Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A inhibitors interacting with Tyr683 in the PDE10A selectivity pocket

Article abstract of DOI:10.1016/j.bmc.2016.05.049

Utilizing structure-based drug design techniques, we designed and synthesized phosphodiesterase 10A (PDE10A) inhibitors based on pyridazin-4(1H)-one. These compounds can interact with Tyr683 in the PDE10A selectivity pocket. Pyridazin-4(1H)-one derivative

Full text of DOI:10.1016/j.bmc.2016.05.049

Accepted Manuscript
Design and synthesis of potent and selective pyridazin-4(1H)-one-based
PDE10A inhibitors interacting with Tyr683 in the PDE10A selectivity pocket
Masato Yoshikawa, Takenori Hitaka, Tomoaki Hasui, Makoto Fushimi, Jun
Kunitomo, Hironori Kokubo, Hideyuki Oki, Kosuke Nakashima, Takahiko
Taniguchi
PII:
S0968-0896(16)30382-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.049
BMC 13033
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 April 2016
21 May 2016
24 May 2016
Please cite this article as: Yoshikawa, M., Hitaka, T., Hasui, T., Fushimi, M., Kunitomo, J., Kokubo, H., Oki, H.,
Nakashima, K., Taniguchi, T., Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A
inhibitors interacting with Tyr683 in the PDE10A selectivity pocket, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.05.049
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Design and synthesis of potent and selective pyridazin-4(1H)-one-based PDE10A inhibitors
interacting with Tyr683 in the PDE10A selectivity pocket
Masato Yoshikawa,* Takenori Hitaka, Tomoaki Hasui, Makoto Fushimi, Jun Kunitomo, Hironori
Kokubo, Hideyuki Oki, Kosuke Nakashima, and Takahiko Taniguchi
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome,
Fujisawa, Kanagawa 251-8555, Japan
*Corresponding author
Masato Yoshikawa
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited
26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
E-mail: masato.yoshikawa@takeda.com; Phone: +81-466-32-1140; Fax: +81-466-29-4454
ABSTRACT
Utilizing structure-based drug design techniques, we designed and synthesized phosphodiesterase
10A (PDE10A) inhibitors based on pyridazin-4(1H)-one. These compounds can interact with
Tyr683 in the PDE10A selectivity pocket. Pyridazin-4(1H)-one derivative 1 was linked with a
benzimidazole group through an alkyl spacer to interact with the OH of Tyr683 and fill the PDE10A
selectivity pocket. After optimizing the linker length, we identified
1-(cyclopropylmethyl)-5-[3-(1-methyl-1H-benzimidazol-2-yl)propoxy]-3-(1-phenyl-1H-pyrazol-5-y
l)pyridazin-4(1H)-one (16f) as having highly potent PDE10A inhibitory activity (IC50 = 0.76 nM)
and perfect selectivity against other PDEs (>13000-fold, IC50 = >10000 nM). The crystal structure
of 16f bound to PDE10A revealed that the benzimidazole moiety was located deep within the
PDE10A selectivity pocket and interacted with Tyr683. Additionally, a bidentate interaction existed
between the 5-alkoxypyridazin-4(1H)-one moiety and the conserved Gln716 present in all PDEs.
Graphical abstract
Keywords: phosphodiesterase 10A; PDE10A; Inhibitors, schizophrenia; SBDD

1. Introduction
Phosphodiesterases (PDEs) constitute a superfamily of enzymes that metabolically inactivate
the ubiquitous intracellular second messenger cyclic adenosine monophosphate (cAMP) and/or
cyclic guanosine monophosphate (cGMP).1 The regulation of intracellular cAMP and/or cGMP
levels can have various therapeutic outcomes depending on the expression site of each PDE.2–4
PDEs are classified into 11 families based on their amino acid sequences, substrate specificities, and
pharmacological properties.1 Phosphodiesterase 10A (PDE10A), which hydrolyzes both cAMP and
cGMP, is expressed at high levels in the medium spiny neurons of the striatum, which is the main
input station of the basal ganglia and is strongly associated with motor and cognitive functions.5–9
Based on this selective expression and its function, PDE10A inhibition is considered to be an
attractive approach for the treatment of diseases related to dysfunctions of the basal ganglia,
particularly schizophrenia and Huntington disease.10–12
Utilizing structure-based drug design (SBDD) techniques to optimize the key lead compound 1,
we previously identified
1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-o
ne (TAK-063, Figure 1A),13 which shows highly potent PDE10A inhibitory activity (IC50 = 0.30
nM) and excellent selectivity against other PDEs (minimum selectivity: >15000-fold). This
compound is currently being evaluated in Phase 2 clinical trials for the treatment of schizophrenia.14
The X-ray crystal structure of TAK-063 bound to PDE10A revealed that it binds to the active site,
forming the following hydrogen bonding interactions (Figure 1B): (i) the methoxy and carbonyl
groups on the pyridazin-4(1H)-one ring form a bidentate interaction with the carboxamide NH of
the conserved Gln716 side chain; (ii) the nitrogen atom of the pyrazole ring interacts with Tyr514
through a water molecule. Furthermore, a pyrazole ring at the 4-position of the fluorine-containing
aromatic ring contributed to the compound's high potency and excellent selectivity by effectively
filling a large space adjacent to Leu625 and Ala626 at the N-terminus of helix H12.15 Interestingly,
the X-ray crystal structure also disclosed that TAK-063 does not interact with the OH group of
Tyr683 in the PDE10A selectivity pocket,16 which was utilized in previously reported PDE10A
inhibitors to ensure PDE selectivity.16–30 As a part of a series of studies on PDE10A inhibitors, we
decided to develop novel pyridazin-4(1H)-one-based PDE10A inhibitors capable of interacting with
Tyr683 to explore a backup clinical candidate with a chemotype structurally different from that of
TAK-063.

A
B
Figure 1. (A) Structures of compound 1 and TAK-063. (B) X-ray crystal structure of TAK-063 in
the PDE10A catalytic domain.
To facilitate interaction with Tyr683 and access the PDE10A selectivity pocket, we focused on
compound 2,31 which was also identified by high-throughput screening using our proprietary
compound library. Compound 2 exhibited potent PDE10A inhibitory activity and excellent
selectivity against other PDEs (PDE10A IC50 = 0.46 nM, other PDEs IC50 = >10000 nM, minimum
selectivity: >20000-fold) (Figure 2A). The X-ray crystal structure of 2 in PDE10A is illustrated in
Figure 2B. The benzimidazole moiety interacts with the OH of Tyr683 and fills the PDE10A
selectivity pocket. Additionally, the nitrogen atom at the 3-position of the pyrrolo[3,5-d]pyrimidine
ring interacts with the carboxamide NH of the conserved Gln716 side chain.
A
B
Figure 2. (A) Structure of compound 2. (B) X-ray crystal structure of 2 in PDE10A.

The design strategy used to develop novel pyridazin-4(1H)-one-based PDE10A inhibitors
capable of interacting with Tyr683 is shown in Figure 3A. Based on an overlay study between
TAK-063 and 2 (Figure 3B), the pyridazin-4(1H)-one moiety was linked with the benzimidazole
ring via a flexible linker. Moreover, the linker was introduced into the alkoxy group at the
5-position of the pyridazin-4(1H)-one ring to maintain the bidentate interaction with the conserved
Gln716. Herein, we describe the development of novel pyridazin-4(1H)-one derivatives capable of
interacting with Tyr683 as potent and selective PDE10A inhibitors.
A
B
Figure 3. (A) Design of novel pyridazin-4(1H)-one derivatives possessing a Tyr683-binding motif.
(B) Overlaid crystal structures of TAK-063 (yellow) and 2 (white) in the PDE10A catalytic domain.

2. Chemistry
Compound 9 was synthesized as described in Scheme 1. Diazotization of aniline 3 with
NaNO2 and HCl aq followed by treatment with methyl 4-methoxy-3-oxobutanoate yielded
hydrazone 4. The pyridazin-4(1H)-one ring was formed by treatment of 4 with dimethylformamide
dimethylacetal (DMFDMA). Following hydrolysis under basic conditions, carboxylic acid 6 was
converted to ketone 8 via the Weinreb amide 7. Heating of the ketone 8 in DMFDMA and
subsequent cyclization to a pyrazole ring afforded compound 9.
Scheme 1. Synthesis of compound 9. Reagents and conditions: (a) (1) NaNO2, HCl aq, 0 °C, (2)
methyl 4-methoxy-3-oxobutanoate, NaOAc, MeOH, 0 °C, 83%; (b) DMFDMA, 90 °C, 95%; (c)
NaOH aq, MeOH, rt, 98%; (d) N,O-dimethylhydroxylamine hydrochloride,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBt, TEA, DMA, rt, 84%; (e)
MeMgBr, THF, −69 °C, 78%; (f) (1) DMFDMA, MeCN, reflux, (2) methylhydrazine, TFA, EtOH,
rt, 55% in 2 steps.
The syntheses of compounds 14a−d are summarized in Scheme 2. After protection of 10
with a benzyl group, a Suzuki–Miyaura coupling32 of 11 with
1-phenyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole gave 12. Deprotection and
alkylation afforded compounds 14a−d.
Scheme 2. Syntheses of compounds 14a−d. Reagents and conditions: (a) NaH, n-Bu4NI, BnBr,
DMF, rt, 53%; (b) 1-phenyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole,
PdCl2[Pt-Bu2(Ph-p-NMe2)]2, K2CO3, toluene, H2O, reflux, 78%; (c) Pd(OH)2/C, H2, THF, MeOH, rt,
81%; (d) alkylating agent (R1OSO2Me, R1Br, or R1OSO2CF3), base (NaH or Cs2CO3), DMF, rt,

1−66%.
Demethylation of compound 14c with chlorotrimethylsilane and sodium iodide gave
hydroxypyridazine 15. Compounds 16a−f were then synthesized by either a Mitsunobu reaction33 or
alkylation (Scheme 3).
Scheme 3. Syntheses of compounds 16a−f. Reagents and conditions: (a) chlorotrimethylsilane, NaI,
MeCN, reflux, 80%; (b) R3OH, di-tert-butyl azodicarboxylate or diethyl azodicarboxylate,
triphenylphosphine, THF, toluene, rt, 46−73%; (c) R3Cl, Cs2CO3, NaI, DMA, 80 °C, 62%.

3. Results and discussion
PDE10A inhibitory activities were measured using a scintillation proximity assay (SPA) based
on the inhibition rate of cGMP hydrolysis. [3H] cGMP was used as a substrate for PDE10A2.
In the course of preparing compounds capable of interacting with the Tyr683 in the PDE10A
selectivity pocket, the number of aromatic rings and the molecular weight of the compound are
expected to increase. Therefore, to retain the physical properties required for CNS drugs,34,35 we
initially tried to remove an aromatic ring. Results for the replacement of each phenyl group in
compound 1 are shown in Table 1. Compound 14a showed more potent PDE10A inhibitory activity
than 9. The phenyl group on the pyrazole ring, which occupied the ribose region of cAMP, made a
bigger contribution to potency than the phenyl group on the pyridazin-4(1H)-one ring. To
compensate for the dramatic loss of potency, we introduced small substituents into the 1-position of
the pyridazin-4(1H)-one ring (substituent R1). Introduction of cyclopropyl (14b),
(cyclopropyl)methyl (14c), or (trifluoromethyl)methyl (14d) groups improved the potency. These
results suggested that potency strongly depends on the presence of a substituent that can occupy the
hydrophobic region defined by the side chains of the Leu625 and Met703 residues (Figure 2A).
From the view point of chemical tractability, the (cyclopropyl)methyl group (14c) was selected as
the preferred group for further optimization because the synthetic yield of the cyclopropyl
derivative 14b was quite low (only 1%, Scheme 2).
Table 1. Optimization of substituents on the pyridazin-4(1H)-one and the pyrazole rings.
compound
R1
R2
PDE10A IC50a (nM)
Ph
Me
Ph
Ph
Ph
2400 (1600−3400)
1000 (880−1200)
130 (100−170)
170 (130−210)
550 (380−790)
9
Me
14a
14b
14c
14d
cyclopropyl
(cyclopropyl)methyl
(trifluoromethyl)methyl Ph
aIC50 values are shown as the mean of duplicate experiments. 95% Confidence intervals are given in
parentheses. All values were rounded to two significant figures.

To fill the selectivity pocket and target the interaction with Tyr683, a benzimidazole ring
was introduced into the pyridazin-4(1H)-one moiety via a linker. In view of the brain
penetration,34,35 the NH of benzimidazole was capped with a methyl group to reduce a
hydrogen-bonding donor. The results of the linker optimization study are summarized in Table 2.
First, the optimal linker length was investigated. Compound 16a, linked with a methylene spacer
between the oxygen and 1-methylbenzimidazole showed weak PDE10A inhibitory activity.
Compound 16b, a two-carbon-linked analogue, exhibited increased potency. Introduction of a
three-carbon propyl linker (16c) resulted in dramatically enhanced PDE10A inhibitory activity.
Finally, a four-carbon-linked analogue (16d) showed a decreased potency compared to 16c. We
identified the three-carbon propyl linker as the most suitable link between the
5-hydroxypyridazin-4(1H)-one and the benzimidazole rings. Heteroatoms were then introduced into
the linker. Introduction of an oxygen atom (16e) resulted in a 5-fold decrease in potency, whereas
cyclization between the linker and the benzimidazole ring (16f) slightly increased PDE10A
inhibitory activity. These results suggested that the direction of a hydrogen-bonding acceptor (HBA)
in the anchor might play an important role in the ability of the compound to inhibit PDE10A.

Table 2. Optimization of the linker
compound
R3
PDE10A IC50a (nM)
2800 (1300−5700)
16a
25 (22−29)
16b
16c
16d
16e
16f
0.81 (0.66−0.98)
760 (550−1000)
4.1 (3.3−5.2)
0.76 (0.64−0.90)
aIC50 values are shown as the mean of duplicate experiments. 95% Confidence intervals are given in
parentheses. All values were rounded to two significant figures.

The crystal structure of compound 16f bound to PDE10A is illustrated in Figure 4. As with the
crystal structure of the complex formed between PDE10A and TAK-063,13 – stacking
interactions between the phenyl rings of Phe719 and Phe686 and the pyridazin-4(1H)-one moiety
were observed. Additionally, the pyrazole ring made a hydrogen bond with Tyr514 via a water
molecule. A lone pair of the propoxy oxygen was directed toward the NH of the Gln716 side chain,
resulting in a bidentate interaction between Gln716 and the 5-alkoxypyridazin-4(1H)-one moiety.
The benzimidazole ring was positioned deep within the PDE10A selectivity pocket, interacting with
Tyr683 as expected. Furthermore, the binding mode of compound 16f resulted in perfect selectivity
against other PDEs (IC50 = >10000 nM , >13000-fold), as shown in Figure 5.
Figure 4. X-ray crystal structure of compound 16f in the PDE10A catalytic domain.
Figure 5. Inhibitory activities of compound 16f against human PDEs
aIC50 values are shown as the mean of duplicate experiments. 95% Confidence intervals are given in
parentheses. All values were rounded to two significant figures.

Conclusion
Utilizing SBDD techniques, we have designed and synthesized novel PDE10A inhibitors based
on pyridazin-4(1H)-one; these inhibitors interact with the Tyr683 residue in the PDE10A selectivity
pocket. A benzimidazole group was selected as the anchor to interact with the OH group of Tyr683
and fill the PDE10A selectivity pocket. The 5-position of the pyridazin-4(1H)-one ring was chosen
to introduce the linker required to access to the PDE10A selectivity pocket. After the optimization
effort, compound 16f was found to display highly potent PDE10A inhibitory activity and perfect
selectivity against other PDEs. The crystal structure of compound 16f with PDE10A revealed that
the benzimidazole moiety was positioned deep within the PDE10A selectivity pocket and interacted
with the OH group of Tyr683. Additionally, the bidentate interaction between Gln716 and the
5-alkoxypyridazin-4(1H)-one moiety was also observed. We succeeded in identifying a highly
potent and selective PDE10A inhibitor with different binding modes based on TAK-063 after the
hybridization of two distinct chemotypes.

5. Experimental procedure
All commercially available solvents and reagents were used without further purification.
Yields were not optimized. Melting points were determined on a B chi melting point apparatus
B-545 or an OptiMelt melting point apparatus MPA100 and were not corrected. 1H NMR spectra
were recorded on Varian Mercury-300 (300 MHz) or Bruker DPX300 (300 MHz) instruments.
Chemical shifts are reported as δ values (ppm) downfield from internal tetramethylsilane (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 doublet of
doublets; quin, quintet; brs, 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 Technologies)
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% 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 ultra-fast liquid chromatography
(UFLC) instrument, equipped with an L-column 2 ODS (3.0 × 50 mm, 2 μm), eluting with a
gradient of 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 1.2 mL/min, with UV detection at 220 nm. Reaction
progress was determined by thin-layer chromatography (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 ϕ 60 μM or NH ϕ 60 μM, Fuji Silysia Chemical, Ltd.).
Preparative HPLC purification was performed by using a Waters Corporation UV purification
system equipped with a Develosil ODS-UG-10 (4.6 × 150 mm, 5μm) column, and eluted with a
gradient of 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-dimethylactamide; AcOH,
acetic acid; DMFDMA, N,N-dimethylformamide dimethylacetal; MeCN, acetonitrile; DIPEA,
N,N-diisopropylethylamine; TEA, triethylamine; diisopropyl ether, IPE; dichloromethane, DCM;
2-propanol, IPA; trifluoroacetic acid, TFA; 1-hydroxy-1H-benzotriazole, HOBt.
5.1. Methyl 4-methoxy-3-oxo-2-(phenylhydrazono)butanoate (4).
A solution of sodium nitrite (55.6 g, 803 mmol) in water (142 mL) was added dropwise to a
solution of 3 (50 g, 539 mmol) in 6 M HCl aq (536 mL) at 0°C. The mixture was added to a
suspension of methyl 4-methoxyacetoacetate (78.5 g, 537 mmol) and sodium acetate (264 g, 3220

mmol) in MeOH (1400 mL) and water (350 mL) at 0°C. After the mixture was stirred at room
temperature for 2 h, the mixture was diluted with water and stirred for 30 min. The formed
precipitate was collected by filtration, washed with water, EtOH/IPE, and IPE, successively, and
dried to give 4 (111.2 g, 83%) as a yellow solid. 1H NMR (CDCl3) δ 3.51 (3H, s), 3.88 (3H, s), 4.69
(2H, s), 7.18-7.25 (1H, m), 7.37-7.48 (4H, m), 14.99 (1H, brs).
5.2. Methyl 5-methoxy-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylate (5).
A mixture of 4 (25.0 g, 100 mmol) and DMFDMA (120 mL) was stirred at 90 °C for 1 h and at
room temperature for 1 h. The formed precipitate was then collected by filtration, washed with
diethyl ether and hexane, and dried to give 5 (24.7 g, 95%) as a pale yellow solid. LC-MS (ESI) m/z
261.1 (M+H)+. 1H NMR (CDCl3) δ 3.96 (3H, s), 3.98 (3H, s), 7.43-7.48 (1H, m), 7.51-7.56 (2H, m),
7.61-7.64 (2H, m), 7.95 (1H, s).
5.3. 5-Methoxy-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxylic acid (6).
1 M NaOH aq (300 mL) was added dropwise to a solution of 5 (39.0 g, 150 mmol) in MeOH (663
mL) at 0 °C. After the mixture was stirred at room temperature for 1 h, 1 M HCl aq (300 mL) was
added to the mixture at 0 °C. The mixture was stirred at 0 °C for 1 h, and the formed precipitate was
collected by filtration. The obtained solid was washed with water, EtOH/IPE, and IPE, successively,
and dried to give 6 (36.1 g, 98%) as a pale yellow solid. LC-MS (ESI) m/z 247.1 (M+H)+. 1H NMR
(DMSO-d6) δ 3.99 (3H, s), 7.55-7.68 (3H, m), 7.87 (2H, d, J = 8.4 Hz), 8.94 (1H, s), 15.25 (1H, s).
5.4. N,5-Dimethoxy-N-methyl-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxamide (7).
To a solution of 6 (40 g, 162 mmol) in DMA (600 mL) were added N,O-dimethylhydroxylamine
hydrochloride (23.7 g, 243 mmol) and HOBt (21.9 g, 162 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (46.6 g, 243 mmol), and TEA (81.4
mL, 584 mmol) at 0 °C. After being stirred at room temperature for 96 h, the mixture was diluted
with EtOAc and the insoluble was filtered off. The filtrate was concentrated under reduced pressure.
The residue was purified by basic silica gel column chromatography with EtOAc/MeOH to give 7
(39.2 g, 84%) as a white solid. LC-MS (ESI) m/z 290.1 (M+H)+. 1H NMR (CDCl3) δ 3.40 (3H, s),
3.71 (3H, s), 3.96 (3H, s), 7.39-7.44 (1H, m), 7.49-7.54 (2H, m), 7.59-7.63 (2H, m), 7.98 (1H, s).
5.5. 3-Acetyl-5-methoxy-1-phenylpyridazin-4(1H)-one (8).
To a solution of 7 (20.0 g, 69.1 mmol) in THF (1000 mL) was added dropwise methylmagnesium
bromide in THF (1 mol/L, 138 mL) at –69 °C, and the mixture was stirred at –69 °C for 1.5 h. The
mixture was quenched with saturated ammonium chloride solution and warmed to room
temperature. The organic layer was washed with brine, dried over MgSO4. The aqueous layer was
extracted with EtOAc. The extract dried over MgSO4 and the organic layer were combined and
concentrated under reduced pressure. The residue was purified by basic silica gel column
chromatography with EtOAc/MeOH to give 8 (13.2 g, 78%) as a yellow solid. LC-MS (ESI) m/z

245.1 (M+H)+. 1H NMR (CDCl3) δ 2.70 (3H, s), 3.96 (3H, s), 7.44-7.49 (1H, m), 7.53-7.58 (2H, m),
7.61-7.65 (2H, m), 7.94 (1H, s).
5.6. 5-Methoxy-3-(1-methyl-1H-pyrazol-5-yl)-1-phenylpyridazin-4(1H)-one (9).
To a suspension of 8 (6.0 g, 24.6 mmol) in MeCN (18 mL) was added DMFDMA (49 mL). After
refluxing for 4.5 h, the mixture was concentrated under reduced pressure. The residue was dissolved
in EtOH (9 mL), and then to the solution was added dropwise a solution of methylhydrazine (2.6
mL, 49.1 mmol) in 10% TFA/EtOH (85 mL) at below 15 °C. After the mixture was stirred at room
temperature for 18 h, the formed precipitate was collected by filtration, washed with EtOH and IPE,
and dried under reduced pressure at 50 °C to give 9 (3.8 g, 55%) as a pale yellow solid. 1H NMR
(CDCl3) δ 3.97 (3H, s), 4.12 (3H, s), 7.24 (1H, d, J = 2.3 Hz), 7.42-7.49 (1H, m), 7.52-7.60 (3H, m),
7.60-7.66 (2H, m), 8.02 (1H, s). LC-MS (ESI) m/z 283.2 (M+H)+. Anal. Calcd for C15H14N4O2: C,
63.82; H, 5.00; N, 19.85. Found: C, 63.69; H, 5.04; N, 19.94.
5.7. 1-Benzyl-3-chloro-5-methoxypyridazin-4(1H)-one (11). To a solution of 10 (10.0 g, 62.3
mmol) in DMF (300 mL) were added NaH (55 wt%, 3.26 g, 74.7 mmol) and nBu4NI (4.60 g, 12.5
mmol) at 0 °C. The mixture was stirred for 10 min at 0 °C, and then benzyl bromide (12.3 g, 71.6
mmol) was added to it at 0 °C. The mixture was stirred for 20 h at room temperature and then
quenched with water and extracted with DCM (twice). The combined organic layer was washed
with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residual
solid was recrystallized from EtOAc/hexane to give 11 (19.8 g, 53%) as a white solid. 1H NMR
(CDCl3) δ 3.82 (3H, s), 5.31 (2H, s), 7.34-7.42 (5H, m), 7.89 (1H, s).
5.8. 1-Benzyl-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (12). A mixture of
11 (13.6 g, 54.3 mmol), 1-phenyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(22.0 g, 81.0 mmol), K2CO3 (51.0 g, 109 mmol) and PdCl2[Pt-Bu2(Ph-p-NMe2)]2 (1.92 g, 2.71
mmol) in toluene (330 mL) and water (33 mL) was refluxed for 24 h under N2 atmosphere. The
mixture was diluted with water and saturated NaHCO3 aqueous solution. The mixture was extracted
with EtOAc, dried over Na2SO4, and concentrated under reduced pressure. The residue was
recrystallized from EtOAc/hexane to give 12 (15.1 g, 78%) as a pale yellow solid. 1H NMR
(DMSO-d6) δ 3.81 (3H, s), 5.10 (2H, s), 6.95 (1H, d, J = 1.6 Hz), 7.05-7.07 (2H, m), 7.24-7.38 (8H,
m), 7.74 (1H, d, J = 1.6 Hz), 8.33 (1H, s).
5.9. 5-Methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4-ol (13). A mixture of 12 (15.0 g, 41.9
mmol) and Pd(OH)2/C (Pd: 20%, 50% wet, 5.88 g, 4.19 mmol) in THF (500 mL) and MeOH (300
mL) was stirred at room temperature for 2 days under H2 atmosphere. After filtration through a
Celite pad, the filtrate was concentrated under reduced pressure. The residual solid was washed with
EtOH/hexane to give 13 (10 g, 81%) as a brown solid. 1H NMR (DMSO-d6) δ 3.74 (3H, s), 6.79
(1H, d, J = 1.6 Hz), 7.27-7.40 (5H, m), 7.76 (1H, d, J = 2.0 Hz), 8.15 (1H, s), 13.4 (1H, brs).

LC-MS (ESI) m/z 269 (M+H)+.
5.10. 5-Methoxy-1-methyl-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (14a).
To a solution of 13 (135 mg, 0.5 mmol) in DMF (2 mL) was added cesium carbonate (361 mg, 1.11
mmol), then, to the mixture was added dimethyl sulfate (0.071 mL, 0.75 mmol) at room temperature.
After being stirred at the same temperature for 16 h, the mixture was poured into water and
extracted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography with EtOAc/MeOH and recrystallized from MeOH/EtOAc to give 14a (47 mg,
33%) as a pale yellow solid. 1H NMR (CDCl3) δ 3.66 (3 H, s), 3.84 (3 H, s), 7.12 (1H, d, J = 1.9
Hz), 7.28 - 7.37 (5H, m), 7.43 (1H, s), 7.76 (1H, d, J = 1.9 Hz). LC-MS (ESI) m/z 283.2 (M+H)+.
Anal. Calcd for C15H14N4O2: C, 63.82; H, 5.00; N, 19.85. Found: C, 63.74; H, 5.01; N, 19.95.
5.11. 1-Cyclopropyl-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (14b).
To a mixture of 13 (1.0 g, 3.7 mmol) and cesium carbonate (2.4 g, 7.5 mmol) in DMF (20 mL) was
added bromocyclopropane (0.60 mL, 7.5 mmol) at room temperature. After stirring at 150 °C in an
autoclave for 30 h, 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 silica gel column chromatography with EtOAc/MeOH and basic silica gel column
chromatography with EtOAc/MeOH to give 14b (15 mg, 1.3%) as a white solid. 1H NMR (CDCl3)
δ 0.51-0.59 (2H, m), 0.69-0.78 (2H, m), 3.37 (1H, tt, J = 7.3, 3.7 Hz), 3.89 (3H, s), 7.24 (1H, d, J =
1.9 Hz), 7.29-7.42 (5H, m), 7.58 (1H, s), 7.75 (1H, d, J = 2.3 Hz). LC-MS (ESI) m/z 309.1 (M+H)+.
5.12. 1-(Cyclopropylmethyl)-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one
(14c).
To a solution of 13 (135 mg, 0.50 mmol) in DMF (2 mL) under argon atmosphere was added 60%
oil suspension of sodium hydride (40.3 mg, 1.01 mmol), and the suspension was stirred for 30 min
at 0 °C. (Bromomethyl)cyclopropane (0.073 mL, 0.75 mmol was added to the mixture, which was
then stirred at the same temperature for 7 h, before MeOH (5 mL) was added to the mixture at
below 15 °C. The mixture was concentrated under reduced pressure, and the residue was purified
by silica gel column chromatography with EtOAc/MeOH and recrystallized from MeOH/EtOAc to
give 14c (103 mg, 63%) as a white solid. 1H NMR (CDCl3) δ 0.17 (2H, q, J = 4.9 Hz), 0.52 (2H, q,
J = 6.2 Hz), 0.83-0.96 (1H, m), 3.59 (2H, d, J = 7.2 Hz), 3.88 (3H, s), 7.18 (1H, d, J = 1.9 Hz),
7.27-7.33 (1H, m), 7.33-7.37 (4H, m), 7.50 (1H, s), 7.76 (1H, d, J = 1.9 Hz). LC-MS (ESI) m/z
323.3 (M+H)+. Anal. Calcd for C18H18N4O2: C, 67.07; H, 5.63; N, 17.38. Found: C, 67.05; H, 5.58;
N, 17.36.
5.13. 5-Methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-1-(2,2,2-trifluoroethyl)pyridazin-4(1H)-one
(14d).

To a solution of 13 (2.0 g, 7.5 mmol) in DMF (40 mL) under argon atmosphere was added 60% oil
suspension of sodium hydride (0.596 g, 14.91 mmol), and the suspension was stirred for 30 min at
0 °C. Then, trifluoromethanesulfonic acid 2,2,2-trifluoroethyl ester (2.6 g, 11.2 mmol) was added.
After the mixture was stirred at the same temperature for 18 h, MeOH (5 mL) was added at below
15°C. This mixture was concentrated under reduced pressure, and the residue was purified by
silica gel column chromatography with EtOAc/hexane to give 14d (1.7 g, 66%) as a white solid. 1H
NMR (CDCl3) δ 3.84 (3H, s), 4.26 (2H, q, J = 8.2 Hz), 7.17 (1H, d, J = 1.9 Hz), 7.30-7.39 (5H, m),
7.44 (1H, s), 7.76 (1H, d, J = 1.9 Hz). LC-MS (ESI) m/z 351.0 (M+H)+. Anal. Calcd for
C16H13F3N4O2: C, 54.86; H, 3.74; N, 15.99. Found: C, 54.78; H, 3.87; N, 15.89.
5.14. 1-(Cyclopropylmethyl)-5-hydroxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (15)
TMSCl (3.2 mL, 25.0 mmol) was added at room temperature to a solution of NaI (3.8 g, 25.0
mmol) in CH3CN (150 mL). After the mixture was stirred for 30 min, 14c (1.6 g, 5.0 mmol) was
added to the resulting suspension. The mixture was stirred for 30 min at room temperature and then
refluxed for 4 h. After being cooled to room temperature, the mixture was poured into water and
stirred for 30 min. The mixture was extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4 and concentrated under reduced pressure. The residue was triturated with EtOAc,
and collected by filtration. The crystals were washed with EtOAc and IPE, and dried under reduced
pressure at 50 °C to give 15 (1.2 g, 80 %) as a pale brown solid. 1H NMR (CDCl3) δ 0.18-0.26 (2H,
m), 0.35-0.44 (2H, m), 0.85-0.98 (1H, m), 3.74 (2H, d, J =7.2 Hz), 6.91 (1H, d, J =1.9 Hz),
7.25-7.34 (3H, m), 7.35-7.43 (2H, m), 7.77 (1H, d, J =1.9 Hz), 8.22 (1H, s). LC-MS (ESI) m/z 309
(M+H)+.
5.15.
1-(Cyclopropylmethyl)-5-[(1-methyl-1H-benzimidazol-2-yl)methoxy]-3-(1-phenyl-1H-pyrazol-
5-yl)pyridazin-4(1H)-one (16a).
To a solution of 15 (100 mg, 0.32 mmol), 2-(1-methyl-1H-benzo[d]imidazol-2-yl)methanol (79 mg,
0.49 mmol) and triphenylphosphine (149 mg, 0.57 mmol) in THF (10 mL) was added
(E)-di-tert-butyl diazene-1,2-dicarboxylate (20% toluene solution, 0.64 mL, 0.49 mmol) at rt. After
stirring at room temperature overnight, the mixture was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography with EtOAc/hexane and recrystallized
from EtOAc to give 16a (72 mg, 49 %) as a white solid. 1H NMR (CDCl3) δ 0.12-0.21 (2H, m),
0.40-0.50 (2H, m), 0.74-0.91 (1H, m), 3.53 (2H, d, J = 7.2 Hz), 3.97 (3H, s), 5.55 (2H, s), 7.18 (1H,
d, J = 2.3 Hz), 7.27-7.43 (8H, m), 7.71-7.79 (2H, m), 8.17 (1H, s). LC-MS (ESI) m/z 453.1 (M+H)+.
Anal. Calcd for C26H24N6O2 + 0.2 H2O: C, 68.47; H, 5.39; N, 18.43. Found: C, 68.51; H, 5.35; N,
18.59.
5.16.
1-(Cyclopropylmethyl)-5-[2-(1-methyl-1H-benzimidazol-2-yl)ethoxy]-3-(1-phenyl-1H-pyrazol-

5-yl)pyridazin-4(1H)-one (16b).
To a solution of 15 (100 mg, 0.32 mmol), 2-(1-methyl-1H-benzo[d]imidazol-2-yl)ethanol (86 mg,
0.49 mmol) and triphenylphosphine (149 mg, 0.57 mmol) in THF (10 mL) was added (E)-diethyl
diazene-1,2-dicarboxylate (0.221 mL, 0.49 mmol) at rt. After being stirred at room temperature for
3 h, the mixture was concentrated under reduced pressure. The residue was purified by silica gel
column chromatography with EtOAc/MeOH to give 16b (71 mg, 47 %) as an off-white solid. 1H
NMR (DMSO-d6) δ 0.15-0.24 (2H, m), 0.30-0.42 (2H, m), 0.78-0.94 (1H, m), 3.38 (2H, t, J = 6.6
Hz), 3.72 (2H, d, J = 7.2 Hz), 3.83 (3H, s), 4.44 (2H, t, J = 6.6 Hz), 6.89 (1H, d, J = 1.9 Hz),
7.10-7.44 (7H, m), 7.47-7.59 (2H, m), 7.75 (1H, d, J = 1.9 Hz), 8.37 (1H, s). LC-MS (ESI) m/z
467.1 (M+H)+. Anal. Calcd for C27H26N6O2 + 0.8 H2O: C, 67.43; H, 5.78; N, 17.47. Found: C,
67.45; H, 5.76; N, 17.22.
5.17.
1-(Cyclopropylmethyl)-5-[3-(1-methyl-1H-benzimidazol-2-yl)propoxy]-3-(1-phenyl-1H-pyrazo
l-5-yl)pyridazin-4(1H)-one (16c).
Compound 16c was obtained (46%) as a white solid in a manner similar to that described for
compound 16a. 1H NMR (CDCl3) δ 0.10-0.18 (2H, m), 0.43-0.51 (2H, m), 0.80-0.93 (1H, m), 2.47
(2H, quin, J =6.5 Hz), 3.13 (2H, t, J =6.8 Hz), 3.55 (2H, d, J =7.2 Hz), 3.75 (3H, s), 4.22 (2H, t, J
=6.2 Hz), 7.17 (1H, d, J =1.9 Hz), 7.20-7.28 (2H, m), 7.29-7.32 (2H, m), 7.32-7.36 (4H, m),
7.65-7.70 (1H, m), 7.71 (1H, s), 7.76 (1H, d, J =1.9 Hz). LC-MS (ESI) m/z 481.3 (M+H)+. Anal.
Calcd for C28H28N6O2 + H2O: C, 67.95; H, 6.29; N, 16.39. Found: C, 68.21; H, 6.26; N, 16.26.
5.18.
1-(Cyclopropylmethyl)-5-[4-(1-methyl-1H-benzimidazol-2-yl)butoxy]-3-(1-phenyl-1H-pyrazol-
5-yl)pyridazin-4(1H)-one (16d).
Compound 16d was obtained (54%) as a white solid in a manner similar to that described for
compound 16b. 1H NMR (CDCl3) δ 0.14-0.25 (2H, m), 0.31-0.43 (2H, m), 0.80-0.96 (1H, m),
1.77-1.97 (4H, m), 2.94 (2H, t, J = 7.2 Hz), 3.65-3.81 (5H, m), 3.94-4.09 (2H, m), 6.89 (1H, d, J =
1.9 Hz), 7.08-7.58 (9H, m), 7.75 (1H, d, J = 1.9 Hz), 8.28 (1H, s). LC-MS (ESI) m/z 495.2 (M+H)+.
Anal. Calcd for C29H30N6O2 + 2 H2O: C, 65.10; H, 6.24; N, 16.27. Found: C, 65.33; H, 6.20; N,
16.26.
5.19.
1-(Cyclopropylmethyl)-5-(2-((1-methyl-1H-benzimidazol-2-yl)oxy)ethoxy)-3-(1-phenyl-1H-pyr
azol-5-yl)pyridazin-4(1H)-one (16e).
Compound 16e was obtained (73%) as a white solid in a manner similar to that described for
compound 16a. 1H NMR (CDCl3) δ 0.09-0.16 (2H, m), 0.43-0.51 (2H, m), 0.76-0.91 (1H, m), 3.50
(2H, d, J = 7.6 Hz), 3.54 (3H, s), 4.67 (2H, dd, J = 5.7, 3.8 Hz), 4.86 (2H, dd, J = 5.7, 3.8 Hz),
7.13-7.18 (3H, m), 7.19 (1H, d, J = 1.9 Hz), 7.27-7.37 (5H, m), 7.46-7.53 (1H, m), 7.75 (1H, s),

7.77 (1H, d, J = 1.9 Hz). LC-MS (ESI) m/z 483.1 (M+H)+. Anal. Calcd for C27H26N6O3 + 0.6 H2O:
C, 65.73; H, 5.56; N, 17.03. Found: C, 65.60; H, 5.55; N, 16.81.
5.20.
1-(Cyclopropylmethyl)-5-[2-(2,3-dihydro-1H-imidazo[1,2-a]benzimidazol-1-yl)ethoxy]-3-(1-ph
enyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (16f).
A mixture of 15 (231 mg, 0.75 mmol),
1-(2-chloroethyl)-2,3-dihydro-1H-imidazo[1,2-a]benzimidazole (249 mg, 1.13 mmol), cesium
carbonate (428 mg, 1.31 mmol), sodium iodide (253 mg, 1.69 mmol) in DMA (10 mL) was stirred
at 80 °C for 13 h. The precipitate was removed by filtration, and the filtrate was concentrated under
reduced pressure. The residue was purified by basic silica gel column chromatography with
EtOAc/hexane to give 16f (231 mg, 62%) as a white solid. 1H NMR (CDCl3) δ 0.11 (2H, q, J = 5.0
Hz), 0.45 (2H, q, J = 6.2 Hz), 0.74-0.87 (1H, m), 3.47 (2H, d, J = 7.2 Hz), 3.84 (2H, t, J = 5.1 Hz),
4.06-4.14 (2H, m), 4.22-4.30 (2H, m), 4.43 (2H, t, J = 5.1 Hz), 7.00-7.11 (3H, m), 7.19 (1H, d, J
=1.9 Hz), 7.27-7.34 (5H, m), 7.38 (1H, d, J = 7.6 Hz), 7.72 (1H, s), 7.77 (1H, d, J = 1.9 Hz).
LC-MS (ESI) m/z 494.4 (M+H)+. Anal. Calcd for C28H27N7O2 + 1.7 H2O: C, 64.16; H, 5.85; N,
18.70. Found: C, 64.13; H, 5.86; N, 18.64.
5.21. Crystallization and Structure Determination. Human PDE10A catalytic domain for
crystallographic study was prepared as previously described.13 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 2 were obtained by immersing apo crystals into the
corresponding reservoir solution containing 0.25 mM inhibitor for 48 hours. As for complex
crystals with compound 16f, apo crystals were soaked into 0.2 mM compound 16f solution for 40
hours. 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 of
2 and 16f were collected from each single crystal at the Advanced Light Source beamline 5.0.2 and
5.0.3 (Berkeley, CA), respectively.
The data were processed using the program HKL2000.36 The
structures were determined by molecular replacement using MOLREP,37 utilizing the previously
reported coordinate of PDE10A with the PDB accession code 3WYM.13 Subsequently, the
structures were refined through an iterative procedure utilizing REFMAC,38 followed by model
building with WinCoot.39 The dictionary files for the inhibitors were prepared using AFITT
(OpenEye Scientific Software). Crystallographic processing and refinement statistics are
summarized in Table 3. The coodinates and structure factors have been deposited in PDB with
accession codes 5B4K and 5B4L for 2 and 16f, respectively.
Table 3. X-ray data collection and refinement statistics

2
16f
Data Collection
X-ray source
ALS BL5.0.2
ALS BL5.0.3
0.97645
Wavelength (Å)
Space group
1.0000
P212121
P212121
Unit cell dimensions (Å)
Resolution (Å)
Unique reflections
Redundancy
a=49.7, b=81.7, c=161.7
2.90
a=49.1, b=81.5, c=159.2
2.40
13970
26587
6.0
5.1
Completeness (%)
I/σ(I)
90.9 (53.1)
18.3 (5.3)
0.082
99.3 (98.4)
10.0 (1.1)
0.123
a
Rsym
Refinement
Reflections used
RMS Bonds (Å)
RMS Angles (°)
Average B value (Å2)
R-valueb
13872
0.004
0.914
51.110
0.181
0.261
25650
0.005
1.013
37.221
0.235
0.282
b
R free
aRsym = ΣhΣj |<I(h)> - I(h)j | / ΣhΣj <I(h)>, where <I(h)> is the mean intensity of symmetry-related
b
reflections. R-value = Σ | |Fobs| - |Fcalc| | / Σ |Fobs|. Rfree for 5% of reflections excluded from refinement.
Values in parentheses are for the highest resolution shell.
5.22. 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 μL of serial diluted compounds
were incubated with 20 μL of PDE enzyme in assay buffer (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 triplicate in 96-well half-area plates (Corning).
To start the reaction, 10 μL of substrate [3H]cGMP (PerkinElmer) for PDE10A2 was added for a
final assay volume of 40 μL. After 60 minutes incubation at rt, 20 μL of 20 mg/mL yttrium SPA
beads containing ZnSO4 was added to terminate the PDE reaction. After being settled for more than
120 min, the assay plate was counted in a scintillation 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 μM 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 (ID Business Solusions, Guildford, UK).
Acknowledgements
We thank 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, and Dr. Takanobu Kuroita for fruitful
discussions and valuable suggestions about medicinal chemistry. The authors are employees of
Takeda Pharmaceutical Company Limited and declare no other competing financial interests.
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