Discovery of a novel melanin concentrating hormone receptor 1 (MCHR1) antagonist with reduced hERG inhibition

Article abstract of DOI:10.1016/j.bmcl.2012.04.006

An initial SAR study resulted in the identification of the novel, potent MCHR1 antagonist 2. After further profiling, compound 2 was discovered to be a potent inhibitor of the hERG potassium channel, which prevented its further development. Additional opt

Full text of DOI:10.1016/j.bmcl.2012.04.006

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3781–3785
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel melanin concentrating hormone receptor 1 (MCHR1)
antagonist with reduced hERG inhibition
a,
⇑
a
a
a
a
b
Jeffrey T. Mihalic , Pingchen Fan , Xiaoqi Chen , Xi Chen , Ying Fu , Alykhan Motani
b
b
c
b
a
b
a
Lingming Liang , Michelle Lindstrom , Liang Tang , Jin-Long Chen , Juan Jaen , Kang Dai , Leping Li
Department of Chemistry, Amgen San Francisco, 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
Department of Biology, Amgen San Francisco, USA
Department of Pharmacokinetics, Amgen San Francisco, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
An initial SAR study resulted in the identification of the novel, potent MCHR1 antagonist 2. After further
profiling, compound 2 was discovered to be a potent inhibitor of the hERG potassium channel, which pre-
vented its further development. Additional optimization of this structure resulted in the discovery of the
potent MCHR1 antagonist 11 with a dramatically reduced hERG liability. The decrease in hERG activity
was confirmed by several in vivo preclinical cardiovascular studies examining QT prolongation. This com-
pound demonstrated good selectivity for MCHR1 and possessed good pharmacokinetic properties across
preclinical species. Compound 11 was also efficacious in reducing body weight in two in vivo mouse
models. This compound was selected for clinical evaluation and was given the code AMG 076.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 February 2012
Accepted 2 April 2012
Available online 7 April 2012
Keywords:
MCHR1
Melanin concentrating hormone
Reducing hERG inhibition
Melanin concentrating hormone (MCH) is a cyclic 19 amino acid
peptide found in the CNS of all vertebrates.1 Recently, several
studies have emerged demonstrating the significance of the MCH
pathway in controlling body weight through modulating food in-
take and energy balance. When MCH is delivered by intracerebro-
ventricular (ICV) injection to rodents, a dose dependent increase in
food intake and body weight gain is observed.2 In transgenic
mouse studies, animals expressing twofold higher than normal
MCH levels are mildly obese and are prone to weight gain and
insulin resistance when fed a high fat diet.3 In contrast, MCH
knockout mice are healthy, lean, consume less food, and are
areas of the CNS.7 The difference in the expression patterns of
MCHR1 and MCHR2 suggests that MCHR2 might not be involved
in feeding behaviors or neuroendocrine function.
The preceding evidence suggests that an MCHR1 antagonist
might be useful as a treatment for obesity. Via a high throughput
screen of our chemical library, we discovered a unique indole lead
1 (Fig. 1). Using a systematic lead optimization approach, a potent
selective MCHR1 inhibitor was then developed (2, Fig. 1).8
i
Compound 2 has a high affinity to MCHR1 (K = 0.3 nM) and is a
potent antagonist of MCH (IC50 = 1 nM). This compound (2) also
possesses good PK in several animal species and is highly CNS
permeable. Pharmacologically, 2 was efficacious in reducing food
consumption in an in vivo MCH cannulated rat model. However,
it was later discovered that 2 was a potent inhibitor of the hERG
(human ether-a-go-go gene) potassium channel which prevented
its further development. The hERG potassium channel plays a cen-
tral role in the repolarization of the cardiac action potential. This
potassium channel is known to be susceptible to inhibition from
a variety of drugs and has the potential to cause QT prolongation
4
hypermetabolic.
There are two GPCR receptors for MCH, MCHR1 and MCHR2.
MCHR1 is ubiquitous to all vertebrates whereas MCHR2 is present
in dogs, ferrets, and primates including humans. Rodents lack a
functional form of MCHR2. MCHR1 is believed to be involved in
the neuronal regulation of food consumption. High levels of this
receptor are found in the ventromedial hypothalamus, arcuate nu-
5
cleus, and zona incerta. These areas of the brain are thought to be
responsible for taste, olfaction, and the positive reward aspects of
feeding and satiety. Targeted disruption of the MCHR1 gene in
mice causes resistance to diet-induced obesity, leanness, reduced
food consumption, hyperactivity, and altered metabolism. Little
is known about the second receptor for MCH, MCHR2. It is
expressed throughout the brain and is enriched in the cortical
6
H
H
H
F3C
N
N
O
N
H
N
H
H
1
2
⇑
Corresponding author. Fax: +1 650 244 2015.
E-mail address: jmihalic@amgen.com (J.T. Mihalic).
Figure 1. MCHR1 antagonist HTS lead and optimized structure.
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.006

3
782
J. T. Mihalic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3781–3785
which can be associated with deadly cardiac arrhythmias known as
torsades de pointes. In fact, it is well known in the literature that
analog 10, most of the MCHR potency is regained and hERG activity
decreases to greater than 5 M. Finally, the replacement of the THP
9
l
many MCHR1 inhibitors also inhibit the hERG potassium chan-
ring with a cyclohexyl ring (11) improves the MCHR1 affinity an
additional 15-fold to 0.6 nM while maintaining the hERG potency
at greater than 5 lM. This compound showed significantly reduced
hERG inhibition over analog 2, while maintaining excellent MCHR1
1
0
nel. Discovering ways to overcome hERG inhibition has become
an important goal for drug discovery in general and specifically
for the MCHR1 research area. In this Letter, we describe the efforts
to remove the hERG liability of 2 while maintaining good potency
towards MCHR1 and preserving good PK, in vivo efficacy, and
selectivity.
affinity.
Compound 11 is a selective MCHR1 antagonist. Affinity to other
receptors and transporters was evaluated for 11 using Panlab’s
lead profile screen of 64 targets and in-house assays. A partial list
of selectivity data for 11 can be found in Table 2. This list includes
some known receptors which likely influence food intake and/or
energy expenditure. When tested with each of these targets, com-
The synthesis of analogs 7, 10, and 11 is outlined in Scheme 1.
The synthesis began with the treatment of the corresponding ester
3
with LDA and allyl bromide to give intermediate 4. This allyl
intermediate 4 was then oxidized using Brown’s hydroboration–
oxidation conditions followed by a Swern oxidation to give alde-
pound 11 showed K
5HT2C, which had a K
of 1.14 M in the functional aequorin assay. Functionally, com-
pound 11 is a potent antagonist to human MCHR1 with an aequo-
rin IC50 of 1.2 nM. Compound 11 also showed potency across
species with IC50 values ranging from 0.6 nM in mouse to 0.8 nM
in rat and rhesus monkey. No activity against MCHR2 was ob-
i
values > 2
l
M with the exception of serotonin
1
1
hyde 5. Intermediate 5 was subsequently reductively aminated
with amine 6 and hydrolyzed with lithium hydroxide to yield ana-
log 7. Amine 6 was synthesized according to published proce-
i
of 1.12
lM in the binding assay and an IC50
l
1
2
dures. The synthesis of analogs 10 and 11 began with the
conversion of allyl intermediate 4 to aldehyde 8 with osmium tet-
raoxide and sodium periodate. Next, the ester group on 8 was
cleaved with lithium hydroxide and then cyclized under acidic
workup to give the oxaspiro intermediate 9. Finally, reductive ami-
nation conditions were utilized to condense amine 6 with interme-
diate 9 to give analogs 10 and 11.
served up to 10 lM.
Analog 11 was further evaluated in mouse, rat, dog (beagle), and
cynomolgus PK. The corresponding pharmacokinetic results can be
found in Table 3. Overall, analog 11 showed low clearance across
species and oral bioavailabilities ranging from 29 to 79%. Com-
pound 11 was also evaluated in rat brain PK, since there was a con-
cern that the carboxylic acid functionality on 11 might prevent
penetration of the compound into the brain. It possessed the fol-
lowing concentration ratios after a 10 mg/kg oral dose: [blood]/
[plasma] = 0.79, [brain]/[plasma] = 0.40, and [CSF]/[plasma] = 0.21.
These data suggested that 11 could sufficiently cross the blood
brain barrier. In general, compound 11 possessed a good pharma-
cokinetic profile.
Because hERG inhibition is a common mechanism for QTc pro-
longation, an extensive cardiac toxicity assessment measuring QTc
prolongation was performed with compound 11 in several species.
In an anesthetized rhesus monkey QT study, compound 11 was
administered as a bolus, followed by an IV infusion for 45 min to
Two assays were initially used to rank order the molecules. The
first assay was a MCH competitive binding assay, and the second
was a hERG rubidium efflux assay.13 MCHR1 antagonism was later
determined by using an aequorin cellular assay measuring the
blockade of intercellular Ca2 mobilization caused by MCH.
+
14
Early exploratory SAR demonstrated that the core indole struc-
ture of 2 was essential for MCHR1 affinity and had a very low tol-
erance to structural change; therefore, modifications to this region
to mitigate the hERG liability were not aggressively pursued.
Attention mostly focused on the THP side chain as a possible site
for modification. The SAR results can be found in Table 1. In the lit-
erature, hERG pharmacophore models suggest incorporation of po-
lar substituents in the periphery or lipophilic binding pockets may
1
5
reduce hERG potency. Furthermore, there are several reports of
successfully reducing hERG potency by adding a carboxyl group
a
steady plasma concentration of ꢀ3000 ng/mL (ꢀ6.5
lM).
16
to the molecule of interest (Fig. 2). This approach was utilized
with our structure. The first molecule synthesized was acid 7.
The tertiary center on the THP side chain was chosen for the car-
boxylic acid placement. With this substitution, a 10-fold loss in
hERG potency was obtained; however, MCHR1 affinity also fell
Throughout the duration of this study, no QTc change was ob-
served. Similarly, no QTc change was observed after PO dosing of
11 at 30 mg/kg in cynomolgus monkey (n = 3). In a 4 h dog study,
no QTc change was observed after PO dosing at 300 mg/kg (Cmax
15,200 ng/mL, ꢀ33
lM). Finally, in a chronic dog study where com-
pound 11 was dosed at 100 mg/kg PO BID for 28 days, no QTc
8
0-fold. If the linker is reduced from three to two carbons, as in
CO Me
MeO C
MeO C
2
2
2
H
H
COOH
a
b
F3C
O
c
N
O
H
H
N
H
X
X
O
F3C
NH
3
4
5
N
H
7
d
6
OH
X
MeO C
O
2
O
H
H
e
f
F3C
O
N
H
COOH
H
H
F C
N
H
X
3
NH
X
9
N
H
8
10 X = -O-
X = -CH -
1
1
2
6
Scheme 1. Reagents and conditions: (a) LDA, allyl bromide, THF; (b) (1) BH O; (3) oxalyl chloride, DMSO, Et
3
ÁTHF complex, THF (2) H
2
O
2
, NaOH, H
2
3
N, DCM; (c) (1)
NaBH(OAc) , amine 6, DCM; (2) LiOH, EtOH, H O; (d) OsO , NaIO , 1,4-dioxane, MeOH; (e) (1) LiOH, THF, H O; (2)HCl; (f) NaBH(OAc) , amine 6, DCM.
3
2
4
4
2
3

J. T. Mihalic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3781–3785
3783
Table 1
a
SAR results of the THP sidechain
Compound
R
MCHR1 IC50
0.0005
(l
m) 125I-MCH displ.
hERG est. IC50
0.03
(l
m) Rb+ efflux
2
O
O
HO
O
O
7
0.04
0.3
>5
>5
HO
O
O
1
1
0
1
0.009
0.0006
HO
a
Values are means of at least two experiments.
HN
O
HN
N
O
HN
O
O
O
N
O
HN
O
Cl
Cl
HO
Et
HN
Cl
HN
Cl
N
N
hERG Ki = 0.016 µM
hERG Ki >10 µM
HO
HO
N
N
HOOC
OH
OH
hERG IC = 0.056 µM
hERG IC = 23 µM
50
5
0
Figure 2. Literature examples of the inhibitory effect of adding a carboxylic acid group on hERG binding and activity.
Table 2
a
Table 3
Selectivity profile for compound 11
a,b
Pharmacokinetic results for analog 11
Receptor/Transporter
Binding K
i
(lM)
Ca2+ mobl. IC50
(lM)
Species
IV
PO
MCHR1 (human)
MCHR1 (mouse)
MCHR1 (rat)
MCHR1 (rhesus monkey)
MCHR2 (human)
Serotonin 5HT1A
Serotonin 5HT2A
Serotonin 5HT2C
Dopamine D1
0.0006
0.0012
0.0006
0.0008
0.0008
>10
Cl (L/h/kg)
V
ss (L/kg)
MRT (h)
%F
Mouse
Rat
Dog (beagle)
Cyno
0.64
0.55
0.53
0.22
3.6
3.8
2.2
2.3
5.8
7.9
4.2
11
79
70
40
29
>2
>2
1.12
>2
a
1.14
n = 3 animals per study.
Dosed at 0.5 mg/kg IV and 3 mg/kg PO.
b
Dopamine D2
3.16
>2
>2
Adrenergic
Adrenergic
a
a
2A
2C
Compound 11 was further evaluated in two established mouse
Opioid
Muscarinic M2
Muscarinic M3
Norepinephrine transporter
Serotonin transporter
l
>2
>2
>2
>2
17
models for obesity. In the first study, C57BL/6 mice were weaned
at 4–5 weeks of age and maintained on a high-fat diet for more
than 24 weeks before the start of dosing. Four groups of mice were
dosed daily PO with either vehicle, Sibutramine (10 mg/kg), or
compound 11 (3 mg/kg or 10 mg/kg dosed as the HCl salt), and
the effect on body weight was measured for 137 days as the mean
>2
a
Experiments were conducted at MDS-Panlabs with the exception of MCHR1 and
MCHR2 experiments. These assays were performed in-house.
Ò
of n = 6–8 mice per group. Sibutramine (Meridia ), a serotonin and
norepinephrine reuptake inhibitor, was chosen as a positive con-
trol since it was reported to show efficacy in rodent models of
obesity and was prescribed as an appetite suppressant until its
changes were observed. Overall, these results suggest a minimal
cardiovascular risk due to hERG inhibition with compound 11.

3
784
J. T. Mihalic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3781–3785
for obesity. Compound 11 was advanced for further clinical evalu-
ation and was given the code AMG 076. A complete in vivo charac-
terization of compound 11 will be the subject of a future
publication.
References and notes
1.
(a) Vaughan, J. M.; Fischer, W. H.; Hoeger, C.; Rivier, J.; Vale, W. Endocrinology
989, 125, 1660; (b) Presse, F.; Nahon, J. L.; Fischer, W. H.; Vale, W. Mol.
1
Endocrinol. 1990, 4, 632; (c) Nahon, J. L.; Presse, F.; Bittencourt, J. C.;
Sawchenko, P. E.; Vale, W. Endocrinology 1989, 125, 2056.
2.
(a) Ludwig, D. S.; Mountjoy, K. G.; Tatro, J. B.; Gillette, J. A.; Frederich, R. C.; Flier,
J. S.; Maratos-Flier, E. Am. J. Physiol. Endocrinol. Metab. 1998, 274, 627; (b) Della-
Zuana, O.; Presse, F.; Ortola, C.; Duhault, J.; Nahon, J. L.; Levens, N. Int. J. Obesity
Figure 3. Changes in body weight in C57BL/6 mice fed high-fat diets. C57BL/6 male
mice were fed a high fat diet (60 kCal% fat, Research Diets Inc. D12492i) for
2
4 weeks prior to start of treatment and throughout the course of treatment. Dosing
2002, 26, 1289; (c) Gomori, A.; Ishihara, A.; Ito, M.; Mashiko, S.; Matsushita, H.;
was performed daily by bolus gavage.
Yumoto, M.; Ito, M.; Tanaka, T.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A.
Am. J. Physiol. Endocrinol. Metab. 2003, 284, 583; (d) Ito, M.; Gomori, A.; Ishihara,
A.; Oda, Z.; Masahiko, S.; Matsushita, H.; Yumoto, M.; Ito, M.; Sano, H.; Tokita,
S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J. Physiol. Endocrinol. Metab. 2003,
removal from the market in 2010.18 Within the graph in Figure 3,
2
84, 940.
which plots body weight versus dose, a statically significant reduc-
tion in body weight (p < 0.01À0.05 vs vehicle) was observed in the
compound 11 treated groups. Both the 3 mg/kg and 10 mg/kg
doses of 11 showed a greater loss in body weight when compared
to Sibutramine. Sibutramine did show a reduction in body weight
when compared to vehicle; however, this reduction was not con-
sidered statistically significant. Food intake measurements were
also obtained within the experiment and dose-related reduction
in food intake in response to 11 was observed throughout the
course of the experiment (data not shown).
3
4
.
.
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.
Qu, D.; Ludwig, D. S.; Gammeltoft, S.; Piper, M.; Pelleymounter, M. A.; Cullen,
M. J.; Mathes, W. F.; Przypek, J.; Kanarek, R.; Maratos-Flier, E. Nature 1996, 380,
243.
5. (a) Viale, A.; Zhixing, Y.; Breton, C.; Pedeutour, F.; Coquerel, A.; Jordan, D.;
Nahon, J. L. Brain Res. Mol. Brain Res. 1997, 46, 243; (b) Bittencourt, J. C.; Presse,
F.; Arias, C.; Peto, C.; Vaughan, J.; Nahon, J. L.; Vale, W.; Sawchenko, P. E. J. Comp.
Neurol. 1992, 319, 218; (c) Skofitsch, G.; Jacobowitz, D. M.; Zamir, N. Brain Res.
Bull. 1985, 15, 635; (d) Nahon, J. L. Crit. Rev. Neurobiol. 1994, 8, 221; (e) Elias, C.
F.; Saper, C. B.; Maratos-Flier, E.; Tritos, N. A.; Lee, C.; Kelly, J.; Tatro, J. B.;
Hoffman, G. E.; Ollmann, M. M.; Barsh, G. S.; Sakurai, T., et al J. Comp. Neurol.
1998, 402, 442.
The second in vivo mouse experiment was designed to confirm
that the weight-loss promoting effects of compound 11 were in-
deed mediated by antagonism of MCHR1 ( Fig. 4). Within this
experiment, two groups of female mice, MCHR1-expressing wild
type and MCHR1 knockout mice, were utilized. Mice maintained
on high-fat diets were dosed either with vehicle or compound 11
6. (a) 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; (b) 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, S. J.; Gopal-Truter, S.;
MacNeil, D. J.; Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Qiang, S.
Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 3240.
7.
An, S.; Cutler, G.; Zhao, J. J.; Huang, S. G.; Tian, H.; Li, W.; Liang, L.; Rich, M.;
Bakleh, A.; Du, J.; Chen, J. L.; Dai, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7576.
(
HCl salt form 100 mg/kg/day ad libitum in the high fat diet) and
their body weights were monitored for 34 weeks. At the end of
the study, compound 11 treated animals showed a statistically sig-
nificant (p < 0.01 vs vehicle) decrease in body weight gain only in
the wild-type mice but not in the MCHR1 knockout mice. This re-
sult suggests that 11 is likely reducing food intake by antagonizing
MCHR1.
In summary, we discovered compound 11, a novel, potent
MCHR1 antagonist with a diminished hERG activity. The decrease
in hERG activity was accompanied by a cleaner cardiovascular
safety profile, as seen in several preclinical QTc studies examining
cardiovascular risk. This compound was selective for MCHR1 and
possessed good PK across several species. Compound 11 was also
efficacious in reducing body weight gain in two in vivo mouse
models. These results suggest that 11 could be a useful treatment
8. Mihalic, J. T.; Chen, X.; Fan, P.; Chen, X.; Fu, Y.; Liang, L.; Reed, M.; Tang, L.;
Chen, J.; Jaen, J.; Li, L.; Dai, K. Bioorg. Med. Chem. Lett. 2011, 21, 7001.
9. Sanguinetti, M. C.; Tristani-Firouzi, M. Nature 2006, 440, 463.
1
0. (a) Sasmal, P. K.; Sasmal, S.; Abbineni, C.; Venkatesham, B.; Rao, P. T.; Roshaiah,
M.; Khanna, I.; Sebastian, V. J.; Suresh, J.; Singh, M. P.; Talwar, R.; Shashikumar,
D.; Reddy, K. H.; Frimurer, T. M.; Rist, O.; Elster, L.; Högberg, T. Med. Chem.
Commun. 2011, 2, 385; (b) Lim, C. J.; Kim, N.; Lee, E. K.; Lee, B. H.; Oh, K.; Yoo, S.;
Yi, K. Y. Bioorg. Med. Chem. Lett. 2011, 21, 2309; (c) Erickson, S. D.; Banner, S.;
Berthel, B.; Conde-Knape, K.; Falcioni, F.; Hakimi, I.; Hennessy, B.; Kester, R. F.;
Kim, K.; Ma, C.; McComas, W.; Mennona, F.; Mischke, S.; Orzechowski, L.; Qian,
Y.; Salari, H.; Tengi, J.; Thakkar, K.; Taub, R.; Tilley, J. W.; Wang, H. Bioorg. Med.
Chem. Lett. 2008, 18, 1402; (d) Fox, B. M.; Natero, R.; Richard, K.; Connors, R.;
Roveto, P. M.; Beckmann, H.; Haller, K.; Golde, J.; Xiao, S.; Kayser, F. Bioorg. Med.
Chem. Lett. 2011, 21, 2460.
11. (a) Brown, H. C.; Zweifel, G. J. Am. Chem. Soc. 1959, 81, 247; (b) Omura, K.;
Swern, D. Tetrahedron 1978, 34, 1651.
1
2. Andersen, D.; Storz, T.; Liu, P.; Wang, X.; Li, L.; Fan, P.; Chen, X.; Allgeier, A.;
Burgos, A.; Tedrow, J.; Baum, J.; Chen, Y.; Crockett, R.; Huang, L.; Syed, R.;
Larsen, R. D.; Martinelli, M. J. Org. Chem. 2007, 72, 9648.
13. (a) MCHR1 binding assay description: material and solutions: Binding-buffer:
5
0 mM Tris pH 7.4, 10 mM MgCl
2
, 2.5 mM EDTA; 0.2% BSA (w/v). Washing-
, 1 mM CaCl , 0.5% BSA (w/v), 0.5 M
NaCl. Mulitiscreen -FB plates (Cat# MAFBN0B50, Millipore USA) were, prior to
use, treated with 100 l of 0.5% BSA (w/v)/H 0 solution. After 2 h at room
temperature the solution was removed by vacuum filtration and the wells
were washed two times with 100 l of binding-buffer. Methods: 50 l of
compound-dilution, made in binding-buffer, was added to 50 l of binding
buffer containing unit MCHR1 membrane preparation (Cat# ES-370M,
Euroscreen; Belgium) and 0.02
00 Ci/mL, PerkinElmer, USA). The 100
buffer: 25 mM Hepes pH 7.4, 5 mM MgCl
2
2
Ò
l
2
l
l
l
1
125
lCi
I-MCH (2200Ci (81.4TBq)/mmol;
ll reaction mixture was incubated
1
l
in a Corning 96-well plate (Cat# 3363, Corning USA) at 37 °C for 2 h on a
shaking platform. Bound and unbound ligand was separated by vacuum
filtration by transferring 80
l
l of the reaction mixture into a corresponding
well of the Mulitiscreen -FB plates. After filtration the wells were washed 4
times with 100 l of washing-buffer before 100 l of Scintillation cocktail
PerkinElmer, USA) was added. Plates were read on a MicroBeta Trilux
Ò
l
l
Ò
(
(
+
PerkinElmer, USA). (b) Rubidium (Rb ) efflux inhibition assay was carried out
as described by Cheng, C.; Alderman, D.; Kwash, J.; Dessaint, J.; Patel, R.; Kay,
M.; Lescoe, M. K.; Kinrade, M. B.; Yu, W. Informa, 2002, 28, 177. Briefly, HEK293
cells expressing hERG were plated in poly-
the next day, the cells were washed with hERG buffer (150 mM NaCl, 2 mM
CaCl , 0.8 mM NaH PO , 1 mM MgCl , 5 mM Glucose, 12 mM HEPES, pH 7.4).
D-lysine coated 96-well plates. On
Figure 4. Effects of 11 on body weight wild-type versus knockout mice. C57BL/6
female mice received compound prepared in a high fat diet (60% kCal fat, Research
Diets Inc. D12492i).
2
2
4
2

J. T. Mihalic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3781–3785
3785
2
00
incubation at 37 °C, compounds were added and the cells incubated at 37 °C for
0 min. The cells were then washed with washing buffer (5 mM KCl in hERG
buffer). 200 L of 50 mM KCl buffer (50 mM KCl in hERG buffer) was added and
l
L of Rb+ loading buffer (5.4 mM RbCl in hERG buffer) was added. After 4 h
Bandoh, K.; Aoki, J.; Hosono, H.; Kobayashi, S.; Kobayashi, T.; Murakami-
Murofushi, K.; Tsujimoto, M.; Arai, H.; Inoue, K. J. Biol. Chem. 1999, 274, 27776.
15. (a) Mitcheson, J. S.; Chen, J.; Lin, M.; Culberson, C.; Sanguinetti, M. C. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 12329; (b) Pearlstein, R. A.; Vas, R. J.; Kang, J.; Chen, X.
L.; Preobrazhenskaya, M.; Shchekotikhin, A. E.; Korolev, A. M.; Lysenkova, L. N.;
Miroshnikova, O. V.; Hendrix, J.; Rampe, D. Bioorg. Med. Chem. Lett. 2003, 1829,
13.
16. Zhu, B.; Jia, Z. J.; Zhang, P.; Su, T.; Huang, W.; Goldman, E.; Tumas, D.; Kadambi,
V.; Eddy, P.; Sinha, U.; Scarborough, R. M.; Song, Y. Bioorg. Med. Chem. Lett.
2006, 16, 5507.
3
l
to activate the hERG channel for 5 min at rt. The supernatant was transferred to
a new 96-well plate and the cells lysed with lysis buffer (1% Triton X-100 in
hERG buffer). Rb concentrations in the supernatant and the cells were
determined by using an atomic absorbance spectrometer. Rb efflux rate (RE)
of compounds was calculated: Rb efflux rate (RE): [Rb supernatant]/[total
+
+
+
+
+
+
Rb ]; Rb efflux efficiency (EE): (RE sample À RE negative control)/(RE positive
control À RE negative control).
17. Rossmeisl, M.; Rim, J. S.; Koza, R. A.; Kozak, L. P. Diabetes 2003, 1958, 52.
18. Day, C.; Bailey, C. J. Int. J. Obes. Relat. Metab. Disord. 1998, 22, 619.
1
4. A description of the aequorin assay can be found in the following references.
(
a) An, S.; Thieu, B.; Yuhua, Z.; Goetzl, E. J. Mol. Pharm. 1998, 54, 881; (b)