A prodrug approach toward cancer-related carbonic anhydrase inhibition

Article abstract of DOI:10.1021/jm401163e

The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs for selectively targeting the extracellular cancer-associated CA enzymes.

Full text of DOI:10.1021/jm401163e

Article
pubs.acs.org/jmc
A Prodrug Approach Toward Cancer-Related Carbonic Anhydrase
Inhibition
Cindy J. Carroux,† Gregory M. Rankin,† Janina Moeker,† Laurent F. Bornaghi,† Kasiram Katneni,‡
Julia Morizzi,‡ Susan A. Charman,‡ Daniela Vullo,§ Claudiu T. Supuran,*,§ and Sally-Ann Poulsen*,†
†Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia
‡Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,
Australia
§Polo Scientifico, Laboratorio di Chimica Bioinorganica, Universita
̀
degli Studi di Firenze, Rm. 188, Via della Lastruccia 3, 50019
Sesto Fiorentino, Florence, Italy
S
* Supporting Information
ABSTRACT: The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and
XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the
design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of
CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl
benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all
compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative
set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key
biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs
for selectively targeting the extracellular cancer-associated CA enzymes.
by hypoxia. CA IX-specific monoclonal antibodies are already in
phase III clinical development as both therapeutic and
diagnostic agents.3 Small molecules can provide improved
pharmacokinetic profiles and lower cost over antibodies,4 and
consequently we expect that small molecules which target CA
IX and XII would be desirable for targeting hypoxic tumors in
vivo. The membrane permeability properties of small molecules
targeting CA IX and XII can in principle be employed to take
advantage of the cell membrane as a barrier between off-target
cytosolic CAs and transmembrane CA IX and XII. CA
inhibitors with limited membrane permeability have been
developed,5 however, these compounds are generally hydro-
philic and so are likely to lack oral bioavailability. Esters are a
common prodrug moiety employed by the pharmaceutical
industry,6 and ester prodrugs of monosaccharides have been
developed to mask polar hydroxyl groups with hydrophobic
acyl groups as an approach to enhance oral bioavailability and
INTRODUCTION
■
There are 12 catalytically active isoforms of CA that have been
characterized in humans, four of these (CA IV, IX, XII, and
XIV) are transmembrane anchored proteins with an extrac-
ellular active site domain. The role of CA IX and XII was
recently shown to be pivotal for pH regulation to support a
microenvironment suited for hypoxic tumor cell survival and
proliferation through the conversion of hypoxic cell generated
carbon dioxide to bicarbonate and a proton. The effect is to
trap protons extracellularly and acidify the extracellular hypoxic
−
microenvironment (supporting tumor invasiveness). HCO3 is
transported back into the cell to maintain intracellular pH
(supporting tumor cell survival).1 The expression of CA IX and
XII is upregulated in many hypoxic tumors, with CA IX usually
absent in the corresponding normal tissue and CA XII
constitutively expressed in a small selection of normal tissues.2
Molecular tools that can selectively bind to or inhibit the
cancer-associated CA isozymes in vivo are required to further
explore the strong association between CA IX/XII expression
and tumor cell survival in the altered microenvironment caused
Received: July 30, 2013
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Variably Acylated Glycoconjugate Primary Sulfonamide Compounds 13−36
a
Reagents and conditions: (i) CuAAC: β-D-glycopyranosyl azide 1−10 (1 equiv), alkyne 11 or 12 (1.1 equiv), CuSO4·5H2O (0.2 equiv), sodium
ascorbate (0.4 equiv), H2O:EtOH (1:1 to 1:5), 60 °C; (ii) 14, 20, 26, or 32, NaOMe in MeOH (25% w/v), MeOH, rt.
enable therapeutic concentrations of the prodrug to reach the
bloodstream.7 When in the blood, the ester moiety is
susceptible to hydrolysis by carboxylesterases to give the
corresponding alcohol and carboxylate, potentially minimizing
passive diffusion into cells within tissues.8
compound as an approach to selectively target the active site of
extracellular CA IX and XII over cytosolic CAs.
RESULTS AND DISCUSSION
■
Compound Design and Synthesis. The 24 glycoconju-
gate compounds (13−36) of this study comprise three core
structural elements: (i) a sugar moiety (glucose or galactose),
(ii) a 1,2,3-triazole moiety, and (iii) a benzene sulfonamide
group (meta- or para- substituted). The sugar moiety provides a
common prodrug template, while the benzene sulfonamide
group is a component of classical small molecule CA inhibitors
wherein the sulfonamide anion is able to coordinate to the CA
active site Zn2+ to block the endogenous reaction.1,16 The
triazole linker serves as a biocompatible, nonlabile covalent
spacer between the sugar and benzene sulfonamide group-
s.15a,17 The 24 library members are in four sublibraries that each
comprises six compounds with an identical sugar and benzene
sulfonamide group but differing in the sugar protecting group.
The sublibraries consist of a fully deprotected compound (13,
19, 25, or 31) and the corresponding acylated compounds,
including acetyl (14, 20, 26 or 32), propionyl (15, 21, 27, or
33), butanoyl (16, 22, 28, or 34), pentanoyl (17, 23, 29, or
35), or 3-methylbutanoyl (18, 24, 30, or 36) acyl groups. The
acylated compounds (14−18, 20−24, 26−30, and 32−36)
were synthesized by the reaction of glycopyranosyl azides 1−10
with either 4-ethynyl benzene sulfonamide (11) or 3-ethynyl
benzene sulfonamide (12) using CuAAC, Scheme 1. Lengthy
reaction times were required for the CuAAC reaction with the
more lipophilic glycopyranosyl azides owing to lower solubility
in the reaction solvent. The synthesis of compounds 1−11 has
been reported previously by ourselves,5h,13 while compound 12
has been reported by others.18 Compounds 13, 19, 25, and 31
Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) to
synthesize triazoles has contributed to a renaissance in the
chemistry of azides as building blocks toward higher complexity
glycoconjugates.9−11 We have recently described the synthesis
of glycopyranosyl azides wherein a monosaccharide moiety was
peracylated to give O-peracylated β-D-glucopyranosyl (1−5)
and β-D-galactopyranosyl azides (6−10), comprising acetyl (1
and 6),12 propionyl (2 and 7), butanoyl (3 and 8), pentanoyl
(4 and 9), and 3-methylbutanoyl (5 and 10) moieties.13 We
have also established that 1,2,3-triazoles are compatible with
commonly employed carbohydrate protecting group ap-
proaches14 and applied CuAAC between the O-peracylated
glycopyranosyl azides and phenylacetylene to synthesize
glycoconjugates as tools to study the effect of different acyl
groups on biopharmaceutical properties. We then demon-
strated that these carbohydrate building blocks were susceptible
to plasma hydrolytic enzymes with serial hydrolysis of the
carbohydrate ester moieties to form a fully deprotected and
hydrophilic glycoconjugate. Other work in our research group
has applied CuAAC to synthesize CA inhibitors.5c,d,f,g,15 In the
present study, we combine the merits of these earlier
investigations by employing the variably acylated glycopyr-
anosyl azide building blocks 1−10 for the first time in a
medicinal chemistry campaign to synthesize glycoconjugates
that target CA. We assess the potential of these compounds as
prodrugs for a hydrophilic glycoconjugate benzene sulfonamide
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Scheme 2. Sequential Hydrolysis of Ester Groups of Glucoconjugates 14−18 and 20−24 to Form the Fully Deacylated
Compounds 13 and 19, Respectively
Table 1. Inhibition Data of hCA Isozymes I, II, IX, and XII with Compounds 11−36 and the Established Drug and CA Inhibitor,
Acetazolamide (AZA)
b
Ki (nM)
a
compd
substituents
hCA I
hCA II
hCA IX
hCA XII
hCA XIV
AZA
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
250
1080
843
12
71.6
66.1
380
9.1
25
54.0
65.7
78.9
120
475
633
951
426
78.1
480
874
583
402
724
126
110
5.1
5.7
44.5
29.7
40.1
67.1
7.4
41
63.9
118
50.3
104
256
119
176
40.1
23.7
45.3
78.4
67.0
137
452
20.1
15.8
10.9
35.6
67.3
15.9
44.5
50.1
58.9
76.0
148
24.9
Glc, OH, para
9.4
Glc, acetyl, para
4400
2020
1795
9226
9441
9450
2245
2216
2936
9165
9833
7950
4300
78.8
Glc, propionyl, para
Glc, butanoyl, para
Glc, pentanoyl, para
Glc, methylbutanoyl, para
Glc, OH, meta
71.1
76.3
573
440
621
261
79.4
181
174
661
6.7
50.2
52.7
52.0
85.4
55.1
73.2
71.9
44.1
61.4
36.3
9.7
Glc, acetyl, meta
Glc, propionyl, meta
Glc, butanoyl, meta
Glc, pentanoyl, meta
Glc, methylbutanoyl, meta
Gal, OH, para
Gal, acetyl, para
8.7
Gal, propionyl, para
Gal, butanoyl, para
Gal, pentanoyl, para
Gal, methylbutanoyl, para
Gal, OH, meta
8.1
5.4
257
6.9
51.8
335
374
590
943
126
69.2
800
254
9.5
2130
3205
6452
3823
2351
83.1
73.6
37.9
69.1
421
253
6.3
31.0
23.3
35.0
61.9
53.3
61.1
43.2
31.4
Gal, acetyl, meta
Gal, propionyl, meta
Gal, butanoyl, meta
Gal, pentanoyl, meta
73.8
8.0
Gal, methylbutanoyl, meta
1876
4781
a
b
Glc = glucose, Gal = galactose. Errors in the range of 5% of the reported value, from three determinations.
are the fully deacylated glycoconjugates that result from the
complete hydrolysis of acyl groups of the corresponding parent
compounds 14−18, 20−24, 26−30, and 32−36, respectively.
These compounds were however synthesized from the
and for the target tumor-associated hCA IX and XII, by analysis
of the hydration activity of carbonic anhydrase,20 Table 1.
Similarly to acetazolamide (AZA), the glycoconjugate sulfona-
mides 13−36 and ethynyl benzene sulfonamides 11 and 12
generally displayed poorest inhibition toward the off-target
hCA I. For most glycoconjugates, hCA I inhibition constants
(Ki) were in the micromolar range and compounds were
typically one order of magnitude weaker inhibitors of this
isozyme than of hCA II, IX, XII, and XIV. The parent
nonglycoconjugate compounds (11 and 12) displayed
submicromolar inhibition of hCA II, IX, XII, and XIV, while
the glycoconjugate sulfonamides (13−36) Ki values spanned
low nanomolar to low micromolar at hCA II, IX, XII, and XIV.
There were no consistent SAR trends for the differing acyl
groups so that any of the acylated glycoconjugates could serve
acetylated analogues 14, 20, 26, and 32 using Zemplen19
́
conditions, Scheme 1. Esterase hydrolysis of compounds 14−
18, 20−24, 26−30, and 32−36 to yield the fully deprotected
compound (13, 19, 25, or 31, respectively) may take place
sequentially, forming multiple triacylated, diacylated, and
monoacylated glycoconjugates as intermediates; this is shown
for the glucoconjugates, Scheme 2.
CA Inhibition. The enzyme inhibition data for ethynyl
benzene sulfonamides 11 and 12, and glycoconjugate
sulfonamides 13−36 were measured for the off-target cytosolic
hCA I and II, for the off-target membrane anchored hCA XIV
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as a prodrug for the corresponding fully deprotected
glycoconjugates. For the glucose series of sulfonamides (13−
24), the deprotected glucose analogues 13 and 19 displayed
selectivity for the target hCA IX over the off-target hCA II, with
notably improved inhibition of hCA IX than for all of the
corresponding parent acylated analogues (15−18, 20−24) and
similar inhibition of hCA XII compared to acylated analogues
15−18, 20−24. This SAR supports the potential viability of an
acyl ester glucoconjugate prodrug approach to selectively target
the inhibition of cancer-related CAs over cytosolic off-target
CAs. The initial acylated glucoconjugate prodrug (15−18, 20−
24) should provide for good oral absorption and low CA
inhibition, while the unmasked prodrug generates a hydrophilic
glucoconjugate (13 or 19) that should be selective toward
overexpressed hCA IX and XII due to a combination of limited
membrane permeability and improved Ki. In contrast, the
deprotected galactose compounds 25 and 31 did not exhibit
this optimal balance of SAR between ester prodrug (26−30,
32−36) and unmasked compound (25 or 31) to achieve a
useful profile in the context of a prodrug approach. The
unmasked compounds 25 and 31 inhibited off-target hCA II
19- and 9-fold better than hCA IX, respectively, while the
acylated compounds inhibited hCA IX and XII an order of
magnitude better (27), 2−4-fold better (28), or similarly (29,
30, 32−36) to the deprotected compounds 25 and 31. This
differing SAR between the glucose and galactose series of
glycoconjugate sulfonamides demonstrates that the structure of
the carbohydrate moiety of compounds, although not directly
impacting on the sulfonamide anion−zinc cation interaction,
can have a profound impact on CA inhibition profile. The
stereochemical difference between these epimers must lead to
an altered interaction(s) of the ligand with the outer residues of
the CA active site. We have previously observed in an unrelated
set of glycoconjugate benzene sulfonamides that glucose and
galactose tail moieties also gave this powerful discrimination
between CA isozyme inhibition and selectivity.5d
In Vitro ADME. As the glucose series of compounds proved
of interest with respect to SAR a representative sublibrary of
these compounds, 13−18 was used for in vitro biopharma-
ceutical property determination. We first determined the in
vitro stability of the sublibrary 13−18 in human plasma at 37
°C, Table 2. The characteristic molecular weight for the
potential hydrolysis intermediates and compound 13 (as a
reference compound for complete hydrolysis) allowed plasma
stability to be monitored using mass spectrometry, Figure 1.
Compound 13 remained stable in human plasma over the
course of the incubation (4 h), indicating stability of the
underlying glycoconjugate benzene sulfonamide scaffold. The
acetyl, propionyl, and butanoyl analogues (14, 15, and 16) were
rapidly degraded, with estimated half-lives of 7, 3, and 25 min,
respectively, while the pentanoyl (17) and the 3-methylbuta-
noyl (18) compounds remained stable in human plasma over
the course of the incubation. The serial hydrolysis of the ester
moieties to variable tri-, di-, and monoacylated ester products,
respectively, formed en route to the fully hydrolyzed compound
13, was confirmed by mass spectrometry, Figure 1. We recently
reported in vitro ADME properties for analogues of 13−18 that
have a phenyl substituent in place of the phenyl sulfonamide
substituent.13 These compounds exhibited the same acyl group
plasma stability profile with estimated half-lives of 55, 26, and
555 min for the acetyl, propionyl, and butanoyl analogues,
respectively. The half-lives are, however, 8−25-fold longer than
Table 2. cLog P, in Vitro Human Plasma Stability,
Chromatographic HSA Binding (cHSA), and in Vitro HLM
Stability Data for Test Glucoconjugate Compounds 13−18
a
HLM stability in
vitro CLint
plasma
stability
(μL/min/mg pro-
c
tein)
d
degradation
cHSA
(%
half-life
(+)
(−)
b
c
compd
Mwt
cLog P
(min)
bound) NADPH NADPH
13
14
15
16
17
18
386.4
554.5
610.6
666.7
722.9
722.9
−1.75
+0.01
+2.81
+4.59
+6.37
+5.74
>250
7
13
30
<7
<7
cnd
cnd
cnd
<7
e
cnd
3
77
cnd
cnd
<7
25
94
f
>250
>250
>99
>99
f
<7
<7
a
Nonstandard abbreviations: cHSA, chromatographic human serum
albumin binding; HLM, human liver microsome; CLint, in vitro
b
c
intrinsic clearance. Calculated using Instant JChem 5.12.0. Values
are the average of duplicate incubations. Values measured using the
d
chromatographic retention times on an immobilized HSA column.
e
cnd = could not be determined owing to rapid degradation in the
f
absence of cofactor. Values are estimates only as the chromatographic
peaks were broad and retention times could not be accurately
determined.
for the corresponding sulfonamides, meaning that the
sulfonamides were more labile toward plasma esterases.
Protein binding values of the test compounds 14−18 were
estimated by comparison of their chromatographic retention
properties on an immobilized human serum albumin (HSA)
column to those for a series of standard compounds, Table 2.21
As expected, the increased protein binding paralleled increased
lipophilicity of the acyl groups of the underlying sugar template.
In addition, the compounds with higher HSA binding (17 and
18, >99% HSA bound) were also the more stable in human
plasma (half-life >250 min). This inverse relationship indicates
that the extent of plasma protein binding may influence the
plasma stability profile observed for this subset of variable acyl
group protected carbohydrate-based sulfonamides. This may be
a desirable characteristic for these molecules, minimizing the
potential for binding to off-target red blood cell hCA I and II
while increasing the circulation half-life. The fully deacylated
compound 13 had low HSA binding (13%).
We then determined the in vitro metabolic stability of
compounds 13−18 in human liver microsomes (HLMs) in
both the presence and absence of NADPH, the cofactor
required for oxidative metabolism by CYP450s, Table 2. No
degradation was detected for the fully deacylated (13) or for
the more lipophilic pentanoyl (17) and 3-methylbutanoyl (18)
compounds; this indicates that 13, 17 and 18 are likely to have
low CYP-mediated metabolic clearance in vivo. In comparison
the acetyl, propionyl, and butanoyl compounds (14, 15, and
16) were degraded in HLMs in both the presence and absence
of cofactor, indicative of non-CYP450 metabolism. The fully
hydrolyzed compound 13 as well as various mono-, di-, and
triacylated intermediates were detected, a finding indicating
that HLM degradation is a result of compound susceptibility to
hydrolytic enzymes such as microsomal esterases.
The cLog P results imply that compounds 13 and 14 (cLog
P = −1.75 and +0.01, respectively) should exhibit poor passive
permeation across gastrointestinal epithelial cells while
compound 15 should exhibit good permeability (cLog P =
+2.81). Caco-2 cell monolayers were used in an attempt to
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Figure 1. In vitro stability of compounds 14−16 in human plasma at 37 °C, monitored by UPLC-MS. (a) Compound 14: (A) parent compound
(m/z 555) at the start of the incubation, (B) monodeacylation products (m/z 513) after 15 min, (C) bis-deacylated products (m/z 471) after 30
min, (D) tris-deacylated products (m/z 429) after 120 min, and (E) complete deacylated product, (equivalent to compound 13) (m/z 387) after 240
min. (b) Compound 15: (A) parent compound (m/z 611) at the start of the incubation, (B) monodeacylation products (m/z 555) after 5 min, (C)
bis-deacylated products (m/z 499) after 30 min, (D) tris-deacylated products (m/z 443) after 120 min, and (E) complete deacylated product
(equivalent to compound 13) (m/z 387) after 240 min. (c) Compound 16: (A) parent compound (m/z 667) at the start of the incubation, (B) tris-
deacylated products (m/z 457) after 120 min, and (C) complete deacylated product, (equivalent to compound 13) (m/z 387) after 240 min. Mono
and bis-deacylated products were not detected.
measure the in vitro permeability of compounds 13−15.
Compounds 16−18 were not tested owing to poor solubility in
the Caco-2 buffer. For compounds 13 and 14, mass balance in
the diffusion chamber was good (100% and 81%, respectively),
however, no test compound was detected in the receptor
chamber. These results are in agreement with expectations of
poor membrane permeability and indicate minimal retention of
compound within the cell monolayer and minimal nonspecific
adsorption. For compound 15, the compound was not detected
in the receptor chamber, however, mass balance was poor
(47%), precluding an accurate assessment of the permeability of
15 and indicating that it is possible that the compound was
retained within the membrane monolayer. This result suggests
that for this class of compound clog P values approaching 3
may be too high for a good passive permeability. In addition to
this, low concentrations of the deacylated compound (13) were
detected in the donor chamber, suggesting that ester hydrolysis
had occurred during the experiment. Overall, these results
indicate that further optimization of the prodrug permeability
properties will be required, however, the unmasked prodrug
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by a solution of CuSO4·5H2O (0.2 equiv) in water (0.5 mL), was
successively added. The bright-yellow suspension was stirred
vigorously at 60 °C, as needed for full consumption of the azide or
no further change as indicated by TLC. The crude product was
filtered, washed with H2O, and redissolved in EtOAc. The organic
solution was dried over MgSO4, filtered, and concentrated under
reduced pressure. Compounds were purified by column chromatog-
raphy as described below.
General Procedure 2: Deacetylation of Carbohydrate
Moieties. Fully deprotected sugar derivatives were prepared by
treating the corresponding acylated precursor in dry MeOH (final
concentration of ∼0.1−0.2 M) with methanolic sodium methoxide
(25% w/v, 100−140 μL; pH 12). The reaction was left to stir until full
deacylation was evident by TLC (CH3CN/H2O 9:1; 30 min to
overnight). Neutralization of the solution by Amberlite IR-120-H+ ion-
exchange resin, followed by filtration (the resin is washed several times
with methanol) and evaporation of the filtrate to dryness, generally
afforded analytically pure compounds.
permeability is as needed to minimize off target binding to
intracellular CAs.
CONCLUSION
■
The ideal profile for an oral prodrug targeting cancer-related
hCA IX and XII is good membrane permeability and poor CA
inhibition, with the compound unmasked once in circulation.
The unmasked compound should then exhibit the opposing
characteristics of poor membrane permeability and good CA
inhibition. Compounds in the glucose series of sulfonamides
(13−18) displayed the needed SAR profile to fulfill these
criteria as evidenced by hCA Ki values. The acetyl, propionyl,
and butanoyl analogues 14−16 were degraded in human
plasma and HLMs, consistent with esterase processing, to form
the fully deacylated compound 13 that remained stable in these
environments. The propionyl analogue 15 has a cLog P value of
+2.81, consistent with expected good membrane permeability,
while compound 13 has a cLog P value of −1.75, consistent
with poor membrane permeability. The measured membrane
permeability for 13 in a Caco-2 cell model confirmed negligible
membrane permeability, while compound 15 may be retained
in the membrane, and future efforts will focus on optimizing
this compound. These findings provide a beneficial guide to the
impact of these acyl groups on glycoconjugate prodrug stability
and performance. In conclusion, the SAR and biopharmaceut-
ical characteristics measured for the panel of 24 glycoconjugate
sulfonamides in this study highlights the potential viability of an
ester-based prodrug approach to selectively target the inhibition
of hCA IX and XII over off-target cytosolic CAs.
4-[4″-(Aminosulfonyl)phenyl]-1-(β-D-glucopyranosyl)-1H-
1,2,3-triazole (13). The title compound 13 was prepared from
compound 14 (185 mg, 0.334 mmol) according to general procedure
2 and isolated as a white solid (111 mg, 93%). Rf = 0.61 (CH3CN/
1
H2O 9:1). H NMR was in agreement with the data reported in the
literature.5h
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-β-
D-glucopyranosyl)-1H-1,2,3-triazole (14). The title compound 14
was prepared from glucopyranosyl azide 1 (338 mg, 1.87 mmol) and
alkyne 11 (696 mg, 1.87 mmol) according to general procedure 1.
Purification of the crude product by flash chromatography (EtOAc/
hexane 7:3 to 4:1) afforded 14 (864 mg, 83% yield) as a white solid.
1H NMR was in agreement with the data reported in the literature.5h
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-propion-
yl-β-D-glucopyranosyl)-1H-1,2,3-triazole (15). The title com-
pound 15 was prepared from glucopyranosyl azide 2 (154 mg, 0.36
mmol) and alkyne 11 according to general procedure 1 in 3.5 h.
Purification of the crude product by flash chromatography (EtOAc/
hexane 3:2) afforded 15 (168 mg, 76%) as an off-white solid; Rf = 0.49
EXPERIMENTAL SECTION
■
General Chemistry. All starting materials and reagents, including
per-O-acetylated sugars, were purchased from commercial suppliers.
All solvents were available commercially dried or dried prior to use.
Reaction progress was monitored by TLC using silica gel-60 F254
plates with detection by short wave UV fluorescence (λ = 254 nm) and
staining with 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 performed using
silica gel 60 Å (230−400 mesh). NMR (1H, 13C {1H}, gCOSY, and
HSQC) spectra were recorded on a 500 MHz spectrometer at 30 °C.
(EtOAc/hexane 3:2); mp 223−225 °C; [α]25 −40 (c = 0.061,
D
CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.11 (s, 1H, CHtriazole),
8.02 (bd, 3J = 8.2 Hz, 2H, CHarom), 7.92 (bd, 3J = 8.2 Hz, 2H, CHarom),
3
7.38 (s, 2H, SO2NH2), 6.44 (d, J1′,2′ = 8.8 Hz, 1H, H1′), 5.69 (dd,
3J2′,3′ = 9.3 Hz, 3J2′,1′ = 8.8 Hz, 1H, H2′), 5.64 (dd, 3J3′,4′ = 9.6 Hz, 3J3′,2′
= 9.3 Hz, 1H, H3′), 5.22 (dd, 3J4′,5′ = 9.7 Hz, 3J4′,3′ = 9.6 Hz, 1H, H4′),
2
3
4.48−4.44 (m, 1H, H5′), 4.22 (dd, J6a′,6b′ = 12.7 Hz, J6a′,5′ = 5.2 Hz,
2
3
1H, H6a′), 4.12 (dd, J6a′,6b′ = 12.7 Hz, J6b′,5′ = 2.0 Hz, 1H, H6b′),
2.39−2.20 (m, 6H, COCH2CH3), 2.12−2.01 (m, 2H, COCH2CH3),
1.03−0.96 (m, 9H, COCH2CH3), 0.78 (t, J = 7.5 Hz, 3H,
1
Chemical shifts 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 = 39.5 ppm) signals, respectively. Chemical shifts for 1H
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); b (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 technique in positive ion and/or
negative ion modes as stated. 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. Purity of all compounds
was ≥95% as determined by HPLC with both UV and ELSD
detection. 1H and 13C NMR spectra of compounds 15−24 and 27−36
are provided in Supporting Information. Glycoconjugates are named in
accordance with the recommendations of the IUPAC−IUBMB Joint
Commission on Biochemical Nomenclature: “Nomenclature of
Carbohydrates (Recommendations 1996)” (http://www.chem.qmul.
ac.uk/iupac/2carb/). The synthesis of the β-D-glycopyranosyl azides
has been reported by us previously.13
COCH2CH3). Assignments were confirmed by H−1H gCOSY. 13C
1
NMR (500 MHz, DMSO-d6): δ = 173.3, 172.8, 172.6, 171.9 (4 ×
COCH2CH3), 145.7 (C1″/4″), 143.6 (C1″/4″), 133.1 (Ctriazole), 126.5 (2
× CHarom), 125.5 (2 × CHarom), 121.6 (CHtriazole), 84.0 (C1′), 73.4
(C5′), 72.0 (C3′), 70.3 (C2′), 67.3 (C4′), 61.5 (C6′), 26.7, 26.69, 26.6,
26.4 (4 × COCH2CH3), 8.9, 8.84 (2C), 8.78 (4 × COCH2CH3).
Assignments were confirmed by 1H−13C gHSQC. LRMS (ESI+): m/z
= 633 [M + Na]+. HRMS (ESI) calcd for C26H34N4O11SNa 633.1837,
found 633.1853.
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-butyryl-
β-D-glucopyranosyl)-1H-1,2,3-triazole (16). The title compound
16 was prepared from glucopyranosyl azide 3 (125 mg, 0.26 mmol)
and alkyne 11 (51 mg, 0.28 mmol) according to general procedure 1
in 4 days. Purification of the crude product by flash chromatography
(EtOAc/hexane 2:3 to 1:1) afforded 16 (85 mg, 49%) as a pale-yellow
solid; Rf = 0.23 (EtOAc/hexane 2:3); mp 155−190 °C (dec.); [α]25
D
−42 (c = 0.083, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.10 (s,
3
3
1H, CHtriazole), 8.00 (bd, J = 8.4 Hz, 2H, CHarom), 7.91 (bd, J = 8.4
3
Hz, 2H, CHarom), 7.34 (s, 2H, SO2NH2), 6.44 (d, J1′,2′ = 8.8 Hz, 1H,
H1′), 5.70 (dd, 3J2′,3′ = 9.2 Hz, 3J2′,1′ = 8.8 Hz, 1H, H2′), 5.65 (dd, 3J3′,4′
General Procedure 1: CuAAC Reaction Conditions. A mixture
of the β-D-glycopyranosyl azide (1.0 equiv) and the alkyne (1.1 equiv)
was suspended in a H2O/EtOH mixture (1:1 to 1:5, 10 mL). A
solution of sodium ascorbate (0.4 equiv) in water (0.5 mL), followed
= 9.6 Hz, 3J3′,2′ = 9.3 Hz, 1H, H3′), 5.23 (dd, 3J4′,5′ = 9.6 Hz, 3J4′,3′ = 9.6
3
3
3
Hz, 1H, H4′), 4.45 (ddd, J5′,4′ = 9.6 Hz, J5′,6a′ = 5.0 Hz, J5′,6b′ = 2.1
2
3
Hz, 1H, H5′), 4.18 (dd, J6a′,6b′ = 12.6 Hz, J6a′,5′ = 5.0 Hz, 1H, H6a′),
F
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4.13 (dd, 2J6a′,6b′ = 12.6 Hz, 3J6b′,5′ = 2.1 Hz, 1H, H6b′), 2.35−2.23 (m,
4H, COCH2CH2CH3), 2.19 (t, J = 7.2 Hz, 2H, COCH2CH2CH3),
2.06−2.01 (m, 2H, COCH2CH2CH3), 1.56−1.44 (m, 6H,
COCH2CH2CH3), 1.29−1.23 (m, 2H, COCH2CH2CH3), 0.90−0.80
(m, 9H, COCH2CH2CH3), 0.56 (t, J = 7.4 Hz, 3H, COCH2CH2CH3).
Assignments were confirmed by 1H−1H gCOSY. 13C NMR (500
MHz, DMSO-d6): δ = 172.4, 171.8, 171.6, 170.6 (4 ×
COCH2CH2CH3), 145.7 (C1″/4″), 143.7 (C1″/4″), 133.1 (Ctriazole),
126.5 (2 × CHarom), 125.5 (2 × CHarom), 121.6 (2 × CHtriazole), 84.1
(C1′), 73.4 (C5′), 71.8 (C3′), 70.2 (C2′), 67.3 (C4′), 61.4 (C6′), 35.14,
35.08, 35.06, 34.8 (4 × COCH2CH2CH3), 17.71, 17.67 (3C) (4 ×
COCH2CH2CH3), 13.28, 13.26, 13.2, 12.7 (COCH2CH2CH3).
(4 × COCH2CH(CH3)2), 25.1, 25.0, 24.88, 24.85 (4 × COCH2CH-
(CH3)2), 22.01 (4C), 21.98, 21.95, 21.53, 21.45 (8 × COCH2CH-
1
(CH3)2). Assignments were confirmed by H−13C gHSQC. LRMS
(ESI+): m/z = 745 [M + Na]+. HRMS (ESI) calcd for
C34H20N4O11SNa 745.3089, found 745.3082.
4-[3″-(Aminosulfonyl)phenyl]-1-(β-D-glucopyranosyl)-1H-
1,2,3-triazole (19). The title compound 19 was prepared from
triazole 20 (60 mg, 0.11 mmol) according to general procedure 2 and
isolated as a white solid (41 mg, 98%); Rf = 0.62 (CH3CN/H2O 9:1);
1
mp 210−213 °C; [α]21 −63 (c = 0.12, CH3OH). H NMR (500
D
MHz, DMSO-d6): δ = 8.97 (s, 1H, CHtriazole), 8.40 (bs, 1H, H2″), 8.06
3
3
(bd, J4″/6″,5″ = 7.7 Hz, 1H, H4″ or 6″), 7.87 (bd, J4″/6″,5″ = 7.9 Hz, 1H,
4″ or 6″), 7.66 (t, 3J5″,4″/6″ = 7.8 Hz, 1H, H5″), 7.42 (bs, 2H, SO2NH2),
1
Assignments were confirmed by H−13C gHSQC. LRMS (ESI+): m/
H
3
z = 689 [M + Na]+. HRMS (ESI) calcd for C30H42N4O11SNa
689.2463, found 689.2482.
5.60 (d, J1′,2′ = 9.3 Hz, 1H, H1′), 5.43 (d, J = 5.9 Hz, 1H, OH), 5.31
(d, J = 4.7 Hz, 1H, OH), 5.16 (d, J = 5.4 Hz, 1H, OH), 4.61 (t, J = 5.6
Hz, 1H, OH), 3.84−3.78 (m, 1H, H2′), 3.75−3.69 (m, 1H, H6′), 3.52−
3.40 (m, 3H, H3′, H5′, H6′), 3.31−3.24 (m, 1H, H4′). Assignments were
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-pentyryl-
β-D-glucopyranosyl)-1H-1,2,3-triazole (17). The title compound
17 was prepared from glucopyranosyl azide 4 (155 mg, 0.290 mmol)
and alkyne 11 (91 mg, 0.50 mmol) according to general procedure 1
in 6 days. Purification of the crude product by flash chromatography
(EtOAc/hexane 3:7 to 2:3) afforded 17 (118 mg, 57%) as a white
solid; Rf = 0.08 (EtOAc/hexane 3:7); mp 215−217 °C; [α]25D −29 (c
confirmed by H−1H gCOSY. 13C NMR (500 MHz, DMSO-d6): δ =
1
145.2 (C1″ or 3″), 144.9 (C1″ or 3″), 131.3 (Ctriazole), 129.7 (C5″), 128.2
(C4″ or 6″), 124.9 (C4″ or 6″), 122.1 (C2″), 121.2 (CHtriazole), 87.7 (C1′),
79.9 (C3′ or 5′), 76.8 (C3′ or 5′), 72.2 (C2′), 69.6 (C4′), 60.7 (C6′).
Assignments were confirmed by 1H−13C gHSQC. LRMS (ESI+): m/z
= 409 [M + Na]+. HRMS (ESI) calcd for C14H18N4O7SNa 409.0788,
found 409.0790.
1
= 0.056, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.09 (s, 1H,
3
3
CHtriazole), 8.01 (bd, J = 8.4 Hz, 2H, CHarom), 7.92 (bd, J = 8.4 Hz,
2H, CHarom), 7.39 (s, 2H, SO2NH2), 6.44 (d, 3J1′,2′ = 8.9 Hz, 1H, H1′),
5.70 (dd, 3J2′,3′ = 9.2 Hz, 3J2′,1′ = 8.9 Hz, 1H, H2′), 5.65 (dd, 3J3′,4′ = 9.4
Hz, 3J3′,2′ = 9.2 Hz, 1H, H3′), 5.23 (dd, 3J4′,5′ = 9.6 Hz, 3J4′,3′ = 9.4 Hz,
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-β-
D-glucopyranosyl)-1H-1,2,3-triazole (20). The title compound 20
was prepared from glucopyranosyl azide 1 (168 mg, 0.45 mmol) and
alkyne 12 (90 mg, 0.50 mmol) according to general procedure 1 in 5
h. Purification of the crude product by flash chromatography (EtOAc/
hexane 1:1) afforded 20 (199 mg, 80%) as a white solid; Rf = 0.18
3
3
3
1H, H4′), 4.44 (ddd, J5′,4′ = 9.6 Hz, J5′,6a′ = 5.1 Hz, J5′,6b′ = 1.9 Hz,
2
3
1H, H5′), 4.17 (dd, J6a′,6b′ = 12.7 Hz, J6a′,5′ = 5.1 Hz, 1H, H6a′), 4.12
(dd, J6a′,6b′ = 12.7 Hz, J6b′,5′ = 1.9 Hz, 1H, H6b′), 2.39−2.22 (m, 4H,
COC H 2 C H2 CH 2 C H3 ), 2. 21 (t, 7. 3 Hz, 2H,
2
3
(EtOAc/hexane 1:1); mp 103−105 °C; [α]26 −57 (c = 0.089,
J
=
D
CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.11 (s, 1H, CHtriazole),
COCH2CH2CH2CH3), 2.10−1.99 (m, 2H, COCH2CH2CH2CH3),
1.53−1.39 (m, 6H, COCH2CH2CH2CH3), 1.33−1.14 (m, 8H,
COCH2CH2CH2CH3), 0.98−0.88 (m, 2H, COCH2CH2CH2CH3)
0.88−0.80 (m, 9H, COCH2CH2CH2CH3), 0.63 (t, J = 7.3 Hz, 3H,
COCH2CH2CH2CH3). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 172.5, 171.9, 171.8,
171.1 (4 × COCH2CH2CH2CH3), 145.7 (C1″/4″), 143.6 (C1″/4″),
133.1 (Ctriazole), 126.4 (2 × CHarom), 125.4 (2 × CHarom), 121.6
(CHtriazole), 84.0 (C1′), 73.4 (C5′), 71.9 (C3′), 70.2 (C2′), 67.4 (C4′),
61.4 (C6′), 33.0, 32.9 (2C), 32.7 (4 × COCH2CH2CH2CH3), 26.32,
26.27 (3C) (4 × COCH2CH2CH2CH3), 21.47, 21.45 (2C), 21.1 (4 ×
3
8.33 (bs, 1H, H2″), 8.04 (bd, J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.82
3
3
(bd, J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.68 (t, J5″,4″/6″ = 7.8 Hz, 1H,
3
H5″), 7.43 (bs, 2H, SO2NH2), 6.42 (d, J1′,2′ = 9.0 Hz, 1H, H1′), 5.68
3
3
3
(dd ≈ t, J3′,2′ = 9.3 Hz, J2′,3′ = 9.0 Hz, 1H, H2′), 5.61 (t, J3′,2′ = 9.3
Hz, 3J3′,4′ = 9.3 Hz, 1H, H3′), 5.19 (dd ≈ t, 3J4′,5′ = 9.7 Hz, 3J4′,3′ = 9.3
3
3
3
Hz, 1H, H4′), 4.43 (ddd, J5′,4′ = 9.7 Hz, J5′,6a′ = 5.4 Hz, J5′,6b′ = 1.7
2
3
Hz, 1H, H5′), 4.18 (dd, J6a′,6b′ = 12.6 Hz, J6a′,5′ = 5.4 Hz, 1H, H6a′),
2
3
4.11 (dd, J6b′,6a′ = 12.6 Hz, J6b′,5′ = 1.7 Hz, 1H, H6b′), 2.04 (s, 3H,
COCH3), 2.01 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.81 (s, 3H,
COCH3). Assignments were confirmed by 1H−1H gCOSY. 13C NMR
(500 MHz, DMSO-d6): δ = 170.0, 169.5, 169.4, 168.6 (4 × COCH3),
145.8 (C1″ or 3″), 145.0 (C1″ or 3″), 130.7 (Ctriazole), 129.9 (C5″), 128.3
(C4″ or 6″), 125.3 (C4″ or 6″), 122.2 (C2″), 121.2 (CHtriazole), 84.1 (C1′),
73.3 (C5′), 72.0 (C3′), 70.4 (C2′), 67.6 (C4′), 61.8 (C6′), 20.5
(COCH3), 20.4 (COCH3), 20.2 (COCH3), 19.9 (COCH3). Assign-
ments were confirmed by 1H−13C gHSQC. LRMS (ESI+): m/z = 555
[M + H]+, 577 [M + Na]+. HRMS (ESI) calcd for C22H26N4O11SNa
577.1211, found 57.1235.
COCH2CH2CH2CH3), 13.48, 13.46, 13.41, 13.3 (4
×
COCH2CH2CH2CH3). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 723 [M + H]+. HRMS (ESI) calcd
for C34H50N4O11SNa 745.3089, found 745.3061.
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-(3-meth-
ylbutyryl)-β-D-glucopyranosyl)-1H-1,2,3-triazole (18). The title
compound 18 was prepared from glucopyranosyl azide 5 (260 mg,
0.48 mmol) and alkyne 11 (96 mg, 0.53) according to general
procedure 1 in 3 days. Purification of the crude product by flash
chromatography (EtOAc/hexane 3:7 to 1:1) afforded 18 (65 mg,
23%) as a white solid; Rf = 0.08 (EtOAc/hexane 3:7); mp >240 °C;
[α]25D −49 (c = 0.072, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ =
9.08 (s, 1H, CHtriazole), 8.00 (bd, 3J = 8.4 Hz, 2H, CHarom), 7.91 (bd, 3J
= 8.4 Hz, 2H, CHarom), 7.39 (s, 2H, SO2NH2), 6.44 (d, 3J1′,2′ = 8.8 Hz,
1H, H1′), 5.71 (dd, 3J2′,3′ = 9.2 Hz, 3J2′,1′ = 8.8 Hz, 1H, H2′), 5.66 (dd,
3J3′,4′ = 9.3 Hz, 3J3′,2′ = 9.2 Hz, 1H, H3′), 5.23 (dd, 3J4′,5′, = 9.6 Hz, 3J4′,3′
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-propion-
yl-β-D-glucopyranosyl)-1H-1,2,3-triazole (21). The title com-
pound 21 was prepared from glucopyranosyl azide 2 (123 mg, 0.29
mmol) and alkyne 12 (57 mg, 0.31 mmol) according to general
procedure 1 in 5.5 h. Purification of the crude product by flash
chromatography (EtOAc/hexane 2:3) afforded 21 (118 mg, 67%) as a
white solid; Rf = 0.13 (EtOAc/hexane 2:3); mp 78−85 °C (dec.);
[α]26D −42 (c = 0.067, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ =
9.13 (s, 1H, CHtriazole), 8.33 (bs, 1H, H2″), 8.03 (bd, 3J4″/6″,5″ = 7.8 Hz,
= 9.3 Hz, 1H, H4′), 4.44 (ddd, 3J5′,4′ = 9.6 Hz, 3J5′,6a′ = 4.8 Hz, 3J5′,6b′
=
3
1H, H4″ or 6″), 7.82 (bd, J4″/6″,5″ = 7.8 Hz, 1H, H4″ or 6″), 7.68 (t,
2.6 Hz, 1H, H5′), 4.18−4.09 (m, 2H, H6a′/6b′) 2.27−2.22 (m, 1H,
COCH2CH(CH3)2), 2.20−2.14 (m, 3H, COCH2CH(CH3)2), 2.10 (d,
J = 6.9 Hz, 2H, COCH2CH(CH3)2), 1.99−1.84 (m, 5H,
COCH2CH(CH3)2), 1.68−1.58 (m, 1H, COCH2CH(CH3)2), 0.91−
0.88 (m, 6H, COCH2CH(CH3)2), 0.85 (d, J = 6.6 Hz, 12H,
COCH2CH(CH3)2), 0.57 (d, J = 6.6 Hz, 6H, COCH2CH(CH3)2).
Assignments were confirmed by 1H−1H gCOSY. 13C NMR (500
MHz, DMSO-d6): δ = 171.8, 171.1, 171.0, 170.3 (4 × COCH2CH-
(CH3)2), 145.8 (C1″/4″), 143.6 (C1″/4″), 133.2 (Ctriazole), 126.5 (2 ×
CHarom), 125.5 (2 × Carom), 121.7 (CHtriazole), 84.1 (C1′), 73.4 (C5′),
71.8 (C3′), 70.2 (C2′), 67.4 (C4′), 61.4 (C6′), 42.29, 42.25, 42.18, 42.0
3
3J5″,4″/6″ = 7.8 Hz, 1H, H5″), 7.43 (bs, 2H, SO2NH2), 6.44 (d, J1′,2′
=
9.1 Hz, 1H, H1′), 5.72 (dd ≈ t, 3J2′,3′ = 9.3 Hz, 3J2′,3′ = 9.1 Hz, 1H, H2′),
3
3
5.64 (dd ≈ t, J3′,2′ = 9.3 Hz, J3′,4′ = 9.5 Hz, 1H, H3′), 5.23 (dd ≈ t,
3J4′,5′ = 9.7 Hz, J4′,3′ = 9.5 Hz, 1H, H4′), 4.45 (ddd, J5′,4′ = 9.7 Hz,
3J5′,6a′ = 5.3 Hz, 3J5′,6b′ = 1.7 Hz, 1H, H5′), 4.22 (dd, 2J6a′,6b′ = 12.6 Hz,
3J6a′,5′ = 5.3 Hz, 1H, H6a′), 4.12 (dd, 2J6b′,6a′ = 12.6 Hz, 3J6b′,5′ = 1.7 Hz,
1H, H6b′), 2.40−2.27 (m, 4H, COCH2CH3), 2.26−2.20 (m, 2H,
COCH2CH3), 2.13−2.01 (m, 2H, COCH2CH3), 1.04−0.95 (m, 9H,
COCH2CH3), 0.78 (t, J = 7.5 Hz, 3H, COCH2CH3). Assignments
were confirmed by 1H−1H gCOSY. 13C NMR (500 MHz, DMSO-d6):
3
3
G
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
δ = 173.3, 172.8, 172.6, 172.0 (4 × COCH2CH3), 145.8 (C1″ or 3″),
145.0 (C1″ or 3″), 130.7 (Ctriazole), 129.9 (C5″), 128.3 (C4″ or 6″), 125.3
(C4″ or 6″), 122.1 (C2″), 121.2 (CHtriazole), 84.1 (C1′), 73.4 (C5′), 72.0
(C3′), 70.3 (C2′), 67.3 (C4′), 61.5 (C6′), 26.8, 26.7, 26.6, 26.4 (4 ×
COCH2CH3), 8.91, 8.88, 8.86, 8.8 (4 × COCH2CH3). Assignments
gHSQC. LRMS (ESI+): m/z = 723 [M + H]+. HRMS (ESI) calcd
for C34H50N4O11SNa 745.3089, found 745.3106.
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-(3-meth-
ylbutyryl)-β-D-glucopyranosyl)-1H-1,2,3-triazole (24). The title
compound 24 was prepared from glucopyranosyl azide 5 (260 mg,
0.48 mmol) and alkyne 12 (96 mg, 0.53 mmol) according to general
procedure 1 in 7.5 h . Purification of the crude product by flash
chromatography (EtoAc/hexane 3:7) afforded 24 (258 mg, 74%) as a
1
were confirmed by H−13C gHSQC. LRMS (ESI+): m/z = 611 [M +
H]+. HRMS (ESI) calcd for C26H34N4O11SNa 633.1837, found
633.1826.
white solid; Rf = 0.28 (EtoAc/hexane 2:3); mp 152−153 °C; [α]25
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-butyryl-
β-D-glucopyranosyl)-1H-1,2,3-triazole (22). The title compound
22 was prepared from glucopyranosyl azide 3 (74 mg, 0.15 mmol) and
alkyne 12 (30 mg, 0.17 mmol) according to general procedure 1 in 1
day. Purification of the crude product by flash chromatography
(EtOAc/hexane 2:3) afforded 22 (80 mg, 80%) as a white solid; Rf =
D
−46 (c = 0.072, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.11 (s,
3
1H, CHtriazole), 8.31 (bs, 1H, H2″), 8.00 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.82 (bd, 3J4″/6″,5″ = 7.8 Hz, 1H, H4″ or 6″), 7.68 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.44 (bs, 2H, SO2NH2), 6.43 (d, 3J1′,2′ = 9.1 Hz, 1H,
H1′), 5.74 (dd ≈ t, 3J2′,3′ = 9.3 Hz, 3J2′,3′ = 9.1 Hz, 1H, H2′), 5.67 (dd ≈
t, 3J3′,2′ = 9.3 Hz, 3J3′,4′ = 9.4 Hz, 1H, H3′), 5.24 (dd ≈ t, 3J4′,5′ = 9.7 Hz,
0.24 (EtOAc/hexane 2:3); mp 57−75 °C (dec.); [α]25 −59 (c =
D
3
3
3J4′,3′ = 9.4 Hz, 1H, H4′), 4.44 (ddd, J5′,4′ = 9.7 Hz, J5′,6a′ = 4.7 Hz,
3J5′,6b′ = 2.9 Hz, 1H, H5′), 4.18−4.10 (m, 2H, H6a′/6b′), 2.28−2.21 (m,
1H, COCH2CH(CH3)2), 2.20−2.13 (m, 3H, COCH2CH(CH3)2),
2.10 (d, J = 6.9 Hz, 2H, COCH2CH(CH3)2), 2.00−1.85 (m, 5H,
COCH2CH(CH3)2), 1.68−1.60 (m, 1H, COCH2CH(CH3)2), 0.92−
0.88 (m, 6H, COCH2CH(CH3)2), 0.85 (d, J = 6.6 Hz, 12H,
COCH2CH(CH3)2), 0.58 (d, J = 6.6 Hz, 6H, COCH2CH(CH3)2).
Assignments were confirmed by 1H−1H gCOSY. 13C NMR (500
MHz, DMSO-d6): δ = 171.8, 171.1, 171.0, 170.3 (4 × COCH2CH-
(CH3)2), 145.8 (C1″ or 3″), 145.0 (C1″ or 3″), 130.7 (Ctriazole), 129.8
(C5″), 128.2 (C4″ or 6″), 125.3 (C4″ or 6″), 122.1 (C2″), 121.2 (CHtriazole),
84.1 (C1′), 73.4 (C5′), 71.7 (C3′), 70.2 (C2′), 67.4 (C4′), 61.4 (C6′),
42.3, 42.23, 42.15, 42.2 (4 × COCH2CH(CH3)2), 25.02, 24.97, 24.84,
24.81 (4 × COCH2CH(CH3)2), 21.98 (2C), 21.97 (2C), 21.95, 21.9,
21.53, 21.47 (8 × COCH2CH(CH3)2). Assignments were confirmed
1
0.056, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.12 (s, 1H,
3
CHtriazole), 8.32 (bs, 1H, H2″), 8.02 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.82 (bd, 3J4″/6″,5″ = 7.8 Hz, 1H, H4″ or 6″), 7.68 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.44 (bs, 2H, SO2NH2), 6.43 (d, 3J1′,2′ = 9.1 Hz, 1H,
H1′), 5.73 (dd ≈ t, 3J2′,3′ = 9.3 Hz, 3J2′,3′ = 9.1 Hz, 1H, H2′), 5.66 (dd ≈
t, 3J3′,2′ = 9.3 Hz, 3J3′,4′ = 9.5 Hz, 1H, H3′), 5.24 (dd ≈ t, 3J4′,5′ = 9.7 Hz,
3J4′,3′ = 9.5 Hz, 1H, H4′), 4.45 (ddd, J5′,4′ = 9.7 Hz, J5′,6a′ = 5.2 Hz,
3
3
3J5′,6b′ = 2.0 Hz, 1H, H5′), 4.18 (dd, 2J6a′,6b′ = 12.6 Hz, 3J6a′,5′ = 5.2 Hz,
2
3
1H, H6a′), 4.13 (dd, J6b′,6a′ = 12.6 Hz, J6b′,5′ = 2.0 Hz, 1H, H6b′),
2.36−2.21 (m, 4H, COCH2CH2CH3), 2.19 (t, J = 7.2 Hz, 2H,
COCH2CH2CH3), 2.06−2.01 (m, 2H, COCH2CH2CH3), 1.57−1.43
(m, 6H, COCH2CH2CH3), 1.32−1.20 (m, 2H, COCH2CH2CH3),
0.90−0.80 (m, 9H, COCH2CH2CH3), 0.57 (t, J = 7.4 Hz, 3H,
COCH2CH2CH3). Assignments were confirmed by H−1H gCOSY.
1
13C NMR (500 MHz, DMSO-d6): δ = 172.4, 171.8, 171.6, 170.9 (4 ×
1
by H−13C gHSQC. LRMS (ESI+): m/z = 723 [M + H]+, 745 [M +
COCH2CH2CH3), 145.8 (C1″ or 3″), 145.0 (C1″ or 3″), 130.7 (Ctriazole),
129.8 (C5″), 128.2 (C4″ or 6″), 125.3 (C4″ or 6″), 122.1 (C2″), 121.2
(CHtriazole), 84.1 (C1′), 73.4 (C5′), 71.8 (C3′), 70.2 (C2′), 67.3 (C4′),
61.4 (C6′), 35.14, 35.08, 35.06, 34.8 (4 × COCH2CH2CH3), 17.70
(2C), 17.67 (2C) (4 × COCH2CH2CH3), 13.27, 13.26, 13.2, 12.8 (4
× COCH2CH2CH3). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 689 [M + Na]+. HRMS (ESI) calcd
for C30H42N4O11SNa 689.2463, found 689.2482.
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-pentyryl-
β-D-glucopyranosyl)-1H-1,2,3-triazole (23). The title compound
23 was prepared from glucopyranosyl azide 4 (142 mg, 0.260 mmol)
and alkyne 12 (81 mg, 0.45 mmol) according to general procedure 1
in 4 h. Purification of the crude product by flash chromatography
(CH2Cl2 to CH2Cl2/MeOH 99:1) afforded 23 (104 mg, 55%) as a
Na]+. HRMS (ESI) calcd for C34H50N4O11SNa 745.3089, found
745.3065.
4-[4″-(Aminosulfonyl)phenyl]-1-(β-D-galactopyranosyl)-1H-
1,2,3-triazole (25). The title compound 25 was prepared from
compound 26 (86 mg, 0.16 mmol) according to general procedure 2
and isolated as a white solid (60 mg, 98%); Rf = 0.58 (CH3CN/H2O
9:1). 1H NMR was in agreement with the data reported in the
literature.5h
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-β-
D-galactopyranosyl)-1H-1,2,3-triazole (26). The title compound
26 was prepared from galactopyranosyl azide 6 (168 mg, 0.45 mmol)
and alkyne 11 (90 mg, 0.50 mmol) according to general procedure 1.
Purification of the crude product by flash chromatography (EtOAc/
1
hexane 3:2) afforded 26 (189 mg, 76% yield) as a white solid. H
white solid; Rf = 0.26 (CH2Cl2/MeOH 99:1); mp 107−111 °C; [α]25
NMR was in agreement with the data reported in the literature.22
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-propion-
yl-β-D-galactopyranosyl)-1H-1,2,3-triazole (27). The title com-
pound 27 was prepared from galactopyranosyl azide 7 (178 mg, 0.42
mmol) and alkyne 11 (81 mg, 0.45 mmol) according to general
procedure 1 in 5 h. Purification of the crude product by flash
chromatography (EtOAc/hexane 1:1) afforded 27 (181 mg, 71%
yield) as a white foam; Rf = 0.10 (EtOAc/hexane 2:3); [α]25D −45 (c =
D
−34 (c = 0.056, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.11 (s,
3
1H, CHtriazole), 8.32 (bs, 1H, H2″), 8.02 (bd, J4″/6″,5″ = 7.8 Hz, 1H,
H
4″ or 6″), 7.81 (bd, 3J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.68 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.43 (bs, 2H, SO2NH2), 6.43 (d, 3J1′,2′ = 9.1 Hz, 1H,
H1′), 5.72 (dd ≈ t, 3J2′,3′ = 9.3 Hz, 3J2′,3′ = 9.1 Hz, 1H, H2′), 5.65 (dd ≈
t, 3J3′,2′ = 9.3 Hz, 3J3′,4′ = 9.5 Hz, 1H, H3′), 5.23 (dd ≈ t, 3J4′,5′ = 9.7 Hz,
3J4′,3′ = 9.5 Hz, 1H, H4′), 4.44 (ddd, J5′,4′ = 9.7 Hz, J5′,6a′ = 5.1 Hz,
3
3
3J5′,6b′ = 2.1 Hz, 1H, H5′), 4.17 (dd, 2J6a′,6b′ = 12.6 Hz, 3J6a′,5′ = 5.1 Hz,
1
0.089, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.03 (s, 1H,
2
3
3
3
1H, H6a′), 4.12 (dd, J6b′,6a′ = 12.6 Hz, J6b′,5′ = 2.1 Hz, 1H, H6b′),
2.38−2.23 (m, 4H, COCH2CH2CH2CH3), 2.21 (t, J = 7.4 Hz, 2H,
COCH2CH2CH2CH3), 2.08−2.01 (m, 2H, COCH2CH2CH2CH3),
1.52−1.40 (m, 6H, COCH2CH2CH2CH3), 1.30−1.17 (m, 8H,
COCH2CH2CH2CH3), 0.99−0.89 (m, 2H, COCH2CH2CH2CH3)
0.88−0.79 (m, 9H, COCH2CH2CH2CH3), 0.64 (t, J = 7.3 Hz, 3H,
COCH2CH2CH2CH3). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 172.5, 171.9, 171.7,
171.1 (4 × COCH2CH2CH2CH3), 145.8 (C1″ or 3″), 145.0 (C1″ or 3″),
130.7 (Ctriazole), 129.8 (C5″), 128.2 (C4″ or 6″), 125.3 (C4″ or 6″), 122.1
(C2″), 121.1 (CHtriazole), 84.1 (C1′), 73.4 (C5′), 71.9 (C3′), 70.2 (C2′),
CHtriazole), 8.11 (bd, J = 8.6 Hz, 2H, CHarom), 7.91 (bd, J = 8.6 Hz,
2H, CHarom), 7.38 (s, 2H, SO2NH2), 6.37 (d, 3J1′,2′ = 9.2 Hz, 1H, H1′),
5.68 (dd ≈ bt, 3J2′,3′ = 10.1 Hz, 3J2′,1′ = 9.2 Hz, 1H, H2′), 5.56 (dd, 3J3′,2′
= 10.1 Hz, 3J3′,4′ = 3.5 Hz, 1H, H3′), 5.48 (bd, 3J4′,3′ = 3.4 Hz, 1H, H4′),
3
3
4.68 (dd ≈ bt, J5′3,6a′ = 7.2 Hz, J5′,6b′ = 5.6 Hz, 1H, H5′), 4.18 (dd,
2J6a′,6b′ = 11.5 Hz, J6a′,5′ = 5.6 Hz, 1H, H6b′), 4.07 (dd, J6a′,6b′ = 11.5
Hz, J6a′,5′ = 7.2 Hz, 1H, H6a′), 2.56−2.51 (m, 2H, COCH2CH3),
2
3
2.33−2.26 (m, 2H, COCH2CH3), 2.24−2.51 (m, 2H, COCH2CH3),
2.18−2.02 (m, 2H, COCH2CH3), 1.15 (t, J = 7.5 Hz, 3H,
COCH2CH3), 1.01−0.95 (m, 6H, 2 × COCH2CH3), 0.78 (t, J = 7.5
Hz, 3H, COCH2CH3). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 173.22, 173.16, 172.4,
172.0 (4 × COCH2CH3), 145.7 (C1″/4″), 143.6 (C1″/4″), 133.1
(Ctriazole), 126.4 (2 × CHarom), 125.6 (2 × Carom), 121.8 (CHtriazole),
84.5 (C1′), 73.0 (C5′), 70.4 (C3′), 67.8 (C2′), 67.1 (C4′), 61.2 (C6′),
26.8, 26.7, 26.54, 26.47 (4 × COCH2CH3), 9.2, 8.9, 8.8, 8.7 (4 ×
67.4 (C4′), 61.5 (C6′), 33.0, 32.93, 32.91, 32.7 (4
COCH2CH2CH2CH3), 26.32, 26.29 (2C), 26.28 (4
COCH2CH2CH2CH3), 21.48, 21.45 (2C), 21.1 (4
COCH2CH2CH2CH3 ), 13.49, 13.47, 13.4, 13.3 (4
×
×
×
×
COCH2CH2CH2CH3). Assignments were confirmed by 1H−13C
H
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
COCH2CH3). Assignments were confirmed by 1H−13C gHSQC.
LRMS (ESI+): m/z = 633 [M + Na]+. HRMS (ESI) calcd for
C26H34N4O11SNa 633.1837, found 633.1811.
(219 mg, 0.40 mmol) and alkyne 11 (96 mg, 0.53 mmol) according to
general procedure 1 in 7.5 h. Purification of the crude product by flash
chromatography (EtOAc/hexane 3:7) afforded 30 (93 mg, 32% yield)
as an off-white solid; Rf = 0.09 (EtOAc/hexane 3:7); mp 152−154 °C;
[α]26D −53 (c = 0.072, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ =
9.00 (s, 1H, CHtriazole), 8.09 (d, 3J = 8.2 Hz, 2H, CHarom), 7.91 (d, 3J =
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-butyryl-
β-D-galactopyranosyl)-1H-1,2,3-triazole (28). The title compound
28 was prepared from galactopyranosyl azide 8 (181 mg, 0.37 mmol)
and alkyne 11 (74 mg, 0.41 mmol) according to general procedure 1
in 5 h. Purification of the crude product by flash chromatography
(EtOAc/hexane 2:3) afforded 28 (181 mg, 71% yield) as a white solid;
Rf = 0.10 (EtOAc/hexane 2:3); mp 78−88 °C (dec.); [α]25D −42 (c =
3
8.2 Hz, 2H, CHarom), 7.39 (s, 2H, SO2NH2), 6.37 (d, J1′,2′ = 9.2 Hz,
1H, H1′), 5.72 (dd ≈ bt, 3J2′,3′ = 10.1 Hz, 3J2′,1′ = 9.3 Hz, 1H, H2′), 5.57
(dd, 3J3′,2′ = 10.1 Hz, 3J3′,4′ = 3.1 Hz, 1H, H3′), 5.49 (bd, 3J4′,3′ = 3.1 Hz,
1H, H4′), 4.66 (dd ≈ bt, 3J5′,6a′ = 6.1 Hz, 3J5′,6b′ = 6.1 Hz, 1H, H5′), 4.11
1
3
0.072, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.01 (s, 1H,
(d, J6a′,5′ = 6.1 Hz, 2H, H6a′/6b′), 2.40 (m, 2H, COCH2CH(CH3)2),
3
3
CHtriazole), 8.10 (bd, J = 8.4 Hz, 2H, CHarom), 7.90 (bd, J = 8.4 Hz,
2H, CHarom), 7.38 (s, 2H, SO2NH2), 6.36 (d, 3J1′,2′ = 9.2 Hz, 1H, H1′),
5.69 (dd ≈ bt, 3J2′,3′ = 10.2 Hz, 3J2′,1′ = 9.2 Hz, 1H, H2′), 5.56 (dd, 3J3′,2′
= 10.2 Hz, 3J3′,4′ = 3.4 Hz, 1H, H3′), 5.48 (bd, 3J4′,3′ = 3.4 Hz, 1H, H4′),
2.15 (d, J = 7.9 Hz, COCH2CH(CH3)2), 2.13−2.04 (m, 3H,
COCH2CH(CH3)2, COCH2CH(CH3)2), 1.98−1.86 (m, 4H, 2 ×
COCH2CH(CH3)2, COCH2CH(CH3)2), 1.70−1.59 (m, 1H,
COCH2CH(CH3)2), 1.02−0.98 (m, 6H, COCH2CH(CH3)2), 0.88−
0.81 (m, 12H, 2 × COCH2CH(CH3)2), 0.63−0.55 (m, 6H,
COCH2CH(CH3)2). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 171.7, 171.6, 171.0,
170.2 (4 × COCH2CH(CH3)2), 145.7 (C1″/4″), 143.5 (C1″/4″), 133.2
(Ctriazole), 126.4 (2 × CHarom), 125.6 (2 × CHarom), 121.8 (CHtriazole),
84.6 (C1′), 73.1 (C5′), 70.3 (C3′), 67.6 (C2′), 67.1 (C4′), 61.2 (C6′),
42.32, 42.29, 42.2, 42.0 (4 × COCH2CH(CH3)2), 25.2, 25.11, 25.07,
24.8 (4 × COCH2CH(CH3)2), 21.98 (2C), 21.92 (4C), 21.5, 21.4 (8
× COCH2CH(CH3)2). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 723 [M + H]+. HRMS (ESI) calcd for
C34H50N4O11SNa 745.3089, found 745.3087.
3
3
4.67 (dd ≈ bt, J5′3,6a′ = 7.2 Hz, J5′,6b′ = 5.5 Hz, 1H, H5′), 4.18 (dd,
2J6a′,6b′ = 11.5 Hz, J6a′,5′ = 5.5 Hz, 1H, H6b′), 4.07 (dd, J6a′,6b′ = 11.5
Hz, J6a′,5′ = 7.2 Hz, 1H, H6a′), 2.49−2.45 (m, 2H, COCH2CH2CH3),
2
3
2.25 (t, J = 7.4 Hz, 2H, COCH2CH2CH3), 2.22−2.13 (m, 2H,
COCH2CH2CH3), 2.13−1.99 (m, 2H, COCH2CH2CH3), 1.70−1.61
(m, 2H, COCH2CH2CH3), 1.53−1.43 (m, 4H, 2 × COCH2CH2CH3),
1.34−1.20 (m, 2H, COCH2CH2CH3), 0.97 (t, J = 7.4 Hz, 3H,
COCH2CH2CH3), 0.87−0.82 (m, 6H, 2 × COCH2CH2CH3), 0.57 (t,
J = 7.4 Hz, 3H, COCH2CH2CH3). Assignments were confirmed by
1H−1H gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 172.33, 172.29,
171.6, 171.0 (4 × COCH2CH2CH3), 145.7 (C1″/4″), 143.6 (C1″/4″),
133.2 (Ctriazole), 126.4 (2 × CHarom), 125.7 (2 × CHarom), 121.8
(CHtriazole), 84.6 (C1′), 73.1 (C5′), 70.3 (C3′), 67.7 (C2′), 67.1 (C4′),
61.2 (C6′), 35.3, 35.2, 35.1, 34.8 (4 × COCH2CH2CH3), 18.1, 17.79,
17.78, 17.6 (4 × COCH2CH2CH3), 13.3, 13.2 (2C), 12.8 (4 ×
4-[3″-(Aminosulfonyl)phenyl]-1-(β-D-galactopyranosyl)-1H-
1,2,3-triazole (31). The title compound 31 was prepared from
triazole 32 (58 mg, 0.11 mmol) according to general procedure 2 and
isolated as a white solid (38 mg, 93%); Rf = 0.65 (CH3CN/H2O 9:1);
1
COCH2CH2CH3). Assignments were confirmed by H−13C gHSQC.
1
mp 124−126 °C; [α]22 +7 (c = 0.013, CH3OH). H NMR (500
D
LRMS (ESI+): m/z = 689 [M + Na]+. HRMS (ESI) calcd for
C30H42N4O11SNa 689.2463, found 689.2435.
MHz, DMSO-d6): δ = 8.90 (s, 1H, CHtriazole), 8.40 (bs, 1H, H2″), 8.10
3
3
(bd, J4″/6″,5″ = 7.5 Hz, 1H, H4″ or 6″), 7.79 (bd, J4″/6″,5″ = 7.8 Hz, 1H,
H
4″ or 6″), 7.65 (t, 3J5″,4″/6″ = 7.5 Hz, 1H, H5″), 7.41 (bs, 2H, SO2NH2),
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-pentyryl-
β-D-galactopyranosyl)-1H-1,2,3-triazole (29). The title compound
29 was prepared from galactopyranosyl azide 9 (158 mg, 0.29 mmol)
and alkyne 11 (91 mg, 0.50 mmol) according to general procedure 1
in 5 h. Purification of the crude product by flash chromatography
(EtOAc/hexane 3:7) afforded 29 (144 mg, 68% yield) as a white solid;
Rf = 0.07 (EtOAc/hexane 3:7); mp 76−79 °C; [α]27D −47 (c = 0.089,
CHCl3). 1H NMR (600 MHz, DMSO-d6): δ = 9.01 (s, 1H, CHtriazole),
5.55 (d, 3J1′,2′ = 9.2 Hz, 1H, H1′), 5.30−5.26 (m, 1H, OH), 5.08−5.03
(m, 1H, OH), 4.71−4.64 (m, 2H, 2 × OH), 4.13−4.07 (m, 1H, H2′),
3.82−3.78 (m, 1H, H5′), 3.78−3.75 (m, 1H, H4′), 3.62−3.49 (m, 3H,
H
3′, 6a′, 6b′). Assignments were confirmed by 1H−1H gCOSY. 13C
NMR (500 MHz, DMSO-d6): δ = 145.2 (C1″ or 3″), 144.9 (C1″ or 3″),
131.3 (Ctriazole), 129.6 (C5″), 128.3 (C4″ or 6″), 124.8 (C4″ or 6″), 122.1
(C2″), 121.0 (CHtriazole), 88.4 (C1′), 78.5 (C4′), 73.6 (C3′), 69.4 (C2′),
68.4 (C5′), 60.4 (C6′). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 409 [M + Na]+. HRMS (ESI) calcd for
C14H18N4O7SNa 409.0788, found 409.0786.
3
3
8.10 (d, J = 8.6 Hz, 2H, CHarom), 7.91 (d, J = 8.6 Hz, 2H, CHarom),
3
7.39 (s, 2H, SO2NH2), 6.36 (d, J1′,2′ = 9.3 Hz, 1H, H1′), 5.70 (dd ≈
bt, 3J2′,3′ = 10.2 Hz, 3J2′,1′ = 9.3 Hz, 1H, H2′), 5.56 (dd, 3J3′,2′ = 10.2 Hz,
3J3′,4′ = 3.5 Hz, 1H, H3′), 5.48 (bd, 3J4′,3′ = 3.5 Hz, 1H, H4′), 4.66 (dd ≈
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-acetyl-β-
D-galactopyranosyl)-1H-1,2,3-triazole (32). The title compound
32 was prepared from galactopyranosyl azide 6 (122 mg, 0.33 mmol)
and alkyne 12 (65 mg, 0.36 mmol) according to general procedure 1
in 4 h. Purification of the crude product by flash chromatography
(EtOAc/hexane 3:2) afforded 32 (151 mg, 83%) as a white solid; Rf =
3
3
2
bt, J5′,6a′ = 7.3 Hz, J5′,6b′ = 5.6 Hz, 1H, H5′), 4.13 (dd, J6a′,6b′ = 11.6
Hz, 3J6a′,5′ = 5.6 Hz, 1H, H6b′), 4.07 (dd, 2J6a′,6b′ = 11.6 Hz, 3J6a′,5′ = 7.3
Hz, 1H, H6a′), 2.53−2.50 (m, 2H, COCH2CH2CH2CH3), 2.29−2.25
(m, 2H, COCH2 CH2 CH2 CH3 ), 2.22−2.17 (m, 2H,
COCH2CH2CH2CH3), 2.12−2.02 (m, 2H, COCH2CH2CH2CH3),
1.65−1.59 (m, 2H, COCH2CH2CH2CH3), 1.49−1.41 (m, 4H, 2 ×
COCH2CH2CH2CH3), 1.41−1.36 (m, 2H, COCH2CH2CH2CH3),
1.27−1.20 (m, 6H, 2 × COCH2CH2CH2CH3, COCH2CH2CH2CH3),
0.95−0.90 (m, 5H, COCH2CH2CH2CH3, COCH2CH2CH2CH3), 0.83
(t, J = 7.4 Hz, 3H, COCH2CH2CH2CH3), 0.80 (t, J = 7.4 Hz, 3H,
0.21 (EtOAc/hexane 3:2); mp 120−145 °C (dec.); [α]26 −25 (c =
D
0.1, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.05 (s, 1H,
3
CHtriazole), 8.42 (bs, 1H, H2″), 8.13 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.81 (bd, 3J4″/6″,5″ = 7.8 Hz, 1H, H4″ or 6″), 7.67 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.43 (bs, 2H, SO2NH2), 6.35 (d, 3J1′,2′ = 9.2 Hz, 1H,
H1′), 5.64 (dd ≈ t, 3J2′,3′ = 10.1 Hz, 3J2′,3′ = 9.2 Hz, 1H, H2′), 5.52 (dd,
3J3′,2′ = 10.1 Hz, 3J3′,4′ = 3.2 Hz, 1H, H3′), 5.46 (bd, 3J4′,3′ = 3.2 Hz, 1H,
COC H2 C H2 C H2 CH 3 ), 0. 63 (t,
J = 7. 4 Hz, 3H,
COCH2CH2CH2CH3). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 172.40, 172.37,
171.7, 171.0 (COCH2CH2CH2CH3), 145.7 (C1″/4″), 143.5 (C1″/4″),
133.2 (Ctriazole), 126.3 (2 × CHarom), 125.6 (2 × Carom), 121.8
(CHtriazole), 84.5 (C1′), 73.0 (C5′), 70.3 (C3′), 67.7 (C2′), 67.1 (C4′),
61.1 (C6′), 33.04, 32.99, 32.9, 32.7 (4 × COCH2CH2CH2CH3), 26.6,
26.38, 26.35, 26.2 (4 × COCH2CH2CH2CH3), 21.5, 21.44, 21.41, 21.0
(4 × COCH2CH2CH2CH3), 13.52, 13.45, 13.40, 13.3 (4 ×
COCH2CH2CH2CH3). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 723 [M + H]+, 745 [M + Na]+.
HRMS (ESI) calcd for C34H50N4O11SNa 745.3089, found 745.3089.
4-[4″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-(3-meth-
ylbutyryl)-β-D-galactopyranosyl)-1H-1,2,3-triazole (30). The
title compound 30 was prepared from galactopyranosyl azide 10
3
3
H4′), 4.65 (dd ≈ bt, J5′,6a′ = 7.3 Hz, J5′,6b′ = 5.0 Hz, 1H, H5′), 4.17
(dd, J6a′,6b′ = 11.6 Hz, J6a′,5′ = 5.0 Hz, 1H, H6b′), 4.07 (dd, J6a′,6b′
2
3
2
=
3
11.6 Hz, J6a′,5′ = 7.3 Hz, 1H, H6a′), 2.23, 2.00, 1.96, 1.83 (4 × s, 4 ×
3H, 4 × COCH3). Assignments were confirmed by H−1H gCOSY.
1
13C NMR (500 MHz, DMSO-d6): δ = 170.0, 169.88, 169.4, 168.6 (4 ×
COCH3), 145.7 (C1″ or 3″), 144.9 (C1″ or 3″), 130.7 (Ctriazole), 129.7
(C5), 128.5 (C4″ or 6″), 125.2 (C4″ or 6″), 122.3 (C2″), 121.3 (CHtriazole),
84.6 (C1′), 73.0 (C5′), 70.0 (C3′), 68.0 (C2′), 67.3 (C4′), 61.5 (C6′),
20.44, 20.40, 20.3, 20.0 (4 × COCH3). Assignments were confirmed
by H−13C gHSQC. LRMS (ESI+): m/z = 555 [M + H]+, 577 [M +
1
Na]+. HRMS (ESI) calcd for C22H26N4O11SNa 577.1211, found
577.1221.
I
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-propion-
yl-β-D-galactopyranosyl)-1H-1,2,3-triazole (33). The title com-
pound 33 was prepared from galactopyranosyl azide 7 (177 mg, 0.41
mmol) and alkyne 12 (82 mg, 0.45 mmol) according to general
procedure 1 in 4 h. Purification of the crude product by flash
chromatography (EtOAc/hexane 1:1) afforded 33 (178 mg, 71%) as a
white solid; Rf = 0.27 (EtOAc/hexane 1:1); mp 78−89 °C (dec.);
[α]26D −22 (c = 0.067, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ =
9.03 (s, 1H, CHtriazole), 8.41 (bs, 1H, H2″), 8.12 (bd, 3J4″/6″,5″ = 7.8, Hz,
H1′), 5.70 (dd ≈ t, 3J2′,3′ = 10.2 Hz, 3J2′,3′ = 9.2 Hz, 1H, H2′), 5.56 (dd,
3J3′,2′ = 10.2 Hz, 3J3′,4′ = 3.2 Hz, 1H, H3′), 5.48 (bd, 3J4′,3′ = 3.1 Hz, 1H,
3
3
H4′), 4.66 (dd ≈ bt, J5′,6a′ = 6.3 Hz, J5′,6b′ = 6.3 Hz, 1H, H5′), 4.16−
4.05 (m, 2H, H6a′, H6b′), 2.53−2.50 (m, 2H, COCH2CH2CH2CH3),
2.27 (t, J = 7.2 Hz, 2H, COCH2CH2CH2CH3), 2.23−2.16 (m, 2H,
COCH2CH2CH2CH3), 2.13−2.02 (m, 2H, COCH2CH2CH2CH3),
1.66−1.57 (m, 2H, COCH2CH2CH2CH3), 1.50−1.42 (m, 4H, 2 ×
COCH2CH2CH2CH3), 1.42−1.34 (m, 2H, COCH2CH2CH2CH3),
1.29−1.17 (m, 6H, 2 × COCH2CH2CH2CH3, COCH2CH2CH2CH3),
1.00−0.93 (m, 2H, COCH2CH2CH2CH3), 0.92 (t, J = 7.3 Hz, 3H,
3
1H, H4″ or 6″), 7.81 (bd, J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.67 (t,
3
3J5″,4″/6″ = 7.8 Hz, 1H, H5″), 7.42 (bs, 2H, SO2NH2), 6.36 (d, J1′,2′
=
C O C H 2 C H 2 C H 2 C H 3 ) , 0 . 8 6 − 0 . 7 6 ( m , 6 H ,
2
×
3
3
9.2 Hz, 1H, H1′), 5.68 (dd ≈ t, J2′,3′ = 10.1 Hz, J2′,3′ = 9.2 Hz, 1H,
C O C H2 CH2 C H2 CH 3 ) , 0 . 6 4 ( t , = 7 . 3 Hz, 3H,
J
3
3
COCH2CH2CH2CH3). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 172.42, 172.40,
171.7, 171.1 (4 × COCH2CH2CH2CH3), 145.7 (C1″ or 3″), 144.9
(C1″ or 3″), 130.7 (Ctriazole), 129.7 (C5″), 128.6 (C4″ or 6″), 125.2
(C4″ or 6″), 122.2 (C2″), 121.3 (CHtriazole), 84.5 (C1′), 73.0 (C5′), 70.3
(C3′), 67.7 (C2′), 67.1 (C4′), 61.1 (C6′), 33.02, 32.99, 32.90, 32.7 (4 ×
H2′), 5.56 (dd, J3′,2′ = 10.1 Hz, J3′,4′ = 3.3 Hz, 1H, H3′), 5.48 (bd,
3J4′,3′ = 3.3 Hz, 1H, H4′), 4.68 (dd ≈ bt, J5′,6a′ = 7.1 Hz, J5′,6b′ = 5.6
3
3
2
3
Hz, 1H, H5′), 4.18 (dd, J6a′,6b′ = 11.5 Hz, J6a′,5′ = 5.6 Hz, 1H, H6b′),
2
3
4.07 (dd, J6a′,6b′ = 11.5 Hz, J6a′,5′ = 7.1 Hz, 1H, H6a′), 2.56−2.51 (m,
2H, COCH2CH3), 2.32−2.27 (m, 2H, COCH2CH3), 2.26−2.17 (m,
2H, COCH2CH3), 2.17−2.02 (m, 2H, COCH2CH3), 1.15 (t, J = 7.5
Hz, 3H, COCH2CH3), 1.03−0.94 (m, 6H, COCH2CH3), 0.79 (t, J =
COCH2 CH2CH2CH3), 26.6, 26.38, 26.36, 26.2 (4
COCH2CH2CH2CH3), 21.5, 21.44, 21.41, 21.1 (4
COCH2CH2CH2CH3), 13.54, 13.46, 13.41, 13.3 (4
×
×
×
1
7.5 Hz, 3H, COCH2CH3). Assignments were confirmed by H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 173.23, 173.16, 172.4,
172.0 (4 × COCH2CH3), 145.7 (C1″ or 3″), 144.9 (C1″ or 3″), 130.7
(Ctriazole), 129.7 (C5″), 128.6 (C4″ or 6″), 125.2 (C4″ or 6″), 122.2 (C2″),
121.3 (CHtriazole), 84.5 (C1′), 73.0 (C5′), 70.3 (C3′), 67.9 (C2′), 67.1
(C4′), 61.2 (C6′), 26.8, 26.7, 26.6, 26.5 (4 × COCH2CH3), 9.2, 8.9, 8.8,
8.7 (4 × COCH2CH3). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 633 [M + Na]+. HRMS (ESI) calcd for
C26H34N4O11SNa 633.1837, found 633.1851.
COCH2CH2CH2CH3). Assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 723 [M + H]+. HRMS (ESI) calcd
for C34H50N4O11SNa 745.3089, found 745.3104.
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-(3-meth-
ylbutyryl)-β-D-galactopyranosyl)-1H-1,2,3-triazole (36). The
title compound 36 was prepared from galactopyranosyl azide 10
(255 mg, 0.47 mmol) and alkyne 12 (96 mg, 0.53 mmol) according to
general procedure 1 in 4 h. Purification of the crude product by flash
chromatography (EtOAc/hexane 3:7) afforded 36 278 mg, 82%) as an
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-butyryl-
β-D-galactopyranosyl)-1H-1,2,3-triazole (34). The title compound
34 was prepared from galactopyranosyl azide 8 (181 mg, 0.37 mmol)
and alkyne 12 (76 mg, 0.42 mmol) according to general procedure 1
in 4 h. Purification of the crude product by flash chromatography
(EtOAc/hexane 2:3) afforded 34 (195 mg, 79%) as a white solid; Rf =
off-white solid; Rf = 0.30 (EtOAc/hexane 2:3); mp 76−80 °C; [α]27
D
−34 (c = 0.089, CHCl3). 1H NMR (500 MHz, DMSO-d6): δ = 9.01 (s,
3
1H, CHtriazole), 8.39 (bs, 1H, H2″), 8.10 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.81 (bd, 3J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.67 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.43 (bs, 2H, SO2NH2), 6.36 (d, 3J1′,2′ = 9.2 Hz, 1H,
H1′), 5.72 (dd ≈ t, 3J2′,3′ = 10.2 Hz, 3J2′,1′ = 9.2 Hz, 1H, H2′), 5.57 (dd,
3J3′,2′ = 10.2 Hz, 3J3′,4′ = 3.1 Hz, 1H, H3′), 5.49 (bd, 3J4′,3′ = 3.1 Hz, 1H,
H4′), 4.66 (dd ≈ bt, 3J5′,6a′ = 6.3 Hz, 3J5′,6b′ = 6.3 Hz, 1H, H5′), 4.11 (d,
3J6a′/6b′,5′ = 6.3 Hz, 2H, H6a′/6b′), 2.42−2.38 (m, 2H, COCH2CH-
0.22 (EtOAc/hexane 2:3); mp 58−70 °C (dec.); [α]26 −35 (c =
D
1
0.072, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.02 (s, 1H,
3
CHtriazole), 8.40 (bs, 1H, H2″), 8.11 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.81 (bd, 3J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.67 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.43 (bs, 2H, SO2NH2), 6.36 (d, 3J1′,2′ = 9.2 Hz, 1H,
H1′), 5.69 (dd ≈ t, 3J2′,3′ = 10.1 Hz, 3J2′,3′ = 9.2 Hz, 1H, H2′), 5.57 (dd,
3J3′,2′ = 10.1 Hz, 3J3′,4′ = 3.2 Hz, 1H, H3′), 5.49 (bd, 3J4′,3′ = 3.2 Hz, 1H,
(CH3)2), 2.18−2.06 (m, 5H,
2 × COCH2CH(CH3)2,
COCH2CH(CH3)2), 1.98−1.87 (m, 4H, 2 × COCH2CH(CH3)2,
COCH2CH(CH3)2), 1.70−162 (m, 1H, COCH2CH(CH3)2), 1.00
(dd, J = 6.6 Hz, J = 2.7 Hz, 6H, COCH2CH(CH3)2), 0.87−0.82 (d, J =
6.6 Hz, 12H, 2 × COCH2CH(CH3)2), 0.60 (d, J = 6.6 Hz, J = 2.7 Hz,
6H, COCH2CH(CH3)2). Assignments were confirmed by 1H−1H
gCOSY. 13C NMR (500 MHz, DMSO-d6): δ = 171.7, 171.6, 171.0,
170.2 (4 × COCH2CH(CH3)2), 145.8 (C1″ or 3″), 144.9 (C1″ or 3″),
130.8 (Ctriazole), 129.7 (C5″), 128.6 (C4″ or 6″), 125.2 (C4″ or 6″), 122.2
(C2″), 121.3 (CHtriazole), 84.6 (C1′), 73.1 (C5′), 70.3 (C3′), 67.6 (C2′),
67.1 (C4′), 61.2 (C6′), 42.30, 42.28, 42.2, 42.0 (COCH2CH(CH3)2),
25.2, 25.11, 25.08, 24.8 (4 × COCH2CH(CH3)2), 22.4 (2C), 21.9
(4C), 21.53, 21.47 (8 × COCH2CH(CH3)2). Assignments were
confirmed by 1H−13C gHSQC. LRMS (ESI+): m/z = 745 [M + Na]+.
HRMS (ESI) calcd for C34H50N4O11SNa 745.3089, found 745.3079.
CA Inhibition Assay. An Applied Photophysics stopped-flow
instrument was used for assaying the CA-catalyzed CO2 hydration
activity as previously reported.5c,20 IC50 values were obtained from
dose response curves working at seven different concentrations of test
compound, by fitting the curves using PRISM (www.graphpad.com)
and nonlinear least-squares methods, values represent the mean of at
least three different determinations. The inhibition constants (Ki) were
then derived by using the Cheng−Prusoff equation,23 as follows: Ki =
IC50/(1 + [s]/Km), where [S] represents the CO2 concentration at
which the measurement was carried out and Km the concentration of
substrate at which the enzyme activity is at half-maximal.
3
3
H4′), 4.67 (dd ≈ bt, J5′,6a′ = 7.2 Hz, J5′,6b′ = 5.5 Hz, 1H, H5′), 4.14
(dd, J6a′,6b′ = 11.4 Hz, J6a′,5′ = 5.5 Hz, 1H, H6b′), 4.09 (dd, J6a′,6b′
2
3
2
=
3
11.4 Hz, J6a′,5′ = 7.2 Hz, 1H, H6a′), 2.50−2.45 (m, 2H,
COCH2CH2CH3), 2.29−2.22 (m, 2H, COCH2CH2CH3) 2.20−2.16
(m, 2H, COCH2CH2CH3), 2.11−2.01 (m, 2H, COCH2CH2CH3),
1.70−1.61 (m, 2H, COCH2CH2CH3), 1.54−1.44 (m, 4H,
COCH2CH2CH3), 1.35−1.21 (m, 2H, COCH2CH2CH3), 0.87−0.80
(m, 9H, COCH2CH2CH3), 0.59 (t, J = 7.4 Hz, 3H, COCH2CH2CH3).
Assignments were confirmed by 1H−1H gCOSY. 13C NMR (500
MHz, DMSO-d6): δ = 172.3, 171.6, 171.0, 170.3 (4 ×
COCH2CH2CH3), 145.7 (C1″ or 3″), 144.9 (C1″ or 3″), 130.7 (Ctriazole),
129.7 (C5″), 128.6 (C4″ or 6″), 125.2 (C4″ or 6″), 122.2 (C2″), 121.3
(CHtriazole), 84.6 (C1′), 73.0 (C5′), 70.3 (C3′), 67.7 (C2′), 67.1 (C4′),
61.1 (C6′), 35.2, 35.1, 35.0, 34.8 (4 × COCH2CH2CH3), 18.1, 17.8
(2C), 17.6 (4 × COCH2CH2CH3), 13.3, 13.2 (2C), 12.8 (4 ×
1
COCH2CH2CH3). Assignments were confirmed by H−13C gHSQC.
LRMS (ESI+): m/z = 667 [M + Na]+, 689 [M + Na]+. HRMS (ESI)
calcd for C30H42N4O11SNa 689.2463, found 689.2494.
4-[3″-(Aminosulfonyl)phenyl]-1-(2′,3′,4′,6′-tetra-O-pentyryl-
β-D-galactopyranosyl)-1H-1,2,3-triazole (35). The title compound
35 was prepared from galactopyranosyl azide 9 (148 mg, 0.27 mmol)
and alkyne 12 (85 mg, 0.47 mmol) according to general procedure 1
in 4 h. Purification of the crude product by flash chromatography
(EtOAc/hexane 3:7) afforded 35 (104 mg, 53%) as a transparent gum;
Rf = 0.15 (EtOAc/hexane 3:7); mp 41−51 °C (dec.); [α]27D −26 (c =
In Vitro Metabolic Stability. Human liver microsomes
(Xenotech, Tokyo, Japan) were suspended in 0.1 M phosphate buffer
(pH 7.4) at a final protein concentration of 0.4 mg/mL and incubated
with compounds (1 μM) at 37 °C. An NADPH-regenerating system
(1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, 1 U/mL glucose-6-
1
0.089, CHCl3). H NMR (500 MHz, DMSO-d6): δ = 9.01 (s, 1H,
3
CHtriazole), 8.40 (bs, 1H, H2″), 8.11 (bd, J4″/6″,5″ = 7.8, Hz, 1H,
H
4″ or 6″), 7.81 (bd, 3J4″/6″,5″ = 7.8, Hz, 1H, H4″ or 6″), 7.67 (t, 3J5″,4″/6″
=
7.8 Hz, 1H, H5″), 7.42 (bs, 2H, SO2NH2), 6.36 (d, 3J1′,2′ = 9.2 Hz, 1H,
J
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
phosphate dehydrogenase) and MgCl2 (0.67 mg/mL) was added to
initiate the metabolic reactions, which were subsequently quenched
with ice-cold acetonitrile at time points ranging from 2−60 min.
Samples were also incubated in the absence of cofactor to monitor for
noncytochrome P450-mediated metabolism in the microsomal matrix.
Samples were then centrifuged and the concentration of parent
compound remaining in the supernatant monitored by LCMS. The
first-order rate constant for substrate depletion was determined by
fitting the data to an exponential decay function and these values were
used to calculate the in vitro intrinsic clearance (CLint).
AUTHOR INFORMATION
Corresponding Authors
*For S.-A.P.: phone, +61 7 3735 7825; e-mail, s.poulsen@
griffith.edu.au.
*For C.T.S.: phone, +39 055 457 3005; e-mail, claudiu.
supuran@unifi.it.
Notes
■
The authors declare no competing financial interest.
In Vitro Plasma Stability. Human plasma was separated from
whole blood procured from the Australian Red Cross Blood Service
and was stored frozen at −80 °C. On the day of the experiment, frozen
plasma was thawed in a water bath maintained at 37 °C and plasma
from three different donors was pooled prior to confirmation of
plasma esterase activity by monitoring the conversion of p-nitrophenyl
acetate to p-nitrophenol. Each test compound was spiked into a
separate aliquot of human plasma to a nominal concentration of 5000
ng/mL (final DMSO and acetonitrile concentrations were 0.2 and
0.4% (v/v), respectively). Spiked plasma was incubated at 37 °C for 4
h, and at various time points, duplicate plasma samples were taken and
immediately snap-frozen in dry ice. All plasma samples were stored
frozen (−20 °C) until analysis by UPLC-MS (using a Waters/
Micromass Xevo triple quadrupole mass spectrometer) relative to
calibration standards prepared in blank human plasma. At each sample
time, the average concentration of test compound (based on duplicate
samples) was expressed as a % compound remaining, and these data
were used to calculate the apparent degradation half-life by fitting to an
exponential decay function.
Chromatographic Human Serum Albumin Binding. Protein
binding characteristics were estimated by correlation of their
chromatographic retention properties on an immobilized human
serum albumin (HSA) column against the characteristics of a series of
standard compounds with known protein binding properties. The
method employed is a gradient HPLC method based on the approach
developed by Valko.21 Chromatographic retention was assessed using
a ChromTech Chiral HSA column (3.0 mm × 50 mm, 5 μm), with
mobile phase comprising a mixture of aqueous ammonium acetate
(pH 7.4) and 2-propanol. The assay uses 13 standard compounds of
known plasma protein binding (PPB) values ranging from 5.4 to
99.5% bound.
Permeability Measurements. Caco-2 cells (passage 31) were
seeded onto 0.3 cm2 polycarbonate filter transwells at a density of
60000 cells/well. Confluent cell monolayers were obtained 21 days
postseeding. The integrity of the cell monolayers was determined by
measuring the transepithelial electrical resistance (TEER), and only
monolayers with TEER values of >300 Ω.cm2 were utilized. The
permeability of nadolol and propranolol (low and high permeability
markers, respectively) was also assessed using a subset of wells from
the same batch as those used to assess the test compounds.
Permeability experiments were performed using Hank’s Balanced
Salt Solution containing 20 mM HEPES (pH 7.4) in both the apical
and basolateral chambers, and permeability was assessed in the apical
to basolateral direction in the absence and presence of 4% BSA in the
basolateral chamber using an initial donor solution concentration of 20
μM for 13, 15 μM for 14, and 8 μM for 15. Test compound solubility
in the transport buffer was confirmed prior to the experiment.
Compound flux was determined over 90 min, with samples taken from
the acceptor chamber at 5, 15, 30, 45, 60, and 90 min. At each sample
time, the volume of acceptor solution removed was replaced with
blank transport buffer, and acceptor concentrations were corrected for
this dilution. Donor samples were taken at the start and completion of
the experiment. The amount of compound transported was
quantitated by LC-MS.
ACKNOWLEDGMENTS
■
This research was financed by the Australian Research Council
(grant no. DP110100071 to S.-A.P. and S.A.C., FT10100185 to
S.-A.P.), Griffith University (Student Scholarship to C.J.C.),
and an EU grant of the seventh framework program (Metoxia
project to C.T.S.).
ABBREVIATIONS USED
■
CuAAC, copper-catalyzed azide alkyne cycloaddition; CA,
carbonic anhydrase; Ki, inhibition constant; HLMs, human
liver microsomes; HSA, human serum albumin; CYP450,
cytochrome P450; cHSA, chromatographic human serum
albumin binding; CLint, in vitro intrinsic clearance; TEER,
transepithelial electrical resistance
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ASSOCIATED CONTENT
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S
* Supporting Information
1H and 13C NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
K
dx.doi.org/10.1021/jm401163e | J. Med. Chem. XXXX, XXX, XXX−XXX

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