2-(2-Phenylmorpholin-4-yl)pyrimidin-4(3H)-ones; A new class of potent, selective and orally active glycogen synthase kinase-3β inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.09.020

A series of 2-(2-phenylmorpholin-4-yl)pyrimidin-4(3H)-ones was synthesized and examined for their inhibitory activity against glycogen synthase kinase-3β (GSK-3β). We found 21, 29 and 30 to possess potent in vitro GSK-3β inhibitory activity with good in vitro PK profiles. 21 demonstrated significant decrease of tau phosphorylation after oral administration in mice and excellent PK profiles.

Full text of DOI:10.1016/j.bmcl.2013.09.020

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6933–6937
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-(2-Phenylmorpholin-4-yl)pyrimidin-4(3H)-ones; A new class of
potent, selective and orally active glycogen synthase kinase-3b
inhibitors
Kenji Fukunaga, Fumiaki Uehara, Keiichi Aritomo , Aya Shoda à, Shinsuke Hiki §, Masahiro Okuyama,
⇑
,
Yoshihiro Usui –, Kazutoshi Watanabe , Koichi Yamakoshi –, Toshiyuki Kohara, Tokushi Hanano à,
Hiroshi Tanaka, Susumu Tsuchiya k, Shinji Sunada, Ken-Ichi Saito , Jun-ichi Eguchi àà, Satoshi Yuki,
Shoichi Asano, Shinji Tanaka, Akiko Mori §§, Keiji Yamagami ––, Hiroshi Baba kk, Takashi Horikawa ,
Masatake Fujimura
Mitsubishi Tanabe Pharma Corporation, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2013
Revised 19 August 2013
Accepted 5 September 2013
Available online 21 September 2013
A series of 2-(2-phenylmorpholin-4-yl)pyrimidin-4(3H)-ones was synthesized and examined for their
inhibitory activity against glycogen synthase kinase-3b (GSK-3b). We found 21, 29 and 30 to possess
potent in vitro GSK-3b inhibitory activity with good in vitro PK profiles. 21 demonstrated significant
decrease of tau phosphorylation after oral administration in mice and excellent PK profiles.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Glycogen synthase kinase-3b
Alzheimer’s disease
Tau protein
Hyperphosphorylation
Alzheimer’s disease (AD) is a neurodegenerative disorder de-
fined by the presence of extracellular plaques of b-amyloid (Ab),
and intracellular neurofibrillary tangles (NFTs). NFTs are composed
of abnormally hyperphosphorylated microtubule-associated pro-
tein tau and their temporal and spatial distribution correlate well
with severity of clinical symptoms, memory loss and cognitive
impairment.1
⇑
Corresponding author. Tel.: +81 48 433 2641; fax: +81 48 433 2729.
E-mail address: Watanabe.Kazutoshi@mb.mt-pharma.co.jp (K. Watanabe).
Present address: Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-
50, Kawagishi, Toda-shi, Saitama 335-8505, Japan.
Present address: Pharmacovigilance & Quality Assurance Division, Mitsubishi
Tanabe Pharma Corporation, 17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-
8405, Japan.
Present address: Development Division, Mitsubishi Tanabe Pharma Corporation,
17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-8405, Japan.
Present address: Tanabe R&D Service Co., Ltd, Kamoshida-cho, Aoba-ku, Yoko-
hama 227-0033, Japan.
Present address: Mitsubishi Tanabe Pharma Factory Ltd, 588-3, Kubota-cho,
Ashikaga-shi, Tochigi 326-0324, Japan.
Present address: Mitsubishi Chemical Holdings Corporation, 1-1, Marunouchi 1-
choume, Chiyoda-ku Tokyo 100-8251, Japan.
Present address: The KAITEKI Institute, Inc., 1-1, Marunouchi 1-chome, Chiyoda-
ku, Tokyo 100-8251, Japan.
Present address: Global Product Strategy Department, Mitsubishi Tanabe Pharma
Corporation, 17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-8405, Japan.
Glycogen synthase kinase-3b (GSK-3b) is a serine/threonine ki-
nase and is thought to be one of the key enzymes for aberrant tau
phosphorylation.2 Pathogenic activation of GSK-3b causes hyper-
phosphorylation of tau proteins, which induce their detachment
from microtubules leading to microtubule de-polymerization and
destabilization of neural cell processes. In parallel, hyperphospho-
rylated tau released from microtubules tends to misfold and aggre-
gate into paired helical filaments (PHFs) inside the cells to form
NFTs in the course of aging.3 A conditional GSK-3b overexpressing
transgenic mouse exhibits persistent tau hyperphosphorylation,
pretangle-like somatodendritic localization of tau, neuronal death
in hippocampus and cognitive deficits.4 These studies suggest that
GSK-3b is associated with AD progression and that GSK-3b inhibi-
tion is expected to be a promising therapeutic approach for AD.5
In the previous studies,6 we obtained 1 as a lead compound
which showed potent in vitro inhibitory activity against GSK-3b.
However, 1 showed no significant decrease of tau phosphorylation
after oral administration. We hypothesized that the N–H group,
carbonyl group and linear connection of linker between the phenyl
à
§
–
k
àà
§§
––
Present address: Translational Research Center, The University of Tokyo Hospital,
The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
kk
Present address: Research Division, Mitsubishi Tanabe Pharma Corporation, 1-1-
1, Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan.
Present address: National Institute for Minamata Disease, 4058-18 Hama,
Minamata City, Kumamoto 867-0008, Japan.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.020

6934
K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6933–6937
N
N
N
HS
H3C N
O
a
b
N
5
O
O
Ar COOH
Ar
Ar
Ar
OEt
N
1
N
3
IC50; 8.9 nM
weak in vivo activity
at 100 mg/kg, i.p.
4
N
H
O
N
2
N O
O
CH3
O
CH3
Cl
H3C N
O
c
N
Figure 1. Design for in vivo active compound.
6
group and the pyrimidin-2-one moiety would prevent the increase
of brain concentration by hydrogen bonding donation, moderate
metabolic stability or its flexible conformation. In order to confer
better in vivo inhibitory activity, we chose to transform the 2-
oxo-ethylamine moiety into a morpholine group in the expectation
that increases of metabolic stability and cell permeability would
result in an increase of brain concentrations (cell permeability of
1 was better than that of propranolol in Caco-2 permeability and
good brain concentration by intraperitoneal administration)
(Fig. 1). A molecular docking study suggested that 2 would have
protein-ligand interaction with GSK-3b as an possible inhibitor.7
The scaffold possesses two hydrogen bonding interactions with
GSK-3b, one between the nitrogen atom on the pyridine moiety
and the H–N bond of Val135, and the other between the carbonyl
oxygen atom of the pyrimidone moiety and the side chain of Lys85.
The morpholine moiety fills the space between the N- and C-termi-
Scheme 1. Reagent and conditions: (a) potassium monoethyl malonate, carbonyl
diimidazole, MgCl2, THF, 50 °C; (b) N-methylthiourea, DBU, EtOH, 80 °C; (c) POCl3,
DMF, dichloroethane, 60 °C.
borohydride11 yielded 2-phenylmorpholine 13, and removal of
the benzyl group by using 1-chloroethyl chloroformate12 yielded
2-(S)-phenylmorpholine hydrochloride 14. Desired compounds
15–31 were obtained by treatment of 6 with appropriate morphol-
ines 14 in the presence of triethylamine at room temperature
(Scheme 2). Phenylmorpholines containing a cyano group was
prepared by substitution of the corresponding N-protected
bromomorpholine by zinc cyanide catalyzed by tetrakis(triphenyl-
phosphine)palladium or tris(dibenzylideneacetone)dipalladium(0)
- 1,10-bis(diphenylphosphino)ferrocene in N,N-dimethylformam-
ide. 2-(S)-(2,6-Disubstituted-phenyl)morpholines were prepared
by separation of the racemate by chiral HPLC because of poor
enantioselectivity in an asymmetric reduction of 2,6-disubstituted
acetophenones. (R)-15 was synthesized using (R)-CBS instead of
(S)-CBS.
Preliminary results of cyclization of the linker are shown in
Table 1.13 Racemic phenylmorpholine 2 showed a severalfold de-
crease in inhibitory activity with maintained in vitro metabolic sta-
bility and improved CYP450 inhibition profiles. We thought that
the deletion of the carbonyl group would afford moderate micro-
somal clearance by increasing the electron density of the phenyl
moiety, and a fluorine atom was introduced at the 4-position.
Racemic 15 showed increased inhibitory activity and metabolic
stability with maintained CYP450 inhibition. (S)-Enantiomer of
15 had threefold more potent inhibitory activity against GSK-3b
than its (R)-enantiomer, which was consistent with the prediction
of the docking study for 2. From these results we thought that the
transformation including deletion of a hydrogen bonding donor as
nal domains, and the phenyl moiety makes a cation-p interaction
with Arg141 in the hydrophobic site of GSK-3b. The docking study
suggests only the (S)-isomer makes these interactions (Fig. 2).
2-(2-Phenylmorpholin-4-yl)-pyrimidin-4-ones 15–30 was syn-
thesized by the condensation of the appropriate 2-phenylmorpho-
line and 2-chloropyrimidin-4-one.8 b-Keto esters 4 were prepared
from aryl carboxylic acid 3 with potassium monoethylmalonate,
carbonyl diimidazole and magnesium chloride.9 Condensation of
the resulting keto esters 4 and N-methylthiourea afforded pyrimi-
dine-4(3H)-one 5, and successive treatment with phosphorous
oxychloride and N,N-dimethylformamide yielded key intermedi-
ates 6 (Scheme 1).
2-(S)-Phenylmorpholine 14 was prepared from the correspond-
ing 2-chloroacetophenone 7 in six steps. An asymmetric catalytic
reduction of 7 with BH3ꢀTHF and (S)-CBS afforded (S)-alcohol 8
with high enantioselectivity,10 and successive treatment of 8 with
potassium hydroxide yielded epoxide 9. After reaction of 9 and
benzylamine, the resulting amino alcohol 10 was acylated with
chloroacetyl chloride followed by cyclization with potassium
hydroxide to afford morpholin-2-one 12. Reduction of 12 with
in situ generated borane by chlorotrimethylsilane and lithium
a
b
R2
R2
R2
Cl
Cl
O
O
OH
7
8
9
O
R2
R2
c
d
e
Cl
N
H
Ph
N
OH
OH
11
Ph
10
HCl
NH
f
R2
g
R2
R2
N
Ph
N
Ph
O
O
O
O
13
12
14
Ar
N
h
N
R2
N
O
O
CH3
15-30
Scheme 2. Reagent and conditions: (a) (S)-CBS (20 mol %), BH3ꢀTHF, ꢁ30 °C; (b)
KOH, H2O, Et2O, r.t.; (c) benzylamine, 80 °C; (d) chloroacetyl chloride, NaOH, H2O,
CH2Cl2, r.t.; (e) KOH, 2-PrOH, r.t.; (f) LiBH4, TMSCl, THF, r.t.; (g) 1-chloroethyl
chloroformate, dichloroethane, r.t., then MeOH, reflux; (h) 6, Et3N, THF, r.t..
Figure 2. Predicted binding mode of 2 with GSK-3b by docking calculation.

K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6933–6937
6935
Table 1
Preliminary result of the effects of the linker transformation
N
N
2 (X=H)
X
N
15
(X=F)
N
O
O
CH3
CLb (ml/min/mg)
CYP450 inhibitionc (IC50
2D6
; lM)
a
Compd
GSK-3b IC50 (nM)
1A2
3A4
1
8.9
51
27
22
77
0.172
0.155
0.104
n.t.
5.70
23.1
23.9
17.1
n.t.
>50.0
47.4
35.8
44.2
n.t.
>50.0
>50.0
>50.0
>50.0
n.t.
(RS)-2
(RS)-15
(S)-15
(R)-15
n.t.
a
b
c
See Ref. 13 for inhibition assay conditions for human GSK-3b cell free enzyme.
Rat microsome.
Recombinant human CYP450 isoform inhibition against 1A2, 2D6 and 3A4.
Table 2
GSK-3b inhibition and recombinant human CYP450 inhibition
Ar
N
F
N
N
O
O
CH3
CYP450 inhibitionb (IC50
2D6
; lM)
a
Compd
Ar
GSK-3b IC50 (nM)
R1
1A2
3A4
(S)-15
16
17
18
19
20
H
3-F
3-Cl
3-CH3
3-OCH3
2-CI
22
7.4
430
2466
791
903
17.1
5.5
n.t.
n.t.
n.t.
n.t.
44.2
22.1
n.t.
n.t.
n.t.
n.t.
>50.0
>50.0
n.t.
n.t.
n.t.
n.t.
N
R1
N
21
12
29.4
39.7
>50.0
N
n.t.: not tested.
a
See Ref. 13 for inhibition assay conditions for human GSK-3b cell free enzyme.
Recombinant human CYP450 isoform inhibition against 1A2, 2D6 and 3A4.
b
well as a metabolically labile group and conformational restriction
would afford desired compounds by further modifications. Further
optimization of 15 was conducted using (S)-enantiomer.
CYP 2D6 inhibition. Introduction of a methoxy group increased
the inhibitory activity. 4-Methoxy derivative 23 and 3-methoxy
derivative 24 showed a moderate increase of inhibitory activity,
and more than 10-fold increase of activity was observed in 2-
methoxy derivative 26. Further introduction of a methoxy group
at the ortho position gave 2,6-dimethoxy analogue 28, which
was the most potent compound, showing a nearly 50-fold in-
crease of inhibitory activity. Effects of the introduction of sub-
stituents at the 2-position were also observed in the case of
the fluorine atom containing compound, regardless of its elec-
tronic profile, which showed a several fold increase of GSK-3b
inhibitory activity (21 vs 25). Although no effect was observed
against impaired microsomal human intrinsic clearance of meth-
oxy analogues by introducing an electron-withdrawing fluorine
atom at the 4-position (27), further introduction of an elec-
tron-withdrawing substituent to the 2-fluorophenyl moiety re-
sulted in 29 and 30 with increased in vitro activity and
desirable in vitro pharmacokinetic profiles.
Effects of the nitrogen atom of the pyridine moiety was sum-
marized in Table 2. Reduced GSK-3b inhibitory activity of pyri-
din-2-yl and 3-yl derivatives (data not shown) suggested that
the nitrogen atom at the 4-position of pyridine was essential,
and introduction of a fluorine atom at the 3-position of pyridine
(16) showed an increase in inhibitory activity together with in-
creased CYP 1A2 inhibition. Other substituents at the 3-position,
such as chlorine (17), methyl (18), methoxy (19) decreased the
activity independently of their electronic features. 2-Chloropyri-
dine derivative 20 showed a further decrease in in vitro activity
compared to that of 3-chloro isomer 17. These facts suggested
that the steric bulkiness of the substituent on the pyridine moi-
ety perturbed the inhibitory activity. Transformation of the pyr-
idine moiety into pyrimidine increased the inhibitory activity
against GSK-3b with improved in vitro pharmacokinetic profiles
(21). Based on this compound, we further examined the effect
of substituents on the phenyl group of the 2-phenylmorpholine
moiety (Table 3). 22, having an electron-withdrawing cyano
group at the 4-position maintained in vitro activity with potent
From these studies, 21, 29 and 30 proved to have preferable
in vitro GSK-3b inhibitory activity and in vitro PK profiles. Among
them, 21 was selected for further examinations of pharmacological
and pharmacokinetic profiles.

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K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6933–6937
Table 3
GSK-3b inhibition and recombinant human CYP450 inhibition
N
N
N
N
R2
N
O
O
CH3
b
Compd
R2
GSK-3b; IC50a(nM)
CLint,
(mL/min/mg)
CYP inhibitionc (IC50
2D6
; lM)
human
1A2
3A4
21
22
23
24
25
26
27
28
29
30
4-F
4-CN
12
13
2.9
4.6
0.06
0.03
0.11
0.13
0.10
0.19
0.18
0.20
0.03
0.07
29.4
39.7
4.5
6.9
26.6
32.4
16.0
18.0
>50.0
26.7
32.3
>50.0
n.t.
n.t.
>50.0
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
>50.0
>50.0
>50.0
23.3
15.7
16.5
>50.0
>50.0
19.5
4-OCH3
3-OCH3
2-F
2-OCH3
2-OCH3, 4-F
2,6-di-OCH3
2-F, 4-CN
2-F, 6-Cl
3.2
0.87
0.47
0.23
6.5
0.64
n.t.: not tested.
a
Conditions for human GSK-3b cell free enzyme inhibition assay, see Ref. 13.
Hunan liver microsome assay.
Recombinant human CYP450 isoform inhibition for 1A2, 2D6 and 3A4.
b
c
Table 4
Pharmacokinetic properties of 21
a
b
a
b
b
c
CLtot (L/h/kg)
T1/2 (h)
Vdss (L/kg)
Cmax (ng/L)
AUC0–1 (ngꢀh/ml)
Kp0ꢁ1
F (%)d
Caco-2 (ꢂ10ꢁ7 cm/s)
Pgp (MDR1/LLC)
2.9
1.7
2.4
221
1305
3.1
37
583
1.0
a
b
c
SD rats were given intravenously at 1.0 mg/kg (n = 5, 6 W, female, 0.5% HPMC suspension).
SD rats were given orally at 10 mg/kg (n = 5, 6W, female, 0.5% HPMC suspension).
Kp was measured at 1.0 mg/kg oral administration in ICR mice (n = 4, 7 W, male, 0.5% Tween suspension).
d
Bioavailability in rats.
Figure 3. (a) Dose–response of 21 for decrease of phosphorylated tau protein after 2 h. N: male normal mice, V: vehicle-treated mice, LiCl: LiCl (20 mmol/kg, i.p. 8 h). ⁄⁄p
<0.01 versus vehicle, Dunnett’s test ##p <0.01 versus vehicle, t-test, n = 6. (b) Time course for 21 (30 mg/kg, p.o.)-evoked decrease phosphorylated tau protein after p.o.
administration in normal mice. N: male normal mice. LiCl: LiCl (20 mmol/kg, i.p. 8 h) ⁄p <0.05, ⁄⁄p <0.01 versus normal, Dunnett’s test ##p <0.01 versus normal, t-test, n = 6.

K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6933–6937
6937
4. (a) Lucas, J. J.; Hernandez, F.; Gomez-Ramos, P.; Moran, M. A.; Hen, R.; Avila, J.
EMBO J. 2001, 20, 27; (b) Hernandez, F.; Borrell, J.; Guaza, C.; Avila, J.; Lucas, J. J.
J. Neurochem. 2002, 83, 1529; (c) Gomez de Barreda, E.; Perez, M.; Gomez-
Ramos, P.; Cristobal, J.; Martin-Maestro, P.; Moran, A.; Dawson, H. N.; Vitek, M.
P.; Lucas, J. J.; Hernandez, F.; Avila J. Neurobiol. Dis. 2010, 37, 622.
5. (a) Mazanetz, M. P.; Fischer, P. M. Nat. Rev. Drug. Disc. 2007, 6, 464; (b) Bhat, R.
V.; Haeberlein, S. L. B.; Avila, J. J. Neurochem. 2004, 89, 1313; (c) Cohen, P.;
Goedert, M. Nat. Rev. Drug Disc. 2004, 3, 479; (d) Hernandez, F.; Gomez de
Barreda, E.; Fuster-Matanzo, A.; Lucas, J. J.; Avila J. Exp. Neurol. 2010, 223, 322;
(e) Morales-Garcia, J. A.; Luna-Medina, R.; Alonso-Gil, S.; Sanz-SanCristobal, M.;
Palomo, V.; Gil, C.; Santos, A.; Martinez, A.; Perez-Castillo, A. ACS Chem.
Neurosci. 2012, 3, 963.
6. Uehara, F.; Shoda, A.; Aritomo, K.; Fukunaga, K.; Watanabe, K.; Ando, R.;
Shinoda, M.; Ueno, H.; Kubodera, H.; Sunada, S.; Saito, K.; Kaji, T.; Asano, S.;
Eguchi, J.; Yuki, S.; Tanaka, S.; Yoneyama, Y.; Niwa, Y. Bioorg. Med. Chem. Lett. in
press, http://dx.doi.org/10.1016/j.bmcl.2013.09.021.
7. Compound 2 was docked into the X-ray structures of GSK-3b (PDB code 1Q3W)
using Schrödinger’s GLIDE. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T.
A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.;
Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47, 1739. We searched
appropriate predictive binding pose by flexible docking calculation with main
hydrogen bondings with Val135 and Lys85. In the docking calculation, we also
prepared some rotamers of Arg141 because of its flexibility.
Rats and mice PK experiments of 21 were performed and
pharmacokinetic parameters are summarized in Table 4. Caco-2
permeability was high and no effects were observed on P-glyco-
protein (P-gp) efflux. The systemic clearance of 21 after iv dosing
averaged 2.9 L/h/kg with volume of distribution of 2.4 L/kg and
the AUC after p.o. dosing of 1305 ng h/mL in rats. 21 demonstrated
moderate oral bioavailability (37%) with reasonable half-lives in
rats, and excellent blood-brain-barrier penetration (brain/plasma
3.1) in mice for CNS drugs.
A kinase selectivity study revealed that 21 had more than 1000-
fold selectivity over 50 kinases except CK1 (IC50 = 1.5 lM). Results
of the inhibitory effects on in vivo Ser396 tau phosphorylation in
mice14 are shown in Figure 3. A dose response study showed 21
significantly decreased tau phosphorylation two hours after
administration at a dose of 30 mg/kg or higher doses and a time
course study showed 21 effectively inhibited in vivo tau phosphor-
ylation for two hours after oral administration at a dose of
30 mg/kg.
In conclusion, transformation of the 2-oxoethylamine moiety
into a morpholine moiety provided potent inhibitors of GSK-3b
with good in vitro pharmacokinetic profile such as 21, 29 and 30.
Among them, 21 was orally active, inhibited in vivo tau phosphor-
ylation in mice and showed moderate bioavailability in rats with
high kinase selectivity. A detailed profile of 21 will be published
elsewhere.
8. Okuyama, M.; Uehara, F.; Iwamura, H.; Watanabe, K. WO 2007/011065 A2; PCT
Int. Appl.
9. Brooks, D. W.; Lu, L. D.-L.; Masamune, S. Angew. Chem., Int. Ed. Engl. 1979, 18,
72.
10. Corey, E. J.; Helal, C. J. Angew. Chem. Int. Ed. 1998, 37, 1986. In the case of R2 is 4-
fluoro group, enantioselectivity of the corresponding chlorohydrin 8 is 95% ee
(Chiralcel OJ, mobile phase; hexane–ethanol (97/3), flow rate; 1.0 ml/min,
detection; UV 254 nm, temperature; 30 °C). Optical purity of assayed
compounds is up to 93% ee by HPLC analysis.
11. Giannis, A.; Sandhoff, K. Angew. Chem., Int. Ed. Engl. 1989, 28, 218.
12. Olofson, R. A.; Martz, J. M.; Senet, J.-P.; Piteau, M.; Malfroot, T. J. Org. Chem.
1984, 49, 2081.
Acknowledgment
13. Human GSK-3b cell free enzyme inhibition assay; 7.5
GS1 peptide and 10
32P]ATP were incubated in 50 mM N-(2-
hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES)-sodium
lM of prephosphorylated
l
M [c-
We gratefully thank Sanofi-Aventis R&D CNS Research Depart-
ment (formerly Sanofi-Synthelabo R&D, Bagneux, France) for a
fruitful collaboration.
hydroxide (pH 7.2), 1 mM dithiothreitol (DTT), 1 mM magnesium chloride,
0.02% Tween-20 buffer for 1 h at room temperature in the presence of human
recombinant GSK-3b. The reaction was stopped with 0.1 volume of 21%
perchloric acid. An aliquot of the reaction mixture was then transferred onto
Whatman P81 cation exchange filters and the filters were washed 3 times with
75 mM phosphoric acid solution, once in water and once in acetone.
Incorporated 32P radioactivity was determined by liquid scintillation
spectrometry. The prephosphorylated GS1 peptide had the following
sequence; NH2-YRRAAVPPSPSLSRHSSPHQS(P)EDEE-COOH. IC50 values are the
mean of at least two experiments.
References and notes
1. (a) Grundke-Iqbal, I.; Iqbal, K.; Tung, Y. C.; Quinlan, M.; Wisniewski, H. M.;
Binder, L. I. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4913; (b) Friedhoff, P.; von
Bergen, M.; Mandelkow, E.-M.; Mandelkow, E. Biochim. Biophys. Acta 2000,
1502, 122; (c) Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
14. ICR mice (male, 5–6 w) were orally treated with 21 (10–100 mg/kg, 0.5%
Tween80 suspension). Each group was then sacrificed at a given time, from 1
up to 6 h after drug administration. Prefrontal cortexes were sonicated with
homogenization buffer (62.5 mM Tris–HCl pH 6.8, 2.3% sodium dodecyl sulfate
(SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol
tetraacetic acid (EGTA), 1 mM DTT, Protease inhibitor cocktail and Phosphatase
inhibitor cocktail), boiled for 5 min and centrifuged at 15000g for 15 min at
4 °C. Protein content was measured and samples were anti-pS396-tau antibody
and bands were quantified by using a fluorescent scanner and ImageQuant
software. Data were analyzed mixed with Laemmli buffer, then applied on
Western blot. Phosphorylated tau protein was detected by Dunnett’s test and t-
test.
2. (a) Ishiguro, K.; Takamatsu, M.; Tomizawa, K.; Omori, A.; Takahashi, M.; Arioka,
M.; Uchida, T.; Imahori, K. J. Biol. Chem. 1992, 267, 10897; (b) Sperber, B. R.;
Leight, S.; Goedert, M.; Lee, V. M. Neurosci. Lett. 1995, 197, 149; (c) Ali, A.;
Hoeflich, K. P.; Woodgett, J. R. Chem. Rev. 2001, 101, 2527; (d) Rayasam, G. V.;
Tulasi, V. K.; Sohdi, R.; Davis, J. A.; Ray, A. Br. J. Pharmacol. 2009, 156, 885; (e)
Phukan, S.; Babu, V. S.; Kannoji, A.; Hariharan, R.; Balaji, V. N. Br. J. Pharmacol.
2010, 160, 1; (f) Martin, L.; Lapypova, X.; Wilson, C. M.; Magnaudeix, A.; Perrin,
M.-L.; Yardin, C.; Terro, F. Ageing Res. Rev. 2013, 12, 289.
3. (a) Planel, E.; Sun, X.; Takashima, A. Drug Dev. Res. 2002, 56, 491; (b) Avila, J.
FEBS Lett. 2006, 580, 2922; (c) Hooper, C.; Killick, R.; Lovestone, S. J. Neurochem.
2008, 104, 1433; (d) Gendron, T. F.; Petrucelli, L. Mol. Neurodegener. 2009, 4, 13;
(e) Hanger, D. P.; Anderton, B. H.; Noble, W. Trends Mol. Med. 2009, 15, 112; (f)
Martin, L.; Latypova, X.; Terro, F. Neurochem. Int. 2011, 58, 458.