Identification of 2-(4-benzyloxyphenyl)-N-[1-(2-pyrrolidin-1-yl-ethyl)-1H- indazol-6-yl]acetamide, an orally efficacious melanin-concentrating hormone receptor 1 antagonist for the treatment of obesity

Article abstract of DOI:10.1021/jm0490890

Optimization of a high-throughput screening hit against melanin-concentrating hormone receptor 1 (MCHr1) led to the discovery of 2-(4-benzyloxy-phenyl)-N-[1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6-yl]acetamide (7a). This compound was found to be a high-aff

Full text of DOI:10.1021/jm0490890

1318
J. Med. Chem. 2005, 48, 1318-1321
Identification of
2-(4-Benzyloxyphenyl)-N-
[1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-
6-yl]acetamide, an Orally Efficacious
Melanin-Concentrating Hormone
Receptor 1 Antagonist for the Treatment
of Obesity
Andrew J. Souers,* Ju Gao, Michael Brune,
Eugene Bush, Dariusz Wodka, Anil Vasudevan,
Andrew S. Judd, Mathew Mulhern, Sevan Brodjian,
Brian Dayton, Robin Shapiro, Lisa E. Hernandez,
Kennan C. Marsh, Hing L. Sham,
Figure 1. Indole-based MCHr1 antagonists. (a) Values
represent an average of at least two determinations with a
deviation of (45% of the mean value shown. (b) Results are
for 10 mg/kg, po in DIO mice, and interanimal variability was
less than 25% for all values.
Christine A. Collins, and Philip R. Kym
Metabolic Disease Research, Abbott Laboratories,
100 Abbott Park Road, Abbott Park, Illinois 60064
tional antagonism of MCH-mediated Ca2+ release. When
dosed orally at 10 mg/kg in DIO mice, 1 demonstrated
significant drug levels in plasma and brain.
Received November 11, 2004
Abstract: Optimization of a high-throughput screening hit
against melanin-concentrating hormone receptor 1 (MCHr1)
led to the discovery of 2-(4-benzyloxy-phenyl)-N-[1-(2-pyrro-
lidin-1-yl-ethyl)-1H-indazol-6-yl]acetamide (7a). This com-
pound was found to be a high-affinity ligand for MCHr1 and
a potent inhibitor of MCH-mediated Ca2+ release, showed good
plasma and CNS exposure upon oral dosing in diet-induced
obese mice, and is the first reported MCHr1 antagonist that
is efficacious upon oral dosing in a chronic model of weight
loss.1
Melanin-concentrating hormone2 (MCH) is a cyclic 19-
amino-acid peptide that is synthesized in cell bodies in
the lateral hypothalamus and zona incerta of the central
nervous system (CNS). The MCH peptide is understood
to play a major role in body weight regulation in
rodents2,3 as a single injection of MCH into the CNS
stimulates food intake in rats4 and chronic administra-
tion leads to increased body weight.5 Transgenic mice
overexpressing the MCH gene are susceptible to insulin
resistance and obesity,6 while mice lacking the gene
encoding MCH are hypophagic, lean, and maintain
elevated metabolic rates.7 Consistent with this pheno-
type, genetically altered mice that lack the gene encod-
ing the MCH receptor maintain elevated metabolic rates
and thus remain lean despite hyperphagia on a normal
diet.8,9 These data indicate that successful antagonism
of the MCHr1 system could lead to weight loss, and the
observation that chronic intraperitoneal administration
of a small-molecule antagonist leads to the reduction
of food intake and body weight10 provides further
validation of MCHr1 blockade as a novel target for
antiobesity pharmacotherapy.11
Initial medicinal chemistry efforts to improve upon
the binding affinity of 1 by modifying the 6-positional
substituent were largely unsuccessful as the incorpora-
tion of several linear and branched glycine-based side
chains resulted in compounds of comparable or weaker
activity. Since deletion of the tertiary amine from the
1-(2-pyrrolidin-1-ylethyl) substituent was also deleteri-
ous to activity, we focused on the removal of the
6-positional glycine-based side chain in favor of nonbasic
amides. The structure-activity relationship (SAR) of the
resultant batch of analogues was similarly unforgiving,
affording primarily inactive compounds. One exception
was the 6-benzyloxyphenylacetic amide 2, which was
10-fold more active in the binding assay and showed
improved functional antagonism relative to lead 1.
However, indole 2 demonstrated less efficient CNS
penetration when evaluated in DIO mice (10 mg/kg, po).
The difficulty in obtaining positive SAR trends with
respect to the amide side chain along with the disap-
pointing pharmacokinetic profile of 2 prompted the
investigation of benzimidazole and indazole surrogates
of the indole core.
The general synthetic approach to the 2-pyrrolidin-
1-ylethylindazole amides is outlined in Scheme 1. The
4-nitroindazole was synthesized from the corresponding
3-nitro-o-tolylamine, while the 5- and 6-nitroindazoles
were obtained from commercial sources. Alkylaton of the
nitroindazole cores (4) with 2-chloroethylpyrrolidine
hydrochloride afforded a mixture of N1 and N2-alky-
lated isomers in ratios that varied by the position of the
nitro substituent (see Supporting Information). After
chromatographic separation of the isomers, iron-medi-
ated reduction afforded the individual anilines 5 and
amide bond formation furnished the final products 7a-c
and 8a-c. A similar strategy was taken for the benz-
imidazole analogues 6a,b, starting with the alkylation
of 5-nitrobenzimidazole followed by separation of the
isomers before reduction and amide coupling.
High-throughput screening of the Abbott compound
collection against MCHr1 resulted in the identification
of the 6-substituted 2-pyrolidinylethylindole 1 (Figure
1). This compound displayed moderate affinity in a
receptor-binding assay using MCHr1 obtained from
human neuronal IMR-32 cells and showed low-micro-
molar activity in an assay designed to measure func-
Table 1 summarizes the SAR of the MCHr1 binding
and functional activities of the heterocyclic analogues.
The 6-substituted benzimidazole analogue 6a demon-
* To whom correspondence should be addressed. Phone: 847-937-
5312. Fax: 847-938-1674. E-mail: andrew.souers@abbott.com.
10.1021/jm0490890 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/15/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1319
Scheme 1a
Table 2. Selected Pharmacokinetic Parameters of
Benzimidazole and Indazole Analoguesa
plasma AUC brain AUC brain Cmax
brain C12h
compd
(µg‚h/g)b
(µg‚h/g)b
(ng/g)b
(ng/g)c
6a
7a
7b
7c
8c
0.937
2.12
0.244
1.33
54.4 ( 0.3
177 ( 16
0.0
29.0 ( 1.8
8.51
10.4
4261 ( 656 76.0 ( 15.2
0.611
2.09
1.18
0.214
464 ( 40
214 ( 31
8.11 ( 1.58
12.3 ( 3.8
a All values are mean values ( SEM (n ) 3 unless specified
otherwise). Compounds are dosed in DIO mice at 10 mg/kg, po, in
a vehicle containing 1% Tween-80 and water. b The three mice
with highest plasma and brain concentrations were averaged to
provide the peak plasma and brain concentrations Cmax ( SEM,
respectively. The mean plasma or brain concentration data were
submitted to multiexponential curve fitting using WinNonlin. The
area under the mean concentration-time curve from 0 to t hours
a Reagents and conditions: (a) NaNO2, AcOH, room temp, 95%;
(b) chloroethylpyrolidine hydrochloride, K2CO3, DMF, 60 °C, then
SiO2 chromatography, 27-67%; (c) Fe, NH4Cl, 65 °C; (d) 4-ben-
zyloxy-4-phenylacetic acid, EDCI, HOBt, DMF, NMM, 56-89%.
(time of the last measurable concentration) after dosing (AUC0-t
)
was calculated using the linear trapezoidal rule for the concentra-
tion-time profile. The residual area was extrapolated to infinity,
determined as the final measured mean concentration (Ct) divided
by the terminal elimination rate constant (â) and was added to
Table 1. SAR of Heterocylic Analogues of 2a
c
AUC0-t to produce the total area under the curve (AUC0-∞). C12h
) brain concentration taken at 12 h postdose.
combination of 5-amino substitution and N2-alklyation
led to a predictable decrease in binding and functional
activity as 8b was the least active of the compounds
shown. Interestingly, the N2-alkylated 4-amino isomer
8c showed similar binding affinity to 7c, indicating a
greater tolerance of the receptor for the 4-amino sub-
stituted analogues. However, the preference for N1-
alkylation in the functional assay was further demon-
strated by 8c, which dropped over 4-fold in activity
relative to 7c.
Several of these analogues were dosed orally in DIO
mice to investigate the structural requirements for brain
and plasma exposure (Table 2). This set included the
N1-alkylated analogues 7a-c in order to assess the
effect of transposing the indazole amide side chain on
the pharmacokinetic profile. Similarly, the most potent
representatives from the benzimidazole (6a) and N2-
alkylated subseries (8c) were included for further
comparison of the differing heterocyclic cores. The
pharmacokinetic profile of the benzimidazole analogue
6a was characterized by moderate plasma exposure and
poor CNS penetration. Indazole analogue 7a showed
improved pharmacokinetic properties relative to 6a and
a nearly 4-fold increase in tissue exposure with respect
to lead indole 2. Although the brainAUC/plasmaAUC ratio
was less than 1, the compound maintained brain levels
that provided coverage of the functional IC50 for at least
12 h. Interestingly, each of the N1-substituted indazole
isomers 7a-c demonstrated good brain penetration. The
5-substituted indazole 7b resulted in the highest levels
of parent compound in the brain at the maximal
concentration (Cmax) and at 12 h (C12h). The N2-alky-
lated indazole 8c was less efficient in penetrating the
CNS and had a brainAUC/plasmaAUC ratio of 0.1.
MCHr1 binding IC50
(nM)b,d
Ca2+ release IC50
compd
position
(µM)c,d
6a
6b
7a
7b
7c
8a
8b
8c
6
5
6
5
4
6
5
4
161 ( 91
772 ( 317
1.40 ( 0.40
558 ( 79
24.5 ( 10.4
146 ( 59
>10
>10
0.011 ( 0.002
>10
0.071 ( 0.035
0.874 ( 0.157
>10
1985 ( 346
41.4 ( 14.1
0.287 ( 0.148
a All compounds were >95% pure by HPLC and characterized
by 1H NMR and HRMS. b Displacement of [125I]-MCH from MCHr1
expressed in IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25
nM, Bmax ) 0.40 ( 0.08 pmol/mg). c Inhibition of MCH-mediated
Ca2+ release in whole IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM).
d All values are mean values ( SEM and are derived from at least
three independent experiments (all duplicates).
strated moderate MCHr1 affinity but showed only weak
functional antagonism at the assayed concentration. A
similar result was observed for the 5-amino isomer 6b.
In contrast, incorporation of the 6-aminoindazole core
imparted significant improvements in binding affinity
and functional potency relative to indole 2 as analogue
7a showed low-nanomolar inhibition of MCH binding
and MCH-mediated Ca2+ release. Transposition of the
amide side chain to the 5-position (7b) of the indazole
core imparted an over 500-fold decrease in binding
affinity. However, binding activity was rescued to a
significant degree when the amide side chain was moved
to the 4-position to afford 7c, which was also a potent
inhibitor of Ca2+ release.
To probe the positional requirements of the 2-pyrro-
lidin-1-ylethyl substituent, the N2-alkylated nitroinda-
zoles were elaborated to the corresponding final prod-
ucts (Scheme 1, 8a-c). While the binding activity of
6-amino isomer 8a decreased over 140-fold relative to
the N1-alkylated 7a, it is noteworthy that indazole 8a
retained moderate antagonism of Ca2+ release. The
Compound 7a, which showed the best combination of
binding affinity, functional antagonism, and CNS ex-
posure, was selected for further evaluation. In receptor
selectivity panels, 7a showed weak affinity (IC50 > 10
µM) for other peptide receptors that are structurally
related to MCHr1, including MCHr2,12 somatostatin,
and the opioid receptors.13 We next explored the effects
of administration of 7a in a study measuring food intake
and body weight in DIO mice. For a 2-week period, DIO

1320 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Letters
Figure 2. Effect of 7a (dosed at 10 and 30 mg/kg, po, bid in
1% Tween-80 in water) and D-fenfluramine (D-fen, 10 mg/kg,
po, qd) on the body weight of DIO mice. Change is registered
as the number of grams of body weight difference for each
measurement time point relative to day zero. All values are
mean values ( SEM for n ) 12: (//) p < 0.05 for comparisons
against vehicle group.
Figure 3. Effect of 7a (10 and 30 mg/kg, po, bid, dosed in 1%
Tween-80 in water) and D-fenfluramine (D-fen, 10 mg/kg, po,
qd) on daily and cumulative food intake of DIO mice. All values
are mean values ( SEM for n ) 12: (/) p < 0.05 for
comparisons against vehicle group.
Table 4. Exposure Levels of 7a at 1 h (Cmax) and 17 h (C17h
)
after Final Dose on Day 14
Table 3. Body Weight (BW) of All Animal Groups at Days 0
and 14 and Percent Change of the Drug-Treatment Groups
Relative to Vehiclea
dose (mg/kg, po, bid) time (h) plasma (ng/mL)a brain (ng/g)a
10
10
30
30
1
17
1
168 ( 25.8
0.390 ( 0.220
824 ( 270
115 ( 67
7.02 ( 2.66
233 ( 115
5.35 ( 1.18
groupb
BWday0 (g) BWday14 (g) % change from vehicle
17
2.23 ( 0.280
lean
vehicle
31.7 ( 0.8 31.8 ( 0.8
45.3 ( 0.8 46.9 ( 0.8
a Drug concentrations in plasma and in brain were determined
1 or 17 h after the final dose. All values are mean values ( SEM
(n ) 3).
D-fen, 10 mg/kg 43.2 ( 1.5 40.4 ( 1.6
-10.1 ( 1.1
-8.25 ( 0.92
-15.2 ( 0.9
7a, 10 mg/kg
7a, 30 mg/kg
44.7 ( 0.5 42.6 ( 0.7
45.0 ( 0.7 39.7 ( 0.8
a All values are mean values ( SEM (n ) 12). b All doses were
given in 4 mL/kg body weight volume of vehicle (1% Tween-80 in
water). Compound 7a was administered po by gavage at doses of
10 and 30 mg/kg, bid, and D-fenfluramine was administered at a
dose of 10 mg/kg, po, qd. Food and body weights were determined
on the first day and periodically thereafter for 14 days.
yet were resistant to weight gain as a result of increased
energy expenditure.8,9
The exposure levels for 7a at 1 h (Cmax) and 17 h (C17h
)
following the final dose of the chronic study were
determined (Table 4). The plasma Cmax for the 10 mg/
kg treatment group is low (therapeutic plasma concen-
tration of 0.37 µM), which bodes well for the establish-
ment of a therapeutic safety index upon chronic dosing.
Additionally, it was apparent that a Cmax concentration
of 115 ( 67 ng/g in the brain was sufficient to deliver
significant chronic weight loss in this model.
mice fed a high-fat diet ad libitum were dosed orally
with 7a (10 or 30 mg/kg, bid), D-fenfluramine (10 mg/
kg, qd), or vehicle. Food intake and body weight were
measured at days 1, 4, 7, 11, and 14 for each group. The
vehicle group continued to gain weight throughout the
study (Figure 2), while D-fenfluramine caused a rapid
decrease in body weight followed by a slight rebound
starting on day 7.
Compound 7a caused a dose-dependent decrease in
body weight throughout the duration of the treatment.
At the end of the study, mice treated with D-fenflu-
ramine weighed 10.1 ( 1.1% less than vehicle-treated
mice (Table 3, p < 0.01), whereas the mice dosed with
indazole 7a weighed 8.25 ( 0.92% (p < 0.01) and 15.2
( 0.9% (p < 0.01) less for the 10 and 30 mg/kg groups,
respectively.
Interestingly, the cumulative food intake was not
significantly altered in the mice treated with 7a (Figure
3) at either dose (41.9 ( 3.3 and 44.7 ( 4.8 g, respec-
tively) compared to vehicle-treated controls (41.1 ( 1.3
g). In contrast, treatment with D-fenfluramine caused
a significant decrease in food intake over the first 7 days
(15.7 ( 1.2 g, p < 0.05 vs 20.6 ( 0.8 g for vehicle-treated
controls) and lower total food consumption by the end
of the 2-week study (36.0 ( 1.7 g). These results suggest
that the weight loss observed upon treatment with
MCHr1 antagonist 7a could be the result of an alter-
ation in energy expenditure. Similar findings have been
reported in MCHr1 -/- mice, which consumed the same
amount of food on a normal diet as the wild-type mice,
No significant changes in locomotor activity were
observed upon treatment with 7a at either dose, indi-
cating that treatment did not cause any overt behavioral
effects (data not shown). Finally, analysis of body
composition following the 2-week treatment was per-
formed with dual-energy X-ray absorptiometry14 (DEXA)
to determine the contributions of changes in fat and lean
mass relative to the observed decrease in body weight.
Fat mass was significantly reduced by treatment with
7a for both groups (fat massday14 ) 16.6 ( 1.7 and 13.1
( 0.3 g, p < 0.05, for 10 and 30 mg/kg, respectively)
relative to vehicle-treated controls (22.1 ( 0.1 g), while
no loss of lean mass was apparent. Administration of
D-fenfluramine also resulted in a significant decrease
in fat mass (fat massday14 ) 12.3 ( 1.0 g, p < 0.05) with
no change in lean mass.
In summary, optimization of the high-throughput
screening lead structure 1 led to the identification of
7a, an MCHr1 antagonist that binds with high affinity
to the MCHr1 receptor and potently inhibits Ca2+
mobilization in IMR-32 cells. This compound shows good
brain and plasma exposure upon oral dosing in DIO
mice and is the first MCHr1 antagonist reported to date
that is efficacious upon oral dosing in a chronic model

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1321
of weight loss.1 Furthermore, the dose-dependent an-
orectic effects of 7a were achieved at low concentrations
in the plasma and brain. A contribution from the
activation or antagonism of other receptors cannot be
ruled out, and further studies are needed to delineate
the full actions of treatment with 7a. However, given
the excellent potency and good CNS penetration of this
compound, it is likely that MCHr1 antagonism plays a
major role in the observed weight loss.
(8) 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. Targeted disruption of the melanin-concentrating
hormone receptor-1 results in hyperphagia and resistance to
diet-induced obesity. Endocrinology 2002, 143, 2469-2477.
(9) 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. T.; Qian,
S. Melanin-concentrating hormone 1 receptor-deficient mice are
lean, hyperactive, and hyperphagic and have altered metabolism.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3240-3245.
Acknowledgment. The authors thank Christopher
Ogiela, Dennis Fry, and Doug Falls for preparing the
IMR-32 cells and aiding the execution of the binding
and functional assays, Paul Richardson and J. J. Jiang
for MCH production, and Brian Droz and Victoria
Knourek-Segel for help with compound dosing.
(10) (a) Borowsky, B.; Durkin, M. M.; Ogozalek, K.; Marzabadi, M.
R.; DeLeon, J.; Lagu, B.; Heurich, R.; Lichtblau, H.; Shaposhnik,
Z.; Daniewska, I.; Blackburn, T. P.; Branchek, T. A.; Gerald, C.;
Vaysse, P. J.; Forray, C. Antidepressant, anxiolytic and anorectic
effects of a melanin-concentrating hormone-1 receptor antago-
nist. Nat. Med. 2002, 8, 779-781. (b) An orally active small-
molecule MCHr1 antagonist in an acute feeding model was
reported in the following: Takekawa, S.; Asami, A.; Ishihara,
Y.; Terauchi, J.; Kato, K.; Shimomura, Y.; Mori, M.; Murakoshi,
H.; Kato, K.; Suzuki, N.; Nishimura, O.; Fujino, M. T-226296:
Supporting Information Available: Experimental pro-
cedures including characterization data for compounds and
procedures for in vitro and in vivo assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
A
novel, orally active and selective melanin-concentrating
hormone receptor antagonist. Eur. J. Pharmacol. 2002, 438 (3),
129-135.
(11) Multiple patents describing small-molecule MCHr1 antagonists
have been published recently. For excellent reviews on the
subject, see the following: (a) Kowalski, T. J.; McBriar, M. D.
Therapeutic potential of melanin-concentrating hormone-1 re-
ceptor antagonists for the treatment of obesity. Expert Opin.
Invest. Drugs 2004, 13 (9), 1113-1122. (b) Browning, A. Recent
developments in the discovery of melanin-concentrating hormone
antagonists: novel antiobesity agents. Expert Opin. Ther. Pat.
2004, 14 (3), 313-325. (c) Collins, C. A.; Kym, P. R. Prospects
for obesity treatment: MCH receptor antagonists. Curr. Opin.
Invest. Drugs 2003, 4 (4), 386-394.
(12) Functional MCHr2 is not expressed in rodents. For the identi-
fication and species characterization of MCHr2, respectively, see
the following references: (a) An, S.; Cutler, G.; Zhao, J. J.;
Huang, S.; Tian, H.; Li, W.; Liang, L.; Rich, M.; Bakleh, A.; Du,
J.; Chen, J.; Dai, K. Identification and characterization of a
melanin-concentrating hormone receptor. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98 (13), 7576-7581. (b) Tan, C. P.; Sano, H.;
Iwaasa, H.; Pan, J.; Sailer, A. W.; Hreniuk, D. L.; Feighner, S.
D.; Palyha, O. C.; Pong, S.; Figueroa, D. J.; Austin, C. P.; Jiang,
M. M.; Yu, H.; Ito, J.; Ito, M.; Ito, M.; Guan, X. M.; MacNeil, D.
J.; Kanatani, A.; Van der Ploeg, L. H. T.; Howard, A. D. Melanin-
concentrating hormone receptor subtypes 1 and 2: species-
specific gene expression. Genomics 2002, 79 (6), 785-792.
(13) Cross-reactivity assays were performed by CEREP (www.cerep-
.com).
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