Identification of a novel series of benzimidazoles as potent and selective antagonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2007.06.010

A novel series of benzimidazoles was identified and optimized, leading to the discovery of potent and selective antagonists of the human melanocortin-4 receptor. In addition, compound 5i was shown to cross the blood-brain barrier after intravenous dosing

Full text of DOI:10.1016/j.bmcl.2007.06.010

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4464–4470
Identification of a novel series of benzimidazoles as potent
and selective antagonists of the human melanocortin-4 receptor
a
a
b
b
Lydie Poitout,a, Valerie Brault, Carole Sackur, Sonia Bernetiere, Jose Camara,
*
´
`
´
Pascale Plasc and Pierre Roubertc
aDepartment of Medicinal Chemistry, Ipsen Research Laboratories, Institut Henri Beaufour, 5 Avenue du Canada,
91966 Les Ulis Cedex, France
bDepartment of Biopharmaceutical Profiling and Structural Chemistry, Ipsen Research Laboratories, Institut Henri Beaufour,
5 Avenue du Canada, 91966 Les Ulis Cedex, France
cDepartment of Endocrinology, Ipsen Research Laboratories, Institut Henri Beaufour, 5 Avenue du Canada,
91966 Les Ulis Cedex, France
Received 20 March 2007; revised 31 May 2007; accepted 2 June 2007
Available online 8 June 2007
Abstract—A novel series of benzimidazoles was identified and optimized, leading to the discovery of potent and selective antagonists
of the human melanocortin-4 receptor. In addition, compound 5i was shown to cross the blood–brain barrier after intravenous dos-
ing in rats.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The melanocortin receptors are a family of five G pro-
tein-coupled receptor subtypes. They are activated by
a-, b- and c-melanocyte stimulating hormones (MSH)
and adrenocorticotropin (ACTH), all of which are de-
rived from a single precursor peptide, proopiomelano-
cortin (POMC).1 Of these five subtypes, the
melanocortin receptor subtype 4 (MC4-R), which is dis-
tributed in multiple sites in the brain, plays a key role in
the regulation of food intake and energy homeostasis.
Activation of MC4-R reduces food intake and body
weight, whereas inhibition of MC4-R activity induces
increased feeding and weight gain.2
Several small molecule MC4-R antagonists have already
been reported (Fig. 1). Compound 1 binds to the human
MC4 (hMC4) receptor with high affinity (Ki = 7.9 nM).5
The benzamidine 2a, which exhibits a Ki of 160 nM on
the hMC4 receptor, was shown to reduce tumour-in-
duced weight loss in mice following peripheral adminis-
tration.6a A related series with similar in vitro binding
affinity, where the amidine is replaced by an imidazole
such as 2b, was also reported.6b Finally, a series of pipe-
razinebenzylamines were found to be potent MC4
antagonists. For example, compound 3a exhibited a Ki
value of 3.2 nM.7a Further optimization led to 3b
(NBI-12i; Ki = 6.3 nM) which increased food intake in
mice over a 24-h period, when orally administered.7b,c
Recently, a related propionylpiperazine series was dis-
closed,7d 3c for example, binds to the hMC4 receptor
with a Ki value of 25 nM.
Cachexia is a wasting syndrome and a major cause of
morbidity and mortality that occurs in many chronic
diseases such as cancer, AIDS, heart or renal failure,
lung disease, liver cirrhosis or rheumatoid arthritis.3
However, no effective treatment is currently available
to reverse the loss of body weight.
Screening of our compound collection, by competitive
binding experiments8 using the radiolabelled ligand
Small molecule MC4 antagonists which cross the blood–
brain barrier are believed to be potentially useful for the
treatment of cachexia related syndromes.4
[
125I]NDP-a-MSH in CHO-K1 cells, led to the identifi-
cation of several leads. Among them, an amino-benz-
imidazole series (Fig. 2) was found to bind selectively
to the hMC4 receptor with moderate affinity (4a,
Ki = 1.0 lM; 5a, Ki = 0.77 lM). These encouraging re-
sults prompted us to initiate a structure–activity rela-
tionship (SAR) study of the amide group, the basic
Keywords: Melanocortin-4 receptor; Antagonist; Benzimidazole.
*
Corresponding author. Tel.: +33 1 6092 2000; fax: +33 1 6907
3802; e-mail: lydie.poitout@ipsen.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.010

L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
4465
F
O
Br
O
Br
N
N
N
N
N
N
NH
N
F
O
F
1 (MCL0129)
2a (ML253764)
2b
Cl
Cl
F
Cl
Cl
N
Cl
N
NH2
N
N
HN
S
NH
N
O
O
NH
N
O
O
O
O
O
NH2
3b (NBI-12i)
3a
3c
Figure 1. Small molecule antagonists of the human MC4 receptor.
O
O
O
O
N
N
N
N
N
H
N
H
N
N
O
O
H2N
H2N
4a
Figure 2. Lead compounds coming from screening.
5a
chain and the right hand side group of the lead
compounds.
using manganese dioxide gave the corresponding ben-
zoyl derivative 17 (Scheme 2).
Benzimidazole derivatives were prepared according to
Scheme 1. The synthesis started from 3-fluoro-4-nitro-
benzoic acid 7,9 which was coupled with various amines
under standard conditions (EDC, HOBt) to afford the
corresponding amides 8. Displacement of the fluorine
atom by various primary amines led to the derivatives
9 followed by hydrogenation of the nitro group using
Pd/C. Conversion of the dianilines 10 to amino-benz-
imidazoles 4–6 was performed using various isothiocya-
nates in the presence of cyclodesulfurization agents such
as DCC10a,b or mercury(II) oxide.10c Preparation of the
corresponding benzimidazole analogues 11 was carried
out by condensation of dianilines 10 with aldehydes in
nitrobenzene used as solvent and oxidant,11a or by
acid-promoted cyclisation and dehydration in acetic
acid11b of the amide intermediate, resulting from the
condensation of dianilines 10 with acid chlorides or car-
boxylic acids using coupling reagents. When N-Boc pro-
tected amines were used for R3, such as N-Boc
propylamino group, the resulting compounds were
deprotected under acidic conditions to generate the de-
sired products. Oxidation of the benzyl group of 11h
To explore the role of the amide group, an alternative
synthetic route was used as depicted in Scheme 3. Thus,
3-fluoro-4-nitro-benzoic acid 7 was converted to the cor-
responding methyl ester 12 by treatment with a solution
of trimethylsilyldiazomethane. Displacement of the fluo-
rine atom by a primary amine followed by hydrogena-
tion of the nitro group led to the dianilines 14.
Conversion to amino-benzimidazoles 15 was performed
using the same conditions as described for Scheme 1, fol-
lowed by saponification of the methyl ester to yield the
corresponding carboxylic acid 16. This, in turn, was sub-
jected to coupling reactions with various amines to af-
ford compounds 4–6.
We first investigated the replacement of the N,N-dibutyl
amide in compound 4a (Table 1). Shortening the alkyl
chains (compound 4c) caused a drop in affinity, whereas
extending it by one carbon maintained comparable activ-
ity (4e, Ki = 0.64 lM). Ramification of the lipophilic
chains, for example, with diisopentyl groups led to the
most active compounds in the series (4b, Ki = 0.42 lM).
Introduction of this active pharmacophore in the other

4466
L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
NO2
NO2
R2
N
NO2
F
R2
N
b
a
R1
NH
R3
HO
R1
F
O
O
O
7
9
8
c
NH2
R2
N
R1
NH
R3
O
10
d, f
e, f
R2
N
N
N
R2
N
N
H
N
R4
R4
R1
N
R1
R3
R3
O
4-6
O
11
Scheme 1. Reagents and conditions: (a) R1R2NH2, EDC, HOBt, DCM, rt; (b) R3NH2, K2CO3, CH3CN, reflux; (c) H2, Pd/C, AcOEt/EtOH; (d)
R4NCS, •-DCC,10b THF, reflux; or R4NCS, HgO, S, EtOH, reflux; (e) R4CHO, nitrobenzene, 130 ꢁC; or R4COCl, AcOH, 100 ꢁC; or R4COOH,
TBTU, DIEPA then AcOH, 100 ꢁC; (f) for R3 containing BocNH, TFA/DCM or HCl/dioxane.
O
O
chain provided optimal binding (5b), compounds with
an aminoethyl or aminobutyl chain exhibiting lower
binding affinity (5c, Ki = 220 nM; 5d, Ki = 130 nM).
R2
N
N
N
R2
N
N
N
R1
R1
MnO2, dioxane
reflux, 4d
O
Substitution of the primary amine by a tert-butyloxycar-
bonyl group or its replacement by a hydroxy function
resulted in a loss of activity, demonstrating that the
basic site is crucial for binding. Incorporation of lipo-
philic alkyl groups on the primary amine (5f and 5g)
as well as introduction of heterocycloalkyl groups such
as pyrrolidine or piperidine resulted in a significant in-
crease of binding affinity, leading to potent compounds
with nanomolar affinity for the hMC4 receptor (5h,
Ki = 4.9 nM; 5i, Ki = 2.0 nM). Morpholine and pipera-
zine rings however gave less potent compounds (5j and
5k). Introduction of conformational constraints to the
R3
R3
O
O
11h
17
Scheme 2.
lead 5a (Fig. 2) led to a more active compound 5b with a
Ki = 60 nM. Thus, further SAR was carried out on this
compound.
A study of the linker between the nitrogen and the benz-
imidazole ring (Table 2) revealed that the propylene
NO2
F
NO2
F
NO2
NH2
a
c
b
O
HO
O
O
NH
R3
NH
R3
O
O
O
O
7
13
12
14
d
R2
N
N
N
N
N
N
N
H
N
H
N
H
e
f, g
N
R4
R4
R1
R4
O
HO
R3
R3
R3
O
O
O
16
4-6
15
Scheme 3. Reagents and conditions: (a) TMSCH2N2, MeOH, 0 ꢁC; (b) R3NH2, K2CO3, CH3CN, reflux; (c) H2, Pd/C, AcOEt/EtOH; (d) R4NCS,
•-DCC,10b THF, reflux; or R4NCS, HgO, S, EtOH, reflux; (e) LiOH, THF, H2O, reflux; (f) R1R2NH2, EDC, HOBt, DCM, rt; (g) for R3 containing
BocNH, TFA/DCM or HCl/dioxane.

L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
Table 1. Binding affinity of amides 4 at the human MC4 receptora Table 2. SAR of the basic chain at the human MC4 receptora
4467
O
O
O
O
R2
N
N
N
N
N
H
N
H
N
N
R1
R3
O
O
5b-p
4a-e
Compound
R3
pKi SEMb
Ki (nM)
H2N
N
5b
7.22 0.075
60
220
130
51
H2N
Compound
R1R2N
pKi SEMb
Ki (lM)
5c
5d
5e
5f
6.66 0.11
6.89 0.061
7.29 0.032
7.93 0.53
8.10 0.071
8.31 0.038
8.71 0.081
7.49 0.12
7.34 0.19
6.14 0.12
7.30 0.013
4.07 0.027
5.64 0.18
H2N
H2N
4a
5.98 0.23
1.0
H
N
N
4b
6.38 0.12
0.42
12
N
N
N
5g
5h
5i
7.9
4c
4d
4.75 0.14
17
11
N
4.9
2.0
N
4.93 0.094
N
O
N
5j
33
N
4e
6.20 0.033
0.64
N
5k
5l
45
730
N
a Binding affinity at the human melanocortin-4 receptor stably
expressed in CHO-K1 cells, using [125I]NDP-a-MSH as the radiola-
belled ligand.
N
N
b pKi are arithmetic means of three-independent measurements.
5m
5o
5p
50
propyl linker, such as gem-dimethyl substituents, dis-
played lower potency (5l, Ki = 730 nM) and replacement
of the acyclic chain by a piperidine ring decreased the
activity (5m, Ki = 50 nM). The N-piperidine propyl
chain gave the compound with the best binding activity
in this series (5i, Ki = 2.0 nM).
H
N
O
>10,000
O
2300
HO
a Binding affinity at the human melanocortin-4 receptor stably
expressed in CHO-K1 cells, using [125I]NDP-a-MSH as the radiola-
belled ligand.
A comprehensive survey of the substitution of the ami-
no-benzimidazole 6 on the right hand side with different
groups: substituted phenyls, aryls, heteroaryls, substi-
tuted alkyl chains (Table 3) showed that all compounds
possess high binding affinity. However some slight dif-
ferences can be noted. Investigation of the phenyl substi-
tuent revealed a preference for 4- and 3-substitution
compared to 2-substitution as exemplified by com-
pounds 5i and 6a versus 6b. Similar binding activity
was obtained either with electron donating substituents
such as methoxy (6d, Ki = 5.0 nM) or electron-with-
b pKi are arithmetic means of three-independent measurements.
drawing groups such as carbonyl, ester or nitro. Thus,
5i, 6i and 6j had Ki values of 2.0, 0.90 and 2.2 nM,
respectively, on the hMC4 receptor. Halogenated phe-
nyls gave compounds with slightly reduced potency
(6k–6m). Increasing the size of the phenyl substituents
such as phenyl, phenoxy, phenylketone, piperidine or

4468
L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
Table 3. SAR of amino-benzimidazole 6 at the human MC4 receptora
Table 4. SAR of benzimidazoles 11 and 17 at the human MC4
receptora
R4
N
N
N
R4
N
H
N
N
N
O
O
5i, 6a-z
N
11a-h, 17
N
Compound R4
pKi SEMb Ki (nM)
Compound
R4
pKi SEMb
Ki (nM)
5i
4-Me(O)C–Ph–
3-Me(O)C–Ph–
2-Me(O)C–Ph–
Ph–
8.71 0.081
8.35 0.024
6.96 0.077
8.14 0.11
8.30 0.015
8.57 0.16
8.48 0.08
8.19 0.12
8.43 0.046
9.05 0.089
8.67 0.061
8.08 0.054
8.06 0.066
8.12 0.10
8.28 0.050
8.69 0.12
8.76 0.066
8.01 0.037
2.0
4.5
6a
6b
6c
6d
6e
6f
11a
11b
11c
11d
11e
11f
11g
11h
17
4-MeC(O)Ph–
4- MeO–Ph–
7.70 0.035
7.43 0.13
7.35 0.059
8.15 0.083
8.49 0.047
8.53 0.02
8.41 0.012
7.55 0.054
9.02 0.015
20
37
110
7.3
5.0
2.7
3.3
6.5
3.7
0.90
2.2
8.3
8.8
7.5
5.2
2.1
1.7
9.7
4.0
4.2
4-MeOC(O)–Ph–
3-Indolyl–
45
4-MeO–Ph–
7.0
3.2
2.9
3.9
28
4-Et(O)C–Ph–
4-MeNH(O)C–Ph–
4-H2NC(O)–Ph–
4-MeC(O)NH–Ph–
4-MeOC(O)–Ph–
4-NO2–Ph–
3-N-methyl-indolyl
2-Benzothienyl
4-MeC(O)Ph–CH2–
4-MeO–Ph–CH2–
4-MeO–Ph–C(O)–
6g
6h
6i
0.96
6j
a Binding affinity at the human melanocortin-4 receptor stably
expressed in CHO-K1 cells, using [125I]NDP-a-MSH as the radiola-
belled ligand.
6k
6l
4-F–Ph–
4-Cl–Ph–
6m
6n
6o
6p
6q
6r
6s
6t
4-Br–Ph–
4-Ph–Ph–
b pKi are arithmetic means of three-independent measurements.
4-PhO–Ph–
4-PhC(O)–Ph–
4-Piperidine–Ph–
the amino derivatives 5i, 6h and 6i. However, replace-
ment of the NH group by a methylene group gave com-
pounds with better binding affinity (11g, Ki = 3.9 nM)
and replacement by a carbonyl group led to the most po-
tent compound in this series (17, Ki = 0.96 nM).
4-(1H-Imidazolyl)–Ph– 8.40 0.046
3-Pyridyl–
4-Indolyl–
8.38 0.080
7.85 0.11
7.48 0.030
7.76 0.11
8.24 0.14
7.51 0.13
7.11 0.13
7.77 0.13
14
6u
6v
6w
6x
6y
6z
3-Thienyl
Ph–CH2–
33
18
4-MeC(O)–Ph–CH2–
Me(O)CNH–(CH2)2–
MeO–(CH2)2–
Pyrrolidinone–(CH2)3–
5.8
Interestingly, all these results summarized in Tables 3
and 4 showed that this region of the molecule tolerated
groups with different properties. Thus, it offers an
opportunity for optimization of this series of com-
pounds towards desirable physicochemical properties.
31
78
17
a Binding affinity at the human melanocortin-4 receptor stably
expressed in CHO-K1 cells, using [125I]NDP-a-MSH as the radiola-
belled ligand.
Selected compounds were tested for their selectivity over
the other melanocortin receptor subtypes.8 Compounds
5i, 6f, 6z, 11g and 17 exhibited a 40- to 1000-fold selec-
tivity of MC4-R over the MC3 receptor subtype and in
addition all compounds show weak binding activity for
the other MC receptors (Table 5).
b pKi are arithmetic means of three-independent measurements.
imidazolyl (6n–6r) gave rise to compounds with compa-
rable activity to 6c where the phenyl ring is
unsubstituted.
Functional antagonist and agonist assays were per-
formed on the same set of compounds, using CHO-K1
cells stably transfected with the human MC4-R. While
derivatives 5i, 6f, 6z, 11g and 17 were unable to stimu-
late cyclic AMP release at 10 lM, they inhibit NDP-a-
MSH-stimulated cAMP production in a dose-dependent
manner,12 demonstrating their antagonist properties
(Table 5). All the compounds tested increased the
EC50 of NDP-a-MSH in a dose-dependent manner with-
out affecting the maximal inhibition observed, indicating
the competitive nature of the antagonism. Schild regres-
sion analysis of the data led to the determination of a KB
value (concentration of compound that shifts NDP-a-
MSH dose–response twofold). Compound 6f exhibited
enhanced functional potency (KB = 3.2 nM, Fig. 3) as
compared to the other analogues (Table 5), however it
Replacement of the phenyl ring by heteroaromatic rings
such as indolyl (6t) or thienyl (6u) slightly lowered bind-
ing affinity, whereas pyridine exhibited a similar effect to
the phenyl moiety in binding (6c, Ki = 7.3 nM; 6s,
Ki = 4.2 nM). Benzyl groups turned out to be slightly
less potent than phenyl analogues (6v, Ki = 18 nM; 6w,
Ki = 5.8 nM vs 6c and 5i, respectively).
Changing the aryl substituent to alkyl groups bearing
various functional groups such as amide, methoxy or
pyrrolidinone (6x–6z) led to less potent compounds
(Ki = 31, 78 and 17 nM, respectively).
Benzimidazole analogues 11a–11c (Table 4) displayed
more than a 10-fold reduction in binding, compared to

L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
4469
Table 5. Binding affinity (Ki, nM) at the melanocortin receptor subtypesa,b and functional antagonist potency (KB, nM)c of selected compounds at
the human MC4-R
c
KB (nM)
Compound
Kib (nM)
hMC1-R
hMC3-R
hMC4-R
hMC5-R
hMC4-R
5i
5200
9300
400
130
2.0
3.3
2200
880
77
3.2
11
14
44
6f
6z
11g
17
>10,000
>10,000
7500
3800
4500
540
17
>10,000
5900
1600
3.9
0.96
a Human melanocortin-1,-3,-4 and -5 receptors stably expressed in CHO-K1 cells and [125I]NDP-a-MSH as radiolabelled ligand in the competitive
binding assay.
b Ki are logarithmic means of three-independent measurements.
c KB were determined using Schild regression analysis and are logarithmic means of three-independent measurements.
250
200
150
100
50
Further optimization studies of compounds in this series
as well as in vivo evaluation of analogues in food intake
experiments will be reported in due course.
2
1.5
1
Acknowledgments
0.5
0
´
We thank Denis Giraud, Herve Gaudry, Gilles Mario
and Karine Bremard for analytical support.
-7.5
-7
-8
-6.5
´
Log [antagonist]
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.06.010.
0
1e-13
1e-11
1e-9
1e-7
References and notes
NDP-alpha-MSH : [M]
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IDrugs 2005, 8, 314; (b) Scarlett, J. M.; Marks, D. L.
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Figure 3. Effect of compound 6f on NDP-a-MSH-stimulated cAMP
production. cAMP levels were measured in presence of 10 nM (s),
30 nM (j), 100 nM (h), 300 nM (m) or without (d) compound 6f. The
inset shows the Schild regression analysis of the data.
5. Chaki, S.; Hirota, S.; Funakoshi, T.; Suzuki, Y.; Suetake,
S.; Okubo, T.; Ishii, T.; Nakazato, A.; Okuyama, S. J.
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possessed lower selectivity towards the MC3 receptor
subtype.
6. (a) Vos, T. J.; Caracoti, A.; Che, J.; Dai, M.; Farrer, C. A.;
Forsyth, N. E.; Drabic, S. V.; Horlick, R. A.; Lamppu, D.;
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Tran, J. A.; Fleck, B. A.; Madan, A.; Dyck, B. P.; Parker,
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Among the most active compounds, a representative
member of the amino-benzimidazole series, 5i, was
selected to be further evaluated for its ability to cross
the blood–brain barrier, an important parameter to
assess, since the MC4-R is centrally localized. Plasma
and brain levels were determined following intravenous
administration at 4.5 lmol/kg to rats.13 Compound 5i
displayed a half-life of 5.0 h and was shown to penetrate
into the brain yielding to an AUC(brain/blood) (area under
the curve) ratio of 2.9.
In conclusion, a novel class of small molecule MC4-R
antagonists has been identified and optimized for
potency. Selected compounds display good to high selec-
tivity over other melanocortin receptors and were shown
to be potent antagonists at the MC4 receptor. Further-
more, one analogue 5i showed a good brain penetration.

4470
L. Poitout et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4464–4470
C.; Brookhart, G. B.; Hesse, A.; Hoare, S. R. J.; Fleck, B.
12. Cyclic AMP assay: Intracellular cyclic AMP (cAMP)
levels were determined by an electro-chemiluminescence
(ECL) assay (Meso Scale Discovery, MSD). CHO-K1 cells
stably expressing the human MC4 receptors were sus-
pended in RMPI 1640 containing 0.5 mM IBMX and
0.2% protein cocktail (MSD). They were dispensed (7000
cells/well) in multi-array plates containing integrated
carbon electrodes and coated with anti-cAMP antibody.
Concentration–response experiments of NDP-a-MSH
were carried out in the presence of increasing concentra-
tions of the tested compound by incubating the cells for
40 min at 37 ꢁC. Then, the cells were lysed and 2.5 nM
TAG ruthenium-labelled cAMP was added. After 90 min,
cAMP levels were determined by ECL detection using
sector imager 6000 reader (MSD). cAMP data were
analysed by computer-assisted non-linear regression anal-
ysis (XL fit; IDBS). The Kb values were determined by
Schild regression analysis.
13. Fed male Sprague–Dawley rats were dosed in groups of
three animals (for each kinetic point). A solution of
4.5 lmol/kg in 1 ml/kg of a 10% DMA/90% propanediol
solution was given by intravenous administration via the
penile vein. Then, rats were deeply anaesthetised under
Isofluraneꢂ and blood was taken from the vena cava (at 1,
15, 30, 60, 90 and 120 min) and centrifuged (2000g,
15 min, 4 ꢁC). The supernatants were mixed with cold
acetonitrile to precipitate the proteins. After a further
centrifugation (2000g, 15 min, 4 ꢁC), the samples were
analysed by HPLC (UV detection) or frozen at ꢀ 80ꢁ
pending analysis. After blood withdrawal, tissues were
rapidly washed by intracardiac perfusion with 50 ml of
saline. The brains were then removed, frozen in liquid
nitrogen and weighed. After mixing the brains with
methanol for 40 s with an Ultraturaxꢂ, brain homoge-
nates were centrifuged (25,000g, 15 min, 4 ꢁC) and the
supernatants were analysed by HPLC.
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Arellano, M.; Wen, J.; Fleck, B. A.; Marinkovic, D.;
White, N. S.; Pontillo, J.; Saunders, J.; Madan, A.; Foster,
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8. MC Receptor binding assay: membranes were prepared
from CHO-K1 cells stably expressing the human melano-
cortin receptor subtypes MC1, MC3, MC4 and MC5: they
were incubated at 1–10 lg protein/well in 50 mM Tris–
HCl, pH 7.4, containing 0.2% BSA, 5 mM MgCl2, 1 mM
CaCl2 and 0.1 mg/mL bacitracin, with increasing concen-
trations of the tested compound and 0.1–0.3 nM
[
125I]NDP-a-MSH for 90–120 min at 37 ꢁC, depending
on the receptor subtype. Bound from free [125I]NDP-a-
MSH was separated by filtration through GF/C glass fibre
filters presoaked with 0.1% (w/v) PEI. Filters were washed
three times with 50 mM Tris–HCl, pH 7.4, at 0–4 ꢁC and
assayed for radioactivity using Perkin-Elmer Topcount
counter. Binding data were analysed by computer-assisted
non-linear regression analysis (XL fit; IDBS).
9. (a) Schmelkes, F. C.; Rubin, M. J. Am. Chem. Soc. 1944,
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imide, N0-meꢂthyl polystyrene resin (purchased from
Novabiochem ) was used instead of N,N0-dicyclohexyl-
carbodimide to facilitate by-products removal; (c) Jans-
sens, F.; Torremans, J.; Janssen, M.; Stokbroekx, R. A.;
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