Piperazinebenzylamines as potent and selective antagonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2004.08.055

SAR studies of a series of piperazinebenzylamines resulted in the discovery of potent antagonists of the human melanocortin-4 receptor. Compounds 11c, 11d, and 11l, which had K_i values of 21, 14, and 15 nM, respectively, possessed low efficacy

Full text of DOI:10.1016/j.bmcl.2004.08.055

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5605–5609
Piperazinebenzylamines as potent and selective antagonists of
the human melanocortin-4 receptor
Joseph Pontillo,a Joseph A. Tran,a Beth A. Fleck,b Dragan Marinkovic,a
Melissa Arellano,a Fabio C. Tucci,a Marion Lanier,a Jodie Nelson,a Jessica Parker,a
John Saunders,a Brian Murphy,b 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 June 2004; revised 20 August 2004; accepted 26 August 2004
Available online 18 September 2004
Abstract—SAR studies of a series of piperazinebenzylamines resulted in the discovery of potent antagonists of the human melano-
cortin-4 receptor. Compounds 11c, 11d, and 11l, which had Ki values of 21, 14, and 15nM, respectively, possessed low efficacy in
cAMP stimulation (ꢀ15% of a-MSH maximal level) mediated by MC4R, and functioned as antagonists in inhibition of a-MSH-
stimulated cAMP release in a dose-dependent manner (11l, IC50 = 36nM).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
(2) binds to the human MC4 receptor with high affinity
(Ki = 7.9nM), exhibiting both anxiolytic-like and
antidepressant-like activity in several animal models.6b
A recent publication demonstrated that an MC4R
Five subtypes of melanocortin receptors, MC1-5R,
members of the class A G-protein-coupled receptor
(GPCR) superfamily,1 are associated with various bio-
logical activities.2 The melanocortin peptides, the natu-
ral ligands for the MCRs, consist of the melanotropins
a-MSH, b-MSH, and c-MSH, and the adrenocorticot-
ropin ACTH. In addition to these agonists, two endog-
enous antagonists, agouti protein, and agouti related
protein (AgRP) have been identified. Because of the
importance of the MC4 receptor in feeding behavior,
metabolism, and energy homeostasis, selective MC4R
agonists or antagonists may be useful in treatment of
human diseases including obesity and cachexia.3 While
many synthetic peptide ligands have been known for
decades,4 small molecule agonists5 and antagonists6 of
the melanocortin-4 receptor have been discovered more
recently. Compound 1, first reported to be an inhibitor
of AgRP binding to the MC4 receptor (IC50 = 52nM),
also exhibits moderate inhibition (IC50 = 220nM) of
NDP-MSH binding and reduces a-MSH-stimulated
cAMP production at high concentration.6a MCL0129
antagonist, compound
3 (Ki = 160nM), protected
against tumor-induced weight loss in mice following
peripheral administration.6c SAR studies conducted on
a related series (compound 4, Ki = 180nM) have also
been reported (Fig. 1).6d
Previously we described a series of substituted phenyl-
piperazines (e.g., 5c, EC50 = 3.8nM),7 derived from an
earlier lead that acts as a potent MC4R agonist (5a,
EC50 = 80nM).5e Herein we report our continued efforts
on the optimization of this series of compounds, with
the goal of developing potent and selective antagonists
of the human MC4 receptor.
2. Chemistry
The synthesis (Scheme 1) started from piperazinebenzal-
dehyde 6, which was subjected to a reductive amination
with 2-thienylethylamine and sodium triacetoxyborohy-
dride. The resulting secondary amine was protected with
a Boc-group, generating intermediate 8 with an overall
yield of 82%. Deprotection of 8 by palladium-catalyzed
hydrogenation provided a free amine, which was
Keywords: Melanocortin; MC4; Antagonist; Piperazinebenzylamine.
*
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7619; e-mail: cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.055

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J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5605–5609
F
F
Br
O
N
N
N
NH
N
N
N
N
N
N
F
O
O
3 (ML253764)
1
2 (MCL0129)
Cl
N
N
5a: R =
5b: R =
5c: R =
N
R
N
Br
O
N
O
N
N
S
N
H
N
Cl
H
O
HN
NH
N
H
4
5a-c
Figure 1. Some small molecule MC4 antagonists.
c
b
a
N
N
N
N
1
NCbz
Boc
NCbz
NCbz
N
R
O
HN
S
N
S
HN
S
O
6
8
9,10
7
d
Cl
Cl
e
N
N
Boc
N
N
N
S
NHFMOC
HN
S
NH
O
O
2
O
R
11
15
Scheme 1. Reagents and conditions: (a) 2-thienylethylamine/NaBH(OAc)3/CH2Cl2; (b) (Boc)2O, 82%, two steps; (c) i. H2/Pd–C, 36%, ii. R1COOH/
EDC/HOBt, iii. TFA/CH2Cl2; (d) H2/Pd–C, 36%, then N-FMOC-D-(4-Cl)Phe-OH/EDC/HOBt, 93%; (e) i. Et2NH/CH2Cl2, 94%, ii. R2COOH/EDC/
HOBt, (iii) TFA/CH2Cl2.
coupled under the standard peptide coupling conditions
(EDC/HOBt) with a various phenylacetic or phenyl-
propionic acids to afford the desired products 9 or 10,
after deprotection with TFA. Compound 8 was also
coupled with N-Fmoc-D-(4-Cl)Phe-OH to give 15 in
93% yield. After deprotection with diethylamine, the
free amine derived from 15 was coupled with various
carboxylic acids, including N-Boc-amino acids to give
the final compounds 11, after TFA-deprotection.
The 2-piperazinephenyl aldehyde and ketones 17
(Scheme 2) were obtained from 16. A coupling reaction
of the free amine of 17 (obtained by TFA-deprotection)
with the dipeptide 19 under standard conditions (EDC/
HOBt) afforded the ketones 18. Reductive aminations of
18 with various amines gave the desired products
12–14. A pair of diastereoisomers could be detected dur-
ing HPLC purification in some cases but were not
separated.
Cl
Cl
b
a
c
F
N
N
3
N
3
4
NBoc
N
R
3
N
3
O
R
O
R
N
R
NH
O
R
NH
5
O
O
R
O
NH
O
NHBoc
2
16a: R3 = H
16b: R3 = Me
16c: R3 = Et
17
12-14
18
Scheme 2. Reagents and conditions: (a) N-Boc-piperazine/DMF/120°C; (b) i. TFA/CH2Cl2, ii. N-Boc-b-Ala-D-(4-Cl)Phe-OH (19)/EDC/HOBt/
DCM; (c) i. R4R5NH/NaBH(OAc)3/CH2Cl2 (for 18a) or R4R5NH/TiCl4/NaBH4/CH2Cl2 (for 18b,c), ii. TFA/CH2Cl2.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5605–5609
5607
3. Results and discussion
hydroisoquinoline-3-carbonyl (Tic) group of 5b resulted
in 50-fold loss in binding (10a, Ki = 600nM). All varia-
tions of 10a gave analogs (10b–h) with similar or lower
affinity. None of the phenylpropionyl analogs other
than 10a (IA = 85%, EC50 = 8.7lM) were able to stimu-
late cAMP release to 50% of the a-MSH level at 10lM
concentration. While these results suggest the Tic group
is very important for receptor binding, the role of its
amide functionality is not clear at this point.
The competition binding experiments, functional antag-
onist and agonist assays were performed using HEK293
cells stably transfected with the human melanocortin-4
receptor as previously described.8
[
125I]-NDP-MSH
was used as radiolabeled ligand in the binding assay.
a-MSH was used to stimulate cAMP release in the func-
tional antagonist assay, and as a standard for calcula-
tion of intrinsic activity in the agonist assay.
We then examined a series of amide derivatives of 4-
chlorophenylalanine. The results are summarized in
Table 2. The binding affinity of the glycine derivative
11a (Ki = 44nM) increased 15-fold from 10a, and was
comparable to 5b. This compound still stimulated a sig-
nificant level of cAMP production (EC50 = 580nM,
IA = 77%). The b-alanine analog 11c, however, exhib-
ited slightly better binding affinity (Ki = 21nM) than
11a but much lower level of cAMP stimulation (14%
at 10lM concentration). The c-aminopropionyl analog
The observation that most reported MC4 ligands with
good binding affinity possess a 4-chlorophenyl group
led to the examination of chlorophenyl derivatives of
compounds 9 and 10 (Table 1). The substituted 4-chlo-
rophenylacetyl derivatives (9a–d) exhibited poor binding
affinity (Ki > 1.8lM). Deletion of the (R)-1,2,3,4-tetra-
Table 1. SAR of the chlorophenyl carboxylic derivatives 9–10 at the
hMC4Ra
Compound
R1
Ki (nM)b
Table 2. SAR of amides of (R)-(4-chloro)phenylalanine 11a
5b
12
Cl
9a
1800
Compound
R2
Ki (nM)a
EC50, nM (IA, %)b
NH2
OH
5b
12
320 (100)
Cl
HN
9b
9c
>10,000
>10,000
11a
11b
NH2
44
38
580 (77)
Cl
Cl
1400 (45)
N
11c
11d
21
14
(14)
(15)
NH2
NH2
9d
3800
NH2
11e
11f
66
30
1500 (80)
2600 (45)
NH2
10a
10b
600c
3900
NH2
Cl
NH2
11g
11h
11i
11j
40
46
48
50
1800 (57)
(12)
N
H
Cl
NH2
NH2
10c
10d
1000
5400
Cl
NH2
1627 (47)
1900 (56)
HN
HN
Cl
NH2
10e
10f
6400
1000
11k
75
1100 (60)
Cl
Cl
HN
NH
NH
11l
15
26
(12)
(39)
H
N
11m
S
CF3
10g
410
O
O
Cl
Cl
O
NH2
11n
11o
70
41
(16)
(5)
O
10h
290
O
N
a Human melanocortin-4 receptor stably expressed in HEK 293 cells,
and [125I]-NDP-MSH as radioligand. Data are average of two or
more independent measurements.
a Human melanocortin-4 receptor stably expressed in HEK 293 cells,
and [125I]-NDP-MSH as radioligand. Data are average of three or
more independent measurements.
b Intrinsic activity: percentage of a-MSH-stimulated cAMP level at
10lM concentration.
b Less than 45% of a-MSH-stimulated cAMP level at 10lM
concentration.
c EC50 = 8.7lM, IA = 85%.

5608
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5605–5609
11d had similar profile to 11c. As a matter of fact, many
amide derivatives with a small aminoalkyl group exhib-
ited similar levels of affinities, however, a-amino amides
possessed higher levels of cAMP stimulation (11a, 11e,
and 11k, IA P 60%) than other amide analogs (11c
and 11l, IA 6 14%). The piperizin-3-yl compound 11l
possessed a Ki value of 15nM. These results suggest that
the tetrahydroisoquinoline moiety of the Tic group is
critical for receptor activation, but less important for
receptor binding.
We also examined the effect of the side-chain at the benz-
ylamino group while the b-Ala-D-(4-Cl)Phe-group was
held constant (12–14, Table 3). Previously we found that
incorporation of a methyl group at the a-position of the
benzylamine of 5 increased binding affinity in most
cases.7 However, either N-methylation or a-alkylation
of 11c resulted in a decrease in affinity (12: Ki = 220nM,
13a: Ki = 110nM). Many other alkylamine derivatives
(13d–i) exhibited similar or lower binding affinities.
Diamines such as 13h from this set had poor cAMP
stimulation. Interestingly, the 2-aminobenzyl analog
13c (Ki = 120nM, EC50 = 364nM, IA = 100%) pos-
Figure 2. Dose response curves of a-MSH-stimulated cAMP produc-
tion in HEK 293 cells expressing the human MC4 receptor, and
inhibition by compound 11l.
Table 4. Selectivity and functional antagonist potency of 11c, 11d, and
11la
Compound
Binding affinities (Ki, nM)
MC1 MC3 MC4 MC5 IC50 (nM)
(50%)c 1900
MC4 functionb
d
11a
11c
11d
11l
44
21
14
15
41
990
1000
1000
720
1300
1200
2300
1000
1400
1400
90
66
36
Table 3. SAR of the substitution at the benzylamines 12–14a
Compound
R3
R4NR5
Ki (nM)
EC50, nM
(IA, %)b
11o
(23%)c 1800
730
880
a Human melanocortin receptors stably expressed in HEK 293 cells,
and [125I]-NDP-MSH as radioligand. Data are average of two or
more independent measurements.
b Inhibition of a-MSH-stimulated cAMP production.
c Percentage of inhibition at 10lM concentration.
d Agonist with an EC50 value of 580nM (IA = 77%).
H
N
11c
12
H
H
21
(14)
(32)
S
N
220
S
H
N
13a
13b
Me
Me
110
140
(32)
S
sessed high efficacy and moderate agonist potency in
stimulation of cAMP release.
1872 (99)
O
NH
N
Selected compounds were tested and found to be func-
tional antagonists in dose-dependent inhibition of a-
MSH-stimulated cAMP production in HEK 293 cells
expressing the MC4 receptor. For example, 11l had an
IC50 value of 36nM in this functional assay (Fig. 2).
This compound also exhibited good selectivity over
other melanocortin subtypes. It possessed Ki values of
2.3, 1.4, and 0.72lM at the human MC1, MC3, and
MC5 receptors, respectively. Selectivity and functional
antagonism data of 11a, 11c–d, 11l, and 11o are summa-
rized in Table 4.
13c
Me
120
364 (100)
H
H2N
H
N
13d
13e
Me
Me
470
290
2717 (56)
(22)
H2N
H
N
N
H
13f
Me
Me
N
N
N
430
86
(35)
13g
N
471 (59)
H2N
4. Conclusion
13h
13i
14
Me
Me
Et
150
210
140
590 (64)
(41)
N
H
In summary, a series of piperazinebenzylamines bearing
the (R)-(4-Cl)Phe moiety were synthesized and tested as
melanocortin-4 ligands. Structure–activity relationship
studies of the (R)-(4-Cl)-Phe amides revealed several po-
tent MC4 antagonists such as 11c (Ki = 21nM) and 11l
(Ki = 15nM). The replacement of the Tic group of an
early MC4 ligand 5b with a small aminoalkyl carbox-
amide such as b-analine diminished its ability to stimu-
late cAMP production in cells expressing the human
MC4 receptor, but retained binding affinity to this
N
HN
H
N
(29)
S
a Human melanocortin-4 receptor stably expressed in HEK 293 cells,
and [125I]-NDP-MSH as radioligand.
b Intrinsic activity: percentage of a-MSH-stimulated cAMP level at
10lM concentration.

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