Pharmacological and pharmacokinetic characterization of 2-piperazine-α-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2008.03.072

A series of 2-piperazine-α-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compo

Full text of DOI:10.1016/j.bmc.2008.03.072

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5606–5618
Pharmacological and pharmacokinetic characterization of
2-piperazine-a-isopropyl benzylamine derivatives as
melanocortin-4 receptor antagonists
Chen Chen,a,* Fabio C. Tucci,a,ꢀ Wanlong Jiang,a Joe A. Tran,a Beth A. Fleck,b
Sam R. Hoare,b Jenny Wen,c Takung Chen,c Michael Johns,c Stacy Markison,d
Alan C. Foster,d Dragan Marinkovic,a Caroline W. Chen,a Melissa Arellano,a
John Harman,a John Saunders,a,ꢁ Haig Bozigiane and Daniel Markse
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
eDepartment of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA
Received 6 March 2008; revised 25 March 2008; accepted 28 March 2008
Available online 1 April 2008
Abstract—A series of 2-piperazine-a-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 recep-
tor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting
compounds 8–12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists.
These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver
microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They
also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic
properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations.
Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model.
After intraperitoneal (ip) administration of 1 mg/kg dose, mice treated with 10d had significantly more food intake and weight gain
than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d
was dosed orally at 20 mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in
the treatment of cancer cachexia.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
obstructive pulmonary disease, and renal failure. Cancer
cachexia significantly impairs quality of life and
response to anti-neoplastic therapies and ultimately
increases morbidity and mortality of cancer patients. It
is estimated to be responsible for over 20% of all can-
cer-related deaths.1 Unfortunately, currently available
drugs are not very effective.2
Cachexia, characterized by weight loss, wasting of mus-
cle, loss of appetite, and general debility, often coincides
with chronic disease states including cancer, chronic
Keywords: Melanocortin-4 receptor; Antagonist; Pharmacokinetics;
Cachexia; SAR.
The melanocortin-4 receptor (MC4R), a member of the
class A G-protein coupled receptor superfamily, is
expressed in the hypothalamus, brain stem, and many
other brain regions and plays a significant role in feeding
behavior and energy homeostasis in animals and
humans.3 While MC4R agonists have demonstrated
efficacy in suppressing food intake and reducing body
*
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7602; e-mail: cchen@neurocrine.com
ꢀ
Present address: Department of Medicinal Chemistry, Tanabe
Research Laboratories, USA, Inc., 4540 Towne Centre Ct., San
Deigo, CA 92121, USA.
ꢁ
Present address: Vertex Pharmaceuticals Inc., 11010 Torreyana
Road, San Diego, CA 92121, USA.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.072

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5607
Cl
Cl
F3C
O
Cl
N
NH
O
N
N
Br
N
N
O
O
NH
NH2
F
O
O
O
NH2
2
1
3a
F
Cl
F
Cl
Cl
Cl
N
N
N
N
NH
N
N
NH
N
NH
N
O
O
O
O
O
O
NH2
HN
N
3b
10d
4
Figure 1. Chemical structures of some MC4R antagonists 1–3, 10d, and agonist 4.
weight in animals suggesting their potential in the treat-
ment of obesity,4 MC4R antagonists promote food in-
take and increase weight gain.5 Recent studies have
demonstrated that cachexia brought about by a variety
of illnesses can be attenuated or reversed by blocking
MC4R activation within the central nervous system.6
activity relationship studies led to the identification of
potent MC4R antagonists such as 3b (Ki = 0.94 nM),
which demonstrates efficacy in the murine cachexia
model.16 However, 3b has low brain penetration in mice
that is probably caused by its low permeability and high
efflux ratio associated with its high polar surface area.
Based on these leads, we started a research effort to
improve their properties in several categories including
potency and pharmacokinetics. Here we report the
synthesis and characterization of a series of a-isopropyl
benzylamine derivatives, exemplified by 1-{2-[(1S)-2-
(1-(dimethylaminopropionyl)amino)-2-methylpropyl]-6-
fluorophenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)propi-
onyl]piperazine (10d, Fig. 1), as MC4R antagonists.§
Non-peptide MC4R antagonists have been sought and
identified in recent years.7 Because of their ability to
penetrate the blood–brain barrier, small molecules with
appropriate pharmacokinetic properties have been
peripherally administered to demonstrate the pharmaco-
logical role of the MC4 receptor. One example is the 2-
phenylimidazoline 1 (Fig. 1). After subcutaneous (sc)
administration twice daily at 15 mg/kg for 13 days to
C57BL6 mice-bearing tumors induced by inoculation
with Lewis lung carcinoma (LLC) cells, this functional
MC4R antagonist (IC50 = 103 nM)8 stimulated food in-
take during the light-phase. During a 21-day experi-
2. Chemistry
The target compounds 8–13 were synthesized from
the protected piperazinebenzylamines 517 as shown in
Scheme 1. Compounds 5 were coupled with (2R)-methyl-
3-(4-chlorophenyl)propionic acids18 to give amides 6,
which were deprotected with HCl in methanol to afford
benzylamines 7. Reductive alkylation of 7 with methyl-
(2-oxoethyl)carbamic acid tert-butyl ester provided dia-
mines 8–9 after Boc-deprotection. Alternatively, coupling
reactions of 7 with N,N-dimethylamino-b-alanine, or with
an N-Boc-amino acid followed by TFA treatment, affor-
ded 10–13.
ment, the tumor-bearing mice treated with
1
maintained their lean body mass while vehicle-treated
controls showed a reduction.9 The binding affinity
(IC50 = 160 nM) of this compound at MC4R is only
moderate, and it might interact with the melanocortin-
3 receptor which is also believed to play a role in feeding
regulation.10 We have shown that a b-alanine-2,4-
dichlorophenylalanine dipeptide derivative 2 (Fig. 1) is
a potent functional MC4R antagonist with negligible
affinity at MC3R.11 In addition, 2 has good brain pene-
tration. Intraperitoneal (ip) administration of 2 effec-
tively stimulates daytime food intake as well as
decreases basal metabolic rate in normal animals. Fur-
thermore, this compound attenuates cachexia and pre-
serves lean body mass in a murine cancer model.12
These data have demonstrated the potential of small
molecule MC4R antagonists in the treatment of cancer
cachexia.13
3. Results and discussion
Compounds 7–13 were tested for their binding affinity at
the human melanocortin-4 receptor (hMC4R) using a
binding assay as previously described,19 and the results
are summarized in Table 1. Functional antagonism
was determined by their dose-dependent inhibition of
a-MSH-stimulated cAMP production in CHO cells sta-
Previously, we reported the discovery of a series of pip-
erazinebenzylamines such as 3a (Ki = 6.5 nM) as MC4R
antagonists.14,15 While this highly lipophilic compound
(clogP = 6.1) displayed metabolic instability in human
liver microsomes (CLsys = 17 mL/min kg), structure–
§
Part of this work has been published as a communication, see Ref.
24.

5608
C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
Cl
O
S
O
S
H
N
X
X
X
HO
N
H
N
H
N
H
Cl
Cl
O
Y
N
N
N
N
N
NH
a
O
Y
O
Y
5a: X = 6-F
5b: X = 4-Me
5c: X = 4-Cl
6a-h
8: X = 6-F, Y = H
9: X = 4-Me, Y = F
6d: X= 4-Me, Y = H
c
b
O
X
X
d or e
N
H
R
O
NH2
N
Cl
Cl
N
N
N
Y
O
Y
7a-c: X = 6-F
7d-f: X = 4-Me
7g: X= 4-Cl
7h: X = H
10a-e: X = 6-F
11a-f: X = 4-Me
12a-f: X = 4-Cl
13: X = H
Scheme 1. Reagents and conditions: (a) i—SOCl2/reflux, ii—Et3N/CH2Cl2/rt, 8 h or EDC/HOBt/DIEA/DMF/rt, 3 h, 40–80%; (b) HCl/MeOH/rt,
1 h, 60–90%; (c) N-BocNMeCH2CHO/NaBH(OAc)3/CH2Cl2/rt, 8–14 h; then TFA/CH2Cl2/rt, 1–2 h, ꢀ20%; (d) N-Boc-amino acid/EDC/HOBt/
NaHCO3/DMF/CH2Cl2/rt, 8 h; or HBTU/DIEA/DMF/rt, 8 h; then TFA/CH2Cl2/rt 1 h, 30–60%; (e) Me2NCH2CH2COOH/EDC/HOBt/Et3N/
CH2Cl2/rt, 8 h, 70–90%.
Table 1. SAR of MC4R antagonists at the human melanocortin-4 receptora
c
IC50 (nM)
Compound
X
R
Y
Kib (nM)
7a
7b
6-F
6-F
H
83
26
n.d.d
n.d.d
n.d.d
2400
n.d.d
n.d.d
750
n.d.d
480
660
340
390
n.d.d
270
42
MeO
Cl
7c
7d
6-F
27
69
4-Me
4-Me
4-Me
4-Cl
H
H
7e
F
35
7f
7g
Me
H
9.7
210
180
23
7h
8
H
6-F
H
9
4-Me
6-F
6-F
F
6.0
10
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
13
CH2NHMe
CH2NHMe
CH2NHMe
H
MeO
Cl
3.4
5.3
7.5
3.1
2.8
5.9
5.7
2.8
2.2
0.6
4.1
1.1
0.8
3.3
1.3
5.4
24
6-F
6-F
CH2CH2NMe2
Azetadin-4-yl
CH2NHMe
H
6-F
MeO
F
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
410
280
87
CH2CH2NH2
CH2CH2NHMe
CH2CH2NMe2
CH2CH2NMe2
CH2CH2NMe2
CH2NHMe
H
H
H
35
F
190
92
Me
H
300
290
150
150
n.d.d
160
480
CH2NHMe
MeO
MeO
H
R-CH(Me)NH2
CH2CH2NMe2
Azetadin-4-yl
1-Methyl-azetadin-4-yl
CH2CH2NMe2
MeO
H
H
a Data are average of two or more independent measurements.
b Binding affinity was obtained using [125I]NDP-MSH as the radiolabeled ligand.
c Dose-dependent inhibition of a-MSH-stimulated cAMP production.
d Not determined.
bly expressing hMC4R. For the binding affinity of the
primary benzylamines 7a–c, the 2-methoxy and 2-chloro
derivatives 7b and 7c were much more potent than the 2-
unsubstituted compound 7a. For 7d–f, the 2-methyl 7f
was about 7-fold more potent than the 2-unsubstituted
analog 7d and 4-fold more potent than the 2-fluoro 7e.

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5609
These results indicate that a small group at the ortho-po-
sition of this 4-chlorophenyl group is favored, probably
due to a steric effect to steer the phenyl ring to a desir-
able orientation for receptor interactions. In compari-
son, a small substituent at the left side phenyl ring had
a minimal effect on binding affinity of these compounds
(7a, 7d, 7g, and 7h), only the 4-methyl compound 7d dis-
played about 2-fold improvement over the non-substi-
tuted 7h.
the reason for a lack of clear correlation between these
two parameters is unknown.
Compounds 10–12 were also tested for their binding
affinity at the other melanocortin receptor subtypes
(Table 2). In general, these compounds had little interac-
tion with MC1R and displayed much lower affinity at
MC3R than MC4R. For example, 10d and 11d had over
60- and 100-fold selectivity, respectively, at MC4R over
MC3R or MC5R. Several compounds (10b, 12b, and
Incorporating a methylaminoethyl side-chain to benzyl-
amine 7a (Ki = 83 nM) increased the binding affinity
about 4-fold (8, Ki = 23 nM). Similarly, a 6-fold
improvement was observed for diamine 9 (Ki = 6 nM)
compared to 7e. The sarcosine derivative 10a (Ki =
10 nM) possessed slightly higher affinity than the meth-
ylaminoethyl 8, and this was supported by the compar-
ison between 9 and 11a. These results indicate that the
additional amino group in 8 and 10a is able to function
as the benzylamine of 7a to further increase affinity. The
12c) bearing
a
2-methoxy-4-chlorophenylpropionyl
group exhibited high binding affinity at MC5R. For
example, compound 12b had a Ki value of 13 nM at
MC5R, while its binding affinity at MC4R was
1.1 nM. The wider receptor selectivity of compounds
10d and 11d was tested at a concentration of 10 lM in
a panel of radioligand binding assays for approximately
70 receptors, ion channels, enzymes and transporters as
described previously.12 Significant activity was seen only
at the ghrelin receptor with Ki values of 240 and 680 nM
for 10d and 11d, respectively. In a functional assay, 10d
had negligible activation at the ghrelin receptor (8%
stimulation at 10 lM concentration based on calcium
flux). Because of the low affinity of 10d and 11d for
the ghrelin receptor compared to MC4R (32- and 200-
fold selectivity, respectively), the contribution of the
ghrelin component to the actions of 10d and 11d should
be minimal.20 Consequently, both compounds show
excellent selectivity for MC4R.
3-(N,N-dimethylamino)propionyl
derivative
10d
(Ki = 7.5 nM) was 11-fold more potent in binding affin-
ity than its parent 7a. Similar results were also obtained
for 7d–f/11d–f, 7g/12d, and 7h/13 modifications. For the
sarcosine derivatives 10a–c, a chloro (10c) or a methoxy
group (10b) at the 2-position of the phenyl ring in the
2R-methyl-3-phenylpropionyl moiety improved affinity
by 2–3 times. Compound 10e, which bears a highly basic
azetadine functionality, also exhibited potent binding
affinity.
The X-ray crystal structure of 6d was resolved (Fig. 3).
In this solid state, the 4-chlorophenyl ring of 6d was al-
most parallel to the piperazine ring. Interestingly, this
precursor of antagonist 7d possessed a very similar con-
formation to that of an MC4R agonist 4 (THIQ, Fig. 1)
found in its X-ray crystal structure,20 suggesting the
framework of an antagonist structure is not much differ-
ent than that of an agonist. This result indirectly indi-
cates that the Tic-group of 4, which is missing from
antagonist 7d, is the primary contributor to receptor
activation. This conformation was further demonstrated
by the X-ray crystal structure of 11e as a mesylate salt,
Like 11d (Ki = 2.8 nM), the N,N-dimethyl-b-alanine
derivative 12d (Ki = 3.3 nM) also possessed potent bind-
ing affinity. Compound 13 (Ki = 24 nM), however,
exhibited moderate potency. None of the compounds
8–12 had significant stimulation of cAMP production
at 10 lM concentration (Emax 6 15%) except 7h (38%).
All of the compounds tested in the functional antagonist
assay showed dose-dependent inhibition of a-MSH-
stimulated cAMP production (Table 1). For example,
11d exhibited an IC50 value of 35 nM in this assay
(Fig. 2). The discrepancy between the Ki and IC50 values
is probably due to different assay conditions, however,
Table 2. Selectivity profiles (Ki, nM) of MC4R antagonists at the
human melanocortin receptor subtypesa
110
90
70
50
30
10
Compound
hMC1Rb
hMC3R
hMC4R
hMC5R
10a
10b
10d
11a
11b
11c
11d
12a
12b
12c
12d
12f
(49%)
(11%)
(34%)
n.d.c
570
190
490
230
270
710
590
200
160
220
450
380
10
1100
67
3.4
7.5
2.8
5.9
5.7
2.8
4.1
1.1
0.8
3.3
5.4
1100
180
320
340
1200
110
13
(42%)
(43%)
(30%)
(28%)
(19%)
(40%)
6200
-10
-9
-8
-7
-6
-5
-10
log [11d] (M)
34
390
400
Figure 2. Functional activity of compound 11d. (Antagonism of a-
MSH-stimulated cAMP production, where 100% cAMP is defined as
stimulation of cAMP production by 10 nM a-MSH (ꢀEC50) in the
absence of antagonist. Assay was performed in CHO cells transfected
with hMC4R, and cAMP was measured using an ELISA assay. Data
are average of two independent experiments.)
8200
a Data are average of two and more independent measurements.
b Percentage inhibition at 10 lM concentration is indicated in
parentheses.
c Not determined.

5610
C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
Table 3. Physicochemical properties of MC4R antagonistsa
Compound MW logDb clogD clogP pKa
2
˚
PSA (A )
7d
8
428
489
503
503
533
537
531
545
516
499
513
527
545
541
519
549
549
547
561
545
n.d.c
2.6
2.5
2.4
3.1
4.3
2.4
2.6
4.0
2.4
2.7
3.4
3.5
4.0
4.3
3.5
4.5
4.0
2.3
3.0
2.3
2.0
1.5
3.1
3.3
3.7
3.4
1.7
2.8
2.5
1.9
3.4
3.0
3.9
3.5
3.6
3.8
3.7
2.0
3.0
4.7
4.5
4.2
3.5
3.7
4.2
4.4
3.8
3.3
3.7
4.0
4.5
4.1
4.9
3.9
4.1
4.4
4.7
4.1
4.4
9.8
50
10, 5.8 48
10, 6.2 48
9
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
8.0
8.0
8.0
8.9
9.8
8.0
8.9
9.6
8.9d
8.9
8.9
8.0
8.0
8.2
8.9
9.8
9.1
65
74
65
56
74
65
79
65
56
56
56
65
74
88
56
74
56
Figure 3. X-ray crystal structures of 6d.
which possessed a similar arrangement between the 2-
fluoro-4-chlorophenyl ring and the piperazine group
(Fig. 4).
a Calculated values were obtained using ACD software.
b Measured using a shake-flask method at pH of 7.4.
c Not determined.
All of the potent compounds possessed high molecular
weights (489–561). This feature presents a challenge
for good oral bioavailability and brain penetration.
Most of the compounds possessed moderate to high
lipophilicity based on the measured logD values at pH
of 7.4 using a shake-flask method (Table 3). These num-
bers matched reasonably well with the calculated values
using ACD software, although for some highly basic
amines such as 9 and 10e, the difference was as high as
one log unit. The measured pKa value of N,N-dimethyla-
minopropionamide 11d was 7.8, about one log unit low-
er than the calculated 8.9, which might explain the
discrepancy for the more basic compounds. The polar
surface area (PSA) for these compounds was also calcu-
lated in an attempt to correlate them with permeabil-
ity.21 Most compounds exhibited moderate values
d The measured pKa value was 7.8.
protein (P-gp) transporter activity, and the results are
summarized in Table 4. Most compounds displayed
low to moderate permeability from the apical to basolat-
eral direction (a to b), presumably due to their high
molecular weights. Many compounds had high perme-
ability from basolateral to apical direction (b to a), indic-
ative of an efflux mechanism due to high P-gp activity.
Benzylamine 7d showed moderate permeability
Table 4. Permeability in Caco-2 cells and metabolic stability of 8–12a
c
2
˚
Compound
Papp
(10À6 cm/s)
Efflux ratio
(b to a/a to b)
CLsys
(mL/min kg)
between 50 and 60 A (Table 3).
These MC4R antagonists were also tested in an in vitro
Caco-2 assay measuring cell permeability and P-glyco-
7d
8
5
3.7
61
13.4
13.5
n.d.b
13.3
16.5
14.7
11.9
14
0.4
3
9
26
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
1
15.4
8.3
14
2
2
10
0.2
8
4.4
740
7.3
25.8
7.2
4.5
3
12.4
9.2
3
12
41
37
16
n.d.b
n.d.b
3
4.8
12.2
16.1
16.6
13.1
16.1
15.5
15.9
12.3
11.6
4.5
n.d.b
n.d.b
6.9
11
9
0.3
2
120
14.4
a Digoxin was used as a control in the Caco-2 assay, and it showed on
average Papp of 1.0 · 10À6 cm/s and efflux ratio of 20–30.
b Not determined.
c Determined using human liver microsomes in an in vitro assay.
Figure 4. X-ray crystal structures of 11e mesylate.

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5611
(Papp = 5.0 · 10À6 cm/s) and a relatively low efflux ratio
(b to a/a to b = 3.7). Methylaminoethyl 9 exhibited lower
permeability and higher efflux ratio (Table 4). In
comparison, the monobasic sarcosine 11a showed an
improved permeability profile compared to 9. The
dosing, respectively), presumably due to P-gp activity
impairing its penetration through the blood–brain bar-
rier.23 In the in vitro Caco-2 assay, 8 exhibited poor per-
meability (Papp = 0.4 · 10À6 cm/s) and a very high efflux
ratio of 61 (Table 4), indicating strong P-gp activity.
2
˚
3-(N,N-dimethylamino)propionyl
side-chain
(11e),
Interestingly, the calculated PSA value of 48 A was
however, increased permeability from 11a. For the three
b-alanine derivatives 11b–d, the primary amine 11b had
lowest permeability and highest efflux, while the
monomethylamine 11c displayed an improvement. The
high permeability of 11d might be associated with its
high logD value of 3.4 and/or lower polar surface area
moderate. The three sarcosine derivatives 10a–c dis-
played high plasma clearance (39–52 mL/min kg) and
high volume of distribution (13–29 L/kg). The t1/2 for
these compounds ranged from 3.8 to 6.5 h, and their
oral bioavailabilities were moderate (12–23%). The least
lipophilic compound 10a (logD = 2.4) had the highest
brain concentrations at 1 and 4 h postdosing. The 3-
(N,N-dimethylamino)propionyl 10d had a moderate Vd
of 5.2 L/kg, a short t1/2 of 2.3 h, and penetrated into
the brain with a brain/plasma (b/p) ratio of 0.6 and 0.8
at the 1 and 4 h time points, respectively. Compound
10d also had good plasma exposure after oral adminis-
tration, resulting in a 42% oral bioavailability. In con-
trast, the highly basic azatadine 10e had lower brain
penetration and much lower oral bioavailability com-
pared to 10d. In the Caco-2 assay, 10d had a moderate
Papp value (10 · 10À6 cm/s) and a relatively low efflux
ratio (4.4) compared to Papp of 0.2 · 10À6 cm/s and an
efflux ratio of 740 for 10e. Therefore, the difference in
permeability and P-gp activity between these two com-
pounds might be the key determinants of their differing
oral absorption and brain penetration profiles.
2
˚
(56 A ) than the secondary and primary amines 11b–c
2
(PSA = 65 and 79 A , respectively).
˚
When incubated with human liver microsomes in an
in vitro assay, all compounds exhibited moderate meta-
bolic stability, which might be associated in part with
their high lipophilicity or/and high molecular weight
(Table 4). For example, the 3-(N,N-dimethylamino)pro-
pionyl 11d (CLsys = 12.2 mL/min kg) had a much higher
systemic clearance than its less lipophilic monomethyl
analog 11c (CLsys = 4.8 mL/min.kg).
3.1. Pharmacokinetic properties
Compounds 8, 10a–e, 11a, 11d, and 12a–f were studied
in mice for their pharmacokinetic properties, and the
results are summarized in Table 5. Whole brain concen-
trations were sampled at 1 and 4 h after intravenous
administration in these studies. Diamine 8 had a moder-
ate plasma clearance (CL = 36 mL/min kg) and a very
large volume of distribution (Vd = 52 L/kg), which
might be associated with its dibasic structure,22 resulting
in a very long half-life (t1/2) of 17 h. Oral bioavailability
(F% = 47) was good in this species. Despite its high tis-
sue distribution, the compound had moderate whole
brain concentrations (53 and 62 ng/g at 1 and 4 h post-
The 4-methylphenyl compounds 11a and 11d had mod-
erate oral bioavailability but low brain penetration.
Among the 4-chlorophenyl derivatives 12a–f, 12b, and
12d had high brain concentrations, while 12f had high
oral bioavailability. The low permeability and high
P-gp activity of 12f may have impaired its brain penetra-
tion but not intestinal absorption. It is worth noting that
2
˚
the calculated PSA (56 A ) of 12f was similar to that of
12d.
Table 5. Pharmacokinetic parameters of MC4R antagonists in micea
Compound CL (mL/min kg) Vd (L/kg) t1/2 (h) Cb (ng/g) at 1, 4 hb b/p ratio at 1, 4 h Tmax (h) Cmax (ng/mL) Oral AUC F%
(ng/mL h)
8
36
42
52
39
26
28
24
29.6
52
19
29
13
17
5.2
53, 62
0.4, 1.2
0.6, 1.1
0.5, 1.7
0.3, 0.5
0.6, 0.8
0.1, 0.4
0.1, 0.2
0.1, 0.1c,e
0.7, 1.5
1.3, 1.3
0.4, 1.0
1.9, 2.3
0.1, 0.4
0.1, 0.1
0.5
0.5
0.5
2.0
0.5
2.0
2
166
165
114
172
308
104
632
946
88
1629
813
47
21
12
23
42
10a
10b
10c
10d
10e
11a
11d
12a
12b
12c
12d
12e
12f
231, 126
165, 107
85, 71
6.5
3.8
2.3
4.4
1.4
1.6
1.8
1.8
1.9
1.9
4.2
3.2
374
1014
2690
336
5.2
322, 175
51, 32
10
3
5.6
59, 42
2290
2530
232
30
32
13
4.1
19, 18c,d
131, 62
334, 102
95, 59
0.5
0.5
0.5
0.25
1
102
16
11
16
11
69
74
64
23
11
55
208
397
8.0
143
109
151
762
16
19
580, 176
61, 39
79, 77
600
422
8.2
3
2
5.8
58
6
9163
a Three animals were dosed intravenously at 5 mg/kg and orally at 10 mg/kg.
b Brain concentrations were taken from iv dose.
c Brain samples were taken from oral administration.
d Brain Cmax = 91 ng/g, Tmax = 0.3 h, AUC = 367 ng/g h.
e b/p (Cmax) = 0.1; b/p (AUC) = 0.15.

5612
C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
The brain concentration versus time relationship of 11d
in mice was studied in a separate experiment. After a
10 mg/kg oral dosing, a Cmax of 91 ng/g appeared at
0.3 h time point. The area under the curve (AUC) was
367 ng/g h (Fig. 5). The brain/plasma ratio was 0.1
based on Cmax values or 0.15 based on AUCs. These
results confirmed the finding from 1 and 4 h sampling
(Table 5).
which were similar to that of 11d. The half-life of 10d
was shorter in mice (2.3 h) than in dogs (7.0 h). In com-
parison, 11d exhibited a similar t1/2 of 6.7 h in dogs.
3.2. In vivo efficacy studies
We have previously established a mouse cachexia model
in which the cachectic state is induced by inoculation
with LLC tumor cells.12 Compounds 2 and 3b have
demonstrated efficacy in this model in that they reverse
the weight loss that is observed in the control group.
Since compounds 10d and 11d exhibited superior phar-
macological and pharmacokinetic profiles, they were
also tested in this model. The binding affinity of 10d
was determined at the mouse melanocortin receptors,
and it was found to bind mouse MC4R (Ki = 3.5 nM)
much better than mouse MC3R (Ki = 190 nM). Similar
selectivity was also demonstrated at the rat receptors
(Ki = 5.5 nM for MC4R vs 340 nM for MC3R). There-
fore, no species selectivity in terms of receptor affinity
was observed for this compound.
The brain concentration–time relationship of 11d was
also studied in rats after an oral administration at
10 mg/kg. The maximal whole brain concentration of
11d was 41 ng/g at the 6 h time point, and the AUC
was 254 ng/g h. The brain to plasma ratio was 0.17
based on AUC values.24 These results were consistent
with those obtained in mice for this compound.
Compounds 10d and 11d were studied in rats, monkeys,
and dogs for their pharmacokinetic properties, and the
results for 10d are summarized in Table 6 (data for
11d can be found in Ref. 24).
The PK profile of 10d in rats was very similar to that of
11d. One significant difference was the much slower
brain penetration of 10d compared to that of 11d. The
maximal brain concentration of 10d (52 ng/g) appeared
at 6 h postdosing (2.7 h for 11d). The b/p ratio of 10d
based on AUCs (0.48) was larger than that based on
Cmax values (0.27). 10d had a relatively large Vd value
across all species (5.2–8.7 L/kg). The oral bioavailabili-
ties of 10d were moderate (21–42%) in these species,
For the in vivo study, C57BL/6J male mice were inocu-
lated with Lewis lung carcinoma (LLC) tumor cells.
Beginning 10 days after LLC inoculation, animals were
treated over 4 days with 10d twice daily (0.1, 1.0, or
3.0 mg/kg, ip). At 0.1 mg/kg, 10d showed a moderate ef-
fect on food intake of tumor-bearing mice, but it was
not significantly different from the control animals. How-
ever, 1 and 3 mg/kg of 10d dose-dependently increased
food intake compared with vehicle controls (Fig. 6).
10000
1000
100
10
i.v. plasma (ng/mL)
p.o. plasma (ng/mL)
p.o. brain (ng/g)
8
7
6
5
4
3
2
1
0
10d (bid, i.p.)
5% Crem
0.1 mg/kg
1.0 mg/kg
3.0 mg/kg
1
0
1
2
3
4
5
6
7
8
9
4
5
6
7
8
9
10 11 12 13
Time (h)
Experiment Day
Figure 5. Time–concentration curves of 11d in mice after iv (5 mg/kg)
and po (10 mg/kg) administration.
Figure 6. The effects of 10d given twice daily (0.1, 1, and 3 mg/kg, ip)
on food intake in LLC tumor-bearing mice.
Table 6. Pharmacokinetic parameters of 10d in rats, monkeys, and dogsa
Species
CL (mL/min kg)
Vd (L/kg)
t1/2 (h)
Tmax (h)
Cmax (ng/mL)
oral AUC (ng/mL h)
F%
brain AUC (ng/g h)b
Ratc
27
7.7
5.6
8.7
3.4
4.7
7.0
6
195
200
250
1290
2650
3850
31
21
37
618d,e,f
n.d.g
n.d.g
Monkey
Dog
13.8
15.3
6.7
4.0
a Three animals were dosed intravenously at 5 mg/kg and orally at 10 mg/kg.
b Brain concentrations were taken from po dose.
c The plasma protein binding of 10d was high (>99%) in rats based on an in vitro ultrafiltration study.
d Brain Cmax = 52 ng/g, Tmax = 6.0 h.
e b/p = 0.27 based on Cmax values and 0.48 based on AUCs.
f Cbrain = 6.3 and 38 ng/g at 1 and 4 h, b/p ratio was 0.05 and 0.2, respectively.
g Not determined.

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5613
Body weight and body composition were also signifi-
cantly affected in mice treated with 10d (Fig. 7). Body
weight was increased in animals treated with all doses
of 10d compared to the body weight loss (approximately
3%) which occurred in the vehicle-treated cachectic ani-
mals (Fig. 7A). Analysis of body composition with dual-
energy X-ray absorbtometry (DEXA) demonstrated
that the greater increase of body weight in 10d-treated
mice was probably due to the sparing of lean mass
(LM). Tumor-bearing animals treated with vehicle dem-
onstrated no increase in LM over the course of the 14-
day experiment, whereas tumor-bearing animals treated
with 10d showed a trend toward an increased LM of
ꢀ4%. However, the difference between the dose groups
and vehicle-treated animals was not statistically
significant.
(the binding affinity Ki of 11d at mouse MC4R was
3.2 nM). This study also indicates that the compound
did not accumulate in either plasma or brain after twice
daily dosing in mice.
In conclusion, we conducted a detailed study on a series
of a-isopropyl piperazinebenzylamines bearing an amine
side-chain as MC4R antagonists. These compounds
showed high binding affinity at MC4R and selectivity
over other melanocortin receptor subtypes. They also
functioned as antagonists by dose-dependent inhibition
of a-MSH-stimulated cAMP production. These com-
pounds were profiled in mice for their pharmacokinetic
properties. Several compounds were identified to have
good pharmacokinetics with moderate to high brain
penetration. One analog, 1-{2-[(1S)-2-(1-(dimethylami-
nopropionyl)amino)-2-methylpropyl]-6-fluorophenyl}-4-
[(2R)-methyl-3-(4-chlorophenyl)propionyl]piperazine (10d)
was identified to have good potency and selectivity. It
was profiled for its metabolic and pharmacokinetic
properties in several animal species. Finally, this com-
pound demonstrated efficacy in a mouse cachexia
model.
Compound 11d was also studied in this in vivo model
using oral administration. Beginning 12 days after
LLC inoculation, animals were treated over 4 days with
11d twice daily (5 or 20 mg/kg, po). LLC tumor-bearing
mice treated with 11d at a 20 mg/kg dose showed a sig-
nificant increase in both food intake and weight gain rel-
ative to vehicle-treated tumor-bearing controls.24
However, at a 5 mg/kg dose, 11d did not have an effect
on weight gain, suggesting this dose is not optimal for
efficacy. Studies on the surrogate concentrations of 11d
after 20 mg/kg administration showed that the lowest
concentration before the following dose was 140 ng/
mL for plasma and 11 ng/g for brain (Table 7). The plas-
ma protein binding of 11d was 99% in rats. Assuming
linear PK, a dose of 5 mg/kg po would result in a mini-
mal brain concentration of about 2 ng/mL (ꢀ4 nM),
which might be too low for high receptor occupancy
4. Experimental
4.1. Chemistry
4.1.1. General Methods. NMR spectra were recorded on
a Varian 300 MHz spectrometer with TMS as an inter-
nal standard. Chemical shifts are reported in parts per
million (d), and signals are expressed as s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; or br, broad.
Body Weight Percent Change
A
B
Lean Mass Percent Change
4
7
6
*
*
*
3
2
5
% BW change
1
4
0
3
-1
-2
-3
-4
2
1
0
-1
veh
0.1 mg/kg 1.0 mg/kg 3.0 mg/kg
veh
0.1 mg/kg 1.0 mg/kg 3.0 mg/kg
Figure 7. The effects of 10d after 0.1, 1, and 3 mg/kg ip administration twice per day on body weight (A, left) and lean mass (B, right) in tumor-
bearing mice.
Table 7. Surrogate marker of plasma concentrations of 11d in tumor-bearing micea
Time (h)
Day 1
10
Day 4
10
0
2
12
24b
0
2
12
24c
22
Dose
Cplasma (ng/mL)
Cbrain (ng/g)
·
·
·
·
360
27
430
74
140
11
460
36
405
25
4.5
a Oral administration at 20 mg/kg dose at 8:00 am and 6:00 pm each day for 4 days. Plasma and brain samples were taken from the satellite group.
b Samples taken before the next dose.
c Samples were taken 14 h after the last dose.

5614
C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
Purity measurements were performed on an HP Agilent
1100 HPLC–MS (detection at 220 and 254 nm).
7.20 (m, 2H), 8.12–8.48 (br s, 2H, NH2); MS: 462
(MH+); HRMS (MH+) calcd for C25H33ClFN3O2:
462.2324; found: 462.2320.
4.1.1.1. 1-{2-[(1S)-(S-tert-Butylsulfinyl)amino-2-meth-
ylpropyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(4-chloro-
phenyl)propionyl]piperazine (6a). 1-{2-[(1S)-(S-tert-Butyl-
sulfinyl)amino-2-methylpropyl]-6-fluorophenyl}-4-(tert-
butoxycarbonyl)piperazine (5a, 1.09 g, 2.4 mmol)17 was
dissolved in CH2Cl2 (24 mL) and trifluoroacetic acid
(4.8 mL). The reaction mixture was stirred at rt for
50 min and quenched with aqueous NaHCO3. The or-
ganic layer was separated, washed with saturated NaH-
CO3, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The crude product was obtained
as a yellow foam (610 mg, 72%), which was dissolved in
dichloromethane (24 mL). Triethylamine (1.0 mL) was
added to this solution at 0 ꢁC, followed by (R)-2-
methyl-3-(4-chlorophenyl)propionyl chloride (obtained
from 524 mg (2.64 mmol) of the corresponding acid
and thionyl chloride) in CH2Cl2 (5 mL). The mixture
was allowed to slowly warm to rt overnight and was
quenched with 1 N HCl. The organic layer was sepa-
rated, washed, with saturated aqueous NaHCO3, dried
over anhydrous MgSO4, and concentrated in vacuo to
yield the crude product (1.06 g), which was used in the
next step without further purification. MS: 536 (MH+).
4.1.1.4. 1-{2-[(1S)-Amino-2-methylpropyl]-6-fluoro-
phenyl}-4-[(2R)-methyl-3-(2,4-dichlorophenyl)propion-
yl]piperazine trifluoroacetate (7c). This compound was
synthesized from 5a and (2R)-methyl-3-(2,4-dichloro-
phenyl)propionic acid using the procedure for 7a. Yel-
lowish foam, HPLC purity: 98.8% (220 nm) and 100%
1
(254 nm); H NMR 0.72 (d, J = 5.7 Hz, 3H), 1.06 (d,
J = 5.7 Hz, 3H), 1.17 (d, J = 6.6 Hz, 3H), 2.03–2.20
(m, 1H), 2.46–2.90 (m, 5H), 2.92–3.30 (m, 3H), 3.50–
3.90 (m, 3H), 4.47–4.70 (m, 1H), 6.87–7.07 (m, 1H),
7.08–7.21 (m, 4H), 7.35 (d, J = 8.8 Hz, 1H), 8.00–8.40
(br s, 2H, NH2); MS: 466 (MH+); HRMS (MH+) calcd
for C24H30Cl2FN3O: 466.1828; found: 466.1831.
The synthesis and characterization of compounds 7d–f
can be found in the supporting information of
reference.24
4.1.1.5. 1-{2-[(1S)-Amino-2-methylpropyl]-4-chloro-
phenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)propionyl]-
piperazine trifluoroacetate (7g). This compound was syn-
thesized from 5c using a procedure similar to that for 7a.
1
White solid, HPLC purity: 100% (220 and 254 nm); H
The synthesis and characterization of compounds 6d can
be found in the supporting information of Ref. 24.
NMR (CD3OD): 0.77 (d, J = 6.6 Hz, 3H); 1.14 (d,
J = 6.6 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 2.03–2.17
(m, 1H), 2.46–2.61 (m, 2H), 2.71–2.93 (m, 5H), 3.20–
3.30 (m, 4H), 4.56 (d, J = 9.7 Hz, 1H), 7.24 (d,
J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.40 (d,
J = 2.2 Hz, 1H), 7.42–7.46 (m, 2H); MS: 448 (MH+).
4.1.1.2. 1-{2-[(1S)-Amino-2-methylpropyl]-6-fluoro-
phenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)propionyl]-
piperazine trifluoroacetate (7a). 1-{2-[(1S)-(S-tert-Butyl-
sulfinyl)amino-2-methylpropyl]-6-fluorophenyl}-4-[(2R)-
methyl-3-(4-chlorophenyl)propionyl]piperazine
(6a,
4.1.1.6. 1-{2-[(1S)-Amino-2-methylpropyl]phenyl}-4-
[(2R)-methyl-3-(4-chlorophenyl)propionyl]piperazine tri-
fluoroacetate (7h). This compound was synthesized from
5d using a procedure similar to that for 7a. White solid,
23.3 g, 43.55 mmol) was dissolved in MeOH (436 mL)
and treated with HCl (2 M in ether, 28.3 mL,
56.62 mmol). The reaction mixture was stirred at rt for
about 1 h until all of the starting material had been con-
sumed. The reaction mixture was concentrated in vacuo.
The resulting yellowish foam was dried in high vacuum
for 15 min to provide the crude product as an off-white
foam in quantitative yield (18.8 g). A small sample was
purified using an LC–MS system, and pure compound
was obtained for biological studies. White solid, HPLC
1
HPLC purity: 98.4% (220 nm) and 100% (254 nm); H
NMR (CD3OD): 0.75 (d, J = 6.6 Hz, 3H), 1.15 (d,
J = 6.6 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 2.05–2.21
(m, 2H), 2.46–2.62 (m, 2H), 2.63–2.93 (m, 5H), 3.20–
3.32 (m, 3H), 4.59 (d, J = 9.7 Hz, 1H), 7.20–7.50 (m,
8H); MS: 414 (MH+). HRMS (MH+) calcd for
C24H32ClN3O: 414.2312; found: 414.2310.
1
purity: 100% (220 and 254 nm); H NMR (CD3OD):
0.78 (d, J = 6.6 Hz, 3H), 1.16 (d, J = 6.6 Hz, 3H), 1.18
(d, J = 6.6 Hz, 3H), 2.08–2.23 (m, 1H), 2.45–3.00 (m,
5H), 3.15–3.30 (m, 4H), 4.48–4.46 (m, 2H), 4.74 (d,
J = 9.7 Hz, 1H), 7.15–7.43 (m, 7H); MS: 432 (MH+).
4.1.1.7. 1-{2-[(1S)-(Methylaminoethyl)amino-2-meth-
ylpropyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)
propionyl]piperazine mesylate (8). Sodium triacetoxy-
borohydride (510 mg, 2.4 mmol) was added to a stirred
solution of 1-{2-[(1S)-amino-2-methylpropyl]-6-fluoro-
phenyl}-4-[(2R)-methyl-3-(2-fluoro-4-chlorophenyl)pro-
pionyl]piperazine (7a, 280 mg, 0.60 mmol) and methyl-
(2-oxoethyl)carbamic acid tert-butyl ester (135 mg,
0.78 mmol) in dichloromethane (6 mL), and the reaction
mixture was stirred at rt for 8 h. An aqueous workup
provided a crude intermediate which was dissolved in
1:1 trifluoroacetic acid/CH2Cl2 (6 mL), and the solution
was stirred at rt for 2 h. The crude product was concen-
trated in vacuo and purified on a LC–MS system to give
the titled compound as a TFA salt, which was neutral-
ized with Na2CO3 to provide a free amine (50 mg, 17%
yield), which was converted into the mesylate salt
4.1.1.3. 1-{2-[(1S)-Amino-2-methylpropyl]-6-fluoro-
phenyl}-4-[(2R)-methyl-3-(2-methoxy-4-chlorophenyl)-
propionyl]piperazine trifluoroacetate (7b). This com-
pound was synthesized from 5a and (2R)-methyl-3-(2-
methoxy-4-chlorophenyl)propionic acid using the proce-
dure for 7a. White foam, HPLC purity: 100% (220 and
1
254 nm); H NMR (CDCl3): 0.73 (d, J = 5.3 Hz, 3H),
1.10 (d, J = 5.3 Hz, 3H), 1.12 (d, J = 6.6 Hz, 3H),
2.03–2.20 (m, 1H), 2.50–2.80 (m, 3H), 2.80–2.97 (m,
2H), 3.05–3.28 (m, 3H), 3.58–3.66 (m, 2H), 3.73–3.96
(m, 3H), 3.80 (s, 3H), 4.50–4.70 (m, 1H), 6.92 (d,
J = 6.1 Hz, 1H), 6.99 (dd, J = 1.7, 8.3 Hz, 1H), 7.08–

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5615
(57 mg). White powder, HPLC purity: 98.7% (220 nm)
and 95.1% (254 nm); H NMR (CDCl3): 0.77 (m, 3H),
3.10 (m, 6H), 3.34–3.66 (m, 4H), 3.20–3.32 (m, 3H),
3.84–3.94 (m, 1H), 4.47 (d, J = 9.7 Hz, 1H), 6.91–7.01
(m, 1H), 7.04–7.10 (m, 1H), 7.15–7.22 (m, 1H), 7.27 (s,
1H), 7.22–7.29 (m, 1H), 7.43–7.48 (m, 1H); MS: 537
(MH+); HRMS (MH+) calcd for C27H35Cl2FN4O2:
537.2199; found: 537.2213.
1
1.18 (m, 6H), 2.10 (m, 1H), 2.55–3.35 (m, 16H), 2.68
(s, 3H, MeSO3), 2.79 (s, 3H), 3.77 (m, 1H), 4.43 (m,
1H), 4.62 (d, J = 12 Hz, 1H), 7.01 (m, 1H), 7.13 (dd,
J = 1.8, 8.1 Hz, 1H), 7.25 (s, 4H), 7.26 (m, 1H); MS:
489 (MH+). Anal. Calcd for C27H38ClFN4OÆMeSO3-
HÆ1/2H2O: C, 54.93%; H, 7.41%; N, 9.15%. Found: C,
54.62%; H, 7.34%; N, 9.15%.
4.1.1.11. 1-{2-[(1S)-(3-Dimethylaminopropionamino)-
2-methylpropyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(4-chlo-
rophenyl)propionyl]piperazine mesylate (10d). The crude
1-{2-[(1S)-amino-2-methylpropyl]-6-fluorophenyl}-4-[(2R)-
methyl-3-(4-chlorophenyl)propionyl]piperazine (7a, ꢀ18.8 g,
ꢀ43.5 mmol) was dissolved in dichloromethane
(218 mL) along with 3-dimethylaminopropionic acid
hydrochloride (7.36 g, 47.91 mmol) and diisopropyleth-
ylamine (30.3 mL, 174.2 mmol). The reaction mixture
was stirred at rt for 5 min then HOBt (8.82 g,
65.33 mmol) was added. After 40 min, EDC (4.73 g,
24.67 mmol) was added to the reaction mixture, and stir-
ring was continued for an additional 2 h. The reaction
mixture was washed with saturated aqueous NaHCO3
(2· 250 mL) and brine (250 mL). The organic layer
was collected, dried over anhydrous MgSO4, filtered,
and evaporated in vacuo to give a tan oil. The product
was purified by column chromatography on silica gel
using 3–10% methanol in ethyl acetate, then 90:9.5:0.5
CHCl3/MeOH/NH4OH as the eluent to give a white
foam (30 g, 87% yield) as the free amine.
The synthesis and characterization of compounds 9 can
be found in the supporting information of Ref. 24.
4.1.1.8. 1-{2-[(1S)-(Methylaminoacetamido)-2-methyl-
propyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)
propionyl]piperazine mesylate (10a). HBTU (292 mg,
0.77 mmol) was added to a solution of N-Boc-sarcosine
(104 mg, 0.55 mmol) and DIEA (440 lL) in DMF
(6 mL). The mixture was stirred at rt for 30 min, and
1-{2-[(1S)-amino-2-methylpropyl]-6-fluorophenyl}-4-[(2R)-
methyl-3-(2-fluoro-4-chlorophenyl)propionyl]piperazine
(7a, 233 mg, 0.50 mmol) was added, followed by 3 mL
of DMF. The reaction mixture was stirred at rt for
8 h. After an aqueous workup, the mixture was dis-
solved in TFA/CH2Cl2 (1:1, ꢀ5 mL) and stirred at rt
for 1 h. After concentration in vacuo, the product was
purified by chromatography on silica gel with 400:50:2
CHCl3/MeOH/NH4OH to give the titled compound as
a free amine (80 mg, 32% yield), which was converted
to the mesylate salt. White powder, HPLC purity:
This amine (7.0 g, 13.18 mmol) was dissolved in ethyl
acetate (40 mL) along with methanesulfonic acid
(0.855 mL, 13.18 mmol), and the solution was stirred
at rt for 1 h and then evaporated to dryness in vacuo.
The resulting solid was dissolved in water (ꢀ60 mL), fil-
tered, and lyophilized for 2 days to give a hygroscopic
white solid (7.7 g). HPLC purity: 100% (220 nm) and
1
97.6% (220 nm) and 95% (254 nm); H NMR (CDCl3):
0.75 (m, 3H), 1.01 (m, 3H), 1.16 (m, 3H), 1.98 (m,
1H), 2.69 (s, 6H, 3H for MeSO3), 2.60-3.40 (m, 9H),
3.85 (m, 3H), 4.61 (m, 1H), 5.33 (m, 1H), 6.92 (m,
1H), 7.15 (br s, 4H), 7.27 (m, 2H), 8.41 (br s, 3H);
MS: 503 (MH+). Anal. Calcd for C27H36ClFN4O2ÆMe-
SO3HÆ7/2H2O: C, 50.79%; H, 7.15%; N, 8.46%. Found:
C, 51.07%; H, 7.09%; N, 8.58%.
1
98.9% (254 nm); H NMR (CDCl3, free base): 0.86 (d,
J = 6.6 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 1.17 (d,
J = 6.6 Hz, 3H), 1.86 (m 1H), 2.31 (m, 1H), 2.35 (s,
6H), 2.50–3.40 (m, 12H), 3.80 (m, 1H), 4.65 (d,
J = 12 Hz, 1H), 5.40 (m, 1H), 6.84 (d, J = 8.1 Hz, 1H),
6.90 (m, 1H), 7.12 (m, 3H), 7.26 (m, 2H), 9.11 and
9.21 (d, J = 9.0 Hz, 1H); MS: 531 (MH+). Anal. Calcd
for C29H40ClFN4O2ÆMeSO3HÆ3H2O: C, 52.89%; H,
7.40%; N, 8.22%. Found: C, 53.09%; H, 7.64%; N,
8.50%.
4.1.1.9. 1-{2-[(1S)-(Methylaminoacetamido)-2-meth-
ylpropyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(2-methoxy-4-
chlorophenyl)propionyl]piperazine mesylate (10b). This
compound was synthesized from 7b using the procedure
for 10a. White powder, HPLC purity: 99.0 (220 nm) and
93.6% (254 nm); 1H NMR (DMSO-d6): 0.73 (d,
J = 6.3 Hz, 3H), 0.99 (d, J = 6.3 Hz, 6H), 1.86 (m,
1H), 2.30 (s, 3H), 2.57 (m, 1H), 2.78 (s, 3H), 2.76–2.79
(m, 1H), 3.15 (m, 4H), 3.69 (m, 1H), 3.78 (m, 1H),
3.82 (s, 3H), 3.94 (m, 1H), 4.41 (m, 1H), 5.34 (m, 1H),
6.92 (d, J = 8.1 Hz, 1H), 7.00–7.15 (m, 4H), 7.23 (m,
1H), 8.61 (br s, 2H), 8.77 (d, J = 8.1 Hz, 1H); MS:533
(MH+); HRMS (MH+) calcd for C28H38ClFN4O3:
533.2695; found: 533.2681.
4.1.1.12. 1-{2-[(1S)-(3-Azetidinylcarboxamido)-2-methyl-
propyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(2-methoxy-4-
chlorophenyl)propionyl]piperazine mesylate (10e). This
compound was synthesized from 7b and N-Boc-azeti-
dine-3-carboxylic acid using a procedure similar to that
for 10a. White powder, HPLC purity: 98.6% (220 nm)
1
and 100% (254 nm); H NMR (CDCl3): 0.74 (m, 3H),
4.1.1.10. 1-{2-[(1S)-(Methylaminoacetamido)-2-meth-
ylpropyl]-6-fluorophenyl}-4-[(2R)-methyl-3-(2,4-dichloro-
1.02 (m, 3H), 1.13 (m, 3H), 2.02 (m, 1H), 2.69 (s, 3H),
2.60–3.25 (m, 5H), 3.35 (m, 2H), 3.83 (s, 3H), 3.91 (m,
1H), 4.25 (m, 4H), 4.63 (m, 1H), 5.22 (m, 1H), 6.82–7.24
(m, 6H), 8.06 (br s, 1H), 8.56 (br s, 1H), 8.93 (br s, 1H);
phenyl)propionyl]piperazine
mesylate
(10c).
This
compound was synthesized from 7c using the procedure
for 10a. Light yellow foam, HPLC purity: 99.1%
(220 nm) and 97.6% (254 nm); 1H NMR (CD3OD):
0.75 (d, J = 7.0 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H), 1.20
(d, J = 6.6 Hz, 3H), 1.88–2.02 (m, 1H), 2.56 (s, 3H,
MeSO3), 2.50–2.60 (m, 1H), 2.60–2.70 (m, 1H), 2.80–
MS:545 HRMS
C29H38ClFN4O3 545.2695; found: 545.2681.
(MH+);
(MH+)
calcd
for
The synthesis and characterization of compounds 11a–f
can be found in the supporting information of Ref. 24.

5616
C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
4.1.1.13. 1-{2-[(1S)-(Methylaminoacetamido)-2-meth-
J = 6.3 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 1.02 (d,
J = 6.6 Hz, 3H), 1.78 (m, 1H), 2.35–2.90 (m, 6H), 2.72
(s, 6H), 3.05–3.65 (m, 9H), 5.08 (m, 1H), 6.98 (m, 1H),
7.18–7.42 (m, 6H), 8.48 (d, J = 8.4 Hz, 1H); MS: 547
(MH+). Anal. Calcd for C29H40Cl2N4O2ÆMeSO3HÆH2O:
C, 54.46%; H, 7.01%; N, 8.47%. Found: C, 54.52%; H,
7.47%; N, 8.67%.
ylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(4-chlorophenyl)
propionyl]piperazine dimesylate (12a). This compound
was synthesized from 7g using a procedure similar to
that for 10a. White powder, HPLC purity: 100% (220
1
and 254 nm): H NMR (DMSO-d6, free amine): 0.68
(d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 1.04 (d,
J = 6.6 Hz, 3H), 1.79 (m, 1H), 2.28 (m, 1H), 2.35(s,
3H), 2.46 (m, 2H), 2.54 (m, 2H), 2.63 (m, 1H), 2.82
(dd, J = 8.7, 12.9 Hz, 1H), 3.18 (br s, 2H), 3.30–3.88
(m, 4H), 5.14 (m, 1H), 7.01 (d, J = 8.7 Hz, 1H), 7.26
(m, 3H) 7.35 (m, 3H), 8.64 (br s, 1H), 8.80 (d,
J = 8.4 Hz, 1H); MS: 519 (MH+). Anal. Calcd
for C27H36Cl2N4O2Æ2MeSO3HÆ3/2CH2Cl2.2H2O: C,
41.86%; H, 5.87%; N, 6.40%. Found: C, 41.63%; H,
5.68%; N, 7.15%.
4.1.1.17. 1-{2-[(1S)-(Azetidine-3-carboxamido)-2-
methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(2-meth-
oxy-4-dichlorophenyl)propionyl]piperazine dimesylate (12e).
This compound was synthesized from 1-{2-[(1S)-amino-
2-methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(2-
methoxy-4-chlorophenyl)propionyl]piperazine and N-
Boc-azetidine-3-carboxylic acid using a procedure simi-
lar to that for 10a. White powder, HPLC purity: 97%
(220 nm) and 95% (254 nm); 1H NMR (DMSO-d6):
0.68 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 1.00
(d, J = 6.6 Hz, 3H), 1.78 (m, 1H), 2.27 (m, 1H), 2.58
(m, 1H), 2.74 (m, 1H), 3.16 (m, 2H), 3.25–4.18 (m,
11H), 3.85 (s, 3H), 5.14 (t, J = 9.0 Hz, 1H), 6.95 (dd,
J = 1.8, 7.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.09 (m,
2H), 7.25 (dd, J = 2.4, 8.7 Hz, 1H), 7.29 (d, J = 2.1 Hz,
1H), 8.46 (d, J = 8.7 Hz, 1H), 8.63 (br s, 1H); MS: 561
(MH+). Anal. Calcd for C29H38Cl2N4O3Æ2MeSO3-
HÆCH2Cl2: C, 45.83%; H, 5.77%; N, 6.68%. Found: C,
45.58%; H, 5.72%; N, 6.78%.
4.1.1.14. 1-{2-[(1S)-(Methylaminoacetamido)-2-meth-
ylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(2-methoxy-4-
dichlorophenyl)propionyl]piperazine dimesylate (12b).
This compound was synthesized from 1-{2-[(1S)-ami-
no-2-methylpropyl]-4-chlorophenyl}-4-[(2R)- methyl-3-
(2-methoxy-4-chlorophenyl)propionyl]piperazine using
a procedure similar to that for 10a. HPLC purity:
100% (220 and 254 nm); 1H NMR (DMSO-d6, free
amine): 0.69 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.6 Hz,
3H), 1.00 (d, J = 6.6 Hz, 3H), 1.80 (m, 1H), 2.34 (s,
3H), 2.56 (m, 1H), 2.58 (m, 1H), 2.73 (dd, J = 7.2,
9.9 Hz, 1H), 3.16 (m, 2H), 3.80 (m, 2H), 3.84 (s, 3H),
4.40 (br s, 6H), 5.17 (t, J = 9.0Hz, 1H), 6.95 (dd,
J = 1.8, 7.8 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 7.08 (m,
2H), 7.26 (dd, J = 2.1, 8.7 Hz, 1H), 7.33 (d, J = 2.4 Hz,
1H), 8.62 (br s, 1H), 8.80 (d, J = 9.0 Hz, 1H); MS: 549
(MH+). Anal. Calcd for C28H38Cl2N4O3Æ2MeSO3-
HÆCH2Cl2: C, 45.04%; H, 5.85%; N, 6.78%. Found: C,
45.02%; H, 5.54%; N, 7.00%.
4.1.1.18. 1-{2-[(1S)-(1-Methyl-3-azetadinylcarbonyl)-
amino-2-methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-
(4-chlorophenyl)propionyl]piperazine mesylate (12f).
A solution of 1-{2-[(1S)-(3-azetadinylcarbonyl)amino-
2-methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(4-
chlorophenyl)propionyl]piperazine (154 mg, 0.29 mmol),
synthesized from 7g and N-Boc-azetidine-3-carboxylic
acid using a procedure similar to that for 10a, and form-
aldehyde (37% in water, 3 drops, excess) in methanol
(3 mL) was treated with sodium cyanoborohydride
(100 mg, 1.5 mmol) portionwise. The mixture was stir-
red at rt for 0.5 h and diluted with water (2 mL). The
product was extracted with ethyl acetate (2 · 20 mL),
and the combined extract was dried over Na2SO4, fil-
tered, and concentrated in vacuo. Chromatography on
silica gel using CHCl3/MeOH/NH4OH (200:25:1) gave
the product (69 mg, 44% yield) as a free base which
was converted to the mesylate salt using 1 equiv of
methanesulfonic acid in dichloromethane. White pow-
der, HPLC purity: 100% (220 nm) and 98% (254 nm);
1H NMR (CDCl3, free base): 0.78 (d, J = 6.6 Hz, 3H),
0.98 (d, J = 6.3 Hz, 3H), 1.19 (d, J = 5.7 Hz, 3H), 1.88
(m, 1H), 2.32 (s, 3H), 2.44 (m, 1H), 3.00 (m, 5H), 3.10–
3.68 (m, 9H), 5.14 (t, J = 8.7 Hz, 1H), 6.92 (d, J =
7.5 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 7.05–7.30 (m, 4H),
7.28 (d, J = 7.8 Hz, 2H); MS: 545 (MH+). Anal. Calcd
for C29H38Cl2N4O2ÆMeSO3HÆ2H2O: C, 53.17%; H,
6.84%; N, 8.27%. Found: C, 53.20%; H, 6.43%; N, 8.37%.
4.1.1.15. 1-{2-[(1S)-((2R)-Aminopropionamido)-2-
methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(2-meth-
oxy-4-dichlorophenyl)propionyl]piperazine
mesylate
(12c). This compound was synthesized from 1-{2-[(1S)-
amino-2-methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-
3-(2-methoxy-4-chlorophenyl)propionyl]piperazine and
N-Boc-D-alanine using a procedure similar to that for
10a. White solid, HPLC purity: 97.6% (220 nm) and
96.9% (254 nm); 1H NMR (CD3OD, TFA salt): 0.73
(d, J = 6.6 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H), 1.15 (d,
J = 6.6 Hz, 3H), 1.56 (d, J = 7.0 Hz, 3H), 1.80–1.95
(m, 1H), 2.42–2.55(m, 2H), 2.66–2.87 (m, 3H), 3.10–
3.28 (m, 3H), 3.28–3.40 (m, 1H), 3.58–3.66 (m, 1H),
3.88 (s, 3H), 3.80–3.96 (m, 2H), 5.31 (d, J = 9.2 Hz,
1H), 6.91 (dd, J = 2.2, 10.1 Hz, 1H), 6.88–6.98 (m,
1H), 7.03–7.09 (m, 2H), 7.20 (dd, J = 2.2, 8.3 Hz, 1H),
7.30(d, J = 2.6 Hz, 1H); MS: 549 (MH+); HRMS
(MH+) calcd for C28H38Cl2N4O3: 549.2399; Found:
549.2401.
4.1.1.16. 1-{2-[(1S)-(3-Dimethylaminopropionamino)-
2-methylpropyl]-4-chlorophenyl}-4-[(2R)-methyl-3-(4-
chlorophenyl)propionyl]piperazine mesylate (12d). This
compound was synthesized from 7g using the procedure
for 10d. White powder, HPLC purity: 98.6% (220 nm)
and 97.4% (254 nm); 1H NMR (DMSO-d6): 0.66 (d,
4.1.1.19. 1-{2-[(1S)-(3-Dimethylaminopropionyl) ami-
no-2-methylpropyl]phenyl}-4-[(2R)-methyl-3-(4-chloro-
phenyl)propionyl]piperazine trifluoroacetate (13). This
compound was synthesized from 7h using the procedure
for 10d. Yellowish solid, HPLC purity: 100% (220 nm);
1H NMR (CD3OD): 0.71 (d, J = 7.0 Hz, 3H), 1.00 (d,

C. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5606–5618
5617
J = 6.6 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 1.77–1.84 (m,
1H), 2.41–2.50 (m, 2H), 2.85 (s, 6H), 2.60–2.94 (m, 6H),
3.05–3.30 (m, 4H), 3.30–3.37 (m, 2H), 3.60–3.70 (m,
1H), 5.30 (d, J = 8.8 Hz, 1H), 6.88–7.00 (m, 1H), 7.13
(dd, J = 0.9, 7.5 Hz, 1H), 7.19 (dd, J = 1.8, 7.5 Hz,
1H), 7.21–7.29 (m, 3H), 7.30–7.37 (m, 2H); MS: 513
(MH+); HRMS (MH+) calcd for C29H41ClN4O:
513.2996, found: 513.3008.
Descriptive pharmacokinetics were derived and evalu-
ated based on the mean plasma concentrations (N = 3/
time point). A non-compartmental model in ActivityBase
with linear trapezoidal rule was used to perform all phar-
macokinetic analyses pertaining to this manuscript.
4.4. Efficacy study
C57BL/6J male mice obtained from Jackson Laborato-
ries (Bar Harbor, Maine) were used for the cachexia
studies. Mice were housed individually and maintained
on powdered Purina 5015 chow (13% fat) for at least se-
ven days prior to the start of the study. All studies were
conducted according to the NIH Guide for the Care and
Use of Laboratory Animals and approved by the Animal
Care and Use Committee of the Oregon Health Sciences.
4.2. Pharmacology
Receptor binding was performed on membranes from
HEK293 cells stably expressing the human melanocortin
receptors, using [125I]NDP-MSH as the radiolabeled li-
gand. The cAMP stimulation and inhibition assays were
performed in the same cell lines using a-MSH (as the
standard in agonist assay). The assay conditions were
similar to those previously reported.19
As previously described,3 on day 0, mice were inoculated
subcutaneously into the upper flank with 1 · 106 cells
from a subcloned Lewis lung carcinoma (LLC) cell line.
All experimental animals were found to have a palpable
tumor within five days of the start of the experiment. At
the time of sacrifice, tumors were dissected away from
the surrounding tissue and weighed. There was no statis-
tical difference in tumor size between treatment groups,
and gross examination of organs did not reveal the pres-
ence of metastasis.
4.3. Pharmacokinetic characterization
The pharmacokinetic profiles of selected compounds
were determined in animals (N = 3/time points at a dose
of 10 mg/kg for po or 5 mg/kg for iv). Compounds were
dosed as a mesylate salt in 5% methylcellulose via the
tail vain (iv) or in water with 5% v/v cremophor via oral
gavage (po). Composite sampling was used to collect
samples. Terminal blood samples were taken from trea-
ted mice at nine time points ranging from pre-dose to
24 h (0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h) postdose. Brain
samples were collected at 1 and 4 h post po dosing in
rats for all compounds. Brain samples were also col-
lected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post po dosing
in mice and rats for compound 10d.
On day 10 after tumor inoculation, mice were divided
into four groups and treated twice daily with vehicle,
0.1, 1.0, 3.0 mg/kg 10d (ip). Food intake and body
weight were measured daily. Following 4 days of treat-
ment, animals were sacrificed, tumors were removed
and body composition was determined by dual-energy
X-ray absorbtometry (DEXA, PIXImus mouse densi-
tometer, Lunar Corp.). A baseline measure was made
on the day of inoculation (day 0) also. Animals were
anesthetized prior to the first scan and asphyxiated with
CO2 prior to the tumor dissection and the final scan.
Plasma and brain sample analyses for the quantification
of parent compounds were conducted using a HPLC
equipped with a mass spectrometric detector (LC–MS/
MS). For plasma samples, the compound was extracted
via a protein precipitation assay by adding 130 mL of
acetonitrile (CAN) and 30 mL of internal standard in
50 lL of mouse plasma (EDTA). Standards and QCs
were prepared by drying 50 mL of spiked aqueous solu-
tions and reconstituting with 50 mL of blank mouse
plasma. For brain samples, the whole brain was homog-
enized in 2.0 mL of ACN/H2O/formic acid (v/v,
60:40:0.1) containing 50 mL of internal standard, and
the supernatant was collected and injected into an LC–
MS/MS system for analysis. Brain standards and QCs
were prepared by adding 1.0 mL of spiked aqueous solu-
tions (prepared in 60:40 ACN/H2O), 1.0 mL of ACN/
H2O/formic acid (v/v: 60:40:0.1), and 50 mL of internal
standard into a whole blank mouse brain. Standards
and QCs for both plasma and brain were processed
and analyzed at the same time and in exactly the same
way as the analytical samples. The compound was mea-
sured using a specific and sensitive HPLC/MS assay that
offered linear ranges of 1–1000 ng/mL for iv/po plasma
sample analysis, and 1–500 ng/mL for iv brain sample
analysis. The lower limit of quantitation (LLOQ) was
1 ng/mL for the study. Quantification for both plasma
and brain samples was performed by fitting peak area
ratios to a weighted (1/·) linear calibration curve.
Food intake was averaged over the treatment days. Per-
cent change in body weight, lean mass, and fat mass was
computed from the initial day of the experiment to the
final day. Differences among groups for these variables
were analyzed by one-way ANOVAs with post hoc anal-
ysis. Data sets were analyzed for statistical significance
using SigmaStat (SPSS, Inc).
Acknowledgment
The authors are indebted to Mr. Andrew Fisher for the
technical support in Caco-2 assay and liver microsomes
stability measurements
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