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

Article abstract of DOI:10.1016/j.bmcl.2017.06.078

We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β inhibitors from our promising compounds containing a 2-phenylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3β inhibitors. SAR studies focused on the phenyl moiety revealed that a 4-fluoro-2-methoxy group afforded potent inhibitory activity toward GSK-3β. Based on docking studies, new hydrogen bonding between the nitrogen atom of the piperazine moiety and the oxygen atom of the main chain of Gln185 has been indicated, which may contribute to increased activity compared with that of the corresponding phenylmorpholine analogues. Effect of the stereochemistry of the phenylpiperazine moiety is also discussed.

Full text of DOI:10.1016/j.bmcl.2017.06.078

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 2-(3-phenylpiperazin-1-yl)-pyrimidin-4-ones as
glycogen synthase kinase-3b inhibitors
a
b
⇑
Yoshihiro Usui , Fumiaki Uehara, Shinsuke Hiki, Kazutoshi Watanabe , Hiroshi Tanaka, Aya Shouda ,
Satoshi Yokoshima c, Keiichi Aritomo, Takashi Adachi d, Kenji Fukunaga, Shinji Sunada, Mika Nabeno,
Ken-Ichi Saito e, Jun-ichi Eguchi f, Keiji Yamagami g, Shouichi Asano, Shinji Tanaka, Satoshi Yuki b,
Narihiko Yoshii h, Masatake Fujimura i, Takashi Horikawa b
Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000 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:
We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3b inhibitors from
our promising compounds containing a 2-phenylmorpholine moiety. Transformation of the morpholine
moiety into a piperazine moiety resulted in potent GSK-3b inhibitors. SAR studies focused on the phenyl
moiety revealed that a 4-fluoro-2-methoxy group afforded potent inhibitory activity toward GSK-3b.
Based on docking studies, new hydrogen bonding between the nitrogen atom of the piperazine moiety
and the oxygen atom of the main chain of Gln185 has been indicated, which may contribute to increased
activity compared with that of the corresponding phenylmorpholine analogues. Effect of the stereochem-
istry of the phenylpiperazine moiety is also discussed.
Received 29 March 2017
Revised 16 June 2017
Accepted 29 June 2017
Available online xxxx
Keywords:
glycogen synthase kinase-3
Alzheimer’s disease
phenylpiperazine
Ó 2017 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3 (GSK-3) is a proline-directed ser-
ine-threonine kinase which was initially identified as an enzyme
that was able to deactivate glycogen synthase through phosphory-
forms are ubiquitously expressed, with particularly high levels
observed in the brain and testes.2
It has been reported that GSK-3b is involved in several physio-
logical processes such as cell proliferation, neural function, embry-
onic development, apoptosis, immune response,2d,3 and GSK-3b
would be an attractive drug target for the treatment of
Alzheimer’s disease, Parkinson’s disease, stroke, Huntington’s
disease, amyotrophic lateral sclerosis, type 2 diabetes, cancer and
inflammation.4
lation.1 GSK-3 exists as two isoforms, GSK-3
a (51 kDa) and GSK-3b
(47 kDa) with high homology in their kinase domains. Both iso-
⇑
Corresponding author.
E-mail address: watanabe.kazutoshi@mb.mt-pharma.co.jp (K. Watanabe).
Present address: Ryoto Chemical Industry Co., Ltd., 1410 Takata, Kashiwa City,
a
We previously reported that 6-(4-pyridinyl or pyrimidin-4-yl)-
3-methylpyrimidones with a 2-phenylmorpholine moiety at the 2-
position were found to be potent GSK-3b inhibitors as useful ther-
apeutic drugs for the treatment of Alzheimer’s disease, and that
UDA-680 was discovered as a promising compounds.5 UDA-680
exhibited potent GSK-3b inhibitory activity and in vivo tau phos-
phorylation inhibitory activity by oral administration in mice.
UDA-680 showed excellent cell permeability, and pharmacokinetic
studies indicated that UDA-680 had high brain/plasma ratio with
moderate bioavailability (37%, rat). In order to identify further
promising compounds for clinical studies, we transformed the
morpholine moiety of UDA-680 into a piperazine moiety in order
to improve water solubility of UDA-680 and to evolve a new chem-
ical series (Fig. 1). In this communication, we report synthesis and
results of structure–activity relationship of 3-(substituted phenyl)
piperazine analogues as well as computational docking studies.
Chiba 277-0861, Japan.
b
Present address: Ikuyaku, Integrated Value Development Division, Mitsubishi
Tanabe Pharma Corporation, 17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-
8405, Japan.
c
Present address: Graduate School of Pharmaceutical Sciences, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
d
Corporate Communications Department, Mitsubishi Tanabe Pharma Corporation,
3-2-10, Dosho-machi, Chuo-ku, Osaka 541-8505, Japan.
e
Present address: Life Science Institute, Inc., THE KAITEKI Bldg., 13-4, Uchikanda 1-
chome, Chiyoda-ku, Tokyo 101-0047, Japan.
f
Present address: The KAITEKI Institute, Inc., 1-1, Marunouchi 1-chome, Chiyoda-
ku, Tokyo 100-8251, Japan.
g
Present address: Translational Research Center, The University of Tokyo Hospital,
The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
h
Present address: Development Department, Mitsubishi Tanabe Pharma Korea Co.,
Ltd., 21F MMAA Bldg, 2806, Nambusunhwan-ro, Gangnam-gu, Seoul 135-700,
Republic of Korea.
i
Present address: National Institute for Minamata Disease, 4058-18 Hama,
Minamata City, Kumamoto 867-0008, Japan.
http://dx.doi.org/10.1016/j.bmcl.2017.06.078
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
analogue 14. Pyrimidin-4-yl analogues with chlorine and bromine
groups on the phenyl moiety showed 3 to 5-fold more potent activ-
ity than corresponding 4-pyridinyl analogues (17 versus 18, 19
versus 20 and 21 versus 22 for a chlorine atom and 23 versus 24
for a bromine atom).
No clear tendency of the effect relating the substituent position
was observed. For example, the 2-position was the most preferable
position for a methoxy group with both 4-pyridyl and pyrimidin-4-
yl groups (3, 4), and the 3-position for a 4-pyridyl (13) and both 2-
and 4-positions for a pyrimidin-4-yl (12, 16) was the most prefer-
able position toward GSK-3b inhibition for a fluorine atom. A chlo-
rine atom at the 3- and 4-positions showed comparatively potent
activity for both 4-pyridyl and pyrimidin-4-yl groups (19 and 21,
20 and 22, respectively). Substituents except a fluorine atom
showed a similar substituent effect for the phenyl group between
the 4-pyridyl and the pyrimidin-4-yl analogues.
In vitro pharmacokinetic profiles were examined for methoxy-
substituted phenylpiperazine analogues which showed potent
inhibitory activity against GSK-3b (Table 2). 2- and 3-substituted
analogues 3, 5 showed potent CYP2D6 inhibition and poor meta-
bolic stability, and 4-substituted analogue 7 showed weak CYP
inhibition profile including CYP2D6 and good metabolic stability.
These results indicate that a substituent at the 4-position may
block the possible metabolism of the phenyl moiety and confer
weak CYP inhibition activity, which resulted in preferable in vitro
pharmacokinetic profiles.
Based on the information above, we further introduced sub-
stituents at the 4-position of the phenyl moiety to improve
in vitro pharmacokinetic profiles and at the 2- or 3-positions to
increase GSK-3b inhibitory activity (Table 3). 4-Fluoro-2-methoxy
analogues, 30 and 31 showed potent GSK-3b inhibitory activity,
and further substitution by a fluorine atom at the 6-position (32
and 33) resulted in two-fold potency decrease. On the other hand,
other combinations of substituents did not increase the activity;
thus, 4-fluoro-2-methyl analogue 34 was more than 20-fold less
active compared to the corresponding 4-fluoro-2-methoxy ana-
logue 30, and 3, 4-disubstituted analogues showed no significant
increase of activity compared to 3- or 4-monosubstitued analogues
(35, 36 for methoxy groups and 37, 38 for chlorine groups).
As we successively increased the inhibitory activity by intro-
ducing substituents at the 4- and 2-positions, we resolved 30
and 31 to investigate in vitro pharmacokinetic profiles of both
enantiomers (Table 4). As we expected, 30 and 31 which possessed
a substituent at the 4-position showed good metabolic stability,
Fig. 1. Transformation of morpholine moiety.
First we examined the transformation of the morpholine moiety
into the corresponding piperazine with no substitution on the
nitrogen atom. 4-Pyridinyl and pyrimidin-4-yl groups were
selected as a substituent at the 6-position of the pyrimidone moi-
ety because our previous studies indicated that a 3-fluoropyridin-
4-yl group which was also an effective substituent for the 6-posi-
tion of the pyrimidone generally had potent CYP inhibition activity
together with good GSK-3b inhibitory activity.6 Racemic
phenylpiperazine analogues with various substituents on the phe-
nyl moiety were prepared and their effect toward GSK-3b inhibi-
tory activity was examined (Table 1). This result indicated that
the transformation into phenylpiperazine was preferable com-
pared with IC50 of corresponding phenylmorpholine analogue
(51 nM).5,7 Introduction of a substituent on the phenyl moiety gen-
erally increased the inhibitory activity in the case of compounds
with a 4-pyridinyl group. Methoxy and chlorine groups increased
activity in all substituted positions (3, 5, 7 and 17, 19, 21 respec-
tively) and a fluorine group in 2- and 3-positions (11 and 13) and
a bromine atom at the 2-position (23) also increased inhibitory
activity. A 2-ethoxy group (9) showed no effects toward the activ-
ity despite 10-fold increase of activity by introduction of a 2-meth-
oxy group (3) and further investigation of alkoxy substituents were
not pursued. A cyano group which was generally known as a halo-
gen mimic did not show clear effects toward the inhibitory activity
(28). Phenylpiperazine analogues with a pyrimidin-4-yl moiety
showed comparable or more potent inhibitory activity compared
with those possessing a 4-pyridinyl moiety except 3-fluorophenyl
Table 1
GSK-3b inhibitory activity of substituted phenylpiperazines.
Table 2
In vitro pharmacokinetic profiles of methoxy-substituted phenylpiperazines.
X
Z
GSK-3b inhibition (IC50, nM)1)
Position of the substituent X
2-
3-
4-
H
MeO
CAH
CAH
N
CAH
N
CAH
N
CAH
N
CAH
N
CAH
N
2
3
4
9
10
11
12
17
18
23
24
–
18
1.8
1.8
25
20
11
8.2
9.6
2.6
13
5
6
11
6.7
7
8
4.7
3.5
EtO
F
–
–
–
–
2)
Compound Position GSK-3b
CLint,
CYP inhibition
M)
mouse
13
14
19
20
–
25
–
–
22
12
5.4
1.1
15
16
21
22
26
27
28
29
18
inhibition
(ml/min/mg (IC50
,
l
(IC50, nM)1) protein)
8.6
6.6
1.7
22
8.1
15
1A23) 2D63) 3A43)
Cl
3
5
7
2
3
4
1.8
11
4.7
0.158
0.123
0.078
>50
<0.2
0.4
>50
Br
CN
>50
>50
>534)
454)
>534)
4.3
2.0
1)
2)
3)
4)
All compounds were assayed as corresponding hydrochlorides.
Mouse liver microsome.
–
7.4
1)
Compounds were assayed as corresponding hydrochlorides except 15, 16, 17,
18, 19, 20, 23 and 24.
Recombinant human CYP450.
Human liver microsome.
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Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 3
GSK-3b inhibitory activity of polysubstituted phenylpiperazines.
Aryl
GSK-3b inhibition (IC50, nM)1)
Z
CAH
N
30
31
0.90
0.72
32
33
2.0
1.7
34
–
24
35
36
5.2
7.8
37
38
4.3
2.3
1)
30, 31, 32, 33 and 34 were assayed as corresponding hydrochlorides.
Table 4
In vitro pharmacokinetic profiles of 30, 31 and their enantiomers.
N.D.; not determined.
1)All compounds were assayed as corresponding hydrochlorides.
2)1)
Mouse liver microsome.
3)Human liver microsome.
4)Recombinant human CYP450.
Table 5
GSK-3b inhibitory activity of N-substituted phenylpiperazines.
but both compounds possessed potent CYP2D6 inhibition. Resolu-
tion of stereoisomers indicated that the enantiomers 40 and 42
were more than 100-fold more potent than the antipodes 39 and
41, respectively. No significant difference of CYP2D6 inhibition
was observed between each enantiomer. Both eutomers showed
weaker CYP2D6 inhibition than the corresponding distomers but
their CYP2D6 inhibitory activity was not low enough to make these
compounds a promising compound. As to in vitro metabolic stabil-
ity, there seemed to be a significant difference between 41 and 42.
Effects of a substituent on the nitrogen atom of the piperazine
were also investigated using 4-fluoro-2-methoxy-substituted ana-
logues (Table 5). Introduction of a methyl group showed several
tens of times decrease in activity (43, 44), and introduction of a
benzyl group afforded more than one-hundred fold decrease in
activity (45, 46). An acetyl group also showed large decrease in
activity (47, 48). These results indicated that introduction of a sub-
stituent on the nitrogen atom of the piperazine moiety was intol-
erant regardless of its steric and electric nature.
R
Z
GSK-3b inhibition
(IC50, nM)1)
H
CAH
N
CAH
N
CAH
N
CAH
N
30
31
43
44
45
46
47
48
0.90
0.72
56
Me
26
PhCH2
MeCO
1200
220
1700
1500
1)
In order to analyze the effects of the piperazine moiety, an
All compounds were assayed as corresponding hydrochlorides except 47 and
induced fit docking simulation of 16 with GSK-3b was performed.8
48.
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Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 6
GSK-3b inhibitory activity of both enantiomers of 21 and 22.
Compound
Z
Configuration
GSK-3b inhibition (IC50, nM)1)
49
50
51
52
CAH
CAH
N
(S)
(R)
(S)
(R)
11
110
1.2
26
N
1)
All compounds were assayed as corresponding hydrochlorides.
Fig. 2. Docking model of (S)-isomer of 16 with GSK-3b (PDB code: 3F88) by induced
docking calculation.
Fig. 2 shows that the nitrogen atom at the 1-position of the pyrim-
idin-4-yl group makes hydrogen bonding with the main chain of
Val135 in the hinge region and the carbonyl oxygen of the pyrim-
idone moiety forms hydrogen bonding with the side chain of
Lys85. These hydrogen bonds are common interactions with the
pyrimidone moiety.5,6 In the point of the phenylpiperazine moiety,
Scheme 1. Reagents and conditions: (a) Et3N, THF, r.t. or reflux, or DMF, Et3N, 0 °C
to r.t., 48% (X = 3-MeO, Z = CAH, R = H), quant. (X = 4-MeO, Z = N, R = H), 94% (X = 2-
EtO, Z = CAH, R = H), quant. (X = 2-EtO, Z = N, R = H), 94% (X = 2-F, Z = CAH, R = H),
85% (X = 2-F, Z = N, R = H), 88% (X = 3-F, Z = CAH, R = Boc), 95% (X = 4-F, Z = CAH,
R = H), 88% (X = 4-F, Z = N, R = H), 80% (X = 2-Cl, Z = CAH, R = H), 90% (X = 2-Cl, Z = N,
R = H), 80% (X = 3-Cl, Z = CAH, R = H), 73% (X = 3-Cl, Z = N, R = H), 95% (X = 4-Cl,
Z = N, R = Boc), 29% (X = 2-Br, Z = CAH, R = H), 18% (X = 2-Br, Z = N, R = H), 90%
(X = 3-Br, Z = N, R = H), 62% (X = 4-Br, Z = CAH, R = H), 76% (X = 4-Br, Z = N, R = H),
98% (X = 4-CN, Z = CAH, R = H), 96% (X = 4-CN, Z = N, R = H), 62% (X = 2-MeO-4-F,
Z = CAH, R = H), 88% (X = 2-Me-4-F, Z = CAH, R = H), 44% (X = 3,4-di-MeO, Z = CAH,
R = H), 68% (X = 3,4-di-MeO, Z = N, R = H), 90% (X = 3,4-di-Cl, Z = CAH, R = H), 86%
(X = 3,4-di-Cl, Z = N, R = H), 83% (X = 2-MeO-4-F, Z = CAH, R = PhCH2), 58% (X = 2-
MeO-4-F, Z = N, R = PhCH2), 75% (X = 2-MeO-4-F, X = CAH, R = MeCO), 88% (X = 2-
MeO-4-F, X = N, R = MeCO); (b) 4 N HCl/AcOEt or 4 N HCl/dioxane, AcOEt or CH2Cl2,
69% (2 steps, X = H, Z = CAH, R = H), 95% (2 steps, X = 2-MeO, Z = CAH, R = H), 82% (2
steps, X = 2-MeO, Z = N, R = H), 95% (X = 3-MeO, Z = CAH, R = H), 35% (2 steps, X = 3-
MeO, Z = N, R = H), 67% (2 steps, X = 4-MeO, Z = CAH, R = H), quant. (X = 4-MeO,
Z = N, R = H), quant. (X = 2-EtO, Z = CAH, R = H), 68% (X = 2-EtO, Z = N, R = H), quant.
(X = 2-F, Z = CAH, R = H), quant. (X = 2-F, Z = N, R = H), 77% (X = 3-F, Z = CAH, R = H),
74% (2 steps, X = 4-Cl, Z = CAH, R = H), 73% (X = 4-Cl, Z = N, R = H), quant. (X = 3-Br,
Z = N, R = H), quant. (X = 4-Br, Z = CAH, R = H), quant. (X = 4-Br, Z = N, R = H), 99%
(X = 4-CN, Z = CAH, R = H), 98% (X = 4-CN, Z = N, R = H), quant. (X = 2-MeO-4-F,
Z = CAH, R = H), 86% (2 steps, X = 2-MeO-4-F, Z = N, R = H), 46% (2 steps, X = 2-MeO-
4,6-di-F, Z = N, R = H), 84% (X = 2-Me-4-F, Z = CAH, R = H), 55% (2 steps, X = 2-MeO-
4-F, Z = CAH, R = Me), 25% (2 steps, X = 2-MeO-4-F, Z = N, R = Me), 90% (X = 2-MeO-
4-F, X = CAH, R = PhCH2), 85% (X = 2-MeO-4-F, X = N, R = PhCH2).
only (S)-configuration of 16 makes a cation-p interaction between
the 4-fluorophenyl moiety and Arg141 as well as that of UDA-680,5
and also makes a hydrogen bond between the hydrogen atom of
the amino group of the piperazine and the oxygen atom of the
main chain of Gln185. This information suggests that more potent
inhibitory activity of phenylpiperazine analogues than that of
phenylmorpholine analogues would be attributed to an additional
interaction between piperazine and Gln185, and large decrease of
the activities by introducing the substituents on the nitrogen
atom of the piperazine is explained by the following observation
that the nitrogen atom of the piperazine and the main chain of
Gln185 is so close that there is no space to allow any substituents
on the nitrogen atom. Information that the isomer with (S)-
configuration makes effective interaction with GSK-3b coincides
with the fact that stereoselectively prepared (S)-isomers of 4-
chlorophenylpiperazine analogues 49 and 51 showed more potent
inhibitory activity than the corresponding (R)-isomer 50 and 52,
respectively (Table 6).9
All 3-phenylpiperazine derivatives were synthesized by the
condensation of appropriately substituted phenylpiperazines 54
and 2-chloropyrimidin-4-one 53 under the same conditions as
those used in the preparation of substituted morpholine deriva-
tives (Scheme 1).5,6,10 Triethylamine was used as the base and
the condensation was conducted using tetrahydrofuran as a sol-
vent at room temperature or reflux, or N,N-dimethylformamide
at room temperature. The less hindered nitrogen atom at the
4-position was predominantly reacted when 1,4-unsubstitued
piperazines were used. Most 2-(3-phenylpiperazin-1-yl)-pyrim-
idin-4-ones were obtained as hydrochloric acid salts by treatment
of hydrogen chloride in ethyl acetate or dioxane for the salt forma-
tion or deprotection of tert-butoxycarbonyl group.
by reduction of cyclic imine or amide moiety of the precursors.
Each precursor was prepared by condensation with ethylenedi-
amine and 2-bromophenylacetates, 2-oxophenylacetates or
phenylglyoxals with appropriate substituents on the phenyl
moiety.
Scheme 3 shows the route via 3-phenylpiperazin-2-ones 57
which were synthesized by condensation with ethylenediamine
and methyl 2-bromophenylacetates 56 prepared by bromination
of esters of substituted phenylacetic acid 55 (route 1 in Scheme 2).
1-tert-Butoxycarbonyl-protected 2-phenylpiperazines 59 were
obtained by reduction of the carbonyl moiety of 58 after protection
of the basic nitrogen atom with a tert-butoxycarbonyl group. In the
case of unsubstituted phenylpiperazine 64, a benzyl group was
Substituted 3-phenylpiperazine 54 was synthesized by three
routes (Scheme 2). In every route, the piperazines were prepared
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Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
introduced on the nitrogen atom of the amide moiety, and
then removed by hydrogenation after reduction of the carbonyl
group.
and water11 in good yield. Reduction of a mixture of phenylglyox-
als and ethylendiamine with sodium borohydride afforded 2-
phenylpiperazines 68 in moderate yield (Scheme 5).
Phenylpiperazine synthesis from ethyl phenylglyoxalates is
shown in Scheme 4 (route 2 in Scheme 2). Ethyl phenylglyoxalates
66 prepared by ethyl oxalate and aryl Grignard reagent was
reacted with ethylenediamine, and the resulting 3-aryl-iminolac-
tam 67 was reduced to 2-arylpiperazine 68 by lithium aluminum
hydride. In the case of 2-(4-fluorophenyl)piperazine, ethyl 4-fluo-
rophenylglyoxalete (71) was prepared by Swern oxidation of ethyl
2-hydroxy-(4-fluorophenyl)acetate (70).
In some cases, resulting phenylpiperazines were transformed
into bis-tert-butoxycarbonyl protected ones 75 for purification by
silica gel column chromatography (Scheme 6). In the case of (2-
ethoxyphenyl)piperazine (80), acetophenone 77 was directly
transformed into (2-ethoxyphenyl)glyoxal (78) using hydrobromic
acid and dimethylsulfoxide,12 and sodium triacetoxyborohydride
was used as
a reducing reagent after the reaction with
ethylenediamine.
Phenylpiperazines were also prepared from phenylglyoxals
(Schemes 5–7) (route 3 in Scheme 2). 2-Bromoacetophenones 73
were transformed into phenylglyoxals 74 using dimethylsulfoxide
For the synthesis of 2-(2-methoxy-4,6-difluorophenyl)piper-
azine (86), the starting 20-methoxy-40,60-difluoroacetophenone
(84) was prepared by Fries rearrangement of 1-acetoxy-3,5-difluo-
Scheme 2. Retrosynthetic analysis of 3-arylpiperazines.
Scheme 3. Reagents and conditions: (a) NBS, CCl4, hv; (b) ethylenediamine, Et3N, MeOH, reflux, 17% (R = 3-F, 2 steps); (c) Boc2O, Et3N, CH2Cl2, 70% (R = 3-F), 22% (3 steps,
R = 4-Cl); (d) NaBH4, AcOH, dioxane, heat, 69% (R = 4-Cl), 45% (R = 3-F); (e) Boc2O, Et3N, CH2Cl2, 84%; (f) PhCH2Br, NaH, DMF, 67%; (g) LiAlH4, THF, reflux, 83%; (h) H2, Pd/C,
MeOH.
Scheme 4. Reagents and conditions: (a) Mg, Et2O, or n-BuLi, THF, (COOEt)2, ꢀ78 °C, or ꢀ78 °C to r.t., 20% (R = 4-MeO), 86% (R = 2-MeO), 16% (R = 2-F), 38% (R = 2-Me-4-F), 59%
(R = 2-MeO-4-F); (b) ethylenediamine, EtOH, reflux, 44% (R = 4-MeO), 94% (R = 2-MeO), 47% (R = 2-F), 35% (R = 2-Me-4-F), 79% (R = 2-MeO-4-F), 89% (R = 4-F); (c) LiAlH4, THF
and/or Et2O reflux, quant. (R = 4-MeO), quant. (R = 2-MeO), 90% (R = 2-Me-4-F), 99% (R = 2-MeO-4-F), 39% (R = 4-F); (d) EtI, Cs2CO3, DMF, 50 °C, quant.; (e) DMSO, SOCl2, Et3N,
CH2Cl2, ꢀ78 °C, 85%.
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Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Scheme 5. Reagents and conditions: (a) Br2, CH2Cl2 for R = 3-Br, 2-Br or Me3N+PhBr3- , THF, 0 °C to r.t. for R = 3,4-di-MeO, 3,4,-di-Cl, quant. (R = 3-Br), quant. (R = 2-Br); (b) H2O,
DMSO, 50 °C, for R = 3-Br, 2-Br, 3,4-di-MeO and 3,4-di-Cl, r.t. for R = 4-Br, 86% (R = 3-Br), 89% (R = 2-Br), 35% (2 steps, R = 3,4-di-MeO), 86% (2 steps, R = 3,4-di-Cl), 91% (R = 4-
Br); (c) ethylenediamine, MeOH, THF for R = 3,4-di-MeO and 4-Br, 0 °C for R = 3-Br, 3,4-di-MeO and 3,4-di-Cl or r.t. for R = 2-Br and 4-Br then add NaBH4, 0 °C to r.t., 33%
(R = 3-Br), 31% (R = 2-Br), 22% (R = 3,4-di-MeO), 25% (R = 3,4-di-Cl), 69% (R = 4-Br).
Scheme 6. Reagents and conditions: (a) Br2, CH2Cl2; (b) H2O, DMSO, 50–60 °C, for R = 2-Cl and 3-Cl, r.t. for R = 3-MeO and 4-CN, 74% (R = 3-MeO), 47% (2 steps, R = 2-Cl), 47%
(2 steps, R = 3-Cl), 37% (2 steps, R = 4-CN); (c) ethylenediamine, MeOH for R = 2-Cl, 3-Cl and 3-MeO, MeOH, THF for R = 4-CN, r.t. then add NaBH4, 0 °C for R = 2-Cl and 3-Cl,
0 °C to r.t., for R = 3-MeO and 4-CN; (d) Boc2O, Et3N, CH2Cl2, 12% (2 steps, R = 3-MeO), 37% (2 steps, R = 2-Cl), 11% (2 steps, R = 3-Cl); (e) 4 N HCl/AcOEt, AcOEt, 76% (R = 3-
MeO), 49% (R = 3-Cl), 23% (3 steps, R = 4-CN); (f) aq. NaOH for R = 2-Cl, 47% (2 steps); (g) 48% aq. HBr, DMSO, 80 °C, 47%; (h) ethylenediamine, THF, cat. AcOH, 0 °C, 3 min then
add NaBH(OAc)3, r.t.; (i) Boc2O, Et3N, THF, 29% (2 steps); (j) 4 N HCl/AcOEt, AcOEt, r.t., 76%.
Scheme 7. Reagents and conditions: (a) Ac2O, Py, CH2Cl2, r.t.; (b) AlCl3, ClCH2CH2Cl, 80 °C; (c) MeI, NaH, DMF, r.t., 40% (3 steps); (d) 48% aq. HBr, DMSO, 80 °C, 34%; (e)
ethylenediamine, MeOH, THF, r.t. then add NaBH4, 0 °C to r.t., 31%.
Scheme 8. Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 0 °C, 92%; (b) Ac2O, DMAP, CH2Cl2, r.t., 92%; (c) MeI, NaH, THF, 0 °C to r.t., 79%; (d) PhCH2Br, NaH, THF, r.t., 82%;
(e) 4 N HCl/AcOEt, AcOEt, r.t., quant. (R = Ac), 73% (R = Me), 100% (R = CH2Ph).
robenzene (82) obtained by acetylation of 3,5-difluorophenol (81)
and successive methylation. After transformation into phenylgly-
oxal 85 with hydrobromic acid and dimethylsulfoxide, resulting
phenylpiperazine was obtained after reaction with ethylenedi-
amine and sodium borohydride (Scheme 7).
Synthesis of 1-substituted 2-phenylpiperazines 90 is shown in
Scheme 8. A tert-butoxycarbonyl group was selectively introduced
to the less-hindered amino moiety at the 4-position. 1-Substituted
2-phenylpiperazines 90 were obtained by deprotection of tert-
butoxycarbonyl group after introduction of an appropriate sub-
stituent at the 1-position.
In conclusion, transformation of the morpholine moiety of pre-
viously reported 2-(2-phenylmorpholin-4-yl)pyrimidin-4-one
derivatives into the corresponding piperazine analogue led to
potent inhibition toward GSK-3b by making new hydrogen bond-
ing between the nitrogen atom of the piperazine moiety and the
oxygen atom of the main chain of Gln185; however, no compounds
with preferable in vitro pharmacokinetic profiles were obtained.
We did not pursue further optimization of this analogue by intro-
ducing simple substituents on the phenyl moiety. Further
approach of the transformation of the morpholine moiety into
piperazine will be reported in the following article.
Please cite this article in press as: Usui Y., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.06.078

Y. Usui et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
7
5. Fukunaga K, Uehara F, Aritomo K, et al. Bioorg Med Chem Lett. 2013;23:6933.
6. Fukunaga K, Sakai D, Watanabe K, et al. Bioorg Med Chem Lett. 2015;25:1086.
7. Human GSK-3b cell free enzyme inhibition assay; 7.5 M of prephosphorylated
GS1 peptide and 10
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 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 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. IC50 values are the
mean of at least two experiments.
8. Method of docking study; The X-ray crystal structure of GSK-3b (PDB code:
3F88) was utilized in the docking calculations. The compounds were docked
into GSK-3b 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/.
9. For stereoselective preparation of the piperazines, see: Yokoshima S, Watanabe
F, Uehara F, Usui Y, Tanaka H. Bioorg Med Chem Lett. 2014;24:5749.
10. Uehara F, Shoda A, Aritomo K, et al. Bioorg Med Chem Lett. 2013;23:6928.
11. Floyd MB, Du MT, Fabio PF, Jacob LA, Johnson BD. J Org Chem. 1985;50:5022.
12. Desmond R, Mills S, Volante RP, Shinkai I. Synth Commun. 1989;19:379.
Acknowledgments
l
We gratefully thank Sanofi-Aventis R&D CNS Research Depart-
ment (formerly Sanofi-Synthelabo R&D, Bagneux, France) for a
fruitful collaboration.
lM [c-
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Please cite this article in press as: Usui Y., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.06.078