Screening for cardiovascular safety: A structure-activity approach for guiding lead selection of melanin concentrating hormone receptor 1 antagonists

Article abstract of DOI:10.1021/jm0512286

An inactin-anesthetized rat cardiovascular (CV) assay was employed in a screening mode to triage multiple classes of melanin-concentrating hormone receptor 1 (MCHr1) antagonists. Lead identification was based on a compound profile producing high drug concentration in both plasma (>40 μM) and brain (>20 μg/g) with < 15% change in cardiovascular endpoints. As a result of these stringent requirements, lead optimization activities on multiple classes of MCHr1 antagonists were terminated. After providing evidence that the cardiovascular liabilities were not a function of MCHr1 antagonism, continued screening identified the chromone-substituted aminopiperidine amides as a class of MCHr1 antagonists that demonstrated a safe cardiovascular profile at high drug concentrations in both plasma and brain. The high incidence of adverse cardiovascular effects associated with an array of MCHr1 antagonists of significant chemical diversity, combined with the stringent safety requirements for antiobesity drugs, highlight the importance of incorporating cardiovascular safety assessment early in the lead selection process.

Full text of DOI:10.1021/jm0512286

J. Med. Chem. 2006, 49, 2339-2352
2339
Screening for Cardiovascular Safety: A Structure-Activity Approach for Guiding Lead
Selection of Melanin Concentrating Hormone Receptor 1 Antagonists
Philip R. Kym,*,† Andrew J. Souers,† Thomas J. Campbell,‡ John K. Lynch,† Andrew S. Judd,† Rajesh Iyengar,†
Anil Vasudevan,† Ju Gao,† Jennifer C. Freeman,† Dariusz Wodka,† Mathew Mulhern,† Gang Zhao,† Seble H. Wagaw,§
James J. Napier,§ Sevan Brodjian,† Brian D. Dayton,† Regina M. Reilly,† Jason A. Segreti,‡ Ryan M. Fryer,‡ Lee C. Preusser,‡
Glenn A. Reinhart,‡ Lisa Hernandez, Kennan C. Marsh, Hing L. Sham,† Christine A. Collins,† and James S. Polakowski‡
Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064
ReceiVed December 8, 2005
An inactin-anesthetized rat cardiovascular (CV) assay was employed in a screening mode to triage multiple
classes of melanin-concentrating hormone receptor 1 (MCHr1) antagonists. Lead identification was based
on a compound profile producing high drug concentration in both plasma (>40 µM) and brain (>20 µg/g)
with <15% change in cardiovascular endpoints. As a result of these stringent requirements, lead optimization
activities on multiple classes of MCHr1 antagonists were terminated. After providing evidence that the
cardiovascular liabilities were not a function of MCHr1 antagonism, continued screening identified the
chromone-substituted aminopiperidine amides as a class of MCHr1 antagonists that demonstrated a safe
cardiovascular profile at high drug concentrations in both plasma and brain. The high incidence of adverse
cardiovascular effects associated with an array of MCHr1 antagonists of significant chemical diversity,
combined with the stringent safety requirements for antiobesity drugs, highlight the importance of
incorporating cardiovascular safety assessment early in the lead selection process.
Chart 1
Introduction
Melanin-concentrating hormone (MCH) is a cyclic, 19-amino
acid peptide that is synthesized in cell bodies in the lateral
hypothalamus and zona incerta of the central nervous system
(CNS). MCH-peptide is now understood to play a major role
in body weight regulation in rodents.1,2 A single injection of
MCH into the CNS stimulates food intake in rodents,3 and
chronic administration leads to increased body weight.4 Simi-
larly, transgenic mice altered to increase expression of the MCH
gene are susceptible to insulin resistance and obesity.5 In
contrast, mice lacking the gene encoding MCH are hypophagic,
are lean, and maintain elevated metabolic rates.6 Consistent with
this phenotype, genetically altered animals that lack the gene
encoding the MCH receptor (MCHr1) maintain elevated meta-
bolic rates and thus remain lean, even though they are
hyperphagic on a normal diet.7,8 Finally, the observation that
chronic administration of small-molecule antagonists leads to
the reduction of body weight provides further validation of
MCHr1 blockade as a novel target for antiobesity pharmaco-
therapy.9
Previously, we have identified several structurally diverse
classes of compounds with affinity for MCHr1.10-13 Several of
these unique chemotypes were subsequently optimized to afford
compounds with high affinity for MCHr1, potent functional
inhibition of MCH-mediated Ca2+ release, and good oral
bioavailability in both mouse and rat pharmacokinetic studies.
Furthermore, several analogues from distinct classes of MCHr1
antagonists conferred dose-dependent weight loss in diet-induced
obese (DIO) mice upon oral administration in 14 day efficacy
studies (Chart 1).
Following the identification of orally efficacious MCHr1
antagonists from structurally distinct series, we proceeded to
evaluate several lead compounds in a pentobarbital-anesthetized
dog model of cardiovascular function using an escalating dosing
paradigm.14 Compounds were evaluated for cardiovascular
liabilities dosing up to plasma concentrations 30-fold above the
estimated efficacious plasma Cmax in DIO mice. Previously, we
have described the dog cardiovascular profiles of aminopiperi-
dine coumarin analogues 1 and 2.10 Unfortunately, despite
appearing to be well tolerated upon oral dosing in chronic
efficacy studies, these compounds caused hemodynamic effects
at low multiples of their corresponding therapeutic plasma
concentrations, thus obviating any further development. Interest-
ingly, a structurally related, yet MCHr1 inactive, analogue
demonstrated a similar profile in the pentobarbital-anesthetized
* To whom correspondence should be addressed. Philip R. Kym, Abbott
Laboratories, Department R4MF; Bldg. AP10, 100 Abbott Park Road,
Abbott Park, IL 60064-6099. Tel: (847)-935-4699. Fax: (847)-938-1674.
E-mail: phil.kym@abbott.com.
† Metabolic Disease Research.
‡ Integrative Pharmacology.
§ Process Chemistry.
Exploratory Pharmacokinetics.
10.1021/jm0512286 CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/11/2006

2340 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) Chloroethylpyrrolidine hydrochloride or
chloroethylpiperidine hydrochloride, K2CO3, DMF, 60 °C, then chroma-
tography, 27-67%; (c) Fe, NH4Cl, 65 °C; (d) PhOC6H4NCO, THF, 60 °C,
90%.
a Reagents and conditions: (a) NaNO2, AcOH, rt, 95%; (b) chloro-
ethylpyrrolidine hydrochloride, K2CO3, DMF, 60 °C, then chromatography,
27-67%; (c) Fe, NH4Cl, 65 °C; (d) RCO2H, EDCI, HOBt, DMF, NMM,
56-89%.
Scheme 3a
dog cardiovascular model, suggesting that the observed cardio-
toxicity was linked to the particular structural chemotype and
not to MCHr1 activity.10 The adverse cardiovascular safety
profiles observed in the pentobarbital-anesthetized dog cardio-
vascular model at low drug concentrations revealed a significant
challenge for this program. Furthermore, since considerable time
and resources had been applied to compound optimization prior
to assessment of cardiovascular safety, we identified the need
to screen compounds for cardiovascular liabilities at a much
earlier stage in our lead optimization program. To this end, we
have incorporated a medium throughput, cardiovascular screen-
ing assay in inactin-anesthetized rats to eliminate compounds
with cardiovascular liabilities early in the lead prioritization
process. Over 130 compounds from multiple unique chemical
series were evaluated in the inactin-anesthetized rat cardio-
vascular screening assay, and structure-activity relationships
of the parameters responsible for cardiovascular liabilities were
established. This method of early screening reflects a high
priority on cardiovascular safety and represents a new strategy
for rapidly triaging lead series in a discovery program.
a Reagents and conditions: (a) 1,1′-Dimethoxy-2-bromoethane, K2CO3,
DMF, 60 °C, then chromatography (41-57%); (b) Fe, NH4Cl, 65 °C, 90%;
(c) PhOC6H4NCO, THF, 85%. (d) 2:1 2N HCl:acetone, 60 °C; (e) R1R2NH,
MeOH (1% AcOH), MP-CNBH3.
raphy. Following iron-mediated reduction and urea formation
via isocyanate coupling with the desired aniline (18), the latent
aldehyde was liberated with aqueous acid. Reductive amination
with secondary amines afforded the final compounds.
Representatives of the 4-aminopiperidine classes of MCHr1
antagonists were synthesized from a common precursor as
outlined in Scheme 4. Briefly, 4-N-Boc-aminopiperidine was
reacted with piperonal under reductive amination conditions to
afford the corresponding benzylamine. Deprotection with HCl
afforded the primary amine 22, which could then be coupled
with heterocyclic halides or acids using amide coupling or
palladium-mediated coupling conditions to afford the final
compounds 5a (for synthesis of 5b, see Supporting Information
section III), 6, 24-25, and 23, respectively. To obtain compound
6, 5-isotoic anhydride was reacted with amine 22, followed by
reductive amination to afford the final product. Finally, the
synthesis of compounds 210 and 713 have been reported
elsewhere.
Chemistry
The general synthetic approach to the indazole amides is
outlined in Scheme 1.11 Briefly, 4-nitroindazole (9a) was
synthesized from 3-nitro-o-tolylamine and sodium nitrite, while
the 5- and 6-nitroindazoles (9b and 9c, respectively) were
obtained from commercial sources. Alkylaton of the nitro-
indazole cores (9) with 2-chloroethylpyrrolidine hydrochloride
afforded a mixture of N1 and N2-alkylated isomers in ratios that
varied by the position of the nitro substituent. After chromato-
graphic separation and isolation of each isomer, iron-mediated
reduction afforded the individual anilines 10, and amide bond
formation furnished the final products 3, 4, and 11-13.
The indazole urea analogues were synthesized in an analogous
manner by first alkylating 5-nitroindazole (9b) with the ap-
propriate aminoethyl chloride followed by chromatographic
separation of the N1 and N2 isomers (Scheme 2). Iron-mediated
reduction afforded the corresponding anilines 14a,b, and reaction
with phenoxyphenylisocyanate furnished the urea products 15-
17.
Results and Discussion
Magnitude of the Cardiovascular Problem. Prior to making
significant changes in our lead optimization strategy, we
investigated the option of circumventing the cardiovascular
issues simply by changing the chemotype of our lead series.
The project team selected an efficacious lead compound (4) from
a structurally unique aminoalkylindazole series for cardio-
vascular safety evaluation in the pentobarbital-anesthetized dog
For compounds 19 and 20, a reductive amination strategy
was utilized. As shown in Scheme 3, 5-nitroindazole (9b) was
alkylated with 1,1′-dimethoxy-2-bromoethane, and the desired
N1- or N2-functionalized isomers were isolated via chromatog-

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2341
Scheme 4a
a Reagents and conditions: For 5a: (a) Piperonal, Na2SO4, AcOH, NaB(OAc)3H; (b) TFA, CH2Cl2, then HCl (>100%); (c) i. RCOOH, PS-DCC, HOBt,
DMF, 25 °C, 95%; ii. RNH2, CH3CN, 150 °C, 30 min, microwave, 31%; For 6: (d) 5-Chloroisatoic anhydride, CH3CN, 80 °C, (e) RCHO, NaBH3CN,
MeOH, 50 °C, 21%; For 23: (f) RBr,15 Pd (OAc)2, BINAP, NaOtBu, PhMe, 80 °C, 64%; For 24, 25: (g) RCOOH, EDCI, HOBt, NMM, DMF, 25 °C,
72-81%.
Figure 1. The functional effects of intravenous administration of 4 were assessed in male beagle dogs anesthetized with sodium pentobarbital (35
mg/kg; 6 mg/kg/h maintenance infusion). Animals were intubated, mechanically ventilated, and instrumented for acute measurement of mean
arterial pressure and cardiac contractile function. Following a 30-min baseline period, 4 was infused as a series of three 30-min infusions followed
by a 60-min posttreatment period.
lead optimization process?; and (3) are the observed cardio-
vascular liabilities inherently linked to MCHr1 antagonism?
Cardiovascular Screening: Strategic Considerations. Con-
siderable effort was made to assign the observed cardiovascular
toxicity to cross-reactivity at specific receptors, transporters, and/
or ion channels via CEREP-panel16 and hERG17 affinity
screening. Unfortunately, this effort was unsuccessful, as cross-
reactivity of analogues across different chemotypes and within
subseries proved highly variable. We were unable to identify a
receptor, transporter, or ion channel for which activity of the
Figure 2. Comparison of peak decreases in mean arterial pressure
studied compounds would correlate with observed hemodynamic
(MAP) observed in the pentobarbital-anesthetized dog and inactin-
effects. Consistent with this observation, toxicity could also not
anesthetized rat for indazole 4 (n ) 3 for both species). Data expressed
be attributed to the general state of mast-cell degranulation
as mean ( SEM. Numbers at the bottom of the bar graphs are the
mean plasma concentrations associated with the decreases in MAP.
driven by the release of histamine (data not shown). Finally,
the profiles of aminopiperidine coumarins 1 and 210 and
aminoalkylindazole 4 (Figure 1) in the pentobarbital-anesthetized
dog assay, which produced effects on both the peripheral
vasculature (decrease in MAP) and direct cardiac effects
(changes in dP/dt), suggested that the cardiovascular effects were
not solely attributed to a postsynaptic vasodilatory effect on
vascular smooth muscle. Therefore, an in vitro measurement,
such as an isolated dog femoral artery assay or assay of cross-
reactivity at a single ion channel or receptor would not
adequately predict the in vivo cardiovascular effects of these
model. Once again, despite the absence of observable negative
effects in a 14 day efficacy study, this compound caused
profound reductions in mean arterial pressure and heart con-
tractility during the first 30 min infusion. (Figure 1).
The adverse cardiovascular profiles observed with clearly
distinct chemotypes of MCHr1 antagonists raised several
questions for the project team: (1) what is the source of the
cardiovascular problems?; (2) could we identify an effective
method to screen for adverse cardiovascular events early in our

2342 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
Figure 3. Comparison of MCHr1 active and inactive analogues within the indazole (A) and aminopiperidine (B) chemical series on mean arterial
pressure (MAP) and heart rate (HR) in inactin-anesthetized rats. Tables at the bottom of each figure show in vitro potency (Ca2+ release) and end
of study (EOS) plasma and brain levels for each compound. Data expressed as mean ( SEM.
Chart 2
conducted under anesthesia (inactin) using a series of intrave-
nous infusions and periodic collection of small (150 µL) blood
samples to assess active drug levels, providing concentration-
response functions for clinically relevant endpoints of blood
pressure and heart rate. The model’s predictive value arises from
its ability to identify compounds that exert marked adverse
effects on cardiovascular function at relevant doses or plasma
concentrations, results that would preclude further lead opti-
mization. One significant advantage of the anesthetized rat
model is that it requires significantly less compound (<200 mg)
per assay than the anesthetized dog assay (multigram quantities).
In addition, easier access to animals and reduced instrumentation
contribute to a greater capacity for compound throughput in
the anesthetized rat assay compared to the dog assay. Therefore,
from a technical standpoint, we hypothesized that the inactin-
anesthetized rat model could be used as an effective screening
model to establish structure-activity relationships (SAR)
regarding cardiovascular effects.
Before undertaking a screening effort using the inactin-
anesthetized rat, it was important to determine if the cardio-
vascular effects observed for MCHr1 antagonists would be
similar to those observed in the pentobarbital-anesthetized dog.14
Aminoalkylindazole 4 was selected as an initial compound for
evaluation in both the anesthetized dog and rat models. This
compound represents a potent MCHr1 antagonist with a
functional IC50 (Ca2+ release) of 239 ( 40 nM. Intravenous
infusion of indazole 4 in pentobarbital-anesthetized dogs
produced large decreases in mean arterial pressure (MAP) and
compounds. Thus, our strategic decision was to prioritize
development of a whole-animal screening model that would
identify compounds that had inherently safer cardiovascular
profiles.
Hence, we sought to implement a higher throughput and less
compound-intensive, whole-animal model that could be applied
as an early screening tool. The acutely instrumented anesthetized
rat cardiovascular model18,19 is a predictive in vivo model of
cardiovascular function that effectively interrogates lead mol-
ecules for undesirable cardiovascular effects. Studies are

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2343
Chart 3
Table 1
a All compounds were >95% pure by HPLC and characterized by 1H NMR and passed combustion analysis requirements. b Displacement of [125I]-MCH
from MCHr1 expressed in IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25 nM, Bmax ) 0.40 ( 0.08 pmol/mg). c Inhibition of MCH-mediated Ca2+
release in whole IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM). d All values are mean values ( SEM and are derived from at least three independent
experiments (all duplicates). e Animals were treated with escalating doses of compounds from 0 to 100 mg/kg. The highest doses achieved without cardiovascular
effects are reported. f EOS (end of study) refers to the point at which the infusion was terminated. EOS severity: severity of separation from vehicle at the
termination of the study; V ) <15% change from vehicle; VV ) 15-25% change from vehicle; VVV ) >25% change from vehicle. g NA, not assayed. h Mean
value of two determinations that are provided in parentheses.
cardiac contractility (dP/dt) at relatively low plasma concentra-
tions. Depressor and negative inotropic effects began at plasma
concentrations less than 8.1 ( 0.9 µM and deviation from
baseline continued in a concentration-dependent manner (Figure
1). The infusion of indazole 4 required termination once plasma
concentrations reached 36.3 ( 3.9 µM since the depressor and
contractility effects decreased over 50% compared to baseline
values.19 In the inactin-anesthetized rat cardiovascular screening
paradigm (three rats tested per compound), indazole 4 produced
sharp decreases in MAP and dP/dt at 4.0 ( 0.4 µM µg/mL
plasma concentrations (Figure 2). Hence, escalating doses of
indazole 4 in both anesthetized dogs and rats produced
qualitatively similar decreases in MAP and cardiac contractility
at low plasma concentrations.
Prior to launching an extensive screening campaign to identify
MCHr1 active analogues with an improved cardiovascular
profile, we sought to address the question of whether the
observed cardiovascular toxicity was inherently linked to
MCHr1 blockade. To maximize throughput, the model was
streamlined to focus on MAP and heart rate, clinically relevant
endpoints requiring minimum instrumentation to monitor.
Because MCHr1 is highly concentrated in the lateral hypo-
thalamus, we chose to monitor the end-of-study (EOS) drug
concentration in whole brain in addition to periodic collection
of blood samples to assess plasma drug levels associated with
escalating dosing. We evaluated the cardiovascular profiles of
structurally similar sets of MCHr1 active and inactive analogues
in two different lead series in the inactin-anesthetized rat model.

2344 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
Table 2
a All compounds were >95% pure by HPLC and characterized by 1H NMR and passed combustion analysis requirements. b Displacement of [125I]-MCH
from MCHr1 expressed in IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25 nM, Bmax ) 0.40 ( 0.08 pmol/mg). c Inhibition of MCH-mediated Ca2+
release in whole IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM). d All values are mean values ( SEM and are derived from at least three independent
experiments (all duplicates). e Animals were treated with escalating doses of compounds from 0 to 100 mg/kg. The highest doses achieved without cardiovascular
effects are reported. f EOS (end of study) refers to the point at which the infusion was terminated. EOS severity: severity of separation from vehicle at the
termination of the study; V ) < 15% change from vehicle; VV ) 15-25% change from vehicle; VVV ) > 25% change from vehicle. g NA, Not Assayed.h Mean
value of two determinations that are provided in parentheses. i Animals were treated with escalating doses of compounds from 0 to 300 mg/kg.
In the aminoalkylindazole series, the exceptionally potent
MCHr1 antagonist 311 produced sharp decreases in MAP early
in the dose range (10 mg/kg, Figure 3). Just prior to reaching
the 30 mg/kg dose, MAP fell below the minimum pressure
limit,20 and the infusions were terminated; average drug
concentrations at the end of the study were 23 ( 4 µM (plasma)
and 10 ( 2 µg/g (brain). The structurally related, but MCHr1
inactive, analogue 11 (14% inhibition of MCHr1-mediated Ca2+
release @ 10 µM) also produced a sharp decrease in MAP
leading to termination of the infusion at comparable plasma (39
( 9 µM) and brain (6 ( 2 µg/g) concentrations (Figure 3).
In a second lead series represented by the aminopiperidine
benzamides 5a (Ca2+ release IC50 ) 16 nM) and 5b (30%
inhibition @ 10 µM), similar deleterious effects on cardiovas-
cular function also occurred at comparable drug concentrations.
Escalating doses of the MCHr1 active analogue 5a resulted in
a steep decline in MAP beginning at the 30 mg/kg dose. The
concentrations that led to this effect were 44 ( 19 µM (plasma)
and 88 ( 12 µg/g (brain), while escalating doses of the MCHr1
inactive analogue 5b resulted in cardiovascular effects at
comparable drug concentrations of 61 ( 3 µM (plasma) and
138.1 µg/g (brain). Each of the inactive analogues (11 and 5b)
conferred similar cardiovascular liabilities relative to the cor-
responding lead structures (3 and 5a, respectively), providing
further evidence that the observed hemodynamic effects were
structure related, and not linked to MCHr1 activity.
intense screening effort was initiated using the rat to provide
rapid structure-activity relationships (SAR) with respect to
cardiovascular events. The objective was to eliminate com-
pounds that demonstrated cardiovascular liabilities in the
anesthetized-rat screening assay prior to evaluating compounds
in pharmacokinetic and in vivo efficacy assays (Chart 3). As
an additional filter, since the targeted site of action is the lateral
hypothalamus, we required that analogues demonstrate a safe
cardiovascular profile while attaining high drug concentration
in the brain. Over 130 compounds from multiple unique
chemical series were evaluated in the anesthetized-rat cardio-
vascular screening assay, and structure-activity relationships
of the parameters responsible for cardiovascular liabilities were
established. Thus, in the remaining sections of this paper, we
describe the rat cardiovascular SAR, focusing on MAP and heart
rate (HR) with plasma and brain drug level analysis, for two
distinct series of MCHr1 active antagonists, aminoalkylindazoles
and piperonyl-substituted 4-aminopiperidines.
Aminoalkylindazoles. Several members of aminoalkyl-
indazole series demonstrated excellent in vivo efficacy in a two-
week model for weight loss in DIO mice.11,21 Despite the
absence of any apparent toxicity following semichronic oral
dosing (14 days),11 these compounds produced severe cardio-
vascular effects at low multiples of their corresponding effica-
cious plasma concentrations. Specifically, the occurrence of
hemodynamic effects at low plasma concentrations for 3 and 4
(Table 1), in addition to the effects observed upon infusion of
the inactive analogue 11 indicated that the cardiovascular
toxicity was possibly a result of the structural features of the
indazole amide compounds. Further evidence for this was
Since the inactin-anesthetized rat provided a qualitatively
predictive cardiovascular profile to that observed in the pento-
barbital-anesthetized dog, and our initial data suggested that the
cardiovascular effects were not linked to MCHr1 activity, an

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2345
provided by the infusion of indazole 12, the 5-positional
regioisomer characterized by lesser binding affinity and negli-
gible functional potency. Despite the difference in activity,
indazole 12 demonstrated a similar cardiovascular profile
compared to isomers 3 and 4, with large depressor effects on
MAP and HR becoming apparent during the 30 to 100 mg/kg
infusion period.
The consistent profile of MAP and HR depressor effects at
low plasma concentrations for amides 3, 4, and 11 prompted
us to further explore the effects of minor perturbations to the
side chain and the alkylaminoethyl substituent. As described in
a previous report,11 contraction of the amide side chain from a
benzyloxy to a phenoxy group in 5-substituted analogues (such
as 12 to 13) rescued the in vitro activity to a significant extent
(Table 1). Infusion of this truncated analogue had no deleterious
effects on the MAP or HR of the rats until the 60 mg/kg
infusion. At this time, both parameters rapidly decreased until
all animals dropped below the minimum pressure limit. Interest-
ingly, the EOS plasma concentration was only 2.0 ( 0.2 µM.
The drastic effects at low plasma concentration reiterated the
deleterious effects of amide side chains within the series. This
prompted the cardiovascular assessment of compounds contain-
ing amide replacements, such as urea analogue 15,21 a compound
characterized by good binding affinity and functional potency.
As highlighted in Table 2, MAP was unaffected until the
beginning of the 100 mg/kg infusion, after which it began to
decline steadily. Heart rate was unaffected throughout the study.
Although one animal did fall below the minimum MAP limit
and predicated termination of the infusion, the overall profile
was substantially improved relative to that of the amide
analogues. Additionally, the compound attained an end of study
plasma concentration of 88 ( 18 µM, indicating that this
compound was safer with respect to the measured parameters
when compared to the representatives of the amide series.
Figure 4. Comparison of indazole analogue 3 and optimized analogue
20 on MAP and HR in inactin-anesthetized rats with relative potencies
and achieved EOS plasma and brain levels for each compound. Data
expressed as mean ( SEM.
to-plasma ratio was suboptimal, the increased safety with this
N2-alkylated urea prompted further exploration of analogues of
17. Since the one and two carbon homologues of the N1-
alkylated urea conferred more adverse cardiovascular effects
relative to the parent pyrrolidine 15, we turned our attention to
substituted pyrrolidine analogues. Screening of multiple ureido-
substituted indazoles led to the identification of 20, which was
similar in binding and functional potency to 17. Infusion of this
compound conferred no significant effects on MAP and HR
throughout the experiment (Figure 4), and pharmacokinetic
analysis revealed high drug levels in the plasma (201 ( 53 µM),
albeit with lower levels in the brain (3 ( 1 µg/g).
Analogue 20 demonstrated significantly enhanced cardio-
vascular safety relative to the starting analogue 3 and passed
the critical barrier of conferring no side effects within the limits
of infusion while maintaining high EOS levels of drug in the
plasma. Consequently, this analogue was put forward for
pharmacokinetic analysis in DIO mice in order to assess oral
plasma and brain exposure in preparation for in vivo efficacy
studies. Dosing at 10 mg/kg in the animals and subsequent
analysis, however, revealed negligible brain penetration in DIO
mice (plasma AUC 1.02 µg‚h/mL; brain AUC 0 µg‚h/g), thus
obviating the need to test this compound for weight loss in an
efficacy study. Evaluation of multiple analogues of 20 afforded
similar results, and further SAR efforts to retain the safety profile
while improving the brain penetration were fruitless. Therefore,
the series was deemphasized, and we turned toward alternative
classes of MCHr1 antagonists.
Encouraged by the observation that the urea side chain
conferred heightened tolerability upon intravenous (iv) infusion,
we then explored modifications to the aminoalkyl region of the
compound. Representative examples include the six- and seven-
membered heterocycles 16 and 19, respectively, which are both
of comparable in vitro potency to 15. Interestingly, both
compounds were substantially worse from a tolerability stand-
point than pyrrolidine 15. While piperidine 16 had no effect on
MAP and HR at lower doses, a steady and dose dependent
decrease in MAP started at the 30 mg/kg infusion and continued
throughout the study, culminating in the termination of one
animal’s infusion. A dose dependent decrease in HR occurred
within a similar time frame, although the effects were not as
drastic as for the MAP parameter. Interestingly, the EOS plasma
and brain concentrations were very similar to those of 15,
demonstrating the pronounced differences in in vivo effects
between compounds with very minor structural differences.
Infusion of the seven-membered analogue 19 conferred depres-
sor effects on MAP at an earlier time frame, with significant
decreases appearing within the first infusion.
Because of the improved cardiovascular safety profile of the
indazole urea 15, we next explored the safety profile of the N2-
alkylated regioisomer 17. Similar to 15, no effects on MAP or
HR were observed throughout the first four periods of infusion,
although a steep decline in MAP became evident during the 30
to 100 mg/kg infusion. However, as shown in Table 2, EOS
plasma levels were considerably higher than those observed with
previous analogues (558 ( 178 µM). This result suggested that
very high plasma drug concentration of 17 was required to
confer deleterious effects on MAP or HR. Although the brain-
Aminopiperidines. While the effort to identify analogues
with minimal effects on the rat cardiovascular system at high
drug concentrations in the plasma and brain was compromised
in the aminoalkylindazole lead series due to the low brain

2346 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
Table 3
a All compounds were >95% pure by HPLC and characterized by 1H NMR and passed combustion analysis requirements. b Displacement of [125I]-MCH
from MCHr1 expressed in IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25 nM, Bmax ) 0.40 ( 0.08 pmol/mg). c Inhibition of MCH-mediated Ca2+
release in whole IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM). d All values are mean values ( SEM and are derived from at least three independent
experiments (all duplicates). e Animals were treated with escalating doses of compounds from 0 to 100 mg/kg. The highest doses achieved without cardiovascular
effects are reported. f EOS (end of study) refers to the point at which the infusion was terminated. EOS severity: severity of separation from vehicle at the
termination of the study; V ) <15% change from vehicle; VV ) 15-25% change from vehicle; VVV ) >25% change from vehicle. g NA, not assayed.
exposure of the safest analogues, our experience with this lead
series provided a clear demonstration of the utility of the rat
cardiovascular assay in a screening mode to identify analogues
with improved cardiovascular profiles. Hence, we embarked on
an effort to evaluate the rat cardiovascular profiles of MCHr1
antagonists from multiple chemotypes that had been identified
from both our internal efforts and the external literature. One
of the preferred pharmacophores that has repeatedly appeared
in MCHr1 active compounds is a piperonyl-substituted 4-amino-
piperidine fragment.10,22 We chose to use this fragment to screen
for heterocyclic groups that would lead to potent MCHr1
antagonists demonstrating both novel structure and pharmaco-
logical properties. This strategy led to the rapid identification
of multiple analogues that demonstrated high affinity for MCHr1
and potent functional antagonism (Table 3).
(7) or a 4-subsituted quinoline (23) afforded analogues that were
MCHr1 active but demonstrated deleterious cardiovascular
effects at low doses. Beginning with the 3 mg/kg dose, these
analogues caused severe cardiovascular depressor effects on both
MAP and HR (Table 3). These effects occurred at significantly
lower dose ranges and final plasma concentrations than we had
previously observed with the aminopiperidine coumarin 2.
Next, we investigated the effect of replacing the bicyclic
heterocycle with either the monocyclic o-amino nicotinamide
(5a) or o-amino benzamide (6), taking advantage of the
intramolecular hydrogen bond to mimic the second ring. Once
again, these analogues demonstrated potent MCHr1 antagonism,
yet still caused severe depressor effects on MAP at doses as
low as 3 mg/kg. We were pleased, however, to identify a series
of chromone substituted amides (24 and 25) that were character-
ized by a fundamentally different profile in the rat cardiovascular
assay. The chromone-substituted aminopiperidine amides dem-
onstrate high affinity for MCHr1 and potent functional antago-
nism, but confer only limited effects on the rat cardiovascular
Evaluation of these new classes of heterocycle-substituted
4-aminopiperidine MCHr1 antagonists in the rat cardiovascular
screening assay revealed an interesting SAR. Attempts to replace
the coumarin ring system with either a 3-subsituted indazole

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2347
pared to vehicle responses in order to advance to further lead
optimization. As a result, over 130 compounds representing
multiple structurally distinct series were narrowed down to a
single chemical series that achieved a favorable cardiovascular
profile with high exposures in plasma and brain. This report
highlights the value of positioning in vivo cardiovascular safety
screening early in the lead selection process. Traditional
approaches typically have reserved cardiovascular safety evalu-
ation for advanced stage candidate selection. However, the
current climate of drug discovery requires a thorough under-
standing of the safety profile of compounds as early and as
quickly as possible. In this report, we have shown that creative
application of established experimental approaches can expedite
the discovery of safe compounds and facilitate optimization of
resources for later stage testing.
Experimental Section
General. Unless otherwise specified, all solvents and reagents
were obtained from commercial suppliers and used without further
purification. All reactions were performed under nitrogen atmo-
sphere unless specifically noted. Normal-phase flash chromatog-
raphy was performed using Merck silica gel 60 (230-400 mesh)
from E. M. Science or on a hybrid system employing Gilson
components and Biotage prepacked columns. Following workup,
reaction mixtures were dried over MgSO4 or Na2SO4, filtered
through a fritted glass funnel or a plug of cotton, and concentrated
with a rotary evaporator at ca. 15 mmHg, warming when necessary.
Thin-layer chromatography systems were the same as those used
for column chromatography, with Rf approximately ) 0.3. Analyti-
cal reversed-phase chromatography was performed using a Zorbax
SP-C18 5µM 4.6 × 250 mm column with UV detection analyzed
at 254 nM. Analytical method: (water with 0.1% TFA and CH3CN
gradient) 0-100% CH3CN over 18 min at 1.5 mL/min. Analytical
LC-MS was performed on a Shimadzu HPLC system with a Zorbax
SB-C8, 5 µm 2.1 × 50 mm (Agilent technologies) column
equipped with a PE Sciex, API 150EX single quadropole mass
spectrometer, at a flow rate of 1 mL/min (0.05% NH4OAc-buffer:
CH3CN and 0.05% HCOOH in H2O:CH3CN). UV analysis was
Figure 5. Comparison of indazole-substituted aminopiperidine 7 with
the chromone-substituted aminopiperidine amide 25 on MAP and HR
in inactin-anesthetized rats with relative potencies and achieved EOS
plasma and brain levels for each compound. Data expressed as mean
( SEM.
system even at very high drug concentrations in both the plasma
and the brain. The 6,7-dichlorochromone analogue 24 achieved
an EOS brain concentration of 63 ( 14 µg/g without changing
MAP and causing only a minimal decreases in HR, while the
7-methoxychromone 25 remarkably achieved drug levels of 158
( 13 µg/g in the brain without affecting MAP or HR (Figure
5). Further studies in DIO mice confirmed this excellent profile,
affording excellent overall exposure in the plasma and brain
following a single oral dose of 10 mg/kg (plasma AUC 18.7
µg‚h/mL; brain AUC 4‚4 µg‚h/g). On the basis of its potent
MCHr1 antagonist activity, in addition to its extraordinary safety
against cardiovascular endpoints at high drug concentrations in
the brain, the chromone-substituted aminopiperidine amide
chemotype was selected as a chemical series worthy of further
lead optimization. The strategic shift in lead optimization work
flow (Chart 3) incorporating early assessment of cardiovascular
safety by whole animal screening led to the expedient identifica-
tion of the chromone-substituted aminopiperidine amides as a
series worthy of further lead optimization. A full description
of lead optimization activities focused on this chemotype will
be disclosed in future publications.
1
performed at 220 and 254 nM. H NMR spectra were recorded at
300 MHz unless specified otherwise; all values are referenced to
tetramethylsilane as internal standard and are reported as shift
(multiplicity, coupling constants in Hz, proton count) Mass spectral
analysis is accomplished using fast atom bombardment (FAB-MS),
electrospray (ESI-MS) or direct chemical ionization (DCI-MS)
techniques. Elemental analyses were obtained at QTI Laboratories.
The synthesis and characterization of compounds 1, 2,10 3, 4,11
and 1213 have been reported elsewhere.
Abbreviations. NH4Cl, ammonium chloride; DMF, dimethyl
formamide; Et3N, triethylamine; EtOAc, ethyl acetate; EtOH, ethyl
alcohol; MeOH, methyl alcohol; EDCI, N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole hydrate; NMM,
4-methylmorpholine; AcOH, acetic acid; IPA, isopropyl alcohol;
CH3CN, acetonitrile; CH2Cl2, methylene chloride; CHCl3, chloro-
form; PhMe, toluene; TFA, trifluoracetic acid.
General Procedures. Synthesis of Indazole Amides/Ureas.
Method A. Alkylation of Nitroindazoles. The desired nitroindazole
(18.4 mmol) was dissolved in dry DMF (0.300 M) and K2CO3 (2.95
equiv) was added. The mixture was stirred for 30 min, and then
1-(2-chloroethyl)pyrrolidine hydrochloride (1.54 equiv) was added.
The reaction mixture was heated to 60 °C for 6-8 h and then cooled
to room temperature. The reaction mixture was filtered through a
plug of silica gel and rinsed with EtOAc:Et3N (4:1). The filtrate
was concentrated in vacuo to remove DMF, and the residue was
purified by flash chromatography (60-100% EtOAc (w/5% Et3N)
in hexanes) to afford the individual N1- and N2-alkylated nitro-
indazoles.
Conclusion
Screening for cardiovascular safety early in the lead selection
process using an inactin-anesthetized rat model provided an
efficient means to eliminate MCHr1 antagonists that demon-
strated cardiovascular liabilities. Compounds were triaged by
their effects on cardiovascular endpoints and pharmacokinetic
parameters. Compounds were required to achieve at least 40
µM concentration in plasma and 20 µg/g concentration in brain
with less than 15% change on cardiovascular endpoints com-
Method B. Reduction of Nitroindazoles. The desired alkylated
indazole (7.30 mmol), iron powder (10.0 equiv), and NH4Cl (0.500
equiv) were suspended in a 4:1 solution of EtOH:H2O (0.100 M).

2348 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
The reaction mixture was heated to reflux for 3-5 h and then cooled
to room temperature. The solvent was removed in vacuo, and the
residue was stirred in refluxing EtOAc:Et3N (4:1, 0.2 M) for 15
min, cooled to room temperature, and then filtered through a plug
of silica gel. Rinsing with EtOAc:Et3N (4:1) and removal of the
solvents in vacuo afforded the corresponding aniline, which was
used in the next reaction without further purification.
Method C. Amide Bond Formation. A 100 mL round-bottom
flask was charged with the desired aniline (1.83 mmol), (4-
benzyloxyphenyl)acetic acid (1.05 equiv), and DMF (0.200 M)
under N2. EDCI (1.20 equiv) and HOBt (1.20 equiv) were added,
followed by NMM (2.50 equiv), and the reaction solution was
stirred at room temperature for 4-6 h. Upon completion of the
reaction as determined by TLC analysis, the DMF was removed in
vacuo and the residue dissolved in EtOAc and washed with sat.
NaHCO3 (×3), brine, and water. The organic layer was then dried
and filtered, and the solvents were evaporated in vacuo. The residue
was then purified by column chromatography (0 to 5% MeOH in
CH2Cl2) to afford the corresponding final product.
temperature. After removing the solvents in vacuo, the residue was
taken up and stirred in 4:1 EtOAc:Et3N (30 mL) for 15 min and
then filtered through a plug of silica gel, rinsing with 4:1 EtOAc:
Et3N. The filtrate was concentrated to afford a pale orange oil (3.4
1
g, 90%). H NMR (DMSO-d6) δ 1.35 (m, 2 H), 1.43 (m, 4 H),
2.36 (m, 4 H), 2.64 (t, J ) 6.8 Hz, 2 H), 4.35 (t, J ) 6.8 Hz, 2 H),
4.76 (s, 2H), 6.72 (dd, J ) 2.0, 0.7 Hz, 1 H), 6.79 (dd, J ) 8.8, 2.0
Hz, 1 H), 7.34 (d, J ) 8.8 Hz, 1 H), 7.67 (d, J ) 1.0 Hz, 1 H). MS
(DCI/NH3) m/z 245 [M + H]+.
The title compound was prepared by reacting 4-phenoxyphenyl
isocyanate (50 mg, 0.23 mmol) with 1-(2-piperidin-1-yl-ethyl)-1H-
indazol-5-ylamine (58 mg, 0.23 mmol) at room temperature for 1
h. The solvent was removed in vacuo, and the residue was triturated
in boiling ether. A pale white solid was obtained upon suction
1
filtration (85 mg, 80%). H NMR (DMSO-d6) δ 1.34 (m, 2 H),
1.43 (m, 4 H), 2.39 (s, 4 H), 2.70 (s, 2 H), 4.46 (m, 2 H), 6.94-
7.00 (m, 4 H), 7.06-7.11 (m, 1 H), 7.48 (d, J ) 9.2 Hz, 3 H), 7.58
(d, J ) 8.9 Hz, 1 H), 7.88 (d, J ) 1.5 Hz, 1 H), 7.96 (s, 1 H), 8.61
(s, 1H), 8.66 (s, 1 H). MS (DCI/NH3) m/z 456 [M + H]+. Anal.
(C27H29N5O2) C, H, N.
Method D. Urea Formation. A 250 mL round-bottom flask was
charged with the appropriate aniline (4.34 mmol), 4-phenoxyphenyl
isocyanate (1 equiv), and THF (60.0 mL) and the mixture stirred
at 50 °C for 1 h. The mixture was cooled to room temperature,
and the solvents were removed in vacuo. The resultant solid was
triturated in boiling ether and collected by filtration to provide the
title product.
1-(2,2-Dimethoxy-ethyl)-1H-indazol-5-ylamine (18a). To a 250
mL round-bottom flask with a stir bar were added 3.00 g (18.4
mmol) of 5-nitroindazole, 61 mL of DMF, and 5.08 g (36.9 mmol)
of K2CO3. To this mixture was added 3.42 g (20.2 mmol) of
2-bromo-1,1-dimethoxy-ethane, and the mixture slowly heated to
55 °C and allowed to stir for 12 h. After this time, the reaction
mixture was cooled to room temperature and the contents filtered
over a bed of Celite. The filtrate was then evaporated to dryness
and the residue purified via column chromatography (30-80%
EtOAc:hexanes) to afford 2.00 g of the N1-alkylated nitroindazole
intermediate.
To a 100 mL round-bottom flask with a stir bar were added 2.00
g (7.96 mmol) of this material, NH4Cl (0.210 g, 3.96 mmol), and
80% EtOH (70.0 mL). To this was added 4.40 g (78.4 mmol) of
iron powder, and the mixture was slowly heated to reflux. After
stirring for 1 h, the reaction mixture was cooled to room temper-
ature. The solvents were evaporated, and the residue was taken up
in 10:1 EtOAc:Et3N and filtered over a plug of silica gel, eluting
with the same mixture. Evaporation of the solvents afforded 1.8 g
of a pale yellow oil (38% from 5-nitroindazole). 1H NMR (DMSO-
d6) δ 3.16-3.28 (m, 6 H), 4.35 (d, J ) 5.4 Hz, 2 H), 4.71 (t, J )
5.6 Hz, 1 H), 4.81 (s, 2 H), 6.66-6.76 (m, 1 H), 6.80 (dd, J ) 8.8,
2.0 Hz, 1 H), 7.35 (d, J ) 8.8 Hz, 1 H), 7.72 (s, 1 H). MS (ESI)
m/z 222 [M + H]+.
N-(4-Phenoxyphenyl)-N′-[1-(2-azepan-1-yl-ethyl)-1H-indazol-
5-yl]urea (19). A mixture of 18a (1.00 g, 4.52 mmol), 4-phenoxy-
phenyl isocyanate (0.955 g, 4.52 mmol), and THF (60.0 mL) was
heated to 60 °C for 1 h. The mixture was cooled to room
temperature and concentrated under reduced pressure to provide a
brown solid. The residue was triturated from boiling ether to provide
1.20 g of the urea intermediate. 1H NMR (DMSO-d6) δ 3.25 (s, 6
H), 4.46 (d, J ) 5.4 Hz, 2 H), 4.75 (t, J ) 5.4 Hz, 1 H), 6.89-7.04
(m, 4 H), 7.02-7.14 (m, 1 H), 7.26-7.42 (m, 3 H), 7.43-7.54
(m, 2 H), 7.55-7.65 (m, 1 H), 7.81-7.94 (m,1 H), 7.99 (s, 1 H),
8.52-8.78 (m, 2 H). MS (ESI) m/z 433 [M + H]+.
A solution of this material (0.750 g, 1.74 mmol) in 2 N HCl:
THF (1:1, 16.0 mL) was heated to 60 °C for 6 h after which time
the reaction solution was cooled to room temperature and the
solvents removed in vacuo. The residue was then taken up in PhMe
and concentrated in vacuo (×3). A portion of this residue (0.042
g, 0.109 mmol) was taken up in 1.5 mL of 1:1 CH2Cl2:MeOH (1%
AcOH), and hexamethyleneimine (0.013 g, 0.130 mmol) was added,
followed by macroporous cyanoborohydride resin (155 mg, 2.1
mmol/g, 3 equiv). The reaction mixture was shaken at 40 °C for 3
h, cooled to room temperature, and filtered. The filtrate was
concentrated under reduced pressure and purified by preparative
HPLC to provide the title product (0.022 g, 42%). 1H NMR
(DMSO-d6) δ 1.46 (m, 8 H), 2.60 (m, 4 H), 2.92 (t, J ) 6.6 Hz, 2
H), 4.41 (t, J ) 6.6 Hz, 2 H), 6.94-6.99 (m, 4 H), 7.06-7.11 (m,
1 H), 7.32-7.38 (m, 3 H), 7.48 (d, J ) 9.2 Hz, 2 H), 7.59 (d, J )
2-(4-Methoxyphenyl)-N-[1-(2-pyrrolidin-1-ylethyl)-1H-inda-
zol-5-yl]acetamide (11). The title compound was prepared accord-
ing to Method C, substituting (4-methoxyphenyl)acetic acid for (4-
1
benzyloxyphenyl)acetic acid (0.51 g, 76%). H NMR (500 MHz,
DMSO-d6) δ 1.72-1.88 (m, 2 H), 1.90-2.02 (m, 2 H), 2.93-3.08
(m, 2 H), 3.43-3.56 (m, 2 H), 3.62 (s, 2 H), 3.64-3.71 (m, 2 H),
3.73 (s, 3 H), 4.66 (m, 2 H), 6.83-6.95 (m, 2 H), 7.12 (dd, J )
8.5, 1.5 Hz, 1 H), 7.22-7.32 (m, 2 H), 7.65-7.78 (m, 1 H), 8.08
(s, 1 H), 8.21-8.31 (m, 1 H), 10.38 (s, 1 H). MS (DCI/NH3) m/z
245 [M + H]+. Anal. (C22H26N4O2) C, H, N.
2-(4-Phenoxyphenyl)-N-[1-(2-pyrrolidin-1-ylethyl)-1H-inda-
zol-5-yl]acetamide (13). The title compound was prepared accord-
ing to Method C, substituting (4-phenoxyphenyl)acetic acid for (4-
benzyloxyphenyl)acetic acid (1.8 g, 93%). 1H NMR (DMSO-d6) δ
1.59-1.63 (m, 4 H), 2.41-2.46 (m, 4 H), 2.85 (t, J ) 6.8 Hz, 2
H), 3.64 (s, 2 H), 4.46 (t, J ) 6.8 Hz, 2 H), 6.96-7.01 (m, 4 H),
7.12 (tt, J ) 7.5, 1.0 Hz, 1 H), 7.40-7.35 (m, 4 H), 7.42 (dd, J )
9.2, 2.0 Hz, 1 H), 7.61 (d, J ) 9.2 Hz, 1 H), 7.98 (d, J ) 1.4 Hz,
1 H), 8.10 (d, J ) 1.4 Hz, 1 H), 10.16 (s, 1 H). Anal.
(C27H28N4O2) C, H, N.
N-(4-Phenoxyphenyl)-N′-[1-(2-pyrrolidin-1-ylethyl)-1H-inda-
zol-5-yl]urea (15). The title compound was prepared according to
1
Method D (3.8 g, 79%). H NMR (DMSO-d6) δ 1.56-1.69 (m, 4
H), 2.41-2.48 (m, 4 H), 2.86 (t, J ) 6.6 Hz, 2 H), 4.46 (t, J ) 6.8
Hz, 2 H), 6.91-7.03 (m, 4 H), 7.03-7.14 (m, 1 H), 7.29-7.41
(m, 3 H), 7.44-7.52 (m, 2 H), 7.55-7.63 (m, 1 H), 7.85-8.00
(m, 2 H), 8.55-8.69 (m, 2 H). Anal. (C26H27N5O2) C, H, N.
N-(4-Phenoxyphenyl)-N′-[1-(2-piperidin-1-ylethyl)-1H-inda-
zol-5-yl]urea (16). 5-Nitroindazole (4.00 g, 24.5 mmol) was treated
with K2CO3 (10.1 g, 73.6 mmol) in DMF (60.0 mL) for 30 min,
and then 1-(2-chloroethyl)piperidine hydrochloric acid (6.80 g, 36.8
mmol) was added. The reaction mixture was heated to 60 °C for 6
h and then cooled to room temperature. The reaction mixture was
filtered through a plug of silica gel, rinsed with EtOAc:Et3N (4:1),
and concentrated under vacuum. Purification via column chroma-
tography (EtOAc:Et3N 30:1) afforded 5-nitro-1-(2-piperidin-1-yl-
ethyl)-1H-indazole (4.3 g, 64%). 1H NMR (DMSO-d6) δ 1.35 (m,
6 H), 2.36 (m, 4 H), 2.72 (t, J ) 6.4 Hz, 2 H), 4.58 (t, J ) 6.4 Hz,
2 H), 7.90 (d, J ) 9.5 Hz, 1 H), 8.22 (dd, J ) 9.5, 2.0 Hz, 1 H),
8.40 (s, 1 H), 8.82 (d, J ) 2.0 Hz, 1 H). MS (DCI/NH3) m/z 275
[M + H]+.
A mixture of 5-nitro-1-(2-piperidin-1-yl-ethyl)-1H-indazole (4.30
g, 15.7 mmol), iron powder (8.80 g, 157 mmol), and NH4Cl (423
mg, 7.80 mmol) was suspended in 80% EtOH (70 mL). The reaction
mixture was heated to reflux for 3 h and then cooled to room

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2349
9.2 Hz, 1 H), 7.87 (m, 1 H), 7.95 (d, J ) 0.9 Hz, 1 H), 7.96 (s, 1
H), 8.59 (s, 1 H), 8.63 (s, 1 H). Anal. (C28H31N5O2) C, H, N.
2-(2,2-Dimethoxyethyl)-2H-indazol-5-ylamine (18b). The cor-
responding nitroindazole was isolated via column chromatography
from the reaction described in example 18a (1.08 g). 1H NMR (400
MHz, DMSO-d6) δ 3.31 (s, 6 H), 4.67 (d, J ) 5.5 Hz, 2 H), 4.91
(t, J ) 5.5 Hz, 1 H), 7.81 (m, 1 H), 7.98 (m, 1 H), 8.62 (m, 2 H);
MS (DCI/NH3) m/z 252 [M + H]+.
The title product was prepared via iron reduction according to
example 18a (0.96 g, 23% from 5-nitroindazole). 1H NMR (DMSO-
d6) δ 3.27 (s, 6 H), 4.38 (d, J ) 5.8 Hz, 2 H), 4.81 (m, 3 H), 6.56
(m, 1 H), 6.75 (m, 1 H), 7.33 (m, 1 H), 7.87 (s, 1 H). MS (DCI/
NH3) m/z 222 [M + H]+.
dichloronicotinic acid (0.090 g, 0.47 mmol), EDCI (0.090 g, 0.47
mmol), and HOBt (0.060 g, 0.47 mmol) in CH2Cl2 (10 mL) was
stirred at room temperature for 10 min. 22 (0.10 g, 0.43 mmol)
was added and the solution stirred at room temperature for 12 h.
The reaction was diluted with saturated NaHCO3 (20 mL) and the
organic layer collected, dried over MgSO4, and evaporated to afford
a white solid (0.17 g, 95%). 1H NMR (DMSO-d6) δ 1.55 (m, 2 H),
1.81 (m, 2 H), 2.12 (m, 2 H), 2.72 (m, 2 H), 3.42 (s, 2 H), 3.72 (m,
1 H), 6.00 (s, 2 H), 6.78 (m, 1 H), 6.82 (m, 2 H), 8.14 (s, 1 H),
8.58 (s, 1 H), 8.6 (br s, 1 H). MS (ESI) m/z 408 [M + H]+.
A portion of this material (0.140 g, 0.340 mmol) was dissolved
in CH3CN (5 mL), and 2-isopropoxyethylamine (0.110 g, 1.02
mmol) was added. The reaction solution was heated to 150 °C for
30 min in a CEM Explorer microwave. The solvent was evaporated
and the residue purified via column chromatography on silica gel
using 5% MeOH in CH2Cl2 to afford the title product as a white
N-(4-Phenoxyphenyl)-N′-[2-(2-pyrrolidin-1-ylethyl)-2H-inda-
zol-5-yl]urea (17). The title compound was prepared according to
1
Method D (2.3 g, 51%). H NMR (DMSO-d6) δ 1.66 (m, 4 H),
1
solid. (50 mg, 31%). H NMR, (DMSO-d6) δ 1.08 (d, 6 H), 1.54
2.50 (m, 4 H), 2.96 (m, 2 H), 4.48 (t, J ) 5.9 Hz, 2 H), 6.97 (m,
4 H), 7.11 (m, 2 H), 7.36 (m, 2 H), 7.50 (m, 3 H), 7.88 (m, 1 H),
8.26 (s, 1 H), 8.54 (s, 1 H), 8.64 (s, 1 H). Anal. (C26H27N5O2) C,
H, N.
(m, 2 H), 1.75 (m, 2 H), 1.98 (m, 2 H), 2.79 (m, 2 H), 3.39 (s, 2
H), 3.49 (m, 4 H), 3.55 (m, 1 H), 3.69 (m, 1 H), 5.98 (s, 2 H),
6.72-7.76 (m, 1 H), 6.83-6.85 (m, 2 H), 8.01 (m, 1 H), 8.15 (m,
1 H), 8.34 (s, 1 H), 8.38 (m, 1 H). Anal. (C24H31ClN4O4) C, H, N.
N-(1-Benzo[1,3]dioxol-5-ylmethyl-piperidin-4-yl)-5-chloro-2-
[(thiazol-2-ylmethyl)amino]benzamide (6). A solution of 5-chloro-
isatoic anhydride (0.250 g, 1.26 mmol) and 22 (0.400 g, 1.28 mmol)
were heated at 80 °C for 24 h. The reaction solution was cooled to
room temperature, diluted with water (25 mL), and extracted with
EtOAc (3 × 25 mL). The organic extracts were washed with water
(5 × 20 mL), dried over MgSO4, and evaporated to afford a clear
oil that was carried forward to the next step.
N-{2-[2-(3-(S)-Hydroxypyrrolidin-1-yl)ethyl]-2H-indazol-5-
yl}-N′-(4-phenoxyphenyl)urea (20). A mixture of 18b (0.600 g,
2.71 mmol) and 4-phenoxyphenyl isocyanate (0.573 g, 2.71 mmol)
in THF (36.0 mL) was heated to 60 °C for 1 h. The mixture was
cooled to room temperature and concentrated under reduced
pressure to provide a brown solid. The residue was triturated from
1
boiling ether to provide 1.10 g of the urea intermediate. H NMR
(DMSO-d6) δ 3.29 (s, 6 H), 4.48 (d, J ) 5.4 Hz, 2 H), 4.74-4.92
(m, 1 H), 6.90-7.02 (m, 4 H), 7.04-7.20 (m, 2 H), 7.30-7.40
(m, 2 H), 7.44-7.59 (m, 3 H), 7.84-7.92 (m, 1 H), 8.22 (s, 1 H),
8.46-8.70 (m, 2 H). MS (ESI) m/z 433 [M + H]+.
This material was placed along with thiazole-2-carbaldehyde
(0.170 g, 1.51 mmol) in THF (with 2% AcOH, 15 mL) and heated
at reflux for 15 h. The reaction mixture was then cooled to room
temperature, sodium triacetoxyborohydride (0.830 g, 3.90 mmol)
was added, and the reaction mixture was stirred at room temperature
for 24 h. The reaction was quenched by the addition of saturated
NaHCO3 (25 mL) and water (20 mL) and extracted with EtOAc (3
× 30 mL). The organic extracts were washed with water (20 mL)
and dried over MgSO4. The solvent was evaporated and the residue
purified via reverse phase HPLC to afford a foamy white solid.
This material was taken up in EtOAc and washed with saturated
NaHCO3 (2 × 25 mL). The organic layers were dried over MgSO4
and evaporated to afford a white foam. The foamy material was
crystallized from EtOAc/MeOH to afford the title compound (0.13
g, 21%) as white needles. 1H NMR (500 MHz, DMSO-d6) δ 1.50-
1.60 (m, 2 H), 1.74-1.78 (m, 2 H), 1.99-2.06 (m, 2 H), 2.75-
2.87 (m, 2 H), 3.68-3.81 (m, 1 H), 4.72 (d, J ) 6.1 Hz, 2 H), 5.99
(s, 2 H), 6.66 (d, J ) 8.9 Hz, 1 H), 6.73-6.8 (m, 1 H), 6.82-6.90
(m, 2 H), 7.26 (dd, J ) 8.9, 2.5 Hz, 1 H), 7.61 (d, J ) 3.4 Hz, 1
H), 7.62 (d, J ) 2.5 Hz, 1 H), 7.74 (d, J ) 3.4 Hz, 1 H), 8.33 (m,
1 H), 8.39 (m, 1 H). EIMS: 485.2 (M+ + H). Anal. (C24H25-
ClN4O3S) C, H, N.
A solution of this material (0.750 g, 1.74 mmol) in 2 N HCl:
THF (1:1, 16 mL) was heated to 60 °C for 6 h after which the
solvents were removed in vacuo. A portion of the residue was then
taken up in PhMe and concentrated in vacuo (×3). 1-[2-(2-
Oxoethyl)-2H-indazol-5-yl]-3-(4-phenoxyphenyl)urea (50.0 mg,
0.130 mmol), (S)-3-pyrrolidinol (21.0 µL, 0.26. mmol), 1 M sodium
cyanoborohydride in THF (260 µL, 0.260 mmol), and THF (4.00
mL) were then added, and the mixture shaken at room-temperature
overnight. The solution was concentrated and purified via RP-HPLC
to afford the final product (8.0 mg, 14%). 1H NMR (DMSO-d6) δ
1.45-1.57 (m, 1 H), 1.91 (ddd, J ) 13.1, 7.5, 6.9 Hz, 1 H), 2.32
(dd, J ) 9.5, 3.7 Hz, 1 H), 2.54-2.66 (m, 1 H), 2.73 (dd, J ) 9.5,
6.1 Hz, 1 H), 2.94 (t, J ) 6.4 Hz, 2 H), 3.27-3.29 (s, 1 H), 4.15
(td, J ) 7.0, 3.6 Hz, 1 H), 4.45 (t, J ) 6.4, 2 H), 4.66 (d, J ) 4.4
Hz, 1 H), 6.93-7.02 (m, 4 H), 7.04-7.16 (m, 2 H), 7.32-7.40
(m, 2 H), 7.44-7.56 (m, 3 H), 7.88 (s, 1 H), 8.25 (s, 1 H), 8.53 (s,
1 H), 8.63 (s, 1 H). MS (ESI) m/z 458 [M + H]+. Anal.
(C26H27N5O3) C, H, N.
Synthesis of 4-Aminopiperidine Heterocycles. 1-(1,3-Benzo-
dioxol-5-ylmethyl)piperidin-4-amine (22). AcOH (2.00 mL) was
added to a suspension of 4-N-Boc-aminopiperidine (5.00 g, 25.0
mmol), piperonal (3.75 g, 25.0 mmol), sodium sulfate (7.10 g, 500
mmol), and THF (100 mL). After 20 min, sodium triacetoxyboro-
hydride (10.6 g, 50.0 mmol) was added. After 16 h, MeOH (4.00
mL) was added and the mixture was stirred for 24 h. The solution
was diluted with CH2Cl2 (150 mL), washed with 1 N aqueous
NaOH and brine, dried (MgSO4), filtered, and concentrated under
reduced pressure to provide a white solid (9.00 g). This material
was dissolved in CH2Cl2 (50.0 mL), cooled to 0 °C, and combined
with TFA (25.0 mL). The mixture was stirred for 1 h and
concentrated under reduced pressure and the residue combined with
2 M HCl in ether (30.0 mL) and ether (30.0 mL). The resulting
white precipitate was collected by filtration and air-dried overnight
to provide the title compound as the dihydrochloride salt. 1H NMR
(DMSO-d6) δ 1.94 (m, 2 H), 2.06 (m, 2 H), 2.95 (m, 2 H), 3.21
(m, 2 H), 3.40 (m, 1 H), 4.14 (d, J ) 5.4 Hz, 2 H), 6.07 (s, 2 H),
6.99 (m, 2 H), 7.24 (s, 1 H), 8.28 (s, 2 H); MS (APCI) m/z 235 [M
+ H]+.
(1-Benzo{1,3}dioxol-5-ylmethyl-piperidin-4-yl)(5-chloro-1H-
indazol-3-yl)amine (7). The product was prepared according to
1
ref 13. H NMR (DMSO-d6) δ 1.35-1.57 (m, 2 H), 1.86-2.11
(m, 4 H), 2.70-2.87 (m, 2 H), 3.38 (s, 2 H), 3.41-3.55 (m, 1 H),
5.82 (m, 1 H), 5.98 (s, 2 H), 6.70-6.79 (m, 1 H), 6.80-6.90 (m,
2 H), 7.04-7.34 (m, 2 H), 7.64-7.97 (m, 1 H), 11.52 (s, 1 H).
Anal. (C20H21ClN4O2) C, H, N.
(1-Benzo{1,3}dioxol-5-ylmethylpiperidin-4-yl)(6-methoxyquin-
olin-4-yl)amine (23). A vial was charged with Pd(OAc)2 (0.010
equiv, 0.020 mmol, 5.0 mg), rac-BINAP (0.015 equiv, 0.003 mmol,
20 mg), and PhMe (1.0 mL) and stirred for 15 min. 1-Benzo[1,3]-
dioxol-5-ylmethylpiperidin-4-ylamine (22, 1.10 equiv, 2.30 mmol,
541 mg) was added, and the reaction was stirred for an additional
15 min. An additional 2 mL of PhMe was added, followed by
4-bromo-6-methoxyquinoline (1.00 equiv, 2.10 mmol, 500 mg), and
NaOtBu (1.40 equiv, 2.90 mmol, 282 mg). The reaction was stirred
at 80 °C until the 4-bromo-6-methoxyquinoline was consumed, as
determined by HPLC analysis (1 h), and then cooled to room
temperature. The solids were isolated by filtration (supernatant
levels of product were 1.4 mg/mL), suspended in hot IPA (5 mL),
N-(1-Benzo[1,3]dioxol-5-ylmethyl-piperidin-4-yl)-5-chloro-2-
(2-isopropoxyethylamino)nicotinamide (5a). A solution of 2,5-

2350 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
and water (5 mL) was added dropwise. The resulting suspension
was cooled to room temperature and stirred for 1 h (supernatant )
3.6 mg/mL). The suspension was filtered, and the wet cake was
washed with water and then dried at 50 °C, 20 mmHg. The product
Molecular Devices). Release of Ca2+ from intracellular stores causes
an increase in fluorescence of the dye proportional to the Ca2+
concentration. Briefly, the assays were performed as follows. I3.4.2
cells23 were plated at 100 000 cells/well in poly-D-lysine-coated
96-well plates. After 2 days, cells were loaded with Calcium Assay
Reagent for 1 h at room temperature. Test compounds were prepared
at 60 µM in 6% DMSO. The cell plate was placed in the FLIPR
instrument, and 50 µL/well of test compound was delivered. The
Ca2+ signal was followed for 3 min to assay for potential receptor
agonism by the test compounds. Then, 50 µL/well of 6 µM human
MCH (in D-PBS containing 0.1% BSA) was added, and the ligand-
induced Ca2+ signal was followed for an additional 3 min.
Antagonist activity, as determined by the ability of test compounds
to inhibit 50% of MCH-induced Ca2+ flux, was reported. The
percent inhibition was calculated using the formula below:
1
was obtained as an off-white solid (0.53 g, 64% yield). H NMR
(DMSO-d6) δ 1.54-1.75 (m, 2 H), 1.90-2.03 (m, 2 H), 2.03-
2.16 (m, 2 H), 2.80-2.92 (m, 2 H), 3.37-3.45 (m, 2 H), 3.46-
3.55 (m, 1 H), 3.89 (s, 3 H), 5.99 (s, 2 H), 6.39-6.50 (m, 1 H),
6.50-6.59 (m, 1 H), 6.71-6.80 (m, 1 H), 6.82-6.90 (m, 2 H),
7.24 (dd, J ) 9.2, 2.7 Hz, 1 H), 7.53-7.60 (m, 1 H), 7.63-7.71
(m, 1 H), 8.24 (d, J ) 5.1 Hz, 1 H). Anal. (C23H25N3O3) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6,7-dichloro-
4-oxo-4H-chromene-2-carboxamide (24). A solution of 6,7-
dichloro-4-oxo-4H-chromene-2-carboxylic acid (0.065 g, 0.25
mmol) and 22 (0.062 g, 0.26 mmol) in DMF (1 mL) was charged
with HOBt (0.036 g, 0.26 mmol), Et3N (38 µL, 0.28 mmol), and
EDCI (0.053 g, 0.28 mmol). The resultant homogeneous solution
was stirred at room temperature for 18 h, diluted with EtOAc,
washed with water, 1 M K2CO3, and brine, dried (Na2SO4), and
concentrated to give a beige powder. Purification by flash silica
gel chromatography (0 to 5% MeOH in CH2Cl2) provided the title
compound as a white powder (86 mg, 72%). 1H NMR (DMSO-d6)
δ 1.54-1.69 (m, 2 H), 1.76-1.84 (m, 2 H), 1.95-2.07 (m, 2 H),
2.83 (d, J ) 11.9 Hz, 2 H), 3.37-3.41 (m, 2 H), 3.78 (d, J ) 7.8
Hz, 1 H), 5.99 (s, 2 H), 6.73-6.77 (m, 1 H), 6.83-6.89 (m, 3 H),
8.13 (s, 1 H), 8.16 (s, 1 H), 8.82 (d, J ) 8.1 Hz, 1 H). Anal. (C23H20-
Cl2N2O5) C, H, N.
% inhibition ) [1 - ((fTC - fB)/(fMCH - fB))] × 100
where fTC ) MCH-induced fluorescence in the presence of test
compound, fMCH ) MCH-induced fluorescence in the absence
of test compound, and fB ) baseline fluorescence.
In Vivo Compound Evaluation. Pharmacokinetic Analysis of
Plasma and Brain Exposure in DIO Mice. Male C57Bl/6J mice
are placed on a 60% kcal lard diet for 3-4 months, during which
time they become obese (45 g vs 30 g for lean controls on normal
chow). Compounds are dosed in DIO-mice orally at 10 mg/kg in
a vehicle containing 1% Tween-80 and water. Plasma samples are
drawn, whole brains are harvested at 0.5, 1, 2, 4, 6, 8, 12, and 24
h after the dose, and drug concentrations are determined by mass
spectroscopy analysis in comparison with a standard curve. The
three mice with highest concentrations were averaged to provide
the peak plasma or brain concentration (Cmax) ( SEM; the time
for these three samples was averaged to provide the time to peak
plasma or brain concentration (Tmax) ( SEM. The mean plasma
concentration data were submitted to multiexponential curve fitting
using WinNonlin. The area under the mean plasma concentration-
time curve from 0 to t h (time of the last measurable plasma
concentration) after dosing (AUC0-t) was calculated using the linear
trapezoidal rule for the plasma concentration-time profile. The
residual area was extrapolated to infinity, determined as the final
measured mean plasma concentration (Ct) divided by the terminal
elimination rate constant (â), and added to AUC0-t to produce the
total area under the curve (AUC0-∞).
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-methoxy-
4-oxo-4H-chromene-2-carboxamide (25). A solution of 6-meth-
oxy-4-oxo-4H-chromene-2-carboxylic acid (0.055 g, 0.25 mmol)
and 22 (0.062 g, 0.26 mmol) in 1 mL of DMF was charged with
HOBt (0.036 g, 0.26 mmol), Et3N (38 µL, 0.28 mmol), and EDCI
(0.053 g, 0.28 mmol). The resultant homogeneous solution was
stirred at room temperature for 18 h, diluted with EtOAc, washed
with distilled water, 1 M K2CO3, and brine, dried (Na2SO4), and
concentrated to give an off-white powder. Purification by flash
chromatography (0 to 5% MeOH in CH2Cl2) provided the title
1
compound as a white powder (0.088 g, 81%). H NMR (DMSO-
d6) δ 1.63 (m, 2 H), 1.80 (m, 2 H), 2.00 (m, 2 H), 2.85 (m, 2 H),
3.39 (bs, 2 H), 3.76 (m, 1 H), 3.93 (s, 3 H), 5.98 (s, 2 H), 6.73 (m,
2 H), 6.85 (m, 2 H), 7.10 (dd, J ) 9.0, 2.5 Hz, 1 H), 7.24 (d, J )
2.4 Hz, 1 H), 7.95 (d, J ) 8.8 Hz, 1 H), 8.83 (d, J ) 7.8 Hz, 1 H);
MS (ESI, MeOH/NH4OH) m/z 437 [M + H], 459 [M + Na], 435
[M - H]; Anal. (C24H24N2O6‚0.5H2O) C, H, N.
Compound Evaluation in Inactin-Anesthetized Rat Cardio-
vascular Assay. Male Sprague-Dawley rats 325-375 g were
anesthetized with the long acting barbiturate, Inactin (100 mg/kg,
ip). Catheters (PE-50) were placed in the femoral artery for
measurement of mean arterial pressure (MAP) and heart rate (HR).
Hemodynamic data was monitored continuously and acquired at
250 Hz at a logging rate of every 5 s, which was averaged every
minute using a Ponemah software platform (Gould Instrument
Systems, Valley View, OH). Post-hoc, data was further reduced to
5-min averages for each rat using the Microsoft Excel spreadsheet
application. Additional catheters were placed in the femoral vein
for compound administration (1-2 mL/kg) and saline infusion (10
uL/min) to maintain hydration. A large blood sample was taken
after the highest dose or when MAP decreased below 70 mmHg.
This minimum pressure level was implemented to ensure a
functional cardiovascular system for the collection of the end-of-
study (EOS) blood sample used for drug plasma level determina-
tions. A compromised vasculature can cause venous pooling and
yield unrepresentative plasma results. Escalating doses were given
in half log increments, such that the complete dose was administered
by the end of each 30-min infusion period. The highest doses
infused were either 100 or 300 mg/kg, representing the limits of
solubility achieving the highest possible plasma concentrations. All
compounds were dosed using poly(ethylene glycol) 400 (PEG-400)
as the vehicle. The PEG-400 vehicle produces cardiovascular effects
(increasing MAP and decreasing HR), but these effects are
minimally variable and highly reproducible. All cardiovascular
results are expressed as mean ( SEM (n ) 3 rats per compound)
In Vitro Compound Evaluation. MCHR1 Binding Assay
Using IMR-32 Membrane Preparations. Inhibition of binding
of MCHr1 by MCH was determined using membranes prepared
from IMR-32 cells transfected with the promiscuous G-protein, GR16
(I3.4.2 cells23). In 96-well plates, I3.4.2 membranes (6 µg/well)
were incubated in the presence of test compound in binding buffer
(25 mM HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5%
(w/v) BSA) and 0.05 nM [125I]MCH (PerkinElmer; 2200 Ci/mmol)
per well for 60 min at room temperature. Nonspecific binding
controls consisted of I3.4.2 membranes, 0.05 nM [125I]MCH, and
300 nM human MCH. Total binding controls of I3.4.2 membranes
and 0.05 nM [125I]MCH were also included on each plate. The plates
were centrifuged for 5 min at 1380g in a Beckman GS-6R desktop
centrifuge. The reaction buffer was carefully aspirated from each
well without disturbing the pellet. Wash buffer (25 mM HEPES
pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5 M NaCl) was added
to each well and then transferred to a 0.5% polyethyleneimine-
treated GF/B Filtration Plate (Packard) using a plate Filtermate
Harvester (Packard). The filter plate was washed three times with
wash buffer, Microscint-20 (Packard) was added to each well, and
the plate was read using a TopCount microplate scintillation counter
(Packard).
Assay for Release of Intracellular Ca2+. Activation of MCHr1
by MCH induces the release of Ca2+ from intracellular stores. This
release of intracellular Ca2+ was measured using a fluorometric
imaging plate reader (FLIPR, Molecular Devices) in conjunction
with a Ca2+-sensitive dye reagent (Calcium Assay Reagent,

CardioVascular Screening of MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2351
(7) Chen, Y.; Hu, C.; Hsu, C.-K.; Zhang, Q.; Bi, C.; Asnicar, M.; Hsiung,
H. M.; Fox, N.; Slieker, L. J.; Yang, D. D.; Heiman, M. L.; Sh, Y.
Targeted disruption of the melanin-concentrating hormone receptor-1
results in hyperphagia and resistance to diet-induced obesity. Endo-
crinol. 2002, 143, 2469-2477.
(8) Marsh, D. J.; Weingarth, D. T.; Novi, D. E.; Chen, H. Y.; Trumbauer,
M. E.; Chen, A. S.; Guan, X.-M.; Jiang, M. M.; Feng, Y.; Camacho,
R. E.; Shen, Z.; Frazier, E. G.; Yu, H.; Metzger, J. M.; Kuca, S. J.;
Shearman, L. P.; Gopal-Truter, S.; MacNeil, D. J.; Strack, A. M.;
MacIntyre, D. E.; Van der Ploeg, L. H. T.; Qian, S. Melanin-
concentrating hormone 1 receptor-deficient mice are lean, hyperactive,
and hyperphagic and have altered metabolism. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 3240-3245.
(9) (a) Borowsky, B.; Durkin, M. M.; Ogozalek, K.; Marzabadi, M. R.;
DeLeon, J.; Lagu, B.; Heurich, R.; Lichtblau, H.; Shaposhnik, Z.;
Daniewska, I.; Blackburn, T. P.; Branchek, T. A.; Gerald, C.; Vaysse,
P. J.; Forray, C. Antidepressant, anxiolytic and anorectic effects of
a melanin-concentrating hormone-1 receptor antagonist. Nature Med.
2002, 8, 779-781. (b) An orally active small molecule MCHr1
antagonist in an acute feeding model was reported in the following:
Takekawa, S.; Asami, A.; Ishihara, Y.; Terauchi, J.; Kato, K.;
Shimomura, Y.; Mori, M.; Murakoshi, H.; Kato, K.; Suzuki, N.;
Nishimura, O.; Fujino, M. T-226296: A novel, orally active and
selective melanin-concentrating hormone receptor antagonist. Eur.
J. Pharmacol. 2002, 438, 129-135.
(10) Kym, P. R.; Iyengar, R.; Souers, A. J.; Lynch, J. K.; Judd, A. S.;
Gao, J.; Freeman, J.; Mulhern, M.; Zhao, G.; Vasudevan, A.; Wodka,
D.; Blackburn, Ch.; Brown, J.; Che, J. L.; Cullis, C.; Lai, Su J.;
LaMarche, M.; Marsilje, T.; Roses, J.; Sells, T.; Geddes, B.; Govek,
E.; Patane, M.; Fry, D.; Dayton, B. D.; Brodjian, S.; Falls, D.; Brune,
M.; Bush, E.; Shapiro, R.; Knourek-Segel, V.; Fey, T.; McDowell,
C.; Reinhart, G. A.; Preusser, L. C.; Marsh, K.; Hernandez, L.; Sham,
H. L.; Collins, C. A. Discovery and Characterization of Amino-
piperidinecoumarin Melanin Concentrating Hormone Receptor 1
Antagonists. J. Med. Chem. 2005, 48, 5888-5891.
graphed at 5-min timepoints relative to drug administration as
described above. This level of quantitation was used to define
screening of MCHr1 antagonists for cardiovascular effects.
Compound Evaluation in the Pentobarbital-Anesthetized Dog
Cardiovascular Assay. Male beagle dogs (n ) 3/group) were
anesthetized with pentobarbital (35.0 mg/kg, iv) and immediately
placed on a constant iv infusion (6.0 mg/kg/h, 0.21 mL/min) to
maintain a surgical plane of anesthesia as previously described.14
Animals were intubated with a cuffed endotracheal tube and
ventilated with room air by means of a mechanical respiration pump
(Harvard Apparatus, Model 613). Expiratory CO2 was monitored
with an end-tidal CO2 monitor (Criticare Systems; Model POET
TE) and maintained at 4-5%. Briefly, polyethylene catheters were
inserted into the right femoral vein and artery for infusion of test
agents and collection of blood samples, respectively. A Swan-Ganz
catheter (5.5 F) was advanced into the pulmonary artery via the
right jugular vein for measurement of cardiac output via thermo-
dilution utilizing a cardiac output computer (Abbott Laboratories,
Oximetrix 3). Central venous pressure and pulmonary arterial
pressure were measured through the distal port of the catheter. A
dual tip micromanometer catheter (Millar, Model SPC-770, 7F) was
advanced into the left ventricle of the heart via the right carotid
artery for measurement of left ventricular and aortic blood
pressure. The primary hemodynamic variables were recorded
using a commercial software and a signal processing workstation
(Ponemah, Gould Instrument Systems, Inc). Animals were randomly
divided into one of five treatment or vehicle (PEG-400) groups.
Following the completion of the surgical protocol, animals were
allowed to stabilize for 1 h and baseline data were collected at
5-min intervals.
(11) Souers, A. J.; Gao, J.; Brune, M.; Bush, E.; Wodka, D.; Vasudevan,
A.; Judd, A. S.; Mulhern, M.; Brodjian, S.; Dayton, B.; Shapiro, R.;
Hernandez, L.; Collins, C. A.; Kym, P. R. Identification of
2-(4-benzyloxyphenyl)-N-[1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6-yl]-
acetamide, an orally efficacious melanin-concentrating hormone
receptor 1 antagonist for the treatment of obesity. J. Med. Chem.
2005, 48, 1318-1321.
Compound 4 was administered as a series of three escalating
doses (0.33, 1.00, and 3.33 mg/kg/min) and was dissolved in a
vehicle containing PEG-400 with 1 equiv of HCl. Following
termination of the high dose infusion, the animals were observed
for 1 h.
(12) Vasudevan, A.; LaMarche, M. J.; Blackburn, C.; Che, J. L.; Luchaco-
Cullis, C. A.; Lai, S.; Marsilje, T. H.; Patane, M. A.; Souers, A. J.;
Wodka, D.; Geddes, B.; Chen, S.; Brodjian, S.; Falls, D. H.; Dayton,
B. D.; Bush, E.; Brune, M.; Shapiro, R. D.; Marsh, K. C.; Hernandez,
L. E.; Sham, H. L.; Collins, C. A.; Kym, P. R.. Identification of
ortho-amino benzamides and nicotinamides as MCHr1 antagonists.
Bioorg. Med. Chem. Lett. 2005, 15, 4174-4179.
(13) Vasudevan, A.; Souers, A. J.; Freeman, J. C.; Verzal, M. K.; Gao,
J.; Mulhern, M. M.; Wodka, D.; Lynch, J. K.; Engstrom, K. M.;
Wagaw, S. H.; Brodjian, S.; Dayton, B.; Falls, D. H.; Bush, E.; Brune,
M.; Shapiro, R. D.; Marsh, K. C.; Hernandez, L. E.; Collins, C. A.;
Kym, P. R. Aminopiperidine indazoles as orally efficacious melanin
concentrating hormone receptor-1 antagonists. Bioorg. Med. Chem.
Lett. 2005, 15, 5293-5297.
(14) Fryer R. M.; Preusser L. C.; Calzadilla S. C.; Hu Y.; Xu H.; Marsh
K. C.; Cox B. F.; Lin C. T.; Gopalakrishnan M.; Reinhart G. A.
(-)-(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno-
[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637), a novel ATP-
sensitive potassium channel opener: Hemodynamic comparison to
ZD-6169, WAY-133537 and nifedipine in the anesthetized canine.
J. CardioVasc. Pharmacol. 2004, 44, 137-147.
(15) Raheem, I. T.; Goodman, S. N.; Jacobsen, E. N. Catalytic asymmetric
total syntheses of quinine and quinidine. J. Am. Chem. Soc. 2004,
126, 706-707.
(16) Compounds were tested for % inhibition @ 10 µM in a standard
panel of 78 receptors and ion channels offered from CEREP
(www.cerep.com).
(17) Screening of 130 MCHr1 antagonists for hERG blockade using cell-
based dofetilide binding assays showed no correlation between hERG
blockade and hemodynamic effects in vivo.
Acknowledgment. The authors thank Christopher Ogiela,
Dennis Fry, and Doug Falls for preparing the IMR-32 cells and
aiding with the execution of the binding and functional assays,
Paul Richardson and J.J. Jiang for MCH production, and Brian
Droz and Victoria Knourek-Segel for help with compound
dosing.
Supporting Information Available: Detailed experimental
procedures for synthesis of the compounds described in the paper;
characterization data for the compounds and procedures for both
in vitro and in vivo assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
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For studies in inactin-anesthetized rats, the minimum pressure limit

2352 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Kym et al.
tolerated was 70 mmHg. This minimum pressure level was imple-
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