Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: Assessment of potency, efficacy, and cardiovascular safety

Article abstract of DOI:10.1021/jm060683e

Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat, and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular model compared to other series reported by our group. However, this analogue also led to prolongation of the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional blockade of this ion channel.

Full text of DOI:10.1021/jm060683e

J. Med. Chem. 2006, 49, 6569-6584
6569
Optimization of Chromone-2-carboxamide Melanin Concentrating Hormone Receptor 1
Antagonists: Assessment of Potency, Efficacy, and Cardiovascular Safety
,
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John K. Lynch,* Jennifer C. Freeman, Andrew S. Judd, Rajesh Iyengar, Mathew Mulhern, Gang Zhao, James J. Napier,
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‡
Dariusz Wodka, Sevan Brodjian, Brian D. Dayton, Doug Falls, Christopher Ogiela, Regina M. Reilly, Thomas J. Campbell,
James S. Polakowski, Lisa Hernandez, Kennan C. Marsh, Robin Shapiro, Victoria Knourek-Segel, Brian Droz,
Eugene Bush, Michael Brune, Lee C. Preusser, Ryan M. Fryer, Glenn A. Reinhart, Kathryn Houseman, Gilbert Diaz,
Ann Mikhail, James T. Limberis, Hing L. Sham, Christine A. Collins, and Philip R. Kym
‡
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‡
‡
‡
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‡
‡
‡
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Metabolic Disease Research, IntegratiVe Pharmacology, Process Chemistry, and Exploratory Pharmacokinetics, Abbott Laboratories,
1
00 Abbott Park Road, Abbott Park, Illinois 60064
ReceiVed July 6, 2006
Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists
in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as
having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma
and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and
pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was
orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat,
and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular
model compared to other series reported by our group. However, this analogue also led to prolongation of
the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional
blockade of this ion channel.
Introduction
multiple structurally distinct series of MCHr1 antagonists
revealed one class that achieved a favorable cardiovascular
profile with respect to its effect on mean arterial pressure (MAP)
and heart rate (HR) with high exposures in plasma (>20 µg/
mL, 40 µM) and brain (>20 µg/g). Two chromone-2-carboxa-
mide aminopiperidines in particular, 1 and 2, exhibited potent
MCHr1 antagonism and excellent safety against the chosen
cardiovascular endpoints at very high drug concentrations in
plasma and brain (Table 1). Therefore, the chromone-2-
carboxamide series was selected as a chemical series worthy
of further optimization. Herein, we report the synthesis,
structure-activity relationships, cardiovascular effects, phar-
macokinetics, and in vivo efficacy of this series of MCHr1
antagonists.
Melanin-concentrating hormone (MCH) is a cyclic, 19-amino
acid peptide that is produced predominantly in neurons in the
lateral hypothalamus and zona incerta. This neuropeptide is
understood to have a significant role in the complex control of
food intake and body weight regulation in rodents. Three
general lines of evidence involving experiments with the MCH
peptide, the MCH receptor, and small molecule antagonists have
emerged to suggest that inhibition of the interaction of MCH
with its receptor in the brain would be a potential antiobesity
therapy.
A single injection of the MCH peptide into the lateral
hypothalamus of rodents stimulates food intake and chronic
administration leads to increased body weight. Similarly,
transgenic mice overexpressing the MCH gene are susceptible
1
2
3,4
Chemistry
5
to obesity and insulin resistance. In contrast, mice lacking the
The synthesis of 2-carboxychromones 4 was accomplished
in three steps by acylation of 2′-hydroxyacetophenones 3 with
diethyl oxalate, ring closure by treatment with concentrated
hydrochloric acid and acetic acid, and hydrolysis (Scheme 1,
steps a-c). In the course of our work, it was discovered that
the initial protocol of basic hydrolysis of the ester (step c) led
to ring-opened products in some cases and that an acidic
hydrolysis of the ester was far superior. Optimization of the
chemical route soon revealed that the ring closure and hydrolysis
gene encoding MCH are hypophagic, lean, and maintain
elevated metabolic rates.6
Genetically altered animals that lack the single gene encoding
the MCH receptor in rodents (MCHr1) maintain elevated
metabolic rates and remain lean despite hyperphagia on a normal
7
,8
diet. 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
9-13
for antiobesity pharmacotherapy.
(steps b and c) could be conducted efficiently in one pot. In
Our group recently disclosed a moderate throughput anes-
thetized rat assay that was used to eliminate MCHr1 antagonists
that demonstrated cardiovascular liabilities prior to lead opti-
addition, converting commercially available 2-methoxybenzoic
acids to the 2-hydroxyacetophenones in four steps allowed
access to differentially substituted 2-carboxychromones. The
1
4
mization. Evaluation of over 130 compounds representing
2
-methoxybenzoic acids 5 were treated with oxalyl chloride
followed by methoxymethylamine to produce the Weinreb
amide. Treatment with a Grignard reagent gave 2-methoxyac-
etophenone that was demethylated with boron tribromide to give
*
Corresponding author. Address: Dept R4MF, Bldg AP10/3, Abbott
Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101. Tel:
(
847) 938-0982. Fax: (847) 938-1674. E-mail: john.k.lynch@abbott.com.
†
Metabolic Disease Research.
Integrative Pharmacology.
Process Chemistry.
Exploratory Pharmacokinetics.
3
. However, while this was an efficient route to several
‡
§
2-carboxychromones, it suffered from limited availability of
2-hydroxyacetophenones and 2-methoxybenzoic acids.
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1
0.1021/jm060683e CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/29/2006

6
570 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
Table 1. MCHr1 Activity and Anesthetized Rat Cardiovascular Profile
IC50 (nM)
end of study levels
mean arterial
pressure
heart
rate
effect
MCHr1
binding
Ca2
+
plasma
brain
a,c
b,c
(µg/mL)c
(µg/g)c
effectc
c
compd
R
release
1
2
6,7-dichloro
7-OMe
3 ( 2
11 ( 3
83 ( 10
73 ( 16
20 ( 2
29 ( 2
63 ( 14
158 ( 13
no effect
no effect
10% decreased
no effect
a
125
Displacement of [ I]MCH from MCHr1 expressed in membranes from IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25 nM, Bmax ) 0.40 (
b
2+
c
0
.08 pmol/mg). Inhibition of MCH-mediated Ca release in intact IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM). All values are mean values ( SEM and
d
are derived from at least three independent experiments. At 100 mg/kg.
Scheme 1a
Scheme 3a
a
(a) EDC, HOBt, DMF; (b) TFA; (c) NaBH(OAc)3, ArCHO.
a
(
a) EtO2CCO2Et, NaOEt; (b) glacial AcOH, concd HCl; (c) LiOH, H2O
a
Table 2. Pharmacokinetic Parameters after a 10 mg/kg Oral Dose in
DIO Mice
or AcOH, H2O, ∆; (d) oxalyl chloride, cat. DMF, CH2Cl2; (e) HN(Me)OMe;
f) R1CH2MgX, THF; (g) BBr3, CH2Cl2.
(
plasma
brain
Scheme 2a
compd
Cmax
t1/2
AUC
Cmax
t1/2
AUC
T/P
1
2
33
3596
0.9
3.1
39
18761
0
548
0
4.9
0
0
0.24
4398
a
Cmax (ng/mL or ng/g); t1/2 (h); AUC (ng‚h/mL or ng‚h/g), T/P (tissue
brain) to plasma AUC ratio), n ) 3.
(
processed as shown in Scheme 1 to generate the desired
2
-carboxychromone 8.
The general assembly of these chromone-amide antagonists
was straightforward (Scheme 3). Acylation of a mono-N-
protected-diamine core with the 2-carboxychromone 4 under
standard amide coupling conditions was followed by deprotec-
tion and alkylation of the second nitrogen to give the requisite
analogues 10. In addition, after it was discovered that one of
the best northern fragments was the piperonyl (vide infra), the
intact 4-amino-1-(piperonyl)piperidine piece was coupled to the
carboxychromone 4.
a
(a) Dimethylacetylenedicarboxylate (DMAD); (b) NAOH; (c) P2O5,
CH3SO3H, doxane, ∆; (d) AcCl, Et3N; (e) AlCl3.
A second route to the desired 2-carboxychromones that started
from widely available substituted phenols was accomplished
as described in Scheme 2. Treatment of a phenol 6 with
dimethylacetylenedicarboxylate followed by hydrolysis gave a
mixture of olefin diacids 7. Closure of the ring from 7 was then
affected by heating this mixture with Eaton’s reagent in dioxane
to give 8. This route had the advantage of a large selection of
differentially substituted commercially available phenols but
suffered from low yields for the ring closure of 7 to 8 (only the
cis-olefin closed to the desired product) and generation of a
difficult to separate regioisomeric mixture of substituted chromone
carboxylic acids when starting with 3-substituted or 3,4-
disubstituted phenols.
Results and Discussion
Due to their favorable profiles in the anesthetized rat
cardiovascular assay, compounds 1 and 2 were orally dosed at
10 mg/kg in diet-induced obese (DIO) mice for pharmacokinetic
evaluation (Table 2). The 6,7-dichloro analogue 1 was poorly
absorbed, showed little drug in plasma, and was undetectable
in the brain, which obviously precluded further development.
The 7-methoxy analogue 2 showed a significantly better
pharmacokinetic profile with high levels in the plasma (AUC
) 18.8 µg‚h/mL) as well as good levels in the brain (AUC )
4.4 µg‚h/mL). The half-life in plasma was 3.1 h and in brain
4.9 h with a reasonable but suboptimal brain to plasma AUC
ratio of 0.24.
This regioselectivity problem was solved by acetylation of
the phenol 6 followed by a Fries rearrangement to give
exclusively transfer of the acetyl moiety to the less hindered
position to generate 9. The 2′-hydroxyacetophenone 9 was then
Thus, because of its good pharmacokinetic profile, 2 was
selected for proof-of-concept evaluation in DIO mice that were

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6571
Table 3. SAR of 4-Aminopiperidine Replacement Analoguesa
Figure 1. Effect of 2 and D-fenfluramine (D-fen, 10 mg/kg, po, twice
a day) on the body weight of DIO mice. All values are mean values (
SEM for n ) 12: (**) p < 0.01 for comparisons against vehicle group.
a
nt: not tested.
fed a high fat diet (60% fat) ad libitum and in lean mice that
were fed normal chow (Figure 1). Animals were orally dosed
twice a day with 2, dexfenfluramine as reference weight loss
agent (10 mg/kg), or vehicle. The vehicle-treated DIO mice had
a small weight gain (1%) over the course of 2 weeks, while the
dexfenfluramine-treated group lost weight rapidly then regained
some by the end of the study to ultimately give a cumulative
assay. A 3-aminomethylpyrrolidine analogue (16) retained some
activity (binding IC50 ) 1140 nM, Ca flux IC50 ) 8300 nM),
albeit extremely weak. This brief study, in addition to experience
on related series of MCHr1 antagonists, prompted us to move
forward with 4-aminopiperidine as the most potent core for this
series.
7
% loss in body weight. Treatment with 2 led to dose-dependent
Confident that we had identified the best core, we next
examined substitution on the chromone heterocycle while
keeping the remainder of the molecule intact. A large number
of differentially substituted chromone amide analogues were
constructed that contained the 4-aminopiperidine core with
piperonyl attached to the ring nitrogen (Table 4). A careful
survey of substitution at the 7-position was undertaken first.
Thus, while the 7-hydroxy analogue 17 had very little affinity
for the receptor and no effect in the functional assay, conversion
of the hydroxy to a difluoromethoxy 18 recovered much of the
activity, although this analogue was still significantly weaker
than methoxy analogue 2. Electron-neutral substituents such as
7-ethyl (19) or 7-bromo (20) had moderate activity, but a shift
to electron-withdrawing 7-chloro (21) gave a 2-fold boost to
the binding affinity (IC50 ) 5 nM) versus the methoxy 2 and
was equipotent in the calcium flux assay. The 7-fluoro chromone
(22) had even greater potency in binding affinity (IC50 )3 nM)
as well as an increase in functional potency (IC50 ) 29 nM) to
give the most potent analogue identified thus far in the series.
Alternate electron withdrawing substituents at the 7-position
such as cyano (23) or trifluoromethyl (24) were moderate at
best.
We also explored the 6-position with less beneficial results.
When compared to the 7-substituted analogues, monosubstituted
analogues at the 6-position were generally significantly less
potent as exemplified by analogues 6-chloro (25), 6-trifluoro-
methyl (26), and 6-bromo (27). The 6-methyl (28) and 6-trif-
luoromethoxy (29) also had relatively low affinity for the
receptor but lack direct comparators. Clearly, the best substituent
at the 6-position was fluoro (30), both in terms of affinity (IC50
) 65 nM) and functional potency (IC50 ) 196 nM). However,
compound 30 was only equipotent with the unsubstituted
weight loss of 7.3% and 15.9% at 10 and 30 mg/kg, respectively,
and a cumulative decrease in food intake of 16 and 37%.
Consistent with earlier reports on MCHr1 antagonists from our
group, the observed weight loss was due solely to the loss of
fat mass, as determined by dual-energy X-ray absorptiometry
1
5
(DEXA) imaging. All mice appeared healthy throughout the
study and there were no adverse events observed in a test of
motor coordination (edge-balance test)16 or in a systematic
behavioral assay analogous to that described by Irwin (modified
Irwin test). End of study drug levels (1 h post dose Cmax) for
the lowest efficacious dose of 10 mg/kg were 4.9 µg/g in brain
and 10.1 µg/mL (23 µM) in plasma. The exceptionally high
efficacious peak plasma levels, along with results of a subse-
quent efficacy study that showed no weight loss at lower doses
1
7
(1 and 3 mg/kg, twice a day, data not shown) left us with limited
opportunity to achieve an acceptable therapeutic window with
this analogue. Therefore, we focused our efforts on the
optimization of the chromone amides with particular emphasis
on improving potency and the brain-to-plasma ratio while
maintaining a favorable cardiovascular profile.
In terms of cardiovascular safety in the rat, the chromone
amide had emerged as the best heterocycle from screening a
set of piperonyl-substituted 4-aminopiperidines. In order to
determine if 4-aminopiperidine was the optimal core for this
series, we conducted a survey of alternate diamino-linker
replacements (Table 3). Contraction of the linker to a piperazine
(11), extension to a 4-amino-1-aminomethyl linker (12), or
conversion to 3-aminopiperidine (13), led to a significant loss
of affinity for the MCH receptor. Pyrrolidine linkers also lost
considerable affinity with 3-aminopyrrolidine 14 and 2-ethy-
laminopyrrolidine 15, both having IC50 > 2 µM in our binding

6
572 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
Table 4. SAR of 2-Carboxychromone Amide Analogues
Table 5. SAR of Benzylpiperidine Analoguesa
IC50 (nM)
compd
R
binding
11 ( 3
Ca flux
hERG
2
7-OMe
73 ( 16
>10000
11.5 (3.9
63.7c
b
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
1
4
5
6
7
8
9
7-OH
1480 ( 310
64 ( 20
59 ( 18
32 ( 11
5 ( 2
7-OCHF2
7-Et
160 ( 61
529 ( 263
82 ( 25
79 ( 17
29 ( 7
3.6 ( 1.3
7.4 ( 3.1
2.5 ( 0.6
3.6 ( 0.3
15.1 ( 4.6
5.0 ( 1.2
nt
7-Br
7-Cl
7-F
7-CN
7-CF3
6-Cl
6-CF3
6-Br
3 ( 1
47 ( 11
178 ( 50
734 ( 647
351 ( 142
187 ( 42
393 ( 55
132 ( 2
65 ( 9
137 ( 18
3070 ( 250
2050 ( 100
nt
nt
nt
nt
nt
b
>6000
6-CH3
6-OCF3
6-F
nt
b
>9000
nt
196 ( 30
192 ( 26
4.0 ( 0.8
6.7 ( 1.2
nt
H
5-Cl
66 ( 14
24 ( 9
c
>10000
67 ( 6
3-Me-7-Cl
6,7-diCl
6-F-7-Cl
6-F-7-OMe
6,7-difluoro
6,8-difluoro
5,6-dichloro
6,7,8-trifluoro
2 ( 1
nt
3 ( 2
83 ( 10
71 ( 25
16 ( 1
1.4 ( 0.2
10.5 ( 0.3
15.9 ( 2.3
6.2 ( 1.3
nt
3
3
3
3
3
3
3 ( 1
2 ( 1
2 ( 1
13 ( 1
99 ( 33
75 ( 24
91 ( 23
2720 ( 550
4750 ( 650
418 ( 93
nt
nt
a
All values are mean values ( SEM and are derived from at least three
b
c
independent experiments. nt: not tested. n ) 3. n ) 2.
analogue 31. A 5-chloro-substituted analogue (32) had reason-
able affinity (IC50 ) 24 nM) but no functional potency (IC50
a
All values are mean values ( SEM and are derived from at least three
independent experiments. nt: not tested.
>10 µM). Methyl substitution at the 3-position (33) was
tolerated but did not have a favorable effect on potency (33 vs
enedioxy moiety from the 3,4-position (as in the piperonyl 22)
to the 2,3-position resulted in a dramatic loss in binding affinity
41, IC50 >700 nM). Changing the hydrogens of the 3,4-
methylenedioxy to fluorine (42) led to an almost 20-fold drop
in potency. A chlorine at the 2-position (43) reduced affinity
IC50 ) 22 nM) and functional potency (IC50 ) 266 nM) by
about a factor of 8. Expanding the ring to the benzodioxane
44) had a more significant effect on affinity (>20-fold decrease)
than it did on functional potency (6-fold decrease). Scission of
the five-membered methylenedioxy ring to the 3,4-dimethoxy
2
1). Due to possible beneficial effects on pharmacokinetics,
6
-fluoro substitution was combined with the best moieties at
(
the 7-position to give 6-fluoro-7-chloro, 6-fluoro-7-methoxy,
and 6,7-difluoro analogues 34, 35, and 36, respectively. All three
had exceptional affinity for the receptor (IC50 ) 2-3 nM), but
(
34 had only moderate functional potency (IC50 ) 71 nM) while
35 and 36 had exceptional functional potency as well (IC50 )
16 and 13 nM, respectively). Additional multiply substituted
(
analogues such as 6,8-difluoro (37), 5,6-dichloro (38), or 6,7,8-
trifluoro (39) were significantly less potent.
(
45) led to a dramatic loss in affinity (IC50 ) 427 nM), but the
The more potent analogues were also evaluated for their
affinity for the hERG ion channel using a competitive, radio-
monosubstituted 3-methoxy (46) and 4-methoxy (47) analogues
had reasonable activity, with the 4-methoxy being the more
potent of the two. Systematic monosubstitution of fluorine
around the ring (48-50) showed that 3-fluoro (49) had
reasonable affinity (IC50 ) 12 nM), but all three decreased
significantly in functional potency (IC50 ) 789-2150 nM). The
combination of 4-methoxy with fluorine revealed that 3-fluoro-
4-methoxy (52) was a moderately potent analogue. Other
moieties at the 4-position such as acetyl (53) or acetamide (54)
led to significantly less potent analogues.
3
18
metric, binding assay ([ H]dofetilide). Fortunately, 7-fluoro
and 7-methoxy substitution not only improved MCHr1 potency
but also led to a 2-3-fold decrease in affinity for the hERG
channel (2 and 22 vs 31). Fluorine at the 6-position resulted in
a moderate increase in hERG activity of 1.5-fold (30 vs 31).
Importantly, the most potent analogues from this set (22, 35,
and 36) demonstrated not only a significant improvement in
MCHr1 potency but also possessed a large separation between
hERG affinity and MCHr1 affinity and functional potency.
Finally, the structure-activity relationship of the group
attached to the piperidine nitrogen was examined. A set of
Generally, the compounds shown in Table 5 had only
moderate impact on hERG affinity. Exceptions were the
difluoropiperonyl (42) and 4-methoxy substitution (47), which
led to a 3-fold increase in hERG affinity while one analogue
with a relatively polar acetamide at the 4-position (54) had a
greater than 3-fold decrease in affinity. However, the MCHr1
activity with this analogue (54) had dropped even more
dramatically, resulting in an inferior hERG/MCHr1 ratio. In the
7
5
-fluorochromone-4-aminopiperidines was constructed (Table
) for comparison to piperonyl substituted compound 22
(
binding IC50 ) 3 nM, Ca flux IC50 ) 29 nM). The unsubstituted
benzyl analogue 40 was approximately 20-fold less potent in
terms of affinity and functional potency. Moving the methyl-

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6573
Table 6. Anesthetized Rat Cardiovascular Profile
IC50 (nM)
MCHr1
end of study levels
mean arterial
pressure effect
(infusion range)c
heart rate
effect
(infusion range)
Ca2
+
plasma
brain
a,c
b,c
(mg/mL)c
(mg/g)c
c
compd
R
binding
release
2
3
2
5
7-F
6-F-7-OMe
3 ( 1
2 ( 1
29 ( 7
16 ( 1
62 ( 8
126 ( 17
149 ( 11
no effect
15.3% decrease
no effect
105 ( 16
<15% decrease
(60 mg/kg)
no effect
(
60 mg/kg)
>15% decrease
60 mg/kg)
36
6,7-diF
2 ( 1
13 ( 1
34 ( 6
25 ( 8
(
a
125
Displacement of [ I]MCH from MCHr1 expressed in membarnes from IMR-32 (I3.4.2) cells (MCH binding Kd ) 0.66 ( 0.25 nM, Bmax ) 0.40 (
b
2+
0
.08 pmol/mg). Inhibition of MCH-mediated Ca release in intact IMR-32 cells (MCH EC50 ) 62.0 ( 3.6 nM. c All values are mean values ( SEM and
are derived from at least three independent experiments.
end, piperonyl was found to be the optimal N-linked piperidine,
and analogues 22, 35, and 36 were selected for further
evaluation.
As described in a previous publication, recurring hemody-
namic cardiovascular side effects observed for several different
classes of MCHr1 antagonists led us to move an anesthetized
1
4
rat cardiovascular assay earlier in our triaging process. In this
assay, three male Sprague-Dawley rats are anesthetized with
inactin (100 mg/kg), have catheters placed in the femoral artery
to monitor mean arterial pressure and heart rate, and are
administered iv escalating doses in half log increments such
that the complete dose is administered at the end of each 30-
min infusion period. We took the three most potent analogues
(
22, 35, 36) into this assay. The 7-fluoro analogue 22 had no
effect on mean arterial pressure (MAP) and heart rate (Table
) and drug concentrations reached exceptionally high levels
in the plasma (62 µg/mL, 146 µM) and brain (126 µg/g). The
-fluoro-7-methoxy analogue 35 led to a moderate decrease in
6
6
heart rate, showed a significant effect on MAP (15.3% decrease)
at extremely high plasma concentrations (105 µg/mL, 231 µM),
and all rats went into convulsions six minutes before the end
of the protocol. The 6,7-difluoro analogue 36 had no effect on
heart rate but did cause a significant effect on mean arterial
pressure (>15% decrease), and two out of the three rats dropped
below the minimum allowed mean arterial pressure limit of 70
mmHg during the 60 mg/kg dose.
Figure 2. Pharmacokinetic parameters after a 10 mg/kg oral dose in
DIO mice.
weight loss after administration of 22 had leveled out by day
14 with very little change thereafter.
End of study drug levels for the lowest efficacious dose of
10 mg/kg showed that 22 did have a 3-fold lower peak plasma
level compared to 2 (7.7 µM vs 23 µM). Interestingly, at 10
mg/kg 22 and 2 had nearly identical peak brain levels (Cmax )
4.7 and 4.9 µg/g respectively), but 22 had dramatically lower
trough levels (0.4 vs 2.2 µg/g). This was undoubtedly due to
the longer brain half-life of 2 (4.9 h) versus 22 (2.4 h) and may
explain why the more potent 22 showed the same efficacy as 2
at 10 mg/kg and was not efficacious at lower doses.
On the basis of its superior cardiovascular profile in the rat,
analogue 22 was orally dosed at 10 mg/kg in DIO mice for
pharmacokinetic evaluation (Figure 2). This analogue had a
nearly identical half-life in plasma and brain of about 2.5 h but
rapidly penetrated the brain, resulting in good levels in the
plasma (AUC ) 9.29 µg h/mL) but higher levels in the brain
(AUC ) 12.7 µg h/g) and a very favorable brain-to-plasma ratio
The therapeutic index for this project was initially defined
as the ratio of the highest plasma concentration that was safe
of 1.4. Thus, the analogue 22 was not only 3-fold more potent
than 2 in terms of functional potency but also had a 6-fold
improvement in brain to plasma AUC ratio.
1
9
in our dog cardiovascular model over the peak plasma
concentration required to achieve at least a 5% weight loss in
the DIO mouse model. Due to comparable efficacy at 10 mg/
kg and 3-fold lower plasma Cmax observed for 22 versus 2, we
evaluated 22 in our anesthetized dog cardiovascular model. An
array of clinically relevant endpoints was assessed in the dog,
including blood pressure, heart rate, cardiac contractile function,
pulmonary arterial pressure, and QT interval. Compound 22 was
infused (iv) in three escalating doses at 3, 10, and 30 mg/kg/30
min, which produced peak plasma concentrations of 5.9 ( 0.3,
25.8 ( 1.6, and 85.1 ( 7.9 µM at the end of each dosing period.
This represented 0.8-, 3.3-, and 11.0-fold above the mouse
efficacious Cmax. By the end of the 30 mg/kg infusion period,
Analogue 22 was further evaluated in an efficacy study in
DIO mice (Figure 3). Due to its superior in vitro potency,
compound 22 was dosed orally twice a day at 3 and 10 mg/kg
while 2 had been dosed at 10 and 30 mg/kg. The fluoro-
chromone 22 had no effect on body weight at 3 mg/kg but had
a significant loss of body weight (7.2%) at the 10 mg/kg dose.
Weight loss was due solely to the loss of fat mass as determined
by DEXA imaging, and all mice appeared healthy throughout
the study, displaying no signs of overt toxic effects in the edge-
balance or modified Irwin tests (data not shown). Although the
study with 22 was run for 22 days as opposed to 14 days for 2,

6
574 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
Figure 3. Effect of 22 and sibutramine (10 mg/kg, po, once a day) on
the body weight of DIO mice. All values are mean values ( SEM for
n ) 12: (**) p < 0.01 for comparisons against vehicle group.
22 had produced no physiologically relevant change in mean
arterial pressure (Figure 4C). This constituted a significant
improvement on this parameter versus the best compounds from
earlier publications such as the coumarin 55 and alkyl indazole
5
6 (Figure 4A,B).
However, 22 produced a biphasic effect on heart rate initially
decreasing to -22 ( 1% at 10.94 µg/mL before trending toward
baseline to end at -11 ( 3% at 36.13 ( 3.34 µg/mL (Figure
5
A). Importantly, 22 also produced a pronounced and dose-
dependent increase in the QT-interval corrected for heart rate
QTc; Van de Water correction formula)20 to 9 ( 0.5%, 16 (
(
0
.8%, and 30 ( 1.6% above baseline at the end of each dosing
period (Figure 5B).
The QT prolongation led to the closer evaluation of activity
3
at the hERG channel. A competitive binding assay ([ H]-
dofetilide) had given an IC50 of 15.1 µM for 22. Given the
exceptional potency of this analogue at MCHr1 (IC50 ) 29 nM
Ca flux and 3 nM binding), this compound possessed a
significant in vitro selectivity window versus hERG binding
Figure 4. Effect on mean arterial pressure in anesthetized dog.
(500-5000-fold). However, the high plasma drug levels required
to achieve efficacy (3.27 µg/mL, 7.7 µM, 265-fold above the
IC50) prevented this in vitro selectivity window from translating
into a reasonable in vivo therapeutic index. Furthermore, the
functional effect of 22 on hERG current was evaluated using
HEK 293 cells stably expressing hERG. Drug effects were
evaluated on the basis of tail currents measured during 4-s
repolarization test pulses to -50 mV preceded by a 3-s
depolarizing conditioning pulse to 0 mV. In this assay, 22
demonstrated an IC50 of 1.6 µM and led to 90% current block
when evaluated at the efficacious Cmax (7 µM), thus precluding
the need for further development of this analogue.
Further evaluation of 22 in an anesthetized dog cardiovascular
assay demonstrated significant improvement in effects on mean
arterial pressure compared to earlier disclosed series of MCHr1
antagonists. However, this analogue also had biphasic effects
on heart rate and revealed considerable QT prolongation. The
QT prolongation was linked to activity at the hERG channel
both in terms of binding and functional blockade. While
screening for changes in mean arterial pressure and heart rate
in rat led to an improved cardiovascular profile, clearly
significant hurdles remain. Addressing the hERG activity, along
with efforts to lower efficacious peak plasma drug levels, will
be the topic of future communications from our group.
Conclusion
In conclusion, a series of MCHr1 antagonists was optimized
for potency, triaged for cardiovascular safety in anesthetized
rat, and evaluated for pharmacokinetic profile in DIO mice. This
led to the identification of two analogues (22 and 2) that were
tested in our DIO mouse efficacy model and showed significant,
dose-dependent weight loss with twice-a-day oral dosing.
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)

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6575
0% CH
9
3
CN 6-18 min, 90-0% CH CN 18-20 min). Preparative
3
reversed-phase chromatography was performed using a Zorbax SB-
C18, 7 µM, 21.2 × 250 mm column with UV detection analyzed
3
at 220 and 254 nM (water with 0.1% trifluoracetic acid and CH -
CN with 0.1% trifluoroacetic acid gradient, 5-95% CH
30 min at 15 mL/min).
3
CN over
General Procedures. Method A. Conversion of Phenols to
2′-Hydroxyacetophenones. Acetyl chloride (3.42 mL, 48.1 mmol)
was added slowly to a solution containing the phenol (38.5 mmol),
triethylamine (6.7 mL, 48.1 mmol), and CH Cl (50 mL) at 0 °C.
2
2
The solution was permitted to warm to room temperature as it was
stirred overnight. After 15 h the solution was concentrated, and
the resulting residue was filtered though a plug of silica gel (5 g),
eluting with Et
The solid was added to a slurry of aluminum trichloride (16 g, 118
mmol) in either CH Cl or CS (35 mL). After 1 h at room
2
O, to give a solid after concentration of the filtrate.
2
2
2
temperature, the mixture was heated between 60 and 70 °C and
the solvent removed by distillation. Once the solvent was gone,
the temperature was raised to 140 °C. After 1.5 h, the reaction was
allowed to cool to ambient temperature and combined very carefully
with 50% aqueous HCl (30 mL). The resulting solid was collected
by filtration to provide the desired 2′-hydroxyacetophenone.
Method B. Conversion of 2′-Hydroxyacetophenones to 2-Car-
boxychromones. The 2′-hydroxyacetophenone (32.4 mmol) was
dissolved in diethyl oxalate (22 mL, 162 mmol) and added to
sodium ethoxide (22 wt %) in EtOH (42 mL, 130 mmol). The
yellow, heterogeneous solution was heated at 40 °C for 30 min
before the mixture was cooled to room temperature and filtered to
collect the yellow precipitate. The yellow solid was diluted with
CH
2
Cl
2
, washed with 10% aqueous AcOH and brine, dried
4
), filtered, and concentrated to a yellow solid. This solid
(MgSO
was combined with glacial acetic acid (33 mL) and concentrated
HCl (1 mL) and heated at 80 °C for 14 h before the dark solution
was allowed to cool to room temperature and combined with
distilled water (35 mL). The resulting precipitate, generally a
mixture of the ethyl ester and carboxylic acid, was collected by
filtration and dried in a vacuum oven at 50 °C overnight. Final
conversion to the carboxylic acid was accomplished by heating the
compound with glacial acetic acid (80 mL) and 6 M HCl (40 mL)
at 80 °C for 17 h. The resultant solution was allowed to cool to
ambient temperature, diluted with distilled water (120 mL), cooled
in an ice bath, and the precipitate was collected by filtration.
Alternatively, the initial dark solution from the ring closure step
(at room temperature) was treated directly with glacial acetic acid
(50 mL) and 6 M HCl (40 mL) and heated at 80 °C for 17 h
followed by the same procedure for isolation of precipitate
2-carboxychromone.
Figure 5. Effect of 22 on HR and QTcV interval in anesthetized dog.
from E. M. Science or on Biotage, Analogix or Isolute pre-packed
columns. Following workup, reaction mixtures were dried over
MgSO or Na SO ; filtered through a fritted glass funnel, filter
4 2 4
paper, or a plug of cotton; and concentrated with a rotary evaporator
at ca. 15 mmHg, with warming when necessary. Thin-layer
chromatography systems were the same as those used for column
chromatography, with R approximately ) 0.3. Analytical LC-
f
MS was performed on a Finnigan Navigator mass spectrometer and
Agilent 1100 HPLC system running Xcalibur 1.2 and Open-Access
1.3 software. The mass spectrometer was operated under positive
APCI ionization conditions. The HPLC system comprised an
Agilent Quaternary pump, degasser, column compartment, au-
tosampler, and diode-array detector with a Sedere Sedex 75
evaporative light-scattering detector. The column used was a
Phenomenex Luna Combi-HTS C8(2), 5 µm, 100 Å (2.1 mm ×
Method C. Conversion of 2-Methoxybenzoic Acids to 2′-
Hydroxyacetophenones. 2-Methoxybenzoic acid (15.0 mmol),
DMF (0.25 mL), and CH
to 0 °C. Oxalyl chloride (7.5 mL, 15.0 mmol of a 2 M solution in
CH Cl ) was added carefully dropwise. Gas evolution was observed.
2 2
Cl (60 mL) were combined and cooled
2
2
3
0 mm). A gradient of 10-100% acetonitrile (A) and 0.1%
After 0.5 h at 0 °C, the ice bath was removed. After 1 h at room
temperature, the solution was cooled to 0 °C, and N,O-dimethyl-
hydroxylamine hydrochloride (3.22 g, 33.0 mmol) was added
followed by the slow addition of diisopropylethylamine (5.8 mL,
33.0 mmol). The solution was permitted to slowly warm to room
temperature as it was stirred overnight. After 15 h, the solution
trifluoroacetic acid in water (B) was used, at a flow rate of 1.5
mL/min (0-0.1 min 10% A, 0.1-3.1 min 10-100% A, 3.1-3.9
1
min 100-10% A, 3.9-4.0 min 100-10% A). 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, proton count, coupling constants).
Mass spectral analysis was accomplished using fast atom bombard-
ment (FAB-MS), electrospray (ESI-MS), or direct chemical ioniza-
tion (DCI-MS) techniques. Elemental analyses were obtained at
QTI Laboratories. Routine analytical reversed-phase chromatog-
raphy was performed using a Zorbax SP-C18, 5 µM, 4.6 × 250
mm column with UV detection analyzed at 254 nM (water with
was diluted with CH
brine; dried (MgSO
2
Cl
2
; washed with 1 M HCl, 1 N NaOH, and
4
); filtered; and concentrated to a solid. The
crude Weinreb amide (14.3 mmol) was combined with THF (60
mL) and cooled to -78 °C. Methylmagnesium bromide (5.3 mL,
2
15.8 mmol of a 3 M solution in Et O) was added dropwise. The
mixture was permitted to warm to room temperature slowly as it
was stirred overnight. After 16 h, the reaction was quenched with
2 N HCl and the mixture was diluted with EtOAc; washed with
0
.1% trifluoracetic acid and CH
3
CN gradient, 0-100% CH
3
CN over
18 min at 1.5 mL/min). Additional reversed-phase purity evaluation
3 4
distilled water, saturated NaHCO , and brine; dried (MgSO );
was performed on Phenomenex Luna C18(2), 5 µM (method A)
and Phenomenex 5 µM Phenyl-Hexyl (method B) 4.6 × 250 mm
columns with UV detection at 254 nM (water with 0.1% trifluo-
filtered; and concentrated. Next, boron tribromide (17.4 mL, 17.4
mmol) was added slowly to the 2′-methoxyacetophenone (8.71
mmol) in CH
2 2
Cl (20 mL) at 0 °C. After 1 h, the ice-water bath
roacetic acid and CH
3 3
CN gradient, 0-90% CH CN over 6 min,
was removed and the dark mixture was stirred at ambient temper-

6
576 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
ature for another 0.5 h before the solution was added carefully to
a mixture of ice-cold 2 N aqueous HCl. The aqueous layer was
acetate/acetone, and air-dried to give 7-methoxy-4-oxo-4H-chromene-
2-carboxylic acid ethyl ester (2A) as an off-white solid (10.6 g,
100%). A solution of example 2A (10 g, 40 mmol) in 120 mL of
THF and 40 mL of distilled water was charged with of lithium
hydroxide hydrate (3.4 g, 81 mmol). The resulting brown, homo-
geneous solution was stirred for 2 h and then quenched by slow
extracted with CH
with saturated aqueous NaHCO
2
Cl
2
; the organic layers were combined, washed
and brine, dried (MgSO ), filtered,
3
4
and concentrated. The resulting crude material was purified by
column chromatography, eluting with a gradient of EtOAc in
hexane, to provide the desired 2′-hydroxyacetophenone (2.29 mmol,
2 4
addition of 15 mL of 3 M H SO portionwise via pipet. The
2
6%).
resulting solid was filtered off, washed with distilled water, and
air-dried to give 7-methoxy-4-oxo-4H-chromene-2-carboxylic acid
(2B) as a light yellow powder (7.7 g, 82%). A solution of 2B (7.5
g, 34 mmol) was coupled to 4-amino-1-BOC-piperidine (6.8 g, 34
mmol) using general method E to give 13 g of a brown residue.
This residue was dissolved in 90 mL of dichloromethane, cooled
to 0 °C, and then charged with 45 mL of trifluoroacetic acid in
three portions. After 15 min at 0 °C, the reaction was allowed to
warm to room temperature, stirred for 1.5 h, and then concentrated
to give a brown oil. This oil was dissolved in dichloromethane (300
Method D. Conversion of Phenols to 2-Carboxychromones.
Benzyltrimethylammonium hydroxide (40% weight in water, 0.5
mL, 1.47 mmol) was added to a mixture of the phenol (123 mmol),
dimethylacetylenedicarboxylate (16.6 mL, 135 mmol), and dioxane
(
250 mL), and the solution was heated at 90 °C. After 1.5 h, the
solution was allowed to cool, combined with 20% aqueous NaOH
100 mL), and then heated at 90 °C. After 1 h, the slurry was cooled
(
in an ice bath, 2 M HCl was added until the pH was less than 6,
and the resulting precipitate was isolated by filtration and washed
with distilled water. The crude solid (89.5 mmol) was combined
with Eaton’s reagent (182 mL, 98.4 mmol) and heated at 70 °C for
mL) and washed with 1 M K
with 95/5 CH Cl
/methanol (3 × 300 mL), and the organic extracts
were combined, dried (Na SO ), and concentrated to give 4-[(7-
2 3
CO . The aqueous layer was extracted
2
2
16 h. The solution was cooled to ambient temperature and added
2
4
to ice (1 kg). The resulting white precipitate was collected by
filtration to give the 2-carboxychromone.
Method E. Conversion of Carboxylic Acids to Amides. A
solution of 2-carboxychromone (0.25 mmol) and amine (0.26 mmol)
in DMF (1 mL) was charged with hydroxybenzotriazole (36 mg,
methoxy-4-oxo-4H-chromene-2-carbonyl)amino]piperidine (2C) as
an orange solid (7.8 g, 76%). Compound 2C (7.5 g, 24.8 mmol)
was alkylated with piperonal (3.7 g, 24.8 mmol) using general
method F to give 11.1 g of an orange foam. This foam was dissolved
in a minimal amount of CH
column, and eluted with 0-100% ethyl acetate in hexane (0 to 20
min), 100% ethyl acetate (20-35 min), 100% CH Cl (35-40 min),
Cl Cl (60-
2 2
Cl , placed on a 70-g Isolute silica gel
3
0.26 mmol), Et N (38 uL, 0.28 mmol), and N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 53 mg, 0.28
mmol). The resultant homogeneous solution was stirred at room
temperature for 18 h; diluted with EtOAc; washed with distilled
2
2
0-5% methanol/CH
2
2
(40-60 min), 5/95 methanol/CH
2
2
70 min) at 30 mL/min with detection at 254 nM wavelength to
water, 1 M K
2
CO
3
, and brine; dried (Na
2
SO
4
); and concentrated to
give the title compound 2 as an off-white, amorphous solid (5.3 g,
49%): 1H NMR (300 MHz, DMSO-d
) δ 1.63 (m, 2 H), 1.80 (m,
give a solid. Purification by flash silica gel chromatography (0-
6
5
7
% MeOH in CH
µM, 21.2 × 250 mm column, CH
2
Cl
2
) or reversed phase HPLC (Zorbax SB-C18,
CN, H O with 0.1% TFA)
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
3
2
provided the desired amides. If the amine used in the reaction was
a hydrochloride salt, an extra equivalent of Et
each equivalent of HCl.
3
N was added for
Hz, 1 H), 8.83 (d, J ) 7.8 Hz, 1 H); MS (ESI, MeOH/NH
m/z 437 [M + H], 459 [M + Na], 435 [M - H]; Anal.
‚0.5H O) C, H, N.
4
OH)
Method F. Reductive Alkylation of Amines. Sodium triac-
etoxyborohydride (0.200 mmol) was added to a mixture of the free
amine (0.100 mmol), aldehyde (0.100 mmol), acetic acid (20 µL),
and THF (1 mL). After 18 h, the reaction was quenched with
methanol and/or water. The solution was diluted with EtOAc,
(C24
H
24
N
2
O
6
2
2-(4-Benzo[1,3]dioxol-5-ylmethylpiperazine-1-carbonyl)-7-
methoxychromen-4-one (11). A solution of 7-methoxy-4-oxo-4H-
chromene-2-carboxylic acid (2B, 100 mg, 0.45 mmol) and piperazine-
1-carboxylic acid tert-butyl ester (101 mg, 0.54 mmol) were coupled
in a manner similar to method E, followed by treatment with 4 N
HCl in dioxane to give 7-methoxy-2-(piperazine-1-carbonyl)-
chromen-4-one (11A). Compound 11A and benzo[1,3]dioxole-5-
carbaldehyde (62 mg, 0.41 mmol) were processed in a manner
similar to method F to give the title compound 11 (156 mg, 82%
washed with 1 M NaOH or 1 M K
Na SO ), filtered, and concentrated. Purification by flash silica gel
chromatography (0-5% MeOH in CH Cl ) or reversed phase HPLC
2 3
CO , washed with brine, dried
(
2
4
2
2
as described above provided the desired alkylated amines. If the
amine starting material was a salt, either the free amine was
generated by partitioning between dichloromethane and aqueous
potassium carbonate prior to submission to the reductive alkylation
or 2-4 equiv of KOAc was added to the reaction for each
equivalent of amine salt.
yield): 1H NMR (DMSO-d
6
) δ 2.41 (m, 4H), 3.44 (s, 2H), 3.53
(m, 2H), 3.61 (m, 2H), 5.99 (s, 2 H), 6.44 (s, 1H), 6.76 (dd, J )
7.9 Hz, 1.5 Hz, 1H), 6.85 (d, J ) 7.9 Hz, 1H), 6.88 (d, J ) 1.5 Hz,
1 H), 7.10 (dd, J ) 8.9 Hz, 2.4 Hz, 1 H), 7.21 (d, J ) 2.4 Hz, 1
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6,7-dichloro-
H), 7.95 (d, J ) 8.9 Hz, 1H); MS (ESI, MeOH/NH
4
OH) m/z 422.9
O) C, H, N.
4-oxo-4H-chromene-2-carboxamide (1). 6,7-Dichloro-4-oxo-4H-
[M + H], 444.9 [M + Na]; Anal. (C23 ‚0.25H
H
22
N
2
O
6
2
chromene-2-carboxylic acid (1A) was produced from 3,4-dichlo-
rophenol as detailed in methods A and B. Compound 1A (65 mg,
7-Methoxy-4-oxo-4H-chromene-2-carboxylic Acid (4-{[(Benzo-
[1,3]dioxol-5-ylmethyl)amino]methyl}cyclohexyl)amide (12). A
solution of 2B (99 mg, 0.45 mmol) and 4-[[(benzo[1,3]dioxol-5-
ylmethyl)amino]methyl}cyclohexylamine (118 mg, 0.45 mmol)
0
.25 mmol) and 1-benzo[1,3]dioxol-5-ylmethylpiperidin-4-ylamine
10,14
1B
(62 mg, 0.26 mmol) were processed as detailed in method
E to provide the title compound as a white powder (86 mg, 72%):
were coupled in a manner similar to method E to give the title
1
1
H NMR (300 MHz, DMSO-d
m, 2 H), 1.95-2.07 (m, 2 H), 2.83 (d, J ) 11.9 Hz, 2 H), 3.37-
.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,
OH/NH OH) m/z 475 [M + H],
) C, H, N.
6
) δ 1.54-1.69 (m, 2 H), 1.76-1.84
compound 12 (161 mg, 77% yield): H NMR (DMSO-d
6
) δ 1.46-
(
3
(
1.70 (m, 9H), 1.99 (m, 1H), 2.46 (d, J ) 6.7 Hz, 2H), 3.61 (s, 2H),
3.87-3.88 (bs, 1H), 3.93 (s, 3H), 5.97 (s, 2H), 6.75-6.83 (m, 3H),
6.92 (s, 1H), 7.11 (dd, J ) 8.9 Hz, 2.4 Hz, 1H), 7.24 (d, J ) 2.4
Hz, 1H), 7.95 (d, J ) 8.9 Hz, 1H), 8.60 (d, J ) 7.6 Hz, 1H); MS
J ) 8.1 Hz, 1 H); MS (APCI, CH
3
4
4
73 [M - H]; Anal. (C23
H
20Cl
N
2 2
O
5
(ESI, MeOH/NH
0.5H O) C, H, N.
4 28 2 6
OH) m/z 465.2 [M + H]; Anal. (C26H N O ‚
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-methoxy-
-oxo-4H-chromene-2-carboxamide (2). A solution of com-
2
4
7-Methoxy-4-oxo-4H-chromene-2-carboxylic acid (1-Benzo-
[1,3]dioxol-5-ylmethylpiperidin-3-yl)amide (13). A solution of 2B
(80 mg, 0.36 mmol) and 3-amino-piperidine-1-carboxylic acid tert-
butyl ester (87 mg, 0.43 mmol) were coupled in a manner similar
to method E, followed by treatment with 4 N HCl in dioxane to
give 7-methoxy-4-oxo-4H-chromen-2-carboxylic acid piperidin-3-
ylamide (13A). Compound 13A and benzo[1,3]dioxole-5-carbal-
mercially available 7-hydroxy-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (10 g, 42.7 mmol) in 90 mL of acetone was charged
with K CO (13 g, 93.8 mmol) and iodomethane (3.3 mL, 53.4
2 3
mmol) and the reaction heated at reflux. After 19 h, the reaction
was allowed to cool to room temperature and 50 mL of ethyl acetate
was added. The resultant solid was filtered off, rinsed with 1/1 ethyl

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6577
dehyde (48 mg, 0.32 mmol) were processed in a manner similar to
93%). Compound 17A (800 mg, 3.88 mmol) and 1-(1,3-benzo-
dioxol-5-ylmethyl)piperidin-4-amine dihydrochoride 1B (1.28 g,
4.07 mmol) were processed as detailed in method E to give the
method F to give the title compound 13 (126 mg, 80% yield): 1
H
NMR (DMSO-d ) δ 1.37-1.56 (m, 2H), 1.67-1.86 (m, 2H), 1.91-
6
1
2
3
6
.04 (m, 2H), 2.66-2.86 (m, 2H), 3.49-3.48 (m, 2H), 3.93 (s,
H), 3.94-3.98 (m, 1H), 5.97 (s, 2 H), 6.74-6.77 (m, 2H), 6.83-
.89 (m, 2H), 7.10 (dd, J ) 8.9 Hz, 2.4 Hz, 1 H), 7.21 (d, J ) 2.4
title compound 17 (909 mg, 54%) as an off-white solid: H NMR
(300 MHz, DMSO-d ) δ 1.64-1.74 (m, 2 H), 1.76-1.84 (m, 2
6
H), 2.00-2.16 (m, 2 H), 2.87 (m, 2 H), 3.45 (s, 2 H), 3.72-3.85
Hz, 1 H), 7.95 (d, J ) 8.9 Hz, 1 H), 8.69 (d, J ) 7.6 Hz, 1 H); MS
(m, 1 H), 6.00 (s, 2 H), 6.70 (s, 1 H), 6.74-6.80 (m, 1 H), 6.84-
6.97 (m, 3 H), 7.02 (d, J ) 2.0 Hz, 1 H), 7.88 (d, J ) 8.8 Hz, 1
H), 8.83 (d, J ) 8.1 Hz, 1 H), 10.98 (s, 1 H); MS (ESI, MeOH/
NH OH) m/z 421 [M - H]; HPLC method A (t ) 4.90 min,
(
ESI, MeOH/NH
‚0.5H
-Methoxy-4-oxo-4H-chromene-2-carboxylic Acid (1-Benzo-
1,3]dioxol-5-ylmethylpyrrolidin-3-yl)amide (14). A solution of
B (80 mg, 0.36 mmol) and 3-aminopyrrolidine-1-carboxylic acid
4
OH) m/z 437.2 [M + H], 459.2 [M + Na]; Anal.
2
(C
24
H
24
N O
2 6
O) C, H, N.
7
4
R
[
2
100%), method B (t ) 5.0 min, 98.5%).
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-(difluo-
romethoxy)-4-oxo-4H-chromene-2-carboxamide (18). A solution
of 17 (0.011 g, 0.026 mmol) in DMF (1 mL) at -78 °C was treated
tert-butyl ester (81 mg, 0.43 mmol) were coupled in a manner
similar to method E, followed by treatment with 4 N HCl in dioxane
to give 7-methoxy-4-oxo-4H-chromen-2-carboxylic acid pyrroli-
dine-3-ylamide (14A). Compound 14A and benzo[1,3]dioxole-5-
carbaldehyde (45 mg, 0.30 mmol) were processed in a manner
2 3
with liquid chlorodifluoromethane (1 mL) and K CO (0.011 g,
0.078 mmol). The cold bath was removed, and the suspension was
allowed to warm to ambient temperature and stirred for 2 h. The
similar to method F to give the title compound 14 (100 mg, 66%
reaction mixture was purified by preparative reverse-phase HPLC
1
yield): H NMR (DMSO-d
1
(
(
7
7
6
) δ 1.81-1.89 (m, 1H), 2.14-2.23 (m,
as described in the general section to provide 18: 1H NMR (300
H), 2.47 (m, 2H), 2.62-2.71 (m, 1H), 2.75-2.79 (m, 1H), 3.52
MHz, DMSO-d
6
) δ 1.80 (m, 2 H), 2.07 (m, 2 H), 3.09 (m, 2 H),
s, 2H), 3.93(s, 3H), 4.35-4.43 (m, 1H), 5.98 (s, 2 H), 6.74-6.78
m, 2H), 6.83-6.89 (m, 2H), 7.10 (dd, J ) 8.9 Hz, 2.5 Hz, 1 H),
3
1
7
.43 (m, 2 H), 3.99 (m, 1 H), 4.22 (m, 2 H), 6.09 (s, 2 H), 6.85 (s,
H), 6.96 (m, 1H), 7.02 (dd, 1 H), 7.08 (d, 1 H), 7.35 (dd, 1 H),
.50 (m, 1 H), 7.51 (t, 1 H), 8.10 (d, 1 H), 9.08 (d, 1 H); MS (ESI)
.24 (d, J ) 2.5 Hz, 1 H), 7.95 (d, J ) 8.9 Hz, 1 H), 8.96 (d, J )
.4 Hz, 1 H); MS (ESI, MeOH/NH
‚0.5H
-Methoxy-4-oxo-4H-chromene-2-carboxylic Acid [2-(1-Benzo-
1,3]dioxol-5-ylmethylpyrrolidin-2-yl)ethyl]amide (15). A solu-
4
OH) m/z 423.2 [M + H], 445.2
2
+
m/z 473 [M + H] .
[
M + Na]; Anal. (C23
22
H N
2
O
6
O) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-ethyl-4-
oxo-4H-chromene-2-carboxamide (19). 3-Ethylphenol (5 g, 40.9
mmol), pyridine (8.3 mL, 102 mmol), acetic anhydride (4.3 mL,
7
[
tion of 2B (80 mg, 0.36 mmol) and 2-(2-aminoethyl)pyrrolidine-
-carboxylic acid tert-butyl ester (92 mg, 0.43 mmol) were coupled
4
5 mmol), and dichloromethane (10 mL) were stirred at room
temperature overnight. The solution was washed with water and
brine, dried (Na SO ), and concentrated to dryness. All of the crude
product was suspended in BF -OEt (40 mL) and heated to reflux.
1
in a manner similar to method E, followed by treatment with 4 N
HCl in dioxane to give 7-methoxy-4-oxo-4H-chromen-2-carboxylic
acid (2-pyrrolidin-2-ylethyl)amide (15A). Compound 15A and
benzo[1,3]dioxole-5-carbaldehyde (47 mg, 0.31 mmol) were pro-
cessed in a manner similar to method F to give the title compound
2
4
3
2
After 2 h, the solution was cooled to ambient temperature, diluted
with water (20 mL), and extracted with ethyl acetate (3 × 25 mL).
The combined organics were washed with water and brine, dried
5 (115 mg, 71% yield): 1H NMR (DMSO-d
) δ 1.49-1.71 (m,
1
4
2
(
1
7
6
2 4
(Na SO ), and concentrated to give 1-(4-ethyl-2-hydroxyphenyl)-
H), 1.87-1.99 (m, 2H), 2.06-2.13 (m, 1H), 2.49 (m, 1H), 2.78-
.82 (m, 1H), 3.09-3.12 (m, 1H), 3.34-3.44 (m, 2H), 3.88-3.92
ethanone (19A). The crude 19A was processed according to general
method B to give 7-ethyl-4-oxo-4H-chromene-2-carboxylic acid
m, 4H), 5.90 (d, J ) 12.0 Hz, 2 H), 6.73-6.78 (m, 3H), 6.83 (s,
(19B, 1.9 g, 21% from 3-ethylphenol). Compound 19B (50 mg,
H), 7.06 (d, J ) 2.5 Hz, 1 H), 7.24 (dd, J ) 8.9 Hz, 2.5 Hz, 1 H),
.95 (d, J ) 8.9 Hz, 1 H), 9.18 (t, J ) 5.5 Hz, 1 H); MS (ESI,
0.23 mmol) and 1-benzo[1,3]dioxol-5-ylmethylpiperidin-4-ylamine
dihydrochloric acid salt (70 mg, 0.23 mmol) were processed
according to general method E to give title compound 19 (83 mg,
MeOH/NH
4
OH) m/z 451.2 [M + H], 473.2 [M + Na]; Anal.
‚0.5H O) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)pyrrolidin-3-ylmethyl]-7-
methoxy-4-oxo-4H-chromene-2-carboxamide (16). 7-Methoxy-
-oxo-4H-chromene-2-carboxylic acid (2B) (110 mg, 0.500 mmol)
(
C
H
25 26
N O
2 6
2
1
8
1
3
4
4%): H NMR (300 MHz, DMSO-d
6
) δ 1.19-1.30 (m, 3 H),
.80-1.91 (m, 1 H), 1.99-2.13 (m, 3 H), 2.74-2.84 (m, 2 H),
.00-3.15 (m, 2 H), 3.41 (s, 2 H), 4.01 (d, J ) 11.5 Hz, 1 H),
.21 (d, J ) 4.8 Hz, 2 H), 6.07-6.11 (m, 2 H), 6.96-7.09 (m, 3
H), 7.42 (td, J ) 7.2, 1.5 Hz, 1 H), 7.57-7.61 (m, 1 H), 7.97 (t, J
7.5 Hz, 1 H), 9.08 (d, J ) 7.1 Hz, 1 H), 9.46 (s, 1 H); MS (ESI)
m/z 435 [M + H] , HPLC method A (t
method B (t ) 5.55 min, 100%).
4
and 3-(aminomethyl)-1-N-Boc-pyrrolidine (100 mg, 0.500 mmol)
were processed as described in method E to give (1-N-Boc-
pyrrolidin-3-ylmethyl)-7-methoxy-4-oxo-4H-chromene-2-carboxa-
mide (16A) as a yellow solid (167 mg, 83%). Trifluoroacetic acid
)
+
R
) 5.44 min, 100%),
R
(0.5 mL) was added dropwise to compound 16A (165 mg) and
CH Cl (1 mL) at 0 °C. After 1 h, volatiles were removed, and the
2
2
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-bromo-4-
oxo-4H-chromene-2-carboxamide (20). 3-Bromophenol (5 g, 28.9
mmol), pyridine (5.8 mL, 72 mmol), acetic anhydride (3.0 mL,
31.8 mmol), and dichloromethane (6 mL) were stirred at room
temperature overnight. The solution was washed with water and
resulting yellow oil was triturated with 2 N HCl in ether (1 mL).
The supernatant was removed and the resulting yellow salt was
combined with piperonal (65 mg, 0.46 mmol) and processed as
detailed in method F to give the title compound 16 (30 mg, 17%
over two steps) as a white solid: 1H NMR (300 MHz, DMSO-d
6
)
2 4
brine, dried (Na SO ), and concentrated to dryness. All of the crude
δ 1.43-1.54 (m, 1 H), 1.83-1.95 (m, 1 H), 2.27-2.39 (m, 2 H),
product was suspended in BF
After 2 h, the solution was allowed to cool to room temperature,
3
-OEt
2
(25 mL) and heated to reflux.
2.39-2.49 (m, 2 H), 2.51-2.57 (m, 2 H), 3.23-3.30 (m, 2 H),
3.47 (s, 2 H), 3.92 (s, 3 H), 5.96 (s, 2 H), 6.73-6.86 (m, 3 H),
7.11 (dd, J ) 9.0, 2.5 Hz, 1 H), 7.17 (d, J ) 2.4 Hz, 1 H), 7.95 (d,
diluted with water (20 mL), extracted with ethyl acetate (3 × 25
2 4
mL), washed with water and brine, dried (Na SO ), and concentrated
J ) 8.8 Hz, 1 H), 9.10 (t, J ) 5.6 Hz, 1 H); MS (ESI, MeOH/
NH OH) m/z 437 [M + H], 459 [M + Na], 435 [M - H]; Anal.
‚0.5H O) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-hydroxy-
-oxo-4H-chromene-2-carboxamide (17). Commercially available
ethyl 7-hydroxy-4-oxo-4H-chromene-2-carboxylate (1.00 g, 4.27
mmol), glacial acetic acid (17 mL), and 6 N aqueous HCl (8.5 mL)
were combined and heated at 80 °C for 16 h before the mixture
was allowed to cool to room temperature. The resulting pale yellow
solid was collected by filtration to give 7-hydroxy-4-oxo-4H-
chromene-2-carboxylic acid (17A) as a pale yellow solid (820 mg,
to dryness to give 1-(4-bromo-2-hydroxyphenyl)ethanone (20A, 5.5
g, 89%). The crude 20A was processed according to general method
B to give 7-bromo-4-oxo-4H-chromene-2-carboxylic acid (20B, 2.1
g, 31%). 1-Benzo[1,3]dioxol-5-ylmethyl-piperidin-4-ylamine (37
mg, 0.12 mmol) and 20B (32 mg, 0.12 mmol) were processed
according to general method E to give the title compound 20 (14
4
(
C
24
H
24
N O
2 6
2
4
mg, 24%): 1H NMR (300 MHz, DMSO-d
) δ 1.63 (qd, 2 H, J )
6
11.9, 3.7 Hz), 1.71-1.87 (m, 2 H), 1.96-2.07 (m, 2 H), 2.83 (d,
2 H, J ) 11.2 Hz), 3.39 (s, 2 H), 3.67-3.86 (m, 1 H), 5.99 (s, 2
H), 6.75 (dd, 1 H, J ) 7.9, 1.5 Hz), 6.82 (s, 1 H), 6.85 (d, 1 H, J
) 7.7 Hz), 6.86 (s, 1 H), 7.72 (dd, 1 H, J ) 8.5, 1.8 Hz), 7.96 (d,

6
578 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
H, J ) 8.4 Hz), 8.06 (d, 1 H, J ) 1.8 Hz), 8.80 (d, 1 H, J ) 7.9
Lynch et al.
1
carbonate (8.40 g, 53.5 mmol), and DMF (50 mL), and the system
was heated at 90 °C for 20 h. The resultant milky solution was
+
Hz); MS (ESI) m/z 485 [M + H] . Anal. (C23
H21BrN
2
O
5
) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-chloro-4-
oxo-4H-chromene-2-carboxamide (21). 4-Chloro-2-methoxyben-
zoic acid (5 g, 26.8 mmol) was processed as detailed in method C
followed by method B to provide 7-chloro-4-oxo-4H-chromene-
allowed to cool to room temperature; diluted with Et
washed with distilled water, 1 N aqueous NaOH, 1 N aqueous HCl,
and brine; dried (MgSO ); filtered; and concentrated to give methyl
2
O (250 mL);
4
2-methoxy-4-(trifluoromethyl)benzoate (24A) as an off-white solid
(4.40 g, 77%). Compound 24A was combined with 6 M aqueous
NaOH in methanol (25 mL) and heated at 80 °C for 3 h before the
solution was allowed to cool to room temperature and diluted with
6 M aqueous HCl (16 mL) and distilled water. The resulting white
precipitate was collected by filtration to give 2-methoxy-4-
(trifluoromethyl)benzoic acid (24B) as white crystals (3.35 g, 81%).
Compound 24B was processed in a manner analogous to method
C followed by method B to give 4-oxo-7-(trifluoromethyl)-4H-
chromene-2-carboxylic acid (24C) as a white solid (290 mg).
Compound 24C (287 mg, 1.11 mmol) and 4-amino-1-N-Boc-
piperidine (245 mg, 1.22 mmol) were processed as detailed in
method E to give tert-butyl 4-{[(7-trifluoro-4-oxo-4H-chromen-2-
yl)carbonyl]amino}piperidine-1-carboxylate (24D) as an amber solid
(335 mg, 69%). Trifluoroacetic acid (1.5 mL) was added dropwise
2
(
0
-carboxylic acid (21A) as a beige solid (204 mg). Compound 21A
202 mg, 0.899 mmol) and 4-amino-1-N-Boc-piperidine (189 mg,
.944 mmol) were processed as detailed in method E to give tert-
butyl 4-{[(7-chloro-4-oxo-4H-chromen-2-yl)carbonyl]amino}piper-
idine-1-carboxylate (21B) as a yellow solid (240 mg, 66%).
Trifluoroacetic acid (2 mL) was added dropwise to compound 21B
and CH
the residue was diluted with 1 N aqueous HCl (40 mL) and washed
with Et
O (2 × 20 mL). The aqueous layer was combined with
saturated aqueous K CO until basic and extracted with EtOAc (3
20 mL). Organic extracts and washes were combined, washed
with brine, dried (MgSO ), filtered, and concentrated to give
piperidin-4-yl)-7-chloro-4-oxo-4H-chromene-2-carboxamide (21C)
2 2
Cl (4 mL) at 0 °C. After 1 h, volatiles were removed, and
2
2
3
×
4
(
as an amber solid (120 mg, 67%). Compound 21C (1.19 g, 3.88
mmol) and piperonal (583 mg, 3.88 mmol) were processed as
described in method F to provide the title compound 21 (345 mg,
2
1
2
6
to compound 24D (335 mg, 0.760 mmol) and CH
°C. After 1 h, volatiles were removed, and the resulting dark residue
was diluted with CH Cl (40 mL), washed with aqueous K CO
(40 mL) and brine, dried (Na SO ), filtered, and concentrated to
2 2
Cl (3 mL) at 0
0%) as a white solid: 1H NMR (300 MHz, DMSO-d
6
) δ 1.56-
2
2
2
3
.69 (m, 2 H), 1.76-1.84 (m, 2 H), 1.96-2.06 (m, 2 H), 2.79-
.87 (m, 2 H), 3.39 (s, 2 H), 3.72-3.81 (m, 1 H), 5.99 (s, 2 H),
.73-6.77 (m, 1 H), 6.82-6.87 (m, 3 H), 7.59 (dd, J ) 8.5, 2.0
2
4
give (piperidin-4-yl)-7-trifluoromethyl-4-oxo-4H-chromene-2-car-
boxamide (24E) as an amber solid (84 mg, 32%). Compound 24E
(20 mg, 59 µmol) and piperonal (9 mg, 59 µmol) were processed
Hz, 1 H), 7.90 (d, J ) 1.7 Hz, 1 H), 8.04 (d, J ) 8.8 Hz, 1 H),
.81 (d, J ) 7.8 Hz, 1 H); MS (ESI, MeOH/NH OH) m/z 441 [M
H], 439 [M - H]; Anal. (C23 ) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-fluoro-4-
8
+
4
as detailed in method F to give the title compound 24 as a white
1
H21ClN
2
O
5
solid (12 mg, 43%): H NMR (300 MHz, DMSO-d
6
) δ 1.56-
1.70 (m, 2 H), 1.77-1.85 (m, 2 H), 1.97-2.08 (m, 2 H), 2.80-
2.88 (m, J ) 11.5 Hz, 2 H), 3.40 (s, 2 H), 3.74-3.86 (m, 1 H),
5.99 (s, 2 H), 6.73-6.77 (m, 1 H), 6.83-6.89 (m, 3 H), 7.86 (d, J
oxo-4H-chromene-2-carboxamide (22). 4′-Fluoro-2′-hydroxyac-
etophenone (5.00 g, 32.4 mmol) was processed as described in
method B to give ethyl 7-fluoro-4-oxo-4H-chromene-2-carboxylate
) 8.1 Hz, 1 H), 8.19-8.26 (m, 2 H), 8.88 (d, J ) 8.1 Hz, 1 H);
+
(22A) as a white solid (4.6 g, 68%). Compound 22A (2.00 g, 8.47
MS (ESI) m/z 475 [M + H] , 473 [M - H]; HPLC method A (t
) 5.61 min, 100%), method B (t ) 5.68 min, 100%).
R
mmol) and 4-amino-1-N-Boc-piperidine (1.87 g, 9.32 mmol) were
coupled as described in method E to provide tert-butyl 4-{[(7-
fluoro-4-oxo-4H-chromen-2-yl)carbonyl]amino}piperidine-1-car-
boxylate (22B) as a pale yellow solid (2.9 g, 88%). Trifluoroacetic
acid (7.4 mL) was added dropwise to compound 22B (2.90 g, 7.43
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-chloro-4-
oxo-4H-chromene-2-carboxamide (25). Commercially available
6-chloro-4-oxo-4H-chromene-2-carboxylic acid (2.8 g, 12.5 mmol)
was coupled to 4-amino-1-BOC-piperidine (2.5 g, 12.5 mmol) using
general method E to provide tert-butyl 4-{[(6-chloro-4-oxo-4H-
chromen-2-yl)carbonyl]amino}piperidine-1-carboxylate (25A). To
a solution of 25A (2.5 g, 6.1 mmol) in 16 mL of dichloromethane
at 0 °C was added trifluoroacetic acid (8 mL). The mixture was
allowed to slowly warm to room temperature, stirred for 2 h, and
then concentrated to give a yellow oil. The oil was diluted in 95/5
2 2
mmol) and CH Cl (16 mL) at 0 °C. After 1 h, volatiles were
removed, and the resulting yellow residue was triturated with 2 N
HCl in ether. The supernatant was removed, and the resulting yellow
salt (22C) (450 mg, 1.55 mmol) and piperonal (244 mg, 1.63 mmol)
were processed as described in method F to provide the title
1
compound 22 as a white solid (532 mg, 81%): H NMR (300 MHz,
DMSO-d
6
): δ 1.51-1.72 (m, 2 H), 1.72-1.87 (m, 2 H), 1.92-
dichloromethane/methanol and washed with 1 M K
aqueous layer was extracted with additional dichloromethane, and
the organic extracts were dried (Na SO ) and concentrated to
2 3
CO , the
2
3
6
9
.10 (m, 2 H), 2.83 (d, 2 H, J ) 11.8 Hz), 3.39 (s, 2 H), 3.70-
.85 (m, 1 H), 5.99 (s, 2 H), 6.75 (dd, 1 H, J ) 7.9, 1.4 Hz), 6.81-
.87 (m, 3 H), 7.43 (td, 1 H, J ) 8.7, 2.5 Hz), 7.62 (dd, 1 H, J )
2
4
provide 6-chloro-4-oxo-N-piperidin-4-yl-4H-chromene-2-carboxa-
mide (25B). Reductive alkylation of 25B (31 mg, 0.1 mmol) with
piperonal (15 mg, 0.1 mmol) was accomplished by general method
F. Filtration of semipurified material through a plug of silica gel
(2 g, SepPak), followed by rinsing through with 95/5 dichlo-
romethane/methanol and concentration, provided the title compound
.5, 2.5 Hz), 8.11 (dd, 1 H, J ) 8.9, 6.4 Hz), 8.83 (d, 1 H, J ) 8.1
+
Hz); MS (APCI) m/z 425 [M + H] ; Anal. (C23
N.
H21FN
2
O
5
) C, H,
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-cyano-4-
oxo-4H-chromene-2-carboxamide (23). N-[1-(1,3-Benzodioxol-
25: 1H NMR (300 MHz, DMSO-d
5-ylmethyl)piperidin-4-yl]-7-bromo-4-oxo-4H-chromene-2-carbox-
6
) δ 1.65 (m, 2 H), 1.77 (m, 2
amide (20, 10 mg, 0.02 mmol), zinc cyanide (3 mg, 0.025 mmol),
tetrakis(triphenylphosphine) palladium(0) (10 mg, 0.009 mmol), and
DMF (500 µL) were stirred at 130 °C for 16 h. The reaction was
allowed to cool to room temperature, filtered through Celite, and
purified by preparative reversed-phase HPLC as described in the
H), 1.99 (m, 2 H), 2.83 (m, 2 H), 3.39 (s, 2 H), 3.78 (m, 1 H), 5.97
(s, 1 H), 5.99 (s, 2 H), 6.80 (m, 1 H), 6.90 (m, 2 H), 7.82 (d, J )
8.8 Hz, 1 H), 7.97 (m, 2 H), 8.89 (d, J ) 8.1 Hz, 1 H); MS (ESI,
MeOH/NH
4
OH) m/z 441 [M + H], 439 [M - H]; Anal. (C23
21
H -
ClN ‚0.5H
2
O
5
2
O) C, H, N.
general section to give the title compound 23 (1.5 mg, 17%): 1
H
4-Oxo-7-trifluoromethyl-4H-chromene-2-carboxylic Acid (1-
Benzo[1,3]dioxol-5-ylmethyl-piperidin-4-yl)amide (26). 4-Oxo-
7-trifluoromethyl-4H-chromene-2-carboxylic acid (10 mg, 0.039
mmol, synthesized from 4-trifluoromethylphenol according to
general method D) was coupled to 1-benzo[1,3]dioxol-5-ylmeth-
ylpiperidin-4-ylamine (13 mg, 0.055 mmol, 1B) according to
general method E. Purification by preparative reverse phase
chromatography as described in the general section followed by
NMR (300 MHz, Solvent) δ 1.92-1.99 (m, 2 H), 2.24 (s, 2 H),
.16 (d, J ) 2.4 Hz, 1 H), 3.53-3.62 (m, 3 H), 4.21-4.26 (m, 3
H), 6.03 (s, 2 H), 6.92-6.95 (m, 1 H), 6.98-7.02 (m, 3 H), 7.04
s, 1 H), 7.83 (dd, J ) 8.1, 1.4 Hz, 1 H), 8.13 (s, 1 H), 8.28 (d, J
3
(
+
)
8.1 Hz, 1 H); MS (ESI) m/z 432 [M + H] ; HPLC method A
) 5.24 min, 100%), method B (t ) 5.37 min, 100%).
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-trifluoro-
methyl-4-oxo-4H-chromene-2-carboxamide (24). Iodomethane
3.4 mL, 54 mmol) was added to a stirred solution of 2-hydroxy-
-(trifluoromethyl)benzoic acid (5.00 g, 24.3 mmol), potassium
(
t
R
R
partition between dichloromethane and aqueous 1 M K
2 3
CO
(
4
provided the title compound 26 as an off-white solid (15 mg,
1
6
83%): H NMR (300 MHz, DMSO-d ) δ 1.59-1.72 (m, 2 H)

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6579
1
3
5
.74-1.86 (m, 2 H), 1.94-2.10 (m, 2 H), 2.77-2.91 (m, 2 H),
.27-3.30 (m, 3 H), 3.37-3.44 (m, 2 H), 3.69-3.92 (m, 1 H),
.99 (s, 2 H), 6.71-6.80 (m, 1 H), 6.85 (d, J ) 8.1 Hz, 2 H), 6.91
31 (82 mg, 77%) as a white solid: 1H NMR (300 MHz, DMSO-
d ) δ 1.59-1.72 (m, 2 H), 1.75-1.83 (m, 2 H), 1.96-2.07 (m, 2
6
H), 2.83 (d, J ) 11.9 Hz, 2 H), 3.39 (s, 2 H), 3.73-3.83 (m, 1 H),
5.99 (s, 2 H), 6.73-6.77 (m, 1 H), 6.82-6.87 (m, 3 H), 7.51-
7.56 (m, 1 H), 7.75-7.79 (m, 1 H), 7.90 (ddd, J ) 8.7, 7.0, 1.7
Hz, 1 H), 8.05 (dd, J ) 7.8, 1.4 Hz, 1 H), 8.88 (d, J ) 7.8 Hz, 1
(
s, 1 H), 7.99 (d, J ) 8.5 Hz, 1 H), 8.21-8.33 (m, 2 H), 8.95 (d,
J ) 8.1 Hz, 1 H); MS (ESI, MeOH/NH OH) m/z 475 [M + H],
73 [M - H], Anal. (C24 ‚0.5H O) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-bromo-4-
oxo-4H-chromene-2-carboxamide (27). Commercially available
-bromochromone-2-carboxylic acid (71 mg, 0.263 mmol) was
4
4
H
21
F N
3 2
O
5
2
+
H); MS (APCI) m/z 407 [M + H] , 405 [M - H]; analytical HPLC
method A (t
R
) 5.08 min, 100%), method B (t ) 5.24 min, 100%).
R
6
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-5-chloro-4-
oxo-4H-chromene-2-carboxamide (32). 3-Chlorophenol (10.0 g,
77.8 mmol) was processed as detailed in method D to obtain a
mixture of 5-chloro-2-carboxychromone and 7-chloro-2-carboxy-
chromone (2.36 g). This 2-carboxychromone mixture (0.224 mg,
1.13 mmol) and 4-amino-1-N-Boc-piperidine (340 mg, 1.70 mmol)
were processed according to method E to provide the 5-chloro-
chromone-2-carboxamides and 7-chlorochromone-2-carboxamides
(228 mg, 50%). Trifluoroacetic acid (1 mL) was added to the
coupled to 1-(1,3-benzodioxol-5-ylmethyl)piperidin-4-amine dihy-
drochoride (162 mg, 0.526 mmol) as described in method E to give
the title compound 27 (70 mg, 55%) as a white solid: 1H NMR
(
6
300 MHz, DMSO-d ) δ 1.60-1.70 (m, 2 H), 1.75-1.82 (m, 2
H), 1.96-2.05 (m, 2 H), 2.79-2.86 (m, 2 H), 3.39 (s, 2 H), 3.72-
3
3
2
.83 (m, 1 H), 5.99 (s, 2 H), 6.73-6.77 (m, 1 H), 6.83-6.86 (m,
H), 7.75 (d, J ) 9.2 Hz, 1 H), 8.04-8.08 (m, 1 H), 8.11 (d, J )
.7 Hz, 1 H), 8.89 (d, J ) 7.8 Hz, 1 H); MS (APCI) m/z 485, 487
+
[
M + H] , 483, 485 [M - H]; analytical HPLC method A (t
R
)
carboxamides (224 mg, 0.551 mmol) in CH
After 1 h, the solution was diluted with CH Cl
2
Cl
2
(2 mL) at 0 °C.
5
.47 min, 100%), method B (t
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-methyl-4-
oxo-4H-chromene-2-carboxamide (28). Commercially available
-methylchromone-2-carboxylic acid (54 mg, 0.263 mmol) was
R
) 5.57 min, 98.3%).
2
2
(40 mL) and Et O
2
(15 mL) and extracted with 1 N aqueous HCl (2 × 30 mL). The
aqueous extracts were combined and treated with aqueous K
until basic (pH 10). The aqueous layer was extracted with CH
2
CO
Cl
3
6
2
2
coupled to 1-(1,3-benzodioxol-5-ylmethyl)piperidin-4-amine dihy-
drochoride (162 mg, 0.526 mmol) as described in method E to give
the title compound 28 (56 mg, 51%) as clear, colorless crystals:
(3 × 40 mL); the organic extracts were combined, dried (MgSO
4
),
filtered, and concentrated to a fine yellow solid (83 mg, 49%). The
yellow solid (80 mg, 0.261 mmol) was combined with piperonal
(43 mg, 0.287 mmol) and processed according to method F to obtain
a mixture of regioisomeric products. Purification by preparative
reversed-phase HPLC as described in the general section provided
1
H NMR (300 MHz, DMSO-d
6
) δ 1.61-1.71 (m, 2 H), 1.75-1.82
(
3
m, 2 H), 1.96-2.04 (m, 2 H), 2.44 (s, 3 H), 2.80-2.86 (m, 2 H),
.39 (s, 2 H), 3.71-3.84 (m, 1 H), 5.99 (s, 2 H), 6.73-6.86 (m, 4
title compound 32 (10 mg, 9%): 1H NMR (300 MHz, DMSO-d
)
H), 7.65-7.73 (m, 2 H), 7.83 (br. s, 1 H), 8.85 (d, J ) 7.8 Hz, 1
6
+
H); MS (APCI) m/z 421 [M + H] , 420 [M - H]; analytical HPLC
δ 1.56-1.71 (m, 2 H), 1.72-1.83 (m, 2 H), 1.94-2.05 (m, 2 H),
2.78-2.87 (m, 3.39 Hz, 2 H), 3.39 (s, 2 H), 3.73-3.82 (m, 1 H),
5.99 (s, 2 H), 6.73-6.79 (m, 2 H), 6.81-6.89 (m, 2 H), 7.54 (dd,
J ) 7.8, 1.4 Hz, 1 H), 7.72-7.76 (m, 1 H), 7.78-7.83 (m, 1 H),
method A (t
R
) 5.26 min, 100%), method B (t ) 5.40 min, 100%).
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-trifluoro-
methyl-4-oxo-4H-chromene-2-carboxamide (29). 6-Trifluoro-
methoxychromone-2-carboxylic acid (29 mg, 0.106 mmol) and
+
8.85 (d, J ) 7.5 Hz, 1 H); MS (APCI) m/z 441 [M + H] ; analytical
1
-(1,3-benzodioxol-5-ylmethyl)piperidin-4-amine dihydrochoride
HPLC method A (t
min, 98.9%).
R
) 5.29 min, 100%), method B (t ) 5.41
R
(
21 mg, 0.0683 mmol) were processed as described in method E
1
to give the title compound 29 (16 mg, 20%): H NMR (500 MHz,
pyridine-d
2
1
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-chloro-3-
methyl-4-oxo-4H-chromene-2-carboxamide (33). 7-Chloro-3-
methyl-4-oxo-4H-chromene-2-carboxylic acid 33A was prepared
from 2-methoxy-4-chlorobenzoic acid as described in general
method C, substituting ethylmagnesium bromide for methylmag-
nesium bromide, followed by method B. Compound 33A (480 mg,
2 mmol) and 1-benzo[1,3]dioxol-5-ylmethylpiperidin-4-ylamine
(680 mg, 2.2 mmol) were processed as detailed in method E to
5
) δ 2.00-2.09 (m, 2 H), 2.11-2.17 (m, 2 H), 2.30-
.39 (m, 2 H), 3.09-3.17 (m, 2 H), 3.67 (s, 2 H), 4.31-4.39 (m,
H), 6.00 (s, 2 H), 6.87-6.90 (m, 3 H), 7.07 (s, 1 H), 7.45-7.51
(
(
m, 2 H), 8.16 (d, J ) 1.9 Hz, 1 H), 9.80 (d, J ) 7.8 Hz, 1 H); MS
+
APCI) m/z 491 [M + H] , 489 [M - H].
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-fluoro-4-
oxo-4H-chromene-2-carboxamide (30). Commercially available
-fluoro-4-oxo-4H-chromene-2-carboxylic acid (2.8 g, 12.5 mmol)
6
provide the title compound 33 as a white powder (630 mg, 69%):
1
was coupled to 4-amino-1-BOC-piperidine (2.5 g, 12.5 mmol) using
general method E to provide 4-[(6-fluoro-4-oxo-4H-chromene-2-
carbonyl)amino]piperidine-1-carboxylic acid tert-butyl ester (30A).
A solution of 30A (2.5 g, 6.4 mmol) in 18 mL of dichloromethane
at 0 °C was treated with 9 mL of trifluoroacetic acid, and the
mixture was stirred for 2 h and then concentrated to give an orange
oil. This oil was diluted with 95/5 dichloromethane/methanol and
6
H NMR (500 MHz, DMSO-d ) δ 1.54 (dq, J ) 11.6, 3.3 Hz, 2
H), 1.80 (d, J ) 11.6 Hz, 2 H), 2.01 (t, J ) 11.6 Hz, 2 H), 2.09 (s,
3H), 2.78 (d, J ) 11.6 Hz, 2 H), 3.37 (s, 2 H), 3.71-3.77 (m, 1
H), 5.97 (s, 2 H), 6.73 (dd, J ) 7.9, 1.5 Hz, 1 H), 6.82-6.84 (m,
2 H), 7.54 (dd, J ) 8.6, 1.8 Hz, 1 H), 7.90 (d, J ) 1.8 Hz, 1 H),
8.03 (d, J ) 8.6 Hz, 1 H), 8.83 (d, J ) 7.6 Hz, 1 H); MS (ESI,
MeOH/NH
method A (t
4
OH) m/z 455 [M + H], 453 [M - H]; analytical HPLC
) 5.55 min, 100%), method B (t ) 5.65 min, 100%).
washed with 1 M K
with additional dichloromethane, and the combined organic extracts
were dried (Na SO ), filtered, and concentrated under reduced
2 3
CO . The aqueous washes were back-extracted
R
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7-chloro-6-
fluoro-4-oxo-4H-chromene-2-carboxamide (34). 3-Chloro-4-fluo-
rophenol was processed according to general procedures A and B
to give 7-chloro-6-fluoro-4-oxo-4H-chromene-2-carboxylic acid
(34A). Compound 34A (306 mg, 1.26 mmol) and 1-benzo[1,3]-
dioxol-5-ylmethylpiperidin-4-ylamine dihydrochloric acid salt (407
mg, 1.32 mmol) were coupled using general method E to give the
2
4
pressure to provide 4-[(6-fluoro-4-oxo-4H-chromene-2-carbonyl)-
amino]piperidine (30B, 1.4 g, 75%) Reductive alkylation of 30B
(29 mg, 0.1 mmol) with piperonal (15 mg, 0.1 mmol) according to
general procedure F followed by flash silica gel chromatography
eluting with dichloromethane/methanol provided the title compound
0 (33 mg, 79%): 1H NMR (300 MHz, DMSO-d
) δ 1.64 (m, 2
H), 1.79 (m, 2 H), 1.99 (m, 2 H), 2.85 (m, 2 H), 3.39 (s, 2 H), 3.77
m, 1 H), 5.97 (s, 1 H), 5.99 (s, 2 H), 6.75 (m, 2 H), 6.85 (m, 3 H),
.74 (m, 1 H), 7.85 (m, 1 H), 8.88 (d, 1 H); MS (ESI, MeOH/
NH OH) m/z 425 [M + H], 423 [M - H], 459 [M + Cl]; Anal.
‚0.25H O) C, H, N.
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-4-oxo-4H-
chromene-2-carboxamide (31). Commercially available chromone-
-carboxylic acid (50 mg, 0.263 mmol) was coupled to 1-(1,3-
benzodioxol-5-ylmethyl)piperidin-4-amine dihydrochoride (162 mg,
.526 mmol) as described in method E to give the title compound
title compound 34 (400 mg, 69%): H NMR (300 MHz, DMSO-
1
3
6
6
d ) δ 1.60 (m, 2H), 1.80 (m, 2H), 2.00 (m, 2H), 2.80(m, 2H), 3.40
(
7
(s, 2H), 3.80 (m, 1H), 6.00 (s, 2H), 6.70-6.90 (m, 4H), 7.95 (d,
+
1H), 8.10 (d, 1H), 8.80 (d, 1H); MS (ESI) m/z 459[M + H] ;
4
analytical HPLC method A (t
) 5.60 min, 92.0%).
R
) 5.51 min, 100%), method B (t
R
(C
H21FN O
23 2 5
2
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6-fluoro-7-
methoxy-4-oxo-4H-chromene-2-carboxamide (35). A mixture of
2
1-(4,5-difluoro-2-hydroxyphenyl)ethanone (4.00 g, 23.2 mmol), K
CO (3.84 g, 27.8 mmol), and MOM-Cl (2.12 mL, 27.8 mmol) in
acetone (30 mL) was stirred at room temperature for 16 h. The
2
-
3
0

6
580 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
reaction was then quenched by the addition of H
layers were separated, and the aqueous layer was extracted with
additional Et
O (3 × 20 mL). The combined organic layers were
dried (Na SO ), filtered, and concentrated under reduced pressure.
2
O and Et
2
O. The
general methods A (skip acetylation part of procedure) and B to
give 6,8-difluoro-4-oxo-4H-chromene-2-carboxylic acid (37A, 0.91
g, 69%). Compound 37A (25 mg, 0.11 mmol) was coupled to
1-(1,3-benzodioxol-5-ylmethyl)piperidin-4-amine dihydrochloride
2
2
4
The residue was purified by MPLC (10% EtOAc in hexane to 35%
EtOAc in hexane) to yield 1-(4,5-difluoro-2-methoxymethoxyphe-
nyl)ethanone (35A) as a colorless oil (3.75 g, 75%). To a solution
of compound 35A (3.00 g, 13.9 mmol) in MeOH (30 mL) was
added solid NaOMe (11.3 g, 209 mmol) in three portions. The
resulting solution was heated at 40 °C for 8 h. The cooled reaction
(34 mg) using general method E to give the title compound 37 (12
1
3
mg, 25%): H NMR (300 MHz, CD OD) δ 1.28-1.30 (m, 1 H),
1.75 (dd, J ) 11.9, 3.4 Hz, 1 H), 1.92 (d, J ) 4.4 Hz, 2 H), 2.14-
2.23 (m, 2 H), 2.97 (d, J ) 14.2 Hz, 2 H), 3.49 (s, 2 H), 3.90 (td,
J ) 10.5, 5.4 Hz, 1 H), 5.93 (s, 2 H), 6.75-6.79 (m, 2 H), 6.87 (s,
1 H), 6.97 (s, 1 H), 7.59-7.69 (m, 3 H); MS (APCI) m/z 443 [M
+
was then quenched by addition of H
separated, and the aqueous was extracted with additional EtOAc.
The combined organic layers were dried (Na SO ), filtered, and
concentrated under reduced pressure to yield 1-(5-fluoro-4-methoxy-
-methoxymethoxyphenyl)ethanone (35B) as an oil (2.97 g, 94%)
2
O and EtOAc. The layers were
+ H] ; analytical HPLC method A (t
R
) 5.29 min, 100%), method
B (t ) 5.40 min, 100%).
R
2
4
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-5,6-dichloro-
4-oxo-4H-chromene-2-carboxamide (38). 3,4-Dichlorophenol (20.0
g, 123 mmol) was processed as detailed in method D to provide a
mixture of dichloro-2-carboxychromone isomers (38A) (16.8 g,
53%). Compound 38A (100 mg, 0.386 mmol) and 1-(1,3-benzo-
dioxol-5-ylmethyl)piperidin-4-amine dihydrochoride (237 mg, 0.772
mmol) were processed as described in method E. The title
compound 38 (9 mg, 5%) was separated from the undesired isomer
through preparative reversed-phase HPLC as described in the
2
that was used without further purification. To an ambient solution
of compound 35B (2.97 g, 13.03 mmol) in MeOH (11 mL) and
H
2
O (11 mL) was added TFA (7.28 mL). The reaction was stirred
at room temperature for 16 h and was then diluted with H O and
Et O. The layers were separated, and the aqueous layer was
extracted with additional Et
O (3 × 25 mL). The combined organic
layers were dried (Na SO ), filtered, and concentrated under reduced
pressure. The residue was purified on SiO gel by MPLC (15%
EtOAc in hexane to 50% EtOAc in hexane) to yield 1-(5-fluoro-
-methoxy-2-hydroxyphenyl)ethanone (35C) (1.61 g, 67%). Com-
2
2
2
general section: 1H NMR (300 MHz, DMSO-d ) δ 1.60-1.70 (m,
2
4
6
2
2 H), 1.75-1.82 (m, 2 H), 1.95-2.05 (m, 2 H), 2.79-2.86 (m, 2
H), 3.39 (s, 2 H), 3.71-3.84 (m, 1 H), 5.99 (s, 2 H), 6.73-6.77
(m, 1 H), 6.80 (s, 1 H), 6.85 (d, J ) 7.8 Hz, 2 H), 7.78 (d, J ) 9.2
Hz, 1 H), 8.10 (d, J ) 9.2 Hz, 1 H), 8.87 (d, J ) 7.8 Hz, 1 H); MS
4
pound 35C (1.61 g, 8.75 mmol) was processed according to method
B to give 6-fluoro-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid
(ESI, MeOH/NH OH) m/z 475 [M + H], 473 [M - H]; analytical
4
(35D) as a beige solid (1.50 g, 72%). Compound 35D (1.0 g, 4.20
HPLC method A (t ) 5.59 min, 100%), method B (t ) 5.68
R
R
mmol) and 1-(1,3-benzodioxol-5-ylmethyl)piperidin-4-amine di-
hydrochloride (1.61 g, 5.25 mmol) were processed in a manner
similar to method E to give the title compound 35 as a white solid
min, 98.4%).
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6,7,8-trifluoro-
4-oxo-4H-chromene-2-carboxamide (39). 6,7,8-Trifluoro-4-oxo-
4H-chromene-2-carboxylic acid (39A) was prepared from com-
mercially available 2,3,4-trifluorophenol as detailed in method D.
Compound 39A (75 mg, 0.30 mmol) and 1-benzo[1,3]dioxol-5-
ylmethylpiperidin-4-ylamine (99 mg, 0.32 mmol) were coupled as
detailed in method E to provide the title compound 39 as a tan
(
572 mg, 45%) after purification of the crude residue by recrys-
1
3 6
tallization from hot CH CN: H NMR (300 MHz, DMSO-d ) δ
1
2
4
.53-1.72 (m, 2 H), 1.72-1.87 (m, 2 H), 1.92-2.10 (m, 2 H),
.84 (d, 2 H, J ) 11.2 Hz), 3.39 (s, 2 H), 3.68-3.87 (m, 1 H),
.02 (s, 3 H), 5.99 (s, 2 H), 6.75 (dd, 1 H, J ) 7.9, 1.6 Hz), 6.78
1
(
1
s, 1 H), 6.83-6.87 (m, 2 H), 7.46 (d, 1 H, J ) 7.0 Hz), 7.73 (d,
powder (94 mg, 67%): H NMR (500 MHz, DMSO-d ) δ 1.65
6
H, J ) 10.9 Hz), 8.82 (d, 1 H, J ) 8.0 Hz); MS (ESI) m/z 455
(dq, J ) 11.6, 3.4 Hz, 2 H), 1.77 (d, J ) 11.6 Hz, 2 H), 2.00 (t, J
) 11.6 Hz, 2 H), 2.81 (d, J ) 11.6 Hz, 2 H), 3.38 (s, 2 H), 3.71-
3.80 (m, 1 H), 5.99 (s, 2 H), 6.74 (d, J ) 7.9 Hz, 1 H), 6.83-6.85
(m, 2 H), 6.93 (s, 1H), 7.88 (t, J ) 7.9, 1 H), 8.85 (d, J ) 7.9 Hz,
+
[
M + H] ; analytical HPLC method A (t ) 5.29 min, 100%),
R
method B (t ) 5.40 min, 100%).
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-6,7-difluoro-
-oxo-4H-chromene-2-carboxamide (36). Employing method A,
,4-difluorophenol (5 g, 38.5 mmol) was converted to 4′,5′-difluoro-
′-hydroxyacetophenone (36A), an off-white solid (4.23 g, 64%).
4
3
2
1 H); MS (ESI, MeOH/NH
analytical HPLC method A (t
) 5.53 min, 100%).
4
OH) m/z 461 [M + H], 459 [M - H];
) 5.42 min, 100%), method B (t
R
R
Compound 36A (1.39 g, 8.07 mmol) was converted to 6,7-difluoro-
-carboxychromone (36B), an off-white solid (1.04 g, 57%), though
method B. Compound 36B (1.03 g, 4.55 mmol) and 4-amino-N-
-Boc-piperidine (1.00 g, 5.01 mmol) were coupled as described
in method E to give tert-butyl 4-{[(6,7-difluoro-4-oxo-4H-chromen-
-yl)carbonyl]amino}piperidine-1-carboxylate (36C) as an off-white
solid (900 mg, 48%). Trifluoroacetic acid (3.5 mL) was added
dropwise to compound 36C (0.9 g, 2.2 mmol) and CH Cl (7 mL)
at 0 °C. After 1 h, volatiles were removed and the resulting orange
residue was diluted with CH Cl (100 mL), washed with aqueous
CO
(1 × 100 mL) and with brine (1 × 100 mL), dried (Na
SO ), filtered, and concentrated to N-[1-(1,3-benzodioxol-5-ylm-
N-[1-Benzylpiperidin-4-yl]-7-fluoro-4-oxo-4H-chromene-2-
carboxamide (40). Compound 22C (30 mg, 0.103 mmol) and
benzaldehyde (16 mg, 0.155 mmol) were processed in a manner
analogous to method F to provide the title compound 40 (32 mg,
2
1
82%): 1H NMR (500 MHz, pyridine-d
) δ 1.80-1.88 (m, 2 H),
5
2
2.01-2.08 (m, 4 H), 2.81-2.86 (m, 2 H), 3.40 (s, 2 H), 4.20-
4.29 (m, 1 H), 6.48 (dd, J ) 9.4, 2.2 Hz, 1 H), 7.03-7.08 (m, 1
H), 7.11-7.16 (m, 2 H), 7.26-7.31 (m, 1 H), 7.44 (s, 1 H), 8.28
(dd, J ) 8.9, 6.4 Hz, 1 H), 9.52 (d, J ) 7.5 Hz, 1 H); MS (ESI,
2
2
2
2
MeOH/NH
method A (t
4
OH) m/z 381 [M + H], 379 [M - H]; analytical HPLC
) 5.14 min, 100%), method B (t ) 5.26 min, 100%).
K
2
3
2
-
R
R
4
N-[1-(1,3-Benzodioxol-4-ylmethyl)piperidin-4-yl]-7-fluoro-4-
oxo-4H-chromene-2-carboxamide (41). 2,3-(Methylenedioxy)-
benzaldehyde (13 mg, 0.087 mmol) and compound 22C (19 mg,
0.058 mmol) were processed in a manner analogous to method F
to provide the title compound 41 as the TFA salt after purification
by preparative reversed-phase HPLC as described in the general
ethyl)piperidin-4-yl]-6,7-difluoro-4-oxo-4H-chromene-2-carboxa-
mide (36D) as an orange solid (200 mg, 29%). Compound 36D
(140 mg, 0.454 mmol) and piperonal (75 mg, 0.500 mmol) were
processed as detailed in method F to provide the title compound
1
3
6 (24 mg, 12%) as a pale orange solid: H NMR (300 MHz,
section (16 mg, 47%): 1H NMR (500 MHz, pyridine-d ) δ 2.22-
DMSO-d ) δ 1.52-1.71 (m, 2 H), 1.73-1.87 (m, 2 H), 1.94-2.10
6
5
(
(
(
m, 2 H), 2.76-2.92 (m, 2 H), 3.35-3.46 (m, 2 H), 3.67-3.82
2.27 (m, 2 H), 2.28-2.36 (m, 2 H), 2.52-2.60 (m, 2 H), 3.26-
3.32 (m, J ) 12.2 Hz, 2 H), 3.88 (s, 2 H), 4.29-4.36 (m, 1 H),
6.09 (s, 2 H), 6.91-7.00 (m, 2 H), 7.09 (d, J ) 7.8 Hz, 1 H),
7.33-7.35 (m, 2 H), 7.65 (dd, J ) 9.2, 2.3 Hz, 1 H), 8.31 (dd, J
4
m, 1 H), 5.99 (s, 2 H), 6.77 (s, 1 H), 6.81-6.92 (m, 3 H), 7.91
dd, J ) 10.5, 6.4 Hz, 1 H), 7.95-8.05 (m, 1 H), 8.84 (d, J ) 8.1
+
Hz, 1 H); MS (ESI, MeOH/NH
4
OH) m/z 443 [M + H] , 441 [M
+
-
H] ; analytical HPLC method A (t
R
) 5.34 min, 100%), method
) 8.9, 6.4 Hz, 1 H); MS (ESI, MeOH/NH OH) m/z 425 [M +
+
B (t ) 5.42 min, 97.0%).
R
H] , 423 [M - H]; analytical HPLC method A (t ) 5.20 min,
R
N-[1-(1,3-Benzodioxol-5-ylmethyl)piperidin-4-yl]-7,8-difluoro-
-oxo-4H-chromene-2-carboxamide (37). Commercially available
R
100%), method B (t ) 5.33 min, 100%).
4
N-[1-[(2,2-Difluoro-1,3-benzodioxol-5-yl)methyl]piperidin-4-
yl]-7-fluoro-4-oxo-4H-chromene-2-carboxamide (42). Compound
acetic acid 2,4-difluorophenyl ester was processed according to

Chromone-2-carboxamide MCHr1 Antagonists
2C (29 mg, 0.10 mmol) and 2,2-difluoro-1,3-(methylenedioxy)-
benzaldehyde (19 mg, 0.10 mmol) were processed in a manner
analogous to method F to provide the title compound 42 as a white
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6581
2
7.10-7.15 (m, 1 H), 7.31 (d, J ) 8.7 Hz, 2 H), 7.44 (s, 1 H), 8.28
(dd, J ) 8.7, 6.2 Hz, 1 H), 9.55 (d, J ) 7.5 Hz, 1 H); MS (ESI,
+
MeOH/NH
method A (t
4
OH) m/z 411 [M + H] , 409 [M - H]; analytical HPLC
1
solid: H NMR (300 MHz, DMSO-d
1
6
) δ 1.58-1.71 (m, 2 H),
R
) 5.21 min, 100%), method B (t ) 5.31 min, 98.1%).
R
.76-1.85 (m, 2 H), 1.99-2.10 (m, 2 H), 2.84 (d, J ) 11.2 Hz, 2
N-[1-[(2-Fluorobenzyl)methyl]piperidin-4-yl]-7-fluoro-4-oxo-
4H-chromene-2-carboxamide (48). Compound 22C (19 mg, 0.058
mmol) and 2-fluorobenzaldehyde (11 mg, 0.087 mmol) were
processed in a manner analogous to method F to provide the title
compound 48 as the TFA salt after purification by preparative
H), 3.48-3.52 (m, 2 H), 3.74-3.82 (m, 1 H), 6.82 (s, 1 H), 7.14
(
2
dd, J ) 8.1, 1.4 Hz, 1 H), 7.32-7.37 (m, 2 H), 7.43 (td, J ) 8.7,
.7 Hz, 1 H), 7.61 (dd, J ) 9.5, 2.4 Hz, 1 H), 8.11 (dd, J ) 9.0,
.3 Hz, 1 H), 8.83 (d, J ) 7.8 Hz, 1 H); MS (APCI, MeOH/NH
6
4
-
+
1
OH) m/z 461 [M + H] , 459 [M - H].
reversed-phase HPLC (7 mg, 20% yield): H NMR (500 MHz,
N-{1-[(6-Chloro-1,3-benzodioxol-5-yl)methyl]piperidin-4-yl}-
-fluoro-4-oxo-4H-chromene-2-carboxamide (43). Compound
2C (19 mg, 0.065 mmol) and 6-chloropiperonal (12 mg, 0.065
5
pyridine-d ) δ 2.23 (s, 6 H), 3.10 (s, J ) 11.5 Hz, 2 H), 3.74 (s, 2
7
2
H), 4.20-4.33 (m, 1 H), 7.19-7.25 (m, 1 H), 7.27 (t, J ) 7.5 Hz,
1 H), 7.33-7.37 (m, 2 H), 7.41-7.48 (m, 1 H), 7.54-7.60 (m, 1
mmol) were processed according to general method F to provide
the title compound 43 (14.5 mg, 39%) as the trifluoroacetic acid
salt after purification by preparative reversed-phase HPLC as
described in the general section The TFA salt was dissolved in
H), 7.66 (dd, J ) 9.2, 2.3 Hz, 1 H), 8.31 (dd, J ) 9.1, 6.2 Hz, 1
+
H); MS (APCI, MeOH/NH
4
OH) m/z 399 [M + H] , 397 [M -
H]; analytical HPLC method A (t
(t ) 5.27 min, 100%).
N-[1-[(3-Fluorobenzyl)methyl]piperidin-4-yl]-7-fluoro-4-oxo-
R
) 5.15 min, 97.3%), method B
R
dichloromethane, washed with 1 M K
concentrated to give the free base for elemental analysis: H NMR
300 MHz, DMSO-d ) δ 1.57-1.72 (m, 2 H), 1.76-1.86 (m, 2
2 3 2 4
CO , dried (Na SO ), and
1
4H-chromene-2-carboxamide (49). Compound 22C (30 mg, 0.103
mmol) and 3-fluorobenzaldehyde (19 mg, 0.155 mmol) were
processed in a manner analogous to method F to provide the title
(
6
H), 2.07-2.19 (m, 2 H), 2.85 (d, 2 H, J ) 11.5 Hz), 3.48 (s, 2 H),
compound 49 (28 mg, 68%): 1H NMR (300 MHz, DMSO-d
3
7
9
.74-3.88 (m, 1 H), 6.06 (s, 2 H), 6.83 (s, 1 H), 7.01 (s, 1 H),
6
) δ
.05 (s, 1 H), 7.43 (td, 1 H, J ) 8.7, 2.5 Hz), 7.61 (dd, 1 H, J )
1.66 (qd, J ) 11.9, 3.9 Hz, 2 H), 1.76-1.86 (m, 2 H), 2.02-2.12
(m, 2 H), 2.84 (d, J ) 11.9 Hz, 2 H), 3.52 (s, 2 H), 3.73-3.85 (m,
1 H), 6.82 (s, 1 H), 7.04-7.17 (m, 3 H), 7.33-7.46 (m, 2 H), 7.61
(dd, J ) 9.5, 2.4 Hz, 1 H), 8.11 (dd, J ) 8.8, 6.4 Hz, 1 H), 8.83
.5, 2.5 Hz), 8.12 (dd, 1 H, J ) 8.9, 6.4 Hz), 8.83 (d, 1 H, J ) 7.9
+
Hz); MS (ESI, MeOH/NH
H] ; Anal. (C23
4
OH) m/z 459 [M + H] , 457 [M -
5
+
2
H20ClFN O
2
‚H O) C, H, N.
N-[1-[(2,3-Dihydro-1,4-benzodioxin-6-yl)methyl]piperidin-4-
yl]-7-fluoro-4-oxo-4H-chromene-2-carboxamide (44). Compound
(d, J ) 7.8 Hz, 1 H); MS (ESI, MeOH/NH
4
OH) m/z 399 [M +
H] , 397 [M - H]; analytical HPLC method A (t ) 5.24 min,
100%), method B (t ) 5.34 min, 100%).
+
R
22C (29 mg, 0.10 mmol) and 1,4-benzodioxan-6-carbaldehyde (17
R
mg, 0.10 mmol) were processed in a manner analogous to method
F to provide the title compound 44 as a white solid (17 mg, 31%):
N-[1-[(4-Fluorobenzyl)methyl]piperidin-4-yl]-7-fluoro-4-oxo-
4H-chromene-2-carboxamide (50). Compound 22C (19 mg, 0.058
mmol) and 4-fluorobenzaldehyde (11 mg, 0.087 mmol) were
processed in a manner analogous to method F to provide the title
compound as a TFA salt after purification by preparative reversed-
1
H NMR (300 MHz, DMSO-d ) δ 1.55-1.70 (m, 2 H), 1.75-1.83
6
(
3
4
m, 2 H), 1.95-2.04 (m, 2 H), 2.79-2.86 (m, 2 H), 3.36 (s, 2 H),
.72-3.82 (m, J ) 8.1, 4.1 Hz, 1 H), 4.20 (s, 4 H), 6.72-6.82 (m,
H), 7.43 (td, J ) 8.7, 2.4 Hz, 1 H), 7.61 (dd, J ) 9.5, 2.4 Hz, 1
1
phase HPLC (16 mg, 47%): H NMR (500 MHz, solvent) δ 2.26-
H), 8.11 (dd, J ) 8.8, 6.4 Hz, 1 H), 8.82 (d, J ) 7.8 Hz, 1 H); MS
2.35 (m, 4 H), 2.59-2.67 (m, 2 H), 3.27-3.33 (m, J ) 12.2 Hz,
2 H), 3.94 (s, 2 H), 4.35-4.42 (m, 1 H), 7.20 (t, J ) 8.7 Hz, 2 H),
+
(
APCI, MeOH/NH
analytical HPLC method A (t
5.32 min, 96.02%).
N-[1-[(3,4-Dimethoxybenzyl)methyl]piperidin-4-yl]-7-fluoro-
-oxo-4H-chromene-2-carboxamide (45). Compound 22C (22.5
4
OH) m/z 439 [M + H] , 437 [M - H];
R
) 5.23 min, 100%), method B (t
R
7.33-7.35 (m, 2 H), 7.60-7.66 (m, 3 H), 8.31 (dd, J ) 8.9, 6.4
+
)
Hz, 1 H); MS (APCI, MeOH/NH
+ Cl]; analytical HPLC method A (t
B (t ) 5.33 min, 100%).
4
OH) m/z 399 [M + H] , 434 [M
R
) 5.24 min, 100%), method
4
R
mg, 0.0681 mmol) and 3,4-dimethoxybenzaldehyde (19 mg, 0.102
mmol) were processed in a manner analogous to method F to
provide the title compound 45 (32 mg, 74%): H NMR (500 MHz,
N-[1-[(2-Fluoro-4-methoxybenzyl)methyl]piperidin-4-yl]-7-
fluoro-4-oxo-4H-chromene-2-carboxamide (51). Compound 22C
(29 mg, 0.10 mmol) and 2-fluoro-4-methoxybenzaldehyde (16 mg,
1
pyridine-d
2
4
5
) δ 1.86-1.95 (m, 2 H), 2.03-2.12 (m, 4 H), 2.92-
.98 (m, 2 H), 3.44 (s, 2 H), 3.72 (s, 3 H), 3.77-3.79 (m, 3 H),
.29 (m, 1 H), 6.44 (dd, J ) 9.3, 2.3 Hz, 1 H), 6.92-6.98 (m, 2
0.10 mmol) were processed in a manner analogous to method F to
1
provide the title compound as a white solid (25 mg, 58%):
NMR (300 MHz, DMSO-d
H
6
) δ 1.54-1.68 (m, 2 H), 1.75-1.83
H), 7.15 (td, J ) 8.5, 2.1 Hz, 1 H), 7.46 (s, 1 H), 8.29 (dd, J )
(m, 2 H), 1.99-2.10 (m, 2 H), 2.79-2.87 (m, 2 H), 3.46 (s, 2 H),
3.73 (m, 1 H), 3.76 (s, 3 H), 6.74-6.84 (m, 3 H), 7.28 (t, J ) 8.8
Hz, 1 H), 7.43 (td, J ) 8.7, 2.4 Hz, 1 H), 7.61 (dd, J ) 9.5, 2.4
8
NH
method A (t
.9, 6.4 Hz, 1 H), 9.65 (d, J ) 7.9 Hz, 1 H); MS (ESI, MeOH/
4
+
OH) m/z 441 [M + H] , 439 [M - H]; analytical HPLC
) 5.12 min, 100%), method B (t ) 5.21 min, 97.8%).
R
R
Hz, 1 H), 8.11 (dd, J ) 8.8, 6.4 Hz, 1 H), 8.82 (d, J ) 7.8 Hz, 1
+
N-[1-[(3-Methoxybenzyl)methyl]piperidin-4-yl]-7-fluoro-4-
oxo-4H-chromene-2-carboxamide (46). Compound 22C (30 mg,
H); MS (APCI, MeOH/NH
4
OH) m/z 429 [M + H] , 427 [M -
H]; analytical HPLC method A (t
(t ) 5.36 min, 95.1%).
N-[1-[(3-Fluoro-4-methoxybenzyl)methyl]piperidin-4-yl]-7-
R
) 5.29 min, 100%), method B
0.103 mmol) and 3-methoxybenzaldehyde (21 mg, 0.155 mmol)
R
were processed in a manner analogous to method F to provide the
title compound 46 (35 mg, 83%): 1H NMR (500 MHz, pyridine-
fluoro-4-oxo-4H-chromene-2-carboxamide (52). Compound 22C
d
5
) δ 1.81-1.90 (m, 2 H), 2.02-2.09 (m, 4 H), 2.88-2.93 (m, 2
H), 3.45 (s, 2 H), 3.68 (s, 3 H), 4.21-4.29 (m, 1 H), 6.45 (dd, J )
.4, 2.2 Hz, 1 H), 6.93 (dd, J ) 8.1, 2.5 Hz, 1 H), 7.00 (d, J ) 7.5
Hz, 1 H), 7.09 (s, 1 H), 7.14 (td, J ) 8.6, 2.5 Hz, 1 H), 7.29 (t, J
7.8 Hz, 1 H), 7.44 (s, 1 H), 8.28 (dd, J ) 8.9, 6.4 Hz, 1 H), 9.51
d, J ) 7.5 Hz, 1 H); MS (ESI, MeOH/NH OH) m/z 411 [M +
) 5.24 min, 100%), method
(29 mg, 0.10 mmol) and 3-fluoro-4-methoxybenzaldehyde (16 mg,
0.10 mmol) were processed in a manner analogous to method F to
1
9
provide the title compound as a white solid (22 mg, 51%):
NMR (300 MHz, DMSO-d
H
6
) δ 1.57-1.70 (m, 2 H), 1.80 (m, 2
)
(
H), 1.98-2.07 (m, 2 H), 2.79-2.86 (m, 2 H), 3.42 (s, 2 H), 3.72-
3.81 (m, 1 H), 3.82 (s, 3 H), 6.82 (s, 1 H), 7.03-7.15 (m, 3 H),
7.43 (td, J ) 8.7, 2.5 Hz, 1 H), 7.61 (dd, J ) 9.5, 2.4 Hz, 1 H),
4
+
H] ; analytical HPLC method A (t
B (t ) 5.33 min, 97.1%).
N-[1-[(4-Methoxybenzyl)methyl]piperidin-4-yl]-7-fluoro-4-
oxo-4H-chromene-2-carboxamide (47). Compound 22C and
R
R
8.11 (dd, J ) 9.0, 6.3 Hz, 1 H), 8.82 (d, J ) 8.1 Hz, 1 H); MS
+
(APCI, MeOH/NH
4
OH) m/z 429 [M + H] , 427 [M - H]; Anal.
22 2 2
(C23H F N O4) C, H, N.
3
-methoxybenzaldehyde were processed in a manner analogous to
N-[1-{4-[(4-Aminopiperidin-1-yl)methyl]phenyl}ethanonyl]-
7-fluoro-4-oxo-4H-chromene-2-carboxamide (53). Compound
22C (29 mg, 0.10 mmol) and 4-acetobenzaldehyde (15 mg, 0.10
1
method F to provide the title compound 47 as a solid: H NMR
500 MHz, pyridine-d ) δ 1.82-1.91 (m, 2 H), 2.01-2.10 (m, 4
H), 2.88-2.93 (m, 2 H), 3.41 (s, 2 H), 3.69 (s, 3 H), 4.24-4.30
m, 1 H), 6.43 (d, J ) 9.4 Hz, 1 H), 6.98 (d, J ) 8.4 Hz, 2 H),
(
5
mmol) were processed in a manner analogous to method F to
1
(
provide the title compound as a white solid (25 mg, 58%):
H

6
582 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Lynch et al.
NMR (300 MHz, DMSO-d
6
) δ 1.59-1.73 (m, 2 H), 1.77-1.85
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
(
3
m, 2 H), 2.03-2.13 (m, 2 H), 2.57 (s, 3 H), 2.80-2.87 (m, 2 H),
.57 (s, 2 H), 3.75-3.84 (m, 1 H), 6.82 (s, 1 H), 7.39-7.48 (m, 3
H), 7.61 (dd, J ) 9.5, 2.4 Hz, 1 H), 7.93 (d, J ) 8.1 Hz, 2 H), 8.12
(
dd, J ) 9.0, 6.3 Hz, 1 H), 8.84 (d, J ) 8.1 Hz, 1 H); MS (APCI,
+
MeOH/NH
4 23
OH) m/z 423 [M + H] , 421 [M - H]; Anal. (C24H -
FN ) C, H, N.
2 4
O
N-[1-N-[1-{4-[(4-Aminopiperidin-1-yl)methyl]phenyl}ethanon-
yl]-7-fluoro-4-oxo-4H-chromene-2-carboxamide (54). Compound
22C (29 mg, 0.10 mmol) and 4-acetamidobenzaldehyde (17 mg,
the last measurable plasma concentration) after dosing (AUC0-t)
0
.10 mmol) were processed in a manner analogous to method F to
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 concentra-
tion (C ) divided by the terminal elimination rate constant (â), and
t
added to AUC0-t to produce the total area under the curve
(AUC0-∞).
1
provide the title compound as a white solid (20 mg, 45%):
NMR (300 MHz, DMSO-d
H
6
) δ 1.56-1.70 (m, 2 H), 1.75-1.84
(
3
m, 2 H), 1.95-2.01 (m, 2 H), 2.02 (s, 3 H), 2.79-2.87 (m, 2 H),
.38-3.43 (m, 2 H), 3.72-3.81 (m, 1 H), 6.81-6.82 (m, 1 H),
.21 (d, J ) 8.5 Hz, 2 H), 7.42 (td, J ) 8.7, 2.4 Hz, 1 H), 7.52 (d,
7
J ) 8.5 Hz, 2 H), 7.61 (dd, J ) 9.5, 2.4 Hz, 1 H), 8.11 (dd, J )
Compound Evaluation in Anesthetized Rat Cardiovascular
Assay. Male Sprague-Dawley rats (325-375 g) are anesthetized
with the long-acting barbiturate inactin (100 mg/kg). Catheters (PE-
50) were placed in the femoral artery for measurement of mean
arterial pressure (MAP) and heart rate (HR). Hemodynamic data
was acquired using a Ponemah software platform. (Gould Instru-
ment Systems, Valley View, Ohio). Additional catheters were
placed in the femoral vein for compound administration (1-2 mL/
kg) and saline infusion (10 µL/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 determinations. 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, represent-
ing the limits of solubility achieving the highest possible plasma
concentrations. These high doses required a vehicle of poly(ethylene
glycol) 400 (PEG 400), which produces cardiovascular effects
(increasing MAP and decreasing HR). These effects seen with PEG
400 are minimally variable and highly reproducible. All cardio-
vascular results are expressed as mean ( SEM, (n ) 3 rats per
compound). This level of quantitation was used to define screening
of MCHr1 antagonists for cardiovascular effects.
9
.0, 6.3 Hz, 1 H), 8.82 (d, J ) 7.8 Hz, 1 H), 9.88 (s, 1 H); MS
+
(
APCI, MeOH/NH
analytical HPLC method A (t
5.02 min, 100%).
In Vitro Compound Evaluation. MCHR1 Binding Assay
4
OH) m/z 438 [M + H] , 436 [M - H];
R
) 4.91 min, 100%), method B (t
R
)
Using IMR-32 Membrane Preparations. Inhibition of binding
of the melanin concentrating hormone receptor by MCH was
determined using cell membranes prepared from a subcloned cell
line derived from IMR-32 cells (I3.4.2) and described in our
publication.22 In 96-well plates, I3.4.2 membranes (6 µg/well) were
incubated in the presence of test compound in binding buffer (25
2 2
mM HEPES pH 7.4, 1 mM CaCl , 5 mM MgCl and 0.5% BSA)
and with 0.05 nM [1 I]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 [1 I]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,
25
25
2 2
pH 7.4, 1 mM CaCl , 5 mM MgCl , and 0.5 M NaCl) was added
to each well and then transferred to a 0.5% polyethylenimine-treated
GF/B filtration plate (Packard) using a plate Filtermate Harvester
(Packard). The filter plate was washed three times with wash buffer,
MicroScint 20 (PerkinElmer) was added to each well, and the plate
was read using a Topcount microplate scintillation counter (Perki-
nElmer).
Compound Evaluation in the Anesthetized Dog Cardiovas-
cular Assay.19 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
Assay for Release of Intracellular Calcium. Activation of the
melanin concentrating hormone receptor 1 by MCH induces the
release of Ca2 from intracellular stores. This intracellular Ca2+
release is measured using a fluorometric imaging plate reader
+
14
surgical plane of anesthesia as previously described. Animals were
intubated with a cuffed endotracheal tube and ventilated with room
air by means of a mechanical respiration pump (Harvard Apparatus,
(
FLIPR, Molecular Devices Corp.) in conjunction with the Ca2+
-
sensitive dye reagent (Calcium Assay Reagent, Molecular Devices).
2 2
Model 613). Expiratory CO was monitored with an end-tidal CO
Release of Ca2 from intracellular stores causes an increase in
+
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 thermodilution 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 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.
2+
fluorescence of the dye that is proportional to Ca concentration.
Briefly, the assays are performed as follows. IMR-32 cells
expressing G
6-well plates. After 2 days, cells are loaded with the Calcium
R
16 G protein were plated at 100 000 cells/well in
9
Assay Reagent for 1 h at room temperature. Test compounds are
prepared at 60 µM in 6% DMSO. The cell plate is placed in the
FLIPR and 50 µL/well of test compound is delivered. The calcium
signal is followed for 3 min to assay for potential agonist activity
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
calcium signal was followed for an additional 3 min. Antagonist
activity as determined by the test compound’s ability to inhibit 50%
of MCH-induced Ca2 flux is reported.
+
In Vivo Compound Evaluation. Pharmacokinetic Analysis of
Plasma and Brain Exposure in DIO Mice. Male C57Bl/6J mice
are placed on a diet of 60% kcal from fat (Reseach Diets D12492)
for 3-4 months, during which time they become obese (45 g vs
3
0 g for lean controls on normal chow). Compounds are dosed in
DIO-mice orally at 10 mg/kg in a vehicle containing 1% Tween-
0 and water. Plasma samples are drawn and whole brains are
hERG Affinity Assay.18 Membranes were prepared as described
in our publication; frozen membrane aliquots were thawed and
homogenized again in a glass Dounce homogenizer (approximately
8

Chromone-2-carboxamide MCHr1 Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6583
1
0 passes). The following were added to each 200-µL well: 20
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.
µL of assay buffer, 1 µM astemizole (for nonspecific bounds) or
test compound, 50 µL of [ H]dofetilide, and 130 µL of membrane
homogenate (final protein concentration ) 30 µg/well). The plates
were incubated at 37 °C for 45 min, aspirated onto GF/B filter
plates, and washed with 2 mL of cold wash buffer. The radioactivity
was counted and the ligand concentration was adjusted on the basis
of the calculated K
ratio was calculated using the following equation: (total bounds
3
(
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. Nat. Med.
+
d
at each K concentration. The signal-to-noise
2002, 8, 779-781. (b) 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. (c) McBriar,
M. D.; Guzik, H.; Shapiro, S.; Paruchova, J.; Xu, R.; Palani, A.;
Clader, J. W.; Cox, K.; Greenlee, W. J.; Hawes, B. E.; Kowalski, T.
J.; O’Neill, K.; Spar, B. D.; Weig, B.; Weston, D. J.; Farley, C.;
Cook, J. Discovery of orally efficacious melanin-concentrating
hormone receptor-1 antagonists as antiobesity agents. synthesis, SAR,
and biological evaluation of bicyclo[3.1.0]hexyl ureas. J. Med. Chem.
2006, 49, 2294-2310. (d) Kowalski, T. J.; Spar, B. D.; Weig, B.;
Farley, C.; Cook, J.; Ghibaudi, L.; Frid, S.; O’Neill, K.; Del Veccio,
R. A.; McBriar, M.; Guzik, H.; Clader, J.; Hawes, B. E.; Hwa, J.
Effects of a selective melanin-concentrating hormone 1 receptor
antagonist on food intake and energy homeostasis in diet-induced
obese mice. Eur. J. Pharmacol. 2006, 535, 182-191.
-
nonspecific bounds)/nonspecific bounds.
hERG Ionic Current Studies.21 hERG channels were stably
expressed in HEK 293 cells and maintained in Minimal Eagle
Medium (MEM) supplemented with 10% fetal bovine serum, 1%
penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM nonessential
amino acids, 1 mM sodium pyruvate and 200 mg/mL of G418.
Media were changed every 48 h and cells were passaged weekly.
Currents were recorded using a MultiClamp 700A and pClamp data
acquisition software. Patch pipets were constructed with borosilicate
glass capillary tubes (resistance 1.5-2.9 MΩ). The pipet solution
contained (in mM) K-aspartate, 125; KCl, 20; EGTA, 10; MgCl
; HEPES, 5; MgATP, 5; pH 7.3. The bath solution contained (in
mM) NaCl, 140; KCl, 5; MgCl , 1; CaCl , 2; glucose, 5; HEPES,
0; pH 7.4. Compounds were dissolved in DMSO and then diluted
2
,
1
2
2
2
(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 aminopip-
eridinecoumarin melanin concentrating hormone receptor 1 antago-
nists. J. Med. Chem. 2005, 48, 5888-5891.
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]ac-
etamide, an orally efficacious melanin-concentrating hormone re-
ceptor 1 antagonist for the treatment of obesity. J. Med. Chem. 2005,
in the bath solution. Cells were exposed to a single concentration
of drug. DMSO concentrations never exceeded 0.1% (by volume).
Effects of compounds on hERG current were assessed using a
voltage clamp protocol that stepped to 0 mV for 3 s, followed by
a step to -50 mV for 4 s from a holding potential of -80 mV
once every 15 s. Current measurements were made at 36.5-37 °C.
IC50 values were calculated from the effect of compounds on hERG
tail current at -50 mV corrected for run down/DMSO vehicle
(
(
(
(
effects with a logistic fitting routine using the following equation:
s
y ) [(A
1
- A
2
)/(1 + (x/IC50)) ] + A
2
, where A
1
) initial x value,
A
2
) final x value, and s ) slope.
Acknowledgment. The authors would like to thank Paul
48, 1318-1321.
Richardson and J.J. Jiang for MCH production.
12) Vasudevan, A.; LaMarche, M. J.; Blackburn, C.; Che, J. L.; Luchaco-
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Supporting Information Available: Results from combustion
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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JM060683E