Inhibition of carbonic anhydrase isozymes with benzene sulfonamides incorporating thio, sulfinyl and sulfonyl glycoside moieties

Article abstract of DOI:10.1016/j.bmcl.2009.02.086

A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthesized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four human carbonic anhydrases (hCA): isozymes I,

Full text of DOI:10.1016/j.bmcl.2009.02.086

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2273–2276
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of carbonic anhydrase isozymes with benzene sulfonamides
incorporating thio, sulfinyl and sulfonyl glycoside moieties
Mathilde Singer a, Marie Lopez a, Laurent F. Bornaghi a, Alessio Innocenti b, Daniela Vullo b,
a,
Claudiu T. Supuran b, , Sally-Ann Poulsen
*
*
a Eskitis Institute, Griffith University, Don Young Road, Nathan, Queensland 4111, Australia
b Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthe-
sized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four
human carbonic anhydrases (hCA): isozymes I, II and tumour-associated isozymes IX and XII. The oxida-
tion state of the sulfur in the carbohydrate tail moiety did not influence either enzyme inhibition potency
or isozyme selectivity even though presenting opportunities for differing interactions with the target
isozymes.
Received 12 February 2009
Revised 20 February 2009
Accepted 23 February 2009
Available online 26 February 2009
Keywords:
Carbonic anhydrase
Sulfonamide
Ó 2009 Elsevier Ltd. All rights reserved.
Carbohydrate
Click chemistry
Thio glycoside
Sulfinyl glycoside
Sulfonyl glycoside
Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous zinc metal-
loenzymes spread across the phylogenetic tree.1 There are 16 dif-
ferent CA isozymes presently known in mammals. These
enzymes catalyze a simple, yet critical, reaction: the reversible
hydration of carbon dioxide to bicarbonate anion and a proton.
Hydration does not proceed appreciably under physiological condi-
tions in the absence of CA. The Zn2+ core of CA serves an essential
function: it is a strong Lewis acid that binds to and activates the
hydrating water molecule. CA facilitates deprotonation of water
to generate the strongly basic hydroxide anion (OHÀ) under phys-
iological conditions. Hydroxide anion is the reactive species in the
hydration of CO2 leading to formation and release of HCO3À. The
components of this reaction are known to regulate a broad range
of physiological functions and clinical modulation of CA activity
by inhibitors has proven a reliable treatment for a range of human
disease states.1
matic sulfonamide compounds are the classical CA inhibitors and
compounds with this structural motif have been shown to reverse
or suppress the effects mediated by the cancer-associated CAs:
namely tumour acidification, cancer cell growth and tumour inva-
sion.1–6 Isozymes CA IX and XII also share a spatial localization that
distinguishes them from the physiologically dominant CA I and II.
CA IX and XII are transmembrane proteins that orient their CA cat-
alytic domain extracellularly, while isozymes I and II are soluble
proteins located within the cytosol. The development of CA inhib-
itors with an impaired ability to diffuse through lipid membranes
is one possible and attractive means by which to selectively target
cancer associated CA isozymes.7 Recently our group has demon-
strated that by ‘click-tailing’ sugar moieties to the classic high-
affinity aromatic sulfonamide pharmacophore (ArSO2NH2)—we
were able to deliver glycoconjugate sulfonamide inhibitors that
were potent and selective towards the CA isozyme IX in vitro.8–11
We have reported the synthesis and CA inhibition properties for
a series of benzene sulfonamides containing triazole-O-glycoside
tails.9 The replacement of the naturally occurring O-glycosidic link-
ages by S-glycosides is an approach practiced in the synthesis of
carbohydrate containing compounds as an avenue to enhance the
stability of the glycosidic linkage towards enzymatic hydrolysis
whilst retaining vital molecular recognition interactions with bio-
There are two known cancer-associated CA isozymes for which
elevated gene expression levels are found in a broad spectrum of
solid hypoxic tumour types, these are isozymes IX and XII.2–6 Aro-
* Corresponding authors. Tel.: +61 7 3735 7825; fax: +61 7 3735 7656 (S.-A.P.).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), s.poulsen@griffith.
edu.au (S.-A. Poulsen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.086

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M. Singer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2273–2276
logical targets.12–15 There is an increasing awareness of the biolog-
ical importance of thioglycosides owing to these favourable attri-
butes and several are in clinical use.12–15 Herein we explore
further our very effective ‘click-tailing’ strategy to synthesize a
new class of glycoconjugate sulfonamides through appending S-
propynyl glycosides of three different oxidation states onto the
4-azido benzenesulfonamide scaffold. This library was inspired
by the therapeutic drugs brinzolamide (BRZ) and dorzolamide
(DRZ), used clinically as topically acting CA inhibitors for the treat-
ment of glaucoma. The sulfone moiety of BRZ and DRZ proved crit-
ical to the effective therapeutic properties of these CA inhibitors—
assisting with water solubility yet retaining sufficient lipophilicity
to penetrate the cornea.
of the b-acetate precursors with thiourea to generate the S-glycosyl
isothiourea intermediates which were subsequently transformed
into the S-propynyl glycosides by treatment with Et3N and propar-
gyl bromide.16 Oxidation of 1 and 4 with excess mCPBA in CH2Cl2
gave per-O-acetylated b-sulfonyl propynyl glycosides 3 and 6. Oxi-
dation was generally complete following 3.5 h of stirring at ambient
temperature as evidenced by TLC. The per-O-acetylated b-sulfinyl
propynyl glycosides 2 and 5 were prepared as a mixture of diaste-
reomers (1:1) by mono-oxidation of 1 and 4 with 1.0 equiv mCPBA
in CH2Cl2 at 0 °C for 0.5 h. A number of eluents were tested to sep-
arate the diastereomers by chromatography including EtOAc/hex-
ane, acetone/toluene and MeOH/CH2Cl2 albeit without success.
Although recrystallization of the diastereomeric mixtures from
MeOH could allow enrichment in one of the diastereomers (3:1),
we elected to utilize compounds 2 and 5 as 1:1 diastereomeric mix-
tures in the subsequent synthesis of glycoconjugates. No over oxi-
dation to the sulfone was observed using the conditions described.
Next the library of 12 benzenesulfonamides (compounds 8–19)
containing triazole-tethered glycoside tails was constituted by
Cu(I)-catalyzed 1,3-dipolar cycloaddition.17,18 of the azido scaffold
7 with our panel of per-O-acetylated thiosugar-connected propy-
nyl derivatives (1–6), Scheme 2. Compound 7 was synthesized
from commercially available sulfanilamide by diazo transfer under
neutral conditions as described previously.9,19 Consistent with our
earlier findings the triazole forming reaction proceeded smoothly
when using a catalyst loading of 10 mol % of the Cu(I) source and
20 mol % of sodium ascorbate.8–11 Reactions were complete (as evi-
NHEt
NHEt
S
MeCOHN
SO2NH2
SO2NH2
SO2NH2
N
N
N
S
S
MeO(H2C)3
S
S
O
O
O
O
brinzolamide
(BRZ)
dorzolamide
(DRZ)
acetazolamide
(AZA)
A panel of acetylated S-propynyl, sulfinyl propynyl and sulfonyl gly-
cosides (1–6) were synthesized from commercially available per-O-
acetylated sugars—D-glucose (?1–3) and D-galactose (?4–6),
Scheme 1. The per-O-acetylated b-S-propynyl glycosides 1 and 4
were generated by Lewis acid catalyzed (BF3ÁEt2O) glycosylation
R2
OAc
O
O
S
R1
AcO
AcO
(iii)
(iv)
1
2
2
(R = OAc, R = H)
R2
R1
AcO
R2
OAc
O
OAc
O
5 (R1 = H, R2 = OAc)
(i), (ii)
R1
OAc
AcO
S
AcO
AcO
R2
OAc
1
2
O
1
(R = OAc, R = H)
R1
O
O
4 (R1 = H, R2 = OAc)
AcO
S
AcO
3 (R1 = OAc, R2 = H)
1
2
6
(R = H, R = OAc)
Scheme 1. aReagents and conditions: (i) thiourea, BF3ÁEt2O, CH3CN, reflux, 0.5 h; (ii) Et3N, propargyl bromide, rt, overnight, 55–59%; (iii) 1 equiv mCPBA, CH2Cl2, 0 °C, 0.5 h,
70–73%; (iv) excess mCPBA, CH2Cl2, rt, 3.5 h, 93–95%. bCompounds 2 and 5 are racemic at sulfur.
SO2NH2
SO2NH2
SO2NH2
(ii)
(i) with 1 & 4
O
N
N
O
N
N
S
S
(HO)4
(AcO)4
N
NH2
N
8
14 (Glc)
17 (Gal)
(Glc)
11 (Gal)
SO2NH2
SO2NH2
SO2NH2
O
S
O
S
(ii)
2
5
&
O
(i) with
N
N
O
N
N
(HO)4
(AcO)4
N
N
N3
15 (Glc)
18 (Gal)
9
(Glc)
7
12 (Gal)
SO2NH2
SO2NH2
O
O
S
O
O
(ii)
O
3
6
(i) with
&
N
N
O
N
N
S
(HO)4
(AcO)4
N
N
10
16
(Glc)
(Glc)
13 (Gal)
19 (Gal)
Scheme 2. aReagents and conditions: (i) azide 7 (1.0 equiv; 0.2–0.5 M), glycoside 1–6 (1.0 equiv), CuSO4.5H2O (0.1 equiv), sodium ascorbate (0.2 equiv), 8:2 EtOH/H2O, 40 °C,
2 h, 45–83%; (ii) 1.0 M NaOCH3, CH3OH, rt, overnight, 63–100%. bCompounds 9, 12, 15 and 18 are equimolar mixtures of S-epimers.

M. Singer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2273–2276
2275
Table 1
inhibitors to interact through hydrogen bonding to amino acids
within the various CA active sites and that this would be observed
indirectly by modulating Ki values. In fact, the oxidation state of
the sulfur had virtually no impact on the CA inhibition constants
determined. At hCA I inhibition by 8–19 was typically weaker
(10–20-fold) than for all other isozymes with Kis in the mid-high
nM range (60–117 nM). The oxidation state of the sulfur also did
not effect CA I enzyme inhibition, while galactose-OH compounds
(17–19, Kis 60–62 nM) were marginally stronger inhibitors than
glucose-OH compounds (14–16, Kis 101–114 nM) at hCA I. Com-
parison of CA inhibition of our recently reported O-glycosides
20–23 demonstrates that the replacement of O by S (compounds
8, 11, 14, 17), SO (compounds 9, 12, 15, 18) and SO2 (compounds
10, 13, 16, 19) typically gives weaker inhibitors at hCA I, while at
CA II inhibition is similar, and at CA IX inhibition is generally stron-
ger. This trend indicates that the sulfur analogues may prove supe-
rior for selectively targeting tumour associated hCA IX.
To deliver CA based cancer therapies or diagnostics will benefit
enormously from the development of inhibitors that target the tu-
mour-associated CA isozymes. Selective inhibition among CA iso-
zymes is however challenging owing to conservation of active
site topology within this enzyme class. Using our ‘click-tailing’
strategy we have appended thio, sulfinyl and sulfonyl glycoside tail
moieties onto the benzenesulfonamide CA pharmacophore. This
work presents a new class of glycoconjugate CA inhibitors com-
prising S-linked glycosides in three oxidation states. The antici-
pated stability of these glycoside CA inhibitors towards
endogenous glycosidases combined with the effective enzyme
inhibition properties demonstrated in this study towards CA IX
and XII may render these carbohydrate-based sulfonamides as
valuable candidates for future targeting of CAs for therapeutic
applications in hypoxic tumours. Current research in our labora-
tory is targeted towards identifying the structural features within
the CA active site that explain the tight distribution of inhibition
constants observed for this new class of inhibitors so as to support
future rationale design approach for the synthesis of CA inhibitors.
The minimal impact of the oxidation state at the sulfur atom on CA
enzyme inhibition may appear a profitable finding in future CA
drug development strategies where it is necessary to modify phys-
icochemical properties of inhibitors without impacting on drug-
enzyme molecular recognition interactions. This study is therefore
a valuable step in the ongoing research to improve the characteris-
tics of CA inhibitors.
Inhibition and selectivity ratio data for 1, new glycoconjugate sulfonamides 8–19, O-
glycoside analogues 20–23 and standard inhibitors against human isozymes hCA I, II,
IX and XII
Compound
Kia (nM)
hCA Ib
hCA IIb
hCA IXc
hCA XIIc
AZA
BRZ
DRZ
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
900
450
500
3900
91
103
97
117
80
12
3
9
47
25
47
52
5.7
3.0
3.5
n.d.
9.5
8.4
9.1
9.2
9.5
105
8.6
9.9
9.3
8.6
7.7
7.5
257
9.8
9.5
6.1
8.0
8.1
107
9.7
101
101
5.3
5.4
6.9
9.9
6.8
5.3
5.6
7.8
7.6
6.2
2.9
7.8
46
79
10.9
9.5
114
102
101
60
62
60
1500
6.9
7.0
10.2
10.3
10.3
11.9
9.4
n.d.
n.d.
n.d.
n.d.
6.8
8.7
8.7
7.0
n.d.—not determined.
a
Errors in the range of 5–10% of the reported value, from three determinations.
b
c
Human (cloned) isozymes, by the CO2 hydration method.20–22
Catalytic domain of human (cloned) isozyme, by the CO2 hydration
method.20–22
denced by TLC) after 2 h vigorous stirring. De-O-acetylation of 8–
13 by methanolic sodium methoxide was employed to liberate
the corresponding fully deprotected sugar analogues 14–19,
Scheme 2.
Enzyme inhibition data for the new glycoconjugate
sulfonamides 8–19 were determined by assaying the CA catalyzed
hydration of CO2, Table 1.20 Reference data for clinically used
CA inhibitors acetazolamide (AZA), brinzolamide (BRZ) and
dorzolamide (DRZ) have also been included for comparison with
the compounds reported in this study. Data for O-glucoside and
O-galactoside analogues 20–23, reported recently by our groups,
are included for discussion purposes.9
SO2NH2
SO2NH2
R2
R2
OAc
OH
O
O
R1
AcO
R1
HO
Acknowledgements
N
N
N
N
O
O
AcO
HO
N
N
This work was financed in part by the Australian Research
Council (Grant number DP0877554 to S.-A.P.); the Eskitis Institute
for Cell and Molecular Therapies and an EU grant of the 6th frame-
work programme (DeZnIT project to C.T.S.). We thank Hoan The Vu
for undertaking accurate mass measurements.
(R1 = OAc, R2 = H)
(R1 = OAc, R2 = H)
20
21
22
23
(R1 = H, R2 = OAc)
(R1 = H, R2 = OAc)
The parent azido scaffold 7 had greatest efficacy against hCA II (Ki of
47 nM), approximately twofold weaker inhibition against the tu-
mour-associated hCA IX (105 nM) and 80-fold weaker inhibition
against hCA I (3900 nM). The inhibition profile for 7 was weaker
at all isozymes compared to the clinically used sulfonamide inhibi-
tors AZA, BRZ and DRZ, however 7 did have a similar selectivity pro-
file to AZA. The new glycoconjugates were investigated both in the
per-O-acetylated form (compounds 8–13) and the free hydroxyl
form (compounds 14–19). All glycoconjugates were stronger inhib-
itors than the parent azido compound 7 at the CA isozymes tested.
Glycosides were potent inhibitors of hCA II (Kis 2.9–9.9 nM), IX
(Kis 6.1–9.9 nM) and XII (Kis 8.4–11.9 nM) with Ki values narrowly
distributed—and just one outlier to this trend—compound 14 with
a Ki of 257 nM at hCA IX. The tight distribution of Ki values sur-
prised us as we had expected that the oxidation of the sulfur would
provide an increased opportunity for these small molecule
Supplementary data
Supplementary data (synthetic procedures, compound charac-
terization data and 1H NMR spectra for new compounds) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.02.086.
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