Aryl piperazine melanocortin MC4 receptor agonists

Article abstract of DOI:10.1016/S0960-894X(03)00796-0

Incorporation of substituted phenyl piperazine privileged structures into a known MC4 specific dipeptoid consensus sequence resulted in a series of potent (EC₅₀=24 nM) and selective MC4-R agonists. We report the SAR of this series of compounds

Full text of DOI:10.1016/S0960-894X(03)00796-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3793–3796
Aryl Piperazine Melanocortin MC4 Receptor Agonists
Brian Dyck, Jessica Parker, Teresa Phillips, Lee Carter, Brian Murphy, Robin Summers,
Julia Hermann, Tracy Baker, Mary Cismowski, John Saunders and Val Goodfellow*
Departments of Medicinal Chemistry, Pharmacology, and Molecular Biology, Neurocrine Biosciences Inc.,
10555 Science Center Drive, San Diego, CA, 92121, USA
Received 3 June 2003; revised 25 July 2003; accepted 29 July 2003
Abstract—Incorporation of substituted phenyl piperazine privileged structures into a known MC4 specific dipeptoid consensus
sequence resulted in a series of potent (EC50=24 nM) and selective MC4-R agonists. We report the SAR of this series of com-
pounds using in vitro cAMP functional assays in cells transfected with the MC4 or other melancortin receptors.
# 2003 Elsevier Ltd. All rights reserved.
Melanocortin receptors belong to the seven transmem-
brane spanning G-protein coupled receptor superfamily
and are activated by peptide ligands derived from pro-
teolysis of proopiomelanocortin polypeptide. Melano-
cortin receptor activating peptide ligands include the a,
b, and g melanocyte stimulating hormones and ACTH.1
Recently melanocortin MC3-R and MC4-R receptors
have been the subject of intense interest for the possible
development of therapeutic agents to treat obesity. The
melanocortin MC4-R receptor is found in the hypothal-
amus, a center of the brain long known to control
weight and food intake. Experiments with MC4-recep-
tor knock-out mice and with potent non-selective pep-
tide MC4-R agonists and antagonists suggest that this
receptor plays an important role in energy homeostasis,
metabolism and food intake.2aÀd MC4-R antagonists
are of interest in the treatment of cachexia or wasting
diseases associated with cancer, AIDS, and other con-
ditions.3 MC4-R agonists are of interest as potential
therapeutics for the treatment of obesity and diabetes.
MC4-R specific agonists are also reported to stimulate
penile erections in rodents.7 Non-specific melanocortin
peptide agonists have been shown to stimulate erections
in primates and humans,5,6 therefore MC4-R agonists
are of interest as potential treatments of human sexual
dysfunction. Patents claiming MC4-R agonist activity
have appeared using ‘privileged structures’ formerly
employed in growth hormone secretagogue compounds
attached to a MC4-R consensus dipeptide: d-Tic-d-(p-
Cl)-Phe-X.4a,b For example, the spiroindoline privileged
structure found in MC4-R agonist 1 is a key structural
component of the growth hormone secretagogue MK-
677. In our assays, 1 is a full agonist on the MC4-R
receptor with an EC50 of 190 nM and much weaker
agonist activity on MC1-R, MC3-R and MC5-R. We
have studied the incorporation of several known GPCR
privileged structures, as well as new moieties with MC4-
R activity into the d-Tic-d-(p-Cl)-Phe-X sequence in
order to gain insight into the design of MC4-R novel
ligands. Since the completion of the initial work repor-
ted here, a preliminary report and patent applications
for related compounds have appeared.7À9
Our initial agonist screen measured cAMP accumulation
in HEK cells. These cells were stably transfected with a
plasmid construct expressing human MC4-R from a
cytomegalovirus promoter (Bmax
fmol/mg protein; KD
125I-NDP-MSH]=0.41 nM).
[
125I-NDP-MSH]=40
[
Initial screening was completed by stimulation with a
test compound at a concentration of 30 uM. Activity
was expressed as fold-stimulation over baseline, with a-
MSH (EC50=6 nM) yielding 80-fold stimulation over
baseline. The more active compounds were compared
directly against a-MSH stimulation, and EC50 values
were generated for compounds showing a full dose
*Corresponding author. Tel.: +1-858-658-7684; fax: +1-858-658-
7619; e-mail: vgoodfellow@neurocrine.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00796-0

3794
B. Dyck et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3793–3796
response curve in the concentration range tested. All
active compounds were tested for cAMP accumulation
on HEK cells which were not transfected with the MC4
receptor to evaluate for non-receptor mediated cAMP
production. Key compounds were then tested for selec-
tivity against HEK293 cell lines expressing human
MC1-R, human MC3-R, mouse MC4-R, human MC5-
R, and the cell line Y1(ATCC #CCL-79) endogenously
expressing mouse MC2-R (Table 1).
amide 3 (Scheme 2). The formal replacement of the
sulfonamide with a triazole attached via a methylene
linkage resulted in a compound 21 which was a full
agonist on the MC4 receptor with an EC50 of 80 nM.
Preliminary attempts to increase potency of this mol-
ecule by the simple attachment of electron withdrawing
(22, 23) or donating (29) substituents or by addition of
hydrophobic bulk (25) were not productive. However,
modification of this compound by attachment to the
triazole at the 4-position gave a full agonist 26 with
EC50 of 24 nM. This compound shows greater than 100-
fold specificity for activation at the MC4-R receptor
compared to human MC1-R, MC2-R, MC3-R and
MC5-R as shown in Table 2.
As one of several approaches we explored formal open-
ing of the indoline ring of 1 at position A and replace-
ment of the tetravalent carbon with nitrogen. The
resulting 2-substituted phenyl piperazine4b is also a
known GPCR privilidged structure. The modified
dipeptoid, compound 2 was a full agonist on the MC4-
R with EC50 of 520 nM. Deletion of the N–Me resulted
in compound 3 with similar activity (EC50=380 nM)
(see Scheme 1). Conservative modifications (4–8) of the
sulfonamide resulted in similar or less potent com-
pounds. The receptor tolerated extension of the sulfo-
namide from the aryl ring by one carbon. Extended
analogues with branching one carbon removed from the
nitrogen were tolerated 9 (380 nM) but requirements for
steric interactions were severe with the addition of an
another methyl 10 greatly decreasing agonist activity.
Acyl derivatives (11–20) of the aniline nitrogen reported
here were also weak partial agonists (Scheme 1).
Recently, a patent application7 and preliminary reports8
have appeared for a related agonist 27 which in our
assay has an EC50 of 4 nM and is therefore about 6-fold
more potent than 26 and is nearly equipotent in in vitro
cAMP assays to the endogenous ligand, a-MSH. How-
ever agonist 27 had only a modest short-lived effect in
suppressing food intake in fasted mice compared to the
activity of MT-II or a-MSH when given by icv admin-
istration, and little effect on insulin levels or metabolism
levels measured by oxygen consumption.10 Patent
applications claiming aryl piperazine MC4 ligands with
the d-Tic-d-(p-Cl)-Phe consensus sequence have
recently appeared without reported biological activity.9
Heterocycles were explored which incorporated the
ability to form multidentate hydrogen bonds as in histi-
dine found in MC4-R peptide ligands or the sulfon-
Aryl piperazines were synthesized as described in
Schemes 3 and 4. Deprotection and standard peptide
couplings produced the target compounds. Displace-
ment of the mesylate 34 with the cyanoethyl modified
triazole11 followed by base induced elimination pro-
vided 37 which was incorporated into target structure
26.
Table 1. Agonist activity in cAMP accumulation assay
Compd
EC50 (nM)a
% Stimulationb
Fold
stimulationc
1
2
3
4
190
520
380
4400
440
100
100
100
34
5
85
6
7
8
9
1000
690
2200
380
2400
1900
100
100
100
100
100
64
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
7
1600
55
21
600
650
2100
34
24
68
30
1100
1000
80
470
680
110
170
24
83
62
100
70
100
100
100
100
100
4
aValues are means of three experiments.
bRelative to stimulation observed for a-MSH.
cFold stimulation, @30 mM reported for weak screening hits.
Scheme 1.

B. Dyck et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3793–3796
3795
Scheme 2.
Scheme 3. (a) K2CO3, DMSO 130ꢀ (89%), H2 Raney Ni, EtOH (91%); (b) RSO2Cl, Et3N, CH2Cl2 (63%); (c) RNH2, Na(OAc)3BH (75%);
(d) MeSO2Cl, Et3N, CH2Cl2 (63%); (e) RCOCl, DIEA, DIEA, CH2Cl2; (f) TFA, CH2Cl2; (g) dipeptide, HOBt, EDC, CH2Cl2; (h) TFA, CH2Cl2,
prep-HPLC.
Scheme 4. (a) K2CO3, DMSO 130 ꢀC (93%); (b) NaBH4, MeOH, 0 ꢀC (74%); (c) MsCl, Et3N, 0 ꢀC (96%); (d) triazole sodium salt, CH2Cl2, 25 ꢀC
(55%); (e) CH3CN, 25ꢀ C; (f) aq NaOH (70% over two steps); (g) TFA, CH2Cl2; (h) dipeptide, HOBt, EDC, CH2Cl2; (i) TFA, CH2Cl2, prep-HPLC.

3796
B. Dyck et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3793–3796
Table 2. Selectivity testing for stimulation of melanocortin receptors, in vitro cAMP accumulation in transfected HEK or Y1 cells
Compd
hMC1
EC50 (nM)
mMC2(Y1)
EC50 (nM)
hMC3
EC50 (nM)
hMC4
EC50 (nM)
mMC4
EC50 (nM)
hMC5
EC50 (nM)
a-MSH
21
26
1.6
>3000
>3000
1400
>10,000
>10,000
>10,000
>10,000
1.4
>3000
>3000
1400
6.1
80
24
8.8
59
38
29
>3000
>3000
2700
27
4.1
7.6
4. (a) Bakshi, R. K.; Barakat, K. J.; Nargund, R. P.; Patchet,
A. A.; WO 0074679. (b) Nargund, R. P.; Patchett, A. A.;
Bach, M. A.; Murphy, G.; Smith, R. G. J. Med. Chem. 1998,
41, 3103.
5. Koegler, F. H.; Grove, K. L.; Schiffmacher, A.; Smith,
M. S.; Cameron, J. L. Endocrinology 2001, 142, 2586.
6. Wessels, H.; Fuciarelli, K.; Hensen, J.; Hadley, M. E.;
Hruby, V. J.; Dorr, R.; Levine, N. J. Urol. 1998, 160, 389.
7. Nargund, R. P.; Palucki, B .L.; Bakshi, R. K., Patchet, A.
A.; Van der Ploeg, L. H. T. WO 9964002.
The strategy of incorporating GCPR privileged struc-
tures into dipeptide consensus sequences with affinity
for specific peptide ligand receptors is a viable approach
for producing new series of agonists. Compound 26 and
the related series of phenyl piperazine MC4-R agonists
have provided the starting point and SAR for the
development of two novel series12 of antagonists and
agonists that will be reported in due course.
8. Sebhat, I. K.; Martin, W. J.; Ye, Z.; Barakat, K.; Mosley,
R. T.; Johnston, D. B.; Bakshi, R.; Palucki, B.; Weinberg,
D. H.; MacNeil, T.; Kalyani, R. N.; Tang, R.; Stearns, R. A.;
Miller, R. R.; Strack, A. M.; McGowan, E.; Cashen, D. E.;
Drisko, J. E.; Hom, G. J.; Howard, A. D.; MacIntyre, D. E.;
van der Ploeg, L. H.; Patchet, A. A.; Nargund, R. P. J. Med.
Chem. 2002, 45, 4589.
9. Biggers, C. K.; Briner, K.; Doecke, C. W.; Fisher, M. J. ;
Hertel, L. W.; Mancoso, V.; Martinelli, M. J. ; Mayer, J. P.,
Ornstein, P. L.; Richardson, T. I.; Shah, J. A.; Shi, Q. WO 02/
059108.
10. Cepoi, D. H.; Cismowski, M.; Goodfellow, V. S.; Phillips,
T.; Cone, R. D. Brain Res., In press.
11. Horvath, A. Synthesis 1995, 1183.
12. Dyck, B.P.; Goodfellow, V.S.; Phillips, T.; Parker, J.;
Zhang, X., Chen, C.; Tran, J.A.; Pontillo, J;. Tucci, F.C. WO
03/31410.
References and Notes
1. Eberle, A.N., In Melanocortin Receptors; Cone, R. D., Ed.;
Humana, Totowa, NJ: 2000; p 3.
2. (a) Kask, A.; Rago, L.; Wikberg, J. E. S.; Schioth, H. B.
Eur. J. Pharmacol. 1998, 360, 15. (b) Fan, W.; Boston, B. A.;
Kesterson, R. A.; Hruby, V. J.; Cone, R. D. Nature 1997, 385,
165. (c) Murphy, B.; Nunes, C. N.; Ronen, J. J.; Harper,
C. M.; Beal, M. J.; Henaway, M.; Fairhurst, A. M.; Van der
Ploeg, L. H. T.; MacIntyre, D. E.; Mellin, T. N. Neuropeptides
1998, 32, 491. (d) Huszar, D.; Lynch, C. A.; Fairchild-Hun-
tress, V.; Danmore, J. H.; Fang, Q.; Berkemeier, L. R.; Gu,
W.; Kesterson, R. A.; Boston, B. A.; Cone, R. D.; Smith, F. J.;
Campfield, L. A.; Burn, P.; Lee, F. Cell 1997, 88, 131.
3. Marks, D. L.; Ling, N.; Cone, R. D. Cancer Res. 2001, 61,
1432.