Discovery of (2S)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino] carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl] -4-methyl-2-piperazinecarboxamide (MB243), a potent and selective melanocortin subtype-4 receptor agonist

Article abstract of DOI:10.1016/j.bmcl.2004.10.020

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.

Full text of DOI:10.1016/j.bmcl.2004.10.020

Bioorganic & Medicinal Chemistry Letters 15 (2005) 171–175
Discovery of (2S)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)-
amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-
2-oxoethyl]-4-methyl-2-piperazinecarboxamide (MB243), a
potent and selective melanocortin subtype-4 receptor agonist
Brenda L. Palucki,a,* Min K. Park,a Ravi P. Nargund,a Zhixiong Ye,a Iyassu K. Sebhat,a
Patrick G. Pollard,a Rubana N. Kalyani,b Rui Tang,b Tanya MacNeil,b
David H. Weinberg,b Aurawan Vongs,b Charles I. Rosenblum,b George A. Doss,c
Randall R. Miller,c Ralph A. Stearns,c Qianping Peng,c Constantin Tamvakopoulos,c
Erin McGowan,d William J. Martin,d Joseph M. Metzger,d Cherrie A. Shepherd,d
Alison M. Strack,d D. Euan MacIntyre,d Lex H. T. Van der Ploegb and Arthur A. Patchetta
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
bDepartment of Obesity Research, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
dDepartment of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
Received 31 August 2004; revised 5 October 2004; accepted 5 October 2004
Available online 28 October 2004
Abstract—We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the
melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.
Ó 2004 Elsevier Ltd. All rights reserved.
The five cloned melanocortin receptor subtypes (MC1R-
MC5R) are part of a family of seven-transmembrane
G-protein-coupled receptors. These receptors, which
are located peripherally and centrally, interact with their
endogenous ligands, the melanocortins and corticotro-
pins, to regulate a diverse number of physiological func-
tions, including the control of feeding and sexual
behavior, as well as skin pigmentation, steroidogenesis,
energy metabolism, and exocrine secretion.1 The melano-
cortin subtype-4 receptor (MC4R) is expressed in
the hypothalamus, and both rodent and human genetic
and pharmacological data indicate that inactivation of
this receptor results in obesity.2,3 In addition, rodent
and human pharmacological data indicates that activa-
tion of MC4R modulates erectile function and sexual
behavior.4,5 Consequently, over the past few years,
research efforts in drug discovery groups have focused
on identifying novel MC4R agonists for the potential
treatment of obesity and sexual dysfunction.6
Numerous reports on the identification of structurally
related small-molecule MC4R agonists have emerged
in the literature7,8 as well as structurally distinct
MC4R agonists.9–11 Recent efforts at Merck have lead
to the identification of 1, a potent and selective, small-
molecule agonist of the MC4R. Indeed, 1 was shown
to stimulate erectile activity as well as reduce food intake
in rats.12
Lead compound 1 proved to be a useful tool for study-
ing the pharmacology of an MC4R selective agonist;
however, issues with off-target activity prevented further
development of this compound. We speculated that the
triazole and tetrahydroisoquinoline moieties were con-
tributing to the undesirable off-target activity. Conse-
quently, efforts were focused on identifying suitable
chemical subunits, which can replace the triazole and
tetrahydroisoquinoline moieties to minimize off-target
Keywords: Melanocortin; Obesity; Sexual dysfunction.
*
Corresponding author. Tel.: +1 732 594 6188; fax: +1 732 594
5966; e-mail: brenda_palucki@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.020

172
B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 171–175
activity while maintaining potency. Structure–activity
studies led to the discovery that a t-butylamide group
can replace the triazole,13 and a piperazine can substi-
tute the tetrahydroisoquinoline side chain. In addition,
replacement of the p-chloro-D-phenylalanine amino acid
with a p-fluoro-D-phenylalanine amino acid led to
improved functional potency at the MC4R.14 Herein,
we report the synthesis and evaluation of MC4R agon-
ists containing a piperazine side chain as alternatives to
the tetrahydroisoquinoline moiety. These efforts lead to
the discovery of a potent and selective MC4R agonist,
MB243, which is efficacious in our animal models for
both food intake and erectile dysfunction.
NCbz
O
NH
O
BocN
HN
N
• 2 HCl
a ,b, c, d
O
O
HN
N
N
NH
NH
O
O
F
F
7
12
c
NH
O
NR
O
BocN
HN
HN
HN
• 2 TFA
e or f, a
O
O
N
N
NH
NH
O
O
13 R = Et
14 R = CH2CHF2
15 R = i-Pr
16 R = D-Ala
8
F
F
N
HN
HN
HN
HN
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2; (b) HCHO,
NaBH3CN, NaOAc, MeOH; (c) H2, Pd/C, EtOH; (d) HCl/Et2O,
CH2Cl2; (e) acetaldehyde, or difluoroacetaldehyde ethyl hemiacetal, or
acetone, NaBH3CN, TFA, NaOAc, MeOH; (f) N-Boc-D-Ala, EDC,
HOBt, DIEA, CH2Cl2.
O
O
O
N
N
N
NH
N
N
O
O
Cl
F
1
MB243
acid 4-methylbenzenesulfonate, 3, Boc-protection of the
secondary amine followed by reduction using Rh/Al2O3
as catalyst provided piperidine 4. The carboxylic acid
was then converted to the tert-butylamide followed by
deprotection of the amine to yield piperidine 5. Piper-
idine 5 was coupled to N-Boc-p-fluoro-(D or L)-phenyl-
alanine by standard procedures. Removal of the
Boc-protecting group provided the free amine, which
was then coupled with 1-Boc-4-Cbz-piperazine-2-(S or
R)-carboxylic acid to afford dipeptide 7. Sequential
amine deprotections yielded piperazine 9. Reductive
amination of piperazine 8 and final deprotection with
conversion to the hydrochloride salt, afforded MB243
and its stereoisomers, 10 and 11.
The piperazine compounds were prepared in a linear
fashion as illustrated in Schemes 1 and 2. Starting with
commercially available 4-phenyl-4-piperidine carboxylic
a, b
c, d
O
O
O
+
NH2
NBoc
NH
HO
HO
NH
-
OTs
3
4
5
NCbz
BocN
HN
e, d
f
As illustrated in Scheme 2, various substituted piper-
azine compounds were synthesized from dipeptide 7.
Reversal of the deprotection sequence on the piperazine
ring and reductive amination provided piperazine 12.
Piperazines 13, 14, and 15 were made in a similar fash-
ion to MB243 using the requisite aldehyde precursors
for reductive amination. Likewise, piperazine 16
was synthesized from intermediate 8 by coupling with
N-Boc-D-alanine and final deprotection.
O
O
O
NH2
N
N
NH
NH
O
O
F
F
7
6
NH
O
N
O
BocN
HN
HN
HN
• 2 HCl
g
h, d, i
O
O
N
N
NH
NH
The piperazine compounds (MB243, 9–16) were evalu-
ated in a competitive binding assay (Table 1) and
functional assay (Table 2).15 MB243 is the most promi-
sing compound in terms of overall binding affinity,
functional potency, and receptor subtype selectivity.
Stereochemistry about the p-flurorophenylalanine and
piperazine groups is also very important for binding
and functional activity and selectivity. The 1-(S)-p-fluoro-
phenylalanine analog, 10, shows poor binding affinity
at MC4R, and cAMP levels from the functional assay
suggest that it is only a partial agonist (29% activation).
The 2-(R)-piperazine analog, 11, shows decreased bind-
ing affinity and functional activity compared with the
O
O
F
F
8
MB243 1R, 2S
10 1S, 2S
d
11 1R, 2R
9
1R, 2S
Scheme 1. Reagents and conditions: (a) (Boc)2O, NaOH, dioxane; (b)
Rh/Al2O3, 70psi H2, MeOH, 70°C; (c) (COCl)2, DMF, CH2Cl2, then,
t-BuNH2, CH2Cl2; (d) TFA, CH2Cl2; (e) N-Boc-p-fluoro-(D or L)-Phe,
EDC, HOBt, DIEA, CH2Cl2; (f) 1-Boc-4-Cbz-piperazine-2-(S or R)-
carboxylic acid, EDC, HOBt, DIEA, CH2Cl2; (g) H2, Pd/C, EtOH;
(h) HCHO, NaBH3CN, TFA, NaOAc, MeOH; (i) HCl/Et2O, CH2Cl2.

B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 171–175
173
Table 1. Binding affinity and selectivity of compounds for the human
melanocortin subtype-4 receptora
MB243 was further evaluated in vitro on human, rat,
and mouse melanocortin receptors. The data outlined
in Table 3, shows this compound is >100-fold selective
versus the subtype receptors in binding affinity in
human, rat, and mouse cells. In the functional assays,
MB243 is >32-fold selective versus the other subtype
receptors. Nonetheless, this compound shows good
subtype selectivity on rat and mouse receptors.
b
IC50 (nM)
Compds
MC4R
MC3R/4R
MC5R/4R
a-MSH
19
16
18
2
1
4
1
101
57
3
6
147
63
3
MB243
9
10
6500 1900c
52 44c
11
12
98
84
141
58
46
43
76
63
43
54
36
42
87 37c
20 3c
MB243 was further characterized in pharmacokinetic
(PK) studies with rats, dogs, and monkeys (Table 4).
The oral bioavailability across all three species was
low and was attributed, in part, to incomplete absorp-
tion. Nonetheless, stability data from liver microsome
incubations with MB243 predicted a low clearance in
humans, and the potential for higher oral bioavailability
than in rats, dogs, or monkeys.16
13
14
13
8
3
1
8
15
16
30
a Values represent mean standard error except where indicated. All
data represent at least three determinations except for where
indicated.
b Displacement of [125I]-NDP-a-MSH from human receptors expressed
in CHO cells.
c Values (n = 2) represent mean standard deviation.
The efficacy of MB243 was investigated in our in vivo
rat obesity model. MB243 was administered at 20mg/
kg p.o. bid for four days with vehicle and dexfenfluram-
ine (3mg/kg, bid) as controls, to Sprague–Dawley rats
raised on an energy dense diet. Indeed, the body weights
of the rats treated with MB243 showed an overall de-
crease of À7 5g over the four days (p < 0.01 relative
to vehicle; two tail t-test). The body weights of the
Table 2. Functional activity of compounds at human melanocortin
receptors
EC50 (nM) [% max]b
MC3Rd
a
Compds
MC4Rc
MC5Rc
17 1 [113]
a-MSH
1.9 0.1 [100]
11 1 [87]
13 2 [99]
[29 18]d
1.1 0.1 [102]
[11 5]
[10 4]
MB243
9
10
1900 180 [32]
1800 320 [43]
[3 3]d
Table 3. Binding affinity and functional activity of MB243 at human,
rat, and mouse melanocortin receptorsa
[1 3]
[7 4]
2900 650 [19]c
[3 1]
[6 4]
Receptor
Bindingb
IC50 (nM)
[% inh.]
cAMPc
Activationd at
11
12
82 14 [92]
29 11 [97]
12 3 [90]
52 11 [50]
34 11 [55]
270 82 [64]
2700 600 [39]
3300 800 [19]
840 140 [20]
510 120 [19]
340 53 [22]
2000 640 [27]
EC50 (nM)
[% max]
10lM (%)
13
14
hMC1R
hMC3R
hMC4R
hMC5R
rMC3R
rMC4R
rMC5R
mMC3R
mMC4R
1700 320
1600 300
350 88 [85]
15
16
[4 3]
[12 4]
11
5
16
1
11 1 [87]
1900 180 [32]
990 180 [21]
a Concentration of compound at 50% maximum cAMP accumulation.
b Percentage of cAMP accumulation at 10lM compound relative to
a-MSH.
2400 130
2300 130
8
1
6
1 [76]
c Values represent mean standard error except where indicated. All
data represent at least three determinations.
13,000 1200
1100 100
4
7
1
1
d Values represent mean standard deviation except where indicated.
All data represent at least three determinations.
7
1
16 11 [66]
a All data represent at least three determinations.
b Displacement of [125I]-NDP-a-MSH receptors expressed in CHO
cells. Values represent mean standard error.
c Concentration of compound at 50% maximum cAMP accumulation.
Values represent mean standard error.
2-(S)-piperazine analog, MB243, but more importantly
selectivity versus the MC5R is diminished. Likewise,
methylation at the alternate amine of the piperazine ring
reduces binding affinity at MC4R and subtype selectivity
(12).
d Percentage of cAMP accumulation at 10lM compound relative to
a-MSH. Values represent mean standard deviation.
Table 4. Pharmacokinetic data for MB243
Amine substitution on the piperazine ring revealed a
tight structure–activity relationship. As illustrated in
Tables 1 and 2, the non-substituted piperazine analog, 9,
exhibits similar binding affinity and functional potency
as MB243, but with decreased binding subtype selecti-
vity. Likewise, size and electronic effects of the alkyl sub-
stituent on the piperazine ring (13, 14, and 15) has little
impact on binding affinity and functional activity, yet
imparts a significant effect on MC5R selectivity. Finally,
reducing the basicity of the amine on the piperazine ring
through coupling with D-alanine dramatically reduces
both binding and functional potency and selectivity (16).
PK parameter
Rata
Dogb
Monkeyc
F (%)
Cl (mLminÀ1 kgÀ1
9
17
11
)
48.7
5.4
5.6
1.2
4.1
0.75
15.7
1.2
1.3
5.0
Vdss (LkgÀ1
t1/2 (h)
Tmax (h)
)
1.6
1.54
a Compound dosed in Sprague–Dawley rats as a solution in water at
1mg/kg, iv and 5mg/kg, p.o.
b Compound dosed in beagles as a solution in water at 0.5mg/kg, iv
and 2.0mg/kg, p.o.
c Compound dosed in rhesus as a solution in water at 1mg/kg, iv and
2mg/kg, p.o.

174
B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 171–175
Bednarek, M.; Weinberg, D.; Nargund, R.; Fong, T. M.
60
50
40
30
20
10
0
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-10
-20
5
10
20
40
Dose (mg/kg, p.o.)
Figure 1. Penile erections in conscious rat ex copula model. MB243
dose dependently increased the number of penile erections. Values
represent mean SEM. *p < 0.05, compared to vehicle treatment
(paired t-test). N = 10/dose.
3. For human data, see: (a) Gu, W.; Tu, Z.; Kleyn, P. W.;
Kissebath, A.; Duprat, L.; Lee, J.; Chin, W.; Maruti, S.;
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S.; Yeo, G. S.; Keogh, J. M.; Aminian, S.; Jebb, S. A.;
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Weinberg, D.; Van der Ploeg, L. H. T.; MacIntyre, D. E.;
Martin, W. Int. J. Imp. Res. 2001, 13, S55; (c) Van der
Ploeg, L. H. T.; Martin, W. J.; Howard, A. D.; Nargund,
R. P.; Austin, C. P.; Guan, X.; Drisko, J.; Cashen, D.;
Sebhat, I.; Patchett, A. A.; Figueroa, D. J.; DiLella, A. G.;
Connolly, D. H.; Weinberg, D. H.; Tan, C. T.; Palyha, O.
C.; Pong, S.; MacNeil, T.; Rosenblum, C.; Vongs, A.;
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Gantert, L. T.; Drisko, J. E.; Hom, G. J.; Nargund, R.;
Sebhat, I.; Howard, A. D.; Van der Ploeg, L. H. T.;
MacIntyre, D. E. Eur. J. Pharmacol. 2002, 454, 71.
5. For human data on non-subtype selective MCR agonists,
see: (a) Wessells, H.; Fuciarelli, K.; Hansen, J.; Hadley, M.
E.; Hruby, V. J.; Dorr, R.; Levine, N. J. Urol. 1998, 160,
389; (b) Wessells, H.; Gralnek, D.; Dorr, R.; Hruby, V. J.;
Hadley, M. E.; Levine, N. Urologia 2000, 56, 641.
6. For recent patent reviews, see: (a) Bednarek, M. A.; Fong,
T. M. Expert Opin. Ther. Patents 2004, 14, 1; (b) Speak, J.
D.; Bishop, M. J. Expert Opin. Ther. Patents 2002, 12,
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control rats also changed during the four days with a
decrease of À16 3g in the rats treated with dexfenflur-
amine (p < 0.01), and an increase of +15 2g in vehicle
treated rats.17
Further in vivo studies were performed to evaluate the
erectogenic activity of MB243 using an established
rodent model.4c,d The mean number of erections elicited
over a fifteen-minute time period, determined by a direct
visual count, in vehicle treated rats was 21.6 1.2,
n = 40. Erectogenic effects were investigated using a
within animal design in which each rat served as
its own control where the effect of compound was
compared to that produced by vehicle. Sixty minutes
following p.o. administration of MB243 (5–40mg/kg)
adose-dependent increase in erections was observed
(Fig. 1). The maximal increase in the number of erec-
tions (ꢀ35%) was detected at 20 and 40mg/kg, p.o.
In addition, iv administration of MB243 (3mg/kg) also
produced statistically significant increases in erectile
responses with a mean increase of 21.2 7.9% (p <
0.01; n = 10). The magnitude of the MC4R agonist-
induced erectile activity compared favorably to apomor-
phine (30.9 8.2%, p = 0.02, @ 0.025mg/kg, sc), a
known central initiator of erectile activity.18
In summary, we report the synthesis and evaluation of
MC4R agonists containing a piperazine side chain as
alternatives to the tetrahydroisoquinoline moiety. Struc-
ture–activity studies around the piperazine side chain
lead to the discovery of a potent and selective MC4R
agonist, MB243, which exhibits an excellent off-target
activity profile (IC50 or Ki > 1000nM) based on broad
screening performed at Panlabs versus >100 receptors,
enzymes, and ion channels.19 Finally, MB243 is effica-
cious in our animal models for both food intake and
erectile dysfunction.
7. (a) Dyck, B.; Parker, J.; Phillips, T.; Carter, L.; Murphy,
B.; Summers, R.; Hermann, J.; Baker, T.; Cismowski, M.;
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