Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases

Article abstract of DOI:10.1021/acs.jmedchem.5b01228

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Full text of DOI:10.1021/acs.jmedchem.5b01228

Article
pubs.acs.org/jmc
Phosphate Chemical Probes Designed for Location Specific
Inhibition of Intracellular Carbonic Anhydrases
Gregory M. Rankin,† Daniela Vullo,‡ Claudiu T. Supuran,‡ and Sally-Ann Poulsen*,†
†Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia
‡Polo Scientifico, Laboratorio di Chimica Bioinorganica,Universita
̀
degli Studi di Firenze, Via della Lastruccia 3, Rm. 188, 50019 Sesto
Fiorentino, Florence, Italy
S
* Supporting Information
ABSTRACT: Chemical probes are small molecules designed to
bind to a specific protein and disrupt the proteins function.
Although many inhibitors are reported for human carbonic
anhydrase (CA) enzymes, few may be considered useful as chemical
probes as they exhibit broad action against the 12 catalytically active
CA isozymes. In addition, most do not possess an appropriate
physicochemical profile to discriminate intracellular CA activity from
either global or extracellular CA activity. We report herein the
synthesis of three monophosphate CA proinhibitors (compounds 2,
3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate
and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The
proinhibitors are designed as neutral, membrane-permeable
compounds that once inside the cell may be hydrolyzed by pH-
driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate
compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.
cially available and clinically used CA inhibitor, is commonly
employed as the “go to” CA inhibitor in studies to explore the
biology surrounding CA inhibition (Figure 1). AZA delivers
INTRODUCTION
■
There are 12 catalytically active isozymes of carbonic anhydrase
(CA, EC 4.2.1.1) that have been characterized in humans;
seven have their active site domain in the intracellular space
(CA I, II, III, VII, XIII − cytosolic; CA VA and VB,
mitochondrial), while the remaining five have an extracellular
active site domain (CA IV, IX, XII, and XIV − membrane
anchored; CA VI, secreted).1 CA enzymes catalyze the
reversible hydration of carbon dioxide to bicarbonate and a
proton: CO2 + H2O ⇆ HCO3 + H+. The active site of the
−
different CA isozymes are structurally similar, such that small
molecule inhibitors with CA isozyme selectivity are challenging
to design.2 While there has been some success to address
selectivity, inhibitors with exceptional isozyme selectivity for
the most part remain elusive. The association of CA IX and/or
CA XII with cancer invasion, metastasis, and drug resistance
provides the driving force for the development of compounds
with selectivity for extracellular CAs.3 Our group has
contributed to the development of inhibitors with this
inhibition profile, compound 1, a polar glycoconjugate with
predicted poor membrane permeability, is exemplary in this
respect.4 Compound 1 has >1000-fold selectivity for CA IX and
>100-fold selectivity for CA XII over both intracellular CA I
and CA II. Owing to the combination of physicochemical
properties (poor membrane permeability) with preferential
isozyme selectivity for CA IX and XII, compound 1 has
provided a useful chemical tool to explore the action of
extracellular CA inhibition. Acetazolamide (AZA), a commer-
Figure 1. Known chemical probes for the study of human carbonic
anhydrases (CAs): acetazolamide (AZA) provides global CA
inhibition, and saccharin glycoconjugate 1 provides extracellular CA
inhibition.
broad CA inhibition (except for hCA III) and is membrane
permeable,5 hence it acts to provide a global readout of CA
inhibition (i.e., intracellular + extracellular), Table 1. If
undertaking studies with cell-based models of disease, employ-
ing a panel of CA inhibitors with properties that enable them to
discriminate global, intracellular and extracellular CA inhibition
effects, is desirable. This panel could substantially value add to
the correlation of phenotypic assay readout with localization of
the CA(s) responsible for the phenotype. Inhibitors with
Received: August 3, 2015
© XXXX American Chemical Society
A
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Table 1. Human CA Inhibition Profile of the “Go To” Global CA Inhibitor, Acetazolamide (AZA)
intracellular CAs
extracellular CAs
isozyme I isozyme
II
isozyme
III
isozyme
VA
isozyme
VB
isozyme
VII
isozyme
XIII
isozyme
IV
isozyme
VI
isozyme
IX
isozyme
XII
isozyme
XIV
Ki (nM)
250
12
> 25000
63
54
2.5
17
74
11
25
5.7
41
Figure 2. FDA approved prodrug compounds with activity that is dependent on the intracellular formation and subsequent trapping of a negatively
charged monophosphate.
Figure 3. (A) CycloSal and bis(SATE) glucosyl-6-phosphate proinhibitors 2 and 3, based on the structure of known glucose-based CA inhibitor 4.
(B) Bis(SATE) glucosyl-6-phosphate proinhibitor 5 based on the structure of known glucose-based CA inhibitor 6.9
physicochemical properties that permit them to localize
predominantly intracellularly or predominantly extracellularly
(such as compound 1) are potentially powerful chemical probes
to complement studies investigating the impact of CA in
disease that otherwise employ AZA in isolation.6 While the
development of extracellular CA chemical probes has advanced,
the concomitant development of locality specific intracellular
acting CA chemical probes has not been forthcoming. This
paper focuses on the development of such intracellular acting
CA inhibitors to provide for a comprehensive panel of chemical
probes, alongside AZA (global acting) and compound 1
(extracellular acting), to specifically modulate intracellular CA
activity if employed in cell models.
monophosph(on)ate prodrugs, and second, 18F-fluorodeoxy-
glucose. The success of phosphorus prodrug technology is
evidenced by the FDA approval of the antiviral nucleoside
analogues tenofovir disoproxil fumarate (TDF) and sofosbuvir
(GS/PSI-7977), Figure 2.7 TDF has two bis-
(isopropyloxymethyl) carbonate (POC) phosphate protecting
groups, while GS/PSI-7977 is an aryloxyphosphoramidate
prodrug. The prodrugs can freely enter cells by passive
membrane diffusion, and once inside the cell, their phosphate
groups are unmasked via enzymatic or chemical hydrolysis (or a
combination of both) to generate the active nucleoside
monophosphate.7 The monophosphate is thus delivered and
trapped intracellularly, and the second and third enzymatic-
mediated phosphorylation to a nucleoside triphosphate is
usually efficient,7 allowing for nucleoside prodrugs to elicit their
eventual pharmacological response. The second inspirational
compound for the target compound design of this study is 18F-
Fluorodeoxyglucose (FDG), a PET radiopharmaceutical that is
used to image tumors. Like glucose, FDG is actively
RESULTS AND DISCUSSION
■
Compound Design. The inspiration for the design of
intracellular targeting CA inhibitors is derived from a
combination of two classes of phosphorus-based compounds
that are used in clinical medicine, first, nucleoside
B
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Figure 4. Intracellular hydrolysis mechanisms for cycloSal and bis(SATE) protected monophosphate proinhibitors.
a
Scheme 1. Synthesis of CycloSal Proinhibitor 2 and Bis(SATE) Proinhibitor 3
a
Reagents and conditions: (a) NaOMe, CH2Cl2/MeOH, quant; (b) TBSCl, imidazole, DMF; (c) py, Ac2O, 70%, 2 steps; (d) AcCl, CH2Cl2,
MeOH, 97%; (e) (i) 13, DIPEA, CH3CN, −20 °C, (ii) t-BuOOH, −20 °C to rt, 55%; (f) 14, CuSO4.H2O, sodium ascorbate, EtOH/H2O 3:1, 60
°C, 76%; (g) 14, CuSO4·H2O, sodium ascorbate, tert-butyl alcohol/H2O 1:1, 60 °C, 93%; (h) AcCl, CH2Cl2, MeOH, 90%; (i) (i) 7, 1H-tetrazole,
CH3CN, (ii) m-CPBA, CH2Cl2, −30 °C to rt, 51%.
transported into cells by glucose transport proteins (GLUT).
Once intracellular, glucose and FDG are monophosphorylated
by the enzyme glucose-6-phosphatase. Glucose-6-phosphate
continues along the glycolytic pathway for energy production,
however, 18FDG-6-phosphate is not suited for glycolysis and
instead is effectively trapped intracellularly as a monophosphate
that is deprotonated at physiological pH.8
We have selected two phosphate prodrug approaches in the
design of CA proinhibitors for intracellular CA targeting,
cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl
(SATE) phosphate, Figure 3. The proinhibitors consist of
two glucose-based glycoconjugate CA inhibitors previously
reported by us. Proinhibitors 2 and 3 stem from modification of
the C-6 hydroxyl moiety of the glucoconjugate benzenesulfo-
namide CA inhibitor 4 and comprise cycloSal and bis(SATE)
moieties, respectively.9 Bis(SATE) proinhibitor 5 is based on
modification of the C-6 hydroxyl moiety of the anomeric
glucose sulfonamide CA inhibitor 6.9 All three proinhibitors
additionally comprise acetyl esters on the 2, 3, and 4 glucose
hydroxyl groups. The three acetyl groups increase the
lipophilicity of proinhibitors 2, 3, and 5; to aid cell membrane
permeability, these groups are hydrolyzed by intracellular
esterases to generate a compound with free 2, 3, and 4
hydroxyls.10 We recently reported that the acetyl groups had a
half-life of <1 h in plasma when presented on a glucose scaffold,
consistent with esterase processing.10 The mechanisms of
hydrolysis that operates to cleave the cycloSal and SATE
moieties to release the free monophosphate of prodrugs/
proinhibitors inside the cell are described next.
CycloSal groups are one of the most extensively explored
moieties for the masking of monophosphates, particularly in the
development of antiviral nucleotides.11 The cycloSal com-
C
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a
Scheme 2. Synthesis of Bis(SATE) Proinhibitor 5
a
Reagents and conditions: (a) BrCH(CO2Et)2, NH2PMB, MeOH; (b) KMnO4/CuSO4, CH3CN/H2O, 40% 2 steps; (c) NaOMe, CH2Cl2/MeOH,
quant; (d) TBSCl, imidazole, DMF; (e) py, Ac2O, 60% 2 steps; (f) CAN, CH3CN/H2O, 90%; (g) (i) 7, 1H-tetrazole, CH3CN, (ii) m-CPBA,
CH2Cl2, −30 °C to rt, 89%.
a
Scheme 3. Synthesis of Monophosphate CA Inhibitor 21
a
Reagents and conditions: (a) (i) 23, 1H-tetrazole, CH3CN, (ii) m-CPBA, CH2Cl2, −30 °C to rt, 82%; (b) Pd(OH)2, H2, THF/MeOH, 78%; (c)
NaOMe, MeOH, quant.
pounds are designed as neutral, membrane-permeable com-
pounds that once inside the cell are hydrolyzed by pH-driven
mechanisms to release the negatively charged monophosphate,
which is then trapped intracellularly, Figure 4.12 Under slightly
basic pH conditions, the aryl ester P−O bond is first cleaved to
generate a benzyl phosphate diester intermediate, and this is
followed by spontaneous cleavage of the C−O benzyl ester
bond to release the free phosphorylated drug and salicyl
alcohol.11a The half-life of cycloSal phosphates may be tuned via
substitution on the salicyl alcohol.
In contrast to the pH-mediated delivery of the free
phosphate from the parent cycloSal ester, hydrolysis of the
bis(SATE) phosphotriester moiety is dependent on esterase
activity for activation.7,13 First, esterase hydrolysis of the
thioester generates an unstable thioethyl phosphotriester
intermediate. Next, this intermediate decomposes spontane-
ously via an intramolecular nucleophilic displacement mecha-
nism to release ethylene sulfide with concomitant formation of
the corresponding phosphodiester. The second SATE group is
similarly hydrolyzed to afford the free monophosphorylated
compound.
Chemical Synthesis. The introduction of the cycloSal and
bis(SATE) masked monophosphate moieties occurs by
modification of the primary hydroxyl group on the foundation
inhibitor structure. For cycloSal, this is commonly achieved
using P(III) chemistry,11b,14 while for bis(SATE), it is achieved
with a 1H-tetrazole mediated coupling of a bis(S-pivaloyl-2-
thioethyl) N,N-diisopropylphosphoramidite (7) followed by in
situ oxidation.15 For our target compounds, each route requires
a precursor 2,3,4-triacetylated glucosyl moiety with the C-6
hydroxyl group available for the introduction of the masked
monophosphate moiety. 2,3,4-Tri-O-acetyl-6-O-tert-butyldime-
thylsilyl-β-D-glucopyranosyl azide (8) is a common intermedi-
ate for the synthesis of proinhibitors based on triazole CA
inhibitor 4. Azide 8 was prepared by deacetylation of β-D-
glucopyranosyl azide (9) under Zemplen
́
conditions16 to give
10, followed by silylation at O-6 with tert-butyldimethylsilyl
chloride and subsequent reacetylation of the remaining
hydroxyl groups (O-2, O-3, and O-4) using acetic anhydride
in pyridine, Scheme 1.
To continue to cycloSal proinhibitor 2 required the
preparation of cycolSal glycosyl azide 11. Deprotection of the
tert-butyldimethylsilyl group of 8 using mild acidic conditions
afforded 12, with no acetyl group migration observed.
Compound 11 was prepared in a “one-pot” procedure by
coupling alcohol 12 and saligenyl chlorophosphite 13,11a,17
followed by in situ oxidation with t-BuOOH. The reaction of
the cycolSal glycosyl azide 11 with 4-ethynylbenzenesulfona-
mide (14)18 using Cu(I)-catalyzed azide alkyne cycloaddition
(CuAAC) provided cycloSal prodrug 2 in 76% yield, as a
diastereoisomeric mixture about the phosphorus center,
Scheme 1.
The synthesis of bis(SATE) proinhibitor 3 was achieved in
three steps from glucosyl azide 8. First, CuAAC of azide 8 and
alkyne 14 gave triazole 15 in high yield. Subsequent acidic
cleavage of the silyl ether of 15 afforded alcohol 16 in 90%
yield. The 1H-tetrazole mediated coupling of phosphoramidite
715 with 16, followed by in situ oxidation with m-CPBA,
furnished the target bis(SATE) proinhibitor 3, Scheme 1.
The bis(SATE) protected phosphate proinhibitor 5 was
prepared via an adaptation of the synthesis developed for the
foundation anomeric sulfonamide 6.19 The key modification of
the synthesis to allow for installation of the protected
phosphate moiety is the selective orthogonal protection of
D
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a
Scheme 4. Synthesis of Monophosphate CA Inhibitor 22
a
Reagents and conditions: (a) AcCl, CH2Cl2, MeOH, 96%; (b) (i) 23, 1H-tetrazole, CH3CN, (ii) m-CPBA, CH2Cl2, −30 °C to rt, 90%; (c) CAN,
CH3CN/H2O, 90%; (d) Pd(OH)2, H2, THF/MeOH, 79%; (e) NaOMe, MeOH, quant.
the glucosyl C-6 primary hydroxyl group as a silyl ether (17).
The reaction of glucosyl thioacetate 18 with diethyl
bromomalonate and 4-methoxybenzylamine and subsequent
oxidation afforded 19.19 A sequence of standard carbohydrate
protecting group manipulations of 19, specifically deacetylation,
silylation of the C-6 primary hydroxyl group, and reacetylation,
afforded 17 in 60% yield over three steps. Oxidative cleavage of
the PMB protecting group of 17 using ceric ammonium nitrate
removed the silyl ether to afford 20 in high yield.
Phosphorylation of 20 with phosphoramidite 715 and
subsequent oxidation with m-CPBA gave the target bis(SATE)
proinhibitor 5 in 89% yield, Scheme 2.
With the three target prodrugs in hand, our attention turned
toward the synthesis of the corresponding fully deprotected
monophosphate inhibitors 21 and 22, Schemes 3 and 4,
respectively. These control compounds are needed in order to
establish the CA inhibition profile of inhibitors that would
follow cell-mediated hydrolysis of the cycloSal, bis(SATE), and
acetyl groups of proinhibitors 2, 3, and 5. Our approach toward
the synthesis of 21 and 22 was to use the reactive dibenzyl N,N-
diisopropylphosphoramidite (23) to install the phosphate
moiety. The phosphate triester compounds formed from 23
are lipophilic and amenable to straightforward purification by
normal phase flash chromatography. The benzyl groups may be
conveniently removed when needed by hydrogenolysis. 1H-
Tetrazole mediated coupling of dibenzyl N,N-diisopropylphos-
phoramidite (23) with the C-6 primary hydroxyl group of
precursor glucosyl compounds, 16 and 24, was followed by in
situ oxidation with m-CPBA to provide 25 and 26, respectively.
Oxidative cleavage of the PMB protecting group of anomeric
sulfonamide 26 using ceric ammonium nitrate gave 27.
Hydrogenolysis of the benzyl groups of 25 and 27 afforded
debenzylated monophosphates 28 and 29, respectively. Finally,
Table 2. Human CA Inhibition Profile and cLogP for
Proinhibitors (2, 3, and 5) and the Corresponding
Unmasked Inhibitors (21 and 22)
b
Ki (nM)
intracellular
extracellular
a
compd cLogP
masking groups CA I CA II CA IX CA XII
2
3
5
+0.9
+3.7
+2.6
(i) cycloSal
437
528
860
564
3.7
84.3
51.3
(ii) 3× acetyl
(i) bis(SATE)
(ii) 3× acetyl
(i) bis(SATE)
(ii) 3× acetyl
unmasked
6.3
135
75.1
74.8
154
630
21
22
−2.8
−3.9
1.8
248
213
76.8
90.4
unmasked
922
b
52.3
a
Calculated using ChemDraw Ultra 12. Errors in the range of 5% of
the reported value from three determinations.
The cLogP values for proinhibitor compounds 2, 3, and 5 are
indicative of compounds that are likely to have good membrane
permeability, while the negative cLogP values for the
deprotected compounds 21 and 22 are consistent with
expected poor cell membrane permeability, Table 2.21 All test
compounds have strongest inhibitory activity against intra-
cellular hCA II, with the unmasked inhibitors 21 and 22
experiencing further enhanced inhibition of hCA II compared
to their parent proinhibitors. For example, the bis(SATE)
proinhibitors, 3 and 5, had Kis of 6.3 and 74.8 nM, respectively,
while their unmasked counterparts, 21 and 22, had Kis of 1.8
and 53.3 nM, respectively. This confirms that the proinhibitor
compounds are able to successfully target intracellular CAs. It is
common for primary sulfonamide compounds to experience up
to 3 orders of magnitude weaker inhibition at CA I than at CA
II. This relationship was observed for the test compounds of
this study, with both masked and unmasked inhibitors having Ki
values 437−922 nM. When considered together, these
structure−activity and structure−property relationships estab-
lish that proinhibitors are likely destined for intracellular
localization. The proinhibitors 2, 3, and 5 should readily enter
cells by passive membrane diffusion, unmask intracellularly with
a combination of pH and/or enzyme driven removal of the
acetyl, bis(SATE), and cycloSal protecting groups, with the
hydrolysis of the acetate groups of 28 and 29 under Zemplen
́
conditions16 furnished the target monophosphate inhibitors 21
and 22 in quantitative yield.
CA Inhibition. The CA enzyme inhibition activity for
proinhibitors (2, 3, and 5) and the corresponding unmasked
inhibitors (21 and 22) was measured against cytosolic hCA I
and II, and extracellular hCA IX and XII, using an assay that
measures the hydration activity of carbonic anhydrase,20 Table
2.
E
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spectra of all novel compounds are provided in the Supporting
Information. Glycoconjugates are named in accordance with the
recommendations of the IUPAC-IUBMB Joint Commission on
Biochemical Nomenclature: “Nomenclature of Carbohydrates (Rec-
ommendations 1996)” (http://www.chem.qmul.ac.uk/iupac/2carb/).
β-D-Glucopyranosyl Azide (10).10a,23 Sodium methoxide (prepared
from reacting Na (0.300 g, 13.5 mmol) with MeOH (10 mL)) was
added dropwise to a stirred solution of β-azido-2,3,4,6-tetra-O-acetyl-
D-glucopyranose (9)10a,23 (3.02 g, 8.08 mmol) in MeOH:CH2Cl2 (9:1,
30 mL) until the pH of the solution remained greater than 11. The
reaction mixture was stirred overnight then neutralized by the addition
of Amberlite IR-120 H+ ion-exchange resin. The solution was filtered,
washed with MeOH (50 mL), and the solvent removed in vacuo to
give the title compound 10 (1.70 g, quant) as a colorless oil which was
used without further purification. 1H NMR (500 MHz, D2O) δ 3.33 (t,
J = 9.0 Hz, 1H, H-2), 3.41−3.63 (m, 3H, H-3, H-4, H-5), 3.81 (dd, J =
5.6, 12.4 Hz, 1H, H-6), 3.98 (dd, J = 2.2, 12.5 Hz, 1H, H-6). LRMS-
resulting inhibitors 21 and 22 trapped intracellularly.
Furthermore, as CA I and CA II are two of the most prevalent
intracellular CAs in humans, and the free monophosphate
inhibitors 21 and 22 show good inhibition of these isozymes,
the approach has been successful in providing chemical probes
for location specific inhibition of intracellular CAs. While 21
and 22 are not directly viable as chemical probes to study
intracellular acting CAs, their proinhibitor form, with a
combination of phosphate and hydroxyl protecting groups
and complementary unmasking mechanism (chemical or
enzyme activated), provide suitable characteristics to be
employed as chemical probes.
CONCLUSION
■
It is noteworthy to acknowledge that the “go to” global CA
inhibitor, AZA, entered clinical use several decades in advance
of the discovery and characterization of most human CA
isozymes. As a chemical probe, AZA exhibits global CA
inhibition, with broad action against the 12 catalytically active
human CA isozymes, seven of which are intracellular and five of
which have an extracellular active site domain. A panel of three
CA inhibitors, one acting intracellularly only, one acting
extracellularly only (e.g., compound 1), and a third global
acting probe (e.g., AZA), may provide the needed chemical
probes to interrogate more sophisticated biological hypotheses
associated with CA inhibition. This panel may enhance the
extrapolation from in vitro CA inhibition constants to the more
complex biology of whole cells. In this study, we have
successfully designed and synthesized monophosphate pro-
tected CA proinhibitors 2, 3, and 5. The proinhibitors are
destined for intracellular trapping as the corresponding
monophosphates 4 and 6. The proinhibitors present the
required structure−property and structure−activity profiles
with potential to fulfill the criteria for location specific chemical
probes for intracellular CAs with predicted excellent location
fidelity. In addition to human CA studies, these proinhibitors
may be useful for the study of CAs role in pathogenic
organisms such as bacteria and protozoa.22
1
ESI [M + Na]+ m/z = 228. H NMR was in agreement with the data
reported in the literature.
2,3,4-Tri-O-acetyl-6-O-tert-butyldimethylsilyl-β-D-glucopyranosyl
Azide (8).24 tert-Butyldimethylsilyl chloride (1.47 g, 9.78 mmol) was
added to a stirred solution of β-D-glucopyranosyl azide (10) (1.67 g,
8.15 mmol) and imidazole (1.66 g, 24.5 mmol) in anhydrous DMF
(20 mL) at rt. The reaction mixture was left to stir overnight. The
reaction was quenched by the addition of H2O (2 mL) at 0 °C and the
solvent removed in vacuo to afford 6-O-tert-butyldimethylsilyl-β-D-
glucopyranosyl azide as a pale-yellow oil. The residue was then
dissolved in anhydrous pyridine (15 mL). To this was added acetic
anhydride (10.0 mL, 106 mmol) and the solution stirred at rt for 3 h.
The reaction mixture was quenched by the addition of a few drops of
MeOH and the solvent removed under reduced pressure. The residue
was dissolved into CH2Cl2 (100 mL) and washed with water (40 mL).
The aqueous fraction was re-extracted with CH2Cl2 (2 × 50 mL), then
the combined organic fractions were washed with brine (50 mL), dried
(MgSO4), filtered, and the solvent removed in vacuo. The residue was
purified by column chromatography on silica gel (EtOAc/hexane = 1:9
to 1:2) to give the title compound 8 (2.52 g, 70%, 2 steps) as a white
solid; mp 158−160 °C (EtOAc/hexane); [α]2D4 −17.4 (c = 0.13,
CHCl3). 1H NMR (500 MHz, CDCl3) δ 0.06 (s, 3H, SiCH3), 0.07 (s,
3H, SiCH3), 0.90 (s, 9H, SiC(CH3)3), 2.01 (s, 3H, OCOCH3), 2.02
(s, 3H, OCOCH3), 2.07 (s, 3H, OCOCH3), 3.61−3.65 (m, 1H, H-5),
3.71 (dd, J = 5.0, 11.7 Hz, 1H, H-6), 3.77 (dd, J = 2.0, 11.7 Hz, 1H, H-
6), 4.58 (d, J = 8.8 Hz, 1H, H-1), 4.92 (t, J = 9.2 Hz, 1H, H-2), 5.09 (t,
J = 9.7 Hz, 1H, H-4), 5.21 (t, J = 9.5 Hz, 1H, H-3). 13C NMR (125
MHz, CDCl3) δ −5.34, −5.33 (Si(CH3)2), 18.4 (SiC(CH3)3), 20.6
(OCOCH3), 20.7 (2 × OCOCH3), 25.8 (SiC(CH3)3), 62.1 (C-6),
68.4 (C-4), 70.9 (C-2), 73.1 (C-3), 77.0 (C-5), 87.8 (C-1), 169.2,
169.3, 170.3 (OCOCH3). LRMS-ESI [M + Na]+ m/z = 468.
EXPERIMENTAL SECTION
■
General Chemistry. All starting materials and reagents 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 5% w/v
dodecamolybdophosphoric acid in ethanol or vanillin stain (5 g of
vanillin in a mixture of EtOH:H2O:H2SO4 = 85:10:2.75) with
subsequent heating. Silica gel flash chromatography was performed
using silica gel 60 Å (230−400 mesh). NMR (1H, 13C, 31P, gCOSY,
and HSQC) spectra were recorded on either a 400 or 500 MHz
2,3,4-Tri-O-acetyl-β-D-glucopyranosyl Azide (12).25 Acetyl chlor-
ide (0.05 mL, 0.70 mmol) was added to a solution of 2,3,4-tri-O-acetyl-
6-O-tert-butyldimethylsilyl-β-D-glucopyranosyl azide (8) (0.555 g, 1.25
mmol) in CH2Cl2/MeOH (2:1, 30 mL) and stirred at rt for 30 min.
The reaction mixture was diluted with CH2Cl2 (50 mL) and washed
with NaHCO3 (20 mL). The aqueous layer was re-extracted with
CH2Cl2 (2 × 30 mL), and the combined organic layers were dried
(MgSO4), filtered, and the solvent removed in vacuo. The residue was
purified by column chromatography on silica gel (EtOAc/hexane = 1:2
to 1:1) to give the title compound 12 (0.40 g, 97%) as a white solid;
mp 125−127 °C (EtOAc/hexane); [α]2D4 −33.2 (c = 0.14, CHCl3). 1H
NMR (500 MHz, CDCl3) δ 2.02 (s, 3H, OCOCH3), 2.06 (s, 3H,
OCOCH3), 2.08 (s, 3H, OCOCH3), 2.22 (dd, J = 5.5, 8.1 Hz, 1H,
OH), 3.60−3.67 (m, 2H, H-5, H-6), 3.77−3.82 (m, 1H, 6-H), 4.67 (d,
J = 8.9 Hz, 1H, H-1), 4.93 (t, J = 9.3 Hz, 1H, H-2), 5.07 (t, J = 9.6 Hz,
1H, H-4), 5.27 (t, J = 9.5 Hz, 1H, H-3). 13C NMR (125 MHz, CDCl3)
δ 20.61, 20.62, 20.7 (OCOCH3), 61.2 (C-6), 68.3 (C-4), 70.9 (C-2),
72.6 (C-3), 76.5 (C-5), 88.0 (C-1), 169.3, 170.1, 170.2 (OCOCH3).
LRMS-ESI [M + Na]+ m/z = 354. HRMS-ESI [M + Na]+ calcd for
C12H17N3NaO8, 354.0908; found, 354.0909.
1
spectrometer at 30 °C. H NMR spectra were obtained at 500 MHz
and referenced to the residual solvent peak (CDCl3 δ 7.26 ppm,
DMSO-d6 δ 2.50 ppm). 13C NMR spectra were recorded at 125 MHz
and referenced to the internal solvent (CDCl3 δ 77.0 ppm, DMSO-d6
δ 39.5 ppm). 31P NMR spectra were recorded at 162 MHz with
H3PO4 (δ 0.0 ppm) as the external reference. 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
CycloSal-(1-azido-2,3,4-tri-O-acetyl-β-D-glucopyranosyl-6)-phos-
phate (11). To a solution of alcohol 12 (0.200 g, 0.604 mmol) in
1
was ≥95% as determined by HPLC with UV. H, 13C, and 31P NMR
F
DOI: 10.1021/acs.jmedchem.5b01228
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
anhydrous CH3CN (5 mL) at −20 °C was added DIPEA (0.263 mL,
1.51 mmol). To this solution was added saligenyl chlorophosphite
1317 (0.228 g, 1.21 mmol) dissolved in anhydrous CH3CN (1.0 mL)
dropwise over 5 min. The reaction was left to stir for 30 min. tert-
Butylhydroperoxide (5−6 M in n-decane, 0.252 mL, 1.51 mmol) was
added dropwise at −20 °C and stirred for 30 min before warming to rt
and stirred for 1.5 h. Na2SO3 10% in H2O (0.10 mL) was added to
destroy the excess peroxide. The reaction was diluted with CH2Cl2 (50
mL) and washed with brine (20 mL). The aqueous layer was re-
extracted with CH2Cl2 (3 × 20 mL), and the combined organic
fractions were dried (MgSO4), filtered, and the solvent removed in
vacuo. The residue was purified by column chromatography on silica
gel (EtOAc/hexane = 2:3 to 3:2) to give the title compound 11 (0.165
g, 55%, diastereomeric ratio β1/β2 = 1:1) as a white foam; [α]2D6 −7.1
(0.500 g, 1.12 mmol) and alkyne 1418 (0.203 g, 1.12 mmol) were
suspended in an EtOH/H2O mixture (5:1, 12 mL). To the reaction
mixture was added a solution of sodium ascorbate (0.089 g, 0.449
mmol) in water (0.5 mL), followed by a solution of CuSO4·5H2O
(0.056 g, 0.224 mmol) in water (0.5 mL). The bright-yellow
suspension was stirred vigorously at 60 °C for 3 h. The solvent was
removed in vacuo, and the residue was purified by column
chromatography on silica gel (EtOAc/hexane = 2:3 to 1:1) to give
the title compound 15 (0.653 g, 93%) as a white solid; mp 163−165
°C (EtOAc/hexane); [α]2D6 −41.6 (c = 0.13, MeOH). H NMR (500
1
MHz, (CD3)2SO) δ −0.07 (s, 3H, SiCH3), −0.04 (s, 3H, SiCH3), 0.81
(s, 9H, SiC(CH3)3), 1.82 (s, 3H, OCOCH3), 1.97 (s, 3H, OCOCH3),
2.03 (s, 3H, OCOCH3), 3.66 (dd, J = 2.3, 12.3 Hz, 1H, H-6), 3.74 (dd,
J = 5.2, 12.1 Hz, 1H, H-6), 4.18−4.23 (m, 1H, H-5), 5.16 (t, J = 9.5
Hz, 1H, H-4), 5.57 (t, J = 9.3 Hz, 1H, H-3), 5.62 (t, J = 9.1 Hz, 1H, H-
2), 6.39 (d, J = 8.7 Hz, 1H, H-1), 7.38 (s, 2H, SO2NH2), 7.92 (d, J =
8.2 Hz, 2H, Ar-H), 8.03 (d, J = 8.2 Hz, 2H, Ar-H), 9.05 (s, 1H,
CHtriazole). 13C NMR (125 MHz, (CD3)2SO) δ −5.53, −5.49
(Si(CH3)2), 17.9 (SiC(CH3)3), 19.9, 20.2, 20.4 (OCOCH3), 25.6
(SiC(CH3)3), 61.8 (C-6), 67.7 (C-4), 70.4 (C-2), 72.5 (C-3), 75.9 (C-
5), 84.0 (C-1), 121.6 (CHtriazole), 125.4, 126.4 (Ar-CH), 133.2
(Ctriazole), 143.5, 145.5 (Ar-C), 168.5, 169.0, 169.5 (OCOCH3).
LRMS-ESI [M + Na]+ m/z = 649. HRMS-ESI [M + Na]+ calcd for
C26H38N4NaO10SSi, 649.1970; found, 649.1975.
1
(c = 0.10, CHCl3). H NMR (500 MHz, CDCl3) δ 1.96 (s, 1.5H,
OCOCH3), 1.99 (s, 3H, 2 × OCOCH3), 2.04 (s, 1.5H, OCOCH3),
2.06 (s, 1.5H, OCOCH3), 2.07 (s, 1.5H, OCOCH3), 3.81−3.89 (m,
1H, H-5), 4.21−4.31 (m, 1H, H-6), 4.33−4.40 (m, 1H, 6-H), 4.61 (d,
J = 8.7 Hz, 0.5H, H-1), 4.63 (d, J = 9.2 Hz, 0.5H, H-1), 4.87 (t, J = 9.3
Hz, 0.5H, H-2), 4.92 (t, J = 9.2 Hz, 0.5H, H-2), 5.00 (t, J = 9.9 Hz,
0.5H, H-4), 5.04 (t, J = 10.2 Hz, 0.5H, H-4), 5.17−5.23 (m, 1H, 3-H),
5.30−5.38 (m, 1H, POCH2), 5.40−5.47 (m, 1H, POCH2), 7.06−7.11
(m, 2H, Ar-H), 7.13−7.17 (m, 1H, Ar-H), 7.31−7.36 (m, 1H, Ar-H).
13C NMR (125 MHz, CDCl3) δ 20.5, 20.57, 20.6 (OCOCH3), 66.0 (d,
4-[4-(Aminosulfonyl)phenyl]-1-(2,3,4-tri-O-acetyl-β-D-glucopyra-
nosyl)-1H-1,2,3-triazole (16). Acetyl chloride (0.05 mL, 0.70 mmol)
was added to a solution of 15 (0.400 g, 0.638 mmol) in CH2Cl2/
MeOH (2:1, 15 mL), and the solution was stirred at rt for 30 min. The
reaction mixture was quenched with Et3N (100 μL), stirred for 5 min,
and the solvent removed in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 3:2 to 4:1) to give the
title compound 16 (0.294 g, 90%) as a white solid; mp 224−227 °C
(EtOAc/hexane); [α]2D6 −72.1 (c = 0.12, MeOH). 1H NMR (500
MHz, (CD3)2SO) δ 1.82 (s, 3H, OCOCH3), 1.97 (s, 3H, OCOCH3),
2.04 (s, 3H, OCOCH3), 3.41−3.47 (m, 1H, H-6), 3.52−3.57 (m, 1H,
H-6), 4.12−4.16 (m, 1H, H-5), 4.90 (t, J = 5.7 Hz, 1H, OH), 5.12 (t, J
= 9.5 Hz, 1H, H-4), 5.54−5.62 (m, 2H, H-3, H-2), 6.39 (d, J = 8.5 Hz,
1H, H-1), 7.38 (s, 2H, SO2NH2), 7.92 (d, J = 8.4 Hz, 2H, Ar-H), 8.03
(d, J = 8.4 Hz, 2H, Ar-H), 9.09 (s, 1H, CHtriazole). 13C NMR (125
MHz, (CD3)2SO) δ 19.9, 20.2, 20.4 (OCOCH3), 59.9 (C-6), 67.9 (C-
4), 70.5 (C-2), 72.4 (C-3), 76.4 (C-5), 84.1 (C-1), 121.6 (CHtriazole),
125.4, 126.4, (Ar-CH), 133.2 (Ctriazole), 143.5, 145.6 (Ar-C), 168.5,
169.2, 169.5 (OCOCH3). LRMS-ESI [M + H]+ m/z = 513, [M +
Na]+ m/z = 535. HRMS-ESI [M + Na]+ calcd for C20H24N4NaO10S,
535.1105; found, 535.1106.
1-(4-[4-(Aminosulfonyl)phenyl]-1H-1,2,3-triazole)-2,3,4-tri-O-ace-
tyl-6-O-[[(bis((2-((2,2-dimethylpropionyl)sulfanyl)ethoxy)]-phos-
phoryloxy)-ethyl)]-β-D-glucopyranoside (3). A solution of alcohol 16
(0.100 g, 0.195 mmol) and phosphoramidite 714,26 (0.177 g, 0.390
mmol) in CH2Cl2 (20 mL) was coevaporated to dryness and dried
under high vacuum for 30 min. The mixture was azeotroped from
anhydrous CH3CN (2 × 30 mL) to dryness, then anhydrous CH3CN
(30 mL) was added and the solution reduced to approximately half its
volume. To the stirred reaction mixture was added 1H-tetrazole (0.45
M in CH3CN, 1.30 mL, 0.585 mmol), and the mixture was stirred at rt
for 1.5 h. Anhydrous CH2Cl2 (10 mL) was added, and the reaction
mixture was stirred for a further 10 min before being cooled to −30
°C. m-CPBA (60%, 0.112 g, 0.390 mmol) was added to the cooled
solution and stirred for 10 min, then warmed to rt and stirred for a
further 1 h. The reaction mixture was diluted with CH2Cl2 (50 mL),
quenched with solid NaHCO3 (0.1 g), and diluted with H2O (30 mL)
and 10% Na2SO3 (20 mL). The aqueous phase was extracted with
CH2Cl2 (2 × 40 mL), and the combined organic phases were dried
(MgSO4), filtered, and the solvent removed in vacuo. The residue was
purified by column chromatography on silica gel (CH2Cl2/MeOH =
1:0 to 19:1) to give the title compound 3 (0.088 g, 51%) as a white
solid; mp 145−147 °C (CH2Cl2/MeOH); [α]2D8 −31.3 (c = 0.10,
MeOH). 1H NMR (500 MHz, (CD3)2SO) δ 1.127, 1.132 (2 × s, 18H,
C(CH3)3), 1.82 (s, 3H, OCOCH3), 1.98 (s, 3H, OCOCH3), 2.06 (s,
3H, OCOCH3), 3.00−3.07 (m, 4H, 2 × CH2S), 3.92−4.00 (m, 4H, 2
J = 5.0 Hz, C-6), 66.1 (d, J = 5.1 Hz, C-6), 67.9, 68.0 (C-4), 68.9 (d, J
= 5.5 Hz, POCH2), 69.0 (d, J = 5.4 Hz, POCH2), 70.61, 70.62 (C-2),
72.59, 72.6 (C-3), 74.4 (d, J = 7.3 Hz, C-5), 74.5 (d, J = 7.0 Hz, C-5),
87.9, 88.0 (C-1), 118.78 (d, J = 9.2 Hz, POCCH2), 118.82 (d, J = 9.1
Hz, POCCH2), 120.6, 120.7 (Ar-C), 124.5, 124.52, 125.4, 125.4,
129.92, 129.93, 129.95 (Ar-CH), 150.1, 150.2 (Ar-C), 169.19, 169.21,
169.3, 169.4, 170.10, 170.11 (OCOCH3). 31P NMR (162 MHz,
CDCl3) δ −9.31, −9.34. LRMS-ESI [M + Na]+ m/z = 522. HRMS-
ESI [M + Na]+ calcd for C19H22N3NaO11P, 522.0884; found,
522.0892.
CycloSal-(1-(4-[4-(aminosulfonyl)phenyl]-1H-1,2,3-triazole)-
2,3,4-tri-O-acetyl-β-D-glucopyranosyl-6)-phosphate (2). β-D-Glyco-
pyranosyl azide 11 (0.040 g, 0.080 mmol) and 4-ethynylbenzene-
sulfonamide (14)18 (0.015 g, 0.080 mmol) were suspended in an
EtOH/H2O mixture (3:1, 4 mL). To the reaction mixture was added a
solution of sodium ascorbate (0.013 g, 0.064 mmol) in water (0.25
mL), followed by a solution of CuSO4·5H2O (0.008 g, 0.032 mmol) in
water (0.25 mL). The bright-yellow suspension was stirred vigorously
at 60 °C for 2 h. The solvent was removed in vacuo, and the residue
was purified by column chromatography on silica gel (EtOAc/hexane
= 3:2 to 4:1) to give the title compound 2 (0.042 g, 76%,
diastereomeric ratio β1/β2 = 1:0.8) as a white solid; mp 203−205
°C (EtOAc/hexane); [α]D26 9.3 (c = 0.11, MeOH). H NMR (500
1
MHz, (CD3)2SO) δ 1.81 (s, 3H, 2 × OCOCH3), 1.94 (s, 1.5H,
OCOCH3), 1.95 (s, 1.5H, OCOCH3), 1.97 (s, 1.5H, OCOCH3), 2.04
(s, 1.5H, OCOCH3), 4.14−4.34 (m, 2H, CH2-6), 4.41−4.48 (m, 1H,
H-5), 5.13−5.19 (m, 1H, H-4), 5.38−5.45 (m, 2H, POCH2), 5.57−
5.62 (m, 1H, H-3), 5.64−5.69 (m, 1H, H-2), 6.40 (d, J = 9.1 Hz,
0.55H, H-1), 6.42 (d, J = 9.3 Hz, 0.45H, H-1), 7.00−7.08 (m, 1.5H,
Ar-H), 7.13−7.17 (m, 1H, Ar-H), 7.20−7.24 (m, 0.5H, Ar-H), 7.25−
7.32 (m, 1H, Ar-H), 7.40 (s, 2H, SO2NH2), 7.93 (d, J = 8.2 Hz, 2H,
Ar-H), 7.99−8.04 (m, 2H, Ar-H), 8.96 (s, 0.55H, CHtriazole), 9.06 (s,
0.45H, CHtriazole). 13C NMR (125 MHz, (CD3)2SO) δ 19.8, 20.2, 20.4
(OCOCH3), 65.6 (d, J = 4.1 Hz, C-6), 65.7 (d, J = 5.5 Hz, C-6), 67.0,
67.1 (C-4), 68.37, 68.43 (POCH2), 70.1, 70.2 (C-2), 72.03, 72.07 (C-
3), 73.4 (d, J = 7.8 Hz, C-5), 73.5 (d, J = 7.4 Hz, C-5), 83.9, 84.0 (C-
1), 117.9, 118.0, 118.1 (Ar-CH), 120.8, 120.87, 120.89, 120.97 (Ar-C),
121.47, 121.5 (CHtriazole), 124.2, 124.3, 125.5, 125.8, 126.0, 126.4,
126.5, 129.54, 129.55, 129.58, 129.59 (Ar-CH), 133.1 (Ctriazole), 143.5,
143.6, 145.6, 145.7, 149.2, 149.27 149.33 (Ar-C), 168.5, 169.1, 169.2,
169.5 (OCOCH3). 31P NMR (162 MHz, (CD3)2SO) δ −9.08, −9.39.
LRMS-ESI [M + H]+ m/z = 681, [M + Na]+ m/z = 703.0949. HRMS-
ESI [M + Na]+ calcd for C27H29N4NaO13PS, 703.1082; found,
703.1088.
4-[4-(Aminosulfonyl)phenyl]-1-(2,3,4-tri-O-acetyl-6-O-tert-butyl-
dimethylsilyl-β-D-glucopyranosyl)-1H-1,2,3-triazole (15). Azide 8
G
DOI: 10.1021/acs.jmedchem.5b01228
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
× CH2O), 4.04−4.10 (m, 1H, H-6), 4.13−4.18 (m, 1H, H-6), 4.42−
4.47 (m, 1H, H-5), 5.20 (t, J = 9.7 Hz, 1H, H-4), 5.62 (t, J = 9.4 Hz,
1H, H-3), 5.69 (t, J = 9.3 Hz, 1H, H-2), 6.45 (d, J = 9.0 Hz, 1H, H-1),
7.39 (s, 2H, SO2NH2), 7.91 (d, J = 8.2 Hz, 2H, Ar-H), 8.02 (d, J = 8.2
Hz, 2H, Ar-H), 9.06 (s, 1H, CHtriazole). 13C NMR (125 MHz,
(CD3)2SO) δ 19.8, 20.2, 20.4 (OCOCH3), 26.8 (C(CH3)3), 27.9 (d, J
= 7.6 Hz, CH2S), 28.0 (d, J = 7.6 Hz, CH2S), 45.9 (C(CH3)3), 65.1 (d,
J = 5.1 Hz, C-6), 65.5 (d, J = 5.5 Hz, CH2O), 65.6 (d, J = 5.5 Hz,
CH2O), 67.1 (C-4), 70.2 (C-2), 72.1 (C-3), 73.7 (d, J = 7.8 Hz, C-5),
84.0 (C-1), 121.4 (CHtriazole), 125.5, 126.4 (Ar-CH), 133.1 (Ctriazole),
143.6, 145.7 (Ar-C), 168.5, 169.1, 169.5 (OCOCH3), 204.9, 204.9
(SCOC(CH3)3). 31P NMR (162 MHz, (CD3)2SO) δ −1.34. LRMS-
ESI [M + Na]+ m/z = 903. HRMS-ESI [M + Na]+ calcd for
C34H49N4NaO15PS3, 903.1986; found, 903.1989.
the solvent removed under reduced pressure. The residue was
dissolved into EtOAc (50 mL) and washed with water (30 mL). The
aqueous fraction was re-extracted with EtOAc (3 × 30 mL), then the
combined organic fractions were washed with brine (50 mL), dried
(MgSO4), filtered, and the solvent removed in vacuo. The residue was
purified by column chromatography on silica gel (EtOAc/hexane = 1:9
to 1:2) to give the title compound 17 (0.264 g, 60%, 3 steps) as a
white foam; [α]2D5 −7.9 (c = 0.13, MeOH). H NMR (500 MHz,
1
CDCl3) δ 0.04 (s, 3H, SiCH3), 0.05 (s, 3H, SiCH3), 0.90 (s, 9H,
SiC(CH3)3), 2.02 (s, 3H, OCOCH3), 2.03 (s, 3H, OCOCH3), 2.06 (s,
3H, OCOCH3), 3.56−3.61 (m, 1H, H-5), 3.71 (dd, J = 4.0, 11.7 Hz,
1H, H-6), 3.82 (dd, J = 1.6, 11.5 Hz, 1H, H-6), 3.82 (s, 3H, OCH3),
4.28 (dd, J = 5.0, 14.3 Hz, 1H, N-CH2), 4.36 (d, J = 8.8 Hz, 1H, H-1),
4.41 (dd, J = 6.8, 14.3 Hz, 1H, N-CH2), 4.89 (t, J = 6.0 Hz, 1H, NH),
5.18 (t, J = 9.3 Hz, 1H, H-4),5.25−5.34 (m, 2H, H-2, H-3), 6.90 (d, J
= 8.4 Hz, 2H, Ar-H), 7.27 (d, J = 8.6 Hz, 2H, Ar-H). 13C NMR (125
MHz, CDCl3) δ −5.5, −5.4 (Si(CH3)2), 18.4 (SiC(CH3)3), 20.64
(OCOCH3), 20.65 (OCOCH3), 20.8 (OCOCH3), 25.9 (SiC(CH3)3),
47.6 (N-CH2), 55.4 (OCH3), 61.6 (C-6), 67.9 (C-4), 68.2 (C-2), 73.5
(C-3), 79.3 (C-5), 88.4 (C-1), 114.3 (2 × Ar-CH), 129.1 (Ar-C),
129.3 (2 × Ar-CH), 159.5 (Ar-C), 169.1, 170.2, 170.6 (OCOCH3).
LRMS-ESI [M + Na]+ m/z = 626. HRMS-ESI [M + Na]+ calcd for
C26H41NNaO11SSi, 626.2062; found, 626.2059.
N-(4-Methoxybenzyl)-1-S-(2,3,4,6-tetra-O-acetyl)-D-glucopyrano-
sylsulfonamide (19).19 To a solution of thioacetate derivative 1819
(5.00 g, 12.3 mmol) in anhydrous MeOH (100 mL) under a nitrogen
atmosphere was added diethyl bromomalonate (5.03 mL, 29.5 mmol),
and the solution was stirred at rt for 20 min. 4-Methoxybenzylamine
(8.20 mL, 62.7 mmol) was then added and the reaction mixture stirred
at rt overnight. The solvent was removed in vacuo. The residue was
dissolved into EtOAc (100 mL) and washed with brine (2 × 50 mL).
The aqueous layers were combined and re-extracted with EtOAc (2 ×
30 mL), and the combined organic fractions were dried (MgSO4),
filtered, and the solvent removed in vacuo. The residue was
semipurified by column chromatography on silica gel (EtOAc/hexane
= 1:2 to 2:3, 1% Et3N) to give N-(4-methoxybenzyl)-1-S-(2,3,4,6-tetra-
O-acetyl)-D-glucopyranosylsulfenamide (6.15 g, 12.3 mmol) as a pale-
yellow oil. To the residue dissolved in CH3CN (60 mL), was added a
finely ground mixture of the catalytic oxidative system (KMnO4/
CuSO4·5H2O; 1:1; w/w) (14.3 g, 51.7/12.3 mmol) in H2O (10 mL),
and the solution was stirred at rt for 2 h. The reaction mixture was
filtered through Celite and washed with EtOAc (300 mL). The organic
layer was washed with H2O (100 mL) and brine (100 mL), dried
(MgSO4), and the solvent removed in vacuo. The residue was purified
by column chromatography on silica gel (EtOAc/hexane = 1:4 to 1:2)
to give the title compound 19 (2.63 g, 40%) as a white solid; mp 140−
1-S-(2,3,4-Tri-O-acetyl)-D-glucopyranosylsulfonamide (20). To a
solution of sulfonamide 17 (0.500 g, 0.828 mmol) in CH3CN (30 mL)
and H2O (10 mL) was added ceric ammonium nitrate (1.82 g, 3.31
mmol), and the reaction mixture was stirred at rt for 3 h. Water (30
mL) was added, and the reaction mixture was extracted into EtOAc (3
× 40 mL). The organic extract was washed with brine (40 mL), dried
(MgSO4), and the solvent removed in vacuo. The residue was purified
by column chromatography on silica gel (EtOAc/hexane = 1:1 to 4:1)
to give the title compound 20 (0.274 g, 90%) as a white solid; mp
190−191 °C (EtOAc/hexane); [α]2D6 −10.1 (c = 0.10, MeOH). H
1
NMR (500 MHz, (CD3)2SO) δ 1.93 (s, 3H, OCOCH3), 1.94 (s, 3H,
OCOCH3), 1.99 (s, 3H, OCOCH3), 3.39−3.46 (m, 1H, H-6), 3.49−
3.55 (m, 1H, H-6), 3.87−3.92 (m, 1H, H-5), 4.69−4.73 (m, 2H, H-1,
OH), 4.85 (t, J = 9.8 Hz, 1H, H-4), 5.17 (t, J = 9.5 Hz, 1H, H-2), 5.37
(t, J = 9.4 Hz, 1H, H-3), 7.07 (s, 2H, SO2NH2). 13C NMR (125 MHz,
(CD3)2SO) δ 20.3, 20.36, 20.41 (OCOCH3), 60.2 (C-6), 67.9 (C-2),
68.0 (C-4), 73.1 (C-3), 77.4 (C-5), 86.2 (C-1), 168.6, 169.2, 169.5
(OCOCH3). LRMS-ESI [M − H]− m/z = 368. HRMS-ESI [M + Na]+
calcd for C12H19NNaO10S, 392.0622; found, 392.0618.
142 °C (EtOAc/hexane); [α]2D6 −27.6 (c = 0.12, MeOH). H NMR
1
(500 MHz, CDCl3) δ 2.01 (s, 3H, OCOCH3), 2.03 (s, 3H,
OCOCH3), 2.06 (s, 3H, OCOCH3), 2.08 (s, 3H, OCOCH3), 3.63−
3.67 (m, 1H, H-5), 3.82 (s, 3H, OCH3), 4.16 (dd, J = 2.4, 12.6 Hz, 1H,
H-6), 4.22−4.32 (m, 3H, H-6, H-1, N-CH2), 4.37 (dd, J = 6.0, 14.5
Hz, 1H, N-CH2), 4.98 (t, J = 6.0 Hz, 1H, NH), 5.05 (t, J = 9.7 Hz, 1H,
H-4), 5.25 (t, J = 9.3 Hz, 1H, H-3), 5.31 (t, J = 9.5 Hz, 1H, H-2), 6.91
(d, J = 8.6 Hz, 2H, Ar-H), 7.29 (d, J = 8.6 Hz, 2H, Ar-H). 13C NMR
(125 MHz, CDCl3) δ 20.6 (2 × OCOCH3), 20.71, 20.73 (OCOCH3),
47.5 (N-CH2), 55.4 (OCH3), 61.5 (C-6), 67.7 (C-4), 68.0 (C-2), 73.0
(C-3), 76.4 (C-5), 87.9 (C-1), 114.3 (2 × Ar-CH), 129.1 (Ar-C),
129.3 (2 × Ar-CH), 159.6 (Ar-C), 169.3, 170.0, 170.2, 170.5
(OCOCH3). LRMS-ESI [M − H]− m/z = 530. HRMS-ESI [M +
Na]+ calcd for C22H29NNaO12S, 554.1303; found, 554.1301.
2,3,4-Tri-O-acetyl-6-O-[[(bis((2-((2,2-dimethylpropionyl)sulfanyl)-
ethoxy)]-phosphoryloxy)-ethyl)]-β-D-glucopyranosylsulfonamide
(5). A solution of alcohol 20 (0.025 g, 0.068 mmol) and
phosphoramidite 714,26 (0.080 g, 0.176 mmol) in CH2Cl2 (10 mL)
was coevaporated to dryness and dried under high vacuum for 30 min.
The mixture was azeotroped from anhydrous CH3CN (2 × 10 mL) to
dryness, then anhydrous CH3CN (10 mL) was added and the solution
reduced to approximately half its volume. To the stirred reaction
mixture was added 1H-tetrazole (0.45 M in CH3CN, 0.602 mL, 0.271
mmol), and the mixture was stirred at rt for 1.5 h. Anhydrous CH2Cl2
(5 mL) was added, and the reaction mixture was stirred for a further
10 min before being cooled to −30 °C. m-CPBA (60%, 0.049 g, 0.169
mmol) was added to the cooled solution and stirred for 10 min, then
warmed to rt and stirred for a further 1 h. The reaction mixture was
diluted with CH2Cl2 (50 mL), quenched with solid NaHCO3 (0.05 g),
then diluted with H2O (30 mL) and 10% Na2SO3 (20 mL). The
aqueous phase was extracted with CH2Cl2 (2 × 40 mL), and the
combined organic phases were dried (MgSO4), filtered, and the
solvent removed in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 1:1 to 4:1) to give the
title compound 5 (0.044 g, 89%) as a white foam; [α]2D6 −8.5 (c = 0.12,
N-(4-Methoxybenzyl)-1-S-(2,3,4-tri-O-acetyl-6-O-tert-butyldime-
thylsilyl)-D-glucopyranosylsulfonamide (17). Sodium methoxide
(25% w/v, 0.50 mL, 2.21 mmol) was added dropwise to a stirred
solution of 19 (0.400 g, 0.753 mmol) in MeOH (10 mL), and the
resulting pH of the solution was greater than 11. The reaction mixture
was stirred at rt for 2 h then neutralized by the addition of Amberlite
IR-120 H+ ion-exchange resin. The solution was filtered, washed with
MeOH (50 mL), and the solvent removed in vacuo to give N-(4-
methoxybenzyl)-1-S-D-glucopyranosylsulfonamide as a colorless oil.
To the residue dissolved in anhydrous DMF (5 mL) was added tert-
butyldimethylsilyl chloride (0.131 g, 0.870 mmol) and imidazole
(0.148 g, 2.18 mmol) and the solution stirred at rt overnight. The
reaction was quenched by the addition of H2O (0.5 mL) at 0 °C, and
the solvent was removed in vacuo to afford N-(4-methoxybenzyl)-1-S-
6-O-tert-butyldimethylsilyl-D-glucopyranosylsulfonamide as a pale-
yellow oil, which was used without further purification. Acetic
anhydride (1.5 mL, 15.9 mmol) was added to the residue dissolved
in anhydrous pyridine (4 mL) and stirred at rt for 3 h. The reaction
mixture was quenched by the addition of a few drops of MeOH and
1
MeOH). H NMR (500 MHz, (CD3)2SO) δ 1.19, 1.20 (2 × s, 18H,
C(CH3)3), 1.94 (s, 3H, OCOCH3), 1.95 (s, 3H, OCOCH3), 2.01 (s,
3H, OCOCH3), 3.09−3.14 (m, 4H, 2 × CH2S), 4.01−4.16 (m, 6H, 2
× CH2O, CH2-6), 4.19−4.24 (m, 1H, H-5), 4.78 (d, J = 9.7 Hz, 1H,
H-1), 4.95 (t, J = 9.8 Hz, 1H, H-4), 5.21 (t, J = 9.5 Hz, 1H, H-2), 5.42
(t, J = 9.4 Hz, 1H, H-3), 7.15 (brs, 2H, NH2). 13C NMR (125 MHz,
(CD3)2SO) δ 20.2, 20.3, 20.4 (OCOCH3), 26.8 (C(CH3)3), 28.10 (d,
H
DOI: 10.1021/acs.jmedchem.5b01228
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Journal of Medicinal Chemistry
Article
J = 7.3 Hz, SCH2), 28.14 (d, J = 7.3 Hz, SCH2), 46.0 (C(CH3)3), 65.2
(d, J = 5.2 Hz, C-6), 65.7 (d, J = 5.7 Hz, CH2O), 65.8 (d, J = 5.7 Hz,
CH2O), 67.2 (C-4), 67.7 (C-2), 72.9 (C-3), 74.8 (d, J = 7.4 Hz, C-5),
86.3 (C-1), 168.6, 169.0, 169.5 (OCOCH3), 204.5, 205.0 (SCOC-
(CH3)3). 31P NMR (162 MHz, (CD3)2SO) δ −1.28. LRMS-ESI [M −
H]− m/z = 736. HRMS-ESI [M − H]− calcd for C26H43NO15PS3,
736.1527; found, 736.1531.
was greater than 11. The reaction mixture was stirred at rt for 2 h then
neutralized by the addition of Amberlite IR-120 H+ ion-exchange resin.
The solution was filtered, washed with MeOH (50 mL), and the
solvent removed in vacuo to give the title compound 21 (0.016 g,
1
quant) as a colorless oil; [α]2D8 −13.3 (c = 0.13, MeOH). H NMR
(500 MHz, (CD3)2SO) δ 3.32 (t, J = 9.4 Hz, 1H, H-4), 3.47 (t, J = 8.9
Hz, 1H, H-3), 3.66−3.71 (m, 1H, H-5), 3.82 (t, J = 9.1 Hz, 1H, H-2),
3.85−3.92 (m, 1H, H-6), 4.05−4.11 (m, 1H, H-6), 5.64 (d, J = 9.3 Hz,
1H, H-1), 7.36 (s, 2H, SO2NH2), 7.90 (d, J = 8.4 Hz, 2H, Ar-H), 8.08
(d, J = 8.4 Hz, 2H, Ar-H), 8.98 (s, 1H, CHtriazole). 13C NMR (125
MHz, (CD3)2SO) δ 64.4 (d, J = 3.7 Hz, C-6), 69.3 (C-4), 72.2 (C-3),
76.4 (C-2), 78.0 (d, J = 7.5 Hz, C-5), 87.6 (C-1), 121.6 (CHtriazole),
125.4, 126.4, (Ar-CH), 133.7 (Ctriazole), 143.2, 145.2 (Ar-C). 31P NMR
(162 MHz, (CD3)2SO) δ 0.51. LRMS-ESI [M − H]− m/z = −465.
HRMS-ESI [M + Na]+ calcd for C14H19N4NaO10PS, 489.0452; found,
489.0443.
1-(4-[4-(Aminosulfonyl)phenyl]-1H-1,2,3-triazole)-2,3,4-tri-O-ace-
tyl-β-D-glucopyranosyl-6-O-dibenzyl Phosphate (25). A solution of
alcohol 16 (0.120 g, 0.234 mmol) and dibenzyl N,N-diisopropylphos-
phoramidite 23 (0.121 g, 0.351 mmol) in anhydrous CH2Cl2 (10 mL)
was coevaporated to dryness and dried under high vacuum for 30 min.
The mixture was azeotroped from anhydrous CH3CN (2 × 10 mL) to
dryness, then anhydrous CH3CN (20 mL) was added and the solution
reduced to approximately half its volume. To the stirred reaction
mixture was added 1H-tetrazole (0.45 M in CH3CN, 1.04 mL, 0.468
mmol), and the mixture was stirred at rt for 1.5 h. Anhydrous CH2Cl2
(10 mL) was added, and the reaction mixture was stirred for a further
10 min before being cooled to −30 °C. m-CPBA (60%, 0.168 g, 0.585
mmol) was added to the cooled solution and stirred for 10 min, then
warmed to rt and stirred for a further 1 h. The reaction mixture was
diluted with CH2Cl2 (50 mL), H2O (30 mL), and 10% Na2SO3 (20
mL). The aqueous phase was extracted with CH2Cl2 (3 × 40 mL), and
the combined organic phases were dried (MgSO4), filtered, and the
solvent removed in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 3:2 to 4:1) to give the
title compound 25 (0.149 g, 82%) as a white solid; mp 211−213 °C
(EtOAc/hexane); [α]2D9 −25.7 (c = 0.12, MeOH). 1H NMR (500
MHz, (CD3)2SO) δ 1.82 (s, 3H, OCOCH3), 1.99 (s, 3H, OCOCH3),
2.02 (s, 3H, OCOCH3), 4.06−4.12 (m, 1H, H-6), 4.13−4.18 (m, 1H,
H-6), 4.43−4.48 (m, 1H, H-5), 4.92−4.99 (m, 4H, 2 × PhCH2), 5.23
(t, J = 9.7 Hz, 1H, H-4), 5.63 (t, J = 9.5 Hz, 1H, H-3), 5.71 (t, J = 9.3
Hz, 1H, H-2), 6.47 (d, J = 9.1 Hz, 1H, H-1), 7.24−7.32 (m, 10H, Ph),
7.39 (brs, 2H, SO2NH2), 7.89 (d, J = 8.6 Hz, 2H, Ar-H), 7.94 (d, J =
8.5 Hz, 2H, Ar-H), 9.05 (s, 1H, CHtriazole). 13C NMR (125 MHz,
(CD3)2SO) δ 19.9, 20.2, 20.3 (OCOCH3), 65.1 (d, J = 5.1 Hz, C-6),
67.1 (C-4), 68.5 (d, J = 5.1 Hz, PhCH2), 68.6 (d, J = 5.3 Hz, PhCH2),
70.2 (C-2), 72.1 (C-3), 73.7 (d, J = 7.7 Hz, C-5), 84.0 (C-1), 121.5
(CHtriazole), 125.4, 126.4, 127.6, 127.7, 128.2, 128.26, 128.31, 128.32
(Ar-CH), 133.0 (Ctriazole), 135.7 (d, J = 7.1 Hz, i-Ph), 135.8 (d, J = 6.9
Hz, i-Ph), 143.5, 145.7 (Ar-C), 168.5, 169.2, 169.5 (OCOCH3). 31P
NMR (162 MHz, (CD3)2SO) δ −0.67. LRMS-ESI [M + H]+ m/z =
773, [M + Na]+ m/z = 795. HRMS-ESI [M + Na]+ calcd for
C34H37N4NaO13PS, 795.1708; found, 795.1724.
N-(4-Methoxybenzyl)-1-S-(2,3,4-tri-O-acetyl)-D-glucopyranosyl-
sulfonamide (24). Acetyl chloride (0.05 mL, 0.70 mmol) was added to
a solution of 17 (0.190 g, 0.315 mmol) in CH2Cl2/MeOH (2:1, 15
mL) and stirred at rt for 1 h. The reaction mixture was quenched with
Et3N (100 μL) and stirred for 2 min which neutralized the solution.
The solvent was removed in vacuo. The residue was purified by
column chromatography on silica gel (EtOAc/hexane = 1:1 to 7:3) to
give the title compound 24 (0.148 g, 96%) as a white solid; mp 157−
159 °C (EtOAc/hexane); [α]2D5 −14.2 (c = 0.13, MeOH). H NMR
1
(500 MHz, CDCl3) δ 2.01 (s, 3H, OCOCH3), 2.03 (s, 3H,
OCOCH3), 2.05 (s, 3H, OCOCH3), 2.42−2.50 (m, 1H, OH), 3.42
(ddd, J = 2.1, 5.0, 10.3 Hz, 1H, H-5), 3.52 (dd, J = 4.9, 12.9 Hz, 1H, H-
6), 3.69 (dd, J = 2.2, 13.1 Hz, 1H, H-6), 3.81 (s, 3H, OCH3), 4.24 (d, J
= 9.1 Hz, 1H, H-1), 4.26−4.39 (m, 2H, N-CH2), 4.91 (t, J = 9.5 Hz,
1H, H-4), 5.22−5.29 (m, 2H, H-2, H-3), 5.56−5.62 (m, 1H, NH),
6.90 (d, J = 8.5 Hz, 2H, Ar-H), 7.29 (d, J = 8.5 Hz, 2H, Ar-H). 13C
NMR (125 MHz, CDCl3) δ 20.59, 20.6, 20.7 (OCOCH3), 47.3 (N-
CH2), 55.4 (OCH3), 60.9 (C-6), 68.0 (C-4), 68.1 (C-2), 73.0 (C-3),
78.8 (C-5), 87.5 (C-1), 114.3 (2 × Ar-CH), 129.26 (Ar-C), 129.34 (2
× Ar-CH), 159.5 (Ar-C), 170.0, 170.1, 170.2 (OCOCH3). LRMS-ESI
[M − H]− m/z = 488. HRMS-ESI [M + Na]+ calcd for
C20H27NNaO11S, 512.1197; found, 512.1196.
N-(4-Methoxybenzyl)-1-S-(2,3,4-tri-O-acetyl-6-O-dibenzylphos-
phate)-D-glucopyranosylsulfonamide (26). A solution of alcohol 24
(0.173 g, 0.353 mmol) and dibenzyl diisopropylphosphoramidite 23
(0.244 g, 0.706 mmol) in CH2Cl2 (10 mL) was coevaporated to
dryness and dried under high vacuum for 30 min. The mixture was
azeotroped from anhydrous CH3CN (2 × 10 mL) to dryness, then
anhydrous CH3CN (20 mL) was added and the solution reduced to
approximately half its volume. To the stirred reaction mixture was
added 1H-tetrazole (0.45 M in CH3CN, 1.57 mL, 0.706 mmol), and
the mixture was stirred at rt for 1.5 h. Anhydrous CH2Cl2 (5 mL) was
added, and the reaction mixture was stirred for a further 10 min before
being cooled to −30 °C. m-CPBA (60%, 0.254 g, 0.883 mmol) was
added to the cooled solution and stirred for 10 min, then warmed to rt
and stirred for a further 1 h. The reaction mixture was diluted with
CH2Cl2 (40 mL), H2O (20 mL), and 10% Na2SO3 (20 mL). The
aqueous phase was extracted with CH2Cl2 (3 × 40 mL), and the
combined organic phases were dried (MgSO4), filtered, and the
solvent removed in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/hexane = 1:1 to 3:2) to give the
title compound 26 (0.317 g, 90%) as a white foam; [α]2D5 −3.4 (c =
0.29, MeOH). 1H NMR (500 MHz, CDCl3) δ 1.98 (s, 3H,
OCOCH3), 2.00 (s, 3H, OCOCH3), 2.05 (s, 3H, OCOCH3), 3.43
(ddd, J = 2.4, 5.5, 10.2 Hz, 1H, H-5), 3.79 (s, 3H, OCH3), 3.92−3.99
(m, 1H, H-6), 4.00−4.06 (m, 2H, H-1, H-6), 4.19−4.29 (m, 2H,
NCH2), 4.87 (t, J = 9.7 Hz, 1H, H-4), 4.97−5.11 (m, 4H, 2 ×
PhCH2), 5.14 (t, J = 9.4 Hz, 1H, H-3), 5.28 (t, J = 9.6 Hz, 1H, H-2),
6.08 (t, J = 6.4 Hz, 1H, NH), 6.85 (d, J = 8.5 Hz, 2H, Ph), 7.28 (d, J =
8.5 Hz, 2H, Ph), 7.30−7.38 (m, 10H, Ph). 13C NMR (125 MHz,
CDCl3) δ 20.5, 20.6, 20.7 (OCOCH3), 47.3 (NCH2), 55.4 (OCH3),
65.4 (d, J = 5.4 Hz, C-6), 67.7 (C-4), 67.8 (C-2), 69.7 (d, J = 5.7 Hz,
PhCH2), 70.0 (d, J = 5.8 Hz, PhCH2), 73.1 (C-3), 76.3 (d, J = 5.4 Hz,
C-5), 87.0 (C-1), 114.1 (2 × Ar-CH), 128.1 (Ar-C), 128.2, 128.7,
1-(4-[4-(Aminosulfonyl)phenyl]-1H-1,2,3-triazole)-2,3,4-tri-O-ace-
tyl-β-D-glucopyranosyl-6-O-phosphate (28). Pd(OH)2 (20%, 20 mg)
was added to a solution of 25 (0.113 g, 0.146 mmol) in THF/MeOH
(2:3, 10 mL). The reaction mixture was stirred under an atmosphere
of hydrogen for 2 h at rt, then filtered through Celite and washed with
THF (20 mL) and MeOH (40 mL). The solvent was removed in
vacuo and the residue purified by RP-18 column chromatography
(MeOH/H2O 5:95 to 3:2, product eluting at 1:4) to give the title
compound 28 (0.067 g, 78%) as a white solid; mp 182−183 °C
(MeOH/H2O); [α]2D6 −16.0 (c = 0.18, MeOH). 1H NMR (500 MHz,
(CD3)2SO) δ 1.82 (s, 3H, OCOCH3), 1.98 (s, 3H, OCOCH3), 2.05
(s, 3H, OCOCH3), 3.80−3.93 (m, 2H, CH2−6), 4.35−4.40 (m, 1H,
H-5), 5.12 (t, J = 9.4 Hz, 1H, H-4), 5.58−5.67 (m, 2H, H-2, H-3), 6.42
(d, J = 8.3 Hz, 1H, H-1), 7.39 (s, 2H, SO2NH2), 7.91 (d, J = 8.4 Hz,
2H, Ar-H), 8.03 (d, J = 8.4 Hz, 2H, Ar-H), 9.08 (s, 1H, CHtriazole). 13
C
NMR (125 MHz, (CD3)2SO) δ 19.9, 20.2, 20.4 (OCOCH3), 63.7 (d, J
= 4.0 Hz, C-6), 67.8 (C-4), 70.3 (C-2), 72.1 (C-3), 74.5 (d, J = 8.6 Hz,
C-5), 84.0 (C-1), 121.6 (CHtriazole), 125.5, 126.4, (Ar-CH), 133.1
(Ctriazole), 143.5, 145.7 (Ar-C), 168.6, 169.3, 169.5 (OCOCH3). 31P
NMR (162 MHz, (CD3)2SO) δ −0.67. LRMS-ESI [M − H]− m/z =
591. HRMS-ESI [M + Na]+ calcd for C20H25N4NaO13PS, 615.0769;
found, 615.0761.
1-(4-[4-(Aminosulfonyl)phenyl]-1H-1,2,3-triazole)-β-D-glucopyra-
nosyl-6-O-phosphate (21). Sodium methoxide (25% w/v, 0.150 mL,
2.69 mmol) was added dropwise to a stirred solution of 28 (0.020 g,
0.034 mmol) in MeOH (5 mL), and the resulting pH of the solution
I
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128.72, 128.75 (Ar-CH), 129.4 (Ar-C), 129.6 (2 × Ar-CH), 135.6 (d, J
= 6.6 Hz, i-Ph), 135.7 (d, J = 7.2 Hz, i-Ph), 159.4 (Ar-C), 169.2, 169.7,
170.1 (OCOCH3). 31P NMR (162 MHz, CDCl3) δ −0.73. LRMS-ESI
[M − H]− m/z = 748. HRMS-ESI [M + Na]+ calcd for
C34H40NNaO14PS, 772.1799; found, 772.1803.
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.
1-S-(2,3,4-Tri-O-acetyl-6-O-dibenzylphosphate)-D-glucopyrano-
sylsulfonamide (27). To a solution of 26 (0.100 g, 0.133 mmol) in
CH3CN (5 mL) and H2O (1.5 mL) was added ceric ammonium
nitrate (0.292 g, 0.534 mmol), and the reaction mixture was stirred at
rt for 2 h. H2O (30 mL) was added, and the reaction mixture was
extracted into EtOAc (3 × 40 mL). The organic extract was washed
with brine (40 mL), dried (MgSO4), and the solvent removed in
vacuo. The residue was purified by column chromatography on silica
gel (EtOAc/hexane = 1:1 to 7:3) to give the title compound 27 (0.076
g, 90%) as a colorless oil; [α]2D8 +4.8 (c = 0.16, MeOH). 1H NMR (500
MHz, CDCl3) δ 2.02 (s, 3H, OCOCH3), 2.03 (s, 3H, OCOCH3), 2.07
(s, 3H, OCOCH3), 3.77 (ddd, J = 2.0, 4.0, 10.3 Hz, 1H, H-5), 4.01 (td,
J = 4.1, 12.9, 12.8 Hz, 1H, H-6), 4.16−4.23 (m, 1H, H-6), 4.39 (d, J =
9.6 Hz, 1H, H-1), 4.98−5.15 (m, 5H, H-4, 2 × PhCH2), 5.27−5.33
(m, 2H, H-2, H-3), 5.69 (brs, 2H, SO2NH2), 7.31−740 (m, 10H, Ph).
13C NMR (125 MHz, CDCl3) δ 20.55, 20.6, 20.7 (OCOCH3), 65.5 (d,
J = 6.0 Hz, C-6), 67.5 (C-4), 67.8 (C-2), 70.02 (d, J = 5.8 Hz, PhCH2),
70.08 (d, J = 5.7 Hz, PhCH2), 72.7 (C-3), 76.1 (d, J = 3.4 Hz, C-5),
87.1 (C-1), 128.1, 128.2, 128.7, 128.75, 128.77 (Ph), 135.62 (d, J = 6.5
Hz, i-Ph), 135.63 (d, J = 6.7 Hz, i-Ph), 169.2, 170.0, 170.3
(OCOCH3). 31P NMR (162 MHz, CDCl3) δ −1.41. LRMS-ESI [M
+ H]+ m/z = 630, [M − H]− m/z = 628. HRMS-ESI [M + Na]+ calcd
for C26H32NNaO13PS, 630.1405; found, 630.1406.
1-S-(2,3,4-Tri-O-acetyl-6-O-phosphate)-D-glucopyranosylsulfona-
mide (29). Pd(OH)2 (20%, 25 mg) was added to a solution of 27
(0.120 g, 0.191 mmol) in THF/MeOH (2:3, 10 mL). The reaction
mixture was stirred under an atmosphere of hydrogen for 2 h at rt,
then filtered through Celite and washed with THF (20 mL) and
MeOH (40 mL). The solvent was removed in vacuo and the residue
purified by RP-18 column chromatography (MeOH/H2O 5:95 to 3:2,
product eluting at 1:9) to give the title compound 29 (0.068 g, 79%)
as a white solid; mp 182−184 °C (MeOH/H2O); [α]2D6 +8.8 (c = 0.15,
MeOH). 1H NMR (500 MHz, (CD3)2SO) δ 1.936 (s, 3H, OCOCH3),
1.943 (s, 3H, OCOCH3), 1.99 (s, 3H, OCOCH3), 3.84−3.89 (m, 2H,
H-6), 4.09−4.14 (m, 1H, H-5), 4.74 (d, J = 9.7 Hz, 1H, H-1), 4.86 (t, J
= 9.8 Hz, 1H, H-4), 5.18 (t, J = 9.5 Hz, 1H, H-2), 5.39 (t, J = 9.4 Hz,
1H, H-3), 7.20 (brs, 2H, SO2NH2). 13C NMR (125 MHz, (CD3)2SO)
δ 20.26, 20.3, 20.4 (OCOCH3), 63.4 (d, J = 3.9 Hz, C-6), 67.8 (C-2 or
C-4), 67.9 (C-2 or C-4), 73.0 (C-3), 75.6 (d, J = 7.9 Hz, C-5), 86.3
(C-1). 31P NMR (162 MHz, CDCl3) δ −0.15. LRMS-ESI [M − H]−
m/z = 448; HRMS-ESI [M + Na]+ calcd for C12H20NNaO13PS,
472.0285; found, 472.0281.
1-S-(6-O-Phosphate)-D-glucopyranosylsulfonamide (22). Sodium
methoxide (25% w/v, 0.150 mL, 2.69 mmol) was added dropwise to a
stirred solution of 29 (0.020 g, 0.034 mmol) in MeOH (5 mL), and
the resulting pH of the solution was greater than 11. The reaction
mixture was stirred at rt for 2 h then neutralized by the addition of
Amberlite IR-120 H+ ion-exchange resin. The solution was filtered,
washed with MeOH (50 mL), and the solvent removed in vacuo to
give the title compound 22 (0.016 g, quant) as a colorless oil; [α]D28
+8.5 (c = 0.13, MeOH). 1H NMR (500 MHz, (CD3)2SO) δ 3.12 (t, J
= 9.4 Hz, 1H, H-4), 3.30 (t, J = 8.8 Hz, 1H, H-3), 3.42−3.48 (m, 2H,
H-2, H-5), 3.87−3.94 (m, 1H, H-6), 4.09−4.14 (m, 2H, H-1, H-6),
6.72 (brs, 2H, SO2NH2). 13C NMR (125 MHz, (CD3)2SO) δ 64.9 (d,
J = 5.0 Hz, C-6), 69.1 (C-4), 70.7 (C-2), 76.9 (C-3), 79.2 (d, J = 7.4
Hz, C-5), 90.6 (C-1). 31P NMR (162 MHz, CDCl3) δ 0.70. LRMS-ESI
[M − H]− m/z = 322. HRMS-ESI [M − H]− calcd for C6H13NO10PS,
322.0003; found, 322.0001.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01228.
1H and 13C NMR spectra for compounds 8, 12, 15−17,
1
19, 20, and 24; H, 31P and 13C NMR spectra for
compounds 2, 3, 5, 11, 21, 22, 25, 26, 27, 28, and 29
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +61 7 3735 7825. E-mail: s.poulsen@griffith.edu.au.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Australian Research Council (grant numbers
DP110100071, FT10100185 to S.-A.P.) and two EU grants of
the seventh framework program (Metoxia and Dynano projects
to C.T.S.) for support.
ABBREVIATIONS USED
■
CA, carbonic anhydrase; AZA, acetazolamide; CuAAC, copper-
catalyzed azide alkyne cycloaddition; SAR, structure−activity
relationship; Ki, inhibition constant; cycloSal, cyclosaligenyl;
SATE, S-acyl-2-thioethyl; POC, bis(isopropyloxymethyl) car-
bonate
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