Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel non-basic 1-(2H-indazole-5-yl)pyridin-2(1H)-one derivatives and mitigation of mutagenicity in Ames test

Article abstract of DOI:10.1016/j.bmc.2016.04.013

To develop non-basic melanin-concentrating hormone receptor 1 (MCHR1) antagonists with a high probability of target selectivity and therapeutic window, we explored neutral bicyclic motifs that could replace the previously reported imidazo[1,2-a]pyridine or 1H-benzimidazole motif. The results indicated that the binding affinity of a chemically neutral 2H-indazole derivative 8a with MCHR1 (hMCHR1: IC₅₀ = 35 nM) was comparable to that of the imidazopyridine and benzimidazole derivatives (1 and 2, respectively) reported so far. However, 8a was positive in the Ames test using TA1537 in S9- condition. Based on a putative intercalation of 8a with DNA, we introduced a sterically-hindering cyclopropyl group on the indazole ring to decrease planarity, which led to the discovery of 1-(2-cyclopropyl-3-methyl-2H-indazol-5-yl)-4-{[5-(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one 8l without mutagenicity in TA1537. Compound 8l exerted significant antiobesity effects in diet-induced obese F344 rats and exhibited promising safety profile.

Full text of DOI:10.1016/j.bmc.2016.04.013

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Amine-free melanin-concentrating hormone receptor 1 antagonists:
Novel non-basic 1-(2H-indazole-5-yl)pyridin-2(1H)-one derivatives
and mitigation of mutagenicity in Ames test
a
a
a
a
a
Hideyuki Igawa a, , Masashi Takahashi , Minoru Ikoma , Hiromi Kaku , Keiko Kakegawa , Asato Kina ,
Jumpei Aida a, Shoki Okuda a, Yayoi Kawata a, Toshihiro Noguchi a, Natsu Hotta a, Syunsuke Yamamoto a,
Masaharu Nakayama a, Yasutaka Nagisa b, 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 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:
To develop non-basic melanin-concentrating hormone receptor 1 (MCHR1) antagonists with a high prob-
ability of target selectivity and therapeutic window, we explored neutral bicyclic motifs that could
replace the previously reported imidazo[1,2-a]pyridine or 1H-benzimidazole motif. The results indicated
that the binding affinity of a chemically neutral 2H-indazole derivative 8a with MCHR1 (hMCHR1:
IC50 = 35 nM) was comparable to that of the imidazopyridine and benzimidazole derivatives (1 and 2,
respectively) reported so far. However, 8a was positive in the Ames test using TA1537 in S9ꢀ condition.
Based on a putative intercalation of 8a with DNA, we introduced a sterically-hindering cyclopropyl group
on the indazole ring to decrease planarity, which led to the discovery of 1-(2-cyclopropyl-3-methyl-2H-
indazol-5-yl)-4-{[5-(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one 8l without mutagenicity
in TA1537. Compound 8l exerted significant antiobesity effects in diet-induced obese F344 rats and
exhibited promising safety profile.
Received 1 March 2016
Revised 5 April 2016
Accepted 6 April 2016
Available online xxxx
Keywords:
MCHR1 antagonist
MCH
Antiobesity agent
Indazole
Ames test
Mutagenicity
DNA intercalator
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
particularly under high-fat diet conditions.8–10 Transgenic mice
that overexpressed MCH in the lateral hypothalamus were more
Melanin-concentrating hormone (MCH) is a cyclic 19-amino-
acid peptide expressed predominantly in the lateral hypothalamic
area and zona incerta, and MCH-producing neurons project
throughout the brain.1 Among the two subtypes of the MCH recep-
tor that have been identified,8–12 MCH receptor 1 (MCHR1) is
widely distributed in the brain, including the hypothalamus, thala-
mus, olfactory cortex, amygdala, striatum, and hippocampus, in all
vertebrates, whereas MCH receptor 2 is expressed only in higher
mammals but not in rodents2 with its physiological function
poorly understood.
Many studies have demonstrated that the MCH/MCHR1
pathway plays a key role in the regulation of feeding behavior
and energy expenditure. MCH mRNA and peptide levels were
upregulated in dietary obese rats3 and genetically obese rodents,
including ob/ob mice,4 db/db mice,5 Ay/a (agouti) mice,6 and Zucker
(fa/fa) rats.7 Chronic intracerebroventricular infusion of MCH
induced hyperphagia, body weight gain, and hyperinsulinemia,
susceptible to obesity and insulin resistance when fed a high-fat
diet.11 In contrast, mice that lacked MCH or MCHR1 were lean with
increased metabolic rates and resistance to diet-induced obesity
(DIO).12–15 In human studies, increased levels of MCH expression
were observed in the hypothalamus in obese subjects compared
with lean subjects.16 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.17
In the course of our activities 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 comprise the imidazo[1,2-a]pyridine or
1H-benzimidazole bicyclic motif in their structure, as represented
in compounds 1 and 2 in Figure 1.18,19
Compounds 1 and 2 do not have the aliphatic amine motif
unlike most reported MCHR1 antagonists;20 the bicyclic ring itself
is considered to possess intrinsic binding affinity through putative
interaction of its hydrogen binding acceptor (HBA) with
Asp123 and/or Tyr272 of MCHR1 (Fig. 1). This interaction mode
⇑
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.2016.04.013
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

2
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Asp123 and/or Tyr272
Asp123 and/or Tyr272
Gln127
Gln127
O
Gln127
O
N
N
N
O
O
O
N
N
N
Me
N
N
Me
Me
O
S
Cl
Cl
Cl
2
1
3
IC50 (hMCHR1) = 19 nM
IC50 (hMCHR1) = 26 nM
IC50 (hMCHR1) = 150 nM
Figure 1. Chemical structures of previously reported amine-free MCHR1 antagonists (1 and 2) and indole derivative 3. Dotted lines depict putative interactions with MCHR1.
is supported by indole derivative 3, which drastically decreased the
potency because of the lack of the HBA at the 3 position of 2.
Profiling of these new lead compounds reveal that they exert
significant anorectic effects as well as body weight reduction in
DIO rats based on its MCHR1 antagonistic activity. These new lead
compounds also possess low potential of hERG inhibition and
phospholipidosis induction because of the lack of the aliphatic
amine in its structure.
therapeutic window. Herein, we report a study on the design and
structure–activity relationship (SAR) of novel pyridin-2(1H)-ones
with a chemically neutral 2H-indazol-based bicyclic motif. During
the optimization, compounds including 8a showed a risk for muta-
genicity in the Ames test using a TA1537 strain. In this report, we
also discuss our strategies to decrease putative DNA intercalation,
which successfully led to compound 8l with no mutagenicity risk
but promising antiobesity effects in DIO rats.
Recently, Washburn et al. reported a non-basic MCHR1 antago-
nist21,22 that exhibits low off-target activities such as hERG inhibi-
tion. So far, we have discovered aliphatic amine-free MCHR1
antagonists and successfully decreased the remaining basicity on
the bicyclic motif. Thus, further exploration of non-basic structures
is crucial for ensuring a high probability of target selectivity and
2. Chemistry
Scheme 1 shows a general route to synthesize 4-alkoxypyridone
derivatives 3, 7, 8, and 9, which involves copper coupling reactions
of pyridine-2(1H)-ones 6a–c with the corresponding bicyclic
O
N
N
Me
O
Cl
N
3
O
N
N
c
Me
O
c
O
Cl
7
O
O
N
NH
a
b
N
Me
O
O
N
Cl
O
N
e
R1
R1
4
1 = Cl
1 = F
N
5a R1 = Cl
R
R
6a
6b
Me
1 = F
5b
R
O
6c R1 = H
Cl
9
c, d, or e
N
N
N
O
R2
N
Me
Br
Br
Br
N
Me
N
17
23
Me
O
Me
N
N
R1
N
R2
8a R1 = Cl, R2 = Me
N
= Cl, R2 = Et
Br
1
8h
8i
8j
R
Me
Me
R
R
R
1 = Cl, R2
=
nPr
2 = Me
25
26a
26b
R
R
1 = Cl, R2 = cPr
2 = Et
1 = F, R2 cPr
=
8k
26c R2
26d R2
=
=
nPr
cPr
8m R1 = H, R2 = Me
8n R1 = H, R2
=
cPr
Scheme 1. Synthesis of 4-alkoxypyridone derivatives 3, 7, 8a, 8h–k, 8m, 8n, and 9. Reagents and conditions: (a) corresponding benzyl alcohol, NaH, THF, rt, 5 h, 6–66%; (b)
Ac2O, 140 °C, 2 h, 54–58%; (c) corresponding aryl bromide, CuI, DMEDA, K2CO3, DMSO, 150 °C, 1 h, MW irradiation, 22–67% (for 3, 7, 8j, and 8n); (d) corresponding aryl
bromide, CuI, DMEDA, K2CO3, DMSO, 150 °C, 3 h, 31–70% (for 8a, 8k, and 8m); (e) corresponding aryl bromide, CuI, trans-N,N0-dimethylcyclohexane-1,2-diamine, K2CO3,
dioxane, 110 °C, 16 h, 13–43% (for 8h, 8i, and 9).
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
bromides (17, 23, 25, and 26a–d). SNAr reactions of benzyl alcohols
with 4-chloropyridine-N-oxide 4 yielded intermediates 5a and 5b,
which were subsequently treated with acetic anhydride to give
1-unsubstituted pyridones 6a and 6b. Copper coupling reactions
of 6a–c with corresponding bicyclic bromides afforded the target
compounds.
6-Bromoindole 17 used in Scheme 1 was synthesized as
described in Scheme 3. Phenol 12 was treated with Tf2O to give tri-
flate 13, which was subsequently subjected to Sonogashira cou-
pling to yield alkynylbenzene 14. Reduction of nitro group of 14
yielded the key alkynylaniline intermediate 15. The indole ring
was cyclized in excellent yield using 15 with catalytic amounts
of PdCl2 and FeCl3, which were reported to facilitate in situ reoxi-
dation of Pd0 to PdII.23 This was followed by a methylation reaction
to yield compound 17.
5-Bromopyrazolo[1,5-a]pyridine 23 shown in Scheme 1 was
obtained using the route shown in Scheme 4. The key oxime inter-
mediate 20 was prepared in two steps from 2-methylpyridine 18.
Treatment of 18 with TFAA yielded a di-substituted azirine via
spontaneous rearrangement, and subsequent thermal cyclization
Scheme
2 illustrates an alternative method to obtain
4-alkoxypyridone derivatives 8 using 4-hydroxypyridones (10a
and 10b) or 4-chloropyridone 11 as key intermediates. The interme-
diates 10a and 10b were prepared from compound 8m or 8n by
hydrogenative debenzylation, and subsequent POCl3 chlorination
reaction yielded compound 11. Alkylation of the intermediate 10a
and 10b under basic or Mitsunobu conditions as well as SNAr
reactions of the intermediate 11 yielded the target compounds.
N
O
R2
N
N
N
O
O
R2
N
R2
N
b
a
N
N
N
Me
Me
Me
O
HO
Ar
O
8m R2 = Me
8n R2 = cPr
10a R2 = Me
10b R2 = cPr
2
8b
8c
Ar = 5-CF3-thiophen-2-yl, R = Me
2
Ar = 4-CF3-thiophen-2-y, R = Me
8d Ar = 5-CF3-thiophen-3-yl, R2 = Me
8e Ar = 4-CF3-1,3-thiazole-2-yl, R2 = Me
8f Ar = 2-CF3-1,3-thiazole-4-yl, R2 = Me
8g Ar = 4-F-Ph, R2 = Me
d
c
N
O
N
2
c
8l
Ar = 5-CF3-thiophen-3-yl, R = Pr
N
Me
Cl
11
Scheme 2. Synthesis of 4-alkoxypyridone derivatives 8b–g and 8l. Reagents and conditions: (a) H2 (1 atm), 10% Pd-C, EtOH, rt, 3 h, 78–94%; (b) corresponding benzyl alcohol,
DMEAD, PPh3, THF, 60 °C, 3 h, 13–50% (for 8b–g); (c) POCl3, DMF, 50 °C, 21 h, 54%; (d) 36, KOtBu, DMA, 80 °C, 1.5 h, 67% (for 8l).
OH
OTf
c
d
a
b
Br
Br
NO2
Br
NO2
Br
NO2
14
Br
NH2
15
12
13
e
N
Me
N
H
Br
16
17
Scheme 3. Synthesis of 6-bromoindole 17. Reagents and conditions: (a) Tf2O, pyridine, 0 °C, 3 h, 92%; (b) ethynylcyclopropane, CuI, PdCl2(PPh3)2, triethylamine, rt, 3 h, 93%;
(c) SnCl2, EtOAc, water, rt, reflux, 4 h; (d) PdCl2, FeCl3, EtOH, rt, then 80 °C, 2 h, 94% (2 steps); (e) MeI, NaH, DMF, 0 °C, 3 h, 46%.
OH
c
d
a
b
N
N
N
N
O
N
N
N
Br
Br
Me
N
Br
Br
Br
18
19
20
21
N
e
N
Br
Me
O
22
23
Scheme 4. Synthesis of 5-bromopyrazolo[1,5-a]pyridine 23. Reagents and conditions: (a) NaHMDS, ꢀ78 °C to rt, 1 h, then methyl cyclopropanecarboxylate, ꢀ78 °C to rt, 16 h
56%; (b) hydroxylamine hydrochloride, NaOH, MeOH, 60 °C, 14 h, 53%; (c) TFAA, triethylamine, DME, 0 °C to rt, 30 min, then FeCl2, DME, rt to 80 °C, 2 h, 47%; (d) N-
(chloromethylene)-N-methylmethanaminium chloride, CH3CN, rt, 1 h, 44%; (e) triethylsilane, triethylamine, TFA, rt, 16 h, 95%.
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

4
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
yielded indole 21.24 Formylation at the 3 position followed by
reduction using triethylsilane yielded the target compound 23.
Scheme 5 illustrates the synthesis of N-alkylated indazoles 25
and 26a–c. Alkylation predominantly proceeded at the 1 position
under basic conditions to yield 1H-indazole 25 as a major product
and 2H-indazole 26a–c as byproducts. Therefore, the 2-substituted
indazoles 26a and 26b were synthesized using trialkyloxionium
tetrafluoroborates, whereby the alkylation reaction underwent
selectively at the 2 position.
Scheme 6 describes a novel route for 2-cyclopropylindazole
26d. o-Nitroacetophenone 28 was obtained from commercially
available 27. The indazole ring was constructed by imine formation
using Ti(OiPr)4 followed by a modified Cadogan reaction25 using P
(OEt)3 as a reducing agent to generate nitrene species. Unlike clas-
sical Cadogan reaction using o-nitrobenzaldehyde, our novel
method using o-nitroacetophenone 28 enables us to efficiently
access 3-substituted 2H-indazoles. To our knowledge, this is the
first report on the synthesis of 3-substituted 2H-indazoles bearing
a sterically congesting alkyl group at the 2 position, which cannot
be introduced through alkylation.
Trifluoromethyl-substituted thiophene methanols (32 and 36)
can be obtained as illustrated in Schemes 7 and 8. Non-selective
formylation of 3-trifluoromethylthiophene 29 (Scheme 7) yielded
a mixture of 30 and 31 in a ratio 3:2. Subsequent NaBH4 reduction
followed by silica gel separation gave the desired product 32. A tri-
fluoromethyl group could be introduced through the coupling of
iodothiophene 34 with trifluoromethylcopper species generated
in situ from CuI and methyl 2,2-difluoro-2-(fluorosulfonyl)ac-
etate.26 The resulting 5-trifluoromethylthiophene 35 was sub-
jected to NaBH4 reduction and yielded the target compound 36.
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. Exploration of
non-basic MCHR1 antagonists was initiated to design bicyclic
motifs with lower pKa values than 1 and 2, which possessed the
key nitrogen atom fixed at the X1 position in the general formula
given in Table 1. Since for compounds bearing a pyrazolo[1,5-a]
pyridine ring and a 2H-indazole ring, lower pKa values (7: 4.3
and 8a: 2.9) have been calculated, these compounds were prepared
and evaluated for their in vitro potencies. The pyrazolo[1,5-a]pyr-
idine derivative 7 decreased the binding affinity by two to three-
fold in comparison with compound 1, whereas the neutral 2H-
indazole derivative 8a showed almost identical potency (7:
hMCHR1 IC50 = 69 nM, 8a: hMCHR1 IC50 = 35 nM). These results
indicated that the nitrogen atom basicity did not correlate with
potency, implying that the nitrogen atom forms hydrogen bonding
interaction with Asp123 and/or Tyr272 rather than an ionic inter-
action through a cation generated on the basic nitrogen atom. The
Me
N
N
a
Br
H
N
Me
25
N
b or c
Br
N
Me
N
R2
24
Br
Me
26a R2 = Me
26b
26c
R
R
2 = Et
2
=
nPr
Scheme 5. Synthesis of N-alkylated indazoles 25 and 26a–c. Reagents and
conditions: (a) MeI, NaH, DMF, 0 °C, 4 h 39%; (b) iodopropane, NaH, DMF, 0 °C,
4 h 17% (for 26c); (c) trialkyloxonium tetrafluoroborate, EtOAc, rt, 47–81% (for 26a
and 26b).
1,3-disubstituted-indazole derivative
9 decreased potency by
NO2
Me
N
a
b
N
Me
Br
Br
Br
O
Me
26d
O
27
28
Scheme 6. Synthesis of 2-cyclopropylindazole 26d. Reagents and conditions: (a) HNO3 (fuming), 0 °C, 33%; (b) cyclopropylamine, Ti(OiPr)4, 60 °C, 16 h, then P(OEt)3, 150 °C,
2 h, 45%.
F3C
F3C
F3C
F3C
O
a
b
S
S
S
S
OH
O
32
29
30
31
Scheme 7. Synthesis of [4-(trifluoromethyl)thiophen-2-yl]methanol 32. Reagents and conditions: (a) norpempidine, nBuLi, THF, ꢀ78 °C, 1 h, then DMF, rt, overnight, 50%; (b)
NaBH4, MeOH, 0 °C, 10 min, 37%.
O
O
O
a
b
c
OMe
OH
OH
OMe
F3C
F3C
I
I
S
S
S
S
33
34
35
36
Scheme 8. Synthesis of [5-(trifluoromethyl)thiophen-3-yl]methanol 36. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 1 day, 99%; (b) CuI, methyl 2,2-difluoro-2-
(fluorosulfonyl)acetate, hexamethylphosphoramide, 80 °C, 5 h, 75%; (c) NaBH4, THF, MeOH, 60 °C, 3 h, 99%.
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
indicated that 8a itself induced a mutagenic effect. TA1537 strain
was reported to detect intercalation of test compounds in DNA,
and a number of planar polycyclic aromatic compounds such as
acridine and ellipticine are well known as DNA intercalators that
cause frameshift mutations against TA1537 (Fig. 3).27,28 Since 8a
itself seemed less likely to form covalent bonds with DNA, the
mutagenicity observed by 8a in the absence of S9 fraction was con-
sidered to be a direct mutagenic effect due to DNA intercalation. In
the S9+ condition, a plausible degradation product of 8a could be
the hydroxypyridone derivative 10a since a metabolite analysis
study of 8a analogs predicted the major metabolic site of 8a would
be the benzylic position (Fig. 4). Therefore, a lack of the terminal
aryl ring in the structure of 10a would be a reason for 8a showing
negative in the Ames test with S9 fraction.
In vitro binding affinities and pKa values of compounds 1, 2, 7, 8a, and 9
X1
X5
O
X2
X4
X3
N
O
Cl
X1
IC50
pKa
X5
X4
X2
X3
Compound
(nM)a
value
hMCHR1b
on X1c
N
N
7
69
35
4.3
2.9
Me
On the basis of the aforementioned involvement of the terminal
aryl ring in genotoxicity and a structural overlay between ellip-
ticine and 8a, we hypothesized that the mutagenicity is caused
N
N Me
8a
Me
Me
by the following two major factors: (1) the
the indazole-substituted pyridone substructure (Fig. 5) with a
base-pair of DNA and (2) the additional -stack of the terminal aryl
p–p interaction of
N
p
N
9
1
99
26
—
group on the left hand side. This putative binding mode of 8a to
DNA led to the two strategies to disrupt DNA intercalation: (1)
replacement of the 4-chlorophenyl motif with more electron-
Me
N
O
N
deficient groups to weaken the
p–p interaction with the DNA
N
7.9
Me
base-pair, and (2) introduction of spatially diverse substituents to
break the planarity of the molecule. Albertini et al. proved the
second strategy to be effective in their study of 5HT2c agonists,28
in which geminal dimethyl groups were introduced to the cores
of indeno pyrroles/pyrazoles to reduce planarity and mitigate the
mutagenicity observed in TA1537.
For the first strategy, we replaced the 4-chlorophenyl motif
of 8a with more electron-deficient trifluoromethyl- or fluorine-
substituted group. In the course of this modification, a limited
number of lipophilic aryl rings, which showed good agreement
with the SAR on this position established so far,18,19 were intro-
duced and the mutagenicity risk of the representative compounds
selected by in vitro potency was evaluated in the TA1537 strain
(S9ꢀ). IC50 values and the results of the mutagenicity assay are
summarized in Table 2.
O
Cl
Cl
N
N
O
N
Me
2
19
5.7
O
S
a
IC50 values were calculated using an experiment performed in duplicate, with a
standard deviation of three-fold.
b
Binding affinity for human MCHR1.
c
pKa values on X1 were calculated by ACD Labs ver. 12.0.33
three-fold relative to the 2H-indazole derivative 8a. This result
may be caused by the masking of a lone pair on the nitrogen atom
at the 1 position involved in the putative hydrogen bonding with
the receptor. From these findings, the neutral 2,3-substituted-
indazole ring was found to be a novel bicyclic motif that yielded
the non-basic MCHR1 antagonists.
We first introduced thiophenes (8b–d), which exhibited a
strong binding affinity for MCHR1 in the benzimidazole
derivatives.19 As a result, 2,4-substituted thiophene derivatives
(8c and 8d) exhibited IC50 values in the 10ꢀ8 range for both species,
whereas the 2,5-substituted thiophene derivative 8b decreased
While profiling 8a as a new non-basic lead compound, it was
found to have genotoxic properties as determined by the Ames test
in a TA1537 strain (Fig. 2), although it was negative in the umu test
Me
H
(data not shown). When 250 lg/plate of 8a was tested against
N
Salmonella typhimurium strain TA1537 without metabolic activa-
tion by the S9 fraction, two-fold increase of a number of revertant
colony was observed, whereas mutagenicity was not observed
when 8a was tested under metabolic activation. These results
N
N
Me
acridine
ellipticine
N
Figure 3. Chemical structures of polycyclic aromatic DNA intercalators: acridine
and ellipticine.
O
N Me
N
Me
O
N
O
Cl
N Me
N
8a
Me
HO
Ames test
TA1537, S9-: positive
TA1537, S9+: negative
10a
Figure 2. Result of the Ames test of compound 8a using TA1537.
Figure 4. Chemical structure of a possible degradation product of compound 8a.
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

6
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Me
H
N
N
Me
N
O
N Me
Me
binds to a DNA intercalation site
N
O
π−π stack with a DNA base pair
Cl
8a
Figure 5. Hypothesized binding mode of compound 8a to DNA.
Table 2
Table 3
Biological activities of compounds 8a–g
In vitro binding affinities and results of the Ames test of compounds 8h–l
N
N
O
O
N R2
Me
N Me
N
N
Me
Ar
O
Ar
O
R2
IC50 (nM)
hMCHR1b rMCHR1c
Ames (TA1537,
a
Compound Ar
IC50 (nM)a
Ames (TA1537,
Compound Ar
S9ꢀ)31,32
S9ꢀ)31,32
hMCHR1b rMCHR1c
8a
35
36
90
Positived
NTe
8h
Et
140
65
130
23
NTd
Cl
Cl
8b
170
nPr 210
NT
F3C
8i
S
Cl
F3C
8c
8d
8e
74
90
38
70
76
NT
8j
cPr
cPr
cPr
31
43
38
Negative
Negative
Negative
S
Cl
Positive
NT
8k
35
F3C
F3C
S
F
N
130
8l
26
F3C
S
S
a
IC 50 values were calculated using an experiment performed in duplicate with a
N
8f
230
110
130
76
NT
three-fold standard deviation.
F3C
F
S
b
Binding affinity for human MCHR1.
Binding affinity for rat MCHR1.
Not tested.
c
d
8g
Negative
a
IC50 values were calculated using an experiment performed in duplicate, with a
standard deviation of three-fold.
b
Binding affinity for human MCHR1.
Binding affinity for rat MCHR1.
>Two-fold increase of the revertant colony compared with vehicle control.
Not tested.
potency. Interestingly, the 4-fluorophenyl derivative 8g did not
exhibit mutagenicity in TA1537 up to 5000 lg/plate despite a
slight structural difference to 8a. It is assumed that stronger elec-
c
d
e
tronegativity of the fluorine atom decreases the electron density of
the phenyl ring and weakens the
p–p interaction with DNA. In
addition, we cannot exclude the possibility that a slightly shorter
molecular length in 8g might weaken the interaction of the termi-
nal phenyl group with a DNA backbone as proposed by Ellis et al.29
Next, we examined the second strategy and modified the
methyl group at the 2 position on the indazole ring of 8a (Table 3).
Initially, we prepared ethyl and npropyl derivatives (8h and 8i);
however, they decreased in vitro potency as the substituent
became elongated. Subsequently, we designed 2-cyclopropylinda-
zole derivatives (8j–l). As shown in Figure 6, the cyclopropyl group
rose from a plane on which the indazole ring was placed by
42–68°, indicating the cyclopropyl group effectively reduced the
potency, especially for hMCHR1. Subsequently, we prepared thia-
zole derivatives (8e and 8f) with lower electron density; however,
they exhibited inferior potency compared with the corresponding
thiophene derivatives (8c vs 8e and 8d vs 8f). Among the com-
pounds discussed so far, compound 8d was tested in mutagenicity
assay in TA1537. The result indicates that 313 lg/plate of 8d
caused a two-fold increase in the number of revertant colonies
and 8d was positive. These findings indicate that the introduction
of a trifluoromethyl group does not alleviate mutagenicity, and
reducing the electron density of the terminal aryl ring with azoles
such as thiazoles would not be an effective modification in terms of
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
not induce a risk for mutagenesis in TA1537, were selected for
further evaluation.
Secondary functional cell-based assays for the inhibition of
MCH-stimulated Ca2+ mobilization in CHO cells confirmed that
these two functioned as a MCHR1 antagonist (8j: IC50 = 33 nM,
8l: IC50 = 79 nM), and their pharmacokinetic screening resulted in
reasonable oral availability and plasma exposure (Table 4). Among
these two compounds, 8l, which exhibited a superior anorectic
effect (21.5% and 30.6% at 3 and 10 mg/kg, respectively) in
two-day acute in vivo study using DIO F344 rats fed a high-fat diet
ad libitum,18,19 was selected for a chronic pharmacological study.
The effect of 8l on body weight reduction was assessed using a
two-week repeated dosing study on DIO rats (Fig. 7). A significant
change in body weight was observed from 3 mg/kg, and once daily
oral administration of 5 and 10 mg/kg of 8l resulted in body weight
reductions of 3.9% and 7.6%, respectively, relative to the vehicle
group. The cumulative food intakes of these two cohorts signifi-
cantly reduced by 12.2% and 23.2%, respectively. The trough
Figure 6. The lowest energy conformers of 8l0 calculated using MOE34 (for the
calculation cost, the structure was simplified).
planarity of the molecule. Indeed, these cyclopropyl derivatives
were expected to be promising targets compared to the previously
reported SAR.18,19 For the synthesis of these 2-cyclopropylindazole
derivatives, we successfully overcame the synthetic hurdle and
established the aforementioned novel synthetic route in Scheme 6,
which enabled us to access 3-substituted-2H-indazoles bearing a
sterically hindered alkyl group at the 2 position.
Compounds 8j, 8k, and 8l showed superior potency for MCHR1
relative to the corresponding methyl analogs (8a vs 8j, 8g vs 8k,
and 8d vs 8l), indicating that the cyclopropyl group at the 2 posi-
tion effectively enhanced potency. Furthermore, these cyclopropyl
derivatives were found to be all negative against TA1537 up to
plasma concentration at 10 mg/kg was 3.36 lM, which was equiv-
alent to an unbound drug concentration of 73.9 nM (unbound frac-
tion of 8l in DIO rat plasma was 0.022). These results indicate that
an unbound concentration of more than 2.8 times the IC50 was
maintained in this cohort throughout the study, which was consid-
ered to be the necessary plasma level to afford a body weight
reduction of 7.6%. Compound 8l exhibited good brain exposure
(brain/plasma ratio = 0.66), which suggested that 8l was perme-
able to the blood–brain barrier. The selectivity of the anorectic
property to MCHR1 antagonism was confirmed using MCHR1-
deficient mice, wherein 8l had no effect on the food intake of
MCHR-deficient mice, whereas it significantly reduced food intake
in wild-type mice in a dose-dependent manner (data not shown).
5000 lg/plate. These findings indicated that mutagenicity in
TA1537 was successfully mitigated by utilizing the second strat-
egy, wherein we introduced the spatially diverse cyclopropyl group
on the indazole ring to break planarity of the molecule, which dis-
favors intercalation with DNA.30 Among the compounds discussed
so far, 8j and 8l, which exhibited the best in vitro potency and did
Table 4
Pharmacokinetic parameters of 8j and 8l in ratsa
Compound
Fb (%)
iv (0.1 mg/kg)
po (1 mg/kg)
CLtotal (mL hꢀ1 kgꢀ1
)
Vss (mL kgꢀ1
d
)
Cmax (ng mLꢀ1
)
Tmax (h)
AUC0–8 (ng h mLꢀ1
)
c
e
f
g
h
8j
8l
37
78
207
296
676
949
291
426
4.0
2.7
1813
2879
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).
Figure 7. Results for 8l in a repeated-dose study in DIO F344 rats. (A) Body weight change from the 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 = 6 for each group). (#) p <0.025 versus the vehicle group (Williams test), (⁄⁄) p <0.01 versus the vehicle group (Student’s t-test).
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

8
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
These findings discussed so far indicate that we successfully
identified non-basic MCHR1 antagonist 8l. Compound 8l did not
exhibit reversible inhibition against major CYP isoforms
acetate, water and acetonitrile (1:8:1, v/v/v) and a mixture of
50 mmol/L ammonium acetate and acetonitrile (1:9, v/v), respec-
tively. 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 condition were a mixture of 0.2% formic acid
in 10 mmol/L ammonium formate and 0.2% formic acid in acetoni-
trile, respectively. The ratio of mobile phase B was increased lin-
early 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 analyses within 0.4% of the
calculated values or analytical HPLC. Yields are not optimized.
(1A2/2C8/2C9/2D6/3A4) at 10
inhibition, phospholipidosis potential in an in vitro assay, or hERG
inhibition potential in a patch clamp test (IC50 >10 M). Thus, com-
lM, CYP3A4 time-dependent
l
pound 8l could be a promising antiobesity agent that exerts body
weight-lowering effects based on MCHR1 antagonistic activity.
4. Conclusion
To discover non-basic MCHR1 antagonists with a high probabil-
ity of target selectivity and therapeutic window, we explored neu-
tral bicyclic motifs and discovered a series of 1-(2H-indazole-5-yl)
pyridin-2(1H)-one derivatives. Since the lead compound 8a was
positive in the Ames test using TA1537 in S9ꢀ conditions, we pos-
tulated strategies and designed compounds to avoid mutagenicity
based on a putative binding mode of 8a with DNA. The lead com-
pounds were optimized by the established MCHR1 SAR reported so
far by incrementally checking for mutagenicity in TA1537.
Consequently, decreasing planarity by introducing a sterically
hindering cyclopropyl was found to be effective in prohibiting
putative DNA intercalation, and lead to the discovery of non-
mutagenic compound 8l in TA1537. Despite most DNA intercala-
tors being basic compounds with highly fused ring systems, this
study cautions that non-basic planar compounds without a poly-
cyclic ring system could be a DNA intercalator. Oral administration
of 5 and 10 mg/kg of compound 8l led to significant body weight
reduction in DIO rats, and was expected to be a novel antiobesity
agent based on MCHR1 antagonistic activity.
5.1.1. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-1-methyl-1H-
indol-6-yl)pyridin-2(1H)-one (3)
A mixture of 6a (75 mg, 0.32 mmol), 17 (80 mg, 0.32 mmol),
DMEDA (0.034 mL, 0.32 mmol), CuI (60.9 mg, 0.32 mmol), K2CO3
(133 mg, 0.96 mmol), and DMSO (2 mL) was heated at 150 °C for
1 h under microwave irradiation. 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 0/100) to give the title com-
pound (28.0 mg, 22%) as off-white crystals; mp 210–211 °C. 1H
NMR (400 MHz, DMSO-d6) d 0.70 (2H, d, J = 3.6 Hz), 1.00 (2H, d,
J = 7.4 Hz), 2.03 (1H, br s), 3.74–3.80 (3H, m), 5.15 (2H, s), 5.96
(1H, br s), 6.08 (1H, d, J = 6.8 Hz), 6.14 (1H, s), 6.87 (1H, d,
J = 8.2 Hz), 7.37 (1H, s), 7.41–7.53 (5H, m), 7.57 (1H, d, J = 7.7 Hz).
13C NMR (101 MHz, DMSO-d6) d 6.6, 7.2, 29.6, 68.7, 96.5, 97.9,
99.5, 107.9, 118.0, 119.2, 126.6, 128.5, 129.7, 132.7, 133.9, 135.0,
136.7, 139.9, 145.0, 162.8, 166.5. MS (ESI/APCI) m/z = 405.3
[M+H]+. Anal. Calcd for C24H21ClN2O2: C, 71.19; H, 5.23; N, 6.92.
Found: C, 71.32; H, 5.19; N, 6.93.
5. Experimental section
5.1. Chemistry
5.1.2. 4-[(4-Chlorobenzyl)oxy]pyridine 1-oxide (5a)
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, 4 (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]+.
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 tetramethylsi-
lane (d) as the internal standard in deuterated solvent, and
coupling constants (J) are in Hertz (Hz). Data are reported as
follows: chemical shift, integration, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet,
dd = doublet of doublets, td = triplet 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 determined by thin layer
chromatography (TLC) analysis on Merck Kieselgel 60 F254 plates
or Fuji Silysia NH plates. Chromatographic purification was per-
formed on silica gel columns [(Merck Kieselgel 60, 70–230 mesh
size or 230–400 mesh size, Merck) or (Chromatorex NH-DM
1020, 100–200 mesh size)] or on Purif-Pack (SI or NH, particle size:
5.1.3. 4-[(4-Fluorobenzyl)oxy]pyridine 1-oxide (5b)
The title compound was prepared in 6% yield using
(4-fluorophenyl)methanol in an analogous manner to 5a. Brown
solid. 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]+.
60
lm, Fuji Silysia Chemical, Ltd). LCꢀMS analysis was performed
on a Shimadzu Liquid Chromatography–Mass Spectrometer Sys-
tem, operating in APCI (+ or ꢀ) or ESI (+ or ꢀ) ionization mode.
Analytes were eluted using a linear gradient of 0.05% TFA contain-
ing water/acetonitrile or 5 mM ammonium acetate containing
water/acetonitrile mobile phase and detected at 220 nm. Analitical
HPLC was performed with Corona CAD (Charged Aerosol Detector)
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
5.1.4. 4-[(4-Chlorobenzyl)oxy]pyridine-2(1H)-one (6a)
A mixture of 5a (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
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
68.4, 98.0, 99.1, 128.5, 129.7, 132.7, 135.0, 135.5, 163.9, 167.2. MS
5.1.9. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-{[4-(trifluoromethyl)
thiophen-2-yl]methoxy}pyridin-2(1H)-one (8c)
(ESI/APCI) m/z 236.0 [M+H]+.
The title compound was prepared in 13% yield using 10a and 32
in an analogous manner to 8b. White crystals; mp 199–200 °C. 1H
NMR (300 MHz, DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.38 (2H, s),
6.04–6.11 (2H, m), 7.11 (1H, dd, J = 9.1, 2.0 Hz), 7.50–7.63 (3H, m),
7.63–7.68 (1H, m), 8.29–8.37 (1H, m). 13C NMR (101 MHz, DMSO-
5.1.5. 4-[(4-Fluorobenzyl)oxy]pyridine-2(1H)-one (6b)
The title compound was prepared in 58% yield using 5b in an
analogous manner to 6a. Brown solid. 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).
d6)
d 9.4, 37.4, 63.9, 97.9, 99.6, 116.7, 117.9, 120.1, 122.0
13C NMR (75 MHz, DMSO-d6)
d
68.5, 97.9, 99.1, 115.3 (d,
(q, J = 270.7 Hz), 124.6 (d, J = 2.0 Hz), 125.3, 129.3 (q, J = 35.4 Hz),
129.6 (q, J = 5.1 Hz), 132.7, 133.2, 139.8, 141.4, 145.6, 162.7,
166.2. MS (ESI/APCI) m/z = 420.3 [M+H]+. Anal. Calcd for
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]+.
C
20H16F3N3O2Sꢂ0.15H2O: C, 56.91; H, 3.89; N, 9.95. Found:
5.1.6. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-3-
C, 56.86; H, 3.87; N, 9.90.
methylpyrazolo[1,5-a]pyridin-5-yl)pyridin-2(1H)-one (7)
The title compound was prepared in 55% yield using 6a and 23
in an analogous manner to 3. White crystals; mp 229–231 °C. 1H
NMR (400 MHz, DMSO-d6) d 0.81–1.03 (4H, m), 1.95–2.11 (1H,
m), 2.27 (3H, s), 5.16 (2H, s), 5.99 (1H, d, J = 2.3 Hz), 6.14 (1H, dd,
J = 7.6, 2.4 Hz), 6.65–6.74 (1H, m), 7.50 (4H, s), 7.56 (1H, s), 7.66
(1H, d, J = 7.5 Hz), 8.48 (1H, d, J = 7.3 Hz). 13C NMR (101 MHz,
DMSO-d6) d 6.9, 7.2, 8.1, 68.8, 97.8, 100.4, 104.5, 110.4, 113.2,
128.1, 128.5, 129.7, 132.8, 134.8, 135.2, 138.2, 138.9, 155.2,
162.3, 166.9. MS (ESI/APCI) m/z = 406.3 [M+H]+. Anal. Calcd for
5.1.10. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-{[5-
(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one
(8d)
The title compound was prepared in 43% yield using 10a and 36
in an analogous manner to 8b. White solid; mp 217–218 °C. 1H
NMR (400 MHz, DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.16 (2H,
s), 6.01 (1H, s), 6.09 (1H, d, J = 7.7 Hz) 7.11 (1H, d, J = 9.3 Hz),
7.54 (1H, d, J = 8.9 Hz), 7.60 (1H, d, J = 7.5 Hz), 7.65 (1H, s), 7.81
(1H, s), 8.06 (1H, s). 13C NMR (101 MHz, DMSO-d6) d 9.4, 37.4,
64.5, 97.7, 99.7, 116.6, 117.9, 120.1, 122.4 (q, J = 269.7 Hz), 125.3,
129.6, 129.8 (q, J = 36.4 Hz), 130.3 (q, J = 4.0 Hz), 132.7, 133.3,
137.2, 139.7, 145.6, 162.8, 166.5. MS (ESI/APCI) m/z = 420.3
[M+H]+. Anal. Calcd for C20H16F3N3O2S: C, 57.27; H, 3.85;
N, 10.02. Found: C, 57.34; H, 3.89; N, 10.09.
C23H20ClN3O2: C, 68.06; H, 4.97; N, 10.35. Found: C, 67.78; H,
5.08; N, 10.24.
5.1.7. 4-[(4-Chlorobenzyl)oxy]-1-(2,3-dimethyl-2H-indazol-5-yl)
pyridin-2(1H)-one (8a)
A mixture of 6a (300 mg, 1.27 mmol), 26a (287 mg, 1.27 mmol),
DMEDA (0.137 ml, 1.27 mmol), K2CO3 (528 mg, 3.82 mmol), CuI
(242 mg, 1.27 mmol), and DMSO (10 mL) was heated at 150 °C
for 3 h. The mixture was poured into 28% NH3 solution and
extracted with EtOAc–THF. The extract was washed with brine,
dried over MgSO4, concentrated, and purified by column chro-
matography (silica gel, EtOAc/MeOH = 100/0 to 85/15) followed
by recrystallization from EtOH to give the title compound
5.1.11. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-{[4-
(trifluoromethyl)-1,3-thiazol-2-yl]methoxy}pyridin-2(1H)-one
(8e)
The title compound was prepared in 50% yield using 10a and
[4-(trifluoromethyl)-1,3-thiazol-2-yl]methanol in an analogous
manner to 8b. White solid; mp 213–215 °C. 1H NMR (400 MHz,
DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.56 (2H, s), 6.08 (1H, s),
6.16 (1H, d, J = 9.8 Hz), 7.11 (1H, d, J = 8.9 Hz), 7.54 (1H, d,
J = 8.9 Hz), 7.66 (2H, d, J = 11.7 Hz), 8.61 (1H, s). 13C NMR
(101 MHz, DMSO-d6) d 9.4, 37.4, 66.3, 98.3, 99.4, 116.7, 117.9,
120.1, 120.4 (q, J = 270.7 Hz), 125.0 (q, J = 2.0 Hz), 125.2, 132.8,
133.2, 140.0, 142.4 (q, J = 36.4 Hz), 145.7, 162.6, 165.9, 168.2. MS
(ESI/APCI) m/z = 421.3 [M+H]+. Anal. Calcd for C19H15F3N4O2S: C,
54.28; H, 3.60; N, 13.33. Found: C, 54.20; H, 3.59; N, 13.27.
(150 mg, 31%) as
a
white solid; mp 242–244 °C. 1H NMR
(400 MHz, DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.15 (2H, s), 5.97
(1H, d, J = 2.3 Hz), 6.09 (1H, dd, J = 7.6, 2.3 Hz), 7.10 (1H, d,
J = 9.2 Hz), 7.47–7.55 (5H, m), 7.60 (1H, d, J = 7.5 Hz), 7.65 (1H, s).
13C NMR (101 MHz, DMSO-d6) d 9.4, 37.4, 68.7, 97.9, 99.8, 116.6,
117.8, 120.1, 125.3, 128.5, 129.7, 132.7, 133.3, 134.9, 139.7,
145.6, 162.8, 166.6. MS (ESI/APCI) m/z = 406.3 [M+H]+. Anal. Calcd
for C21H18ClN3O2ꢂ0.27H2O: C, 65.56; H, 4.86; N, 10.92. Found: C,
65.90; H, 4.75; N, 10.97.
5.1.12. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-{[2-
(trifluoromethyl)-1,3-thiazol-4-yl]methoxy}pyridin-2(1H)-one
(8f)
5.1.8. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-{[5-(trifluoromethyl)
thiophen-2-yl]methoxy}pyridin-2(1H)-one (8b)
The title compound was prepared in 24% yield using 10a and
[2-(trifluoromethyl)-1,3-thiazol-4-yl]methanol in an analogous
manner to 8b. White solid; mp 185–188 °C. 1H NMR (400 MHz,
DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.32 (2H, s), 6.05–6.14 (2H,
m), 7.11 (1H, d, J = 8.9 Hz), 7.54 (1H, d, J = 9.2 Hz), 7.61 (1H, d,
J = 7.4 Hz), 7.66 (1H, s), 8.33 (1H, s). 13C NMR (101 MHz,
DMSO-d6) d 9.4, 37.4, 64.7, 97.8, 99.6, 116.6, 117.9, 119.7 (q,
J = 273.7 Hz), 120.1, 124.6, 125.3, 132.7, 133.3, 139.8, 145.6,
152.3, 154.5 (q, J = 40.4 Hz), 162.8, 166.4. MS (ESI/APCI)
m/z = 421.3 [M+H]+. Anal. Calcd for C19H15F3N4O2Sꢂ1.78H2O: C,
50.43; H, 4.13; N, 12.38. Found: C, 50.46; H, 4.03; N, 12.38.
To a suspension of 10a (100 mg, 0.39 mmol), triphenylphos-
phine (308 mg, 1.18 mmol), and [5-(trifluoromethyl)thiophen-2-
yl]methanol (143 mg, 0.78 mmol) in THF (5 mL) at 60 °C was added
DMEAD (275 mg, 1.18 mmol). After stirring 3 h, the mixture was
diluted with EtOAc, washed with water and brine, dried over
MgSO4, and concentrated. The residue was purified by column
chromatography (silica gel, EtOAc/MeOH = 100/0 to 90/10) to give
the title compound (71.2 mg, 43%) as
a white solid; mp
191–193 °C. 1H NMR (400 MHz, DMSO-d6) d 2.61 (3H, s), 4.07
(3H, s), 5.44 (2H, s), 6.03–6.14 (2H, m), 7.11 (1H, dd, J = 9.0,
2.0 Hz), 7.36–7.41 (1H, m), 7.54 (1H, d, J = 8.9 Hz), 7.59–7.71 (3H,
m). 13C NMR (101 MHz, DMSO-d6) d 9.4, 37.4, 64.0, 98.0, 99.6,
116.7, 117.9, 120.1, 122.3 (q, J = 269.7 Hz), 125.3, 128.1, 129.5 (q,
J = 37.4 Hz), 130.0 (q, J = 4.0 Hz), 132.7, 133.2, 139.9, 143.4 (d,
J = 1.5 Hz), 145.6, 162.7, 166.1. MS (ESI/APCI) m/z = 420.3 [M+H]+.
Anal. Calcd for C20H16F3N3O2S: C, 57.27; H, 3.85; N, 10.02. Found:
C, 57.09; H, 3.84; N, 10.00.
5.1.13. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-[(4-fluorobenzyl)
oxy]pyridin-2(1H)-one (8g)
The title compound was prepared in 37% yield using 10a and
(4-fluorophenyl)methanol in an analogous manner to 8b. White
solid. 1H NMR (400 MHz, CDCl3) d 2.62 (3H, s), 4.14 (3H, s),
5.03 (2H, s), 6.06 (1H, dd, J = 7.5, 2.6 Hz), 6.09 (1H, d, J = 2.5 Hz),
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

10
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7.12 (2H, t, J = 8.6 Hz), 7.21(1H, dd, J = 9.1, 1.7 Hz), 7.32 (1H, d,
J = 7.5 Hz), 7.43 (2H, dd, J = 8.2, 5.5 Hz), 7.54 (1H, d, J = 1.0 Hz),
7.70 (1H, d, J = 9.0 Hz). 13C NMR (101 MHz, DMSO-d6) d 9.4, 37.4,
68.9, 97.8, 99.8, 115.3 (d, J = 21.2 Hz), 116.6, 117.8, 120.1, 125.3,
130.2 (d, J = 9.1 Hz), 132.1 (d, J = 4.0 Hz), 132.7, 133.3, 139.6,
145.6, 161.9 (d, J = 245.4 Hz), 162.8, 166.7. MS (ESI/APCI)
m/z = 364.1 [M+H]+.
water was added. After being stirred at rt overnight, the precipitate
was collected by filtration and washed with water, EtOH, and IPE to
give
a solid. The resulting solid was recrystallized from
DMSO–EtOH–water to give the title compound (2.5 g, 67%) as a
solid; mp 215–216 °C. 1H NMR (300 MHz, DMSO-d6) d 1.09–1.19
(2H, m), 1.21–1.31 (2H, m), 2.70 (3H, s), 3.92–4.01 (1H, m), 5.16
(2H, s), 6.01 (1H, d, J = 2.6 Hz), 6.09 (1H, dd, J = 7.6, 2.7 Hz), 7.10
(1H, dd, J = 9.2, 2.0 Hz), 7.53 (1H, d, J = 9.2 Hz), 7.59 (1H, d,
J = 7.6 Hz), 7.65 (1H, d, J = 1.3 Hz), 7.81 (1H, s), 8.06 (1H, d,
J = 1.4 Hz). 13C NMR (101 MHz, DMSO-d6) d 6.9, 9.5, 31.4, 64.5,
97.7, 99.7, 116.9, 117.7, 120.3, 122.4 (q, J = 269.7 Hz), 125.5,
129.6, 129.8 (q, J = 36.4 Hz), 130.3 (q, J = 4.0 Hz), 133.4, 134.1,
137.2, 139.6, 145.2, 162.8, 166.5. MS (ESI/APCI) m/z = 446.2
[M+H]+. Anal. Calcd for C22H18N3F3O2S: C, 59.32; H, 4.07; N, 9.43.
Found: C, 59.30; H, 4.17; N, 9.37.
5.1.14. 4-[(4-Chlorobenzyl)oxy]-1-(2-ethyl-3-methyl-2H-
indazol-5-yl)pyridin-2(1H)-one (8h)
The title compound was prepared in 13% yield using 6a and 26b
in an analogous manner to 9. Off-white solid. 1H NMR (400 MHz,
DMSO-d6) d 0.83–0.87 (3H, m), 1.84–1.91 (2H, m), 2.59 (3H, s),
4.29 (2H, t, J = 7.0 Hz), 7.25 (1H, dd, J = 9.0, 1.8 Hz), 7.48 (1H, d,
J = 9.0 Hz), 7.94 (1H, d, J = 1.6 Hz). MS (ESI/APCI) m/z = 394.2
[M+H]+. Purity 99.4% (HPLC).
5.1.19. 4-(Benzyloxy)-1-(2,3-dimethyl-2H-indazol-5-yl)pyridin-
2(1H)-one (8m)
5.1.15. 4-[(4-Chlorobenzyl)oxy]-1-(3-methyl-2-propyl-2H-
indazol-5-yl)pyridin-2(1H)-one (8i)
The title compound was prepared in 65% yield using 6c and 26a
in an analogous manner to 8a. Pale yellow solid; mp 215–217 °C.
1H NMR (400 MHz, DMSO-d6) d 2.61 (3H, s), 4.07 (3H, s), 5.15
(2H, s), 5.98 (1H, d, J = 2.3 Hz), 6.09 (1H, dd, J = 7.5, 2.2 Hz), 7.11
(1H, d, J = 9.0 Hz), 7.32–7.50 (5H, m), 7.53 (1H, d, J = 9.2 Hz), 7.59
(1H, d, J = 7.5 Hz), 7.65 (1H, s). 13C NMR (101 MHz, DMSO-d6) d
9.4, 37.4, 69.6, 97.8, 99.8, 116.6, 117.8, 120.1, 125.3, 127.8, 128.1,
128.5, 132.7, 133.3, 135.9, 139.6, 145.6, 162.8, 166.8. MS (ESI/APCI)
m/z = 346.3 [M+H]+. Purity 99.9% (HPLC).
The title compound was prepared in 43% yield using 6a and 26c
in an analogous manner to 9. Off-white solid. 1H NMR (400 MHz,
DMSO-d6) d 0.86 (3H, t, J = 7.4 Hz), 1.86–1.92 (2H, m), 2.62 (3H,
s), 4.32 (2H, t, J = 6.9 Hz), 5.15 (2H, s), 5.96 (1H, d, J = 2.6 Hz),
6.08 (1H, dd, J = 7.6, 2.7 Hz), 7.09 (1H, dd, J = 9.0, 1.8 Hz), 7.50
(4H, s), 7.55 (1H, d, J = 9.1 Hz), 7.60 (1H, d, J = 7.6 Hz), 7.65 (1H,
d, J = 1.4 Hz). 13C NMR (101 MHz, DMSO-d6) d 9.3, 10.9, 23.0,
51.1, 68.7, 97.8, 99.8, 116.8, 118.0, 120.0, 125.4, 128.5, 129.7,
132.3, 132.7, 133.3, 135.0, 139.7, 145.8, 162.8, 166.6. MS (ESI/APCI)
m/z = 408.0 [M+H]+. Purity 99.8% (HPLC).
5.1.20. 4-(Benzyloxy)-1-(2-cyclopropyl-3-methyl-2H-indazol-5-
yl)pyridin-2(1H)-one (8n)
5.1.16. 4-[(4-Chlorobenzyl)oxy]-1-(2-cyclopropyl-3-methyl-2H-
indazol-5-yl)pyridin-2(1H)-one (8j)
The title compound was prepared in 67% yield using 6c and 26d
in an analogous manner to 3. White crystals; mp 194–195 °C. 1H
NMR (400 MHz, DMSO-d6) d 1.11–1.19 (2H, m), 1.26 (2H, d,
J = 3.0 Hz), 2.70 (3H, s), 3.96 (1H, dt, J = 7.3, 3.6 Hz), 5.15 (2H, s),
5.97 (1H, d, J = 2.5 Hz), 6.09 (1H, dd, J = 7.5, 2.5 Hz), 7.10 (1H, dd,
J = 9.1, 1.8 Hz), 7.34–7.50 (5H, m), 7.52 (1H, d, J = 9.2 Hz), 7.58
(1H, d, J = 7.5 Hz), 7.65 (1H, s). 13C NMR (101 MHz, DMSO-d6) d
6.9, 9.5, 31.4, 69.6, 97.8, 99.8, 116.9, 117.7, 120.3, 125.5, 127.8,
128.1, 128.5, 133.5, 134.1, 135.9, 139.6, 145.1, 162.8, 166.8. MS
(ESI/APCI) m/z = 372.3 [M+H]+. Purity >99.9% (HPLC).
The title compound was prepared in 39% yield using 6a and 26d
in an analogous manner to 3. White crystals; mp 222–224 °C. 1H
NMR (300 MHz, DMSO-d6) d 1.09–1.18 (2H, m), 1.22–1.30 (2H,
m), 2.70 (3H, s), 3.96 (1H, dt, J = 7.4, 3.6 Hz), 5.15 (2H, s), 5.97
(1H, d, J = 2.6 Hz), 6.09 (1H, dd, J = 7.6, 2.7 Hz), 7.09 (1H, dd,
J = 9.1, 2.0 Hz), 7.49–7.55 (5H, m), 7.58 (1H, d, J = 7.6 Hz), 7.65
(1H, dd, J = 2.0, 0.7 Hz). 13C NMR (101 MHz, DMSO-d6) d 6.9, 9.5,
31.4, 68.7, 97.8, 99.8, 116.9, 117.7, 120.3, 125.5, 128.5, 129.7,
132.7, 133.4, 134.1, 134.9, 139.6, 145.1, 162.7, 166.6. MS (ESI/APCI)
m/z = 406.3 [M+H]+. Anal. Calcd for C23H20ClN3O2ꢂ0.1H2O: C, 67.76;
H, 4.99; N, 10.31. Found: C, 67.76; H, 4.99; N, 10.31.
5.1.21. 4-[(4-Chlorobenzyl)oxy]-1-(1,3-dimethyl-1H-indazol-5-
yl)pyridin-2(1H)-one (9)
To
a mixture of 25 (200 mg, 0.88 mmol), 6a (168 mg,
5.1.17. 1-(2-Cyclopropyl-3-methyl-2H-indazol-5-yl)-4-[(4-
fluorobenzyl)oxy]pyridin-2(1H)-one (8k)
0.77 mmol), and K2CO3 (368 mg, 2.66 mmol) in dioxane (10 mL)
were added CuI (51 mg, 0.36 mmol) and trans-N,N0-dimethyl-
cyclohexane-1,2-diamine (68 mg, 0.36 mmol). The mixture was
heated at 110 °C for 16 h. The mixture was cooled to rt and concen-
trated. The residue was diluted with DCM, washed with brine,
dried over Na2SO4, and concentrated. The residue was purified by
column chromatography (silica gel, DCM/MeOH = 97/3) to give
the title compound (100 mg, 34%) as a off-white solid; mp
172–173 °C. 1H NMR (400 MHz, DMSO-d6) d 2.47 (3H, s), 3.99
(3H, s), 5.16 (2H, s), 5.97 (1H, d, J = 2.6 Hz), 6.10 (1H, dd, J = 7.6,
2.6 Hz), 7.30 (1H, dd, J = 8.8, 1.7 Hz), 7.49 (4H, s), 7.60 (1H, d,
J = 2.1 Hz), 7.62 (1H, d, J = 3.5 Hz), 7.68 (1H, d, J = 1.4 Hz). 13C
NMR (101 MHz, DMSO-d6) d 11.4, 35.1, 68.7, 97.8, 99.9, 109.6,
118.2, 122.4, 125.5, 128.5, 129.7, 132.7, 133.0, 134.9, 139.4,
139.7, 140.6, 162.8, 166.7. MS (ESI/APCI) m/z = 380.0 [M+H]+. Anal.
Calcd for C21H18ClN3O2: C, 66.40; H, 4.78; N, 11.06. Found:
C, 66.35; H, 4.84; N, 11.09.
The title compound was prepared in 70% yield using 6b and 26d
in an analogous manner to 8a. White crystals; mp 195–196 °C. 1H
NMR (400 MHz, DMSO-d6) d 1.14 (2H, d, J = 5.3 Hz), 1.27 (2H, br s),
2.70 (3H, s), 3.95 (1H, d, J = 3.5 Hz), 5.13 (2H, s), 5.98 (1H, br s), 6.08
(1H, d, J = 7.2 Hz), 7.09 (1H, d, J = 8.9 Hz), 7.26 (2H, t, J = 8.6 Hz),
7.49–7.61 (4H, m), 7.65 (1H, s). 13C NMR (101 MHz, DMSO-d6) d
6.9, 9.5, 31.4, 68.9, 97.8, 99.8, 115.3 (d, J = 21.2 Hz), 116.9, 117.7,
120.3, 125.5, 130.2 (d, J = 9.1 Hz), 132.1 (d, J = 3.0 Hz), 133.5,
134.1, 139.6, 145.1, 161.9 (d, J = 244.4 Hz), 162.8, 166.7. MS
(ESI/APCI) m/z = 390.3 [M+H]+. Anal. Calcd for C23H20FN3O2:
C, 70.94; H, 5.18; N, 10.79. Found: C, 70.92; H, 5.18; N, 10.78.
5.1.18. 1-(2-Cyclopropyl-3-methyl-2H-indazol-5-yl)-4-{[5-
(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one
(8l)
To a solution of 36 (1.82 g, 10.0 mmol) in DMA (25 mL) was
added potassium tert-butoxide (1.12 g, 10.0 mmol) at 0 °C and
the suspension was stirred at same temperature for 15 min. To
the mixture was added 11 (2.5 g, 8.34 mmol) and the mixture
was heated at 80 °C for 1.5 h. The mixture was cooled to rt and
5.1.22. 1-(2,3-Dimethyl-2H-indazol-5-yl)-4-hydroxypyridin-2
(1H)-one (10a)
A mixture of 8m (1.8 g, 5.21 mmol), palladium on carbon
(0.56 g, 5.21 mmol), and EtOH (40 mL) was vigorously stirred
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
under H2 atmosphere at rt for 3 h. The inorganic material was
removed by filtration and the filtrate was concentrated to give
the title compound (1.04 g, 78%) as a solid. 1H NMR (400 MHz,
DMSO-d6) d 2.60 (3H, s), 4.07 (3H, s), 5.64 (1H, d, J = 1.9 Hz), 5.94
(1H, dd, J = 7.5, 2.1 Hz), 7.09 (1H, dd, J = 9.0, 1.5 Hz), 7.52 (2H, d,
J = 8.3 Hz), 7.62 (1H, s), 10.8 (1H, br s).
The organic layer was separated, washed with satd NaHCO3 solu-
tion and brine, dried over MgSO4, and concentrated in vacuo. The
residue was used for next reaction without further purification.
1H NMR (400 MHz, DMSO-d6) d 0.77 (2H, br s), 0.81–0.90 (2H,
m), 1.50–1.60 (1H, m), 5.47 (2H, br s), 6.59 (1H, d, J = 8.2 Hz),
6.85 (1H, s), 6.98 (1H, d, J = 8.3 Hz).
5.1.23. 1-(2-Cyclopropyl-3-methyl-2H-indazol-5-yl)-4-
hydroxypyridin-2(1H)-one (10b)
5.1.28. 6-Bromo-2-cyclopropyl-1H-indole (16)
To a solution of 15 (1.09 g, 4.62 mmol) in EtOH (20 mL) was
added PdCl3 (41 mg, 0.23 mmol) and FeCl2 (37 mg, 0.23 mmol) at
rt, and the mixture was heated at 80 °C for 2 h. The solvent was
evaporated, and the residue was purified by column chromatogra-
phy (NH silica gel, hexane/EtOAc = 100/0 to 70/30) to give the title
compound (1.02 g, 94%) as yellow crystals. 1H NMR (400 MHz,
DMSO-d6) d 0.77 (2H, d, J = 4.8 Hz), 0.97 (2H, d, J = 8.0 Hz), 1.17
(1H, t, J = 7.1 Hz), 1.95–2.05 (1H, m), 6.09 (1H, s), 7.02 (1H, d,
J = 8.3 Hz), 7.31 (1H, d, J = 8.3 Hz), 7.38 (1H, s), 11.02 (1H, br s).
MS (ESI/APCI) m/z = 236.0 [M+H]+.
The title compound was prepared in 94% yield using 8n in an
analogous manner to 10a. Yellow solid. 1H NMR (400 MHz,
DMSO-d6) d 1.06–1.19 (2H, m), 1.26 (2H, d, J = 3.3 Hz), 2.69 (3H,
s), 3.95 (1H, dt, J = 7.2, 3.5 Hz), 5.60 (1H, d, J = 2.1 Hz), 5.92 (1H,
dd, J = 7.5, 2.3 Hz), 7.08 (1H, dd, J = 9.0, 1.8 Hz), 7.49 (2H, t,
J = 7.9 Hz), 7.61 (1H, s), 11.05 (1H, br s). MS (ESI/APCI) m/
z = 282.3 [M+H]+.
5.1.24. 4-Chloro-1-(2-cyclopropyl-3-methyl-2H-indazol-5-yl)
pyridin-2(1H)-one (11)
To a suspension of 10b (8.0 g, 28.4 mmol) in DMF (140 mL) was
added phosphorus oxychloride (3.18 mL, 34.1 mmol) at rt and the
mixture was stirred at 50 °C for 21 h. The mixture was poured into
EtOAc and washed with satd NaHCO3 solution. The aqueous layer
was extracted with EtOAc, and the extracts were washed with
water and brine, dried over Na2SO4, and concentrated. The result-
ing residue was dissolved in EtOAc–THF at 50 °C and NH-silica gel
was added to the mixture. After 1 h, a mixture was purified by col-
umn chromatography (NH silica gel, EtOAc only) to give the crude
product. The crude solid was triturated with IPE and collected by
filtration to give the title compound (4.59 g, 54%) as an off-white
5.1.29. 6-Bromo-2-cyclopropyl-1-methyl-1H-indole (17)
To a solution of 16 (1.0 g, 4.24 mmol) in DMF (5 mL) was added
methyl iodide (0.79 mL, 12.7 mmol) and NaH (40% oil dispersion,
0.17 g, 4.24 mmol) at 0 °C. After 3 h, the mixture was poured into
satd NH4Cl solution and extracted with EtOAc. The organic layer
was separated, washed with water and brine, dried over MgSO4,
and concentrated. The residue was purified by column chromatog-
raphy (silica gel, hexane/EtOAc = 100/0 to 90/10) to give the title
compound (0.48 g, 46%) as pale yellow crystals. 1H NMR
(400 MHz, DMSO-d6)
d 0.68 (2H, d, J = 3.8 Hz), 0.98 (2H, d,
J = 6.5 Hz), 1.99 (1H, br s), 3.30 (1H, s), 3.72–3.79 (3H, m), 6.09
(1H, s), 7.07 (1H, d, J = 8.0 Hz), 7.35 (1H, d, J = 8.3 Hz), 7.62 (1H, s).
solid. 1H NMR (300 MHz, DMSO-d6)
d 1.11–1.20 (2H, m),
1.22–1.31 (2H, m), 2.70 (3H, s), 3.97 (1H, tt, J = 7.4, 3.7 Hz), 6.44
(1H, dd, J = 7.4, 2.5 Hz), 6.65 (1H, d, J = 1.9 Hz), 7.13 (1H, dd,
J = 9.3, 2.1 Hz), 7.56 (1H, d, J = 9.1 Hz), 7.71–7.75 (1H, m), 7.77
(1H, d, J = 7.2 Hz). MS (ESI/APCI) m/z = 300.2 [M+H]+.
5.1.30. 2-(4-Bromopyridin-2-yl)-1-cyclopropylethanone (19)
To a solution of 18 (5.5 g, 32.0 mmol) in THF (40 mL) was added
NaHMDS (1.9 M THF solution, 20.2 mL, 38.4 mmol) at ꢀ78 °C, and
the mixture was stirred at rt for 1 h. Methyl cyclopropanecarboxy-
late (3.27 mL, 32.0 mmol) was added at ꢀ78 °C, and the mixture
was allowed to warm to rt for 16 h. The mixture was quenched
with satd NaHCO3 solution and extracted with EtOAc. The organic
layer was separated, washed with satd NaHCO3 solution and brine,
dried over MgSO4, and concentrated. The residue was purified by
column chromatography (silica gel, hexane/EtOAc = 90/10 to
50/50) to give the title compound (4.3 g, 56%) as a yellow oil. 1H
NMR (400 MHz, CDCl3) d 0.92 (2H, dd, J = 7.5, 3.3 Hz), 1.04–1.14
(2H, m), 2.00–2.10 (1H, m), 4.03 (2H, s), 7.37 (1H, d, J = 5.1 Hz),
7.44 (1H, s), 8.38 (1H, d, J = 5.3 Hz).
5.1.25. 4-Bromo-2-nitrophenyl trifluoromethanesulfonate (13)
To a solution of 12 (9.43 g, 43.3 mmol) in pyridine (200 mL) was
added Tf2O (8.0 mL, 47.6 mmol) at 0 °C and the mixture was stirred
for 1 h. The mixture was quenched with satd NaHCO3 solution and
extracted with EtOAc. The organic layer was separated, washed
with satd NaHCO3 solution and brine, dried over MgSO4, and
concentrated. The residue was purified by column chromatography
(silica gel, hexane/EtOAc = 100/0 to 80/20) to give the title
compound (14.0 g, 92%) as a colorless oil. 1H NMR (300 MHz,
DMSO-d6) d 7.78 (1H, d, J = 8.9 Hz), 8.20 (1H, dd, J = 8.8, 2.5 Hz),
8.56 (1H, d, J = 2.5 Hz).
5.1.31. 2-(4-Bromopyridin-2-yl)-1-cyclopropyl-N–hydroxyetha-
nimine (20)
5.1.26. 4-Bromo-1-(cyclopropylethynyl)-2-nitrobenzene (14)
To a solution of 13 (2.0 g, 5.71 mmol) in THF (20 mL) were
added ethynylcyclopropane (0.58 mL, 6.86 mmol), CuI (0.054 g,
0.29 mmol), PdCl2(PPh3)2 (0.20 g, 0.29 mmol), and TEA (2.4 mL,
17.1 mmol) at rt under N2 atmosphere, and the mixture was stirred
at rt for 3 h. The mixture was concentrated and purified by column
chromatography (silica gel, hexane/EtOAc = 100/0 to 80/20) to give
the title compound (1.42 g, 93%) as a yellow oil. 1H NMR (400 MHz,
DMSO-d6) d 0.79 (2H, br s), 0.90–1.00 (2H, m), 1.61 (1H, d,
J = 4.0 Hz), 7.58 (1H, d, J = 8.3 Hz), 7.89 (1H, d, J = 8.3 Hz),
8.22–8.30 (1H, m).
To a solution of 19 (5.86 g, 24.4 mmol) in MeOH (50 mL) was
added hydroxylamine hydrochloride (8.48 g, 122 mmol) and NaOH
(4.88 g, 122 mmol) at rt, and the mixture was heated at 60 °C for
14 h. The mixture was poured into satd NaHCO3 solution and
extracted with EtOAc. The organic layer was separated, washed
with 1 N NaOH solution and brine, dried over MgSO4, and concen-
trated. The resulting solid was recrystallized from EtOAc–hexane
to give the title compound (3.3 g, 53%) as white crystals. 1H NMR
(400 MHz, DMSO-d6) d 0.55–0.67 (4H, m), 1.46–1.56 (1H, m),
3.66 (2H, s), 7.47–7.55 (2H, m), 8.36 (1H, d, J = 5.9 Hz), 10.55
(1H, s).
5.1.27. 5-Bromo-2-(cyclopropylethynyl)aniline (15)
To a solution of 14 (1.42 g, 5.34 mmol) in EtOAc (20 mL) was
added SnCl2 (1.3 mL, 26.7 mmol) and water (0.96 mL, 53.4 mmol)
at rt, and the mixture was heated at reflux for 4 h. The mixture
was quenched with satd NaHCO3 solution. The insoluble material
was removed by filtration and the filtrate was diluted with EtOAc.
5.1.32. 5-Bromo-2-cyclopropylpyrazolo[1,5-a]pyridine (21)
To a suspension of 20 (395 mg, 1.55 mmol) in DME (4 mL) was
added triethylamine (1.08 mL, 7.74 mmol) at 0 °C. TFAA (0.26 mL,
1.86 mmol) was added to the mixture portionwise at the same
temperature. After 30 min, the mixture was turned to clear yellow
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

12
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
solution. The mixture was quenched with satd NaHCO3 solution
and extracted with EtOAc. The organic layer was separated,
washed with satd NaHCO3 solution and brine, dried over MgSO4,
and concentrated. The residue was diluted with DME (4 mL) and
FeCl2 (19.6 mg, 0.15 mmol) was added to the mixture. The mixture
was heated to 80 °C for 2 h. Then 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. The residue was purified by column
chromatography (silica gel, hexane/EtOAc = 100/0 to 85/15) to give
the title compound (173 mg, 47%) as pale yellow crystals. 1H NMR
(400 MHz, DMSO-d6) d 0.73–0.85 (2H, m), 0.91–1.05 (2H, m),
1.97–2.13 (1H, m), 6.30 (1H, s), 6.87 (1H, dd, J = 7.3, 1.9 Hz), 7.83
(1H, d, J = 1.8 Hz), 8.48 (1H, d, J = 7.4 Hz). MS (ESI/APCI)
m/z = 238.8 [M+H]+.
washed with brine, dried over MgSO4, and concentrated to give the
title compound (3.65 g, 81%) as a solid. 1H NMR (400 MHz, DMSO-
d6) d 2.58 (3H, s), 4.04 (3H, s), 7.25 (1H, d, J = 9.0 Hz), 7.47 (1H, d,
J = 9.0 Hz), 7.94 (1H, s).
5.1.37. 5-Bromo-2-ethyl-3-methyl-2H-indazole (26b)
The title compound was prepared in 47% yield using triethylox-
onium tetrafluoroborate in an analogous manner to 26a. Off-white
solid. 1H NMR (400 MHz, DMSO-d6) d 1.43 (3H, t, J = 7.2 Hz), 2.62
(3H, s), 4.39 (2H, q, J = 7.2 Hz), 5.15 (2H, s), 5.96 (1H, d,
J = 2.6 Hz), 6.08 (1H, dd, J = 7.6, 2.7 Hz), 7.09 (1H, dd, J = 9.1,
1.9 Hz), 7.50 (4H, s), 7.55 (1H, d, J = 9.1 Hz), 7.55 (1H, d,
J = 7.6 Hz), 7.65 (1H, d, J = 1.6 Hz). MS (ESI/APCI) m/z = 240.0
[M+H]+.
5.1.38. 5-Bromo-3-methyl-2-propyl-2H-indazole (26c)
The title compound was prepared in 17% yield using iodopro-
pane in an analogous manner to 25. Yellow oil. 1H NMR
(400 MHz, DMSO-d6) d 0.83–0.87 (3H, m), 1.84–1.91 (2H, m),
2.59 (3H, s), 4.29 (2H, t, J = 7.0 Hz), 7.25 (1H, dd, J = 9.0, 1.8 Hz),
7.48 (1H, d, J = 9.0 Hz), 7.94 (1H, d, J = 1.6 Hz). MS (ESI/APCI)
m/z = 253.0 [M+H]+.
5.1.33. 5-Bromo-2-cyclopropylpyrazolo[1,5-a]pyridine-3-
carbaldehyde (22)
To a solution of 21 (500 mg, 2.11 mmol) in CH3CN (5 mL) was
added N-(chloromethylene)-N-methylmethanaminium chloride
(324 mg, 2.53 mmol) at 0 °C, and the mixture was stirred at rt for
1 h. The mixture was quenched with satd NH4Cl solution and the
suspension was stirred at rt for 30 min. The mixture was poured
into satd NH4Cl solution and extracted with EtOAc. The organic
layer was separated, washed with satd NaHCO3 solution and brine,
dried over MgSO4, and concentrated. The residue was purified by
column chromatography (silica gel, hexane/EtOAc = 100/0 to
75/25) to give the title compound (245 mg, 44%) as white crystals.
1H NMR (400 MHz, DMSO-d6) d 0.99–1.16 (4H, m), 2.58–2.70 (1H,
m), 7.32 (1H, dd, J = 7.1, 1.7 Hz), 8.36 (1H, s), 8.73 (1H, d, J = 7.3 Hz),
10.17 (1H, s).
5.1.39. 5-Bromo-2-cyclopropyl-3-methyl-2H-indazole (26d)
To a solution of 28 (43.0 g, 176 mmol) in toluene (250 mL) were
added cyclopropylamine (24.4 mL, 352 mmol) and titanium iso-
propoxide (105 mL, 352 mmol) at rt, and the mixture was stirred
at 60 °C for 16 h. After removal of solvent, the residue was dis-
solved in triethyl phosphite (91 mL, 529 mmol), and the mixture
was heated to 150 °C for 2 h. The mixture was treated with NH sil-
ica gel (500 g) in EtOAc (500 mL) with stirring overnight. The silica
gel was removed by filtration, washed with EtOAc, concentrated,
and filtered through a silica gel (500 g, hexane/EtOAc = 85/15).
The filtrate was evaporated and the residue was recrystallized from
EtOAc–hexane to give the title compound (19.8 g, 45%) as an
off-white solid. 1H NMR (400 MHz, DMSO-d6) d 1.13 (2H, d,
J = 5.3 Hz), 1.25 (2H, br s), 2.67 (3H, s), 3.87–3.97 (1H, m), 7.25
(1H, d, J = 9.0 Hz), 7.46 (1H, d, J = 9.0 Hz), 7.94 (1H, s). MS (ESI/APCI)
m/z = 251.2 [M+H]+.
5.1.34. 5-Bromo-2-cyclopropyl-3-methylpyrazolo[1,5-a]
pyridine (23)
To a solution of 22 (50 mg, 0.19 mmol) in TFA (3 mL) was added
triethylsilane (0.60 mL, 3.77 mmol) at rt and the mixture was stir-
red vigorously for 16 h. The mixture was poured into 1 N NaOH
solution and extracted with EtOAc. The organic layer was sepa-
rated, washed with sad NaHCO3 solution and brine, dried over
MgSO4, and concentrated. The residue was purified by column
chromatography (silica gel, hexane/EtOAc = 100/0 to 85/15) to give
the title compound (45.2 mg, 95%) as white crystals. 1H NMR
(400 MHz, CDCl3) d 0.92–1.06 (4H, m), 1.91–2.01 (1H, m), 2.25
(3H, s), 6.63 (1H, dd, J = 7.3, 1.9 Hz), 7.46 (1H, d, J = 1.6 Hz), 8.09
(1H, d, J = 7.4 Hz). MS (ESI/APCI) m/z = 252.8 [M+H]+.
5.1.40. 1-(5-Bromo-2-nitrophenyl)ethanone (28)
To nitric acid (fuming, 2 mL, 25.1 mmol) was slowly added sul-
furic acid (2.5 mL, 25.1 mmol) at 0 °C and the mixture was stirred
at same temperature for 15 min. To the solution was slowly added
27 (3.34 mL, 25.1 mmol) at 0 °C with vigorous stirring and the mix-
ture was stirred at same temperature for 1 h. The mixture was
poured into ice-water and extracted with EtOAc. The organic layer
was washed with satd NaHCO3 solution and brine, dried over
MgSO4, concentrated, and purified by column chromatography (sil-
ica gel, hexane/EtOAc = 100/0 to 90/10) to give the title compound
(2.04 g, 33%) as pale yellow crystals. 1H NMR (400 MHz, DMSO-d6)
d 2.58 (3H, s), 7.92–8.00 (H, m), 8.02–8.08 (2H, m).
5.1.35. 5-Bromo-1,3-dimethyl-1H-indazole (25)
To a stirred suspension of NaH (60% oil dispersion, 136 mg,
2.83 mmol) in DMF (10 mL) was added a solution of 24 (400 mg,
1.89 mmol) in DMF (2 mL) at 0 °C, and the mixture was stirred at
the same temperature for 30 min. MeI (400 lL, 2.83 mmol) was
added and the resulting mixture was stirred at 0 °C for 4 h. The
reaction mixture was quenched with water and extracted with
EtOAc. The combined EtOAc layers were washed with brine, dried
over Na2SO4, and concentrated. The residue was purified by col-
umn chromatography (silica gel, hexane/EtOAc = 80/20) to give
the title compound (200 mg, 39%) as a yellow oil. 1H NMR
(400 MHz, DMSO-d6) d 2.45 (3H, s), 3.95 (3H, s), 7.46 (1H, dd,
J = 8.9, 1.8 Hz), 7.54 (1H, d, J = 8.9 Hz), 7.94 (1H, d, J = 1.5 Hz). MS
(ESI/APCI) m/z = 225.0 [M+H]+.
5.1.41. 4-(Trifluoromethyl)thiophene-2-carbaldehyde (30)
To a solution of norpempidine (9.3 mL, 55.1 mmol) in THF
(150 mL) was added nBuLi (34.5 mL, 55.1 mmol) at ꢀ78 °C and
the mixture was stirred at 0 °C for 10 min. Then the mixture was
cooled to ꢀ78 °C and a solution of 3-(trifluoromethyl)thiophene
(6.99 g, 46.0 mmol) in THF (5 mL) was added dropwise over
30 min. Then the mixture was stirred for 1 h at the same temper-
ature. DMF (10.7 mL, 138 mmol) was added, and stirred at rt over-
night. The mixture was quenched with 1 N HCl solution and
extracted with EtOAc. The organic layer was separated, washed
with 1 N HCl solution and brine, dried over MgSO4, and concen-
trated. The residue was purified by column chromatography (silica
gel, hexane/EtOAc = 100/0 to 85/15) to give the title compound
5.1.36. 5-Bromo-2,3-dimethyl-2H-indazole (26a)
To a solution of 24 (4.25 g, 20.1 mmol) in EtOAc (100 mL) was
added trimethyloxonium tetrafluoroborate (4.47 g, 30.2 mmol)
and the mixture was stirred at rt for 5 h. The mixture was poured
into 1 N NaOH solution and extracted with EtOAc. The extract was
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
(4.1 g, 50%) as a light brown oil. 1H NMR (400 MHz, DMSO-d6) d
7.63 (1H, d, J = 5.1 Hz), 8.31 (1H, d, J = 5.1 Hz), 8.37 (1H, s), 8.80
(1H, s), 9.97 (1H, s), 10.09 (1H, s).
100,000 ꢁ g 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.
5.1.42. [4-(Trifluoromethyl)thiophen-2-yl]methanol (32)
To a solution of 30 (7.06 g, 39.2 mmol) in MeOH (50 mL) was
added NaBH4 (1.48 g, 39.2 mmol) at 0 °C and the mixture was
stirred for 10 min. The mixture was quenched with satd NH4Cl
solution and extracted with EtOAc. The organic layer was
separated, washed with satd NH4Cl solution and brine, dried over
MgSO4, and concentrated. The residue was purified by column
chromatography (silica gel, hexane/EtOAc = 100/0 to 85/15) to give
the title compound (2.63 g, 37%) as a pale yellow oil. 1H NMR
(400 MHz, CDCl3) d 1.84–1.93 (1H, m), 4.81–4.87 (2H, m), 7.14
(1H, s), 7.66 (1H, s).
5.2.2. Binding assay
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 was
lL) diluted with an assay buffer was dispensed to a 96-well
lL),
l
l
l
[
5.1.43. Methyl 5-iodothiophene-3-carboxylate (34)
l
A mixture of 33 (3.48 g, 13.7 mmol), methyl iodide (1.29 mL,
20.6 mmol), K2CO3 (2.84 g, 20.6 mmol), and DMF (35 mL) was
stirred at rt for 1 day. The mixture was poured into water and
extracted with EtOAc. The extract was washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified
by column chromatography (silica gel, hexane/EtOAc = 98/2 to
85/15) to give the title compound (3.62 g, 99%) as a white solid.
1H NMR (400 MHz, CDCl3) d 3.86 (3H, s) 7.66 (1H, s) 8.08 (1H, s).
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)
was added at 25 lL/well, and the resulting radioactivity was
measured by TopCount liquid scintillation counter (PerkinElmer).
The binding inhibition rate of the test compound was calculated
by the following formula.
5.1.44. Methyl 5-(trifluoromethyl)thiophene-3-carboxylate (35)
To
a solution of 34 (896 mg, 3.34 mmol), CuI (1.91 g,
10.0 mmol), and hexamethylphosphoramide (3.49 mL, 20.1 mmol)
in DMF (15 mL) was added methyl 2,2-difluoro-2-(fluorosulfonyl)
acetate (2.53 mL, 20.1 mmol) at rt. The mixture was stirred at
80 °C under N2 atmosphere for 5 h. The mixture was neutralized
with satd NaHCO3 solution and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over MgSO4, and
concentrated. The residue was purified by column chromatography
(silica gel, hexane/EtOAc = 99/1 to 85/15) to give the title com-
pound (526 mg, 75%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
d 3.89 (3H, s) 7.86 (1H, s) 8.23 (1H, d, J = 1.0 Hz).
Binding inhibition ð%Þ ¼
100ꢀðradioactivity upon addition of test compound and hot MCHꢀ
radioactivity upon addition of cold MCH and hot MCH solutionÞ=
ðradioactivity upon addition of DMSO solution and hot MCHꢀ
radioactivity upon addition of cold MCH and hot MCH solutionÞꢁ100:
5.3. Measurement of MCH receptor 1 antagonistic activity of test
compound using Ca2+ mobilization assay
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
5.1.45. [5-(Trifluoromethyl)thiophen-3-yl]methanol (36)
To a solution of 35 (523 mg, 2.49 mmol) in THF (10 mL) and
MeOH (1 mL) was added NaBH4 (2.20 g, 58.0 mmol) at rt. The
mixture was stirred at 60 °C for 3 h. The mixture was quenched
with water and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated. The residue was puri-
fied by column chromatography (silica gel, hexane/EtOAc = 90/10
to 50/50) to give the title compound (449 mg, 99%) as a pale yellow
oil. 1H NMR (400 MHz, CDCl3) d 1.71 (1H, t, J = 5.7 Hz), 4.70 (2H, d,
J = 5.6 Hz), 7.39 (1H, s), 7.43 (1H, s).
(Invitrogen). The cells were cultured in selection MEM
a medium
[445 mL of MEM medium without nucleic acid and added with
a
5 mL of penicillin–streptomycin (Invitrogen) and 50 mL of dialyzed
fetal bovine serum]. Colony 24 clones grown in the selection
medium, which were human MCHR1 gene-expressing CHO cell
candidates, 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
integrated 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 incubator
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+
indicator dye reagent (DOJINDO LABORATORIES, Ca screening
5.2. Determination of hMCHR1 competitive inhibitory activity
of test compound using binding assay
5.2.1. Preparation of membrane fraction
Using hMCHR1-expressing CHO cell clone 57,35 MCHR1-
expressing CHO cellular membrane fractions were prepared by
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 400 ꢁ g for 10 min was further centrifuged at
no-wash kit Fluo4) was added at 100 lL/well, and the dye was
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): 1 ꢁ Assay Buffer (DOJINDO LABORATORIES, attached to
Ca screening no-wash kit Fluo4) containing 0.1% BSA] or DMSO
solution was added at 50 lL/well, and then ligand MCH (4–19)
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

14
H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
peptide (final concentration 2 nM) diluted with assay buffer or
DMSO was added at 50 L/well, during which changes in intracellu-
References and notes
l
1. Bittencourt, J. C. Gen. Comp. Endocrinol. 2011, 172, 185.
lar fluorescence were measured at 2 s intervals. The antagonistic
activity of the test compound was calculated by the following for-
mula and shown as an inhibition rate (%) wherein the intracellular
fluorescence activity resulting from the stimulation by the addition
of ligand MCH (4–19) peptide was 100% and that of the well added
with DMSO solution alone was 0%.
2. Sailer, A. W.; Sano, H.; Zeng, Z.; McDonald, T. P.; Pan, J.; Pong, S. S.; Feighner, S.
D.; Tan, C. P.; Fukami, T.; Iwaasa, H.; Hreniuk, D. L.; Morin, N. R.; Sadowski, S. J.;
Ito, M.; Ito, M.; Bansal, A.; Ky, B.; Figueroa, D. J.; Jiang, Q.; Austin, C. P.; MacNeil,
D. J.; Ishihara, A.; Ihara, M.; Kanatani, A.; Van der Ploeg, L. H.; Howard, A. D.; Liu,
Q. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7564.
3. Elliott, J. C.; Harrold, J. A.; Brodin, P.; Enquist, K.; Bäckman, A.; Byström, M.;
Lindgren, K.; King, P.; Williams, G. Brain Res. Mol. Brain Res. 2004, 128, 150.
4. Qu, D.; Ludwig, D. S.; Gammeltoft, S.; Piper, M.; Pelleymounter, M. A.; Cullen,
M. J.; Mathes, W. F.; Przypek, R.; Kanarek, R.; Maratos-Flier, E. Nature 1996, 380,
243.
5. Huang, Q.; Viale, A.; Picard, F.; Nahon, J.; Richard, D. Neuroendocrinology 1999,
69, 145.
6. Hanada, R.; Nakazato, M.; Matsukura, S.; Murakami, N.; Yoshimatsu, H.; Sakata,
T. Biochem. Biophys. Res. Commun. 2000, 268, 88.
7. Stricker-Krongrad, A.; Dimitrov, T.; Beck, B. Brain Res. Mol. Brain Res. 2001, 92,
43.
8. Della-Zuana, O.; Presse, F.; Ortola, C.; Duhault, J.; Nahon, J. L.; Levens, N. Int. J.
Obes. Relat. Metab. Disord. 2002, 26, 1289.
9. Gomori, A.; Ishihara, A.; Ito, M.; Mashiko, S.; Matsushita, H.; Yumoto, M.; Ito,
M.; Tanaka, T.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J. Physiol.
Endocrinol. Metab. 2003, 284, E583.
10. Ito, M.; Gomori, A.; Ishihara, A.; Oda, Z.; Mashiko, S.; Matsushita, H.; Yumoto,
M.; Ito, M.; Sano, H.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J.
Physiol. Endocrinol. Metab. 2003, 284, E940.
11. Ludwig, D. S.; Tritos, N. A.; Mastaitis, J. W.; Kulkarni, R.; Kokkotou, E.; Elmquist,
J.; Lowell, B.; Flier, J. S.; Maratos-Flier, E. J. Clin. Invest. 2001, 107, 379.
12. Shimada, M.; Tritos, N. A.; Lowell, B. B.; Flier, J. S.; Maratos-Flier, E. Nature 1998,
396, 670.
Inhibitory rate ð%Þ ¼
100 ꢀ ½fluorescence activity upon addition
of test compound and MCHð4—19Þpeptide solutionꢀ
fluorescence activity upon addition of DMSO solution onlyꢃ=
½fluorescence activity upon addition of DMSO solution
and MCHð4—19Þpeptide solutionꢀ
fluorescence activity upon addition of DMSO solution onlyꢃ ꢁ 100:
5.4. Animal experiments
All animal experiments were performed in compliance with the
Guidelines for the Care and Use of Laboratory Animals of Takeda
Pharmaceutical Company Ltd.
13. Kokkotou, E.; Jeon, J. Y.; Wang, X.; Marino, F. E.; Carlson, M.; Trombly, D. J.;
Maratos-Flier, E. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005, 289, R117.
14. Chen, Y.; Hu, C.; Hsu, C. K.; Zhang, Q.; Bi, C.; Asnicar, M.; Hsiung, H. M.; Fox, N.;
Slieker, L. J.; Yang, D. D.; Heiman, M. L.; Shi, Y. Endocrinology 2002, 143, 2469.
15. Marsh, D. J.; Weingarth, D. T.; Novi, D. E.; Chen, H. Y.; Trumbauer, M. E.; Chen,
A. S.; Guan, X. M.; Jiang, M. M.; Feng, Y.; Camacho, R. E.; Shen, Z.; Frazier, E. G.;
Yu, H.; Metzger, J. M.; Kuca, S. J.; Shearman, L. P.; Gopal-Truter, S.; MacNeil, D.
J.; Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H.; Qian, S. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 3240.
5.4.1. Pharmacokinetic analysis in rats
Test compounds were administered intravenously (iv, 0.1 mg/
kg) or orally (po, 1 mg/kg, suspended in 0.5% methylcellulose aque-
ous solution) by cassette dosing to fed Sprague–Dawley rats. After
administration, blood samples were collected and centrifuged to
obtain the plasma fraction. The plasma samples were depro-
teinized by mixing with acetonitrile followed by centrifugation.
The compound concentrations in the supernatant were measured
by LC/MS/MS.
16. Cheon, H. G. Handb. Exp. Pharmacol. 2012, 383.
17. Goldstein, C.; Schroeder, J. C.; Fortin, J. P.; Goss, J. M.; Schaus, S. E.; Beinborn,
M.; Kopin, A. S. J. Pharmacol. Exp. Ther. 2010, 335, 799.
18. Igawa, H.; Takahashi, M.; Kakegawa, K.; Kina, A.; Ikoma, M.; Aida, J.; Yasuma,
T.; Kawata, Y.; Ashina, S.; Yamamoto, S.; Kundu, M.; Khamrai, U.; Hirabayashi,
H.; Nakayama, M.; Nagisa, Y.; Kasai, S.; Maekawa, T. J. Med. Chem. 2016, 59,
1116.
5.4.2. Evaluation of anorectic effect using male DIO-F344/Jcl rats
Male DIO-F344/Jcl rats (44 weeks old) fed with a high-fat diet
(D12451: Research Diets) from 5 weeks old were used. From before
the start of experiment, the rats were singly housed, given a pow-
der high-fat diet (D12451M: Research Diets), and habituated to
oral administration with tap water. The rats were grouped based
on both the food intake and the body weight of day 0. The rats were
orally administered vehicle (0.5% methylcellulose solution) or
compounds suspended in vehicle at 2 mL/kg 2 h before the onset
of dark period for 2 days (n = 6 for each group). The food intake
for 2 days from the initial administration was measured. The food
intake inhibition rate of each compound administration group to
the vehicle group was calculated.
19. Igawa, H.; Takahashi, M.; Shirasaki, M.; Kakegawa, K.; Kina, A.; Ikoma, M.; Aida,
J.; Yasuma, T.; Okuda, S.; Kawata, Y.; Noguchi, T.; Yamamoto, S.; Fujioka, Y.;
Kundu, M.; Khamrai, U.; Nakayama, M.; Nagisa, Y.; Kasai, S.; Maekawa, T.
Bioorg. Med. Chem. 2016. http://dx.doi.org/10.1016/j.bmc.2016.04.011. in press.
20. Johansson, A.; Löfberg, C. Expert Opin. Ther. Pat. 2015, 25, 193.
21. Washburn, W. N.; Manfredi, M.; Devasthale, P.; Zhao, G.; Ahmad, S.;
Hernandez, A.; Robl, J. A.; Wang, W.; Mignone, J.; Wang, Z.; Ngu, K.;
Pelleymounter, M. A.; Longhi, D.; Zhao, R.; Wang, B.; Huang, N.; Flynn, N.;
Azzara, A. V.; Barrish, J. C.; Rohrbach, K.; Devenny, J. J.; Rooney, S.; Thomas, M.;
Glick, S.; Godonis, H. E.; Harvey, S. J.; Cullen, M. J.; Zhang, H.; Caporuscio, C.;
Stetsko, P.; Grubb, M.; Maxwell, B. D.; Yang, H.; Apedo, A.; Gemzik, B.; Janovitz,
E. B.; Huang, C.; Zhang, L.; Freeden, C.; Murphy, B. J. J. Med. Chem. 2014, 57,
7509.
22. Devasthale, P.; Wang, W.; Mignone, J.; Renduchintala, K.; Radhakrishnan, S.;
Dhanapal, J.; Selvaraj, J.; Kuppusamy, R.; Pelleymounter, M. A.; Longhi, D.;
Huang, N.; Flynn, N.; Azzara, A. V.; Rohrbach, K.; Devenny, J.; Rooney, S.;
Thomas, M.; Glick, S.; Godonis, H.; Harvey, S.; Cullen, M. J.; Zhang, H.;
Caporuscio, C.; Stetsko, P.; Grubb, M.; Huang, C.; Zhang, L.; Freeden, C.;
Murphy, B. J.; Robl, J. A.; Washburn, W. N. Bioorg. Med. Chem. Lett. 2015, 25,
4412.
23. Terrasson, V.; Michaux, J.; Gaucher, A.; Wehbe, J.; Marque, S.; Prim, D.;
Campagne, J. M. Eur. J. Org. Chem. 2007, 5332.
24. Stevens, K. L.; Jung, D. K.; Alberti, M. J.; Badiang, J. G.; Peckham, G. E.; Veal, J. M.;
Cheung, M.; Harris, P. A.; Chamberlain, S. D.; Peel, M. R. Org. Lett. 2005, 7, 4753.
25. Creencia, E. C.; Kosaka, M.; Muramatsu, T.; Kobayashi, M.; Iizuka, T.;
Horaguchib, T. J. Heterocycl. Chem. 2009, 46, 1309.
26. Duan, J. X.; Chen, Q. Y. J. Chem. Soc., Perkin Trans. 1 1994, 725.
27. Tomosaka, H.; Omata, S.; Hasegawa, E.; Anzai, K. Biosci. Biotechnol. Biochem.
1997, 61, 1121.
28. Albertini, S.; Bös, M.; Gocke, E.; Kirchner, S.; Muster, W.; Wichmann, J.
Mutagenesis 1998, 13, 397.
5.4.3. Evaluation of anti-obesity effect using male DIO-F344/Jcl
rats
DIO-F344 rats (48 weeks old) were habituated and grouped
prior to treatment as described above. The rats were orally admin-
istered vehicle (0.5% methylcellulose solution) or compounds sus-
pended in vehicle at 2 mL/kg for 2 weeks (n = 6 for each group).
Sibutramine was used as a positive control in this study. The com-
pounds were administered after measurement of body weight at
1–3 h before the onset of dark period, and food intake was mea-
sured every 2 or 3 days. The change in body weight was presented
as percentage from initial body weight.
29. Ellis, J. M.; Altman, M. D.; Bass, A.; Butcher, J. W.; Byford, A. J.; Donofrio, A.;
Galloway, S.; Haidle, A. M.; Jewell, J.; Kelly, N.; Leccese, E. K.; Lee, S.; Maddess,
M.; Miller, J. R.; Moy, L. Y.; Osimboni, E.; Otte, R. D.; Reddy, M. V.; Spencer, K.;
Sun, B.; Vincent, S. H.; Ward, G. J.; Woo, G. H.; Yang, C.; Houshyar, H.; Northrup,
A. B. J. Med. Chem. 2015, 58, 1929.
Acknowledgements
We acknowledge Dr. Youichi Nishikawa to drug design. We
thank Dr. Mitsuyuki Shimada for the alliance with TCG Lifesciences
Ltd, and Dr. Tomohiko Suzaki for arrangement of compound
shipment.
30. Elongated molecular length of compounds 8j–l might affect the mutagenic
liability (see Ref. 29).
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013

H. Igawa et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
15
31. Maron, D. M.; Ames, B. N. Mutat. Res. 1983, 113, 173.
34. Molecular Operating Environment (MOE), 2012.10; Chemical Computing
Group Inc.; Montreal, Canada, 2012.
35. Ishihara, Y.; Suzuki, N.; Takekawa, S. Melanin concentrating hormone
antagonists. PCT Int. Appl. WO01/82925 A1, 2001.
32. Gatehouse, D.; Haworth, S.; Cebula, T.; Gocke, E.; Kier, L.; Matsushima, T.;
Melcion, C.; Nohmi, T.; Venitt, S.; Zeiger, E. Mutat. Res. 1994, 312, 217.
33. ACD Labs ver. 12.0, Advanced Chemistry Development Inc, Toronto, Ontario,
Canada, http://www.acdlabs.com.
Please cite this article in press as: Igawa, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.013