6-(4-Pyridyl)pyrimidin-4(3H)-ones as CNS penetrant glycogen synthase kinase-3β inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.09.021

The discovery of a series of 6-(4-pyridyl)pyrimidin-4(3H)-ones derived from a hit compound with low molecular weight and sufficient chemical space is reported. Transformation of substituents led to subnanomolar potent inhibitors with in vivo tau phoshorylation lowering activity.

Full text of DOI:10.1016/j.bmcl.2013.09.021

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6928–6932
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
6-(4-Pyridyl)pyrimidin-4(3H)-ones as CNS penetrant glycogen
synthase kinase-3b inhibitors
Fumiaki Uehara, Aya Shoda , Keiichi Aritomo à, Kenji Fukunaga, Kazutoshi Watanabe à, , Ryoichi Ando,
Masaki Shinoda §, Hiroaki Ueno à, Hideo Kubodera, Shinji Sunada, Ken-Ichi Saito –, Takahide Kaji,
Shoichi Asano, Jun-ichi Eguchi k, Satoshi Yuki, Shinji Tanaka, Yukimi Yoneyama , Takuro Niwa àà
⇑
Research Division, 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 17 September 2013
The discovery of a series of 6-(4-pyridyl)pyrimidin-4(3H)-ones derived from a hit compound with low
molecular weight and sufficient chemical space is reported. Transformation of substituents led to subn-
anomolar potent inhibitors with in vivo tau phoshorylation lowering activity.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Glycogen synthase kinase 3b
Alzheimer’s disease
Tau protein
Hyperphoshorylation
Pyrimidin-4(3H)-one
Glycogen synthase kinase-3 (GSK-3) is a proline-directed ser-
ine-threonine kinase that plays important roles in metabolism, cell
cycle, cell survival and so on. It was initially identified as the en-
zyme that was able to deactivate glycogen synthase through phos-
and phosphorylates tau proteins which regulate microtubule sta-
bility in neural cells.1
Alzheimer’s disease (AD) is a neurodegenerative disorder with
problems of progressive memory loss and cognitive impairment.
AD is defined by the two major histological hallmarks, senile
plaque deposition composed of b-amyloid peptide and intracellular
neurofibrillary tangles (NFTs) composed of hyper-phosphorylated
tau. The temporal and spatial distribution of NFTs correlates well
with the clinical disease severity2 and dysregulation of GSK-3 has
been implicated in Alzheimer disease (AD) and active forms of
GSK-3b localise to pretangle pathology including dystrophic neuri-
tis and neurofibrillarytangles (NFTs) in AD brain.3 Therefore, inhibi-
tion of GSK-3b has been considered to be a potential therapeutic
approach for AD or other tauophathies such as frontotemporal
dementia and Pick’s disease.4 Many chemical series of GSK-3b
inhibitors are reported so far and have been reviewed in the litera-
ture.5 Lithium is the first known inhibitor against GSK-3b which
inhibits GSK-3b directly and indirectly, and is widely used in phar-
macological studies.6 Many groups including SmithKlineBeecham,7
Johnson & Johnson,8 Eli Lilly9 and Roche10 reported maleimide
derivatives with aryl, heteroaryl and aniline groups at the 3- and
4-positions. Oxadiazole,11 pyrimidine,12 oxazole,13 thiadiazolidi-
nones14 and thiazole15 are also used as the monocyclic scaffolds of
GSK-3b inhibitors. As the bicyclic scaffold, many types of substi-
tuted aromatic 6,5- and 6,6-fused heterocycles have been
described such as benzimidazoles,16 purines, 17pyrazolopyridines,18
imidazopyridines,19 pyrazolopyrimidines, 20 pyrazolopyridazines,21
phorylation. GSK-3 is now known to be
a versatile kinase
regulating function of many other substrates including transcrip-
tion factors to modulate downstream signaling. GSK-3 exists as
two isoforms, GSK-3a (51 kDa) and GSK-3b (47 kDa) sharing high
homology in their kinase domains. Both isoforms are ubiquitously
expressed, with particularly high levels observed in the brain and
testes. In most brain areas, GSK-3b is the predominant isoform
⇑
Corresponding author.
E-mail address: Watanabe.Kazutoshi@mb.mt-pharma.co.jp (K. Watanabe).
Present address: Pharmacovigilance & Quality Assurance Division, Mitsubishi
Tanabe Pharma Corporation, 17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-
8405, Japan.
à
Present address: Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-
50, Kawagishi, Toda-shi, Saitama 335-8505, Japan.
§
Present address: API Corporation, 2-3-4, Nihonbashi, Chuo-ku, Tokyo 103-0027,
Japan.
–
Present address: Mitsubishi Chemical Holdings Corporation, 1-1, Marunouchi 1-
chome, Chiyoda-ku, Tokyo 100-8251, Japan.
k
Present address: The KAITEKI Institute, Inc., 1-1, Marunouchi 1-chome, Chiyoda-
ku, Tokyo 100-8251, Japan.
Present address: Pharmacovigilance & Quality Assurance Division, Mitsubishi
Tanabe Pharma Corporation, 2-6-6, Hirano-machi, Chuo-ku, Osaka-shi, Osaka 541-
0046, Japan.
àà
Present address: Japan Bioindustry Association, 2-26-9, Hatchobori, Chuo-ku,
Tokyo 104-0032, Japan.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.021

F. Uehara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6928–6932
6929
Table 1
N
Preliminary SAR of R1 and R2 on the pyrimidone ring
N
6
1 N
5
4O
GSK-3β IC50
:
3 μM
MW:
187.20
2
Me
N
H3
N
R1
N
O
1
R2
Figure 1. Structure of the hit compound 1.
Compd
R1
R2
IC50 (nM)*1
LiCl
1
6530
3000
Me
H
H
∗
∗
2
1300
∗
3
H
H
2100
4
5
2400
1800
∗
∗
H
6
7
H
1300
8900
500
∗
H
8
H
∗
∗
∗
∗
∗
9
Me
500
∗
10
11
12
1900
9800
2200
∗
∗
*1
: Cell free enzyme inhibition assay was performed by second protocol (Ref. 28).
Figure 2. Docking study of hit compound 1 with GSK-3b.
furopyrimidines22 and quinolones.23 GSK-3b inhibitors derived
from natural products has also reported in the literature such as
indirubins,24 paullones,25 hymenialdisines26 and manzamines.27
In this Letter, we report the design and synthesis of brain penetrant
GSK-3b inhibitors for the treatment of AD.
Several candidates for the inhibitor were selected via in silico
screening of our chemical library. Following cell-free enzyme inhi-
bition assay28 of the representative compounds hit compound 1
(Fig. 1) was obtained as a low molecular weight inhibitor with
moderate but more potent activity than lithium chloride (molecu-
Further optimizations of the phenylpropyl side chain were car-
ried out (Table 2).31 Introduction of an oxygen atom at the 2-posi-
tion decreased the activity (13), whilst the NH-linked derivative 14
showed a slight increase in activity compared to 9. Replacement of
the phenyl group by a cyclohexyl group (15) resulted in a twofold
increase of activity compared to 14. Introduction of a hydroxy
group at the benzyl position (16) led to a compound three times
as potent as 14 and phenacyl analogues 17 exhibited a large in-
crease in activity.
lar weight; 187.20, IC50; 3
l
M).
We reasoned that generation of several interactions between
the phenacyl moiety and the enzyme would lead to a significant in-
crease of the in vitro activity. Figure 3 shows the result of the dock-
ing study of 17 with GSK-3b. In this study, we prepared several
Docking study of 1 with the active site of GSK-3b suggested that
the nitrogen atom at the 4-position on the pyridine acted as an
acceptor of hydrogen bonding with amide bond of Val135 in the
hinge region and the oxygen atom of the C2 carbonyl group on
the pyrimidone would be able to form a unique hydrogen bonding
network with Lys85 (Fig 2).29 In addition, the 2-methyl group on
the pyrimidone is located in an external area of the ATP binding
pocket, suggesting that optimizations at this position could en-
hance the in vitro activity and improve in vitro pharmacokinetic
parameters without perturbing the recognition elements inside
the ATP pocket.
protein structures with
a flexible side chain of rotamer of
Arg141, and docked 17 in each structure. The nitrogen atom on
the pyridine ring made a hydrogen bond with the amide of
Val135 and the oxygen atom of the carbonyl moiety in the pyrim-
idone ring made a hydrogen bonding network with Lys85 in the
same mode with 1. An additional interaction was observed be-
tween the phenyl group and the guanidinyl moiety of Arg141
through a cation–p or p–p interaction. No obvious interactions
Preliminary structure activity relationships of the substituents
at the 2- and 3-positions on the pyrimidone are shown in Table 1.30
Introduction of a n-butyl group at the 2-position slightly increased
in vitro activity compared to branched alkyl analogues (2, 3 and 4).
Phenyl derivative 5 also maintained a moderate inhibitory activity
and phenylpropyl analogue 8 was the most potent compound
among phenylalkyl derivatives (6, 7 and 8). Interestingly, N-methyl
analogue 9 had similar activity to 8 (R2 = H), indicating that hydro-
gen bonding at this position was not essential for GSK-3b inhibi-
tion, while ethyl, n-propyl and benzyl analogues (10, 11, and 12,
respectively) showed decreased inhibitory activities.
were observed between the carbonyl moiety of the phenacyl group
and amino acid residues in the predicted binding mode. A hydro-
gen donor moiety was also introduced on the 3-position of pyrim-
idone for further hydrogen bonding with amino acid residues of
the external region in the ATP-binding site. Aminobutyl derivative
18 and aminopentyl derivative 19 showed no increase of activity
compared to 14, suggesting that these aminoalkyl substituents
did not make interactions with any amino acid residues.
Compound 17 showed a satisfactory clearance profile in rat
microsomes but potent inhibition against CYP1A (Table 3).31 There
were no significant changes in CYP1A inhibition for ortho or meta

6930
F. Uehara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6928–6932
Table 2
Table 3
Phenylpropyl analogues at 2-position
Substitution effects on phenyl group toward IC50, clearance and CYP1A inhibition
N
N
N
N
X
R3
N
H
N
O
X
N
O
O
Me
R4
Me
Compd
X
IC50 (nM)*1
CL (ml/min/mg)*2
CYP1A (IC50: lM)
Compd
X
R3
R4
IC50 (nM)*1
90
17
20
21
22
23
24
25
H
8.9
27
25
52
13
38
13
0.172
0.158
0.207
H0.166
H0.262
H0.262
0.133
5.7
3.8
4.2
10.9
0.67
2.2
20-F
9
CH2
30-F
∗
∗
∗
∗
∗
40-F
30-Cl
30-CH3
30-CN
13
14
15
O
N
N
200
70
>53.3
H
H
*1
*2
: Cell free enzyme inhibition assay was performed by second protocol (Ref. 28).
: Rat microsome.
39
120
100
16
17
N
N
H
23
OH
O
H
8.9
∗
*
*
*
80
60
40
20
0
H2N
∗
18
19
N
N
130
150
∗
∗
H2N
∗
***
*1
: Cell free enzyme inhibition assay was performed by second protocol (Ref. 28).
normal 15 min 30 min
1 h
2 h
LiCl
Time after administration
Figure 4. Time course for 17 (100 mg/kg, ip)-evoked decrease phosphorylated tau
⁄⁄⁄
;
protein after ip administration in normal rats. LiCl; LiCl (10 mmol/kg, ip 24 h). ⁄
,
p < 0.05, 0.005 versus normal, respectively, Dunnett’s test (N = 8).
NH
R1
N
O
N
N
NH2
a
b
O
N
N
R1
N
H
O
R1
N
O
OEt
26
R2
9-12
1-8
Scheme 1. Reagents and condition: (a) K2CO3, EtOH, reflux; (b) R2-I or R2-Br, NaH,
DMF.
Figure 3. Docking study of 17 with GSK-3b.
fluoro substituted analogues (20, 21) and introduction of a fluorine
atom at the para position led to a moderate decrease of CYP1A inhi-
bition (22). Both meta-chloro analogue 23 and meta-methyl
analogue 24 showed poor in vitro pharmacokinetic profiles in both
metabolic stability and CYP 1A inhibition. The decrease of lipophil-
icity is generally effective for the improvement of in vitro ADME
profiles and meta-cyano analogue 25 showed no CYP1A inhibition
without loss of in vitro activity and metabolic stability. Pharmaco-
kinetics studies indicated that 17 had a good brain concentration
The synthesis of compounds 1–12 is shown in Scheme 1. Treat-
ment of ethyl 3-oxo-3-(4-pyridyl)propionate (26)33 with amidines
under basic conditions afforded 1–8. Alkylation of phenylpropyl
derivative 8 with appropriate alkyl halides yielded the correspond-
ing N-alkyl analogues 9–12. Preparation of O-linker analogue 13
and N-linker analogue 14–19 is summarized in Scheme 2. 2-Mer-
capto-3-methyl pyrimidone 27 was prepared by condensation
with ketoester 26 and N-methylthiourea. Chlorination of 27 under
Vilsmeier conditions gave 2-chloropyrimidone 28 in good yield.
Condensation of 28 with 2-phenethyl alcohol or appropriate
amines afforded 2-alkoxy and 2-alkylamino analogues 13–17.34
Alkylation of 14 with N-tert-butoxycarbonyl protected 4-iodobu-
tylamine or 5-iodopentylamine in the presence of sodium hydride
and subsequent deprotection yielded 18 and 19, respectively.
(11.2 lM/ml at 0.25 h, 30 mg/kg, ip, rat) and a high central nervous
system penetration (Kp brain; 1.15) so that further pharmacologi-
cal tests were conducted. Intraperitoneal administration at a dose
of 100 mg/kg of 17 induced a decrease by 24% of tau phosphoryla-
tion in the brain compared to vehicle rat (Fig. 4)32

F. Uehara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6928–6932
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a
b
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N
N
c
d, e
N
N
R3X
O
R3R4N
O
N
N
Me
Me
13-17
18, 19
Scheme 2. Reagents and condition: (a) N-methyl thiourea, DBU, EtOH, reflux; (b)
POCl3, DMF, reflux; (c) R3XH, Et3N, THF, rt to reflux; (d) BocNH-(CH2)4-I or BocNH-
(CH2)5-I, NaH, DMF; (e) 4 N-HCl, AcOEt.
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In conclusion, a novel series of pyrimidones as GSK-3b inhibi-
tors from hit compound 1 was developed. Focusing on 2-substi-
tuted derivatives led to phenacyl analogue 17, which showed
subnanomolar inhibitory activity and good brain permeability,
and reduced tau phosphorylation in mouse brain. Further optimi-
zations for orally active compounds from 17 will be reported in
near future.
19. Lee, S.-C.; Kim, H. T.; Park, C.-H.; Lee, D. Y.; Chang, H.-J.; Park, S.; Cho, J. M.; Ro,
S.; Suh, Y.-G. Bioorg. Med. Chem. Lett. 2012, 22, 4221.
Acknowledgments
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H. Q.; Thomson, S. A. Bioorg. Med. Chem. Lett. 2004, 14, 2121; (b) Peat, A. J.;
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D.; Slingsby, B. P.; Smith, D. G.; Ward, R. W. Bioorg. Med. Chem. Lett. 2003, 13,
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Preugschat, F.; Thomson, S. A.; Wang, T. Y.; Zhou, H.-Q. J. Med. Chem. 2004, 47,
4716.
We are grateful to Sanofi-Aventis R&D CNS Research Depart-
ment (formerly Sanofi-Synthelabo R&D, Bagneux, France) for a
fruitful collaboration. In addition, the authors would like to thank
Masako Okamoto, molecular modeling group, for docking studies.
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25. Leost, M.; Schultz, C.; Link, A.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.; Bibb, J.
A.; Snyder, G. L.; Greengard, P.; Zaharevitz, D. W.; Gussio, R.; Senderowicz, A.
M.; Sausville, E. A.; Kunick, C.; Meijer, L. Eur. J. Biochem. 2000, 267, 5983.
26. Meijer, L.; Thunnissen, A. M. W. H.; White, A. W.; Garnier, M.; Nikolic, M.; Tsai,
L. H.; Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu, Y. Z.; Biernat, J.; Mandelkow,
E. M.; Kim, S. H.; Pettit, G. R. Chem. Biol. 2000, 7, 51.
8. (a) Zhang, H.-C.; Boñaga, L. V. R.; Ye, H.; Derian, C. K.; Damiano, B. P.; Maryanoff,
B. E. Bioorg. Med. Chem. Lett. 2007, 17, 2863. and references cited herein; (b)
Shen, L.; Prouty, C.; Conway, B. R.; Westover, L.; Xu, J. Z.; Look, R. A.; Chen, X.;
Beavers, M. P.; Roberts, J.; Murray, W. V.; Demarest, K. T.; Kuo, G.-H. Bioorg.
Med. Chem. 2004, 12, 1239.
27. Hamann, M.; Alonso, D.; Martín-Aparicio, E.; Fuertes, A.; Pérez-Puerto, M. J.;
Castro, A.; Morales, S.; Navarro, M. L.; Monte-Millán, M.; Medina, M.; Pennaka,
H.; Balaiah, A.; Peng, J.; Cook, J.; Wahyuono, S.; Martinez, A. J. Nat. Prod. 2007,
70, 1397.
28. Cell free assay conditions: First protocol; 4.1
lM of prephosphorylated GS1
9. (a) Engler, T. A.; Henry, J. R.; Malhotra, S.; Cunningham, B.; Furness, K.;
Brozinick, J.; Burkholder, T. P.; Clay, M. P.; Clayton, J.; Diefenbacher, C.;
Hawkins, E.; Iversen, P. W.; Li, T.; Lindstrom, T. D.; Marquart, A. L.; McLean, J.;
Mendel, D.; Misener, E.; Briere, D.; O’Toole, J. C.; Porter, W. J.; Queener, S.; Reel,
J. K.; Owens, R. A.; Brier, R. A.; Eessalu, T. E.; Wagner, J. R.; Campbell, R. M.;
peptide and 42 ATP were incubated in 80 mM Mes-NaOH, pH 6.5,
1 mM Mg acetate, 0.5 mM EGTA, 5 mM 2-mercaptoethanol, 0.02% Tween 20,
10% glycerol buffer for 2 h at room temperature in the presence of GSK-3b.
lM
Second protocol; 7.5
lM of prephoshorylated GS1 peptide and 10 lM
[c
32P]ATP were incubated in 50 mM N-(2-hydroxyethyl)piperazine-N0-(2-

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F. Uehara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6928–6932
ethanesulfonic acid) (HEPES)-sodium hydroxide (pH 7.2), 1
(DTT), 1 mM magnesium chloride, 0.02% Tween-20 buffer for 1 hour at room
temperature in the presence of human recombinant GSK-3b.
l
M dithiothreitol
33. (a) Machida, K.; Hirose, Y.; Fuse, S.; Sugawara, T.; Takahashi, T. Chem. Pharm.
Bull. 2010, 58, 87; (b) Almario G. A.; Ando, R.; Arimoto, K.; Uehara, F.; Li, A. T.;
Shoda, A; Frost, J. R.; Watanabe, K. US2003/187004.
In both protocols, 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.
Prephosphorylated GS1 peptides had the following sequence; NH2-
YRRAAVPPSPSLSRHSSPHQS(P)EDEE-COOH. IC50 values are the mean of at
least two experimental data.
34. Synthesis of 17: A solution of ethyl 3-oxo-3-(4-pyridyl)propionate (29.0 g,
150 mmol), N-methyl thiourea (40.6 g, 450 mmol) and 1,8-diazabicyclo[5,4,0]-
7-undecene (22.4 ml, 150 mmol) was refluxed for 4 h and the solution of
methanesulfonic acid (14.4 g, 150 mmol) in water (50 ml) was added after
cooling by ice-water. The precipitate was filtered, washed with water and dried
to afford 2-mercapto-3-methyl-6-pyridin-4-yl-3H-pyrimidin-4-one (23.7 g,
72%). 1H NMR (DMSO-d6) d:3.58 (s, 3H), 6.40 (s, 1H), 7.72 (dd, J = 1.8, 4.5 Hz,
2H), 8.73 (dd, J = 1.8, 4.8 Hz, 2H), 12.92 (brd, 1H). Phosphorous oxychloride
(26.11 g, 170 mmol) was added to N,N-dimethylformamide (180 ml) and
stirred for 20 min. 2-Mercapto-3-methyl-6-pyridin-4-yl-3H-pyrimidin-4-one
(24.15 g, 110 mmol) was added to the solution and stirred at 70 °C for 2 h.
Ethyl acetate (630 ml) was added to the ice-cooled solution and precipitate
was dissolved in water (400 ml) and then pH was adjusted to 10 by aqueous
sodium hydroxide. The precipitate was filtered, washed with water and dried
to afford 2-chloro-3-methyl-6-pyridin-4-yl-3H-pyrimidin-4-one (18.82 g,
77%). 1H NMR (CDCl3) d:3.72 (s, 3H), 6.90 (s, 1H), 7.78 (dd, J = 1.7, 4.5 Hz,
2H), 8.75 (dd, J = 1.6, 4.5 Hz, 2H).
A solution of 2-chloro-3-methyl-6-pyridin-4-yl-3H-pyrimidin-4-one (0.665 g,
3.00 mmol), 2-amino-1-phenyl-ethanone hydrochloride (1.03 g, 6.00 mmol)
and triethylamine (0.80 ml, 6.00 mmol) in tetrahydrofuran was stirred at room
temperature. After stirring for 3 h, water was added to the reaction mixture.
The precipitate was filtered and washed with diethyl ether to afford the title
compound (0.556 g, 68%). 1H NMR (CDCl3) d:3.41 (s, 3H), 4.90 (d, J = 5.1 Hz,
2H), 6.46 (s, 1H), 7.50-7.65 (m, 2H), 7.67-7.80 (m, 3H), 7.90 (t, J = 5.1 Hz, 2H),
8.08 (m, 2H), 8.47 (dd, J = 1.5, 4.8 Hz, 2H). MS[M+H]+:321.
29. Compound 1 was docked into the X-ray structure of GSK-3b (PDB code 1Q3W)
using Schrödinger’s GLIDE 29 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.
30. In vitro assay was conducted by first protocol shown in Ref. 28.
31. In vitro assay was conducted by second protocol shown in Ref. 28.
32. In vivo assay of 17; Wistar rats (male, 7w, N = 8) were orally or
intraperitoneally treated with 17 (100 mg/kg, 0.5% tween80 suspension).
Each group was then sacrificed at a given time, from 15 min up to 2 h after
drug administration. Prefrontal cortexes were sonicated with homogenization
buffer (62.5 mM Tris–HCl pH6.8, 2.3% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, Protease inhibitor cocktail and Phosphatase inhibitor cocktail), boiled for
5 min and centrifuged at 15,000g for 15 min at 4 °C. Protein content was
measured and samples were mixed with Laemmli buffer, then applied on
Western blot. Phosphorylated tau protein was detected with anti-pS396-tau
antibody and bands were quantified by using
ImageQuant software.
a fluorescent scanner and