Fragment-based discovery of pyrimido[1,2-B]indazole PDE10A inhibitors

Article abstract of DOI:10.1248/cpb.c17-00836

In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

Full text of DOI:10.1248/cpb.c17-00836

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Chem. Pharm. Bull. 66, 286–294 (2018)
Vol. 66, No. 3
Regular Article
Highlighted Paper selected by Editor-in-Chief
Fragment-Based Discovery of Pyrimido[1,2-b]indazole PDE10A
Inhibitors
Ayaka Chino,* Ryushi Seo, Yasushi Amano, Ichiji Namatame, Wataru Hamaguchi,
Kazuya Honbou, Takuma Mihara, Mayako Yamazaki, Masaki Tomishima, and Naoyuki Masuda
Drug Discovery Research, Astellas Pharma Inc.; 21 Miyukigaoka, Tsukuba, Ibaraki 305–8585, Japan.
Received October 17, 2017; accepted December 5, 2017
In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A
(
PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine
derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of
PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal
structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by
means of structure–activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimi-
doindazole derivative 13 having good physicochemical properties.
Key words phosphodiesterase10A inhibitor; fragment-based drug discovery; schizophrenia; combinatorial
synthesis; pyrimido[1,2-b]indazole
Schizophrenia is known as a chronic debilitating disorder PDE10A inhibitors might prove useful in treating neurological
1)
6)
affecting the psychic and motor functions of the brain that is and psychiatric disorders.
characterized by a combination of positive symptoms, negative At present, several PDE10A inhibitors are undergoing clini-
2
)
7)
symptoms and cognitive impairments. Current anti-psychotics cal investigation and a large number of studies of PDE10A
8
–16)
are marginally effective for treating the positive symptoms but inhibitors have been reported.
Preclinical evidence sug-
less effective against negative symptoms and cognitive impair- gests that these inhibitors might demonstrate anti-psychotic,
17–21)
ments. Therefore, novel type of drugs which could manage pro-cognitive, and anti-negative symptom efficacy.
negative symptoms and cognitive impairments would represent
We have already reported a potent PDE10A inhibitor,
a significant advance in schizophrenia treatment.
compound 1 (Fig. 1), as the result of the structure–activity
The phosphodiesterases (PDEs) consist of an 11-membered relationship (SAR) studies on the basis of the high-throughput
2
2)
family of enzymes that catalyze the hydrolysis of second- screening (HTS) hit compound,
however, 1 was found
ary signal messengers, such as cAMP and cyclic guanosine to have some issues such as a metabolic instability and the
monophosphate (cGMP). PDE10A is a dual substrate PDE moderate inhibitory activity towards CYP3A4 enzyme. For
that hydrolyzes both cAMP and cGMP. PDE10A is highly ex- the purpose of finding other lead compounds with good physi-
pressed in the brain, particularly in the medium spiny neurons cochemical properties as clinical candidates, the method of
of the striatum. The distribution conserves across mammalian fragment-based drug discovery (FBDD) was chosen as a novel
3
–5)
species.
Inhibition of PDE10A is considered effective in approach.
treating schizophrenia and a range of neurological, psychotic,
anxiety, and movement disorders which could be benefit from actions to one of the binding pockets within the active site.
Fragment hit makes a small number of high-efficiency inter-
2
3)
increasing levels of cAMP and cGMP in neurons. Therefore, Once fragment binders have been identified in the binding
site, the objective in drug discovery is then to design potent
inhibitors by using a range of strategies including fragment
2
4–26)
emerging, linking or growing.
In order to make further
highly efficient interactions in the active site with retaining
the key interactions from the fragment hit, the hit compound
has been evolved into neighboring binding pockets to produce
compact and potent lead compounds. Since crystal structure
of PDE10A enzyme was revealed, co-crystal structure analy-
ses of the enzyme with compounds should be useful to design
some lead compounds having desired physicochemical proper-
ties. Herein, we describe our synthetic studies on optimization
from the fragment-hit compound to achieve creating potent
PDE10A inhibitors by using the method of X-ray crystal struc-
ture analysis.
Fig. 1. Structure of Compound 1
Chemistry
a) Residual activities of human liver microsome (HLM) for metabolism of
midazolam in the presence of test compounds were determined following pre-
incubation for 30min.
The synthesis of target compounds is shown in Charts 1 and
2. Synthesis of pyrazolo[1,5-a]pyrimidin-7-ol derivatives 3–12
*
To whom correspondence should be addressed. e-mail: ayaka.chino@astellas.com
©
2018 The Pharmaceutical Society of Japan

Vol. 66, No. 3 (2018)
Chem. Pharm. Bull.
287
is shown in Chart 1. β-Keto esterification of corresponding the 4-nitrogen of pyrazolopyrimidine accepted a hydrogen
carboxylic acids 19 in the presence of monoethyl potassium bond from Gln726. The X-ray crystal structure also disclosed
malonate, 1,1′-carbonyldiimidazole (CDI) and MgCl followed that 2 does not interact with Tyr693 which was found to be
2
by condensation and cyclization with 20 in AcOH gave 3–12.
The pyrimido[1,2-b]indazole derivatives 13–18 were syn- ies.
important to increase PDE10A selectivity in previous stud-
18,28)
For the purpose of preparing compounds having the
thesized as depicted in Chart 2. Cyclocondensation of amino- interaction with Tyr693, fragment growing approach was
guanidine hydrochloride 21 with succinic anhydride 22 gave adopted and introduction of heteroaromatics into 5-position
a triazolopyrimidine intermediate, followed by condensation of pyrazolopyrimidine ring was performed. In the course of
with acetylacetone to obtain carboxylic acid 23. Pyrimido[1,2- preparing compounds capable of interacting with the Tyr693,
b]indazol-4-ol derivative 13 was prepared from 23 and 3-ami- the molecular weight of the compound would be considered to
noindazole in a manner similar to that described in Chart 1. be increased which should cause the increase in the lipophilic-
Chlorination of 13 with POCl gave 17, which was followed by ity of the compounds. On the other hand, 4-chlorophenyl part
3
displacement of methylamine, methanol or KCN yielded 14, was solvent exposed and was seemed not to exhibit any appre-
15 or 18, respectively. A Suzuki–Miyaura reaction of 17 with ciable contribution to potency of the substrate from the X-ray
trimethylboroxine gave the methyl analogue 16.
co-crystal structure of PDE10A enzyme with compound 2,
so that hydroxyl group was chosen for the substitution on the
7-position of pyrazolopyrimidine ring instead of 4-chloro-
Results and Discussion
The analyses of the X-ray co-crystal structures of phenyl group to reduce lipophilicity and molecular weight
PDE10A enzyme with fragment compounds from in-house of the molecule. In addtion, the replacement with hydroxyl
compounds’ library revealed that 7-(4-chlorophenyl)-2- group is beneficial for quickly optimization of Tyr693 binders
methylpyrazolo[1,5-a]pyrimidine (2) occupied a part of the because of synthetic tractability for combinatorial synthesis.
binding site, showed good ligand efficiency (percent efficiency
2
7)
index (PEI) =0.39), meaning that this compound should be
a good fragment hit (Figs. 2, 3). The pyrazolopyrimidine ring
of 2 was found to occupy the active site of the PDE10A and
Reagents and conditions: (a) Monoethyl potassium malonate, CDI, MgCl , NEt ,
2
3
THF; (b) 20, AcOH, 1,4-dioxane, 11–76% (2 steps).
Chart 1
Fig. 2. Structure of Compound 2
Reagents and conditions: (a) NaOH aq.; (b) acetylacetone, AcOH, EtOH, 68% (2 steps); (c) monoethyl potassium malonate, CDI, MgCl , NEt , THF; (d) 3-aminoinda-
2
3
zole, AcOH, 1,4-dioxane, 11% (2 steps); (e) POCl , 38%; (f) methylamine, THF, 97%; (g) sodium methoxide, MeOH, 82%; (h) trimethylboroxine Pd(dppf)Cl ·CH Cl ,
3
2
2
2
K CO , 1,4-dioxane, 34%; (i) KCN, DABCO, 1,4-dioxane, H O, 37%.
2
3
2
Chart 2

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Chem. Pharm. Bull.
Vol. 66, No. 3 (2018)
Fig. 3. Crystal Structure of 2 (Pink) Bound to PDE10A (PDB Code:
XUJ)
Fig. 4. Crystal Structure of 7 (Cyan) Bound to PDE10A (PDB Code:
5XUI)
5
Table 1. PDE10A Inhibitory Activity of Heterocycles
concentration of 4µM or evaluation of IC50 about each com-
pound.
Initial attempts at accessing the selectivity pocket was
introduction of various hetero-aromatic rings containing a
nitrogen atom expected to interact with Tyr693 (Table 1). In
order to find a potent scaffold efficiently, we used combina-
torial synthesis. Compound 3 which has the ethylene linker
between quinoline and pyrazolopyrimidine showed strong
inhibitory activity. Compound 4 bearing 2-quinoline substitu-
ent directly and compound 5 having methyl thioether as the
linker instead of ethylene in compound 3 showed less inhibi-
tory activity than 3, suggesting that ethylene linker brought
suitable position for the quinoline nitrogen atom to interact
2
9)
with Tyr693. Pyridyl ethyl derivative 6 brought less potency
than quinoline 3, indicating that phenylene part of quinoline
ring would be important for the PDE10A inhibitory activity.
Triazolopyrimidine 7, imidazopyridine 8, imidazopyrimidine
9
and benzimidazole 10 all showed moderate to strong inhibi-
tory activities against PDE10A. Among them, triazolopyrimi-
dine 7 was chosen for the further modification to improve the
inhibitory potency because compound 7 showed inhibitory
activity as strong as compound 3 and was found to be more
hydrophilic than compound 3 (logD7.4 values of 0.3 and 2.5,
respectively).
X-ray co-crystal structure of triazolopyrimidine 7 and
PDE10A enzyme revealed that the triazolopyrimidine unit
could fill in the selectivity pocket of PDE10A enzyme as we
expected, and the 4-nitrogen on triazoropyrimidine ring of 7
formed a hydrogen bond with Tyr693 (Fig. 4).
We next investigated the effect of introducing substituents
on the triazolopyrimidine core and the pyrazolopyrimidine
core of compound 7, respectively (Table 2). 5,7-Dimethyltri-
azolopyrimidine 11 showed inhibitory activity (IC =44nM)
5
0
indicating that introduction of substituents on 5,7-position of
triazolopyrimidine ring should be sterically acceptable. In
contrast, 6-methyltriazolopyrimidine 12 showed weaker inhib-
The in vitro PDE10A inhibitory potency of all synthesized itory activity than compound 11, suggesting that 5,7-position
compounds was assessed by measuring the quantity of cAMP was favorable than 6-position. Pyrimidoindazole 13 surpris-
via the Homogeneous Time-Resolved Fluorescence (HTRF) ingly provided a 20-fold superior inhibitory potency in com-
detection method by using human PDE10A enzyme and the parison with compound 11. As the catalytic pocket of PDE10A
inhibitory activities were shown as the inhibitory ratio at the enzyme was predicted to be well occupied by compound 13,

Vol. 66, No. 3 (2018)
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289
Table 2. PDE10A Inhibitory Activity
Fig. 5. Profiles of Compound 13
a: Residual activities of HLM for metabolism of each substrate in the presence of
test compounds were determined following pre-incubation for 30min. b: Intrinsic
clearance. c: No depletion in this condition.
Table 3. PDE10A Inhibitory Activity for Pyrimidoindazole
physicochemical properties and good metabolical stability.
Conclusion
Optimization of compounds guided by X-ray co-crystal
structure from fragment hit 2 and PDE10A enzyme was held
on for finding PDE10A inhibitors with improved physico-
chemical and pharmacokinetic profiles. Being able to obtain
compound 7 with good inhibitory activity, we moved on to
optimization of the substituents. As a result, the pyrimidoin-
dazole derivative 13 which had potent PDE10A inhibitory ac-
tivity, high metabolic stability and no significant CYPs inhibi-
tory activity was obtained. Further investigation of PDE10A
inhibitors on the basis of compound 13 as the lead compound
will be described in the next report.
Compounds
R
PDE10A IC50 (nM)
13
14
15
16
17
18
OH
NHMe
OMe
Me
2.0
7.8
30
64
Cl
221
606
CN
we proceeded further investigation on the basis of 13.
Table 3 shows the results of replacing the hydroxyl group on Experimental 1
-position of the pyrimidoindazole ring with various substitu- Chemistry H-NMR spectra were recorded on a Var-
4
ents. The methyl amino derivative 14 showed strong inhibitory ian 400-MR and BRUKER AV-III HD500, and chemical
activity as compound 13. Although replacement with methoxy shifts were expressed as δ (ppm) values with tetramethyl-
(15) or methyl (16) group caused decrease in inhibitory activ- silane as an internal reference (s=singlet, d=doublet,
ity (IC50 values of 30, 64nM, respectively), the decrease was t=triplet, m=multiplet, dd=double doublet, dt=double triplet,
not extreme. Replacement with chlorine (17) or cyano group ddd=double double doublet, and br=broad peak). MS were re-
(18) resulted in a much more significant decrease in PDE10A corded on a Waters ultra performance liquid chromatography
inhibitory activity (IC50 values of 221, 606nM, respectively). (UPLC)/SQD and Waters Acquity UPLC/ZQ. Electrospray
The reason why hydroxyl group was the most potent as the ionization (ESI) positive high-resolution (HR)-MS were ob-
4
-substituent was not clear unfortunately, but it was specu- tained using a Thermo EXACTIVE-Plus Waters LCT Premier.
lated that electron donating ability of the 4-substituent of the
2-Methyl-5-[2-(quinolin-2-yl)ethyl]pyrazolo[1,5-a]py-
pyrimidoindazole could be important for the potent PDE10A rimidin-7-ol (3) A mixture of ethyl potassium malo-
inhibitory activity. As a result, we identified hydroxyl deriva- nate (1019mg, 6.00mmol), magnesium chloride (571mg,
tive 13 was the most potent PDE10A inhibitor.
6.00mmol) and triethylamine (1070µL, 7.68mmol) in tetra-
The strong potency of compound 13 as a PDE10A inhibitor hydrofuran (THF) (24mL) was stirred at room temperature
prompted us to evaluate its absorption, distribution, metabo- for 2.5h. One milliliter of the reaction mixture was added
lism, and excretion (ADME) properties (Fig. 5). Compound 13 to another reaction mixture of 3-(quinoline-2-yl)propanoic
showed no metabolical depletion under human and mice liver acid (17.3mg, 0.100mmol) and 1,1′-carbonyldiimidazole
microsomes condition. In addition, the inhibitory activities (CDI; 24.3mg, 0.150mmol) in THF (0.2mL) which had been
of compound 13 against CYP enzymes, CYP1A2, 2C8, 2C9, stirred at 50°C for 2h. The combined reaction mixture was
2C19, 2D6 and 3A4, were found to be all weak, and P-glyco- stirred at 50°C for 15h. After cooling at room temperature,
protein (P-gp) liability of compound 13 was proved to be low. to the reaction mixture were added 1M HCl aqueous solu-
The pyrazoropyrimidine derivative 13 showed strong in- tion (0.5mL), CHCl and saturated NaHCO aqueous solution
3
3
hibitory activity against PDE10A (IC50 value of 2.0nM), good (0.5mL). The mixture was through the phase separator. The

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Chem. Pharm. Bull.
Vol. 66, No. 3 (2018)
organic layer was concentrated in vacuo. To the residue was d6) δ: 2.27 (s, 3H), 3.05–3.13 (m, 2H), 3.23–3.30 (m, 2H),
added 3-methyl-1H-pyrazole-5-amine (9.70mg, 0.100mmol) 5.57 (s, 1H), 5.94 (s, 1H), 7.31 (dd, 1H, J=6.8, 4.3Hz), 8.83
in 1,4-dioxane (0.100mL) and AcOH (5.0µL, 87µmol), and (dd, 2H, J=4.3, 1.9Hz), 9.33 (dd, 1H, J=6.8, 1.9Hz), 12.20
+
the mixture was stirred at 90°C for overnight. After cool- (s, 1H); MS (ESI) m/z 296 [M+H] ; HR-MS (ES+) Calcd for
+
ing at room temperature, the mixture was concentrated in C H ON [M+H] 296.1254; Found, 296.1257.
14
14
7
vacuo. The residue was purified by preparative HPLC (Waters
5 - [2 - ( I m i d a z o [1 , 2 - a] p y r i d i n - 2 - y l ) e t h y l ] - 2 -
SunFire™ Column, C18, 5µm, 19×100mm, 10–95% MeOH methylpyrazolo[1,5-a]pyrimidin-7-ol (8) Compound 8 was
in 0.1% (v/v) formic acid aqueous solution) to give 3 (5.4mg, prepared from 3-(imidazo[1,2-a]pyridin-2-yl)propanoic acid
1
1
7%) as a pale brown solid. H-NMR (DMSO-d ) δ: 2.27 (s, in a manner similar to that described for compound 6, with a
3
1
1
6
+
H), 3.09 (dd, 2H, J=9.0, 6.8Hz), 3.32–3.36 (m, 2H), 5.57 (d, yield of 30%. MS (ESI) m/z 294 [M+H] .
H, J=1.4Hz), 5.95 (s, 1H), 7.49 (d, 1H, J=8.4Hz), 7.56 (ddd, 5 -[2 - ( Im ida zo[1, 2 - a]py r i m id i n-2 -yl)ethyl]-2 -
H, J=8.0, 6.9, 1.2Hz), 7.74 (ddd, 1H, J=8.4, 6.9, 1.5Hz), methylpyrazolo[1,5-a]pyrimidin-7-ol (9) Compound 9 was
7
.93–7.96 (m, 2H), 8.30 (d, 1H, J=8.3Hz), 12.23 (s, 1H); MS prepared from 3-(imidazo[1,2-a]pyrimidin-2-yl)propanoic acid
+
(
[
ESI) m/z 305 [M+H] ; HR-MS (ES+) Calcd for C H ON
in a manner similar to that described for compound 6, with a
yield of 10%. MS (ESI) m/z 295 [M+H] .
5-[2-(1H-Benzimidazol-2-yl)ethyl]-2-methylpyrazolo[1,5-
18
17
4
+
+
M+H] 305.1397; Found, 305.1398.
2
-Methyl-5-(quinolin-2-yl)pyrazolo[1,5-a]pyrimidin-7-ol
(4) Compound 4 was prepared from quinaldic acid in a man- a]pyrimidin-7-ol (10) Compound 10 was prepared from
ner similar to that described for compound 3, with a yield 3-(1H-benzimidazole-2-yl)propanoic acid in a manner simi-
1
of 12%. H-NMR (DMSO-d ) δ: 2.34 (s, 3H), 6.22 (s, 1H), lar to that described for compound 3, with a yield of 27%.
6
1
6
.72 (s, 1H), 7.75 (ddd, 1H, J=8.1, 7.0, 0.99Hz), 7.92 (ddd,
H, J=8.4, 6.9, 1.4Hz), 8.12 (d, 1H, J=7.5Hz), 8.24 (d, 1H, 3.23 (t, 2H, J=7.3Hz), 5.54 (s, 1H), 5.95 (s, 1H), 7.08–7.16
H-NMR (DMSO-d ) δ: 2.27 (s, 3H), 3.09 (t, 2H, J=7.5Hz),
6
1
J=8.4Hz), 8.37 (d, 1H, J=8.8Hz), 8.62 (d, 1H, J=8.8Hz), (m, 2H), 7.43–7.53 (m, 2H), 12.30 (brs, 2H); MS (ESI) m/z
+
+
+
1
2.53 (s, 1H); MS (ESI) m/z 277 [M+H] ; HR-MS (ES+) 294 [M+H] ; HR-MS (ES+) Calcd for C H ON [M+H]
16 16 5
+
Calcd for C H ON [M+H] 277.1084; Found, 277.1084.
294.1349; Found, 249.1347.
3-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)pro-
16
13
4
2
-Methyl-5-[(quinolin-2-ylsulfanyl)methyl]pyrazolo[1,5-
a]pyrimidin-7-ol (5) Compound
5
was prepared from panoic Acid (23) A mixture of aminoguanidine hydrochlo-
(
quinolin-2-ylsulfanyl)acetic acid in a manner similar to that ride 21 (25.0g, 226mmol) and succinic anhydride 22 (25.0g,
1
described for compound 3, with a yield of 27%. H-NMR 250mmol) was stirred at 190°C for 1h. After cooling at room
DMSO-d ) δ: 2.26 (s, 3H), 4.52 (s, 2H), 5.84 (s, 1H), 6.00 (s, temperature, to the reaction mixture were added sodium
(
6
1
H), 7.47 (d, 1H, J=8.6Hz), 7.54 (ddd, 1H, J=8.0, 7.0, 1.1Hz), hydroxide (27.0g, 675mmol) and H O (67mL). The mixture
2
7
.76 (ddd, 1H, J=8.4, 7.0, 1.4Hz), 7.90–7.98 (m, 2H), 8.23 (d, was stirred at 120°C for 1.5h. After cooling, to the reaction
+
1H, J=8.6Hz), 12.43 (brs, 1H); MS (ESI) m/z 323 [M+H] ; mixture were added conc. HCl aqueous solution (37mL) and
+
HR-MS (ES+) Calcd for C H ON S [M+H] 323.0961; H O (25mL) at room temperature, and the mixture was stood
17
15
4
2
Found, 323.0961.
overnight. The resulting precipitate under ice-bath cooling
2
-Methyl-5-[2-(pyridin-2-yl)ethyl]pyrazolo[1,5-a]py- was collected by filtration. To the residue were added EtOH
rimidin-7-ol (6) A mixture of ethyl potassium malo- (400mL), acetylacetone (20.0mL, 194mmol) and AcOH
nate (2145mg, 12.6mmol), magnesium chloride (1428mg, (2.00mL, 35.0mmol), and the mixture was stirred under
15.0mmol) and triethylamine (2676µL, 19.2mmol) in THF reflux for 7h. After cooling at room temperature, to the mix-
(
60mL) was stirred at room temperature for 2.5h. Half mil- ture were added H O (30mL) and sodium hydroxide (10.0g,
2
liliter of the reaction mixture was added to another reaction 250mmol), and the mixture was stirred at room temperature
mixture of 3-(pyridin-2-yl)propanoic acid (7.56mg, 50.0µmol) for 2h. The mixture was filtered and the filtrate was concen-
and CDI (12.2mg, 75µmol) in THF (0.2mL) which had been trated in vacuo lightly. To the solution was added conc. HCl
stirred at 50°C for 2h. The combined reaction mixture was aqueous solution (20mL) and the mixture was cooled under
stirred at 50°C for overnight. After cooling at room tem- ice-bath. The resulting precipitate was collected by filtra-
1
perature, to the reaction mixture were added 1M HCl aque- tion to give 23 (33.7g, 68%) as a colorless solid. H-NMR
ous solution (0.5mL), CHCl (2.5mL) and saturated NaHCO3 (DMSO-d ) δ: 2.55 (s, 3H), 2.69 (d, 3H, J=0.9Hz), 2.76 (t, 2H,
3
6
aqueous solution (0.5mL). The mixture was through the phase J=7.3Hz), 3.05 (t, 2H, J=7.4Hz), 7.10 (d, 1H, J=0.9Hz), 12.20
+
separator. The organic layer was concentrated in vacuo. To the (brs, 1H); MS (ESI) m/z 221 [M+H] .
residue was added 3-methyl-1H-pyrazole-5-amine (4.90mg,
5-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-
0µmol) in 1,4-dioxane (0.100mL) and AcOH (5.0µL, yl)ethyl]-2-methylpyrazolo[1,5-a]pyrimidin-7-ol (11) To a
7µmol), and the mixture was stirred at 90°C for overnight. mixture of 23 (661mg, 3.00mmol) in THF (10mL) was added
5
8
After cooling at room temperature, the mixture was con- CDI (584mg, 3.60mmol), and the mixture was stirred at 50°C
centrated in vacuo. The residue was purified by preparative for 1h. To the reaction mixture were added ethyl potassium
HPLC (Waters SunFire™ Column, C18, 5µm, 19×100mm, malonate (1.02g, 6.00mmol), magnesium chloride (571mg,
1
0–95% MeOH in 0.1% (v/v) formic acid aqueous solution) to 6.00mmol), triethylamine (1.04mL, 7.50mmol), and the mix-
+
give 6 (5.3mg, 42%). MS (ESI) m/z 255 [M+H] .
ture was stirred at 50°C for 12h. After cooling at room tem-
2
-Methyl-5-[2-([1,2,4]triazolo[1,5-a]pyrimidin-2-yl)eth- perature, to the reaction mixture was added 1M HCl aqueous
yl]pyrazolo[1,5-a]pyrimidin-7-ol (7) Compound
7
was solution, and the mixture was stirred at ambient temperature
prepared from 3-([1,2,4]triazolo[1,5-a]pyrimidine-2-yl)propano for 1h. The mixture was extracted with EtOAc. The organic
ic acid in a manner similar to that described for compound 3, layer was washed with brine, dried over anhydrous Na SO ,
2
4
1
with a yield of 40% as a pale brown solid. H-NMR (DMSO- filtered and concentrated in vacuo. To the residue were added

Vol. 66, No. 3 (2018)
Chem. Pharm. Bull.
291
1,4-dioxane (10mL), 5-methyl-1H-pyrazol-3-amine (200mg, 13 (280mg, 0.779mmol) and phosphorous oxychloride (3.35g,
2
.06mmol) and AcOH (1.18mL, 20.6mmol), and the mixture 21.8mmol) was stirred at 100°C for 1d. After cooling at room
was stirred at 90°C for 12h. After cooling at room tempera- temperature, the mixture was concentrated in vacuo. The
ture, the mixture was diluted with Et O, and the resulting pre- residue was diluted with saturated NaHCO aqueous solution
2
3
cipitate was collected by filtration to give 11 (614mg, 92%) and extracted with CHCl . The organic layer was washed
3
1
as a beige solid. H-NMR (DMSO-d ) δ: 2.27 (s, 3H), 2.56 (s, with brine, dried over anhydrous MgSO , filtered, and con-
6
4
3
2
H), 2.68 (d, 3H, J=1.0Hz), 3.01–3.11 (m, 2H), 3.20–3.28 (m, centrated in vacuo. The residue was purified by flash column
H), 5.57 (s, 1H), 5.94 (s, 1H), 7.12 (d, 1H, J=0.8Hz), 12.20 chromatography (silica gel, 0–3% MeOH in CHCl ) to give
3
+
1
(
br-s, 1H); MS (ESI) m/z 324 [M+H] ; HR-MS (ES+) Calcd 17 (112mg, 38%) as a yellow solid. H-NMR (DMSO-d ) δ:
6
+
for C H ON [M+H] 324.1567; Found, 324.1569.
2.55 (s, 3H), 2.68 (d, 3H, J=0.9Hz), 3.36–3.58 (m, 4H), 7.10
16
18
7
2
-Methyl-5-[2-(6-methyl[1,2,4]triazolo[1,5-a]pyrimidin- (d, 1H, J=0.9Hz), 7.32 (ddd, 1H, J=8.3, 6.7, 0.7Hz), 7.67 (ddd,
2
of
-yl)ethyl]pyrazolo[1,5-a]pyrimidin-7-ol (12) To a mixture 1H, J=8.7, 6.7, 1.1Hz), 7.87 (dt, 1H, J=8.6, 0.9Hz), 7.98 (s,
+
3-(6-methyl[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)propanoic 1H), 8.23 (dt, 1H, J=8.3, 1.0Hz); MS (ESI) m/z 378 [M+H] ;
+
acid (619mg, 3.00mmol) in THF (10mL) was added CDI HR-MS (ES+) Calcd for C H N Cl [M+H] 378.1228;
19
17
7
(584mg, 3.60mmol), and the mixture was stirred at 50°C Found, 378.1230.
for 1h. To the reaction mixture were added ethyl potassium
2-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-
malonate (1.02g, 6.00mmol), magnesium chloride (571mg, yl)ethyl]-N-methylpyrimido[1,2-b]indazol-4-amine (14) To
.00mmol), triethylamine (1.04mL, 7.50mmol), and the mix- a solution of 17 (96.0mg, 0.254mmol) in THF (0.96mL) was
6
ture was stirred at 50°C for 12h. After cooling at room tem- added methylamine solution (381µL, 0.762mmol) and the
perature, to the reaction mixture was added 1M HCl aqueous mixture was stirred at room temperature for 17h. The mixture
solution, and the mixture was stirred at ambient temperature was concentrated in vacuo, and the residue was purified by
for 1h. The mixture was extracted with EtOAc. The organic flash column chromatography (silica gel, 0–20% MeOH in
1
layer was washed with brine, dried over anhydrous Na SO , CHCl ) to give 14 (92.0mg, 97%) as a yellow solid. H-NMR
2
4
3
filtered and concentrated in vacuo. To the residue were added (DMSO-d ) δ: 2.56 (s, 3H), 2.70 (d, 3H, J=0.7Hz), 3.04 (d,
6
1,4-dioxane (10mL), 5-methyl-1H-pyrazol-3-amine (200mg, 3H, J=4.9Hz), 3.33–3.43 (m, 4H), 6.58 (s, 1H), 7.05–7.14 (m,
2
9
.06mmol) and AcOH (1.18mL, 20.6mmol), and stirred at 2H), 7.43–7.58 (m, 1H), 7.66 (d, 1H, J=8.6Hz), 8.05–8.13 (m,
+
0°C for 12h. After cooling at room temperature, the mixture 1H), 8.14–8.23 (m, 1H); MS (ESI) m/z 373 [M+H] ; HR-MS
+
was diluted with Et O, and the resulting precipitate was col- (ES+) Calcd for C H N [M+H] 373.1884; Found, 373.1886.
2
20 21
8
lected by filtration to give 12 (487mg, 76%) as a beige solid.
2-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-
1
H-NMR (DMSO-d ) δ: 2.26 (s, 3H), 2.36 (d, 3H, J=0.8Hz), yl)ethyl]-4-methoxypyrimido[1,2-b]indazole (15) To a so-
6
3
.01–3.11 (m, 2H), 3.20–3.29 (m, 2H), 5.55 (s, 1H), 5.93 (s, lution of 17 (100mg, 0.265mmol) in MeOH (1.00mL) was
1
H), 8.73 (d, 1H, J=2.4Hz), 9.17 (dd, 1H, J=2.3, 1.1Hz), 12.20 added sodium methoxide (28.6mg, 0.529mmol) and the mix-
+
(s, 1H); MS (ESI) m/z 310 [M+H] ; HR-MS (ES+) Calcd for ture was stirred at room temperature for 3h. The reaction
+
C H ON [M+H] 310.1411; Found, 310.1414.
mixture was diluted with H O, and the resulting precipitate
15
16
7
2
2
-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2- was collected by filtration. The residue was purified by flash
yl)ethyl]pyrimido[1,2-b]indazol-4-ol (13) To a mixture of column chromatography (silica gel, 0–10% MeOH in CHCl3).
2
3 (2.20g, 10.0mmol) in THF (33mL) was added CDI (1.95g, The residue was washed with EtOAc to give 15 (81.0mg,
1
12.0mmol), and the mixture was stirred at 50°C for 1h. To the 82%) as a yellow solid. H-NMR (DMSO-d ) δ: 2.56 (s, 3H),
6
reaction mixture were added ethyl potassium malonate (3.40g, 2.70 (d, 3H, J=0.9Hz), 3.39–3.55 (m, 4H), 4.28 (s, 3H), 7.11
0.0mmol), magnesium chloride (1.90g, 20.0mmol), triethyl- (d, 1H, J=0.9Hz), 7.14–7.29 (m, 2H), 7.56 (ddd, 1H, J=8.8,
amine (3.48mL, 25.0mmol), and the mixture was stirred at 6.7, 1.1Hz), 7.72 (dt, 1H, J=8.7, 0.9Hz), 8.15 (dt, 1H, J=8.3,
2
+
5
0°C for 12h. After cooling at room temperature, to the reac- 1.0Hz); MS (ESI) m/z 374 [M+H] ; HR-MS (ES+) Calcd for
+
tion mixture was added 1M HCl aqueous solution (50mL), and C H ON [M+H] 374.1724; Found, 374.1726.
2
0
20
7
the mixture was stirred at ambient temperature for 1h. The
mixture was extracted with EtOAc. The organic layer was yl)ethyl]-4-methylpyrimido[1,2-b]indazole
washed with brine, dried over anhydrous Na SO , filtered and stirred mixture of 17 (50.0mg, 0.132mmol), trimethylborox-
2-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-
(16) To
a
2
4
concentrated in vacuo. To a half of the residue were added ine (49.8mg, 0.397mmol) and [1,1′-bis(diphenylphosphino)-
,4-dioxane (15mL), 1H-indazol-3-amine (610mg, 4.58mmol) ferrocene]palladium(II) dichloride dichloromethane adduct
and AcOH (4mL), and stirred at 95°C for 18h. After cooling (Pd(dppf)Cl ·CH Cl ; 32.4mg, 39.7µmol) in 1,4-dioxane
1
2
2
2
at room temperature, the mixture was concentrated in vacuo. (1.00mL) was added K CO (110mg, 0.794mmol) under argon
2
3
The residue was diluted with EtOAc and saturated NaHCO3 atmosphere, and the mixture was stirred at 90°C for 1d.
aqueous solution, and the resulting precipitate was col- After cooling at room temperature, the mixture was diluted
lected by filtration to give 13 (194mg, 11%) as a beige solid. with water and extracted with CHCl . The organic layer was
3
1
H-NMR (DMSO-d ) δ: 2.56 (s, 3H), 2.70 (d, 3H, J=0.7Hz), washed with brine, dried over anhydrous MgSO , filtered
.03–3.13 (m, 2H), 3.21–3.27 (m, 2H), 5.76 (s, 1H), 6.82 (ddd, and concentrated in vacuo. The residue was purified by flash
6
4
3
1
H, J=7.9, 6.8, 0.8Hz), 7.09 (d, 1H, J=0.9Hz), 7.24–7.33 (m, column chromatography (silica gel, 0–5% MeOH in CHCl )
3
1
1
H), 7.44 (d, 1H, J=8.6Hz), 7.90 (dt, 1H, J=8.2, 1.0Hz); MS to give 16 (16.0mg, 34%) as a pale yellow solid. H-NMR
+
(
[
ESI) m/z 360 [M+H] ; HR-MS (ES+) Calcd for C H ON
M+H] 360.1567; Found, 360.1568.
(DMSO-d ) δ: 2.55 (s, 3H), 2.69 (d, 3H, J=0.7Hz), 2.88 (d,
19
18
7
6
+
3H, J=0.7Hz) 3.36–3.54 (m, 4H), 7.10 (d, 1H, J=0.9Hz), 7.24
4
-Chloro-2-[2-(5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimi- (ddd, 1H, J=8.2, 6.7, 0.8Hz), 7.43–7.65 (m, 2H), 7.70–7.90 (m,
+
din-2-yl)ethyl]pyrimido[1,2-b]indazole (17) A mixture of 1H), 8.20 (dt, 1H, J=8.3, 0.9Hz); MS (ESI) m/z 358 [M+H] ;

2
92
Chem. Pharm. Bull.
Vol. 66, No. 3 (2018)
+
HR-MS (ES+) Calcd for C H N [M+H] 358.1775; Found, with Cryptate, Cisbio). After 60min, the fluorescence transfer
2
0
20
7
3
58.1776.
-[2-(5,7-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2- at lex. 320nm, lem. 620nm and lem. 665nm using an Envi-
yl)ethyl]pyrimido[1,2-b]indazole-4-carbonitrile (18) sion plate reader (PerkinElmer, Inc., U.S.A.) and signal ratio
corresponding to the amount of residual cAMP was measured
2
A
mixture of 17 (50.0mg, 0.132mmol), potassium cya- (665/620) was calculated. The ratio determined in the absence
nide (18.0mg, 0.276mmol) and 1,4-diazabicyclo[2.2.2]octane of enzyme was subtracted from all data. The obtained results
(
DABCO; 18.0mg, 0.160mmol) in 1,4-dioxane (2.0mL) and were converted to inhibition percentage relative to an uninhib-
H O (0.20mL) was stirred at room temperature for 5h. The ited control.
2
residue was diluted with H O. The resulting precipitate was
PDE10A Inhibition Assay (for Inhibition Assay of Com-
2
collected by filtration and purified by flash column chro- pounds 11–18) Inhibitory activity of compounds on human
matography (silica gel, 0–5% MeOH in CHCl ) to give 18 PDE10A2 was assessed by measuring the residual amount
3
1
(
18.0mg, 37%) as a bright red solid. H-NMR (DMSO-d ) of cAMP, substrate for PDE10A2, by the HTRF detection
6
8)
δ: 2.55 (s, 3H), 2.68 (s, 3H), 3.40–3.60 (m, 4H), 7.10 (s, 1H), method (cisbio). The obtained results were converted to ac-
.36–7.50 (m, 1H), 7.73 (ddd, 1H, J=8.4, 7.0, 1.0Hz), 7.94 (d, tivity relative to an uninhibited control (100%) and IC50 values
7
1
H, J=8.6Hz), 8.26–8.36 (m, 1H), 8.40 (s, 1H); MS (ESI) were calculated using Prism software (GraphPad Software,
+
+
m/z 369 [M+H] ; HR-MS (ES+) Calcd for C H N [M+H]
3
Inc., U.S.A.).
2
0
17
8
69.1571; Found, 369.1571.
Crystallization and Structural Analysis PDE10A crys-
tals were obtained as previously reported. Briefly, crystals
3
0)
PDE10A Enzyme Assay Protocol
were precipitated by sitting drop vapor diffusion methods.
Expression and Purification of PDE10A Protein (for The reservoir solution contains 0.05M bis-tris propane (pH
Inhibition Assay of Compounds 2–10) Human PDE10A 6.0), 0.1M magnesium sulfate, 15–18% (w/v) PEG3350. Com-
phosphodiesterase domain (amino acids 449–789) was cloned pounds were soaked into the apo PDE10A crystal. An apo
between the NdeI and XhoI sites of pET28a vector to express crystal was transferred to a mother liquor containing com-
target protein as a 6×histidine (His)-tagged protein. This pound (5mM final concentration) and incubated at 4°C for
plasmid was transformed in Escherichia coli BL21(DE3) and three days. X-ray diffraction data were collected at AR-NE3A
31)
overexpressed. The cells were disrupted by sonication and beamline at the Photon Factory in the National Laboratory
the supernatant was collected by centrifugation at 4°C. The for High Energy Physics (KEK), Tsukuba, Japan. Diffraction
32)
target protein was precipitated from the supernatant by adding data were indexed, integrated, and scaled using HKL2000.
ammonium sulfate to 60% and was collected by centrifuga- Crystal structures of PDE10A were determined by the mo-
33)
30)
tion. The precipitate was dissolved with nickel–nitrilotriacetic lecular replacement method using AMoRe with 2OUN as
acid (Ni–NTA) wash buffer (25mM Tris–HCl pH 8.0, 500mM a search model. An initial refinement was performed using
3
4)
NaCl, 20mM imidazole) and was applied onto an Ni–NTA REFMAC. Compounds were fitted into electron densities
column. After washing with Ni–NTA wash buffer, the bound observed in initial Fo-Fc maps using AFITT (OpenEye Sci-
protein was eluted with 25mM Tris–HCl pH 8.0, 500mM entific Software, NM, U.S.A.). Water placements and further
35)
NaCl, 250mM imidazole. Eluate was collected and the buffer refinements were performed using Coot and REFMAC, and
exchanged with 25mM Tris–HCl pH 8.0, 500mM NaCl. The
amino-terminal His tag of PDE10A (449–789) was removed
by incubation with thrombin protease for 12h at 4°C. After
Table 4. Data Processing and Refinement Statistics
digestion, protein solution was passed through Benzamidine
Sepharose 6B column (GE Healthcare, U.S.A.) and again ap-
plied onto an Ni–NTA column to remove thrombin and un-
cleaved His-tag protein. Further purification was performed by
passing through Q Sepharose column (GE Healthcare) equili-
brated with 25mM Tris–HCl pH 8.0, 150mM NaCl followed
by Superdex 75pg 16/60 (GE Healthcare) in 25mM N-(2-
hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-
Na pH 7.5, 100mM NaCl.
Compound
2
7
PDB ID
Space group
a (Å)
5XUJ
5XUI
P2 2 2
P2 2 2
1 1 1
1
1
1
50.3
81.3
157.6
90
49.3
81.4
158.7
90
b (Å)
c (Å)
α (°)
β (°)
90
90
γ (°)
90
90
PDE10A Inhibition Assay (for Inhibition Assay of
Compounds 2–10) Inhibition of human PDE10A enzyme
activity was assessed by measuring the quantity of cAMP
via the Homogeneous Time-Resolved Fluorescence (HTRF)
detection method. The assay was performed in 20µL samples
containing an optimal amount of the PDE10A enzyme do-
Resolution (Å)
Multiplicity
50.0–2.44 (2.48–2.44) 50.0–2.77 (2.82–2.77)
4.8 (5.0)
31.7 (5.8)
7.3 (35.6)
21730 (1567)
92.0 (91.7)
19.8
5.5 (5.1)
25.2 (4.1)
8.7 (41.9)
15497 (1103)
96.6 (98.2)
18.5
Average I/σ(I)
a)
R
merge (%)
No. of reflections
Completeness (%)
Rworkb) (%)
main, a buffer (50mM Tris–HCl pH 8.0; 2mM MgCl , 0.1%
2
Rfreec) (%)
28.0
27.2
BSA), 0.1µM cAMP and various concentrations of compounds
2
Average B factor (Å )
RMSD bond length (Å)
RMSD bond angle (°)
52.4
57.6
(0.03nM to 40µM). After compounds were mixed with the
0.011
0.010
enzyme, the reaction was initiated by adding the substrate
cAMP and the mixture was incubated for 30min at room tem-
perature with agitation. The reaction was terminated on addi-
tion of the fluorescence acceptor (cAMP labeled with the dye
d2) and the fluorescence donor (anti-cAMP antibody labeled
1.569
1.461
Values for the outer shell are given in parentheses. a) Rmerge=∑ ∑ |I −I|/∑ ∑ I ,
hkl
i
i
hkl
i i
where I is the intensity of an individual reflection and I is the mean intensity obtained
i
from multiple observations of symmetry related reflections. b) Rwork=∑hkl||Fobs|−|Fcalc||
∑hkl|Fobs|. c) Five percent randomly omitted reflections were used for Rfree.
/

Vol. 66, No. 3 (2018)
Chem. Pharm. Bull.
293
the final models were determined. Data collection and refine- for 1h. After aspiration of the transport buffer, the donor
ment statistics are summarized in Table 4. solution (transport buffer (0.5% dimethyl sulfoxide (DMSO))
Mouse and Human Liver Microsomal Assays Pooled containing the test compound (1µM) and Texas Red (1µM))
mouse or human liver microsomes (Xenotech LLC., U.S.A.) was added to the apical or basolateral side for the influx or
were diluted in 100mM KH PO /K HPO buffer (pH 7.4) con- efflux transport study, respectively, and the receiver solution
2
4
2
4
taining 0.1mM ethylenediaminetetraacetic acid (EDTA). The (transport buffer (0.5% DMSO)) was added to the opposite
incubation mixtures (270µL total volume), which contained side. After incubation for 3h, the test compound in both sides
0.2mg/mL of microsomal proteins, and 0.2µM of substrates was analyzed by LC/MS/MS and the apparent permeability
were pre-incubated for approximately 15min at 37°C. Reac- was determined. Efflux ratio (ER) was calculated by dividing
tions were initiated by the addition of 1mM reduced nicotin- the apparent permeability in the direction from the basolateral
amide adenine dinucleotide phosphate (NADPH) (30µL). to the apical side by that in the opposite direction. Net efflux
After the appropriate incubation time (0, 15, 30, 45min), 40µL ratio (NER) was the ratio of ER of LLC-PK1-MDR1 to LLC-
of incubation mixture was transferred into 80% acetonitrile PK1-WT. Texas Red was used for the estimation of the appar-
containing internal standard (200nM methyltestosterone, ent permeability via para cellular transport.
250µL), stood at 4°C for 20min, and centrifuged for 20min
at 2800rpm. The supernatant (200µL) was prepared and ana-
Acknowledgments The authors wish to thank to Dr.
lyzed via LC-MS/MS with UPLC system (Waters) and Xevo Takeshi Shimada for performing pharmacological evaluations,
TQ (Waters). The in vitro intrinsic clearance (CLint, vitro) was Mr. Hiroyuki Moriguchi for his helpful support in preparing
calculated using Eq. 1, which is based on the time course of this manuscript, and the staff of Astellas Research Technolo-
3
6)
the residual ratio of the compounds.
gies Co., Ltd. for conducting the CYP inhibition screening,
metabolic clearance assay, partition coefficient assay, elemen-
(1) tal analysis, and spectral measurements.
Ke (1/min)×MS content (mg/kg)
CLint,vitro (mL/min/kg)=
MS protein conc. (mg/mL)
where the Ke is the disappearance rate constant.
Conflict of Interest All authors were employees of Astel-
CYPs Inhibitory Assays Time-dependent inhibition las Pharma Inc. when the study was conducted and have no
assay for CYP activity was performed in two steps, a pre- further conflicts of interest to declare.
incubation step where the test compound was incubated with
human liver microsomes and the secondary incubation period References
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