Inhibition of carbonic anhydrases with glycosyltriazole benzene sulfonamides

Article abstract of DOI:10.1021/jm701426t

A library of glycoconjugate benzene sulfonamides have been synthesized and investigated for their ability to inhibit the enzymatic activity of physiologically relevant human carbonic anhydrase (hCA) isozymes: hCA I, II, and tumor-associated IX. Our synthetic strategy directly links the known CA pharmacophore (ArSO₂NH₂) to a sugar tail moiety through a rigid 1,2,3-triazole linker unit using the Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction or click chemistry . Many of the glycoconjugates were potent CA inhibitors and exhibited some isozyme selectivity. In particular, the methyl-D-glucuronate triazoles 6 and 14 were potent inhibitors of hCA IX (K_iS 9.9 and 8.4 nM, respectively) with selectivity also favoring this isozyme. Other exceptional compounds included the deprotected β-D-ribofuranosyl triazole 15 and α-D-mannosyl triazole 17, which were potent and selective hCA II inhibitors (K_i 7.5 nM and K_i 2.3 nM, respectively). Collectively, the results confirm that modification of ring size, stereochemical configuration, and chain length in the sugar tail moiety of glycoconjugate CA inhibitors permits tunable potency and selectivity that may constitute an important avenue for the future development of efficacious and selective CA-based therapeutics.

Full text of DOI:10.1021/jm701426t

J. Med. Chem. 2008, 51, 1945–1953
1945
Inhibition of Carbonic Anhydrases with Glycosyltriazole Benzene Sulfonamides
Brendan L. Wilkinson,† Alessio Innocenti,‡ Daniela Vullo,‡ Claudiu T. Supuran,*,‡ and Sally-Ann Poulsen*,†
Eskitis Institute for Cell and Molecular Therapies, Griffith UniVersity, 170 Kessels Road, Nathan, Queensland 4111, Australia, and Polo
Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, UniVersità degli Studi di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy
ReceiVed NoVember 12, 2007
A library of glycoconjugate benzene sulfonamides have been synthesized and investigated for their ability
to inhibit the enzymatic activity of physiologically relevant human carbonic anhydrase (hCA) isozymes:
hCA I, II, and tumor-associated IX. Our synthetic strategy directly links the known CA pharmacophore
(ArSO2NH2) to a sugar “tail” moiety through a rigid 1,2,3-triazole linker unit using the Cu(I)-catalyzed
1,3-dipolar cycloaddition reaction or “click chemistry”. Many of the glycoconjugates were potent CA inhibitors
and exhibited some isozyme selectivity. In particular, the methyl-D-glucuronate triazoles 6 and 14 were
potent inhibitors of hCA IX (Kis 9.9 and 8.4 nM, respectively) with selectivity also favoring this isozyme.
Other exceptional compounds included the deprotected ꢀ-D-ribofuranosyl triazole 15 and R-D-mannosyl
triazole 17, which were potent and selective hCA II inhibitors (Ki 7.5 nM and Ki 2.3 nM, respectively).
Collectively, the results confirm that modification of ring size, stereochemical configuration, and chain length
in the sugar tail moiety of glycoconjugate CA inhibitors permits tunable potency and selectivity that may
constitute an important avenue for the future development of efficacious and selective CA-based therapeutics.
Introduction
loss, depression,6 arteriosclerosis, and renal pathology.7 The
The carbonic anhydrases (CA,a EC 4.2.1.1) are ubiquitous
zinc(II) metalloenzymes that are found in higher vertebrates,
green plants, algae, bacteria, and archaea.1 CA catalyzes the
reversible hydration of carbon dioxide (CO2) to give bicarbonate
(HCO3-) and a proton (H+), a fundamental physiological
reaction that underpins a multitude of essential cellular processes
associated with respiration and transport of CO2/HCO3- between
metabolizing tissues and the lungs, photosynthesis in higher
modulation of CA activity through inhibition (or activation) is
therefore a promising avenue for the treatment of a wide range
of acquired and inherited diseases.2
Since the original report by Mann and Keilin concerning the
inhibition of CA with sulfanilamide (SA),8 followed some time
later with the pioneering work of Krebs,9 primary aryl and
heteroaryl sulfonamide CA inhibitors have become a clinical
mainstay as antihypertensive, antiglaucoma, antithyroid and
hypoglycemic drugs. The aryl- and heteroaryl sulfonamides
acetazolamide (AZA), methazolamide (MZA), ethoxazolamide
(EZA), and the bis-sulfonamide, dichlorophenamide (DCP) have
been used clinically for over 40 years as systemic CA inhibitors
(Figure 1). Acetazolamide AZA is the first nonmercurial diuretic
used clinically.1a In addition to orally available CA inhibitor-
based drugs, the topically acting sulfonamides dorzolamide
(DRZ) and the structurally related brinzolamide (BRZ) are
potent inhibitors of hCA II and hCA XII within the ciliary
processes of the eye and are used for the treatment of open-
angle glaucoma.10 Furthermore, the fructopyranose sulfamate
ester, topiramate (TPZ) has been assessed as a powerful inhibitor
of hCA II and hCA VII in vitro and, apart from its well
established anticonvulsant activity, its inhibition of brain CA
isozymes also makes it a promising candidate for the treatment
of memory disorders and depression.11
-
plants, provision of HCO3 for biosynthetic pathways (gluco-
neogenesis, lipogenesis and ureagenesis), pH regulation and CO2
homeostasis, electrolyte and fluid secretion, as well as bone
resorption and calcification.1,2 In mammals, 16 different R-CA
isozymes and CA related proteins (CARP) have been identified
and characterized to date, 15 of these are present in humans
(designated hCA; CA XV is absent in humans and primates
but is present in rodents and other higher mammals). hCA
isozymes exhibit variable enzyme kinetics, tissue distribution,
expression levels, and subcellular locations.1,2 Several hCA
isozymes are cytosolic (hCA I-III, hCA VII, hCA XIII), four
are membrane bound or transmembrane proteins (hCA IV, IX,
XII and XIV), two are mitochondrial (hCA VA and VB), and
one is secreted into the saliva and milk (hCA VI). Many hCA
isozymes are quite recent discoveries compared with the
physiologically abundant and widely distributed isozymes hCA
I and hCA II (known since the 1930s, with isozymes acknowl-
edged in the 1960s). The dysregulation of hCA expression is
now implicated in a host of pathophysiological processes such
as oncogenesis,3 elevated intraocular pressure,4 obesity,5 memory
Furthermore, several important developments within the CA-
based disciplines have also provided new and viable targets for
molecular medicine. Of particular relevance to cancer chemo-
therapy is the overexpression of transmembrane isozyme hCA
IX on certain tumor cells (primarily carcinomas) within tissues
not normally known to express hCA IX, including carcinomas
of the lung, breast, colon, esophagus, and cervix. This hCA IX
is found ectopically expressed in many hypoxic tissues through
the transcriptional activation of hypoxia-induced factor 1 (HIF-
1).12 Also, hCA IX is a high activity CA isozyme responsible
for the extracellular acidification of the tumor microenvironment,
leading to multiple downstream effects, including tumor pro-
gression and poor prognosis.13 The differential expression
* To whom correspondence should be addressed. Tel.: +61 7 3735
7825 (S.-A.P.); +39 055 457 3005(C.T.S.). Fax: +61 7 3735 7656 (S.-
A.P.); +39 055 457 3385 (C.T.S.). E-mail: s.poulsen@griffith.edu.au (S.-
A.P.); claudiu.supuran@unifi.it. (C.T.S.).
†
Griffith University.
Università degli Studi di Firenze.
‡
a Abbreviations: CA, carbonic anhydrase; 1,3-DCR, 1,3-dipolar cycload-
dition reaction; AZA, acetazolamide; MZA, methazolamide; EZA, ethox-
zolamide; DCP, dichlorophenamide; DRZ, dorzolamide; BRZ, brinzolamide;
IND, indisulamm; TPZ, topiramate.
10.1021/jm701426t CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

1946 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Wilkinson et al.
Figure 1. Known human CA inhibitors including some clinically used drugs.
patterns of hCA IX has the potential to function as a cancer
biomarker for the early detection of gastric cancer and certain
other carcinomas. It is known that hCA IX displays high affinity
for primary aryl and heteroaryl sulfonamides and thus its
inhibition can reverse the acidification of the tumor microen-
vironment, thus retarding tumor growth and reducing the
chemoresistance to weakly basic frontline drugs such as
paclitaxel, topotecan, and mitoxantrone.3,9 The bis-sulfonamide
indisulam (IND) is in phase II clinical trials for the treatment
of solid tumors and is a potent inhibitor of hCA IX in vitro (Ki
24 nM).14
containing natural products possessing antimicrobial and anti-
cancer properties, such as the aminoglycosides, saponins, and
anthracyclines.18 Furthermore, carbohydrate prodrugs of cytoxic
agents are a promising avenue for the selective targeting of
anticancer drugs to the desired site of action.19
The Cu(I)-catalyzed ligation of a terminal acetylene to an
organic azide to form regiospecifically a 1,4-disubstituted-1,2,3-
triazole has emerged as a venerable synthetic tool in the drug
discovery/medicinal chemistry and biotechnology sectors.20 The
transformation, now known as “click chemistry” occurs under
ambient conditions, has a high degree of biocompatibility, and
has been shown to be orthogonal to pre-existing synthetic
methodologies.21 Furthemore, the methodology has also applied
to the synthesis and screening potent CA II inhibitors in situ.22
We have recently demonstrated the versatility of the click
chemistry methodology as an expedient method of generating
1,4-disubstituted-1,2,3-triazole sulfonamide glycoconjugates.23–25
These compounds contain a benzene sulfonamide moiety linked
via a 1,2,3-triazole to a sugar tail. In our first and second
generation glycoconjugates the triazole linker was in combina-
tion with either an ester, amide or O-glycoside functionality
(Figure 2). These compounds have so far been evaluated for
their inhibition in vitro of hCA I, II, and IX and, in some cases,
also for transmembrane CAs XII and XIV. The impressive
potency and selectivity of several inhibitors within these libraries
reflects not only the significance of the carbohydrate tail at
differentiating isozyme selectivity, but also for the fact that the
high affinity aryl sulfonamide “anchor” moiety more than
compensates for the potential loss of energy on binding to the
carbohydrate moiety.
Despite the widespread use of sulfonamides in the clinic, their
systemic administration often causes side effects due to the
indiscriminate inhibition of CA isozymes in other tissues. This
is prompting the development of tissue specific delivery systems
and isozyme specific inhibitors with the aim of producing
therapeutics with improved tolerability and efficacy. In the latter
respect, it has become imperative to target subtle differences in
active site topology and structure, a challenging process given
the high degree of conservation among the CA isozymes. One
promising strategy that has emerged in recent years to dif-
ferentiate transmembrane hCA IX from the physiologically
dominant cytosolic isozymes hCA I and II, is to develop
inhibitors with polar or charged tails, thus impairing their ability
to diffuse through lipid membranes.15,16 In this respect, carbo-
hydrates offer unique advantages to this fragment-based ap-
proach, whereby a high degree of polyfuntionality and hydro-
philicity can be imparted onto the sulfonamide pharmacophore.
The fine-tuning of the stereochemical arrangement of the
carbohydrate tail and physicochemical properties of CA inhibitor
can potentially allow for the differentiation of subtle differences
in CA active site topology, as well as impairing membrane
diffusion for the selective targeting of transmembrane hCA
isozymes of clinical interest. Carbohydrates are powerful
structural motifs for attenuating the magnitude and selectivity
of binding to native and non-native protein receptors,17 and their
presence within many natural products is often a prerequisite
for biological activity and can thus heavily influence the
pharmacokinetics, drug targeting, and mechanism of action.
Indeed, the literature is replete with examples of carbohydrate-
Encouraged by these earlier results, we have recently
undertaken the expedient synthesis of sulfonamide glycocon-
jugates using click chemistry, differing in this new generation
of compounds is that the carbohydrate tail is linked directly to
the benzene sulfonamide fragment through a rigid 1,2,3-triazole
unit. The removal of the carboxy ester, amide or O-glycoside
linker functionality endows these third generation inhibitors with
potentially enhanced stability toward esterase, protease or
glycosidase hydrolysis in the in vivo environment.26 In addition
to the glycoconjugate sulfonamides previously reported by our

Inhibition of Carbonic Anhydrases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1947
Figure 2. Generation I and II benzene sulfonamide glycoconjugates.23–25
Scheme 1. Synthesis of 4-Ethynyl Benzene Sulfonamide (1) CA
Anchor Fragment
phase extraction (Scheme 2). The Cu(I) catalyst loading was
achieved by employing 10 mol% of CuSO4 and 20 mol% of
sodium ascorbate reductant. The O-acetate and O-benzoate
groups of 2-9 were subsequently removed using methanolic
sodium methoxide (Zemplen conditions) to liberate the fully
deprotected sugar analogues 10-17 in near quantitative yields
(Scheme 2).
Carbonic Anhydrase Inhibition. hCA I, II, and IX enzyme
inhibition data for the new glycoconjugate sulfonamides were
determined by assaying the impact of these compounds on the
31
CA-catalyzed hydration of CO2 (Table 1). The selectivity
group, the inclusion now of a conformationally rigid library
provides a useful comparative surveillance of the active sites
of clinically relevant CA isozymes, a necessary step toward the
discovery of novel CA-based therapeutics and diagnostics
displaying improved potency and selectivity.
ratios for inhibition of isozyme IX compared to I and II are
also given in Table 1. Data for clinically used CA inhibitors
are included for comparison.
Isozyme hCA I. At hCA I the glycoconjugates with acyl-
protected sugar tails (2–9) were generally weaker inhibitors than
the parent alkyne 1 (Ki 1.1 µM) with two notable exceptions,
the R-D-mannosyl triazole 8 and R-D-arabinopyranosyl triazole
9, which showed potent inhibition (Ki 1.6 nM and 10.5 nM,
respectively). In addition, the R-D-configured deprotected
analogues 16 (Ki 2.8 nM) and 17 (Ki 2.5 nM) were also excellent
hCA I inhibitors, similarly to their per-O-acetylated counterparts
8 and 9. In this respect, the R-configuration at the anomeric
center may be a significant stereochemical feature toward
inhibitory potency at hCA I, with these compounds up to 3
orders of magnitude more potent than the parent alkyne 1. Of
the remaining deprotected sugar analogues 10–15, compounds
12–15 were micromolar inhibitors similar to the corresponding
per-O-acylated analogues 3–7, while potent inhibition was
detected for the D-gluco- and D-galacto-configured triazoles 10
(Ki 9.4 nM) and 11 (Ki 9.3 nM), being ∼500-fold stronger
inhibitors than their per-O-acetylated counterparts 2 and 3,
respectively. Interestingly, inhibition data for the N-acetyl
glucosamine derivative 13 (Ki 4.4 µM) when compared with
data observed for the corresponding D-gluco-configured ana-
logue 10 (Ki 9.4 nM) showed a significant loss in hCA I
inhibitory potency due the presence of the 2-acetamido group.
Collectively, these results demonstrate that subtle structural
differences in the sugar tail can indeed discriminate the hCA I
isozyme active site topology to substantially influence enzyme
inhibition characteristics.
Results and Discussion
To facilitate our synthetic strategy, it was necessary to design
and synthesize an aryl sulfonamide anchor fragment with reliable
hCA recognition and with the complementary reactive func-
tionality (i.e., an acetylene moiety) for the expedient 1,3-DCR
ligation to a panel of acyl-protected glycosyl azide fragments
(a-h). Sharpless and colleagues have previously employed
4-ethynyl benzenesulfonamide (1) as the reactive fragment for
developing bovine CA inhibitors in situ.22 This fragment was
selected by ourselves as it represents a minimal fragment for
the facile parallel synthesis of the proposed glycoconjugate hCA
inhibitor library. Compound 1 was prepared via a Sonagashira
coupling of 4-iodobenzene sulfonamide with trimethylsilyl
acetylene, followed by fluoride mediated silyl deprotection
(Scheme 1).27
The azide panel employed for ligation to 1 was comprised
of carbohydrates with varying ring size, chain length and
relative stereochemistry so as to access variable physico-
chemical properties across the downstream inhibitor library
to allow for efficient interrogation of CA active site
architecture. In this respect, a panel of eight peracylated
glycosyl azides a-h derived from (a) glucose, (b) galactose,
(c) N-acetyl glucosamine, (d) maltose, (e) glucuronic acid,
(f) ribose, (g) mannose, and (h) arabinose were synthesized
(Figure 3). Glycosyl azides (a-e, h) were synthesized by
the stereoselective nucleophilic displacement of the respective
glycosyl halides with azide.28 The glycosyl halide precursors
were themselves prepared from the corresponding per-O-
acetates or purchased from commercial sources. The ꢀ-D-
ribosyl azide (f) and R-D-mannosyl azide (g) were prepared
by the Lewis acid (SnCl4)-promoted azidolysis of the
respective 1-O-acetate precursors.29
Isozyme hCA II. The parent alkyne 1 had a Ki of 5.1 nM at
hCA II, ∼200-fold more potent than at hCA I. The incorporation
of the eight different sugar tails onto the benzene sulfonamide
scaffold 1 had a variable effect on the inhibition of hCA II in
vitro with Ki values in the range of 2.1–440 nM for the protected
sugar series 2–9 and 2.3–460 nM for the deprotected sugar series
10–17. As for hCA I, the R-configuration at the anomeric center
gave the most potent hCA II inhibitors: the per-O-acetylated
R-D-mannosyl triazole 8 (Ki 2.1 nM) and the deprotected R-D-
arabinopyranosyl triazole 17 (Ki 2.3 nM). There were several
library members with hCA II inhibition less than 10 nM (2, 3,
8, 9, 11, 15, 16, 17), many of these were comparable with
By employing a modified click chemistry methodology,30
fragment 1 was reacted with the azido sugar panel a-h to
generate a series of acyl-protected 1,4-disubsituted glycosyl-
triazole benzene sulfonamides 2–9 in moderate to good yields
(69–88%) following flash chromatography or normal phase solid

1948 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Wilkinson et al.
Figure 3. Azido sugar “tail” fragment panel: a-e, g, h (R ) Ac); f (R ) Bz); and a′-h′ (R ) H).
Scheme 2. Synthesis of Glycoconjugate Benzene Sulfonamide Library (2–17) from 1 and Sugar Panel a-ha
a Reagents and conditions: (i) a-h (1.0 equiv), CuSO4 (0.1 equiv), sodium ascorbate (0.2 equiv), 1:1 tert-butanol–water, 40 °C, 30 min–1 h, 69–88%;
(ii) NaOCH3, CH3OH, rt, 15 min–2 h, quantitative.
corresponding hCA I activity, while a few were quite selective
for hCA II over hCA I (2, 3, and 15). Noteworthy, the
deprotected ribose analogue 15 displayed high selectivity for
the hCA II isozyme compared with both hCA I (573-fold) and
IX (16-fold). Interestingly, the removal of the acetate groups
from the disaccharide motif of 4 (Ki 235 nM) to afford 12 (Ki
432 nM) and the O-acetates from the GlcNAc derivative 5 (Ki
440 nM) to afford 13 (Ki 460 nM) did not result in enhanced
inhibitory potency at hCA II, a pattern not mirrored by the other
glycoconjugates within the series. This observation is in contrast
with the removal of the bulky benzoate groups from the
D-ribofuranosyl triazole 7 (Ki 427 nM) to afford 15 (Ki 7.5 nM),

Inhibition of Carbonic Anhydrases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1949
Table 1. Inhibition and Isozyme Selectivity Ratio Data for Clinically
Used CA Inhibitors, 4-Ethynyl Benzenesulfonamide 1, and the 16 New
Glycoconjugates 2–17 against Human Isozymes hCA I, II, and IX
the human isozymes does however make this a challenging
process. Within this study, we have prepared of a library of
hydrolytically stable benzene sulfonamides with triazole-tethered
carbohydrate tails as a dual isozyme differentiating and solu-
bilizing strategy. The evaluation of in vitro inhibition at cytosolic
isozymes hCA I, II, and the cancer-related and membrane-
associated extracellular hCA IX has demonstrated the successful
interrogation of CA active site topology via the carbohydrate
tail moiety. The facile conjugation of carbohydrate tails to a
primary aryl sulfonamide fragment using click chemistry has
provided an expedient and powerful means to generate these
glycoconjugate CA inhibitors. The stereochemical and structural
variability of the carbohydrate “tail” region allows for explora-
tion of active site architecture and the potential development
of neutral, water soluble CA inhibitors for drug delivery
applications, and as a means of specifically targeting extracel-
lular and clinically relevant CA isozymes due to impaired
permeation of the plasma membrane. This strategy has suc-
cessfully led to the identification of several potent and selective
inhibitors of the pharmacologically relevant isozymes hCA II
and hCA IX. In particular, the methyl-D-glucuronate triazoles
6 and 14 were shown to be potent inhibitors of hCA IX (Ki
values 9.9 and 8.4 nM, respectively), and the ꢀ-D-ribofuranosyl
triazole 15 and R-D-mannosyl triazole 17 exhibited potent and
selective inhibition of hCA II (Ki 7.5 nM and Ki 2.3 nM,
respectively). Such compounds may constitute important leads
for the development of safe and efficacious CA inhibitor-based
therapeutics, thus further highlighting the significance of gly-
coconjugates in CA-based inhibitor research.
Kia (nM)
selectivity ratiosb
cmpd hCA Ic hCA IIc hCA IXd hCA I/hCA IX hCA II/hCA IX
AZA
MZA
EZA
BRZ
DRZ
DCP 1200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
250
50
25
450
500
12
14
8
3
9
38
5.1
9.1
8.7
235
440
13
427
2.1
8.2
380
8.8
432
460
13
7.5
7.6
2.3
25
27
34
47
52
50
10
18.5
0.74
9.6
9.6
0.48
0.52
0.24
0.06
0.17
0.76
0.63
0.08
0.08
1.96
4.36
1.3
4.27
0.42
0.06
4.3
0.07
3.32
5.41
1.6
24
1080
4400
4300
2500
5000
48
8.1
133.3
36.7
39.1
20.8
49.5
4.84
53.0
0.32
120
110
120
101
9.9
100
5.0
134
89
120
130
85
5300
1.6
10.5
9.4
9.3
5000
4400
560
4300
0.09
0.11
0.08
38.5
51.8
50.8
35.5
0.52
0.09
8.4
121
5.4
28
0.06
1.41
0.08
2.8
2.5
a Errors in the range of (5–10% of the reported value, from three
determinations. b The Ki ratios are indicative of isozyme selectivity. c Human
(cloned) isozymes, by the CO2 hydration method.31 d Catalytic domain of
human (cloned) isozyme, by the CO2 hydration method.31
resulting in a 57-fold increase in inhibitory potency, the steric
demands of the hCA II active site may account for this
observation.
Experimental Section
General. Glycosyl azide precursors were prepared by phase
transfer nucleophilic displacement of corresponding peracetylated
R-glycosylbromide.28 All reagents were purchased from commercial
sources and were used without further purification. All solvents
were available commercially dried or freshly dried and distilled
prior to use. Reaction progress was monitored by TLC using silica
gel-60 F254 plates with detection by short wave UV fluorescence
(λ ) 254 nm) and staining with 10% (v/v) H2SO4 in ethanol char.
Flash chromatography was conducted using Merk flash silica gel
60 (60–240 mesh). Solid phase extraction was performed using
Phenomenex Strata cartridges prepacked with silica (SI-1). 1H NMR
(400 or 300 MHz), 13C{1H} NMR (100 or 75 MHz), 2D gCOSY,
gHSQC, and gHMBC spectra were recorded on a Varian NMR
spectrometers with chemical shift values given in ppm (δ) using
deuterated solvent as specified. 13C NMR spectra were referenced
to either δ 77 ppm (CDCl3) or δ 39.5 ppm (DMSO-d6). Melting
points were recorded on a GallenKamp Variable Temperature
Apparatus by capillary method and are reported as uncorrected.
Mass spectra were recorded on a Fisons VG platform II and a
Waters Micromass ZQ4000 spectrometer employing a single quad
dual source and using electrospray as the ionization technique in
positive and negative ion modes. High resolution electrospray
ionization mass spectra were recorded on a Bruker Daltonix 4.7T
Fourier transform ion cyclotron resonance mass spectrometer
(FTICR MS) fitted with an Apollo ESI source in positive ion or
negative ion as stated. All MS analysis samples were prepared as
solutions in methanol.
Isozyme hCA IX. The parent alkyne 1 had a Ki of 8.1 nM
at hCA IX. Many of the glycoconjugates (2-5, 7, 9-13, 15,
and 17) exhibited clustered inhibition of hCA IX but were ∼10-
fold weaker inhibitors than the parent acetylene 1. The methyl
D-glucuronates (6 and 14) were exceptional, however, and
exhibited potent inhibition of hCA IX (Ki values 9.9 nM
and 8.4 nM, respectively) as were the R-D-mannosyl triazole 8
and 16 (Ki values 5.0 nM and 5.4 nM, respectively), these are
among the most potent inhibitors of hCA IX reported to date.
In addition to their potent hCA IX inhibition, triazoles 6 and
14 also exhibited mild selectivity for hCA IX with respect to
both hCA I and II. Noteworthy, these compounds exhibit up to
56-fold stronger inhibition potencies at hCA IX than the hitherto
describedcarboxyesterandamide-linkedglucuronatetriazoles.23,25
The parent fragment 1 displays no selectivity for hCA IX with
respect to hCA II, while a number of our moderate hCA IX
inhibitors (4, 5, 7, 12, and 13) were selective for hCA IX with
respect to isozymes I and II, representing an important result
for future therapeutic applications. Collectively, these results
confirm our earlier observations that by tethering a sugar triazole
tail onto the CA anchor pharmacophore it is possible to generate
hCA IX selective inhibitors over the physiologically abundant
hCA I and II isozymes.
General Procedure 1: Synthesis of Glycoconjugate Ben-
zene Sulfonamides (2–9). A mixture of azide (1.0 equiv.) and
acetylene (1.0 equiv.) was suspended in a tert-butyl alcohol and
distilled water (1:1, 0.2–0.5 M final concentration). A solution of
sodium ascorbate (0.2 equiv) in water, followed by a solution of
CuSO4 ·5H2O (0.1 equiv) in water was successively added. The
bright yellow heterogeneous mixture was stirred vigorously at 40
°C until TLC indicated reaction completion (generally within 2 h).
The mixture was evaporated under reduced pressure and the
resulting residue was purified by flash chromatography to yield
Conclusions
To maximize the benefits and safety of future CA-based
therapies, it has become a matter of acute importance to develop
isozyme selective CA inhibitors and/or drug delivery systems
so as to avoid side effects associated with indiscriminate
inhibition of CAs present in multiple tissue sites. The poly-
pharmacology and strong conservation of amino acid sequence
and three-dimensional architecture of the catalytic site among

1950 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Wilkinson et al.
analytically pure material. The 1,4-regioselectivity of the reaction
144.23 (Ar C), 146.39 (triazole C), 169.36 (OAc), 170.16 (OAc),
170.62 (OAc), 170.68 (OAc). HRMS (ESI) calcd for C22-
H26N4O11SNa+, 577.12110; found, 577.12222. Anal. (C22H26-
N4O11S) C, H, S. N: calcd, 10.10; found, 9.67.
1
was verified by H and 13C NMR chemical shifts of the triazole
moiety in products obtained using DMSO-d6 and D2O and are in
agreement with literature values.30,32
General Procedure 2: Deprotection of Glycoconjugate
Benzene Sulfonamides (2–9 f 10–17). Deprotected triazoles
10–17 were prepared by treating the corresponding per-O-acylated
precursor 2–9 with anhydrous methanolic sodium methoxide (final
concentration of ∼0.1–0.2 M, pH 9–12) at room temperature.
Reactions were found to be complete within 30 min by TLC.
Neutralization of the methoxide by Amberlite IR-120 acidic ion-
exchange resin, followed by filtration and evaporation of the filtrate
4-[4-(Aminosulfonyl)phenyl]-1-(ꢀ-D-galactopyranosyl)-1H-
1,2,3-triazole (11). The title compound was prepared from 3
according to general procedure 2 and isolated as pale yellow
crystalline solid (140 mg, 0.36 mmol, ∼100%). Rf 0.12 (1:9 H2O-
1
CH3CN); mp 171–172 °C (decomp). H NMR (400 MHz, ∼1%
D2O in DMSO-d6) δ 3.45–3.52 (m, 2H, H6′, H6′′), 3.56 (dd, 3J3′-2′
3
) 9.2 Hz, J3′-4′ ) 3.2 Hz, 1H, H3′), 3.72–3.75 (m, 1H, H5′),
3.76–3.77 (m, 1H, H4′), 4.04-4.09 (m, 1H, H2′), 5.52 (d, 3J1′-2′
)
1
afforded pure material by H NMR and 13C NMR spectroscopy.
9.2 Hz, 1H, H1′), 7.36 (br s, 2H, SO2NH2), 7.86–8.08 (m, 4H, Ar
H), 8.90 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz, 1% D2O
in DMSO-d6) δ 61.04 (C6′), 68.97 (C5′), 69.97 (C2′), 74.11 (C3′),
79.14 (C4′), 88.03 (C1′), 122.17 (triazole CH), 126.13 (Ar CH),
127.10 (Ar CH), 143.76 (triazole C or Ar C), 145.84 (triazole C or
Ar C). HRMS (ESI) calcd for C14H18N4O7S+, 409.07902; found,
409.07817.
Special precaution was required for the deprotection of methyl
D-glucuronate 6 to prevent saponification of the methyl ester using
sodium methoxide in methanol. In this case, the pH was kept as
near as possible to 8–9 with a lengthened reaction time (∼1 h).
4-Ethynyl Benzene Sulfonamide (1). Prepared by Sonagashira
coupling according to a literature procedure.27 Rf 0.37 (1:1
1
EtOAc-hexanes); mp 174–175 °C. H NMR (400 MHz, DMSO-
4-[4-(Aminosulfonyl)phenyl]-1-(hepta-O-acetyl-ꢀ-D-maltopy-
ranosyl)-1H-1,2,3-triazole (4). The title compound was prepared
according to the general procedure 1 and isolated as white solid
(210 mg, 0.25 mmol, 83%). Rf 0.26 (3:7 hexanes-EtOAc); mp
241–242 °C (decomp). 1H NMR (400 MHz, DMSO-d6) δ 1.75 (s,
3H, OAc), 1.94 (s, 3H, OAc), 1.95 (s, 3H, OAc), 1.97 (s, 3H, OAc),
1.99 (s, 3H, OAc), 2.00 (s, 3H, OAc), 2.03 (s, 3H, OAc), 3.89–4.03
(m, 2H, GlcR H5′, GlcR H6′), 4.09–4.18 (m, 3H, Glcꢀ H4′, Glcꢀ
H6′, GlcR H6′′), 4.38 (ddd, 3J5′-4′ ) 9.6 Hz, 3J5′-6′ ) 5.6 Hz, 3J5′-6′′
) 2.0 Hz, 1H, Glcꢀ H5′), 4.44 (dd, 2J6′′-6′ ) 12.4 Hz, 3J6′′-5′ ) 2.4
Hz, 1H, Glcꢀ H6′′), 4.90 (dd, 3J2′-3′ ) 10 Hz Hz, 3J2′-1′ ) 4.0 Hz,
1H, GlcR H2′), 4.97–5.02 (m, 1H, GlcR H4′), 5.23 (dd, 3J3′-2′ ) 10
Hz, 3J3′-4′ ) 9.6 Hz, 1H, GlcR H3′), 5.36 (d, 3J1′-2′ ) 4.0 Hz, 1H,
GlcR H1′), 5.50–5.54 (m, 1H, Glcꢀ H2′), 5.61–5.66 (m, 1H, Glcꢀ
d6) δ 4.41 (s, 1H, CtCH), 7.41 (br s, 2H, SO2NH2), 7.63–7.79
(m, 4H, Ar H); 13C{1H} NMR (100 MHz, DMSO-d6) δ 82.98
(CtCH), 84.05 (CtCH), 125.72 (Ar C), 126.63 (Ar CH), 132.92
(Ar CH), 144.78 (Ar C).
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-ꢀ-D-
glucopyranosyl)-1H-1,2,3-triazole (2). The title compound was
prepared according to the general procedure 1 and isolated as white
solid (102 mg, 0.18 mmol, 69%). Rf 0.29 (3:7 hexanes-EtOAc);
1
mp 218–219 °C. H NMR (400 MHz, DMSO-d6) δ 1.78 (s, 3H,
OAc), 1.95 (s, 3H, OAc), 1.98 (s, 3H, OAc), 2.01 (s, 3H, OAc),
2
3
4.07 (dd, 1H, J6′-6′′ ) 12.8 Hz, J6′-5′ ) 2.4 Hz, 1H, H6′), 4.14
(dd, 2J6′′-6′ ) 12.8 Hz, 3J6′′-5′ ) 5.6 Hz, 1H, H6′′), 4.41 (ddd, 3J5′-4′
3
3
) 10 Hz, J5′-6′′ ) 5.6 Hz, J5′-6′ ) 2.4 Hz, 1H, H5′), 5.10–5.16
(m, 1H, H4′), 5.56–5.61 (m, 1H, H3′), 5.62–5.67 (m, 1H, H2′), 6.41
(d, 3J1′-2′ ) 8.8 Hz, 1H, H1′), 7.37 (br s, 2H, SO2NH2), 7.87–8.01
(m, Ar), 8.68 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz,
DMSO-d6) δ 20.56 (OAc), 20.92 (OAc), 21.07 (OAc), 21.20 (OAc),
62.43 (C6′), 68.21 (C4′), 70.99 (C3′), 72.68 (C2′), 73.88 (C5′), 84.69
(C1′), 122.34 (triazole CH), 126.21 (Ar CH), 127.18 (Ar CH), 133.81
(Ar C), 144.27 (triazole C or Ar C), 146.40 (triazole C or Ar C),
169.30 (OAc), 170.07 (OAc), 170.24 (OAc), 170.70 (OAc). HRMS
(ESI) calcd for C22H25N4O11S1-, 553.12460; found, 553.12368.
Anal. (C22H26N4O11S.2H2O) C, N, S. H: calcd, 5.12; found, 4.64.
4-[4-(Aminosulfonyl)phenyl]-1-(ꢀ-D-glucopyranosyl)-1H-1,2,3-
triazole (10). The title compound was prepared from 2 according
to general procedure 2 and isolated as white crystalline solid (32
mg, 0.08 mmol, ∼100%); mp 243–244 °C (decomp). 1H NMR (400
MHz, 2% D2O in DMSO-d6) δ 3.20–3.25 (m, 1H, H4′), 3.38–3.49
(m, 3H, H3′, H5′ H6′), 3.67–3.69 (m, 1H, H6′′), 3.74–3.78 (m, 1H,
H2′), 5.57 (d, 3J1′-2′ ) 9.2 Hz, 1H, H1′), 7.86–8.05 (m, 4H, Ar CH),
8.94 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz, 1% D2O in
DMSO-d6) δ 61.28 (C6′), 70.10 (C4′), 72.81 (C2′), 77.23 (C3′), 80.53
(C5′), 88.37 (C1′), 122.38 (triazole CH), 126.11 (Ar CH), 127.15
(Ar CH), 134.41 (Ar C), 143.76 (triazole C or Ar C), 145.83 (triazole
C or Ar C). HRMS (ESI) calcd for C14H17N4O7S-, 387.09689;
found, 387.09752.
3
H3′), 6.37 (d, J1′-2′ ) 9.2 Hz, 1H, Glcꢀ H1′), 7.37 (br s, 2H,
SO2NH2), 7.87–8.02 (m, 4H, Ar), 9.87 (s, 1H, triazole CH); 13C{1H}
NMR (100 MHz, DMSO-d6) δ 20.61 (OAc), 20.95 (OAc), 21.03
(2 × OAc), 21.12 (OAc), 21.20 (OAc), 21.25 (OAc), 62.08 (GlcR
C6′), 63.48 (Glcꢀ C6′), 68.38 (GlcR C4′), 68.87 (Glcꢀ C4′), 69.59
(GlcR C3′), 70.12 (GlcR C2′), 71.47 (Glcꢀ C2′), 74.13 (GlcR C5′),
74.59 (Glcꢀ C5′), 74.92 (Glcꢀ C3′), 84.27 (Glcꢀ C1′), 96.44 (GlcR
C1′), 122.42 (triazole CH), 126.23 (Ar CH), 127.15 (Ar CH), 133.79
(Ar C), 144.26 (triazole C or Ar C), 146.31 (triazole C or Ar C),
169.48 (OAc), 169.85 (OAc), 170.20 (OAc), 170.37 (OAc), 170.55
(OAc), 170.68 (OAc), 170.80 (OAc). HRMS (ESI) calcd for
C34H42N4O19S1Na+, 865.20562; found, 865.20611. Anal. (C34-
H42N4O19S) C, H, N, S.
4-[4-(Aminosulfonyl)phenyl]-1-(ꢀ-D-maltopyranosyl)-1H-1,2,3-
triazole (12). The title compound was prepared from 4 according
to general procedure 2 and isolated as white solid (45 mg, 0.08
mmol, 92%). Rf 0.14 (1:9 H2O-CH3CN); mp 228–230 °C (de-
comp). 1H NMR (400 MHz, D2O) δ 3.28–3.33 (m, 1H, GlcR H3′/
3
3
H4′ or Glcꢀ H3′/H4′), 3.48 (dd, J2′-3′ ) 10.0 Hz, J2′-1′ ) 3.6 Hz,
1H, GlcR H2′), 3.57–3.85 (m, 8H, GlcR H3/H4′, GlcR H5′, GlcR
H6, GlcR H6′′, Glcꢀ H3′/H4′, Glcꢀ H5′, Glcꢀ H6′ and Glcꢀ H6′′),
3.87–3.86 (m, 2H, Glcꢀ H2′, Glcꢀ H3′), 3.35 (d, 1H, GlcR H1′),
3
5.68 (d, J1′-2′ ) 8.0 Hz, 1H, Glcꢀ H1′), 7.72–7.79 (m, 4H, Ar),
8.46 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz, D2O) δ 60.65
(GlcR C6′ and Glcꢀ C6′), 69.49, 71.86, 72.40, 72.95, 73.06, 76.01,
76.54, 77.72 (GlcR C2′, GlcR C3′, GlcR C4′, GlcR C5′ and Glcꢀ C2′,
Glcꢀ C3′, Glcꢀ C4′ Glcꢀ C5′), 87.59 (Glcꢀ C1′), 99.85 (GlcR C1),
122.51 (triazole CH), 126.49 (Ar CH), 126.77 (Ar CH), 133.74 (Ar
C), 141.07 (triazole C or Ar C), 146.25 (triazole C or Ar C). HRMS
(ESI) calcd for C20H28N4O12S1Na+, 571.13166; found, 571.13127.
4-[4-(Aminosulfonyl)phenyl]-1-(2′-acetamido-2′-deoxy′-3′,4′,6′-
tri-O-acetyl-ꢀ-D-glucopyranosyl)-1H-1,2,3-triazole (5). The title
compound was prepared according to general procedure 1 and
isolated as white solid (195 mg, 3.52 mmol, 88%). Rf 0.42 (1:9
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-ꢀ-D-
galactopyranosyl)-1H-1,2,3-triazole (3). The title compound was
prepared according to general procedure 1 and isolated as white
solid (255 mg, 0.46 mmol, 86%). Rf 0.21 (1:4 hexanes-EtOAc);
mp 219–220 °C (decomp). 1H NMR (400 MHz, DMSO-d6) δ 1.81
(s, 3H, OAc), 1.93 (s, 3H, OAc), 1.97 (s, 3H, OAc), 2.19 (s, 3H,
OAc), 4.03 (dd, 2J6′-6′′ ) 11.6 Hz, 3J6′-5′ ) 7.2 Hz, 1H, H6′), 4.13
2
3
(dd, J6′′-6′ ) 11.2 Hz, J6′-5 ) 4.8 Hz, 1H, H6′), 4.61–4.64 (m,
1H, H5′), 5.42–4.53 (m, 1H, H4′), 5.49 (dd, 3J3′-2′ ) 10.4 Hz, 3J3′-4′
3
) 3.6 Hz, 1H, H3′), 5.58–5.63 (m, 1H, H2′), 6.32 (d, J1′-2′ ) 9.2
Hz, 1H, H1′), 7.37 (br s, 2H, SO2NH2), 7.87–8.10 (m, 4H, Ar),
8.75 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz, DMSO-d6) δ
20.67 (OAc), 20.99 (OAc), 21.11 (OAc), 21.15 (OAc), 62.27 (C6′),
67.95 (C4′), 68.59 (C3′), 71.00 (C2′), 73.72 (C5′), 85.23 (C1′), 122.51
(triazole CH), 126.33 (Ar CH), 127.05 (Ar CH), 133.85 (Ar C),
1
CH3OH-EtOAc); mp 249.6 °C (decomp). H NMR (400 MHz,
DMSO-d6) δ 1.57 (s, 3H, NHAc), 1.93 (s, 3H, OAc), 1.98 (s, 3H,
OAc), 2.00 (s, 3H, OAc), 4.06 (dd, 2J6′-6′′ ) 12.4 Hz, 3J6′-5′ ) 2.0
2
3
Hz, 1H, H6′), 4.15 (dd, J6′′-6′ ) 12.5 Hz, J6′′-5′ ) 5.2 Hz, 1H,

Inhibition of Carbonic Anhydrases
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1951
3
3
3
H6′′), 4.28 (ddd, J5′-4′ ) 10.4 Hz, J5′-6′′ ) 5.2 Hz, J5′-6′ ) 2.4
(CdO). HRMS (ESI) calcd for C34H28N4O9SNa+, 691.14692;
found, 691.14614. Anal. (C34H28N4O) H, N, S. C: calcd, 61.07;
found, 60.61.
Hz, 1H, H5′), 4.58–4.67 (m, 1H, H2′), 5.06–5.11 (m, 1H, H4′),
3
5.34–5.39 (m, 1H, H3′), 6.14 (d, 1H, J1′-2′ ) 10.0 Hz, 1H, H1′),
7.37 (br s, 2H, SO2NH2), 7.87–8.00 (m, 4H, Ar), 8.10 (d, 3JNH-2
)
4-[4-(Aminosulfonyl)phenyl]-1-(ꢀ-D-ribofuranosyl)-1H-1,2,3-
triazole (15). The title compound prepared from 7 according to
general procedure 2 and isolated as white solid (84 mg, 0.24 mmol,
∼100%). Rf 0.16 (1:9 CH3OH-EtOAc); mp 219–220 °C. 1H NMR
9.2 Hz, 1H, NHAc NH), 8.98 (s, 1H, triazole H); 13C{1H} NMR
(100 MHz, DMSO-d6) δ 20.96 (OAc), 21.10 (OAc), 21.20 (OAc),
22.98 (NHAc), 53.05 (C2′), 62.43 (C6′), 68.70 (C4′), 72.91 (C3′),
74.10 (C5′), 85.68 (C1′), 122.81 (triazole CH), 126.14 (Ar CH),
127.17 (Ar CH), 134.01 (Ar C), 144.16 (triazole CH or Ar C),
145.98 (triazole CH or Ar C), 170.06 (CdO), 170.21 (CdO), 170.27
(CdO), 170.71 (CdO). HRMS (ESI) calcd for C22H26N5O10S-,
577.12110; found, 577.12222. Anal. (C22H27N5O10S) C, H, S. N:
calcd, 12.65; found, 12.18.
2
(400 MHz, 1% D2O in DMSO-d6) δ 3.51 (dd, J5′-5′′ ) 12.0 Hz,
2
3
3J5′-4′ ) 4.4 Hz, 1H, H5′), 3.62 (dd, J5′′-5′ ) 12.4 Hz, J5′′-4′ )
4.0 Hz, 1H, H5′′), 3.87–4.00 (m, 1H, H4′), 4.12–4.15 (m, 1H, H3′),
4.40–4.43 (m, 1H, H2′), 5.96 (d, J1′-2′ ) 4.4 Hz, 1H, H1′), 3.73
3
(br s, 2H, SO2NH2), 7.87–8.03 (m, 4H, Ar), 8.88 (s, 1H, triazole
CH); 13C NMR (100 MHz, 1% D2O in DMSO-d6) δ 61.88 (C5′),
70.86 (C3′), 75.63 (C2′), 86.57 (C4′), 92.90 (C1′), 121.72 (triazole
CH), 126.15 (Ar CH), 127.14 (Ar CH), 134.34 (Ar C), 143.89
(triazole C), 146.06 (Ar C). HRMS (ESI) calcd for C13H17N4O6S+,
357.08650; found, 357.08299.
4-[4-(Aminosulfonyl)phenyl]-1-(2′-acetamido-2′-deoxy-ꢀ-D-
glucopyranosyl)-1H-1,2,3-triazole (13). The title compound was
prepared from 5 according to general procedure 2 and isolated as
1
white solid (116 mg, 0.27 mmol, ∼100%); mp 215–217 °C. H
NMR (400 MHz, 1% D2O in DMSO-d6) δ 1.59 (s, 3H, NHAc),
3.24–3.23 (m, 1H, H4′), 3.43–3.49 (m, 2H, H6′, H6′′), 3.55–3.59 (m,
1H, H3′), 3.68–3.73 (m, 1H, H5′), 4.04–4.09 (m, 1H, H2′), 5.73 (d,
3J1′-2′ ) 10.0 Hz, 1H, H1′), 7.85–8.02 (m, 4H, Ar H), 8.81 (s, 1H,
triazole CH); 13C{1H} NMR (100 MHz, DMSO-d6) δ 23.28 (NHAc
CH3), 55.46 (C2′), 61.32 (C6′), 70.55 (C4′), 74.25 (C5′), 80.74 (C3′),
86.99 (C1′), 122.02 (triazole CH), 126.10 (Ar CH), 127.10 (Ar CH),
134.35 (Ar C), 143.86 (triazole C or Ar C), 145.59 (triazole C or
Ar C), 169.92 (NHAc CdO). HRMS (ESI) calcd for C16H20N5O7S-,
426.10889; found, 426.10768.
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-r-D-
mannopyranosyl)-1H-1,2,3-triazole (8). The title compound was
prepared according to general procedure 1 and isolated as white
glassyfoam(189mg,0.34mmol,51%).Rf 0.37(3:7hexanes-EtOAc).
1H NMR (400 MHz, DMSO-d6) δ 1.98 (s, 3H OAc), 2.00 (s, 3H,
3
OAc), 2.01 (s, 3H, OAc), 2.04 (s, 3H, OAc), 3.88 (ddd, J5′-4′
)
9.2 Hz, 3J5′-6′ ) 5.6 Hz, 3J5′-6′′ ) 3.2 Hz, 1H, H5′), 4.06 (dd, 2J6′′-6′
) 17.2 Hz, 3J6′′-5 ) 3.6 Hz, 1H, H6′′), 4.24 (dd, 2J6′-6′′ )16.8 Hz,
3J6′-5′ ) 6.4 Hz, 1H, H6′), 5.25–5.31 (m, 1H, H4′), 5.23 (dd, 3J3′-4′
3
3
) 12.8 Hz, J3′-2′ ) 5.2 Hz, 1H, H4′), 5.88 (dd, J2′-3′ ) 5.2 Hz,
3J2′-1′ ) 3.2 Hz, 1H, H2′), 6.47 (d, 3J1′-2′ ) 3.0 Hz, 1H, H1′), 7.41
(br s, 2H, SO2NH2), 7.91–8.12 (m, 4H, Ar), 8.94 (s, 1H, triazole
CH). HRMS (ESI) calcd for C22H26N4O11SNa+, 577.12109; found,
577.11897.
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′-tri-O-acetyl-ꢀ-D-glu-
curonic acid methyl ester)-1H-1,2,3-triazole (6). The title com-
pound was prepared according to general procedure 1 and isolated
as white solid (145 mg, 0.27 mmol, 87%). Rf 0.63 (1:9
CH3OH-CH2Cl2); mp 249–251 °C (decomp). 1H NMR (400 MHz,
DMSO-d6) δ 1.79 (s, 3H, OAc), 1.98 (s, 3H, OAc), 2.01 (s, 3H,
4-[4-(Aminosulfonyl)phenyl]-1-(r-D-mannopyranosyl)-1H-
1,2,3-triazole (16). The title compound prepared from 8 according
to general procedure 2 and isolated as white solid (110 mg, 0.28
mmol, ∼100%). Rf 0.12 (1:9 CH3OH-EtOAc); mp 239–240 °C.
1H NMR (400 MHz, 1% D2O in DMSO-d6) δ 3.40–3.45 (m, 2H,
3
OAc), 3.61 (s, 3H, COCH3), 4.84 (d, J5′-4′ ) 10.4 Hz, 1H, H5′),
5.19–5.24 (m, 1H, H4′), 5.63–5.74 (m, H2′ and H3′), 6.46 (d, 3J1′-2′
) 8.8 Hz, 1H, H1′), 7.37 (br s, 2H, SO2NH2), 7.88–8.01 (m, 4H,
Ar CH), 9.15 (s, 1H, triazole CH); 13C{1H} NMR (100 MHz,
DMSO-d6) δ 20.57 (OAc), 20.90 (OAc), 20.95 (OAc), 53.39
(OCH3), 69.13 (C4′), 70.65 (C2′), 72.03 (C3′), 73.59 (C5′), 84.58
(C1′), 122.52 (triazole CH), 126.22 (Ar CH), 127.21 (Ar CH), 133.73
(Ar C), 144.32 (Ar C), 156.52 (triazole C), 167.22 (CdO), 164.24
(OAc), 170.02 (OAc), 170.20 (OAc). HRMS (ESI) calcd for
C21H24N4O11SNa+, 563.10545; found, 563.10376.
3
H6′′, H5′), 3.57–3.63 (m, 2H, H6′, H4′), 3.85 (dd, J3′-4′ ) 9.9 Hz,
3
3J3′-2′ ) 3.3 Hz, 1H, H3′), 4.43–4.46 (m, 1H, H2′), 5.95 (d, J1′-2′
) 4.8 Hz, 1H, H1′), 7.87–8.07 (m, 4H, Ar), 8.85 (s, 1H, triazole
CH). 13C{1H} NMR (75 MHz, 1% D2O in DMSO-d6) δ 61.19 (C6′),
68.29 (C4′), 68.59 (C2′), 71.82 (C3′), 79.18 (C5′), 86.55 (C1′), 122.98
(triazole CH), 126.18 (Ar CH), 127.13 (Ar CH), 134.35 (Ar C),
143.93 (Ar C), 145.81 (triazole C). HRMS (ESI) calcd for
C14H18N4O7SNa+, 409.07884; found, 409.07856.
4-[4-(Aminosulfonyl)phenyl]-1-(ꢀ-D-glucuronic acid methyl
ester)-1H-1,2,3-triazole (14). The title compound was prepared
from 6 according to general procedure 2 and isolated as pale yellow
foam (22 mg, 0.05 mmol, ∼100%). 1H NMR (400 MHz, 2% D2O
in DMSO-d6) δ 3.45-3.54 (m, 2H, H3′ and H4′), 3.63 (s, 3H, OCH3),
3.85–3.89 (m, 1H, H2′), 4.19 (d, 3J5′-4′ ) 8.8 Hz, 1H, H5′), 5.79 (d,
3J1′-2′.06 (m, 4H, Ar CH), 8.98 (s, 1H, triazole CH); 13C{1H} NMR
(100 MHz, 2% D2O in DMSO-d6) δ 52.78 (OCH3), 71.80 (C3′),
72.31 (C4′), 76.39 (C2′), 78.02 (C5′), 87.95 (C1′), 122.51 (triazole
CH), 126.16 (Ar CH), 127.14 (Ar CH), 134.28 (Ar C), 143.86 (Ar
C), 145.97 (triazole C), 169.39 (CdO). HRMS (ESI) calcd for
C15H18N4O8S+, 437.07376; found, 437.07397.
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′-tri-O-benzoyl-ꢀ-D-ri-
bofuranosyl)-1H-1,2,3-triazole (7). The title compound was
prepared according to general procedure 1 and isolated as white
solid (343 mg, 0.51 mmol, 88%). Rf 0.52 (2:3 hexanes-EtOAc);
mp 168–169 °C. 1H NMR (400 MHz, DMSO-d6) δ 4.58 (dd, 2J5′-5′′
) 12.0 Hz, 3J5′-4′ ) 4.4 Hz, 1H, H5′), 4.70 (dd, 2J5′′-5′ ) 12.4 Hz,
3J5′′-4′ ) 3.6 Hz, 1H, H5′′), 4.96–5.00 (m, 1H, H4′), 6.13 (dd, 3J3′-4′
) 6.0 Hz, J3′-2′ ) 5.2 Hz, 1H, H3′), 6.33 (dd, J2′-3′ ) 5.2 Hz,
3J2′-1′ ) 2.8 Hz, 1H, H2′), 6.78 (d, 3J1′-2′ ) 2.8 Hz, 1H, H1′), 7.39
(br s, 2H, SO2NH2), 7.40–7.50 (m, 6H, Ar), 7.56–7.68 (m, 3H, Ar),
7.87–8.01 (m, 10H, Ar), 8.97 (s, 1H, triazole CH); 13C{1H} NMR
(100 MHz, DMSO-d6) δ 63.86 (C5′), 71.69 (C3′), 75.19 (C2′), 80.61
(C4′), 90.25 (C1′), 123.24 (triazole CH), 126.29, 127.09, 129.31,
129.43, 129.55, 129.78, 129.93, 130.07, 130.15, 133.88, 134.18,
134.61, 134.77 (Ar CH and Ar C), 144.20 (triazole C or Ar C),
146.33 (triazole C or Ar C), 165.15 (CdO), 165.37 (CdO), 166.08
4-[4-(Aminosulfonyl)phenyl]-1-(2′,3′,4′-tri-O-acetyl-r-D-ara-
binopyranosyl)-1H-1,2,3-triazole (9). The title compound was
prepared according to general procedure 1 and isolated as white
solid (154 mg, 0.32 mmol, 60%). Rf 0.52 (1:1 CH2Cl2-EtOAc);
1
mp 229–230 °C. H NMR (300 MHz, DMSO-d6) δ 1.84 (s, 3H,
2
OAc), 1.97 (s, 3H, OAc), 2.20 (s, 3H, OAc), 4.09 (dd, J5′-5′′
)
3
2
13.2 Hz, J5′-4′ ) 1.8 Hz, 1H, H5′), 4.24 (dd, 1H, J5′-5′′ ) 12.3
3
Hz, J5′′-4′ ) 1.3 Hz, 1H, H5′′), 5.34–5.35 (m, 1H, H4′), 5.46 (dd,
3J3′-2′ ) 10.2 Hz, J3′-4′ ) 3.6 Hz, 1H, H3′), 5.58–5.65 (m, 1H,
3
3
H2′), 6.22 (d, J1′-2′ ) 9.0 Hz, 1H, H1′), 7.40 (br s, 2H, SO2NH2),
7.88–8.13 (m, 4H, Ar H), 9.03 (s, 1H, triazole H); 13C {1H} NMR
(75 MHz, DMSO-d6) δ 20.02 (OAc), 20.41 (OAc), 20.75 (OAc),
66.54 (C5′), 67.86 (C4′), 68.21 (C3′), 70.22 (C2′), 85.11 (C1′), 121.71
(triazole CH), 125.63 (Ar CH), 126.38 (Ar CH), 133.26 (Ar C),
143.51 (triazole C or Ar C), 145.62 (triazole C or Ar C), 168.72
(CdO), 168.73 (CdO), 169.57 (CdO). HRMS (ESI) calcd for
C19H22N4O9SNa+, 505.09998; found, 505.09996.
4-[4-(Aminosulfonyl)phenyl]-1-(r-D-arabinopyranosyl)-1H-
1,2,3-triazole (17). The title compound prepared from 9 according
to general procedure 2 and isolated as clear oil (76 mg, 0.21 mmol,
∼100%). 1H NMR (300 MHz, 2% D2O in DMSO-d6) δ 3.59 (dd,
3J3′-2′ ) 9.6 Hz, 3J3′-4′ ) 3.3 Hz, 1H, H3′), 3.78–3.84 (m, 2H, H4′,
3
3
3
H5′′), 4.06–4.12 (m, 1H, H2′), 5.48 (d, 1H, J1′-2′ ) 9.0 Hz, 1H,
H1′), 7.88–8.10 (m, 4H, Ar H), 8.90 (s, 1H, triazole H); 13C {1H}
NMR (75 MHz, DMSO-d6) δ 68.28 (C4′ or C5′), 69.43 (C4′ or C5′),
69.44 (C2′), 73.04 (C3′), 88.72 (C1′), 121.57 (triazole CH), 125.57
(Ar CH), 126.54 (Ar CH), 132.37 (Ar C), 133.90 (Ar C), 145.22

1952 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Wilkinson et al.
(triazole C). HRMS (ESI) calcd for C13H16N4O6SNa+, 379.0683;
found, 379.06931.
Anhydrase Inhibitors: Topically Acting Antiglaucoma Sulfonamides
Incorporating Esters and Amides of 3- and 4-Carboxybenzolamide.
Bioorg. Med. Chem. Lett. 2003, 13 (17), 2867–2873. (c) Mincione,
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ides and 4-Chloro-3-sulfamoyl-benzenecarboxamides with Strong
Topical Antiglaucoma Properties. Bioorg. Med. Chem. Lett. 2001, 11
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CarbonicAnhydraseCatalytic/InhibitionAssay.AnSX.18MV-R
Applied Photophysics stopped-flow instrument has been used for
assaying the CA I, II, and IX CO2 hydration activity.31 Phenol red
(at a concentration of 0.2 mM) has been used as indicator, working
at the absorbance maximum of 557 nm, with 10 mM Hepes (pH
7.5) as buffer, 0.1 M NaClO4 (for maintaining constant ionic
strength, this anion is not inhibitory), following the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s. Saturated CO2
solutions in water at 20 °C were used as substrate. Stock solutions
of inhibitors were prepared at a concentration of 10–50 mM (in
the assay buffer) and dilutions up to 1 nM done with the assay
buffer mentioned above. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay,
to allow for the formation of the E-I complex. The inhibition
constants were obtained by nonlinear least-squares methods using
PRISM 3. The curve-fitting algorithm allowed us to obtain the IC50
values (working at the lowest concentration of substrate of 1.7 mM)
from which Ki values were calculated by using the Cheng-Prusoff
equation. The catalytic activity (in the absence of inhibitors) of
these enzymes was calculated from Lineweaver–Burk plots, as
reported earlier, and represent the mean from at least three different
determinations.33–35 Enzyme concentrations were 10 nM for CA I
and CA II and 14 nM for CA IX. Kinetic parameters and inhibition
constants were calculated as described previously.33–35 Enzymes
used here were recombinant ones, prepared and purified as described
earlier.
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Acknowledgment. This work was financed in part by the
Australian Research Council (Grant Numbers F00103312 and
DP0343419), the Eskitis Institute for Cell and Molecular
Therapies and the School of Science (Griffith University, S.-
A.P.), and an EU grant of the 6th framework programme
EUROXY project (University of Florence, C.T.S.). We thank
Hoan The Vu for carrying out accurate mass measurements and
Dr. Sue Boyd for assistance with NMR spectroscopy.
Supporting Information Available: Elemental analysis data and
1H and 13C NMR spectra for compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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