Atropisomeric 4-phenyl-4 H -1,2,4-triazoles as selective glycine transporter 1 inhibitors

Article abstract of DOI:10.1021/jm400383w

We report on the optimization of 4H-1,2,4-triazole derivatives to increase their activity and selectivity as glycine transporter 1 (GlyT1) inhibitors. Structure-activity relationship exploration resulted in the identification of a 3-[3-ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-methylbenzonitrile (14u) compound with markedly higher selectivity for GlyT1. Physiochemical studies revealed that 14u exists as a stable pair of atropisomers under physiological conditions. We successfully separated the atropisomers to obtain active enantiomer (R)-14u, which displayed favorable pharmacokinetic properties, as well as positive results in the mice Y-maze test.

Full text of DOI:10.1021/jm400383w

Article
pubs.acs.org/jmc
Atropisomeric 4‑Phenyl‑4H‑1,2,4-triazoles as Selective Glycine
Transporter 1 Inhibitors
Takashi Sugane,*,† Takahiko Tobe,† Wataru Hamaguchi,† Itsuro Shimada,† Kyoichi Maeno,†
Junji Miyata,† Takeshi Suzuki,† Tetsuya Kimizuka,† Shuichi Sakamoto,† and Shin-ichi Tsukamoto†
†Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
S
* Supporting Information
ABSTRACT: We report on the optimization of 4H-1,2,4-
triazole derivatives to increase their activity and selectivity as
glycine transporter 1 (GlyT1) inhibitors. Structure−activity
relationship exploration resulted in the identification of a 3-[3-
ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-methyl-
benzonitrile (14u) compound with markedly higher selectivity
for GlyT1. Physiochemical studies revealed that 14u exists as a
stable pair of atropisomers under physiological conditions. We
successfully separated the atropisomers to obtain active enantiomer (R)-14u, which displayed favorable pharmacokinetic
properties, as well as positive results in the mice Y-maze test.
INTRODUCTION
■
In the central nervous system, specific sodium/chloride-
dependent transporters regulate glycine levels in synapses.
Two such high-affinity glycine transporters, glycine transporters
1 (GlyT1, Slc6a9) and 2 (GlyT2, Slc6a5), terminate glycine
activity by mediating its uptake.1,2 GlyT1 is expressed in
forebrain areas, such as the hippocampus and cerebral cortex,
where the transporter is believed to modulate N-methyl-D-
aspartate (NMDA) receptor activity by regulating glycine
levels, which acts as a coagonist with glutamate.
Figure 1. Structure of two GlyT1 inhibitors.
Hypofunction of NMDA receptors is believed to be a
synthesized and characterized.2,12−15 More recently, however,
contributing factor for the development of a number of human
diseases, particularly schizophrenia.3 A few clinical studies have
nonsubstrate-based GlyT1 inhibitors have attracted attention,
and a wide variety of compounds without a carboxylate group
have been disclosed.2,12−15 Among these compounds, [4-(3-
fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methane-
sulfonyl-2-((S)-2,2,2-trifluoro-1-methylethoxy)phenyl]-
methanone (2)16 was reported to have beneficial effects in
schizophrenic patients in a recent phase II clinical trial.
Our group is also actively studying a novel series of GlyT1
inhibitors derived from 3-biphenyl-4-yl-4-phenyl-4H-1,2,4-
triazole (3, Figure 2), which was identified using a high-
throughput screening (HTS) approach.17 Modification of the
4-position of the triazole increased the GlyT1 inhibitory activity
of the obtained compounds (4 and 5). Further modification of
5 by the introduction of a heterocycle at the 4-position led to
the formation of isoquinoline analogue 6, which exhibited 38-
fold selectivity between GlyT1 and GlyT2 and effectively
antagonized (+)-3-amino-1-hydroxypyrrolid-2-one
[(+)-HA966]-induced hyperlocomotion in mice upon oral
administration at 3 mg/kg.18
demonstrated that positive, negative, and cognitive symptoms
in schizophrenic patients can be ameliorated by the
administration of glycine, D-serine (glycine site agonist of
NMDA receptors), and sarcosine (N-methyl glycine, weak
GlyT1 inhibitor) in conjunction with conventional therapy.4−6
These clinical findings suggest that potentiating NMDA
receptor function may be a useful approach for treating
schizophrenia. In addition to their role in disease induction,
NMDA receptors are involved in memory formation and
learning.7 Activation of NMDA receptors mediates long-term
potentiation of synaptic transmission, which underlies memory
formation and learning at the neuronal level.8 Thus, agents that
inhibit the activity of GlyT1 are expected to modulate NMDA
receptor activity and may be useful for treating schizophrenia,
dementia, and related disorders.
N-[(3R)-3-(4′-Fluorophenyl)-3-(4′-phenylphenoxy)propyl]-
sarcosine (1),9 an analogue of sarcosine, was reported as a
selective inhibitor of GlyT1 (Figure 1), with preclinical in vivo
studies in rodents demonstrating that 1 has a similar efficacy as
clinically approved antipsychotics.10,11 Due to these reported
properties, a number of sarcosine analogues have been
Received: March 15, 2013
Published: July 9, 2013
© 2013 American Chemical Society
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Figure 2. Structure of 1,2,4-triazole-based GlyT1 inhibitors.
a
Scheme 1. Synthesis of 1,2,4-Triazole Derivatives
a
Reagents and conditions: (a) Lawesson’s reagent, toluene, 110 °C; (b) MeI, K2CO3, CH3CN, 50 °C; (c) R2CONHNH2, TsOH·H2O, DMF, 120
°C; (d) (i) n-BuLi, THF, −78 °C, (ii) B(OMe)3; (e) ArBr, Pd(PPh3)4, Na2CO3, DME, reflux; (f) NBS, CCl4, AcOH, reflux; (g) 4-phenylpiperidine
or 1-phenylpiperadine.
In this paper, we describe the further optimization of this
series of 1,2,4-triazole derivatives to improve selectivity for
GlyT1 by modification of the 3- and 4-positions of triazole. In
addition, we characterized the physicochemical properties of
the optimized compound 14u, which exist as separable
atropisomers at physiological conditions. We also report the
pharmacokinetic properties and in vivo behavioral effects of the
optically active atropisomer of 14u.
thioimidates 9a,b. These compounds were coupled with acyl
hydrazides and heated under acidic conditions to produce 1,2,4-
triazole derivatives 10a,e−g. Pyridine derivatives 10b−d were
prepared by the reaction of arylboronic acid 11 with the
corresponding aryl bromides under Suzuki coupling con-
ditions.19 Piperidine 10i and piperadine 10j were prepared by
bromination of 10h, followed by substitution with the
corresponding cyclic amines.
The second general synthesis method for 1,2,4-triazole
derivatives utilized the reaction of 1,3,4-oxadiazoles 13a,b with
the corresponding anilines, as shown in Scheme 2.20 13a and
13b were heated with a variety of anilines under acidic
conditions to yield 14a,b,e,f,k−p,r,t. Aniline 14c was prepared
by the hydrogenation of the nitro derivative 14r, which was
subsequently methylated to give 14d. Benzyl ether 14f was
converted to methanol 14g using BCl3, followed by
chlorination to give intermediate 14h. Methyl ether 14i,
CHEMISTRY
■
The 1,2,4-triazole derivatives examined in this study were
prepared as illustrated in Schemes 1 and 2. Scheme 1 shows the
initial general synthesis method in which the thioimidates 9a,b
were utilized as intermediates for the synthesis of the target
1,2,4-triazole derivatives 10a,e−g. Amides 8a,b were first
reacted with Lawesson’s reagent to afford thioamides, which
were then treated with MeI and K2CO3 in acetonitrile to obtain
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a
Scheme 2. Synthesis of 1,2,4-Triazole Derivatives
a
Reagents and conditions: (a) anilines, TsOH·H2O, 160 °C; (b) Fe, 1 M HCl, EtOH, H2O, 70 °C; (c) HCHO, NaBH3CN, AcOH; (d) BCl3,
CHCl3; (e) SOCl2, CHCl3; (f) NaOMe, MeOH, reflux; (g) Me2NH, CH3CN; (h) POCl3, 100 °C; (i) KCN, 18-crown-6, CH3CN, reflux.
benzylamine 14j, and phenyl acetonitrile 14s were readily
prepared by the reaction of 14h with the corresponding
nucleophiles. Nitriles 14q,u were obtained from carboxamides
14o,t using POCl3 as a dehydrating agent.
GlyT2.16,23,24 Therefore, we first modified the biphenyl moiety
by incorporating aromatic and aliphatic amines and then
assayed for GlyT1 inhibitory activity, as presented in Table 1.
Replacement of the terminal benzene ring of 4 with pyridine-
2-yl groups resulted in a slight loss of GlyT1 inhibitory activity
(10d, IC50 = 0.86 μM). In addition, a marked reduction of
activity was observed upon replacement of the benzene ring
with pyridine-3-yl and pyridine-4-yl groups (10b, IC50 = 5.9
μM; 10c, IC50 = 7.0 μM). These results suggested that the
terminal pyridines adopted an unfavorable structure for potent
GlyT1 inhibitory activity. In contrast, the in vitro potency of
was increased with translocation of a pyridine ring adjacent to
the 1,2,4-triazole ring (10e,10f). In particular, 10e showed
equipotent GlyT1 inhibitory activity (IC50 = 0.12 μM) to the
corresponding biphenyl analogue 5. Interestingly, a slight
reduction of GlyT2 inhibitory activity was observed for 10e but
resulted in a 6.6-fold increase in the selectivity compared to 5.
Aliphatic amines (10g,i,j), however, decreased GlyT1 inhibitory
activity (IC50 = 2.6−6.6 μM), suggesting that the aromaticity of
the biphenyl moiety is essential for potency.
RESULTS AND DISCUSSION
■
The GlyT1 inhibitory activities of the synthesized 4-phenyl-4H-
1,2,4-triazoles were assayed by measuring [3H]glycine uptake
into rat C6 glioma cells according to reported protocols.21 This
assay method was validated using previously characterized
GlyT1 inhibitors, including compound 1, which showed potent
inhibitory activity (IC50 = 0.0089 μM). GlyT2 inhibitory
activities were evaluated in a similar manner to the GlyT1 assay
using rat brainstem cells. The well-characterized GlyT2
i n h i b i t o r , 4 - b e n z y l o x y - 3 , 5 - d i m e t h o x y - N - [ 1 -
(dimethylaminocyclopentyl)methyl]benzamide (Org-25543),22
displayed potent activity (IC50 = 0.022 μM) in this assay,
confirming the reliability of the assay.
We first attempted to modify the biphenyl group at the 3-
position of triazole in compounds 4 and 5 to increase the
GlyT1 inhibitory activity and selectivity over GlyT2. For
exploration of the structure−activity relationship (SAR) of the
biphenyl moiety, we postulated that a basic amine structure
may improve the potency and selectivity for GlyT1, as a wide
variety of cyclic amines were incorporated into the non-
sarcosine-based GlyT1 inhibitors with high selectivity over
In addition to a slight improvement of selectivity for 10e, we
observed a marked increase in solubility of this compound
(10e, solubility: 31 μM in pH 6.8 aqueous buffer) compared to
the corresponding biphenyl derivative (5, solubility: <1 μM in
pH 6.8 aqueous buffer).
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incorporating a variety of R-functional groups could increase
both the GlyT1 inhibitory activity and selectivity (Figure 3).
We therefore synthesized a variety of 2-methylbenzene
analogues of 6 and determined their GlyT1 inhibitory activities
(Table 2).
Table 1. In Vitro GlyT1 Inhibitory Activities of 4,5, and
10b−i
Table 2. In Vitro GlyT1 Inhibitory Activities of 6,14a−e,g,i−
s
rGlyT1 IC50
rGlyT2 IC50
a
a
b
compd
R
(μM)
(μM)
selectivity
6
0.37 0.012
0.30 0.030
0.94 0.030
0.32 0.040
3.1 0.26
0.34 0.046
0.25 0.080
2.4 0.29
29 3.8
14 5.3
0.70 0.24
1.2 0.24
0.34 0.026
38
2.3
1.3
1.1
14a
14b
14c
14d
14e
14g
14i
H
Me
NH2
c
c
NMe2
OH
ND
ND
d
1.3
3.8
12
d
CH2OH
CH2OMe
CH2NMe2
F
3.1
c
c
ND
ND
c
c
14j
ND
ND
14k
14l
0.63 0.05
2.2 0.18
2.2 0.33
5.1 0.79
3.9 0.74
0.66 0.028
1.0
c
c
Cl
ND
ND
c
c
14m
14n
14o
14p
14q
14r
14s
Br
ND
ND
c
c
CO2Me
CONH2
CF3
ND
ND
c
c
ND
ND
e
c
c
>10 (38%)
ND
ND
f
CN
0.49 0.040
0.80 0.023
2.8 0.15
>30 (43%)
>61
16
NO2
13 2.3
c
c
a
b
CH2CN
ND
ND
Values are the mean standard error for three experiments. Ratio of
a
b
c
Values are the mean standard error for three experiments. Ratio of
the IC50(μM) values rGlyT2/rGlyT1. Values are the means for two
experiments. ND: not determined.
c
d
d
the IC50(μM) values rGlyT2/rGlyT1. ND: not determined. Values
are the mean for two experiments. % inhibition at 10 μM (solubility
limit). % inhibition at 30 μM (solubility limit).
e
f
In addition to modification of the biphenyl moiety at the 3-
position of triazole, we also attempted to optimize 4-position to
obtain more potent and selective GlyT1 inhibitors. This
position was targeted based on the results of our previous SAR
study, in which a marked improvement of selectivity was
observed following the introduction of isoquinoline at the 4-
position of triazole. The nitrogen atom in isoquinoline plays an
important role in GlyT1 inhibitory activity and selectivity, as
demonstrated by the potent GlyT1 inhibitory activity and
highest selectivity (38-fold) exhibited by isoquinoline 6 (Figure
3) among all examined N-substitution patterns of quinolines
and isoquinolines.18 On the basis of the above findings for 6, we
hypothesized that a 2-methyl-3-substituted phenyl group would
mimic the structure of isoquinoline and, further, that
A wide range of substituents resulted in potent GlyT1
inhibitory activity. Both acidic (14e) and basic (14c)
substituents led to potent GlyT1 inhibitors, which were
equipotent to isoquinoline 6 (14c, IC50 = 0.32 μM; 14e, IC50
= 0.34 μM). An unsubstituted analogue (14a) also retained
potent inhibitory activity (14a, IC50 = 0.30 μM). A slight
decrease in activity was observed when methyl, fluoro, cyano,
and nitro groups were substituted as functional groups;
however, the corresponding compounds retained moderate
and acceptable GlyT1 inhibitory activities (14b, IC50 = 0.94
μM; 14k, IC50 = 0.63 μM; 14q, IC50 = 0.49 μM; and 14r, IC50
= 0.80 μM). In contrast, introduction of halogens (14l,m),
methyl ester (14n), carboxamide (14o), trifluoromethyl (14p),
and cyanomethyl (14s) groups led to weakly active analogues
(IC50 > 2 μM). Considering that potent GlyT1 inhibitory
activity was obtained from both electron-withdrawing and
-donating groups, the electrostatic properties of substituents
appear to be unrelated to the inhibitory activity.
Although the aniline 14c exhibited potent GlyT1 inhibitory
activity, a 10-fold reduction in activity was observed for the
dimethylamino derivative 14d (IC50 = 3.1 μM). Moreover,
introduction of a dimethylaminomethyl group resulted in a
marked reduction of potency (14j, IC50 = 29 μM), which we
Figure 3. Synthetic plan for 2-methylbenzene analogues of isoquino-
line.
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Table 3. In Vitro and In Vivo Profiles of 6, 14q, and 14u
a
a
b
c
compd
rGlyT1 IC50 (μM)
rGlyT2 IC50 (μM)
selectivity
38
(+)-HA966-induced hyperlocomotion in mice % inhibition, dose
6
0.37 0.040
14 5.3
82%, 3 mg/kg
8%, 1 mg/kg
44%, 3 mg/kg
91%, 3 mg/kg
14q
14u
0.49 0.040
0.16 0.030
>30
>30
>61
>187
56%, 1 mg/kg
a
b
c
Values are the mean standard error for three experiments. Ratio of the IC50(μM) values rGlyT2/rGlyT1. Compound given orally, n = 16.
speculated was due to steric hindrance in the binding region of
GlyT1. Interestingly, homologation of the hydroxyl group of
14e retained the potent activity (14g, IC50 = 0.25 μM), whereas
substitution of 14g with a methyl group resulted in 10-fold
reduction of activity (14i, IC50 = 2.4 μM). Together, these
results suggested that the introduction of hydrophilic functional
groups had little effect on GlyT1 inhibitory activity.
Compounds 14a−c,e,g,k,q,r, which displayed equipotent
GlyT1 inhibitory activity to that of isoquinoline 6, were next
assessed for GlyT2 inhibitory activity. Unsubstituted derivative
14a and compounds with amino (14c), hydroxyl (14e), fluoro
(14k), and methyl (14b) groups, exhibited potent GlyT2
inhibitory activity and poor selectivity (GlyT2/GlyT1 < 5).
However, introduction of polar substituents, including
hydroxymethyl (14g), cyano (14q), and nitro (14r) groups,
led to a favorable reduction in GlyT2 inhibitory activity and
resulted in higher GlyT1 selectivity. Notably, the nitrile 14q
exhibited a significant loss in GlyT2 inhibitory activity (14q,
IC50 > 30 μM) and displayed excellent GlyT1 selectivity (>61-
fold).
14u. Compared to the biphenyl derivative 14q, 14u exhibited
potent and selective GlyT1 inhibitory activity. In addition, a
significant improvement in solubility (14u; 98 μM) was
observed as expected, and 14u showed better liver microsome
clearances compared to those of 6 and 14q. Lower lipophilicity
of 14u may have contributed to these beneficial drug properties
of 14u because measured LogD7.4 of 14u was lower than those
of 6 and 14q. Furthermore, the in vivo potency of 14u was also
increased to 56% inhibition at a dose of 1 mg/kg, a value that
was much potent than that of 6 and 14q.
Despite the favorable drug properties of 14u, its develop-
ment as a potential candidate of GlyT1 inhibitor was
complicated by the structure of 14u. The adjacent spaces to
the bond between triazole and the 3-cyano-2-methylphenly
moiety were occupied with pyridine, ethyl, and methyl groups,
which resulted in two enantiomeric rotational isomers of 14u
(Figure 4). Atropisomer chirality can have a significant impact
Having obtained the 3-cyano-2-methylphenyl derivative 14q
with potent and selective GlyT1 inhibitory activity, we next
evaluated the aqueous solubility and in vivo behavioral effects of
14q using a (+)-HA966-induced hyperlocomotion assay in
mice (Tables 3 and 4).25,26 Unfortunately, compound 14q
showed poor solubility in a pH 6.8 aqueous solution (<1 μM)
and only inhibited hyperlocomotion by 44% at an oral dose of 3
mg/kg (po).
Figure 4. Compound 14u with hindered rotation around the NC
bond that creates a chiral axis.
on drug discovery and must be managed appropriately when
assessing the efficacy, safety, and pharmacokinetic profiles of
drug candidates.27−29 Furthermore, racemization is often a
concern in the manufacturing process, particularly when high
temperature is necessary for the preparative and purification
processes. Although only a few rotamers of 4-phenyl-1,2,4-
triazoles have been reported to date,30 numerous unsymmetri-
cally substituted biphenyl derivative have been well-docu-
mented as separable atropisomers at a wide range temper-
atures.31 It was suggested that atropisomers that equilibrate
slowly (rotational barrier >30 kcal/mol) can be treated similarly
to compounds with stereocenter-based chirarity.28,29 Here, we
therefore attempted to separate and determine the rotational
barrier of the enantiomers of 14u to decide whether 14u should
be treated as a single enantiomer or racemate in further
preclinical studies.
Table 4. Properties of 6, 14q, and 14u
a
a
solubility
(μM) pH
6.8
CLint, in vitro
human
(mL/min/kg)
CLint, in vitro
mouse
compd
LogD7.4
(mL/min/kg)
6
5
<1
98
4.1
4.1
3.0
374
126
48
>1000
>1000
467
14q
14u
a
In vitro clearance using liver microsomes.
As these properties were inferior compared with compound
6, we further modified 14q. As shown in the SAR of the
compounds in Table 1, we observed an improvement of
solubility with a slight increase of GlyT1 selectivity. As we
anticipated that these beneficial effects could be applied to 14q,
the biphenyl moiety of 14q was replaced with a 2-phenyl-
pyridine-5-yl group at the 3-positon of 1,2,4-triazole to generate
The optical resolution of each atropisomer of 14u was
performed using chiral high-pressure liquid chromatography
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(HPLC). An iterative screening of available chiral HPLC
columns, and conditions resulted in the separation of individual
atropisomers of 14u at 40 °C, as shown in Figure 5. The
On the basis of the high selectivity and activity of (R)-14u,
PK study of the promising GlyT1 inhibitor were conducted in
mice and monkeys (Table 6). (R)-14u showed moderate and
acceptable oral bioavailability in both species (mouse, 32%;
monkey, 23%). The in vitro metabolic stability of (R)-14u in
liver microsomes and unbound drug ratio in plasma were also
evaluated (Table 7). As the CLint value in humans was
markedly lower than those from the other two species, we
speculate that (R)-14u would exhibit good absorption on oral
administration to humans. The unbound (R)-14u ratio in
human and mouse plasma was similar (12% vs 11%,
respectively), whereas slightly lower binding was observed in
monkey (25% unbound).
The ability of 6, 14q, 14u, and (R)-14u to act as substrates of
P-glycoprotein (P-gp)-mediated transport (encoded by multi-
drug registrant gene [MDR1]) were evaluated in MDR1-
expressing cells (LLC-GA5-COL150 cells) and LLC-PK1
control cells (Table 8). It was found that (R)-14u had excellent
permeability profile (LLC-PK1 Papp AB = 35.4 × 10−6 cm/s)
coupled with low potential for P-gp efflux as evidenced by low
efflux ratio of 1.5 in MDR1-transfected LLC-PK1 cells.
Compounds 6, 14q, and 14u also showed excellent
permeability with low P-gp liability, indicating that this series
of GlyT1 inhibitors have favorable profiles for CNS agents.
Actually, 14u showed good total brain/plasma ratio of 0.7−1.4
in mouse.
Additional in vitro profiling revealed that (R)-14u inhibited
hGlyT1 with IC50 value of 0.59 μM using human neuro-
blastoma SK-N-MC cells. Further, (R)-14u showed low activity
toward non-GlyT1 targets, including receptors (NMDA,
AMPA, GABA, strychnine-sensitive Gly, dopamine D2, 5-
HT1A/1B/2A, muscarine, and vasopressin V1A receptors) and
transporters (glutamate, GABA, dopamine, norepinephrine,
and 5-HT transporters), with less than half of the affinity or
inhibitory activity at a concentration of 10 μM in all assays.
With regard to drug−drug interactions, compound (R)-14u
showed no significant inhibition of the major human CYP450
enzymes, such as hCYP1A2, 2D6, 2C9, 2C19, and 3A4 (IC50
>35 for all enzymes).
The favorable profiles of (R)-14u prompted us to evaluate
the effect of this compound on spatial working memory using
the Y-maze test.36,37 Spontaneous alternation behavior has been
proposed as a rudimentary animal model of working memory.38
Hypofunction of NMDA receptors has been proposed as a
causative factor underlying many schizophrenia symptoms,
including working memory, and animals treated with (+)-5-
methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine
maleate (MK-801, a noncompetitive NMDA receptor antago-
nist) exhibit impaired spontaneous alternation in the Y-maze
test.39 Thus, MK-801-induced working memory deficit in the Y-
maze test can be regarded as an animal model of cognitive
impairment in schizophrenia. In addition, the total number of
Figure 5. HPLC of atropisomers of 14u. Column, CHIRALPAK AD
4.6 mm × 250 mm (Daicel Corporation); mobile phase, n-hexane/
EtOH = 85/15; flow rate, 1.0 mL/min; and temperature, 40 °C. UV
detection at 254 nm.
collected atropisomers, (R)-14u and (S)-14u,32 appeared to be
stable against racemization at room temperature over a period
of months in the solution used in the HPLC analysis.
Having separated both atropisomers and confirming their
thermal stability against racemization at ambient temperature,
we next assessed the rate of interconversion at elevated
temperatures. To evaluate the thermal stability of interconver-
sion, a solution of (S)-14u in nitrobenzene was maintained at
79, 100, 112, 120, and 134 °C, and racemization was monitored
by HPLC. Although racemization was observed at all examined
temperatures, the process was quite slow.33 The Gibbs energy
of activation for racemization was estimated to be 31.4 kcal/
mol at 25 °C using the Eyring equation.33 On the basis of the
estimated activation energy, the half-life of racemization at 37
°C was calculated to be greater than 21 years. Solvent
dependency was not observed when 1,2-dichlorobenzene was
used as a solvent (Gibbs energy: 31.2 kcal/mol at 25 °C).
These results enabled (R)-14u and (S)-14u to be treated as
single enantiomers, similar to the case of molecules with chiral
carbon atoms.
We next evaluated the in vitro and in vivo profiles of each
isomer of 14u, as shown in Table 5. The (R)-isomer was found
to be an active enantiomer with an IC50 value of 0.064 μM for
rGlyT1, whereas the corresponding (S)-isomer displayed
markedly less activity (previously reported data).32 The
GlyT1 selectivity of (R)-14u was greater than 469-fold, which
was the highest selectivity among the compounds prepared in
this series. In addition, we observed superior in vivo efficacy to
the corresponding racemic compound, displaying 82%
inhibition of hyperlocomotion induced by (+)-HA966.
Table 5. In Vitro and In Vivo Profiles of Atropisomers of 14u
c
rGlyT1 IC50
rGlyT2 IC50
solubility (μM) pH
(+)-HA966-induced hyperlocomotion in mice % inhibition,
dose
a
a
b
compd
(μM)
(μM)
selectivity
6.8
LogD7.4
(RS)-14u
(R)-14u
(S)-14u
0.16 0.030
0.064 0.023
20 4.8
>30
>30
>30
>187
>469
>1.5
98
95
94
3.0
3.0
56%, 1 mg/kg
82%, 1 mg/kg
d
d
ND
ND
a
b
c
d
Values are the mean standard error for three experiments. Ratio of the IC50(μM) values rGlyT2/rGlyT1. Compound given orally, n = 16. ND:
not determined.
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a
Table 6. Pharmacokinetic Parameters for Compound (R)-14u
b
c
species
mouse
route
dose (mg/kg)
t1/2 (h)
AUC (ng·h/mL)
Vd (L/kg)
CL (mL/min·kg)
Cmax (ng/mL)
F (%)
iv
1
3
1
3
0.22
248
235
875
522
1.0
67
po
iv
319
141
32
23
monkey
2.4
3.4
21
po
a
b
c
Each value is the mean for n = 3 measurements. Volume of distribution. Total plasma clearance.
compounds with different mode of action when investigated in
cognitive test.40−42 For instance, previous studies have
suggested that proper cognitive function requires dopamine
levels be optimum, otherwise cognition is impaired.43 Similarly,
GlyT1 inhibition by agent as (R)-14u may also have an
optimum level. Glycine levels in the brain after administration
of (R)-14u were not measured, preventing us from drawing
definitive conclusions on the effect of (R)-14u in Y-maze test.
Detailed analysis using microdialysis is required to address the
mode of action of (R)-14u in this model.
Table 7. CLint Values and Plasma Protein Binding Ratio of
(R)-14u
a
CLint in liver microsomes
(mL/min/kg)
unbound ratio in plasma drug
concentration (% unbound)
species
human
mouse
monkey
55
787
162
12
11
25
a
Assays were performed in duplicate.
Table 8. Permeability Studies Using LLC-PK1 Cells
CONCLUSIONS
parameters
6
14q
14u
(R)-14u
■
a
LLC-PK1 Papp AB (10−6 cm/s)
25.6
0.9
27.5
1.0
29.6
1.0
35.4
0.8
Through iterative chemical modifications of our previously
identified series of GlyT1 inhibitors, we successfully improved
the GlyT1 inhibitory activity and selectivity of several of these
optimized compounds, leading to the identification of
compound 14u. Our analyses also revealed that 14u exists as
a mixture of stable atropisomers at ambient temperature,
enabling them to be treated as single enantiomers, similar to
chiral molecules with an asymmetric quaternary carbon. The
active atropisomer (R)-14u displayed good PK profiles and also
had the most potent in vivo effects in the (+)-HA966-induced
hyperlocomotion assay in mice of all tested compounds in this
series of GlyT1 inhibitors. Additionally, (R)-14u effectively
attenuated working memory deficit and reduced total arm entry
in the Y-maze test, suggesting that (R)-14u may have beneficial
effects for the treatment of cognitive impairment and positive
symptoms of schizophrenia based on the hypoglutamatergic
theory.
b
LLC-PK1 ER (BA/AB)
c
b
LLC-GA5-COL150 ER (BA/AB)
1.5
1.2
1.6
1.5
a
Apparent membrane permeability (Papp) were calculated as the
transported amount divided by initial donor concentration. The efflux
ratio (ER) was the ratio of Papp from apical to basal to that from basal
b
c
to apical. LLC-GA5-COL150 cells were established by transfection of
human MDR1 cDNA into LLC-PK1 cells.34,35
arm entries in the Y-maze is correlated with locomotor activity.
Because MK-801-induced hyperlocomotion is often used as an
indicator of positive symptoms, the increase in total arm entries
in the Y-maze induced by MK-801 could be used as an animal
model of positive symptoms.
As shown in Figure 6, (R)-14u significantly improved MK-
801-induced memory deficit at a dose range of 0.03−0.3 mg/kg
po. In addition, (R)-14u reduced the total number of arm
entries at a dose of 0.3 mg/kg po. These results suggest that
(R)-14u may have beneficial effects on both cognitive
impairment and positive symptoms in schizophrenia. We
noted that the dose−response curve of (R)-14u is an inverted
U-shape in this model, a characteristic shared by many other
EXPERIMENTAL SECTION
Chemistry: General. Melting points were determined using Buchi
■
̈
B-545 or Yanaco MP-500D micromelting apparatus and were
1
uncorrected. H NMR spectra were recorded on a JEOL JNM-LA-
Figure 6. Effects of (R)-14u on MK-801-induced working memory deficit in the mouse Y-maze test. Left panel shows the alternation rate. Right
panel shows the total number of arm entries. (R)-14u was administered orally, followed 10 min later by intraperitoneal injection of MK-801 (0.12
mg/kg). Then 20 min after MK-801 administration, each mouse was placed at the end of one arm and allowed to freely explore the apparatus for 8
min. Each value is the mean SEM (n = 12). N, normal group treated with saline; C, control group treated with MK-801. ##p < 0.01; statistically
significant compared with the normal group, as assessed by the Student’s t test. *p < 0.05, **p < 0.01; statistically significant compared with the
control group, as assessed by Dunnett’s multiple comparison test.
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300 or JEOL JNM-EX-400 spectrometer, and the chemical shifts were
expressed in δ (ppm) values using 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 (EI) or JEOL JMS-LX2000 spectrometer (FAB). The
purities of tested compounds were determined by elemental or HPLC
analyses and were found to be above 95%, except for compound 10b.
HPLC analyses were conducted on a Hitachi D-7500 system using a
TSKgel ODS-80TM column with UV-254 nm detection. Acetonitrile/
[10 mmol/L KH2PO4 aq] (70:30) was used as eluent (flow rate, 1
mL/min; column temperature, 40 °C). Elemental analyses were
performed with a Yanaco MT-5 CHN analyzer (C, H, N), Elementar
Vario EL III elemental analyzer [or consider “instrument”] (C, H, N),
and a Dionex DX-500 system (S, halogene) and were within 0.4% of
theoretical values.
Methyl N-(2-Fluorophenyl)ethanimidothioate (9a). To a
suspension of 2-fluoroacetanilide (20.3 g, 133 mmol) in toluene
(500 mL) was added Lawesson’s reagent (26.8 g, 66.3 mmol), and the
resultant mixture was stirred at 110 °C for 1.5 h. After cooling the
mixture to room temperature, the solvent was evaporated in vacuo and
the remaining residue was purified by column chromatography on
silica gel (CHCl3/MeOH = 20/1) to give crude 2-fluorothioacetanilide
(13.0g, 56%) as a brown solid. The obtained solid (13.0 g, 75.0 mmol)
was mixed with K2CO3 (10.5 g, 75.0 mmol) in CH3CN (200 mL), to
which was then added MeI (7.00 mL, 112 mmol), and the resultant
mixture was stirred at 50 °C for 0.5 h. After cooling the mixture to
room temperature, the mixture was evaporated in vacuo and the
obtained residue was partitioned between EtOAc and water. The
organic layer was washed with water, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/EtOAc = 20/1) to give the
title compound 9a (10.4 g, 76%) as a pale-yellow oil. 1H NMR
(DMSO-d6) δ 1.99 (3H, s), 2.36 (3H, s), 6.81−7.29 (4H, m). MS (EI)
m/z 183 [M]+.
mL) and then refluxed for 2.5 h under an argon atmosphere. After
cooling the mixture to room temperature, it was filtered through a
Celite pad, and the filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (CHCl3/MeOH =
98/2) to give the title compound 10c (140 mg, 28%) as a colorless
powder; mp 156−158 °C. 1H NMR (DMSO-d6) δ 2.26 (3H, s), 7.44−
7.56 (5H, m), 7.63−7.70 (1H, m), 7.74−7.79 (3H, m), 8.07−8.11
(1H, m), 8.56−8.60 (1H, m), 8.88−8.92 (1H, m). MS (FAB) m/z 331
[(M + H)+]. Anal. (C20H15N4F·0.3H2O·0.1C4H8O2) C, H, N, F.
2-{4-[4-(2-Fluorophenyl)-5-methyl-4H-1,2,4-triazol-3-yl]-
phenyl}pyridine (10d). Compound 10d was prepared from 10a and
2-bromopyridine according to the procedure described for 10c. The
title compound 10d (200 mg, 27%) was obtained as a white solid; mp
1
156−158 °C. H NMR (DMSO-d6) δ 2.27 (3H, s), 7.34−7.38 (1H,
m), 7.43−7.54 (4H, m), 7.63−7.69 (1H, m), 7.73−7.78 (1H, m),
7.86−7.91 (1H, m), 7.95−7.99 (1H, m), 8.06−8.11 (2H, m), 8.64−
8.68 (1H, m). MS (FAB) m/z 351 [(M + H)+]. Anal.
(C20H15N4F·0.1H2O) C, H, N, F.
5-[5-Ethyl-4-(2-fluorophenyl)-4H-1,2,4-triazol-3-yl]-2-phe-
nylpyridine (10e). Compound 10e was prepared from 9b and 6-
phenylnicotinic acid hydrazide according to the procedure described
for 10a. The title compound 10e (198 mg, 43%) was obtained as a
1
colorless crystal; mp 108−109 °C. H NMR (DMSO-d6) δ 1.19 (3H,
t, J = 7.6 Hz), 2.50−2.72 (2H, m), 7.43−7.58 (5H, m), 7.65−7.73
(1H, m), 7.77−7.82 (2H, m), 8.01 (1H, d, J = 8.4 Hz), 8.05−8.12
(2H, m), 8.64 (1H, d, J = 2.0 Hz). MS (FAB) m/z 345 [(M + H)+].
Anal. (C21H17N4F) C, H, N, F.
2-[5-Ethyl-4-(2-fluorophenyl)-4H-1,2,4-triazol-3-yl]-5-phe-
nylpyridine (10f). Compound 10f was prepared from 9b and 6-
phenylnicotinic acid hydrazide according to the procedure described
for 10a. The title compound 10f (16 mg, 3%) was obtained as a white
1
solid; mp 130−131 °C. H NMR (DMSO-d6) δ 1.18 (3H, t, J = 7.6
Hz), 2.48−2.66 (2H, m), 7.36−7.51 (5H, m), 7.57−7.65 (2H, m),
7.70−7.76 (2H, m), 8.17−8.26 (2H, m), 8.57−8.60 (1H, m). MS
(FAB) m/z 345 [(M + H)+]; 98% pure by HPLC.
Methyl N-(2-Fluorophenyl)propanimidothioate (9b). Com-
pound 9b was prepared from 2-fluoropropionanilide according to the
procedure described for 9a. Compound 9b (25.7g, 90% in two steps)
4-[5-Ethyl-4-(2-fluorophenyl)-4H-1,2,4-triazol-3-yl]-1-phe-
nylpiperidine (10g). Compound 10g was prepared from 9b and 1-
phenylpyperidine-4-carboxylic acid hydrazide according to the
procedure described for 10a. The title compound 10g (154 mg,
33%) was obtained as a white solid; mp 141−142 °C. 1H NMR
(DMSO-d6) δ 1.09 (3H, t, J = 7.6 Hz), 1.63−1.92 (4H, m), 2.35−2.70
(5H, m), 3.60−3.74 (2H, m), 6.73 (1H, t, J = 7.2 Hz), 6.90 (2H, d, J =
7.6 Hz), 7.18 (2H, t, J = 7.6 Hz), 7.47 (1H, t, J = 8.0 Hz), 7.59 (1H, t,
J = 9.2 Hz), 7.65−7.77 (2H, m). MS (FAB) m/z 351 [(M + H)+].
Anal. (C21H23N4F) C, H, N, F.
3-Ethyl-4-(2-fluorophenyl)-4H-1,2,4-triazole (10h). Com-
pound 10h was prepared from 9b and formic hydrazide according
to the procedure described for 10a. The title compound 10h (10.4 g,
81%) was obtained as a white solid. 1H NMR (DMSO-d6) δ 1.15 (3H,
t, J = 7.5 Hz), 2.60 (2H, q, J = 7.5 Hz), 7.40−7.70 (4H, m), 8.69 (1H,
d, J = 0.8 Hz). MS (FAB) m/z 192 [(M + H)+].
1
was obtained as a pale-yellow oil. H NMR (DMSO-d6) δ 0.96−1.25
(3H, m), 2.25−2.80 (5H, m), 6.76−7.26 (4H, m). MS (EI) m/z 197
[M]+.
3-(4-Bromophenyl)-4-(2-fluorophenyl)-5-methyl-4H-1,2,4-
triazole (10a). A mixture of 9a (500 mg, 2.73 mmol), 4-
bromobenzoic hydrazide (705 mg, 3.28 mmol), p-toluenesulfonic
acid monohydrate (52.0 mg, 0.270 mmol), and N,N-dimethylforma-
mide (DMF; 10 mL) was stirred at 120 °C for 1 h. The mixture was
cooled to room temperature and then concentrated in vacuo. The
obtained residue was purified by column chromatography on silica gel
(CHCl3/MeOH = 97/3) to give a crude solid, which was then
triturated with EtOAc/hexane to give the title compound 10a (550
1
mg, 61%) as a colorless solid. H NMR (DMSO-d6) δ 2.25 (3H, s),
7.28−7.75 (8H, m). MS (FAB) m/z 332 [(M + H)+].
1-[5-Ethyl-4-(2-fluorophenyl)-4H-[1,2,4]triazol-3-yl]-4-phe-
nylpiperidine (10i). A mixture of 12 (1.1 g, 4.1 mmol) and 4-
phenylpiperidine (5.80 g, 36 mmol) was stirred at 170 °C for 3 h,
cooled to room temperature, and then partitioned between CHCl3 and
saturated NaHCO3 aqueous solution. The organic phase was washed
with saturated NaHCO3 aqueous solution, dried over MgSO4, and
concentrated in vacuo. The obtained residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 30/1) to give crude
10i as a solid, which was then recrystallized with iPr2O to give the title
compound 10i (856 mg, 60%) as a colorless crystal; mp 108−109 °C.
1H NMR (DMSO-d6) δ 1.07 (3H, t, J = 7.6 Hz), 1.40−1.58 (2H, m),
1.60−1.74 (2H, m), 2.40 (2H, q, J = 7.6 Hz), 2.56−2.70 (1H, m),
2.78−2.94 (2H, m), 3.17−3.26 (1H, m), 3.27−3.34 (1H, m), 7.14−
7.22 (3H, m), 7.25−7.31 (2H, m), 7.44 (1H, t, J = 8.0 Hz), 7.55 (1H,
t, J = 8.8 Hz), 7.60−7.70 (2H, m). MS (FAB) m/z 351 [(M + H)+].
Anal. (C21H23N4F) C, H, N, F.
4-{4-[4-(2-Fluorophenyl)-5-methyl-4H-1,2,4-triazol-3-yl]-
phenyl}pyridine (10b). Compound 10b was prepared from 10a and
4-bromopyridine according to the procedure described for 10c. The
title compound 10b (460 mg, 62%) was obtained as a white solid; mp
1
169−172 °C. H NMR (DMSO-d6) δ 2.26 (3H, s), 7.44−7.86 (10H,
m), 8.61−8.67 (2H, m). MS (FAB) m/z 351 [(M + H)+]; 92% pure
by HPLC.
3-{4-[4-(2-Fluorophenyl)-5-methyl-4H-1,2,4-triazol-3-yl]-
phenyl}pyridine (10c). To a solution of 10a (500 mg, 1.51 mmol) in
THF (15 mL) was added n-BuLi (1.57 M in hexane, 1.2 mL, 1.8
mmol) dropwise at −78 °C. After stirring for 20 min, trimethyl borate
(0.50 mL, 4.5 mmol) was added to the reaction mixture at −78 °C,
and the mixture was stirred for 3 h at room temperature, followed by
the addition of 2 M HCl to a final pH of 2. The mixture was extracted
with CHCl3, and the combined organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo to give crude 11 as a
pale-yellow oil. The obtained crude 11, 3-bromopyridine (0.15 mL, 1.5
mmol), Pd(PPh3)4 (87 mg, 57 μmol), and 2 M Na2CO3 aqueous
solution (1.5 mL, 3.0 mmol) were mixed in 1,2-dimethoxyethane (5
1-[5-Ethyl-4-(2-fluorophenyl)-4H-[1,2,4]triazol-3-yl]-4-phe-
nylpiperazine (10j). Compound 10j was prepared from 12 and 1-
phenylpiperadine according to the procedure described for 10i. The
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1
title compound 10j (50 mg, 12%) was obtained as a pale-brown
crystal; mp 108−112 °C. 1H NMR (DMSO-d6) δ 1.07 (3H, t, J = 7.5
Hz), 2.40 (2H, q, J = 7.5 Hz), 2.99−3.14 (8H, m), 6.77 (1H, t, J = 7.5
Hz), 6.89 (2H, d, J = 7.5 Hz), 7.18 (2H, t, J = 7.5 Hz), 7.44 (1H, t, J =
7.5 Hz), 7.55 (1H, t, J = 7.5 Hz), 7.60−7.71 (2H, m). MS (FAB) m/z
352 [(M + H)+]. Anal. (C20H22N5F) C, H, N, F.
298−300 °C. H NMR (DMSO-d6) δ 1.16 (3H, t, J = 7.6 Hz), 1.63
(3H, s), 2.44−2.50 (2H, m), 6.96 (1H, d, J = 7.8 Hz), 7.02 (1H, d, J =
7.8 Hz), 7.25 (1H, t, J = 7.8 Hz), 7.34−7.40 (1H, m), 7.42−7.49 (4H,
m), 7.64−7.69 (4H, m), 10.03 (1H, s). MS (FAB) m/z 356 [(M +
H)+]. Anal. (C23H21N3O·0.3H2O) C, H, N.
4-{3-[(Benzyloxy)methyl]-2-methylphenyl}-3-(biphenyl-4-
yl)-5-ethyl-4H-1,2,4-triazole (14f). Compound 14f was prepared
from 13a and 3-[(benzyloxy)methyl]-2-methylaniline according to the
procedure described for 14a. The title compound 14f (799 mg, 90%)
3-Bromo-5-ethyl-4-(2-fluorophenyl)-4H-[1,2,4]triazole (12).
To a solution of 10h (1.29 g, 6.75 mmol) in CCl4 (30 mL)−AcOH
(30 mL) was added NBS (1.8 g, 10.0 mmol), and the resulting mixture
was refluxed for 1.5 h. After cooling to room temperature, the mixture
was diluted with CH2Cl2, washed with 10% K2CO3 aq, dried over
MgSO4, and concentrated in vacuo. The residue was recrystallized with
EtOAc−hexane to give the title compound 12 (955 mg, 52%) as a
1
was obtained as a solid. H NMR (DMSO-d6) δ 1.15 (3H, t, J l 7.5
Hz), 1.77 (3H, s), 2.40−2.52 (2H, m), 4.40−4.65 (4H, m), 7.26−7.65
(17H, m). MS (FAB) m/z 460 [(M + H)+].
{3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-
methylphenyl}methanol (14g). To a solution of 14f (4.4 g, 9.6
mmol) in CHCl3 (130 mL) was added 1 M BCl3 solution in heptane
(14 mL) dropwise at 0 °C. After it was stirred at room temperatrure
for 3 h, an additional batch of BCl3 (1 M solution in heptane, 24 mL)
was added dropwise at 0 °C, and the resultant mixture was stirred
overnight at room temperature. The reaction mixture was then poured
into a 1 M aqueous NaOH solution (150 mL) at 0 °C and stirred for 1
h. The organic layer was separated, washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH = 96/4) to give
1
yellow crystal. H NMR (DMSO-d6) δ 1.12 (3H, t, J = 7.5 Hz), 2.54
(2H, q, J = 7.5 Hz), 7.48 (1H, t, J = 7.1 Hz), 7.56−7.65 (1H, m),
7.67−7.77 (2H, m). MS (FAB) m/z 270 [(M + H)+].
3-Biphenyl-4-yl-5-ethyl-4-(2-methylphenyl)-4H-1,2,4-tria-
zole (14a). To a mixture of 2-(biphenyl-4-yl)-5-ethyl-1,3,4-oxadiazole
13a (230 mg, 0.92 mmol) and 2-methylaniline (250 mg, 2.33 mmol)
was added p-toluenesulfonic acid monohydrate (30 mg, 0.16 mmol),
and the resultant mixture was stirred at 160 °C for 3 h. After cooling
the mixture to room temperature, it was purified by column
chromatography on silica gel (AcOEt) to give a crude solid, which
was then triturated with EtOAc/hexane to give the title compound 14a
(198 mg, 63%) as a white solid; mp 195−197 °C. 1H NMR (CDCl3) δ
1.27 (3H, t, J = 7.6 Hz), 1.93 (3H, s), 2.58 (2H, q, J = 7.6 Hz), 7.26−
7.56 (13H, m). MS (FAB) m/z 340 [(M + H)+]. Anal.
(C23H21N3·0.1H2O) C, H, N.
1
the title compound 14g (2.32g, 66%) as a solid; mp 199−201 °C. H
NMR (DMSO-d6) δ 1.15 (3H, t, J = 7.3 Hz), 1.76 (3H, s), 2.45 (2H,
q, J = 7.3 Hz), 4.48−4.58 (2H, m), 5.30 (1H, t, J = 5.4 Hz), 7.34−7.47
(7H, m), 7.60−7.68 (5H, m). MS (FAB) m/z 370 [(M + H)+]. Anal.
(C24H23N3O·0.3H2O) C, H, N.
3-(Biphenyl-4-yl)-4-[3-(chloromethyl)-2-methylphenyl]-5-
ethyl-4H-1,2,4-triazole (14h). To a solution of 14g (1.0 g, 2.71
mmol) in CHCl3 (20 mL) was added SOCl2 (0.9 mL), and the
resultant mixture was stirred for 30 min. The mixture was then
evaporated in vacuo, and the obtained residue was diluted with CHCl3,
washed with saturated aqueous NaHCO3 solution, dried over MgSO4,
and concentrated in vacuo to give the title compound 14h (1.04 g,
99%) as a solid. 1H NMR (DMSO-d6) δ 1.15 (3H, t, J = 7.5 Hz), 1.89
(3H, s), 2.40−2.50 (2H, m), 4.86 (2H, s), 7.35−7.70 (12H, m); MS
(FAB) m/z 388 [(M + H)+].
3-(Biphenyl-4-yl)-5-ethyl-4-[3-(methoxymethyl)-2-methyl-
phenyl]-4H-1,2,4-triazole (14i). To a mixture of 14h (280 mg, 0.72
mmol), MeOH (15 mL), and THF (5 mL) was added NaOMe (390
mg, 7.2 mmol), and the reaction mixture was refluxed for 2 h. After it
was cooled to room temparature, a 1 M aqueous solution of HCl (6.5
mL) was added to mixture, which was then concentrated in vacuo. The
obtained residue was diluted with CHCl3, washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was recrystallized
with hexane/EtOAc/MeCN to give the title compound 14i (120 mg,
43%) as a colorless powder; mp 152−154 °C. 1H NMR (DMSO-d6) δ
1.17 (3H, t, J = 7.6 Hz), 1.79 (3H, s), 2.45−2.51 (2H, m), 3.28 (3H,
s), 4.43−4.51 (2H, m), 7.35−7.40 (1H, m), 7.42−7.48 (5H, m), 7.51−
7.59 (2H, m), 7.64−7.68 (4H, m). MS (FAB) m/z 384 [(M + H)+].
Anal. (C25H25N3O·0.1H2O) C, H, N.
3-Biphenyl-4-yl-4-(2,3-dimethylphenyl)-5-ethyl-4H-[1,2,4]-
triazole (14b). Compound 14b was prepared from 13a and 2,3-
dimethylaniline according to the procedure described for 14a. The title
compound 14b (71 mg, 14%) was obtained as a white solid; mp 180−
181 °C. 1H NMR (DMSO-d6) δ 1.15 (3H, t, J = 7.6 Hz), 1.75 (3H, s),
2.30 (3H, s), 2.40−2.47 (2H, m), 7.31−7.47 (8H, m), 7.62−7.68 (4H,
m). MS (FAB) m/z 354 [(M + H)+]. Anal. (C24H23N3) C, H, N.
3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-methyla-
niline (14c). To a mixture of 14r (1.6 g, 4.1 mmol), 1 M aqueous HCl
(0.6 mL), EtOH (20 mL), and water (20 mL) was added Fe (powder,
2.9 g, 53 mmol), and the reaction mixture was stirred at 70 °C for 2 h.
After the mixture was cooled to room temperature, it was diluted with
CHCl3 and filtered through a Celite pad. The obtained filtrate was
mixed with 1 M aqueous NaOH (3.0 mL), and the organic layer was
then separated, washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 99/1) to give a
crude solid, which was then recrystallized with EtOAc to give the title
compound 14c (330 mg, 23%) as a colorless powder; mp 200−202
1
°C. H NMR (DMSO-d6) δ 1.15 (3H, t, J = 7.6 Hz), 1.55 (3H, s),
2.43−2.49 (2H, m), 5.28−5.33 (2H, m), 6.62 (1H, d, J = 7.3 Hz), 6.81
(1H, d, J = 8.3 Hz), 7.11 (1H, t, J = 8.1 Hz), 7.34−7.39 (1H, m),
7.42−7.52 (4H, m), 7.63−7.69 (4H, m). MS (FAB) m/z 355 [(M +
H)+]. Anal. (C23H22N4) C, H, N.
1-{3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-meth-
ylphenyl}-N,N-dimethylmethanamine dihydrochloride (14j).
To a solution of 14h (287 mg, 0.74 mmol) in MeCN (12 mL) was
added dimethylamine aqueous solution [50%(w/w), 330 mg, 3.7
mmol], and the resultant mixture was stirred for 1 h. After it was
concentrated in vacuo, the residue was diluted with CHCl3, washed
with saturated NaHCO3 aqueous solution, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 90/10) to give a
solid, which was then dissolved in EtOAc (2 mL), followed by addition
of 4 M HCl solution in EtOAc (0.45 mL). The resulting precipitate
was filtered off, and it was recrystallized from EtOH/hexane to give the
title compound 14j (209 mg, 56%) as a colorless powder; mp 134−
137 °C. 1H NMR (DMSO-d6) δ 1.22 (3H, t, J = 7.3 Hz), 2.06 (3H, s),
2.54−2.69 (5H, m), 2.73 (3H, d, J = 4.4 Hz), 4.32−4.48 (2H, m),
7.36−7.50 (5H, m), 7.57 (1H, t, J = 7.8 Hz), 7.63−7.74 (5H, m), 7.92
(1H, d, J = 7.8 Hz). MS (FAB) m/z 397 [(M + H)+]. Anal.
(C26H28N4·2HCl·2H2O) C, H, N, Cl.
3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-N, N, 2-tri-
methylaniline (14d). To a solution of 14c (248 mg, 0.70 mmol) and
folmaldehyde (37% w/w in water, 0.13 mL) in AcOH (5 mL) was
added NaBH3CN (53 mg, 0.84 mmol), and the reaction mixture was
stirred for 3 h before diluting with CHCl3. The organic layer then was
separated, washed with saturated NaHCO3 aqueous solution, dried
over MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH = 99/1) to give
a crude solid, which was recrystallized with iPr2O to give the title
compound 14d (71 mg, 27%) as a colorless powder; mp 134−135 °C.
1H NMR (DMSO-d6) δ 1.16 (3H, t, J = 7.6 Hz), 1.73 (3H, s), 2.45−
2.52 (2H, m), 2.61 (6H, s), 7.17−7.20 (1H, m), 7.26 (1H, d, J = 7.9
Hz), 7.34−7.48 (6H, m), 7.62−7.68 (4H, m). MS (FAB) m/z 383
[(M + H)+]. Anal. (C25H26N4) C, H, N.
3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-methyl-
phenol (14e). Compound 14e was prepared from 13a and 3-amino-
2-methylphenol according to the procedure described for 14a. The
title compound 14e (36 mg, 7%) was obtained as a white solid; mp
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3-Biphenyl-4-yl-5-ethyl-4-(3-fluoro-2-methylphenyl)-4H-
[1,2,4]triazole (14k). Compound 14k was prepared from 13a and 3-
fluoro-2-methylaniline according to the procedure described for 14a.
The title compound 14k (195 mg, 37%) was obtained as a solid; mp
mmol) and 18-crown-6 (401 mg, 1.52 mmol), and the resultant
mixture was refluxed for 2 h. After cooling the mixture to room
temperature, it was concentrated in vacuo and then diluted with
CHCl3. The resultant organic solution was washed with a saturated
NaHCO3 aqueous solution, dried over MgSO4, and concentrated in
vacuo. The obtained residue was purified by column chromatography
on silica gel (CHCl3/MeOH = 98/2) to give a crude solid, which was
recrystallized from iPr2O/AcOEt to give the title compound 14s (39
mg, 14%) as a colorless solid; mp 150−152 °C. 1H NMR (DMSO-d6)
δ 1.15 (3H, t, J = 7.6 Hz), 1.81 (3H, s), 2.41−2.48 (2H, m), 4.13 (2H,
s), 7.34−7.53 (6H, m), 7.56−7.67 (6H, m). MS (FAB) m/z 379 [(M
+ H)+]. Anal. (C25H22N4) C, H, N.
1
161−162 °C. H NMR (DMSO-d6) δ 1.17 (3H, t, J = 7.6 Hz), 1.77
(3H, d, J = 2.0 Hz), 2.46−2.53 (2H, m), 7.34−7.40 (1H, m), 7.42−
7.55 (7H, m), 7.64−7.70 (4H, m). MS (FAB) m/z 358 [(M + H)+].
Anal. (C23H20N3F·0.1H2O) C, H, N, F.
3-Biphenyl-4-yl-5-ethyl-4-(3-chloro-2-methylphenyl)-4H-
[1,2,4]triazole (14l). Compound 14l was prepared from 13a and 3-
chloro-2-methylaniline according to the procedure described for 14a.
The title compound 14l (182 mg, 31%) was obtained as a solid; mp
1
168−169 °C. H NMR (DMSO-d6) δ 1.17 (3H, t, J = 7.4 Hz), 1.88
3-[3-Ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-
methylbenzamide (14t). Compound 14t was prepared from 13b
and 3-amino-2-methylbenzamide according to the procedure described
for 14a. The title compound 14t (550 mg, 89%) was obtained as a
(3H, s), 2.44−2.51 (2H, m), 7.35−7.40 (1H, m), 7.41−7.52 (5H, m),
7.60−7.64 (1H, m), 7.65−7.73 (5H, m). MS (FAB) m/z 374 [(M +
H)+]. Anal. (C23H20N3Cl) C, H, N, Cl.
1
3-Biphenyl-4-yl-4-(3-bromo-2-methylphenyl)-5-ethyl-4H-
1,2,4-triazole (14m). Compound 14m was prepared from 13a and 3-
bromo-2-methylaniline according to the procedure described for 14a.
The title compound 14m (1.40 g, 28%) was obtained as a solid; mp
solid. H NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.2 Hz), 1.90 (3H, s),
2.40−2.55 (2H, m), 7.42−7.53 (4H, m), 7.59−6.61 (2H, m), 7.67
(1H, d, J = 7.6 Hz), 7.82 (1H, dd, J = 2.0, 8.0 Hz), 7.96 (1H, brs), 7.99
(1H, d, J = 8.8 Hz), 8.05−8.09 (2H, m), 8.60 (1H, d, J = 2.4 Hz). MS
(FAB) m/z 384 [(M + H)+].
1
173−174 °C. H NMR (DMSO-d6) δ 1.17 (3H, t, J = 7.8 Hz), 1.90
(3H, s), 2.47 (2H, q, J = 7.4 Hz), 7.35−7.47 (6H, m), 7.63−7.70 (5H,
m), 8.87 (1H, d, J = 8.3 Hz). MS (FAB) m/z 418 [(M + H)+]. Anal.
(C23H20N3Br) C, H, N, Br.
3-[3-Ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-
methylbenzonitrile (14u). Compound 14u was prepared from 14t
according to the procedure described for 14q. The title compound
1
Methyl 3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-
methylbenzoate (14n). Compound 14n was prepared from 13a
and methyl 3-amino-2-methylbenzoate according to the procedure
described for 14a. The title compound 14n (46 mg, 9%) was obtained
14u (242 mg, 46%) was obtained as a solid; mp 181−182 °C. H
NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.5 Hz), 2.08 (3H, s), 2.41−2.59
(2H, m), 7.43−7.52 (3H, m), 7.63 (1H, dd, J = 7.8, 8.3 Hz), 7.76 (1H,
dd, J = 2.0, 8.3 Hz), 7.98−8.02 (1H, m), 7.99 (1H, d, J = 8.3 Hz),
8.05−8.12 (3H, m), 8.64 (1H, d, J = 2.0 Hz). MS (FAB) m/z 366 [(M
+ H)+]. Anal. (C23H19N5) C, H, N.
1
as a solid; mp 194−196 °C. H NMR (DMSO-d6) δ 1.21 (3H, t, J =
7.5 Hz), 2.03 (3H, s), 2.50 (2H, q, J = 7.5 Hz), 3.88 (3H, s), 7.39−
7.52 (5H, m), 7.63 (1H, t, J = 7.9 Hz), 7.69−7.73 (4H, m), 7.89 (1H,
d, J = 7.8 Hz), 8.05 (1H, d, J = 7.8 Hz). MS (FAB) m/z 398 [(M +
H)+]. Anal. (C25H23N3O2) C, H, N.
(R)-3-[3-Ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-
2-methylbenzonitrile [(R)-14u] and (S)-3-[3-Ethyl-5-(6-phenyl-
pyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-methylbenzonitrile [(S)-
14u]. 14u was separated on a preparative CHIRALPAK AD column
(20 mm × 250 mm, flow rate: 5 mL/min wavelength: 254 nm) using
n-hexane/EtOH (1:1) as an eluent to give (R)-14u as the first eluting
product (retention time, 30 min) and (S)-14u as the second eluting
product (retention time, 37 min).
3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-methyl-
benzamide (14o). Compound 14o was prepared from 13a and 3-
amino-2-methylbenzamide according to the procedure described for
14a. The title compound 14o (597 mg, 39%) was obtained as a solid;
1
mp 240−242 °C. H NMR (DMSO-d6) δ 1.18 (3H, t, J = 7.3 Hz),
25
1.88 (3H, s), 2.40−2.50 (2H, m), 7.34−7.52 (6H, m), 7.56−7.68 (7H,
m), 7.96 (1H, br). MS (FAB) m/z 383 [(M + H)+]. Anal.
(C24H22N4O) C, H, N.
(R)-14u: white solid, 100% ee, [α]D −211° (c 1.0, CHCl3), mp
1
162−163 °C. H NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.4 Hz), 2.08
(3H, s), 2.41−2.59 (2H, m), 7.43−7.52 (3H, m), 7.66 (1H, t, J = 7.8
Hz), 7.76 (1H, dd, J = 1.5, 8.3 Hz), 7.97−8.02 (2H, m), 8.05−8.12
(3H, m), 8.64 (1H, d, J = 1.9 Hz). MS (FAB) m/z 366 [(M + H)+].
Anal. (C23H19N5) C, H, N.
3-Biphenyl-4-yl-5-ethyl-4-[2-methyl-3-(trifluoromethyl)-
phenyl]-4H-1,2,4-triazole (14p). Compound 14p was prepared
from 13a and 3-amino-2-methylbenzotrifluoride according to the
procedure described for 14a. The title compound 14p (177 mg, 30%)
was obtained as a solid; mp 174−175 °C. 1H NMR (DMSO-d6) δ 1.78
(3H, t, J = 7.6 Hz), 1.92 (3H, s), 2.44−2.51 (2H, m), 7.35−7.48 (5H,
m), 7.65−7.73 (5H, m), 7.98 (2H, dd, J = 8.3, 3.0 Hz). MS (FAB) m/z
408 [(M + H)+]. Anal. (C24H20N3F) C, H, N, F.
(S)-14u: white solid, 99.7% ee, [α]D25 +213° (c 1.0, CHCl3), 100%
ee, mp 159−160 °C. 1H NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.4 Hz),
2.08 (3H, s), 2.41−2.59 (2H, m), 7.43−7.52 (3H, m), 7.66 (1H, J =
7.8 Hz), 7.76 (1H, dd, J = 2.4, 8.3 Hz), 7.98−8.02 (2H, m), 8.05−8.12
(3H, m), 8.64 (1H, d, J = 1.9 Hz). MS (FAB) m/z 366 [(M + H)+].
Anal. (C23H19N5) C, H, N.
3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-methyl-
benzonitrile (14q). A mixture of 14o and POCl3 was stirred at 100
°C for 1 h. The mixture was evaporated in vacuo, and the residue was
diluted with CHCl3. The solution was washed with a saturated
NaHCO3 aqueous solution, dried over MgSO4, and concentrated in
vacuo. The residue was recrystallized with EtOAc to give the title
compound 14q (532 mg, 44%) as a colorless powder; mp 199−200
Kinetic Measurements. The kinetics of rotamer interconversion
of (S)-14u to its racemate in nitrobenzene were followed at 78.5, 100,
112, 120, and 134 °C by HPLC using the chromatographic conditions
described in Figure 5. Fitting first-order kinetics gave rate constants for
racemization at each temperature. The activation energy of
racemizaton was calcurated using Eyling plots of the rate constant at
each temperature.33
1
°C. H NMR (DMSO-d6) δ 1.18 (3H, t, J = 7.8 Hz), 2.05 (3H, s),
2.45−2.52 (2H, m), 7.35−7.48 (5H, m), 7.64−7.70 (5H, m), 7.98
(1H, d, J = 7.8 Hz), 8.07 (1H, d, J = 7.9 Hz). MS (FAB) m/z 365 [(M
+ H)+]. Anal. (C24H20N4) C, H, N.
[3H]Glycine Uptake Assay (GlyT1). The inhibitory effect of the
compounds on rat GlyT1 was examined as previously described21 with
some modifications. Rat glioma C6 cells, which endogenously express
GlyT1, were plated at a density of 2 × 104 cells per well in a white 96-
well CulturPlate (PerkinElmer Inc.) and incubated for two days in a
CO2 incubator (37 °C, 5% CO2). After washing with 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
[NaCl (150 mmol/L), KCl (5 mmol/L), MgCl2 (1 mmol/L), CaCl2
(1 mmol/L), HEPES (10 mmol/L), D-glucose (10 mmol/L), and L-
alanine (5 mmol/L), pH 7.4], the buffer was replaced with HEPES
buffer containing the test compounds (for concentration ranges, see
Supporting Information), and plates were incubated at 30 min at 37
°C. After incubation, [3H]glycine (final concentration of [3H]glycine:
3-Biphenyl-4-yl-5-ethyl-4-(2-methyl-3-nitrophenyl)-4H-
1,2,4-triazole (14r). Compound 14r was prepared from 13a and 2-
methyl-3-nitroaniline according to the procedure described for 14a.
The title compound 14r (799 mg, 33%) was obtained as a solid; mp
1
174−176 °C. H NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.6 Hz), 1.97
(3H, s), 2.44−2.54 (2H, m), 7.35−7.40 (1H, m), 7.42−7.48 (4H, m),
7.65−7.75 (5H, m), 8.02 (1H, d, J = 6.9 Hz), 8.18−8.22 (1H, m). MS
(FAB) m/z 385 [(M + H)+]. Anal. (C23H20N4O2) C, H, N.
{3-[3-(Biphenyl-4-yl)-5-ethyl-4H-1,2,4-triazol-4-yl]-2-
methylphenyl}acetonitrile (14s). To a solution of 14h (287 mg,
0.74 mmol) in CH3CN (10 mL) were added KCN (243 mg, 3.7
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0.14 μM) was added, and the plate was further incubated for 10 min at
37 °C. After incubation, the wells were aspirated and washed three
times with ice-cold phosphate-buffered saline (PBS). After solubilizing
the cells with 17 μL of 0.1 mol/L NaOH, 100 μL of scintillant
(MicroScint PS; PerkinElmer Inc.) was added to the wells, and the
plate was counted in a Top Count microplate scintillation counter
(Hewlett-Packard Company; Palo Alto, CA, USA). Nonspecific uptake
was detected using 3 mmol/L sarcosine. We evaluated the effect of
sarcosine on [3H]glycine uptake in parallel as a reference GlyT1
inhibitor. The drug concentrations required to inhibit 50% of the
specific [3H]glycine uptake (IC50) were obtained using nonlinear
regression analysis and SAS software (SAS Institute Inc.; Cary, NC,
bottom and top, respectively. Mice were acclimated in the
experimental room for at least 1 h prior to testing. Test compounds
were orally administered, and animals were returned to their home
cages. Ten minutes after administration of (R)-14u, 0.12 mg/kg of
MK-801 was administered intraperitoneally and animals were again
returned to their home cages. Twenty minutes after administration of
MK-801, each mouse was placed at the end of one arm and allowed to
freely explore the apparatus for 8 min. The sequence and number of all
arm entries were recorded for each animal throughout the test period.
The sequence triads, in which all three arms were represented
(including ABC, ACB, BAC, BCA, CAB, and CBA), were calculated as
successful alternations to evaluate normal perception and working
memory of the last arm entered. Alternation percentage was defined as
entries into all three arms on consecutive occasions using the following
formula:
USA), with the mean
experiments.
SEM obtained from three independent
[3H]Glycine Uptake Assay (GlyT2). The inhibitory effect of the
compounds on rat GlyT2 was examined using [3H]glycine uptake into
primary cultured cells, which endogenously express GlyT2. Pregnant
rats (Wistar, 17 days gestation, Japan SLC) were euthanized under
ether anesthesia by exsanguination by severing the carotid artery, and
the infants were then obtained from the pregnant rats and their
brainstems isolated. After digestion with papain, brainstem cells were
dispersed into culture medium and plated at a density of 5 × 104 cells
per well in white 96-well plates coated with poly-L-lysine and then
incubated for 14−21 days in a CO2 incubator (37 °C, 5% CO2).
On the day of the assay, the cells were washed once 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 10 mmol/L HEPES, pH 7.4), and each well was then
filled with 100 μL of assay buffer. After preincubation at 37 °C for 10
min, the assay buffer was then replaced with the reaction mixture
containing the test compounds (for concentration ranges, see
Supporting Information) and [3H]glycine (final concentration of
[3H]glycine: 0.14 μM), and the plate was further incubated for 10 min
at 37 °C, The reaction was then stopped by washing the cells four
times with ice-cold buffer. After solubilizing the cells with 17 μL of 0.1
M NaOH, 100 μL of scintillant was added and the plate was counted
using a TopCount microplate scintillation counter (Hewlett-Packard
Company). Nonspecific [3H]glycine uptake was determined with 3
mmol/L unlabeled glycine. The drug concentrations required to
inhibit 50% of the specific [3H]glycine uptake (IC50) were determined
using nonlinear regression analysis and SAS software (SAS Institute
Inc.). We evaluated glycine in parallel as the reference, and the mean
SEM from three independent experiments are reported as the final
inhibition values.
(+)-HA966-Induced Enhancement of Locomotor Activity in
Mice. Locomotor activity was assessed using a previously reported
method with minor modifications.44 Animal, male ICR mice (Japan
SLC; age 5−7 weeks); drug, reserpine (Apoplon injection, 1 mg/mL;
Daiichi Pharmaceuticals Co., Ltd., Tokyo, Japan), (+)-HA966, α-
methyl-p-tyrosine methyl ester (Sigma, Inc.); equipment, Supermex
(Muromachi Machine). Method. (1) Pharmaceutical drug-treated
groups (n = 16) were defined as follows: [artificial cerebrospinal fluid
(ACSF) + vehicle] group, [(+)-HA966 80 μg/mouse (icv) + vehicle]
group, [(+)-HA966 80 μg/mouse (icv) + test compound] group. (2)
Nineteen hours before (+)-HA966 administration, reserpine (10 mg/
kg) was administered intraperitoneally. (3) Thirty minutes before
(+)-HA966 administration, α-methyl-p-tyrosine methyl ester (250
mg/kg) was administered intraperitoneally. (4) Twenty minutes
before (+)-HA966 administration, a test compound was administered
orally. (5) (+)-HA966 was acutely administered bilaterally into the
lateral ventricule using a two-step needle. Immediately after
administration, each animal was placed in the measuring cage of an
activity measurement apparatus. Activity per 90 min was measured
immediately upon placing the animal in the cage. The integral value of
locomotor activity per 90 min was selected as the data.
alternation(%) = 100 × (number of alternations
/(number of total arm entries − 2))
Data were eliminated in cases where the number of total arm entries
was less than 10 or where the mouse escaped from the Y-maze field.
ASSOCIATED CONTENT
* Supporting Information
Supplemental data for in vitro rGlyT1/2 assay, synthesis of the
intermediates, plasma time curve, experimental method for PK
studies, combustion analyses or HPLC data for the tested
compound, and kinetic data for racemization. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-29-863-6678. Fax: +81-29-852-5387. E-mail:
takashi.sugane@astellas.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We sincerely thank Takuma Morita, Atsuyuki Kohara, Masaki
Aota, Hitoshi Doihara, Kyouko Saita, Katsuya Harada, and
Junko Yarimizu for performing pharmacological evaluations.
We also thank the staff of the Analysis and Pharmacokinetics
Research Laboratories for performing spectral measurements.
ABBREVIATIONS USED
■
ACSF, artificial cerebrospinal fluid; GlyT, glycine transporter;
NMDA, N-methyl-D-aspartate; SAR, structure−activity rela-
tionship
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