Substituted 2-thioxoimidazolidin-4-ones and imidazolidine-2,4-diones as fatty acid amide hydrolase inhibitors templates

Article abstract of DOI:10.1021/jm050977k

The demonstration of the essential role of fatty acid amide hydrolase (FAAH) in hydrolyzing endogenous bioactive fatty acid derivatives has launched the quest for the discovery of inhibitors for this enzyme. Along this line, a set of 58 imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one derivatives was evaluated as FAAH inhibitors. Among these compounds, 3-substituted 5,5′-diphenylimidazolidine-2,4-dione and 3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one derivatives were previously described as CB₁ cannabinoid receptor ligands. In the present study, we synthesized several derivatives exhibiting interesting FAAH inhibitory activity and devoid of affinity for the CB₁ and CB₂ cannabinoid receptors. For instance, 3-heptyl-5,5′-diphenylimidazolidine- 2,4-dione (14) and 5,5′-diphenyl-3-tetradecyl-2-thioxo-imidazolidin-4-one (46) showed p/₅₀ values of 5.12 and 5.94, respectively. In conclusion, it appears that even though several 3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one and 3-substituted 5,5′-diphenylimidazolidine-2,4-dione derivatives have been previously shown to behave as CB₁ cannabinoid receptor ligands, appropriate substitutions of these templates can result in FAAH inhibitors devoid of affinity for the cannabinoid receptors.

Full text of DOI:10.1021/jm050977k

J. Med. Chem. 2006, 49, 417-425
417
Substituted 2-Thioxoimidazolidin-4-ones and Imidazolidine-2,4-diones as Fatty Acid Amide
Hydrolase Inhibitors Templates
Giulio G. Muccioli,† Nicola Fazio,† Gerhard K. E. Scriba,‡ Wolfgang Poppitz,§ Fabio Cannata,† 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 30, 2005
The demonstration of the essential role of fatty acid amide hydrolase (FAAH) in hydrolyzing endogenous
bioactive fatty acid derivatives has launched the quest for the discovery of inhibitors for this enzyme. Along
this line, a set of 58 imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one derivatives was evaluated as
FAAH inhibitors. Among these compounds, 3-substituted 5,5′-diphenylimidazolidine-2,4-dione and
3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one derivatives were previously described as CB1
cannabinoid receptor ligands. In the present study, we synthesized several derivatives exhibiting interesting
FAAH inhibitory activity and devoid of affinity for the CB1 and CB2 cannabinoid receptors. For instance,
3-heptyl-5,5′-diphenylimidazolidine-2,4-dione (14) and 5,5′-diphenyl-3-tetradecyl-2-thioxo-imidazolidin-4-
one (46) showed pI50 values of 5.12 and 5.94, respectively. In conclusion, it appears that even though several
3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one and 3-substituted 5,5′-diphenylimidazolidine-2,4-
dione derivatives have been previously shown to behave as CB1 cannabinoid receptor ligands, appropriate
substitutions of these templates can result in FAAH inhibitors devoid of affinity for the cannabinoid receptors.
Introduction
The cloning of the first cannabinoid receptor,1 named CB1,
in 1990 was rapidly followed by the report of the discovery of
its first endogenous ligand, arachidonoylethanolamide,2 or
anandamide (AEA), which acts as a partial agonist.3,4 Nowadays,
several other compounds are considered as endocannabinoids,
i.e. endogenous ligands of the cannabinoid receptors. Among
them, 2-arachidonoylglycerol5,6 (2-AG), the most abundant
endocannabinoid in the brain,7 is a full CB1 cannabinoid receptor
agonist.8 The characterization of anandamide and other endo-
cannabinoids launched the quest for the discovery of the
enzyme(s) responsible(s) for their degradation. For anandamide,
the question was partially answered with the characterization
of an enzyme, now widely called fatty acid amide hydrolase9-11
(FAAH). This membrane-bound enzyme was cloned12 in 1996
and crystallized13 in 2002, by Cravatt’s group. FAAH is
responsible for the hydrolysis of arachidonoylethanolamide to
yield arachidonic acid and ethanolamine. Later on, 2-AG was
shown to be a substrate for FAAH, at least in vitro.14,15
Moreover, FAAH is also involved in the hydrolysis of other
bioactive fatty acid amides, such as the sleep-inducing agent
oleamide,16,17 the anorexigenic compound N-oleoylethanol-
amide18 (OEA), and the antiinflammatory agent N-palmitoyl-
ethanolamide15 (PEA).
Figure 1. Structures of four FAAH inhibitors: ATMFK (1), PMSF
(2), OL-135 (3), and URB-597 (4).
levels22 as well as a CB1 cannabinoid receptor-dependent
analgesia phenotype.23 Thus, inhibition of fatty acid amide
hydrolase, enhancing fatty acid amides effects, is considered
as a very promising therapeutic target.24
An increasing number of compounds were described as
FAAH inhibitors (Figure 1).25 Among them are, for instance,
arachidonoyl trifluoromethyl ketone (1, ATFMK) and phenyl-
methylsulfonyl fluoride (2, PMSF). Interestingly, more recent
inhibitors, such as OL-13526,27 (3) or URB-59720 (4), induce
analgesia after in vivo administration. OL-135, as well as the
recent 2-keto-5-(2-pyridyl)-1,3,4-oxadiazoles,28 were shown to
be highly selective for FAAH over other serine hydrolase
enzymes using a proteome screening approach.29 The therapeutic
potential of FAAH inhibitors, e.g. to treat pain and anxiety,
further highlights the interest in the synthesis and characteriza-
tion of new FAAH inhibitors.
Since its discovery, several pharmacological effects were
reported for anandamide. Among these were antinociception,19
modulation of anxiety,20 and control of reproduction.21 In
addition, mice lacking FAAH showed elevated anandamide
In this connection, we have found in a preliminary screening
assay that some 3-substituted 5,5′-diphenylimidazolidine-2,4-
dione are able to inhibit FAAH activity. The 3-substituted 5,5′-
diphenylimidazolidines-2,4-diones30,31 and their thioxo ana-
logues the 3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-
* Corresponding author D. M. Lambert: Phone: +32 2 7647347, Fax:
+32 2 7647363, 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/jm050977k CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/13/2005

418 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Muccioli et al.
Scheme 1. Synthesis of the
Scheme 3. Synthesis of the 3-Substituted
5,5′-Diphenylimidazolidine-2,4-dione (X′ ) O, 7-28) and
5,5′-Diphenyl-2-thioxoimidazolidin-4-one (X′ ) S, 29-54)
Derivativesa
5-Phenylimidazolidine-2,4-dione (X′ ) O, 58) and
2-Thioxoimidazolidin-4-one Derivatives (X′)S, 59 and 60)a
a Reagents and conditions: (a) stirred for 24 h in pyridine at 40 °C.
After evaporation of pyridine, the residue is refluxed in 2 N HCl.
a Reagents and conditions: (a) DMSO/aq KOH, nine microwaves pulses
(750 W).
Scheme 4. Synthesis of the 1,5-Diphenyl Derivatives (63 and
64)a
Scheme 2. Synthesis of the
3,5,5′-Triphenylimidazolidine-2,4-dione (55)a
a Reagents and conditions: (a) refluxed for 4 h in CH3COOH/HCl.
Scheme 5. Synthesis of the 3-Substituted
a Reagents and conditions: (a) stirred for 24 h in DMF/CH3COOH at rt
in the presence of H2O2.
Imidazolidine-2,4-dione (X′ ) O, 69-71) and
2-Thioxoimidazolidin-4-one (X′ ) S, 72 and 73) Derivativesa
one32 derivatives were previously described as CB1 cannabinoid
receptor ligands. In this paper, we report the ability of such
compounds to inhibit FAAH. Preliminary structure-activity
relationships, as well as the inhibition mode, are discussed.
These derivatives represent new interesting FAAH reversible
inhibitors.
a Reagents and conditions: (a) stirred for 4 h in CHCl3; (b) following
evaporation of CHCl3, refluxed for 3 h in ethanol and HCl (10 N) (1:1,
v/v).
Results and Discussion
acetic acid with hydrochloric acid as catalyst, allowed the
synthesis of both compounds in good yields.
Chemistry. 5,5′-Diphenylimidazolidine-2,4-dione and 5,5′-
diphenyl-2-thioxoimidazolidin-4-one derivatives were synthe-
sized from the respective benzil (5) and urea (6, X′ ) O) or
thiourea (6, X′ ) S) derivatives using a microwave-enhanced
method previously described33 (Scheme 1). The method allowed
the rapid synthesis of the target derivatives (7-54) in moderate
to good yields. However, when phenylurea is used, the resulting
compound is 1-benzhydryl-3-phenylurea instead of the target
3-substituted imidazolidine-2,4-dione.34 Therefore, 3,5,5′-tri-
phenylimidazolidine-2,4-dione (55) was obtained, in high yield,
from the corresponding 2-thioxoimidazolidin-4-one (54) upon
reaction with hydrogen peroxide in DMF-acetic acid (Scheme
2). This reaction was already used for N-alkyl derivatives33and
has been extended to the N-phenyl derivatives.
3-Substituted 5-phenylimidazolidine-2,4-dione (58) and 3-sub-
stituted 5-phenyl-2-thioxoimidazolidin-4-one (59 and 60) de-
rivatives were synthesized in two steps by reacting phenyl-
glycine methyl ester (56) with the desired phenyl or alkyl
isocyanate/isothiocyanate (57, X′ ) O or S), respectively
(Scheme 3). The first step consisted of the condensation of the
amino acid derivative with a phenyl or alkyl iso(thio)cyanate
in pyridine, leading to 3-substituted (thio)ureido-phenyl acetic
acid. The second step allowed the cyclization of the acid
derivative upon refluxing in acidic water.
3-Heptyl-1,5-diphenylimidazolidine-2,4-dione (65) and 1,3-
diheptyl-5,5′-diphenylimidazolidine-2,4-dione (66) were ob-
tained by alkylation in DMF of the corresponding 3-unsubsti-
tuted derivatives (63 and 14, respectively) using chloroheptane.
The compounds bearing no substituent in position 5 of the
central moiety were obtained following a method described by
Ryczek36 (Scheme 5). Glycine ethyl ester isocyanate (67, X′ )
O) and the desired amine (68) were refluxed in chloroform to
afford the 3-substituted ureidoacetic acid ethyl ester, which upon
refluxing in ethanol/hydrochloric acid cyclized to form the
imidazolidine-2,4-dione nucleus bearing the desired substituent
in position 3 (69-71). Following the same procedure, but
starting from glycine ethyl ester isothiocyanate (67, X′ ) S)
the 3-substitituted imidazolidine-2,4-dione derivatives (72, 73)
were obtained in good yields.
FAAH Inhibition and Primary Structure-Activity Rela-
tionships. An initial screening, performed at 10 µM, using rat
brain homogenates as source of FAAH and [3H]-anandamide
as substrate, revealed that some 3-substituted 5,5′-diphenylimid-
azolidine-2,4-dione derivatives inhibited FAAH activity. There-
fore, a set of 21 3-substituted 5,5′-diphenylimidazolidine-2,4-
dione derivatives and a set of 26 thioxo analogues have been
challenged in a FAAH inhibition assay, to establish structure-
activity relationships.
1,5-Diphenylimidazolidine-2,4-dione (63) and 1,5-diphenyl-
2-thioxoimidazolidin-4-one (64) were obtained, as previously
described by Joshi et al.,35 by reacting phenylglyoxal (61) with
phenylurea (62, X′ ) O) and phenylthiourea (62, X′ ) S),
respectively (Scheme 4). The synthesis, carried out in glacial
The results, expressed as pI50 values, obtained for the
3-substituted 5,5′-diphenylimidazolidine-2,4-dione derivatives
are summarized in Table 1 (pI50 ) -log IC50). The influence

Fatty Acid Amide Hydrolase Inhibitors Templates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 419
Table 1. 3-Substituted 5,5′-Diphenylimidazolidine-2,4-dione Derivatives
(7-28, 55)a
Table 2. 3-Substituted 5,5′-Diphenyl-2-thioxoimidazolidin-4-one
Derivatives (29-50, 54)a
% of displacement
% of displacement
hCB1
receptor
hCB2
receptor
hCB1
receptor
hCB2
receptor
compd
X
R
pI50
compd
X
R
pI50
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
54
51
52
53
H
H
H
Cl
H
C2H5
n-C4H9
n-C4H9
3.27 ( 0.03 <15
<10
7
8
9
H
H
H
Cl
Br
H
H
H
H
<2
<20
<20
3.96 ( 0.05 40.0 ( 5.03
4.68 ( 0.03 42.8 ( 4.8b
4.34 ( 0.09 72.7 ( 4.6
4.09 ( 0.18 73.8 ( 5.5b
3.85 ( 0.09 34.6 ( 3.4b
<10b
<10b
<20
C2H5
C4H9
C4H9
C4H9
3.76 ( 0.02 <20
<20
4.29 ( 0.02 <20b
<20b
<20
23.1 ( 5.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
55
26
27
28
4.99 ( 0.03 51.2 ( 7.1
4.71 ( 0.09 69.2 ( 6.5
Br n-C4H9
<20b
<10b
<20b
<20b
<20
C5H11
C6H13
C7H15
C7H15
C7H15
C8H17
C8H17
C8H17
C8H17
C8H17
4.53 ( 0.03 21.3 ( 5.5b <20b
5.02 ( 0.02 29.9 ( 4.5b <20b
H
H
H
H
H
H
Cl
i-C4H9
(CH2)3OCH3 3.43 ( 0.07 29.4 ( 3.8b
(CH2)3OH
C5H11
C6H13
C7H15
C7H15
<2
20.8 ( 5.3b
5.12 ( 0.03 23.4 ( 5.7
4.31 ( 0.15 42.7 ( 5.1
<20
<20
4.91 ( 0.08 27.6 ( 3.4
5.35 ( 0.02 21.9 ( 2.9
5.49 ( 0.02 28.8 ( 5.1b
3.75 ( 0.08 67.8 ( 4.5b
3.62 ( 0.05 34.2 ( 5.2b
5.52 ( 0.02 38.8 ( 4.3b
3.72 ( 0.12 36.6 ( 5.0b
3.23 ( 0.11 23.4 ( 3.7b
5.89 ( 0.03 <10
Cl
Br
H
4.56 ( 0.12 44.8 ( 6.6b <20b
4.87 ( 0.03 25.1 ( 5.2b <20b
4.89 ( 0.05 40.1 ( 4.3b <20b
<20
<20b
<20b
<20b
<20b
<10b
<20b
<20
F
Br C7H15
H
Cl
Cl
Br
Me
3.62 ( 0.05 39.5 ( 6.4
<20
3.30 ( 0.04 45.1 ( 5.0b 21.1 ( 4.2b
C8H17
C8H17
3.79 ( 0.04 24.5 ( 4.3
3.52 ( 0.10 31.2 ( 5.3
4.31 ( 0.08 <20
<20
<20
<20
<20
<20
<20
<20
<20
<20
Br C8H17
OMe C8H17
H
H
H
H
H
H
H
H
Cl
H
C10H21
C14H29
C16H33
C18H37
C18H35
C6H11
C6H5
CH2C6H5
CH2C6H5
(CH2)2C6H5
H
H
H
H
H
Cl
H
C10H21
C14H29
C6H11
C6H5
CH2C6H5
CH2C6H5
5.94 ( 0.05 <10
<20
3.98 ( 0.11 <20
4.28 ( 0.07 < 10
<10
3.88 ( 0.17 24.3 ( 6.5
4.13 ( 0.05 28.5 ( 6.6
4.73 ( 0.05 33.7 ( 6.1
5.00 ( 0.12 72.2 ( 6.8
3.11 ( 0.06 < 10
<10
4.21 ( 0.05 < 10
<10
4.19 ( 0.09 38.5 ( 3.8b
4.53 ( 0.18 32.6 ( 3.5
4.91 ( 0.10 44.7 ( 5.3b
4.34 ( 0.04 74.2 ( 2.5b
4.61 ( 0.09 29.3 ( 5.5b
<20b
<20
(CH2)2C6H5 4.30 ( 0.07 <20
<20b
<15b
<20b
a Inhibition potential toward FAAH (rat brain homogenate, pI50 ( SEM)
and affinity for the hCB1 and hCB2 cannabinoid receptor expressed in CHO
cells (% of displacement of bound [3H]-SR141716A and [3H]-CP-55,940,
for the hCB1 and hCB2 cannabinoid receptor, respectively, obtained with
10 µM of competitor, mean ( SEM). b From ref 30.
a Inhibition potential toward FAAH (rat brain homogenate, pI50 ( SEM)
and affinity for the hCB1 and hCB2 cannabinoid receptor expressed in CHO
cells (% of displacement of bound [3H]-SR141716A and [3H]-CP-55,940,
for the hCB1 and hCB2 cannabinoid receptor, respectively, obtained with
10 µM of competitor, mean ( SEM). b From ref 32.
resulted in less potent inhibitors as illustrated by compounds
17 (X ) H), 19 (X ) Cl), and 20 (X ) Br), possessing pI50
values of 4.87 ( 0.03, 3.62 ( 0.05, and 3.30 ( 0.04,
respectively. The methyl (21) and methoxy (22) moieties were
also responsible for a decreased potency with pI50 values of
3.79 ( 0.04 and 3.52 ( 0.10, respectively. Interestingly, the
fluoro derivative 18 retained the activity of the unsubstituted
compound (17) (pI50 ) 4.89 ( 0.05), suggesting that the drop
of potency observed with other halogens is more likely due to
a steric effect rather than to an electronic one.
When the substituent in position 3 is a phenyl or an
alkylphenyl, the highest inhibitory activity was obtained with
the benzyl substituent (26, pI50 ) 4.73 ( 0.05) followed by the
ethylphenyl (28, pI50 ) 4.30 ( 0.07) and the phenyl (55, pI50
) 4.13 ( 0.05) substituents.
Similar structure-activity relationships were partially found
for the 3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one
derivatives (Table 2 and Figure 2). As observed for the
3-substituted 5,5′-diphenylimidazolidine-2,4-dione derivatives,
the activity increases with the length of the alkyl substituent.
Compound 30, bearing an ethyl moiety, possesses a pI50 value
of 3.96 ( 0.05, while compounds 31 (n-butyl) and 38 (n-hexyl)
have pI50 values of 4.68 ( 0.03 and 5.35 ( 0.02, respectively.
A decreased activity is also observed for the thio derivatives
substituted on the phenyl rings. This is well illustrated by
Figure 2. Influence of the N3 substituent length of the 5,5′-
diphenylimidazolidine-2,4-dione (b) and 5,5′-diphenyl-2-thioxoimid-
azolidin-4-one (2) derivatives on the FAAH inhibition (pI50 ( SEM).
of the alkyl chain length on the enzyme activity is illustrated in
Figure 2. The inhibitory potency is the highest for derivatives
bearing alkyl chains of six (13) or seven (14) carbon atoms.
Shorter or longer alkyl substituents resulted in loss of activity.
For instance, on one hand, 5,5′-diphenylimidazolidine-2,4-dione
(7), better known as phenytoin, which is unsubstituted in position
3, was unable to inhibit FAAH activity. On the other hand, 5,5′-
diphenyl-3-tetradecylimidazolidine-2,4-dione (24), which has a
rather long substituent, showed a reduced activity (pI50 ) 3.98
( 0.11) compared to 13 or 14.
Considering the 3-octyl derivatives (17-22), substitution in
para position of the phenyl ring by chlorine or bromine atoms

420 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Muccioli et al.
compounds 39 (X ) H), 40 (X ) Cl), and 41 (X ) Br),
exhibiting pI50 value of 5.49 ( 0.02, 3.75 ( 0.08, and 3.62 (
0.05, respectively. And finally, as for the oxo derivatives, a
benzyl substituent (51, pI50 ) 4.91 ( 0.10) is preferred to an
ethylphenyl (53, pI50 ) 4.61 ( 0.09) or phenyl (54, pI50 ) 4.53
( 0.18) substituent. However, with the thio derivatives, the drop
in the activity is observed with much longer N3 substituents in
contrast to what is observed with the 3-substituted 5,5′-
diphenylimidazolidine-2,4-dione derivatives (Figure 2). Indeed,
the highest inhibitory activities were obtained for compounds
45 (pI50 ) 5.89 ( 0.03) and 46 (pI50 ) 5.94 ( 0.05), possessing
a decyl and tetradecyl substituent, respectively, and the activity
is reduced for compounds 47 (pI50 ) 4.28 ( 0.07) and 48 (pI50
) 3.11 ( 0.06), possessing an hexadecyl and octadecyl
substituent, respectively. One could argue that this activity
enhancement when the substituent length increases is due to an
overall lipophilic effect. However, this is overruled by com-
pounds 31 and 34, which are both substituted with a butyl alkyl
chain and thus have the same lipophilicity. Compound 31,
substituted with a n-butyl chain, possesses a pI50 value of 4.68
( 0.03, whereas compound 34, substituted with an isobutyl
chain, has a pI50 value of 3.85 ( 0.09. Interestingly enough, a
different evolution of the inhibitory potencies is observed for
the oxo and thio derivatives when the alkyl chain length is
increased (Figure 2). Previous studies revealed that the differ-
ences observed in the electrostatic (molecular electrostatic
potential) and lipophilic properties (CLOGP) of the oxo and
thio derivatives are so small, that it could hardly explain the
differences observed with the in vitro data.32 More in-depth
investigations, such as docking studies, could possibly answer
this question.
(60, pI50 ) 4.29 ( 0.05) and 3-heptyl-5,5′-diphenyl-2-thioxo-
imidazolidin-4-one (39, pI50 ) 5.49 ( 0.02).
Three 1,5-diphenyl derivatives were also synthesized (63-
65, Table 3). 1,5-Diphenylimidazolidine-2,4-dione (63) and 1,5-
diphenyl-2-thioxoimidazolidin-4-one (64) exhibited very low
pI50 values (<2 and 3.78 ( 0.02, respectively). In addition, 1,5-
diphenyl-3-heptylimidazolidine-2,4-dione (65) showed a pI50
value of 2.40 ( 0.07, which is much lower than the value
obtained for 3-heptyl-5,5′-diphenylimidazolidine-2,4-dione (14,
pI50 ) 5.12 ( 0.03).
As illustrated above, when derivatives differing in the phenyl
rings positions, but bearing the same substituent in position 3,
are compared, it appears that in the vast majority of the cases
the preferred substitution for FAAH inhibition is the 5,5′-
diphenyl one. To confirm the essential role of the phenyl rings
in position 5 of the heterocycle, five derivatives (69-73) without
phenyl rings in that position were synthesized (Table 4). None
of them displayed a significant inhibition of FAAH hydrolytic
activity. For instance, 3-heptylimidazolidine-2,4-dione (70)
exhibited a pI50 of 2.73 ( 0.04 compared to the 5.12 ( 0.03
obtained for compound 14 possessing two phenyls in position
5.
Mode of Inhibition. The mode of inhibition of FAAH
activity by these derivatives was investigated using 3-octyl-5,5′-
diphenyl-2-thioxoimidazolidin-4-one (42) as representative com-
pound. Michaelis-Menten curves were constructed, in the
presence (12.6 µM) and in the absence of the inhibitor, using
increasing concentrations of anandamide (0-70 µM) (Support-
ing Information). On one hand, the Vmax values obtained in the
presence and absence of the inhibitor (6511 and 6346 pmol/
min/mg protein, respectively) are of similar magnitude. On the
other hand, Km values are significantly increased in the presence
of the inhibitor (9.6 and 19.9 µM, respectively). These results,
representative of two experiments performed in triplicate,
illustrate a competitive mode of inhibition, as Vmax values remain
constant while the Km value is increased in the presence of the
inhibitor. Although potent competitive inhibitors of FAAH have
already been described (e.g. OL135, or the 2-keto-5-(2-pyridyl)-
1,3,4-oxadiazoles), competitive inhibition is not the most
common mode of inhibition described so far for FAAH
inhibitors.25 Competitive inhibition of FAAH by 5,5′-diphenyl-
2-thioxoimidazolidin-4-one derivatives could represent an ad-
vantage compared to more potent, but irreversible, inhibitors
of the enzyme. Indeed, due to their mechanism of action, i.e. a
reactive electrophilic center, the irreversible FAAH inhibitors
are more likely to interact with other serine hydrolase compared
to compounds lacking this kind of electrophilic moiety.
It is known that unsaturations in the side chain of FAAH
inhibitors bearing a fatty acid chain can result in an increased
inhibitory activity; therefore, an unsaturation was introduced
in the alkyl side chain of derivative 48, resulting in a compound
bearing a octadec-9-enyl substituent (49). As expected, the
unsaturated side chain induced an increase of the activity (49,
pI50 ) 4.21 ( 0.05) compared to the octadecyl substituent (48,
pI50 ) 3.11 ( 0.06).
Another interesting point is the drop of activity observed for
more polar substituents such as methoxypropyl (35, pI50 ) 3.43
( 0.07) and, even more dramatically, with hydroxypropyl (36,
pI50 < 2). The activities of these two compounds should be
compared with the activity of 3-butyl-5,5′-diphenyl-2-thioxo-
imidazolidin-4-one (31), which bears a substituent of similar
length (pI50 ) 4.68 ( 0.03).
Next the structre-activity relationships of changing the
substitution pattern around the imidazolidine-2,4-dione and
2-thioxoimidazolidin-4-one rings was evaluated. Compounds
possessing only one phenyl ring in position 5 (58-65) or even
no phenyl at all (69-73) were synthesized and their activity
toward FAAH hydrolytic activity was evaluated (Tables 3 and
4).
It has been established that the optimum pH value for FAAH
activity is 9.11 Therefore, we assayed two imidazolidine-2,4-
diones (13, 14) and two 2-thioxoimidazolidin-4-ones (45, 46)
at pH 9. The results obtained and summarized in Table 5 suggest
that the FAAH inhibition by the imidazolidine-2,4-dione and
2-thioxoimidazolidin-4-one derivatives is not influenced by the
pH of the medium.
The question whether the 2-thioxoimidazolidin-4-one and
imidazolidine-2,4-dione derivatives inhibit FAAH hydrolytic
activity by being hydrolyzed instead of anandamide (competing
substrates) was also investigated. Preincubations (10 and 20 min)
of 3-octyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (42) in the
presence of brain homogenate prior to anandamide incubation
(10 min) were performed. The pI50 values obtained were 5.55
( 0.02 and 5.53 ( 0.03 for 10 and 20 min of preincubation,
respectively, and are similar to the value obtained when the
homogenate was preincubated alone prior to anandamide
Three compounds substituted with a phenyl in position 5 and
with a phenyl ring or a heptyl chain in position 3 were obtained
(58-60, Table 3). The activity of 3,5-diphenylimidazolidine-
2,4-dione (58, pI50 ) 3.34 ( 0.04) is significantly lower than
the activity of the corresponding 5,5′-diphenyl derivative (55,
pI50 ) 4.13 ( 0.05). In a similar way, the inhibitory activity of
3,5-diphenyl-2-thioxoimidazolidin-4-one (59, pI50 ) 3.79 (
0.04) is lower than the activity of the 5,5′-diphenyl-2-thioxo
derivative (54, pI50 ) 4.53 ( 0.18). The same trend was
observed when the substituent in position 3 is an alkyl chain,
as in the case of 3-heptyl-5-phenyl-2-thioxoimidazolidin-4-one

Fatty Acid Amide Hydrolase Inhibitors Templates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 421
Table 3. Substituted Imidazolidine-2,4-dione and 2-Thioxoimidazolidin-4-one Derivatives (58-60, 63-66)a
a Inhibition potential toward FAAH (rat brain homogenate, pI50 ( SEM) and affinity for the hCB1 and hCB2 cannabinoid receptor expressed in CHO cells
(% of displacement of bound [3H]-SR141716A and [3H]-CP-55,940, for the hCB1 and hCB2 cannabinoid receptor, respectively, obtained with 10 µM of
competitor, mean ( SEM). Compounds 58-60, 63-65 were tested as racemic mixtures.
Table 4. Substituted 5,5′-Diphenylimidazolidine-2,4-dione and
5,5′-Diphenyl-2-thioxoimidazolidin-4-one Derivatives (69-73)a
Table 5. Influence of the pH on the FAAH Inhibition by
Imidazolidine-2,4-dione (13, 14) and 2-Thioxoimidazolidin-4-one (45,
46) Derivatives
FAAH inhibition
compd
X
R
pH 7.6
pH 9.0
13
14
45
46
O
O
S
C6H13
C7H15
C10H21
C14H29
5.02 ( 0.02
5.12 ( 0.03
5.89 ( 0.03
5.94 ( 0.05
5.00 ( 0.02
5.08 ( 0.05
5.87 ( 0.04
6.05 ( 0.04
% of displacement
S
hCB1
hCB2
compd
X
R
pI50
receptor
receptor
for 0, 10, or 40 min, and analyzed by HPLC the residue obtained
after lyophylization. This procedure allowed an easy and rapid
analysis of the entire incubation medium. A phosphate buffer,
instead of the usual TRIS-HCl/EDTA buffer, was used in this
assay due to UV absorption of TRIS buffers.37 The pI50 value
obtained for 42 using the phosphate buffer was similar to the
value obtained with the TRIS-HCl/EDTA buffer. The time of
retention (3.35 min) and the peak area of compound 42 were
found similar, whatever the incubation time. Indeed, no
significant differences were found between the peak area values
obtained after 0, 10, and 40 min of incubation (one-way
ANOVA analysis followed by a Tuckey post-test; data not
69
70
71
72
73
O
O
O
S
C4H9
C7H15
C6H5
C4H9
C6H5
<10% @ 100 µM
2.73 ( 0.04
23% @ 100 µM
<10% @ 100 µM
25% @ 100 µM
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
S
a Inhibition potential toward FAAH (rat brain homogenate) and affinity
for the hCB1 and hCB2 cannabinoid receptor expressed in CHO cells (%
of displacement of bound [3H]-SR141716A and [3H]-CP-55,940, for the
hCB1 and hCB2 cannabinoid receptor, respectively, obtained with 10 µM
of competitor, mean ( SEM).
adjunction (pI50 ) 5.52 ( 0.03). In addition to the preincubation
assays, we have incubated the membranes with compound 42,

422 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Muccioli et al.
from NEN Life Science (Zaventem, Belgium). HU-210 was
purchased from Tocris (Bristol, UK).
shown). Moreover, no significant additional peak was detected
after 10 or 40 min of incubation, compared to those detected
without incubation. Taken together, the results obtained in these
two assays suggest that the 3-substituted 5,5′-diphenyl-2-
thioxoimidazolidin-4-one derivatives do not act as competing
substrates of the enzyme.
The microwave oven used was a commercial household micro-
wave oven (frequency 2450 MHz). Melting points (mp) were
determined in open capillaries using a Electrothermal 9100 ap-
paratus 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 tetra-
methylsilane 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; 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 of
original compounds 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 the
3-Substituted 5,5′-Diphenylimidazolidine-2,4-diones. These com-
pounds were obtained following a microwave-enhanced method
previously described.33 Briefly, to a solution of benzil (9.9 mmol)
and substituted urea (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 microwave 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.
FAAH Inhibition versus the Cannabinoid Receptor Rec-
ognition. 3-Substituted 5,5′-diphenylimidazolidine-2,4-dione and
3-substituted 5,5′-diphenyl-2-thioxoimidazolidin-4-one deriva-
tives were previously described as CB1 cannabinoid receptor
selective ligands.30-32 Therefore, all the new derivatives were
screened at 10 µM for competitive binding to the hCB1 and
hCB2 cannabinoid receptors using membranes of Chinese
hamster ovarian (CHO) cells selectively expressing either the
hCB1 or the hCB2 cannabinoid receptors. None of the com-
pounds displayed significant binding to the hCB2 cannabinoid
receptors, as they displaced less than 20% of the radioligand
([3H]-CP-55,940) bound to the receptor (Tables 1-4). The
competitive binding to the hCB1 cannabinoid receptor, expressed
as the displacement percentages of [3H]-SR141716A from its
binding site, are listed in Tables 1-4. We have shown
previously that the hCB1 cannabinoid receptor affinity of these
derivatives is enhanced by para substitution of the phenyl rings
by halogens and by substituting nitrogen 3 with a short alkyl
chain (e.g. butyl).31,32 In the present study, substitution of the
phenyls afforded less active inhibitors, and long alkyl chains
(from 7 to 14 carbons for the 2-thioxoimidazolidin-4-ones)
produced higher FAAH inhibitor activity than the short alkyl
chains. This is confirmed, for instance, by compounds 45 and
46, which displayed a high inhibition of FAAH activity while
having no affinity for the hCB1 cannabinoid receptor. The
structure-activity relationships found here for FAAH inhibition,
i.e. unsubstituted phenyls and long alkyl chain, are the opposite
of the structure-activity relationships found at the CB1 can-
nabinoid receptor, i.e. substituted phenyls and short alkyl chain.
It is therefore possible to favor either the affinity for the CB1
cannabinoid receptor or the FAAH inhibition potential of the
imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one by
modulating the nature and position of the substituents around
the central scaffold.
Compounds 9-13, 15-18, 20, 25, 26, and 28 were previously
described by us.30,33,34
3-Ethyl-5,5′-diphenylimidazolidine-2,4-dione (8). Yield: 60%.
Mp: 137.9-139.2 °C. MS (EI): 280 [M]+. 13C NMR (DMSO-
d6): δ 13.37 (CH3), 33.29 (CH2), 67.14 (C), 126.72, 128.27, 128.72,
139.79 (Carom and CHarom), 155.31 (CdO), 173.11 (CdO).
3-Heptyl-5,5′-diphenylimidazolidine-2,4-dione (14). Yield: 55%.
Mp: 82.3-83.4 °C. MS (EI): 350 [M]+. 13C NMR (DMSO-d6):
δ 13.78 (CH3), 21.74 (CH2), 25.72 (CH2), 27.19 (CH2), 27.90 (CH2),
30.94 (CH2), 37.86 (CH2), 68.90 (C), 126.46, 128.02, 128.44, 139.60
(Carom and CHarom), 155.31 (CdO), 173.09 (CdO). Anal. Calcd
for C22H26N2O2: C, H, N.
In conclusion, we have described for the first time the
inhibitor potential toward FAAH activity of imidazolidine-2,4-
dione and 2-thioxoimidazolidin-4-one derivatives. The latter are
more potent inhibitors than the corresponding oxo derivative.
They act as competitive inhibitors of FAAH activity without
being hydrolyzed by the enzyme. Among them, 5,5′-diphenyl-
3-tetradecyl-2-thioxoimidazolidin-4-one (46) showed the highest
activity (pI50 ) 5.94) for the enzyme, while displaying no
affinity for the cannabinoid receptors. Actually, unsubstituted
phenyls combined with N3 long alkyl chains enhance the
inhibitory activity for the enzyme, while halogen substitu-
tion on the phenyls along with N3 short alkyl chains enhance
CB1 cannabinoid receptor affinity. Finally, 3-substituted 5,5′-
diphenyl-2-thioxoimidazolidin-4-one could constitute an inter-
esting template for the development of more potent reversible
and competitive FAAH inhibitors.
5,5′-Bis(4-chlorophenyl)-3-octylimidazolidine-2,4-dione (19).
Yield: 47%. Mp: 129.4-130.3 °C. MS (EI): 432 [M]+. 13C NMR
(DMSO-d6): δ 13.78 (CH3), 21.93 (CH2), 25.81 (CH2), 27.11
(CH2), 28.27 (CH2), 28.40 (CH2), 38.40 (CH2), 38.11 (CH2), 68.06
(C), 128.42, 128.62, 133.15, 138.26 (Carom and CHarom), 155.15 (Cd
O), 172.61 (CdO). Anal. Calcd for C23H26Cl2N2O2: C, H, N.
5,5′-Bis(4-methylphenyl)-3-octylimidazolidine-2,4-dione (21).
Yield: 45%. Mp: 107.8-108.9 °C. MS (EI): 393 [M]+. 13C NMR
(DMSO-d6): δ 14.07 (CH3), 21.06 (2xCH3), 22.64 (CH2), 24.54
(CH2), 26.62 (CH2), 28.01 (CH2), 29.11 (CH2), 31.73 (CH2), 39.08
(CH2), 68.54 (C), 126.74, 129.43, 136.51, 138.36 (Carom and CHarom),
155.45 (CdO), 172.83 (CdO). Anal. Calcd for C25H32N2O2: C,
H, N.
5,5′-Bis(4-methoxyphenyl)-3-octylimidazolidine-2,4-dione (22).
Yield: 48%. Mp: 87.8-88.9 °C. MS (EI): 424 [M]+. 13C NMR
(DMSO-d6): δ 14.01 (CH3), 22.17 (CH2), 25.98 (CH2), 27.47
(CH2), 28.44 (CH2), 28.57 (CH2), 31.16 (CH2), 38.02 (CH2), 55.29
(2xCH3), 68.29 (C), 113.91, 128.01, 132.03 (Carom and CHarom),
155.51 (CdO), 159.07 (Carom), 173.89 (CdO). Anal. Calcd for
C25H32N2O4: C, H, N.
Experimental Section
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]-AEA
(60 Ci/mmol) was purchased from American Radiolabeled Chemical
(St Louis, MO), [3H]-SR141716A (52 Ci/mol) from Amersham
(Roosendaal, The Netherlands), and [3H]-CP-55,940 (101 Ci/mol)
3-Decyl-5,5′-diphenylimidazolidine-2,4-dione (23). Yield: 48%.
Mp: 72.4-73.8 °C. MS (EI): 392 [M]+. 13C NMR (CDCl3): δ
13.85 (CH3), 21.93 (CH2), 25.75 (CH2), 27.17 (CH2), 28.27 (CH2),
28.53 (CH2), 28.73 (CH2), 31.12 (CH2), 37.85 (CH2), 68.91 (C),
126.49, 127.98, 128.53, 139.75 (C and CH arom.), 155.23 (CdO),
173.17 (CdO). Anal. Calcd for C25H32N2O2: C, H, N.

Fatty Acid Amide Hydrolase Inhibitors Templates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 423
5,5′-Diphenyl-3-tetradecylimidazolidine-2,4-dione
(24).
41.61 (CH2), 72.09 (C), 126.30, 128.11, 128.69, 137.94 (Carom and
CHarom), 173.47 (CdO), 182.53 (CdS). Anal. Calcd for C33H48N2OS:
C, H, N.
Yield: 30%. Mp: 69.1-70.5 °C. MS (EI): 449 [M + H]+. 13C
NMR (CDCl3): δ 14.11 (CH3), 22.65 (CH2), 26.59 (CH2), 27.06
(CH2), 27.95 (CH2), 28.53 (CH2), 29.05 (CH2), 29.16 (CH2), 29.32
(CH2), 29.45 (CH2), 29.64 (CH2), 30.03 (CH2), 31.90 (CH2), 39.09
(CH2), 70.01 (C), 126.82, 128.44, 128.76, 139.31 (Carom and CHarom),
156.84 (CdO), 173.34 (CdO). Anal. Calcd for C29H40N2O2: C,
H, N.
3-((Z)-Octadec-9-enyl)-5,5′-diphenyl-2-thioxoimidazolidin-4-
one (49). Yield: 21%. Oily compound. MS (EI): 518 [M]+. 13C
NMR (CDCl3): δ 13.44 (CH3), 23.04 (CH2), 26.46 (CH2), 26.99
(CH2), 27.34 (CH2), 27.56 (CH2), 27.96 (CH2), 28.65 (CH2), 29.19
(CH2), 30.03 (CH2), 32.29 (CH2), 32.93 (CH2), 41.99 (CH2), 72.34
(C), 127.27, 127.66, 129.08 (Carom and CHarom), 129.28 (CH), 138.40
(Carom), 173.98 (CdO), 181.49 (CdS). Anal. Calcd for C33H46N2OS:
C, H, N.
3-Benzyl-5,5′-bis(4-chlorophenyl)imidazolidine-2,4-dione (27).
Yield: 52%. Mp: 156.8-157.6 °C. MS (EI): 412 [M + H]+. 13
C
NMR (DMSO-d6) δ 41.03 (CH2), 68.56 (C), 126.39, 127.63, 127.88,
128.47, 128.86, 129.70, 129.89, 130.15, 133.51, 133.64, 136.62,
138.30 (Carom and CHarom), 155.25 (CdO), 172.66 (CdO). Anal.
Calcd for C22H16Cl2N2O2: C, H, N.
3,5,5′-Triphenylimidazolidine-2,4-dione (55). 3,5,5′-Triphenyl-
2-thioxoimidazolidin-4-one (54) was dissolved in a mixture of
DMF (10 mL) and acetic acid (1 mL). After addition of hydrogen
peroxide (30%, 2.5 mL), the solution was stirred for 24 h at room
temperature and poured onto ice. The precipitate was collected,
dried, and crystallized from ethanol. Yield: 80%. 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 (Carom and CHarom), 154.28 (CdO), 172.46 (CdO).
3,5-Diphenylimidazolidine-2,4-dione (58). Phenylglycine meth-
yl ester (56; 6.6 mmol) and phenyl isocyanate (57, X′ ) O; 6.6
mmol) were vigorously stirred for 2 h at room temperature in
pyridine (30 mL). The resulting mixture was evaporated to dryness
under reduced pressure. Aqueous HCl (30 mL, 2 N) was added to
the residue and the mixture refluxed for 4 h. A precipitate appeared
upon cooling and was recrystallized from hot water. Yield: 25%.
Mp: 192.3-192.8 °C. MS (DCI): 253 [M + H]+. 13C NMR
(DMSO-d6): δ 59.82 (CH), 126.64, 126.95, 127.69, 128.34, 128.66,
135.58, (Carom and CHarom), 155.57 (CdO), 171.49 (CdO).
3,5-Diphenyl-2-thioxoimidazolidin-4-one (59). The compound
was synthesized as 58 using phenyl isothiocyanate (57, X′ ) S)
instead of phenyl isocyanate. Yield: 30%. Mp: 232.1-233.6 °C.
MS (DCI): 269 [M + H]+. 13C NMR (DMSO-d6): δ 62.80 (CH),
127.34, 128.85, 128.99, 129.13, 129.66, 133.58, (Carom and CHarom),
172.97 (CdO), 182.91 (CdS).
3-Heptyl-5-phenyl-2-thioxoimidazolidin-4-one (60). The com-
pound was synthesized as 58 using heptyl isothiocyanate (57, X′
) S) instead of phenyl isocyanate. Yield: 25%. Mp: 98.6-99.2
°C. MS (EI): 290 [M]+. 13C NMR (DMSO-d6) δ 14.44 (CH3), 22.91
(CH2), 27.05 (CH2), 27.96 (CH2), 29.19 (CH2), 32.10 (CH2), 42.06
(CH2), 63.02 (CH), 127.01, 127.14, 129.79, 133.68 (Carom and
CHarom), 172.75 (CdO), 184.92 (CdS). Anal. Calcd for C16H22N2OS:
C, H, N.
General Procedure for the Synthesis of the 1,5-Diphenyl
Derivatives. These compounds were synthesized following a
method adapted from Joshi et al.35 Briefly, phenylglyoxal hydrate
(61; 4.5 mmol) and phenylurea (62, X′ ) O) or phenylthiourea
(62, 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 in water and the resulting
precipitate filtered off. The residue was subsequently crystallized
from ethanol.
1,5-Diphenylimidazolidine-2,4-dione (63). Yield: 68%. Mp:
197.6-198.9 °C. MS (EI): 252 [M]+. 13C NMR (DMSO-d6): δ
64.73 (CH), 121.15, 124.45, 127.49, 128.85, 128.98, 129.17, 134.35,
136.81 (Carom and CHarom), 155.12 (CdO), 171.81 (CdO).
1,5-Diphenyl-2-thioxoimidazolidin-4-one (64). Yield: 70%.
Mp: 234.1-235.6 °C. MS (EI): 268 [M]+. 13C NMR (DMSO-
d6): δ 63.19 (CH), 126.14, 127.56, 128.21, 128.34, 129.24, 129.44,
133.77, 134.87 (Carom and CHarom), 173.11 (CdO), 183.20 (CdS).
3-Heptyl-1,5-diphenylimidazolidine-2,4-dione (65). This com-
pound was obtained by alkylation of 63. Briefly, 63 (2.5 mmol)
and K2CO3 (5 mmol) were stirred in DMF (4 mL) for 30 min prior
to addition of chloroheptane (3.25 mmol). The resulting mixture
was stirred for 24 h and poured in water. The solution was extracted
with CHCl3, and the organic layer was washed with HCl, NaOH,
and brine. The resulting organic phase was dried over MgSO4 and
evaporated to dryness. Yield: 35%. Mp: 66.5-67.8 °C. MS (EI):
350 [M]+. 13C NMR (DMSO-d6): δ 13.62 (CH3), 21.71 (CH2),
25.72 (CH2), 27.08 (CH2), 27.86 (CH2), 30.84 (CH2), 62.92 (CH),
General Procedure for the Synthesis of the 3-Substituted 5,5′-
Diphenyl-2-thioxoimidazolidin-4-ones. These compounds were
obtained similarly to the 3-substituted 5,5′-diphenylimidazolidine-
2,4-dione derivatives starting from the corresponding thiourea.
Syntheses of compounds 30, 31, 33-36, 39-44, 50-53,32 and 5434
were described elsewhere. For compounds 46-49, the residue
obtained following the reaction was extracted with CHCl3, and the
organic layer was subsequently washed with HCl, NaOH, and brine.
The resulting organic phase was dried over MgSO4 and evaporated
to dryness.
5,5′-Bis(4-chlorophenyl)-3-butyl-2-thioxoimidazolidin-4-one
(32). Yield: 51%. Mp: 153.8-154.6 °C. MS (EI): 393 [M]+. 13
C
NMR (DMSO-d6): δ 13.63 (CH3), 19.38 (CH2), 29.35 (CH2), 40.61
(CH2), 70.24 (C), 128.59, 129.1, 133.70, 136.87 (Carom and CHarom),
173.11 (CdO), 181.52 (CdS). Anal. Calcd for C19H18Cl2N2OS:
C, H, N.
3-Pentyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one
(37).
Yield: 71%. Mp: 108.3-109.1 °C. MS (EI): 338 [M]+. 13C NMR
(DMSO-d6): δ 13.56 (CH3), 21.45 (CH2), 26.56 (CH2), 27.92
(CH2), 41.31 (CH2), 70.01 (C), 126.46, 128.34, 128.60, 138.04
(Carom and CHarom), 173.37 (CdO), 181.13 (CdS). Anal. Calcd for
C20H22N2OS: C, H, N.
3-Hexyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one
(38).
Yield: 69%. Mp: 108.6-109.7 °C. MS (EI): 352 [M]+. 13C NMR
(DMSO-d6): δ 13.56 (CH3), 21.71 (CH2), 25.40 (CH2), 26.82
(CH2), 30.51 (CH2), 40.51 (CH2), 70.01 (C), 126.01, 126.46, 128.34,
138.04 (Carom and CHarom), 173.37 (CdO), 181.13 (CdS). Anal.
Calcd for C21H24N2OS: C, H, N.
3-Decyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one
(45).
Yield: 15%. Mp: 54.4-55.5 °C. MS (EI): 408 [M]+. 13C NMR
(CDCl3): δ 14.34 (CH3), 22.49 (CH2), 26.31 (CH2), 27.47 (CH2),
28.89 (CH2), 29.09 (CH2), 29.22 (CH2), 29.57 (CH2), 31.68 (CH2),
40.85 (CH2), 71.53 (C), 126.98, 128.86, 129.12, 138.69 (Carom and
CHarom), 173.88 (CdO), 181.71 (CdS). Anal. Calcd for C25H32N2OS:
C, H, N.
5,5′-Diphenyl-3-tetradecyl-2-thioxoimidazolidin-4-one (46).
Yield: 17%. Mp: 46.5-47.7 °C. MS (EI): 464 [M]+. 13C NMR
(CDCl3): δ 13.75 (CH3), 21.90 (CH2), 25.72 (CH2), 26.82 (CH2),
27.65 (CH2), 28.24 (CH2), 28.63 (CH2), 28.85 (CH2), 28.98 (CH2),
29.22 (CH2), 29.41 (CH2), 31.16 (CH2), 31.27 (CH2), 39.09 (CH2),
70.95 (C), 126.20, 127.50, 128.60, 138.04 (Carom and CHarom),
173.37 (CdO), 181.13 (CdS). Anal. Calcd for C29H40N2OS: C,
H, N.
3-Hexadecyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (47).
Yield: 19%. Mp: 63.7-64.6 °C. MS (EI): 492 [M]+. 13C NMR
(CDCl3): δ 14.50 (CH3), 23.11 (CH2), 27.06 (CH2), 27.65 (CH2),
28.02 (CH2), 28.24 (CH2), 28.63 (CH2), 28.85 (CH2), 28.98 (CH2),
29.19 (CH2), 29.51 (CH2), 29.77 (CH2), 29.90 (CH2), 30.09 (CH2),
32.36 (CH2), 41.13 (CH2), 72.40 (C), 127.34, 127.79, 129.34, 138.40
(Carom and CHarom), 173.92 (CdO), 183.04 (CdS). Anal. Calcd for
C31H44N2OS: C, H, N.
3-Octadecyl-5,5′-diphenyl-2-thioxoimidazolidin-4-one (48).
Yield: 16%. Mp: 64.1-65.0 °C. MS (EI): 520 [M]+. 13C NMR
(CDCl3): δ 14.04 (CH3), 22.65 (CH2), 26.60 (CH2), 26.92 (CH2),
27.33 (CH2), 27.57 (CH2), 27.76 (CH2), 27.91 (CH2), 28.35 (CH2),
28.57 (CH2), 28.73 (CH2), 29.06 (CH2), 29.64 (CH2), 31.90 (CH2),

424 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Muccioli et al.
120.70, 124.13, 126.98, 128.40, 128.53, 128.73, 133.64, 136.17
(Carom and CHarom), 155.15 (CdO), 170.07 (CdO).
pH 7.6 or 9) were incubated at 37 °C for 10 min, in 200 µL final
volume. Reactions were stopped by rapidly placing the tubes in
ice and adding 400 µL of cold chloroform/methanol (1:1 v/v)
followed by vigorous mixing. Phases were separated by centrifuga-
tion 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.
HPLC Assay. The HPLC system consisted of a Perkin-Elmer
10 pump, a Perkin-Elmer LC-85B UV detector, and a Perkin-Elmer
LCI-100 integrator. The separations were performed using a 250
× 4.6 mm HPLC column from Bio-Rad (RoSil C18 HL 5 µm).
The mobile phase was MeOH (analytical grade) delivered at 1 mL/
min. UV detection was carried out at 285 nm, which is the λmax for
compound 42 under the assay conditions. Membranes, compound
42 or DMSO (10 µL), and assay buffer (H2PO4--HPO42-, 0.1%
BSA, pH 7.6) were incubated at 37 °C for 0, 10, or 40 min in 200
µL final volume. Reactions were stopped by placing the tubes in a
dry ice-2-propanol mixture. The frozen solutions were subse-
quently lyophilized. Methanol (200 µL) was added to each tube
prior to injection of a 20 µL aliquot into the HPLC.
Competition Binding Assay. The assay was performed as
previously described.30 Briefly, the competitive binding experiments
were performed using [3H]-SR141716A (1 nM) or [3H]-CP-55,940
(1nM) 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 albumine, pH 7.4). The test compounds were present at a
10 µM concentration 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 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 of shaking and 3 h of resting. Assays were
performed at least in triplicate. Final DMSO concentrations in the
assay were less than 0.1%.
1,3-Diheptyl-5,5′-diphenylimidazolidine-2,4-dione (66). Com-
pound 66 was obtained by alkylation of 5,5′-diphenylimidazolidine-
2,4-dione (7). The 5,5′-diphenylimidazolidine-2,4-dione (4 mmol)
and K2CO3 (12 mmol) were stirred in DMF (40 mL) for 30 min
prior to addition of chloroheptane (8 mmol). The resulting mixture
was stirred for 24 h and poured in water. The solution was extracted
with CHCl3, and the organic layer was washed with HCl, NaOH,
and brine. The resulting organic phase was dried over MgSO4 and
evaporated to dryness to yield a colorless oil. Yield: 16%. MS
(EI): 448 [M]+. 13C NMR (CDCl3): δ 13.75 (CH3), 13.80 (CH3),
21.79 (CH2), 25.75 (CH2), 26.32 (CH2), 27.24 (CH2), 27.29 (CH2),
27.88 (CH2), 27.97 (CH2), 30.76 (CH2), 31.01 (CH2), 38.33 (CH2),
41.26 (CH2), 73.98 (C), 127.99, 128.69, 137.16, 138.74 (Carom and
CHarom), 154.92 (CdO), 173.01 (CdO). Anal. Calcd for C29H40N2O2:
C, H, N.
General Procedure for the Synthesis of the 3-Substituted
Derivatives. To the glycine ethyl ester isothiocyanate or isocyanate
(7 mmol) dissolved in CHCl3 (25 mL) was added, over a 10 min
period, the amine (7 mmol) in 15 mL of CHCl3. After 4 h of stirring,
the solution was evaporated to dryness. The resulting residue was
refluxed for 3 h in 50 mL of a 1:1 mixture of ethanol and HCl (10
M). After cooling, the solution was evaporated to dryness and the
resulting solid crystallized from hexane.
3-Butylimidazolidine-2,4-dione (69). Analytical data are in
accordance with those reported by Ryczek.36 Yield: 74%. Mp:
97.1-98.2 °C. MS (EI): 157 [M + H]+. 13C NMR (DMSO-d6):
δ 13.39 (CH3), 19.28 (CH2), 29.56 (CH2), 37.20 (CH2), 45.74 (CH2),
157.70 (CdO), 171.97 (CdO).
3-Heptylimidazolidine-2,4-dione (70). Analytical data are in
accordance with those reported by Ryczek.36Yield: 77%. Mp:
81.1-81.9 °C. MS (EI): 198 [M]+. 13C NMR (DMSO-d6): δ 14.01
(CH3), 22.10 (CH2), 26.17 (CH2), 27.66 (CH2), 28.31 (CH2), 31.22
(CH2), 37.63 (CH2), 45.91 (CH2), 157.77 (CdO), 172.14 (CdO).
3-Phenylimidazolidine-2,4-dione (71). Analytical data are in
accordance with those reported by Ryczek.36 Yield: 67%. Mp:
158.2-159.5 °C. MS (EI): 176 [M]+. 13C NMR (DMSO-d6): δ
46.17 (CH2), 126.8, 127.76, 128.79, 132.41 (Carom and CHarom),
156.61 (CdO), 171.17 (CdO).
3-Butyl-2-thioxoimidazolidin-4-one (72). Analytical data are
in accordance with those reported by Ryczek.36 Yield: 64%. Mp:
109.8-110.7 °C. MS (EI): 172 [M]+. 13C NMR (DMSO-d6): δ
13.69 (CH3), 19.51 (CH2), 29.47 (CH2), 38.86 (CH2), 48.44 (CH2),
172.78 (CdO), 183.59 (CdS).
3-Phenyl-2-thioxoimidazolidin-4-one (73). Analytical data are
in accordance with those reported by Ryczek.36 Yield: 60%. Mp:
258.3-259.7 °C. MS (EI): 192 [M]+. 13C NMR (DMSO-d6): δ
49.27 (CH2), 128.66, 128.79, 128.92, 133.64 (Carom and CHarom),
172.26 (CdO), 183.53 (CdS).
Pharmacology. Brain Membrane Preparation. 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).
Following decapitation, brains were carefully dissected on ice. All
the manipulations were performed at 0-4 °C. Adult rat brain was
homogenized in homogenization buffer (20 mM HEPES, 1 mM
MgCl2, pH 7.0) using a potter and subsequently centrifuged for 20
min at 36 000g. The pellet was resuspended in homogenization
buffer and centrifuged again for 20 min at 36 000g. 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). The protein content of the preparation was
determined,38 and the membranes aliquots were stored at -80 °C
until use.
Data Analysis. Inhibition curves of FAAH were performed at
least three times in duplicate. IC50 values were determined by
nonlinear regression analysis performed using the GraphPad prism
4.0 program (GraphPad Software, San Diego) and expressed as pI50
(pI50 ) -log IC50). Competitive binding assays on the cannabinoid
receptors (CB1 and CB2) were performed at least three times in
duplicate. Results are expressed as mean ( SEM.
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 fellowship. This work was
partially funded by a FRSM grant (Belgian National Fund for
Scientific Research) and a FSR grant (Universite´ catholique de
Louvain). The authors wish to warmly thank Geoffray Labar
for constructive discussions during the preparation of this
manuscript. N.F. and F.C. are exchange pregraduate students
from Universita` degli Studi di Bologna.
Supporting Information Available: Characteristic IR peaks,
1H NMR and elemental analysis data, and the Michaelis-Menten.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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