Synthesis of and a comparative study on the inhibition of muscle and liver glycogen phosphorylases by epimeric pairs of D-gluco- and D-xylopyranosylidene-spiro-(thio)hydantoins and N-(D-glucopyranosyl) amides

Article abstract of DOI:10.1021/jm010892t

D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the key step of the syntheses C-(1-bromo-1-deoxy-β-D-glycopyranosyl)formami

Full text of DOI:10.1021/jm010892t

J . Med. Chem. 2001, 44, 2843-2848
2843
Syn th esis of a n d a Com p a r a tive Stu d y on th e In h ibition of Mu scle a n d Liver
Glycogen P h osp h or yla ses by Ep im er ic P a ir s of D-Glu co- a n d
D-Xylop yr a n osylid en e-sp ir o-(th io)h yd a n toin s a n d N-(D-Glu cop yr a n osyl) Am id es
La´szlo´ Somsa´k,*,† La´szlo´ Kova´cs,† Marietta To´th,† Erzse´bet ?Osz,† La´szlo´ Szila´gyi,† Zolta´n Gyo¨rgydea´k,†
Zolta´n Dinya,† Tibor Docsa,‡ Be´la To´th,‡ and Pa´l Gergely‡
Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary, and
Department of Medical Chemistry, University of Debrecen, H-4026 Debrecen, Bem te´r 18/ B, Hungary
Received April 2, 2001
D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from
the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the
key step of the syntheses C-(1-bromo-1-deoxy-â-D-glycopyranosyl)formamides were reacted with
cyanate ion to give spiro-hydantoins with a retained configuration at the anomeric center as
the major products. On the other hand, thiocyanate ions gave spiro-thiohydantoins with an
inverted anomeric carbon as the only products. On the basis of radical inhibition studies, a
mechanistic rationale was proposed to explain this unique stereoselectivity and the formation
of C-(1-hydroxy-â-D-glycopyranosyl)formamides as byproducts. Enzyme assays with a and b
forms of muscle and liver glycogen phosphorylases showed spiro-hydantoin 12 and spiro-
thiohydantoin 14 to be the best and equipotent inhibitors with Ki values in the low micromolar
range. The study of epimeric pairs of D-gluco and D-xylo configurated spiro-hydantoins and
N-(D-glucopyranosyl)amides corroborated the role of specific hydrogen bridges in binding the
inhibitors to the enzyme.
In tr od u ction
glycogen phosphorylase are weakly hypoglycemic, the
concept of inhibition of this enzyme as a potential tool
for controlling hyperglycemia was put forward.3
To find potent glucose analogue inhibitors of glyco-
gen phosphorylase, extensive synthetic,3-6 crystallo-
graphic,7-11 molecular modeling,3,8,12-14 and enzymo-
logical3-5,7,12,14 studies have been carried out. As a result
of these investigations, glucopyranosylidene-spiro-hy-
dantoin 12 emerged as the strongest glucose analogue
inhibitor of muscle glycogen phophorylase b known to
date.4 However, the published synthetic routes to 12
were rather lengthy on one hand or gave the much less
efficient inhibitor 20 as the major product on the
other.4-6
In this paper we disclose full experimental details of
a simple, short, and highly chemo-, regio-, and stereo-
selective procedure for the preparation of D-glucopyra-
nosylidene-spiro-(thio)hydantoins,15 which was also ap-
plied to the synthesis of their D-xylo configurated
analogues. Kinetic data obtained for the first time with
liver glycogen phosphorylases for glucose analogue
inhibitors demonstrate that such compounds may pos-
sess hypoglycemic activity. Furthermore, investigation
of D-xylo configurated compounds as well as other N-
and C-glycosyl amide derivatives shed light on the
importance of some particular H bonds between the
enzyme and the inhibitor.
Diabetes mellitus and its complications afflict 3% of
the population of Europe and North America and 140
million people worldwide.1 The disease is characterized
by chronic elevated blood sugar levels for which several
reasons like defects of insulin action in tissues or
impairment of pancreatic insulin secretion may be
responsible. Excessive hepatic glucose production can
also significantly contribute to diabetic hyperglycemia.
The non-insulin-dependent diabetes mellitus (NIDDM
or type II diabetes) appears in ∼75% of the diabetic
patients. Hyperglycemia in type II diabetics can be
controlled by dietary regulation and exercise, which can
be combined with the use of oral hypoglycemic agents.
However, this treatment is not entirely satisfactory
because blood glucose concentration cannot be regulated
as efficiently as under normal physiological conditions.
This can result in complications occurring in the later
life of the diabetic patient.2 Therefore, there is an
obvious need for novel agents and/or therapeutic strate-
gies that could act more closely to the physiological
regulation of blood sugar level.
Hepatic glucose output is regulated by a complex
system of enzymes that is under hormonal, neuronal,
and metabolic control. The main regulatory enzyme of
this system is glycogen phosphorylase, which catalyzes
the first step of glycogen breakdown. The inhibition of
this enzyme may help to shift the balance between
glycogen degradation and synthesis in favor of the
latter. On the basis of the fact that weak inhibitors of
Resu lts a n d Discu ssion
Syn th esis of D-Glycop yr a n osylid en e-sp ir o-(th io)-
h yd a n t oin s a n d N-D-Glu cop yr a n osyla m id es. The
spiro heterocycles were synthesized by adapting and
improving our recently published protocol for the prepa-
ration of D-galacto- and D-arabinopyranosylidene-spiro-
* To whom correspondence should be addressed. Phone: +36-52-
512-900/2348. Fax: +36-52-453-836. E-mail: somsak@tigris.klte.hu.
† Department of Organic Chemistry.
‡ Department of Medical Chemistry.
10.1021/jm010892t CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/20/2001

2844 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Somsa´k et al.
Sch em e 1. Preparation of Glycopyranosylidene-
the yield of 13 to 79%, and only 4% of 5 was obtained;
the respective yields for 17 and 8 were 64% and 15%.
See the mechanistic proposal below to rationalize the
use of the above conditions.
spiro-(thio)hydantoinsa
The acetylated compounds 5, 8, 11, 13, 15, 17, 19,
and 21 were deprotected by the Zemple´n procedure to
give 6, 9, 12, 14, 16, 18, 20, and 22, respectively, in high
yields.
N-Acyl-D-glucopyranosylamines 27,18 28,19 29, and 30
were obtained from the corresponding per-O-acetylated
compounds20 by the Zemple´n protocol.
Str u ctu r e Elu cid a tion of th e New Com p ou n d s.
The structure of the new compounds was unequivocally
established from NMR data. The presence of the car-
boxamido group in 4-9 was indicated by two broad
singlets in the proton spectra and a carbon resonance
around 167-170 ppm. An additional broad singlet was
characteristic for the free hydroxyl group in 5 and 8.
For each hydantoin the 13C NMR spectra exhibited
two resonances at chemical shifts characteristic21-23 of
the “amide” [δC-10 167-175 ppm], the “urea” [δC-8 155-
160 ppm], or the “thiourea” [δC-8 182-186 ppm] type
1
of (thio)carbonyl carbons. The H chemical shifts of the
NH protons are similar to those found in simple (thio)-
hydantoins,24,25 the H-9 resonances appearing at lower
field (10.4-10.8 ppm in hydantoins; 11.4-11.9 ppm in
thiohydantoins) than those for H-7 (7.7-8.4 ppm in
hydantoins; 9.8-10.5 ppm in thiohydantoins). Long-
range 13C-1H or 15N-1H connectivities detected in the
respective HMBC spectra have provided further confir-
mation of the spiro-(thio)hydantoin structures as fol-
low: 13C-1H correlations through two bonds, C-6/
NH-7, C-8/NH-7, C-8/NH-9, C-10/NH-9; 13C-1H cor-
relations through three bonds: C-6/NH-9, C-10/H-5,
C-10/NH-7; and 15N-1H correlations through three
bonds, N-7/H-5, N-7/NH-9, and N-9/NH-7.
a
(a) TiCl4, H2O (1 equiv), AcOH, 0 °C f room temp; (b) Ag2O,
H2O (1 equiv), DMSO, room temp; (c) NaOMe (cat.), MeOH, room
temp; (d) AgOCN (4 equiv), CH3NO2, 80 °C; (e) KSCN (4 equiv),
S8, CH3NO2, 80 °C, N2 atm.
The conformations of the pyranose rings were shown
to be 4C1 by vicinal 1H-1H coupling constants (see
Supporting Information) in all cases. This ensures that
H-2 or H-5 atoms on the pyranose rings are in the axial
position. Therefore, the configurations of the anomeric
carbons (C-1 or C-6) could be deduced from the values
of heteronuclear three-bond coupling constants16,26
(thio)hydantoins.16 Thus, acetylated 1-bromo-1-deoxy-
â-D-glycopyranosyl cyanides (1 or 2, Scheme 1) readily
obtained by radical-mediated bromination17 from per-
O-acetylated â-D-glycopyranosyl cyanides were treated
with TiCl4 in AcOH in the presence of 1 equiv of H2O
to give, after chromatographic purification, C-(1-bromo-
1-deoxy-â-D-glycopyranosyl)formamides 4 (68%) and 7
(66%). Reaction of 4 with AgOCN in CH3NO2 at 80 °C
gave spiro-hydantoins 11 (4%) and 19 (25%) together
with hydroxyamide 5 (53%). Similar reaction of 7 gave
spiro-hydantoins 15 and 21 and hydroxyamide 8 in a
1:1:2 ratio (1H NMR). Under the same conditions as
those for a mixture of 7 and 10 obtained by partial
hydrolysis of 2 and 3, the product mixture of the
photobromination 15, 21, and 8 were isolated in 7%,
15%, and 27% yield, respectively. Compounds 5 (85%)
and 8 (83%) were also prepared from 4 and 7, respec-
tively, by Ag2O-promoted hydrolysis in DMSO in the
presence of 1 equiv of H2O. When 4 or 7 was reacted
with AgSCN or KSCN in CH3NO2 at 80 °C, spiro-
thiohydantoins 13 (57%) and 17 were the only cyclized
products accompanied by 5 (19%) and 8, respectively.
Carrying out these reactions in the presence of a small
amount of elemental sulfur under nitrogen atmosphere
in order to suppress radical-mediated pathways raised
3
3J H-2,CONH2 and J H-5,C-10. The configurational assign-
ments were supported by 1H chemical shift rules
recently established for spiro-hydantoins.16
A Mech a n istic P r op osa l. The main peculiarities of
the (thio)hydantoin forming reactions in the D-gluco and
D-xylo configurations described in this work, as well as
in the D-galacto and D-arabino ones,16 can be sum-
marized as follows: (1) the stereoselectivity of the ring-
closing step at the anomeric center depends on the
nucleophile, while with silver cyanate the highly selec-
tive formation of a retention product (like 19 and 21)
was observed; ring closure with both silver and potas-
sium thiocyanate gave thiohydantoins (like 13 and 17)
with inversion of the anomeric configuration exclusively.
(2) The formation of C-(1-hydroxy-D-glycosyl)formamides
(like 5 and 8) was unavoidable in each of these trans-
formations.
To rationalize these observations and to improve the
selectivity of the reactions, numerous modifications and
inhibition experiments were carried out (see Supporting
Information) and we propose the following mechanism

Inhibition of Muscle and Liver Glycogen Phosphorylases
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2845
oxygen.30-32 Formation of D on this pathway is not a
chain reaction; therefore, small amounts of radical traps
do not exert very efficient inhibition.30
Sch em e 2. Proposed Mechanism of
Glycopyranosylidene-spiro-(thio)hydantoin Formation
As a result of these studies, we have obtained tools
for suppressing hydroxyamide formation, as was dem-
onstrated by the enhanced yield for the D-glycopyrano-
sylidene-spiro-thiohydantoins 13 and 17 (see above).
Kin etic Stu d ies. The kinetic studies with glycogen
phosphorylases were performed as described previ-
ously.15 Inhibitor constants (Ki) determined with muscle
glycogen phosphorylase b for compounds with D-glucose
or D-xylose skeletons are given in Table 1. The glucopy-
ranosylidene-spiro-hydantoin 12 and its 8-thio analogue
14 have been identified as the highest affinity inhibitors
of muscle GPb4,15 (entries 1 and 2). These findings are
supported by X-ray studies: analysis of the structure
of muscle GPb-12 complex pointed out the significance
in binding of a H bond between N-7-H of the hydantoin
ring and the peptide backbone oxygen of His377;9 this
important H bridge has been shown to be present in
the muscle GPb-14 complex as well.33 The epimeric 20
is obviously devoid of this interaction with the enzyme,
and in agreement with published data,5 it proved to be
a less efficient inhibitor (entry 3). The importance of this
H bond to the protein is further corroborated by the loss
of inhibition in the R-configurated 26 compared to its
â-anomer 25 (entries 6 and 7). Such a comparison was
not possible earlier because of the lack of a suitable
synthetic method20 to produce N-(R-D-glycosyl) amides.
The presence of the trifluoromethyl group in 25 caused
a significant weakening in the inhibition compared to
that in 23 (entry 4). The reason for this is not obvious:
CH3 and CF3 groups are similar in size; the H-bond
donating capacity of the NH moiety in the trifluoro-
acetamido group can be higher than that of the acet-
amido group, and this might predict a stronger binding;
the hydrophobicity is much higher for the CF3 moiety34
with respect to the CH3 group; however, the presence
of the much more hydrophobic phenyl group in 24 (entry
5) led to a smaller decrease of the inhibitory activity.
Further studies to clear this point are in progress.
Hydroxyamide 6 proved to be a weak inhibitor (entry
8). This seems to be surprising when compared to R-D-
glucose (28) and â-carboxamide 29, which show stronger
inhibition and have the hydroxyl and the carboxamido
groups in the same position as in 6 (entries 10 and 11,
respectively). The joint presence of these moieties at the
anomeric center is clearly highly detrimental for the
binding. A possible reason for this may be an intramo-
lecular H bond between the anomeric OH and CONH2
groups in 6. A similar intramolecular H bond has
recently been observed in C-(1-azido-1-deoxy-D-glyco-
pyranosyl)formamides in solution35 and in the solid
phase.36 Such a H bond may alter the conformational
position of the groups involved, thereby preventing
formation of favorable interactions between these groups
and the protein, which are present in the complexes
GPb-283 or GPb-29.7 This observation also needs
further investigations. Changing the configuration of the
sugar ring from D-gluco to D-xylo by removal of the
hydroxymethyl side chain brought about a decrease of
4 orders of magnitude in the inhibition (entries 13-15).
This is in keeping with earlier data obtained with
6-deoxy-D-glucose37 and clearly indicates the importance
(Scheme 2). A highly probable general reaction that may
proceed independent of solvent and cation can be started
by a single-electron transfer (SET) from (thio)cyanate
to bromoamide A to give radical anion I. From I a
bromide can split off to give radical II, which may
combine with the SCN• radical in the solvent cage,27
followed by a tautomeric ring closure furnishing C. This
route is indistinguishable from a bimolecular substitu-
tion, and C (X ) S) may indeed be formed by the
classical SN2 route (A f C) with complete inversion. If
radical II escapes from the solvent cage, it may react
with a cyanate ion to give IV. Because of well-known
stereoelectronic reasons,28 axial attack of the OCN- ion
may be favored as shown in III (major) leading to B
after ring closure. Isomers C can also be formed in these
reactions via III (minor) as byproducts. Oxidation of
radical anion IV may proceed by the starting material
A, thereby giving rise to a chain reaction. This is in
keeping with the finding that significantly lower than
stoichiometric amounts of radical traps or radical anion
traps could completely inhibit the reaction. A similar
mechanism was proposed for the formation 1-azido-1-
deoxy-D-glycopyranosyl cyanides.26
Out of the solvent cage radical II may also be trapped
by triplet oxygen to give the hydroperoxyl radical V.
This can then abstract a hydrogen from the solvent and
undergo known thermal decomposition29 to D. This
possibility is corroborated by several reports on the
formation of alcohols via radicals trapped by molecular

2846 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Somsa´k et al.
Ta ble 1. Inhibitor Constants (Ki) Determined for Muscle Glycogen Phosphorylase b
anomeric substituent
compound
entry
1
sugar skeleton
no.
R
â
Ki [µM]
3.1
ref
4
12
CONH-CONH
4.2
5.1
320
105
this work
this work
5
2
3
14
20
CONH-CSNH
NHCO-NHCO
this work
4
5
23
24
H
H
H
NHCOCH3
NHCOC6H5
32
31
81
144
8
this work
8
this work
this work
this work
this work
3
3
7
7
6
7
8
25
26
6
27
28
29
30
NHCOCF3
H
CONH2
OH
H
CONH2
H
710
NHCOCF3
OH
H
OH
H
CONH2
no inhibition
3100
7400
1700
440
9
10
11
12
370
13
16
CONH-CONH
11500
this work
14
15
16
18
22
9
CONH-CSNH
NHCO-NHCO
CONH2
>10000a
this work
this work
this work
>10000a
OH
no inhibition
a
Because of insufficient sample quantity, no precise value could be obtained.
uncorrected. Optical rotations were determined with a Perkin-
Elmer 241 polarimeter at room temperature. NMR spectra
were recorded with Bruker WP 200 SY (200/50 MHz for 1H/
13C) or Avance DRX 500 (500/125/50 MHz for 1H/13C/15N)
spectrometers. Chemical shifts are referenced to Me4Si (1H),
to the residual solvent signals (13C), or to NH4Cl as external
standard (15N). Fast-atom bombardment (FAB) mass spectra
were obtained using a VG-7070MS mass spectrometer. TLC
was performed on DC-Alurolle Kieselgel 60 F254 (Merck), and
the plates were visualized by gentle heating. For column
chromatography Kieselgel 60 (Merck, particle size 0.063-0.200
mm) was used. Organic solutions were dried over anhydrous
MgSO4 and concentrated in vacuo at 40-50 °C (water bath).
Nitromethane was distilled from P4O10 directly in the reaction
flask or was stored over 3 Å molecular sieves.
in binding of an optimal hydrogen bond between the side
chain of His377 in the enzyme and the oxygen of the
hydroxymethyl side chain in the inhibitor revealed by
X-ray studies.9,33
Potential antidiabetic compounds should exert their
inhibitory action on the active a form of glycogen
phosphorylase, and especially important is their effect
on the hepatic enzymes. For the best inhibitors Ki values
[µM] were also determined with liver glycogen phos-
phorylases b (12, 12.8 ( 1.1; 14, 7.0 ( 1.0) and a (12,
16.5 ( 1.3; 14, 29.8 ( 2.9) as well as with muscle
phosphorylase a (12, 26.0 ( 2.4; 14, 10.9 ( 1.0) to show
similar potency for both compounds.15
Gen er a l P r oced u r e I for th e P r ep a r a tion of C-(P er -
O -a c e t y l-1-b r o m o -1-d e o x y -D -g ly c o p y r a n o s y l)fo r m -
a m id es (4 or 7). To a suspension of a glycosyl cyanide (1 or
2) in AcOH (1-1.5 mL/mmol), cooled in an ice bath, were added
with stirring TiCl4 (2-4 equiv) and H2O (1 equiv). After 30
min the ice bath was removed and the mixture was stirred
for 6 days at room temperature. Then the solution was poured
with continuous stirring into a mixture of ice and water and
the crude product was extracted with CHCl3 (3×). The
combined extracts were washed with cold saturated NaHCO3
solution and H2O and dried, and the solvent was evaporated.
The crude product was sufficiently pure for further transfor-
mations.
Gen er a l P r oced u r e II for th e P r ep a r a tion of C-(P er -
O-a cetyl-1-h yd r oxy-D-glycop yr a n osyl)for m a m id es (5 or
8). To a solution of 4 or 7 in dry DMSO (18 mL/mmol) were
added with stirring Ag2O (1 equiv) and H2O (1 equiv). The
reaction mixture was stirred at room temperature until
disappearance of the starting material (TLC, eluent: EtOAc/
hexane 1:1). After filtration the solvent was evaporated under
reduced pressure and the residue was dissolved in EtOAc and
passed through a 3 cm layer of silica gel. Evaporation of the
solvent left a syrupy crude product processed as given for the
particular compounds.
Con clu sion
The synthetic procedure described in this paper allows
the stereoselective preparation of glycopyranosylidene-
spiro-(thio)hydantoins in six steps from the correspond-
ing free sugar. Although the peculiar stereoselectivity
of the hydantoin-forming reaction disfavored obtention
of the efficient glycogen phosphorylase inhibitor 12, the
high-yielding preparation of its thio analogue 14 pro-
vides an easy access to an equipotent inhibitor of both
b and a forms of muscle and liver glycogen phosphory-
lases. Further improvements38 of the synthetic sequence
have facilitated acquisition of 14 in gram quantities.
This has allowed us to demonstrate that 14 is effective
in inhibiting glycogen phosphorylase a activity in he-
patic cells both in vitro and in vivo,39 indicating that
this compound and its analogues can be useful bio-
chemical tools for the study of the importance of
glycogen phosphorylases in the regulation of hypergly-
cemia.
Gen er a l P r oced u r e III for th e P r ep a r a tion of P er -O-
a cetyla ted d -glycop yr a n osylid en e-sp ir o-(th io)h yd a n to-
in s 11, 13, 15, 17, 19, 21. To a solution of 4 or 7 in dry CH3NO2
(15 mL/mmol) were added molecular sieves (3 Å) and an
Exp er im en ta l Section
Gen er a l Con sid er a tion s. Melting points were measured
in open capillary tubes or on a Kofler hot stage and are

Inhibition of Muscle and Liver Glycogen Phosphorylases
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2847
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MXCN salt (4 equiv of KSCN or freshly prepared dry AgSCN
or AgOCN). The reaction mixture was stirred at 80 °C under
nitrogen atmosphere for 3-4 h. After filtration and evapora-
tion of the solvent the crude product was purified by column
chromatography using ethyl acetate/hexane (1:2 to 1:1) as
eluent.
Gen er a l P r oced u r e IV for th e Rem ova l of Acetyl
P r otectin g Gr ou p s. To the solution of an acetylated com-
pound in dry MeOH (10-15 mL/mmol) were added a few drops
of a 1 M methanolic NaOMe solution, and the mixture was
stirred at room temperature until disappearance of the start-
ing material (TLC, eluent: chloroform/methanol 1:1). After
neutralization with a cation-exchange resin (H+ form) and
filtration, the solvent was removed to furnish the product,
which was further purified if necessary.
Ack n ow led gm en t. This work was supported by the
Hungarian Scientific Research Fund (Grants OTKA
T23138, T26541, T29024, T32124), the Ministry of
Education (Grant FKFP 423/2000), the Ministry of
Health (Grant ETT 01/2000), and the Zsigmond Diabe-
tes Foundation.
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Amides: A Combined Study by NMR Spectroscopy, X-Ray
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of Ammonia with Some Acetylated and Benzoylated Monosac-
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395-404.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR chemical
shift rules for the determination of configuration in the spiro-
hydantoins, modifications of the reaction conditions and
inhibition experiments supporting the mechanistic proposal,
experimental data (compound characterization, details of
inhibition experiments, and enzyme assays), and additional
references. This material is available free of charge via the
Internet at http://pubs.acs.org.
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