2-Piperazinecarboxamides as potent and selective melanocortin subtype-4 receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2005.02.068

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by covale

Full text of DOI:10.1016/j.bmcl.2005.02.068

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1993–1996
2-Piperazinecarboxamides as potent and selective
melanocortin subtype-4 receptor agonists
a,
a
b
b
Brenda L. Palucki,a,* Min K. Park, Ravi P. Nargund, Rui Tang, Tanya MacNeil,
*
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
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
Received 26 January 2005; revised 18 February 2005; accepted 22 February 2005
Abstract—We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the
melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by
covalent binding in microsome preparations.
Ó 2005 Elsevier Ltd. All rights reserved.
The five cloned melanocortin receptor subtypes
(MC1R–MC5R) are a part of a family of seven-trans-
membrane G-protein-coupled receptors. These recep-
tors, which are located peripherally and centrally,
interact with their endogenous ligands, the melanocor-
tins and corticotropins, to regulate a diverse number
of physiological functions, including the control of feed-
ing and sexual behavior, as well as skin pigmentation,
steroidogenesis, energy metabolism, and exocrine secre-
tion.1 Over the past few years, research efforts in drug
discovery groups are focusing on identifying novel
MC4R agonists for the potential treatment of obesity
and sexual dysfunction.2
occurring via initial oxidation at C-5 on the piperazine
ring. Consequently, we speculated that blocking the site
of activation via alkylation on the piperazine ring
should reduce bioactivation. Herein, we report the syn-
thesis and evaluation of alkyl-substituted 2-piperazine-
carboxamides, which are potent and selective MC4R
agonists with significantly reduced bioactivation.
R2
R1
HN
R3
N
O
5
2
N
3
HN
HN
O
O
HN
O
N
N
NH
NH
Recent efforts at Merck have led to the identification of
MB243, a potent and selective, small-molecule agonist
of the MC4R. Indeed, MB243 was shown to stimulate
erectile activity as well as reduce food intake in rats.3
Nonetheless, further development of this compound
was discontinued due to the potential for bioactivation
as seen in vitrothrough extensive covalent binding in
rat and human liver microsomes.4 NMR analysis of
the isolated adducts suggested that bioactivation was
O
O
F
F
MB243
1
The 5-alkylated piperazine compounds were prepared in
a linear fashion as illustrated in Scheme 1. Starting with
commercially available methyl N-b-Boc-L-a,b-diamino-
propionate hydrochloride, 2, conversion to diamine 3
was achieved via alkylation with various substituted
a-bromoketones.5 Removal of the Boc group fol-
lowed by intramolecular reductive amination provided
Keywords: Melanocortin; Piperazinecarboxamide.
*
Corresponding authors. Tel.: +1 732 594 6188; fax: +1 732 594
5966; e-mail: brenda_palucki@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.068

1994
B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1993–1996
• HCl
NH2
O
O
O
O
O
O
R2
NH
O
H
N
a
Bn
N
b
BnN
O
a
b
H
N
HN
O
NH
O
O
O
R2
O
O
NHBoc
4
NHBoc
20
NHBoc
3
O
NHBoc
21
4
2
R2
c, d
e, f
g, h, i
HN
BocN
HO
MeO
N
N
NH
O
HN
HN
O
O
R2
NBoc
c, d
e, f
BocN
HO
22
23
O
N
NH
O
O
5
7
N
O
N
O
HN
HN
HN
HN
• 2 HCl
and
• 2 HCl
F
R2
R2
O
O
N
N
NH
NH
NH
• 2 HX
NH
O
O
O
HN
HN
HN
HN
• 2 HX
g
O
O
O
and
F
F
N
N
NH
NH
24
25
O
O
Scheme 2. Reagents and conditions: (a) PhCHO, Na(OAc)3BH, DCE
(100%); (b) TFA, CH2Cl2, then, Na(OAc)3BH, AcOH, DCE (72%); (c)
HCHO, NaCNBH3, NaOAc, TFA, MeOH (71%); (d) H2, Pd/C, HCl,
EtOH (55%); (e) (Boc)2O, TEA, CH2Cl2 (83%); (f) NaOH, MeOH,
H2O (100%); (g) 6, EDC, HOBt, DIEA, CH2Cl2 (84%); (h) TFA,
CH2Cl2 (98%); (i) preparative HPLC.
F
F
8
9
R2 = Me
R2 = Et
14 R2 = Et
15 R2 = i-Pr
10 R2 = i-Pr
16 R2 = cyclopropyl
17 R2 = t-Bu
18 R2 = 1-methyl-1-cyclopropyl
19 R2, R2 = spirocyclopropyl
11 R2 = cyclopropyl
12 R2 = t-Bu
13 R2 = 1-methyl-1-cyclopropyl
a 6-alkylated-2-piperazincarboxamide. The synthesis of
piperazine 30, is depicted in Scheme 3. The commer-
cially available N-a-Z-L-2,3-diaminopropionic acid, 26,
was converted to the methyl ester and then alkylated
with a-bromo-3-methyl-2-butanone5 togive 27. Depro-
tection via hydrogenation and concomitant intramole-
cular reductive amination provided piperazine ester 28.
Protection of both secondary amines of the piperazine
ring and hydrolysis yielded acid 29. Coupling, deprotec-
tion, and separation via preparative reverse-phase
HPLC afforded piperazine 30 as a single diastereomer.8
Scheme 1. Reagents and conditions: (a) a-bromoketone, DIEA, DMF
(12–60%); (b) TFA, CH2Cl2, then, Na(OAc)3BH, AcOH, DCE; (c)
(Boc)2O, TEA, CH2Cl2 (45–74% for two steps); (d) NaOH, MeOH,
H2O (88–100%); (e) 6, EDC, HOBt, DIEA, CH2Cl2 (46–93%); (f)
TFA, CH2Cl2 (98–100%); (g) preparative HPLC.
piperazine 4. Protection of both secondary amines of the
piperazine ring followed by hydrolysis of the ester
yielded the 5-alkylated-2-piperazinecarboxylic acids, 5.
Coupling of piperazine 5 with 1-[(2R)-2-amino-3-(4-fluoro-
phenyl)-1-oxopropyl]-4-cyclohexyl- N-(1,1-dimethyl-
ethyl)-4-piperidine carboxamide (6)6 and deprotection
provided dipeptide 7 as a 2:1–10:1 mixture of trans:cis
diastereomers. The diastereomers were separated using
preparative reverse-phase HPLC togive the trans-piper-
azines 8–13, and cis-piperazines 14–18, as either the
ditrifluoroacetic acid or dihydrochloride salts.7 Pipe-
razine 19 contains a spirocyclopropyl group at C-5,
and consequently, preparative HPLC separation was
not necessary.
The piperazine compounds (8–19, 24–35, 30) were eval-
uated in a competitive binding assay (Table 1) and
O
O
NHCbz
N
NHCbz
NH2
a, b
c
HN
O
HO
O
NH
H
O
O
28
27
26
Scheme 2 illustrates the synthesis of compounds 24 and
25. Starting with diamine 4, benzyl protection of the a-
amine, Boc-deprotection, and intramolecular reductive
amination provided piperazine 21. Reductive amination
of the piperazine amine with formaldehyde followed by
hydrogenation yielded the methylated piperazine 22. Fi-
nal Boc-protection and hydrolysis provided piperazine
acid 23. Coupling of piperazine acid 23 with amine 6,6
deprotection, and separation via preparative reverse-
phase HPLC provided the trans-piperazine 24 and cis-
piperazine 25.
*
NH
O
HN
• 2 TFA
d, e
f, g, h
BocN
HO
O
HN
NBoc
N
NH
O
O
29
30
F
Scheme 3. Reagents and conditions: (a) TMSCl, MeOH (98%); (b) a-
bromo-3-methyl-2-butanone, DIEA, DMF (49%); (c) H2, Pd/C, EtOH
(93%); (d) (Boc)2O, TEA, CH2Cl2 (62%); (e) NaOH, MeOH, H2O
(53%); (f) 6, EDC, HOBt, DIEA, CH2Cl2 (48%); (g) TFA, CH2Cl2
(97%); (h) preparative HPLC.
In an effort to further understand bioactivation of 2-pip-
erazinecarboxamides, we also synthesized and examined

B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1993–1996
Table 3. Covalent binding to rat liver microsomal proteinsa
1995
Table 1. Binding affinity and selectivity of compounds for the human
melanocortin subtype-4 receptora
Compound
+ NADPH (pmol equiv/mg protein)
b
IC50 (nM)
Compds
MC4R
MB243
8
2153 266b
50
MC3R/4R
MC5R/4R
10
11
30
107
103
216
a-MSH
8
19
6
2
1
86
6
87
1
9
3
2c
1
152
140
119
116
99
126
116
59
a The numbers represent values for covalent binding after 30 min
incubations of [3H]-compound11 with rat liver microsomes in the
presence of NADPH.
10
11
12
13
14
15
16
17
18
19
24
25
30
4
8
1c
5
1
39
40
b Mean standard deviation (n = 3).
5
1
40
20
5
59
61
75
49
6
25 10
42
48
65
26
receptor subtype selectivity. Interestingly, the 6-isopro-
pylpiperazine compound, 30, is a potent and selective
MC4R agonist with very little activation on the
MC5R subtype (10%).
29
24
17
3
9
5
49
42
23
61
3
1c
5
116
36
192
49
46
11 1c
A representative number of the alkylated piperazine
compounds were evaluated for potential bioactivation.10
Indeed, alkyl substitution on C-5 of the piperazine ring
dramatically reduced covalent binding in comparison to
MB243 (Table 3). Likewise, the 6-isopropylpiperazine
analog, 30, also afforded a 10-fold decrease in covalent
binding as compared to MB243. Thus, as predicted,
alkylation at either C-5 or C-6 of the piperazine ring
blocks oxidation and further bioactivation.
81
105
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.
functional assay (Table 2).9 The trans-piperazine com-
pounds (8–13, 24, 30) are the best compounds in terms
of overall binding affinity, functional potency, and
receptor subtype selectivity. In addition, N-methylation
on the piperazine ring, does not affect potency or selec-
tivity (24 vs 9). The spirocyclopropylpiperazine com-
pound, 19, shows a 5-fold loss in functional potency in
comparison to the trans-methyl analog, 8, with reduced
In summary, we report the synthesis and evaluation of
MC4R agonists containing a 5- or 6-alkylated piper-
azine side chain. Structure–activity studies show the
trans-5-alkylpiperazine diastereomers are potent and
selective MC4R agonists, while the corresponding cis-
isomers exhibit moderate potency and receptor subtype
selectivity. More importantly, the 5- and 6-alkylated
piperazine side-chain analogs show significantly reduced
covalent binding in comparison to MB243.
Table 2. Functional activity of compounds at human melanocortin
receptors
a
Compds
EC50 (nM) [% max]b
References and notes
MC4Rc
MC3Rd
MC5Rc
1. For recent reviews on melanocortin system, see: (a)
Holder, J. R.; Haskell-Luevano, C. Med. Res. Rev. 2004,
24, 325; (b) Voisey, J.; Carroll, L.; van Daal, A. Curr.
Drug Targ. 2003, 4, 586; (c) Sebhat, I.; Ye, Z.; Bednarek,
M.; Weinberg, D.; Nargund, R.; Fong, T. M. Ann. Rep.
Med. Chem. 2003, 38, 31; (d) Gantz, I.; Fong, T. M. Am. J.
Physiol. Endocrinol. Metab. 2003, 284, E468; (e) Zimanyi,
I. A.; Pelleymounter, M. A. Curr. Pharm. Des. 2003, 9,
1381; (f) Yang, Y. K.; Harmon, C. M. Obesity Rev. 2003,
4, 239.
2. 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,
1631; (c) Wikberg, J. E. S. Expert Opin. Ther. Patents
2001, 11, 61; (d) Anderson, P. M.; Boman, A.; Seifert, E.;
Skottner, A.; Torbjorn, L. Expert Opin. Ther. Patents
2001, 11, 1583.
3. Palucki, B. L.; Park, M. K.; Nargund, R. P.; Ye, Z.;
Sebhat, I. K.; Pollard, P. G.; Kalyani, R. N.; Tang, R.;
MacNeil, T.; Weinberg, D. H.; Vongs, A.; Rosenblum, C.
I.; Doss, G. A.; Miller, R. R.; Stearns, R. A.; Tamvako-
poulos, C.; Cashen, D. E.; Martin, W. J.; McGowan, E.;
Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.;
Patchett, A. A. Bioorg. Med. Chem. Lett. 2005, 15, 171.
a-MSH
8
1.9 0.1 [100]
1.1 0.1 [102]c
[6 3]
[9 4]
17 1 [113]
880 100 [42]
710 270 [43]
340 29 [49]
760 200 [44]
360 58 [46]
300 48 [62]
[45 4]d
8
5
3
5
7
7
2 [79]
1 [89]
1 [82]
1 [102]
2 [99]
2 [95]
9
10
11
12
13
14
15
16
17
18
19
24
25
30
[8 2]
[9 4]
[11 3]
[10 3]
[6 2]
150 73 [69]
42 10 [77]
36 10 [67]
91 25 [77]
93 23 [69]
41 16 [53]
[6 2]
[4 3]
1400 210 [41]
1400 280 [33]
1000 170 [54]
920 210 [56]
1900 220 [36]
450 51 [56]
2400 1000 [36]
[10 1]d
[5 3]
[5 4]
[6 4]
2
1 [85]
150 21 [29]c
[8 2]
[4 1]
330 170 [58]
18 2 [92]
a Concentration of compound at 50% maximum cAMP accumulation.
b Percentage of cAMP accumulation at 10 lM compound relative to
a-MSH.
c Values represent mean standard error except where indicated. All
data represent at least three determinations.
d Values represent mean standard deviation except where indicated.
All data represent at least three determinations.

1996
B. L. Palucki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1993–1996
4. Doss, G. A.; Miller, R. R.; Zhang, Z.; Teffera, Y.;
8. Absolute stereochemistry was not determined and minor
diastereomer could not be isolated.
Nargund, R. P.; Palucki, B.; Park, M. K.; Tang, Y. S.;
Evans, D. C.; Baillie, T. A.; Stearns, R. A. Chem. Res.
Toxicol. 2005, 18, 271.
9. For a detailed description of the assay protocols, see: (a)
Bednarek, M. A.; MacNeil, T.; Kalyani, R. N.; Tang, R.;
Van der Ploeg, L. H. T.; Weinberg, D. H. J. Med. Chem.
2001, 44, 3665; (b) Bednarek, M. A.; Siva, M. V.; Arison,
B.; MacNeil, T.; Kalyani, R. N.; Huang, R.-R. C.;
Weinberg, D. H. Peptides 1999, 20, 401.
10. For a detailed description of the assay protocols, see Ref.
4.
11. The tritium group is on the p-fluorophenyl ring of each
compound.
5. The a-bromoketones were either commercially available
or prepared via bromination of ketone using bromine in
methanol.
6. See Ref. 3 for the synthesis of this intermediate.
7. NMR studies were carried out to determine the stereo-
chemistry of the piperazine ring, and the major isomer
was assigned trans based on the observed coupling
constants.