicinal chemists,
notably that 90% of drug leads had LE > 12.4 while 90% of drugs
had LE > 14.7. All sulfamates of this study have LE similar to or
far exceeding these thresholds at the cancer-associated CAs
(Table 3). Compound 14, a free sugar, represents the core
scaffold of all the novel sulfamates of this study and has the
highest LE (>30) at both CA IX and CA XII. The other free sugar
of this study, compound 18, was also a very efficient ligand, with
LE > 20 at both CA IX and CA XII. The precursors to 14,
compounds 8 and 9, have LE values of >14 and >13, respectively,
indicative of good drug leads and supporting our strategy with
masking of the hydroxyl groups.
Ki (nM)a
selectivity ratiod
compd CA Ib CA IIb CA IXc CA XIIc I/IX I/XII II/IX II/XII
19
20
21
22
23
24
4530 4500
3900 4910
4340 4350
3710 4550
4120 4980
4150 4100
3910
4050
4150
4190
4080
4220
4660
4690
4710
4800
4610
4840
1.15 0.97 1.15 0.97
0.96 0.83 1.21 1.05
1.05 0.92 1.05 0.92
0.89 0.77 1.09 0.95
1.01 0.89 1.22 1.08
0.98 0.86 0.97 0.85
a Errors in the range of (5-10% of the reported value, from three
determinations. b Human (cloned) isozymes. c Catalytic domain of
human (cloned) isozymes. d These are Ki ratios and are indicative of
isozyme selectivity in vitro.
’ CONCLUSIONS
Our study has delivered novel, potent, and selective carbohy-
drate-based sulfamates that inhibit cancer-associated CAs. Com-
pounds comprised a sulfamate zinc binding group appended to
the C-6 hydroxyl of a panel of monosaccharides or the C-60
hydroxyl of maltose. The SAR and SPR properties for these
compounds, with variable acyl protecting groups, have provided
a new approach to CA inhibitor development. Parent sulfamates
are predicted to have good oral bioavailability, yet target selec-
tively the active site of extracellular CAs IX and XII over
intracellular CAs once processed by esterases. Collectively this
library of carbohydrate-based sulfamates exhibits biopharmaceu-
tical properties that render these compounds potentially valuable
for applications as chemical probes in medicinal chemistry.
Table 3. Calculated Properties for TPM and Glucose Sulfa-
mate Derivatives 7-9, 13, 14, and 18
compd cLogP a TPSA (Å2)b MW (Da) LE(CA IX)
LE(CA XII)
c
c
TPM
7
þ0.04
-0.72
þ1.40
þ3.52
-0.79
-3.30
-2.67
116
184
184
184
167
160
149
339.4
427.4
483.5
539.6
399.4
259.2
273.3
21.3
16.7
14.7
13.1
17.9
31.1
20.2
24.8
16.6
14.5
13.1
20.4
31.4
20.3
8
9
13
14
18
a cLogP data calculated using InstantJChem, version 3.0.4, from Che-
mAxon. b TPSA calculated using ChemBioDraw Ultra, version 11.0. c LE
calculated using equation LE = pKi/MW (MW in kDa).38
’ EXPERIMENTAL SECTION
barrier diffusion. Molecules with a TPSA > 140 Å2 are likely to
have a low capacity for penetrating cell membranes, while those
with TPSA e 60 Å2 typically have good passive permeability
properties.35,36 For the glucose sulfamates the calculated TPSA
values ranged from 160 to 184 Å2, which is above the upper limit
(140 Å2) for good passive permeability properties. cLogP values
of <0 are indicative of molecules with poor membrane perme-
ability. The cLogP values are consistent with the compound
design and provide a broad range that extends from -3.3 for
compound 14 (four free hydroxyl groups) to þ3.52 for com-
pound 9 (four butyryl protected hydroxyl groups). This again is
noteworthy given that the underlying active core structure is
retained. The per-O-acetylated compounds 7 and 13, despite no
free hydroxyl groups, still have cLogP values of <0 (values of -
0.72 and -0.79, respectively), while the propionyl and butyryl
protected compounds 8 and 9 have cLogP values of þ1.40 and
þ3.52, respectively. The implications of these values may prove
useful for the provision of compounds suited for oral adminis-
tration. Compounds 8 and 9 are predicted to be orally bioavail-
able, yet once absorbed, their physicochemical properties altered
Chemistry. All starting materials and reagents were purchased
from commercial suppliers. Solvents were dried prior to use or pur-
chased anhydrous from Sigma-Aldrich. All reactions were monitored by
TLC using Merck F60254 silica plates with visualization of product bands
by UV fluorescence (λ = 254 nm) and charring with orcinol stain (1 g of
orcinol monohydrate in a mixture of EtOH/H2O/H2SO4 72.5:22.5:5).
Silica gel flash chromatography was performed using silica gel (Davisil)
60 Å (230-400 mesh). NMR (1H, 13C {1H}, gCOSY, and HSQC)
spectra were recorded on a Varian 500 MHz spectrometer at 30 °C. For
1H and 13C NMR acquired in CDCl3 chemical shifts (δ) are reported in
ppm relative to the solvent residual peak: proton (δ = 7.27 ppm) and
1
carbon (δ = 77.2 ppm), respectively. Chemical shifts for H and 13C
NMR acquired in DMSO-d6 are reported in ppm relative to residual
solvent proton (δ = 2.50 ppm) and carbon (δ = 39.5 ppm) signals,
respectively. Multiplicity is indicated as follows: s (singlet); d (doublet); t
(triplet); m (multiplet); dd (doublet of doublet); ddd (doublet of
doublet of doublet); br s (broad singlet). Coupling constants are
reported in hertz (Hz). Melting points measured on a Cole Parmer
instrument are reported uncorrected. Low resolution mass spectra were
acquired on an Applied Biosystems Pty Ltd. Mariner ESI-TOF mass
spectrometer using electrospray as the ionization technique in positive
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ARTICLE
ion and/or negative ion modes as stated. High resolution mass spectra
were performed in positive ion mode on an Apex III Bruker Daltonics
4.7 T Fourier transform mass spectrometer fitted with an Apollo
electrospray ionization source. All MS analysis samples were prepared
as solutions in methanol. Optical rotations were measured on a Jasco
P-1020 polarimeter at 25 °C with Na 589 nm wavelength and a 100 mm
cell and reported as an average of 10 measurements. The purities of
isolated products were determined by HPLC obtained on an LCMS
instrument (MS-ZQ Waters; HPLC-Alliance Waters) using a HPLC
column (Ascentis, C18 3 μm, 5 cm ꢀ 4.6 mm). Elution was performed
with a gradient of water/methanol (containing 1% formic acid) from
95:5 to 0:100 for 10 min at a flow rate of 1 mL/min. UV (200-400 nm)
and ELSD (evaporative light scattering detection, Alltech) detection
were used. Purity of all compounds proved to be g95%. The synthesis of
all intermediates was adapted from literature methods and is described in
Supporting Information.
crude product (1:9 acetone/toluene) afforded the title compound as a
mixture of R- and β-anomers. A further recrystallization from EtOH
afforded the β-anomer 9 (26% yield) as white crystals. Mp = 109 °C.
1
[R]25 þ9 (c 1.0, chloroform). Rf = 0.25 (1:9 acetone/toluene). H
D
NMR (500 MHz, DMSO-d6): δ = 7.55 (s, 2H, NH2); 6.02 (d, J = 8.5 Hz,
1H, H-1); 5.50 (t, J = 9.5 Hz, 1H, H-3); 4.99 (t, J = 9.5 Hz, 2H, H-2,
H-4); 4.30 (m, 1H, H-5); 4.03-3.99 (m, 2H, H-6a, H-6b); 2.31, 2.28,
2.22, 2.16 (4 ꢀ m, 8H, OCOCH2); 1.54-1.44 (m, 8H, CH2CH3); 0.86,
0.84, 0.83, 0.82 (4 ꢀ t, J = 7.5 Hz, 12H, CH2CH3); assignments were
confirmed by 1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ =
171.7, 171.5, 171.3, 171.0 (4 ꢀ OCOPr); 90.6 (C-1); 71.5, 71.4 (C-3,
C-5); 69.8 (C-4); 67.7 (C-2); 66.6 (C-6); 35.1, 35.1, 35.0, 35.0 (4 ꢀ
OCOCH2); 17.8, 17.7, 17.6, 17.5 (4 ꢀ CH2CH3); 13.3, 13.2, 13.1, 13.1
(4 ꢀ CH2CH3); assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 562 [M þ Na]þ. HRMS: calcd for
C22H37N1O12SNaþ 562.1929; found 562.1941.
General Procedure 1. Synthesis of Sulfamates 7-13.29,39 The
carbohydrate precursor (1.0 equiv) was dried under vacuum and solubilized
in anhydrous N,N-dimethylacetamide (15 equiv) under argon. Sulfamoyl
chloride (3.8 equiv) was prepared from formic acid and chlorosufonyl
isocyanate at 0 °C in a dry two-neck flask connected to a bubbler and under
an anhydrous atmosphere of argon. The alcohol solution was added slowly to
the flask containing sulfamoyl chloride. Then the mixture was allowed to
warm to room temperature and stirred under argon for 1 h. The crude
reaction mixture was diluted in EtOAc, washed with H2O (ꢀ1) and brine
(ꢀ2). The aqueous fractions were back-extracted with EtOAc (ꢀ2), then the
organic fractions combined, dried over MgSO4, filtered, and concentrated.
1,2,3,4-Tetra-O-acetyl-6-sulfamoyl-β-D-glucopyranose
(7). The title compound 7 was prepared from 1,2,3,4-tetra-O-acetyl-β-
D-glucopyranose according to general procedure 1. Recrystallization of
the crude product from EtOH afforded 7 (67% yield) as white crystals.
Mp = 126 °C. [R]25D þ8 (c 1.0, chloroform). Rf = 0.58 (3:1 EtOAc/
hexane). 1H NMR (500 MHz, DMSO-d6): δ = 7.56 (s, 2H, NH2); 5.98
(d, J = 8.5 Hz, 1H, H-1); 5.44 (t, J = 9.5 Hz, 1H, H-3); 4.95 (t, J = 10.0
Hz, 1H, H-2); 4.94 (t, J = 9.5 Hz, 1H, H-4); 4.28 (ddd, J = 8.5, 5.5, 2.5
Hz, 1H, H-5); 4.04 (dd, J = 11.5, 2.5 Hz, 1H, H-6a); 4.00 (dd, J = 11.5,
5.5 Hz, 1H, H-6b); 2.07, 2.01, 2.00, 1.94 (4 ꢀ s, 12H, OCOCH3);
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 169.5, 169.2, 169.1, 168.7 (4 ꢀ OCOCH3); 90.8 (C-1);
71.8 (C-3); 71.4 (C-5); 70.7 (C-4); 67.8 (C-2); 66.6 (C-6); 20.5, 20.4,
1,2,3,4-Tetra-O-acetyl-6-sulfamoyl-β-D-galactopyranose
(10). The title compound 10 was prepared from 1,2,3,4-tetra-O-acetyl-
β-D-galactopyranose according to general procedure 1. Purification of
the crude product by flash chromatography (1:1 EtOAc/hexane)
afforded 10 (48% yield) as a white solid. Mp = 154 °C. [R]25 þ14
D
(c 1.0, chloroform). Rf = 0.64 (3:1 EtOAc/hexane). 1H NMR (500 MHz,
DMSO-d6): δ = 7.60 (s, 2H, NH2); 5.91 (d, J = 8.5 Hz, 1H, H-1); 5.35
(dd, J = 10.0, 3.5 Hz, 1H, H-3); 5.33 (br d, J = 3.5 Hz, 1H, H-4); 5.09
(dd, J = 9.5, 8.5 Hz, 1H, H-2); 4.47 (br t, J = 6.0 Hz, 1H, H-5); 4.04-3.99
(m, 2H, H-6a, H-6b); 2.13, 2.07, 2.02, 1.93 (4 ꢀ s, 12H, OCOCH3);
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 169.8, 169.5, 169.3, 168.8 (4 ꢀ OCOCH3); 91.3
(C-1); 71.0 (C-5); 69.9 (C-3); 67.6 (C-2); 67.2 (C-4); 66.2 (C-6); 20.5,
20.4, 20.4, 20.3 (4 ꢀ OCOCH3); assignments were confirmed
by 1H-13C HSQC. LRMS (ESIþ): m/z = 445 [M þ NH4]þ, 450 [M þ
Na]þ. HRMS: calcd for C14H21NO12SNaþ 450.0677; found 450.0678.
1,2,3,4-Tetra-O-acetyl-6-sulfamoyl-β-D-mannopyranose
(11). The title compound 11 was prepared from 1,2,3,4-tetra-O-acetyl-
β-D-mannopyranose according to general procedure 1. Purification by
flash chromatography (1:1 EtOAc/hexane) afforded 5 (15% yield) as a
colorless oil. [R]25 -15 (c 1.0, chloroform). Rf = 0.58 (3:1 EtOAc/
D
hexane). 1H NMR (500 MHz, DMSO-d6): δ = 7.57 (s, 2H, NH2); 6.12
(br s, 1H, H-1); 5.38 (dd, J = 10.0, 3.5 Hz, 1H, H-3); 5.37 (br d, J = 3.5
Hz, 1H, H-2); 5.02 (t, J = 10.0 Hz, 1H, H-4); 4.15 (ddd, J = 10.0, 5.5, 2.5
Hz, 1H, H-5); 4.05-4.00 (m, 2H, H-6a, H-6b); 2.15 (s, 3H, OCOCH3);
2.04 (s, 6H, 2 ꢀ OCOCH3); 1.94 (s, 3H, OCOCH3); assignments were
confirmed by 1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ =
169.8, 169.4, 169.4, 168.1 (4 ꢀ OCOCH3); 90.1 (C-1); 71.6 (C-5); 69.8
(C-3); 67.9 (C-2); 66.8 (C-6); 65.2 (C-4); 20.7, 20.4, 20.4, 20.3 (4 ꢀ
1
20.3, 20.3 (4 ꢀ OCOCH3); assignments were confirmed by H-13C
HSQC. LRMS (ESIþ): m/z = 450 [M þ Na]þ. HRMS: calcd for
C14H21NO12SNaþ 450.0677; found 450.0657.
1,2,3,4-Tetra-O-propionyl-6-sulfamoyl-β-D-glucopyranose
(8). The title compound 8 was prepared from 1,2,3,4-tetra-O-propio-
nyl-β-D-glucopyranose according to general procedure 1. Purification of
the crude product by flash chromatography (2:3 EtOAc/hexane)
1
OCOCH3); assignments were confirmed by H-13C HSQC. LRMS
(ESIþ): m/z = 445 [M þ NH4]þ, 450 [M þ Na]þ. HRMS: calcd for
afforded 8 (70% yield) as a white solid. Mp =119 °C. [R]25 = þ11
C14H21NO12SNaþ 450.0677; found 450.0660.
D
1
(c = 1.0, chloroform). Rf = 0.21 (1:2 EtOAc/hexane). H NMR (500
1,2,20,3,30,40,6-Hepta-O-acetyl-60-sulfamoyl-β-maltose
(12). The title compound 12 was prepared from 1,2,20,3,30,40,6-hepta-
O-acetyl-β-maltose according to general procedure 1. The crude ma-
terial was purified by flash chromatography (2:1 EtOAc/hexane) to give
pure β-anomer 12 as a white solid (50% yield). Rf = 0.52 (2:1 EtOAc/
hexane). Mp = 98-99 °C. [R]25D þ40 (c 1.0, chloroform). 1H NMR
(500 MHz, DMSO-d6): δ = 7.51 (s, 2H, NH2); 5.89 (d, J = 8.5 Hz, 1H,
H-1); 5.43 (t, J = 9.5 Hz, 1H, H-3); 5.31 (d, J = 4.0 Hz, 1H, H-10); 5.20 (t,
J = 10.0 Hz, 1H, H-30); 4.95 (t, J = 9.5 Hz, 1H, H-40); 4.85 (dd, J = 10.5,
3.5 Hz, 1H, H-20); 4.80 (dd, J = 9.5, 8.5 Hz, 1H, H-2); 4.37 (dd, J = 13.0,
3.5 Hz, 1H, H-6a); 4.12 (m, 2H, H-6b, H-5); 4.06-4.01 (m, 3H, H-50,
H-6a0, H-6b0); 3.99 (t, J = 9.5 Hz, 1H, H-4); 2.06, 2.04, 1.98, 1.97, 1.95,
1.94, 1.93 (7 ꢀ s, 21H, OCOCH3); assignments were confirmed by
1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 170.4, 170.1,
169.8, 169.8, 169.5, 169.3, 168.9 (7 ꢀ OCOCH3); 95.4 (C-10); 90.8 (C-
1); 73.9, (C-3); 73.2 (C-50); 72.3 (C-5); 70.9 (C-2); 69.5 (C-20); 69.1
MHz, DMSO-d6): δ = 7.55 (s, 2H, NH2); 6.02 (d, J = 8.5 Hz, 1H, H-1);
5.48 (t, J = 9.5 Hz, 1H, H-3); 4.98 (t, J = 10.0 Hz, 1H, H-2); 4.97
(t, J = 9.5 Hz, 1H, H-4); 4.31 (ddd, J = 9.5, 5.0, 2.5 Hz, 1H, H-5); 4.03
(dd, J = 11.0, 2.5 Hz, 1H, H-6a); 4.00 (dd, J = 11.0, 5.5 Hz, 1H, H-6b);
2.36, 2.29, 2.25, 2.19 (4 ꢀ m, 8H, OCOCH2CH3); 1.01, 1.00, 0.98, 0.96
(4 ꢀ br t, J = 7.5 Hz, 12H, OCOCH2CH3); assignments were confirmed
by 1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 169.5, 169.2,
169.1, 168.7 (4 ꢀ OCOEt); 91.2 (C-1); 72.1 (C-3); 71.9 (C-5); 70.4
(C-4); 68.2 (C-2); 67.1 (C-6); 27.2, 27.2, 27.1, 27.1 (4 ꢀ
OCOCH2CH3); 9.5, 9.4, 9.3, 9.0 (4 ꢀ OCOCH2CH3); assignments
were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z = 506 [M þ
Na]þ. HRMS: calcd for C18H29N1O12SNaþ 506.1303; found 506.1290.
1,2,3,4-Tetra-O-butyryl-6-sulfamoyl-β-D-glucopyranose
(9). The title compound 9 was prepared from 1,2,3,4-tetra-O-butyryl-
β-D-glucopyranose according to general procedure 1. Purification of the
1486
dx.doi.org/10.1021/jm101525j |J. Med. Chem. 2011, 54, 1481–1489

Journal of Medicinal Chemistry
ARTICLE
(C-30); 68.1 (C-4); 67.8 (C-40); 66.6 (C-60); 62.6 (C-6); 20.9, 20.7,
20.7, 20.6, 20.5, 20.4, 20.4 (7 ꢀ OCOCH3); assignments were con-
firmed by 1H-13C HSQC and 1H-13C HMBC. LRMS (ESIþ): m/z =
738 [M þ Na]þ. HRMS: calcd for C26H37N1O20SNaþ 738.1522; found
738.1548.
J = 5.0, 3.5 Hz, 0.6H, H-4β); 3.24 (m, 0.4H, H-2β); assignments were
confirmed by 1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ =
97.4 (C-1β); 92.7 (C-1R); 75.8 (C-4R); 73.1 (C-4β); 72.1 (C-5β); 71.8
(C-3β); 69.4 (C-5R); 69.2 (C-2R); 69.0 (C-3R); 68.8 (C-2β); 67.9 (C-
6β) 67.3 (C-6R); assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 282 [M þ Na]þ. HRMS: calcd for
C6H13NO8SNaþ 282.0254; found 282.0268.
Methyl 2,3,4-Tri-O-acetyl-6-sulfamoyl-r-D-glucopyranose
(13). The title compound 13 was prepared from methyl 2,3,4-tri-O-
acetyl-R-D-glucopyranose according to general procedure 1. Purification
of the crude product by flash chromatography (1:2 acetone/hexane)
afforded the 11 (61% yield) as a colorless gum. Rf = 0.29 (1:1 EtOAc/
hexane). 1H NMR (500 MHz, DMSO-d6): δ = 7.47 (s, 2H, NH2); 5.30
(t, J = 10.0 Hz, 1H, H-3); 4.94 (d, J = 3.5 Hz, 1H, H-1); 4.94 (t, J = 10.0
Hz, 1H, H-4); 4.84 (dd, J = 10.0, 3.5 Hz, 1H, H-2); 4.07-4.04 (m, 2H,
H-6a, H-6b); 3.98 (m, 1H, H-5); 3.35 (s, 3H, OCH3); 2.02, 1.99, 1.96 (3
ꢀ s, 9H, OCOCH3); assignments were confirmed by 1H-1H gCOSY.
13C NMR (125 MHz, DMSO-d6): δ = 169.6, 169.2, 169.1 (3 ꢀ
OCOCH3); 96.0 (C-1); 69.8 (C-2); 69.4 (C-3); 68.2 (C-5); 66.9 (C-
4); 66.8 (C-6); 54.9 (OCH3); 20.2, 20.3, 20.3 (3 ꢀ OCOCH3);
assignments were confirmed by 1H-13C HSQC. LRMS (ESIþ):
m/z = 422 [M þ Na]þ. HRMS: calcd for C13H21N1O11SNaþ
422.0728; found 422.0726.
General Procedure 2. Synthesis of Sulfamates 14-
18. Fully deprotected sulfamates were prepared by treating a solution
of the per-O-acetylated parent compound (1.0 equiv) in anhydrous
MeOH at 0 °C with methanolic sodium methoxide (0.05 M final
concentration), final pH 12. The mixture was warmed to room
temperature and left to stir until full deprotection was evident by TLC
(30 min to 2 h). The solution was neutralized with Amberlite IR-120
[Hþ], filtered and the resin washed several times with methanol. The
solvent was evaporated under reduced pressure and the product
lyophilized to dryness.
6-Sulfamoyl-r,β-D-mannopyranose (16). The title com-
pound 16 was prepared from 11 according to general procedure 1.
Purification of the crude product by flash chromatography (95:5
CH3CN/H2O) afforded 16 (36% yield, R/β 4/6) as a colorless oil. Rf
= 0.32 (95:5 CH3CN/H2O). 1H NMR (500 MHz, DMSO-d6): δ = 7.42
(s, 0.8H, NH2R); 7.40 (s, 1.2H, NH2β); 6.44 (d, J = 4.5 Hz, 0.6H, OH-
1β); 6.32 (d, J = 8.0 Hz, 0.4H, OH-1R); 4.96 (d, J = 3.0 Hz, 0.4H, OH-
4R); 4.92 (d, J = 4.5 Hz, 0.6H, OH-4β); 4.88 (d, J = 4.0 Hz, 0.6H, H-1β);
4.67 (d, J = 4.0 Hz, 0.6H, OH-2β); 4.65 (br s, 0.4H, OH-2R); 4.60 (d, J =
8.0 Hz, 0.4H, H-1R); 4.57 (d, J = 6.0 Hz, 0.6H, OH-3β); 4.54 (d, J = 4.5
Hz, 0.4H, OH-3R); 4.29 (br d, J = 10.5 Hz, 0.4H, H-6aR); 4.26 (dd, J =
10.0, 1.5 Hz, 0.6H, H-6aβ); 4.03 (dd, J = 10.0, 7.0 Hz, 0.6H, H-6bβ); 4.00
(m, 0.4H, H-6bR); 3.76 (m, 0.6H, H-5β); 3.57-3.53 (m, 1.6H, H-2β,
H-3R, H-3β); 3.36 (m, 0.4H, H-4β); 3.31-3.30 (m, 1.2H, H-2R, H-4R,
H-5R); assignments were confirmed by 1H-1H gCOSY. 13C NMR (125
MHz, DMSO-d6): δ = 94.2 (C-1R); 94.1 (C-1β); 74.0 (C-5R); 73.5 (C-
3R); 71.5 (C-2R); 71.3 (C-3β); 70.5 (C-2β); 70.4 (C-5β); 69.3 (C-6β);
69.1 (C-6R); 67.0 (C-4β); 66.6 (C-4R); assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 282 [M þ Na]þ. HRMS: calcd
for C6H13NO8SNaþ 282.0254; found 282.0258.
60-Sulfamoyl-r,β-maltose (17). The title compound 17 was
prepared from 12 according to general procedure 2. Purification of the
crude product by flash chromatography (95:5 CH3CN/H2O) afforded
17 (37% yield, R/β 4/6) as a hygroscopic white solid. Rf = 0.32 (95:5
1
CH3CN/H2O). H NMR (500 MHz, DMSO-d6): δ = 7.40 (s, 2H,
6-Sulfamoyl-r,β-D-glucopyranose (14). The title compound
14 was prepared from the 7 according to general procedure 2 and
obtained as a hygroscopic white solid (91% yield, R/β 4/6). Rf = 0.53
(9:1 CH3CN/H2O). 1H NMR (500 MHz, DMSO-d6): δ = 7.43
(s, 1.2H, NH2β); 7.40 (s, 0.8H, NH2R); 6.72 (d, J = 6.5 Hz, 0.6H,
OH-1β); 6.41 (d, J = 4.5 Hz, 0.4H, OH-1R); 5.13 (d, J = 5.5 Hz, 0.6H,
OH-4β); 5.08 (d, J = 5.5 Hz, 0.4H, OH-4R); 4.95 (br s, 0.6H, OH-3β);
4.92 (t, J = 3.5 Hz, 0.4H, H-1R); 4.92 (br s, 0.6H, OH-2β); 4.76 (br s,
0.4H, OH-3R); 4.54 (d, J = 5.0 Hz, 0.4H, OH-3R); 4.32 (br d, J = 7.0 Hz,
0.6H, H-1β); 4.28 (dd, J = 10.5, 1.5 Hz, 0.6H, H-6aβ); 4.22 (dd, J = 10.0,
1.5 Hz, 0.4H, H-6aR); 4.02 (dd, J = 10.5, 6.5 Hz, 0.4H, H-6bR); 3.99 (dd,
J = 10.5, 7.0 Hz, 0.6H, H-6bβ); 3.82 (m, 0.4H, H-5R); 3.44 (br t, J = 9.0
Hz, 0.4H, H-3R); 3.40 (m, 0.6H, H-5β); 3.16 (m, 0.6H, H-3β); 3.13 (m,
0.4H, H-2R); 3.04-3.02 (m, 1H, H-4R, H-4β); 2.91 (m, 0.4H, H-2β);
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 96.9 (C-1β); 92.3 (C-1R); 76.5 (C-3β); 74.7 (C-2β);
73.5 (C-5β); 73.0 (C-3R); 72.1 (C-2R); 70.4 (C-4R); 70.0 (C-4β); 69.2
(C-6R); 69.0 (C-5R); 68.9 (C-6β); assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 282 [M þ Na]þ. HRMS: calcd
for C6H13NO8SNaþ 282.0254; found 282.0251.
6-Sulfamoyl-r,β-D-galactopyranose (15). The title com-
pound 15 was prepared from 10 according to general procedure 2.
Purification of the crude product by flash chromatography (95:5
CH3CN/H2O) afforded 4 (71% yield, R/β 4/6) as a colorless oil. Rf
= 0.28 (95:5 CH3CN/H2O). 1H NMR (500 MHz, DMSO-d6): δ = 7.45
(s, 1.2H, NH2β); 7.43 (s, 0.8H, NH2R); 6.64 (d, J = 6.5 Hz, 0.6H, OH-
1β); 6.29 (d, J = 4.5 Hz, 0.4H, OH-1R); 4.96 (t, J = 4.0 Hz, 0.4H, H-1R);
4.74 (d, J = 4.5 Hz, 1H), 4.72 (d, J = 5.5 Hz, 0.4H), 4.62 (d, J = 4.5 Hz,
0.6H), 4.56 (m, 0.6H), 4.32 (d, J = 5.5 Hz, 0.4H) (OH-2, OH-3, OH-4);
4.27 (t, J = 7.0 Hz, 0.6H, H-1β); 4.12-4.06 (m, 2H, H-6aR, H-6aβ,
H-6bR, H-6bβ); 3.70 (br t, J = 3.5 Hz, 0.4H, H-5R); 3.66 (br t, J = 6.0 Hz,
0.6H, H-3β); 3.62 (br t, J = 3.5 Hz, 0.6H, H-5β); 3.59 (m, 0.4H, H-3R);
3.52 (m, 0.4H, H-2R); 3.31 (dd, J = 5.0, 3.5 Hz, 0.4H, H-4R); 3.29 (dd,
NH2); 6.68, 6.34, 5.43, 5.15 (4 ꢀ br s, 4H, OH); 5.08, 5.04 (2 ꢀ d, J = 3.5
Hz, 1H, H-10R, H-10β); 4.97 (br s, 1H, OH); 4.91 (br s, 0.4H, H-1R);
4.54, 4.44 (2 ꢀ br s, 2H, OH); 4.31 (d, J = 7.5 Hz, 0.6H, H-1β); 4.19 (br
d, J = 10.5 Hz, 1H, H-6a0); 4.10 (dd, J = 10.5, 5.5 Hz, 1H, H-6b0); 3.73
(m, 1H, H-50); 3.69-3.64 (m, 2H, H-6a, H-4); 3.62-3.58 (m, 1H,
H-6b); 3.50 (m, 0.4H, H-3R); 3.40-3.37 (m, 2.2H, H-3β, H-30, H-5β);
3.32 (m, 0.4 H, H-5R); 3.25-3.18 (m, 1.4H, H-2R, H-20); 3.09 (t, J = 9.5
Hz, 1H, H-40); 2.95 (t, J = 0.6H, H-2β); assignments were confirmed by
1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 100.8, 100.6 (C-
10R, C-10β); 96.8 (C-1β); 92.2 (C-1R); 80.2 (C-5R); 79.5 (C-5β); 76.6
(C-3β); 75.2 (C-2R); 74.5 (C-2β); 73.3, 73.2 (C-30R, C-30β); 73.0 (C-4R
or C-4β); 72.5, 72.4 (C-20R, C-20β); 72.0 (C-3R); 70.5 (C-50R, C-50β);
70.4 (C-4β or C-4R); 69.6, 69.5 (C-40R, C-40β); 68.4, 68.4 (C-60R,
C-60β); 60.7, 60.6 (C-6R, C-6β); assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 444 [M þ Na]þ, LRMS
(ESI-): m/z = 420 [M - H]-, 455 [M þ Cl]-. HRMS: calcd for
C12H23N1O13SNaþ 444.0782; found 444.0797.
Methyl-6-sulfamoyl-r-D-glucopyranose (18). The title com-
pound 18 was prepared from compound 13 according to general
procedure 2. The crude product was purified by flash chromatography
(9:1 CH3CN/H2O) affording 18 (94% yield) as a hygroscopic white
solid. [R]25D þ30 (c 1.0, methanol). Rf = 0.50 (9:1 CH3CN/H2O). 1H
NMR (500 MHz, DMSO-d6): δ = 7.43 (NH2); 5.14 (d, J = 6.0 Hz, 1H,
OH-4); 4.86 (d, J = 5.0 Hz, 1H, OH-3); 4.78 (d, J = 6.0 Hz, 1H, OH-2);
4.56 (d, J = 4.0 Hz, 1H, H-1); 4.26 (m, 1H, H-6a); 4.05 (m, 1H, H-6b);
3.58 (m, 1H, H-5); 3.39 (m, 1H, H-3); 3.27 (s, 3H, OCH3); 3.23 (m,
1H, H-2), 3.04 (m, 1H, H-4); assignments were confirmed by 1H-1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 99.7 (C-1); 73.2 (C-3);
71.7 (C-5); 70.1 (C-4); 69.5 (C-5); 68.6 (C-6); 54.5 (OCH3); assign-
ments were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z = 296
[M þ Na]þ; HRMS: calcd for C7H15N1O8SNaþ 296.0411; found
296.0424.
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Journal of Medicinal Chemistry
ARTICLE
’ ASSOCIATED CONTENT
(11) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti,
A.; Vullo, D.; Supuran, C. T.; Poulsen, S.-A. Carbonic anhydrase
inhibitors: inhibition of isozymes I, II, and IX with triazole-linked
O-glycosides of benzene sulfonamides. J. Med. Chem. 2007, 50, 1651–
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(12) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.;
Vullo, D.; Supuran, C. T.; Poulsen, S.-A. Inhibition of membrane-
associated carbonic anhydrase isozymes IX, XII and XIV with a library
of glycoconjugate benzenesulfonamides. Bioorg. Med. Chem. Lett. 2007,
17, 987–992.
Supporting Information. 1H and 13C NMR spectra for
S
b
sulfamates 7-18 and novel intermediates and the synthesis of all
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
(13) Wilkinson, B. L.; Innocenti, A.; Vullo, D.; Supuran, C. T.;
Poulsen, S.-A. Inhibition of carbonic anhydrases with glycosyltriazole
benzene sulfonamides. J. Med. Chem. 2008, 51, 1945–1953.
(14) Lopez, M.; Bornaghi, L. F.; Innocenti, A.; Vullo, D.; Charman,
S. A.; Supuran, C. T.; Poulsen, S.-A. Sulfonamide linked neoglycocon-
jugates: a new class of inhibitors for cancer-associated carbonic anhy-
drases. J. Med. Chem. 2010, 53, 2913–2926.
(15) Singer, M.; Lopez, M.; Bornaghi, L. F.; Innocenti, A.; Vullo, D.;
Supuran, C. T.; Poulsen, S.-A. Inhibition of carbonic anhydrase
isozymes with benzene sulfonamides incorporating thio, sulfinyl and
sulfonyl glycoside moieties. Bioorg. Med. Chem. Lett. 2009, 19, 2273–
2276.
(16) Meutermans, W.; Le, G. T.; Becker, B. Carbohydrates as
scaffolds in drug discovery. ChemMedChem 2006, 1, 1164–1194.
(17) Boltje, T. J.; Buskas, T.; Boons, G.-J. Opportunities and
challenges in synthetic oligosaccharide and glycoconjugate research.
Nat. Chem. 2009, 1, 611–622.
(18) Winum, J.-Y.; Poulsen, S.-A.; Supuran, C. T. Therapeutic
applications of glycosidic carbonic anhydrase inhibitors. Med. Res. Rev.
2009, 29, 419–435.
*For C.T.S.: þ39-055-4573005; fax, þ39-055-4573385; e-mail,
claudiu.supuran@unifi.it. For S.-A.P.: phone, þ61-7-3735-7825;
fax, þ61-7-3735-7656; e-mail, s.poulsen@griffith.edu.au.
’ ACKNOWLEDGMENT
This work was financed by the Australian Research Council
(Grant DP0877554 to S.-A.P.) and in part an EU grant of the 7th
Framework Programme (METOXIA Project to C.T.S.). We
thank Associate Professor Courtney Aldrich for guidance with
the preparation and handling of sulfamoyl chloride.
’ ABBREVIATIONS USED
CA, carbonic anhydrase; TPM, topiramate; pHe, extracellular
pH; pHi, intracellular pH; cLogP, calculated log P; TPSA, total
polar surface area; LE, ligand efficiency; SAR, structure-activity
relationship; SPR, structure-property relationship
(19) Lopez, M.; Paul, B.; Hofmann, A.; Morizzi, J.; Wu, Q. K.;
Charman, S. A.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S.-A.
S-Glycosyl primary sulfonamides, a new structural class for selective
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(20) Maryanoff, B. E. Pharmaceutical “gold” from neurostabilizing
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(21) Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.; Scozzafava,
A.; Pastorekovꢁa, S.; Supuran, C. T. Carbonic anhydrase inhibitors.
Inhibition of the transmembrane isozyme XII with sulfonamides—
a new target for the design of antitumor and antiglaucoma drugs? Bioorg
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