Sulfonamide linked neoglycoconjugates-A new class of inhibitors for cancer-associated carbonic anhydrases

Article abstract of DOI:10.1021/jm901888x

The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to optimize both SAR (structure-activity relationship) and SPR (structure-property relationship) characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors (K_is in the low nanomolar range) that target cancer-associated CAs.

Full text of DOI:10.1021/jm901888x

J. Med. Chem. 2010, 53, 2913–2926 2913
DOI: 10.1021/jm901888x
Sulfonamide Linked Neoglycoconjugates-A New Class of Inhibitors for Cancer-Associated
Carbonic Anhydrases
Marie Lopez,† Laurent F. Bornaghi,† Alessio Innocenti,§ Daniela Vullo,§ Susan A. Charman,‡
Claudiu T. Supuran,*,§ and Sally-Ann Poulsen*,†
†Eskitis Institute for Cell and Molecular Therapies, Griffith University, 170 Kessels Road, Nathan, Queensland 4111, Australia,
‡Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade,
§
Parkville, Victoria 3052, Australia, and Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Universitaꢀ degli Studi di Firenze,
Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
Received December 21, 2009
The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and
progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In
this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to
selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe
the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise
both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of
our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to
optimize both SAR (structure-activity relationship) and SPR (structure-property relationship)
characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors
(Kis in the low nanomolar range) that target cancer-associated CAs.
Introduction
Tumor cells also experience elevated metabolism and acid
production compared to normal cells.7,8 If this acid-load is
allowed to accumulate, then intracellular pH (pHi) would fall
to dangerously low levels and disrupt critical biological func-
tions.7,8 Tocounter this, tumor cells have evolved mechanisms
to extrude acid into the extracellular environment. Recent
evidence has demonstrated that it is cell-generated CO2 that is
primarily responsiblefor the rapid removal of acid equivalents
from tumor cells and not lactic acid, which has been the
popular belief until now.7,8 Cell-derived CO2 is freely mem-
brane permeable, so provided there is sufficient outward
gradient this CO2 diffuses to the extracellular space, where
in poorly vascularized hypoxic tumor regions, it is hydrated to
bicarbonate (HCO3-) and a proton (Hþ): CO2 þ H2O a
In solid tumors, vasculature is poor; this limits blood supply
to the tumor mass and in turn reduces delivery of O2 to
the tumor cells. At a molecular level, low O2, known as hypoxia
(ꢀ 0.1-5% O2), induces the activation of a transcription
factor, appropriately named hypoxia-inducible factor
(HIFa).1 High levels of HIF regulate a signaling cascade,
involving ∼hundred genes, that adapt cellular functions to
allow solid tumor cells to not only survive hypoxia but to
proliferate and metastasize.1 Carbonic anhydrase IX (CA IX) is
one of the most highly induced HIF responsive genes and is
overexpressed and sustained in a number of solid tumors
including breast, brain (glioblastoma), clear cell renal, color-
ectal, head and neck, bladder, and nonsmall cell lung
carcinomas.2-4 CA IX is a multidomain protein consisting of
an N-terminal proteoglycan like domain, a transmembrane
domain, a short intracellular domain and an extracellular
catalytic domain.5 The catalytic domain of CAs contains an
active site Zn2þ. This metal cation is a strong Lewis acid that
binds to and activates a substrate H2O molecule to catalyze the
-
HCO3 þ Hþ. The net effect is to trap acid extracellularly to
lower extracellular pH (pHe ∼6.9-7.0) and maintain pHi
-
(∼7.2) with HCO3 recycled back into the cell, where it
combineswitha proton to give CO2 (catalyzed by intracellular
CA II) to continue this cycle, Figure 1.8 The contribution of
CA IX (and CA XII, which is also HIF activated) to hypoxic
tumor growth and progression has long been debated, how-
ever the intensity of recent research has provided convergent
evidence that affirms that these transmembrane CAs, with
their extracellular oriented active sites, are the key molecules
for regulating cancer cell pHi during hypoxia and thus are
major tumor prosurvival enzymes.7,8 Very recently, it was
reported that “knockout” of CA IX and CA XII using gene
silencing technology led to a remarkable 85% tumor growth
retardation in colorectal cancer xenograft models;this re-
search has advanced our understanding of the pivotal role of
CAs in cancer and implicates cancer-associated CAs as a new
and promising drug target for oncology therapies.7 Of the 12
-
reversible reaction: CO2 þ H2O a HCO3 þ Hþ.6 The
hydration of CO2 does not proceed at an appreciable rate
under physiological conditions in the absence of CA enzymes.6
*To whom correspondence should be addressed. For S.-A.P.: phone,
þ61 7 3735 7825; fax, þ61 7 3735 6001; E-mail, s.poulsen@griffith.edu.
au. For C.T.S.: phone, þ39 055 457 3005; fax, þ39 055 457 3385; E-mail,
claudiu.supuran@unifi.it.
a Abbreviations: CA, carbonic anhydrase; AZA, acetazolamide;
TPM, topiramate; SAR, structure-activity relationship; SPR, struc-
ture-property relationship; HIF, hypoxia-inducible factor; PAMPA,
parallel artificial membrane permeability assay; Pe, apparent in vitro
effective permeability.
r
2010 American Chemical Society
Published on Web 03/04/2010
pubs.acs.org/jmc

2914 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
sulfonamide-linked glycoconjugate. We also present the CA
inhibition profile of our library, comprising 30 new com-
pounds 3-32, against the cancer-associated (hCA IX and XII)
and physiologically dominant (hCA I and II) CA isozymes
(h = human). We show that our approach to targeting cancer-
associated CAs is achieved with minimal SAR penalties.
In principle, anomeric sulfonyl chlorides through reaction
with myriad primary amines could serve as precursors to a
multitude of carbohydrate-based S-glycosyl sulfonamides.
However, it has been shown that anomeric sulfonyl chlorides
are unstable and rapidly convert to the corresponding anome-
ric chlorides by loss of SO2.18 This lack of stability eliminates
these compounds as precursors to sulfonamides, and as a
result, S-glycosyl primary sulfonamides have remained an
unknown class of compounds, quite remarkable given the
broad utility of the sulfonamide functional group across
medicinal chemistry.19 We recently developed a simple and
efficient synthesis for S-glycosyl primary sulfonamides, based
on oxidation of S-glycosyl sulfenamides and subsequent re-
moval of the acetate protecting group.20 The primary sulfon-
amide functional group -SO2NH2 of these compounds is
attached directly to the anomeric position of the carbohydrate.
In this study, we discovered that anomeric S-glycosyl sulfena-
mides could be synthesized by reaction of sugar thioacetates
with either primary (RNH2) or secondary amines (R2NH) to
give sulfenamides [sugar-S-NHR] and [sugar-SNR2], respec-
tively; this reaction had previously been reported as being only
suited with secondary amines.21 Herein we postulate that our
synthetic strategy could be readily adapted for the synthesis of
hitherto unknown S-glycosyl secondary sulfonamides [sugar-
SO2NH-R] to give a non-native glycoconjugate covalent
linkage with potential for enzymatic resistance. Interestingly,
we have found reported only a small number of monosacchar-
ide tertiary S-glycosyl sulfonamides [sugar-SO2NR2]18,22,23 in
addition to our own reported compounds.20 Herein we explore
this novel synthetic opportunity as an avenue to append
carbohydrate “tail” moieties to the well established aromatic
sulfonamide CA inhibitor pharmacophore to generate robust,
sulfonamide-linked, neoglycoconjugate CA inhibitors of the
type [sugar-SO2NH-aromatic-SO2NH2].
Per-O-acetylated sugar precursors derived from monosac-
charides D-glucose, D-galactose, and D-glucuronic acid methyl
ester, and disaccharides lactose and maltose, were reacted
with boron trifluoride diethyl etherate and thiourea to synthe-
size the corresponding glycosyl thioacetates a-e.24 The para-
substituted aminobenzyl and aminophenethyl sulfonamides 1
and 2 have a primary aliphaticamino functional groupintheir
structure and are known CAinhibitors. These primary amines
were each reacted with the sugar thioacetate panel a-e to give
10 per-O-acetylated sulfenamide glycoconjugates, 3-7 and
18-22 (Scheme 1).25 Oxidation of the sulfenamides with
excess mCPBA in CH2Cl2 gave per-O-acetylated glycosylsul-
fonamidyl benzenesulfonamides 8-12 and 23-27(Scheme1).
Oxidation was typically complete following 1 h of stirring at
room temperature as evidenced by TLC. The O-acetate
groups of the carbohydrate moiety were next removed using
Zemplen’s conditions26 to liberate the fully deprotected sugar
analogues 13-17 and 28-32 in a quantitative or nearly
quantitative yield (Scheme 1).
Figure 1. Schematic of CA IX mediated pHi regulation of a hy-
poxic tumor cell microenvironment. CA IX is a transmembrane
enzyme with an extracellular oriented active site domain.
catalytically active CAs known, only the membrane bound
CA IX and CA XII isozymes are associated with cancer.6
The CA-mediated pHi regulation of hypoxic tumor cells
underpins a compelling case for the development of small-
molecule CA inhibitors as chemical probes and as lead
molecules to appraise the potential of cancer-associated
CA IX and XII for therapeutic intervention. In the past
few years, our group has established a novel approach to
targeting CA IX and XII with small molecule inhibitors.9-15
Our inhibitors comprise a carbohydrate “tail” moiety teth-
ered to the well characterized, high-affinity aromatic sulfon-
amide CA pharmacophore [aromatic-SO2NH2], leading to
glycoconjugates with the triple motif [carbohydrate-aro-
matic-SO2NH2]. The -SO2NH2 group is a Zn2þ binding
function that anchors the small molecule inhibitor molecule
within the CA enzyme active site.6 We and others have
shown that the CA active site is tolerant to diverse structural
characteristics within the tail moiety of inhibitors such as
size, shape and charge, polar and hydrophobic groups, etc.6,9-15
This structural tolerance permits a flexible ligand design
approach to allow finetuning of biopharmaceutical and tox-
icological properties of the inhibitor through structural ma-
nipulation. In our experience, and consistent with this
premise, the tail group of the CA inhibitor can impact the
compound’s physicochemical properties, i.e. structure-property
relationships (SPR), more so than structure-activityrelation-
ships (SAR). Recently, we demonstrated that a selection of
our glycoconjugates was unable to passively diffuse through
an artificial lipid membrane barrier (a mimic of the cell
membrane); this observation was consistent with calculated
Log P values.10 The results confirmed that selectively targeting
extracellular CA active sites over intracellular CAs is possible
by altering membrane permeability properties through delib-
erate structural changes of the inhibitor compounds. This
combined SAR-SPR characteristics of our general inhibitor
motif thus offers excellent potential for the development of
isozyme selective inhibitors of cancer-associated CAs in vivo.
Results and Discussion
Artificial glycoconjugates already constitute a diverse family
of clinically used therapeutics, including small molecule anti-
bacterial glycoconjugates.16 The continuing need for new
therapeutic neoglycoconjugates requires the development of
straightforward synthetic methodology toward stable, novel
glycoconjugate linkers in place of native, enzyme labile cova-
lent linkages.17 Here we outline the targeting of transmem-
brane CAs over cytosolic CAs by incorporation of a
carbohydrate tail group into the CA inhibitor structure in
accord with our combined SPR-SAR strategy. We describe
the application of novel chemistry toward this new class of
Carbonic Anhydrase Inhibition
The enzyme inhibition characteristics for the 30 new glycocon-
jugates 3-32 were determined by assaying the CA catalyzed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2915
Scheme 1. Synthesis of S-Glycosyl Sulfenamides (3-7, 18-22) and S-Glycosyl Sulfonamides (8-17, 23-32) from Glycosyl
Thioacetates (a-e) and Amino Sulfonamide Scaffolds (1 and 2)a
a Glycosyl thioacetates a-e are employed to incorporate a carbohydrate “tail” moiety to give novel sulfonamide-linked neoglycoconjugate CA
inhibitors.
hydration of CO2, Table 1.27 Isozyme selectivity ratios for
3-32 are in Table 2. Reference data for clinically used CA
inhibitors acetazolamide (AZA) and topiramate (TPM) as
well as for aminosulfonamide precursors 1 and 2 are included
for comparison with the new glycoconjugates reported in this
study.
Off-Target CA Isozymes. Fourteen of the 20 glycoconju-
gates with O-acetate protected carbohydrate tail moieties are
low micromolar inhibitors of hCA I; the exceptions are
compounds 4, 5, 8, 9, 20, and 23, which displayed slightly
stronger inhibition with Kis of 90-120 nM. The SAR of
the acetylated compounds were independent of the linker
type (sulfenamide compared to sulfonamide) or the parent
compound (1 or 2). The 10 deprotected sugar analogues
(13-17 and 28-32) all had closely grouped hCA I inhibition
constants that ranged from 81 to 107 nM, which is similar to
the stronger inhibitors of the acetylated series. The incor-
poration of the five different sugar tails onto the benzene
sulfonamide scaffolds 1 and 2 via a sulfenamide or a sulfon-
amide revealed some notable SAR trends at hCA II. All
10 deprotected compounds (13-17 and 28-32) exhibited
potent hCA II inhibition (Kis < 10 nM). All acetylated
compounds were slightly less potent (with Kis > 10 nM).
The acetylated sulfenamides (3-7 and 18-22) exhibited
a narrow range of Kis, from 12.0 to 18.1 nM, while the
acetylated sulfonamides had Kis in two distinct groupings:
14.8-16.3 nM for 8, 9, 23, and 27, and 56.2-73.8 nM
for compounds 10-12, 24-26. These two groupings are
again spread across both scaffolds (1 and 2) and across sugar
types (a-e).

2916 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
Table 1. Inhibition Data for New Sulfonamide-Linked Glycoconjugates 3-32, Precursors 1 and 2, and the Established Drugs and CA Inhibitors
AZA and TPM against Human Isozymes hCA I, II, IX, and XIIa
Ki (nM)c
compd
Log Pb
Pe (10-6 cm s-1
)
hCA Id
hCA IIe
hCA IXe
hCA XIIe
AZA
TPM
1
-1.0
<0.15g
nd
nd
250
250
12
5
25
58
5.7
3.8
0.3
3.2
11.5
15.5
15.6
9.8
8.5
9.3
9.9
9.8
10.4
9.1
8.3
8.7
8.9
8.9
8.2
13.6
16.7
9.5
8.7
8.8
9.5
9.4
9.7
9.5
8.8
7.6
7.8
9.5
9.1
7.3
þ0.04
-0.73f
-0.47f
-0.09
-0.09
-0.07
-0.54
-0.54
-1.40
-1.40
-1.38
-1.85
-1.85
-3.17
-3.17
-2.85
-4.94
-4.94
þ0.20
þ0.20
þ0.22
-0.25
-0.25
-1.11
-1.11
-1.15
-1.56
-1.56
-2.88
-2.88
-2.56
-4.65
-4.65
25000
21000
4160
120
170
160
14.8
13.4
18.1
15.5
13.1
14.8
16.3
65.8
58.8
62.5
9.1
103
33
2
nd
nd
3
105
91
4
nd
nd
5
110
102
100
84
6
nd
nd
<0.05 g
nd
nd
6830
3460
120
7
8
79
89
9
110
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2400
4930
2040
102
89
nd
nd
<0.05 g
nd
nd
102
88
95
99
87
81
7.8
7.1
97
nd
nd
107
101
5.0
4.8
106
98
nd
nd
3890
4100
110
14.5
13.3
16.2
12.0
15.1
15.4
73.8
62.7
56.2
15.2
7.8
97
107
90
nd
nd
8040
6750
90
97
94
nd
nd
95
96
nd
nd
4710
3210
3460
5570
88
95
97
nd
nd
93
84
nd
nd
82
97
7.6
4.7
100
96
nd
nd
78
102
4.6
5.2
100
81
nd
a The calculated value for compound Log P is also included as well as Pe for compounds AZA, 8 and 13. b Log P data calculated using InstantJChem
3.0.4 from ChemAxon.28 c Errors in the range of (5-10% of the reported value, from three determinations. d Human (cloned) isozymes. e Catalytic
domain of human (cloned) isozymes. fCalculated for neutral compound. g The concentration of test compound in the acceptor chamber was lower than
the LLQ. Upper limits for the Pe values were estimated using the respective analytical LLQ values as the maximum concentration in the acceptor
chamber. nd not determined.
Cancer-Associated CA Isozymes. The 30 glycoconjugates
were good hCA IX inhibitors and exhibited a clustered
inhibition profile at hCA IX (Kis 79-106 nM). This inhibi-
tion is however ∼10-fold weaker inhibition than for hCA II
(or XII; see below) and was either similar to, or selective over,
inhibition compared to the hCA I isozyme. All glycoconju-
gates were very good inhibitors of hCA XII; the 20 sulfon-
amide-linked glycoconjugates had Kis e 10 nM, while the 10
sulfenamide compounds had Kis that ranged from 8.5 to
16.7 nM. Many compounds (3, 6, 7, 10-12, 18, 19, 21, 22,
24-27) had quite notable (several hundred-fold) selectivity
compared to the physiologically abundant hCA I isozyme,
with compound 21 3 orders of magnitude (924-fold) hCA
XII selective. Although not as striking in magnitude, several
of the compounds also had some selectivity compared for
hCA II (compounds 10-12, 24-26).
Comparison with Other Glycoconjugate CA Inhibitors. We
have previously observed that the CA active sites are tolerant
to diverse structural characteristics within the tail moiety of
inhibitors.9-15 Table 3 presents data for a selection of
glucoconjugate CA inhibitors wherein the glucosyl moiety
is either a free sugar (33a-37a) or a peracetylated sugar
(33b-37b) (Figure 2). These inhibitors differ by the nature of
the linkage between the sugar and the sulfonamide zinc
binding function and have been selected for comparison to
glucosides 8 and 13 of the present study. The inhibitor
structures encompass 1,2,3-triazoles with O-glucoside (33a,
33b), sulfonyl glucoside (34a, 34b), amide (35a, 35b), and
ester (36a, 36b) covalent linkages, as well as anomeric
S-glucosyl sulfonamides (37a, 37b) in which the sulfonamide
and sugar are directly linked. With the exception of the
S-glucosyl sulfonamides (37a, 37b) that are micromolar inhi-
bitors for CA I, II, IX, and XII, the glucoconjugates exhibit
similarities in their isozyme inhibition profiles. The gluco-
conjugates are typically weak CA I inhibitors (micromolar Kis)
and good CA II and XII inhibitors (low nanomolar Kis).
We do however observe variation at CA IX, with the Kis for
this selection of inhibitors ranging from low nM to micro-
molar. The observed trends demonstrate that the com-
pounds of this study are consistent with our premise
regarding the active site tolerance of CAs to variable inhi-
bitor structures, although interestingly have allowed us
to identify that the cancer-associated CA IX isozyme is
the least tolerant to structural changes. This observation
should prove a useful aid in defining the opportunities for
future medicinal chemistry efforts to selectively target this

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2917
Table 2. Cancer-Associated CA Isozyme Selectivity Ratio Data for New Sulfonamide-Linked Glycoconjugates 3-32, Precursors 1 and 2, and the
Established Drugs and CA Inhibitors Acetazolamide (AZA) and Topiramate (TPM)
selectivity ratiosa
compd
hCA I/ hCA IX
hCA I/ hCA XII
hCA II/ hCA IX
hCA II/ hCA XII
AZA
TPM
1
10
4.3
43.9
65.8
0.48
0.09
1.7
2.1
1.3
243
636
39.6
1.3
83330
6563
361.7
7.7
566.7
50.0
1.3
2
4.9
3
0.14
0.15
0.18
0.16
0.16
0.19
0.18
0.74
0.58
0.71
0.10
0.08
0.07
0.05
0.05
0.15
0.12
0.18
0.12
0.16
0.16
0.77
0.66
0.58
0.16
0.09
0.08
0.05
0.05
0.06
4
0.86
1.2
5
1.1
7.1
6
68.3
41.2
1.5
696.9
407.1
12.9
1.6
1.5
7
8
1.6
1.7
9
1.2
11.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
27.0
48.3
23.2
1.1
244.9
474.0
224.2
12.3
6.7
5.7
6.9
1.1
0.9
0.8
10.0
9.1
0.90
0.80
0.56
0.59
1.1
1.0
1.0
12.0
12.3
40.1
38.3
1.2
286.0
245.5
11.6
0.80
1.7
82.9
71.8
0.95
49.1
33.8
35.7
59.9
1.1
924.1
767.1
9.5
1.4
1.7
1.6
7.9
501.1
330.9
364.2
633.0
11.6
6.5
5.9
1.7
1.0
0.8
1.0
10.5
10.2
0.97
0.49
0.51
0.71
0.8
1.3
8.6
14.0
a The Ki ratios are indicative of isozyme selectivity in vitro and are calculated using the Ki values in Table 1.
Table 3. Inhibition Data for hCA Isozymes I, II, IX and XII with
Compounds 8 and 13 of the Present Study and with a Selection of
Previously Reported Glucoconjugate CA Inhibitors (33a-37a, 33b-
37b)
in the development of compounds that target hCA IX and
XII in vivo, the passive diffusion across biological membrane
barriers is likely to be central. In general, passive diffusion
across a biological membrane correlates very well with the
lipophilicity of the small molecule, with membrane perme-
ability generally decreasing with increasing small molecule
polarity or with capacity for hydrogen bonding. Our strategy
to target the extracellular catalytic domain of cancer-asso-
ciated CAs is to deliberately manipulate physicochemical
properties of the inhibitor so as to impart membrane im-
permeability. Log P represents intrinsic lipophilicity, and
compounds with Log P<0 typically have good solubility
but poor lipid bilayer permeability (although paracellular
permeability may be a contributor for very small hydrophilic
molecules).29,30a Table 1 includes calculated Log P values for
the glycoconjugates of this study. These calculated values are
a very useful guide in the prediction of passive membrane
permeability, with Log P prediction software one of the most
reliable in silico tools for property profiling.30b Calculated
Log P values of compounds generated from 1 and 2 differ in
magnitude by 0.29, with scaffold 2 more hydrophobic
(higher Log P) than 1 owing to one additional methylene
group. The Log P trend is acetylated sulfenamides >
acetylated sulfonamides > free sugar sulfonamides. This is
consistent with (i) the incorporated acetate groups increasing
the lipophilicity and (ii) the sulfonamide oxygens increasing
Ki (nM)a
compd
hCA Ib
hCA IIb
hCA IXc
hCA XIIc
8
13
120
102
14.8
9.1
79
95
9.3
8.3
33a
33b
34a
34b
35a
35b
36a
36b
37a
37b
7.0
8.7
101
107
9.5
nd
nd
1500
101
46
7.6
6.9
10.3
9.1
97
9.3
2000
5600
4400
2300
3900
4530
8.2
384
7.0
442
430
183
1238
4050
3910
11.4
4.3
7.1
7.7
119
4910
4500
4690
4660
a Errors in the range of (5-10% of the reported value, from three
determinations. b Human (cloned) isozymes. c Catalytic domain of hu-
man (cloned) isozymes. nd not determined.
important tumor-associated CA isozyme with drug-like
small molecule inhibitors.
Passive Membrane Permeability. The physical barrier of
the cell membrane is not a contributing factor in CA inhibi-
tion studies that utilize pure recombinant enzymes, however,

2918 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
Figure 2. Structures of previously reported glucoconjugate CA inhibitors (33a-37a R = H, 33b-37b R = Ac).
the polarity of the glycoconjugates. The calculated Log P
values for the acetylated sulfonamides range from -1.11 to
-1.85, while the more hydrophilic free sugar sulfonamides
have log P values that range from -2.56 to -4.94. All
sulfonamides have Log P values that fall within the range
indicative of molecules with poor membrane permeability, i.e.,
Log P < 0.
with the exception of methyl 1,2,3,4-tetra-O-acetyl-β-D-gluco-
pyranuronate, which was synthesized as described in the litera-
ture.36 Solvents were dried prior to use, or purchased anhydrous
from Sigma-Aldrich. All reactions were monitored by TLC
using Merck F60254 silica plates with visualization of product
bands by UV fluorescence (λ = 254 nm) and charring by
sulphuric 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 performed
using silica gel 60 A (230-400 mesh). NMR (1H, 13C {1H},
gCOSY, and HSQC) spectra were recorded on a Varian 500
MHz spectrometer at room temperature using DMSO-d6
solvent unless otherwise stated. Chemical shifts are reported in
δ (ppm). Chemical shifts for 1H and 13C NMR run in DMSO are
reported in ppm relative to residual solvent proton (δ = 2.50
ppm) and carbon (δ = 39.5 ppm) signals, respectively. For 1H
NMR run in D2O chemical shifts are reported in ppm relative to
the solvent residual peak: proton (δ = 4.80 ppm). Multiplicity
is indicated as follows: s (singlet), d (doublet), t (triplet), m
(multiplet), dd (doublet of doublet), ddd (doublet of doublet
of doublet), br s (broad singlet). Coupling constants are re-
ported in hertz (Hz). Melting points were acquired on a Gallen-
kamp melting point apparatus and are reported as uncorrected.
Low resolution mass spectra were acquired in positive ion
mode on an Applied Biosystems Pty Ltd. Mariner ESI-TOF
mass spectrometer. High resolution mass spectra were per-
formed in positive ion mode on an Apex III Bruker Daltonics
4.7T Fourier transform mass spectrometer (FTMS) fitted with
an Apollo electrospray ionization source. All MS analysis
samples were prepared as solutions in methanol. Optical rota-
tion were measured at 25 °C and reported as an average of
10 measurements. The purities of isolated products were deter-
mined by HPLC obtained on an LCMS instrument (MS-ZQ
Waters; HPLC-Alliance Waters) using a HPLC column
(Ascentis, C18 3 μm, 5 cm ꢁ 4.6 mm). Elution was performed
with a gradient of water:methanol (containing 1% formic acid)
from 95:5 to 0:100 for 10 min at a flow rate of 1 mL/min. UV
(200-400 nm) and evaporative light scattering detection
(ELSD-Alltech) detection were used. Purity of all compounds
proved to be g95%.
Permeability measurements through artificial lipid mem-
brane biolayers have been extensively used to classify small
molecules for their passive membrane permeability char-
acteristics.31-33 We have previously used this technique to
measure the apparent in vitro effective permeability (Pe) of
anomeric sulfonamides.10 Typical Pe values for high perme-
ability compounds are >3 ꢁ 10-6 cm s-1, while for low
permeability compounds are <3 ꢁ 10-6 cm s-1 34,35
.
The Pe
of per-O-acetylated glucoconjugate (8), fully deprotected
glucoconjugate (13), AZA, and control compounds were
determined using a parallel artificial membrane permeability
assay (PAMPA)31-33 as previously described.10 The experi-
mental values of Pe at pH 7.4 using the PAMPA assay are
included in Table 1. For the compounds tested (AZA, 8
and 13), the concentrations in the acceptor chamber were
below the analytical lower limits of quantitation (LLQ),
suggesting that these compounds have very low permeability
in this assay. A theoretical estimate of the maximum Pe value
for each compound was calculated using the respective LLQ
values, Table 1. The PAMPA results for these representative
neoglycoconjugates (that include an example of a sugar-
OAc and sugar-OH moiety) confirm that this class of
compound would be expected to have poor passive mem-
brane permeability and thus may preferentially inhibit the
transmembrane CAs IX and XII over cytosolic CAs in the
in vivo environment.
To ensure that the intrinsically low permeability charac-
teristics of the CA inhibitors is unlikely to limit bioavail-
ability for compounds that may progress to animal studies
we envisage either intravenous administration (the free
sugars have good aqueous solubility) or for oral delivery
a prodrug strategy that utilizes ester groups on the sugar
hydroxyls to improve intestinal absorption, with ester
hydrolysis to follow absorption.
General Procedure 1. To a solution of per-O-acetylated
derivative (1.0 equiv) in acetonitrile was added thiourea (1.3
equiv) and boron trifluoride diethyl etherate (2.1 equiv). The
reaction mixture was refluxed until starting material was
consumed (∼30 min) then cooled to room temperature (rt).
Acetic anhydride (2.5 equiv) and triethylamine (4.5 equiv) were
added and the solution stirred at rt overnight in the absence of
light. The reaction mixture was concentrated and the residue
diluted in dichloromethane (CH2Cl2) before washing with
Experimental Section
Chemistry. All starting materials and reagents, including per-
O-acetylated sugars, were purchased from commercial suppliers

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2919
aqueous 5% HCl (ꢁ1) and brine (ꢁ2). The aqueous extracts
were combined and back extracted with CH2Cl2 (ꢁ2). The
organic extracts were combined, dried over MgSO4, filtered,
and evaporated.
Synthesis of Sulfenamides 3-7 and 18-22: General Procedure 2.
A solution of thioacetate (a-e, 1.0 equiv) under nitrogen was
prepared in anhydrous methanol. Diethylbromomalonate (2.5
equiv) was added and the reaction was maintained under stirring
for 20 min at rt, after which the benzene sulfonamide derivative
was added (3.0 equiv), either with no other reactant in the case of
4-aminoethylbenzenesulfonamide 2 or with diisopropylethyla-
mine (3.0 equiv) when 4-aminomethylbenzenesulfonamide hy-
drochloride (hydrochloride salt of 1) is used. The mixture was
stirred at rt under nitrogen until completion, typically 1.5 h. The
methanol was evaporated and the residue solubilized in CH2Cl2
and washed with brine (ꢁ3). Each aqueous phase was back
extracted with CH2Cl2 (ꢁ2), the organic phases were combined,
dried over MgSO4, filtered, concentrated, and purified.
2,3,4,6-Tetra-O-acetyl-1-S-acetyl-1-thio-β-D-glucopyranose (a).
Thioacetate a was prepared from per-O-acetylated D-glucose
using General Procedure 1. Recrystallization from ethanol
afforded off-white crystals (77% yield); Rf = 0.67 (1:1 EtOAc/
hexane); mp = 105-108 °C; [R]25D = þ9 (c = 1.0, chloroform).
1H NMR (500 MHz, CDCl3): δ = 5.28 (t, J = 9.5 Hz, 1H, H-3),
5.26 (d, J = 10.0 Hz, 1H, H-1), 5.13 (t, J = 9.5 Hz, 1H, H-2),
5.11 (t, J = 9.5 Hz, 1H, H-4), 4.27 (dd, J = 12.5, 4.5 Hz, 1H,
H-6a), 4.11 (dd, J = 12.5, 2.0 Hz, 1H, H-6b), 3.84 (ddd, J =
10.0, 4.5, 2.0 Hz, 1H, H-5), 2.39 (s, 3H, SCOCH3), 2.08, 2.04,
2.03, 2.01 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed
by 1H-1H gCOSY. LRMS (ESIþ): m/z = 429 [M þ Na]þ.
2,3,4,6-Tetra-O-acetyl-1-S-acetyl-1-thio-β-D-galactopyranose
(b). Thioacetate b was prepared from per-O-acetylated D-galac-
tose using General Procedure 1. Recrystallization from ethanol
afforded off-white crystals (75% yield); Rf = 0.67 (1:1 EtOAc/
hexane); mp = 99-103 °C; [R]25D = þ29 (c = 1.0, chloroform).
1H NMR (500 MHz, CDCl3): δ = 5.46 (d, J = 3.5 Hz, 1H, H-4),
5.32 (t, J = 10.0 Hz, 1H, H-2), 5.25 (d, J = 10.5 Hz, 1H, H-1),
5.12 (dd, J = 3.5, 10.0 Hz, 1H, H-3), 4.15-4.05 (m, 3H, H-5, H-
6a, H-6b), 2.40 (s, 3H, SCOCH3), 2.16, 2.04, 2.03, 1.99 (4 ꢁ s,
12H, OCOCH3), assignments were confirmed by 1H-1H
gCOSY. LRMS (ESIþ): m/z = 429 [M þ Na]þ.
N-4-(Aminosulfonyl)benzyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-β-
D-glucopyranosyl)sulfenamide (3). Sulfenamide 3 was prepared
from 4-aminomethylbenzenesulfonamide hydrochloride and
thioacetate derivative a according to General Procedure 2. The
expected compound was obtained after flash chromatography
(1:1 EtOAc/hexane) as a slightly yellow solid (94% yield); Rf =
0.27 (1:1 EtOAc/hexane); mp = 122-129 °C; [R]25 = -117
D
1
(c = 1.2, chloroform). H NMR (500 MHz, DMSO-d6): δ =
7.77 (d, J = 8.0 Hz, 2H, Harom), 7.45 (d, J = 8.0 Hz, 2H, Harom),
7.28 (s, 2H, NH2), 5.34 (d, J = 9.5 Hz, 1H, H-3), 4.97 (t, J = 10.0
Hz, 1H, H-2), 4.93 (t, J = 9.5 Hz, 1H, H-4), 4.76 (d, J = 10.0 Hz,
1H, H-1), 4.44 (t, J = 10.5 Hz, 1H, NH), 4.16 (m, 1H, H-6a), 4.11
(m, 1H, H-6b), 4.07 (m, 2H, CH2), 4.02 (m, 1H, H-5), 2.01, 2.00,
1.99, 1.95 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed
by 1H-1H gCOSY. 13C NMR (125MHz, DMSO-d6): δ = 170.0,
169.5, 169.3, 169.2 (OCOCH3), 144.3, 142.6 (Carom), 128.3, 125.4
(CHarom), 87.4 (C-1), 74.3 (C-5), 73.2 (C-3), 68.1 (C-4), 67.5 (C-
2), 62.0 (C-6), 55.8 (CH2), 20.5, 20.4, 20.3, 20.2 (OCOCH3),
assignments were confirmed by 1H-13C HSQC. LRMS (ESIþ):
m/z = 549 [M þ H]þ; 571 [M þ Na]þ. LRMS (ESI-): m/z = 547
[M - H]-, 583 [M þ Cl]-. HRMS: calcd for C21H28N2O11S2Na,
571.1027; found, 571.1039.
Methyl 2,3,4-Tetra-O-acetyl-1-S-acetyl-1-thio-β-D-glucopyr-
anuronate (c). Thioacetate c was prepared from methyl 1,2,3,4-
tetra-O-acetyl-β-D-glucopyranuronate using General Procedure 1.
Recrystallization from methanol afforded off-white crystals
(47% yield); Rf = 0.57 (1:1 EtOAc/hexane); mp = 149-150 °C;
[R]25 = þ19 (c = 1.1, chloroform). 1H NMR (500 MHz,
D
CDCl3): δ = 5.34 (t, J = 9.5 Hz, 1H, H-3), 5.30 (t, J = 10.0 Hz,
1H, H-1), 5.20 (d, J = 9.5 Hz, 1H, H-4), 5.15 (t, J = 10.0 Hz, 1H,
H-2), 4.17 (d, J = 10.0 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.39 (s,
3H, SCOCH3), 2.04, 2.03 (2 ꢁ s, 12H, OCOCH3), assignments
were confirmed by 1H-1H gCOSY. LRMS (ESIþ): m/z = 415
[M þ Na]þ.
N-4-(Aminosulfonyl)benzyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-β-
D-galactopyranosyl)sulfenamide (4). Sulfenamide 4 was prepared
from 4-aminomethylbenzenesulfonamide hydrochloride and
thioacetate derivative b according to the General Procedure 2.
The expected compound was obtained after flash chromatog-
raphy (1:1 EtOAc/hexane) as a white, highly hygroscopic solid
(58% yield); Rf = 0.22 (1:1 EtOAc/hexane); [R]25D = -82 (c =
1.2, chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 7.77 (d,
J = 8.0 Hz, 2H, Harom), 7.60 (d, J = 8.5 Hz, 2H, Harom), 7.29 (s,
2H, NH2), 5.32 (d, J = 3.5 Hz, 1H, H-4), 5.26 (dd, J = 3.5,
10.0 Hz, 1H, H-3), 5.07 (t, J = 10.0 Hz, 1H, H-2), 4.75 (d, J =
9.5 Hz, 1H, H-1), 4.45 (t, J = 6.0 Hz, 1H, NH), 4.24 (m, 1H, H-
5), 4.11 (m, 2H, CH2), 4.06-4.04 (m, 2H, H-6a, H-6b), 2.13,
2.01, 2.00, 1.93 (4 ꢁ s, 12H, OCOCH3), assignments were
2,20,3,30,40,6,60-Hepta-O-acetyl-1-S-acetyl-1-thio-β-lactose (d).
Thioacetate d was prepared from per-O-acetylated lactose using
General Procedure 1. Purification by flash chromatography (1:1
EtOAc/hexane) afforded a colorless oil (79% yield); Rf = 0.25
1
(1:1 EtOAc/hexane); [R]25 = -3 (c = 1.3, chloroform). H
D
NMR (500 Mz, CDCl3): δ = 5.36 (d, J = 3.0 Hz, 1H, H-40), 5.26
(t, J = 9.0 Hz, 1H, H-3), 5.22 (d, J = 10.5 Hz, 1H, H-1), 5.12
(dd, J = 8.0, 10.5 Hz, 1H, H-20), 5.05 (t, J = 10.5 Hz, 1H, H-2),
4.95 (dd, J = 3.0, 10.5 Hz, 1H, H-30), 4.46 (d, J = 8.0 Hz, 1H,
H-10), 4.45 (dd, J = 2.0, 10.0 Hz, 1H, H-6a0), 4.15-4.07 (m, 3H,
H-6a, H-6b0, H-50), 3.87 (m, 1H, H-6b), 3.83 (t, J = 9.0 Hz, 1H,
H-4), 3.76 (m, 1H, H-5), 2.38 (s, 3H, SCOCH3), 2.16, 2.12, 2.08,
2.05, 2.05, 2.03, 1.97 (7 ꢁ s, 21H, OCOCH3), assignments were
confirmed by 1H-1H COSY. LRMS (ESIþ): m/z = 713 [M þ
NH4]þ, 717 [M þ Na]þ.
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 169.9 (2C), 169.6, 169.4 (OCOCH3), 144.2, 142.7
(Carom), 128.3, 125.5 (CHarom), 88.2 (C-1), 73.3 (C-5), 71.1 (C-
3), 67.6 (C-4), 65.1 (C-2), 61.7 (C-6), 55.8 (CH2), 20.5 (2C), 20.4,
20.3 (OCOCH3), assignments were confirmed by 1H-13C
HSQC. LRMS (ESIþ): m/z = 571 [M þ Na]þ. LRMS (ESI-):
m/z = 547 [M - H]-, 583 [M þ Cl]-. HRMS: calcd for C21H29-
N2O11S2, 549.1207; found, 549.1234.
2,20,3,30,40,6,60-Hepta-O-acetyl-1-S-acetyl-1-thio-β-maltose (e).
The title compound was prepared from per-O-acetylated mal-
tose using General Procedure 1. Recrystallization from metha-
nol afforded off-white crystals (68% yield); Rf = 0.48 (1:1
EtOAc/hexane); mp = 139-141 °C; [R]25 = þ57 (c = 1.0,
Methyl N-4-(Aminosulfonyl)benzyl-S-(2,3,4-tri-O-acetyl-1-
thio-β-D-glucopyranuronoyl)sulfenamide (5). Sulfenamide 5 was
prepared from 4-aminomethylbenzenesulfonamide hydrochlor-
ide and thioacetate derivative c according to the General
Procedure 2. The expected compound was obtained after flash
chromatography (3:2 EtOAc/hexane) as a white solid (88%
yield); Rf = 0.40 (2:1 EtOAc/hexane); mp = 152-156 °C;
[R]25D = -109 (c = 1.1, chloroform). 1H NMR (500 MHz, DM-
SO-d6): δ = 7.75 (d, J = 8.0 Hz, 2H, Harom), 7.45 (d, J = 8.0 Hz,
2H, Harom), 7.28 (s, 2H, NH2), 5.40 (t, J = 9.5 Hz, 1H, H-3), 4.98
(t, J = 9.5 Hz, 1H, H-2), 4.96 (t, J = 9.5 Hz, 1H, H-4), 4.83
(d, J = 10.0 Hz, 1H, H-1), 4.50 (t, J = 5.0 Hz, 1H, NH), 4.45 (d,
D
chloroform). 1H NMR (500 Mz, CDCl3): δ = 5.40 (d, J = 3.5 Hz,
1H, H-10), 5.36 (t, J = 10.0 Hz, 1H, H-30), 5.33 (t, J = 9.0 Hz,
1H, H-3), 5.29 (d, J = 10.5 Hz, 1H, H-1), 5.06 (t, J = 10.0 Hz,
1H, H-40), 4.99 (t, J = 9.5 Hz, 1H, H-2), 4.87 (dd, J = 4.0, 10.5
Hz, 1H, H-20), 4.45 (dd, J = 2.0, 12.5 Hz, 1H, H-6a), 4.24 (dd,
J = 4.5, 12.5 Hz, 1H, H-6b), 4.21 (dd, J = 4.5, 12.5 Hz, 1H, H-
6a0), 4.04 (dd, J = 2.0, 12.5 Hz, 1H, H-6b0), 4.00 (t, J = 9.5 Hz,
1H, H-4), 3.95(m, 1H, H-50), 3.83 (m, 1H, H-5), 2.38 (s, 3H,
SCOCH3), 2.13, 2.10, 2.06, 2.03, 2.01, 2.00 (6 ꢁ s, 21H,
OCOCH3), assignments were confirmed by 1H-1H COSY.
LRMS (ESIþ): m/z = 713 [M þ NH4]þ, 717 [M þ Na]þ.

2920 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
J = 10.0 Hz, 1H, H-5), 4.08 (m, 2H, CH2), 3.65 (s, 3H, OCH3),
1.98, 1.97, 1.96 (3 ꢁ s, 9H, OCOCH3), assignments were
NH2), 5.34 (d, J = 9.5 Hz, 1H, H-3), 4.92 (t, J = 9.5 Hz, 1H,
H-2), 4.89 (t, J = 9.5 Hz, 1H, H-4), 4.71 (d, J = 10.0 Hz, 1H, H-1),
4.12 (dd, J = 5.0, 12.0 Hz, 1H, H-6a), 4.05 (m, 1H, H-6b),
4.01(m, 1H, H-5), 3.92 (t, J = 10.0 Hz, 1H, NH), 3.09 (m, 2H,
NHCH2), 2.82 (t, J = 7.0 Hz, NHCH2CH2), 2.01, 1.99, 1.95,
1.89 (4 ꢁ s, 12H, OCOCH3), assignments were confirmed by
1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 169.9,
169.5, 169.3, 169.2 (OCOCH3), 144.0, 141.8 (Carom), 129.0,
125.6 (CHarom), 87.4 (C-1), 74.2 (C-5), 73.2 (C-3), 68.0 (C-4),
67.5 (C-2), 61.9 (C-6), 54.0 (NHCH2), 35.7 (NHCH2CH2), 20.4,
20.3 (2C), 20.2, (OCOCH3), assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 563 [M þ H]þ, 585 [M þ
Na]þ. LRMS (ESI-): m/z = 561 [M - H]-. HRMS: calcd for
C22H31N2O11S2, 563.1364; found, 563.1392.
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 169.7, 169.5, 169.5 (OCOCH3), 144.3, 142.7 (Carom),
128.6, 125.7 (CHarom), 87.5 (C-1), 74.5 (C-5), 72.5 (C-3), 69.3 (C-
4), 67.4 (C-2), 55.8 (CH2), 52.7 (OCH3), 20.5, 20.4, 20.3
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 535 [M þ H]þ. LRMS (ESI-): m/z = 533
[M - H]-. HRMS: calcd for C20H27N2O11S2, 535.1051; found,
535.1061.
N-4-(Aminosulfonyl)benzyl-S-(2,20,3,30,40,6,60-hepta-O-acet-
yl-1-thio-β-maltosyl)sulfenamide (6). Sulfenamide 6 was pre-
pared from 4-aminomethylbenzenesulfonamide hydrochloride
and thioacetate derivative d according to the General Procedure 2.
The expected compound was obtained after flash chromatography
(2:1 EtOAc/hexane) as a white solid (93% yield); Rf = 0.32 (2:1
N-4-(Aminosulfonyl)phenethyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-
β-D-galactopyranosyl)sulfenamide (19). Sulfenamide 19 was pre-
pared from 4-aminoethylbenzenesulfonamide and thioacetate
derivative b according to the General Procedure 2. The expected
compound was obtained after flash chromatography (1:1
EtOAc/hexane) as a yellow gum (46% yield); Rf = 0.33 (1:1
EtOAc/hexane); [R]25D = -82 (c = 1.1, chloroform). 1H NMR
(500 MHz, DMSO-d6): δ = 7.72 (d, J = 8.0 Hz, 2H, Harom), 7.38
(d, J = 8.5 Hz, 2H, Harom), 7.25 (s, 2H, NH2), 5.30 (d, J = 3.0
Hz, 1H, H-4), 5.26 (dd, J = 3.5, 10.0 Hz, 1H, H-3), 5.03 (t, J =
10.0 Hz, 1H, H-2), 4.69 (d, J = 10.0 Hz, 1H, H-1), 4.23 (m, 1H,
H-5), 4.02 (m, 2H, H-6a, H-6b), 3.96 (t, J = 5.0 Hz, 1H, NH),
3.12 (m, 2H, NHCH2), 2.86 (t, J = 7.0 Hz, NHCH2CH2), 2.07,
2.04, 1.99, 1.92 (4 ꢁ s, 12H, OCOCH3), assignments were
EtOAc/hexane); mp = 99-102 °C; [R]25 = -80 (c = 1.0,
D
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 7.75 (d,
J = 8.0 Hz, 2H, Harom), 7.42 (d, J = 8.5 Hz, 2H, Harom), 7.29
(s, 2H, NH2), 5.22 (d, J = 3.5 Hz, 1H, H-40), 5.19 (t, J = 9.0 Hz,
1H, H-3), 5.13 (dd, J = 3.5, 9.5 Hz, 1H, H-30), 4.85 (t, J = 10.0,
1H, H-20), 4.83 (t, J = 9.0 Hz, 1H, H-2), 4.74 (d, J = 8.0 Hz, 1H,
H-10), 4.68 (d, J = 10.0 Hz, 1H, H-1), 4.37 (t, J = 5.5 Hz, 1H,
NH), 4.36 (m, 1H, H-6a), 4.20 (br t, J = 7.0 Hz, 1H, H-50),
4.05-4.00 (m, 5H, H-6b, H-6a0, H-6b0, CH2), 3.80 (m, 1H, H-5),
3.77 (t, J = 10.0 Hz, 1H, H-4), 2.08, 2.05, 1.99, 1.97, 1.96, 1.89
(6 ꢁ s, 21H, OCOCH3), assignments were confirmed by 1H-1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 170.7, 170.4,
170.3, 169.9 (2C), 169.8, 169.6, (OCOCH3), 144.6, 142.8 (Carom),
128.7, 125.8 (CHarom), 100.2 (C-10), 87.5 (C-1), 76.3 (C-4), 75.9
(C-5), 73.7 (C-3), 70.6 (C-30), 70.0 (C-50), 69.2 (C-2), 68.1 (C-20),
67.3 (C-40), 62.7 (C-6), 61.1 (C-60), 55.9 (CH2), 20.9, 20.7 (3C),
20.6, 20.5 (2C) (OCOCH3), assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 837 [M þ H]þ, 859
[M þ Na]þ. LRMS (ESI-): m/z = 835 [M - H]-. HRMS: calcd
for C33H44N2O19S2Na, 859.1872; found, 859.1872.
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 169.9 (2C), 169.6, 169.4 (OCOCH3), 144.0, 141.9
(Carom), 129.0, 125.6 (CHarom), 88.4 (C-1), 73.3 (C-5), 71.1 (C-3),
67.7 (C-4), 65.1 (C-2), 61.7 (C-6), 54.0 (NHCH2), 35.7
(NHCH2CH2), 20.5, 20.4, 20.3 (2C) (OCOCH3), assignments
were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z = 563
[M þ H]þ, 585 [M þ Na]þ. LRMS (ESI-): m/z = 561 [M - H]-.
HRMS: calcd for C22H31N2O11S2, 563.1364; found, 563.1364.
Methyl N-4-(Aminosulfonyl)phenethyl-S-(2,3,4-tri-O-acetyl-
1-thio-β-D-glucopyranuronoyl)sulfenamide (20). Sulfenamide 20
was prepared from 4-aminoethylbenzenesulfonamide and
thioacetate derivative c according to the General Procedure 2.
The expected compound was obtained after flash chromatog-
raphy (3:2 EtOAc/hexane) as a slightly yellow solid (88% yield);
N-4-(Aminosulfonyl)benzyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-lactosyl)sulfenamide (7). Sulfenamide 7 was prepared
from 4-aminomethylbenzenesulfonamide hydrochloride and
thioacetate derivative e according to the General Procedure 2.
The expected compound was obtained after flash chromatog-
raphy (2:1 EtOAc/hexane) as a white solid (48% yield); Rf =
0.12 (1:1 EtOAc/hexane); mp = 92-94 °C; [R]25D = -16 (c =
1.1, chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 7.75 (d,
J = 8.5 Hz, 2H, Harom), 7.42 (d, J = 8.5 Hz, 2H, Harom), 7.29 (s,
2H, NH2), 5.36 (t, J = 8.5 Hz, H-3), 5.29 (d, J = 3.5 Hz, 1H,
H-10), 5.22 (t, J = 10.0 Hz, 1H, H-30), 4.98 (t, J = 10.0 Hz, 1H,
H-40), 4.86 (dd, J = 3.0, 10.5 Hz, 1H, H-20), 4.86 (t, J = 10.0, 1H,
H-2), 4.72 (d, J = 10.0 Hz, 1H, H-1), 4.45 (m, 1H, H6a), 4.37 (t,
J = 6.0 Hz, 1H, NH), 4.18-4.14 (m, 2H, H-6a0, H-6b), 4.07 (dd,
J = 6.0, 14.0 Hz, 1H, H-6b0), 4.02-3.96 (m, 4H, H-5, H-50, CH2)
3.93 (t, J = 8.5 Hz, 1H, H-4), 2.04, 2.01, 1.98, 1.97, 1.94 (5 ꢁ s,
21H, OCOCH3), assignments were confirmed by 1H-1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 170.4, 170.3,
170.1, 169.9, 169.8 (2C), 169.4, (OCOCH3), 144.5, 142.7 (Carom),
128.5, 125.6 (CHarom), 95.6 (C-10), 87.2 (C-1), 75.5 (C-3), 75.2
(C-5), 73.8 (C-4), 69.6 (C-20), 69.0 (C-30), 68.3 (C-2), 68.2 (C-50),
67.9 (C-40), 63.1 (C-60), 61.6 (C-6), 56.0 (CH2), 20.7 (2C), 20.6
(2C), 20.5, 20.4, 20.3 (OCOCH3), assignments were confirmed
by 1H-13C HSQC. HRMS: calcd for C33H44N2O19S2Na,
859.1872; found, 859.1873.
Rf = 0.16 (1:1 EtOAc/hexane); mp = 124-128 °C; [R]25
=
D
1
-88 (c = 1.2, chloroform). H NMR (500 MHz, DMSO-d6):
δ = 7.72 (d, J = 8.5 Hz, 2H, Harom), 7.37 (d, J = 8.0 Hz, 2H,
H
arom), 7.25(s, 2H, NH2),5.40(t, J= 9.5 Hz, 1H, H-3), 4.94 (t, J=
10.0 Hz, 1H, H-4), 4.93 (t, J = 10.0 Hz, 1H, H-2), 4.79 (d, J =
10.0 Hz, 1H, H-1), 4.45 (d, J = 10.0 Hz, 1H, H-5), 3.95 (t, J =
5.0 Hz, 1H, NH), 3.62 (s, 3H, OCH3), 3.08 (m, 2H, NHCH2),
2.82 (t, J = 7.0 Hz, NHCH2CH2), 2.00, 1.97, 1.96 (3 ꢁ s, 9H,
OCOCH3), assignments were confirmed by 1H-1H gCOSY. 13
C
NMR (125 MHz, DMSO-d6): δ = 169.7, 169.5, 169.4, 167.5
(COCH3), 144.2, 141.9 (Carom), 129.2, 125.7 (CHarom), 87.4
(C-1), 74.5 (C-5), 72.5 (C-3), 69.2 (C-4), 67.4 (C-2), 55.8
(NHCH2), 52.6 (OCH3), 35.7 (NHCH2CH2), 20.6, 20.4, 20.3
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 549 [M þ H]þ. LRMS (ESI-): m/z = 547
[M - H]-. HRMS: calcd for C21H29N2O11S2, 549.1207; found,
549.1224.
N-4-(Aminosulfonyl)phenethyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-lactosyl)sulfenamide (21). Sulfenamide 21 was prepared
from 4-aminoethylbenzenesulfonamide and thioacetate deriva-
tive d according to the General Procedure 2. The expected
compound was obtained after flash chromatography (2:1
EtOAc/hexane) as a white solid (88% yield); Rf = 0.44 (2:1
EtOAc/hexane); mp = 91-93 °C; [R]25D = -70 (c = 1.0, chlo-
N-4-(Aminosulfonyl)phenethyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-
β-D-glucopyranosyl)sulfenamide (18). Sulfenamide 18 was pre-
pared from 4-aminoethylbenzenesulfonamide and thioacetate
derivative a according to the General Procedure 2. Expected
compound was obtained after flash chromatography (1:1
EtOAc/hexane) as a white solid (66% yield); Rf = 0.23 (1:1
1
roform). H NMR (500 MHz, DMSO-d6): δ = 7.70 (d, J =
EtOAc/hexane); mp = 124-125 °C; [R]25 = -102 (c = 1.2,
8.5 Hz, 2H, Harom), 7.35 (d, J = 8.5 Hz, 2H, Harom), 7.26 (s, 2H,
NH2), 5.21 (d, J = 3.5 Hz, 1H, H-40), 5.18 (t, J = 9.5 Hz, 1H,
H-3), 5.12 (dd, J = 3.5, 10.5 Hz, 1H, H-30), 4.83 (dd, J = 8.0,
D
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 7.72 (d, J =
8.0 Hz, 2H, Harom), 7.37 (d, J = 8.5 Hz, 2H, Harom), 7.25 (s, 2H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2921
10.0 Hz, 1H, H-20), 4.80 (t, J = 9.5 Hz, 1H, H-2), 4.73 (d, J = 8.0
Hz, 1H, H-1), 4.63 (d, J = 10.5 Hz, 1H, H-10), 4.32 (br d, J =
11.0 Hz, 1H, H6a0), 4.19 (br t, J = 6.5 Hz, 1H, H-50), 4.01-3.98
(m, 3H, H-6a, H-6b, H-6b0), 3.86 (br t, J = 5.0 Hz, 1H, NH),
3.81 (m, 1H, H-5), 3.73 (t, J = 9.5 Hz, 1H, H-4), 3.05 (m, 2H,
NHCH2), 2.79 (t, J = 7.0 Hz, NHCH2CH2), 2.08, 1.99, 1.98,
1.97, 1.91, 1.88 (6 ꢁ s, 21H, OCOCH3), assignments were
2.00, 1.96, 1.95 (4 ꢁ s, 12H, OCOCH3), assignments were
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 170.1, 169.6, 169.3, 168.7 (OCOCH3), 143.0, 142.6
(Carom), 127.6, 125.7 (CHarom), 86.3 (C-1), 74.6 (C-5), 72.8 (C-3),
67.6, 67.5 (C-4, C-2), 61.6 (C-6), 46.1 (CH2), 20.5, 20.4, 20.3, 20.2
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 603 [M þ Na]þ. LRMS (ESI-): m/z =
579 [M - H]-. HRMS: calcd for C21H28N2O13S2Na, 603.0925;
found, 603.0942.
N-4-(Aminosulfonyl)benzyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-β-
D-galactopyranosyl)sulfonamide (9). Sulfonamide 9 was pre-
pared from sulfenamide 4 according to the General Procedure 3.
Following flash chromatography using solid addition (2:1
EtOAc/hexane), the expected derivative was obtained as a slightly
1
confirmed by H-1H gCOSY. 13C NMR 125 MHz, DMSO-
d6): δ = 170.4, 170.1 (2C), 169.8, 169.7, 169.6, 169.3 (OCOCH3),
144.3, 141.9 (Carom), 129.3, 125.8 (CHarom), 100.1 (C-10), 87.5
(C-1), 76.2 (C-4), 75.7 (C-5), 73.6 (C-3), 70.5 (C-30), 69.9 (C-50),
69.1 (C-20), 68.0 (C-2), 67.2 (C-40), 62.6 (C-60), 61.0 (C-6), 54.1
(NHCH2), 35.8 (NHCH2CH2), 20.7, 20.6 (3C), 20.5 (2C), 20.4
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 873 [M þ Na]þ. HRMS: calcd for C34H46-
N2O19S2Na, 873.2028; found, 873.2063.
N-4-(Aminosulfonyl)phenethyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-maltosyl)sulfenamide (22). Sulfenamide 22 was pre-
pared from 4-aminoethylbenzenesulfonamide and thioacetate
derivative e according to the General Procedure 2. The expected
compound was obtained after flash chromatography (1:1
EtOAc/hexane) as a slightly yellow gum (49% yield); Rf =
0.18 (1:1 EtOAc/hexane); [R]25D = -2 (c = 1.3, chloroform). 1H
NMR (500 MHz, DMSO-d6): δ = 7.72 (d, J = 8.5 Hz, 2H,
yellow oil (46% yield); Rf = 0.31 (2:1 EtOAc/hexane); [R]25
=
D
-28 (c = 1.3, chloroform). 1H NMR (500 MHz, DMSO-d6): δ =
8.26 (t, J = 6.0 Hz, 1H, NH), 7.80 (d, J = 8.5 Hz, 2H, Harom), 7.50
(d, J = 8.0 Hz, 2H, Harom), 7.31 (s, 2H, NH2), 5.41 (t, J = 9.5 Hz,
1H, H-3), 5.34 (br d, J = 3.5 Hz, 1H, H-4), 5.30 (dd, J = 3.5,
9.5 Hz, 1H, H-3), 4.85 (d, J = 9.5 Hz, 1H, H-1), 4.37 (br t, J =
6.0 Hz, 1H, H-5), 4.30 (br d, J = 4.5 Hz, 2H, CH2), 4.06 (br d, J =
6.0 Hz, 2H, H-6a, H-6b), 2.13, 2.00, 1.98, 1.93 (4 ꢁ s, 12H,
OCOCH3), assignments were confirmed by 1H-1H gCOSY. 13
C
NMR (125 MHz, DMSO-d6): δ = 169.9, 169.8, 169.4, 168.6
(OCOCH3), 142.9, 142.5 (Carom), 127.5, 125.7 (CHarom), 86.8
(C-1), 74.9 (C-5), 70.7 (C-3), 67.3 (C-4), 64.7 (C-2), 61.4 (C-6),
46.0 (CH2), 20.5 (2C), 20.4, 20.3 (OCOCH3), assignments were
H
arom), 7.36 (d, J = 8.0 Hz, 2H, Harom), 7.24 (s, 2H, NH2), 5.38
(t, J = 9.0 Hz, H-3), 5.29 (d, J = 4.0 Hz, 1H, H-10), 5.22 (t, J =
10.0 Hz, 1H, H-30), 4.98 (t, J = 9.5 Hz, 1H, H-40), 4.87 (dd, J =
3.5, 10.5 Hz, 1H, H-20), 4.80 (t, J = 9.5, 1H, H-2), 4.70 (d, J =
10.0 Hz, 1H, H-1), 4.41 (dd, J = 3.0, 12.0 Hz, 1H, H6a0), 4.16
(dd, J = 4.5, 12.5 Hz, 1H, H-6a), 4.13 (dd, J = 4.5, 12.5 Hz, 1H,
H-6b0), 4.02 (m, 1H, H-6b), 4.00-3.97 (m, 2H, H-5, H-50), 3.89
(t, J = 9.5 Hz, 1H, H-4), 3.88 (m, 1H, NH), 3.06 (m, 2H,
NHCH2), 2.80 (t, J = 6.5 Hz, NHCH2CH2), 2.00, 1.99, 1.98,
1.97, 1.95, 1.94 (6 ꢁ s, 21H, OCOCH3), assignments were
1
confirmed by H-13C HSQC. LRMS (ESIþ): m/z = 603 [M þ
Na]þ. HRMS: calcd for C21H28N2O13S2Na, 603.0925; found,
603.0944.
Methyl N-4-(Aminosulfonyl)benzyl-S-(2,3,4-tri-O-acetyl-1-
thio-β-D-glucopyranuronoyl)sulfonamide (10). Sulfonamide 10
was prepared from sulfenamide 5 according to the General
Procedure 3. The expected derivative was obtained after flash
chromatography (2:1 EtOAc/hexane) as a white solid (40%
yield); Rf = 0.29 (2:1 EtOAc/hexane); mp = 235-236 °C;
confirmed by H-1H gCOSY. 13C NMR 125 MHz, DMSO-
1
d6): δ = 170.0, 169.9, 169.8, 169.6, 169.4 (2C), 169.1 (OCOCH3),
144.5, 141.8 (Carom), 129.0, 125.5 (CHarom), 95.4 (C-10), 87.0 (C-1),
75.3 (C-3), 74.8 (C-50), 73.6 (C-4), 69.4 (C-20), 68.8 (C-30), 68.2
(C-2), 68.0 (C-5), 67.8 (C-40), 63.0 (C-60), 61.4 (C-6), 53.9
(NHCH2), 35.7 (NHCH2CH2), 20.5, 20.4 (2C), 20.3 (3C), 20.2
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 873 [M þ Na]þ. HRMS: calcd for
C34H46N2O19S2Na, 873.2028; found, 873.2043.
Oxidation of Sulfenamide Derivatives: General Procedure 3.
The sulfenamide derivative (1.0 equiv) was solubilized in
CH2Cl2. meta-Chloroperoxybenzoic acid (7.0 equiv) was added
dropwise as a solution in CH2Cl2 over ∼20 min. The reaction
was maintained under stirring at rt until the full disappearance
of the starting material, as evidenced by TLC, typically 1 h. The
reaction mixture was then diluted in CH2Cl2 and quenched with
NaHCO3 satd. The resulting mixture was next filtered through
celite. After extraction with CH2Cl2, the organic layer was
washed with NaHCO3 satd (ꢁ1) and brine (ꢁ1). The aqueous
phases were back extracted with CH2Cl2 (ꢁ2), organic phases
were combined, dried with MgSO4, filtered, and concentrated.
Expected sulfonamides were purified as described for each
compound, 8-12 and 23-27.
[R]25 = -25 (c = 1.0, chloroform). 1H NMR (500 MHz,
D
DMSO-d6): δ = 8.36 (t, J = 6.0 Hz, 1H, NH), 7.78 (d, J = 8.0 Hz,
2H, Harom), 7.48 (d, J = 8.0 Hz, 2H, Harom), 7.31 (s, 2H, NH2),
5.47 (t, J = 9.5 Hz, 1H, H-3), 5.26 (t, J = 9.5 Hz, 1H, H-2), 5.03
(d, J = 9.5 Hz, 1H, H-1), 4.97 (t, J = 9.5 Hz, 1H, H-4), 4.56 (d,
J = 10.0 Hz, 1H, H-5), 4.32 (m, 2H, CH2), 3.65 (s, 3H, OCH3),
2.00, 1.97, 1.96 (3 ꢁ s, 9H, OCOCH3), assignments were
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 169.4, 169.2, 168.6, 166.6 (OCOCH3), 142.9, 142.5
(Carom), 127.5, 125.6 (CHarom), 86.1 (C-1), 73.9 (C-5), 71.9 (C-3),
68.5 (C-4), 67.4 (C-2), 52.7 (OCH3), 46.1 (CH2), 20.3, 20.2, 20.1
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESI-): m/z = 565 [M - H]-. HRMS: calcd for C20H26-
N2O13S2Na, 589.0769; found, 589.0769.
N-4-(Aminosulfonyl)benzyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-maltosyl)sulfonamide (11). Sulfonamide 11 was pre-
pared from sulfenamide 6 according to the General Procedure
3. Expected derivative was obtained after flash chromatography
(2:1 EtOAc/hexane) as a white solid (64% yield); Rf = 0.23 (2:1
EtOAc/hexane); mp = 151-152 °C; [R]25 = -19 (c = 1.4,
D
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 8.23 (t, J =
6.0 Hz, 1H, NH), 7.79 (d, J = 8.0 Hz, 2H, Harom), 7.47 (d, J =
8.5 Hz, 2H, Harom), 7.31 (s, 2H, NH2), 5.27 (t, J = 9.5, 1H, H-3),
5.23 (d, J = 3.5 Hz, 1H, H-40), 5.16 (d, J = 9.5 Hz, 1H, H-2),
5.16 (dd, J= 3.5, 10.0 Hz, 1H, H-30), 4.90(d, J= 9.5 Hz, 1H, H-1),
4.85 (dd, J = 8.0, 10.0 Hz, 1H, H-20), 4.77 (d, J = 8.0 Hz, 1H, H-
10), 4.32 (br d, J = 10.5 Hz, 1H, H-6a0), 4.26 (d, J = 6.0 Hz, 2H,
CH2), 4.22 (t, J = 7.0 Hz, 1H, H-6a), 4.09 (dd, J = 7.0, 12.0 Hz,
1H, H-6b0), 4.01 (m, 3H, H-5, H-50, H-6b), 3.84 (t, J = 9.5 Hz,
1H, H-4), 2.10, 2.06, 2.01, 1.98, 1.94, 1.90 (7 ꢁ s, 24H,
OCOCH3), assignments were confirmed by 1H-1H gCOSY.
13C NMR (125 MHz, DMSO-d6): δ = 170.3, 170.0 (2C), 169.5,
169.4, 169.2, 168.8 (OCOCH3), 143.0, 142.6 (Carom), 127.6,
125.8 (CHarom), 100.0 (C-10), 86.2 (C-1), 75.8 (C-4), 75.7
N-4-(Aminosulfonyl)benzyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-β-
D-glucopyranosyl)sulfonamide (8). Sulfonamide 8 was prepared
from sulfenamide 3 according to the General Procedure 3.
Expected derivative was obtained as white crystals follow-
ing recrystallization from ethanol (63% yield); Rf = 0.13 (1:1
EtOAc/hexane); mp = 182-185 °C; [R]25 = -32 (c = 1.1,
D
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 8.29 (t, J =
6.0 Hz, 1H, NH), 7.79 (d, J = 8.5 Hz, 2H, Harom), 7.49 (d, J =
8.5 Hz, 2H, Harom), 7.31 (s, 2H, NH2), 5.39 (t, J = 9.5 Hz, 1H,
H-3), 5.23 (t, J = 9.5 Hz, 1H, H-2), 4.95 (d, J = 9.0 Hz, 1H, H-1),
4.94 (t, J = 9.0 Hz, 1H, H-4), 4.29 (d, J = 5.5 Hz, 2H, CH2), 4.20
(dd, J = 5.5, 12.5 Hz, 1H, H-6a), 4.13 (ddd, J = 2.0, 5.5,
10.0 Hz, 1H, H-5), 4.02 (dd, J = 1.5, 12.0 Hz, 1H, H-6b), 2.01,

2922 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
(C-50), 73.0 (C-3), 70.4 (C-30), 69.8 (C-5), 68.9 (C-20), 67.8 (C-2),
67.1 (C-40), 62.2 (C-60), 60.9 (C-6), 46.1 (CH2), 20.6, 20.5 (2C),
20.4 (3C), 20.3 (OCOCH3), assignments were confirmed by
1H-13C HSQC. LRMS (ESIþ): m/z = 891 [M þ Na]þ. LRMS
(ESI-): m/z = 867 [M - H]-. HRMS: calcd for C33H44N2O21S2-
Na, 891.1770; found, 891.1786.
70.8 (C-3), 67.4 (C-4), 64.7 (C-2), 61.7 (C-6), 44.0 (NHCH2), 35.8
(NHCH2CH2), 20.5, 20.4, 20.3 (2C) (OCOCH3), assignments were
1
confirmed by H-13C HSQC. LRMS (ESIþ): m/z = 617 [M þ
Na]þ. HRMS: calcd for C22H30N2O13S2Na, 617.1082; found,
617.1097.
Methyl N-4-(Aminosulfonyl)phenethyl-S-(2,3,4-tri-O-acetyl-
1-thio-β-D-glucopyranuronoyl)sulfonamide (25). Sulfonamide
25 was prepared from sulfenamide 20 according to the General
Procedure 3. The expected derivative was obtained after flash
chromatography (3:2 EtOAc/hexane) as a white solid (61%
yield); Rf = 0.26 (3:2 EtOAc/hexane); mp = 203-204 °C;
N-4-(Aminosulfonyl)benzyl-S-(2,20,3,30,40,6,60-hepta-O-acet-
yl-1-thio-β-lactosyl)sulfonamide (12). Sulfonamide 12 was pre-
pared from sulfenamide 7 according to the General Procedure 3.
The expected derivative was obtained after flash chromatogra-
phy (2:1 EtOAc/hexane) as a white solid (37% yield); Rf = 0.24
(2:1 EtOAc/hexane); mp = 112-115 °C; [R]25D = þ37 (c = 1.3,
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 8.23 (t, J =
6.0 Hz, 1H, NH), 7.80 (d, J = 8.5 Hz, 2H, Harom), 7.48 (d, J =
8.0 Hz, 2H, Harom), 7.31 (s, 2H, NH2), 5.44 (t, J = 9.0 Hz, 1H, H-
3), 5.30 (d, J = 4.0 Hz, 1H, H-10), 5.23 (t, J = 10.0 Hz, 1H, H-30),
5.13 (t, J = 9.5 Hz, 1H, H-2), 5.00 (t, J = 10.0 Hz, 1H, H-40),
4.95 (d, J = 10.0 Hz, 1H, H-1), 4.89 (dd, J = 4.0, 10.0 Hz, 1H,
H-20), 4.43 (br d, J = 10.0 Hz, 1H, H-6a), 4.27 (d, J = 6.0 Hz,
2H, CH2), 4.21 (dd, J = 6.0, 12.5 Hz, 1H, H-6b), 4.17 (dd, J =
4.0, 12.0 Hz, 1H, H-6a0), 4.14 (m, 1H, H-5) 4.02 (br d, J =
12.0 Hz, 1H, H-6b0), 4.00 (m, 1H, H-50) 3.98 (t, J = 9.0 Hz, 1H,
H-4), 2.06, 2.02, 1.99, 1.98, 1.95, 1.93 (6 ꢁ s, 24H, OCOCH3),
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125
MHz, DMSO-d6): δ = 170.3, 170.1, 170.0, 169.8, 169.6, 169.3,
169.0 (OCOCH3), 143.0, 142.7 (Carom), 127.6, 125.8 (CHarom),
95.8 (C-10), 86.4 (C-1), 75.3 (C-5), 74.8 (C-3), 73.6 (C-4), 69.5 (C-
20), 69.0 (C-30), 68.3 (C-2), 68.2 (C-50), 67.8 (C-40), 62.8 (C-6),
61.5 (C-60), 46.2 (CH2), 20.7, 20.6, 20.5 (2C), 20.4 (2C), 20.3
(OCOCH3), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 891 [M þ Na]þ. LRMS (ESI-): m/z =
867 [M - H]-. HRMS: calcd for C33H44N2O21S2Na, 891.1770;
found, 891.1770.
[R]25 = -18 (c = 1.0, chloroform). 1H NMR (500 MHz,
D
DMSO-d6): δ = 7.78 (t, J = 6.0 Hz, 1H, NH), 7.75 (d, J =
8.0 Hz, 2H, Harom), 7.39 (d, J = 8.0 Hz, 2H, Harom), 7.27 (s, 2H,
NH2), 5.45 (t, J = 9.5 Hz, 1H, H-3), 5.23 (t, J = 9.5 Hz, 1H, H-
2), 5.00 (d, J = 9.5 Hz, 1H, H-1), 4.97 (t, J = 9.5 Hz, 1H, H-4),
4.62 (d, J = 10.0 Hz, 1H, H-5), 3.63 (s, 3H, OCH3), 3.28 (m, 2H,
NHCH2), 2.83 (t, J = 7.5 Hz, 2H, NHCH2CH2), 2.00, 1.97, 1.96
(3 ꢁ s, 9H, OCOCH3), assignments were confirmed by 1H-1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 169.5, 169.2,
168.6, 166.7 (OCOCH3), 142.8, 142.2 (Carom), 129.1, 125.6
(CHarom), 86.0 (C-1), 73.9 (C-5), 71.9 (C-3), 68.5 (C-4), 67.4
(C-2), 52.7 (OCH3), 44.1 (NHCH2), 35.8 (NHCH2CH2), 20.4,
20.2, 20.1 (OCOCH3), assignments were confirmed by 1H-13C
HSQC. LRMS (ESI-): m/z = 579 [M - H]-. HRMS: calcd for
C21H28N2O13S2Na, 603.0925; found, 603.0895.
N-4-(Aminosulfonyl)phenethyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-lactosyl)sulfonamide (26). Sulfonamide 26 was pre-
pared from sulfenamide 21 according to the General Procedure 3.
The expected derivative was obtained after flash chromato-
graphy (2:1 EtOAc/hexane) as a off-white solid (37% yield);.
Rf = 0.24 (2:1 EtOAc/hexane); mp = 105-107 °C; [R]25
=
D
1
-22 (c = 1.3, chloroform). H NMR (500 MHz, DMSO-d6):
δ = 7.75 (d, J = 8.5 Hz, 2H, Harom), 7.70 (t, J = 5.5 Hz, 1H, NH),
7.39 (d, J = 8.5 Hz, 2H, Harom), 7.28 (s, 2H, NH2), 5.27 (t, J = 9.5
Hz, 1H, H-3), 5.23 (d, J = 3.5 Hz, 1H, H-40), 5.16 (d, J = 9.5 Hz,
1H, H-2), 5.16 (dd, J = 3.5, 10.0 Hz, 1H, H-30), 4.90 (d, J = 9.5 Hz,
1H, H-1), 4.85 (dd, J = 8.0, 10.0 Hz, 1H, H-20), 4.77 (d, J = 8.0 Hz,
1H, H-10), 4.31 (br d, J = 10.5 Hz, 1H, H-6a), 4.21 (t, J = 6.5 Hz,
1H, H-50), 4.08 (m, 1H, H-6b), 4.06 (m, 1H, H-5), 4.02 (m, 2H, H-
6a0, H-6b0), 3.83 (t, J = 9.5 Hz, 1H, H-4), 3.23 (m, 2H, NHCH2),
2.84 (t, J = 7.5 Hz, 2H, NHCH2CH2), 2.10, 2.00, 1.99, 1.97, 1.96,
1.94, 1.89 (7 ꢁ s, 21H, OCOCH3), assignments were confirmed by
1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 170.2,
169.9 (2C), 169.5, 169.4, 169.0, 168.7 (OCOCH3), 142.9, 142.2
(Carom), 129.1, 125.7 (CHarom), 99.9 (C-10), 85.6 (C-1), 75.8 (C-4),
75.6 (C-5), 73.0 (C-3), 70.3 (C-30), 69.8 (C-50), 68.9 (C-20), 67.8
(C-2), 67.1 (C-40), 62.4 (C-6), 60.9 (C-60), 44.0 (NHCH2), 35.8
(NHCH2CH2), 20.5, 20.4 (3C), 20.3 (2C), 20.2 (OCOCH3),
assignments were confirmed by 1H-13C HSQC. LRMS
(ESIþ): m/z = 891 [M þ Na]þ. LRMS (ESI-): m/z = 867 [M -
H]-. HRMS: calcd for C34H46N2O21S2Na, 905.1927; found,
905.1944.
N-4-(Aminosulfonyl)phenethyl-S-(2,20,3,30,40,6,60-hepta-O-acetyl-
1-thio-β-maltosyl)sulfonamide (27). Sulfonamide 27 was pre-
pared from sulfenamide 22 according to the General Procedure
3. The expected derivative was obtained after flash chromatog-
raphy (2:1 EtOAc/hexane) as a white solid (37% yield); Rf =
0.19 (2:1 EtOAc/hexane); mp = 100-103 °C; [R]25D = þ32 (c =
1.1, chloroform). 1H NMR bad spectra (500 MHz, DMSO-d6):
δ = 7.75 (d, J = 8.0 Hz, 2H, Harom), 7.70 (t, J = 6.0 Hz, 1H,
NH), 7.40 (d, J = 8.0 Hz, 2H, Harom), 7.28 (s, 2H, NH2), 5.43 (t,
J = 9.0 Hz, 1H, H-3), 5.28 (d, J = 4.0 Hz, 1H, H-10), 5.21 (t, J =
10.0 Hz, 1H, H-30), 5.09 (t, J = 9.5 Hz, 1H, H-2), 4.99 (t, J = 9.5
Hz, 1H, H-40), 4.95 (d, J = 9.5 Hz, 1H, H-1), 4.88 (dd, J = 4.0,
10.0 Hz, 1H, H-20), 4.40 (m, 1H, H-6a), 4.19 (m, 1H, H-6b), 4.17
(m, 1H, H-5), 4.15 (m, 1H, H6a0), 4.02 (m, 1H, H-6b0), 3.97 (m,
1H, H-50), 3.95 (t, J = 9.0 Hz, H-4), 3.24 (br q, J = 8.0 Hz, 2H,
NHCH2), 2.84 (t, J = 7.5 Hz, 2H, NHCH2CH2), 2.01, 1.99,
1.98, 1.96, 1.95, 1.92 (6 ꢁ s, 21H, OCOCH3), assignments were
N-4-(Aminosulfonyl)phenethyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-
β-D-glucopyranosyl)sulfonamide (23). Sulfonamide 23 was pre-
pared from sulfenamide 18 according to the General Procedure
3. Expected derivative was obtained after recrystallization from
ethanol as white crystals (57% yield); Rf = 0.29 (2:1 EtOAc/
hexane); mp = 112-115 °C; [R]25 = -32 (c = 1.1, chlo-
D
roform). 1H NMR (500 MHz, DMSO-d6): δ = 7.74 (d, J = 8.0
Hz, 2H, Harom), 7.73 (t, J = 6.0 Hz, 1H, NH), 7.40 (d, J = 8.0
Hz, 2H, Harom), 7.28 (s, 2H, NH2), 5.39 (t, J = 9.5 Hz, 1H, H-3),
5.20 (t, J = 9.5 Hz, 1H, H-2), 4.97 (d, J = 9.5 Hz, 1H, H-1), 4.92
(t, J = 9.5 Hz, 1H, H-4), 4.18 (m, 2H, H-5, H-6a), 4.04 (m, 1H,
H-6b), 3.26 (m, 2H, NHCH2), 2.84 (t, J = 7.5 Hz, 2H,
NHCH2CH2), 1.99, 1.95, 1.94, 1.93 (4 ꢁ s, 12H, OCOCH3),
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125
MHz, DMSO-d6): δ = 169.9, 169.5, 169.2, 168.6 (OCOCH3),
142.8, 142.2 (Carom), 129.1, 125.6 (CHarom), 85.7 (C-1), 74.5 (C-
5), 72.7 (C-3), 67.5 (2C) (C-4, C-2), 61.8 (C-6), 44.0 (NHCH2),
35.6 (NHCH2CH2), 20.4, 20.3 (2C), 20.2 (OCOCH3), assign-
ments were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z =
612 [M þ NH4]þ, 617 [M þ Na]þ. HRMS: calcd for
C22H30N2O13S2Na, 617.1082; found, 617.1084.
N-4-(Aminosulfonyl)phenethyl-S-(2,3,4,6-tetra-O-acetyl-1-thio-
β-D-galactopyranosyl)sulfonamide (24). Sulfonamide 24 was pre-
pared from sulfenamide 19 according to the General Procedure 3.
Expected derivative was obtained after flash chromatography
(2:1 EtOAc/hexane) as a white solid (52% yield); Rf = 0.21 (2:1
EtOAc/hexane); mp = 88-92 °C; [R]25 = -17 (c = 1.3,
D
chloroform). 1H NMR (500 MHz, DMSO-d6): δ = 7.75 (d, J =
8.5 Hz, 2H, Harom), 7.72 (t, J = 6.0 Hz, 1H, NH), 7.41 (d, J = 8.0
Hz, 2H, Harom), 7.28 (s, 2H, NH2), 5.39 (t, J = 9.5 Hz, 1H, H-2),
5.34 (d, J = 2.5 Hz, 1H, H-4), 5.28 (dd, J = 3.5, 10.0 Hz, 1H, H-3),
4.89 (d, J = 9.5 Hz, 1H, H-1), 4.40 (br t, J = 6.0 Hz, 1H, H-5), 4.06
(m, 2H, H-6a, H-6b), 3.26 (m, 2H, NHCH2), 2.86 (t, J = 7.5 Hz,
2H, NHCH2CH2), 2.11, 1.97, 1.93 (4 ꢁ s, 12H, OCOCH3),
assignments were confirmed by 1H-1H gCOSY. 13C NMR (125
MHz, DMSO-d6): δ = 169.9, 169.8, 169.4, 168.6 (OCOCH3),
142.8, 142.2 (Carom), 129.1, 125.7 (CHarom), 86.3 (C-1), 73.9 (C-5),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2923
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 170.0, 169.9, 169.8, 169.6, 169.4, 169.1, 168.8
(OCOCH3), 142.8, 142.2 (Carom), 129.1, 125.6 (CHarom), 95.6
(C-10), 85.6 (C-1), 75.0 (C-5), 74.7 (C-3), 73.6 (C-50), 69.4 (C-20),
68.8 (C-30), 68.8 (C-2), 68.0 (C-4), 67.7 (C-40), 62.9 (C-6), 61.4
(C-60), 44.0 (NHCH2), 35.8 (NHCH2CH2), 20.5, 20.4 (2C), 20.3
(3C), 20.2 (OCOCH3), assignments were confirmed by 1H-13C
HSQC. LRMS (ESIþ): m/z = 883 [M þ H]þ, 906 [M þ Na]þ.
LRMS (ESI-): m/z = 881 [M - H]-. HRMS: calcd for
C34H46N2O21S2Na, 905.1927; found, 905.1921.
NMR (125 MHz, DMSO-d6): δ = 169.7 (CO2H), 142.8
(Carom), 127.7, 125.6 (CHarom), 90.2 (C-1), 78.9 (C-5), 76.9
(C-3), 71.0 (C-4), 70.4 (C-2), 46.0 (CH2), assignments were
confirmed by 1H-13C HSQC. LRMS (ESI-): m/z = 425
[M - H]-. HRMS: calcd for C13H18N2O10S2Na, 449.0295;
found, 449.0305.
N-4-(Aminosulfonyl)benzyl-S-(1-thio-β-maltosyl)sulfonamide
(16). Sulfonamide 16 was prepared from sulfonamide 11 accord-
ing to the General Procedure 4. The expected derivative was
obtained after freeze-drying as a white solid (85% yield); Rf =
0.24 (95:5 CH3CN/H2O); mp = 162-164 °C; [R]25D = -5 (c =
1.1, methanol). 1H NMR (500 MHz, DMSO-d6): δ = 7.79 (d,
J = 8.5 Hz, 2H, Harom), 7.67 (br s, 1H, NH), 7.52 (d, J = 8.0 Hz,
2H, Harom), 7.31 (s, 2H, NH2), 5.38 (d, J = 4.5 Hz, 1H, OH-2),
5.08 (d, J = 4.0 Hz, 1H, OH-20), 4.83 (d, J = 1.5 Hz, 1H, OH-3),
4.77 (d, J = 4.0 Hz, 1H, OH-30), 4.64 (t, J = 5.0 Hz, 1H, OH-60),
4.58 (t, J = 6.0 Hz, 1H, OH-6), 4.50 (d, J = 4.5 Hz, 1H, OH-40),
4.29 (m, 2H, CH2), 4.26 (d, J = 9.5 Hz, 1H, H-1), 4.22 (d, J = 7.0
Hz, 1H, H-10), 3.80 (dd, J = 6.0, 10.5 Hz, 1H, H-6a), 3.62 (m,
2H, H-6b, H-40), 3.52 (m, 3H, H-2, H-6a0, H-6b0), 3.43 (m, 3H,
H-3, H-4, H-50), 3.32 (m, 3H, H-20, H-30, H-5), assignments were
Deacetylation of Peracetylated Compounds: General Proce-
dure 4. Fully deprotected compounds were prepared by treating
a solution of the per-O-acetylated compound (1.0 equiv) in
anhydrous MeOH at 0 °C with methanolic sodium methoxide
(0.05 M final concentration), final pH = 12. The reaction was
warmed to rt and left to stir until full deprotection was evident
by TLC (∼2 h). The solution was neutralized with Amberlite IR-
120 [Hþ], filtered and the resin washed several times with
methanol. The solvent was evaporated under reduced pressure
and lyophilized to dryness to afford fully deprotected com-
pounds 13-17 and 28-32.
N-(4-Aminosulfonyl)benzyl-S-(1-thio-β-D-glucopyranosyl)sul-
fonamide (13). Sulfonamide 13 was prepared from sulfonamide 8
according to the General Procedure 4. The expected derivative
was obtained after freeze-drying as a white solid (100% yield);
Rf = 0.31 (95:5 CH3CN/H2O); mp = 185 °C; [R]25D = -9 (c =
1.1, methanol). 1H NMR (500 MHz, DMSO-d6): δ = 7.78 (d,
J = 8.5 Hz, 2H, Harom), 7.52 (d, J = 8.0 Hz, 2H, Harom), 7.30 (br
s, 2H, NH2), 5.17 (br s, 1H, OH), 5.09 (br s, 1H, OH), 4.55 (br s,
1H, OH), 4.29 (t, J = 6.0 Hz, 1H, NHCH2), 4.27 (t, J = 6.0 Hz,
1H, NHCH2), 4.14 (d, J = 9.5 Hz, 1H, H-1), 3.70 (br d, J = 12.0
Hz, 1H, H-6a), 3.48 (m, 1H, H-6b), 3.46 (t, J = 9.0 Hz, 1H, H-2),
3.23 (m, 2H, H-3, H-5), 3.07 (t, J = 9.0 Hz, 1H, H-4), assign-
ments were confirmed by 1H-1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 142.9, 142.8 (Carom), 127.8, 125.6 (CHarom),
89.8 (C-1), 81.2 (C-5), 77.5 (C-3), 70.5 (C-2), 69.6 (C-4), 61.1 (C-
6), 46.0 (CH2), assignments were confirmed by 1H-13C HSQC.
LRMS (ESIþ): m/z = 413 [M þ Na]þ, 435 [M þ Na]þ. HRMS:
calcd for C13H20N2O9S2Na, 435.0502; found, 435.0524.
N-(4-Aminosulfonyl)benzyl-S-(1-thio-β-D-galactopyranosyl)-
sulfonamide (14). Sulfonamide 14 was prepared from sulfonam-
ide 9 according to the General Procedure 4. Expected derivative
was obtained after freeze-drying as a white solid (78% yield);.
Rf = 0.50 (9:1 CH3CN/H2O); mp = 181-183 °C; [R]25D = -3
(c = 1.1, methanol). 1H NMR (500 MHz, DMSO-d6): δ = 7.79
(d, J = 8.0 Hz, 2H, Harom), 7.63 (br t, J = 6.0 Hz, 1H, NH), 7.52
(d, J = 8.0 Hz, 2H, Harom), 7.30 (s, 2H, NH2), 4.95 (d, J =
5.5 Hz, 1H, OH-2), 4.92 (d, J = 5.5 Hz, 1H, OH-3), 4.60 (t, J =
5.5 Hz, 1H, OH-6), 4.57 (d, J = 3.5 Hz, 1H, OH-4), 4.27 (m, 2H,
CH2), 4.10 (d, J = 9.0 Hz, 1H, H-1), 3.81 (dt, J = 5.5, 9.0 Hz,
1H, H-2), 3.71 (br t, J = 3.5 Hz, 1H, H-4), 3.55 (m, 2H, H-6a, H-
6b), 3.49 (t, J = 6.0 Hz, 1H, H-5), 3.38 (m, 1H, H-3), 3.05 (td,
J = 5.5, 9.5 Hz, 1H, H-4), 3.25 (br t, J = 7.0 Hz, 1H,
NHCH2CH2), 2.84 (t, J = 7.0 Hz, 2H, NHCH2CH2), assign-
ments were confirmed by 1H-1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 142.9, 142.8 (Carom), 127.8, 125.6 (CHarom),
90.8 (C-1), 79.8 (C-5), 74.0 (C-3), 68.0 (C-4), 67.2 (C-2), 60.4
(C-6), 46.0 (CH2), assignments were confirmed by 1H-13C
HSQC. LRMS (ESIþ): m/z = 411 [M - H]-. HRMS: calcd
for C13H20N2O9S2Na, 435.0502; found, 435.0510.
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 142.9 (2C) (Carom), 127.8, 125.6 (CHarom), 103.7 (C-10),
89.4 (C-1), 79.8 (C-5), 79.1 (C-50), 75.7 (C-40), 75.5 (C-3), 73.2
(C-30), 70.5 (C-20), 70.4 (C-2), 68.1 (C-4), 60.4 (C-6, C-60), 46.0
(CH2), assignments were confirmed by 1H-13C HSQC. LRMS
(ESIþ): m/z = 573 [M - H]-. HRMS: calcd for C19H30N2O14S2-
Na, 597.1031; found, 597.1059.
N-4-(Aminosulfonyl)benzyl-S-(1-thio-β-lactosyl)sulfonamide
(17). Sulfonamide 17 was prepared from sulfonamide 12 accord-
ing to the General Procedure 4. The expected derivative was
obtained after freeze-drying as a white highly hygroscopic solid
(84% yield); Rf = 0.52 (9:1 CH3CN/H2O); [R]25D = þ53 (c =
1.0, methanol). 1H NMR (500 MHz, DM SO-d6): δ = 7.79 (d,
J = 8.5 Hz, 2H, Harom), 7.68 (t, J = 6.0 Hz, 1H, NH), 7.52 (d,
J = 8.0 Hz, 2H, Harom), 7.31 (s, 2H, NH2), 5.65 (br s, 1H, OH-
40), 5.40 (d, J = 6.0 Hz, 1H, OH-20), 5.32 (d, J = 5.0 Hz, 1H,
OH-2), 5.05 (d, J = 3.5 Hz, 1H, H-10), 4.90 (d, J = 5.5 Hz, 1H,
OH-3), 4.87 (d, J = 4.5 Hz, 1H, OH-30), 4.53 (t, J = 6.0 Hz, 1H,
OH-6), 4.52 (t, J = 6.0 Hz, 1H, OH-60), 4.29 (m, 2H, CH2), 4.26
(d, J = 9.5 Hz, 1H, H-1), 3.76 (dd, J = 6.5, 11.0 Hz, 1H, H-6a),
3.64 (m, 1H, H-6a0), 3.59 (m, 1H, H-6b), 3.55 (m, 1H, H-2), 3.53
(m, 1H, H-40), 3.48 (m, 1H, H-4), 3.45 (m, 1H, H-6b0), 3.40 (m,
1H, H-5), 3.38 (m, 1H, H-30), 3.35 (m, 1H, H-50), 3.24 (m, 1H, H-
20), 3.07 (m, 1H, H-3), assignments were confirmed by 1H-1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 142.9, 142.8
(Carom), 127.8, 125.6 (CHarom), 100.7 (C-10), 89.6 (C-1), 79.3 (C-
5), 78.8 (C-50), 77.1 (C-40), 73.5 (C-4), 73.2 (C-30), 72.4 (C-20),
70.2 (C-2), 69.9 (C-3), 60.8(C-60), 60.6 (C-6), 46.0 (CH2), assign-
ments were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z =
573 [M - H]-. HRMS: calcd for C19H30N2O14S2Na, 597.1031;
found, 597.1043.
N-(4-Aminosulfonyl)phenethyl-S-(1-thio-β-D-glucopyranosyl-
sulfonamide (28). Sulfonamide 28 was prepared from sulfonam-
ide 23 according to the General Procedure 4. The expected
derivative was obtained after freeze-drying as a white highly
hygroscopic solid (96% yield); Rf = 0.38 (95:5 CH3CN/H2O);
[R]25D = -16 (c = 1.0, methanol). 1H NMR (500 MHz, DMSO-
d6): δ = 7.74 (d, J = 8.5 Hz, 2H, Harom), 7.41 (d, J = 8.0 Hz, 2H,
H
arom), 7.27 (s, 2H, NH2), 7.05 (t, J = 6.0 Hz, 1H, NH), 5.12 (d,
N-(4-Aminosulfonyl)benzyl-S-(1-thio-β-D-glucopyranuronoyl)-
sulfonamide (15). Sulfonamide 15 was prepared from sulfonam-
ide 10 according to the General Procedure 4. Expected deriva-
tive was obtained after freeze-drying as a slightly yellow gum
(40% yield); Rf = 0.15 (9:1 CH3CN/H2O); [R]25D = -13 (c =
1.1, methanol). 1H NMR (500 MHz, D2O): δ = 7.98 (d, J = 8.0
Hz, 2H, Harom), 7.68 (d, J = 8.0 Hz, 2H, Harom), 4.57 (d, J =
16.0 Hz, 1H, CHH), 4.49 (d, J = 16.0 Hz, 1H, CHH), 4.23 (d,
J = 9.5 Hz, 1H, H-1), 3.84 (m, 1H, H-5), 3.55 (m, 3H, H-2, H-3,
H-4), assignments were confirmed by 1H-1H gCOSY. 13C
J = 4.5 Hz, 1H, OH), 5.11 (d, J = 1.0 Hz, 1H, OH), 5.04 (d, J =
6.5 Hz, 1H, OH), 4.49 (t, J = 6.0 Hz, 1H, OH-6), 4.24 (d, J = 9.5
Hz, 1H, H-1), 3.69 (dd, J = 1.5, 12.0 Hz, 1H, H-6a), 3.46 (m, 1H,
H-6b), 3.42 (m, 1H, H-2), 3.28-3.23 (m, 4H, H-3, H-5,
NHCH2CH2), 3.05 (dt, J = 5.5, 9.5 Hz, 1H, H-4), 2.84 (t, J =
7.0 Hz, 2H, NHCH2CH2), assignments were confirmed by
1H-1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 142.2,
142.1 (Carom), 129.2, 125.6 (CHarom), 89.2 (C-1), 81.1 (C-5), 77.4
(C-3), 70.5 (C-2), 69.7 (C-4), 61.1 (C-6), 43.8 (NHCH2CH2), 35.7
(NHCH2CH2), assignments were confirmed by 1H-13C HSQC.

2924 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Lopez et al.
LRMS (ESIþ): m/z = 449 [M þ Na]þ. HRMS: calcd for
C14H28N2O9S2Na, 449.0659; found, 449.0680.
NH2), 7.11 (t, J = 6.0 Hz, 1H, NH), 5.62 (d, J = 3.5 Hz, 1H,
OH-40), 5.39 (d, J = 6.0 Hz, 1H, OH-20), 5.26 (d, J = 6.0 Hz, 1H,
OH-2), 5.04 (d, J = 3.5 Hz, 1H, H-10), 4.89 (d, J = 6.0 Hz, 1H,
OH-3), 4.86 (d, J = 5.0 Hz, 1H, OH-30), 4.52 (t, J = 5.0 Hz, 1H,
OH-60), 4.48 (t, J = 6.0 Hz, 1H, OH-6), 4.32 (d, J = 8.5 Hz, 1H,
H-1), 3.75 (dd, J = 7.0, 10.5 Hz, 1H, H-6a), 3.63 (dd, J = 5.5, 9.5
Hz, 1H, H-6a0), 3.57 (m, 1H, H-6b), 3.52-3.47 (m, 2H, H-2, H-
40), 3.48-3.44 (m, 3H, H-4, H-50, H-6b0), 3.38-3.34 (m, 2H, H-
5, H-30), 3.27-3.22 (m, 3H, H-20, NHCH2CH2), 3.06 (m, 1H, H-
3), 2.84 (t, J = 7.5 Hz, 2H, NHCH2CH2), assignments were
N-(4-Aminosulfonyl)phenethyl-S-(1-thio-β-D-galactopyranosyl)-
sulfonamide (29). Sulfonamide 29 was prepared from sulfonam-
ide 24 according to the General Procedure 4. The expected
derivative was obtained after freeze-drying as a white solid
(75% yield); Rf = 0.53 (9:1 CH3CN/H2O); mp = 185 °C;
[R]25 = -2 (c = 1.0, methanol). 1H NMR (500 MHz,
D
DMSO-d6): δ = 7.74 (d, J = 8.0 Hz, 2H, Harom), 7.41 (d, J =
8.0 Hz, 2H, Harom), 7.27 (s, 2H, NH2), 7.06 (br s, 1H, NH), 4.90
(d, J = 5.5 Hz, 1H, OH-3), 4.87 (d, J = 4.5 Hz, 1H, OH-2), 4.57
(m, 1H, OH-6), 4.56 (m, 1H, OH-4), 4.17 (d, J = 9.5 Hz, 1H, H-
1), 3.69 (dt, J = 4.5, 9.0 Hz, 1H, H-2), 3.69 (t, J = 3.5 Hz, 1H, H-
4), 3.53 (m, 3H, H-5, H-6a, H-6b), 3.40 (m, 1H, H-3), 3.25 (br t,
J = 7.0 Hz, 1H, NHCH2CH2), 2.84 (t, J = 7.0 Hz, 2H,
NHCH2CH2), assignments were confirmed by 1H-1H gCOSY.
13C NMR (125 MHz, DMSO-d6): δ = 142.2, 142.1 (Carom),
129.2, 125.6 (CHarom), 90.2 (C-1), 79.7 (C-5), 74.0 (C-3), 68.0 (C-
4), 67.3 (C-2), 60.4 (C-6), 43.9 (NHCH2CH2), 35.7 (NHCH2-
CH2), assignments were confirmed by 1H-13C HSQC. LRMS
(ESIþ): m/z = 425 [M - H]-. HRMS: calcd for C14H22N2O9S2Na,
449.0659; found, 449.0668.
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 143.1, 142.1 (Carom), 129.2, 125.6 (CHarom), 100.7
(C-10), 88.8 (C-1), 79.2 (C-50), 79.0 (C-5), 77.0 (C-40), 73.5
(C-4), 73.2 (C-30), 72.4 (C-20), 70.2 (C-2), 69.9 (C-3), 60.8 (C-
60), 60.7 (C-6), 43.8 (NHCH2CH2), 35.7 (NHCH2CH2), assign-
ments were confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z =
587 [M - H]-. HRMS: calcd for C20H32N2O14S2Na, 611.1187;
found, 611.1188.
Carbonic Anhydrase Inhibition Assay. An SX.18MV-R Ap-
plied Photophysics stopped-flow instrument was used for assay-
ing the CA I, II, and IX CO2 hydration activity.27 Phenol red (at
a concentration of 0.2 mM) was 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 the ionic
strength;this anion is not inhibitory), following the CA-cata-
lyzed CO2 hydration reaction for a period of 10-100 s. Satu-
rated 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
performed with the assay buffer mentioned above. Inhibitor and
enzyme solutions were preincubated together for 15 min at room
temperature prior to assay in order 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 and
represent the mean from at least three different determinations.
Enzyme concentrations were 10 nM for CA I and CA II, 14 nM
for CA IX, and 0.11 μM for hCA XII. Kinetic parameters and
inhibition constants were calculated as described previously.37,38
Enzymes used here were recombinant ones, prepared and puri-
fied as described earlier.37,38
N-4-(Aminosulfonyl)phenethyl-S-(1-thio-β-maltosyl)sulfonamide
(30). Sulfonamide 30 was prepared from sulfonamide 25 accord-
ing to the General Procedure 4. The expected derivative was
obtained after freeze-drying as a yellow gum (60% yield); Rf =
0.12 (9:1 CH3CN/H2O); [R]25D = -23 (c = 1.1, methanol). 1H
NMR (500 MHz, D2O): δ = 7.94 (d, J = 8.0 Hz, 2H, Harom),
7.57 (d, J = 8.5 Hz, 2H, Harom), 4.46 (d, J = 9.5 Hz, 1H, H-1),
3.98 (d, J = 9.5 Hz, 1H, H-5), 3.78 (br t, J = 9.0 Hz, 1H, H-2),
3.62 (m, 1H, H-4), 3.59 (m, 1H, H-3), 3.57 (m, 2H, NHCH2-
CH2), 3.02 (t, J = 8.5 Hz, 2H, NHCH2CH2), assignments were
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 169.7 (CO2H), 143.1, 142.1 (Carom), 129.1, 125.6
(CHarom), 89.9 (C-1), 78.9 (C-5), 76.8 (C-3), 71.0 (C-4), 70.4
(C-2), 43.9 (NHCH2CH2), 35.8 (NHCH2CH2), assignments
were confirmed by 1H-13C HSQC. LRMS (ESI-): m/z = 439
[M - H]-. HRMS: calcd for C14H20N2O10S2Na, 463.0452;
found, 463.0460.
N-4-(Aminosulfonyl)phenethyl-S-(1-thio-β-maltosyl)sulfonamide
(31). Sulfonamide 31 was prepared from sulfonamide 26 accord-
ing to the General Procedure 4. The expected derivative was
obtained after freeze-drying as a slightly yellow gum (77%
yield); Rf = 0.15 (9:1 CH3CN/H2O); [R]25D = 0 (c = 1.0, meth-
anol). 1H NMR (500 MHz, DMSO-d6): δ = 7.74 (d, J = 8.0 Hz,
2H, Harom), 7.41 (d, J = 8.0 Hz, 2H, Harom), 7.27 (s, 2H, NH2),
7.08 (t, J = 6.0 Hz, 1H, NH), 5.33 (d, J = 6.0 Hz, 1H, OH-2),
5.08 (d, J = 4.0 Hz, 1H, OH-20), 4.83 (d, J = 1.5 Hz, 1H, OH-3),
4.76 (d, J = 4.0 Hz, 1H, OH-30), 4.68 (t, J = 5.0 Hz, 1H, OH-60),
4.58 (t, J = 6.0 Hz, 1H, OH-6), 4.50 (d, J = 4.0 Hz, 1H, OH-40),
4.32 (d, J = 9.5 Hz, 1H, H-1), 4.20 (d, J = 7.0 Hz, 1H, H-1), 3.80
(dd, J = 6.0, 10.0 Hz, 1H, H-6a), 3.62 (m, 2H, H-6b, H-40),
3.52-3.45 (m, 6H, H-2, H-3, H-4, H-50, H-6a0, H-6b0),
3.32-3.28 (m, 3H, H-20, H-30, H-5), 3.25 (m, 2H, NHCH2CH2),
2.84 (t, J = 8.0 Hz, 2H, NHCH2CH2), assignments were
Conclusions
Recent experimental evidence has implicated CAs as a drug
target with promising potential for the development of much
needed personalized cancer therapies targeting hard-to-treat
hypoxic tumors. Here we have developed a novel and straight-
forward method for the stereoselective synthesis of small
molecule sulfonamide-linked neoglycoconjugates. In these
compounds, the carbohydrate fragment is linked to the classical
aromatic sulfonamide CA pharmacophore to target inhibition
of cancer-associated CAs. The synthesis has been achieved by
reaction of S-glycosyl acetates with either an aminobenzyl or
aminophenethyl sulfonamide scaffold to form S-glycosylsulfe-
namide glycoconjugates, which are then oxidized to give robust
S-glycosyl sulfonamide linked glycoconjugates.
The CA inhibitors of this study are designed in accord with
a combined SAR-SPR strategy and compounds were very
good CA IX inhibitors and potent CA XII inhibitors. The role
of the carbohydrate fragment is of most relevance in the
context of selectively targeting the extracellular active sites
of CA IXand XII. The sugar group takes advantage of the cell
membranes lipophilic properties as a physical barrier to
1
confirmed by H-1H gCOSY. 13C NMR (125 MHz, DMSO-
d6): δ = 143.2, 142.1 (Carom), 129.3, 125.7 (CHarom), 103.8 (C-
10), 88.7 (C-1), 80.0 (C-5), 79.1 (C-50), 75.7 (C-40), 75.6 (C-3),
73.3 (C-30), 70.6 (C-20), 70.4 (C-2), 68.2 (C-4), 60.5 (2C) (C-6, C-
60), 43.9 (NHCH2CH2), 35.8 (NHCH2CH2), assignments were
confirmed by 1H-13C HSQC. LRMS (ESIþ): m/z = 587 [M -
H]-. HRMS: calcd for C20H32N2O14S2Na, 611.1187; found,
611.1201.
N-4-(Aminosulfonyl)phenethyl-S-(1-thio-β-lactosyl)sulfonamide
(32). Sulfonamide 32 was prepared from sulfonamide 27 accord-
ing to the General Procedure 4. The expected derivative was
obtained after freeze-drying as a slightly yellow gum (84%
yield); Rf = 0.17 (9:1 CH3CN/H2O); [R]25 = þ46 (c = 1.1,
D
methanol). 1H NMR (500 MHz, DMSO-d6): δ = 7.75 (d, J =
8.5 Hz, 2H, Harom), 7.41 (d, J = 8.5 Hz, 2H, Harom), 7.27 (s, 2H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2925
minimize passive membrane permeability of the polar small
molecule inhibitors, this in turn can promote the preferential
inhibition of extracellular CAs. Our approach has delivered
neutral, water-soluble CA inhibitors that have excellent po-
tential as isozyme selective inhibitors of cancer-associated
CAs in vivo and represents an important outcome for inves-
tigating future therapeutic applications of CA inhibitors.
Finally, our results also showcase new chemistry toward
S-glycosyl secondary sulfonamides. This chemistry is straight-
forward and synthetically tractable, needing only a primary
amine functional group in the molecule of interest and a
S-glycosyl acetate partner. We believe this chemistry may
readily be applied toward the synthesis of diverse range of
therapeutically relevant neoglycoconjugates and thus will
have significant impact across broad areas of medicinal
chemistry and chemistry.
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Acknowledgment. This work was financed in part by the
Australian Research Council (grant no. DP0877554 to S.-A.P.),
the Eskitis Institute for Cell and Molecular Therapies and an
EU grant of the 6th framework programme (DeZnIT project
to C.T.S.), and an EU grant of the 7th framework programme
(METOXIA project to C.T.S.). We thank Hoan The Vu for
carrying out accurate mass measurements.
Supporting Information Available: 1H and 13C NMR spectra
for new compounds 3-32. This material is available free of
charge via the Internet at http://pubs.acs.org.
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