Glucose-based spiro-heterocycles as potent inhibitors of glycogen phosphorylase

Article abstract of DOI:10.1016/j.bmc.2009.05.080

Glucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cyclization of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogous glucopyranosylidene-spiro-1,2,4-oxadiazolines, with a nitrogen atom instead of the sulphur, attempted cyclizations resulted in aromatization of the heterocycle with opening of the pyranosyl ring. Enzymatic measurements showed that some of the glucose-based inhibitors were active in the micromolar range. The 2-naphthyl-substituted 1,4,2-oxathiazole displayed the best inhibition against RMGPb (K_i = 160 nM), among glucose-based inhibitors known to date.

Full text of DOI:10.1016/j.bmc.2009.05.080

Bioorganic & Medicinal Chemistry 17 (2009) 5696–5707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Glucose-based spiro-heterocycles as potent inhibitors
of glycogen phosphorylase
Veronika Nagy a, Mahmoud Benltifa b,c,d,e, Sébastien Vidal b,c,d,e, Eszter Berzsényi a, Cathie Teilhet b,c,d,e
,
b,c,d,e,
Katalin Czifrák a, Gyula Batta f, Tibor Docsa g, Pál Gergely h, László Somsák a, , Jean-Pierre Praly
*
*
a Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary
b Université de Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires associé au CNRS, UMR 5246, Laboratoire de Chimie Organique 2,
Bâtiment Curien, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
c Université Lyon 1, F-69622 Villeurbanne, France
d CNRS, UMR5246, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Laboratoire de Chimie Organique 2, Bâtiment Curien,
43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
e CPE-Lyon, F-69616 Villeurbanne, France
f Department of Biochemistry, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
g Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences at the Department of Medical Chemistry, Medical and Health Science Centre,
University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
h Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cycli-
zation of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogous glucopyr-
Received 12 January 2009
Revised 25 May 2009
Accepted 31 May 2009
Available online 6 June 2009
anosylidene-spiro-1,2,4-oxadiazolines, with
a nitrogen atom instead of the sulphur, attempted
cyclizations resulted in aromatization of the heterocycle with opening of the pyranosyl ring. Enzymatic
measurements showed that some of the glucose-based inhibitors were active in the micromolar range.
The 2-naphthyl-substituted 1,4,2-oxathiazole displayed the best inhibition against RMGPb (Ki = 160 nM),
among glucose-based inhibitors known to date.
Keywords:
Glucosyl-hydroximothioates
Spiro-cyclization
Ó 2009 Elsevier Ltd. All rights reserved.
Spiro-compounds
Glucosylidene-spirooxathiazoles
Inhibitors
Glycogen phosphorylase
Carbohydrates
Glycomimics
1. Introduction
Hepatic glucose output is elevated in type 2 diabetic patients
and current evidence indicates that glycogenolysis (release of
Type 2 diabetes mellitus1 is currently estimated to affect more
than 5% of the adult population in Western societies, and its inci-
dence is expected to increase considerably in the future.2,3 This is
in particular due to the dramatic increase in obesity even among
young adults and children.4–6 Striking enhancements in the preva-
lence of the disease is predicted for the developing countries of
Africa, Asia, and South America.2 Current preventive and therapeu-
tic strategies do not achieve adequate control of blood glucose to
diminish chronic morbidity, and there is a need to develop novel
healthcare interventions to address this substantial biomedical
challenge.7
monomeric glucose from the glycogen polymer storage form) is
an important contributor to the abnormally high production of glu-
cose by the liver. Glycogen phosphorylase (GP) is the rate limiting
enzyme in the liver responsible for glycogen breakdown to pro-
duce glucose and related metabolites for energy supply.8 Due to
its key role in modulation of glycogen metabolism, pharmacologi-
cal inhibition of GP has been regarded as an effective therapeutic
approach to treating type 2 diabetes.9–11
Several types of compounds for inhibition of GP have been re-
ported,10–13 and among them derivatives of
D-glucose represent,
as ligands of the catalytic site, the most populated class (Chart
1).12–15 There are three mammalian GP isoenzymes termed as
‘brain’, ‘muscle’, or ‘liver’ GP depending on the tissue in which they
are preferentially expressed. All isoenzymes can be converted from
the inactive form (GPb) into the active GPa form through the
phosphorylation of Ser-14. Most accessible isoforms of GP can be
* Corresponding authors. Tel.: +36 52512900x22348; fax: +36 52453836 (L.S.);
tel.: +33 4 72 43 11 61; fax: +33 4 72 44 83 49 (J.-P.P.).
E-mail addresses: somsak@tigris.unideb.hu (L. Somsák), jean-pierre.praly@
univ-lyon1.fr (J.-P. Praly).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.080

V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
5697
OH
OH
O
H
N
HO
HO
O
H
N
HO
HO
R
X
HO
O
OH
N
H
O
R
X
O
A
B
C
D
CH3
CF3
3216
7524
4925
8116
14426
G
3.128,29
4.226,30
5.126,30
CH2N3
C6H5
H
S
H
N
His-377
HN
N
H
O
E
C10H7 (2-naphthyl)
O
H
N
O
HO
HO
1027
R
OH
O
F
CH=CH-C10H7
3.527
H-bridges between His-377 and N-acyl-β-D-
glucopyranosylamine type inhibitors in the catalytic
site of GP
(2-naphthyl)
OH
O
H
N
H
N
HO
HO
R
OH
O
O
OH
R
O
HO
HO
S
N
I
CH3
C6H5
30531
4.631
HO
O
J
N
26
K
L
4-tBu-C6H4
0.720
OH
O
H
N
HO
HO
C10H7 (1-naphthyl)
1520
HO
Ar
O
N
O
M
C10H7 (2-naphthyl)
0.3520
Chart 1. Inhibitory constants (Ki [l
M]) for some representative inhibitors of rabbit muscle glycogen phosphorylase.24,25,27
obtained from rabbit skeletal muscles (RMGPa/RMGPb) or rat liver
(RLGPa/RLGPb). Because of a ꢀ80% homology between the liver
and muscle isoforms of GP, it is a general practice to use the more
readily available RMGP (most frequently in the unphosphorylated
b form) for preliminary kinetic studies (Chart 1), even though, for
in vivo experiments, the existence of isoforms raises the question
of the selectivity of inhibition.13 The functional form of GP is a
homodimer having a C-2 symmetry. The catalytic site of GP is bur-
ied at the centre of the monomers, and is accessible to the bulk sol-
vent through a 15 Å long channel. The site has been extensively
investigated with glucose analogue inhibitors which promote the
less active T (tensed) state through stabilisation of the closed
position of the 280s loop. Upon transition from the T state to the
R (relaxed) state, the 280s loop becomes disordered and displaced,
resulting in the activation of the enzyme. Crystallographic
investigations have shown the existence of an empty pocket in
with the N-acyl-b-D-glucopyranosylamine structure (e.g., B–F) was
synthesized, tested, and also investigated by X-ray crystallogra-
phy.10 These studies revealed that a H-bridge between the amide
NH and main chain CO of His377 (Chart 1) significantly contributed
to the binding. This H-bridge was also observed in GP crystal com-
plexes of the low micromolar inhibitor spiro-hydantoins G17 and
H,18 and was, thereby, considered to be an essential feature for
strong inhibition. The rigidity of the spiro-bicyclic structure of
the latter compounds with a pyranoid sugar and a five-membered
heterocyclic ring was also considered to be a decisive factor for the
efficiency.17 On the other hand, N-b- -glucopyranosyl-N0-acyl ur-
D
eas12,13,19 (e.g., I–M) with large aromatic acyl moieties (K, M) prop-
erly oriented so as to interact with the side chains of the so-called
b-channel next to the catalytic site, exhibit nanomolar inhibi-
tion12,13,20 despite the lack of the H-bond mentioned above. There-
fore, interactions in the so called b-channel of the enzyme must
play a more important role than that particular H-bond.
the direction of the b-anomeric substituent of D-glucopyranose,
lined by both polar and non-polar group, and named the
Based on the binding peculiarities of these inhibitors, we envis-
aged to prepare compounds which might unify the structural fea-
tures of the best inhibitors, that is, spiro junction between the
b-channel.10
N-Acetyl-b-
cient glucose analogue inhibitors of GP. A large array of compounds
D
-glucopyranosylamine16 A was one of the first effi-
anomeric carbon of D-glucopyranose and a five-membered hetero-

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V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
cycle, as well as a large aromatic substituent capable of interac-
tions in the b-channel. This design was supported by the finding
that spiro-oxathiazole N, available from earlier synthetic stud-
ies,21,22 proved to be a micromolar inhibitor. Therefore, synthesis
of further compounds of this series was carried out23 and, in order
to facilitate the formation of an additional H-bond, preparation of
spiro-oxadiazolines of type O was also attempted.
spiro-cyclization is believed to proceed through a 1,5-hydrogen
transfer to alkoxyl radicals to create predominantly oriented
a
C–O bonds.34,35 Spiro-cyclization of 1f was not selective under
the applied conditions (heating for 1 h with a lamp), yielding a
multicomponent mixture. Among the four fractions obtained by
column chromatography, only the third one was pure enough to
get exploitable NMR data corresponding probably to the (1R)-3f
spiro compound, because of the observed pattern of the proton sig-
nals resonating at d = 5.59, 5.10, 5.27 and 4.10 ppm for protons H-
2, H-3, H-4 and H-5, respectively (in CDCl3).
2. Results and discussion
Treatment of 1i with NBS (2 equiv) in boiling chloroform for
30 min resulted in the formation of two major and two minor com-
pounds (TLC). Column chromatography afforded only one homoge-
neous fraction identified as (1R)-3i (30%), based on NMR data and
the fact that (1R)-epimers were found to be significantly more dex-
trorotatory than the (1S)-epimers.22,33 Since spiro-cyclization
afforded in this case a higher proportion of the (1R)-epimer, we
wondered whether this was due to the basic properties of pyridine.
Therefore, the spiro-cyclization of 1a (Ar = phenyl) was carried out
in the presence of DBU (1 equiv) affording 2a (34% yield) in a 1:1
(1S)/(1R) ratio after repeated column chromatographies. As com-
pared to the selectivity normally observed (60% yield, 5:1 (1S)/
(1R) ratio), this change suggested a variation along the possible
reaction pathways.
2.1. Preparation of glucosyl-hydroximothioates
The
D
-glucosyl hydroximothioates 1a–j were prepared from
-glucopyranose and in situ formed ni-
per-O-acetylated 1-thio-b-
D
trile oxides used in slight excess (1.2 equiv), following a procedure
reported previously by Rollin32 (Scheme 1, Table 1). The synthesis
of 1i (Ar = 4-pyridyl) was optimized by increasing the amount of 4-
pyridyl-hydroximoyl chloride used for producing the correspond-
ing nitrile oxide with yields growing as follows: 6% (1.4 equiv),
48% (3.8 equiv), 78% (5 equiv). The prepared hydroximothioates
which appeared to be stable under normal conditions were deacet-
ylated by standard procedures (NaOMe/MeOH or MeOH/H2O/Et3N)
to afford the corresponding unprotected products in high yields
(Scheme 1).
The Zemplén deacetylation was achieved in high yields to ob-
tain the desired spiro-oxathiazoles 4b,c,e,g,h. The final products
had a very poor solubility in H2O, MeOH, EtOH (less than 0.1 mg/
mL) but were soluble in DMF or DMSO.
In an attempt to prepare molecules susceptible to bind more
tightly at the active site of GP through non covalent interactions
involving a polar group near the anomeric center of the ligand,
we tried to oxidize compound 1i (Ar = 4-pyridyl) with m-CPBA,
as applied earlier for the synthesis of glucosyl-phenyl-sulfoxides.33
The oxidation was not selective, since the reaction mixture was
shown by TLC to contain a mobile compound, and two major polar
products. One of them was shown to be the N-oxide 1j (Ar = 4-pyr-
idyl-N-oxide), isolated in ca 30% yield.
2.3. Attempted preparations of glucosylidene-spiro-1,2,4-
oxadiazolines
The synthesis of glucosylidene-spiro-1,2,4-oxadiazolines was
planned as nitrogen-containing spiro analogues by applying again
a NBS-mediated cyclization reaction to N-b-D-glucosyl-amidox-
2.2. NBS-mediated cyclization of hydroximothioates
imes 8 (Scheme 2). A detailed retrosynthetic analysis and the prep-
aration of the amidoximes36 are presented in the Supporting
Information. Preparation of the required amidoxime 8 from per-
O-acetylated glucopyranosyl bromide 5 or glucopyranosylamine
6 failed. Fortunately, in the presence of trimethylphosphine, acety-
The spiro-cyclization of phenyl hydroximothioate 1a proceeded
in CHCl3 with the same selectivity as already observed in CCl4,22,33
affording a 5:1 mixture of the (1S)/(1R)-epimers of 2a (Scheme 1).
The reaction of hydroximothioates 1b,c,e,g,h with N-bromosuccin-
imide (NBS) in freshly distilled CCl4 or CHCl3 was stopped when
TLC monitoring indicated complete conversion of the substrates,
ca 45 min at reflux under irradiation with a tungsten lamp
(60 W). This afforded the spiro-bicycles 3b,c,e,g,h as a mixture of
epimers, with the predominance of the (1S)-configured com-
pounds. Separation by flash column chromatography afforded first
the pure (1S)-epimers. The fractions collected next contained epi-
meric mixtures whose composition was estimated by 1H NMR to
permit calculation of the yield for each epimer (Table 1). This
lated b-D-glucopyranosyl azide 7 was transformed into the corre-
sponding phosphinimine derivative37 which reacted readily with
hydroximoyl chlorides38–40 and gave the target molecules 8a–c,h.
The NMR spectra of the crude products indicated the presence of
two species (ꢀ1:10 ratio) assumed to be the E and Z-configured
isomers. Due to the poor stability of some of these compounds,
only 8b and 8c could be deacetylated by the Zemplén method to
give 9b and 9c.
The oxidative photocyclization of 4-nitrobenzamidoxime 8b
was carried out under conditions similar to those applied in the
synthesis of spiro-oxathiazoles 3. A chloroform solution of 8b
and NBS was irradiated with a heating lamp (60 or 375 W). NBS
(4 equiv, twice as much as for the oxathiazoles) had to be added
at once or portion wise to completely transform the starting mate-
rial. Three experiments afforded mixtures which did not contain
the expected spiro-molecule IV (Scheme 2) but separation by col-
umn chromatography afforded 10b, 11b, and 12b in variable pro-
portions (Table 2). Nitroso compound 10b should result from 8b
by oxidation without cyclization. However, formation of 11b and
12b clearly indicated that spiro-cyclization must have occurred
followed by ring opening of the pyranosyl ring to allow aromatiza-
tion of the oxadiazole ring. Compound 12b displayed in addition a
carbonyl due to oxidation of the liberated hydroxyl at C-5. The
product distribution was highly dependent on the applied condi-
tions which were not optimized, although it was possible to obtain
12b as a single product.
AcO
O
2,3,4,6-tetra-O-acetyl-
AcO
SH
AcO
β-
D
-glucopyranose
OAc
a
RO
RO
O
1a-j R = Ac
2a-i R = H
S
Ar
RO
b
b
RO
N
HO
c
RO
RO
O
3a-i R = Ac
4a-h R = H
S
N
RO
RO
Ar
O
Scheme 1. Synthesis of
conditions: (a) ArC(Cl) = NOH, Et3N; (b) NaOMe, MeOH, or MeOH, Et3N, H2O, rt; (c)
NBS, h , 45 min, in refluxing CCl4 or CHCl3.
D-glucopyranosylidene-spiro-oxathiazoles. Reagents and
m

V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
5699
Table 1
Preparation of hydroximothioates 1–2 and glucosylidene-spiro-oxathiazoles 3–4
Ar=
Compound/yielda (%)
Ph
p-NO2Ph
p-CNPh
1a/9032
1b/6032
1c/48
2a/9432
2b/9132
2c/86
(1S)-3a/46
(1S)-3b/33
(1R)-3a/11
(1R)-3b/16
(1R)-3c/7
(1S)-4a/9022
(1S)-4b/71
(1S)-4c/84
(1S)-4d/79
(1S)-4e/79
(1S)-3c/15
p-FPh
1d/6532
1e/7132
1f/85
2d/9532
2e/9732
(1S)-3d/3022
(1S)-3e/69
(1R)-3d/1622
(1R)-3e/17
p-MeOPh
3,4,5-triMeOPh
p-PhPh
2-Naphthyl
4-Pyridyl
4-Pyridyl-N-oxide
Unselective reaction
(1S)-3g/61
(1S)-3h/36
(1S)-3i/0
1g/40
1h/78
1i/78
2g/90
2h/85
2i/90
(1R)-3g/18
(1R)-3h/16
(1R)-3i/30
(1S)-4g/97
(1S)-4h/94
(1R)-4i/95
1j/30
a
Calculated yields given in italics take into account the ratio of anomers estimated by 1H NMR in mixed fractions after purification.
OAc
O
OAc
O
AcO
AcO
AcO
AcO
NH2
AcO
AcO
Br
6
5
Ar
8 (%)
57
9 (%)
--
83
81
--
OAc
O
OR
O
a C6H5
a
H
AcO
AcO
RO
RO
b 4-NO2-C6H4
c 4-CN-C6H4
h C10H7
64
N3
N
Ar
60
6
AcO
RO
N
8 R = Ac
(2-naphthyl)
7
OH
b
c
9 R = H
OAc
O
H
N
AcO
AcO
OAc
O
OAc
OH
OAc
AcO
Ar
O
O
AcO
AcO
AcO
AcO
AcO
N
N
Ar
N
N
N
N
IV
AcO
+
+
AcO
AcO
AcO
Ar
Ar
O
O
N
O
11b
12b
10b
Scheme 2. Attempted preparations of glucosylidene-spiro-oxadiazolines IV. Reagents and conditions: (a) i) PMe3, ii) ArC(Cl) = NOH, Et3N; (b) NaOMe, MeOH; (c) see
conditions in Table 2.
Table 2
(N2CAr) and 154.2 ppm (NCO) characteristic of the oxadiazole
N-oxide ring.44 Formation of an aromatic oxadiazole ring with
opening of the pyrane ring was in analogy with our previous obser-
vations from attempted spiro-cyclization of benzamidoximes.
Photolysis of 8b under various conditions
Lamp (W)
NBS (equiv)
Yield of 10b (%)
Yield of 11b (%)
Yield of 12b (%)
60
60
375
4
31
0
0
0
0
20
ꢀ3
45
10
4 ꢁ 1
4
2.4. Inhibition of glycogen phosphorylase
Enzymatic tests (Table 3) with deprotected hydroximothioates
2, benzamidoximes 9b,c, and the (1R)-configured spiro-oxathiazole
(1R)-4i showed no significant inhibition of rabbit muscle glycogen
phosphorylase (RMGP) b. In contrast, several (1S)-configured spiro-
compounds (1S)-4 showed inhibition in the low micromolar range,
except for compounds (1S)-4b,c having polar substituents (4-nitro
and 4-cyano, respectively). The most efficient was the 2-naphthyl
derivative (1S)-4h with a nanomolar Ki which appears to be the
best glucose-based inhibitor known at the moment. This shows
conclusively that a rigid spiro-bicyclic structure, attached to a
properly oriented large apolar aromatic group as the 2-naphthyl
are favorable structural features for strong binding at the catalytic
site of GP. Further exploitation of these findings is in progress for
the design of more potent inhibitors.
1,3-Dipolar cycloaddition of nitrile oxides to benzyl-protected
glucosyl-hydroximolactone 1341,42 was envisaged as another ap-
proach to analogous glycopyranosylidene-spiro-1,2,4-oxadiazo-
lines. Cycloadditions with aryl nitrile oxides derived from
chloro-benzaldoxime, -chloro-4-methoxybenzaldoxime, and
a
a
-
-
a
chloro-4-nitrobenzaldoxime proved to be disappointing since 13
underwent partial conversion after 24 h in boiling toluene in the
presence of 8 equiv of 4-methoxybenzonitrile oxide. Among the
isolated products (Scheme 3), we could identify the N-oxide dimer
V
43 deriving from the nitrile oxide used and 15 (13% isolated yield).
Based on the conversion of 13, the calculated yield for 15 was 22%.
The low reactivity of 13 was confirmed by attempted reactions
with either trimethylsilyldiazomethane in refluxing dioxane, or
with bromonitrile oxide, which resulted only in slow and unselec-
tive transformations. Isolation of V (m/z = 299.1 [M+H]+) proved
the existence, in the reaction mixture, of the 4-methoxybenzoni-
trile oxide which reacted slowly with 13. Mass spectrometry
(ESI) confirmed that cycloaddition took place but it was not appro-
priate to discriminate between structures 14 or 15 which both
might correspond to the observed ions (m/z = 703 [M+H]+). 13C
NMR spectroscopy indicated chemical shifts at 140.8 ppm
3. Conclusion
The recent identification of glycogen phosphorylase as a biolog-
ical target for the treatment of hyperglycemia led to the design of a
large collection of glucose-based inhibitors displaying inhibitory
activities in the micromolar range.10,12–15 Among them, the gluco-
pyranosylidene-spiro-hydantoins28–30 and N-acyl-N0-b-
D
-glucopyr-
anosyl urea20,31 derivatives were shown to be the best inhibitors.

5700
V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
OBn
O
BnO
13
BnO
BnO
N
OH
+
MeO
C
N
O
OBn
OH
OBn
H3CO
O
OH
O
BnO
BnO
BnO
BnO
O
N
N
+
N
BnO
OMe
OMe
BnO
O
OMe
O
N
N
O
N
V
14
15
Scheme 3. Attempted cyclization of glucosyl-hydroximolactone 13.
23 °C using Bruker DRX300or DRX500spectrometerswith theresid-
ual solvent as the internal standard. The following abbreviations are
used to explain the observed multiplicities: s, singlet; d, doublet; dd,
doublet of doublet; ddd, doublet of doublet of doublet; t, triplet; td,
triplet of doublet; q, quadruplet; m, multiplet; br, broad; p, pseudo.
Structure elucidation was deduced from 1D and 2D NMR spectros-
copy which allowed, in most cases, complete signal assignments
based on COSY, HSQC, and HMBC correlations. Atom numbering fol-
lows that of the parent carbohydrate for the description of the sugar
signals in the NMR spectra. Uncertain assignments are indicated by
an asterisk. NMR solvents were purchased from Euriso-Top (Saint
Aubin, France). HRMS (LSIMS) mass spectra were recorded in the po-
sitive mode using a Thermo Finnigan Mat 95 XL spectrometer. MS
(ESI) mass spectra were recorded in the positive mode (unless stated
otherwise) using a Thermo Finnigan LCQ spectrometer. Optical rota-
tions were measured using a Perkin Elmer polarimeter. A collection
of compounds 1 and 2 have been previously described except for
Table 3
Inhibition of rabbit muscle glycogen phosphorylase (RMGP) b
Compound
Inhibition (
l
M)
Compound
Inhibition (lM)
2b
2c
2e
2g
2h
2i
IC50 > 10 000
(1S)-4a
(1S)-4b
(1S)-4c
(1S)-4d
(1S)-4e
(1S)-4g
(1S)-4h
(1R)-4i
Ki = 26
No inhibition at 625
No inhibition at 312.5
Insoluble in DMSO
Insoluble in DMSO
No inhibition at 625
No inhibition at 625
Ki = 1 800
IC50 = 250
IC50 = 700
Ki = 48
Ki = 8.2
IC50 = 250
Ki = 0.16
9b
9c
No inhibition at 625
Based on the binding peculiarities of these types of inhibitors as re-
vealed by X-ray crystallography, we set up a new design principle
towards better inhibitors.23 To validate this approach, we designed
a synthesis of properly substituted glucopyranosylidene-spiro-
oxathiazoles by NBS-mediated spiro-cyclization at the anomeric
center of glucopyranosyl-hydroximothioate precursors. Attempted
preparations of the corresponding aza-analogs, in which the sul-
phur atom is replaced by a nitrogen, failed and led to heteroaro-
matic structures with an acyclic carbohydrate moiety. The 2-
naphthyl substituted glucopyranosylidene-spiro-oxathiazole dis-
played the strongest inhibition with a Ki value of 160 nM. The pres-
ent study provides the best inhibitor of GP in the glucose-based
family and strengthens the need for additional investigations to-
wards inhibition of this enzyme.
their 13C NMR data.32 1-Thio-b-
D-glucopyranosyl hydroximothio-
ates 2a,b,d,e were previously reported.32 Compound (1S)-3d was
previously reported.22
4.1.1. General procedure A for the preparation
of O-peracetylated hydroximothioates 1
Preparation adapted from previously reported procedures.21,32
2,3,4,6-Tetra-O-acetyl-1-thio-b-D-glucopyranose (1 mmol) and
hydroximoyl chloride (1.2 mmol, 1.2 equiv) were dissolved in
CH2Cl2 (5 mL). After adding triethylamine (3 mmol, 3 equiv), the
mixture was stirred at room temperature and TLC monitoring
showed completion of the reaction. The solvent was evaporated
and the residue purified by flash silica gel column chromatography
(PE/EtOAc 1:1, then 2:3) to afford the desired hydroximothioates 1.
4. Experimental
4.1. General methods
Dichloromethane, chloroform and carbon tetrachloride were
washed with water (3 times), then dried (CaCl2) prior distillation
over CaH2, and kept away from light. Triethylamine and water were
distilled twice. Thin-layer chromatography (TLC) was carried out on
aluminum sheets coated with Silica Gel 60 F254 (Merck). TLC plates
were inspected by UV light (k = 254 nm) and developed by treat-
ment with a mixture of 10% H2SO4 in EtOH/H2O (1/1 v/v) followed
by heating. Brominated compounds were visualized on TLC plates
by spraying first a 0.1% w/v fluorescein solution in absolute MeOH,
then a 1:1 mixture of H2O2 (30% in water) and AcOH. Upon gentle
heating, bromo-compounds gave pink-colored spots. Silica gel col-
umn chromatography was performed with GeduranÒ Silica Gel Si
4.1.2. General procedure B for the preparation of deacetylated
compounds 2i and (1R)-4i
The acetylated compound (0.25 mmol) was dissolved in a mix-
ture of water (5 mL), MeOH (5 mL) and triethylamine (1 mL). After
stirring overnight at room temperature, TLC showed deacetylation
was complete. Evaporation of the volatiles under reduced pressure
afforded a residue corresponding to pure deacetylated product.
4.1.3. General procedure C for the preparation of spiro-
oxathiazoles 3
A solution of hydroximothioate 1 (1 mmol) and N-bromosuccin-
imide (2 mmol) in CCl4 (20 mL, for 3b–e,g,h) or CHCl3 (20 mL for
3a,f,i) was boiled and illuminated by a 60 W heat lamp for
45 min. The reaction was diluted with EtOAc (150 mL) and the or-
ganic layer was washed with 5% aqueous Na2SO3 (2 ꢁ 100 mL) and
water (2 ꢁ 100 mL), dried (Na2SO4), filtered and evaporated under
60 (40–63 lm) purchased from Merck (Darmstadt, Germany). Pre-
parative reversed phase chromatography (RP-18) was performed
using a 15 ꢁ 150 mm column of fully endcapped Silica Gel 100 C18
(>400 mesh, Fluka). 1H and 13C NMR spectra were recorded at

V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
5701
reduced pressure. The residue was purified by flash column chro-
matography (PE then PE/EtOAc 65:35) to afford the desired acety-
lated spiro-oxathiazole 3.
4.2.3. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-4-methoxybenzhydroximoyl-
1-thio-b- -glucopyranose 1e
From tetra-O-acetyl-1-thio-b-
D
D
-glucopyranose (1.63 g, 4.47 mmol)
according to general procedure A yielding 1e (1.17 g, 2.28 mmol,
4.1.4. General procedure D for the Zemplén deacetylation
A solution of an acetylated compound 1 or 3 (150 mg) and
NaOMe (5 mg) in MeOH (15 mL) was stirred at rt for 3 h. The reac-
tion was neutralized to pH 5–6 with Amberlite IR-120 resin (H+
form) and the resin washed with MeOH (2 ꢁ 10 mL). The filtrate
was evaporated under reduced pressure to afford the desired
hydroximothioates 2 and spiro-oxathiazoles 4, respectively.
51%) as a white crystalline product.
Rf = 0.27 (PE/EtOAc, 1:1); ½a D25
ꢂ
+29 (c 0.44, CH2Cl2); lit.32
[a]
D
+13 (c 1, CHCl3); mp = 157–161 °C lit.32 mp = 150 °C; 1H NMR
(360 MHz, CDCl3) d 9.46 (s, 1H, OH), 7.46 (d, 2H, J = 7.9 Hz, H-ar),
6.93 (d, 2H, J = 7.9 Hz, H-ar), 5.10–4.99 (m, 3H, H-2 H-3 H-4),
4.50 (d, 1H, J = 10.5 Hz, H-1), 4.12 (dd, 1H, J = 3.6 and 11.9 Hz, H-
6), 3.98 (dd, 1H, J = 1.2 Hz, H-60), 3.86 (s, 3H, OCH3), 3.13 (ddd,
1H, H-5), 2.10, 2.04 (2s, 6H, CH3CO), 1.96 (s, 6H, CH3CO); 13C
NMR (90 MHz, CDCl3) d 170.6, 170.2 (2s, CH3CO), 169.2 (s, 2C,
CH3CO), 160.8 (C@N), 152.2, 130.3, 124.5 (2s, CH-ar), 113.7 (2s,
CH-ar), 81.4 (C-1), 75.3, 73.7, 69.4, 67.8 (4s, C-2 C-3 C-4 C5), 61.8
(C-6), 55.3 (OCH3), 20.6, 20.5, 20.4, 20.3 (4s, CH3CO); Anal. Calcd
for C22H27NO11S (513.52): C, 51.46; H, 5.30; N, 5.51; S, 6.24. Found:
C, 51.62; H, 5.46; N, 5.50; S, 6.01.
4.1.5. General procedure E for the synthesis of N-glycosyl-
amidoximes 8
Trimethyl phosphine (2.68 mL, 1 M in toluene) was added with
stirring to a solution of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl
azide 7 (1 g, 2.68 mmol) in dry dichloromethane (10 mL). After
gas evolution had ceased and TLC showed complete transformation
of the starting azide 7 (10–15 min), a hydroximoyl chloride
(1 equiv) was added. After 20–30 min, the reaction mixture was
poured into water, and the organic layer was separated. The aque-
ous layer was extracted with dichloromethane (2 ꢁ 20 mL). The or-
ganic layers were combined, washed with 10% aq sodium
carbonate and water (5 ꢁ 20 mL), dried (Na2SO4), filtered and
evaporated. The residue was purified by flash column chromatog-
raphy (EtOAc/hexane, 1:3) or crystallization. An optimized synthe-
sis of 8c is also presented below.
4.2.4. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-3,4,5-trimethoxybenzhydr-
oximoyl-1-thio-b-
D-glucopyranose 1f
Tetra-O-acetyl-1-thio-b-
D
-glucopyranose (553 mg, 1.57 mmol)
dissolved in CH2Cl2 (30 mL) was reacted with 3,4,5-trim-
ethoxybenzhydroximoyl chloride (4.92 mmol, 3 equiv) upon stir-
ring at room temperature in the presence of NEt3 (3 mL,
22 mmol, 14 equiv) and under argon. After the reaction was over
(ꢀ30 min), aqueous washings and concentration afforded a crude
oil which was purified by column chromatography (PE/EtOAc
1:1, then 2:3), yielding 1f (770 mg, 1.35 mmol, 85% yield) as a col-
orless foam.
4.2. Synthesis of O-peracetylated hydroximothioates 1
Rf = 0.23 (PE/EtOAc, 1:1); ½a D20
ꢂ
+27.8 (c 1, CH2Cl2); 1H NMR
4.2.1. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-4-nitrobenzhydroximoyl-1-
thio-b-
D-glucopyranose 1b
(300 MHz, CDCl3) d 9.12 (s, 1H, OH), 6.75 (s, 2H, H-ar), 5.03 (m,
3H, H-2 H-3 H-4), 4.68 (d, 1H, J = 9.5 Hz, H-1), 4.06 (dd, 1H,
J = 3.8 and 12.5 Hz, H-6), 3.93 (dd, 1H, J = 2.3 Hz, H-60), 3.84 (s,
9H, OMe), 3.19 (ddd, 1H, H-5), 2.00, 1.99, 1.94, 1.92 (4s, 12H,
CH3CO); 13C NMR (75 MHz, CDCl3) d 171.1, 170.7, 170.2, 169.7
(4s, CH3CO), 153.6 (s, 2C), 151.8 (C@N), 140.0, 128.4, 106.8 (s, 2C,
CH-ar), 82.1 (C-1), 76.2 (C-5), 74.1, 70.8, 68.1 (3s, C-2 C-3 C-4),
From tetra-O-acetyl-1-thio-b-
D
-glucopyranose (1.8 g, 5.3 mmol)
according to general procedure A yielding 1b (1.38 g, 2.61 mmol,
53%) as a yellow crystalline product.
Rf = 0.48 (PE/EtOAc, 1:1); ½a D25
ꢂ
+7.5 (c 0.57, MeOH), lit.32
[a]
D
+8.0 (c 1, CHCl3); mp = 156–157 °C; 1H NMR (360 MHz, CDCl3) d
9.40 (s, 1H, OH), 8.30 (d, 2H, J = 7.9 Hz, H-ar), 7.62 (d, 2H,
J = 7.9 Hz, H-ar), 5.11–5.02 (m, 3H, H-2 H-3 H-4), 4.63 (d, 1H,
J = 9.2 Hz, H-1), 4.12 (dd, 1H, J = 3.8 and 11.9 Hz, H-6), 4.01 (dd,
1H, J = 2.6 Hz, H-60), 3.24 (ddd, 1H, H-5), 2.11, 2.07 (2s, 6H, CH3CO),
1.99 (s, 6H, CH3CO); 13C NMR (90 MHz, CDCl3) d 170.6, 170.2 (2s,
CH3CO), 169.3 (s, 2C, CH3CO), 149.4 (C=N), 148.6, 139.2 (2s, CH-
ar), 129.8 (s, 2C, CH-ar), 123.5 (s, 2C, CH-ar), 81.3 (C-1), 75.8,
73.4, 70.1, 67.8 (4s, C-2 C-3 C-4 C-5), 61.8 (C-6), 20.7, 20.6 (2s,
*
61.8 (C-6), 21.0, 20.92, 20.91, 20.8 (4s, CH3CO); Anal. Calcd for
C24H31NO13S (573.57): H, 5.45; N, 2.44; O, 36.26. Found: H, 5.20;
N, 2.25; O, 35.46.
4.2.5. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-(4-phenyl)-benzhydroxi-
moyl-1-thio-b-
D
-glucopyranose 1g
-glucopyranose (1.94 g, 5.32 mmol)
From tetra-O-acetyl-1-thio-b-
D
2C, CH3CO), 20.4 (s, 2C, CH3CO); Anal. Calcd for C21H24N2O12
S
according to general procedure A yielding 1g (1.20 g, 2.14 mmol,
40%) as a white crystalline product.
(528.50): C, 47.73; H, 4.58; N, 5.30; S, 6.07. Found: C, 47.51; H,
4.76; N, 5.65; S, 5.99.
Rf = 0.28 (PE/EtOAc, 1:1); ½a D25
ꢂ
+5.7 (c 0.17, MeOH); mp = 101–
103 °C; 1H NMR (360 MHz, CDCl3) d 9.53 (s, 1H, OH), 7.72–7.63
(m, 6H, H-ar), 7.55–7.41 (m, 3H, H-ar), 5.16–5.00 (m, 3H, H-2 H-
3 H-4), 4.55 (d, 1H, J = 9.2 Hz, H-1), 4.25 (dd, 1H, J = 3.6 and
11.9 Hz, H-6), 4.15 (dd, 1H, J = 2.6 Hz, H-60), 3.15 (ddd, 1H, H-5),
2.12, 2.09, 2.01, 1.98 (4s, 12H, CH3CO); 13C NMR (90 MHz, CDCl3)
d 170.6, 170.2, 169.2 (4s, CH3CO), 151.9 (C@N), 142.7, 139.8,
131.9, 129.3, 128.9 (s, 2C), 127.8, 127.6, 127.3, 127.0 (s, 2C),
126.9 (CH-ar), 81.3 (C-1), 75.8, 73.7, 69.9, 67.8 (4s, C-2 C-3 C-4
C-5), 61.2 (C-6), 20.6, 20.5, 20.4, 20.3 (4s, CH3CO); Anal. Calcd for
C27H29NO10S (559.60): C, 57.95; H, 5.22; N, 2.50; S, 5.73. Found:
C, 57.71; H, 4.99; N, 2.66; S, 5.52.
4.2.2. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-4-cyanobenzhydroximoyl-
1-thio-b-D-glucopyranose 1c
From tetra-O-acetyl-1-thio-b-
D
-glucopyranose (1.5 g, 4.1 mmol)
according to general procedure A yielding 1c (900 mg, 1.77 mmol,
48%) as a white crystalline product.
Rf = 0.19 (PE/EtOAc, 2:1); ½a D25
ꢂ
+23 (c 0.28, MeOH); mp = 165–
167 °C; 1H NMR (360 MHz, CDCl3) d 10.10 (s, 1H, OH), 7.80–7.70
(m, 4H, H-ar), 5.10–5.00 (m, 3H, H-2 H-3 H-4), 4.62 (d, 1H,
J = 9.2 Hz, H-1), 4.12 (dd, 1H, J = 3.8 and 11.9 Hz, H-6), 3.98 (dd,
1H, J = 2.6 Hz, H-60), 3.22 (ddd, 1H, H-5), 2.09, 2.05 (2s, 6H, CH3CO),
1.99 (s, 6H, CH3CO); 13C NMR (90 MHz, CDCl3) d 170.6, 170.1, (2s,
CH3CO), 169.3 (s, 2C, CH3CO), 149.0 (C=N), 137.5, 132.1 (2s, CH-
ar), 129.4 (s, 2C, CH-ar), 117.9 (s, 2C, CH-ar), 113.4 (CN), 81.1 (C-
1), 75.6, 73.4, 70.0, 67.7 (4s, C-2 C-3 C-4 C5), 61.7 (C-6), 20.6,
4.2.6. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-2-naphthoyl-hydroximoyl-
1-thio-b-
D
-glucopyranose 1h23
From
tetra-O-acetyl-1-thio-b-
D
-glucopyranose
(875 mg,
20.4 (2s, CH3CO), 20.3 (s, 2C, CH3CO); Anal. Calcd for C21H24N2O10
S
2.40 mmol) according to general procedure
(999 mg, 1.87 mmol, 78%) as a white crystalline product.
A
yielding 1h
(508.11): C, 51.96; H, 4.76; N, 5.51; S, 6.31. Found: C, 51.71; H,
4.88; N, 5.32; S, 6.11.

5702
V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
4.2.7. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-isonicotinoyl-hydroximoyl-
1-thio-b- -glucopyranose 1i
2,3,4,6-Tetra-O-acetyl-1-thio-b-
2.66 (ddd, 1H, H-5); 13C NMR (90 MHz, D2O) d 154.5 (C@N), 149.9,
140.1, 131.6 (s, 2C, CH-ar), 125.0 (s, 2C, CH-ar), 84.4 (C-1), 81.5,
78.5, 73.6, 70.3 (4s, C-2 C-3 C-4 C-5), 61.7 (C-6); Anal. Calcd for
C13H16N2O8S (360.34): C, 43.33; H, 4.48; N, 7.77; S, 8.90. Found: C,
43.44; H, 4.26; N, 7.16; S, 8.70.
D
D
-glucopyranose (107 mg, 0.29
mmol) and 4-pyridyl-hydroximoyl chloride (250 mg, 1.59 mmol,
5.6 equiv) were introduced in a 25 mL flask. After the flask was
flushed with argon, anhydrous dichloromethane (6 mL) and trieth-
ylamine (0.53 mL, 3.77 mmol, 13 equiv) were added with stirring.
After the dark brown solution was stirred for 30 min at room tem-
perature, the reaction was finished (TLC). The mixture, diluted with
dichloromethane (25 mL), was washed with a satd aq NaHCO3
solution (25 mL), then with water (25 mL). The organic layer was
dried (MgSO4) and concentrated under reduced pressure to afford
a crude yellow oil (245 mg) which was subjected to column chro-
matography (PE/EtOAc 2:3 gradually changed to 0:1, with 0.1%
Et3N) to afford an orange-colored solid. After treatment of a dichlo-
romethane solution with charcoal, 1i was obtained a white solid
upon filtration and evaporation (112 mg, 0.23 mmol, 78% yield).
4.3.2. 1-S-(Z)-4-Cyanobenzhydroximoyl-1-thio-b-D-glucopyranose
2c
From compound 1c (200 mg, 0.39 mmol) according to general
procedure D yielding (122 mg, 0.36 mmol, 86%) as a slightly col-
ored crystalline product.
Rf = 0.74 (CHCl3/MeOH, 7:3);
½
a 2D0
ꢂ
+23 (c 0.27, MeOH);
7.80 (d, 2H,
mp = 156–160 °C; 1H NMR (360 MHz, D2O)
d
J = 7.9 Hz, H-ar), 7.72 (d, 2H, J = 7.9 Hz, H-ar), 4.24 (d, 1H,
J = 9.2 Hz, H-1), 3.62 (dd, 1H, J = 5.3 and 13.2 Hz, H-6), 3.52 (dd,
1H, J = 1.3 Hz, H-60), 3.40–3.10 (m, 3H, H-2 H-3 H-4), 2.75 (ddd,
1H, H-5); 13C NMR (90 MHz, D2O) d 148.3 (C@N) 139.7, 132.8
(2s, CH-ar), 132.6 (2s, CH-ar), 119.5, 111.0 (CN), 83.3 (C-1), 80.0,
77.2, 72.6, 69.2 (4s, C-2 C-3 C-4 C-5), 60.4 (C-6); Anal. Calcd for
C14H16N2O6S (340.36): C, 49.41; H, 4.74; N, 8.23; S, 9.42. Found:
C, 49.24; H, 4.66; N, 8.42; S, 9.12.
Rf = 0.12 (PE/EtOAc, 2:3); ½a D20
ꢂ
+9.3 (c 1, CH2Cl2); mp = 139–
140 °C; 1H NMR (300 MHz, CD3COCD3) d 11.61 (s, 1H, OH), 8.69
(dd, 2H, J = 1.6 and 4.4 Hz, H-ar), 7.56 (dd, 2H, H-ar), 5.23 (t, 1H,
J = 9.0 Hz H-3), 5.01 (t, 1H, H-2), 4.98 (t, 1H, H-4), 4.95 (d, 1H,
J = 10.0 Hz, H-1), 4.12 (dd, 1H, J = 5.5 and 12.5 Hz, H-6), 3.98 (dd,
1H, J = 2.3 Hz, H-60), 3.56 (ddd, 1H, H-5), 2.06, 2.02, 1.94, 1.92 (4s,
12H, CH3CO); 13C NMR (75 MHz, CD3COCD3) d 171.6, 171.1,
170.9, 170.8 (4s, CH3CO), 152.0 (s, 2C, CH-ar), 149.9 (C@N), 143.0,
124.9 (s, 2C, CH-ar), 82.5 (C-1), 77.3 (C-5), 75.0 (C-3), 72.1, 70.0
4.3.3. 1-S-(Z)-4-Methoxybenzhydroximoyl-1-thio-b-D-glucopyr-
anose 2e
From compound 1e (300 mg, 0.58 mmol) according to general
procedure D yielding 2e (172 mg, 0.50 mmol, 86%) as a white crys-
talline product.
*
(2s, C-2 C-4), 63.8 (C-6), 21.6, 21.5, 21.4, 21.3 (4s, CH3CO); HRMS
(FAB > 0) C20H25N2O10S [M+H]+ Calcd 485.1230, found 485.1233;
Anal. Calcd for C20H24N2O10S (484.28): C, 49.58; H, 4.99; N, 5.78;
O, 33.02; S, 6.62. Found: C, 49.51; H, 4.96; N, 5.70; O, 32.95.
Rf = 0.70 (CHCl3/MeOH, 7:3); ½a D25
ꢂ
– 8.5 (c 0.31, MeOH) lit.32
½ ꢂ
a 2D5
ꢃ7 (c 1, MeOH); mp = 146–148 °C; 1H NMR (360 MHz, D2O) d 7.45
(d, 2H, J = 7.9 Hz, H-ar), 7.04 (d, 2H, J = 7.9 Hz, H-ar), 4.22 (d, 1H,
J = 9.2 Hz, H-1), 3.85 (s, 3H, OCH3), 3.20–3.44 (m, 3H, H-2 H-3 H-
4), 3.68 (dd, 1H, J = 3.9 and 13.2 Hz, H-6), 3.56 (dd, 1H, J = 2.6 Hz,
H-60), 2.75 (ddd, 1H, H-5); 13C NMR (90 MHz, D2O) d 159.5
(C@N), 151.0, 130.9 (2s, CH-ar), 126.7 (2s, CH-ar), 113.8, 83.2 (C-
1), 79.9, 77.3, 72.3, 69.1 (4s, C-2 C-3 C-4 C-5), 60.4 (C-6), 55.6
(OCH3); Anal. Calcd for C14H19NO7S (345.37): C, 48.69; H, 5.55; N,
4.06; S, 9.28. Found: C, 48.44; H, 5.36; N, 4.26; S, 9.02.
4.2.8. 2,3,4,6-Tetra-O-acetyl-1-S-(Z)-isonicotinoyl-hydroximoyl-
1-thio-b-D-glucopyranose-N-oxide 1j
A solution of 1i (195 mg, 0.4 mmol) in chloroform (10 mL) was
cooled down to 0 °C before the addition of m-chloroperbenzoic
acid (139 mg, 0.8 mmol, 2 equiv). After 8 h, TLC monitoring
showed two main polar products. After concentration under re-
duced pressure, the crude mixture was purified by flash column
chromatography (EtOAc containing 0.1% Et3N gradually enriched
in MeOH from 0% to 6%) affording 1j (62 mg, 0.12 mmol, 30% yield)
as an orange solid. When the reaction was carried out with a lower
amount of m-CPBA (1 equiv), the yield was 25%.
4.3.4. 1-S-(Z)-(4-Phenyl)-benzhydroximoyl-1-thio-b-D-glucopyr-
anose 2g
From compound 1g (300 mg, 0.53 mmol) according to general
procedure D yielding (190 mg, 0.48 mmol, 90%) as a yellow oil.
Rf = 0.20 (EtOAc/MeOH, 9:1); ½a D20
ꢂ
– 17.7 (c 0.53, MeOH); mp =
Rf = 0.58 (CHCl3/MeOH, 7:3); ½a D25
ꢂ
+5.7 (c 0.17, MeOH); 1H NMR
152 °C darkening, 182 °C decomposition; 1H NMR (300 MHz,
CD3COCD3) d 11.49 (s, 1H, OH), 8.07 (d, 2H, J = 7.2 Hz, H-ar), 7.50
(d, 2H, H-ar), 5.14 (t, 1H, J = 9.2 Hz, H-3), 4.98 (d, 1H, J = 10.1 Hz,
H-1), 4.89 (dd, 1H, J = 9.0 Hz, H-2), 4.88 (t, 1H, J = 9.7 Hz, H-4),
4.04 (dd, 1H, J = 5.5 and 12.4 Hz, H-6), 3.93 (dd, 1H, J = 2.3 Hz, H-
60), 3.68 (ddd, 1H, H-5), 1.92, 1.89, 1.83, 1.80 (4s, 12H, CH3CO);
13C NMR (75 MHz, CD3COCD3) d 170.2, 169.7, 169.4, 169.3 (4s,
CH3CO), 147.2 (C@N), 139.3 (s, 2C, CH-ar), (CIV not seen), 126.3
(s, 2C, CH-ar), 81.3 (C-1), 75.7 (C-5), 73.5 (C-3), 70.6 (C-2), 68.5
(C-4), 62.5 (C-6), 20.0 (m, 4C, CH3CO).
(360 MHz, DMSO-d6) d 8.40 (s, 1H, OH), 7.52–7.20 (m, 9H, H-ar),
4.98–4.51 (m, 4H, OH), 4.15 (d, 1H, J = 9.2 Hz, H-1), 4.24 (dd, 1H,
J = 5.8 and 13.2 Hz, H-6), 4.17 (dd, 1H, J = 2.6 Hz, H-60) 4.16–3.72
(m, 3H, H-2 H-3 H-4), 3.24 (ddd, 1H, H-5); 13C NMR (90 MHz,
DMSO-d6) d 151.1 (C@N), 140.9, 140.1, 129.9, 129.7, 129.6, 129.5,
129.3, 128.2, 128.1, 127.1, 126.9, 126.7, 84.4 (C-1), 80.4, 79.5, 73.8,
70.2 (4s, C-2 C-3 C-4 C-5), 60.9 (C-6); Anal. Calcd for C19H21NO6S
(391.45): C, 58.30; H, 5.41; N, 3.58; S, 8.19. Found: C, 58.14; H,
5.16; N, 3.12; S, 8.02.
4.3.5. 1-S-(Z)-2-Naphthoyl-hydroximoyl-1-thio-b-D-glucopyr-
4.3. Deacetylation of O-peracetylated hydroximothioates
anose 2h
From compound 1h (343 mg, 0.64 mmol) according to general
4.3.1. 1-S-(Z)-4-Nitrobenzhydroximoyl-1-thio-b-
2b
D
-glucopyranose
procedure D yielding (200 mg, 0.55 mmol, 85%) as a yellow oil.
Rf = 0.68 (CHCl3/MeOH, 7:3); ½a D25
ꢂ
+4.4 (c 0.3, MeOH); mp = 150–
From compound 1b (200 mg, 0.38 mmol) according to general
procedure D yielding 2b (111 mg, 0.3 mmol, 82%) as a white crys-
talline product.
152 °C; 1H NMR (360 MHz, D2O) d 8.04–7.54 (m, 7H, H-ar), 4.24 (d,
1H, J = 10.6 Hz, H-1), 3.56 (dd, 1H, J = 3.9 and 13.2 Hz, H-6), 3.44
(dd, 1H, J = 2.6 Hz, H-60) 3.36, 3.28, 3.12 (3t, 3H, J = 9.2 Hz, H-2 H-3
H-4), 2.52 (ddd, 1H, H-5); 13C NMR (90 MHz, D2O) d 148.3 (C@N),
139.7, 132.8, 132.7, 132.6 (s, 2C), 129.8 (s, 2C), 129.6, 119.6, 111.5,
83.3(C-1), 80.1, 77.2, 72.6, 69.2 (4s, C-2C-3 C-4C-5), 60.5(C-6);Anal.
Calcd for C17H19NO6S (365.41): C, 55.88; H, 5.24; N, 3.83; S, 8.77.
Found: C, 55.74; H, 5.06; N, 3.52; S, 8.55.
Rf = 0.12 (CHCl3/MeOH, 9:1); ½a D25
ꢂ
+7.5 (c 0.56, MeOH), lit.32
½ ꢂ
a 2D5
+5 (c 1, MeOH); mp = 184–187 °C; 1H NMR (360 MHz, D2O) d 8.27 (d,
2H, J = 7.9 Hz, H-ar), 7.72 (d, 2H, J = 7.9 Hz, H-ar), 4.24 (d, 1H,
J = 9.2 Hz, H-1), 3.58 (dd, 1H, J = 1.2 and 11.9 Hz, H-6), 3.51 (dd, 1H,
J = 3.9 Hz, H-60), 3.36, 3.28, 3.20 (3t, 3H, J = 9.2 Hz, H-2 H-3 H-4),

V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
5703
4.3.6. 1-S-(Z)-Isonicotinoyl-hydroximoyl-1-thio-b-
glucopyranose 2i
A solution composed of 1i (90 mg, 0.18 mmol) in a mixture of
MeOH (2 mL), water (2 mL) and NEt3 (0.4 mL) was treated accord-
ing to general procedure B. The residue was purified (RP-18: water)
to afford 2i (53 mg, 0.16 mmol, 90% yield) as a white solid.
D
-
170.4, 169.6, 169.29, 169.26, (4s, CH3CO), 154.5 (C@N), 132.6 (s,
2C, CH-ar), 131.2, 128.4 (s, 2C, CH-ar), 123.3 (C-1), 117.7, 115.1
(C„N), 70.84 (C-2 or C-3), 70.80 (C-5), 67.9 (C-2 or C-3), 67.3 (C-
4), 60.9 (C-6), 20.6 (CH3CO), 20.4 (s, 3C, CH3CO); MS (ESI >0) m/
z = 529.0 [M+Na]+, 1034.5 [2M+Na]+; HRMS (ESI > 0) m/z =
C22H22N2NaO10S [M+Na]+ calcd 529.0893, found 529.0898.
Rf = 0.48 (EtOAc/MeOH, 4:1); ½a D20
ꢂ
– 1.6 (c 1, H2O); mp = 140 °C
darkening, 175 °C decomposition; 1H NMR (300 MHz, D2O) d 8.62
(dd, 2H, J = 1.5 and 5.5 Hz, H-ar), 7.57 (dd, 2H, H-ar), 4.33 (d, 1H,
J = 9.8 Hz, H-1), 3.60 (dd, 1H, J = 2.4 and 12.7 Hz, H-6), 3.52 (dd,
1H, J = 4.9 Hz, H-60), 3.40 (t, 2H, J = 9.2 Hz, H-2), 3.31 (t, 1H,
J = 9.0 Hz, H-4), 3.25 (t, 1H, J = 8.7 Hz, H-3), 2.75 (ddd, 1H, H-5);
13C NMR (75 MHz, D2O) d 152.7 (C@N), 149.6 (s, 2C, CH-ar),
141.6, 124.3 (s, 2C, CH-ar), 83.1 (C-1), 80.3 (C-5), 77.3 (C-3), 72.5,
4.4.4. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-glucitol-
spiro[1.5]-3-(4-methoxyphenyl)-1,4,2-oxathiazole (1S)-3e
A solution composed of 1d (690 mg, 1.34 mmol) and NBS
(478 mg, 2.68 mmol)wastreatedaccordingto thegeneralprocedure
C to afford a first crop of pure (1S)-3e (372 mg) as a white foam and
an additional crop (218 mg) of a mixture of (1S)-3e and (1R)-3e in a
45:55 ratio, respectively, (1H NMR). The calculated yields were,
respectively, 69% and 17% for the (1S)- and (1R)-epimers.
*
69.2 (2s, C-2, C-4), 60.5 (C-6); Anal. Calcd for C12H16N2O6S, 0.5
H2O: C, 44.30; H, 5.27; N, 8.61; O; 31.97; S, 9.86. Found: C,
44.34; H, 5.26; N, 8.62; O, 32.45.
Rf = 0.43 (PE/EtOAc, 3:2); ½a D20
ꢂ
+52 (c 1, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 7.61 (d, 2H, J = 8.9 Hz, CH-ar), 6.94 (d, 2H,
J = 8.9 Hz, CH-ar), 5.62 (m, 2H, H-2 H-3), 5.26 (m, 1H, H-4), 4.41
(ddd, 1H, J = 2.0, 3.6 and 10.3 Hz, H-5), 4.34 (dd, 1H, J = 3.6 and
12.6 Hz, H-60), 4.07 (dd, 1H, J = 2.0 and 12.6 Hz, H-6), 3.85 (s, 3H,
OMe), 2.09, 2.08, 2.05, 2.03, (4s, 12H, CH3CO); 13C NMR (75 MHz,
CDCl3) d 170.6, 169.6, 169.5, 169.4 (4s, CH3CO), 162.2, 155.9
(C@N), 129.6 (s, 2C, CH-ar), 122.1 (C-1), 119.3, 114.3 (s, 2C, CH-
ar), 71.1 (C-2 or C-3), 70.5 (C-5), 67.9 (C-2 or C-3), 67.5 (C-4),
61.1 (C-6), 55.4 (OMe), 20.7 (CH3CO), 20.5 (s, 3C, CH3CO); MS
(ESI >0) m/z = 511.8 [M+H]+, 534.0 [M+Na]+, 1022.4 [2M+H]+,
1044.6 [2M+Na]+; HRMS (ESI >0) m/z = C22H25NNaO11S [M+Na]+
calcd 534.1046, found 534.1049.
4.4. Spiro-cyclization
4.4.1. (1S)- and (1R)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-
glucitol-spiro[1.5]-3-(phenyl)-1,4,2-oxathiazole (1S)-3a and
(1R)-3a
When the spiro-cyclization was carried out with 1a (278 mg) in
CHCl3 in the presence of NBS (2 equiv) and DBU (1 equiv) according
to the general procedure C, TLC suggested a ꢀ1:1 ratio of the (1S)/
(1R)-epimers. Separation by repeated column chromatographies
with a gradient of PE/EtOAc as the mobile phase afforded (1S)-3a
(44 mg, 16%) and (1R)-3a (51 mg, 18%).
4.4.5. (1R)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-glucitol-
4.4.2. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-
spiro[1.5]-3-(4-nitrophenyl)-1,4,2-oxathiazole (1S)-3b
D
-glucitol-
spiro[1.5]-3-(3,4,5-trimethoxyphenyl)-1,4,2-oxathiazole (1R)-3f
Spiro-cyclization of 1f (580 mg) was attempted as before (NBS:
360 mg, 2 equiv; CHCl3, 25 mL) upon heating with a 60 W tungsten
lamp for 1 h. TLC showed that the transformation was not selec-
tive, yielding a multicomponent mixture. Workup and flash col-
umn chromatography (PE then PE/EtOAc 1:1) of the crude
product (827 mg) afforded four fractions (90, 33, 21, 41 mg). Only
the third one was pure enough and led to exploitable NMR spectra
(COSY, HSQC), concluding to the presence of (1R)-3f.
A solution composed of 1b (827 mg, 1.56 mmol) and NBS
(557 mg, 3.13 mmol)wastreatedaccordingtothegeneralprocedure
C to afford a first crop of pure (1S)-3b (259 mg) as a white foam and
an additional crop (144 mg) of a mixture of (1S)-3b and (1R)-3b in a
7:93 ratio, respectively, (1H NMR). The calculated yields were,
respectively, 33% and 16% for the (1S)- and (1R)-epimers.
Rf = 0.62 (PE/EtOAc, 3:2); ½a D20
ꢂ
+33 (c 1, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 8.31 (d, 2H, J = 9.0 Hz, H-ar), 7.87 (d, 2H,
J = 9.0 Hz, H-ar), 5.64 (m, 2H, H-2 H-3), 5.29 (m, 1H, H-4), 4.44
(ddd, 1H, J = 2.1, 3.6 and 10.3 Hz, H-5), 4.35 (dd, 1H, J = 3.6 and
12.7 Hz, H-60), 4.10 (dd, 1H, J = 2.1 and 12.7 Hz, H-6), 2.05, 2.07,
2.09, 2.10 (4s, 12H, CH3CO); 13C NMR (75 MHz, CDCl3) d 170.4
169.5, 169.3, 169.2, (4s, CH3CO), 154.4 (C@N), 149.3, 132.8, 128.8
(s, 2C, CH-ar), 124.1 (s, 2C, CH-ar), 123.4 (C-1), 70.9 (C-2 or C-3),
70.8 (C-5), 67.8 (C-2 or C-3), 67.2 (C-4), 60.9 (C-6), 20.6 (s, CH3CO),
20.4 (s, 3C, CH3CO); MS (ESI >0) m/z = 549.0 [M+Na]+, 1074.5
[2M+Na]+; HRMS (ESI >0) m/z = C21H22N2NaO12S [M+Na]+ calcd
549.0791, found 549.0792.
1H NMR (300 MHz, CDCl3) d 6.93 (s, 2H, CH-ar), 5.59 (d, 1H,
J = 10.2 Hz, H-2), 5.27 (t, 1H, H-4), 5.10 (dd, 1H, J = 9.5 and
10.2 Hz, H-3), 4.35 (dd, 1H, J = 3.3 and 12.3 Hz, H-6), 4.10 (m, 2H,
H-5 H-60), 3.90 (s, 9H, OMe), 2.08, 2.07 (2s, 6H, CH3CO), 2.05 (s,
6H, CH3CO); 13C NMR (75 MHz, CDCl3) d 171.0, 170.3, 169.6,
168.9, (4s, CH3CO), 155.6 (C@N), 153.8 (s, 2C), 141.4, 127.4 (C-1),
122.8, 105.7 (s, 2C, CH-ar), 73.7 (C-3), 71.0 (C-5), 68.7 (C-2), 67.5
(C-4), 61.42 (C-6), 61.40 (OMe), 56.7 (s, 2C, OMe), 20.7 (CH3CO),
20.6 (s, 2C, CH3CO), 20.5 (CH3CO).
4.4.6. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-
spiro[1.5]-3-[(4-phenyl)-phenyl]-1,4,2-oxathiazole (1S)-3g
A solution composed of 1g (200 mg, 357 mol) and NBS
(127 mg, 715 mol) was treated according to the general proce-
D-glucitol-
4.4.3. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-
D
-glucitol-
l
spiro[1.5]-3-(4-cyanophenyl)-1,4,2-oxathiazole (1S)-3c
A solution composed of 1c (680 mg, 1.34 mmol) and NBS
(476 mg, 2.67 mmol) was treated according to the general proce-
dure C to afford a first crop of pure (1S)-3c (103 mg, 15%) as a white
foam and an additional crop (48 mg) of a mixture of (1S)-3c and
(1R)-3c in a 5:95 ratio, respectively, (1H NMR). The calculated
yields were, respectively, 15% and 7% for the (1S)- and (1R)-
epimers.
l
dure C to afford a first crop of pure (1S)-3g (65 mg) as a white foam
and an additional crop (90 mg) of a mixture of (1S)-3g and (1R)-3g
in a 3:2 ratio, respectively. The calculated yields were, respectively,
61% and 18% for the (1S)- and (1R)-epimers.
Rf = 0.52 (PE/EtOAc, 3:2); ½a D20
ꢂ
+45 (c 1, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 7.74 (d, 2H, J = 8.5 Hz, CH-ar), 7.65 (d, 2H,
J = 8.5 Hz, CH-ar), 7.57–7.63 (m, 2H, CH-ar), 7.37–7.50 (m, 3H,
CH-ar), 5.64 (m, 2H, H-2 H-3), 5.28 (m, 1H, H-4), 4.44 (ddd, 1H,
J = 2.0, 3.7 and 10.3 Hz, H-5), 4.36 (dd, 1H, J = 3.7 and 12.6 Hz, H-
60), 4.09 (dd, 1H, J = 2.0 and 12.6 Hz, H-6), 2.09, 2.06 (2s, 6H,
CH3CO), 2.04 (2s, 6H, CH3CO); 13C NMR (75 MHz, CDCl3) d 170.5,
169.6, 169.5, 169.4 (4s, CH3CO), 156.1 (C@N), 144.5, 139.6, 128.9
(s, 2C, CH-ar), 128.4 (s, 2C, CH-ar), 128.1 (CH-ar), 127.5 (s, 2C,
Rf = 0.49 (PE/EtOAc, 3:2); ½a D20
ꢂ
+35 (c 1, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 7.79 (d, 2H, J = 8.7 Hz, CH-ar), 7.74 (d, 2H,
J = 8.7 Hz, CH-ar), 5.28 (m, 1H, H-4), 5.64 (m, 2H, H-2 H-3), 4.44
(ddd, 1H, J = 2.1, 3.6 and 10.3 Hz, H-5), 4.33 (dd, 1H, J = 3.6 and
12.7 Hz, H-60), 4.10 (dd, 1H, J = 2.1 and 12.7 Hz, H-6), 2.09 (s, 6H,
CH3CO), 2.04, 2.06 (2s, 6H, CH3CO); 13C NMR (75 MHz, CDCl3) d

5704
V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
CH-ar), 127.1 (s, 2C, CH-ar), 125.8, 122.4 (C-1), 71.0 (C-2 or C-3),
70.6 (C-5), 68.0 (C-2 or C-3), 67.5 (C-4), 61.1 (C-6), 20.7 (CH3CO),
20.5 (s, 3C, CH3CO); MS (ESI >0) m/z = 557.8 [M+H]+, 580.0
[M+Na]+, 1114.4 [2M+H]+, 1136.7 [2M+Na]+; HRMS (ESI >0) m/z =
C27H27NNaO10S [M+Na]+ calcd 580.1253, found 580.1252.
3.89 (d, 1H, J = 9.6 Hz, H-2), 3.81 (dd, 1H, J = 2.3 and 12.3 Hz, H-
60), 3.70–3.78 (m, 2H, H-3 H-6), 3.51 (t, 1H, J = 9.0 Hz, H-4); 13C
NMR (75 MHz, CD3OD) d 155.8 (C@N), 134.0 (s, 2C, CH-ar), 133.7,
129.4 (s, 2C, CH-ar), 128.8 (C-1), 119.0, 115.6 (CN), 77.6 (C-5),
76.1 (C-3), 72.7 (C-2), 70.6 (C-4), 61.9 (C-6); MS (ESI <0) m/z =
372.9 [M+Cl]ꢃ; HRMS (ESI <0) m/z = C14H14ClN2O6S [M+Cl]ꢃ calcd
373.0261, found 373.0264.
4.4.7. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-glucitol-
spiro[1.5]-3-(2-naphthyl)-1,4,2-oxathiazole (1S)-3h23
A solution composed of 1h (655 mg, 1.23 mmol) and NBS
(437 mg, 2.46 mmol) was treated according to the general proce-
dure C to afford a first crop of pure (1S)-3h (181 mg) as a white
foam and an additional crop (160 mg) of a mixture of (1S)-3h
and (1R)-3h in a 1:2 ratio, respectively. The calculated yields were,
respectively, 36% and 16% for the (1S)- and (1R)-epimers.
4.5.3. (1S)-1,5-Anhydro-D-glucitol-spiro[1.5]-3-(4-fluorophen-
yl)-1,4,2-oxathiazole (1S)-4d
Prepared by general procedure D from (1S)-3d (500 mg) to af-
ford (1S)-4d (392 mg, 79%) as a pale yellow solid.
Rf = 0.69 (CHCl3/MeOH, 7:3); ½a D20
ꢂ
+51 (c 1.1, DMSO); mp = 192–
193 °C; 1H NMR (360 MHz, DMSO-d6) d 7.73–7.69 (m, 2H, H-ar),
7.38–7.33 (m, 2H, H-ar), 3.72–3.62 (m, 3H, H-6 H-60 H-1), 3.53–
3.23 (m, 3H, H-2 H-3 H-5); 13C NMR (90 MHz, DMSO-d6) d 163.2,
154.7 (C@N), 130.8, 130.7, 128.3, 117.5, 117.2, 108.8 (C-1), 77.7,
75.3, 72.1, 70.1 (C-2 C-3 C-4 C-5), 61.2 (C-6); Anal. Calcd for
C13H14NO6S (331.32): C,47.13; H, 4.26; N, 4.23; S, 9.68. Found: C,
47.33; H, 4.09; N, 4.52; S, 9.45.
4.4.8. (1R)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-D-glucitol-
spiro[1.5]-3-(4-pyridyl)-1,4,2-oxathiazole (1R)-3i
A solution composed of 1i (636 mg, 1.31 mmol) and NBS
(467 mg, 2.62 mmol) in anhydrous chloroform (30 mL) was treated
with for 30 min according to the general procedure C. TLC monitor-
ing showed the appearance of two main products (more mobile
than 1i) and two minor products. Workup and flash chromatogra-
phy with petroleum ether EtOAc (containing 0.1% Et3N) 7:3 then
2:3 afforded a mixture (107 mg) with 3 components (NMR) some
being brominated (as shown by TLC using a specific staining re-
agent with fluorescein), and (1R)-3i (190 mg, 0.39 mmol, 30%
yield) as a transparent solid which appeared labile at room temper-
ature and was kept at 0 °C.
4.5.4. (1S)-1,5-Anhydro-D-glucitol-spiro[1.5]-3-(4-methoxyphe-
nyl)-1,4,2-oxathiazole (1S)-4e
A solution composed of (1S)-3e (370 mg) was treated according
to general procedure D to afford (1S)-4e (196 mg, 79%) as a pale
yellow solid.
Rf = 0.33 (EtOAc/MeOH, 85:15);
½
a 2D0
ꢂ
+43.4 (c 1, DMSO);
D2O) d 7.57 (d,
mp = 192–194 °C; 1H NMR (300 MHz, DMSO-d6+
e
Rf = 0.63 (EtOAc); mp = 60–61 °C; ½a D20
ꢂ
+181.7 (c 0.9, CH2Cl2); 1H
2H, J = 8.7 Hz, H-ar), 7.04 (d, 2H, J = 8.7 Hz, H-ar), 3.80 (s, 3H,
NMR (300 MHz, CDCl3) d 8.75 (dd, 2H, J = 1.6 and 4.4 Hz, H-ar), 7.57
(dd, 2H, H-ar), 5.63 (d, 1H, J = 10.2 Hz, H-2), 5.28 (t, 1H, J = 9.7 Hz,
H-4), 5.11 (dd, 1H, J = 9.5 Hz, H-3), 4.34 (dd, 1H, J = 4.2 and 12.9 Hz,
H-6), 4.12 (m, 2H, H-5 H-60), 2.08, 2.07, 2.06, 2.05 (4s, 12H, CH3CO);
13C NMR (75 MHz, CDCl3) d 170.9, 170.3, 169.5, 168.9 (4s, CH3CO),
154.2 (C@N), 151.0 (s, 2C, CH-ar), 135.2, 128.3 (C-1), 122.0 (s, 2C,
CH-ar), 73.5 (C-3), 71.1 (C-5), 68.6 (C-2), 67.3 (C-4), 61.3 (C-6),
OMe), 3.43–3.71 (m, 5H), 3.24 (t, 1H, J = 9.4 Hz); 13C NMR
(75 MHz, DMSO-d6+eD2O) d 161.6, 154.6, 129.2 (s, 2C, CH-ar),
126.8, 119.8, 114.8 (s, 2C, CH-ar), 76.6, 74.3, 71.0, 69.1, 60.3 (C-6),
55.6 (OMe); MS (ESI <0) m/z = 377.9 [M+Cl]ꢃ; MS (ESI >0) m/z =
365.9 [M+Na]+, 708.8 [2M+Na]+; HRMS (ESI >0) m/z =
C14H17NNaO7S [M+Na]+ calcd 366.0623, found 366.0621.
21.1, 21.0, 20.90, 20.92 (4s, CH3CO); Anal. Calcd for C20H22N2O10
S
4.5.5. (1S)-1,5-Anhydro-D-glucitol-spiro[1.5]-3-[(4-phenyl)-
(482.46): C, 49.79; H, 4.60; N, 5.81; O, 33.16. Found: C, 50.07; H,
4.68; N, 5.55; O, 33.41.
phenyl]-1,4,2-oxathiazole (1S)-4g
A solution composed of (1S)-3g (65 mg) was treated according
to general procedure D to afford (1S)-4g (44 mg, 97%) as a pale yel-
low foam.
4.5. Deacetylation of spiro-oxathiazoles
Rf = 0.32(EtOAc/MeOH, 85:15);½a D20
ꢂ
+39.4(c0.6, DMSO); 1H NMR
4.5.1. (1S)-1,5-Anhydro-
D
-glucitol-spiro[1.5]-3-(4-nitrophenyl)-
(500 MHz, CD3OD) d 7.79 (d, 2H, J = 8.3 Hz, CH-ar), 7.73 (d, 2H,
J = 8.3 Hz, H-ar), 7.65–7.69 (m, 2H, H-ar), 7.36–7.52 (m, 3H, CH-ar),
3.92 (ddd, 1H, J = 2.3, 4.6 and 9.9 Hz, H-5), 3.88 (d, 1H, J = 9.9 Hz,
H-2), 3.82 (dd, 1H, J = 2.3 and 12.3 Hz, H-60), 3.73–3.79 (m, 2H, H-3
H-6), 3.52 (t, 1H, J = 9.9 Hz, H-4), 13C NMR (125 MHz, CD3OD) d
62.0 (C-6), 70.8 (C-4), 72.8 (C-2), 76.2 (C-3), 77.5 (C-5), 127.8 (C-1),
128.1 (s, 2C, CH-ar), 128.5 (s, 2C, CH-ar), 129.2 (s, 2C, CH-ar), 129.3
(s, 2C, CH-ar), 130.1 (s, 2C, CH-ar), 133.8, 141.1, 145.4, 157.2
(C@N); MS (ESI <0) m/z = 423.9 [M+Cl]ꢃ; HRMS (ESI <0) m/z =
C19H19ClNO6S [M+Cl]ꢃ calcd 424.0622, found 424.0623.
1,4,2-oxathiazole (1S)-4b
A solution composed of (1S)-3b (255 mg) was treated according
to general procedure D to afford (1S)-4b (124 mg, 71%) as a pale
yellow solid.
Rf = 0.43 (EtOAc/MeOH, 85:15);
½
a 2D0
+58.8 (c 1, DMSO);
ꢂ
mp = 182–183 °C; 1H NMR (300 MHz, CD3OD, 50 °C) d 8.30 (d,
2H, J = 8.9 Hz, CH-ar), 7.90 (d, 2H, J = 8.9 Hz, CH-ar), 3.92 (ddd,
1H, J = 2.3, 4.7 and 9.5 Hz, H-5), 3.90 (d, 1H, J = 9.5 Hz, H-2), 3.82
(dd, 1H, J = 2.3 and 12.2 Hz, H-60), 3.78 (t, 1H, J = 9.5 Hz, H-3),
3.76 (dd, 1H, J = 4.7 and 12.2 Hz, H-6), 3.53 (t, 1H, J = 9.5 Hz, H-
4); 13C NMR (75 MHz, CD3OD, 50 °C) d 154.5 (C@N), 150.7, 135.3,
129.8 (s, 2C, CH-ar), 129.0, 125.2 (s, 2C, CH-ar), 77.7 (C-5), 76.2
(C-3), 73.0 (C-2), 70.8 (C-4), 62.2 (C-6); MS (ESI <0) m/z = 392.9
[M+Cl]ꢃ, 750.5 [2M+Cl]ꢃ; HRMS (ESI <0) m/z = C13H14ClN2O8S
[M+Cl]ꢃ calcd 393.0159, found 393.0158.
4.5.6. (1S)-1,5-Anhydro-D-glucitol-spiro[1.5]-3-(2-naphthyl)-
1,4,2-oxathiazole (1S)-4h23
A solution composed of (1S)-3h (180 mg) was treated according
to general procedure D to afford (1S)-4h (116 mg, 94%) as a pale
yellow solid.
4.5.2. (1S)-1,5-Anhydro-
cyanophenyl)-1,4,2-oxathiazole (1S)-4c
D
-glucitol-spiro[1.5]-3-(4-
4.5.7. (1R)-1,5-Anhydro-
1,4,2-oxathiazole (1R)-4i
(1R)-3i (115 mg, 0.24 mmol) was treated according to general
procedure (NEt3, MeOH, H2O) to afford (1R)-4i (71 mg,
D-glucitol-spiro[1.5]-3-(4-pyridyl)-
A solution composed of (1S)-3c (100 mg) was treated according
to general procedure D to afford (1S)-4c (56 mg, 84%) as a pale yel-
low foam.
B
0.22 mmol, 95% yield) as a white solid.
Rf = 0.35 (EtOAc/MeOH, 85:15); ½a D20
ꢂ
+36.3 (c 1, DMSO); 1H NMR
Rf = 0.40 (EtOAc/MeOH, 4:1); ½a D20
ꢃ4 (c 0.3, DMF); mp = 174–
ꢂ
(300 MHz, CD3OD) d 7.85 (m, 4H, CH-ar), 3.86–3.93 (m, 1H, H-5),
175 °C; 1H NMR (300 MHz, D2O) d 8.63 (dd, 2H, J = 1.8 and 4.5 Hz,

V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
5705
H-ar), 7.70 (dd, 2H, H-ar), 3.94 (d, 1H, J = 10.2 Hz, H-2), 3.81 (m, 3H,
4.7. Deacetylation of N-glycosyl-amidoximes
4.7.1. N-(b- -Glucopyranosyl)-4-nitrobenzamidoxime 9b
H-5 H-6 H-60), 3.60 (t, 1H, J = 9.1 Hz, H-4), 3.40 (dd, 2H, J = 9.0 Hz, H-
3); 13C NMR (75 MHz, D2O) d 156.2 (C@N), 150.0 (s, 2C, CH-ar),
135.9, 130.8 (C-1), 122.6 (s, 2C, CH-ar), 76.7 (C-3), 75.5 (C-5), 71.6
(C-2), 68.5 (C-4), 60.2 (C-6); HRMS (FAB > 0) m/z = C12H15N2O6S
[M+H]+ calcd 315.0651, found 315.0655.
D
A few drops of NaOMe (2.5 M in MeOH) were added to a solu-
tion of 8b (100 mg, 0.19 mmol) in dry methanol (7 mL). The reac-
tion mixture was stirred at rt for 5 min before the addition of
cation exchange resin Amberlyst 15 (H+ form). The resin was fil-
tered off and the solvent was removed. The residue was purified
by crystallization to give 56 mg (83%) of 9b.
4.6. Synthesis of N-glycosyl-amidoximes
4.6.1. N-(2,3,4,6-Tetra-O-acetyl-b-
D-glucopyranosyl)-
[a
]
D
ꢃ56 (c 1.3, DMSO); mp = 165–168 °C; 1H NMR (300 MHz,
benzamidoxime 8a
DMSO-d6) d 10.39 (s, 1H, OH), 8.22 (d, 2H, J = 8.4 Hz, H-ar), 7.91
(s, 2H, J = 8.2 Hz, H-ar), 6.40 (d, 1H, J = 9.84 Hz, NH), 5.1–4.65 (m,
4H, OH), 4.00–2.80 (m, 7H, H-1 H-2 H-3 H-4 H-5 H-6 H-60); 13C
NMR (75 MHz, DMSO-d6) d 152.0 (CNO2), 147.6 (N = CN), 138.5
(N2C–C), 129.6, 123.1, 83.8 (C-1), 78.1, 77.3, 72.8, 70.1 (4s, C-2 C-
3 C-4 C-5), 61.0 (C-6); Anal. Calcd for C13H17N3O8 (343.30): C,
45.48; H, 4.99; N, 12.24. Found: C, 46.02; H, 5.25; N, 11.87.
Prepared according to general procedure E to afford 8a in 57%
yield as a syrup. The product decomposed rapidly during the
NMR spectroscopic measurement.
4.6.2. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-4-
nitrobenzamidoxime 8b
Prepared according to general procedure
(880 mg) in 64% yield.
E to afford 8b
4.7.2. N-(b-D-Glucopyranosyl)-4-cyanobenzamidoxime 9c
[
a]
D
+20 (c 1.2, CHCl3); mp = 183–185 °C; 1H NMR (360 MHz,
A few drops of NaOMe (2.5 M in MeOH) were added to a solu-
tion of 8c (90 mg, 0.18 mmol) in dry methanol (9 mL). The reaction
mixture was stirred at rt for 25 min before the addition of cation
exchange resin Amberlyst 15 (H+ form). The resin was filtered off
and the solvent was removed to give 48 mg (81%) of 9c as a syrup.
CDCl3) d 9.36 (s, 1H, OH), 8.25 (d, 2H, J = 9.1 Hz, H-ar), 7.72 (d,
2H, J = 8.4 Hz, H-ar), 6.20 (d, 1H, NH), 5.12 (t, 1H, J = 9.6 Hz, H-2),
5.03 (t, 1H, J = 9.4 Hz, H-3), 4.97 (t, 1H, J = 9.2 Hz, H-4), 4.31 (t,
1H, J = 9.9 Hz, H-1), 4.12 (dd, 1H, J = 5.1 Hz, H-6), 4.02 (dd, 1H,
J = 11.8 Hz, H-60), 3.38 (ddd, 1H, J = 2.8 Hz, H-5), 2.09, 2.05, 1.97,
1.95 (4s, 12H, CH3CO); 13C NMR (90 MHz, CDCl3) d 170.4, 170.1,
170.0, 169.4 (4s, CH3CO), 151.9 (NHC(@NOH)Ar), 148.7 (CNO2),
136.5 (N2C–C), 129.5, 123.6, 82.5 (C-1), 72.9, 72.5, 70.6, 68.3 (4s,
C-2 C-3 C-4 C-5), 62.1 (C-6), 20.5, 20.6, 20.4 (3s, 4C, CH3CO); Anal.
Calcd for C21H25N3O12 (511.45): C, 49.32; H, 4.93; N, 8.22. Found: C,
50.02; H, 5.03; N, 8.16.
[
a
]
ꢃ132 (c 0.6, MeOH); 1H NMR (360 MHz, CD3OD) d 7.65 (d,
D
2H, J = 9.2 Hz, H-ar), 7.39 (d, 2H, J = 7.9 Hz, H-ar), 3.55 (t, 1H,
J = 11.8 Hz), 3.40–3.1 0(m, 5H); 13C NMR (90 MHz, CD3OD) d
156.4 (N–C@N), 134.6 (N2C–C), 124.9, 119.9 (NC–C), 108.1 (CN),
84.6 (C-1), 79.6, 78.6, 74.2, 71.5 (4s, C-2 C-3 C-4 C-5), 62.9 (C-6);
Anal. Calcd for C14H17N3O6 (323.31): C, 52.01; H, 5.30; N, 13.00.
Found: C, 52.52; H, 5.23; N, 12.65.
4.6.3. N-(2,3,4,6-Tetra-O-acetyl-b-
D-glucopyranosyl)-4-
4.8. N-(Phenyl-nitrosomethylene)-2,3,4,6-tetra-O-acetyl-b-D-
cyanobenzamidoxime 8c
glucopyranosyl amine 10b
Prepared according to general procedure E to afford 8c (245 mg)
in 18% yield.
Photolysis of 8b with a 60 W heating lamp using 4 equiv of NBS in
one portion: A solution of 8b (200 mg, 0.39 mmol) in dry chloro-
form (8 mL) was placed in a 100 mL erlenmeyer flask equipped
with a reflux condenser and NBS (278 mg, 1.56 mmol) was added
in one portion. The mixture was irradiated with a 60 W white heat-
ing lamp from a distance of 1 cm. After 15 min, chloroform was
added (30 mL), the mixture was washed with 5% aq Na2SO3, satd
aq NaHCO3 and water. The organic layer was dried (Na2SO4), fil-
tered and the solvent was removed under vacuo. The residue was
purified by column chromatography (EtOAc/hexane, 1:4) affording
10b as syrup in 30% yield (58 mg). Traces of 12b (ꢀ3%) were also
detected in the crude product by NMR spectroscopy.
Optimised synthesis of 8c: 2,3,4,6-Tetra-O-acetyl-b-D-glucopyr-
anosyl azide 7 (100 mg, 0.26 mmol) was dissolved in dry dichloro-
methane (1 mL) and trimethyl phosphine (0.268 mL, 1 M in
toluene) was added with stirring. During gas evolution, the reagent
solution was freshly prepared by adding triethylamine to a solu-
tion of p-cyanophenyl hydroximinoyl chloride (153 mg,
0.26 mmol) in dry dichloromethane (1 mL) causing the formation
of triethylamine hydrochloride precipitate. When TLC showed
complete transformation of the starting material 7 (ꢀ15 min),
the reagent solution was added to the mixture. After 10 min, the
reaction mixture was poured into water and was worked up as de-
scribed in general procedure E to afford 8c (78 mg, 60% yield).
Compound 10b: [
a]
+22 (c 0.4, CHCl3); 1H NMR (300 MHz,
D
[
a]
D
+9.8 (c 0.2, CDCl3); mp = 120–123 °C; 1H NMR (300 MHz,
CDCl3) d 8.39 (d, 2H, J = 8.3 Hz, H-ar), 8.05 (d, 2H, J = 8.9 Hz, H-
ar), 5.32 (d, 1H, J = 9.8 Hz, H-1), 5.23 (t, 1H, J = 9.5 Hz, H-3), 4.83
(t, 2H, J = 9.4 Hz, H-2 H-4), 4.44 (dd, 1H, J = 12.9 Hz, H-6), 4.13
(dd, 1H, J = 6.0 Hz, H-60), 3.90 (ddd, 1H, J = 1.9 Hz, H-5), 2.16,
2.01, 1.99, 1.93 (4s, 12H, CH3CO); 13C NMR (75 MHz, CDCl3) d
170.2, 169.5, 169.4, 169.2 (4s, CH3CO), 157.3 (N2C–C), 156.6
(NC@N), 150.1 (CNO2), 131.5, 123.7, 82.0 (C-1), 75.3, 72.2, 68.7,
66.8 (C-2 C-3 C-4 C-5), 61.0 (C-6), 20.7, 20.4, 20.3, 20.2 (4s, CH3CO);
Anal. Calcd for C21H23N3O12 (509.43): C, 49.51; H, 4.55; N, 8.25.
Found: C, 49.48; H, 4.76; N 8.22.
CDCl3) d 8.50 (s, 1H, OH), 7.71 (d, 2H, J = 8.6 Hz, H-ar), 7.65 (d,
2H, J = 8.3 Hz, H-ar), 6.16 (d, 1H, J = 10.4 Hz, NH), 5.14 (t, 1H,
J = 9.4 Hz, H-3 or H-4), 5.03 (t, 1H, J = 8.8 Hz, H-2), 4.99 (t, 1H,
J = 9.4 Hz, H-3 or H-4), 4.31 (dd, 1H, J = 8.8 Hz, H-1), 4.10 (d, 1H,
J = 6.6 Hz, H-6), 4.02 (dd, 1H, J = 12.5 Hz, H-60), 3.39 (ddd, 1H,
J = 2.7 Hz, H-5), 2.09, 2.06, 2.02, 1.99 (4s, 12H, CH3CO); 13C NMR
(75 MHz, CDCl3) d 170.4, 170.2, 170.0 (3s, 4C, CH3CO), 152.3
(NHC(@NOH)Ar), 134.7 (N2C–C), 132.3, 129.2, 128.9 (C–CN),
113.9 (CN), 82.5 (C-1), 72.9 (C-5), 72.4, 70.7, 68.3 (C-2 C-3 C-4),
62.1 (C-6), 21.0, 20.7, 20.6, 20.5 (4s, CH3CO); Anal. Calcd for
C22H25N3O10 (491.46): C, 53.77; H, 5.13; N, 8.55. Found: C, 53.43;
H, 5.09; N, 8.62.
4.9. 3-(4-Nitrophenyl)-5-(D-gluco-1,2,3,5-tetraacetoxy-4-
hydroxypentyl)-1,2,4-oxadiazole 11b
4.6.4. N-(2,3,4,6-Tetra-O-acetyl-b-
naphthyl)-amidoxime 8h
Prepared according to the general procedure E to afford 8h
(92 mg) in 6% yield as a brownish foam which decomposed rapidly.
D
-glucopyranosyl)-C-(2-
Photolysis of 8b with a 375 W heating lamp using 4 equiv of NBS in
one portion: A solution of 8b (200 mg, 0.39 mmol) in dry chloro-
form (12 mL) was placed in a 100 mL erlenmeyer flask equipped
with a reflux condenser and NBS (278 mg, 1.56 mmol, 4 equiv)

5706
V. Nagy et al. / Bioorg. Med. Chem. 17 (2009) 5696–5707
was added in one portion. The mixture was irradiated with a
375 W white heating lamp from a distance of 1 cm. After 2 h, the
mixture was diluted with chloroform (30 mL), washed with 5%
aq Na2SO3, satd aq NaHCO3 and water. The organic layer was dried
(Na2SO4), filtered and the solvent was removed under vacuo. The
residue was purified by column chromatography (EtOAc/hexane,
1:4) affording 12b (13 mg, 13%) and 11b (20 mg, 20%) as a syrup.
(125 MHz, CDCl3) d 156.3, 154.2 (NCO), 140.8 (NC@N), 137.8,
137.4, 137.0, 136.6, 130.1, 127.0–128.6 (m, 16C, CH-ar), 121.4 (s,
2C, CH-ar), 114.2 (s, 2C, CH-ar), 80.8 (C-3), 77.2 (C-4), 77.1 (C-5),
73.4 (CH2Ph), 73.0 (CH2Ph), 72.9 (C-2), 71.7 (CH2Ph), 71.0 (CH2Ph),
67.7 (C-6), 55.5 (OCH3); MS (ESI >0) m/z = 703.0 [M+H]+, 725.0
[M+Na]+, 1404.9 [2M+H]+, 1426.9 [2M+Na]+; HRMS (ESI >0) m/z =
C42H43N2O8 [M+H]+ calcd 703.3019; found 703.3022.
Compound 11b: [a]
+26.2 (c 0.26, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d 8.33 (d, 2H, J = 8.9 Hz, H-ar), 8.24 (d, 2H, J = 8.8 Hz, H-
ar), 6.16 (d, 1H, J = 6.7 Hz, H-2), 5.73 (dd, 1H, J = 3.1 and 7.3 Hz,
H-4), 5.20–5.17 (m, 1H, H-5), 3.53 (dd, 1H, J = 3.6 and 11.9 Hz, H-
6), 3.39 (dd, 1H, J = 6.4 Hz, H-60), 2.18, 2.11, 2.10, 2.08 (4s, 12H,
CH3CO); 13C NMR (100 MHz, CDCl3) d 174.3 (C-1), 169.5, 169.4,
169.3, 169.0 (4s, CH3CO), 166.9 (NC@N), 149.6 (CNO2), 131.8 (C–
CN2), 128.5, 124.1, 69.6, 69.2, 68.6 (C-2 C-3 C-4), 65.7 (C-6), 30.2
(C-5), 20.6, 20.5, 20.3, 20.2 (4s, CH3CO); MS (FAB > 0) m/z = 663.7
[M+dihydroxybenzoic acid]+; Anal. Calcd for C21H23N3O12
(509.43): C, 49.51; H, 4.55; N, 8.25. Found: C, 49.76, H 4.58, N 8.17.
4.12. Enzymatic methods
Glycogen phosphorylase b was prepared from rabbit skeletal
muscle according to the method of Fischer and Krebs,45 using dithi-
othreitol instead of L-cysteine, and recrystallized at least three
times before use. Kinetic experiments were performed in the direc-
tion of glycogen synthesis as described previously.18 Kinetic data
for the inhibition of rabbit skeletal muscle glycogen phosphorylase
were collected using different concentrations of
a-D-glucose-1-
phosphate (2–20 mM), constant concentrations of glycogen (1%
w/v) and AMP (1 mM), and various concentrations of inhibitors.
Inhibitors were dissolved in dimethyl sulfoxide (DMSO) and di-
luted in the assay buffer (50 mM triethanolamine, 1 mM EDTA
and 1 mM dithiothreitol) so that the DMSO concentration in the as-
say should be lower than 1%. The enzymatic activities were pre-
sented in the form of double-reciprocal plots (Lineweaver–Burk)
applying a nonlinear data analysis program. The inhibitor con-
stants (Ki) were determined by Dixon plots, by replotting the
slopes from the Lineweaver-Burk plots against the inhibitor con-
centrations.26,30 The means of standard errors for all calculated ki-
netic parameters averaged to less than 10%.
4.10. 3-(4-Nitrophenyl)-5-(D-xylo-1,2,3,5-tetraacetoxy-4-
oxopentyl)-1,2,4-oxadiazole 12b
Photolysis of 8b with a 60 W heating lamp using 4 equiv of NBS in 4
portions: A solution of 8b (1 g, 1.96 mmol) in dry chloroform
(40 mL) was placed in a 250 mL erlenmeyer flask equipped with
a reflux condenser and NBS (348 mg, 1.96 mmol, 1 equiv) was
added. The mixture was irradiated with a 60 W white heating lamp
from a distance of 1 cm. After 30 min, NBS (348 mg, 1.96 mmol,
1 equiv) was added to the reaction mixture. An additional 2 equiv
of NBS were added in two portions after 105 min and 135 min,
respectively. After 2 h 45 min total reaction time, the mixture
was diluted with chloroform (120 mL), washed with 5% aq Na2SO3,
satd aq NaHCO3 and water. The organic layer was dried (Na2SO4),
filtered and the solvent was removed under vacuo. The residue
was purified by column chromatography (EtOAc/hexane, 1:4)
affording 12b as a sole syrupy product in 45% yield (447 mg).
Acknowledgements
Support of our bilateral collaboration by the French Ministry of
Foreign Affairs (PAI Balaton projects 03767RJ and 11061PE), by the
CNRS (Project No. 7885 and PICS No.4576), as well as support by
Région Rhône-Alpes are gratefully acknowledged. The synthetic
work in Debrecen was supported by the Hungarian Scientific Re-
search Fund (OTKA 46081 and 61336). V.N. thanks the French Em-
bassy in Budapest for financial support of her co-tutored PhD
thesis. M.B. thanks the Région Rhone-Alpes for financing his co-tu-
tored PhD thesis (MIRA Mobilité).
Compound 12b: [a]
+19.4 (c 0.3, CHCl3); 1H NMR (300 MHz,
D
CDCl3) d 8.32 (d, 2H, J = 10.1 Hz, H-ar), 8.22 (d, 2H, J = 9.1 Hz, H-
ar), 6.27 (d, 1H, J = 5.9 Hz, H-2), 5.90 (dd, 1H, J = 3.7 Hz, H-3),
5.54 (d, 1H, H-4), 4.87 (d, 1H, J = 17.3 Hz, H-6), 4.72 (d, 1H, H-60),
2.19, 2.13, 2.12, 2.01 (4s, 12H, CH3CO); 13C NMR (75 MHz, CDCl3)
d 197.1 (C-5), 174.3 (C-1), 169.7, 169.4, 169.2, 168.9 (4s, CH3CO),
167.0 (N2CAr), 149.6 (CNO2), 131.7 (N2C–C), 128.5, 124.1, 73.7 (C-
3), 69.5 (C-4), 66.4 (C-6), 65.4 (C-2), 23.1, 20.2, 20.1 (3s, 4C,
CH3CO); MS (FAB > 0) m/z = 508 [M+H]+; Anal. Calcd for
C21H21N3O12 (507.41): C, 49.71; H, 4.17; N, 8.28. Found: C, 49.58;
H, 4.23; N, 8.24.
Supplementary data
Supplementary data (attempted preparations of glucopyranosy-
lidene-spiro-oxadiazolines, separation of amidoximes, structural
elucidation of amidoximes and oxidation products) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.05.080.
4.11. (1R,2S,3R,4R)-1,2,3,5-Tetra-O-benzyl-1-C-[3-(4-methoxy-
phenyl)-4N-oxido-1,2,4-oxadiazole-5-yl]pentitol 15
References and notes
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