Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase kinase-3β inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.077

We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β inhibitors from our promising compounds containing a 3-methylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GS

Full text of DOI:10.1016/j.bmcl.2017.06.077

Accepted Manuscript
Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as gly-
cogen synthase kinase-3β inhibitors
Toshiyuki Kohara, Kazuki Nakayama, Kazutoshi Watanabe, Shin-ichi Kusaka,
Daiki Sakai, Hiroshi Tanaka, Kenji Fukunaga, Shinji Sunada, Mika Nabeno,
Ken-Ichi Saito, Jun-ichi Eguchi, Akiko Mori, Shinji Tanaka, Tomoko Bessho,
Keiko Takiguchi-Hayashi, Takashi Horikawa
PII:
S0960-894X(17)30692-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.077
BMCL 25111
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 March 2017
16 June 2017
29 June 2017
Please cite this article as: Kohara, T., Nakayama, K., Watanabe, K., Kusaka, S-i., Sakai, D., Tanaka, H., Fukunaga,
K., Sunada, S., Nabeno, M., Saito, K-I., Eguchi, J-i., Mori, A., Tanaka, S., Bessho, T., Takiguchi-Hayashi, K.,
Horikawa, T., Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase
kinase-3β inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.06.077
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as
glycogen synthase kinase-3β inhibitors
Toshiyuki Koharaa, Kazuki Nakayama, Kazutoshi Watanabe*, Shin-ichi Kusaka, Daiki Sakai, Hiroshi
Tanaka, Kenji Fukunaga, Shinji Sunada, Mika Nabeno, Ken-Ichi Saitob, Jun-ichi Eguchic, Akiko Morid,
Shinji Tanaka, Tomoko Besshoe, Keiko Takiguchi-Hayashi f and Takashi Horikawag
Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β
inhibitors from our promising compounds containing
a 3-methylmorpholine moiety.
Revised
Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3β
inhibitors. SAR studies focused on the nitrogen atom of the piperazine moiety revealed that a
phenyl group afforded potent inhibitory activity toward GSK-3β. Docking studies indicated that
the phenyl group on the piperazine nitrogen atom and the methyl group on the piperazine make
cation-π and CH-π interactions with GSK-3β respectively. 4-Methoxyphenyl analogue 29
showed most potent inhibitory activity toward GSK-3β with good in vitro and in vivo
pharmacokinetic profiles, and 29 demonstrated a significant decrease in tau phosphorylation
after oral administration in mice.
Accepted
Available online
Keywords:
Glycogen synthase kinase-3
Alzheimer’s disease
alkylpiperazine
Keyword_4
2009 Elsevier Ltd. All rights reserved
.
Keyword_5
a Present address: Sales & Marketing Division, Mitsubishi Tanabe Pharma
Corporation, 3-2-10, Dosho-machi, Chuo-Ku, Osaka 541-8505, Japan.
b Present address: Life Science Institute, Inc., THE KAITEKI Bldg., 13-4,
Uchikanda 1-chome, Chiyoda-ku, Tokyo 101-0047, Japan.
potent GSK-3β inhibitory activity together with a certain level of
CYP inhibition probably due to the 3-fluoropyridin-4-yl group at
the 6-position of the pyrimidone moiety. Based on the
information about hydrogen bonding of the piperazine moiety,
we hypothesize that transformation of the alkylmorpholine
moiety into the corresponding piperazine moiety would introduce
new hydrogen bonding toward the enzyme indicated by the
docking study of the phenylpiperazine, and that a potential
c Present address: The KAITEKI Institute, Inc., 1-1, Marunouchi 1-chome,
Chiyoda-ku, Tokyo 100-8251, Japan.
d Present address: Medicines Development Unit Japan, Eli Lilly Japan K.K.
7-1-5, Isogamidori, Chuo-ku, Kobe 651-0086, Japan.
e Present address: Business Development Department, Mitsubishi Tanabe
Pharma Corporation,17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-
8405, Japan
increase in activity by this interaction would enable a
transformation of the 3-fluoropyridin-4-yl moiety into a pyridine-
4-yl or a pyrimidin-4-yl moiety which would lead to evolution of
a new chemical series with decreased CYP inhibition. Namely,
we designed 2-methylpiperazine analogues by transforming the
morpholine moiety of 3-methylmorpholine analogues such as 1
shown in Fig. 1.2 In this paper we describe results of the
transformation and successive optimization of alkylpiperazine
derivatives 2.
f Present address: Mitsubishi Chemical Cleansui Corporation, Gate City
Osaki East Tower, 1-11-2, Osaki, Shinagawa-Ku, Tokyo 141-0032, Japan
g
Present address: Ikuyaku, Integrated Value Development Division,
Mitsubishi Tanabe Pharma Corporation, 17–10, Nihonbashi-Koamicho,
Chuo-ku, Tokyo 103-8405, Japan.
In a previous paper, we described the transformation of the
morpholine moiety of previously reported GSK-3β inhibitors
with
a 2-(2-phenylmorpholin-4-yl)-pyrimidin-4-one skeleton
such as UDA-680 into the corresponding piperazine analogues. A
docking study indicated that potent inhibitory activity of
phenylpiperazine analogues toward GSK-3β was attributed to
hydrogen bonding between the nitrogen atom of the piperazine
moiety and the oxygen atom of the main chain of Gln185 which
was not observed for the phenylmorpholine analogues.1 We also
reported that the alkylmorpholine analogues generally showed
Fig. 1. Transformation of morpholine moiety.

Preliminary results of the transformation of the 3-
methylmorpholine moiety into a 2-methylpiperazine moiety are
shown in Table 1.4 Simple transformation of the oxygen atom of
the (R)-3-methylmorpholine into a nitrogen atom was well
tolerated and 3 and 5 showed good inhibitory activity, nearly 2-
fold more active for a 4-pyrimidinyl analogue than for a 4-
pyridyl analogue. Introduction of a methyl group on the nitrogen
atom of the piperazine showed a 2- to 3-fold decrease in activity
(3 vs 4, 5 vs 6). The effect of the (R)-2-methyl group was clearly
observed by comparing it with that of des-methyl piperazine
analogue 11, indicating that introduction of a (R)-methyl group
afforded a nearly 30-fold increase in activity. The methyl group
with (S)-configuration generally showed a large decrease in
activity. For example, 7 and 9 showed a 6 to 30-fold decrease in
activity compared with the corresponding (R)-isomers, 3 and 5,
respectively, and N-methyl analogues 8 and 10 lost the activity.
The (S)-2-methyl group seemed detrimental to the inhibitory
activity compared with des-methyl analogue 11 which still had
weak inhibitory activity.
with the main chain of Val135 in the hinge region, and the
carbonyl oxygen atom of the pyrimidone moiety forms hydrogen
bond with the side chain of Lys85.2,5 The methyl group of 3-(R)-
methylmorpholine of 1 was buried in a hydrophobic pocket of the
active site in the enzyme.2 The amino moiety of the piperazine of
5 makes hydrogen bonding with the oxygen atom of the main
chain of Gln185. This hydrogen bonding is the same interaction
as that of phenylpiperazine.1 And the methyl group with (R)-
configuration on the piperazine makes a CH-π interaction with
Phe67. We consider that the interaction between the piperazine
and Gln185 would rotate the piperazine moiety, which would
result in making a CH-π interaction between the methyl group of
the piperazine and Phe67.
Table 1
Preliminary structure-activity relationship of 2 and/or 4-
substituted piperazine derivatives.
Fig. 3. Docking model of 5 (cyan) and 9 (magenta) with
GSK-3β.
The effect of the configuration of the methyl group was also
analyzed using docking simulation (Fig. 3). The (R)-enantiomer,
5 makes a CH-π interaction with Phe67 and hydrogen bonding
with Gln185. The docking study suggests that there are no
effective interactions between the enzyme and the piperazine
moiety of 9 which have (S)-configuration at the methyl group.
We think lack of effective interactions between the piperazine
moiety of 9 and the enzyme leads to decreased inhibitory activity
of 9.
Table 2
Effect of the substituent on the nitrogen atom of the
piperazine moiety (1).
Fig. 2. Docking model of 1 (yellow) and 5 (cyan) with
GSK-3β.
In order to analyze the effects of the piperazine moiety, an
induced fit docking simulation of 1 and 5 with GSK-3β was
performed.4 Fig. 2 shows that the pyrimidone moiety of both
compounds makes interactions which are common to our
pyrimidine-type GSK-3β inhibitors. The nitrogen atom at the 1-
position of the pyrimidin-4-yl group makes hydrogen bonding

Preliminary in vitro pharmacokinetic studies indicated that 5 had
no CYP450 inhibition (>50 µM, recombinant human CYP1A2,
2D6 and 3A4). From these results, 2-((R)-2-methylpiperazin-1-
yl)-pyrimidin-4-one analogues with a 4-pyridyl or pyrimidin-4-yl
group at the 6-position seemed to have preferable profiles for
GSK-3β inhibitory activity and CYP inhibition, and we selected
6 as a lead compound for further optimization because 6 had
acceptable inhibitory activity and no hydrogen bonding donor
which was preferable structural profile as a CNS-acting drug.
piperazine makes a CH-π interaction with Phe67. The methyl
substituent on the piperazine is bound to the hydrophobic pocket
of the enzyme composed of the side chains of Cys199 and
Asn186. In this case, no hydrogen bonding interaction between
the piperazine and Gln185 is observed, and the cation-π and CH-
π interactions observed above would afford potent inhibitory
activity toward GSK-3β.
Effect of the position of the methyl group was studied using
N-benzyl analogues (Table 3). A methyl group at the 3-position
was not tolerant regardless of its stereochemistry (20, 21). Effect
of a larger alkyl group was also studied. An ethyl group (22)
afforded a 5-fold decrease in activity. From these results, we
concluded that the 2-(R)-methyl group was preferable as the
substituent of the ethylene moiety of the piperazine.
Table 4
Effect of the substituent on the nitrogen atom of the
piperazine moiety (2).
Fig. 4. Docking model of 14 with GSK-3β.
Table 2 shows the results of the optimization of the substituent
on the nitrogen atom of the piperazine. Introduction of alkyl
groups such as isopropyl (12) and benzyl (13) showed a moderate
increase in activity. The phenyl analogue 14 was over 10-fold
more active than 6 and showed moderate metabolic stability and
CYP2D6 inhibition. In the case of acyl, alkoxycarbonyl and
sulfonyl groups, the substituents containing a phenyl group such
as benzoyl (16) and 4-toluenesulfonyl (19) were tolerant and
those without a phenyl group showed decreased activity (15, 18).
Among the compounds possessing a phenyl group, 16 which had
moderate in vitro activity and 19 which showed poor metabolic
stability were not attractive for further optimization toward drug
candidates, and 14 which had potent inhibitory activity was
chosen for further optimization.
Table 3
Effect of the alkyl group on the piperazine moiety.
Results of the optimization of the substituent on the phenyl
group of 14 are shown in Table 4. Halogen atoms such as a
fluorine atom and a chlorine atom, a methoxy and a cyano group
were introduced as the substituents. Except the cyano group, the
substituents on the phenyl group generally increased the activity,
especially the fluorine and methoxy groups afforded potent
inhibitory activity regardless their position. No clear relationship
between the activity and the position of the substituent was
observed. For example, the compound with a substituent at the 2-
position showed weaker activity than that of the 3- and 4-position
in the case of methoxy (0.8 nM of 27 vs 0.2, 0.1 nM of 28, 29,
respectively) and cyano (4.2 nM of 30 vs 2.2, 2.7 nM of 31, 32,
respectively) groups, and slightly potent activity in the case of a
fluorine atom (0.2 nM of 23 vs 0.5, 0.4 nM of 24, 25,
respectively). Among the compounds, 4-methoxy analogue 29
showed most potent inhibitory activity. Most of the compounds
showed poor metabolic stability regardless of their electronic
nature and the position of the substituent, and many compounds
showed moderate one or two CYP isoforms inhibition. No clear
relationship between those profiles and structural profiles of the
The result of a docking study of 14 with GSK-3β is shown in
Fig. 4. The phenyl group on the piperazine moiety makes a
cation-π interaction with Arg141 and the ethylene moiety of the

substituent was observed. In the case of electron-withdrawing
substituents, a cyano group afforded increased metabolic stability
for 3- and 4- substituted analogues 31 and 32 and no
improvement of metabolic stability was observed for 2-
substituted analogue 30 compared with unsubstituted analogue
14. The compounds with the fluorine group at the 3- and 4-
positions, 24 and 25 respectively, afforded nearly the same level
of metabolic stability as the unsubstituted analogue 14, and
decreased metabolic stability was observed for 2-substituted
analogue 23. In the case of electron donating methoxy group, 3-
substituted analogue 28 showed poor metabolic stability and
good metabolic stability was observed for 4-substituted analogue
29. Cell permeability of the compounds we tested was excellent.
4-Substituted-2-(R)-methyl-piperazine
prepared by alkylation or acylation of unsubstituted 2-(R)-
methyl-piperazine (Scheme 2). The benzyl group was
introduced by reductive alkylation with benzaldehyde. Alkylation
with isopropylbromide afforded N-isopropyl analogue 12. The
acyl and sulfonyl groups were introduced by acylation or
sulfonylation with the corresponding acyl or sulfonyl halide.
analogues
were
5
Pyridyl and pyrimidinyl groups were also introduced as the
aromatic substituent on the nitrogen atom of the piperazine
moiety. Compounds possessing pyridyl groups showed nearly 3-
fold weaker activity than those with a phenyl group. A
pyrimidinyl groups largely decreased the activity regardless of its
substituted position. All pyridyl and pyrimidinyl compounds
showed weak CYP inhibition and some compounds had
preferable metabolic stability (33, 37).
Scheme 2. Reagents and conditions: a) PhCHO,
NaBH(OAc)3, cat. AcOH, r.t., 29% (R = PhCH2); b) i-PrBr,
K2CO3, DMF, 80 ºC (R = i-Pr); c) acyl or sulfonyl chloride,
Et3N, THF, r.t., 66% (R = PhCO), 56% (R = MeCO), 64% (R
= MeSO2), 60% (R = 4-Me-PhSO2); d) ClCOOMe, Et3N,
60% (R = MeOCO).
Compound 29, which was the most potent compound,
possessed good metabolic stability and cell permeability together
with acceptable CYP inhibition profiles, and we checked its
mouse in vivo pharmacokinetic profiles. Cmax of 29 was 248, 528
ng/ml in plasma and brain, respectively one hour after oral
administration at a dose of 10 mg/kg, and AUC (0-4 hr) was 770,
1614 ngh/mL in plasma and brain, respectively. Kp brain for both
Cmax and AUC was 2.1. These data indicated that 29 had higher
brain concentration than plasma concentration and preferable
pharmacokinetic profiles as a CNS-acting drug. In vivo Ser396
tau phosphorylation inhibition of 29 in mice was also evaluated.6
Compound 29 significantly demonstrated 60% of decrease of tau
phosphorylation one hour after oral administration at a dose of 10
mg/kg. The positive control UDA-6807 at 100 mg/kg one hour
after oral administration showed 73% of inhibition of tau
phospholylation.
Scheme 3 shows the preparation of 4-aryl derivatives. 4-Aryl-
2-(R)-methyl-piperazine analogues were prepared by arylation of
unsubstituted 2-(R)-methyl-piperazine 5 with the corresponding
aryl
chloroform adduct was used as a catalyst, and following two sets
of ligand and base were used; BINAP (2,2’-
bis(diphenylphosphino)-1,1’-binaphthalene) and sodium tert-
butoxide, or Xphos (2-dicyclohexylphosphino-2',4',6'-
bromide.
Tris(dibenzylideneacetone)dipalladium(0)-
a
a
triisopropylbiphenyl) and potassium phosphate. Introduction of
the 2-pyrimidinyl group was performed by nucleophilic
substitution of 5 with 2-chloropyrimidine. 4-(4-Cyanophenyl)-2-
methylpiperazine (46) was prepared by nucleophilic substitution
with 4-fluorobenzonitrile and 1-tert-butoxycarbonyl-2-(R)-
methyl-piperazine (44) followed by deprotection of the tert-
butoxycarbonyl group, and 46 was reacted with 2-chloro-3-
methyl-6-(pyrimidin-4-yl)-pyrimidine-4-one (38) (Z = N) to
afford 32.
2-(2-(R)- or (S)-methylpiperazin-1-yl)-pyrimidin-4-ones 3‒10
were synthesized by condensation of commercially available 2-
(R)- or (S)-methyl-4-(tert-butoxycarbonyl)-piperazine and the
corresponding 2-chloropyrimidin-4-one 38 with triethylamine in
tetrahydrofuran as a solvent (Scheme 1).2,5,8 After deprotection of
the tert-butoxycarbonyl group with hydrogen chloride in ethyl
acetate, the methyl group was introduced by reductive alkylation
with formaldehyde for 4, 6, 8 and 10.
Scheme 3. Reagents and conditions: a) ArBr, tert-BuONa,
Pd2(dba)3•CHCl3, BINAP, Tol, dioxane, 90 ºC, 36% (Ar =
Ph), 28% (Ar = 2-F-Ph), 28% (Ar = 3-F-Ph), 19% (Ar = 4-F-
Ph), 25% (Ar = 2-MeO-Ph), 11% (Ar = 4-MeO-Ph); b) ArBr,
Pd2(dba)3• CHCl3, Xphos, K3PO4, DME, reflux, 21% (Ar = 4-
Cl-Ph), 58% (Ar = 2-CN-Ph), 21% (Ar = 3-CN-Ph), 26% (Ar
= 2-Py), 60% (Ar = 3-Py), 56% (Ar = 4-Py), 21% (Ar = 5-
pyrimidinyl); c) 2-chloropyrimidine, Et3N, THF, 85 ºC
(sealed tube), 71%; d) 4-fluorobenzonitrile, K2CO3, DMSO,
120 ºC, 83%; e) 4N HCl/AcOEt then basification; f) 2-
chloro-3-methyl-6-(pyrimidin-4-yl)-pyrimidine-4-one, Et3N,
THF, 85 ºC (sealed tube), 55%.
Scheme 1. Reagents and conditions: a) (R)- or (S)-2-methyl-
4-tert-butoxycarbonylpiperazine, Et3N, THF, 65 ºC, 48%
((R)-isomer, Z = C-H), 52% ((R)-isomer, Z = N), 52% ((S)-
isomer, Z = C-H), 28% ((S)-isomer, Z = N); b) 4N
HCl/AcOEt, CH2Cl2 or AcOEt, r.t. then 1N NaOH, 68%
((R)-isomer, Z = C-H), 49% ((R)-isomer, Z = N), 77% ((S)-
isomer, Z = C-H), 73% ((S)-isomer, Z = N); c) HCHO,
AcOH, NaBH(OAc)3, MeOH, CH2Cl2, r.t., 57% ((R)-isomer,
Z = C-H), 47% ((R)-isomer, Z = N), 44% ((S)-isomer, Z = C-
H), 44% ((S)-isomer, Z = N).

Preparation of 2-(R)-ethyl analogue 22 and 3-methyl
analogues, 21 and 20, are depicted in Scheme 4. 2-(R)-Ethyl-4-
benzylpiperazine (51) was prepared by one-pot tandem reductive
amination – transamidation – cyclization reported by Beshore
and Dinsmore.9 N-Benzyl-N-(2-oxoethyl)trifluoeoacetamide (49),
prepared by allylation of N-benzyltrifluoroacetamide (47)
followed by oxidative cleavage of the allyl moiety, and methyl 2-
demonstrated a significant decrease of tau phosphorylation in
mice by oral administration at a dose of 10 mg/kg. Further
optimization of this series will be published elsewhere.
Acknowledgments
(R)-aminobutylate
hydrochloride
was
reacted
with
We gratefully thank Sanofi-Aventis R&D CNS Research
Department (formerly Sanofi-Synthelabo R&D, Bagneux,
France) for a fruitful collaboration.
triacetoxyborohydride. After deprotection of the trifluoroacetyl
group, reduction of the resulting piperazinone with lithium
aluminum hydride afforded 51. 1-Benzyl-2-(R)-methylpiperazine
(53) for 3-(R)-methyl analogue 21 was prepared by benzylation
of 4-tert-butoxy-2-(R)-methylpiperazine (52) followed by
deprotection of the tert-butoxycarbonyl group. Resulting
piperazines were reacted with 2-chloro-3-methyl-6-(pyrimidin-4-
yl)-pyrimidine-4-one (38) (Z = N) to afford 21. In the case of 3-
(S)-methyl analogue 20, commercially available (S)-2-
methylpiperazine was reacted with 2-chloro-3-methyl-6-
(pyrimidin-4-yl)-pyrimidine-4-one (38) (Z = N) which was
reacted at less-hindered nitrogen atom at the 4-position to afford
des-benzyl analogue 55, and successive N-benzylation afforded
20.
References and notes
1. Usui, Y.; Tanaka, H.; Watanabe, K.; Shouda, A.; Uehara, F.; Hiki,
S.; Sunada, S.; Aritomo, K.; Adachi, T.; Yokoshima, S.; Mabeno,
M.; Saito, K,; Eguchi, J.; Yamagami, K.; Asano, S.; Tanaka, S.;
Yuki, S.; Yoshii, N.; Fujimura, M.; Horikawa, T. Bioorg. Med.
Chem. Lett., in press.
2. Fukunaga, K.; Sakai, D.; Watanabe K.; Nakayama, K.; Kohara, T.;
Tanaka, H.; Sunada, S.; Nabeno, M.; Okamoto, M.; Saito, K.;
Eguchi, J.; Mori, A.; Tanaka, S.; Inazawa, K.; Horikawa, T.
Bioorg. Med. Chem. Lett. 2015, 25, 1086.
3. Human GSK-3β cell free enzyme inhibition assay; 7.5 µM of
prephosphorylated GS1 peptide and 10 µM [γ-32P]ATP were
incubated in 50 mM N-(2-hydroxyethyl)piperazine-N-(2-
ethanesulfonic acid) (HEPES)-sodium hydroxide (pH 7.2), 1 mM
dithiothreitol (DTT), 1 mM magnesium chloride, 0.02% Tween-20
buffer for 1 hr at room temperature in the presence of human
recombinant GSK-3β. 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 three times with 75 mM phosphoric acid
solution, once with water and once with acetone. Incorporated 32P
radioactivity was determined by liquid scintillation spectrometry.
The prephosphorylated GS1 peptide had the following sequence;
NH2-YRRAAVPPSPSLSRHSSPHQS(P)EDEE-COOH.
values are the mean of at least two experiments.
IC50
4. Method of docking study; The X-ray crystal structure of GSK-3β
(PDB code: 3F88) was utilized in the docking calculations. The
compounds were docked into GSK-3β using Glide, version 6.7
(*). Induced fit docking simulation was performed using Glide,
version 6.7 and Prime, version 4.0 (*).
*Schrodinger, LLC, New York, NY. 2015.
http://www.schrodinger.com/.
5. Fukunaga, K.; Uehara, F.; Aritomo, K.; Shoda, A.; Hiki, S.;
Okuyama, M.; Usui, Y.; Watanabe, K.; Yamakoshi, K.; Kohara,
T.; Hanano, T.; Tanaka, H.; Tsuchiya, S.; Sunada, S.; Saito, K.;
Eguchi, J.; Yuki, S.; Asano, S.; Tanaka, S.; Mori, A.; Yamagami,
K.; Baba, H.; Horikawa, T.; Fujimura, M. Bioorg. Med. Chem.
Lett. 2013, 23, 6933.
Scheme 4. Reagents and conditions: a) allyl bromide, K2CO3,
acetone, reflux, 68%; b) H5IO6, K2OsO4•2 H2O, THF, H2O, 0
ºC to r.t.; c) methyl 2-(R)-aminobutylate hydrochloride,
NaBH(OAc)3, MeCN, AcOH, molecular sieves 4A, 0 ºC to
r.t., 42%, 2 steps; d) K2CO3, MeOH, H2O, 0 ºC; e) LiAlH4,
cyclopentyl methyl ether, reflux, 75%, 2 steps; f) 2-chloro-3-
methyl-6-(pyrimidin-4-yl)-pyrimidine-4-one, Et3N, THF, 100
ºC, sealed tube, 13%; g) benzyl bromide, K2CO3, DMF, 70
ºC; h) 4N HCl/AcOEt, AcOEt, r.t., 61%, 2 steps; i) 2-chloro-
3-methyl-6-(pyrimidin-4-yl)-pyrimidine-4-one, Et3N, THF,
r.t., 25%; j) 2-chloro-3-methyl-6-(pyrimidin-4-yl)-
6. Inhibitory activity on tau phosphorylation in vivo; Test compound
was administrated to male CD-1 mice of 5–6 weeks weighing 25–
35
g (Charles River Japan, Inc.) at 10 mg/kg p.o. (0.5%
Tween/H2O suspension) and after 1 hr, mice were decapitated and
cortex was promptly removed, followed by being frozen in liquid
N2. Cortex was directly homogenized with 2.3% SDS
homogenization buffer (62.5 mM Tris–HCl, 2.3% SDS, 1 mM
each of EDTA, EGTA and DTT, protease inhibitor cocktail
pyrimidine-4-one, Et3N, THF, r.t., 56%; k) benzyl bromide,
K2CO3, DMF, 70 ºC, 60%.
(sigma
P2714)
containing
0.2
µM
4-(2-
aminoethyl)benzenesulfonylfluoride (AEBSF), 13 µM bestatin,
1.4 µM E-64, 0.1 mM leupeptin, 30 nM aprotinin, pH 6.8) and
centrifuged at 15000g for 15 min at 4 ºC. Protein concentrations
were determined using DC protein assay kit (BIO-RAD).
Supernatants were diluted with sample buffer (62.5 mM Tris–HCl,
25% glycerol, 2% SDS, 0.01% bromophenol blue, pH 6.8) to
adjust the protein concentrations around 0.5–2 mg/mg and then
boiled for 5 min. 10 µg of samples were applied on 10% SDS–
PAGE mini slab gels and transferred onto PVDF membranes.
Membranes were incubated with PBS containing 5% non-fat milk
for 1 hr at room temperature and then probed with pS396 anti-
body (BIOSOURCE) overnight at 4 ºC. Anti-rabbit IgG HRP-
conjugated anti-body (Promega) was used as secondary anti-body.
Membranes were visualized by ECL kit (Amersham Bioscience)
In conclusion, the transformation of the 3-(R)-
methylmorpholine moiety of 1 into the corresponding 2-(R)-
methylpiperazine moiety was well tolerated and the optimization
of the substituent on the nitrogen atom of the piperazine led to
the discovery of a novel N-aryl-2-(R)-methylpiperazine series of
potent GSK-3β inhibitors. Among several potent compounds 29
exhibited the most potent GSK-3β inhibitory activity with good
in vitro and in vivo pharmacokinetic profiles, especially high cell
permeability and brain/plasma concentration ratio. 29 also

and detected by LAS 1000 (Fuji Photo Film). We have already
checked that the amount of total tau was not changed by the
compounds which were analogue of UDA-680 assayed in this
screening. Therefore, we did not measure the amount of total tau
but measured pS396 phosphorylated tau normalized with total
protein concentration for compound screening in vivo.
K.; Baba, H.; Horikawa, T.; Fujimura, M. Bioorg. Med. Chem.
Lett. 2013, 23, 6933.
8. 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. 2013, 23, 6928.
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7. Fukunaga, K.; Uehara, F.; Aritomo, K.; Shoda, A.; Hiki, S.;
Okuyama, M.; Usui, Y.; Watanabe, K.; Yamakoshi, K.; Kohara,
T.; Hanano, T.; Tanaka, H.; Tsuchiya, S.; Sunada, S.; Saito, K.;
Eguchi, J.; Yuki, S.; Asano, S.; Tanaka, S.; Mori, A.; Yamagami,

Table 1
piperazine compound
Z
R'
GSK-3β
inhibition
(IC50, nM)
29
3
4
5
C-H
C-H
N
H
Me
H
80
13
6
7
8
9
N
Me
H
Me
22
920
>1000
76
C-H
C-H
N
H
10
11
N
N
Me
Me
>1000
630

Table 2
a)
compound
R'
GSK-
3β inhibition
(IC50, nM)
CYP inhibition (IC50,
Caco-2
CLint, human
µM) b)
(x10-7 cm/s)
(mL/min/mg)
1A2
-----
>50
>50
>50
-----
>50
-----
-----
>50
2D6
-----
>50
21
3A4
-----
>50
>50
>50
-----
N.D.
-----
-----
1.4
6
Me
i-Pr
PhCH2
Ph
MeCO
PhCO
MeOCO
MeSO2
4-
22
13
8.3
1.4
49
21
70
46
19
-----
N.D.
0.18
0.23
-----
0.05
-----
-----
-----
N.D.
-----
-----
-----
333
-----
-----
566
12
13
14
15
16
17
18
19
23
-----
>50
-----
-----
34
0.37
MePhSO2
N.D.; not determined
a) human liver microsome.
b) Recombinant human CYP450.
Table 3
compound
13
piperazine
GSK-
3β inhibition
(IC50, nM)
8.3
20
21
22
780
>1000
43

Table 4
a)
compound
Ar
CYP inhibition (IC50,
Caco-2
(x10-
GSK-3β
inhibition
(IC50, nM)
CLint, human
µM) b)
(mL/min/mg)
7cm/s)
-----
645
511
620
-----
450
630
610
-----
N.D.
-----
-----
-----
-----
-----
1A2
2D6
3A4
14
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Ph
2-F-Ph
3-F-Ph
4-F-Ph
4-Cl-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
2-CN-Ph
3-CN-Ph
4-CN-Ph
4-Py
1.4
0.2
0.5
0.4
1.2
0.8
0.2
0.1
4.2
2.2
2.7
3.8
3.9
4.1
24
0.23
0.43
0.24
0.26
0.14
N.D.
0.38
0.09
0.27
0.02
0.11
0.01
0.13
0.40
-----
>50
42
41
43
30
23
19
10
10
7.6
10
>50
N.D.
>50
>50
>50
>50
9.6
41
28
19
18
>25
27
15
25
>50
8.3
>50
>50
>50
>50
-----
N.D.
>50
N.D.
>50
N.D.
>50
-----
24
>50
>50
40
3-Py
2-Py
2-
-----
pyrimidinyl
37
5-
14
0.04
>50
>50
N.D.
-----
pyrimidinyl
N.D.; not determined
a) human liver microsome.
b) Recombinant human CYP450.

Graphical Abstract
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Discovery of novel 2-(4-aryl-2-
methylpiperazin-1-yl)-pyrimidin-4-ones as
glycogen synthase kinase-3β inhibitors
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Toshiyuki Kohara, Kazuki Nakayama, Kazutoshi Watanabe*, Shin-ichi Kusaka, Daiki Sakai, Hiroshi Tanaka,
Kenji Fukunaga, Shinji Sunada, Mika Nabeno, Ken-Ichi Saito, Jun-ichi Eguchi, Akiko Mori, Shinji Tanaka,
Tomoko Bessho, Keiko Takiguchi-Hayashi and Takashi Horikawa