An unprecedented reversible mode of action of β-lactams for the inhibition of human fatty acid amide hydrolase (hFAAH)

Article abstract of DOI:10.1016/j.ejmech.2012.11.035

A series of compound was prepared to clarify the reversible mechanism of β-lactamic hFAAH inhibitors on the one hand, and to modulate some of their physicochemical parameters on the other hand. In particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad. Reversibility studies showed that these two compounds retain the reversible mode of inhibition, suggesting a noncovalent interaction between our β-lactams and hFAAH. Finally, pharmacological evaluations of bioisosteres of the lead compound (4a, IC₅₀ = 5.3 nM) revealed that log P values and PSA could be optimized without altering the FAAH inhibition (IC₅₀ values from 3.65 nM to 70.9 nM).

Full text of DOI:10.1016/j.ejmech.2012.11.035

European Journal of Medicinal Chemistry 60 (2013) 101e111
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
An unprecedented reversible mode of action of
b
-lactams for the inhibition
of human fatty acid amide hydrolase (hFAAH)
,
,
Marion Feledziak a b, Catherine Michauxc, Didier M. Lambert b, Jacqueline Marchand-Brynaert a
*
a Laboratoire de Chimie Organique et Médicinale, Institute of Condensed Matter and Nanosciences (IMCN), Université catholique de Louvain (UCL), Bâtiment Lavoisier,
Place Louis Pasteur L4.01.02, B-1348 Louvain-La-Neuve, Belgium
b Unité de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute (LDRI), Université catholique de Louvain (UCL), Avenue E. Mounier 73.40,
B-1200 Bruxelles, Belgium
c Unité de Chimie Physique Théorique et Structurale, Facultés Universitaires Notre Dame de la Paix (FUNDP), Rue de Bruxelles 61, B-5000 Namur, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compound was prepared to clarify the reversible mechanism of b-lactamic hFAAH inhibitors
Received 20 August 2012
Received in revised form
21 November 2012
Accepted 23 November 2012
Available online 29 November 2012
on the one hand, and to modulate some of their physicochemical parameters on the other hand. In
particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the
crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad.
Reversibility studies showed that these two compounds retain the reversible mode of inhibition, sug-
gesting a noncovalent interaction between our b-lactams and hFAAH. Finally, pharmacological evalua-
tions of bioisosteres of the lead compound (4a, IC50 ¼ 5.3 nM) revealed that log P values and PSA could be
optimized without altering the FAAH inhibition (IC50 values from 3.65 nM to 70.9 nM).
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
b
-Lactam
Azetidinone
Reversible inhibition
Noncovalent inhibition
Fatty acid amide hydrolase
Serine hydrolase
Endocannabinoid system
Anandamide
1. Introduction
in vitro and/or in vivo assays confirmed their potential therapeutic
interest. FAAH inhibitors were demonstrated to increase the level of
Over the last ten years, the selective inhibition of FAAH is one of
the most extensive research fields in the world of endocannabinoids
[1e6]. Different kinds of electrophilic functions have been broadly
anandamide, an endogenous ligand of cannabinoid receptors CB1
and CB2, by blocking its FAAH-catalyzed degradation [8,10e13].
Rising the amount of anandamide leads to prolonged beneficial
physiological effects due to activation of the cannabinoid receptors.
In contrast, it was demonstrated that the cannabinoid receptors
studied in view of designing pharmacophores such as a-keto-oxa-
zole (1, Chart 1) [7], carbamate (2, Chart 1) [8] and urea (3, Chart 1)
[9]. Structureeactivity relationship (SAR) studies performed on
a large number of published lead compounds allowed improve-
ments in activity and selectivity of FAAH inhibitors. Moreover,
activation by exogenous ligand, e.g. cannabinoids like
D
9-THC
(tetrahydrocannabinol) or synthetic agonists, maximizes the side
effects arising from central nervous system disturbance [14,15]. This
is why, taking advantage of the endocannabinoid system, FAAH
inhibitors are presently in development for treating inflammation
[16,17] and pain [18e20], sleep disorders [21] and CNS diseases [22].
Recently, some clinical trials were achieved with the best repre-
sentatives of some families, notably PF-04457845 (3b) [23]. This
compound is involved in a phase II study aimed to collect pharma-
codynamic, pharmacokinetic and toxicity data, on the one hand and
to evaluate its analgesic efficiency in knee osteoarthritis and its
effect on sleep, on the other hand.
Abbreviations: SAR, structureeactivity relationship; LC, liquid chromatography;
MS, mass spectrometry; hFAAH, human fatty acid amide hydrolase; hMAGL, human
monoacylglycerol lipase; CB1, cannabinoid receptor subtype-1; CB2, cannabinoid
receptor subtype-2; AEA, anandamide; 2-AG, 2-arachidonoylglycerol; 2-OG, 2-
oleoylglycerol; CNS, central nervous system; PSA, polar surface area; TBDMS, tert-
butyldimethylsilyl; THF, tetrahydrofuran; DCM, dichloromethane; DMF, dime-
thylformamide; ACN, acetonitrile; DMSO, dimethyl sulfoxide; DCC, dicyclohex-
ylcarbodiimide; DMAP, dimethylaminopyridine; HMDS, hexamethyldisilazane.
* Corresponding author. Tel.: þ32 10 47 27 40; fax: þ32 10 47 41 68.
Our recent results prompt us to continue investigations about
b-
lactamic derivatives for FAAH inhibition. Indeed, we have described
E-mail address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.035

102
M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
Chart 1. Most studied FAAH inhibitors (Covalent inhibitors).
a new class of inhibitors based on the azetidin-2-one (i.e.
b
-lactam)
may cause several effects considering lipophilicity, conformational
arrangement and the potential occurrence of hydrogen bonds. In
addition, they also may change the chemical reactivity and there-
fore the mode of inhibition. Indeed, compound 4b for instance,
presents a potential leaving group on the exocyclic carbonyl. In the
case of a nucleophilic attack of the active serine onto the exocyclic
carbonyl, this feature could drive to the formation of a stable acyl-
enzyme intermediate, by releasing the allylic alcohol moiety. Thus,
we will be able to conclude whether our inhibitors are covalently
bound to FAAH via the exocyclic carbonyl e in the case of an irre-
versible inhibition e or not e in the case of a still reversible inhi-
bition. To further develop this idea of possibly transforming
a reversible inhibitor into an irreversible one, an additional
compound was designed (4e, Chart 2) which presents a better
leaving group than 4b and can be easily prepared from a commer-
cial reagent (namely propyl chlorothioformate).
template and disclosed the lead compound 4a (i.e. 1-(pent-4-
enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-azetidin-2-one,
Chart 2) which exhibits a nanomolar activity, and an unusual
competitive, fully reversible mode of action [24]. From SAR studies
and LC/HRMS analyses, we could establish that the exocyclic
carbonyl of the imide function is more essential for the inhibition
than the
reversibility of inhibition results from the processing of our inhib-
itors by FAAH [25]. Thus, -lactamic inhibitors such as 4a are not
b-lactam carbonyl, and we could exclude that the
b
slow substrates of FAAH; they are remaining unchanged in the test
solutions. To further explore the reversible mode of inhibition, we
designed new compounds based on bioisosteric modifications (4be
4e, Chart 2). With the aim of modulating the log P and polar surface
area (PSA) values, two parameters related to solubility and
permeability respectively, we synthesized
a small library of
analogues of 4a. For that purpose, heteroatoms were inserted in our
lead structure 4a (Chart 2). Because of the great importance of the
exocyclic carbonyl of the imide function, carbamate (4b), urea (4c)
and thiourea (4d) functions were investigated. These modifications
Finally, to complete the SAR study, the ester side-chain was also
modified by introducing heteroatoms at various positions (5e7,
Chart 3).
The effects of all these structural modifications on FAAH inhi-
bition were evaluated in vitro, in a competitive hydrolytic assay on
human FAAH, and in reversibility assays to evaluate not only the
activity but also the mode of inhibition. The selectivity for FAAH
versus MAGL was also examined.
2. Results
2.1. Chemistry
Compounds synthesis started with precursor 8, described in our
previous publication, which is obtained in two steps from
commercially available (3R,4R)-4-acetoxy-3-[(R)-1-(tert-butyldi-
methylsilyloxy)ethyl]azetidin-2-one. We established a three-step
sequence to prepare our library: i) N-functionalization, ii) depro-
tection of silyl ether and iii) O-functionalization. Compounds 9aee
Chart 2. Bioisosteric structures of the imide function.
Chart 3. Bioisosteric structures of the ester chain.

M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
103
were obtained by a N-acylation step adapted to the nature of each
chain (Scheme 1). Compound 9a was prepared using 4-pentenoyl
chloride in the presence of pyridine in refluxing DCM (80%) while
9bee were obtained by reaction with allyl chloroformate (9b, 82%),
allyl isocyanate (9c, 57%), allyl isothiocyanate (9d, 79%) and propyl
chlorothioformate (9e, 49%), after deprotonation of 8 in the pres-
ence of LiHMDS in THF at ꢀ78 ꢁC. Silyl ether protection was
removed in acidic conditions leading to compounds 10aee in
moderate to good yields (45e89%, Scheme 1).
The resulting alcohols were engaged in the last step to give the
final compounds (Scheme 2). On the one hand, each alcohol (10aee)
was esterified by one or two usual methods of esterification: i) 4b
(63%), 4c (88%), 4d (89%) and 4e (76%) were synthesized in the pres-
ence of pyridine with 4-phenylbutanoyl chloride at room tempera-
ture; ii) 5aed and 7awere obtained by using 2-(benzyloxy)acetic acid
or 4-(pyridin-4-yl)butanoic acid in the presence of dicyclohex-
ylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) in good
yields (5a (89%), 5b (58%), 5c (93%), 5d (74%), 7a (64%), Scheme 2). On
the other hand, compounds 10aed were functionalized with phe-
nethyl isocyanate using very mild conditions. Indeed, the conversion
into the corresponding alkoxide was not possible and any basic
conditionscould not beused because of the acidityof H-3proton. This
property drove to a rearrangement which gave in fine compound 11
(Scheme 3) by an elimination step followed by azetidinone ring
opening. Thus, the carbamate bond was created in the presence of
Ti(OtBu)4 as a Lewis acid which catalyzed the nucleophilic attack of
the hydroxyl function upon the isocyanate carbonyl function [26]. By
this method, compounds were obtained with good yields, in 1 h at
room temperature (6a (81%), 6b (64%), 6c (70%), 6d (84%), Scheme 2).
Scheme 2. Reagents and conditions: (a) 4-phenylbutanoyl chloride, pyridine, DCM, rt,
15 h; (b) 2-(benzyloxy)acetic acid, DCC, DMAP, DCM, rt, 15 h; (c) phenethyl isocyanate,
Ti(OtBu)4, DCM, rt, 1 h, (d) 4-(pyridin-4-yl)butanoic acid, DCC, DMAP, DCM, rt, 15 h.
All the novel b-lactam derivatives were characterized by the usual
spectroscopies; representative 1H and 13C NMR spectra are provided
in the Supporting information. Compounds 4aee, 5aed, 6aed and 7a
are soluble in dichloromethane, tetrahydrofuran, ethyl acetate,
acetonitrile, chloroform, acetone, methanol, dimethylformamide and
dimethyl sulfoxide (DMSO). They are soluble in aqueous media (Trise
EDTA or phosphate buffers, pH 7.2e7.4) with 5% DMSO.
2.2.1. hFAAH inhibition
Human recombinant enzyme, developed in our laboratory [27],
was used in a competitive hydrolytic assay using [3H]-AEA as
substrate. Tested compounds, enzyme and [3H]-AEA were incu-
bated at 37 ꢁC during 10 min. The extent of inhibition was evaluated
by liquid scintillation counting of [3H]-ethanolamine resulting from
the hydrolysis of labelled AEA. Considering the results summarized
in Table 1, it appeared clearly that the insertion of a sulfur atom
(thiocarbonyl) replacing the exocyclic oxygen atom of the imide
function, leads to a loss of activity compared to the corresponding
bioisosteres (4d compared to 4aec, 5d to 5aec and 6d to 6aec,
IC50 ¼ 145.8, 1042 and 959 nM, respectively). This structural
moiety suspected to interact with the active serine, is thus less
active as a thiocarbonyl function. Comparatively to our previous hit
(4a, IC50 ¼ 5.32 nM, Table 1 and Chart 2), the other bioisosteres
were also active in the nanomolar range. They appeared to be good
to excellent inhibitors of FAAH, from 70.9 nM (5c) to 3.65 nM (4b),
except 7a, which exhibited a moderate activity (IC50 ¼ 310.1 nM).
Insertion of heteroatoms results in fluctuations of the log P
values: increased ones with sulfur atoms (notably 4d and 4e, 3.47
2.2. Pharmacology
The library of compounds was tested for FAAH inhibition using
human recombinant FAAH as source of enzyme. Some of them were
also evaluated as potential MAGL inhibitors to check the selectivity
and to study the effect of the exocyclic C]S function of compounds
4e6d. Indeed, MAGL is a serine hydrolase involving three cysteine
residues in its active site. According to the HSAB (hard and soft
acids and bases) theory, a privileged interaction could occur
between the C]S (electrophile) and SH (nucleophile) functions. In
addition, the inhibition mode was investigated in the case of two
compounds (4b and 4e, Chart 2) which present a potential good
leaving group. All the collected results were analyzed taking the
relative log P and PSA calculated values in consideration (Table 1).
Scheme 1. Reagents and conditions: (a) 4-pentenoyl chloride, pyridine, DCM, 45 ꢁC, 24 h or allyl chloroformate, allyl isocyanate, allyl isothiocyanate or propyl chlorothioformate,
LiHMDS, THF, ꢀ78 ꢁC, 4 h; (b) HCl, AcOH, ACN, 0 ꢁC, 3 h.

104
M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
Scheme 3. Proposed mechanism for the formation of 11 (see Supporting information).
and 4.11, respectively) and equalled (4b, 3.31) or lowered ones with
nitrogen or oxygen atoms (from 2.65 to 1.41, 6d and 5c, respec-
tively). We could not correlate inhibitory potency and log P values
but we could observe that fluctuations in the lipophilicity of some
compounds did not really alter their activity compared to 4a: for
instance, 4c presents a similar activity with a lower log P value
(IC50 ¼ 5.32 and 5.56 nM, and log P ¼ 3.25 and 2.59, respectively);
5aeb and 6aec differ by a log P decrease of one logarithmic unit at
least and retain excellent activities (e.g. 6c, IC50 ¼ 28.6 nM and
log P ¼ 1.76) and finally, compound 5c displays the lowest log P
value and a good activity (IC50 ¼ 70.9 nM and log P ¼ 1.41).
Inversely to log P values, PSAs tend to increase with heteroatoms
insertion. But, alike log P fluctuations, no direct correlation could be
done. We observed good to excellent activities for different
modulations of PSA.
2.2.3. Mode of inhibition
To address the reversibility question, we selected two inhibitors
featuring good leaving-groups near the exocyclic carbonyl (4b and
4e, Chart 2). Wash-out experiments were undertaken to measure
the recovery of enzyme activity after a rapid and large dilution of
the inhibitoreenzyme mixtures. Results were collected after 0, 30
and 90 min.
The enzyme activity should be almost totally recovered if the
inhibitor is reversible, but the enzyme should remain largely
inhibited in the case of an irreversible inhibition. Here, the rapid
and large dilution led to the recovery of activity for 4b and 4e,
similarly to 1c (CAY-10402, Chart 1) [30]. As a further control we
used two known irreversible FAAH inhibitors, 2 [31] (URB-597,
Chart 1) and 3a (PF-750, Chart 1) [32]; we found that the enzyme
activity is still largely inhibited after the dilution experiment
(Fig. 1).
2.2.2. hMAGL inhibition
Human recombinant enzyme, developed in our laboratory [29],
was used in a competitive hydrolytic assay using [3H]-2-OG as
substrate. Tested compounds, enzyme and [3H]-2-OG were pre-
incubated at room temperature during 30 min and incubated at
37 ꢁC during 10 min. The extent of inhibition was evaluated by
liquid scintillation counting of [3H]-glycerol resulting from the
hydrolysis of labelled 2-OG. All the tested compounds presented
a very low activity, those with a thiocarbonyl function (4e6d) and
2.2.4. Docking studies
Recent publications have shown the different binding modes of
inhibitors into the FAAH active site thanks to the X-ray diffraction
analysis of co-crystals obtained from an engineered humanized rat
FAAH (h/rFAAH). Molecules 2 (URB-597, Chart 1) [33] and 3a (PF-
750, Chart 1) [12] are trapped as stable acyl-enzyme intermediates
after expelling their respective leaving-group. Molecule 1a (OL-135,
Chart 1) is quenched as the tetrahedral intermediate [19,34,35].
Interestingly, noncovalent and covalent states of the same inhibitor
1b (Chart 1) into h/rFAAH could be observed: the catalytic residues
Ser241, Ser217 and Lys142 occupy essentially identical positions in
both complexes [36]. A noncovalent complex between the revers-
ible inhibitor 12 (Chart 4) and rFAAH was also disclosed [37,38].
The hFAAH enzyme we used is not yet crystallized. In order to
bring out a potential binding mode of the present inhibitors in
the other ones (4b, 4c and 5c). IC50 values ranged from 10.3 mM (4b)
to 121.7 mM (5d) and compounds 4d and 6d inhibited 60% and 25%
of MAGL at 10ꢀ3 M, respectively. Comparatively to the lead
compound (4a), there is a high selectivity for the inhibition of
FAAH: for instance, compounds 4b and 4c are respectively 2800
and 18,000 times more potent against FAAH.
Table 1
Determination of inhibitory potential on hFAAH and hMAGL, log
parameters.
P and PSA
150
0 min
Compound log Pa PSAa,b
pI50
IC50 on
hFAAH
IC50c on
hMAGL
c
30 min
(hFAAH)
4a
4b
4c
4d
4e
5a
5b
5c
5d
6a
6b
6c
6d
7a
3.25
3.31
2.59
3.47
4.11
2.08
2.14
1.41
2.30
2.43
2.49
1.76
2.65
2.03
76.57 ꢀ8.27 ꢂ 0.05
72.91 ꢀ8.44 ꢂ 0.03
75.71 ꢀ8.26 ꢂ 0.03
90.73 ꢀ6.84 ꢂ 0.03
88.98 ꢀ7.92 ꢂ 0.07
72.91 ꢀ7.51 ꢂ 0.02
82.14 ꢀ7.52 ꢂ 0.06
84.94 ꢀ7.15 ꢂ 0.02
5.32 4060
3.65 10,300
5.56 98,410
90 min
100
145.8
12.1
30.9
30.1
70.9
60% inh at 10ꢀ3 M
18,960
nd
nd
50
56,850
99.96 ꢀ5.98 ꢂ 0.02 1042
121,700
nd
75.71 ꢀ7.48 ꢂ 0.03
84.94 ꢀ7.92 ꢂ 0.02
87.74 ꢀ7.54 ꢂ 0.02
102.76 ꢀ6.02 ꢂ 0.05
76.57 ꢀ6.51 ꢂ 0.02
33.2
12.1
28.6
959
310.1
nd
nd
25% inh at 10ꢀ3 M
nd
0
4e
4b
a
PF750
Calculated via Marvinsketch.
URB597
b
Calculated by the atom-based method (topological PSA or TPSA) of Ertl, Rohde
CAY10402
and Seltzer [28]. It consists in the summation of tabulated values corresponding to
commonly used polar fragments (N and O) and slightly less one (S).
Fig. 1. Test of reversibility: influence of a rapid and large dilution on the recovery of
hFAAH activity (studies after 0, 30 and 90 min following the rapid and large dilution).
c
In nM, from three independent experiments.

M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
105
a high affinity noncovalent complex between the enzyme and the
-lactamic inhibitor (step 1), eventually leading to a covalent
b
tetrahedral intermediate by nucleophilic attack of the active serine
Ser241 on the C]X bond of the inhibitor (step 2). In the absence of
good leaving-group on this intermediate (Y is CH2), the step 2 could
be reversible. Such modes of action (i.e. steps 1 and 2) have been
recently demonstrated with co-crystal structures: reversible cova-
Chart 4. Noncovalent inhibitors of FAAH.
lent interaction with OL-135 (1a) and other
a-keto-oxazoles
[19,34,35] and reversible noncovalent interaction with ketobenzi-
midazole 12 (Chart 4) [37,38]. We speculated that the presence of
a potential leaving group on the tetrahedral intermediate (Y is
a heteroatom), could draw the reaction towards the acyl-enzyme
intermediate (step 3). Depending on the stability of this interme-
diate versus hydrolysis, the resulting inhibition could be irrevers-
ible or slowly reversible, when the inhibitor behaves as a bad
substrate.
Irreversible inhibition of hFAAH, due to the formation of a stable
acyl-enzyme intermediate, is the recognized mode of action of
URB-597 (2, Chart 1) [33,49], PF-04457845 (3b, Chart 1) [12,32,50]
and related carbamates and ureas. Independently of the presence of
hFAAH, docking studies were performed in the catalytic site of the
enzyme, inspired by the h/rFAAH crystallographic data [36]. This
active pocket is formed by a hydrophobic tunnel, called the acyl
chain binding channel (ACB), leading from the membrane-bound
surface to the hydrophilic catalytic triad (Ser241, Ser217, and
Lys142). A second tunnel, the cytoplasmic access channel (CA), is
exposed to the solvent. A third channel composed of three
phenylalanine residues (Phe388, Phe381, and Phe192), called the
“phenyl pocket”, lies close to the ACB channel. We docked the
compounds into the crystal structure of the humanized rat FAAH as
the Michaelis complexes. The emerging binding mode, pictured
here for 4b, 4c and 4e, is the same as the one previously described
for 4a (Fig. 3) [24]. The phenyl chain lies in the “phenyl pocket” and
interacts with the three phenylalanines Phe192, Phe381, and
Phe388. The catalytic serine Ser241 is close to the lactam and imide
carbonyls. The alkene chain lies at the beginning of ACB channel.
Hydrogen bonds are observed between Ser241 and the exocyclic
CO, between Thr236 and the lactam CO, between the ester CO and
the NH group of Val270 and Cys269.
a potential leaving group in their structures, our b-lactamic inhib-
itors act systematically as reversible inhibitors. Our results strongly
suggest a similar mode of action as ketobenzimidazole 12 (Chart 4)
[38] or benzothiazole 13 (Chart 4) [51], i.e. an inhibition due only to
the high affinity of our b-lactams with aminoacid residues of the
active site (step 1). By docking experiment we can propose
a possible binding mode where the enzymeeinhibitor Michaelis
complex is stabilized by hydrogen bondings of the exocyclic and b-
lactam carbonyls with, respectively, the Ser241 and Thr236 resi-
dues of the active site, on the one hand and by hydrophobic
interactions of the 4-phenyl-butanoyl chain in the “phenyl pocket”,
on the other hand (Fig. 3). As a matter of fact, we observed
a decrease of activity when the phenyl ring of this ester chain is
replaced by a pyridine ring (7a). This observation suggests a loss of
affinity in the hydrophobic cavity where pyridine, probably
protonated, does not stabilize the interaction. All the tested
compounds were highly selective for the hFAAH inhibition versus
hMAGL. The hypothesis of a favourable interaction between the
3. Discussion and conclusion
The competitive, reversible inhibition of our inhibitors appears
unique in the
Whereas -lactams are widely known to inhibit traditional serine
hydrolases (i.e. serine enzymes featuring the Ser-Asp-His catalytic
triad) [39] in an irreversible manner (DD-peptidases [40], -lacta-
b-lactam literature, to the best of our knowledge.
b
b
mases [41,42], elastases [43e47]), a reversible mechanism occurs
between our compounds and hFAAH of which the catalytic pocket
involves the unusual Ser-Ser-Lys catalytic triad [48]. We have firmly
thiocarbonyl group of b-lactams 4d, 5d and 6d and cysteine resi-
dues of the hMAGL catalytic pocket, was not verified.
Finally, we could not correlate log P values, PSAs and inhibition
potency. Modification of these physicochemical parameters did not
seem to influence greatly the inhibition of FAAH. However, we
showed that it is possible to improve physicochemical parameters,
i.e. solubility and permeability factors, without altering the inhibi-
tion of hFAAH. Indeed, the best representatives of this study
conserved a nanomolar activity against hFAAH with lower log P
value and PSA. This information is important for candidates selec-
established that our b-lactams are not processed by the enzyme
[25] and that, after rapid and large dilution of enzymeeinhibitor
mixtures, the enzyme recovers its full activity.
From our previous studies, we know that the key moiety for
interaction with hFAAH is the exocyclic carbonyl group of the imide
function [25]. As a matter of fact, the replacement of this carbonyl
by a thiocarbonyl led to an important decrease of the inhibition
activity (compounds 4d, 5d and 6d). As shown in Fig. 2, the
reversible inhibition of hFAAH could result from the formation of
tioninviewof invivo experiments withb-lactamic hFAAH inhibitors.
Fig. 2. Possible mechanisms of inhibition (X ¼ O, S; Y ¼ CH2, NH, O, S).

106
M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
4.2. General procedure for coupling chloro(thio)formate with amide
function (9b and 9e)
To a stirred solution of azetidinone (1 equiv) 8 in dry THF (7 mL/
mmol) cooled at ꢀ78 ꢁC, was dropwise added a solution of LiHMDS
(1.1 equiv) in dry THF (1 M), under argon atmosphere. The mixture
was stirred for 30 min at ꢀ78 ꢁC and the chloro(thio)formate
(1.1 equiv) was added. After 1 h at ꢀ78 ꢁC, the solutionwas allowed to
warm up to r.t. and additionally stirred for 1 h. The reaction was
quenched, at low temperature, with brine and diluted with DCM. The
aqueous layers were extracted with DCM three times. The organic
layers were combined, dried over MgSO4, filtered and concentrated
under vacuum. After purification by flash chromatography (cyclo-
hexane/AcOEt), colourless oils were obtained (9b and 9e).
4.2.1. 1-(Propyl-3-enoxy)carbonyl-(3S)-3-[(1R)-(tert-
butyldimethylsilyloxy)ethyl]azetidin-2-one (9b)
Purification by flash chromatography (cyclohexane/AcOEt 5:2)
gave 9b (112 mg, 82%) as a colourless oil: Rf ¼ 0.54 (cyclohexane/
AcOEt, 5:3); 1H NMR (500 MHz, CDCl3):
0.81 (s, 9H), 1.15 (d, J ¼ 6.3 Hz, 3H), 3.20 (m, 1H), 3.55 (m, 1H), 3.70
(dd, J ¼ 3.5 Hz, J ¼ 6.5 Hz, 1H), 4.26 (m, 1H), 4.60e4.76 (m, 2H),
5.20e5.38 (m, 2H), 5.84e5.96 ppm (m, 1H); 13C NMR (125 MHz,
d
¼ 0.02 (s, 3H), 0.04 (s, 3H),
CDCl3):
d
¼ ꢀ5.1, ꢀ4.2, 14.2, 17.9, 22.3, 25.7, 39.7, 57.2, 64.8, 66.9,
119.1, 131.5, 149.1, 165.8 ppm; IR:
n
¼ 2852e2976, 1805, 1730, 1375,
Fig. 3. Proposed binding mode of 4b (light blue), 4c (white) and 4e (magenta) in the
active site of the humanized rat FAAH. H atoms were removed for clarity. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
1325,1277, 1259, 839 cmꢀ1; MS (ESI): m/z (%): 314.09 (16) [M þ H]þ,
336.13 (41) [M þ Na]þ, 648.93 (100) [2M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C15H28NO4Si: 314.17821, found: 314.17859.
4. Experimental section
4.2.2. 1-(Propyl-3-sulfanyl)carbonyl-(3S)-3-[(1R)-(tert-
butyldimethylsilyloxy)ethyl]azetidin-2-one (9e)
4.1. Chemistry
Purification by flash chromatography (cyclohexane/AcOEt 5:1)
gave 9e (71 mg, 49%) as a colourless oil: Rf ¼ 0.63 (cyclohexane/
All solvents, including anhydrous solvents, and reagents were
purchased from Acros Organics, Alfa Aesar, Cayman chemical, Fluka,
SigmaeAldrich or VWR, and used without purifications. [3H]-AEA
(60 Ci/mmol) was purchased from American Radiolabeled Chemical
(St Louis, MO). UltimaGoldscintillation liquid wasboughtfromPerkin
Elmer. All reactions under dry conditions were performed under
argon atmosphere in flame-dried glassware. Nuclear Magnetic
Resonance (1H NMR and 13C NMR) spectra were recorded at 300 MHz
for protonand 75MHz forcarbon (Bruker Avance300)or500 MHzfor
protonand 125MHz forcarbon(BrukerAvance500)using deuterated
chloroform. Chemical shifts are reportedinppm relativetothe signals
of residual non-deuterated solvent (CDCl3 7.26 and 77.16 ppm). NMR
coupling constants (J) are reported in hertz. Melting points (mp) were
determined on a Büchi B-540 apparatus calibrated with caffeine,
vanillin and phenacetin. Rotations were recorded on Atago Ap-100
polarimeter, at 25 ꢁC, in CHCl3. Concentrations are given in
percentage (g/100 mL). Low resolution mass spectra were acquired
using a Thermo Finnigan LCQ spectrometer in negative mode of
electrospray ionisation (ESI). High Resolution Mass Spectrometry
(HRMS) analyses were performed using a QExactive (Thermo Scien-
tific) spectrometer. Infrared (IR) spectra were recorded using FTIR-
8400S Shimadzu apparatus. Products were analyzed as thin films
deposited on a SeeZn crystal by evaporation of CH2Cl2 solutions. TLC
analysis was performed on Merck silica gel 60F254 with detection
under UVlight, and flash chromatography wasperformed on silicagel
(40e60 mesh) purchased from Rocc (Belgium). Purity of tested
compounds was assessed by HPLC on chiralpak IA column
AcOEt, 5:2); 1H NMR (500 MHz, CDCl3):
d
¼ 0.04 (s, 3H), 0.05 (s, 3H),
0.82 (s, 9H), 0.97 (t, J ¼ 7.4 Hz, 3H),1.17 (d, J ¼ 6.3 Hz, 3H),1.65 (m, 2H),
2.85e2.99 (m, 2H), 3.23 (m, 1H), 3.61 (m, 1H), 3.75 (dd, J ¼ 3.5 Hz,
J ¼ 6.8 Hz, 1H), 4.26e4.33 ppm (m, 1H); 13C NMR (125 MHz, CDCl3):
d
¼ ꢀ5.1, ꢀ4.2, 13.3, 17.9, 22.3, 23.0, 25.7, 31.1, 40.0, 56.7, 64.7, 165.5,
165.7 ppm; IR:
n
¼ 2856e2949, 1786, 1668, 1306, 1252, 1136, 1076,
1018, 839 cmꢀ1; MS (ESI): m/z (%): 332.17 (60) [M þ H]þ, 354.15 (89)
[M þ Na]þ, 685.32 (100) [2M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd
for C15H30NO3SSi: 332.17102, found: 332.17127.
4.3. General procedure for coupling iso(thio)cyanate with amide
function (9c and 9d)
To a stirred solution of azetidinone (1 equiv) 8 in dry THF (7 mL/
mmol) cooled at ꢀ78 ꢁC, was dropwise added a solution of LiHMDS
(1.1 equiv) in dry THF (1 M), under argon atmosphere. The mixture
was stirred for 30 min at ꢀ78 ꢁC and the iso(thio)cyanate (1.1 equiv)
was added. After 1 h at ꢀ78 ꢁC, the solution was allowed to warm up
to r.t. and additionally stirred for 1 h. The reaction was quenched, at
low temperature, with brine and diluted with DCM. The aqueous
layers wereextracted with DCM three times. The organic layers were
combined, dried over MgSO4, filtered and concentrated under
vacuum. After purification by flash chromatography (cyclohexane/
AcOEt), colourless oils were obtained (9c and 9d).
4.3.1. 1-(Propyl-3-enamino)carbonyl-(3S)-3-[(1R)-(tert-
butyldimethylsilyloxy)ethyl]azetidin-2-one (9c)
(4.6 mm ꢃ 250 mm, 5
eluant (95:5), at a flow rate of 1.0 mL/min and on symmetry C18
m particle size) using a gradient of
acetonitrile/H2O eluant (50:50 to 100:0), at a flow rate of 1.2 mL/min
(purity ꢄ 95%).
m
m particle size) using hexane/isopropanol
Purification by flash chromatography (DCM/AcOEt 98:2) gave 9c
(78 mg, 57%) as a colourless oil: Rf ¼ 0.42 (cyclohexane/AcOEt, 5:3);
column (4.6 mm ꢃ 250 mm, 5
m
1H NMR (500 MHz, CDCl3):
d
¼ ꢀ0.04 (s, 3H), ꢀ0.02 (s, 3H), 0.75 (s,
9H), 1.10 (d, J ¼ 6.3 Hz, 3H), 3.17 (m, 1H), 3.51 (m, 1H), 3.63 (dd,
J ¼ 3.1 Hz, J ¼ 6.3 Hz, 1H), 3.74e3.80 (m, 1H, AB system), 3.82e3.89

M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
107
(m, 1H, AB system), 4.20 (m, 1H), 5.00e5.13 (m, 2H), 5.74 (m, 1H),
6.52 ppm (br t, J ¼ 5.7 Hz, 1H); 13C NMR (125 MHz, CDCl3):
2H), 5.89 (m, 1H), 8.43 ppm (br s, 1H); 13C NMR (125 MHz, CDCl3):
d
¼ 21.7, 43.1, 47.2, 54.5, 64.8, 117.9, 132.1, 166.1, 178.2 ppm; IR:
d
¼ ꢀ5.3, ꢀ4.3, 17.3, 22.1, 25.5, 38.9, 41.9, 56.5, 64.5, 116.0, 133.9,
n
¼ 2934, 1749, 1539, 1340, 1238, 1134 cmꢀ1; MS (ESI): m/z (%):
150.4, 168.2 ppm; IR:
n
¼ 2856e2957, 1765, 1699, 1531, 1464, 1337,
215.09 (25) [M þ H]þ, 237.07 (100) [M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C9H15N2O2S: 215.08487, found: 215.08501.
1259, 1140, 1076, 839 cmꢀ1; MS (ESI): m/z (%): 313.19 (100)
[M þ H]þ, 625.38 (38) [2M þ H]þ; HRMS-ESI: m/z [M þ H]þ calcd for
C15H29N2O3Si: 313.19420, found: 313.19431.
4.4.4. 1-(Propyl-3-sulfanyl)carbonyl-(3S)-3-[(1R)-hydroxyethyl]
azetidin-2-one (10e)
4.3.2. 1-(Propyl-3-enamino)thiocarbonyl-(3S)-3-[(1R)-(tert-
butyldimethylsilyloxy)ethyl]-azetidin-2-one (9d)
Purification by flash chromatography (AcOEt 100%) gave 10e
(21 mg, 45%) as a colourless oil: Rf ¼ 0.21 (cyclohexane/AcOEt 5:3); 1H
Purification by flash chromatography (cyclohexane/AcOEt 5:1)
gave 9d (113 mg, 79%) as a colourless oil: Rf ¼ 0.69 (chex/AcOEt, 5:3);
NMR (500 MHz, CDCl3):
d
¼ 0.98 (t, J ¼ 7.4 Hz, 3H),1.29 (d, J ¼ 6.4 Hz,
3H),1.66 (m, 2H), 2.02 (br s,1H), 2.94 (m, 2H), 3.30 (m,1H), 3.68e3.73
1H NMR (500 MHz, CDCl3):
1.17 (d, J ¼ 6.3 Hz, 3H), 3.17 (m,1H), 3.72 (dd, J ¼ 6.9 Hz, J ¼ 6.0 Hz 1H),
3.84 (dd, J ¼ 3.2 Hz, J ¼ 6.9 Hz,1H), 4.13e4.21 (m,1H), 4.23e4.34 (m,
2H), 5.13e5.24 (m, 2H), 5.83 (m, 1H), 8.41 ppm (br s, 1H); 13C NMR
d
¼ 0.01 (s, 3H), 0.03 (s, 3H), 0.79 (s, 9H),
(m, 2H), 4.26 ppm (m, 1H); 13C NMR (125 MHz, CDCl3):
d
¼ 13.4, 21.7,
23.0, 31.2, 40.9, 56.2, 64.8,165.2, 165.8 ppm; IR:
n
¼ 2959, 1780,1664,
1306,1238,1130cmꢀ1; MS(ESI):m/z (%):218.08(12)[Mþ H]þ, 240.06
(100) [M þ Na]þ, 457.14 (75) [2M þ Na]þ; HRMS-ESI: m/z [M þ H]þ
calcd for C9H16NO3S: 218.08454, found: 218.08466.
(125MHz, CDCl3):
117.4, 132.2, 166.5, 178.3 ppm; IR:
d
¼ꢀ5.2, ꢀ4.1,17.8, 22.2, 25.6, 42.3, 46.9, 55.1, 64.7,
n
¼ 2858e2953, 1755, 1533, 1342,
1323, 1254, 1140, 1074, 1020, 839 cmꢀ1; MS (ESI): m/z (%): 329.17
(100) [M þ H]þ, 351.15 (80) [M þ Na]þ; HRMS-ESI: m/z [M þ H]þ
calcd for C15H29N2O2SSi: 329.17135, found: 329.17153.
4.5. General procedure for esterification with acyl chloride (4bee)
To a stirred solution of alcohol precursor (1 equiv) in dry
dichloromethane (20 mL/mmol), at 20 ꢁC, were added pyridine
(2 equiv) and 4-phenylbutanoyl chloride (2 equiv) under argon
atmosphere. After stirring overnight, the mixture was diluted in
DCM and the excess of acyl chloride was quenched by 10% aqueous
Na2CO3. The organic layer was washed with 3 N HCl and brine,
dried over MgSO4, filtered and concentrated under vacuum. After
purification by flash chromatography (DCM/EtOAc), a colourless oil
was obtained in all cases.
4.4. General procedure for silyl ether deprotection (10aee)
To a stirred suspension of silyl ether (1 equiv) in acetonitrile
(30 mL/mmol) at ꢀ5 ꢁC was added dropwise 12 N HCl (5 equiv) and
17 N AcOH (7 equiv). The mixture was stirred for 30 min at ꢀ5 ꢁC,
and for 3 h at 0 ꢁC. Acetonitrile was removed under vacuum, and
the oily residue was diluted in ethyl acetate. The organic layer was
washed with 10% NaHCO3 and brine, dried over MgSO4, filtered and
concentrated under vacuum. After purification by flash chroma-
tography (ethyl acetate 100%) a white solid (10c) or colourless oils
(10b, dee) were obtained.
4.5.1. 1-(Propyl-3-enoxy)carbonyl-(3S)-3-[(1R)-(4-
phenylbutanoyloxy)ethyl]azetidin-2-one (4b)
Purification by flash chromatography (DCM/AcOEt 98:2) gave 4b
(44 mg, 63%) as a colourless oil: [
a
]
¼ ꢀ0.32 (c ¼ 0.60); Rf ¼ 0.43
D
4.4.1. 1-(Propyl-3-enoxy)carbonyl-(3S)-3-[(1R)-hydroxyethyl]
azetidin-2-one (10b)
(DCM/AcOEt 95:5); 1H NMR (300 MHz, CDCl3):
d
¼ 1.34 (d,
J ¼ 6.4 Hz, 3H), 1.92 (m, 2H), 2.31 (m, 2H), 2.62 (m, 2H), 3.40 (m, 1H),
3.59 (dd, J ¼ 3.6 Hz, J ¼ 7.0 Hz, 1H), 3.70 (m, 1H), 4.69 (d, J ¼ 5.7 Hz,
2H), 5.21e5.42 (m, 3H), 5.92 (m, 1H), 7.12e7.32 ppm (m, 5H); 13C
Purification by flash chromatography (AcOEt 100%) gave 10b
(241 mg, 76%) as a colourless oil: Rf ¼ 0.57 (AcOEt 100%); 1H NMR
(500 MHz, CDCl3):
d
¼ 1.24 (d, J ¼ 6.4 Hz, 3H), 2.85 (br s, 1H), 3.22e
NMR (75 MHz, CDCl3):
67.34, 119.4, 126.1, 128.5, 128.6, 131.2, 141.3, 149.0, 163.7, 172.5 ppm;
d
¼ 18.5, 26.5, 33.7, 35.1, 41.4, 54.5, 67.26,
3.31 (m, 1H), 3.60e3.71 (m, 2H), 4.22 (m, 1H), 4.67 (m, 2H), 5.20e
5.41 (m, 2H), 5.91 (m, 1H); 13C NMR (125 MHz, CDCl3):
¼ 21.5,
¼ 3447, 2912e
d
IR:
n
¼ 2912e2959, 1805, 1798, 1728, 1454, 1377, 1327, 1186,
40.6, 56.8, 64.6, 67.1, 119.3, 131.3, 149.0, 165.8; IR:
n
1126 cmꢀ1; MS (ESI): m/z (%): 346.04 (3) [M þ H]þ, 368.19 (100)
[M þ Na]þ; HRMS-ESI: m/z [M þ Na]þ calcd for C19H23NO5Na:
368.1474, found: 368.1464.
2988, 1790, 1732, 1379, 1319, 1252, 1128, 1047 cmꢀ1; MS (ESI): m/z
(%): 199.98 (2) [M þ H]þ, 222.10 (16) [M þ Na]þ, 420.81 (100)
[2M þ Na]þ; HRMSeCI: m/z [M þ H]þ calcd for C9H14NO4:
200.09288, found: 200.09274.
4.5.2. 1-(Propyl-3-enamino)carbonyl-(3S)-3-[(1R)-(4-
phenylbutanoyloxy)ethyl]azetidin-2-one (4c)
4.4.2. 1-(Propyl-3-enamino)carbonyl-(3S)-3-[(1R)-hydroxyethyl]
azetidin-2-one (10c)
Purification by flash chromatography (DCM/AcOEt 98:2) gave 4c
(61 mg, 88%) as a colourless oil: [
a
]
¼ ꢀ0.12 (c ¼ 0.51); Rf ¼ 0.36
D
Purification by flash chromatography (AcOEt 100%) gave 10c
(cyclohexane/AcOEt 5:3); 1H NMR (500 MHz, CDCl3):
d
¼ 1.32 (d,
(24 mg, 47%) as a colourless oil: Rf ¼ 0.29 (AcOEt 100%); 1H NMR
J ¼ 6.4 Hz, 3H),1.90 (m, 2H), 2.28 (m, 2H), 2.60 (m, 2H), 3.40 (m,1H),
3.57 (dd, J ¼ 3.2 Hz, J ¼ 6.8 Hz,1H), 3.69 (m, 1H), 3.86 (m, 2H), 5.08e
5.20 (m, 2H), 5.26 (m, 1H), 5.71e5.88 (m, 1H), 6.51 (br t, J ¼ 5.6 Hz,
(500 MHz, CDCl3):
d
¼ 1.27 (d, J ¼ 6.4 Hz, 3H), 2.73 (br s,1H), 3.28 (m,
1H), 3.65 (m, 2H), 3.82e3.92 (m, 2H), 4.20 (m,1H), 5.10e5.21 (m, 2H),
5.81 (m,1H), 6.58 ppm (br s,1H); 13C NMR (125 MHz, CDCl3):
¼ 21.7,
¼ 3354,
d
1H), 7.10e7.29 ppm (m, 5H); 13C NMR (125 MHz, CDCl3):
d
¼ 18.5,
40.0, 42.2, 56.3, 64.7, 116.6, 133.8, 150.6, 168.2 ppm; IR:
n
26.6, 33.8, 35.1, 40.6, 42.2, 54.0, 67.1, 116.6, 126.2, 128.5, 128.6, 133.8,
2926e2961, 1755, 1678, 1528, 1331, 1286, 1265, 1138 cmꢀ1; MS (ESI):
m/z (%): 199.11 (34) [M þ H]þ, 221.09 (100) [Mþ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C9H15N2O3: 199.10772, found: 199.10769.
141.2, 150.3, 166.4, 172.4 ppm; IR:
n
¼ 2868e2918, 1769, 1734, 1703,
1533, 1339, 1136 cmꢀ1; MS (ESI): m/z (%): 345.18 (7) [M þ H]þ,
367.16 (100) [M þ Na]þ, 711.8 (6) [2M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C19H25N2O4: 345.18088, found: 345.18124.
4.4.3. 1-(Propyl-3-enamino)thiocarbonyl-(3S)-3-[(1R)-
hydroxyethyl]azetidin-2-one (10d)
4.5.3. 1-(Propyl-3-enamino)thiocarbonyl-(3S)-3-[(1R)-(4-
phenylbutanoyloxy)ethyl]azetidin-2-one (4d)
Purification by flash chromatography (AcOEt 100%) gave 10d
(66 mg, 89%) as a white solid: Rf ¼ 0.26 (cyclohexane/AcOEt 5:3);
Purification by flash chromatography (DCM/AcOEt 99:1) gave 4d
1H NMR (500 MHz, CDCl3):
1H), 3.25 (m, 1H), 3.82 (m, 2H), 4.20e4.32 (m, 3H), 5.18e5.29 (m,
d
¼ 1.30 (d, J ¼ 6.4 Hz, 3H), 2.10 (br s,
(48 mg, 89%) as a colourless oil: [
a]
¼ 0.31 (c ¼ 0.48); Rf ¼ 0.56
D
(cyclohexane/AcOEt 5:2); 1H NMR (300 MHz, CDCl3):
d
¼ 1.35 (d,

108
M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
J ¼ 6.4 Hz, 3H), 1.92 (m, 2H), 2.31 (m, 2H), 2.62 (m, 2H), 3.33e3.43
(m, 1H), 3.71e3.79 (m, 1H), 3.88 (m, 1H), 4.12e4.34 (m, 3H), 5.14e
5.37 (m, 2H), 5.78 (m, 1H), 7.12e7.33 (m, 5H), 8.38 ppm (br s, 1H);
4.6.3. 1-(Propyl-3-enamino)carbonyl-(3S)-3-[(1R)-(2-(benzyloxy)
acetoyloxy)ethyl]azetidin-2-one (5c)
Purification by flash chromatography (DCM/AcOEt 98:2) gave 5c
13C NMR (75 MHz, CDCl3):
d
¼ 18.5, 26.6, 33.7, 35.1, 43.8, 47.2, 52.3,
(35 mg, 93%) as a colourless oil: [
a
]
¼ ꢀ0.36 (c ¼ 1.27); Rf ¼ 0.27
D
67.0, 117.9, 126.1, 128.5 (2C), 132.0, 141.2, 164.6, 172.4, 178.0 ppm; IR:
(chex/AcOEt 5:2); 1H NMR (300 MHz, CDCl3):
d
¼ 1.38 (d, J ¼ 6.3 Hz,
n
¼ 2912e2932, 1755, 1734, 1533, 1339, 1321, 1132 cmꢀ1; MS (ESI):
3H), 3.43 (m, 1H), 3.58 (dd, J ¼ 3.0 Hz, J ¼ 6.9 Hz, 1H), 3.72 (m, 1H),
3.89 (m, 2H), 4.06 (s, 2H), 4.60 (s, 2H), 5.10e5.23 (m, 2H), 5.35 (m,
1H), 5.76e5.88 (m, 1H), 6.52 (br s, 1H), 7.28e7.39 ppm (m, 5H); 13C
m/z (%): 361.16 (16) [M þ H]þ, 383.14 (100) [M þ Na]þ, 743.29 (6)
[2M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd for C19H25N2O3S:
345.18088, found: 345.18124.
NMR (75 MHz, CDCl3):
d
¼ 18.4, 40.7, 42.3, 53.8, 67.1, 68.1, 73.5,
116.7, 128.2, 128.2, 128.7, 133.8, 137.0, 150.3, 166.1, 169.6 ppm; IR:
4.5.4. 1-(Propyl-3-sulfanyl)carbonyl-(3S)-3-[(1R)-(4-
phenylbutanoyloxy)ethyl]azetidin-2-one (4e)
n
¼ 2912,1767,1703,1528,1340,1275,1202,1124 cmꢀ1; MS (ESI): m/
z (%): 347.16 (26) [M þ H]þ, 369.14 (100) [M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C18H23N2O5: 347.16015, found: 347.16032.
Purification by flash chromatography (DCM/AcOEt 99:1) gave 4d
(27 mg, 76%) as a colourless oil: [
a
]
¼ 0.47 (c ¼ 0.17); Rf ¼ 0.50
D
(cyclohexane/AcOEt 5:2); 1H NMR (300 MHz, CDCl3):
d
¼ 1.00 (t,
4.6.4. 1-(Propyl-3-enamino)thiocarbonyl-(3S)-3-[(1R)-(2-
(benzyloxy)acetoyloxy)ethyl]azetidin-2-one (5d)
J ¼ 7.4 Hz, 3H), 1.35 (d, J ¼ 6.4 Hz, 3H), 1.67 (m, 2H), 1.93 (m, 2H),
2.30 (m, 2H), 2.63 (m, 2H), 2.94 (m, 2H), 3.42 (m, 1H), 3.62 (dd,
J ¼ 3.6 Hz, J ¼ 7.3 Hz, 1H), 3.75 (dd, J ¼ 6.5 Hz, J ¼ 7.2 Hz, 1H), 5.28
Purification by flash chromatography (DCM/AcOEt 98:2) gave 5d
(37 mg, 74%) as a colourless oil: [
a
]
¼ 0.08 (c ¼ 1.78); Rf ¼ 0.43
D
(m, 1H), 7.15e7.31 ppm (m, 5H); 13C NMR (75 MHz, CDCl3):
d
¼ 13.4,
(chex/AcOEt 5:2); 1H NMR (300 MHz, CDCl3):
d
¼ 1.37 (d, J ¼ 6.3 Hz,
18.5, 23.0, 26.5, 31.2, 33.7, 35.1, 41.7, 54.0, 67.3, 126.1, 128.5, 128.6,
3H), 3.37 (m, 1H), 3.71 (dd, J ¼ 3.2 Hz, J ¼ 7.5 Hz, 1H), 3.87 (m, 1H),
4.06 (s, 2H), 4.19e4.29 (m, 2H), 4.59 (s, 2H), 5.15e5.27 (m, 2H), 5.34
(m, 1H), 5.87 (m, 1H), 7.26e7.44 (m, 5H), 8.36 ppm (br s, 1H); 13C
141.3, 163.4, 165.8, 172.4 ppm; IR:
n
¼ 2925e2962, 1788, 1732, 1663,
1454, 1308, 1240, 1130 cmꢀ1; MS (ESI): m/z (%): 364.16 (4) [M þ H]þ,
386.14 (100) [M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd for
C19H26NO4S: 364.15771, found: 364.15797.
NMR (75 MHz, CDCl3):
d
¼ 18.2, 43.7, 47.0, 52.0, 66.9, 67.8, 73.3,
117.8, 127.9, 128.0, 128.4, 131.8, 136.7, 164.0, 169.3, 177.8 ppm; IR:
n
¼ 2920, 1755, 1531, 1339, 1244, 1196, 1122 cmꢀ1; MS (ESI): m/z (%):
4.6. General procedure for esterification with carboxylic acid (5be
d and 7a)
363.14 (94) [M þ H]þ, 385.12 (100) [M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C18H23N2O4S: 363.13730, found: 363.13710.
To a stirred solution of alcohol precursor, DCC (1.1 equiv) and
DMAP (0.1 equiv) in dry DCM (13 mL/mmol), at 20 ꢁC, was added
a solution of the suitable carboxylic acid (1.1 equiv) in dry
dichloromethane (7 mL/mmol) under argon atmosphere. After
stirring overnight, the mixture was cooled in an ice-bath for
precipitation of urea, filtered and concentrated under vacuum.
After purification by flash chromatography (DCM/EtOAc), a colour-
less oil was obtained in all cases.
4.6.5. 1-(Pent-4-enoyl)-(3S)-3-[(1R)-(4-(pyridin-4-yl)butanoyl)
ethyl]azetidin-2-one (7a)
Purification by flash chromatography (DCM/AcOEt 9:1) gave 7a
(45 mg, 64%) as a colourless oil: [
a
]
¼ 0.10 (c ¼ 0.50); Rf ¼ 0.36
D
(DCM/MeOH 9:1); 1H NMR (500 MHz, CDCl3):
d
¼ 1.32 (d, J ¼ 6.4 Hz,
3H), 1.90 (m, 2H), 2.28 (t, J ¼ 7.4 Hz, 2H), 2.35 (m, 2H), 2.60 (m, 2H),
2.73 (m, 2H), 3.39 (m, 1H), 3.50 (dd, J ¼ 3.7 Hz, J ¼ 7.7 Hz, 1H), 3.63
(dd, J ¼ 6.8 Hz, J ¼ 7.6 Hz, 1H), 4.93e5.06 (m, 2H), 5.25 (m, 1H), 5.77
(m, 1H), 7.07 (d, J ¼ 6.0 Hz, 2H), 8.46 ppm (d, J ¼ 6.0 Hz, 2H); 13C
4.6.1. 1-(Pent-4-enoyl)-(3S)-3-[(1R)-(2-(benzyloxy)acetoyloxy)
ethyl]azetidin-2-one (5a)
NMR (125 MHz, CDCl3):
d
¼ 18.3, 25.3, 27.9, 33.4, 34.2, 35.8, 39.8,
53.5, 67.4, 115.9, 123.9, 136.3, 149.8, 150.2, 164.4, 170.3, 171.9 ppm;
Purification by flash chromatography (DCM/AcOEt 98:2) gave 5a
IR:
n
¼ 2922,1786,1734,1701,1603,1313,1238,1132 cmꢀ1; MS (ESI):
(78 mg, 89%) as a colourless oil: [
a
]
¼ ꢀ0.15 (c ¼ 0.52);Rf ¼ 0.38
m/z (%): 345.18 (100) [M þ H]þ, 367.16 (80) [M þ Na]þ; HRMS-ESI:
m/z [M þ H]þ calcd for C19H25N2O4: 345.18088, found: 345.18088.
D
(chex/AcOEt 5:3); 1H NMR (300 MHz, CDCl3):
d
¼ 1.39 (d, J ¼ 6.4 Hz,
3H), 2.39 (m, 2H), 2.76 (m, 2H), 3.42 (m, 1H), 3.52 (dd, J ¼ 3.7 Hz,
J ¼ 7.8 Hz, 1H), 3.66 (dd, J ¼ 6.7 Hz, J ¼ 7.8 Hz, 1H), 4.06 (s, 2H), 4.60
(s, 2H), 5.02 (m, 2H), 5.35 (m, 1H), 5.81 (m, 1H), 7.21e7.41 ppm (m,
4.7. General procedure for coupling isocyanate with alcohol
function (6aed)
5H); 13C NMR (75 MHz, CDCl3):
68.2, 73.4, 115.9, 128.1, 128.2, 128.6, 136.4, 136.9, 164.1, 169.5,
d
¼ 18.4, 27.9, 35.9, 40.0, 53.5, 67.1,
To a stirred solution of the alcohol precursor (1 equiv) in dry
DCM (7 mL/mmol) at r.t., was dropwise added the phenethyl
isocyanate (1.1 equiv) under argon atmosphere, and finally
Ti(OtBu)4 (0.1 equiv). After 1 h, the solution was quenched, at low
temperature, with a saturated aqueous solution of NH4Cl and
diluted with DCM. The aqueous layers were several times extracted
with DCM and the organic layers were combined, dried over
MgSO4, filtered and concentrated under vacuum. After purification
by flash chromatography (cyclohexane/AcOEt), white solids (6b
and 6d) or colourless oils were obtained (6a and 6c).
170.3 ppm; IR:
n
¼ 2849e2957, 1788, 1701, 1339, 1317, 1190,
1115 cmꢀ1; MS (ESI): m/z (%): 346.20 (4) [M þ H]þ, 368.16 (100)
[M þ Na]þ; HRMS-ESI: m/z [M þ Na]þ calcd for C19H23NO5Na:
368.1474, found: 368.1469.
4.6.2. 1-(Propyl-3-enoxy)carbonyl-(3S)-3-[(1R)-(2-(benzyloxy)
acetoyloxy)ethyl]azetidin-2-one (5b)
Purification by flash chromatography (DCM/AcOEt 98:2) gave 5b
(31 mg, 58%) as a colourless oil: [
a
]
¼ ꢀ0.45 (c ¼ 1.27); Rf ¼ 0.16
D
(chex/AcOEt 5:2); 1H NMR (300 MHz, CDCl3):
d
¼ 1.38 (d, J ¼ 6.4 Hz,
3H), 3.42 (m, 1H), 3.58 (dd, J ¼ 3.6 Hz, J ¼ 7.1 Hz, 1H), 3.70 (m, 1H),
4.06 (s, 2H), 4.59 (s, 2H), 4.68 (d, J ¼ 5.7 Hz, 2H), 5.18e5.44 (m, 3H),
5.82e5.99 (m, 1H), 7.21e7.37 ppm (m, 5H); 13C NMR (75 MHz,
4.7.1. 1-(Pent-4-enoyl)-(3S)-3-[(1R)-((2-phenylethylamino)
carbonyloxy)-ethyl]azetidin-2-one (6a)
Purification by flash chromatography (DCM/AcOEt 9:1) gave 6a
CDCl3):
d
¼ 18.2, 41.3, 54.2, 66.9, 67.1, 68.1, 73.3, 119.3, 127.9, 128.0,
(61 mg, 81%) as a colourless oil: [
a
]
¼ ꢀ0.19 (c ¼ 0.77); Rf ¼ 0.71
D
128.4, 130.9, 136.8, 148.7, 163.2, 169.3 ppm; IR:
n
¼ 2932, 1811, 1759,
(AcOEt); 1H NMR (500 MHz, CDCl3):
d
¼ 1.33 (d, J ¼ 6.3 Hz, 3H),
1734, 1377, 1329, 1261, 1124 cmꢀ1; MS (ESI): m/z (%): 348.14 (2)
[M þ H]þ, 370.13 (100) [M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd
for C18H22NO6: 348.14416, found: 348.14460.
2.29e2.48 (m, 2H), 2.79 (m, 4H), 3.25e3.46 (m, 4H), 3.62 (m, 1H),
4.74 (br s, 1H), 4.95e5.10 (m, 2H), 5.15 (m, 1H), 5.82 (m, 1H), 7.04e
7.42 ppm (m, 5H); 13C NMR (125 MHz, CDCl3):
d
¼ 18.7, 28.0, 35.8,

M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
109
36.0, 40.0, 42.2, 53.8, 67.9, 115.9, 126.6, 128.7, 128.8, 136.5, 138.7,
subtracted. Using these conditions, URB-597 (2, Chart 1) inhibits
hFAAH with an IC50 value of 40 nM.
155.2, 164.6, 170.4 ppm; IR:
n
¼ 2922, 1786, 1699, 1529, 1379, 1315,
1240, 1196, 1134 cmꢀ1; MS (ESI): m/z (%): 345.18 (4) [M þ H]þ,
367.16 (100) [M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd for
C19H25N2O4: 345.18088, found: 345.18129.
4.9. In vitro assays for human MGL activity
Tubes containing purified enzyme [29] (10 mM TriseHCl, 1 mM
4.7.2. 1-(Propyl-3-enoxy)carbonyl-(3S)-3-[(1R)-((2-
phenylethylamino)carbonyloxy)-ethyl]-azetidin-2-one (6b)
Purification by flash chromatography (DCM/AcOEt 9:1) gave 6b
EDTA, 0.1% (w/v) BSA, pH 8.0, 165
DMSO alone for controls (10
L) and [3H]-2-OG (50,000 dpm, 2
final concentration, 25
L) were preincubated at 20 ꢁC for 30 min
and incubated at 37 ꢁC for 10 min. Reactions were stopped by
rapidly placing the tubes in ice and adding 400 L of ice-cold
chloroform/methanol (1:1 v/v) followed by vigorous mixing. Pha-
ses were separated by centrifugation at 850 g, and aliquots (200 L)
of the lower chloroform phase were counted for radioactivity by
liquid scintillation counting. In all experiments, tubes containing
buffer only were used as control for chemical hydrolysis (blank) and
this value was systematically subtracted.
mL), test compounds in DMSO or
m
mM
m
(22 mg, 64%) as a white solid: [
a
]
¼ ꢀ0.51 (c ¼ 1.32); Rf ¼ 0.78
D
(AcOEt); 1H NMR (500 MHz, CDCl3):
d
¼ 1.35 (d, J ¼ 6.3 Hz, 3H), 2.81
m
(t, J ¼ 6.9 Hz, 2H), 3.36 (m, 1H), 3.43 (m, 2H), 3.58 (dd, J ¼ 3.5 Hz,
J ¼ 6.9 Hz, 1H), 3.68 (m, 1H), 4.71 (m, 3H), 5.14 (m, 1H), 5.25e5.44
(m, 2H), 5.94 (m, 1H), 7.14e7.20 (m, 2H), 7.21e7.26 (m, 1H), 7.27e
m
7.34 ppm (m, 2H); 13C NMR (125 MHz, CDCl3):
d
¼ 18.7, 36.1, 41.6,
42.3, 54.8, 67.3, 68.0, 119.5, 126.7, 128.8, 128.9, 131.3, 138.7, 149.0,
155.3, 163.9 ppm; IR:
n
¼ 2912e2932, 1807, 1717, 1705, 1518, 1379,
1327, 1259, 1128 cmꢀ1; MS (ESI): m/z (%): 347.16 (3) [M þ H]þ,
369.14 (49) [M þ Na]þ, 715.29 (100) [2M þ Na]þ; HRMS-ESI: m/z
[M þ H]þ calcd for C18H23N2O5: 347.16015, found: 347.16044.
4.10. Reversibility studies
In a total volume of 15 mL, human FAAH (27.5 mg) and inhibitors
4.7.3. 1-(Propyl-3-enamino)carbonyl-(3S)-3-[(1R)-((2-
phenylethylamino)carbonyloxy)-ethyl]-azetidin-2-one (6c)
Purification by flash chromatography (DCM/AcOEt 9:1) gave 6c
(or DMSO for controls) at concentrations allowing inhibition of the
enzyme before dilution and no inhibition after the 100-fold dilu-
tion, were preincubated during 1 h at room temperature. The
mixtures were then diluted 100-fold by adding assay buffer.
(29 mg, 70%) as a colourless oil: [
a
]
¼ ꢀ0.73 (c ¼ 1.17); Rf ¼ 0.45
D
(chex/AcOEt 5:3); 1H NMR (300 MHz, CDCl3):
d
¼ 1.33 (d, J ¼ 6.4 Hz,
Immediately after, an aliquot (175
m
L) was taken and [3H]-AEA
L) was added. Two
3H), 2.80 (t, J ¼ 6.9 Hz, 2H), 3.34e3.47 (m, 3H), 3.56 (dd, J ¼ 3.0 Hz,
J ¼ 6.8 Hz, 1H), 3.68 (m, 1H), 3.95 (m, 2H), 4.72 (br s, 1H), 5.03e5.27
(m, 3H), 5.71e5.93 (m, 1H), 6.56 (br s, 1H), 7.08e7.36 ppm (m, 5H);
(50,000 dpm, 2 M final concentration, 25 m
m
samples were taken at 30 and 90 min after the dilution too. Each
aliquots was incubated at 37 ꢁC for 30 min and reactions were
13C NMR (75 MHz, CDCl3):
d
¼ 18.7, 36.1, 40.7, 42.3 (2C), 54.2, 67.7,
stopped by rapidly placing the tubes in ice and adding 400 mL of ice-
cold chloroform/methanol (1:1 v/v) followed by vigorous mixing.
Phases were separated by centrifugation at 850 g, and aliquots
(200 mL) of the upper methanol/buffer phase were counted for
radioactivity by liquid scintillation counting. In all experiments,
tubes containing buffer only were used as control for chemical
hydrolysis (blank) and this value was systematically subtracted.
116.7, 126.7, 128.8, 128.9, 133.8, 138.7, 150.4, 155.3, 166.7 ppm; IR:
n
¼ 2980, 1765, 1699, 1645, 1531, 1497, 1454, 1337, 1248, 1136 cmꢀ1
;
MS (ESI): m/z (%): 346.18 (9) [M þ H]þ, 368.16 (100) [M þ Na]þ;
HRMS-ESI: m/z [M þ H]þ calcd for C18H24N3O4: 346.17613, found:
346.17636.
4.7.4. 1-(Propyl-3-enamino)thiocarbonyl-(3S)-3-[(1R)-((2-
phenylethylamino)carbonyloxy)-ethyl]-azetidin-2-one (6d)
Purification by flash chromatography (DCM/AcOEt 95:5) gave 6d
4.11. Docking studies
(56 mg, 84%) as a white solid: [
a
]
¼ 0.25 (c ¼ 0.60); Rf ¼ 0.35
The crystal structure of humanized rat FAAH (PDB code:
3PPM) was used [36]. Docking of the inhibitors into the active
site of FAAH was performed using the GOLD program [52]. GOLD
is based on a genetic algorithm, performing docking of flexible
ligands into proteins with partial flexibility in the neighbourhood
of the active site. Default settings were used for the genetic
algorithm parameters. Twenty solutions were generated and
ranked by GOLD score. The GOLD fitness function is made up of
four components: proteineligand hydrogen bond energy,
proteineligand van der Waals energy, ligand internal van der
Waals energy, and ligand torsional strain energy. The figures
were produced using PyMOL [53].
D
d
(AcOEt); 1H NMR (300 MHz, CDCl3):
¼ 1.33 (d, J ¼ 6.4 Hz, 3H), 2.80
(t, J ¼ 6.9 Hz, 2H), 3.26e3.36 (m, 1H), 3.37e3.48 (m, 2H), 3.72 (dd,
J ¼ 3.2 Hz, J ¼ 7.4 Hz, 1H), 3.84 (m, 1H), 4.20e4.34 (m, 2H), 4.75 (br s
1H), 5.09e5.34 (m, 3H), 5.76e5.98 (m, 1H), 7.09e7.36 (m, 5H),
8.42 ppm (br s, 1H); 13C NMR (75 MHz, CDCl3):
d
¼ 18.7, 36.0, 42.2,
43.8, 47.2, 52.5, 67.5, 117.9, 126.6, 128.7, 128.9, 132.0, 138.6, 155.2,
164.8, 178.1 ppm; IR:
n
¼ 2976, 1749, 1715, 1697, 1524, 1497, 1337,
1242, 1130 cmꢀ1; MS (ESI): m/z (%): 362.15 (16) [M þ H]þ, 384.13
(100) [M þ Na]þ; HRMS-ESI: m/z [M þ H]þ calcd for C18H24N3O3S:
362.15329, found: 362.15359.
4.8. In vitro assays for human FAAH
Acknowledgement
Tubes containing the enzyme [27] (10 mM TriseHCl, 1 mM
The UCL (Université catholique de Louvain) and the F. R. S.-FNRS
(Fonds de la Recherche Scientifique, Belgium) are gratefully
acknowledged for financial support of this work (FRFC grant, no.
2.4.654.06 F). This work is partially supported by the Interuniver-
sity Attraction Pole program (IAP P6/19 PROFUSA). J.M.-B. and C.M.
are senior research associate of the Belgian F. R. S.-FNRS. The
authors wish to thank Kaneka Corporation (Japan), in particular Dr.
Claudio Salvagnini, for providing the starting azetidinone ((3R,4R)-
3-[(R)-10-(tert-butyldimethylsilyloxy)-ethyl]-4-acetoxy-2-
EDTA, 0.1% (w/v) BSA, pH 7.4, 165
DMSO alone for controls (10
L) and [3H]-AEA (50,000 dpm, 2
final concentration, 25
L) were incubated at 37 ꢁC for 10 min.
Reactions were stopped by rapidly placing the tubes in ice and
adding 400 L of ice-cold chloroform/methanol (1:1 v/v) followed
by vigorous mixing. Phases were separated by centrifugation at
850 g, and aliquots (200 L) of the upper methanol/buffer phase
mL), test compounds in DMSO or
m
mM
m
m
m
were counted for radioactivity by liquid scintillation counting. In all
experiments, tubes containing buffer only were used as control for
chemical hydrolysis (blank) and this value was systematically
azetidinone). Dr Geoffray Labar is acknowledged for the prepara-
tion of hFAAH, and Bouazza Es Saadi for technical assistance.

110
M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
[20] J.A. Palmer, E.S. Higuera, L. Chang, S.R. Chaplan, Fatty acid amide hydrolase
Appendix A. Supplementary material
inhibition enhances the anti-allodynic actions of endocannabinoids in
a model of acute pain adapted for the mouse, Neuroscience 154 (2008)
1554e1561.
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.11.035.
[21] B.F. Cravatt, O. Prospero-Garcia, G. Siuzdak, N.B. Gilula, S.J. Henriksen,
D.L. Boger, R.A. Lerner, Chemical characterization of a family of brain lipids
that induce sleep, Science (New York, NY) 268 (1995) 1506e1509.
[22] S.G. Kinsey, S.T. O’Neal, J.Z. Long, B.F. Cravatt, A.H. Lichtman, Inhibition of
endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the
marble burying assay, Pharmacol. Biochem. Behav. 98 (2011) 21e27.
[23] G.L. Li, H. Winter, R. Arends, G.W. Jay, V. Le, T. Young, J.P. Huggins, Assessment
of the pharmacology and tolerability of PF-04457845, an irreversible inhibitor
of fatty acid amide hydrolase-1, in healthy subjects, Br. J. Clin. Pharmacol. 73
(2012) 706e716.
References
[1] M. Seierstad, J.G. Breitenbucher, Discovery and development of fatty acid
amide hydrolase (FAAH) inhibitors, J. Med. Chem. 51 (2008) 7327e7343.
[2] S. Vandevoorde, Overview of the chemical families of fatty acid amide
hydrolase and monoacylglycerol lipase inhibitors, Curr. Top. Med. Chem. 8
(2008) 247e267.
[3] M. Feledziak, D.M. Lambert, J. Marchand-Brynaert, G.G. Muccioli, Inhibitors of
the endocannabinoid-degrading enzymes, or how to increase endocannabi-
noid’s activity by preventing their hydrolysis, Recent Pat. CNS Drug Discov. 7
(2012) 49e70.
[24] M. Feledziak, C. Michaux, A. Urbach, G. Labar, G.G. Muccioli, D.M. Lambert,
J. Marchand-Brynaert,
b-Lactams derived from a carbapenem chiron are
selective inhibitors of human fatty acid amide hydrolase versus human
monoacylglycerol lipase, J. Med. Chem. 52 (2009) 7054e7068.
[25] M. Feledziak, G.G. Muccioli, D.M. Lambert, J. Marchand-Brynaert, SAR and LC/MS
studies of b-lactamic inhibitors of human fatty acid amide hydrolase (hFAAH):
evidence of a nonhydrolytic process, J. Med. Chem. 54 (2011) 6812e6823.
[26] C. Spino, M.-A. Joly, C. Godbout, M. Arbour, Ti-catalyzed reactions of hindered
isocyanates with alcohols, J. Org. Chem. 70 (2005) 6118e6121.
[4] A. Minkkila, S.M. Saario, T. Nevalainen, Discovery and development of
endocannabinoid-hydrolyzing enzyme inhibitors, Curr. Top. Med. Chem. 10
(2010) 828e858.
[27] G. Labar, F.V. Vliet, J. Wouters, D.M. Lambert, A MBP-FAAH fusion protein as
a tool to produce human and rat fatty acid amide hydrolase: expression and
pharmacological comparison, Amino Acids 34 (2008) 127e133.
[5] K. Otrubova, C. Ezzili, D.L. Boger, The discovery and development of inhibitors
of fatty acid amide hydrolase (FAAH), Bioorg. Med. Chem. Lett. 21 (2011)
4674e4685.
[28] P. Ertl, B. Rohde, P. Selzer, Fast calculation of molecular polar surface area as
a sum of fragment-based contributions and its application to the prediction of
drug transport properties, J. Med. Chem. 43 (2000) 3714e3717.
[6] I.K. Khanna, C.W. Alexander, Fatty acid amide hydrolase inhibitors e progress
and potential, CNS Neurol. Disord. Drug Targets 10 (2011) 545e558.
[7] D.L. Boger, H. Miyauchi, W. Du, C. Hardouin, R.A. Fecik, H. Cheng, I. Hwang,
M.P. Hedrick, D. Leung, O. Acevedo, C.R.W. Guimaraes, W.L. Jorgensen,
B.F. Cravatt, Discovery of a potent, selective, and efficacious class of reversible
alpha-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as
analgesics, J. Med. Chem. 48 (2005) 1849e1856.
[8] D. Piomelli, G. Tarzia, A. Duranti, A. Tontini, M. Mor, T.R. Compton, O. Dasse,
E.P. Monaghan, J.A. Parrott, D. Putman, Pharmacological profile of the selective
FAAH inhibitor KDS-4103 (URB597), CNS Drug Rev. 12 (2006) 21e38.
[9] D.S. Johnson, C. Stiff, S.E. Lazerwith, S.R. Kesten, L.K. Fay, M. Morris, D. Beidler,
M.B. Liimatta, S.E. Smith, D.T. Dudley, N. Sadagopan, S.N. Bhattachar,
S.J. Kesten, T.K. Nomanbhoy, B.F. Cravatt, K. Ahn, Discovery of PF-04457845:
a highly potent, orally bioavailable, and selective urea FAAH inhibitor, ACS
Med. Chem. Lett. 2 (2010) 91e96.
[10] S.D. Roughley, H. Browne, A.T. Macias, K. Benwell, T. Brooks, J. D’Alessandro,
Z. Daniels, S. Dugdale, G. Francis, B. Gibbons, T. Hart, T. Haymes, G. Kennett,
S. Lightowler, N. Matassova, H. Mansell, A. Merrett, A. Misra, A. Padfield,
R. Parsons, R. Pratt, A. Robertson, H. Simmonite, K. Tan, S.B. Walls, M. Wong,
Fatty acid amide hydrolase inhibitors. 3: tetra-substituted azetidine ureas
with in vivo activity, Bioorg. Med. Chem. Lett. 22 (2012) 901e906.
[11] M.J. Meyers, S.A. Long, M.J. Pelc, J.L. Wang, S.J. Bowen, B.A. Schweitzer,
M.V. Wilcox, J. McDonald, S.E. Smith, S. Foltin, J. Rumsey, Y.-S. Yang, M.C. Walker,
S. Kamtekar, D. Beidler, A. Thorarensen, Discovery of novel spirocyclic inhibitors
of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane
urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase
(FAAH) for pain, Bioorg. Med. Chem. Lett. 21 (2011) 6545e6553.
[12] K. Ahn, D.S. Johnson, M. Mileni, D. Beidler, J.Z. Long, M.K. McKinney,
E. Weerapana, N. Sadagopan, M. Liimatta, S.E. Smith, S. Lazerwith, C. Stiff,
S. Kamtekar, K. Bhattacharya, Y. Zhang, S. Swaney, K. Van Becelaere, R.C. Stevens,
B.F. Cravatt, Discovery and characterization of a highly selective FAAH inhibitor
that reduces inflammatory pain, Chem. Biol. 16 (2009) 411e420.
[29] G. Labar, C. Bauvois, G.G. Muccioli, J. Wouters, D.M. Lambert, Disulfiram is an
inhibitor of human purified monoacylglycerol lipase, the enzyme regulating
2-arachidonoylglycerol signaling, ChemBioChem 8 (2007) 1293e1297.
[30] D.L. Boger, H. Sato, A.E. Lerner, M.P. Hedrick, R.A. Fecik, H. Miyauchi,
G.D. Wilkie, B.J. Austin, M.P. Patricelli, B.F. Cravatt, Exceptionally potent
inhibitors of fatty acid amide hydrolase: the enzyme responsible for degra-
dation of endogenous oleamide and anandamide, Proc. Natl. Acad. Sci. U.S.A.
97 (2000) 5044e5049.
[31] M. Mor, S. Rivara, A. Lodola, P.V. Plazzi, G. Tarzia, A. Duranti, A. Tontini,
G. Piersanti, S. Kathuria, D. Piomelli, Cyclohexylcarbamic acid 30- or 40-
substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors:
synthesis, quantitative structureeactivity relationships, and molecular
modeling studies, J. Med. Chem. 47 (2004) 4998e5008.
[32] K. Ahn, D.S. Johnson, L.R. Fitzgerald, M. Liimatta, A. Arendse, T. Stevenson,
E.T. Lund, R.A. Nugent, T.K. Nomanbhoy, J.P. Alexander, B.F. Cravatt, Novel
mechanistic class of fatty acid amide hydrolase inhibitors with remarkable
selectivity, Biochemistry 46 (2007) 13019e13030.
[33] M. Mileni, S. Kamtekar, D.C. Wood, T.E. Benson, B.F. Cravatt, R.C. Stevens,
Crystal structure of fatty acid amide hydrolase bound to the carbamate
inhibitor URB597: discovery of a deacylating water molecule and insight into
enzyme inactivation, J. Mol. Biol. 400 (2010) 743e754.
[34] M. Mileni, J. Garfunkle, J.K. DeMartino, B.F. Cravatt, D.L. Boger, R.C. Stevens,
Binding and inactivation mechanism of
a humanized fatty acid amide
hydrolase by -ketoheterocycle inhibitors revealed from cocrystal structures,
a
J. Am. Chem. Soc. 131 (2009) 10497e10506.
[35] M. Mileni, J. Garfunkle, C. Ezzili, F.S. Kimball, B.F. Cravatt, R.C. Stevens,
D.L. Boger, X-ray crystallographic analysis of
a-ketoheterocycle inhibitors
bound to a humanized variant of fatty acid amide hydrolase, J. Med. Chem. 53
(2010) 230e240.
[36] M. Mileni, J. Garfunkle, C. Ezzili, B.F. Cravatt, R.C. Stevens, D.L. Boger, Fluoride-
mediated capture of a noncovalent bound state of a reversible covalent
enzyme inhibitor: X-ray crystallographic analysis of an exceptionally potent
[13] E. de Lago, S. Petrosino, M. Valenti, E. Morera, S. Ortega-Gutierrez,
J. Fernandez-Ruiz, V. Di Marzo, Effect of repeated systemic administration of
selective inhibitors of endocannabinoid inactivation on rat brain endo-
cannabinoid levels, Biochem. Pharmacol. 70 (2005) 446e452.
a
-ketoheterocycle inhibitor of fatty acid amide hydrolase, J. Am. Chem. Soc.
133 (2011) 4092e4100.
[14] F.A. Moreira, M. Grieb, B. Lutz, Central side-effects of therapies based on CB1
cannabinoid receptor agonists and antagonists: focus on anxiety and
depression, Best Pract. Res. Clin. Endocrinol Metab. 23 (2009) 133e144.
[15] G. Gerra, A. Zaimovic, M.L. Gerra, R. Ciccocioppo, A. Cippitelli, G. Serpelloni,
L. Somaini, Pharmacology and toxicology of Cannabis derivatives and endo-
cannabinoid agonists, Recent Pat. CNS Drug Discov. 5 (2010) 46e52.
[16] J. Schlosburg, S. Kinsey, A. Lichtman, Targeting fatty acid amide hydrolase
(FAAH) to treat pain and inflammation, AAPS J. 11 (2009) 39e44.
[17] K. Ahn, S.E. Smith, M.B. Liimatta, D. Beidler, N. Sadagopan, D.T. Dudley,
T. Young, P. Wren, Y. Zhang, S. Swaney, K. Van Becelaere, J.L. Blankman,
D.K. Nomura, S.N. Bhattachar, C. Stiff, T.K. Nomanbhoy, E. Weerapana,
D.S. Johnson, B.F. Cravatt, Mechanistic and pharmacological characterization
of PF-04457845: a highly potent and selective fatty acid amide hydrolase
inhibitor that reduces inflammatory and noninflammatory pain, J. Pharmacol.
Exp. Ther. 338 (2011) 114e124.
[37] D.J. Gustin, Z. Ma, X. Min, Y. Li, C. Hedberg, C. Guimaraes, A.C. Porter,
M. Lindstrom, D. Lester-Zeiner, G. Xu, T.J. Carlson, S. Xiao, C. Meleza, R. Connors,
Z. Wang, F. Kayser, Identification of potent, noncovalent fatty acid amide
hydrolase (FAAH) inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 2492e2496.
[38] X. Min, S.T. Thibault, A.C. Porter, D.J. Gustin, T.J. Carlson, H. Xu, M. Lindstrom,
G. Xu, C. Uyeda, Z. Ma, Y. Li, F. Kayser, N.P.C. Walker, Z. Wang, Discovery and
molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase
(FAAH), Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 7379e7384.
[39] L. Hedstrom, Serine protease mechanism and specificity, Chem. Rev. 102
(2002) 4501e4524.
[40] M. Lee, D. Hesek, M. Suvorov, W. Lee, S. Vakulenko, S. Mobashery, A mechanism-
based inhibitor targeting the D-D-transpeptidase activity of bacterial penicillin-
binding proteins, J. Am. Chem. Soc. 125 (2003) 16322e16326.
[41] J.-E. Hugonnet, J.S. Blanchard, Irreversible inhibition of the Mycobacterium
tuberculosis b-lactamase by clavulanate, Biochemistry 46 (2007) 11998e12004.
[18] L.J. Alvarez-Jaimes, J.A. Palmer, The role of endocannabinoids in pain modu-
lation and the therapeutic potential of inhibiting their enzymatic degradation,
Curr. Pharm. Biotechnol. 10 (2011) 1644e1659.
[19] C. Ezzili, M. Mileni, N. McGlinchey, J.Z. Long, S.G. Kinsey, D.G. Hochstatter,
R.C. Stevens, A.H. Lichtman, B.F. Cravatt, E.J. Bilsky, D.L. Boger, Reversible
[42] S. Vanwetswinkel, J. Fastrez, J. Marchand-Brynaert, Synthesis of new
sulfonylamido-penicillanic acid sulfones inhibitors of beta-lactamases,
J. Antibiot. 47 (1994) 1041e1051.
[43] W.B. Knight, A.L. Maycock, B.G. Green, B.M. Ashe, P. Gale, H. Weston,
P.E. Finke, W.K. Hagmann, S.K. Shah, J.B. Doherty, Mechanism of inhibition of
human leukocyte elastase by two cephalosporin derivatives, Biochemistry 31
(1992) 4980e4986.
competitive a-ketoheterocycle inhibitors of fatty acid amide hydrolase con-
taining additional conformational constraints in the acyl side chain: orally
active, long-acting analgesics, J. Med. Chem. 54 (2011) 2805e2822.

M. Feledziak et al. / European Journal of Medicinal Chemistry 60 (2013) 101e111
111
[44] J.B. Doherty, B.M. Ashe, L.W. Argenbright, P.L. Barker, R.J. Bonney,
G.O. Chandler, M.E. Dahlgren, C.P. Dorn, P.E. Finke, R.A. Firestone, D. Fletcher,
W.K. Hagmann, R. Mumford, L. O’Grady, A.L. Maycock, J.M. Pisano, S.K. Shah,
K.R. Thompson, M. Zimmerman, Cephalosporin antibiotics can be modified to
inhibit human leukocyte elastase, Nature 322 (1986) 192e194.
[49] J.P. Alexander, B.F. Cravatt, Mechanism of carbamate inactivation of FAAH:
implications for the design of covalent inhibitors and in vivo functional probes
for enzymes, Chem. Biol. 12 (2005) 1179e1187.
[50] M. Mileni, D.S. Johnson, Z. Wang, D.S. Everdeen, M. Liimatta, B. Pabst,
K. Bhattacharya, R.A. Nugent, S. Kamtekar, B.F. Cravatt, K. Ahn, R.C. Stevens,
Structure-guided inhibitor design for human FAAH by interspecies active site
conversion, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 12820e12824.
[51] X. Wang, K. Sarris, K. Kage, D. Zhang, S.P. Brown, T. Kolasa, C. Surowy, O.F. El
Kouhen, S.W. Muchmore, J.D. Brioni, A.O. Stewart, Synthesis and evaluation of
benzothiazole-based analogues as novel, potent, and selective fatty acid
amide hydrolase inhibitors, J. Med. Chem. 52 (2009) 170e180.
[45] J. Mulchande, R.C. Guedes, W.-Y. Tsang, M.I. Page, R. Moreira, J. Iley, Azetidine-
2,4-diones (4-oxo-b-lactams) as scaffolds for designing elastase inhibitors,
J. Med. Chem. 51 (2008) 1783e1790.
[46] C. Beauve, G. Tjoens, R. Touillaux, J. Lamotte-Brasseur, J. Marchand-Brynaert,
J. Fastrez, 1-Alkoxycarbonyl-3-bromoazetidin-2-ones as potential elastase
inhibitors, Eur. J. Org. Chem. 1999 (1999) 1441e1447.
[47] M.I. Konaklieva, Beta-lactams as inhibitors of serine enzymes, Curr. Med.
Chem. Anti-infect. Agents 1 (2002) 215e238.
[48] M.K. McKinney, B.F. Cravatt, Structure and function of fatty acid amide
hydrolase, Annu. Rev. Biochem. 74 (2005) 411e432.
[52] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and validation
of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997) 727e748.
[53] W.L. De Lano, The PyMOL Molecular Graphics System, 0.99, DeLano Scientific,
San Carlos, 2002.