Glucose-based spiro-oxathiazoles as: In vivo anti-hyperglycemic agents through glycogen phosphorylase inhibition

Article abstract of DOI:10.1039/c9ob01190k

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (K_i = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ～36% at 30 mg kg^-1 and ～43% at 60 mg kg^-1. The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

Full text of DOI:10.1039/c9ob01190k

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DOI: 10.1039/C9OB01190K
ARTICLE
Glucose-based spiro-oxathiazoles as in vivo anti-hyperglycemic
agents through glycogen phosphorylase inhibition
David Goyard,a Bálint Kónya,b Katalin Czifrák,b Paolo Larini,a Fanny Demontrond,a Jérémy Leroy,c
Sophie Balzarin,c Michel Tournier,c Didier Tousch,c Pierre Petit,c Cédric Duret,d,e Patrick Maurel,d,e
Tibor Docsa,f Pál Gergely,f László Somsák,b Jean-Pierre Praly,a* Jacqueline Azay-Milhau,c and
Sébastien Vidala*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a
better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been
demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been
elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the
spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor
according to Fairbanks’ protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-
naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (Ki = 160 nM
against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human
hepatocytes demonstrated that compound 5h is a druggable anti-hyperglycaemic compound performing slightly better than
DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the
potency of compound 5h since it lowered blood glucose levels by ~36% at 30 mg/kg and ~43% at 60 mg/kg. The present
study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such
drug candidates.
to the insulin action to promote uptake of blood glucose. Due
to the constantly high levels of glycaemia, short and especially
long term complications are developed being frequent causes
of death, thereby diabetes is one of the main contributors to
global mortality. At present no causal therapy is known,
treatment regimens aim at maintaining blood sugar levels
around the normoglycemic value of ~6.1 mM by applying
various medications.1, 2
Hepatic glucose output is elevated in T2DM patients. This
glucose production consists of glycogenolytic and
gluconeogenetic components and the latter is known to be
cycled through the glycogen pool.3, 4 Therefore, liver glycogen
phosphorylase, the rate limiting enzyme of glycogen
degradation has become a validated target to find potential
new therapeutical possibilities against T2DM.4, 5
Introduction
Diabetes mellitus is one of the most severe global health
problems of the 21st century. According to the International
Diabetes Federation (IDF) 425 million people, ~9% of the adult
population suffer from this disease, many of them may even
remain undiagnosed. The number of diabetic patients is
predicted to reach close to 700 million by 2045 with the most
significant increase in low and medium income territories of
Asia, Africa and South-America. In 2017 USD ~727 billion was
spent for diabetes treatments representing more than 12% of
the global health expenditures. Most of the diabetic patients
(>90%) belong to the so-called type 2 diabetes mellitus (T2DM)
when the organism does not produce insulin in sufficient
quantities and/or the peripheral cells are more or less resistant
A broad variety of synthetic compounds and natural products
have been tested as inhibitors of glycogen phosphorylase (GP)6-
12
a. Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR 5246,
CNRS, Université Claude Bernard Lyon 1, Bâtiment Lederer, 1 Rue Victor Grignard,
F-69622 Villeurbanne, France. Fax: +33 472 448 109; Tel: +33 472 448 349; E-
mail: sebastien.vidal@univ-lyon1.fr
mainly with the most easily available rabbit muscle GPb
(RMGPb).13 Many of these compounds’ complexes with the GP
enzyme were also studied by X-ray crystallography. From the so
discovered seven GP binding sites the most investigated one is
the catalytic (or active) site which can accommodate D-glucose
b. Department of Organic Chemistry, University of Debrecen, POB 400, H-4002
Debrecen, Hungary
c. Montpellier University, EA7288, Biocommunication in cardiometabolism (BC2M),
Montpellier, France
(the physiological inhibitor of the enzyme) and a large array of
d. INSERM U1040, Montpellier, France
15
glucose derivatives.14,
The most efficient glucose derived
e. Montpellier University, UMR-1040, Montpellier, France
f. Institute of Medical Chemistry, University of Debrecen, POB 7, Nagyerdei krt. 98,
H-4012 Debrecen, Hungary
inhibitors belong to three main compound categories:
glucopyranosylidene-spiro-heterocycles,14-17
N-acyl-N’-β-D-
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
15
glucopyranosyl ureas,14,
and C-β-D-glucopyranosyl
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heterocycles.18-26 In each category several inhibitors have expanded aromatic moieties for better interactions of the
View Article Online
submicromolar and even low nanomolar inhibitor constants. aglycons in the -pocket of the enzyme’sDcOatI:a1l0y.t1i0c3s9i/tCe9.OB01190K
Some of these glucose analog GP inhibitors (GPIs) were
subjected to various physiological investigations to show
Ar
N
Method A
Cl
OH
significant blood sugar diminishing effects in streptozotocin-
induced diabetic rats,27 restoration of whole body insulin
sensitivity,28 triggering of mitochondrial oxidation and mTORC2
(mammalian target of rapamycin complex 2) signaling,29 and
improvement of pancreatic β-cell function.30
Et3N
Et2O or CH2Cl2
AcO
AcO
AcO
AcO
AcO
AcO
O
O
SH
S
Ar
OAc
OAc
N
1
2
HO
NBS /  / h
CHCl3 or CCl4
Among the anomeric spirocycles the most efficient inhibitors
are the glucopyranosylidene-spiro-isoxazolines31, 32 (Ki = 0.63
μM against RMGPb for a 2-naphthyl derivative) and the
glucopyranosylidene-spiro-oxathiazoles33 (Ki = 0.16 μM against
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
S
N
+
N
Ar
AcO
AcO
S
O
4
RMGPb for
a
2-naphthyl derivative) while their
Ar
xylopyranosylidene counterparts remained inactive.34 The
glucose derived isoxazolines were shown to have significant
inhibitory effects in rat and human hepatocytes, and also
diminished hepatic glucose production in Zucker fa/fa rats by
single dose oral administration.35 In this paper a new synthesis
of the glucopyranosylidene-spiro-oxathiazoles, their in vitro
enzymatic evaluation, as well as cellular and in vivo evaluations
of the best inhibitor as an antihyperglycaemic agent are
described.
3
5
R = Ac
R = H
NaOMe
MeOH
Previously reported pharmacophores
NO2
CN
F
OMe
a
b
OMe
c
d
e
N
OMe
OMe
g
f
h
i
Additional pharmacophores
N
Synthesis
CO2H
j
k
l
Our previously explored strategy36 was developed for the
synthesis of glycosylidene-spiro-oxathiazoles. The key step was
the oxidative spirocyclization of O-peracetylated glycosyl
hydroximothioates37 (e.g. 2) to glycosylidene-spiro-oxathiazoles
3 and 4 (Scheme 1), a transformation achieved readily upon
treatment with NBS in refluxing halogenated solvents (CCl4,
CHCl3). When the hydroximothioate moiety displayed an aryl
substituent, our earlier study showed that the cyclization
proceeded well whatever the configuration of the pyranose ring
(D-gluco, D-galacto) or that of the anomeric centre (/).
Moreover, the cyclization was stereoselective, yielding
preferentially 1S-spiro-oxathiazoles in the D-gluco, D-galacto
series. Later on, this methodology was applied for preparing D-
gluco33, 38 or D-xylo34 analogues. In this last series, the cyclization
was shown to occur with the opposite stereoselectivity, thus
favouring formation of the 1R-configured spiro-oxathiazoles.
The compounds studied in more details displayed the aromatic
pharmacophores a-i (Scheme 1).33, 38 For the first step of the
sequence, earlier studies37 showed that, under basic conditions,
36-38
Scheme 1. Synthesis of glucose-based spiro-oxathiazoles33,
spirocyclization.
by oxidative
Glucosyl hydroximothioates precursors 2j-l were obtained in
variable yields (Table 1), their spirocyclization proceeded as
generally observed, yielding preferentially the 1S-configured
spirobicycles 3j-l. The anomeric configurations for compounds
3j-l were deduced from NMR spectroscopy which revealed
significantly different chemical shifts for the H3 and H5
pyranosylidene protons, and the C1 spiro carbon.33, 34, 36, 38 As
noted earlier for 1S-configured analogs, these signals were
respectively close to 5.65, 4.45, and 122.5 ppm. The protected
1S-spirobicylces were deacetylated in high yield under Zemplén
conditions.
Enzyme kinetic studies (IC50 and/or Ki measurements) were then
performed to evaluate whether the prepared spirobicycles
were GP inhibitors. Then, the most potent candidates were
further evaluated through in vitro and in vivo biological assays
to demonstrate their pharmacological interest. Therefore,
substantial quantities of material were needed, typically gram-
to multi-gram-scale. This called for the design and development
of an unprecedented route to D-glucose based spiro-
oxathiazoles, with the hope that a stereoselectivity higher than
that of the NBS-mediated cyclization could be achieved.
2,3,4,6-tetra-O-acetyl-1-thio--D-glucopyranose
1
reacted
readily with hydroximoyl chlorides to afford the corresponding
glucosyl hydroximothioates 2. For improving access to
glucosinolates,39 a procedure involving the in situ formation of
an oximoyl chloride from the oxime using inexpensive bleach,
which is then reacted directly under basic conditions with
thioglycopyranose has been reported. However, comparison of
these procedures showed that the conventional method to
hydroximothioates resulted in higher yields.34 Therefore, the
two-step conventional method has been used to prepare new
analogs in the present work (Ar = p-CO2H-C6H4, 2-quinolinyl, 9-
phenanthryl) with either hydrogen bond donors or acceptors or
2 | J. Name., 2012, 00, 1-3
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group of Fairbanks.46, 47 1-Thiosugars reacting wiVthiewtAerrtitc-lebOuntlyinle-
DOI: 10.1039/C9OB01190K
sulfinyl chloride in the presence of triethylamine were
Table 1: Stereoselective cyclization of -D-glucosyl thiohydroximates 2 to glucose-based
spiro-oxathiazoles 3-4 and their deacetylation to 5.33, 36, 38
isolated yields (%)a
converted into glycosyl S-tert-butyl thiosulfinates (within 10
min at room temperature), which upon thermolysis (10-20 min
in refluxing toluene) yielded various glycono-thionolactones,
and in particular D-glucono--thionolactone 12 in a notable 85%
yield.47 Glycono--thionolactones have been shown to react
with dienes, diazoalkanes, and carbenoids,48 and the
thermolysis of the obtained dihydro-1,2,3-, and -1,3,4-
thiadiazoles has been investigated.49
Ar
a
b
c
2
3
4
5
9036
71
84
79
79
-
9037
6037
48
4636
33a
15a
3036
69a
11
16a
7a
We developed this new route for synthesizing compound 5h on
the multi-gram scale considering its remarkable inhibitory
properties. The direct synthesis of dithiocarbonate 8 from the
commercially available hemiacetal 6 using tosyl chloride and
potassium O-ethyl dithiocarbonate under phase-transfer
conditions50 gave in our hands poor results. However, the two-
step protocol in one-pot reported by Vasella51 (treatment of
hemiacetal 6 with CCl4 and HMPT, then with potassium O-ethyl
dithiocarbonate at -40°C, for which basic conditions dictate the
choice of base-stable protective groups) afforded the desired
dithiocarbonate 8 via the intermediate chloride 7 (Scheme 2).
Subsequent methanolysis delivered the 2,3,4,6-tetra-O-benzyl-
1-thio-D-glucopyranose 9 in a 85% overall yield. Thiol 9 was
reacted with tert-butyl-sulfinyl chloride47 to afford the -
configured thiosulfinate 10 in 90% yield as a ~1:1 S(S),S(R)
diastereoisomeric mixture. While mass spectrometry displayed
a main peak as expected (m/z = 661 [M+H]+), the formed
diastereoisomers 10 were visible as two close spots on TLC
plates. They were purified by column chromatography but
could not be fully separated in pure form. The 1H NMR spectra
of this mixture displayed two singlets for the diastereotopic
tert-butyl groups resonating at 1.40 and 1.58 ppm, respectively.
d
e
f
6537
7137
85
1636
17a
unselective reaction
g
h
i
40
61a
36a
0
18a
16a
30
-
97
94
-
78
78
j
53
50
36
49
73
88
>95%
k
l
35
-
86
-
a Yields were calculated based on the isolated yields and on the amount of product
present in the 1RS-mixtures (ratio measured by 1H NMR) unresolved by column
chromatography.
A new route to spiro-oxathiazoles by 1,3-dipolar cycloaddition of
nitrile oxides to gluconothionolactone
The design of a new access to spiro-oxathiazoles was inspired
by our reported synthesis of glycosylidene-spiro-isoxazolines
based on 1,3-dipolar cycloaddition of nitrile oxides to exo-
methylene glycals (pyranoid exo-glucals).31,
CCl4 / HMPT
-40°C / CH2Cl2
KSC(S)OEt
-40°C / CH2Cl2
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
S
OEt
32, 35
OH
Cl
BnO
BnO
OBn
Concerted
6
7
8
S
Na / MeOH
RT
85% from 6
reactions are advantageous as regard to atom economy,
experimental conditions and yield, so they are of particular
interest in carbohydrate chemistry. We and others could
demonstrate that such cycloadditions occurred with a high
stereocontrol, the dipole attacking the exo-methylene
carbohydrate from the -face of the D-glucopyranosylidene
ring,40 while for steric and electronic reasons, regioselectivity
was equally high, favouring attack of the anomeric carbon by
the oxygen atom of nitrile oxides in all cases studied. The more
complex cycloaddition of nitrones (E and/or Z configurations,
endo and/or exo transition states) to pyranoid exo-glycals led to
mixtures of cis/trans and / products, but the -face approach
usually predominated, or was even exclusive in some cases.41, 42
Based on this 1,3-dipolar cycloaddition analogy, the known D-
glucono--thionolactone 1243-45 appeared as the required
precursor towards spiro-oxathiazoles. Moreover, benzylated D-
glucono--thionolactone 12 appeared to be a readily accessible
substrate, based on the one-pot two-step synthetic route by the
BnO
BnO
BnO
10 (1:1)
BnO
BnO
BnO
tBuSOCl / Et3N
RT / PhMe
O
O
S
S
110°C
PhMe
90%
SH
OBn
BnO
9
O
diastereoisomeric
/ 15:85
90%
mixture
Cl
RO
RO
RO
BnO
BnO
BnO
BnO
BnO
HO
N
O
O
O
Et3N / THF
BnO
S
+
isolated yields:
S
13 85%
O
RO
BnO
BnO
O
N
R isomer < 10%
45%
45%
11
12
13 R = Bn
BCl3
-60°C
CH2Cl2
65%
5h R = H
Scheme 2. New access to 1S-glucopyranosylidene-spiro-oxathiazole by nitrile oxide-
thionolactone 1,3-dipolar cycloaddition
Following Fairbanks’ procedure, thermal elimination was
carried out in refluxing toluene with either the crude
thiosulfinate 10 (two-step one-pot process) or the purified
diastereoisomeric mixture, but disappointingly, the yields
recorded for the thionolactone 12 were around 45%,
significantly lower than the 85% yield reported.47 As a
byproduct, the glucono--lactone 11 was identified and actually
This journal is © The Royal Society of Chemistry 20xx
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isolated in an equal amount when the reaction time was or total debenzylation, fragments corresponding to M+14
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increased thus allowing for the complete conversion of both indicated the oxidation of benzyls to beDnOzoI:a10te.1s0,39w/Ch9ilOeBo0t1h19e0rKs
thiosulfinate diastereoisomers 10. Formation of an undesired (M+16 and M+32) suggested oxidation to sulfoxides and
analogous -lactone was observed by Fairbanks (~20% yield) sulfones. Fortunately, debenzylation could be achieved by
during the thermal elimination at 120°C of a rhamno-configured treatment with BCl3 at –60°C55 with careful TLC monitoring to
thiosulfinate.46 Presence of di-tert-butylthiosulfinate as a quench the reaction immediately after disappearance of the
byproduct (vide infra, text and Fig. 3, Fig. 4) has been noted as starting material to avoid further unwanted reactions.
another limitation of the method.47 These disappointing and Purification by column chromatography delivered the desired
intriguing observations led to repeated trials for enhancing the final O-unprotected spiro-oxathiazole 5h (815 mg, 65% yield).
yield of thiolactone 12, but without success. TLC monitoring of In conclusion, the designed 1,3-dipolar cycloaddition approach
the reaction revealed that the more mobile thiosulfinate between 2-naphthyl nitrile oxide and perbenzylated D-glucono-
diastereoisomer 10 was converted faster than the other one, thionolactone 12 was most efficient for synthesizing the
and while its spot decreased visually on TLC plates, that of the corresponding D-glucose-based 1S-3-(2-naphthyl)-spiro-1,4,2-
thionolactone 12 increased. Seemingly, after complete oxathiazole 13. Both regio-, and stereo-selectivities were
conversion of the more reactive thiosulfinate, there was no excellent. In contrast to literature reports, the limiting step of
increase in the amount of D-glucono--thionolactone 12 from this approach was the synthesis of tetra-O-benzyl D-glucono--
the qualitative analysis of TLC, and disappointingly, the isolated thionolactone 12 by thermolysis of glucopyranosyl tert-butyl
yields never exceeded 45%. Analysis of MS data collected for sulfinate precursors 10. The discrepancies between our results
the fractions recovered from column chromatography and those of Fairbanks as reported and detailed in an internal
confirmed the formation of D-gluconolactone 11 (m/z [M+Na]+ document prompted the investigation of the thermolysis
= 561), and showed the presence of an unidentified product mechanism by DFT calculations to better understand the
(m/z = 607 M+H+) (vide infra, text and Fig. 1). After inquiring possible path to the desired thionolactone.
about these discrepancies, Prof. A.J. Fairbanks kindly provided
us with the investigator’s original report. It appeared that the Proposed mechanisms for the thermal elimination of
85% yield reported47 for the thionolactone 12 was thiosulfinates 10 to thionolactone 12
overestimated as, from the report, it corresponded to a crude Various possible modes of reaction were envisaged (Scheme 3)
product « contaminated with tert-butyl species, possibly di-tert- since tert-butyl thiosulfinates may undergo thermal elimination
butyl thiosulfinate. Yields projected to approximately 54-56% ». by hydrogen atom abstraction through five-membered cyclic
Presence of byproducts, allegedly mixed disulfides or transition states (TS). Glycono-thionolactones being reportedly
47
disproportionation products, was also supposed, but not obtained sometimes in excellent yields,46,
elimination
1
proven. A note clearly stated that the yield estimated by H involving the anomeric proton (path a) was privileged
NMR for the thionolactone 12 were 54 and 56% (close to the (anomeric effect), thus delivering the desired thionolactone 12
43-53% yields observed in the D-galacto series),47 so that these and tert-butyl sulfenic acid. However, elimination might also
data corroborated ours, suggesting the occurrence of at least involve one of the nine equivalent hydrogen atoms of the tert-
one unwanted reaction detrimental to the yield of the desired butyl moiety (path b), which would afford isobutene and the 3-
D-glucopyranosyl-thiosulfoxylic acid 1456 (other name: 3-D-
D-Glycono-thionolactones are subject to epimerization47 at C-2 glycopyranosyl-1-oxa-trisulfane). Considering early56 and
glycono-thionolactone.
58
or 2,3-elimination43 under basic conditions. Therefore, D- recent57,
reports on the thermolysis of di-tert-
glucono--thionolactone 12 was often used without further butylthiosulfinate (also termed di-tert-butyldisulfane-S-oxide,
purification for the 1,3-dipolar cycloaddition. With the crude tBuS(O)StBu),57 these speculations were reasonable, but the
thionolactone 12, this step proceeded smoothly in the presence total weight of lactones 11 and 12 (ca 90%, 1:1 ratio) measured
of 2-naphthyl hydroximoyl chloride to afford the spiro- after full conversion of both thiosufinates indicated that path b
oxathiazole 13 in high yield (~95%) and stereoselectivity (9:1 S/R was only a marginal process.
mixture). The diastereoisomeric mixture could be resolved by
silica gel column chromatography to afford the desired spiro-
oxathiazole 13 with a S-configuration at the spiro-anomeric
center (85% isolated yield) and the minor R-epimer (<10%
yield).
Not surprisingly, the final debenzylation step proved to be
problematic, and standard hydrogenolysis in the presence of
52
palladium did not proceed at all, while Et3SiH/I2 resulted in
partial deprotection only. Treatments with bromine or NBS and
water under various free radical conditions (NBS, CCl4, h, with
or without CaCO3; NaBrO3/Na2S2O4) resulted in complex
mixtures, due to partial debenzylations,53, 54 and/or formation
of oxidized products as evidenced by mass spectrometry: in
addition to the expected fragments corresponding to the partial
4 | J. Name., 2012, 00, 1-3
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gluconolactone byproduct (hydrolysis versVuiesw ArttichleeOrmnlinael
rearrangement followed by elimination).DOI: 10.1039/C9OB01190K
DFT Calculations
As a representative model, the tetra-O-methyl analog of 10 was
chosen (See supporting information for further details) to have
reasonable computational time. The numbering of molecules
applied is the same for both O-benzyl- (synthetic work) and O-
methyl-protected (computational approach) analogues, but
using italicized types in the last case.
Our calculations addressed first the three reaction pathways
mentioned above (Scheme 3), namely the two possible direct
internal eliminations (Ei) (path a, path b), then path c, in which
an internal O,S-rearrangement by 1,3-shift was supposed to
occur prior to the Ei elimination. The stereochemistry at the
sulfur atom did not influence the calculations and the results
are presented here for a single diastereoisomer at the sulfur
atom.
Scheme 3: Possible reaction paths for the thermolysis of D-glucosyl tert-butyl
thiosulfinates 10
Starting from 10, the two divergent internal eliminations (Ei)
discussed above were envisaged (Fig. 1). Deprotonation of the
anomeric position (path a) was found to be easy (TS-a, ΔG# =
21.2 kcal.mol-1), leading to the D-glucono--thionolactone 12
and formation of tert-butyl sulfenic acid. In the case of path b,
which involves one of the nine hydrogen atoms of the tert-butyl
groups, and leads to product 14 and isobutene, a higher
activation barrier of ΔG# = 24.9 kcal.mol-1 (TS-b) was calculated.
This is in agreement with the synthetic work, as the thermal -
elimination delivered the thionolactone 12 in moderate yield
while the benzyl-protected product 14 was not observed.
The puzzling formation of D-gluconolactone 11 might be due to
hydrolysis by water present in trace amount59 or to adventitious
reactions, as discussed recently,60 but since 11 was detected on
TLC plates even at the initial stage of the reaction, and as use of
a purified mixture of diastereomeric thiosulfinates 10 led to
similar lactone distribution, this hypothesis was uncertain. As
the glucopyranosyl tert-butyl thiosulfinates 10 must achieve a
favourable 5-membered cyclic TS for the thermal elimination to
proceed, and because of probable steric clashes between the
tert-butyl group and the 2-O-benzyl group for the S(S)-
diastereoisomer, most probably the S(R)- and S(S)-tert-butyl
thiosulfinates underwent elimination at different rates, the (R)-
diastereoisomer reacting faster to deliver thionolactone 12
through path a. Still, another hypothesis was needed to explain
the presence of glyconolactone 11. The thermal rearrangement
of -configured glycopyranosyl xanthates to -glycosyl
dithiocarbonates has been reported to proceed without
inversion of configuration at moderate temperatures (50-
65°C).61 This reported example of the Freudenberg-Schönberg
rearrangement62, 63 was supposed to occur by an intramolecular
1,3-shift through a 4-membered cyclic TS.61 An analogous
thermal rearrangement could occur with the less reactive S(S)
thiosulfinate (path c), and the rearranged product with a O-
glycosidic bond [OS(S) moiety] would be susceptible to afford
by thermal elimination the lactone 11 and tert-butyl-
thiosulfenic acid (tBuSSH). Interestingly, the relative enthalpies
for thiosulfinic acid isomers [HS(O)SH, and HS(S)OH] estimated
by theoretical studies at 298 K in the gas phase were found to
be almost the same,64, 65 suggesting that isomers 10 and 10’
might have similar energies. While the rearrangement of
xanthates to dithiocarbonates is stability driven, the proposed
pathway to gluconolactone 11 might benefit from the activation
of the anomeric center (rearrangement favoured) and, in
lactone 11, the stabilization of C=O bond (162 kcal.mol-1) by ca.
50 kcal.mol-1 compared to the C=S thiocarbonyl bond (115
kcal.mol-1) in thionolactone 12.66 Eventually, calculations were
carried out to identify the reaction pathway leading to the
TS-a
G#= 21.2
TS-b
G#= 24.9


MeO
MeO
MeO
MeO
MeO
MeO
O
S
O
S
S
t-Bu
S
OMe
OMe
H
H
O
O
H
MeO
MeO
MeO
O
(R
)
S
t-Bu
path a
path b
S
OMe
H
O
10
MeO
MeO
MeO
MeO
MeO
MeO
O
HO
S
O
OMe
+
+
SSOH
S
OMe
H
12
14
syntheses carried out with

G= -12
O-benzyl protected analogs
G= -5.9
Figure 1. Calculations for Ei eliminations, involving either anomeric or tert-butyl
protons (paths a,b). ΔG in kcal.mol-1
As a possibility for the puzzling formation of 11 from 10, path c
was envisioned (Fig. 2). For this two-step pathway, a 1,3 shift of
the tert-butyl thiosulfinate group led first to 10’ (Ts-c, ΔG# = 36.3
kcal.mol-1), which subsequently could react through an Ei
elimination (TS-d, ΔG# = 10.9 kcal.mol-1) leading to 11 and tert-
butyl thiosulfenic acid. Due to the high activation energy of TS-
c, this pathway can thus be excluded.
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Journal Name
TS-f
G#= 41.5
View Article Online

OH
MeO
DOI: 10.1039/C9OB01190K
O
MeO
S
+
MeO
MeO
MeO
MeO
S
O
S
OMe
MeO
MeO
MeO
1 eq.
O
12
TS-c
G#= 36.3
MeO

syntheses carried out with
O-benzyl protected analogs
O
O
MeO
MeO
MeO
11
O
OMe
S
S
S
0.5 eq. each
S
S
O
t-Bu
S
OMe
H
t-BuSSH
+
+
O
S
H2O
0.5 eq.
S
S
TS-d
Dismutation
+
syntheses carried out with
O-benzyl protected analogs
G#= 10.9
MeO
MeO
MeO
MeO
MeO

MeO
MeO
MeO

G= -4.4
0.5 eq.
G= -28.8
O
O
O
path c
S
O
MeO
O
t-Bu
t-Bu
Figure 4. Dismutation of tert-butyl sulfenic acid, and di-tert-butyl thiosulfinate attacking
thionolactone 12. ΔG in kcal.mol-1
S
S
OMe
OMe
H
OMe
H
O
S
10

G= -5.6
10'
11

G= -49.3
Figure 2. 1,3 Shift of the thiosulfinate group in 10, followed by Ei elimination (path c). ΔG
in kcal.mol-1
In summary, these calculations showed a good agreement with
the synthetic results, as they indicated that the thermal
elimination via 5-membered transition states preferentially
involved the anomeric proton rather than one of the nine tert-
butyl hydrogen atoms, thus yielding the target
At this point, since the formation of the experimentally
observed D-gluconolactone 11 cannot be explained through
path c, we wondered whether lactone 11 could be formed due
to hydrolysis of D-glucono-thionolactone 12. In this event, since
the reaction was conducted under anhydrous conditions, water
had to be formed as the reaction proceeded. Interestingly,
Block et al.67 reported the dismutation of tert-butyl sulfenic
acid, giving rise to tert-butyl-2-methyl-propane-2-sulfinothioate
(or thiosulfinate) and water (0.5 eq. each, Fig. 3). The computed
thermodynamics for the dismutation (ΔG = -4.4 kcal mol-1) were
in agreement with the experimental observations of Block.67
Subsequently, water, assisted by the coordination of the
thiosulfinate, can trigger the hydrolysis of compound 12
through TS-e (ΔG# = 30.6 kcal.mol-1), providing gluconolactone
11 and hydrogen sulfide (0.5 eq. each). This proposed
mechanism is in agreement with the experimental formation of
lactones 11 and 12 in a 1:1 ratio.
gluconothionolactone.
Simultaneous
formation
of
gluconolactone through a 1,3-shift of the thiosulfinate moiety,
followed by Ei can be ruled out. Based on the reported
dismutation of tert-butyl sulfenic acid which yielded di-tert-
butyl thiosulfinate and water (0.5 eq. each), the calculations
predicted that 12 can be hydrolysed into 11 (0.5 eq.). Thus, in
order to optimize the yield of the target gluconothionolactone
12, water, formed while the thermal elimination proceeds, has
to be trapped by efficient means, for example by adding
activated molecular sieves to the reaction medium.46, 47
Enzyme kinetics
Previous structure-activity relationship studies15, 33, 38 revealed
that phenyl oxathiazole 5a was a moderate inhibitor of GP (Ki =
26 µM). Other para-substituted phenyl analogues (R = phenyl,
NO2, CN) exhibited lower inhibitory properties with IC50 values
in the high micromolar range for 5b,c,g. The para-fluoro and
para-methoxy analogues 5d and 5e had similar and slightly
better inhibitory potential than 5a (Ki = 28 and 8 µM,
respectively). As observed for several other classes of glucose-
based GP inhibitors,14, 15, 32, 35, 68 the 2-naphthyl oxathiazole
derivative 5h was the best inhibitor among the series of
compounds studied, and one of the best among glucose-based
GP inhibitors. Substitution by a para-carboxy group on the
phenyl (5j) resulted in a good inhibitory potency (Ki = 238 µM),
while the 2-quinolinyl analogue 5k was more potent (Ki = 26
µM), although much less potent than the 2-naphthyl analogue
5h. In spite of the size of the 9-phenanthryl residue, compound
5l displayed moderate inhibitory properties.
TS-e

G#= 30.6
OH
MeO
MeO
MeO
O
S
+
MeO
MeO
MeO
S
O
S
OMe
1 eq.
MeO
MeO
MeO
12
O
MeO
O
H
O
H
OMe
O
S
O
+
H2O
0.5 eq.
S
S
S
0.5 eq.
0.5 eq.
11
0.5 eq.
G= -4.4
H2S
+
syntheses carried out with
O-benzyl protected analogs
Dismutation

G= -18.1
Figure 3. Dismutation of tert-butyl sulfenic acid, followed by hydrolysis of
thionolactone 12. ΔG in kcal.mol-1
To further confirm that lactone 11 came from the hydrolysis of
thionolactone 12 (Fig. 4), we computed the attack of di-tert-
butyl thiosulfinate on substrate 12 (Fig. 4). TS-f (ΔG# = 41.5
kcal.mol-1) could lead indeed to the formation of 11 and tert-
butyl-2-methylpropane-2-sulfinodithioate (or dithiosulfinate)
without the assistance of a water molecule. Since the ΔΔG#
between TS-e and TS-f was 10.9 kcal.mol-1 in favor of the
former, water hydrolysis pathway was confirmed to be the most
energetically reasonable route.
6 | J. Name., 2012, 00, 1-3
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IC50 Values calculated for glucose release (product of GP-
Table 2: Kinetic data measured for spiro-oxathiazoles 5a-e,g,h,j-l for the inhibition of
RMGPb
View Article Online
mediated glycogen depolymerisation) orDiOntI:r1a0c.e10ll3u9l/aCr9gOlBy0c1o1g9e0nK
content (substrate of GP-mediated glycogen depolymerisation)
are similar (2-5 M, Table 3) in human hepatocytes. In the
studied cellular model, this result demonstrates that compound
5h affected glycogenolysis via GP inhibition.
Compound
Structure
IC50 or Ki values
(µM)
HO
O
HO
HO
5a
5b
Ki = 26 ± 2.138
S
OH
O
N
HO
Table 3: In vitro IC50 for compound 5h based on glucose release and intracellular glycogen
content after glucagon stimulation in rat and human primary hepatocytes in primary
cultures
O
HO
HO
IC50 = 25033
S
OH
NO2
CN
F
O
N
HO
Compound
Glucose release
IC50 (µM)a
Intracellular glycogen
IC50 (M)b
O
HO
HO
HO
S
HO
5c
5d
5e
IC50 = 70033
Ki = 28 ± 2.838
Ki = 8 ± 0.938
OH
O
N
Rats
6.7
8
Humans
5h
2.08 ± 1.51
2.17 ± 1.30
4.88 ± 1.20
1.10 ± 1.11
HO
O
DAB
S
HO
OH
a
O
IC50 values are average of three to five measurements (see supporting
N
information).
HO
b
IC50 values are average triplicate measurements for three human hepatocyte
O
S
HO
cultures (see supporting information).
OH
O
O
O
N
The concentration-response curves obtained for glucose
release (Fig. 5) and observed intracellular glycogen content (Fig.
6) provided a basis for a comparison against DAB. Compound 5h
performed slightly better than DAB in terms of concentrations
to obtain 50% inhibition on glucose release in rat and human
hepatocytes. Such concentrations are compatible with
potential pharmacological applications.
HO
HO
HO
S
5g
5h
IC50 = 25033
OH
O
N
HO
O
HO
HO
S
OH
O
Ki = 0.16 ± 0.0433, 38
N
HO
O
HO
S
HO
OH
CO2H
5j
5k
5l
Ki = 238.5
Ki = 26.02
O
N
HO
O
HO
HO
N
S
OH
O
N
HO
O
HO
HO
S
OH
IC50 = 32.4 ±1.1
O
N
In vitro pharmacological evaluations
Guided by the kinetic results and for an in depth evaluation of
his inhibitory property, the glucose-based GP inhibitor 5h with
the best Ki value was assayed in vitro with rat and human
hepatocytes in primary culture (see Table S1). The potent in
vitro GP inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB, Ki =
400 nM)69 was selected as the reference compound for its
established in vivo activity in a GP-dependent glycaemia study.3,
69, 70 An evaluation of glucose release after glucagon stimulation
in primary rat hepatocytes was performed. Further evaluation
in human hepatocytes for the candidate 5h was accomplished
by measuring both glucose release and intracellular glycogen
for an assessment of species specificity.
Figure 5: Glucose release from rat and human hepatocytes after glucagon stimulation in
vitro measured in the presence of compound 5h versus DAB as the reference compound.
Top: Rat hepatocytes, bottom: human hepatocytes
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Liver glucose output kinetics (Fig. 7A) and their areas under the
View Article Online
DOI: 10.1039/C9OB01190K
curves (AUC) for 45 min (Fig. 7B) pointed to a dose-dependent
decrease in liver glucose production ranging from 7.5 to 60
mg/kg (Fig. 7B). The rate of endogenous glucose production
being elevated in type 2 diabetes renders this effect on liver
output relevant for therapeutic applications.71
Compound 5h was then selected for a subchronic oral
administration with a dose corresponding to the first
significantly effective in the acute in vivo glucagon challenge
test (30 mg/kg). In a glucagon challenge test performed after 4
days of treatment, a nearly 33% reduction of hepatic glucose
production (p<0.05) was observed (Fig. 8A-B). It is worth
pointing out that the activity of glycogen phosphorylase was
preserved, in spite of the subchronic treatment, since hepatic
glucose production was not further reduced.
Figure 6: Intracellular glycogen content in human hepatocytes after glucagon stimulation
in vitro measured in the presence of compounds 5h versus DAB as the reference
compound
In vivo pharmacological evaluations
The glucagon challenge test in the Zucker fa/fa rat model was
performed to measure the glucose-lowering effect in vivo with
for compound 5h. This animal model, displaying hyperphagia
and insulin resistance with hyperinsulinemia, was chosen due
to its high hepatic glycogen content. We used glucagon (200
µg/kg, in a single subcutaneous “SC” administration) as
hyperglycemic agent. A dose-dependent effect was clearly
observed when compound 5h was introduced orally in a single
administration (Figure 7).
Figure 8. Plasma glucose concentration of the unprotected compound 5h obtained in
Zucker fa/fa rats subjected to in vivo glucagon challenge after subchronic
administrations. (A) Kinetics of glucose output for 45 min after acute glucagon
administration in mmol/L. (B) Area Under the Curve (AUC) corresponding to glucose
release for 45 min after acute glucagon administration. Plasma glucose concentrations
were expressed as variations (delta), meaning that the basal value of glycaemia was
substracted for each individual value and for each rat.
Figure 7. Hepatic glucose production of the unprotected compound 5h obtained in
Zucker fa/fa rats in acute in vivo glucagon challenge. (A) Kinetics of glucose output for 45
min after acute glucagon administration in mmol/L. (B) Area Under the Curves (AUC)
corresponding to glucose release for 45 min after acute glucagon administration.
*p<0.05 for 15 mg/kg, ***p<0.001 for 30 mg/kg and **p<0.01 for 60 mg/kg
As a summary, the in vitro and in vivo pharmacological effects
of glucose-based spiro-oxathiazole 5h were analyzed and they
validated its potent GP inhibition both in vitro, in cell assays and
in a diabetic rat model (in vivo). In rat and human hepatocytes,
compound 5h reduced glucagon-stimulated glucose output
through glycogenolysis inhibition (Table 3, Fig. 5). No species
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specificity could be observed since the potency of compound 5h candidate, multigram-scale preparations for in viVvieowvAartlicidleaOtniloinne
DOI: 10.1039/C9OB01190K
for inhibition of glucose output were comparable between rat called for a robust and faster synthetic route to the target
and human cells (Table 3) as previously reported.72,
Compound 5h was efficient in vivo in the Zucker fa/fa rat model
of insulin resistance and a dose-dependent decrease of hepatic
glucose output to a maximal 43% was observed (Fig. 7). This
glucose lowering effect allows for the design of therapeutic
applications due to a high rate of endogenous glucose
production in type 2 diabetes.71 Indeed, hepatic glucose
production tends to match the values usually obtained in Wistar
normal rat.
The complete suppression of glucagon-induced glucose output
in the presence of DAB appears in sharp contrast with the
moderate level of inhibition of GP.70 The incomplete inhibition
of GP by compound 5h is advantageous for limiting potential
side effects. In addition, the absence tissue-specificity between
liver and muscle GP isoforms could impair exercise-mediated
metabolism of muscle glycogen. Preserving a minimum level of
activity will limit this risk, although the selection of a liver-
specific inhibitor preferred for long-term therapy remains a
valuable research challenge.74 Even though hypoglycaemia
could be a detrimental consequence of GP inhibition, hepatic
glucose production was not further reduced after repeated
administrations of compound 5h, indicating a residual activity
of GP thus limiting the risk of hypoglycaemia (Fig. 7).
glucose-based spiro-oxathiazole.
73
Conflicts of interest
There are no conflicts to declare
Acknowledgements
Financial support from CNRS and the Hungarian Academy of
Sciences, University Claude Bernard Lyon 1, the French Agence
Nationale de la Recherche (ANR-08-BLAN-0305) as well as the
Hungarian National Research, Development and Innovation
Office (OTKA K109450), and the EU co-financed by the European
Regional Development Fund under the project GINOP-2.3.2-15-
2016-00008 are gratefully acknowledged. We are grateful to
Prof. A.J. Fairbanks for providing the internal report of B.
Wilkinson (January 2008) on the research carried out under his
supervision. CCIR-ICBMS (UCBL) is gratefully acknowledged for
the allocation of computational resources.
Rat hepatocytes in primary culture were isolated in situ from rat
liver. Human hepatocytes were isolated from pieces of liver
resection after surgery for medical purpose. Informed consents
were obtained from human participants of this study. Use of
this animal or human material was approved by our local and
national ethics committee and legal instances (MESR DC-2008-
531). The same approval was provided for the use of animal
models in accordance with the guidelines for care and use of
laboratory animals.
Conclusion
Type 2 diabetes is a major public health problem and design of
glycogen phosphorylase inhibitors targeting the catalytic site of
the enzyme appears as a promising strategy for a better control
of hyperglycaemia. An especially potent group of GP inhibitors
is represented by some glucopyranosylidene-spiro-
heterocycles, among them spiro-oxathiazoles. A new synthesis
has now been elaborated for this valuable class of compounds
by the 1,3-dipolar cycloaddition of an aryl nitrile oxide to a
glucono-thionolactone affording in one step the spiro-
oxathiazole moiety. The thionolactone was obtained from the
thermal rearrangement of a thiosulfinate precursor according
to Fairbanks’ protocols, although with a revisited outcome and
also based on DFT calculations. The 2-naphthyl substituted
glucose-based spiro-oxathiazole 5h has recently been identified
as one of the most potent GP inhibitors with a Ki of 160 nM
against RMGPb. Further evaluation in vitro using rat and human
hepatocytes demonstrated that compound 5h was promising as
a potential anti-hyperglycaemic druggable compound as it
performed better than DAB used as a positive control.
Investigation in Zucker fa/fa rat model in acute and subchronic
assays further confirmed the potency of compound 5h since it
lowered blood glucose levels by ~36% at 30 mg/kg and ~43% at
60 mg/kg. This indicates that compound 5h can be considered
as a powerful anti-hyperglycemic agent in the context of type 2
diabetes. The present study is one of the few in vivo
investigations for glucose-based GP inhibitors and provides data
in animal models for such drug candidates. This study is also an
example of molecular diversity-oriented strategies to identify
the fittest inhibitor of GP and from the identification of this
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DOI: 10.1039/C9OB01190K
OP
O
OH
O
S
2 steps
PO
PO
HO
HO
S
Ar
PO
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Synthesis of the
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1
glucothionolactone
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at 30 mg/kg
2
Zucker fa/fa rats