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glycaemia by intensive lifestyle intervention as a primary treat-
ment, with adequate diet and exercise, along with pharmacological
therapies [1,2].
Biguanides and a-glycosidase inhibitors have therapeutic value
as they limit, respectively, hepatic glucose output, and intestinal
absorption of carbohydrates. Sulfonylureas and incretin mimetic
drugs act by directly or indirectly increasing insulin release from
the b-cells in the pancreas, while thiazolidinediones (TZD) activate
peroxisome proliferator-activated receptors (PPARs). Metformin is
a biguanide now believed to be the most widely prescribed anti-
diabetic drug. As pharmacological treatments are inadequate for
30e40% of T2DM patients, combination therapy is frequently
applied. With increasing severity of diabetes, insulin administra-
tion is prescribed and many patients progress to insulin therapy
with time. In spite of substantial progress in the management of
diabetic pathologies, the limitations or adverse effects of current
treatments are incentive for improving diabetes management.
Glycogen phosphorylase (GP) inhibition is one of the pharmaco-
logical approaches currently investigated [3].
GP isozymes [4] have been identified and characterized in a
large number of organisms (bacteria, fungi, yeast, plants, insects,
animals) and in mammalian tissues. GP is expressed mainly in the
muscles, liver and brain where it permits the breakdown by
phosphorolysis of glycogen to glucose-1-phosphate. GPa and GPb
represent, respectively, the phosphorylated (active) and unphos-
phorylated (less active) isoforms [5]. This enzyme has been thor-
oughly studied by kinetic investigations and X-ray diffraction
analysis of enzymeeligand complexes. These studies provided ev-
idence on the binding site and binding mode of ligands to the
enzyme. The accumulated information forms a rational basis for the
kinetic data, but more significantly, provides a detailed view of the
GP structural features with identification of several binding sites
such as the active site, the inhibitor site, the allosteric and the new
allosteric sites, the glycogen storage site, the phosphorylation site
and an understanding of their roles at molecular level [6]. As
glycogenolysis is a key component to hepatic glucose production,
generally observed excessive in T2DM, a large variety of synthetic
molecules has been investigated as GP inhibitors, as a possible
pharmacological control of glycaemia. The studied inhibitors
mainly target the allosteric [7] and new allosteric site [8], and the
active site which accommodates glucose and glucose-based and
related analogs [9e14], as can be seen also from general reviews
[3,15e20]. NMR spectroscopy has been shown recently through the
fragment-based approach to offer additional techniques for prob-
ing, in solution, the binding pockets of GP, and investigating
cooperativity between the various binding sites [21].
Fig. 1. Various types of spiro-anomeric carbohydrate derivatives: structures, bio-
activities, or inhibitory properties against rabbit muscle glycogen phosphorylase b
(RMGPb).
induced and obese diabetic rats has been investigated. This showed
the coordinated regulation of glycogen phosphorylase and synthase
by 50 mM A in liver extracts of Wistar rats, resulting in the activation
of synthase by a shortening of the latency compared to control
animals. Compound A was also effective in lowering blood glucose
levels and restoring hepatic glycogen content in streptozotocin-
induced diabetic rats. Furthermore, intravenous administration of
A to Zucker Diabetic Fatty (ZDF) rats significantly decreased hepatic
GPa levels, and the activation of synthase was initiated without any
delay [27].
GP inhibition is a therapeutic approach to limit the pathogenic
consequences of chronic hyperglycaemia in T2DM but also possibly
tumor growth [28,29], or cerebral ischemia [30]. Nevertheless, little
is known about the potential of glucose-based molecules that bind
at the catalytic site at cellular level, and only a few have been
studied in detail in hepatocytes [27,31,32]. This is why on the basis
of our preliminary studies [33,34], the synthesis of additional
glucose-based spiro-isoxazolines followed by kinetic investigations
were performed. The more potent molecules (Ki in the low
mM
range) were selected for cellular assays with primary cultures of rat
or human hepatocytes. The most promising molecules identified
from these in vitro cellular assays were further investigated in an
animal model (i.e. Zucker hyperinsulinemic rat).
The glucopyranose-based analog B of hydantocidin (Fig. 1)
prepared by Fleet's group was found to be a potent inhibitor of
glycogen phosphorylase [35]. This was an incentive for investi-
gating chemical synthesis, kinetic measurements and crystallo-
graphic analysis of enzymeeligand complexes. The kinetic and
crystallographic data obtained showed that spiro-compounds A
[36], B [35], C [35], and D [37] are competitive inhibitors and are
bound at the enzyme catalytic site through a network of stabilizing
interactions. By exploiting stereoselective 1,3-dipolar cycloaddi-
tions, we have also synthesized glucopyranose-based spiro-iso-
xazolines [33,34] (e.g. E) and spiro-oxathiazoles [38e40] (e.g. F)
which were found among the best inhibitors of GP targeting the
catalytic site [39]. Glucopyranosylidene-spiro-iminothiazolidinone
derivatives G were reported recently as good GP inhibitors [41],
while spiro-oxazolidinones H proved practically inactive against
the enzyme [42].
Even though a large set of data has been reported from in vitro
enzymatic experiments, much need to be clarified through phar-
macological studies to get a better understanding of the in vivo
specific response of a drug under evaluation. Its effects, which
depend on pharmacokinetic and pharmacodynamic properties, are
unpredictable and sometimes difficult to rationalize. For example,
the inhibitory effects of indole-site effectors has been reported to
be modulated by endogenous small-molecular-weight effectors of
GPa activity, although at higher concentrations, indole-site GP in-
hibitors almost completely inhibit phosphorylase activity and
retain a glucose concentration dependence [22,23]. A series of
benzamide derivatives, presumed to bind at the new allosteric site
of GP (dimer interface) as suggested by molecular docking simu-
lation, was found to simultaneously inhibit GP and activate gluco-
kinase [24]. GP inhibitors can not only interfere with glycogenesis,
as mentioned, but also with gluconeogenesis, and the in vivo effects
of AMP and indole site inhibitors have been reviewed [18,25,26].
The present report discloses further synthetic and kinetic, as
well as in vitro and in vivo pharmacological evaluation of new
representatives of type E compounds to determine the properties
Recently, the effect of
D-glucopyranosylidene-spiro-thiohydantoin
(Fig. 1A) on glycogen metabolism in liver tissues of streptozotocin-
Goyard, David
