SAR and LC/MS studies of β-lactamic inhibitors of human fatty acid amide hydrolase (h FAAH): Evidence of a nonhydrolytic process

Article abstract of DOI:10.1021/jm200723m

The endocannabinoid hydrolyzing enzyme FAAH uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of Ser-Asp-His) to cleave its endogenous substrates. Because inhibiting FAAH has a clear therapeutic potential, we previously developed β-lactam-t

Full text of DOI:10.1021/jm200723m

ARTICLE
pubs.acs.org/jmc
SAR and LC/MS Studies of β-Lactamic Inhibitors of Human Fatty Acid
Amide Hydrolase (hFAAH): Evidence of a Nonhydrolytic Process
Marion Feledziak,†,§ Giulio G. Muccioli,*,‡ Didier M. Lambert,§ and Jacqueline Marchand-Brynaert*,†
†Laboratoire de Chimie Organique et Mꢀedicinale, Institute of Condensed Matter and Nanosciences, Universitꢀe catholique de Louvain,
B^atiment Lavoisier, Place Louis Pasteur L4.01.02, B-1348 Louvain-La-Neuve, Belgium
‡Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, Louvain Drug Research Institute, Universitꢀe catholique de Louvain,
Avenue E. Mounier 72, B1.72.01, B-1200 Bruxelles, Belgium
§Unitꢀe de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute, Universitꢀe catholique de Louvain,
Avenue E. Mounier 73.40, B-1200 Bruxelles, Belgium
S Supporting Information
b
ABSTRACT: The endocannabinoid hydrolyzing enzyme FAAH
uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of
Ser-Asp-His) to cleave its endogenous substrates. Because inhibit-
ing FAAH has a clear therapeutic potential, we previously devel-
oped β-lactam-type inhibitors of hFAAH. Here, we report the
synthesis of five novel derivatives (5ꢀ9) of our lead compound
1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-
azetidin-2-one (4, IC50 = 5 nM) obtained via the systematic replacement of one to three carbonyls by methylene groups. The SAR results
showed that the imide, but not the lactam, function is essential to the inhibition of hFAAH. We also performed LC/MS analysis following
incubation of our inhibitors with hFAAH or mouse liver. We demonstrated that hFAAH interacts with these β-lactam-type inhibitors but,
unexpectedly, does not open the β-lactam moiety. This mechanism seems to be unique to FAAH because the β-lactam function of the
inhibitors is hydrolyzed when they are incubated in the presence of the serine hydrolases expressed in the mouse liver. Finally, we
confirmed these results by showing that a highly selective FAAH inhibitor (PF-750) does not prevent this hydrolysis by liver homogenates.
’ INTRODUCTION
studied.17,18 This serine amidase possesses an unusual Ser-Ser-Lys
catalytic triad, with substrate hydrolysis involving the nucleophilic
attack of the Ser241 residue on the amide carbonyl group leading
to an unstable acyl-enzyme intermediate. To date, small molecules
featuring a wide diversity of electrophilic functions have been
investigated as potent FAAH inhibitors for therapeutic applica-
tions or as pharmacological tools.19ꢀ22 For most of them, the
modes of inhibition have been studied and elucidated. Thus,
different types of mechanisms have been listed, depending on
the interaction between the inhibitor and the active serine: (i) a
covalent interaction that leads to a stable acyl-enzyme complex,
(ii) a covalent interaction that leads to a reversible tetrahedral
intermediate, and (iii) a noncovalent interaction based on strong
affinity. Thus, URB-597 (1, Chart 1)23 and PF-750 (2, Chart 1)24
were demonstrated to covalently bind FAAH by carbamylation.
Indeed, the aniline and the phenol moieties, respectively, were
shown by MALDI-MS mapping after trypsinization to be expelled
as leaving groups, leading to stable and inactive acyl-enzyme
complexes. On the contrary, OL-135 (3, Chart 1) covalently binds
to FAAH but in a reversible manner. X-ray crystal structures of the
carbamoyl intermediates formed by Ser241 acylation with 125 and
2,26,27 as well as the tetrahedral intermediate resulting from Ser241
The endogenous cannabinoid (CB) system has been exten-
sively explored these last two decades due to its involvement in a
lot of therapeutically promising biological effects.1ꢀ3 Composed
of two G-protein coupled receptors (GPCR) named CB1 and
CB2 and their endogenous ligands called endocannabinoids (N-
arachidonoylethanolamine (AEA) also named anandamide and
2-arachidonoylglycerol (2-AG), for the most studied),4ꢀ6 the
endocannabinoid system is involved in numerous physiological
and pathological processes depending on the ligand, receptor,
and tissue localization, which are considered.7 Three main
strategies were investigated to take advantage of the beneficial
effects of CB1 and/or CB2 activation. First, a large number of
synthetic agonists were considered, which allow the enhancement
of the effects directly resulting from receptor activation.8ꢀ12
However, most of the time psychoactive effects (due to CB1
activation in the CNS) also occurred.13,14 More recently, allosteric
modulators of cannabinoid receptors were explored as a second
strategy that would increase the effect of the endogenous ligands
without displaying their characteristic side effects.15,16 The third
strategy was to design inhibitors of endocannabinoid-degrading
enzymes as a potential alternative to direct agonist administration
with the hope of reducing side effects associated with agonist
administration. In this research line, fatty acid amide hydrolase
(FAAH), the main anandamide-degrading enzyme, is the most
Received: June 7, 2011
Published: September 08, 2011
r
2011 American Chemical Society
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Chart 1. Chemical Structure of Previously Described FAAH-
Inhibitors
Chart 2. Analogues of Compound 4
nucleophilic addition on the carbonyl function of 3,28,29 confirmed
the covalent irreversible and reversible FAAH inhibitions, respectively.
We have recently described a novel template designed for
FAAH inhibition, which is based on the β-lactam ring. From that
study, a lead (i.e., 1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyl-
oxy)-ethyl]-azetidin-2-one 4, Chart 1) emerged as a promising
nanomolar inhibitor.30 Preliminary experiments to determine the
inhibition mode showed a fully reversible, competitive inhibition,
a rather surprising result for a β-lactam-based inhibitor. Indeed,
β-lactamic inhibitors of serine hydrolases usually lead to irrever-
sible inhibition via the formation of stable acyl-enzyme com-
plexes, resulting possibly from a suicide-type mechanism.31 Slow
reversible inhibition can also occur when the β-lactam ring is in
fine hydrolyzed as a bad substrate, after being covalently attached
to the active serine via its acyl-enzyme complex. Other possibi-
lities could explain a reversible inhibition: the tetrahedral inter-
mediate cannot evolve toward a stable acyl-enzyme complex, or
the inhibitor does not interact covalently with the enzyme but has
a very good affinity for the catalytic site. In order to elucidate the
mode of action of FAAH β-lactamic inhibitors such as 4, we
undertook (I) a structureꢀactivity relationship (SAR) study,
based on the successive deletions of one to three carbonyls from
the parent β-lactam 4. This aims at identifying the electrophilic
function(s) susceptible to interact with the FAAH active serine
residue. Thus, we synthesized and evaluated five molecules
(5ꢀ9) as potential FAAH inhibitors. Then, (II) we performed
analyses by mass spectrometry coupled with liquid chromatog-
raphy (LC/MS) to identify the products resulting from the
interaction of the inhibitors with FAAH.
to independently modulate the two key positions N1 and C5-O
thanks to the silyl ether protection of the commercial starting
material ((3S)-[1(R)-(tbutyldimethylsilyloxy)-ethyl]-azetidin-2-
one (Scheme 1)). Unfortunately, drawbacks inherent to the
reduction of azetidinone into azetidine and to the O-alkylation
step ultimately guided the synthesis of compounds 8 and 9 via
longer ways. Actually, their synthetic routes evolved according to
the following experimental considerations: (i) the silyl ether
protection did not resist to reducing conditions such as AlH2Cl
treatment, which caused the degradation of the β-lactam ring;
(ii) the azetidinone reduction was not selective versus ester,
amide, or imide motifs, which implied that solely the carbonyl of
the lactam ring should be present on the precursors to be
reduced; and (iii) the O-alkylation reaction did not occur when
the N1 position was acylated because the anionic intermediate
resulted in a rearrangement; this implied that the N1 position had
to be protected with an alkyl group in the case of treatment with a
strong base.
Briefly, compounds 5 and 6 were prepared from the starting
material in three steps by alkylation of the N1 position, deprotec-
tion of the silyl ether group, and functionalization of the C5-O
position (Scheme 1). The starting material was N-alkylated by
reaction with 4-pentenyl bromide and KOH, in the presence of a
phase transfer agent, giving compound 11 (76%).32 Then, the
silyl ether was deprotected in an acidic solution of tetrabutylam-
monium fluoride in tetrahydrofuran. The corresponding alcohol
12 (93%) was acylated by reaction with 4-phenylbutanoyl
chloride in the presence of pyridine or alkylated using sodium
hydride and 4-phenylbutyl bromide to furnish compounds 5
(80%) and 6 (92%), respectively. Azetidinone 6 was then
reduced into azetidine 7 (99%) by reaction with AlH2Cl formed
in situ from a mixture of AlCl3 and LiAlH4.33ꢀ36 Unfortunately, a
similar sequence of reactions was not applicable for the synthesis
’ RESULTS
Chemistry. On the basis of the structure of our lead com-
pound 4, potentially six derivatives can be imagined that would
possess two (5, 9, 10), one (6, 8), or zero (7) carbonyl(s)
(Chart 2) instead of the three carbonyls of 4. Among these six
analogues that we planned to synthesize, 10 was not obtained
despite huge synthetic efforts, while the other ones were obtained
with various difficulties. We first thought to investigate the
synthetic scheme used in our previous report for the synthesis
of compounds 5 to 9.30 Indeed, this strategy offers the possibility
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Scheme 1a
a Reagents and conditions: (a) 5-bromopent-1-ene, KOH, Bu4NHSO4, KI, THF, rt, 24 h; (b) TBAF, AcOH, THF, rt, 1 h; (c) 4-phenylbutanoyl chloride,
pyridine, DCM, rt, 15 h; (d) 4-phenyl-1-butyl bromide, KI, NaH, DMF, 0 °C to rt, 4 h; (e) LiAlH4, AlCl3, Et2O, 0 to 40 °C, 4 h.
Scheme 2a
a Reagents and conditions: (a) 4-pentenoyl chloride, pyridine, DCM, 45 °C, 24 h; (b) HCl, AcOH, ACN, 0 °C, 3 h; (c) 4-phenyl-1-butyl bromide, KI,
NaH, DMF, 0 °C to rt, 4 h.
Scheme 3. Proposed Mechanism for the Formation of 15 in Anhydrous Conditions
of 8 and 9. Indeed, the precursor 14, resulting from N-acylation
of the starting material (80%, 13) and silyl ether deprotection
with HCl (95%, 14), could not be O-alkylated without β-lactam
degradation (Scheme 2).
Accordingly, we decided to protect the nitrogen atom of the
β-lactam ring with an alkyl group to avoid the rearrangement
previously observed. The paramethoxybenzyl (PMB) group37,38
was selected and introduced by reacting the starting material with
paramethoxybenzyl bromide under phase transfer conditions
(16, 79%). After tbutyldimethylsilyl deprotection, the alcohol
17 (99%) was alkylated by the same procedure as that described
above, to furnish compound 18 (91%) (Scheme 4). Azetidinone
18 was then reduced in azetidine 19 (73%) using monochlor-
ohydroalane as the reductive agent. Finally, the paramethoxy-
benzyl group was removed by an oxidative treatment with cerium
ammonium nitrate. The resulting azetidine 20 (86%) was acylated
by a method of peptidic coupling using PyBOP and pentenoic
acid, which resulted in compound 8 (54%).
After treatment with a strong base (NaH) and 4-phenyl-1-
butyl bromide, we recovered a complex mixture. The only
isolated product 15, in moderate yield (47%), resulted from
the four-membered ring-opening. As proposed in Scheme 3, the
driving force should be the acidity of the H3 proton due to the
electron-withdrawing effect of the imide function. The N1ꢀC2
bond of the carbanionic intermediate (Scheme 3a) might be
cleaved to furnish the hydroxyketene intermediate (Scheme 3b)
leading to the α,β-unsaturated carboxylate (Scheme 3c) trapped
in fine by the alkyl bromide reagent (see Supporting In-
formation). As a matter of fact, compound 12, devoid of exocyclic
carbonyl function, was readily O-alkylated into 6 under the same
experimental conditions (see Scheme 1). We conclude that
the O-alkylation step must occur before the N-acylation step.
Attempt to N-deprotect azetidinone 18, similarly to azetidi-
none 19, failed because the oxidized intermediate, i.e., hydroxyl
group on the benzylic position, is stabilized by intramolecular
hydrogen bonding and does not decompose further.39 Thus, we
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Scheme 4a
a Reagents and conditions: (a) PMBBr, KOH, Bu4NHSO4, KI, THF, rt, 24 h; (b) TBAF, AcOH, THF, rt, 1 h; (c) 4-phenylbutyl bromide, KI, NaH,
DMF, 0 °C to rt, 4 h; (d) LiAlH4, AlCl3, Et2O, 0 to 40 °C, 4 h; (e) CAN, H2O, ACN, 70 °C, 1 h; (f) 4-pentenoic acid, DIEA, PyBOP, DMF, rt, 15 h.
Scheme 5a
a Reagents and conditions: (a) bromoanisole, N,N-dimethylethylenediamine, CuI, K2CO3, dioxane, 105 °C, 24 h; (b) TBAF, AcOH, THF, rt, 1 h; (c)
4-phenylbutyl bromide, KI, NaH, DMF, 0 °C to rt, 4 h; (d) CAN, H2O, ACN, ꢀ15 °C, 15 min; (e) 4-pentenoyl chloride, pyridine, DCM, 45 °C, 24 h.
had to modify our synthetic route, and the paramethoxyphenyl
(PMP) substituent was considered as the N-protecting group
(Scheme 5).40,41 Compound 21 (61%) was obtained by orga-
nometallic coupling between the starting material and para-
methoxyphenyl bromide42 and then O-deprotected as already
described. The corresponding alcohol 22 (99%) was alkylated
under standard conditions. Compound 23 (37%) was obtained
in moderate yield and with some contamination with a diaster-
eoisomer (partial epimerization at the C3 position) that could
not be separated. PMP was easily removed using cerium ammo-
nium nitrate, and compound 24 (80%) was N-acylated with
4-pentenoyl chloride in the presence of pyridine giving azetidi-
none 9 (47%) (Scheme 5).
The synthesized analogues of compound 4 and all intermedi-
ates were fully characterized by the usual spectroscopies (see
Experimental Section). Of note, we observed particular features
concerning azetidine 1H and 13C NMR spectra due to the flipping
of the small ring. Indeed, spectra of compounds 7, 8, and 20
revealedsplit, broad, or missingsignals inCDCl3 or C6D6 at25 °C,
especially for protons or carbons at the positions 2, 4, and 5 of the
β-lactamic core. Experiments with 8 in C6D6 at 25 °C showed that
these signals were split into two but that a coalescence of signals
occurred when raising the temperature to 75 °C, confirming the
presence of conformers (see Supporting Information).
Biochemical Evaluations. The inhibition activity of com-
pounds 5 to 9 was evaluated on human recombinant FAAH.
Then, in order to determine whether our azetidinones are hydro-
lyzed by FAAH or not, incubation mixtures were analyzed by
HPLC/MS using a high resolution mass spectrometer (LTQ-
orbitrap) as LC detector. First, hydrolytic products from compounds
Despite numerous efforts, azetidine 10 (see Chart 2) could
not be prepared starting from the different available potential
precursors 4, 17, 22, and the commercial starting material by
applying synthetic routes inspired from Schemes 1, 4, and 5.
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Table 1. Determination of the Inhibition Activity Towards
Human FAAH
90 min. The reactions were stopped with cold acetonitrile. After
the addition of the internal standard (25, see Chart 3C) and
centrifugation, the supernatants were concentrated and injected
in the HPLC/MS system. In order to avoid artifacts due to matrix
effects, the blank controls (for chemical hydrolysis) were per-
formed with heat-denatured enzymes (either hFAAH or liver
homogenate depending on the assay). For comparison, the
enzymatic hydrolysis of anandamide (AEA) was similarly exam-
ined by HPLC/MS, using N-palmitoylethanolamine (PEA) as
the internal standard (Chart 3D). All compounds (native in-
hibitors/substrates, hydrolysis products, and internal standards,
see Chart 3) are separated on the HPLC reverse phase column
and detected by MS as [M + H]+ positive ions. Chart 3 gathers
the species detected and the corresponding m/z values. Results
are given as the area under curve (AUC) ratios of measured
compounds to the respective internal standard.
Assay with Liver Homogenate. The mammalian liver con-
tains a number of hydrolases, including FAAH. Thus, we
considered murine liver homogenate as a good model to assess
the stability of compounds 4 and 9 under physiological condi-
tions. The β-lactam ring-opening by hydrolases is a general
reaction that we expected to occur, as well as the ester hydrolysis
in the case of compound 4, leading to the cleavage of the C5 side
chain. Moreover, hydrolysis of the exocyclic N1—CdO bond
cannot be excluded. Accordingly, after the incubation of 4 and 9
with liver homogenate (10ꢀ5 M final concentration, 37 °C, 90 min),
we mainly detected by HPLC/MS the ions at m/z = 362.19675
and m/z = 348.21748 corresponding to the hydrolytic ring-
opening of 4 and 9, respectively. For compound 4, the ion at
m/z = 198.11302, which results from the cleavage of the ester
function, was also visible (see Supporting Information). Addi-
tionally, m/z = 262.14432 (Chart 3A) and m/z = 248.16505
(Chart 3B) ions, which correspond to the imide bond cleavage,
were also detected but in tenuous amounts (see Supporting
Information). Figure 1 shows the disappearance of the parent
compounds 4 and 9 under enzymatic hydrolysis ((+) liver
homogenate) by measuring the AUC for the peaks correspond-
ing to the ions at m/z = 344.18618 and m/z = 330.20692,
respectively. The data are expressed relative to the signal
obtained by incubating the parent compounds in the presence
of heat-inactivated liver homogenate ((ꢀ) liver homogenate)
(see Supporting Information). Following 90 min of incubation in
the presence of liver homogenate, more than 80% of β-lactams 9
and 4 (10ꢀ5 M) were hydrolyzed. Note, however, that 9 is twice
as stable as 4, probably most due to the replacement of the ester
bond (at the C5-O position) with an ether bond.
a
compd
X
Y
Z
pI50
IC50
4
5
6
7
8
9
O
O
O
8.27 ( 0.05
4.08 ( 0.03
4.05 ( 0.05
<3.5
0.005
82.8
O
H, H
H, H
H, H
O
O
O
H, H
H, H
H, H
H, H
88.6
H, H
H, H
O
>1000
2.9
5.54 ( 0.06
8.09 ( 0.06
O
0.008
a IC50 in μM (from three independent experiments performed in
duplicate).
4 and 9 were analyzed after incubation in the presence of murine
liver homogenate. Then, compound 9 was incubated with
murine liver homogenate after a preincubation with URB-597
or PF-750 (see 1 and 2, Chart 1), known for being irreversible
and selective FAAH inhibitors. The results were then compared
to similar experiments performed in the presence of human
recombinant FAAH.
FAAH Inhibition. Human recombinant enzyme, developed in
our laboratory,43 was used in a competitive hydrolytic assay using
[3H]-AEA as the substrate. Tested compounds, enzyme, and
[3H]-AEA were incubated at 37 °C during 10 min. The extent of
inhibition was evaluated by liquid scintillation counting of the
[3H]-ethanolamine resulting from the hydrolysis of the labeled
AEA. Regarding the inhibition potencies summarized in Table 1,
the relative importance of the three carbonyls (X, Y, and Z) can
be discussed as follows: (i) a significant loss of activity occurs
when the N-acyl chain is replaced by a N-alkyl chain indepen-
dently of the nature of the C5-O chain (compare compounds 5
and 6 to 4); (ii) the loss of activity is total when the carbonyl of
the β-lactam ring is also deleted (compound 7); (iii) the activity
is linked to the exocyclic N-carbonyl and not so much to the
β-lactam carbonyl (compare compounds 6 and 8); and (iv) the
deletion of the C5-O carbonyl has no influence on the activity
(compare compounds 9 and 4). On the basis of these observa-
tions, and contrary to what could be expected, we conclude that
the β-lactam carbonyl is not the main electrophilic group
responsible for FAAH inhibition. Indeed, its sole preservation
leads to a 40-times less potent inhibitor (6) than the analogue in
which the exocyclic N-carbonyl was exclusively preserved (8).
However, the inhibition potency is completely preserved
when both N-carbonyls are kept from the original structure,
which clearly indicates that the imide function is the most
important, at the expense of the ester one, for the inhibition of
human FAAH.
With these results, we have validated the HPLC/MS method
for further detection of the potential hydrolytic products of our
compounds under FAAH processing. The next experiments were
conducted with β-lactam 9 using anandamide (AEA) for
comparison.
To determine whether the hydrolysis of compound 9 by liver
homogenate is FAAH-dependent, the same experiment was
performed in the presence of FAAH inhibitors 1 and 2 (see
Chart 1). First of all, we confirmed the presence of FAAH activity
in our liver homogenate preparation, by measuring the hydrolysis
of anandamide (AEA) in the same conditions. We found a strong
decrease in AEA (10ꢀ5 M) following a 90 min incubation, which
was completely prevented when the liver homogenate was
preincubated with FAAH inhibitors 1 or 2 (Figure 2A). This
clearly shows that the FAAH activity in the liver is sufficient for a
total hydrolysis of its substrate and that we can inhibit this activity
Mechanistic Studies by HPLC/MS Analysis. We used a high
accuracy mass spectrometer coupled to a high performance
liquid chromatography apparatus (HPLC) to study the crude
mixtures obtained by incubating the β-lactams 4 and 9 (the most
active compounds of Table 1) with mouse liver homogenate
and with recombinant hFAAH, in Tris-HCl buffer at 37 °C for
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Chart 3. Ions Detected in the HPLC/MS Analysisa
aStructure and exact mass for the [M + H]+ ions detected by HPLC/MS analysis of incubation media containing 4 (A) or 9 (B) and native (or denatured)
hFAAH or liver homogenate. (C) Compound 25, 1-(pent-4-enoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-ethyl]-azetidin-2-one,30 was used as the internal
standard for the experiments performed with 4 and 9. (D) Structure and exact mass for anandamide (AEA) and N-palmitoylethanolamine (PEA) (used
as the internal standard for experiments involving anandamide) [M + H]+ ions.
was almost not detected in the presence of 1 (Figure 2C). These
results, at first surprising, may be explained by the reported lower
selectivity of 1 toward FAAH compared to that for 224,44 and
suggest that 9 is not hydrolyzed by FAAH but by other hydrolase(s)
present in the liver.
Assay with Recombinant hFAAH. To confirm the above-
mentioned results and to firmly establish that FAAH was not
responsible for the hydrolysis of 9, we performed a similar set of
experiments but using recombinant human FAAH instead of
liver homogenate.
First, we used AEA to confirm the suitability of our experi-
mental conditions. As expected, an almost total AEA hydrolysis
occurred in the presence of hFAAH, and this hydrolysis was fully
Figure 1. HPLC/MS analysis of 4 and 9 hydrolysis by liver homo-
blocked in the presence of the irreversible inhibitors 1 and 2
genate. Detection of m/z = 344.18618 and m/z = 330.20692 ions
(Figure 3A). Confirming what was suggested with the liver
homogenates, β-lactam 9 was not hydrolyzed at all, including
in the absence of inhibitors 1 or 2 (Figure 3B). We performed
additional experiments using larger amounts of hFAAH (150 μg/
tube versus 90 μg/tube), and even in this case, no hydrolytic
processing was observed (data not shown).
corresponding to the [M + H]+ ions of 4 and 9, respectively, after
incubation with mouse liver homogenate. The data are reported to the
signal obtained for the chemical hydrolysis ((ꢀ) liver homogenate), which
was determined using heat-inactivated liver homogenate. (***) P < 0.0001
compared to that of denatured liver (Student's t test). Data are shown as
the mean ( SEM of 3 independent experiments performed in duplicate.
’ DISCUSSION
using irreversible FAAH inhibitors such as 1 and 2. When analyzing
the fate of β-lactam 9, a difference between the effect of inhibitors 1
and 2 was observed, considering either the disappearance of
the native compound at m/z = 330.20692 (Figure 2B) or the
appearance of the hydrolytic product at m/z = 348.21748
(Figure 2C). From Figure 2B, it clearly appears that 1 completely
prevents the hydrolysis of 9, whereas 2 had no effect since 9 was
hydrolyzed to the same extent as that in the control. Accordingly,
the ion due to the azetidinone ring-opening (m/z = 348.21748),
was detected in a large amount in the presence of 2, whereas it
To date, only a few published FAAH inhibitors are described
as acting in a reversible manner. Their structures feature benzo-
thiazole,45 (thio)hydanthoin,46 and oxime carbamate47 moieties,
whose interaction with a FAAH catalytic pocket could not be
experimentally established. Indeed, short-lived and transient phe-
nomena most often prove to be hardly detectable by usual protein
analysis by MS and X-ray diffraction methods. Note, however, that
very recently, ketobenzimidazoles described as reversible inhibitors
were demonstrated to act by a noncovalent mechanism.48 Indeed,
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Figure 2. Study of the influence of FAAH inhibitors on the liver homogenate hydrolysis of AEA and 9. AEA (m/z = 348.29025) (A) and 9 (m/z =
344.18618) (B) were incubated at 10ꢀ5 M with liver homogenates in the presence (or not) of the FAAH inhibitors 1 and 2 (5.10ꢀ5 M). Heat-
denaturated liver homogenate was used as the control for chemical hydrolysis and used to normalize the data. PEA and 25 were used as internal standards
for the HPLC/MS analysis in A and B, respectively. (C) The relative quantification of the azetidinone ring-opening product during the incubation of 9.
(**) P < 0.01 compared to that of the denatured liver (C) or control (A and B) (ANOVA one-way, Dunnett’s post-test). Data are shown as the mean (
SEM of 3 independent experiments performed in duplicate.
homogenate showed a dramatic difference between the inhibi-
tors 1 and 2. Several hydrolases from liver were inhibited in the
presence of 1, whereas 2 only inhibited FAAH. Consequently,
β-lactam 9 was fully protected from hydrolases in the presence of
1 and was almost completely hydrolyzed in the presence of 2.
Additionally, a similar experiment was performed with recombi-
nant hFAAH. In this case, 9 did not undergo any processing,
independently of the presence of inhibitors 1 or 2. Taken
together, these results bear strong evidence for a nonhydrolytic
Figure 3. Study of the role of FAAH in the hydrolysis of 9. AEA (m/z =
348.29025) (A) and 9 (m/z = 344.18618) (B) were incubated at 10ꢀ5 M
with recombinant hFAAH in the presence (or not) of the FAAH
inhibitors 1 and 2 (5.10ꢀ5 M). Heat-denaturated enzyme preparation
was used as the control for chemical hydrolysis and used to normalize the
data. PEA and 25 were used as internal standards for the HPLC/MS
analysis in A and B, respectively. (**) P < 0.01 compared to that of
denatured hFAAH (ANOVA one-way, Dunnett’s post-test). Data are
shown as the mean ( SEM of 2 (AEA) or 3 (9) independent
experiments performed in duplicate.
interaction between our β-lactamic inhibitor and FAAH. More-
over, we have indirectly demonstrated that PF-750 (2) is a
selective inhibitor of FAAH, while URB-597 (1) is not selective
for FAAH since it inhibits other hydrolases present in a liver
homogenate. This problem has recently been commented on in
the literature.24,44 Because we demonstrated that 9 is not a slow
substrate for FAAH, the mechanism by which it reversibly
inhibits FAAH is questionable. Indeed, the fact that a β-lactam
ring is left intact in the presence of a serine hydrolase is quite
unusual. The experiments we performed with liver homogenates,
i.e., in the presence of a wide variety of serine hydrolases, confirm
that a β-lactam ring is easily processed by the classical Ser-Asp-
His catalytic triad. However, FAAH is a member of a distinct
serine hydrolase family, featuring the amidase signature and
possessing its own catalytic Ser-Ser-Lys triad. Thus, here, it
seems that the β-lactam ring is not targeted by the active serine
of this unaccustomed triad. However, although this carbonyl is
not the key electrophilic function, the β-lactam ring appears to be
necessary for an efficient FAAH inhibition. Indeed, azetidine 8 is
a 300-fold less potent inhibitor compared to 9, demonstrating
that both the exo and the endo carbonyls of the imide function
are essential. These results suggest that the ensemble of endo-
and exocyclic carbonyls, which constitute the imide function, is
the pharmacophore in FAAH inhibition and not the β-lactam
ring alone. Indeed, the β-lactam ring is probably not attacked by
the active serine because in this case, the resulting tetrahedral
intermediate would evolve toward the C2ꢀN1 bond cleavage
with the release of the four-membered cyclic strain as the driving
force. Thus, we hypothesize here that the β-lactam scaffold
correctly presents the exocyclic carbonyl of the imide function
to the active serine of hFAAH. Two explanations may be pro-
posed for the mechanism of FAAH inhibition: (i) the exocyclic
carbonyl, which is directed toward the active serine Ser241 (from
previous modelization studies),30 may undergo nucleophilic
from X-ray studies of cocrystal structures, the authors concluded
that a noncovalent inhibition mechanism exists because of the
absence of covalent interaction between Ser241 and the inhibitors.49
The present study has established that our β-lactamic inhibitors of
hFAAH are not substrates of this enzyme, although they can be
degraded by other hydrolases present in mammalian liver. A SAR
study based on the systematic replacement of CdO functions of the
lead compound 4 with CH2 units confirmed that the azetidinone
carbonyl is not essential for activity but rather reinforces the effect of
the exocyclic carbonyl. Thus, the imide function clearly appears to
be essential for an efficient hFAAH inhibition: compounds 8 and 9
exhibit good to excellent activities as does 4, whereas analogues 5
and 6 show significantly lower activities. Mass spectrometry is now
recognized as an accurate analytical tool in medicinal chemistry.50
Thus, we have validated a HPLC/MS assay to analyze crude
mixtures from enzymatic reactions and follow (i) the disappearance
of native inhibitors 4 and 9 and (ii) the appearance of their res-
pective potential hydrolysis products. Two different sources of
FAAH were considered: a mouse liver homogenate in which a
wide diversity of serine hydrolases is present and a human
recombinant FAAH. For the identification of the components
exclusively due to the reaction with FAAH, we preincubated both
enzyme sources with URB-597 (1) and PF-750 (2), two standard
irreversible FAAH inhibitors. Interestingly, the assay with liver
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Figure 4. Schematic proposed binding mode between 9 and FAAH.
attack (Figure 4a). Then, the resulting tetrahedral intermediate is
reversed, probably because of the absence of proton transfer to
N1 as required to be a good leaving group; (ii) there is no
nucleophilic attack at all. A high affinity between the N-acyl-β-
lactam and the catalytic site occurs (Figure 4b). Because of the
limited purity and stability of recombinant hFAAH and the
absence of X-ray data, the real structure of the inhibitor-hFAAH
complex formed with our β-lactamic inhibitor is not experimen-
tally accessible for discriminating between the two mechanisms
of reversible, nonprocessing inhibition.
on Merck silica-gel 60F254 with detection under UV light, and flash
chromatography was performed on silica gel (40ꢀ60 mesh) purchased
from Rocc (Belgium). Purity of tested compounds was assessed by
HPLC on a chiralpak IA column (4.6 mm ꢁ 250 mm, 5 μm particle size)
using a hexane/isopropanol eluent (95:05), at a flow rate of 1.0 mL/min,
and on a symmetry C18 column (4.6 mm ꢁ 250 mm, 5 μm particle size)
using a gradient of acetonitrile/H2O eluent (50:50 to 100:0), at a flow
rate of 1.2 mL/min (purity g97%). For compound 9, contamination
with the C3 diastereoisomer is less than 20%.
General Procedure for N-Alkylation (11 and 16). To a stirred
solution of the starting azetidinone (1 equiv) in tetrahydrofuran
(9.2 mL/mmol) at r.t. were added tetrabutylammonium hydrogen
sulfate (0.2 equiv), sodium iodide (4 equiv), potassium hydroxide
(2 equiv), and the suitable alkyl bromide (4 equiv). The mixture was
stirred for 15 h, the inorganic precipitate was filtered off and washed with
tetrahydrofuran, and the filtrate was concentrated under vacuum. After
purification by flash chromatography (cyclohexane/ethyl acetate), a
colorless oil was obtained (11 and 16).
’ CONCLUSIONS
We have disclosed a novel class of potent hFAAH reversible
inhibitors, featuring an imide function, which is part of a β-lactam
ring that is not hydrolyzed by the enzyme. The behavior of β-
lactams 4 and 9 in the presence of hFAAH and other hydrolases
from the liver was accurately analyzed thanks to a HPLC/MS
method validated with a set of reference compounds (AEA
(substrate), URB-597, and PF-750 (1 and 2, irreversible in-
hibitors)). Works are in progress to further exploit the imide
pharmacophore embedded into various cyclic templates for the
search of new FAAH inhibitors of pharmacological interest.
1-(Pent-4-enyl)-3(S)-[1(R)-(tert-butyldimethylsilyloxy)-ethyl]-azeti-
din-2-one (11). Yield: 76% (198 mg from 0.87 mmol of the starting
material). [α]D = ꢀ36.9 (c = 3.0). Rf = 0.29 (cyclohexane/ethyl acetate:
1
5/2). MS (ESI): m/z 298.16 ((M + H)+), 320.17 ((M + Na)+). H
NMR (300 MHz, CDCl3): δ = ꢀ0.06 (s, 3H), ꢀ0.05 (s, 3H), 0.74
(s, 9H), 1.05 (d, 3H, J = 6.3 Hz), 1.50 (m, 2H), 1.95 (m, 2H), 2.94ꢀ3.19
(m, 5H), 4.06 (m, 1H), 4.80ꢀ4.95 (m, 2H), 5.65 (ddt, 1H, J = 10.5 Hz,
J = 17.5 Hz, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3): δ = ꢀ5.0, ꢀ4.6,
17.8, 22.5, 25.6, 26.8, 31.0, 40.89, 40.92, 57.0, 65.0, 115.2, 137.2, 168.1.
IR (cmꢀ1): υ = 2856ꢀ2953, 1747, 1641, 1472, 1404, 1252, 835. HRMS:
C16H31NO2SiNa calculated, 320.2022; found, 320.2036.
’ EXPERIMENTAL SECTION
Chemistry. All solvents, including anhydrous solvents and reagents
were purchased from Acros Organics, Alfa Aesar, Cayman chemical,
Fluka, Sigma-Aldrich, or VWR and used without any further purification.
[3H]-AEA (60 Ci/mmol) was purchased from American Radiolabeled
Chemical (St Louis, MO). UltimaGold scintillation liquid was bought
from Perkin-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 proton and 75 MHz for carbon (Bruker Avance 300) or 500 MHz for
proton and 125 MHz for carbon (Bruker Avance 500) using deuterated
chloroform, methanol, or benzene. Chemical shifts are reported in
ppm relative to the signals of residual nondeuterated solvents (CDCl3,
7.26 and 77.16 ppm; CD3OD, 3.31 and 49.00 ppm; C6D6, 7.16 and
128.06 ppm). NMR coupling constants (J) are reported in hertz.
Melting points (mp) were determined on a B€uchi B-540 apparatus
calibrated with caffeine, vanillin, and phenacetin. Rotations were re-
corded on a Perkin-Elmer 241 MC polarimeter, at 20 °C, in CHCl3,
except for compound 20 in CH3OH. 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 ionization (ESI). High Resolution Mass Spectrometry
(HRMS) analyses were performed at the University College London
(UK). Infrared (IR) spectra were recorded using a FTIR-8400S Shimadzu
apparatus. Products were analyzed as thin films deposited on a SeꢀZn
crystal by evaporation from CH2Cl2 solutions. TLC analysis was performed
General Procedure for Silyl Ether Deprotection (12, 17,
and 22). To a stirred solution of silyl ether (1 equiv) in dry tetra-
hydrofuran (33 mL/mmol) at r.t. was added, dropwise, a solution of
tetrabutyl ammonium fluoride in tetrahydrofuran (5 equiv). The solu-
tion was stirred for 1 h, and then acetic acid was added (2.2 equiv). The
solution was stirred for an additional 15 min and then extracted three
times with dichloromethane. The organic layers were combined, washed
with brine and water, dried over MgSO4, filtered, and concentrated under
vacuum. After purification by flash chromatography (ethyl acetate/
methanol), a colorless oil (12) or a white solid (17 and 22) was obtained.
1-(Pent-4-enyl)-3(S)-[1(R)-hydroxyethyl]-azetidin-2-one (12). Yield:
93% (135.5 mg from 0.79 mmol of 11). [α]D = ꢀ22.4 (c = 1.1). Rf = 0.33
(ethyl acetate/methanol: 99/1). MS (ESI): m/z 184.20 ((M + H)+),
1
206.15 ((M + Na)+). H NMR (300 MHz, CDCl3): δ = 1.20 (d, 3H,
J = 6.3 Hz), 1.57 (m, 2H), 2.02 (m, 2H), 2.98ꢀ3.23 (m, 5H), 3.25 (broad
s, 1H), 4.07 (m, 1H), 4.82ꢀ5.04 (m, 2H), 5.72 (ddt, 1H, J = 10.5 Hz,
J = 17.2 Hz, J = 6.5Hz). 13C NMR (75MHz, CDCl3): δ = 21.4, 26.7, 31.0,
41.1, 41.6, 56.7, 64.8, 115.4, 137.3, 168.6. IR (cmꢀ1): υ = 3402, 2928,
1717, 1641, 1418, 1238. HRMS: C10H18NO2 calculated, 184.13375;
found, 184.13297.
1-(Pent-4-enyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-
azetidin-2-one (5). To a stirred solution of 12 (1 equiv) in dry
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dichloromethane (20 mL/mmol) at r.t. were added pyridine (2 equiv)
and 4-phenylbutanoyl chloride (2 equiv) under argon atmosphere. After
stirring overnight, the mixture was diluted in dichloromethane, and the
excess of acyl chloride was quenched by a 10% aqueous solution of
Na2CO3. The organic layer was washed with 3 N aqueous solution of
HCl and brine, dried over MgSO4, filtered, and concentrated under
vacuum. After purification by flash chromatography (dichloromethane/
ethyl acetate), a colorless oil was obtained. Yield: 80% (33 mg from 0.12
mmol of 12). [α]D = ꢀ2.1 (c = 1.8). Rf = 0.34 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z 330.18 ((M + H)+), 352.15 ((M + Na)+).
1H NMR (300 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.3 Hz), 1.61 (m,
2H), 1.93 (m, 2H), 2.07 (m, 2H), 2.30 (t, 2H, J = 7.5 Hz), 2.63 (t, 2H, J =
7.6 Hz), 3.07ꢀ3.33 (m, 5H), 4.90ꢀ5.08 (m, 2H), 5.21 (m, 1H), 5.77
azetidin-2-one). After 4 h, the reaction mixture was cooled, and water
was added. The aqueous layer was extracted with dichloromethane. The
organic layers were combined, dried over MgSO4, filtered, and concen-
trated under vacuum. After purification by flash chromatography
(dichloromethane/methanol), a colorless oil was obtained in all cases.
1-(Pent-4-enyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-azetidine (7).
Yield: 99% (148 mg from 0.50 mmol of 6). [α]D = ꢀ26.0 (c = 3.0). Rf =
0.21 (dichloromethane/methanol: 9/1). MS (ESI): m/z 302.17 ((M +
H)+). 1H NMR (500 MHz, CDCl3, 25 °C): δ = 0.97 (d, 3H, J = 6.3 Hz),
1.51ꢀ1.72 (m, 4H), 2.04 (m, 2H), 2.59 (m, 2H), 2.90 (m, 3H), 3.29 (m,
1H), 3.41 (broad signal, 1H), 3.60 (m, 2H), 3.70 (broad signal, 1H),
4.16 (broad signal, 2H), 4.80ꢀ4.95 (m, 2H), 5.64 (ddt, 1H, J = 10.5 Hz,
J = 17.2 Hz, J = 6.5 Hz), 7.05ꢀ7.30 (m, 5H). 13C NMR (125 MHz,
CDCl3, 25 °C): δ = 16.4, 23.3, 28.2, 29.5, 30.3, 34.8, 35.6, 53.6 (broad
signal), 54.1, 54.5, 68.5, 71.8 (broad signal), 116.5, 125.8, 128.3, 128.3,
136.0, 142.1. IR (cmꢀ1): υ = 2854ꢀ2930, 1641, 1452, 1377, 1157, 1088.
HRMS: C20H32NO calculated, 302.2484; found, 302.2469.
1-(4-Methoxybenzyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-azetidine
(19). Yield: 73% (64 mg from 0.25 mmol of 18). [α]D = ꢀ19.8 (c = 1.0).
Rf = 0.21 (dichloromethane/methanol: 94/6). MS (ESI): m/z 354.13
((M + H)+), 390.29 ((M + Na)+). 1H NMR (500 MHz, CDCl3, 25 °C):
δ = 0.90 (d, 3H, J = 6.2 Hz), 1.54ꢀ1.80 (m, 4H), 2.56 (t, 2H, J = 7.2 Hz),
2.75 (m, 1H), 3.24 (td, 1H, J = 6.2 Hz, J = 12.4 Hz, AB system), 3.34 (m,
1H), 3.47 (m, 1H), 3.52ꢀ3.62 (m, 2H), 3.67 (s, 3H), 3.84ꢀ3.98 (m,
4H), 6.76 (d, 2H, J = 8.6 Hz), 7.06ꢀ7.20 (m, 5H), 7.26 (d, 2H, J = 8.6
Hz). 13C NMR (125 MHz, CDCl3, 25 °C): δ = 16.5, 28.4, 29.8, 34.8,
35.7, 53.0, 53.7, 55.3, 57.8, 68.6, 72.6, 114.6, 122.2, 125.9, 128.3, 128.40,
128.43, 131.4, 133.7, 142.2, 160.4. IR (cmꢀ1): υ = 2841ꢀ2928, 1612,
1516, 1454, 1375, 1252, 1180, 1088, 1030. HRMS: C23H32NO2
calculated, 354.2433; found, 354.2424.
3(S)-[1(R)-(4-Phenylbutoxy)-ethyl]-azetidine (20). To a stir-
red solution of 19 (1 equiv) in acetonitrile (23 mL/mmol of 19) was
added dropwise a solution of cerium ammonium nitrate (4 equiv) in
water (2 mL/mmol of CAN). The solution was stirred at 70 °C during
1 h, and then water was added (13 mL/mmol of 19). The aqueous layer
was extracted three times with ethyl acetate. The organic layers were
combined, dried over MgSO4, filtered, and concentrated under vacuum.
After purification byflash chromatography (dichloromethane/methanol),
a colorless oil was obtained. Yield: 86% (31 mg from 0.15 mmol of 19).
[α]D = ꢀ21.6 (c = 3.1). Rf = 0.20 (dichloromethane/methanol: 94/6).
MS (ESI): m/z 234.19 ((M + H)+). 1H NMR (500 MHz, MeOD): δ =
1.05 (d, 3H, J = 6.2 Hz), 1.58ꢀ1.77 (m, 4H), 2.64 (m, 2H), 2.90 (m, 1H),
3.40 (td, 1H, J = 6.3 Hz, J = 12.6 Hz, AB system), 3.56 (m, 1H), 3.65 (td,
1H, J = 6.5 Hz, J = 12.9 Hz, AB system), 3.95 (dd, 1H, J = 7.2 Hz, J = 10.6
Hz), 4.01ꢀ4.09 (m, 3H), 7.08ꢀ7.27 (m, 5H). 13C NMR (125 MHz,
MeOD, 25 °C): δ = 16.4, 29.3, 30.6, 36.7, 38.9, 69.8, 74.9, 126.7, 129.3,
129.4, 143.7 (two carbons are not visible because they are masked by the
deuterated methanol signal). 13C NMR (125 MHz, C6D6, 25 °C): δ =
16.5, 28.5, 30.0, 36.2, 38.0, 49.5, 49.8, 69.0, 75.2, 126.1, 128.7, 129.0, 143.0
(all carbons are visible). IR (cmꢀ1): υ = 2859ꢀ2932, 1643, 1339, 1254.
HRMS: C15H24NO calculated, 234.1858; found, 234.1863.
1-(Pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-
azetidine (8). To a stirred solution of 4-pentenoic acid (1.5 equiv) in
dimethylformamide (12 mL/mmol) was added N,N-diisopropylethyla-
mine (3 equiv) and PyBOP (1.5 equiv) under argon atmosphere. The
solution was stirred for 10 min, then a solution of compound 20 in
dimethylformamide (12 mL/mmol) was added, and the mixture was
stirred overnight. The reaction mixture was diluted with water and diethyl
ether, and the aqueous phase was extracted three times with diethyl ether.
The organiclayers were combined, washedtwicewith anaqueous solution
of 3 N HCl, dried over MgSO4, filtered, and concentrated under vacuum.
After purification by flash chromatography (cyclohexane/ethyl acetate), a
colorless oil was obtained. Yield: 54% (23 mg from 0.13 mmol of 20). Rf =
0.18 (cyclohexane/ethyl acetate: 1/1). MS (ESI): m/z 316.20 ((M + H)+),
(ddt, 1H, J = 10.5 Hz, J = 17.2 Hz, J = 6.5 Hz), 7.12ꢀ7.31 (m, 5H). 13
C
NMR (75 MHz, CDCl3): δ = 18.7, 26.6, 26.8, 31.0, 33.9, 35.1, 41.3, 42.7,
54.5, 68.6, 115.7, 126.1, 128.5, 128.6, 137.3, 141.4, 166.7, 172.7. IR
(cmꢀ1): υ = 2862ꢀ2930, 1744, 1728, 1641, 1454, 1413, 1240, 1134.
HRMS: C20H27NO3Na calculated, 352.1889; found, 352.1900.
General Procedure for O-Alkylation (6 and 18). To a stirred
suspension of sodium hydride (4 equiv) in dry dimethylformamide
(6 mL/mmol of alcohol precursor) at 0 °C was added, dropwise, the
alcohol precursor (1 equiv) in dry dimethylformamide (6 mL/mmol of
alcohol precursor) under argon atmosphere. The suspension was stirred
for 30 min at 0 °C, and then freshly dried potassium iodide (3 equiv) and
4-phenyl-1-butyl bromide (3 equiv) were added. The suspension was
stirred for an additional 30 min and then allowed to warm up to r.t. After
4 h, the reaction was quenched, at low temperature, with an aqueous
saturated solution of NH4Cl, and the aqueous layer was extracted several
times with diethyl ether. The organic layers were combined, dried over
MgSO4, filtered, and concentrated under vacuum. After purification by
flash chromatography (cyclohexane/ethyl acetate), a colorless oil was
obtained in all cases.
1-(Pent-4-enyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-azetidin-2-one
(6). Yield: 92% (196.1 mg from 0.68 mmol of 12). [α]D = ꢀ25.7 (c =
1.1). Rf = 0.47 (cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z 316.16
((M + H)+), 338.23 ((M + Na)+). 1H NMR (300 MHz, CDCl3): δ =
1.21 (d, 3H, J = 6.3 Hz), 1.51ꢀ1.72 (m, 4H), 2.07 (m, 2H), 2.61 (t, 2H, J
= 7.5 Hz), 3.04ꢀ3.29 (m, 4H), 3.40 (td, 1H, J = 6.3 Hz, J = 12.5 Hz, AB
system), 3.57 (td, 1H, J = 6.3 Hz, J = 12.5 Hz, AB system), 3.72 (m, 1H),
4.95ꢀ5.08 (m, 2H), 5.77 (ddt, 1H, J = 10.5 Hz, J = 17.2 Hz, J = 6.5 Hz),
7.12ꢀ7.31 (m, 5H). 13C NMR (75 MHz, CDCl3): δ =18.5, 26.8, 28.1,
29.7, 31.0, 35.7, 41.1, 42.1, 55.7, 68.9, 72.7, 115.4, 125.7, 128.3, 128.4,
137.4, 142.5, 168.4. IR (cmꢀ1): υ = 2860ꢀ2932, 1747, 1641, 1452,
1407, 1103. HRMS: C20H29NO2Na calculated, 338.2096; found,
338.2107.
1-(4-Methoxybenzyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-azetidin-
2-one (18). Yield: 91% (156 mg from 0.46 mmol of 17). [α]D = 8.0 (c =
1.9). Rf = 0.23 (cyclohexane/ethyl acetate: 1/1). MS (ESI): m/z 368.24
((M + H)+), 390.29 ((M + Na)+). 1H NMR (300 MHz, CDCl3): δ =
1.20 (d, 3H, J = 6.2 Hz), 1.48ꢀ1.77 (m, 4H), 2.62 (t, 2H, J = 7.4 Hz),
3.04ꢀ3.24 (m, 3H), 3.39 (td, 1H, J = 6.2 Hz, J = 12.4 Hz, AB system),
3.56 (td, 1H, J = 6.2 Hz, J = 12.4 Hz, AB system), 3.75 (m, 1H), 3.79 (s,
3H), 4.21 (d, 1H, J = 14.9 Hz, AB system), 4.39 (d, 1H, J = 14.9 Hz, AB
system), 6.85 (d, 2H, J = 8.6 Hz), 7.12ꢀ7.31 (m, 7H). 13C NMR (75
MHz, CDCl3): δ = 18.5, 28.2, 29.9, 35.9, 42.1, 45.1, 55.3, 56.0, 69.0, 72.7,
114.1, 125.8, 128.4, 128.5, 129.5, 142.6, 159.3, 168.5. IR (cmꢀ1): υ =
2860ꢀ2932, 1747, 1610, 1512, 1452, 1402, 1246. HRMS: C23H29NO3-
Na calculated, 390.2045; found, 390.2056.
General Procedure for the Reduction of Azetidin-2-one (7
and 19). To a stirred suspension of aluminum chloride (3 equiv) in dry
diethyl ether (12 mL/mmol of azetidin-2-one) at 0 °C was added lithium
aluminum hydride (3 equiv) under argon atmosphere. The suspension
was stirred for 10 min, then refluxed for 30 min, and finally, the azetidin-
2-one (1 equiv) was added dropwise in dry diethyl ether (6 mL/mmol of
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1
338.13 ((M + Na)+). H NMR (500 MHz, CDCl3, 25 °C): δ = 1.08
acetate), a colorless oil was obtained. Yield: 47% (30 mg from 0.19 mmol
of 24). Rf = 0.23 (cyclohexane/ethyl acetate: 7/1). MS (ESI): m/z 330.25
((M + H)+), 352.23 ((M + Na)+). 1H NMR (500 MHz, CDCl3): δ =
1.22 (d, 3H, J = 6.3 Hz), [1.31 (d, 3H, J = 6.1 Hz)], 1.53ꢀ1.77 (m, 4H),
2.40 (m, 2H), 2.60 (t, 2H, J = 7.3 Hz), 2.71ꢀ2.82 (m, 2H), 3.25 (m, 1H),
3.33 (m, 1H), 3.55ꢀ3.64 (m, 3H), 3.81 (m, 1H), 4.97ꢀ5.11 (m, 2H),
5.82 (m, 1H), 7.13ꢀ7.30 (m, 5H). Chemical shifts of the minor C-3
diastereoisomer are given in parentheses. 13C NMR (125 MHz, CDCl3):
δ = 18.0 (17.1), 28.01 (2C) (28.04), 29.6 (29.5), 35.7, 35.8, 39.5 (39.4),
55.0 (54.0), 69.0 (69.1), 71.9 (72.1), 115.7, 125.8, 128.36, 128.44, 136.6,
142.4, 166.3 (166.0), 170.4. IR (cmꢀ1): υ = 2856ꢀ2980, 1786, 1697,
1452, 1387, 1312. HRMS: C20H27NO3Na calculated, 352.1889; found,
352.1880.
Enzymes Preparation. hFAAH was prepared in our laboratory as
previously described.43 Mouse liver homogenates were prepared by
homogenizing a mouse liver in Tris buffer (pH 7.4, 4 mL) before cen-
trifuging it at 800g (15 min). The supernatant was recovered and
aliquots stored until use. Aliquots of hFAAH or liver homogenate were
boiled (45 min, in a water bath) and used as control to account for the
chemical hydrolysis of our compounds of interest (blank).
IC50 Determination Using hFAAH. Tubes containing the
enzyme43 (6 μg in 10 mM Tris-HCl, 1 mM EDTA, and 0.1% (w/v)
BSA, pH 7.4, 165 μL), test compounds in DMSO or DMSO alone for
controls (10 μL), and [3H]-AEA (50,000 dpm, 2 μM final concentra-
tion, 25 μL) were incubated at 37 °C for 10 min. Reactions were stopped
by rapidly placing the tubes in ice and adding 400 μL of ice-cold
chloroform/methanol (1:1, v/v) followed by vigorous mixing. Phases
were separated by centrifugation at 850g, and aliquots (200 μL) of the
upper methanol/buffer phase were counted for radioactivity by liquid
scintillation counting. In all experiments, tubes containing only buffer
were used as the control for chemical hydrolysis (blank), and this value
was systematically subtracted. Using these conditions, URB-597 inhibits
hFAAH with an IC50 value of 40 nM.
Incubations for the HPLC/MS Analyses. Test compounds
(4, 9, or anandamide) at 2.10ꢀ4 M or their vehicle alone for controls
(acetonitrile 10 μL) were incubated in TE buffer (10 mM Tris-HCl,
1 mM EDTA, and 0.1% (w/v) BSA, pH 7.4, 190 μL) for 90 min at 37 °C
in the presence of hFAAH (90 μg/tube) or liver homogenate (22.5 μg/
tube). Reactions were stopped by rapidly placing the tubes in ice and
adding 200 μL of ice-cold acetonitrile at which point an internal standard
(10 μL of 2.10ꢀ4 M solution in acetonitrile) was added followed by
vigorous mixing. Compound 25 and N-palmitoylethanolamine were used
as internal standards for the experiments with 4 and 9 and anandamide,
respectively. Proteins were further precipitated by centrifugation at
10000g, and aliquots (300 μL) of the supernatants were concentrated
under reduced pressure. The residues were recovered in 30 μL of
acetonitrile (4 and 9) or chloroform/methanol (1:1) (anandamide) for
HPLC/MS analysis. In all experiments, tubes containing compounds and
denatured enzymes (either hFAAH or liver homogenate) were used as a
controlforchemicalhydrolysis in buffer (blank). Whenneeded, PF-750or
URB-597 (1.10ꢀ3 M, 10 μL in acetonitrile) was used as FAAH inhibitors
and preincubated for 5 min with the enzyme preparation (180 μL) before
adding the test compounds.
(d, 3H, J = 6.1 Hz), 1.54ꢀ1.76 (m, 4H), 2.15 (m, 2H), 2.36 (m, 2H),
2.57 (m, 1H), 2.63 (m, 2H), 3.31 (td, 1H, J = 6.4 Hz, J = 12.8 Hz, AB
system), 3.46 (m, 1H), 3.60 (td, 1H, J = 6.4 Hz, J = 12.5 Hz, AB system),
3.75 (broad signal, 1H), 3.92 (broad signal, 1H), 4.05 (broad signal,
2H), 4.96ꢀ5.14 (m, 2H), 5.84 (ddt, 1H, J = 10.5 Hz, J = 17.2 Hz, J =
6.5 Hz), 7.14ꢀ7.34 (m, 5H). 13C NMR (125 MHz, CDCl3, 25 °C):
δ = 16.6, 28.2, 29.0, 29.80, 29.83, 30.7, 34.4, 35.8, 50.7 (broad signal,
2C), 68.9, 76.4, 115.4, 125.9, 128.4, 128.5, 137.4, 142.5, 172.6. IR
(cmꢀ1): υ = 2858ꢀ2926, 1628, 1454, 1373, 1335, 1113, 1088. HRMS:
C20H29NO2Na calculated, 338.2096; found, 338.2087.
1-(4-Methoxyphenyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-
azetidin-2-one (23). To a stirred suspension of sodium hydride (1.1
equiv) in dry dimethylformamide (6 mL/mmol of alcohol precursor) at
0 °C was added, dropwise, the alcohol precursor (1 equiv) in dry
dimethylformamide (6 mL/mmol of alcohol precursor) under argon
atmosphere. The suspension was stirred for 30 min at 0 °C, and then
freshly dried potassium iodide (3 equiv) and 4-phenyl-1-butyl bromide
(3 equiv) were added. The suspension was stirred for an additional 30
min and then allowed to warm up to r.t. After 4 h, the reaction was
quenched, at low temperature, with an aqueous saturated solution of
NH4Cl, and the aqueous layer was extracted several times with diethyl
ether. The organic layers were combined, dried over MgSO4, filtered,
and concentrated under vacuum. After purification by flash chromato-
graphy (cyclohexane/ethyl acetate), a colorless oil was obtained. Yield:
37% (85 mg from 0.64 mmol of 22). Rf = 0.50 (cyclohexane/ethyl
acetate: 1/1). MS (ESI): m/z 354.12 ((M + H)+), 376.29 ((M + Na)+).
1H NMR (500 MHz, CDCl3): δ = 1.28 (d, 3H, J = 6.3 Hz), 1.48ꢀ1.72
(m, 4H), 2.57 (t, 2H, J = 7.5 Hz), 3.29 (m, 1H), 3.41 (m, 1H), 3.49ꢀ3.64
(m, 3H), 3.77 (s, 3H), 3.81 (m, 1H), 6.86 (d, 2H, J = 9.0 Hz), 7.04ꢀ7.36
(m, 7H). Chemical shifts of the minor C-3 diastereoisomer are given in
parentheses. 13C NMR (125 MHz, CDCl3): δ = 18.7 (16.6), 28.0 (28.1),
29.7 (29.6), 35.68 (35.73), 41.7 (40.5), 55.2 (53.7), 55.5, 69.0 (68.9), 72.9
(72.2), 114.4, 117.5, 125.7 (125.7), 128.3, 128.4, 132.23 (132.17), 142.5,
156.0, 164.9 (164.7). IR (cmꢀ1): υ = 2860ꢀ2930, 1738, 1512, 1454,
1246. HRMS: C22H28NO3 calculated, 354.20692; found, 354.20643.
3(S)-[1(R)-(4-Phenylbutoxy)-ethyl]-azetidin-2-one (24). To
a stirred solution of 23 (1 equiv) in acetonitrile (23 mL/mmol of 23) at
ꢀ15 °C was added dropwise a solution of cerium ammonium nitrate (4
equiv) in water (2 mL/mmol of CAN). The solution was stirred at low
temperature for 15 min, and then water was added (13 mL/mmol of 23).
The aqueous layer was extracted three times with ethyl acetate. The
organic layers were combined, dried over MgSO4, filtered, and concen-
trated under vacuum. After purification by flash chromatography
(cyclohexane/ethyl acetate), a colorless oil was obtained. Yield: 80%
(47 mg from 0.24 mmol of 23). Rf = 0.23 (cyclohexane/ethyl acetate:
1/1). MS (ESI): m/z 354.12 ((M + H)+), 376.29 ((M + Na)+). H
1
NMR (300 MHz, CDCl3): δ = 1.26 (d, 3H, J = 6.3 Hz), 1.52ꢀ1.75 (m,
4H), 2.62 (d, 2H, J = 7.0 Hz), 3.19ꢀ3.51 (m, 4H), 3.52ꢀ3.62 (m, 1H),
3.66ꢀ3ꢀ80 (m, 1H), 5.74 (broad s, 1H), 7.08ꢀ7.33 (m, 5H). Chemical
shifts for the minor C-3 diastereoisomer are given in parentheses. 13C
NMR (125 MHz, CDCl3): δ = 18.5 (16.5), 28.1 (28.2), 29.8 (29.7),
35.8, 39.0 (37.8), 58.0 (56.5), 68.9, 72.9 (72.0), 125.8, 128.4, 128.5,
142.6, 169.5. IR (cmꢀ1): υ = 3309, 2853ꢀ2935, 1726, 1452, 1101.
HRMS: C15H21NO2Na calculated, 270.1470; found, 270.1467.
1-(Pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutoxy)-ethyl]-azeti-
din-2-one (9). To a stirred solution of 24 (1 equiv) in dry dichlor-
omethane (8.6 mL/mmol) at r.t. were added pyridine (2 equiv) and
4-pentenoyl chloride (2 equiv) under argon atmosphere. The reaction
mixture was refluxed for 24 h then diluted in dichloromethane, and the
excess of acyl chloride was quenched by 10% aqueous solution of
Na2CO3. The organic layer was washed with 3 N aqueous solution of
HCl and brine, dried over MgSO4, filtered, and concentrated under
vacuum. After purification by flash chromatography (cyclohexane/ethyl
HPLC/MS Analyses of the Incubation Medium. The residues
obtained following hFAAH or liver incubation were analyzed by HPLC/
MS using an LTQ Orbitrap mass spectrometer (ThermoFischer Scien-
tific) coupled to an Accela HPLC system (ThermoFischer Scientific).
Separation of the hydrolytic products was performed on a C-18
Supelguard precolumn and a Supelcosil LC-18 column (3 μM, 4 ꢁ
150 mm) (Sigma-Aldrich). Chromatographic conditions (0.5 mL/min)
were as follows: (i, for 4 and 9) from A (methanol/H2O/acetic acid;
60:40:0.1) to B (methanol/acetic acid; 100:0.1) in 15 min, followed by a
5 min plateau with B and 8 min re-equilibration with A; and (ii, for
anandamide) from A (methanol/H2O/acetic acid; 75:25:0.1) to B
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Journal of Medicinal Chemistry
ARTICLE
(methanol/acetic acid; 100:0.1) in 15 min, followed by a 5 min plateau
with B and 8 min re-equilibration with A, using a flow rate of 0.5 mL/
min. The MS analyses were performed in the positive mode with an
APCI ionization source. The capillary and APCI vaporizer temperatures
were set at 250 and 400 °C, respectively.
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from Simple to Complex. Drug Discovery Today 2010, 15 (11ꢀ12), 474–
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’ ASSOCIATED CONTENT
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Supporting Information. Experimental procedures and
b
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As an Emerging Target of Pharmacotherapy. Pharmacol. Rev. 2006, 58
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spectroscopic details of compounds 15, 16, 17, 21, and 22;
discussion about the rearrangement which resulted in 15; 1H and
13C NMR spectra of azetidines 7, 8, and 20; and complementary
figures of HPLC/MS analysis. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Receptor Agonists As Medicines. Br. J. Pharmacol. 2009, 156 (3),
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Cannabis Derivatives and Endocannabinoid Agonists. Recent Pat. CNS
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’ AUTHOR INFORMATION
Corresponding Author
(10) Ashton, J. C.; Wright, J. L.; McPartland, J. M.; Tyndall, J. D. A.
Cannabinoid CB1 and CB2 Receptor Ligand Specificity and the
Development of CB2-Selective Agonists. Curr. Med. Chem. 2008,
15 (14), 1428–1443.
*Phone: +32 10 47 27 40. Fax: +32 10 47 41 68. E-mail: jacqueline.
marchand@uclouvain.be (J.M.B.); giulio.muccioli@uclouvain.be
(G.G.M.).
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Advances in Cannabinoid Receptor Agonists. Expert Opin. Ther. Pat.
2009, 19 (12), 1647–1673.
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Ligands of the Endocannabinoid System. Curr. Med. Chem. 2010, 17
(14), 1341–1359.
(13) Moreira, F. A.; Grieb, M.; Lutz, B. Central Side-Effects of
Therapies Based on CB1 Cannabinoid Receptor Agonists and Antago-
nists: Focus on Anxiety and Depression. Best Pract. Res., Clin. Endocrinol.
Metab. 2009, 23 (1), 133–144.
(14) Karst, M.; Wippermann, S. Cannabinoids against Pain. Efficacy
and Strategies to Reduce Psychoactivity: a Clinical Perspective. Expert
Opin. Invest. Drugs 2009, 18 (2), 125–133.
(15) Ross, R. A. Allosterism and Cannabinoid CB1 Receptors: The
Shape of Things to Come. Trends Pharmacol. Sci. 2007, 28 (11), 567–572.
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Coupled Receptors. Eur. J. Pharm. Sci. 2004, 21 (4), 407–420.
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Characterisation to Therapeutics. Chem. Biodiversity 2007, 4, 1882–1902.
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Acid Amide Hydrolase. Annu. Rev. Biochem. 2005, 74, 411–432.
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’ ACKNOWLEDGMENT
The UCL (Universitꢀe catholique de Louvain) and the F.R.S.-
FNRS (Fonds de la Recherche Scientifique, Belgium) are grate-
fully acknowledged for financial support of this work (FRFC grant,
no. 2.4.654.06 F). This work is partially supported by the Inter-
university Attraction Pole program (IAP P6/19 PROFUSA). J.M.-B.
is senior research associate of the Belgian F.R.S.-FNRS. We wish
to warmly thank Kaneka Corporation (Japan), in particular
Dr. Claudio Salvagnini, for providing the starting azetidinone
((3R,4R)-3-[(R)-10-(tbutyldimethylsilyloxy)-ethyl]-4-acetoxy-
2-azetidinone). Dr Geoffray Labar is also acknowledged for the
preparation of hFAAH.
’ ABBREVIATIONS USED
SAR, structureꢀactivity relationship; LC, liquid chromatogra-
phy; MS, mass spectrometry; hFAAH, human fatty acid amide
hydrolase; CB, cannabinoid; GPCR, G-protein coupled recep-
tor; CB1, cannabinoid receptor subtype-1; CB2, cannabinoid
receptor subtype-2; AEA, anandamide; 2-AG, 2-arachidonoyl-
glycerol; CNS, central nervous system; MALDI, matrix-assisted
laser desorption/ionization; TBDMS, tert-butyldimethylsilyl;
THF, tetrahydrofurane; TBAF, tetra-n-butylammonium fluor-
ide; DCM, dichloromethane; DMF, dimethylformamide; ACN,
acetonitrile; PMB, paramethoxybenzyl; PMP, paramethoxyphenyl;
PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate; CAN, ceric ammonium nitrate; DIEA, N-N,
diisopropylethylamine; DMSO, dimethyl sulfoxide; PEA, N-pal-
mitoylethanolamine; AUC, area under curve
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