Pyrrolidinones as orally bioavailable antagonists of the human melanocortin-4 receptor with anti-cachectic activity

Article abstract of DOI:10.1016/j.bmc.2007.05.026

A series of pyrrolidinones derived from phenylalanines were synthesized as potent antagonists of the human melanocortin-4 receptor. These compounds showed high potencies and selectivities, and several of them had good oral bioavailabilities. In addition,

Full text of DOI:10.1016/j.bmc.2007.05.026

Bioorganic & Medicinal Chemistry 15 (2007) 5166–5176
Pyrrolidinones as orally bioavailable antagonists of the
human melanocortin-4 receptor with anti-cachectic activity
Joe A. Tran,a Fabio C. Tucci,a Wanlong Jiang,a Dragan Marinkovic,a Caroline W. Chen,a
Melissa Arellano,a Stacy Markison,c Beth A. Fleck,b Jenny Wen,d Nicole S. White,a
Joseph Pontillo,a John Saunders,a Daniel Marks,e Sam R. Hoare,b Ajay Madan,d
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
dDepartment of Preclinical Development, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
eDepartment of Pediatrics, Oregon Health & Science University, Portland, OR 97239, USA
Received 7 November 2006; revised 3 May 2007; accepted 10 May 2007
Available online 17 May 2007
Abstract—A series of pyrrolidinones derived from phenylalanines were synthesized as potent antagonists of the human melanocor-
tin-4 receptor. These compounds showed high potencies and selectivities, and several of them had good oral bioavailabilities. In
addition, 12e demonstrated in vivo efficacy in a murine cachexia model.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The melanocortin-4 receptor (MC4R) is a member of
the G-protein-coupled receptor (GPCR) superfamily
and it plays an important role in regulating feeding
behavior and energy homeostasis.1 Recent studies have
shown that MC4R antagonists can increase food intake,
and more importantly, reverse lean body mass loss in
animal models of cachexia.2,3 Therefore, a potent and
selective MC4R antagonist with good oral bioavailabil-
ity might be useful in clinical treatment of cachexia.4
prove the potency and selectivity of these compounds,
we have conducted a study by using a nitrogen-contain-
ing moiety to replace the a-methyl group of 3a. These
modifications increase potency from the initial phenyl
propionyl compounds.9 Here we report the discovery
of a series of potent and selective MC4R antagonists
with good oral bioavailability in mice.
Compounds S-7, 9–10 were synthesized from the key
intermediates S-5 according to the sequence described
in Scheme 1. Thus, coupling reactions of S-410 with N-
Boc-D(2,4-Cl)phenylalanine, followed by selective
deprotection with TFA, gave the primary amines S-5
in good yields. These compounds were then acetylated
with acetic anhydride, or coupled with N-Boc-b-alanine
and N-Boc-glycine, to afford intermediates that were
fully deprotected using hydrochloric acid to provide
compounds S-7, 9–10. Alternatively, coupling reactions
of S-4 with preformed N-Boc-b-Ala-D(2,4-Cl)Phe-OH
or N-Boc-Gly-D(2,4-Cl)Phe-OH, followed by deprotec-
tion, afforded S-9–10. A similar procedure was used to
prepare R-7–10 from the corresponding R-4. Deprotec-
tion of S-5d (X = 4-CF3) with HCl gave the diamine 6.
S-5d was also converted to the piperazinone 11 by the
following reaction sequence: reductive alkylation with
N-Boc-glycinaldehyde; treatment with chloroacetyl
Potent and selective antagonists of the melanocortin-4
receptor from several chemical classes have been discov-
ered.5 For example, the amidine 1 (Fig. 1) has been
shown by Vos and coworkers, as a MC4R antagonist
with CNS penetration, to protect rodent from weight
loss induced by tumor.6 The dipiperazine 2 is also a po-
tent MC4 antagonist, which exhibits efficacy in a anxiety
model.7 We have previously identified a series of a-ben-
zylpropionylpiperazines such as 3a (Ki = 25 nM) as
MC4R antagonists with moderate oral bioavailability
and high brain penetration in rodents.8 To further im-
Keywords: Pyrrolidinone; Synthesis; Melanocortin-4 receptor; Anta-
gonist; Pharmacokinetics; Cachexia; Animal model.
*
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7967; e-mail: cchen@neurocrine.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.026

J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5167
F
X
Y
Cl
N
N
NH
NH2
N
N
N
N
N
O
O
F
O
3a: X = Me, Y = H, Ki = 25 nM
3b: X = CF3, Y = Cl, Ki = 26 nM
1
2
Figure 1. Examples of MC4R antagonists.
X
F3C
Cl
Cl
Cl
Cl
F3C
Cl
Cl
NH2
N
NH2
N
NH2
N
N
N
N
N
NH
NH2
O
NH
O
O
O
O
R
S
-7, 9-10
11
6
c
e
b
d
O
S
O
S
X
X
X
Cl
Cl
Cl
a
N
H
N
NH2
N
H
N
N
N
a: X = 6-F
b: X = 6-CF3
c: X = 4-F
d: X = 4-CF3
e: X = H
NH
h
N
Y
NH2
NH
O
O
O
R
R
-7-10
S
-4a-e
S
-5
g
f
Y
Cl
X
Y
Cl
F3C
Cl
NH2
NH2
N
HO
N
N
N
O
N
O
N
N
O
O
14a: Y = Cl
14b: Y = H
O
O
12
13
Scheme 1. Reagents and conditions: (a) N-Boc-D(2-Y,4-Cl)Phe-OH, HBTU, DIEA, DMF, rt, then TFA, CH2Cl2, 50–75% over two steps; (b) N-Ala-
D(2,4-Cl)Phe-OH, HBTU, DIEA, DMF, rt, then HCl, MeOH, about 65% over two steps; (c) RCOOH, EDC, HOBt, rt or Ac2O, CH2Cl2, DIEA,
DMAP cat., then HCl, MeOH; (d) HCl, MeOH; (e) i—Boc-NHCH2CHO, NaBH(OAc)3, CH2Cl2, rt, 59%; ii—ClCH2COCl, EtOAc, aq NaHCO3, rt;
iii—HCl, MeOH, rt, 57% over three steps; (f) i—HOOCCH2CH2CHO, NaBH(OAc)3, H2O, THF, rt; ii—THF, Ac2O, rt; iii—HCl, MeOH, 20–50%;
(g) R-14, HBTU, DIEA, DMF, rt, then HCl, MeOH, rt, about 60% over two steps; (h) S-14b, HBTU, DIEA, DMF, rt, then HCl, MeOH, rt, 60%
over two steps.
chloride under basic conditions; full deprotection under
acidic conditions (HCl in MeOH), followed by treat-
ment with aqueous Na2CO3 to promote a cyclization.
by Ac2O-promoted lactamization and tert-butanesulfin-
amide deprotection. Compounds 12 could be prepared
by reactions of S-4 with pre-assembled pyrrolidinones
R-1411 under standard peptide coupling conditions. The
S-configured 4-chlorophenylalanine derivative 13 was
prepared by using the D-phenylalanine S-14b.12 These
compounds were then tested for their binding affinity at
Pyrrolidinones 12a–d were obtained from reductive alky-
lations of the amines S-5d with succinic semialdehyde in
the presence of sodium acetoxyborohydride, followed

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J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
the human MC4 receptor using
[
125I]-NDP-MSH
S-10c had a Ki of 0.8 nM, which matched that of S-7c,
but R-10c was 3-fold less potent than its S-isomer
S-10c. These results might suggest that the 6-fluoro
affects the dihedral angle between the piperazine plane
and the phenyl ring, which has an orthogonal relation-
ship for a closely related analog,7 and this relationship
could be important for the pharmacophore of an active
compound.
{[Nle4,D-Phe7]a-melanocyte-stimulating hormone} radio-
labled ligand as previously reported.13
In comparison with 3-(2,4-dichlorophenyl)propionyl-
piperazine 3b (Ki = 26 nM), the 2,4-chlorophenylalanine
compound 6 (Ki = 39 nM) exhibited similar binding
affinity (Table 1). Acetylation of 5 increased its binding
affinity by over 20-fold (S-7a, Ki = 1.8 nM). Incorpora-
tion of an amine group (S-7b–d) improved potency
slightly from S-7a. This was different from some other
published series, in which a b-alanine seems to be impor-
tant in this area.14 These results suggest that an addi-
tional amine moiety in the current set of compounds
plays a minimal role, probably due to a strong interac-
tion with the receptor from the optimized ‘western-side’
of the molecule. In addition, R-7c (Ki = 10 nM) was
about 10 times less active than its S-isomer, demonstrat-
ing a stereo preference at this site of the molecules.
While compounds such as S-7b, S-7c, S-9c, S-10c, and
R-10c displayed high binding affinity, they generally
possessed high polar surface due to the polar amine
and the secondary amide, which is undesirable for a
CNS agent.15 We chose to retain the amide functionality
by using a cyclized form. Thus, the piperazinone 11,
derived from the glycine S-7b via an ethylene bridge,
exhibited a Ki of 4.5 nM, which was only about 2-fold
less potent than its parent. Similarly, the pyrrolidinone
12a (Ki = 4.5 nM) also possessed good binding affinity.
Moving the trifluoromethyl group of S-7 from the 4-po-
sition of the phenyl ring to the 6-position (S-9) resulted
in only slight loss in potency, and the ratio between the
S- and R-isomers of 9 was similar to that of 7. For
example, R-9c (Ki = 29 nM) was about 12-fold less
potent than S-9c (Ki = 2.5 nM).
The substitution at the western-side phenyl ring of the
pyrrolidones (12a–d) had a minimal effect on binding
affinity except for the 4-fluoro group which decreased
potency (12b, Ki = 26 nM, Table 2). The 4-chlorophe-
nylalanine 12e on the 4-trifluoromethyl template
displayed only 2-fold reduction in potency from the
2,4-dichloro analog 12a, however, the monochloro com-
pound 12f was 6-fold less potent than the dichloro ana-
log 12c, suggesting that the lipophilic 4-trifluoromethyl
group of 12e plays a role in maintaining its potency.
Interestingly, the L-(4-chlorophenyl)alanine derivative
13 (Ki = 8.8 nM) possessed similar binding affinity to
its D-analog 12e (Ki = 11 nM). This lack of stereo-pref-
erence might indicate that the pyrrolidinone moiety does
not have a direct interaction with the receptor. Instead,
it helps to orient the 4-chlorophenyl group to be located
in a preferred position for its receptor interactions.16,17
A ‘Y’ shape conformation for the Tic-D(4-Cl)Phe piper-
azine of the THIQ MC4R agonist has been demon-
strated by its solid-state structure.18
The 6-fluorophenyl compounds 10 showed some inter-
esting structure-activity relationships. The S-acetamide
S-10a had a Ki of 7.5 nM, which was significantly less
potent than the b-alanine S-10c. Interestingly, R-10a
exhibited a similar Ki value to S-10a. In comparison,
Table 1. SAR of amides at the human MC4R
X
X
Cl
Cl
Cl
Cl
NH2
N
NH2
N
N
N
NH
NH
O
O
O
R
O
R
S
-7, 9-10
R-7-10
Table 2. SAR of heterocycles at the human MC4R
Compound
X
R
Kia (nM)
6
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-F
39
X
Y
Cl
S-7a
S-7b
S-7c
R-7c
S-7d
R-8a
R-9a
S-9b
R-9b
S-9c
R-9c
S-10a
R-10a
S-10b
R-10b
S-10c
R-10c
Me
1.8
1.8
1.0
10
NH2
N
CH2NH2
CH2CH2NH2
CH2CH2NH2
4-Piperidinyl
Me
N
N
0.8
26
11: Z = NHCH2
12: Z = CH2
O
Z
O
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-F
Me
47
CH2NH2
CH2NH2
CH2CH2NH2
CH2CH2NH2
Me
3.3
49
Compound
X
Y
Kia (nM)
2.5
29
11
4-CF3
4-CF3
4-F
Cl
Cl
Cl
Cl
Cl
H
4.5
4.5
26
12a
12b
12c
12d
12e
13
7.5
6.0
3.3
7.1
0.8
2.3
6-F
6-F
Me
H
6-F
6,4
9.7
11
CH2NH2
CH2NH2
CH2CH2NH2
CH2CH2NH2
6-F
6-F
4-CF3
4-CF3
H
H
8.8
38
6-F
12f
H
a Data are average of two or more independent measurements.
a Data are average of two or more independent measurements.

J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5169
Several compounds were further tested for their binding
affinities at the other melanocortin receptor subtypes
and found to be highly selective. For example, S-10c
had Ki values of 450, 0.8, and 240 nM at the MC3R,
MC4R, and MC5R, respectively (Table 3). Compound
12e had the best selectivity (ꢀ100-fold) among all ana-
logs tested, and it did not have appreciable binding affin-
ity at the MC1 receptor. None of the compounds listed
in Tables 1 and 2 exhibited significant stimulation of
cAMP release in cells expressing the MC4 receptor at
a 10 lM concentration, demonstrating the lack of func-
tional agonist activity of these compounds. Instead,
these compounds showed dose-dependent inhibition of
a-MSH-stimulated cAMP production. For example, 11
and 12e had IC50 values of 210 and 510 nM, respec-
tively, in this assay.
binding.19 Another pyrrolidinone 12e (measured
logD = 3.1) had a moderate plasma CL of 31 mL/
min kg, a moderate Vd of 5.8 L/kg, although its t1/2 of
2.2 h was short in this species. At the 1- and 4-h time
points post-dosing, the whole brain concentrations were
484 and 154 ng/g, resulting in brain/plasma ratios of
1.27 and 0.81, respectively.
After an oral dose of 10 mg/kg, 11 reached a maximal
concentration of 214 ng/mL at 0.5 h to give an
AUC of 1527 ng/mL h, which resulted in an absolute
bioavailability of 19.4%. For 12a, although it displayed
high plasma concentrations (Cmax = 560 ng/mL,
AUC = 1400 ng/mL h), its absolute oral bioavailability
was only 4.3%. In comparison, 12e reached a maximal
plasma concentration of 270 ng/mL at 0.5 h to give an
AUC of 2271 ng/mL h, resulting in a high absolute bio-
availability of 42.4% (Fig. 2).
Compounds 11, 12a, and 12e were profiled for their
pharmacokinetic properties in mice (Table 4). After an
intravenous injection at 5 mg/kg, the moderately lipo-
philic piperazinone 11 (measured logD = 2.8) exhibited
a moderate plasma clearance (CL = 20 mL/min kg)
and a high volume of distribution (Vd = 13 L/kg), which
resulted in a long half-life (t1/2 = 7.7 h) in this species.
The high Vd might be associated with its dibasic nature,
although the piperazinone moiety might only be weakly
basic. The whole brain concentration was only about
40 ng/g at 1 h post-dosing, and the brain-to-plasma ratio
judged by area under curve (AUC) values was 32%. In
comparison, the lipophilic pyrrolidinone 12a (measured
logD of 3.5 vs 2.8 for 11) had a low plasma CL of
5.1 mL/min kg, a low Vd of 1.8 L/kg, and a moderate
t1/2 of 4.1 h presumably due to its high plasma protein
Compound 12e displayed favorable pharmacokinetic
properties and therefore it was further studied in a
mouse cancer cachexia model as previously described.3a
It was found that 12e possessed high binding affinity (Ki)
of 5.5 nM at the mouse MC4 receptor in vitro (9.1 nM,
rat MC4R). For the in vivo study, C57BL/6J male mice
were inoculated with Lewis lung carcinoma (LLC)
tumor cells. Beginning 12 days after LLC inoculation,
animals were treated over 4 days with 12e twice daily
(3 or 9 mg/kg ip). LLC tumor bearing mice treated with
12e showed a significant increase in food intake relative
to vehicle-treated tumor bearing controls (Fig. 3a).
Body weight was also significantly increased in mice
treated with 12e (Fig. 3b). Analysis of body composition
with dual-energy X-ray absorptiometry (DEXA) dem-
onstrated that the greater increase of body weight in
12e treated mice was due to sparing of lean mass as well
as body fat (Fig. 4a and b). Tumor bearing vehicle trea-
ted animals demonstrated only a 3% increase in LBM
over the course of the 15-day experiment, whereas
tumor-bearing animals treated with 12e increased their
LBM by ꢀ20%. Fat mass actually decreased by ꢀ3%
in vehicle treated animals but increased by ꢀ10% in mice
treated with the MC4 antagonist. The difference between
the 3 and 9 mg/kg groups was not statistically signifi-
cance. It is worth noting that 12e was a very weak
Table 3. Selectivity profiles of MC4R ligands
Compound
Kia (nM)
MC1R
MC3R
MC4R
MC5R
S-10c
11
n.d.
450
950
0.8
4.5
4.5
11
240
360
250
990
(23%)
(21%)
(31%)
12a
12e
640
1,800
a Data are average of two or more independent measurements.
Table 4. Pharmacokinetic parameters of compounds 10, 12a, and 12e
in micea
10000
Oral Plasma (ng/mL)
Compound
10
12a
12e
IV Plasma (ng/mL)
IV Brain (ng/g)
1000
iv dose (mg/kg)
CL (mL/min kg)
Vd (L/kg)
5
20
5
5.1
5
31
13
7.7
1.8
4.1
5.8
2.2
100
10
1
t1/2 (h)
AUC (ng/mL h)
Cbrain (ng/g)@1, 4 h
Cbrain/Cplasma
po dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
3943
41, 46b
0.07, 0.24c
10
16,300
397, 115
0.12, 0.13
10
2678
484, 154
1.27, 0.81
10
214
0.5
560
0.5
270
0.5
0.1
AUC (ng/ml h)
F (%)
1527
19.4
1400
4.3
2271
42.4
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hour)
a Data are average of three animals.
b Brain AUC (0–24 h): 1262 ng/g h.
c Ratio of 0.32 based on AUCs.
Figure 2. Time-concentration curve of 12e in mice after 5 mg/kg
intravenous injection and 10 mg/kg oral gavage administration.

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J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5
4
3
2
1
0
20
15
10
5
0
0
3
9
0
3
9
12e dose (mg/kg, i.p.)
12e dose (mg/kg, i.p.)
Figure 3. Food intake and body weight changes in LLC tumor-bearing mice in comparison with controls after administration of compound 12e.
25
20
15
10
5
15
10
5
0
0
-5
3
3
0
0
9
9
12e dose (mg/kg, i.p.)
12e dose (mg/kg, i.p.)
Figure 4. Lean and fat mass changes in LLC tumor-bearing mice in comparison with controls after administration of compound 12e.
partial ghrelin agonist, with an EC50 value of 4.1 lM
(47% intrinsic activity) in an in vitro assay, therefore,
the contribution of the ghrelin component to the ob-
served efficacy should be minimal.20
1.1.2. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-4-trifluoro-
methylphenyl]-4-[(2R)-amino-3-(2,4-dichlorophenyl)propi-
onyl]}-piperazine (6). To a solution of 4-{2-[(1S)-((S)-2-
methylpropanesulfinylamino)-3-methylbutyl]-4-trifluoro-
methylphenyl}-piperazine-1-carboxylic acid tert-butyl
ester (Boc-S-4d,7 5.0 g, 9.6 mmol) in CH2Cl2 (80 mL)
was added TFA (20 mL). The reaction mixture was
stirred at room temperature for 1 h, quenched with sat-
urated NaHCO3 aqueous solution, and extracted with
ethyl acetate (3 · 100 mL). The combined organic layers
were dried over Na2SO4. The solvents were removed in
vacuo. The residue was dissolved in 20 mL of DMF/
DCM (1:3) followed by the addition of N-Boc-D(2,4-
Cl)Phe-OH (3.54 g, 10.6 mmol), HOBt (1.95 g,
14.4 mmol), EDC (2.77 g, 14.4 mmol), and NaHCO3
(2.42 g, 28.9 mmol). The reaction mixture was stirred
at room temperature overnight, diluted with 100 mL of
ethyl acetate, and washed with 1 N HCl aqueous
solution, saturated NaHCO3, and brine. The organic
layer was dried over Na2SO4, and the solvents were
removed in vacuo. The product was purified using
flash column chromatography on silica gel (30–40%
ethyl acetate in hexanes) to give 1-{4-[2-(1S)-((S)-2-meth-
ylpropanesulfinylamino-3-methylbutyl)-4-trifluoromethyl-
phenyl]-4-[(2R)-tert-butoxycarbonylamino-3-(2,4-dichloro-
phenyl)propionyl]}-piperazine (Boc-S-5d) as a white foam
(5.24 g, 74% yield).
In conclusion, a series of phenylalaninepiperazine
derivatives were synthesized as potent and selective
antagonists of the melanocortin-4 receptor. In addi-
tion, several compounds were profiled for their phar-
macokinetic properties in mice. Compound 12e was
found to have favorable pharmacokinetics and dem-
onstrated to be efficacious in a mouse cachexia
model.
1. Experimental
1.1. Chemistry
1.1.1. General methods. NMR spectra were recorded on
a Varian 300 MHz spectrometer with TMS as an inter-
nal standard, and 13C NMR spectra were recorded at
75 MHz. Chemical shifts are reported in parts per mil-
lion (d), and signals are expressed as s (singlet), d (dou-
blet), t (triplet), q (quartet), m (multiplet) or br (broad).
High resolution mass spectra were measured at the
Scripps Center for Mass Spectrometry using MALDI-
FTMS. Purity measurements were performed on an
HP Agilent 1100 HPLC-MS (detection at 220 and
254 nm).
The above white foam (30 mg, 0.04 mmol) was dissolved in
5 mL of MeOH and treated with 4 N HCl in 1,4-dioxane

J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5171
(27 lL, 0.11 mmol). The reaction mixture was stirred at
room temperature for 1 h. MeOH and excess of HCl were
removed in vacuo, and the residue was treated with 5 mL
of 30% TFA in CH2Cl2. After stirring for 1 h, excess of
TFA and CH2Cl2 were removed in vacuo, and the product
was purified using flash silica chromatograph column
(10–15% MeOH/CH2Cl2) to give the titled compound as
colorless oil (15 mg). HPLC purity: 97.8% (220 nm),
95% (254 nn). 1H NMR (DMSO-d6): 7.82 (d,
J = 1.8 Hz, 1H), 7.59 (d, J = 1.3 Hz, 1H), 7.48 (dd,
J = 1.8, 8.3 Hz, 1H), 7.30–7.38 (m, 2H), 7.12 (d,
J = 8.3 Hz, 1H), 4.28–4.36 (m, 1H), 3.96–4.06 (m, 1H),
3.04–3.64 (m, 8H), 2.92–2.02 (m, 1H), 2.80–2.86 (m,
1H), 1.10–1.60 (m, 3H), 0.86 (d, J = 6.4 Hz, 3H), 0.84
(d, J = 6.4 Hz, 3H); 13C NMR: 173.6, 153.9, 144.7,
135.8, 135.0, 134.3, 132.6, 129.2, 127.8, 125.5 (q,
J = 31.3 Hz), 125.2 (q, J = 271.6 Hz), 124.7, 124.3,
121.3, 53.0, 49.9 (2C), 49.6, 47.3, 45.9, 42.5, 25.3, 23.9,
22.8 (2C); HRMS (MH+) calcd for C25H31Cl2F3N4O
531.1900, found 531.1875.
Compounds R-8a, R-9a, R-10a, and S-10a were synthe-
sized using a procedure similar to that for S-7a from the
corresponding R-5c, R-5d, R-5a, and S-5a,
respectively.10
1.1.4. 1-{4-[(1R)-2-(1-Amino-3-methylbutyl)-4-fluorophe-
nyl]-4-[(2R)-acetamido-3-(2,4-dichlorophenyl)propionyl]}-
piperazine (R-8a). White foam, HPLC purity: 100% (220
and 254 nm). 1H NMR (DMSO-d6): 8.42 (d, J = 8.8 Hz,
1H), 7.55–7.58 (m, 1H), 7.28–7.40 (m, 2H), 7.26 (dd,
J = 2.6, 9.6 Hz, 1H), 6.98–7.10 (m, 1H), 6.95 (dd,
J = 3.0, 8.3 Hz, 1H), 5.00–5.12 (m, 1H), 4.29–4.38 (m,
1H), 2.80–3.40 (m, 8H), 2.98–3.08 (m, 1H), 2.82–2.94
(m, 1H), 1.76 (s, 3H), 1.14–1.62 (m, 3H), 0.85 (d,
J = 6.1 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H); 13C NMR:
169.8, 169.4, 160.3 (d, J = 240.4 Hz), 147.2, 146.4,
135.1, 134.7 (d, J = 2.8 Hz), 134.1, 133.0, 129.3, 127.9,
123.0 (d, J = 7.8 Hz), 113.9 (2C, d, J = 20.2 Hz), 53,
49.6 (2C), 47.6, 47.3, 46.4, 43, 36.0, 25.3, 23.9, 22.9
(2C); MS: 523 (MH+).
1.1.3. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-4-trifluoro-
methylphenyl]-4-[(2R)-acetamido-3-(2,4-dichlorophenyl)pro-
pionyl]}-piperazine (S-7a). To a solution of 1-{4-[2-(1S)-
((S)-2-methylpropanesulfinylamino-3-methylbutyl)-4-tri-
fluoromethylphenyl]-4-[(2R)-tert-butoxycarbonylamino-
3-(2,4-dichlorophenyl)propionyl]}-piperazine (Boc-S-
5d, 1.47 g, 2.8 mmol) in CH2Cl2 (18 mL) was added
TFA (2 mL). The reaction mixture was stirred at room
temperature for 1 h, quenched with saturated NaHCO3
aqueous solution, and extracted with ethyl acetate
(3 · 50 mL). The combined organic layers were dried over
Na2SO4 and solvents were removed in vacuo to give 1-{4-
[(1S)-2-((S)-2-methylpropanesulfinylamino-3-methylbu-
tyl)-4-trifluoromethylphenyl]-4-[(2R)-amino-3-(2,4-dichlor-
ophenyl)propionyl]}-piperazine S-5d without further
purification.
1.1.5. 1-{4-[(1R)-2-(1-Amino-3-methylbutyl)-6-trifluoro-
methylphenyl]-4-[(2R)-acetamido-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine (R-9a). Colorless foam, HPLC
purity: 100% (220 and 254 nm). 1H NMR (DMSO-
d6): 8.36-8.44 (m, 1H), 7.75–7.84 (m, 1H), 7.50–7.60
(m, 2H), 7.30–7.44 (m, 3H), 4.98 (m, 1H), 4.00–4.20
(m, 1H), 3.76–3.92 (m, 1H), 2.8–3.42 (m, 9H), 1.72
(s, 3H), 1.14–1.62 (m, 3H), 0.87 (d, J = 6.1 Hz, 3H),
0.85 (d, J = 6.1 Hz, 3H); 13C NMR: 169.7, 169.4,
151.8, 146.3, 146.2, 134.9, 133.9, 133.4, 132.9 (q,
J = 7.8 Hz), 130.3 (q, J =28.4 Hz), 129.2, 127.8 (2 C),
125.9, 125.0 (q, J = 272.1 Hz), 52.3, 51.5 (2C), 47.9,
47.7, 46.6, 43.0, 35.9, 25.1, 24.5, 22.9, 22.3; MS: 574
(MH+).
1.1.6. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-6-fluorophe-
nyl]-4-[(2R)-acetamido-3-(2,4-dichlorophenyl)propionyl]}-
piperazine trifluoroacetate (S-10a). Purified on HPLC,
To a solution of S-5d (50 mg) in CH2Cl2 (2 mL) was
added acetic anhydride (1 mL, excess). The mixture
was stirred at room temperature for 1 h and then con-
centrated in vacuo. The crude product was then dis-
solved in 2 mL of MeOH and treated with 4 N HCl in
1,4-dioxane (27 lL, 0.11 mmol). The reaction mixture
was stirred at room temperature for 1 h. MeOH and
excess of HCl were removed in vacuo, and the product
was purified using flash column chromatography on sil-
ica gel (30–40% ethyl acetate in hexanes) to give the
titled compound as colorless oil, HPLC purity: 100%
(220 and 254 nm). 1H NMR (DMSO-d6): 8.43 (d,
J = 8.3 Hz, 1H, NH), 7.81 (d, J = 1.3 Hz, 1H), 7.60 (d,
J = 1.7 Hz, 1H), 7.48 (dd, J = 1.7, 8.3 Hz, 1H), 7.32–
7.40 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 5.07 (dd,
J = 7.9 Hz, 1H), 4.28–4.36 (m, 1H), 3.10–3.70 (m, 6H),
2.98–3.08 (m, 1H), 2.86–2.95 (m, 1H), 2.60–2.74 (m,
2H), 1.74 (s, 3H), 1.14–1.60 (m, 3H), 0.86 (d,
J = 6.4 Hz, 3H), 0.84 (d, J = 6.4 Hz, 3H); 13C NMR:
169.8, 169.4, 153.7, 144.9, 135.1, 134.8, 134.1, 132.9,
129.3, 127.8, 125.5 (q, J = 31.7 Hz), 125.2 (q,
J = 271.6 Hz), 124.7, 124.5, 121.3, 64.2, 53.0, 49.7
(2C), 47.7, 47.3, 46.2, 42.7, 35.9, 25.3, 23.9, 23.3, 22.8
(2C); HRMS (MH+) calcd for C27H33Cl2F3N4O2
573.2005, found 573.1998.
1
white solid, HPLC purity: 100% (220 and 254 nm). H
NMR (DMSO-d6): 8.41-8.50 (b, 1H), 7.56 (dd, J = 1.8,
4.8 Hz, 1H), 7.26–7.44 (m, 4H), 7.15 (dd d,
JH,F = 12.3 Hz, J = 2.6, 7.9 Hz, 1H), 5.00–5.14 (m,
1H), 4.76–4.84 (m, 1H), 4.30–4.46 (m, 1H), 3.86–4.04
(m, 1H), 3.20–3.42 (m, 4H), 2.60–3.18 (m, 4H), 1.74 (s,
3H), 1.58–1.72 (m, 1H), 1.40–1.57 (m, 2H), 0.89 (d,
J = 6.4 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H); 13C NMR:
169.8, 169.4, 162.0 (d, J = 250.5 Hz), 142.3, 136.5,
134.9, 134.5, 134.0, 132.9, 129.2, 128.7, 127.8, 123.4,
116.7, 51.8, 51.0, 47.5, 46.6, 45.7, 43.0, 36.0, 26.4, 24.9,
23.2, 23.1, 22.8; MS: 523 (MH+).
1.1.7. 1-{4-[(1R)-2-(1-Amino-3-methylbutyl)-6-fluorophe-
nyl]-4-[(2R)-acetamido-3-(2,4-dichlorophenyl)propionyl]}-
piperazine trifluoroacetate (R-10a). Purified on HPLC,
white solid, HPLC purity: 100% (220 nm). 1H NMR
(DMSO-d6): 8.42-8.50 (b, 1H), 7.56 (dd, J = 1.8,
3.5 Hz, 1H), 7.30–7.42 (m, 4H), 7.18–7.28 (m, 1H),
5.01–5.13 (m, 1H), 4.86–4.98 (m, 1H), 4.32–4.46 (m,
1H), 3.82–4.04 (m, 1H), 2.83–3.20 (m, 6H), 2.60–2.80
(m, 2H), 1.75 (s, 3H), 1.64–1.76 (m, 1H), 1.48–1.62 (m,
1H), 1.30–1.48 (m, 1H), 0.89 (d, J = 6.6 Hz, 3H), 0.87
(d, J = 6.6 Hz, 3H); MS: 523 (MH+).

5172
J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
1.1.8. 1-{4-[(1S)-2-(Amino-3-methylbutyl)-6-fluorophenyl]-
4-[(2R)-(3-aminopropionylamido)-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine (S-10c). To a solution of 4-{2-
[(1S)-((S)-2-methylpropanesulfinylamino)-3-methylbu-
tyl]-6-fluorophenyl}-piperazine-1-carboxylic acid tert-
butyl ester (Boc-S-4a,7 43.0 mg, 0.092 mmol) in 2 mL
of CH2Cl2 was added 0.5 mL of TFA. The reaction mix-
ture was stirred for 0.5 h, basified with saturated aque-
ous NaHCO3, and extracted with EtOAc (3 · 10 mL).
After removal of solvents, the residue was dissolved in
2 mL of DMF/CH2Cl2 (1:4) followed by the addition
of N-Boc-b-Ala-(2,4-Cl)Phe-OH (44.5 mg, 0.11 mmol),
NaHCO3 (23.1 mg, 0.28 mmol), HOBt (24.8 mg,
0.18 mmol), and EDCI (35.1 mg, 0.18 mmol) sequen-
tially. The reaction mixture was stirred overnight.
EtOAc/saturated aqueous NaHCO3 (50 mL/20 mL)
workup followed by flash silica chromatograph column
(50–60% EtOAc/hexanes) purification gave the product
as white foam (54.8 mg, 79%). This white foam was dis-
solved in 5 mL of MeOH and treated with 4 N HCl in
1,4-dioxane (27 lL, 0.1085 mmol). The reaction mixture
was stirred at room temperature for 1 h. MeOH and ex-
cess of HCl were removed in vacuo to give white foam
which was treated with 5 mL of 30%TFA in CH2Cl2.
After stirring for 1 h, excess of TFA and CH2Cl2 were
removed in vacuo and the product was purified with
flash silica chromatograph column (10–15% MeOH/
CH2Cl2) to give the titled compound as oil (34.0 mg,
85%), HPLC purity: 100% (220 nm). 1H NMR
(DMSO-d6): 8.6 (m, 1H), 7.5 (br s, 1H), 7.24–7.38 (m,
3H), 7.10–7.22 (m, 1H), 6.91–7.00 (m, 1H), 5.04–5.16
(m, 1H), 4.28–4.48 (m, 2H), 3.84–3.98 (m, 1H), 2.80–
3.20 (m, 6H), 2.56–2.80 (m, 4H), 2.10–2.34 (m, 2H),
1.42–1.70 (m, 1H), 1.18–.44 (m, 2H), 0.80–0.92 (m,
6H) 13C NMR: 171.2, 169.9, 161.9 (d, J = 249.4 Hz),
149.5 (d, J = 3.4 Hz), 135.7 (d, J = 10.3 Hz), 135.0,
134.5, 134.0, 133.1, 129.2 (d, J = 6.1 Hz), 128.0, 127.9,
123.1, 114.7 (d, J = 21.2 Hz), 52.0, 49.2, 48.2, 47.8,
43.1, 38.3, 38.2, 38.0, 36.1, 35.6, 25.3, 24.0, 22.9. HRMS
(MH+): calcd for C27H36Cl2FN5O2 552.2303, found
552.2309.
(DMSO-d6): 8.53 (d, J = 8.3 Hz, 1H), 7.81 (d,
J = 1.8 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.47 (dd,
J = 1.8, 8.3 Hz, 1H), 7.28–7.38 (m, 2H), 7.13 (d,
J = 8.3 Hz, 1H), 5.04–5.15 (m, 1H), 4.26–4.33 (m, 1H),
3.20–3.68 (m, 2H), 2.78–3.28 (m, 8H), 2.56–2.74 (m,
2H), 2.10–2.20 (m, 2H), 1.46–1.61 (m, 1H), 1.32–1.46
(m, 1H), 1.18–1.32 (m, 1H), 0.86 (d, J = 6.6 Hz, 3H),
0.84 (d, J = 6.6 Hz, 3H); MS: 602 (MH+).
1.1.11. 1-{4-[(1S)-2-(Amino-3-methylbutyl)-6-trifluoro-
methylphenyl]-4-[(2R)-(3-aminopropionylamido)-3-(2,4-
dichlorophenyl)propionyl]}-piperazine trifluoroacetate
(S-9c). Purified on HPLC, white powder, HPLC purity:
1
97% (220 nm), 100% (254 nm). H NMR (DMSO-d6):
8.64–8.76 (b, 1H), 7.82–7.90 (m, 1H), 7.73–7.80 (m,
1H), 7.54–7.68 (m, 2H), 7.29–7.42 (m, 2H), 5.04–5.20
(m, 1H), 4.48–4.68 (m, 1H), 3.64–3.82 (m, 2H), 3.50–
3.63 (m, 2H), 3.32–3.46 (m, 2H), 2.75–3.20 (m, 6H),
2.36–2.46 (m, 2H), 1.46–1.96 (m, 1H), 1.59–1.75 (m,
1H), 1.42–1.59 (m, 1H), 0.90 (d, J = 6.1 Hz, 3H), 0.87
(d, J = 6.1 Hz, 3H); MS: 602 (MH+).
1.1.12. 1-{4-[(1R)-2-(Amino-3-methylbutyl)-6-trifluoro-
methylphenyl]-4-[(2R)-(3-aminopropionylamido)-3-(2,4-dichlor-
ophenyl)propionyl]}-piperazine (R-9c). Colorless oil,
HPLC purity: 100% (220 and 254 nm). 1H NMR
(DMSO-d6): 8.59 (d, J = 8.8 Hz, 1H), 7.74–7.90 (m,
1H), 7.48–7.62 (m, 2H), 7.26–7.46 (m, 3H), 5.00–5.20
(m, 1H), 4.00–4.24 (m, 1H), 3.70–3.94 (m, 1H), 3.20–
3.70 (m, 6H), 3.00–3.20 (m, 2H), 2.80–3.00 (m, 2H),
2.70–2.78 (m, 1H), 2.14–2.38 (m, 2H), 1.46–1.61 (m,
1H), 1.32–1.46 (m, 1H), 1.18–1.32 (m, 1H), 0.87 (d,
J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H); MS: 602
(MH+).
1.1.13. 1-{4-[(1R)-2-(Amino-3-methylbutyl)-6-fluorophe-
nyl]-4-[(2R)-(3-aminopropionylamido)-3-(2,4-dichlorophe-
nyl)propionyl]}-piperazine
trifluoroacetate
(R-10c).
Purified on HPLC, white powder, HPLC purity: 100%
(220 nm). 1H NMR (DMSO-d6): 8.67–8.77 (b, 1H),
7.58 (dd, J = 2.2, 4.3 Hz, 1H), 7.28–7.42 (m, 4H), 7.22
(dd d, JH,F = 12.3 Hz, J = 2.2, 7.5 Hz, 1H), 5.05–5.18
(m, 1H), 4.88–4.97 (m, 1H), 4.32–4.45 (m, 1H), 3.79–
4.02 (m, 1H), 2.58–3.20 (m, 10H), 2.39–2.48 (m, 2H),
1.65–1.78 (m, 1H), 1.47–1.62 (m, 1H), 1.32–1.46 (m,
1H), 0.88 (d, J = 6.1 Hz, 6H); MS: 552 (MH+).
Compounds S-7c, R-7c, S-9c, R-9c, and R-10c were syn-
thesized using a procedure similar to that of S-10c from
the corresponding S-4d, R-4d, S-4b, R-4b, and R-4a,
respectively.10
1.1.9. 1-{4-[(1S)-2-(Amino-3-methylbutyl)-4-fluorophenyl]-
4-[(2R)-(3-aminopropionylamido)-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine (S-7c). White foam, HPLC purity:
1.1.14. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-4-trifluoro-
methylphenyl]-4-[(2R)-aminoacetamido-3-(2,4-dichloro-
phenyl)propionyl]}-piperazine (S-7b). This compound
was prepared using a procedure similar to that of S-
10c, but replacing N-Boc-Ala-D(2,4-Cl)Phe-OH with
N-Boc-Gly-D(2,4-Cl)Phe-OH.
1
99% (220 and 254 nm). H NMR (DMSO-d6): 8.63 (d,
J = 8.3 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.61 (d,
J = 1.7 Hz, 1H), 7.49 (dd, J =1.8, 8.3 Hz, 1H), 7.30–
7.38 (m, 2H), 7.14 (d, J = 8.3 Hz, 1H), 5.02–5.16 (m,
1H), 4.26–4.34 (m, 1H), 3.40–3.64 (m, 4H), 2.80–3.26
(m, 6H), 2.58–2.80 (m, 2H), 2.12–2.18 (m, 2H), 1.32–
1.61 (m, 2H), 1.20–1.32 (m, 1H), 0.86 (d, J = 6.6 Hz,
3H), 0.84 (d, J = 6.6 Hz, 3H); MS: 602 (MH+).
White foam, HPLC purity: 99% (220 nm), 100%
(254 nm). 1H NMR (DMSO-d6): 8.26–8.38 (b, 1H,
NH), 7.80–7.84 (br s, 1H), 7.60 (d, J = 1.8 Hz, 1H),
7.46 (dd, J = 1.8, 8.8 Hz, 1H), 7.28–7.40 (m, 2H), 7.13
(d, J = 8.3 Hz, 1H), 5.06–5.20 (m, 1H), 4.26–4.38 (m,
1H), 3.40–3.64 (m, 2H), 2.80–3.40 (m, 6H), 2.20–2.36
(m, 2H), 2.60–2.70 (m, 2H), 1.84–1.90 (m, 1H), 1.14–
1.62 (m, 2H), 0.86 (d, J = 6.1 Hz, 3H), 0.84 (d,
J = 6.1 Hz, 3H); 13C NMR: 169.5 (2C), 153.7, 144.9,
1.1.10. 1-{4-[(1R)-2-(Amino-3-methylbutyl)-4-fluorophe-
nyl]-4-[(2R)-(3-aminopropionylamido)-3-(2,4-dichlorophe-
nyl)propionyl]}-piperazine (R-7c). White foam, HPLC
purity: 91% (220 nm), 92% (254 nm). 1H NMR

J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5173
135.1, 134.5, 134.2, 133.0, 129.3, 127.9, 125.5 (q,
J = 31.3 Hz), 125.2 (q, J = 271.6 Hz), 124.7, 124.3,
121.3, 64.2, 53.0, 47.4, 47.3, 46.2, 42.7, 36.1, 29.7, 25.3,
23.9, 22.8, 18.6; HRMS (MH+) calcd for
C27H34Cl2F3N5O2 588.2114, found 588.2101.
ylpropanesulfinylamino-3-methylbutyl)-4-trifluorometh-
ylphenyl]-4-[(2R)-amino-3-(2,4-dichlorophenyl)propio-
nyl]}-piperazine (S-5d, 50 mg, 0.079 mmol) in DMF/
CH2Cl2 (2 mL) was added N-Boc-isonipecotic acid
(23 mg, 0.1 mmol), followed by HOBt (20 mg,
0.14 mmol), EDCI (28 mg, 0.14 mmol), and NaHCO3
(24 mg, 0.3 mmol). The mixture was stirred at rt over-
night and then concentrated in vacuo. The crude prod-
uct was then dissolved in 5 mL of MeOH and treated
with 4 N HCl in 1,4-dioxane (27 lL, 0.1085 mmol),
stirred at rt for 1 h, and concentrated in vacuo, and
the residue was treated with 30% TFA/CH2Cl2 at rt
for 1 h. The product was then purified using HPLC
and obtained as white powder, HPLC purity: 100%
(220 and 254 nm). 1H NMR (DMSO-d6): 8.49 (d,
J = 8.3 Hz, 1H), 7.93 (d, J = 1.3 Hz, 1H), 7.68 (dd,
J = 1.3, 8.8 Hz, 1H), 7.59 (s, 1H), 7.35 (br s, 2H), 7.33
(d, J = 8.8 Hz, 1H), 5.03–5.14 (m, 1H), 4.70–4.80 (m,
1H), 4.04–4.16 (m, 1H), 3.12–3.40 (m, 9H), 3.02–3.12
(m, 1H), 2.90–3.00 (m, 1H), 2.74–2.89 (m, 2H),
2.56–2.70 (m, 1H), 1.62–1.80 (m, 3H), 1.30–1.62 (m,
3H), 0.90 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H);
13C NMR: 173.3, 169.7, 154.7, 137.4, 135.1, 134.7,
134.3, 132.9, 129.2, 127.7, 126.7 (q, J = 5.6 Hz),
126.3 (q, J = 32.2 Hz), 125.0 (q, J = 271.7 Hz), 124.9,
122.9, 53.0, 49.2, 47.6, 47.0, 46.0, 45.8, 43.0 (2C), 42.6,
39.2, 35.7, 25.9, 25.6, 24.7, 23.0 (2C); HRMS
(MH+) calcd for C31H40Cl2F3N5O2 642.2584, found
642.2583.
Compounds S-9b, R-9b, S-10b, and R-10b were synthe-
sized using a procedure similar to that of S-7b from
S-4b, R-4b, S-4a, and R-4a, respectively.10
1.1.15. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-6-trifluoro-
methylphenyl]-4-[(2R)-aminoacetamido-3-(2,4-dichloro-
phenyl)propionyl]}-piperazine trifluoroacetate (S-9b).
Purified on HPLC, white powder, HPLC purity: 95%
1
(220 and 254 nm). H NMR (DMSO-d6): 8.86–9.00 (b,
1H), 7.87 (d, J = 1.8 Hz, 1H), 7.72–7.79 (m, 1H), 7.53–
7.63 (m, 2H), 7.28–7.42 (m, 2H), 5.13–5.24 (m, 1H),
4.50–4.67 (m, 1H), 3.67–3.80 (m, 1H), 3.47–3.65 (m,
3H), 3.32–3.46 (m, 1H), 3.04–3.16 (m, 2H), 2.81–3.03
(m, 4H), 2.70–2.80 (m, 1H), 1.79–2.00 (m, 1H), 1.59–
1.78 (m, 1H), 1.45–1.59 (m, 1H), 0.89 (d, J = 6.5 Hz,
3H), 0.87 (d, J = 6.5 Hz, 3H); MS: 588 (MH+).
1.1.16. 1-{4-[(1R)-2-(1-Amino-3-methylbutyl)-6-trifluo-
romethylphenyl]-4-[(2R)-aminoacetamido-3-(2,4-dichloro-
phenyl)propionyl]}-piperazine (R-9b). White solid, HPLC
1
purity: 100% (220 and 254 nm). H NMR (DMSO-d6):
8.30–8.42 (b, 1H, NH), 7.76–7.90 (m, 1H), 7.50–7.58
(m, 2H), 7.22–7.46 (m, 3H), 5.06–5.20 (m, 1H), 4.06–
4.20 (m, 1H), 3.74–3.94 (m, 2H), 3.40–3.64 (m, 2H),
2.80–3.40 (m, 6H), 3.00–3.16 (m, 1H), 2.84–2.98 (m,
1H), 1.80–1.90 (m, 1H), 1.14–1.62 (m, 2H), 0.87 (d,
J = 6.2 Hz, 3H), 0.85 (d, J = 6.2 Hz, 3H); MS: 588
(MH+).
1.1.20. 1-[(R)-2-{4-[(1S)-2-(1-Amino-3-methylbutyl)-4-tri-
fluoromethylphenyl]piperazin-1-yl}-1-(2,4-dichlorobenzyl)-2-
oxoethyl]-piperazin-2-one dihydrochloride (11). A solu-
tion of 1-{4-[(1S)-2-((S)-2-methylpropanesulfinylamino-
3-methylbutyl)-4-trifluoromethylphenyl]-4-[(2R)-amino-
3-(2,4-dichlorophenyl)propionyl]}-piperazine (S-5d, 739
mg, 1.2 mmol) in CH2Cl2 (12 mL) was treated with
glacial acetic acid (1 drop) and tert-butyl-N-(2-oxoeth-
yl)carbamate (372 mg, 2.34 mmol). Stirring was contin-
ued at ambient temperature for 0.5 h, and then
Na(OAc)3BH (1.24 g, 5.85 mmol) was added portion-
wise. The resulting suspension was stirred at room
temperature for 14 h, after which time the reaction
was deemed complete by LCMS. The reaction mixture
was transferred to a separatory funnel, diluted with
CH2Cl2 (50 mL), and washed with saturated NaHCO3
aqueous solution (2 · 50 mL). The organic layers were
dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by flash column chro-
matography on silica gel, eluting with ethyl acetate, and
the product was obtained as white foam (534 mg, 59%
yield).
1.1.17.
1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-6-fluoro-
phenyl]-4-[(2R)-aminoacetamido-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine trifluoroacetate (S-10b). Purified
on HPLC, white solid, HPLC purity: 100% (220 nm).
1H NMR (DMSO-d6): 8.60–8.72 (b, 1H), 7.57 (dd,
J = 1.8, 6.1 Hz, 1H), 7.28–7.42 (m, 4H), 7.11–7.21 (m,
1H), 5.10–5.21 (m, 1H), 4.81–4.90 (m, 1H), 4.31–4.45
(m, 1H), 3.86–4.03 (m, 1H), 3.22–3.52 (m, 6H), 2.88–
3.10 (m, 2H), 2.58–2.83 (m, 2H), 1.60–1.73 (m, 1H),
1.36–1.56 (m, 2H), 0.86 (d, J = 6.1 Hz, 6H); MS: 538
(MH+).
1.1.18. 1-{4-[(1R)-2-(1-Amino-3-methylbutyl)-6-fluoro-
phenyl]-4-[(2R)-aminoacetamido-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine trifluoroacetate (R-9b). Purified on
HPLC, white powder, HPLC purity: 100% (220 nm). 1H
NMR (DMSO-d6): 8.96 (d, J = 8.3 Hz, 1H), 7.57–7.62
(m, 1H), 7.28–7.43 (m, 4H), 7.22 (dd d, JH,F = 12.3 Hz,
J = 2.2, 7.5 Hz, 1H), 5.12–5.24 (m, 1H), 4.85–4.98 (m,
1H), 4.36–4.46 (m, 1H), 3.80–4.03 (m, 1H), 3.44–3.60
(m, 2H), 2.60–3.14 (m, 8H), 1.64–1.78 (m, 1H), 1.46–
1.62 (m, 1H), 1.32–1.46 (m, 1H), 0.88 (d, J = 6.1 Hz,
6H); MS: 538 (MH+).
The above compound (368 mg, 0.47 mmol) was dis-
solved in ethyl acetate (3 mL) and treated with saturated
NaHCO3 aqueous solution (3 mL). The resulting bipha-
sic system was stirred vigorously and chloroacetyl chlo-
ride (57 lL, 0.71 mmol) was added dropwise. After 1 h
the reaction was complete according to TLC analysis
(EtOAc). The organic layer was then separated and
evaporated to give a crude product as white foam
(340 mg, 84%). This foam was dissolved in CH2Cl2
(5 mL) and treated with HCl (360 lL of a 4.0 M solution
1.1.19. 1-{4-[(1S)-2-(1-Amino-3-methylbutyl)-4-trifluoro-
methylphenyl]-4-[(2R)-(piperidin-4-ylcarboxamido)-3-(2,
4-dichlorophenyl)propionyl]}-piperazine trifluoroacetate
(S-7d). To
a solution of 1-{4-[(1S)-2-((S)-2-meth-

5174
J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
1
127.04, 126.52, 124.83, 124.55 (q, HC,F = 234 Hz),
in 1,4-dioxane, 1.42 mmol). After 2 h at room tempera-
ture, the volatiles were removed in vacuo and the crude
residue was purified by preparative TLC plate (500 lm),
eluting with a 400:50:2 v/v mixture of CHCl3, MeOH,
and NH4OH, respectively. The free base (39 mg,
0.06 mmol, 13%) was converted to the respective hydro-
chloride salt, by treatment with an HCl solution in
diethyl ether. The salt was obtained as a white solid
(45 mg).
123.26, 50.06, 47.29, 45.88, 45.27, 43.59, 42.65, 40.36,
39.78, 39.76 (MeSO3H), 39.69, 30.68, 24.66, 23.02,
22.98, 18.56; MS: 599.1 (MH+); Anal. calcd for
C29H35Cl2F3N4O2ÆMeSO3 HÆ2.5H2O: C 48.65%, H
5.99%, N 7.56%; found: C 48.66%, H 5.96%, N, 7.59%.
Compounds 12b, 12c, 12d, and 12f were synthesized
using a procedure similar to that of 12a from S-4c, S-
4f, S-4a, and S-4e, respectively.10
1
Free base: H NMR (CDCl3): 7.65 (d, J = 1.8 Hz, 1H),
7.45 (dd, J1 = 1.8 Hz, J2 = 8.1 Hz, 1H), 7.42 (d,
J = 1.5 Hz, 1H), 7.26–7.18 (m, 2H), 7.02 (d,
J = 9.0 Hz, 1H), 5.99 (t, J = 7.7 Hz, 1H), 4.50 (t,
J = 7.2 Hz, 1H), 3.72–3.57 (br s, 3H), 3.54–3.47 (m,
1.1.22. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)-4-fluoro-
phenyl]piperazin-1-yl}-1-(2,4-dichlorobenzyl)-2-oxoeth-
yl]pyrrolidin-2-one (12b). White foam, HPLC purity:
1
100% (220 and 254 nm); H NMR (CDCl3): 7.35–7.42
4H),
3.23
(ABX,
J1 = 8.1 Hz,
J2 = 13.5 Hz,
(m, 1H), 7.10–7.30 (m, 4H), 6.90–7.01 (m, 1H), 5.38–
5.53 (m, 1H), 4.74–4.90 (m, 1H), 3.40–3.64 (m, 2H),
3.22–3.34 (m, 1H), 3.06–3.21 (m, 1H), 2.44–2.90 (m,
3H), 2.20–2.38 (m, 2H), 1.90–2.10 (m, 2H), 1.36–1.90
(m, 5H), 1.18–1.36 (m, 2H), 0.98–1.10 (m, 1H), 0.91
(d, J = 7.0 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H); MS: 549
(MH+).
Dm = 46.8 Hz, 2H), 3.09 (t, J = 5.2 Hz, 2H), 2.93–2.89
(m, 1H), 2.66 (br s, 3 H), 1.71 (br s, 2 H), 1.61–1.50
(m, 2H), 1.47–1.40 (m, 1H), 0.92 (dd, J = 4.6 Hz, 6H);
MS:
614.5
(MH+).
Anal.
calcd
for
C29H36Cl2F3N5O2Æ2HClÆCH2Cl2: C 46.65%, H 5.22%,
N 9.07%; found: C 46.79%, H 4.98%, N 9.01%.
1.1.21. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)-4-tri-
fluoromethylphenyl]piperazin-1-yl}-1-(2,4-dichlorobenzyl)-
2-oxoethyl]pyrrolidin-2-one mesylate (12a). 1-{4-[(1S)-2-
((S)-2-Methylpropanesulfinylamino-3-methylbutyl)-4-tri-
fluoromethylphenyl]-4-[(2R)-amino-3-(2,4-dichlorophenyl)-
propionyl]}-piperazine (S-5d, 2.86 g, 4.5 mmol) was
dissolved in 15 mL of 1,2-dichloroethane followed by
the addition of succinic semialdehyde (15% in water,
3 mL, 4.8 mmol), HOAc (274 lL, 4.8 mmol), and NaB-
H(OAc)3 (1.02 g, 4.8 mmol). The reaction mixture was
stirred at room temperature overnight, followed by the
addition of acetic anhydride (375 lL, 4.8 mmol). The
reaction mixture was stirred for another 2 h diluted with
100 mL of EtOAc, washed with saturated NaHCO3
aqueous solution and brine. The organic layer was dried
over anhydrous Na2SO4, and solvents were removed in
vacuo. The residue was purified with flash silica column
chromatograph (50% EtOAc in hexanes) to give a sulfin-
imide as white foam (2.06 g, 65%).
1.1.23. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)phenyl]-
piperazin-1-yl}-1-(2,4-dichlorobenzyl)-2-oxoethyl]pyrroli-
din-2-one (12c). White foam, HPLC purity: 90%
1
(220 nm), 97% (254 nm); H NMR (CDCl3): 7.35–7.42
(m, 2H), 7.10–7.24 (m, 4H), 6.95 (d, J = 8.3 Hz, 1H),
5.48 (dd, J = 7.4 Hz, 1H), 4.44–4.58 (m, 1H), 3.00–3.84
(m, 6H), 3.22–3.32 (m, 1H), 3.10–3.20 (m, 1H), 2.44–
2.90 (m, 3H), 2.20–2.36 (m, 2H), 1.90–2.10 (m, 2H),
1.36–1.62 (m, 3H), 0.92–1.06 (m, 1H), 0.90 (d,
J = 6.1 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H); 13C NMR:
174.7, 167.7, 150.0, 135.5, 133.8, 133.5 (2C), 132.7,
129.7, 128.0, 127.4, 127.0, 125.9, 121.2, 53.7, 49.4,
48.6, 47.6, 46.6, 44.0, 43.0, 40.0, 33.1, 30.9, 25.5, 23.2,
22.7, 18.6; MS: 531 (MH+).
1.1.24. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)-6-fluo-
rophenyl]piperazin-1-yl}-1-(2,4-dichlorobenzyl)-2-oxoeth-
yl]pyrrolidin-2-one (12d). White foam, HPLC purity:
1
100% (220 nm); H NMR 300 MHz (DMSO-d6): 7.54
(br s, 1H), 7.13–7.40 (m, 3H), 6.97 (dd d, JH,F = 12.7 Hz,
J = 1.8, 8.3 Hz, 1H), 5.18–5.33 (m, 1H), 4.32–4.46 (m,
1H), 3.71–3.83 (m, 1H), 3.47–3.65 (m, 1H), 2.95–3.40
(m, 6H), 2.60–2.90 (m, 4H), 2.04–2.18 (m, 2H), 1.82–
2.00 (m, 2H), 1.52–1.64 (m, 2H), 1.10–1.44 (m, 2H),
0.88 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H); MS:
549 (MH+).
The above intermediate (1.05 g, 1.5 mmol) was dissolved
in 50 mL of MeOH and treated with HCl (4 N in 1,4-
dioxane, 0.48 mL, 1.92 mmol). The reaction mixture
was stirred for 1 h, MeOH and excess HCl were re-
moved in vacuo. The crude product was purified with
flash silica column chromatograph (8% MeOH in
CH2Cl2) to give 12a as white foam (0.70 g, 78% yield),
HPLC purity: 99.3% (220 nm) and 99.1% (254 nm).
The free base was converted into a mesylate salt using
a standard procedure. 1H NMR (CDCl3): 7.94 (d,
1.1.25. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)-4-tri-
fluoromethylphenyl]-piperazin-1-yl}-1-(4-dichlorobenzyl)-
2-oxoethyl]pyrrolidin-2-one mesylate (12e). A solution of
(R)-3-(4-chloro-phenyl)-2-(2-oxo-pyrrolidin-1-yl)-propi-
onic acid (R-14b,12 139 mg, 0.52 mmol) in DMF (5 mL)
was treated with diisopropylethylamine (180 lL, 1.04
mmol) and HBTU (256 mg, 0.68 mmol) under N2. The
resulting mixture was stirred at room temperature for
0.5 h and then treated with a solution of 4-{4-trifluorom-
ethyl-2-[(S)-3-methyl-1-(2-methylpropane-2-sulfinylamino)-
butyl]-phenyl}-piperazine (S-4d, 217 mg, 0.52 mmol),
obtained from 4-{4-trifluoromethyl-2-[(S)-3-methyl-1-
(2-methylpropane-2-sulfinylamino)butyl]-phenyl}-piper-
3
3JH,H = 1.4 Hz, 1H), 7.56 (d, JH,H = 1.8 Hz, 1H),
3
7.67–7.74 (m, 1H), 7.34 (d, JH,H = 1.8 Hz, 1H), 7.30–
7.42 (m, 2H), 5.20–5.30 (m, 1H), 4.70–4.86 (m, 1H),
3.36–3.64 (m, 5H), 3.16–3.30 (m, 2H), 3.02–3.14 (m,
2H), 2.80–3.02 (m, 2H), 2.60–2.74 (m, 1H), 2.31 (s,
3H, MeSO3H), 2.00–2.14 (m, 2H), 1.82–1.98 (m, 2H),
1.70–1.84 (m, 1H), 1.46–1.60 (m, 1H), 1.26–1.42 (m,
1H), 0.91 (t, 3JH,H = 6.4 Hz, 3H), 0.83 (t,
3JH,H = 6.4 Hz, 3H); 13C NMR: 174.04, 167.55, 154.83,
136.17, 135.10, 134.92, 133.82, 132.77, 129.24, 127.83,

J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
5175
azine-1-carboxylic acid tert-butyl ester (Boc-S-4d,
292 mg, 0.56 mmol) with TFA treatment as previously
described, in DMF (1 mL). The reaction was deemed
complete after 2 h at room temperature, monitored by
LC/MS. The mixture was diluted with EtOAc (50 mL)
and placed in a separatory funnel. The organic layer
was washed with 0.1 N HCl aqueous (50 mL), saturated
NaHCO3 (100 mL), and brine (50 mL), dried over anhy-
drous MgSO4, filtered, and evaporated in vacuo to give
the crude product as a tan film, which was used without
further purification.
HPLC purity: 100% (220 and 254 nm); 1H NMR
(DMSO-d6): 7.82 (d, J = 1.8 Hz, 1H), 7.50 (dd,
J = 1.8, 8.8 Hz, 1H), 7.22–7.33 (m, 4H), 7.20 (d,
J = 8.8 Hz, 1H), 5.10–5.18 (m, 1H), 4.33–4.41 (m,
1H), 3.40–3.72 (m, 2H), 3.20–3.32 (m, 4H), 2.75–3.07
(m, 6H), 2.60–2.72 (m, 1H), 2.00–2.27 (m, 1H), 1.76–
2.00 (m, 2H), 1.39–1.58 (m, 2H), 1.24–1.36 (m, 1H),
0.86 (d, J = 6.1 Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H); MS:
565 (MH+).
1.2. Receptor binding
The above compound (347 mg, 0.52 mmol) was dis-
solved in MeOH (5 mL) and treated with HCl (340 lL
of a 2.0 M solution in Et2O, 0.68 mmol). The resulting
solution was stirred at ambient temperature for 1 h.
The volatiles were removed in vacuo and the residue
was purified by preparative HPLC/MS. The free base
was isolated in 60% yield (187 mg, 0.33 mmol). HPLC
purity: 99.4% (220 nm) and 100% (254 nm). The com-
pound was converted to the mesylate salt in CH2Cl2
using one equivalent of methanesulfonic acid.
Receptor binding was performed on HEK293 cells sta-
bly expressing the human melanocortin receptors, using
[
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 previ-
ously reported.13
125I]-NDP-MSH as the radiolabeled ligand. The cAMP
1.3. Pharmacokinetic characterization
The pharmacokinetic profile of 11, 12a, and 12e was
determined in male mice (N = 3/time points at a dose
of 10 mg/kg for po or 5 mg/kg for iv). Compounds were
dosed as a dihydrochloride salt (11) or mesylate salt (12a
and 12e) 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. Termi-
nal blood samples were taken from treated mice at 9
time points ranging from pre-dose to 24 h (0, 0.25, 0.5,
1, 2, 4, 6, 8, and 24 h) post-dose. Brain samples were col-
lected at 1 and 4 h post-iv dosing.
1
Free base: H NMR (DMSO-d6): 7.82 (d, J = 1.8 Hz,
1H), 7.49 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 1H), 7.28 (dd,
J1 = 8.4 Hz, J2 = 15.9 Hz, 4H), 7.17 (d, J = 8.4 Hz,
1H), 5.13 (t, J = 8.4 Hz, 1H), 4.36 (t, J = 7.5 Hz, 1H),
3.50–3.37 (m, 2H), 3.29–3.21 (m, 4H), 3.06–2.83 (m,
6H), 2.63 (br, 1H), 2.17–2.01 (m, 1H), 1.92–1.78 (m,
2H), 1.55–1.42 (m, 2 H), 1.33–1.21 (m, 1H), 0.84 (dd,
J = 6.6 Hz, 6H); 13C NMR: 174.0, 167.9, 153.8,
143.9, 137.3, 131.9, 131.3 (d, J = 29.2 Hz), 128.8, 125.5
(d, J = 31.4 Hz), 125.2 (d, J = 270.3 Hz), 124.7, 121.5,
53.5, 52.8, 51.9, 49.3, 47.2, 45.9, 43.6, 42.7, 34.4,
30.8, 25.2, 23.7, 22.8, 18.6; MS: 565.2 (MH+). Anal.
calcd for C29H36ClF3N4O2ÆCH3SO3 HÆ2/3 CH2Cl2: C
51.31, H 5.80, N 7.81; found: C 50.97%, H 5.74%, N
7.64%.
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
solutions (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. Stan-
dards and QCs for both plasma and brain were pro-
cessed and analyzed at the same time and in exactly
the same way as the analytical samples. The compound
was measured 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/x) linear calibration
curve.
1.1.26. 1-[(R)-2-{4-[2-((S)-1-Amino-3-methylbutyl)phenyl]-
piperazin-1-yl}-1-(4-dichlorobenzyl)-2-oxoethyl]pyrrolidin-
2-one mesylate (12f). This compound was prepared using
a procedure similar to that of 12e from S-4e.
Free base as white foam, HPLC purity: 97% (220 nm),
96% (254 nm); 1H NMR (CDCl3): 7.37 (dd,
J = 1.8 Hz, J = 7.5 Hz, 1H), 7.08–7.32 (m, 6H), 6.94
(d, J = 7.0 Hz, 1H), 5.31 (dd, J = 7.3 Hz, 1H), 4.44–
4.58 (m, 1H), 3.15–3.84 (m, 5H), 3.20–3.33 (m, ABX,
1H), 2.5–2.95 (m, ABX, 1H), 2.66–2.86 (m, 2H), 2.46–
2.64 (m, 1H), 1.82–2.40 (m, 6H), 1.40–1.62 (m, 2H),
0.96–1.12 (m, 1H), 0.89 (d, J = 6.1 Hz, 3H), 0.87 (d,
J = 6.1 Hz, 3H); 13C NMR: 174.8, 167.7, 150.0, 135.6
(2C), 133.0, 131.1 (2C), 128.9 (2C), 128.0, 126.9, 125.9,
121.3, 53.7, 51.3, 48.6, 47.6, 46.6, 44.1, 43.6, 43.0, 35.3,
31.0, 25.5, 23.2, 22.7, 18.6; HRMS (MH+) calcd for
C28H37ClN4O2 497.2678, found 497.2667.
1.1.27. 1-[(S)-2-{4-[2-((S)-1-Amino-3-methylbutyl)-4-tri-
fluoromethylphenyl]-piperazin-1-yl}-1-(4-dichlorobenzyl)-
2-oxoethyl]pyrrolidin-2-one trifluoroacetate (13). This
compound was prepared using a procedure similar to
that of 12e, by replacing R-14b with S-14b. White foam,

5176
J. A. Tran et al. / Bioorg. Med. Chem. 15 (2007) 5166–5176
Marks, D. L.; Ling, N.; Cone, R. D. Cancer Res. 2001, 61,
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Descriptive pharmacokinetics was derived and evalu-
ated based on the mean plasma concentrations (N = 3/
time point). A non-compartmental model in Activity-
Base with linear trapezoidal rule was used to perform
all pharmacokinetic analyses pertained to this
manuscript.
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C57BL/6 J 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 Animal and approved by the Animal
Care and Use Committee of the Oregon Health
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As previously described,3a on day 0, mice were inocu-
lated 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 5 days of the start of
the experiment. At the time of sacrifice, tumors were dis-
sected away from surrounding tissue and weighed. There
was no statistical difference in tumor size between treat-
ment groups and gross examination of organs did not
reveal the presence of metastasis.
On day 11 after tumor inoculation, mice were divided
into 3 groups and treated twice daily with vehicle, 3,
or 9 mg/kg 12e (ip). Food intake and body weight were
measured daily. Following 4 days of treatment, animals
were sacrificed, tumors were removed, and body compo-
sition was determined by dual-energy X-ray absorbtom-
etry (DEXA, PIXImus mouse densitometer, Lunar
corp.). A baseline measure was made on the day of inoc-
ulation (day 0) also. Animals were anesthetized prior to
the first scan, and asphyxiated with CO2 prior to the tu-
mor dissection and the final scan.
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.).
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References and notes
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