Synthesis of novel melanocortin 4 receptor agonists and antagonists containing a succinamide core

Article abstract of DOI:10.1016/j.bmcl.2003.10.056

A novel series of piperazines appended to a succinamide backbone were synthesized and found to have a high affinity for the melanocortin-4 receptor (IC₅₀s ranging from <0.1 to 200 nM). Both agonists and antagonists of MC4R were prepared by modi

Full text of DOI:10.1016/j.bmcl.2003.10.056

Bioorganic & Medicinal Chemistry Letters 14 (2004) 377–381
Synthesis of novel melanocortin 4 receptor agonists and
antagonists containing a succinamide core
Ning Xi,a,* Clarence Hale,b Michael G. Kelly,a,y Mark H. Norman,a Markian Stec,a
Shimin Xu,a James W. Baumgartnerb andChristopher Fotsch a
aDepartment of Chemistry Research & Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
bDepartment of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
Received21 August 2003; revised17 October 2003; accepted31 October 2003
Abstract—A novel series of piperazines appended to a succinamide backbone were synthesized and found to have a high affinity for
the melanocortin-4 receptor (IC50s ranging from <0.1 to 200 nM). Both agonists andantagonists of MC4R were preparedby
modifying the groups attached to the right-hand side of the succinamide. This series also exhibits a high level of selectivity (up to
7000-fold) over mouse MC1R and human MC3R.
# 2003 Elsevier Ltd. All rights reserved.
The five melanocortin receptor subtypes (MC1–MC5R)
that have been identified and cloned1 belong to the
superfamily of seven transmembrane-spanning G-pro-
tein-coupledreceptors (GPCRs). These receptors are
activated by peptides derived from the prohormone
proopiomelanocortin (POMC), such as a-, b-, and g-
melanocyte-stimulating hormones (MSH) andadreno-
corticotropic hormome (ACTH).2 The MCR subtypes
differ in their tissue distribution and ligand binding
specificity. One of the receptor subtypes, MC4R, is
expressedin the hypothalamus, andis believedto play a
sequence ‘His-Phe-Arg-Trp’ foundin peptide ligands
for the MCRs.11 In the course of our work, we also
identified a small molecule agonist of MC4R that con-
tainedthe piperazine anda
p-chloro-d-phenylalanine
(e.g., compound 3 in Fig. 2).12 We searchedfor alter-
natives to the d-phenylalanine subunit to expandthe
scope of these piperazine analogues. To that end, we
considered compounds with the succinamide core (see
14a in Fig. 2). Compound 14a has the same connectivity
as compound 3 except that the amide is reversed on the
right side of the compound. The result of this change is
that the NH on p-chloro-d-phenylalanine is replaced
with a CH2 group, but an amide carbonyl is still pre-
sent. Herein, we describe the synthesis of these succina-
mides and their activities toward melanocortin
receptors.
3
role in controlling foodintake andenergy homeostasis.
MC4R has also been implicatedin normal stimulation
of sexual arousal.4 Therefore, small molecule agonist of
MC4R may be useful for the treatment of diseases and
disorders, such as obesity and sexual dysfunction.5
Much of the research on MC4R agonists has focusedon
peptide analogues of the endogenous ligands (e.g., a-
MSH), such as cyclic lactam MTII.6 More recently,
however, many groups have identified agonists contain-
ing the piperidine and piperazine cores7,8 (see, for
example, 19 and 210 in Fig. 1). A component that many
of these agonists share is a central phenylalanine, which
may mimic the same amino acidin the message
The construction of the chiral succinic acidwas
achievedusing Evan’s chiral oxazolidinone as outlined
* Corresponding author. Tel.: +1-805-447-2285; fax: +1-805-480-
3016; e-mail: nxi@amgen.com
y
Current address: Renovis, 270 Littlefield Avenue, South San Fran-
cisco, CA 94080, USA.
Figure 1. Examples of non-peptide MCR agonists.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.056

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N. Xi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 377–381
Figure 2. Concept for replacing the p-chloro-d-phenylalanine on
compound 3 with a succinamide backbone.
Scheme 2. (a) NaH, DMF, cyclopropyl methyl bromide, 90%; (b) H2,
Pd/C in EtOAc, 100%; (c) EDC, HOAt, Et3N, CH2Cl2, 90%; (d)
TFA/CH2Cl2, 100%; (e) (i) 4-Boc-piperazine, EDC, HOAt, CH2Cl2;
(ii) TFA/CH2Cl2; (iii) when X=H2, R1CHO, NaBH3CN, AcOH,
MeOH; when X=O, R1(C¼O)Cl, Et3N, CHCl3.
Scheme 1. (a) THF, À78 ꢀC, 70%; (b) KHMDS, BrCH2CO2t-Bu,
THF, À78 ꢀC, 75%; (c) (i) LiOH–H2O, H2O2, THF, 0 ꢀC; (ii) 2 N HCl,
80%.
in Scheme 1.13 The imide 6, acquiredthrough coupling
of acidchloride 5 andlithium salt of ( R)-4-benzyl-2-
oxazolidinone (4), was alkylatedwith t-butyl bromo-
acetate to affordsuccinamide 7. The chiral auxiliary was
removed under standard condition to furnish acid 8.
The absolute configuration of this key intermediate was
Scheme 3. (a) EtNH2, NaBH4, MeOH, 70%; (b) (i) Ac2O, Et3N,
CH2Cl2, 99%; (ii) satdHCl in EtOAc, 100%; (c) EDC, HOAt, Et 3N,
CH2Cl2; (d) TFA/CH2Cl2; (e) R1R2NH, EDC, HOAt, CH2Cl2.
14
confirmedby X-ray crystallography.
The preparation of the final products is illustrated in
Schemes 2 and3 . Sulfonamide 915 was alkylatedwith
bromomethyl cyclopropane to furnish compound 10.
Removal of benzyl group under hydrogenation condi-
tions ledto piperazine 11. Coupling of 11 with succinic
acid 8 provided amide 12, which was deprotected to
furnish acid 13. This compoundwas coupledwith 4-
Boc-piperazine, which following deprotection, was
either substituted with aldehydes in a reductive amina-
tion step to give 14, where X=H2, or acylatedwith acid
chlorides to give 14, where X=O (see Table 1 for spe-
cific examples).
with 19 to provide amides 20 (see Table 2 for specific
examples).
All analogues were testedin binidng andfunctional
assays against the human MC4R by the methoddescri-
17
bedpreviously,
andthe results are summarizedin
Tables 1 and2 . We first examinedthe SAR of succina-
mides containing a six-membered ring that would mimic
the isonipecotic acidfoundon the right-handside of
analogue 3. Succinamde 14a hadan IC 50=220 nM,
which was comparable to the activity of 3 (IC50=130
nM). In the functional assay 14a hadan EC 50=2000
nM, which is in sharp contrast to an EC50 <0.1 nM
(>90% activation) measuredfor analogue 3. The dis-
parity in functional activities between 14a and 3 sug-
gests that the NH d-phenylalanine portion of 3 makes
significant contribution in activating the receptor for
this class of compounds.
The acetamide analogues 20 were preparedin the simi-
lar fashion (Scheme 3). Reductive amination of com-
pound 1516 with ethylamine gave compound 16, which
was acetylatedanddeprotectedto affordcompound
17.
Intermediate 19 was preparedfrom 17 using the same
steps as described for the conversion of 11 to 13 in
Scheme 2. Various amines were subsequently coupled

N. Xi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 377–381
379
Table 1. Biological activity of 14
CompdR
MC4R IC
(nM)a
MC4Rb EC50 (nM) (activation)c
50
MTIId
1d
14a
14b
14c
NA
NA
H
Ethyl
n-Propyl
i-Pentyl
1.4ꢂ0.3
66ꢂ16
<0.01
5.2ꢂ1.6 (100%)
2000ꢂ1400 (70%)
>10,000 (0%)
>10,000 (0%)
>10,000 (0%)
>10,000 (0%)
>10,000 (0%)
>10,000 (0%)
>10,000 (0%)
83ꢂ31 (55%)
74ꢂ48 (45%)
220ꢂ10
9.5ꢂ7.3
8.9ꢂ8.8
4.8ꢂ4.3
3.0ꢂ2.9
3.3ꢂ2.4
1.4ꢂ1.3
11ꢂ3
14d
14e
14f
14g
14h
14i
14j
Cyclopropyl-methyl
C(O)OtBu
C(O)OEt
C(O)CH3
C(O)Ph
C(O)CH2tBu
4.4ꢂ1.1
<0.1
NA, not applicable.
a 125I-NDP-a-MSH binding to the melanocortin-4 receptor, SEM of at least two IC50s determined over six dilutions.
b Intracellular levels of cAMP in cells expressing the melanocortin-4 receptor, SEM of two EC50s determined over six dilutions.
c Percentage of maximal response with respect to a-MSH.
d Cyclic lactam MTII andcompound 1 were testedas benchmarks for the assays.
Table 2. Biological activity of 20
a
CompdR
20a
MC4R
IC50 (nM)
MC4Rb EC50 (nM) (activation)c
530ꢂ510 (80%)
9.4ꢂ6.4
20b
19ꢂ2
13ꢂ1
32ꢂ22
5.8ꢂ3.1
2d
340ꢂ240 (90%)
20c
20d
20e
20f
89ꢂ44 (100%)
37ꢂ4 (95%)
15ꢂ6 (100%)
>10,000 (0%)
a 125I-NDP-a-MSH binding to the melanocortin-4 receptor, SEM of at least two IC50s determined over six dilutions.
b Intracellular levels of cAMP in cells expressing the melanocortin-4 receptor, SEM of two EC50s determined over six dilutions.
c Percentage of maximal response with respect to a-MSH.
d One determination
We then examinedderivatives of 14a where the second-
ary amine was substitutedwith alkyl andacyl groups.
Alkyl derivatives (14b–e) all hadbetter binding affinity
(IC50 <10 nM) than compound 14a, but they did not
activate MC4R at 10 mM in the functional assay. Car-
bamate and amide analogues were also prepared, and
they were also more potent (IC50 ꢁ10 nM) than the
parent compound 14a. Apparently the basic nitrogen

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N. Xi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 377–381
foundon
14a–e is not requiredfor high affinity
the known MC4R agonists. We have preparedboth
potent agonists andantagonists for MC4R within this
series by modifying the pendant groups. Compounds
from series 14 have high affinity but are poor activators
of the receptor in the functional assay. Series 20 analo-
gues, on the other hand, have both a high affinity and
high functional activity. Many of the most potent com-
pounds at MC4R exhibited a high degree of selectiv-
ity over mouse MC1R andhuman MC3R, with
selectivity ratios ranging from 40 to 7,000. Unlike many
MC4R agonists or antagonists known in the current
literature, our succinamides lack the central amino acid,
which provides a novel alternative to the known MC4R
agonists.
(IC50<15 nM) since the amides and carbamates, 14f–j,
are just as active. Functional activities were >10,000
nM for analogues 14f–h, but analogues 14i and 14j had
EC50s ꢃ80 nM with ca. 50% activation. The difference
in functional activity is especially interesting for amide
14j andthe relatedcarbamate 14f. The only difference
between these two compounds is a methylene and an
oxygen atom, yet 14j activates the receptor at 50% while
14f does not activate the receptor at 10 mM.
In addition to the arylsulfonamide on the left side of the
succinamide core, we used the tertiary acetamide moi-
ety, which was also employedin our d-phenylalanine
series.12 Rather than trying to mimic the isonipecotic
acid, we selected a more diverse set of amines as
appendages to the succinamide core. (Table 2). Com-
pounds 20a–e have goodbinding affinities at the recep-
tor (IC50<30 nM), but unlike the previously set of
succinamides, 20a–e have robust activation responses in
the functional assay (80–100% of the response produced
by a-MSH). Compounds 20a–e have EC50s between 15
and530 nM andshow little correlation with the binding
activity. These results are consistent with the notion that
binding does not necessarily correlate with functional
efficacy.18 To make a comparison between the two ser-
ies, compound 20f was prepared, which is an analogue
of 14f. Both compounds showed no activation at 10
mM, which suggests that the modifications on the right-
handside of 20a–e were responsible for increasing the
functional activity within this series of compounds.
Apparently, the larger piperazine-Boc group of 20f
binds to MC4R in such a way that it no longer activates
the receptor. Compound 20f illustrates how a subtle
change in structure can adversely affect functional
activity.19
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Selectivity for many of the succinamide analogues were
determined at two receptor sub-types: MC1R (mouse)
andMC3R (human) ( Table 3). Of those compounds
tested, they all showed a high degree of selectivity versus
the other receptor sub-types whether they are from the
sulfonamide series or the tertiary acetamide series.
In summary, we have shown that the succinamide ana-
logues have a high affinity to the MC4R, andare a use-
ful alternative to the d-phenylalanine foundon many of
Table 3. MC1R andMC3R binding assay results for selectedsucci-
namides
CompdMouse MC1R
IC50 (nM)
Human MC3R
IC50 (nM)
Human MC4R
IC50 (nM)
MTII
1
0.06ꢂ0.01
153ꢂ36
44ꢂ3
2900ꢂ400
>8000
>10,000
>3500
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1.4ꢂ0.3
66ꢂ16
220ꢂ10
8.9ꢂ8.8
3.3ꢂ2.4
1.4ꢂ1.3
11ꢂ3
14a
14c
14f
14g
14h
14j
20b
20c
20e
>8000
>10,000
>10,000
>10,000
>10,000
>1800
>1400
>1600
>600
<0.1
19ꢂ2
13ꢂ1
5.8ꢂ3.1

N. Xi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 377–381
381
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ane andits structure was determinedby a Rigaku AFC7R
diffractometer with graphite monochromated Cu-Ka
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monoclinic; lattice type, primitive; lattice parameters:
a=12.429(1) A; b=8.947(1) A; c=14.910(1) A;
b=96.137(7); V=1648.4(3) A3; space group, P21 (#4);
residuals: R, 0.118; Rw, 0.109.
15. For the preparation of compound 9, see: Elliott, J. M.;
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in the structure of their MCR agonist, MTII, leadto loss
in functional activity. The d-phenylalanine on MTII was
replacedwith the bulkier
d-1-napthylalanine, which
resultedin a peptide that was an antagonist (SHU9119):
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14. The single crystal was obtainedfrom 50% EtOAc in hex-