Design and synthesis of novel benzimidazole derivatives as phosphodiesterase 10A inhibitors with reduced CYP1A2 inhibition

Article abstract of DOI:10.1016/j.bmc.2013.10.035

A novel class of phosphodiesterase 10A (PDE10A) inhibitors with reduced CYP1A2 inhibition were designed and synthesized starting from 2-{[(1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (1). Introduction of an isopropyl group at the 2-position and a methoxy group at the 5-position of the benzimidazole ring of lead compound 1 resulted in the identification of 2-{[(2-isopropyl-5-methoxy-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (25b), which exhibited potent PDE10A inhibitory activity with reduced CYP1A2 inhibitory activity compared to compound 1.

Full text of DOI:10.1016/j.bmc.2013.10.035

Bioorganic & Medicinal Chemistry 21 (2013) 7612–7623
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel benzimidazole derivatives as
phosphodiesterase 10A inhibitors with reduced CYP1A2 inhibition
⇑
Wataru Hamaguchi , Naoyuki Masuda, Mai Isomura, Satoshi Miyamoto, Shigetoshi Kikuchi,
Yasushi Amano, Kazuya Honbou, Takuma Mihara, Toshihiro Watanabe
Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, 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:
A novel class of phosphodiesterase 10A (PDE10A) inhibitors with reduced CYP1A2 inhibition were
designed and synthesized starting from 2-{[(1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (1).
Introduction of an isopropyl group at the 2-position and a methoxy group at the 5-position of the benz-
imidazole ring of lead compound 1 resulted in the identification of 2-{[(2-isopropyl-5-methoxy-1-phe-
nyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (25b), which exhibited potent PDE10A inhibitory
activity with reduced CYP1A2 inhibitory activity compared to compound 1.
Received 27 September 2013
Revised 22 October 2013
Accepted 23 October 2013
Available online 31 October 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
PDE10A inhibitor
Schizophrenia
Benzimidazole
CYP1A2
1. Introduction
rodent behavioral models of psychiatric disorders.5 Especially a
couple of literatures suggested that these PDE10A inhibitors also
Schizophrenia is a chronic and devastating psychiatric disorder
that is estimated to affect approximately 1% of the world’s popula-
tion.1 Current antipsychotic drugs are effective in treating positive
symptoms of schizophrenia such as hallucination and delusion, but
have a only limited efficacy on negative symptoms such as social
withdrawal, and cognitive impairments. In addition, these existing
antipsychotics frequently induce adverse effects such as extrapyra-
midal syndrome, weight gain, diabetes and QT prolongation,2 high-
lighting the unmet medical needs for drugs less prone to such side
effects.
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that
regulate intracellular signaling by hydrolyzing cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP). The PDE superfamily of enzymes is classified into 11 fam-
ilies, namely PDE1–PDE11, in mammals. PDE10A enzyme is a dual
substrate (cAMP/cGMP) phosphodiesterase that is highly ex-
pressed in the brain, particularly in the medium spiny neurons of
the mammalian striatum.3 Inhibition of PDE10A may enhance the
intracellular second messenger signaling and striatal output sug-
gested to be impaired in the schizophrenic patients.4 Furthermore,
papaverine and MP-10 (Fig. 1) which were reported as potent
selective PDE10A inhibitors, showed potent efficacies in several
have pharmacological effects in animal models of cognitive deficits
and negative symptoms in schizophrenia.5d,e Thus, PDE10A inhibi-
tors have gathered attention as a new therapeutic approach for the
treatment of schizophrenia.6
Our high-throughput screening approach identified new benz-
imidazole analog 1 (Fig. 1) as a PDE10A inhibitor with an IC50 value
of 309 nM, but this lead compound 1 also had potent inhibitory
activity against human cytochrome P450 1A2 (CYP1A2). Since
CYP1A2 metabolizes some clinically used drugs such as theophyl-
line,7 propranolol,8 and clozapine,9 the inhibition of CYP1A2 can
cause unfavorable drug-drug metabolizing interaction.
Before the modification of lead compound 1, we focused our
attention on the structural similarity between MP-10 and com-
pound 1. That is, both MP-10 and our lead compound 1 contain
the quinolin-2-ylmethoxy unit, and the quinoline ring in MP-10
was reported to occupy the PDE10A selectivity pocket and form a
hydrogen bond to Tyr693 of PDE10A enzyme.5e Therefore, we at-
tempted to modify the 1-phenyl-benzimidazol moiety of com-
pound
1 to increase PDE10A inhibitory activity and avoid
CYP1A2 inhibition on the assumption that the quinoline ring of
compound 1 occupied the selectivity pocket of PDE10A, as in the
case of MP-10.
In this paper, we report the synthesis and the successful devel-
opment of novel PDE10A inhibitors with reduced CYP1A2 inhibi-
tory activity.
⇑
Corresponding author. Tel.: +81 29 863 6743; fax: +81 29 852 5387.
E-mail address: wataru.hamaguchi@astellas.com (W. Hamaguchi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.035

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7613
O
O
N
N
N
N
N
N
O
N
O
O
O
N
1
MP-10
papaverine
Figure 1. Structures of papaverine, MP-10, and compound 1.
O
O
N
2. Chemistry
N
a
N
O
HO
The synthesis of target compounds is shown in Schemes 1–6.
The ipso-substitution reaction of reagent 2 with aniline followed
by alkylation yielded compound 4, which was converted to dia-
mine 6 by reduction of the nitro group. Cyclization of diamine 6
with various orthoesters, carboxylic acids, acid chloride, carboxilic
acid anhydride, or phosgene iminium chloride gave phenyl benz-
imidazole derivatives 1 and 7a–i. 5-Fluoro-benzimidazole ana-
logue 8 was also synthesized from 2,5-difluoro-4-nitrophenol 3
using a similar procedure (Scheme 1).
Scheme 2 shows the synthesis of indole and indazole deriva-
tives 14a and 14b. Pd-catalyzed coupling between compound 9
and bromobenzene generated compound 10, which after deprotec-
tion afforded intermediate 13a. Hydroxyindazole derivative 13b
was synthesized via a cyclization reaction between benzaldehyde
11 and phenylhydrazine followed by the deprotection of methoxy
group. Compounds 13a and 13b were reacted with 2-(chloro-
methyl)quinoline to give 14a and 14b, respectively. Benzoxazole
analogue 16 was also synthesized in a similar manner from its pre-
cursor 15 as described in Scheme 3.10
16
15
Scheme 3. Reagents and conditions: (a) 2-(chloromethyl)quinoline hydrochloride,
KI, K2CO3, DMF.
5-Methoxybenzimidazole analogues 25a and 25b were pre-
pared using the synthesis depicted in Scheme 5. Compound 2211
was converted to amine intermediate 23 using a Buchwald–Har-
twig cross coupling reaction. Reduction of compound 23 followed
by cyclization with orthoesters yielded benzimidazole derivatives
24a and 24b. Deprotection of the benzyl group and subsequent
alkylation generated compounds 25a and 25b.
The synthesis of 1-substituted benzimidazole analogues 31a–i
and 32 are shown in Scheme 6. Phenol 26 was reacted with 2-
(chloromethyl)quinoline to give compound 27, which was con-
verted to the intermediate 30 using Buchwald-Hartwig coupling
or Ullmann coupling when the R group is an aromatic ring. Inter-
mediate 30 with cyclohexyl ring or 4-pyridylmethyl group as the
R group were prepared using an ipso-substitution reaction be-
tween amine and compound 29, which was synthesized by the
alkylation of phenol 28. Reduction of the nitro group of compounds
30a–i gave diamines, followed by cyclization with orthoformic acid
methyl ester yielded benzimidazole analogues 31a–i. Intermediate
Azabenzimidazole analogue 21 was prepared in five steps as
outlined in Scheme 4. Ipso-substitution of chloropyridine 17 with
aniline gave compound 18. Reduction of the nitro group followed
by cyclization with orthoester yielded intermediate 19. Deprotec-
tion of the methoxy group of 19 gave compound 20, which was
alkylated under Mitsunobu-type condition to afford compound 21.
N
NO2
OH
NO2
NH
X
NH2
R
F
d, e, f, or g
N
c
a, b
N
N
O
N
O
NH
O
(X = H)
X
1, 7a, 7b, 7d-i
6
4 (X = H)
5 (X = F)
h
2 (X = H)
3 (X = F)
c, d
N
N
F
(X = F)
N
N
N
N
O
N
O
7c
8
Scheme 1. Reagents and conditions: (a) aniline; (b) 2-(chloromethyl)quinoline hydrochloride, KI, K2CO3, DMF; (c) Fe, NH4Cl, EtOH, H2O; (d) RC(OMe)3 or RC(OEt)3, TsOHꢀH2O,
THF; (e) RCOCl, pyridine, THF then AcOH; (f) RCO2H, WSCꢀHCl, HOBt, DMF then AcOH; (g) (RCO)2O, THF then AcOH; (h) phosgene iminium chloride, 1,2-dichloroethane.
a
N
N
b
O
N
H
O
X
X
e
N
N
N
O
HO
10
12
9
14a (X = CH)
14b (X = N)
d
O
13a (X = CH)
13b (X = N)
N
c
MeO
H
F
MeO
11
Scheme 2. Reagents and conditions: (a) bromobenzene, Pd(OAc)2, Xphos, K3PO4, toluene; (b) Pd(OH)2/C, HCO2NH4, MeOH, H2O, THF; (c) phenylhydrazine, Cs2CO3, NMP; (d)
BBr3, CH2Cl2; (e) 2-(chloromethyl)quinoline hydrochloride, K2CO3, KI, DMF.

7614
W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
N
N
N
N
NO2
NH
N
N
NO2
Cl
e
d
N
b, c
a
O
N
HO
N
MeO
N
MeO
N
MeO
N
17
18
19
20
21
Scheme 4. Reagents and conditions: (a) aniline, K2CO3, DMF; (b) Pd(OH)2/C, H2, EtOH–DMF; (c) HC(OEt)3, TsOHꢀH2O, THF; (d) pyridine hydrochloride; (e) 2-
quinolinylmethanol, ADDP, nBu3P, THF.
MeO
O
MeO
O
N
N
N
N
NO2
NH
MeO
O
NO2
Br
MeO
O
R
R
d, e
b, c
a
N
23
25a (R = H)
25b (R = i Pr)
22
24a (R = H)
24b (R = i Pr)
Scheme 5. Reagents and conditions: (a) aniline, Pd2(dba)3, BINAP, Cs2CO3, toluene; (b) Fe, NH4Cl, EtOH, H2O; (c) HC(OEt)3 or iPrCH(OMe)3, TsOHꢀH2O, THF; (d) anisole, TFA;
(e) 2-(chloromethyl)quinoline hydrochloride, Cs2CO3, KI, DMF.
N
NO2
N
NO2
Br
N
R
O
a
a
g, h
N
N
b, c, or d
e or f
Br
O
NO2
HO
HO
31a-i
N
27
26
28
NH
R
O
N
N
g, i
NO2
F
N
O
30a-i
NO2
F
O
N
32
29
Scheme 6. Reagents and conditions: (a) 2-(chloromethyl)quinoline hydrochloride, KI, K2CO3, DMF; (b) R-NH2, Pd2(dba)3, BINAP, tBuONa, toluene; (c) R-NH2, Pd2(dba)3,
Xantphos, Cs2CO3, toluene or NMP; (d) R-NH2, CuI, N,N0-dimethylethylenediamine, K3PO4, DMSO; (e) R-NH2, K2CO3, DMF; (f) R-NH2, DMF; (g) Fe, NH4Cl, EtOH, H2O; (h)
HC(OMe)3, TsOHꢀH2O, THF; (i) iPrC(OMe)3, TsOHꢀH2O, THF.
30b was also converted to isopropyl benzimidazole analogue 32
using a similar procedure.
a decrease in activity (7h and 7i) compared with isopropyl ana-
logue 7g. The X-ray co-crystal structure of compound 7g in the
catalytic domain of PDE10A enzyme confirmed that the quinolinyl
3. Results and discussion
Table 1
PDE10A inhibitory potencies of newly synthesized compounds
were tested using in vitro inhibition of human recombinant
PDE10A catalyzed cAMP hydrolysis. The lead compound 1 had
moderate PDE10A inhibitory activity with an IC50 of 309 nM but
displayed potent CYP1A2 inhibitory activity with an IC50 value of
PDE10A potency and CYP1A2 inhibition for core heterocycles
A
A
A
A
A
A
A
N
A
O
A
0.38 lM.
To determine the optimal ring of the core heterocycle, the benz-
imidazole ring of compound 1 was replaced with other 5,6-mem-
bered hetroaromatic ring (Table 1). Indole, indazole and
benzisoxazole analogues showed no PDE10A inhibitory activity
up to 1000 nM concentration (14a, 14b, and 16), which suggested
that the nitrogen atom at the 3-position of the benzimidazole ring
is important for PDE10A inhibitory activity. Imidazopyridine ana-
logue 21 had improved PDE10A inhibitory activity, although the
CYP1A2 inhibition of 21 was extremely potent. The introduction
of fluorine atom into the 5-position of the benzimidazole ring led
to a slight decrease in activity (8), but the methoxy group at the
5-position contributed to a slight increase in PDE10A inhibitory
activity with decreased CYP1A2 inhibition (25a).
We also optimized substituents at the 2-position of the benz-
imidazole ring of compound 1 (Table 2). Although the introduction
of the electron-withdrawing trifluoromethyl group resulted in a
loss of activity (7a), the electron-donating methoxymethyl and
dimethylamino groups caused an increase in activity (7b and 7c).
When we added several alkyl groups to the structure, PDE10A
inhibitory activities increased in order of steric bulkiness up to
the size of the isopropyl group (7d–g). However, the introduction
of bulkier substituents such as n-propyl or isobutyl groups led to
Compd
A
PDE10A IC50 (nM)
309
CYP1A2 IC50
0.38
(lM)
N
N
1
14a
14b
16
>1000
>1000
>1000
NTa
NTa
NTa
N
N
N
N
O
N
N
21
8
89
<0.31
NTa
N
F
N
494
214
N
MeO
N
N
25a
7.9
a
Not tested.

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7615
Table 2
4-pyrimidyl analogue 31h had similar potency to 4-pyridyl ana-
logue 31b. The pyridyl nitrogen atom of 31b and pyrimidyl N-1
nitrogen atom of 31h may form a hydrogen bond to a water mol-
ecule inside the PDE10A catalytic site, as in the case with MP-
10.5e Fortunately, the CYP1A2 inhibitory activities of 31b and
31h were twofold weaker than that of compound 1. The thiazole
derivative 31i showed the most potent PDE10A inhibitory activity
in Table 3, but also had extremely potent CYP1A2 inhibitory activ-
Effect of C-2 substituents on PDE10A potency and CYP1A2 inhibition
N
R
N
N
O
Compd
R
PDE10A IC50 (nM)
CYP1A2 IC50 (lM)
ity (IC50 <0.31 lM). The logarithm of the calculated partition coef-
1
H
CF3
CH2OMe
NMe2
Me
309
>1000
166
152
305
208
180
92
0.38
NTa
5.1
7.3
0.63
1.6
ficient between n-octanol and water (ClogP) of compounds 31i,
31b and 31h was 3.60, 1.92 and 1.87, respectively.13 Thus, the
higher ClogP value of 31i compared with those of 31b and 31h
may contribute to the strong CYP1A2 inhibition.14
We successfully obtained compounds 25a, 7g and 31b, which
had increased PDE10A inhibitory activities with reduced CYP1A2
inhibition compared to lead compound 1. We therefore decided
to combine the methoxy group found in 25a with the isopropyl
group found in 7g, which gave successful results regarding PDE10A
inhibitory activity (25b, IC50 = 25 nM) and CYP1A2 inhibition
7a
7b
7c
7d
7e
7f
7g
7h
7i
Et
Cyclopropyl
Isopropyl
n-Propyl
Isobutyl
3.3
2.8
299
849
3.3
NTa
a
Not tested.
(IC50 = 18 lM), as shown in Table 4. Compound 25b had reduced
CYP1A2 inhibitory activity, even compared with MP-10 that
showed moderate CYP1A2 inhibitory activity in our assay
(IC50 = 3.2 lM). We also combined the 4-pyridyl group found in
31b with the isopropyl group found in 7g. Unfortunately, PDE10A
inhibitory activity of the combined compound 32 was reduced to
one-sixth of that of compounds 31b and 7g, and was also weaker
than even that of lead compound 1. Although the reason for the re-
duced activity of 32 is unclear, the isopropyl group may cause the
nitrogen atom formed a hydrogen bond to Tyr693 of PDE10A en-
zyme as in the case with MP-10, and the isopropyl group was
found to occupy a hydrophobic pocket in the catalytic domain of
PDE10A (Fig 2). The benzimidazole ring of 7g was sandwiched by
Phe729 and Phe696, and it formed
Phe729 and CH– interaction with Phe696. In addition, the N-1
phenyl ring probably formed CH– interaction with Ile692. As for
p–p stacking interaction with
p
p
the CYP1A2 inhibition of our compounds, any substituent at the
2-position was effective in reducing inhibition. We assumed that
the substituents at the 2-position may directly block the interac-
tion between the benzimidazole and CYP1A2 enzyme. We also as-
sumed that the co-planer structure between the benzimidazole
and the phenyl ring might cause the CYP1A2 inhibition,12 therefore
substituents at the 2-position increase the dihedral angle between
the benzimidazole and the phenyl ring to attenuate CYP1A2
inhibition.
Table 3
Effect of N-1 substituents on PDE10A potency and CYP1A2 inhibition
N
N
N
R
O
The influence of the phenyl ring on the 1-position of the benz-
imidazole ring of compound 1 was then investigated (Table 3).
Nonaromatic cyclohexyl analogue 31a lost PDE10A inhibitory
activity. The replacement of the phenyl ring with 4-pyridyl ring re-
sulted in a threefold increase in activity (31b), but the insertion of
the methylene unit between the benzimidazole ring and 4-pyridyl
moiety of compound 31b was found to be detrimental to activity
(31c; IC50 >1000 nM). We also examined other heteroaromatic
rings. Although analogues including pyrazole (31d and 31e), pyrid-
azine (31f), and 2-pyrimidine (31g) had decreased activity,
Compd
R
PDE10A IC50 (nM)
309
CYP1A2 IC50
0.38
(lM)
1
31a
>1000
NTa
31b
31c
31d
99
0.80
NTa
NTa
N
>1000
774
N
N
N
Me
N
N
31e
>1000
NTa
Me
31f
31g
31h
>1000
>1000
NTa
NTa
N
N
N
N
S
N
72
16
0.70
N
N
31i
<0.31
a
Not tested.
Figure 2. Crystal structure of 7g (green) bound to the PDE10A (PDB code 3WI2).

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W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
Table 4
Combination effect on PDE10A potency and CYP1A2 inhibition
Compd
Structure
PDE10A IC50 (nM)
CYP1A2 IC50 (lM)
MeO
O
N
N
N
25b
25
18
N
N
N
O
32
601
NTa
N
a
Not tested.
pyridyl nitrogen atom to locate at an unsuitable position for a pos-
sible hydrogen bond to a water molecule inside the PDE10A cata-
lytic domain.
We succeeded in finding novel benzimidazole derivatives such
as 25b with improved PDE10A inhibitory potency and reduced
CYP1A2 inhibition. Further optimization and biological evaluation
of benzimidazole analogues is being conducted at present.
and the mixture was stirred at 60 °C for 3 h. After cooling at room
temperature, the mixture was concentrated in vacuo, and the res-
idue was partitioned between EtOAc and water. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. The res-
idue was washed with hexane–EtOAc to give 4 (12.1 g, 82%) as a
yellow solid. 1H NMR (DMSO-d6) d 5.38 (s, 2H), 6.61–6.65 (m,
2H), 7.11–7.23 (m, 5H), 7.56 (d, 1H, J = 8.5 Hz), 7.61–7.66 (m,
1H), 7.77–7,82 (m, 1H), 7.93 (d, 1H, J = 8.5 Hz), 8.00 (d, 1H,
J = 8.0 Hz), 8.12–8.16 (m, 1H), 8.40 (d, 1H, J = 8.5 Hz), 8.58 (s,
1H); MS (ESI) m/z 372 [M+H]+.
4. Conclusions
Here, on the assumption that the quinoline ring of lead com-
pound 1 occupied the selectivity pocket of PDE10A, the phenyl
benzimidazole moiety of 1 was modified to increase PDE10A inhib-
itory activity and avoid CYP1A2 inhibition. Accordingly, the isopro-
pyl group at the 2-position of the benzimidazole ring increased
PDE10A inhibitory activity with reduced CYP1A2 inhibition, and
the quinolinyl nitrogen atom of 7g was found to form a hydrogen
bond to Tyr693 of PDE10A enzyme, as in the case of MP-10. The
methoxy group at the 5-position of the benzimidazole ring was
also effective in increasing PDE10A inhibitory activity and avoiding
CYP1A2 inhibition. The combination of the isopropyl group at the
2-position and the methoxy group at the 5-position resulted in
success, and led to compound 25b being a relatively potent
PDE10A inhibitor with reduced CYP1A2 inhibition.
5.1.2. 4-Fluoro-2-nitro-N-phenyl-5-(quinolin-2-ylmethoxy)
aniline (5)
Compound 5 was prepared from 2,5-difluoro-4-nitrophenol (3)
and aniline in a manner similar to that described for compound 4,
with a yield of 66% as a yellow solid. 1H NMR (CDCl3) d 5.31 (s, 2H),
6.69 (d, 1H, J = 7.4 Hz), 6.98–7.02 (m, 2H), 7.13–7.17 (m, 3H), 7.58–
7.62 (m, 2H), 7.73–7.79 (m, 1H), 7.86 (d, 1H, J = 8.1 Hz), 7.95 (d, 1H,
J = 8.5 Hz), 8.00 (d, 1H, J = 11.4 Hz), 8.22 (d, 1H, J = 8.5 Hz), 9.65 (br
s, 1H); MS (ESI) m/z 390 [M+H]+.
5.1.3. N2-Phenyl-4-(quinolin-2-ylmethoxy)benzene-1,2-diamine (6)
To a mixture of 4 (12.1 g, 32.6 mmol) and iron powder (9.10 g,
163 mmol) in EtOH (200 mL) and water (75 mL) was added NH4Cl
(871 mg, 16.3 mmol), and the mixture was refluxed for 3 h. After
cooling at room temperature, the mixture was diluted with CHCl3
and filtered through celite pad, and the filtrate was concentrated in
vacuo. The residue was partitioned between water and EtOAc, and
the organic layer was dried over Na2SO4, filtered and concentrated
in vacuo to give 6 (10.9 g, 98%) as a brown solid. 1H NMR (CDCl3) d
3.41 (s, 2H), 5.28 (s, 2H), 5.30 (br s, 1H), 6.63–6.67 (m, 1H), 6.72–
6.90 (m, 5H), 7.14–7.20 (m, 2H), 7.51–7.57 (m, 1H), 7.65 (d, 1H,
J = 8.5 Hz), 7.69–7.75 (m, 1H), 7.80–7,84 (m, 1H), 8.05 (d, 1H,
J = 8.3 Hz), 8.17 (d, 1H, J = 8.5 Hz); MS (APCI/ESI) m/z 342 [M+H]+.
5. Experimental section
5.1. Chemistry
1H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-
LA400, or JEOL JNM-AL400 and the chemical shifts were expressed
in d (ppm) values with trimethylsilane as an internal reference
(s = singlet, d = doublet, t = triplet, q = quartet, sep = septet,
m = multiplet, dd = doublet of doublets, and br = broad peak). Mass
spectra (MS) were recorded on Thermo Electron LCQ Advantage or
Agilent 6140. Elemental analyses were performed using 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.
5.1.4. 2-{[(1-Phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline
dihydrochloride (1)
To a solution of 6 (230 mg, 0.67 mmol) in THF (6 mL) were
added triethyl orthoformate (0.25 mL, 1.50 mmol) and p-toluene-
sulfonic acid monohydrate (13 mg, 0.07 mmol), and the mixture
was refluxed for 2 h. The mixture was then cooled at room temper-
ature and concentrated in vacuo. The residue was partitioned be-
tween saturated aqueous sodium bicarbonate and CHCl3. The
organic layer was washed with brine, dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (10–50% EtOAc in hexane) to give a
brown oil, which was dissolved in CHCl3 (5 mL) and 4 M HCl/EtOAc
(0.43 mL, 1.72 mmol) was added to the solution. The mixture was
concentrated in vacuo, and the residue was washed with
diisopropylether to give 1 (239 mg, 84%) as a pale pink solid. 1H
5.1.1. 2-Nitro-N-phenyl-5-(quinolin-2-ylmethoxy)aniline (4)
A mixture of 3-fluoro-4-nitrophenol (2, 10.0 g, 63.7 mmol) and
aniline (17.0 mL, 187 mmol) was stirred at 140 °C for 3 h. After
cooling at room temperature, the mixture was diluted with EtOAc
and washed with 1 M hydrochloric acid. The organic layer was
dried over MgSO4, filtered and concentrated in vacuo. The residue
was washed with hexane–EtOAc to give 3-anilino-4-nitrophenol
(9.14 g, 62%) as a red solid. To a mixture of the above-obtained
3-anilino-4-nitrophenol and 2-(chloromethyl)quinoline hydro-
chloride (9.35 g, 43.7 mmol) in DMF (200 mL) were added K2CO3
(12.1 g, 87.3 mmol) and potassium iodide (659 mg, 3.97 mmol),

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7617
NMR (DMSO-d6) d 5.61 (s, 2H), 7.42–7.45 (m, 2H), 7.66–7.79
5.1.9. 2-({[2-(Methoxymethyl)-1-phenyl-1H-benzimidazol-6-
(m, 6H), 7.86 (d, 1H, J = 8.5 Hz), 7.88–7.93 (m, 2H), 8.09–8.16
(m, 2H), 8,64 (d, 1H, J = 8.5 Hz), 9.76 (s, 1H); MS (ESI) m/z
352 [M+H]+; Anal. Calcd for C23H17N3Oꢀ2HClꢀ3.0H2O: C, 57.75;
H, 5.27; N, 8.78; F, 14.82. Found: C, 58.03; H, 5.24; N, 8.78; F,
14.56.
yl]oxy}methyl)quinoline dihydrochloride (7b)
To a solution of 6 (341 mg, 1.00 mmol) and methoxyacetic acid
(90 lL, 1.20 mmol) in DMF (15 mL) were added 1H-benzotriazol-1-
ol (135 mg, 1.00 mmol) and 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (230 mg, 1.20 mmol), and the mixture
was stirred at room temperature for 17 h. The mixture was concen-
trated in vacuo, and the residue was partitioned between EtOAc
and water. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The residue was dissolved in acetic acid
(10 mL) and refluxed for 1 h before cooling at room temperature.
The mixture was concentrated in vacuo, and the residue was par-
titioned between EtOAc and saturated aqueous sodium bicarbon-
ate. The organic layer was concentrated in vacuo, and the residue
was purified by silica gel column chromatography (0–5% MeOH
in CHCl3) to give the free form of the title compound, which was
then dissolved in EtOH (10 mL), and 4 M HCl/EtOAc (1.00 mL,
4.00 mmol) was added to the mixture. The mixture was concen-
trated in vacuo, and the residue was recrystalized from
EtOH–Et2O to give 7b (288 mg, 62%) as a beige solid. 1H NMR
(DMSO-d6) d 3.34 (s, 3H), 4.70 (s, 2H), 5.46 (s, 2H), 7.05 (d, 1H,
J = 2.1 Hz), 7.33 (dd, 1H, J = 9.0, 2.3 Hz), 7.59–7.71 (m, 6H), 7.75
(d, 1H, J = 8.6 Hz), 7.80 (d, 1H, J = 8.9 Hz), 7.82–7.88 (m, 1H), 7.99
(d, 1H, J = 8.4 Hz), 8.04 (d, 1H, J = 8.9 Hz), 8.51 (d, 1H, J = 8.5 Hz);
MS (ESI) m/z 396 [M+H]+; Anal. Calcd for C25H21N3O2ꢀ2HClꢀ0.5H2O:
C, 62.90; H, 5.07; N, 8.80; Cl, 14.85. Found: C, 62.80; H, 5.08; N,
8.85; Cl, 14.73.
5.1.5. 2-{[(2-Methyl-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}
quinoline dihydrochloride (7d)
Compound 7d was prepared from 6 and triethyl orthoacetate in
a manner similar to that described for compound 1, with a yield of
55% as a colorless solid. 1H NMR (DMSO-d6) d 2.64 (s, 3H), 5.51 (s,
2H), 7.06 (d, 1H, J = 2.2 Hz), 7.38 (dd, 1H, J = 9.1, 2.3 Hz), 7.64–7.76
(m, 6H), 7.79 (d, 1H, J = 8.6 Hz), 7.84 (d, 1H, J = 8.6 Hz), 7.86–7.92
(m, 1H), 8.07 (dd, 2H, J = 8.7, 8.7 Hz), 8.58 (d, 1H, J = 8.5 Hz); MS
(ESI) m/z 366 [M+H]+; Anal. Calcd for C24H19N3Oꢀ2HClꢀ3.9H2O: C,
56.68; H, 5.71; N, 8.26; F, 13.94. Found: C, 56.81; H, 5.60; N,
8.20; F, 13.81.
5.1.6. 2-{[(2-Ethyl-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}
quinoline dihydrochloride (7e)
Compound 7e was prepared from 6 and triethyl orthopropionate
in a manner similar to that described for compound 1, with a yield
of 47% as a pale pink solid. 1H NMR (DMSO-d6) d 1.30 (t, 3H,
J = 7.5 Hz), 2.94 (q, 2H, J = 7.5 Hz), 5.49 (s, 2H), 7.02 (d, 1H,
J = 2.3 Hz), 7.39 (dd, 1H, J = 9.0, 2.3 Hz), 7.64–7.80 (m, 7H), 7.83–
7.91 (m, 2H), 8.02–8.09 (m, 2H), 8.56 (d, 1H, J = 8.5 Hz); MS
(ESI+) m/z 380 [M+H]+; Anal. Calcd for C25H21N3Oꢀ2HClꢀ1.7H2O:
C, 62.17; H, 5.51; N, 8.70; F, 14.68. Found: C, 62.33; H, 5.49; N,
8.57; F, 14.58.
5.1.10. 2-{[(1-Phenyl-2-propyl-1H-benzimidazol-6-yl)oxy]
methyl}quinoline dihydrochloride (7h)
Compound 7h was prepared from 6 and butyric acid in a man-
ner similar to that described for compound 7b, with a yield of 49%
as a pale yellow solid. 1H NMR (DMSO-d6) d 0.87 (t, 3H, J = 7.4 Hz),
1.66–1.76 (2H, m), 2.89 (t, 2H, J = 7.6 Hz), 5.43 (s, 2H), 7.02 (d, 1H,
J = 2.2 Hz), 7.36 (dd, 1H, J = 9.0, 2.3 Hz), 7.62–7.77 (m, 7H), 7.80–
7.86 (m, 2H), 7.94 (d, 1H, J = 8.4 Hz), 8.01 (d, 1H, J = 7.9 Hz), 8.45
(d, 1H, J = 8.5 Hz); MS (ESI) m/z 394 [M+H]+; Anal. Calcd for C26H23-
N3Oꢀ2HClꢀ0.4H2O: C, 65.94; H, 5.49; N, 8.87; Cl, 14.97. Found: C,
66.04; H, 5.43; N, 8.91; Cl, 14.96.
5.1.7. 2-{[(2-Isopropyl-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}
quinoline dihydrochloride (7g)
Compound 7g was prepared from 6 and trimethyl orthoisobuty-
rate in a manner similar to that described for compound 1, with a
yield of 81% as a pale orange solid. 1H NMR (DMSO-d6) d 1.37 (d,
6H, J = 6.9 Hz), 3.06–3.18 (m, 1H), 5.48 (s, 2H), 6.98 (s, 1H), 7.40
(d, 1H, J = 9.0 Hz), 7.67–7.80 (m, 7H), 7.83–7.90 (m, 2H),
8.00–8.08 (m, 2H), 8.55 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 394
[M+H]+; Anal. Calcd for C26H23N3Oꢀ2HClꢀ1.1H2Oꢀ0.1CHCl3: C,
62.93; H, 5.52; N, 8.44; Cl, 16.74. Found: C, 62.85; H, 5.58; N,
8.36; Cl 16.45.
5.1.11. 2-{[(2-Isobutyl-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}
quinoline dihydrochloride (7i)
Compound 7i was prepared from 6 and 3-methylbutanoic acid
in a manner similar to that described for compound 7b, with a
yield of 31% as a yellow solid. 1H NMR (DMSO-d6) d 0.84 (d, 6H,
J = 6.7 Hz), 1.98–2.10 (m, 1H), 2.86 (d, 2H, J = 7.3 Hz), 5,47 (s, 2H),
7.03 (d, 1H, J = 2.2 Hz), 7.39 (dd, 1H, J = 9.0, 2.3 Hz), 7.66–7.79 (m,
7H), 7.83–7.89 (m, 2H), 8.00 (d, 1H, J = 8.5 Hz), 8.05 (d, 1H,
J = 8.0 Hz), 8.54 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 408 [M+H]+; Anal.
Calcd for C27H25N3Oꢀ1.9HClꢀ0.6H2O: C, 66.51; H, 5.81; N, 8.62; Cl,
13.81. Found: C, 66.67; H, 5.69; N, 8.69; Cl, 13.53.
5.1.8. 2-({[1-Phenyl-2-(trifluoromethyl)-1H-benzimidazol-6-yl]
oxy}methyl)quinoline hydrochloride (7a)
To a solution of 6 (101 mg, 0.30 mmol) in THF (3 mL) cooled
with ice-water bath was added trifluoroacetic anhydride (60 lL,
0.43 mmol), and the mixture was stirred at room temperature for
30 min. The mixture was concentrated in vacuo, and the residue
was dissolved in acetic acid (3 mL) and stirred at 90 °C for 6 h. After
cooling at room temperature, the mixture was concentrated in
vacuo. The residue was partitioned between EtOAc and saturated
aqueous sodium bicarbonate, and the organic layer was washed
with brine, dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(0–30% EtOAc in CHCl3) to give a yellow solid, which was then
dissolved in CHCl3 (5 mL), and 4 M HCl/EtOAc (0.29 mL,
1.16 mmol) was added to the solution. The mixture was concen-
trated to give 7a (29 mg, 22%) as a orange solid. 1H NMR (DMSO-
d6) d 5.41 (s, 2H), 6.83 (d, 1H, J = 2.1 Hz), 7.23 (dd, 1H, J = 8.9,
2.2 Hz), 7.52 (d, 2H, J = 7.0 Hz), 7.60–7.70 (m, 4H), 7.75 (d, 1H,
J = 8.5 Hz), 7.82–7.88 (m, 2H), 7.99 (d, 1H, J = 8.5 Hz), 8.04 (d, 1H,
J = 8.2 Hz), 8.51 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 420 [M+H]+; Anal.
Calcd for C24H16F3N3OꢀHClꢀ0.6C4H8O2ꢀ0.05CHCl3: C, 61.72; H, 4.28;
N, 8.16; Cl, 7.92; F, 11.07. Found: C, 61.52; H, 3.98; N, 8.04; Cl, 8.14;
F, 10.69.
5.1.12. 2-{[(2-Cyclopropyl-1-phenyl-1H-benzimidazol-6-
yl)oxy]methyl}quinoline dihydrochloride (7f)
To a solution of 6 (100 mg, 0.29 mmol) and pyridine (35
0.43 mmol) in THF (3 mL) cooled with ice-water bath was added
cyclopropanecarbonyl chloride (30 L, 0.33 mmol), and the mix-
lL,
l
ture was stirred at room temperature for 30 min. The mixture
was concentrated in vacuo, and the residue was dissolved in acetic
acid (3 mL) and stirred at 90 °C for 18 h. After cooling at room tem-
perature, the mixture was concentrated in vacuo. The residue was
partitioned between EtOAc and saturated aqueous sodium bicar-
bonate, and the organic layer was washed with brine, dried over
MgSO4, filtered and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (10–60% EtOAc in hex-
ane) to give an oil, which was then dissolved in CHCl3 (3 mL),

7618
W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
and 4 M HCl/EtOAc (0.29 mL, 1.16 mmol) was added to the mix-
ture. The mixture was concentrated to give 7f (26 mg, 19%) as a
pale orange solid. 1H NMR (DMSO-d6) d 1.19–1.26 (m, 2H), 1.43–
1.49 (m, 2H), 1.98–2.06 (m, 1H), 5.46 (s, 2H), 7.01 (d, 1H,
J = 2.2 Hz), 7.34 (dd, 1H, J = 9.0, 2.3 Hz), 7.65–7.77 (m, 8H), 7.82–
7.88 (m, 1H), 8.00 (d, 1H, J = 8.5 Hz), 8.04 (d, 1H, J = 8.0 Hz), 8.51
(d, 1H, J = 8.5 Hz); MS (ESI) m/z 392 [M+H]+; Anal. Calcd for C26H21
N3Oꢀ2HClꢀ3.0H2Oꢀ0.2C4H8O2: C, 60.05; H, 5.75; N, 7.84; Cl, 13.23.
Found: C, 60.24; H, 5.78; N, 7.73; Cl, 13.01.
saturated aqueous sodium bicarbonate, and concentrated in vacuo.
The residue was purified by silica gel column chromatography (0–
5% MeOH in CHCl3) to give 10 (1.83 g, 61%) as a colorless oil. 1H
NMR (DMSO-d6) d 5.11 (s, 2H), 6.61 (d, 1H, J = 3.2 Hz), 6.87 (dd,
1H, J = 8.6, 2.3 Hz), 7.12 (d, 1H, J = 2.2 Hz), 7.29–7.47 (m, 6H),
7.49 (d, 1H, J = 3.2 Hz), 7.51–7.61 (m, 5H); MS (ESI) m/z 300
[M+H]+.
5.1.16. 1-Phenyl-1H-indol-6-ol (13a)
To a mixture of 10 (1.83 g, 6.11 mmol) and ammonium formate
(3.86 g, 61.1 mmol) in water (10 mL), MeOH (30 mL) and THF
(30 mL) was added 20% Pd(OH)2 on carbon (429 mg), and the mix-
ture was refluxed for 3 h. After cooling at room temperature, the
mixture was filtered through celite pad, and concentrated in vacuo.
The residue was partitioned between EtOAc and water, and the or-
ganic layer was concentrated in vacuo. The residue was purified by
silica gel column chromatography (10–50% EtOAc in hexane) to
give 13a (1.28 g, quant) as a pale green oil. 1H NMR (DMSO-d6) d
6.55 (d, 1H, J = 3.3 Hz), 6.65 (dd, 1H, J = 8.5, 2.1 Hz), 6.93 (d, 1H,
J = 1.8 Hz), 7.35–7.43 (m, 3H), 7.52–7.59 (m, 4H), 9.09 (br s, 1H);
MS (EI) m/z 209 [M]+.
5.1.13. N,N-Dimethyl-1-phenyl-6-(quinolin-2-ylmethoxy)-1H-
benzimidazol-2-amine dihydrochloride (7c)
To a solution of 6 (512 mg, 1.50 mmol) in 1,2-dichloroethane
(10 mL) was added phosgene iminium chloride (366 mg,
2.25 mmol), and the mixture was stirred at 50 °C for 5 h. After cool-
ing at room temperature, the mixture was partitioned between
CHCl3 and saturated aqueous sodium bicarbonate. The organic
layer was concentrated in vacuo, and the residue was purified by
silica gel column chromatography (0–5% MeOH in CHCl3) to give
the free form of the title compound, which was then dissolved in
CHCl3 (10 mL), and 4 M HCl/EtOAc (2.0 mL, 8.0 mmol) was added
to the mixture. The mixture was concentrated in vacuo to give 7c
(488 mg, 70%) as a beige solid. 1H NMR (DMSO-d6) d 2.93 (s, 6H),
5.37 (s, 2H), 6.66 (d, 1H, J = 2.3 Hz), 7.11 (dd, 1H, J = 8.7, 2.4 Hz),
7.47 (d, 1H, J = 8.8 Hz), 7.63–7.72 (m, 7H), 7.81–7.87 (m, 1H),
7.97–8.04 (m, 2H), 8.47 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 395
[M+H]+; Anal. Calcd for C25H22N4Oꢀ2HClꢀ4.0H2O: C, 55.66; H,
5.98; N, 10.39; Cl, 13.14. Found: C, 55.68; H, 6.10; N, 10.10; Cl,
13.19.
5.1.17. 6-Methoxy-1-phenyl-1H-indazole (12)
To a solution of 2-fluoro-4-methoxybenzaldehyde (11, 2.31 g,
15.0 mmol) in DMF (60 mL) were added phenylhydrazine (1.62 g,
15.0 mmol) and Cs2CO3 (9.77 g, 30.0 mmol), and the mixture was
stirred at 150 °C for 3 h. After cooling at room temperature, the
mixture was diluted with EtOAc, washed with saturated aqueous
sodium bicarbonate and brine, and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography (5 to 20%
EtOAc in hexane) to give 12 (640 mg, 19%) as a yellow oil. 1H
NMR (DMSO-d6) d 3.86 (s, 3H), 6.90 (dd, 1H, J = 8.8, 1.9 Hz), 7.18
(s, 1H), 7.40 (t, 1H, J = 7.4 Hz), 7.57–7.64 (m, 2H), 7.73–7.81 (m,
3H), 8.24 (s, 1H); MS (ESI) m/z 225 [M+H]+.
5.1.14. 2-{[(5-Fluoro-1-phenyl-1H-benzimidazol-6-
yl)oxy]methyl}quinoline dihydrochloride (8)
To a suspension of 5 (835 mg, 2.14 mmol) and NH4Cl (57 mg,
1.07 mmol) in EtOH (15 mL) and water (4.5 mL) was added iron
powder (599 mg, 10.7 mmol), and the mixture was refluxed for
1 h. After cooling at room temperature, the mixture was diluted
with CHCl3 and water, and then filtered through celite pad. The or-
ganic layer of the filtrate was dried over Na2SO4, filtered and con-
centrated in vacuo to give a brown syrup (790 mg). To a solution of
the above-obtained brown syrup (300 mg) in THF (6 mL) were
added triethyl orthoformate (0.35 mL, 2.08 mmol) and p-toluene-
sulfonic acid monohydrate (16 mg, 0.08 mmol), and the mixture
was refluxed for 3 h. The mixture was cooled at room temperature
and concentrated in vacuo. The residue was partitioned between
saturated aqueous sodium bicarbonate and EtOAc. The organic
layer was washed with brine, dried over MgSO4, filtered and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (0–5% MeOH in CHCl3) to give a solid, which
was suspended in EtOH (10 mL), and the mixture was treated with
4 M HCl/EtOAc (0.50 mL, 2.00 mmol). The precipitate was collected
by filtration and washed with diethylether to give 8 (189 mg, 51%)
as a pale brown solid. 1H NMR (DMSO-d6) d 5.67 (s, 2H), 7.51–7.68
(m, 5H), 7.73–7.82 (m, 3H), 7.93–8.00 (m, 2H), 8.18 (d, 1H,
J = 8.3 Hz), 8.27 (d, 1H, J = 8.6 Hz), 8.80 (d, 1H, J = 8.6 Hz), 9.19 (s,
1H); MS (ESI+) m/z 370 [M+H]+; Anal. Calcd for C23H16FN3-
Oꢀ2HClꢀ0.5H2O: C, 61.21; H, 4.24; N, 9.31; Cl, 15.71; F, 4.21. Found:
C, 61.11; H, 4.27; N, 9.36; Cl, 15.69; F, 4.28.
5.1.18. 1-Phenyl-1H-indazol-6-ol (13b)
To a solution of 12 (1.36 g, 6.06 mmol) in CH2Cl2 (50 mL) cooled
with ice-water bath was slowly added 1 M BBr3 solution in CH2Cl2
(18.3 mL, 18.3 mmol), and the mixture was stirred at room tem-
perature for 16 h. To the resultant mixture cooled with ice-water
bath was slowly added saturated aqueous sodium bicarbonate
and the mixture was extracted with CHCl3. The organic layer was
concentrated in vacuo, and the residue was purified by silica gel
column chromatography (0–10% MeOH in CHCl3) to give 13b
(1.27 g, quant) as a colorless solid. 1H NMR (DMSO-d6) d 6.78 (dd,
1H, J = 8.7, 1.7 Hz), 7.09 (s, 1H), 7.38 (t, 1H, J = 7.4 Hz), 7.58 (dd,
2H, J = 7.8, 7.8 Hz), 7.65–7.72 (m, 3H), 8.17 (s, 1H), 9.82 (s, 1H);
MS (ESI) m/z 211 [M+H]+.
5.1.19. 2-{[(3-Phenyl-1,2-benzoxazol-5-yl)oxy]methyl}
quinoline hydrochloride (16)
To a mixture of 3-phenyl-1,2-benzoxazol-5-ol10 (15, 140 mg,
0.66 mmol)
and
2-(chloromethyl)quinoline
hydrochloride
(142 mg, 0.66 mmol) in DMF (5 mL) were added K2CO3 (202 mg,
1.46 mmol) and potassium iodide (110 mg, 0.66 mmol), and the
mixture was stirred at 80 °C for 6 h. After cooling at room temper-
ature, the mixture was concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3) to give a
beige solid, which was dissolved in MeOH (5 mL) and 4 M HCl/
EtOAc (0.25 mL, 1.00 mmol) was added to the solution. The
mixture was concentrated in vacuo, and the residue was washed
with 2-propanol to give 16 (98 mg, 38%) as a beige solid. 1H NMR
(DMSO-d6) d 5.59 (s, 2H), 7.21 (dd, 1H, J = 8.9, 2.5 Hz), 7.53
(d, 1H, J = 2.5 Hz), 7.58–7.66 (m, 3H), 7.71–7.77 (m, 2H),
7.87–7.95 (m, 2H), 8.11–8.23 (m, 4H), 8.68 (d, 1H, J = 8.6 Hz); MS
(ESI) m/z 353 [M+H]+. Anal. Calcd for C23H16N2O2ꢀHClꢀ0.2H2O: C,
5.1.15. 6-(Benzyloxy)-1-phenyl-1H-indole (10)
To a mixture of 6-(benzyloxy)-1H-indole (9, 2.23 g, 10.0 mmol),
bromobenzene (1.88 g, 12.0 mmol), palladium(II) acetate (47 mg,
0.21 mmol) and 2-(dicyclohexylphosphino)-20,40,60-triisopropylbi-
phenyl (Xphos, 239 mg, 0.50 mmol) in toluene (27 mL) was added
K3PO4 (3.19 g, 15.0 mmol), and the mixture was stirred at 100 °C
for 16 h under argon atmosphere. After cooling at room tempera-
ture, the mixture was diluted with EtOAc and washed with

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7619
70.39; H, 4.47; N, 7.14; Cl, 9.03. Found: C, 70.39; H, 4.58; N, 7.05;
Cl, 9.07.
free form of the title compound, which was dissolved in CHCl3
(10 mL) and 4 M HCl/EtOAc (1.0 mL, 4.0 mmol) was added to the
solution. The mixture was concentrated in vacuo and the residue
was recrystalized from EtOH–Et2O to give a beige solid. 1H NMR
(DMSO-d6) d 5.88 (s, 2H), 7.19 (d, 1H, J = 8.8 Hz), 7.35–7.45 (m,
3H), 7.65–7.71 (m, 2H), 7.78–7.84 (m, 1H), 7.90 (d, 1H,
J = 8.6 Hz), 7.99–8.05 (m, 1H), 8.19 (d, 1H, J = 8.2 Hz), 8.30 (d, 1H,
J = 8.8 Hz), 8.33 (d, 1H, J = 8.5 Hz), 8.81 (d, 1H, J = 8.6 Hz), 9.13 (s,
1H); MS (ESI) m/z 353 [M+H]+; Anal. Calcd for C22H16N4-
Oꢀ2HClꢀ2H2Oꢀ0.08CHCl3: C, 56.32; H, 4.73; N, 11.90; Cl, 16.86.
Found: C, 56.32; H, 4.77; N, 11.87; Cl, 16.83.
5.1.20. 2-{[(1-Phenyl-1H-indazol-6-yl)oxy]methyl}quinoline
hydrochloride (14b)
Compound 14b was prepared from 13b in a manner similar to
that described for compound 16, with a yield of 23% as a brown so-
lid. 1H NMR (DMSO-d6) d 5.58 (s, 2H), 7.08 (dd, 1H, J = 8.7, 2.0 Hz),
7.38–7.42 (m, 2H), 7.55 (dd, 2H, J = 7.9, 7.9 Hz), 7.66–7.72 (m, 3H),
7.78–7.91 (m, 3H), 8.08 (d, 1H, J = 8.2 Hz), 8.13 (d, 1H, J = 8.5 Hz),
8.25 (s, 1H), 8.58 (d, 1H, J = 8.5 Hz); MS (ESI) m/z 352 [M+H]+. Anal.
Calcd for C23H17N3OꢀHClꢀ0.1H2O: C, 70.89; H, 4.71; N, 10.78; Cl,
9.10. Found: C, 70.82; H, 4.68; N, 10.61; Cl, 8.90.
5.1.25. 5-(Benzyloxy)-4-methoxy-2-nitro-N-phenylaniline (23)
To a mixture of 1-(benzyloxy)-5-bromo-2-methoxy-4-nitroben-
zene11 (22, 1.00 g, 2.96 mmol), aniline (413 mg, 4.44 mmol),
tris(dibenzylideneacetone)dipalladium (0) (Pd2(dba)3, 135 mg,
0.15 mmol) and (+/ꢁ)-2,20-bis(diphenylphosphoino)-1,10-binaph-
thyl (BINAP, 138 mg, 0.22 mmol) in toluene (20 mL) was added
Cs2CO3 (1.93 g, 5.91 mmol), and the mixture was stirred at 85 °C
for 2 h under an argon gas atmosphere. After cooling at room tem-
perature, the mixture was concentrated in vacuo, and the residue
was purified by silica gel column chromatography (20–100% EtOAc
in hexane) to give 23 (1.04 g, quant) as a yellow solid. 1H NMR
(CDCl3) d 3.90 (s, 3H), 5.08 (s, 2H), 6.58 (s, 1H), 6.66–6.70 (m,
1H), 7.02 (d, 2H, J = 7.7 Hz), 7.18–7.37 (m, 7H), 7.65 (s, 1H), 9.77
(br s, 1H); MS (ESI) m/z 351 [M+H]+.
5.1.21. 6-Methoxy-3-nitro-N-phenylpyridin-2-amine (18)
To a mixture of 2-chloro-6-methoxy-3-nitropyridine (17, 10.0 g,
53.0 mmol) and aniline (7.25 mL, 79.6 mmol) in DMF (200 mL) was
added K2CO3 (7.33 g, 53.0 mmol), and the mixture was stirred at
110 °C for 5 h. After cooling at room temperature, the mixture
was concentrated in vacuo. The residue was partitioned between
EtOAc and water, and the organic layer was dried over Na2SO4, fil-
tered and concentrated in vacuo. The residue was washed with
hexane–EtOAc to give 18 (9.15 g, 70%) as a yellow solid. 1H NMR
(DMSO-d6) d 3.89 (s, 3H), 6.38 (d, 1H, J = 9.1 Hz), 7.17 (t, 1H,
J = 7.4 Hz), 7.37–7.43 (m, 2H), 7.72 (d, 2H, J = 7.7 Hz), 8.44 (d, 1H,
J = 9.1 Hz), 10.45 (br s, 1H); MS (FAB) m/z 246 [M+H]+.
5.1.22. 6-Methoxy-N2-phenylpyridine-2,3-diamine (19)
5.1.26. 6-(Benzyloxy)-5-methoxy-1-phenyl-1H-benzimidazole
(24a)
To a stirred mixture of 18 (6.50 g, 26.5 mmol) in EtOH (100 mL)
and DMF (25 mL) was added 20% Pd(OH)2 on carbon (50% wet,
102 mg), and the mixture was stirred at room temperature for
16 h under 1 atm of hydrogen gas atmosphere. The mixture was fil-
tered through celite pad, and the filtrate was concentrated in vacuo
to give a black oil. To the above-obtained black oil in THF (30 mL)
were added triethyl orthoformate (13.2 mL, 79.5 mmol) and p-
toluenesulfonic acid monohydrate (5.55 g, 29.2 mmol), and the
mixture was refluxed for 5 h. The mixture was cooled at room tem-
perature and concentrated in vacuo. The residue was partitioned
between saturated aqueous sodium bicarbonate and CHCl3. The
organic layer was washed with brine, dried over Na2SO4, filtered
and concentrated in vacuo to give 19 (5.97 g, quant) as a brown
oil. 1H NMR (DMSO-d6) d 3.91 (s, 3H), 6.81 (d, 1H, J = 8.6 Hz),
7.42–7.48 (m, 1H), 7.58–7.64 (m, 2H), 7.96–8.00 (m, 2H), 8.10
(d, 1H, J = 8.6 Hz), 8.68 (s, 1H); MS (ESI) m/z 226 [M+H]+.
To a suspension of 23 (3.10 g, 8.85 mmol) and NH4Cl (47 mg,
0.89 mmol) in EtOH (55 mL) and water (17 mL) was added iron
powder (2.47 g, 44.2 mmol), and the mixture was refluxed for
3 h. After cooling at room temperature, the mixture was diluted
with CHCl3 and water, and then filtered through celite pad. The or-
ganic layer of the filtrate was dried over MgSO4, filtered and con-
centrated in vacuo to give a purple solid. To a solution of the
above-obtained purple solid in THF (57 mL) were added triethyl
orthoformate (3.67 mL, 22.1 mmol) and p-toluenesulfonic acid
monohydrate (168 mg, 0.88 mmol), and the mixture was refluxed
for 3 h. The mixture was then cooled at room temperature and con-
centrated in vacuo. The residue was partitioned between saturated
aqueous sodium bicarbonate and EtOAc. The organic layer was
washed with brine, dried over MgSO4, filtered and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (0–5% MeOH in CHCl3) to give 24a (1.92 g, 66%) as a red
solid. 1H NMR (CDCl3) d 3.99 (s, 3H), 5.15 (s, 2H), 7.01 (s, 1H),
7.31–7.46 (m, 9H), 7.51–7.75 (m, 2H), 7.96 (s, 1H); MS (ESI) m/z
331 [M+H]+.
5.1.23. 3-Phenyl-3H-imidazo[4,5-b]pyridin-5-ol (20)
A mixture of 19 (5.97 g, 26.5 mmol) and pyridine hydrochloride
(30.6 g, 265 mmol) was stirred at 200 °C for 2 h. After cooling at
room temperature, the mixture was basified with saturated aque-
ous sodium bicarbonate and extracted with CHCl3–MeOH for 10
times. The combined organic layer was concentrated in vacuo,
and the residue was purified by silica gel column chromatography
(0–10% MeOH in CHCl3) to give 20 (4.33 g, 77%) as a pale yellow so-
lid. 1H NMR (DMSO-d6) d 6.63 (d, 1H, J = 8.6 Hz), 7.42–7.47 (m, 1H),
7.56–7.62 (m, 2H), 7.86–7.90 (m, 2H), 8.01 (d, 1H, J = 8.6 Hz), 8.54
(s, 1H), 10.84 (br s, 1H); MS (ESI) m/z 212 [M+H]+.
5.1.27. 6-(Benzyloxy)-5-methoxy-1-phenyl-1H-benzimidazole
(24b)
Compound 24b was prepared from 23 and trimethyl orthoi-
sobutyrate in a manner similar to that described for compound
24a, with a yield of 37% as a pale brown solid. 1H NMR (CDCl3) d
1.32 (d, 6H, J = 6.8 Hz), 3.04 (sep, 1H, J = 6.8 Hz), 3.93 (s, 3H), 5.03
(s, 2H), 6.59 (s, 1H), 7.25–7.41 (m, 8H), 7.49–7.58 (m, 3H); MS
(ESI) m/z 373 [M+H]+.
5.1.24. 2-{[(3-Phenyl-3H-imidazo[4,5-b]pyridin-5-yl)oxy]methyl}
quinoline dihydrochloride (21)
5.1.28. 2-{[(2-Isopropyl-5-methoxy-1-phenyl-1H-benzimidazol-
6-yl)oxy]methyl}quinoline dihydrochloride (25b)
To a mixture of 20 (211 mg, 1.00 mmol) and 2-quinolinylmeth-
anol (260 mg, 1.63 mmol) in THF (10 mL) were added 1,10-(azodi-
A mixture of 24b (982 mg, 2.64 mmol) and anisole (5.71 mL,
52.5 mmol) in trifluoroacetic acid (37 mL) was stirred at 80 °C
overnight. After cooling at room temperature, the mixture was
concentrated in vacuo and the residue was purified by silica gel
column chromatography (0–5% MeOH in CHCl3) to give a purple
solid (754 mg). To a mixture of the above-obtained purple solid
carbonyl)dipiperidine
(ADDP,
378 mg,
1.50 mmol)
and
tributylphosphine (0.37 mL, 1.50 mmol), and the mixture was stir-
red at 60 °C for 15 h. After cooling at room temperature, the mix-
ture was concentrated in vacuo, and the residue was purified by
silica gel column chromatography (0–5% MeOH in CHCl3) to give

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W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
(250 mg) and 2-(chloromethyl)quinoline hydrochloride (227 mg,
1.06 mmol) in DMF (5 mL) were added Cs2CO3 (692 mg,
2.13 mmol) and potassium iodide (15 mg, 0.09 mmol), and the
mixture was stirred at 60 °C for 2 h followed by 80 °C for 2 h. After
cooling at room temperature, the mixture was diluted with EtOAc
and washed with water and brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 0–5% MeOH in CHCl3 then NH silica
gel, 0–40% EtOAc in hexane) to give the free form of the title
compound, which was dissolved in EtOH (10 mL), and 4 M HCl/
EtOAc (0.50 mL, 2.00 mmol) was added to the solution. The
mixture was concentrated in vacuo and the residue was triturated
with 2-propanol. The precipitate was collected by filtration
and dried in vacuo to give 25b (120 mg, 27%) as a pale yellow solid.
1H NMR (DMSO-d6) d 1.37 (d, 6H, J = 7.0 Hz), 3.09 (sep, 1H,
J = 7.0 Hz), 3.95 (s, 3H), 5.44 (s, 2H), 7.06 (s, 1H), 7.39 (s, 1H),
7.63–7.79 (m, 7H), 7.85–7.91 (m, 1H), 7.98 (d, 1H, J = 8.5
Hz), 8.07 (d, 1H, J = 8.2 Hz), 8.57 (d, 1H, J = 8.5 Hz); MS (ESI) m/z
424 [M+H]+; Anal. Calcd for C27H25N3O2ꢀ2HClꢀ0.5H2O: C, 64.16;
H, 5.58; N, 8.31; Cl, 14.03. Found: C, 63.90; H, 5.75; N, 8.14; Cl,
14.04.
5.1.33. N-Cyclohexyl-2-nitro-5-(quinolin-2-ylmethoxy)aniline
(30a)
To a solution of 29 (895 mg, 3.00 mmol) in DMF (30 mL) was
added cyclohexylamine (1.03 mL, 9.00 mmol), and the mixture
was stirred at 80 °C for 6 h. After cooling at room temperature,
the mixture was diluted with water. The precipitate was collected
by filtration and dried in vacuo to give 30a (1.12 g, 99%) as a yellow
solid. 1H NMR (CDCl3) d 1.21–1.37 (m, 6H), 1.61–1.70 (m, 2H),
1.84–1.90 (m, 2H), 3.29–3.37 (m, 1H), 5.44 (s, 2H), 6.28 (d, 1H,
J = 2.5 Hz), 6.32 (dd, 1H, J = 9.5, 2.6 Hz), 7.55–7.62 (m, 2H), 7.73–
7.78 (m, 1H), 7.84 (d, 1H, J = 8.1 Hz), 8.08 (d, 1H, J = 8.5 Hz), 8.12
(d, 1H, J = 9.5 Hz), 8.21 (d, 1H, J = 8.5 Hz), 8.31 (br d, 1H,
J = 7.1 Hz); MS (ESI) m/z 378 [M+H]+.
5.1.34. N-[2-Nitro-5-(quinolin-2-ylmethoxy)phenyl]pyridin-4-
amine (30b)
To a mixture of 27 (300 mg, 0.84 mmol), 4-aminopyridine
(80 mg, 0.85 mmol), Pd2(dba)3 (38 mg, 0.04 mmol) and BINAP
(52 mg, 0.08 mmol) in toluene (10 mL) was added sodium tert-
butoxide (120 mg, 1.25 mmol), and the mixture was stirred at
85 °C for 2 h under argon gas atmosphere. After cooling at room
temperature, the mixture was concentrated in vacuo. The residue
was purified by silica gel column chromatography (40–100% EtOAc
in CHCl3) to give 30b (224 mg, 72%) as a yellow solid. 1H NMR
(CDCl3) d 5.43 (s, 2H), 6.69 (dd, 1H, J = 9.3, 2.7 Hz), 6.85–6.88 (m,
2H), 7.03 (d, 1H, J = 2.4 Hz), 7.56–7.63 (m, 2H), 7.77–7.82 (m,
1H), 7,87 (d, 1H, J = 7.8 Hz), 8.04 (d, 1H, J = 8.3 Hz), 8.19–8.25 (m,
4H), 9.62 (br s, 1H); MS (ESI) m/z 373 [M+H]+.
5.1.29. 2-{[(5-Methoxy-1-phenyl-1H-benzimidazol-6-
yl)oxy]methyl}quinoline dihydrochloride (25a)
Compound 25a was prepared from 24a in a manner similar to
that described for compound 25b, with a yield of 6.1% as a yellow
solid. 1H NMR (DMSO-d6) d 3.96 (s, 3H), 5.55 (s, 2H), 7.45 (s, 1H),
7.48 (s, 1H), 7.68–7.76 (m, 6H), 7.80 (d, 1H, J = 8.5 Hz), 7.85–7.89
(m, 1H), 8.02–8.09 (m, 2H), 8.58 (1H, d, J = 8.5 Hz), 9.74 (s, 1H);
MS (ESI) m/z 382 [M+H]+; Anal. Calcd for C24H19N3O2ꢀ2HClꢀ2.0H2-
Oꢀ0.2 C2H6O: C, 58.66; H, 5.29; N, 8.41; Cl, 14.19. Found: C,
58.73; H, 5.30; N, 8.33; Cl, 13.92.
5.1.35. 2-Nitro-N-(pyridin-4-ylmethyl)-5-(quinolin-2-ylmethoxy)
aniline (30c)
To a solution of 29 (300 mg, 1.01 mmol) and 4-picolylamine
(0.12 mL, 1.20 mmol) in DMF (3 mL) was added K2CO3 (209 mg,
1.50 mmol), and the mixture was stirred at 80 °C for 1 h. After cool-
ing at room temperature, the mixture was partitioned between
EtOAc and saturated aqueous sodium bicarbonate. The organic
layer was washed with brine, dried over MgSO4, filtered and con-
centrated in vacuo to give 30c (370 mg, 95%) as a yellow solid.
1H NMR (CDCl3) d 4.47 (d, 2H, J = 6.0 Hz), 5.32 (s, 2H), 6.16 (d,
1H, J = 2.5 Hz), 6.42 (dd, 1H, J = 9.5, 2.5 Hz), 7.14–7.17 (m, 2H),
7.56–7.62 (m, 2H), 7.74–7.79 (m, 1H), 7.82–7.86 (m, 1H), 8.04 (d,
1H, J = 8.5 Hz), 8.14 (d, 1H, J = 8.5 Hz), 8.19 (d, 1H, J = 9.5 Hz),
8.42–8.45 (m, 2H), 8.70 (br t, 1H, J = 6.0 Hz); MS (ESI) m/z 387
[M+H]+.
5.1.30. 2-[(3-Bromo-4-nitrophenoxy)methyl]quinoline (27)
To a mixture of 3-bromo-4-nitrophenol (26, 4.91 g, 22.5 mmol),
2-(chloromethyl)quinoline hydrochloride (5.79 g, 27.0 mmol) and
potassium iodide (374 mg, 2.25 mmol) in DMF (50 mL) was added
K2CO3 (7.47 g, 54.1 mmol), and the mixture was stirred at 60 °C for
90 min. After cooling at room temperature, the mixture was di-
luted with EtOAc, and washed with water and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The resi-
due was washed with EtOAc–hexane to give 27 (6.89 g, 85%) as a
pale yellow solid. 1H NMR (CDCl3) d 5.44 (s, 2H), 7.03–7.07 (m,
1H), 7.42 (d, 1H, J = 2.4 Hz), 7.55–7.62 (m, 2H), 7.74–7.80 (m,
1H), 7.85 (d, 1H, J = 8.3 Hz), 7.96 (d, 1H, J = 9.3 Hz), 8.09 (d, 1H,
J = 8.8 Hz), 8.23 (d, 1H, J = 8.3 Hz); MS (ESI) m/z 359, 361 [M+H]+.
5.1.36. 1-Methyl-N-[2-nitro-5-(quinolin-2-ylmethoxy)phenyl]-
1H-pyrazol-3-amine (30e)
5.1.31. 2-{[(1-Phenyl-1H-indol-6-yl)oxy]methyl}quinoline (14a)
Compound 14a was prepared from 13a in a manner similar to
that described for compound 27, with a yield of 18% as a pale yel-
low solid. 1H NMR (DMSO-d6) d 5.39 (s, 2H), 6.61 (dd, 1H, J = 3.3,
0.7 Hz), 6.95 (dd, 1H, J = 8.6, 2.2 Hz), 7.20 (d, 1H, J = 2.2 Hz),
7.35–7.39 (m, 1H), 7.44–7.52 (m, 5H), 7.56 (d, 1H, J = 8.6 Hz),
7.59–7.64 (m, 1H), 7.70 (d, 1H, J = 8.5 Hz), 7.78–7.82 (m, 1H),
7.96–8.04 (m, 2H), 8.39 (d, 1H, J = 8.5 Hz); MS (ESI) m/z
351[M+H]+; Anal. Calcd for C24H18N2Oꢀ0.1H2O: C, 81.84; H, 5.21;
N, 7.95. Found: C, 81.67; H, 5.20; N, 7.79.
To a mixture of 27 (160 mg, 0.45 mmol), 1-methyl-1H-pyrazol-
3-amine (48 mg, 0.49 mmol), Pd2(dba)3 (38 mg, 0.024 mmol) and
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(Xantphos,
27 mg, 0.047 mmol) in toluene (5 mL) was added Cs2CO3
(220 mg, 0.68 mmol), and the mixture was stirred at 85 °C for 2 h
under an argon gas atmosphere. After cooling at room tempera-
ture, the mixture was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (0–30% EtOAc in CHCl3)
to give 30b (145 mg, 87%) as an orange solid. 1H NMR (CDCl3) d
3.78 (s, 3H), 5.41 (s, 2H), 5.91 (d, 1H, J = 2.2 Hz), 6.51 (dd, 1H,
J = 9.5, 2.7 Hz), 7.21 (d, 1H, J = 2.2 Hz), 7.48 (d, 1H, J = 2.6 Hz),
7.54–7.59 (m, 1H), 7.62 (d, 1H, J = 8.5 Hz), 7.73–7.78 (m, 1H),
7.83–7.86 (m, 1H), 8.06 (d, 1H, J = 8.6 Hz), 8.18–8.23 (m, 2H),
10.02 (s, 1H); MS (ESI) m/z 376 [M+H]+.
5.1.32. 2-[(3-Fluoro-4-nitrophenoxy)methyl]quinoline (29)
Compound 29 was prepared from 3-fluoro-4-nitrophenol (28)
in a manner similar to that described for compound 27, with a
yield of 98% as a yellow solid. 1H NMR (CDCl3) d 5.55 (s, 2H),
7.12 (dd, 1H, J = 9.3, 1.7 Hz), 7.35 (1H, dd, J = 13.6, 2.5 Hz), 7.61–
7.67 (m, 1H), 7.70 (d, 1H, J = 8.6 Hz), 7.77–7.83 (m, 1H), 7.99–
8.04 (m, 1H), 8.18 (dd, 1H, J = 9.1, 9.1 Hz), 8.46 (d, 1H, J = 8.5 Hz);
MS (ESI) m/z 299 [M+H]+.
5.1.37. 1-Methyl-N-[2-nitro-5-(quinolin-2-ylmethoxy)phenyl]-
1H-pyrazol-4-amine (30d)
Compound 30d was prepared from 29 and 1-methyl-1H-pyra-
zol-4-amine dihydrochloride in a manner similar to that described

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7621
for compound 30e, with a yield of 30% as a yellow solid. 1H NMR
5.1.42. 2-{[(1-Cyclohexyl-1H-benzimidazol-6-yl)oxy]
(CDCl3) d 3.87 (s, 3H), 5.31 (s, 2H), 6.44 (dd, 1H, J = 9.5, 2.6 Hz),
6.52 (d, 1H, J = 2.6 Hz), 7.27 (s, 1H), 7.41 (s, 1H), 7.55–7.60 (m,
2H), 7.73–7.78 (m, 1H), 7.83–7.86 (m, 1H), 8.06 (d, 1H,
J = 8.6 Hz), 8.17 (d, 1H, J = 9.5 Hz), 8.21 (d, 1H, J = 8.4 Hz), 9.26 (s,
1H); MS (ESI) m/z 376 [M+H]+.
methyl}quinoline dihydrochloride (31a)
Compound 31a was prepared from 30a in a manner similar
to that described for compound 8, with a yield of 22% as a col-
orless solid. 1H NMR (DMSO-d6) d 2.06–2.16 (m, 6H), 3.60–3.69
(m, 2H), 4.00–4.07 (m, 2H), 5.00–5.10 (m, 1H), 5.80 (s, 2H), 7.43
(dd, 1H, J = 9.1, 2.3 Hz), 7.78–7.85 (m, 2H), 7.99–8.04 (m, 2H),
8.08 (d, 1H, J = 2.3 Hz), 8.21 (d, 1H, J = 8.3 Hz), 8.36 (d, 1H,
J = 8.6 Hz), 8.87 (d, 1H, J = 8.6 Hz), 9.80 (s, 1H); MS (ESI) m/z
358 [M+H]+. Anal. Calcd for C23H23N3Oꢀ2HClꢀ3.1H2O: C, 56.82;
H, 6.47; N, 8.64; Cl, 14.58. Found: C, 56.97; H, 6.81; N, 8.70;
Cl, 14.54.
5.1.38. N-[2-Nitro-5-(quinolin-2-ylmethoxy)phenyl]pyrimidin-
4-amine (30h)
To a mixture of 27 (500 mg, 1.39 mmol), 4-aminopyrimidine
(199 mg, 2.09 mmol), Pd2(dba)3 (138 mg, 0.070 mmol) and Xant-
phos (81 mg, 0.14 mmol) in N-methylpyrrolidone (4 mL) was
added Cs2CO3 (680 mg, 2.09 mmol), and the mixture was stirred
at 160 °C for 10 min under microwave irradiation. After cooling
at room temperature, the mixture was filtered through celite
pad, and the filtrate was partitioned between EtOAc and water.
The organic layer was washed with brine, dried over Na2SO4, fil-
tered and concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel, 20–100% EtOAc in hexane then
NH silica gel, 20–50% CHCl3 in hexane) to give 30h (97 mg, 19%) as
a yellow solid. 1H NMR (CDCl3) d 5.53 (s, 2H), 6.75–6.80 (m, 2H),
7.75–7.61 (m, 1H), 7.66 (d, 1H, J = 8.5 Hz), 7.76–7.80 (m, 1H),
7.85 (d, 1H, J = 8.2 Hz), 8.10 (d, 1H, J = 8.6 Hz), 8.22–8.28 (m, 2H),
8.38 (d, 1H, J = 5.8 Hz), 8.70 (s, 1H), 8.77 (d, 1H, J = 2.7 Hz), 10.60
(br s, 1H); MS (ESI) m/z 374 [M+H]+.
5.1.43. 2-({[1-(1-Methyl-1H-pyrazol-3-yl)-1H-benzimidazol-6-yl]
oxy}methyl)quinoline dihydrochloride (31e)
To a suspension of 30e (145 mg, 0.39 mmol) and NH4Cl (13 mg,
0.24 mmol) in EtOH (9 mL) and water (3 mL) was added iron pow-
der (110 mg, 1.97 mmol), and the mixture was refluxed for 1 h.
After cooling at room temperature, the mixture was diluted with
CHCl3 and filtered through celite pad. The organic layer of the fil-
trate was washed with saturated aqueous sodium bicarbonate and
brine, dried over MgSO4, filtered and concentrated in vacuo to
give a black oil. To a solution of the above-obtained black oil in
THF (5 mL) were added triethyl orthoformate (0.10 mL,
0.87 mmol) and p-toluenesulfonic acid monohydrate (10 mg,
0.05 mmol), and the mixture was refluxed for 2 h. The mixture
was cooled at room temperature and partitioned between satu-
rated aqueous sodium bicarbonate and CHCl3. The organic layer
was washed with brine, dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (0–50% EtOAc in CHCl3) to give an oil, which
was dissolved in CHCl3 (5 mL) and the mixture was treated with
4 M HCl/EtOAc (0.40 mL, 1.60 mmol). The precipitate was col-
lected by filtration and washed with 2-propanol to give 31e
(75 mg, 45%) as a colorless solid. 1H NMR (DMSO-d6) d 3.94 (s,
3H), 5.67 (s, 2H), 6.84 (d, 1H, J = 2.3 Hz), 7.40 (dd, 1H, J = 9.1,
2.4 Hz), 7.73–7.78 (m, 1H), 7.83–7.87 (m, 2H), 7.90–7.97 (m,
2H), 8.00 (d, 1H, J = 2.3 Hz), 8.14 (d, 1H, J = 8.2 Hz), 8.24 (d, 1H,
J = 8.5 Hz), 8.71 (d, 1H, J = 8.5 Hz), 9.72 (s, 1H); MS (ESI) m/z 356
[M+H]+. Anal. Calcd for C21H17N5Oꢀ2HClꢀ1.2H2O: C, 56.06; H,
4.79; N, 15.57; Cl, 15.76. Found: C, 56.10; H, 4.77; N, 15.51; Cl,
15.74.
5.1.39. N-[2-Nitro-5-(quinolin-2-ylmethoxy)phenyl]pyrimidin-
2-amine (30g)
Compound 30g was prepared from 27 and pyrimidin-2-amine
in a manner similar to that described for compound 30h, with a
yield of 44% as a yellow solid. 1H NMR (CDCl3) d 5.52 (s, 2H),
6.72 (dd, 1H, J = 9.4, 2.7 Hz), 6.86 (t, 1H, J = 4.8 Hz), 7.56–7.61 (m,
1H), 7.68 (d, 1H, J = 8.5 Hz), 7.74–7.80 (m, 1H), 7.84–7.87 (m,
1H), 8.10 (d, 1H, J = 8.5 Hz), 8.23 (d, 1H, J = 8.5 Hz), 8.27 (d, 1H,
J = 9.4 Hz), 8.45 (d, 2H, J = 4.8 Hz), 8.83 (d, 1H, J = 2.7 Hz), 10.31
(br s, 1H); MS (ESI) m/z 374 [M+H]+.
5.1.40. N-[2-Nitro-5-(quinolin-2-ylmethoxy)phenyl]-1,3-
thiazol-2-amine (30i)
Compound 30i was prepared from 27 and 1,3-thiazol-2-amine
in a manner similar to that described for compound 30e, with a
yield of 90% as a yellow solid. 1H NMR (CDCl3) d 5.50 (s, 2H),
6.70 (dd, 1H, J = 9.4, 2.7 Hz), 6.85 (d, 1H, J = 3.7 Hz), 7.33 (d, 1H,
J = 3.7 Hz), 7.56–7.61 (m, 1H), 7.67 (d, 1H, J = 8.5 Hz), 7.75–7.79
(m, 1H), 7.84–7.88 (m, 1H), 8.10 (d, 1H, J = 7.8 Hz), 8.22–8.27
(m, 2H), 8.52 (d, 1H, J = 2.7 Hz), 10.66 (s, 1H); MS (ESI) m/z 379
[M+H]+.
5.1.44. 2-({[1-(Pyridin-4-yl)-1H-benzimidazol-6-
yl]oxy}methyl)quinoline trihydrochloride (31b)
Compound 31b was prepared from 30b in a manner similar
to that described for compound 31e, with a yield of 57% as a
pink solid. 1H NMR (DMSO-d6) d 5.69 (s, 2H), 7.26–7.31 (m,
1H), 7.72–7.78 (m, 2H), 7.81 (d, 1H, J = 8.9 Hz), 7.90–7.97 (m,
2H), 8.11–8.16 (m, 1H), 8.20–8.27 (m, 1H), 8.34–8.39 (m, 2H),
8.68–8.75 (m, 1H), 9.02–9.10 (m, 3H); MS (ESI) m/z 353
[M+H]+. Anal. Calcd for C22H16N4Oꢀ2.6HClꢀ2.4H2O: C, 53.88; H,
4.81; N, 11.42; Cl, 18.80. Found: C, 54.15; H, 4.84; N, 11.44;
Cl, 18.55.
5.1.41. N-[2-Nitro-5-(quinolin-2-ylmethoxy)phenyl]pyridazin-
4-amine (30f)
To a mixture of 27 (500 mg, 1.39 mmol), 4-aminopyridazine
(159 mg, 1.67 mmol) and K3PO4 (355 mg, 1.67 mmol) in DMSO
(8 mL) were added CuI (318 mg, 1.67 mmol) and N,N0-dimethyleth-
ylenediamine (0.18 mL, 1.67 mmol), and the mixture was stirred at
110 °C for 30 min under an argon gas atmosphere. After cooling at
room temperature, the mixture was diluted with water and 28%
aqueous ammonia solution, and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (0–5% MeOH in CHCl3) to give 30f
(138 mg, 27%) as a yellow solid. 1H NMR (CDCl3) d 5.47 (s,
2H), 6.80 (dd, 1H, J = 9.4, 2.6 Hz), 6.90 (dd, 1H, J = 5.9, 2.9 Hz),
7.07 (d, 1H, J = 2.6 Hz), 7.57–7.64 (m, 2H), 7.78–7.84 (m, 1H),
7.87 (d, 1H, J = 8.1 Hz), 8.05 (d, 1H, J = 8.6 Hz), 8.22–8.27 (m, 2H),
8.59 (br s, 1H), 9.08 (br s, 1H), 9.58 (s, 1H); MS (ESI) m/z 374
[M+H]+.
5.1.45. 2-({[1-(Pyridin-4-ylmethyl)-1H-benzimidazol-6-
yl]oxy}methyl)quinoline trihydrochloride (31c)
Compound 31c was prepared from 30c in a manner similar to
that described for compound 8, with a yield of 36% as a yellow so-
lid. 1H NMR (DMSO-d6) d 5.47 (s, 2H), 6.00 (s, 2H), 7.37 (dd, 1H,
J = 9.0, 2.3 Hz), 7.59 (d, 1H, J = 2.3 Hz), 7.65–7.70 (m, 1H), 7.73 (d,
1H, J = 8.5 Hz), 7.75–7.79 (m, 2H), 7.81–7.87 (m, 2H), 8.00–8.07
(m, 2H), 8.49 (d, 1H, J = 8.5 Hz), 8.73–8.76 (m, 2H), 9.60 (s, 1H);
MS (ESI) m/z 367 [M+H]+. Anal. Calcd for C23H18N4Oꢀ3.1HClꢀ3.6H2
O: C, 50.75; H, 5.24; N, 10.29; Cl, 20.19. Found: C, 50.66; H, 5.32;
N, 10.30; Cl, 20.13.

7622
W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
5.1.46. 2-({[1-(1-methyl-1H-pyrazol-4-yl)-1H-benzimidazol-6-
yl]oxy}methyl)quinoline dihydrochloride (31d)
J = 8.9, 2.2 Hz), 7.77 (dd, 1H, J = 7.5, 7.5 Hz), 7.84–7.91 (m, 2H),
7.93–8.03 (m, 3H), 8.15 (d, 1H, J = 8.1 Hz), 8.20 (d, 1H, J = 8.5 Hz),
8.74 (d, 1H, J = 8.5 Hz), 9.06–9.10 (m, 2H); MS (ESI) m/z 395
[M+H]+. Anal. Calcd for C25H22N4Oꢀ3HClꢀ1.2H2Oꢀ0.2C3H8O: C,
57.21; H, 5.44; N, 10.42; Cl, 19.79. Found: C, 57.17; H, 5.26; N,
10.64; Cl, 19.64.
Compound 31d was prepared from 30d and in a manner similar
to that described for compound 31e, with a yield of 40% as a pale
pink solid. 1H NMR (DMSO-d6) d 4.00 (s, 3H), 5.59 (s, 2H), 7.39
(dd, 1H, J = 9.0, 2.4 Hz), 7.47 (d, 1H, J = 2.2 Hz), 7.67–7.72 (m, 1H),
7.82–7.91 (m, 3H), 8.00 (d, 1H, J = 0.7 Hz), 8.07–8.13 (m, 2H),
8.48 (s, 1H), 8.59 (d, 1H, J = 8.5 Hz), 9.59 (s, 1H); MS (ESI) m/z
356 [M+H]+. Anal. Calcd for C21H17N5Oꢀ2HClꢀ0.1H2O: C, 58.64; H,
4.50; N, 16.28; Cl, 16.49. Found: C, 58.77; H, 4.50; N, 16.34; Cl,
16.28.
5.2. PDE10A enzyme assay protocol
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 hu-
man 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 enzymes,
BamHI/HindIII, and this digested product was inserted into a pFast-
Bac1 vector (Invitrogen. Inc.).
5.1.47. 2-({[1-(Pyridazin-4-yl)-1H-benzimidazol-6-yl]oxy}
methyl)quinoline trihydrochloride (31f)
Compound 31f was prepared from 30f and in a manner similar
to that described for compound 8, with a yield of 39% as a colorless
solid. 1H NMR (DMSO-d6) d 5.68 (s, 2H), 7.28 (dd, 1H, J = 8.9,
2.4 Hz), 7.72–7.78 (m, 2H), 7.82 (d, 1H, J = 8.9 Hz), 7.92–7.98 (m,
2H), 8.15 (d, 1H, J = 8.1 Hz), 8.25 (d, 1H, J = 8.5 Hz), 8.28 (dd, 1H,
J = 5.8, 2.8 Hz), 8.74 (d, 1H, J = 8.5 Hz), 9.14 (s, 1H), 9.52 (dd, 1H,
J = 5.8, 1.0 Hz), 9.85 (dd, 1H, J = 2.8, 1.0 Hz); MS (ESI) m/z 354
[M+H]+. Anal. Calcd for C21H15N5Oꢀ3HClꢀ1.7H2Oꢀ0.2C2H6O: C,
51.14; H, 4.53; N, 13.93; Cl, 21.16. Found: C, 51.43; H, 4.54; N,
14.30; Cl, 20.78.
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.
5.1.48. 2-({[1-(Pyrimidin-2-yl)-1H-benzimidazol-6-yl]oxy}
methyl)quinoline dihydrochloride (31g)
Compound 31g was prepared from 30g in a manner similar to
that described for compound 8, with a yield of 57% as a pale brown
solid. 1H NMR (DMSO-d6) d 5.68 (s, 2H), 7.28 (dd, 1H, J = 8.9,
2.5 Hz), 7.57 (t, 1H, J = 4.9 Hz), 7.74–7.80 (m, 2H), 7.94–8.00 (m,
2H), 8.16 (d, 1H, J = 8.3 Hz), 8.29 (d, 1H, J = 8.5 Hz), 8.34 (d, 1H,
J = 2.5 Hz), 8.76 (d, 1H, J = 8.5 Hz), 8.98 (d, 2H, J = 4.9 Hz), 9.34 (s,
1H); MS (ESI) m/z 354 [M+H]+. Anal. Calcd for C21H15N5-
Oꢀ2HClꢀ1.6H2O: C, 55.42; H, 4.47; N, 15.39; Cl, 15.58. Found: C,
55.30; H, 4.61; N, 15.45; Cl, 15.76.
5.2.3. PDE10A2 inhibition assay
Inhibition of compounds on human PDE10A enzyme activity
was assessed by measuring the amount of cAMP by the Homoge-
neous Time-Resolved Fluorescence (HTRF) detection method. The
assay was performed in 12
amount of the 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 preincu-
lL samples containing a optimal
l
l
5.1.49. 2-({[1-(Pyrimidin-4-yl)-1H-benzimidazol-6-yl]oxy}
methyl)quinoline trihydrochloride (31h)
bated for 30 min with the enzyme, the reaction was initiated by
adding the substrate cAMP and the mixture was incubated for
60 min at room temperature with agitation. The reaction was ter-
minated by the addition of the fluorescence acceptor (cAMP la-
beled with the dye d2) and the fluorescence donor (anti-cAMP
antibody labeled with Cryptate, Cisbio). After 60 min, the fluores-
cence 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 (PerkinElmer) and signal ratio (665:620)
was calculated. The ratio determined in the absence of enzyme
was subtracted from all data. The obtained results were converted
to activity relative to an uninhibited control (100%) and IC50 values
were calculated using Prism software (GraphPad Software, Inc.).
Compound 31h was prepared from 30h in a manner similar to
that described for compound 8, with a yield of 65% as a pale yellow
solid. 1H NMR (DMSO-d6) d 5.64 (s, 2H), 7.24 (dd, 1H, J = 8.8,
2.5 Hz), 7.72–7.77 (m, 2H), 7.92–7.97 (m, 2H), 8.12–8.16 (m, 2H),
8.26 (d, 1H, J = 8.5 Hz), 8.27 (d, 1H, J = 2.5 Hz), 8.72 (d. 1H.
J = 8.5 Hz), 8.97 (d, 1H, J = 5.8 Hz), 9.20 (d, 1H, J = 0.9 Hz), 9.22 (s,
1H); MS (ESI) m/z 354 [M+H]+. Anal. Calcd for C21H15N5-
Oꢀ2.6HClꢀ2.6H2O: C, 50.95; H, 4.64; N, 14.15; Cl, 18.62. Found: C,
50.84; H, 4.48; N, 14.01; Cl, 18.57.
5.1.50. 2-({[1-(1,3-Thiazol-2-yl)-1H-benzimidazol-6-yl]oxy}
methyl)quinoline dihydrochloride (31i)
Compound 31i was prepared from 30i in a manner similar to
that described for compound 31e, with a yield of 25% as a pale yel-
low solid. 1H NMR (DMSO-d6) d 5.67 (s, 2H), 7.25 (dd, 1H, J = 8.9,
2.4 Hz), 7.73–7.83 (m, 4H), 7.93 (d, 1H, J = 2.4 Hz), 7.95–8.00 (m,
2H), 8.17 (d, 1H, J = 8.0 Hz), 8.28 (d, 1H, J = 8.5 Hz), 8.78 (d, 1H,
J = 8.5 Hz), 8.98 (s, 1H); MS (ESI) m/z 359 [M+H]+. Anal. Calcd for
5.3. CYP1A2 inhibition
Using a 96-well plate, 3-cyano-7-ethoxycoumarin (5
test compound (from 0.16 to 20 M), and the enzyme (0.026 pmol)
were incubated at 37 °C for 20 min in 100 L of total volume of
100 mM phosphate buffer (pH 7.4) containing 8.2
M NADP+,
lM), each
l
l
l
C
20H14N4OSꢀ1.9HClꢀ0.5H2O: C, 55.01; H, 3.90; N, 12.83; S, 7.34;
0.41 mM glucose-6-phosphate, 0.41 mM MgCl2 and 0.4 Units/mL
glucose-6-phosphate dehydrogenase. Thereafter, the reaction was
stopped by adding 0.5 M 2-amino-2-hydroxymethyl-1,3-propane-
diol aqueous solution containing 80% acetonitrile, and the fluores-
cence intensity (excitation wavelength; 409 nm, fluorescence
wavelength; 460 nm) was measured using a fluorescence plate
reader. The residual activity was calculated based on the following
formula, and concentration of each test compound by which the
residual activity becomes 50% (IC50) was obtained.
Cl, 15.42. Found: C, 55.15; H, 3.99; N, 12.72; S, 7.15; Cl, 15.41.
5.1.51. 2-({[2-Isopropyl-1-(pyridin-4-yl)-1H-benzimidazol-6-yl]
oxy}methyl)quinoline trihydrochloride (32)
Compound 32 was prepared from 30b in a manner similar to
that described for compound 31e, with a yield of 56% as a pink so-
lid. 1H NMR (DMSO-d6) d 1.39 (d, 6H, J = 6.9 Hz), 3.20 (sep, 1H,
J = 6.9 Hz), 5.58 (s, 2H), 7.21 (d, 1H, J = 2.2 Hz), 7.40 (dd, 1H,

W. Hamaguchi et al. / Bioorg. Med. Chem. 21 (2013) 7612–7623
7623
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The residual activityð%Þ ¼ ðC1 ꢁ B1Þ=ðC0 ꢁ B1Þ ꢂ 100
C1: fluorescence intensity in the presence of test compound
having known concentration, enzyme, and substrate.
C0: fluorescence intensity in the absence of test compound and
in the presence of enzyme and substrate.
B1: fluorescence intensity of blank well.
Acknowledgments
The authors wish to thank Dr. Takeshi Shimada for his helpful
support in preparing this manuscript, Dr. Atsushi Suzuki for per-
forming pharmacological evaluations, and the staff of Astellas Re-
search Technologies Co., Ltd, for conducting CYP inhibition
screening, elemental analysis and spectral measurements.
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