Targeting hypoxic tumor cell viability with carbohydrate-based carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1021/jm200892s

Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of ne

Full text of DOI:10.1021/jm200892s

ARTICLE
pubs.acs.org/jmc
Targeting Hypoxic Tumor Cell Viability with Carbohydrate-Based
Carbonic Anhydrase IX and XII Inhibitors
Jason C. Morris,† Johanna Chiche,§ Caroline Grellier,† Marie Lopez,† Laurent F. Bornaghi,†
Alfonso Maresca,‡ Claudiu T. Supuran,‡ Jacques Pouyssꢀegur,§ 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, Universitꢁa degli Studi di Firenze, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy
§Institute of Developmental Biology and Cancer, CNRS UMR654 3, University of Nice, Centre Antoine Lacassagne, 33 av. de
Valombrose 06189 Nice, France
S Supporting Information
b
ABSTRACT:
Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system
that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of
new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model
systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of
which inhibit CA IX and XII within a narrow range of low nanomolar Ki values (5.3ꢀ11.2 nM). We evaluate for the first time
carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor
microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have
potential for development as in vivo cancer cell selective inhibitors.
’ INTRODUCTION
generate a bicarbonate anion (HCO3ꢀ) and a proton (H+).5 CA
IX and XII differ from cytosolic CAs in that they are transmem-
brane proteins with an extracellular catalytic domain.5 A char-
acterized role of CA IX and XII in cancer is the hydration of
tumor cell-generated CO2; the net effect is to trap H+ extra-
cellularly, lowering the pH outside the cell (pHo) while main-
taining normal pHi.3,6 CA IX and XII expression is upregulated in
a wide variety of human tumors under the control of HIF-1, CA
IX is commonly absent in corresponding normal tissue (except
the stomach and GI tract), while CA XII is expressed in a
selection of normal tissues.7 The impact of modulating CA
activity on tumor growth was recently affirmed in a set of
in vivo studies in mice.4 These studies demonstrated that
silencing of ca9 and ca12 genes using shRNA significantly
reduced the rate of xenograft growth, and this outcome was
Tumor cells alter their metabolism when in a low oxygen
(hypoxic) microenvironment to survive, proliferate, and
metastasize.1 The hypoxia-inducible transcription factor (HIF-1)
regulates gene expression of proteins that are responsible for
alterations in hypoxic tumor cell metabolism—one core aspect of
which is the shift from aerobic to fermentative glycolysis.2
Fermentative glycolysis leads to elevated lactic acid production;
hence, a drop in the intracellular pH (pHi) of hypoxic tumor cells
is expected unless pH homeostasis is established.3 Carbonic
anhydrase (CA, EC 4.2.1.1) enzymes, specifically isozymes CA
IX and CA XII, have been shown to underpin a robust pH-
regulating system by maintaining a pHi range compatible with
cell viability and proliferation, while they contribute to the
extracellular acidification of the tumor microenvironment.
These enzymes confer a survival advantage to tumor cells
growing in a hypoxic and acidic microenvironment.4 CAs
catalyze the reversible hydration of carbon dioxide (CO2) to
Received: July 7, 2011
Published: August 18, 2011
r
2011 American Chemical Society
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Figure 1. (a) S-Glycoside 1,4-disubstituted-1,2,3-triazole CA inhibitor motif. (b) S-Glycoside 5-iodo-1,4-disubstituted-1,2,3-triazole CA inhibitor
motif.
Scheme 1. Synthesis of Target S-Glycoside 5-Iodo-1,4-disubstituted-1,2,3-triazole and Glycoside 5-H-1,4-Disubstituted-1,2,3-
triazole CA Inhibitor Compoundsa
a Reagents and conditions: (a) Peracetylated β-S-propynyl glycoside (2ꢀ4, 1.0 equiv), azide (1, 1.2 equiv), CuI (1.2 equiv), DIPEA (1.2 equiv), NBS
(1.2 equiv), THF, room temperature, 16 h. (b) m-CPBA (7.0 equiv), anhydrous CH2Cl2, 0 °Cꢀroom temperature, 2 h. (c) NaOMe in MeOH (0.05 M,
pH 12), anhydrous MeOH, 0 °Cꢀroom temperature, 30 minꢀ16 h.
attributed to slowed proliferation in the absence of membrane-
associated CAs. In humans, studies targeting CA IX-expressing
cells using immunotherapy with the monoclonal antibody G250
have reached clinical trials for the treatment of patients with
metastatic renal cell carcinoma.8 On the basis of the in vivo
mouse results using genetic silencing and success with immu-
notherapy in human cancer patients, the modulation of CA IX
and XII with small molecule inhibitors represents a mechan-
istically novel opportunity with potential for anticancer therapy
intervention. From the perspective of targeted cancer therapeu-
tics, this hypothesis has generated scientific interest that so far
remains to be comprehensively explored. To date, only a few
small molecules have been characterized in CA-relevant cell and
animal model systems.9 CA inhibitors that incorporate a hydro-
philic moiety such as a cationic pyridinium10,11 and “Cp*Ru(II)”
salts12 or carbohydrate moieties13ꢀ17 have been developed to
have limited membrane permeability and so may better target CA
IX and XII over CA II.
To characterize the potential effect of inhibition of CA IX
and XII in tumor hypoxia, there is an implied need to develop
small molecule, cell impermeable CA inhibitors for use in cell-
based models of these extracellular, cancer-linked CAs in tumor
hypoxia. Once characterized, these molecules should have value
as probes to further aid experimental advances in targeted cancer
therapies associated with CA and tumor pH homeostasis.4,17 Our
group's research has focused on small molecule medicinal
chemistry of CA inhibitors, and we recently reported a library
of S-glycosides that were potent inhibitors of CA isozymes IX and
XII in vitro.18 Encouraged by the nanomolar activity of these
compounds, we now elaborate this novel compound class and
evaluate for the first time carbohydrate-based CA inhibitors in
cell-based models that emulate the role of CA IX in an acidic
tumor microenvironment.
’ RESULTS AND DISCUSSION
Compound Design. Sugar-based molecules have proven
valuable drug leads, often presenting desirable activity and
safety profiles.19 Many of the sugar-based drugs in clinical use
are antiviral, antibacterial, or antifungal20 and benefit from
improved solubility, tolerability, stability, and/or targeting
when compared to noncarbohydrate drugs. Work in our
research group has demonstrated the versatility of the Cu(I)-
catalyzed 1,3-dipolar cycloaddition reaction (1,3-DCR or “click
chemistry”) of organic azides and acetylenes to generate 1,2,3-
triazole glycoconjugates.13 We have developed small molecule
membrane impermeable carbohydrate-based CA inhibitors
from alkynyl sugars and p-azidobenzene sulfonamide,14,18 as
well as the “reverse polarity” inhibitors from azido sugars and p-
ethynylbenzene sulfonamide.15 These compounds were de-
signed to selectively target cancer relevant CA isozymes (with
an extracellular active site) over other (intracellular) CA
isozymes.13b,14ꢀ16 Compounds comprise a hydrophilic sugar
moiety tethered to an aromatic sulfonamide CA pharmaco-
phore through an intervening triazole ring to provide a selection
of membrane impermeable small molecule CA inhibitors.
The replacement of the naturally occurring O-glycoside
bond by a S-glycoside bond is an approach practiced in the
synthesis of carbohydrate-containing compounds for down-
stream biological applications. This isosteric replacement seeks
to enhance the stability of the small molecule glycoside toward
enzymatic hydrolysis of the glycosidic bond while retaining vital
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Scheme 2. Synthesis of Target S-Glycoside-1,4,5-trisubstituted-1,2,3-triazole CA Inhibitors, Compounds 27 and 28a
a Reagents and conditions: CuI (1.0 equiv), triethylamine (1.0 equiv), propargyl alcohol (1.0 equiv) in THF, nitrogen atmosphere, room temperature,
30 min; then Pd(PPh3)4 (1.0 equiv in THF) is added, 50 °C, 30 min.
molecular recognition interactions with the biological target.21
We have reported the synthesis and CA enzyme inhibition of S-
linked glucose and galactose moieties tethered to a triazole-
ArSO2NH2 motif (Figure 1a).18 Our intention here is to build
structureꢀactivity relationships (SARs) and structureꢀproperty
relationships (SPRs) around this compound class that can value
add to the interpretation of CA inhibitor behavior in cell-based
assays. The target compounds of the present study were thus
designed to incorporate a synthetic handle that would allow
further elaboration of the structure to 1,4,5-trisubstituted-1,2,3-
triazoles. Specifically, we chose to incorporate an iodine atom at
the 5-position of the triazole moiety of the target compounds
(Figure 1b); this modification is key to further developing our
molecular library.
(10 mol %) and K2CO3 reporting both the ease and the efficiency
of the reaction (71ꢀ99%).28 Here, we explored this chemistry
with our iodo substrates, compounds 5 and 7, and the alkyne,
propargyl alcohol. This short chain alkyne was chosen as it
provides a hydroxyl functional group that is capable of picking up
additional H-bonding interactions with the target CA protein
with minimal additional steric bulk. A variety of Sonogashira
reaction conditions were explored, many proceeded with either
no conversion of the starting material or starting material
decomposition. The coupling reaction between the 5-iodo
triazoles and alkyne was however achieved when using Pd-
(PPh3)4, CuI, and Et3N to give 1,4,5-trisubstituted triazoles 27
and 28 (Scheme 2). This synthetic strategy has provided an
additional layer of diversity within this class of CA inhibitor.
CA Inhibition. The enzyme inhibition data for 5ꢀ28 as well as
acetazolamide (AZA) were obtained for the physiologically
dominant CA I and II and tumor-associated CA IX and XII
using a stopped flow assay that monitors the physiological
reaction (CA catalyzed hydration of CO2) (Table 1).29 AZA is
a clinically used CA inhibitor. Similarly to AZA, the S-glycosides
5ꢀ26 were potent inhibitors of hCA II, IX, and XII, most within
a range of narrowly distributed Ki values (5.3ꢀ11.2 nM) with Ki
selectivity ratios close to unity, with only a few exceptions. The
tight distribution of Ki values on inhibitor activity for CA
enzymes is consistent with our previously reported observations
with this enzyme class.16a,30 This flat SAR showing minimal effect
of compound structural variation on inhibition provides a valu-
able set of compounds as the presence of underlying consistent
and high potency enzyme inhibition characteristics allows the
relationship between physicochemical properties of the inhibitor
and the performance in cell-based assays to be readily garnished.
At hCA I, inhibition by 5ꢀ26 was typically weaker (10ꢀ20-fold)
than for all other isozymes with Ki values in the high nanomolar
to low micromolar range and is also consistent with earlier
studies with this compound class.16a The two trisubstituted
compounds, 27 and 28, both with a short hydroxyl bearing
group at the 5-position on the triazole, demonstrated some
noteworthy SAR regarding isozyme selectivity for the cancer-
associated CAs. Compound 28 has much lower inhibition for
both cytosolic CAs as compared to all other compounds of this
study; as a consequence, it has much better selectivity for the
cancer-associated CAs, particularly CA XII, where the Ki is
9.0 nM and selectivity over CA II is 24-fold.
Chemistry. Both Meldal22 and Sharpless23 discovered that
catalytic Cu(I) leads to a dramatic rate enhancement (up to 107-
fold) of the 1,3-DCR of organic azides (RꢀN3) with alkynes
(R0ꢀCtCH) with exclusive synthesis of the 1,4-disubstituted-
1,2,3-triazole product. The reaction is now referred to as the
copper-catalyzed azideꢀalkyne cycloaddition (CuAAC) and has
rapidly become the foremost click chemistry reaction.24,25
A
recent variation to CuAAC involves the combination of a
CuI/NBS catalytic system.26 This variation allows the in situ
generation of I+ and facilitates the formation of the 5-iodo-1,4-
disubstituted 1,2,3-triazole product. We have applied this meth-
odology to sugar alkyne and p-azidobenzene sulfonamide sub-
strates (Scheme 1). In our hands, this system generates two
reaction products in similar yields, these are the 5-iodo-
(Figure 1b) and the 5-H-triazoles (Figure 1a). We reconcile
the formation of the two products to have resulted from
competing reaction pathways, one in which the triazole-Cu
intermediate is captured by I+ (giving the 5-iodo-product) and
the second where this intermediate is protonated (giving the
5-H-product). This click chemistry variant thus gave useful
quantities of two products both of which were valuable to the
subsequent SAR and SPR investigations of CA inhibition.
We synthesized a set of triazole-based glycoconjugate sulfo-
namides 5ꢀ10 from p-azidobenzene sulfonamide 1 and perace-
tylated β-S-propynyl glycoside derivatives 2ꢀ4 in the presence of
CuI and NBS (Scheme 1). Oxidation of the 5-iodo- (5, 7, and 9)
and 5-H- (6, 8, and 10) 1,2,3-triazole benzenesulfonamide
glycoconjugates to the corresponding sulfonyl derivatives
11ꢀ16 was achieved with an excess of m-CPBA. Deacetylation
of the sugar moieties of glycoconjugate compounds was con-
ducted using the standard Zemplꢀen conditions27 of 0.05 M
sodium methoxide in methanol, to afford compounds 17ꢀ26.
Chen et al. has recently demonstrated the application of the
Sonogashira reaction to introduce an alkyne onto the 5-iodo-1,
4-disubstituted triazole scaffold catalyzed by PdCl2(PPh3)2
Cell-Based Screening of CA Inhibitors. To evaluate the
impact of the CA inhibitors in cells, we used our previously
described in vitro systems of fibroblasts lacking endogenous
expression of CA IX (CCL39-nhe1ꢀ-pev, also named PS120-pev)
and fibroblasts that overexpress the human form of CA IX
(CCL39-nhe1ꢀ-pca9, also named PS120-pca9).4 Exposure of
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Table 1. Inhibition and Isozyme Selectivity Ratio Data of CA
Isozymes I, II, IX, and XII with 5ꢀ28 and the Established
Drug and CA Inhibitor, AZA
cross-section of SAR and SPR. Compounds incorporate a spread
of 5-iodo- (5, 11, 17, and 21) versus 5-H- (6, 12, 18, and 22)
triazoles, acetylated (5, 6, 11, and 12) versus deacetylated (17,
18, 21, and 22) sugars, and sulfur in two oxidation states: ꢀSꢀ
(5, 6, 17, and 18) and ꢀSO2ꢀ (11, 12, 21, and 22). These
compounds also encompass a range of calculated Log P (cLog P)
values, Table 2. cLog P represents intrinsic lipophilicity and
compounds with Log P < 0 (such as 17, 18, 12, 21, and 22)
typically have poor passive membrane permeability. All S-gluco-
sides were potent CA II inhibitors (Ki range, 5.3ꢀ10.6 nM) with
only compound 21 slightly weaker, yet still reasonably potent
(Ki = 49.2 nM). Similarly, all S-glucosides were potent CA XII
inhibitors (Ki range, 5.3ꢀ10.7 nM) inhibitors, while 5, 6, 17, 12,
and 22 were potent CA IX inhibitors (Ki < 10 nM), with 18, 11,
and 21 less potent but still good CA IX inhibitors.
The effect of the S-glucoside CA inhibitors on cell viability on
the two fibroblastic cell lines expressing or not CA IX is
summarized in Table 2. Cells remained viable in the presence
of the 5-H-triazole compounds (6, 18, 12, and 22) at the highest
concentration tested (300 μM) when grown at pHo 7.5. These
compounds also had no impact on the CA IX-mediated cell
viability at pHo 6.4, except for compound 6, which reduced cell
viability only at 300 μM. Of the 5-iodo-substituted compounds
(5, 11, 17, and 21), cells grown at pHo 7.5 remained viable in the
presence of 17 at the highest concentration tested (300 μM), in
the presence of 21 at 100 μM, in the presence of 5 at 60 μM, and
in the presence of compound 11 at 30 μM. Compound 21
reduced cell viability of CA IX-expressing cells in acidic condi-
tions (pHo 6.4) only at 300 μM, while compound 17 reduced
viability of these cells at 100 μM. At a lower concentration
(60 μM), only compound 5 was not able to sustain viability at
the acidic environment (pHo 6.4). AZA reduced viability only at
300 μM; however, there was also no cell type or pHo selectivity.
Among the eight S-glycoside inhibitors tested, we identified
two inhibitors (compounds 5 and 17) that demonstrated selec-
tive toxicity for PS120-pca9 cells growing in a pHo 6.4 in vitro.
The concentration of compound 17 needed to block CA IX-
induced survival in the acidic media was however high (100 μM),
and 17 was less suited to follow-up in vitro and in vivo studies
than compound 5. Compound 5 blocked the CA IX-mediated
survival in the acidic environment at 60 μM without general
toxicity at pHo 7.5 in both cell lines (Table 2 and Figure 2A).
Compound 5 (60 μM for 30 min) reduced membrane-associated
CA activity similarly to the level obtained in cells lacking CA IX
(Figure 2B). Notably, 60 μM AZA was not as effective as
compound 5 at either abolishing CA IX-mediated cell survival
in extracellular acidic conditions (Figure 2A) or in reducing CA
IX activity (Figure 2B). In addition, compound 5 (60 μM for
60 min) reduced the pHi of CA IX expressing PS120-pca9 cells
exposed to a pHo of 6.4 by 0.2 units. Under the same conditions,
60 μM AZA had no impact (Figure 2C).
Ki valuesa
Ki selectivity ratiosb
compd CA I CA II CA IX CA XII CA II/CA IX CA II/CA XII
AZA 250
12
25
5.7
0.48
0.85
0.62
0.12
1.15
2.1
5
112
91
5.3
6.2
8.6
95
10.0
9.6
0.53
0.55
1.3
6
5.3
7
115
117
ND
ND
103
97
11.2
9.9
8.9
8
8.6
ND
ND
71
9.2
1.08
9
ND
ND
10.6
6.9
ND
ND
8.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
27
26
27
28
0.15
0.74
0.40
0.71
0.50
0.72
0.85
0.02
1.47
1.02
0.81
0.80
0.43
0.96
0.88
0.68
0.17
2.4
1.2
9.3
97
9.1
0.76
4.2
4070 38.5
9.1
79
5.3
7.5
99.6
61.9
6.5
257
7.3
6.1
61
10.9
59.2
53.1
10.7
9.5
0.48
1.2
101
184
119
114
119
60
49.8
44.5
5.5
0.84
0.51
0.59
1.4
5.6
10.7
6.2
7.8
10.3
5.3
0.60
9.3
3920 49.2
101 7.6
3730 37.9
9.5
89
10.3
9.4
0.74
4.0
60
7.8
8.1
68.0
71.2
72
9.4
0.83
5.1
142
632
105
59.5
48.2
11.9
11.7
56.0
7.9
0.86
1.5
6860 219
91
9.0
24.3
a Errors in the range of (5% of the reported value, from three
determinations. b The Ki ratios are indicative of isozyme selectivity
for cancer-associated CAs in vitro and are calculated as Ki CA II/Ki
CA IX or XII.
both of these cell types to nominally HCO3ꢀ-free media at two
external pHo (7.5 or 6.4) can discriminate their viability. Ex-
posure at pHo 7.5 does not affect cell viability, while exposure at
pHo 6.4 sharply discriminates PS120 cells that are expressing or
not CA IX. Indeed, at this acidic external pHo 6.4, PS120-pev cells
die while the PS120-pca9 remain viable.4 This differential
survival under physiological pH versus acidic conditions is
directly linked to the capacity of CA IX expressing cells to
maintain a more alkaline pHi ensuring cell survival. PS120-pev
cells (lacking CA IX expression) when grown in the neutral
media system provide a control cell-based assay that allows us to
evaluate the S-glycoside CA inhibitors for generic cytotoxicity.
PS120-pca9 cells (overexpressing CA IX) when grown in the
acidic media system provide a cell-based assay that allows us to
evaluate the impact of S-glycoside CA inhibition on CA IX-
mediated cell viability. Together, these two cell lines provide a
cell-based model to study the likely effect of inhibition of CA IX
in tumors with small molecules.4
Next, we investigated compound 5 in our human colon
carcinoma cell model LS174Tr cells where we have previously
demonstrated the in vitro and in vivo effects of knocking-down
ca9 and ca12 genes via shRNA.4 Control LS-shca9/ctl ꢀTet cells
induced both CA IX and CA XII in hypoxia (Figure 2A). CA IX
expression was reduced with a specific shRNA targeting ca9 in
control LS-shca9/ctl +Tet cells and in ca12-silenced LS-shca9/
ca12ꢀ +Tet cells upon tetracycline (Tet) treatment. Following
a 48 h treatment with 5 (60 μM), we observed reduced levels
of mRNA (data not shown) and protein for CA IX in the control
(ca9-nonsilenced) cell line LS-shca9/ctl ꢀTet in normoxia
We focused arbitrarily on the S-glucoside panel of com-
pounds (5, 6, 11, 12, 17, 18, 21, and 22) and the classical CA
inhibitor drug AZA to study in the cell-based systems. The subset
of eight S-glucoside compounds allows us to evaluate the full
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Table 2. Cell Viability Assays on CA IX-Expressing Cell Model PS120-pca9 vs CA IX Lacking Cell Model PS120-pev Treated with
S-Glucoside Compounds and AZAa
cell viability (pHo 7.5)
300/100/60/30 μMc,e
cell viability (pHo 6.4)
300/100/60/30 μMd,e
compd
X
Y
R
cLog Pb
Ki at CA IX
AZA
5
ꢀ0.98
+1.98
+1.10
ꢀ0.16
ꢀ1.04
+0.54
ꢀ0.34
ꢀ1.60
ꢀ2.48
25
6.2
8.6
6.5
257
71
ꢀ/+/+/+
ꢀ/ꢀ/+/+
+/+/+/+
+/+/+/+
+/+/+/+
ꢀ/ꢀ/ꢀ/+
+/+/+/+
ꢀ/+/+/+
+/+/+/+
ꢀ/+/+/+
ꢀ/ꢀ/ꢀ/+
ꢀ/+/+/+
ꢀ/ꢀ/+/+
+/+/+/+
ꢀ/ꢀ/ꢀ/+
+/+/+/+
ꢀ/+/+/+
+/+/+/+
S
S
S
S
I
Ac
Ac
H
6
H
I
17
18
11
12
21
22
H
I
H
SO2
SO2
SO2
SO2
Ac
Ac
H
H
I
9.3
61
H
H
9.5
a The calculated Log P value is also included. b cLog P data calculated using CS ChemDraw Ultra. c Viability in PS120-pev and PS120-pca9 cells exposed
to a pHo 7.5. d Viability in PS120-pca9 cells exposed to a pHo 6.4. e + indicates cells are viable; ꢀ indicates cells are not viable.
(N, 21% O2) and hypoxia (H, 1% O2) (Figure 3A). We also
observed the concomitant upregulation of CA XII expression in
these cells at the mRNA and protein level (Figure 3A). The effect
of compound 5 on CA IX and CA XII expression mirrors exactly
that obtained by us earlier with tet-inducible shRNA targeting
ca9 (LS-shca9/ctl +Tet) (Figure 3A). The lowest level of CA IX
and CA XII ever observed in hypoxia was obtained in ca9 and
ca12 double silenced cells (LS-shca9/ca12ꢀ +Tet) incubated
with 60 μM compound 5. Under the same conditions, 60 μM
AZA had no impact either on mRNA or protein levels of CA IX
and CA XII (data not shown). Treatment of control colon LS-
shca9/ctl ꢀTet cells with 5 (60 μM) in hypoxia (1% O2) showed
a 60% decrease in cell proliferation after 3 days and an additional
16% reduction on double-silenced LS-shca9/ca12ꢀ +Tet cells,
consistent with the levels of CA IX and CA XII observed in these
conditions (Figure 3B).
Our strategy to target the extracellular catalytic domain of
cancer-associated CAs is to deliberately manipulate the physico-
chemical properties of the inhibitor to limit membrane perme-
ability. Compound 5 has a clog P value >0 (+1.98) and thus is
predicted to have some cell membrane permeability, while the
cLog P for 17 is <0 (ꢀ0.16) and thus is predicted to be
membrane impermeable. Despite their physicochemical differ-
ences, these compounds were both identified as candidates
that target CA IX in the cell models investigated. One explana-
tion for the findings for compound 5 is that its high potency at
CA IX (Ki = 6.2 nM) demonstrates that it is a strong binder to CA
IX, and even though it is likely to be membrane permeable, CA IX
may act as an extracellular trap or “sink” for compound 5 as a
consequence of the high binding affinity. In the absence of CA IX
overexpression, compound 5 is likely to enter cells through
passive diffusion; however, when CA IX is overexpressed at the
cell surface compound 5 may still bind and have the desired effect.
effective in vivo for therapeutically targeting hypoxic tumors.
Small molecule CA IX and XII inhibitors thus represent a
mechanistically novel opportunity with potential for anticancer
therapy intervention. From the perspective of targeted cancer
therapeutics, this hypothesis so far remains to be comprehen-
sively explored, and druglike small molecule tools are needed to
maximize the potential benefit of chemotherapies involving CA-
mediated hypoxic tumor cell survival. We have designed and
synthesized a new class of carbohydrate-based small molecule
that very effectively inhibits both CA IX and XII enzymes. These
inhibitors were studied in two fibroblast cell lines—one lacking
endogenous expression of CA IX and one overexpressing CA IX.
These in vitro systems allowed the discrimination of the protec-
tive role of CA IX in an acidic tumor microenvironment versus
general cytotoxicity. Our findings identified two inhibitors
(compounds 5 and 17) that selectively blocked CA IX-induced
survival. Extended studies with compound 5 showed that it
reduced membrane-associated CA activity to a similar level as
that obtained in cells lacking CA IX. Compound 5 was also
investigated in our human colon carcinoma cell model (LS174Tr
cells) where we have previously demonstrated the beneficial in
vitro and in vivo effects of knocking-down either ca9 or ca12 or
both ca9 and ca12 genes with shRNA. Compound 5 reduced
levels of mRNA and protein for CA IX in control (ca9-non-
silenced) colon carcinoma cells in normoxia and hypoxia, while
CA XII expression was upregulated. Notably, the effect of
compound 5 mirrors exactly that obtained by us earlier with
shRNA targeting ca9 gene expression. The lowest level of CA IX
and CA XII ever observed in hypoxia was obtained in ca9 and
ca12 double-silenced colon carcinoma cells incubated with
compound 5. Consistent with the levels of CA IX and CA XII
expression observed, colon carcinoma cells in hypoxia treated
with 5 showed a 60% decrease in cell proliferation after 3 days
and an additional 16% decrease in cell proliferation in ca9/ca12
double-silenced cells. Notably, AZA, the clinically used CA
inhibitor, was not as effective as compound 5 at either abolishing
CA IX-mediated cell survival, reducing CA IX activity, or reducing
’ CONCLUSIONS
Both genetic silencing and immunotherapy targeting the
modulation of CA IX and/or XII activity has been shown
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were able to effectively discriminate cell viability in CA IX/XII cell-
based models. We have shown that compound 5 is a candidate
small molecule mimetic of the shRNA targeting ca9 that we have
used in our previous studies. We conclude that small molecule CA
inhibitors have potential for development as effective in vivo cancer
cell selective inhibitors.
’ EXPERIMENTAL SECTION
Chemistry—Materials and Methods. All starting materials and
reagents, including per-O-acetylated sugars, were purchased from com-
mercial suppliers. All reactions were monitored by TLC. TLC plates
were visualized with UV light, sulfuric acid stain (5% H2SO4 in ethanol),
and/or orcinol stain (1 g of orcinol monohydrate in a mixture of EtOH:
H2O:H2SO4 72.5:22.5:5). Silica gel flash chromatography was per-
formed using silica gel 60 Å (230ꢀ400 mesh). 1H NMR spectra were
acquired at 500 MHz and 13C NMR spectra at 125 MHz. Chemical shifts
1
for H and 13C NMR spectra obtained in DMSO-d6 are reported in
ppm relative to residual solvent proton (δ = 2.50 ppm) and carbon (δ =
1
39.5 ppm) signals, respectively. Chemical shifts for H NMR spectra
obtained in CDCl3 are reported in ppm relative to residual solvent
proton (δ = 7.26 ppm). Multiplicity is indicated as follows: s (singlet), d
(doublet), t (triplet), m (multiplet), dd (doublet of doublet), ddd
(doublet of doublet of doublet), and br (broad). Coupling constants
are reported in Hertz (Hz). Melting points are uncorrected. Mass
spectra (low and high resolution) were recorded using electrospray as
the ionization techniqueinpositiveion or negativeionmodes asstated. All
MS analysis samples were prepared as solutions in methanol. Optical
rotations were measured at 25 °C and reported as an average of 10
measurements. All compounds were analyzed for purity by HPLC with
both UV (200ꢀ400 nm) and ELSD (evaporative light scattering
detection) detection used. The purity of all compounds was g95%.
General Procedure 1. Synthesis of Peracetylated β-S-
Propynyl Glycosides. To a solution of the per-O-acetylated sugar
(1.0 equiv) in anhydrous CH3CN at room temperature were added
thiourea (1.5 equiv) and boron trifluoride diethyletherate (1.5 equiv).
The resulting mixture was refluxed (∼1 h), and full consumption of the
sugar starting material was evidenced by TLC (1:1 EtOAc/hexane). The
reaction mixture was cooled to room temperature, triethylamine
(1.5 equiv) and propargyl bromide (1.5 equiv) were added, and the reaction
was stirred at room temperature until completion (∼4 h), evidenced by
TLC (1:1 EtOAc/hexane). The solvent was evaporated, the resulting
syrup was diluted with CH2Cl2 and washed with 5% HCl (ꢁ1) and brine
(ꢁ2), and aqueous phases were back extracted with CH2Cl2 (ꢁ2). The
organic extracts were combined, dried with MgSO4, and filtered, and the
solvent was removed in vacuo. The crude mixture was purified by flash
chromatography as detailed to afford pure glycosides 2ꢀ4.
Figure 2. (A) Cell viability assays for PS120-pev and PS120-pca9 cells in
normoxia (21% O2) as a function of the pHo. Once attached cells were
incubated in a HCO3ꢀ-free media adjusted to pHo 6.4 or 7.5 for 24 h in a
CO2-free atmosphere in the absence (DMSO control) or presence of
60 μMꢀof CA inhibitors AZA or compound 5. Fresh media containing
HCO3 (40 mM) at pHo 7.5 were then added, and the assay was
maintained for 4 days in a 5% CO2 incubator before staining to visualize
the cell density. (B) Rate of extracellular acidification in response to a
CO2-load on normoxic (21% O2) PS120-pev and PS120-pca9 cells. The
rate of acidification reflects the CA activity at the cell surface evaluated in
the absence (DMSO) or in the presence of 60 μM CA inhibitors (AZA
or compound 5) 30 min prior CO2-loading. (C) Compound 5 (60 μM)
but not AZA (60 μM) reduces the resting pHi of CA IX-expressing
PS120-pca9 cells in an acidic HCO3ꢀ-free media adjusted to pHo 6.4 but
not pHo 7.5.
General Procedure 2. Dual Synthesis of 5-Iodo- and 5-H-
1,2,3-Triazole Benzenesulfonamide Glycoconjugates.26
A
solution of peracetylated β-S-propynyl glycoside (1.0 equiv) and
4-azidobenzene sulfonamide (1, 1.2 equiv) in THF was prepared. CuI
(1.2 equiv) and DIPEA (1.2 equiv) were added, followed by dropwise
addition of a solution of NBS (1.2 equiv) in THF. The reaction was
stirred at room temperature overnight. The solvent was removed in
vacuo leaving a brown residue. This crude material was redissolved in
CH2Cl2 and washed with 5% HCl (ꢁ1) and brine (ꢁ2), followed by
back extraction of the aqueous extracts with CH2Cl2 (ꢁ2). The organic
extracts were then combined, dried with MgSO4, and filtered. The
solvent was removed in vacuo to produce a brown solid. Purification by
flash chromatography, detailed below, afforded both the target 5-iodo
substituted 5, 7, and 9 and the unsubstituted 6, 8, and 10 1,2,3-triazole
benzensulfonamide glycoconjugates.
colon carcinoma cell proliferation. In summary, among the inhibi-
tors tested herein two compounds were identified in this study that
General Procedure 3. Oxidation of 1,2,3-Triazole Benze-
nesulfonamide Glycoconjugates. A solution of the 1,2,3-triazole
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Figure 3. (A) Impact of compound 5 (60 μM) treatment on CA IX and CA XII expression in colon carcinoma cells: control cells (LS-shca9/ctl ꢀTet)
or Tet-inducible ca9-silenced cells (LS-shca9/ctl +Tet) or double ca9- and ca12-silenced cells (LS-shca9/ca12ꢀ +Tet). Stable LS-shca9/ctl and LS-
shca9/ca12ꢀ cells were incubated with (+Tet) or without (ꢀTet) tetracyline for 4 days before incubation in either normoxia (N, 21% O2) or hypoxia
(H, 1% O2) for 48 h in the absence (ꢀ) or in the presence (+) of 60 μM compound 5. Total cell extracts were analyzed by immunoblotting with
antibodies against HIF-1α, CA IX, CA XII, and Hsp90. The latter was used as a loading control. (B) Proliferation index of control LS-shca9/ctl ꢀTet
cells or double silenced LS-shca9/ca12ꢀ +Tet cells exposed to hypoxia (H, 1% O2) for 48 h in the absence (DMSO) or in the presence of 60 μM of CA
inhibitors (AZA or compound 5). The total cell number was counted every 24 h during 3 days.
benzenesulfonamide glycoconjugate (1.0 equiv) in anhydrous CH2Cl2
was prepared and cooled to 0 °C. A solution of mCPBA (7.0 equiv) in
anhydrous CH2Cl2 was added dropwise, and the reaction was allowed to
warm to room temperature. Oxidation of the starting material was
complete after 2 h, with the reaction progress monitored by TLC. The
reaction mixture was washed with NaHCO3 (ꢁ1) and brine (ꢁ2), dried
with MgSO4, and filtered, and the solvent was then removed in vacuo.
The crude product was purified by flash chromatography, described
below, to afford target compounds 11ꢀ16.
temperature under an inert nitrogen atmosphere. A second solution,
comprising CuI (1.0 equiv), triethylamine (1.0 equiv), and propargyl
alcohol (1.0 equiv) in THF, was stirred under an inert nitrogen atmo-
sphere at room temperature. After 30 min, the two solutions were
combined, and the reaction was stirred at 50 °C until complete
consumption of the starting material was evidenced by TLC (∼30 min).
The reaction mixture was filtered through a prepacked silica SPE
cartridge and eluted with EtOAc. The solvent was removed from the
crude product in vacuo, and the residue was redissolved in CH2Cl2 and
washed with 5% HCl (ꢁ1) and brine (ꢁ2). The aqueous extracts were
back extracted with CH2Cl2 (ꢁ2). The organic extracts were combined,
dried with MgSO4, and filtered, and the solvent was removed in vacuo.
The crude product was purified by flash chromatography as described to
afford the substituted triazole derivatives 27 and 28.
p-Azidobenzene Sulfonamide (1). Sulfanilamide (10.0 g,
58.1 mmol, 1.0 equiv) was dissolved in CH3CN (300 mL), and the solution
was cooled to 0 °C. tert-Butyl nitrite (t-BuONO) (10.3 mL, 86.6 mmol,
1.5 equiv) was added dropwise followed by dropwise addition of
azidotrimethylsilane (TMSN3) (11.5 mL, 86.6 mmol, 1.5 equiv). The
reaction mixture was stirred at room temperature, and the reaction
progress was monitored by TLC (1:1 EtOAc/hexane). After full
conversion of the starting material, the solvent was evaporated, and
the resulting syrup was diluted with EtOAc (100 mL) and washed with
General Procedure 4. Deprotection of Glycoconjugates.
Fully deprotected sugars of glycoconjugates were prepared by treating
the corresponding acetylated precursors (1.0 equiv) in dry MeOH at
0 °C with methanolic sodium methoxide (0.05 M final concentration;
pH 12). The reaction was warmed to room temperature and left to stir
until full deacetylation was evident by TLC (CH3CN:H2O 9:1; 30 min,
overnight). Neutralization of the solution with Amberlite IR-120-H+ ion-
exchange resin, followed by filtration (the resin washed several times
with methanol) and evaporation of the filtrate to dryness, afforded pure
compounds 17ꢀ26.
General Procedure 5. Synthesis of 5-Substituted-1,2,3-
triazole Benzenesulfonamide Glycoconjugates. A solution of
5-iodo-1,2,3-triazole benzenesulfonamide glycoconjugate (1.0 equiv)
and Pd(PPh3)4 (1.0 equiv) in THF was prepared and stirred at room
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ARTICLE
water (ꢁ1) and brine (ꢁ2). The aqueous extracts were combined and
back extracted with EtOAc (ꢁ2). The combined organic extracts were
dried with MgSO4 and filtered, and the solvent was removed in vacuo
to produce an orange solid. The solid was redissolved in EtOAc (150 mL)
and reprecipitated with the slow addition of hexane (350 mL) to afford
the title compound as a pale yellow solid (9.33 g, 47.1 mmol, 81% yield).
1H NMR (500 MHz, CDCl3): δ (ppm) = 7.93 (d, J = 8.5 Hz, 2H,
Harom.), 7.15 (d, J = 8.5 Hz, 2H, Harom.), 4.84 (s, 2H, SONH2),
(3:1 EtOAc/hexane) afforded compound 5 (43%) and 6 (45%) as light
brown solids.
Compound 5: mp = 97ꢀ100 °C. [α]D25 = ꢀ60 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.06, 7.85 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.58 (s, 2H, NH2), 5.32 (t, J = 9.0 Hz, 1H, H-30), 4.97 (t,
J = 10.0 Hz, 1H, H-40), 4.96 (d, J = 9.5 Hz, 1H, H-10), 4.94 (t, J = 9.0 Hz,
1H, H-20), 4.16 (dd, J = 12.5, 5.0 Hz, 1H, H-6a0), 4.07ꢀ4.00 (m, 4H,
H-5, H-6b0, SCH2), 2.01, 2.00, 1.99, 1.95 (4 ꢁ s, 12H, OCOCH3),
1
1
assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz;
assignments were confirmed by 1Hꢀ H gCOSY. LRMS (ESI+):
DMSO): δ (ppm) = 170.0, 169.5, 169.2, 169.0 (OCOCH3), 148.4
(Carom.), 145.3 (Ctriazole), 139.0 (Carom.), 127.0, 127.0, 126.6, 126.6
(CHarom.), 85.2 (CItriazole), 81.1 (C-10), 74.4 (C-50), 72.9 (C-30), 69.6
(C-20 or C-40), 68.1 (C-40 or C-20), 61.9 (C-60), 23.2 (SCH2), 20.5, 20.4,
m/z = 221 [M + Na]+.
2,3,4,6-Tetra-O-acetyl-S-propynyl-1-thio-β-D-glucopyra-
noside (2). The title compound 2 was prepared from commercially
purchased per-O-acetylated β-D-glucose according to general proce-
dure 1 and isolated as a clear oil (76% yield) by flash chromatography
(1:1 EtOAc/hexane). 1H NMR (500 MHz, CDCl3): δ (ppm) = 5.26 (t,
J = 9.5 Hz, 1H, H-3), 5.10 (t, J = 9.5 Hz, 1H, H-4), 5.06 (t, J = 9.5 Hz,
1H, H-2), 4.77 (d, J = 10.0 Hz, 1H, H-1), 4.25 (dd, J = 12.0, 4.5 Hz,
H-6a), 4.15 (dd, J = 12.5, 2.0 Hz, 1H, H-6b), 3.73 (ddd, J = 10.0, 4.5, 2.0
Hz, 1H, H-5), 3.58 (dd, J = 16.5, 2.5 Hz, 1H, SCHa), 3.29 (dd, J = 16.0,
2.5 Hz, 1H, SCHb), 2.26 (t, J = 2.5 Hz, 1H, CtCH), 2.08, 2.06, 2.03,
13
20.3, 20.2 (OCOCH3), assignments were confirmed by 1Hꢀ C
HSQC. LRMS (ESI+): m/z = 727 [M + H]+, 749 [M + Na]+. HRMS:
Calcd for C23H28IN4O11S2, 727.0235. Found, 727.0216.
Compound 6: mp = 91ꢀ95 °C. [α]D25 = ꢀ44 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.75 (s, 1H, CHtriazole),
8.10, 8.02 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.51 (s, 2H, NH2), 5.31 (t, J =
9.5 Hz, 1H, H-30), 4.98 (d, J = 10.0 Hz, 1H, H-10), 4.97 (t, J = 9.5 Hz, 1H,
H-20), 4.93 (t, J = 10.0 Hz, 1H, H-40), 4.16 (dd, J = 12.5, 5.0 Hz, 1H,
1
2.01 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed by 1Hꢀ H
H-6a0), 4.05ꢀ4.01 (m, 4H, H-5, H-6b0, SCH2), 1.98, 1.97, 1.95 (3 ꢁ s,
gCOSY. 13C NMR (125 Mz, CDCl3): δ (ppm) = 170.8, 170.4, 169.6,
169.5 (OCOCH3), 82.3 (C-1), 78.9 (CH2CtCH), 76.2 (C-5), 74.0
(C-3), 72.1 (CH2CtCH), 70.0 (C-2), 68.5 (C-4), 62.2 (C-6), 20.9,
20.8, 20.8, 20.7 (OCOCH3), 17.7 (CH2CtCH), assignments were
1
12H, OCOCH3), assignments were confirmed by 1Hꢀ H gCOSY. 13
C
NMR (500 MHz; DMSO): δ (ppm) = 170.0, 169.5, 169.2, 169.1
(OCOCH3), 148.4 (Ctriazole), 143.8, 138.6 (Carom.), 127.4, 127.4
(CHarom.), 121.8 (CHtriazole), 120.2, 120.2 (CHarom.), 81.0 (C-10),
74.5 (C-50), 72.9 (C-30), 69.5 (C-20 or C-40), 68.0 (C-40 or C-20),
61.8 (C-60), 23.2 (SCH2), 20.4, 20.3, 20.3, 20.2 (OCOCH3), assign-
13
confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 425 [M + Na]+.
Analytical data are consistent with values reported in the literature.11
2,3,4,6-Tetra-O-acetyl-S-propynyl-1-thio-β-D-galactopyr-
anoside (3). The title compound 3 was prepared from commercially
purchased per-O-acetylated β-D-galactose according to general proce-
dure 1 and isolated as a clear oil (82% yield) by flash chromatography
(1:1 EtOAc/hexane). 1H NMR (500 MHz, CDCl3): δ (ppm) = 5.45 (d,
J = 3.5 Hz, 1H, H-4), 5.26 (t, J = 10.0 Hz, 1H, H-2), 5.09 (dd, J = 10.0, 3.5
Hz, 1H, H-3), 4.74 (d, J = 10.0 Hz, 1H, H-1), 4.19ꢀ4.08 (m, 2H, H-6a,
H-6b), 3.96 (m, 1H, H-5), 3.57 (dd, J = 16.5, 2.5 Hz, 1H, SCHa), 3.31
(dd, J = 16.5, 2.5 Hz, 1H, SCHb), 2.26 (t, J = 2.5 Hz, 1H, CtCH), 2.16,
2.07, 2.05, 1.99 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed by
ments were confirmed by Hꢀ C HSQC. LRMS (ESI+): m/z = 601
[M + H]+, 623 [M + Na]+. HRMS: Calcd for C23H29N4O11S2, 601.1269.
Found, 601.1276. Analytical data are consistent with values reported in
the literature.18
1
13
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-galactopyranosyl]thio-
methyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfonamide
(7) and p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-galactopyranosyl]-
thiomethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide (8). The
title compounds 7 and 8 were prepared from azide 1 and alkyne 3 according
to general procedure 2. Purification by flash chromatography (3:1 EtOAc/
hexane) afforded compound 7 (45%) and 8 (47%) as light brown solids.
Compound 7: mp = 98ꢀ99 °C. [α]D25 = ꢀ37 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.07, 7.84 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.59 (s, 2H, NH2), 5.32 (d, J = 3.0 Hz, 1H, H-40), 5.22
(dd, J = 10.0, 3.5 Hz, 1H, H-30), 5.03 (t, J = 10.0 Hz, 1H, H-20), 4.90 (d,
J = 10.0 Hz, 1H, H-10), 4.22 (m, 1H, H-50), 4.06ꢀ3.98 (m, 4H, H-6a0,
H-6b0, SCH2), 2.13, 2.01, 2.00, 1.92 (4 ꢁ s, 12H, OCOCH3), assign-
1
1Hꢀ H gCOSY. 13C NMR (125 Mz, CDCl3): δ (ppm) = 170.6, 170.4,
170.2, 169.9 (OCOCH3), 82.8 (C-1), 79.0 (CH2CtCH), 74.8 (C-5),
72.1 (C-3), 72.0 (CH2CtCH), 67.5 (C-4), 67.4 (C-2), 61.6 (C-6), 21.0,
20.9, 20.9, 20.8 (OCOCH3), 17.7 (CH2CtCH), assignments were
13
confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 425 [M + Na]+.
Analytical data are consistent with values reported in the literature.11
2,20,3,30,40,6,60-Hepta-O-acetyl-S-propynyl-1-thio-β-mal-
toside (4). The title compound 5 was prepared from 1,2,20,3,30,
40,6,60-hepta-O-acetyl-β-maltose according to general procedure 1
and isolated as a white solid (37% yield) by flash chromatography
(1:1.2 EtOAc/hexane). Rf = 0.48 (1:1 EtOAc/hexane). 1H NMR (500
MHz, CDCl3): δ (ppm) = 5.35 (m, 1H, H-3), 5.27 (d, J = 4.0 Hz, 1H,
H-10), 5.21 (t, J = 10.0 Hz, 1H, H-30), 4.98 (t, J = 9.5 Hz, 1H, H-40),
4.97 (d, J = 9.5 Hz, 1H, H-1), 4.87 (dd, J = 10.5, 4.0 Hz, 1H, H-20), 4.78
(t, J = 9.5 Hz, 1H, H-2), 4.39 (m, 1H, H-6a), 4.16 (m, 2H, H-6a0, H-6b),
4.02 (m, 1H, H-6b0), 4.00 (m, 1H, H-5), 3.97 (m, 2H, H-50, H-4), 3.44
(br s, 2H, SCH2), 3.18 (br s, 1H, CtCH), 2.07, 2.02, 2.02, 1.99, 1.99,
1.98, 1.95 (7 ꢁ s, 21H, OCOCH3), assignments were confirmed by
1
ments were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz;
DMSO): δ (ppm) = 170.1, 170.0, 169.5, 169.4 (OCOCH3), 148.5
(Carom.), 145.4 (Ctriazole), 139.1 (Carom.), 127.1, 126.7 (CHarom.), 85.2
(CIriazole), 81.9 (C-10), 73.5 (C-50), 71.0 (C-30), 67.5 (C-40), 67.3 (C-20),
61.3 (C-60), 24.3 (SCH2), 20.6, 20.6, 20.5, 20.4 (OCOCH3), assign-
1
13
ments were confirmed by Hꢀ C HSQC. LRMS (ESI+): m/z = 727
[M + H]+, 749 [M + Na]+. HRMS: Calcd for C23H27IN4O11S2Na,
749.0055. Found, 749.0047.
Compound 8: mp = 189ꢀ190 °C. [α]D25 = ꢀ34 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.74 (s, 1H, CHtriazole),
8.11, 8.02 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.51 (s, 2H, NH2), 5.32 (d, J =
3.5 Hz, 1H, H-40), 5.22 (dd, J = 10.0, 3.5 Hz, 1H, H-30), 5.04 (t, J = 10.0
Hz, 1H, H-20), 4.93 (d, J = 10.0 Hz, 1H, H-10), 4.25 (m, 1H, H-6a0), 4.08
(d, J = 14.0 Hz, 1H, SCHa), 4.04ꢀ4.01 (m, 3H, H-5, H-6b0 SCHb), 2.13,
2.00, 1.97, 1.92 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed by
1
1Hꢀ H gCOSY. LRMS (ESI+): m/z = 708 [M + NH4]+, 713 [M +
Na]+.
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-glucopyranosyl]thio-
methyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfonamide
(5) and p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-glucopyranosyl]-
thiomethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide (6).
Title compounds 5 and 6 were prepared from azide 1 and alkyne 2
according to general procedure 2. Purification by flash chromatography
1
1Hꢀ H gCOSY. 13C NMR (500 MHz; DMSO): δ (ppm) = 169.9, 169.8,
169.4, 169.3 (OCOCH3), 145.0 (Ctriazole), 143.8, 138.5 (Carom.), 127.4,
127.4 (CHarom.), 121.7 (CHtriazole), 120.2, 120.2 (CHarom.), 81.8 (C-10),
73.4 (C-50), 70.9 (C-30), 67.5 (C-40), 67.2 (C-20), 61.4 (C-60), 23.5
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(SCH2), 20.4, 20.4, 20.3, 20.3 (OCOCH3), assignments were confirmed
(OCOCH3), 144.1 (Ctriazole), 138.3, 135.5 (Carom.), 127.5, 127.5
(CHarom.), 124.2 (CHtriazole), 120.5, 120.5 (CHarom.), 84.9 (C-10),
74.9 (C-50), 72.5 (C-30), 67.2 (C-40), 65.7 (C-20), 61.4 (C-60), 47.0
(SCH2), 20.5, 20.3, 20.2, 20.1 (OCOCH3), assignments were confirmed
13
by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 601 [M + H]+, 623 [M + Na]+.
HRMS: Calcd for C23H29N4O11S2, 601.1269. Found, 601.1252. Analy-
tical data are consistent with values reported in the literature.18
p-(4-{[20,200,30,300,40,60,600-Hepta-O-acetyl-β-maltoside]thio-
methyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfonamide
(9) and p-(4-{[20,200,30,300,40,60,600-Hepta-O-acetyl-β-maltoside]-
thiomethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide
(10). Compounds 9 and 10 were prepared from azide 1 and alkyne 4
according to general procedure 2. Purification by flash chromatography
(1:1 then 2:1 EtOAc/hexane) afforded compounds 9 (35%) and 10
(47%) as light yellow solids. These compounds were carried through to
oxidation.
13
by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 655 [M + Na]+. HRMS: Calcd
for C23H28N4O13S2Na, 655.0987. Found, 655.0966. Analytical data are
consistent with values reported in the literature.18
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-galactopyranosyl]sulfonyl-
methyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfonamide
(13). The title compound 13 was prepared from compound 7 according
to the general procedure 3. Purification by flash chromatography
(3:1 EtOAc/hexane) afforded the title compound 13 (78% yield) as a
1
white foam. H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.09, 7.88
Compound 9: Rf = 0.36 (2:1 AcOEt/hexane). 1H NMR (500 MHz,
DMSO-d6): δ (ppm) = 8.07, 7.85 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.58 (s,
2H, NH2), 5.35 (m, 1H, H-30), 5.29 (d, J = 3.5 Hz, 1H, H-100), 5.23 (t, J =
10.0 Hz, 1H, H-300), 4.99 (t, J = 9.5 Hz, 1H, H-400), 4.95 (d, J = 10.0 Hz,
1H, H-10), 4.87 (dd, J = 10.5, 3.5 Hz, 1H, H-200), 4.80 (t, J = 9.5 Hz, 1H,
H-20), 4.42 (m, 1H, H-6a0), 4.16 (m, 2H, H-6a00, H-6b0), 4.03 (m, 1H,
H-6b00), 3.97 (m, 5H, H-40, H-50, H-50, SCH2), 2.09, 2.02, 2.01, 1.99,
1.98, 1.96, 1.95 (7 ꢁ s, 21H, OCOCH3), assignments were confirmed
(2 ꢁ d, J = 8.5 Hz, 2H, Harom.), 7.60 (s, 2H, NH2), 5.55 (t, J = 9.5 Hz, 1H,
H-20), 5.38 (m, 2H, H-30, H-40), 5.16 (d, J = 9.5 Hz, 1H, H-10), 4.78 (d, J
= 14.5 Hz, 1H, SCHa), 4.59 (d, J = 14.5 Hz, 1H, SCHb), 4.46 (br t, J =
6.0 Hz, 1H, H-50), 4.23 (dd, J = 11.5, 7.0 Hz, 1H, H-6a0), 4.12 (dd, J =
11.5, 5.5 Hz, 1H, H-6b0), 2.17, 2.01, 1.97, 1.94 (4 ꢁ s, 3H, OCOCH3),
1
assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz;
DMSO): δ (ppm) = 169.9, 169.9, 169.3, 168.5 (OCOCH3), 145.5
(Ctriazole), 140.5, 138.8 (Carom.), 127.1, 127.1, 126.7, 126.7 (CHarom.),
89.4 (CItriazole), 85.7 (C-10), 74.5 (C-50), 70.6 (C-30), 67.1 (C-40), 62.9
(C-20), 61.0 (C-60), 47.4 (SCH2), 20.5, 20.4, 20.3, 20.2 (OCOCH3),
1
by 1Hꢀ H gCOSY. LRMS (ESI+): m/z = 1016 [M + H]+, m/z = 1038
[M + Na]+.
13
assignments were confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 759
Compound 10: Rf = 0.25 (2:1 AcOEt/hexane). 1H NMR (500 MHz,
DMSO-d6): δ (ppm) = 8.72 (s, 1H, CHtriazole), 8.11, 8.03 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.51 (s, 2H, NH2); 5.35 (t, J = 8.5 Hz, 1H, H-30), 5.28
(d, J = 3.5 Hz, 1H, H-100), 5.22 (t, J = 10.0 Hz, 1H, H-300), 4.98 (t, J = 10.0
Hz, 1H, H-400), 4.97 (d, J = 10.0 Hz, 1H, H-10), 4.87 (dd, J = 11.0, 4.0 Hz,
1H, H-200), 4.81 (t, J = 10.0 Hz, 1H, H-20), 4.39 (dd, J = 12.0, 1.5 Hz, 1H,
H-6a0), 4.15 (m, 2H, H-6a00, H-6b0), 4.07 (m, 1H, H-6b00), 4.00 (m, 5H,
H-40, H-50, H-500, SCH2), 2.03, 2.02, 2.02, 1.98, 1.95, 1.95, 1.94 (7 ꢁ s,
[M + H]+, 781 [M + Na]+. HRMS: Calcd for C23H28IN4O13S2,
759.0133. Found, 759.0119.
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-galactopyranosyl]sulfo-
nylmethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide (14). The
title compound 14 was prepared from compound 8 according to the
general procedure 3. Purification by flash chromatography (3:1 EtOAc/
hexane) afforded the title compound 14 (82% yield) as a white solid. mp =
206ꢀ208 °C. [α]D25 = ꢀ17 (c = 1.0, chloroform). 1H NMR (500 MHz,
DMSO-d6): δ (ppm) = 8.91 (s, 1H, CHtriazole), 8.16 (2 ꢁ d, J = 8.5 Hz, 4H,
Harom.), 7.53 (s, 2H, NH2), 5.52 (t, J = 10.0 Hz, 1H, H-20), 5.37 (d, J = 3.5
Hz, 1H, H-40), 5.34 (dd, J = 10.0, 3.5 Hz, 1H, H-30), 5.12 (d, J = 10.0 Hz,
1H, H-10), 4.84 (d, J = 14.5 Hz, 1H, SCHa), 4.73 (d, J = 15.0 Hz, 1H,
SCHb), 4.45 (br t, J = 6.0 Hz, 1H, H-50), 4.21 (dd, J = 11.5, 7.0 Hz, 1H,
H-6a0), 4.10 (dd, J = 11.5, 5.0 Hz, 1H, H-6b0), 2.16, 2.02, 1.95, 1.93 (4 ꢁ s,
1
21H, OCOCH3), assignments were confirmed by 1Hꢀ H gCOSY.
LRMS (ESI+): m/z = 890 [M + H]+, m/z = 912 [M + Na]+.
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-glucopyranosyl]sulfo-
nylmethyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfona-
mide (11). The title compound 11 was prepared from compound 5
according to the general procedure 3. Purification by flash chroma-
tography (3:1 EtOAc/hexane) afforded compound 11 (78%) as a
white solid. mp = 234ꢀ236 °C. [α]D25 = ꢀ86 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.09, 7.87 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.60 (s, 2H, NH2), 5.44 (m, 2H, H-20, H-30), 5.19 (d,
J = 9.5 Hz, 1H, H-10), 5.07 (t, J = 9.5 Hz, 1H, H-40), 4.74 (d, J = 15.0 Hz,
1H, SCHa), 4.66 (d, J = 15.0 Hz, 1H, SCHb), 4.26ꢀ4.21 (m, 3H, H-50,
H-6a0, H-6b0), 2.04, 2.01, 1.96 (3 ꢁ s, 12H, OCOCH3), assignments
1
1
12H, OCOCH3), assignments were confirmed by Hꢀ H gCOSY. 13C
NMR (500 MHz; DMSO): δ (ppm) = 169.9, 169.8, 169.4, 168.5
(OCOCH3), 144.1 (Ctriazole), 138.3, 135.6 (Carom.), 127.5, 127.5
(CHarom.), 124.2 (CHtriazole), 120.5, 120.5 (CHarom.), 85.0 (C-10), 74.4
(C-50), 70.6(C-30), 67.1(C-40), 62.8(C-20), 61.3 (C-60), 47.1 (SCH2), 20.5,
1
13
20.4, 20.3, 20.2 (OCOCH3), assignments were confirmed by Hꢀ C
HSQC. LRMS (ESI+): m/z = 655 [M + Na]+. HRMS: Calcd for
C23H28N4O13S2Na, 655.0987. Found, 655.0962. Analytical data are con-
sistent with values reported in the literature.18
1
were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz; DMSO): δ
(ppm) = 170.1, 169.5, 169.2, 168.6 (OCOCH3), 145.5 (Carom.), 140.4
(Ctriazole), 138.9 (Carom.), 127.1, 127.1, 126.8, 126.8 (CHarom.), 89.4
(CItriazole), 85.6 (C-10), 75.1 (C-50), 72.6 (C-30), 67.3 (C-40), 65.8
(C-20), 61.3 (C-60), 47.3 (SCH2), 20.6, 20.3, 20.3, 20.2 (OCOCH3),
p-(4-{[20,200,30,300,400,60,600-Hepta-O-acetyl-β-maltosyl]sulf-
onylmethyl}-5-iodo-1-H-1,2,3-triazol-1-yl)benzenesulfona-
mide (15). The title compound 15 was prepared from compound 9
according to the general procedure 3. Purification by flash chromatog-
raphy (2:1 EtOAc/hexane) afforded the title compound 15 (80% yield)
as a white solid. mp = 112ꢀ115 °C. [α]D25 = +25 (c = 1.0, chloroform).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.09, 7.88 (2 ꢁ d, J = 9.0
Hz, 4H, Harom.), 7.60 (s, 2H, NH2), 5.46 (t, J = 9.0 Hz, 1H, H-30), 5.33
(d, J = 2.0 Hz, 1H, H-100), 5.30 (t, J = 9.0 Hz, 1H, H-20), 5.24 (t, J = 10.0
Hz, 1H, H-300), 5.16 (d, J = 10.0 Hz, 1H, H-10), 5.01 (t, J = 10.0 Hz, 1H,
H-400), 4.90 (dd, J = 11.0, 4.0 Hz, 1H, H-200), 4.70 (d, J = 15.0 Hz, 1H,
SCHH), 4.62 (d, J = 15.0 Hz, 1H, SCHH), 4.59 (m, 1H, H-6a0), 4.28
(dd, J = 13.0, 5.0 Hz, 1H, H-6b0), 4.22 (m, 1H, H-50), 4.19 (dd, J = 12.5,
4.5 Hz, 1H, H-6a00), 4.10 (t, J = 9.0 Hz, 1H, H-40), 4.06 (m, 1H, H-6b00),
4.02 (m, 1H, H-500), 2.10, 2.03, 2.00, 1.99, 1.97, 1.96, 1.93 (7 ꢁ s, 21H,
13
assignments were confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z =
759 [M + H]+, 776 [M + NH4]+, 781 [M + Na]+. HRMS: Calcd for
C23H28IN4O13S2, 759.0133. Found, 759.0104.
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-glucopyranosyl]sulfo-
nylmethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide (12).
The title compound 12 was prepared from compound 6 according to the
general procedure 3. Purification by flash chromatography (3:1 EtOAc/
hexane) afforded the title compound 12 (83% yield) as a white solid. 1H
NMR (500 MHz, DMSO-d6): δ (ppm) = 8.91 (s, 1H, CHtriazole), 8.17,
8.05 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.53 (s, 2H, NH2), 5.41 (m, 2H,
H-20, H-30), 5.18 (d, J = 9.5 Hz, 1H, H-10), 5.03 (t, J = 9.5 Hz, 1H, H-40),
4.82 (d, J = 15.0 Hz, 1H, SCHa), 4.76 (d, J = 15.0 Hz, 1H, SCHb),
4.26ꢀ4.21 (m, 3H, H-50, H-6a0, H-6b0), 2.02, 2.01, 1.95, 1.94 (4 ꢁ s,
1
COCH3), assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR
1
12H, OCOCH3), assignments were confirmed by 1Hꢀ H gCOSY. 13
C
NMR (500 MHz; DMSO): δ (ppm) = 170.0, 169.5, 169.1, 168.5
(125 MHz, DMSO-d6): δ (ppm) = 170.9, 170.7, 170.5, 170.3, 170.2,
6913
dx.doi.org/10.1021/jm200892s |J. Med. Chem. 2011, 54, 6905–6918

Journal of Medicinal Chemistry
ARTICLE
169.8, 169.4 (OCOCH3), 146.2 (Carom.), 146.0 (Ctriazole), 139.6
(Carom.), 127.9, 127.9, 127.5, 127.5 (CHarom.), 95.3 (C-100), 90.1
(CItriazole), 86.0 (C-10), 76.4 (C-50), 75.3 (C-30), 73.7 (C-40), 70.1 (C-
200), 69.6 (C-300), 68.8 (C-500), 68.4 (C-400), 63.1 (C-20), 63.0 (C-60),
61.1 (C-600), 47.8 (SCH2), 24.5, 21.4, 21.2, 21.1, 21.0, 21.0,
143.6, 138.6 (Carom.), 127.5, 127.5 (CHarom.), 121.8 (CHtriazole), 120.0,
120.0 (CHarom.), 84.2 (C-10), 81.1 (C-50), 78.1 (C-30), 73.1 (C-40), 70.1
(C-20), 61.3 (C-60), 23.0 (SCH2), assignments were confirmed by
13
1Hꢀ C HSQC. LRMS (ESI+): m/z = 433 [M + H]+, 450 [M +
NH4]+, 455 [M + Na]+. HRMS: Calcd for C15H20N4O7S2Na, 455.0665.
Found, 455.0657. Analytical data are consistent with values reported in
the literature.18
13
21.0 (OCOCH3), assignments were confirmed by 1Hꢀ C HSQC
1
13
and Hꢀ C HMBC. LRMS (ESI+): m/z = 1047 [M + H]+, 1069
[M + Na]+. HRMS: Calcd for C35H44I1N4O21S2, 1047.0979. Found,
1047.0991.
p-(4-{(β-D-Galactopyranosyl)thiomethyl}-5-iodo-1-H-1,2,3-
triazol-1-yl)benzenesulfonamide (19). The title compound 19
was prepared from compound 7 according to the general procedure 4.
Lyophilization afforded the title compound 19 (91% yield) as a highly
hygroscopic whitesolid. [α]D25 =ꢀ57(c= 1.0, methanol). 1H NMR(500
MHz, DMSO-d6): δ (ppm) = 8.06, 7.85 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.),
7.58 (s, 2H, NH2), 4.37 (d, J = 9.5 Hz, 1H, H-10), 4.02 (d, J = 14.0 Hz, 1H,
SCHa), 3.91 (d, J = 14.0 Hz, 1H, SCHb), 3.73 (d, J = 3.0 Hz, 1H, H-40),
3.54ꢀ3.50 (m, 2H, H-50, H-6a0), 3.42 (br d, J = 10.5 Hz, 1H, H-6b0), 3.41
(t, J = 9.5 Hz, 1H, H-20), 3.29 (dd, J = 9.5, 3.0 Hz, 1H, H-30), assignments
p-(4-{[20,200,30,300,400,60,600-Hepta-O-acetyl-β-maltosyl]sulf-
onylmethyl}-1-H-1,2,3-triazol-1-yl)benzenesulfonamide (16).
The title compound 16 was prepared from compound 10 according to
the general procedure 3. Purification by flash chromatography (3:1 EtOAc/
hexane) afforded the title compound 16 (57% yield) as a white foam. mp =
114ꢀ116 °C. [α]D25 = +67 (c = 1.0, chloroform). 1H NMR (500 MHz,
DMSO-d6): δ (ppm) = 8.88 (s, 1H, CHtriazole), 8.17, 8.05 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.52 (s, 2H, NH2), 5.45 (t, J = 9.0 Hz, 1H, H-30), 5.30 (d,
J= 3.5 Hz, 1H, H-100), 5.27 (t, J= 9.5 Hz, 1H, H-20), 5.23(t, J= 10.0 Hz, 1H,
H-300), 5.14 (d, J = 10.0 Hz, 1H, H-10), 5.00 (t, J = 10.0 Hz, 1H, H-400), 4.89
(dd, J = 11.0, 4.0 Hz, 1H, H-200), 4.79 (d, J = 15.0 Hz, 1H, SCHH), 4.72 (d,
J= 15.0 Hz, 1H, SCHH), 4.55 (dd, J= 12.0, 2.5 Hz, 1H, H-6a0), 4.26 (dd, J=
12.0, 5.5 Hz, 1H, H-6b0), 4.20 (m, 1H, H-50), 4.17 (m, 1H, H-6a00), 4.05(m,
1H, H-6b00), 4.03 (m, 1H, H-40); 4.00 (m, 1H, H-500), 2.08, 2.03, 2.02, 1.99,
1.98, 1.95, 1.90 (7 ꢁ s, 21H, COCH3), assignments were confirmed by
1
were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz; DMSO): δ
(ppm) = 150.0 (Carom.), 145.3 (Ctriazole), 139.1 (Carom.), 127.1, 127.1,
126.7, 126.7 (CHarom.), 85.0 (CItriazole), 84.8 (C-10), 79.1 (C-50), 74.8
(C-30), 70.0 (C-40), 68.3 (C-20), 60.4 (C-60), 23.8 (SCH2), assignments
13
were confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 573 [M + H]+,
590 [M + NH4]+, 595 [M + Na]+. HRMS: Calcd for C15H20IN4O7S2,
558.9813. Found, 558.9793.
1
1Hꢀ H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 170.2,
p-(4-{(β-D-Galactopyranosyl)thiomethyl}-1-H-1,2,3-tria-
zol-1-yl)benzenesulfonamide (20). The title compound 20 was
prepared from compound 8 according to the general procedure 4.
Lyophilization afforded the title compound 20 (96% yield) as a highly
hygroscopic white solid. [α]D25 = ꢀ49 (c = 1.0, methanol). 1H NMR
(500 MHz, DMSO-d6): δ (ppm) = 8.82 (s, 1H, CHtriazole), 8.10, 8.01
(2 ꢁ d, J = 9.0 Hz, 4H, Harom.), 7.50 (s, 2H, NH2), 5.00 (d, J = 6.0 Hz,
1H, OH), 4.79 (d, J = 5.5 Hz, 1H, OH), 4.73 (t, J = 5.0 Hz, 1H, OH-6),
4.47 (d, J = 4.5 Hz, 1H, OH), 4.28 (d, J = 9.5 Hz, 1H, H-10), 4.08 (d, J =
14.0 Hz, 1H, SCHa), 3.93 (d, J = 14.0 Hz, 1H, SCHb), 3.70 (m, 1H,
H-40), 3.55ꢀ3.52 (m, 2H, H-6a0, H-6b0), 3.44ꢀ3.41 (m, 2H, H-20,
170.0, 169.8, 169.6, 169.5, 169.1, 168.7 (OCOCH3), 144.1, 138.4 (Carom.),
135.5 (Ctriazole), 127.7, 127.7 (CHarom.), 125.9 (CHtriazole), 120.5, 120.5
(CHarom.), 95.6 (C-100), 84.5 (C-10), 75.5 (C-50), 74.6 (C-30), 73.1 (C-40),
68.9 (C-300), 68.4 (C-200), 68.1 (C-500), 67.8 (C-400), 66.4 (C-20), 62.5 (C-
60), 61.5 (C-600), 47.0 (SO2CH2), 20.6, 20.5, 20.4, 20.3, 20.3, 20.2, 20.2
13
(OCOCH3), assignments were confirmed by 1Hꢀ C HSQC and
13
1Hꢀ C HMBC. LRMS (ESI+): m/z = 921 [M + H]+, 943 [M + Na]+.
HRMS: Calcd for C35H44N4O21S2Na1, 943.1837. Found, 943.1842.
p-(4-{(β-D-Glucopyranosyl)thiomethyl}-5-iodo-1-H-1,2,3-
triazol-1-yl)benzenesulfonamide (17). The title compound 17
was prepared from compound 5 according to the general procedure 4.
Lyophilization afforded the title compound 17 (92% yield) as a highly
1
1
H-50), 3.30 (m, 1H, H-30), assignments were confirmed by Hꢀ H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 146.2 (Carom.),
143.6 (Ctriazole), 138.6 (Carom.), 127.5, 127.5 (CHarom.), 121.8
(CHtriazole), 120.0, 120.0 (CHarom.), 84.6 (C-10), 79.4 (C-50), 74.6
(C-30), 69.8 (C-40), 68.6 (C-20), 60.9 (C-60), 22.9 (SCH2), assign-
1
hygroscopic white solid. [α]D25 = ꢀ64 (c = 1.0, methanol). H NMR
(500 MHz, DMSO-d6): δ (ppm) = 8.06 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.),
7.58 (s, 2H, NH2), 5.15 (d, J = 6.0 Hz, 1H, OH), 5.01 (br s, 1H, OH),
4.92 (br s, 1H, OH), 4.50 (t, J = 5.5 Hz, 1H, OH-6), 4.42 (d, J = 9.5 Hz,
1H, H-1), 4.05 (d, J = 14.0 Hz, 1H, SCHa), 3.91 (d, J = 14.0 Hz, 1H,
SCHb), 3.70 (dd, J = 11.0, 5.5 Hz, 1H, H-6a), 3.49 (m, 1H, H-6b), 3.18
(m, 3H, H-3, H-4, H-5), 3.06 (m, 1H, H-2), assignments were confirmed
13
ments were confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 433
[M + H]+, 450 [M + NH4]+, 455 [M + Na]+. HRMS: Calcd for
C15H20N4O7S2Na, 455.0665. Found, 455.0645. Analytical data are
consistent with values reported in the literature.18
1
by 1Hꢀ H gCOSY. 13C NMR (500 MHz; DMSO): δ (ppm) = 150.0
p-(4-{(β-D-Glucopyranosyl)sulfonylmethyl}-5-iodo-1-H-
1,2,3-triazol-1-yl)benzenesulfonamide (21). The title com-
pound 21 was prepared from compound 11 according to the general
procedure 4. Lyophilization afforded the title compound 21 (93%
(Carom.), 145.2 (Ctriazole), 139.0 (Carom.), 127.0, 127.0, 126.6, 126.6
(CHarom.), 84.7 (CItriazole), 84.3 (C-10), 80.8 (C-50), 78.2 (C-30), 73.1
(C-40), 69.9 (C-20), 61.1 (C-60), 23.6 (SCH2), assignments were
1
13
confirmed by Hꢀ C HSQC. LRMS (ESI+): m/z = 559 [M + H]+,
576 [M + NH4]+, 781 [M + Na]+. HRMS: Calcd for C15H20IN4O7S2,
558.9813. Found, 558.9811.
25
yield) as a highly hygroscopic white solid. [α]D = ꢀ19 (c = 1.0,
1
methanol). H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.08, 7.88
(2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.60 (s, 2H, NH2), 5.53 (d, J = 6.0 Hz, 1H,
OH), 5.22 (d, J = 5.5 Hz, 1H, OH), 5.11 (d, J = 5.5 Hz, 1H, OH), 4.75
(t, J = 6.0 Hz, 1H, OH), 4.76 (d, J = 14.5 Hz, 1H, SCHa), 4.66 (d, J =
14.5 Hz, 1H, SCHb), 4.56 (d, J = 9.5 Hz, 1H, H-10), 3.76 (dd, J = 11.0,
7.0 Hz, 1H, H-6a0), 3.61 (m, 1H, H-20), 3.52 (m, 1H, H-6b0),
3.38ꢀ3.28 (m, 2H, H-30, H-50), 3.14 (m, 1H, H-40), assignments
p-(4-{(β-D-Glucopyranosyl)thiomethyl}-1-H-1,2,3-triazol-
1-yl)benzenesulfonamide (18). The title compound 18 was pre-
pared from compound 6 according to the general procedure 4. Lyophi-
lization afforded the title compound 18 (97% yield) as a highly
1
hygroscopic white solid. [α]D25 = ꢀ60 (c = 1.0, methanol). H NMR
(500 MHz, DMSO-d6): δ (ppm) = 8.83 (s, 1H, CHtriazole), 8.11, 8.02 (2
ꢁ d, J = 9.0 Hz, 4H, Harom.), 7.51 (s, 2H, NH2), 5.16 (d, J = 6.0 Hz, 1H,
OH), 5.02 (d, J = 4.5 Hz, 1H, OH), 4.95 (d, J = 5.5 Hz, 1H, OH), 4.76 (t,
J = 5.5 Hz, 1H, OH-6), 4.34 (d, J = 10.0 Hz, 1H, H-10), 4.10 (d, J = 14.5
Hz, 1H, SCHa), 3.95 (d, J = 14.0 Hz, 1H, SCHb), 3.76 (m, 1H, H-6a0),
3.47 (m, 1H, H-6b0), 3.19 (m, 1H, H-50), 3.14 (m, 1H, H-30), 3.08 (m,
1
were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz; DMSO): δ
(ppm) = 145.5 (Carom.), 141.6 (Carom.), 138.9 (Ctriazole), 127.1, 126.8;
(CHarom.), 89.8 (CItriazole), 89.2 (C-10), 81.9 (C-50), 77.7 (C-30),
69.3 (C-40), 69.3 (C-20), 60.9 (C-60), 48.8 (SCH2), assignments were
13
confirmed by 1Hꢀ C HSQC. LRMS(ESI+): m/z = 608 [M + NH4]+,
613 [M + Na]+. HRMS: Calcd for C15H19IN4O9S2, 612.9530. Found,
612.9538.
1
1H, H-40), 3.05 (m, 1H, H-20), assignments were confirmed by 1Hꢀ H
gCOSY. 13C NMR (500 MHz; DMSO): δ (ppm) = 146.2 (Ctriazole),
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p-(4-{(β-D-Glucopyranosyl)sulfonylmethyl}-1-H-1,2,3-
triazol-1-yl)benzenesulfonamide (22). The title compound
22 was prepared from compound 12 according to the general
procedure 4. Lyophilization afforded the title compound 22 (93%
J = 4.0 Hz, 1H, H-100), 4.90 (m, 2H, OH), 4.90 (m, 2H, OH, SCHa),
4.67 (d, J = 14.5 Hz, 1H, SCHb), 4.60 (d, J = 9.5 Hz, 1H, H-10), 4.53 (br
t, J = 5.5 Hz, 1H, OH), 3.82 (m, 1H, H-6a0), 3.69 (m, 4H, H-20, H-40,
H-6a00, H-6b00), 3.46 (m, 6H, H-200, H-30, H-300, H-50, H-500, H-6b0),
25
1
3.07 (m, 1H, H-400), assignments were confirmed by 1Hꢀ H gCOSY.
yield) as a highly hygroscopic white solid. [α]D = ꢀ14 (c = 1.0,
methanol). 1H NMR (500 MHz, DMSO-d6): δ (ppm) = 9.05 (s, 1H,
CHtriazole), 8.12, 8.03 (2 ꢁ d, J = 9.0 Hz, 4H, Harom.), 7.52 (s, 2H,
NH2), 5.48 (d, J = 6.0 Hz, 1H, OH), 5.20 (d, J = 5.0 Hz, 1H, OH),
5.14 (d, J = 5.0 Hz, 1H, OH), 5.04 (t, J = 5.5 Hz, 1H, OH), 4.82 (d,
J = 14.5 Hz, 1H, SCHa), 4.67 (d, J = 15.0 Hz, 1H, SCHb), 4.49 (d, J =
9.5 Hz, 1H, H-10), 3.82 (dd, J = 12.0, 5.0 Hz, 1H, H-6a0), 3.60 (m,
1H, H-20), 3.44 (m, 1H, H-6b0), 3.42 (m, 1H, H-50), 3.29 (m, 1H,
13C NMR (125 MHz, DMSO-d6): δ (ppm) = 145.6 (Carom.), 141.4
(Ctriazole), 138.8 (Carom.), 127.0, 127.0, 126.7, 126.7 (CHarom.), 100.7
(C-100), 89.5 (C-10), 89.3 (CItriazole), 80.1 (C-50), 78.4 (C-30), 77.3 (C-
40), 73.6 (C-200), 73.2 (C-300), 72.4 (C-500), 69.9 (C-400), 69.1 (C-20),
60.8 (C-60), 60.4 (C-600), 48.8 (SCH2), assignments were confirmed by
13
13
1Hꢀ C HSQC and 1Hꢀ C HMBC. LRMS (ESI+): m/z = 775 [M +
Na]+.
1
1
H-30), 3.07 (m, 1H, H-40), assignments were confirmed by Hꢀ H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 143.9, 138.4
(Carom.), 136.7 (Ctriazole), 127.5, 127.5 (CHarom.), 124.2 (CHtriazole),
120.3, 120.3 (CHarom.), 88.7 (C-10), 81.7 (C-50), 77.4 (C-30), 69.5
(C-40), 69.1 (C-20), 61.1 (C-60), 48.3 (SCH2), assignments were con-
p-(4-{[β-Maltosyl]sulfonylmethyl}-1-H-1,2,3-triazol-1-yl)-
benzenesulfonamide (26). The title compound 26 was prepared
from compound 16 according to the general procedure 4. Lyophilization
afforded the title compound 26 (76% yield) as a highly hygroscopic
25
1
white solid. [α]D = +11 (c = 1.0, methanol). H NMR (500 MHz,
DMSO-d6): δ (ppm) = 9.02 (s, 1H, CHtriazole), 8.12, 8.03 (2 ꢁ d, J = 9.5
Hz, 4H, Harom.), 7.52 (br s, 2H, NH2), 5.70 (d, J = 3.5 Hz, 1H, OH), 5.62
(d, J = 6.5 Hz, 1H, OH), 5.46 (d, J = 6.0 Hz, 1H, OH), 5.06 (d, J = 4.0 Hz,
1H, H-100), 5.01 (br t, J = 6.0 Hz, 1H, OH), 4.91 (d, J = 5.5 Hz, 1H, OH),
4.87 (d, J = 5.0 Hz, 1H, OH), 4.84 (d, J = 15.0 Hz, 1H, SCHa), 4.68 (d,
J = 14.5 Hz, 1H, SCHb), 4.56 (d, J = 9.0 Hz, 1H, H-10), 4.53 (br t, J = 5.5
Hz, 1H, OH), 3.87 (m, 1H, H-6a0), 3.63 (m, 5H, H-20, H-40, H-50, H-6a0,
H-6a00, H-6b0), 3.46 (m, 2H, H-200, H-6b00), 3.37 (m, 2H, H-30, H-300),
3.26 (m, 1H, H-500), 3.07(m, 1H, H-400), assignments were confirmed by
13
firmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 482 [M + NH4]+, 487
[M + Na]+. HRMS: Calcd for C15H20N4O9S2Na, 487.0564. Found,
487.0561. Analytical data are consistent with values reported in the
literature.18
p-(4-{(β-D-Galactopyranosyl)sulfonylmethyl}-5-iodo-1-H-
1,2,3-triazol-1-yl)benzenesulfonamide (23). The title com-
pound 23 was prepared from compound 13 according to the general
rocedure 4. Lyophilization afforded the title compound 23 (86% yield)
as a highly hygroscopic white solid. [α]D25 = ꢀ10 (c = 1.0, methanol).
1H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.08, 7.88 (2 ꢁ d, J = 8.5
Hz, 4H, Harom.), 7.59 (s, 1H, NH2), 5.35 (d, J = 6.5 Hz, 1H, OH), 5.00
(d, J = 6.0 Hz, 1H, OH), 4.71 (d, J = 14.5 Hz, 1H, SCHa), 4.71 (m, 1H,
OH), 4.66 (d, J = 4.0 Hz, 1H, OH), 4.62 (d, J = 14.0 Hz, 1H, SCHb), 4.49
(d, J = 9.0 Hz, 1H, H-10), 3.95 (m, 1H, H-20), 3.75 (m, 1H, H-6a0),
3.63ꢀ3.56 (m, 3H, H-30, H-50, H-6b0), 3.46 (m, 1H, H-40), assignments
1
1Hꢀ H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 144.0,
138.4 (Carom.), 136.6 (Ctriazole), 127.5, 127.5 (CHarom.), 124.2
(CHtriazole), 120.4, 120.4 (CHarom.), 100.7 (C-100), 88.3 (C-10), 79.8
(C-50), 78.7 (C-30), 77.1 (C-40), 73.6 (C-200), 73.2 (C-300), 72.3 (C-500),
69.9 (C-400), 68.8 (C-20), 60.8 (C-60), 60.7 (C-600), 48.3 (SCH2),
13
13
assignments were confirmed by 1Hꢀ C HSQC and 1Hꢀ C HMBC.
1
were confirmed by 1Hꢀ H gCOSY. 13C NMR (125 MHz, DMSO-d6):
LRMS (ESI+): m/z = 649 [M + Na]+.
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-glucopyranosyl]thio-
methyl}-5-(1-hydroxy-2-propyn)-1-H-1,2,3-triazol-1-yl)ben-
zenesulfonamide (27). The title compound 27 was prepared from
compound 5 according to the general procedure 5. Purification by flash
chromatography (3:1 EtOAc/hexane) afforded the title compound
δ (ppm) = 145.4, 141.6 (Carom.), 138.9 (Ctriazole), 127.1, 126.8
(CHarom.), 90.5 (CItriazole), 89.2 (C-10), 80.4 (C-50), 74.2 (C-30), 68.0
(C-40), 66.2 (C-20), 60.4 (C-60), 48.8 (SCH2), assignments were
13
confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 608 [M +
NH4]+, 613 [M + Na]+. HRMS: Calcd for C15H19IN4O9S2, 612.9530.
Found, 612.9508.
1
27 (56% yield) as a white solid. H NMR (500 MHz, DMSO-d6): δ
p-(4-{(β-D-Galactopyranosyl)sulfonylmethyl}-1-H-1,2,3-
triazol-1-yl)benzenesulfonamide (24). The title compound 24
was prepared from compound 14 according to the general procedure
4. Lyophilization afforded the title compound 24 (86% yield) as a highly
hygroscopic white solid. [α]D25 = 0 (c = 1.0, methanol). 1H NMR (500
MHz, DMSO-d6): δ (ppm) = 9.07 (s, 1H, CHtriazole), 8.10, 8.02 (2 ꢁ d, J
= 9.0 Hz, 4H, Harom.), 7.50 (s, 2H, NH2), 4.79 (d, J = 14.5 Hz, 1H,
SCHa), 4.63 (d, J = 14.5 Hz, 1H, SCHb), 4.39 (d, J = 9.0 Hz, 1H, H-10),
3.92 (t, J = 9.5 Hz, 1H, H-20), 3.71 (d, J = 2.5 Hz, 1H, H-40), 3.68ꢀ3.66
(m, 2H, H-50, H-6a0), 3.57 (m, 1H, H-6b0), 3.44 (dd, J = 9.0, 3.0 Hz, 1H,
(ppm) = 8.05, 8.01 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.57 (s, 2H, NH2),
5.53 (t, J = 6.0 Hz, 1H, CH2OH), 5.31 (t, J = 9.0 Hz, 1H, H-30), 4.99 (d,
J = 10.0 Hz, 1H, H-10), 4.94 (t, J = 10.0 Hz, 1H, H-20), 4.93 (t, J = 10.0
Hz, 1H, H-40), 4.40 (d, J = 6.0 Hz, 2H, CH2OH), 4.13 (dd, J = 13.0, 5.0
Hz, 1H, H-6a0), 4.10 (d, J = 14.5 Hz, 1H, SCHa), 4.02 (d, J = 14.5 Hz,
1H, SCHb), 3.99 (m, 2H, H-5, H-6b0), 1.99, 1.98, 1.96, 1.95 (4 ꢁ s, 12H,
1
OCOCH3), assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR
(500 MHz; DMSO): δ (ppm) = 170.0, 169.5, 169.3, 169.2 (OCOCH3),
147.9 (Carom.), 144.8 (CtriazoleCH2), 138.0 (Carom.), 127.2, 127.2, 123.8,
123.8 (CHarom.), 118.7, 104.0 (CtC), 81.4 (C-10), 74.5 (C-50), 73.0
(C-30), 69.6 (C-40), 68.5 (C-20), 68.0 (CtriazoleCtC), 61.7 (C-60), 49.6
(CH2OH), 22.8 (SCH2), 20.4, 20.4, 20.4, 20.3 (OCOCH3), assignments
1
H-30), assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR (125
MHz, DMSO-d6): δ (ppm) = 144.2, 138.3 (Carom.), 136.8 (Ctriazole),
127.5, 127.5 (CHarom.), 124.3 (CHtriazole), 120.3, 120.3 (CHarom.), 89.5
(C-10), 80.5 (C-50), 73.9 (C-30), 68.3 (C-40), 66.0 (C-20), 61.0 (C-60),
13
were confirmed by 1Hꢀ C HSQC. LRMS (ESI+): m/z = 655 [M + H]+,
677 [M + Na]+. HRMS: Calcd for C26H30N4O12S2Na, 677.1194. Found,
677.1162.
13
48.3 (SCH2), assignments were confirmed by 1Hꢀ C HSQC. LRMS
(ESI+): m/z = 482 [M + NH4]+, 487 [M + Na]+. HRMS: Calcd for
C15H20N4O9S2Na, 487.0564. Found, 487.0566. Analytical data are
consistent with values reported in the literature.18
p-(4-{[20,30,40,60-Tetra-O-acetyl-β-D-galactopyranosyl]thio-
methyl}-5-(1-hydroxy-2-propyn)-1-H-1,2,3-triazol-1-yl)ben-
zenesulfonamide (28). The title compound 28 was prepared from
compound 7 according to the general procedure 5. Purification by flash
chromatography (3:1 EtOAc/hexane) afforded the title compound 28
(49% yield) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ (ppm) =
8.03, 8.01 (2 ꢁ d, J = 8.5 Hz, 4H, Harom.), 7.57 (s, 2H, NH2), 5.56 (t, J =
6.0 Hz, 1H, CH2OH), 5.30 (t, J = 3.5 Hz, 1H, H-40), 5.21 (dd, J = 10.0,
3.5 Hz, 1H, H-30), 5.01 (t, J = 10.0 Hz, 1H, H-20), 4.94 (d, J = 10.0 Hz,
1H, H-10), 4.39 (d, J = 6.0 Hz, 2H, CH2OH), 4.20 (m, 1H, H-50), 4.09
p-(4-{[β-Maltosyl]sulfonylmethyl}-5-iodo-1-H-1,2,3-tria-
zol-1-yl)benzenesulfonamide (25). The title compound 25 was
prepared from compound 15 according to the general procedure 4.
Lyophilization afforded the title compound 25 (72% yield) as a highly
hygroscopic white solid. [α]D25 = +20 (c = 1.0, methanol). 1H NMR
(500 MHz, DMSO-d6): δ (ppm) = 8.08, 7.88 (2 ꢁ d, J = 9.0 Hz, 4H,
H
arom.), 7.59 (s, 2H, NH2), 5.75, 5.69, 5.44 (3 ꢁ br s, 3H, OH), 5.08 (d,
6915
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Journal of Medicinal Chemistry
ARTICLE
(d, J = 14.0 Hz, 1H, SCHa), 4.03 (d, J = 14.0 Hz, 1H, SCHb), 3.99 (m,
2H, H-6a0, H-6b0), 2.10, 1.99, 1.97, 1.91 (4 ꢁ s, 12H, OCOCH3),
compound 5 at 60 μM for 30 min. A 0.1 volume of this cell suspension
was added to a 3 mM HEPES-buffered solution (HBS) adjusted to pHo
8.2 before rapid addition to a CO2-saturated nonbuffered solution, and
the extracellular pH was determined over time (microelectrode, Schott
Instrument) to monitor the rapid hydration of CO2 to carbonic acid in
response to the membrane-associated CAs activity.
1
assignments were confirmed by 1Hꢀ H gCOSY. 13C NMR (500 MHz;
DMSO): δ (ppm) = 170.1, 170.0, 169.5, 169.5 (OCOCH3), 147.9
(Carom.), 144.8 (CtriazoleCH2), 138.0 (Carom.), 131.6, 131.5 128.9, 128.8
(CHarom.), 118.7, 104.1 (CtC), 82.4 (C-10), 73.6 (C-50), 71.0 (C-30),
68.4 (C-40), 68.5 (C-20), 68.3 (CtriazoleCtC), 61.2 (C-60), 49.6
(CH2OH), 23.4 (SCH2), 20.5, 20.5, 20.4, 20.4 (OCOCH3), assignments
Resting pHi Measurement [14C]-Benzoic Acid. The pHi was
measured using the technique of distribution of the weak acid [7-14C]-
benzoic acid (Amersham Biosciences) in intracellular and extracellular
spaces for exponentially growing cells.4 One hour of preincubation in the
absence (DMSO) or in the presence of 60 μM AZA or compound 5 was
used before addition of the radioactive ligand.
1
13
were confirmed by Hꢀ C HSQC. LRMS (ESI+): m/z = 655 [M +
H]+, 677 [M + Na]+. HRMS: Calcd for C26H31N4O12S2, 655.1374.
Found, 655.1351.
CA Catalytic Inhibition Assay. An SX.18MV-R Applied Photo-
physics stopped-flow instrument has been used for assaying the CA I, II,
IX, and XII CO2 hydration activity.29 Phenol red (at a concentration of
0.2 mM) has been used as an indicator, working at the absorbance
maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer and 0.1 M
NaClO4 (for maintaining constant the ionic strength—this anion is not
inhibitory), following the CA-catalyzed CO2 hydration reaction for a
period of 10ꢀ100 s. Saturated CO2 solutions in water at 20 °C were used
as a substrate. Stock solutions of inhibitors were prepared at a concen-
tration of 10ꢀ50 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 EꢀI 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 ChengꢀPrusoff
equation. The catalytic activity (in the absence of inhibitors) of these
enzymes was calculated from LineweaverꢀBurk plots and represent the
mean from at least three different determinations. Enzyme concentra-
tions were 10.3 nM for CA I and CA II, 12 nM for CA IX, and 15 nM for
hCA XII. Enzymes used here were recombinant ones.
Cell Culture and Hypoxic Exposure. Chinese hamster lung
CCL39 fibroblasts (American Type Culture Collection) and the
CCL39-derived mutant PS120 cells, lacking the amiloride-sensitive
Na+/H+ exchanger,31 were maintained in DMEM (Sigma) supplemen-
ted with 7.5% FCS in a humidified atmosphere of 5% CO2, 95% air, or
100% air at 37 °C. The colon adenocarcinoma cell lines LS174Tr,
expressing the tetracycline (Tet) repressor, were maintained in DMEM
supplementedwith 10%FCSandblasticidin(10μg/mL, Invitrogen). The
incubation in hypoxia at 1% O2 was carried out at 37 °C in 95% humidity
and 5% CO2/94% N2 in a sealed anaerobic workstation (Ruskinn).
Stable Transgenic Cells. PS120 cells were transfected with the
pca9 vector as previously described.4 LS174Tr cells were transfected
with Tet (10 μg/mL)-inducible short hairpin RNA targeting ca9 (shca9)
and transduced with lentiviral particles containing nontarget shRNA
(ctl) and shRNA-ca12 (ca12ꢀ) to constitutively silence ca12, as pre-
viously described.4 These cells are named, respectively, LS-shca9/ctl and
LS-shca9/ca12ꢀ cells.
Cell-Based Screening of CA IX Inhibitors. Fibroblasts expres-
sing CA IX (PS120-pca9 cells) and control fibroblasts (PS120-pev cells)
were seeded onto 12-well plates. Once attached, the medium was
replaced by HCO3ꢀ-free DMEM buffered at either pHo 6.4 (30 mM
MES) or pHo 7.5 (30 mM HEPES), supplemented with 10% dialyzed
serum, 0.1 mM hypoxanthine, and 0.1 mM uridine triphosphate for 24 h
of growth in a CO2/HCO3ꢀ-free atmosphere without (DMSO) or with
60 μM CA inhibitors. Plates were then returned to 5% CO2 in a regular
media for 7 days before staining with Giemsa (Fluka).
Proliferation Assay. Tetracycline (1 μg/mL) was added in the
medium of LS-shca9/ca12ꢀ cells 4 days before the assay. LS-shca9/ctl
ꢀTet and LS-shca9/ca12ꢀ +Tet cells (6 ꢁ 104) were seeded onto 6-well
plates in the absence or presence of Tet. Twenty-four hours after
seeding, cells were incubated in hypoxia (1% O2) in the presence or
in the absence of CA inhibitors. Every 24 h during 3 days, cells were
detached and counted with Coulter Z1 (Beckman). The proliferation
index was calculated by dividing the total cell number obtained each day
by the cell number obtained 24 h after seeding.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectra of com-
S
b
pounds 5ꢀ28. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +61 7 3735 7825. Fax: +61 7 3735 7656. E-mail: s.poulsen@
griffith.edu.au.
’ ACKNOWLEDGMENT
This research was financed in part by the Australian Research
Council (Grant Number DP110100071), by a grant of the 7th FP
of EU (Metoxia project), and Ligue Nationale Contre le Cancer
(JP, equipe labellisꢀee).
Immunoblotting. Cells were lysed in a SDS sample buffer.
Proteins (40 μg) were separated on 7.5% SDS polyacrylamide gels
and transferred onto polyvinylidene difluoride membranes (Millipore).
Membranes were blotted with the monoclonal M75 antibody to CA IX
(Bayer), a polyclonal antibody to recombinant CA XII (Sigma), a
polyclonal antibody HIF-1α prepared and validated in our laboratory
and Hsp90 (Abcam). Immunoreactive bands were detected with a
horseradish peroxidase (HRP) antimouse or antirabbit antibody
(Promega) by enhanced chemiluminescence (Amersham Biosciences).
In Cellulo Determination of CA Activity. Exponentially grow-
ing cells were scraped into ice-cold PBS to obtain intact cells expressing
membrane-bound CAs. The cell suspension was centrifuged and
resuspended in a HCO3ꢀ-free media (Sigma) buffered at pHo of 7.5
with 30 mM HEPES in the presence or absence of the AZA or
’ ABBREVIATIONS USED
CA, carbonic anhydrase; HIF, hypoxia-inducible transcrip-
tion factor; pHo, extracellular pH; pHi, intracellular pH; SARs,
structureꢀactivity relationships; SPRs, structureꢀproperty rela-
tionships; 1,3-DCR, 1,3-dipolar cycloaddition reaction; cLog P,
calculated Log P; ELSD, evaporative light scattering detection;
Tet, tetracycline
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