Optimization of piperazinebenzylamines with a N-(1-methoxy-2-propyl) side chain as potent and selective antagonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2005.06.058

Piperazinebenzylamines bearing a small N-(1-methoxy-2-propyl) side chain were found to be potent and selective antagonists of the human melanocortin-4 (MC4) receptor. Compound 7b, having K_i values of 6.9 and 2800 nM at the human MC4 and MC3 receptors, respectively, has moderate oral bioavailability in mice, which is improved relative to the arylethyl analogues.

Full text of DOI:10.1016/j.bmcl.2005.06.058

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4615–4618
Optimization of piperazinebenzylamines with
a N-(1-methoxy-2-propyl) side chain as potent and selective
antagonists of the human melanocortin-4 receptor
Joseph Pontillo,a Dragan Marinkovic,a Joe A. Tran,a Melissa Arellano,a
Beth A. Fleck,b Jenny Wen,c Fabio C. Tucci,a Jodie Nelson,a John Saunders,a
Alan C. Fosterd 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 Preclinical Development, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
dDepartment of Neuroscience, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 13 May 2005; accepted 10 June 2005
Available online 18 August 2005
Abstract—Piperazinebenzylamines bearing a small N-(1-methoxy-2-propyl) side chain were found to be potent and selective antag-
onists of the human melanocortin-4 (MC4) receptor. Compound 7b, having Ki values of 6.9 and 2800 nM at the human MC4 and
MC3 receptors, respectively, has moderate oral bioavailability in mice, which is improved relative to the arylethyl analogues.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
selective antagonist 3 (Ki = 3.2 and 950 nM, respective-
ly, at MC4R and MC3R) stimulates food intake in sati-
ated mice when given by intracerebroventricular
administration. Here, we report our efforts to identify
potent and selective MC4R antagonist derivatives with
improved metabolic stability and suitable physicochem-
ical properties for possible peripheral administration.
The melanocortin-4 receptor (MC4R) is known to be a
major mediator for feeding behavior, metabolism
and energy homeostasis, and non-selective peptide
antagonists such as SHU9119 and AgRP have been
demonstrated, after central administration, to have a
profound effect in stimulating food-intake in rodent.1
Importantly, recent studies have shown that MC4R
antagonists prevent tumor-bearing mice from weight
loss.2 Thus, potent and selective MC4R antagonists3
may have potential utility in the treatment of cachexia.4
We recently reported a series of substituted piperazineb-
enzylamines exemplified by 1–3 as potent and selective
MC4R antagonists (Fig. 1).5 For example, 2 possesses
a Ki value of 1.8 nM in competitive binding with radio-
labeled NDP-MSH and a pA2 value of 7.9 in the inhibi-
tion of a-MSH-stimulated cAMP production. It alone
does not stimulate cAMP release in cells expressing the
human MC4R. We also demonstrated that the MC4R-
The metabolic stabilities of 2 and 3 were initially deter-
mined in human liver microsomes in vitro. While 2
exhibits good stability (CLint = 23 ml/min kg),
3
(CLint = 870 ml/min kg) has high clearance in this assay.
Following oral administration (10 mg/kg) in rats, plas-
ma concentrations of 2 were low due to poor absorp-
Keywords: Melanocortin-4; Antagonist; Piperazinebenzylamine;
Synthesis.
*
Corresponding author. Tel.: +1 8586177634; fax: +1 8586177634;
e-mail: cchen@neurocrine.com
Figure 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.058

4616
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4615–4618
tion, probably associated with its high hydrophilicity
(log D = 1.1) and dibasic structure. Incubation of 3 or
its close analogues with liver microsomes resulted in ra-
pid degradation, which appears to be related to the thi
enylethylamino group. We postulated that the large
and flexible nature of this side chain of 3 may contribute
at least in part to the poor metabolic stability of lipo-
philic derivatives (calculated log D value of 3 was 4.0).6
Previous structure–activity studies on the N-side chain
of the benzylamines have shown that a diamine such
as 3-aminopiperazine provides compounds with high
binding affinity.8 However, compounds bearing a dia-
mine side chain possess high residual agonistic activity.
Alternatively, 2-thienylethylamine was found to be a
side chain at the basic nitrogen for compounds with high
binding affinity but low cAMP stimulation. On the basis
of these early results, a study was conducted to find a
replacement for the lipophilic and flexible 2-thienylethyl
moiety of 1–3. In addition to small alkyls such as the
cyclopropyl group, a set of alkoxyalkyls were evaluated
because of their lower lipophilicity, which led to the
identification of potent analogue 4a (Ki = 60 nM)
(Table 1). The non-branched methoxyethyl analogue
4b (Ki = 190 nM) displayed a 3-fold reduction in bind-
ing. Larger isopropoxyethyl (4c) and methoxy-2-butyl
(4e) derivatives were slightly less potent. The additional
methoxy group of 4d (Ki = 280 nM) resulted in further
loss of potency.
4-Chlorophenylalanine derivatives of 4 were synthesized
in a manner similar to that used for the preparation of
3.5a In brief, piperazinebenzaldehyde 8, after trifluoro-
acetic acid (TFA) deprotection, was coupled with N-
Boc-R-(4-chlorophenyl)alanine under standard coupling
conditions to give 9. Reductive amination of 9 with a set
of alkylamines in the presence of sodium triacetoxyboro-
hydride afforded the desired products 4a–f (Scheme 1).
Alternatively, the piperazinebenzaldehyde 8 was subject-
ed to reductive amination with 1-methoxy-2-propyl-
amine using sodium triacetoxyborohydride, followed
by protection of the resulting secondary amine with an
Fmoc group to give the protected diamine intermediate
10. Selective deprotection with TFA in dichlorometh-
ane, followed by peptide coupling with N-Boc-2,4-
dichlorophenylalanine using a standard protocol, gave,
after TFA deprotection, the amide 11. Reaction of 11
with alkyl chloroformates under basic conditions, fol-
lowed by Fmoc-deprotection with diethylamine, provid-
ed carbamates 5a–d. Similarly, reaction of 11 with
isocyanates in dichloromethane, and then with diethyl-
amine provided the ureas 6a–e. Alternatively, the ureas
6f–h were obtained from the reaction of 11 with phos-
gene in toluene, followed by the addition of alkylamines.
Finally, coupling of 11 with various carboxylic acids
gave the amides 7a–i after Fmoc deprotection. Final
products were purified by a HPLC equipped with a mass
detector as previously described.5b Compounds were
tested in the competitive binding experiments using
We have previously shown that the 2,4-dichlorophenyl-
alanine derivative 2 is about 10-times more potent than
the monochloro analogue
1 (Ki = 21 nM, IC50 =
90 nM).5 As expected, replacing the 4-chlorophenylala-
nine moiety of 4a with a 2,4-dichloro analogue increased
the binding affinity by 10-fold (5a, Ki = 5.4 nM) (Table
2). To further optimize this compound, a number of car-
bamates, ureas, and amides were prepared in the
1-methoxy-2-propylamine series. As shown in Table 2,
Table 1. SAR of N-alkoxyalkyl benzylamines 4a–f at MC4R
Compound
R1
Ki (nM)a
4a
4b
4c
4d
4e
4f
CH(Me)CH2OMe
CH2CH2OMe
60
190
110
280
120
210
i
CH2CH2 OPr
CH2CH(OMe)2
CH(Et)CH2OMe
S-CH(Bn)CH2OMe
[
125I]-NDP-MSH as the radiolabeled ligand and in the
a Binding affinity at the human melanocortin-4 receptor (MC4R) sta-
bly transfected in HEK 293 cells, using [125I]-NDP-MSH as the
radiolabeled ligand.
antagonist assay in HEK293 cells stably transfected with
the human melanocortin-3 and -4 receptors as previous-
ly described.7
Scheme 1. Reagents and conditions: (a) TFA/CH2Cl2; (b) N-Boc-(R)-(4-Cl)Phe-OH/EDC/HOBt/DCM/rt, 80%; (c) R1NH2/NaBH(OAc)3/DCM/rt;
(d) CH3CH(NH2)CH2OCH3/NaBH(OAc)3/DCM; (e) Fmoc-Cl, 82%; (f) N-Boc-(R)-(2,4-Cl)Phe-OH/EDC/HOBt/DCM, 80%; (g) ROCOCl/Et3N/
CH2Cl2/rt, or RNC/ CH2Cl2/rt, or COCl2/THF/rt then RNH2, or R2COOH/EDC/HOBt/DCM; (h) Et2NH/DCM.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4615–4618
4617
Table 2. Binding affinity (Ki, nM) of compounds 5–7 at MC3R and
MC4Ra
Table 3. Binding affinity (Ki, nM) at the melanocortin receptor
subtypes of selected compoundsa
Compound MC1R MC3R MC4R MC5R IC50(nM)d
6a
6b
6e
6g
7b
7f
—
—
2600
2900
1400
4.9
4.8
4.7
4.8
6.9
1.2
8.8
1400 NDc
800 NDc
620 150
(9%)b
(13%)b 3100
(16%)b 2800
(36%)b
200
1100 220
(49%)b 170
400 180
890
4900
7g
580 120
Compound
R2
MC4Rb
MC3R
a Binding affinity at the human melanocortin receptors stably trans-
fected in HEK 293 cells, using [125I]-NDP-MSH as the radiolabeled
ligand.
5a
5b
5c
OtBu
OiBu
OEt
5.4
7.7
17
2800
2600
—
b Percentage of inhibition at 10 lM concentration.
c ND, not determined.
5d
6a
6b
6d
6e
OMe
NHiPr
NHsBu
NHPh
19
—
4.9
4.8
5.5
4.7
3.5
5.3
4.8
7.2
27
2600
2900
6600
1400
1600
3000
3100
3100
—
d Inhibition of a-MSH-stimulated cAMP production in CHO cells
expressing the human melanocortin-4 receptor.
NHCH2CH2OH
NHBn
6e
6f
metabolism, we tested all of the potent MC4R com-
pounds in the MC3R binding assay. None of these com-
pounds possessed high binding affinity at this receptor
(Ki > 900 nM, Table 2), demonstrating high MC4R
selectivity of these compounds. For example, 7b dis-
played 400-fold selectivity with Ki values of 6.9 and
2800 nM, respectively, at MC4R and MC3R. High
selectivity was also demonstrated at the other melano-
cortin receptor subtypes for compounds 6a, b, e, g, 7b,
f, and g (Table 3).
1-Piperidinyl
1-Pyrrolidinyl
NMe2
6g
6h
7a
7b
7c
H
Et
nBu
6.9
5.3
3.1
2.2
1.2
8.8
5.6
88
2800
3700
3700
1900
890
7d
7e
Cyclohexyl
CH2-2-thienyl
2-Thienyl
CH2CH2NH2
CH2CH2NH2
CH2CH2NH2
7f
7g
R-7g
S-7g
2
4900
3400
—
All compounds derived from 2,4-dichlorophenylalanine
were unable to stimulate significant levels of cAMP
accumulation at 10 lM concentration (<3% of a-MSH
maximal levels, data not shown) in cells expressing the
human MC4 receptor, demonstrating that these com-
pounds were not functional MC4 agonists. Moreover,
selected compounds (6e, g, 7b, f; and g, Table 3) were
shown to dose dependently inhibit a-MSH-stimulated
cAMP production. For example, 7b and g exhibited
IC50 values of 170 and 120 nM, respectively, in this
assay.
1.8
640
a Binding affinity at the human melanocortin-4 (MC4) or melanocor-
tin-3 (MC3) receptor stably transfected in HEK 293 cells, using [125I]-
NDP-MSH as the radiolabeled ligand.
b Data are average of three or more independent measurements and
SEM is <25% of the average.
carbamates with a smaller alkyl group exhibited slightly
reduced potency (5c–d) from the tert- and iso-butyl
analogues 5a–b. Ureas 6a–g possessed Ki values of
3.5–7.2 nM regardless of the size of the N-alkyl group.
Various amides 7a–g also displayed high binding affini-
ty. Among them, the formyl 7a (Ki = 27 nM) was the
least potent, and the 2-thienyl analogue 7f had the best
Ki value of 1.2 nM, suggesting a lipophilic amide is pre-
ferred. The compound derived from b-alanine 7g exhib-
ited a Ki of 8.8 nM, which had similar binding affinity to
the des-amino analogue 7b (Ki = 6.9 nM).
Compound 7b had a measured log D value of 1.8,
which is a very desirable value for a CNS agent.9
Therefore, this compound was further studied in rats
for oral bioavailability and brain penetration (Table
4). After intravenous administration of 5 mg/kg, as
an aqueous solution of the hydrochloride salt, 7b dis-
played moderate volume of distribution (Vd = 6.2 l/kg)
Next, we determined the potency of the individual dia-
stereomer of N-(1-methoxy-2-propyl) 7g. The two dia-
stereoisomers (R-7g and S-7g) were separated using
HPLC. The stereochemistry of S-7g was established by
chemical synthesis from commercially available S-1-
methoxy-2-propylamine using the procedure described
in Scheme 1. The R-isomer R-7g possessed a Ki value
of 5.6 nM, which was much better than the correspond-
ing S-7g (Ki = 88 nM). In comparison with the more
lipophilic 2-thienylethyl analogue 2, R-7g is only 3-fold
less potent.
Table 4. Pharmacokinetic profile of 7b in rats (N = 3)
iv (5 mg/kg)
CL (ml/min kg)
Vd (l/kg)
t1/2 (h)
52
6.2
1.4
280
Cbrain at 1 h (ng/g)
Cbrain/Cplasma at 1 h
0.5
po (10 mg/kg)
Tmax (h)
6.0
Cmax (ng/ml)
AUC0–8 (ng/ml h)
F (%)
47
250
8
Since the MC3 receptor is also centrally localized and
may also play a role in the control of food intake and

4618
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4615–4618
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Figure 2. Pharmacokinetic profile of compound 7b in rats (5 mg/kg, iv,
10 mg/kg, po, N = 3).
and high clearance (Cl = 52 ml/min kg), which resulted
in a short half-life (t1/2 = 1.4 h). At the 1-h time point,
the concentration of 7b in the brain was 280 ng/g,
which reflected a brain/plasma ratio of 0.5. Oral dose
at 10 mg/kg resulted in a maximal concentration
(Cmax) of 47 ng/ml at the 6-h time point (Tmax), and
an area under curve (AUC0–8) value of 250 ng/ml.h.
The absolute bioavailability (F) thus was calculated
to be 8% in this species (Fig. 2). Although the abso-
lute bioavailability of compound 7b in rats was mod-
erate based on this 8-h PK study, it displayed
reasonably good brain penetration.
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J.; Saunders, J.; Murphy, B.; Foster, A. C.; Chen, C.
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In conclusion, we have identified several piperazineben-
zylamines bearing an N-(1-methoxy-2-propyl) group as
potent and selective antagonists of the human MC4
receptor. In addition, many compounds possessed suit-
able lipophilicity for potential oral administration.
Compound 7b, having a desirable log D value of 1.8,
exhibited moderate oral bioavailability and blood–brain
barrier penetration in rats.
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