C-Glucosylated malonitrile as a key intermediate towards carbohydrate-based glycogen phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.07.033

Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxadiazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phosphorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range. In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydropyrimidin-2-one may bind at the catalytic site.

Full text of DOI:10.1016/j.bmc.2012.07.033

Bioorganic & Medicinal Chemistry 20 (2012) 5592–5599
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C-Glucosylated malonitrile as a key intermediate towards
carbohydrate-based glycogen phosphorylase inhibitors
Sophie Feuillastre a, Aikaterini S. Chajistamatiou b, Constantinos Potamitis b, Maria Zervou b,
a,
a,
Panagiotis Zoumpoulakis b, Evangelia D. Chrysina b, , Jean-Pierre Praly , Sébastien Vidal
⇑
⇑
⇑
a Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique 2 – Glycochimie, UMR 5246, CNRS, Université Claude Bernard Lyon 1,
43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
b Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens GR-11635, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target
for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single
C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxa-
diazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phos-
phorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range.
In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydro-
pyrimidin-2-one may bind at the catalytic site.
Received 12 March 2012
Revised 14 June 2012
Accepted 13 July 2012
Available online 2 August 2012
Keywords:
Carbohydrate
Glycogen phosphorylase
1,3-Dipolar cycloaddition
Malonitrile
Ó 2012 Elsevier Ltd. All rights reserved.
Pyrimidinone
1. Introduction
Control of hyperglycaemia is one of the major challenges in the
prevention of type 2 diabetes complications. A large series of bio-
logical targets have been identified for the treatment of type 2 dia-
betes over the past decades1–3 and glycogen phosphorylase4 (GP)
has been recognized as a possible macromolecular target for the
control of hyperglycaemia.1,5 This dimeric enzyme presents five
different binding sites for a variety of small molecule inhibi-
tors.6–9 Glycogen phosphorylase inhibitors with high affinity for
the catalytic site—the most extensively studied site of this en-
zyme—are mostly D-glucopyranose-based molecules with various
substituents introduced at the anomeric position forming distinct
classes.6–10 The catalytic site of GP comprises a large cavity with
high specificity for the
two additional pockets lying below and above the anomeric center
of the carbohydrate moiety, that is, pointing into the and b direc-
tions, respectively. Both subsites in the and b directions have
been explored with different substituents with the aim to identify
the determinants of binding at this site.5,6,9,10 Based on the well
established homology between species and tissues isoforms of
D-glucopyranose moiety (Fig. 1) but also
Figure 1. X-ray crystallographic structure of the catalytic site of GP (PDB code:
2PYD) in complex with -glucopyranose and visualization of the -subsite (below
glucose and left to the 280s loop) and b-channel (on the right side of glucose and
a
a-
D
a
a
above the 280s loop).
GP,11 and because unphosphorylated GP (GPb) plays a crucial role
in the reciprocal control between glycogenesis and glycogenoly-
sis,5–7 it is a common practice to use rabbit muscles GPb (RMGPb)
for kinetic studies and inhibition evaluation.
Previous binding studies of GP in complex with potential inhib-
itors targeting the catalytic site revealed that the most potent ones
had aromatic groups pointing towards the direction of b-pock-
⇑
Corresponding authors. Tel.: +33 472 431 161; fax: +33 472 432 752 (J.-P.P.);
tel.: +30 210 7273851; fax: +30 210 7273831 (E.D.C.).
E-mail addresses: echrysina@eie.gr (E.D. Chrysina), jean-pierre.praly@univ-
lyon1.fr (J.-P. Praly).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.033

S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
5593
OH
O
OH
O
HO
Het
β-Pocket
HO
HO
Ar
HO
OH
OH
OH
β
-Pocket
O
HO
α-Pocket
HO
Ar
Het
HO
N
O
O
OH
OH
Ar
Ar
O
O
HO
HO
HO
HO
N
N
H
OH
OH
HN
O
N
N
O
Ar
Ar
Figure 2. Structures of known (top right) and potential (bottom) GP inhibitors.
et.7,12–20 The most suitable
D-glucose-based scaffolds are substi-
2. Results and discussion
tuted heterocycles with either a C-glucosidic bond or a spiro-bicy-
clic framework (Fig. 2). These structural designs provided potent
inhibitors of GP with inhibition constants (Ki) in the low micromo-
lar to sub-micromolar range. Such scaffolds improved the inhibi-
tory potency of the ligands designed by taking advantage of the
space occupied by water molecules in the b-channel and by form-
ing additional contacts stabilizing the closed (inactive) conforma-
tion of the 280s loop. With the aim to make the most of both the
unoccupied space and the 280s loop flexibility, a second aromatic
moiety could be introduced in the vicinity of the anomeric position
2.1. Synthesis of GP inhibitors
The C-glucosylated malonitrile precursor 2 was readily pre-
pared according to a known procedure21 from acetobromoglucose
1 and malonitrile (Scheme 1). The 1,3-dipolar cycloaddition with
nitrile oxides, generated in situ from the corresponding a-chlorox-
imes 3a–b,18 was then performed and provided the corresponding
acetylated bis-oxadiazoles 4a–b in good yields. Subsequent deacet-
ylation afforded the desired hydroxylated C-glucosyl compounds
5a–b. The moderate yields sometimes observed in these two syn-
theses were probably due to the acidity of the methine proton
of the carbohydrate pointing towards the
a-plane of the sugar
(Fig. 2). The putative interactions of this additional substituent
next to the anomeric center, with the residues lining the 280s loop
of the enzyme could provide complementary contacts and
strengthen binding, enhancing inhibitor potency. To this end, we
have designed a general synthetic route from a C-glucosylated
malonitrile precursor for the synthesis of bis-oxadiazole and bis-
amide inhibitors as well as a C-glucosylated tetrahydropyrimi-
din-2-one.
(H-a) between two electron withdrawing groups being either ni-
triles (for compound 2) or 5-(1,2,4-oxadiazolyl) rings13,18 (for com-
pounds 4a–b). The basic conditions applied for the 1,3-dipolar
cycloaddition with in situ generation of nitrile oxides and/or the
deacetylation step could lead to deprotonated intermediates prone
to undergo ring-opening by cleavage of the endocyclic C–O bond
and eventually formation of side products which were neither iso-
lated nor characterized. The reduction of the C-glucosylated malo-
Ar
N
4a
R = Ac, Ar = 2-Naphthyl (78%)
N
O
OR
O
OAc
O
OAc
O
Et3N / PhMe
110ºC
CN
MeOH
H2O
Et3N
4b R = Ac, Ar = p-toluyl
(50%)
NC
CN
AcO
AcO
AcO
AcO
RO
RO
N
OH
5a
CN
R = H, Ar = 2-Naphthyl (40%)
Ar
NaH / DMF
then AcOH 10%
N
AcO
OAc
OR
5b R = H, Ar = p-toluyl
(73%)
Br
O
2
N
Ar
Cl
1
H2 / PtO2
CHCl3
EtOH
71%
99%
3a Ar = 2-Naphthyl
Ar
O
3b
Ar = p-toluyl
7a
R = Ac, Ar = 2-Naphthyl (39%)
NH2
NH2
NH
OAc
O
OR
O
7b R = Ac, Ar = Ph
(70%)
ArCOCl
MeONa
MeOH
OH
O
H
N
AcO
RO
RO
H
N
Ar
HO
HO
AcO
Me
8a
R = H, Ar = 2-Naphthyl (29%)
C5H5N
OAc
OR
HO
O
8b R = H, Ar = Ph
(53%)
Inhibitors used for
O
O
A
6
in silico studies
NH2
O
OH
O
H
H
N
H
N
H
N
N
O
O
OAc
O
OH
O
HO
HO
N
N
N
N
MeOH / H2O
OH
AcO
AcO
HO
HO
O
O
B
NH
NH
(Table 1)
38%
Et3N
99%
OAc
OH
9
10
Scheme 1. Synthesis of the C-glucosylated bis-oxadiazoles 5a–b, bis-amides 8a–b and tetrahydropyrimidin-2-one 10 and structure of inhibitors A–B used for the in silico
studies.

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S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
(Fig. 3). In CDCl3, this might be due to the formation of an intramo-
lecular hydrogen bond between the NH protons and the oxygen
atom of the carbonyl acetate at the 2 and/or 6 position of the car-
bohydrate (Fig. 4) thus creating either a 9- or a 11-membered ring
between the glucose unit and the aglycon part. Another intramo-
lecular hydrogen bond appears possible between one of the NH
protons and the oxygen atom of the other amide moiety. This net-
work of intramolecular interactions should entail restricted rota-
tion in 7b, thereby explaining the existence of several signals for
the NH protons and for the methylene b-protons in CDCl3, while
in DMSO-d6, intermolecular H-bonding between 7b and the sol-
vent should lead to more mobile species.
Finally, upon treatment with 1,10-carbonyldiimidazole (CDI),
the bis-amine 6 was converted into the tetrahydropyrimidin-2-
one 9 that was deacetylated to afford the hydroxylated C-glucosyl
derivative 10 (Scheme 1). Synthetic assays first attempted in DMF
at 80 °C (Table 1, entry 1) afforded the desired tetrahydropyrimi-
din-2-one 9 in poor yield. The nucleophilicity of amines can be im-
Figure 3. Partial 1H NMR spectrum (293 K, 300 MHz) of acetylated bis-amide 7b in
(a) CDCl3 and (b) DMSO-d6.
nitrile 2 afforded the bis-amine 621 (Scheme 1) and subsequent
acylation provided the corresponding bis-amides 7a–b. Deprotec-
tion under Zemplén conditions gave the hydroxylated bis-amides
8a–b in high yields.
proved by using AlMe3 to generate
a highly nucleophilic
aluminum–nitrogen complex with enhanced reactivity towards
the addition to the carbonyl of CDI.22 Moreover, the electrophilicity
of the CDI carbonyl group can be enhanced by chelation with Lewis
acids such as AlMe3. Both of these possibilities most probably ex-
plain the better yield obtained when the reaction was performed
in the presence of excess of AlMe3 in refluxing dichloromethane
(Table 1, entry 2). The formation of oligomers or polymers arising
from the intermolecular addition of two amines from two different
molecules of 6 might explain the limited improvement of the yield
when the reaction was performed at 12 mM concentration of bis-
amine 6. In keeping with this hypothesis, the synthesis under di-
luted conditions afforded 9 in an acceptable 38% yield (1.2 mM, Ta-
ble 1, entry 3).
Interestingly, the 1H NMR data collected for the acetylated bis-
amide 7b dissolved in CDCl3 (Fig. 3a) displayed two signals for the
NH protons (d = 7.89 and 6.90 ppm). In addition, the signals of the
methylene protons attached to the amide moieties (H-b) were also
separated into three different signals: one multiplet (d = 4.03) cor-
responding to two protons and two additional multiplets (d = 3.47
and 3.22 ppm) for one proton each. The NMR spectrum recorded
from a DMSO-d6 solution (Fig. 3b) showed a single signal for both
NH protons (d = 8.50 ppm) and another signal resonating at
d = 3.45 ppm for the four methylene protons which merged into a
single signal. Restricted rotation of the N–C(O) bond leading to sep-
arate amide rotamers being observable in the NMR time scale ap-
pears to be influenced by the medium. The hydrogen bond pattern
could explain our observation as proposed in the model shown in
Figure 4. The protons at position 2, 5 and 6 of the glucopyranosyl
ring have their chemical shifts most affected by changing the sol-
vent, while protons at positions 1, 3 and 4 are roughly unchanged
2.2. Enzyme kinetics
The five glucose-based compounds 5a–b, 8a–b and 10 were
evaluated for their potency as GP inhibitors (Table 2). Rabbit skel-
etal muscle glycogen phosphorylase b (RMGPb) was used as the
model enzyme.5–7 The bis-oxadiazole 5a was poorly soluble in
Me
O
Ph
H
H
N
O
Ph
H
Me
O
S
Ph
O
O
Me
Me
Me
N
N
N
OAc
O
O
OAc
O
O
S
H
H
DMSO-d6
CDCl3
O
AcO
AcO
AcO
AcO
AcO
AcO
N
Ph
N
Ph
O
O
OAc
OAc
O
O
O
Me
H
7b
No diastereotopic amide
2 signals = 1 NH and 1 H-
11-membered ring
2 diastereotopic amides
6 signals = 2 NH and 4 H-
Ph
9-membered ring
2 diastereotopic amides
6 signals = 2 NH and 4 H-
β
β
β
Figure 4. Possible H-bonded network for bis-amide 7b influenced by the solvent used (CDCl3 or DMSO-d6).
Table 1
Synthesis of the tetrahydropyrimidin-2-one 9
Entry
Reagents
Solvent
Concentrationa (mM)
Temperature (°C)
Yield (%)
1
2
3
CDI
CDI/AlMe3
CDI/AlMe3
DMF
CH2Cl2
CH2Cl2
12
12
1.2
80
40
40
7
18
38
b
b
a
Concentration of bis-amine 6.
CDI (1.1 equiv), AlMe3 (3.5 equiv).
b

S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
5595
Table 2
used for the docking studies of compound 10 only. Our initial ap-
proach was to maintain the water network at the catalytic site.
The docking procedure included the application of Glide XP with
core constraint in order to ‘lock’ the glucopyranose moiety and
subsequent redocking of the top ranked poses using QPLD-XP ap-
proach.12,26 The docking protocol was validated using the x-ray
crystal structures of both RMGPb complexes as well as a series of
inhibitors with known binding affinities.26
Inhibition of RMGPb measured for the glucose-based compounds 5a–b, 8a–b and 10
Compounds
Inhibition measured (lM)
5a
5b
8a
8b
10
38% at 250a
39% at 1000
44% at 300
21% at 1000
8.5% at 1000
a
Partial solubility even in the presence of 20% DMSO in water; hence the inhi-
bition is indicative and underestimated.
2.3.1. Docking studies with 10 at the catalytic site of the
formamide A/RMGPb complex
Results showed that compound 10 may bind at the active site of
RMGPb in a way similar to the formamide A ligand (Fig. 5). The
hydrogen bond interactions formed between the glucopyranose
moiety and the residues of the catalytic site were maintained;
water even with 20% DMSO, as the two naphthyl groups present
displaced the hydrophobic/hydrophilic balance towards highly
hydrophobic properties. Therefore, the inhibitory potency de-
scribed for compound 5a might be slightly higher since only the
supernatant of a saturated solution was used. Compounds 5b, 8b
and 10 were poor inhibitors even at high concentration (1 mM).
Nevertheless, the inhibition of RMGPb by the bis-naphthylamide
namely,
His377:O6,
Asn484:O6,
Gly675:O4,
Asn484:O4,
Ser674:O3, Glu672:O3, Glu672:O2 and a water-mediated hydro-
gen bond between O4 and Thr676. No additional electrostatic
interaction was observed for the pyrimidinone ring. Furthermore,
an unfavorable van der Waals interaction (4.18 kcal molꢀ1) was
observed due to the close contact of the pyrimidone ring with
the side chain of Asn284. The corresponding interaction in
RMGPb-formamide docked complex though, is energetically favor-
able (ꢀ2.16 kcal molꢀ1). The absence of electrostatic interactions
(apart from the glucose ring) coupled with the presence of unfavor-
able van der Waals interactions may provide an explanation for the
low inhibition of enzyme activity by compound 10 (8.5% at
8a is quite significant with 44% inhibition at 300 lM. Bis-oxadiaz-
oles 5a–b compare unfavorably to their related mono-oxadiazole
counterparts, found from previous studies to exhibit both IC50
and Ki values in the low micromolar range18 for the 2-naphthyl
substituted compound. Both bulkiness and the presence of an addi-
tional carbon atom between the anomeric position and the oxadi-
azole rings could alter the binding of 5a–b to the catalytic site of
GP. Compared to some related C-glucopyranosyl pyrimidine
nucleosides,23,24 the C-glucosylated tetrahydropyrimidin-2-one
10 exhibited a much weaker inhibition against RMGPb.
1000 lM).
2.3.2. Docking studies at the catalytic site of 2-naphthoyl urea B/
RMGPb complex
2.3. Docking results
The most prominent changes in the 2-naphthoyl urea/RMGPb
complex structure involve the distal arrangement of Asn284 and
the loss of the close contact formed between the side chain atoms
of Asn284 with O2 of the sugar coupled with dislocation of Phe285.
These changes result in creating a wider cavity for ligand binding.
Docking of the voluminous compounds 5a, 5b, 8a, 8b failed to re-
port any poses in the binding pocket of 2-naphthoyl urea B/RMGPb
complex. Compound 10 was also tested for docking at the catalytic
site of this complex. Avoiding the steric clashes with the 280s loop,
the synthesized molecule could bind in a similar mode with the 2-
naphthoyl urea derivative B at the catalytic site of RMGPb but its
pyrimidinone ring failed again to develop any additional electro-
static interaction (data not shown).
The binding mode of compounds listed in Table 2 was further
investigated by in silico studies to account for the differences ob-
served in their inhibitory potency. Our aim was also to compre-
hend better the unfavorable structural features in the vicinity of
the anomeric center that would allow the rational design of im-
proved inhibitors. Given the flexibility of the 280s loop and the
structural dissimilarity among the studied compounds, two differ-
ent crystal structures of RMGPb were used as starting models: (a)
that found for RMGPb with the inhibitor C-(1-acetamido-
a-D-glu-
copyranosyl) formamide (Scheme 1, A), bearing both - and b-ori-
a
ented substituents at the anomeric carbon atom and found to bind
at the enzyme catalytic site (PDB code: 1P4H); (b) that found for
RMGPb in complex with a bulkier inhibitor, 2-naphthoyl urea
(Scheme 1, B) whose binding has already been associated with a
shift of the 280s loop (residues 282–287).9,25 The first model was
In order to investigate whether the bulkier compounds 5a, 5b,
8a and 8b could dock in a less hindered pocket, all the 415 water
Figure 6. The best docked pose of 8b (shown in grey) at the catalytic site of the
modified 2-naphthoyl urea B/RMGPb complex (in the absence of five water
molecules) superimposed onto 2-naphthoyl urea (shown in green). Hydrogen bond
interactions of 8b with the residues lining the binding site are depicted in dotted
lines.
Figure 5. The best docked pose of 10 (shown in grey) at the catalytic site of the
formamide A/RMGPb complex (PDB code: 1P4H) superimposed onto formamide A
(shown in green). Direct and water-mediated hydrogen bond interactions of 10
with the residues lining the binding site are depicted in dotted lines.

5596
S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
molecules of the 2-naphthoyl urea/RMGPb complex structure were
excluded. This approach resulted in acceptable docking poses that
maintained the position of glucopyranose moiety only for 8b.
Moreover, one of its side chains (b branch) was oriented towards
the b-channel and smoothly overlaid the 2-naphthoyl urea moiety
towards RMGPb with two of the compounds responding in the
sub-millimolar range concentration. In silico studies indicated that
10 may bind at the catalytic site; however, the unfavorable van der
Waals interactions identified with Asn284 of the formamide A/
RMGPb complex binding site might account for the poor inhibition
observed. In the case of the branched compound 8b, theoretical
calculations showed that it could be accomodated at the wider
binding site of the 2-naphthoyl urea B/RMGPb complex. Binding
of 8b seems to require disruption of the solvent network and con-
comitant loss of interactions stabilizing the complex that could
possibly explain the weak inhibition. The slightly improved inhib-
itory potency of 8a taking into account that it failed to be accom-
modated at the catalytic site might be attributed to binding at a
different site of RMGPb. In the absence of structural evidence, the
poor inhibition observed for all compounds synthesized to target
the catalytic site could be interpreted in terms of the limited space
of compound B, while the other chain (a-like branch) laid on a
plane parallel to the glucose ring.
To further explore the role of the solvent structure, the water
molecules that hindered the binding of 8b at the catalytic site of
RMGPb, were identified by inspecting the 8b docked pose in the
absence of all water molecules. Then, the RMGPb crystal structure
was subjected to a stepwise deletion of five water molecules, lying
in close proximity to the a-like branch of 8b, leading to its success-
ful binding at the catalytic site (Fig. 6). Comparison of QPLD-XP
docking results for 8b with the crystal structure of the complex
with 2-naphthoyl urea B showed that crucial hydrogen bonding
interactions between the glucopyranose moiety and the residues
of the catalytic site are maintained; namely, Asn484:O6,
Gly675:O4, Gly675:O3, Ser674:O3 and Glu672:O3. Further to this,
8b specific binding features are the loss of two hydrogen bonds be-
tween His377:O6 and Asn484:O4 as well as between two water
molecules and O2 (one of the water molecules has been deleted).
In addition, both carbonyl groups of 8b side chains form water-
mediated electrostatic interactions with residues in the vicinity
in the vicinity of the
a-subsite and steric hindrance induced by
bulky substituents. Despite the unsatisfactory potency exhibited,
the docking results of these molecules could provide guiding infor-
mation in a structure-based inhibitor design approach.
4. Experimental section
4.1. General methods
(b branch carbonyl group with Glu88, Gly134, Gly137 and
branch carbonyl group with Asp339). Furthermore, the conforma-
tion adopted by -like branch is induced by the interactions
between 8b phenyl group and Tyr573.
a-like
All reagents and solvents used for syntheses were commercial
and used without further purification. Solvents were distilled over
CaH2 (CH2Cl2), Mg/I2 (MeOH), or purchased dry. Reactions were
performed under argon atmosphere. NMR spectra were recorded
at 293 K, unless stated otherwise, using a 300, 400 or 500 MHz
spectrometer. Shifts are referenced relative to deuterated solvent
residual peaks. Low and high resolution mass spectra were re-
corded in the positive mode using a Bruker MicroTOF-Q II XL spec-
trometer. 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 solution of 10% H2SO4 in EtOH/H2O (1:1 v/v) or KMnO4
in H2O or ninhydrin in n-BuOH/H2O followed by heating. Silica gel
column chromatography was performed with silica gel Si 60 (40–
a
p–p
Application of MM-GBSA post docking scoring protocol calcu-
lated a
D
Gbind of ꢀ124.4 kcal molꢀ1 for 8b. The corresponding free
energy value for 2-naphthoyl urea B in the absence of the same
water molecules was
D
Gbind = ꢀ102.8 kcal molꢀ1 (compared to
D
Gbind = ꢀ130.5 kcal molꢀ1 when all waters were included). This
difference could be attributed to the lack of several favorable elec-
trostatic interactions, induced by the disruption of a stable water
network, involving loss of crucial water interactions with O2 of
glucopyranose as well as with the amide groups of the urea moiety.
MM-GBSA binding site strain energy calculation, upon deletion of
the aforementioned five water molecules, gave an increased value
by 13 kcal molꢀ1 as compared to the strain energy when all water
molecules were present. This could be an indication that the cata-
lytic site is in a thermodynamically less favorable state after water
deletion, which possibly accounts for the reduced inhibitory po-
63 lm). Optical rotations were measured using a Perkin Elmer
polarimeter and values are given in deg dmꢀ1 gꢀ1 cm3.
4.2. General procedure A for 1,3-dipolar cycloaddition
tency of 8b (21% at 1000 lM).
Additional efforts to accommodate compounds 5a, 5b and 8a at
the catalytic site involved docking studies with induced flexibility
of the receptor (IFD protocol). No favorable results were obtained
for none of the compounds since the geometry had to be consider-
ably distorted to get accommodated to the catalytic site. Since this
finding is not in accordance with the kinetic results obtained for
8a, it suggests that there might be weak binding of this compound
at a different RMGPb site.
A solution of C-glucosyl malonitrile 2 (0.25 mmol) and hydroxi-
moyl chloride 3a–b18 (2.2 mmol, 9 equiv) in toluene (10 mL) was
stirred at 110 °C under argon. Triethylamine (1.3 mmol, 5 equiv)
was dissolved in toluene (1 mL) and slowly added over 12 h with
a syringe pump. The solvent was evaporated to dryness. The resi-
due was purified by flash silica gel column chromatography
(EtOAc/PE 3:7) to afford the desired bis-oxadiazoles 4a–b.
4.3. General procedure B for deacetylation of bis-oxadiazoles
3. Conclusion
A
solution of the acyl-protected bis-oxadiazoles 4a–b
Short routes to potential glycogen phosphorylase inhibitors
were designed from a C-glucosylated malonitrile precursor. 1,3-
Dipolar cycloaddition with nitrile oxides provided the correspond-
ing bis-oxadiazoles, while reduction of the nitrile moieties afforded
a bis-amine, and subsequently bis-amides, by N-acylation. 1,10-
Carbonyldiimidazole reacted with the bis-amine in the presence
of trimethylaluminium and under diluted conditions so as to form
a tetrahydropyrimidin-2-one derivative by cyclization. Five inhib-
itors were obtained and their inhibition towards RMGPb was eval-
uated. The enzymatic data attested to poor inhibitory properties
(0.2 mmol) in MeOH (7 mL), extra pure water (1.5 mL) and trieth-
ylamine (1.5 mL) was stirred at room temperature for 3 days. The
solution was then evaporated to dryness and the residue was puri-
fied by flash silica gel column chromatography (CH2Cl2/MeOH 9:1)
to afford the hydroxylated bis-oxadiazoles 5a–b.
4.4. General procedure C for preparation of bis-amides
A
solution of bis-amine
6 (0.2 mmol) and aryl chloride
(0.48 mmol, 4 equiv) in dry pyridine (5 mL) was stirred at room

S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
5597
temperature during 2 h. The reaction was then poured into EtOAc
(50 mL) and the organic layer was washed with HCl 1 N
(2 ꢁ 35 mL), saturated NaHCO3 (2 ꢁ 35 mL) and saturated NaCl
(35 mL), dried (MgSO4), filtered and evaporated to dryness. The
residue was purified by flash silica gel column chromatography
(EtOAc/PE 3:7 to EtOAc).
CDCl3): d (ppm) = 7.97 (d, 4H, J = 8.3 Hz, H-ar), 7.29 (dd, 4H,
J = 3.5, 8.3 Hz, H-ar), 5.34 (dd, 1H, J = 9.2, 9.6 Hz, H-2), 5.25 (dd,
1H, J = 9.2, 9.6 Hz, H-3), 5.14 (d, 1H, J = 7.2 Hz, H-a), 5.08 (dd, 1H,
J = 9.5, 9.6 Hz, H-4), 4.67 (dd, 1H, J = 7.2, 9.6 Hz, H-1), 4.11 (dd,
1H, J = 5.9, 12.3 Hz, H-6a), 4.01 (dd, 1H, J = 2.3, 12.3 Hz, H-6b),
3.82 (ddd, 1H, J = 2.3, 5.9, 9.5 Hz, H-5), 2.42 (s, 6H, PhMe), 2.02,
1.97, 1.88, 1.74 (4s, 12H, COCH3). 13C NMR (100 MHz, CDCl3): d
(ppm) = 172.5, 172.4 (2s, C-5-oxa), 171.0, 170.7, 169.8, 169.4 (4s,
COCH3), 142.6, 142.4 (2s, C-3-oxa), 130.2, 130.1, 130.0 (3s, 4C,
CH-ar), 123.8 (1s, 2C, CIV-ar), 77.7 (C-5), 77.0 (C-1), 74.6 (C-3),
4.5. General procedure D for Zemplén deacylation
A solution of the acyl-protected carbohydrate derivatives 7a–b
or 9 (0.05 mmol) and MeONa (0.025 mmol, 0.5 equiv) in a solution
of freshly distilled MeOH (1 mL) and CH2Cl2 (5 mL) was stirred at
room temperature for 2 days. The solution was then evaporated
to dryness and the residue was purified by C-18 reverse-phase
chromatography (H2O/CH3CN 5:5) to afford the desired hydroxyl-
ated products.
71.2 (C-2), 68.7 (C-4), 62.3 (C-6), 41.0 (C-a), 22.1 (1s, 2C, PhMe),
21.5, 21.0, 20.9, 20.6 (CH3). HR-ESI-MS (positive mode): m/z Calcd
for C33H35N4O11 [M+H]+ 663.2299. Found 663.2297.
4.9. (b-D-Glucopyranosyl)-di-[3-(2-naphthyl)-1,2,4-oxadiazol-5-
yl)]methane (5a)
4.6. 2-(2,3,4,6-Tetra-O-acetyl-b-
D
-glucopyranosyl)-malonitrile
Prepared according to general procedure B from 4a (145 mg,
0.19 mmol) to afford 5a (44.4 mg, 40%) as a beige foam. Rf = 0.20
(2)21
(CH2Cl2/MeOH 9:1).
(400 MHz, CD3OD): d (ppm) = 8.66 (s, 2H, H-ar), 8.13 (m, 2H, H-
½
a 2D0 = +3.6 (c = 1.00, CH3OH). 1H NMR
ꢂ
To a solution of malonitrile (1.2 g, 18.3 mmol, 5 equiv) in DMF
(6 mL) was added NaH (0.51 g, 21.2 mmol, 5.8 equiv) by portions.
ar), 8.02-7.98 (m, 6H, H-ar), 7.58 (m, 4H, H-ar), 4.41 (d, 1H,
After gas evolution ceased, 2,3,4,6-tetra-O-acetyl-
a
-D
-glucopyran-
J = 9.6 Hz, H-a), 3.80 (dd, 1H, J = 2.1, 12.2 Hz, H-6a), 3.63 (dd, 1H,
osyl bromide 1 (1.5 g, 3.65 mmol) in DMF (3 mL) was added drop-
wise at 0 °C. The reaction mixture was stirred for 2 h at 0 °C and
then 10% aqueous AcOH (60 mL) was added and the solution was
stirred overnight at room temperature. The precipitate was col-
lected, then dissolved in CH2Cl2, dried (MgSO4) and evaporated to
dryness to obtain 2 (1.02 g, 71%) as a white foam. Rf = 0.13 (PE/
EtOAc 7:3). 1H NMR (300 MHz, CDCl3): d (ppm) = 5.29 (dd, 1H,
J = 9.2, 9.5 Hz, H-3), 5.17 (dd, 1H, J = 9.5, 9.5 Hz, H-4), 5.14 (dd,
1H, J = 9.2, 9.2 Hz, H-2), 4.28 (dd, 1H, J = 4.9, 12.5 Hz, H-6a), 4.18
J = 5.6, 12.2 Hz, H-6b), 3.56 (dd, 1H, J = 9.0, 9.6 Hz, H-1), 3.48 (dd,
1H, J = 8.8, 9.0 Hz, H-2), 3.43 (ddd, 1H, J = 2.1, 5.6, 9.5 Hz, H-5),
3.33-3.28 (m overlap with CD3OD, 2H, H-3, H-4). 13C NMR
(100 MHz, CD3OD): d (ppm) = 176.0, 175.3 (2s, C-5-oxa), 170.0
(1s, 2C, C-3-oxa), 136.2, 134.5, 128.89 (3s, 6C, CIV-ar), 130.0,
129.94, 129.89, 129.2, 128.9, 128.1, 128.0, 124.6 (8s, 14C, CH-ar),
82.3 (C-5), 79.8 (C-a), 79.4 (C-2), 73.0 (C-1), 71.2 (C-3 or C-4),
62.4 (C-6), 48.8 (C-3 or C-4). HR-ESI-MS (positive mode): m/z Calcd
for C31H27N4O7 [M+H]+ 567.1874. Found 567.1890.
(dd, 1H, J = 2.4, 12.5 Hz, H-6b), 4.02 (d, 1H, J = 4.3 Hz, H-a), 3.97
(dd, 1H, J = 4.3, 9.2 Hz, H-1), 3.86 (ddd, J = 2.4, 4.9, 9.5 Hz, 1H, H-
5), 2.12, 2.11, 2.06, 2.04 (4s, 12H, COCH3). 13C NMR (100 MHz,
CDCl3): d (ppm) = 172.7, 172.2, 170.7, 170.2 (4s, COCH3), 109.1 (s,
2C, C„N), 77.4 (C-5), 74.7 (C-1), 73.1 (C-3), 70.0 (C-4), 67.6 (C-2),
4.10. (b-D-Glucopyranosyl)-di-[3-(p-tolyl)-1,2,4-oxadiazol-5-
yl]methane (5b)
Prepared according to general procedure B from 4b (200 mg,
0.30 mmol) to afford 5b (109 mg, 73%) as a beige foam. Rf = 0.70
61.5 (C-6), 26.9 (C-
a), 21.2, 20.8, 20.7 (3s, 4C, CH3).
(CH2Cl2/MeOH 9:1).
(400 MHz, CD3OD): d (ppm) = 7.96 (dd, 4H, J = 2.4, 8.0 Hz, H-ar),
½
a 2D0
ꢂ
ꢀ1.4 (c = 0.5, CH3OH). 1H NMR
4.7. (2,3,4,6-Tetra-O-acetyl-b-
D-glucopyranosyl)-di-[3-(2-
naphthyl)-1,2,4-oxadiazol-5-yl]methane (4a)
7.34 (dd, 4H, J = 4.3, 8.0 Hz, H-ar), 5.51 (d, 1H, J = 4.6 Hz, H-a),
4.32 (dd, 1H, J = 4.6, 9.3 Hz, H-1), 3.76 (dd, 1H, J = 2.2, 12.2 Hz, H-
6a), 3.59 (dd, 1H, J = 5.6, 12.2 Hz, H-6b), 3.46 (m, 2H, H-2, H-3),
3.38 (ddd, 1H, J = 2.2, 5.6, 9.6 Hz, H-5), 3.27 (m overlap with
CD3OD, 1H, H-4), 2.41 (d, 6H, J = 1.4 Hz, PhMe). 13C NMR
(100 MHz, CD3OD): d (ppm) = 169.81, 169.78 (2s, C-5-oxa), 143.4,
143.3 (2s, C-3-oxa), 130.73, 130.70, 128.4 (3s, 4C, CH-ar), 124.9,
124.8 (2s, CIV-ar), 82.5 (C-5), 79.9 (C-1), 79.6 (C-3), 73.2 (C-2),
Prepared according to the general procedure A from 2 (100 mg,
0.25 mmol) to afford 4a (145.1 mg, 78%) as a brown oil. Rf = 0.65
(PE/EtOAc 7:3). ½a D20
ꢂ
ꢀ3.0 (c = 1.00, CHCl3). 1H NMR (400 MHz,
CDCl3): d (ppm) = 8.65 (s, 2H, H-ar), 8.14 (d, 2H, J = 8.5 Hz, H-ar),
7.97-7.88 (m, 6H, H-ar), 7.59-7.55 (m, 4H, H-ar), 5.37 (dd, 1H,
J = 9.2, 9.6 Hz, H-2), 5.30 (dd, 1H, J = 9.1, 9.2 Hz, H-3), 5.25 (d, 1H,
J = 7.2 Hz, H-
a
), 5.12 (dd, 1H, J = 9.5, 9.7 Hz, H-4), 4.76 (dd, 1H,
71.4 (C-4), 62.7 (C-6), 41.2 (C-a), 21.5 (1s, 2C, PhMe). HR-ESI-MS
J = 7.2, 9.6 Hz, H-1), 4.13 (dd, 1H, J = 5.9, 12.3 Hz, H-6a), 4.04 (dd,
1H, J = 2.2, 12.3 Hz, H-6b), 3.87 (ddd, 1H, J = 2.2, 5.9, 9.5 Hz, H-5),
2.04, 1.99, 1.87, 1.77 (4s, 12H, COCH3). 13C NMR (100 MHz, CDCl3):
d (ppm) = 172.5, 172.3 (2s, C-5-oxa), 170.6, 170.4 (2s, C-3-oxa),
169.5, 169.2, 169.1, 169.0 (4s, COCH3), 135.0, 134.9, 133.1 (3s, 6C,
CIV-ar), 129.1, 129.07, 129.01, 128.6, 128.5, 128.05, 128.04,
127.98, 127.86, 127.07, 126.99, 123.83, 123.75 (13s, 14C, CH-ar),
77.4 (C-5), 76.7 (C-1), 74.2 (C-3), 71.0 (C-2), 68.4 (C-4), 62.0 (C-6),
(positive mode): m/z Calcd for C25H26N4NaO7 [M+Na]+ 517.1694.
Found 517.1670.
4.11. 2-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-1,3-
diaminopropane (6)21
Compound 2 (50 mg, 0.13 mmol) and PtO2 (14 mg, 0.06 mmol,
0.5 equiv) were suspended in ethanol (25 mL) and CHCl3 (5 mL)
and the suspension was stirred vigorously under H2 atmosphere
(1 atm.) overnight. The catalyst was filtered off over celite then
the filtrate was evaporated to dryness to afford compound 6 as a
40.8 (C-
a), 20.4, 20.6, 20.7, 22.8 (CH3). HR-ESI-MS (positive mode):
m/z Calcd for C39H35N4O11 [M+H]+ 735.2297. Found 735.2273.
4.8. (2,3,4,6-Tetra-O-acetyl-b-
D-glucopyranosyl)-di-[3-(p-tolyl)-
white foam (61.5 mg, 99%). 1H NMR (300 MHz, CD3OD):
d
1,2,4-oxadiazol-5-yl]methane (4b)
(ppm) = 5.31 (dd, 1H, J = 9.3, 9.3 Hz, H-3), 5.14 (m, 2H, H-2, H-4),
4.30 (dd, 1H, J = 5.4, 12.6 Hz, H-6a), 4.21 (dd, 1H, J = 2.4, 12.6 Hz,
H-6b), 3.92 (m, 2H, H-1, H-5), 3.32-3.22 (m overlap with CD3OD,
1H, H-b), 2.31 (br s, 1H, H-a), 2.11, 2.07, 2.03, 2.00 (4s, 12H,
COCH3).
Prepared according to general procedure A from 2 (250 mg,
0.63 mmol) to afford 4b (211.5 mg, 50%) as a brown oil. Rf = 0.40
(PE/EtOAc 7:3). ½a D20
ꢂ
ꢀ7.4 (c = 0.50, CHCl3). 1H NMR (400 MHz,

5598
S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
4.12. 2-(2,3,4,6-Tetra-O-acetyl-b-
D
-glucopyranosyl)-1,3-di-(2-
ar), 3.89 (m, 1H, H-6a), 3.75, 3.70, 3.65 (3 m, 3H, H-6, H-b), 3.58
naphthamido)propane (7a)
(dd, 1H, J = 6.0, 13.7 Hz, H-b), 3.50, 3.47, 3.43 (3 m, 3H, H-1 or H-2
or H-3 or H-4, H-b), 3.35 (m overlap with CD3OD, 2H, H-1 or H-2
or H-3 or H-4), 3.28 (m overlap with CD3OD, 1H, H-5), 2.51 (m,
Prepared according to general procedure C from 6 (50 mg,
0.12 mmol) and 2-naphthoyl chloride (92 mg, 0.48 mmol) to afford
7a (33.5 mg, 39%) as a white foam. Rf = 0.05 (EtOAc/PE 3:7).
1H, H-a
). 13C NMR (100 MHz, CD3OD): d (ppm) = 170.5, 170.4 (2s,
CONH), 135.6 (1s, 2C, CIV-ar), 132.7, 129.6, 128.3 (3s, 10C, CH-ar),
82.2 (C-5), 81.0, 80.1, 72.3, 71.8 (4s, C-1, C-2, C-3, C-4), 63.1 (C-6),
½
a 2D0 = +8.7 (c = 0.6, CHCl3). 1H NMR (400 MHz, CDCl3):
d
ꢂ
(ppm) = 8.53 (s, 1H, H-ar), 8.36 (s, 1H, H-ar), 8.15 (m, 1H, NH),
8.00 (dd, 2H, J = 8.0, 25.7 Hz, H-ar), 7.89 (m, 7H, H-ar), 7.57(m,
3H, H-ar), 7.14 (m, 1H, NH), 5.25 (m, 2H, H-2, H-3), 5.12 (m, 1H,
H-4), 4.31 (m, 1H, H-6a), 4.17 (m, 1H, H-6b), 3.77 (m, 1H, H-1),
3.72 (m, 1H, H-5), 4.12, 3.57, 3.34 (3 m, 4H, H-b), 2.13 (m overlap
39.7 (C-
a), 41.1, 38.2 (2s, C-b). HR-ESI-MS (positive mode): m/z
Calcd for C23H29N2O7 [M+H]+ 445.1965. Found 445.1965.
4.16. 5-(2,3,4,6-Tetra-O-acetyl-b-D-
glucopyranosyl)tetrahydropyrimidin-2(1H)-one (9)
with OAc signals, 1H, H-a), 2.16, 2.10, 2.07, 2.01 (4s, 12H, COCH3).
13C NMR (100 MHz, CDCl3): d (ppm) = 170.7, 170.4, 170.3, 169.7
(4s, COCH3), 168.7, 167.7 (2s, CONH), 135.0, 132.9, 132.8, 131.5,
131.3 (5s, 6C, CIV-ar), 129.3, 129.1, 128.64, 128.55, 128.1, 128.0,
127.9, 127.8, 127.7, 127.0, 126.7, 123.8, 123.3 (13s, 14C, CH-ar),
80.2 (C-1), 76.5 (C-5), 74.3 (C-3), 69.4 (C-2), 68.3 (C-4), 62.3 (C-
A solution of bis-amine 6 (50 mg, 0.12 mmol) in freshly distilled
CH2Cl2 (100 mL) was cooled at 0 °C. Then, AlMe3 (0.42 mmol,
3.5 equiv) was added slowly at 0 °C and the mixture was stirred dur-
ing 15 min. 1,10-Carbonyldiimidazole (0.13 mmol, 1.1 equiv) diluted
in CH2Cl2 (2 mL) was then added at room temperature and the mix-
ture was stirred at 45 °C overnight. Water (50 mL) was added slowly
at room temperature and the aqueous layer was extracted with
CH2Cl2 (3 ꢁ 150 mL). The organic layers were combined, washed
with saturated NaCl (5 ꢁ 30 mL), dried (MgSO4), filtered and evapo-
rated to dryness. The residue was purified by flash silica gel column
chromatography (EtOAc/MeOH 9:1) to give compound 9 as a white
6), 39.0 (C-a), 36.2, 40.2 (2s, C-b), 20.9, 20.8, 20.7 (3s, 4C, CH3).
HR-ESI-MS (positive mode): m/z Calcd for C39H41N2O11 [M+H]+
713.2705. Found 713.2704.
4.13. 2-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-1,3-
dibenzamidopropane (7b)
foam (21 mg, 38%). Rf = 0.21 (EtOAc/MeOH 85:15). ½a D20
ꢀ13.0
ꢂ
Prepared according to general procedure C from 6 (50 mg,
(c = 0.4, CHCl3). 1H NMR (400 MHz, CDCl3): d (ppm) = 5.23 (br s,
1H, NH), 5.18 (dd, 1H, J = 9.2, 9.6 Hz, H-3), 4.99 (m, 2H, H-2, H-4),
4.93 (br s, 1H, NH), 4.18 (dd, 1H, J = 2.3, 9.9 Hz, H-6a), 4.08 (dd, 1H,
J = 5.0, 9.9 Hz, H-6b), 3.61 (ddd, 1H, J = 2.3, 5.0, 9.9 Hz, H-5), 3.52
(m, 1H, H-1), 3.37 (m, 2H, H-b), 3.20 (m, 2H, H-b), 2.11 (m overlap
0.12 mmol) and benzoyl chloride (56
lL, 0.48 mmol) to afford 7b
(51.5 mg, 70%) as a white foam. Rf = 0.76 (EtOAc). ½a D20
ꢀ3.8
ꢂ
(c = 0.4, CHCl3). 1H NMR (400 MHz, CDCl3): d (ppm) = 7.95 (m,
2H, H-ar), 7.89 (m, 1H, NH), 7.80 (m, 2H, H-ar), 7.48 (m, 6H, H-
ar), 6.90 (m, 1H, NH), 5.20 (m, 2H, H-1, H-3), 5.10 (m, 1H, H-4),
4.26 (dd, 1H, J = 4.8, 12.5 Hz, H-6a), 4.11 (m, 1H, H-6b), 3.73 (m,
1H, H-2), 3.69 (ddd, 1H, J = 4.8, 7.1, 9.9 Hz, H-5), 4.03, 3.47, 3.22
with OAc signals, 1H, H-a), 2.10, 2.07, 2.04, 2.03 (4s, 12H, COCH3).
13C NMR (100 MHz, CDCl3): d (ppm) = 170.7, 169.6 (2s, 4C, COCH3),
156.8 (NHCONH), 76.6 (C-1), 76.2 (C-5), 74.4 (C-3), 69.6 (C-2), 68.4
(3 m, 4H, H-b), 2.10 (m overlap with OAc signals, 1H, H-
a), 2.09,
(C-4), 62.2 (C-6), 42.7, 39.5 (2s, C-b), 32.6 (C-a), 20.9, 20.7 (2s, 4C,
2.05, 2.02, 1.95 (4s, 12H, COCH3). 13C NMR (100 MHz, CDCl3): d
(ppm) = 170.7, 170.33, 170.30, 169.7 (4s, COCH3), 168.7, 167.7
(2s, CONH), 134.3, 134.2 (2s, CIV-ar), 132.0, 131.7, 128.8, 128.7,
127.3, 127.0 (6s, 10C, CH-ar), 80.3 (C-2), 76.4 (C-5), 74.3 (C-3),
CH3). HR-ESI-MS (positive mode): m/z Calcd for C18H27N2O10
[M+H]+ 431.1660. Found 431.1663.
4.17. 5-(b-D-Glucopyranosyl)tetrahydropyrimidin-2(1H)-one
69.4 (C-1), 68.3 (C-4), 62.3 (C-6), 38.7 (C-
a
), 35.9, 39.6 (2s, C-b),
(10)
20.7, 20.8, 20.9 (3s, 4C, CH3). HR-ESI-MS (positive mode): m/z Calcd
for C31H37N2O11 [M+H]+ 613.2392. Found 613.2385.
Prepared according to general procedure D from 9 (10 mg,
0.02 mmol) to afford 10 (6 mg, 99%) as a white foam. Rf = 0.66
4.14. 2-(b-
D
-Glucopyranosyl)-1,3-di-(2-naphthamido)propane
(CH2Cl2/MeOH/H2O 6:4:0.5). ½a D20
ꢂ
ꢀ0.5 (c = 0.2, CH3OH). 1H NMR
(8a)
(500 MHz, CD3OD): d (ppm) = 3.82 (dd, 1H, J = 1.9, 12.0 Hz, H-6a),
3.60 (dd, 1H, J = 5.5, 12.0 Hz, H-6b), 3.42 (m, 1H, H-b), 3.33 (m,
2H, H-b, H-1 or H-2 or H-3 or H-4 or H-5), 3.29 (m, 2H, H-b),
Prepared according to general procedure D from 7a (33.5 mg,
0.05 mmol) to afford 8a (7.3 mg, 29%) as a white foam. Rf = 0.90
3.19 (m, 4H, H-1 or H-2 or H-3 or H-4 or H-5), 2.34 (m, 1H, H-a).
(CH2Cl2/MeOH/H20 4:6:0.5). ½a D20
ꢂ
= +5.8 (c = 0.14, CH3OH). 1H NMR
13C NMR (125 MHz, CD3OD): d (ppm) = 159.7 (C=O), 82.0, 80.0,
(400 MHz, CD3OD): d (ppm) = 8.41 (d, 2H, J = 4.9 Hz, H-ar), 7.94
(m, 8H, H-ar), 7.56 (m, 4H, H-ar), 3.94 (d, 1H, J = 11.3 Hz, H-6a),
3.84 (m, 2H, H-b), 3.70 (m, 2H, H-b, H-6b), 3.59 (dd, 1H, J = 7.1,
14.1 Hz, H-b), 3.53 (m, 2H, H-1 or H-2 or H-3 or H-4 or H-5), 3.37
(m overlap with CD3OD, 3H, H-1 or H-2 or H-3 or H-4 or H-5), 2.61
79.6, 73.2, 71.7 (5s, C-1, C-2, C-3, C-4, C-5), 63.1 (C-6), 43.6, 40.7
(2s, C-b), 34.1 (C-
C
a). HR-ESI-MS (positive mode): m/z Calcd for
10H19N2O6 [M+H]+ 263.1238. Found 263.1235.
4.18. Determination of inhibition of RMGPb
(m, 1H, H-a
). 13C NMR (100 MHz, CD3OD): d (ppm) = 170.5 (1s, 2C,
CONH), 136.3, 134.1, 132.8 (3s, 6C, CIV-ar), 130.09, 130.05, 129.4,
129.2, 128.82, 128.80, 128.75, 127.8, 124.9 (9s, 14C, CH-ar), 82.3,
81.2, 80.1, 72.3, 71.8 (5s, C-1, C-2, C-3, C-4, C-5), 61.1 (C-6), 39.7
RMGPb was isolated from rabbit skeletal muscle according to
Fischer & Krebs,27 using 2-mercaptoethanol. Kinetic experiments
at different inhibitor concentrations were conducted in the direc-
tion of glycogen synthesis, at 30 °C in the presence of 2 mM glu-
cose-1-phosphate (G-1-P) and 1 mM AMP. The phosphate
released was calculated with Fiske & Subbarow28 and Saheki, Tak-
eda & Shimazu29 methods.
(C-
a), 41.5, 34.9 (2s, C-b). HR-ESI-MS (positive mode): m/z Calcd
for C31H33N2O7 [M+H]+ 545.2282. Found 545.2280.
4.15. 1,3-Dibenzamido-2-(b-D-glucopyranosyl)propane (8b)
Prepared according to general procedure D from 7b (50 mg,
0.08 mmol) to afford 8b (7.5 mg, 53%) as a white foam. Rf = 0.85
4.19. Docking calculations
(CH2Cl2/MeOH/H20 4:6:0.5). ½a D20
ꢂ
ꢀ6.7 (c = 0.34, CH3OH). 1H NMR
In silico studies were performed using SCHRÖDINGER Suite
(400 MHz, CD3OD): d (ppm) = 7.86 (m, 4H, H-ar), 7.49 (m, 6H, H-
2011.30 The crystal structure of RMGPb in complex with the C-(1-

S. Feuillastre et al. / Bioorg. Med. Chem. 20 (2012) 5592–5599
5599
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was initially selected as a starting model. The protein was pre-trea-
ted using Protein Preparation Wizard. Water molecules lying at a
distance beyond 5 Å of the ligand were deleted. Bond orders were
assigned, hydrogens were added and assigned with an exhaustive
sampling including water orientations. The system was optimized
with minimization procedure using convergence criterion RMSD of
0.3 Å to heavy atoms (Force field OPLS2005). The crystal structure
of RMGPb in complex with the inhibitor 2-naphthoyl urea B9,25 was
subsequently used as an initial model in which the 280s loop is
subjected to extended shifts to allow binding of bulkier ligands
at the catalytic site. The protein was pre-treated as previously de-
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were performed using GLIDE module (v. 5.7) in extra-precision
(XP) mode. Core constraint was applied on the ligand to ‘lock’
the glucopyranose ring atoms. Best docking poses were re-docked
by applying quantum polarized ligand docking procedure (QPLD-
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Financial supports from CNRS, University Claude-Bernard Lyon
1 and the French Agence Nationale de Recherche (project GPdia
N° ANR-08-BLAN-0305) are gratefully acknowledged. This work
was supported by the FP7 Capacities coordination and support ac-
tions REGPOT-2008-1-No 230146 ‘EUROSTRUCT’ and REGPOT-
2009-1-No 245866 ‘ARCADE’. Dr. F. Albrieux, C. Duchamp and N.
Henriques are gratefully acknowledged for mass spectrometry
analyses.
Supplementary data
Supplementary data (1H and 13C NMR spectra for compounds 2,
4a–b, 5a–b, 6, 7a–b, 8a–b, 9 and 10) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2012.07.033.
References and notes
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