Design and synthesis of 1-(1-benzothiophen-7-yl)-1H-pyrazole, a novel series of G protein-coupled receptor 52 (GPR52) agonists

Article abstract of DOI:10.1016/j.bmc.2018.02.005

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-D-aspartate receptors via intracellular cAMP accumulation. Therefore, G

Full text of DOI:10.1016/j.bmc.2018.02.005

Accepted Manuscript
Design and Synthesis of 1-(1-Benzothiophen-7-yl)-1H-Pyrazole, a Novel Series
of G Protein-coupled Receptor 52 (GPR52) Agonists
Takashi Nakahata, Kazuyuki Tokumaru, Yoshiteru Ito, Naoki Ishii, Masaki
Setoh, Yuji Shimizu, Toshiya Harasawa, Kazunobu Aoyama, Teruki Hamada,
Masakuni Kori, Kazuyoshi Aso
PII:
S0968-0896(17)32105-3
https://doi.org/10.1016/j.bmc.2018.02.005
BMC 14191
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 October 2017
24 January 2018
5 February 2018
Please cite this article as: Nakahata, T., Tokumaru, K., Ito, Y., Ishii, N., Setoh, M., Shimizu, Y., Harasawa, T.,
Aoyama, K., Hamada, T., Kori, M., Aso, K., Design and Synthesis of 1-(1-Benzothiophen-7-yl)-1H-Pyrazole, a
Novel Series of G Protein-coupled Receptor 52 (GPR52) Agonists, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.02.005
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Design and Synthesis of 1-(1-Benzothiophen-7-yl)-1H-Pyrazole, a Novel
Series of G Protein-coupled Receptor 52 (GPR52) Agonists
a
a
a
a
Takashi Nakahata a,c*, Kazuyuki Tokumaru , Yoshiteru Ito , Naoki Ishii , Masaki Setoh , Yuji
a
a
b
Shimizu , Toshiya Harasawa , Kazunobu Aoyama a,d, Teruki Hamada a,d, Masakuni Kori and
Kazuyoshi Aso a,d*
aPharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-higashi
2-chome, Fujisawa, Kanagawa 251-8555, Japan
bPharmaceutical Sciences, Takeda Pharmaceutical Company Ltd., 17-85, Jusohonmachi-2-chome,
Yodogawa-ku, Osaka 532-8686, Japan
*Corresponding authors. Tel.: +81 466 32 1126; fax: +81 466 29 4473.
E-mail: takashi.nakahata1@takeda.com (T. Nakahata).
kazuyoshi.aso@axcelead-ddp.com (K. Aso).
cCurrent address: Cardurion Pharmaceuticals K.K., 26-1, Muraoka-Higashi 2-Chome, Fujisawa,
Kanagawa 251-8555, Japan
dCurrent address: Axcelead Drug Discovery Partners, Inc., 26-1, Muraoka-Higashi 2-Chome,
Fujisawa, Kanagawa 251-0012, Japan

ABSTRACT
G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled
receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-D-
aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to
alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as
GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been
reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor
aqueous solubility still remained as issues for candidate selection. To resolve these issues,
replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as
pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated
logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives,
introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52
agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and
further introduction of hydroxy group to lower logD led to significant improvement of solubility
while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole
derivative 17 (GPR52 EC50 = 21 nM, Emax = 103%, logD = 2.21, Solubility at pH 6.8 = 21 μg/mL)
with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore,
this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in
mice.

Key words: GPCR; GPR52 agonist; Benzothiophene; 1H-Pyrazole; Solubility; Methamphetamine
(MAP)-induced hyperlocomotion.
Abbreviations: GPR52, G protein-coupled receptor 52; SAR, structure–activity relationship; cAMP,
3',5'-cyclic adenosine monophosphate; EC50, half maximal effective concentration; MAP,
methamphetamine; NMDA, N-methyl-D-aspartate; PK, pharmacokinetic; CHO, Chinese hamster
ovary; SEM, standard error of the mean; AUC, area under the curve.
1. Introduction
G protein-coupled receptor 52 (GPR52) is an orphan Gs-coupled GPCR,1 and a surrogate ligand
of GPR52 has been reported by our research group.2,3 Localization of GPR52 mRNA in rat brain,
which was studied by in situ hybridization test, indicated that GPR52 was highly expressed in the
mesolimbic system and co-localized with the dopamine D2 receptors in the nucleus accumbens.
GPR52 was also partially co-localized with the dopamine D1 receptors in the medial prefrontal
cortex.2 Moreover, GPR52 transgenic mice showed anti-psychiatric-like behaviors, whereas GPR52
knockout mice displayed psychosis-related behaviors.2 These findings demonstrate great potential
that activation of GPR52 would ameliorate the positive symptoms of schizophrenia4,5 by
antagonizing D2 receptor signaling as well as enhancing cognitive function by activation of D1/N-
methyl-D-aspartate (NMDA) function via intracellular cAMP accumulation. Thus, GPR52 agonists

are expected to become a novel class of antipsychotics to satisfy the unmet medical needs of the
current typical and atypical agents.
The benzothiophene derivatives 1a-c were discovered as the first GPR52 agonists in our
laboratory (Figure 1).6 Recently, we also reported thiazole derivative 2 and 1,2,4-triazole derivative
3 with potent GPR52 agonistic activity as different chemotypes.7,8
Figure 1. Reported GPR52 agonists 1a-c, 2 and 3.
2. Design and synthesis
2.1. Design
We previously examined impacts of a core structure on GPR52 agonistic activity,6 providing not
only identification of the first small molecule GPR52 agonists 1a-c, but also a key strategy to
enhance GPR52 agonistic activity. Discovery of 3-(1-benzothiophen-7-yl)benzamide derivatives
opened the way to the development of a novel class of antipsychotic drugs. The compound 1a and its
derivatives, however, still have some issues as drug candidates of central nervous system (CNS) in

terms of physicochemical and pharmacokinetic (PK) profiles. Especially, high lipophilicity and poor
aqueous solubility of the compounds 1a (logD pH7.4 = 7.05, Solubility at pH 6.8 = <0.05 μg/mL), 1b
(logD
= 7.17, Solubility at pH 6.8 = <0.06 μg/mL), and 1c (logD
= 6.37, Solubility at pH
pH7.4
pH7.4
6.8 = 0.11 μg/mL) needed to be improved for drug discovery. High lipophilicity is considered as a
primary reason of poor aqueous solubility and lowering of oral absorption as well as posing several
toxicity issues.9,10,11,12 In addition, lipophilicity is one of the key structural properties that greatly
affect the passive transcellular Blood-Brain Barrier (BBB) permeation of compounds. As LogD
value in the range of 1-3 would be generally preferred for CNS drugs,13,14 we focused on lowering
lipophilicity of compound 1b for suitability as a CNS drug by replacing 7-phenyl group of 1b with
heterocyclic rings. First, we speculated that the carbonyl moiety and the benzene ring in the 2-benzyl
group on benzothiophene of compound 1b were important for GPR52 agonistic activity based on our
accumulated knowledge of structure-activity relationship (SAR) around benzothiophene derivatives.
In addition, the benzene ring at the 7-position of compound 1b was considered to fill the large space
as a linker that connected between the carbonyl moiety and the 2-benzylbenzothiophene moiety of
compound 1b for enhancement of the GPR52 agonistic activity. On the basis of these hypotheses, we
planned introduction of heterocycle rings instead of the benzene ring at the 7-position of compound
1b.
Introduction of heterocyclic core B-F as a linker in structure A reduced calculated logD value
compared with that of compound 1b (Figure 2). Among them, the pyrazole derivative B showed the

reduced logD value (9; X = OMe, logD = 4.51). We expected that the pyrazole derivative B could
improve solubility due to its low lipophilicity. In addition, because the pyrazole derivatives have
several substituent patterns at 3-, 4-, and 5-position (s), we examined SAR on the pyrazole ring.
Superimposition analysis performed using MOE software15 between compound 1b and compound B
(9) indicated that the carbonyl group of compound B (9) successfully overlapped with that of the lead
compound 1b (Figure 3). Furthermore, the benzene ring in the 2-benzyl group on benzothiophene 1b
also overlapped with that on benzothiophene B (9). In the previous report,6 we reported that location
of the benzene ring in the 2-benzyl group on compound 1b would impact enhancement of GPR52
agonistic activity. Thus, pyrazole derivatives (10-18) with various substituent patterns were
designed. The pyrazole derivatives (11-15) introduced a methyl group at the 3- and/or 5-position (s)
on the pyrazole ring B (9) or 10 were designed to clarify the tolerability of the substituent sites for
the GPR52 agonistic activity. The pyrazole derivative 16 was designed to examine regiochemistry
effects of a pyrazole ring compared with compound 11. The pyrazole derivatives 17 and 18 were
designed to further lower lipophilicity, aiming at lowering logD by introducing a hydroxy group at
the 3- or 5-position on the pyrazole ring. Finally, the N-2-methoxyethyl group and the N-2-
hydroxyethyl group, which showed potent activity as reported in the previous paper,6 were selected
as an amide moiety of the pyrazole derivatives (9-18).

Figure 2. Design of pyrazole derivatives.
a logD at pH7.4 was calculated using ACD/LogD, version 12.
b The second fluid for the disintegration test is described in the Japanese Pharmacopoeia 15th edition
(pH 6.8).
Figure 3. Superimposition model of compounds 1b (lime green) and B = 9 (magenta). This image
was generated using MOE 2013.08.

Herein, we describe discovery of a novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as
GPR52 agonists and their synthesis and physicochemical parameters. Biological and
pharmacological effects of the representative compounds are also discussed.
2.2. Chemistry
The designed pyrazole compounds 9, 10, 14, and 15 were synthesized by a common synthetic
route (Scheme 1). Preparation of starting boronic ester 4 as a key intermediate is described in
Scheme 5. Copper-promoted coupling reaction16 of the boronic ester 4 with ethyl 1H-pyrazole-4-
carboxylate or ethyl 3,5-dimethyl-1H-pyrazole-4-carboxylate afforded pyrazole esters 5 and 6,
respectively. Basic hydrolysis of the esters 5 and 6 gave corresponding carboxylic acids 7 and 8,
followed by condensation with amines (R4NH2) to afford the desired amide derivatives 9, 10, 14 and
15.
Scheme 1a

o
a Reagents and conditions: (a) pyrazoles, Cu(OAc)2, pyridine, 50 C; (b) NaOH aq., THF-EtOH, 80
oC; (c) R4NH2, HOBt, EDCI, DMF.
The boronic ester 4 was treated with commercially available ethyl 3-methyl-1H-pyrazole-4-
carboxylate 19 to afford esters 20 and 21 (ca.6:1) as a regioisomeric mixture, which were separated
by silica gel column chromatography (Scheme 2). The methyl-substituted position on pyrazole ring
of the ester 20 was determined by two-dimensional (2D) nuclear magnetic resonance (NMR). In
compound 20, a nuclear overhauser effect (NOE) was observed between the proton at C6-
position of the benzothiophene core and the proton at C5-position of the pyrazole ring. After
hydrolysis of the esters 20 and 21 under basic condition, corresponding carboxylic acids were
condensed with 2-methoxyethyl amine or 2-aminoethanol to afford the desired pyrazol derivatives 11
and 12. The minor regioisomer 21 was converted into the only methoxyethyl amide 13.

Scheme 2a
a
o
o
Reagents and conditions: (a) Cu(OAc)2, pyridine., 50 C; (b) NaOH aq., THF-EtOH, 80 C; (c)
R1NH2, HOBt, EDCI, i-Pr2NEt, DMF; (d) MeO(CH2)2NH2, HOBt, EDCI, DMF.
Synthesis of hydroxymethyl pyrazole derivatives 17 and 18 is shown in Scheme 3. Copper-
promoted coupling of the boronic ester 4 with reported pyrazole derivative 2217 gave esters 23 and
24 (ca.1:1) as a regioisomeric mixture. After separation of the regioisomers 23 and 24 by silica gel
column chromatography, the substituted position of tert-buthoxymethyl on the pyrazole ring of the
esters 23 and 24 was also determined by 2D NMR. In compound 23, NOE was observed between
the proton at C6-position of the benzothiophene core and the methylene protons at C5-position

of the pyrazole ring. On the other hand, NOE was observed in compound 24 between the
proton at C6-position of the benzothiophene core and the methyl group at C5-position of the
pyrazole ring. After removal of the tert-buthyl group under acidic condition and basic hydrolysis of
the ethyl esters of 23 and 24, corresponding carboxylic acids were condensed with 2-aminoethanol to
afford the desired pyrazol derivatives 17 and 18.
Scheme 3a
a Reagents and conditions: (a) Cu(OAc)2, pyridine., 50 oC; (b) 4 M HCl/EtOAc; (c) NaOH aq., THF-
o
EtOH, 80 C; (d) H2N(CH2)2OH, HOBt, EDCI, DMF; (e) 4 M HCl/EtOAc; (f) NaOH aq., THF-
EtOH, 80 oC; (g) H2N(CH2)2OH, HOBt, EDCI, DMF.

N-Methyl pyrazole ester 26 was prepared by Suzuki-Miyaura coupling18 of the boronic ester 4
with the reported triflate 25.19 Basic hydrolysis of the ester 26 gave the corresponding carboxylic
acid, which was condensed with 2-aminoethanol to afford the pyrazole derivative 16 (Scheme 4).
Scheme 4a
a Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DMF, 95 oC, 25%; (b) NaOH aq., THF-EtOH, 80
oC; (c) NH2(CH2)2OH, HOBt, EDCI, i-Pr2NEt, DMF.
Preparation of the key intermediate boronic ester 4 described above is depicted in Scheme 5. The
boronic ester 4 was prepared by concise methods. Hydroxylmethyl benzothiophene 27 was treated
with methanesulfonyl chloride to afford mesylate 28. Suzuki-Miyaura coupling reaction of the
mesylate 28 with 3-trifluoromethylphenylboronic acid regioselectively gave bromide 29 without
other coupling adducts. Palladium-catalyzed cross-coupling reaction of the bromide 29 with
Bis(pinacolato)diboron afforded boronic ester 4.

Scheme 5a
a
o
Reagents and conditions: (a) Et3N, MsCl, THF, 0 C to rt; (b) m-CF3PhB(OH)2, Cs2CO3,
PdCl2(dppf)·CH2Cl2, THF-H2O, 60 oC; (c) (pin-B)2, PdCl2(dppf)·CH2Cl2, AcOK, DMF, 85 oC.
3. Biological results and discussion
3.1. In vitro assay
GPR52 agonistic activity of the synthesized compounds 9-18 was determined from the cAMP
production by using human GPR52 expressed on Chinese hamster ovary (CHO) cell lines. The EC50
values were calculated from the concentration-activity curve of each agonist producing 50%
response of 1 μM of the reference compound (N-(2-hydroxyethyl)-3-[2-[3-(trifluoromethyl)benzyl]-
1-benzofuran-4-yl]benzamide)6 (Table 1).
The 3,5-unsubstituted pyrazole derivatives 9 and 10, and the 5-methyl pyrazole derivative 13
showed moderate GPR52 agonistic activities with EC50 values of 62 nM, 87 nM, and 62 nM,
respectively. The 5-methyl group on the pyrazole ring (13) did not affect the enhancement of the
GPR52 agonistic activity compared with compound 9. On the other hand, introduction of a methyl
group at the 3-position on the pyrazole ring (11 and 12) remarkably increased GPR52 agonistic
activity (EC50 = 9.6 nM, Emax = 89% for 11; 5.7 nM, Emax = 102% for 12) compared with the 3,5-

unsubstituted pyrazole derivatives 9 (EC50 = 62 nM, Emax = 81%) and 10 (EC50 = 87 nM, Emax
=
73%). Furthermore, the 3,5-dimethyl pyrazole derivatives 14 and 15 (EC50 = 17 nM, Emax = 104% for
14; 15 nM, Emax = 110% for 15) also showed a higher potency than the 3,5-unsubstituted pyrazole
derivatives (9 and 10). Based on these results, introduction of the 3-methyl group on the pyrazole
ring (11 and 14) clearly increased the potency compared with the 3,5-unsubstituted pyrazole
derivative (9) and the 5-methyl pyrazole derivative (13). To examine effect of the 3-methyl group on
the pyrazole ring, we conducted superimposition studies of compounds 11, 14, and 1b using the
MNDO-PM3 (MOPAC version 7.01) method in MOE (Figure 4). The 3-methyl group of compounds
11 and 14 successfully occupied the hydrophobic space located around the 7-benzene ring of 1b.
These data suggested that introduction of the 3-methyl group into the pyrazole ring filled the spatial
gap of ring size observed between the pyrazole ring of compound 9 and the benzene ring of
compound 1b. On the other hand, the 5-methyl group of the 3,5-dimethylpyrazole derivative 14
would have no impact on the GPR52 agonistic activity, because the 5-methylpyrazole derivative 13
was as potent as the 3,5-unsubstituted pyrazole derivative 9. The 5-methyl group of compound 14
was located outside the 7-benzene ring of 1b, as observed in Figure 4.
As a result of SAR study of pyrazole derivatives (9-15), the N-2-hydroxyethyl group as an amide
moiety showed a potent GPR52 agonistic activity and also decreased logD value by 0.81 compared
with the N-2-methoxyethyl group. Therefore, the N-2-hydroxyethyl group was selected as an amide
moiety for further investigation. The GPR52 agonistic activity of compound 16 (EC50 = 45 nM) was

diminished compared with compound 12 (EC50 = 5.2 nM). The methyl group on the polar nitrogen
atom of the pyrazole ring in compound 16 could be intolerable for the GPR52 agonistic activity.
Hydrophilic substituent effect at the 3-position or the 5-position on the pyrazole ring in compound 15
was examined by introduction of a hydroxy group. The GPR52 agonistic activity of the 5-
hydroxymethyl-3-methyl pyrazole derivative 17 (EC50 = 21 nM, Emax = 103%) was as potent as that
of 15, whereas the 3-hydroxymethyl-5-methyl pyrazole derivative 18 (EC50 = 146 nM) showed
weaker GPR52 agonistic activity than compounds 15 and 17. The hydroxy group at the 5-position on
the pyrazole ring 15 was fully tolerated for the GPR52 agonistic activity, while 3-hydroxymethyl
group on the pyrazole ring 18 was intolerable.
3.2. Solubility
Aqueous Solubility of the synthesized compounds 9-18 is also shown in Table 1. Compounds 9-
12 and 16 showed poor solubility, whereas the 5-methyl pyrazole derivative 13 (solubility at pH 6.8
= 1.3 μg/mL) showed better solubility than the 3,5-unsubstituted pyrazole derivative 9 (solubility at
pH 6.8 = <0.08 μg/mL). Furthermore, the 3,5-dimethyl pyrazole derivatives 14 and 15 (solubility at
pH 6.8 = 2.9 μg/mL and 12 μg/mL, respectively) provided much better solubility than the 3,5-
unsubstituted pyrazole derivatives 9 and 10 (solubility at pH 6.8 = <0.09 μg/mL) or the 3-methyl
pyrazole derivatives 11 and 12 (solubility at pH 6.8 = <0.1 μg/mL and <0.1 μg/mL, respectively).
Improvement of the solubility of compounds 13, 14, and 15 was considered to be caused by reducing

their logD values. However, solubility of the 5-methyl pyrazole derivative 13 was greatly improved
compared with compound 11 even though the logD value of both compounds is 4.29. In addition, the
3-methyl pyrazole derivative 12 showed poorer solubility than compounds 13 and 14, although the
logD value of compound 12 (logD = 3.48) was lower than those of compounds 13 and 14 (logD =
4.08). These results suggested that the methyl group at the 5-position on the pyrazole ring in
compounds 13, 14, and 15 plays an important role to improve the solubility independently of the
logD value as a lipophilic parameter.
We performed global minimum conformational analyses of compounds 11 and 14 in MOE to
confirm an effect of the methyl group at the 5-position on the pyrazole ring of compound 14 (Figure
5). The dihedral angle between benzothiophene core of compound 11 and the 3-methyl pyrazole ring
at 7-position was nearly planar (3.7o), as shown in Figure 5 (left image), while the dihedral angle
between benzothiophene core of compound 14 and the 3,5-dimethyl pyrazole ring at 7-position was
30.6o, because of the steric hindrance by the 5-methyl group on the pyrazole ring, as shown in
Figure 5 (right image). These analyses suggested that introduction of the 5-methyl group on the
pyrazole ring of compound 14 caused disruption of molecular planarity and improvement of the
solubility in addition to the decrease in logD value.20
Solubility of the compounds 17 and 18 (logD = 2.21 and 2.21, respectively) with a hydroxyl
group at 3-position or 5-position in the pyrazole ring was increased because of lower logD values
than for compound 15 (logD = 3.27).

Table 1. GPR52 agonistic activities and physicochemical properties of pyrazole derivatives.
a EC50 values are derived from the mean curves of the experiments (n =2-4). Numbers in parentheses
represent the 95% confidence interval.
b The second fluid for the disintegration test is described in the Japanese Pharmacopoeia 15th edition
(pH 6.8).
c logD at pH 7.4 was calculated using ACD/LogD, version 12.

Figure 4. Superimposition model of compounds 1b (lime green), 11 (cyan), and 14 (yellow). This
image was generated using MOE 2013.08.
Figure 5. Minimized structures 11 (left image) and 14 (right image). These images were generated
using MOE 2013.08.
3.3. Pharmacokinetic Profiles
Compound 17 showed highly potent GPR52 agonistic activity, good solubility, and acceptable
logD values in the range 1-3 favored for CNS drugs13,14 among synthesized pyrazole derivatives (9-

18, Table 1), and therefore compound 17 was selected for further PK evaluations. It was found that
compound 17 possessed good oral absorption (Cmax = 83.0 ng/mL; Tmax = 1.7 h; AUC0−8h = 405.5
ng·h/mL; F (bioavailability) = 29.9%, mouse cassette dosing at 0.1 mg/kg, iv and 1 mg/kg, p.o.) and
the plasma protein binding value in mice at 1 M concentration was 98% (Table 2). In addition to its
moderate PK profile in mice, the multidrug resistance (MDR) efflux ratio of compound 17 was 1.7.
These results led to expectation that compound 17 would be a non-substrate of p-glycoprotein1 and
show a high brain penetration as oral administration.
In our previous study, we reported that compound 1a (EC50 = 30 nM, Emax = 79.8%), used for in
vivo pharmacological evaluation, possessed oral PK profile [Cmax =108.1 ng/mL; Tmax = 2 h;
AUC0−8h = 613.7 ng·h/mL; F (bioavailability) = 73%, mouse cassette dosing at 0.1 mg/kg, iv and 1
mg/kg, po] and good brain penetration (brain/plasma AUC ratio B/P = 0.94, in mice after oral
administration of 30 mg/kg).6 As compared to compound 1a, GPR52 agonistic activity and PK
profile of compound 17 were similar to compound 1a, whereas the solubility of compound 17 was
much higher than that of compound 1a. Moreover, MDR efflux ratio of compound 17 was suitable
for use as an in vivo tool compound. Based on these results, compound 17 was found to be suitable
as a biological in vivo tool compound.
Table 2. Pharmacokinetic profile in mice of compound 17.

a Mice cassette dosing. ICR mice (n=3). Dose: 0.1 mg/kg, i.v.; 1 mg/kg, p.o. Data are presented as
averages for 3 mice.
b Maximum drug concentration.
c Time of maximum drug concentration observed.
d Area under the curve from 0 to 8 h.
e Clearance.
f Bioavailability.
3.4. MAP-induced hyperlocomotion
Thus, compound 17, with acceptable PK profile, was evaluated for antipsychotic efficacy by
methamphetamine (MAP)-induced hyperlocomotion21 test in mice. Compound 17 dose-dependently
suppressed MAP-induced hyperlocomotion at 10 and 30 mg/kg after oral administration (Figure 6).
Haloperidol, currently employed clinically as an antipsychotic, also suppressed MAP-induced
hyperlocomotion, at 0.3 mg/kg. In addition, no nonspecific effect, such as sedation, general malaise,
or compound toxicity, was observed during a 60-minute period after pretreatment of compound 17.
These data indicate that compound 17 has a potent antipsychotic-like effect in mice.

Figure 6. Dose-dependent inhibition of MAP-induced hyperlocomotion by compound 17 in mice.
Mice were administered compound 17 (10, 30 mg/kg, p.o.) and haloperidol (0.3 mg/kg, p.o.) and
placed in activity chambers for 60 min. After this period, MAP (2 mg/kg, s.c.) was injected and
measurement was started. Data are expressed as mean ± S.E.M. (n = 6–7 per group). Data were
analyzed by Williams’ test, *P < 0.025 versus the control group treated with vehicle and MAP.
4. Conclusions
A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole GPR52 agonists was designed and
synthesized to improve high lipophilicity and poor solubility of the compound 1b. The newly
designed pyrazole derivatives (9-18) were efficiently synthesized by synthetic routes using Suzuki-

Miyaura coupling and Chan-Lam-Evans coupling conditions as key reactions. In the evaluation of
these pyrazole derivatives, we found introduction of the methyl substituent at the 3-position of the
pyrazole ring led to enhancement of the GPR52 agonistic activity. Moreover, substituents at the 5-
position on the pyrazole ring would play an important role to improve the solubility. As a result, we
identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 with highly potent GPR52 agonistic
activity (EC50 = 21 nM, Emax = 103%), good solubility (21 μg/mL at pH 6.8), and acceptable
bioavailability (F = 29.9%). Furthermore, this compound 17 also suppressed methamphetamine-
induced hyperlocomotion in mice after oral administration (10 and 30 mg/kg, p.o.). These results
indicate that the newly designed pyrazole GPR52 agonists have potential as a next generation
antipsychotics.
5. Experimental protocols
5.1.1. General
The proton nuclear magnetic resonance (1H NMR) spectra were determined on a Bruker
1
AVANCE II (300 MHz) or Varian INOVA- 400 (400 MHz) instruments. Chemical shifts for H
NMR were reported in parts per million (ppm) downfield from tetramethylsilane (δ) as the internal
standard in deuterated solvent and coupling constants (J) are in Hertz (Hz). The following
abbreviations are used for spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet and br = broad. Elemental analyses were carried out by Takeda Analytical Research

Laboratories, Ltd. In the following experimental, melting points ere determined on a hi -545
and were uncorrected. Analytical thin layer chromatography (TLC) was performed on silica gel 60
F254 plates (Merck) or NH TLC plate (Fuji Silysia Chemical Ltd.). Column chromatography was
R
○
carried out on a silica gel column (Chromatorex
NH-DM1020, 100–200 mesh, Fuji Silysia
chemical) or on Purif-Pack (SI / 60 lM or NH / 60 lM, Fuji Silysia Chemical, Ltd.). Liquid
chromatography-mass spectrometry (LC/ MS) analysis was performed on the following methods;
(A) an Agilent 1200, equipped with a L-column2 ODS (3.0 × 50 mm I.D., 3 lm-particle size, CERI,
Japan), eluting with 5 mM ammonium acetate (AcONH4) in ultrapure water/acetonitrile = 90/10
(Mobile phase A), and 5 mM AcONH4 in ultrapure water/acetonitrile = 10/90 (Mobile phase B),
using the following elution gradient of 5% B to 90% B over 0.9 min followed by 90% B isocratic
over 1.1 min at a flow rate of 1.5 mL/min (detection at 220 nm or 254 nm). MS spectra were
recorded using an Agilent 6130 with electrospray ionization. (B) a Shimadzu LC-20AD, equipped
with a L-column2 ODS (3.0 × 50 mm I.D., 3 lm-particle size, CERI, Japan), eluting with 5 mM
AcONH4 in ultrapure water/acetonitrile = 90/10 (Mobile phase A), and 5 mM AcONH4 in ultrapure
water/acetonitrile = 10/90 (Mobile phase B), using the following elution gradient of 5% B to 90% B
over 0.9 min followed by 90% B isocratic over 1.1 min at a flow rate of 1.5 mL/min (detection at
220 nm or 254 nm). MS spectra were recorded using a Shimadzu LCMS-2020 with electrospray
ionization. (C) a Shimadzu LC- 20AD, equipped with a L-column2 ODS (3.0 × 50 mm I.D., 3
lmparticle size, CERI, Japan), eluting with 0.05% TFA in ultrapure water (Mobile phase A), and

0.05% TFA in acetonitrile (Mobile phase B), using the following elution gradient of 5% B to 90% B
over 0.9 min followed by 90% B isocratic over 1.1 min at a flow rate of 1.5 mL/min (detection at
220 nm). MS spectra were recorded using a Shimadzu LCMS-2020 with electrospray ionization. (D)
a Waters 2795, equipped with a L-column2 ODS (3.0 × 50 mm I.D., 3 lmparticle size, CERI, Japan),
eluting with 0.05% TFA in ultrapure water (Mobile phase A), and 0.05% TFA in acetonitrile (Mobile
phase B), using the following elution gradient of 5% B to 90% B over 2 min followed by 90% B
isocratic over 1.5 min at a flow rate of 1.2 mL/min (detection at 220 nm or 254 nm). MS spectra
were recorded using a Waters ZQ2000 with electrospray ionization. Preparative LC was performed
on a Waters 2525 separations module (L-column2 ODS (20 × 150 mm I.D., CERI, Japan); 0.1%
TFA in distilled water/acetonitrile gradient; MS spectra were recorded using a Waters ZQ2000 with
electrospray ionization. The purities of all compounds tested in biological systems were assessed as
being >95% using analytical high-performance liquid chromatography (HPLC). Purity data were
collected by a HPLC with Corona CAD (Charged Aerosol Detector) or photo diode array detector.
The column was a Capcell Pak C18AQ (50 mm × 3.0 mm I.D., Shiseido, Japan) or L-column 2 ODS
o
(30 mm × 2.0 mm I.D., CERI, Japan) with a temperature of 50 C and a flow rate of 0.5 mL/min.
Mobile phase A and B under a neutral condition were a mixture of 50 mmol/L AcONH4, water and
acetonitrile (1:8:1, v/v/v) and a mixture of 50 mmol/L AcONH4 and acetonitrile (1:9, v/v),
respectively. The ratio of mobile phase B was increased linearly from 5% to 95% over 3 min, 95%
over the next 1 min. Mobile phase A and B under an acidic condition were a mixture of 0.2% formic

acid in 10 mmol/L ammonium formate and 0.2% formic acid in acetonitrile, respectively. The ratio
of mobile phase B was increased linearly from 14% to 86% over 3 min, 86% over the next 1 min. All
commercially available solvents and reagents were used without further purification. Yields were not
optimized. Abbreviations: (pin-B)2, bis(pinacolato) diboron; Pd(Ph3P)4, tetrakis(triphenylphosphine)-
palladium(0); PdCl2(dppf)-CH2Cl2, dichloro [1,10-bis(diphenylphosphino)ferrocene] palladium(II)
CH2Cl2 adduct; Na2CO3, sodium carbonate; NaHCO3, sodium hydrogen carbonate; NaOH, sodium
hydroxide; K2CO3, potassium carbonate; Cs2CO3, cesium carbonate; AcOK, potassium acetate;
MgSO4, magnesium sulfate; TFA, trifluoroacetic acid; NH4Cl, ammonium chloride; MsCl,
methanesulfonyl chloride; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
HOBt, 1-hydroxybenzotriazole; i-Pr2NEt, di-isopropylethylamine; Et3N, triethylamine; HCl,
hydrochloric acid; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; EtOAc, ethyl acetate;
MeOH, methyl alcohol; EtOH, ethyl alcohol; THF, tetrahydrofuran.
5.1.2. Ethyl 1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-carboxylate (5)
To a solution of 4,4,5,5-tetramethyl-2-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-
1,3,2-dioxaborolane (4) (1.25 g, 3.0 mmol) and ethyl 1H-pyrazole-4-carboxylate (840 mg, 6.0 mmol)
in pyridine (10 mL) was added Cupric acetate, monohydrate (1.19 g, 6.0 mmol) at room temperature.
The mixture was stirred at 50 ºC under a dry atmosphere (CaCl2 tube) overnight. The mixture was
quenched with water at room temperature and extracted with EtOAc. The organic layer was

separated, washed with 1 M HCl and brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, eluted with 0% - 25% EtOAc in hexane)
to give 5 (557 mg, 43%) as colorless crystals.
1H-NMR (300 MHz, CDCl3) δ: 1.39 (3H, t, J = 7.1 Hz), 4.29 (2H, s), 4.36 (2H, q, J = 7.1 Hz),
7.08−7.11 (1H, m), 7.38−7.57 (6H, m), 7.68 (1H, dd, J = 6.6, 2.2 Hz), 8.18 (1H, s), 8.50 (1H, s).
5.1.3. Ethyl 3,5-dimethyl-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-
carboxylate (6)
Compound 6 was prepared in a manner similar to that desired for 5 in 55% yield.
1H-NMR (300 MHz, CDCl3) δ: 1.39 (3H, t, J = 7.1 Hz), 2.46 (3H, s), 2.52 (3H, s), 4.26 (2H, s), 4.34
(2H, q, J = 7.1 Hz), 7.08 (1H, t, J = 1.1 Hz), 7.20 (1H, dd, J = 7.6, 1.0 Hz), 7.37−7.55 (5H, m), 7.73
(1H, dd, J = 8.0, 1.1 Hz).
5.1.4. N-(2-methoxyethyl)-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-
carboxamide (9)
To a solution of ethyl 1-(2-(3-(trifluoromethyl)benzyl)benzo[b]thiophen-7-yl)-1H-pyrazole-4-
carboxylate (5) (557 mg, 1.29 mmol) in THF (5 ml)/EtOH (5.00 ml) was added 8 M NaOH aq.
(0.324 ml, 2.59 mmol) at room temperature. The mixture was stirred at 80 ºC under a dry atmosphere
(CaCl2 tube) overnight. 6 M HCl was added to bring the pH of the solution to 2-3 and extracted with

EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4 and
concentrated in vacuo. This product was subjected to the next reaction without further purification.
To a solution of 1-(2-(3-(trifluoromethyl)benzyl)benzo[b]thiophen-7-yl)-1H-pyrazole-4-carboxylic
acid (7) (200 mg, 0.50 mmol), WSC (HCl) (191 mg, 0.99 mmol) and iPr2EtN (0.260 ml, 1.49 mmol)
in DMF (5 ml) was added HOBt (Anhydrous) (134 mg, 0.99 mmol) at room temperature. After being
stirred at room temperature for 10 min, 2-methoxyethanamine (74.7 mg, 0.99 mmol) was added to
the reaction mixture. The mixture was stirred at room temperature under N2 overnight. The mixture
was quenched with sat. NH4Cl aq. at room temperature and extracted with EtOAc. The organic layer
was separated, washed with water and brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, eluted with 0% - 60% EtOAc in hexane
and 0% - 5% MeOH in EtOAc) and crystallized from EtOAc-hexane to give 9 (420 mg, 71%) as
colorless crystals.
1H-NMR (300 MHz, CDCl3) δ: 3.41 (3 H, s), 3.53 - 3.69 (4 H, m), 4.29 (2 H, s), 6.22 (1 H, br s),
7.08 - 7.11 (1 H, m), 7.40 - 7.58 (6 H, m), 7.66 - 7.70 (1 H, m), 8.02 (1 H, d, J = 0.8 Hz), 8.47 (1 H,
d, J = 0.8 Hz). 13C NMR (75 MHz, CDCl3) δ: 36.4, 39.2, 58.8, 71.2, 114.6, 120.2, 122.0, 122.3,
123.7 (q, JC–F = 3.7 Hz), 124.9, 125.4 (q, JC–F = 3.7 Hz), 127.6 (q, JC–F = 270 Hz), 129.2, 129.3,
131.0 (d, JC–F = 32 Hz), 131.2, 132.1, 134.2, 138.7, 140.0, 142.6, 146.5, 162.1. mp: 157 – 159 ºC.
MS (ESI+) m/z = 460.1 [M+H]+. Chemical purity 100%.

5.1.5. N-(2-methoxyethyl)-3,5-dimethyl-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-
1H-pyrazole-4-carboxamide (14)
Compound 14 was prepared from 6 in a manner similar to that described for 9 in 85% yield. 1H-
NMR (300 MHz, CDCl3) δ: 2.43 (3 H, s), 2.50 (3 H, s), 3.40 (3 H, s), 3.53 - 3.68 (4 H, m), 4.25 (2 H,
s), 6.06 - 6.16 (1 H, m), 7.05 - 7.09 (1 H, m), 7.18 (1 H, dd, J = 7.7, 0.8 Hz), 7.35 - 7.54 (5 H, m),
13
7.71 (1 H, dd, J = 8.0, 0.8 Hz). C NMR (75 MHz, CDCl3) δ: 11.9, 14.1, 36.6, 39.0, 58.8, 71.3,
114.6, 121.4, 122.1, 123.4, 123.8 (q, JC–F = 3.9 Hz), 124.7, 125.5 (q, JC–F = 3.9 Hz), 127.6 (q, JC–F
=
272.9 Hz), 129.2, 131.0 (d, JC–F = 31.9 Hz), 132.2, 133.0, 137.0, 139.9, 142.0, 142.8, 145.6, 147.6,
164.5. MS (ESI+) m/z = 488.1 [M+H]+. Chemical purity 100%.
5.1.6. N-(2-hydroxyethyl)-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-
carboxamide (10)
Compound 10 was prepared in a manner similar to that desired for 9 in 54% yield.
1H-NMR (300 MHz, CDCl3) δ: 3.27 - 3.37 (2 H, m), 3.45 - 3.59 (2 H, m), 4.33 - 4.47 (2 H, m), 4.69
- 4.82 (1 H, m), 7.28 - 7.38 (1 H, m), 7.41 - 7.83 (7 H, m), 8.16 - 8.35 (2 H, m), 8.95 - 9.07 (1 H, m).
mp: 226 - 228 ºC. MS (ESI+) m/z = 446.1 [M+H]+.
5.1.7. N-(2-hydroxyethyl)-3,5-dimethyl-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-
1H-pyrazole-4-carboxamide (15)

Compound 15 was prepared from 6 in a manner similar to that desired for 9 in 60% yield.
1H-NMR (300 MHz, CDCl3) δ: 2.44 (3H, s), 2.52 (3H, s), 2.63 (1H, t, J = 4.9 Hz), 3.59−3.68 (2H,
m), 3.81−3.89 (2H, m), 4.26 (2H, s), 6.15 (1H, br s), 7.09 (1H, t, J = 1.1 Hz), 7.19 (1H, dd, J = 7.5,
0.8 Hz), 7.38−7.55 (5H, m), 7.74 (1H, dd, J = 7.9, 1.1 Hz). mp: 137−139 ºC．MS (ESI+) m/z =
474.1 [M+H]+. Chemical purity 100%. Anal. Calcd for C24H22N3O2SF3: C, 60.88; H, 4.68; N, 8.87.
Found: C, 60.81; H, 4.70; N, 8.87.
5.1.8.
Ethyl
3-methyl-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-
carboxylate (20)
and ethyl 5-methyl-1-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-1H-pyrazole-4-
carboxylate (21)
To a solution of 4,4,5,5-tetramethyl-2-(2-(3-(trifluoromethyl)benzyl)-1-benzothiophen-7-yl)-
1,3,2-dioxaborolane (4) (1.25 g, 3.0 mmol) and ethyl 3-methyl-1H-pyrazole-4-carboxylate (554 mg,
3.6 mmol) in pyridine (15 mL) was added Cupric acetate, monohydrate (300 mg, 1.5 mmol) at room
temperature. The mixture was stirred at 50 ºC under a dry atmosphere (CaCl2 tube) overnight. The
mixture was quenched with water at room temperature and extracted with EtOAc. The organic layer
was separated, washed with 1 M HCl aq. and brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel, eluted with 0% - 35% EtOAc in
hexane) to give 20 (884 mg, 66%) as colorless crystals and 21 (150 mg, 11%) as a brown oil.