Regioselective sulfamoylation at low temperature enables concise syntheses of putative small molecule inhibitors of sulfatases

Article abstract of DOI:10.1039/c5ob00211g

Regioselective sulfamoylation of primary hydroxyl groups enabled a 5-step synthesis (overall yield 17%) of the first reported small molecule inhibitor of sulfatase-1 and 2, ((2S,3R,4R,5S,6R)-4,5-dihydroxy-2-methoxy-6-((sulfamoyloxy)methyl)tetrahydro-2H-pyran-3-yl)sulfamic acid, which obviated the use of hydroxyl protecting groups and is a marked improvement on the reported 9-step synthesis (overall yield 9%) employing hazardous trifluoromethylsulfonyl azide. The sulfamoylation methodology was used to prepare a range of derivatives of 1, and inhibition data was generated for Sulf-2, ARSA and ARSB.

Full text of DOI:10.1039/c5ob00211g

Organic &
Biomolecular Chemistry
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Regioselective sulfamoylation at low temperature
enables concise syntheses of putative small
molecule inhibitors of sulfatases†
Cite this: Org. Biomol. Chem., 2015,
13, 5279
Duncan C. Miller,*a Benoit Carbain,a Gary S. Beale,b Sari F. Alhasan,b Helen L. Reeves,b
Ulrich Baisch,c,d David R. Newell,b Bernard T. Golding*a and Roger J. Griffin‡a
Regioselective sulfamoylation of primary hydroxyl groups enabled a 5-step synthesis (overall yield 17%) of
the first reported small molecule inhibitor of sulfatase-1 and 2, ((2S,3R,4R,5S,6R)-4,5-dihydroxy-2-
methoxy-6-((sulfamoyloxy)methyl)tetrahydro-2H-pyran-3-yl)sulfamic acid, which obviated the use of
hydroxyl protecting groups and is a marked improvement on the reported 9-step synthesis (overall yield
9%) employing hazardous trifluoromethylsulfonyl azide. The sulfamoylation methodology was used to
prepare a range of derivatives of 1, and inhibition data was generated for Sulf-2, ARSA and ARSB.
Received 2nd February 2015,
Accepted 31st March 2015
DOI: 10.1039/c5ob00211g
www.rsc.org/obc
see Scheme 1), a derivative of glucosamine sulfate, which
bears an O-sulfamate at the 6-position and a N-sulfate on the
Introduction
The human sulfatase enzymes, sulfatase-1 (Sulf-1) and sulfa- amino function. The reported IC50 values for 1 are 95 μM and
tase-2 (Sulf-2), were first cloned in 2002 1 and have been associ- 130 μM against Sulf-1 and Sulf-2, respectively.
ated with both the FGF and wnt signalling pathways.2 The FGF
signalling pathway is known to affect cell proliferation, inva-
sion, migration and angiogenesis,3 while the wnt signalling
pathway has been implicated in cell growth and proliferation.4
Sulf-1 and Sulf-2 have selectivity for removing sulfates from
the 6-position of glucosamine residues in heparan sulfate pro-
teoglycans (HSPGs), which consist of a protein core conjugated
to multiple heparan sulfate polysaccharide chains. The HSPG
chains are composed to a large extent of repeating disacchar-
ide units of hexuronic acid (glucuronic acid or iduronic acid)
and glucosamine. Weakly active small molecule dual inhibi-
tors of Sulf-1 and Sulf-2 have been reported.5 The most active
inhibitor was ((2S,3R,4R,5S,6R)-4,5-dihydroxy-2-methoxy-6-
((sulfamoyloxy)methyl)tetrahydro-2H-pyran-3-yl)sulfamic acid,
aNewcastle Cancer Centre, Northern Institute for Cancer Research, School of
Scheme 1 Synthesis of 1a, 1b, 10, 11 and 16 from D-glucosamine.
Reagents and conditions: (a) CbzCl, NaHCO3, H2O, 92%; (b) HCl–MeOH,
80 °C, 18 h, 73%; (c) Method 1 H2NSO2Cl, DMA–toluene, −15 °C, 43%;
Method 2 H2NSO2Cl, DMF–CH3CN, −40 °C, 55%; Method 3 H2NSO2Cl,
10% DMA–MeCN, −40 °C, 19%. (d) H2/Pd/C, MeOH, 40 °C, 2 h, 99%;
(e) R1 = SO3NH4: (i) Py·SO3, H2O, pH 9–10, (ii) EtOAc–MeOH–NH4OH,
Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU,
UK. E-mail: bernard.golding@newcastle.ac.uk; Fax: +44 (0)191 2226929;
Tel: +44 (0)191 2226647
bNorthern Institute for Cancer Research, Paul O’Gorman Building, Medical School,
Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
cSchool of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne,
43%; R1 = MeSO2: MeSO2Cl, Et2iPrNH, CH2Cl2, 0 °C-r.t., 1 h, 28%; R1
=
NE1 7RU, UK
CF3SO2: (CF3SO2)2O, Et3N, CH2Cl2–dioxane, 0 °C, 1 h, 33%. R1 = CO-
(CH2)2CO2H: succinic anhydride, H2O–dioxane, r.t., 18 h, 18%; (f) 1,1,1-
trichloroethylsulfonyl imidazolium tetrafluoroborate (1 eq.), THF, 60 °C,
18 h, 61%; (g) R1 = SO3Na (i) Zn (15 eq.), MeOH, H2O, 60 °C. 1 h; (ii) ion
exchange chromatography on Dowex 50W8 × 200 Na form, H2O, 93%.
dDepartment of Chemistry, University of Malta, Msida, MSD 2080, Malta
†Electronic supplementary information (ESI) available. CCDC 1043886. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c5ob00211g
‡Deceased 24 September 2014.
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The published synthesis5 from (D)-(+)-glucosamine 2 pro-
Organic & Biomolecular Chemistry
vided compound 1 by a 9-step route in 9% overall yield. The
amino group of 2 was converted into azido through a diazo
transfer reaction with triflic azide, a reagent with a well-docu-
mented explosion hazard.6 The use of this reagent in the pres-
ence of dichloromethane further exacerbates the risk, as this
combination can form diazidomethane, another high energy
compound with an inherent explosion hazard.7 The anomeric
methoxy group was installed, followed by a sequence of 3 steps
requiring benzyl protection of the 3- and 4-hydroxy groups.
After sulfamate formation on the unprotected 6-hydroxyl
group, the azido group was reduced to an amino function,
which was converted to an N-sulfate. Finally, deprotection of
the 3- and 4-benzyl ethers provided 1.
The developed conditions were applied for sulfamoylation of
4. After 18 hours at −40 °C, a further 0.15 equivalent of
H2NSO2Cl was added to each reaction and stirring was contin-
ued for a further 3 hours. Chromatographic purification led to
isolation of 55% product 5 using Method 2, but only 19% with
Method 3. Full details of sulfamoylation methods 1–3 are
given in the general procedures in the ESI.†
The Cbz protecting group was removed from 5 in quantitat-
ive yield under palladium-catalysed flow hydrogenation con-
ditions in methanol to give 6. Addition of sulfur trioxide-
pyridine complex to 6 in an aqueous medium (pH 9–10),
resulted in chemoselective sulfation of the amino group to
give 1 in 43% yield.11 Thus, the target monosaccharide 1 was
obtained in a 17% overall yield in only five steps (Scheme 1).
An alternative N-sulfation process via a trichloroethyl pro-
tected sulfate provided 1 in a superior yield of 56% over two
steps from 6 via 9 (Scheme 1). Incorporation of this sulfation
protocol into the synthesis of 1 improved the overall yield for
the preparation of this key Sulf-2 inhibitor to 21% over six
steps.
Improved synthesis of 1
In order to prepare inhibitor 1 as a tool for further study of the
biological implications of inhibition of Sulf-1 and Sulf-2, we
investigated an alternative route that avoids triflic azide and
minimises the use of protecting groups, thus reducing the
number of steps and improving the efficiency of the synthesis.
We sought a strategy to compound 1 that dispensed with pro-
tection/deprotection at the 3- and 4-positions of glucosamine.5
Selective sulfamoylation of the primary hydroxyl groups in
di- and tri-hydroxylated compounds using (N-(tert-butoxycarbo-
nyl)-N-[(triethylenedi-ammonium) sulfonyl]azanide) gave only
moderate yields and regioselectivity on simple alkanediols,
required the synthesis of the reagent and a subsequent acidic
deprotection step to liberate the primary sulfamate.8 We there-
fore investigated an alternative approach using sulfamoyl
chloride as the sulfamoylating reagent, exploiting the differen-
tial reactivity of primary versus secondary alcohols to achieve
direct regioselective sulfamoylation of the O6-position
(Scheme 1).
Synthesis of analogues of 1
From the homology model of F. heparinium sulfatase, it has
been proposed that the sulfate on N2 of the glucosamine
residue in heparan sulfate may affect binding solely by enhan-
cing the hydrogen bond donor ability of the N2-hydrogen
atom.12 To explore this hypothesis for Sulf-2, sulfonamides 10
and 11 that would affect NH acidity were synthesised as shown
in Scheme 1.
Protection of glucosamine 2 with a benzylcarbamate (Cbz)
group under standard conditions,9 gave intermediate 3 in 92%
yield. Heating 3 in methanolic hydrogen chloride gave a 5 to 1
ratio of α to β anomers under thermodynamic control. The
desired α anomer 4 was easily separated in 73% yield.
Slow evaporation of a dilute methanolic solution of 10 pro-
vided a crystal suitable for small molecule X-ray crystallo-
graphy. The structure (Fig. 1) confirmed the assignment of the
O6-sulfamate regiochemistry and that as expected, the pyra-
nose core adopts a chair conformation. The amino hydrogen
atoms of N1 and N2 and the oxygen atoms of the sulfonyl
Regioselective sulfamoylation was initially attempted using
sulfamoyl chloride (H2NSO2Cl) prepared by reaction of sulf-
amoyl isocyanate with formic acid.10 Suitable solvent systems
for sulfamate formation were identified using 4-nitrophenethyl
alcohol 7 as a model substrate. For optimisation studies it was
preferable to generate H2NSO2Cl in solution in acetonitrile,
prior to addition to substrate in DMA, to allow control of gas
evolution on scale-up. Reaction of the acetonitrile solution of
H2NSO2Cl with 7 in DMA (Method 1) resulted in efficient con-
version to 8 within 5 minutes at room temperature. Further
studies showed that DMF (Method 2) and 10% DMA in aceto-
nitrile (Method 3) were also suitable for the sulfamoylation of
7 to 8, and allowed the reaction to be performed at −40 °C,
giving high conversions after 24 hours and similar reaction
profiles.
Fig. 1 Small molecule X-ray crystal structure of 10.
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groups are in ideal positions to form moderate to strong hydro-
From consideration of the structure of heparan sulfate, it
gen bonds. Sulfonamides are amongst the strongest H-bond was concluded that the binding site of Sulf-2 accommodates
donors, with N(H)⋯O distances ranging between 2.7 Å (270 further saccharide units at the reducing end of the mono-
pm) and 3.2 Å.13 Molecules of 10 form a 2-dimensional saccharide template. In an effort to develop SARs for this
network of double layered sheets comprising hydrogen bonds region, alternative anomeric substituents were investigated.
(O–H⋯O and NH⋯O) with donor–acceptor distances of Thus, reaction of 3 with isopropyl alcohol and 4 M HCl–
2.85–3.10 Å. The H-bond distances are in the range reported dioxane gave predominantly α-anomer 27 in 73% isolated
for amido-functionalised monosaccharides.14
yield. Sulfamoylation using Method 1 gave a 37% yield of 28,
Succinamide derivative 16 was prepared by reaction of 6 which was progressed through deprotection/sulfation steps
with succinic anhydride (Scheme 1). The importance of the (Scheme 3) to provide target 30.
anomeric stereochemistry of the monosaccharide template was
The anomeric position of glucosamine in HSPGs is linked
investigated by preparing β anomer 20 (see ESI: Scheme S1†). to an iduronic acid residue. Hence, polar groups at this posi-
Reaction of 3 with a 1.25 M methanolic HCl solution for tion may be able to mimic interactions of the polar functional-
1 hour gave a 3 : 2 ratio of α to β anomers, allowing isolation of ity of the iduronate residue with the Sulf-2 protein. The
a 37% yield of 4 and 21% of 17. Sulfamoylation of 17 using allyloxy group was introduced into the anomeric position
Method 1 gave a 19% yield of 18, which was converted into using allyl alcohol and 4 M HCl–dioxane at 70 °C for 18 h, to
target 20 with no epimerisation at the anomeric centre.
give a 52% yield of α anomer 31, together with 20% of the β
To assess whether the N-sulfate could be replaced by a anomer, which were readily separable. Sulfamate formation
hydroxyl group, analogues were prepared in a single step from using Method 1 on 31 gave a 40% yield of 32. Reduction of the
commercially available methyl α-D-glucopyranoside 12 and alkene was achieved concurrently with hydrogenolysis of the
methyl α-D-mannopyranoside 14 using sulfamoylation Method Cbz-protected amine to give 33, which was sulfated to provide
2. The reaction did not proceed cleanly and isolated yields of 34 (Scheme 3). Ozonolysis of 32, with reductive work-up, gave
only 25% of 13 and 9% of 15 were obtained. Sulfamoylation of 35, which was carried through the standard deprotection/sulfa-
the 2-position is a likely confounding factor in this reaction, tion methodology to provide 37.
consistent with the empirically observed order of reactivity of
the hydroxyl groups of glucopyranosides (6 > 2 > 3 > 4) in reac-
tions with benzoyl chloride.15
For removal of the anomeric substituent of 1 (Scheme 2),
treatment of 3 with acetyl chloride16 gave 21. Radical hydro-
dechlorination16 with tributyltin hydride and AIBN gave a 47%
isolated yield of 22. Tributyltin hydride could be replaced by
the less toxic tris(trimethylsilyl)silane,17 resulting in a cleaner
reaction profile and a straightforward purification on silica,
leading to an improved isolated yield of 90% for 22. Deprotec-
tion of the acetoxy groups under Zemplén conditions18 pro-
ceeded in high yield to triol 23. The use of sulfamoylation
Method 1, gave a 34% yield of 24, which was progressed using
the conditions described for the previous analogues, to
provide 26.
Scheme 3 Reagents and conditions: R1 = R2
=
iPr: (a) HCl–dioxane,
IPA, 60 °C, 4 h, 73%; (b) ClSO2NH2, Tol–DMA, −15 °C, 2 h, 37%; (c) H2/
10% Pd/C, MeOH–CH2Cl2, 40 °C, 1 h, 100%; (f) SO3·Py, H2O, pH 9–10,
r.t., 90 min, 27%; R1 = OCH2CHvCH2, R2
=
nPr: (a) allyl alcohol, HCl–
dioxane, 60 °C, 4 h, 52%; (b) ClSO2NH2, Tol–DMA, −15 °C, 2.5 h, 40%;
(c) H2/10% Pd/C, MeOH–CH2Cl2, 40 °C, 2 h, 100%; (f) SO3·Py, H2O, pH
9–10, r.t. 90 min, 39%; R1 = OCH2CHvCH2, R2 = CH2CH2OH: (a) allyl
alcohol, HCl–dioxane, 60 °C, 4 h, 52%; (b) ClSO2NH2, Tol–DMA, −15 °C,
2.5 h, 40%; (d) (i) O3/MeOH, −78 °C, 30 min; (ii) NaBH4, 1 h, 69%; (e) H2/
10% Pd/C, MeOH–CH2Cl2, 40 °C, 3 h, 78%; (f) SO3·Py, H2O, pH 9–10,
r.t., 1 h, 30%. R1 = R2 = O(CH2)3OBn: (a) 3-(benzyloxy)propan-1-ol, HCl–
dioxane, 75 °C, 5 h, 31%; (b) ClSO2NH2, DMF, −40 °C, 18 h, 57%; (c) H2/
5% Pd/C, EtOH, 20 °C, 1 h, 75%; (f) SO3·Py, H2O, pH 9–10, r.t., 18 h, 62%.
R1 = O(CH2)3OBn, R2 = O(CH2)3OH: (a) 3-(benzyloxy)propan-1-ol, HCl–
dioxane, 75 °C, 5 h, 31%; (b) ClSO2NH2, DMF, −40 °C, 18 h, 57%; (c) H2/
5% Pd/C, AcOH, 20 °C, 1 h, 83%; (f) SO3·Py, H2O, pH 9–10, r.t., 1 h, 41%.
Scheme 2 Reagents and conditions: (a) AcCl, r.t., 48 h, 55%; (b) (i)
Bu3SnH–AIBN, 110 °C, 1.5 h, 47%, or (ii) (TMS)3SiH–AIBN, 110 °C, 1.5 h,
92%; (c) NaOMe (cat.), MeOH, r.t., 2 h, 85%; (d) ClSO2NH2, Tol–DMA,
−15 °C, 2 h, 34%; (e) H2/10% Pd/C, MeOH–CH2Cl2, 40 °C, 2 h, 98%; (f)
SO3·Py, H2O, pH 9–10, r.t., 2 h, 24%.
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Incorporation of 3-benzyloxypropanol at the C1-position of
3 gave a 31% yield of α anomer 46 (Scheme 3). Sulfamoylation
using Method 2 gave 47 in 57% yield. Palladium catalysed
hydrogenation allowed deprotection of both O-benzyl and
N-Cbz groups to give 48, which was sulfated on nitrogen to
yield target 49. Chemoselective removal of the carbamate from
47 was achieved by flow hydrogenation over 5% palladium on
carbon at room temperature, providing 50, which was sulfated
using the pH-controlled sulfation conditions to give benzyloxy-
propyl derivative 51 in 62% yield (Scheme 3).
Scheme 5 Reagents and conditions: (a) PhCH(OMe)2, p-TsOH, DMF,
75 °C, 3 h, 79%; (b) (i) CS(Im)2, toluene, 110 °C, 3 h; (ii) TMS3SiH, AIBN,
110 °C, 1 h, 83%; (c) (i) p-TSA (cat), MeOH–CH2Cl2, μW, 80 °C, 20 min;
(ii) 10% K2CO3 (aq), 72%; (d) ClSO2NH2, DMF, −40 °C, 24 h, 36%; (e) H2/
10% Pd/C, AcOH, 40 °C, 2 h, 97%; (f) SO3·Py, H2O, pH 9–10, r.t., 2 h,
21%.
Oxidative cleavage of the allyl group of 38 using NaIO4 with
19
catalytic RuCl3
afforded 39 (Scheme 4). Alkylation of the
carboxylic acid 39 gave ester 40. Deprotection of the acetate
groups using Zemplén conditions proceeded in high yield to
41, which was sulfamoylated to 42 in 67% yield using Method
2. Hydrolysis of ester 42 to the corresponding carboxylic acid
43 prior to deprotection of the amino functionality afforded
44. The latter was sulfamoylated to acid 45.
An approach employing Barton–McCombie radical deoxy-
genation to allow preparation of C3- and C4-methylene targets
57 and 63 was inspired by a similar strategy described for
the synthesis of a series of activators of the glmS-riboswitch of
Staphylococcus aureus.20 The 4- and 6-positions of 4 were selec-
tively protected as benzylidene acetal 52 (Scheme 5)21 which
gave an isolated yield of 79%. Radical deoxygenation of 52
under modified Barton–McCombie conditions22,23 using tris
(trimethylsilyl)-silane17 gave 53 in 83% isolated yield.24 Com-
plete removal of the benzylidene acetal from 53 afforded diol
54. Regioselective sulfamate formation with Method 2, fol-
lowed by hydrogenation and N-sulfation gave 57. Hydrogen-
ation followed by N-sulfation, gave 57. The synthesis of
C4-methylene derivative 63 required selective protection of the
3- and 6-hydroxyl groups. The order of reactivity of the
hydroxyl groups of methyl-D-glucopyranoside15 is conserved in
D-glucosamine systems, enabling selective protection at the
3- and 6-positions.20 Using the literature conditions at room
temperature resulted in no selectivity, with only 7% of 58
being obtained along with 69% of tris(benzoyl) product. Redu-
cing the reaction temperature to −40 °C allowed 58 to be iso-
lated in a 72% yield (Scheme 6). Radical deoxygenation of 58
provided 59, which was deprotected to give a 68% yield of diol
Scheme 6 Reagents and conditions: (a) BzCl (2.2 eq.), CH2Cl2–pyri-
dine, −40 °C, 3 h, 72%; (b) (i) CS(Im)2, toluene, 110 °C, 3 h; (ii) TMS3SiH,
AIBN, 110 °C, 1 h, 79%; (c) NaOMe (cat), MeOH, r.t., 18 h, 68%; (d)
ClSO2NH2, DMF, −40 °C, 36 h, 51%; (e) H2/10% Pd/C, AcOH, 40 °C, 2 h,
70%; (f) SO3·Py, H2O, pH 9–10, r.t., 2 h, 8%; (g) benzyl-2,2,2-trichloro-
acetimidate (8 eq.), TfOH, dioxane, 60 °C, 4 h, 78%; (h) 3 eq. LiAlH4, 0 °C,
2 h, 69%; (i) ClSO2NH2, Tol–DMA, −40 °C, 22 h, 52% ( j) 10 bar H2/5%
Pd/C, MeOH, r.t. 35 min, 100% (k) SO3·Py, H2O, pH 9–10, r.t., 2 h, 25%.
Scheme 4 Reagents and conditions: (a) pyridine, Ac2O, r.t. 8 h, 96%; (b)
RuCl3, NaIO4, MeCN, CH2Cl2, H2O, r.t. 30 min, 56–62%. (c) MeI–
Cs2CO3–CH3CN, r.t., 18 h, 77–95%; (d) NaOMe (cat.), MeOH, r.t., 1 h,
100%; (e) ClSO2NH2, DMF, −40 °C, 18 h, 67%; (f) 2 M NaOH(aq.), THF, r.t.,
2 h, 82%; (g) H2/10% Pd/C, MeOH, 40 °C, 3 h, 100%; (h) SO3·Py, H2O, pH
9–10, r.t., 24 h, 38%.
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Table 1 Sulfatase inhibition data
Cmpd
Sulf-2a
ARSAa
ARSBa
R1
R2
R3
R4
1
0
0
0
0
0
0
n.t.
0
0
0
n.t.
0
0
0
7
0
0
0
0
0
70
0
0
0
40
0
0
0
29
0
0
86
0
0
0
0
0
0
32
67
0
0
0
77
19
0
12
55
33
44
3
11
14
12
21
0
α-OMe
NHSO3Na
NHSO3NH4
NHSO3Na
NHSO3Na
NHSO3NH4
NHSO3Na
NHSO3NH4
NHSO3NH4
NHSO3NH4
NHSO2Me
NHSO2CF3
NHCO(CH2)2CO2H
NHCbz
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
26
20
37
49
45
51
30
34
10
11
16
6
H
β-OMe
α-O(CH2)2OH
α-O(CH2)3OH
α-CH2CO2H
α-O(CH2)2OBn
α-OiPr
α-OnPr
α-OMe
α-OMe
α-OMe
α-OMe
5
α-OMe
NH2
13
15
57
63
68
α-OMe
OHeq
α-OMe
OHax
α-OMe
NHSO3Na
NHSO3Na
NHSO3Na
α-OMe
OH
OH
0
α-OMe
OBn
a % inhibition at 1 mM; n.t. = not tested.
60. Finally, sulfamoylation, N-deprotection and sulfate for- cated at the concentration of 1 tested, 1 mM, there was no
mation provided target 63. significant Sulf-2 inhibition. All analogues prepared also dis-
Introduction of alkoxy substituents at the 4-position of 1 played poor inhibition of Sulf-2.
was investigated starting from 3,6-dibenzoyl protected inter-
Only two compounds exhibited significant inhibition of
mediate 58. Benzylation using Bundle’s reagent (benzyl-2,2,2- ARSA. The β anomer of 1 inhibited 70% of ARSA activity at
trichloroacetimidate) with catalytic triflic acid25,26 provided O4- 1 mM, and also inhibited ARSB to a similar extent (67%
benzyl ether 64 in 78% yield (Scheme 6). Reaction of 64 with inh@1 mM). The unsubstituted 2-amino derivative of 1 was
lithium aluminium hydride allowed isolation of 65 in 69% selective for ARSA (86% inh@1 mM) over ARSB (3%
yield. Sulfamoylation using Method 2 gave 66 in 52% yield, inh@1 mM). The N-Cbz derivative of 1 exhibited some degree
which was deprotected selectively in the presence of the benzyl of selectivity for inhibition of ARSB (44% inh@1 mM) with no
ether under mild palladium-catalysed flow hydrogenation con- inhibition of ARSA at this concentration.
ditions, to give 67 which was sulfated to 68.
Conclusions
Biological data
A short synthesis of the purported inhibitor 1 of Sulf-1 and
Sulf-2 has been developed. Optimised low temperature con-
ditions were developed with a model substrate and applied to
a monosaccharide template, resulting in the first regioselective
sulfamoylation of a carbohydrate. The developed route allows
access to 1 in five steps and 17% overall yield compared to 9
steps and 9% yield for the previously published procedure. A
range of analogues has been prepared using the regioselective
sulfamate formation methodology, exploring diversification at
the 1, 2, 3, and 4-positions of the glucosamine template. Com-
pound 1, and all derivatives prepared were found to have
minimal inhibition of Sulf-2, in contrast to claims in ref. 5.
Inhibition of Sulf-2, and counter-screening against aryl sulfa-
tases A (ARSA) and B (ARSB) was assessed. Compounds were
assayed for their ability to inhibit the desulfation of 4-methyl-
umbelliferyl sulfate (4-MUS) to the fluorescent phenol,
4-methylumbelliferone (4-MU), at a single concentration of
1 mM. In this assay format, lead monosaccharide sulfamate 1
exhibited less than 10% inhibition of Sulf-2 activity, and also
demonstrated no inhibition of ARSA or ARSB (Table 1). The
measured Sulf-2 inhibition of 1 was in contrast to the reported
literature value (IC50 = 130 μM), which may reflect differences
in the assay procedures. Specifically, the assay employed by
Schelwies and colleagues5 involved pre-incubation of the
inhibitor and Sulf-2 followed by a 10-fold dilution into the
assay mixture prior to determination of the residual enzyme
activity; however, the IC50 cited relates to the inhibitor concen-
Acknowledgements
tration in the undiluted sample and not in the final enzymatic The authors thank: Cancer Research UK and Pfizer Global
reaction. In the data presented here, all values relate to inhibi- Research and Development for financial support, the EPSRC
tor concentrations in the final enzymatic assay and as indi- National Mass Spectrometry Service at the University of Wales
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(Swansea) for mass spectrometric determinations, Drs C. Cano 12 J. R. Myette, V. Soundararajan, Z. Shriver, R. Raman and
and I. R. Hardcastle for helpful discussions and
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