Discovery of a 7-arylaminobenzimidazole series as novel CRF1receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2016.08.005

A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing factor 1 (CRF₁) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of 1, predicted to occupy a large lipophilic pocket of a CRF₁receptor, with an aryl group. During the course of this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhibitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog 16g, showed potent CRF binding inhibitory activity against a human CRF₁receptor and human CRF₁receptor antagonistic activity (IC₅₀?=?27?nM, 56?nM, respectively). This compound exhibited ex vivo¹²⁵I-Tyr⁰(¹²⁵I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland at 20?mg/kg after oral administration. In this report, we discuss the structure–activity-relationship of these 7-arylamino-1H-benzimidazoles and their synthetic method.

Full text of DOI:10.1016/j.bmc.2016.08.005

Bioorganic & Medicinal Chemistry 24 (2016) 4675–4691
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a 7-arylaminobenzimidazole series as novel CRF1
receptor antagonists
b
a
c
a
a
Michiyo Mochizuki a, , Masakuni Kori , Mitsunori Kono , Takahiko Yano , Yuu Sako , Maiko Tanaka ,
⇑
Naoyuki Kanzaki a, Albert C. Gyorkos d, Christopher P. Corrette d, Kazuyoshi Aso a
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Ltd, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
c Taisho Pharmaceutical Company Ltd, 403, Yoshino-cho 1-chome, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
d Array BioPharma Inc., 3200 Walnut Street, Boulder, CO 80301, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing
factor 1 (CRF1) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of
1, predicted to occupy a large lipophilic pocket of a CRF1 receptor, with an aryl group. During the course of
this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel
synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhi-
bitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog
16g, showed potent CRF binding inhibitory activity against a human CRF1 receptor and human CRF1
receptor antagonistic activity (IC50 = 27 nM, 56 nM, respectively). This compound exhibited ex vivo
Received 8 June 2016
Revised 2 August 2016
Accepted 3 August 2016
Available online 4 August 2016
Keywords:
Corticotropin-releasing factor
CRF1 receptor antagonist
7-Arylaminobenzimidazole
125I-Tyr0 125I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland
(
at 20 mg/kg after oral administration. In this report, we discuss the structure–activity-relationship of
these 7-arylamino-1H-benzimidazoles and their synthetic method.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
stress-related disorders. Moreover, CRF plays an important role
in the brain as a neurotransmitter that mediates stress-related
Corticotropin-releasing factor (CRF) is a 41-amino acid neu-
ropeptide and mediates its actions through two Gs-coupled G pro-
tein-coupled receptor subtypes, CRF1 and CRF2.1,2 CRF is considered
to be the main regulator of the hypothalamus–pituitary–adreno-
cortical (HPA) axis via CRF1 receptors.3,4 After exposure to stress,
secretion of CRF increases in the neurons of the paraventricular
nucleus of the hypothalamus and stimulates the release of adreno-
corticotropic hormone (ACTH) from the anterior pituitary gland.5,6
In healthy individuals not suffering from life-threatening events, a
negative feedback system against the activation of the HPA-axis is
operating; in contrast, the system collapses in patients with
behaviors.4,7
In our previous study, we designed unique benzazole derivatives
with a single-atom linker between the core and the pendant phenyl
group as novel CRF1 receptor antagonists.8 Initial structure-activity-
relationship (SAR) studies revealed that lead compound 1 shown in
Figure 1 demonstrated potent in vitro CRF1 receptor binding activity
with an IC50 value of 12 nM8 and ex vivo CRF binding inhibitory
activity in the brain after oral administration in mice. Compound
1 has a di-alkylamino moiety located in a para orientation of the
hydrogen-bonding acceptor (HBA) of the N-methylbenzimidazole
core to fit into a large lipophilic pocket of the CRF1 receptor. We
were also interested in placing an alkyl-arylamino group at the
same position of the benzimidazole core and whether it would bind
more effectively in the lipophilic pocket. In addition, previous SAR
studies indicated that a normal propyl group can be replaced with
an isopropyl group8 and the branched alkyl group can be expected
to effectively occupy the large three-dimensional pocket compared
with a linear group. Therefore, compounds in which these alkyl
groups were replaced with an aryl group and a branched alkyl group
were designed and examined.
Abbreviations: CRF, corticotropin-releasing factor; HPA, hypothalamus–pitui-
tary–adrenocortical; ACTH, adrenocorticotropic hormone; SAR, structure–activity-
relationship; HBA, hydrogen-bonding acceptor; CDI, N,N0-carbonyldiimidazole;
o-biphenylPCy2, o-biphenyl(dicyclohexyl)phosphine; S-Phos, 2-dicyclohexylphos-
phino-20,60-dimethoxybiphenyl; X-Phos, 2-dicyclohexylphosphino-20,40,60-triiso-
propylbiphenyl; PMB, p-methoxybenzyl; CHO, Chinese hamster ovary.
⇑
Corresponding author.
E-mail address: michiyo.mochizuki@takeda.com (M. Mochizuki).
http://dx.doi.org/10.1016/j.bmc.2016.08.005
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

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M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
described in Table 1. The coupling reaction of p-bromoanisole, p-
(N)
chlorobromobenzene, and 2-bromo-5-methylpyridine using tris
(dibenzylideneacetone)dipalladium and o-biphenyl(dicyclohexyl)
phosphine (o-biphenylPCy2) afforded the desired compounds 6a,
6e, and 6k, respectively (entries 2, 7, and 16), while the reaction
of p-bromophenyl methyl sulfone and p-bromobenzonitrile failed
(entries 8 and 11). The phosphine ligands with a bulky biphenyl
group, 2-dicyclohexylphosphino-20,60-dimethoxybiphenyl (S-Phos)
and 2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl (X-
Phos), have been reported to increase the activity and stability of
the catalytic system in comparison with simpler phosphine ligands
such as 2-(dicyclohexylphosphino)-20-methylbiphenyl.10,11 In our
case, using S-Phos as a ligand also provided the desired coupled
compounds having the methylsulfonyl and the cyano group, 6f
and 6h (entries 9 and 12). The yield of the reaction with p-bro-
moanisole also increased when X-Phos was used instead of o-
biphenylPCy2 (entries 2 vs 3). To summarize these results, bulkier
phosphine ligands, S-Phos or X-Phos, worked better than o-
biphenylPCy2 in these coupling reactions with both electron-rich
and electron-deficient phenyl halides, and were especially effective
for the reaction of electron-deficient phenyl halides when no
desired compounds coupled by o-biphenylPCy2 were provided.
The chemistry also worked well with meta substituents, as exem-
plified with compounds 6 wherein an alkoxy, an alkyl and a
sulfonyl group, 6b, 6d, 6g, and 6j (entries 4, 6, 10, and 15), as
well as a pyridine analog 6l (entry 17) were obtained in 20–67%
yields. While the reaction of neutral type of bromobenzene with
X-Phos mainly gave the diphenyl adduct 7 (entry 1), and m-cyano
analog 6i could not be prepared even with m-iodobenzonitrile
(entry 14), p-iodobenzonitrile provided the target compound 6h
(entry 13). Therefore, the reactants were reversed such that
7-arylaminobenzimidazole 5 was replaced with 7-bromo-1,3-
dihydro-2H-benzimidazol-2-one 8 and subsequently coupled with
an aniline, as illustrated in Scheme 2. The 7-bromobenzimidazole 8
was prepared from the 7-aminobenzimidazole 5 by a modified
Sandmeyer reaction using copper(II) bromide and tert-butylnitrate.
A desired monophenylamino coupled compound 6m was obtained
in good yield by the optimized coupling reaction of 7-bromo
analog 8 with X-Phos by microwave irradiation. Furthermore, this
condition also enabled the reaction of 8 and m-aminobenzonitrile
to provide the desired product 6i in 37% yield, a significant
improvement relative to original attempt (<5%) described in Table 1
(entry 14).
R2
N
N
7
N
N
N
N
NH OMe
Br
NH OMe
Z
2
HBA
HCl
1
R2 = alkyl
Z = Cl, Br
IC50 = 12 nM
Figure 1. Design of a novel 7-arylaminobenzimidazole series.
Flexible alignment of 7-isopropylphenylaminobenzimidazole
16 with the lead compound 1 was performed using MOE.9 It was
found that the aryl group of compound 16, exemplified as a yellow
circle in Figure 2, overlapped well with the corresponding alkyl
chain of compound 1 as well as the other key functional groups
including an HBA and a pendant aryl group exemplified as white
circles. In addition, the aryl group of compound 16 might be able
to accommodate three-dimensionally compared with the alkyl
group of compound 1. That means that the aryl group would
occupy unknown space and enhance potency. This superimposi-
tion study suggested that the newly designed compounds should
exhibit potent inhibitory activity.
The aryl amino series, having diverse size of substituents with
electron-deficient or electron-donating characteristics, was tar-
geted for SAR studies. In this study, another anilino group at the
2-position was fixed in 4-chloro or bromo-2-methoxy-6-methy-
lanilines, because 7-dialkylamino-1H-benzimidazoles having these
groups exhibited potent and comparable CRF1 receptor binding
activity.8 We also investigated novel synthetic methods to prepare
2,7-diarylaminobenzimidazoles. In this report, the synthesis and
the biological activities of a novel series of 7-arylamino-1H-benz-
imidazoles, as well as SAR study results are discussed.
2. Results and discussion
2.1. Chemistry
Synthesis of the 7-anilinobenzimidazole derivatives is
illustrated in Schemes 1–6 and Table 1. Preparation of 7-amino-
1,3-dihydro-2H-benzimidazol-2-one
5
was performed from
commercially available 2-chloro-1,3-dinitrobenzene 2 as described
in Scheme 1. The chlorobenzene 2 was treated with methylamine
to afford the corresponding N-methylaniline 3, which was followed
by reduction to give triamine 4. The key intermediate 5 was pro-
vided by cyclization of the triamine 4 with N,N0-carbonyldiimida-
zole (CDI).
Our initial efforts were focused on preparation of the desired 7-
arylaminobenzimidazoles 6 by a palladium-catalyzed coupling
reaction of 7-aminobenzimidazole 5 and aryl halide (ArX) as
7-Arylaminobenzimidazol-2-ones 6 with both electron-donating
and electron-withdrawing groups were easily converted to 2-
chloro-1,3-dihydro-2H-benzimidazoles 9 using phosphorus oxychlo-
ride, and the common precursors10weresmoothly synthesized from
the 2-chlorobenzimidazoles 9 with isopropylbromide in good yields
as shown in Scheme 3. However, chlorination of electron-rich 7-
aminobenzimidazol-2-ones 6, according to substitution pattern of
an aryl group at the 7-N-position, gave no desired 2-chlorobenzimi-
dazoles 9. The electron-rich 7-aminobenzimidazole core might be
Figure 2. Superimposition of 1 (pink) and 16 (green).

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4677
NO2
NO2
NH2
NH2
c
a
b
Cl
NHMe
NO2
NHMe
NH2
N
O
99%
77%
(two steps)
N
H
NO2
2
3
4
5
Scheme 1. Reagents and conditions: (a) MeNH2, MeOH, rt; (b) H2, Pd–C, THF, rt; (c) CDI, THF, rt.
R1
NH2
Br
NH
a
b
N
N
N
O
O
O
42%
N
H
N
H
N
H
5
8
R1
Yield (%)
6m
6i
H
CN
64
37
Scheme 2. Reagents and conditions: (a) CuBr2, tBuONO, DMF, rt; (b) aniline or 3-cyanoaniline, Pd2dba3, S-Phos or X-Phos, NaOtBu, 1,4-dioxane, microwave irradiation,
100–120 °C.
Ar
Ar
Ar
NH
N
NH
b or c
a
a
N
N
N
N
N
Cl
Cl
O
N
H
6
9
10
Putative structures
In the case that
Ar is an electron-rich group
Ar
PO3H2
Ar
Ar
PO2HCl
N
Ar
N
NH
N
NH
N
N
N
N
N
O
O
O
O
N
H
N
H
12a
or
or
or
PO3H2
PO2HCl
11a
11b
12b
6
9
9
10
Ar
C6H5
m-MeOC6H4
m-iPrOC6H4
p-NCC6H4
m-NCC6H4
m-NCMe2CC6H4
6-MeO-3-pyridyl
Yield (%)
32
Ar
Yield (%)
85
70
66
87
75
59
9a
9b
9c
9d
9e
9f
10a
10b
C6H5
m-MeOC6H4
44
58
45
38
81
41
10c m-iPrOC6H4
10d
10e
p-NCC6H4
m-NCC6H4
10f m-NCMe2CC6H4
10g
9g
6-MeO-3-pyridyl
90
i
i
i
Scheme 3. Reagents and conditions: (a) POCl3, 100 °C; (b) PrBr or PrI, NaH, (nBu4NI,) DMF, rt; (c) PrI, NaH, DMF, rt.
formed with phosphorus oxychloride to afford a stable complex 11 or
12. As such, an alternative route was envisioned wherein benzimida-
zol-2-ones 6 wouldbe converted tothe2-chlorobenzimidazoles 10 as
described in Scheme 4. The key point was selection of a stable protec-
tive group under the conditions of the first three steps, regioselective
protection of the benzimidazol-2-ones 6 at the 3-position, alkylation
or acylation at the 7-N-position, and removal of the protective group.
A p-methoxybenzyl (PMB)-protecting group was thus selected and
introduced at the 3-position of the benzimidazol-2-ones 6. Subse-
quent alkylation or acylation at the 7-N-position was performed
without issue on an electron-rich 7-aminobenzimidazole analog
bearing p-alkoxy phenyl groups, 14b and 14c. The PMB group in the
7-N-alkylated or 7-N-acylated compounds 14 could be removed by
heating in TFA. Finally, chlorination by phosphorus oxychloride suc-
cessfully gave the desired 2-chlorobenzimidazoles 10. This route
was also acceptable for other analogs with electron-deficient
aryl group such as 13d and 13e and hetero aromatic analog 13f,
suggesting that this route was more amenable to the synthesis of
more diverse compounds in comparison to the route described in
Scheme 3.
Coupling reaction of the resulting 2-chlorobenzimidazoles 10
and 4-halogeno-2-methoxy-6-methylanilines 17 gave the target
compounds 16 by heating or under microwave irradiation
(Scheme 5).
A m-cyano group in compound 16n was subsequently converted
to a carboxamide in 16p by oxidation with hydrogen peroxide under
basic conditions (Scheme 6). Methyl ester 16o was also prepared
from 16n with methanol under acidic conditions. Hydrolysis of the
methyl ester 16o provided to the corresponding carboxylic acid
16x and was subsequently condensed with methylamine or
dimethylamine to afford 16q and 16r, respectively. The 2-car-
bamoyl-2-propyl analog 16s was prepared from the corresponding
carboxylic acid 16y obtained from the cyano analog 16v in a method
similar to that for preparing the amide analogs, 16q and 16r.
As mentioned above, we established novel synthetic methods
for preparing 2,7-diarylaminobenzimidazoles, employing two key

4678
M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
Ar
7
Ar
Ar
R2
Ar
R2
Ar
R2
NH
NH
N
N
N
b or c
d
e
a
N
N
N
N
N
N
N
N
O
O
O
O
Cl
N
H
N
H
3
PMB
PMB
15
6
13
14
10
6
13
13
14
Yield (%)
Yield (%)
>99
95
67
49
71
87
Ar
13a p-ClC6H4
13b p-MeOC6H4
13c
Ar
R2
14a p-ClC6H4
iPr
62
80
71
84
73
74
32
89
81
60
14b p-MeOC6H4
14c
p-iPrOC6H4
p-iPrOC6H4
13d p-MeSO2C6H4
13e m-MeSO2C6H4
14d p-MeSO2C6H4
14e m-MeSO2C6H4
13f
14f
5-Me-2-pyridyl
5-Me-2-pyridyl
14g p-ClC6H4
CH2CMe3
COMe
CH2CH2OMe
14h
14i
14j
CH2-3-tetrahydrofuranyl
14
15
15
10
Yield (%)
Ar
R2
iPr
Ar
10l p-ClC6H4
R2
iPr
Yield (%)
72
78
18 (2 steps)
47
23 (2 steps)
64
73
69
80
15a p-ClC6H4
88
>99
-
75
-
39
80
73
71
91
15b
10m
p-MeOC6H4
p-MeOC6H4
15c p-iPrOC6H4
10n p-iPrOC6H4
10o p-MeSO2C6H4
15d p-MeSO2C6H4
15e
10p
m-MeSO2C6H4
m-MeSO2C6H4
15f 5-Me-2-pyridyl
10q 5-Me-2-pyridyl
15g p-ClC6H4
CH2CMe3
COMe
CH2CH2OMe
CH2-3-tetrahydrofuranyl
10r p-ClC6H4
CH2CMe3
COMe
CH2CH2OMe
15h
15i
15j
10s
10t
10u
CH2-3-tetrahydrofuranyl 56
Scheme 4. Reagents and conditions: (a) PMBCl, K2CO3, (nBu4NI,) DMF, rt–70 °C; (b) R2Br or R2I or R2OMs, K2CO3 or NaH, (nBu4NI,) DMF, rt–70 °C; (c) Ac2O, pyridine, 120 °C;
(d) TFA, 65–70 °C; (e) POCl3, 80–100 °C.
Ar
R2
HCl
Z
Yield (%)
53
22
53
33
53
27
65
10
36
26
36
64
9.5
69
16a p-ClC6H5
iPr
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Br
16b
16c
16d
16e
CH2CMe3
COMe
CH2CH2OMe
CH2-3-tetrahydrofuranyl
iPr
HCl
HCl
HCl
Ar
R2
Ar
R2
a
N
N
N
N
N
N
Cl
NH OMe
Z
16f
C6H5
16g p-MeOC6H4
m-MeOC6H4
p-iPrOC6H4
16j m-iPrOC6H4
(HCl)
16
16h
16i
10
16k
16l
p-MeSO2C6H4
m-MeSO2C6H4
16m p-NCC6H4
16n
16t
m-NCC6H4
6-MeO-3-pyridyl
32
41
74
16u 5-Me-2-pyridyl
16v
m-NCMe2CC6H4
Scheme 5. Reagents and conditions: (a) i) 4-halogeno-2-methoxy-6-methylaniline 17 (NMP), (microwave irradiation), 100–120 °C, (ii) 2 M HCl/Et2O).
approaches. The first involved selection of an appropriately bulky
phosphine ligand, S-Phos or X-Phos, in the palladium-catalyzed
coupling reactions of 7-aminobenzimidazole 5 or 7-bromobenzim-
idazole 8. The second strategy was to adopt the alternate synthetic
route described in Scheme 4, to prepare 2-chlorobenaimidaole 10,
based on the electron density of the benzimidazole core 6, thus
avoiding the difficulties presented by the route shown in Scheme 3.
expressed in Chinese hamster ovary (CHO) cellular membranes.
This SAR study initiated from p-chloroanilino derivatives because
a phenyl ring with a substituent at the para position is advanta-
geous for metabolic stability.12 The results of the 7-(p-chloroani-
lino) series are listed in Table 2. The isopropyl analog 16a
exhibited potent CRF1 receptor binding activity comparable to
7-(dialkylamino)benzimidazole 1 with an IC50 value of 12 nM. It
was revealed that the 7-(p-chloroanilino) group was able to access
the lipophilic pocket and demonstrated the feasibility of replacing
an alkyl group of 1 with an aromatic moiety. In addition, the com-
pounds could be further diversified via introduction of an alkyl
group of the 7-(p-chloroanilino) series, because they still retained
2.2. Human CRF1 receptor binding study
The synthesized compounds 16a–u were screened for their
ovine 125I-CRF binding inhibitory activity to human CRF1 receptors

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4679
R1
R1
R1
a or b
c
N
N
N
N
N
N
N
N
N
NH OMe
Br
NH OMe
NH OMe
Br
Br
R1
R1
16o CO2Me
Yield (%)
R1
16x CO2H
16y
CMe2CO2H
Yield (%)
16n CN
16v CMe2CN
77
-
75
16w
CMe2CO2Et
49 (2 eteps)
d
e
38%
f
R1
CONH2
N
CONH2
N
69%
N
N
N
N
N
NH OMe
Br
NH OMe
NH OMe
Br
N
N
R1
Yield (%)
Br
16q
CONHMe
16r CONMe2
52
59
16p
16s
Scheme 6. Reagents and conditions: (a) i) HCl, MeOH, rt; ii) H2O, THF; (b) EtOH, H2SO4, reflux; (c) NaOH, THF, (MeOH,) H2O, rt–70 °C; (d) H2O2, NaOH, EtOH, H2O, reflux;
i
(e) MeNH2 or Me2NH, HBTU, Pr2NEt, THF, rt; (f) NH4OH, WSC, HOBt, DMF, rt.
Table 1
Effect of phosphine ligands on Pd-catalyzed coupling reaction of 7-amino-1-methylbenzimidazol-2-one with aryl halides
Ar
NH2
NH
PCy2
PCy2
PCy2
ArX (X = Br, I)
N
MeO
OMe iPr
iPr
N
N
N
O
O
Pd2dba3, NaOtBu
O
N
H
N
H
iPr
N
H
S-Phos or X-Phos / dioxane
6
5
100oC (microwave)
S-Phos
o-biphenylPCy2
X-Phos
7
or o-biphenylPCy2
/ THF 60oC or 1,4-dioxane reflux
Entry
Ar
Phosphine ligand
6 Yield (%)
Compd No.
b
1
C6H4
X-Phos
—
7
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
p-MeOC6H4
p-MeOC6H4
m-MeOC6H4
p-iPrOC6H4
m-iPrOC6H4
p-ClC6H4
p-MeSO2C6H4
p-MeSO2C6H4
m-MeSO2C6H4
p-NCC6H4
o-biphenylPCy2
X-Phos
X-Phos
X-Phos
X-Phos
o-biphenylPCy2
o-biphenylPCy2
S-Phos
S-Phos
o-biphenylPCy2
S-Phos
S-Phos
S-Phos
X-Phos
o-biphenylPCy2
X-Phos
42
61
57
22
20
44
6a
6a
6b
6c
6d
6e
-
6f
6g
-
6h
6h
6i
6j
6k
6l
a
b
—
61
65
b
—
p-NCC6H4
p-NCC6H4
m-NCC6H4
19
74
<5
67
41
32
a
a
m-NCMe2CC6H4
5-Me-2-pyridyl
6-MeO-3-pyridyl
a
X = I.
b
No identification of desired compounds.
potency. The methoxyethyl analog 16d (IC50 = 50 nM) exhibited a
little less CRF1 receptor binding activity than isopropyl analog
16a (IC50 = 25 nM). The series with bulkier alkyl groups such as
neopentyl or (tetrahydrofuranyl)methyl analogs, 16b and 16e,
showed less potent activity than 16a and 16d, respectively, sug-
gesting that the tolerable position for a bulky group is limited.
The acetyl-substituted analog 16c was six-fold less potent than
the isopropyl analog 16a. Compounds having a lipophilic group
in R2 exhibited greater potency as opposed to polar groups in bind-
ing to the CRF1 receptors.
Effects of substitution with different sizes and diverse electronic
characteristics of the 7-N-aryl groups are shown in Table 3. The

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M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
Table 2
Table 4
Effects of alkyl groups at the 7-N-position on hCRF1 receptor binding activity
Ex vivo 125I-CRF binding inhibitory activitya
Frontal cortex (%)
Olfactory bulb (%)
Pituitary (%)
Cl
Vehicle
16g
1
0
80
79
0
82
72
0
89
65
R2
N
N
NH OMe
N
The values are% inhibition of ovine 125I-CRF binding to mouse frontal cortex,
olfactory bulb, and pituitary homogenates. Tissues were collected by decapitation
1 h after oral administration of 20 mg/kg of test compounds (n = 10), respectively.
Homogenates of each brain area were prepared from 10 brains for each compound.
a
Cl
Compound No.
Additive
HCl
R2
Bindinga (IC50, nM)
25 (20–31)
16g versus 16h, and the cyano analogs, 16m versus 16n. These
results suggest that the binding pocket of CRF1 receptor for the
para site is not so large in comparison with that for the meta site.
Regarding the meta position, electron-donating substituents such
as a methoxy (16h) and electron-withdrawing groups such as
methylsulfonyl (16l) and cyano (16n) exhibited potent binding
activity with IC50 values of 52, 90, and 69 nM, respectively. Neutral
groups such as a carboxamide (16p), amides (16q and 16r), or an
alkyl (16s) group on the 7-anilino group also showed activity with
IC50 values of 53–120 nM. These results indicate that binding activ-
ity is independent of the electron density of the 7-anilino groups
and the size of substituents at the meta position, except for iso-
propyl analog 16j. Furthermore, it was revealed that polar groups
were acceptable in this region of the binding pocket of CRF1 recep-
tors. Pyridyl analogs 16t and 16u, with IC50 values of 25 and 69 nM,
respectively, were as potent as the phenyl analogs 16f and 16g.
As demonstrated in the SAR studies noted above, introduction
of aniline groups in place of the alkyl groups in compound 1 in
the benzimidazole series gave compounds exhibiting high potency
in binding to the CRF1 receptors. The results of the SAR studies sug-
gested that the binding activity was independent of the electron
density of the anilino group and the size of substituents at the
meta position of the anilino group. On the other hand, the binding
pocket of CRF1 receptor for the para site of the anilino group is
small in comparison with that for the meta site. Polar groups such
as pyridyl moiety instead of an anilino one and amide substituents
on the anilino group were also tolerable. Small and lipophilic alkyl
groups as R2 were better than bulky or polar groups in binding to
CRF1 receptors.
16a
16b
4400 (2600–7200)
O
16c
16d
HCl
HCl
160 (100–240)
50 (30–85)
OMe
O
16e
1
220 (160–310)
HCl
12 (7.7–17)
a
IC50 values and 95% confidential intervals were calculated from the concen-
tration–response curves.
chloro- and methoxy-analogs, 16a, 16g, and 16h, were as potent as
the non-substituted phenyl analog 16f and the lead compound 1
with IC50 values of the binding activity of 10ꢀ8 M order. Com-
pounds having bulkier substituents at the meta position of the 7-
anilino moiety showed 3–12-fold more potent binding activity
than the p-substituted compounds, for instance, the isopropyloxy
analogs, 16i versus 16j, and the methylsulfonyl analogs, 16k versus
16l. On the other hand, compounds substituted by smaller groups
showed almost equal activity, for instance, the methoxy analogs,
Table 3
Effects of alkyl groups at the 7-N-position on hCRF1 receptor binding activity
Y2
Y1
R1
Among these 7-N-arylbenzimidazoles, compound 16g demon-
strated better mouse metabolic stability as well as potent activity
in the CRF1 receptor-binding assay. Compound 16g also inhibited
luciferase levels responding to cAMP released by human CRF with
IC50 values of 56 (40–77) nM in vitro CRF1 antagonistic activity,
N
N
NH OMe
N
indicating that compound 16g behaves as
antagonist.
a CRF1 receptor
Z
Compound No.
R1
Y1
Y2
Z
Bindinga (IC50, nM)
2.3. Ex vivo binding study in mice
16a
16f
16g
16h
16i
16j
16k
16l
16m
16n
16p
16q
16r
16s
16t
16u
p-Cl
H
p-MeO
m-MeO
p-iPrO
m-iPrO
p-MeSO2
m-MeSO2
p-NC
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
C
CH
C
CH
C
CH
C
C
C
C
C
N
CH
Cl
Cl
Cl
Br
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
25 (20–31)
13 (8.7–20)
27 (18–39)
52 (38–69)
1800 (920–3500)
580 (280–1200)
1100 (570–2200)
90 (62–130)
84 (63–110)
69 (48–100)
120 (84–160)
53 (41–69)
The penetration into the brain and the CRF1 receptor binding
activity of compound 16g one hour after 20 mg/kg oral administra-
tion was evaluated in ex vivo studies in mice. The results are listed
in Table 4. Compound 16g significantly inhibited 125I-CRF binding
in frontal cortex, olfactory bulb, and pituitary gland. The inhibitory
rates of 16g were around 80%, which showed activity as potent as
that of compound 1. These results suggested that compound 16g
was orally administered, well absorbed, and easily penetrated into
the brain.
m-NC
m-H2NCO
m-MeHNCO
m-Me2NCO
m-H2NCOCMe2
6-MeO
75 (56–101)
120 (76–190)
25 (18–33)
3. Conclusion
5-Me
69 (50–94)
1
12 (7.7–17)
In this study, a novel series of 7-arylamino-1H-benzimidazole
was synthesized and evaluated as CRF1 receptor antagonists. The
aromatic groups at the 7-N-position of the benzimidazole were
a
IC50 values and 95% confidential intervals were calculated from the
concentration–response curves.

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4681
introduced by palladium-catalyzed coupling reactions of 7-
aminobenzimidazole 5 or 7-bromobenzimidazole 8 in the presence
of bulky phosphine ligands, S-Phos or X-Phos. The synthetic route
of the key intermediates, 2-chlorobenzimidazole 10, can be
selected depending on the electron density of the benzimidazole
core. These investigations successfully provided a novel synthetic
method for preparing 2,7-diarylaminobenzimidazoles. The synthe-
sized target compounds were evaluated in an in vitro CRF1 recep-
tor-binding assay and the SAR study was examined. The results
suggested that diverse substituents on the anilino group at the 7-
position were tolerable for binding activity, and small alkyl groups
at the 7-N-position were better than bulky groups. It was revealed
that an aryl group at the 7-N-position of a benzimidazole core
could replace an alkyl group and occupy the lipophilic pocket of
the CRF1 receptor. The selected compound having potent human
CRF1 receptor binding inhibition activity, 16g, also showed human
CRF1 antagonistic activity in cAMP accumulation assay. Further-
more, this compound exhibited ex vivo CRF binding inhibitory
activity in mice, suggesting that this compound can be well
absorbed orally and easily penetrate the brain. It was found that
the novel benzimidazole series with an aryl group as well as an
alkyl group at the 7-N-position resulted in compounds exhibiting
potent activity as CRF1 receptor antagonists.
cal shifts are shown in parts per million (ppm), and tetramethylsi-
lane was used as an internal standard. Abbreviations used are as
follows: s = singlet, d = doublet, t = triplet, q = quartet, quin = quin-
tet, sxt = sextet, m = multiplet, dd = doublets of
a doublet,
br = broad, and br s = broad singlet. Coupling constants (J values)
are given in hertz (Hz). The acidic protons of diketones, carboxylic
acids, alcohols, or anilines were not frequently observed in the 1H
NMR spectra. 13C NMR spectra were recorded on a Bruker Avance II
+ 600 (600 MHz). Chemical shifts are given in parts per million
(ppm), with tetramethylsilane used as an internal standard. The
purities were assessed using elemental analyses or analytical HPLC.
Elemental analyses were performed by Takeda Analytical Research
Laboratories, Ltd. HPLC analyses were performed using a Varian
ProStar [YMC ODS-AQ (4.6 ꢁ 150 mm I.D.); 1% i-PrOH and 10 mM
NH4OAc in
a distilled H2O/MeCN gradient; UV detection at
220 nm or 254 nm] or a Shimadzu UFLC instrument [L-column2
ODS (3.0 ꢁ 50 mm, I.D.); 0.1% TFA in a distilled H2O/MeCN gradi-
ent; UV detection at 220 nm]. HPLC analysis showed %purity in
terms of the area under the curve of a main peak.
4.2. Synthesis
4.2.1. N-Methyl-2,6-dinitroaniline (3)
To
a
suspension of 2-chloro-1,3-dinitrobenzene (200 g,
987 mmol) in methanol (300 mL) was added methylamine (40%
solution in methanol; 314 mL, 2960 mmol), and the mixture was
stirred at room temperature for 3 h. The solvent was evaporated
in vacuo, and the residue was dissolved in ethyl acetate and satu-
rated aqueous sodium hydrogen carbonate. The aqueous layer was
separated and extracted with ethyl acetate. The organic layer was
washed with water, brine, passed through celite and concentrated
in vacuo to give the title compound (191.8 g, 99%) as a yellow solid.
1H NMR (CDCl3) d 2.89 (3H, d, J = 5.4 Hz), 6.76 (1H, t, J = 8.1 Hz),
8.18 (2H, d, J = 8.1 Hz), 8.49 (1H, br s).
4. Experimental
4.1. General
All reactions were performed using commercially available
starting materials, reagents and solvents, without further purifica-
tion. Reactions were monitored using thin-layer chromatography
with silica gel 60 F254 plates (Merck) or liquid chromatography–
mass spectrometry (LC/MS). LC/MS analysis was performed using
three methods. The first method was performed using an HP-
1100 (Agilent Technologies) separations module [CAPCELL PAK
UG-120 ODS (2.0 ꢁ 50 mm I.D., Shiseido Co., Ltd, Japan); 0.1% TFA
in a distilled H2O/MeCN gradient; UV detection at 220 nm or
254 nm]. MS spectra were recorded using a Micromass ZMD with
electrospray ionization. The second method was performed using
4.2.2. N2-Methylbenzene-1,2,3-triamine (4)
To a solution of compound 3 (31.6 g, 150 mmol) in tetrahydro-
furan with stabilizer (470 mL) was added palladium on carbon
(3.5 g), and the mixture was stirred at room temperature for 7 h
under hydrogen pressure. The catalyst was removed by filtration,
and the filtrate was concentrated in vacuo to give the title com-
pound, which was used without purification. 1H NMR (CDCl3) d
1.83–1.87 (3H, m), 3.72–3.77 (5H, m), 6.22 (2H, d, J = 7.8 Hz),
6.75 (1H, t, J = 7.8 Hz).
a
Shimadzu LC-20AD separations module [L-column2 ODS
(3.0 ꢁ 50 mm I.D., CERI, Japan); 5 mM AcONH4 in an ultrapure
H2O/MeCN gradient; UV detection at 220 nm or 254 nm]. MS spec-
tra were recorded using a Shimadzu LCMS-2020 system with elec-
trospray ionization. The third method was performed using a
Waters 2795 separations module [L-column2 ODS (3.0 ꢁ 50 mm
I.D., CERI, Japan); 0.05% TFA in an ultrapure H2O/MeCN gradient;
UV detection at 220 nm or 254 nm]. MS spectra were recorded
using a Waters ZQ2000 with electrospray ionization. Magnesium
sulfate or sodium sulfate was used as a desiccant for organic
extracts. Chromatographic purification was performed using a sil-
ica gel column (Kieselgel 60, 0.063–0.22 mm, Merck) or Purif-Pack
4.2.3. 7-Amino-1-methyl-1,3-dihydro-2H-benzimidazol-2-one (5)
To a solution of compound 4 (42.0 g, 306 mmol) in tetrahydro-
furan (450 mL) was added CDI (25.5 g, 158 mmol), and the mixture
was stirred at room temperature for 24 h. The solvent was evapo-
rated in vacuo, and the residual solid was washed with dichloro-
methane to give the title compound (20.1 g, 77%) as a solid. 1H
NMR (DMSO-d6) d 3.50 (3H, br s), 4.84 (2H, s), 6.29 (1H, dd,
J = 7.8, 0.9 Hz), 6.34 (1H, dd, J = 7.8, 0.9 Hz), 6.67 (1H, t, J = 8.1 Hz).
(SI 60 lm or NH 60 lm, Fuji Silysia, Ltd). Preparative TLC purifica-
tion was performed using a TLC plate (silica gel 60, Merck). Prepar-
ative HPLC purification was performed using a Gilson pumping
system with a photodiode array detector (Hewlett Packard 1100
series) [YMC ODS-A (20 ꢁ 50 mm I.D.); 0.1% TFA in a distilled
H2O/MeCN gradient; UV detection at 220 nm]. Separations were
achieved using a YMC packed column. Synthesized compounds
were analyzed as described below. Melting points were deter-
mined on a Yanaco micro melting point apparatus and were uncor-
rected. LC/MS analysis for the detection of mass ion peaks was
performed as described above. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded using a Varian Mercury-300
(300 MHz) and a Bruker DPX-300 (300 MHz) NMR system. Chemi-
4.2.4. 7-[(4-Methoxyphenyl)amino]-1-methyl-1,3-dihydro-2H-
benzimidazol-2-one (6a) (entry 2)
To
a mixture of compound 5 (0.183 g, 1.12 mmol), o-
biphenylPCy2 (0.037 g, 0.11 mmol), sodium tert-butoxide
(0.237 g, 2.47 mmol) and tris(dibenzylidineacetone)dipalladium
(0.041 g, 0.045 mmol) in tetrahydrofuran (6 mL) was added 4-bro-
moanisole (0.14 mL, 1.12 mmol) and heated to 60 °C for 18 h. The
reaction mixture was diluted with ethyl acetate, and passed
through celite. The filtrate was purified by silica gel column chro-
matography eluting with a 97% methanol/dichloromethane mix-
ture to give the title compound (0.126 g, 42%) as a tan powder.

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M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4.2.5. 7-[(4-Methoxyphenyl)amino]-1-methyl-1,3-dihydro-2H-
benzimidazol-2-one (6a) (entry 3)
(2H, d, J = 8.6 Hz), 6.83 (1H, d, J = 8.0 Hz), 6.93 (1H, d, J = 8.0 Hz),
7.02 (1H, t, J = 8.0 Hz), 7.64 (2H, d, J = 8.6 Hz), 8.53 (1H, s), 11.01
(1H, s).
To a mixture of compound 5 (2.00 g, 12.3 mmol), X-Phos
(0.292 g, 0.613 mmol), sodium tert-butoxide (2.94 g, 30.6 mmol)
and tris(dibenzylidineacetone)dipalladium (0.224 g, 0.245 mmol)
in 1,4-dioxane (25 mL) was added 4-bromoanisole (1.6 mL,
12.9 mmol) and heated to 100 °C for 2 h. The reaction mixture
was poured into water and extracted with ethyl acetate. The
organic layer was washed with water and concentrated in vacuo.
The residue was powdered from diethyl ether to give the title com-
pound (2.02 g, 61%). MS calcd: 269; Found: 270 (M+H). 1H NMR
(DMSO-d6) d 3.29 (3H, s), 3.66 (3H, s), 6.62 (2H, d, J = 8.8 Hz),
6.70–6.83 (4H, m), 6.91 (1H, t, J = 7.8 Hz), 7.30 (1H, s), 10.85 (1H, s).
4.2.11. 1-Methyl-7-[(3-methylsulfonyl)phenylamino]-1,3-dihydro-
2H-benzimidazol-2-one (6g)
Compound 6g (0.545 g, 65%) was prepared from 3-bro-
mophenylmethylsulfone (0.590 g, 1.35 mmol) in a manner similar
to that described in compound 6f. 1H NMR (DMSO-d6) d 3.13 (3H,
s), 3.29 (3H, s), 6.82–6.91(3H, m), 7.01 (1H, t, J = 8.0 Hz), 7.13 (1H,
s), 7.18 (1H, d, J = 8.0 Hz), 7.38 (1H, t, J = 8.0 Hz), 8.23 (1H, s), 11.00
(1H, br s).
4.2.12. 4-[(1-Methyl-2-oxo-1,3-dihydro-2H-benzimidazol-7-yl)
amino]benzonitrile (6h) (entry 13)
4.2.6. 7-[(3-Methoxyphenyl)amino]-1-methyl-1,3-dihydro-2H-
benzimidazol-2-one (6b)
Compound 6h (0.036 g, 74%) was prepared from 4-iodobenzoni-
trile (0.051 g, 0.22 mmol) in a manner similar to that described in
compound 6f. 1H NMR (DMSO-d6) d 3.23 (3H, s), 6.69 (2H, d,
J = 8.8 Hz), 6.82 (1H, d, J = 8.0 Hz), 6.93 (1H, d, J = 8.0 Hz), 7.02
(1H, t, J = 8.0 Hz), 7.52 (2H, d, J = 8.8 Hz), 8.55 (1H, s), 11.01 (1H, s).
Compound 6b (0.283 g, 57%) was prepared from 3-iodoanisole
(0.230 mL, 1.93 mmol) in a manner similar to that described in
compound 6a (entry 3). Solid. 1H NMR (CDCl3) d 3.27 (3H, s),
3.65 (3H, s), 6.15–6.19 (2H, m), 6.25–6.28 (1H, m), 6.77–6.84
(2H, m), 6.96 (1H, t, J = 7.8 Hz), 7.02 (1H, t, J = 7.8 Hz), 7.67 (1H,
s), 10.93 (1H, s).
4.2.13. 3-[(1-Methyl-2-oxo-1,3-dihydro-2H-benzimidazol-7-yl)
amino]benzonitrile (6i)
4.2.7. 1-Methyl-7-[[4-(1-methylethyl)oxyphenyl]amino]-1,3-
dihydro-2H-benzimidazol-2-one (6c)
Compound 6i (0.043 g, 37%) was prepared from compound 8
(0.100 g, 0.44 mmol), the preparation is described below, and 3-
aminobenzonitrile (0.062 g, 0.53 mmol) in a manner similar to that
described in compound 6f. 1H NMR (CDCl3) d 3.45 (3H, s), 5.53 (1H,
s), 6.84–6.89 (3H, m), 7.02–7.10 (4H, m), 9.03 (1H, s).
Compound 6c (0.370 g, 22%) was prepared from 1-bromo-4-(1-
methylethoxy)benzene (0.801 mL, 6.43 mmol) in a manner similar
to that described in compound 6a (entry 3). Solid. 1H NMR (DMSO-
d6) d 1.20 (6H, d, J = 6.3 Hz), 3.30 (3H, s), 4.34–4.44 (1H, m), 6.58–
6.62 (2H, m), 6.71–6.81 (4H, m), 6.88–6.94 (1H, m), 7.33 (1H, s).
4.2.14. 2-Methyl-2-{3-[(3-methyl-2-oxo-2,3-dihydro-1H-benz-
imidazol-4-yl)amino]phenyl}propanenitrile (6j)
4.2.8. 1-Methyl-7-[[3-(1-methylethyl)oxyphenyl]amino]-1,3-
dihydro-2H-benzimidazol-2-one (6d)
Compound 6j (0.930 g, 67%) was prepared from 2-(3-bro-
mophenyl)-2-methylpropanenitrile (1.11 g, 4.95 mmol) in a man-
ner similar to that described in compound 6a (entry 3). Solid. MS
calcd: 306; Found: 307 (M+H). 1H NMR (CDCl3) d 1.70 (6H, s),
3.50 (3H, s), 5.47 (1H, s), 6.52 (1H, dd, J = 8.4, 2.1 Hz), 6.80–6.95
(2H, m), 6.95–7.15 (2H, m), 7.17 (1H, d, J = 8.1 Hz), 9.37(1H, s).
Compound 6d (0.370 g, 20%) was prepared from 1-bromo-3-
[(1-methylethyl)oxy]benzene (1.38 g, 6.44 mmol) in a manner
similar to that described in compound 6a (entry 3). Solid. 1H
NMR (CDCl3) d 1.29 (6H, d, J = 6.4 Hz), 3.49 (3H, s), 4.42–4.52
(1H, m), 5.32 (1H, br s), 6.15–6.17 (1H, m), 6.22–6.27 (1H, m),
6.34–6.39 (1H, m), 6.85–6.90 (1H, m), 6.94–7.12 (3H, m), 9.37
(1H, br s).
4.2.15. 1-Methyl-7-(5-methylpyridin-2-ylamino)-1,3-dihydro-
2H-benzimidazol-2-one (6k)
Compound 6k (1.29 g, 41%) was prepared from 2-bromo-5-
methylpyridine (2.21 g, 12.0 mmol) in a manner similar to that
described in compound 6e. MS calcd: 254; Found: 255 (M+H). 1H
NMR (CDCl3) d 2.13 (3H, s), 3.32 (3H, s), 6.44 (1H, d, J = 8.4 Hz),
6.75–6.90 (2H, m), 6.90–7.00 (1H, m), 7.33 (1H, d, J = 8.4 Hz),
7.83 (1H, s), 8.20 (1H, s), 10.88 (1H, s).
4.2.9. 7-[(4-Chlorophenyl)amino]-1-methyl-1,3-dihydro-2H-
benzimidazol-2-one (6e)
To
a mixture of compound 5 (5.00 g, 30.6 mmol), o-
biphenylPCy2 (0.537 g, 1.53 mmol), sodium tert-butoxide (7.40 g,
2.50 mmol) and tris(dibenzylidineacetone)dipalladium (0.56 g,
0.61 mmol) and 1,4-dioxane (80 mL) were added 4-chlorobro-
mobenzene (6.16 g, 32.2 mmol), and the mixture was refluxed for
22 h. The reaction mixture was cooled, poured into water
(200 mL) and adjusted to pH 8 with saturated aqueous ammonium
chloride. The precipitate was filtered, washed with water and
dried. Recrystallization from ethanol gave the title compound
(3.69 g, 44%) as a tan crystal. MS calcd: 273; Found: 274 (M+H).
4.2.16. 7-[(6-Methoxypyridin-3-yl)amino]-1-methyl-1,3-dihydro-
2H-benzimidazol-2-one (6l)
Compound 6l (0.525 g, 32%) was prepared from 5-bromo-2-
methoxypyridine (0.833 mL, 6.43 mmol) in a manner similar to
that described in compound 6a (entry 3). Solid. 1H NMR (DMSO-
d6) d 3.34 (3H, s), 3.76 (3H, s), 6.67–6.72 (2H, m), 6.77 (1H, d,
J = 7.2 Hz), 6.92 (1H, t, J = 7.8 Hz), 7.10–7.13 (1H, m), 7.43 (1H, s),
7.60–7.61 (1H, m), 10.92 (1H, br s).
4.2.10. 1-Methyl-7-[(4-methylsulfonyl)phenylamino]-1,3-dihydro-
2H-benzimidazol-2-one (6f)
To a mixture of compound 5 (0.300 g, 1.84 mmol), S-Phos
(0.0377 g, 0.0919 mmol), sodium tert-butoxide (0.350 g, 3.70 mmol),
tris(dibenzylidineacetone)dipalladium (0.170 g, 0.180 mmol) and
1,4-dioxane (0.5 mL) was added 4-bromophenylmethylsulfone
(0.520 g, 2.20 mmol), and the mixture was refluxed for 4 h. The
reaction mixture was cooled, poured into water, and extracted
with ethyl acetate and ethyl acetate-tetrahydrofuran. The organic
layer was concentrated in vacuo. The resulting solid was washed
with ethyl acetate to give the title compound (0.357 g, 61%)
as crystals. 1H NMR (CDCl3) d 3.09 (3H, s), 3.25 (3H, s), 6.70
4.2.17. 7-Anilino-1-methyl-1,3-dihydro-2H-benzimidazol-2-one
(6m)
Compound 6m (0.402 g, 64%) was prepared from compound 8
(0.600 g, 2.64 mmol), the preparation is described below, and ani-
line (0.253 mL, 2.77 mmol) in a manner similar to that described in
compound 6a (entry 3). MS calcd: 239; Found: 240 (M+H). 1H NMR
(CDCl3) d 3.48 (3H, s), 5.35 (1H, s), 6.64–6.67 (2H, m), 6.83 (1H, t,
J = 7.2 Hz), 6.85–6.89 (1H, m), 6.97 (1H, t, J = 6.6 Hz), 7.03 (1H, t,
J = 7.8 Hz), 7.17–7.23 (2H, m), 9.23 (1H, br s).

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4683
4.2.18. 7-Bromo-1-methyl-1,3-dihydro-2H-benzimidazol-2-one
(8)
compound 9d. Oil. MS calcd: 324; Found: 325 (M+H). 1H NMR
(CDCl3) d 1.69 (6H, s), 3.83 (3H, s), 5.58 (1H, s), 6.46 (1H, dd,
J = 8.4, 2.4 Hz), 6.85–6.95 (1H, m), 7.00–7.12 (2H, m), 7.16 (1H, d,
J = 8.1 Hz), 7.20–7.30 (1H, m), 7.59 (1H, d, J = 8.1 Hz).
To a suspension of compound 5 (6.10 g, 37.4 mmol) and copper
(II) bromide (4.20 g, 19.0 mmol) in N,N-dimethylformamide
(30 mL) was added tert-butyl nitrite (5.43 mL, 41.1 mmol) at 0 °C,
and the mixture was stirred at room temperature for 8 h. The reac-
tion mixture was passed through celite, and the filtrate was
extracted with ethyl acetate. The organic layer was washed with
brine and concentrated in vacuo. The resulting solid was washed
with ethanol-diethyl ether to give the title compound (3.53 g,
42%). 1H NMR (DMSO-d6) d 3.56 (3H, s), 6.91 (1H, t, J = 8.0 Hz),
6.98 (1H, d, J = 8.0 Hz), 7.15 (1H, d, J = 8.0 Hz), 11.17 (1H, s).
4.2.25. 2-Chloro-N-(6-methoxypyridin-3-yl)-1-methyl-1H-benz-
imidazol-7-amine (9g)
Compound 9g (0.228 g, 41%) was prepared from compound 6l
(0.520 g, 1.92 mmol) in a manner similar to that described in com-
pound 9d. Oil. 1H NMR (CDCl3) d 3.86 (3H, s), 3.88 (3H, s), 5.45 (1H,
br s), 6.63–6.66 (1H, m), 6.93–6.95 (1H, m), 7.01–7.05 (1H, m), 7.17
(1H, t, J = 8.4 Hz), 7.47–7.50 (1H, m), 7.64–7.65 (1H, m).
4.2.19. 4-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)amino]
benzonitrile (9d)
4.2.26. 2-Chloro-1-methyl-N-(1-methylethyl)-N-phenyl-1H-benz-
imidazol-7-amine (10a)
A mixture of compound 6h (0.137 g, 0.518 mmol) and phospho-
rous oxychloride (1.5 mL) was refluxed for 3 h. The mixture was
concentrated in vacuo. The residue was neutralized with saturated
aqueous sodium bicarbonate and extracted with ethyl acetate. The
organic layer was washed with brine and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with a 25% ethyl acetate/hexane mixture to give the title
compound (0.066 g, 45%). MS calcd: 282; Found: 283 (M+H). 1H
NMR (CDCl3) d 3.79 (3H, s), 5.97 (1H, s), 6.62 (2H, d, J = 8.6 Hz),
7.09 (1H, d, J = 7.8 Hz), 7.28 (1H, t, J = 7.8 Hz), 7.46 (2H, d,
J = 8.6 Hz), 7.66 (1H, d, J = 7.8 Hz).
To a suspension of compound 9a (0.130 g, 0.504 mmol) in N,N-
dimethylformamide (2.5 mL) was added sodium hydride (60% dis-
persion in oil; 0.202 g, 5.04 mmol) at 0 °C, and the mixture was
stirred at room temperature for 5 min. 2-Iodopropane (0.504 mL,
5.04 mmol) was added at 0 °C, and the mixture was stirred at room
temperature for 1.5 h. The reaction mixture was poured into water
and extracted with ethyl acetate. The organic layer was washed
with water and brine and concentrated in vacuo. The residue was
purified by silica gel column chromatography eluting with a 20%
ethyl acetate/hexane mixture to give the title compound (0.128 g,
85%) as a syrup. MS calcd: 299; Found: 300 (M+H). 1H NMR (CDCl3)
d 0.96 (3H, br s), 1.40 (3H, br s), 3.63 (3H, s), 4.35–4.44 (1H, m),
6.49 (2H, dd, J = 8.7, 0.9 Hz), 6.68–6.73 (1H, m), 7.05 (1H, dd,
J = 7.5, 0.9 Hz), 7.13–7.18 (2H, m), 7.30 (1H, t, J = 8.1 Hz), 7.69
(1H, dd, J = 8.1, 0.9 Hz).
4.2.20. 2-Chloro-1-methyl-N-phenyl-1H-benzimidazol-7-amine
(9a)
Compound 9a (0.138 g, 32%) was prepared from compound 6m
(0.400 g, 1.67 mmol) in a manner similar to that described in com-
pound 9d. MS calcd: 257; Found: 258 (M+H). 1H NMR (CDCl3) d
3.81 (3H, s), 5.20 (1H, br s), 6.61–6.65 (2H, m), 6.82–6.87 (1H,
m), 7.04 (1H, d, J = 7.5 Hz), 7.16–7.25 (3H, m), 7.57 (1H, t,
J = 7.5 Hz).
4.2.27. 2-Chloro-N-(3-methoxyphenyl)-1-methyl-N-(1-methyl-
ethyl)-1H-benzimidazol-7-amine (10b)
Compound 10b (0.368 g, 70%) was prepared from compound 9b
(0.336 g, 1.17 mmol) in a manner similar to that described in com-
pound 10a. Oil. 1H NMR (CDCl3) d 0.83–0.94 (3H, m), 1.36–1.44
(3H, m), 3.64 (3H, s), 3.71 (3H, s), 4.26–4.39 (1H, m), 6.06–6.12
(2H, m), 6.26–6.29 (1H, m), 7.01–7.15 (2H, m), 7.28 (1H, t,
J = 7.2 Hz), 7.67 (1H, d, J = 6.9 Hz).
4.2.21. 2-Chloro-N-(3-methoxyphenyl)-1-methyl-1H-benzimida-
zol-7-amine (9b)
Compound 9b (0.336 g, 44%) was prepared from compound 6b
(0.715 g, 2.66 mmol) in a manner similar to that described in com-
pound 9d. Oil. 1H NMR (CDCl3) d 3.73 (3H, s), 3.82 (3H, s), 5.48 (1H,
br s), 6.15–6.17 (1H, m), 6.21–6.24 (1H, m), 6.38–6.42 (1H, m),
7.03–7.13 (2H, m), 7.22 (1H, t, J = 7.8 Hz), 7.57–7.60 (1H, m).
4.2.28. 2-Chloro-1-methyl-N-(1-methylethyl)-N-[3-(1-methyl-
ethyl)oxyphenyl]-1H-benzimidazol-7-amine (10c)
Compound 10c (0.0690 g, 66%) was prepared from compound 9c
(0.0930 g, 0.294 mmol) in a manner similar to that described in
compound 10a. Oil. 1H NMR (CDCl3) d 0.93 (3H, d, J = 6.6 Hz), 1.27
(6H, d, J = 6.0 Hz), 1.40 (3H, d, J = 6.6 Hz), 3.65 (3H, s), 4.30–4.40
(1H, m), 4.37–4.47 (1H, m), 6.01–6.06 (2H, m), 6.25–6.28 (1H, m),
7.00–7.06 (2H, m), 7.28 (1H, t, J = 7.8 Hz), 7.66–7.69 (1H, m).
4.2.22. 2-Chloro-1-methyl-N-(3-(1-methylethyl)oxyphenyl)-1H-
benzimidazol-7-amine (9c)
Compound 9c (0.180 g, 58%) was prepared from compound 6d
(0.295 g, 0.992 mmol) in a manner similar to that described in
compound 9d. Oil. 1H NMR (CDCl3) d 1.27 (6H, d, J = 6.0 Hz), 3.82
(3H, s), 4.39–4.49 (1H, m), 5.43 (1H, br s), 6.12–6.14 (1H, m),
6.19–6.22 (1H, m), 6.37–6.40 (1H, m), 7.03–7.11 (2H, m), 7.22
(1H, t, J = 8.4 Hz), 7.56–7.59 (1H, m).
4.2.29. 4-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)(1-methylethyl)
amino]benzonitrile (10d)
To a suspension of compound 9d (0.064 g, 0.226 mmol), tetra-
butylammonium iodide (0.0084 g, 0.023 mmol) and sodium
hydride (90% dry; 0.0181 g, 0.679 mmol) in N,N-dimethylfor-
mamide (0.5 mL) was added 2-bromopropane (0.0723 mL,
0.679 mmol), and the mixture was stirred at room temperature
for 12 h. The reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was washed with
brine and concentrated in vacuo. The residue was purified by silica
gel column chromatography eluting with a 50% ethyl acetate/
hexane mixture to give the title compound (0.064 g, 87%).
MS calcd: 324; Found: 325 (M+H). 1H NMR (CDCl3) d 0.96
(3H, d, J = 6.6 Hz), 1.43 (3H, d, J = 6.6 Hz), 3.58 (3H, s), 4.30–4.43
(1H, m), 6.49 (2H, d, J = 8.2 Hz), 7.02 (1H, d, J = 8.0 Hz), 7.34 (1H,
t, J = 8.0 Hz), 7.42 (2H, d, J = 8.2 Hz), 7.75 (1H, d, J = 8.0 Hz).
4.2.23. 3-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)amino]
benzonitrile (9e)
Compound 9e (0.557 g, 38%) was prepared from compound 6i
(1.38 g, 5.22 mmol) in a manner similar to that described in com-
pound 9d. MS calcd: 282; Found: 283 (M+H). 1H NMR (CDCl3) d
3.83 (3H, s), 5.76 (1H, s), 6.83–6.86 (2H, m), 7.07–7.12 (2H, m),
7.26–7.30 (2H, m), 7.65 (1H, dd, J = 8.0, 0.8 Hz).
4.2.24. 2-[3-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)amino]
phenyl]-2-methylpropanenitrile (9f)
Compound 9f (0.789 g, 81%) was prepared from compound 6j
(0.920 g, 3.00 mmol) in a manner similar to that described in

4684
M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4.2.30. 3-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)(1-methyl-
ethyl)amino]benzonitrile (10e)
compound 13b. Solid. 1H NMR (CDCl3) d 1.29 (6H, d, J = 6.0 Hz),
3.53 (3H, s), 3.76 (3H, s), 4.34–4.46 (1H, m), 5.01 (2H, s), 5.19
(1H, br s), 6.62–6.66 (2H, m), 6.71–6.95 (7H, m), 7.28–7.31 (2H, m).
Compound 10e (0.480 g, 75%) was prepared from compound 9e
(0.556 g, 1.97 mmol) in a manner similar to that described in com-
pound 10d. MS calcd: 324; Found: 325 (M+H). 1H NMR (CDCl3) d
0.97 (3H, d, J = 6.4 Hz), 1.41 (3H, d, J = 6.4 Hz), 3.61 (3H, s), 4.30–
4.38 (1H, m), 6.62 (1H, d, J = 8.0 Hz), 6.78 (1H, s), 6.98 (1H, d,
J = 8.0 Hz), 7.01 (1H, d, J = 8.0 Hz), 7.20 (1H, t, J = 8.0 Hz), 7.33
(1H, t, J = 8.0 Hz), 7.74 (1H, d, J = 8.0 Hz).
4.2.36. 1-(4-Methoxybenzyl)-3-methyl-4-[[(4-methylsulfonyl)
phenyl]amino]-1,3-dihydro-2H-benzimidazol-2-one (13d)
Compound 13d (0.595 g, 49%) was prepared from compound 6f
(0.887 g, 2.80 mmol) in a manner similar to that described in com-
pound 13b. 1H NMR (CDCl3) d 3.00 (3H, s), 3.47 (3H, s), 3.77 (3H, s),
5.01 (2H, s), 6.31 (1H, s), 6.69 (1H, d, J = 8.8 Hz), 6.84–6.89 (4H, m),
7.01 (1H, t, J = 8.0 Hz), 7.29 (2H, d, J = 8.8 Hz), 7.69 (2H, d,
J = 8.8 Hz).
4.2.31. 2-[3-[(2-Chloro-1-methyl-1H-benzimidazol-7-yl)(1-methyl-
ethyl)amino]phenyl]-2-methylpropanenitrile (10f)
Compound 10f (0.520 g, 59%) was prepared from compound 9f
(0.780 g, 2.13 mmol) in a manner similar to that described in com-
pound 10a. Solid. Mp 105–106 °C. MS calcd: 366; Found: 367 (M
+H). 1H NMR (CDCl3) d 0.97 (3H, d, J = 7.2 Hz), 1.42 (3H, d,
J = 7.2 Hz), 1.66 (6H, s), 3.64 (3H, s), 4.35–4.50 (1H, m), 6.24 (1H,
d, J = 7.2 Hz), 6.76 (1H, d, J = 7.2 Hz), 6.78 (1H, s), 7.05 (1H, d,
J = 8.0 Hz), 7.10 (1H, t, J = 7.2 Hz), 7.31 (1H, t, J = 8.0 Hz), 7.70 (1H,
d, J = 8.0 Hz).
4.2.37. 1-(4-Methoxybenzyl)-3-methyl-4-[[(3-methylsulfonyl)
phenyl]amino]-1,3-dihydro-2H-benzimidazol-2-one (13e)
Compound 13e (0.980 g, 71%) was prepared from compound 6g
(1.00 g, 3.15 mmol) in a manner similar to that described in com-
pound 13b. 1H NMR (CDCl3) d 3.03 (3H, s), 3.50 (3H, s), 3.78 (3H,
s), 5.03 (2H, s), 5.63 (1H, s), 6.81–6.87 (5H, m), 7.00 (1H, t,
J = 8.0 Hz), 7.29–7.35 (5H, m).
4.2.32. 2-Chloro-N-(6-methoxypyridin-3-yl)-1-methyl-N-(1-methyl-
ethyl)-1H-benzimidazol-7-amine (10g)
4.2.38. 1-(4-Methoxybenzyl)-3-methyl-4-(5-methylpyridin-2-yl-
amino)-1,3-dihydro-2H-benzimidazol-2-one (13f)
Compound 10g (0.221 g, 90%) was prepared from compound 9g
(0.228 g, 0.790 mmol) in a manner similar to that described in
compound 10a. Oil. 1H NMR (CDCl3) d 0.98–1.43 (6H, m), 3.72
(3H, s), 3.85 (3H, s), 4.24–4.34 (1H, m), 6.58 (1H, dd, J = 9.0,
0.9 Hz), 6.79 (1H, dd, J = 9.0, 3.3 Hz), 7.05 (1H, dd, J = 7.5, 0.9 Hz),
7.28 (1H, t, J = 7.5 Hz), 7.53 (1H, d, J = 3.3 Hz), 7.67 (1H, dd,
J = 7.5, 0.9 Hz).
Compound 13f (1.60 g, 87%) was prepared from compound 6k
(1.25 g, 4.92 mmol) in a manner similar to that described in com-
pound 13b. Oil. MS calcd: 374; Found: 375 (M+H). 1H NMR (CDCl3)
d 2.21 (3H, s), 3.52 (3H, s), 3.78 (3H, s), 5.02 (2H, s), 6.22 (1H, s),
6.28 (1H, d, J = 8.4 Hz), 6.80–6.90 (2H, m), 6.90 (1H, d, J = 8.0 Hz),
6.98 (1H, t, J = 8.0 Hz), 7.30 (2H, d, J = 8.4 Hz), 7.25-.35 (1H, m),
7.98 (1H, s).
4.2.33. 4-[(4-Chlorophenyl)amino]-1-(4-methoxybenzyl)-3-methyl-
1,3-dihydro-2H-benzimidazol-2-one (13a)
4.2.39. 4-[(4-Chlorophenyl)(1-methylethyl)amino]-1-(4-methoxy-
benzyl)-3-methyl-1,3-dihydro-2H-benzimidazol-2-one (14a)
To a mixture of compound 13a (0.118 g, 0.30 mmol), 2-bromo-
propane (0.056 mL, 0.60 mmol), tetrabutylammonium iodide (one
spatula) and N,N-dimethylformamide (2 mL) was added sodium
hydride (90% dry; 0.016 g, 0.60 mmol). The mixture was stirred
at 60 °C for 6 h. The mixture was diluted with water and extracted
with ethyl acetate. The organic layer was washed with water and
concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 20–33% ethyl acetate/hexane gradi-
ent mixture to give the title compound (0.0806 g, 62%) as an oil.
MS calcd: 435; Found: 436 (M+H). 1H NMR(CDCl3) d 0.96 (3H, d,
J = 6.0 Hz), 1.33 (3H, d, J = 6.0 Hz), 3.30 (3H, s), 3.79 (3H, s), 4.20–
4.35 (1H, m), 5.02 (2H, s), 6.40 (2H, d, J = 9.2 Hz), 6.77 (1H, d,
J = 8.0 Hz), 6.87 (2H, d, J = 8.4 Hz), 6.92 (1H, d, J = 8.0 Hz), 7.05 (1H,
d, J = 8.0 Hz), 7.09 (2H, d, J = 9.2 Hz), 7.32 (2H, d, J = 8.4 Hz).
A mixture of compound 6e (0.270 g, 1.00 mmol), 4-methoxy-
benzyl chloride (0.17 mL, 1.20 mmol), potassium carbonate
(0.210 g, 1.50 mmol) and N,N-dimethylformamide (1 mL) was stir-
red at 70 °C for 100 min. The mixture was diluted with water
(20 mL) and extracted with ethyl acetate (30 mL). The organic layer
was washed with water and concentrated in vacuo. The residue
was purified by silica gel column chromatography eluting with a
15% ethyl acetate/hexane mixture to give the title compound
(0.39 g, >99%) as a powder. MS calcd: 393; Found: 394 (M+H). 1H
NMR (CDCl3) d 3.49 (3H, s), 3.78 (3H, s), 5.02 (2H, s), 5.30 (1H, s),
6.56 (2H, d, J = 8.4 Hz), 6.80–6.95 (4H, m), 6.97 (1H, t, J = 8.0 Hz),
7.13 (2H, d, J = 8.4 Hz), 7.30 (2H, d, J = 8.0 Hz).
4.2.34. 1-(4-Methoxybenzyl)-4-[(4-methoxyphenyl)amino]-3-
methyl-1,3-dihydro-2H-benzimidazol-2-one (13b)
A mixture of compound 6a (1.53 g, 5.68 mmol), 4-methoxyben-
zyl chloride (0.924 mL, 6.82 mmol), potassium carbonate (1.57 g,
11.4 mmol), tetrabutylammonium iodide (0.0315 mg, 0.085 mmol)
and N,N-dimethylformamide (20 mL) was stirred at room temper-
ature for 24 h. The reaction mixture was poured into water and
extracted with dichloromethane. The organic layer was washed
with water and concentrated in vacuo. The residue was powdered
from diethyl ether to give the title compound (2.10 g, 95%) as a
powder. MS calcd: 389; Found: 390 (M+H). 1H NMR (CDCl3) d
3.53 (3H, s), 3.75 (3H, s), 3.77 (3H, s), 5.01 (2H, s), 5.20 (1H, br s),
6.64–6.68 (2H, m), 6.71–6.70 (4H, m), 6.84–6.95 (3H, m), 7.28–
7.31 (2H, m).
4.2.40. 4-[(4-Chlorophenyl)(2,2-dimethylpropyl)amino]-1-(4-
methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzimidazol-2-one
(14g)
To a solution of compound 13a (0.118 g, 0.300 mmol), neopen-
tyl iodide (0.119 g, 0.600 mmol) in N,N-dimethylformamide (3 mL)
was added sodium hydride (90% dry; 0.016 g, 0.600 mmol), and the
mixture was stirred at 60 °C for 5 h. The reaction mixture was
diluted with water and extracted with ethyl acetate. The organic
layer was washed with water and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography eluting with
a 20% ethyl acetate/hexane mixture to give the title compound
(0.045 g, 32%). MS calcd: 463; Found: 464 (M+H). 1H NMR
(CDCl3) d 0.95 (9H, s), 3.28 (3H, s), 3.37 (1H, d, J = 14.8 Hz), 3.79–
3.82 (4H, m), 4.95–5.10 (2H, m), 6.52 (2H, d, J = 8.8 Hz), 6.80–
6.90 (3H, m), 6.95–7.05 (2H, m), 7.05 (2H, d, J = 8.8 Hz), 7.31 (2H,
d, J = 8.4 Hz).
4.2.35. 1-(4-Methoxybenzyl)-3-methyl-4-[[4-(1-methylethyl)
oxyphenyl]amino]-1,3-dihydro-2H-benzimidazol-2-one (13c)
Compound 13c (0.860 g, 67%) was prepared from compound 6c
(0.920 g, 3.09 mmol) in a manner similar to that described in

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4685
4.2.41. 1-(4-Methoxybenzyl)-4-[4-(methoxyphenyl)(1-methyl
ethyl)amino]-3-methyl-1,3-dihydro-2H-benzimidazol-2-one
(14b)
bicarbonate and concentrated in vacuo. The residue was purified
by silica gel column chromatography eluting with a 40–50% ethyl
acetate/hexane gradient mixture to give the title compound
(0.388 g, 89%) as an oil. MS calcd: 435; Found: 436 (M+H). 1H
NMR (CDCl3) d 2.05 (3H, s), 3.47 (3H, s), 3.79 (3H, s), 4.99 (1H, d,
J = 15.6 Hz), 5.05 (1H, d, J = 15.6 Hz), 6.87 (2H, d, J = 8.4 Hz), 6.80–
6.90 (1H, m), 6.90–7.10 (2H, m), 7.31 (2H, d, J = 8.4 Hz), 7.20–
7.40 (4H, m).
Compound 14b (1.80 g, 80%) was prepared from compound 13b
(2.02 g, 1.35 mmol) and 2-iodopropane (7.18 mL) according to the
method described in the preparation of compound 14g. MS calcd:
431; Found: 432 (M+H). 1H NMR (CDCl3) d 0.80–1.30 (6H, m), 3.36
(3H, s), 3.73 (3H, s), 3.79 (3H, s), 4.20–4.35 (1H, m), 5.02 (2H, s),
6.46 (2H, d, J = 8.8 Hz), 6.75 (2H, d, J = 8.8 Hz), 6.80 (1H, d,
J = 8.0 Hz), 6.85–6.95 (3H, m), 7.01 (1H, t, J = 8.0 Hz), 7.32 (2H, d,
J = 8.8 Hz).
4.2.47. 4-[(4-Chlorophenyl)(2-methoxyethyl)amino]-1-(4-meth
oxybenzyl)-3-methyl-1,3-dihydro-2H-benzimidazol-2-one (14i)
Compound 14i (0.560 g, 81%) was prepared from compound
13a (0.601 g, 1.53 mmol) and 2-bromoethyl methyl ether
(0.215 mL, 2.29 mmol) in a manner similar to that described in
compound 14a. MS calcd: 451; Found: 452 (M+H). 1H NMR (CDCl3)
d 3.29 (6H, s), 3.55–3.65 (2H, m), 3.79 (3H, s), 3.70–4.00 (2H, m),
4.95–5.10 (2H, m), 6.49 (2H, d, J = 8.4 Hz), 6.80–6.95 (4H, m),
7.04 (1H, t, J = 8.0 Hz), 7.10 (2H, d, J = 8.4 Hz), 7.32 (2H, d,
J = 8.4 Hz).
4.2.42. 1-(4-Methoxybenzyl)-3-methyl-4-[(1-methylethyl)[4-(1-
methylethyl)oxyphenyl]amino]-1,3-dihydro-2H-benzimidazol-
2-one (14c)
Compound 14c (0.674 g, 71%) was prepared from compound
13c (0.860 g, 2.06 mmol) and 2-iodopropane (2.06 mL, 20.6 mmol)
in a manner similar to that described in compound 14g. Solid. 1H
NMR (CDCl3) d 1.04–1.33 (6H, m), 1.27 (6H, d, J = 6.0 Hz), 3.35
(3H, s), 3.79 (3H, s), 4.19–4.29 (1H, m), 4.29–4.39 (1H, m), 5.02
(2H, s), 6.41–6.45 (2H, m), 6.70–6.76 (2H, m), 6.79–6.82 (1H, m),
6.85–6.89 (3H, m), 7.00 (1H, t, J = 7.5 Hz), 7.32 (2H, d, J = 8.7 Hz).
4.2.48. 4-[(4-Chlorophenyl)(tetrahydrofuran-3-ylmethyl)amino]-
1-(4-methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzimidazol-2-
one (14j)
4.2.43. 1-(4-Methoxybenzyl)-3-methyl-4-[(1-methylethyl)[4-
(methylsulfonyl)phenyl]amino]-1,3-dihydro-2H-benzimidazol-
2-one (14d)
Compound 14j (0.220 g, 60%) was prepared from compound 13a
(0.300 g, 0.762 mmol) and (tetrahydrofuran-3-yl)methyl methane-
sulfonate (0.310 g, 1.50 mmol) in
a manner similar to that
Compound 14d (0.545 g, 84%) was prepared from compound
13d (0.590 g, 1.35 mmol) in a manner similar to that described in
compound 14a. 1H NMR (CDCl3) d 0.99 (3H, d, J = 6.2 Hz), 1.26
(3H, d, J = 6.2 Hz), 3.01 (3H, s), 3.25 (3H, s), 3.79 (3H, s), 4.32–
4.39 (1H, m), 5.01 (1H, d, J = 15.4 Hz), 5.06 (1H, d, J = 15.4 Hz),
6.56 (2H, d, J = 8.8 Hz), 6.77 (1H, d, J = 8.0 Hz), 6.84–6.89 (2H, m),
6.98 (1H, d, J = 8.0 Hz), 7.08 (1H, t, J = 8.0 Hz), 7.32 (2H, d,
J = 8.8 Hz), 7.68 (2H, d, J = 8.8 Hz).
described in compound 14a. Oil. MS calcd: 477; Found: 478 (M
+H). 1H NMR (CDCl3) d 1.50–1.65 (1H, m), 1.95–2.10 (1H, m),
2.60–2.75 (1H, m), 3.26 (3H, s), 3.20–3.60 (2H, m), 3.79 (3H, s),
3.65–3.95 (4H, m), 4.95–5.10 (2H, m), 6.48 (2H, d, J = 8.8 Hz),
6.80–6.95 (4H, m), 7.02 (1H, t, J = 8.0 Hz), 7.11 (2H, d, J = 8.8 Hz),
7.32 (2H, d, J = 8.8 Hz).
4.2.49. 7-[(4-Methoxyphenyl)(1-methylethyl)amino]-1-methyl-
1,3-dihydro-2H-benzimidazol-2-one (15b)
4.2.44. 1-(4-Methoxybenzyl)-3-methyl-4-[(1-methylethyl)[3-
(methylsulfonyl)phenyl]amino]-1,3-dihydro-2H-benzimidazol-
2-one (14e)
Compound 14e (0.782 g, 73%) was prepared from compound
13e (0.977 g, 2.23 mmol) in a manner similar to that described in
compound 14a. 1H NMR (CDCl3) d 0.98 (3H, d, J = 6.4 Hz), 1.37
(3H, d, J = 6.4 Hz), 3.03 (3H, s), 3.28 (3H, s), 3.79 (3H, s), 4.34–
4.39 (1H, m), 5.04 (2H, s), 6.57 (1H, d, J = 8.0 Hz), 6.77 (1H, d,
J = 8.0 Hz), 6.89 (2H, d, J = 8.6 Hz), 6.96 (1H, d, J = 8.0 Hz), 7.06
(1H, t, J = 8.0 Hz), 7.18–7.29 (3H, m), 7.32 (2H, d, J = 8.6 Hz).
To a mixture of compound 14b (1.80 g, 4.17 mmol) and trifluo-
roacetic acid (45 mL) was stirred at 65 °C for 4 days. The reaction
mixture was concentrated in vacuo. The residue was diluted with
ethyl acetate, washed with aqueous sodium bicarbonate and water
and concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with a 50% ethyl acetate/hexane
mixture and preparative HPLC to give the title compound
(0.408 g, 31%) as an oil. MS calcd: 311; Found: 312 (M+H). 1H
NMR (CDCl3) d 0.90–1.20 (6H, br), 3.33 (3H, s), 3.73 (3H, s), 4.23–
4.28 (1H, m), 6.47–6.69 (2H, m), 6.75–6.80 (3H, m), 6.71–7.07
(2H, m), 8.94 (1H, s).
4.2.45. 1-(4-Methoxybenzyl)-3-methyl-4-[(1-methylethyl)(5-
methylpyridin-2-yl)amino]-1,3-dihydro-2H-benzimidazol-2-
one (14f)
4.2.50. 7-[(4-Chlorophenyl)(1-methylethyl)amino]-1-methyl-
1,3-dihydro-2H-benzimidazol-2-one (15a)
Compound 14f (0.254 g, 74%) was prepared from compound 13f
(0.310 g, 0.828 mmol) and 2-bromopropane (1.31 mL, 1.66 mmol)
in a manner similar to that described in compound 14g. Oil. MS
calcd: 416; Found: 417 (M+H). 1H NMR (CDCl3) d 0.94 (3H, d,
J = 6.8 Hz), 1.35 (3H, d, J = 6.8 Hz), 2.17 (3H, s), 3.27 (3H, s), 3.79
(3H, s), 4.95–5.10 (1H, m), 5.02 (2H, s), 5.86 (1H, d, J = 8.8 Hz),
6.83 (1H, d, J = 8.0 Hz), 6.87 (2H, d, J = 8.4 Hz), 6.93 (1H, d,
J = 8.4 Hz), 7.06 (1H, t, J = 8.4 Hz), 7.09 (1H, d, J = 8.4 Hz), 7.32
(2H, d, J = 8.8 Hz), 8.04 (1H, s).
Compound 15a (0.558 g, 88%) was prepared from compound
14a (0.880 g, 2.03 mmol) in a manner similar to that described in
compound 15b. Oil. MS calcd: 315; Found: 316 (M+H). 1H NMR
(CDCl3) d 0.98 (3H, d, J = 6.4 Hz), 1.36 (3H, d, J = 6.4 Hz), 3.28 (3H,
s), 4.20–4.35 (1H, m), 6.43 (2H, d, J = 8.8 Hz), 6.78–6.85 (1H, m),
7.05–7.20 (4H, m), 9.09 (1H, s).
4.2.51. 1-Methyl-7-[(1-methylethyl)[4-(methylsulfonyl)phenyl]
amino]-1,3-dihydro-2H-benzimidazol-2-one (15d)
Compound 15d (0.333 g, 75%) was prepared from compound
14d (0.590 g, 1.23 mmol) in a manner similar to that described in
compound 15b. 1H NMR (CDCl3) d 0.98 (3H, d, J = 6.3 Hz), 1.41
(3H, d, J = 6.3 Hz), 3.01 (3H, s), 3.21 (3H, s), 4.33–4.39 (1H, m),
6.56 (2H, d, J = 8.8 Hz), 6.80–6.83 (1H, m), 7.14–7.26 (2H, m),
7.69 (2H, d, J = 8.8 Hz), 9.67 (1H, s).
4.2.46. N-(4-Chlorophenyl)-N-[1-(4-methoxybenzyl)-3-methyl-
2-oxo-2,3-dihydro-1H-benzimidazol-4-yl]acetamide (14h)
A mixture of compound 13a (0.393 g, 1.00 mmol), pyridine
(0.1 mL) and acetic anhydride (10 mL) was heated at 120 °C for
4 days. The mixture was concentrated in vacuo. The residue
was diluted with ethyl acetate, washed with saturated sodium

4686
M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4.2.52. 1-Methyl-7-[(1-methylethyl)(5-methylpyridin-2-yl)
amino]-1,3-dihydro-2H-benzimidazol-2-one (15f)
6.43–6.49 (2H, m), 6.72–6.78 (2H, m), 7.04–7.07 (1H, m), 7.28
(1H, t, J = 7.8 Hz), 7.64–7.67 (1H, m).
Compound 15f (0.070 g, 39%) was prepared from compound 14f
(0.250 g, 0.600 mmol) in a manner similar to that described in
compound 15b. MS calcd: 296; Found: 297 (M+H). 1H NMR (CDCl3)
d 0.96 (3H, d, J = 6.8 Hz), 1.37 (3H, d, J = 6.8 Hz), 2.16 (3H, s), 3.26
(3H, s), 5.00–5.15 (1H, m), 5.90 (1H, d, J = 8.4 Hz), 6.87 (1H, t,
J = 4.0 Hz), 7.00–7.15 (3H, m), 8.06 (1H, s), 9.58 (1H, s).
4.2.59. 2-Chloro-1-methyl-N-(1-methylethyl)-N-[4-(1-methyl-
ethyl)oxyphenyl]-1H-benzimidazol-7-amine (10n)
Compound 10n (0.0943 g, 18% in two steps) was prepared from
compound 15c, prepared from compound 14c (0.674 g, 1.47 mmol)
in a manner similar to that described in compound 15b, in a man-
ner similar to that described in compound 10l. Oil. 1H NMR (CDCl3)
d 1.00–1.38 (6H, m), 1.28 (6H, d, J = 6.0 Hz), 3.68 (3H, s), 4.30–4.39
(2H, m), 6.40–6.45 (2H, m), 6.71–6.77 (2H, m), 7.04–7.07 (1H, m),
7.27 (1H, t, J = 7.8 Hz), 7.64–7.67 (1H, m).
4.2.53. 7-[(4-Chlorophenyl)(2,2-dimethylpropyl)amino]-1-
methyl-1,3-dihydro-2H-benzimidazol-2-one (15g)
Compound 15g (0.052 g, 80%) was prepared from compound
14g (0.088 g, 0.190 mmol) in a manner similar to that described
in compound 15b. MS calcd: 343; Found: 344 (M+H). 1H NMR
(CDCl3) d 0.96 (9H, s), 3.25 (3H, s), 3.40 (1H, d, J = 16.0 Hz), 3.83
(1H, d, J = 16.0 Hz), 6.54 (2H, d, J = 8.2 Hz), 6.96 (1H, d, J = 6.0 Hz),
7.04 (1H, d, J = 8.2 Hz), 7.10–7.20 (3H, m), 8.06 (1H, s).
4.2.60. 2-Chloro-1-methyl-N-(1-methylethyl)-N-[4-(methyl-
sulfonyl)phenyl]-1H-benzimidazol-7-amine (10o)
Compound 10o (0.148 g, 47%) was prepared from compound
15d (0.300 g, 0.835 mmol) in a manner similar to that described
in compound 10l. 1H NMR (CDCl3) d 0.97 (3H, d, J = 6.6 Hz), 1.45
(3H, d, J = 6.6 Hz), 3.02 (3H, s), 3.60 (3H, s), 4.38–4.46 (1H, m),
6.55–6.57 (2H, m), 7.02–7.05 (1H, m), 7.34–7.38 (1H, m), 7.65–
7.79 (3H, m).
4.2.54. N-(4-Chlorophenyl)-N-[3-methyl-2-oxo-2,3-dihydro-1H-
benzimidazol-4-yl]acetamide (15h)
Compound 15h (0.200 g, 73%) was prepared from compound
14h (0.380 g, 0.872 mmol) in a manner similar to that described
in compound 15b. 1H NMR (CDCl3) d 2.07 (3H, s), 3.44 (3H, s),
6.85–6.95 (1H, m), 7.05–7.15 (2H, m), 7.25–7.30 (4H, m), 9.25
(1H, br s).
4.2.61. 2-Chloro-1-methyl-N-(1-methylethyl)-N-[3-(methyl-
sulfonyl)phenyl]-1H-benzimidazol-7-amine (10p)
Compound 15e, used for the next step without further purifica-
tion, was prepared from compound 14e (0.780 g, 1.63 mmol) in a
manner similar to that described in compound 15b. MS calcd:
4.2.55. 7-[(4-Chlorophenyl)(2-methoxyethyl)amino]-1-methyl-
1,3-dihydro-2H-benzimidazol-2-one (15i)
359; Found: 360 (M+H). 1H NMR (CDCl3)
d 0.99 (3H, d,
Compound 15i (0.404 g, 71%) was prepared from compound 14i
(0.550 g, 1.22 mmol) in a manner similar to that described in com-
pound 15b. MS calcd: 331; Found: 332 (M+H). 1H NMR (CDCl3) d
3.26 (3H, s), 3.30 (3H, s), 3.55–3.70 (2H, m), 3.70–4.00 (2H, m),
6.51 (2H, d, J = 8.8 Hz), 6.90 (1H, d, J = 8.0 Hz), 7.02 (1H, d,
J = 8.0 Hz), 7.05–7.20 (3H, m), 8.81 (1H, s).
J = 6.2 Hz), 1.39 (3H, d, J = 6.2 Hz), 3.03 (3H, s), 3.24 (3H, s), 4.33–
4.40 (1H, m), 6.58 (1H, d, J = 8.0 Hz), 6.81–6.83 (1H, m), 7.12–
7.29 (5H, m), 9.54 (1H, s). Compound 10p (0.153 g, 23% in two
steps) was prepared from compound 15e in a manner similar to
that described in compound 10l. MS calcd: 377; Found: 378 (M
+H). 1H NMR (CDCl3) d 0.97 (3H, d, J = 6.6 Hz), 1.42 (3H, d,
J = 6.6 Hz), 3.05 (3H, s), 3.62 (3H, s), 4.40–4.47 (1H, m), 6.46–6.49
(1H, m), 7.03 (1H, d, J = 8.0 Hz), 7.24–7.27 (3H, m), 7.33 (1H, t,
J = 8.0 Hz), 7.74 (1H, d, J = 8.0 Hz).
4.2.56. 7-[(4-Chlorophenyl)(tetrahydrofuran-3-ylmethyl)amino]-
1-methyl-1,3-dihydro-2H-benzimidazol-2-one (15j)
Compound 15j (0.150 g, 91%) was prepared from compound 14j
(0.220 g, 0.460 mmol) in a manner similar to that described in
compound 15b. MS calcd: 357; Found: 358 (M+H). 1H NMR (CDCl3)
d 1.80–2.00 (2H, m), 2.60–2.80 (1H, m), 3.25 (3H, s), 3.45–3.60 (2H,
m), 3.60–4.00 (4H, m), 5.50 (2H, d, J = 8.8 Hz), 6.80–7.00 (1H, m),
7.05 (1H, t, J = 8.0 Hz), 7.05–7.20 (3H, m), 9.68 (1H, s).
4.2.62. 2-Chloro-1-methyl-N-(1-methylethyl)-N-(5-methylpyridin-
2-yl)-1H-benzimidazol-7-amine (10q)
Compound 10q (0.048 g, 64%) was prepared from compound
15f (0.070 g, 0.236 mmol) in a manner similar to that described
in compound 10l. Crystal. MS calcd: 314; Found: 315 (M+H). 1H
NMR (CDCl3) d 0.93 (3H, d, J = 6.8 Hz), 1.40 (3H, d, J = 6.8 Hz),
2.17 (3H, s), 3.61 (3H, s), 5.10–5.20 (1H, m), 5.79 (1H, d,
J = 8.4 Hz), 7.08 (2H, d, J = 8.0 Hz), 7.31 (1H, t, J = 8.0 Hz), 7.70
(1H, d, J = 8.0 Hz), 8.07 (1H, s).
4.2.57. 2-Chloro-N-(4-chlorophenyl)-1-methyl-N-(1-methyl-
ethyl)-1H-benzimidazol-7-amine (10l)
A mixture of compound 15a (0.042 g, 0.130 mmol) and phos-
phorous oxychloride (1.5 mL) was stirred at 80 °C for 1.5 h. The
mixture was concentrated in vacuo. The residue was neutralized
with saturated aqueous sodium bicarbonate and extracted with
ethyl acetate. The organic layer was washed with water and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 17% ethyl acetate/hexane mixture
to give the title compound (0.032 g, 72%) as an oil. MS calcd:
4.2.63. 2-Chloro-N-(4-chlorophenyl)-N-(2,2-dimethylpropyl)-1-
methyl-1H-benzimidazol-7-amine (10r)
Compound 10r (0.053 g, 73%) was prepared from compound
15g (0.050 g, 0.145 mmol) in a manner similar to that described
in compound 10l. MS calcd: 361; Found: 362 (M+H). 1H NMR
(CDCl3) d 0.95 (9H, s), 3.47 (1H, d, J = 14.8 Hz), 3.62 (3H, s), 3.93
(1H, d, J = 14.8 Hz), 6.51 (2H, d, J = 8.8 Hz), 7.08 (2H, d, J = 8.8 Hz),
7.20–7.40 (2H, m), 7.60 (1H, d, J = 8.0 Hz).
333; Found: 334 (M+H). 1H NMR (CDCl3)
d 0.95 (3H, d,
J = 6.0 Hz), 1.39 (3H, d, J = 6.0 Hz), 3.63 (3H, s), 4.30–4.40 (1H, m),
6.40 (2H, d, J = 8.8 Hz), 7.03 (1H, d, J = 8.0 Hz), 7.09 (2H, d,
J = 8.8 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.70 (1H, d, J = 8.0 Hz).
4.2.64. N-(2-Chloro-1-methyl-1H-benzimidazol-7-yl)-N-(4-chloro-
phenyl)acetamide (10s)
4.2.58. 2-Chloro-N-(4-methoxyphenyl)-1-methyl-N-(1-methyl-
ethyl)-1H-benzimidazol-7-amine (10m)
Compound 10m (0.394 g, 78%) was prepared from compound
15b (0.480 g, 1.54 mmol) in a manner similar to that described in
compound 10l. MS calcd: 329; Found: 330 (M+H). 1H NMR (CDCl3)
d 1.20–1.22 (6H, m), 3.69 (3H, s), 3.73 (3H, s), 4.28–4.37 (1H, m),
Compound 10s (0.146 g, 69%) was prepared from compound
15h (0.200 g, 0.633 mmol) in a manner similar to that described
in compound 10l. MS calcd: 333; Found: 334 (M+H). 1H NMR
(CDCl3) d 2.05 (3H, s), 3.78 (3H, s), 7.05–7.25 (1H, m), 7.25–7.45
(5H, m), 7.75 (1H, d, J = 8.0 Hz).

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4687
4.2.65. 2-Chloro-N-(4-chlorophenyl)-N-(2-methoxyethyl)-1-
methyl-1H-benzimidazol-7-amine (10t)
ether-hexane to give the title compound (0.0074 g, 9.5%). Solid. MS
calcd: 503; Found: 504 (M+H). 1H NMR (CDCl3) d 1.03 (3H, d,
J = 6.6 Hz), 1.42 (3H, d, J = 6.6 Hz), 2.18 (3H, s), 3.49 (3H, s), 3.81
(3H, s), 4.34–4.40 (1H, m), 5.82 (1H, s), 6.55 (2H, d, J = 8.6 Hz),
6.80 (1H, d, J = 7.8 Hz), 6.93 (1H, s), 7.06 (1H, s), 7.16 (1H, t,
J = 7.8 Hz), 7.42 (2H, d, J = 8.6 Hz), 7.55 (1H, d, J = 7.8 Hz). HPLC:
>99% purity.
Compound 10t (0.240 g, 80%) was prepared from compound 15i
(0.285 g, 0.859 mmol) in a manner similar to that described in
compound 10l. MS calcd: 349; Found: 350 (M+H). 1H NMR (CDCl3)
d 3.27 (3H, s), 3.50–3.65 (2H, m), 3.62 (3H, s), 3.80–4.00 (2H, m),
6.50 (2H, d, J = 8.6 Hz), 7.05–7.20 (3H, m), 7.31 (1H, t, J = 8.0 Hz),
7.65 (1H, d, J = 8.0 Hz).
4.2.70. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(4-chloro-
phenyl)-1-methyl-N7-(1-methylethyl)-1H-benzimidazole-2,7-
diamine (16a)
4.2.66. 2-Chloro-N-(4-chlorophenyl)-1-methyl-N-(tetrahydro-
furan-3-ylmethyl)-1H-benzimidazol-7-amine (10u)
Compound 10u (0.089 g, 56%) was prepared from compound
15j (0.150 g, 0.419 mmol) in a manner similar to that described
in compound 10l. Oil. MS calcd: 375; Found: 376 (M+H). 1H NMR
(CDCl3) d 1.50–1.70 (1H, m), 1.95–2.10 (1H, m), 2.60–2.75 (1H,
m), 3.59 (3H, s), 3.50–3.65 (2H, m), 3.65–3.80 (2H, m), 3.80–4.00
(2H, m), 6.49 (2H, d, J = 8.8 Hz), 7.05–7.20 (3H, m), 7.32 (1H, t,
J = 8.0 Hz), 7.66 (1H, d, J = 8.0 Hz).
Compound 16a (0.148 g, 53%) was prepared from compound
10l (0.200 g, 0.598 mmol) and compound 17a (0.310 g, 1.80 mmol)
in a manner similar to that described in compound 16m. Solid. MS
calcd: 468; Found: 469 (M+H). 1H NMR (CDCl3) d 1.02 (3H, d,
J = 4.5 Hz), 1.39 (3H, d, J = 4.5 Hz), 2.19 (3H, s), 3.55 (3H, s), 3.81
(3H, s), 4.25–4.40 (1H, m), 5.81 (1H, s), 6.47 (2H, d, J = 8.8 Hz),
6.78 (1H, s), 6.81 (1H, d, J = 8.0 Hz), 6.90 (1H, s), 7.10 (1H, d,
J = 8.8 Hz), 7.13 (1H, t, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz).
4.2.67. 4-Chloro-2-methoxy-6-methylaniline (17a)
To a solution of 4-chloro-2-methylaniline (98.8 g, 697 mmol) in
acetonitrile (400 mL) was added N-bromosuccinimide (137 g,
767 mmol), and the mixture was stirred at 0 °C for 90 min. The
reaction mixture was concentrated in vacuo. The residue was
diluted with saturated aqueous sodium hydrogen carbonate and
extracted with ethyl acetate. The organic layer was washed with
brine and concentrated in vacuo to give 2-bromo-4-chloro-6-
methylaniline (154 g, >99%) as a brown powder. MS calcd: 219;
Found: 220 (M+H). 1H NMR (CDCl3) d 2.18 (3H, s), 4.04 (2H, br s),
6.99 (1H, d, J = 1.2 Hz), 7.29 (1H, d, J = 1.2 Hz). To a solution of 2-
bromo-4-chloro-6-methylaniline (45.0 g, 204 mmol) in methanol
(30 mL) were added sodium methoxide (28% solution in methanol,
225 mL) and copper iodide (44.7 g, 234 mmol), and the mixture
was stirred at 100 °C for 2 h. The reaction mixture was diluted with
saturated aqueous ammonium chloride and extracted with ethyl
acetate. The organic layer was washed with brine and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluting with a 0–10% ethyl acetate/hexane gradient mixture
to give the title compound (26.4 g, 75%) as a brown solid. MS calcd:
171; Found: 172 (M+H). 1H NMR (CDCl3) d 2.13 (3H, s), 3.70 (2H, br
s), 3.82 (3H, s), 6.66 (1H, d, J = 2.1 Hz), 6.69 (1H, d, J = 2.1 Hz).
4.2.71. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(4-chloro-
phenyl)-N7-(2,2-dimethylpropyl)-1-methyl-1H-benzimidazole-
2,7-diamine Hydrochloride (16b)
A mixture of compound 10r (0.039 g, 0.106 mmol) and com-
pound 17a (0.055 g, 0.320 mmol) was stirred at 120 °C for 26 h.
The reaction mixture was dissolved in ethyl acetate, washed with
saturated sodium hydrogen carbonate and water and concentrated
in vacuo. The residue was purified by preparative HPLC. The frac-
tion was concentrated in vacuo. The residue was dissolved in
methanol (5 mL), treated with 2 M hydrochloride in diethyl ether
(2 mL) and concentrated in vacuo to give the title compound
(0.012 g, 22%). Solid. MS calcd: 496; Found: 497 (M+H). 1H NMR
(CDCl3) d 0.93 (9H, s), 2.38 (3H, s), 3.10 (3H, s), 3.42 (1H, d,
J = 14.8 Hz), 3.62 (3H, s), 3.90 (1H, d, J = 14.8 Hz), 6.51 (2H, d,
J = 8.0 Hz), 6.73 (1H, s), 6.87 (1H, s), 7.10 (2H, d, J = 8.0 Hz), 7.20–
7.40 (1H, m), 7.42 (1H, s), 10.47 (1H, s), 13.35 (1H, s).
4.2.72. N-[2-[(4-Chloro-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl]-N-(4-chlorophenyl)acetamide
hydrochloride (16c)
Compound 16c (0.116 g, 53%) was prepared from compound
10s (0.145 g, 0.434 mmol) in a manner similar to that described
in compound 16b. Colorless solid. Mp: 176–178 °C. MS calcd:
468; Found: 469 (M+H). 1H NMR (CDCl3) d 2.11 (3H, s), 2.37 (3H,
s), 3.48 (3H, s), 3.65 (3H, s), 6.76 (1H, s), 6.82 (1H, s), 7.00–7.20
(1H, m), 7.20–7.30 (2H, m), 7.30–7.45 (3H, m), 7.50–7.65 (1H, m),
10.80 (1H, s). HPLC: >96% purity.
4.2.68. 4-Bromo-2-methoxy-6-methylaniline (17b)
To a solution of 2-methoxy-6-methylaniline (25.0 g, 182 mmol)
in acetic acid (30 mL) and methanol (60 mL) was added a solution
of bromine (9.34 mL, 182 mmol) in acetic acid (60 mL) at 0 °C, and
the mixture was stirred at room temperature for 2 h. The precipi-
tate was collected by filtration, washed with diethyl ether and dis-
solved in ethyl acetate and saturated aqueous sodium hydrogen
carbonate. The organic layer was separated, and the aqueous layer
was extracted with ethyl acetate. The combined organic layer was
washed with brine and concentrated in vacuo to give the title com-
pound (20.7 g, 53%) as a brown solid. MS calcd: 215; Found: 216
(M+H). 1H NMR (CDCl3) d 2.14 (3H, s), 3.83 (3H, s), 6.80 (1H, s),
6.84 (1H, s).
4.2.73. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(4-chloro-
phenyl)-N7-(2-methoxyethyl)-1-methyl-1H-benzimidazole-2,7-
diamine Hydrochloride (16d)
Compound 16d (0.059 g, 33%) was prepared from compound
10t (0.120 g, 0.343 mmol) in a manner similar to that described
in compound 16b. Colorless solid. Mp: 131–133 °C. MS calcd:
484; Found: 485 (M+H). 1H NMR (CDCl3) d 2.38 (3H, s), 3.07 (3H,
s), 3.25 (3H, s), 3.50–3.60 (2H, br), 3.61 (3H, s), 3.80–4.00 (2H,
m), 6.49 (2H, d, J = 8.0 Hz), 6.71 (1H, s), 6.86 (1H, s), 7.12 (2H, d,
J = 8.0 Hz), 7.14 (1H, d, J = 8.0 Hz), 7.35–7.50 (2H, m), 10.59 (1H,
s). HPLC: >98% purity.
4.2.69. 4-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]benzonitrile
(16m)
A mixture of compound 10d (0.050 g, 0.154 mmol) and com-
pound 17b (0.100 g, 0.460 mmol) was stirred at 120 °C for 3 days.
The mixture was dissolved in ethyl acetate, washed with saturated
aqueous sodium bicarbonate and water and concentrated in vacuo.
The residue was purified by silica gel chromatography eluting with
a 33% ethyl acetate/hexane mixture. The desired fraction was con-
centrated in vacuo, and the residual solid was washed with diethyl
4.2.74. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(4-chloro-
phenyl)-1-methyl-N7-(tetrahydrofuran-3-ylmethyl)-1H-benzimi-
dazole-2,7-diamine (16e)
Compound 16e (0.061 g, 53%) was prepared from compound
10u (0.085 g, 0.226 mmol) and compound 17a (0.12 g,
0.680 mmol) in a manner similar to that described in compound

4688
M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
16m. Colorless crystal. Mp: 212–214 °C. MS calcd: 510; Found: 511
(M+H). 1H NMR (CDCl3) d 1.50–1.70 (1H, m), 2.00–2.15 (1H, m),
2.18 (3H, s), 2.65–2.80 (1H, m), 3.51 (3H, s), 3.50–3.70 (2H, m),
3.81 (3H, s), 3.65–4.00 (4H, m), 5.81 (1H, s), 6.56 (2H, d,
J = 8.0 Hz), 6.79 (1H, s), 6.85–7.00 (2H, m), 7.13 (2H, d, J = 8.0 Hz),
7.10–7.20 (1H, m), 7.47 (1H, d, J = 6.8 Hz). Anal. Calcd for C27H28N4-
O2Cl2: C, 63.41; H, 5.52; N, 10.95. Found: C, 63.28; H, 5.57; N, 10.68.
in compound 16g. Solid. mp: 202–203 °C. MS Calcd: 492; Found:
493 (M+H). 1H NMR (CDCl3) d 1.09–1.28 (6H, m), 1.29 (6H, d,
J = 6.6 Hz), 2.18 (3H, s), 3.60 (3H, s), 3.81 (3H, s), 4.25–4.41 (2H,
m), 5.82 (1H, br s), 6.48–6.51 (2H, m), 6.72–6.78 (3H, m), 6.84–
6.89 (2H, m), 7.11 (1H, t, J = 7.8 Hz), 7.47 (1H, d, J = 7.8 Hz).
4.2.79. N2-(4-Chloro-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-[3-(1-methylethyl)oxyphenyl]-1H-benzimi-
dazole-2,7-diamine (16j)
4.2.75. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(4-methoxy-
phenyl)-1-methyl-N7-(1-methylethyl)-1H-benzimidazole-2,7-
diamine (16g)
Compound 16j (0.0237 g, 26%) was prepared from compound
10c (0.0670 g, 0.187 mmol) in a manner similar to that described
in compound 16g. Solid. Mp: 192–193 °C. MS calcd: 492; Found:
493 (M+H). 1H NMR (CDCl3) d 0.99 (3H, d, J = 6.6 Hz), 1.25–1.29
(6H, m), 1.39 (3H, d, J = 6.6 Hz), 2.18 (3H, s), 3.56 (3H, s), 3.80
(3H, s), 4.28–4.47 (2H, m), 5.82 (1H, br s), 6.10–6.13 (2H, m),
6.23–6.26 (1H, m), 6.77–6.78 (1H, m), 6.84 (1H, d, J = 7.8 Hz),
6.88–6.89 (1H, m), 7.07 (1H, t, J = 9.0 Hz), 7.11 (1H, t, J = 8.1 Hz),
7.48 (1H, d, J = 8.1 Hz).
A mixture of compound 10m (0.291 g, 0.881 mmol), compound
17a (0.454 g, 2.64 mmol) and N-methyl-2-pyrrolidinone (0.3 mL)
was stirred at 120 °C for 18 h. The mixture was poured into saturated
aqueous sodium bicarbonate and extracted with ethyl acetate. The
organic layer was washed with water and concentrated in vacuo.
The residue was purified by silica gel column chromatography elut-
ing with a 50% ethyl acetate/hexane mixture. The desired fraction
was concentrated in vacuo. The residue was powdered from diethyl
ether to give the title compound (0.359 g, 65%) as a colorless powder.
mp: 213–214 °C. MS calcd: 464; Found: 465 (M+H). 1H NMR (CDCl3)
d 1.21–1.27 (6H, m), 2.18 (3H, s), 3.61 (3H, s), 3.74 (3H, s), 3.81 (3H,
s), 4.25–4.35 (1H, m), 5.82 (1H, br s), 6.50–6.54 (2H, m), 6.74–6.78
(3H, m), 6.83–6.89 (2H, m), 7.11 (1H, t, J = 7.5 Hz), 7.47 (1H, d,
J = 7.2 Hz). 13C NMR (DMSO-d6) d 155.4, 153.3, 150.8, 144.1, 142.9,
137.9, 132.7, 129.9, 126.5, 126.0, 122.0, 121.7, 121.0, 114.7, 114.6,
109.6, 55.9, 55.1, 48.2, 29.9, 20.6, 17.7. HPLC: 97% purity.
4.2.80. N2-(4-Bromo-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-[4-(methylsulfonyl)phenyl]-1H-benzimidazole-
2,7-diamine (16k)
Compound 16k (0.0555 g, 36%) was prepared from compound
10o (0.104 g, 0.275 mmol) in a manner similar to that described
in compound 16m. Colorless solid. Mp: 154–155 °C. MS calcd:
556; Found: 557 (M+H). 1H NMR (CDCl3)
d 1.04 (3H, d,
J = 6.4 Hz), 1.44 (3H, d, J = 6.4 Hz), 2.19 (3H, s), 3.01 (3H, s), 3.49
(3H, s), 3.81 (3H, s), 4.38–4.46 (1H, m), 5.83 (1H, s), 6.61 (2H, d,
J = 8.8 Hz), 6.80 (1H, d, J = 8.0 Hz), 6.93 (1H, s), 7.06 (1H, s), 7.17
(1H, t, J = 8.0 Hz), 7.55 (1H, d, J = 8.0 Hz), 7.69 (2H, d, J = 8.8 Hz).
Anal. Calcd for C26H29N4O3SBr: C, 56.01; H, 5.24; N, 10.05. Found:
C, 56.18; H, 5.49; N, 9.68.
4.2.76. N2-(4-Chloro-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-phenyl-1H-benzimidazole-2,7-diamine (16f)
Compound 16f (0.0427 g, 27%) was prepared from compound 10a
(0.111 g, 0.367 mmol) in a manner similar to that described in com-
pound 16g. Colorless solid. Mp: 230–231 °C. MS calcd: 434; Found:
435(M+H). 1H NMR (CDCl3) d 1.03 (3H, br s), 1.39 (3H, br s), 2.18
(3H, s), 3.56 (3H, s), 3.80 (3H, s), 4.32–4.42 (1H, m), 5.82 (1H, br s),
6.56 (2H, d, J = 8.4 Hz), 6.67–6.71 (1H, m), 6.77–6.78 (1H, m), 6.84
(1H, d, J = 7.5 Hz), 6.88–6.89 (1H, m), 7.10–7.19 (3H, m), 7.49 (1H,
d, J = 7.5 Hz). Anal. Calcd for C25H27N4OClꢂ0.3H2O: C, 68.19; H,
6.32; N, 12.72. Found: C, 68.19; H, 6.22; N, 12.51. HPLC: 96% purity.
4.2.81. N2-(4-Bromo-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-[3-(methylsulfonyl)phenyl]-1H-benzimidazole-
2,7-diamine (16l)
Compound 16l (0.143 g, 64%) was prepared from compound
10p (0.150 g, 3.97 mmol) in a manner similar to that described in
compound 16m. Colorless solid. Mp: 240–242 °C. MS calcd: 556;
Found: 557 (M+H). 1H NMR (CDCl3) d 1.03 (3H, d, J = 6.2 Hz), 1.42
(3H, d, J = 6.2 Hz), 2.18 (3H, s), 3.05 (3H, s), 3.53 (3H, s), 3.81 (3H,
s), 4.39–4.45 (1H, m), 5.83 (1H, s), 6.58 (1H, d, J = 7.8 Hz), 6.80
(1H, d, J = 7.8 Hz), 6.92 (1H, s), 7.05 (1H, s), 7.16 (1H, t,
J = 7.8 Hz), 7.22–7.29 (3H, m), 7.53 (1H, d, J = 7.8 Hz). Anal. Calcd
for C26H29N4O3SBr: C, 56.01; H, 5.24; N, 10.05. Found: C, 55.96;
H, 5.31; N, 9.87. HPLC: >99% purity.
4.2.77. N2-(4-Bromo-2-methoxy-6-methylphenyl)-N7-(3-meth-
oxyphenyl)-1-methyl-N7-(1-methylethyl)-1H-benzimidazole-
2,7-diamine (16h)
A mixture of compound 10b (0.100 g, 0.303 mmol), compound
17b (0.197 g, 0.910 mmol) and N-methyl-2-pyrrolidinone (1.0 mL)
was stirred at 120 °C for 2 h under microwave irradiation. The mix-
ture was poured into water and extracted with ethyl acetate. The
organic layer was washed with water and concentrated in vacuo.
The residue was purified by preparative HPLC. The desired fraction
was diluted with saturated aqueous sodium hydrogen carbonate
and extracted with ethyl acetate. The organic layer was washed with
water and concentrated in vacuo. The residue was crystallized from
diethyl ether to give the title compound (0.0155 g, 10%) as a solid.
MS calcd: 508; Found: 509 (M+H). 1H NMR (CDCl3) d 1.00 (3H, d,
J = 6.6 Hz), 1.39 (3H, d, J = 6.6 Hz), 2.17 (3H, s), 3.56 (3H, s), 3.72
(3H, s), 3.81 (3H, s), 4.30–4.36 (1H, m), 5.85 (1H, br s), 6.14–6.16
(2H, m), 6.26–6.28 (1H, m), 6.83 (1H, d, J = 7.8 Hz), 6.91–6.92 (1H,
m), 7.04–7.15 (2H, m), 7.26 (1H, s), 7.48–7.61 (1H, m).
4.2.82. 3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]benzo-
nitrile (16n)
Compound 16n (0.507 g, 69%) was prepared from compound
10e (0.475 g, 1.46 mmol) in a manner similar to that described in
compound 16m. Purple solid. Mp: 248–250 °C. MS calcd: 503;
Found: 504 (M+H). 1H NMR (CDCl3) d 1.03 (3H, t, J = 6.4 Hz), 1.40
(3H, t, J = 6.4 Hz), 2.20 (3H, s), 3.52 (3H, s), 3.81 (3H, s), 4.29–4.35
(1H, m), 5.84 (1H, s), 6.71 (1H, d, J = 7.6 Hz), 6.79 (1H, d,
J = 7.6 Hz), 6.83 (1H, s), 6.93 (1H, s), 6.96 (1H, d, J = 7.6 Hz), 7.06
(1H, s), 7.14–7.22 (2H, m), 7.54 (1H, d, J = 7.6 Hz). Anal. Calcd for
C
26H26N5OBrꢂ0.5H2O: C, 60.82; H, 5.30; N, 13.64. Found: C, 61.03;
H, 5.20; N, 13.29.
4.2.78. N2-(4-Chloro-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-[4-(1-methylethyl)oxyphenyl]-1H-benzimi-
dazole-2,7-diamine (16i)
4.2.83. N2-(4-Chloro-2-methoxy-6-methylphenyl)-N7-(6-methoxy-
pyridin-3-yl)-1-methyl-N7-(1-methylethyl)-1H-benzimidazole-2,
7-diamine (16t)
Compound 16i (0.0266 g, 36%) was prepared from compound
10n (0.0543 g, 0.152 mmol) in a manner similar to that described
Compound 16t (0.0319 g, 32%) was prepared from compound
10g (0.070 g, 0.212 mmol) in a manner similar to that described

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4689
in compound 16g. Colorless solid. Mp: 215–216 °C. MS calcd: 465;
Found: 466 (M+H). 1H NMR (CDCl3) d 1.10–1.35 (6H, m), 2.19 (3H,
s), 3.63 (3H, s), 3.81 (3H, s), 3.86 (3H, s), 4.21–4.33 (1H, m), 5.83
(1H, br s), 6.57–6.60 (1H, m), 6.78–6.91 (4H, m), 7.11 (1H, t,
J = 8.1 Hz), 7.47 (1H, d, J = 8.1 Hz), 7.58–7.59 (1H, m). HPLC: 97%
purity.
mixture was diluted with water and extracted with ethyl acetate.
The combined organic layer was washed with brine and concen-
trated in vacuo. The resulting solid was collected by filtration
and washed with dichloromethane to give the title compound
(0.016 g). The filtrate was concentrated in vacuo, and the residue
was purified by silica gel column chromatography eluting with a
5% methanol/dichloromethane mixture. The desired fraction was
concentrated in vacuo. The resulting solid was washed with
dichloromethane to give the title compound (0.014 g). The filtrate
was concentrated in vacuo, and the residue was crystallized from
dichloromethane to give the title compound (0.006 g) as a crystal.
Total: 0.035 g, 38%. MS calcd: 521; Found: 522 (M+H). 1H NMR
(DMSO-d6) d 0.93 (3H, d, J = 5.8 Hz), 1.31 (3H, d, J = 5.8 Hz), 2.08
(3H, s), 3.47 (3H, s), 3.74 (3H, s), 4.43–4.48 (1H, m), 5.95 (1H, s),
6.49 (1H, d, J = 6.8 Hz), 6.69 (1H, d, J = 8.0 Hz), 7.03–7.19 (6H, m),
7.25 (1H, s), 7.84 (1H, s), 7.93 (1H, s). Anal. Calcd for C26H28N5O2-
Brꢂ0.3H2O: C, 59.16; H, 5.46; N, 13.27. Found: C, 59.40; H, 5.43;
N, 12.89.
4.2.84. N2-(4-Chloro-2-methoxy-6-methylphenyl)-1-methyl-N7-
(1-methylethyl)-N7-(5-methylpyridin-2-yl)-1H-benzimidazole-
2,7-diamine (16u)
Compound 16u (0.028 g, 41%) was prepared from compound
10q (0.047 g, 0.149 mmol) and compound 17a (0.077 g,
0.449 mmol) in a manner similar to that described in compound
16m. Colorless solid. Mp: 245–246 °C. MS Calcd: 449; Found:
450 (M+H). 1H NMR (CDCl3) d 1.00 (3H, d, J = 6.8 Hz), 1.39 (3H, d,
J = 6.8 Hz), 2.18 (6H, s), 3.53 (3H, s), 3.80 (3H, s), 5.10–5.20 (1H,
m), 5.91 (1H, d, J = 8.8 Hz), 5.90 (1H, br s), 6.78 (1H, s), 6.87 (1H,
d, J = 8.0 Hz), 6.89 (1H, s), 7.11 (1H, d, J = 8.0 Hz), 7.16 (1H, t,
J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz), 8.07 (1H, s). Anal. Calcd for
C
25H28N5OCl: C, 66.73; H, 6.27; N, 15.56. Found: C, 66.51; H,
4.2.88. 3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]benzoic
acid (16x)
6.36; N, 15.40.
4.2.85. 2-[3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-
1-methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]phenyl]-
2-methylpropanenitrile (16v)
A mixture of compound 16o (0.266 g, 0.495 mmol), 1 N aqueous
sodium hydroxide (3 mL) and tetrahydrofuran (3 mL) was stirred
at room temperature for 24 h. The reaction mixture was diluted
with water and adjusted to pH 4.5 with hydrochloric acid. The
aqueous layer was extracted with ethyl acetate. The organic layer
was washed with brine and concentrated in vacuo. The residue
was purified by silica gel column chromatography eluting with a
10% methanol/dichloromethane mixture to give the title com-
pound (0.195 g, 75%) as a solid. MS calcd: 522; Found: 523 (M
+H). 1H NMR (CDCl3) d 0.94 (3H, d, J = 6.0 Hz), 1.32 (3H, d,
J = 6.0 Hz), 2.08 (3H, s), 3.47 (3H, s), 3.73 (3H, s), 4.40–4.45 (1H,
m), 6.70–6.74 (2H, m), 6.92–7.10 (4H, m), 7.10–7.23 (3H, m),
7.95 (1H, s), 12.72 (1H, s).
Compound 16v (0.270 g, 74%) was prepared from compound
10f (0.250 g, 0.680 mmol) and compound 17b (0.440 g, 2.04 mmol)
in a manner similar to that described in compound 16g. Solid. Mp:
227–229 °C. MS calcd: 545; Found: 546 (M+H). 1H NMR (CDCl3) d
1.04 (3H, d, J = 6.6 Hz), 1.42 (3H, d, J = 6.6 Hz), 1.65 (3H, s), 1.67
(3H, s), 2.16 (3H, s), 3.55 (3H, s), 3.81 (3H, s), 4.35–4.50 (1H, m),
5.82 (1H, s), 6.35 (1H, d, J = 6.9 Hz), 6.75–6.80 (2H, m), 6.83 (1H,
d, J = 8.4 Hz), 6.92 (1H, s), 7.09 (1H, s), 7.05–7.20 (2H, m), 7.52
(1H, d, J = 8.1 Hz).
4.2.86. Methyl 3-[[2-[(4-bromo-2-methoxy-6-methylphenyl)
amino]-1-methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]
benzoate (16o)
4.2.89. 3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]-N-methyl-
benzamide (16q)
To a solution of compound 16n (0.337 g, 0.668 mmol) in metha-
nol (5 mL) was bubbled hydrogen chloride gas for 10 min., and the
mixture was stirred at room temperature for 15 h. After hydrogen
chloride and the solvent were removed in vacuo, the residue was
diluted with tetrahydrofuran and water. The mixture was stirred
at room temperature for 24 h. The reaction mixture was diluted
with water and extracted with ethyl acetate. The organic layer
was washed with brine and concentrated in vacuo. The residue
was purified by silica gel column chromatography eluting with a
3% methanol/dichloromethane mixture to give the title compound
(0.359 g, 77%) as a solid. MS calcd: 536; Found: 537 (M+H). 1H NMR
(CDCl3) d 1.03 (3H, d, J = 6.2 Hz), 1.41 (3H, d, J = 6.2 Hz), 2.18 (3H,
s), 3.54 (3H, s), 3.81(3H, s), 3.88 (3H, s), 4.41–4.47 (1H, m), 5.81
(1H, s), 6.56 (1H, d, J = 8.0 Hz), 6.83 (1H, d, J = 8.0 Hz), 6.92 (1H,
s), 7.05 (1H, s), 7.13–7.17 (2H, m), 7.37 (1H, d, J = 8.0 Hz), 7.42
(1H, s), 7.52 (1H, d, J = 8.0 Hz). Anal. Calcd for C27H29N4O3Br: C,
60.34; H, 5.44; N, 10.42. Found: C, 60.34; H, 5.40; N, 10.57.
To a suspension of compound 16x (0.080 g, 0.153 mmol) in
tetrahydrofuran (1 mL), were added O-(benzotriazol-1-yl)-N,N,N0,
N0-tetramethyluronium hexafluorophosphate (HBTU) (0.120 g,
0.310 mmol) and diisopropylethylamine (0.027 mL, 0.153 mmol),
and the mixture was stirred at room temperature for 30 min. Addi-
tion of 2 M methylamine in tetrahydrofuran (0.11 mL, 0.23 mmol)
was followed by stirring at room temperature for 5 h. The reaction
mixture was diluted with water and extracted with ethyl acetate.
The organic layer was and concentrated in vacuo. The residue
was purified by silica gel column chromatography eluting with a
10% methanol/dichloromethane mixture. The desired fraction
was concentrated in vacuo. The resulting crystals were washed
with dichloromethane-diethyl ether to give the title compound
(0.043 g, 52%). Colorless solid. Mp: 261–263 °C. MS calcd: 535;
Found: 536 (M+H). 1H NMR (CDCl3) d 1.01 (3H, d, J = 6.2 Hz), 1.40
(3H, d, J = 6.2 Hz), 2.18 (3H, s), 2.98 (3H, d, J = 4.8 Hz), 3.53 (3H,
s), 3.80 (3H, s), 4.42–4.49 (1H, m), 5.86 (1H, br s), 5.98 (1H, br s),
6.53 (1H, d, J = 8.0 Hz), 6.83 (1H, d, J = 8.0 Hz), 6.92 (1H, s), 6.96
(1H, d, J = 8.0 Hz), 7.05 (1H, s), 7.12–7.16 (3H, m), 7.51 (1H, d,
J = 8.0 Hz). Anal. Calcd for C27H30N5O2Brꢂ0.5H2O: C, 59.45; H,
5.73; N, 12.84. Found: C, 59.65; H, 5.56; N, 12.83. HPLC: 96% purity.
4.2.87. 3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]benzamide
(16p)
To a solution of compound 16n (0.090 g, 0.178 mmol) in etha-
nol (1 mL) were added 30% aqueous hydrogen peroxide
(0.064 mL, 0.624 mmol) and 10 N aqueous sodium hydroxide
(0.00428 mL, 0.0428 mmol), and the mixture was refluxed for
24 h. Addition of 30% aqueous hydrogen peroxide (0.064 mL,
0.624 mmol) and 10 N aqueous sodium hydroxide (0.00428 mL,
0.0428 mmol) was followed by refluxing for 60 h. The reaction
4.2.90. 3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-1-
methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]-N,N-dimethyl-
benzamide (16r)
To a suspension of compound 16x (0.080 g, 0.153 mmol) in
tetrahydrofuran (1 mL) were added HBTU (0.120 g, 0.310 mmol)

4690
M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
and diisopropylethylamine (0.040 mL, 0.23 mmol), and the mix-
ture was stirred at room temperature for 30 min. Addition of 2 M
dimethylamine in tetrahydrofuran (0.11 mL, 0.23 mmol) was fol-
lowed by stirring at room temperature for 5 h. The reaction mix-
ture was diluted with water and extracted with ethyl acetate.
The organic layer was and concentrated in vacuo. The resulting
crystals were washed with ethyl acetate to give the title compound
(0.050 g, 59%). Colorless solid. Mp: 271–272 °C. MS calcd: 549;
Found: 550 (M+H). 1H NMR (CDCl3) d 1.01 (3H, d, J = 6.6 Hz), 1.39
(3H, d, J = 6.6 Hz), 2.19 (3H, s), 2.91 (3H, s), 3.06 (3H, s), 3.56 (3H,
s), 3.81 (3H, s), 4.34–4.43 (1H, m), 5.82 (1H, s), 6.52 (1H, d,
J = 8.0 Hz), 6.92 (1H, s), 6.72 (1H, d, J = 8.0 Hz), 6.82 (1H, d,
J = 8.0 Hz), 6.92 (1H, s), 7.04 (1H, s), 7.12 (1H, d, J = 8.0 Hz), 7.16
(1H, d, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz). Anal. Calcd for C28H32N5-
O2Br: C, 61.09; H, 5.86; N, 12.72. Found: C, 60.94; H, 5.77; N, 12.65.
HPLC: 96% purity.
and 50 pM of 125I-CRF in binding assay buffer. The reaction was
performed at rt for 1.5 h, the membrane was collected on a glass
filter (UniFilter plate GF-C/Perkin Elmer) using suction filtration
and a cell harvester (Perkin Elmer), and it was washed with ice-
cold 50 mM Tris–HCl (pH 7.5). After drying the glass filter, a liquid
scintillation cocktail (Microscinti; Perkin Elmer) was added, and
the radioactivity of 125I-CRF remaining on the glass filter was mea-
sured using a TopCount (Perkin Elmer). The percent inhibition was
calculated using the following equation:
%Inhibition ¼ ðBound ꢀ NSBÞ=ðTB ꢀ NSBÞ ꢁ 100;
where Bound is the radioactivity when a compound is added, TB is
the total binding radioactivity, and NSB is the non-specifically
bound radioactivity. IC50 values and 95% confidence intervals were
calculated using GraphPad Prism software.
4.3.2. CRF1 antagonistic activity
CRF1 antagonistic activity was obtained by measuring inhibition
of adenylate cyclase using a CRE-luciferase reporter assay. CHO
cells expressing the human CRF1 receptor and the CRE-luciferase
gene were added to the wells of a 96-well plate (40,000 cells/well)
and incubated for 24 h. After cultivation, the culture medium was
removed, and the cells were treated for 4 h with various drug con-
4.2.91. 2-[3-[[2-[(4-Bromo-2-methoxy-6-methylphenyl)amino]-
1-methyl-1H-benzimidazol-7-yl](1-methylethyl)amino]phenyl]-
2-methylpropanamide (16s)
A mixture of compound 16v (0.100 g, 0.183 mmol), ethanol
(10 mL) and concentrated sulfuric acid (4 mL) was refluxed for
48 h. The reaction mixture was poured into aqueous sodium
hydrogen carbonate and extracted with ethyl acetate. The organic
layer was washed with water and concentrated in vacuo. The resi-
due was dissolved in methanol (5 mL) and tetrahydrofuran (2 mL),
followed by addition of 8 N aqueous sodium hydroxide (1 mL).
After stirring at 70 °C for 3 h, the reaction mixture was acidified
with 1 N hydrochloric acid and extracted with ethyl acetate. The
organic layer was washed with water and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with a 5% methanol/dichloromethane mixture to give com-
pound 16y (0.051 g, 49%) as a solid. MS Calcd: 564; Found: 565 (M
+H). To a solution of compound 16y (0.047 g, 0.081 mmol) in N,N-
dimethylformamide (0.5 mL) were added 1-[3-(dimethylamino)
propyl]-3-ethylcarbodiimide hydrochloride (WSC) (0.029 g,
0.166 mmol) and 1-hydroxybenzotriazole monohydrate (HOBT)
(0.029 g, 0.166 mmol), and the mixture was stirred at room tem-
perature for 3 h. The reaction mixture was diluted with dichloro-
methane and aqueous ammonia, and the mixture was stirred for
1 h. The reaction mixture was diluted with ethyl acetate. The
organic layer was washed with water and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with a 5% methanol/dichloromethane mixture to give the
title compound (0.032 g, 69%) as a colorless solid. Mp: 234–
236 °C. MS calcd: 564; Found: 565 (M+H). 1H NMR (CDCl3) d 1.04
(3H, d, J = 6.3 Hz), 1.40 (3H, d, J = 6.3 Hz), 1.58 (6H, s), 2.16 (3H,
s), 3.52 (3H, s), 3.81 (3H, s), 4.30–4.45 (1H, m), 5.15–5.30 (2H,
m), 5.75–5.90 (1H, m), 6.37 (1H, d, J = 7.5 Hz), 6.63 (1H, s), 6.73
(1H, d, J = 8.1 Hz), 6.83 (1H, d, J = 7.5 Hz), 6.92 (1H, s), 7.04
(1H, s), 7.05–7.20 (2H, m), 7.50 (1H, d, J = 7.8 Hz). Anal. Calcd for
centrations in 100 lL of assay buffer [20 mM HEPES, Ham F-12, and
0.1% BSA (pH 7.2)] containing 1 nM human CRF. After exposure to
the test compounds, the cells were lysed and luciferase activity
was measured using a Steady-GloÒ luciferase assay system (Pro-
mega). Light emission was detected using an ARVO-SX (Wallac).
IC50 values and 95% confidence intervals were calculated using
GraphPad Prism software.
4.4. Ex vivo study
4.4.1. Preparation of brain membrane homogenates
Mice were sacrificed by decapitation, and their brains were
rapidly removed and homogenized at 4 °C using a Physcotron
homogenizer (setting, 10 s) in lysis buffer [50 mM Tris–HCl (pH
7.0), 10 mM MgCl2, 2 mM EDTA, and 100 KU/mL aprotinin]. The
frontal cortex was diluted with lysis buffer to a final concentration
of 5 mg wet tissue/mL. The olfactory bulb was homogenized in
5 mL of lysis buffer and diluted to 1/5 with lysis buffer. The pitu-
itary was homogenized in 2.5 mL of lysis buffer and diluted with
lysis buffer to a final concentration of 5 mg wet tissue/mL.
Animals were handled according to the procedures approved by
the Animal Experiment Ethics Committee of Takeda Pharmaceuti-
cal Company Ltd.
4.4.2. Drugs
The compounds were suspended in 0.5% methyl cellulose (MC;
ShinEtsu) in water and administered orally in a volume of 10 mL/
kg for use in the ex vivo binding assay described below.
C29H34N5O2Br: C, 61.70; H, 6.07; N, 12.41. Found: C, 61.35; H,
4.4.3. Ex vivo binding assay in mice
6.02; N, 12.17.
Test compounds or the corresponding vehicle was orally admin-
istered to mice (10 per group) 1 h before decapitation and organ
(frontal cortex, olfactory bulb, and pituitary) removal. The tissues
were homogenized in ice-cold lysis buffer using a Physcotron
homogenizer and diluted as described above. 125I-CRF (ovine)
binding was performed with membrane homogenates in the pres-
ence of 100 pM of 125I-CRF (ovine) in lysis buffer containing 0.1%
4.3. In vitro study
4.3.1. Measurement of the rate of inhibition of CRF1 binding
A receptor-binding experiment was performed using a human
CRF1 receptor expressing a CHO cell membrane fraction and ovine
CRF, 125I-Tyr0 125I-CRF). Various concentrations of a test com-
(
BSA, 0.5% DMSO, and 0.05% CHAPS in a final volume of 200 lL.
pound were incubated with 1 lg of the CHO membrane fraction
and 50 pM of 125I-CRF in binding assay buffer [50 mM Tris–HCl,
5 mM EDTA, 10 mM MgCl2, 0.05% CHAPS, 0.1% BSA, 0.5 mM PMSF,
After incubation at rt for 2 h, the incubation mixture was filtered
on a Whatman GF/C filter presoaked in 0.3% polyethylenimine.
The filters were washed six times with ice-cold wash buffer (PBS
containing 0.05% CHAPS and 0.01% Triton X-100) and dried.
Radioactivity was determined using a gamma scintillation counter.
0.1
suring non-specific binding (NSB), 0.1
cortin was incubated with 1 g of the CHO cell membrane fraction
lg/mL pepstatin, and 20
l
g/mL leupeptin (pH 7.5)]. For mea-
lM of unlabeled human uro-
l

M. Mochizuki et al. / Bioorg. Med. Chem. 24 (2016) 4675–4691
4691
The results were expressed as an inhibitory rate of 125I-CRF (ovine)
binding, with in vitro determination of NSB using 1 lM of com-
These data include MOL files and InChiKeys of the most important
compounds described in this article.
pound 1.
References and notes
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The authors thank Dr. Takanobu Kuroita for helpful discussions
during the preparation of the manuscript. The authors also thank
Dr. Katsumi Kobayashi, Mr. Takuto Kojima and Mr. Yoshirou Tomi-
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.08.005.