Synthesis of saccharin-glycoconjugates targeting carbonic anhydrase using a one-pot cyclization/deprotection strategy

Article abstract of DOI:10.1016/j.carres.2019.03.001

Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity obtained applying the “tail-approach” to derivatize SAC with a methylene triazole linker that connected to a “tail” beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative approach to the synthesis of these compounds featuring a gentle “one pot” deprotection/cyclization as the final synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.

Full text of DOI:10.1016/j.carres.2019.03.001

CarbohydrateResearch476(2019)65–70
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of saccharin-glycoconjugates targeting carbonic anhydrase using a
one-pot cyclization/deprotection strategy
Akilah B. Murraya,1, Marta Quadrib,1, Haoxi Lib, Robert McKennaa, Nicole A. Horensteinb,∗
a Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
b Department of Chemistry, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL, 32611, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors
due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have
shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity
obtained applying the “tail-approach” to derivatize SAC with a methylene triazole linker that connected to a
“tail” beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards
CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in
the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl
saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative
approach to the synthesis of these compounds featuring a gentle “one pot” deprotection/cyclization as the final
synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.
Click chemistry
Benzosulfimide
Saccharin
Carbonic anhydrase
Glucose
Galactose
Inhibitor
1. Introduction
phenomenon results in increased lactic acid being exported from the
cell, decreasing the extracellular pH and increasing the expression of
Carbonic anhydrase IX (CA IX) belongs to a family of zinc me-
talloenzymes that catalyze the interconversion of carbon dioxide and
water to bicarbonate and a proton [1–3]. These enzymes play roles in a
broad range of physiological processes including pH regulation, ion
transport, and signal transduction [4,5]. There are 15 human CA iso-
forms expressed in specific subcellular locations including cytosolic,
mitochondrial and membrane-bound forms [6]. All enzymatically ac-
tive CAs contain a highly conserved active site consisting of a zinc ion
coordinated by three histidine residues (H94, H96, H119, CA II num-
bering) and a zinc-bound solvent (ZBS, H2O/eOH) [7,8]. Due to their
association with various diseases such as glaucoma, epilepsy and
cancer, many of these CA isoforms have become drug targets [9–13].
CA IX is a membrane-bound isoform with an extracellular catalytic
site, and has been associated with metastatic tumors [14,15]. With
these tumor types, rapidly proliferating cancer cells outgrow the blood
supply, causing a reduction in extracellular oxygen availability and
acidification of the extracellular environment, also known as tumor
hypoxia [16,17]. This hallmark of metastatic cancers leads to an al-
teration in cell metabolism from mitochondrial oxidative phosphor-
ylation to anaerobic glycolysis, termed the Warburg effect [18]. This
pH regulators, such as CA IX, to buffer the pH of the tumor micro-
environment [14,15,18]. As such, inhibition of CA IX has been shown to
reduce tumor cell survival and proliferation making it a good anticancer
target [19–21]. Due to this association with tumor hypoxia, CA IX has
also been identified as a biomarker for metastatic cancers [22,23].
Currently, there are over 20 clinically used CA inhibitors, but due to
the highly conserved active site across the isoforms these inhibitors lack
specificity. There is a need to develop isoform-selective inhibitors that
target CA IX as potential anticancer chemotherapeutics [24]. Structural
differences between the isoforms have been exploited to develop more
isoform-specific inhibitors [10,25,26]. For example, CA IX has a wider
active site entrance when compared to CA II, a ubiquitously expressed
isoform, due to a residue variation of valine in CA IX and phenylalanine
in CA II at the 131 position. This difference allows bulkier inhibitors to
bind within the CA IX active site. Also, differing residues ∼15 Å from
the catalytic zinc have been identified, and define a region known as
the “selective pocket” [27]. One of the most successful approaches to
target this region has been the “tail-approach” which includes a zinc
binding group (ZBG) to anchor the compound to the catalytic zinc, a
linker region to extend the compound out of the active site and a tail
Abbreviations: CA, Carbonic Anhydrase; ZBS, zinc-bound solvent; ZBG, zinc-binding group; SAC, saccharin; SBC, saccharin-based compound
∗ Corresponding author.
E-mail address: horen@chem.ufl.edu (N.A. Horenstein).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.carres.2019.03.001
Received 20 December 2018; Received in revised form 23 February 2019; Accepted 2 March 2019
Availableonline19March2019
0008-6215/©2019PublishedbyElsevierLtd.

A.B. Murray, et al.
CarbohydrateResearch476(2019)65–70
was performed with MestReNova 8.1.1. Mass spectra were obtained on
a Hewlett-Packard 5988 A spectrometer or on an Agilent 6220 ESI TOF
(Santa Clara, CA) mass spectrometer equipped with electrospray and
DART sources operated in positive ion mode. The mass spectroscopic
data analyses were performed by the UF Chemistry Department
(Funding from NIH S10 OD021758-01A1).
Methyl 4-nitro-2-sulfamoylbenzoate (3). Following is a variation
of the previously reported method [45]. In an oven dried round bottom
flask flushed with argon, nitro-saccharin (2) (1 g, 4.38 mmol, 1 equiv)
was dissolved in 50 mL dry MeOH, cooled to 0 °C and 0.5 mL of dry TFA
was added dropwise. The ice-bath was then removed, and the reaction
mixture was allowed to warm to rt before refluxing it at 70 °C for 18 h.
Upon no further reaction progress (TLC in hexane/EtOAc 1:1), the re-
action mixture was cooled to rt and the solvent was evaporated under
reduced pressure. The resulting crude was separated from unreacted
starting material by recrystallization from methanol. White crystals
(511 mg, 45%). mp 191–193 °C. 1H NMR (300 MHz, (CD3)2SO) δ 8.74
(d, J = 2.3 Hz, 1H), 8.50 (dd, J = 8.5, 2.3 Hz, 1H), 7.94 (d, J = 8.4 Hz,
1H), 7.76 (br s, 2H), 3.88 (s, 3H); 13C NMR (75 MHz, (CD3)2SO) δ
166.4, 148.3, 142.9, 136.1, 130.6, 126.8, 122.6, 53.4; HRMS (ESI) of
C8H8N2O6S [39]: calcd for [M + Na]+ 283.0001, found 283.0007.
Methyl 4-amino-2-sulfamoylbenzoate (4). [45] Under anhydrous
and inert argon atmosphere, methyl-4-nitro-2-sulfamoylbenzoate (3)
(716 mg, 2.75 mmol, 1 equiv) was dissolved in 50 mL dry MeOH and
hydrogenated at 1 atm over 10% Pd/C (50 mg) at rt. The reaction was
complete by TLC after 1 h (hexane/EtOAc 3:7). The reaction mixture
was filtered through a Celite pad, which was then washed with MeOH.
Evaporation of the solvent under reduced pressure quantitatively af-
forded the desired compound 4, used without further purification. Light
yellow solid (627 mg, yield 99%). 1H NMR (300 MHz, (CD3)2CO) δ 7.72
(d, J = 8.5 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.5, 2.4 Hz,
1H), 6.63 (br s, 2H), 5.84 (br s, 2H), 3.85 (s, 3H); 13C NMR (75 MHz,
(CD3)2CO) δ 168.3, 153.3, 145.8, 134.5, 116.0, 115.5, 114.4, 52.7;
HRMS (ESI) of C8H10N2O4S [M]: calcd for [M + Na]+ 253.0253, found
253.0261.
Fig. 1. Structures of saccharin-glycoconjugates.
moiety to interact with unique residues within the selective pocket
[28–31].
Previous studies have shown that certain artificial sweeteners bind
selectively to CA IX, some with nanomolar affinities [32–35]. The ar-
tificial sweetener saccharin has been used as a ZBG in drug develop-
ment using the “tail-approach” [36,37] along with a methylene triazole
linker and glucose tail moiety (compound 1a, Fig. 1) [38]. The sac-
charin-based compound (SBC) displayed over 1000-fold selectivity for
CA IX over off-target CA isoforms CA I and CA II [38]. This increased
specificity could be attributed to interactions made by the 2′eOH of the
glucose moiety with Gln 67 and Gln 92, as well as an added hydro-
phobic interaction with an O-methylene of the tail sugar and Leu 91,
which is hypothesized to aid in selectivity for CA IX [26,27].
The high selectivity displayed by the saccharin-glycoconjugate 1a
[39] led us to the design of new derivatives characterized by the pre-
sence of a saccharin moiety connected to a sugar with an intervening
triazole spacer, assembled by a Cu-catalyzed click reaction [40–44]. We
focused our attention on modifications affecting the nature of the sugar
scaffold by replacing β-glucose with β-galactose together with in-
creasing the length of the triazole spacer by introducing a two-carbon
ethyl linker between the glyosidic oxygen and the triazole ring (com-
pounds 1b and 1c, Fig. 1). With these modifications, our idea was to
control the binding interactions of the linker and tail region of the
saccharin-sugar compound with CA IX residues.
Methyl 4-azido-2-sulfamoylbenzoate (5). Methyl 4-amino-2-sul-
famoylbenzoate (4) (250 mg, 1.09 mmol, 1 equiv) was dissolved in dry
CH3CN (3 mL) and cooled to 0 °C. Tert-butyl nitrite (194 μl, 1.63 mmol,
1.5 equiv) and trimethylsilyl azide (TMS-N3) (172 μl, 1.30 mmol, 1.2
equiv) were subsequently added dropwise at 0 °C. The resulting mixture
was stirred at 0 °C for 1 h, then let warm to room temperature for 14 h,
at which time TLC showed complete consumption of the starting ma-
terial (hexane/EtOAc 2:1). The solvent was evaporated under vacuum
and the crude product was purified on a silica gel column (hexane/
EtOAc 2:1 to 1:1) to afford the pure compound as a light yellow solid
(259 mg, yield 93%). IR (solid sample, ATR cell) 2126 cm−1 (strong)
ArN3; 1H NMR (300 MHz, CD3OD) δ 7.87 (d, J = 8.3 Hz, 1H), 7.71 (d,
J = 2.4 Hz, 1H), 7.37 (dd, J = 8.3, 2.4 Hz, 1H), 3.94 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 167.0, 144.8, 144.1, 133.2, 125.3, 122.1, 119.5,
53.6; mp 137.5–138.3 °C. HRMS (ESI) of C8H8N4O4S [M]: calcd for [M
+ Na]+ 279.0158, found 279.0167.
2. Materials and methods
2.1. Chemistry
All reagents for chemical synthesis were purchased from Fisher
Scientific (Pittsburg, PA) and Sigma-Aldrich (St. Louis, MO).
Trifluoroacetic acid (TFA) was freshly distilled from phosphorous
pentoxide prior to use. Unless otherwise specified, all reactions were
carried out in anhydrous conditions and under an inert atmosphere of
argon or nitrogen. Reactions were monitored by thin-layer chromato-
graphy (TLC) on glass plates precoated with silica gel 60 (F-254, EMD
Millipore), and visualized with UV light at λ = 254 nm. Glassware was
flame dried or oven dried prior to use. The synthesized compounds
were purified by flash chromatography on silica gel (230–400 mesh
particle size, pore size 60 Å, Sigma-Aldrich). Melting points were ob-
tained on an MFB-595010 M Gallenkamp apparatus equipped with a
digital thermometer. NMR Spectra (1H and 13C) were recorded on a
Varian Mercury-300 (300 and 75.0 MHz) instrument using CDCl3,
(CD3)2CO, (CD3)2SO or D2O as solvent at 20 °C. Chemical shifts are
reported in parts per million (ppm) relative to the peak internal TMS
standard (δ = 0.00 ppm) for CDCl3, CD3OD, (CD3)2CO and (CD3)2SO
and relative to the peak of the solvent for D2O in 1H NMR spectra and
relative to the central peak of the solvent (δ = 77.16 ppm for CDCl3,
29.84 ppm for (CD3)2CO and 39.52 ppm for (CD3)2SO) in 13C NMR
spectra. Abbreviations used for peak multiplicities in 1H NMR are: app,
apparent; s, singlet; br s, broad singlet; d, doublet; t, triplet; m, multi-
plet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of
doublets; ddd, doublet of doublet of doublets. Processing of the spectra
2-Propynyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (7a).
[46] 3 Å molecular sieves (1 g) were placed in an oven dried round
bottom flask flushed with argon. β-D-glucose pentaacetate (5a) (1 g,
2.56 mmol, 1 equiv) was added and dissolved in 18 mL dry CH2Cl2,
followed by addition of propargyl alcohol (298 μL, 5.12 mmol, 2 equiv).
The resulting mixture was cooled to 0 °C and BF3·Et2O (2.53 mL,
20.5 mmol, 8 equiv) was added dropwise. After 16 h at 40 °C the TLC
(hexane/EtOAc 3:2) showed complete consumption of the starting
material. The reaction mixture was diluted with 30 mL CH2Cl2, quen-
ched by addition of solid NaHCO3 (3 g) and stirred at room temperature
for 20 min. Molecular sieves were filtered off and the solution was
subsequently washed with 10% aq. NaHCO3 (x 1). The aqueous bi-
carbonate solution was back-extracted with CH2Cl2 and the combined
organic phases were washed with brine (x 1). The organic phase was
dried over anhydrous Na2SO4, filtered and concentrated under vacuum.
66

A.B. Murray, et al.
CarbohydrateResearch476(2019)65–70
The crude product was purified by silica chromatography eluting with a
step gradient of hexane/EtOAc 4:1 to 1:1. A white solid (590 mg, yield
60%) was obtained after removal of solvents. 1H NMR (300 MHz,
CDCl3) δ 5.25 (app t, J = 9.4 Hz, 1H), 5.10 (app t, J = 9.6 Hz, 1H), 5.02
(dd, J = 9.4, 8.0 Hz, 1H), 4.78 (d, J = 7.9 Hz, 1H), 4.38 (d, J = 2.3 Hz,
2H), 4.32–4.24 (m, 1H), 4.15 (dd, J = 12.3, 2.3 Hz, 1H), 3.73 (ddd,
J = 9.9, 4.5, 2.4 Hz, 1H), 2.47 (t, J = 2.3 Hz, 1H), 2.09 (s, 3H), 2.06 (s,
3H), 2.03 (s, 3H), 2.01 (s, 3H) 1H NMR agreed with the reported data
[46]. HRMS (ESI) of C17H22O10 [M]: calcd for [M + NH4]+ 404.1551,
found 404.1562.
the reaction mixture was diluted with H2O and extracted with CH2Cl2
(x 2). The combined organic phases were dried over anhydrous Na2SO4,
filtered and concentrated under vacuum. The crude was purified by
silica gel column chromatography (CH2Cl2/MeOH 98:2). The product
was obtained as a white solid (142 mg, 95%). 1H NMR (300 MHz,
CDCl3) δ 8.45 (d, J = 2.1 Hz, 1H), 8.19–8.13 (m, 2H), 8.07 (d,
J = 8.4 Hz, 1H), 5.92 (s, 2H), 5.23 (app t, J = 9.5 Hz, 1H), 5.17–4.91
(m, 4H), 4.73 (d, J = 7.9 Hz, 1H), 4.24 (dd, J = 12.3, 4.6 Hz, 1H), 4.17
(dd, J = 12.1, 1.9 Hz, 1H), 4.04 (s, 3H), 3.79–3.71 (m, 1H), 2.08 (s,
3H), 2.03 (s, 6H), 2.01 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.9,
170.2, 169.6, 169.5, 166.8, 145.8, 144.0, 138.9, 132.9, 129.3, 123.1,
121.4, 119.9, 100.3, 72.7, 72.0, 71.3, 68.3, 62.9, 61.9, 53.8, 20.8, 20.7,
20.6 (2C); mp 82.6–85.4 °C; HRMS (ESI) of C25H30N4O14S [M]: calcd
for [M + H]+ 643.1552, found 643.1559; calcd for [M +Na]+
665.1371, found 665.1376.
2-Propynyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (7b).
3 Å molecular sieves (1 g) were placed in an oven dried round bottom
flask flushed with argon. β-D-galactose pentaacetate (5b) (1.5 g,
3.84 mmol, 1 equiv) was added and dissolved in 27 mL dry CH2Cl2,
followed by addition of propargyl alcohol (447 μL, 7.68 mmol, 2 equiv).
The resulting mixture was cooled to 0 °C and BF3·Et2O (3.79 mL,
30.7 mmol, 8 equiv) was added dropwise. After 16 h at rt, TLC (hexane/
EtOAc 1:1) showed complete consumption of the starting material. The
reaction mixture was diluted with 45 mL CH2Cl2, then quenched by
addition of solid NaHCO3 (4.5 g) followed by stirring at room tem-
perature for 20 min. Molecular sieves were filtered off and the solution
was subsequently washed with 10% aq. sol. NaHCO3 (x 1) and brine (x
2). The organic phase was dried over anhydrous Na2SO4, filtered and
concentrated under vacuum. The crude product was purified by silica
gel column chromatography, eluting in hexane/EtOAc 1:1. After re-
moval of solvents, a syrup was obtained which solidified upon standing
(1.17 g, yield 79%). 1H NMR (300 MHz, CDCl3) δ 5.43–5.37 (m, 1H),
5.22 (dd, J = 10.2, 8.2 Hz, 1H), 5.10–5.02 (m, 1H), 4.74 (d, J = 7.9 Hz,
1H), 4.41–4.36 (m, 2H), 4.24–4.08 (m, 2H), 3.94 (t, J = 6.6 Hz, 1H),
2.47 (app s, 1H), 2.15 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H)
(1H NMR matched literature reported [47] spectra); HRMS (ESI) of
C17H22O10 [M]: calcd for [M + NH4]+ 404.1551, found 404.1571;
calcd for [M + Na]+ 409.1105, found 409.1115.
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
triazol-4-yl} methyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside
(8b). In a round bottom flask under nitrogen, 7b (75 mg, 0.19 mmol, 1
equiv) was dissolved in 1.8 mL THF. Azide 5 (50 mg, 0.19 mmol, 1
equiv) was added to the stirred suspension, followed by 0.1 M aq. sol.
sodium ascorbate (16 mg, 0.08 mmol, 0.4 equiv) and 0.1 M aq. sol.
CuSO4·5H2O (10 mg, 0.04 mmol, 0.2 equiv). The resulting suspension
was stirred at rt for 16 h. Upon completion (TLC in CH2Cl2/MeOH 9:1)
the reaction mixture was diluted with H2O and extracted with CH2Cl2
(x 2). The organic phases were combined, dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. The crude product
was purified by silica gel column chromatography, eluting in a step
gradient with hexane/EtOAc 1:1 to 1:3. The product was obtained as a
colorless oil (104 mg, yield 76%). 1H NMR (300 MHz, CDCl3) δ 8.42
(app s, 1H), 8.17 (s, 1H), 8.13 (app d, J = 8.4 Hz, 1H), 8.06 (d,
J = 8.3 Hz, 1H), 6.05 (s, 2H), 5.42 (d, J = 2.5 Hz, 1H), 5.26 (dd,
J = 10.2, 8.1 Hz, 1H), 5.09–5.01 (m, 2H), 4.93 (d, J = 13.0 Hz, 1H),
4.71 (d, J = 7.8 Hz, 1H), 4.24–4.07 (m, 2H), 4.03 (s, 3H), 3.98 (t,
J = 6.6 Hz, 1H), 2.17 (s, 3H), 2.03 (s, 6H), 1.99 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ 170.6, 170.3, 170.2, 169.8, 166.8, 145.9, 144.0,
138.9, 132.9, 129.3, 123.1, 121.3, 119.8, 100.7, 71.0, 70.8, 68.8, 67.1,
3-butynyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (7c).
[48] 3 Å molecular sieves (1 g) were placed in an oven dried round
bottom flask flushed with argon. β-D-galactose pentaacetate (5b) (1 g,
2.56 mmol, 1 equiv) was added and dissolved in 18 mL dry CH2Cl2,
followed by addition of 3-butyn-1-ol (388 μL, 5.12 mmol, 2 equiv). The
resulting mixture was cooled to 0 °C and BF3·Et2O (2.53 mL, 20.5 mmol,
8 equiv) was added dropwise. After 12 h at rt the TLC (hexane/EtOAc
1:1) showed complete consumption of the starting material. The reac-
tion mixture was diluted with 30 mL CH2Cl2, quenched by addition of
solid NaHCO3 (3 g) and stirred at room temperature for 20 min. Mole-
cular sieves were filtered off and the solution was subsequently washed
with 10% aq. NaHCO3 (x 1) and brine (x 1). The organic phase was
dried over anhydrous Na2SO4, filtered and concentrated under vacuum.
The crude product was purified by silica gel column chromatography,
eluting with a step gradient of hexane/EtOAc 2:1 to 1:1 to afford the
product as a colorless solid. (681 mg, 66% yield). 1H NMR (300 MHz,
CDCl3) δ 5.39 (d, J = 3.4 Hz, 1H), 5.21 (dd, J = 10.5, 7.9 Hz, 1H), 5.03
(dd, J = 10.5, 3.4 Hz, 1H), 4.55 (d, J = 7.9 Hz, 1H), 4.23–4.08 (m, 2H),
4.00–3.89 (m, 2H), 3.68 (dt, J = 9.8, 7.3 Hz, 1H), 2.48 (td, J = 6.8,
2.5 Hz, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01–1.96 (m, 4H);
13C NMR (75 MHz, CDCl3) δ 170.4, 170.3, 170.2, 169.5, 101.4, 80.7,
70.9, 70.8, 69.6, 68.7, 67.9, 67.1, 61.3, 20.9, 20.7, 20.7, 20.6, 19.9 (1H
and 13CNMR matched literature reported spectra) [48,49]; mp
89.8–90.2 °C. HRMS (ESI) of C18H24O10 [M]: calcd for [M + NH4]+
418.1708, found 418.1695.
62.8, 61.4, 53.8, 20.9, 20.8, 20.7, 20.6. HRMS (ESI) of C25H30N4O14
S
[M]: calcd for [M + H]+ 643.1552, found 643.1539; calcd for [M +
Na]+ 665.1371, found 665.1363.
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
triazol-4-yl} ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside
(8c). In an oven dried round bottom flask under nitrogen, 7c (96 mg,
0.24 mmol, 1 equiv) was dissolved in 1.5 mL dry THF. Azide 5 (62 mg,
0.24 mmol, 1 equiv) was added to the stirring suspension, followed by
0.1 M aq. sol. sodium ascorbate (20 mg, 0.10 mmol, 0.4 equiv) and
0.1 M aq. sol. CuSO4·5H2O (12 mg, 0.05 mmol, 0.2 equiv). The resulting
suspension was heated at 40 °C for 20 h. Upon completion (TLC in
CH2Cl2/MeOH 9:1) the reaction mixture was cooled to rt and extracted
with CH2Cl2 (x 2). The organic phases were combined, dried over an-
hydrous Na2SO4, filtered and concentrated under vacuum to afford the
pure desired compound as a yellow oil (123 mg, yield 78%). 1H NMR
(300 MHz, CDCl3) δ 8.49 (d, J = 2.2 Hz, 1H), 8.25 (dd, J = 8.6, 2.1 Hz,
1H), 8.17 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 6.00 (s, 2H), 5.42 (d,
J = 2.3 Hz, 1H), 5.32–5.20 (m, 1H), 5.02 (dd, J = 10.5, 3.0 Hz, 1H),
4.52 (d, J = 8.0 Hz, 1H), 4.28–4.07 (m, 3H), 4.03 (s, 3H), 3.99–3.87
(m, 2H), 3.14 (t, J = 5.8 Hz, 2H), 2.17 (s, 3H), 2.02 (s, 3H), 1.99 (s,
3H), 1.93 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.6, 170.3, 170.1,
169.8, 166.8, 146.3, 144.0, 139.1, 132.7, 128.9, 122.6, 120.9, 119.4,
101.2, 70.7, 70.7, 68.9, 68.3, 67.0, 61.4, 53.6, 26.3, 20.7 (3C), 20.5.
HRMS (ESI) of C26H32N4O14S [M]: calcd for [M + H]+ 657.1708,
found 657.1724; calcd for [M +Na]+ 679.1528, found 679.1545.
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
triazol-4-yl} methyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside
(8a). In a round bottom flask under nitrogen, 7a (90 mg, 0.23 mmol, 1
equiv) was dissolved in 2.2 mL THF. Azide 5 (60 mg, 0.23 mmol, 1
equiv) was added to the stirred suspension, followed by 0.1 M aq. so-
dium ascorbate (19 mg, 0.09 mmol, 0.4 equiv) and 0.1 M aq.
CuSO4·5H2O (12 mg, 0.05 mmol, 0.2 equiv). The resulting suspension
was stirred at rt for 13 h. Upon completion (TLC in CH2Cl2/MeOH 9:1)
triazol-4-yl} methyl-β-D-glucopyranoside (1a). [39] In an oven dried
round bottom flask under nitrogen, cycloaddition product 8a (50 mg,
0.078 mmol, 1 equiv), 6 mL dry MeOH and crushed K2CO3 (125 mg,
0.9 mmol) were added. The resulting suspension was stirred at room
67

A.B. Murray, et al.
CarbohydrateResearch476(2019)65–70
temperature for 2.5 h. Upon completion (TLC in CH2Cl2/MeOH 3:2),
the crude product was filtered through a neutral alumina column,
eluting with 3:2 MeOH/H2O. A silica gel column was applied to remove
the remaining salt, eluting with a step gradient of 4:1 CH2Cl2/MeOH to
3:2 CH2Cl2/MeOH. Finally, an aqueous solution of 1a was filtered
through a small Dowex 50Wx8 cation exchange column (H+ form) for
further desalting. The product 1a was obtained as an amorphous white
solid, mp 190–193 °C (d) (25.1 mg, yield 73%). 1H NMR (300 MHz,
D2O) δ 8.62 (s, 1H), 8.33 (app s, 1H), 8.15 (app d, J = 8.3 Hz, 1H), 7.97
(d, J = 8.2 Hz, 1H), 5.08 (d, J = 13.0 Hz, 1H), 4.97 (d, J = 12.7 Hz,
1H), 4.62 (d, J = 7.8 Hz, 1H), 3.93 (d, J = 12.0 Hz, 1H), 3.73 (dd,
J = 12.0, 5.6 Hz, 1H), 3.55–3.46 (m, 2H), 3.41 (d, J = 9.1 Hz, 1H), 3.33
(t, J = 8.7 Hz, 1H); 13C NMR (75 MHz, D2O) δ 167.7, 144.7, 142.5,
139.9, 130.8, 125.7, 125.7, 123.6, 112.8, 101.5, 75.9, 75.6, 73.0, 69.6,
61.8, 60.7 (1H and 13CNMR agreed with literature reported spectra)
[39]; HRMS (ESI) of C16H18N4O9S [M]: calcd for [M + H]+ 443.0867,
found 443.0871; calcd for [M + Na]+ 465.0687, found 465.0693.
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
Scheme 2. Synthesis of acetylenic glycosides.
[2 M + Na]+ 935.1794, found 935.1774.
2.2. Inhibition assays
The inhibitory activity of compounds 1a-c were estimated by their
ability to inhibit the soluble form of CA IX (0.1 mg/mL) and CA II
(0.1 mg/mL) [50] via monitoring hydrolysis of 0.8 mM p-nitrophenyl
acetate in assay buffer (50 mM Tris-HCl, pH 7.8) at 37 °C. The con-
centrations of 1a-c were varied to produce 50% inhibition and the re-
ported values represent an IC50. Inhibition assays with CA II used 2 μM
concentrations of 1a-c, and showed no inhibition.
triazol-4-yl} methyl-β-D-galactopyranoside (1b). In an oven dried
round bottom flask under nitrogen, cycloaddition product 8b (34.5 mg,
0.054 mmol, 1 equiv), 3 mL dry MeOH and crushed K2CO3 (58.8 mg,
0.43 mmol) were added. The resulting suspension was stirred at room
temperature for 2.5 h. Upon completion (TLC in CH2Cl2/MeOH 3:2),
the crude product was filtered through a neutral alumina column
eluting in MeOH/H2O 3:2. A silica gel column was applied to remove
the remaining salt eluting with a step gradient of 7:3 CH2Cl2/MeOH to
3:2 CH2Cl2/MeOH. The product was obtained as a translucent white
glass (20.1 mg, yield 85%). 1H NMR (300 MHz, D2O) δ 8.59 (s, 1H),
8.21 (app s, 1H), 8.05 (dd, J = 8.0, 1.7 Hz, 1H), 7.84 (d, J = 8.3 Hz,
1H), 5.10 (d, J = 12.8 Hz, 1H), 4.98 (d, J = 12.9 Hz, 1H), 4.57 (d,
J = 7.7 Hz, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.88–3.73 (m, 3H), 3.69 (dd,
J = 9.9, 3.3 Hz, 1H), 3.59 (dd, J = 9.9, 7.7 Hz, 1H); 13C NMR (75 MHz,
D2O) δ 170.6, 144.6, 143.8, 139.3, 132.4, 125.3, 125.2, 123.6, 112.5,
102.0, 75.2, 72.7, 70.6, 68.6, 61.7, 60.9. HRMS (ESI) of C16H18N4O9S
[M]: calcd for [M + H]+ 443.0867, found 443.0874; calcd for [M +
Na]+ 465.0687, found 465.0691.
3. Results and discussion
3.1. Chemistry
The key assembly step for the synthesis of the saccharin-sugar de-
rivatives involved the reported [39] copper mediated click reaction
between a propargyl glucoside and 6-azido saccharin protected on the
lactam nitrogen with a tert-butyl group. Given the acidity of the sulfi-
mide NH group, protection was considered important to avoid inter-
ference with the cycloaddition reaction via coordination of copper [51]
with the electron rich benzosulfimidate. In our studies which involved
multiple attempts and variations, we were unable to satisfactorily re-
move the tert-butyl protecting group in refluxing TFA under the re-
ported conditions [25]; we typically observed considerable decom-
position resulting in complex mixtures. This led us to design an
alternative synthetic approach, featuring a protection/deprotection
strategy that was mild. We hypothesized that the ring opened version of
saccharin with a sulfonamide group would be compatible with the cy-
cloaddition chemistry, and provide entry back to the saccharin ring
system after alkaline cyclization. Here we report new saccharin-glyco-
conjugates, obtained in good yields, under mild conditions that feature
simultaneous alkaline cyclization to the saccharin ring system and
sugar deprotection.
{1-[4-(methoxycarbonyl)-3-sulfamoylphenyl]-1H-1,2,3-
triazol-4-yl} ethyl-β-D-galactopyranoside (1c). In an oven dried
round bottom flask under argon, cycloaddition product 8c (181 mg,
0.276 mmol, 1 equiv) was dissolved in 5 mL dry MeOH and crushed
K2CO3 (50 mg) was added. The resulting suspension was stirred at room
temperature for 40 min. Upon completion (TLC in CH2Cl2/MeOH 1:1),
the crude product was filtered through a neutral alumina column
eluting in MeOH/H2O 3:2. The K salt of the product was obtained as a
colorless oil (104 mg, yield 76%). 1H NMR (300 MHz, D2O) δ 8.35 (s,
1H), 8.15 (app s, 1H), 7.99 (app d, J = 8.1 Hz, 1H), 7.81 (d, J = 8.3 Hz,
1H), 4.48 (d, J = 7.8 Hz, 1H), 4.31–4.19 (m, 1H), 4.08–3.97 (m, 1H),
3.94 (d, J = 2.8 Hz, 1H), 3.86–3.63 (m, 4H), 3.54 (dd, J = 9.8, 8.1 Hz,
1H), 3.12 (t, J = 6.4 Hz, 2H); 13C NMR (75 MHz, D2O) δ 170.1, 145.9,
143.6, 138.7, 131.8, 124.8, 124.0, 121.4, 111.3, 102.9, 75.1, 72.8,
70.7, 68.7, 68.3, 60.9, 25.3. HRMS (ESI) of C17H20N4O9S [M]: calcd for
[M + H]+ 457.1024, found 457.1016; calcd for [M + Na]+ 479.0843,
found 479.0836; calcd for [2 M + H]+ 913.1975, found 913.1960;
We considered saccharin derivative 5 suitable for click reaction with
different peracetylated alkynyl glycosides (Schemes 1 and 2). The de-
sired key intermediate azide 5 was obtained in a 3 step synthesis from
the commercially available nitro-saccharin (Scheme 1). First, nitro-
saccharin was refluxed with TFA in methanol to promote solvolytic ring
opening [52] and form 3 in 45% yield. Compound 3 was then converted
calcd for [2 M
+
NH4]+ 930.2240, found 930.2224; calcd for
Scheme 1. Synthesis of the key intermediate methyl 4-azido-2-sulfamoylbenzoate 5.
68

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CarbohydrateResearch476(2019)65–70
Scheme 3. Cycloaddition and cyclization/deprotection sequence for saccharin-glycoconjugates.
linker has allowed better interaction of the sugar with the selectivity
pocket, despite the greater flexibility anticipated for a longer linker.
Table 1
Inhibition and selectivity ratio for CA II and CA IX with 1a-c, and AZM.
Compound
IC50 (nM)
selectivity ratioa
3.3. Conclusions
CA II
CA IX
CA II:CA IX
We have demonstrated that saccharin nitrogen protection with the
tert-butyl group can be successfully avoided, and report a useful and
mild route to glucosyl and galactosyl saccharin conjugates. The new
compounds display selective nM inhibition of CA IX, while not in-
hibiting CA II (an off-target CA) up to 2 μM. These compounds show
increased isoform specificity with at least 22-fold selectivity for CA IX
compared to the classically used CA inhibitor acetazolamide (AZM)
which does not display selective inhibition for CA IX over CA II. These
characteristics help develop the rules for a SAR for CA IX inhibitors
with glycosyl tails.
1a
> 2000
> 2000
> 2000
12
197
206
93
> 10
> 10
> 22
0.5
1b
1c
AZMb
25
a
Selectivity is determined by the ratio of IC50s for the cytosolic CA II relative
to the targeted CA IX isoform.
b
AZM = acetazolamide.
into the corresponding amino derivative 4by catalytic hydrogenation in
presence of palladium on carbon (99% yield). The amino group of 4 was
smoothly converted into the azide by reacting it with tert-butyl nitrite
and trimethylsilyl azide to achieve compound 5 in 93% yield. The
syntheses of the alkynyl sugar scaffolds 7 are depicted in Scheme 2. The
readily available β-D-galactose or β-D-glucose pentaacetates 6a,b were
reacted with propargylic alcohol or 3-butyn-1-ol in the presence of BF3
etherate and 3 Å molecular sieves to afford 7a-c in 60–79% yields after
purification (Scheme 2).
Acknowledgement
Mass spectrometry was supported by NIH S10 OD021758-01A1, and
ABM was supported by training grant T32AI0007110 from the NIH.
Appendix A. Supplementary data
The copper(I)-catalyzed cycloaddition of compound 5 and alkynes
7a-c afforded the protected triazoles 8a-c in 76–95% yields (Scheme 3).
These results indicate the free sulfonamide and methyl ester groups are
indeed compatible with the cycloaddition chemistry, obviating the need
for the tert-butyl protecting group. With the molecule assembled it re-
mained to close the saccharin ring and deprotect the sugar. We sus-
pected that reaction of adjacent sulfonamide and ester groups to form
the saccharin ring system would be facile in alkaline conditions
[53,54], suggesting that cyclization and methanolysis of the sugar
acetyl protecting group removal could be combined into a single one-
pot reaction.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.carres.2019.03.001.
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