Melanocortin subtype-4 receptor agonists containing a piperazine core with substituted aryl sulfonamides

Article abstract of DOI:10.1016/j.bmcl.2005.01.060

The biological activity for a set of melanocortin-4 receptor (MC4R) agonists containing a piperazine core with an ortho-substituted aryl sulfonamide is described. Compounds from this set had binding and functional activities at MC4R less than 30 nM. The most selective compound in this series was >25,000-fold more potent at MC4R than MC3R, and 490-fold more potent at MC4R than MC5R. This compound also reduced food intake after oral dosing at 25, 50, and 100 mg kg^-1 in fasted mice.

Full text of DOI:10.1016/j.bmcl.2005.01.060

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1623–1627
Melanocortin subtype-4 receptor agonists containing a
piperazine core with substituted aryl sulfonamides
Christopher Fotsch,* Nianhe Han, Premilla Arasasingham, Yunxin Bo,
Michelle Carmouche, Ning Chen, James Davis, Martin H. Goldberg, Clarence Hale,
Feng-Yin Hsieh, Michael G. Kelly, Qingyian Liu, Mark H. Norman,
Duncan M. Smith, Markian Stec, Nuria Tamayo, Ning Xi, Shimin Xu,
Anthony W. Bannon and James W. Baumgartner
Departments of Chemistry Research and Discovery and Metabolic Disorders, Amgen Inc., One Amgen Center Drive,
Mailstop 29-1-B, Thousand Oaks, CA 91320, USA
Received 13 December 2004; revised 24 January 2005; accepted 25 January 2005
Abstract—The biological activity for a set of melanocortin-4 receptor (MC4R) agonists containing a piperazine core with an ortho-
substituted aryl sulfonamide is described. Compounds from this set had binding and functional activities at MC4R less than 30 nM.
The most selective compound in this series was >25,000-fold more potent at MC4R than MC3R, and 490-fold more potent at
MC4R than MC5R. This compound also reduced food intake after oral dosing at 25, 50, and 100 mg kgꢀ1 in fasted mice.
Ó 2005 Elsevier Ltd. All rights reserved.
The melanocortin-4 receptor (MC4R) is a G-protein
coupled receptor expressed in the hypothalamus, which
has been shown to regulate feeding behavior.1,2 Agonists
to the receptor have been shown to decrease feeding in
rodents.3 Conversely, feeding increased when rodents
were treated with MC4R antagonists.4,5 In addition,
mice lacking the gene encoding MC4R are obese and
hyperphagic.2 In humans, MC4R polymorphisms that
impair receptor function have been linked to obesity.6
More recently, experiments have been conducted that
link MC4R to sexual arousal.7,8 Agonists to MC4R
cause an increase in the number of penile erection in
rats.7 In addition, administration of a potent melanocor-
tin receptor peptide agonist increases the number of
erections in men with erectile dysfunction (ED).8 Be-
cause of the evidence supporting MC4R as a key medi-
ator in two physiological functions, we set out to
identify small molecule agonists for MC4R that might
be used as therapies for either obesity or ED.
In the past few years, several patent applications9 and
articles in the literature10 have appeared that contain
MC4R agonists with either the piperidine or piperazine
core (see Fig. 1). In many of the published structures,
the piperidine or piperazine is appended with two amino
acids: para-chloro-D-phenylalanine (pCl-D-Phe) and
D-1,2,3,4-tetrahydro-3-isoquinoline (D-Tic). In 2002, a
research group at Merck published agonists that
contained this dipeptide appended onto a piperidine
core.10a Compound 1 from this report was a low-nano-
molar functional agonist and was several fold more
selective for MC4R over two other receptor subtypes,
MC3R and MC5R. In addition, compound 1 showed
both anti-feeding effects and increased the number of
erections in rats. Scientists at Lilly10b and Neurocrine
Biosciences10c–e also independently identified potent
and selective MC4R agonists that contained a piper-
azine appended to the pCl-D-Phe, D-Tic dipeptide. In
our own studies, we identified MC4R agonists that con-
tained the piperazine core with the pCl-D-Phe, and L-Tic
dipeptide (compound 2). The piperazine on 2 is ap-
pended with a ortho-substituted aryl sulfonamide group.
Compound 2 was potent at MC4R and selective
over MC3R, but 2 was only 21-fold selectivity for
MC4R over MC5R. We wanted to further explore the
Keywords: Melanocortin subtype 4 receptor; Piperazine; Feeding.
*
Corresponding author. Tel.: +1 805 447 7746; fax: +1 805 480 1337;
e-mail: cfotsch@amgen.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.060

1624
C. Fotsch et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1623–1627
Figure 1. MC4R agonists containing a piperidine or a piperazine core (see Ref. 10).
structure–activity relationship of this compound by
substituting the sulfonamide and replacing the Tic
group to see if potency and selectivity could be im-
proved. In addition, once we obtained a more potent
and selective compound, we wanted to study its in vivo
activity in rodent behavioral models that measured
feeding.
(
125I-NDP-MSH). They were also tested in a whole cell
functional assay where intracellular levels of adeno-
sine 30,50-cyclic phosphate (cAMP) were measured.12
The level of cAMP for the compounds tested was
90–100% of the level achieved with the endogenous
ligand, a-melanocyte stimulation hormone (a-MSH),
unless stated otherwise. The values in Tables 1–4 are
the average of at least two separate experiments re-
ported with the standard error of the mean. The
pCl-D-Phe group was retained in all compounds since
we had found that this group was preferred for MC4R
binding and functional activity for this series of
compounds.
Compounds for the SAR studies were derived from ami-
nophenyl piperazine 3 (Scheme 1). Sulfonylation of
compound 311 followed by an N-alkylation of the sul-
fonamide gave compound 4. Deprotection of 4, followed
by amino acid coupling with pCl-D-Phe, and then a sec-
ond deprotection gave the key intermediate 5. Com-
pounds 6, 7, and 8 were all derived from intermediate
5 as outlined in the Scheme 1.
We first examined the effects of substituting the sulfon-
amide group by increasing the size of R1 and R2 (see
Table 1). Larger groups at R1, like the benzyl analog 6a
and phenyl analog 6b, did not improve activity. How-
ever, if substituents were added to the nitrogen of the
Compounds were tested in a competition binding
assay with 125I-NDP-melanocyte stimulation hormone
Scheme 1. Reagents and conditions: (a) R1SO2Cl, i-Pr2NEt, ClCH2CH2Cl (DCE); (b) NaH, R2–I, DMF; (c) trifluoroacetic acid/CH2Cl2 or HCl in
EtOAc; (d) Boc protected amino acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (EDC), 1-hydroxybenzotriazole (HOBT), DCE and/or
DMF; (e) (hetero)aryl carboxylic acid, EDC, HOBT, DCE, and/or DMF; (f) ketone or aldehyde, NaBH(OAc)3, DCE.

C. Fotsch et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1623–1627
1625
Table 1.
sulfonamide, activity improved (see 6d–f). Interestingly,
compound 6e and 6f have very different function-
alities at R2, an ethyl amino group, and a propyl group,
respectively, but their binding and functional activities
were similar (IC50s <40 nM and EC50s <1–4 nM).
Compound 6d containing a methylene cyclopropyl
group on R2 was slightly less potent that 6f in the bind-
ing assay (IC50 = 61 vs 25 nM), but functional activity
was nearly the same (EC50 = 0.58 vs 0.44 nM).
#
R1
R2
hMC4
receptor
IC50 (nM)
hMC4R
functional
EC50 (nM)
While investigating the SAR at the Tic position, we
kept the left side of the molecule constant with a one
of our more potent sulfonamides, the methylene cyclo-
propyl group (see Tables 2 and 3). Compounds that
were studied in Table 2 contained two fused 6-mem-
bered rings, which we thought might be good surro-
gates for Tic ring system. Compound 7a, with the
isoquinoline group, was ca. 7-fold less potent in the
binding assay than the corresponding Tic analog, com-
pound 6d. The naphthyl derivative 7b had a similar
binding affinity to 7a, and the 3-quinoline and 6-quin-
oline derivatives, 7c and 7d, were 4–5-fold more potent
than 7a. The trend for receptor binding and functional
activity diverged in this group of compounds, however.
In this set, compound 7a was the least potent in the
binding assay, but it was the most potent in the func-
tional assay. Compounds 7b and 7c differed in potency
by ca. 3-fold (IC50 = 280 vs 94 nM), but their func-
tional activities were similar (EC50 ca. 60 nM). The
functional activity for compound 7d was nearly identi-
cal to its binding affinity (IC50 and EC50 both ca.
30 nM), but 7d could only produce 50% of the total re-
sponse. None of the compounds in Table 2 were func-
tionally as potent as 6d, so these analogs were not
further pursued.
2
CH3
Bn
H
340 30
720 160
730 60
210 70
11 3
65 23
6a
6b
6c
6d
6e13
6f
H
Ph
H
CH3
120 50
4
CH3
CH3
CH3
CH3
4
CH2(cyclopropyl)
(CH2)2NH2
(CH2)2CH3
61
35
25
8
7
3
0.58 0.15
<0.1
0.44 0.21
Table 2.
#
X
Y
Z
hMC4R
receptor
IC50 (nM)
hMC4R
functional
EC50 (nM)
7a
7b
7c
7d
N
CH2
CH2
N
CH2
CH2
CH2
N
410 37
280 100
94 24
18
63 30
3
CH2
CH2
CH2
60 16
27 10 (50%)
CH2
29
8
We then looked at a set of piperidines and azetidines as
replacements for the Tic group. A disparity between the
trends in binding and functional activities was observed
for this set of compounds too (see Table 3). In the bind-
ing assay, the rank order for potency was
8d > 8c > 8b > 8a. Compound 8d, with the N-cyclohexyl-
azetidine group, had the best binding and functional
activity for this set of compounds (IC50 = 67 nM,
EC50 = 1.8 nM). However, the rank order potency in
the functional assay for the remaining compounds was
8b > 8a > 8c. Apparently, the factors responsible for
functional activity are subtly different from those ele-
ments that affect binding to the receptor for this region
of the molecule.14 Since we were interested in agonists
for MC4R, we chose compounds with the best func-
tional activity for further profiling.
Table 3.
#
n
R4
hMC4R
receptor
IC50 (nM)
hMC4R
functional
EC50 (nM)
8a
8b
8c
8d
1
1
0
0
Methyl
Ethyl
tert-Amyl
120 40
110 30
83 30
67 10
12
1
8.6 1.5
49 13
1.8 1.7
Cyclohexyl
Table 4. Selectivity at human melanocortin receptors
#
hMC4R IC50 (nM)
hMC4R EC50 (nM)
hMC3R EC50 (nM)
hMC5R EC50 (nM)
MC5R/MC4R
MTII
2
6d
1.48 0.23
340 30
<0.01
11
<0.01
>10,000
7000
0.01 0.006
230 50
3
21
280
ꢁ79
490
61
35
25
9
7
3
0.6 0.2
<0.1
0.4 0.2
170 20 (50%)
79 50 (60%)
210 10
6e
6f
>10,000
>10,000

1626
C. Fotsch et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1623–1627
Table 5. In vitro potency and pharmacokinetics of compound 6f in micea
MC4R IC50
(nM)
MC4R EC50
(nM)
Cl/F
(mL minꢀ1 kgꢀ1
Vz/F
(L kgꢀ1
Tmax
Cmax
(ng mLꢀ1
t1/2
%F
Cbrain
(ng gꢀ1) at Tmax
)
)
(h)
)
(h)
0.1
0.1
30
19
0.25
1400
0.44
20%
130
a Compound formulated in PBS with 1% HMBC; 1% Tween 80. Dosed in fed male CD-1 mice at 50 mg kgꢀ1 (p.o.).
We next chose to examine the selectivity of our most po-
tent compounds at receptor subtypes MC3R and MC5R
(see Table 4).12 For reference purposes, we also included
the activity of MT-II, a known potent peptide agonist
for the melanocortin receptors.15 Our lead compound
2 and three of our most potent analogs, 6d–f, all had
IC50s over 10 lM in the human MC3R functional assay.
At the human MC5 receptor, our lead compound 2 was
only modestly selectivity for MC4R over MC5R (20-
fold). On the other hand, compounds 6d, 6e, and 6f
were, respectively, 280, ꢁ79, and 490-fold more selective
for MC4R versus MC5R. Only partial activation of
MC5R (50–60%) was seen for compound 6d and 6e.
Compound 6f was chosen for additional in vivo phar-
macology experiments since it was the most selective
and did not show partial agonism for MC5R.
Figure 2. Effects of oral administration of 6f on cumulative food
intake in fasted male C57BL/6 mice. Values represent mean SEM
(n = 10/group); significantly different from vehicle; *p < 0.05;
**p < 0.01; ***p < 0.001 based on ANOVA followed by Fishers
protected least significant difference test where appropriate.
Compound 6f was then run in a pharmacology model
that measured acute feeding behavior in mice. Prior to
this study, however, we tested 6f for in vitro potency
and determined the pharmacokinetic profile in mice.
Compound 6f was very potent at the mouse receptor
(IC50 and EC50 below 1 nM, see Table 5). In the PK
experiments, we dosed compound 6f orally at
50 mg kgꢀ1. The concentration of 6f in the brain was
measured since we were looking for exposure in the tis-
sue where MC4R presumably affects feeding. While the
Tmax was under an hour, compound 6f had plasma and
brain concentrations well above the IC50 and EC50 of
the receptor, so we felt that this compound would be
useful in evaluating the effects of MC4R agonism in
mice.
cific or toxic side effects. Compound 6f was screened
against two other receptors that are implicated in reg-
ulating feeding behavior: the neuropeptide Y5 and
melanocyte stimulating hormone receptors. In both
cases, compound 6f had IC50s > 10,000 nM in a recep-
tor binding assay for these two receptors. In addi-
tion, no overt motor deficiencies were observed in
any of the animals that showed a reduction in feeding,
indicating that the feeding effects of compound 6f
were not caused by a decrease in locomotor activity.
Our experience is that overtly toxic agents will pro-
duce decreases in locomotor activity. Feeding inhibi-
To examine the feeding behavior in mice, we measured
the amount of food consumed in animals that were
fasted for 24 h. Since rodents normally eat during the
night, mice were acclimated to a reversed light-cycle
(lights on at night, lights off during the day) so their
feeding behavior could be measured during normal
working hours. Mice were given compound 6f, by oral
gavage, and then given a pre-weighed amount of chow.
Efficacy was measured by comparing the amount of
food consumed in mice treated with compound to those
animals treated only with the vehicle. Compound 6f
showed a dose dependent decrease in food intake at
25, 50, and 100 mg kgꢀ1 after 1 h (Fig. 2). Animals trea-
ted at 50 mg kgꢀ1 showed a significant reduction in food
intake that extended for 4 h, and the 100 mg kgꢀ1 group
showed a significant reduction in feeding that lasted 6 h.
After 24 h, however, cumulative food intake in all dose
groups returned to levels observed in the control
animals.
tion through
a
mechanism other than MC4R
agonism still cannot be completely ruled out. How-
ever, the evidence shown here, and the fact that 6f
is a potent MC4R agonist that accesses the target tis-
sue, suggests that the feeding effects are mediated
through the MC4R.
By preparing analogs at the sulfonamide and Tic posi-
tions we were able to identify compounds that were
more potent at MC4R than our lead compound 2. Ana-
logs with larger groups on the sulfonamide nitrogen, for
example, 6d–f, were also more selective than the lead.
We were able to show that one of these compounds,
6f, achieved brain levels above its EC50, and reduced
food consumption in fasted mice. These effects are con-
sistent with the observations that MC4R agonism re-
duces feeding. Additional in vivo pharmacology
experiments with 6f that study the erections in rodents
are being conducted and will be reported in a future
publication.
We next ran experiments to address whether the inhi-
bition of food intake with 6f was a result of non-spe-

C. Fotsch et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1623–1627
1627
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Summers, R.; Hermann, J.; Baker, T.; Cismowski, M.;
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11. Compound 3 was prepared according to the procedure
found in Ref. 9h.
12. For a description of the in vitro assays see: Fotsch, C.;
Smith, D. M.; Adams, J. A.; Cheetham, J.; Croghan, M.;
Doherty, E. M.; Hale, C.; Jarosinski, M. A.; Kelly, M. G.;
Norman, ; Mark, H.; Tamayo, N. A.; Xi, N.; Baumgart-
ner, J. W. Bioorg. Med. Chem. Lett. 2003, 13, 2337.
13. Compound 6e was prepared by a route not outlined in
Scheme 1. The ethylamino group was protected with a Boc
group, and Fmoc protected amino acids were used in the
coupling steps.
14. For a recent review discussing GPCR binding and
functional phenomena see: Kenakin, T. Nat. Rev. Drug
Disc. 2002, 1, 103.
15. Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH2.
Obeidi, F.; Hadley, M. E.; Pettitt, B. M.; Hruby, V. J.
Al-
J. Am. Chem. Soc. 1989, 111, 3413.