Substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one as CB 1 cannabinoid receptor ligands: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1021/jm049263k

A set of 30 substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one (thiohydantoins) derivatives was synthesized, and their affinity for the human CB₁ cannabinoid receptor has been evaluated. These compounds are derived from the previously described cannabinoid ligands 5,5′- diphenylimidazolidine-2,4-dione (hydantoins). The replacement of the oxygen by a sulfur leads to an increase of the affinity while the function-i.e., inverse agonism-determined by [³⁵S]-GTPγS experiments remains unaffected. Finally, to evaluate the molecular parameters that could influence the affinity of the thiohydantoins, molecular electrostatic potential as well as lipophilicity calculations were undertaken on representative thiohydantoins and hydantoins derivatives. In conclusion, 5,5′-bis-(4-iodophenyl)-3-butyl-2- thioxoimidazolidin-4-one (31) and 3-allyl-5,5′-bis(4-bromophenyl)-2- thioxoimidazolidin-4-one (32) possess the highest affinity for the CB ₁ cannabinoid receptor described to date for the hydantoin and thiohydantoins series when compared in a same bioassay.

Full text of DOI:10.1021/jm049263k

J. Med. Chem. 2005, 48, 2509-2517
2509
Substituted 5,5′-Diphenyl-2-thioxoimidazolidin-4-one as CB1 Cannabinoid
Receptor Ligands: Synthesis and Pharmacological Evaluation
Giulio G. Muccioli,† Diana Martin,† Gerhard K. E. Scriba,‡ Wolfgang Poppitz,§ Jacques H. Poupaert,†
Johan Wouters,| 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; Department of Pharmaceutical Chemistry,
University of Jena, Philosophenweg 14, D-07743 Jena, Germany; Department of Inorganic and Analytical Chemistry,
University of Jena, August-Bebel-Strasse 2, D-07743 Jena, Germany; and Laboratoire de Chimie Biologique Structurale,
Faculte´ des Sciences, Faculte´s Universitaires Notre Dame de la Paix, Rue de Bruxelles 61, 5000 Namur, Belgium
Received September 10, 2004
A set of 30 substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one (thiohydantoins) derivatives
was synthesized, and their affinity for the human CB1 cannabinoid receptor has been evaluated.
These compounds are derived from the previously described cannabinoid ligands 5,5′-
diphenylimidazolidine-2,4-dione (hydantoins). The replacement of the oxygen by a sulfur leads
to an increase of the affinity while the functionsi.e., inverse agonismsdetermined by [35S]-
GTPγS experiments remains unaffected. Finally, to evaluate the molecular parameters that
could influence the affinity of the thiohydantoins, molecular electrostatic potential as well as
lipophilicity calculations were undertaken on representative thiohydantoins and hydantoins
derivatives. In conclusion, 5,5′-bis-(4-iodophenyl)-3-butyl-2-thioxoimidazolidin-4-one (31) and
3-allyl-5,5′-bis(4-bromophenyl)-2-thioxoimidazolidin-4-one (32) possess the highest affinity for
the CB1 cannabinoid receptor described to date for the hydantoin and thiohydantoins series
when compared in a same bioassay.
Introduction
Notably, the control of the motor activity by this system
suggests that compounds targeting the cannabinoid
system could be useful for the treatment of motor
disorders, such as Parkinson’s disease,13 Tourette’s
syndrome,14 or choreas.15 The involvement of can-
nabimimetics in airway dilatation,16 intraocular pres-
sure,17 and intestinal motility18 represents further
potential therapeutic applications for compounds tar-
geting the cannabinoid system. Antagonists of the
CB1 receptor also possess very useful applications.
For instance, CB1 antagonists such as rimonabant
(SR141716A, Acomplia)19,20 could be of particular inter-
est for treating obesity21,22 and may be a helpful tool in
smoking cessation.23 These therapeutic goals have trig-
gered the synthesis of new compounds able to modulate
the activity of cannabinoid receptors. Several pharma-
ceutical companies have developed their own CB1 recep-
tor antagonist scaffolds, among them the diarylpyrazole
derivatives, such as SR141716A or the recently de-
scribed SR14777824 by Sanofi, substituted benzofurane
derivatives25 described by Eli Lilly (LY-320135), or 4,5-
dihydro-1H-pyrazole derivatives26,27 developed by Solvay
(SLV326) (Figure 1). Other derivatives such as imida-
zoles28 and triazoles29 have also been recently described.
We previously reported the synthesis and pharmaco-
logical characterization of several 3-substituted 5,5′-
diphenylimidazolidinedione derivatives, also known as
3-substituted 5,5′-diphenylhydantoins, as cannabinoid
ligands.30 These compounds exhibit moderate affinity
for the CB1 cannabinoid receptor and act as antagonists.
We recently demonstrated that they act as neutral
antagonists on rat brain membrane preparations and
as inverse agonists on human CB1 cannabinoid recep-
tors expressed in CHO cells, independently of the
Cannabinoids, either plant-derived or of synthetic
origin, mainly exert their properties by binding to two
G-protein-coupled receptors, the CB1 and the CB2 can-
nabinoid receptors. The cloning and pharmacological
characterization of these receptors,1-3 as well as the
discovery of their endogenous ligands4,5 along with the
enzymes responsible for their inactivation,6,7 have per-
mitted a better understanding of the so-called endocan-
nabinoid system.8
The CB1 cannabinoid receptor acts upon activation
by coupling primarily to a Gi/o G-protein. Gs coupling
has been described under certain conditions by Calan-
dra et al.9 The highest density of the receptor is found
in the cerebellum, the basal ganglia, the substantia
nigra pars compacta, and in some regions of the globus
pallidus. It is also present in peripheral organs such as
the adrenal glands, bone marrow, lungs, testis, and
uterus.10 In contrast to the CB1 cannabinoid receptor,
the CB2 cannabinoid receptor is limited essentially to
cells associated with the immune system, like leuko-
cytes, spleen, thymus, and tonsils.8 In addition, it was
recently reported that some specific human cerebellum
microglial cells do express the CB2 cannabinoid recep-
tor.11
The knowledge of the endocannabinoid system has led
to considerable interest in its therapeutic potential.12
* Corresponding author. Phone and Fax: +32 2 7647347. E-mail:
lambert@cmfa.ucl.ac.be.
† Universite´ Catholique de Louvain.
‡ Department of Pharmaceutical Chemistry, University of Jena.
§ Department of Inorganic and Analytical Chemistry, University of
Jena.
| Faculte´s Universitaires Notre Dame de la Paix.
10.1021/jm049263k CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/25/2005

2510 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Muccioli et al.
Figure 1. Structures of SR141716A, LY-320135, SLV326, and DML20, four CB1 cannabinoid receptor antagonists.
Scheme 1. Synthesis of the Alkylthiourea Derivatives
receptor density at the cell surface.31 Despite their
modest affinity, the 5,5′-diphenylhydantoins represent
(2a-2l)a
an original structure for cannabinoid receptor recogni-
tion. Preliminary structure-affinity relationships re-
vealed that introduction of a para substituent on the
phenyl rings increases the affinity of the 3-alkyl-5,5′-
diphenylhydantoins for the CB1 receptor in the order
H < Me < F < OMe < Br.32 In addition, substitution of
the nitrogen in position 3 is mandatory for the affinity
towards the CB1 cannabinoid receptor.
In the present study, this class of ligands was further
evaluated by replacement of the oxygen in position 2 of
the hydantoin ring by a sulfur atom, leading to 3-sub-
a Method A reagents and conditions: (a) NH4SCN, bromoben-
zene, reflux under nitrogen stream, 90 min. After cooling, the
product is precipitated in water. Method B reagents and condi-
tions: (a) CS2, NaOH, reflux, 2 h; (b) ClCOOC2H5, rt, 1.5 h. The
isothiocyanate derivative separates on cooling; (c) isothiocyanate
derivative distilled under reduced pressure; (d) NH3, rt.
stituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one deriva-
tives.33 These compounds can be prepared by reaction
of benzil with a substituted thiourea derivative.34 This
reaction was optimized by using the same microwave-
enhanced method we applied previously to the synthesis
of 3-substituted 5,5′-diphenylhydantoin derivatives.35
The pharmacological activity of the thiohydantoin de-
rivatives at the CB1 cannabinoid receptor was deter-
mined, and structure-activity relationships were es-
tablished.
Table 1. Alkylthiourea Derivatives (2a-2l) Synthesized,
Methods Used, and Synthetic Yields
compd
R
method
% yield
Results and Discussion
2a
2b
2c
2d
2e
2f
2g
2h
2i
C2H5
B
B
B
B
A
A
B
B
B
B
A
B
40
35
30
40
45
45
60
40
45
50
40
35
Chemistry. Benzoin condensation36 was used to
synthesize the dibromo- and dichlorobenzils. Despite the
moderate yields obtained, this route proved to be the
easiest for the benzil derivatives.
i-C3H7
n-C4H9
i-C4H9
C7H15
C8H17
C6H11
The substituted thioureas were synthesized by two
methods, both starting from the corresponding amine.
The first method (Scheme 1, method A) was used for
the preparation of thiourea derivatives 2e, 2f, and 2k
from amines with a high boiling point due to the high
temperatures applied during the reaction of the amine
with ammonium isothiocyanate in bromobenzene at
reflux. The second method, as illustrated in Scheme 1
(method B), is more general, although it requires two
steps. The isothiocyanate derivative of the correspond-
ing amine was first synthesized by following the pro-
cedure described by Moore.37 The amine was heated in
the presence of carbon disulfide and sodium hydroxide
with subsequent addition of ethyl chlorocarbonate. After
completion of the reaction, the isothiocyanate derivative,
which separates on the top of the solution, was collected
and further purified by distillation under reduced
pressure, resulting in an approximately 90% pure
product which was used in the second step. The sub-
stituted thioureas 2a-d,g-j,l were obtained by reaction
of the respective isothiocyanate derivatives with am-
(CH2)3OCH3
CH2CHdCH2
CH2C6H5
(CH2)2C6H5
furan-2-ylmethyl
2j
2k
2l
monia. Comparable yields were achieved by the two
methods (A or B) as listed in Table 1.
The target 5,5′-diphenyl-2-thioxoimidazolidin-4-one
derivatives were obtained by reaction of the respective
benzil and thiourea derivatives in moderate to good
yields (Scheme 2, Table 2) following a microwave-
enhanced method, previously described for the synthesis
of several 3-alkyl-5,5′-diphenylhydantoin derivatives.32
This procedure combines the advantages of a selective
synthesis, since one of the thiourea nitrogens is already
bearing the desired substituent, and an enhanced yield
due to the microwaves applied during the reaction.
Thus, compounds 3-32 selectively substituted at N-3
were obtained by reaction of benzil derivatives (X ) H,
Cl, Br, I), obtained from the corresponding benzalde-

CB1 Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2511
dione series,32 either under classical reflux conditions
or using microwave-enhanced conditions. The size of the
substituent on the thiourea nitrogen does not seem to
affect the reaction yield.
Scheme 2. Synthesis of the
5,5′-Diphenyl-2-thioxoimidazolidin-4-one Derivatives
(3-32)a
It should be noted that both methods described here
(methods A and B) do not allow the synthesis of
3-hydroxypropylthiourea. Interestingly, previous at-
tempts made to obtain the 3-hydroxypropylurea have
also failed. Therefore, the N-hydroxypropyl thiohydan-
toin derivative (11) was synthesized starting from the
respective N-methoxypropyl derivative (10) followed by
demethylation with boron tribromide (BBr3). The latter
reaction is nearly quantitative and, therefore, only
slightly reduces the overall yield for 3-(3-hydroxypro-
pyl)-5,5′-diphenyl-2-thioxoimidazolidin-4-one (11).
Pharmacology. The 5,5′-diphenyl-2-thioxoimidazo-
lidin-4-one derivatives (3-32) were screened at 10 µM
concentrations for their affinity and selectivity towards
the human CB1 and CB2 (hCB1 and hCB2) cannabinoid
receptors in a competitive binding experiment. Mem-
branes from Chinese hamster ovarian (CHO) cells
expressing either the hCB1 or the hCB2 cannabinoid
receptor were used in these experiments. [3H]SR141716A
and [3H]CP-55,940 at concentrations of 1 nM were used
as radioligands for the hCB1 and the hCB2 cannabinoid
receptor, respectively. The results expressed as the
displacement percentages of the radioligand from its
binding site are summarized in Table 2.
None of the compounds displayed significant affinity
towards the hCB2 receptor, as none of the tested
compounds displaced more than 20% of [3H]CP-55,940
bound to the receptor. However, good affinity was noted
towards the hCB1 receptor (Table 2). Substitution of the
phenyl rings with chlorine, bromine, or iodine led to an
increased affinity for the hCB1 receptor. The radioactiv-
ity displacement is below 50% for the majority of the
unsubstituted compounds (3-15), while higher displace-
ment was observed for the respective p-halogenated
compounds. This effect is well-illustrated by compounds
6, 16, and 24 or by compounds 13, 19, and 29. Increas-
ing the affinity for the hCB1 receptor by para substitu-
tion with chlorine or bromine was previously shown for
the hydantoins derivatives.29
a Reagents and conditions: (a) DMSO/aq KOH, nine microwaves
pulses.
Table 2. Synthetic Yield of the
5,5′-Diphenyl-2-thioxoimidazolidin-4-one Derivatives (3-32)
and Percentages of Displacement of [3H]SR141716A and
[3H]CP-55,940, Respectively, by Compounds 3-32 (10 µM) on
hCB1 and hCB2 Cannabinoid Receptorsa
% of displacement
%
yield
CB1
receptor
CB2
receptor
compd
X
R
3
H
H
H
H
H
H
H
H
H
H
H
H
H
C2H5
70
65
80
75
77
75
85
70
65
65
70
76
75
45
35
37
30
35
30
25
35
30
35
50
32
35
30
38
35
34
40.0 ( 5.0
42.8 ( 4.2
42.8 ( 4.8
34.6 ( 3.4
28.8 ( 5.1
38.8 ( 4.3
38.5 ( 3.8
29.4 ( 3.8
20.8 ( 5.3
40.0 ( 5.4
44.7 ( 5.3
29.3 ( 5.5
59.2 ( 4.5
76.1 ( 4.3
67.8 ( 4.5
36.6 ( 5.0
74.2 ( 2.5
79.4 ( 5.3
90.9 ( 4.4
91.7 ( 3.1
73.8 ( 5.5
76.3 ( 4.6
34.2 ( 5.2
23.4 ( 3.7
45.5 ( 5.5
87.2 ( 5.6
76.0 ( 4.7
54.6 ( 4.1
77.4 ( 5.0
94.2 ( 4.7
<10
<10
<10
<10
<10
<20
<10
<15
<10
<15
<10
<10
<15
<20
<10
<10
<15
<15
<20
<15
<15
<20
<10
<10
<10
<15
<10
<10
<20
<20
4
i-C3H7
n-C4H9
i-C4H9
C7H15
C8H17
C6H11
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
(CH2)3OCH3
(CH2)3OH
CH2CHdCH2
CH2C6H5
(CH2)2C6H5
furan-2-ylmethyl
Cl i-C4H9
Cl C7H15
Cl C8H17
Cl CH2C6H5
Cl (CH2)2C6H5
Br C2H5
Br i-C3H7
To study structure-affinity relationships, 11 com-
pounds (16, 19-24, 28, 29, and 31, 32) were selected
for the determination of the inhibition constant, Ki, by
measuring the radioligand ([3H]SR141716A, 1 nM)
displaced by increasing concentrations of each com-
Br n-C4H9
Br i-C4H9
Br C7H15
Br C8H17
Br C6H11
pound (10-8-10-5 M). Concentrations above 10-5
M
Br CH2CHdCH2
Br CH2C6H5
Br (CH2)2C6H5
could not be evaluated due to the poor aqueous solubility
of the compounds. The results of the experiments are
summarized in Table 3, and illustrated in Figure 2. The
Ki values are expressed for a Kd of 1.13 nM and obtained
from IC50 values following the Cheng and Prusoff
method.38
I
I
n-C4H9
CH2CHdCH2
a Mean ( SEM of at least four experiments performed in
duplicate.
hydes, with substituted thioureas. The ORTEP diagram
of compound 28 supporting the structure of these
compounds is given in the Supporting Information.
Not surprisingly, yields tend to decrease when the size
of the benzil substituent is enlarged (Table 2). Yields
were up to 80% when an unsubstituted benzil was used
in the reaction but fell to 30-35% when the benzil was
substituted by chlorine, bromine, or iodine atoms. This
is also the case in the 5,5′-diphenylimidazolidine-2,4-
A bromine substituent in the para position of the
phenyl ring slightly increases the receptor affinity
compared to chlorine substitution. Thus, Ki values of
2188 ( 101 and 993 ( 43 nM were determined for
compounds 19 and 29, respectively, differing only in the
para phenyl substituent. Further, an iodine in the same
position slightly enhanced the affinity for the cannab-
inoid receptor compared to the bromine substitution. Ki
values of 1412 ( 65 and 724 ( 52 nM were obtained

2512 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Muccioli et al.
Table 3. Affinities of Selected Derivatives (compounds 16,
19-24, 28, 29, 31, 36) and of SR141716A, WIN-55,212-2, and
HU-210 at the hCB1 Cannabinoid Receptora
compd
X
X
R
Ki (nM)
Thiohydantoins
16
19
20
21
22
23
24
28
29
31
32
S
S
S
S
S
S
S
S
S
S
S
Cl
i-C4H9
2089 ( 96
2188 ( 101
3801 ( 175
2193 ( 98
3630 ( 167
1412 ( 65
1778 ( 82
871 ( 40
Cl
Cl
Br
Br
Br
Br
Br
Br
I
CH2C6H5
(CH2)2C6H5
C2H5
i-C3H7
n-C4H9
i-C4H9
CH2CHdCH2
CH2C6H5
n-C4H9
993 ( 43
724 ( 52
589 ( 49
I
CH2CHdCH2
Hydantoins
33
34
35
36
O
O
O
O
Br
Br
I
n-C4H9
5495 ( 253
3467 ( 163
1820 ( 251
1738 ( 82
CH2C6H5
n-C4H9
CH2CHdCH2
I
Reference Cannabinoids
SR141716A
WIN55,212-2
HU-210
5.4 ( 0.2
3802 ( 158
18.6 ( 1.7
Figure 2. Effect of 16 (9), 19 (2), 20 (]), 21 (b), and 22 (0)
on [3H]SR141716A binding on hCB1-CHO cells. Data are the
mean of at least four experiments, and vertical lines show the
SEM. Effect of 23 (4), 24 (1), 28 (2), 29 (9), 31 (]), and 32
(×) on [3H]SR141716A binding on hCB1-CHO cells. Data are
the mean of at least four experiments, and vertical lines show
the SEM.
a The 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.
for compounds 23 and 31, respectively, substituted with
a butyl in position 3 and differing only in the halogen
substituent of the phenyl ring. With respect to N3-alkyl
substitution, increasing the chain length over six carbon
atoms caused a significant loss of affinity, as the
displacement of the radioligand dropped below 40% for
compounds 25 and 26. An alkylaryl substituent like
benzyl (19 or 29) increased the affinity of these com-
pounds for the hCB1 receptor, while the additional
methylene group of the ethylphenyl substituent de-
creased the affinity as illustrated by compounds 19 and
20 (Ki values of 2188 ( 101 and 3801 ( 175 nM,
respectively) or by compounds 29 and 30 (Ki values 993
( 43 and >10 000 nM, respectively). Finally, a short
unsaturated alkyl chain such as the allyl group in 28
and 32 appears to be favorable for the affinity of this
series of compounds.
The effect of the sulfur-oxygen exchange in position
2 of the hydantoin nucleus, leading to the thiohydantoin
derivatives, may be estimated from a comparison of
the 5,5′-bis(4-bromophenyl)-3-butylimidazolidine-2,4-
dione (33), 3-benzyl-5,5′-bis(4-bromophenyl)imidazoli-
dine-2,4-dione (34), 3-butyl-5,5′-bis(4-iodophenyl)imida-
zolidine-2,4-dione (35), and 3-allyl-5,5′-bis(4-iodophenyl)-
imidazolidine-2,4-dione (36) with the respective thiohy-
dantoins 23, 29, 31, and 32. The Ki values for deriva-
tives 33 to 36 are approximately 2-4 times higher than
the Ki values of the corresponding thiohydantoin deriva-
tives (Table 3). For instance, compound 29 has a Ki
value of 993 ( 43 nM compared to 3467 ( 163 nM for
the corresponding hydantoin 34. Thus, replacement of
the oxygen in position 2 of the hydantoin ring by a sulfur
atom resulted in an increase of affinity for the hCB1
receptor.
The question whether the thiohydantoin derivatives
possess the same functionality compared to the hydan-
toin derivatives was also investigated by using a [35S]-
GTPγS assay.39 This assay constitutes a functional
measure of the interaction of the receptor and the
G-protein, the first step in activation of the G-protein-
coupled receptors. It is a useful tool to distinguish
between agonists (increasing the nucleotide binding),
inverse agonists (decreasing the nucleotide binding), and
neutral antagonists (not affecting the nucleotide bind-
ing). The known cannabinoid agonist HU-210, the
inverse agonist SR141716A, and thiohydantoins 16, 19-
24, 28, 29, 31, and 32 showing higher affinity for the
receptor, as well as the four hydantoins 33-36 were
screened at a concentration of 10 µM in this assay. As
expected, 10 µM HU-210 induced an increase in the [35S]-
GTPγS binding (130% compared to basal level), while
SR141716A at the same concentration decreased [35S]-
GTPγS binding (-85% compared to basal level). The test
compounds induced a significant decrease in the [35S]-
GTPγS binding, ranging from -52% to -74% compared
to the basal level at a concentration of 10 µM, revealing
inverse agonist properties at the hCB1 cannabinoid
receptor (Figure 3). Thus, the 5,5′-diphenylthiohydan-
toins derivatives, like the 5,5′-diphenylhydantoins de-
rivatives, possess inverse agonist properties at the
human CB1 cannabinoid receptor. The substitution of
the oxygen atom by a sulfur atom does not affect the

CB1 Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2513
Figure 4. Attractive electrostatic potential zones (isocontour
of -0.3 e/Å3) of the molecular electrostatic potential calculated
for compounds 28 (A), 32 (B), and 36 (C). The ab initio
calculated MEP isocontour was plotted using the GopenMol
graphical interface.
The lipophilicity of the compounds was calculated
using the CLOGP program. The CLOGP values ob-
tained for the thiohydantoins derivatives 23 and 29
(5.86 and 6.04, respectively) were slightly higher than
those obtained for the corresponding hydantoin deriva-
tives 33 and 34 (5.56 and 5.64, respectively). However,
the thiohydantoin 28 having a CLOGP value of 5.05
possesses an affinity for the hCB1 cannabinoid receptor
comparable to the affinity of the thiohydantoin 29. In
contrast, the hydantoin 34, characterized by a higher
CLOGP value compared to the thiohydantoin 28, pos-
sesses a lower affinity for the hCB1 receptor. Conse-
quently, although an enhanced overall lipophilicity
(CLOGP value) is not solely responsible for the better
affinity of the thiohydantoin derivatives, a local increase
in lipophilicity around carbon 2 compared to the hy-
dantoins could be a favorable parameter for binding of
the ligands to the receptor.
In conclusion, the 5,5′-diphenyl-2-thioxoimidazolidin-
4-one derivatives bind to the hCB1 cannabinoid receptor
with good selectivity. The most active compounds pos-
sess an iodine atom in para position of the phenyl rings
as well as an alkyl chain on N-3 having less than six
carbon atoms. Preferably, the alkyl chain is unsatur-
ated. The bioisosteric replacement of the oxygen in the
hydantoin nucleus by a sulfur atom resulting in thio-
hydantoins led to an increase in the affinity of the
compounds without affecting their functional charac-
teristics. Finally, compounds 5,5′-bis(4-iodophenyl)-3-
butyl-2-thioxoimidazolidin-4-one (31) and 3-allyl-
5,5′-bis(4-bromophenyl)-2-thioxoimidazolidin-4-one (32)
possess the highest affinity for the CB1 cannabinoid
receptor described to date for the hydantoin and thio-
hydantoins series when compared in the same bioassay.
Figure 3. Influence of 10 µM HU-210, SR141716A, and 16,
19, 20, 21, 22, 23, and 24 on [35S]GTPγS binding on hCB1-
CHO cells membranes. Data are the mean ( SEM of three
experiments performed in duplicate, with one-way ANOVA
followed by a Dunett post-test (**P < 0.01). Influence of 10
µM 28, 29, 31, 32, 33, 34, 35, and 36 on [35S]GTPγS binding
on hCB1-CHO cells membranes. Data are the mean ( SEM of
three experiments performed in duplicate, with one-way
ANOVA followed by a Dunett post-test (**P < 0.01).
functionality of the compounds. Moreover, an EC50 value
of 282 ( 35 nM was obtained for the inhibition of [35S]-
GTPγS binding by derivative 32. This value is in good
accordance with the Ki value of the compound (Ki ) 589
( 49 nM).
To estimate the molecular parameters that could
influence the affinity of thiohydantoins, the electrostatic
and lipophilic properties of both series, the thiohydan-
toin and hydantoin derivatives, were calculated.
The MIDI basis set developed by Truhlar et al. used
for the molecular electrostatic potential calculations
applies to H, C, F, S, Cl, Br, and I, and includes
polarization functions.40 Therefore, in contrast to many
other classical basis sets, MIDI is well-suited to study
the electronic properties of the selected bromo- and
iodohydantoins and thiohydantoins. Molecular electro-
static potential maps calculated for 28, 32, and 36 show
distinct patterns of attractive potentials generated by
the sulfur or oxygen atom of the (thio)hydantoin ring
(Figure 4). In particular, the thiocarbonyl appears less
prone to interact via hydrogen bonds compared to the
carbonyl of the hydantoin. This may suggest that
hydantoin and thiohydantoin derivatives bind in a
slightly different way to the receptor. However, in
contrast to the biological data, there is no significant
influence of the halogen substituent of the phenyl
moiety (bromo or iodo) on the molecular electrostatic
potential and atomic charges on the sulfur and/or
oxygen atoms of the (thio)hydantoin analogues.
Experimental Section
General Procedures. All reagents were purchased from
commercial sources (Sigma-Aldrich or Acros) and were used
without further purification. Solvents were of analytical grade.
The microwave oven used was a commercial household mi-
crowave oven (frequency 2450 MHz).
Melting points (mp) were determined in open capillaries
using a Electrothermal 9100 apparatus and are reported
uncorrected. Infrared (IR) spectra were recorded using a
Perkin-Elmer FT-IR 286 spectrometer, and values are reported
as ν in cm-1 (see the 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 tetramethylsilane peak set
at 0.00 ppm. In the case of multiplets, the signals are reported
as intervals. Signals were abbreviated as s, singlet; d, doublet;
t, triplet; and m, multiplet. Coupling constants are expressed
in hertz. Mass spectra were recorded on a Finnigan MAT 44S,

2514 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Muccioli et al.
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.
Synthesis. General Procedure for the Synthesis of
Substituted Thiourea Derivatives. Method A. The amine
hydrochloride (0.1 mol) and ammonium isothiocyanate (0.1
mol) were refluxed in bromobenzene (25 mL) under nitrogen
for 90 min. Upon cooling to 5 °C the precipitate was placed in
300 mL of water with shaking. The resulting powder was
filtered and used without further purification.
Method B. Carbon disulfide (12.2 mL, 0.2 mol) and sodium
hydroxide (0.2 mole) were shaken at 4 °C before addition of
the amine (0.2 mol). Subsequently, the solution was refluxed
for 2 h. After cooling to room temperature, ethyl chloroformate
(0.2 mol) was added and the resulting mixture was stirred for
an additional 30 min. The isothiocyanate derivative floating
on the top of the solution was separated and dried over MgSO4.
Further purification was performed by distillation under
reduced pressure. The isothiocyanate was reacted with an
excess of ammonia at room temperature to afford the thiourea
derivative. The excess of ammonia was removed under reduced
pressure and the solid washed twice with water.
General Procedure for the Synthesis of the 2-Thi-
oxoimidazolidin-4-one Compounds (Except Compound
11). The synthesis was performed as described for hydantoin
derivatives.34 Briefly, to a solution of benzil (9.9 mmol) and a
substituted thiourea (19.9 mmol) in 25 mL of DMSO was added
15 mL of a 1.2 M KOH solution with stirring. Following an
initial 90-s microwave irradiation (750 W), the mixture was
stirred for 5 min. Eight 30-s microwaves pulses were applied
at the time points of 6, 9, 12, 15, 18, 21, 24, and 30 min. The
mixture was stirred between pulses. At the end of the sequence
the mixture was poured onto crushed ice and the precipitate
filtered off. The residue was subsequently crystallized.
3-Octyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (8).
Mp: 96.6-97.4 °C. MS (EI): 381 [M + H]+. 1H NMR (300
MHz): δ 0.83 (t, J ) 7.35, 3H), 1.76 (m, 10H), 1.58-1.60 (m,
2H), 3.75 (t, J ) 7.35, 2H), 7.30-7.45 (m, 10H), 11.65 (s, 1H).
13C NMR (300 MHz): δ 13.84 (CH3), 21.93 (CH2), 25.81 (CH2),
26.97 (CH2), 28.40 (CH2), 30.99 (CH2), 38.44 (CH2), 40.38 (CH2),
71.10 (C), 126.55 (CHarom), 128.49 (CHarom), 128.68 (CHarom),
138.13 (Carom), 173.52 (CdO), 181.28 (CdS). Anal. Calcd for
C23H28N2OS: C, H, N.
3-Cyclohexyl-5,5′-diphenyl-2-thioxoimidazolidin-4-
one (9). Mp: 237.5-238.3 °C. MS (EI): 350 [M]+. 1H NMR
(300 MHz): δ 1.08-1.32 (m, 3H), 1.58-1.61 (m, 3H), 1.76-
1.80 (m, 2H), 2.06-2.13 (m, 2H), 4.47-4.55 (m, 1H), 7.21-
7.45 (m, 10H), 11.59 (s, 1H). 13C NMR (300 MHz): δ 24.71
(CH2), 25.36 (CH2), 28.08 (CH2), 54.09 (CH), 70.07 (C), 126.55
(CHarom), 128.42 (CHarom), 128.68 (CHarom), 138.32 (Carom),
173.59 (CdO), 181.54 (CdS). Anal. Calcd for C21H22N2OS: C,
H, N.
3-(3-Methoxypropyl)-5,5′-diphenyl-2-thioxoimidazoli-
1
din-4-one (10). Mp: 145.5-146.3 °C. MS (EI): 340 [M]+. H
NMR (300 MHz): δ 1.81 (m, 2H), 3.08 (s, 3H), 3.28 (t, J )
7.35, 2H), 3.82 (t, J ) 7.35, 2H), 7.30-7.43 (m, 10H), 11.03 (s,
1H). 13C NMR (300 MHz): δ 28.65 (CH2), 41.98 (CH2), 57.88
(CH3), 69.97 (CH2), 71.05 (C), 126.08 (CHarom), 128.45 (CHarom),
129.02 (CHarom), 138.71 (Carom), 170.95 (CdO), 181.35 (CdS).
Anal. Calcd for C19H20N2O2S: C, H, N.
3-(3-Hydroxypropyl)-5,5′-diphenyl-2-thioxoimidazoli-
din-4-one (11). To a solution of 10 in dry 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 tem-
perature and stirred overnight. The resulting solution was
evaporated to dryness under reduced pressure and the result-
ing solid washed with water. After filtration, the compound
was crystallized from ethanol. Mp: 227.2-227.8 °C. MS (EI):
1
326 [M]+. H NMR (300 MHz): δ 2.12 (m, 2H), 3.18 (m, 2H),
3-Ethyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (3).
Mp: 188.3-189.4 °C. MS (EI): 296 [M]+. 1H NMR (300
MHz): δ 1.13 (t, J ) 7.35, 3H), 3.78 (q, J ) 7.35, 2H), 7.30-
7.43 (m, 10H), 10.98 (s, 1H). 13C NMR (300 MHz): δ 13.23
(CH3), 36.14 (CH2), 71.05 (C), 126.98 (CHarom), 128.92 (CHarom),
129.24 (CHarom), 138.56 (Carom), 173.69 (CdO), 181.39 (CdS).
Anal. Calcd for C17H16N2OS: C, H, N.
3.57-3.62 (m, 2H), 7.27-7.38 (m, 10H), 11.59 (s, 1H), 11.67
(s, 1H). 13C NMR (300 MHz): δ 27.15 (CH2), 40.66 (CH2), 55.72
(CH2), 71.96 (C), 127.06 (Carom), 128.45 (CHarom), 128.98
(CHarom), 139.23 (Carom), 170.96 (CdO), 181.36 (CdS). Anal.
Calcd for C18H18N2O2S: C, H, N.
3-Allyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (12).
Mp: 178.7-179.2 °C. MS (EI): 308 [M]+. 1H NMR (300
MHz): δ 4.39-4.40 (d, J ) 4.41, 2H), 4.94-5.00 (d, J ) 17.64,
1H), 5.08-5.12 (d, J ) 10.29, 1H), 5.76-5.89 (m, 1H), 7.32-
7.46 (m, 10H), 11.75 (s, 1H). 13C NMR (300 MHz): δ 42.37
(CH2), 71.23 (C), 116.71 (CH2), 126.48 (CHarom), 128.42 (CHarom),
128.68 (CHarom), 131.34 (Carom), 138.13 (CH), 173.00 (CdO),
180.90 (CdS). Anal. Calcd for C18H16N2OS: C, H, N.
3-Benzyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (13).
Mp: 155.1-156.0 °C. MS (EI): 358 [M]+. 1H NMR (300
MHz): δ 5.00 (s, 2H), 7.23-7.43 (m, 15H), 11.77 (s, 1H). 13C
NMR (300 MHz): δ 43.90 (CH2), 71.40 (C), 126.65 (CHarom),
127.36 (CHarom), 127.56 (CHarom), 128.53(CHarom), 128.66 (CHarom),
128.85 (CHarom), 136.16 (Carom), 138.17 (Carom), 173.49 (CdO),
181.32 (CdS). Anal. Calcd for C22H18N2OS: C, H, N.
3-Isopropyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one
1
(4). Mp: 215.9-216.5 °C. MS (EI): 310 [M]+. H NMR (300
MHz): δ 1.36 (m, 6H), 4.83-4.92 (m, 1H), 7.28-7.45 (m, 10H),
11.74 (s, 1H). 13C NMR (300 MHz): δ 26.86 (2 × CH3), 54.55
(CH), 78.29 (C), 134.78 (CHarom), 136.59 (CHarom), 136.91
(CHarom), 146.49 (Carom), 181.75 (CdO), 189.58 (CdS). Anal.
Calcd for C18H18N2OS: C, H, N.
3-Butyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (5).
Mp: 132.5-133.5 °C. MS (EI): 324 [M]+. 1H NMR (300
MHz): δ 0.85 (t, J ) 7.35, 3H), 1.17-1.29 (m, 2H), 1.52-1.62
(m, 2H), 3.77 (t, J ) 7.35, 2H), 7.32-7.45 (m, 10H), 11.68 (s,
1H). 13C NMR (300 MHz): δ 13.45 (CH3), 19.21 (CH2), 29.24
(CH2), 37.75 (CH2), 71.10 (C), 126.55 (CHarom), 128.42 (CHarom),
128.68 (CHarom), 138.19 (Carom), 173.46 (CdO), 181.22 (CdS).
Anal. Calcd for C19H20N2OS: C, H, N.
3-Phenethyl-5,5′-diphenyl-2-thioxoimidazolidin-4-
1
one (14). Mp: 175.2-176.1 °C. MS (EI): 372 [M]+. H NMR
3-Isobutyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (6).
Mp: 173.6-174.3 °C. MS (EI): 324 [M]+. 1H NMR (300
MHz): δ 0.78-0.80 (m, 6H), 2.09-2.23 (m, 1H), 3.58-3.60 (d,
J ) 7.35, 2H), 7.25-7.50 (m, 10H), 11.68 (s, 1H). 13C NMR
(300 MHz): δ 19.60 (2 × CH3), 26.52 (CH), 47.42 (CH2), 71.04
(C), 126.48 (CHarom), 128.36 (CHarom), 128.68 (CHarom), 138.13
(Carom), 173.65 (CdO), 181.54 (CdS). Anal. Calcd for C19H20N2-
OS: C, H, N.
(300 MHz): δ 2.97 (t, J ) 7.35, 2H), 4.01 (t, J ) 7.35, 2H),
7.19-7.38 (m, 15H), 11.02 (s, 1H). 13C NMR (300 MHz): δ
32.73 (CH2), 41.53 (CH2), 71.10 (C), 126.03 (CHarom), 126.29
(CHarom), 126.55 (CHarom), 127.13 (CHarom), 128.30 (CHarom),
128.75 (CHarom), 137.48 (Carom), 138.07 (Carom), 173.20 (CdO),
180.83 (CdS). Anal. Calcd for C23H20N2OS: C, H, N.
3-(Furan-2-ylmethyl)-5,5′-diphenyl-2-thioxoimidazoli-
1
din-4-one (15). Mp: 178.2-179.1 °C. MS (EI): 348 [M]+. H
NMR (300 MHz): δ 5.01 (s, 2H), 6.20-6.39 (m, 3H), 7.31-
7.56 (m, 10H), 11.79 (s, 1H). 13C NMR (300 MHz): δ 37.33
(CH2), 71.23 (C5), 108.11 (CH), 110.50 (CH), 126.55 (CHarom),
128.42 (CHarom), 128.68 (CHarom), 137.93 (Carom), 142.46 (C),
148.80 (CH), 172.87 (CdO), 180.44 (CdS). Anal. Calcd for
C20H16N2OS: C, H, N.
3-Heptyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (7).
Mp: 110-110.8 °C. MS (EI): 367 [M + H]+. 1H NMR (300
MHz): δ 0.81 (t, J ) 7.35, 3H), 1.18 (m, 8H), 1.57-1.61 (m,
2H), 3.76 (t, J ) 7.35, 2H), 7.31-7.45 (m, 10H), 11.65 (s, 1H).
13C NMR (300 MHz): δ 13.78 (CH3), 21.80 (CH2), 25.75 (CH2),
26.98 (CH2), 28.01 (CH2), 30.99 (CH2), 38.10 (CH2), 71.10 (C),
126.55 (CHarom), 128.42 (CHarom), 128.68 (CHarom), 138.19
(Carom), 173.52 (CdO), 181.12 (CdS). Anal. Calcd for C22H26N2-
OS: C, H, N.
5,5′-Bis(4-chlorophenyl)-3-isobutyl-2-thioxoimidazoli-
1
din-4-one (16). Mp: 187.6-188.4 °C. MS (EI): 392 [M]+. H
NMR (300 MHz): δ 0.78 (m, 6H), 2.09-2.19 (m, 1H), 3.59 (d,

CB1 Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2515
J ) 7.35, 2H), 7.31-7.34 (d, J ) 8.82, 4H), 7.52-7.55 (d, J )
8.82, 4H), 11.73 (s, 1H). 13C NMR (300 MHz): δ 19.60 (2 ×
CH3), 26.52 (CH), 47.61 (CH2), 70.07 (C), 128.36 (Carom), 128.94
(CHarom), 133.54 (CHarom), 136.77 (Carom), 173.20 (CdO), 181.74
(CdS). Anal. Calcd for C19H18Cl2N2OS: C, H, N.
5,5′-Bis(4-chlorophenyl)-3-heptyl-2-thioxoimidazolidin-
4-one (17). Mp: 159.5-160.0 °C. MS (EI): 434 [M]+. 1H NMR
(300 MHz): δ 0.81 (t, J ) 7.35, 3H), 1.16 (m, 8H), 1.56-1.61
(m, 2H), 3.76 (t, J ) 7.35, 2H), 7.32-7.35 (d, J ) 8.82, 4H),
7.51-7.54 (d, J ) 8.82, 4H), 11.72 (s, 1H). 13C NMR (300
MHz): δ 13.23 (CH3), 22.03 (CH2), 25.91 (CH2), 27.14 (CH2),
28.31 (CH2), 31.22 (CH2), 38.44 (CH2), 70.23 (C), 127.49 (Carom),
129.76 (CHarom), 133.77 (CHarom), 136.94 (Carom), 173.17 (Cd
O), 181.52 (CdS). Anal. Calcd for C19H18Cl2N2OS‚1/3H2O: C,
H, N.
5,5′-Bis(4-chlorophenyl)-3-octyl-2-thioxoimidazolidin-
4-one (18). Mp: 133.0-133.8 °C. MS (EI): 449 [M + H]+. 1H
NMR (300 MHz): δ 0.83 (m, 3H), 1.16 (m, 10H), 1.57 (m, 2H),
3.75 (m, 2H), 7.30-7.33 (d, J ) 8.82, 4H), 7.52-7.54 (d, J )
8.82, 4H), 11.71 (s, 1H). 13C NMR (300 MHz): δ 13.78 (CH3),
21.93 (CH2), 25.75 (CH2), 26.85 (CH2), 28.33 (CH2), 30.92 (CH2),
38.69 (CH2), 40.54 (CH2), 70.07 (C), 128.36 (Carom), 128.88
(CHarom), 133.54 (CHarom), 136.71 (Carom), 173.00 (CdO), 181.35
(CdS). Anal. Calcd for C23H26Cl2N2OS: C, H, N.
3-Benzyl-5,5′-bis(4-chlorophenyl)-2-thioxoimidazolidin-
4-one (19). Mp: 182.3-182.8 °C. MS (EI): 426 [M]+. 1H NMR
(300 MHz): δ 4.98 (s, 1H), 7.21-7.61 (m, 13H), 11.86 (s, 1H).
13C NMR (300 MHz): δ 43.86 (CH2), 70.26 (C), 127.19 (Carom),
127.45 (CHarom), 128.42 (CHarom), 128.94 (CHarom), 133.42
(CHarom), 133.60 (CHarom), 135.86 (Carom), 136.64 (Carom), 172.84
(CdO), 181.28 (CdS). Anal. Calcd for C22H16Cl2N2OS: C, H,
N.
5,5′-Bis(4-chlorophenyl)-3-phenethyl-2-thioxoimidazo-
lidin-4-one (20). Mp: 203.4-204.2 °C. MS (EI): 440 [M]+.
1H NMR (300 MHz): δ 2.96 (t, J ) 7.35, 2H), 3.99 (t, J ) 7.35,
2H), 7.12-7.48 (m, 13H), 11.63 (s, 1H). 13C NMR (300 MHz):
δ 32.60 (CH2), 41.60 (CH2), 70.07 (C), 126.35 (Carom), 128.23
(Carom), 128.49 (CHarom), 128.75 (CHarom), 129.23 (CHarom),
133.47 (CHarom), 136.64 (Carom), 137.42 (Carom), 172.68 (CdO),
181.02 (CdS). Anal. Calcd for C23H18Cl2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-ethyl-2-thioxoimidazolidin-
4-one (21). Mp: 203.9-204.5 °C. MS (EI): 454 [M]+. 1H NMR
(300 MHz): δ 1.12 (t, J ) 7.35, 3H), 3.79 (q, J ) 7.35, 2H),
7.23-7.25 (d, J ) 8.82, 4H), 7.64-7.67 (d, J ) 8.82, 4H), 11.70
(s, 1H). 13C NMR (300 MHz): δ 12.68 (CH3), 35.84 (CH2), 70.19
(C), 122.21 (Carom), 128.68 (CHarom), 131.85 (CHarom), 137.03
(Carom), 172.55 (CdO), 181.02 (CdS). Anal. Calcd for C17H14-
Br2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-isopropyl-2-thioxoimidazo-
lidin-4-one (22). Mp: 214.0-214.8 °C. MS (EI): 468 [M]+.
1H NMR (300 MHz): δ 1.36 (m, 6H), 4.81-4.90 (m, 1H), 7.20-
7.23 (d, J ) 8.82, 4H), 7.64-7.67 (d, J ) 8.82, 4H), 11.69 (s,
1H). 13C NMR (300 MHz): δ 25.98 (2 × CH3), 53.85 (CH), 77.89
(C), 122.98 (Carom), 129.41 (CHarom), 132.78 (CHarom), 137.70
(Carom), 173.49 (CdO), 182.10 (CdS). Anal. Calcd for C18H16-
Br2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-butyl-2-thioxoimidazolidin-
4-one (23). Mp: 177.7-178.3 °C. MS (EI): 482 [M]+. 1H NMR
(300 MHz): δ 0.85 (t, J ) 7.35, 3H), 1.16-1.28 (m, 2H), 1.51-
1.61 (m, 2H), 3.76 (t, J ) 7.35, 2H), 7.24-7.27 (d, J ) 8.82,
4H), 7.64-7.67 (d, J ) 8.82, 4H), 11.67 (s, 1H). 13C NMR (300
MHz): δ 14.07 (CH3), 19.90 (CH2), 29.80 (CH2), 38.44 (CH2),
70.88 (C), 122.90 (Carom), 129.37 (CHarom), 132.54 (CHarom),
137.78 (Carom), 173.49 (CdO), 182.10 (CdS). Anal. Calcd for
C19H18Br2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-heptyl-2-thioxoimidazolidin-
4-one (25). Mp: 161.8-162.4 °C. MS (EI): 525 [M + H]+. 1H
NMR (300 MHz), δ 0.81 (t, J ) 7.35, 3H), 1.15 (m, 8H), 1.58
(m, 2H), 3.75 (t, J ) 7.35, 2H), 7.24-7.27 (d, J ) 8.82, 4H),
7.64-7.67 (d, J ) 8.82, 4H), 11.71 (s, 1H). 13C NMR (300
MHz): δ 13.78 (CH3), 21.80 (CH2), 25.75 (CH2), 26.85 (CH2),
28.01 (CH2), 30.99 (CH2), 38.44 (CH2), 70.19 (C), 122.15 (Carom),
128.68 (CHarom), 131.85 (CHarom), 137.09 (Carom), 172.87 (Cd
O), 181.35 (CdS). Anal. Calcd for C22H24Br2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-octyl-2-thioxoimidazolidin-
4-one (26). Mp: 147.8-148.3 °C. MS (EI): 538 [M]+. 1H NMR
(300 MHz): δ 0.82 (t, J ) 7.35, 3H), 1.15 (m, 10H), 1.55 (m,
2H), 3.75 (t, J ) 7.35, 2H), 7.24-7.27 (d, J ) 8.82, 4H), 7.64-
7.67 (d, J ) 8.82, 4H), 11.70 (s, 1H). 13C NMR (300 MHz): δ
13.78 (CH3), 21.93 (CH2), 25.75 (CH2), 26.85 (CH2), 28.33 (CH2),
30.92 (CH2), 38.43 (CH2), 40.37 (CH2), 70.19 (C), 122.15 (Carom),
128.68 (CHarom), 131.79 (CHarom), 137.09 (Carom), 172.87 (Cd
O), 181.35 (CdS). Anal. Calcd for C23H26Br2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-cyclohexyl-2-thioxoimida-
zolidin-4-one (27). Mp: 227.1-228.0 °C. MS (EI): 508 [M]+.
1H NMR (300 MHz): δ 1.24 (m, 3H), 1.59 (m, 3H), 1.75 (m,
2H), 2.08 (m, 2H), 4.49 (m, 1H), 7.20-7.23 (d, J ) 8.82, 4H),
7.64-7.66 (d, J ) 8.82, 4H), 11.70 (s, 1H). 13C NMR (300
MHz): δ 24.71 (CH2), 25.29 (CH2), 28.01 (CH2), 54.15 (CH),
69.16 (C), 122.15 (Carom), 128.68 (CHarom), 131.85 (CHarom),
137.22 (Carom), 172.87 (CdO), 181.61 (CdS). Anal. Calcd for
C21H20Br2N2OS: C, H, N.
3-Allyl-5,5′-bis(4-bromophenyl)-2-thioxoimidazolidin-
4-one (28). Mp: 181.3-181.9 °C. MS (EI): 466 [M]+. 1H NMR
(300 MHz), δ 4.38 (d, J ) 4.40, 2H), 4.91-4.97 (d, J ) 17.62,
1H), 5.08-5.12 (d, J ) 10.29, 1H), 5.75-5.86 (m, 1H), 7.24-
7.27 (d, J ) 8.82, 4H), 7.65-7.68 (d, J ) 8.82, 4H), 11.78 (s,
1H). 13C NMR (300 MHz): δ 42.57 (CH2), 70.32 (C), 116.84
(CH2), 122.71 (Carom), 128.68 (CHarom), 131.21 (CH), 131.85
(CHarom), 137.03 (Carom), 172.42 (CdO), 181.03 (CdS). Anal.
Calcd for C18H14Br2N2OS: C, H, N.
3-Benzyl-5,5′-bis(4-bromophenyl)-2-thioxoimidazolidin-
4-one (29). Mp: 162.1-162.8 °C. MS (EI): 516 [M]+. 1H NMR
(300 MHz): δ 4.98 (s, 2H), 7.23-7.67 (m, 13H), 11.85 (s, 1H).
13C NMR (300 MHz): δ 43.86 (CH2), 70.32 (C), 122.21 (Carom),
127.13 (CHarom), 127.39 (CHarom), 128.36 (CHarom), 128.62
(CHarom), 131.85 (CHarom), 135.80 (Carom), 136.96 (Carom), 172.68
(CdO), 181.28 (CdS).
5,5′-Bis(4-bromophenyl)-3-phenethyl-2-thioxoimidazo-
lidin-4-one (30). Mp: 189.1-190.0 °C. MS (EI): 530 [M]+.
1H NMR (300 MHz): δ 2.95 (t, J ) 7.35, 2H), 3.99 (t, J ) 7.35,
2H), 7.06-7.62 (m, 13H), 11.63 (s, 1H). 13C NMR (300 MHz):
δ 33.10 (CH2), 42.09 (CH2), 70.62 (C), 122.51 (Carom), 126.78
(CHarom), 128.66 (CHarom), 129.17 (CHarom), 131.99 (CHarom),
132.15(CHarom), 137.46 (Carom), 137.91 (Carom), 173.04 (CdO),
181.03 (CdS). Anal. Calcd for C23H18Br2N2OS: C, H, N.
5,5′-Bis(4-iodophenyl)-3-butyl-2-thioxoimidazolidin-4-
1
one (31). Mp: 163.6-165.3 °C. MS (EI): 576 [M]+. H NMR
(300 MHz): δ 0.85 (t, J ) 7.35, 3H), 1.14-1.26 (m, 2H), 1.49-
1.59 (m, 2H), 3.73 (t, J ) 7.35, 2H), 7.05-7.82 (m, 8H), 11.66
(s, 1H). 13C NMR (300 MHz): δ 13.61 (CH3), 19.38 (CH2), 29.28
(CH2), 38.25 (CH2), 70.62 (C), 95.60 (Carom), 128.85 (Carom),
137.65 (CHarom), 137.85 (CHarom), 172.98 (CdO), 181.46 (Cd
S). Anal. Calcd for C19H18I2N2OS: C, H, N.
3-Allyl-5,5′-bis(4-iodophenyl)-2-thioxoimidazolidin-4-
1
one (32). Mp: 223.6-224.3 °C. MS (EI): 560 [M]+. H NMR
(300 MHz), δ 4.37 (m, 2H), 4.91-4.97 (d, J ) 17.62, 1H), 5.07-
5.11 (d, J ) 10.29, 1H), 5.75-5.84 (m, 1H), 7.08-7.68 (m, 8H),
11.70 (s, 1H). 13C NMR (300 MHz): δ 42.99 (CH2), 71.01 (C),
95.92 (Carom), 117.27 (CH2), 129.11 (Carom), 131.70 (CH), 137.91
(CHarom), 138.17 (Carom), 172.78 (CdO), 181.46 (CdS). Anal.
Calcd for C18H14I2N2OS: C, H, N.
5,5′-Bis(4-bromophenyl)-3-isobutyl-2-thioxoimidazoli-
1
din-4-one (24). Mp: 210.6-211.0 °C. MS (EI): 382 [M]+. H
5,5′-Bis(4-bromophenyl)-3-butylimidazolidine-2,4-di-
one (33). The synthesis of this compound was previously
described by us.34 Mp: 151.0-152.2 °C. MS DCI (H2O): 467
[M + H]+. 1H NMR (300 MHz): δ 0.8183-0.8673 (m, 3H),
1.1565-1.2300 (m, 2H), 1.4701-1.5191 (m, 2H), 3.4106-
3.4596 (m, 2H), 7.2719 (d, J ) 8.82 Hz, 4H), 7.6345 (d, J )
8.82 Hz, 4H), 9.6975 (s, 1H). 13C NMR (300 MHz): δ 13.2320
NMR (300 MHz): δ 0.76-0.79 (m, 6H), 2.09-2.18 (m, 1H),
3.57-3.59 (d, J ) 7.35, 2H), 7.24-7.27 (d, J ) 8.82, 4H), 7.65-
7.68 (d, J ) 8.82, 4H), 11.74 (s, 1H). 13C NMR (300 MHz): δ
19.53 (CH3), 26.52 (CH), 47.55 (CH2), 70.13 (C), 122.15 (Carom),
128.62 (CHarom), 131.79 (CHarom), 137.09 (Carom), 173.07 (Cd
O), 181.67 (CdS). Anal. Calcd for C19H18Br2N2OS: C, H, N.

2516 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Muccioli et al.
(CH3), 19.0549 (CH2), 29.3422 (CH2), 37.7531 (CH2), 68.1619
(C), 121.6685 (Carom), 128.6560 (CHarom), 131.5028 (CHarom),
138.6198 (Carom), 155.1182 (CdO), 172.4576 (CdO). Anal. Calcd
for: C19H18Br2N2O2: C, H,N.
3-Benzyl-5,5′-bis(4-bromophenyl)imidazolidine-2,4-di-
one (34). The synthesis of this compound was previously
described by us elsewhere.41 Mp: 181.1-182.2. MS DCI: 500
[M + H]+. 1H NMR (300 MHz): δ 4.63 (s, 2H), 7.18-7.64 (m,-
13H), 9.84 (s, 1H).13C NMR (300 MHz): δ 41.76 (CH2), 68.55
(C), 121.99, 127.36, 127.69, 128.72, 128.85, 131.83, 136.42,
138.62, 155.12 (CdO), 172.46 (CdO). Anal. Calcd for C22H16-
Br2N2O2: C, H, N.
binding buffer without serum albumin. The radioactivity on
the filters was measured in a Pharmacia Wallac 1410 â-counter
using 10 mL of Aqualuma, after 10 s of shaking and 3 h of
resting. Assays were performed at least in triplicate.
[
35S]GTPγS Assay. [35S]GTPγS (1173 Ci/mmol) was from
Amersham (Roosendaal, The Netherlands). The binding ex-
periments were performed 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 10 µM of the test compounds. The assay was initiated by
the addition of [35S]GTPγS (0.05 nM, final concentration). 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 suspen-
sion 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.
3-Butyl-5,5′-bis(4-iodophenyl)imidazolidine-2,4-di-
one (35). This compound was synthesized with the same
microwave-enhanced procedure described previously.34 Mp:
1
147.3-148.3 °C. MS (EI): 560 [M]+. H NMR (300 MHz): δ
0.83 (t, J ) 7.35, 3H), 1.14-1.21 (m, 2H), 1.46-1.50 (m, 2H),
3.39-3.77 (m, 2H), 7.1-7.79 (m, 8H), 9.66 (s, 1H). 13C NMR
(300 MHz): δ 13.08 (CH3), 19.64 (CH2), 29.86 (CH2), 38.28
(CH2), 68.88 (C), 95.47 (Carom), 129.24 (CHarom), 137.91 (CHarom),
139.46 (Carom), 155.64 (CdO), 172.91 (CdO). Anal. Calcd for
C19H18I2N2O2: C, H, N.
Data Analysis. IC50 values were determined by nonlinear
regression analysis performed using the GraphPad prism 4.0
program (GraphPad Software, San Diego). The Ki values were
calculated based on the Cheng-Prusoff equation: Ki ) IC50/
(1 + L/Kd). The statistical significance of [35S]GTPγS assay
results was assessed using an one-way ANOVA followed by a
Dunett post-test.
Molecular Electrostatic Potential and CLOGP Calcu-
lations. The crystal structure of 28 served as starting point
for the geometry optimization of compounds 28, 32, and 36
using molecular mechanics (Discover program). The internal
coordinates served as input for ab initio calculation of the
electronic properties (Gaussian94 program). The molecular
electrostatic potential isocontour was plotted using the Gopen-
Mol graphical interface.43
3-Allyl-5,5′-bis(4-iodophenyl)imidazolidine-2,4-dione
(36). This compound was synthesized with the same microwave-
enhanced procedure described previously.34 Mp: 187.0-189
1
°C. MS (EI): 544 [M]+. H NMR (300 MHz), δ 4.04 (m, 2H),
4.92-4.98 (d, J ) 17.64, 1H), 5.07-5.10 (d, J ) 10.29, 1H),
5.73-5.84 (m, 1H), 7.12-7.81 (m, 8H), 9.71 (s, 1H). 13C NMR
(300 MHz): δ 40.51 (CH2), 68.75 (C), 95.14 (Carom), 116.56
(CH2), 128.92 (Carom), 132.03 (CH), 137.65 (CHarom), 139.08
(Carom), 154.87 (CdO), 172.27 (CdO). Anal. Calcd for
C18H14I2N2O2: C, H, N.
Cell Culture and Preparation of hCB1- or hCB2-
Transfected CHO Cell Membranes. 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, respec-
tively (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 strep-
tomycin, and 400 µg/mL G418. Once at confluence, the cells
were trypsinized and collected by centrifugation at 100g for
10 min. The following steps were performed in ice. The 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. The protein content was
determined as described by Bradford42 using Coomasie Blue
(Biorad, Belgium) with bovine serum albumin as standard.
Competition Binding Assay. [3H]SR141716A (52 Ci/mol)
was from Amersham (Roosendaal, The Netherlands), [3H]CP-
55,940 (101 Ci/mol) was from NEN Life Science (Zaventem,
Belgium), and HU-210 was from Tocris (Bristol, UK). Glass
fiber filters were purchased from Whatman (Maidstone, UK),
while Aqualuma was from PerkinElmer (Schaesberg, The
Netherlands). Stock solutions of the compounds were prepared
in DMSO and further diluted (100×) with the binding buffer
to the desired concentration. Final DMSO concentrations in
the assay were less than 0.1%.
Values of lipophilicity were calculated using the CLogP
program implemented in the ChemDraw Ultra 8.0 package
(CambridgeSoft).
Acknowledgment. The authors acknowledge Ber-
nadette Norberg for the X-ray analysis. 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 from the Belgian
National Fund for Scientific Research and a FSR grant
from the Universite´ catholique de Louvain.
Supporting Information Available: General procedure
for the synthesis of benzil derivatives, characteristic IR peaks
of the alkylthiourea derivatives, X-ray crystallographic data
of compound 28, characteristic IR peaks, and elemental
analysis of compounds 3-36. This material is available free
of charge via the Internet at http://pubs.acs.org.
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JM049263K