Design and synthesis of a novel 2-oxindole scaffold as a highly potent and brain-penetrant phosphodiesterase 10A inhibitor

Article abstract of DOI:10.1016/j.bmc.2015.10.002

Highly potent and brain-penetrant phosphodiesterase 10A (PDE10A) inhibitors based on the 2-oxindole scaffold were designed and synthesized. (2-Oxo-1,3-oxazolidin-3-yl)phenyl derivative 1 showed the high P-glycoprotein (P-gp) efflux (efflux ratio (ER) = 6.

Full text of DOI:10.1016/j.bmc.2015.10.002

Bioorganic & Medicinal Chemistry 23 (2015) 7138–7149
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of a novel 2-oxindole scaffold as a highly potent
and brain-penetrant phosphodiesterase 10A inhibitor
a
a
a
a
Masato Yoshikawa a, , Haruhi Kamisaki , Jun Kunitomo , Hideyuki Oki , Hironori Kokubo ,
Akihiro Suzuki b, Tomomi Ikemoto b, Kosuke Nakashima a, Naomi Kamiguchi a, Akina Harada a,
Haruhide Kimura a, Takahiko Taniguchi a
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Highly potent and brain-penetrant phosphodiesterase 10A (PDE10A) inhibitors based on the 2-oxindole
scaffold were designed and synthesized. (2-Oxo-1,3-oxazolidin-3-yl)phenyl derivative 1 showed the high
P-glycoprotein (P-gp) efflux (efflux ratio (ER) = 6.2) despite the potent PDE10A inhibitory activity
(IC50 = 0.94 nM). We performed an optimization study to improve both the P-gp efflux ratio and
PDE10A inhibitory activity by utilizing structure-based drug design (SBDD) techniques based on the
X-ray crystal structure with PDE10A. Finally, 1-(cyclopropylmethyl)-4-fluoro-5-[5-methoxy-4-oxo-
3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-1(4H)-yl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-one (19e) was
identified with improved P-gp efflux (ER = 1.4) and an excellent PDE10A inhibitory activity
(IC50 = 0.080 nM). Compound 19e also exhibited satisfactory brain penetration, and suppressed PCP-
induced hyperlocomotion with a minimum effective dose of 0.3 mg/kg by oral administration in mice.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 11 August 2015
Revised 1 October 2015
Accepted 4 October 2015
Available online 9 October 2015
Keywords:
Phosphodiesterase 10A (PDE10A)
Inhibitors
Schizophrenia
P-gp-mediated efflux
1. Introduction
rons of the striatum.8,9 Based on this selective expression and its
function, PDE10A inhibition is considered to be an attractive
Phosphodiesterases (PDEs) comprise a superfamily of enzymes
encoded by 21 genes and are subdivided into 11 distinct families
according to their structural and functional properties. These
enzymes metabolically inactivate the ubiquitous intracellular sec-
ond messengers cyclic adenosine monophosphate (cAMP) and cyc-
lic guanosine monophosphate (cGMP). PDEs selectively catalyze
the hydrolysis of the 30-phosphodiester bond, forming the inactive
50-monophosphate.1
Both cAMP and cGMP are involved in the regulation of virtually
every physiological process.2 In neurons, these second messengers
play an important role in the regulation of synaptic transmission as
well as in neuronal differentiation and survival.3 Intracellular
cAMP and cGMP concentrations appear to be temporally, spatially,
and functionally compartmentalized via the regulation of adenyl
and guanyl cyclases in response to extracellular signaling and their
degradation by PDEs.4
approach for the treatment of basal-ganglia-related diseases,
including schizophrenia, Parkinson’s disease and Huntington’s dis-
ease.10–12 Several companies are actively developing PDE10A inhi-
bitor programs, and MP-10 (Pfizer)13 and FRM-6308 (Forum) are
currently under evaluation in human clinical trials. We recently
reported the discovery of pyridazinone-based PDE10A inhibitors;14
among these, TAK-063 has potent inhibitory activity (IC50 = 0.30
nM), excellent selectivity (>15,000-fold selectivity over other
PDEs), and favorable pharmacokinetics in mice, including high
brain penetration. In addition, TAK-063 is currently being
evaluated in clinical trials for the treatment of schizophrenia.
In the course of studies to identify TAK-063, we found 1-[2-flu-
oro-4-(2-oxo-1,3-oxazolidin-3-yl)phenyl]-5-methoxy-3-(1-phe-
nyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (1)14 (PDE10A IC50
=
0.94 nM) as described in Figure 1. Despite having potent PDE10A
inhibitory activity, compound 1 exhibited high efflux (efflux
ratio(B to A/A to B) = 6.2) in an in vitro P-gp-overexpressing cell line.
To achieve appropriate blood–brain barrier (BBB) penetration, it is
critically important that central nervous system (CNS) drugs are
not P-gp substrates. There are several drug-binding sites on
P-gp,15–17 which are large and hydrophobic cavities.18 The affinity
of substrates for the binding sites is related to P-gp-mediated
Phosphodiesterase 10A (PDE10A), which was discovered in
1999 by three independent groups,5–7 hydrolyzes both cAMP and
cGMP, and is predominantly expressed in the medium spiny neu-
⇑
Corresponding author. Tel.: +81 466 32 1140.
E-mail address: masato.yoshikawa@takeda.com (M. Yoshikawa).
http://dx.doi.org/10.1016/j.bmc.2015.10.002
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
7139
critical pharmacophore.14 In contrast, a large hydrophobic space,
which is defined by the side chains of Leu625, Ala626, Phe629,
and Met703, lies around the 3- and 4-positions of the phenyl
group at the 1-position of the pyridazin-4(1H)-one. The pendant
1,3-oxazolidin-2-one ring, which occupies a part of the hydrophobic
space in the solvent-accessible region, is thought to interact with
Ala626 but not have a specific hydrogen-bonding interaction
with the amino acid (Fig. 2B). It suggests that modification of the
1,3-oxazolidin-2-one moiety, which is oriented toward the sol-
vent-accessible region, would allow for potent PDE10A inhibitory
activity and could adjust the ADME-Tox profiles including the
P-gp ER. Herein, we described our synthetic studies on modifica-
tion of the 1,3-oxazolidin-2-one moiety to achieve good BBB pen-
etration while enhancing PDE10A inhibitory activity. Moreover,
the pharmacological profiles of the potent and brain-penetrant
PDE10A inhibitor 19e are also described.
Figure 1. TAK-063 and lead compound 1.
efflux; therefore, eliminating or disrupting interactions between
substrates and the drug-binding sites on P-gp would weaken the
recognition of substrates by P-gp and improve the P-gp ER.19,20
However, there are limitations on what can be done to block the
interaction because of the necessity of maintaining potency and
favorable ADME-Tox profiles such as metabolic stability. Thus, it
is important to identify the pharmacophores contributing to each
chemotype.
To clarify the appropriate positions to modify in our PDE10A
inhibitors, the X-ray crystal structure of compound 1 bound to
PDE10A is illustrated in Figure 2. Compound 1 binds to the active
site, forming three critical interactions (Fig. 2A): (i) the pyri-
2. Chemistry
5-Methoxypyridazin-4(1H)-one derivatives 3a and 3b were
prepared by Ullmann-type coupling reactions21 or the Buchwald–
Hartwig reaction22 of 2,14 with the corresponding heterocycles as
shown in Scheme 1.
The synthesis of anilines 12a–e is summarized in Scheme 2.
Compounds 8a and 8b were prepared from commercially available
2,3-difluoronitrobenzene (4) via the insertion of diethyl malonate,
decarboxylation, esterification, and alkylation. Reduction of nitro
groups in compounds 8a and 8b directly afforded cyclized 2-oxin-
doles 9a and 9b. After regioselective nitration, N-alkylation and
subsequent reduction of the nitro group resulted in anilines 12a–e.
Compound 19a was synthesized as described in Scheme 3.
Diazotization of the aniline 12a with NaNO2 and HCl aq followed
by treatment with methyl 4-methoxy-3-oxobutanoate yielded
hydrazone 13. The pyridazin-4(1H)-one ring was constructed using
dazin-4(1H)-one ring has face-to-face and edge-to-face p–p stack-
ing interactions with the phenyl rings of Phe719 and Phe686
residues, respectively; (ii) one nitrogen atom of the pyrazole ring
interacts with Tyr514 through a water molecule; and (iii) the
methoxy and carbonyl groups on the pyridazinone ring form a
bidentate interaction with the carboxamide NH of the conserved
Gln716 side chain. As previously described, the 5-methoxy-3-
(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one moiety is the
Figure 2. X-ray crystal structures of compound 1 in the PDE10A catalytic domain. (A) Right perspective view. Leu625, Ala626, Phe629, and Met703 were omitted for
simplicity. (B) Left perspective view. Phe686, Phe719, and Tyr514 were omitted for simplicity.
Scheme 1. Synthesis of 5-methoxypyridazin-4(1H)-one derivatives 3a and 3b. Reagents and conditions: (a) pyrrolidin-2-one, CuI, trans-1,2-diaminocyclohexane, K3PO4, 1,
4-dioxane, 90 °C, 33%; (b) pyrrolidine, Pd2(dba)3, Xantphos, Nat-OBu, 1,4-dioxane, 90 °C, 49%.

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M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
Scheme 2. Synthesis of anilines 12a–e. Reagents and conditions: (a) diethyl malonate, K2CO3, DMF, 65 °C, 90%; (b) HCl aq, AcOH, reflux, 96%; (c) SOCl2, MeOH, rt, 100%; (d)
iodomethane or 1,3-dibromopropane, NaH, DMF, rt, 33–76%; (e) zinc, AcOH, 100 °C, 93–98%; (f) fuming nitric acid, sulfuric acid, 40 °C, 66–77%; (g) iodomethane, 2-
bromopropane or (bromomethyl)cyclopropane as an alkyl halide, NaH or Cs2CO3 as a base, DMF, rt, 62–89%; (h) cyclobutanol, diethyl azodicarboxylate, triphenylphosphine,
THF, rt, 54%; (i) Pd/C, H2, MeOH, rt, 63–95%.
O
O
O
N
O
O
N
O
O
F
O
F
O
F
O
O
OH
NH2
NH
N
N
N
F
a
c
b
HN
O
NH
13
N
N
12a
O
O
O
14
15
O
N
O
O
N
O
O
N
O
O
N
N
O
F
O
O
F
O
O
N
N
N
N
N
N
d
e
f
g
N
F
F
N
N
N
N
O
O
O
O
18
19a
16
17
Scheme 3. Synthesis of 2-oxindole derivative 19a. Reagents and conditions: (a) (1) NaNO2, HCl aq, 0 °C; (2) methyl 4-methoxy-3-oxobutanoate, NaOAc, MeOH, 0 °C, 96%; (b)
DMFDMA, reflux, 56%; (c) NaOH aq, MeOH, rt, 100%; (d) N,O-dimethylhydroxylamine hydrochloride, 1,10-carbonyldiimidazole, DIPEA, THF, rt, 61%; (e) MeMgBr, THF, ꢀ78 °C,
66%; (f) DMFDMA, reflux; (g) phenylhydrazine, TFA, EtOH, rt, 27% in 2 steps.
dimethylformamide dimethylacetal (DMFDMA). During heating in
DMFDMA, N-methylation of the 2-oxindole ring also occurred.
After hydrolysis under basic conditions, the carboxylic acid 15
was converted to the ketone 17 via the Weinreb amide 16. Heating
of the ketone 17 in DMFDMA and subsequent cyclization to a pyra-
zole ring afforded compound 19a.
The synthetic method shown in Scheme 3 required sequential
multistep reactions, and it was inefficient for synthesizing multiple
compounds possessing varying substituents on the nitrogen atom
of the 2-oxindole ring because the nitrogen atom was methylated
during cyclization using DMFDMA. Therefore, we explored an
alternative synthetic route to ensure a diversity of substituents
for R2.
An alternative synthetic route via compound 22 as a key inter-
mediate is shown in Scheme 4. Treatment of 1-phenylpyrazole
with n-BuLi followed by 2-(methoxymethyl)oxirane afforded the
alcohol 21. The key intermediate 22 was obtained by a variant of
the Swern oxidation.23 Diazotization of anilines 12b–e and subse-
quent treatment with the key intermediate ketone 22 afforded cor-
responding hydrazones 23b–e. Cyclization with DMFDMA afforded
the desired compounds 19b–e.
3. Results and discussion
PDE10A inhibitory activities were measured using a scintilla-
tion proximity assay based on the inhibition rate of cGMP

M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
7141
Scheme 4. Alternative synthetic route for 2-oxindole derivatives 19b–e. Reagents and conditions: (a) n-BuLi, 2-(methoxymethyl)oxirane, THF, ꢀ78 °C, 51%; (b) DMSO,
(CF3CO)2O, TEA, THF, ꢀ42 °C, 43%; (c) anilines 12b–e, NaNO2, HCl aq, NaOAc, MeOH, 0 °C; (d) DMFDMA, MeCN, reflux, 6–42% in 2 steps.
pyrrolidine-2-one (3a) resulted in a significantly reduced ER (2.0)
without sacrificing potent PDE10A inhibitory activity. This
suggests that the oxygen atom at the 1-position of the 1,3-oxazo-
lidin-2-one ring may interact with the drug-binding sites on
P-gp. Compound 3a has a small topological polar surface area
(TPSA)24 value (82 Å2) compared to 1 (91 Å2). This is compatible
with the fact that TPSA is one of the most dominating determinants
for defining the P-gp substrates.25,26 Furthermore, the pyrrolidine
analog 3b with a lower TPSA (65 Å2) exhibited a more favorable
ER and enhanced the PDE10A inhibitory activity, presumably
reflecting that an alkylene moiety of the pyrrolidine ring would
be located in a closer and more desirable position to interact with
Ala626 because a planarity of the pyrrolidine ring was reduced due
to the loss of the carbonyl group in the pyrrolidin-2-one ring.27
However, compound 3b displayed poor in vitro metabolic stability
in human hepatic microsomes. Previous studies revealed that low
electron density on the ring A and the presence of a carbonyl group
in substituent R1 were advantageous for metabolic stability.14 The
carbonyl group of the pyrrolidin-2-one in compound 3a is
supposed to contribute to the metabolic stability as well as the pre-
vious studies. Accordingly, the pyrrolidin-2-one analog 3a had
most balanced profiles among the compounds listed in Table 1.
In our further optimization, we designed a 2-oxindole scaffold
by fusing the pyrrolidin-2-one ring with ring A of compound 3a
with retention of the TPSA value (Fig. 3). The strategies of this
design are as follows: (i) introduction of substituents R3 to protect
the metabolically labile benzyl position and to add hydrophobic
interactions with Phe629 and/or Met703; (ii) introduction of sub-
stituent R2 to fill the space that is originally occupied by the pyrro-
lidine-2-one ring which interacts with Ala626; and (iii) retention of
the carbonyl group in the 2-oxindole ring to adjust the electron
density of ring A and avoid interaction of the assumed metabolic
sites with the heme moiety of CYPs.
Table 1
Optimization of substituents at 4-position of ring A
O
N
O
N
N
N
F
A
4
R1
Compound R1
PDE10A IC50
(nM)
TPSAb
(Å2)
ERc
HLMd
L/min/mg)
a
(l
∗
N
0.94
O
1
91
6.2
2.0
<1
33
(0.72–1.2)
O
1
∗
N
0.79
3a
82
65
O
(0.63–0.99)
∗
N
0.23
3b
0.56 242
(0.20–0.26)
a
IC50 values and 95% confidence intervals (given in parentheses) were calculated
by nonlinear regression analysis of percent inhibition data (n = 2). All values were
rounded to two significant digits.
b
Calculated by daylight.
MDR1 efflux ratios in P-gp-overexpressing cells.
Metabolic stability in human liver microsomes.
c
d
hydrolysis. [3H]-cGMP was used for PDE10A2 as a substrate. ERs
were evaluated in P-gp-overexpressing cells.
The results of the transformation of substituents at the
4-position in ring A are summarized in Table 1. First, we confirmed
whether the 1,3-oxazolidin-2-one moiety is related to the
P-gp-mediated efflux. Substitution of 1,3-oxazolidin-2-one with
Table 2 summarizes the profiles of the various 2-oxindole
derivatives 19a–e. The ring fusion in compound 19a resulted in
improved metabolic stability and increased PDE10A inhibitory
Figure 3. Design of the fused 2-oxindole derivatives.

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M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
Table 2
Table 3
PDE10A inhibitory activities and P-gp ERs of various 2-oxindole derivatives 19a–e
Pharmacokinetic parameters of compound 19e in micea
Plasma
349
0.50
2819
4.78
Brain
O
N
O
Cmax (ng/mL or ng/g)
Tmax (h)
46
1.0
558
6.58
0.20
N
N
N
AUC0–24h,po (ng h/mL or ng h/g)
MRTb (h)
F
c
Kpbrain
R3
a
b
c
Administered orally at 1 mg/kg (n = 3).
Mean residence time.
Brain-to-plasma AUC0–24h ratio.
R3
N
R2
O
Compound R2
R3
PDE10A
IC50 (nM)
TPSAb
(Å2)
ERc HLMd
L/min/mg)
The X-ray crystal structures of 19e with PDE10A are displayed
a
(l
in Figure 4A and B. The 2-oxindole moiety fills a large space sand-
wiched between the Phe629, Met703 and Ala626 residues, and
gem-dimethyl groups are positioned 4.4 Å from the SdMe moiety
of Met703 and 4.0 Å from the Cf atom of Phe629. The distance
between the gem-dimethyl groups and each residue suggests that
the gem-dimethyl groups have hydrophobic interactions between
the side chains of Phe629 and Met703.28 When comparing
the binding modes between 1 and 19e (Fig. 4B), we found that
compound 19e is located closer to Met703 than compound 1,
presumably reflecting the interaction with Met703. The
cyclopropylmethyl group occupies the same space as the 1,3-oxa-
zolidin-2-one of compound 1, keeping the lipophilic interaction
19a
19b
19c
19d
19e
Me
Me
0.22
(0.17–0.28)
0.13
(0.12–0.15)
0.11
(0.093–0.13)
0.12
(0.11–0.14)
0.080
(0.046–0.14)
82
82
82
82
82
4.0 <1
4.6
1.9 64
Me
iPr
–(CH2)3–
Me
8
cBu
Me
0.9 216
1.4 <1
CH2cPr Me
a
IC50 values and 95% confidence intervals (given in parentheses) were calculated
by nonlinear regression analysis of percent inhibition data (n = 2). All values were
rounded to two significant digits.
with the C and Cb atoms of Ala626 at distances of 3.7 and 3.8 Å,
b
a
Calculated by daylight.
MDR1 efflux ratios in P-gp-overexpressing cells.
Metabolic stability in human liver microsomes.
c
respectively. The highly potent PDE10A inhibitory activity of
compound 19e can be explained by the hydrophobic interactions
with Phe629, Met703, and Ala626 in addition to the known
interactions we previously reported.14 Compound 19e, which
displayed the best balance of potency, ER, and metabolic stability,
was selected for pharmacokinetic study and further evaluation in
an animal model of schizophrenia.
The results of a pharmacokinetic study of compound 19e are
shown in Table 3. Oral administration of compound 19e to mice
at 1 mg/kg resulted in good oral absorption and Kpbrain
value. Moreover, the mean residence time in the brain was
suggestive of potent and long-lasting efficacy. Subsequently, the
antipsychotic-like effect of compound 19e was assessed by the
suppression of phencyclidine (PCP)-induced hyperlocomotion,
which is commonly used as a rodent model for acute psychosis
of schizophrenia10,29 (Fig. 5). Compound 19e (0.1, 0.3, 1.0, or
3.0 mg/kg, p.o.) produced dose-dependent suppression of
PCP-induced hyperlocomotion in mice with a minimum effective
dose of 0.3 mg/kg.
d
activity versus 3a. However, compound 19a showed a higher ER
than compound 3a despite having the same TPSA value (82 Å2).
Relocation of the carbonyl group may induce an interaction with
any of the drug-binding sites on P-gp. Thus, we introduced various
bulky and lipophilic substituents that were expected to disrupt the
interaction between the carbonyl group and P-gp. Replacement of
gem-dimethyl groups with the spiro-cyclobutyl group (19b)
slightly increased PDE10A inhibitory activity but did not improve
ER. As for the substituent on the nitrogen atom of the 2-oxindole,
more bulky isopropyl (19c), cyclobutyl (19d), and cyclopropyl-
methyl (19e) groups remarkably improved ER while maintaining
the potent PDE10A inhibitory activity. Among these, compound
19e exhibited both the most potent PDE10A inhibitory activity
(IC50 = 0.080 nM) and the best metabolic stability (<1 lL/min/mg).
Figure 4. (A) X-ray crystal structure of 19e in the PDE10A catalytic domain. The hydrophobic interactions between Phe629, Met703, and Ala626 are highlighted with red
dashed lines. The bidentate interactions with Gln716, the water-mediated interaction with Tyr514, and the interactions with Phe719 and Phe686 were omitted for
simplicity. (B) Overlaid crystal structures of compounds 1 (white) and 19e (yellow) in the PDE10A catalytic domain. Phe719, Phe686, and Phe629 were omitted for simplicity.
p–p

M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
7143
of theoretical values. All of the final products undergoing biological
testing were >95% pure as demonstrated by analysis carried out
using analytical high-performance liquid chromatography (HPLC).
The HPLC analyses were performed using a Shimadzu UFLC
instrument, equipped with a L-column 2 ODS (3.0 ꢁ 50 mm,
2 lm) column, eluting with a gradient of 5–90% solvent B in sol-
vent A (solvent A was 0.1% TFA in water, and solvent B was 0.1%
TFA in acetonitrile), at a flow rate of 1.2 mL/min, with UV detection
at 220 nm. Reaction progress was determined by TLC analysis on
Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Column
chromatography was performed using silica gel (Merck Kieselgel
60, 70–230 mesh), basic silica gel (Chromatorex NH-DM 1020,
100–200 mesh, Fuji Silysia Chemical, Ltd), or Purif-Pack (SI /
60
purification was performed by using a Waters Corporation UV
purification system equipped with Develosil ODS-UG-10
(4.6 ꢁ 150 mm, 5 m) column, and eluted with a gradient of
lM or NH / 60 lM, Fuji Silysia Chemical, Ltd). Preparative HPLC
Figure 5. Effect of compound 19e on PCP-induced hyperlocomotion in mice. PCP
(5 mg/kg) was administered subcutaneously 60 min after oral administration of
compound 19e to ICR mice. Accumulated activity counts during the 120 min
following PCP treatment were calculated and are indicated as the mean SEM
(n = 6–7); #p 6 0.025 versus vehicle + PCP group by one-tailed Williams’ test.
a
l
5–90% solvent B in solvent A (solvent A was 0.1% TFA in water,
and solvent B was 0.1% TFA in acetonitrile), at a flow rate of
150 mL/min, with UV detection at 220 nm. Abbreviations of
solvents are used as follows: CDCl3, deuterated chloroform;
DMSO-d6, dimethyl sulfoxide-d6; EtOAc, ethyl acetate; DMF,
N,N-dimethylformamide; MeOH, methanol; THF, tetrahydrofuran;
EtOH, ethanol; DMSO, dimethyl sulfoxide; DMA, N,N-dimethylac-
etamide; AcOH, acetic acid; DMFDMA, N,N-dimethylformamide
dimethylacetal; MeCN, acetonitrile; DIPEA, N,N-diisopropylethy-
lamine; TEA, triethylamine.
4. Conclusion
To improve the P-gp ER of lead compound 1, we performed an
optimization study to decrease the affinity for the drug-binding
sites on P-gp while maintaining the high affinity for PDE10A by
using SBDD techniques. Replacement of the 1,3-oxazolidin-2-one
with the pyrrolidine-2-one or pyrrolidine resulted in a significant
improvement of ER without affecting the potent PDE10A inhibitory
activity, although room for improvements in metabolic stability
remains. In the course of further optimization to obtain well-
balanced PDE10A inhibitors regarding potency, ER, and metabolic
stability, the ring fusion to the 2-oxindole resulted in increased
PDE10A inhibitory activity. Finally, compound 19e was identified
with 10-fold increased PDE10A inhibitory activity (IC50 = 0.080
nM) compared to lead compound 1 and an acceptable P-gp ER
(1.4). The X-ray crystal structure of compound 19e with PDE10A
revealed that gem-dimethyl groups interact with Phe629 and
Met703 and the cyclopropyl group interacts with Ala626 in
addition to the known pharmacophore we reported previously.
Compound 19e also achieved satisfactory brain penetration
and suppressed PCP-induced hyperlocomotion with a minimum
effective dose of 0.3 mg/kg, p.o. in mice. Further pharmacological
studies of 19e will be reported in due course.
5.1. 1-[2-Fluoro-4-(2-oxopyrrolidin-1-yl)phenyl]-5-methoxy-3-
(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (3a)
A
suspension of 2 (244 mg, 0.50 mmol), 2-pyrrolidinone
(0.046 mL, 0.60 mmol), CuI (9.5 mg, 0.050 mmol), trans-1,2-
diaminocyclohexane (0.012 mL, 0.10 mmol), and K3PO4 (212 mg,
1.0 mmol) in 1,4-dioxane (2 mL) was stirred at 90 °C for 6 h under
Ar atmosphere. The mixture was poured into water and extracted
with EtOAc. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue
was purified by basic silica gel column chromatography with THF
and recrystallized from MeOH/water to give 3a (74 mg, 33%) as a
pale yellow solid. Mp 200–202 °C. 1H NMR (CDCl3) d 2.16–2.26
(2H, m), 2.62–2.68 (2H, m), 3.82–3.87 (2H, m), 3.90 (3H, s), 6.40
(1H, t, J = 9.0 Hz), 7.10 (1H, ddd, J = 9.0, 2.3, 1.1 Hz), 7.30 (1H, d,
J = 1.9 Hz), 7.35–7.45 (5H, m), 7.77–7.83 (3H, m); LC–MS (ESI)
m/z 446 (M+H)+. Anal. Calcd for C24H20FN5O3ꢂ0.25H2O: C, 64.06;
H, 4.59; N, 15.59. Found: C, 64.08; H, 4.57; N, 15.49.
5. Experimental procedure
All commercially available solvents and reagents were used
without further purification. Yields were not optimized. Melting
points were determined on a Büchi melting point apparatus B-
545 or an OptiMelt melting point apparatus MPA100 and were
not corrected. 1H NMR spectra were recorded on Varian Mer-
cury-300 (300 MHz) or Bruker DPX300 (300 MHz) instruments.
Chemical shifts are reported as d values (ppm) downfield from
internal TMS of the indicated organic solution. Peak multiplicities
are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet;
dd, doublet of doublets; td, triplet of doublets; ddd, doublet of dou-
blet of doublets; quin, quintet; br s, broad singlet; m, multiplet.
Coupling constants (J values) are given in hertz (Hz). LC–MS was
performed on a Waters liquid chromatography–mass spectrometer
system, using a CAPCELL PAK UG-120 ODS column (2.0 mm
i.d. ꢁ 50 mm, Shiseido Co., Ltd) with a 5–95% gradient of CH3CN
in water containing 0.04% TFA and an HP-1100 (Agilent Technolo-
gies) apparatus for monitoring at 220 nm. Elemental analyses were
carried out by Takeda Analytical Laboratories and Sumika
Chemical Analysis Service, Ltd, and the results were within 0.4%
5.2.
1-[2-Fluoro-4-(pyrrolidin-1-yl)phenyl]-5-methoxy-3-(1-
phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (3b)
A suspension of 2 (244 mg, 0.50 mmol), pyrrolidine (0.050 mL,
0.60 mmol), Pd2(dba)3 (18 mg, 0.020 mmol), Xantphos (0.046 mg,
0.080 mmol), and Nat-OBu (67 mg, 0.70 mmol) in 1,4-dioxane
(2.5 mL) was stirred at 90 °C for 2 h under Ar atmosphere. The mix-
ture was poured into water and extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by basic silica gel
column chromatography with hexane/EtOAc and recrystallized
from EtOAc to give 3b (106 mg, 49%) as a yellow solid. 1H NMR
(CDCl3) d 1.89–2.03 (4H, m), 3.16–3.30 (4H, m), 3.76 (3H, s), 6.29
(1H, dd, J = 8.7, 2.6 Hz), 6.44 (1H, dd, J = 14.4, 2.6 Hz), 6.78–6.88
(1H, m), 6.91 (1H, d, J = 1.9 Hz), 7.26–7.48 (5H, m), 7.77 (1H, d,
J = 1.5 Hz), 8.33 (1H, d, J = 1.9 Hz); LC–MS (ESI) m/z 432 (M+H)+.
Anal. Calcd for C24H22FN5O2: C, 66.81; H, 5.14; N, 16.23. Found:
C, 66.89; H, 5.20; N, 16.16.

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M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
5.3. Diethyl (2-fluoro-6-nitrophenyl)malonate (5)
mixture of 2,3-difluoronitrobenzene (10.0 g, 62.9 mmol),
(1H, m), 2.37–2.54 (3H, m), 2.71–2.86 (2H, m), 3.77 (3H, s),
7.28–7.41 (2H, m), 7.52–7.58 (1H, m). LC–MS (ESI) m/z 253 (M)+.
A
diethyl malonate (10.5 mL, 69.1 mmol), and K2CO3 (16 g,
113 mmol) in DMF (200 mL) was stirred at 65 °C overnight. The
mixture was acidified with 6 M HCl aq (50 mL) and extracted with
EtOAc. The organic layer was washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography with hexane/EtOAc
(7:3) to give 5 (16.9 g, 90%) as a pale yellow oil. 1H NMR (CDCl3)
d 1.28 (6H, t, J = 7.2 Hz), 4.27 (4H, q, J = 7.2 Hz), 5.23 (1H, d,
J = 1.1 Hz), 7.39–7.47 (1H, m), 7.48–7.57 (1H, m), 7.87–7.91
(1H, m).
5.8. 4-Fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one (9a)
A mixture of 8a (4.2 g, 17.5 mmol) and zinc (25 g, 382 mmol) in
AcOH (40 mL) was stirred at 100 °C for 2 days. The mixture was fil-
tered, and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography with
hexane/EtOAc (8:2) to give 9a (2.9 g, 93%) as a white solid. 1H NMR
(CDCl3) d 1.51 (6H, s), 6.65–6.78 (2H, m), 7.11–7.22 (1H, m), 8.84
(1H, br s). LC–MS (ESI) m/z 180 (M+H)+.
5.9. 40-Fluorospiro[cyclobutane-1,30-indol]-20(10H)-one (9b)
5.4. (2-Fluoro-6-nitrophenyl)acetic acid (6)
A mixture of 8b (11.3 g, 44.6 mmol) and zinc (58.4 g, 892 mmol)
in AcOH (150 mL) was stirred at 100 °C for 4 h. The mixture was fil-
tered, and the filtrate was concentrated under reduced pressure.
The residue was dissolved in EtOAc and washed with saturated
NaHCO3 aqueous solution. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography with hexane/EtOAc
(7:3) to give 9b (8.0 g, 94%) as a white solid. 1H NMR (CDCl3) d
2.28–2.43 (2H, m), 2.58–2.67 (4H, m), 6.64–6.81 (2H, m), 7.16
(1H, td, J = 8.1, 5.7 Hz), 8.34 (1H, br s). LC–MS (ESI) m/z 192 (M+H)+.
A mixture of 5 (16.9 g, 56.5 mmol), 6 M HCl aq (200 mL), and
AcOH (100 mL) was refluxed for 6 h. The mixture was concentrated
under reduced pressure. The residue was dissolved in EtOAc and
washed with brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure to give 6 (10.8 g, 96%) as a
pale yellow solid. 1H NMR (CDCl3) d 3.92 (2H, d, J = 1.5 Hz),
7.54–7.77 (2H, m), 7.88–8.00 (1H, m), 12.77 (1H, br s).
5.5. Methyl (2-fluoro-6-nitrophenyl)acetate (7)
5.10.
one (10a)
4-Fluoro-3,3-dimethyl-5-nitro-1,3-dihydro-2H-indol-2-
To a solution of 6 (10.8 g, 54.2 mmol) in MeOH (200 mL) was
added dropwise thionyl chloride (5.9 mL, 81 mmol) at 0 °C. After
stirring at rt for 3 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in EtOAc and filtered through
basic silica gel. The filtrate was concentrated under reduced pres-
sure to give 7 (11.6 g, 100%) as a colorless oil. 1H NMR (CDCl3) d
3.74 (3H, s), 4.07 (2H, d, J = 1.1 Hz), 7.36–7.52 (2H, m), 7.90–7.95
(1H, m).
A solution of nitric acid (1.0 mL, 23.9 mmol) in sulfuric acid
(10 mL) was added dropwise to a solution of 9a (4.1 g, 22.7 mmol)
in sulfuric acid (60 mL) at ꢀ30 °C, and the mixture was stirred at
ꢀ30 °C to 0 °C for 30 min. The mixture was poured onto ice. The
formed precipitate was collected by filtration, washed with water,
and dissolved in EtOAc. The organic solution was washed with
brine, dried over MgSO4, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with hexane/EtOAc (1:1) to give 10a (2.2 g, 43%) as an
off-white solid. 1H NMR (CDCl3) d 1.57 (6H, s), 6.84 (1H, d,
J = 8.7 Hz), 8.10 (1H, dd, J = 8.7, 7.5 Hz), 8.76 (1H, br s).
5.6. Methyl 2-(2-fluoro-6-nitrophenyl)-2-methylpropanoate
(8a)
To a solution of 7 (5.0 g, 23.5 mmol) in DMF (75 mL) was added
portionwise NaH (2.3 g, 58.6 mmol) at 0 °C, and the mixture was
stirred at 0 °C for 5 min. To the mixture was added dropwise a
solution of iodomethane (7.3 mL, 117 mmol) in THF (50 mL) at
0 °C. After stirring at 0 °C for 2 h, the mixture was allowed to warm
to rt and stirred at rt overnight. The mixture was quenched with
water and extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with hexane/EtOAc (9:1) to give 8a (4.3 g, 76%) as a pale yel-
low oil. 1H NMR (CDCl3) d 1.69 (3H, s), 1.70 (3H, s), 3.67 (3H, s),
7.21–7.45 (3H, m).
5.11.
one (10b)
40-Fluoro-50-nitrospiro[cyclobutane-1,30-indol]-20(10H)-
A solution of nitric acid (1.8 mL, 43.9 mmol) in sulfuric acid
(15 mL) was added dropwise to a solution of 9b (8.0 g, 41.8 mmol)
in sulfuric acid (80 mL) at ꢀ40 °C, and the mixture was warmed up
to rt. After stirring for 4 h, the mixture was poured onto ice and
extracted with EtOAc. The organic layer was washed with satu-
rated NaHCO3 aqueous solution and brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was dissolved
in THF and the solution was filtered through basic silica gel. The fil-
trate was concentrated under reduced pressure. The residue was
recrystallized from EtOAc/hexane to give 10b (8.0 g, 81%) as a
white solid. 1H NMR (300 MHz, DMSO-d6) d 2.20–2.34 (2H, m),
2.39–2.67 (4H, m), 6.80–6.90 (1H, m), 8.05–8.16 (1H, m), 11.13
(1H, br s).
5.7. Methyl 1-(2-fluoro-6-nitrophenyl)cyclobutanecarboxylate
(8b)
A solution of 7 (7.9 g, 37 mmol) in DMF (50 mL) was added
dropwise to
a suspension of NaH (3.3 g, 81 mmol) in DMF
(150 mL) at 0 °C, and the mixture was stirred at 0 °C for 15 min.
To the mixture was added dropwise a solution of 1,3-dibromo-
propane (4.5 mL, 44 mmol) in THF (10 mL) at 0 °C. After stirring
at 0 °C for 3 h, the mixture was neutralized with saturated NH4Cl
aqueous solution at 0 °C and extracted with EtOAc. The organic
layer was washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography with hexane/EtOAc (9:1) to
give 8b (3.1 g, 33%) as a white solid. 1H NMR (CDCl3) d1.75–1.91
5.12. 40-Fluoro-10-methyl-50-nitrospiro[cyclobutane-1,30-indol]-
20(10H)-one (11b)
To a mixture of 10b (300 mg, 1.27 mmol) and NaH (37 mg,
1.52 mmol) in DMF (10 mL) was added iodomethane (0.395 mL,
6.35 mmol) at 0 °C. After stirring at rt for 1 h, the mixture was
neutralized with saturated NH4Cl aqueous solution at 0 °C and
extracted with EtOAc. The organic layer was washed with water

M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
7145
and brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with hexane/EtOAc (3:2) to give 11b (282 mg, 89%) as a
white solid. 1H NMR (CDCl3) d 2.34–2.49 (2H, m), 2.56–2.75 (4H,
m), 3.24 (3H, s), 6.68 (1H, d, J = 8.7 Hz), 8.14 (1H, dd, J = 8.7,
7.5 Hz). LC–MS (ESI) m/z 250 (M+H)+.
1.40–1.47 (12H, m), 3.53 (2H, br s), 4.58 (1H, quin, J = 7.1 Hz),
6.57–6.70 (2H, m).
5.19. 5-Amino-1-cyclobutyl-4-fluoro-3,3-dimethyl-1,3-dihydro-
2H-indol-2-one (12d)
Compound 12d was obtained (86%) as a white solid in a manner
similar to that described for compound 12a. LC–MS (ESI) m/z 249
(M+H)+.
5.13. 4-Fluoro-1-isopropyl-3,3-dimethyl-5-nitro-1,3-dihydro-
2H-indol-2-one (11c)
Compound 11c was obtained (62%) as colorless crystals in a
manner similar to that described for compound 11b. 1H NMR
(CDCl3) d 1.47–1.52 (12H, m), 4.62 (1H, dt, J = 14.1, 7.1 Hz), 6.87
(1H, d, J = 9.1 Hz), 8.11 (1H, dd, J = 8.9, 7.7 Hz).
5.20.
5-Amino-1-(cyclopropylmethyl)-4-fluoro-3,3-dimethyl-
1,3-dihydro-2H-indol-2-one (12e)
Compound 12e was obtained (94%) as a white solid in a manner
similar to that described for compound 12a. 1H NMR (CDCl3) d
0.30–0.40 (2H, m), 0.44–0.55 (2H, m), 1.07–1.19 (1H, m), 1.43–
1.48 (6H, m), 3.41–3.67 (4H, m), 6.53 (1H, d, J = 7.9 Hz), 6.64–
6.72 (1H, m). LC–MS (ESI) m/z 249 (M+H)+.
5.14. 1-Cyclobutyl-4-fluoro-3,3-dimethyl-5-nitro-1,3-dihydro-
2H-indol-2-one (11d)
Diethyl azodicarboxylate in toluene solution (1.48 mL,
3.75 mmol) was added dropwise to a solution of 10a (300 mg,
1.34 mmol), cyclobutanol (0.136 mL, 1.74 mmol) and triph-
enylphosphine (702 mg, 2.68 mmol) in THF (15 mL) at 0 °C. After
stirring at rt under N2 atmosphere for 5 h, the mixture was
quenched with water at 0 °C and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography with hexane/EtOAc (1:1) to give 11d
(200 mg, 54%) as a colorless oil. LC–MS (ESI) m/z 279 (M+H)+.
5.21. Methyl 2-[(4-fluoro-3,3-dimethyl-2-oxo-2,3-dihydro-1H-
indol-5-yl)hydrazono]-4-methoxy-3-oxobutanoate (13)
A solution of sodium nitrite (0.081 g, 1.17 mmol) in water
(1 mL) was added dropwise to
a solution of 12a (0.19 g,
0.978 mmol) in 6 M HCl aq (2 mL) at 0 °C. After stirring at 0 °C
for 15 min, the mixture was added to a suspension of methyl
4-methoxyacetoacetate (0.127 mL, 0.978 mmol) and sodium acet-
ate (1.0 g, 12.19 mmol) in MeOH (5 mL) at 0 °C. The formed precip-
itate was dissolved in EtOAc, and the solution was washed with
water and saturated NaHCO3 aqueous solution. The organic layer
was dried over MgSO4, and concentrated under reduced pressure
to give 13 (0.25 g, 73%) as a yellow solid. 1H NMR (CDCl3) d 1.54
(6H, s), 3.51 (3H, s), 3.88 (3H ꢁ 1/2, s), 3.93 (3H ꢁ 1/2, s), 4.66
(2H ꢁ 1/2, s), 4.69 (2H ꢁ 1/2, s), 6.78 (1H, d, J = 8.3 Hz), 7.48
(1H ꢁ 1/2, t, J = 7.9 Hz), 7.72 (1H ꢁ 1/2, t, J = 8.1 Hz), 7.88
(1H ꢁ 1/2, br s), 7.92 (1H ꢁ 1/2, br s), 13.18 (1H ꢁ 1/2, s), 15.12
(1H ꢁ 1/2, s).
5.15. 1-(Cyclopropylmethyl)-4-fluoro-3,3-dimethyl-5-nitro-1,3-
dihydro-2H-indol-2-one (11e)
Compound 11e was obtained (85%) as a colorless oil in a man-
ner similar to that described for compound 11b. 1H NMR (CDCl3) d
0.35–0.43 (2H, m), 0.52–0.61 (2H, m), 1.08–1.20 (1H, m), 1.53 (6H,
s), 3.63 (2H, d, J = 6.8 Hz), 6.81 (1H, d, J = 8.7 Hz), 8.14 (1H, dd,
J = 8.7, 7.5 Hz). LC–MS (ESI) m/z 279 (M+H)+.
5.16. 5-Amino-4-fluoro-3,3-dimethyl -1,3-dihydro-2H-indol-2-
one (12a)
5.22. Methyl 1-(4-fluoro-1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-
indol-5-yl)-5-methoxy-4-oxo-1,4-dihydropyridazine-3-
carboxylate (14)
A mixture of 10a (0.23 g, 1.03 mmol) and 5% Pd/C (0.15 g) in
MeOH (20 mL) was stirred at rt overnight under H2 atmosphere.
The catalyst was removed by filtration, and the filtrate was concen-
trated under reduced pressure to give 12a (0.19 g, 95%) as a pale
red solid. 1H NMR (CDCl3) d 1.49 (6H, s), 3.52 (2H, br s), 6.52 (1H,
d, J = 8.3 Hz), 6.64 (1H, t, J = 8.3 Hz), 8.18 (1H, br s). LC–MS (ESI)
m/z 195 (M+H)+.
A mixture of 13 (4.02 g, 11.44 mmol) and DMFDMA (8.0 mL,
53.8 mmol) in MeCN (40 mL) was refluxed overnight. The mixture
was concentrated under reduced pressure. The residue was puri-
fied by basic silica gel column chromatography with EtOAc/MeOH
(95:5) to give 14 (2.40 g, 56%) as pale yellow crystals. 1H NMR
(CDCl3) d 1.53 (6H, s), 3.27 (3H, s), 3.93 (3H, s), 3.98 (3H, s), 6.78
(1H, d, J = 8.3 Hz), 7.52 (1H, dd, J = 8.3, 7.5 Hz), 7.72 (1H, d,
J = 2.3 Hz).
5.17.
50-Amino-40-fluoro-10-methylspiro[cyclobutane-1,30-
indol]-20(10H)-one (12b)
5.23. 1-(4-Fluoro-1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-
5-yl)-5-methoxy-4-oxo-1,4-dihydropyridazine-3-carboxylic
acid (15)
Compound 12b was obtained (63%) as a white solid in a manner
similar to that described for compound 12a. 1H NMR (CDCl3) d
2.27–2.43 (2H, m), 2.50–2.69 (4H, m), 3.13 (3H, s), 3.57 (2H, br
s), 6.39 (1H, d, J = 8.3 Hz), 6.64–6.73 (1H, m). LC–MS (ESI) m/z
221 (M+H)+.
To a solution of 14 (2.60 g, 6.93 mmol) in MeOH (30 mL) was
added 1 M NaOH aq (14 mL, 14 mmol). After stirring at rt for 2 h,
the mixture was acidified with 1 M HCl aq, and partitioned
between EtOAc and water. The organic layer was dried over MgSO4,
and concentrated under reduced pressure to give 15 (2.50 g, 100%)
as a colorless oil. 1H NMR (DMSO-d6) d 1.43 (6H, s), 3.22 (3H, s),
3.90 (3H, s), 7.15 (1H, d, J = 8.3 Hz), 7.75 (1H, t, J = 8.1 Hz), 8.88
(1H, s).
5.18. 5-Amino-4-fluoro-1-isopropyl-3,3-dimethyl-1,3-dihydro-
2H-indol-2-one (12c)
Compound 12c was obtained (79%) as a white solid in a manner
similar to that described for compound 12a. 1H NMR (CDCl3) d

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M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
5.24. 1-(4-Fluoro-1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-
5-yl)-N,5-dimethoxy-N-methyl-4-oxo-1,4-dihydropyridazine-3-
carboxamide (16)
chromatography with hexane/EtOAc to give 21 (12.20 g, 51%) as
a light brown oil. 1H NMR (CDCl3) d 2.33 (1H, d, J = 4.1 Hz), 2.88
(2H, dd, J = 6.6, 3.2 Hz), 3.19–3.28 (1H, m), 3.33 (3H, s), 3.34–3.40
(1H, m), 3.94–4.06 (1H, m), 6.30–6.38 (1H, m), 7.34–7.53 (5H,
m), 7.63 (1H, s).
To a suspension of 15 (0.50 g, 1.38 mmol) in THF (10 mL) was
added 1,10-carbonyldiimidazole (0.247 g, 1.52 mmol) at rt, and
the mixture was stirred at 50 °C for 2 h. To the mixture were added
N,O-dimethylhydroxylamine hydrochloride (0.20 g, 2.08 mmol)
and DIPEA (0.363 mL, 2.08 mmol) at rt. After stirring at rt over-
night, the mixture was acidified with 1 M HCl aq and partitioned
between EtOAc and water. The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure
to give 16 (0.34 g, 61%) as a yellow solid. 1H NMR (CDCl3) d 1.52
(6H, s), 3.26 (3H, s), 3.39 (3H, s), 3.73 (3H, s), 3.93 (3H, s), 6.76
(1H, d, J = 8.3 Hz), 7.52 (1H, t, J = 7.9 Hz), 7.75 (1H, d, J = 1.9 Hz).
5.29. 1-Methoxy-3-(1-phenyl-1H-pyrazol-5-yl)acetone (22)
To a solution of DMSO (14.2 mL, 200 mmol) in THF (100 mL)
was added trifluoroacetic anhydride (8.5 mL, 60 mmol) at ꢀ42 to
ꢀ46 °C over 15 min. The mixture was stirred at ꢀ46 °C for
15 min. To the solution was added a solution of 21 (9.3 g, 40 mmol)
in THF (66 mL) dropwise at ꢀ42 to ꢀ46 °C over 1 h, and the mix-
ture was stirred at 0 °C for 15 min. To the mixture was added
TEA (22.3 ml, 160 mmol) dropwise at 0–10 °C over 15 min, and
the mixture was stirred at 0 °C for 1 h. The mixture was quenched
with 10% Na2CO3 aqueous solution (160 mL) and extracted with
EtOAc. The organic layer was concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy with hexane/EtOAc to give 22 (4.0 g, 43%) as a light brown
oil. 1H NMR (CDCl3) d 3.33 (3H, s), 3.88 (2H, s), 3.95 (2H, s), 6.34
(1H, d, J = 1.9 Hz), 7.35–7.52 (5H, m), 7.66 (1H, d, J = 1.9 Hz).
5.25. 5-(3-Acetyl-5-methoxy-4-oxopyridazin-1(4H)-yl)-4-fluoro-
1,3,3-trimethyl-1,3-dihydro-2H-indol-2-one (17)
To a solution of 16 (0.34 g, 0.841 mmol) in THF (10 mL) was
added dropwise methylmagnesium bromide in THF (1 mol/L,
4.0 mL, 4.00 mmol) at ꢀ78 °C, and the mixture was stirred at
ꢀ78 °C for 2 h. The mixture was quenched with 1 M HCl aq at
ꢀ78 °C, and partitioned between EtOAc and water. The organic
layer was dried over MgSO4 and concentrated under reduced pres-
sure to give 17 (0.20 g, 66%) as a yellow solid. 1H NMR (CDCl3) d
1.53 (6H, s), 2.69 (3H, s), 3.27 (3H, s), 3.93 (3H, s), 6.80 (1H, d,
J = 8.3 Hz), 7.53 (1H, t, J = 7.9 Hz), 7.72 (1H, d, J = 2.3 Hz).
5.30. 40-Fluoro-50-{2-[3-methoxy-2-oxo-1-(1-phenyl-1H-pyra-
zol-5-yl)propylidene]hydrazino}-10-methylspiro[cyclobutane-
1,30-indol]-20(10H)-one (23b)
Compound 23b was obtained by the reaction of 12b with 22 in a
manner similar to that described for compound 13. This product
was used in a next step without further purification. LC–MS (ESI)
m/z 462 (M+H)+.
5.26.
5-{3-[3-(Dimethylamino)prop-2-enoyl]-5-methoxy-4-
oxopyridazin-1(4H)-yl}-4-fluoro-1,3,3-trimethyl-1,3-dihydro-
2H-indol-2-one (18)
5.31. 4-Fluoro-1-isopropyl-5-{2-[3-methoxy-2-oxo-1-(1-phenyl-
1H-pyrazol-5-yl)propylidene]hydrazino}-3,3-dimethyl-1,3-
dihydro-2H-indol-2-one(23c)
A mixture of 17 (0.20 g, 0.557 mmol) and DMFDMA (1.0 mL,
7.47 mmol) in MeCN (5 mL) was refluxed for 1 h. The mixture
was concentrated under reduced pressure to give 18 as a brown
oil. This product was used in
purification.
a
next step without further
Compound 23c was obtained by the reaction of 12c with 22 in a
manner similar to that described for compound 13. This product
was used in a next step without further purification. LC–MS (ESI)
m/z 478 (M+H)+.
5.27. 4-Fluoro-5-[5-methoxy-4-oxo-3-(1-phenyl-1H-pyrazol-5-
yl)pyridazin-1(4H)-yl]-1,3,3-trimethyl-1,3-dihydro-2H-indol-2-
one (19a)
5.32.
1-Cyclobutyl-4-fluoro-5-{2-[3-methoxy-2-oxo-1-(1-
phenyl-1H-pyrazol-5-yl)propylidene]hydrazino}-3,3-dimethyl-
1,3-dihydro-2H-indol-2-one (23d)
To a suspension of 18 in EtOH (10 mL) was added dropwise a
solution of phenylhydrazine (0.061 mL, 0.613 mmol) in 10% TFA/
EtOH (10 mL) at 0 °C. After stirring at rt overnight, the mixture
was concentrated under reduced pressure. The residue was puri-
fied by basic silica gel column chromatography with EtOAc/MeOH
(95:5) and recrystallized from EtOAc/heptane to give 19a (70 mg,
27% in 2 steps) as a pale yellow solid. Mp 236–238 °C. 1H NMR
(DMSO-d6) d 1.37 (6H, s), 3.17 (3H, S), 3.78 (3H, s), 6.92–6.96
(2H, m), 7.14 (1H, t, J = 8.1 Hz), 7.28–7.47 (5H, m), 7.78 (1H, d,
J = 1.5 Hz), 8.45 (1H, d, J = 1.5 Hz). LC–MS (ESI) m/z 460 (M+H)+.
Anal. Calcd for C25H22FN5O3: C, 65.35; H, 4.83; N, 15.24. Found:
C, 65.22; H, 4.86; N, 15.19.
Compound 23d was obtained by the reaction of 12d with 22 in a
manner similar to that described for compound 13. This product
was used in a next step without further purification.
5.33. 1-(Cyclopropylmethyl)-4-fluoro-5-{2-[3-methoxy-2-oxo-
1-(1-phenyl-1H-pyrazol-5-yl)propylidene]hydrazino}-3,3-dime-
thyl-1,3-dihydro-2H-indol-2-one (23e)
Compound 23e was obtained by the reaction of 12e with 22 in a
manner similar to that described for compound 13. This product
was used in a next step without further purification. LC–MS (ESI)
m/z 490 (M+H)+.
5.28. 1-Methoxy-3-(1-phenyl-1H-pyrazol-5-yl)propan-2-ol (21)
To a solution of 20 (13.75 mL, 104 mmol) in THF (450 ml) was
added dropwise 1.6 M n-butyllithium in hexane (98 mL,
156 mmol) at ꢀ78 °C. The mixture was stirred at ꢀ78 °C for 1 h.
To the mixture was added 2-(methoxymethyl)oxirane (27.8 mL,
312 mmol) at ꢀ78 °C, and the mixture was stirred at rt for 1 h.
The mixture was neutralized with 1 M HCl aq (150 mL) and
extracted with EtOAc. The organic layer was concentrated under
reduced pressure. The residue was purified by silica gel column
5.34. 40-Fluoro-50-[5-methoxy-4-oxo-3-(1-phenyl-1H-pyrazol-5-
yl)pyridazin-1(4H)-yl]-10-methylspiro[cyclobutane-1,30-indol]-
20(10H)-one (19b)
Compound 19b was obtained (35%) as an off-white solid in a
manner similar to that described for compound 14. 1H NMR
(DMSO-d6) d 2.19–2.32 (2H, m), 2.40–2.60 (4H, m), 3.14 (3H, s),
3.79 (3H, s), 6.87 (1H, d, J = 8.3 Hz), 6.97 (1H, d, J = 1.5 Hz), 7.08

M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
7147
Table 4
(1H, t, J = 7.9 Hz), 7.29–7.47 (5H, m), 7.79 (1H, d, J = 1.9 Hz), 8.49
(1H, d, J = 1.5 Hz). LC–MS (ESI) m/z 472 (M+H)+. Anal. Calcd for
26H22FN5O3: C, 66.23; H, 4.70; N, 14.85. Found: C, 66.27; H,
4.93; N, 14.59.
X-ray data collection and refinement statistics
19e*
C
1
Data collection
X-ray source
Wavelength (Å)
Space group
ALS BL5.0.3
ALS BL5.0.3
0.97645
P212121
a = 49.7,
b = 81.8,
c = 159.8
1.77
0.97645
P212121
a = 45.8,
b = 81.8,
c = 159.7
1.95
5.35.
4-Fluoro-1-isopropyl-5-[5-methoxy-4-oxo-3-(1-phenyl-
1H-pyrazol-5-yl)pyridazin-1(4H)-yl]-3,3-dimethyl-1,3-dihydro-
2H-indol-2-one (19c)
Unit cell dimensions (Å)
Compound 19c was obtained (35%) as an off-white solid in a
manner similar to that described for compound 14. Mp 225–
226 °C. 1H NMR (DMSO-d6) d 1.43–1.49 (12H, m), 3.91 (3H, s),
4.58 (1H, quin, J = 7.0 Hz), 6.34–6.44 (1H, m), 6.61 (1H, d,
J = 8.7 Hz), 7.24 (1H, d, J = 1.9 Hz), 7.34–7.48 (5H, m), 7.72 (1H, d,
J = 2.3 Hz), 7.78 (1H, d, J = 1.9 Hz). LC–MS (ESI) m/z 488 (M+H)+.
Anal. Calcd for C27H26FN5O3: C, 66.52; H, 5.38; N, 14.37. Found:
C, 66.45; H, 5.36; N, 14.31.
Resolution (Å)
Unique reflections
Redundancy
45,993
6.5
59,305
6.4
Completeness (%)
95.0 (58.6)
13.2 (1.4)
0.077
92.4 (91.4)
11.5 (1.7)
0.071
I/r(I)
a
Rsym
Refinement
Reflections used
RMS bonds (Å)
RMS angles (°)
Average B value (Å2)
R-valueb
43,643
0.008
1.210
38.9
0.179
0.234
56,318
0.007
1.172
36.4
0.173
0.211
5.36. 1-Cyclobutyl-4-fluoro-5-[5-methoxy-4-oxo-3-(1-phenyl-
1H-pyrazol-5-yl)pyridazin-1(4H)-yl)-3,3-dimethyl-1,3-dihydro-
2H-indol-2-one (19d)
b
Rfree
a
Rsym
=
R
h
R
j|<I(h)> ꢀ I(h)j|/
R
h
R
j<I(h)>, where <I(h)> is the mean intensity of
symmetry-related reflections.
b
Compound 19d was obtained (6%) as a white solid in a manner
similar to that described for compound 14. 1H NMR (CDCl3) d 1.44
(6H, s), 1.80–2.02 (2H, m), 2.29–2.45 (2H, m), 2.69–2.87 (2H, m),
3.91 (3H, s), 4.66 (1H, quin, J = 8.9 Hz), 6.43 (1H, t, J = 8.3 Hz),
6.68 (1H, d, J = 8.7 Hz), 7.25 (1H, d, J = 1.9 Hz), 7.35–7.46 (5H, m),
7.72 (1H, d, J = 2.3 Hz), 7.78 (1H, d, J = 1.9 Hz). LC–MS (ESI) m/z
500 (M+H)+.
R-value =
R
||Fobs| ꢀ |Fcalc||/
R|Fobs|. Rfree for 5% of reflections excluded from
refinement. Values in parentheses are for the highest resolution shell.
*
Crystal structure in complex with 19e was obtained by cross-soaking of 1 and
19e. In only monomer A of two monomers in asymmetric unit, 1 was replaced with
19e.
building with WinCoot.33 The dictionary files for the inhibitors
were prepared using AFITT (OpenEye Scientific Software). Crystal-
lographic processing and refinement statistics are summarized in
Table 4. The coordinates and structure factors have been deposited
in PDB with accession codes 5AXP and 5AXQ for 1 and 19e,
respectively.
5.37. 1-(Cyclopropylmethyl)-4-fluoro-5-[5-methoxy-4-oxo-3-(1-
phenyl-1H-pyrazol-5-yl)pyridazin-1(4H)-yl)-3,3-dimethyl-1,3-
dihydro-2H-indol-2-one (19e)
Compound 19e was obtained (42%) as a white solid in a manner
similar to that described for compound 14. Mp 175–176 °C. 1H
NMR (CDCl3) d 0.33–0.42 (2H, m), 0.50–0.59 (2H, m), 1.04–1.17
(1H, m), 1.47 (6H, s), 3.59 (2H, d, J = 7.2 Hz), 3.92 (3H, s), 6.37–
6.45 (1H, m), 6.52–6.58 (1H, m), 7.35–7.47 (6H, m), 7.73 (1H, d,
J = 2.3 Hz), 7.78 (1H, d, J = 1.9 Hz). LC–MS (ESI) m/z 500 (M+H)]+.
Anal. Calcd for C28H26FN5O3: C, 67.32; H, 5.25; N, 14.02. Found:
C, 67.26; H, 5.36; N, 13.84.
5.39. Enzyme assay protocol
Human PDE10A2 was generated from COS-7 cells transfected
with the full-length gene. The enzyme was stored at ꢀ70 °C until
use. PDE10A activity was measured using a SPA (PerkinElmer).
To evaluate the inhibitory activity, 10
pounds were incubated with 20 L of PDE enzyme in assay buf-
lL of serial diluted com-
l
fer (50 mM HEPES–NaOH, 8.3 mM MgCl2, 1.7 mM EGTA, 0.1% BSA
(pH 7.4)) for 30 min at rt. Final concentration of DMSO in the
assay was 1% as compounds were tested in duplicate or tripli-
cate in 96-well half-area plates (Corning). To start the reaction,
5.38. Crystallization and structure determination
Human PDE10A catalytic domain for crystallographic study was
prepared as previously described.14 Crystals were obtained using
the sitting-drop vapor-diffusion method by mixing 50 nL of protein
solution (5–10 mg/mL PDE10A catalytic domain in TBS (pH 7.4),
0.5 mM DTT, 1 mM EDTA, and 10% glycerol) and 50 nL of reservoir
solution containing 0.1 M HEPES (pH 7.5–8.0), 24–32% PEG 3350,
and 200 mM MgCl2 at 4 °C. Complex crystals for compound 1 were
obtained by immersing apo crystals into the corresponding reser-
voir solution containing 0.2 mM inhibitor for 40 h. As for complex
crystals with compound 19e, apo crystals were soaked into 0.2 mM
compound 1 solution for 9 h and subsequently soaked in 0.1 mM
compound 19e solution for 15 h. Prior to data collection, complex
crystals were treated with the reservoir solution containing 25%
ethylene glycol as a cryoprotectant and were flash-frozen in liquid
nitrogen. Diffraction data were collected from a single crystal at
the Advanced Light Source beamline 5.0.3 (Berkeley, CA) and pro-
cessed using the program HKL2000.30 The structures were deter-
mined by molecular replacement using MOLREP,31 utilizing the
previously reported coordinate of PDE10A with the PDB accession
code 3WYM.14 Subsequently, the structures were refined through
an iterative procedure utilizing REFMAC,32 followed by model
10
added for a final assay volume of 40
at rt, 20 L of 20 mg/mL yttrium SPA beads containing ZnSO4
l
L of substrate [3H]cGMP (PerkinElmer) for PDE10A2 was
lL. After 60 min incubation
l
was added to terminate the PDE reaction. After being settled
for more than 120 min, the assay plate was counted in a scintil-
lation counter (PerkinElmer) to allow calculation of inhibition
rate. Inhibition rate was calculated on the basis of 0% control
wells with DMSO and 100% control wells with enzyme plus
10 lM papaverine (Wako Pure Chemical Industries) for PDE10A2.
IC50 values were determined by the least squares non-linear
regression with logistic. The calculation was carried out using
XLfit software (IDBS, Guildford, UK).
5.40. Metabolic clearance assay
In vitro oxidative metabolic studies of the tested compounds
were carried out using hepatic microsomes obtained from humans.
The reaction mixture with a final volume of 0.05 mL consists of
0.2 mg/mL hepatic microsome in 50 mM KH2PO4–K2HPO4

7148
M. Yoshikawa et al. / Bioorg. Med. Chem. 23 (2015) 7138–7149
phosphate buffer (pH 7.4) and 1
l
M test compound. The reaction
water were provided ad libitum. Mice used in the experiments
were 7 weeks old and had completed an acclimation period of at
least 1 week. The care and use of the animals and the experimental
protocols used in this research were approved by the Experimental
Animal Care and Use Committee of Takeda Pharmaceutical Com-
pany Limited.
was initiated by the addition of an NADPH-generating system
containing 50 mM MgCl2, 50 mM glucose 6-phosphate, 5 mM
b-NADP+, and 15 unit/mL glucose 6-phosphate dehydrogenase at
10% volume of reaction mixture. After the addition of the
NADPH-generating system, the mixture was incubated at 37 °C
for 0, 15, and 30 min. The reaction was terminated by the addition
of an equivalent volume of CH3CN. After the samples were mixed
and centrifuged, the supernatant fractions were analyzed using liq-
uid chromatography tandem mass spectrometry. For metabolic
clearance determinations, chromatograms were analyzed for par-
ent compound disappearance rate from the reaction mixtures. All
incubations were made in duplicate.
5.43.2. Measurements
Compound 19e was suspended in 0.5% (w/v) methylcellulose in
distilled water. Compound 19e was administered orally (p.o.). PCP
hydrochloride (Lot No. 010M4010) purchased from Sigma Aldrich,
Inc. (USA) was dissolved in saline and was administered (s.c.). All
compounds were dosed in a volume of 20 mL/kg body weight.
Hyperlocomotion was measured using
a spontaneous motor
5.41. Transcellular transport study using transporter-
expression system
analyzer MDC system (BrainScience Idea. Co. Ltd, Japan). Mice were
placed in locomotor chambers for more than 60 min for
habituation. Animals were removed from each chamber and
treated with either vehicle or compound 19e, and then quickly
returned to the chamber. The following doses were used: 0.1, 0.3,
1.0, and 3.0 mg/kg, p.o. Sixty minutes after treatment with com-
pound 19e, animals were again removed from the chambers and
treated with either vehicle (saline) or PCP (5 mg/kg as a salt, s.c.),
and then quickly transferred to the test chamber. Activity counts
were recorded in successive 1-min bins and the total number
counts were determined for the 120 min period after PCP
administration.
Human MDR1-expressing LLC-PK1 cells were cultured with
minor modification as reported previously.34 The transcellular
transport study was performed as reported previously.35 In brief,
the cells were grown for 7 days in HTS TranswellÒ 96 well perme-
able support (pore size 0.4 l
m, 0.143 cm2 surface area) with poly-
ethylene terephthalate membrane (Corning Life Sciences, Lowell,
MA, USA) at a density of 1.125 ꢁ 105 cells/well. The cells were
preincubated with M199 at 37 °C for 30 min. Subsequently, tran-
scellular transport was initiated by the addition of M199 either
to apical compartments (75
(250 L) containing 10 M digoxin, 200
marker for the tightness of monolayer, and 10
and terminated by the removal of each assay plate after 2 h. The
aliquots (25 L) in the opposite compartments were mixed with
CH3CN containing alprenolol and diclofenac as an internal standard
and then centrifuged. The supernatants were diluted with 10 mM
ammonium formate/formic acid (500: 1, v/v) and measured in a
LC–MS/MS analysis (API4000, AB SCIEX, Foster City, CA, USA). The
apparent permeability (Papp) of test compounds in the receiver
wells was determined and the ER for MDR1 membrane permeabil-
ity test was calculated using the following equation:
l
L) or to the basolateral compartments
M lucifer yellow as a
M test compounds
l
l
l
Acknowledgements
l
We thank Maki Miyamoto for pharmacokinetic study of 19e,
Yumi Zama for preparation of human PDE10A catalytic domain
for crystallographic study, Dr. Gyorgy Snell for data collection
and processing at the Advanced Light Source, Dr. Tsuyoshi Ishii
for in vitro enzyme assays, Dr. Kazunori Suzuki for discussion
about in vivo experiments, and Dr. Takanobu Kuroita for fruitful
discussions and valuable suggestions about medicinal chemistry.
l
References and notes
ER ¼ Papp; B to A=Papp; A to B
where Papp, A to B is the apical-to-basal passive permeability-surface
area product and Papp, B to A is the basal-to-apical passive permeabil-
ity-surface area product.
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