Synthesis and activity of 1,3,5-triphenylimidazolidine-2,4-diones and 1,3,5-triphenyl-2-thioxoimidazolidin-4-ones: Characterization of new CB 1 cannabinoid receptor inverse agonists/antagonists

Article abstract of DOI:10.1021/jm050484f

Obesity and metabolic syndrome, along with drug dependence (nicotine, alcohol, opiates), are two of the major therapeutic applications for CB ₁ cannabinoid receptor antagonists and inverse agonists. In the present study, we report the synthesis and structure-affinity relationships of 1,5-diphenylimidazolidine-2,4-dione and 1,3,5-triphenylimidazolidine-2,4-dione derivatives. These new 1,3,5-triphenylimidazolidine-2,4-dione derivatives and their thio isosteres were obtained by an original pathway and exhibited interesting affinity and selectivity for the human CB₁ cannabinoid receptor. A [³⁵S]-GTPγS binding assay revealed the inverse agonist properties of the compounds at the CB₁ cannabinoid receptor. Furthermore, molecular modeling studies were conducted in order to delineate the binding mode of this series of derivatives into the CB₁ cannabinoid receptor. 1,3-Bis(4-bromophenyl)-5-phenylimidazolidine-2,4-dione (25) and 1,3-bis(4-chlorophenyl)-5-phenylimidazolidine-2,4-dione (23) are the imidazolidine-2,4-dione derivatives possessing the highest affinity for the human CB₁ cannabinoid receptor reported to date.

Full text of DOI:10.1021/jm050484f

872
J. Med. Chem. 2006, 49, 872-882
Synthesis and Activity of 1,3,5-Triphenylimidazolidine-2,4-diones and
1,3,5-Triphenyl-2-thioxoimidazolidin-4-ones: Characterization of New CB1 Cannabinoid
Receptor Inverse Agonists/Antagonists
Giulio G. Muccioli,† Johan Wouters,‡ Caroline Charlier,‡ Gerhard K. E. Scriba,§ Teresa Pizza,† Pierluigi Di Pace,†
Paolo De Martino,† Wolfgang Poppitz,| Jacques H. Poupaert,† and Didier M. Lambert*,†
Unite´ de Chimie pharmaceutique et de Radiopharmacie, Ecole de Pharmacie, Faculte´ de Me´decine, UniVersite´ catholique de LouVain,
AVenue E. Mounier 73, UCL-CMFA 7340, B-1200 Bruxelles, Belgium, Laboratoire de Chimie Biologique Structurale, Faculte´ des Sciences,
UniVersity of Namur, Rue de Bruxelles 61, 5000 Namur, Belgium, Department of Pharmaceutical Chemistry, UniVersity of Jena,
Philosophenweg 14, D-07743 Jena, Germany, and Department of Inorganic and Analytical Chemistry, UniVersity of Jena,
August-Bebel-Strasse 2, D-07743 Jena, Germany
ReceiVed May 24, 2005
Obesity and metabolic syndrome, along with drug dependence (nicotine, alcohol, opiates), are two of the
major therapeutic applications for CB1 cannabinoid receptor antagonists and inverse agonists. In the present
study, we report the synthesis and structure-affinity relationships of 1,5-diphenylimidazolidine-2,4-dione
and 1,3,5-triphenylimidazolidine-2,4-dione derivatives. These new 1,3,5-triphenylimidazolidine-2,4-dione
derivatives and their thio isosteres were obtained by an original pathway and exhibited interesting affinity
and selectivity for the human CB1 cannabinoid receptor. A [35S]-GTPγS binding assay revealed the inverse
agonist properties of the compounds at the CB1 cannabinoid receptor. Furthermore, molecular modeling
studies were conducted in order to delineate the binding mode of this series of derivatives into the CB1
cannabinoid receptor. 1,3-Bis(4-bromophenyl)-5-phenylimidazolidine-2,4-dione (25) and 1,3-bis(4-chlo-
rophenyl)-5-phenylimidazolidine-2,4-dione (23) are the imidazolidine-2,4-dione derivatives possessing the
highest affinity for the human CB1 cannabinoid receptor reported to date.
Introduction
The wide application for CB1 cannabinoid receptor antago-
nists or inverse agonists as pharmacological agents prompted
the synthesis and characterization of new classes of com-
pounds.21 Indeed, in addition to the biarylpyrazoles (SR141716A,
SR14777822), several other heterocyclic compounds such as
triazoles,23,24 thiazoles,24 pyrazolines,25 imidazoles,24,26 and
pyridines27 have been described.
The cloning of the cannabinoid CB1 and CB2 receptors in
the early 1990s1,2 followed by the discovery and characterization
of their endogenous ligandssi.e. anandamide3 (AEA) and
2-arachidonoylglycerol4,5 (2-AG)sas well as the characterization
of the enzymes responsible for endocannabinoid degradations
i.e. fatty acid amide hydrolase,6 monoglyceride lipase,7 and
N-acylethanolamine-hydrolyzing acid amidase8soffered new
areas for therapeutic interventions.9 Clinical trials involving
either cannabinoid receptor ligands or Cannabis satiVa extracts10
demonstrated pharmacological activities. For instance, ∆9-
tetrahydrocannabinol (∆9-THC) was recently shown to be
effective in the treatment of Tourette’s syndrome tics11 and in
the relief of pain.12
Along this line, our group previously reported the synthe-
sis28,29 and pharmacological characterization of 3-alkyl-5,5′-
diphenylimidazolidine-2,4-dione28,30 and 3-alkyl-5,5′-diphenyl-
2-thioxoimidazolidin-4-one31 derivatives as cannabinoid ligands.
These compounds, exhibiting moderate affinity, act as selective
inverse agonists at the CB1 cannabinoid receptor. The structure-
affinity relationships obtained to date revealed three important
features to enhance the affinity toward the receptor: (i) the N3
nitrogen must be substituted, (ii) introduction of an halogen
substituent in para position of the phenyl rings increases the
affinity, (iii) thio derivatives are more active than the corre-
sponding oxo derivatives.
In this field, CB1 cannabinoid receptor antagonists or inverse
agonists exhibited interesting therapeutic potential while being
devoid of psychotropic side effects.13-15 The first reported CB1
cannabinoid receptor inverse agonist, rimonabant16 (SR141716A,
Acomplia), was successfully tested in humans as treatment for
obesity17 (and associated metabolic syndrome) and smoking
cessation (Figure 1).18 Another promising application for such
drugs is the treatment of drug dependence (alcohol, opiates, ∆9-
THC) as indicated by both animal and human studies.19 Finally,
recent studies in rats using the two inverse agonists SR141716A
and LY320135 suggested that CB1 cannabinoid receptor inverse
agonists may be effective in reducing brain injury caused by
focal ischemia.20
In the present study, new substitution patterns of the imida-
zolidine-2,4-dione ring, leading to the 1,5-diphenylimidazoli-
dine-2,4-dione and to the 1,3,5-triphenylimidazolidine-2,4-dione
series, were explored as new potential human CB1 cannabinoid
receptor ligands. The latter series of compounds was synthesized
in good yields by a previously unreported method from
phenylglyoxal and 1,3-diphenylurea. The pharmacological
properties of 1,5-diphenylimidazolidine-2,4-dione (6-18), 1,3,5-
triphenylimidazolidine-2,4-dione (20-29), and 1,3,5-triphenyl-
2-thioxoimidazolidin-4-one (30-38) derivatives at the human
CB1 cannabinoid receptor were characterized. Finally, the results
allowed the establishment of structure-activity relationships,
aiming at the characterization of this new series of CB1
cannabinoid receptor inverse agonists.
* Corresponding author. Phone: +32 2 7647347. Fax: +32 2 7647363.
lambert@cmfa.ucl.ac.be.
† Universite´ catholique de Louvain.
‡ University of Namur.
§ Department of Pharmaceutical Chemistry, University of Jena.
| Department of Inorganic and Analytical Chemistry, University of Jena.
10.1021/jm050484f CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/13/2006

New CB1 Receptor InVerse Agonists/Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 873
Figure 1. Structures of derivative 25 and of SR141716, WIN-55,212-2, and HU-210.
Scheme 1. Synthesis of the
Scheme 2. Synthesis of the
1,5-Diphenylimidazolidine-2,4-dione (6, 8-18) and
1,3,5-Triphenylimidazolidine-2,4-dione (20-29) and
1,3,5-Triphenyl-2-thioxoimidazolidin-4-one (30-38)
Derivativesa
1,5-Diphenyl-2-thioxoimidazolidin-4-one (7) Derivativesa
a Reagents and conditions: (a) SeO2, dioxane-water (70:4), 55 °C,
overnight; (b) CH3COOH/H2O, NaOCN, rt; (b′) acetone, NH4OH, rt; (c)
CH3COOH-HCl (15:0.5), reflux, 4 h; (d) CH2Cl2, pyridine, COCl-R, rt,
overnight; (e) DMF, K2CO3, tetrabutylammonium bromide, Cl-R, 3 × 1.5
min microwave pulses (100 W).
Results and Discussion
Chemistry. Substituted phenylglyoxal derivatives (2, X1 )
Cl, Br) were synthesized from the corresponding acetophenones
(1) using selenium dioxide as oxidant according to a method
described by Riley and Gray32 (Scheme 1). Distillation of the
resulting oil (substituted phenylglyoxal) followed by recrystal-
lization from water was usually sufficient to remove selenium
contaminants from substituted phenylglyoxal.
Substituted phenylurea derivatives (5, X′ ) O) were easily
obtained in high yields by reacting the corresponding aniline
(3) with sodium cyanate in an acetic acid-water mixture33
(Scheme 1). The reaction was almost complete, and the resulting
phenylureas were used without further purification. The phe-
nylthiourea (5, X′ ) S) was obtained in high yields by reacting
phenyl isothiocyanate (4, X′ ) S) with ammonia.
The 1,5-diphenylimidazolidine-2,4-dione derivatives (6, 8-14,
X′ ) O) were synthesized from phenylglyoxal and phenylurea
by slightly modifying the procedure described by Joshi et al.34
(Scheme 1). Four hours of reflux in an acetic acid-hydrochloric
acid mixture yielded the 1,5-diphenylimidazolidine-2,4-dione
derivatives in good yields (59-64%). The same procedure using
phenythiourea afforded the 1,5-diphenyl-2-thioxoimidazolidin-
4-one (7, X′ ) S).
a Reagents and conditions: (a) acetone, rt, 6 h; (b) CH3COOH-HCl
(20:0.5), reflux, 6 h.
The target 1,3,5-triphenylimidazolidine-2,4-dione derivatives
(20-29) were obtained by an original pathway starting from
1,3-diphenylurea (19, X′ ) O) and phenylglyoxal (2) as
described in Scheme 2. This reaction represents a simple one-
pot procedure to obtain in moderate to good yields 20-29.
However, it has to be mentioned that this reaction pathway does
not allow for the synthesis of enantiopure compounds. The
structure of the triphenylimidazolidine-2,4-dione products was
confirmed by the X-ray diffraction of compound 22 (Figure 2).
Interestingly, spontaneous resolution was observed upon crystal-
lization. Indeed, the crystallization conditions of 22 yielded
crystals presenting a preferential enrichment of the R enantiomer
(3:1 ratio for the R/S enantiomers) starting from a racemic
mixture of the product. Similar spontaneous resolution of
racemic mixtures, although rarely observed in the natural work,
has been reported in other series of molecules, in particular for
1,3,5-triphenyl-2-pyrazoline.36
No classical H-bond is observed within the crystal packing
of 22. This is explained by the lack of any suitable H-bond
donor within the molecule. In particular, substitution of N1 by
a phenyl eliminates this center as sole potential H-bond donor,
in contrast with what is observed with N1 unsubstituted
analogues.29,31 Interestingly, short intramolecular C-H···O bonds
(2.6-3.0 Å) involve the oxygen atoms in position O1 and the
H atoms in the ortho positions of the aromatic rings substituting
both N atoms. These intramolecular interactions influence the
conformation and relative orientation of the aromatics around
Derivatives 15 and 16 were obtained in high yields by
acylation of 6 with benzoyl chloride and hexanoyl chloride,
respectively, in a CH2Cl2-pyridine mixture (Scheme 1).
Derivatives 17 and 18 were prepared by alkylation of 6 using
a microwave-assisted procedure adapted from Bogdal et al.35
(Scheme 1).
Reacting the aniline 3 with phenyl isocyanate (4, X′ ) O)
and phenyl isothiocyanate (4, X′ ) S) afforded in good yields
the 1,3-diphenylurea (19, X′ ) O) and 1,3-diphenylthiourea (19,
X′ ) S) derivatives, respectively (Scheme 2).

874 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Muccioli et al.
Scheme 3. Synthesis of
1,3-Dicyclohexyl-5-phenylimidazolidine-2,4-dione (40)a
a Reagents and conditions: (a) CH3COOH-HCl (20:0.5), reflux, 10 h.
Table 1. Percentages of Displacement of [3H]-SR141716A and
[3H]-CP-55,940, Respectively, by Synthesized
1,5-Diphenylimidazolidine-2,4-diones (10 µM) on hCB1 and hCB2
Cannabinoid Receptorsa
% displacement
Figure 2. ORTEP diagram of 22 representing one molecule of the
asymmetric unit. Both enantiomers were present in the crystal.
hCB1
hCB2
compd
X′
X1
X2
R
receptor
receptor
the imidazolidine-2,4-dione scaffold and could play a role during
the binding to the receptor.
6
7
8
O
S
H
H
H
H
H
H
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
O
O
O
O
O
O
O
O
O
O
O
Cl
Br
I
OMe
H
Cl
Br
H
<15
Crystal cohesion of 22 mainly results from stacking interac-
tions that involve the π-system of the aromatic rings. All three
phenyl rings are involved in these intermolecular interactions.
In addition, the central imidazolidine-2,4-dione ring is engaged
in T-shape perpendicular interactions with neighboring mol-
ecules. The CO bond of one imidazolidine-2,4-dione ring points
to the center of an imidazolidine-2,4-dione ring of a symmetry-
related molecule.
9
25.2 ( 1.2
<20
10
11
12
13
14
15
16
17
18
<10
<15
<15
<15
CO-C6H5
CO-C5H11
C5H11
<10
H
H
H
<10
41.1 ( 1.2
45.6 ( 3.5
C7H15
Taken together, the crystallographic data obtained on 22
confirm the structure of the compound and the presence of both
enantiomers in the mixture. They further suggest that the central
imidazolidine-2,4-dione ring could influence the relative orienta-
tion of the aromatic substituents within the molecule (through
intramolecular H-bonds), allowing them to form proper π-stack-
ing interactions with residues of the protein during binding to
a receptor. The conformation of 22 obtained by X-ray crystal-
lography is also an excellent starting point for modeling of the
different 1,3,5-triphenylimidazolidine-2,4-dione derivatives (20-
29) within the CB1 cannabinoid receptor, as discussed further
in the text.
Using 1,3-diphenylthiourea (19, X′ ) S) instead of the urea
derivative, the 1,3,5-triphenyl-2-thioxoimidazolidin-4-one de-
rivatives were similarly obtained (30-38, Scheme 2). Except
for the iodo derivative (35), yields were not influenced by the
phenyl substituents (X1 and X2). 1,3-Bis(4-hydroxyphenyl)-5-
phenyl-2-thioxoimidazolidin-4-one (38) was obtained in high
yield from the methoxy derivative (37) by demethylation using
boron tribromide.
a Mean ( SEM of at least four experiments performed in duplicate.
derivatives (6-14). None of the compounds (6-14), assayed
as racemic mixtures, displayed significant competitive binding
toward either the hCB1 or hCB2 cannabinoid receptor. Therefore,
on the basis of the structure-affinity relationships obtained with
the 5,5′-diphenylimidazolidine-2,4-dione and 5,5′-diphenyl-2-
thioxoimidazolidin-4-one derivatives which showed the impor-
tance of the substitution of the nitrogen in position 3,28,30-31
four N3-substituted compounds were synthesized and assayed
for their affinity (15-18). The acylated derivatives 15 (N3-
benzoyl) and 16 (N3-hexanoyl) were both devoid of affinity for
the cannabinoid receptors. However, as expected, the introduc-
tion of an alkyl moiety (17 and 18) resulted in an increased
affinity for the hCB1 cannabinoid receptor with displacement
percentages around 40%.
In a second group of compounds, derivatives possessing an
additional phenyl ring in position 3 were synthesized, leading
to the 1,3,5-triphenylimidazolidine-2,4-dione series of deriva-
tives (20-29, X′ ) O). Screening results for this series of
compounds are summarized in Table 2. The hCB1 cannabinoid
receptor affinity was greatly enhanced by the presence of an
additional phenyl ring, as the radioactivity displacement is above
50% for all the compounds. This is further illustrated by
compounds 6 and 20 (<10% and 57% displacement at 10 µM,
respectively) and by compounds 12 and 22 (<15% and 75%
displacement at 10 µM, respectively). It has to be mentioned
that the hCB2 cannabinoid receptor affinity was also improved,
although to a much lower extent than the hCB1 cannabinoid
receptor affinity.
1,3-Dicyclohexyl-5-phenylimidazolidine-2,4-dione (40) was
synthesized from 1,3-dicyclohexylurea (39) and phenylglyoxal
(2) as illustrated in Scheme 3.
Pharmacology-Affinity toward the Cannabinoid Recep-
tors. All the compounds were screened at 10 µM concentrations
for competitive binding to the human cannabinoid receptors CB1
and CB2 in a competitive binding experiment, using membranes
of Chinese hamster ovarian (CHO) cells selectively expressing
either the human CB1 (hCB1) or CB2 (hCB2) cannabinoid
receptor. [3H]-SR141716A and [3H]-CP-55,940 were used as
radioligands (1 nM) in these experiments for the hCB1 and hCB2
cannabinoid receptor, respectively. Table 1 summarizes the
results expressed as the displacement percentages of the
radioligand from its binding site for the 1,5-diphenylimidazo-
lidine-2,4-dione and 1,5-diphenyl-2-thioxoimidazolidin-4-one
The 1,3,5-triphenyl-2-thioxoimidazolidin-4-one derivatives
(30-38), which are the thio isosteres of the 1,3,5-triphenylimi-
dazolidine-2,4-diones, were also obtained and assayed for their
affinity at the cannabinoid receptors (Table 2). In this series,

New CB1 Receptor InVerse Agonists/Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 875
Table 2. Percentages of Displacement of [3H]-SR141716A and
[3H]-CP-55,940, Respectively, by
Table 3. Affinities of 20-37 and Reference Cannabinoids SR141716A,
WIN-55,212-2, and HU-210 at the hCB1 Cannabinoid Receptora and
1,3,5-Triphenylimidazolidine-2,4-diones and
Determination of the Potency (EC50) and Maximal Stimulation (Emax
for Compounds 23-25, 27, 29, 32, 34, 35 and for HU-210 and
SR141716A on hCB1 Cannabinoid Receptorsb
)
1,3,5-Triphenyl-2-thioxoimidazolidin-4-ones (10 µM) on hCB1 and hCB2
Cannabinoid Receptorsa and Percentages of FAAH Activity (rat brain
homogenate) Inhibition by 1,3,5-Triphenylimidazolidine-2,4-diones and
1,3,5-Triphenyl-2-thioxoimidazolidin-4-ones (10 µM)a
activity
affinity
Ki (nM)
EC50
(nM)
Emax
(%)
% displacement
compd
X′ X1
X2
hCB1
receptor
hCB2
receptor
% FAAH
inhibition
Imidazolidine-2,4-dione Derivatives
compd X′ X1
X2
20
21
22
23
24
25
26
27
28
29
O
O
O
O
O
O
O
O
O
O
H
Cl
Br H
H
H
H
H
Cl 4-Cl
Cl 3-Cl
Br 4-Cl
H
H
6296 ( 354 /
6661 ( 386 /
4027 ( 276 /
/
/
/
Imidazolidine-2,4-dione Derivatives
20
21
22
23
24
25
26
27
28
29
O
O
O
O
O
O
O
O
O
O
H
Cl
Br
H
H
H
H
H
H
H
57.1 ( 1.5
60.2 ( 1.8
75.4 ( 2.1
101.1 ( 1.5 21.6 ( 2.3 <10
76.0 ( 2.2 32.1 ( 1.6 <10
102.1 ( 1.9 35.6 ( 1.9 <10
57.8 ( 3.1
71.6 ( 2.1
67.4 ( 2.4
79.1 ( 3.1
<10
<15
<15
<10
13.7 ( 3.5
14.5 ( 4.4
4-Cl
3-Cl
4-Br
353 ( 34
309 ( 31
-74.4 ( 1.3
2110 ( 99 936 ( 74
-50.9 ( 1.4
4-Cl
3-Cl
4-Br
4-OMe
243 ( 18
195 ( 24
-75.1 ( 1.2
4-OMe 6456 ( 354 /
/
1877 ( 87 1115 ( 86 -45.2 ( 1.6
31.4 ( 2.1 <10
28.1 ( 2.3 <10
24.8 ( 3.1 <10
3312 ( 167 /
905 ( 42
/
Cl 4-Cl
Cl 3-Cl
Br 4-Cl
1234 ( 64 -48.6 ( 1.7
2-Thioxoimidazolidin-4-one Derivatives
<20
15.4 ( 2.5
30
31
32
33
34
35
36
37
S
S
S
S
S
S
S
S
H
Br H
H
>8000
/
/
/
2-Thioxoimidazolidin-4-one Derivatives
6309 ( 304 /
30
31
32
33
34
35
36
37
38
S
S
S
S
S
S
S
S
S
H
Br
H
H
H
H
H
H
H
H
H
61.7 ( 1.7
85.1 ( 3.7
93.7 ( 3.1
<10
<10
<10
<10
11.8 ( 3.6
19.1 ( 2.5
11.4 ( 2.4
22.9 ( 1.7
<10
H
H
H
H
H
H
4-Cl
2163 ( 84 2103 ( 174 -51.9 ( 2.5
3,4-diCl 2001 ( 73
4-Br
3-I
/
/
4-Cl
1447 ( 54 1624 ( 123 -35.7 ( 1.7
692 ( 34
4968 ( 264 /
3,4-diCl 78.7 ( 1.5
461 ( 43
-19.3 ( 0.8
/
/
4-Br
3-I
4-Me
4-OMe
4-OH
100.1 ( 1.3 <15
4-Me
81.2 ( 1.8
80.1 ( 2.1
65.6 ( 3.1
<20
<15
<20
<20
<10
17.4 ( 2.4
<10
4-OMe 7033 ( 321 /
Reference Cannabinoids
<10
SR141716A
WIN-55,212-2
HU-210
5.4 ( 0.2
10.1 ( 0.7 -84.1 ( 1.7
31.5 ( 2.3
3802 ( 158 /
/
a Mean ( SEM of at least four experiments performed in duplicate.
18.6 ( 1.7 0.6 ( 0.04 201.2 ( 14.1
a Ki values were obtained from nonlinear analysis of competition curves
using [3H]-SR141716A as radioligand. Mean ( SEM of at least four
experiments done in duplicate. b Mean ( SEM of at least four experiments
performed in duplicate.
the affinity was also enhanced by the phenyl in position 3, as
demonstrated by compounds 7 and 30 (<10% and 62% displace-
ment at 10 µM, respectively). Meanwhile, the hCB2 cannabinoid
receptor affinity was not affected by this modification.
To estimate the influence of the aromatic ring in positions 1
and 3, 1,3-dicyclohexyl-5-phenylimidazolidine-2,4-dione (40)
was tested (at 10 µM) in a binding assay. This compound
displaced around 25% of the [3H]-SR141716, compared to the
57% displacement obtained with 20, confirming the important
role of the phenyl rings in positions 1 and 3.
32 (X2 ) Cl), and 34 (X2 ) Br), showing Ki values of >8000,
2163 ( 84, and 1447 ( 54 nM, respectively. A methyl
substituent (36, X2 ) Me) induces a slight affinity increase but
to a lower extent compared to a halogen substituent. The highest
affinity in this series was obtained with 1,3-bis(3-iodophenyl)-
5-phenyl-2-thioxoimidazolidin-4-one (35), exhibiting a Ki value
of 692 ( 34 nM.
To study the structure-affinity relationships, the inhibition
constants (Ki) of the derivatives showing the highest radioligand
displacement (>55%) were determined. The Ki values are
summarized in Table 3. With respect to the imidazolidine-2,4-
dione series, a bromine or a chlorine substituent in the para
position of the N1 and N3 phenyl rings (X2, Scheme 2 and Table
3) strongly enhances the affinity, as illustrated by derivatives
20 (X2 ) H), 23 (X2 ) Cl), and 25 (X2 ) Br), exhibiting Ki
values of 6296 ( 354 nM, 353 ( 34 nM, and 243 ( 18 nM,
respectively. A methoxy substituent in the same position (26,
X2 ) OMe) has no effect on the affinity (Ki ) 6456 ( 354
nM). Further, it appears that substitution at the para position is
preferred to the meta position, as revealed by compounds 23
and 24 bearing a chlorine substituent (X2) in the para and meta
positions, respectively (Ki values of 353 ( 34 nM and 2110 (
99 nM for 23 and 24, respectively). Moreover, chlorine and
bromine substituents in the para position of the C5 phenyl ring
(X1) are responsible for a decreased affinity. For example,
derivatives 27 (X1 ) Cl) and 29 (X1 ) Br) exhibited lower Ki
values than derivative 23 (X1 ) H). The highest affinities were
obtained for 1,3-bis(4-chlorophenyl)-5-phenylimidazolidine-2,4-
dione (23) and 1,3-bis(4-bromophenyl)-5-phenylimidazolidine-
2,4-dione (25) with Ki values of 353 ( 34 and 243 ( 18 nM,
respectively.
The structure-affinity relationships among the 3-alkyl-5,5′-
diphenyl derivatives have demonstrated an affinity increase
induced by replacement of the carbonyl by a thiocarbonyl in
position 2 of the imidazolidine-2,4,dione ring.31 In the 1,3,5-
triphenyl derivatives series (20-38) the opposite situation
occurred. For instance, the oxo derivative 1,3-bis(4-chlorophe-
nyl)-5-phenylimidazolidine-2,4-dione (23) showed a higher
affinity than its thio homologue 1,3-bis(4-chlorophenyl)-5-
phenyl-2-thioxoimidazolidin-4-one (32) (Ki values of 353 ( 34
and 2163 ( 84 nM, respectively), and 1,3-bis(4-bromophenyl)-
5-phenylimidazolidine-2,4-dione (25) exhibited a lower Ki value
than 1,3-bis(4-bromophenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (34) (Ki values of 243 ( 18 and 1447 ( 54 nM,
respectively).
Pharmacology. CB1 Cannabinoid Receptor Recognition
and Fatty Acid Amide Hydrolase Inhibition. The demonstra-
tion of fatty acid amide hydrolase (FAAH) inhibition by several
2-thioxoimidazolidin-4-one and imidazolidine-2,4-dione deriva-
tives37 prompted us to determine the potential of 1,3,5-
triphenylimidazolidine-2,4-dione and 1,3,5-triphenyl-2-thiox-
oimidazolidin-4-one derivatives for inhibition of FAAH.
Compounds 20-38 were screened at 10 µM concentrations for
their ability to inhibit tritiated anandamide degradation in the
presence of rat brain homogenates. The results are summarized
in Table 2. Imidazolidine-2,4-dione derivatives were not able
In the 2-thioxoimidazolidin-4-one series, the X2 substituent
effect is conserved as illustrated by compounds 30 (X2 ) H),

876 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Muccioli et al.
to inhibit FAAH activity, as inhibition percentages were lower
than 15%. Thus, the presence of three phenyl rings around the
imidazolidine-2,4-dione moiety enhances CB1 cannabinoid
receptor affinity, without affecting FAAH activity. 2-Thiox-
oimidazolidin-4-one derivatives also exhibited poor activity as
FAAH inhibitors, further confirming the selectivity achieved
with the 1,3,5-triphenyl substitution pattern for the CB1 can-
nabinoid receptor.
Functionality at the CB1 Cannabinoid Receptors. 3-Alkyl-
5,5′-diphenylimidazolidine-2,4-dione and 3-alkyl-5,5′-diphenyl-
2-thioxoimidazolidin-4-one derivatives were previously shown
to behave as inverse agonists at the hCB1 cannabinoid receptor.
Therefore, the functionality of 1,3,5-triphenylimidazolidine-2,4-
dione (20-29) and 1,3,5-triphenyl-2-thioxoimidazolidin-4-one
(31-36) derivatives was explored using a [35S]-GTPγS assay.38
GDP-GTP exchange is an early event in the signal transduction
mechanism of a G-protein-coupled receptor (GPCR). Thus,
measuring the [35S]-GTPγSsa radiolabeled nonhydrolyzable
analogue of GTPsbinding provides a direct measurement of
the interaction between the receptor and the G protein upon
binding of an agonist to the GPCR. Actually, agonist binding
to a receptor will increase the guanine nucleotide binding, a
neutral antagonist will not influence the nucleotide binding, and
finally, an inverse agonist will decrease the [35S]-GTPγS binding
to the G protein.
Figure 3. (A) [35S]-GTPγS binding stimulation assay of 1,3,5-
triphenylimidazolidine-2,4-dione (20-26) derivatives, and of SR141716A
(SR1) and HU-210 (10 µM) on the hCB1 cannabinoid receptor. Data
are expressed as the mean ( SEM of at least three experiments
performed in duplicate. Statistical significance assessed by one-way
ANOVA followed by a Dunett post-test (**P < 0.01). (B) [35S]-GTPγS
binding stimulation assay of 1,3,5-triphenylimidazolidine-2,4-dione
(27-29) derivatives and of 1,3,5-triphenyl-2-thioxoimidazolidin-4-one
(31-36) derivatives (10 µM) on the hCB1 cannabinoid receptor. Data
are expressed as the mean ( SEM of at least three experiments
performed in duplicate. Statistical significance assessed by one-way
ANOVA followed by a Dunett post-test (**P < 0.01).
Derivatives possessing a Ki value at the hCB1 cannabinoid
receptor lower than 7000 nM were screened at a 10 µM concen-
tration, along with HU-210 and SR141716A, a known agonist
and an inverse agonist, respectively. As expected, 10 µM
SR141716A and HU-210 induced a decrease (-79% compared
to basal) and an increase (129% compared to basal level),
respectively, in the [35S]-GTPγS binding (Figure 3). The 1,3,5-
triphenylimidazolidine-2,4-dione (20-29) and 1,3,5-triphenyl-
2-thioxoimidazolidin-4-one (31, 32, 34-36) derivatives induced
a significant decrease in [35S]-GTPγS binding, ranging from
-12% to -70%, with the notable exception of derivative 33,
which did not induced any significant variation in nucleotide
binding (Figure 3). The observed decrease in [35S]-GTPγS
binding revealed the inverse agonist properties of the series of
imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one deriva-
tives at the hCB1 cannabinoid receptor. Thus with respect to
the imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one
substitution pattern, the 1,3,5-triphenyl derivatives behaved as
the 3-alkyl-5,5′-diphenyl derivatives at the hCB1 cannabinoid
receptor. The activity of compounds 32 and 33 deserves some
attention. Indeed, while both compounds exhibited similar
affinity (Ki ∼ 2100 nM), the [35S]-GTPγS assay evidenced dif-
ferent activities, i.e. inverse agonism for 32 and antagonism for
33. The additional chlorine substituent in the meta position in-
duced an activity change, revealing a modified binding position
to the receptor. This finding should be helpful in further studies
aimed at understanding the molecular parameters inducing the
inverse agonism and/or antagonism of this series of compounds.
at the hCB1 cannabinoid receptor with EC50 values of 309 (
31, 195 ( 24, and 461 ( 43 nM, respectively.
As observed in the affinity studies, the thio derivatives
exhibited lower potency and efficacy compared to the imida-
zolidine-2,4-dione derivatives. This is well-demonstrated by
compounds 23 and 32 (EC50 values of 309 ( 31 and 2103 (
174 nM, respectively; Emax values of -74.4 ( 1.3 and -51.9
( 2.5%, respectively) or by compounds 25 and 34 (EC50 values
of 195 ( 24 nM and 1624 ( 123 nM, respectively; Emax values
of -75.1 ( 1.2 and -35.7 ( 1.7%, respectively).
3-Alkyl-5,5′-bis(4-bromophenyl)imidazolidine-2,4-dione
derivatives reported to act as inverse agonists at the human
CB1 cannabinoid receptor expressed in CHO cells (hCB1-CHO)
were also shown to behave as neutral antagonists at the rat
CB1 cannabinoid receptor expressed in cerebellum homog-
enates (rCB1).39,28 Thus, to determine if this behavior is shared
by the 1,3,5-triphenylimidazolidine-2,4-diones, the affinity and
functionality of derivatives 23, 25, 27, and 29 at
the murine CB1 receptor were determined. On one hand, Ki
values at the rCB1 cannabinoid receptor (Table 4) are of the
same magnitude compared to those obtained at the hCB1
cannabinoid receptor (Table 3). On the other hand, derivatives
23, 25, 27, and 29 at 10 µM concentrations did not influence
the [35S]-GTPγS binding, thus revealing neutral antagonistic
properties at the rCB1 cannabinoid receptor (Figure 4). In
general, 1,3,5-triphenylimidazolidine-2,4-dione derivatives ex-
hibited the same behavior as 3-alkyl-5,5′-bis(4-bromophenyl)-
imidazolidine-2,4-diones at the human and murine CB1 can-
nabinoid receptors.
To further explore the inverse agonist properties, potencies
of imidazolidine-2,4-diones 23-25, 27, and 29 and 2-thioxo-
imidazolidin-4-ones 32, 34, and 35 were determined by measur-
ing the decrease in nucleotide binding induced by increasing
concentrations of test compounds. The respective EC50 and Emax
values are summarized in Table 3. The compounds possessing
the highest affinity, 1,3-bis(4-chlorophenyl)-5-phenylimidazo-
lidine-2,4-dione (23), 1,3-bis(4-bromophenyl)-5-phenylimida-
zolidine-2,4-dione (25), and 1,3-bis(3-iodophenyl)-5-phenyl-2-
thioxoimidazolidin-4-one (35), exhibited also the highest potency

New CB1 Receptor InVerse Agonists/Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 877
Table 4. Determination of the Affinities of 23, 25, 27, and 29 at the
rCB1 Cannabinoid Receptor, Compared to Those of Reference
Cannabinoids SR141716A and HU-2106
rCB1
rCB1
(cerebellum)
Ki (nM)
(cerebellum)
Ki (nM)
compd
compd
23
25
27
247 ( 25
311 ( 21
2618 ( 104
29
1744 ( 91
1.23 ( 0.1
2.75 ( 0.2
SR141716A
HU-210
a Ki values were obtained from nonlinear analysis of competition curves
using [3H]-SR141716A as radioligand. Mean ( SEM of at least four
experiments done in duplicate.
Figure 5. Superimposition of the complex of SR141716A and the CB1
cannabinoid receptor model (colored in green and blue, respectively)
with the complex of S-25 and the CB1 cannabinoid receptor model
(colored in gray and cyan, respectively). The representation of the
Connolly surface (probe 1.4 Å) defines the accessible binding pocket.
Figure 4. [35S]-GTPγS binding stimulation assay of 1,3,5-triphenyl-
imidazolidine-2,4-dione (23, 25, 27, 29) derivatives and of SR141716A
(SR1) and HU-210 (10 µM) on the rCB1 cannabinoid receptor (rat
cerebellum homogenate). Data are expressed as the mean ( SEM of
at least three experiments performed in duplicate. Statistical significance
assessed by one-way ANOVA followed by a Dunett post-test (**P <
0.01)
Molecular Modeling. To assess the influence of stereochem-
istry on the hCB1 cannabinoid receptor affinity, the binding
modes of both enantiomers of 25, the most active compound in
the imidazolidine-2,4-dione series, have been explored and
compared with that of the reference inverse agonist SR141716A.
The binding of the latter has recently been modeled inside the
hCB1 receptor40 and correlates well with the established
mutagenesis studies.41 It involves direct aromatic stacking
interactions with F3.36, Y5.39, W5.43, and W6.48, as well as
a hydrogen bond with K3.28.
We manually docked SR141716A into the inactive R-state
of the CB1 receptor model in order to reproduce the interaction
pattern described in the literature. Then, the R- and S-
enantiomers of 25, which act as inverse agonists too, were
docked into the inactive R-state of the cannabinoid receptor.
As we had no a priori knowledge on their mode of interaction,
we explored their binding with the help of the automated GOLD
docking software.42 All the receptor-ligand complexes were
finally refined by molecular mechanics,43 allowing the active
site’s side chains to accommodate the ligand.
As illustrated in Figures 5 and 6, both enantiomers of 25 adopt
a similar orientation within the CB1 cannabinoid receptor. The
interaction pattern of 25 seems to be governed by the binding
of the two bromophenyls to the aromatic microdomain. Indeed,
these two rings form several direct stacking interactions with
the aromatic microdomain and especially with F3.36, Y5.39,
and W5.43. Furthermore, in both cases, the unsubstituted phenyl
interacts strongly through an aromatic contact with F6.32.
Orientation of the two N-substituent phenyl rings is stabilized
by intramolecular C-H···O bonds involving a carbonyl oxygen
of the imidazolidine-2,4-dione moiety. Similar interactions were
suggested on the basis of the crystal structure of 22 and are
probably a characteristic of this series of molecules.
Figure 6. Receptor-based alignment of SR141716A (in green), S-25
(in gray), and R-25 (in orange). Dashed green lines represent H-bonds.
parison with the S-enantiomer and prevents the formation of a
hydrogen bond with K3.28. In contrast, the S-enantiomer realizes
a hydrogen bond with K3.28 similarly to SR141716A, which
also interacts through hydrogen bonding with this residue. This
particular interaction has been shown to account for the higher
affinity of SR141716A for the inactive R-state, leading to its
inverse agonism.41 This binding difference between R- and
S-enantiomers suggests a strong influence of stereochemistry
on CB1 cannabinoid receptor affinity for 25.
Compared to SR141716A, the imidazolidine-2,4-dione de-
rivatives fit only partially the shape of the binding site. The
lipophilic pocket bordered by F2.67, F2.64, H2.65, and F2.61
is left empty, whereas in the case of SR141716A, the piperidine
ring is favorably stabilized within that cavity (Figure 6). This
might account for the lower CB1 cannabinoid receptor affinity
of these compounds in comparison with SR141716A.
Conclusion
The results reported herein further confirm the interest of the
imidazolidine-2,4-dione ring as central scaffold for cannabinoid
receptors ligands. Indeed, albeit the 1,5-diphenylimidazolidine-
2,4-dione derivatives showed no affinity, the 1,3,5-triphenylimi-
dazolidine-2,4-dione derivatives and the 1,3,5-triphenyl-2-
thioxoimidazolidin-4-one derivatives behaved as selective CB1
cannabinoid receptor ligands. Further, the [35S]-GTPγS assay
demonstrated their inverse agonists properties at the human CB1
However, this orientation involves, for the R-enantiomer,
inversion of the central imidazolidine-2,4-dione ring in com-

878 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Muccioli et al.
1-(4-Bromophenyl)-5-phenylimidazolidine-2,4-dione (9). Yield:
cannabinoid receptor. The most active compounds possess a
chlorine or bromine substituent in the para position of the N1
and N3 phenyl rings, while additional substitution on the C5
phenyl ring resulted in a decreased affinity. Finally, the
compounds 1,3-bis(4-bromophenyl)-5-phenylimidazolidine-2,4-
dione (25) and 1,3-bis(4-chlorophenyl)-5-phenylimidazolidine-
2,4-dione (23) are the imidazolidine-2,4-dione derivatives
possessing the highest affinity for the human CB1 cannabinoid
receptor reported to date.
1
60%. Mp: 216.2-216.8 °C. MS (EI): 332 [M + H]+. H NMR
(DMSO-d6): δ 5.94 (s, 1H), 7.34-7.46 (m, 9H), 11.50 (s, 1H).
13C NMR (DMSO-d6): δ 64.74 (CH), 116.56, 123.03, 127.56,
129.05, 129.31, 131.90, 134.10, and 136.36 (C and CHarom.), 155.18
(CdO), 171.68 (CdO).
1-(4-Iodophenyl)-5-phenylimidazolidine-2,4-dione (10). Yield:
58%. Mp: 236.3-237.2 °C. MS (EI): 378 [M]+. 1H NMR (DMSO-
d6): δ 5.94 (s, 1H), 7.32-7.64 (m, 9H), 11.53 (s, 1H). 13C NMR
(DMSO-d6): δ 64.67 (CH), 88.67, 123.23, 127.56, 129.05, 129.37,
134.09, 136.87, and 137.78 (C and CHarom.), 155.12 (CdO), 171.62
(CdO). Anal. Calcd for C15H11IN2O2: C, H, N.
Experimental Section
1-(4-Methoxyphenyl)-5-phenylimidazolidine-2,4-dione (11).
Yield: 64%. Mp: 151.7-152.6 °C. MS (EI): 282 [M]+. 1H NMR
(DMSO-d6): δ 3.70 (s, 3H), 5.92 (s, 1H), 6.87-7.42 (m, 9H), 11.35
(s, 1H). 13C NMR (DMSO-d6): δ 55.55 (CH3), 65.45 (CH), 114.36,
123.74, 127.69, 128.85, 129.24, 129.76, 134.55, and 156.61 (C and
CHarom.), 155.25 (CdO), 172.07 (CdO).
5-(4-Bromophenyl)-1-phenylimidazolidine-2,4-dione (12).
Yield: 60%. Mp: 203.1-204.2 °C. MS (EI): 332 [M]+. 1H NMR
(DMSO-d6): δ 6.00 (s, 1H), 7.02-7.56 (m, 9H), 11.50 (s, 1H).
13C NMR (DMSO-d6): δ 64.29 (CH), 121.29, 122.26, 124.65,
129.18, 129.82, 132.22, 133.90, and 136.81 (C and CHarom.), 155.12
(CdO), 171.49 (CdO).
5-(4-Bromophenyl)-1-(4-chlorophenyl)imidazolidine-2,4-di-
one (13). Yield: 64%. Mp: 205.1-205.9 °C. MS (EI): 366 [M]+.
1H NMR (DMSO-d6): δ 5.98 (s, 1H), 7.32-7.58 (m, 8H), 11.55
(s, 1H). 13C NMR (DMSO-d6): δ 64.41 (CH), 122.16, 122.99,
129.31, 130.08, 132.54, 133.77, and 136.04 (C and CHarom.), 155.32
(CdO), 171.49 (CdO).
1,5-Bis(4-bromophenyl)imidazolidine-2,4-dione (14). Yield:
62%. Mp: 242.1-243.0 °C. MS (EI): 410 [M]+. 1H NMR (DMSO-
d6): δ 5.97 (s, 1H), 7.31-7.57 (m, 8H), 11.54 (s, 1H). 13C NMR
(DMSO-d6): δ 64.41 (CH), 122.64, 123.29, 130.02, 132.28, 132.54,
133.77, and 136.49 (C and CHarom.), 155.32 (CdO), 171.49 (Cd
O).
General Procedures. All reagents were purchased from com-
mercial sources (Sigma-Aldrich or Acros) and were used without
further purification. Solvents were of analytical grade. [3H]-
SR141716A (52 Ci/mol) was acquired from Amersham (Roosendaal,
The Netherlands), [3H]-CP-55,940 (101 Ci/mol) from NEN Life
Science (Zaventem, Belgium), and [3H]-AEA (60 Ci/mmol) from
American Radiolabeled Chemical (St Louis, MO). HU-210 was
obtained from Tocris (Bristol, UK), while WIN-55,212-2 was
purchased from Research Biochemicals International (Boornem,
Belgium). SR141716A was a generous gift from Dr. Barth, Sanofi-
Synthe´labo Research (Montpellier, France).
The microwave oven used was a commercial household micro-
wave oven (frequency 2450 MHz). Melting points (mp) were
determined in open capillaries using the Electrothermal 9100
apparatus and are reported uncorrected. Infrared (IR) spectra of
compounds dispersed in KBr were recorded using a Perkin-Elmer
FT-IR 286 spectrometer, and values are reported as ν in cm-1 (see
Supporting Information). Nuclear magnetic resonance (1H NMR,
13C NMR) spectra were recorded on a Bruker AM-300 spectrometer
at room temperature and analyzed using the WIN NMR software
package. Chemical shifts (δ) are reported relative to the tetram-
ethylsilane peak set at 0 ppm. In the case of multiplets, the signals
are reported as intervals. Signals are abbreviated as s, singlet; d,
doublet; t, triplet; m, multiplet. Coupling constants are expressed
in hertz. Mass spectra were recorded on a Finnigan MAT 44S, with
an ionization voltage of 70 eV. Elemental analyses were performed
on a Carlo Erba EA 1108 analyzer (Carlo Erba, Milano, Italy) and
are within (0.4% of the theoretical values.
A suitable crystal of compound 22 obtained by recrystallization
from ethanol was mounted with a quartz fiber on a goniometer
head of a CAD4 Nonius diffractometer. After determination of the
cell parameter using 25 well-centered reflections, complete dif-
fraction data sets were collected. The structure was solved using
direct methods and refined by full matrix least squares on F2 using
the program Shelxl97.44 All non-hydrogen atoms were treated
anisotropically while a riding model was applied for hydrogen
atoms. Analytical correction for absorption was introduced.
Crystallographic Data for Compound 22. Colorless plate (0.08
× 0.28 × 0.35 mm); triclinic; P1h: a ) 10.079 (2) Å, b ) 17.079
(2) Å, c ) 21.679 (3) Å, R ) 73.4°, â ) 81.9°, γ ) 86.5°; V )
3622.0 Å3; Z ) 8, µ ) 3.24 mm-1; Dx ) 1.494 g/cm3; λ (Cu KR)
) 1.54178 Å; F(000) ) 1648; T ) 293 K; 9882 unique reflections
(Rint ) 0.009); 937 refined parameters, R1 ) 0.0403 for 9067 Fo
>4σ (Fo), R1 ) 0.0445 for all data and wR2 ) 0.1049, GOF ) S
) 1.03; ∆Fmin ) -1.09 e/ Å3; ∆Fmax ) 0.85 e/ Å3.
Synthesis of 3-Benzoyl-1,5-diphenylimidazolidine-2,4-dione
(15). 1,5-Diphenylimidazolidine-2,4-dione (6) (1.98 mmol) was
dissolved in a mixture of freshly distilled dichloromethane (25 mL)
and pyridine (0.2 mL) under nitrogen prior to the addition of
benzoyl chloride (1.98 mmol). The resulting mixture was stirred
overnight at room temperature and subsequently washed by
NaHCO3 5%, citric acid 5%, and brine. The resulting organic layer
was dried over MgSO4 and evaporated to dryness. The resulting
solid was crystallized from ethanol. The formation of 15 was
1
confirmed by the disappearance of the NH peak of 6 in H NMR
(δ ) 11.49 ppm) and by the appearance of one additional carbonyl
peak in 13C NMR (δ ) 166.45 ppm). Yield: 75%. Mp: 183.5-
1
183.9 °C. MS (EI): 356 [M]+. H NMR (DMSO-d6): δ 6.20 (s,
1H), 7.10-8.04 (m, 15H). 13C NMR (DMSO-d6): δ 64.41 (CH),
122.19, 125.43, 127.76, 128.98, 129.24, 130.54, 131.96, 133.26,
135.07, and 135.78 (C and CHarom.), 151.24 (CdO), 166.45 (Cd
O), 168.58 (CdO).
Synthesis of 3-Hexanoyl-1,5-diphenylimidazolidine-2,4-dione
(16). This derivative was synthesized as 15, using hexanoyl chloride
instead of benzoyl chloride. Yield: 73%. Mp: 133.7-134.6 °C.
1
MS (EI): 350 [M]+. H NMR (DMSO-d6): δ 0.87 (s, 3H), 1.29
(m, 4H), 1.60 (m, 2H), 2.96 (t, J ) 7.3, 2H), 6.01 (s, 1H), 7.10-
7.35 (m, 10H). 13C NMR (DMSO-d6): δ 14.14 (CH3), 22.17, 23.46,
30.90, and 38.01 (CH2), 63.51 (CH), 121.28, 122.77, 125.75,
127.56, 128.14, 129.24, 133.71, and 135.97 (C and CHarom.), 151.37
(CdO), 168.58 (CdO), 171.75 (CdO). Anal. Calcd for C21H22-
N2O3: C, H, N.
Synthesis of 3-Pentyl-1,5-diphenylimidazolidine-2,4-dione (17)
and 3-Heptyl-1,5-diphenylimidazolidine-2,4-dione (18). To a
mixture of 6 (1.98 mmol), K2CO3 (7.9 mmol), and tetrabutylam-
monium bromide (0.19 mmol) in DMF (2 mL) was added the
chloroalkyl derivative (2.6 mmol) under stirring. The resulting
mixture (in a open flask) was placed in a microwave oven and
three 1.5-min pulses (100 W) were applied. After cooling, the
mixture was poured into water and extracted with CHCl3. The
General Procedure for the Synthesis of the 1,5-Diphenyl
Derivatives (8-14). The synthesis of 6 and 7 was previously
described.37 The 1,5-diphenyl derivatives were synthesized follow-
ing a method adapted from Joshi et al.34 Phenylglyoxal (2, 4.5
mmol) and phenylurea (5, X′ ) O) or phenylthiourea (5, X′ ) S)
(4.5 mmol) were refluxed for 4 h in 15 mL of glacial acetic acid
in the presence of 0.5 mL of hydrochloric acid. After cooling, the
mixture was poured into water and the resulting precipitate filtered
off. The residue was subsequently crystallized from ethanol.
1-(4-Chlorophenyl)-5-phenylimidazolidine-2,4-dione (8). Yield:
64%. Mp: 213.1-214.0 °C. MS (EI): 286 [M]+. 1H NMR (DMSO-
d6): δ 5.95 (s, 1H), 7.33-7.55 (m, 9H), 11.49 (s, 1H). 13C NMR
(DMSO-d6): δ 64.87 (CH), 122.77, 127.56, 128.53, 129.05, 129.30,
and 134.10, (C and CHarom.), 155.18 (CdO), 171.69 (CdO).

New CB1 Receptor InVerse Agonists/Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 879
organic layer was washed with HCl, NaOH, and brine. The resulting
organic phase was dried over MgSO4 and evaporated to dryness.
The yields obtained (55 and 59%) were higher than those we
previously obtained using another alkylating procedure, i.e. DMF,
K2CO3, 24 h of stirring (yield 35%).37
3-Pentyl-1,5-diphenylimidazolidine-2,4-dione (17). Yield: 59%.
Mp: 98.1-99.0 °C. MS (EI): 323 [M]+. 1H NMR (DMSO-d6): δ
0.82-0.87 (m, 3H), 1.27-1.28 (m, 4H), 1.57-1.62 (m, 2H), 3.51
(t, J ) 7.4, 2H), 6.02 (s, 1H), 7.04-7.43 (m, 10H). 13C NMR
(DMSO-d6): δ 13.72 (CH3), 21.54, 26.98, 28.90, and 38.36 (CH2),
63.15 (CH), 120.85, 124.29, 127.13, 128.62, 128.62, 128.75, 128.88,
133.66 (C and CHarom.), 154.31 (CdO), 170.22 (CdO). Anal. Calcd
for C20H22N2O2: C, H, N.
(DMSO-d6): δ 3.68 (s, 3H), 3.80 (s, 3H), 6.05 (s, 1H), 6.87-7.46
(m, 13H). 13C NMR (DMSO-d6): δ 55.31 (2×OMe), 63.92 (CH),
114.07, 123.71, 124.41, 127.65, 128.36, 128.62, 128.81, 129.01,
133.86, and 133.95 (C and CHarom.), 153.66 (CdO), 156.44 and
158.90 (Carom.), 169.77 (CdO). Anal. Calcd for C23H20N2O4: C,
H, N.
1,3,5-Tris(4-chlorophenyl)imidazolidine-2,4-dione (27). Yield:
50%. Mp: 187.1-187.9 °C. MS DEI: 430 [M]+. 1H NMR (DMSO-
d6): δ 6.18 (s, 1H), 7.40-7.61 (m, 12H). 13C NMR (DMSO-d6):
δ 62.63 (CH), 122.86, 122.98, 126.23, 128.44, 128.65, 128.88,
129.66, 130.43, 132.18, 132.82, 133.67, and 134.90 (C and CHarom.),
152.95 (CdO), 168.73 (CdO). Anal. Calcd for C21H13Cl3N2O2:
C, H, N.
3-Heptyl-1,5-diphenylimidazolidine-2,4-dione (18). Yield: 57%.
Mp: 66.5-67.8 °C. MS (EI): 350 [M]+. 1H NMR (DMSO-d6): δ
0.81-0.83 (m, 3H), 1.23-1.26 (m, 8H), 1.56-1.59 (m, 2H), 3.50
(t, J ) 7.3, 2H), 6.01 (s, 1H), 7.06-7.53 (m, 10H). 13C NMR
(DMSO-d6): δ 13.62 (CH3), 21.71, 25.72, 27.08, 27.86, and 30.84
(CH2), 62.92 (CH), 120.70, 124.13, 126.98, 128.40, 128.53, 128.73,
133.64, and 136.17 (C and CHarom.), 155.15 (CdO), 170.07 (Cd
O). Anal. Calcd for C22H26N2O2: C, H, N.
General Procedure for the Synthesis of 1,3,5-Triphenylimi-
dazolidine-2,4-dione Derivatives (20-29). The phenylglyoxal (2)
(5 mmol) and 1,3-diphenylurea (19, X′ ) O) (5 mmol) were
refluxed with stirring in concentrated acetic acid (20 mL) and
concentrated hydrochloric acid (0.5 mL). After 6 h, the solution
was allowed to cool and then poured into water. The resulting
precipitate was filtered, dried, and recrystallized from ethanol.
1,3,5-Triphenylimidazolidine-2,4-dione (20). Yield: 60%.
Mp: 119.9-120.9 °C. MS DEI: 328 [M]+. 1H NMR (DMSO-d6):
δ 6.71 (s, 1H), 7.31-7.61 (m, 15H). 13C NMR (DMSO-d6): δ 63.64
(CH), 121.54, 124.84, 127.30, 127.75, 128.05, 128.46, 128.92,
129.11, 129.54, 131.96, 133.97, and 136.42 (C and CHarom.), 153.51
(CdO), 169.62 (CdO).
1,3-Bis(3-chlorophenyl)-5-(4-chlorophenyl)imidazolidine-2,4-
dione (28). Yield: 48%. Mp: 80.8-81.4 °C. MS DEI: 430 [M]+.
1H NMR (DMSO-d6): δ 6.21 (s, 1H), 7.18-7.76 (m, 12H). 13C
NMR (DMSO-d6): δ 62.99 (CH), 120.06, 121.54, 125.04, 126.27,
127.43, 128.85, 129.37, 130.22, 130.92, 132.61, 133.32, 133.64,
134.16, and 137.85 (C and CHarom.), 153.31 (CdO), 169.10 (Cd
O). Anal. Calcd for C21H13Cl3N2O2: C, H, N.
5-(4-Bromophenyl)-1,3-bis(4-chlorophenyl)imidazolidine-2,4-
dione (29). Yield: 46%. Mp: 197.6-198.5 °C. MS DEI: 476 [M]+.
1H NMR (DMSO-d6): δ 6.18 (s, 1H), 7.39-7.62 (m, 12H). 13C
NMR (DMSO-d6): δ 63.25 (CH), 122.84, 123.36, 126.72, 129.11,
129.31, 130.47, 130.99, 132.03, 132.35, 133.13, 133.38, and 135.45
(C and CHarom.), 153.44 (CdO), 169.16 (CdO). Anal. Calcd for
C21H13BrCl2N2O2: C, H, N.
General Procedure for the Synthesis of 1,3,5-Triphenyl-2-
thioxoimidazolidin-4-one Derivatives (30-38). These compounds
were synthesized similarly to the 1,3,5-triphenylimidazolidine-2,4-
diones using 1,3-diphenylthiourea (19, X′ ) S) instead of 1,3-
diphenylurea (19, X′ ) O).
1,3,5-Triphenyl-2-thioxoimidazolidin-4-one (30). Yield: 59%.
Mp: 169.1-170.2 °C. MS DEI: 344 [M]+. 1H NMR (DMSO-d6):
δ 6.28 (s, 1H), 7.22-7.60 (m, 15H). 13C NMR (DMSO-d6): δ 68.49
(CH), 127.04, 127.62, 128.27, 129.11, 129.24, 133.25, 133.96, and
137.33 (C and CHarom.), 171.36 (CdO), 181.78 (CdS). Anal. Calcd
for C21H16N2OS: C, H,N.
5-(4-Bromophenyl)-1,3-diphenyl-2-thioxoimidazolidin-4-
one (31). Yield: 57%. Mp: 193.3-193.7 °C. MS DEI: 423 [M]+.
1H NMR (DMSO-d6): δ 6.28 (s, 1H), 7.23-7.68 (m, 14H). 13C
NMR (DMSO-d6): δ 68.26 (CH), 126.81, 127.39, 128.04, 128.62,
128.88, 129.01, 133.02, 133.73, 134.10, 134.64, and 137.10 (C and
CHarom.), 171.06 (CdO), 181.55 (CdS). Anal. Calcd for C21H15-
BrN2OS: C, H, N.
1,3-Bis(4-chlorophenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (32). Yield: 58%. Mp: 214.9-215.3 °C. MS DEI: 413 [M]+.
1H NMR (DMSO-d6): δ 6.24 (s, 1H), 7.35-7.63 (m, 13H). 13C
NMR (DMSO-d6): δ 68.36 (CH), 128.41, 128.72, 128.98, 129.17,
129.31, 129.44, 131.05, 132.02, 132.67, 132.86, 133.83, and 136.10
(C and CHarom.), 171.04 (CdO), 181.52 (CdS). Anal. Calcd for
C21H14Cl2N2OS: C, H, N.
1,3-Bis(4,3-dichlorophenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (33). Yield: 52%. Mp: 172.6-173.7 °C. MS DEI: 482 [M]+.
1H NMR (DMSO-d6): δ 6.29 (s, 1H), 7.37-7.97 (m, 11H). 13C
NMR (DMSO-d6): δ 68.43 (CH), 127.49, 128.85, 129.11, 129.57,
129.82, 129.96, 131.12, 131.38, 131.57, 132.80, 133.40, 133.84,
and 137.27 (C and CHarom.), 170.85 (CdO), 181.65 (CdS). Anal.
Calcd for C21H12Cl4N2OS: C, H, N.
1,3-Bis(4-bromophenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (34). Yield: 54%. Mp: 233.4-234.5 °C. MS DEI: 502 [M]+.
1H NMR (DMSO-d6): δ 6.23 (s, 1H), 7.35-7.77 (m, 13H). 13C
NMR (DMSO-d6): δ 68.30 (CH), 120.51, 122.45, 128.40, 128.98,
129.31, 129.44, 131.31, 131.90, 132.16, 132.80, 133.13, and 136.49
(C and CHarom.), 170.91 (CdO), 181.39 (CdS). Anal. Calcd for
C21H14Br2N2OS: C, H, N.
5-(4-Chlorophenyl)-1,3-diphenylimidazolidine-2,4-dione (21).
Yield: 51%. Mp: 158.1-158.7 °C. MS DEI: 362 [M]+. 1H NMR
(DMSO-d6): δ 6.21 (s, 1H), 7.11-7.60 (m, 14H). 13C NMR
(DMSO-d6): δ 63.19 (CH), 121.87, 125.17, 125.49, 127.49, 128.73,
129.24, 129.37, 129.95, 132.16, 133.19, 133.97, and 136.55 (C and
CHarom.), 153.70 (CdO), 169.52 (CdO). Anal. Calcd for C21H15-
ClN2O2: C, H, N.
5-(4-Bromophenyl)-1,3-diphenylimidazolidine-2,4-dione (22).
1
Yield: 53%. Mp: 174.4-175.1 °C. MS DEI: 408 [M + H]+. H
NMR (DMSO-d6): δ 6.20 (s, 1H), 7.09-7.60 (m, 14H). 13C NMR
(DMSO-d6): δ 62.99 (CH), 121.55, 122.32, 124.91, 127.30, 128.53,
128.98, 130.02, 131.64, 131.90, 132.03, 133.32, and 136.29 (C and
CHarom.), 153.44 (CdO), 169.23 (CdO). Anal. Calcd for C21H15-
BrN2O2: C, H, N.
1,3-Bis(4-chlorophenyl)-5-phenylimidazolidine-2,4-dione (23).
Yield: 48%. Mp: 190.8-191.8 °C. MS DEI: 396 [M]+. 1H NMR
(DMSO-d6): δ 6.13 (s, 1H), 7.34-7.58 (m, 13H). 13C NMR
(DMSO-d6): δ 63.83 (CH), 123.36, 126.59, 126.78, 128.08, 129.05,
129.37, 130.99, 133.26, 133.71, 134.16, 135.52, and 135.84 (C and
CHarom.), 153.44 (CdO), 169.49 (CdO). Anal. Calcd for C21H14-
Cl2N2O2: C, H, N.
1,3-Bis(3-chlorophenyl)-5-phenylimidazolidine-2,4-dione (24).
Yield: 48%. Mp: 117.5-118.6 °C. MS DEI: 396 [M]+. 1H NMR
(DMSO-d6): δ 6.19 (s, 1H), 7.16-7.79 (m, 13H). 13C NMR
(DMSO-d6): δ 63.76 (CH), 120.06, 121.54, 124.97, 126.27, 127.43,
128.21, 128.79, 129.37, 130.86, 133.38, 133.64, and 137.98 (C and
CHarom.), 153.38 (CdO), 169.36 (CdO). Anal. Calcd for C21H14-
Cl2N2O2: C, H, N.
1,3-Bis(4-bromophenyl)-5-phenylimidazolidine-2,4-dione (25).
Yield: 54%. Mp: 211.4-212.5 °C. MS DEI: 486 [M]+. 1H NMR
(DMSO-d6): δ 6.14 (s, 1H), 7.36-7.76 (m, 13H). 13C NMR
(DMSO-d6): δ 63.83 (CH), 117.40, 121.80, 123.80, 126.91, 128.21,
129.44, 129.63, 131.44, 132.16, 132.41, 133.71, and 136.08 (C and
CHarom.), 153.44 (CdO), 169.55 (CdO). Anal. Calcd for C21H14-
Br2N2O2: C, H, N.
1,3-Bis(3-iodophenyl)-5-phenyl-2-thioxoimidazolidin-4-one (35).
Yield: 49%. Mp: 182.3-182.9 °C. MS DEI: 596 [M]+. 1H NMR
(DMSO-d6): δ 6.21 (s, 1H), 7.14-8.01 (m, 13H). 13C NMR
(DMSO-d6): δ 68.42 (CH), 94.04, 126.52, 128.53, 128.79, 129.24,
129.44, 130.80, 130.99, 132.87, 135.00, 135.52, 136.43, 137.52,
1,3-Bis(4-methoxyphenyl)-5-phenylimidazolidine-2,4-dione (26).
Yield: 56%. Mp: 140.4-141.3 °C. MS DEI: 388 [M]+. 1H NMR

880 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Muccioli et al.
137.91, and 138.43 (C and CHarom.), 170.98 (CdO), 181.95 (Cd
S). Anal. Calcd for C21H14I2N2OS: C, H, N.
conditions. Protein concentration was measured as described for
the CHO membranes.
1,3-Bis(4-methylphenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (36). Yield: 63%. Mp: 191.2-192.0 °C. MS DEI: 372 [M]+.
1H NMR (DMSO-d6): δ 2.24 (s, 3H), 2.37 (s, 3H), 6.19 (s, 1H),
7.14-7.44 (m, 13H). 13C NMR (DMSO-d6): δ 20.68 (Me), 20.69
(Me), 68.43 (CH), 126.07, 126.78, 128.21, 128.85, 129.24, 129.37,
129.57, 131.38, 133.32, 134.80, 137.07, and 138.63 (C and CHarom.),
171.43 (CdO), 181.91 (CdS). Anal. Calcd for C23H20N2OS: C,
H, N.
1,3-Bis(4-methoxyphenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (37). Yield: 58%. Mp: 188.0-188.9 °C. MS DEI: 404 [M]+.
1H NMR (DMSO-d6): δ 3.71 (s, 3H), 3.81 (s,3H), 6.13 (s, 1H),
6.85-7.44 (m, 13H). 13C NMR (DMSO-d6): δ 55.12 (OMe), 55.32
(OMe), 68.45 (CH), 113.80, 114.00, 126.29, 128.04, 128.43, 128.95,
129.53, 129.98, 130.24, 133.15, 158.06, and 159.29 (C and CHarom.),
171.39 (CdO), 182.13 (CdS). Anal. Calcd for C23H20N2O3S: C,
H, N.
1,3-Bis(4-hydroxyphenyl)-5-phenyl-2-thioxoimidazolidin-4-
one (38). To a solution of 37 in freshly distilled CH2Cl2 (1 mM)
kept in an ice bath was added boron tribromide (1.2 mM) under
stirring. The solution was then allowed to reach room temperature
and stirred overnight. The resulting solution was evaporated to
dryness under reduced pressure and the resulting solid washed with
water. After filtration, the compound was crystallized from ethanol.
Yield: 75%. Mp: 199.2-200.0 °C. MS DEI: 376 [M]+. 1H NMR
(DMSO-d6): δ 6.05 (s, 1H), 6.66-7.44 (m, 13H), 9.61 (s, 1H),
9.87 (s, 1H). 13C NMR (DMSO-d6): δ 68.68 (CH), 115.34, 115.53,
125.10, 128.21, 128.40, 128.66, 129.17, 130.15, 133.54, 156.67,
and 157.84 (C and CHarom.), 171.75 (CdO), 182.49 (CdS). Anal.
Calcd for C21H16N2O3S: C, H, N.
Competition Binding Assay. The assay was performed using
CHO cells membranes (or rat cerebellum homogenate) as previously
described.31 Briefly, the competitive binding experiments were
performed using [3H]-SR141716A (1 nM) or [3H]-CP-55,940 (1
nM) as radioligands for the hCB1 and the hCB2 cannabinoid
receptors, respectively, at 30 °C in plastic tubes, and 40 µg of
membranes per tube resuspended in 0.5 mL (final volume) binding
buffer (50 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 0.5% bovine
serum albumin, pH 7.4). The test compounds were present at
varying concentrations, and the nonspecific binding was determined
in the presence of 10 µM HU-210. After 1 h the incubation was
stopped, and solutions were rapidly filtered through 0.5% PEI
pretreated GF/B glass fiber filters (Whatman, Maidstone, UK) on
a M-48T Brandell cell harvester and washed twice with 5 mL of
ice-cold binding buffer without serum albumin. The radioactivity
on the filters was measured in a Pharmacia Wallac 1410 â-counter
using 10 mL of Aqualuma (PerkinElmer, Schaesberg, The Neth-
erlands), after 10 s shaking and 3 h resting. Assays were performed
at least in triplicate. Final DMSO concentrations in the assay were
less than 0.1%.
Under these conditions, using [3H]-SR141716A, the Bmax value
was 57 pmol/mg of protein and the Kd value was 1.13 ( 0.13 nM
for the hCB1 cannabinoid receptor.
The competition binding assays performed on the rat cerebellum
were performed in the presence of 100 µg of protein per tube. Under
these conditions, using [3H]-SR141716A, the Bmax value was 3.30
pmol/mg of protein and the Kd value was 3.11 ( 0.15 nM for the
hCB1 cannabinoid receptor.
[35S]-GTPγS Assay. [35S]-GTPγS (1173 Ci/mmol) was obtained
from Amersham (Roosendaal, The Netherlands). The binding
experiments were performed at 30 °C in plastic tubes containing
40 µg of protein (CHO cells homogenate or rat cerebellum
homogenate) in 0.5 mL (final volume) of binding buffer (50 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 100 mM NaCl, 0.1% bovine
serum albumin, pH 7.4) supplemented with 20 µM GDP and
varying concentrations of the test compounds. The assay was
initiated by the addition of [35S]-GTPγS (0.05 nM, final concentra-
tion). The tubes were incubated for 1 h. The incubations were
terminated by the addition of 5 mL of ice-cold washing buffer (50
mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 100 mM NaCl). The
suspension was immediately filtered through GF/B filters using a
48-well Brandell cell harvester and washed twice with the same
ice-cold buffer. The radioactivity on the filters was counted as
mentioned above. Nonspecific binding was measured in the
presence of 100 µM Gpp(NH)p. Assays were performed in
triplicate.
Brain Membrane Preparation for FAAH Assay. Adult rat
brains were homogenized at 4°C in homogenization buffer (20mM
HEPES, 1 mM MgCl2, pH 7.0) using a tissue grinder and
subsequently centrifuged for 20 min at 36 000 g. The pellet was
resuspended in homogenization buffer and centrifuged again for
20 min at 36 000 g. The latter operation was performed twice. The
resulting pellet was stored in a conservation buffer (50 mM
Tris.HCl, 1 mM EDTA, 3 mM MgCl2, pH 7.6). Membranes aliquots
were stored at -80 °C until use. The protein content was determined
as for CHO cells membranes.
Fatty Acid Amide Hydrolase Inhibition Assay. Compounds
were assayed as described by Jonsson et al.45 Membranes, test
compounds or DMSO (10 µL), [3H]-AEA (50.000 dpm; 2 µM final
concentration), and assay buffer (10 mM Tris-HCl, 1 mM EDTA,
0.1% (w/v) BSA, pH 7.6) were incubated at 37 °C for 10 min.
Reactions were stopped by rapidly placing the tubes in ice and
adding 400 µL of chloroform/methanol (1:1 v/v) followed by
vigorous mixing. Phases were separated by centrifugation at 850g
and aliquots (200 µL) of the upper methanol/buffer phase were
counted for radioactivity by liquid scintillation counting (Pharmacia
Wallac 1410 â-counter). Blanks contained buffer instead of the
homogenate preparations.
1,3-Dicyclohexyl-5-phenylimidazolidine-2,4-dione (40) was
synthesized as derivatives 20-29, with 1,3-dicyclohexylurea (39)
instead of 1,3-diphenylurea, and recrystallized from hexane-
acetone. Yield: 46%. Mp: 99.2-99.8 °C. MS DEI: 340 [M]+. 1H
NMR (DMSO-d6): δ 0.68-2.09 (m, 20H), 3.49-3.58 (m, 1H),
3.73-3.81 (m, 1H), 5.17 (s, 1H), 7.28-7.36 (m, 5H). 13C NMR
(DMSO-d6): δ 24.95, 25.34, 35.47, 29.22, 29.93, and 31.41 (CH2),
50.89, 53.22, and 61.63 (CH), 127.62, 128.73, 128.99, and 136.43
(C and CHarom.), 153.44 (CdO), 169.16 (CdO). Anal. Calcd for
C21H28N2O2: C, H, N.
Cell Culture and Preparation of hCB1- or hCB2-Transfected
CHO Cell Membranes. Chinese hamster ovarian (CHO) cells
stably transfected with the cDNA sequences encoding either the
human CB1 or the human CB2 cannabinoid receptors were kindly
donated by Dr. M. Detheux and Dr. P. Nokin, respectively
(Euroscreen s.a., Gosselies, Belgium). Cells were grown in Ham’s
F12 nutrient mixture supplemented with 10% FBS, 2.5 µL/mL
fungizone, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400
µg/mL G418. Once at confluence, cells were trypsinized and
collected by centrifugation at 100g for 10 min. The following steps
were performed on ice. Pellet was lysed in ice-cold 50 mM Tris-
HCl, pH 7.4, and the homogenate was centrifuged at 15 000g for
10 min. The resulting pellet (membranes) was washed twice with
the same solution under identical conditions. Protein content was
determined as described by Bradford using Coomasie Blue (Bio-
Rad, Belgium) with bovine serum albumin as standard.
Preparation of Rat Cerebellum Membranes. Male Wistar rats
(250-300 g) were purchased from IFFA-CREDO (Les Oncins,
France). All experiments on animals were approved by the
institutional ethics committee and the housing conditions were as
specified by the Belgian Law of November 14, 1993, on the
protection of laboratory animals (agreement no. LA 1230315).
Cerebella were carefully dissected on ice. All the manipulations
were performed at 0-4°C. Rat cerebellum membranes were
prepared in 50 mM Tris-HCl pH 7.4, with a Potter-Elvejhem and
a Dounce tissue grinder, and the suspension was centrifuged at 400g
for 10 min. The supernatant was collected and centrifuged at
39 000g for 10 min. The resulting pellet was resuspended in 50
mM Tris-HCl pH 7.4, homogenized, and centrifuged again at 39 000
g for 10 min. The pellet was washed twice more under the same

New CB1 Receptor InVerse Agonists/Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 881
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Molecular Modeling. All computational experiments were
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Salo. The original model40 was modified to the inactive R-state,
and several residues had to be rotated in order to enable the accurate
docking of SR141716A and of our derivatives.
Docking of both enantiomers of 25 was carried out using the
genetic algorithm GOLD (version 3.0).42 It performs docking of
flexible ligands into proteins with partial flexibility in the neighbor-
hood of the active site. The torsion angles of Ser, Thr, and Tyr
hydroxyl groups as well as Lys NH3+ moieties are optimized during
the run so that hydrogen-bond formation is favored. Defaults settings
were used for the genetic algorithm parameters. To take protein
flexibility into account, the complexes were then refined with
Discover3 (CVFF force field, dielectric constant ) r).43 The
energetic minimization process consists of two sequential steps:
the Steepest Descent algorithm, reaching a final convergence of
10.0 kcal mol-1 Å-1, followed by the Conjugate Gradient algorithm
to reach a final convergence of 0.01 kcal mol-1 Å-1. First, all the
protein’s atoms were held fixed, and only the orientation of the
ligand was optimized. Then, the R-C atoms of the binding site’s
residues (sphere of 18.0 Å around the nitrogen atom of K3.28) were
relaxed. A tethering restraint was applied on these atoms, to keep
them from moving too far from their original positions. This
restraint had a quadratic form with a constant force of 20 kcal Å-2
and was progressively decreased (scale factors of 0.5 and 0.25).
Data Analysis. IC50 values were determined by nonlinear
regression analysis performed using the GraphPad prism program
(GraphPad Software, San Diego, CA). The Ki values were calculated
following the Cheng-Prusoff equation: Ki ) IC50/(1 + L/Kd). The
statistical significance of [35S]-GTPγS results was assessed using
one-way ANOVA followed by a Dunett post-test.
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Acknowledgment. The authors wish to thank Franc¸ois
Abbeloos for technical assistance. T.P., P.D.P., and P.D.M. are
exchange pregraduate students from Universita` degli Studi di
Salerno (Italy). One of us (G.G.M.) is very indebted to “Fonds
pour la formation a` la Recherche dans l’Industrie et l’Agriculture
(FRIA)” for the award of a research fellowship. This work was
partially funded by a FRSM grant (Belgian National Fund for
Scientific Research) and a FSR grant (Universite´ catholique de
Louvain).
(20) Muthian, S.; Rademacher, D. J.; Roelke, C. T.; Gross, G. J.; Hillard,
C. J. Anandamide Content is Increased and CB1 Cannabinoid
Receptor Blockade Is Protective During Transient, Focal Cerebral
Ischemia. Neuroscience 2004, 129, 743-750.
Supporting Information Available: Procedures for the syn-
thesis of substituted phenylglyoxal and substituted urea and thiourea
derivatives, characteristic IR peaks, elemental analysis results of
original compounds, and X-ray structure data for 22. This material
is available free of charge via the Internet at http://pubs.acs.org.
(21) Muccioli, G. G.; Lambert, D. M. Current Knowledge on the
Antagonists and Inverse Agonists of Cannabinoid Receptors. Curr.
Med. Chem. 2005, 12, 1361-1394.
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