Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.119

New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed good potency in enzyme and cell-based assays (IC₅₀ = 111 nM, EC₅₀ = 1.78 μM). Moreover, it has demonstrated desirable water solubility, PK profile, and moderate brain penetration.

Full text of DOI:10.1016/j.bmcl.2013.03.119

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3983–3987
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]
pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3)
inhibitors
Kwangwoo Chun a, Ji-Seon Park a, Han-Chang Lee a, Young-Ha Kim a, In-Hea Ye a, Kang-Jeon Kim a,
Il-Whea Ku a, Min-Young Noh b, Goang-Won Cho c, Heejaung Kim b, Seung Hyun Kim b, , Jeongmin Kim a,
⇑
⇑
a Division of New Drug Development, R&D Center, Jeil Pharmaceutical Co., Ltd. 117-1, Keungok-Ri, Baekam-Myun, Cheoin-Gu, Yongin-City, Kyunggi-Do 449-861, Republic of Korea
b Department of Neurology, Hanyang University College of Medicine, Seoul 133-791, Republic of Korea
c Department of Biology, College of Natural Sciences, Chosun University, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-
one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed
Received 24 January 2013
Revised 18 March 2013
Accepted 27 March 2013
Available online 4 April 2013
good potency in enzyme and cell-based assays (IC50 = 111 nM, EC50 = 1.78
lM). Moreover, it has demon-
strated desirable water solubility, PK profile, and moderate brain penetration.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Glycogen synthase kinase-3
GSK-3 inhibitor
8-Amino-[1,2,4]triazolo[4,3-a]pyridin-
3(2H)-one derivatives
Glycogen synthase kinase-3 (GSK-3), a serine/threonine pro-
tein kinase, is known to be highly expressed in the brain.1 GSK-
3 was identified as an enzyme that inhibited activation of glyco-
gen synthase (GS) as well as a large number of proteins.2–4 GSK-3
associated with these various targets has significant potency in
the treatment of many diseases, including Alzheimer’s disease,
Huntington’s disease, Parkinson’s disease, amyotrophic lateral
sclerosis (ALS), diabetes, and diverse cancers.5–10
gen bond between the Val135 and the inhibitors is essential as
shown in Figure 1.
We are describing synthesis, structure-activity relationship
(SAR) study results, and biological evaluations of 8-amino-
[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as potential
GSK-3 inhibitors which were designed by a binding mode analysis
of Staurosporine crystal structure (Fig. 2).18 The backbone of the
Asp133 and Val135 located on hinge region will be able to create
hydrogen bonds with the triazolopyridinone ring. The binding
mode was similar to that of the Staurosporine. Polar residues of
GSK-3 were located on the binding cavity: Lys85, Glu97, Asp200,
Gln185 and Arg141. These residues play an important role in bind-
ing activity and selectivity against other kinases, and for this rea-
son various C-6 and C-8 substituents of the triazolopyridinone
were designed.19
Recently, GSK-3 inhibitors, having various structures, are being
developed to treat cancers, Alzheimer’s disease, and diabetes.8,10–
16
Herein, we designed and synthesized new triazolopyridine
derivatives as small molecule GSK-3 inhibitors that can be used
in the treatment of various diseases caused by over expression
of GSK-3. On the basis of the known crystallographic structures
obtained from Protein Data Bank (PDB) database, interactions be-
tween protein and inhibitors were analyzed to design novel GSK-3
inhibitors (PDB code: 1J1B, 1Q3D, 1Q3W, 1Q41, 1Q4L, and
1Q5K).12,13,16–18 The analysis of hinge bindings was shown in Fig-
ure 1. The amino acids located in the hinge region of GSK-3 and
the inhibitors form four hydrogen bonds.18 In particular, a hydro-
8-Amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one
derivatives
were synthesized as outlined in Schemes 1–3. Nitration of pyri-
din-2-ol 1 gave 3-nitropyridin-2-ol 2 which was halogenated to
generate 2-chloro-3-nitropyridine 3. 8-Nitro-pyridone 5 was syn-
thesized by introduction of hydrazine to the compound 3 followed
by cyclization using triphosgene. Palladium was employed for syn-
thesis of compound 6a–b and 6d, but did not work for the case of
compound 6c. It was found that tin(II) chloride was employed for
successful reduction of 6-chloro-pyridone 5c to produce an amine
6c. Compound 10 was afforded by Mitsunobu protocol from com-
⇑
Corresponding authors. Tel.: +82 2 2290 8371; fax: +82 2 2296 8370 (S.H.K.);
tel.: +82 31 332 4457; fax: +82 31 333 0337 (J.K.).
E-mail addresses: kimsh1@hanyang.ac.kr (S.H. Kim), jminkim@jeilpharm.co.kr (J.
Kim).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.119

3984
K. Chun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3983–3987
ASP133
Tyr134
ASP133
Tyr134
ASP133
Tyr134
O2N
O
O
O
H
H
HN
HN
N
HN
N
N
N
O
OMe
O
O
O
O
O
O
H
N H
O
NH
N H
O
NH
N
N
Val135
Pro136
Val135
Pro136
Val135
Pro136
NHCH3
OH
H
N
N
O
O
N
N
O
O
PDB code: 1Q3D (Staurosporine)
PDB code: 1Q4I (Indirubin-3'-monoxime)
PDB code: 1Q3W (Alsterpaullone)
ASP133
Tyr134
ASP133
Tyr134
ASP133
O
Cl
O
O
O
HN
H
HN
H-donor
H-acceptor
H-donor
HN
N
NO2
Tyr134
O
O
O
N
O
N H
O
HN
N H
O
S
NH
Cl
COOH
Val135
Pro136
Val135
Pro136
Val135
Pro136
N
H
O
O
N
N
O
N
N
H
H-donor
O
O
O
PDB code: 1Q4L (I-5)
Figure 1. Hinge binding analysis of GSK-3 crystal structure and binding mode of GSK-3 inhibitors.
LYS85
PDB code: 1Q5K (AR-A014418)
Binding mode of GSK-3 in hibitors
ASP133
GLU97
O
R2
NH2
ASP133
Tyr134
HN
H
O
R1
N
N
N
O
Tyr134
N
HO
OH
H
HN
O
N
O
6-7 A
N
O
N H
O
OMe
O
O
Val135
Pro136
O
R3
ASP200
N H
O
N
Val135
Pro136
NHCH3
N
11 A
O
N
O
9 A
O
NH2
H2N
NH2
Staurosporine
GLN185
NH
ARG141
Figure 2. Design of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one GSK-3 inhibitors.
HO
HO
N
Cl
N
H2NHN
N
NO2
NO2
NO2
c
a
b
d
N
R3
R3
R3
R3
4a-d
3a-d
2a-d
1a-d
N
N
NO2
NH2
R3
HN
HN
e or f
R3
N
N
O
O
c: Cl
d: CF3
a: H
R3
b: Me
5a-d
6a-d
Scheme 1. Reagents and conditions: (a) H2SO4, HNO3, H2O, 60 °C, 18 h; (b) SOCl2, DMF, 110 °C, 3 h; (c) H2NNH2ÁH2O, MeOH, 80 °C, 1 2 h; (d) triphosgene, THF, 60 °C, 30 min;
(e) SnCl2ÁH2O, HCl, EtOH/EtOAc, reflux, 3 h; (f) Pd/C, H2, MeOH, 48 h or Pd(OH)2, H2, THF/EtOH, 2 days.
O
N
N
N
N
NH2
NH2
N
HN
pound 6a (Scheme 2). The compound 6a–d reacted with di-t-butyl
dicarbonate in the presence of 4-(dimethylamino)pyridine to give
Boc-protected compound 7a–d. Reductive amination of 7a–d with
an appropriate aldehyde in the presence of sodium cyanoborohy-
dride afforded compound 8a–d.20 Additionally, N-methylation
with sodium hydride yielded N-methylated compound 9b. Finally,
deprotection of the amine under acidic condition afforded 11a,
a
O
O
O
6a
10
Scheme 2. Reagents and conditions: (a) 2-(4-methoxy-phenoxy)-ethanol, PPh3,
DIAD, DMF, 0 °C, 1 h.

K. Chun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3983–3987
3985
Boc
N
N
Boc
N
N
NH2
R3
HN
R3
N
N
R2
N
N
HN
NH2
R3
R2
N
HN
N
HN
a
b or c
e
O
O
O
O
R3
6a-d
7a-d
8a-d
11-26
d
Boc
N
N
N
N
N
R2
R2
HN
N
e
N
O
O
R3
R3
9b
14b
Scheme 3. Reagents and conditions: (a) (Boc)2O, DMAP, THF, 2 h; (b) aldehyde, NaCNBH3, ZnCl2, 1.25 N HCl, MeOH, 12 h; (c) acyl chloride, pyridine, 1 h; (d) MeI, NaH, DMF,
0 °C, 1 h; (e) 4.0 N HCl in 1,4-dioxane, 12 h.
Table 1
Table 2 (continued)
Enzymatic activities of the synthesized compounds
a
b
Compound
R1
R2
R3
IC50
(
lM)
EC50 (lM)
R2
R4
R1
N
N
N
N
N
N
O
17b
18b
H
H
Me
Me
0.607
0.774
6.23
12.1
R3
Compound R1
R2 R3 R4
IC50
M)
a
Cl
(
l
N
19b
H
Me
0.641
9.11
6a
10
H
H
H
H
H
H
H
>50
O
NH2
O
>50
20b
21b
22b
H
H
H
Me
Me
Me
1.01
1.36
>50
18.4
13.3
NDc
O
O
N
N
N
11a
12a
H
H
H
H
H
H
>50
13
NH
O
O
a
Enzymatic assay results were obtained using a commercially available human
O
recombinant GSK-3 enzyme (upstate, Cat No. 14-306) and phospho-glycogen syn-
thase peptide-2 (GS2, Cat No. 12-241) in 96 well plates. The numbers are the mean
of triplicate experiments.
H4IIE (ATCC, CRL154) was used for the cell-based assay. The numbers are the
mean of triplicate experiments.
a
Enzymatic assay results were obtained using a commercially available human
recombinant GSK-3 enzyme (upstate, Cat No. 14-306) and phospho-glycogen syn-
thase peptide-2 (GS2, Cat No. 12-241) in 96 well plates. The numbers are the mean
of triplicate experiments.
b
c
ND; not determined.
Table 3
Table 2
Enzymatic and cellular activities of the synthesized compounds
Enzymatic and cellular activities of the synthesized compounds
R2
H
O
1
R2
N
N
N
R
N
H
O
1
N
N
N
R
N
R3
R2
R3
a
b
Compound
R1
R3
IC50
(lM)
EC50
NDc
(lM)
a
b
Compound R1
R2 R3 IC50
EC50
M)
(
l
M)
(
l
O
12b
H
Me
7.01
15c
H
Cl 0.520
1.68
N
O
16c
17c
23c
H
H
H
Cl 0.342
Cl 0.111
Cl 0.818
2.74
1.78
7.03
13b
14b
H
Me
Me
1.20
21.1
NDc
N
N
N
N
Me
ꢀ50
N
N
15b
16b
H
H
Me
Me
0.354
0.309
5.44
1.25
N
N
18c
H
Cl 0.199
2.63
Cl
(continued on next page)

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K. Chun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3983–3987
Table 4
Table 3 (continued)
Rat PK profile of 17c and 17da, n = 3
a
b
Compound R1
R2 R3 IC50
EC50
M)
Compound
17c
17d
(l
M)
(
l
Administration route
Dose (mg/kg)
iv
3
2489.5
1.2
0.40
—
po
5
3455.7
—
1.2
83
iv
3
1492.3
2.0
0.20
—
po
5
1736.0
—
0.40
70
N
24c
25c
26c
H
H
Cl 0.249
Cl 0.190
2.36
2.71
b
AUC0–inf (h ng/ml)
NH2
O
CLc (L/h/kg)
d
t1/2 (h)
Fe (%)
a
b
c
O
The values were determined by LC/MS/MS analysis after administration.
AUC means area under the concentration-time curve.
CL means clearance.
t1/2 Means half-life of elimination.
F means bioavailability.
H
H
Cl 0.247
3.84
1.32
N
O
d
e
15c
17c
Cl 0.0800
N
N
H
H
Cl 0.0630
Cl 0.133
1.75
1.10
26c
Table 5
a
Plasma and brain concentrations after an iv administration (30 mg/kg) in ICR mice,
Enzymatic assay results were obtained using a commercially available human
recombinant GSK-3 enzyme (upstate, Cat No. 14-306) and phospho-glycogen syn-
thase peptide-2 (GS2, Cat No. 12-241) in 96 well plates. The numbers are the mean
of triplicate experiments.
17c (n = 3)a
Time (h)
Plasma (ng/ml)
Brain (ng/g)
Cbrain/Cplasma
b
0.25
0.75
634.00
1047.00
214.13
106.80
0.34
0.1
H4IIE (ATCC, CRL154) was used for the cell-based assay. The numbers are the
mean of triplicate experiments.
a
The values were calculated at 0.25 and 0.76 h by LC/MS/MS analysis.
study of 17b in the catalytic domain of human GSK-3b (PDB code:
1Q5K) can clearly explain the results (Fig. 3). The hydrogen of the
amine at C-8 position formed a bridge hydrogen bond with the
Asp200 mediated by a water molecule. As a result, the inhibitor
can be stabilized.25 m- or p-Pyridinylpropylamino derivatives at
C-8 position (15b–17b, 15c–17c, 15d, and 17d) were 2- to 3-fold
more active than o-pyridinylpropylamino derivatives (13b and
14b) because of hydrogen bonds between the pyridine and
Phe67. Also, m-substituted aryl propylamino derivatives (18b–
19b, 18c, 23c–26c, and 26d) retained enzymatic and cellular
activities.
12a–b, 25c, and others (HCl salt form: 13b–22b in Table 2, 15c–24c
and 26c–26d in Table 3).21
Enzyme and cell-based activities of various 8-amino-
[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives were summa-
rized in Tables 1–3.22–24 Compound 10 that do not fit well on
GSK-3 binding site showed poor enzymatic activities because of
disturbing hydrogen bond with the Asp133. N-(4-methoxy-
phenyl)ethyl derivative at C-8 position (12a) displayed moderate
activity. Methyl substituted compound 12b was twofold more ac-
tive than compound 12a because of hydrophobic interactions be-
tween the methyl group at C-6 position and Ile62, Val70, Thr138,
and Leu188 (Fig. 3). SAR analysis at C-6 position showed the fol-
lowing trend of CF3 ꢁ Cl > Me in activity. On the other hand, amide
11a and tertiary amine 14b demonstrated poor activity. A docking
Based on the results, we examined pharmacokinetic (PK) char-
acteristics (Table 4), brain/plasma concentrations (Table 5), and
off-target effect against a set of commercially available enzyme pa-
Figure 3. Docking of compound 17b (ball and stick) in the catalytic domain of human GSK-3b (PDB code: 1Q5K).

K. Chun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3983–3987
3987
Table 6
12. Bhat, R.; Xue, Y.; Berg, S.; Hellberg, S.; Ormo, M.; Nilsson, Y.; Radesater, A. C.;
Jerning, E.; Markgren, P. O.; Borgegard, T.; Nylof, M.; Gimenez-Cassina, A.;
Hernandez, F.; Lucas, J. J.; Diaz-Nido, J.; Avila, J. J. Biol. Chem. 2003, 278, 45937.
13. Lescot, E.; Bureau, R.; Sopkova-de Oliveira Santos, J.; Rochais, C.; Lisowski, V.;
Lancelot, J. C.; Rault, S. J. Chem. Inf. Model. 2005, 45, 708.
14. Meijer, L.; Flajolet, M.; Greengard, P. Trends Pharmacol. Sci. 2004, 25, 471.
15. Sereno, L.; Coma, M.; Rodriguez, M.; Sanchez-Ferrer, P.; Sanchez, M. B.; Gich, I.;
Agullo, J. M.; Perez, M.; Avila, J.; Guardia-Laguarta, C.; Clarimon, J.; Lleo, A.;
Gomez-Isla, T. Neurobiol. Dis. 2009, 35, 359.
16. Kramer, T.; Schmidt, B.; Lo Monte, F. Int. J. Alzheimers Dis. 2012, 2012, 381029.
17. Gadakar, P. K.; Phukan, S.; Dattatreya, P.; Balaji, V. N. J. Chem. Inf. Model. 2007,
47, 1446.
18. Bertrand, J. A.; Thieffine, S.; Vulpetti, A.; Cristiani, C.; Valsasina, B.; Knapp, S.;
Kalisz, H. M.; Flocco, M. J. Mol. Biol. 2003, 333, 393.
Off-target screening results of 17c at 10 l
Ma
Targets
AMPK
CDK2/cyclinA (human)
CDK5/p25 (human)
CHK1 (human)
CK2 (human)
CSK (human)
GSK3
GSK3b (human)
JNK1 1 (human)
Inhibition (%)
Targets
Inhibition (%)
a
1 (human)
À1
54
53
21
5
p70S6K (human)
PDK1 (human)
14
10
25
0
12
18
14
27
34
À1
1
PhKc2 (human)
PKA (human)
PKB
PKC
a
a
(human)
(human)
À29
99
90
9
a
(human)
PRAK (human)
ROCK-II (human)
Rsk2 (human)
SAPK2a (human)
SAPK2b (human)
SAPK3 (human)
SAPK4 (human)
SGK (human)
a
19. Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Myrianthopoulos, V.;
Mikros, E.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.; Leost, M.;
Greengard, P.; Meijer, L. J. Med. Chem. 2004, 47, 935.
Lck (human)
19
15
19
9
MAPK1 (human)
MAPKAP-K2 (human)
MEK1 (human)
MSK1 (human)
5
2
22
20. Young, J. R.; Huang, S. X.; Walsh, T. F.; Wyvratt, M. J.; Yang, Y. T.; Yudkovitz, J.
B.; Cui, J.; Mount, G. R.; Ren, R. N.; Wu, T. J.; Shen, X.; Lyons, K. A.; Mao, A. H.;
Carlin, J. R.; Karanam, B. V.; Vincent, S. H.; Cheng, K.; Goulet, M. T. Bioorg. Med.
Chem. Lett. 2002, 12, 827.
9
a
This experiment was carried out by Millipore (www.millipore.com).
21. Compound 17C: 1H NMR (400 MHz, DMSO-d6) d 12.59(s, 1H), 8.80 (d, J = 6.6 Hz,
2H), 7.97 (d, J = 6.6 Hz, 2H), 7.22 (s, 1H), 6.70 (br, 1H), 5.90 (s, 1H), 3.21 (t,
J = 6.8 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 2.00–1.94 (m, 2H); LC–MS (ESI+) m/z
304.1 [M+H+].
nel (Table 6).25,26 Pharmacokinetic study has shown that 17c has
better properties than 17d in terms of oral bioavailability (F), clear-
ance (CL), half life (t1/2), and area under the curve (AUC). In addi-
tion, 17c has good water solubility (>10 mg/ml). A blood-brain
barrier (BBB) permeability study using SD rat revealed that 17c
can penetrate into the brain. The off-target screening results re-
vealed that inhibition percentages of 17c against CDK2 and CDK5
22. de Raemy-Schenk, A. M.; Trouble, S.; Gaillard, P.; Page, P.; Gotteland, J. P.;
Scheer, A.; Lang, P.; Yeow, K. Assay Drug Dev. Technol. 2006, 4, 525.
23. The inhibitory effect of GSK-3 inhibitors was determined using human
recombinant GSK-3 enzyme (upstate, Cat No. 14-306) and Phospho-Glycogen
Synthase Peptide-2 (GS2, upstate, Cat No. 12-241) according to the
manufacturer’s recommendations. Briefly, GSK-3 (1 ng/well) was pre-
incubated with the kinase reaction buffer [12 mM MOPS (pH 7.0), 1 mM DTT,
1 mM EDTA, 10 mM MgCl2] supplemented with 6.67 lM Phospho-Glycogen
Synthase Peptide-2 (GS2, YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE) and enzyme
buffer [2 mM MOPS (pH 7.0), 0.01 mM EDTA, 0.001% Brij-35, 0.5% glycerol,
0.1 mg BSA, 0.01% 2-mercaptoethanol] for test compound (DMSO, 1% final
concentration) in a 96 well round bottom plate (corning, Cat No. 3365). We
also prepared a negative control without GSK-3 inhibitor, providing a 100%
were 54% and 53% at a concentration of 10 lM, respectively, be-
cause the hinge region interactions of GSK-3 are similar to those
described for CDK inhibitors.14 CDK2 is known not to be expressed
in the brain and CDK5, a protein involved in tau phosporylation, is
implicated in Alzheimer’s disease.11,19
activity reference point. After 10 min of incubation at 30 °C,
1 lM non-
radioactive ATP or 0.2 Ci 33P-gamma-ATP were added to reaction mixtures.
l
In conclusion, we reported synthesis and biological evaluation
of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as
glycogen synthase kinase-3 (GSK-3) inhibitors. Compound 17c,
having desirable oral PK and water solubility, was found to be po-
tent in enzyme and cell-based assays (IC50 = 111 nM,
The plate was incubated at 30 °C for 30 min and then was stopped by adding of
3% phosphoric acid. All of the terminated kinase reaction was transferred to
P81 Ion Exchange Papers (Whatman, Cat No. 3698-915) using a cell harvester
(Inotech, Cat No. IH-110). Plates were washed 4 times with 0.5% phosphoric
acid solution and 1 time with acetone. 3 ml of scintillation cocktail was added,
and readout was performed using the Liquid Scintillation counter (Wallac, Cat
No. 1409). The result analysis was performed using SigmaPlot 10 (Systat
Software Inc., USA) to calculate IC50 values of the compounds. All mean values
were obtained from triplicate experiments.
EC50 = 1.78 lM). We are planning further studies to find out thera-
peutic candidate for the treatment of Alzheimer’s disease, cancers,
24. Cell culture: H4IIE cells were cultured routinely in grown as monolayers in
growth medium at an atmosphere of 95% air and 5% CO2 equilibrated at 37 °C.
The growth medium consisted of Dulbecco’s Modified Eagle’s Medium
and so on.
Acknowledgments
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine and 1%
penicillin–streptomycin solution. Cells were passaged every 3–4 days when
they reached 75% confluence.
Cell-based assay: Cells were plated on 96-well tissue culture plates at a density
This study was supported by a Grant from the Korea Healthcare
Technology R&D Project, Ministry for Health, Welfare & Family Af-
fairs, Republic of Korea (A091049-1222-0000300).
of 100,000 cells/well in a volume of 100
for approximately 3 h. Cells were washed in phosphate-buffered saline and
100 l of glucose production medium (glucose- and serum-free Dulbecco’s
ll of growth medium and left to attach
l
Modified Eagle’s Medium, containing 20 mM sodium lactate and 2 mM sodium
pyruvate solution) was added. The cells were left overnight at 37 °C in 5% CO2.
References and notes
The next day, the medium was replaced with 90
medium. Dilutions of compounds were performed in glucose production
medium and a 10 l volume was added to the cells. The treated cells were left
overnight and the following day a volume of 10 l medium was pipetted into
the 384-well black plate. 10
l of Amplex RedÒ working solution was then
added to each well and the plate was left in the dark for 30 min. The Amplex
RedÒ glucose/glucose oxidase assay kit is used to measure the modulation of
glucose production in H4IIE Cells. The fluorescence was measurement using
Wallac EnVisionTM plate reader (PerkinElmer Oy, Turku, Finland) at 560 nm
(ex) and 615 nm (em). Analysis of the results was performed using SigmaPlot
10 (Systat Software Inc., USA) to calculate EC50 values of the compounds. All
mean values were obtained from triplicate experiments.
ll of fresh glucose production
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