The efficacy and cardiac evaluation of aminomethyl tetrahydronaphthalene ketopiperazines: A novel class of potent MCH-R1 antagonists

Article abstract of DOI:10.1016/j.bmc.2006.12.028

The design, synthesis, and biological studies of a novel class of MCH-R1 antagonists based on an aminotetrahydronaphthalene ketopiperazine scaffold is described. Compounds within this class promoted significant body weight reduction in mouse diet induced obesity studies. The potential for hERG blockage activity and QT interval studies in anesthetized dogs are discussed.

Full text of DOI:10.1016/j.bmc.2006.12.028

Bioorganic & Medicinal Chemistry 15 (2007) 2092–2105
The efficacy and cardiac evaluation of aminomethyl
tetrahydronaphthalene ketopiperazines: A novel class
of potent MCH-R1 antagonists
*
´
´
Jose L. Mendez-Andino, Anny-Odile Colson, Kenneth M. Meyers, Maria C. Mitchell,
Karen Hodge, Jeremy M. Howard, Nicholas Kim, David C. Ackley, Jerry K. Holbert,
Scott W. Mittelstadt, Martin E. Dowty, Cindy M. Obringer, Paula Suchanek,
Ofer Reizes,à X. Eric Hu§ and John A. Wos
Procter & Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, Mason, OH 45039, USA
Received 12 September 2006; revised 18 November 2006; accepted 15 December 2006
Available online 20 December 2006
Abstract—The design, synthesis, and biological studies of a novel class of MCH-R1 antagonists based on an aminotetrahydronaph-
thalene ketopiperazine scaffold is described. Compounds within this class promoted significant body weight reduction in mouse diet
induced obesity studies. The potential for hERG blockage activity and QT interval studies in anesthetized dogs are discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ture.4–6 Therefore, small molecule MCH-R1 antagonists
are being heavily pursued by many laboratories trying to
find an effective drug molecule for the treatment of
obesity.7,8
The World Health Organization considers obesity a ma-
jor health concern for many countries, especially the US.
Estimates indicate that more than 30% of the US adult
population is overweight, and that obesity-related health
care cost is in the range of $100 billion per year.1 These
trends have promoted increased research activity in the
pharmaceutical industry directed to find anti-obesity
therapies.2
Structures 1, 2 and 3 (Fig. 1) exemplify classes of potent
MCH-R1 antagonists that have been disclosed in recent
patent literature.9 All these compounds are similar in
structure by having a biphenyl carboxy group and a ter-
tiary amine linked via an aniline or aminotetralin unit.
Aided by molecular modeling, we utilized putative key
receptor contact points of these classes of compounds
to design and synthesize additional lead classes of
MCH-R1 antagonists.
The G-protein-coupled melanin-concentrating hormone
receptor 1 (MCH-R1) has received significant attention
in recent years as a potential target for effective anti-
obesity therapies.3 It has been suggested that CNS-locat-
ed MCH-R1 is involved in biological processes related
to mammal feeding behaviors and energy expendi-
1.1. Structure-based design of biphenyl carboxy group
replacements
Keywords: Melanin-concentrating hormone; MCH R1 antagonists;
Obesity; hERG; QT interval prolongation.
Extensive SAR developed in our laboratory suggests
that the tertiary amine, the amide bond, and the termi-
nal aromatic ring of the biphenylcarboxy group are
key pharmacophoric points for biological activity, as
also observed by others.10 Docking experiments per-
formed in our h-MCH-R1 homology model11 suggest
that compounds of the type shown in Figure 1 are
embedded in the transmembrane region between helices
3, 6, and 7. Our model suggests that the basic amine
*
Corresponding author. Tel.: +1 513 622 0304; fax: +1 513 622
1433; e-mail: mendezandino.jl@pg.com
Present address: Boehringer Ingelheim Pharmaceuticals, 900 Ridge-
bury Road, Ridgefield, CT 06877, USA.
à
Present address: Lerner Research Institute, Cleveland Clinic Foun-
dation, 9500 Euclid Avenue-NC10, Cleveland, OH 44195, USA.
Present address: Genome Research Institute, University of
Cincinnati, 2180 E. Galbraith Road, Cincinnati, OH 45237, USA.
§
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.028

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2093
O
N
O
N
N
R
3
N
H
1
X
X
O
N
R = H or Me
X = H, halogen or OMe
N
R
2
X
Figure 1. Benchmark MCH-R1 antagonist.
putatively interacts deep in the receptor with the con-
served Asp123 (helix 3), while the biaryl system is in-
volved in pi-stacking and pi-cationic interactions closer
to the receptor surface. In addition, the amide bond ap-
pears to be involved in a hydrogen bonding interaction
with Gln279 and Arg197 (Fig. 2), with the carbonyl oxy-
gen acting as the hydrogen bond acceptor, and both
Arg197 and Gln279 acting as hydrogen bond donors.
The methyl amide analog 5 (Fig. 3) displayed similar
MCH-R1 antagonist activity compared to 4 which fur-
ther confirms the fact that the amide N–H bond might
not be involved in any key binding interactions. Inver-
sion of the amide bond in compound 4 led to the synthe-
sis of compound 6 which was equipotent (Fig. 3). This
observation is in agreement with our hypothesis that a
glutamine might be involved in this interaction, as sim-
ple bond rotation within its side chain allows Gln279 to
act as a H-bond acceptor toward compound 6 without
changing the overall MCH-R1-binding capabilities of
the compound. Significant losses in biological activity
were observed when the amide carbonyl was reduced
to a methylene group resulting in a secondary amine
linker (compound 7).
As mentioned above, our model suggests that the biaryl
moiety interacts with the extracellular region of the
receptor. Indeed, we observe the formation of pi-cation-
ic interactions between Arg284 (extra-cellular loop 3)
and the external phenyl ring of our compounds, while
interactions with the internal phenyl ring are dominated
by steric interactions such as a weak pi-stacking with
Phe 289 (helix 7). Based on this observation, we hypoth-
esized that the terminal phenyl ring of the biaryl unit
plays a more critical role in MCH-R1 binding than its
internal counterpart whose role may only be as a linker
to position the distal ring for optimal interactions.
The model-based observation described heretofore led
us to consider the possibility to utilize the amide bond
as a handle for a new linker unit or ring structure to pro-
duce potent MCH-R1 antagonists lacking the benzam-
ide unit. Among several proposed scaffolds designed to
fulfill this hypothesis, structures 8 and 9 (Fig. 4) emerged
as a series of synthetically accessible compounds.
1.2. Chemistry
Compounds 14 and 15 were prepared from commercial-
ly available starting materials as depicted in Scheme 1.
The synthesis of compound 14 was initiated with the
N-allylation of commercially available N-(tert-butoxy-
carbonyl)glycine methyl ester 10 followed by the ozonol-
ysis of the alkene unit to produce acetaldehyde 12. The
resulting material was used in a reductive alkylation
reaction with 4-(1-piperidinylmethyl)-phenylethan-
amine. Under the present reaction conditions, the result-
ing secondary amine lactamized to produce
ketopiperazine 13 in modest yield. Removal of the car-
bamate group followed by coupling of the resulting sec-
ondary amine with 4-fluorophenylacetic acid produced
Figure 2. Compound 2 (X = H, R = H): main interactions for MCH-
R1 binding.

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2094
Gln279
H
O
H
Gln279
H
O
H
N
N
O
N
O
N
N
N
H
5
4
IC50 = 3 nM
Ki = 7 nM
IC50 = 31 nM
Ki = 19 nM
F
F
NH2
O
Gln279
N
H
N
N
N
H
O
6
7
F
F
IC50 = 31 nM
Ki = 25 nM
IC50 = 2 μM
Ki = 2 μM
Figure 3. Biological data supporting pharmacophore model and the role of the amide functionality in MCH-R1 binding.
R
R
O
N
R
O
N
R
N
N
N
N
R = alkyl
X = halogen
Y = O or H2
X
X
Y
Y
9
8
Figure 4. Proposed in silico MCH-R1 antagonist scaffolds.
the desired compound 14. Compound 15 was obtained
from the synthetic intermediate 13 via a two-step proto-
col which involved the removal of the carbamate group
followed by a reductive alkylation of the resulting sec-
ondary amine with phenylacetaldehyde.
proved to be challenging and inefficient. Several at-
tempts to satisfactorily purify nitrile intermediates (20)
were ineffective and repeatedly led to low overall yields
of the desired products 21a–k. An alternative synthetic
route was designed to address the need for multi-gram
preparation of these compounds for further biological
evaluation.
The desired compounds 21a–k were initially prepared as
depicted in Scheme 2. The synthesis departed from the
commercially available (S)-6-amino-5,6,7,8-tetrahydro-
naphthalene-2-carbonitrile hydrochloride 16. Reductive
alkylation of 16 with chloroacetaldehyde followed by
reaction of the resulting secondary amine with chloro
acetylchloride led to the key intermediate 18. This mate-
rial reacted with primary amines under basic conditions
to produce the ketopiperazine compounds 20 in poor–
modest yields. Finally, the desired compounds were ob-
tained by converting the nitrile functionality into a
diethyl benzyl amine via a two-step protocol that con-
sisted of a Raney nickel reduction followed by reductive
alkylation with two equivalents of acetaldehyde. This
synthetic route was effective to produce small amounts
(100–200 mg) of these compounds for the purpose of
in vitro biological testing and SAR development. Per-
forming this synthetic protocol on multi-gram scale
In order to overcome some of the limitations of our
initial synthetic approach, we envisioned that the keto-
piperazine unit of 20 could be built in a single synthet-
ic operation via
a novel multi-step ring-forming
transformation (Scheme 3). The synthetic precursors
are a methyl 2-(2-chloroethyl-phenethylamino)-acetate
(23) unit and (S)-6-amino-5,6,7,8-tetrahydronaphtha-
lene-2-carbonitrile (16). Compounds 23 were easily
prepared directly from the corresponding phenethyl
amine 19 and without isolating intermediate 22. Under
microwave conditions (Biotage Personal Chemistry
System) and with catalytic amounts of sodium iodide,
the ketopiperazine compound 20 was consistently ob-
tained in moderate yields. The reaction did not work
without the addition of sodium iodide (recovered start-
ing material and thermal decomposition). Attempts

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2095
O
O
O
O
O
a
c
O
b
O
NH
O
N
O
N
O
O
O
O
10
11
12
O
N
O
N
N
N
d
N
IC50 = 3.7 μM
Ki = 1.2 μM
O
N
13
O
O
F
14
e
O
N
N
IC50 = 20 nM
Ki = 11 nM
N
15
Scheme 1. Synthesis of MCH-R1 antagonists 14 and 15. Reagents and conditions: (a) NaH, allyl bromide, 0 ꢁC, 90 min, 70%; (b) i—O3, pyridine,
MeOH, CH2Cl2, ꢀ78 ꢁC, 90 min; ii—methyl sulfide, ꢀ78 to 25 ꢁC, 16 h, 80%; (c) 4-(1-piperidinylmethyl)-phenylethanamine, NaCNBH3, AcOH,
MeOH, 25 ꢁC, 16 h, 55%; (d) i—DCM, TFA, 25 ꢁC, 2 h; ii—p-fluorophenylacetic acid, EDCI, HOBT, NMM, DMF, 25 ꢁC, 16 h, 78%; (e) i—DCM,
TFA, 25 ꢁC, 2 h; ii—phenylacetaldehyde, NaCNBH3, AcOH, MeOH, 25 ꢁC, 16 h, 22%.
CN
CN
O
CN
a
b
Cl
N
HN
ClH3N
18
16
17
Cl
Cl
CN
O
O
N
c
d
N
N
NH2
X
X
N
N
19
X
21
20
Scheme 2. Initial synthesis of MCH-R1 antagonists 21. Reagents and conditions: (a) chloroacetaldehyde, NaCNBH3, MeOH, AcOH, 4 h, 75%;
(b) chloroacetic anhydride, DIPEA, CH3CN, ꢀ20 ꢁC to rt, 30 min; (c) NaI, MW 2-W, 70 ꢁC , 10 min, 20% over two steps; (d) i—Raney nickel,
H2, NH4OH, DMF, 16 h; ii—acetaldehyde, NaCNBH3, MeOH, AcOH, 15 min, 40% overall yield from 20.
to promote this process using conventional heat
(60–100 ꢁC) produced limited amounts of halogen
displacement and did not promote lactamization to
produce 20. The reaction worked well whenever car-
ried out at high concentrations, which allowed for
the production of 5 g of material in a 20-mL reaction
vessel in about 20 min (see Section 3). Multi-gram
preparations for selected compounds 21 (those includ-
ed in Table 2) were achieved in batch-mode by
employing an automated-sample microwave system
(see Section 3).
1.3. Biological results and discussion
Testing of compound 14 in the MCH-R1 binding
assay12 revealed a mediocre activity (IC50 = 3.7
9 lM, Ki = 1.2 1 lM), which is 100-fold lower than
that of our benchmark (compound 2). Although dock-
ing of this compound in the homology model satisfied
interactions with Gln279, Arg197, Asp123, and
Arg284 described above, the newly added distal
carbonyl interacted unfavorably with the phenyl ring
of Phe289. Since this was the only unfavorable

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2096
O
O
NH2
a
b
O
O
X
NH
22
N
Cl
X
X
19
23
16, c
CN
O
N
O
N
N
d
N
N
X
X
21
20
Scheme 3. Scale-up synthesis of MCH-R1 antagonists 21. Reagents and conditions: (a) methyl chloroacetate, diisopropylethylamine, CH3CN, 0 ꢁC;
(b) chloroacetaldehyde, NaCNBH3, MeOH, AcOH, 56% over two steps; (c) (S)-amino-5,6,7,8-tetrahydronaphthalene-2-carbonitrile hydrochloride
(16), diisopropylethylamine, CH3CN, microwave, 130 ꢁC, 30 min, 53–66%; (d) i—Raney Ni, H2, NH4OH, DMF, 16 h; ii—acetaldehyde, NaCNBH3,
MeOH, AcOH, 15 min, 87–93% from 20.
pharmacophoric point when compared to the pharma-
cophore requirements of compound 2 described above,
it was suggested to remove this carbonyl unit and syn-
thesize the phenethyl analog 15.
Table 1. SAR-phenethyl halogen substitution and MCH-R1 binding
affinity
O
N
N
This phenethyl ketopiperazine 15 displayed improved
MCH-R1 in vitro activity compared to compound
14. Contrary to the biaryl-containing MCH-R1 antag-
onists, compounds 15 had very high-water solubility
(>45 mg/mL) allowing us to easily formulate it for
in vivo studies. However despite its high in vitro
MCH-R1 antagonist activity, 15 did not promote sig-
nificant weight loss in a mouse diet induced obesity
study (mDIO). We hypothesized that its lack of effica-
cy in our 4-day mDIO weight loss model was due to
limited blood–brain barrier penetration. This hypothe-
sis was supported by Caco-2 cell assay13 data identify-
ing 15 as a potential efflux substrate (A–B/B–A = 11/
21).14 In addition, the hydrophilic nature of 15
(clogP = 2.1)15 could also have contributed to its sus-
pected inability to effectively cross the blood–brain
barrier. This led us to consider making compounds
21 with increased lipophilicity (clogP range = 2.6–
3.2) and less free bond rotation in an attempt to ad-
dress the blood–brain barrier penetration hypothesis
postulated with compound class 15. The subsequent
synthesis of 21a–k (see chemistry section) represents
a new class of potent MCH-R1 antagonists.
N
X
21a-k
Compound
X
MCH-R1 Kia (nM)
21a
21b
21c
21d
21e
21f
21g
21h
21i
H
4-F
16
3
3-F
2-F
10
12
4
4-Cl
3-Cl
2-Cl
4-Br
19
194
4
4-CF3
3-CF3
2-CF3
42
93
374
21j
21k
a n = 2 for all compounds except for 21b and 21i where n = 4.
In contrast to compound 15, molecule 21b promoted
weight loss and decreased food intake in mice. Other
compounds within this novel class of MCH-R1 antago-
nists showed similar results (Table 2). It is interesting to
note that, in agreement with the in vitro data, the meta-
substituted 21j (3-trifluoromethyl) appeared to be less
active in vivo than its para-substituted counterpart 21i
(4-trifluoromethyl). All the para-halogenated com-
pounds displayed similar in vivo activity with the chlo-
ro-containing compound 21d appearing to be sligthly
better. In order to corroborate these preliminary results
and further our understanding or the MCH-R1 antago-
nist activity of this class, we choose 21b as an early lead
and submitted it to a more comprehensive biological
evaluation.
Compounds 21a–k proved to have high binding affin-
ity for MCH-R1 (Table 1). Halogen variations at the
phenethyl unit suggested that, for a single halogen
substitution, the para-position was optimal while the
ortho-position produced less active compounds (see
21d–f and 21h–j). We observed that this effect in-
creased with the increased size of the halogen atom
or group. The effect was most notable for the trifluo-
romethyl group, intermediate for chlorine and negligi-
ble for fluorine.

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2097
Table 2. 4-day mouse DIO studies (20 mg/kg dose BID)
O
N
N
N
X
21a-k
Compound
X
MCH-R1
Kia (nM)
MCH-R1
a
IC50 (nM)
hERGb,16
% body weight loss in
4-d MDIO (vehicle)
% food intake reduction
in 4-d MDIO
[drug in serum]/lMc
at 2 h at 5 h
IC50 (lM)
21b
21e
21i
21j
4-F
4-Cl
3
4
1
10
—
38
—
3
7
8.1
3.3
—
7.0 0.3 (2.5 0.6)
9.3 0.7 (1.5 0.4)
7.8 0.4 (2.5 0.6)
4.7 0.7 (1.5 0.4)
30
45
30
18
5
3
3
4
10
2
7
1
1a
6.5 0.8
5.5 0.7
4.4 0.7
5.1 0.3
4.4 0.3
3.0 0.6
4-CF3
3-CF3
42 12
93 17a
1.7
a n = 4 for 21b and 21i; n = 2 for 21e and 21j.
b Patch–clamp hERG IC50 determinations were carried out as described in Ref. 16.
c Levels of drug in serum measured at day 5 of dosing.
Table 3. Body weight change in 10-day mDIO study for 21ba
1.4. Lead evaluation
Group
Day 0 BW
(g SEM)
Day 10 BW
(g SEM)
% change
(% SEM)
Both in vitro and in vivo evaluations were performed to
assess the ability of compound 21b to cross the blood–
brain barrier. The Caco-2 cell assay13 predicted 21b to
passively permeate across the blood–brain barrier (A–
B/B–A = 10/16). Serum and brain concentrations of
21b at 2 h after a single po dose in mice at 20 mg/kg were
1.1 0.1 mg/mL and 3.2 0.6 mg/g, respectively. These
results supported the idea that compound 21b had the
ability to penetrate the blood–brain barrier in order to
antagonize the CNS-located MCH-R1.
Vehicle
44.5 0.9
44.8 1.1
44.7 0.8
44.8 1.1
43.6 0.8
43.3 1.4
42.0 0.9
41.5 1.3
ꢀ2.1 1.0
ꢀ3.3 1.0
ꢀ6.0 0.8
ꢀ7.5 1.0
2 mg/kg
7 mg/kg
20 mg/kg
a Body weight (BW) of all groups of mice treated with vehicle or
compound 21b at days 0 and 10 of the study with percent change in
BW relative to baseline.
ꢀ2.1 1.0% change from their initial body weight
(Table 3). The effect of compound 21b on body weight
was associated with a significant effect on food intake
compared to vehicle treated mice (Fig. 6). Compound
21b dosed at 20 mg/kg and 7 mg/kg had an effect on
weight with significant reduction in food intake, while
In a 10-day mouse diet induced obesity study, MCH-R1
antagonist 21b was dosed once a day in at 2, 7, and
20 mg/kg leading to ꢀ3.3 1.0%, ꢀ6.0 0.8%, and
ꢀ7.5 1.0% reductions in body weight, respectively
(Fig. 5). The vehicle treated mice displayed
a
30
2
0
Vehicle
2 mg/kg
7 mg/kg
20 mg/kg
25
20
Vehicle
2 mg/kg
-2
-4
*
*
*
*
*
*
*
*
15
10
5
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
7 mg/kg
-6
*
*
*
*
*
*
*
20 mg/kg
*
-8
*
*
0
-10
0
1
2
3
4
5
6
7
8
9
10
0
2
4
6
8
10
Time (Days)
Time (Days)
Figure 5. Percent change in body weight from baseline of compound
21b tested at 2, 7, and 20 mg/kg, and vehicle (2% Tween 80/saline) at
5 mL/kg po QD. All values are mean values SEM for n = 8. Asterisk
(*) indicates statistical significance at p 6 0.05 compared to vehicle
treated mice.
Figure 6. Cumulative food intake of DIO mice treated with compound
21b at 2, 7, and 20 mg/kg, and vehicle (2% Tween 80/saline) at 5 mL/kg
po QD. Food intake was measured daily throughout the study. All
values are mean values SEM for n = 8. Asterisk (*) Indicates
statistical significance at p 6 0.05 compared to vehicle treated mice.

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2098
it did not show a significant reduction in food intake
when dosed at 2 mg/kg. The cumulative food intake
amounts for compound 21b treated mice were
26.1 1.3, 23.8 0.7, and 23.5 1.3 g for mice dosed
with 2, 7, and 20 mg/kg of the compound, respectively,
while cumulative food intake for vehicle treated mice
was 26.5 0.6 g. This resulted in a ꢀ1.7 4.9%,
ꢀ10.2 2.7% and ꢀ11.3 4.8% inhibition of food
intake compared to vehicle treated mice at 2, 7, and
20 mg/kg, respectively.
0
-1
-2
-3
-4
-5
-6
-7
-8
Magnetic Resonance Relaxometer (MRR) analysis17
(performed as part of the 10-day mDIO) revealed a
reduction in fat mass for mice treated with compound
21b at 7 and 20 mg/kg compared to vehicle treated mice
with ꢀ1.7 0.5 and ꢀ2.4 0.6 fat gram changes,
respectively, though the data did not reach significance
(Fig. 7). Mice treated with 2 mg/kg of compound 21b
showed no reduction in fat mass compared to vehicle
treated mice with a change of ꢀ0.9 0.7 fat grams.
Vehicle treated mice showed a reduction of ꢀ0.9 0.6
fat grams. MRR data revealed no significant reduction
in lean mass for mice treated with compound 21b at 2,
7, and 20 mg/kg compared to vehicle treated mice with
ꢀ0.6 0.4, ꢀ1.3 0.6, and ꢀ1.0 0.4 lean gram chang-
es, respectively (Fig. 8). Vehicle treated mice showed a
reduction of ꢀ0.5 0.4 lean grams. The reduction in
fat and lean mass was determined by comparing the final
to initial MRR fat and lean mass.
Vehicle
2 mg/kg
7 mg/kg 20 mg/kg
Treatment (mg/kg)
Figure 8. Lean mass loss determination: MRR for mice treated with
compound 21b at 2, 7, and 20 mg/kg, and vehicle (2% Tween 80/saline)
at 5 mL/kg po QD.
chromosome aberrations in CHO cells. Amazingly,
cytotoxic levels were determined to be the order of
1.25 mg/mL, which represent a 25-fold improvement
when compared to the cytotoxic level range (0.040–
0.050 mg/mL) observed for the benchmark MCH-R1
antagonists (1, 2, 3).
Additional biological data were collected on compound
21b to assess its potential as an effective orally dosed
MCH-R1 antagonist. Compound 21b was estimated to
have moderate bioavailability (40%) following oral dos-
ing in male Sprague–Dawley rats. Peak plasma levels
were observed at 0.5 h with an estimated half-life of
approximately 4 h. In addition, compound 21b was test-
ed for chromosomal aberrations and was concluded to
be negative for the induction of structural and numerical
Despite the attractive in vitro profile and in vivo results,
further development of 21b and its analogs as MCH-R1
antagonists was compromised by significant hERG
channel binding in vitro. This class of ketopiperazine-
aminotetralin compounds proved to be effective hERG
blockers at concentrations comparable to those of drug
in serum at administered doses (Table 2). hERG block-
ers can induce QT interval prolongation which is fre-
quently associated with potentially lethal arrhythmias
known as torsades de pointes (TdP), which has led to
the removal of several drugs from the market.18 At this
point, it was necessary to assess if the observed hERG
activity was indicative of the potential for compound
21b to induce QT interval prolongation.
0
-1
-2
-3
-4
-5
-6
-7
-8
Unfortunately, we found that 21b promotes a dose-de-
pendent increase in QT interval in anesthetized dogs
(Fig. 9).19 A statistically significant dose-dependent de-
crease in heart rate and an increase in QT interval at
10 mg/kg were observed. The heart rate decreased from
111 10 beats/min at the end of the vehicle infusion to
93 6 beats/min at the end of the 10 mg/kg infusion. QT
interval increased from 330 22 ms at the end of the
vehicle infusion to 368 36 ms at the end of the
10 mg/kg infusion. The plasma concentrations of 21b
after 1 mg/kg and 10 mg/kg were 1190 218 ng/ml and
9280 973 ng/mL, respectively. This study demonstrat-
ed that 21b prolonged QT interval in the anesthetized
dogs at plasma concentrations of 9280 ng/mL. This con-
centration was of the same order of magnitude as that
which caused a 50% inhibition of hERG (IC50 = 8.1 lM
ꢁ 3500 ng/mL).
Vehicle
2 mg/kg 7 mg/kg 20 mg/kg
Treatment
Figure 7. Fat mass loss determination: MRR for mice treated with
compound 21b at 2, 7, and 20 mg/kg, and vehicle (2% Tween 80/saline)
at 5 mL/kg po QD.

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2099
425
400
375
350
325
300
RP-HPLC on a Varian ProStar Prep HPLC (column:
Polaris C18-A 10 lm, 250 · 500 mm. Mobile phase:
95% H2O (1% TFA), 5% ACN for 30 s then linear gra-
dient to 100% ACN over 40 min followed by a 16 min
hold at 100% ACN. Flow rate 60 mL/min) Analytical
RP-HPLC analysis was performed on an Agilent 1100
series instrument or a Spectra Physics AS3000 (Column:
Polaris 3 lm C18-A 250 · 4.6 mm. Mobile phase: 80%
H2O (0.1% H3PO4), 20% ACN linear gradient over
20 min to 100% ACN followed by a hold for 5 min at
100% ACN. Flow rate 1 mL/min).
Vehicle
1 mg/kg
10 mg/kg
*
* p<0.05 vs Vehicle
Figure 9. Effect of 21b on QT interval (ms) in the anesthetized dog.
Plots of mean and SD (n = 4) measurements for incremental concen-
trations. Measurements were made during the last minute of a 15-min
perfusion of each concentration. *Significantly different (p < 0.05)
from vehicle.
3.2. Modeling
A model of the MCH-R1 receptor was constructed
by mapping the sequence of the MCH-R1 receptor onto
20
˚
the 2.8 A resolution crystal structure of rhodopsin.
Several intermediate models were produced using the
in-house protein engineering software PROTENG
whose core revolves around a simplified energy function
and libraries derived from scans of conformations of
both side chains and backbones for mutated residues.
The energy function is simplified for rapid calculation
of packing, hydrogen bonding, and salt bridges. The
model with the best packing quality was selected for
subsequent validation which was performed using muta-
genesis data obtained for both the receptor21 and the
melanin-concentrating hormone generated in-house.
The model was subsequently refined iteratively as SAR
data generated across various compound classes in our
laboratory became available. Coordinates of the result-
ing model are published.11
Currently, our efforts are focused on the design of
com-
aminotetrahydronaphthalene
ketopiperazine
pounds analogous to 21b with lower hERG blockage
activity for which we have recently founds compounds
with improved hERG profiles which will be discussed
in a later report.
2. Conclusions
In this work, medicinal chemistry efforts in tandem with
molecular modeling studies have led to the synthesis of
aminotetrahydronaphthalene ketopiperazine 21b which
acts as a novel class of potent MCH-R1 antagonists.
Compound 21b promoted significant body weight
reduction in a pair of mouse diet induced obesity stud-
ies. In addition, Magnetic Resonance Relaxometer body
weight composition analysis of the mice in both studies
revealed a significant reduction in fat mass with negligi-
ble changes in lean mass. Despite the attractive in vitro
profile and in vivo results, further development of com-
pond 21b as an MCH-R1 antagonist was compromised
by its hERG blockage activity leading to an observed
dose-dependent increase in QT interval in anesthetized
dogs at serum concentrations comparable to those
obtained at efficacious doses.
3.3. Chemistry
3.3.1. N-Allyl-N-(tert-butoxycarbonyl)glycine methyl ester
(11). To a 0 ꢁC solution of N-(tert-butoxycarbonyl)gly-
cine methyl ester (1.8 mL, 12 mmol) in 30 mL DMF
was added allyl bromide (1.6 mL, 18 mmol, 1.5 equiv)
followed by sodium hydride (0.64 g, 18 mmol,
1.5 equiv). The reaction was maintained at 0 ꢁC for
90 min. Saturated ammonium chloride solution and
EtOAC were added. The layers were separated and the
aqueous layer was extracted with fresh EtOAC. The
organic layers were combined, washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The
crude product was purified by silica gel flash chromatog-
1
3. Experimental
raphy to yield 2.0 g of a colorless oil (70%). H NMR
(300 MHz, CDCl3) (mixture of rotational isomers) d
5.85–5.72 (m, 1H), 5.19–5.10 (m, 2H), 3.98–3.94 (m,
2H), 3.89 (d, J = 5.6 Hz, 1H), 3.86–8.82 (m, 1H), 3.73
(s, 3H), 1.47–1.43 (m, 9H); 13C NMR (75 MHz, CDCl3)
(mixture of rotational isomers) d 170.8, 155.8, 155.3,
133.9, 133.8, 117.8, 17.0, 80.6, 52.1, 51.0, 50.5, 48.1,
47.7, 28.5. LRMS (M+H): 230.2, 174.1.
3.1. General methods
All solvents and reagents unless otherwise noted were
purchased from commercial sources and used without
further purification. Reactions were carried-out under
argon or nitrogen in oven-dried glassware. Mass spectra
were recorded using an electro-spray ionization mass
detector from Micromass. H and 13C{1H} NMR spec-
3.3.2.
N-Acetaldehyde,N-(tert-butoxycarbonyl)glycine
1
tra were recorded on a Varian or Bruker NMR spec-
trometer operating at the frequencies indicated, and
the chemical shifts (given in ppm) were referenced
against the residual protons of the deuterated solvents.
Products were purified by flash column chromatography
using Aldrich silica gel 60, Biotage Flash 40 or by
methyl ester (12). A solution of N-allyl,N-(tert-butoxy-
carbonyl)glycine methyl ester (11) (2.7 g, 11.8 mmol) in
50 mL dichloromethane, 6 mL methanol, and 1 mL pyr-
idine was cooled to ꢀ78 ꢁC. Ozone was bubbled through
this solution for 45 min. Oxygen was then bubbled
through the blue solution until it became colorless.

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2100
Methyl sulfide (5 mL) was added and the reaction mix-
ture was allowed to warm to room temperature over
16 h. The reaction mixture was concentrated in vacuo,
and the crude product was purified by silica gel flash
chromatography to yield 2.1 g of a colorless oil (80%).
1H NMR (300 MHz, CDCl3): (mixture of rotational iso-
mers) d 9.67–9.65 (m, 1H), 4.12 (s, 1H), 4.05 (s, 1H),
4.01 (s, 1H), 3.90 (s, 1H), 3.75 (s, 3H), 1.45 (s, 9H);
13C NMR (75 MHz, CDCl3): (mixture of rotational iso-
mers) d 199.5, 199.1, 170.5, 170.3, 155.5, 155.2, 82.1,
81.8, 58.3, 58.2, 52.4, 50.3, 49.8, 28.3. LRMS (M+H):
232.1, 176.1.
133.3, 132.8, 132.7, 131.2, 131.0, 128.7, 126.6, 61.6,
54.1, 47.8, 47.5, 44.1, 40.8, 40.4, 33.9, 24.2, 22.9; HRMS:
calcd for C27H33F3N3O2 (M+H+) 488.2525, found
488.2548.
3.3.5. 1-(4-(Piperidin-1-ylmethyl)phenethyl)-4-phenethyl-
piperazin-2-one (15). To
a solution of tert-butyl
4-(4-(piperidin-1-ylmethyl)phenethyl)-3-oxopiperazine-1-
carboxylate (13) (402 mg, 1.0 mmol) in dichloromethane
(5 mL) was added trifluoroacetic acid (2 mL). The reac-
tion mixture was stirred at 25 ꢁC for 90 min. The solvent
was removed in vacuo. The crude product was dissolved
in 1 mL methanol and several drops of acetic acid. Sodi-
um cyanoborohydride (192 mg, 3.0 mmol) was added
followed by excess phenylacetaldehyde. The reaction
mixture was stirred for 16 h when it was poured into eth-
yl acetate and water. The layers were separated and the
aqueous layer was extracted with fresh EtOAC. The
organic layers were combined, washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The
crude product was purified by preparative HPLC. Pure
fractions were combined, treated with 1 N HCl, and
concentrated in vacuo to give 98 mg (22%) of product
3.3.3. tert-Butyl 4-(4-(piperidin-1-ylmethyl)phenethyl)-3-
oxopiperazine-1-carboxylate (13). Sodium cyanoborohy-
dride (378 mg, 6.0 mmol) was added to a solution of
N-acetaldehyde,N-(tert-butoxycarbonyl)glycine methyl
ester (12) (924 mg, 4.0 mmol) and 2-(4-(piperidin-1-ylm-
ethyl)phenyl)ethanamine (1.3 g, 6.0 mmol) in methanol
(20 mL). After 2 h, additional sodium cyanoborohy-
dride (378 mg, 6.0 mmol) was added, and the mixture
was stirred overnight at 25 ꢁC. Solvent was removed in
vacuo and the crude residue was diluted with 1 N NaOH
and EtOAc. The layers were separated and the aqueous
layer was extracted with fresh EtOAC. The organic lay-
ers were combined, washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude
product was purified by preparative reverse-phase
HPLC to yield 883 mg (55%) as a thick light yellow
1
as a white powder. H NMR: (300 MHz, dmso d6) d
7.53 (d, J = 8.0 Hz, 2H), 7.38–7.33 (m, 4H), 7.28 (d,
J = 7.1 Hz, 3H), 4.21–4.19 (m, 2H), 3.85–3.74 (m, 4H),
3.58–3.22 (series of m, 8H), 3.11–3.06 (m, 2H), 2.86–
2.81 (m, 4H), 1.77–1.75 (m, 5H), 1.38–1.33 (m, 1H);
LRMS (M+H+): 406.3.
oil. 1H NMR (300 MHz, CDCl3):
d
7.32 (d,
J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.11 (s, 2H),
4.02 (s, 2H), 3.59 (t, J = 7.5 Hz, 2H), 3.55–3.48 (m,
4H), 3.21 (t, J = 5.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H),
2.85–2.81 (m, 2H), 2.57–2.50 (m, 2H), 1.90–1.83 (m,
4H), 1.44 (s, 9H); 13C NMR (75 MHz, CD3OD): d
168.1, 155.6, 142.7, 132.7, 131.0, 128.7, 82.3, 61.6,
54.0, 47.9, 33.9, 28.7, 24.2, 22.9; LRMS (M+H+):
402.33.
3.3.6.
(S)-6-(2-Chloroethylamino)-5,6,7,8-tetrahydro-
naphthalene-2-carbonitrile (17). To a solution of (S)-
6-amino-5,6,7,8-tetrahydronaphthalene-2-carbonitrile
hydrochloride (16) (1.0 g, 4.8 mmol) in 50 mL MeOH and
0.3 mL AcOH was added chloroacetaldehyde ꢂ 50 wt.%
solution in water (1.13 mL, 1.5 equiv) followed by sodium
cyanoborohydride (0.9 g, 3.0 equiv). The reaction mix-
ture was stirred for 4 h when it was concentrated in vacuo.
The crude product was purified by prep HPLC to yield a
white solid (0.84 g, 75%). 1H NMR (300 MHz, CDCl3) d
7.54 (s, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz,
1H), 3.96 (t, J = 5.7 Hz, 2H), 3.72–3.63 (m, 1H), 3.61 (d,
J = 5.7 Hz, 2H), 3.43–3.36 (m, 2H), 3.10–2.92 (m, 3H),
2.42–2.38 (m, 1H), 1.95–1.83 (m, 1H); 13C NMR
(75 MHz, CDCl3): d 139.5, 138.1, 133.7, 131.6, 130.9,
119.8, 111.8, 55.7, 47.9, 40.4, 33.1, 28.2, 26.5; LRMS
(M+H+): 235.15.
3.3.4. 1-(4-(Piperidin-1-ylmethyl)phenethyl)-4-(2-(4-triflu-
oromethylphenyl)acetyl) piperazin-2-one (14). To
a
solution of tert-butyl 4-(4-(piperidin-1-ylmethyl)-
phenethyl)-3-oxopiperazine-1-carboxylate (13) (200 mg,
0.5 mmol) in dichloromethane (4 mL) was added triflu-
oroacetic acid (1 mL). The reaction mixture was stirred
at 25 ꢁC for 90 min. The solvent was removed in vacuo.
The crude residue was dissolved in 1 mL DMF. p-Fluor-
ophenylacetic acid (77 mg, 0.5 mmol), EDCI (106 mg,
0.55 mmol), HOBT (74 mg, 0.55 mmol), and 4-meth-
ylmorpholine (0.2 mL, excess) were added, and the mix-
ture was stirred at 25 ꢁC for 16 h. The reaction mixture
was poured into water and the resulting precipitate fil-
tered off. The crude precipitate was purified by prepara-
tive HPLC. Pure fractions were combined, treated with
1 N HCl, and concentrated in vacuo to give 203 mg
(78%) of product as a white powder. 1H NMR:
(300 MHz, CD3OD) (mixture of rotational isomers) d
7.65–7.59 (m, 2 H), 7.45–7.34 (series of m, 6H), 4.25
(s, 2H), 4.09 (s, 0.4 · 2H), 3.92 (s, 0.6 · 2H), 3.88–3.58
(series of m, 4H), 3.42–3.34 (m, 6H), 2.97–2.89
(m, 4H), 1.94–1.69 (series of m, 5H), 1.51–1.47
(m, 1H); 13C NMR: (300 MHz, CD3OD) (mixture of
3.4. Representative experimental protocol for the prepa-
ration of compounds 21a–k according to Scheme 2 (the
example below focuses on compound 21j, X = 3-CF3)
To a ꢀ20 ꢁC suspension of (S)-6-(2-chloroethylamino)-
5,6,7,8-tetrahydronaphthalene-2-carbonitrile (17) (235
mg, 1.0 mmol) and N,N-diisopropylethylamine (0.35 mL,
2 mmol) in 1 mL acetonitrile was added a solution of
chloroacetic anhydride (258 mg, 1.5 mmol) in 1 mL aceto-
nitrile. The reaction mixture was allowed to warm to room
temperature over 30 min. 3-(Trifluoromethyl)phenethyl-
amine (378 mg, 2 mmol) and sodium iodide (75 mg,
0.5 mmol) were added. The suspension was microwaved
at 70 ꢁC and 2 W for 10 min. The crude reaction mixture
is filtered through a short pad of silica-gel eluting with a
rotational isomers)
d 171.4, 167.8, 142.5, 140.9,

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2101
solvent mixture (chloroform/ethyl acetate/methanol/
diisopropyl ethyl amine = 75:24:0.5:0.5). The filtrates were
concentrated under reduced pressure and crude product
(90 mg, ꢂ0.2 mmol, ꢂ20% yield) was pushed into the next
step without further purification.
3.4.3. (S)-1-(6-((Diethylamino)methyl)-1,2,3,4-tetrahydro-
naphthalen-2-yl)-4-phenethylpiperazin-2-one (21a). Yield:
29%, H NMR (300 MHz, CD3OD): d 7.39–7.22 (m,
1
8H), 4.80–4.70 (m, 1H), 4.28 (s, 2H), 4.08 (s, 2H),
3.79–3.75 (m, 4H), 3.55–3.50 (m, 2H), 3.24–2.90 (series
of m, 10H), 2.04–2.03 (m, 2H), 1.35 (t, J = 7.3 Hz,
6H); 13C NMR (75 MHz, CD3OD): d 162.8, 138.3,
138.0, 137.4, 132.5, 131.5, 130.2, 130.0, 129.6, 128.9,
128.6, 59.0, 57.1, 54.1, 52.2, 50.3, 48.0, 39.5, 32.3, 31.4,
To a suspension of 100 mg Raney nickel slurry in 4 mL
DMF under a hydrogen atmosphere was added 0.5 mL
ammonium hydroxide and a solution of the crude (S)-6-
(4-(3-(trifluoromethyl)phenethyl)-2-oxopiperazin-1-yl)-
5,6,7,8-tetrahydronaphthalene-2-carbonitrile (20j, 90
mg, ꢂ0.2 mmol) in 0.5 mL DMF. The reaction mixture
was stirred at room temperature under a hydrogen
atmosphere for 16 h. The suspension was filtered
through a pad of Celite and the filtrate was concentrated
in vacuo. At this point, a 10-mg cut of the primary
amine intermediate was purified by preparatory HPLC
for the purpose of characterization while the remainder
of the crude was utilized in the next step without further
purification.
30.1, 27.1, 9.2; HRMS calcd for C27H38N3O (M+H+):
20
365
420.3015, found 420.3008; ½aꢃ
ꢀ101.2ꢁ (c 1.0,
methanol).
3.4.4. (S)-4-(4-Fluorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
1
one (21b). Yield: 21%, H NMR (300 MHz, CDCl3): d
7.27–7.12 (m, 4H), 7.05 (d, J = 7.7 Hz, 1H), 6.97 (t,
8.4 Hz, 2 H), 4.79–4.68 (m, 1H), 4.19–4.00 (m, 4H),
3.94–3.65 (m, 2H), 3.47–2.90 (series of m, 14H), 1.87–
1.70 (m, 2H), 1.37 (t, J = 6.4 Hz, 6 H); 13C NMR
(75 MHz, CD3OD) d 164.0 (d, J = 244 Hz), 162.9,
138.6, 138.3, 133.5, 132.8, 132.1 (d, J = 8 Hz, 2C),
131.8, 129.9, 129.2, 117.1 (d, J = 22 Hz, 2C), 68.5 (2C),
59.1, 57.4, 54.3, 52.5, 39.8, 32.6, 30.8, 30.4, 27.4, 9.4
(2C). 19F NMR (282 MHz, CD3OD) d 45.2; LRMS
found: 438.36, HRMS found: 438.2925; Calcd:
438.2921 for C27H36FN2O+H+.
3.4.1. [4-(3-(Trifluoromethyl)phenethyl)-1-(6-(aminometh-
yl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-one. H
1
NMR (300 MHz, TFA-d4) d 7.70–7.60 (m, 2H), 7.33–
7.20 (series of m, 5H), 5.15–5.00 (m, 1H), 4.79 (d,
J = 16.2 Hz, 1H), 4.44 (s, 2H), 4.30–4.26 (m, 3H),
3.95–3.65 (series of m, 4H), 3.47–3.41 (m, 2H), 3.15–
2.94 (series of m, 4H), 2.24–2.04 (m, 2H). 13C NMR
(75 MHz, MeOH-d4) d 162.8, 138.8, 137.3, 136.9,
134.0, 132.2, 131.1, 130.8, 130.5, 127.6, 126.8, 126.7,
125.1, 58.2, 53.9, 52.0, 44.2, 44.1, 39.4, 32.2, 30.9, 30.0,
27.1. 19F NMR (282 MHz, MeOD-d4) d 99.35. HRMS
3.4.5. (S)-4-(3-Fluorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
1
one (21c). Yield: 32%, H NMR (300 MHz, CD3OD): d
7.43–7.36 (m, 1H), 7.31–7.24 (m, 3H), 7.20–7.14 (m,
2H), 7.08–7.02 (m, 1H), 4.77–4.70 (m, 1H), 4.29 (s,
2H), 4.12–4.08 (m, 2H), 3.80–3.76 (m, 4H), 3.58–3.52
(m, 2H), 3.36–3.30 (m, 2H), 3.23–2.92 (series of m,
8H), 2.06–2.05 (m, 2H), 1.36 (t, J = 7.2 Hz, 6H); 13C
NMR (75 MHz, CD3OD): d 164.6 (d, J = 245 Hz),
162.8, 140.2, 138.3, 138.0, 132.5, 132.0 (d, J = 8.5 Hz),
131.5, 129.6, 128.9, 126.0, 116.9 (d, J = 22 Hz), 115.3
(d, J = 21 Hz), 58.6, 57.2, 54.1, 52.2, 50.1, 48.0, 39.5,
calcd 432.2263; found 432.2281 for C24H29F3N3O.
ꢀ40.4ꢁ (c 1.0,
20
D
LRMS: 415.29 [MꢀNH2]+. ½aꢃ
methanol)].
The crude product was dissolved in 1 mL methanol
and several drops of acetic acid. Sodium cyanoboro-
hydride (3 equiv) was added followed by excess acetal-
dehyde. The reaction mixture was stirred for 30 min
when it was poured into ethyl acetate and water.
The layers were separated and the aqueous layer was
extracted with EtOAc. The organic layers were com-
bined, washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The crude product was
purified by prep HPLC. Pure fractions were com-
bined, treated with 1 N HCl, and concentrated in vac-
uo to give 21j, as a white powder (40 mg, 40% from
crude 20j).
32.2, 31.0, 30.1, 27.1, 9.2; HRMS calcd for C27H37FN3O
20
365
(M+H+): 438.2921, found 438.2934; ½aꢃ
ꢀ106.0ꢁ (c
1.0, methanol).
3.4.6. (S)-4-(2-Fluorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
1
one (21d). Yield: 27%, H NMR (300 MHz, CD3OD) d
7.42 (t, J = 7.4 Hz, 1H), 7.38–7.29 (m, 3H), 7.24–7.11
(m, 3H), 4.75–4.70 (m, 1H), 4.28 (s, 2H), 4.13 (s, 2H),
3.81–3.77 (m, 4H), 3.56–3.51 (m, 2H), 3.26–3.13 (m,
6H), 3.07–2.90 (m, 4H), 2.05–2.03 (m, 2H), 1.35 (t,
J = 7.1 Hz, 6H); 13C NMR (75 MHz, CD3OD) d 162.9
(d, J = 243 Hz), 163.0, 138.5, 138.2, 132.8 (2C), 131.8,
131.2 (d, J = 8 Hz), 129.9, 129.2, 126.4, 124.4 (d,
J = 15 Hz), 117.0 (d, J = 22 Hz), 57.6, 57.4, 54.3, 52.5,
50.2, 48.3 (2C), 39.8, 32.6, 30.4, 27.4, 25.3, 9.5 (2C);
3.4.2. 4-(3-(Trifluoromethyl)phenethyl)-1-(6-((diethylami-
no)methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-
2-one (21j). Yield: 40%, 1H NMR (300 MHz, CD3OD) d
7.68 (s, 1H), 7.63–7.52 (m, 3H), 7.28–7.19 (m, 3H), 4.74–
4.68 (m, 1H), 4.26 (s, 2H), 4.10 (s, 2H), 3.78–3.76 (m,
4H), 3.55–3.51 (m, 2H), 3.49–3.10 (series of m, 6H),
3.05–2.88 (m, 4H), 2.03–1.95 (m, 2H), 1.32 (t,
J = 7.2 Hz, 6H); 13C NMR (75 MHz, CD3OD) d
163.1, 139.1, 138.6, 134.3, 132.8, 131.8, 131.3, 129.9,
129.2, 127.1, 125.6, 58.7, 57.4, 54.3, 52.5, 50.3, 48.3,
39.8, 32.6, 31.3, 30.4, 27.4, 9.4; HRMS calcd
HRMS calcd for C27H37FN3O (M+H+): 438.2921,
20
365
found 438.2906; ½aꢃ ꢀ110.2ꢁ (c 1.1, methanol).
3.4.7. (S)-4-(4-Chlorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
for C28H37F3N3O (M+H+): 488.2889, found 488.2892;
one (21e). Yield: 42%, H NMR (300 MHz, CD3OD): d
1
20
365
½aꢃ ꢀ90.8ꢁ (c 1.0, methanol).
7.37 (s, 4H), 7.31–7.23 (m, 3H), 4.76–4.70 (m, 1H), 4.29

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2102
(s, 2H), 4.10 (s, 2H), 3.80–3.76 (m, 4H), 3.56–3.52 (m,
2H), 3.27–2.93 (series of m, 10H), 2.06–2.04 (m, 2H),
1.36 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CD3OD)
d 163.0, 138.6, 138.3, 136.5, 134.7, 132.8, 132.0, 131.8,
130.5, 129.9, 129.2, 58.9, 57.4, 54.3, 52.5, 50.3, 48.3,
39.9, 32.6, 30.9, 30.4, 27.4, 9.5; HRMS calcd for
3.4.12. (S)-4-(2-(Trifluoromethyl)phenethyl)-1-(6- ((dieth-
ylamino)methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)pip-
erazin-2-one (21k). Yield: 41%, 1H NMR (300 MHz,
CD3OD): d 7.73 (d, J = 7.7 Hz, 1H), 7.65–7.59 (m,
2H), 7.49 (t, J = 7.0 Hz, 1H), 7.29–7.22 (m, 3H), 4.75–
4.68 (m, 1H), 4.27 (s, 2H), 4.11 (s, 2H), 3.79–3.75 (m,
4H), 3.52–3.31 (series of m, 4H), 3.25–2.95 (series of
m, 8H), 2.05–2.03 (m, 2H), 1.34 (t, J = 7.0 Hz, 6H);
HRMS calcd for C28H37F3N3O (M+H+): 488.2889,
found 488.2882.
C27H37ClN3O (M+H+): 454.2625, found 454.2646;
20
365
½aꢃ ꢀ107.3ꢁ (c 1.0, methanol).
3.4.8. (S)-4-(3-Chlorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
1
one (21f). Yield: 37%, H NMR (300 MHz, CD3OD): d
3.5. Representative experimental protocol forthepreparation
of compounds 23b, 23e, 23i, and 23j as outlined in Scheme
3 (the specific example below relates to 23e, X = 4-Cl)
7.41 (s, 1H), 7.37–7.21 (m, 6H), 4.79–4.68 (m, 1H),
4.27 (s, 2H), 4.07 (s, 2H), 3.75–3.73 (m, 4H), 3.54–
3.48 (m, 2H), 3.23–2.89 (series of m, 10H), 2.04–2.02
(m, 2H), 1.34 (t, J = 7.3 Hz, 6H); 13C NMR
(75 MHz, CD3OD): d 162.9, 139.8, 138.3, 138.0,
135.9, 132.5, 131.7, 131.5, 130.2, 129.6, 128.9, 128.7,
128.6, 58.5, 57.1, 54.1, 52.2, 50.0, 48.0, 39.6, 32.3,
Methyl chloroacetate (2.49 mL, 28.3 mmol) is added to
a
solution of 4-chlorophenethyl amine (4.55 g
29.2 mmol) in diisopropylethylamine (15 mL) and ace-
tonitrile (5 mL) at 0 ꢁC (internal temperature). After
stirring at 0 ꢁC for 1 h and the formation of a white
precipitate, the resulting suspension is allowed to stir
at room temperature for 15 min. Ether (100 mL) and
water (50 mL) are added and stirring continued for
5 min. The reaction mixture is poured into a separatory
funnel, the layers separated, and the aqueous layer
extracted with ether (40 mL). The combined organics
are washed with brine and concentrated under reduced
pressure. A 100-mg cut of the resulting material is
purified by preparatory HPLC of the purpose of
characterization.
31.0, 30.1, 27.1, 9.2; HRMS calcd for C27H37ClN3O
20
365
(M+H+): 454.2625, found 454.2621; ½aꢃ
ꢀ106.8ꢁ (c
1.0, methanol).
3.4.9.
(S)-4-(2-Chlorophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
one (21g). 26%, 1H NMR (300 MHz, CD3OD): d
7.46–7.40 (m, 2H), 7.34–7.20 (m, 5H), 4.79–4.68 (m,
1H), 4.27 (s, 2H), 4.12 (s, 2H), 3.79–3.75 (m, 4H),
3.52–3.46 (m, 2H), 3.30–2.89 (series of m, 10H), 2.04–
2.01 (m, 2H), 1.33 (t, J = 7.2Hz, 6H); 13C NMR
(75 MHz, CD3OD): d 162.8, 138.3, 138.0, 135.2, 135.0,
132.5, 131.5, 131.1, 130.6, 129.6, 129.0, 128.9, 57.1,
57.0, 54.0, 52.2, 50.0, 48.0, 39.5, 32.3, 30.1, 29.3, 27.1,
3.5.1. Methyl 2-(4-chlorophenethylamino)acetate (22e).
1H NMR (300 MHz, CDCl3): d 7.23–7.10 (m, 2H), 6.98
(t, J = 8.6 Hz, 2H), 3.85 (s, 2H), 3.75 (s, 3H), 3.32–3.20
(m, 2H), 3.10–3.04 (m, 2H). 13C NMR (75 MHz,
CDCl3): d 166.5, 162.0 (d, J = 245 Hz), 131.7 (d,
9.2; HRMS calcd for C27H37ClN3O (M+H+):
20
365
454.2625, found 454.2614; ½aꢃ
ꢀ85.4ꢁ (c 1.4,
methanol).
J = 2 Hz), 130.2 (d, J = 8 Hz,
2
C), 115.8 (d,
3.4.10. (S)-4-(4-Bromophenethyl)-1-(6-((diethylamino)-
methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)piperazin-2-
J = 22 Hz, 2 C), 53.0, 49.1, 47.3, 31.8. 19F NMR
(292 MHz, CDCl3) 47.5. HRMS calcd for C11H15NO2F,
212.1087; found 212.1081. LRMS: 212.12 (M+H).
1
one (21h). Yield: 33%, H NMR (300 MHz, CD3OD): d
7.53 (d, J = 8.3 Hz, 2H), 7.30–7.23 (m, 5H), 4.78–4.70
(m, 1H), 4.28 (s, 2H), 4.07 (s, 2H), 3.77–3.73 (m, 4H),
3.54–3.48 (m, 2H), 3.24–2.90 (series of m, 10H), 2.05–
2.03 (m, 2H), 1.35 (t, J = 7.3 Hz, 6H); 13C NMR
(75 MHz, CD3OD): d 162.8, 138.3, 138.0, 136.6, 133.3,
132.5, 132.0, 131.5, 129.6, 128.9, 122.4, 58.6, 57.1,
54.2, 52.2, 50.1, 48.0, 39.5, 32.3, 30.8, 30.1, 27.2, 9.2;
HRMS calcd for C27H37BrN3O (M+H+): 498.2120,
found 498.2140.
The crude product is redissolved in methanol (200 mL)
and acetic acid (15 mL). Sodium triacetoxyborohydride
(10 g, 47 mmol) is added and the reaction flask placed in
a water bath at room temperature (water jacket). Aque-
ous (50%) chloroacetaldehyde (10 mL, excess) is added
over 5 min while keeping the reaction temperature below
40 ꢁC. The reaction mixture is allowed to stir for 30 min
and saturated aqueous NaHCO3 (120 mL) is carefully
added. After stirring for 15 min, the reaction mixture
is extracted with ether (3· 100 mL). The combined
organics are washed with brine, dried over MgSO4, fil-
tered, and concentrated under reduced pressure. The
residue is chromatographed in silica gel using 20%
ether/hexanes as eluent to yield 4.25 g (56% overall) of
the desired product as a clear oil.
3.4.11. (S)-4-(4-(Trifluoromethyl)phenethyl)-1-(6-((dieth-
ylamino)methyl)-1,2,3,4-tetrahydronaphthalen-2-yl)pip-
erazin-2-one (21i). Yield: 58%, 1H NMR (300 MHz,
CDCl3) d 7.68 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz,
2H), 7.32–7.23 (m, 3H), 4.29 (s, 2H), 4.13 (m, 2H),
3.82–3.78 (m, 2H), 3.68–3.57 (m, 2H), 3.26–3.14 (series
of m, 7H), 3.10–2.97 (m, 4H), 2.16–2.05 (m, 4H), 1.36
(t, J = 7.3 Hz, 6H). 13C NMR (75 MHz, CDCl3) d
163.0, 142.3, 138.6, 138.3, 132.8, 131.8, 131.1, 129.9,
129.2, 127.3, 127.2, 79.9, 58.6, 57.4, 55.3, 54.3, 52.5,
39.8, 32.5, 31.3, 30.4, 27.4, 9.5. 19F NMR (282 MHz,
CDCl3) d 98.7. LRMS found: 488.37; calcd: 488.2889
for C28H36F3N3O+H+.
3.5.2. Methyl 2-[(4-chlorophenethyl)(2-chloroethyl)ami-
no]acetate (23e). Overall yield: 56%, 1H NMR
(300 MHz, CDCl3): d 7.26 (d, J = 8.4 Hz, 2H), 7.14 (d,
J = 8.4 Hz, 2H), 3.72 (s, 3H), 3.53–3.49 (series of m,
4H), 3.06 (t, J = 7.0 Hz, 2H), 2.97–2.92 (m, 2H), 2.75–
2.72 (m, 2H). 13C NMR (75 MHz, CDCl3): d 171.9,

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2103
138.5, 130.6 (2C) 128.7 (2C), 56.5, 56.4, 55.3, 51.7, 42.4,
34.4. HRMS calcd for C13H18NO2Cl2, 290.0710; found
290.0715. LRMS: 290.14 (M+H), 292.15 294.16.
3.6.2. (S)-6-(4-(4-Chlorophenethyl)-2-oxopiperazin-1-yl)-
5,6,7,8-tetrahydronaphthalene-2-carbonitrile (20e). Yield:
53%, white solid, H NMR (300 MHz, CDCl3): d 7.40–
1
7.38 (m, 2H), 7.29–7.26 (m, 2H), 7.17–7.26 (series of m,
3H), 4.91–4.83 (m, 1H), 3.97–3.30 (m, 2H), 2.29–2.27
(m, 2H), 2.99–2.92 (series of m, 4H), 2.81–2.71 (series
of m, 4H), 2.68–2.63 (m, 2H), 2.02–1.87 (m, 2H). 13C
NMR (75 MHz, CDCl3): d 166.6, 140.8, 137.9, 136.7,
132.4, 132.0, 130.1, 129.9 (2 C), 129.2, 128.6 (2 C),
119.0, 109.9, 59.0, 57.6, 50.0, 48.6, 41.1, 32.7, 31.8,
28.9, 25.9. HRMS calcd for C23H25N3OCl, 394.1686;
3.5.3. 2-[(4-Fluorophenethyl)(2-chloroethyl)amino]acetate
(23b). Yield: 41% overall yield, faint yellow oil. 1H
NMR (300 MHz, CDCl3) d 7.15 (dd, J = 8.6, 5.5 Hz,
2H), 6.95 (t, J = 8.6 Hz, 2H), 3.69 (s, 3H), 3.55–3.39
(series of m, 4H), 3.04 (t, J = 7.0 Hz, 2H), 2.95 (m,
2H), 2.74–2.63 (m, 2H). 13C NMR (75 MHz, CDCl3) d
172.1, 161.6 (d, J = 244 Hz), 135.7, 130.4 (d, J = 8 Hz,
2C), 115.4 (d, J = 22 Hz, 2 C), 56.6, 56.4, 55.3, 51.8,
45.2, 42.3. 19F NMR (292 MHz, CDCl3) 45.6. HRMS
calcd for C13H18ClFNO2, 274.1010; found 290.1010.
LRMS: 274.18 (M+H), 276.17 (MH+2).
found 394.1699. LRMS: 394.27 (M+H), 396.26
20
D
(M+H+2). ½aꢃ ꢀ52.0ꢁ (c 1.1, chloroform).
3.6.3. (S)-6-(4-(4-Fluorophenethyl)-2-oxopiperazin-1-yl)-
5,6,7,8-tetrahydronaphthalene-2-carbonitrile (20b). Yield:
1
3.5.4.
2-[(4-Trifluoromethylphenethyl)-(2-chloroethyl)-
66%, pale yellow solid. H NMR (300 MHz, CDCl3): d
amino]acetate (23i). Overall yield: 30%, light yellow oil.
1H NMR (300 MHz, CDCl3): d 7.51 (d, J = 7.9 Hz,
2H), 7.29 (d, J = 7.9 Hz, 2H), 3.67 (s, 3H), 3.59–3.37
(series of m, 4 H), 3.04 (t, J = 6.9 Hz, 2H), 2.99–2.91
(m, 2H), 2.87–2.78 (m, 2H). 13C NMR (75 MHz,
CDCl3) d 171.4, 143.9, 129.0 (3 C), 125.1, 125.0, 56.0,
55.7, 54.7, 51.3, 41.9, 34.4. 19F NMR (292 MHz, CDCl3)
100.4. HRMS calcd for C14H18NOClF3, 324.0978;
found 324.0989. LRMS: 324.23 (M+H), 326.24
(MH+2).
7.32–7.30 (m, 2H), 7.15 (dd, J = 8.3, 5.2 Hz, 2H), 7.07
(d, J = 8.4 Hz, 1H), 6.93 (t, J = 8.4 Hz, 2H), 4.77 (m,
1H), 3.95 (s, 2H), 3.66–3.60 (m, 2H), 3.55–3.50 (m, 2H),
3.36–3.31 (m, 2H), 3.08–3.03 (m, 2H), 2.89–2.80 (series
of m, 4H), 1.98–1.80 (m, 2H). 13C NMR (75 MHz,
CDCl3) d 162.0 (d, J = 245 Hz), 160.7, 139.9, 132.3,
130.7 (d, J = 2 Hz), 130.1 (J = 8 Hz), 130.0, 129.3,
118.8, 115.9 (d, J = 22 Hz), 109.9, 57.9, 52.9, 49.9, 48.7,
37.8, 31.3, 29.4, 28.5, 25.5. LRMS: 378.3 (M + H).
3.6.4. (S)-6-(4-(4-Trifluoromethylphenethyl)-2-oxopipera-
zin-1-yl)-5,6,7,8-tetrahydronaphthalene-2-carbonitrile
3.5.5. 2-[(3-Trifluoromethylphenethyl)(2-chloroethyl)ami-
no]acetate (23j). Overall yield: 62%, colorless oil. 1H
NMR (300 MHz, CDCl3): d 7.55–7.30 (m, 4H), 3.70
(s, 3H), 3.57–3.46 (series of m, 4H), 3.06 (t, J = 7.0 Hz,
2H), 3.00–2.91 (m, 2H), 2.89–2.80 (m, 2H). 13C NMR
(75 MHz, CDCl3) d 171.6, 140.7, 132.2, 128.7, 125.4
(d, J = 3 Hz) 123.0, 122.9, 56.1, 55.9, 54.9, 51.4, 42.1,
34.6. HRMS calcd for C14H18NOClF3, 324.0972; found
324.0978. LRMS: 324.18 (M+H), 326.18 (MH+2).
1
(20i). Yield: 54%, off-white solid. H NMR (300 MHz,
CDCl3): d 7.58 (d, J = 8.2 Hz, 2H), 7.39–7.33 (m, 4H),
7.15 (d, J = 8.2 Hz, 1H), 4.89–4.78 (m, 1H), 3.68 (s,
2H), 3.56 (t, J = 5.3 Hz, 2H), 3.27–3.22 (m, 2H), 3.15–
3.05 (m, 4H), 2.99–2.85 (series of m, 4H), 2.03–1.86
(m, 2H). 13C NMR (75 MHz, CDCl3) d 162.4, 140.7,
140.1, 136.4, 132.4, 130.1, 129.9, 129.4, 129.0, 125.9(d,
J = 2 Hz), 118.9, 110.1, 58.0, 54.4, 49.5, 49.2, 38.9,
31.7, 31.2, 28.7, 25.8. 19F NMR (292 MHz, CDCl3)
100.3. HRMS calcd for C24H25N3OClF3, 428.1950;
found 428.1952. LRMS: 428.33 (M+H).
3.6. Representative experimental protocol for the prepa-
ration of compounds 20b, 20e, 20i, and 20j as outlined in
Scheme 3 (the specific example below relates to 20e,
X = 4-Cl)
3.6.5.
(S)-6-(4-(3-(Trifluoromethyl)phenethyl)-2-oxopi-
perazin-1- yl)-5,6,7,8-tetrahydronaphthalene-2-carbonitri-
le (20j). Yield: 65%, off-white solid. 1H NMR (300 MHz,
CDCl3): d 7.51–7.44 (m, 2H), 7.42–7.33 (series of m,
4H), 7.14 (d, 8.5 Hz, 1H) 4.92–4.80 (m, 1H), 3.36–3.27
(series of m, 4H) 2.98–2.84 (series of m, 6H), 2.81–2.65
(series of m, 4H). 2.01–1.82 (m, 2H). 13C NMR
(75 MHz, CDCl3) d 166.6, 140.4 (d, J = 31 Hz), 136.8,
132.4, 132.0, 130.1, 129.3, 126.0, 123.2, 119.0, 109.9,
58.7, 57.7, 50.0, 48.6, 41.1, 33.2, 31.8, 28.9, 25.9. HRMS
3.6.1. (S)-6-(4-(4-Chlorophenethyl)-2-oxopiperazin-1-yl)-
5,6,7,8-tetrahydronaphthalene-2-carbonitrile (20e).
slurry-mixture containing methyl 2-[(4-chloropheneth-
yl)(2-chloroethyl)amino]acetate (23e) (5.78 g,
A
20.0 mmol), (S)-amino-5,6,7,8-tetrahydronaphthalene-
2-carbonitrile hydrochloride (4.16 g, 20.0 mmol), sodi-
um iodide (6 g, 40 mmol), diisopropylethylamine
(8 mL), acetonitrile (4 mL), and dimethyl formamide
(4 mL) were heated using microwave (Microwave Appa-
ratus = Biotage-Personal Chemistry System; absorp-
tion = high, temperature = 130 ꢁC, time = 30 min). The
crude reaction mixture was transferred into a separatory
funnel with the aid of ethyl acetate (100 mL). The reac-
tion solution was washed with water (25 mL) and brine
(2 · 25 mL), dried over MgSO4, filtered, and concentrat-
ed under reduced pressure. The residue was chromato-
calcd for C24H25N3OF3, 428.1950; found 428.1946.
20
D
LRMS: 428.2 (M+H). ½aꢃ ꢀ33.5ꢁ (c 1.1, chloroform).
3.7. Representative experimental protocol for the prepa-
ration of compounds 21b, 21e, 21i, 21j as alternatively
outlined in Scheme 3 (the specific example below relates to
20b, X = 4-F)
graphed in silica gel using
a
mixture of
A heterogeneous mixture of 20b (5.55 g, 14.7 mmol),
Raney nickel (5 g) in a solvent mixture of ammonium
hydroxide (10 mL), DMF (10 mL), and ethanol
(180 mL) was stirred overnight at room temperature
diisopropylethylamine/methanol/ethyl acetate/chloro-
form (0.2:0.3:20:50) as eluent to yield 4.17 g (53%) of
the desired product 20e as a white solid.

´
J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2104
and under H2-atmosphere. The crude reaction mixture
was filtered through a pad of Celite with the aid of eth-
anol. The filtrates were concentrated under reduced
pressure and the residue dissolved in 5%AcOH/metha-
nol (100 mL). The resulting solution was treated with
sodium cyanoborohydride (3 g, excess) and acetalde-
hyde (3 mL, excess). After stirring for 15 min, the sol-
vents were removed under reduced pressure and the
resulting material partitioned between ethyl acetate
(150 mL) and 1 N aqueous sodium hydroxide (50 mL).
The layers were separated, and the aqueous layer was
extracted with ethyl acetate (50 mL). The combined
organics were washed with brine, dried over magnesium
sulfate, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatogra-
phy (silica gel) using diisopropylethylamine/methanol/
ethyl acetate (0.5:10:89.5) as eluent to yield 6.9 g (93%)
of product as a light yellow gel.
tained at 6 weeks of age and provided a high fat diet
(High fat, 45% Kcal, Research Diets, Inc.) for a 16-week
period. The animals were singly housed in cages on a re-
versed light cycle (11:00 a.m. lights off; 11:00 p.m. lights
on). This research was conducted in accordance with
Procter & Gamble’s policy on research involving ani-
mals with strict oversight for care and welfare, and ap-
proved by the Animal Care and Use Committee.
Mice were randomized into groups (n = 8) based on
body weight as well as fat composition, determined
using magnetic resonance relaxometry (MRR) (Echo-
MRI, Houston, TX). The obese mice were dosed with
compound · or vehicle (2% Tween 80/saline) via oral ga-
vage once daily for a period of 10 days. On day 11, four
mice from each dosing group were given a single dose
and blood samples were then collected approximately
2 h after dosing to determine serum concentrations.
Blood samples from the remaining four mice were col-
lected approximately 24 h after dosing from the previous
day. The blood was put into serum separator microtain-
er tubes (Becton–Dickinson, Franklin Lakes, NJ) and
the tubes were centrifuged after sitting at least 60 min.
The serum was poured off into a tube and frozen until
prepared for analysis. Food intake and body weights
were measured daily and body composition was mea-
sured at the end of the study. Statistical analysis of mea-
sured parameters was performed to determine
significance of the compound’s effect using Student’s t-
test.
Spectral data for 21b are presented previously within
this experimental section (see above).
Employing the same protocol produced compounds
21e, 21i, and 21j in 89, 92, and 87 percent yield, respec-
tively. Spectral data for 21e, 21i, and 21j are presented
previously within this experimental section (see above).
3.8. Biology
3.8.1. 4-day mDIO study-methods. Male C57Bl/6J (Jack-
son Laboratory, Bar Harbor Maine) mice were obtained
at 6 weeks of age and provided a high fat diet (High fat,
45% kcal, Research Diets, Inc.) for a 16 week period.
The animals were singly housed in cages on a reversed
light cycle (11:00 a.m. lights off; 11:00 p.m. lights on).
This research was conducted in accordance with
P&G’s policy on research involving animals with strict
oversight for care and welfare, and approved by the Ani-
mal Care and Use Committee.
3.9. QTc prolongation experiment-methods
3.9.1. Approvals. The animal components of this study
were approved by the Institutional Animal Care and
Use Committee and conformed to the Guide for the
Care and Use of Laboratory Animals published by the
U.S. National Institute of Health (NIH Publication
No. 85–23, revised 1996).
Mice were randomized into groups (n = 8) based on
body weight as well as fat composition, determined
using magnetic resonance relaxometry (MRR) (Echo-
MRI, Houston, TX). The obese mice were dosed with
compound X or vehicle (2% Tween 80/saline) via oral
gavage once daily for a period of 4 days. On day 5, mice
(n = 8) from each dosing group were given a single dose
and blood samples were then collected approximately
2 h after dosing from four of the mice to determine ser-
um concentrations. Blood samples from the remaining
four mice were collected approximately 5 h after dosing.
The blood was put into serum separator microtainer
tubes (Becton–Dickinson, Franklin Lakes, NJ) and the
tubes were centrifuged after sitting at least 60 min. The
serum was poured off into a tube and frozen until pre-
pared for analysis. Food intake and body weights were
measured daily and body composition was measured
at the end of the study. Statistical analysis of measured
parameters was performed to determine significance of
the compound’s effect using Student’s t-test.
3.9.2. Experimental protocol. Experiments using male
(n = 4) mongrel dogs (11 – 13 kg) were conducted to
assess the effects of 21b on ECG parameters in anes-
thetized dogs. The animals were anesthetized initially
with intravenous administration of sodium pentothal
(25 mg/kg). After intubation with a cuffed endotrache-
al tube, anesthesia was maintained with 1–3% isoflu-
rane vaporized with oxygen.
A
catheter for
measurement of arterial blood pressure was inserted
through the femoral artery and positioned in the
abdominal aorta. Surface lead II ECG was obtained
from limb leads. Each dog was dosed 0, 1, and
10 mg/kg, intravenously in a rising dose design. Each
dose was infused over 15 min with a 30-min period
between doses. ECGs were monitored continuously
throughout the study and data were analyzed during
the last minute of each infusion. Blood samples were
also taken at the end of the infusion to assess plasma
concentrations of 21b. Acquisition and analysis of
ECG data was conducted using a Notocord data
acquisition system (Croissy, France). QTc was calcu-
lated using the cube root method of Fridericia (QTcF)
(Spence et al., 1998).19b
3.8.2. 10-day mDIO study-methods. Male C57Bl/6J
(Jackson Laboratory, Bar Harbor Maine) mice were ob-

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J. L. Mendez-Andino et al. / Bioorg. Med. Chem. 15 (2007) 2092–2105
2105
R.; Knourek-Segel, V.; Droz, B.; Bush, E.; Brune, M.;
Preusser, L. C.; Fryer, R. M.; Reinhart, G. A.; Houseman,
K.; Diaz, G.; Mikhail, A.; Limberis, J. T.; Sham, H. L.;
Collins, C. A.; Kym, P. R. J. Med. Chem. 2006, 49, 6569–
6584.
3.9.3. Statistical analysis. Mean values were calculated
for each dose and statistical comparisons were made be-
tween treatment groups using parametric statistics
(repeated measures analysis of variance). Statistical sig-
nificance was considered at p < 0.05. Data are reported
as means SD.
9. (a) Hu, X. E. US 2005075324, 2005; (b) Stenkamp, D.;
Lehmann-Lintz, T.; Mueller, S.; Rudolf, K.; Lustenberger,
P.; Arndt, K.; Lotz, R.; Wieland, H.; Lenter, M. DE
10238865, 2004; (c) Kato, K.; Terauchi, J.; Mori, M.;
Suzuki, N.; Shimomura, Y.; Takekawa, S.; Ishihara, Y.
WO 2001021577, 2001.
10. Ulven, T.; Frimurer, T. M.; Receveur, J.-M.; Little, P. B.;
Rist, Ø.; Nørregaard, P. K.; Ho¨gberg, T. J. Med. Chem.
2005, 48, 5684.
Acknowledgment
We acknowledge Dr. Paul Correa for making PROT-
ENG available to us during the course of this work.
11. Mieling, G. E.; Mieling, K. K.; Bush, R. D.; Colson, A. O.
US 2005170433 A1 2005.
References and notes
12. MCH-R1 binding using a Flashplate radioligand binding
assay was performed by Perkin-Elmer Biosignal (Toronto,
Ontario). Full competition curves were generated with
compound concentration varied from 100 lM to 11 fM.
Potency (ki) and maximal efficacy were determined and
used to define structure–activity relationship.
1. The Surgeon General’s Call to Action to Prevent and
Decrease Overweight and Obesity 2001, Public Health
Service: Office of the Surgeon General, U.S. Department
of Health and Human Sevices: Rockville, MD, 2001;
www.surgeongeneral.gov/topics/obesity.
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