1-Benzhydryl-3-phenylurea and 1-benzhydryl-3-phenylthiourea derivatives: New templates among the CB1 cannabinoid receptor inverse agonists

Article abstract of DOI:10.1021/jm0503906

New 1-benzhydryl-3-phenylurea derivatives and their 1-benzhydryl-3- phenylthiourea isosteres were synthesized and evaluated for their human CB ₁ and CB₂ cannabinoid receptor affinity. These compounds proved to be selective CB₁ cannabinoid receptor ligands, acting as inverse agonists in a [³⁵S]-GTPγS assay. The affinity of 3,5,5′-triphenylimidazolidine-2,4-dione and 3,5,5′-triphenyl-2- thioxoimidazolidin-4-one derivatives, possessing the 1-benzhydryl-3-phenylurea and 1-benzhydryl-3-phenylthiourea moiety, respectively, was also evaluated. In conclusion, the 1-benzhydryl-3-phenylurea scaffold seems to be a new interesting template of CB₁ cannabinoid receptor inverse agonists.

Full text of DOI:10.1021/jm0503906

7486
J. Med. Chem. 2005, 48, 7486-7490
1-Benzhydryl-3-phenylurea and 1-Benzhydryl-3-phenylthiourea Derivatives:
New Templates among the CB1 Cannabinoid Receptor Inverse Agonists
Giulio G. Muccioli,† Johan Wouters,# Gerhard K. E. Scriba,‡ 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 April 25, 2005
New 1-benzhydryl-3-phenylurea derivatives and their 1-benzhydryl-3-phenylthiourea isosteres
were synthesized and evaluated for their human CB1 and CB2 cannabinoid receptor affinity.
These compounds proved to be selective CB1 cannabinoid receptor ligands, acting as inverse
agonists in a [35S]-GTPγS assay. The affinity of 3,5,5′-triphenylimidazolidine-2,4-dione and
3,5,5′-triphenyl-2-thioxoimidazolidin-4-one derivatives, possessing the 1-benzhydryl-3-pheny-
lurea and 1-benzhydryl-3-phenylthiourea moiety, respectively, was also evaluated. In conclusion,
the 1-benzhydryl-3-phenylurea scaffold seems to be a new interesting template of CB1
cannabinoid receptor inverse agonists.
Scheme 1a
Introduction
The synthesis of the first potent CB1 cannabinoid
receptor antagonist/inverse agonist SR141716A1 (ri-
monabant) and the breeding of knock-out CB1 mice2,3
confirmed the high therapeutic potential for modulating
CB1 cannabinoid receptor activity. Several CB1 cannab-
inoid receptor inverse agonists were shown to reduce
food intake,4,5 ethanol consumption,6 and nicotine re-
ward7,8 in animals. Other studies suggested a beneficial
effect on septic shock in rats9 and in gastrointestinal
disorders.10 Subsequent phase II or III clinical trials
confirmed the usefulness of such compounds in treating
obesity11,12 and nicotine11 addictions. Among the can-
nabinoid receptor antagonists/inverse agonists avail-
able, most are constructed around a central heterocyclic
ring (pyrazole, triazole, indole, imidazolidinedione, or
thioxoimidazolidinone) bearing further substituted aro-
matic rings.13
In this paper, we report the CB1 cannabinoid receptor
inverse agonist properties of new compounds that are
not built around such a central ring. The 1-benzhydryl-
3-phenylurea derivatives described herein exhibited
interesting affinities and potencies for the CB1 cannab-
inoid receptor. The 1-benzhydryl-3-phenylthiourea isos-
teres also showed affinity and inverse agonist properties
at the CB1 cannabinoid receptor. The compounds were
then compared with the corresponding 3-phenyl-5,5′-
diphenylimidazolidine-2,4-dione and 3-phenyl-5,5′-di-
phenyl-2-thioxoimidazolidin-4-one derivatives.
a All X2 substituents are in the para position except that for 8
(X2 is in ortho). Reagents and conditions: (i1) H2O/EtOH, NaCN,
reflux 3 h, CHCl3 extraction of the oily benzoin intermediate; (i2)
HNO3, reflux, 2 h; (i′) CH3COOH/H2O, NaCNO, room temp; (i′′)
acetone, NH4OH, room temp; (ii) DMSO/aqueous KOH, nine
microwave pulses.
the substituted benzils (1). Phenylurea (2, X′ ) O) and
phenylthiourea (2, X′ ) S) derivatives were easily
synthesized from the corresponding aniline and phe-
nylisothiocyanate, respectively (Scheme 1). Target 1-ben-
zhydryl-3-phenylurea derivatives (3-21) were obtained
by reaction of the respective benzil (1) and phenylurea
(2, X′ ) O), following a microwave-enhanced method
previously described14 (Scheme 1). The corresponding
thio derivatives (22-25) were obtained similarly by
reacting 1 and phenylthiourea (2, X′ ) S). The synthesis
of the thio derivatives, reported here for the first time,
will allow the oxo and thio isostere comparison at the
CB1 and CB2 cannabinoid receptors. The 3,5,5′-triph-
enylimidazolidine-2,4-dione (26-29) and 3,5,5′-triphen-
yl-2-thioxoimidazolidin-4-one derivatives (30, 31) were
obtained by reaction of 1 and phenylurea (2, X′ ) O) or
phenylthiourea (2, X′ ) S) in ethanol in the presence of
ethanolate (Scheme 2).
Results and Discussion
Chemistry. Benzoin condensation starting from sub-
stituted benzhaldehydes was used for the synthesis of
* To whom correspondence should be addressed. Phone: +32 2
7647347. Fax: +32 2 7647363. E-mail: lambert@cmfa.ucl.ac.be.
† Universite´ Catholique de Louvain.
# University of Namur.
Pharmacology. An initial screening, performed at
10 µM, using human CB1 and CB2 cannabinoid recep-
tors expressed in Chinese hamster ovarian (CHO) cells
‡ Department of Pharmaceutical Chemistry, University of Jena.
§ Department of Inorganic and Analytical Chemistry, University of
Jena.
10.1021/jm0503906 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/21/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7487
Table 1. Percentage Displacement of [3H]-SR141716A and
[3H]-CP-55,940 Specific Binding (10 µM) and Affinities (Ki, If
Given) for 3-31a on hCB1 and hCB2 Cannabinoid Receptorsb
Scheme 2a
% displacement
hCB1
hCB2
hCB1
Ki (nM)
compd
X′ X1
X2
receptor receptor
a Reagents and conditions: EtOH, Na, reflux 12 h.
and either [3H]-SR141716 or [3H]-CP-55,940 as radio-
ligands for the CB1 or CB2 cannabinoid receptors,
respectively, allowed the establishment of some pre-
liminary structure-affinity relationships (Table 1).
First, the benzhydryl moiety must be substituted to
achieve CB1 cannabinoid receptor affinity. Indeed com-
pounds 3, 4, and 6-9 displaced less than 25% of [3H]-
SR141716A specific binding. Furthermore, the preferred
benzhydryl substitutents are chlorine (12) and bromine
(13). Second, comparing the radioligand displacements
obtained with 14-21 revealed that chlorine and bro-
mine are the preferred substituents for the phenyl
moiety as well. Compounds 3-21 showed only poor
displacement of the CB2 cannabinoid receptor bound
radioligand ([3H]-CP-55,940). Ki values of 12-18, which
showed the highest radioligand displacement at 10 µM
at the CB1 cannabinoid receptor, were obtained (Table
1). Substitution of the phenyl moiety (X2 in Scheme 1)
greatly increased the affinity for the CB1 cannabinoid
receptor as illustrated by 12 (Ki ) 7050 ( 550 nM) and
14 (Ki ) 1100 ( 100 nM) or by 13 (Ki ) 2900 ( 250
nM) and 16 (Ki ) 650 ( 50 nM). With respect to the
bromobenzhydryl derivatives (13, 16, 17, 19-21), the
bromine substituent (X2 in Scheme 1) proved to be the
preferred substituent at the para position of the phenyl
ring (17, Ki ) 500 ( 50 nM). Smaller and larger halogen
atoms resulted in decreased affinity, as illustrated by
16 (Ki ) 650 ( 50 nM) and 19 (32 ( 2% of displacement
at 10 µM), respectively. Replacing the bromobenzhydryl
(X1 in Scheme 1) by a chlorobenzhydryl (15, Ki ) 1250
( 100 nM) or an iodobenzhydryl (18, Ki ) 750 ( 100
nM) moiety also decreased the affinity. Four 1-benzhy-
dryl-3-phenylthioureas (22-25) were obtained to esti-
mate the effect of the oxygen-sulfur substitution (Table
1). The affinity of the unsubstituted 3-phenyl derivatives
was increased by the replacement of the oxygen atom
by the sulfur as illustrated by 12 (Ki ) 7050 ( 550 nM)
and 22 (Ki ) 2900 ( 250 nM) or by 13 (Ki ) 2900 ( 250
nM) and 23 (Ki ) 1800 ( 150 nM). However, the affinity
of the substituted 3-phenyl derivatives (X2 in Scheme
1) is decreased for the thio derivatives as shown by 14
(Ki ) 1100 ( 100 nM) and 24 (Ki ) 1900 ( 200 nM) or
by 16 (Ki ) 650 ( 50 nM) and 25 (Ki ) 1500 ( 150 nM).
Thus, replacement of the oxygen by a sulfur atom in
1-[bis(4-bromophenyl)methyl]-3-(4-chlorophenyl)urea (16)
resulted in decreased affinity.
3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
F
Cl
Br
Cl Cl
Cl Br
Br Cl
Br Br
H
F
Cl
Br
I
2-F
OMe
C6H13
H
<20
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
4
23 ( 2 <10
43 ( 2 <20
5
6
<20
<20
<10
<10
<20
24 ( 2
<10
7
<10
<20
<20
8
9
10
11
12
13
14
15
16
17
18
19
20
21
55 ( 2
<20
70 ( 3
70 ( 2
95 ( 1
80 ( 2
83 ( 1
75 ( 3
60 ( 2
32 ( 2
H
H
23 ( 2 7050 ( 550
38 ( 3 2900 ( 250
24 ( 2 1100 ( 100
29 ( 3 1250 ( 100
31 ( 2 650 ( 50
22 ( 2 500 ( 50
19 ( 2 750 ( 100
I
Br
Br
I
34 ( 1
ND
ND
ND
Br CH2OH
Br C6H13
49 ( 2 <20
<20 <20
22
23
24
25
S
S
S
S
Cl
Br
Cl Cl
Br Cl
H
H
77 ( 4
86 ( 2
85 ( 1
71 ( 3
23 ( 2 2900 ( 250
29 ( 3 1800 ( 150
23 ( 2 1900 ( 200
29 ( 4 1500 ( 150
26
27
28
29
30
31
O
O
O
O
S
H
H
Br
H
H
H
28 ( 3 < 20
ND
H
< 20
< 20
27 ( 3 < 20
29 ( 4 < 20
73 ( 2 <20
62 ( 2 <20
ND
F
ND
Br
Br
ND
3450 ( 300
2750 ( 250
S
Br Cl
Reference Compounds
ND
SR141716A
WIN-552122
HU-210
ND
ND
ND
5.4 ( 0.2
3800 ( 150
19 ( 2
ND
ND
a All X2 substituents are in the para position except that for 8
(X2 is in ortho). b Mean ( SEM of at least four experiments
performed in duplicate. The Ki values were obtained from non-
linear analysis of competition curves using [3H]-SR141716A as
radioligand.
10 µM, 3,5,5′-triphenylimidazolidine-2,4-dione (26), 3-(4-
bromophenyl)-5,5′-diphenylimidazolidine-2,4-dione (27),
5,5′-bis(4-fluorophenyl)-3-phenylimidazolidine-2,4-di-
one (28), and 5,5′-bis(4-bromophenyl)-3-phenylimidazo-
lidine-2,4-dione (29), displaced less than 30% of the [3H]-
SR141716A specifically bound to the CB1 cannabinoid
receptor. The 2-thioxoimidazolidin-4-ones, 5,5′-bis(4-
bromophenyl)-3-phenyl-2-thioxoimidazolidin-4-one (30)
and 5,5′-bis(4-bromophenyl)-3-(4-chlorophenyl)-2-thiox-
oimidazolidin-4-one (31), exhibited Ki values of 3450 (
300 and 2750 ( 250 nM, respectively. When these
values are compared with those obtained for the corre-
We previously described the structure-affinity rela-
tionships at the CB1 cannabinoid receptor of alkyl and
alkylaryl 3-substituted 5,5′-diphenylimidazolidine-2,4-
diones15 and 5,5′-diphenyl-2-thioxoimidazolidin-4-ones16
as selective CB1 cannabinoid receptor inverse agonists.
Therefore, four 3,5,5′-triphenylimidazolidine-2,4-diones
(26-29) and two 3,5,5′-triphenyl-2-thioxoimidazolidin-
4-one derivatives (30, 31) were synthesized (Scheme 2)
and assayed for their CB1 cannabinoid receptor affinity
(Table 1). The four imidazolidine-2,4-diones assayed at

7488 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Brief Articles
Table 2. Determination of the Potency (EC50) and Percentages
of Basal Maximal Stimulation (Emax) of [35S]-GTPγS Binding at
hCB1 Cannabinoid Receptors for 14-17, 25, HU-210, and
SR141716Aa
compd
EC50 (nM)
Emax (% above basal)
Urea Derivatives
1050 ( 100
700 ( 50
14
15
16
17
-74 ( 2
-66 ( 1
-72 ( 2
-61 ( 1
450 ( 50
500 ( 50
Thiourea Derivatives
1250 ( 100
25
-61 ( 3
Reference Compounds
10.1 ( 0.7
Figure 1. [35S]-GTPγS binding stimulation assay of selected
compounds and reference cannabinoid ligands (10 µM) on hCB1
cannabinoid receptor. Data are the mean ( SEM of at least
three experiments performed in duplicate. Statistical signifi-
cance was assessed by one-way ANOVA followed by a Dunett
post-test: (//) P < 0.01.
SR141716A
HU-210
-84 ( 2
200 ( 15
0.6 ( 0.04
a Mean ( SEM of at least three experiments performed in
duplicate. Statistical signification of [35S]-GTPγS assay results was
assessed using a one-way ANOVA followed by a Dunett post-test.
sponding 1-benzhydryl-3-phenylurea and thiourea de-
rivatives, it appears that the orientation of the 1-ben-
zhydryl-3-phenylurea and thiourea phenyl rings imposed
by the additional carbonyl led to a decrease in the
affinity for the CB1 cannabinoid receptor. This is il-
lustrated by 13 and 29 (70% and 29% of displacement
at 10 µM, respectively) or by 25 and 31 (Ki of 1500 (
150 and 2750 ( 250 nM, respectively).
substitution of the phenyl rings is also observed in the
structure-affinity relationships of SR141716A and
other CB1 cannabinoid receptor inverse agonists.13
Finally, despite a lower affinity compared to the affinity
of the reference inverse agonist SR141716A, the 1-ben-
zhydryl-3-phenylurea structure constitutes a new, in-
teresting template for CB1 cannabinoid receptor inverse
agonists. Indeed the noncyclic core scaffold CB1 can-
nabinoid receptor inverse agonist described herein dif-
fers from most of the reported CB1 cannabinoid receptor
antagonists/inverse agonists, which are built around a
cyclic central unit.
The 1-benzhydryl-3-phenylurea and thiourea deriva-
tives were further characterized in a functional [35S]-
GTPγS assay as previously described.17 The assay relies
on the binding of [35S]-GTPγS, a radiolabeled nonhy-
drolyzable GTP analogue, to the G protein upon activa-
tion by an agonist of the G-protein-coupled receptor.18
The assay distinguishes between agonists (increasing
nucleotide binding), antagonists (not affecting the bind-
ing), and inverse agonists (decreasing nucleotide bind-
ing). HU-210, a potent cannabinoid agonist, the inverse
agonist SR141716A, and the 1-benzhydryl-3-phenylurea
and thiourea derivatives 12-18 and 22-25 were
screened at 10 µM. The cannabinoid receptor agonist
HU-210 induced an increase in the [35S]-GTPγS binding
(200 ( 15% compared to basal level), while SR141716A
and the tested compounds significantly decreased the
nucleotide binding, thus acting as inverse agonists of
the human CB1 cannabinoid receptor (Figure 1). For
instance, 1-[bis(4-bromophenyl)methyl]-3-(4-chlorophe-
nyl)urea (16) and its thio derivative 1-[bis(4-bromophe-
nyl)methyl]-3-(4-chlorophenyl)thiourea (25) decreased
[35S]-GTPγS binding by 68 ( 1% and 61 ( 1% compared
to basal level, respectively. To further explore the
inverse agonist properties, the potency of 14-17 and
25 was determined (Table 2). The 1-benzhydryl-3-
phenylureas 14-17 and the thiourea derivative 25 dose-
dependently decreased the [35S]-GTPγS binding, show-
ing EC50 values of the same magnitude compared to
their respective Ki values.
Experimental Section
All reagents, used without further purification, were pur-
chased from Sigma-Aldrich or Acros. Solvents were of analyti-
cal grade. A commercial household microwave oven (2.45 Ghz)
was used. Melting points (uncorrected) were determined in
1
open capillaries using an Electrothermal 9100 apparatus. H
NMR and 13C NMR spectra were recorded on a Bruker AM-
300 spectrometer at room temperature and analyzed using
WIN-NMR software. Chemical shifts (δ) are reported relative
to the tetramethylsilane peak set at 0.00 ppm. Signals were
abbreviated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. For multiplets, signals are reported as intervals.
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. Infrared (IR) spectra (ν in
cm-1) were recorded on a Perkin-Elmer FT-IR 286 spectrom-
eter (Supporting Information).
Synthesis: General Procedure for the Synthesis of
1-Benzhydryl-3-phenylurea Derivatives. Compounds 3, 5,
7, 11-13, 15, 17, and 19 were previously described.14 The other
1-benzhydryl-3-phenylurea derivatives were similarly ob-
tained. To a solution of benzil (7.15 mmol) and phenylurea
(14.3 mmol) in 25 mL of DMSO was added with stirring 25
mL of 1.2 M aqueous KOH. Following 90 s of 750 W microwave
irradiation, the mixture was stirred for an additional 5 min.
Additional 30 s pulses were applied at 6, 9, 12, 15, 18, 21, 24,
and 30 min. Between pulses, the mixture was stirred. After
the completion of the sequence, the mixture was poured onto
cold water and the resulting precipitate was filtered, dried,
and crystallized. All microwave irradiations were carried out
in an open system.
In conclusion, the 1-benzhydryl-3-phenylurea and
1-benzhydryl-3-phenylthiourea derivatives reported here
selectively bind to the CB1 cannabinoid receptor acting
as inverse agonists. The structure-affinity relationships
highlighted the importance of halogen substituents,
especially bromine. Thus, 1-[bis(4-bromophenyl)methyl]-
3-(4-bromophenyl)urea (17) exhibited the highest affin-
ity for the CB1 cannabinoid receptor within this series
of compounds. Interestingly, the crucial role of halogen
1-Benzhydryl-3-(4-fluorophenyl)urea (4). Yield: 52%.
Mp 229.1-230.2 °C. MS DEI (Desorption Electron Impact):
320 [M]+. 13C NMR (DMSO-d6) δ 56.71 (CH), 114.88, 115.20,
119.02, 126.78, 127.43, 128.34, 136.43, 143.02 (C and CH
arom), 154.21 (CdO). Anal. (C20H17FN2O) C, H, N.

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7489
1-Benzhydryl-3-(4-bromophenyl)urea (6). Yield: 50%.
Mp 233.4-234.1 °C. MS DEI: 381 [M]+. 13C NMR (DMSO-d6) δ
56.97 (CH), 112.68, 119.67, 127.04, 128.59, 131.57, 143.16 (C
and CH arom), 154.28 (CdO). Anal. (C20H17BrN2O) C, H, N.
1-Benzhydryl-3-(2-fluorophenyl)urea (8). Yield: 55%.
Mp 229.5-230.2 °C. MS DEI: 320 [M]+. 13C NMR (DMSO-d6)
δ 56.87 (CH), 114.58, 114.84, 119.69, 121.44, 121.57, 124.35,
126.81, 126.94, 128.43, 142.99 (C and CH arom), 153.01 (Cd
O), 153.92.
1-Benzhydryl-3-(4-methoxyphenyl)urea (9). Yield: 51%.
Mp 215.0-215.9°C. MS DEI: 332 [M]+. 13C NMR (DMSO-d6)
δ 55.06 (CH3), 56.74 (CH), 113.87, 119.11, 126.81, 128.36,
133.28, 143.25 (C and CH arom), 153.92 (CdO), 154.44. Anal.
(C21H20N2O2) C, H, N.
1-Benzhydryl-3-(4-hexylphenyl)urea (10). Yield: 48%.
Mp 172.1-173.2 °C. MS DEI: 386 [M]+. 13C NMR (DMSO-d6)
δ 13.84, 21.99, 28.14, 31.06, 34.35, and 40.12 (CH2), 56.74 (CH),
117.56, 126.81, 128.36, 135.05, 137.74, 143.18 (C and CH
arom), 154.31 (CdO). Anal. (C26H30N2O) C, H, N.
1-[Bis(4-chlorophenyl)methyl]-3-(4-chlorophenyl)-
urea (14). Yield: 46%. Mp >300 °C. MS DEI: 406 [M]+. 13C
NMR (DMSO-d6) δ 56.20 (CH), 119.60, 120.70, 125.29, 128.99,
129.70, 131.89, 139.47, 142.38 (C and CH arom), 154.54 (Cd
O). Anal. (C20H15Cl3N2O) C, H, N.
resulting solution was refluxed for 12 h and after cooling was
poured onto ice. The precipitate was filtered, dried, and
recrystallized from ethanol.
3,5,5′-Triphenylimidazolidine-2,4-dione (26). Yield: 62%.
Mp 204.2-204.9 °C. MS DCI: 329 [M + H]+. 13C NMR (DMSO-
d6): δ 69.21 (C), 117.66, 121.24, 126.97, 128.33, 128.59, 128.79,
129.04, 131.82, 139.66 (C and CH arom), 154.28 (CdO), 172.46
(CdO).
3-(4-Bromophenyl)-5,5′-diphenylimidazolidine-2,4-di-
one (27). Yield: 58%. Mp 214.4-215.1 °C. MS DEI: 408 [M +
H]+. 13C NMR (DMSO-d6): δ 69.65 (C), 121.54, 127.23, 128.79,
129.11, 129.24, 131.44, 132.35, 139.86 (C and CH arom), 154.22
(CdO), 172.52 (CdO). Anal. (C21H15Br2N2O2) C, H, N.
5,5′-Bis(4-fluorophenyl)-3-phenylimidazolidine-2,4-di-
one (28). Yield: 57%. Mp 242.9-244.0 °C. MS DEI: 364 [M]+.
13C NMR (DMSO-d6): δ 68.03 (C), 115.27, 115.6, 126.72,
128.07, 128.72, 128.85, 131.44, 135.45, 160.11, 163.34 (C and
CH arom), 153.83 (CdO), 172.01 (CdO). Anal. (C21H14F2N2O2)
C, H, N.
5,5′-Bis(4-bromophenyl)-3-phenylimidazolidine-2,4-di-
one (29). Yield: 58%. Mp 162.5-163.7 °C. MS DEI: 486 [M]+.
13C NMR (DMSO-d6): δ 68.45 (C), 121.94, 126.56, 128.46,
128.96, 129.54, 129.87, 131.78, 138.45 (C and CH arom), 154.05
(CdO), 171.76 (CdO). Anal. (C21H14Br2N2O2) C,H,N.
Synthesis of 3-Substituted 5,5′-Diphenyl-2-thioxoimi-
dazolidin-4-one Derivatives. These derivatives were ob-
tained similarly to the 3-substituted 5,5′-diphenylimidazolidine-
2,4-dione derivatives starting from the corresponding phenyl-
thiourea.
5,5′-Bis(4-bromophenyl)-3-phenyl-2-thioxoimidazolidin-
4-one (30). Yield: 40%. Mp 293.0-293.9 °C. MS DEI: 502
[M]+. 13C NMR (DMSO-d6): δ 70.89 (C), 122.26, 128.79, 128.98,
131.83, 132.67, 136.94 (C and CH arom), 172.33 (CdO), 181.00
(CdS). Anal. (C21H14Br2N2OS) C, H, N.
5,5′-Bis(4-bromophenyl)-3-(4-chlorophenyl)-2-thiox-
oimidazolidin-4-one (31). Yield: 41%. Mp >300 °C. MS
DEI: 536 [M]+. 13C NMR (DMSO-d6): δ 71.53 (C), 122.83,
129.37, 131.18, 132.02, 132.41, 134.22, 137.40 (C and CH
arom), 172.72 (CdO), 181.19 (CdS). Anal. (C21H13Br2ClN2OS)
C, H, N.
1-[Bis(4-bromophenyl)methyl]-3-(4-chlorophenyl)-
urea (16). Yield: 49%. Mp >300 °C. MS DEI: 494 [M]+. 13C
NMR (DMSO-d6) δ 55.81 (CH), 119.34, 124.97, 128.21, 128.66,
131.90, 139.20, 141.73 (C and CH arom), 154.28 (CdO). Anal.
(C20H15Br2ClN2O) C, H, N.
1-[Bis(4-iodophenyl)methyl]-3-(4-bromophenyl)urea
(18). Yield: 43%. Mp 246.1-247.0 °C. MS DEI: 633 [M]+. 13
C
NMR (DMSO-d6) δ 56.13 (CH), 93.20, 112.74, 119.73, 129.43,
131.38, 131.51, 137.39, 137.65, 139.59, 142.51 (C and CH
arom), 154.21 (CdO). Anal. (C20H15I2BrN2O) C, H, N.
1-[Bis(4-bromophenyl)methyl]-3-(4-hydroxymethylphe-
nyl)urea (20). Yield: 52%. Mp 213.4-214.3 °C. MS DEI: 490
[M]+. 13C NMR (DMSO-d6) δ 56.19 (CH), 60.98 (CH2), 120.31,
121.86, 127.49, 128.07, 129.44, 131.57, 137.91, 142.38 (C and
CH arom), 154.67 (CdO). Anal. (C21H18Br2N2O2*1/2H2O) C, H,
N.
1-[Bis(4-bromophenyl)methyl]-3-(4-hexylphenyl)-
urea (21). Yield: 49%. Mp 202.8-203.3 °C. MS DEI: 544 [M]+.
13C NMR (DMSO-d6) δ 14.08, 18.67, 22.17, 28.37, 31.16, and
34.59 (CH2), 55.87 (CH), 117.92, 120.31, 128.53, 129.31, 131.57,
135.39, 137.78, 142.31 (C and CH arom), 154.47 (CdO). Anal.
(C26H28Br2N2O) C, H, N.
Synthesis of 1-Benzhydryl-3-phenylthiourea Deriva-
tives. The procedure described for the synthesis of 1-benzhy-
dryl-3-phenylurea derivatives was used except that phenylth-
iourea was substituted for phenylurea.
1-[Bis(4-chlorophenyl)methyl]-3-phenylthiourea (22).
Yield: 43%. Mp 185.1-185.8 °C. MS DEI: 387 [M]+. 13C NMR
(DMSO-d6): δ 59.23 (CH), 122.64, 123.94, 128.27, 128.40,
129.11, 131.76, 139.34, 140.50 (C and CH arom), 180.23 (Cd
S). Anal. (C20H16Cl2N2S) C, H, N.
Competition Binding Assay. CHO cells stably expressing
the human CB1 or the human CB2 cannabinoid receptors were
donated by Drs. M. Detheux and P. Nokin, respectively
(Euroscreen s.a., Gosselies, Belgium). Cells membranes were
obtained as previously described.16 Stock solutions of the
compounds were prepared in DMSO and further diluted
(100×) with the binding buffer to the desired concentration.
Under these conditions Bmax was 57 pmol/mg protein and Kd
was 1.13 ( 0.13 nM for the hCB1 cannabinoid receptor ([3H]-
SR141716A). The competitive binding experiments were per-
formed using [3H]-SR141716A (1 nM, 52 Ci/mol, Amersham,
Roosendaal, The Netherlands) or [3H]-CP-55940 (1 nM, 101
Ci/mol, from NEN Life Science, Zaventem, Belgium) as radio-
ligands for the hCB1 and the hCB2 cannabinoid receptor,
respectively, at 30 °C in plastic tubes, and 40 µg of membranes
per tube was resuspended in 0.5 mL (final volume) of binding
buffer (50 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 0.5%
bovine serum albumine, pH 7.4). Test compounds were present
at varying concentrations, and nonspecific binding was deter-
mined in the presence of 10 µM HU-210 (Tocris, Bristol, U.K.).
After 1 h of incubation, solutions were rapidly filtered through
0.5% PEI pretreated GF/B glass fiber filters (Whatman,
Maidstone, U.K.) on a M-48T Brandell cell harvester and
washed twice with 5 mL of ice-cold binding buffer without
serum albumin. Radioactivity was measured in a Pharmacia
Wallac 1410 â-counter in 10 mL of Aqualuma (PerkinElmer,
Schaesberg, The Netherlands) after 10 s of shaking and 3 h of
resting. Assays were performed at least in triplicate.
1-[Bis(4-bromophenyl)methyl]-3-phenylthiourea
(23).Yield: 44%. Mp 206.8-207.5 °C. MS DEI: 476 [M]+. 13
C
NMR (DMSO-d6): δ 59.37 (CH), 120.25, 122.64, 123.94, 128.27,
129.44, 131.31, 139.34, 140.89 (C and CH arom), 180.23 (Cd
S). Anal. (C20H16Br2N2S) C, H, N.
1-[Bis(4-chlorophenyl)methyl]-3-(4-chlorophenyl)thio-
urea (24). Yield: 41%. Mp 200.8-201.4 °C. MS DEI: 422 [M]+.
13C NMR (DMSO-d6): δ 59.30 (CH), 119.03, 124.20, 127.69,
128.40, 129.12, 131.77, 138.36, 140.37 (C and CH arom), 180.22
(CdS). Anal. (C20H15Cl3N2S) C, H, N.
1-[Bis(4-bromophenyl)methyl]-3-(4-chlorophenyl)thio-
urea (25). Yield: 43%. Mp 230.0-230.3 °C. MS DEI: 511 [M]+.
13C NMR (DMSO-d6): δ 59.43 (CH), 120.31, 124.20, 127.69,
128.07, 129.05, 131.31, 138.30, 140.67 (C and CH arom), 180.22
(CdS). Anal. (C20H15Br2ClN2S) C, H, N.
[
35S]-GTPγS Assay. Binding experiments were performed
General Procedure for the Synthesis of 3-Substituted
5,5′-Diphenylimidazolidine-2,4-dione Derivatives. So-
dium (1 g) was stirred in ethanol (50 mL) for 15 min before
benzil (9.5 mmol) and phenylurea (19 mmol) were added. The
at 30 °C in plastic tubes containing 40 µg of protein 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 the appropriate

7490 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Brief Articles
(6) Colombo, G.; Vacca, G.; Serra, S.; Carai, M. A.; Gessa, G. L.
Suppressing Effect of the Cannabinoid CB1 Receptor Antagonist,
SR141716, on Acohol’s Motivational Properties in Alcohol-
Preferring Rats. Eur. J. Pharmacol. 2004, 498, 119-123.
(7) Cohen, C.; Perrault, G.; Voltz, C.; Steinberg, R.; Soubrie, P.
SR141716, a Central Cannabinoid (CB1) Receptor Antagonist,
Blocks the Motivational and Dopamine-Releasing Effects of
Nicotine in Rats. Behav. Pharmacol. 2002, 13, 451-463.
(8) Le Foll, B.; Goldberg, S. R. Cannabinoid CB1 Receptor Antago-
nists as Promising New Medications for Drug Dependence. J.
Pharmacol. Exp. Ther. 2005, 312, 875-883.
(9) Kadoi, Y.; Hinohara, H.; Kunimoto, F.; Kuwano, H.; Saito, S.;
Goto, F. Effects of AM281, a Cannabinoid Antagonist, on
Systemic Haemodynamics, Internal Carotid Artery Blood Flow
and Mortality in Septic Shock in Rats. Br. J. Anaesth. 2005, 94,
563-568.
(10) Mascolo, N.; Izzo, A. A.; Ligresti, A.; Costagliola, A.; Pinto, L.;
Cascio, M. G.; Maffia, P.; Cecio, A.; Capasso, F.; Di Marzo, V.
The Endocannabinoid System and the Molecular Basis of
Paralytic Ileus in Mice. FASEB J. 2002, 16, 1973-1975.
(11) Cleland, J. G.; Ghosh, J.; Freemantle, N.; Kaye, G. C.; Nasir,
M.; Clark, A. L.; Coletta, A. P. Clinical Trials Update and
Cumulative Meta-Analyses from the American College of Car-
diology: WATCH, SCD-HeFT, DINAMIT, CASINO, INSPIRE,
STRATUS-US, RIO-Lipids and Cardiac Resynchronisation
Therapy in Heart Failure. Eur. J. Heart Failure 2004, 6, 501-
508.
(12) Van Gaal, L. F.; Rissanen, A. M.; Sheen, A. J.; Ziegler, O.;
Ro¨ssner, S. Effects of the Cannabinoid-1 Receptor Blocker
Rimonabant on Weight Reduction and Cardiovascular Risk
Factors in Overweight Patients: 1-Year Experience from the
RIO-Europe Study. Lancet 2005, 365, 1389-1397.
concentration of test compounds. The assay was initiated by
the addition of [35S]-GTPγS (0.05 nM, final concentration, 1173
Ci/mmol, Amersham, Roosendaal, The Netherlands). Following
1 h of incubation, a total of 5 mL of ice-cold washing buffer
(50 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 100 mM NaCl)
was added. 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. Radioactivity was
counted as mentioned above. Nonspecific binding was mea-
sured in the presence of 100 µM Gpp(NH)p. Assays were
performed in triplicate.
Data Analysis. IC50 and EC50 values were determined by
nonlinear regression analysis performed using the GraphPad
Prism 4.0 software (GraphPad Software, San Diego). The Ki
values were calculated on the basis of the Cheng-Prusoff
equation: Ki ) IC50/(1 + L/Kd).
Acknowledgment. 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 fel-
lowship. This work was partially funded by a FRSM
grant (Belgian National Fund for Scientific Research)
and a FSR grant (Universite´ catholique de Louvain).
Supporting Information Available: General procedures
for the synthesis of benzils, phenylureas, and phenylthioureas,
1H NMR and IR data, and results from elemental analysis of
the synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(13) Muccioli, G. G.; Lambert, D. M. Current Knowledge on the
Antagonists and Inverse Agonists of Cannabinoid Receptors.
Curr. Med. Chem. 2005, 12, 1361-1394.
(14) Muccioli, G. G.; Wouters, J.; Poupaert, J. H.; Norberg, B.;
Poppitz, W.; Scriba, G. K. E.; Lambert, D. M. Versatile Access
to Benzhydryl-Phenylureas through an Unexpected Rearrange-
ment during Microwave-Enhanced Synthesis of Hydantoins.
Org. Lett. 2003, 5, 3599-3602.
(15) Ooms, F.; Wouters, J.; Oscari, O.; Happaerts, T.; Bouchard, G.;
Carrupt, P.-A.; Testa, B.; Lambert, D. M. Explorations of the
Pharmacophore of 3-Alkyl-5-arylimidazolidinediones as New CB1
Cannabinoid Receptor Ligands and Potential Antagonists: Syn-
thesis, Lipophilicity, Affinity, and Molecular Modeling. J. Med.
Chem. 2002, 45, 1748-1756.
(16) Muccioli, G. G.; Martin, D.; Scriba, G. K. E.; Poppitz, W.;
Poupaert, J. H.; Wouters, J.; Lambert, D. M. Substituted 5,5′-
Diphenyl-2-thioxo-imidazolidin-4-one as CB1 Cannabinoid Re-
ceptor Ligands: Synthesis and Pharmacological Evaluation. J.
Med. Chem. 2005, 48, 2509-2517.
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