Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition

Article abstract of DOI:10.1016/j.bmc.2016.04.011

Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF₃ group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats.

Full text of DOI:10.1016/j.bmc.2016.04.011

Bioorganic & Medicinal Chemistry 24 (2016) 2486–2503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Amine-free melanin-concentrating hormone receptor 1 antagonists:
Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and
design to avoid CYP3A4 time-dependent inhibition
a
a
a
a
Hideyuki Igawa a, , Masashi Takahashi , Mikio Shirasaki , Keiko Kakegawa , Asato Kina ,
Minoru Ikoma a, Jumpei Aida a, Tsuneo Yasuma b, Shoki Okuda a, Yayoi Kawata a, Toshihiro Noguchi a,
Syunsuke Yamamoto a, Yasushi Fujioka a, Mrinalkanti Kundu c, Uttam Khamrai c, Masaharu Nakayama a,
Yasutaka Nagisa d, Shizuo Kasai a, Tsuyoshi Maekawa a
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., Shonan Research Center, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Co., Ltd., 17-85, Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan
c TCG Lifesciences Ltd., Block BN, Plot 7, Saltlake Electronics Complex, Sector V, Kolkata 700091, India
d CVM Marketing Japan Pharma Business Unit, Takeda Pharmaceutical Co., Ltd., 12-10, Nihonbashi 2-Chome, Chuo-ku, Tokyo 103-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug
discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We
recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally
featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with
lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-
2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a
1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopy-
ridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q–u); however,
most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of
thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI,
we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring
or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-
2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified
as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile
including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344
rats.
Received 23 February 2016
Revised 4 April 2016
Accepted 5 April 2016
Available online 6 April 2016
Keywords:
MCHR1 antagonist
MCH
Antiobesity agent
Benzimidazole
Thiophene
Bioactivation
CYP3A4 time-dependent inhibition
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
in the form of body fat. This fat releases numerous inflammatory
adipokines and contributes to the development of various
Obesity mainly results from an imbalance between energy
intake and expenditure, leading to the storage of excess energy
disorders such as diabetes, hypertension, dyslipidemia, depression,
coronary artery disease, and cancer.1,2 The rate of obesity is
markedly increasing worldwide, resulting in a serious public
health issue.3,4 The FDA recently approved several new antiobesity
drugs such as lorcaserin, combined phentermine and topiramate,
combined bupropion and naltrexone, and liraglutide; however,
these drugs have still not fully met doctors’ and patients’ needs
for antiobesity treatment due to concerns regarding their efficacy,
safety, and cost.2–5
Abbreviations: MCH, melanin-concentrating hormone; MCHR1, melanin-con-
centrating hormone receptor 1; CYP3A4, cytochrome P450 3A4; TDI, time-depen-
dent inhibition; DIO, diet-induced obesity; hERG, human ether-a-go-go related
gene; DMEDA, N,N0-dimethylethylenediamine; HATU, (1-[bis(dimethylamino)
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate);
DIPEA, N,N-diisopropylethylamine; TFAA, trifluoroacetic anhydride; ADDP, 1,10-
(azodicarbonyl)dipiperidine; MW, microwave; CHO, Chinese Hamster Ovary; TM,
transmembrane; GSH, glutathione; HLM, human liver microsomes.
Melanin-concentrating hormone (MCH) is a cyclic 19-amino-
acid peptide, which is expressed predominantly in the lateral
hypothalamic area and zona incerta, and MCH-producing neurons
⇑
Corresponding author. Tel.: +81 466 32 1057; fax: +81 466 29 4468.
E-mail address: hideyuki.igawa@takeda.com (H. Igawa).
http://dx.doi.org/10.1016/j.bmc.2016.04.011
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2487
project throughout the brain.6,7 Among the two subtypes of the
MCH receptor that have been identified,8–12 MCH receptor 1
(MCHR1) is widely distributed in the brain, including the hypo-
thalamus, thalamus, olfactory cortex, amygdala, striatum, and
hippocampus, in all vertebrates,13,14 whereas MCH receptor 2 is
expressed only in higher mammals15–20 with its physiological
function poorly understood.
Many studies have demonstrated that the MCH/MCHR1 path-
way plays a key role in the regulation of feeding behavior and
energy expenditure. MCH mRNA and peptide levels were upregu-
lated in dietary obese rats21 and genetically obese rodents, includ-
ing ob/ob mice,22 db/db mice,23 Ay/a (agouti) mice,24 and Zucker
(fa/fa) rats.25 Chronic intracerebroventricular infusion of MCH
induced hyperphagia, body weight gain, and hyperinsulinemia,
particularly under high-fat diet conditions.26–28 Transgenic mice
that overexpressed MCH in the lateral hypothalamus were more
susceptible to obesity and insulin resistance when fed a high-fat
diet.29 In contrast, mice that lacked MCH or MCHR1 were lean with
increased metabolic rates and resistance to diet-induced obesity
(DIO).30–33 In human studies, increased levels of MCH expression
were observed in the hypothalamus in obese subjects compared
with lean subjects.34 Two loss-of-function MCHR1 mutants
(R210H and P377S) were identified in markedly underweight
subjects, indicating the possibility that the lean phenotype could
be linked to deficient MCHR1 signaling.35
In the course of our efforts to discover MCHR1 antagonists as
novel antiobesity agents with higher therapeutic window, we
recently reported novel pyridine-2(1H)-ones as amine-free MCHR1
antagonists that structurally featured an imidazo[1,2-a]pyridine-
based bicyclic motif.36 Compound 1 does not have an aliphatic
amine motif, unlike most existing MCHR1 antagonists,37 and the
imidazopyridine ring itself is considered to possess intrinsic bind-
ing affinity through the putative interaction of its nitrogen atom
with Asp123 and/or Tyr272 of MCHR1 (Fig. 1). Profiling of this
new lead compound revealed that 1 had low potential for inhibi-
tion of the human ether-a-go-go related gene (hERG) or induction
of phospholipidosis and showed potent MCHR1 antagonistic activ-
ity as well as a significant body-weight-lowering effect in DIO F344
rats after two weeks of oral administration.
providing possible solutions to alleviate thiophene-induced
CYP3A4 TDI.
2. Chemistry
Scheme 1 illustrates a general route to synthesize 4-alkoxypyri-
done derivatives 5 and 6, which involves copper coupling reactions
of pyridine-2(1H)-ones 4a–c with corresponding bicyclic bromides
7a–c or 8a–h. SNAr reactions of various benzyl alcohols with
4-chloropyridine-N-oxide 2 gave intermediates 3a–c, which were
subsequently treated with acetic anhydride to yield 1-unsubsti-
tuted pyridones 4a–c. Copper coupling reactions of 4a–c with
bicyclic bromides 7a–c or 8a–h afforded the target compounds.
For these coupling reactions, we selected N,N0-dimethylethylenedi-
amine (DMEDA) or trans-N,N0-dimethylcyclohexane-1,2-diamine
as a ligand and used stoichiometric amounts of CuI owing to the
low reactivity of 4a–c.
Scheme 2 shows an alternative method to obtain 4-alkoxypyri-
done derivatives 6 for the screening of the 4 position on the
pyridine-2(1H)-one ring using 4-hydroxypyridones 9a and 9b or
4-bromopyridone 10 as key intermediates. The intermediates 9a
and 9b were prepared from compounds 6l or 6y by hydrogenative
debenzylation, and subsequent bromination of 9a using POBr3 gave
compound 10. Alkylation of the intermediates 9a and 9b under
basic or Mitsunobu conditions as well as SNAr reactions of the
intermediate 10 afforded the target compounds.
The substituents on the 2 position of the benzimidazole ring can
be efficiently modified using the route described in Scheme 3. An
SNAr reaction of 11 with methylamine gave the p-nitrobromoben-
zene derivative 12a, which was subjected to a copper coupling
reaction followed by reduction of the nitro moiety by iron to give
the diamine intermediate 14. Amidation of 14 with 1 equivalent
of carboxylic acids using HATU gave the corresponding amide
intermediates, which were subsequently cyclized in acetic acid to
afford the target compounds.
Schemes 4 and 5 illustrate the synthesis of azaimidazopyridines
7a–c. Imidazopyridazine 7a and imidazopyrimidine 7c were
synthesized by a one-pot alkylation–cyclization reaction using
the corresponding anilines 15a and 15b, and 2-bromo-1-cyclo-
propylpropan-1-one. In contrast, imidazopyrazine 7b was synthe-
sized in a stepwise manner, as illustrated in Scheme 5. Aniline 16
was protected by a tosyl group and alkylated with 2-bromo-
1-cyclopropylpropan-1-one to give the intermediate 18, which
was treated with trifluoroacetic anhydride (TFAA) to give the target
compound 7b.
Although we successfully discovered the aliphatic-amine-free
MCHR1 antagonist 1, it showed a tendency to inhibit cytochrome
P450 3A4 (CYP3A4) at a concentration of 10 lM, which was con-
sidered to be due to the coordinating basic nitrogen atom on the
imidazopyridine ring. Therefore, we continued a further medicinal
chemistry campaign to investigate imidazopyridine variants with
lower basicity, and various bicyclic motifs with hydrogen-bonding
acceptors were introduced instead. Herein, we report the design of
novel pyridine-2(1H)-ones bearing a low-basicity 1H-benzimida-
zole ring as a new bicyclic motif and subsequent optimization
studies that led to the discovery of a preclinical candidate 6s with
excellent affinity for MCHR1 as well as a potent body weight-
lowering effect in DIO rats based on its MCHR1 antagonistic activity.
In the course of this study, compounds with thiophene as a terminal
aryl ring showed time-dependent inhibition (TDI) of CYP3A4. We
also discuss bioactivation mechanisms that cause CYP3A4 TDI,
which were analyzed by the glutathione adduct formation study,
Scheme 6 depicts the synthesis of 6-bromobenzimidazoles
8a–h. p-Nitrobromobenzene derivatives 12a–c were obtained as
described in Scheme 3. Reduction of the nitro moiety of 12a–c
gave diamine intermediates, which were subsequently heated with
the corresponding carboxylic acids in POCl3 or subjected to the
conditions described in Scheme 3 to afford the target compounds
(paths A and B). Alternatively, the p-nitrobromobenzene derivative
12a was subjected to amidation prior to reduction of the nitro
moiety (path C), wherein the resulting amides 20a and 20b were
subsequently heated with zinc in acetic acid to afford the target
compounds.
Asp123 and/or Tyr272
(unutilized)
Asp123 and/or Tyr272
Gln127
Gln127
Me
N
O
O
N
Me
N
Asn294
N
Asn294
N
H
Me
(unutilized)
O
Cl
F
T-226296
1
Figure 1. Chemical structures of T-226296 and the previously reported amine-free MCHR1 antagonist 1. Dotted lines depict putative interactions with MCHR1.

2488
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
X
N
O
Y
O
N
O
O
c
N
Z
N
b
NH
a
N
Me
O
O
O
R1
R1
Cl
Cl
2
R
R
R
1 = 4-Cl
1 = 3-Cl
1 = 4-F
5a X = CH, Y = CH, Z = N
5b X = CH, Y = N, Z = CH
3a
3b
3c
4a R1 = 4-Cl
4b R1 = 3-Cl
4c R1 = 4-F
X
Z
N
Y
5c
X = N, Y = CH, Z = CH
N
Br
Me
d-f
7a X = CH, Y = CH, Z = N
6a R1 = 4-Cl, R2 = Me, R3
=
cPr
7b
X = CH, Y = N, Z = CH
7c X = N, Y = CH, Z = CH
6b R1 = 4-F, R2 = Me, R3 = Me
6e R1 = 4-F, R2 = Me, R3
=
=
cPr
N
N
6f R1 = 4-F, R2 = Me, R3
cBu
O
R3
R3
1 = 4-F, R2 = Me, R3 = CH2cPr
6h
6i
R
N
N
Br
N
R
R
1 = 4-F, R2 = Me, R3 = CH2tBu
R2
R2
6j
O
1 = 4-Cl, R2 = Et, R3 cPr
=
R1
8a R2 = Me, R3
=
cPr
cPr
8e R2 = Me, R3 = CH2cPr
8f R2 = Me, R3 = CH2tBu
R
1 = 4-Cl, R2
=
nPr, R3
cPr
=
cPr
cPr
6k
8b R2 = Et, R3
=
cPr
6l R1 = H, R2 = Me, R3
=
2
8c
8d
8g
8h
R
=
nPr, R3
=
R
R
2 = Me, R3 cBu
=
6m R1 = 3-Cl, R2 = Me, R3
6y R1 = H, R2 = Me, R3 = Et
=
R
2 = Me, R3 = Me
2 = Me, R3 = Et
Scheme 1. Synthesis of 4-alkoxypyridone derivatives 5a–c and 6a, 6b, 6e, 6f, 6h–m, and 6y. Reagents and conditions: (a) corresponding benzyl alcohol, NaH, THF, rt, 5 h, 6–
66%; (b) Ac2O, 140 °C, 2 h, 49–58%; (c) 7a–c, CuI, DMEDA, K2CO3, DMSO, 150 °C, 1 h, MW irradiation, 1–42%; (d) 8, CuI, trans-N,N0-dimethylcyclohexane-1,2-diamine, K2CO3,
dioxane, 110 °C, 16 h, 18–87% (for 6a, 6f, 6l, and 6m); (e) 8, CuI, DMEDA, K2CO3, DMSO, 120 °C, 1 h, MW irradiation, 11–48% (for 6h, 6j, and 6k); (f) 8, CuI, DMEDA, K2CO3,
DMSO, 150 °C, 2 h, 22–87% (for 6b, 6e, 6f, 6i, 6l, 6m, and 6y).
N
O
N
N
R3
N
N
O
O
R3
R3
N
Me
a
b or c
N
=
N
N
Me
Me
6c R3 = Et, R4 = 4-F-C 6H4
O
R4
O
HO
6n R3
6o R3
6z R3
=
=
=
=
=
=
=
=
=
=
=
cPr, R4 = 2-Cl-C6H4
cPr, R4 = 5-Cl-pyridin-2-yl
cPr, R4 = CN
6l R3
cPr
9a R3 cPr
=
R
3 = Et
3 = Et
6y
9b
R
f
g
R
cPr, R4 = C(NH)NH2.HCl
cPr, R4 = 5-Cl-pyrimidin-2-yl
cPr, R4 = thiophen-2-yl
e
3
6aa
3
3
d
6p
6q
6r
R
R
3
R
cPr, R4 = thiophen-3-yl
N
O
6s R3
6t R3
cPr, R4 = 5-Cl-thiophen-2-yl
cPr, R4 = 4-Cl-thiophen-2-yl
cPr, R4 = 5-Cl-thiophen-3-yl
cPr, R4 = 5-CF3-thiophen-2-yl
N
Me
N
6u R3
Br
6v R3
10
3
6w
6x
R
=
=
cPr, R4 = 4-CF3-thiophen-2-yl
cPr, R4 = 5-CF3-thiophen-3-yl
3
R
Scheme 2. Synthesis of 4-alkoxypyridone derivatives 6c, 6n–x, 6z, and 6aa. Reagents and conditions: (a) H2 (1 atm), 10% Pd–C, MeOH, 3 h, 86–99%; (b) corresponding alkyl
halide, K2CO3, DMF, 14–76% (for 6c, 6o, and 6z); (c) corresponding benzyl alcohol, ADDP, PBu3, THF, 60 °C, 3 h, 9–42% (for 6n and 6q–v); (d) POBr3, DMF, 50 °C, 9 h, 49%; (e)
corresponding benzyl alcohol, NaH, DMA, 120 °C, 10 min, 26–51% (for 6w and 6x); (f) NaOMe, MeOH, rt, 4 h, then NH4Cl, rt, overnight, quant.; (g) 2-chloro-1,3-bis
(dimethylamino)trimethinium hexafluorophosphate, NaOMe, MeOH, rt, 1 h, 51%.
NO2
NH2
O
O
NO2
NO2
F
a
b
c
N
NH
Me
N
NH
Me
Br
O
3
NH
Me
Br
O
O
F
F
11
12a
13
14
N
N
R3
d
N
Me
O
nPr
cPen
F
6d
6g
R
=
=
3
R
Scheme 3. Synthesis of 4-alkoxypyridone derivatives 6d and 6g. Reagents and conditions: (a) MeNH2 (40% MeOH solution), EtOH, rt, 1 h, 94%; (b) 4c, CuI, DMEDA, K2CO3,
DMSO, 120 °C, 1 h, MW irradiation, 44%; (c) Fe, CaCl2, EtOH, water, 70 °C, 3 h, 94%; (d) corresponding carboxylic acids, HATU, DIPEA, DMF, 1 h, then AcOH, 90 °C, 1 h, 18–65%.
alternative imidazopyridine motifs with lower basicity, various
bicyclic motifs with lower pKa values bearing a nitrogen atom, as
in the 1 position in the imidazopyridine ring of compound 1, were
prepared (Table 1). First, we systematically introduced a nitrogen
atom into the six-membered ring and prepared a series of azaimi-
dazopyridines 5a–c. Thus, the imidazopyrazine derivative 5b
showed an almost two-fold decrease in binding affinity relative
3. Results and discussion
The binding affinities of the compounds prepared herein to
human and rat MCHR1 (hMCHR1 and rMCHR1, respectively) were
evaluated in a binding assay using radiolabeled [125I]MCH (4–19)
and membrane fractions prepared from Chinese hamster ovary
(CHO) cell lines that stably expressed MCHR1. To investigate

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2489
X
Z
derivative 6b (hMCHR1: IC50 = 77 nM) and increased with the ethyl
derivative 6c and npropyl derivative 6d almost two-fold. When a
cycloalkyl group was introduced at the 2 position (6e, 6f, and
6g), the cyclopropyl derivative 6e showed a slight increase in
potency (6e vs. 6c). The introduction of other cyclic substituents
at the 2 position, however, decreased the affinity as the ring size
increased (6e > 6f > 6g). In contrast, the introduction of a cyclo-
propylmethyl group at the 2 position caused an almost three-fold
decline in binding affinity (6h vs. 6d), whereas a neopentyl group
was found to lead to a deterioration in potency (6i). These findings
indicated that the 2 position on the benzimidazole ring was
directed toward a narrow lipophilic space, wherein sterically less
hindering substituents with a length of two or three carbon atoms
were preferred. The structure–activity relationship (SAR) study
that has been discussed so far (see Table 2) revealed that a cyclo-
propyl group was optimal for a substituent at the 2 position.
We subsequently briefly investigated the SAR at the 1 position
on the benzimidazole ring (6j and 6k in Table 2). The ethyl deriva-
tive 6j showed a slight decrease in binding affinity to rMCHR1 and
further elongation of the chain resulted in a significant decline in
potency, as indicated by compound 6k. These results indicated that
the 1 position possessed limited capacity to accommodate a sub-
stituent and a methyl group was optimal.
We next examined the influence of the terminal benzene ring
on potency (Table 3). The unsubstituted derivative 6l showed good
binding affinities (hMCHR1: IC50 = 45 nM). The introduction of flu-
orine and chlorine atoms at the 4 position enhanced potency (6e
and 6a), and the 4-chloro derivative 6a showed a two-fold increase
in affinity for rMCHR1 relative to the unsubstituted derivative 6l.
In contrast, the 3-chloro and 2-chloro derivatives (6m and 6n)
showed significant decreases in binding affinity. Replacement of
the terminal benzene ring with polar heteroaromatic rings (6o
and 6p) caused a decline in potency in a lipophilicity-dependent
manner (6o: ClogP = 3.56, 6p: ClogP = 2.56).38 These findings are
consistent with the results of our previous studies on dihydro-
naphthalenes41 and quinolines,42,43 wherein the left-hand side of
X
Z
NH2
N
a or b
N
N
Br
Br
Me
15a X = CH, Z = N
15b X = N, Z = CH
7a X = CH, Z = N
7c X = N, Z = CH
Scheme 4. Synthesis of azaimidazopyridines 7a and 7c. Reagents and conditions: (a)
2-bromo-1-cyclopropylpropan-1-one, NaHCO3, DMA, 80 °C, 16 h, 76% (for 7a); (b)
2-bromo-1-cyclopropylpropan-1-one, DMF, 100 °C, 24 h, 13% (for 7c).
to 1 (5b: IC50 = 44 nM) and approximately 10- and 50-fold declines
in potency were observed in the imidazopyridazine derivative 5a
and the imidazopyrimidine derivative 5c, respectively (5a:
IC50 = 380 nM, 5c: IC50 > 1000 nM). These results indicated that
the introduction of a nitrogen atom into the six-membered ring
of the imidazopyridine system was basically not an appropriate
approach, owing to the decrease in potency. Encouraged by the fact
that the potency did not correlate with the pKa value, we then
designed compound 6a with a low-basicity 1H-benzimidazole ring,
which was regarded as a substructure with less risk of general
safety concerns.39,40 Compound 6a showed as potent a binding
affinity as compound 1 (6a: IC50 = 35 nM) and its pKa value was
5.71, indicating the compound 6a was considered as a novel
low-basicity lead compound for further optimization.
The optimization study was initiated with the 2 position on the
benzimidazole ring (Table 2). During this study, 4-fluorophenyl
was selected as the terminal aryl ring, which provided identical
potency to the 4-chlorophenyl analog. In a previous study on imi-
dazopyridine derivatives,36 the introduction of a polar substituent
at this position was found to be unfavorable for potency. This trend
was also true with the current benzimidazole derivatives, and the
introduction of a polar substituent such as an alkoxy group, alco-
hol, ketone, or amide decreased the affinity (data not shown).
Therefore, we examined various aliphatic substituents to optimize
lipophilic interaction. Investigation of the size of the substituent
(6b–d) revealed that the potency was moderate with the methyl
Ts
N
H
N
NH2
N
N
c
a
b
N
N
Ts
N
O
N
N
N
N
Br
Br
Br
Br
Me
Me
18
16
17
7b
Scheme 5. Synthesis of azaimidazopyridine 7b. Reagents and conditions: (a) TsCl, pyridine, rt, overnight, 51%; (b) 2-bromo-1-cyclopropylpropan-1-one, NaH, DMF, rt,
overnight, NaHCO3, 46%; (c) TFAA, THF, 0 °C, then 60 °C, 3 h, 80%.
path A
b
NH2
Br
NH
Me
c
19
NO2
F
NO2
N
N
path B
a
d or e
R3
Br
Br
NH
R2
Br
R2
path C
f or g
h
11
2 = Me, R3
=
=
cPr
NO2
8a
8b
8c
8d
R
R
R
R
2 = Me
12a
12b
12c
R
R
R
2 = Et, R3
2
=
cPr
2 = Et
2
Me
nPr, R3
cPr
Br
N
=
nPr
=
2 = Me, R3 = Me
O
3
R3
8e R2 = Me, R3 = CH2cPr
8f R2 = Me, R3 = CH2tBu
20a
20b
R
R
= Me
3 = CH2tBu
8g R2 = Me, R3
=
cBu
8h R2 = Me, R3 = Et
Scheme 6. Synthesis of benzimidazoles 8a–h. Reagents and conditions: (a) corresponding amine, EtOH, rt, 1 h, 76–94%; (b) Zn, NH4Cl, MeOH, water, rt, 1 h, 93%; (c)
cyclobutanecarboxylic acid, POCl3, 120 °C, 4 h, 45% (for 8g); (d) Zn, NH4Cl, MeOH, water, rt, 1 h, then corresponding carboxylic acid, POCl3, 120 °C, 3 h, 45–72% (for 8a, 8c, and
8g in two steps); (e) Zn, AcOH, rt, 30 min, then corresponding carboxylic acid, HATU, DIPEA, DMF, rt, 1 h, then AcOH, 80 °C, 1 h, 37–96% (for 8b, 8e, and 8h in three steps); (f)
acetyl chloride, toluene, 90 °C, 15 h, 97% (for 20a); (g) 3,3-dimethylbutanoyl chloride, NaH, DMF, 70 °C, overnight, 50% (for 20b); (h) Zn, AcOH, 90 °C, 4 h, 65–78% (for 8d and
8f).

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H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
Table 1
Table 3
In vitro binding affinities and pKa values of compounds 1, 5a–c, and 6c
In vitro binding affinities of compounds 6a, 6e, and 6l–p
O
N
N
Me
O
Ar
N
N
R4
O
O
Cl
Compound
R4
IC50 (nM)a
c
hMCHR1b
rMCHR1c
Compound
Ar
IC50 (nM)a
hMCHR1b
pKa
6l
45
40
43
28
N
N
1
26
7.85
6.35
5.35
6.35
5.71
6e
Me
N
F
6a
35
21
72
N
5a
5b
5c
6a
380
44
Cl
N
Me
N
6m
100
N
N
N
Cl
Me
6n
6o
160
73
140
47
N
N
Cl
N
>1000
35
Me
Cl
Cl
N
N
6p
>1000
900
N
Me
N
a
IC50 values were calculated using an experiment performed in duplicate with a
a
IC50 values were calculated using an experiment performed in duplicate with a
three-fold standard deviation.
three-fold standard deviation.
b
Binding affinity for human MCHR1.
Binding affinity for rat MCHR1.
b
Binding affinity for human MCHR1.
c
c
pKa values were calculated using ACD Labs ver. 12.0.38
Table 2
Table 4
In vitro binding affinities of compounds 6b–k
In vitro binding affinities and CYP3A4 TDI risk of compounds 6q–u
N
N
N
O
O
R3
N
N
N
R2
Me
R4
O
O
R1
R2
Compound R4
IC50 (nM)a
hMCHR1b rMCHR1c
CYP3A4TDId (% remaining)
Compound
R1
R3
IC50 (nM)a
hMCHR1b
rMCHR1c
6q
76
28
120
29
NTe
74
S
6b
6c
6d
6e
6f
6g
6h
6i
F
F
F
F
F
F
F
F
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
Et
77
48
34
40
240
600
90
>1000
37
65
38
39
28
210
460
140
>1000
34
6r
nPr
S
cPr
S
S
Cyclobutyl
Cyclopentyl
CH2cPr
CH2tBu
cPr
6s
19
11
83
Cl
Cl
6t
17
14
18
25
44
S
6j
6k
Cl
Cl
Cl
6u
9.3
nPr
cPr
85
110
a
a
IC50 values were calculated using an experiment performed in duplicate with a
IC50 values were calculated using an experiment performed in duplicate with a
three-fold standard deviation.
three-fold standard deviation.
b
b
Binding affinity for human MCHR1.
Binding affinity for rat MCHR1.
Binding affinity for human MCHR1.
c
c
Binding affinity for rat MCHR1.
d
CYP3A4 time-dependent inhibition assay (n = 2). The remaining activity of
CYP3A4 after pre-incubation with a test compound was determined.
e
Not tested.
the molecule was expected to be surrounded by a hydrophobic
environment formed by Phe213, Ala216, and Phe217 on TM5 and
Tyr273 on TM6,41 which suggests that the terminal benzene ring
is in a narrow hydrophobic pocket of the receptor and only the 4
position can accommodate a substituent.
In a previous report, we found that thiophene as the terminal
aryl ring generally afforded better binding affinities than benzene
or other heteroaromatic rings,36 which prompted us to introduce
thiophene rings into the current chemotype to increase potency
(Table 4). The thiophen-2-yl derivative 6q showed two- and
three-fold weaker potencies compared with the corresponding
benzene analog 6l for hMCHR1 and rMCHR1, whereas the
thiophen-3-yl derivative 6r possessed better potencies relative to
6l. The introduction of a chlorine atom onto these isomeric thio-
phenes revealed that both thiophen-2-yl derivatives (6s and 6t)
and a thiophen-3-yl derivative (6u) showed successful increases
in potency and their binding affinities were superior to those of
the lead compound 6a. Among these, the 5-chlorothiophen-3-yl
derivative 6u showed the best potency for both species (rMCHR1:
IC50 = 9.3 nM). Thus, the in vitro potency could be successfully

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2491
N
N
O
N
N
N
O
O
Glutathione
Cl
N
S
GSH
HLM
Me
N
N
N
O
Me
Me
O
S
O
Cl
Cl
O
S
S
O
6t
estimated structure of GSH adduct
[M+H]+ = 735
estimated structure of oxidative reactive metabolite
[M+H]+ = 412
N
O
N
N
N
Me
O
O
Cl
Cl
O
N
N
N
Glutathione
Cl
O
O
N
S
S
or
O
N
Me
GSH
HLM
N
S
Me
N
N
O
O
Cl
S
S
Me
6u
O
estimated structure of GSH adduct
[M+H]+ = 717
[M+H]+ = 412
estimated structure of oxidative reactive metabolite
Figure 2. Glutathione adduct formation by incubation of test compounds 6t and 6u with GSH and human liver microsomes (HLM). The test compounds (30
incubated with HLM (1.0 mg/mL) in the presence of GSH (1 mM) at 37 °C for 60 min. The structures of the GSH adducts were estimated by LC/MS/MS analysis.
lM) were
Table 5
drugs, CYP3A4 TDI may preclude the drug development from the
perspective of drug–drug interactions. Given the fact that benzene
and pyridine derivatives (6e and 6o) did not show potential for
CYP3A4 TDI, we hypothesized that the thiophene substructure
was responsible for the risk of CYP3A4 TDI. This hypothesis was
also supported by the reported recognition that thiophenes were
regarded as toxicophores in some cases owing to the potential risk
of oxidative bioactivation leading to the formation of electrophilic
species, which are linked to toxicity caused by conjugation with
biomolecules and a mechanism-based inactivation of CYP3A4,
resulting in clinical drug–drug interactions.44,45 To investigate this
assumption, compounds 6t and 6u were tested in glutathione
(GSH) trapping experiments. When 6t and 6u were incubated in
human liver microsomes with GSH followed by analysis using
liquid chromatography coupled with mass spectrometry (LC/MS),
accurate masses corresponding to ‘‘parent mass + GSH + O” and
‘‘parent mass + GSH ꢀ H2” were observed with 6t and 6u, respec-
tively (Fig. 2). Furthermore, from the product ion spectra of the
GSH adducts it appeared that the GS moiety was substituted on
the thiophene ring. These findings suggested that the formation
of a GSH adduct of 6t would be caused by the generation of a sul-
foxide by microsomal oxidation followed by 1,4-addition of GSH to
yield a GSH adduct at the b position to the sulfur atom. In contrast,
6u would be oxidized to an epoxide, followed by nucleophilic
attack of GSH and subsequent loss of water to generate the GSH
adduct. These mechanistic analyses of the formation of GSH
adducts of 6t and 6u suggested that CYP3A4 TDI that was observed
with thiophene derivatives was triggered by oxidation of the
In vitro binding affinities and CYP3A4 TDI risk of compounds 6v–x
N
O
N
Me
N
R4
O
Compound R4
IC50 (nM)a
CYP3A4TDId (% remaining)
hMCHR1b rMCHR1c
S
S
6v
16
13
97
F3
C
F3C
6w
6x
34
41
17
29
87
96
S
F3C
a
IC50 values were calculated using an experiment performed in duplicate with a
three-fold standard deviation.
b
Binding affinity for human MCHR1.
Binding affinity for rat MCHR1.
CYP3A4 time-dependent inhibition assay (n = 2). The remaining activity of
c
d
CYP3A4 after pre-incubation with a test compound was determined.
increased by the replacement of the 4-chlorophenyl moiety in 6a
with various chloro-substituted thiophenes.
Further evaluation of these thiophene derivatives, however,
revealed that compounds 6r, 6t, and 6u caused CYP3A4 TDI.
Because CYP3A4 is the most abundant CYP isoform and is respon-
sible for the oxidative metabolism of a wide variety of clinical
Table 6
Pharmacokinetic parameters of 6s and 6v in rats.a
Compound
Fb (%)
iv (0.1 mg/kg)
po (1 mg/kg)
CLtotal (mL hꢀ1 kgꢀ1
)
Vss (mL kgꢀ1
d
)
Cmax (ng mLꢀ1
)
Tmax (h)
AUC0–8h (ng h mLꢀ1
)
c
e
f
g
6s
6v
23
57
450
285
920
979
164.7
297.8
1.0
2.7
514.9
2015.6
a
b
c
d
e
f
n = 3; SD rats (male, eight weeks old).
Bioavailability.
Total clearance.
Volume of distribution at steady state.
Maximal plasma concentration.
Time of maximal concentration.
g
Area under the plasma concentration–time curve (0–8 h).

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H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
on the risk of CYP3A4 TDI by introducing the strongly electron-
withdrawing trifluoromethyl group and prepared a series of
trifluoromethyl-substituted thiophene derivatives (Table 5). The
5-trifluoromethylthiophen-2-yl derivative 6v showed comparable
potency to the corresponding chloro analog 6s for both species;
however, the 4-trifluoromethylthiophen-2-yl derivative 6w
showed a slight decrease in potency for hMCHR1 relative to 6t.
The 5-trifluoromethylthiophen-3-yl derivative 6x showed
a
three-fold reduction in affinity compared with the chloro analog
6u. Evaluation of CYP3A4 TDI of these thiophene derivatives
showed that not only compound 6v, which had both
a carbons
substituted, but also compounds 6w and 6x, which had one
a
carbon unsubstituted, gave negative results. Considering the fact
that the corresponding chloro-substituted analogs 6t and 6u were
CYP3A4 TDI-positive, this clearly indicated that decreasing the
electron density of the thiophene ring by replacement with a
trifluoromethyl group was effective in reducing the risk of
CYP3A4 TDI and that trifluoromethyl-substituted thiophenes are
considered to be less susceptible to bioactivation and safer alterna-
tives to chloro-substituted thiophenes.
Among the compounds discussed so far, we selected 6s and 6v,
which showed potent in vitro binding affinities for both MCHR1s
and did not possess the potential for CYP3A4 TDI. Secondary
functional cell-based assays of the inhibition of MCH-stimulated
Ca2+ mobilization in CHO cells confirmed that these two com-
pounds functioned as MCHR1 antagonists (6s: IC50 = 24 nM, 6v:
IC50 = 18 nM). Pharmacokinetic screening of 6s and 6v resulted in
reasonable oral availability and plasma exposure (Table 6).
To evaluate their pharmacological effects, compounds 6s and
6v (3 and 10 mg/kg) were orally administered to DIO F344 rats
fed a high-fat diet ad libitum. The results of a two-day in vivo
study are shown in Figure 3. Compound 6s significantly and
dose-dependently suppressed food intake in DIO rats by 11.6%
and 36.5% at 3 and 10 mg/kg, respectively. Compound 6v showed
an almost equal anorectic effect at 3 mg/kg; however, its efficacy
at 10 mg/kg was a slightly weaker than that of 6s (20.3%). It was
Figure 3. Results for 6s and 6v in a two-day food intake study in DIO F344 rats.
Inhibition of cumulative food intake over two days in DIO F344 rats. The
compounds were administered once daily, and food intake from the initial
administration to two days later was measured. The cumulative food intake
inhibition rate was calculated by dividing the average food intake of each treatment
group by that of the vehicle group (n = 6 for each group). (#) p < 0.025 vs. the
vehicle group (Williams test).
thiophenes to form reactive species such as electrophilic sulfoxides
or epoxides. Therefore, we assumed that the risk of CYP3A4 TDI
could be reduced by the following two strategies: (1) preventing
the oxidation of thiophenes with increased steric influence around
the sulfur atom and (2) decreasing the electron density of thio-
phenes. Indeed, compound 6s, which had substituents on both car-
bons in positions
a to the sulfur atom, did not show a risk of TDI,
assumed that the low solubility of 6v (0.25
lg/mL at pH 6.8)
which would indicate the effectiveness of the strategy of masking
relative to 6s (2.3 g/mL at pH 6.8) caused a non-linear pharmaco-
l
the sulfur atom to prevent sulfoxide formation.
We then investigated the second strategy and examined the
influence of decreasing the electron density of the thiophene ring
kinetic profile and caused lower plasma exposure at a dose of
10 mg/kg of 6v. Given these results, we selected 6s for further
pharmacological evaluation.
Figure 4. Results for 6s in a repeated-dose study in DIO F344 rats. (A) Body weight change from initial value during two weeks of dosing. (B) Cumulative food intake for two
weeks of dosing. The compounds were administered once daily for two weeks and the body weight and food intake were measured before drug administration. Each data
point represents mean SD (n = 5 or 6 for each group). (#) p < 0.025 vs. the vehicle group (Williams test), (⁄⁄) p < 0.01 vs. the vehicle group (Student’s t-test).

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2493
The effect of 6s on body weight reduction was assessed using a
two-week repeated dosing study on DIO rats (Fig. 4). A significant
change in body weight was observed from 3 mg/kg, and once daily
oral administration of 3 and 10 mg/kg of 6s resulted in body
weight reductions of 2.2% and 4.1%, respectively, relative to the
vehicle group. The cumulative food intakes of these two cohorts
were significantly reduced by 11.6% and 20.9%, respectively. It
was noteworthy that 10 mg/kg of 6s showed a more potent
antiobesity effect relative to 1 mg/kg of sibutramine (4.1% vs
3.1%), whereas the anorectic effects in these two cohorts were
almost the same (20.9% vs 18.7%). The involvement of MCHR1
antagonists in energy expenditure has been reported,46 which
would make an additional contribution to the antiobesity effect
of 6s, resulting in greater loss of body weight relative to sibu-
tramine. The trough plasma concentration at 10 mg/kg was
(IC50 > 10 lM), providing better safety profile in terms of drug-
induced cardiovascular risk. Therefore, compound 6s could be a
promising antiobesity agent that exerted a body-weight-lowering
effect based on its MCHR1 antagonistic activity.
4. Conclusion
To investigate imidazopyridine variants with lower basicity and
potential for CYP3A4 inhibition, we designed pyridine-2(1H)-ones
bearing various less basic bicyclic motifs. Among these, a lead com-
pound 6a bearing a 1H-benzimidazole motif showed comparable
binding affinity for MCHR1 to the corresponding imidazopyridine
derivative 1. During optimization studies, a series of potent thio-
phene derivatives were shown to induce CYP3A4 TDI via bioactiva-
tion of the thiophene ring. We found that blockage of the
a carbon
atoms of thiophenes as well as the introduction of the bulky and
strongly electron-withdrawing trifluoromethyl group were effec-
tive in preventing this bioactivation mechanism, which led to the
identification of 6s and 6v as potent MCHR1 antagonists without
the risk of CYP3A4 TDI. Note that the trifluoromethyl group
possessed a beneficial effect in preventing CYP3A4 TDI of thio-
0.79 lM, which was equivalent to an unbound drug concentration
of 15.8 nM (unbound fraction of 6s in DIO rat plasma was 0.02).
These results indicated that a unbound concentration of more
than 1.44 times the IC50 was maintained in this cohort throughout
the study, which was considered to be the necessary plasma
level to afford a body weight reduction of 4.7%. Compound 6s
was confirmed to show acceptable brain exposure (brain/plasma
ratio = 0.5), which suggested that 6s was blood–brain barrier-
permeable.
To clarify that the anorectic property was linked to MCHR1
antagonism, the effect of 6s in MCHR1-deficient and wild-type
mice was examined. Compound 6s exerted no body-weight-lower-
ing effect in MCHR1-deficient mice, whereas it significantly
reduced body weight in wild-type mice in a dose-dependent
manner (Fig. 5). Compound 6s also exhibited a trend toward
decreasing cumulative food intake in a dose-dependent manner
in MCHR1-deficient mice. These results indicated that the
anorectic effect of 6s and the resulting weight loss were based
on its MCHR1 antagonistic activity.
phenes even though one
a carbon atom was left unsubstituted,
as in 6w and 6x, which indicated that trifluoromethyl-substituted
thiophenes could be used in drug design as safer building blocks
without the risk bioactivation. In summary, the preclinical candi-
date 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-
1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s), which exhibited a
significant body-weight-lowering effect in DIO rats on oral admin-
istration at 5 and 10 mg/kg, was expected to be a new antiobesity
agent based on its MCHR1 antagonistic activity.
5. Experimental
5.1. Chemistry
The findings discussed so far indicated that we have success-
fully identified a low-basicity MCHR1 antagonist, 6s. Compound
6s possessed a much lower potential for reversible CYP3A4
inhibition, which prevented previously reported imidazopyridine
derivatives from proceeding for development, and did not display
potential for phospholipidosis in an in vitro assay. Furthermore,
compound 6s did not inhibit hERG channel in a patch clamp test
Melting points were determined on a Yanaco melting point
apparatus Mp-500D and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker AVANCE III (300 MHz) or a Bruker
Advance III plus (400 MHz) spectrometer. Chemical shifts are given
in parts per million (ppm) downfield from tetramethylsilane (d) as
the internal standard in deuterated solvent, and coupling constants
Figure 5. Results for 6s in a three-day study in MCHR1-deficient (KO) and wild-type (WT) mice. The mice were fed a high-fat diet. The body weight change (A) and
cumulative food intake (B) were measured for three days. Each data point represents mean + SD (n = 6 for each group). (#) p < 0.025 vs. the vehicle group (Williams test).

2494
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
(J) are in hertz (Hz). Data are reported as follows: chemical shift,
integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quar-
tet, quint = quintet, m = multiplet, dd = doublet of doublets, td = tri-
plet of doublets, and br s = broad signal), and coupling constants.
Reagents and solvents were obtained from commercial sources
and used without further purification. Reaction progress was deter-
mined by thin layer chromatography (TLC) analysis on Merck Kiesel-
gel 60 F254 plates or Fuji Silysia NH plates. Chromatographic
purification was performed on silica gel columns [(Merck Kieselgel
60, 70–230 mesh size or 230–400 mesh size, Merck) or (Chroma-
torex NH-DM 1020, 100–200 mesh size)] or on Purif-Pack (SI or
5.1.4. 4-[(4-Chlorobenzyl)oxy]pyridine-2(1H)-one (4a)
A mixture of 3a (54.3 g, 230 mmol) and acetic anhydride
(540 mL, 5.71 mol) was stirred at 140 °C for 2 h. After concentra-
tion of the mixture, the residue was dissolved in MeOH (300 mL).
Water (450 mL) was added to the mixture, followed by stirring at
rt for 1 h. The resulting precipitate was collected by filtration,
washed with IPA, and dried to give the title compound (29.3 g,
54%) as a gray solid. 1H NMR (300 MHz, CDCl3) d 4.99 (2H, s),
5.93 (1H, d, J = 2.3 Hz), 6.03 (1H, dd, J = 7.4, 2.5 Hz), 7.23 (1H, d,
J = 7.2 Hz), 7.29–7.44 (4H, m). 13C NMR (75 MHz, DMSO-d6) d
68.4, 98.0, 99.1, 128.5, 129.7, 132.7, 135.0, 135.5, 163.9, 167.2.
MS (ESI/APCI) m/z 236.0 [M+H]+.
NH, particle size: 60
lm, Fuji Silysia Chemical, Ltd.). LCꢀMS analysis
was performed on a Shimadzu liquid chromatography–mass spec-
trometer system, operating in APCI (+ or ꢀ) or ESI (+ or ꢀ) ionization
mode. Analytes were eluted using a linear gradient of 0.05% TFA con-
taining water/acetonitrile or 5 mM ammonium acetate containing
water/acetonitrile mobile phase and detected at 220 nm. Analytical
HPLC was performed with Corona Charged Aerosol Detector (CAD)
or photo diode array detector. The column was a Capcell Pak
C18AQ (50 mm ꢁ 3.0 mm I.D., Shiseido, Japan) or L-column 2 ODS
(30 mm ꢁ 2.0 mm I.D., CERI, Japan) with a temperature of 50 °C
and a flow rate of 0.5 mL/min. Mobile phase A and B under a neutral
condition were a mixture of 50 mmol/L ammonium acetate, water
and acetonitrile (1:8:1, v/v/v) and a mixture of 50 mmol/L ammo-
nium acetate and acetonitrile (1:9, v/v), respectively. The ratio of
mobile phase B was increased linearly from 5% to 95% over 3 min,
95% over the next 1 min. Mobile phase A and B under an acidic con-
dition were a mixture of 0.2% formic acid in 10 mmol/L ammonium
formate and 0.2% formic acid in acetonitrile, respectively. The ratio of
mobile phase B was increased linearly from 14% to 86% over 3 min,
86% over the next 1 min. The purities of compounds submitted for
biological evaluation were >95% as determined by elemental analy-
ses within 0.4% of the calculated values or analytical HPLC. Yields
are not optimized.
5.1.5. 4-[(3-Chlorobenzyl)oxy]pyridine-2(1H)-one (4b)
The title compound was prepared in 49% yield using 3b in an
analogous manner to 4a. Off-white solid. 1H NMR (400 MHz,
DMSO-d6) d 5.07 (2H, s), 5.76 (1H, d, J = 2.4 Hz), 5.92 (1H, dd,
J = 7.2 2.4 Hz), 7.25 (1H, d, J = 7.3 Hz), 7.34–7.45 (3H, m), 7.50
(1H, br s), 11.10 (1H, br s). MS (ESI/APCI) m/z = 236.0 [M+H]+.
5.1.6. 4-[(4-Fluorobenzyl)oxy]pyridine-2(1H)-one (4c)
The title compound was prepared in 58% yield using 3c in an
analogous manner to 4a. 1H NMR (300 MHz, DMSO-d6) d 5.04
(2H, s), 5.78 (1H, d, J = 2.3 Hz), 5.90 (1H, dd, J = 7.2, 2.7 Hz),
7.17–7.28 (3H, m), 7.43–7.53 (2H, m), 11.10 (1H, br s). 13C NMR
(75 MHz, DMSO-d6) d 68.5, 97.9, 99.1, 115.3 (d, J = 21 Hz), 130.2
(d, J = 8.3 Hz), 132.2 (d, J = 3 Hz), 135.4, 161.9 (d, J = 242.3 Hz),
164.0, 167.3. MS (ESI/APCI) m/z = 220.1 [M+H]+.
5.1.7. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-3-
methylimidazo[1,2-b]pyridazin-6-yl)pyridin-2(1H)-one (5a)
The title compound was prepared in 28% yield using 4a and 7a
in an analogous manner to 5c. Pale yellow crystals; mp
234–236 °C. 1H NMR (400 MHz, DMSO-d6) d 0.87–1.08 (4H, m),
2.17 (1H, br s), 2.52 (3H, br s), 5.19 (2H, s), 6.04 (1H, s), 6.23 (1H,
d, J = 7.8 Hz), 7.30 (1H, d, J = 9.3 Hz), 7.50 (4H, s), 7.82 (1H, d,
J = 7.8 Hz), 8.02 (1H, d, J = 9.4 Hz). 13C NMR (75 MHz, DMSO-d6) d
7.6, 8.29, 8.31, 69.0, 97.7, 101.1, 115.6, 120.9, 124.0, 128.6, 129.8,
132.8, 134.7, 136.5, 137.8, 146.2, 148.1, 162.5, 167.5. MS (ESI/APCI)
m/z = 407.3 [M+H]+. Anal. Calcd for C22H19ClN4O2: C, 64.94; H,
4.71; N, 13.77. Found: C, 64.86; H, 4.63; N, 13.70.
5.1.1. 4-[(4-Chlorobenzyl)oxy]pyridine 1-oxide (3a)
A solution of (4-chloropheny)methanol (49.5 g, 347 mmol) in
THF (200 mL) was added dropwise to a suspension of NaH (60%
oil dispersion, 16.7 g, 419 mmol) in THF (200 mL) at 0 °C. After
the mixture was stirred at 0 °C for 30 min, 2 (45.0 g, 347 mmol)
was added portionwise to the reaction mixture. After completion
of the addition, the mixture was stirred at rt for 5 h. The mixture
was quenched with water (400 mL) at 0 °C and extracted with
EtOAc/THF (1:1) four times. The organic layers were combined,
passed through NH-silica gel pad (EtOAc/MeOH) and concentrated.
The filtrate was concentrated, and the residual solid was washed
with IPE and dried to give the title compound (54.3 g, 66%) as a
brown solid. 1H NMR (400 MHz, CDCl3) d 5.17 (2H, s), 7.08 (2H,
d, J = 6.9 Hz), 7.48 (4H, s), 8.10 (2H, d, J = 7.0 Hz). MS (ESI/APCI)
m/z 236.0 [M+H]+.
5.1.8. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-3-
methylimidazo[1,2-a]pyrazin-6-yl)pyridin-2(1H)-one (5b)
The title compound was prepared in 42% yield using 4a and 7b
in an analogous manner to 5c. Pale yellow crystals; mp
221–222 °C. 1H NMR (400 MHz, DMSO-d6) d 0.92–1.04 (4H, m),
2.14–2.24 (1H, m), 2.54 (3H, s), 5.18 (2H, s), 6.01 (1H, d,
J = 2.4 Hz), 6.17 (1H, dd, J = 7.7, 2.6 Hz), 7.50 (4H, s), 7.78 (1H, d,
J = 7.7 Hz), 8.68 (1H, s), 8.83 (1H, s). 13C NMR (101 MHz, DMSO-
d6) d 7.5, 8.1, 8.7, 68.8, 97.7, 100.4, 115.1, 119.4, 128.5, 129.7,
132.8, 134.8, 135.8, 138.0, 138.3, 138.9, 149.2, 162.5, 166.9. MS
(ESI/APCI) m/z = 407.4 [M+H]+. Purity 97.8% (HPLC).
5.1.2. 4-[(3-Chlorobenzyl)oxy]pyridine 1-oxide (3b)
The title compound was prepared in 45% yield using (3-chloro-
phenyl)methanol in an analogous manner to 3a. Brown solid. 1H
NMR (400 MHz, DMSO-d6) d 5.18 (2H, s), 7.08 (2H, dd, J = 5.6,
1.8 Hz), 7.42–7.43 (3H, m), 7.53 (1H, br s), 8.10 (2H, dd, J = 5.6,
1.8 Hz). MS (ESI/APCI) m/z 236.0 [M+H]+.
5.1.9. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-3-
methylimidazo[1,2-a]pyrimidin-6-yl)pyridin-2(1H)-one (5c)
A mixture of 4a (83 mg, 0.35 mmol), 7c (89 mg, 0.35 mmol),
DMEDA (0.075 mL, 0.71 mmol), CuI (66.6 mg, 0.35 mmol), K2CO3
(146 mg, 1.06 mmol), and DMSO (3 mL) was heated at 150 °C for
1 h under microwave irradiation. The mixture was poured into
28% NH3 solution at rt and extracted with EtOAc. The organic layer
was separated, washed with 0.1 N NaOH solution and brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified
by column chromatography (NH silica gel, hexane/EtOAc = 50/50)
to give the title compound (1.1 mg, 0.77%) as white crystals. 1H
5.1.3. 4-[(4-Fluorobenzyl)oxy]pyridine 1-oxide (3c)
The title compound was prepared in 6% yield using (4-fluo-
rophenyl)methanol in an analogous manner to 3a. 1H NMR
(300 MHz, DMSO-d6) d 5.15 (2H, s), 7.04–7.13 (2H, m), 7.19–7.30
(2H, m), 7.46–7.57 (2H, m), 8.07–8.14 (2H, m). MS (ESI/APCI) m/z
220.1 [M+H]+.

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2495
NMR (400 MHz, DMSO-d6) d 0.95 (4H, d, J = 2.0 Hz), 2.06–2.17 (1H,
m), 2.45 (3H, s), 5.18 (2H, s), 6.01–6.06 (1H, m), 6.16–6.24 (1H, m),
7.50 (4H, s), 7.68–7.75 (1H, m), 8.34–8.41 (1H, m), 8.87–8.94 (1H,
m). MS (ESI/APCI) m/z = 407.4 [M+H]+. Purity 99.2% (HPLC).
vacuo. The residue was dissolved with AcOH (2.0 mL) and stirred
at 90 °C for 1 h. After evaporating, the residue was purified by col-
umn chromatography (NH silica gel, hexane/EtOAc = 90/10 to
0/100). The residual solid was recrystallized from EtOAc–MeOH
to give the title compound (48.7 mg, 47%) as an off-white solid;
mp 217–219 °C. 1H NMR (300 MHz, DMSO-d6) d 1.00 (3H, t,
J = 7.4 Hz), 1.74–1.88 (2H, m), 2.86 (2H, t, J = 7.6 Hz), 3.74 (3H, s),
5.13 (2H, s), 5.98 (1H, s), 6.09 (1H, dd, J = 7.6, 3.0 Hz), 7.06 (1H,
dd, J = 8.5, 2.1 Hz), 7.21–7.31 (2H, m), 7.50–7.56 (3H, m),
7.56–7.61 (2H, m). 13C NMR (101 MHz, DMSO-d6) d 13.7, 20.1,
28.4, 29.6, 68.8, 97.8, 99.8, 108.7, 115.3 (d, J = 21.2 Hz), 118.0,
120.3, 130.2 (d, J = 8.1 Hz), 132.1 (d, J = 3.0 Hz), 134.6, 135.6,
135.6, 139.7, 141.5, 156.7, 161.9 (d, J = 245.4 Hz), 162.7, 162.8,
166.6. MS (ESI/APCI) m/z = 392.2 [M+H]+. Anal. Calcd for
5.1.10. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-methyl-1H-
benzimidazol-6-yl)pyridin-2(1H)-one (6a)
To a stirred degassed mixture of 8a (502 mg, 2.0 mmol), 4a
(470 mg, 2.0 mmol), and K2CO3 (552 mg, 4.0 mmol) in dioxane
(15 mL) were added CuI (76 mg, 0.4 mmol) and trans-N,N0-
dimethyl-cyclohexane-1,2-diamine (56 mg, 0.4 mmol). The reac-
tion vessel was sealed and heated at 110 °C for 16 h. The reaction
mixture was cooled to rt and concentrated. The resulting residue
was diluted with DCM (250 mL), washed with brine (100 mL),
dried over Na2SO4, and concentrated. The residue was purified by
column chromatography (silica gel, DCM/MeOH = 97/3 to 96/4)
to give the title compound (150 mg, 18%) as a white solid. 1H
NMR (400 MHz, CD3OD) d 1.14–1.20 (4H, m), 2.24 (1H, m), 3.90
(3H, s), 5.16 (2H, s), 6.09 (1H, d, J = 2.6 Hz), 6.27 (1H, dd, J = 7.6,
2.7 Hz), 7.15 (1H, dd, J = 8.5, 2.0 Hz), 7.41–7.50 (5H, m), 7.69 (2H,
t, J = 8.3 Hz). MS (ESI/APCI) m/z = 406.0 [M+H]+. Anal. Calcd for
C
23H22FN3O2ꢂ0.1H2O: C, 70.25; H, 5.69; N, 10.69. Found: C, 70.28;
H, 5.57; N, 10.71.
5.1.14. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-[(4-
fluorobenzyl)oxy]pyridin-2(1H)-one (6e)
To a solution of 4c (2.44 g, 11.2 mmol), 8a (2.8 g, 11.15 mmol),
K2CO3 (4.62 g, 33.5 mmol), and DMEDA (1.20 mL, 11.15 mmol) in
DMSO (56 mL) was added CuI (2.12 g, 11.2 mmol), and the mixture
was stirred at 150 °C under Ar atmosphere for 2 h. After cooling to
0 °C, 28% NH3 solution (56.0 mL) was added, and the mixture was
allowed to warm to rt for 2 h. The precipitate was collected by fil-
tration, washed with water and IPE, dissolved in THF (500 mL), and
filtered through a short NH silica-gel column (EtOAc). The filtrate
was concentrated and the residue was purified by column chro-
matography (NH silica gel, hexane/EtOAc = 90/10 to 0/100), fol-
lowed by recrystallized from EtOH–water to give the title
compound (1.60 g, 37%) as an off-white solid; mp 221–223 °C. 1H
NMR (300 MHz, CDCl3) d 0.99–1.15 (4H, m), 2.20–2.33 (1H, m),
3.85 (3H, s), 5.13 (2H, s), 5.98 (1H, d, J = 2.6 Hz), 6.09 (1H, dd,
J = 7.7, 2.8 Hz), 7.00–7.09 (1H, m), 7.21–7.32 (2H, m), 7.43–7.67
(5H, m). 13C NMR (101 MHz, DMSO-d6) d 7.0, 8.3, 29.6, 68.8, 97.8,
99.8, 108.6, 115.3 (d, J = 22.2 Hz), 117.6, 120.5, 130.2 (d,
J = 9.1 Hz), 132.1 (d, J = 3.0 Hz), 134.5, 135.7, 139.6, 141.0, 158.2,
161.9 (d, J = 245.4 Hz), 162.7, 166.6. MS (ESI/APCI) m/z = 390.2
[M+H]+. Anal. Calcd for C23H20FN3O2ꢂ0.1H2O: C, 70.61; H, 5.20; N,
10.74. Found: C, 70.53; H, 5.19; N, 10.69.
C23H20ClN3O2: C, 68.06; H, 4.97; N, 10.35. Found: C, 67.96; H,
5.01; N, 10.30.
5.1.11. 1-(1,2-Dimethyl-1H-benzimidazol-6-yl)-4-[(4-fluorobenzyl)
oxy]pyridin-2(1H)-one (6b)
The title compound was prepared in 25% yield using 4c and 8d
in an analogous manner to 6e. White solid; mp 256–258 °C. 1H
NMR (300 MHz, DMSO-d6) d 2.55 (3H, s), 3.73 (3H, s), 5.14 (2H,
s), 5.99 (1H, s), 6.09 (1H, d, J = 6.8 Hz), 7.06 (1H, d, J = 8.5 Hz),
7.26 (2H, t, J = 8.7 Hz), 7.45–7.64 (5H, m). 13C NMR (101 MHz,
DMSO-d6)
d 13.4, 29.7, 68.8, 97.8, 99.8, 108.6, 115.4 (d,
J = 22.2 Hz), 117.8, 120.2, 130.2 (d, J = 8.1 Hz), 132.1 (d,
J = 3.0 Hz), 134.6, 135.6, 139.7, 141.5, 141.5, 153.6, 161.9 (d,
J = 245.4 Hz), 162.7, 166.6. MS (ESI/APCI) m/z = 364.3 [M+H]+. Anal.
Calcd for C21H18FN3O2: C, 69.41; H, 4.99; N, 11.56. Found: C, 69.29;
H, 5.04; N, 11.45.
5.1.12. 1-(2-Ethyl-1-methyl-1H-benzimidazol-6-yl)-4-[(4-fluoro-
benzyl)oxy]pyridin-2(1H)-one (6c)
A suspension of 9b (5.00 g, 18.6 mmol), 1-(chloromethyl)-4-
fluorobenzene (5.37 g, 37.1 mmol), K2CO3 (7.70 g, 55.7 mmol),
and DMF (50 mL) was stirred at rt for 19 h. The resulting precipi-
tate was collected by filtration, and the solid was washed with
IPE and water successively to give a crude product (3.89 g). Other
two bathes using 35 g and 50 g of 9b gave 33.2 g and 50.0 g of
crude product, respectively. Three lots were combined and recrys-
tallized from MeOH–water to give the title compound (71.4 g, 57%,
three bathes) as a white solid; mp 228–229 °C. 1H NMR (300 MHz,
CDCl3) d 1.46 (3H, t, J = 7.6 Hz), 2.93 (2H, q, J = 7.5 Hz), 3.73 (3H, s),
5.02 (2H, s), 6.01–6.11 (2H, m), 7.05–7.16 (3H, m), 7.24–7.45 (6H,
m), 7.77 (1H, d, J = 8.5 Hz). 13C NMR (75 MHz, DMSO-d6) d 11.3,
20.1, 29.5, 68.9, 97.8, 99.8, 108.8, 115.4 (d, J = 21.0 Hz), 118.1,
120.3, 130.3 (d, J = 8.3 Hz), 132.2 (d, J = 3.0 Hz), 134.7, 135.7,
139.7, 141.5, 157.8, 162.0 (d, J = 243.0 Hz), 162.8, 166.7. MS (ESI/
APCI) m/z = 378.3 [M+H]+. Anal. Calcd for C22H20FN3O2: C, 70.01;
H, 5.34; N, 11.13. Found: C, 69.90; H, 5.26; N, 11.12.
5.1.15. 1-(2-Cyclobutyl-1-methyl-1H-benzimidazol-6-yl)-4-[(4-
fluorobenzyl)oxy]pyridin-2(1H)-one (6f)
The title compound was prepared in 22% yield using 4c and 8g
in an analogous manner to 6a. White solid; mp 246–249 °C. 1H
NMR (400 MHz, CDCl3) d 1.91–1.94 (1H, m), 2.05–2.12 (1H, m),
2.39–2.46 (4H, m), 3.66 (3H, s), 3.89 (1H, m), 5.13 (2H, s), 5.98
(1H, d, J = 2.8 Hz), 6.08 (1H, dd, J = 7.6, 2.8 Hz), 7.07 (1H, dd,
J = 8.4, 2.0 Hz), 7.26 (2H, t, J = 8.8 Hz), 7.51–7.55 (3H, m), 7.57–
7.63 (2H, m). 13C NMR (101 MHz, DMSO-d6) d 18.1, 26.4, 29.4,
31.5, 68.8, 97.8, 99.8, 108.7, 115.3 (d, J = 21.2 Hz), 118.2, 120.3,
130.2 (d, J = 9.1 Hz), 32.1 (d, J = 3.0 Hz), 134.7, 135.9, 139.6,
141.4, 159.2, 161.9 (d, J = 245.4 Hz), 162.7, 166.6. MS (ESI/APCI)
m/z = 404.0 [M+H]+. Anal. Calcd for C24H22FN3O2ꢂ0.11H2O: C,
71.10; H, 5.52; N, 10.36. Found: C, 71.14; H, 5.42; N, 10.32.
5.1.16. 1-(2-Cyclopentyl-1-methyl-1H-benzimidazol-6-yl)-4-[(4-
fluorobenzyl)oxy]pyridin-2(1H)-one (6g)
5.1.13. 4-[(4-Fluorobenzyl)oxy]-1-(1-methyl-2-propyl-1H-benzi-
midazol-6-yl)pyridin-2(1H)-one (6d)
The title compound was prepared in 22% yield using cyclopen-
tanecarboxylic acid in an analogous manner to 6d. White solid; mp
262–263 °C. 1H NMR (300 MHz, DMSO-d6) d 1.59–1.85 (4H, m),
1.88–1.99 (2H, m), 2.07 (2H, br s), 3.45 (1H, t, J = 7.7 Hz), 3.76
(3H, s), 5.13 (2H, s), 5.99 (1H, d, J = 2.6 Hz), 6.09 (1H, dd, J = 7.5,
2.6 Hz), 7.06 (1H, dd, J = 8.5, 2.1 Hz), 7.26 (2H, t, J = 8.9 Hz), 7.46–
7.64 (5H, m). 13C NMR (101 MHz, DMSO-d6) d 25.3, 29.6, 31.1,
36.2, 68.8, 97.8, 99.8, 108.7, 115.3 (d, J = 21.2 Hz), 118.1, 120.2,
The mixture of 14 (90 mg, 0.27 mmol), HATU (106 mg,
0.28 mmol), nbutyric acid (0.024 mL, 0.27 mmol), DIPEA
(0.136 mL, 0.80 mmol), and DMF (2 mL) was stirred at ambient
temperature for 1 h. The mixture was quenched with water and
extracted with EtOAc. The organic layer was separated, washed
with water and brine, dried over MgSO4, and concentrated in

2496
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
130.2 (d, J = 8.1 Hz), 132.1 (d, J = 3.0 Hz), 134.6, 135.9, 139.7, 141.3,
160.3, 161.9 (d, J = 245.4 Hz), 162.7, 166.6. MS (ESI/APCI) m/
z = 418.1 [M+H]+. Anal. Calcd for C25H24FN3O2ꢂ0.14H2O: C, 71.49;
H, 5.83; N, 10.00. Found: C, 71.48; H, 5.67; N, 10.04.
5.1.21. 4-(Benzyloxy)-1-(2-cyclopropyl-1-methyl-1H-benzimida-
zol-6-yl)pyridin-2(1H)-one (6l)
The title compound was prepared in 87% yield using 4-(benzy-
loxy)pyridin-2(1H)-one and 8a in an analogous manner to 6a.
White solid; mp 210–211 °C. 1H NMR (300 MHz, CDCl3) d 0.95–
1.19 (4H, m), 2.18–2.34 (1H, m), 3.85 (3H, s), 5.15 (2H, s), 5.98
(1H, d, J = 3.0 Hz), 6.10 (1H, dd, J = 7.6, 2.6 Hz), 6.96–7.11 (1H, m),
7.29–7.66 (8H, m). 13C NMR (101 MHz, DMSO-d6) d 7.1, 8.3, 29.5,
69.6, 97.8, 99.8, 108.5, 117.7, 120.3, 127.8, 128.1, 128.5, 134.5,
135.8, 135.9, 139.6, 141.4, 158.3, 162.8, 166.7. MS (ESI/APCI)
m/z = 372.0 [M+H]+. Anal. Calcd for C23H21N3O2: C, 74.37; H, 5.70;
N, 11.31. Found: C, 74.19; H, 5.76; N, 11.16.
5.1.17. 1-[2-(Cyclopropylmethyl)-1-methyl-1H-benzimidazol-6-
yl]-4-[(4-fluorobenzyl)oxy]pyridin-2(1H)-one (6h)
The title compound was prepared in 37% yield using 4c and 8e
in an analogous manner to 6j. Pale yellow solid; mp 206–214 °C. 1H
NMR (300 MHz, CDCl3) d 0.27–0.37 (2H, m), 0.57–0.68 (2H, m),
1.12–1.25 (1H, m), 2.87 (2H, d, J = 6.4 Hz), 3.75 (3H, s), 5.02 (2H,
s), 5.96–6.13 (2H, m), 7.04–7.16 (3H, m), 7.31 (1H, d, J = 7.2 Hz),
7.35–7.45 (3H, m), 7.79 (1H, d, J = 8.3 Hz). 13C NMR (101 MHz,
DMSO-d6) d 4.5, 8.9, 29.8, 31.1, 68.8, 97.8, 99.8, 108.8, 115.3 (d,
J = 21.2 Hz), 118.1, 120.3, 130.2 (d, J = 8.1 Hz), 132.1 (d,
J = 3.0 Hz), 134.7, 135.6, 139.7, 141.6, 156.4, 161.9 (d,
J = 245.4 Hz), 162.7, 166.6. MS (ESI/APCI) m/z = 404.2 [M+H]+. Anal.
Calcd for C24H22FN3O2: C, 71.45; H, 5.50; N, 10.42. Found: C, 70.88;
H, 5.57; N, 10.13.
5.1.22. 4-[(3-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-methyl-1H-
benzimidazol-6-yl)pyridin-2(1H)-one (6m)
The title compound was prepared in 29% yield using 4b and 8a
in an analogous manner to 6a. White solid; mp 220–222 °C. 1H
NMR (400 MHz, DMSO-d6) d 1.02–1.11 (4H, m), 2.25–2.29 (1H,
m), 3.85 (3H, s), 5.18 (2H, s), 5.97 (1H, d, J = 2.4 Hz), 6.12 (1H, dd,
J = 7.5, 2.5 Hz), 7.04 (1H, dd, J = 8.5, 1.6 Hz), 7.43–7.48 (3H, m),
7.49–7.55 (3H, m), 7.59 (1H, d, J = 7.6 Hz). 13C NMR (75 MHz,
DMSO-d6) d 7.1, 8.4, 29.6, 68.6, 97.9, 99.8, 108.6, 117.8, 120.4,
126.4, 127.5, 128.1, 130.5, 133.1, 134.4, 135.8, 138.5, 139.8,
141.4, 158.3, 162.8, 166.5. MS (ESI/APCI) m/z = 405.8 [M+H]+. Anal.
Calcd for C23H20ClN3O2: C, 68.06; H, 4.97; N, 10.35. Found: C,
67.94; H, 4.91; N, 10.31.
5.1.18. 1-[2-(2,2-Dimethylpropyl)-1-methyl-1H-benzimidazol-
6-yl]-4-[(4-fluorobenzyl)oxy]pyridin-2(1H)-one (6i)
The title compound was prepared in 34% yield using 4c and 8f
in an analogous manner to 6e. White solid; mp 238–239 °C. 1H
NMR (300 MHz, DMSO-d6) d 1.04 (9H, s), 2.81 (2H, s), 3.77 (3H,
s), 5.14 (2H, s), 5.99 (1H, d, J = 2.6 Hz), 6.09 (1H, dd, J = 7.6,
2.6 Hz), 7.08 (1H, dd, J = 8.5, 2.1 Hz), 7.20–7.32 (2H, m), 7.48–
7.56 (3H, m), 7.61 (2H, dd, J = 7.9, 5.3 Hz). 13C NMR (101 MHz,
DMSO-d6) d 29.4, 30.4, 32.4, 39.0, 68.8, 97.8, 99.8, 109.0, 115.3
(d, J = 21.2 Hz), 118.1, 120.4, 130.2 (d, J = 8.1 Hz), 132.1 (d,
J = 3.0 Hz), 134.6, 135.3, 139.7, 141.6, 155.0, 161.9 (d,
J = 245.4 Hz), 162.8, 166.7. MS (ESI/APCI) m/z = 420.2 [M+H]+. Anal.
Calcd for C25H26FN3O2: C, 71.58; H, 6.25; N, 10.02. Found: C, 71.46;
H, 61.7; N, 9.97.
5.1.23. 4-[(2-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-methyl-1H-
benzimidazol-6-yl)pyridin-2(1H)-one (6n)
To a suspension of 9a (100 mg, 0.36 mmol) and (2-chloro-
phenyl)methanol (101 mg, 0.71 mmol) in THF (2 mL) were
added tributylphosphine (0.266 ml, 1.07 mmol) and ADDP
(269 mg, 1.07 mmol) at 60 °C, and the mixture was stirred at
the same temperature for 3 h. After solvent was removed by
evaporation, the residue was purified by column chromatogra-
phy (silica gel, hexane/EtOAc = 90/10 to 0/100, then EtOAc/
MeOH = 100/0 to 85/15), followed by column chromatography
(NH silica gel, hexane/EtOAc = 90/10 to 0/100, then EtOAc/
MeOH = 100/0 to 85/15). The residual solid was recrystallized
by EtOH–hexane to give the title compound (60.0 mg, 42%) as
an off-white solid; mp 199–201 °C. 1H NMR (400 MHz, DMSO-
d6) d 1.01–1.13 (4H, m), 2.23–2.31 (1H, m), 3.85 (3H, s), 5.20
(2H, s), 6.02 (1H, d, J = 2.6 Hz), 6.10 (1H, dd, J = 7.5, 2.5 Hz),
7.06 (1H, dd, J = 8.6, 1.6 Hz), 7.40–7.48 (2H, m), 7.50–7.57 (3H,
m), 7.59 (1H, d, J = 7.5 Hz), 7.61–7.66 (1H, m). 13C NMR
(75 MHz, DMSO-d6) d 7.1, 8.4, 29.6, 67.3, 97.7, 99.6, 108.6,
117.8, 120.4, 127.5, 129.5, 130.4, 130.7, 133.0, 133.1, 134.4,
135.8, 139.8, 141.4, 158.3, 162.8, 166.7. MS (ESI/APCI) m/z =
406.1 [M+H]+. Anal. Calcd for C23H20ClN3O2: C, 68.06; H, 4.97;
N, 10.35. Found: C, 68.11; H, 4.88; N, 10.31.
5.1.19. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-ethyl-1H-
benzimidazol-6-yl)pyridin-2(1H)-one (6j)
A mixture of 4a (100 mg, 0.42 mmol), 8b (124 mg, 0.47 mmol),
CuI (81 mg, 0.42 mmol), DMEDA (0.048 mL, 0.42 mmol), K2CO3
(147 mg, 1.06 mmol), and DMSO (2.5 mL) was heated 120 °C for
1 h under microwave irradiation. The mixture was quenched with
28% ammonia solution at rt and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over MgSO4, and con-
centrated in vacuo. The residue was purified by column chromatog-
raphy (NH silica gel, hexane/EtOAc = 75/25 to 0/100). The solid was
crystallized from IPA–hexane to give the title compound (85 mg,
48%) as a light pink solid; mp 203–204 °C. 1H NMR (300 MHz, CDCl3)
d 1.08–1.19 (2H, m), 1.20–1.31 (2H, m), 1.46 (3H, t, J = 7.4 Hz), 1.93–
2.05 (1H, m), 4.29 (2H, q, J = 7.2 Hz), 5.02 (2H, s), 6.01–6.08 (2H, m),
7.10 (1H, dd, J = 8.7, 1.9 Hz), 7.28–7.42 (6H, m), 7.70 (1H, d,
J = 8.3 Hz). 13C NMR (75 MHz, DMSO-d6) d 7.0, 7.1, 8.4, 15.0, 37.7,
68.7, 97.9, 99.8, 108.4, 117.8, 120.4, 128.5, 129.7, 132.7, 134.5,
134.8, 135.0, 139.8, 141.5, 157.6, 162.8, 166.6. MS (ESI/APCI) m/
z = 420.1 [M+H]+. Anal. Calcd for C24H22ClN3O2: C, 68.65; H, 5.28;
N, 10.01. Found: C, 68.53; H, 5.29; N, 9.73.
5.1.24. 4-[(5-Chloropyridin-2-yl)methoxy]-1-(2-cyclopropyl-1-
methyl-1H-benzimidazol-6-yl)-pyridin-2(1H)-one (6o)
The title compound was prepared in 14% yield using 9a and (5-
chloropyridin-2-yl)methanol in an analogous manner to 6c. White
solid; mp 234–236 °C. 1H NMR (400 MHz, DMSO-d6) d 1.04–1.09
(4H, m), 2.26–2.32 (1H, m), 3.84 (3H, s), 5.23 (2H, s), 5.96 (1H, s),
6.12 (1H, dd, J = 7.7, 2.6 Hz), 7.04 (1H, m), 7.50–7.52 (2H, m),
7.59–7.61 (2H, m), 8.02–8.04 (1H, m), 8.67 (1H, s). 13C NMR
(75 MHz, DMSO-d6) d 7.1, 8.4, 29.6, 69.8, 98.0, 99.7, 108.5, 117.8,
120.4, 123.5, 130.5, 130.5, 134.4, 135.8, 136.9, 139.9, 141.4,
147.8, 154.1, 158.3, 162.7, 166.5. MS (ESI/APCI) m/z = 407.4 [M
+H]+. Anal. Calcd for C22H19ClN4O2: C, 64.94; H, 4.71; N, 13.77.
Found: C, 64.77; H, 4.83; N, 13.50.
5.1.20. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-propyl-1H-
benzimidazol-6-yl)pyridin-2(1H)-one (6k)
The title compound was prepared in 11% yield using 4a and 8c
in an analogous manner to 6j. Off-white solid. 1H NMR (300 MHz,
CDCl3) d 1.00 (3H, t, J = 7.5 Hz), 1.08–1.20 (2H, m), 1.23–1.33 (2H,
m), 1.85–1.96 (2H, m), 1.96–2.04 (1H, m), 4.20 (2H, t, J = 7.3 Hz),
5.02 (2H, s), 6.00–6.13 (2H, m), 7.01–7.16 (3H, m), 7.28–7.36 (2H,
m), 7.37–7.45 (2H, m), 7.70 (1H, d, J = 8.3 Hz). MS (ESI/APCI)
m/z = 434.2 [M+H]+.

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2497
5.1.25. 4-[(5-Chloropyrimidin-2-yl)methoxy]-1-(2-cyclopropyl-
1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6p)
m), 2.26 (1H, m), 3.85 (3H, s), 5.32 (2H, s), 6.03–6.08 (2H, m), 7.04
(1H, dd, J = 8.5, 1.8 Hz), 7.28 (1H, s), 7.50–7.52 (2H, m), 7.58 (1H, d,
J = 7.5 Hz), 7.64 (1H, s). 13C NMR (101 MHz, DMSO-d6) d 7.1, 8.3,
29.5, 64.0, 97.9, 99.6, 108.5, 117.8, 120.3, 122.1, 123.0, 127.8,
134.4, 135.8, 139.4, 139.8, 141.4, 158.3, 162.7, 166.1. MS (ESI/APCI)
m/z = 412.2 [M+H]+. Purity > 99.9% (HPLC).
A
mixture of 6aa (100 mg, 0.27 mmol), 2-chloro-1,3-bis
(dimentylamino)trimethinium hexafluorophosphate (98 mg,
0.32 mmol), sodium methoxide (43.4 mg, 0.80 mmol), and MeOH
(3 mL) was stirred at rt for 1 h. The mixture was concentrated
and the residue was diluted with water. The aqueous phase was
extracted with EtOAc. The combined organic layers were washed
with water and brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography (NH
silica gel, hexane/EtOAc = 90/10 to 0/100) to give the title com-
pound (56.0 mg, 51%) as a white solid; mp 217–219 °C. 1H NMR
(400 MHz, DMSO-d6) d 0.94–1.17 (4H, m), 2.26 (1H, br s), 3.85
(3H, s), 5.36 (2H, s), 5.87 (1H, br s), 6.13 (1H, d, J = 9.0 Hz), 7.04
(1H, d, J = 9.0 Hz), 7.51 (2H, br s), 7.59 (1H, d, J = 6.9 Hz), 9.02
(2H, s). 13C NMR (101 MHz, DMSO-d6) d 7.0, 8.3, 29.5, 69.5, 97.9,
99.6, 108.5, 117.7, 120.3, 129.8, 134.4, 135.8, 139.8, 141.4, 156.1,
158.3, 162.5, 162.6, 166.6. MS (ESI/APCI) m/z = 408.3 [M+H]+.
5.1.30. 4-[(5-Chlorothiophen-3-yl)methoxy]-1-(2-cyclopropyl-
1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6u)
The title compound was prepared in 9% yield using 9a and
(5-chlorothiophen-3-yl)methanol in an analogous manner to 6n.
White solid; mp 236–237 °C. 1H NMR (400 MHz, DMSO-d6) d
1.02–1.11 (4H, m), 2.24–2.32 (1H, m), 3.85 (3H, s), 5.05 (2H, s),
5.98 (1H, d, J = 2.6 Hz), 6.06 (1H, dd, J = 7.6, 2.7 Hz), 7.03 (1H, dd,
J = 8.4, 1.8 Hz), 7.20 (1H, d, J = 1.4 Hz), 7.51 (2H, dd, J = 5.4,
3.5 Hz), 7.56–7.58 (2H, m). 13C NMR (101 MHz, DMSO-d6) d 7.1,
8.3, 29.5, 65.0, 97.7, 99.7, 108.5, 117.7, 120.3, 124.2, 127.0, 128.8,
134.4, 135.8, 136.5, 139.7, 141.4, 158.3, 162.7, 166.5. MS (ESI/APCI)
m/z = 412.0 [M+H]+. Anal. Calcd for C21H18ClN3O2S: C, 61.23; H,
4.40; N, 10.20. Found: C, 61.19; H, 4.39; N, 10.17.
5.1.26. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-
(thiophen-2-ylmethoxy)pyridin-2(1H)-one (6q)
The title compound was prepared in 30% yield using 9a and
thiophen-2-ylmethanol in an analogous manner to 6n. White
solid; mp 222–223 °C. 1H NMR (400 MHz, DMSO-d6) d 1.04–1.11
(4H, m), 2.24–2.28 (1H, m), 3.85 (3H, s), 5.34 (2H, s), 6.05–6.07
(2H, m), 7.03–7.08 (2H, m), 7.27 (1H, d, J = 2.8 Hz), 7.50–7.52
(2H, m), 7.57 (1H, d, J = 7.2 Hz), 7.61 (1H, d, J = 4.9 Hz). 13C NMR
(101 MHz, DMSO-d6) d 7.1, 8.3, 29.5, 64.4, 97.8, 99.7, 108.5,
117.8, 120.4, 126.9, 127.3, 128.3, 134.4, 135.8, 137.8, 139.7,
141.4, 158.3, 162.7, 166.3. MS (ESI/APCI) m/z = 377.8 [M+H]+.
Purity 99.4% (HPLC).
5.1.31. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-
{[5-(trifluoromethyl)thiophen-2-yl]methoxy}pyridin-2(1H)-one
(6v)
The title compound was prepared in 35% yield using 9a and [5-
(trifluoromethyl)thiophen-2-yl]methanol in an analogous manner
to 6n. White solid; mp 256–257 °C. 1H NMR (400 MHz, DMSO-d6)
d 0.99–1.14 (4H, m), 2.22–2.31 (1H, m), 3.85 (3H, s), 5.45 (2H, s),
6.06 (1H, d, J = 2.5 Hz), 6.10 (1H, dd, J = 7.5, 2.7 Hz), 7.05 (1H, dd,
J = 8.5, 1.8 Hz), 7.38 (1H, d, J = 2.8 Hz), 7.49–7.54 (2H, m), 7.60
(1H, d, J = 7.5 Hz), 7.69 (1H, d, J = 2.8 Hz). 13C NMR (101 MHz,
DMSO-d6) d 7.0, 8.3, 29.5, 64.0, 98.0, 99.5, 108.5, 117.8, 120.3,
125.0 (q, J = 269.7 Hz), 128.1, 129.4 (q, J = 37.4 Hz), 130.0 (q,
J = 4.0 Hz), 134.4, 135.8, 139.9, 141.4, 143.4 (d, J = 2.0 Hz), 158.3,
162.6, 166.0. MS (ESI/APCI) m/z = 446.1 [M+H]+. Anal. Calcd for
5.1.27. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-(thio
phen-3-ylmethoxy)pyridin-2(1H)-one (6r)
The title compound was prepared in 37% yield using 9a and
thiophen-3-ylmethanol in an analogous manner to 6n. Off-white
solid; mp 223–225 °C. 1H NMR (400 MHz, DMSO-d6) d 1.02–1.09
(4H, m), 2.24–2.28 (1H, m), 3.85 (3H, s), 5.13 (2H, s), 5.99 (1H, d,
J = 2.6 Hz), 6.06 (1H, dd, J = 7.5, 2.6 Hz), 7.04 (1H, dd, J = 8.4,
1.7 Hz), 7.19 (1H, d, J = 4.2 Hz), 7.50–7.52 (2H, m), 7.56–7.60 (2H,
m), 7.64 (1H, m). 13C NMR (101 MHz, DMSO-d6) d 7.1, 8.3,
29.5, 65.1, 97.6, 99.8, 108.5, 117.8, 120.4, 124.7, 126.8, 127.6,
134.5, 135.8, 136.7, 139.6, 141.4, 158.3, 162.8. MS (ESI/APCI)
m/z = 378.2 [M+H]+. Anal. Calcd for C21H19N3O2Sꢂ0.12H2O: C,
66.44; H, 5.11; N, 11.07. Found: C, 66.49; H, 5.09; N, 11.08.
C22H18F3N3O2S: C, 59.32; H, 4.07; N, 9.43. Found: C, 59.43; H,
4.10; N, 9.42.
5.1.32. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-
{[4-(trifluoromethyl)thiophen-2-yl]methoxy}pyridin-2(1H)-one
(6w)
NaH (60% oil dispersion, 87 mg, 2.18 mmol) was added to a
solution of [4-(trifluoromethyl)thiophen-2-yl]methanol (397 mg,
2.18 mmol) in DMA at 0 °C. After being stirred at the same temper-
ature for 30 min, 10 (500 mg, 1.45 mmol) was added to the
reaction mixture. The mixture was stirred at 120 °C for 10 min.
The mixture was quenched with water at 0 °C and extracted with
EtOAc. The organic layer was separated, washed with water and
brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography (NH silica gel, hexane/
EtOAc = 100/0 to 0/100), followed by preparative HPLC (L-Column
2 ODS, eluted with H2O in acetonitrile containing 0.1% TFA). The
desired fraction was neutralized with satd NaHCO3 solution and
extracted with EtOAc. The organic layer was separated, dried over
MgSO4, and concentrated in vacuo to give the title compound
(330 mg, 51%) as a pale yellow solid; mp 218–219 °C. 1H NMR
(400 MHz, DMSO-d6) d 0.95–1.14 (4H, m), 2.21–2.33 (1H, m),
3.85 (3H, s), 5.39 (2H, s), 6.05–6.10 (2H, m), 7.05 (1H, dd, J = 8.5,
1.2 Hz), 7.52 (2H, dd, J = 5.0, 3.2 Hz), 7.56–7.67 (2H, m), 8.33 (1H,
s). 13C NMR (101 MHz, DMSO-d6) d 7.0, 8.3, 29.5, 63.9, 97.9, 99.6,
108.5, 117.8, 120.3, 122.0 (q, J = 270.7 Hz), 124.57, 124.59, 129.3
(q, J = 35.4 Hz), 129.6 (q, J = 4.0 Hz), 134.4, 135.8, 139.8, 141.4,
158.3, 162.7, 166.1. MS (ESI/APCI) m/z = 446.1 [M+H]+. Anal. Calcd
for C22H18F3N3O2Sꢂ0.25H2O: C, 58.72; H, 4.14; N, 9.34. Found: C,
58.89; H, 4.21; N, 9.29.
5.1.28. 4-[(5-Chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-
1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s)
The title compound was prepared in 33% yield using 9a and (5-
chlorothiophen-2-yl)methanol in an analogous manner to 6n.
White solid; mp 218–220 °C. 1H NMR (400 MHz, DMSO-d6) d
1.02–1.11 (4H, m), 2.23–2.28 (1H, m), 3.85 (3H, s), 5.29 (2H, s),
6.03–6.07 (2H, m), 7.02 (1H, dd, J = 8.5, 1.8 Hz), 7.08 (1H, d,
J = 3.7 Hz), 7.16 (1H, d, J = 3.8 Hz), 7.51 (2H, dd, J = 5.4, 3.5 Hz),
7.57 (1H, d, J = 7.4). 13C NMR (75 MHz, DMSO-d6) d 7.1, 8.4, 29.6,
64.4, 97.9, 99.6, 108.5, 117.8, 120.4, 126.6, 128.3, 129.1, 134.4,
135.8, 137.3, 139.8, 141.4, 158.3, 162.7, 166.1. MS (ESI/APCI)
m/z = 412.3 [M+H]+. Anal. Calcd for C21H18ClN3O2S: C, 61.23; H,
4.40; N, 10.20. Found: C, 61.34; H, 4.43; N, 10.21.
5.1.29. 4-[(4-Chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-
1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6t)
The title compound was prepared in 24% yield using 9a and
(4-chlorothiophen-2-yl)methanol in an analogous manner to 6n.
Off-white solid. 1H NMR (400 MHz, DMSO-d6) d 1.04–1.09 (4H,

2498
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
5.1.33. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-
{[5-(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one
(6x)
with EtOAc. The organic layer was separated, washed with water
and brine, dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by column chromatography (silica gel, hexane/
EtOAc = 100/0 to 70/30) to give the title compound (1.10 g, 76%)
as yellow crystals. 1H NMR (400 MHz, DMSO-d6) d 0.87–1.01 (4H,
m), 2.14 (1H, br s), 2.51–2.53 (3H, s), 7.28 (1H, d, J = 9.3 Hz), 7.90
(1H, d, J = 9.3 Hz). MS (ESI/APCI) m/z = 252.0 [M+H]+.
The title compound was prepared in 26% yield using 10 and [5-
(trifluoromethyl)thiophen-3-yl]methanol in an analogous manner
to 6x. White solid; mp 236–237 °C. 1H NMR (400 MHz, DMSO-d6)
d 1.02–1.12 (4H, m), 2.22–2.31 (1H, m), 3.85 (3H, s), 5.16 (2H, s),
6.01 (1H, d, J = 2.5 Hz), 6.09 (1H, dd, J = 7.6, 2.4 Hz), 7.04 (1H, dd,
J = 8.5, 1.3 Hz), 7.48–7.55 (2H, m), 7.59 (1H, d, J = 7.5 Hz), 7.81
(1H, s), 8.06 (1H, s). 13C NMR (101 MHz, DMSO-d6) d 7.0, 8.3,
29.5, 64.5, 97.7, 99.7, 108.5, 117.8, 120.3, 122.4 (q, J = 270.0 Hz),
129.6, 129.8 (q, J = 37.4 Hz), 130.2 (q, J = 3.0 Hz), 134.4, 135.8,
137.2, 139.7, 141.4, 158.3, 162.7, 166.4. MS (ESI/APCI)
m/z = 446.3 [M+H]+. Anal. Calcd for C22H18F3N3O2S: C, 59.32; H,
4.07; N, 9.43. Found: C, 59.36; H, 4.25; N, 9.34.
5.1.38. 6-Bromo-2-cyclopropyl-3-methylimidazo[1,2-a]pyrazine
(7b)
To a solution of 18 (1.24 g, 2.92 mmol) in THF (10 mL) was
added TFAA (0.826 mL, 5.84 mmol) at 0 °C, and the mixture was
heated at 60 °C for 3 h. The mixture was poured into satd NaHCO3
solution at rt and extracted with EtOAc. The organic layer was sep-
arated, washed with 1 N NaOH solution and brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by col-
umn chromatography (silica gel, hexane/EtOAc = 100/0 to 50/50)
to give the title compound (0.59 g, 80%) as pale yellow crystals.
1H NMR (400 MHz, DMSO-d6) d 0.81–1.07 (4H, m), 2.15 (1H, t,
J = 4.8 Hz), 2.53 (3H, s), 8.61 (1H, s), 8.68 (1H, s). MS (ESI/APCI)
m/z = 252.2 [M+H]+.
5.1.34. 4-(Benzyloxy)-1-(2-ethyl-1-methyl-1H-benzimidazol-6-
yl)pyridin-2(1H)-one (6y)
The title compound was prepared in 47% yield using 4-(benzy-
loxy)pyridin-2(1H)-one and 8h in an analogous manner to 6e.
White solid. 1H NMR (300 MHz, CDCl3) d 1.46 (3H, t, J = 7.5 Hz),
2.93 (2H, q, J = 7.6 Hz), 3.73 (3H, s), 5.06 (2H, s), 6.03–6.13 (2H,
m), 7.13 (1H, dd, J = 8.4, 1.9 Hz), 7.28–7.48 (7H, m), 7.77 (1H, d,
J = 8.4 Hz). 13C NMR (75 MHz, DMSO-d6) d 11.3, 20.1, 29.5, 69.6,
97.8, 99.9, 108.8, 118.1, 120.3, 127.9, 128.2, 128.5, 134.7, 135.7,
135.9, 139.7, 141.5, 157.8, 162.8, 166.8. MS (ESI/APCI)
m/z = 360.3 [M+H]+. Anal. Calcd for C22H21N3O2ꢂ0.92H2O: C,
70.28; H, 6.12; N, 11.18. Found: C, 70.18; H, 5.72; N, 11.13.
5.1.39. 6-Bromo-2-cyclopropyl-3-methylimidazo[1,2-a]pyrimidine
(7c)
To a solution of 15b (500 mg, 2.87 mmol) in DMF (10 mL) was
added 2-bromo-1-cyclopropylpropan-1-one (0.70 mL, 5.75 mmol)
at rt, and the mixture was stirred at 100 °C for 24 h. The mixture
was poured into 1 N NaOH solution and extracted with EtOAc.
The organic layer was separated, washed with 1 N NaOH solution
and brine, dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by column chromatography (NH silica gel, hex-
ane/EtOAc = 100/0 to 50/50) to give the title compound (89 mg,
12%) as pale yellow crystals. 1H NMR (400 MHz, DMSO-d6) d
0.84–1.02 (4H, m), 2.04–2.15 (1H, m), 2.50 (3H, br s), 8.41 (1H,
s), 8.98 (1H, s).
5.1.35. {[1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-2-oxo-
1,2-dihydropyridin-4-yl]oxy}acetonitrile (6z)
A
mixture of 9a (1.00 g, 3.55 mmol), bromoacetonitrile
(0.27 mL, 3.91 mmol), K2CO3 (1.47 g, 10.7 mmol), and DMF
(10 mL) was stirred at 80 °C for 2 h. The mixture was poured into
water and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated. The residue was puri-
fied by column chromatography (NH silica gel, hexane/
EtOAc = 97/3 to 0/100) to give the title compound (0.87 g, 76%)
as a white solid; mp 184–186 °C. 1H NMR (400 MHz, DMSO-d6) d
0.97–1.20 (4H, m), 2.27 (1H, br s), 3.85 (3H, s), 5.25 (2H, s), 6.08
(1H, br s), 6.14 (1H, d, J = 7.65 Hz), 7.07 (1H, d, J = 8.5 Hz), 7.49–
7.59 (2H, m), 7.67 (1H, d, J = 7.4 Hz). 13C NMR (101 MHz, DMSO-
d6) d 7.1, 8.4, 29.5, 53.3, 98.2, 98.9, 108.6, 115.7, 117.8, 120.3,
134.2, 135.8, 140.4, 141.5, 158.4, 162.4, 164.9. MS (ESI/APCI)
m/z = 321.3 [M+H]+.
5.1.40. 6-Bromo-2-cyclopropyl-1-methyl-1H-benzimidazole (8a)
A mixture of 12a (4.20 g, 18.2 mmol), zinc (5.94 g, 90.9 mmol),
NH4Cl (9.7 g, 182 mmol), MeOH (50 mL), and water (25 mL) was
stirred at rt for 3 h. After MeOH was removed by evaporation,
the mixture was neutralized with satd NaHCO3 solution and
extracted with EtOAc. The organic layer was separated, washed
with water and brine, dried over MgSO4, and concentrated in
vacuo. Then the residue was dissolved in POCl3 (1.68 mL,
18.0 mmol) and cyclopropanecarboxylic acid (2.86 mL, 36.0 mmol)
was added to the mixture at rt. The mixture was stirred at 120 °C
for 3 h. After cooling to 0 °C, ice water and satd NaHCO3 solution
were carefully added, and the mixture was extracted with EtOAc.
The extract was washed with brine, dried over MgSO4, concen-
trated to give a brown solid. This solid was dissolved in 1 N HCl
solution and washed with EtOAc. The aqueous layer was basified
with 4 N NaOH solution and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concentrated
to give the title compound (3.3 g, 72%) as a brown solid. 1H NMR
(300 MHz, DMSO-d6) d 0.95–1.14 (4H, m), 2.23 (1H, tt, J = 7.9,
5.1 Hz), 3.83 (3H, s), 7.24 (1H, dd, J = 8.5, 2.1 Hz), 7.41 (1H, d,
J = 8.7 Hz), 7.75 (1H, d, J = 1.9 Hz). 13C NMR (101 MHz, DMSO-d6)
d 7.0, 8.4, 29.5, 112.4, 113.4, 119.6, 123.9, 137.2, 141.1, 157.9. Anal.
Calcd for C11H11BrN2: C, 52.61; H, 4.42; N, 11.16. Found: C, 52.37;
H, 4.31; N, 11.14.
5.1.36. 2-{[1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-
2-oxo-1,2-dihydropyridin-4-yl]oxy}ethanimidamide hydrochloride
(6aa)
Sodium methoxide (2.53 mg, 0.050 mmol) was added to a solu-
tion of 6z (300 mg, 0.94 mmol) in MeOH (4 mL) and the mixture
was stirred at rt for 4 h. To the solution was added ammonium
chloride (52.6 mg, 0.98 mmol) and the mixture was stirred at rt
overnight. The solvent was evaporated to give the title compound
(368 mg, quant.) as a light brown solid. 1H NMR (400 MHz, DMSO-
d6) d 1.19 (4H, m), 2.27 (1H, br s), 3.86 (3H, s), 4.99 (2H, s), 5.87
(1H, s), 6.16 (1H, d, J = 5.3 Hz), 7.04 (1H, d, J = 7.8 Hz), 7.48–7.57
(2H, m), 7.68 (1H, d, J = 7.5 Hz), 9.04 (3H, br s). MS (ESI/APCI)
m/z = 321.3 [M+H]+.
5.1.37. 6-Bromo-2-cyclopropyl-3-methylimidazo[1,2-b]pyridazine
(7a)
To a solution of 15a (1.0 g, 5.75 mmol) in DMA (10 mL) was
added 2-bromo-1-cyclopropylpropan-1-one (1.40 mL, 11.5 mmol)
and NaHCO3 (0.97 g, 11.5 mmol) at rt, and the mixture was stirred
at 80 °C for 16 h. The mixture was poured into water and extracted
5.1.41. 6-Bromo-2-cyclopropyl-1-ethyl-1H-benzimidazole (8b)
Zinc (8.0 g, 122 mmol) was added to a solution of 12b (3.0 g,
12.2 mmol) in AcOH (60 mL) at rt. The mixture was stirred at
ambient temperature for 30 min. The insoluble material was
removed by filtration and the filtrate was concentrated in vacuo.

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2499
The mixture was neutralized with satd NaHCO3 solution
and extracted with EtOAc. The organic layer was separated,
washed with water and brine, dried over MgSO4, and concentrated
in vacuo to give an intermediate 4-bromo-N2-ethylbenzene-1,2-
diamine.
5.1.45. 6-Bromo-2-(2,2-dimethylpropyl)-1-methyl-1H-benzimi
dazole (8f)
The title compound was prepared in 78% yield using 20b in an
analogous manner to 8d. White solid. 1H NMR (300 MHz, CDCl3) d
1.02 (9H, s), 2.77 (2H, s), 3.75 (3H, s), 7.28 (1H, dd, J = 8.7, 1.9 Hz),
7.51 (1H, d, J = 8.3 Hz), 7.76 (1H, d, J = 1.9 Hz). MS (ESI/APCI)
m/z = 281.0 [M+H]+.
HATU (4.89 g, 12.9 mmol) was added to a solution of the inter-
mediate 4-bromo-N2-ethylbenzene-1,2-diamine, DIPEA (6.40 mL,
36.7 mmol),
and
cyclopropanecarboxylic
acid
(0.98 mL,
12.2 mmol) in DMF (40 mL), and the mixture was stirred at rt for
1 h. The mixture was quenched with water and extracted
with EtOAc. The organic layer was separated, washed with water
and brine, dried over MgSO4, and concentrated in vacuo. The
residue was dissolved in AcOH (40 mL) and the mixture was stirred
at 80 °C for 1 h. After concentration of the mixture, the residue
was neutralized with satd NaHCO3 solution and extracted with
EtOAc. The organic layer was separated, washed with water and
brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, hexane/
EtOAc = 100/0 to 0/100) to give the title compound (1.2 g, 37%)
as a pale yellow solid. 1H NMR (300 MHz, CDCl3) d 1.03–1.18
(2H, m), 1.19–1.30 (2H, m), 1.46 (3H, t, J = 7.2 Hz), 1.96 (1H, tt,
J = 8.2, 4.9 Hz), 4.25 (2H, q, J = 7.3 Hz), 7.29 (1H, dd, J = 8.3,
1.9 Hz), 7.43 (1H, d, J = 1.5 Hz), 7.50 (1H, d, J = 8.3 Hz). 13C NMR
(101 MHz, DMSO-d6) d 7.0, 8.4, 14.9, 37.6, 112.3, 113.5, 119.7,
123.9, 136.1, 141.2, 157.3. MS (ESI/APCI) m/z = 265.1 [M+H]+. Anal.
Calcd for C12H13BrN2: C, 54.36; H, 4.94; N, 10.57. Found: C, 54.29;
H, 4.87; N, 10.58.
5.1.46. 6-Bromo-2-cyclobutyl-1-methyl-1H-benzimidazole (8g)
To a mixture of cyclobutanecarboxylic acid (352 lL, 3.73 mmol)
and 19 (500 mg, 2.48 mmol) was added POCl3 (10 mL) and the
mixture was heated under reflux for 4 h. The mixture was cooled
to rt and poured into ice-cold satd NaHCO3 solution (100 mL).
The mixture was extracted with EtOAc, and the organic layer was
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The reside was purified by column chromatogra-
phy (silica gel, hexane/EtOAc = 70/30) to afford the title compound
(300 mg, 45%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) d
1.89–1.95 (1H, m), 2.01–2.12 (1H, m), 2.37–2.46 (4H, m), 3.64 (3H,
s), 3.80–3.88 (1H, m), 7.27 (1H, dd, J = 8.4, 1.6 Hz), 7.51 (1H, d,
J = 8.4 Hz), 7.75 (1H, d, J = 1.6 Hz). MS (ESI/APCI) m/z = 266.8
[M+H]+.
5.1.47. 6-Bromo-2-ethyl-1-methyl-1H-benzimidazole (8h)
The title compound was prepared in 57% yield using 12a and
propanoic acid in an analogous manner to 8e. 1H NMR (400 MHz,
CDCl3) d 1.45 (3H, t, J = 7.5 Hz), 2.89 (2H, q, J = 7.5 Hz), 3.69 (3H,
s), 7.32 (1H, dd, J = 8.5, 1.8 Hz), 7.44 (1H, d, J = 1.5 Hz), 7.57 (1H,
d, J = 8.5 Hz). MS (ESI/APCI) m/z = 239.0 [M+H]+.
5.1.42. 6-Bromo-2-cyclopropyl-1-propyl-1H-benzimidazole (8c)
The title compound was prepared in 56% yield using 12c in an
analogous manner to 8a. Pink solid. 1H NMR (300 MHz, CDCl3) d
1.00 (4H, t, J = 7.4 Hz), 1.07–1.16 (2H, m), 1.21–1.30 (2H, m),
1.80–2.01 (3H, m), 4.13–4.21 (2H, m), 7.26–7.32 (1H, m), 7.42
(1H, d, J = 1.9 Hz), 7.50 (1H, d, J = 8.7 Hz). MS (ESI/APCI)
m/z = 279.1 [M+H]+.
5.1.48. 1-(2-Cyclopropyl-1-methyl-1H-benzimidazol-6-yl)-4-
hydroxypyridin-2(1H)-one (9a)
A mixture of 6l (2.4 g, 6.46 mmol), 10% Pd–C (1.2 g), and MeOH
(60 mL) was hydrogenated under H2 atmosphere (1 atm) at rt for
3 h. The inorganic material was removed by filtration and the
filtrate was concentrated in vacuo to give the title compound
(1.57 g, 86%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6)
d 0.99–1.13 (4H, m), 2.21–2.30 (1H, m), 3.85 (3H, s), 5.65 (1H, br
s), 5.95 (1H, d, J = 7.4 Hz), 7.02 (1H, dd, J = 8.4, 2.0 Hz), 7.40–7.58
(3H, m), 10.78 (1H, br s). 13C NMR (101 MHz, DMSO-d6) d 7.1,
8.4, 29.6, 108.4, 109.0, 117.9, 120.0, 121.9, 133.8, 135.3, 135.8,
140.2, 141.7, 158.6, 160.4. MS (ESI/APCI) m/z = 282.1 [M+H]+. Anal.
Calcd for C16H15N3O2ꢂ0.11H2O: C, 67.84; H, 5.42; N, 14.83. Found:
C, 67.80; H, 5.42; N, 14.81.
5.1.43. 6-Bromo-1,2-dimethyl-1H-benzimidazole (8d)
To a solution of 20a (30.0 g, 109 mmol) in AcOH (300 mL) was
added zinc powder (35.9 g, 549 mmol) at rt. After being stirred
over 90 °C for 4 h, the reaction mixture was allowed to cool to rt,
and the zinc dust was removed by filtration with Celite pad and
washed with EtOAc. The filtrate was concentrated and partitioned
between EtOAc and satd NaHCO3 solution. The resulting precipi-
tate was removed by filtration with Celite pad, and the filtrate
was extracted with EtOAc. The organic layer was separated,
washed with water and brine, dried over MgSO4, and concentrated.
The residue was purified by chromatography (NH silica gel, hex-
ane/EtOAc = 100/0 to 0/100) to give the title compound (16.0 g,
65%) as a purple solid. 1H NMR (400 MHz, CDCl3) d 2.56–2.61
(3H, m), 3.69 (3H, s), 7.32 (1H, dd, J = 8.5, 1.3 Hz), 7.43 (1H, s),
7.53 (1H, d, J = 8.4 Hz). 13C NMR (101 MHz, DMSO-d6) d 13.4,
29.8, 112.6, 113.6, 119.6, 123.8, 137.1, 141.3, 153.3. MS (ESI/APCI)
m/z = 225.1 [M+H]+. Anal. Calcd for C9H9BrN2: C, 48.02; H, 4.03; N,
12.45. Found: C, 48.12; H, 3.99; N, 12.52.
5.1.49. 1-(2-Ethyl-1-methyl-1H-benzimidazol-6-yl)-4-hydroxy
pyridin-2(1H)-one (9b)
The title compound was prepared in 99% yield using 6y in an
analogous manner to 9a. Pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 1.33 (3H, t, J = 7.5 Hz), 2.90 (2H, q, J = 7.5 Hz), 3.74
(3H, s), 5.65 (1H, d, J = 2.5 Hz), 5.96 (1H, dd, J = 7.5, 2.5 Hz), 7.04
(1H, dd, J = 8.4, 2.0 Hz), 7.49–7.62 (3H, m), 10.88 (1H, br s). 13C
NMR (101 MHz, DMSO-d6) d 11.3, 20.0, 29.5, 98.4, 100.2, 108.7,
117.9, 120.3, 135.0, 135.7, 139.9, 141.4, 157.7, 163.0, 167.2. MS
(ESI/APCI) m/z = 470.2 [M+H]+. Anal. Calcd for C15H15N3O2ꢂ1.35H2O:
C, 61.36; H, 6.08; N, 14.31. Found: C, 61.49; H, 5.73; N, 14.39.
5.1.44. 6-Bromo-2-(cyclopropylmethyl)-1-methyl-1H-benzimida
zole (8e)
The title compound was prepared in 96% yield using 12a and
cyclopropylacetic acid in an analogous manner to 8b. 1H NMR
(300 MHz, CDCl3) d 0.31 (2H, d, J = 6.0 Hz), 0.63 (2H, dd, J = 7.9,
1.1 Hz), 1.08–1.27 (1H, m), 2.83 (2H, d, J = 6.4 Hz), 3.72 (3H, s),
7.34 (1H, d, J = 1.9 Hz), 7.45 (1H, d, J = 1.9 Hz), 7.58 (1H, d,
J = 8.7 Hz). MS (ESI/APCI) m/z = 265.1 [M+H]+.
5.1.50. 4-Bromo-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)
pyridin-2(1H)-one (10)
To a solution of 9a (1.0 g, 3.55 mmol) in DMF (15 mL) was
added phosphoryl tribromide (1.22 g, 4.27 mmol) at ambient tem-
perature. The mixture was stirred at 50 °C for 9 h. The mixture was
poured into satd NaHCO3 solution and extracted with EtOAc. The

2500
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
organic layer was separated, washed with water and brine, dried
over MgSO4, and concentrated in vacuo. The residual solid was
recrystallized from EtOH–hexane to give the title compound
(600 mg, 49%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) d
0.99–1.16 (4H, m), 2.22–2.32 (1H, m), 3.85 (3H, s), 6.54 (1H, dd,
J = 7.3, 2.0 Hz), 6.84 (1H, d, J = 2.0 Hz), 7.09 (1H, dd, J = 8.4,
1.9 Hz), 7.50–7.61 (2H, m), 7.67 (1H, d, J = 7.3 Hz). 13C NMR
(101 MHz, DMSO-d6) d 7.1, 8.3, 29.5, 98.5, 99.9, 108.5, 117.7,
120.4, 134.7, 135.8, 140.0, 141.3, 158.2, 162.9, 166.8. MS (ESI/APCI)
m/z = 345.0 [M+H]+. Anal. Calcd for C16H14BrN3O: C, 53.78; H, 4.36;
N, 11.76. Found: C, 53.85; H, 4.10; N, 11.94.
water and brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, hexane/
EtOAc = 100/0 to 50/50) to give the title compound (1.92 g, 51%) as
white crystals. 1H NMR (400 MHz, DMSO-d6) d 2.36 (3H, s), 7.40 (2H,
d, J = 8.2 Hz), 7.82 (2H, d, J = 8.3 Hz), 8.18 (1H, s), 8.44 (1H, s), 11.67
(1H, br s). MS (ESI/APCI) m/z = 328.0 [M ꢀ H]ꢀ.
5.1.57. N-[(2E)-5-Bromo-1-(1-cyclopropyl-1-oxopropan-2-yl)
pyrazin-2(1H)-ylidene]-4-methylbenzenesulfonamide (18)
To a solution of 17 (1.73 g, 5.27 mmol) in DMF (20 mL) was
added NaH (60% oil dispersion, 0.32 g, 7.9 mmol) at 0 °C, and the
mixture was stirred at rt for 30 min. To the mixture was added
2-bromo-1-cyclopropylpropan-1-one (1.87 g, 10.5 mmol) at rt
and the resulting mixture was stirred overnight. The mixture was
poured into water and extracted with EtOAc. The organic layer
was separated, washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, hexane/EtOAc = 100/0 to 50/50) to give
the title compound (1.02 g, 46%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 0.74–1.00 (4H, m), 1.71 (3H, t, J = 7.3 Hz),
2.21–2.29 (1H, m), 2.36 (3H, s), 5.63 (1H, d, J = 7.3 Hz), 7.34 (2H,
d, J = 8.0 Hz), 7.69 (2H, d, J = 8.2 Hz), 8.31 (1H, s), 8.76 (1H, s).
5.1.51. 5-Bromo-N-methyl-2-nitroaniline (12a)
To a solution of 11 (25.0 g, 114 mmol) in EtOH (100 mL) was
added methylamine (40% in MeOH, 34.8 mL, 341 mmol) at rt. The
mixture was stirred at rt for 1 h and then cooled to 0 °C. The pre-
cipitate was collected by filtration, and washed with EtOH and
IPE successively to give the title compound (24.8 g, 94%) as a yel-
low solid. 1H NMR (300 MHz, DMSO-d6) d 2.95 (3H, d, J = 4.9 Hz),
6.83 (1H, dd, J = 9.1, 1.9 Hz), 7.17 (1H, d, J = 1.9 Hz), 7.98 (1H, d,
J = 9.1 Hz), 8.23 (1H, br s).
5.1.52. 5-Bromo-N-ethyl-2-nitroaniline (12b)
The title compound was prepared in 80% yield using ethylamine
in an analogous manner to 12a. Pale yellow solid. 1H NMR
(300 MHz, CDCl3) d 1.38 (3H, t, J = 7.2 Hz), 3.33 (2H, qd, J = 7.2,
5.1 Hz), 6.75 (1H, dd, J = 9.1, 1.9 Hz), 7.01 (1H, d, J = 1.9 Hz), 7.98
(1H, br s), 8.03 (1H, d, J = 9.1 Hz).
5.1.58. 4-Bromo-N2-methylbenzene-1,2-diamine (19)
A
solution of 12a (350 mg, 1.51 mmol), zinc (495 mg,
7.57 mmol), and NH4Cl (810 mg, 15.15 mmol) in MeOH (4 mL)/
water (2 mL) was stirred at rt for 1 h. The insoluble material was
removed by filtration and neutralized satd NaHCO3 solution. The
mixture was concentrated and extracted with EtOAc. The organic
layer was separated, washed with water and brine, dried over
MgSO4, and concentrated in vacuo to give the title compound as
a brown solid (282 mg, 93%). 1H NMR (400 MHz, DMSO-d6) d
2.68 (3H, d, J = 4.9 Hz), 4.60 (2H, s), 4.87 (1H, d, J = 4.9 Hz), 6.32–
6.58 (3H, m). MS (ESI/APCI) m/z = 202.09 [M+H]+.
5.1.53. 5-Bromo-2-nitro-N-propylaniline (12c)
The title compound was prepared in 76% yield using npropy-
lamine in an analogous manner to 12a. Orange solid. 1H NMR
(300 MHz, CDCl3) d 1.06 (3H, t, J = 7.5 Hz), 1.77 (2H, m), 3.25 (2H,
td, J = 7.1, 5.1 Hz), 6.74 (1H, dd, J = 9.0, 1.9 Hz), 7.01 (1H, d,
J = 1.9 Hz), 7.92–8.11 (2H, m).
5.1.59. N-(5-Bromo-2-nitrophenyl)-N-methylacetamide (20a)
To a solution of 12a (50.0 g, 216 mmol) in toluene (500 mL) was
added acetyl chloride (30.8 mL, 432 mmol) at rt. After being stirred
at 90 °C for 15 h, acetyl chloride (7.69 mL, 108 mmol) was added
and the mixture was stirred at 90 °C for further 5 h. The reaction
mixture was cooled to rt, poured into EtOAc, washed with satd
NaHCO3 solution and brine, dried over Na2SO4, and concentrated.
The residual solid was recrystallized from EtOAc–hexane to give
5.1.54. 4-[(4-Fluorobenzyl)oxy]-1-[3-(methylamino)-4-nitrophenyl]
pyridin-2(1H)-one (13)
The title compound was prepared in 44% yield using 4c and 12a
in an analogous manner to 6a. Yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 2.96 (3H, d, J = 4.9 Hz), 5.13 (2H, s), 6.00 (1H, d,
J = 2.3 Hz), 6.14 (1H, dd, J = 7.7, 2.8 Hz), 6.69 (1H, dd, J = 9.0,
1.9 Hz), 6.96 (1H, d, J = 1.9 Hz), 7.25 (2H, t, J = 8.9 Hz), 7.52 (2H,
dd, J = 8.3, 5.7 Hz), 7.64 (1H, d, J = 7.5 Hz), 8.13 (1H, d, J = 9.0 Hz),
8.27 (1H, d, J = 4.9 Hz). MS (ESI/APCI) m/z = 370.1 [M+H]+.
the title compound (57 g, 97%) as
a
yellow solid. 1H NMR
(400 MHz, DMSO-d6) d 1.65–2.26 (3H, m), 3.00–3.52 (3H, m),
7.67–8.20 (3H, m). MS (ESI/APCI) m/z = 272.9 [M+H]+.
5.1.55. 1-[4-Amino-3-(methylamino)phenyl]-4-[(4-fluorobenzyl)
oxy]pyridin-2(1H)-one (14)
5.1.60. N-(5-Bromo-2-nitrophenyl)-N,3,3-trimethylbutanamide
(20b)
A mixture of 13 (90 mg, 0.24 mmol), iron (54.4 mg, 0.97 mmol),
calcium chloride (54.1 mg, 0.49 mmol), EtOH (1.5 mL), and water
(1.5 mL) was heated at 70 °C for 3 h. The inorganic material was
removed by filtration, and the filtrate was concentrated. The resi-
due was neutralized with satd NaHCO3 solution and extracted with
EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated to give the title compound (78 mg, 94%) as an brown
solid. 1H NMR (300 MHz, CDCl3) d 2.85 (3H, s), 3.29–3.41 (2H, m),
4.99 (2H, s), 5.90–6.08 (2H, m), 6.52–6.65 (2H, m), 6.74 (1H, d,
J = 7.9 Hz), 7.09 (2H, t, J = 8.7 Hz), 7.23 (1H, s), 7.39 (2H, dd,
J = 8.7, 5.3 Hz). MS (ESI/APCI) m/z = 340.1 [M+H]+.
To a mixture of 12a (300 mg, 1.30 mmol), 3,3-dimethylbutanoyl
chloride (0.45 mL, 3.25 mmol), and DMF (5 mL) was added NaH
(60% oil dispersion, 57.1 mg, 1.43 mmol), and the mixture was
heated at 70 °C overnight. The mixture was poured into water
and extracted with EtOAc. The extract was washed with brine,
dried over MgSO4, concentrated, and purified by column chro-
matography (silica gel, hexane/EtOAc = 100/0 to 75/25) to give
the title compound (214 mg, 51%) as a solid. 1H NMR (400 MHz,
DMSO-d6) d 0.86–1.06 (9H, m), 1.67–2.39 (2H, m), 3.02–3.45 (3H,
m), 7.68–8.13 (3H, m). MS (ESI/APCI) m/z = 329.0 [M+H]+.
5.1.56. N-(5-Bromopyrazin-2-yl)-4-methylbenzenesulfonamide
(17)
5.2. Determination of hMCHR1 competitive inhibitory activity
of test compound using binding assay
To a solution of 16 (2.0 g, 11.5 mmol) in pyridine (40 mL) was
added TsCl (3.29 g, 17.2 mmol) at rt, and the mixture was stirred
at rt overnight. The solvent was evaporated, and the residue was
poured into satd NH4Cl solution, extracted with EtOAc, washed with
5.2.1. Preparation of membrane fraction
Using hMCHR1-expressing CHO cell clone 57,47 MCHR1-
expressing CHO cellular membrane fractions were prepared by

H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
2501
the following method. In phosphate buffered saline (pH 7.4) sup-
plemented with 5 mM EDTA (ethylenediaminetetraacetic acid)
were respectively suspended human MCHR1-expressing CHO cells
(1 ꢁ 108 cells) and centrifuged. Homogenate buffer (10 mL, 10 mM
NaHCO3, 5 mM EDTA, pH 7.5, 0.5 mM PMSF (phenylmethylsulfonyl
fluoride), 20 mg/L leupeptin, 4 mg/L E-64, 1 mg/L pepstatin A) was
added to the pellets of the cells and, using Polytron homogenizer,
the mixture was homogenated. The supernatant obtained after
centrifugation at 400ꢁg for 10 min was further centrifuged at
100,000ꢁg for 1 h to give precipitate of the membrane fraction.
The precipitate were suspended in 2 mL of assay buffer [20 mM
Tris–HCl (pH 7.5), 5 mM EDTA, 0.5 mM PMSF, 20 mg/L leupeptin,
4 mg/L E-64, 1 mg/L pepstatin A]. The membrane fractions were
suspended in assay buffer to a protein concentration of 2 mg/mL,
and after dispensing, preserved at ꢀ80 °C and used upon thawing
each time when in use.
indicator dye reagent (DOJINDO LABORATORIES, Ca screening
no-wash kit Fluo4) was added at 100 L/well, and the dye was
l
allowed to penetrate into the cell for 30 min in an incubator at
5% CO2, 37 °C. The plate was set on a plate reader. First, a test
compound solution diluted with an assay buffer [10 mM HEPES
(pH 7.4): 1ꢁ Assay Buffer (DOJINDO LABORATORIES, attached to
Ca screening no-wash kit Fluo4) containing 0.1% BSA] or DMSO
solution was added at 50
peptide (final concentration 2 nM) diluted with assay buffer or
DMSO was added at 50 L/well, during which changes in
lL/well, and then ligand MCH (4–19)
l
intracellular fluorescence were measured at 2 s intervals. The
antagonistic activity of the test compound was calculated by the
following formula and shown as an inhibition rate (%) wherein
the intracellular fluorescence activity resulting from the stimula-
tion by the addition of ligand MCH (4–19) peptide was 100% and
that of the well added with DMSO solution alone was 0%.
Inhibitory rateð%Þ ¼ 100
5.2.2. Binding assay
ꢀ½fluorescence activity upon addition of test compound
and MCHð4—19Þpeptide solutionꢀfluorescence activity
upon addition of DMSO solution onlyꢃ=½fluorescence activity
upon addition of DMSO solution and MCHð4—19Þpeptide solution
ꢀfluorescence activity upon addition of DMSO solution onlyꢃ
ꢁ100:
An MCHR1-expressing CHO cellular membrane fraction
(173
polypropylene plate (3363, Corning). DMSO solution (2
33 M cold MCH (1–19) diluted with DMSO solution (2 L), or a
test compound solution diluted with DMSO solution to various
concentrations (2 L) was added, and last,
125I]-MCH(4–19)
diluted with assay buffer (hereinafter, sometimes to be referred
to as ‘‘hot MCH”, 25 L) was added to each well. The mixture
l
L) diluted with an assay buffer was dispensed to a 96-well
l
L),
l
l
l
[
l
5.4. Evaluation of time-dependent inhibition (TDI) of CYP3A4
(single-point assay)
was reacted with stirring at room temperature for 1 h, and the
plate was set on FilterMate harvester (PerkinElmer). Using a
polyethylenimine-treated glass filter plate (GF/C, PerkinElmer),
which had been previously set, the plate was suction-filtered and
washed three times with washing buffer (50 mM Tris–HCl buffer
pH 7.5). The glass filter plate was dried, MicroScint 0 (PerkinElmer)
Human liver microsomes were purchased from Xenotech, LLC
(Lenexa, KS). A mixture of a test compound (30 lM) and micro-
somes in phosphate buffer (pH 7.4) was preincubated at 37 °C in
the presence of an NADPH-generating system containing MgCl2,
glucose-6-phosphate, b-NADP+, and glucose-6-phosphate dehydro-
genase. After preincubation, enzymatic activity of CYP3A4 in the
incubation mixture was determined by measuring 6b-hydrox-
ytestosterone in the reaction with testosterone by UPLC. The activ-
ity (% of control) for each preincubation time was calculated to the
following: {(activity with test compound)/(activity with DMSO)} ꢁ
100. The remaining activity (% remaining) after preincubation was
calculated to the following: {activity with preincubation (% of con-
trol)}/{activity without preincubation (% of control)} ꢁ 100.
was added at 25 lL/well, and the resulting radioactivity was mea-
sured by TopCount liquid scintillation counter (PerkinElmer). The
binding inhibition rate of the test compound was calculated by
the following formula.
Binding inhibitionð%Þ ¼ 100
ꢀ ðradioactivity upon addition of test compound and hot MCH
ꢀ radioactivity upon addition of cold MCH and hot MCH solutionÞ
=ðradioactivity upon addition of DMSO solution and hot MCH
ꢀ radioactivity upon addition of cold MCH and hot MCH solutionÞ ꢁ 100:
5.5. GSH trapping experiment
5.3. Measurement of MCH receptor 1 antagonistic activity of test
compound using Ca2+ mobilization assay
5.5.1. Instrument
LC/MS system consisted of UPLC system (Waters, Milford, MA)
and SYNAPT Q-TOF mass spectrometer (Waters) equipped with
an electrospray ionization source.
Using an expression vector plasmid introduced with human
MCHR1 gene for expression in animal cells, human MCHR1 gene
was introduced into CHO cells (CHO dhfrꢀ) by Lipofectamine LTX
(Invitrogen). The cells were cultured in selection MEM
a
medium
5.5.2. Microsomal incubation with GSH
[445 mL of MEM medium without nucleic acid and added with
a
For the GSH trapping experiments each test compound (30 lM)
5 mL of penicillin–streptomycin (Invitrogen) and 50 mL of dialyzed
fetal bovine serum]. Colony 24 clones grown in the selection med-
ium, which were human MCHR1 gene-expressing CHO cell candi-
dates, were selected. From these clones, clone no.4 which
showed the highest response to the change of Ca2+ concentration
on stimulation by the addition of 25 nM ligand MCH (4–19) was
selected by Ca2+ mobilization assay. In the following test, this
human MCHR1-expressing CHO cell (clone no.4) was used. An inte-
grated dispensing function fluorometer (CellLux, PerkinElmer) was
used for Ca2+ mobilization assay. The CHO cells were sown in a 96-
well plate (type 3904, Corning) with a black wall and clear well
bottom at a density of 20000 cells/well and cultured in an incuba-
tor for about 24 h at 5% CO2, 37 °C. The medium was removed, and
the cells were washed with phosphate buffered saline (PBS). A Ca2+
was incubated with human liver microsomes (final protein concen-
tration 1.0 mg/mL; XenoTech, LLC. Lenexa, KS) in the presence of
an NADPH-regenerating system and GSH (1 mM) in phosphate
buffer (pH 7.4) at 37 °C. The reaction was terminated after
60 min by the addition of an equal volume of acetonitrile. After
centrifugation at 15000 rpm for 10 min, 5
injected into LC/MS system.
lL of supernatant was
5.5.3. LC/MS/MS analysis
Microsomal incubation mixtures were separated on a BEH C18
column (1.7
l
m, 2.1 ꢁ 100 mm; Waters) using solvent A (5%
acetonitrile in 5 mM aqueous ammonium acetate) and solvent B
(90% acetonitrile in 50 mM aqueous ammonium acetate). At a flow
rate of 0.5 mL/min, the initial elution gradient was 98% solvent A

2502
H. Igawa et al. / Bioorg. Med. Chem. 24 (2016) 2486–2503
and 2% solvent B with a linear gradient to 70% solvent B over
10 min and returned to initial condition. The column was allowed
to equilibrate at 2% solvent B for 5 min before the next injection.
The column temperature was 40 °C and the eluents were moni-
tored with a PDA detector. The mass spectrometry was run in pos-
itive ion mode. The source settings were 1.20 kV capillary voltage,
35 V sampling corn voltage, 120 °C source temperature, 350 °C
desolvation temperature. GSH adducts were analyzed based on
their product ion spectra of the protonated molecules upon CID
ramped from 15 V to 40 V.
Acknowledgement
We thank Dr. Mitsuyuki Shimada for the alliance with TCG
Lifesciences Ltd., and Dr. Tomohiko Suzaki for arrangement of
compound shipment.
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