Synthesis and biological evaluation of (4H-1,2,4-triazol-4-yl)isoquinoline derivatives as selective glycine transporter 1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.038

To identify novel glycine transporter 1(GlyT1) inhibitors with greater selectivity relative to GlyT2 and improved aqueous solubility, we synthesized a series of 4H-1,2,4-triazole derivatives with heteroaromatic rings at the 4-position and investigated the

Full text of DOI:10.1016/j.bmc.2011.11.038

Bioorganic & Medicinal Chemistry 20 (2012) 34–41
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of (4H-1,2,4-triazol-4-yl)isoquinoline
derivatives as selective glycine transporter 1 inhibitors
Takashi Sugane a, , Takahiko Tobe a, Wataru Hamaguchi a, Itsuro Shimada a, Kyoichi Maeno a,
⇑
Junji Miyata a, Takeshi Suzuki a, Tetsuya Kimizuka b, Takuma Morita c, Shuichi Sakamoto d,
Shin-ichi Tsukamoto a
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
b Intellectual Property, Astellas Pharma Inc., 2-3-11 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8411, Japan
c Astellas Research Technologies Co., Ltd, 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
d Technology, Astellas Pharma Inc., 3-17-1 Hasune, Itabashi-ku, Tokyo 174-8612, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify novel glycine transporter 1(GlyT1) inhibitors with greater selectivity relative to GlyT2 and
improved aqueous solubility, we synthesized a series of 4H-1,2,4-triazole derivatives with heteroaro-
matic rings at the 4-position and investigated their structure–activity relationships. Replacement of
the 2-fluorophenyl group of lead compound 5 with various aromatic groups led to the identification of
5-(3-biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquinoline (15) with 38-fold selectivity between
GlyT1 and GlyT2. 15 also showed improved aqueous solubility and in vivo efficacy on (+)-HA966-induced
hyperlocomotion in mice over the lead compound.
Received 12 October 2011
Revised 17 November 2011
Accepted 18 November 2011
Available online 30 November 2011
Keywords:
GlyT1
GlyT2
Ó 2011 Elsevier Ltd. All rights reserved.
The NMDA receptors
Schizophrenia
Triazole
1. Introduction
apy.4 These previous findings suggest that potentiating NMDA
receptor function may be useful in treating schizophrenia.
In the central nervous system (CNS), specific sodium/chloride-
dependent transporters regulate glycine levels in the synapse.
Glycine activity here is terminated by reuptake via two high-affin-
ity glycine transporters: glycine transporters 1 (GlyT1) and 2
(GlyT2).1,2 GlyT1 has widespread distribution in forebrain areas
such as the cortex and hippocampus. GlyT1 expressions is ob-
In addition to its above association with disease induction, the
NMDA receptors are also known to be associated with memory for-
mation and learning.5 Activation of the NMDA receptors is involved
in long-term potentiation (LTP), which is considered a mechanism
of memory formation and learning at the neuronal level.6 In addi-
tion, improvement of the NMDA receptor function via glycine-
binding sites rescues neurons from falling victim to the cellular
mechanisms thought to underlie age-related deficits in learning
and memory.7 Taken together, these previous findings strongly
suggest that memory and cognitive dysfunction may be amelio-
rated by activation of the NMDA receptors. As such, medications
which inhibiting the activity of GlyT1 and thereby activating the
function of the NMDA receptors may be useful as therapeutic
agents for treating schizophrenia, dementia, and related disorders.
A previous study identified N-[(3R)-3-(4’-fluorophenyl)-3-(4’-
phenylphenoxy)propyl]sarcosine (1), an analogue of sarcosine, as
a glycine transporter inhibitor (Fig. 1),8 with preclinical in vivo
studies in rodents further demonstrating 1 to have efficacy similar
to that of clozapine in prepulse inhibition (PPI) and latent inhibition
(LI) mouse models.9,10 In light of these findings, a number of sarco-
sine analogues have been subsequently synthesized and reported in
the literature.2,11–14 However, pharmaceutical companies have re-
cently launched new efforts to investigate non-substrate-based
served in brain areas rich in the N-methyl-D-aspartate (NMDA)
receptors, and is believed to modulate the NMDA receptor func-
tion. In contrast, expression of GlyT2 is limited to the spinal cord,
brain stem, and cerebellum, and GlyT2 is therefore thought to con-
trol the function of the strychnine-sensitive glycine receptor.
The hypofunction of the NMDA receptors is believed to be asso-
ciated with a number of human diseases. For example, the func-
tional deterioration of this receptor may have a role in inducing
schizophrenia,3 with clinical studies demonstrating that positive,
negative, and cognitive symptoms in schizophrenic patients can
be ameliorated with administration of glycine, D-serine (glycine
site agonist of the NMDA receptors), and sarcosine (N-methyl gly-
cine, weak GlyT1 inhibitor) in conjunction with conventional ther-
⇑
Corresponding author. Tel.: +81 29 863 6678; fax: +81 29 852 5387.
E-mail address: takashi.sugane@astellas.com (T. Sugane).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.038

T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
35
Table 1
Me
In vitro activity and solubility of 1,2,4-triazole derivertives 4 and 5
O
O
CF3
2
1
N
N
N
3
5
O
N
N
F
N
R
F
OH
4
N
Me
O S O
Me
CF3
O
F
4 R = i-Pr
5 R = Et
2 RG1678
1 ALX-5407
M)a
rGlyT2 IC50
(
l
M)a
Solubility pH 6.8 (lM)
Figure 1. Structures of ALX-5407 (1) and RG1678 (2).
Compd
rGlyT1 IC50
(l
4
5
0.37 0.020
0.22 0.050
3.3 0.053
0.23 0.092
<1
<1
inhibitors, with a wide variety of compounds subsequently being
disclosed.2,11–14 Among these compounds, [4-(3-fluoro-5-trifluo-
romethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-
2,2,2-trifluoro-1-methylethoxy)phenyl]methanone (2) was found
to exert a beneficial effect in schizophrenic patients in a recent
phase II clinical trial.15
Our team has also contributed to this field of study with our
recent report on a novel series of GlyT1 inhibitors derived from
the high-throughput screening (HTS) hit, 3-biphenyl-4-yl-4-(2-
a
Values are means standard error for three experiments.
We were unable to obtain the isoquinoline derivative 19 from
reaction with intermediate 7 and 1-aminoisoquinoline (25) under
acidic conditions, likely due to the low nucleophilicity of 25. Com-
pound 19 was thus synthesized via the reaction shown in Scheme
3. Briefly, 25 was first reacted with propionyl chloride to produce
amide 26, and then was treated with Lawesson’s reagent to give
thioamide (27). This compound was then methylated in aqueous
methanol to produce thioimidate (28), which was coupled with
acyl hydrazide (6) under acidic conditions to give the desired
1,2,4-triazole derivative 19.
Scheme 4 shows the preparation of the isoquinoline analogues
20 and 21. Compound 15 was first converted into N-oxide (20)
using m-chloroperbenzoic acid (mCPBA), followed by the heating
in Ac2O to give the 1-acetoxy derivative 29. Isoquinoline-1-one
(21) was then obtained by hydrolysis of 29. Lastly, 1,2,3,4-tetrahy-
droisoquinoline analogue 22 was obtained by the reduction of the
corresponding isoquinoline (15) using NaBH3CN in acetic acid
(Scheme 5).
methoxyphenyl)-5-methyl-4H-1,2,4-triazole (3) with
a novel
structure (Fig. 2).16 We have established the structure–activity
relationships (SARs) in this series of GlyT1 inhibitors, demonstrat-
ing that the 3-bipheny-4-yl-4-phenyl-4H-1,2,4-triazole moiety is
an essential core for this novel class of non-sarcosine-derived
GlyT1 inhibitors. Among the compounds prepared previously, 4
showed good oral bioavailability and brain permeability. Com-
pound 4 also effectively antagonized (+)-3-amino-1-hydroxypyrro-
lid-2-one [(+)-HA966]-induced hyperlocomotion, ameliorating
learning impairment in the passive avoidance task in mice. How-
ever, despite this attractive profile, the selectivity of 4 between
GlyT1 and GlyT2 was moderate, and its aqueous solubility was ex-
tremely poor, precluding further preclinical development of the
compound and its related analogues (Table 1).
3. Result and discussion
Here, we report our efforts to improve the GlyT2/GlyT1 selectiv-
ity and aqueous solubility of this series of GlyT1 inhibitors by
replacing the 2-fluorophenyl moiety of 4 with various heteroaro-
matic rings.
GlyT1 and GlyT2 inhibitory activities were assessed according
to reported protocols utilizing [3H]glycine uptake into rat C6 gli-
oma cells17 and rat brainstem cells, respectively. In our previous
SAR study,16 we reported that while introduction of an isopropyl
group at the 5-position of 1,2,4-triazole effectively improved selec-
tivity between GlyT1 and GlyT2 inhibition, this modification also
reduced aqueous solubility. Given these previous findings, we con-
ducted an SAR study with compound 5 as the lead to improve both
selectivity and solubility.
Our previous examination of the SAR using a substituted phenyl
group found that ortho substituted phenyl groups at the 4-positon
of triazoles were well tolerated, exerting potent GlyT1 inhibitory
activities. However, we have never investigated the SAR using het-
eroaromatic rings, particularly with bicyclic rings such as indoles
and quinolines. Working under the belief that these nitrogen-con-
taining structures would not only modulate affinities for both
GlyTs, but also improve physiochemical properties, we decided to
incorporate hetero-bicyclic rings into the compounds to hopefully
obtain GlyT1-selective compounds with improved solubility over
currently available compounds.
2. Chemistry
The 1,2,4-triazole derivatives were prepared as shown in
Schemes 1–5. Scheme 1 describes the synthesis of 1,2,4-triazole
derivatives 8, 10–18, 23, and 24. Commercially available phen-
ylbenzoyl hydrazide (6) was first acylated, followed by cyclization
using POCl3 to obtain the common intermediate 7. Derivatives 8,
10–18, 23, and 24 were then obtained by addition-elimination
reactions of 7 with various aromatic amines under acidic condi-
tions. N-Me indole derivative 9 was synthesized by methylation
of 8 with MeI under basic conditions (Scheme 2).
N
N
N
N
Me
Me
N
Me
OMe
N
F
We first investigated the SAR of compounds following introduc-
tion of an indole, indazole, and benzimidazole (Table 2). Replace-
ment of the 2-fluorophenyl moiety with an indole resulted in
3
4
HTS hit
reduced activity (8, IC50 = 22
methyl group at the 1-position of the indole showed better activity
(9, IC50 = 3.0 M), its activity was 14-fold less potent than that of
lM), and while introduction of a
IC50 (GlyT1) = 0.37 µM
IC50 (GlyT2) = 3.3 µM
IC50 (GlyT1) = 1.8 µM
IC50 (GlyT2) = 1.7 µM
l
compound 5. We also examined other nitrogen-containing
5,6-membered heterocyclic rings, such as indazole and benzimid-
Figure 2. In our previous work, modification of HTS hit 3 led to identification of
compound 4.

36
T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
N
N
O
N
N
NH2
N
R
Et
c
N
H
a, b
O
Et
8, 10-18,
23,24
6
7
Scheme 1. Reagents and conditions: (a) EtCOCl, Et3N, DMF; (b) POCl3, 100 °C, (c) R-NH2, p-TsOHꢀH2O, 160–200 °C.
quinoline and isoquinoline derivatives shown in Table 3, examin-
ing all N-substitution patterns. Interestingly, all quinoline and iso-
quinoline derivatives retained their GlyT1 inhibitory activity, and
the introduction of a nitrogen atom into the 1–4 positions (see
the positions on structure 13) was found to be particularly effec-
tive for GlyT1 inhibitory activity. Of the compounds examined,
the quinoline-8-yl (13) and isoquinoline-5-yl (15) analogues
showed potent GlyT1 inhibitory activity comparable to that of
N
N
N
N
Et
Me
Et
N
N
a
H
N
N
9
8
Scheme 2. Reagents: (a) MeI, KOH, DMF.
compound 5 (13, IC50 = 0.30
noteworthy, however, is that 15 showed weak GlyT2 inhibitory
activity (IC50 = 14 M) and a selectivity ratio (GlyT2/GlyT1) of
lM; 15, IC50 = 0.37 lM). Perhaps more
azole, but found the in vitro potencies of these compounds to be
weak, similar to that for indole derivatives (IC50 = 2.8–7.5 M).
l
l
38-fold, values much higher than observed with 5.
We next turned our attention to the SAR of 6,6-membered
heterocyclic derivatives such as quinolines. We evaluated the
In light of our finding that the isoquinoline-5-yl fragment is use-
ful for improving selectivity (GlyT2/GlyT1), we next synthesized
Me
O
S
S
NH2
HN
Et
HN Et
N
Et
c
b
a
N
N
N
N
25
26
27
28
N
N
O
NH2
N
Et
N
H
d
N
6
19
Scheme 3. Reagents and conditions: (a) EtCOCl, pyridine, THF; (b) Lawesson’s reagent, pyridine, 100 °C; (c) MeI, NaOH, EtOH; (d) 28, p-TsOHꢀH2O, xylene, 130 °C.
N
N
N
N
N
Et
a
N
Et
N+
N
O
20
15
N
N
N
N
N
Et
O
N
Et
c
b
20
N
N
OAc
29
21
Scheme 4. Reagents and conditions: (a) mCPBA, CH2Cl2; (b) Ac2O, 120 °C; (c) NaOH, MeOH.
N
N
N
N
N
Et
N
Et
a
NH
N
22
15
Scheme 5. Reagents: (a) NaBH3CN, AcOH.

T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
37
Table 2
improved solubility may have contributed to the potent in vivo
efficacy of 15.
In vitro GlyT1 inhibitory activities of 1,2,4-triazole derivatives 5, 8–12
N
N
4. Conclusion
N
R
Et
Through the iterative processes of synthesis and biological eval-
uation starting from lead compound 5, we demonstrated that
replacement of the 2-fluorophenyl group of 5 with a heteroaro-
matic group such as quinoline or isoquinoline maintained GlyT1
inhibitory activity. From these studies, we successfully derived iso-
quinoline derivative 15, which showed 38-fold selectivity and im-
proved aqueous solubility over lead compound 5. Compound 15
also reversed (+)-HA966-induced hyperlocomotion at 3 mg/kg po,
which was better in vivo activity than obtained with 4. Further
pharmaceutical characterization of 15 is ongoing.
a
Compd
R
rGlyT1 IC50 (lM)
F
5
0.22 0.050
H
N
8
9
22 3.5
5. Experiment
5.1. Chemistry
Me
N
3.0b
5.6b
Uncorrected melting points (Mps) were determined using
BÜCHI B-545 or Yanaco MP-500D micro melting apparatuses. 1H
NMR spectra were recorded on a JEOL JMN-LA-300 or JMN-EX-
400, and the chemical shifts were expressed in d (ppm) values with
trimethylsilane as an internal reference (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, and br = broad peak). Mass
spectra (MS) were recorded on a Hitachi M-80 or JEOL JMS-
LX2000 spectrometer. Elemental analyses were performed using
a Yanaco MT-5 (C, H, N), Elementar Vario EL III (C, H, N), and Dionex
DX-500 (S, halogene) and were within 0.4% of theoretical values.
H
N
10
N
N
11
12
2.8b
N
H
5.1.1. 2-(Biphenyl-4-yl)-5-ethyl-1,3,4-oxadiazole (7)
Propionyl chloride (4.37 g, 47.2 mmol) at 0 °C was added to a
solution of biphenyl-4-carboxylic hydrazide (6, 10.0 g, 47.2 mmol)
and triethylamine (4.96 g, 49.1 mmol) in N,N-dimethylformamide
(DMF) (200 mL), and the mixture was stirred at room temperature
for 2 h before being concentrated in vacuo. EtOAc and water was
then added, and the mixture was further stirred for 10 min. The
precipitate was collected by filtration and washed with water
and EtOAc, and the wet solid was dried in vacuo to give N’-propi-
onylbiphenyl-4-carbohydrazide (8.20 g, 65%) as a colorless pow-
der. A solution of N’-propionylbiphenyl-4-carbohydrazide (11.5 g,
42.9 mmol) in phosphorus oxychloride (50 mL) was stirred at
100 °C for 1 h. After cooling at room temperature, the mixture
was concentrated in vacuo, and the residue was then partitioned
between EtOAc and water. The organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo, while the
residue was purified using column chromatography on silica gel
(hexane/EtOAc = 3/1) to give 7 (8.21 g, 97%) as a white solid. 1H
NMR (DMSO-d6) d 1.46 (3H, t, J = 7.5 Hz), 2.98 (2H, q, J = 7.5 Hz),
7.35–7.52 (3H, m), 7.61–7.66 (2H, m), 7.71 (2H, dt, J = 2.0,
8.6 Hz), 8.10 (2H, dt, J = 1.8, 8.6 Hz); MS (FAB) m/z 251 [M+H]+.
H
N
7.5 0.80
N
a
Values are means standard error for three experiments.
Values are means for two experiments.
b
derivatives of 15 as described in Table 4—attempts which unfortu-
nately resulted in loss of activity. For example, compounds 20 and
21, both containing oxygen atoms, showed complete loss of GlyT1
inhibitory activity, as did tetrahydroisoquinoline and pyridine ana-
logues 22–24. These results suggest that either or both the aromatic-
ity of isoquinoline or the conformational restrictions are essential to
retaining GlyT1 inhibitory activity.
Given our successful improvement of selectivity for GlyT1 over
GlyT2 utilizing the isoquinoline moiety, we next evaluated the
aqueous solubility of 15 and compared it with the solubility of
compounds 4 and 5 (Table 5). Compound 15 showed higher solu-
bility than either 4 or 5 in both pH 1.2 and 6.8 aqueous solutions.
The related analogues 13, 14, and 16 also showed improved solu-
bility in both pH 1.2 and 6.8 aqueous solutions, probably due to
the contribution of basic and hydrophilic properties of the quino-
line and isoquinoline moiety.
Lastly, we assessed the in vivo efficacy of 15 on (+)-HA966 (an
antagonist for Gly binding sites on the NMDA receptors)18 -in-
duced hyperlocomotion in monoamine-depleted mice (Table 5).
Compound 15 showed 82% inhibition of hyperlocomotion at a dose
of 3 mg/kg orally (po).19 This attenuation of the locomotor activity
indicates that 15 increases the extracellular level of glycine, which
indirectly displaces the (+)-HA966 from Gly binding sites on the
NMDA receptors. Considering that compound 4 showed 70% inhi-
bition at a dose of 10 mg/kg intraperitoneally (ip), the in vivo effi-
cacy of 15 was judged to be much improved over that of 4. The
5.1.2. 5-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-
yl)isoquinoline (15)
To the mixture of 7 (500 mg, 2.0 mmol) and 5-aminoisoquino-
line (651 mg, 4.52 mmol) was added p-toluenesulfonic acid mono-
hydrate (50 mg, 0.26 mmol), and the resultant mixture was stirred
at 160 °C for 24 h. After cooling at room temperature, the mixture
was purified using column chromatography on silica gel (CHCl3/
MeOH = 98/2) to give crude 15 as a brown solid. The solid was
recrystallized from EtOAc to give 15 (300 mg, 40%) as a colorless
powder. Mp: 228–230 °C; 1H NMR (DMSO-d6) d 1.09 (3H, t,
J = 7.6 Hz), 2.33–2.50 (2H, m), 7.11 (1H, d, J = 5.8 Hz), 7.31–7.42
(5H, m), 7.53–7.58 (4H, m), 7.89–7.91 (1H, t, J = 7.9 Hz), 8.17 (1H,
d, J = 7.4 Hz), 8.42 (1H, d, J = 8.3 Hz), 8.54 (1H, d, J = 6.3 Hz), 9.51

38
T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
Table 3
In vitro GlyT1 inhibitory activities of 1,2,4-triazole derivatives of 5, 13–16
N
N
N
R
Et
a
a
Compd
R
rGlyT1 IC50
(
lM)
rGlyT2 IC50
(l
M)
Selectivity (rGlyT2/rGlyT1)
1.0
F
5
0.22 0.050
0.30 0.060
0.23 0.092
0.38 0.11
1
N
8
2
3
7
13
1.3
6
5
4
14
15
N
0.80 0.021
0.37 0.012
4.2 0.87
14 5.3
5.3
38
N
16
17
0.82 0.17
1.7 0.033
2.7 0.47
3.3
N
NDb
NDb
N
18
19
3.7 0.52
1.4 0.10
NDb
NDb
NDb
NDb
N
N
a
Values are means standard error for three experiments.
ND, not determined.
b
(1H, s); MS (FAB) m/z 377 [M+H]+; Anal. Calcd for C25H20N4: C,
79.76; H, 5.35; N, 14.88. Found: C, 79.73; H, 5.30; N, 14.85.
Compounds 8, 10–14, 16–18, 23 and 24 were prepared by a
method similar to that described for 15. Yields refer to the final
substitution step.
5.1.5. 4-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-1H-
indazole (11) (22%)
Mp: 164–167 °C; 1H NMR (DMSO-d6) d 1.11 (3H, t, J = 7.5 Hz),
2.51–2.57 (2H, m), 7.28–7.45 (6H, m), 7.48–7.64 (5H, m), 7.76
(1H, d, J = 8.0 Hz), 7.83 (1H, s), 13.50–13.57 (1H, brs); MS (FAB)
m/z 366 [M+H]+; Anal. Calcd for C23H19N5: C, 75.59; H, 5.24; N,
19.16. Found: C, 75.65; H, 5.15; N, 19.13.
5.1.3. 7-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-1H-
indole (8) (35%)
Mp: 254–256 °C; 1H NMR (DMSO-d6) d 1.11 (3H, t, J = 7.4 Hz),
2.38–2.44 (2H, q, J = 7.4 Hz), 6.60 (1H, s), 7.12–7.16 (2H, m),
7.31–7.44 (6H, m), 7.52–7.62 (4H, m), 7.73–7.78 (1H, m), 11.5
(1H, s); MS (FAB) m/z 365 [M+H]+; Anal. Calcd for C24H20N4: C,
79.10; H, 5.53; N, 15.37. Found: C, 79.17; H, 5.54; N, 15.38.
5.1.6. 7-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-1H-
benzimidazole (12) (46%)
Mp: >300 °C; 1H NMR (DMSO-d6) d 1.10 (3H, t, J = 7.6 Hz),
2.41–2.48 (2H, m), 7.26 (1H, d, J = 7.3 Hz), 7.32–7.47 (6H, m),
7.55 (2H, d, J = 8.3 Hz), 7.60 (2H, d, J = 7.8 Hz), 7.75 (1H, d,
J = 8.3 Hz), 8.32 (1H, s), 12.9 (1H, s); MS (FAB) m/z 366 [M+H]+;
Anal. Calcd for C23H19N5: C, 75.59; H, 5.24; N, 19.16. Found: C,
75.65; H, 5.14; N, 19.17.
5.1.4. 7-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-1H-
indazole (10) (47%)
Mp: 294–296 °C; 1H NMR (DMSO-d6) d 1.12 (3H, t, J = 7.5 Hz),
2.43–2.49 (2H, m), 7.27 (1H, t, J = 7.8 Hz), 7.32–7.43 (5H, m), 7.49
(1H, d, J = 7.0 Hz), 7.55–7.61 (4H, m), 8.00 (1H, d, J = 8.0 Hz), 8.26
(1H, s), 13.5 (1H, s); MS (FAB) m/z 366 [M+H]+; Anal. Calcd for
5.1.7. 8-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)quino-
line (13) (79%)
Mp: 205–207 °C; 1H NMR (CDCl3) d 1.21 (3H, t, J = 7.5 Hz), 2.39–
2.66 (2H, m), 7.21–7.65 (12H, m), 8.01 (1H, dd, J = 2.2, 7.3 Hz), 8.28
(1H, dd, J = 1.8, 7.3 Hz), 8.92 (1H, dd, J = 1.8, 2.2 Hz); MS (FAB) m/z
C23H19N5: C, 75.59; H, 5.24; N, 19.16. Found: C, 75.62; H, 5.31; N,
19.18.

T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
39
Table 4
In vitro GlyT1 inhibitory activities of 1,2,4-triazole derivatives of 15, 20–24
5.1.9. 5-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)- quino-
line (16) (18%)
Mp: 202–203 °C; 1H NMR (DMSO-d6) d 1.09 (3H, t, J = 7.6 Hz),
2.33–2.51 (2H, m), 7.30–7.42 (5H, m), 7.53–7.59 (5H, m),
7.65–7.68 (1H, m), 7.96–7.80 (2H, m), 8.26–8.31 (1H, m), 9.00
(1H, dd, J = 3.9, 1.4 Hz); MS (FAB) m/z 377 [M+H]+; Anal. Calcd for
N
N
N
R
Et
C25H20N4: C, 79.76; H, 5.35; N, 14.88. Found: C, 79.58; H, 5.37; N,
14.76.
a
Compd
R
rGlyT1 IC50 (lM)
5.1.10. 4-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)- quino-
line (17) (32%)
Mp: 189–191 °C; 1H NMR (DMSO-d6) d 1.10 (3H, t, J = 7.8 Hz),
2.35–2.52 (2H, m), 7.32–7.35 (2H, m), 7.38–7.42 (4H, m), 7.55–
7.59 (4H, m), 7.66 (1H, t, J = 7.3 Hz), 7.87 (1H, t, J = 7.3 Hz), 7.96
(1H, d, J = 4.4 Hz), 8.20 (1H, d, J = 8.3 Hz), 9.16 (1H, d, J = 7.8 Hz);
MS (FAB) m/z 377 [M+H]+; Anal. Calcd for C25H20N4.0.2H2O: C,
79.01; H, 5.41; N, 14.74. Found: C, 78.86; H, 5.66; N, 14.51.
15
0.37 0.012
N
b
20
21
>10 (28%)
N+
O
5.1.11. 4-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)- iso-
quinoline (18) (29%)
Mp: 209–210 °C; 1H NMR (DMSO-d6) d 1.10 (3H, t, J = 7.8 Hz),
2.35–2.52 (2H, m), 7.29–7.35 (2H, m), 7.38–7.42 (4H, m),
7.54–7.59 (4H, m), 7.79–7.89 (2H, m), 8.35 (1H, d, J = 7.8 Hz),
8.85 (1H, s), 9.57 (1H, s); MS (FAB) m/z 377 [M+H]+; Anal. Calcd
for C25H20N4: C, 79.76; H, 5.35; N, 14.88. Found: C, 79.83; H,
5.41; N, 15.09.
b
>10 (16%)
NH
NH
O
N
b
22
23
24
>10 (25%)
5.1.12. 4-[(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-
methyl]pyridine (23) (48%)
Mp: 205–206 °C; 1H NMR (DMSO-d6) d 1.24 (3H, t, J = 7.3 Hz),
2.65 (2H, q, J = 7.3 Hz), 5.40 (2H, s), 6.99 (2H, d, J = 5.9 Hz), 7.39
(1H, t, J = 5.4 Hz), 7.48 (2H, t, J = 7.8 Hz), 7.62 (2H, d, J = 8.3 Hz),
7.70 (2H, d, J = 6.8 Hz), 7.78 (2H, d, J = 8.3 Hz), 8.53 (2H, d,
J = 5.3 Hz); MS (FAB) m/z 377 [M+H]+; Anal. Calcd for C22H20N4:
C, 77.62; H, 5.92; N, 16.46. Found: C, 77.50; H, 5.87; N, 16.47.
b
>10 (15%)
b
>10 (36%)
Me
N
5.1.13. 4-[1-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-
ethyl]pyridine (24) (6%)
a
Values are means standard error for three experiments.
b
% Inhibition at 10 lM.
Mp: 175–176 °C; 1H NMR (DMSO-d6) d 1.17 (3H, t, J = 7.8 Hz),
1.82 (3H, d, J = 6.5 Hz), 2.23–2.34 (1H, m), 2.55–2.66 (1H, m),
5.61 (1H, q, J = 7.3 Hz), 7.09 (2H, d, J = 6.4 Hz), 7.40 (1H, t,
J = 7.3 Hz), 7.46–7.55 (4H, m), 7.71 (2H, d, J = 7.4 Hz), 7.79 (2H, d,
J = 8.3 Hz), 8.56 (2H, d, J = 5.9 Hz); MS (FAB) m/z 355 [M+H]+; Anal.
Calcd for C23H22N4.0.2H2O: C, 77.15; H, 6.31; N, 15.65. Found: C,
77.35; H, 6.15; N, 15.37.
Table 5
Aqueous solubility and effects on (+)-HA966 induced hyperlocomotion for 1,2,4-
triazole derivertives 4, 5, and 13–16
Compd
Solubility pH
Solubility pH
6.8 (lM)
(+)-HA966-induced
hyperlocomotion
1.2 (l
M)
in mice inhibitiona (%)
5.1.14. 7-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-
1-methyl-1H-indole (9)
4
5
13
14
15
16
5
96
>100
>100
>100
>100
<1
<1
23
11
5
70% @ 10 mg / kg ip
NDb
NDb
CH3I (0.023 ml, 0.37 mmol) and K2CO3 (65 mg, 0.47 mmol) were
added to a solution of 8 (110 mg, 0.30 mmol) in DMF (5.0 mL), and
the mixture was stirred at room temperature. After 3 h, a second
batch of CH3I (0.015 ml, 0.24 mmol) and KOH (30 mg, 0.54 mmol)
was added, and the mixture was stirred for 19 h. The reaction mix-
ture was partitioned between EtOAc and water, and the organic
layer was separated and dried over anhydrous MgSO4. The solvent
was removed via evaporation, and the residue was purified using
column chromatography on silica gel (CHCl3/MeOH = 98/2) to give
crude 9. The crude solid was recrystallized from diisopropyl ether
to give 9 (74 mg, 65%) as a colorless powder. Mp: 185–186 °C; 1H
NMR (DMSO-d6) d 1.16 (3H, t, J = 7.6 Hz), 2.45–2.51 (2H, m), 3.16
(3H, s), 6.57 (1H, d, J = 2.9 Hz), 7.22 (1H, t, J = 7.6 Hz), 7.32–7.37
(3H, m), 7.39–7.44 (2H, m), 7.46–7.50 (2H, m), 7.57–7.64 (4H,
m), 7.79 (1H, d, J = 7.3 Hz); MS (FAB) m/z 378 [M+H]+; Anal. Calcd
for C25H22N4: C, 79.34; H, 5.86; N, 14.80. Found: C, 79.37; H,
6.03; N, 14.72.
NDb
82% @ 3 mg/kg po
9
NDb
a
n = 16 experiments.
ND, not determined.
b
377 [M+H]+; Anal. Calcd for C25H20N4: C, 79.76; H, 5.35; N, 14.88.
Found: C, 79.73; H, 5.35; N, 14.91.
5.1.8. 8-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquino-
line (14) (22%)
Mp: 197–198 °C; 1H NMR (DMSO-d6) d 1.11 (3H, t, J = 7.6 Hz),
2.38–2.55 (2H, m), 7.30–7.43 (5H, m), 7.52–7.60 (4H, m),
7.98–8.07 (3H, m), 8.24–8.28 (1H, m), 8.59–8.63 (2H, m); MS
(FAB) m/z 377 [M+H]+; Anal. Calcd for C25H20N4: C, 79.76; H,
5.35; N, 14.88. Found: C, 79.91; H, 5.44; N, 14.83.

40
T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
5.1.15. 1-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)iso-
quinoline (19)
on silica gel (CHCl3/MeOH = 20/1) to give 29. 1 M NaOH solution
(5 ml) was added to a solution of 29 in MeOH (5 ml), and the mix-
ture was stirred for 10 min. The reaction mixture was concentrated
in vacuo, and the residue was purified using column chromatog-
raphy on Al2O3 (CHCl3/MeOH = 20/1) to obtain a solid, which was
then washed with hexane/EtOAc (1:2) to give 21 (730 mg, 72%)
as a pale-yellow solid. Mp: 257–259 °C; 1H NMR (DMSO-d6) d
1.13 (3H, t, J = 7.5 Hz), 2.33–2.50 (2H, m), 5.71 (1H, d, J = 7.3 Hz),
7.23 (1H, t, J = 6.0 Hz), 7.32–7.45 (5H, m), 7.60–8.02 (5H, m), 7.99
(1H, d, J = 7.5 Hz), 8.40 (1H, d, J = 8.2 Hz), 11.57 (1H, d, J = 5.5 Hz);
MS (FAB) m/z 393 [M+H]+; Anal. Calcd for C25H20N4O.0.2H2O: C,
75.81; H, 5.19; N, 14.15. Found: C, 75.86; H, 5.17; N, 14.17.
Propionyl chloride (3.34 ml, 38.5 mmol) solution in THF (20 ml)
at 0 °C was added to a solution of 1-aminoisoquinoline (25, 5.04 g,
35.0 mmol) in pyridine (100 ml), and the mixture was stirred for
11 h at room temperature. MeOH (50 ml) was then, and the solu-
tion was concentrated in vacuo. The residue was then partitioned
between EtOAc and water, and the organic layer was separated
and dried over anhydrous Na2SO4. The solvent was removed via
evaporation to give N-isoquinolin-1-ylpropanamide (26, 5.22 g,
74%).
Lawesson’s reagent (1.70 g, 4.2 mmol) was added to a suspen-
sion of 26 (1.40 g, 7.0 mmol) in pyridine (30 ml), and the mixture
was stirred at 100 °C for 2 h. After cooling at room temperature,
the reaction mixture was partitioned between EtOAc and water,
and the organic layer was separated and dried over anhydrous
MgSO4. The solvent was removed via evaporation, and the residue
was purified using column chromatography on silica gel (hexane/
EtOAc = 1/1) to give N-isoquinolin-1-ylpropanethioamide (27,
1.39 g, 92%) as a pale-yellow solid.
CH3I (1.36 g, 9.57 mmol) and 1 M NaOH solution (13 ml) were
added to a solution of 27 (1.38 g, 6.38 mmol) in EtOH (20 ml),
and the resultant mixture was stirred at room temperature for
0.5 h. The mixture was then evaporated in vacuo, and the residue
was partitioned between EtOAc and water. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified using column chromatography
on silica gel (hexane/EtOAc = 2/1) to give methyl N-isoquinolin-
1-ylpropanimidothioate (28, 1.29 g, 88%) as a pale-yellow oil. A
mixture of 28 (500 mg, 2.17 mmol), 6 (424 mg, 2.0 mmol), p-tolu-
enesulfonic acid monohydrate (35 mg, 0.18 mmol), and xylene
(5 mL) was stirred at 130 °C for 4 h. After it was cooled at room
temperature, the residue was purified using column chromatogra-
phy on silica gel (CHCl3/MeOH = 20/1) to give a crude solid, which
was then purified by column chromatography on Al2O3 (CHCl3) to
give 19 (20 mg, 3%) as a brown solid. Mp: 179–181 °C; 1H NMR
(CDCl3) d 1.21 (3H, t, J = 7.5 Hz), 2.59 (2H, q, J = 7.5 Hz), 7.26–7.46
(10H, m), 7.55 (1H, t, J = 7.7 Hz), 7.74 (1H, t, J = 7.7 Hz), 7.88 (1H,
d, J = 5.9 Hz), 7.95 (1H, d, J = 8.1 Hz), 8.61 (1H, d, J = 5.8 Hz); MS
(FAB) m/z 377 [M+H]+; Anal. Calcd for C25H20N4: C, 79.76; H,
5.35; N, 14.88. Found: C, 79.99; H, 5.41; N, 15.12.
5.1.18. 5-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)-1,2,3,4-
tetrahydroisoquinoline (22)
NaBH3CN (500 mg. 7.96 mmol) was added to a solution of 15
(520 mg, 1.38 mmol) in acetic acid (20 ml), and the mixture was
stirred at room temperature for 2 h and partitioned between EtOAc
and K2CO3 solution. The organic layer was separated, washed with
NaHCO3 (aqueous) and brine, and dried over anhydrous MgSO4.
The solvent was removed via evaporation, and the residue was
purified using column chromatography on silica gel (CHCl3/
MeOH = 10/1) to give crude 22. The crude product was washed
with hexane/EtOAc (2:1) to give 22 (276 mg, 53%) as a white solid.
Mp: 155–156 °C; 1H NMR (CDCl3) d 1.27 (3H, t, J = 7.5 Hz), 2.16 (2H,
t, J = 5.7 Hz), 2.56 (2H, q, J = 7.5 Hz), 2.97 (2H, t, J = 5.8 Hz), 4.07
(2H, s), 7.15–7.56 (12H, m); MS (FAB) m/z 381 [M+H]+; Anal. Calcd
for C25H24N4: C, 78.92; H, 6.36; N, 14.73. Found: C, 78.87; H, 6.47;
N, 14.72.
5.2. Aqueous solubility
Small volumes of the DMSO solutions of test compounds were
diluted to 130 lM by adding the aqueous buffer solutions of
pH1.2 and pH6.8. After incubation at 25 °C for 20 h, precipitates
were separated by filtration. The solubility was determined by
HPLC analysis of each filtrate.
5.3. Biology
5.3.1. [3H]Glycine uptake assay (GlyT1)
Gomenza et al. previously reported the expression of rat GlyT1
in C6 cells.17 In the present study, C6 cells were maintained in
growth medium (D-MEM containing 10% fetal calf serum) at
37 °C in humidified air with 5% CO2. Two days before the [3H]gly-
cine uptake experiments, C6 cells were plated at a density of
2 ꢁ 104 cells per well in a 96-well white CulturPlate (PerkinElmer
5.1.16. 5-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquin-
oline 2-oxide (20)
mCPBA (1.80 g, 8.0 mmol) was added to a solution of 15 (1.95 g,
5.2 mmol) in CH2Cl2 (20 mL), and the mixture was stirred at room
temperature for 6 days. K2CO3 was then added, and the solution
was subjected to extraction with CHCl3-MeOH (10:1). The organic
layer was dried over anhydrous MgSO4 and concentrated in vacuo,
and the residue was purified using column chromatography on sil-
ica gel (CHCl3/MeOH = 20/1) to give 20 (1.42 g, 70%) as a white so-
lid. Mp: 287–289 °C; 1H NMR (DMSO-d6) d 1.12 (3H, t, J = 7.6 Hz),
2.40–2.55 (2H, m), 7.21 (1H, d, J = 7.2 Hz), 7.31–7.44 (5H, m),
7.56–7.62 (4H, m), 7.84–7.88 (1H, m), 7.90–7.94 (1H, m),
8.09–8.14 (2H, m), 9.13 (1H, d, J = 1.2 Hz); MS (FAB) m/z 393
[M+H]+; Anal. Calcd for C25H20N4O: C, 76.51; H, 5.14; N, 14.28.
Found: C, 76.58; H, 5.16; N, 14.56.
Inc.). The assays were performed at 37 °C in 100 lL of 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
containing NaCl (150 mmol/L), KCl (5 mmol/L), MgCl2 (1 mmol/L),
CaCl2 (1 mmol/L), HEPES (10 mmol/L),
-alanine (5 mmol/L) at pH 7.4. The growth medium was removed,
and after being washed with HEPES buffer, the cells were incu-
D-glucose (10 mmol/L), and
L
bated for 30 min with 50
compounds.
l
L of HEPES buffer containing the test
Uptake was initiated by adding 50
l
L of 0.167 mmol/L [3H]gly-
cine (53.3 Ci/mmol). Non-specific uptake was detected using
3 mmol/L sarcosine. After incubating for 10 min at 37 °C, the wells
were aspirated and washed three times with ice-cold phosphate-
5.1.17. 5-(3-Biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)iso-
quinolin-1(2H)-one (21)
buffered saline (PBS). After solubilizing the cells with 17
lL of
A solution of 20 (1.01 g, 2.57 mmol) in acetic anhydride (15 ml)
was stirred at 120 °C for 14 h. The reaction mixture was then evap-
orated, and the residue was partitioned between EtOAc and K2CO3
solution. The organic layer was separated, washed with brine, and
dried over anhydrous MgSO4. The solvent was removed by evapo-
ration, and the residue was purified using column chromatography
0.1 mol/L NaOH, 100 L of scintillant (Micro Scint PS; PerkinElmer
l
Inc.) was added to the wells, and the plate was counted in a Top
Count (Hewlett-Packard Company; Palo Alto, CA, USA). We evalu-
ated sarcosine in parallel as a reference GlyT1 inhibitor. The drug
concentrations required to inhibit 50% of the specific [3H]glycine

T. Sugane et al. / Bioorg. Med. Chem. 20 (2012) 34–41
41
uptake (IC50) were obtained using nonlinear regression analysis
and SAS software (SAS Institute Inc.; Cary, NC, USA), with the mean
SEM obtained from three independent experiments.
5) (+)-HA966 was acutely administered bilaterally into the lat-
eral ventricule (free-handed using two-step needle). Imme-
diately after administration, each animal was placed in the
measuring cage of an activity measurement apparatus.
6) Activity per 90 min was measured immediately upon plac-
ing the animal in the cage.
7) The integral value of locomotor activity per 90 min was
selected as the data, and the inhibition(%) of hyperlocomo-
tion with the test compounds was determined as follows:
5.3.2. [3H]Glycine uptake assay (GlyT2)
A primary cell culture of rat brain was prepared for a [3H]glycine
uptake assay. Pregnant rats (Wistar at 17 days’ gestation; Japan SLC,
Shizuoka, Japan) were euthanized under ether anesthesia by exsan-
guination through severing the carotid artery. The fetuses were then
obtained and their brainstems isolated. After digestion by papain,
brainstem cells were dispersed into the culture medium and plated
at a density of 5.0 ꢁ 104 cells per well in 96-well white plates coated
with poly-l-lysine, at which point the plates were incubated for
14–21 days in a CO2 incubator (37 °C, 5% CO2).
100 ꢁ ðfactivity of ½ðþÞ ꢃ HA966 þ Vehicleꢄgroup
ꢃ activity of ðACSF þ VehicleÞgroupg ꢃ factivity of ½ðþÞ
ꢃ HA966 þ test compoundꢄgroup ꢃ activity ofðACSF
þ VehicleÞgroupgÞ=factivity of ½ðþÞ ꢃ HA966
On the day of the assay, after being washed with assay buffer
(150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2,
10 mmol/L glucose, 5 mmol/L l-Alanine, 3 mmol/L sarcosine, and
þ Vehicleꢄgroup ꢃ activity ofðACSF þ VehicleÞgroupg:
Acknowledgments
10 mmol/L HEPES, pH 7.4), each well was filled with 100 lL of as-
say buffer and preincubated at 37 °C. After 10 min preincubation,
The authors deeply acknowledge Atsuyuki Kohara, Masaki Aota,
Hitoshi Doihara, and Kyouko Saita for performing pharmacological
evaluations. We also thank the staff of the Analysis and
Pharmacokinetics Research Laboratories for performing spectral
measurements.
the incubation buffer was replaced with reaction mixture contain-
ing the test compounds and approximately 0.3 l
M [3H]glycine, the
plate was then incubated for 10 min at 37 °C, and the reaction was
stopped by washing the cells four times with ice-cold buffer. After
solubilizing the cells with 17 lL of 0.1 mol/L NaOH, 100 lL of scin-
tillant was added and the plate was counted using Top Count
(Hewlett-Packard Company; Palo Alto, CA, USA). In each experi-
ment, specific [3H]glycine uptake was estimated based on the up-
take inhibition of 3 mmol/L glycine. The drug concentrations
References and notes
1. Broer, S.; Cavanaugh, J. A.; Rasko, J. E. J. Biochem. Soc. Trans. 2005, 33, 233.
2. Sur, C.; Kinney, G. G. Curr. Drug Targets 2007, 8, 643.
3. (a) Shim, S. S.; Hammonds, M. D.; Kee, B. S. Eur. Arch. Psych. Clin. Neurosci. 2008,
258, 16; (b) Javitt, D. C. Curr. Opin. Drug Discovery Dev. 2009, 12, 468.
4. Tsai, G. E.; Lin, P. Y. Curr. Pharm. Des. 2010, 16, 522.
5. Riedel, G.; Platt, B.; Micheau, J. Behav., Brain Res. 2003, 140, 1.
6. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Baudry, M. Nature 1986, 319, 774.
7. Benvenga, M. J.; Spaulding, T. C. Pharmacol. Biochem. Behav. 1988, 30, 205.
8. Atkinson, B. N.; Bell, S. C.; De Vivo, M.; Kowalski, L. R.; Lechner, S. M.;
Ognyanov, V. I.; Tham, C. S.; Tsai, C.; Jia, J.; Ashton, D.; Klitenick, M. A. Mol.
Pharmacol. 2001, 60, 1414.
9. Lipina, T.; Labrie, V.; Weiner, I.; Roder, J. Psychopharmacology 2005, 179, 54.
10. Kinney, G. G.; Sur, C.; Burno, M.; Mallorga, P. J.; Williams, J. B.; Figueroa, D. J.;
Wittmann, M.; Lemaire, W.; Conn, P. J. J. Neurosci. 2003, 23, 7586.
11. Lindsley, C. W.; Wolkenberg, S. E.; Kinney, G. G. Curr. Top. Med. Chem. 2006, 6,
1883.
required to inhibit 50% of the specific [3H]glycine uptake (IC50
)
were obtained using nonlinear regression analysis and SAS soft-
ware (SAS Institute Inc.; Cary, NC, USA). We evaluated glycine in
parallel as the reference, and the mean SEM from three indepen-
dent experiments gave the final inhibition values.
5.3.3. (+)-HA966-induced enhancement of locomotor activity in
mice
The method reported previously20 was used to conduct the
experiment, with some modification.
12. Harsing, L. G.; Juranyi, Z.; Gacsalyi, I.; Tapolicsanyi, P.; Czompa, A.;
Matyus, P. Curr. Med. Chem. 2006, 13, 1017.
ꢂ Animal: male ICR mice (Japan SLC; age 5–7 weeks)
ꢂ Drug: reserpine (Apoplon injection, 1 mg/ml; Daiichi Pharma-
ceuticals Co., Ltd., Tokyo, Japan), (+)-HA966, a-methyl-para-
tyrosine methyl ester (Sigma, Inc.)
13. Bridges, T. M.; Williams, R.; Lindsley, C. W. Curr. Opin. Mol. Ther. 2008, 10, 591.
14. Wolkenberg, S. E.; Sur, C. Curr. Top. Med. Chem. 2010, 10, 170.
15. Pinard, E.; Alanine, A.; Alberati, D.; Bender, M.; Borroni, E.; Bourdeaux, P.;
Brom, V.; Burner, S.; Fischer, H.; Hainzl, D.; Halm, R.; Hauser, N.; Jolidon, S.;
Lengyel, J.; Marty, H.-P.; Meyer, T.; Moreau, J.-L.; Mory, R.; Narquizian, R.;
Nettekoven, M.; Norcross, R. D.; Puellmann, B.; Schmid, P.; Schmitt, S.; Stalder,
H.; Wermuth, R.; Wettstein, J. G.; Zimmerli, D. J. Med. Chem. 2010, 53, 4603.
16. Sugane, T.; Tobe, T.; Hamaguchi, W.; Shimada, I.; Maeno, K.; Miyata, J.; Suzuki,
T.; Kimizuka, T.; Kohara, A.; Morita, T.; Doihara, H.; Saita, K.; Aota, M.; Furutani,
M.; Shimada, Y.; Hamada, N.; Sakamoto, S.; Tsukamoto, S. J. Med. Chem. 2011,
54, 387.
17. Gomeza, J.; Zafra, F.; Olivares, L.; Gimenez, C.; Aragon, C. Biochim. Biophys. Acta,
Biomembr. 1995, 1233, 41.
18. Singh, L.; Donald, A. E.; Foster, A. C.; Hutson, P. H.; Iversen, L. L.; Iversen, S. D.;
Kemp, J. A.; Leeson, P. D.; Marshall, G. R.; Oles, R. J. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 347.
ꢂ Equipment: Supermex (Muromachi Machine)
ꢂ Methods:
1) Pharmaceutical drug-treated groups were defined as fol-
lows, with 16 mice per group: [artificial cerebrospinal fluid
(ACSF) + Vehicle] group, [(+)-HA966 80 lg/mouse (intracer-
ebroventricular) + Vehicle] group, [(+)-HA966 80
lg/mouse
(intracerebroventricular) + test compound] group
2) Nineteen hours before (+)-HA966 administration, reserpine
(10 mg/kg) was administered intraperitoneally.
3) Thirty minutes before (+)-HA966 administration, a-methyl-
para-tyrosine methyl ester (250 mg/kg) was administered
intraperitoneally.
19. We did not observe any significant change in locomotor activity of the naive
mice treated only by compound 15 (100 mg/kg po).
20. Slusher, B. S.; Rissolo, K. C.; Jackson, P. F.; Pullan, L. M. J. Neural Transm. 1994,
97, 175.
4) Twenty minutes before (+)-HA966 administration, a test
compound was administered orally or intraperitoneally.