Benzodioxoles: Novel cannabinoid-1 receptor inverse agonists for the treatment of obesity

Article abstract of DOI:10.1021/jm701487t

The application of the evolutionary fragment-based de novo design tool TOPology Assigning System (TOPAS), starting from a known CB1R (CB-1 receptor) ligand, followed by further refinement principles, including pharmacophore compliance, chemical tractabili

Full text of DOI:10.1021/jm701487t

J. Med. Chem. 2008, 51, 2115–2127
2115
Benzodioxoles: Novel Cannabinoid-1 Receptor Inverse Agonists for the Treatment of Obesity
Leo Alig, Jochem Alsenz, Mirjana Andjelkovic, Stefanie Bendels, Agnès Bénardeau, Konrad Bleicher, Anne Bourson,
Pascale David-Pierson, Wolfgang Guba, Stefan Hildbrand, Dagmar Kube, Thomas Lübbers, Alexander V. Mayweg,
Robert Narquizian, Werner Neidhart, Matthias Nettekoven, Jean-Marc Plancher,* Cynthia Rocha, Mark Rogers-Evans,
Stephan Röver, Gisbert Schneider, Sven Taylor, and Pius Waldmeier
F. Hoffmann-La Roche Ltd, Pharmaceuticals DiVision, B092/4.18C, CH-4070 Basel, Switzerland
ReceiVed NoVember 29, 2007
The application of the evolutionary fragment-based de novo design tool TOPology Assigning System
(TOPAS), starting from a known CB1R (CB-1 receptor) ligand, followed by further refinement principles,
including pharmacophore compliance, chemical tractability, and drug likeness, allowed the identification
of benzodioxoles as a novel CB1R inverse agonist series. Extensive multidimensional optimization
was rewarded by the identification of promising lead compounds, showing in vivo activity. These
compounds reversed the CP-55940-induced hypothermia in Naval Medical Research Institute (NMRI)
mice and reduced body-weight gain, as well as fat mass, in diet-induced obese Sprague-Dawley rats.
Herein, we disclose the tools and strategies that were employed for rapid hit identification, synthesis
and generation of structure-activity relationships, ultimately leading to the identification of (+)-[(R)-
2-(2,4-dichloride-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-benzo[1,3]dioxol-5-yl]-morpholin-4-yl-metha-
none (R)-14g. Biochemical, pharmacokinetic, and pharmacodynamic characteristics of (R)-14g are
discussed.
Introduction
(2) Several side-effects arise either from the mechanism of
action itself or from a lack of selectivity, or both.
These facts highlight the shortcoming of current therapies
and the need for safer and more effective treatments of obesity,
possibly as an add-on to existing therapies.
The Cannabinoid-1 Receptor and Food Intake. Among the
constellation of pharmacological effects due to hashish con-
sumption are the alteration of taste and appetite, which were
already reported in 1845 and scientifically investigated in 1970.8
Several experimental studies have shown that 3 ((-)-∆9-
tetrahydrocannabinol) (see Figure 1), the psycho-active constitu-
ent of marijuana, as well as endocannabinoids such as 2 (2-
arachidonoyl glycerol) stimulate appetite and food intake in
animal models.9,10
Scale of the Problem. Presently, the obesity epidemic
represents a significant healthcare burden. As a result of the
massive expansion in the prevalence of obesity in almost all
societies, the World Health Organization officially declared, 10
years ago, human obesity to be one of the most significant health
problems that mankind is facing.1 In the United States, for
example, the prevalence of obesity tripled during the last 25
years.2 Such an obesity pandemic may potentially lead to a
decline of life expectancy in the 21st century.3
Obesity is indeed a major risk factor in the development of
associated diseases such as hypertension, hyperglycemia, dys-
lipidemia, coronary artery disease, and cancer.4,5 The total cost
in the United States for all obesity-related health problems
exceeds $200 billion. More alarmingly, each year, 300 000
people die prematurely in the United States from obesity-related
complications. Consequently, a tremendous need for efficient
therapies to combat obesity and its associated comorbidities
exists. The two main causes for the global rise in prevalence of
obesity are increasingly sedentary lifestyles and high-fat, energy-
rich diets.
Although lifestyle modifications may be the preferred ap-
proach for the treatment of obesity, patients often find diet and
exercise insufficient or unsustainable. The first generation of
centrally mediated antiobesity treatments either act by promoting
neuroreceptor release and/or inhibiting reuptake mechanisms or
act directly on neurotransmitter receptors.6,7 However, these
drugs have had limited success mainly for two reasons:
(1) The discrepancy between patient expectations of up to
30% body-weight loss in the first year, possibly without diet,
and current therapy performance, typically 5-10% body-weight
loss over the same period of time, with specific diet (compared
to a placebo group under the same diet), is hampering patient
compliance.
Furthermore, cannabinoid-1 receptor (CB1R)a subtype knock-
out mice resulted in markedly lower daily food intake and body
weight in mice, even when they were fed with high-fat diet
(HFD).11
These findings suggested the involvement of the endocan-
nabinoid system in the neuronal pathway controlling appetite,
food intake, and ultimately body weight. The CB1R is consti-
tutively active, and therefore, most of the antagonists are
appropriately classified as inverse agonist.12
In the past 10 years, several CB1R antagonist/inverse agonists
including5(SR141716A),13,14 6(SLV319),15 and7(MK-0364)16,17
have been reported to be efficacious in various animal models
a Abbreviations: 5-HT2B, 5-hydroxytryptamine 2B receptor subtype;
CB1R, cannabinoid-1 receptor subtype; CB2R, cannabinoid-2 receptor
subtype; DIO, diet-induced obesity; DMF, N,N-dimethylformamide; FBS:
fetal bovine serum; Fassif, fasted state simulated intestinal fluid; Fessif,
fed state simulated intestinal fluid; IBMX, 3-isobutyl-1-methylxanthine; h,
human; HFD, high-fat diet; HRMS, high-resolution mass spectrum; LYSA,
lyophilization solubility assay; MAB, maximum achievable bioavailability;
MEM, modified Eagle’s medium; MRI, magnetic resonance imaging;
NADPH, reduced nicotinamide adenine dinucleotide phosphate; NMR,
nuclear magnetic resonance; NMRI, Naval Medical Research Institute; PBS,
phosphate buffered saline; SAR, structure-activity relationship; THF,
tetrahydrofuran; TOPAS, topology assigning system.
* To whom correspondence should be addressed. Tel: 41 61 68 86 725.
Fax: 41 61 68 88 714. E-mail: jean-marc.plancher@roche.com.
10.1021/jm701487t CCC: $40.75
2008 American Chemical Society
Published on Web 03/13/2008

2116 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Alig et al.
Figure 1. Some endogenous, natural and synthetic CB1R agonists.
Figure 2. Selected CB1R antagonists/inverse agonists.
Scheme 1. Preparation of 5-H Phenyl Amides and Sulfonamidesa
a Reagents and conditions: (a) N-bromosuccinimide, chloroform; (b) n-BuLi, Et2O, then SO2 (g); (c) N-chlorosuccinimide, chloroform; (d) iPr2NEt, 4-(4-
fluorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride, dichloromethane; (e) tBuOK, tetrahydrofuran (THF), piperidine; (f) n-BuLi, THF, then CO2 (s); (g)
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide (DMF); (h) piperidine, acetonitrile, rt; (i) morpholine,
acetonitrile, rt; (j) trifluoroacetic acid, triethylsilane, rt; (k) 18b, neat, 180 °C.
of obesity and later in patients (see Figure 2). 7 is currently
undergoing phase III clinical trials for obesity and is also under
evaluation as an aid for smoking cessation, whereas 5 has been
approved in the European Union for the treatment of obesity.18
However, the FDA Advisory Committee did not recommend
approval of 5 for use in obese and overweight patients with
associated risk factors, and consequently, the New Drug
Application was withdrawn in the United States.19
Bromination of 2,2-diphenyl-benzo[1,3]dioxole 1920 furnished
preferentially 20,21,22 which was metallated and subsequently
reacted with gaseous sulfur dioxide. The sulfinic acid lithium
salt was subjected to oxidative chlorination leading to 21.
Reaction with various amines led to the desired 2,2-diphenyl-
benzo[1,3]dioxole-5-sulfonic acid amides 11a and 11b (see
Scheme 1).
From the common intermediate 20, the lithiated species was
reacted with solid carbon dioxide to afford the lithium carboxy-
late 22, which was activated as its stable oxybenzotriazole
Chemistry. A general synthetic route for CB1R ligand
preparation is outlined in Scheme 1.

Benzodioxoles: NoVel CB1R InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2117
Scheme 2. Synthesis of Substituted Dichlorodiphenylmethane Derivativesa
a Reagents and conditions: (a) tetrachloromethane, AlCl3, 30 °C; (b) AlCl3, 1,2-dichloroethane, 0 °C; (c) thionyl chloride, DMF, 90 °C.
derivative 2321,23 and subsequently reacted with several amines
to afford amides 12a and 12b. The unsubstituted diphenyl-
methane motif was exchanged by hydrogenolyis and reaction
with the substituted dichlorodiphenylmethane 18b, which was
prepared as shown in Scheme 2 (vide infra). Diphenyldioxo-
methane motifs, either used here as a catechol protecting group
or as a crucial bioactive structural motif, were introduced by
heating the free catechol and the dichlorodiphenylmethane
without solvent at 180 °C for 20 min.20
ring, the use of potassium carbonate during the ketal formation
did not lead to better yield.
Starting from common building block 29, a modular synthetic
scheme was developed for the preparation of the 5-fluoro-phenyl
amide 14a-l and sulfonamide subseries 15a-c (see Scheme
4). Demethylation of 4-fluoroveratrole 29 with BBr3, followed
by selective bromination of the catechol, led to 30, which was
protected with dichlorodiphenylmethane under neat conditions.
Bromine-lithium exchange of 31, facilitated by the presence
of the ortho-fluorine, offered the possibility to use either solid
carbon dioxide or chlorocarbamoyl derivatives as the electro-
phile to afford the carboxylic acid or amides in good yields.
Compounds 14a and 14b were further refined by the exchange
of the diphenylmethane region (see Scheme 4), allowing
structure-activity relationship (SAR) exploration of the left-
hand part. Reduction of the amide 14k to amine 16 was achieved
under standard reduction conditions.
Three different approaches were conveniently used to generate
the required substituted dichlorodiphenylmethanes (see Scheme
2). (1) Friedel-Crafts double addition of halobenzenes onto
tetrachloromethane, catalyzed by AlCl3,24 led quantitatively to
the desired symmetrical compound 18a, with excellent regi-
oselectivity. (2) Friedel-Crafts alkylation of trifluoromethyl-
benzene derivatives with halobenzenes by using AlCl3 afforded
unsymmetrical dichlorodiphenylmethanes. Although strongly
activating and ortho-directing substituents such as methoxy led
to the desired product 18d without detectable amount of
regioisomers, fluorobenzene reacted with 2,4-dichloro-1-triflu-
oromethyl-benzene to give around 20% of 18c along with the
desired material 18b. Dichlorodiphenylmethane derivatives were
found to be sensitive to hydrolysis and were thus used without
further purification. Therefore, separation of the regioisomers
was completed at the following ketal stage. End-products derived
from 18c (see 14l, Table 1), obtained initially as side products,
proved to be biologically interesting, and 18c was obtained
regioselectively by using 2-fluoro-1-trifluoromethyl-benzene and
2,4-dichlorobenzene in the Friedel-Crafts alkylation step. (3)
Alternatively, when a substituted diphenylketone was com-
mercially available, SOCl2-mediated chlorination was the
method of choice to prepare the corresponding dichlorodiphe-
nylmethanes.
Capitalizing on the orthogonal reactivity of the left and right
sides of the benzodioxoles, early introduction of a functionalized
ketal motif, followed by metalation of 32, allowed late stage
derivatization of the right-hand moiety, including amides 14c-l
and sulfonamides 15c.
Alternatively, direct sulfonylation, followed by chlorination27
of the fluoroveratrole 29, leading to 34, shortened the synthesis
of sulfonamides from six to four linear chemical steps.
Investigation on the critical 6-position (benzodioxole numbering)
was accomplished by oxidation of 6-chloropiperonal 3628 and
reaction of the activated acid with morpholine, yielding compound
37 (see Scheme 5). Reaction with BCl3 unlocked the catechol
functionality, which was further reacted with dichlorodiphenyl-
methane under standard conditions to afford 17a.
In turn, 4-methyl catechol 38 proved to be a versatile reagent,
allowing the introduction of various substituents on the 6-posi-
tion. Thus, selective bromination and protection afforded 39.
The 6-methyl derivative 17d was obtained through metalation
of the bromide, whereas oxidation of the methyl group by using
potassium permanganate and subsequent activation of the
resulting acid afforded 17b, containing a bromine functionality
in the 6-position which was subjected to further manipulation,
for example, to introduce a 6-nitrile functionality as in 17c.
4,5-Dihydroxy-pyridine-2-carboxylic acid 27 was obtained
from kojic acid by known procedures (see Scheme 3).25,26 The
routinely applied neat process for the ketal formation, involving
gaseous HCl release, led to very poor yield, and the desired
product 13 was found unstable on silica gel. Although it was
postulated that such unstability, even in weakly acidic media
(data not shown), might be due to the protonation of the pyridine

2118 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Table 1. Activity of Benzodioxolesa
Alig et al.
hCB1R hCB1R EC50 Cl human micros. Cl rat micros.
hypothermia
no. R1
R2
NR3
X
Y
Ki (nM)
(cAMP)
µL/min/mg
µL/min/mg
assay
4-(4-fluoro-phenyl)-1,2,3,
6-tetrahydro-pyridine
piperidinyl
piperidinyl
morpholinyl
morpholinyl
piperidinyl
morpholinyl
piperidinyl
11a
11b H
12a
12b H
H
H
C-H SO2
13
n.t.
n.t.
n.t.
inactive at 30 mg/kg ip
H
H
H
C-H SO2
C-H CO
C-H CO
C-H CO
38
28
50
30
5
15
11
7
2
1
4
4
7
6
46
18
4
9
25
3
n.t.
n.t.
784
156
n.t.
26
1.2
72
5
2
5
11
4
2
466
n.t.
2
3
11
3
n.t.
n.t.
n.t.
n.t.
n.t.
137
53
n.t.
19
n.t.
33
50
48
34
34
31
549
673
n.t.
4
n.t.
272
486
81
88
122
89
22
30
inactive at 30 mg/kg ip
inactive at 30 mg/kg ip
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
ID50: 8 mg/kg po
n.t.
n.t.
ID50: 5 mg/kg po
ID50: 8 mg/kg po
n.t.
n.t.
H
12c 2,4-Cl 4′-F
13
14a
H
H
H
H
H
4′-F
N
CO
C-F CO
C-F CO
C-F CO
C-F CO
C-F CO
C-F CO
C-F CO
C-F CO
C-F CO
14b H
14c 4-F
piperidinyl
14d 2,4-Cl 4′-OMe piperidinyl
14e 2,4-F 2′,4′-F piperidinyl
piperidinyl
morpholinyl
14h 2,4-F 2′,4′-F morpholinyl
14i 2,4-F 2′,4′-F (4,4-difluoro-piperidinyl
14f 2,4-Cl 4′-F
14g 2,4-Cl 4′-F
10
96
17
20
5
46
14j 2,4-F 2′,4′-F (2S)-hydroxymethyl-pyrrolidinyl C-F CO
14k 4-F
14l 2-F
15a 2,4-Cl 4′-F
4′-F
2′,4′-Cl morpholinyl
morpholinyl
morpholinyl
C-F CO
C-F CO
C-F SO2
C-F SO2
C-F SO2
n.t.
21
20
102
271
n.t.
44
n.t.
n.t.
n.t.
n.t.
123
53
149
287
n.t.
388
n.t.
n.t.
n.t.
n.t.
ID50: 6 mg/kg po
ID50: 16 mg/kg po
inactive at 30 mg/kg po
inactive at 30 mg/kg po
n.t.
n.t.
n.t.
n.t.
n.t.
15b 2,4-F 2′,4′-F morpholinyl
15c 2,4-F 2′,4′-F 4,4-difluoro-piperidinyl
16 4-F
17a
17b H
17c
17d H
4′-F
H
H
H
H
morpholinyl
morpholinyl
morpholinyl
morpholinyl
morpholinyl
C-F CH2 >1000
H
C-Cl CO
C-Br CO
C-CN CO
C-Me CO
216
873
277
509
H
a Ki and EC50 were calculated from dose-response curves. n.t.: not tested.
Scheme 3. Synthesis of 4,5-Dihydroxy-pyridine-2-carboxylic Acid Derivativea
a Reagents and conditions: (a) 1,1′-carbonyldiimidazole, piperidine, DMF, 90 °C; (b) dichlorodiphenylmethane, potassium carbonate, DMF, 110 °C.
Those compounds presenting a stereocenter and having an
interesting in vitro profile were routinely resolved by using
preparative chiral HPLC. Attempts to circumvent the chromato-
graphic purification of racemic 14g by crystallization were unsuc-
cessful because no crystals of (S)- or (R)-14g could be obtained.
In contrast, the precursor bromoacetal 32c was a highly crystalline
compound and suitable for X-ray crystallography (see Scheme 6).
Therefore, the intermediate 32c was resolved by using chiral HPLC
and (R)-32c was crystallized in a methanol/water solution. X-ray
diffraction of this single crystal unambiguously attributed the
absolute configuration as being (R). (R)-32c was converted in a
single step to (S)-14g with complete retention of the configuration.
Comparison of the retention time in analytical chiral HPLC of this
compound with the two previously obtained enantiomers of 14g
allowed for the attribution of their absolute configurations (specific
rotation at 20 °C: 589R ) + 7.33 for (R)-14g and 589R ) - 6.62
for (S)-14g). (R)-14g was confirmed as being the bioactive
stereoisomer (vide infra).
the evolutionary fragment-based de novo design tool TOPAS.29,30
In a first step, the TOPAS’ algorithm generated molecular
fragments out of 1381 known GPCR modulators by using a
computer-based retro-synthesis fragmentation scheme. In a
second step, these fragments were reassembled in silico to
molecular structures which are enriched with GPCR ligand
motifs. These virtual compounds were ranked on the basis of
their topological pharmacophore similarity to the seed molecule
(see Figure 3).
The selection of the seed structure was particularly critical
for this effort. Compound 8, derived from the pyrrole series
described by Huffman and co-workers,31 was selected among
others as a seed on the basis of its affinity (hCB1R Ki ) 110
nM), rigidity, comparatively low lipophilicity (as described by
clog P ) 5.68), and molecular weight (420 g ·mol-1).
In addition to the predicted similarity to the seed, other
parameters (including chemical tractability and patentability)
were used to further bias the output generated by using the
TOPAS system. From this exercise, design 9 attracted our
attention and was explored. Of the nine closely related series
which were investigated, only 10 retained significant affinity
to the receptor (hCB1R Ki ) 1.5 µM). Hit evolution, supported
Results and Discussion
Lead Identification. Our aim in this already crowded field
was to rapidly identify novel CB1R ligands by making use of

Benzodioxoles: NoVel CB1R InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2119
Scheme 4. Preparation of 5-Fluoro-phenyl Derivativesa
a Reagents and conditions: (a) BBr3, dichloromethane, -78 to 0 °C; (b) Br2, tetrachloromethane/chloroform/dichloromethane, rt; (c) dichlorodiphenylmethane,
neat, 180 °C; (d) n-BuLi, then CO2 (s), Et2O, -78 °C to rt; (e) 1,1′-carbonyldiimidazole, HNR3, DMF, rt then 90 °C; (f) n-BuLi, then chlorocarbonylamine,
Et2O, -78 °C to rt; (g) trifluoroacetic acid, triethylsilane, 0 °C to rt; (h) 18a-e, neat, 180 °C; (i) LiAlH4, THF, rt; (j) SO3, DMF, 1,2-dichloroethane, 85 °C,
then SOCl2, 85 °C; (k) HNR3, dichloromethane or Et2O, rt; (l) BBr3, dichloromethane, 0 °C to rt; (m) n-BuLi, then SO2 (g), Et2O, -78 to rt; (n)
N-chlorosuccinimide, chloroform, rt; (o) 4,4-difluoropiperidine, Et2O, rt.
by a pharmacophore model and rapid parallel synthesis, led to
a second hit structure 11a.32 Remarkably, only four months were
needed from the seed selection to the hit identification (see
Figure 4).
demonstrated that fluorine was optimal for binding affinity
as well as for functional activation as inverse agonist,
although its effect in improving metabolic stability was less
pronounced (see 14g vs 12c, Table 1).
Although compound 11a complied with our needs in terms
of novelty, affinity (hCB1R Ki ) 13 nM), and selectivity
(hCB2R Ki > 10 µM), the molecular weight and lipophilicity
Diphenylmethyleneketals are known as protecting groups for
catechols and can be cleaved by acidic hydrolysis.33 To
minimize such hydrolysis occurring in our CB1R inverse
agonists, either during their preparation or more critically in
biological fluids, most of the optimization on the phenyl ketal
motif was focused on electron-withdrawing groups: among
these, mainly fluorine and chlorine were introduced, and similar
substitution patterns occurred on the aryl ring decoration of the
5 series. Symmetric 4,4′-difluoro substitution (see 14c) vastly
improved metabolic stability in rat microsomal preparation, most
probably by shielding the metabolically sensitive para-position.
Furthermore, an optimization of the lipophilic interactions in
the CB1R binding pocket was achieved by the addition of
chlorine in the 2- and 4-positions.
Lead Optimization: Biology and Physicochemistry. Ap-
plication of a CB1R agonist (e.g. 4/CP-55940, see Figure 1)
in mice led to a rapid reduction of body temperature
(hypothermia).34 This reduction in body temperature was
antagonized with a CB1R antagonist/inverse agonist, although
the application of our CB1R antagonists/inverse agonists
alone showed no significant effect. Thus, in vivo first-line
screening was based on the antagonist/inverse agonist rever-
sion of the hypothermia induced by 4 in mice. As expected
from microsomal stability and CB1R potency data, applica-
tion of 14d led to a significant activity in the hypothermia
assay (ID50 ) 8 mg/kg po).
were still rather high for a hit compound (MW ) 514 g ·mol-1
clog P ) 7.27).
,
Lead Optimization: Biochemistry. Molecular modeling of
11a and known CB1 ligands revealed that the para-fluorophenyl
residue was not essential for binding and was therefore stripped
off with a modest loss of binding affinity (see Figure 5, 11b).
Replacing the sulfonyl linker (e.g., 11b, hCB1R Ki ) 38 nM)
by a carbonyl linker (e.g., 12a, hCB1R Ki ) 28 nM), in spite
of expected major conformational changes, helped restore the
binding affinity. In contrast, reduction of the amide to the amine
(see 16 vs. 14k, Table 1) led to a dramatic loss of affinity (>1
µM).
Exchanging the phenyl central core by a pyridinyl (see 13,
Table 1), with the expectation to reduce the lipophilicity, led
to an improvement in terms of binding affinity and solubility,
assessed by the lyophilisation solubility assay (LYSA ) 64
mg/L at pH ) 6.5 in aqueous buffer). However, the poor
chemical stability of 13 in weakly acid media prevented further
investigations within this subseries. Replacement of the pyridinyl
by fluorophenyl (see 14a, Table 1) led to systematic and
significant affinity and potency improvements (see 14a vs
12b and 14g vs 12c, Table 1). Investigations around the
6-position (see 12b, 14a, and 17a-d, Table 1) rapidly

2120 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Scheme 5. 6-Position Derivativesa,b
Alig et al.
a Reagents and conditions for 6-chloro derivative: (a) NaClO2, phosphoric acid, H2O2, acetone, H2O, then HCl 3M; (b) 1-(2-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride, N-hydroxybenzotriazole, morpholine, acetonitrile, rt; (c) BCl3, dichloromethane, 0 °C to rt; (d) dichlorodiphenylmethane,
neat, 180 °C. b Reagents and conditions for 6-methyl, 6-bromo, and 6-cyano derivatives: (a) Br2, dichloromethane/chloroform, rt; (b) dichlorodiphenylmethane,
neat, 180 °C, 20 min; (c) KMnO4, pyridine/water, 110 °C; (d) 1,1′-carbonyldiimidazole, DMF, morpholine, rt to 90 °C; (e) CuCN, N-methylpyrrolidone, 190
°C; (f) n-BuLi, THF, then CO2 (s), -78 °C to rt; (g) O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, morpholine, DMF, rt, 20 h.
Scheme 6. Identification of the Absolute Configuration of 14g Enantiomers
The water-octanol partitioning coefficient (log D) for most
of our CB1R ligands exceeded 4 and hence our measurable
window. Consequently, a calculated log P was routinely used
to estimate lipophilicity. Plotting hCB1R binding against clog
P revealed a correlation within several subseries, highlighting
again the preference for compounds of higher lipophilicity in
the receptor binding pocket. An optimization of binding affinity
while closely monitoring physicochemical properties, particu-
larly lipophilicity, was critical in our progress. Thus, binding
affinity (hCB1R Ki) was balanced against other properties, such
as calculated log P, LYSA solubility, and microsomal clearance
(MAB: maximum achievable bioavailability, an inverse function
of microsomal clearance, normalized to rat liver blood flow).
Figure 6 represents a multidimensional visualization. In the
bottom right quadrant, all compounds are characterized by an
excellent affinity (<10 nM) but a rather poor MAB (<40%)
and were therefore of limited interest. In contrast, the carbonyl-
4-morpholine substituent appeared to be an interesting com-
promise between these four first-line screening parameters and
was therefore preferentially kept in the next investigations.
Molecular modeling of the 6-fluorobenzodioxole analogue
(R)-14g predicted a dihedral angle between the amide function

Benzodioxoles: NoVel CB1R InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2121
point was rapidly identified by using the de novo design tool
TOPAS and rapid parallel synthesis. After SAR development
and extensive multidimensional optimization, (R)-14g proved
to have the best overall characteristics for advancement. In an
indirect acute pharmacodynamic model, (R)-14g reversed
dose-dependently the hypothermia induced by 4. Furthermore,
in a 16-day DIO rat model, (R)-14g demonstrated a robust
reduction in body-weight gain, correlated with a reduction
of food intake. In this model, (R)-14g proved to be equipotent
to 5. On the basis of its efficacy and selectivity profile,
compound (R)-14g was selected for further evaluation for
the treatment of obesity.
Experimental Section
Chemistry. General. Proton nuclear magnetic resonance (NMR)
spectra were obtained on a Bruker 250, 300 or 400 MHz instrument
with chemical shifts (δ) reported relative to tetramethylsilane as
an internal standard. Elemental analyses were performed by Solvias
AG (Mattenstrasse, Postfach, CH-4002 Basel). Column chroma-
tography was carried out on silica gel 60 (32-60 mesh, 60 Å) or
on prepacked columns (Isolute Flash Si). Mass spectra were
recorded on SSQ 7000 (Finnigan-MAT) for electron impact
ionization. High-resolution mass spectrum (HRMS) were recorded
on Nanospray Bruker Reflex.
Figure 3. TOPology-Assigning System (TOPAS): Principle.
and the central aromatic ring of approximately 70°, therefore
rather close to the angle estimated for the sulfonamide analogue
11b (∼90°). A re-evaluation of the sulfonamide subseries,
combined with the 6-fluoro substituent, was conducted, leading
indeed to potent compounds. However, metabolic stability was
generally lower for the sulfonamides. Consistent with this
liability is the reduced efficacy during the hypothermia assay
of the sulfonamide subseries vs the amide analogues (see Table
1, 14g vs 15a and 14h vs 15b).
Characterization of (R)-14g. In the hypothermia assay, 14g
demonstrated high efficacy with an ID50 of 5 mg/kg po (see
Table 1), even when applied in its racemic form. Resolution
was performed by using chiral preparative HLPC,35 a method
which proved to be suitable up to the kilogram scale. Despite
the remote stereogenic elements, the binding affinity of the
enantiomer differed by about 1 logarithmic unit (hCB1R Ki )
3 nM for (R)-14g vs 35 nM for the enantiomer (S)-14g).
Selectivity for the CB2R exceeded 1000-fold and was over 300-
fold on a selected panel of 67 receptors, enzymes, and channels.
Figure 7 shows some key characteristics of (R)-14g.
Highly representative for CB1R inverse agonists, (R)-14g is
characterized by a high lipophilicity (clog P ) 5.82) and, despite
optimization efforts, a low solubility in aqueous media (<1 mg/L
in pH ) 6.5 buffer). However, solubility in fasted-state and
fed-state simulated intestinal fluids (Fassif and Fessif) was
considerably higher (129 and 445 mg/L, respectively). Cell
membrane permeation determined in Caco2 cells led to a good
apical-to-basolateral permeation (Pe ) 10.6 ×10-6 cm/s).36
Oral application of (R)-14g reversed the hypothermia induced
by the application of 4 with an ID50 of 4 mg/kg, (see Figure 8).
This value was directly comparable to the performance of 5
used as a positive control.
Detailed Description. 5-Bromo-2,2-diphenyl-benzo[1,3]dioxole
(20). To a mixture of 2,2-diphenyl-benzo[1,3]dioxole20 (27.4 g, 100
mmol) in chloroform (80 mL) was added N-bromosucinimide (18.75
g, 105 mmol). The reaction mixture was refluxed for 20 h, cooled,
and filtered. The filtrate was washed with water, dried over sodium
sulfate, and concentrated in vacuo. The residue was recrystallized
from ethanol to afford the desired product as white crystals (14.15
1
g, 40%). H NMR (400 MHz, DMSO-d6) δ 7.01 (d, J ) 8 Hz,
1H); 7.07 (dd, J ) 8 Hz, <1 Hz, 1H); 7.30 (d, J < 1 Hz, 1H);
7.40-7.50 (m, 6H); 7.50-7.56 (m, 4H). MS (EI) m/e 352 (M+•).
mp: 88-89 °C.
1-(2,2-Diphenyl-benzo[1,3]dioxole-5-sulfonyl)-4-(4-fluoro-phen-
yl)-1,2,3,6-tetrahydro-pyridine (11a). To a mixture of 4-(4-
fluorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (2.56 g, 12
mmol) in dichloromethane (150 mL) was added N-ethyldiisopro-
pylamine (4.2 mL, 25 mmol), and the reaction was stirred for 10
min at room temperature. 2,2-Diphenyl-benzo[1,3]dioxole-5-sul-
fonyl chloride (20) (3.72 g, 10 mmol) was added, and the reaction
was stirred 2 h at room temperature. The solvent was evaporated
in vacuo, and the residue was purified by column chromatography
on silica gel (dichloromethane as eluant). The product was
crystallized from hexane to afford the desired product as white
1
crystals (3.8g, 75%). H NMR (300 MHz DMSO-d6) δ 2.50 (m,
2H); 3.23 (t, J ) 5.6 Hz, 2H); 3.68 (bs, 2H); 6.03 (bs, 1H); 7.09
(t, J ) 8.9 Hz, 2H); 7.26 (d, J ) 8.2 Hz, 1H); 7.36-7.40 (m, 3H);
7.44-7.48 (m, 7H); 7.50-7.54 (m, 4H). MS m/e (IPS) 514.3 (M
+ H+). Anal. (C30H24FNO4S) C, H, N, O, F, S. HRMS C30H24F-
NO4S: 514.14841 (M + H+).
1-(2,2-Diphenyl-benzo[1,3]dioxole-5-sulfonyl)-piperidine (11b).
To a mixture of 2,2-diphenyl-benzo[1,3]dioxole-5-sulfonyl chloride
(20) (2.0 g, 5.4 mmol) and potassium tert-butoxide (840 mg, 7.5
mmol) in THF (20 mL) was added piperidine (1.17 mL, 11.8 mmol).
The dark reaction mixture was stirred 20 h at room temperature
and partitioned between dichloromethane and 0.5 M aqueous
hydrochloric acid. The aqueous phase was extracted with dichlo-
romethane, and the combined organic phases were washed with
saturated aqueous solution of sodium hydrogencarbonate and brine,
concentrated in vacuo, and purified by column chromatography on
silica gel (cyclohexane/ethyl acetate 9:1 as eluant) to afford the
To evaluate (R)-14g in a disease-relevant model, male
Sprague-Dawley rats were fed with a HFD (43% of energy
from fats) 10 weeks prior to the treatment period and during
dosing. Chronic application of (R)-14g (1, 3, and 10 mg/kg)
and 5 (10 mg/kg) for 16 days in this diet-induced obesity (DIO)
rat model led to a slower progression of body weight in all
groups, statistically significant for the 3 and 10 mg/kg groups
with (R)-14g and for the 10 mg/kg group for 5 (see Figure 9).
This dose-dependent body-weight reduction was associated with
a reduction in body fat, measured by magnetic resonance
imaging (MRI).
1
desired product as a white solid (737 mg, 33%). H NMR (300
MHz, CDCl3) δ 1.35-1.50 (m, 2H); 1.60-1.70 (m, 4H); 2.97 (dd,
J ) 5.4, 5.4 Hz, 4H); 6.95 (d, J ) 8.4 Hz, 1H); 7.25 (s, 1H); 7.28
(dd, J ) 8.0, 0.8 Hz, 1H); 7.35-7.50 (m, 6H); 7.53-7.58 (m, 4H).
MS (EI) m/e 422.2 (M + H)+. Anal. (C24H23NO4S) C, H, N, O, S.
HRMS C24H23NO4S: 422.14204 (M + H+).
Conclusion
A series of benzodioxole derivatives was identified as a novel
class of CB1R inverse agonists.23,35,37,38 A chemistry starting

2122 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Alig et al.
Figure 4. From seed to hit within four months by using TOPAS and rapid parallel synthesis.
Figure 5. Modeled overlay of sulfonamide 11a and 5. para-Fluorophenyl (in the orange circle) did not appear essential.
(2,2-Diphenyl-[1,3]dioxolo[4,5-c]pyridin-6-yl)-piperidin-1-yl-
methanone (13). A mixture of 4,5-dihydroxy-2-pyridinecarboxylic
acid25,26 (116 mg, 0.75 mmol) and carbonyldiimidazole (122 mg,
0.75 mmol) in DMF (5 mL) was stirred at room temperature
overnight. Piperidine (75 µL, 0.75 mmol) was added, and the
reaction was heated 5 h at 90 °C. After cooling, the reaction was
poured onto diethyl ether. A solid precipitated. The liquid phase
was decanted from the precipitated solid, and the residue was dried
in vacuo to afford (4,5-Dihydroxy-pyridin-2-yl)-piperidin-1-yl-
methanone 28 (110 mg, 69%) as an off-white solid which was used
without further purification.
extracted with ethyl acetate. The combined organic phases were
washed with saturated aqueous sodium hydrogenocarbonate
solution and brine. The combined organic phases were dried over
sodium sulfate and evaporated in vacuo to afford the desired
product as a brown oil (25 g, 46%) which was used without
further purification. 1H NMR (300 MHz, CDCl3) δ 5.64 (bs,
1H); 6.09 (bs, 1H); 6.49 (ddd, J ) 8.8, 8.6, 2.7 Hz, 1H); 6.64
(dd, J ) 9.3, 2.7 Hz, 1H); 6.78 (dd, J ) 8.8, 5.3 Hz, 1H). MS
(EI) m/e 129.1 (M + H)+.
4-Bromo-5-fluoro-benzene-1,2-diol (30). To a solution of
4-fluoro-benzene-1,2-diol (25 g, 117 mmol) in chloroform (228 mL)
and dichloromethane (46 mL) was added a solution of bromine
(24 g, 150 mmol) in tetrachloromethane (27 mL) over 40 min. The
reaction mixture was stirred 3 h at room temperature, and volatiles
were evaporated in vacuo. Purification was performed by using
column chromatography on silica gel (dichloromethane/ethyl acetate
19:1 as eluant) to afford the desired product as a brown oil (30 g,
(4,5-Dihydroxy-pyridine-2-yl)-piperidin-1-yl-methanone 28 (110
mg, 0.5 mmol) was dissolved in DMF (5 mL) and heated to 100
°C. Potassium carbonate (76 mg, 0.55 mmol) and dichlorodiphe-
nylmethane (0.1 mL, 0.55 mmol) were added, and the reaction was
stirred overnight at 110 °C. After cooling, the mixture was
partitioned between water (50 mL) and ethyl acetate. The aqueous
phase was extracted with ethyl acetate (three times), and the
combined organic phases were washed with water and brine. The
solvents were removed in vacuo, and the residue was purified by
column chromatography on silica gel (dichloromethane/ethyl acetate
1:0-0:1 as eluant) to afford the desired product as white wax (5
1
92%). H NMR (400 MHz, CDCl3) δ 5.34 (s, 1H); 5.66 (s, 1H);
6.73 (d, J ) 12 Hz, 1H); 7.01 (d, J ) 8 Hz, 1H). MS (EI) m/e
207.2 (M + H)+.
5-Bromo-6-fluoro-2,2-diphenyl-benzo[1,3]dioxole (31). A mix-
ture of 4-bromo-5-fluoro-benzene-1,2-diol 30 (102 g, 0.49 mol) and
dichlorodiphenylmethane (140 g, 0.59 mol) was heated at 180 °C
under a stream of nitrogen. After 20 min, the brown mixture was
removed from the oil bath and allowed to cool. The residue was
diluted with methanol (400 mL) under rapid stirring. The product
precipitated after a few minutes. The product was filtered, dried,
and recrystallized from methanol to afford the product as a light-
1
mg, 3%). H NMR (250 MHz, DMSO-d6) δ 1.44-1.59 (m, 6H);
3.32 (m, 2H); 3.56 (m, 2H); 7.33 (s, 1H); 7.45-7.59 (m, 10H);
8.26 (s, 1H). MS m/e (IPS) 387.3 (M + H+).
4-Fluoro-benzene-1,2-diol. To a cold (acetone/dry-ice bath)
solution of boron tribromide in dichloromethane (1 M, 132 mL,
738 mmol) was added the 1-fluoro-3,4-dimethoxybenzene (34 mL,
256 mmol) over 40 min. After the addition, the reaction mixture
was allowed to warm up to room temperature and was stirred for
an additional 3 h. The brown reaction mixture was partioned
between ice-water (1 L) and ethyl acetate (300 mL) and then
1
brown solid (116.5 g, 63%). H NMR (400 MHz, CDCl3) δ 6.72
(d, J ) 7 Hz, 1H); 7.00 (d, J ) 7 Hz, 1H); 7.35-7.45 (m, 6H);
7.5-7.6 (m, 4H). MS m/e 370 (M)+.

Benzodioxoles: NoVel CB1R InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2123
Figure 6. Multidimensional (4D) visualization. Calculated log P and binding affinity tend to correlate in a defined subseries, requiring a compromise
with other properties, i.e., calculated log P, solubility in LYSA, and microsomal clearance expressed as MAB.
Figure 7. Some key characteristics of (R)-14g.
hexane (1.6 M solution, 170 mL, 272 mmol) at a rate such that the
temperature did not exceed -72 °C. The addition was completed
after 30 min, and the mixture was stirred 1 h at -78 °C to afford
a yellow solution. 4-Morpholinecarbonyl chloride (42.7 mL, 371
mmol) was added at a rate such that the temperature did not exceed
-67 °C. A precipitate formed. The cooling mechanism was
removed, and the suspension was allowed to warm by 10 °C (about
1.5 h). The reaction mixture was quenched by the addition of
saturated aqueous sodium bicarbonate solution (200 mL, saturated
solution), and the mixture was extracted with diethyl ether. The
combined organic phases were washed with saturated sodium
bicarbonate solution (300 mL) and brine (500 mL) and dried over
sodium sulfate, and the solution was concentrated to ∼400 mL.
The product crystallized as a white solid (49.0 g, 60%). mp:
1
174-177 °C. H NMR (400 MHz, CDCl3) δ 3.37 (bs, 2H); 3.63
(bs, 2H); 3.75 (bs, 4H); 6.63 (d, J ) 8.4 Hz, 1H); 6.87 (d, J ) 5.6
Hz, 1H); 7.38 (m, 6H); 7.54 (m, 4H). MS (ISP) m/e 406.4 (M +
H)+. Anal. (C24H20FNO4) C, H, N, O, F. HRMS C24H20FNO4:
406.14491 (M + H+).
2,4-Dichloro-1-[dichloro-(4-fluoro-phenyl)-methyl]-benzene
(18b). To a cold (ice bath) suspension of aluminum trichloride
(129.3 g, 0.96 mol) in 1,2-dichloroethane (450 mL) was slowly
added 2,4-dichlorobenzotrifluoride (70 g, 0.32 mol) in 60 min.
Fluorobenzene (30 mL, 0.32 mol) was slowly added to the dark-
red mixture in 60 min. The reaction mixture was stirred 5 h at 0
°C and poured onto crushed ice. The aqueous phase was extracted
with dichloromethane, and the combined organic phases were
washed with water and brine, dried over sodium sulfate, and dried
in vacuo to afford the desired product as a dark-yellow oil (110.4
Figure 8. Reversion of 4-induced hypothermia in mice by using (R)-
14g and 5.
(6-Fluoro-2,2-diphenyl-benzo[1,3]dioxol-5-yl)-morpholin-4-yl-
methanone (14a). To a cooled (-78 °C) solution of 5-bromo-6-
fluoro-2,2-diphenyl-1,3-benzodioxole 31 (91.8 g, 247 mmol) in
diethyl ether (1500 mL) was added a solution of n-butyl lithium in

2124 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Alig et al.
Figure 9. Chronic application of (R)-14g and 5 in a DIO rat model. Dose-dependent reduction of body weight was observed in correlation with
body fat loss.
g, quantitative) which was used without further purification. 1H
NMR (300 MHz, CDCl3) δ 7.0-7.10 (t, 2H, J ) 8.8 Hz);
7.35-7.55 (m, 4H); 8.28 (d, J ) 8.7 Hz, 1H). MS (EI) m/e 325.0
(M + H)+.
5-Bromo-2-(2,4-dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxole (32c). A mixture of 4-bromo-5-fluoro-benzene-
1,2-diol 30 (15 g, 72 mmol) and 2,4-dichloro-1-[dichloro-(4-fluoro-
phenyl)-methyl]-benzene 18b (25.8 g, 80 mmol) was heated 20 min
at 180 °C under a stream of nitrogen. The resulting brown mixture
was purified by column chromatography on silica gel (cyclohexane/
dichloromethane 1:0-19:1 as eluant) to afford the desired product
as a light-yellow oil (21.1 g, 64%). 1H NMR (300 MHz, CDCl3) δ
6.75 (d, J ) 5.7 Hz, 1H); 7.03-7.1 (m, 3H); 7.33 (dd, J ) 8.4, 2.1
Hz, 1H); 7.40-7.45 (m, 3H); 7.72 (d, J ) 8.4 Hz, 1H). MS m/e
(EI) 456, 458 (M).
(R)-5-Bromo-2,2-bis-(2,4-difluoro-phenyl)-6-fluoro-
benzo[1,3]dioxole ((R)-32c). A total of 28 mg of racemic 5-bromo-
2-(2,4-dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-benzo[1,3]-
dioxole 32c was separated into its enantiomers by semipreparative
HPLC by using an analytical Chiralpak AD-H column (250 ×
4.6 mm, 5 µm) in the polar-organic mode by using MeCN/EtOH
96:4 as mobile phase (flow: 1.0 mL/min, detection at 210 nm),
affording approximately 14 mg of each enantiomer. The earlier
eluting enantiomer 32c (tR ) 4.33 min) was then dissolved in
MeOH (1 mL) and treated at room temperature with water (0.1
mL). After 30 min, crystals suitable for X-ray analysis formed.
The absolute configuration of this enantiomer was determined
to be R.
(dd, J ) 8.4, 2.1 Hz, 1H); 7.41-7.51 (m, 4H); 7.73 (d, J ) 8.7
Hz, 1H). MS m/e (EI) 422 (M - H)-.
[2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone (14g). A solu-
tion of 2-(2,4-dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-benzo-
[1,3]dioxole-5-carboxylicacid(4.03g,9.2mmol)andcarbonyldiimida-
zole (1.95 g, 12.0 mmol) in DMF (60 mL) was stirred at room
temperature overnight. Morpholine was added (2 mL, 23 mmol),
and the reaction mixture was stirred 8 h at 90 °C. The reaction
mixture was partitioned between ethyl acetate and aqueous sodium
hydrogenocarbonate solution. The aqueous phase was extracted with
ethyl acetate, and the combined organic phases were washed with
brine, dried over sodium sulfate, filtered, and evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(cyclohexane/ethyl acetate 1:1 as eluant) to afford the product as
1
an off-white foam (4.24 g, 93%). H NMR (300 MHz, CDCl3) δ
3.10 (bs, 2H); 3.38 (bs, 2H); 3.38 (bs, 4H); 6.68 (d, J ) 8.4 Hz,
1H); 6.91 (d, J ) 5.4 Hz, 1H); 7.07 (t, J ) 8.4 Hz, 2H); 7.32 (dd,
J ) 8.7, 2.1 Hz, 1H); 7.40-7.46 (m, 3H); 7.75 (d, J ) 8.7 Hz,
1H). MS m/e (EI) 492.1 (M + H)+. Anal. (C24H17F2Cl2NO4) C, H,
N, O, F, Cl. HRMS C24H17Cl2F2NO4: 492.05748 (M + H+).
(S) and (R) [2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-
phenyl)-benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone ((S)-
and (R)-14g). Preparative HPLC: racemic 2-(2,4-dichloro-phenyl)-
6-fluoro-2-(4-fluoro-phenyl)-benzo[1,3]dioxole-5-carboxylic acid 14
g (250 mg/run) was dissolved in ethanol/heptane (1:1, 4 mL) and
injected onto a preparative chiral HPLC (column, 20 µm ChiralPak
AD, Daicel Chemical Industries, Ltd.; 5 × 50 cm; flow, 35 mL/
min; 15 bar; solvents, heptane/isopropanol 90:10). UV detection
was performed at 225 nm.
2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxole-5-carboxylic Acid. To a cold mixture (dry ice/
acetone bath) of 5-bromo-2-(2,4-dichloro-phenyl)-6-fluoro-2-(4-
fluoro-phenyl)-benzo[1,3]dioxole 32c (16.5 g, 36 mmol) in
diethylether (250 mL) was slowly added a solution of n-butyllithium
in hexane (1.6 M, 23 mL, 37 mmol). The reaction mixture was
stirred 1 h at -78 °C, and solid carbon dioxide (approximately
50 g) was added to the dark solution. The reaction mixture was
allowed to warm up to room temperature to form a copious
precipitate. After 4 h at room temperature, the reaction mixture
was partitioned between aqueous 0.5 N hydrochloric acid solution
and ethyl acetate. The aqueous phase was extracted three times
with ethyl acetate. The combined organic phases were concentrated
in vacuo, and the residue was purified by column chromatography
on silica gel (dichloromethane/methanol 19:1 as eluant) to afford
Analytical HPLC: (column, 20 µm ChiralPak AD, Daicel
Chemical Industries Ltd.; 0.46 × 25 cm; flow, 1 mL/min; 28 bar;
solvents, heptane/isopropanol 90:10).
(S)-(-)-[2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone ((S)-14g).
Yield: 31% (off-white solid). Retention time: 240 min (preparative
HPLC); 31.0 min (analytical HLPC). Enantiomeric ratio (analytical
HPLC): 94%.
Specific rotation: -6.62 °·mL·g-1 ·dm-1 at 589 nm in MeOH;
-37.26 ° · mL · g-1 · dm-1 at 365 nm in MeOH. Anal. (C24H17-
F2Cl2NO4) calc. C ) 58.55%, H ) 3.48%, N ) 2.85%, O )13.00%,
F ) 7.72%, Cl ) 14.40%; found: C ) 58.25%, H ) 3.56%, N )
3.07%, O ) 13.43%, F ) 7.72%, Cl ) 14.04%. HRMS
C24H17Cl2F2NO4: 492.05761 (M + H+).
1
the product as a yellow solid (4.48 g, 29%). H NMR (300 MHz,
CDCl3) δ 6.73 (d, J ) 9.9 Hz, 1H); 7.08 (t, J ) 8.5 Hz, 2H); 7.33

Benzodioxoles: NoVel CB1R InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2125
(R)-(-)-[2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone ((R)-14g).
Yield: 43% (off-white solid). Retention time: 295 min (preparative
HPLC); 35.0 min (analytic HLPC). Enantiomeric ratio (analytical
HPLC): 99%. Specific rotation: +7.33 °·mL·g-1 ·dm-1 at 589 nm
in MeOH; +41.83 °·mL·g-1 ·dm-1 at 365 nm in MeOH. Anal.
(C24H17F2Cl2NO4) C, H, N, O, F, Cl. HRMS C24H17Cl2F2NO4:
492.05759 (M + H+).
under a stream of nitrogen. The resulting brown mixture was
purified by column chromatography on silica gel (heptane/ethyl
acetate 3:1 as eluant) to afford the product as an off-white foam
(374 mg, 87%). 1H NMR (400 MHz, CDCl3) δ 3.16 (m, 4H); 3.74
(m, 4H); 6.80 (d, J ) 9 Hz, 1H); 7.07-7.11 (m, 2H); 7.30-7.35
(m, 2H); 7.40-7.46 (m, 3H); 7.71 (d, J ) 8 Hz, 1H). MS m/e
528.1 (M+H)+. Anal. (C23H17F2Cl2NO5S) C, H, N, O, F, Cl, S.
HRMS C23H17Cl2F2NO5S: 528.02479 (M + H+).
In Vivo Assays. LYSA. Samples were prepared in duplicate from
10 mM DMSO stock solutions. After evaporation (1 h) of DMSO
with a centrifugal vacuum evaporator (Genevac Technologies), the
compounds were dissolved in 50 mM phosphate buffer (pH 6.5),
stirred for 1 h, and shaken 2 h. After one night, the solutions were
filtered by using a microtiter filter plate (Millipore MSDV N65),
and the filtrate and its 1/10 dilution were analyzed by direct UV
measurement or by HPLC-UV. In addition, a four-point calibration
curve was prepared from the 10 mM stock solutions and used for
the solubility determination of the compounds. The results are
expressed in µg/mL. Starting from a 10 mM stock solution, the
measurement range for MW 500 was 0-666 µg/mL. In the case
where the percentage of sample measured in solution after evapora-
tion divided by the calculated maximum of sample amount was
larger than 80%, the solubility was reported as larger than this value.
Solubility in Simulated Intestinal Fluids. Approximately 1-2
mg of each compound was added in excess into about 300 mL of
50 mM buffer (either Fassif or Fessif buffer) at room temperature.
Composition of Fassif buffer: pH 6.5; KH2PO4, 3.9 g/L; KCl, 7.7
g/L; sodium taurocholate, 1.6 g/L; lecithin, 0.6 g/L. Composition
of Fessif buffer: pH 5.0; acetic acid, 8.64 g/L; KCl, 15.2 g/L;
sodium taurocholate, 8.08 g/L; lecithin, 2.92 g/L. Each sample was
placed in a microanalysis tube, sonicated 1 h, and agitated 2 h. All
suspensions were left overnight. The following day, pH was
measured with a pH-meter, and the samples were filtered with a
micronic filterplate (MSGVN2250) to separate any solid material
from the solution. All solutions were analyzed by HPLC. The
calibration line was established from five different dilutions of the
compound in a DMSO stock solution (concentration approximately
1 mg/mL). From this regression equation, the solubility of the
compound was determined. In cases where the drug was completely
dissolved in the buffer, the value for equilibrium solubility was
assumed to be higher than the value determined by HPLC and
reported as such.
Rat and Human Microsomal Clearances. Human or rat
microsome incubations were conducted by an automated procedure
implemented on a Genesis workstation (Tecan, Switzerland).
Compounds (2 µM) were incubated in microsomes at 0.5 mg protein
per mL in a 50 mM potassium phosphate buffer, pH 7.4, at 37 °C.
Cofactor (reduced nicotinamide adenine dinucleotide phosphate,
NADPH) was produced by a generating system (glucose-6-
phosphate 3.2 mM, ꢀ-nicotinamide adenine dinucleotide phosphate
2.6 mM, MgCl2 6.5 mM). Addition of the NADPH generating
system to the prewarmed microsomes containing the test
compound started the reaction. Aliquots (50 µL) were taken at
six defined time points within 30 min and transferred into 100
µL of methanol containing an internal standard. Concentration
of each compound was analyzed by LC-MS/MS by using a
Synergy-4-Polar RP 18 column (Phenomenex, Torrance, CA).
Quantitative detection was achieved on a SCIEX 2000 instrument
(MDS Sciex, Concord, ON, Canada) by using electron spray
ionization. Concentrations were determined by the ratio of test-
compound and internal-standard peaks and given as a percentage
of the concentration measured at the first time point (substrate
depletion). Intrinsic clearance (CLint, in µL min-1 mg-1 mi-
crosomal protein) is the rate constant of the first-order decay of
the test compound, normalized for the protein concentration in
the incubation. Two values were calculated to normalize this
measured intrinsic clearance to an allometric scale:
(S)-[2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxol-5-yl]-morpholin-4-yl-methanone ((S)-14g). A
solution of (R)-5-bromo-2,2-bis-(2,4-difluoro-phenyl)-6-fluoro-
benzo[1,3]dioxole (R)-32c (2.0 mg, 4.4 µmol) in THF (16 µL) was
treated at 0-5 °C with a solution of isopropyl magnesium chloride
in THF (2 M, 22 µL, 44 µmol), and the resulting mixture was stirred
1 h at 0 °C. To the clear solution was added 4-morpholine carbonyl
chloride (10.3 µL, 88 µmol). The reaction mixture was diluted with
THF (2 mL) and quenched with water (100 µL). The resulting
mixture was dried over MgSO4 and evaporated in vacuo. The
residue was purified by column chromatography on silica gel (1 g
silica gel, heptane/ethyl acetate 10:1 as eluant), and the resulting
product was analyzed by enantioselective HPLC and identical to
the sample prepared above by chiral HPLC resolution of 14g.
2-Fluoro-4,5-dimethoxy-benzenesulfonyl chloride (34). To a
suspension of sulfur trioxide/DMF complex (4.108 g, 22.4 mmol)
in 1,2-dichloroethane (10 mL) was added 4-fluoroveratrole (3.49
g, 26.8 mmol) dropwise. The mixture was slowly heated to 85 °C
in an oil bath, and heating was continued for 7 h. The oil bath was
removed, and thionyl chloride (1.95 mL, 32 mmol) was added
dropwise. The mixture was heated 4 h at 85 °C and allowed to
cool to room temperature. The solution was poured into water and
extracted with dichloromethane. The combined organic phases were
washed with water, dried over magnesium sulfate, and evaporated
to afford the product as an off-white solid (5.5 g, 96%) which was
1
used without further purification. H NMR (400 MHz, CDCl3) δ
3.93 (s, 3H); 3.97 (s, 3H); 7.31 (d, J ) 6 Hz, 1H); 7.79 (d, J ) 11
Hz, 1H). MS m/e 254.0 (M)+.
4-(2-Fluoro-4,5-dimethoxy-benzenesulfonyl)-morpholine. To
a solution of 2-fluoro-4,5-dimethoxy-benzenesulfonyl chloride 34
(5.3 g, 20.8 mmol) in dichloromethane (30 mL) was added
morpholine (3.81 mL, 43.7 mmol) dropwise. The mixture was
stirred overnight at room temperature, filtered through a pad of silica
gel, and eluted with ethyl acetate. The solvent was evaporated under
reduced pressure to afford the product as a pale-yellow solid (5.99
g, 94%) which was used without further purification. A small
sample was purified by column chromatography on silica gel (10
g, heptane/ethyl acetate 1:1 as eluant) to afford the product as a
colorless solid. 1H NMR (400 MHz, CDCl3) δ 3.16 (m, 4H); 3.75
(m,4H); 3.90 (s, 3H); 3.94 (s, 3H); 6.73 (d, J ) 11 Hz, 1H); 7.21
(d, J ) 6 Hz, 1H). MS m/e 305.1 (M)+.
4-Fluoro-5-(morpholine-4-sulfonyl)-benzene-1,2-diol (35). To
a cooled (ice bath) solution of 4-(2-fluoro-4,5-dimethoxy-benze-
nesulfonyl)-morpholine (5.1 g, 16.7 mmol) in dichloromethane (100
mL) was added boron tribromide (1 M in dichloromethane, 50 mL,
50 mmol) dropwise over 30 min, and the mixture was stirred
overnight at room temperature. The brown solution was slowly
added to 1 M aqueous KH2PO4 (200 mL), and the mixture was
stirred 1 h at room temperature. The phases were separated, and
the aqueous phase was extracted with ethyl acetate. The combined
organic phases were washed with brine, dried over magnesium
sulfate, and evaporated. The resulting brown oil was purified by
column chromatography on silica gel (dichloromethane/ethanol/
acetic acid 18:1:0.1-10:1:0.1 as eluant) to afford the product as a
light-brown oil (3.55 g, 76%) which solidified upon standing in
1
the refrigerator. H NMR (400 MHz, DMSO-d6) δ 2.93 (m, 4H);
3.62 (m, 4H); 6.76 (d, J ) 12 Hz, 1H); 7.04 (d, J ) 7 Hz, 1H); 9.7
(br s, 1H); 10.5 (br s, 1H). MS m/e 276.0 (M - H)-.
4-[2-(2,4-Dichloro-phenyl)-6-fluoro-2-(4-fluoro-phenyl)-
benzo[1,3]dioxole-5-sulfonyl]-morpholine (15a). A mixture of
4-fluoro-5-(morpholine-4-sulfonyl)-benzene-1,2-diol 35 (225 mg,
0.81 mmol) and 2,4-dichloro-1-[dichloro-(4-fluoro-phenyl)-methyl]-
benzene 18b (329 mg, 1.01 mmol) was heated 20 min at 180 °C
hepatic clearance ) (hepatic blood flow × CLint × protein
concentration in microsome incubation × liver weight per kg body
weight)/(hepatic blood flow + CLint × protein concentration in
microsome incubation × liver weight per kg body weight)

2126 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Alig et al.
MAB ) 100 × (1 - (hepatic clearance/hepatic blood flow))
(R)-14g Counterscreening. (R)-14g counterscreening on a panel
of 67 receptors, enzymes, and channels was done by Cerep
Laboratories (Le bois l’Eveque, F-86 600 Celle L’Evescault, France)
at a maximal concentration of 10 µM (see Supporting Information).
In Vivo Assays. Hypothermia Assay in NMRI Mice. Animal.
Male NMRI mice were used in this study and were obtained from
Research Consulting Company Ltd. of Füllinsdorf (Switzerland).
A total of 64 mice, weighing 29-32 g, were used in this study.
Ambient temperature was 20-21 °C, and relative humidity was
55-65%. A 12-h light-dark cycle was maintained in the rooms
with all tests being performed during the light phase. Access to
tap water and food was ad libitum.
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Body temperature was measured:
- 20 min before administration of either vehicle or tested
substance.
- Immediately before the injection of either vehicle or CP 55940.
- 20 min after administration of either vehicle or CP 55940.
Either vehicle or tested substance was administered 90 min before
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catalogue no. 0949, batch no. 6/23039) were dissolved in 0.3%
Tween 80. The specific volume of administration was 10 mL/kg.
DIO Model. Animal. A group of 3-week old Sprague-Dawley
rats (Iffa Credo/Charles River, France), with homogeneous body-
weight values, was fed either with HFD (KLIBA 2157, 43% of
energy, 19% fat, n ) 90) or with a chow diet (n ) 10). The chow-
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and before the randomization. After a period of 10 weeks, obese
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to rats for which the difference between their average body weight
and that of chow-diet rats was at least 2 times the standard deviation
of the animals from the chow-diet group [body weight (obese) -
body weight (chow) > 2 SD (chow)].
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during the growing period. After 3 days of gavage adaptation, the
rats received a daily dose of compounds per gavage at 5:00 p.m.
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measured daily. The total body fat mass and the lean mass content
were measured in the MRI laboratory before and at the end of the
treatment.
Drug. Test substances were dissolved in water with 7.5% of
gelatine and 0.62% of sodium chloride. The specific volume of
administration was 2 mL/kg.
Acknowledgment. Authors wish to thank Beat Frei and
Daniel Zimmerli for their contributions to the chiral separation.
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