Identification of 2-aminobenzimidazoles as potent melanin-concentrating hormone 1-receptor (MCH1R) antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.04.147

A series of 2-aminobenzimidazole-based MCH1R antagonists was identified by core replacement of the aminoquinoline lead 1. Subsequent modification of the 2- and 5-positions led to improvement in potency and intrinsic clearance. Compound 25 exhibited good p

Full text of DOI:10.1016/j.bmcl.2009.04.147

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3568–3572
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2-aminobenzimidazoles as potent melanin-concentrating
hormone 1-receptor (MCH1R) antagonists
*
Minoru Moriya , Hiroyuki Kishino, Shunji Sakuraba, Toshihiro Sakamoto, Takuya Suga, Hidekazu Takahashi,
Takao Suzuki, Masahiko Ito, Junko Ito, Ryuichi Moriya, Norihiro Takenaga, Hisashi Iwaasa,
Akane Ishihara, Akio Kanatani, Takehiro Fukami
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo-3, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-aminobenzimidazole-based MCH1R antagonists was identified by core replacement of the
aminoquinoline lead 1. Subsequent modification of the 2- and 5-positions led to improvement in potency
and intrinsic clearance. Compound 25 exhibited good plasma and brain exposure, and attenuated MCH
induced food intake at 30 mg/kg PO in rats.
Received 25 February 2009
Revised 27 April 2009
Accepted 29 April 2009
Available online 5 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MCHIR-antagonist
Melanin-concentrating hormone
Obesity
GPCR
Melanin-concentrating hormone (MCH) is a cyclic nonadeca-
peptide with a disulfide bond that is expressed predominantly in
the lateral hypothalamus. MCH was originally isolated from sal-
mon pituitaries as a factor of skin color alteration.1 Thus far, sev-
eral lines of evidence have shown that MCH is an important
mediator of energy homeostasis. Up-regulation of prepro-MCH
mRNA has been reported in several obese animals.2–4 Chronic
intracerebroventricular infusion of MCH causes obesity with
hyperphagia.5,6 Targeted disruption of the prepro-MCH gene re-
duces food intake and increases the metabolic rate, generating a
lean phenotype.7 In contrast, overexpression of the prepro-MCH
gene causes moderate obesity in mice.8 MCH binds to and activates
two types of G-protein-coupled receptors, MCH1R9,10 and
MCH2R.11 MCH1R is expressed in rodents and higher mammals,
whereas MCH2R is expressed in ferrets, dogs, rhesus monkeys,
and humans, but not in rodents. Recently, we showed that chronic
administration of a peptidic MCH1R antagonist suppresses food in-
take and body weight gain in diet-induced obesity (DIO) rats and
mice.12,13 Thus, the MCH1R antagonist could be a potential thera-
peutic agent for the treatment of obesity.14
and virtual screening techniques,17 was subsequently published
by other laboratories. This 2-aminoqunoline series exhibits excel-
lent binding activity to MCH1R; however, no orally available com-
pound has been reported in this class. We speculated that the
lipophilic quinoline core might be detrimental to metabolic stabil-
ity and solubility, resulting poor bioavailability in this series. In
addition, we conjectured that the nitrogen atom at the 1-position
in the quinoline ring may be involved in a key interaction with
the receptor, and that the basicity coming from the nitrogen atom
in the quinoline moiety is critical for potency. These hypotheses
led us to attempt to create potent and orally available MCH1R
antagonists by replacing the quinoline core with hydrophilic bio-
isostere, which possesses a basic site capable of interacting with
the receptor. A bioisosteric 2-aminobenzimidazole template was
found to show potent MCH1R binding activity, and subsequent
structure–activity relationship (SAR) studies led to the identifica-
tion of potent and in vivo active compounds exemplified by 25.18
F3C
Discovery of 2-aminoquinoline-6-carboxamide MCH1R antago-
nists, exemplified by 1, was reported first by DeVita et al. (Fig. 1).15
Identification of the related compounds series, using a homology
model of MCH1R based on the crystal structure of rhodopsin16
H
N
Me
O
N
N
Me
Me
1
* Corresponding author. Tel.: +81 29 877 2218; fax: +81 29 877 2029.
E-mail address: minoru_moriya@merck.com (M. Moriya).
Figure 1. Aminoquinoline MCH1R antagonist.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.147

M. Moriya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3568–3572
3569
Boc
N
H
N
H
N
a
b
O2N
O2N
O2N
N
+
Cl
Cl
Cl
Cl
N
N
N
N
Boc
5
3
4
2
c
3
H
N
H
H
N
H
N
R2
H2N
R3
O
O2N
R2
R2
f
N
N
e
N
N
N
R1
R1
N
R1
d
N
4 and 5
6
7
8-25
Scheme 1. Reagents and conditions: (a) 70% HNO3, H2SO4; (b) Boc2O, DMAP, THF; (c) R1R2NH (8–50 equiv), neat or in dioxane, sealed tube, 130 °C; (d) R1R2NH or HCl salt
(2.2–3.3 equiv), DIEA, dioxane or DMSO, 80–100 °C; (e) H2, Pd(OH)2–C, MeOH; (f) R3COOH, HATU, DIEA, CH2Cl2 or R3COCl, Et3N, CH2Cl2.
In this Letter, we report the development of a series of 2-amino-
benzimidazole-5-carboxamide MCH1R antagonists.
The synthesis of the 2-aminobenzimidazole-5-carboxamide
derivatives is described in Scheme 1. Commercially available 2-
chlorobenzimidazole (2) was converted to 5-nitro derivative 3 by
treatment with 70% HNO3 and H2SO4. Displacement of the chloro
substituent with desired amino groups was accomplished by reac-
tion of 3 with a large excess amount of the corresponding amines
in a sealed tube at 130 °C, providing 2-aminobenzimidazole 6.
Alternatively N-Boc-protected 2-chloro-5-nitrobenzimidazole can
be used as a mixture of regioisomers (4 and 5) instead of 3, In this
case, the substitution reaction proceeded under milder conditions,
O
Cl
N
a
b
c
Cl
N
N
Me
Me
MeO
Me
Me
N
N
N
Cl
N
N
Me
Me
26
27
28
F3C
O
H
H
H2N
N
N
d, e
N
f, g
N
N
N
Me
Me
Boc
Me
Me
Me
Me
H
O
N
N
Me
N
N
Me
N
N
Me
29
30
31
Scheme 2. Reagents and conditions: (a) N-methylisopropylamine (10 equiv), dioxane, sealed tube, 120 °C; (b) CO (140 psi), Pd(dppf)Cl2ꢀCH2Cl2, NaHCO3, MeOH/dioxane,
100 °C; (c) NH2NH2ꢀH2O, MeOH; (d) NaNO2, 1 N aq HCl, 0 °C; (e) tert-BuOH/dioxane, reflux; (f) CF3COOH; (g) RCOCl, pyridine, CHCl3.
O2N
O2N
Br
t
O2N
CO2 Bu
a
b
c
N
N
H
O
N
NH2
N
NH2
F3C
32
33
34
H
N
O2N
d, e, f
Me
Me
Me
O
N
N
Cl
N
N
N
35
36
Scheme 3. Reagents and conditions: (a) tert-butyl acrylate, Pd(OAc)2, (o-tol)3P, Et3N/DMF, 130 °C; (b) 5 N aq HCl, sealed tube, 130 °C; (c) POCl3, reflux; (d) N-
methylisopropylamine, K2CO3, DMF, 80 °C; (e) H2, Pd–C, MeOH; (f) RCOCl, Et3N, CH2Cl2.
F3C
Me
N
H
N
Me
N
a
b, c
O2N
O2N
Me
Me
S
N
S
N
S
N
Cl
O
Me
Me
37
38
39
Scheme 4. Reagents and conditions: (a) N-methylisopropylamine (3 equiv), K2CO3, DMF, 80 °C; (b) H2, Pd–C, MeOH/THF; (c) RCOOH, 2-chloro-1,3-dimethylimidazolidinium
chloride, pyridine, CHCl3.

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M. Moriya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3568–3572
Table 1
Table 2
MCH1R binding and calculated pKa data for bioisosteric derivatives
MCH1R binding and function data for 2-aminobenzimidazole-5-carboxamides
Compound Structure
Binding Calculated
F3C
b
assay
pKa
H
N
H
N
a
R2
IC50
(nM)
N
O
R1
N
F3C
Me
N
H
N
H
N
Compound
NR1R2
Binding assay
IC50 (nM)
Functional assay
IC50 (nM)
8
Me
5.9
7.8
5.1
5.3
3.9
a
a
O
O
N
Me
F3C
Me
H
N
N
8
5.9
12
19
24
21
nd
nd
nd
N
N
Me
Me
31
375
Me
N
N
Me
Me
F3C
Me
H
N
9
N
Me
36
365
Me
O
N
N
Me
Me
F3C
Me
Me
H
N
10
11
N
39
Me
>750
S
N
Et
N
O
Me
Me
a
The data represent means of at least two experiments.
N
pKa values were calculated by the SPARC on-line calculator.21
b
namely 4 and 5 reacted with 2.2–3.3 equiv of the desired amine at
a lower temperature of 80–100 °C. Under the present reaction con-
ditions, the Boc group at the resulting 2-amino-benzimidazole was
removed to afford 6. Hydrogenation of the 6-nitro group in 6 fol-
lowed by a coupling reaction with carboxylic acid in the presence
of O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate (HATU) or acyl chloride afforded the target com-
pounds 8–25.
Scheme 2 shows the synthesis of 1,5-naphthyridine derivative
31. Mono-amination of commercially available 2,6-dic-
hloronaphthyridine (26) followed by palladium catalyzed methoxy
carbonylation provided methyl ester 28. Compound 28 was con-
densed with hydrazine to give hydrazide 29, which was then sub-
jected to diazotization and subsequent Curtius rearrangement in
the presence of tert-butanol to give compound 30. Treatment of
30 with trifluoroacetic acid followed by acylation afforded com-
pound 31.
Synthesis of 1,8-naphthyridine derivative 36 was performed as
shown in Scheme 3. Commercially available 2-amino-3-bromo-5-
nitropyridine (32) was converted to naphthyridinone 34 by Heck
reaction employing tert-butyl acrylate, subsequent cyclization un-
der acidic conditions. Treatment of 34 with POCl3 provided 2-
chloro-2-nitronaphthyridine (35), which was converted to com-
pound 36 through a similar fashion (amination, reduction, and
then acylation) to the synthesis for benzimidazoles 8–25.
Benzothiazole derivative 39 was prepared from commercially
available 2-chloro-6-nitrobenzothiazlole (37) through a similar
fashion (amination, reduction, and then acylation) to the synthesis
for benzimidazoles 8–25 as shown in Scheme 4.
Compounds were evaluated for their binding affinity to the
membranes of CHO-K1 cells expressing human MCH1R (hMCH1R)
in a competition binding assay with [125I]-MCH as the radioli-
gand.19 Antagonistic activity was estimated by the inhibitory effect
of compounds on intracellular calcium mobilization induced by
MCH using a FLIPR in CHO-K1 cells expressing human MCH1R.20
Me
N
12
13
14
15
16
17
4
22
27
nd
nd
nd
Me
N
6.7
27
30
23
Me
N
O
N
Me
N
O
Me
N
NMe
Et
N
135
428
nd
nd
18
N
O
a
The data represent means of at least two experiments (nd = not determined).

M. Moriya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3568–3572
3571
Related compounds possessing the bioisostere for quinoline
were prepared and evaluated for MCH1R binding activity (benzim-
dazole 8, 1,5-naphthyridine 31, 1,8-naphthyridine 36, and benzo-
thiazole 39). Compounds 31, 36, and 39 exhibited modest
binding affinity. Interestingly, benzimidazole 8 showed excellent
The hMCH1R binding affinities and functional inhibitory poten-
cies of compounds with a variety of amino substituents at the 2-
position are shown in Table 2. Replacement of the iso-propyl group
on the amino group in 8 with (cyclo) lower alkyl groups such as
methyl, ethyl, and cyclopropyl groups led to a decrease in binding
affinity (9, 10, and 11). Introduction of larger cycloalkyl groups
such as cyclopentyl and cyclohexyl groups retained the binding
affinity (12 and 13), although introduction of oxygen- or nitro-
gen-containing cycloalkyl groups was deleterious to binding activ-
ity as seen in 14, 15, and 16. Replacing the methyl group in 13 with
a larger alkyl group, an ethyl group, produced a 20-fold less active
compound 17 (IC50: 135 nM). The combination of a methyl group
and a bulky group on the amine provided potent MCH1R antago-
nists. Interestingly, morpholine 18 (IC50: 428 nM) showed a signif-
icant loss in binding activity.
binding affinity (IC50: 5.9 nM) and functional activity (IC50:
21 nM). The calculated pKa values of compounds 31, 36, and 39
are less than 5.3.21 In contrast, the calculated pKa value of com-
pound 8 is 7.8,21 supporting our hypothesis that appropriate basi-
city at the 1-position is critical for potency in this series (Table 1).
Discovery of benzimidazole 8 with excellent MCH1R binding activ-
ity encouraged us to explore further SAR studies of this series.
Table 3
MCH1R binding and function data for 2-aminobenzimidazole-5-carboxamides
Compound 8 was then evaluated for its metabolic stability
using rat hepatocytes.22 Compound 8 was found to show modest
clearance (32 mL/min/kg). We turned our attention to expand
SAR to the carboxamide moiety to improve binding affinity and
intrinsic clearance value (Table 3). Replacement of the terminal
phenyl ring with a 2-pyridyl ring 19 (IC50: 41 nM) showed a seven-
fold decrease in binding activity compared to the parent 8. The eth-
H
N
H
N
R
Me
N
O
N
Me
Me
ylene linkage in the dihydrocinnamyl moiety,
a potentially
Compound
R
Binding assay
IC50 (nM)
Rat hepatic clearance
CLb (mL/min/kg)
a
metabolically labile moiety, was replaced with aromatic rings to
give p-biaryl compound 20 (IC50: 3.1 nM, clearance: 16 mL/min/
kg) with significant improvement in binding affinity (compound
19 vs 20) and clearance (compound 8 vs. 20). Therefore, we inves-
tigated a series of biaryl analogs. Azine rings such as pyridine,
pyrimidine, and pyrazine rings were well tolerated. Incorporation
of a fluorine atom to the phenyl group in 21 provides a positive im-
pact on the binding activity and clearance as seen in 22. The meta-
F3C
8
5.9
41
32
nd
16
12
F3C
F3C
19
20
21
N
N
Table 4
Pharmacokinetic data of 25 in rats
3.1
8.8
25
Fa (%)
35
11
1.2
2.3
3.9
1.4
CLa (mL/min/kg)
Vda (L/kg)
a
N
T1/2 (h)
Plasma levelb,c
(lM)
Brain levelb,c (nmol/g)
a
The rats were dosed at 1 mg/kg IV and 3 mg/kg PO (mean values, n = 3).
The rats were dosed at 30 mg/kg PO (mean values, n = 3).
Plasma and brain levels were determined at 2 h post-dose.
b
F
c
22
23
24
5.5
8
N
N
10
8
900
nd
F
F
**
6
4
2
0
15
9
N
N
aCSF
Vehicle
10
30
F
Compound 25 (mg/kg, PO)
MCH (5 µg, ICV)
N
N
25
2.7
10
Figure 2. Effect of compound 25 on MCH-induced feeding response in SD rats.
Values are the mean SEM of 15–20 rats per group. **P <0.01 versus vehicle/MCH
group.
b
Intrinsic clearance measured in rat hepatocytes.22
The data represent means of at least two experiments (nd = not determined).
a

3572
M. Moriya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3568–3572
10. Saito, Y.; Nothacker, H. P.; Wang, Z. W.; Lin, S. H. S.; Leslie, F.; Civelli, O. Nature
1999, 400, 265.
orientated biaryl analog 23 (IC50: 900 nM) displayed a significant
decrease in affinity. The pyrazinecarboxamide derivative 25 (IC50
2.7 nM, clearance: 10 mL/min/kg) showed the best balance of po-
tency and clearance.
We evaluated the pharmacokinetic properties of compound 25
in rats as shown in Table 4. Compound 25 demonstrated a low
clearance (11 mL/min/kg) and good oral bioavailability (35%). Fur-
:
11. Sailer, A. W.; Sano, H.; Zeng, Z. 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. P.; Austin, C. P.;
MacNeil, D. J.; Ishihara, A.; Ihara, M.; Kanatani, A.; Van der Ploeg, L. H. T.;
Howard, A. D.; Liu, Q. Y. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7564.
12. Borowsky, B.; Durkin, M. M.; Ogozalek, K.; Marzabadi, M. R.; DeLeon, J.;
Heurich, R.; Lichtblau, H.; Shaposhnik, Z.; Daniewska, I.; Blackburn, T. P.;
Branchek, T. A.; Gerald, C.; Vaysse, P. J.; Forray, C. Nat. Med. 2002, 8, 825.
13. Mashiko, S.; Ishihara, A.; Gomori, A.; Moriya, R.; Ito, M.; Iwaasa, H.; Matsuda,
M.; Feng, Y.; Shen, Z.; Marsh, D. J.; Bednarek, M. A.; MacNeil, D. J.; Kanatani, A.
Endocrinology 2005, 146, 3080.
thermore, its levels in plasma and brain at 2 h were 3.9 lM and
1.4 nmol/g, respectively (brain/plasma ratio: 0.36) at an oral dose
of 30 mg/kg. We also examined the effect of compound 25 on the
MCH-induced feeding response in SD rats as shown in Figure 2.
Oral dosing at 10 and 30 mpk dose-dependently suppressed food
intake by 32% and 72% for 2 h, respectively, compared to the vehi-
cle treatment group.23
In summary, we discovered that 2-aminobenzimidazoles were
potent MCH1R antagonists with bioisosteric replacement of the
quinoline core. Subsequent optimization of 2-amino and 6-carbox-
amide positions led to identification of compound 25 that has
demonstrated excellent in vitro activity and good brain penetrabil-
ity. Compound 25 also significantly suppressed MCH induced food
intake after oral dosing in rats.
14. General reviews on MCH1R antagonists: (a) Luithin, D. R. Life Sci. 2007, 81, 423;
(b) Rokosz, L. L. Expert Opin. Drug Discov. 2007, 10, 1301; (c) Dyke, H. J.; Ray, N.
C. Expert Opin. Ther. Patents 2005, 15, 1303.
15. (a) DeVita, R. J.; Chang, L.; Chaung, D.; Hoang, M.; Jiang, J.; Lin, P.; Sailer, A. W.;
Young, J. R. WO03045313, 2003; (b) Jiang, J. L.; Hoang, M.; Young, J. R.; Chaung,
D.; Eid, R.; Turner, C.; Lin, P.; Tong, X. C.; Wang, J. Y.; Tan, C.; Feighner, S.;
Palyha, O.; Hreniuk, D. L.; Pan, J.; Sailer, A. W.; MacNeil, D. J.; Howard, A.;
Shearman, L.; Stribling, S.; Camacho, R.; Strack, A.; Van der Ploeg, L. H. T.;
Goulet, M. T.; DeVita, R. J. Bioorg. Med. Chem. Lett. 2006, 16, 5270.
16. Ulven, T.; Frimurer, T. M.; Receveur, J. M.; Little, P. B.; Rist, O.; Norregaard, P. K.;
Hogberg, T. J. Med. Chem. 2005, 48, 5684.
17. Clark, D. E.; Higgs, C.; Wren, S. P.; Dyke, H. J.; Wong, M.; Norman, D.; Lockey, P.
M.; Roach, A. G. J. Med. Chem. 2004, 47, 3962.
18. Moriya, M.; Kanatani, A.; Iwaasa, H.; Ishihara, A.; Fukami, T. WO2004011440,
2004.
19. Membranes from CHO-K1 cells stably expressing human MCH1R were
incubated with
compounds in binding buffer (50 mM Tris, 10 mM MgCl2, 2 mM EDTA, 50
mL bacitracin, 0.2% BSA, pH 7.4) at room temperature for 2 h. The membranes
were filtrated onto GF/C filter plates and dried. The radioactivity was counted
using a microplate scintillation counter (TopCount, PerkinElmer). Non-specific
[
125I]-MCH (PerkinElmer, Waltham, MA, USA) and test
l
g/
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binding was determined by including 1
reaction.
lM unlabeled MCH in the binding
20. CHO-K1 cells stably expressing human MCH1R were seeded into 96-well
culture plates at 3.0 ꢁ 104 cells per well and cultured in culture medium
(Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) (Invitrogen, Carlsberg, CA, USA), 10% Hepes, 1 mg/mL G418,
100 U/ml penicillin, and 100 g/ml streptomycin) for 24 h. The cells were loaded
with Fluo-4 AM in DMEM containing 10% FBS and 2.5 mM probenecid at 37 °C
for 1 h. Then the cells were washed with assay buffer (Hanks’ balanced salt
solution containing 20 mM Hepes, 0.5% BSA, and 2.5 mM probenecid, pH 7.4)
and analyzed using a FLIPR system (Molecular Devices, Sunnyvale, CA) to
measure the mobilization of intracellular Ca2+ in response to MCH. The cells
were treated with the test compounds for 5 min before addition of MCH.
21. pKa values were calculated by the SPARC on-line calculator (http://
ibmlc2.chem.uga.edu/sparc/).
22. Shibata, Y.; Takahashi, H.; Ishii, Y. Drug Metab. Dispos. 2000, 28, 1518.
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23.
A 26-gauge guide cannula was stereotaxically implanted into the third
ventricle of male SD rats as described previously (Endocrinology 146:3080-
3086, 2005). After at least 1 week recovery, rats were fed a high fat diet for
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J.; Lowell, B.; Flier, J. S.; Maratos-Flier, E. J. Clin. Invest. 2001, 107, 379.
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One hour after the drug administration, MCH (5 lg/lL/head, dissolved in
artificial cerebrospinal fluid) was injected ICV and food intake for 2 h was
measured. MCH injection was performed 1 h before the onset of the dark cycle.