Design, synthesis, and SAR studies on a series of 2-pyridinylpiperazines as potent antagonists of the melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2006.04.069

A series of 2-pyridinylpiperazines derived from β-Ala-(2,4-Cl)Phe dipeptide was synthesized for the study of their SARs and possible interactions with the MC4 receptor. Compounds such as 11k (K_i = 6.5 nM) possessed high potency.

Full text of DOI:10.1016/j.bmcl.2006.04.069

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3693–3696
Design, synthesis, and SAR studies on a series
of 2-pyridinylpiperazines as potent antagonists
of the melanocortin-4 receptor
Joe A. Tran,a Joseph Pontillo,a Beth A. Fleck,b Dragan Marinkovic,a Melissa Arellano,a
Fabio C. Tucci,a Marion Lanier,a John Saunders,a Wanlong Jiang,a
Caroline W. Chen,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 6 February 2006; revised 21 April 2006; accepted 21 April 2006
Available online 11 May 2006
Abstract—A series of 2-pyridinylpiperazines derived from b-Ala-(2,4-Cl)Phe dipeptide was synthesized for the study of their SARs
and possible interactions with the MC4 receptor. Compounds such as 11k (Ki = 6.5 nM) possessed high potency.
Ó 2006 Elsevier Ltd. All rights reserved.
We have previously demonstrated that the basic nitro-
gen of the benzylamines 1 (Fig. 1), which possibly inter-
acts with Asp122 of the human melanocortin-4 receptor
(hMC4R), is very important for high affinity binding.1
Thus, the benzylamines bearing an alkyl side chain such
as 2-thienylethyl (1a, Ki = 1.8 nM) or 1-methoxy-2-pro-
pyl group (1b, Ki = 8.8 nM) possess potent affinity in a
competition binding assay.2 Since the phenyl ring of
the benzylamines 1 could reside in an area close to the
transmembrane domain seven (TM-7) of hMC4R, and
interacts with several lipophilic residues such as
Phe284, Leu288, and Ile289 based on a computational
receptor model,1 we designed and synthesized a series
of cyclohexyl derivatives exemplified by 2a and 2b as po-
tent hMC4R antagonists.3 Interestingly, while com-
pound 2a (Ki = 11 nM) bearing a basic side chain
exhibits good binding affinity as expected, compound
2b (Ki = 4.2 nM), with an amide group, also possesses
high potency. One possible explanation for these results
is that, instead of the charge-charge attraction between
the basic amine of 2a and an acidic residue of the recep-
tor, the amide of 2b may pick up the interaction with
Asp122 through hydrogen-bonding. However, the piper-
azine of 2 is weakly basic (estimated pKa value is 6.8 on
the basis of calculation), it might participate in the inter-
action of 2b with the receptor. Therefore, we decided to
explore the role of the weakly basic piperazine by install-
ing the more basic 2-piperazinepyridine moiety (calcu-
lated pKa is 9.2). In addition, the more hydrophilic
pyridine will reduce the lipophilicity of its phenyl
analogs such as 3 (clogD = 3.1). Here we report the syn-
thesis and structure–activity relationship studies of a
series of 2-pyridinylpiperazines, derived from b-Ala-D-
(2,4-Cl)-Phe dipeptide, bearing an amine or amide side
chain, as potent hMC4R antagonists.
The synthesis of the targeted compounds started from
the 2-chloro-3-pyridinylcarboxaldehyde 4, which was
condensed with N-Boc-piperazine at an elevated temper-
ature to give the 2-aminopyridine 5. Deprotection of 5
with trifluoroacetic acid in dichloromethane, followed
by a standard peptide coupling protocol with D-N-
[N-(tert-butoxycarbonyl)-b-alanine]-2,4-dichlorophenyl-
alanine, or D-N-acetyl-2,4-dichlorophenylalanine, afforded
the key intermediate 6a, or 6b. Reductive amination of
the aldehyde 6a with ammonia provided the primary
benzylamine 7, which was subjected to coupling reac-
tions with various carboxylic acids to give the amides
8a–l after TFA-deprotection. Coupling reactions of
7 with different chloroformates in the presence of
triethylamine, followed by TFA treatment, afforded
Keywords: Melanocortin; Antagonist; 2-Pyridinylpiperazine; Synthesis.
*
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7967; e-mail: cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.069

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J. A. Tran et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3693–3696
Cl
Cl
Cl
Cl
Cl
Cl
N
N
N
HN
2
N
N
N
HN
S
NH
NH
R
HN
R
NH
1
O
O
O
O
O
NH
2
O
NH
2
1
2
1a: R = 2-thienylethyl;
2a: R = CH CH Ph
2 2
3
1
2
1b: R = 1-methoxy-2-propyl;
2b: R = COCH C H OMe-4
2
6 4
1
2
1c: R = 2'-methoxyphenethyl;
2c: R = COC H OMe-4
6
4
2
2d: R = CONHC H OMe-4
6
4
Figure 1. Small molecule antagonists of the melanocortin-4 receptor.
the carbamates 9a–c. Condensations of 7 with several al-
kyl or arylisocyanates provided, after deprotection, the
ureas 10a–h in excellent yields (Scheme 1).
(Ki = 36 nM) of cyclohexylpiperazine had about 9-fold
reduction in potency, suggesting the conformation of this
side chain is an important factor for 2. These results indi-
cate that the aromatic ring in the side chain of 8–10 did
not participate in the interaction of these compounds
with the receptor. They are clearly different from the
cyclohexyl compounds such as 2a–d in which the pheny-
lacetamides (i.e., 2b) are much better than the benzoyla-
mides (i.e., 2c) and arylureas (i.e., 2d), but the
phenethylamine 2a had comparable binding value to 2b.3
Alternatively, reductive aminations of 6a with various
primary amines afforded the secondary amines 11a–l
after TFA treatment. Similarly, compounds 12a–c were
obtained from 6b.
The synthesized compounds were tested for their ability
to compete with [125I]-NDP-MSH at hMC4R expressed
in HEK 293 cells in a binding assay as previously
described.4 The amides 8a–l displayed Ki values of
310–2800 nM regardless of the R3-group, implying this
group is not in the contact with the receptor. For
example, the 4-methoxybenzoyl 8e (Ki = 1500 nM) and
the 4-methoxyphenylacetyl 8k (Ki = 550) displayed simi-
lar binding affinity. In contrast, cyclohexylpiperazine
analogs 2c (Ki = 250 nM) and 2b (Ki = 4.2 nM) exhibited
about 40-fold difference in potency, suggesting the
aromatic ring of 2b, but not 2c, offers extra binding
energy. To further explore this point, several carbamates
9a–c and a series of ureas 10a–h were synthesized and
examined. The carbamates 9a–c exhibited Ki values of
440–1,800 nM, and the ureas 10a–h displayed Ki values
of 560–1300 nM (Table 1). These results were no better
than that of the carboxamides 8a–l, including acetamide
8a (Ki = 2800 nM). In comparison, the urea 2d
A series of amines 11a–l then was studied. We have previ-
ously demonstrated that a proper N-alkyl group can im-
prove binding affinity of a primary benzylamine over
60-fold.1 While an aromatic ring connected by an ethylene
such as 2-thienylethylamine is an optimal side chain for
both benzylamine 1a and cyclohexylmethylamine 2a, a
smaller 1-methoxy-2-propyl group is found to be a
suitable replacement for 1a. Thus, the racemic compound
1b (Ki = 8.8 nM) is a potent hMC4R antagonist. Howev-
er, while this group was incorporated into the pyridine
series, the corresponding analog 11c (Ki = 87 nM) exhib-
ited 10-fold reduction in binding from 1b, although it was
still better than the lipophilic trifluoroethyl 11a (Ki =
170 nM) and the hydrophilic hydroxyethyl 11b (Ki =
520 nM). Its O-demethylated analog 11d (Ki = 420 nM)
was only moderately active, and an additional methyl
group on 11d did not alter its binding affinity (11e,
Cl
Cl
Cl
Cl
N
N
O
N
O
N
O
d
a
b,c
N
N
N
Cl
N
N
N
O
H N
2
NH
O
NH
O
O
O
O
N
H
O
O
R
4
5
6a: R = CH2CH2NHBoc
6b: R = CH3
7
e,b
f
f,b
Cl
Cl
Cl
Cl
Cl
Cl
N
N
N
N
N
N
N
N
N
HN
NH
HN
R
NH
HN
R
NH
4
4
O
3
O
O
O
R
O
NH
2
O
O
NH
2
8-10
12
11
Scheme 1. Reagents and condition: (a) N-Boc-piperazine/DMSO/D; (b) TFA/CH2Cl2; (c) N-(RCO)-D-(2,4-Cl)Phe-OH/EDC/HOBt; (d) NH3/
NaBH(OAc)3; (e) R3COOH/EDC; or R3COCl/Et3N or isocyanate; (f) R4NH2/NaBH(OAc)3.

J. A. Tran et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3693–3696
Table 1. SAR of amides, carbamates and ureas at hMC4R Table 2. SAR of amine at hMC4R
3695
Cl
Cl
Cl
Cl
Cl
Cl
N
N
N
N
N
N
N
N
N
HN
R4
NH
HN
R4
NH
HN
NH
O
O
O
R3
O
NH2
O
NH2
O
O
12
11
8-10
Compound
R4
Ki (nM)
Compound
R3
Ki (nM)
11a
11b
11c
11d
11e
11f
11g
11h
11i
CF3CH2–
HOCH2CH2–
170
520
87
8a
8b
Me–
2800
2800
1800
2200
1500
2600
1800
450
MeOCH2–
Ph–
4-FC6H4–
MeOCH2CH(Me)–
HOCH2CH(Me)–
HOCH2C(Me)2–
MeOCH2CH(Et)–
HOCH2CH(Et)–
(HOCH2)2CH–
8c
8d
420
480
200
710
1500
130
12
8e
4-MeOC6H4–
2-Furanyl–
2-Thienyl–
8f
8g
8h
8i
2-FC6H4CH2–
2-FC6H4CH2–
2-FC6H4CH2CH2–
2-MeOC6H4CH2–
4-FC6H4CH2–
4-MeOC6H4CH2–
2-ThienylCH2–
PhO–
360
11j
8j
1000
550
11k
11l
2-MeOC6H4CH2CH2–
2-ThienylCH2CH2–
MeOCH2CH(Me)–
2-MeOC6H4CH2CH2–
2-ThienylCH2CH2–
6.7
15
8k
8l
310
12a
12b
12c
180
10
9a
9b
1200
1800
440
2-MeOC6H4O–
BnO–
i-PrNH–
21
9c
10a
10b
10c
10d
10e
10f
10g
10h
1300
710
PhNH–
1a–c were only moderately less potent. For example,
the Ki values of 11k (Ki = 6.7 nM) and 11l (Ki = 15 nM)
were 4- and 8-fold higher than that of 1c (Ki = 1.6 nM)
and 1a (Ki = 1.8 nM), respectively. Computational anal-
ysis of the conformations of piperazinebenzylamine and
the corresponding pyridine analog indicates that the al-
most planar acylpiperazine ring is orthogonal to the aro-
matic ring, while the dihedral angle of the pyridine
analog could be slightly smaller (Fig. 2). This difference
in conformations could partly explain the different
binding affinities of these two series of compounds.
Alternatively, the pyridine ring is less lipophilic and p-
electron-deficient in comparison with benzene, there-
fore, its interaction with an aromatic residue through
p-stacking, or aliphatic side chains of the receptor via
van der Walls interaction, would be less favored.5 One
aromatic acidic acid Phe284 of hMC4R is located at
the top TM-7, which is involved in the interaction with
a small molecule agonist based on mutagenesis studies.4a
2-FC6H4NH–
2-MeOC6H4NH–
BnNH–
1100
1300
1000
970
2-FC6H4CH2NH–
2-MeOC6H4CH2NH–
2-ThienylCH2NH–
560
750
Ki = 480 nM). The 1-methoxy-2-butyl analog 11f
(Ki = 200 nM) exhibited slightly reduced potency from
11c, and its O-demethylated derivative 11g (Ki = 710 nM)
again displayed over 3-fold reduction in binding. As
expected, the more hydrophilic 11h had further reduction
in binding affinity, and the 2-fluorobenzyl 11i, however,
showed similar potency to that of 11c. Further extension
of the 2-fluorobenzyl group of 11i resulted in compound
11j with 10-fold improvement (Ki = 12 nM). The 2-meth-
oxyphenethyl 11k (Ki = 6.7 nM), exhibiting the best affin-
ity of this series, was slightly better than that of the
2-thienylethyl 11l (Ki = 15 nM) which had similar binding
affinity to 11j. However, the binding affinity of these two
compounds was 4- and 10-fold, respectively, lower than
that of benzylamines 1a (Ki = 1.8 nM) and 1c
(Ki = 1.6 nM) (Table 2).
While the orthogonal conformation of the phenyl- and
pyridinyl-piperazines could account for the difference
in binding affinity between these two series, the lower
affinity of amides such as 8i than the amine such as
11k needs further understanding. The lack of SAR of
the amides could not be simply explained by the loss
of a charge–charge interaction of the amides 8–10 since
it has been demonstrated the aromatic ring on the
N-side chain plays a role in receptor binding. A reason-
able explanation is the lack of cation–p interaction.6
Thus, in the case of a 2-pyridinylpiperazine with an
amine side chain, such as 11k, the protonated amine
under physiological conditions has a strong interaction
with the p-system of the aromatic pyridine ring. This
interaction might position the phenethyl side chain of
11k to a conformation required for the pharmacophore.
It will cost energy for 8i to possess a similar conforma-
Similarly, the acetamides 12b (Ki = 10 nM) and 12c
(Ki = 21 nM) exhibited Ki values close to that of 11k
and 11l, while 12a (Ki = 180 nM) was slightly less potent
than the b-alanine derivative 11c. The pyridine com-
pound 12c was again about 9-fold less active than the
benzene analog 3 (Ki = 2.4 nM).
Apparently, the 2-piperazinepyridine bearing an amide
side chain at the 3-position such as 8k (Ki = 550 nM)
could not mimic the piperazinecyclohexane attached
by an amide at the 1-position such as 2b (Ki = 4.2 nM).
In contrast, the pyridine analogs 11 of benzylamines

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J. A. Tran et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3693–3696
of 390 ml/min kg, which would give an oral bioavailabil-
ity of 15% assuming complete absorption. The poor
metabolic stability prevents the further development of
these compounds.
In conclusion, a series of 2-pyridinylpiperazines was syn-
thesized to study the detailed structure–activity relation-
ships and their interactions with the MC4 receptor.
While these compounds possessing a basic moiety dis-
played high binding affinity, similar to the benzylamine
analogs, analogs bearing an amide side chain were much
less potent. These results are different from the cyclo-
hexylpiperazines, in which an amide side chain resulted
in a compound with similar to or better than that with
an amine group in binding affinity. Compounds from
this series possessed high binding affinity. For example,
12b had a Ki value of 10 nM at hMC4R. While it was
Figure 2. Orthogonal relationship between pyridine and piperazine
represented by 5 (generated by DS ViewerPro 5.0, Accelrys).
tion due to a much weaker amide-p interaction.7 In the
cyclohexyl series, the amine nitrogen of 2a presumably
resides at the top of the cyclohexyl ring as an active con-
formation, and the amide functionality of 2b could be
slightly favored to do so, because of the weak interac-
tion of the partially positively charged protons of the
cyclohexyl ring with the electron-negative amide, which,
in combination with an amide-acid hydrogen bond,
could compensate for the loss of a strong charge–charge
attraction. Thus, the amide 2b possessed slightly better
binding affinity than the amine 2a. In comparison to
2b (Ki = 4.2 nM), in which the 2-methoxyphenyl group
is connected through a methylene to the amide, the
directly connected 2c (Ki = 250 nM) and the NH-linked
2d (Ki = 36 nM) push the aromatic ring to a less favored
position for receptor interaction. One of the amino acid
residues of the receptor interacting with this aromatic
moiety could be Phe184 at the top of TM-4 based on
a receptor model. This residue has been demonstrated
to interact with a-MSH based on mutagenesis studies.8
slightly less potent than its phenyl analog
3
(Ki = 2.4 nM),9 the reduced lipophilicity caused by the
hydrophilic pyridine provided 12b with a desirable
calculated logD value of 2.7.
References and notes
1. 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. 2004, 47, 6821.
2. Pontillo, J.; Marinkovic, D.; Pontillo, J.; Tran, J. A.;
Arellano, M.; Fleck, B. A.; Wen, J.; Tucci, F. C.; Nelson, J.;
Saunders, J.; Foster, A. C.; Chen, C. Bioorg. Med. Chem.
Lett. 2005, 15, 4615.
3. Tran, J. A.; Pontillo, J.; Arellano, M.; Fleck, B. A.; Tucci,
F. C.; Marinkovic, D.; Chen, C. W.; Saunders, J.; Foster,
A. C.; Chen, C. Bioorg. Med. Chem. Lett. 2005, 15, 3434.
4. (a) Nickolls, S. A.; Cismowski, M. I.; Wang, X.; Wolff, M.;
Conlon, P. J.; Maki, R. A. J. Pharmacol. Exp. Ther. 2003,
304, 1217; (b) Fleck, B. A.; Chen, C.; Yang, W.; Huntley,
R.; Markison, S.; Nickolls, S. A.; Foster, A. C.; Hoare, S.
R. Biochemistry 2005, 44, 14494.
5. (a) Janiak, C. J. Chem. Soc., Dalton Trans. 2000, 3885; (b)
Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. J.
Chem. Soc., Perkin Trans. 2 2001, 651.
6. Ma, J.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303.
7. Steiner, T.; Koellner, G. J. Mol. Biol. 2001, 305, 535.
8. Haskell-Luevano; Cone, R. D.; Monck, E. K.; Wan, Y-P.
Biochemistry 2001, 40, 6164.
9. Pontillo, J.; Tran, J. A.; Markison, S.; Joppa, M.; Fleck, B.
A.; Marinkovic, D.; Arellano, M.; Tucci, F. C.; Lanier, M.;
Nelson, J.; Saunders, J.; Hoare, S. R. J.; Foster, A. C.;
Chen, C. Bioorg. Med. Chem. Lett. 2005, 15, 2541.
Compounds such as 11k from this series were also
highly selective. Thus, 11k had Ki values of 5000, 6.7,
and 2100 nM at hMC3R, hMC4R, and hMC5R, respec-
tively. All compounds had no significant stimulation of
cAMP production at the human MC4 receptor
expressed in HEK 293 cells at 10 lM concentration.
However, despite the reduction in lipophilicity, com-
pounds such as 12b possessed low metabolic stability
possibly due to their structural features such as high
flexibility. For example, 12b had an intrinsic clearance
CLint of greater than 3500 ml/min kg in an in vitro rat
liver microsomal assay, predicting a zero percentage of
bioavailability in this species. In comparison, the dibasic
molecule 11k with a calculated logD of 1.1 had a CLint