Identification and characterization of a selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R)

Article abstract of DOI:10.1016/j.bmcl.2009.03.102

We have developed and characterized [³⁵S]4a as a potent and selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R). Compound [³⁵S]4a showed appreciable specific signals in brain slices prepared from wild-type mice

Full text of DOI:10.1016/j.bmcl.2009.03.102

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2835–2839
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and characterization of a selective radioligand
for melanin-concentrating hormone 1-receptor (MCH1R)
b
b
a
b
Masahiko Ito a, , Toshihiro Sakamoto , Takao Suzuki , Shinichiro Egashira , Minoru Moriya ,
Akane Ishihara c, Hisashi Iwaasa a, Hiroko Matsushita c, Kazuho Nakase a, Michael A. Wallace d,
Dennis Dean d, Nagaaki Sato b, Shigeru Tokita a, Akio Kanatani a
*
a Department of Metabolic Disorder, Tsukuba Research Institute, BANYU Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
b Department of Medicinal Chemistry, Tsukuba Research Institute, BANYU Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
c Department of Pharmacology, Tsukuba Research Institute, BANYU Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
d Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
We have developed and characterized [35S]4a as a potent and selective radioligand for melanin-concen-
trating hormone 1-receptor (MCH1R). Compound [35S]4a showed appreciable specific signals in brain
slices prepared from wild-type mice but not from MCH1R deficient mice, confirming the specificity
and utility of [35S]4a as a selective MCH1R radioligand for ex vivo receptor occupancy assays.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 19 February 2009
Revised 18 March 2009
Accepted 21 March 2009
Available online 26 March 2009
Keywords:
Melanin-concentrating hormone 1-receptor
Antagonist
Radioligand
Receptor occupancy
Melanin-concentrating hormone (MCH) is a cyclic neuropeptide
that is predominantly expressed in the central nervous system
(CNS). Although MCH neurons are exclusively found in the hypo-
thalamus, they project widely throughout the CNS, suggesting that
MCH plays a variety of functions in CNS. Two G-protein coupled
receptors (GPCRs), named MCH1R and MCH2R, are identified as
specific receptors for MCH in mammals.1–5 Higher species (e.g., hu-
man, rhesus, and dog) have both MCH1R and 2R while rodents
(e.g., rat and mouse) have only MCH1R, indicating that physiolog-
ical functions of MCH are mediated solely through MCH1R in ro-
dents.6 In rodents, consistent with the widespread projections of
MCH neurons, MCH1Rs are widely expressed in various brain re-
gions.7 Accumulated evidence has demonstrated that MCH plays
a variety of physiological roles and provides therapeutic opportu-
nities for several CNS disorders such as obesity, depression and
anxiety.8–11 Regarding the roles of MCH in obesity, it is reported
that continuous central administration of MCH potently stimulated
feeding behavior resulting in the development of obesity and insu-
lin resistance in rodents.12–15 In addition, MCH1R-deficient mice
are resistant to diet-induced obesity (DIO).16,17 Furthermore,
several MCH1R antagonists with diverse structures (i.e.,
T-22629618,19 and SNAP-794120,21 in Fig. 1) displayed anti-obesity
efficacy in DIO rats. Collectively, these genetic and pharmacological
evidences strongly support the idea that MCH1R might be a prom-
ising therapeutic target for the treatment of obesity. Currently,
GW856464 (structure undisclosed),22 AMG-07623 ( Fig. 1) and
NGD-4715 (structure undisclosed)24 have entered clinical trials
for the treatment of obesity.
From a drug discovery point of view, a selective radioligand is
an essential tool for pharmacodynamic analyses of antagonists
(i.e., assessment of relationship between target occupancy levels
and pharmacological efficacy). It could facilitate pre-clinical and
clinical studies.25 As radioligands for MCH1R, [125I]-S3605726,27
(a peptide agonist) (Fig. 2) and [3H]-SNAP-7941 have been identi-
fied and used for ex vivo receptor occupancy (RO) studies.28 How-
ever, the selectivity of these radioligands in vivo has not been
validated in MCH1R deficient mice (i.e., loss of specific signal in
MCH1R deficient mice).
In this study, we report the discovery and characterization of a
potent and selective [35S]-radioligand for MCH1R that showed spe-
cific signals in an ex vivo RO study using wild-type mice brains but
not MCH1R deficient mice brains.
Compounds 1–5 were synthesized as outlined in Scheme 1.
Commercially available ester 7 was brominated to give 8. The bro-
mo group of 8 was displaced by 4-(methylsulfonyl)piperazin-2-one
or tert-butyl 3-oxopiperazine-1-carboxylate in the presence of base
followed by hydrolysis of the ester group to afford carboxylic acid
* Corresponding author. Tel.: +81 29 877 2000; fax: +81 29 877 2027.
E-mail address: masahiko_ito2@merck.com (M. Ito).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.102

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M. Ito et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2835–2839
Figure 1. Structures of MCH1R antagonists that were tested in a clinical trial.
4a without racemization (Scheme 2), establishing the synthetic
route of [35S]4a from the corresponding precursor 5a using
[
Figure 2. Amino acid sequence of [125I]-S36057. a8-Amino-3,6-dioxyoctanoyl.
35S]methanesulfonyl chloride as a radiolabel donor.
We previously reported that compound 1 is a significant human
and mouse P-gp substrate; hence, further development of 1 was
9a, b. Preparation of 9c was reported previously from our labora-
tory.29 Carboxylic acids 9a–c were coupled with amines 10a, b,
or c to afford target compounds 1–5. Racemates 4 and 5 were re-
solved by HPLC to give single isomers 4a, b and 5a, b, respec-
tively.30 Cleavage of the tert-butylcarbonyl group of the active
isomer 5a provided 6a. Mesylation of 6a gave the active isomer
abandoned.29 However, the relatively low logD value (logD7.4
=
2.2) and potent intrinsic activity (IC50 = 2.7 nM) of 1 prompted us
to utilize compound 1 as a template for the identification of radio-
ligands for rodent RO studies if a sulfur atom is appropriately
incorporated into the molecule. The result of the present struc-
Scheme 1. Reagents and conditions: (a) NBS, HBr, CCl4, reflux, 17 h, 98%; (b) 4-(methylsulfonyl)piperazin-2-one, NaH, DMF–THF, 0 °C, 30 min, 31%; (c) tert-butyl
3-oxopiperazine-1-carboxylate, n-BuLi, THF, 0 °C, 30 min, 46%; (d) 4 N NaOH aq, MeOH, rt, 2 h, 99%; (e) EDCIÁHCl, HOBtÁH2O, Et3N, CHCl3, DMF, rt, 17 h, 20–68%.

M. Ito et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2835–2839
2837
Scheme 2. Reagents and conditions: (a) TFA, rt, 10 min, 85%; (b) MsCl, NaHCO3, THF, rt, 10 min, 99%.
Table 1
ture–activity relationship study is shown in Table 1. Ethyl deriva-
Human MCH1R binding of compounds 1–5a
tive 2 (R2 = Et) showed improved activity (IC50 = 1.0 nM), and the
F
substitution of the right hand spiropiperidine portion with a fluo-
O
R3
rine atom (R3 = F) as in
3
additionally improved potency
(IC50 = 0.71 nM). Subsequently, we found that 4-(methanesulfo-
nyl)-piperidin-2-one-1-yl structure was tolerated as in
F
N
R2
N
R1
4
(IC50 = 0.71). After the resolution of racemate 4, the active isomer
4a was found to have an IC50 value of 0.36 nM. Thus, compound
4a was identified as a markedly potent radioligand candidate that
is amenable to 35S labeling. In addition, corresponding tert-butyl-
carbonyl derivatives 5a and precursor 6a were shown to retain
MCH1R potency. However, compound 4a was a significant human
and mouse P-gp substrate. Therefore, the radioligand might be use-
ful for ex vivo RO study, but it might be difficult to use for in vivo
RO assay.
In vitro binding profiles of 4a are summarized in Table 2. Com-
pound 4a showed high affinity to human MCH1R (Ki = 0.42 nM)
while having negligible affinity to MCH2R. In addition, the Ki value
of compound 4a for mouse MCH1R was comparable to that for hu-
man MCH1R, indicating no significant species differences in recep-
tor binding. In the cellular functional assay using CHO cells
expressing human MCH1R (i.e., FLIIPR), compound 4a inhibited
MCH-induced [Ca2+]i mobilization, indicating the antagonistic
activity of 4a. Compound 4a was selective over a panel of 60 unre-
O
Compound
R1
R2
R3
H
hMCH1Rb (nM)
2.7
1
Me
Et
N
N
N
O
2
3
H
F
1.0
O
O
Et
0.71
N
O
S
4
Et
F
0.71
N
O
O
lated receptors, enzymes and transporters (IC50 > 1 lM for all the
items tested). The binding profile of [35S]4a (specific activity:
1200 mCi/mmol) at human and mouse MCH1Rs was examined
and summarized in Figure 3. The CHO cell membranes over-
expressing human or mouse MCH1R were mixed with various con-
centrations of [35S]4a in the presence or absence of an excessive
amount of cold compound 4a to determine non-specific or total
binding, respectively. After 1 h incubation at room temperature,
the membranes were filtrated, and the residual radioactivities on
the filters were measured by microplate scintillation counter. Com-
pound [35S]4a bound to human MCH1R with a Kd value of
N
N
N
N
O
S
4a
4b
5a
5b
Et
Et
Et
F
F
F
0.36
480
0.24
N
O
O
O
O
O
O
S
N
O
N
N
O
0.18 0.02 nM and
(Fig. 3A and B). The receptor density (i.e., the Bmax value) deter-
mined by
35S]4a was comparable to that obtained using
125I]MCH (24 4 pmol/mg protein) (Fig. 3C and D). No specific
a Bmax value of 23 1 pmol/mg protein
O
O
[
Et
Et
F
F
145
O
[
binding of [35S]4a to CHO cell membranes was observed (data
not shown), suggesting that [35S]4a recognized MCH1R in a specific
manner. Next, we investigated the kinetic profile of [35S]4a (i.e.,
association and dissociation rates) for human MCH1R in order to
further assess the potential of [35S]4a as a radioligand for receptor
occupancy assays. Non-specific binding was determined in the
presence of an excess amount of compound 4a. As a result, the spe-
N
6a
0.87
NH
O
Inhibitory activities of compounds 1–5 against [125I]MCH binding to human
MCH1R in CHO cells were determined.
a
b
The values represent the mean for n P 2.

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M. Ito et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2835–2839
Table 2
In vitro profile of compound 4a
Binding assaya Ki (nM)
[Ca2+]i mobilizationb
IC50 (nM)
Human
MCH1R
Mouse
MCH1R
Human
MCH2R
Human MCH1R
Compound 4a 0.42 0.02
0.50 0.08
>9700 100 3.0 0.3
MCH
0.034 0.009 0.052 0.009 1.6 0.6
a
Ki values of the compound 4a and human MCH for human and mouse MCH1R
receptors were determined by ligand binding assay. The values are mean SD from
three independent tests.
b
Inhibition activity of compound 4a on MCH-induced [Ca2+]i increase in CHO
cells expressing human MCH1R was determined by FLIPR.
Figure 4. Kinetic profiles of [
35S]4a for human MCH1R. The association and
dissociation kinetic studies were performed with 300 pM [35S]4a as described in
Figure 2 with slight modification. The association rate (Kon) was determined by
analyzing time-dependent changes of the specific binding of [35S]4a to human
MCH1R (from 1 min to 15 min). The non-specific binding was determined in the
cific binding of [35S]4a to MCH1R reached equilibrium within
5 min (Fig. 4). The kinetic parameters, kon and koff, were determined
to be 2.3 1.0 nM minÀ1 and 0.54 0.06 minÀ1, respectively. These
data suggest that [35S]4a binds rapidly to MCH1R when compared
to [125I]MCH that requires 1 h for equilibrium (data not shown).
Finally, we examined the binding of [35S]4a to brain coronal
slices prepared from wild type and MCH1R-knockout mice
(C57BL/6j background). In the RO assay, striatum was used as a tar-
get area because MCH1R is abundantly and widely expressed in
this region, thus making it feasible to perform the RO assay.7,25
Mice coronal brain sections including striatum were incubated
with 500 pM of [35S]4a for 5 min at room temperature. After wash-
ing the treated brain slices, the radioactivities on the striatum area
were imaged and measured by BAS5000. As shown in Figure 5,
presence of an excessive amount of cold 4a (1
lM). Regarding the dissociation rate,
the membranes were incubated with [35S]4a for 1 h and then mixed with cold 4a
(1 lM) to initiate the dissociation step. The dissociation rate was determined by
analyzing time-dependent changes of the specific binding of [35S]4a to human
MCH1R (from 1 min to 10 min). The binding experiment follows the procedure
described in the legend of Figure 3.
brains. These results demonstrate the selectivity of [35S]4a not only
in in vitro but also in vivo, which strongly suggest that [35S]4a is a
very useful tool for the RO assay.
In summary, we developed and characterized a potent and
selective radioligand [35S]4a that rapidly and specifically binds to
MCH1R in both in vitro and in vivo systems. The use of [35S]4a will
[
35S]4a gave appreciable signals in the striatum region of wild-type
brains while showing negligible signals in the knockout mice
Figure 3. Saturation binding of [35S]4a to human and mouse MCH1R CHO cell membranes. Membranes from CHO cells expressing human or mouse MCH1R were incubated
with several concentrations of [35S]4a in the presence of DMSO (total binding) or 1
M compound 4a in assay buffer (pH 7.4, 50 mM Tris–HCl, 10 mM MgCl2, 2 mM EDTA,
50 g/ml bacitracin, 0.2% bovine serum albumin (BSA)) at room temperature for 1 h. The membranes were filtrated onto GF/C filter and washed with washing buffer (pH 7.4,
50 mM Tris–HCl, 10 mM MgCl2, 2 mM EDTA, 0.02% Tween-20) three times. The residual radioactivities on the dried filter were measured by microplate scintillation counter.
l
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