Bioactivity of glycogen phosphorylase inhibitors that bind to the purine nucleoside site

Article abstract of DOI:10.1016/j.bmc.2006.07.060

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor s

Full text of DOI:10.1016/j.bmc.2006.07.060

Bioorganic & Medicinal Chemistry 14 (2006) 7835–7845
Bioactivity of glycogen phosphorylase inhibitors that bind
to the purine nucleoside site
Laura J. Hampson,a Catherine Arden,a Loranne Agius,a,* Minas Ganotidis,b,c
Magda N. Kosmopoulou,b Costas Tiraidis,b Yiannis Elemes,c Constantinos Sakarellos,c
Demetres D. Leonidasb and Nikos G. Oikonomakosb,d,
*
aSchool of Clinical Medical Sciences-Diabetes, Newcastle University, The Medical School, Newcastle upon Tyne NE2 4HH, UK
bInstitute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation,
48, Vas. Constantinou Ave. 116 35 Athens, Greece
cDepartment of Chemistry, University of Ioannina, Ioannina 45110, Greece
dInstitute of Biological Research and Biotechnology, The National Hellenic Research Foundation,
48, Vas. Constantinou Ave. 116 35 Athens, Greece
Received 4 April 2006; revised 25 July 2006; accepted 28 July 2006
Abstract—The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site
and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site
has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavo-
piridol (1–4) that bind to the purine site. Flavopiridol and 1–4 counteracted the activation of phosphorylase in hepatocytes caused
by AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole
carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rath-
er than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they coun-
teracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase.
Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles
of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on
glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
peutic strategy for controlling blood glucose in Type-2
diabetes.1
Increased hepatic glucose production by glycogenolysis
and gluconeogenesis is a contributing factor to the
hyperglycaemia in Type-2 diabetes. Inhibition of hepatic
glycogen phosphorylase, the flux-generating enzyme in
glycogenolysis, is therefore considered a potential thera-
Hepatic glycogen phosphorylase is regulated by covalent
modification (phosphorylation of a serine residue at the
N-terminus) and by allosteric mechanisms.2 The
dephosphorylated form of liver glycogen phosphorylase
(phosphorylase-b) is essentially inactive in liver in vivo
and only the phosphorylated form (phosphorylase-a)
contributes to glycogenolysis.3 Phosphorylation is catal-
ysed by phosphorylase kinase which is activated by
cAMP and elevated calcium ion concentration and
dephosphorylation is catalysed by the catalytic unit of
protein phosphatase-1 in association with glycogen tar-
geting proteins.4 Phosphorylase exists as either a relaxed
(R) or a tense (T) conformation representing active and
inactive conformations. Phosphorylation and certain
ligands (AMP, phosphate and glucose 1-P) favour the
Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide 1-b-D-ribo-
furanoside; DAB, 1,4-dideoxy-1,4-imino-D-arabinitol; DMEM, Dul-
becco’s modified Eagle’s medium; glucose 1-P, glucose 1-phosphate;
glucose 6-P, glucose 6-phosphate; MEM, Minimum essential medium;
ZMP, 5-aminoimidazole-4-carboxamide 1-b-D-ribofuranotide.
Keywords: Type-2 diabetes; Glycogen phosphorylase; Flavopiridols;
Inhibitors; Glycogen metabolism; Hepatocytes.
*
Corresponding authors. Tel.: +44 191 2227033; fax: +44 191
2220723. (L.A.); tel.: +30 210 7273 884; fax: +30 210 7273831
(N.G.O.); e-mail addresses: Loranne.agius@ncl.ac.uk; ngo@eie.gr
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.060

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L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
O
OH
OH
O
O
R-conformation, whereas glucose and glucose 6-P fa-
vour the T-conformation, which is a better substrate
for dephosphorylation by protein phosphatase-1.5–7
R
HO
R
HO
O
In the liver the phosphorylated form of phosphorylase is
a potent inhibitor of glycogen synthase phosphatase
through interaction with a high-affinity allosteric site
on the glycogen targeting protein GL, which binds pro-
tein phosphatase-1.2 Depletion of phosphorylase-a by
dephosphorylation induced by glucose, glucose 6-P or
pharmacological ligands that favour the T-conforma-
tion, results in activation of glycogen synthase through
reversal of the inhibition of glycogen synthase phospha-
tase.8–10
OH
R= 2-Cl Phenyl
1: R= 2-Cl Phenyl
2: R= 3-Cl Phenyl
3: R= 4-Cl Phenyl
4: R= Phenyl
N
N
CH3
CH3
Figure 1. Flavopiridol (left) and its olefin analogues.
chloride catalysed by DMAP gave the ester 7a–d,
(Fig. 2). Reaction of the derivative 7 with NaH/THF
and H2SO4/AcOH provided the dimethoxy flavone 8a–
d, (Fig. 2). These were completely demethylated to the
desired final products, 1–4, upon treatment with pyri-
dinium hydrochloride. The alternative synthetic route
starting with 9,25 has been followed as well (Scheme
2). However, acid-catalysed condensation between 3,5-
dimethoxy phenol and 1-methyl-4-piperidone afforded,
in addition to the desired compound, 9, the product of
double substitution at the phenyl ring, 10, in a 19% yield
(Scheme 3).
Various ligand binding sites have been identified from
crystallography of either rabbit muscle or human liver
phosphorylase that bind physiological or pharmacolog-
ical ligands, they include the catalytic site, an allosteric
AMP-activator site, a purine nucleoside inhibitor site
(caffeine-binding site), an indole carboxamide site and
a glycogen storage site.5,11,13 The biological actions in
hepatocytes of high-affinity ligands of the catalytic site,
the activator site and the indole carboxamide site have
been reported.8–10,14–17 Although the caffeine-binding
site is well characterised from crystallographic studies,
less is known about its physiological role or pharmaco-
logical potential. Uric acid, xanthine, hypoxathine and
allopurinol bind at the purine nucleoside site, but with
weaker affinity than caffeine, whereas riboflavin binds
with higher affinity.18 Whether these or other endoge-
nous ligands have a physiological role in regulating
hepatic glycogenolysis is not known.19 Flavopiridol, a
potent inhibitor of cyclin-dependent kinases with anti-
proliferative effects on tumour cells, was recently identi-
fied as an inhibitor of glycogen phosphorylase and high-
affinity ligand of the purine nucleoside site.20,21 Flavo-
piridol is therefore a potential tool to determine the
pharmacological actions of the purine-inhibitor site of
glycogen phosphorylase in comparison with other inhib-
itor sites. In this study, we compared the mode of action
of flavopiridol and four olefin derivatives of flavopiridol
with the effects of an indole carboxamide that has been
well characterised previously from in vivo and in vitro
studies.9,10,22 The results show distinct differences in
the mode of action of purine nucleoside and indole car-
boxamide site inhibitors.
2.2. Structure and inhibitor potency of flavopiridol deriv-
atives on muscle glycogen phosphorylase
Previous work showed that flavopiridol (Fig. 1) binds to
muscle phosphorylase at the purine nucleoside inhibitor
site21 and is a potent inhibitor of the AMP-activated
muscle phosphorylase-b but is less inhibitory to phos-
phorylase-a.20 We tested the potency of four olefin
derivatives of flavopiridol (1–4) at inhibiting muscle
phosphorylase-b. The Ki values on muscle glycogen
phosphorylase-b were between 0.83 and 1.89 lM (Table
1). To elucidate the mechanism of inhibition, we
have determined the crystal structures of the four
analogues 1–4 complexed with rabbit muscle glycogen
phosphorylase b. The structures showed that the inhib-
itors bind at the allosteric inhibitor site, where flavopir-
idol binds (Ganotidis et al., unpublished results).
2.3. Effects of flavopiridols on glycogenolysis in
hepatocytes
The efficacy of flavopiridol and the olefin analogues 1–4 at
inhibiting glycogenolysis in hepatocytes was tested at con-
centrations of 2.5–25 lM and compared with two previ-
ously characterised phosphorylase inhibitors, DAB16
and the indole carboxamide, CP 91149.9 Glycogenolysis
was determined in a glucose-free medium without or
with 100 lM dibutyryl cAMP, which caused a 2.5-fold
2. Results
2.1. Chemistry
stimulation of glucose production (from 47
9
a
The olefin analogues of flavopiridol (1–4, Fig. 1) were
synthesized according to Murthi et al.23 with minor
modifications (Scheme 1). Compound 5 was prepared
as described by Naik et al.24 In general, 1-methyl-4-
(2,4,6 trimethoxyphenyl)-1,2,3,6-tetrahydropyridin, 5,
was treated with BF3OEt2 and Ac2O to afford 1-meth-
yl-4-(2-hydroxy-3-acetyl-4,6-dimethoxyphenyl)-1,2,3,6-
tetrahydropyridine, 6, with selective demethylation,
acylation and Lewis acid-promoted Fries rearrange-
ment. Benzoylation of 6 with the appropriate acid
to 115 19 nmol mgÀ1 protein). DAB caused
similar fractional inhibition of glycogenolysis irrespective
of the absence or presence of dibutyryl-cAMP (Fig. 3A),
whereas CP-91149 was more potent in the absence of
dibutyryl-cAMP (Fig. 3B). Compounds flavopiridol, 2
and 3 (at 25 lM) inhibited glycogenolysis by less than
35% (results not shown). In contrast 1 and 4 caused a con-
centration-dependent inhibition of glycogen degradation
with maximum inhibition of 49% and 54% in the absence

L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
7837
OMe
OMe
O
CH3
i
MeO
OMe
MeO
OH
ii
RCOCl
N
N
OMe
O
O
CH3
CH3
6
5
CH3
O
MeO
R
N
CH3
iii
OH
O
OMe
O
O
7a-d
HO
O
R
MeO
R
iv
N
N
CH3
CH3
8a-d
1-4
Scheme 1. Reagents: (i) BF3ÆOEt2, Ac2O, dry CH2Cl2; (ii) 10% DMAP, dry pyridine; (iii) a—NaH, dry THF; b—H2SO4, AcOH; (iv) pyridine
hydrochloride.
OCH3
OCH3
O
O
O
O
200 lM AICAR at inhibitor concentrations of 25 lM
(Fig. 4B) and also at 2.5 and 10 lM (P < 0.05, in all
cases, results not shown). The effects of varying inhibitor
concentrations are shown for CP-91149 (Fig. 4C) and 4
(Fig. 4D).
CH3
O
CO
R
H3
CO
H3
R
7a: R= 2-Cl Phenyl
7b: R= 3-Cl Phenyl
7c: R= 4-Cl Phenyl
7d: R= Phenyl
8a: R= 2-Cl Phenyl
8b: R= 3-Cl Phenyl
8c: R= 4-Cl Phenyl
8d: R= Phenyl
2.5. Analogue 4 inhibits glycogenolysis by both allosteric
inhibition and inactivation of phosphorylase-a
N
N
CH3
CH3
To determine the relative contributions of inactivation
of phosphorylase as distinct from allosteric inhibition,
we correlated the effects of CP-91149 and des-chloro 4
on glycogenolysis with the activation state of phosphor-
ylase being determined from the activity of phosphory-
lase-a (data from Figs. 3 and 4, respectively). The rate
of glycogenolysis decreased with increasing CP-91149
concentration (2.5–25 lM) and correlated with deple-
tion of phosphorylase-a (Fig. 5A) as shown previously.17
However, in the case of analogue 4 the rates of glycogen-
olysis were lower than could be explained by inactiva-
tion of phosphorylase when compared with the data
for CP-91149 (Fig. 5B). In the absence of AICAR or
dibutyryl cAMP, inhibition of glycogenolysis was pre-
dominantly due to allosteric inhibition as shown by
the lack of depletion of phosphorylase-a, whereas in
the presence of AICAR or dibutyryl cAMP inhibition
of glycogenolysis was in part due to inactivation of
phosphorylase and in part to allosteric inhibition as
shown by the distribution of the data points below the
correlation line corresponding to CP-91149 (Fig. 5B).
Figure 2. Intermediate products 7a–d and 8a–d.
of dibutyryl cAMP, and 61% and 69% in the presence of
dibutyryl cAMP (Figs. 3C and D).
2.4. Effects of flavopiridols on inactivation of phosphor-
ylase-a
Since CP-91149 and DAB inhibit glycogenolysis by dif-
ferent mechanisms (dephosphorylation of phosphory-
lase-a and allosteric inhibition, respectively),17 we
tested the effects of flavopiridols (2.5–25 lM) on the
activation state of phosphorylase (Fig. 4). Unlike
CP-91149, none of the flavopiridols caused significant
depletion (dephosphorylation) of phosphorylase-a in
the absence of dibutyryl-cAMP stimulation at concen-
trations up to 25 lM (Fig. 4A). However, flavopiridol,
1, 2 and 4 (25 lM) significantly counteracted (by 14%,
27%, 18% and 41%, respectively) the activation caused
by dibutyryl cAMP (Fig. 4A). This inactivation was less
than that caused by CP-91149 (70%). To test whether
the flavopiridols counteract the activation of phosphor-
ylase by an AMP analogue, we used AICAR which is
metabolised by hepatocytes to the AMP analogue,
5-aminoimidazole-4-carboxamide 1-b-D-ribofuranotide
(ZMP).7,26,27 All flavopiridols tested including 3 signifi-
cantly counteracted the activation of phosphorylase by
2.6. Lack of stimulation of glycogen synthesis by
flavopiridols
The indole carboxamide, CP-91149, caused a 6.5-fold
increase in glycogen synthesis in incubations with

7838
L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
OMe
OMe
O
CH3
i
MeO
OH
MeO
OH
ii
RCOCl
N
N
OMe
O
O
CH3
CH3
6
9
CH3
O
MeO
R
N
CH3
iii
OH
O
OMe
O
O
7a-d
HO
O
R
MeO
R
iv
N
N
CH3
CH3
8a-d
1-4
Scheme 2. Reagents: (i) BF3ÆOEt2, Ac2O, dry CH2Cl2; (ii) 10% DMAP, dry pyridine; (iii) a—NaH, dry THF; b—H2SO4, AcOH; (iv) pyridine
hydrochloride.
CH3
OMe
OMe
N
OMe
O
i
MeO
OH
MeO
OH
+
+
MeO
OH
N
CH3
N
N
CH3
CH3
10
9
Scheme 3. Reagents: (i) HCl(g), AcOH.
explained by either inhibition of glucokinase or by
non-specific cytotoxicity.
Table 1. Inhibition studies of flavopiridol and compounds 1–4 on
rabbit muscle phosphorylase-b
Compound
Ki (lM)
Flavopiridol
1.16 ( 0.01)
0.99 ( 0.16)
0.83 ( 0.06)
1.89 ( 0.06)
1.28 ( 0.01)
3. Discussion
1
2
3
4
The activity of phosphorylase-a and also its dephos-
phorylation by protein phosphatase-1 are regulated
by multiple interacting allosteric sites. The main phys-
iological regulators of hepatic phosphorylase are glu-
cose and glucose 6-P which act synergistically in
promoting inactivation (dephosphorylation).6,7,28 Glu-
cose inhibits purified phosphorylase synergistically
with pharmacological ligands of the purine nucleoside
site, the indole carboxamide site and the AMP-
site.5,9,29,30 Glucose 6-P promotes inactivation of phos-
phorylase in hepatocytes synergistically with an inhib-
itor of the indole carboxamide site.31 Although
intermediates of the purine catabolism are low-affinity
inhibitors of the purine nucleoside site,18,19 whether
they have a physiological role in the control of either
activity or activation state of phosphorylase is not
15 mM glucose (130 31 vs 19.9 7.1 nmol/3 h mgÀ1),
and a 2-fold activation of glycogen synthase (results
not shown) in agreement with previous findings.10
The ortho analogue
P < 0.02) stimulation
1
caused
of glycogen
a
small (34%,
synthesis
(26.8 6.7 vs 19.9 7.1 nmol/3 h mgÀ1), whereas the
other flavopiridols had no effect on glycogen synthesis
(results not shown). We confirmed that the flavopirid-
ols did not affect glucose phosphorylation as deter-
mined from the metabolism of [2-3H] glucose (results
not shown). This indicates that the lack of stimulation
of glycogen synthesis by flavopiridols cannot be

L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
7839
Figure 3. Inhibition of glycogenolysis. Hepatocytes were pre-cultured in MEM containing 25 mM glucose and 100 nM insulin for 16 h and were then
incubated in glucose-free DMEM in either the absence (open bars) or presence (solid bars) of 100 lM dibutyryl cAMP (db-cAMP) and the inhibitors
indicated: A, DAB; B, CD-91149; C, Compound 1; D, Compound 4. Rates of glucose production in incubations with inhibitors are expressed as
percentageofcontrolwithoutinhibitor. Resultsaremeans SEforfiveexperiments. *P < 0.05;**P < 0.005presenceversusabsenceofdibutyrylcAMP.
known. Flavopiridol,
a
cyclin-dependent kinase
catalyses conversion of phosphorylase b to phosphory-
lase-a and the indole carboxamide which makes phos-
phorylase-a a better substrate for dephosphorylation.
inhibitor, is the highest-affinity pharmacological ligand
of the purine nucleoside site reported.20,21 Derivatives
of flavopiridol are therefore potential tools to compare
the mechanism of action of the purine nucleoside site
with the high-affinity ligands of other allosteric sites
such as the catalytic site16 and the indole carboxamide
site.9,11,12 In this study, we report the synthesis
and bioactivity of four new olefin derivatives of
flavopiridol.
We show in this study that four derivatives of flavopir-
idol, with similar Ki values of 1–2 lM, affect the activa-
tion state of glycogen phosphorylase and glycogenolysis
by different mechanisms from the indole carboxamide or
DAB. Unlike the indole carboxamide they had negligi-
ble effect on the activation state of glycogen phosphory-
lase in the absence of AICAR or dibutyryl cAMP, and
accordingly they had negligible effect on glycogen syn-
thesis, since stimulation by indole carboxamides is ex-
plained by depletion of phosphorylase-a which causes
sequential activation of glycogen synthase.10 However,
all five flavopiridols fully blocked the activation of phos-
phorylase caused by AICAR and they partially reversed
the activation caused by dibutyryl cAMP. The latter ef-
fects suggest suppression of phosphorylase kinase activ-
ity through either a substrate-independent mechanism as
suggested by Kaiser et al.20 or alternatively through a
Previous studies comparing the mechanism of action of
the indole carboxamide inhibitor, CP-91149,9 with
DAB16 showed that the latter compound inhibited gly-
cogenolysis exclusively by allosteric inhibition, whereas
the indole carboxamide inhibited predominantly by
dephosphorylation of phosphorylase.17 Furthermore,
the indole carboxamide, but not DAB, showed a lower
potency at inhibiting glycogenolysis in the presence of
glucagon. This was explained by opposing effects
of phosphorylase kinase (activated by cAMP) which

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L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
Figure 4. Effects of inhibitors on conversion of phosphorylase-a to phosphorylase-b in hepatocytes. Hepatocytes pre-cultured as in Figure 3 were pre-
incubated with inhibitors (flavopiridol, F or compounds 1–4) for 30 min in glucose-free DMEM, then dibutyryl cAMP or AICAR was added as
indicated and incubations continued for a further 30 min before snap-freezing in liquid nitrogen for determination of phosphorylase-a. (A and B)
Hepatocytes were incubated without (0) or with 25 lM inhibitor followed by the absence (open bars) or presence (solid bars) of 100 lM dibutyryl-
cAMP (A) or 200 lM AICAR (B). (C and D) Effects of varying concentration of CP-91149 (C) or analogue 4 (B) followed by the absence (O) or
presence of 100 lM dibutyryl-cAMP (filled circle) or 200 lM AICAR (filled square). Results are means SE for four experiments. *P < 0.05;
**P < 0.005 relative to no inhibitor.
substrate-directed mechanism by favouring the T-state
as suggested for glucose 6-P.32,33 AICAR is metabolised
by hepatocytes to ZMP, an AMP analogue,26 and pro-
motes the conversion of phosphorylase-b to phosphory-
lase-a by favouring the R-conformation, which is a
better substrate for phosphorylase kinase.27 AMP is
known to bind to both the allosteric activation site
and the purine nucleoside site.5 Accordingly, flavopirid-
ols may counteract the effect of AICAR by inhibiting
ZMP binding to the purine nucleoside site.
The inhibition of glycogenolysis by analogues 1 and 4
was greater than could be explained by depletion of
phosphorylase-a indicating an important role for allo-
steric inhibition of phosphorylase-a. There was an
apparent lower affinity of the flavopiridols on glycogen-
olysis as compared with counteraction of phosphorylase
activation by AICAR. Since the latter most likely repre-
sents binding of the flavopiridols to phosphorylase-b,
this difference in affinity can be best explained by the
higher affinity of flavopiridols for phosphorylase-b than
phosphorylase-a.20
Of the five flavopiridols tested analogues 1 and 4 were
the most potent at inhibiting glycogenolysis in a glu-
cose-free medium. In liver, unlike in muscle, phosphor-
ylase-b is catalytically inactive in physiological
conditions and only phosphorylase-a is involved in
glycogenolysis.3 Inhibition of glycogenolysis by
phosphorylase inhibitors may therefore involve two
mechanisms: depletion of phosphorylase-a by dephos-
phorylation and allosteric inhibition of phosphorylase-a.
The difference in potency between the various flavop-
iridols on glycogenolysis (1,4 > 2, 3, F) could not be
explained by the affinity of the inhibitor for either
purified muscle phosphorylase-b or phosphorylase-a,
assayed in the direction of glycogen degradation
(unpublished results). A possible explanation is that
liver phosphorylase shows greater differential selectivi-
ty for the flavopiridol derivatives than the muscle

L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
7841
Figure 5. Correlation between glycogenolysis and the amount of phosphorylase-a. Experimental details for determination of glycogenolysis and
phosphorylase-a were as in given Figures 3 and 4. Rates of glycogenolysis and phosphorylase-a activity are expressed as percentage of the values in
the presence of dibutyryl cAMP without inhibitor (% max). (A) Values for incubations with CP-91149 (2.5–25 lM) and (B) values for incubations
with analogue 4 (2.5–25 lM).
isoform. Kasvinsky et al.29 showed that the endoge-
nous ligands, inosine, FMN and folic acid, inhibited
the muscle but not liver isoform of phosphorylase,
indicating greater specificity of the purine nucleoside
site of the liver isoform. Further work is required to
explore the selectivity of the purine nucleoside site of
the liver isoform.
gift from Novo Nordisk A/S or from Sigma. AI-
CAR and dibutyryl cAMP were obtained from Sig-
ma. Reagents and solvents were obtained from
commercial suppliers and used without further puri-
fication. Solvents used were of analytical grade.
Dichloromethane was distilled from P2O5 under Ar
atmosphere and kept under Ar and MS 4 A. Pyri-
dine was first left overnight at stirring with KOH,
and then distilled from BaO under N2 atmosphere
˚
This study demonstrates three distinguishing features
between the mechanism of action of flavopiridols and
indole carboxamides on glycogen metabolism. First,
allosteric inhibition of phosphorylase was a significant
component of the mechanism of action of flavopiridols
but not of the indole carboxamide. Second, the potency
of the indole carboxamide was decreased in the presence
of dibutyryl-cAMP, whereas that of analogues 1 and 4
was unchanged or slightly increased by dibutyryl-cAMP
because of the additional role of enzyme inactivation
mediated by binding to phosphorylase-b. Third, the
flavopiridols caused negligible stimulation of glycogen
synthesis when compared with the indole carboxamide
at concentrations that cause comparable inhibition of
glycogenolysis, consistent with the lack of depletion of
phosphorylase-a in basal conditions.
˚
and kept under N2 with MS 4 A. THF was distilled
from Na, with benzophenone as an indicator, under
Ar atmosphere.
4.2. General chemistry
Melting points were measured on a Barloworld Scientif-
1
ic-SMP11 apparatus and are given uncorrected. H and
13C NMR spectra were recorded on a Bruker Avance
250 MHz spectrometer. Chemical shifts are given in
parts per million (d) relative to TMS and coupling con-
stants are in hertz. Mass spectra were obtained by a
Micromass UK Limited Platform LC spectrometer in
positive ion spray mode with a flow rate of 10 lL/min.
IR- spectra were recorded on a Shimadzu FT-IR
8400S spectrometer.
Two issues have implications in terms of therapeutic use
for treatment of Type-2 diabetes. First, flavopiridols are
predicted to inhibit glycogenolysis in the postabsorptive
state without promoting glycogen storage in the absorp-
tive state, similar to DAB but unlike indole carboxa-
mides. Second, ligands of the purine nucleoside site
may enable better differential selectivity between muscle
and liver isoforms.
4.2.1. 1-Methyl-4-(2-hydroxy-3-acetyl-4,6-dimethoxyphe-
nyl)-1,2,3,6-tetrahydropyridine (6). In a three-necked,
round-bottomed flask, equipped with a reflux condenser
and magnetic stirring bar, under N2 atmosphere, 1 g
(3.8 mmol) of compound 5 was dissolved in 16 ml of
dry CH2Cl2, and the flask was cooled in an ice bath.
To this solution, 3.3 ml (27 mmol) of BF3ÆOEt2 was add-
ed followed by a dropwise addition of 3.6 ml (19 mmol)
Ac2O. The resulted solution was then allowed to stir
overnight at rt. Then an aqueous solution of 20%
Na2CO3 was added until pH 8. The organic phase was
separated, the crude product extracted from the aqueous
phase with dichloromethane and the combined organic
extracts were dried (MgSO4) and evaporated under re-
duced pressure. The crude product was purified with
4. Materials and methods
4.1. Materials
CP-91149 was a gift from Prizer Global Research &
Development, Groton Laboratories, and DAB was a

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L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
SiO2 column chromatography (eluant: CH2Cl2/MeOH/
NH4OH, 90:9:1, v/v/v) and gave 6 as a yellow solid
(610 mg, 55% yield). Mp: 119–122 °C; 1H NMR
(250 MHz, CDCl3) d: 13.90 (s, 1H), 5.94 (s, 1H), 5.53
(m, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 3.10 (m, 2H), 2.67
(t, 2H, J = 5 Hz), 2.58 (s, 3H), 2.38 (s, 3H), 2.34 (br
m, 2H); 13C NMR (250 MHz, CDCl3) d: 203.3, 163.3,
162.9, 162.4, 128.6, 125.1, 112.0, 105.8, 85.9, 55.6,
55.4, 54.6, 52.2, 45.8, 33.2, 29.3; MS (ESI):
[M+H]+ = 292; FT-IR (KBr pellet): 3430, 2951, 2849,
3H), 3.84 (s, 3H), 2.95 (m, 2H), 2.59 (m, 2H), 2.46 (s,
3H), 2.40 (m, 2H), 2.32 (s, 3H); 13C NMR (250 MHz,
CDCl3) d 199.5, 164.2, 159.7, 157.9, 146.3, 140.0,
131.4, 130.9, 128.9, 127.9, 124.9, 118.1, 116.9, 93.0,
55.9, 55.8, 53.7, 51.6, 44.7, 32.0, 28.3; MS (ESI) m/z
431 [M+H+]; IR (KBr) 2940, 2779, 1728, 1675, 1600,
1559, 1460, 1330, 1247, 1100, 755 cmÀ1
.
4.2.5. 2-Acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6-tetra-
hydropyridin-4-yl)phenyl benzoate (7d). As described
above for the preparation of compound 7a, we followed
the same procedure using benzoyl chloride. Product 7d
was isolated as a brown solid in 75% yield (505 mg).
1631, 1599, 1440, 1270, 1270, 900, 802 cmÀ1
.
4.2.2. 2-Acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6-tetra-
hydropyridin-4-yl)phenyl 2-chlorobenzoate (7a). 2-Chlo-
robenzoyl chloride (0.65 ml; 5.1 mmol) was added
dropwise to a solution of compound 6 (500 mg,
1.7 mmol) and DMAP (20 mg, 0.2 mmol) in 7 ml of
dry pyridine, and the solution was stirred for 45 min, un-
der nitrogen atmosphere, at rt. Then, the resulted mix-
ture was poured into a saturated aqueous solution of
NaHCO3 (15 ml) and stirred for few minutes. The
organic phase was separated, the crude product was
extracted from the aqueous phase with dichloromethane
and the combined organic extracts were dried and evap-
orated under reduced pressure. The crude product was
purified with SiO2 column chromatography (eluant:
CH2Cl2/MeOH, 1:4, v/v) and gave 7a as a brown solid
(533 mg, 73% yield). Mp: 138–142 °C; 1H NMR
(250 MHz, CDCl3) d: 7.99 (m, 1H), 7.44 (m, 2H), 7.32
(m, 1H), 6.39 (s, 1H), 5.57 (s, 1H), 3.88 (s, 3H), 3.83
(s, 3H), 2.93 (m, 2H), 2.52 (t, 2H, J = 5 Hz), 2.47 (s,
3H), 2.34 (br s, 2H), 2.28 (s, 3H); 13C NMR
(250 MHz, CDCl3) d: 199.7, 163.6, 159.7, 157.7, 145.8,
133.8, 132.8, 131.9, 130.9, 129.0, 128.5, 126.6, 125.6,
118.5, 116.8, 92.9, 55.8, 55.7, 54.3, 52.0, 45.5, 31.9,
29.1; MS (ESI): [M+H+] = 431; FT-IR (KBr pellet):
2943, 2778, 1720, 1680, 1606, 1562, 1463, 1328, 1246,
1
Mp: 156–160 °C; H NMR (250 MHz, CDCl3) d 8.07
(d, 2H, J = 7.5 Hz), 7.59 (tr, 1H, J = 7.5 Hz), 7.45 (tr,
2H, J = 7.5 Hz), 6.39 (s, 1H), 5.54 (br s, 1H), 3.89 (s,
3H), 3.83 (s, 3H), 2.82 (m, 2H), 2.48 (m, 2H), 2.46 (s,
3H), 2.37 (m, 2H), 2.24 (s, 3H); 13C NMR (250 MHz,
CDCl3) d 199.7, 165.1, 169.6, 157.6, 146.4, 133.4,
130.1, 129.2, 128.6, 128.4, 125.6, 118.4, 117.3, 92.9,
55.9, 55.8, 54.2, 52.0, 45.3, 32.0, 28.8; MS (ESI) m/z
396 [M+H+]; IR (KBr) 2938, 2777, 1728, 1677, 1603,
1562, 1464, 1333, 1252, 1108, 700 cmÀ1
.
4.2.6. 2-(2-Chlorophenyl)-5,7-dimethoxy-8-(1-methyl-
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (8a).
Compound 7a (800 mg, 1.9 mmol) was dissolved in
dry THF (20 ml) and stirred under nitrogen at rt.
Then 170 mg NaH (60% in paraffin oil, 4.2 mmol)
was added and the mixture was stirred at 60 °C for
1 h. Then the mixture was left to cool down and treat-
ed with MeOH to quench the excess of NaH. The sol-
vents were removed under vacuum. The crude product
was dissolved in 12 ml of glacial acetic acid, 100 ll
concentrated H2SO4 was added and the solution was
stirred at 100 °C for 1 h. Solvents were removed under
reduced pressure. The residue was dissolved in water
and the pH was adjusted to 8 with aqueous 15%
Na2CO3 solution. The crude product was extracted
from the aqueous phase with dichloromethane and
the organic extracts were combined, dried and evapo-
rated under reduced pressure. The crude product was
purified with SiO2 column chromatography (eluant:
CH2Cl2/MeOH/NH4OH, 90:9:1, v/v/v) and afforded
8a as a yellow solid (430 mg, yield 55%); Mp: 145–
152 °C; 1H NMR (250 MHz, CDCl3) d: 7.60 (m,
1H), 7.48 (m, 1H), 7.38 (m, 2H), 6.56 (s, 1H), 6.42
(s, 1H), 5.63 (br s, 1H), 4.00 (s, 3H), 3.90 (s, 3H),
3.10 (m, 2H), 2.64 (t, 2H, J = 7.5 Hz), 2.38 (br s,
5H); 13C NMR (250 MHz, CDCl3) d: 177.7, 160.9,
160.3, 159.7, 156.1, 132.6, 131.6, 131.4, 130.8, 130.6,
127.4, 126.9, 126.3, 113.8, 112.4, 108.6, 91.7, 56.4,
55.9, 54.5, 52.1, 45.6, 29.5; MS (ESI): [M+H+] = 413;
FT-IR (KBr pellet): 2960, 2798, 1649, 1596, 1479,
1100, 755 cmÀ1
.
4.2.3. 2-Acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6- tetra-
hydropyridin-4-yl)phenyl 3-chlorobenzoate (7b). As de-
scribed above for the preparation of compound 7a, we
followed the same procedure using 3-chlorobenzoyl chlo-
ride. Product 7b was isolated as a brown solid in 69% yield
(504 mg). Mp: 53–59 °C; 1H NMR (250 MHz, CDCl3) d
8.02 (s, 1H), 7.95 (m, 1H), 7.55 (m, 1H), 7.38 (m, 1H),
6.39 (s, 1H), 5.53 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 2.91
(m, 2H), 2.52 (m, 2H), 2.45 (s, 3H), 2.36 (m, 2H), 2.28
(s, 3H); 13C NMR (250 MHz, CDCl3) d 199.4, 163.9,
159.7, 158.0, 146.2, 134.6, 133.5, 130.9, 130.0, 129.8,
128.7, 128.1, 124.8, 118.1, 116.8, 93.0, 55.9, 55.8, 53.7,
51.5, 44.6, 31.9, 28.2; MS (ESI) m/z 431 [M+H+]; IR
(KBr) 2940, 2777, 1738, 1680, 1601, 1557, 1460, 1331,
1247, 1103, 755 cmÀ1
.
1380, 1331, 1210, 1122, 765 cmÀ1
.
4.2.4. 2-Acetyl-3,5-dimethoxy-6-(1-methyl-1,2,3,6-tetra-
hydropyridin-4-yl)phenyl 4-chlorobenzoate (7c). As de-
scribed above for the preparation of compound 7a, we
followed the same procedure using 4-chlorobenzoyl
chloride. Product 7c was isolated as a brown solid in
64% yield (468 mg). Mp: 89–92 °C; 1H NMR
(250 MHz, CDCl3) d 7.97 (d, 2H, J = 7.5 Hz), 7.37 (d,
2H, J = 7.5 Hz), 6.40 (s, 1H), 5.53 (br s, 1H), 3.90 (s,
4.2.7. 2-(3-Chlorophenyl)-5,7-dimethoxy-8-(1-methyl-
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (8b).
As described above for the preparation of 8a, we fol-
lowed the same procedure using 7b. Product 8b was iso-
lated as a dark yellow solid in 45% yield (353 mg). Mp:
170–178 °C; 1H NMR (250 MHz, CDCl3) d 7.89 (m,
1H), 7.67 (d, 1H, J = 7.5 Hz), 7.41 (m, 2H), 6.63

L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
7843
(s, 1H), 6.40 (s, 1H), 5.72 (br s, 1H), 3.98 (s, 3H), 3.90 (s,
4.3. Supplementary data
3H), 3.21 (m, 2H), 2.74 (tr, 2H, J = 5 Hz), 2.45 (br s,
5H); 13C NMR (250 MHz, CDCl3) d 177.8, 161.0,
159.9, 158.6, 155.3, 135.1, 133.3, 130.9, 130.1, 127.9,
126.6, 126.1, 123.7, 112.3, 108.6, 108.3, 91.6, 56.3,
55.8, 54.8, 52.4, 45.9, 29.8; MS (ESI) m/z 413 [M+H+];
IR (KBr): 2936, 2790, 1656, 1593, 1475, 1376, 1330,
4.3.1. 1-Methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetra-
hydropyridine (5). 13C NMR (250 MHz, CDCl3) d 159.8,
158.2, 129.1, 124.4, 113.2, 90.6, 55.7, 55.1, 54.7, 52.3,
45.9, 29.8; IR (KBr): 2879, 2783, 1604, 1460, 1411,
1203, 1132, 1037, 980, 810 cmÀ1
.
1120, 1115, 801, 699 cmÀ1
.
4.3.2.
2-(3-Chlorophenyl)-5,7-dihydroxy-8-(1-methyl-
4.2.8. 2-(4-Chlorophenyl)-5,7-dimethoxy-8-(1-methyl-
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (2).
13C NMR (250 MHz, CDCl3) d 182.4, 161.5, 161.3,
161.2, 154.1, 135.2, 133.0, 131.5, 131.4, 128.2, 128.0,
126.1, 124.0, 108.0, 105.8, 104.7, 99.9, 54.5, 52.3, 45.1,
29.5; IR (KBr): 3440, 2924, 1653, 1581, 1423, 1375,
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (8c).
As described above for the preparation of 8a, we fol-
lowed the same procedure using 7c. Product 8c was iso-
lated as a yellow solid in 51% yield (400 mg). Mp: 182–
186 °C; 1H NMR (250 MHz, CDCl3) d 7.77 (d, 2H,
J = 5 Hz), 7.45 (d, 2H, J = 5 Hz), 6.61 (s, 1H), 6.41 (s,
1H), 5.69 (br s, 1H), 3.99 (s, 3H), 3.90 (s, 3H), 3.17
(m, 2H), 2.71 (m, 2H), 2.45 (s, 3H), 2.43 (br m, 2H);
13C NMR (250 MHz, CDCl3) d 177.9, 160.9, 159.9,
159.3, 155.5, 137.2, 130.1, 129.2, 127.9, 127.1, 126.5,
112.5, 108.6, 108.0, 91.6, 56.3, 55.9, 54.9, 52.5, 45.9,
29.8; MS (ESI) m/z 413 [M+H+]; IR (KBr): 2935,
2799, 1651, 1594, 1480, 1364, 1324, 1278, 1126,
1275, 1186, 1105, 760 cmÀ1
.
4.3.3.
2-(4-Chlorophenyl)-5,7-dihydroxy-8-(1-methyl-
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (3).
13C NMR (250 MHz, CDCl3) d 183.8, 164.0, 163.3,
161.7, 155.5, 139.1, 130.9, 130.4, 129.3, 128.7, 126.2,
113.1, 109.5, 105.9, 100.2, 54.8, 52.8, 45.2, 29.3; IR
(KBr): 3440, 2921, 1581, 1419, 1375, 1275, 1188, 1103,
831 cmÀ1
.
820 cmÀ1
.
4.3.4. 2-Phenyl-5,7-dihydroxy-8-(1-methyl-1,2,3,6-tetra-
hydropyridin-4-yl)-4H-benzopyran-4-one (4). 13C NMR
(250 MHz, CDCl3) d 182.7, 163.6, 161.3, 160.6, 154.3,
131.7, 131.2, 129.0, 128.1, 127.6, 125.9, 108.1, 105.4,
104.7, 99.7, 54.4, 52.6, 45.0, 29.4; IR (KBr): 3440,
2943, 2775, 1641, 1583, 1423, 1279, 1184, 1101, 773,
4.2.9. 2-Phenyl-5,7-dimethoxy-8-(1-methyl-1,2,3,6-tetra-
hydropyiridin-4-yl)-4H-benzopyran-4-one (8d). As de-
scribed above for the preparation of 8a, we followed
the same procedure using 7d. Product 8d was isolated
as a yellow solid in 44% yield (316 mg). Mp: 184–
190 °C; 1H NMR (250 MHz, CDCl3) d 7.80 (br s,
2H,), 7.45 (br s, 3H), 6.62 (s, 1H), 6.38 (s, 1H), 5.58
(br s, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 3.14 (m, 2H),
2.68 (m, 2H), 2.42 (br s, 5H); 13C NMR (250 MHz,
CDCl3) d 178.0, 160.7, 160.3, 159.8, 155.5, 131.5,
130.9, 128.7, 127.7, 126.4, 125.7, 112.2, 108.5, 107.8,
91.4, 56.5, 55.7, 54.8, 52.4, 45.9, 29.7; MS (ESI) m/z
378 [M+H+]; IR (KBr): 2930, 2795, 1642, 1593, 1465,
692 cmÀ1
.
4.3.5.
2,6-Di(1,2,3,6-tetrahydropyridin-4-yl)-3,5-dime-
1
thoxy phenol (10). H NMR (250 MHz, CDCl3) d 6.04
(s, 1H), 5.54 (br s, 2H), 3.76 (s, 6H), 3.08 (m, 4H),
2.64 (t, 4H, J = 5 Hz), 2.38 (br s, 10H); MS (ESI) m/z
345 [M+H+], 173 [(M+2H+)/2]
1380, 1332, 1116, 780, 679 cmÀ1
.
4.4. Phosphorylase kinetics
4.2.10. 2-(2-Chlorophenyl)-5,7-dihydroxy-8-(1-methyl-
Rabbit glycogen phosphorylase-b was isolated, purified,
recrystallised, and assayed as described.34 Kinetic exper-
iments with flavopiridol and analogues were performed
in the direction of glycogen synthesis in the presence
of constant concentrations of glycogen (0.2% w/v),
AMP (1 mM), and various concentrations of Glc-1-P
(1.5–20 mM) and inhibitors (0.25–10 lM), in 30 mM
imidazole/HCl buffer (pH 6.8), 60 mM KCl, 0.6 mM
EDTA, 0.6 mM dithiothreitol, and 0.1% DMSO.
1,2,3,6-tetrahydropyridin-4-yl)-4H-benzopyran-4-one (1).
Compound 8a (25 mg, 0,06 mmol) was placed in a
sealed tube together with hydrochloric pyridine
(300 mg, 2.6 mmol) and heated to 180 °C. The solution
then was stirred for 2 h. The mixture was left to cool
down and a saturated aqueous solution of NaHCO3
was added. The crude product was extracted from the
aqueous phase with dichloromethane/methanol (9:1, v/
v) and the organic extracts were combined, dried and
evaporated under reduced pressure. The crude product
was purified with SiO2 column chromatography (eluant:
CH2Cl2/MeOH/NH4OH, 90:9:1, v/v/v) and afforded 1 as
4.5. Hepatocyte isolation and culture
Hepatocytes were isolated by collagenase perfusion of
the liver10 of male Wistar rats (body wt 200–250 g) ob-
tained from B&K, Hull, UK. All experiments were con-
ducted in accordance with EC Council Directive (86/
609/EEC). The hepatocytes were suspended in MEM
supplemented with 5% new born calf serum and plated
in 24-well plates for determination of glycogen synthesis
and phosphorylase-a activity and in gelatine coated
96-well plates for determination of glycogenolysis. After
cell attachment (approximately 4 h) the medium was
replaced by serum-free MEM containing 10 nM
1
a green solid (13 mg, yield 56%); Mp: 205–211 °C; H
NMR (250 MHz, CDCl3) d 7.39–7.58 (m, 4H), 6.51 (s,
1H), 6.28 (s, 1H), 5.74 (br s, 1H), 3.25 (br s, 2H), 2.82
(m, 2H), 2.57 (br s, 5H) [data in agreement with the lit-
erature3]; 13C NMR (250 MHz, CDCl3) d 182.3, 163.0,
161.4, 160.7, 154.8, 132.6, 131.4, 131.9, 130.9, 130.6,
128.0, 127.4, 127.1, 110.9, 108.2, 104.7, 99.8, 54.1,
51.9, 44.8, 29.2; MS (ESI) m/z 385 [M+H+]; IR (KBr):
3440, 2921, 1655, 1577, 1419, 1369, 1275, 1186, 1101,
760 cmÀ1
.

7844
L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
2. Bollen, M.; Keppens, S.; Stalmans, W. Biochem. J. 1998,
336, 19.
3. Stalmans, W.; Gevers, G. Biochem. J. 1981, 200, 327.
4. Newgard, C. B.; Brady, M. J.; O’Doherty, R. M.; Saltiel,
A. R. Diabetes 2000, 49, 1967.
5. Newgard, C. B.; Hwang, P. K.; Fletterick, R. J. Crit. Rev.
Biochem. Mol. Biol. 1989, 24, 69.
6. Aiston, S.; Andersen, B.; Agius, L. Diabetes 2003, 52, 1333.
7. Aiston, S.; Green, A.; Mukhtar, M.; Agius, L. Biochem. J.
2004, 377, 195.
dexamethasone, 100 nM insulin and 25 mM glucose for
subsequent determination of glycogenolysis and activity
of phosphorylase-a and in medium containing 10 nM
dexamethasone and 5 mM glucose for subsequent deter-
mination of glycogen synthesis.
4.6. Glycogenolysis and phosphorylase-a activity
Hepatocyte monolayers in 96-well plates were pre-cul-
tured for 16 h in MEM containing 100 nM insulin,
25 mM glucose and [U–14C]glucose (2 lCi/ml). They
were then washed three times with 150 mM NaCl and
incubated for 3 h in glucose-free DMEM (Invitrogen,
Life Technologies) containing the inhibitor concentra-
tions and other additions indicated. On termination
of the incubation the medium was collected for deter-
mination of glucose spectrometrically by coupling to
hexokinase and glucose 6-phosphate dehydrogenase17
and the cells were extracted in 0.1 M NaOH and radio-
labelled glycogen was determined by ethanol precipita-
tion.17 Glycogenolysis was determined from the
fractional decrease in radiolabelled glycogen during
the 3 h incubation. For determination of phosphory-
lase-a parallel incubations were performed on hepato-
cytes in 24-well plates incubated under similar
conditions as for glycogenolysis except for omission
of the radiolabelled glucose. On termination of the
incubations, the plates were snap-frozen in liquid nitro-
gen and the activity of phosphorylase-a was determined
spectrometrically by coupling to phosphoglucomutase
and glucose 6-phosphate dehydrogenase as described
previously.10 Activities are expressed as m units/mg cell
protein.
8. Bergans, N.; Stalmans, W.; Goldmann, S.; Vanstapel, F.
Diabetes 2000, 49, 1419.
9. Martin, W. H.; Hoover, D. J.; Armento, S. J.; Stock, I. A.;
McPherson, R. R.; Danley, D. E.; Stevenson, R. W.;
Barrett, E. J.; Treadway, J. L. Proc. Natl. Acad. Sci. 1998,
95, 1776.
10. Aiston, S.; Hampson, L.; Gomez-Foix, A. M.; Guinovart,
J. J.; Agius, L. J. Biol. Chem. 2001, 276, 23858.
11. Rath, V. L.; Ammirati, M.; Danley, D. E.; Ekstrom, J. L.;
Gibbs, E. M.; Hynes, T. R.; Mathiowetz, A. M.; McPh-
erson, R. K.; Olson, T. V.; Treadway, J. L.; Hoover, D. J.
Chem. Biol. 2000, 7, 677.
12. Oikonomakos, N. G.; Skamnaki, V. T.; Tsitsanou, K. E.;
Gavalas, N. G.; Johnson, L. N. Structure 2000, 8, 575–584.
13. Zographos, S. E.; Oikonomakos, N. G.; Tsitsanou, K. E.;
Leonidas, D. D.; Chrysina, E. D.; Skamnaki, V. T.;
Bischoff, H.; Goldmann, S.; Watson, K. A.; Johnson, L.
N. Structure 1997, 5, 1413.
14. Board, M.; Bollen, M.; Stalmans, W.; Kim, Y.; Fleet, G.
W.; Johnson, L. N. Biochem. J. 1995, 311, 845.
15. Massillon, D.; Bollen, M.; De Wulf, H.; Overloop, K.;
Vanstapel, F.; Van Hecke, P.; Stalmans, W. J. Biol. Chem.
1995, 270, 19351.
16. Andersen, B.; Rassov, A.; Westergaard, N.; Lundgren, K.
Biochem J. 1999, 342, 545.
17. Latsis, T.; Andersen, B.; Agius, L. Biochem. J. 2002, 368,
309.
18. Ekstrom, J. L.; Pauly, T. A.; Carty, M. D.; Soeller, W. C.;
Culp, J.; Danley, D. E.; Hoover, D. J.; Treadway, J. L.;
Gibbs, E. M.; Fletterick, R. J.; Day, Y. S.; Myszka, D. G.;
Rath, VL. Chem. Biol. 2002, 9, 915.
19. Ercan-Fang, N. G.; Nuttall, F. Q.; Gannon, M. C. Am. J.
Physiol. 2001, 280, E248.
20. Kaiser, A.; Nishi, K.; Gorin, F. A.; Walsh, D. A.;
Bradbury, E. M.; Schnier, J. B. Arch. Biochem. Biophys.
2001, 386, 179.
21. Oikonomakos, N. G.; Schnier, J. B.; Zographos, S. E.;
Skamnaki, V. T.; Tsitsanou, K. E.; Johnson, L. N. J. Biol.
Chem. 2000, 275, 34566.
22. Gustafson, L. A.; Neeft, M.; Reijngoud, D. J.; Kuipers,
F.; Sauerwein, H. P.; Romijn, J. A.; Herling, A. W.;
Burger, H. J.; Meijer, A. J. Biochem. J. 2001, 358, 665.
23. Murthi, K. K.; Dubay, M.; McClure, C.; Brizuela, L.;
Boisclair, M. D.; Worland, P. J.; Mansuri, M. M.; Pal, K.
Bioorg. Med. Chem. Lett. 2000, 10, 1037.
4.7. Glycogen synthesis
Hepatocyte monolayers in 24-well plates were pre-cul-
tured for 16 h in MEM containing 5 mM glucose. They
were then incubated for 3 h in fresh MEM containing
15 mM glucose and [U–14C]glucose (2 lCi/ml) and other
additions as indicated. On termination of the incuba-
tions the hepatocytes were washed in 150 mM NaCl
and extracted in 0.1 M NaOH. Radiolabelled glycogen
was determined as previously described10 and rates of
glycogen synthesis are expressed as nmol of glucose
incorporated into glycogen/3 h mgÀ1 cell protein.
Acknowledgments
24. Naik, R. G.; Kattige, S. L.; Bhat, S. V.; Alreja, N. J.; de
Souza, N. J.; Rupp, R. H. Tetrahedron 1988, 44, 2081.
25. Mansuri, M. M.; Murthi K. K.; Pal K. Inhibitors of cyclin
dependent kinases. WO9716447.
26. Vincent, M. F.; Marangos, P. J.; Gruber, H. E.; Van den
Berghe, G. Diabetes 1991, 40, 1259.
This work was supported by Greek General Secretariat
for Research and Technology through PENED-204/
2001 (N.G.O.), and Scientific and Technological cooper-
ation between Greece and USA (2005–2006) (N.G.O.),
an EMBO short-term fellowship (M.N.K.), and by
equipment grant support from Diabetes UK. We thank
Dr. Judith Treadway for the kind gift of CP-91149.
27. Shang, J.; Lehrman, M. A. J. Biol. Chem. 2004, 279,
12076.
28. Harndahl, L. B.; Schmoll, D.; Herling, A. W.; Agius, L.
FEBS J. 2006, 273, 336.
29. Kasvinski, P. J.; Shechosky, S.; Fletterick, R. J. J. Biol.
Chem. 1978, 253, 9102.
References and notes
30. Kristiansen, M.; Andersen, B.; Iversen, L. F.; Westerg-
aard, N. J. Med. Chem. 2004, 47, 3537.
1. Treadway, J. L.; Mendys, P.; Hoover, D. J. Exp. Opin.
Invest. Drugs 2001, 10, 439.

L. J. Hampson et al. / Bioorg. Med. Chem. 14 (2006) 7835–7845
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31. Hampson, L. J.; Agius, L. Diabetes 2005, 54, 617.
32. Krebs, E. G.; Love, D. S.; Bratfold, G. E.; Trayser, K. A.;
Meyer, W. L.; Fischer, E. H. Biochemistry 1964, 3, 1022.
33. Tu, J.-U.; Graves, D. J. Biochem. Biophys. Res. Commun.
1973, 53, 59.
34. Anagnostou, E.; Kosmopoulou, M. N.; Chrysina, E. D.;
Leonidas, D. D.; Hadjiloi, T.; Tiraidis, C.; Zographos, S.
E.; Gyorgydeak, Z.; Somsak, L.; Docsa, T.; Gergely, P.;
Kolisis, F. N.; Oikonomakos, N. G. Bioorg. Med. Chem.
2006, 14, 181.