Structure-activity relationship of a series of cyclohexylpiperidines bearing an amide side chain as antagonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2005.05.017

A series of cyclohexylpiperazines was synthesized as potent and selective antagonists of the human MC4 receptor. Compound 14t displayed binding affinity (K_i) of 4.2 and 1100 nM at MC4R and MC3R, respectively.

Full text of DOI:10.1016/j.bmcl.2005.05.017

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3434–3438
Structure–activity relationship of a series of
cyclohexylpiperidines bearing an amide side chain as antagonists
of the human melanocortin-4 receptor
Joseph A. Tran,a Joseph Pontillo,a Melissa Arellano,a Beth A. Fleck,b
Fabio C. Tucci,a Dragan Marinkovic,a Caroline W. Chen,a John Saunders,a
Alan C. Fosterc and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Pharmacology, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Neuroscience, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 25 March 2005; revised 29 April 2005; accepted 3 May 2005
Available online 9 June 2005
Abstract—A series of cyclohexylpiperazines was synthesized as potent and selective antagonists of the human MC4 receptor.
Compound 14t displayed binding affinity (Ki) of 4.2 and 1100 nM at MC4R and MC3R, respectively.
Ó 2005 Elsevier Ltd. All rights reserved.
Among the five melanocortin receptors (MC1–5R),
MC3R and MC4R are centrally expressed and are
believed to play important roles in mediating feeding
behavior, metabolism, and energy homeostasis.1 While
it has been well documented that peptide MC3R/
MC4R antagonists such as SHU9119 and AgRP have
a profound effect in inducing feeding in animals,2 recent
discovery of selective peptide MC4R antagonists such as
MBP10 and HS131 has made it possible to define the
biological role of the melanocortin-4 receptor.3 More
importantly, additional studies have showed that
MC4R antagonists can protect weight loss in tumor-
bearing mice.4 Recent studies have also provided evi-
dences that MC4R may have a role in anxiety5 and
pain.6 Thus, a potent and selective MC4R antagonist
could be useful in treatment of human diseases such as
cachexia, anxiety, and neuropathic pain.
inhibits NDP-MSH binding to hMC4R with a Ki value
of 7.9 nM, dose-dependently blocks a-MSH-stimulated
cAMP release.8 This compound also exhibits anxioly-
tic-like and antidepressant-like activity in several animal
models.
Another
antagonist,
compound
3
(Ki = 160 nM), has been demonstrated to be effective
in protecting tumor-induced weight loss in mice follow-
ing peripheral administration9 (Fig. 1).
We have previously reported a series of piperazineben-
zylamine derivatives as MC4R-selective ligands which,
interestingly, possesses both agonistic and antagonistic
activities with only subtly different chemical structures.10
For example, molecule 4 exhibits a Ki of 1.8 nM in com-
petitive binding and a pA2 value of 7.9 in inhibition of
MSH-stimulated cAMP release.11,12 Here, we report
the synthesis and structure–activity relationship of a ser-
ies of cyclohexylpiperazines bearing an amine or amide
side chain as potent antagonists of the human melano-
cortin-4 receptor.
In addition to agonists such as 1,7 several small molecule
antagonists for the human melanocortin-4 receptor
(hMC4R) have been discovered. MCL0129 (2), which
The synthesis of the target compounds started from
cyclohexanone 5, which was converted to aminonitrile
6 using a Strecker reaction (N-Bn-piperizine/Na2S2O5/
KCN) in quantitative yield (Scheme 1). Reduction of
the cyano group in 6 (LiAlH4/Et2O) at room tempera-
ture, followed by protection of the resulting primary
Keywords:
Antagonist.
Melanocortin-4
receptor;
Cyclohexylpiperazine;
*
Corresponding author. Tel.: +1 8586177634; fax: +1 8586177967;
e-mail: cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.017

J. A. Tran et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3434–3438
3435
Figure 1. Some small molecule MC4R ligands.
Scheme 1. Reagents and conditions: (a) 4-Bn-piperazine/Na2S2O5/KCN/H2O/rt, 16 h, quant.; (b) LiAlH4/Et2O/rt, 0.5 h, 94%, then TFAA/Et3N/
CH2Cl2/0 °C to rt, quant.; (c) NH4COOH/Pd-C/MeOH/65 °C, 2 h, 86%; then N-(N-Boc-b-Ala)-(R)-(2,4-Cl)Phe-OH (8)/HBTU/EtN(iPr)2/DMF/rt,
8 h, 54%; (d) K2CO3/MeOH-H2O/65 °C, 8 h, 86%; (e) R1CHO/NaBH4/MeOH/rt, 1–8 h; (f) TFA/CH2Cl2/rt, 0.5 h; then HPLC purification; (g)
R1COCl/Et3N/THF/rt, 8 h; (h) R1NCO/Et3N/THF/rt, 8 h.
amine with trifluoroacetic anhydride, afforded the
protected triamine 7 in about 94% yield. Debenzylation
of 7 under catalytic conditions (HCOONH4/Pd-C), fol-
lowed by a peptide coupling reaction of the free amine
with N-(N-Boc-b-Ala)-(2,4-Cl)Phe-OH (8) under stan-
dard conditions (HBTU/DIEA/DMF), gave the amide
9 in 46% yield. The key intermediate 11 was obtained
in 86% yield from deprotection of the trifluoroacetyl
group in 9 with potassium carbonate in methanol at
65 °C. Deprotection of the Boc-group in 9 and 11 with
TFA/CH2Cl2 afforded the amines 10 and 12, respec-
tively. Reductive alkylation of 11 with various aldehydes

3436
J. A. Tran et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3434–3438
(NaBH4/MeOH) and deprotection provided the second-
ary amines 13a–o as the desired products. Acylation of
11 with a set of acyl chlorides (Et3N/THF) gave the
amides 14a–v. And finally, the reaction of 11 with aryl
isocyanates afforded the ureas 15a–f.13 In each series,
the final deprotection was achieved in almost quantita-
tive yield by exposure to TFA in dichloromethane. All
final products were purified with a preparative HPLC
instrument, as previously described, and the purity of
these compounds was greater than 95%.11
cyclohexyl moiety could retain or improve the interac-
tion between the ligand and the receptor. A series of
cyclohexylpiperazines bearing an alkylamine side chain
13 was, therefore, synthesized and tested in the MC4R
binding assay.
Incorporation of a small lipophilic alkyl group (com-
pound 13a–c) to the nitrogen of the primary amine 12
(Ki = 290 nM) had little effect on binding affinity, while
a phenyl group seemed to have an impact (13d,
Ki = 60 nM, Table 1). A study of the linker (13d–f) be-
tween the nitrogen and the phenyl ring revealed that
the ethylene connection provided the optimal binding
(13e, Ki = 11 nM), although the shorter methylene link-
age maintained comparable activity (13f, Ki = 18 nM).
A brief survey on the substitution of this phenyl ring
with different functionality (13g–l) showed no obvious
improvement. A heteroaromatic ring such as thiophene
and pyridine exhibited a similar effect to the phenyl
moiety in binding. Overall, despite the distinct advan-
tage of an aromatic ring over a plain aliphatic side
chain, the electronic preference of this aromatic group
is unclear.
Competition binding experiments were performed using
HEK293 cells stably transfected with the human mela-
nocortin receptors, as previously described, using
[
125I]-NDP-MSH as a radiolabeled ligand.14 The func-
tional antagonist activity of selected compounds was
measured for their ability to inhibit a-MSH-stimulated
cAMP production in the same cell lines.
Previously we have shown that the basic benzylamine
functionality of 4 and its analogues is important for
binding of this series of compounds to MC4R.11 On
the basis of our receptor model, the phenyl moiety of
the benzylamine is seen to interact with several lipophilic
residues such as Phe284, Leu288, Ile289, Ile291, and
Met292 of transmembrane helix 7 (TM-7), while the
basic nitrogen may pair up with Asp122 of TM-3. Since
most of these amino acid residues of the top TM-7 pro-
jecting into the putative binding pocket are non-aro-
matic, replacing the phenyl ring of 4 with an aliphatic
The above results are quite similar to those of the benzyl-
amines exemplified by 4, in which an N-side chain with
an ethylene linker connecting an aromatic ring such as
phenyl or thienyl moiety to the nitrogen is optimal for
Table 2. SAR of amides 14a–v at hMC4Ra
Table 1. SAR of amines 13a–o at hMC4R
Compound R1
Ki (nM) Stimulation (%)
10
CF3-
Ph-
490
220
190
210
240
250
350
400
120
270
340
520
260
230
220
150
0.9
5.1
7.9
Compound
R1
Ki (nM)a
Stimulation (%)b
14a
14b
14c
14d
14e
14f
14g
14h
14i
12
290
180
340
220
60
0.5
0.7
3.6
5.1
2.8
0.4
5.1
4.7
6.4
5.0
4.2
4.1
2.4
8.1
4.9
3.0
4-FC6H4-
4-ClC6H4-
4-BrC6H4-
13a
13b
13c
13d
13e
13f
13g
13h
13i
iPr-
10
12
7.7
t-BuCH2-
Cyclohexyl-
PhCH2CH2-
PhCH2-
4-MeOC6H4-
2-MeOC6H4-
4-CF3OC6H4-
4-NO2C6H4-
4-MeC6H4-
6.5
8.9
6.7
7.8
8.7
8.2
4.5
5.1
4.3
4.4
1.6
2.3
1.4
1.8
0.6
2.7
3.4
11
18
Ph-
2-FC6H4-
4-FC6H4-
4-NCC6H4-
4-(Me2N)C6H4-
4-CF3C6H4-
4-CF3OC6H4-
2-Thienyl-
2-Thiazolyl-
2-Pyridinyl-
12
61
14j
4-CF3C6H4-
4-t-BuC6H4-
2-Furanyl-
92
14k
14l
13j
110
45
13k
13l
14m
14n
14o
14p
14q
14r
14s
14t
14u
14v
2-Thienyl-
22
12
3-Pyridinyl-
4-Pyridinyl-
C6H5CH2-
13m
13n
13o
49
36
8.8
4-FC6H4CH2-
4-ClC6H4CH2-
3-MeOC6H4CH2-
4-MeOC6H4CH2-
3,4-(MeO)2C6H3CH2-
2-ThienylCH2-
21
23
a Inhibition of [125I]-NDP-MSH binding to the human melanocortin-4
receptor stably expressed in HEK293 cells, and as radioligand in the
competitive binding assay. Data are average of two to three inde-
pendent measurements.
b Stimulation of cAMP levels in the same cell line at 10 lM ligand
concentration, in comparison with that of a-MSH.
7.6
4.2
18
8.4
a See footnotes of Table 1.

J. A. Tran et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3434–3438
3437
binding,10c and appear to indicate that the phenethyl-
amino side chain in these two series binds to the same
site of the receptor. Therefore, the role of cyclohexyl
moiety of 13 is similar to that of the phenyl group of
4, even though 13 exhibited about 10-fold reduced affin-
ity. We have shown that the basic nitrogen of the benzyl-
amine is very important for high affinity binding, and
this seems to agree with the results from a series of ben-
zoylamides 14a–k (Table 2). Thus, the benzoyl analogue
14a (Ki = 220 nM), having a similar binding affinity to
the TFA intermediate 10 (Ki = 490 nM), was much less
potent than the corresponding benzyl derivative 13f
(Ki = 18 nM). While these data might suggest an impor-
tant role of the basic nitrogen, the 12-fold reduction in
binding for the amide 14a could be the result of a con-
formational change induced by the carbonyl group,
since substitution on the phenyl ring had little effect in
binding (14b–k), and heteroaromatic derivatives exhib-
ited similar potency (14l–o). These results actually point
out that this aromatic ring is no longer in place to inter-
act with the receptor. Replacing the benzoyl moiety with
a phenylacetyl group, however, resulted in over a 20-fold
increase in potency (14p, Ki = 8.8 nM). Based on the
limited data, the substitution at the phenyl ring had no
significant effect (14q–u). Thus, thienylacetyl compound
14v had a Ki value of 8.4 nM, and the 4-methoxyphenyl-
acetyl compound (14t) possessed a Ki of 4.2 nM.
Replacement of the phenylacetyl amide with
a
phenylurea resulted in reduction of potency for these
compounds (15a–f, Table 3). Thus, the urea 15a
(Ki = 210 nM) was about 10-fold less potent than the
acetyl analogue 14q (Ki = 21 nM). This set of data
clearly showed that an electron-donating substituent at
the phenyl ring was favored. Thus, the 4-methoxyphenyl
compound 15c (Ki = 36 nM) was about 15-fold better
than the 4-nitrophenyl analogue 15e (Ki = 540 nM) in
binding affinity.
In this study, we demonstrated that, for this series of
cyclohexylpiperidines bearing a functional side chain,
an aromatic group and the linker connecting the nitro-
gen to this aromatic ring are important for receptor
binding. The basic nitrogen could be replaced by an
amide moiety, which presumably forms hydrogen-bond-
ing interaction with an acidic residue of the MC4
receptor.
Further evaluation of selected compounds from this ser-
ies demonstrated that these compounds were MC4R-se-
lective. They were at least 80-fold selective over MC1R,
MC3R, and MC5R (Table 4). For example, 14t pos-
sessed Ki values of 5700, 1100, 4.2, and 540 nM at the
MC1R, MC3R, MC4R, and MC5R, respectively. These
compounds displayed similar affinity in displacement of
both NDP-MSH and AgRP binding (Ki = 2.8 nM for
14t) to the MC4 receptor (Table 4). None of the com-
pounds were able to stimulate significant level of cAMP
release at 10 lM concentration (less than 12% of the a-
MSH level), suggesting they were not efficacious ago-
nists. Instead, selected compounds were demonstrated
to be potent antagonists in dose-dependent inhibition
of a-MSH-stimulated cAMP release (Table 4). For
example, 14t exhibited an IC50 value of 150 nM in this
cAMP assay.
Table 3. SAR of ureas 15a–f at hMC4Ra
Compound R1
Ki (nM) Stimulation (%)
15a
15b
15c
15d
15e
15f
4-FC6H4-
4-ClC6H4-
210
420
36
5.5
5.9
1.0
2.7
4-MeOC6H4-
4-(Me2N)C6H4-
4-NO2C6H4-
In conclusion, a series of cyclohexylpiperidines bearing
an amine or amide side chain was synthesized and found
to possess potent antagonist activity at the human mel-
anocortin-4 receptor. Results from this study indicate
that the phenyl ring of the benzylamine moiety of 4
62
540
11
12
4-(MeOCO)C6H4- 260
a See footnotes of Table 1.
Table 4. Binding affinity (Ki, nM) at the melanocortin receptor subtypes and functional activity at MC4R of selected compoundsa
Compound
MC1R
MC3R
MC5R
MC4R
MC4Rb
IC50 (nM)c
13e
13g
13m
14p
14s
14t
(44%)d
(45%)d
4500
3200
2300
2400
710
1200
1400
1900
920
11
10
NDe
13
430
760
12
NDe
6.4
NDe
260
(47%)d
8.8
7.6
4.2
8.4
2800
5700
650
470
540
NDe
2.8
NDe
NDe
150
1100
1100
14v
4600
480
NDe
a Human melanocortin-1, -3, -4, and -5 receptors stably expressed in HEK293 cells, and [125I]-NDP-MSH as radioligand in the competitive binding
assay.
b
[
125I]-AgRP(83–132) as radioligand in the competitive binding assay.
c Dose-dependent inhibition of cAMP production stimulated by 10 nM a-MSH in the same cell line.
d Percentage of inhibition at 10 lM concentration.
e ND—not determined.

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Stearns, R. A.; Miller, R. R.; Tamvakopoulos, C.; 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. T.; Patchett, A. A.; Nargund, R. P. J. Med. Chem.
2002, 45, 4589.
might interact with lipophilic residues of the receptor
since it is replaceable by a non-aromatic cyclohexyl moi-
ety, which resides in an area near TM-7 based on a
receptor model. These compounds are highly selective
over the human melanocortin-3 receptor, as well as
other melanocortin receptor subtypes.
8. Arasasingham, P. A.; Fotsch, C.; Ouyang, X.; Norman,
M. H.; Kelly, M. G.; Stark, K. L.; Karbon, B.; Hale, C.;
Baumgartner, J. W.; Zambrano, M.; Cheetham, J.;
Tamayo, N. A. J. Med. Chem. 2003, 46, 9.
References and notes
9. (a) Vos, T. J.; Caracoti, A.; Che, J.; Dai, M.; Farrer, C.
A.; Forsyth, N. E.; Drabic, S. V.; Horlick, R. A.;
Lamppu, D.; Yowe, D. L.; Balani, S.; Li, P.; Zeng, H.;
Joseph, I. B. J. K.; Rodriguez, L. E.; Claiborne, C. F. J.
Med. Chem. 2004, 47, 1602; (b) Marsilje, T. H.; Roses,
J. B.; Calderwood, E. F.; Stroud, S. G.; Forsyth, N. E.;
Blackburn, C.; Yowe, D. L.; Miao, W.; Drabic, S. V.;
Bohane, M. D.; Daniels, J. S.; Li, P.; Wu, L.; Patanea,
M. A.; Claiborne, C. F. Bioorg. Med. Chem. Lett. 2004,
14, 3721.
10. (a) Dyck, B.; Parker, J.; Phillips, T.; Carter, L.; Murphy,
B.; Summers, R.; Hermann, J.; Baker, T.; Cismowski, M.;
Saunders, J.; Goodfellow, V. Bioorg. Med. Chem. Lett.
2003, 13, 3793; (b) Pontillo, J.; Tran, J. A.; Arellano, M.;
Fleck, B. A.; Huntley, R.; Marinkovic, D.; Lanier, M.;
Nelson, J.; Parker, J.; Saunders, J.; Tucci, F. C.; Jiang, W.;
Chen, C. W.; White, N. S.; Foster, A.; Chen, C. Bioorg.
Med. Chem. Lett. 2004, 14, 4417; (c) Pontillo, J.; Tran, J.
A.; Fleck, B. A.; Marinkovic, D.; Arellano, M.; Tucci, F.
C.; Lanier, M.; Nelson, J.; Parker, J.; Sauders, J.; Murphy,
B.; Foster, A. C.; Chen, C. Bioorg. Med. Chem. Lett. 2004,
14, 5605.
11. Chen, C.; Pontillo, J.; Fleck, B. A.; Gao, Y.; Wen, J.;
Tran, J. A.; Tucci, F. C.; Marinkovic, D.; Foster, A. C.;
Saunders, J. J. Med. Chem. 2005, 47, 6821.
12. Several phenylpiperazines bearing an amide side chain
have been reported to be potent and selective antagonists
of the melanocortin-4 receptor. See: Xi, N.; Hale, C.;
Kelly, M. G.; Norman, M. H.; Stec, M.; Xu, S.;
Baumgartner, J. W.; Fotsch, C. Bioorg. Med. Chem. Lett.
2004, 14, 377.
1. (a) Gantz, I.; Fong, T. M. Am. J. Physiol. Endocrinol.
Metab. 2003, 284, E468; (b) Goodfellow, V.; Saunders, J.
Curr. Top Med. Chem. 2003, 3, 855.
2. Adage, T.; Scheurink, J. W.; de Boer, S. F.; de Vries, K.;
Konsman, J. P.; Kuipers, F.; Adan, R. A. H.; Baskin, D. G.;
Schartz, M. W.; Van Dijk, G. Neuroreport 2001, 12, 1281.
3. (a) Foster, A. C.; Joppa, M.; Markinson, S.; Gogas, K. R.;
Fleck, B. A.; Murphy, B. J.; Wolff, M.; Cismowski, M. J.;
Ling, N.; Goodfellow, V. S.; Chen, C.; Saunders, J.;
Conlon, P. J. Ann. NY Acad. Sci. 2003, 994, 103; (b)
Schioth, H. B.; Kask, A.; Mutulis, F.; Muceniece, R.;
Mutule, I.; Mutule, I.; Mandrika, I.; Wikberg, J. E. S.
Biochem. Biophys. Res. Commun. 2003, 301, 399.
4. (a) Marks, D. L.; Ling, N.; Cone, R. Cancer Res. 2001, 61,
1432; (b) Wisse, B. E.; Frayo, R. S.; Schwartz, M. W.;
Cummings, D. E. Endocrinology 2001, 142, 3292.
5. (a) Chaki, S.; Ogawa, S.; Todaa, Y.; Funakoshi, T.;
Okuyama, S. Eur. J. Pharmacol. 2003, 474, 95; (b) Chaki,
S.; Hirota, S.; Funakoshi, T.; Suzuki, Y.; Suetake, S.;
Okubo, T.; Ishii, T.; Nakazato, A.; Okuyama, S. J.
Pharmacol. Exp. Ther. 2003, 304, 818; (c) Nakazato, A.,
Ishii, T., Nozawa, S., Tomisawa, K., Okubo, T., Ueki, T.,
Okuyama, S., Chaki, S. Book of Abstracts, 228th Amer-
ican Chemical Society National Meeting, August 22–26,
2004, Philadelphia, PA; MEDI055.
6. (a) Bellasio, S.; Nicolussi, E.; Bertorelli, R.; Reggiani, A.
Eur. J. Pharmacol. 2003, 482, 127; (b) Beltramo, M.;
Campanella, M.; Tarozzo, G.; Fredduzzi, S.; Corradini,
L.; Forlani, A.; Bertorelli, R.; Reggiani, A. Mol. Brain
Res. 2003, 118, 111; (c) Bertorelli, R.; Fredduzzi, S.;
Tarozzo, G.; Campanella, M.; Grundy, R.; Beltramo, M.;
Reggiani, A. Behav. Brain Res. 2005, 157, 55.
13. For experimental details, see: Dyck, B. P., Goodfellow, V.,
Phillips, T., Parker, J., Zhang, X., Chen, C., Tran, J. A.,
Pontillo, J., Tucci, F. C. WO 03/031410, 2003.
7. Sebhat, I. K.; Martin, W. J.; Ye, Z.; Barakat, K.; Mosley,
R. T.; Johnston, D. B. R.; Bakshi, R.; Palucki, B.;
Weinberg, D. H.; MacNeil, T.; Kalyani, R. N.; Tang, R.;
14. Nickolls, S. A.; Cismowski, M. I.; Wang, X.; Wolff, M.;
Conlon, P. J.; Maki, R. A. J. Pharmacol. Exp. Ther. 2003,
304, 1217.