Discovery of N -[(4 R)-6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2- dimethyl-3,4-dihydro-2 H -pyrano[2,3- b ]pyridin-4-yl]-5-methyl-1 H -pyrazole-3-carboxamide (MK-5596) as a novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity

Article abstract of DOI:10.1021/jm100023j

This paper describes the discovery of N-[(4R)-6-(4-chlorophenyl)-7-(2,4- dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl] -5-methyl-1H-pyrazole-3-carboxamide (MK-5596, 12c) as a novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity. Structure-activity relationship (SAR) studies of lead compound 3, which had off-target hERG (human ether-a-go-go related gene) inhibition activity, led to the identification of several compounds that not only had attenuated hERG inhibition activity but also were subject to glucuronidation in vitro providing the potential for multiple metabolic clearance pathways. Among them, pyrazole 12c was found to be a highly selective CB1R inverse agonist that reduced body weight and food intake in a DIO (diet-induced obese) rat model through a CB1R-mediated mechanism. Although 12c was a substrate of P-glycoprotein (P-gp) transporter, its high in vivo efficacy in rodents, good pharmacokinetic properties in preclinical species, good safety margins, and its potential for a balanced metabolism profile in man allowed for the further evaluation of this compound in the clinic.

Full text of DOI:10.1021/jm100023j

4028 J. Med. Chem. 2010, 53, 4028–4037
DOI: 10.1021/jm100023j
Discovery of N-[(4R)-6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-
pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-carboxamide (MK-5596) as a Novel
Cannabinoid-1 Receptor (CB1R) Inverse Agonist for the Treatment of Obesity
Lin Yan,*,† Pei Huo,† John S. Debenham,† Christina B. Madsen-Duggan,† Julie Lao,‡ Richard Z. Chen,‡ Jing Chen Xiao,‡
Chun-Pyn Shen,‡ D. Sloan Stribling,§ Lauren P. Shearman,§ Alison M. Strack,§ Nancy Tsou,^ Richard G. Ball,^
Junying Wang, Xinchun Tong, Thomas J. Bateman, Vijay B. G. Reddy, Tung M. Fong,‡,# and Jeffrey J. Hale†
Departments of †Medicinal Chemistry, ‡Metabolic Disorders, §Pharmacology, Drug Metabolism and Pharmacokinetics, and
^Pharmaceutical Research and Development, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065.
# Current address: Forest Research Institute, Jersey City, New Jersey 07311.
Received January 8, 2010
This paper describes the discovery of N-[(4R)-6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-
3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-carboxamide (MK-5596, 12c) as a
novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity. Structure-activity
relationship (SAR) studies of lead compound 3, which had off-target hERG (human ether-a-go-go
related gene) inhibition activity, led to the identification of several compounds that not only had
attenuated hERG inhibition activity but also were subject to glucuronidation in vitro providing the
potential for multiple metabolic clearance pathways. Among them, pyrazole 12c was found to be a
highly selective CB1R inverse agonist that reduced body weight and food intake in a DIO (diet-induced
obese) rat model through a CB1R-mediated mechanism. Although 12c was a substrate of P-glycopro-
tein (P-gp) transporter, its high in vivo efficacy in rodents, good pharmacokinetic properties in
preclinical species, good safety margins, and its potential for a balanced metabolism profile in man
allowed for the further evaluation of this compound in the clinic.
Introduction
technology have made energy-dense food readily available and
led to lifestyles requiring less physical activity. While obesity
can be potentially treated via a low calorie diet, physical
exercise, and a balanced lifestyle, the mechanism of energy
homeostasis can make it difficult for overweight and obese
people to lose weight by voluntarily eating less.3 Although
regulation of energy homeostasis is complex and many aspects
of it are still unknown, the endocannabinoid system, particu-
larly cannabinoid-1 receptor (CB1R), has been indicated to
play an important role in regulating energy balance.4
Cannabinoid-1 receptor is a G-protein-coupled receptor
widely expressed in the central nervous system (CNS) and also
in peripheral tissues. Both preclinical and clinical studies have
demonstrated that blocking CB1R activity with antagonists
or inverse agonists can reduce body weight and food intake.5
Consequently, the development of CB1 antagonists and
inverse agonists as therapeutic agents has been the subject of
intense research in the pharmaceutical industry. Merck Re-
search Laboratories have previously disclosed several CB1R
inverse agonists.6 A novel acyclic CB1R inverse agonist, tara-
nabant (MK-0364, 1), was evaluated in clinical trials as a
potential treatment for obesity (Figure 1).7 Compound 1 is a
potent CB1R inverse agonist and reduces body weight and
food intake in both human and rodents through CB1R-
based mechanism. Compound 1 is primarily metabolized by
CYP3A4enzymeandthereforehasthe potentialtobeaffected
by CYP3A4 inhibitors. The objective for the backup program is to
develop a CB1R inverse agonist that has CB1R pharmacological
Obesity is a global epidemic.1 A World Health Organiza-
tion (WHOa) 2006 survey of the United States found that
67% of adultsaged20andabove areoverweight(25eBMI <
30) or obese (BMI g 30). Globally, obesity is alarmingly on
the rise, particularly in developing countries. There are app-
roximately 400 million obese people aged 15 and above and
over 1.6 billion overweight in 2005. Obesity is a major risk
factor for a number of chronic diseases, including diabetes,
cardiovascular diseases, and some forms of cancer. The treat-
ment cost of obesity- and overweight-related illnesses has
placed a substantial burden on health care resources: In
2008 the United States alone spent $147 billion.2
The fundamental cause of obesity is the excess consump-
tion versus expenditure of calories. Advances in science and
*To whom correspondence should be addressed. Phone: (732) 594-
5419. Fax: (732) 594-2200. E-mail: lin_yan@merck.com.
aAbbreviations: WHO, World Health Organization; BMI, body mass
index; CB1R, cannabinoid-1 receptor; CB2R, cannabinoid-2 receptor;
CNS, central nervous system; DIO, diet-induced obese; GABA, γ-amino-
butyric acid; SAR, structure-activityrelationship; hERG, human ether-a-
go-go related gene; PyBOP, benzotriazol-1-oxytripyrrolidinophospho-
nium hexafluorophosphate; ee, enantiomeric excess; CHO, Chinese ham-
ster ovary; CP-55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-
phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; cAMP, cyclic adenosine
monophosphate; MK499, N-[10-(6-cyano-1,2,3,4-tetrahydro-2R-naphthal-
enyl)-3,4-dihydro-4R-hydroxyspiro(2H-1-benzopyran-2,40-piperidin)-6-yl]-
methanesulfonamide; HEK, human embryonic kidney; d-Fen, dexfen-
fluramine; MED, minimal efficacious dose; P-gp, P-glycoprotein efflux
transporter.
r
pubs.acs.org/jmc
Published on Web 04/27/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4029
Scheme 1a
a (a) (1) 2-methylpropenylmagnesium bromide, THF, 50 ꢀC, 16 h; (2)
pH ∼ 3, room temp, 5 h (78% over two steps); (b) H2NOH HCl,
3
NaOAc, Et3N, THF, CH3OH, 70 ꢀC, 16 h; (c) Zn, HOAc, 95 ꢀC, 2 h
(70% over two steps); (d) (1) RCO2H, PyBOP, Et3N, CH2Cl2, room
temp (70-100%); (2) chiral HPLC separation.
Figure 1. Merck early leads of CB1R inverse agonists.
profile similar to 1 but is less dependent on CYP3A4 for
clearance.8
Scheme 2a
Recently, two new series of CB1R inverse agonists bearing
fused pyridine bicyclic cores were discovered by Merck scien-
tists.9 Furanopyridine 2 is a potent CB1R inverse agonist and
induced overnight body weight loss and food intake suppres-
sion in DIO rats (MED ≈ 0.3 mg/kg); however, it caused mild
to pronounced hyperactivity, hyperreactivity, and ataxia at
doses of 30-100 mg/kg in conscious mice, possibly because of
its partial agonism of R1β3γ2 GABAA receptor subtype. SAR
studies of 2 led to the identification of several fused bicyclic
pyridine series of CB1R inverse agonists. Among them,
pyranopyridine 3 was a potent CB1R inverse agonist and
efficacious in DIO rats. Although devoid of GABAA off-
target activity, compound 3 had potent off-target inhibition of
hERG potassium channel. In this paper, we will describe the
SAR studies of 3 that lead to the discovery of a potent and
efficacious CB1R inverse agonist 12c (MK-5596)10 with atte-
nuated off-target hERG activity. This compound has a CB1R
activity profile similar to that of 1 but with a more balanced
metabolic profile.
a (a) Ru-(1S,2S)TsDPEN, IPA, CH2Cl2 room temp, 16 h (69%); (b)
Zn(N3)2(pyridine)2, PPh3, imidazole, DIAD, CH2Cl2, room temp
(92%); (c) P(CH3)3, H2O, THF, room temp (80%); (d) RCO2H, PyBOP,
Et3N, CH2Cl2, room temp (76% to quant).
and (1S,2S)-N-(p-toluenesulfonyl)-1,2-diphenylethylenedi-
amine, ketone 5 was reduced to S-alcohol 9 in >99% ee.12
S-Alcohol 9 was then stereospecifically converted to R-azide
10, which was subsequently reduced to amine 11 in >98%
ee.13,14 Similarly, a variety of acids were readily coupled to
amine 11 using PyBOP.
Chemistry
Pyranopyridine compounds discussed in this paper were
prepared according to two general methods: a racemic syn-
thetic route (Scheme 1)9b and anasymmetric synthetic method
(Scheme 2). At the earlier stage of SAR studies, pyranopyr-
idines were prepared according to the racemic synthetic meth-
od. Treatment of 5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)-
2-oxo-1,2-dihydropyridine-3-carbonitrile with 2-methyl-1-propenyl-
magnesium bromide followed by acidic hydrolysis gave ketone
5 (Scheme 1). Condensation of 5 with hydroxylamine provided
oxime 6, which was subsequently reduced by zinc dust in acetic
acid to amine 7. A variety of acids were coupled to 7 using
PyBOP as the coupling reagent, and the resulting mixture of
amides 8 were then separated by chiral chromatography to give
two enantiomers or four diastereomers, if a chiral acid
was used. For clarity, only the more active enantiomer or
diastereomers will be discussed in this paper. To expedite the
SAR study, an asymmetric synthesis of the penultimate
amine was developed using Noyori’s asymmetric hydrogen
transfer reaction (Scheme 2).11 By use of ruthenium catalyst,
i.e., Ru-(1S,2S)TsDPEN prepared from [RuCl2(η6-arene)]2
Results and Discussion
Synthesizing compounds that are cleared by multiple meta-
bolic enzymes could minimize the potential for drug-drug
interactions in humans.15 Phase II metabolism may occur
directly on the parent compounds that contain appropriate
functional groups. It is therefore conceivable that by incor-
poration of such functional groups, drug clearance will be less
dependent upon phase I metabolism, which is often mediated
by cytochrome P450 enzymes, for clearance, making them less
susceptible to CYP450 mediated drug-drug interactions.
Previous studies have shown that the hydroxyl group of
glycolic amide in 2 undergoes rapid glucuronidation (one
class of phase II metabolism) in dog; however, no formation
of glucuronide conjugate of 3 was detected. It was thus
hypothesized thatthe appropriateamide group in an analogue

4030 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yan et al.
of 3 might lead to the identification of functional groups that
undergo direct glucuronidation across species as well as
minimize off-target hERG activity.
Table 1. Effects of Compound’s Chirality on Binding Affinities of
CB1R and CB2R and Inhibition of hERG Potassium Channel Are
Exemplified by Two Pairs of Pyranopyridine Enantiomersd
The CB1R and CB2R binding affinities (IC50) of test
compounds were determined in competitive binding assays
with membranes of Chinese hamster ovary (CHO) cells stably
expressing recombinant human CB1R and CB2R using
[3H]CP-55,940 as ligand.16 The CB1R and CB2R functional
potencies (EC50) as inverse agonists were measured by inhibi-
tion of forskolin-stimulated intracellular cAMP increase of
CB1R and CB2R expressing CHO cells. All compounds
discussed in this paper were found to be CB1R inverse
agonists. Off-target hERG inhibition of test compounds was
measured by competitive inhibition of [35S]MK499 binding to
hERG channel expressed in HEK-293 cell membranes.17 The
relative propensity of test compounds to undergo phase II
metabolism was tested by the detection of metabolites follow-
ing incubation with rat and human hepatocytes.18 Com-
pounds exhibiting direct conjugation (i.e., glucuronidation)
as a major metabolic pathway were predicted to be less
vulnerable to CYP mediated drug-drug interactions. Active
compounds, especially those without off-target activities and/
or capable of glucuronidation, were tested in an acute phar-
macodynamic model (usually at a dose of 3 mg/kg) for
pharmacological efficacy to evoke overnight body weight loss
and food intake suppression in DIO rats.
During the initial phase of SAR studies, compounds were
prepared using a racemic synthesis followed by chiral HPLC
separation. Comparison of the values of CB1R inhibition
(IC50) of these enantiomers generally showed at least greater
than 5-fold difference. In order to expedite the SAR study, an
asymmetric synthesis was developed. Table 1 compares the
CB1R binding affinities and hERG inhibition activities of two
exemplarypairsofenantiomers. Compound13, anR-enantio-
mer, was a potent CB1R inverse agonist with 3 orders of
magnitude selectivity over CB2R, while its corresponding
S-enantiomer 14 was about 50 times less potent as CB1R
inhibitor. The discrepancy of CB1R binding affinities was
much greater between compounds 15 and 16. Although
compound 15 showed effectively no detectable hERG binding
at the highest testing concentration (30 μM), no formation
of a glucuronide conjugate of 15 was detected. These com-
parisons established that the higher CB1R binding activity
of pyranopyridine series was largely associated with the
R-configuration.
a Competitive CB1R and CB2R binding against CP-55,950. Data
are reported as the mean for n = 2 measurements. SD is generally
within (20% of the average. b Competitive hERG binding against
MK499. Data are reported as the mean for n = 2 measurements (n = 1
when IC50 > 30 μM). SD is generally within (20% of the average. c Not
determined. d The chiral center is marked by an asterisk (/).
profiles similar to 3 but did not form a glucuronide conjugate.
Formation of a glucuronide conjugate of the parent was
detected following incubation with both human and rat
hepatocytes for compound 8b, which had a cyclopropane
group replacing the geminal dimethyl group of 8a; however,
8b was still a potent hERG inhibitor (IC50 = 300 nM).
Replacement of the lactamide methyl group of 3 with a
trifluoromethyl group provided compound 8c that was cap-
able of forming a glucuronide conjugate of the parent follow-
ing incubation with both rat and human hepatocytes. The
strong electron-withdrawing effect of trifluoromethyl
group was expected to increase the acidity of the hydroxyl
group and thus might enhance its potential for undergoing
glucuronidation.
Compound 8c was a potent CB1R inverse agonist with
good selectivity against CB2R and exhibited robust overnight
food intake suppression and body weight loss in DIO rats at a
dose of 1 mg/kg. However, it still inhibited hERG channel at
low concentration. The other active diastereomer 8d also
showed similar potent hERG inhibition. Increasing steric
hindrance around the hydroxyl group of 8d by introducing a
methyl group led to 8e that did not improve its selectivity
against hERG inhibition, although it remained as a potent
CB1R inverse agonist. By movement of the trifluoroethanol
group one carbon away from the amide carbonyl of 8c and 8e,
compound 8f was a 10-fold less potent hERG inhibitor than 3
while still forming a glucuronide conjugate of the parent.
Compound 8f was about 4 times more potent as CB1R inverse
agonist than 3, but it showed less significant in vivo efficacy at
a dose of 3 mg/kg, although it had good pharmacokinetics in
rat (t1/2 = 8.2 h and F = 41%). The other active diastereomer
8g was also less active in acute DIO rat study. Finally, the
hydroxyl group of 2,2-difluoro-3-hydroxypropanamide in 8h
also allowed for glucuronidation, but it was still a potent
A series of compounds containing functional groups with
the potential for glucuronidation were designed and synthe-
sized. Their biological activities were compared with those of
the lead compound 3 in CB1R/CB2R selectivity, off-target
hERG activity, potential of glucuronidation, and acute over-
night body weight loss and food intake suppression in DIO
rats (Table 2). Lead compound 3 was a potent CB1R inverse
agonist with greater than 1000-fold selectivity against CB2R
and had good pharmacokinetics in rat (t1/2 = 7.9 h and F =
100%). Oral administration of 3 at doses of 1 and 3 mg/kg to
DIO rats resulted in body weight loss of -6 and -16 g and
food intake suppression of -28% and -72% vs vehicle
treated animals (þ5 g weight gain and 0%), respectively.
Compound 3, however, was a potent hERG inhibitor
(IC50 = 120 nM), and its hydroxyl group did not form a
glucuronide conjugate following incubation with both rat and
human hepatocytes. Increasing the lipophilicity around the
lactamide of 3 by introducing a methyl group led to com-
pound 8a, which had in vitro and in vivo CB1R activity

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4031
Table 2. Comparison of CB1R and CB2R Binding Activities, Off-Target hERG Binding Activities, Potential for Glucuronidation, and Effects of
Test Compounds on the Acute Overnight Body Weight Change (ΔBW) and Food Intake (FI) Suppression vs Vehicle (veh.) in Diet-Induced Obese
(DIO) Ratsi
a Competitive CB1R and CB2R binding against CP-55,950. Data are reported as the mean for n g 2 measurements. SD is generally within (20% of
the average. b Competitive hERG binding against MK499. Data are reported as the mean for n = 2 measurements (n = 1 when IC50 > 30 μM). SD is
generally within (20% of the aveage. c Glucuronidation of parent compound. Trace describes glucuronidation levels that are detectable by mass
spectrographic analysis but do not represent a significant metabolic pathway. d Overnight body weight gain and food intake suppression in DIO rat (n =
6-8 rats per group). e All p < 0.05 unless otherwise noted. f Only racemic mixture was tested. g Not determined. h Experiments were performed on
racemic mixtures. i The chiral center is marked by an asterisk (/).
hERG inhibitor. By modulation of the steric and electronic
effects around the lactamide of 3, most of the compounds were
still potent CB1R inverse agoinsts. Several compounds were
found to form glucuronide conjugates of the parent and also
attenuate off-target hERG activity; however, none of them
showed in vivo activities comparable to 3 in the acute phar-
macodymanic model.
glucuronidation. Phenol of compound 12a, as expected,
formed a glucuronide conjugate by both human and rat
hepatocytes, but it only exhibited low in vivo activity,
probably due to its poor bioavailability in rat (t1/2
=
1.9 h and F = 1%). Formation of a glucuronide conjugate
of the anilinic amine group in 12b was also detected. Like
12a, compound 12b did not induce significant in vivo
Next we examined the replacement of the lactamide
of 3 with aryl groups containing functional groups for
biology activity because of its low bioavailability in rat (t1/2 =
7.4 h and F = 5%). Replacing phenyl group with pyrazole

4032 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yan et al.
Figure 2. X-ray crystallography analysis confirmed the absolute
stereochemistry of compound 12c as R.
resulted in compound 12c, which is a potent CB1R inverse
agonist, and reduced food intake and body weight at a dose
of 3 mg/kg in DIO rats. In addition, compound 12c was a
weak hERG inhibitor (IC50 = 1 μM) and formation of a
glucuronide conjugate of the parent was detected following
incubation with both human and rat hepatocytes. By
extension of methyl at C4 to ethyl, 12d was slightly less
active than 12c in the acute PD model. Transposing the
methyl group from C4 to C5 gave 12e, which was 5-fold
more potent and 5-fold more selective against hERG than
12c, suggesting substitution at C5 might enhance selectivity
against hERG. Formation of a glucuronide conjugate of
12e was detected, and in vivo efficacy in DIO rats was
similar to that of 12c at a dose of 3 mg/kg. Compounds 12f
and 12g with substitution on C5 were more selective
against hERG but less efficacious in lowering body weight
and suppressing food intake in DIO rats than 3. Interest-
ingly, no formation of a glucuronide conjugate of the
pyrazole 12h was detected, while 12i and 12j with substitu-
tions on the pyrazole ring generated glucuronide conju-
gates of parent. Both compounds were potent CB1R
inverse agonists and had similar efficacy as that of 12c at
a dose of 3 mg/kg. Conversion of 12c into triazole 12k did
not result in compounds with good pharmacodynamic
efficacy. Finally, formation of glucuronide conjugates of
indazole 12l was detected in the presence of rat hepato-
cytes, while no glucuronide conjugate formation for in-
dazole 12m, and 12n was detected. Although 12l was a
potent CB1R inverse agonist, it did not induce body weight
loss and food intake suppression comparable to 12c.
It was possible to differentiate among these four new active
compounds (12c, 12d, 12h, and 12i) based on their ability to
drive a pharmacodynamic response, i.e., reduction of body
weight and food intake, at lower dose (1 mg/kg). Compound
12c emerged as the most potent compound to cause overnight
body weight loss and food intake suppression at a dose of 1
mg/kg. It has greater than 1000-fold of selectivity of CB1R
over CB2R in both human and rat (rCB1R and rCB2R
IC50 of 0.7 and 1500 nM, respectively) and is an inverse
agonist (EC50 = 5.8 nM, -111% inverse agonism) of human
CB1R. The absolute structure of 12c was confirmed to be R by
X-ray crystallographic analysis (Figure 2). Its corresponding
S-enantiomer was prepared and found to be less potent as a
CB1R inverse agonist (IC50 = 7.65 nM) and more potent as a
hERG inhibitor (MK499 IC50 = 0.67 μM) than 12c. On the
Figure 3. Chronic efficacy studies of compound 12c dosed at 0.3
and 1 mg/kg in DIO rats: (A) daily body weight change; (B)
cumulative food intake. Fresh food was given on day 8. Last
measurement was conducted 18 h after dosing of test compound
on day 13. In all graphs, data represent the mean ( SEM (n = 7-8
rats per group).
basis of its potent CB1R binding affinity, attenuated inhibi-
tion of off-target hERG potassium channel, acute pharmaco-
dynamic efficacy, and potential for phase II metabolism in
human, compound 12c was chosen for further pharmaco-
kinetic and pharmacodynamic characterization and metabo-
lism profiling in preclinical species.
Chronic efficacy of compound 12c was evaluated by com-
paring body weight loss and food intake suppression of 12c-
treated group of DIO rats to those of vehicle- and d-Fen-
treated groups over a period of 13 days. Compound 12c
produced a dose-dependent lowering of body weight
(Figure 3A). Average body weight gain of the vehicle-treated
group over 13 days was 10 ( 1 g. Cumulative body weight
changes from starting weight in the 0.3 and 1 mg/kg groups
were -0 ( 5 and -19 ( 4 g, respectively. The final relative
body weight loss compared with vehicle was 1.6% and 4.5%
for the 0.3 and 1 mg/kg groups, respectively. As a positive
control, the 3 mg/kg d-Fen-treated group produced an incre-
mental body weight loss up to day 7 and then gradually body
weight loss decreased. In comparison, 12c at a dosage of 1 mg/kg
generated a sustained body weight loss up to day 13 with the
final body weight loss comparable to that of the 3 mg/kg
d-Fen group. Compound 12c also generated a dose-dependent

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4033
Figure 4. Comparison of effects of 12c on food intake and body weight gain between lean wide type (WT) mice and CB1R knockout (KO)
C57BL/6 mice. Effects of compound 12c on ad libitum food intake of WT C57BL/6 mice (A) and overnight body weight gain in WT mice (B).
Ad libitum fed lean mice (6-7 months of age, BW ≈ 29 g) were dosed orally ∼30 min prior to dark phase. Diet was switched from regular chow
to medium high fat diet (D12266) upon dosing. Mice were weighed 4 and 18 h (overnight) after dosing. Mean and SEM are shown (n = 6-7 per
group): (/) P < 0.05 or (//) P < 0.01 vs vehicle (one-way ANOVA and Dunnett’s test for multiple comparison). Effects of 12c (30 mg/kg) on
food intake of WT mice (6-7 months of age, BW ≈ 28 g) or KO mice (6-7 months of age, BW ≈ 25 g) (C) and overnight body weight gain of
WT or KO mice (D). Mean and SEM are shown (n = 10 per group): (//) P < 0.01 vs corresponding vehicle (unpaired t test).
food intake suppression in DIO rats (Figure 3B). In compar-
ison with the vehicle-treated group, the reductions in cumula-
tive food intake of 12c-treated group were 10 ( 4% and 19 (
4% for the 0.3 and 1 mg/kg groups, respectively. The 1 mg/kg
12c-treated group exhibited cumulative food intake suppres-
sion similar to that of the 3 mg/kg d-Fen-treated group. The
minimal efficacious dose (MED) of the 13-day chronic study
was determined to be 0.3 mg/kg. At the end of the chronic
efficacy study, the levels of compound 12c (18 h after the last
dose, 0.3 mg/kg) were determined to be 180 ( 40 nM in plasma
and 110 ( 30 nM in brain, respectively, and the corresponding
ratio of brain and plasma exposure was calculated to be 0.63.
For comparison, a 13-day parallel pharmacokinetics study was
carried out in DIO rats and the exposures of compound 12c
(24 h after the last dose, 0.3 mg/kg) were determined to be 135 (
59 nM in plasma and 74 (15 nM in brain, respectively, and thus
the brain and plasma ratio was 0.55. This chronic study
Figure 5. Representative radioprofiles of [3H]12c following incu-
bation (1 μM) with cryohepatocytes (3 million cells/mL) from (A)
rat and (B) human.
demonstrated that compound 12c had good brain penetration
and required low plasma exposure to drive in vivo efficacy to
reduce body weight and food intake in DIO rats.
The antiobesity effect of 12c was examined in CB1R knock-
out mice.19 In ad libitum fed wild-type lean mice (C57BL/6),
compound 12c caused a dose-dependent reduction in food
intake (Figure 4A) and a reduced overnight body weight gain
or even a reduction in body weight (Figure 4B). The MED for
12c in lean mice was determined to be 3 mg/kg. In contrast,
compound 12c did not show significant effects on food intake
(Figure 4C) or body weight (Figure 4D) at a dose of 30 mg/kg
in CB1R knockout mice. The lack of in vivo efficacy in CB1R-
deficient mice suggested that the anorectic effects observed for
12c were due to a CB1R-mediated mechanism.
shown). In the presence of anti-CYP3A4 antibodies, incuba-
tion of 12c did not yield M2 in human liver microsomes,
indicating that the oxidative metabolism of this compound
was CYP3A4 dependent. Additional studies were conducted
in both rat and human hepatocytes to observe both phase I
and phase II metabolism of 12c. Following incubation with
rat hepatocytes the predominant metabolic route of 12c was
oxidation to metabolite M2, consistent with microsomal data
(Figure 5). In human hepatocytes, however, metabolism of
12c to both the oxidative metabolite M2 and the direct
conjugation with glucuronic acid, designated M1, was equally
prevalent. Dispositionand metabolism of compound12c were
Incubation of 12c in both human and rat liver microsomes
produced predominately oxidative metabolite M2 (data not

4034 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yan et al.
Figure 6. Metabolism profile of [3H]12c in rat. Compound was formulated in Imwitor 742/Tween-80 (1:1) and dosed to bile duct cannulated
rats po at 2 mg/kg. Bile was collected over a period of 72 h.
also evaluated in bile-duct-cannulated rats (Figure 6). Follow-
ing oral dosing, the percentages of 12c related radioactivity
recovered over 72 h in feces, bile, and urine were 48%, 28%,
and <1%, respectively, indicating that renal clearance was
insignificant for this compound in the rat. The major radio-
active component detected in bile was the intact parent
(∼30% of the absorbed dose), the parent glucuronide (M1,
∼4%), and the oxidative metabolites M2 and M3. The major
radioactive component in feces was the unabsorbed parent
compound plus small amount of M2. Following the oral
dosing, the major radioactive compound in rat plasma and
the brain was the parent compound. The low levels of direct
glucuronidation of parent observed in vivo in rats were
consistent with data obtained in vitro in rat hepatocytes.
The consistent metabolism profiles observed in vitro in hepato-
cytes as well as in vivo in rats suggested the potential for mixed
metabolism in humans, where oxidative and direct glucuro-
nidation pathways were comparable following incubation
with human hepatocytes. The excretion of intact parent, likely
due to Pgp-mediated efflux (see below), in rodents provided
some additional support that clearance in man would not rely
entirely on CYP mediated metabolism.
Susceptibility of compound 12c to P-glycoprotein (P-gp)
efflux transporter was evaluated in vivo by comparing levels
of compound12c in the brainof wildtypemouse(Mdr1aþ/þ)
and P-gp deficient CF-1 mouse (Mdr1a-/-). After intrave-
nous administration of 12c at a dose of 1 mg/kg, the levels of
parent (4 h postdose) were determined to be 410 nM in plasma
and 270 nM in brain in wild type and to be 300 nM in plasma
and 1400 nM in brain in P-gp deficient mouse. These data
(brain to plasma ratio of 0.66 in wild type vs 4.66 in P-gp
deficient CF-1 mouse) suggested that compound 12c is a P-gp
substrate.
The pharmacokinetic properties of compound 12c were
examinedinthree preclinicalspecies(Table3). Compound12c
showed low systemic plasma clearance (1.1-4.5 (mL/min)/
kg) across species and good elimination half-life in rat and
monkey and long half-life in dog. Good oral bioavailability
was observed in all species, with monkey the lowest at 46%.
Finally, compound 12c was a moderate inhibitor of
CYP3A4, CYP2C8, CYP2C9, and CYP2D6 with IC50 values
of3.3, 11, 18, and6.0 μM, respectively, and was anactivator of
human pregnane X receptor (PXR) with an EC50 of ∼5 μM
and 55% activation response (at 5 μM) relative to rifampicin.
Because of the structural resemblance to2, compound12c was
evaluated in conscious mice for abnormal activity on CNS
function at a single dose of 100 mg/kg. CNS assessments were
Table 3. Pharmacokinetics of Compound 12c after Intravenous and
Oral Administration in Rat, Dog, and Monkeya
species
pharmacokinetic parameter
rat
dog
monkey
Clp ((mL/min)/kg)
Vdss (L/kg)
4.5
3.0
8.5
1.03
91
1.1
5.7
60
2.3
2.4
15
t
1/2 (h)
Cmax (μM)
F (%)
0.15
65
0.09
46
oral AUCN (μM h kg/mg)
11
18
6.1
3
3
a Pharmacokinetics were determined at doses of 1 mg/kg iv and 2 mg/
kg po in male Sprague-Dawley rats and 0.2 mg/kg iv and 0.4 mg/kg po
in male beagle dogs and rhesus monkeys. Intravenous pharmacokinetics
were determined using a solution formulation in EtOH/PEG400/water
(20:50:30, v/v/v) for rats and EtOH/PEG400/water (20:40:40 v/v/v) for
dogs and monkeys. Oral pharmacokinetics were determined using a
solution formulation in Imwitor742/Tween-80 (1:1 w/w) for rats, dogs,
and monkeys.
conducted at 0.5, 1, 2, 5, and 24 h after dosing, and body
temperature was measured at 2 h after dosing. Compound 12c
had no effect on CNS functions including gross behavior,
neural reflexes, spontaneous activity, and thermoregulation
during the 24h period after dosing. Since12chas a hERG IC50
of 1 μM, its potential cardiovascular effect was examined by
intravenously infusing 12c evenly over 30 min with rising
cumulativedosesof 1, 3, and 10mg/kgin anesthetized dogs. In
comparison to vehicle-treated dogs, compound 12c caused a
modest reduction (∼20%) in mean arterial pressure only at
the highest dose. No changes in heart rat, electrocardio-
graphic PR, ORS, and QTc cardiac intervals were observed
in 12c-treated dogs over this dose range. The peak plasma
concentrations reached 1.63 ( 0.19, 3.26 ( 0.30, and 6.96 (
0.33 μM during the administration of doses of 1, 3, and 10
mg/kg, respectively. Since 12c was highly bound to plasma
proteins (>99%) of rat, dog, monkey, and human at a
concentration of 0.2 μM and only required low plasma
concentration (<200 nM) to drive pharmacodynamic effi-
cacy, compound 12c should have a sufficient safety margin in
a clinical study.
In conclusion, a series of pyranopyridines containing func-
tional groups suitable for glucuronidation of the parent
compound were designed and synthesized as potent and
selective CB1R inverse agonists. Several functional groups,
such as trifluoroacetamide and substituted pyrazole groups,
were identified to form glucuronide conjugates of the parent.
Many of these compounds showed good pharmacodynamic
efficacy to reduce overnight body weight and food intake in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4035
DIO rats similar to lead compound 3. Despite being a sub-
strate of P-gp, compound 12c, as a potent and selective CB1R
inverse agonist, emerged as one of the most efficacious
compounds in this series with attenuated off-target hERG
activity. Compound 12c demonstrated CB1R-related pharma-
cological efficacy similar to that of 1, good pharmaco-
kinetic profiles in preclinical species, acceptable safety margins,
and a potentially balanced metabolism profile in man, allow-
ing for further clinical evaluation as an antiobesity agent
with lower potential of CYP3A4 mediated drug-drug inter-
actions. Unfortunately, the development of several CB1 an-
tagonists/inverse agonists, such as rimonabant (Sanofi-
Aventis) and taranabant (1, Merck), was discontinued on the
basis of concerns about adverse psychiatric effects,
i.e., increased anxiety and depression or suicidal intentions.20
Pfizer soon decided to discontinue the phase III development
program for its investigational CB1 antagonist, i.e., CP-
945,598, citing changing regulatory perspectives on the risk/
benefit profile of the CB1 class.21 In addition, the contra-
dictory study results of rimonabant in psychiatric behavior
animal models make it difficult to guide the selection of a
preclinical candidate for development on the anticipation of
improved psychiatric profile based on preclinical animal mod-
els.22 Under such circumstances, clinical evaluation of 12c as
an antiobesity agent was no longer in progress. Nevertheless,
the discovery of 12c demonstrated the successful optimization
of the metabolism profile of a CB1R inverse agonist without
impacting its pharmacological properties.
OD, or Chiralcel OJ column (20 mm ꢀ 250 mm) (Daicel
Chemical Industries, Ltd.) with the desired isocratic solvent
systems identified on chiral analytical chromatography. The
purity of all final compounds was determined by LC-MS to be
g95%. Preparation of compound 12c represents a typical
procedure used for the synthesis of chiral compounds described
therein.
6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-2,3-dihydro-
4H-pyrano[2,3-b]pyridin-4-one(5). To a solutionof 6-(2,4-dichlo-
rophenyl)-5-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-car-
bonitrile (4)9 (15.7 g, 41.7 mmol) in THF (100 mL) was added
2-methyl-1-propenylmagnesium bromide (0.5 M in THF, 250 mL,
125 mmol). After being stirred at 50 ꢀC overnight, the mixture was
cooled and the reaction was quenched with 20 mL of H2O. To the
resulting mixture was added 100 mL of 2 N HCl, and the reaction
mixture was stirred at room temperature for 20 min. The reaction
mixture was poured into Et2O (100 mL). The organic layer was
separated and washed with H2O (100 mL), saturated aqueous
NaHCO3 (2 ꢀ 100 mL), and brine. They organic layer was
separated, dried over MgSO4, and concentrated. The residue was
purified by flash chromatography on a silica gel gradient, eluting
with 0-25% EtOAc in hexane to afford the title compound (14.1 g,
78%): 1H NMR δ 1.60 (s, 6H), 2.86 (s, 2H), 7.03-7.30 (m, 7H),
8.25 (s, 1H).
(4S)-6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-
3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-ol (9). A mixture of com-
pound 5 (14.1 g, 32.5 mmol) and [Ru(II)(η6-arene)]-(S,S)-
TsDPEN11 (998 mg, 1.62 mmol) in IPA (20 mL) in CH2Cl2
(10 mL) was stirred at room temperature. After 16 h, the
reaction mixture was concentrated to give the product, which
was taken to the next step without further purification (9.8 g,
69.4%). 1H NMR δ 1.43 (s, 3H), 1.56 (s, 3H), 1.96 (dd, J = 9.7,
13.4, 1H), 2.06 (d, J = 7.3, 1H), 2.28 (dd, J = 6.2, 13.5, 1H), 5.00
(m, 1H), 7.03 (d, J = 8.5, 2H), 7.18-7.26 (m, 5H), 7.90 (s, 1H).
(4R)-4-Azido-6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-
dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine (10). To a mixture
of 9 (9.8 g, 22.5 mmol), Zn(N3)2/bis-pyridine complex13 (13.9 g,
45.1 mmol), triphenylphosphine (11.8 g, 45.1 mmol), and imida-
zole (6.1 g, 90 mmol) in 100 mL of CH2Cl2 was added diisopropyl
azodicarboxylate (8.8 mL, 45.1 mmol) dropwise at room tempera-
ture. The mixture was allowed to stir at room temperature for
16 h. The supernatant was separated and washed with diluted HCl
(1.0 N, 3 ꢀ 50 mL), saturated aqueous NaHCO3 (3 ꢀ 50 mL), and
brine (50 mL). The organic layer was separated, dried over
MgSO4, and concentrated. Chromatography on Biotage 40þM
cartridges using 3:17 v/v EtOAc/hexanes as the eluant afforded the
product (9.5 g, 92%): 1H NMR δ 1.47 (s, 3H), 1.59 (s, 3H), 2.11
(dd, J = 9.6, 13.5, 1H), 2.32 (dd, J = 6.1, 13.6, 1H), 4.75 (dd, J =
6.0, 9.6, 1H), 7.04 (d, J= 8.5, 2H), 7.19-7.28 (m, 5H), 7.78 (s, 1H).
(4R)-6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-
3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-amine (11). To a solution
of 10 (9.5 g, 20.7 mmol) in 50 mL of THF was added 2.5 mL
of H2O and 31.0 mL of trimethylphosphine in THF (1.0 M,
31.0 mmol). After the mixture was stirred at room temperature
for 3 h, the solvent was evaporated. Chromatography on a
Biotage 40þM cartridge using 1:19 v/v CH3OH/CH2Cl2 as the
eluant gave the product (7.2 g, 80%): 1H NMR δ 1.42 (s, 3H),
1.54 (s, 3H), 1.78 (t, J = 12.5, 1H), 2.18 (dd, J = 5.9, 13.4, 1H),
4.16 (dd, J = 6.0, 11.7, 1H), 7.04 (d, J = 8.5, 2H), 7.17-7.26 (m,
5H), 7.93 (s, 1H).
Experimental Section
General. Unless noted otherwise, all materials were purchased
from commercial sources and used without further purification.
Reactions sensitive to moisture or air were performed under
nitrogen or argon using anhydrous solvents and reagents. The
progress of reactions was determined by either analytical
thin layer chromatography (TLC), performed with E. Merck
precoated TLC plates, silica gel 60F-254, and layer thickness of
0.25 mm, or liquid chromatography-mass spectrometry
(LC-MS). Mass analysis was performed on a Waters Micro-
mass ZQ with electrospray ionization in positive ion detection
mode. High performance liquid chromatography (HPLC) was
conducted on an Agilent 1100 series HPLC (LC-1), on Waters
C18 XTerra 3.5 μm, 3.0 ꢀ 50 mm column with gradient 10:90 to
98:2 v/v CH3CN/H2O þ v 0.05% TFA over 3.75 min then held
at 98:2 v/v CH3CN/H2O þ v 0.05% TFA for 1.75 min, with flow
rate of 1.0 mL/min, UV wavelength of 254 nm, or (LC-2) on
Waters C18 XTerra 3.5 μm, 2.1 mm ꢀ 20 mm column with
gradient 10:90 to 98:2 v/v CH3CN/H2O þ v 0.05% TFA over
3.25 min, then held at 98:2 v/v CH3CN/H2O þ v 0.05% TFA for
0.75 min, with flow rate of 1.5 mL/min, UV wavelength 254 nm.
Flash chromatography was performed using a Biotage flash
chromatography apparatus (Dyax Corp.) on silica gel (32-
˚
63 mM, 60 A pore size) in prepacked cartridges of the size
1
noted. H NMR spectra were acquired at 500 MHz spectro-
meters in CDCl3 solutions unless otherwise noted. Chemical
shifts were reported in parts per million (ppm). Tetramethyl-
silane (TMS) was used as internal reference in CD3Cl solutions,
and residual CH3OH peak or TMS was used as internal
reference in CD3OD solutions. Coupling constants (J) were
reported in hertz (Hz). Chiral analytical chromatography was
performed on a Chiralpak AS, Chiralpak AD, Chiralcel OD, or
Chiralcel OJ column (4.6 mm ꢀ 250 mm) (Daicel Chemical
Industries, Ltd.) with noted percentage of either ethanol in
hexane (%Et/Hex) or isopropanol in heptane (%IPA/Hep) as
isocratic solvent systems. Chiral preparative chromatography
was conducted on a Chiralpak AS, Chiralpak AD, Chiralcel
N-[(4R)-6-(4-Chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-
3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-
carboxamide (12c). A mixture of 11 (300 mg, 0.69 mmol),
5-methylpyrazole-3-carboxylic acid (105 mg, 0.83 mmol), PyBOP
(540 mg, 1.04 mmol), and Et3N (0.19 mL, 1.38 mmol) in 20 mL of
CH2Cl2 was stirred at room temperature. After 16 h, the reaction
mixture was diluted with Et2O (50 mL) and washed with saturated
aqueous NaHCO3 (3 ꢀ 50 mL) and brine (3 ꢀ 50 mL). The
organiclayer was separated, driedoverMgSO4, and concentrated.
Chromatography on a Biotage 40þS cartridge using 1:1 v/v

4036 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yan et al.
EtOAc/hexanes as the eluant afforded the desired product
(344 mg, 92%): H NMR δ 1.44 (s, 3H), 1.51 (s, 3H), 1.94 (t,
J = 12.5, 1H), 2.26-2.30 (m, 4H), 5.62 (m, 1H), 6.58 (s, 1H),
6.96 (d, J = 8.5, 2H), 7.12-7.29 (m, 5H), 7.69 (s, 1H). The
product purity was determined by LC-MS to be greater than 98%.
Compound formula C27H23Cl3N4O2, Mr = 541.840, orthorhombic,
D. E.; Van der Ploeg, L. H. T.; Goulet, M. T. Synthesis of functionalized
1,8-naphthyridinones and their evaluation as novel, orally active CB1
receptor inverse agonists. Bioorg. Med. Chem. Lett. 2006, 16, 681–
685. (c) Lin, L. S.; Lanza, T. J.; Jewell, J. P.; Liu, P.; Shah, S. K.; Qi, H.;
Tong, X.; Wang, J.; Xu, S. S.; Fong, T. M.; Shen, C.-P.; Lao, J.; Xiao, J.
C.; Shearman, L. P.; Stribling, D. S.; Rosko, K.; Strack, A.; Marsh, D. J.;
Feng, Y.; Kumar, S.; Samuel, K.; Yin, W.; Van der Ploeg, L. H. T.;
Goulet, M. T.; Hagmann, W. K. Discovery of N-[(1S,2S)-3-(4-
chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifl-
uoromethyl)pyridine-2-yl]oxy}propanamide (MK-0364), a novel, acyclic
cannabinoid-1 receptor inverse agonist for the treatment of obesity.
J. Med. Chem. 2006, 49, 7584–7587. (d) Armstrong, H. E.; Galka, A.;
Lin, L. S.; Lanza, T. J.; Jewell, J. P.; Shah, S. K.; Guthikonda, R.; Truong,
Q.; Chang, L. L.; Quaker, G.; Colandrea, V. J.; Tong, X.; Wang, J.; Xu,
S. S.; Fong, T. M.; Shen, C.-P.; Lao, J.; Chen, J.; Shearman, L. P.; Stribling,
D. S.; Rosko, K.; Strack, A.; Ha, S.; Van der Ploeg, L. H. T.; Goulet, M. T.;
Hagmann, W. K. Substituted acyclic sulfonamides as human cannabinoid-
1 receptor inverse agonists. Bioorg. Med. Chem. Lett. 2007, 17, 2184–
2187. (e) Liu, P.; Lin, L. S.; Hamill, T. G.; Jewell, J. P.; Lanza, T. J.;
Gibson, R. E.; Krause, S. M.; Ryan, C.; Eng, W.; Sanabria, S.; Tong, X.;
Wang, J.; Levorse, D. A.; Owens, K. A.; Fong, T. M.; Shen, C.-P.; Lao, J.;
Kumar, S.; Yin, W.; Payak, J. F.; Springfield, S. A.; Hargreaves, R.; Burns,
H. D.; Goulet, M. T.; Hagmann, W. K. Discovery of N-{(1S,2S)-2-(3-
cyanophenyl)-3-[4-(2-[18F]fluoroethoxy)phenyl]-1-methylpropyl}-2-meth-
yl-2-[(5-methylpyridin-2-yl)oxy]propanamide, a cannabinoid-1 receptor
positron emission tomography tracer suitable for clinical use. J. Med.
Chem. 2007, 50, 3427–3430. (f) Debenham, J. S.; Madsen-Duggan, C. B.;
Wang, J.; Tong, X.; Lao, J.; Fong, T. M.; Schaeffer, M.-T.; Xiao, C. J.;
Huang, C. C. R.-R.; Shen, C.-P.; Stribling, D. S.; Shearman, L. P.; Strack,
A. M.; MacIntyre, D. E.; Hale, J. J.; Walsh, T. F. Pyridopyrimidine based
cannabinoid-1 receptor inverse agonists: synthesis and biological evalua-
tion. Bioorg. Med. Chem. Lett. 2009, 19, 2591–2594. (g) Vachal, P.;
Fletcher, J. M.; Fong, T. M.; Huang, C. C. R.-R.; Lao, J.; Xiao, C. J.; Shen,
C.-P.; Strack, A.; Shearman, L.; Stribling, S.; Chen, R. Z.; Frassetto, A.;
Tong, X.; Wang, J.; Ball, R,. G.; Tsou, N. N.; Hickey, G. J.; Thompson,
D. F.; Faidley, T. D.; Nicolich, S.; Achanfuo-Yeboah, J.; Hora, D. F.; Hale,
J. J.; Hagmann, W. K. 1-Sulfonyl-4-acylpiperazines as selective cannabi-
noid-1 receptor (CB1R) inverse agonists for the treatment of obesity.
J. Med. Chem. 2009, 52, 2550–2558. (h) Du, W.; Jewell, J. P.; Lin, L. S.;
Colandrea, V. J.; Xiao, J. C.; Lao, J.; Shen, C.-P.; Bateman, T. J.; Reddy,
V. B. G.; Ha, S. N.; Shah, S. K.; Fong, T. M.; Hale, J. J.; Hagmann, W. K.
Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cy-
anophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human
cannabinoid-1 receptor (CB1R) inverse agonists. Bioorg. Med. Chem.
Lett. 2009, 19, 5195–5199.
1
˚
˚
˚
P212121, a = 11.1695(8) A, b = 11.2252(8) A, c = 20.0044(14) A,
V = 2508.1(3) A , Z = 4, Dx = 1.435 g cm-3, monochromatized
3
˚
radiation λ(Mo) = 0.710 73 A, μ = 0.40 mm-1, F(000) = 1120,
˚
T = 100 K. Data were collected on a Bruker CCD diffract-
ometer to a θ limit of 28.64ꢀ which yielded 34 470 reflections.
There are 6426 unique reflections with 5500 observed at the 2σ
level. The structure was solved by direct methods (SHELXS-97;
Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473) and
refined using full-matrix least-squares on F2 (SHELXL-97;
Sheldrick, G. M. SHELXL-97. Program for the Refinement of
€
€
Crystal Structures; University of Gottingen: Gottingen,
Germany). The final model was refined using 322 parameters
and all 6426 data. All non-hydrogen atoms were refined with
isotropic thermal displacements. The final agreement statistics
are as follows: R = 0.043 (based on 5500 reflections with I >
2σ(I)), wR = 0.090, S= 1.17 with (Δ/σ)max = 0.01. The maximum
peak height in a final difference Fourier map is 0.293 e A-3, and
˚
this peak is without chemical significance. CCDC contains the
supplementary crystallographic data for this paper (CCDC deposi-
tion number 760723). These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
Acknowledgment. The authors thank Dr. William K.
Hagmann for useful discussions about the manuscript,
Dr. Vincent J. Colandrea for useful discussion regarding
the Noyori chemistry, and Dr. Hugo Vargas and Patricia
Bunting (Department of Safety Assessment, Merck Research
Laboratories, West Point, PA 19486) for CV dog and CNS
mice studies.
Supporting Information Available: 1H NMR, LC-MS, and
chiral analytical HPLC data for compounds 3, 13-16, 8a-8h,
12a, 12b, 12d-n; crystallographic data in CIF format. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(7) (a) Hagmann, W. K. The discovery of taranabant, a selective
cannabinoid-1receptor inverse agonist for the treatment of obesity.
Arch. Pharm. (Weinheim, Ger.) 2008, 341, 405–411. (b) Gantz, I.;
Erondu, N.; Suryawanshi, S.; Musser, B.; Nayee, J.; Johnson-Levonas,
A. O.; Heymsfield, S.; Amatruda, J. A Two-Year Study To Assess the
Efficacy, Safety, and Tolerability of Taranabant in Obese Patients: 52
Week Results. Presented at the 57th Annual Scientific Session, Periph-
eral Arterial Disease; Pharmacology/Hormones;Basic and Clinical
1021-220, American College of Cardiology, Chicago, IL, March 31
through April 1, 2008.
(8) (a) Schwartz, J. I.; Dunbar, S.; Yuan, J.; Li, S.; Miller, D. L.;
Rosko, K.; Johnson-Levonas, A. O.; Lasseter, K. C.; Wagner, J. A.
Influence of taranabant, an orally active, highly selective, potent
cannabinoid-1 receptor (CB1R) inverse agonist, on ethinyl estra-
diol and norelegestromin plasma pharmacokinetics. J. Clin. Phar-
macol. 2009, 49, 72–79. (b) Addy, C.; Rothenberg, P.; Li, S.;
Majumdan, A.; Agrawal, N.; Li, H.; Zhong, L.; Yuan, J.; Maes, A.;
Dunbar, S.; Cote, J.; Rosko, K.; Van Dyck, K.; De Lepeleire, I.; de
Hoon, J.; Van Hecken, A.; Depre, M.; Knops, A.; Gottensdiener, K.;
Stoch, A.; Wagner, J. Multiple-dose pharmacokinetics, pharmacody-
namics, and safety of taranabant, a novel selective cannabinoid-1
receptor inverse agonist, in healthy male volunteers. J. Clin. Pharma-
col. 2008, 48, 734–744.
(9) (a) Debenham, J. S.; Madsen-Duggan, C. B.; Toupence, R. B.;
Walsh, T. F.; Wang, J.; Tong, X.; Kumar, S.; Lao, J.; Fong,
T. M.; Xiao, J. C.; Huang, C. R.-R. C.; Shen, C. P.; Feng, Y.;
Marsh, D. J.; Stribling, D. S.; Shearman, L. P.; Strack, A. M.;
Goulet, M. T. Furo[2,3-b]pyridine-based cannabinoid-1 receptor
inverse agonists: synthesis and biological evaluation part 1.
Bioorg. Med. Chem. Lett. 2010, 20, 1448–1452. (b) Madsen-Duggan,
C. B.; Debenham, J. S.; Walsh, T. F.; Yan, L.; Huo, P.; Wang, J.;
Tong, X. S.; Lao, Z. J.; Fong, T. M.; Xiao, J. C.; Huang, R. C.; Shen, C.;
Stribling, D. S.; Shearman, L. P.; Strack, A. M.; Goulet, M. T.;
Hale, J. J. Dihydro-pyrano[2,3-b]pyridines and tetrahydro-1,8-
naphthyridines as CB1 receptor inverse agonists: synthesis, SAR
and biological evaluation. Bioorg. Med. Chem. Lett., manuscript
accepted.
References
(1) World Health Organization. Obesity Fact Sheet. http://www.who.
int/mediacentre/factsheets/fs311/en/index.html (accessed January
6, 2010).
(2) Finkelstein, E. A.; Trogdon, J. G.; Cohen, J. W.; Dietz, W. Annual
medical spending attributable to obesity: payer- and service-
specific estimates. Health Affairs 2009, 28, w822–w831.
(3) Crowley, V. E. F. Overview of human obesity and central mechan-
isms regulating energy homeostasis. Ann. Clin. Biochem. 2008, 45,
245–255.
(4) (a) Woods, S. C.; D’Alessio, D. A. Central control of body weight
and appetite. J. Clin. Endocrinol. Metab. 2008, 93, S37–S50. (b)
Kunos, G.; Osei-Hyiaman, D.; Liu, J.; Godlewski, G.; Batkai, S.
Endocannabinoids and the control of energy homeostasis. J. Biol.
Chem. 2008, 283, 33021–33025.
(5) (a) Akbas, F.; Gasteyger, C.; Sjodin, A.; Astrup, A.; Larsen, T. M.
A critical review of the cannabinoid receptor as a drug target for
obesity management. Obes. Rev. 2009, 10, 58–67. (b) Janero, D. R.;
Makriyannis, A. Cannabinoid receptor antagonists: pharmacological
opportunities, clinical experience, and translational prognosis. Expert
Opin. Emerging Drugs 2009, 14, 43–65.
(6) (a) Plummer, C. W.; Finke, P. E.; Mills, S. G.; Wang, J.; Tong, X.;
Doss, G. A.; Fong, T. M.; Lao, J. Z.; Schaeffer, M.-T.; Chen, J.;
Shen, C.-P.; Stribling, D. S.; Shearman, L. P.; Strack, A. M.; Van
der Ploeg, L. H. T. Synthesis and activity of 4,5-diarylimidazoles as
human CB1 receptor inverse agonist. Bioorg. Med. Chem. Lett.
2005, 15, 1441–1446. (b) Debenham, J. S.; Madsen-Duggan, C. B.;
Walsh, T. F.; Wang, J.; Tong, X.; Doss, G. A.; Lao, J.; Fong, T. M.;
Schaeffer, M.-T.; Xiao, J. C.; Huang, C. R.-R. C.; Shen, C.-P.; Feng, Y.;
Marsh, D. J.; Stribling, D. S.; Shearman, L. P.; Strack, A. M.; MacIntyre,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4037
(10) Debenham, J. S.; Hale, J. J.; Huo, P.; Madsen-Duggan, C. B.;
Walsh, T. F.; Yan, L. Substituted Pyrano[2,3-b]pyridine Deriva-
tives as Cannabinoid-1 Receptor Modulators and Their Prepara-
tion, Pharmaceutical Compositions and Use in the Treatment of
Diseases. WO 2008094473 A1 20080807, 2008.
(11) Haack, K.-J.; Hashiguchi, S.; Fujii, A.; Ikariya, T.; Noyori, R. The
catalyst precursor, catalyst, and intermediate in the RuII-promoted
asymmetric hydrogen transfer between alcohols and ketones.
Angew. Chem., Int. Ed. Engl. 1997, 36, 285–288.
(12) For better separation on chiral HPLC, S-alcohol 9 and its
R-enantiomer were converted to their corresponding benzoate
esters. The enantiomeric enrichment was determined by chiral
HPLC on ChiralCel OJ 250 mm ꢀ 4.6 mm i.d. and 10 μm silica
gel column with isocratic 5% EtOH in hexane over 40 min, flow
rate of 0.75 mL/min, wavelength of 254 nm.
(13) Viaud, M. C.; Rollin, P. Zinc azide mediated Mitsunobu substitu-
tion, an expedient method for the one-pot azidation of alcohols.
Synthesis 1990, 130–132.
(14) R-Amine 11 and its S-enantiomer were converted to their corre-
sponding acetamides. The enantiomeric enrichment was deter-
mined by chiral HPLC on ChiralPak AS 250 mm ꢀ 4.6 mm i.d.
and 10 μm silica gel column with isocratic 7% EtOH in hexane over
40 min, flow rate of 0.75 mL/min, and wavelength of 254 nm.
(15) (a) Rowland, M.; Matin, S. B. Kinetics of drug-drug interactions.
J. Pharmacokinet. Biopharm. 1973, 1, 553–567. (b) Ito, K.; Hallifax,
D.; Obach, R. S.; Houston, J. B. Impact of parallel pathways of drug
elimination and multiple cytochrome P450 involvement on drug-drug
interactions: CYP2D6 paradigm. Drug Metab. Dispos. 2005, 33, 837.
(c) Shipkova, M.; Wieland, E. Glucuronidation in therapeutic drug
monitoring. Clin. Chim. Acta 2005, 358, 2–23.
(16) For detailed procedures for in vitro pharmacology and in vivo food
intake and body weight studies in mouse and DIO rats, reference to
the following: Fong, T. M.; Guan, X.-M.; Marsh, D. J.; Shen,
C.-P.; Stribling, D. S.; Rosko, K. M.; Lao, J.; Yu, H.; Feng, Y.;
Xiao, J. C.; Van der Pleog, L. H. T.; Goulet, M. T.; Hagmann,
W. K.; Lin, L. S.; Lanza, T. J.; Jewell, J. P.; Liu, P.; Shah, S. K.; Qi,
H.; Tong, X.; Wang, J.; Xu, S. S.; Francis, B.; Strack, A. M.;
MacIntyre, D. E.; Shearman, L. P. Antiobesity efficacy of a novel
cannabinoid-1 receptor inverse agonist, N-[(1S,2S)-3-(4-chloro-
phenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluo-
romethyl)pyridine-2-yl]oxy}propanamide (MK-0364), in rodents.
J. Pharmacol. Exp. Ther. 2007, 321, 1013–1022.
(17) Wang, J.; Della Penna, K.; Wang, H.; Karczewski, J.; Connolly,
T. M.; Koblan, K. S.; Bennett, P. B.; Salata, J. J. Functional and
pharmacological properties of canine ERG potassium channels.
Am. J. Physiol.: Heart Circ. Physiol. 2003, 284, H256–H267.
(18) Metabolism studies were analyzed on a Shimadzu HPLC coupled
to a Thermo Finnagan LTQ mass spectrometer, equipped with a
Packard flow scintillation analyzer.
(19) (a) Chen, R. Z.; Frassetto, A.; Lao, J. Z.; Huang, R.-R. C.; Xiao,
J. C.; Clements, M. J.; Walsh, T. F.; Hale, J. J.; Wang, J.; Tong, X.;
Fong, T. M. Pharmacological evaluation of LH-21, a newly
discovered molecule that binds to cannabinoid CB1 receptor.
Eur. J. Pharmacol. 2008, 584, 338–342. (b) Zimmer, A.; Zimmer,
A. M.; Hohmann, A. G.; Herkenham, M.; Bonner, T. I. Increased
mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor
knockout mice. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5786–5790.
ꢀ
(20) Di Marzo, V.; Despres, J.-P. CB1 antagonists for obesity;what
lessons have we learned from rimonabant? Nat. Rev. Endocrinol.
2009, 5, 633–638.
(21) Griffith, D. A.; Hadcock, J. R.; Black, S. C.; Iredale, P. A.;
Carpino, P. A.; DaSilva-Jardine, P.; Day, R.; DiBrino, J.; Dow,
R. L.; Landis, M. S.; O’Connor, R. E.; Scott, D. O. Discovery of
1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethyla-
mino-piperidine-4-carboxylic acid amide hydrochloride (CP-
945,598), a novel, potent, and selective cannabinoid type 1 receptor
antagonist. J. Med. Chem. 2009, 52, 234–237.
(22) (a) Griebel, G.; Stemmelin, J.; Scatton, B. Effects of the cannabi-
noid CB1 receptor antagonist rimonabant in models of emotional
reactivity in rodents. Biol. Psychiatry 2005, 57, 261–267. (b) Wiley,
J. L.; Martin, B. R. Cannabinoid pharmacological properties common to
other centrally acting drugs. Eur. J. Pharmacol. 2003, 471, 185–193.
ꢀ
~
(c) Navarro, M.; Hernandez, E.; Munoz, R. M.; del Arco, I.; Villanꢀua,
M. A.; Carrera, M. R. A.; de Fonseca, F. R. Acute administration of the
CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like
responses in the rat. NeuroReport 1997, 8, 491–496.