Novel benzimidazole derivatives as phosphodiesterase 10A (PDE10A) inhibitors with improved metabolic stability

Article abstract of DOI:10.1016/j.bmc.2014.04.023

In this study, we report the identification of potent benzimidazoles as PDE10A inhibitors. We first identified imidazopyridine 1 as a high-throughput screening hit compound from an in-house library. Next, optimization of the imidazopyridine moiety to improve inhibitory activity gave imidazopyridinone 10b. Following further structure-activity relationship development by reducing lipophilicity and introducing substituents, we acquired 35, which exhibited both improved metabolic stability and reduced CYP3A4 time-dependent inhibition.

Full text of DOI:10.1016/j.bmc.2014.04.023

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel benzimidazole derivatives as phosphodiesterase 10A (PDE10A)
inhibitors with improved metabolic stability
⇑
Ayaka Chino , Naoyuki Masuda, Yasushi Amano, Kazuya Honbou, Takuma Mihara, Mayako Yamazaki,
Masaki Tomishima
Institute for Drug Discovery Research, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we report the identification of potent benzimidazoles as PDE10A inhibitors. We first iden-
tified imidazopyridine 1 as a high-throughput screening hit compound from an in-house library. Next,
optimization of the imidazopyridine moiety to improve inhibitory activity gave imidazopyridinone
10b. Following further structure–activity relationship development by reducing lipophilicity and intro-
ducing substituents, we acquired 35, which exhibited both improved metabolic stability and reduced
CYP3A4 time-dependent inhibition.
Received 7 March 2014
Revised 11 April 2014
Accepted 12 April 2014
Available online xxxx
Keywords:
Ó 2014 Published by Elsevier Ltd.
PDE10A inhibitor
Schizophrenia
Benzimidazole
Imidazopyridine
Metabolic stability
1. Introduction
Inhibition of PDE10A is hypothesized to be effective in treating
schizophrenia and a range of neurological, psychotic, anxiety, and
Schizophrenia is a chronic debilitating disorder affecting the
psychic and motor functions of the brain1 that is typically diag-
nosed in individuals in their early to mid-twenties. The symptoms
normally manifest in three broad categories: positive symptoms,
negative symptoms, and cognitive impairments. Current anti-
psychotics are marginally effective for treating the positive
symptoms but less effective against negative symptoms and
cognitive impairments. New therapies with improved efficacy against
negative symptoms and cognitive impairments would therefore
represent a significant advance in schizophrenia treatment.
The phosphodiesterases (PDEs) are an 11-membered family of
enzymes that catalyze the hydrolysis of secondary signal messen-
gers, cyclic adenosine monophosphate (cAMP) and cyclic guano-
sine monophosphate (cGMP). Phosphodiesterase 10A (PDE10A) is
a dual substrate PDE that hydrolyzes both cAMP and cGMP.
Expression of PDE10A is highest in the brain, particularly in the
medium spiny neurons of the striatum, a distribution conserved
across mammalian species and is limited in peripheral tissues.2
movement disorders which benefit from increasing levels of cAMP
and cGMP within neurons. Therefore, agents that inhibit PDE10A
might prove useful in treating neurological and psychiatric
disorders.3
To date, a large number of studies of PDE10A inhibitors have
been reported.4 One PDE10A inhibitor, MP-10, was recently under
clinical evaluation for the treatment for schizophrenia.5 Preclinical
evidence further suggests that these inhibitors might demonstrate
anti-psychotic, pro-cognitive, and negative symptom efficacy.5a
We recently conducted structure–activity relationship (SAR) stud-
ies on novel quinoline derivatives as potent inhibitors of PDE10A6
and are currently conducting studies on a related series of com-
pounds. We therefore decided to explore new scaffold.
In our high-throughput screening (HTS) study, we identified 1
(PDE10A IC50 = 1700 nM) as a low-micromolar PDE10A inhibitor
(Fig. 1). Results of a focused SAR study showed that the introduc-
tion of a phenyl ring to the N-1 position on benzimidazole greatly
improved inhibitory activity. In particular, 14a, which has a methyl
group at the 5-position on benzimidazole, was approximately 3
times more active than 1 in the enzyme assay (PDE10A IC50
=
⇑
Corresponding author at present address: Medicinal Chemistry IV, Chemistry
590 nM). We therefore initiated further optimization of compound
14a. In the present study, we refer to the benzimidazole analogue
as the left part and the other heterocycle as the right part in each
compound.
Research Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc.,
21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Tel.: +81 (0)29 829 6261; fax:
+81 (0)29 854 1519.
E-mail address: ayaka.chino@astellas.com (A. Chino).
http://dx.doi.org/10.1016/j.bmc.2014.04.023
0968-0896/Ó 2014 Published by Elsevier Ltd.
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A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
were treated under catalytic hydrogenation conditions to give
2. Chemistry
23a–b. Amidation of 23a–b with carboxylic acid 9 followed by
cyclization in AcOH gave 24a–b.
The synthesis of target compounds is shown in Schemes 1–6.
The synthetic route of imidazo[4,5-b]pyridin-2-one derivatives
10a–f is outlined in Scheme 1. A Buchwald–Hartwig reaction of
bromonitrobenzene 2–4 with corresponding aryl amine gave the
nitro aniline analogues 5a–f. Catalytic hydrogenation of 5a–f
yielded the phenylenediamines 6a–f. Compound 9 was prepared
with Michael addition of 77 with ethyl acrylate and following
hydrolysis. Phenylenediamines 6a–f were condensed with 9 and
cyclized in AcOH to give 10a–f.
As shown in Scheme 5, synthesis of imidazo[4,5-b]pyrazin-2-
one analogue 29 started with a coupling reaction of methylamine
to 3-chloropyrazin-2-amine (25) to give the N-methylpyrazine-
2,3-diamine (26). Cyclization of 26 by 1,10-carbonylbis-1H-imidaz-
ole (CDI) followed by alkylation with ethyl acrylate gave the ester
27. Hydrolysis of 27 with NaOH aqueous solution yielded the
carboxylic acid 28. Amidation of 28 with diaminopyridine 23a
followed by cyclization in AcOH gave 29.
As shown in Scheme 6, synthesis of 3,7-dimethyl-imidazo[4,5-
b]pyridin-2-one 35 started with displacement of b-alanine ethyl
ester with 2-chloro-4-methyl-3-nitropyridine (30) which gave 3-
nitropyridin-2-amine 31. Catalytic hydrogenation and condensa-
tion with CDI of 31 gave imidazopyridine 32. Methylation of 32
followed by hydrolysis with NaOH aqueous solution gave carbox-
ylic acid 34. Amidation with 34 and diaminopyridine 23a followed
by cyclization in AcOH gave 35.
The synthesis of imidazopyridazine analogues 14a–d and 16 is
illustrated in Scheme 2. Condensation of the phenylenediamine
analogue 6b with mono-methyl succinate and cyclization in AcOH
gave 11, which was then hydrolyzed with NaOH aqueous solution
to give carboxylic acid 12. Condensation of 12 with 2-aminometh-
ylheteroaryl (13a–d) in the presence of propylphosphonic anhy-
dride (T3P)8 gave cyclized 14a–d. Bromination of 14d with NBS
gave 15, which was followed by a Suzuki–Miyaura cross coupling
reaction with trimethylboroxine to give 16.
Pyrazolo[3,4-b]pyridine 20a was synthesized in accordance
with the procedures outlined in Scheme 3. Michael addition of
17a9 with ethyl acrylate gave 18a. Ester 18a was hydrolyzed with
NaOH aqueous solution to give the carboxylic acid 19a. Compound
19a was condensed with the phenylenediamine derivative 6b in
the presence of HATU and DIPEA and then cyclized in AcOH to give
20a. Pyrido[2,3-b]pyrazin-3(4H)-one 20b was prepared from 17b10
in a manner similar to that described for 20a.
The azabenzimidazole analogues 24a–b were synthesized as
depicted in Scheme 4. A coupling reaction of 2-chloropyridine
21a with aniline gave the 2-aminopyridine analogue 22a. On the
other hand, a reaction of the 2-chloropyridine 21b with an excess
of aniline under microwave irradiation gave the 2-aminopyridine
derivative 22b. The 2-amino-3-nitropyridine derivatives 22a–b
3. Results and discussion
PDE10A inhibitory potencies of newly synthesized compounds
were evaluated using in vitro inhibition of human recombinant
PDE10A catalyzed cAMP hydrolysis. Compound 14a exhibited
moderate PDE10A inhibitory activity with an IC50 of 590 nM.
Referring to the literature,4d we speculated that the nitrogen of
benzimidazole interacts with Tyr693 of the binding pocket while
the 1-nitrogen of the imidazopyridine interacts with Gln726. Initial
SAR evaluation focused on the in vitro potency of PDE10A. Since we
considered that a benzimidazole unit was required for selectivity
from other PDEs,5b we first optimized the imidazopyridine ring.
The SAR results for optimization of the substitutions and the
different numbers and positions of nitrogen atoms are summarized
in Table 1. Removal of the 1-methyl substituent of 14a diminished
the inhibitory activity, indicating that this substituent of imidazo-
pyridine is important (14b). It was speculated that the electron
density of the 2-nitrogen atom might influence the binding
between the 2-nitrogen atom of imidazopyridine and Gln726. We
therefore evaluated the effect of heterocycles in the right part by
introducing an additional nitrogen atom. Incorporation of this
nitrogen atom (14c and 16) led to an increase in in vitro potency.
Interestingly, despite only a small structural difference between
14c and 16, a 20-fold improvement in IC50 value was noted to
16. To clarify the differences in inhibitory activity, we obtained
N
Me
N
N
N
N
N
O
N
N
H
1
14a
Figure 1. Structures of compounds 1 and 14a.
R
R
NO2
R
NH2
a
NO2
Br
b
NH
Ar
NH
Ar
R
N
N
O
2: R = 3-MeO
3: R = 4-Me
4: R = 4-MeO
5a: R = 3-MeO, Ar = Ph
5b: R = 4-Me, Ar = Ph
5c: R = 4-MeO, Ar = Ph
6a: R = 3-MeO, Ar = Ph
e, f
6b: R = 4-Me, Ar = Ph
N
6c: R = 4-MeO, Ar = Ph
N
5d: R = 4-MeO, Ar = pyridin-2-yl
5e: R = 4-MeO, Ar = pyridin-3-yl
5f : R = 4-MeO, Ar = pyridin-4-yl
6d: R = 4-MeO, Ar = pyridin-2-yl
6e: R = 4-MeO, Ar = pyridin-3-yl
6f : R = 4-MeO, Ar = pyridin-4-yl
Ar
N
10a: R = 4-MeO, Ar = Ph
10b: R = 5-Me, Ar = Ph
10c: R = 5-MeO, Ar = Ph
EtO
O
HO
O
O
c
d
10d: R = 5-MeO, Ar = pyridin-2-yl
10e: R = 5-MeO, Ar = pyridin-3-yl
10f : R = 5-MeO, Ar = pyridin-4-yl
N
N
N
O
N
O
N
HN
N
N
N
7
8
9
Scheme 1. Reagents and conditions: (a) Ar-NH2, Pd2(dba)3, BINAP, Cs2CO3, toluene, 33–97%; (b) H2, Pd(OH)2-C, EtOH, 14–79%; (c) ethyl acrylate, DBU, THF, 65%; (d) NaOH aq,
EtOH, 99%; (e) HATU, DIPEA, CH2Cl2; (f) AcOH, 21–99% (2 steps).
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A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
NH2
NH
N
N
N
c
a, b
O
O
N
OMe
OH
12
6b
11
N
N
N
X
H2N
d
X
O
N
N
+
N
Z
N
Z
OH
Y
Y
12
13a: X = Me, Y = CH, Z = CH
13b: X = H, Y = CH, Z = CH
13c: X = Me, Y = N, Z = CH
13d: X = H, Y = CH, Z = N
14a: X = Me, Y = CH, Z = CH
14b: X = H, Y = CH, Z = CH
14c: X = Me, Y = N, Z = CH
14d: X = H, Y = CH, Z = N
e
f
15 : X = Br, Y = CH, Z = N
16 : X = Me, Y = CH, Z = N
Scheme 2. Reagents and conditions: (a) mono-methyl succinate, HATU, DIPEA, CH2Cl2; (b) AcOH, 96% (2 steps); (c) NaOH aq, MeOH, 81%; (d) propylphosphonic anhydride,
EtOAc, 33–82%; (e) N-bromosuccinimide, CH3CN, CH2Cl2, 81%; (f) trimethylboroxine, PdCl2(dppf)2, K2CO3, 1,4-dioxane, 35%.
N
N
O
HN
N
N
O
O
b
HN
a
c, d
N
R
or
EtO
HO
R
R
N
17a
18a-b
19a-b
20a-b
17b
O
N
N
N
N
N
R
or
=
N
a
b
Scheme 3. Reagents and conditions: (a) ethyl acrylate, DBU, THF, 28–71%; (b) NaOH aq, EtOH, 79–89%; (c) 6b, HATU, DIPEA, CH2Cl2; (d) AcOH, 77–91% (2 steps).
R
R
R
NO2
NH
NH2
NH
N
N
O
R
a or b
c
d, e
NO2
Cl
N
N
N
N
N
N
N
21a: R = 4-MeO
21b: R = 5-Me
22a: R = 4-MeO
22b: R = 5-Me
23a: R = 4-MeO
23b: R = 5-Me
24a: R = 7-MeO
24b: R = 6-Me
Scheme 4. Reagents and conditions: (a) aniline, 47%; (b) aniline, Pd2(dba)3, BINAP, Cs2CO3, toluene, 74%; (c) H2, Pd–C, EtOH, 23–98%; (d) 9, HATU, DIPEA, CH2Cl2; (e) AcOH,
63–72% (2 steps).
OMe
O
O
O
O
O
N
N
O
Cl
HN
a
b, c
d
e, f
N
N
H2N
HO
N
N
N
H2N
N
N
N
N
N
N
N
N
N
N
N
N
25
26
27
28
29
Scheme 5. Reagents and conditions: (a) methylamine, CuSO4, 80%; (b) CDI, THF; (c) ethyl acrylate, DBU, THF, 76% (2 steps); (d) NaOH aq, EtOH, 89%; (e) 23a, HATU, DIPEA,
CH2Cl2; (f) AcOH, 76% (2 steps).
crystal structures of 14c and 14d (which is an analogue of 16)
Each 3-nitrogen atom of the benzimidazole core of 14c and 14d
bound to the catalytic site of the PDE10A enzyme (Figs. 2 and 3).
accepted a hydrogen bond from Tyr693. In addition, the right rings
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A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
OMe
N
O
O
O
O
N
N
O
O
N
+ O
O
N
+ O
R
e
f, g
N
a
N
b, c
N
N
H
N
EtO
HO
N
N
N
Cl
EtO
N
O
N
N
N
30
31
32: R = H
33: R = Me
34
35
d
Scheme 6. Reagents and conditions: (a) b-alanine ethyl ester, NEt3, EtOH, 82%; (b) H2, Pd(OH)2-C, EtOH; (c) CDI, THF, 44% (2 steps); (d) MeI, Cs2CO3, DMF, 99%; (e) NaOH aq,
EtOH, 85%; (f) 23a, HATU, DIPEA, CH2Cl2; (g) AcOH, 68% (2 steps).
Table 1
PDE10A inhibitory activity and metabolic stability of heterocycles (right)
Me
R
N
N
Compds
R
PDE10A IC50 (nM) hCLint (mL/min/kg) logD7.4
Me
N
N
14a
590
6800
210
92
>1000
NTa
NTa
NTa
NTa
4.5
N
N
14b
14c
14d
16
Me
N
N
N
N
N
>1000
>1000
>1000
7
N
N
1
N
N
N
N
Figure 2. Crystal structure of 14c (green) bound to PDE10A (PDB code: 3WS8).
Me
Me
N
9.1
5.0
N
N
N
N
20a
10b
8.3
9.2
910
330
4.9
3.6
N
O
Me
N
N
N
O
N
N
Me
N
20b
28
>1000
4.4
a
Not tested.
of 14c and 14d respectively were sandwiched by Phe729 and
Ile692, and they probably formed stacking interaction with
Phe729 and CH– interaction with Ile692. Interestingly, these
N
N
6
p–p
N
p
N
N
crystal structures revealed that the binding modes of 14c and
14d to Gln726 differ. The 7-nitrogen of imidazopyrimidine core
of 14c formed a hydrogen bond with Gln726. In contrast, the 1-
nitrogen of imidazopyridazine core of 14d accepted a hydrogen
bond from Gln726, revealing that the binding mode of 14d was
more suitable as a PDE10A inhibitor than that of 14c. We therefore
considered the 1-nitrogen atom to be important in obtaining a
suitable conformation against the PDE10A enzyme.
1
Figure 3. Crystal structure of 14d (purple) bound to PDE10A (PDB code: 3WS9).
Although 16 exhibited potent inhibitory activity, it also had
high human liver microsomal intrinsic clearance (hCLint) and
required optimization. On the basis that metabolic stability
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A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
PDE10A inhibitory activity and metabolic stability for N-1 ring
1
R
N
N
O
N
N
2
R
N
Compds
R1
R2
PDE10A IC50 (nM)
hCLint (mL/min/kg)
logD7.4
10b
10c
10d
10e
10f
Me
Ph
Ph
9.2
6.3
220
37
330
320
310
310
180
3.6
3.6
1.8
1.4
1.5
MeO
MeO
MeO
MeO
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
97
Table 3
reduced lipophilicity. Further, these derivatives had decreased
inhibitory activity, indicating that pyridine groups are not toler-
ated at this position.
PDE10A inhibitory activity and metabolic stability for benzimidazole
R 4
N
N
O
5
We next introduced a heteroatom onto the benzimidazole ring
(Table 3). We first synthesized 24b, which was substituted
nitrogen for carbon at the 7-position and had a methyl substituent
at the 5-position. However, this compound exhibited less inhibi-
tory activity than 10c. In contrast, exchanging the 5-methoxy sub-
stituent of 10c with a 4-methoxy substituent retained inhibitory
activity (10a), and substitution from carbon to nitrogen at the
7-position of 10a gave 24a, which exhibited relatively maintained
inhibitory activity and good metabolic stability compared with
10c.
N
N
X
N
Compds
R
X
PDE10A IC50 (nM) hCLint (mL/min/kg) logD7.4
10c
24b
10a
24a
5-MeO CH 6.3
5-Me 200
4-MeO CH 7.3
4-MeO 28
320
480
460
130
3.6
2.0
2.8
1.6
N
N
The promising in vitro profile of 24a encouraged us to evaluate
other pharmacological profiles. Unfortunately, 24a exerted time-
dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 enzyme,
with only 20% of total CYP3A4 activity remaining after 30 min pre-
incubation with 24a. Given that the pyridine nitrogen atom might
be a CYP3A4 inhibition site,13 we attempted to convert the imidaz-
opyridinone part (Table 4). The replacement of a carbon atom at
the 7-position of imidazopyridinone of 24a with a nitrogen atom
gave 29. Although 29 exerted significantly weaker CYP3A4 TDI
than 24a, it is a pity that the activity was decreased. It was consid-
ered this decreased activity of 29 as possibly being due to its lower
basicity and decreased coordination to the PDE10A enzyme. In
contrast, the incorporation of a methyl group at the 7-position of
imidazopyridinone produced 35, which exhibited weaker CYP3A4
TDI and improved inhibitory activity relative to 24a. Although
we have no data about metabolite identification studies, these
findings suggest that imidazopyridinone might generate a reactive
metabolite at the 7-position and that conjugate addition to the
metabolite influenced CYP3A4 TDI. Therefore, improved TDI might
be observed when the 7-position is replaced by a nitrogen or
correlates with lipophilicity,11 we focused on reducing lipophilic-
ity. Given that imidazopyridazine 16 showed high lipophilicity
(logD7.4 = 5.0), we investigated new scaffolds with the 1-nitrogen
atom and reduced lipophilicity, for example, 20a (logD7.4 = 4.9),
10b (logD7.4 = 3.6) and 20b (logD7.4 = 4.4). It was observed that
replacing the imidazopyridazine with an imidazopyridinone12 gave
10b, which exhibited improved metabolic stability while maintain-
ing potent inhibitory activity.
We next focused on the derivation of the left part to address the
insufficient metabolic stability of 10b (Table 2). First, replacement
of the methyl group at 5-position of benzimidazole with methoxy
group gave 10c. The inhibitory activity of 10c was slightly
increased compared with 10b, but the metabolic stability
remained unchanged. The phenyl substituent at the 1-position of
10c was then converted to aza-derivatives, that is, 10d
(logD7.4 = 1.8), 10e (logD7.4 = 1.4) and 10f (logD7.4 = 1.5). However,
replacement of the phenyl ring at the N-1 position of benzimid-
azole with pyridine had no effect on a metabolic stability despite
Table 4
PDE10A inhibitory activity, metabolic stability and CYP3A4 TDI for imidazopyridinone
O
N
O
N
N
N
N
X
N
Compds
X
PDE10A IC50 (nM)
hCLint (mL/min/kg)
CYP3A4 TDIa (% activity remaining)
logD7.4
24a
29
35
CH
N
CMe
28
111
9.5
130
100
260
20
91
73
1.6
1.3
1.9
TDI, time-dependent inhibition.
a
Time-dependent inhibition of CYP3A4 activity, expressed as percent midazolam activity remaining following 30 min of pre-incubation.
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A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
methyl substituent. In addition, compound 35 showed satisfactory
metabolic stability with no inhibition against the other four iso-
forms of the CYP enzymes (CYP1A2, CYP2C9, CYP2C19 and
CYP2D6)—therefore compound 35 did embody the best compound
with potent inhibitory activity.
We succeeded in identifying a novel benzimidazole derivative
(35) with improved PDE10A inhibitory potency and metabolic sta-
bility. Further optimization and biological evaluation of this com-
pound is being conducted at present.
5.1.4. N-(4-Methoxy-2-nitrophenyl)pyridin-2-amine (5d)
Compound 5d was prepared from 4 and pyridin-2-amine in a
manner similar to that described for compound 5a, with a yield
of 33% as a red oil. 1H NMR (CDCl3) d 3.85 (s, 3H), 6.88–6.92 (m,
2H), 7.22 (dd, 1H, J = 9.4, 3.1 Hz), 7.60 (ddd, 1H, J = 8.7, 7.4,
2.0 Hz), 7.67 (d, 1H, J = 3.1 Hz), 8.29–8.31 (m, 1H), 8.69 (d, 1H,
J = 9.4 Hz), 9.92 (s, 1H); MS (ESI) m/z 246 [M+H]+.
5.1.5. N-(4-Methoxy-2-nitrophenyl)pyridin-3-amine (5e)
Compound 5e was prepared from 4 and pyridin-3-amine in a
manner similar to that described for compound 5a, with a yield
of 64% as a red solid. 1H NMR (DMSO-d6) d 3.81 (s, 3H), 7.25 (s,
1H), 7.26 (s, 1H), 7.34 (ddd, 1H, J = 7.6, 4.7, 0.7 Hz), 7.56–7.59 (m,
2H), 8.25 (dd, 1H, J = 4.7, 1.5 Hz), 8.46 (d, 1H, J = 2.2 Hz), 8.97 (s,
1H); MS (ESI) m/z 246 [M+H]+.
4. Conclusion
Optimization of the imidazopyridine moiety of HTS hit com-
pound 1 for improved inhibitory activity gave imidazopyridinone
10b. We then attempted to improve the metabolic stability of
10b by reducing lipophilicity. Being able to obtain 24a with
improved metabolic stability, we moved on to reducing CYP3A4
TDI activity by introducing substituents to imidazopyridine. The
incorporation of a methyl group at the 7-position of imidazopyrid-
inone gave 35, which has potent PDE10A inhibitory activity, good
metabolic stability and reduced inhibition of CYP enzymes. Further
investigation of this novel class of PDE10A inhibitors will be
reported in the near future.
5.1.6. N-(4-Methoxy-2-nitrophenyl)pyridin-4-amine (5f)
Compound 5f was prepared from 4 and pyridin-4-amine in a
manner similar to that described for compound 5a, with a yield
of 44% as a red oil. 1H NMR (DMSO-d6) d 3.85 (s, 3H), 6.81 (dd,
2H, J = 4.8, 1.6 Hz), 7.32 (dd, 1H, J = 9.0, 3.0 Hz), 7.51 (d, 1H,
J = 9.0 Hz), 7.58 (d, 1H, J = 3.0 Hz), 8.20 (dd, 2H, J = 4.8, 1.6 Hz),
8.77 (s, 1H); MS (ESI) m/z 246 [M+H]+.
5.1.7. 3-Methoxy-N1-phenylbenzene-1,2-diamine
dihydrochloride (6a)
5. Experimental
5.1. Chemistry
To a solution of 5a (3.19 g, 13.1 mmol) in EtOH (20 mL) was
added 10% Pd(OH)2-C (183 mg, 0.26 mmol) and stirred at room
temperature for 24 h under hydrogen atmosphere. The mixture
was filtered through a pad of Celite, and the filtrate was concen-
trated in vacuo and diluted with 4 M HCl in EtOAc (10 mL,
40.0 mmol) to give 6a (2.97 g, 79%) as a purple solid. 1H NMR
(DMSO-d6) d 3.88 (s, 2H), 3.89 (s, 3H), 8.08–8.25 (m, 8H), 9.57 (s,
1H); MS (ESI) m/z 215 [M+H]+.
1H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-
LA400 or JEOL JNM-AL400 and chemical shifts were expressed as d
(ppm) values with tetramethylsilane as an internal reference
(s = singlet, d = doublet, t = triplet, m = multiplet, dd = double dou-
blet, dt = double triplet, tt = triple triplet, q = quartet, ddd = double
double doublet, and br = broad peak). Mass spectra (MS) were
recorded on a Waters UPLC/SQD[413W]-LC/MS system. Elemental
analyses were performed using a Yanaco MT-6 (C, H, N), Elementar
Vario EL III (C, H, X), and Dionex ICS-3000 (S, halogene) and were
within 0.4% of theoretical values. Electrospray ionization positive
high-resolution mass spectra (HRMS) were obtained using a
Waters LCT Premier.
5.1.8. 4-Methyl-N1-phenylbenzene-1,2-diamine
dihydrochloride (6b)
Compound 6b was prepared from 5b in a manner similar to that
described for compound 6a, with a yield of 49% as a purple solid. 1H
NMR (DMSO-d6) d 2.26 (s, 3H), 3.46 (br s, 3H), 6.78 (tt, 1H, J = 7.2,
1.0 Hz), 6.84 (dd, 2H, J = 8.6, 1.0 Hz), 6.98 (d, 1H, J = 8.1 Hz), 7.05 (s,
1H), 7.16–7.21 (m, 3H); MS (ESI) m/z 199 [M+H]+.
5.1.1. 3-Methoxy-2-nitro-N-phenylaniline (5a)
To a mixture of 2 (3.00 g, 12.9 mmol) in toluene (30 mL) were
added aniline (3.55 mL, 38.9 mmol), Tris(dibenzylideneace-
tone)dipalladium(0) (Pd2(dba)3; 1.18 g, 1.29 mmol), 2,20-Bis
(diphenylphosphino)-1,10-binaphthyl (BINAP; 1.20 g, 1.93 mmol)
and Cs2CO3 (8.42 g, 25.8 mmol), and stirred at 110 °C for 24 h under
argon atmosphere. After cooling at room temperature, the mixture
was filtered through a pad of Celite, and the filtrate was then concen-
trated in vacuo. The residue was purified by flash column chroma-
tography (silica gel, 0–50% EtOAc in n-hexane) to give 5a (1.90 g,
60%) as an orange oil. 1H NMR (DMSO-d6) d 3.85 (s, 3H), 6.77 (dd,
1H, J = 8.4, 0.9 Hz), 6.84 (dd, 1H, J = 8.4, 0.9 Hz), 6.92–7.07 (m, 3H),
7.23–7.48 (m, 3H), 7.96 (s, 1H); MS (ESI) m/z 245 [M+H]+.
5.1.9. 4-Methoxy-N1-phenylbenzene-1,2-diamine
dihydrochloride (6c)
Compound 6c was prepared from 5c in a manner similar to that
described for compound 5a, with a yield of 63% as a purple solid.
1H NMR (DMSO-d6) d 3.73 (s, 3H), 6.69–6.72 (m, 4H), 6.79 (d, 1H,
J = 2.6 Hz), 7.12–7.18 (m, 3H); MS (ESI) m/z 215 [M+H]+.
5.1.10. 4-Methoxy-N1-(pyridin-2-yl)benzene-1,2-diamine
dihydrochloride (6d)
Compound 6d was prepared from 5d in a manner similar to that
described for compound 6a, with a yield of 14% as a brown solid. 1H
NMR (DMSO-d6) d 3.73 (s, 3H), 6.38 (dd, 1H, J = 8.7, 2.8 Hz), 6.56 (d,
1H, J = 2.8 Hz), 6.92–7.09 (m, 2H), 7.89 (d, 1H, J = 6.1 Hz), 7.96 (ddd,
1H, J = 8.7, 7.0, 1.7 Hz), 10.15 (s, 1H); MS (ESI) m/z 216 [M+H]+.
5.1.2. 4-Methyl-2-nitro-N-phenylaniline (5b)
Compound 5b was prepared from 3 in a manner similar to that
described for compound 5a, with a yield of 97% as a red oil. 1H NMR
(DMSO-d6) d 2.27 (s, 3H), 7.13–7.17 (m, 2H), 7.27–7.47 (m, 5H),
7.92 (dd, 1H, J = 1.8, 0.8 Hz), 9.21 (s, 1H); MS (ESI) m/z 229 [M+H]+.
5.1.11. 4-Methoxy-N1-(pyridin-3-yl)benzene-1,2-diamine
dihydrochloride (6e)
Compound 6e was prepared from 5e in a manner similar to that
described for compound 6a, with a yield of 64% as a brown solid.
1H NMR (DMSO-d6) d 3.71 (s, 3H), 3.82–4.15 (br s, 2H), 6.27 (dd,
1H, J = 8.6, 2.8 Hz), 6.45 (d, 1H, J = 2.8 Hz), 7.50 (ddd, 1H, J = 7.7,
2.8, 1.1 Hz), 7.72 (dd, 1H, J = 8.6, 5.3 Hz), 7.81 (d, 1H, J = 2.8 Hz),
8.07 (d, 1H, J = 5.3 Hz), 8.54 (s, 1H); MS (ESI) m/z 216 [M+H]+.
5.1.3. 4-Methoxy-2-nitro-N-phenylaniline (5c)
Compound 5c was prepared from 4 in a manner similar to that
described for compound 5a, with a yield of 80% as an orange solid.
1H NMR (CDCl3) d 3.83 (s, 3H), 6.98–7.64 (m, 8H), 9.33 (s, 1H); MS
(ESI) m/z 245 [M+H]+.
Please cite this article in press as: Chino, A.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.023

A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
5.1.12. 4-Methoxy-N1-(pyridin-4-yl)benzene-1,2-diamine
dihydrochloride (6f)
J = 6.3 Hz), 4.23 (t, 2H, J = 6.3 Hz), 6.98 (dd, 1H, J = 7.7, 5.3 Hz),
7.19 (d, 1H, J = 8.4 Hz), 7.35 (dd, 1H, J = 8.4, 1.0 Hz), 7.40–7.45 (m,
3H), 7.57–7.60 (m, 3H), 7.62 (dd, 1H, J = 5.3, 1.3 Hz), 7.67–7.68
(m, 1H); MS (ESI) m/z 384 [M+H]+ Anal. Calcd for C23H21N5-
Oꢀ2.6HClꢀ0.8H2O: C, 56.07; H, 5.16; N, 14.22; Cl, 18.71. Found: C,
56.01; H, 5.00; N, 14.12; Cl, 18.48.
Compound 6f was prepared from 5f in a manner similar to that
described for compound 6a, with a yield of 37% as a brown solid.
1H NMR (DMSO-d6) d 3.34–3.57 (br s, 2H), 3.70 (s, 3H), 6.21 (dd,
1H, J = 8.8, 2.8 Hz), 6.40 (d, 1H, J = 2.8 Hz), 6.91 (d, 1H, J = 8.6 Hz),
7.07 (s, 1H), 8.08–8.25 (m, 2H), 9.78 (s, 1H), 13.38 (s, 1H); MS
(ESI) m/z 216 [M+H]+.
5.1.17. 3-[2-(5-Methoxy-1-phenyl-1H-benzimidazol-2-yl)ethyl]-
1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (10c)
To a mixture of 6c (390 mg, 1.36 mmol) in CH2Cl2 (6 mL) were
5.1.13. Ethyl 3-(1-methyl-2-oxo-1,2-dihydro-3H-imidazo[4,5-
b]pyridin-3-yl)propanoate (8)
added HATU (770 mg, 1.49 mmol), DIPEA (930 lL, 5.43 mmol), and
To a stirred mixture of 7 (5.25 g, 35.2 mmol) in acetonitrile
(100 mL) were added ethyl acrylate (7.65 mL, 70.4 mmol) and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 2.63 mL, 17.6 mmol),
and stirred at 50 °C for 12 h. The mixture was concentrated in
vacuo, and the residue was purified by flash column chromatogra-
phy (silica gel, 0–5% MeOH in CHCl3) to give 8 (5.72 g, 65%) as a
pale yellow oil. 1H NMR (DMSO-d6) d 1.10 (t, 3H, J = 7.1 Hz), 2.77
(t, 2H, J = 7.2 Hz), 3.34 (s, 3H), 4.00 (t, 2H, J = 7.1 Hz), 4.11 (t, 2H,
J = 7.2 Hz), 7.08 (dd, 1H, J = 7.7, 5.2 Hz), 7.49 (dd, 1H, J = 7.7,
1.4 Hz), 7.98 (dd, 1H, J = 5.2, 1.4 Hz); MS (ESI) m/z 250 [M+H]+.
9 (300 mg, 1.36 mmol), and the mixture was stirred at room temper-
ature for 18 h. The mixture was diluted with saturated NaHCO3
aqueous solution and extracted with CHCl3. The organic layer was
washed with brine, dried over anhydrous Na2SO4, filtered and con-
centrated in vacuo. To the residue was added AcOH (6 mL), and
the mixture was stirred at 100 °C for 8 h. The resulting mixture
was concentrated in vacuo, and the residue was purified by flash col-
umn chromatography (silica gel, 0–10% MeOH in CHCl3). The residue
was washed with EtOAc to give 10c (213 mg, 39%) as a purple solid.
1H NMR (DMSO-d6) d 3.21 (t, 2H, J = 7.4 Hz), 3.27 (s, 3H), 3.79 (s, 3H),
4.24 (t, 2H, J = 7.4 Hz), 6.81 (dd, 1H, J = 8.8, 2.4 Hz), 6.97 (d, 1H,
J = 8.8 Hz), 7.02 (dd, 1H, J = 7.7, 5.3 Hz), 7.19 (d, 1H, J = 2.4 Hz),
7.39–7.56 (m, 6H), 7.86 (dd, 1H, J = 5.3, 1.4 Hz); MS (ESI) m/z 400
[M+H]+ Anal. Calcd for C23H21N5O2ꢀ0.2H2O: C, 68.54; H, 5.35; N,
17.38. Found: C, 68.41; H, 5.19; N, 17.33.
5.1.14. 3-(1-Methyl-2-oxo-1,2-dihydro-3H-imidazo[4,5-
b]pyridin-3-yl)propanoic acid (9)
To a solution of 8 (4.18 g, 16.7 mmol) in EtOH (50 mL) was added
1 M NaOH aqueous solution (30 mL, 30.0 mmol), and the mixture
was stirred at room temperature for 2 h. The resulting mixture
was concentrated in vacuo. The residue was neutralized with 1 M
HCl aqueous solution (30 mL). The resulting precipitate was col-
lected by filtration and washed with water. The solid was dried in
vacuo to give 9 (3.67 g, 99%) as a colorless solid. 1H NMR (DMSO-
d6) d 2.68 (t, 2H, J = 7.5 Hz), 3.05–3.52 (br s, 1H), 3.33 (s, 3H), 4.06
(t, 2H, J = 7.5 Hz), 7.07 (dd, 1H, J = 7.7, 5.2 Hz), 7.47 (dd, 1H, J = 7.7,
1.3 Hz), 7.98 (dd, 1H, J = 5.2, 1.3 Hz); MS (ESI) m/z 222 [M+H]+.
5.1.18. 3-{2-[5-Methoxy-1-(pyridin-2-yl)-1H-benzimidazol-2-
yl]ethyl}-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one (10d)
To a suspension of 6d (390 mg, 1.36 mmol) in CH2Cl2 (6 mL)
were added HATU (770 mg, 1.49 mmol), DIPEA (930 lL,
5.43 mmol), and 9 (300 mg, 1.36 mmol), and the mixture was stir-
red at room temperature for 18 h. The mixture was diluted with
saturated NaHCO3 aqueous solution and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. To the residue was
added AcOH (6 mL), and the mixture was stirred at 130 °C for
48 h. The resulting mixture was concentrated in vacuo. The residue
was purified by flash column chromatography (NH silica gel, 0–10%
MeOH in CHCl3) to give 10d (120 mg, 22%) as a colorless solid. 1H
NMR (DMSO-d6) d 3.26 (s, 3H),3.42 (t, 2H, J = 7.2 Hz), 3.81 (s, 3H),
4.26 (t, 2H, J = 7.2 Hz), 6.85 (dd, 1H, J = 8.8, 2.4 Hz), 7.02 (dd, 1H,
J = 7.7, 2.4 Hz), 7.21 (d, 1H, J = 2.4 Hz), 7.25 (d, 1H, J = 8.8 Hz),
7.39–7.44 (m, 2H), 7.49 (dt, 1H, J = 8.1, 0.7 Hz), 7.86 (dd, 1H,
J = 5.2, 1.3 Hz), 8.00 (td, 1H, J = 7.7, 2.0 Hz), 8.52 (ddd, 1H, J = 5.2,
2.0, 1.1 Hz); MS (ESI) m/z 401 [M+H]+; HRMS (ES+) calcd for
5.1.15. 3-[2-(4-Methoxy-1-phenyl-1H-benzimidazol-2-yl)ethyl]-
1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one
dihydrochloride (10a)
To a suspension of 6a (250 mg, 0.871 mmol) in CH2Cl2 (5 mL)
were added 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazol-
o[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU; 500 mg,
1.32 mmol), N,N0-diisopropylethylamine (DIPEA, 600
lL, 3.51 mmol),
and 9 (200 mg, 0.904 mmol), and the mixture was stirred at room
temperature for 12 h. The mixture was diluted with saturated
NaHCO3 aqueous solution and extracted with CHCl3. The organic
layer was washed with brine, dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. To the residue was added AcOH (5 mL),
and the mixture was stirred at 100 °C for 24 h. The resulting mixture
was concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, 0–10% MeOH in CHCl3) to give the free
form of the title compound, which was dissolved in EtOAc
(2.5 mL), and 4 M HCl in EtOAc (0.218 mL, 0.870 mmol) was added
to the solution. The mixture was concentrated in vacuo to give 10a
(87.0 mg, 21%) as a colorless solid. 1H NMR (DMSO-d6) d 3.25 (s,
3H), 3.40 (t, 2H, J = 6.5 Hz), 4.04 (s, 3H), 4.21 (t, 2H, J = 6.5 Hz), 6.81
(d, 1H, J = 7.9 Hz), 7.00 (dd, 1H, J = 7.7, 5.2 Hz), 7.13 (d, 1H,
J = 8.1 Hz), 7.38–7.46 (m, 4H), 7.58–7.60 (m, 3H), 7.67 (dd, 1H,
J = 5.2, 1.3 Hz); MS (ESI) m/z 400 [M+H]+; HRMS (ES+) calcd for
C
22H20N6O2 [M+H]+ 401.1726; found, 401.1725.
5.1.19. 3-{2-[5-Methoxy-1-(pyridin-3-yl)-1H-benzimidazol-2-
yl]ethyl}-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one (10e)
Compound 10e was prepared from 6e in a manner similar to that
described for compound 10d, with a yield of 25% as a beige solid. 1H
NMR (DMSO-d6) d 3.23 (t, 2H, J = 7.2 Hz), 3.26 (s, 3H), 3.79 (s, 3H),
4.22 (t, 2H, J = 7.2 Hz), 6.83 (dd, 1H, J = 8.8, 2.3 Hz), 6.99–7.04 (m,
2H), 7.21 (d, 1H, J = 2.3 Hz), 7.42 (dd, 1H, J = 7.9, 1.2 Hz), 7.57 (dd,
1H, J = 7.9, 4.8 Hz), 7.85 (dd, 1H, J = 5.2, 1.2 Hz), 7.91–7.95 (m,
1H), 8.65–8.67 (m, 2H); MS (ESI) m/z 401 [M+H]+; HRMS (ES+) calcd
for C22H20N6O2 [M+H]+ 401.1726; found, 401.1733.
C
23H21N5O2 [M+H]+ 400.1773; found, 400.1779.
5.1.16. 1-Methyl-3-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one
dihydrochloride (10b)
Compound 10b was prepared from 6b in a manner similar to
that described for compound 10a, with a yield of 99% as a colorless
solid. 1H NMR (DMSO-d6) d 2.50 (s, 3H), 3.24 (s, 3H), 3.50 (t, 2H,
5.1.20. 3-{2-[5-Methoxy-1-(pyridin-4-yl)-1H-benzimidazol-2-
yl]ethyl}-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one (10f)
Compound 10f was prepared from 6f in a manner similar to that
described for compound 10c, with a yield of 23% as a colorless
Please cite this article in press as: Chino, A.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.023

8
A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
solid. 1H NMR (DMSO-d6) d 2.26 (s, 3H), 3.32 (t, 2H, J = 7.4 Hz), 3.80
(s, 3H), 4.23 (t, 2H, J = 7.4 Hz), 6.85 (dd, 1H, J = 7.8, 2.5 Hz), 7.02 (dd,
1H, J = 7.8, 2.5 Hz), 7.14 (d, 1H, J = 8.9 Hz), 7.22 (d, 1H, J = 2.5 Hz),
7.42 (dd, 1H, J = 7.8, 1.4 Hz), 7.49 (dd, 2H, J = 4.4, 1.6 Hz), 7.85
(dd, 1H, J = 5.2, 1.4 Hz), 8.70 (dd, 2H, J = 4.4, 1.6 Hz); MS (ESI) m/z
401 [M+H]+; HRMS (ES+) calcd for C22H20N6O2 [M+H]+ 401.1726;
found, 401.1716.
6.3, 1.2 Hz), 6.70 (ddd, 1H, J = 9.2, 6.3, 0.7 Hz), 6.96–7.02 (m, 2H),
7.20 (d, 1H, J = 0.7 Hz), 7.44–7.63 (m, 7H), 8.07 (dd, 1H, J = 7.2,
1.0 Hz); MS (ESI) m/z 353 [M+H]+; HRMS (ES+) calcd for C23H20N4
[M+H]+ 353.1766; found, 353.1761.
5.1.25. 8-Methyl-6-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]imidazo[1,5-a]pyrimidine dihydrochloride (14c)
To a mixture of 12 (336 mg, 1.20 mmol) in EtOAc (10 mL) were
added propylphosphonic anhydride (1.7 M solution in EtOAc;
4.00 mL, 6.85 mmol) and 13c (163 mg, 1.10 mmol) at room tem-
perature, and the mixture was stirred at 80 °C for 24 h. The mixture
was diluted with saturated NaHCO3 aqueous solution and
extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was purified by flash column chromatography (silica
gel, 0–5% MeOH in CHCl3) to give the free form of the title com-
pound, which was dissolved in EtOAc (10 mL), and 4 M HCl in
EtOAc (1.20 mL, 4.80 mmol) was added to the solution. The mix-
ture was concentrated in vacuo and the residue was triturated with
2-propanol and isopropyl ether. The resulting precipitate was col-
lected by filtration and dried in vacuo to give 14c (528 mg, 53%) as
a yellow solid. 1H NMR (DMSO-d6) d 1.63 (s, 3H), 1.65 (s, 3H), 2.73
(t, 2H, J = 7.3 Hz), 3.09 (t, 2H, J = 7.3 Hz), 6.25 (dd, 1H, J = 3.8,
7.9 Hz), 6.34 (d, 1H, J = 8.5 Hz), 6.48 (dd, 1H, J = 8.5, 0.9 Hz), 6.76–
6.90 (m, 6H), 7.56 (dd, 1H, J = 3.8, 1.4 Hz), 8.26 (dd, 1H, J = 7.4,
1.4 Hz); MS (ESI) m/z 368 [M+H]+ Anal. Calcd for C23H21N5-
ꢀ2HClꢀ1.5H2Oꢀ0.1C6H14O: C, 59.35; H, 5.78; N, 14.85; Cl, 18.80.
Found: C, 59.43; H, 5.59; N, 14.56; Cl, 18.97.
5.1.21. Methyl 3-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)propanoate (11)
To a mixture of 5b (2.18 g, 8.05 mmol) in CH2Cl2 (33 mL) were
added HATU (4.59 g, 12.1 mmol), DIPEA (5.50 mL, 32.2 mmol)
and 4-methoxy-4-oxobutanoic acid (1.12 g, 8.45 mmol), and stir-
red at room temperature for 12 h. The mixture was diluted with
saturated NaHCO3 aqueous solution and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. To the residue was
added AcOH (44 mL), and stirred at 90 °C for 24 h. The reaction
mixture was concentrated in vacuo. The residue was diluted with
saturated NaHCO3 aqueous solution and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was puri-
fied by flash column chromatography (silica gel, 0–5% MeOH in
CHCl3) to give 11 (2.23 g, 96%) as a colorless solid. 1H NMR
(DMSO-d6) d 2.41 (s, 3H), 2.86–2.90 (m, 2H), 2.93–2.97 (m, 2H),
3.56 (s, 3H), 6.98 (dd, 1H, J = 8.2, 0.7 Hz), 7.01 (dd, 1H, J = 8.2,
1.2 Hz), 7.43–7.45 (m, 1H), 7.51–7.67 (m, 5H); MS (ESI) m/z 295
[M+H]+.
5.1.22. 3-(5-Methyl-1-phenyl-1H-benzimidazol-2-yl)propanoic
acid (12)
5.1.26. 7-[2-(5-Methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]imidazo[1,5-b]pyridazine (14d)
To a mixture of 11 (2.17 g, 7.37 mmol) in EtOH (22 mL) was
added 1 M NaOH aqueous solution (15 mL, 15.0 mmol), and the
mixture was stirred at room temperature for 20 h. The resulting
mixture was concentrated in vacuo. The residue was neutralized
with 1 M HCl aqueous solution (13 mL). The resulting precipitate
was collected by filtration and washed with water. The solid was
dried in vacuo to give 12 (1.67 g, 81%) as a colorless solid. 1H
NMR (DMSO-d6) d 2.41 (s, 3H), 2.79 (t, 2H, J = 7.0 Hz), 2.91 (t, 2H,
J = 7.0 Hz), 6.97–7.02 (m, 2H), 7.03–7.67 (m, 6H), 12.17 (s, 1H);
MS (ESI) m/z 281 [M+H]+.
Compound 14d was prepared from 12 and 13d in a manner
similar to that described for compound 14a, with a yield of 82%
as a pale yellow solid. 1H NMR (DMSO-d6) d 2.41 (s, 3H), 3.27
(dd, 2H, J = 8.3, 6.5 Hz), 3.52 (dd, 2H, J = 8.3, 6.5 Hz), 6.65 (dd, 1H,
J = 9.3, 4.3 Hz), 6.95–7.02 (m, 2H), 7.34 (s, 1H), 7.43–7.61 (m, 6H),
8.03 (dd, 1H, J = 9.3, 1.5 Hz), 8.20 (dd, 1H, J = 3.0, 1.5 Hz); MS
(ESI) m/z 354 [M+H]+; HRMS (ES+) calcd for C22H19N5 [M+H]+
354.1718; found, 354.1719.
5.1.27. 5-Bromo-7-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]imidazo[1,5-b]pyridazine (15)
5.1.23. 5-Methyl-2-[2-(1-methylimidazo[1,5-a]pyridin-3-
yl)ethyl]-1-phenyl-1H-benzimidazole (14a)
To a mixture of 14d (300 mg, 0.849 mmol) in MeCN (3.0 mL)
was added N-bromosuccinimide (181 mg, 1.02 mmol) in CH2Cl2
(0.60 mL) at 0 °C, and the mixture was stirred at the same temper-
ature for 1 h under argon atmosphere. The mixture was diluted
with H2O and extracted with CHCl3. The organic layer was washed
with saturated NaHCO3 aqueous solution and brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, 0–5%
MeOH in CHCl3) to give 15 (297 mg, 81%) as a pale yellow solid.
1H NMR (DMSO-d6) d 2.41 (s, 3H), 3.26 (dd, 2H, J = 8.3, 6.3 Hz),
3.51 (dd, 2H, J = 8.3, 6.3 Hz), 6.76 (dd, 1H, J = 9.3, 4.3 Hz), 6.96–
7.02 (m, 2H), 7.43–7.61 (m, 6H), 7.88 (dd, 1H, J = 9.3, 1.6 Hz),
8.26 (dd, 1H, J = 4.3, 1.6 Hz); MS (ESI) m/z 432, 434 [M+H]+.
To a mixture of 12 (336 mg, 1.20 mmol) in EtOAc (10 mL) were
added propylphosphonic anhydride (1.7 M solution in EtOAc;
1.96 mL, 3.35 mmol) and 13a (161 mg, 1.32 mmol) at room tem-
perature, and the mixture was stirred at 80 °C for 12 h. The mixture
was diluted with saturated NaHCO3 aqueous solution and
extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was purified by flash column chromatography (silica
gel, 0–5% MeOH in CHCl3). The product was recrystallized from
EtOAc/n-hexane to give 14a (160 mg, 36%) as a beige solid. 1H
NMR (DMSO-d6) d 2.30 (s, 3H), 2.41 (s, 3H), 3.22 (dd, 2H, J = 8.3,
6.8 Hz), 3.40 (dd, 2H, J = 8.3, 6.8 Hz), 6.48 (ddd, 1H, J = 6.2, 4.2,
1.1 Hz), 6.56 (dd, 1H, J = 9.2, 5.9 Hz), 6.96–7.02 (m, 2H), 7.38–
7.63 (m, 7H), 7.92 (d, 1H, J = 7.2 Hz); MS (ESI) m/z 367 [M+H]+;
HRMS (ES+) calcd for C24H22N4 [M+H]+ 367.1923; found, 367.1928.
5.1.28. 5-Methyl-7-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]imidazo[1,5-b]pyridazine (16)
To a mixture of 15 (120 mg, 0.277 mmol) in dioxane (2.4 mL)
were added trimethylboroxine (0.116 mL, 0.833 mmol), [1,10-
Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (PdCl2
5.1.24. 2-[2-(Imidazo[1,5-a]pyridin-3-yl)ethyl]-5-methyl-1-
phenyl-1H-benzimidazole (14b)
(dppf)2; 68.0 mg, 83.2 lmol) and K2CO3 (230 mg, 1.66 mmol), and
Compound 14b was prepared from 12 and 13b in a manner
similar to that described for compound 14a, with a yield of 33%
as a beige solid. 1H NMR (DMSO-d6) d 2.41 (s, 3H), 3.26 (dd, 2H,
J = 9.0, 4.8 Hz), 3.46 (dd, 2H, J = 9.0, 4.8 Hz), 6.59 (ddd, 1H, J = 7.2,
stirred at 90 °C for 26 h under argon atmosphere. After cooling at
room temperature, the mixture was filtered through a pad of Cel-
ite. The filtrate was concentrated in vacuo, and the residue was
purified by flash column chromatography (silica gel, 0–50% MeOH
Please cite this article in press as: Chino, A.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.023

A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
in CHCl3 then NH silica gel, 0–30% EtOAc in CHCl3) to give 16
(36.0 mg, 35%) as a colorless solid. 1H NMR (DMSO-d6) d 2.33 (s,
3H), 2.41 (s, 3H), 3.25 (dd, 2H, J = 8.5, 5.8 Hz), 3.46 (dd, 2H,
J = 8.5, 5.8 Hz), 6.50 (dd, 1H, J = 9.2, 4.2 Hz), 6.95–7.02 (m, 2H),
7.44–7.61 (m, 6H), 8.00 (dd, 1H, J = 9.2, 1.6 Hz), 8.09 (dd, 1H,
J = 4.2, 1.6 Hz); MS (ESI) m/z 368 [M+H]+; HRMS (ESI+) calcd for
NaHCO3 aqueous solution and extracted with CHCl3. The organic
layer was washed with brine, dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. To the residue was added AcOH
(8 mL), and the mixture was stirred at 90 °C for 12 h. The resulting
mixture was concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, 0–5% MeOH in CHCl3).
The residue was washed with EtOAc/n-hexane to give 20b
(367 mg, 77%) as a pale yellow solid. 1H NMR (DMSO-d6) d 2.38
(s, 3H), 2.41 (s, 3H), 3.15 (t, 2H, J = 7.4 Hz), 4.73 (t, 2H, J = 7.4 Hz),
6.96–7.03 (m, 2H), 7.35 (dd, 1H, J = 7.9, 4.7 Hz), 7.43–7.59 (m,
6H), 8.12 (dd, 1H, J = 7.9, 1.7 Hz), 8.43 (dd, 1H, J = 4.7, 1.7 Hz); MS
(ESI) m/z 396 [M+H]+; HRMS (ES+) calcd for C24H21N5O [M+H]+
396.1824; found, 396.1833.
C
23H21N5 [M+H]+ 368.1875; found, 368.1879.
5.1.29. Ethyl 3-(3-methyl-1H-pyrazolo[3,4-b]pyridin-1-
yl)propanoate (18a)
To a mixture of 17a (3.65 g, 27.4 mmol) in MeCN (25 mL) were
added ethyl acrylate (5.97 mL, 54.9 mmol) and DBU (2.05 mL,
13.7 mmol), and the mixture was stirred at 50 °C for 12 h under
argon atmosphere. The mixture was concentrated in vacuo. The
residue was purified by flash column chromatography (silica gel,
0–20% AcOEt in n-hexane) to give 18a (4.56 g, 71%) as a colorless
oil. 1H NMR (CDCl3) d 1.85 (t, 3H, J = 7.2 Hz), 2.56 (s, 3H), 2.96 (t,
2H, J = 7.2 Hz), 4.12 (q, 2H, J = 7.2 Hz), 4.76 (t, 2H, J = 7.2 Hz), 7.07
(dd, 1H, J = 8.0, 4.6 Hz), 7.97 (dd, 1H, J = 8.0, 1.6 Hz), 8.51 (dd, 1H,
J = 4.6, 1.6 Hz); MS (ESI) m/z 234 [M+H]+.
5.1.35. 4-Methoxy-3-nitro-N-phenylpyridin-2-amine (22a)
Compound 22a was prepared from 21a in a manner similar to
that described for compound 5a, with a yield of 47% as an orange
oil. 1H NMR (DMSO-d6) d 3.94 (s, 3H), 6.52–6.55 (m, 1H), 6.80 (d,
1H, J = 5.9 Hz), 7.25–7.29 (m, 2H), 7.50 (dd, 2H, J = 8.6, 1.1 Hz),
8.18 (d, 1H, J = 5.9 Hz), 8.87 (s, 1H); MS (ESI) m/z 246 [M+H]+.
5.1.30. Ethyl 3-(2-methyl-3-oxopyrido[2,3-b]pyrazin-4(3H)-
yl)propanoate (18b)
5.1.36. 5-Methyl-3-nitro-N-phenylpyridin-2-amine (22b)
To a mixture 21b (3.00 g, 17.4 mmol) in aniline (4.05 mL,
43.5 mmol) was stirred at 120 °C for 3 h under microwave irradia-
tion. The reaction mixture was allowed to cool to room tempera-
ture and diluted with EtOAc. Insoluble fraction was removed by
filtration and the filtrate was concentrated in vacuo. The residue
was washed with n-hexane to give 21b (2.96 g, 74%) as a dark
red solid. 1H NMR (DMSO-d6) d 2.29 (s, 3H), 7.11 (tt, 1H, J = 7.3,
1.1 Hz), 7.31–7.37 (m, 2H), 7.63–7.66 (m, 2H), 8.38–8.40 (m, 2H),
9.85 (s, 1H); MS (ESI) m/z 230 [M+H]+.
Compound 18b was prepared from 17b in a manner similar to
that described for compound 18a, with a yield of 28% as a yellow
solid. 1H NMR (DMSO-d6) d 1.12 (t, 3H, J = 7.1 Hz), 2.46 (s, 3H),
2.69 (t, 2H, J = 7.6 Hz), 4.02 (q, 2H, J = 7.1 Hz), 4.59 (t, 2H,
J = 7.6 Hz), 7.43 (dd, 1H, J = 7.9, 4.7 Hz), 8.18 (dd, 1H, J = 7.9,
1.7 Hz), 8.59 (dd, 1H, J = 4.7, 1.7 Hz); MS (ESI) m/z 262 [M+H]+.
5.1.31. 3-(3-Methyl-1H-pyrazolo[3,4-b]pyridin-1-yl)propanoic
acid (19a)
Compound 19a was prepared from 18a in a manner similar to
that described for compound 12, with a yield of 89% as a colorless
solid. 1H NMR (DMSO-d6) d 2.50 (s, 3H), 2.85 (t, 2H, J = 7.0 Hz), 4.58
(t, 2H, J = 7.0 Hz), 7.17 (dd, 1H, J = 8.0, 4.5 Hz), 8.20 (dd, 1H, J = 8.0,
1.6 Hz), 8.51 (dd, 1H, J = 4.5, 1.6 Hz), 12.30 (s, 1H); MS (ESI) m/z 206
[M+H]+.
5.1.37. 4-Methoxy-N2-phenylpyridine-2,3-diamine
dihydrochloride (23a)
Compound 23a was prepared from 22a in a manner similar to
that described for compound 6a, with a yield of 29% as a green
solid. 1H NMR (DMSO-d6) d 4.03 (s, 3H), 7.05 (d, 1H, J = 7.0 Hz),
7.15–7.22 (m, 3H), 7.36–7.54 (m, 3H), 9.44 (s, 1H); MS (ESI) m/z
216 [M+H]+.
5.1.32. 3-(2-Methyl-3-oxopyrido[2,3-b]pyrazin-4(3H)-
yl)propanoic acid (19b)
5.1.38. 5-Methyl-N2-phenylpyridine-2,3-diamine
dihydrochloride (23b)
Compound 23b was prepared from 22b in a manner similar to
that described for compound 6a, with a yield of 98% as a gray solid.
1H NMR (DMSO-d6) d 2.18 (s, 3H), 7.11 (s, 1H), 7.14 (d, 1H,
J = 1.5 Hz), 7.25 (tt, 1H, J = 7.4, 1.1 Hz), 7.31–7.34 (m, 2H), 7.44–
7.49 (m, 2H), 9.95 (s, 1H); MS (ESI) m/z 200 [M+H]+.
Compound 19b was prepared from 18b in a manner similar to
that described for compound 12, with a yield of 79% as a pale yel-
low solid. 1H NMR (DMSO-d6) d 2.46 (s, 3H), 2.60–2.64 (m, 2H),
4.53–4.57 (m, 2H), 7.42 (dd, 1H, J = 7.9, 4.7 Hz), 8.17 (dd, 1H,
J = 7.9, 1.7 Hz), 8.59 (dd, 1H, J = 4.7, 1.7 Hz), 12.41 (s, 1H); MS
(ESI) m/z 234 [M+H]+.
5.1.33. 3-Methyl-1-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]-1H-pyrazolo[3,4-b]pyridine dihydrochloride (20a)
Compound 20a was prepared from 6b and 19a in a manner sim-
ilar to that described for compound 10a, with a yield of 91% as an
off-white solid. 1H NMR (DMSO-d6) d 2.36 (s, 3H), 2.51 (s, 3H), 3.58
(t, 2H, J = 6.5 Hz), 4.84 (t, 2H, J = 6.5 Hz), 7.12 (dd, 1H, J = 8.0,
4.6 Hz), 7.17 (d, 1H, J = 8.5 Hz), 7.30–7.36 (m, 3H), 7.56–7.65 (m,
3H), 7.70 (t, 1H, J = 0.7 Hz), 8.17 (dd, 1H, J = 8.0, 1.5 Hz), 8.26 (dd,
1H, J = 4.5, 1.5 Hz); MS (ESI) m/z 368 [M+H]+ Anal. Calcd for C23H21-
N5ꢀ2.65HClꢀH2O: C, 57.30; H, 5.36; N, 14.53; Cl, 19.49. Found: C,
57.56; H, 5.29; N, 14.59; Cl, 19.45.
5.1.39. 3-[2-(7-Methoxy-3-phenyl-3H-imidazo[4,5-b]pyridin-2-
yl)ethyl]-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one dihydrochloride (24a)
Compound 24a was prepared from 23a and 9 in a manner sim-
ilar to that described for compound 10a, with a yield of 63% as a
colorless solid. 1H NMR (DMSO-d6) d 3.24 (t, 2H, J = 7.0 Hz), 3.26
(s, 3H), 4.07 (s, 3H), 4.22 (t, 2H, J = 7.0 Hz), 6.93 (d, 1H,
J = 5.6 Hz), 7.01 (dd, 1H, J = 7.7, 5.2 Hz), 7.40–7.55 (m, 6H), 7.81
(dd, 1H, J = 5.2, 1.3 Hz), 8.12 (d, 1H, J = 5.6 Hz); MS (ESI) m/z 401
[M+H]+ Anal. Calcd for C22H20N6O2ꢀ1.8HClꢀ2.87H2O: C, 51.03; H,
5.36; N, 16.23; Cl 12.33. Found: C, 51.31; H, 5.37; N, 15.93; Cl,
12.34.
5.1.34. 2-Methyl-4-[2-(5-methyl-1-phenyl-1H-benzimidazol-2-
yl)ethyl]pyrido[2,3-b]pyrazin-3(4H)-one (20b)
To a mixture of 6b (325 mg, 1.20 mmol) in CH2Cl2 (8 mL) were
added HATU (684 mg, 1.50 mmol), DIPEA (0.820 mL, 4.79 mmol)
and 19b (308 mg, 1.32 mmol), and the mixture was stirred at room
temperature for 12 h. The mixture was diluted with saturated
5.1.40. 1-Methyl-3-[2-(6-methyl-3-phenyl-3H-imidazo[4,5-
b]pyridin-2-yl)ethyl]-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-
one dihydrochloride (24b)
Compound 24b was prepared from 23b and 9 in a manner sim-
ilar to that described for compound 10a, with a yield of 72% as a
Please cite this article in press as: Chino, A.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.023

10
A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
colorless solid. 1H NMR (DMSO-d6) d 2.47 (s, 3H), 3.25 (s, 3H), 3.41
(t, 2H, J = 6.5 Hz), 4.24 (t, 2H, J = 6.5 Hz), 7.00 (dd, 1H, J = 7.7,
5.2 Hz), 7.41–7.45 (m, 3H), 7.52–7.58 (m, 3H), 7.70 (dd, 1H,
J = 7.3, 1.3 Hz), 8.07 (s, 1H), 8.28 (d, 1H, J = 1.3 Hz); MS (ESI) m/z
385 [M+H]+ Anal. Calcd for C22H20N6Oꢀ2HClꢀ0.5H2O: C, 56.66; H,
4.97; N, 18.02; Cl 15.20. Found: C, 56.57; H, 5.02; N, 17.86; Cl,
15.18.
(silica gel, 0–30% EtOAc in n-hexane) to give 31 (6.03 g, 82%) as a
yellow oil. 1H NMR (DMSO-d6) d 1.16 (t, 3H, J = 7.1 Hz), 2.36–2.38
(m, 4H) 2.59 (t, 2H, J = 6.9 Hz), 3.67 (q, 2H, J = 6.9 Hz), 4.05 (q,
2H, J = 7.1 Hz), 6.64 (d, 1H, J = 5.0 Hz), 8.16 (d, 1H, J = 5.0 Hz); MS
(ESI) m/z 254 [M+H]+.
5.1.46. Ethyl 3-(7-methyl-2-oxo-1,2-dihydro-3H-imidazo[4,5-
b]pyridin-3-yl)propanoate (32)
5.1.41. N-Methylpyrazine-2,3-diamine (26)
To a mixture of 31 (765 mg, 3.02 mmol) in EtOH (20 mL) was
added 10% Pd(OH)2-C (38.4 mg, 0.055 mmol) and stirred at room
temperature for 6 h under hydrogen atmosphere. The mixture
was filtered through a pad of Celite. The filtrate was concentrated
in vacuo to give a yellow oil. To a mixture of the residue in THF
(12 mL) was added CDI (343 mg, 2.11 mmol), and the mixture
was stirred at 80 °C for 24 h. The mixture was diluted with H2O
and extracted with CHCl3. The organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, 0–5% MeOH in CHCl3) to give 32 (216 mg, 44%) as a col-
orless solid. 1H NMR (DMSO-d6) d 1.11 (t, 3H, J = 7.1 Hz), 2.29 (s,
3H), 2.75 (t, 2H, J = 7.2 Hz), 4.00 (q, 2H, J = 7.1 Hz), 4.05 (t, 2H,
J = 7.2 Hz), 6.84 (dd, 1H, J = 5.3, 0.6 Hz), 7.82 (d, 1H, J = 5.3 Hz),
11.20 (s, 1H); MS (ESI) m/z 250 [M+H]+.
A mixture of 25 (5.20 g, 40.1 mmol) and CuSO4 (640 mg,
4.01 mmol) in 12 M methanamine aqueous solution (40.0 mL,
480 mmol) was stirred at 120 °C for 3 h under microwave irradia-
tion. The reaction mixture was allowed to cool to room tempera-
ture and concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel, 0–5% MeOH in CHCl3) to give
26 (3.99 g, 80%) as a yellow solid. 1H NMR (DMSO-d6) d 2.81 (d,
3H, J = 4.7 Hz), 5.84 (s, 2H), 6.13 (q, 1H, J = 4.7 Hz), 7.09 (d, 1H,
J = 3.1 Hz), 7.23 (d, 1H, J = 3.1 Hz); MS (ESI) m/z 125 [M+H]+.
5.1.42. Ethyl 3-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-
b]pyrazin-1-yl)propanoate (27)
To a mixture of 26 (3.99 g, 32.1 mmol) in THF (200 mL) was
added carbonyldiimidazole (CDI; 10.4 g, 64.1 mmol), and the mix-
ture was stirred at 70 °C for 5 h. The reaction mixture was allowed
to cool to room temperature and concentrated in vacuo. The resi-
due was purified by flash column chromatography (silica gel, 0–
5% MeOH in CHCl3). The residue was washed with EtOAc/n-hexane.
To the residue in MeCN (100 mL) were added ethyl acrylate
(7.00 mL, 64.4 mmol) and DBU (2.40 mL, 16.4 mmol), and the mix-
ture was stirred at 50 °C for 12 h under argon atmosphere. The
mixture was concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, 0–5% MeOH in CHCl3)
to give 27 (6.11 g, 76%) as a colorless solid. 1H NMR (DMSO-d6) d
1.10 (t, 3H, J = 7.1 Hz), 2.81 (t, 2H, J = 7.1 Hz), 3.34 (s, 3H), 4.00
(q, 2H, J = 7.1 Hz), 4.11 (t, 2H, J = 7.1 Hz), 7.95 (d, 1H, J = 3.3 Hz),
7.96 (d, 1H, J = 3.3 Hz); MS (ESI) m/z 251 [M+H]+.
5.1.47. Ethyl 3-(1,7-dimethyl-2-oxo-1,2-dihydro-3H-
imidazo[4,5-b]pyridin-3-yl)propanoate (33)
To a mixture of 32 (3.21 g, 12.9 mmol) in DMF (50 mL) were
added MeI (1.20 mL, 19.2 mmol) and Cs2CO3 (5.00 g, 15.3 mmol),
and the mixture was stirred at room temperature for 14 h. The
mixture was diluted with H2O and extracted with EtOAc. The
organic layer was washed with brine, dried over anhydrous Na2-
SO4, filtered and concentrated in vacuo. The residue was purified
by flash column chromatography (silica gel, 0–5% MeOH in CHCl3)
to give 33 (3.39 g, 99%) as a yellow oil. 1H NMR (DMSO-d6) d 1.11 (t,
3H, J = 7.1 Hz), 2.55 (s, 3H), 2.74 (t, 2H, J = 7.3 Hz), 3.54 (s, 3H), 4.00
(q, 2H, J = 7.1 Hz), 4.09 (t, 2H, J = 7.3 Hz), 6.85 (dd, 1H, J = 5.3,
0.6 Hz), 7.84 (d, 1H, J = 5.3 Hz); MS (ESI) m/z 264 [M+H]+.
5.1.43. 3-(3-Methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-
b]pyrazin-1-yl)propanoic acid (28)
5.1.48. 3-(1,7-Dimethyl-2-oxo-1,2-dihydro-3H-imidazo[4,5-
b]pyridin-3-yl)propanoic acid (34)
Compound 28 was prepared from 27 in a manner similar to that
described for compound 12, with a yield of 89% as a pale yellow
solid. 1H NMR (DMSO-d6) d 2.75 (t, 2H, J = 7.5 Hz), 3.34 (s, 3H),
4.08 (t, 2H, J = 7.5 Hz), 7.94–7.97 (m, 2H), 12.39 (s, 1H); MS (ESI)
m/z 223 [M+H]+.
Compound 34 was prepared from 33 in a manner similar to that
described for compound 12, with a yield of 85% as a pale yellow
solid. 1H NMR (DMSO-d6) d 2.55 (s, 3H), 2.69 (t, 2H, J = 7.5 Hz),
3.54 (s, 3H), 4.06 (t, 2H, J = 7.5 Hz), 6.86 (dd, 1H, J = 5.2, 0.7 Hz),
7.84 (d, 1H, J = 5.2 Hz), 12.33 (s, 1H); MS (ESI) m/z 236 [M+H]+.
5.1.44. 1-[2-(7-Methoxy-3-phenyl-3H-imidazo[4,5-b]pyridin-2-
yl)ethyl]-3-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyrazin-2-
one hydrochloride (29)
5.1.49. 3-[2-(7-Methoxy-3-phenyl-3H-imidazo[4,5-b]pyridin-2-
yl)ethyl]-1,7-dimethyl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-
2-one (35)
Compound 29 was prepared from 23a and 28 in a manner sim-
ilar to that described for compound 10a, with a yield of 76% as a
beige solid. 1H NMR (DMSO-d6) d 3.25–3.34 (m, 5H), 4.08 (s, 3H),
4.22 (t, 2H, J = 7.0 Hz), 6.95 (d, 1H, J = 5.7 Hz), 7.41–7.54 (m, 5H),
7.78 (d, 1H, J = 3.4 Hz), 7.89 (d, 1H, J = 3.4 Hz), 8.14 (d, 1H,
J = 5.6 Hz); MS (ESI) m/z 402 [M+H]+ Anal. Calcd for C21H19N7O2-
ꢀHClꢀ1.6H2O: C, 54.04; H, 5.01; N, 21.01; Cl, 7.60. Found: C, 54.13;
H, 5.13; N, 20.94; Cl, 7.57.
Compound 35 was prepared from 23a and 34 in a manner sim-
ilar to that described for compound 20b, with a yield of 68% as a
pale yellow solid. 1H NMR (DMSO-d6) d 2.52 (s, 3H), 3.21 (t, 2H,
J = 7.0 Hz), 3.46 (s, 3H), 4.07 (s, 3H), 4.21 (t, 2H, J = 7.0 Hz), 6.78
(d, 1H, J = 5.2 Hz), 6.87 (d, 1H, J = 5.6 Hz), 7.38–7.53 (m, 5H), 7.69
(d, 1H, J = 5.2 Hz), 8.07 (d, 1H, J = 5.6 Hz); MS (ESI) m/z 415
[M+H]+; HRMS (ES+) calcd for C23H22N6O2 [M+H]+ 415.1882;
found, 415.1873.
5.1.45. Ethyl N-(4-methyl-3-nitropyridin-2-yl)-beta-alaninate
(31)
5.2. PDE10A enzyme assay protocol
To a mixture of 30 (5.00 g, 29.0 mmol) in EtOH (50 mL) were
added b-alanine ethyl ester hydrochloride (11.1 g, 72.4 mmol)
and triethylamine (20.2 mL, 145 mmol), and the mixture was stir-
red at 80 °C for 3 days. The mixture was concentrated in vacuo. The
residue was diluted with 50% EtOAc/n-hexane, and the resulting
precipitate was removed by filtration. The filtrate was concen-
trated in vacuo and purified by flash column chromatography
5.2.1. Cloning and vector construction of PDE10A2
The full-length human PDE10A2 was amplified by PCR using the
1st strand cDNA synthesized from the total RNA isolated from
human neuroblastoma TGW cell line. The PCR products were
cloned into a pCR2.1-TOPO vector (Invitrogen. Inc.) to confirm
sequences. The confirmed plasmid was digested with restricted
Please cite this article in press as: Chino, A.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.023

A. Chino et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
enzymes, BamHI/HindIII, and this digested product was inserted
into a pFastBac1 vector (Invitrogen. Inc.).
Each test compound (0.3–100
l
M) was pre-incubated with
human liver microsomes (0.1 mg/mL) and NADPH (1.5 mM) at
37 °C. The pre-incubation times used were 0 and 30 min. Following
the pre-incubation step, each compound was co-incubated with
5.2.2. Preparation of human PDE10A2 enzyme
Human PDE10A2 enzyme protein was expressed in a Spodoptera
frugiperda Sf9 insect cell using the Bac-to-Bac Baculovirus Expres-
sion System (Invitrogen. Inc.). The infected Sf9 cells were collected
by the centrifuge and removed medium. The collected cells were
lysed by sonication in the lysis buffer (50 mM Tris–HCl [pH 8.0],
150 mM NaCl, 3 mM DTT, 0.1% NP-40, 20% glycerol with protease
inhibitors), The lysate was centrifuged and supernatant was col-
lected to obtain the PDE10A2 enzyme solution. We confirmed
the PDE10A2 expression by Western blot analysis.
midazolam (2 lM) at 37 °C for 20 min. At the end of the incubation,
the reaction was terminated by the addition of aqueous solution
containing 80% acetonitrile. The concentration of 10-hydroxymi-
dazolam was determined by LC–MS analysis. The inhibition of
CYP3A4 activity was assessed by comparing the amount of
10-hydroxymidazolam formed in the presence of varying concen-
trations of inhibitor to the amount of 10-hydroxymidazolam
formed in the solvent control. In each study, a CYP3A4 potent
and specific inhibitor, verapamil was used as positive control.
5.2.3. PDE10A2 inhibition assay
Acknowledgments
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
The authors wish thank to Dr. Takeshi Shimada for performing
pharmacological evaluations, Mr. Hiroyuki Moriguchi for his help-
ful support in preparing this manuscript, Mr. Wataru Hamaguchi
for providing language help, and the staff of Astellas Research
Technologies Co., Ltd, for conducting the CYP inhibition screening,
metabolic clearance assay, partition coefficient assay, elemental
analysis, and spectral measurements.
performed in 12
PDE10A enzyme, a buffer (40 mM Tris–HCl pH 7.5; 5 mM MgCl2),
0.1 M cAMP and various concentrations of compounds (0.1 nM
to 10 M). After compounds were preincubated for 30 min with
lL samples containing a optimal amount of the
l
l
the enzyme, the reaction was initiated by adding the substrate
cAMP and the mixture was incubated for 60 min at room temper-
ature with agitation. The reaction was terminated on addition of
the fluorescence acceptor (cAMP labeled with the dye d2) and
the fluorescence donor (anti-cAMP antibody labeled with Cryptate,
Cisbio). After 60 min, the fluorescence transfer corresponding to
the amount of residual cAMP was measured at lex. 320 nm, lem.
620 nm and lem. 665 nm using an Envision plate reader (PerkinEl-
mer) and signal ratio (665/620) was calculated. The ratio deter-
mined in the absence of enzyme was subtracted from all data.
The obtained results were converted to activity relative to an unin-
hibited control (100%) and IC50 values were calculated using Prism
software (GraphPad Software, Inc.).
References and notes
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Pooled human liver microsomes (Xenotech LLC.) were diluted in
100 mM KH2PO4/K2HPO4 buffer (pH 7.4) containing 0.1 mM EDTA.
The incubation mixtures (270
0.2 mg/mL of microsomal proteins, and 1 mM NADPH (30
pre-incubated for approximately 15 min at 37 °C. Reactions were
initiated by the addition of 0.2 M of substrates. After the appro-
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l
L) were
l
l
l
l
tem (Waters) and Xevo TQ (Waters). The in vitro intrinsic clearance
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course of the residual ratio of the compounds.14
Keð1=minÞ ꢁ MS content ðmg=kgÞ
CLint;
ðmL=min=kgÞ ¼
ð1Þ
vitro
MS Protein Concn: ðmg=mLÞ
where the Ke is the disappearance rate constant.
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bation period where CYP3A4 substrate, midazolam, was added to
the preincubate to measure residual CYP3A4 activity. Midazolam
10-hydroxylation was used to monitor the CYP3A4 activity.
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