β-lactams derived from a carbapenem chiron are selective inhibitors of human fatty acid amide hydrolase versus human monoacylglycerol lipase

Article abstract of DOI:10.1021/jm9008532

A library of 30 β-lactams has been prepared from (3R,4R)-3-[(R)- 1′-(tbutyldimethylsilyloxy)-ethyl]-4-acetoxy-2-azetidinone, and the corresponding deacetoxy derivative, by sequential N- and O-functionalizations with various ω-alkenoyl and ω-arylalkanoyl c

Full text of DOI:10.1021/jm9008532

7054 J. Med. Chem. 2009, 52, 7054–7068
DOI: 10.1021/jm9008532
β-Lactams Derived from a Carbapenem Chiron Are Selective Inhibitors of Human Fatty Acid Amide
Hydrolase versus Human Monoacylglycerol Lipase
Marion Feledziak,†,§ Catherine Michaux,‡ Allan Urbach,† Geoffray Labar,§ Giulio G. Muccioli,§,^ Didier M. Lambert,§ and
Jacqueline Marchand-Brynaert*,†
†Uniteꢀ de Chimie Organique et Meꢀdicinale, Universiteꢀ Catholique de Louvain, Ba^timent Lavoisier, Place Louis Pasteur 1, B-1348 Louvain-La-
Neuve, Belgium, §Uniteꢀ de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute, Universiteꢀ Catholique de Louvain,
Avenue E. Mounier 73.40, B-1200 Bruxelles, Belgium, and ‡Laboratoire de Chimie Biologique Structurale, Faculteꢀs Universitaires Notre-Dame de
la Paix, rue de Bruxelles 61, B-5000 Namur, Belgium. ^ Present address: Bioanalysis and Pharmacology of Bioactive Lipids laboratory, Louvain
Drug Research Institute, Universiteꢀ Catholique de Louvain, Avenue E. Mounier 72.30, B-1200 Bruxelles, Belgium.
Received June 11, 2009
A library of 30 β-lactams has been prepared from (3R,4R)-3-[(R)-10-(tbutyldimethylsilyloxy)-ethyl]-4-
acetoxy-2-azetidinone, and the corresponding deacetoxy derivative, by sequential N- and O-functionaliza-
tions with various ω-alkenoyl and ω-arylalkanoyl chains. All compounds were selective inhibitors of
hFAAH versus hMGL, and IC50 values in the nanomolar range (5-14 nM) were recorded for the best
representatives. Fromtime-dependentpreincubation andrapiddilutionstudies, andfromdockinganalyses
in a homology model of the target enzyme, a reversible mechanism of inhibition of hFAAH is proposed.
Introduction
Indeed, the active site differs from traditional enzymes by
the replacement of Ser-His-Asp catalytic triad with Ser-Ser-
Lys triad which constitutes the so-called amidase signature
(AS).8,9 Recently, a second AS enzyme has been discovered
and termed FAAH-2;10 regarding the original FAAH (also
named FAAH-1), this enzyme shares only 20% sequence
identity, but the same amide hydrolyzing activity using a
Ser-Ser-Lys triad. FAAH exerts its activity on substrates
possessing an amide bond, especially endogenous fatty acid
amides (FAA). The principal substrate, and the most studied,
is anandamide (arachidonoylethanolamide, AEA), a partial
agonist of cannabinoid receptors CB1 and CB2.11 Therefore,
FAAH is commonly said to belong to the endocannabinoid
system which consists of different hydrolases: FAAH-1,
FAAH-2, monoacylglycerol lipase (MGL),12 and N-acyletha-
nolamine-hydrolyzing acid amidase (NAAA),13 among
others.14-17 MGL and NAAA preferentially hydrolyze
2-arachidonoylglycerol (2-AG) or 2-oleoylglycerol (2-OG)
and palmitoylethanolamide, respectively. FAAH hydrolyses
anandamide, other endogenous fatty acid amides, but also a
particular class of N-acylamino acids, that is, N-acyl taurines
(NATs) which activate transient receptor potential (TRP)
ions channels,18 and oleamide,19 a fatty acid primary amide
recognized as a sleep-inducing lipid. The actual knowledge of
these bioactive lipids and the role played by FAAH in the
control of their levels open the door to the development of
novel therapeutic agents.20 Indeed, pharmacological investi-
gations in animal models have shown that a large number of
biological benefic effects such as appetite stimulation, anti-
inflammatory effects, sleep induction,21 anxiety release and
analgesia22,23 could be enhanced by controlling FAAH cata-
bolic activity.
The 2-azetidinone template (β-lactam) has been widely
described as a lead structure for the inhibition of serine
hydrolases such as human leukocyte elastase (HLEa),1 pros-
tate specific antigen (PSA),2 thrombin,3 human cytomegalo-
virus,4 and mainly D,D-peptidases and β-lactamases, the
bacterial target-enzymes of penicillin-type drugs used in anti-
biotherapy.5 Generally, enzyme inhibition results from the
interaction between the 2-azetidinone carbonyl and the active
serine of the catalytic triad Ser-His-Asp. This interaction
creates a covalent bond, via a tetrahedral intermediate, lead-
ing to a relatively stable acyl-enzyme complex, and therefore
to the inhibition of the enzyme. Slow hydrolysis of the acyl-
enzyme complex can regenerate the active enzyme, but in the
case of so-called “suicide-substrates”,6 the inhibition is irre-
versible because the acyl-enzyme structure is no more sensitive
toward hydrolysis. Surprisingly, the β-lactam motif has never
been considered for fatty acid amide hydrolase (FAAH)
inhibition, until our preliminary study which disclosed
3-alkenyl-2-azetidinones as micromolar inhibitors.7 Like the
above-mentioned enzymes, FAAH is a serine hydrolase but a
member of a distinct class from the chymotrypsin family.
*Corresponding author. Phone: þ32 10 47 27 40. Fax: þ32 10 47 41
68. E-mail: jacqueline.marchand@uclouvain.be.
a Abbreviations: hFAAH, human fatty acid amide hydrolase; hMGL,
human monoacylglycerol lipase; AS, amidase signature; FAAs, fatty
acid amides; AEA, anandamide; CB1, cannabinoid receptor subtype-1;
CB2, cannabinoid receptor subtype-2; NAAA, N-acylethanolamine
hydrolyzing acid amidase; 2-AG, 2-arachidonoylglycerol; 2-OG, 2-
oleoylglycerol; BTNP, (E)-6-(bromomethylene)tetrahydro-3-(1-naph-
thalenyl)-2H-pyran-2-one; DCM, dichloromethane; ACN, acetonitrile;
DCC, dicyclohexylcarbodiimide; DMAP, dimethylaminopyridine;
HMDS, hexamethyldisilazane; CBz, benzyloxycarbonyl; TBDMS,
tert-butyldimethylsilyl; MAFP, methyl arachidonyl fluorophospho-
nate; rmsd, root-mean-square deviation; ACB, acyl chain binding;
CA, cytoplasmic access; DMSO, dimethyl sulfoxide.
The search of FAAH inhibitors constitutes a domain of
growing interest which has been recently reviewed.24,25 Potent
inhibitors based on different types of electrophilic functions
r
pubs.acs.org/jmc
Published on Web 10/30/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7055
A series of 30 azetidinones was evaluated in vitro for the
inhibition of human FAAH (hFAAH) and human MGL
(hMGL). The most promising compounds were submitted
to a docking study in a new model of hFAAH.
Results and Discussion
Synthesis. Acetoxy-azetidinone 7 is a commercially avail-
able chiral precursor of (carba)penems antibiotics.42 This
molecule offers several advantages: (i) the amide function
can be easily substituted on the N1 position; (ii) after
deprotection of the silyl ether group, the hydroxyl function
of the side-chain can also be substituted (C5-O position);
(iii) the acetoxy substituent (OAc) on the C4 position in-
creases the heterocycle chemical reactivity (N1-C2
cleavage) by its electron withdrawing effect; (iv) OAc is also
a good leaving group. This last structural feature would
make possible the occurrence of an irreversible suicide-type
inhibition, if a serine hydrolase enzyme reacted on the
β-lactam ring. Moreover, the chemical reactivity of the
OAc substituent allows its formal elimination by a two-step
sequence of reactions (substitution/reduction), giving the
less hindered and more stable precursor 8 (Scheme 1).
A first family of lipophilic azetidinones was prepared
from 7, taking inspiration from previously described proto-
cols (Table 1, entries 1-10).43,44 Briefly, 7 was N-acylated by
reaction with hydrocynnamoyl chloride, 4-phenyl-butanoyl
chloride, or 4-pentenoyl chloride, and pyridine, in refluxing
dichloromethane (DCM), to furnish respectively azetidi-
nones 9a (89%), 9b (94%), and 9c (80%). The silyl ether
function was deprotected by treatment with HCl-HOAc at
-5 ꢀC. The resulting alcohols 11a-c (83-99%) were directly
engaged in esterification reactions with hydrocynnamoyl
chloride, 4-phenyl-butanoyl chloride, or 4-pentenoyl chlor-
ide, in the presence of pyridine at room temperature, giving
the following bis-acylated compounds: 13a (99%), 14a
(88%), 13b (70%), 14b (52%), and 15e (90%). The biphenyl-
acetyl side chain was introduced by an alternative method:
the reaction of 11a,b with biphenylacetic acid and dicyclo-
hexylcarbodiimide (DCC), in the presence of dimethylami-
nopyridine (DMAP) as catalyst. Compounds 13d (67%) and
14d (77%) were isolated.
A second family of compounds (Table 1, entries 11 to 31)
was prepared from 8.45 This precursor could be readily
obtained by substitution of 7 with thiophenolate followed
by reduction with tris(trimethylsilyl)silane hydride (see Sup-
porting Information). As above, 8 reacted with hydrocynna-
moyl chloride, 4-phenyl-butanoyl chloride, 5-phenyl-
pentanoyl chloride, 4-pentenoyl chloride, or 5-hexenoyl
chloride to afford respectively the N-acylated azetidinones
10a (88%), 10b (87%), 10c (74%), 10d (95%), and 10e (46%)
(see Scheme 1). After t-butyldimethylsilyl deprotection un-
der acidic conditions, the resulting alcohols 12a-e
(78-94%) were esterified with various acid chlorides and
pyridine (Method A), or with the corresponding carboxylic
acids, DCC and DMAP (Method B). Application of the
Method A to hydrocynnamoyl chloride and 12a,b gave the
azetidinones 16a (79%) and 17a (89%). From 4-phenyl-
butanoyl chloride and 12a-e were obtained respectively
16b (75%), 17b (87%), 18b (63%), 19b (84%), and 20b
(77%). Reaction of 4-pentenoyl chloride with 12d furnished
19e (88%). Applying the Method B to 12a,b and 5-phenyl-
valeric acid, we produced the bis-acylated azetidinones 16c
(59%) and 17c (93%). Similarly, from biphenylacetic acid
Figure 1. Structures of previously described FAAH inhibitors (the
mentioned IC50 is the one reported by the respective authors,
against rat enzyme).
have been published. They are divided into two mechanistic
classes: irreversible carbamates26-31 and ureas32,33 inhibitors,
which include the pharmacological tools 1 (URB-597)34 and
2 (PF-622),33 and the reversible R-keto oxazoles35-39 inhibi-
tors (and other heterocycles) illustrated by 3 (OL-135)23
(Figure 1). Reaction of 1 and 2 with FAAH leads to inactive
and stable acyl-enzymes. Initial proton exchange between
Lys142, Ser217, and Ser241 (catalytic triad) allows the nu-
cleophilic attack of Ser241 on the carbonyl function of the
inhibitor; the resulting tetrahedral intermediate expulses the
leaving group, namely, the phenol moiety of 1 or the aniline
group of 2, along with proton transfer from Ser217, thus
leading to Ser241 covalently modified as a carbamate. The
postulated mechanism of FAAH interaction with 3 starts
similarly, but since the tetrahedral intermediate features no
leaving group, reversible inhibition occurs. Within this family
of covalent reversible inhibitors, SAR studies have clearly
shown that the activity is linked to the electrophilic character
of the ketone.35
Embedding the sensitive carbonyl function into a cyclic
structure appears to be a quite unusual strategy for the design
of FAAH inhibitors. (E)-6-(Bromomethylene)tetrahydro-
3-(1-naphthalenyl)-2H-pyran-2-one (4, Figure 1) was an
early covalent inhibitor of anandamide hydrolysis.40 A uni-
que series of (thio)hydantoin-based FAAH inhibitors, exem-
plified with 3-heptyl-5,50-diphenylimidazolidine-2,4-dione (5,
Figure 1), has been reported by Muccioli et al.41 Such
molecules act as competitive inhibitors without being hydro-
lyzed by the enzyme. Lastly, a few lipophilic β-lactams were
shown to be modest inhibitors of FAAH: 3-(40-pentenyl)-
1-(40-pentenoyl)-2-azetidinone (6, Figure 1) emerged as a
micromolar inhibitor.7 Starting from this preliminary result,
we have investigated the synthesis and the pharmacological
properties of a new family of FAAH inhibitors, derived from
acetoxy-azetidinone 7, in order to possibly improve the
activity. The structures were decorated with different acyl
chains on N1 and C5-O positions, featuring a terminal
phenyl (Ph), biphenyl (biPh), or alkene (Alk) motif as found
on the hydrophobic scaffolds of traditional FAAH inhibitors.

7056 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Scheme 1. Synthesis of Substituted Azetidinonesa
Feledziak et al.
a Reagents and conditions: (a) acyl chloride, pyridine, DCM, 45 ꢀC, 24 h; (b) HCl, AcOH, ACN, -5 ꢀC to rt, 3 h; (c) acyl chloride, pyridine, DCM, rt,
15 h or carboxylic acid, DCC, DMAP, DCM, rt, 15 h; (d) benzyl chloroformate, LiHMDS, -78 ꢀC to rt, 4 h; (e) H2, Pd/C, EtOH/AcOEt, 1 h.
Table 1. Determination of the Inhibitory Potential of Azetidinones Towards Human FAAH and Human MGLa
entry
compound
R1
n
R2
m
R3
IC50 hFAAHb
% inhibition (MGL)c
IC50 hMGLb
1
11a
11b
11c
13a
13b
13d
14a
14b
14d
15e
12a
12b
12c
12d
12e
16a
16b
16c
16d
17a
17b
17c
17d
18b
18d
19b
19d
19e
19f
20b
20d
24
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
H
2
3
2
2
2
2
3
3
3
2
2
3
4
2
3
2
2
2
2
3
3
3
3
4
4
2
2
2
2
3
3
Ph
Ph
223.6
182.8
537.0
2.02
48
61
2
3
Alk
Ph
16
4
2
3
1
2
3
1
2
Ph
Ph
100 (0)
100 (0)
66
5
Ph
Ph
0.96
6
biPh
Ph
0.826
5.12
3.12
7
Ph
Ph
100 (0)
100 (0)
100 (0)
99 (33)
8
8
Ph
9
Ph
biPh
Alk
0.708
1.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Alk
Ph
133
408.7
nd
nd
H
Ph
Ph
nd
nd
H
H
Alk
Alk
Ph
7.9
nd
89 (8)
nd
H
H
2
3
4
1
2
3
4
1
3
1
3
1
2
3
3
1
1
Ph
Ph
0.157
0.049
0.091
0.050
0.057
0.030
0.045
0.032
0.449
0.236
0.005
0.012
0.098
0.032
0.010
0.014
6.5
100 (0)
100 (0)
100 (0)
31
H
Ph
Ph
H
Ph
H
Ph
Ph
biPh
Ph
H
54
100 (0)
59
H
Ph
Ph
Ph
Ph
H
H
Ph
Ph
biPh
Ph
0
39
H
H
Ph
biPh
Ph
25
89
H
Alk
Alk
Alk
Alk
Alk
Alk
4.06
1.84
23.3
4.72
8.51
14.6
H
biPh
Alk
Alk
Ph
91
99
H
H
8
85
H
H
biPh
biPh
67
16
H
a See Supporting Information for the corresponding table of pI50 values and Standard Error. b IC50 in μM (from three independent experiments)
c Percentage of inhibition at 10-4 M. The percentage of inhibition at 10-6 M is stated between brackets.
and 12a-e, we prepared the compounds 16d (93%), 17d
(83%), 18d (81%), 19d (68%), and 20d (66%). Lastly,
reaction of 12d with 5-hexenoic acid gave the azetidinone
19f (84%).
For comparison purposes (see below, enzymatic tests), one
representative azetidinone monosubstituted at the C5-O
position was prepared in four steps (Scheme 1 and Table 1,
entry 32). Amide protection of 8 with a benzyloxycarbonyl

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7057
group (21, 99%), silyl ether deprotection as usual (22, 91%),
esterification with biphenylacetic acid (23, 83%), and N1
deprotection by catalytic hydrogenation afforded the azeti-
dinone 24 (96%; overall yield for four steps, 72%).
previous “hit” (structure 6, Figure 1; IC50 = 4.5 μM), the
activities have been significantly improved. On the basis of
the results reported here, some structure-activity relation-
ships can be drawn. Compounds 16, with N-(3-phenyl-
propanoyl) chain (entries 16-19), and 12, with N-(5-phen-
yl-pentanoyl) chain (entries 24 and 25), are less potent than
their corresponding analogues 17, with N-(4-phenyl-
butanoyl) chain (entries 21-23). Compounds 19, with
N-(4-pentenoyl) chain (entries 26-29), are slightly more
potent than their corresponding analogues 20, with N-(5-
hexenoyl) chain. Within subfamilies, compounds named b,
with O-(4-phenyl-butanoyl) chain (entries 17, 21, 26, 30), and
d, with O-(biphenyl-acetyl) chain (entries 19, 23, 27, 31), are
the best inhibitors. We concluded that similar activities result
from the presence of 4-phenyl-butanoyl (n = 3, R2 = Ph)
and 4-pentenoyl (n = 2, R2 = Alk) substituents at the
N1 position, on the one hand, and from the presence of
4-phenyl-butanoyl (m = 3, R3 = Ph) and biphenylacetyl
(m = 1, R3 = biPh) substituents at the C5-O position, on
the other hand.
All final azetidinones and intermediates were fully char-
acterized by the usual spectroscopies (see Experimental
Section). Typical features are exemplified with 14d (first
1
series, R1 = OAc) and 19b (second series, R1 = H). H
NMR spectrum of 14d shows the vicinal β-lactamic protons
with the trans relationship at 3.28 ppm (H3, dd, J = 6.5 and
1.7 Hz) and 6.46 ppm (H4, d, J = 1.7 Hz); four carbonyl
signals are visible in 13C NMR at 170.4 (O-CO), 169.8 (N-
CO), 169.1 (OAc) and 162.2 (β-lactam carbonyl) ppm; the IR
spectrum shows the carbonyl stretchings at 1803 (β-lactam),
1740 (broad, OAc and ester), and 1717 (imide) cm-1. For
1
19b, the geminal β-lactamic protons H4/H40 appear in H
NMR as a typical ABX pattern at 3.53 ppm (dd, J = 7.7 and
3.7 Hz) and 3.66 ppm (dd, J = 7.7 and 6.6 Hz), while H3
gives a multiplet at 3.40 ppm; the 13C NMR spectrum shows
three carbonyl signals at 172.4 (O-CO), 170.3 (N-CO), and
164.4 (β-lactam CO) ppm, and the IR spectrum shows the
carbonyl stretchings at 1786 (β-lactam), 1734 (ester), and
1703 (imide) cm-1. In both series (R1 = OAc or H), H5
proton of precursors 9,10 (silyl ether) and 11,12 (free alcohol)
gives a multiplet (qd) around 4.3 δ in 1H NMR spectra; after
the O-acylation leading to the final compounds 13-15 and
16-20, a deshielding of about 1 δ is observed (H5 around 5.3
δ). The chemical and enantiomeric purity of all tested
compounds has been controlled by HPLC, using C18 and
AD-H columns, respectively.
MGL Inhibition. Azetidinones 11, 13-14 of the first series
(R1 = OAc) inhibited the enzyme at 10-4 M concentration
(50-100% inhibition), but not at 10-6 M concentration
(Table 1, entries 1 to 10). An IC50 value of 133 μM was
determined for the most active compound 15e (entry 10)
which, however, shows a great selectivity for the inhibition of
FAAH (IC50 = 1.90 μM).
Azetidinones 12, 16-20 of the second series (R1 = H) were
also modest inhibitors of MGL (entries 11-25). IC50 values
of the most active azetidinones 19b-f and 20b,d ranged from
1.84 to 23.3 μM (entries 26-31). Here again, the selectivity
versus FAAH inhibition is high: for instance, 19b (entry 26)
and 20b (entry 30) are respectively 800 and 850 times more
potent against FAAH. For the other compounds, 19d-f and
20d, the selectivities range within 100 and 240.
Biochemical Evaluation
The azetidinones listed in Table 1 have been tested as
potential inhibitors of hFAAH and hMGL. Human recombi-
nant enzymes, developed in our laboratory,46,47 were used in
competitive hydrolytic assays using [3H]-radiolabeled AEA
and [3H]-radiolabeled 2-OG, respectively, as substrates.
Tested compounds, enzymes, and [3H]-substrates were incu-
bated at 37 ꢀC during 10 min. The inhibition rates were
evaluated by liquid scintillation counting (LSC) of the resti-
tual hydrolysis products of the labeled substrates. The results
reported in Table 1 are the means of three independent assays.
FAAH Inhibition. Collected results clearly show that the
azetidinones equipped with only one acyl chain, at N1
position (entries 1-3 and 11-15) or C5-O position (entry
32), are modest or very weak inhibitors of FAAH. Among
the compounds bearing two acyl chains, fixed at N1 and
C5-O positions, the first series (R1 = OAc, entries 4-10)
systematically appears less active than the second one (R1 =
H, entries 16-31). Our initial hypothesis that the C4 acetate
substituent would improve the azetidinone inhibitory
effect - by increasing the chemical reactivity of the hetero-
cycle (electronwithdrawing effect) and/or by initiating an
enzymatic suicide-mechanism (leaving group effect) -
turned out to be contradicted by these first results. Accord-
ingly, the discussion focuses only on the second series of
disubstituted azetidinone inhibitors 16a-d, 17a-d, 18b,d,
19b-f, and 20b,d which are potent FAAH inhibitors. The
studied factors were the chain length (n, m = 1-4) and the
nature of the end group (Ph, biPh, Alk) for both substituted
positions (N1, C5-O). All compounds 16-20 revealed to be
good inhibitors of hFAAH with IC50 values ranging from
0.005 μM (19b) to 0.45 μM (18b). Comparatively to our
Inhibition Mode. To determine the likely mechanism of
FAAH inhibition, two types of experiments were performed,
that is, time-dependent preincubation and rapid dilution
studies, both using azetidinones 19b (IC50 = 0.005 μM)
and 19f (IC50 = 0.032 μM). Concerning the preincubation
study, it is expected with an irreversible-type inhibitor that
the inhibitor potency should increase upon prolonged pre-
incubation time. Conversely, a constant IC50 value upon
preincubation supports a reversible mechanism of inhibi-
tion.48 Thus, 19b and 19f were incubated with the enzyme for
0, 15, 45, or 90 min, prior to substrate addition. As illustrated
in Figure 2, the preincubation had no effect on the inhibiting
activity of the compounds. This suggests an inhibition mode
similar to those of R-keto-oxazoles (see 3, Figure 1) or
hydantoins (see 5, Figure 1).35,41 On the other hand, after
rapid and large dilution of the inhibitor-enzyme mixture,
the recovery of enzymatic activity should be almost total if
the inhibitor is reversible. For the irreversible inhibitors, the
enzyme remains largely inhibited because the inhibitor is
bound to the enzyme. Here, the rapid and large dilution led
to a recovery of enzymatic activity in the case of 19b and 19f,
as for 1-oxazolo[4,5-b]pyridin-2-yl-6-phenyl-1-hexanone
(CAY10402),49 an analogue of 3 (Figure 1). As a further
control we used two irreversible FAAH inhibitors, com-
pound 1 (Figure 1) and methyl arachidonyl fluorophospho-
nate (MAFP)50 and found that the enzyme activity was still
profoundly inhibited after the dilution (Figure 3a).
Furthermore, the mechanism of 19b interaction with hFAAH
was determined by studying the velocity of anandamide

7058 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Feledziak et al.
Figure 2. Determination of the mode of inhibition of 19f (A) and 19b (B). The influence of the time of preincubation (0, 15, 45, and 90 min) on
the inhibition curves of hFAAH was studied resulting in no significant variation of the IC50 values.
Figure 3. (A) 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). (B) Determination of the mechanism of 19b interactions with hFAAH. Michaelis-Menten curves and
rapid dilution graphs were obtained from three independent experiments. The kinetic parameters are shown in the inset (Vmax values are given
as nmol min-1 mg-1 of protein and KM values are in μM).
Figure 4. Representation of the active site of the modeled human FAAH.
metabolism in function of increasing concentration of anand-
amide. The Michaelis-Menten curves (Figure 3b) and resulting
kinetic parameters suggest a competitive inhibition type for this
compound. Indeed, the Vmax values in the presence of 15 or 45
Thereotical Study
Model of the Human FAAH. The crystal structure of
hFAAH is currently not available. But recently, an engi-
neered form of rat FAAH showing the same activity profile
as the human one was crystallized (PDB code 2VYA).51 We
therefore decided to build a model of hFAAH through
homology modeling using this X-ray crystal structure. Their
amino acid sequence shared 80.6% identity. The EsyPred3D
program was used.52 This automated homology model-
ing tool compares results from various multiple alignment
nM of 19b (12.77 ( 0.22 and 11.91 ( 0.50 nmol min-1 mg -1
,
respectively) are similar to the Vmax value obtained in the
absence of inhibitor (13.38 ( 0.25 nmol min-1 mg -1), whereas
the KM values are largely increased in the presence of inhibitor.
On the basis of these data, to collect more information
about the possible enzyme-inhibitor interactions at the
atomic level, a modeling study has been performed.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7059
Figure 5. Proposed binding mode I of 19b into the human FAAH. In the panel on the right, H bonds are depicted by orange dotted lines.
Figure 6. Proposed binding mode II of 19d into the human FAAH. In the panel on the right, H bonds are depicted by orange dotted lines.
algorithms to derive a “consensus” alignment between the
target sequence and the template. Quality verification of the
model was performed with Procheck 3.0 with a pseudoreso-
and emerges at about an 80ꢀ angle from the ACB channel. A
third channel composed of three phenylalanine residues
(Phe388, Phe381, and Phe192), here called the “phenyl
pocket”, lies close to the ACB channel.
lution of 2.8 A.53 The model obtained is reliable based on the
˚
Ramachandran plot, showing 91.2% of the residues in the
core regions and 8.6% in the allowed one (see Supporting
Information). Moreover, 99.1, 94.8, and 100.0% of the main
chain bond lengths, main chain bond angles, and the planar
groups, respectively, are within the standard geometries. The
root mean square deviation (rmsd) for the backbone atoms
Docking Studies. Since the above-described pharmacolo-
gical data suggest that these compounds are competitive
inhibitors we docked the most active inhibitors, 19b and
19d, into the substrate binding site of the modeled hFAAH to
further understand their binding mode. Therefore we used
the GOLD program, which we used for a previous work on
FAAH,54 to dock these compounds into the active site of our
human FAAH model. Recent theoretical and structural
studies showed the planarity of the amide β-lactam bond
and that the two imide carbonyls (called here COlactam and
COexo) can adopt either E or Z configuration, the E con-
figuration being the more stable in the gas phase (ΔE =
3.9 kcal/mol).43 We therefore allowed flipping the imide
˚
between both structures is 0.091 A. The active site of hFAAH
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) (Figure 4). From the membrane, the ACB channel
bifurcates into a lipophilic bulge. A second tunnel, the
cytoplasmic access channel (CA), is exposed to the solvent

7060 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Feledziak et al.
Table 2. Characteristics of the Two Proposed Binding Modes of Azetidinone Compounds Inside the Modelled Human FAAH
˚
distance (A)
compound
binding mode
configuration of the imide carbonyls
H bonds
COexo OH(Ser241)
2.24
3.26
2.95
3.32
3.07
3 3 3
COlactam OH(Thr236)
COester NH(Val270)
19b
19d
I
Z
3 3 3
3 3 3
II
I
Z
E
COester NH(Cys269)
COlactam OH(Ser241)
3 3 3
3 3 3
(phenyl) or m = 1 (biphenyl), and n = 2 (alkenyl), by the
stabilizing π-π interactions between the phenyl/biphenyl or
alkene group and phenylalanine residues of the active site.
Moreover, in both cases, mode I or II, adding an acetate
moiety in the lactam cycle at C4 would lead to steric
hindrance. The same binding modes were also observed for
16b and 16d (results not shown).
The first binding mode (Figure 5) could suggest a mechan-
ism of action similar to that of R-keto heterocycles acting as
reversible, competitive inhibitors presumably via reversible
hemiketal formation with the active serine Ser241.35,55 In this
context, we did a covalent docking of the two putative
tetrahedral intermediates of 19b, binding Ser241 either via
the lactam carbonyl (COlactam) or via the exocyclic imide
carbonyl (COexo). In both cases, the position of the inhibitor
is close to the one of the first binding mode with the phenyl
group interacting with the “phenyl pocket” and the alkene
chain lying in the ACB channel (Figure 7). The anion oxygen
interacts by H bonding with the oxyanion hole, that is, with
the backbone of Ile238, Gly239, and Gly240. For the inter-
mediate via the exocyclic carbonyl, both Z and E configura-
tions are observed with a highest occurrence for E.
Following the second binding mode (Figure 6), the in-
hibitors would rather act as the (thio)hydantoin inhibitors,
described recently, without tetrahedral intermediate.54
As an internal validation of the docking methodology, the
inhibitor N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-car-
boxamide (PF-750)33 covalently attached to the Ser241 and
used to generate the published crystal structure of the
humanized form of rat FAAH,51 was redocked into the
empty catalytic pocket of the crystal structure using the same
docking protocols. The conformation of the top scoring pose
could reproduce the crystal structure conformation (data not
shown), validating the docking methodology.
Figure 7. Binding mode of the putative tetrahedral intermediates of
19b, binding Ser241 either (a) via the exocyclic imide carbonyl
(COexo) or (b) via the lactam carbonyl (COlactam) in the modeled
human FAAH.
Conclusion
bond during the docking run. On the basis of GOLD scoring
function and on the occurrence of the docking poses, two
preferential binding modes were retained (Figures 5 and 6;
Table 2). We observed a Z or E configuration of the imide
carbonyls following the binding mode and the studied
compound. In the first binding mode (I) (Figure 5), the
phenyl or biphenyl 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 and is close to Phe192. The observed H bonds
are described in Table 2. In the second binding mode (II)
(Figure 6), only observed for 19d, the biphenyl and alkene
chains are located in the ACB channel and “phenyl pocket”,
respectively. The biphenyl group interacts with Phe192.
In both binding modes, several amino acids of the active
site are involved in hydrophobic contacts with the inhibitors
(see Supporting Information). From our docking experi-
ments, we can explain the optimal chain length m = 3
Till now, β-lactams were not considered as potential phar-
macologically active compounds to interact with the endo-
cannabinoid system in humans. In 2008, the virtual screening
of a database of about 500000 Shering-Plough compounds by
using a CB1 pharmacophore model as filter, and additional
constraints for drug-like structures, allowed selection of 420
compounds for further in vitro evaluation. Among them, a
series of five diaryl 2-azetidinones emerged, giving an inhibi-
tion rate of g50% at 100 nM in a CB1 competitive binding
assay. From this nonorientated approach, one β-lactam
“lead” compound was identified as novel CB1 receptor an-
tagonist with a Ki value of 53 nM.56
To our knowledge, the design of potentially active β-lactams
in the cannabinoid system was not reported before. Our
approach was simply based on the FAAH inhibition by using
the β-lactam core as electrophilic carbonyl function; this het-
erocycle was equipped with lateral chains mimicking the nat-
ural substrates or the known inhibitors, and susceptible to make
hydrophobic contacts in the active site of the target enzyme.

Article
Starting from the chiral 2-azetidinone 7 traditionally used
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7061
General Procedure for N-Acylation. To a stirred solution of
azetidinone 7 (1 equiv) in dry dichloromethane (8.6 mL/mmol)
at 20 ꢀC were added pyridine (2 equiv) and the suitable acyl
chloride (2 equiv) under argon atmosphere. The mixture was
refluxed during 24 h, then diluted in dichloromethane and the
excess of acyl chloride was quenched by Na2CO3 (10% aqueous
solution; 8.6 mL/mmol). The organic layer was washed with 3 N
HCl and brine, dried over MgSO4, filtered, and concentrated
under vacuum. After purification by flash chromatography
(cyclohexane/ethyl acetate), white solids (9a and 10a-b) or
colorless oils (9b-c and 10c-e) were obtained.
1-(3-Phenylpropanoyl)-(3R,4R)-3-[1(R)-(tert-butyldimethylsily-
loxy)-ethyl]-4-(acetoxy)-azetidin-2-one (9a). Yield: 89% (130.1 mg
from 0.35 mmol of 7). Mp: 70.0-70.5 ꢀC. [R]D = -54.0 (c = 1.0).
Rf = 0.54 (cyclohexane/ethyl acetate: 5/2). MS (ESI): m/z: 442.1
((M þ Na)þ). 1H NMR (500 MHz, CDCl3): δ = 0.05 (s, 3H), 0.10
(s, 3H), 0.84 (s, 9H), 1.34 (d, 3H, J = 6.4 Hz), 2.14 (s, 3H),
2.97-3.06 (m, 4H), 3.15 (dd, 1H, J= 1.5Hz, J =2.5 Hz), 4.31 (m,
1H), 6.62 (d, 1H, J = 1.5 Hz), 7.21-7.34 (m, 5H). 13C NMR (125
MHz, CDCl3): δ = -5.3, -4.1, 17.9, 21.0, 21.9, 25.6, 29.8, 38.3,
64.3, 65.3, 74.3, 126.4, 128.6, 128.6, 140.2, 164.6, 169.1, 169.3. IR
(cm-1): υ = 2854-2952, 1803, 1755, 1714, 1454-1495, 1308,
1251, 837. HRMS: C22H33NO5SiNa: calculated: 442.2026,
found: 442.2040.
1-(4-Phenylbutanoyl)-(3R,4R)-3-[1(R)-(tert-butyldimethylsily-
loxy)-ethyl]-4-(acetoxy)-azetidin-2-one (9b). Yield: 94% (360 mg
from 0.89 mmol of 7). [R]D = -40.2 (c = 1.0). Rf = 0.48
(cyclohexane/ethyl acetate: 5/2). MS (ESI): m/z: 456.2 ((M þ
Na)þ), 888.9 ((2 M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ =
0.03 (s, 3H), 0.08 (s, 3H), 0.82 (s, 9H), 1.31 (d, 3H, J = 6.4 Hz),
1.99 (m, 2H), 2.11 (s, 3H), 2.63-2.78 (m, 4H), 3.12 (m, 1H), 4.29
(m, 1H), 6.59 (d, 1H, J = 1.1 Hz), 7.12-7.38 (m, 5H). 13C NMR
(75 MHz, CDCl3): δ = -5.3, -4.1, 17.8, 20.9, 21.9, 25.3, 25.6,
35.1, 35.9, 64.3, 65.1, 74.2, 126.1, 128.4, 128.5, 141.4, 164.5,
169.1, 169.8. IR (cm-1): υ = 2854-2926, 1805, 1757, 1717, 1462,
1308, 1211-1250, 839. HRMS: C23H35NO5SiNa: calculated:
456.2182, found: 456.2187.
1-(Pent-4-enoyl)-(3R,4R)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-4-(acetoxy)-azetidin-2-one (9c). Yield: 80% (515 mg from
1.74 mmol of 7). Rf = 0.57 (cyclohexane/ethyl acetate: 5/2). MS
(ESI): m/z: 392.1 ((M þ Na)þ), 760.9 ((2 M þ Na)þ). 1H NMR
(500 MHz, CDCl3): δ = 0.03 (s, 3H), 0.07 (s, 3H), 0.82 (s, 9H),
1.31 (d, 3H, J = 6.7 Hz), 2.10 (s, 3H), 2.40 (td, 2H, J = 7.6 Hz,
J = 6.5 Hz), 2.74 (td, 1H, J = 7.6 Hz, J = 16.9 Hz), 2.81 (td, 1H,
J = 7.6 Hz, J = 16.9 Hz), 3.12 (m, 1H), 4.29 (m, 1H), 5.01 (dd,
1H, J = 1.6 Hz, J = 10.5 Hz), 5.08 (dd, 1H, J = 1.6 Hz, J = 17.2
Hz), 5.82 (ddt, 1H, J = 10.5Hz J = 17.2 Hz, J = 6.5Hz), 6.58(d,
1H, J = 1.6 Hz). 13C NMR (125 MHz, CDCl3): δ = -5.5, -4.3,
17.6, 20.8, 21.7, 25.5, 27.4, 35.6, 64.1, 65.0, 74.1, 115.8, 136.2,
164.4, 168.9, 169.1. IR (cm-1): υ = 2857-2961, 1808, 1759, 1721,
1642, 1306, 835. HRMS: C18H31NO5SiNa: calculated:
392.1869, found: 392.1863.
for the synthesis of antibiotics, we generated a variety of
lipophilic derivatives by placing alkenoyl, phenylalkanoyl,
and biphenylacetyl chains on positions N1 and C5-O.
Evaluation of this library of 30 azetidinones against hFAAH
and hMGL revealed good to excellent and selective inhibi-
tors of hFAAH versus hMGL, with IC50 values of 5-14 nM
for the best representatives (19b, 19d, 20b, and 20d). Since the
IC50 values were constant upon prolonged incubation time
and as total recovery of enzymatic activity was observed after
rapid and large dilution, a reversible mechanism of inhibition
can be proposed. In addition, as the Vmax values are not
affected by the presence of 19b while the KM values are
increased, the interaction between 19b and hFAAH is likely
to be of a competitive type. This is a quite unexpected result,
since the β-lactams are prone to form (more or less) stable
acyl-enzyme intermediates with serine hydrolases. Note that
docking studies of two potent inhibitors into a validated
homology model of hFAAH support well the reversible
mechanism, even though they do not allow discriminating
between two binding modes, with either the lactam/imide
carbonyls or the ester carbonyl facing the catalytic triad.
Further studies are in progress to clarify the role played by
each carbonyl function of the inhibitors 16-20 and to identify
the carbonyl function possibly responsible for the formation
of a reversible tetrahedral intermediate by reaction with the
active serine.
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 purifications. (3R,4R)-4-Acetoxy-3-
[(R)-(tert-butyldimethylsilyloxy)ethyl]-2-azetidinone 7 was ob-
tained from Kaneka corporation (Japan). [3H]-AEA (60 Ci/
mmol) and [3H]-2-OG (40 Ci/mmol) were 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 reasonance (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 deuterate chloroform (CDCl3). Chemical shifts are
reported in ppm relative to the solvent signals (CDCl3 7.26
and 77.16 ppm). NMR coupling constants (J) are reported in
€
hertz. Melting points (mp) were determined on a Buchi B-540
apparatus calibrated with caffeine, vanillin, and phenacetin.
Rotations were recorded on Perkin-Elmer 241 MC polarimeter,
at 20 ꢀ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 (ESI).
High resolution mass spectrometry (HRMS) analyses were
performed at the University of Mons Hainaut (Belgium) or at
the University of Oxford (UK). Infrared (IR) spectra were
recorded using FTIR-8400S Shimadzu apparatus. Products
were analyzed as thin films deposited on a Se-Zn crystal by
evaporation from CH2Cl2 solutions. Thin layer chromatogra-
phy (TLC) analysis was performed on Merck silica-gel 60F254
and detected under UV light, and flash chromatography was
performed on silica gel (40-60 mesh) purchased from Rocc
(Belgium). Purity of tested compounds was assessed by HPLC
on chiral AD-H column (2.1 mm ꢀ 150 mm, 5 μm particle size)
using hexane/isopropanol eluant (90:10), at a flow rate of
0.5 mL/min and on Symetry C18 (4.6 mm ꢀ 250 mm, 5 μm
particle size) using acetonitrile/H2O eluant (70:30), at a flow rate
of 1 mL/min (purity g97%).
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-azetidin-2-one (10a). Yield: 97% (617 mg from 1.77 mmol of
8). [R]D = -53.7 (c = 4.1). Rf = 0.53 (cyclohexane/ethyl acetate:
5/3). MS (ESI): m/z: 384.3 ((M þ Na)þ), 744.9 ((2 M þ Na)þ). 1H
NMR (300 MHz, CDCl3): δ = 0.06 (s, 3H), 0.09 (s, 3H), 0.85
(s, 9H), 1.20 (d, 3H, J = 6.3 Hz), 2.95-3.06 (m, 4H), 3.23 (m, 1H),
3.56 (dd, 1H, J = 6.7 Hz, J = 7.2 Hz), 3.70 (dd, 1H, J = 3.6 Hz,
J = 7.2 Hz), 4.31 (m, 1H), 7.11-7.40 (m, 5H). 13C NMR (75 MHz,
CDCl3):δ=-5.1, -4.1, 17.9, 22.2, 25.7, 30.2, 38.3, 38.4, 56.5, 64.8,
126.3, 128.6 (2C), 140.5, 166.5, 170.2. IR (cm-1): υ = 2856-2955,
1786, 1701, 1310, 1252, 839. HRMS: C20H31NO3SiNa: calculated:
384.1971, found: 384.1974.
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-azetidin-2-one (10b). Yield: 87% (286 mg from 0.87 mmol
of 8). Mp: 30.5-31.5 ꢀC. [R]D = -42.3 (c = 1.9). Rf = 0.52
(cyclohexane/ethyl acetate: 5/2). MS (ESI): m/z: 376.2 ((M þ
H)þ), 398.1 ((M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ = 0.06
(s, 3H), 0.09 (s, 3H), 0.85 (s, 9H), 1.20 (d, 3H, J = 6.3 Hz), 2.00

7062 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Feledziak et al.
(m, 2H), 2.61-2.84 (m, 4H), 3.22 (m, 1H), 3.54 (dd, 1H, J = 6.8
Hz, J = 7.2 Hz), 3.69 (dd, 1H, J = 3.6 Hz, J = 7.2 Hz), 4.31 (m,
1H), 7.14-36 (m, 5H). 13C NMR (75 MHz, CDCl3): δ = -5.2,
-4.2, 17.8, 22.2, 25.61, 25.66, 35.2, 36.0, 38.2, 56.3, 64.7, 126.0,
128.4, 128.5, 141.5, 166.4, 170.7. IR (cm-1): υ = 2856-3026,
1784, 1697, 1454-1497, 1389, 1309, 1249, 839. HRMS:
C21H34NO3Si: calculated: 376.2308, found: 376.2295;
C21H33NO3SiNa: calculated: 398.2127, found: 398.2107.
1-(5-Phenylpentanoyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-azetidin-2-one (10c). Yield: 74% (126 mg from 0.43 mmol
of 8). [R]D = -37.2 (c = 1.0). Rf = 0.53 (cyclohexane/ethyl
acetate: 5/2). MS (ESI): m/z: 390.2 ((M þ H)þ), 412.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3):δ= 0.05 (s, 3H), 0.07 (s, 3H),
0.83 (s, 9H), 1.18 (d, 3H, J = 6.3 Hz), 1.56-1.80 (m, 4H),
2.55-2.79 (m, 4H), 3.20 (m, 1H), 3.52 (dd, 1H, J = 6.8 Hz, J =
7.2 Hz), 3.67 (dd, 1H, J = 3.6 Hz, J = 7.2 Hz), 4.30 (m, 1H),
7.07-7.45 (m, 5H). 13C NMR (75 MHz, CDCl3): δ = -5.2, -4.2,
17.9, 22.2, 23.7, 25.6, 30.9, 35.6, 36.3, 38.2, 56.3, 64.7, 125.8, 128.3,
128.4, 142.1, 166.4, 170.9. IR (cm-1): υ = 2856-2951, 1784, 1701,
1454-1496, 1389, 1310, 1250, 839. HRMS: C22H36NO3Si: calcu-
lated: 390.2464, found: 390.2448; C22H35NO3SiNa: calculated:
412.2284, found: 412.2263.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-ethyl]-
azetidin-2-one (10d). Yield: 95% (965 mg from 3.27 mmol of 8).
Rf = 0.76 (cyclohexane/ethyl acetate: 5/2). MS (ESI): m/z: 312.3
((M þ H)þ). 1H NMR (300 MHz, CDCl3): δ = 0.03 (s, 3H), 0.05
(s, 3H), 0.82 (s, 9H), 1.17 (d, 3H, J= 6.1 Hz), 2.38 (m, 2H), 2.75 (m,
2H), 3.21 (m, 1H), 3.52 (dd, 1H, J = 6.6 Hz, J = 6.6 Hz), 3.66 (dd,
1H, J = 3.4 Hz, J = 6.6 Hz), 4.29 (m, 1H), 4.97 (dd, 1H, J = 1.6
Hz, J= 10.2 Hz), 5.05 (dd, 1H, J=1.6Hz, J= 17.0 Hz), 5.80 (ddt,
1H, J = 6.5 Hz, J = 10.2 Hz, J = 17.0 Hz). 13C NMR (75 MHz,
CDCl3):δ=-4.4, -5.4, 17.7, 22.0, 25.4, 27.8, 35.6, 38.1, 56.2, 64.5,
115.5, 136.4, 166.2, 170.1. IR (cm-1): υ = 2858-2930, 1788, 1705,
1311, 840. HRMS: C16H29NO3SiNa: calculated: 334.1814, found:
334.1806.
1-(Hexa-5-enoyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-azetidin-2-one (10e). Yield: 46% (40 mg from 0.27 mmol
of 8). [R]D = -34.6 (c = 1.0). Rf = 0.60 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 326.2 ((M þ H)þ), 348.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 0.05 (s, 3H), 0.07 (s,
3H), 0.83 (s, 9H), 1.19 (d, 3H, J = 6.3 Hz), 1.75 (m, 2H), 2.10
(m, 2H), 2.68 (m, 2H), 3.21 (m, 1H), 3.54 (dd, 1H, J = 6.6 Hz,
J = 6.6 Hz), 3.68 (dd, 1H, J = 3.4 Hz, J = 6.6 Hz), 4.30 (m,
1H), 4.96-5.06 (m, 2H), 5.77 (m, 1H). 13C NMR (75 MHz,
CDCl3): δ = -5.1, -4.1, 18.0, 22.3, 23.3, 25.7, 33.2, 36.0, 38.4,
56.4, 64.8, 115.4, 137.9, 166.5, 171.0. IR (cm-1): υ =
2856-2953, 1786, 1701, 1464, 1389, 1310, 1252, 839. HRMS:
C17H31NO3SiNa: calculated: 348.1971, found: 348.1985.
General Procedure for Deprotection. 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 a 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 con-
centrated under vacuum. After purification by flash chromato-
graphy (dichloromethane/ethyl acetate), a white solid (11a-b
and 12a-b) or a colorless oil (11c, 12c-e, and 22) was obtained.
1-(3-Phenylpropanoyl)-(3R,4R)-3-[1(R)-hydroxyethyl]-4-(ace-
toxy)-azetidin-2-one (11a). Yield: 93% (822 mg from 2.93 mmol
of 9a). Mp: 116.0-117.0 ꢀC. [R]D = -76.1 (c = 2.9). Rf = 0.20
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 328.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.37 (d, 3H, J = 6.3
Hz), 2.16 (s, 3H), 2.62 (br s, 1H), 2.94-3.16 (m, 4H), 3.17 (dd,
1H, J = 1.4 Hz, J = 6.4 Hz), 4.18 (m, 1H), 6.32 (d, 1H, J = 1.4
Hz), 7.16-7.38 (m, 5H). 13C NMR (75 MHz, CDCl3): δ = 21.0,
21.1, 29.9, 38.4, 64.3, 65.3, 75.9, 126.5, 128.6 (2C), 140.0, 163.2,
169.6, 170.4. IR (cm-1): υ = 3504, 2931-2974, 1803, 1755, 1716,
1454-1496, 1313. HRMS: C16H19NO5Na: calculated:
328.1161, found: 328.1174.
1-(4-Phenylbutanoyl)-(3R,4R)-3-[1(R)-hydroxyethyl]-4-(ace-
toxy)-azetidin-2-one (11b). Yield: 99% (83 mg from 0.25 mmol
of 9b). Mp: 81.0-81.5 ꢀC. [R]D = -74.9 (c = 2.9) . Rf = 0.21
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 342.2 ((M þ
1
Na)þ), 660.77 ((2 M þ Na)þ). H NMR (300 MHz, CDCl3):
δ = 1.37 (d, 3H, J = 6.4 Hz), 2.00 (m, 2H), 2.14 (s, 3H), 2.62
(br s, 1H), 2.64-2.88 (m, 4H), 3.13 (dd, 1H, J = 1.6 Hz, J = 5.7
Hz), 4.21 (m, 1H), 6.27 (d, 1H, J = 1.6 Hz), 7.12-7.38 (m, 5H).
13C NMR (75 MHz, CDCl3): δ = 21.0, 21.1, 25.3, 35.1, 36.0,
64.3, 65.2, 75.9, 126.2, 128.5, 128.6, 141.3, 163.2, 170.2, 170.4.
IR (cm-1): υ = 3502, 2931, 1803, 1753, 1716, 1454-1497, 1379,
1308, 1213. HRMS: C17H21NO5Na: calculated: 342.1317,
found: 342.1307.
1-(Pent-4-enoyl)-(3R,4R)-3-[1(R)-(hydroxyethyl]-4-(acetoxy)-
azetidin-2-one (11c). Yield: 83% (490 mg from 2.3 mmol of 9c).
Rf = 0.40 (cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 278.1
((M þ Na)þ). 1H NMR (500 MHz, CDCl3): δ = 1.24 (d, 3H, J =
6.4 Hz), 2.02 (s, 3H), 2.30 (dt, 2H, J = 7.4 Hz, J = 6.8 Hz), 2.70
(m, 1H), 3.08 (dd, 1H, J = 1.4 Hz, J = 5.4 Hz), 3.30 (br s, 1H),
4.13 (qd, 1H, J = 5.4 Hz, J = 6.4 Hz), 4.91 (dd, 1H, J = 1.5 Hz,
J = 10.3 Hz), 4.98 (dd, 1H, J = 1.5 Hz, J = 17.1 Hz), 5.72 (ddt,
1H, J = 10.3 Hz, J = 17.1 Hz, J = 6.8 Hz), 6.33 (d, 1H, J = 1.4
Hz). 13C NMR (125 MHz, CDCl3): δ = 20.7, 20.9, 27.4, 35.6,
63.8, 64.9, 75.2, 115.8, 136.0, 163.3, 169.5, 169.9. IR: υ = 3501,
2932-2978, 1805, 1755, 1718, 1641, 1311. HRMS:
C12H17NO5Na: calculated: 278.1004, found: 278.0992.
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-
2-one (12a). Yield: 78% (297 mg from 1.55 mmol of 10a). Mp:
64.5-65.5 ꢀC. [R]D = -36.7 (c = 1.5). Rf = 0.09 (cyclohexane/
ethyl acetate: 5/3). MS (ESI): m/z: 270.2 ((M þ Na)þ), 516.9 ((2
M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.28 (d, 3H, J =
6.4 Hz), 1.91 (br s, 1H), 2.88-3.12 (m, 4H), 3.25 (m, 1H), 3.60
(m, 2H), 4.22 (m, 1H), 7.12-7.38 (m, 5H). 13C NMR (75 MHz,
CDCl3): δ = 21.7, 30.2, 38.4, 39.2, 55.9, 64.9, 126.4, 128.6,
128.7, 140.4, 166.1, 170.4. IR (cm-1): υ = 3464, 2970, 1782,
1697, 1454-1496, 1387, 1313, 1236. HRMS: C14H17NO3Na:
calculated: 270.1106, found: 270.1109.
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-2-
one (12b). Yield: 86% (165.5 mg from 0.73 mmol of 10b). Mp:
89.5-90.0 ꢀC. [R]D = -38.6 (c = 2.5). Rf = 0.10 (cyclohexane/
ethyl acetate: 5/3). MS (ESI): m/z: 262.1 ((M þ H)þ), 284.15
((M þ Na)þ). 1H NMR (500 MHz, CDCl3): δ = 1.30 (d, 3H,
J = 6.4 Hz), 1.68 (br s, 1H), 2.00 (m, 2H) 2.68 (t, 2H, J = 4.5
Hz), 2.73 (t, 2H, J = 4.5 Hz), 3.27 (m, 1H), 3.60 (m, 2H), 4.27
(m, 1H), 7.15-7.30 (m, 5H). 13C NMR (125 MHz, CDCl3): δ =
21.7, 25.7, 35.2, 36.1, 39.1, 55.8, 64.9, 126.1, 128.5, 128.6, 141.5,
166.1, 171.0. IR (cm-1): υ = 3449, 2970, 1784, 1697, 1454,
1389, 1312, 1250. HRMS: C15H19NO3Na: calculated:
284.1263, found: 284.1261.
1-(5-Phenylpentanoyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-2-
=
one (12c). Yield: 84% (68.6 mg from 0.30 mmol of 10c). [R]D
-13.1 (c = 0.1). Rf = 0.10 (cyclohexane/ethyl acetate: 5/3). MS
(ESI): m/z: 276.2 ((M þ H)þ), 298.1 ((M þ Na)þ). 1H NMR (δ
ppm, 300 MHz, CDCl3): δ = 1.27 (d, 3H, J = 6.4 Hz),
1.55-1.80 (m, 4H), 2.49-2.77 (m, 4H), 3.25 (m, 1H), 3.58 (m,
2H), 4.21 (m, 1H), 7.04-7.33 (m, 5H). 13C NMR (δ ppm, 75
MHz, CDCl3): δ = 21.6, 23.7, 30.8, 35.6, 36.4, 39.1, 55.7, 64.7,
125.8, 128.3, 128.4, 142.1, 166.3, 171.2. IR (ATR-SeZn, cm-1):
3445, 2930, 1784, 1697, 1452, 1389, 1312, 1240. HRMS:
C16H21NO3Na: calculated: 298.1419, found: 298.1405.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-2-one (12d).
Yield: 85% (457 mg from 2.32 mmol of 10d). Rf = 0.38
(cyclohexane/ethyl acetate: 5/4). MS (ESI): m/z: 198.1 ((M þ H)þ),
220.1 ((M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.28 (d, 3H,
J = 6.4 Hz), 2.38 (td, 2H, J = 7.3 Hz, J = 6.8 Hz), 2.78 (t, 2H, J =
7.3 Hz), 3.28 (m, 1H), 3.56 (br s, 1H), 3.61 (m, 2H), 4.24 (m, 1H),
4.99 (dd, 1H, J = 1.5 Hz, J = 10.3 Hz), 5.72 (dd, 1H, J = 1.5 Hz,
J = 17.1 Hz), 5.81 (ddt, 1H, J =10.3 Hz, J = 17.1Hz, J =6.8 Hz).
13C NMR (75 MHz, CDCl3): δ = 21.4, 27.8, 35.6, 39.1, 55.7, 64.6,
115.7, 136.3, 166.2, 170.5. IR (cm-1): υ = 3443, 2928-2972, 1786,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7063
1701, 1641, 1315. HRMS: C10H15NO3Na: calculated: 198.1130,
found: 198.1122.
1-(Hexa-5-enoyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-2-one
10H). 13C NMR (75 MHz, CDCl3): δ = 18.2, 20.8, 25.3, 30.8,
35.0, 35.7, 36.0, 62.7, 65.8, 74.8, 126.1, 126.4, 128.3 (2C), 128.5,
128.6, 140.2, 141.1, 162.3, 169.0, 169.9, 171.8. IR (cm-1): υ =
2935-3028, 1803, 1740, 1717, 1454-1497, 1379, 1310, 1213.
HRMS: C26H29NO6Na: calculated: 474.1893, found:
474.1875.
1-(4-Phenylbutanoyl)-(3R,4R)-3-[1(R)-(4-phenylbutanoyloxy)-
ethyl]-4-(acetoxy)-azetidin-2-one (14b). Yield: 52% (63 mg from
0.26 mmol of 11b). [R]D = -11.6 (c = 2.2). Rf = 0.48
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 488.4 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.40 (d, 3H, J = 6.5
Hz), 1.81-2.03 (m, 4H), 2.12 (s, 3H), 2.31 (m, 2H), 2.57-2.73 (m,
6H), 3.29 (dd, 1H, J = 1.8 Hz, J = 5.6 Hz), 5.31 (m, 1H), 6.49 (d,
1H, J = 1.8 Hz), 7.22-7.33 (m, 10H). 13C NMR (75 MHz,
CDCl3): δ = 18.3, 20.9, 25.4, 26.5, 33.6, 35.0, 35.1, 36.0, 62.7,
65.7, 74.7, 126.1, 126.2, 128.5 (4C), 141.2, 141.3, 162.4, 169.1,
169.9, 172.3. IR (cm-1): υ = 2852-3026, 1803, 1736, 1720,
1454-1496, 1381, 1307, 1213, 1058. HRMS: C27H31NO6Na:
calculated: 488.2049, found: 488.2044.
1-(Pent-4-enoyl)-(3R,4R)-3-[1(R)-(pent-4-enoyloxy)-ethyl]-4-
(acetoxy)-azetidin-2-one (15e). Yield: 90% (950 mg from 3.14
mmol of 11c). [R]D = -21.9 (c = 5.4). Rf = 0.56 (cyclohexane/
ethyl acetate: 5/2). MS (ESI): m/z: 360.0 ((M þ Na)þ). 1H NMR
(300 MHz, CDCl3): δ = 1.39 (d, 3H, J = 6.4 Hz), 2.11 (s, 3H),
2.34 (m, 2H), 2.40 (m, 4H), 2.80 (m, 2H), 3.28 (dd, 1H, J = 1.6
Hz, J = 5.8 Hz), 5.00 (dd, 1H, J = 1.6 Hz, J = 10.3 Hz), 5.02
(dd, 1H, J = 1.6 Hz, J = 10.3 Hz), 5.05 (dd, 1H, J = 1.6 Hz, J =
17.0 Hz), 5.08 (dd, 1H, J = 1.6 Hz, J = 17.0 Hz), 5.29 (qd, 1H,
J = 5.8 Hz, J = 6.4 Hz), 5.78 (ddt, 1H, J = 6.4 Hz, J = 10.3 Hz,
J = 17.0 Hz), 5.82 (ddt, 1H, J = 6.4 Hz, J = 10.3 Hz, J = 17.0
Hz), 6.48 (d, 1H, J = 1.6 Hz). 13C NMR (75 MHz, CDCl3): δ =
18.1, 20.6, 27.4, 28.6, 33.3, 35.7, 62.5, 65.6, 74.5, 115.6, 115.9,
135.9, 136.2, 162.2, 168.8, 169.2, 171.7. IR (cm-1): υ = 2982,
1806, 1742, 1722, 1642, 1313. HRMS: C17H23NO6Na: calcu-
lated: 360.1423, found: 360.1412.
(12e). Yield: 94% (20.5 mg from 0.10 mmol of 10e). [R]D
=
-30.2 (c = 3.0). Rf = 0.13 (cyclohexane/ethyl acetate: 5/3).
MS (ESI): m/z: 212.1 ((M þ H)þ). 1H NMR (300 MHz,
CDCl3): δ = 1.30 (d, 3H, J = 6.4 Hz), 1.75 (m, 2H), 2.00 (br
s, 1H), 2.11 (m, 2H), 2.70 (t, 2H, J = 7.5 Hz), 3.27 (m, 1H), 3.61
(d, 2H, J = 5.1 Hz), 4.26 (m, 1H), 4.96-5.06 (m, 2H), 5.78 (m,
1H). 13C NMR (75 MHz, CDCl3): δ = 21.7, 23.3, 33.1, 36.0,
39.1, 55.8, 64.9, 115.5, 137.8, 166.2, 171.2. IR (cm-1): υ =
3470, 2930, 1786, 1697, 1441-1456, 1389, 1312, 1259. HRMS:
C11H17NO3Na: calculated: 212.12866, found: 212.12837.
1-(Benzoyloxycarbonyl)-(3S)-3-[1(R)-hydroxyethyl]-azetidin-
2-one (22). Yield: 91% (98.3 mg from 0.43 mmol of 21). [R]D
=
-37.0 (c = 4.1). Rf = 0.09 (cyclohexane/ethyl acetate: 5/3). MS
1
(ESI): m/z: 272.1 ((M þ Na)þ). H NMR (300 MHz, CDCl3):
δ = 1.24 (d, 3H, J = 6.4 Hz), 2.65 (br s, 1H), 3.25 (m, 1H),
3.51-3.75 (m, 2H), 4.20 (m, 1H), 5.22 (s, 2H), 7.21-7.54 (m,
5H). 13C NMR (75 MHz, CDCl3): δ = 21.5, 40.6, 56.8, 64.5,
68.1, 128.4, 128.7 (2C), 135.0, 149.1, 165.9. IR (cm-1): υ = 3497,
2972, 1803, 1726, 1456, 1389, 1335. HRMS: C13H15NO4Na:
calculated: 272.0899, found: 272.0888.
General Procedure for Esterification with Acyl Chloride
(13a-b, 14a-b, 15e, 16a-b, 17a-b, 18b, 19b and 19e, 20b). To
a stirred solution of alcohol precursor (1 equiv) in dry dichloro-
methane (20 mL/mmol), at 20 ꢀC, were added pyridine (2 equiv)
and the suitable acyl chloride (2 equiv) under argon atmosphere.
After being stirred overnight, the mixture was diluted in di-
chloromethane 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 a vacuum. After purification by flash chromatography
(dichloromethane/ethyl acetate), a colorless oil was obtained in
all cases.
1-(3-Phenylpropanoyl)-(3R,4R)-3-[1(R)-(3-phenylpropanoylo-
xy)-ethyl]-4-(acetoxy)-azetidin-2-one (13a). Yield: 99% (77 mg
from 0.18 mmol of 11a). [R]D = -16.6 (c = 5.3). Rf = 0.44
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 460.3 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.37 (d, 3H J = 6.9
Hz), 2.15 (s, 3H), 2.64 (m, 2H), 2.90-2.98 (m, 2H), 2.99-3.06
(m, 4H), 3.30 (dd, 1H, J = 1.7 Hz, J = 5.9 Hz), 5.31 (m, 1H),
6.48 (d, 1H, J = 1.7 Hz), 7.17-7.39 (m, 10H). 13C NMR (75
MHz, CDCl3): δ = 18.2, 20.9, 29.8, 30.8, 35.8, 38.3, 62.8, 65.8,
74.8, 126.4, 126.5, 128.3, 128.6, 128.6 (2C), 139.9, 140.2, 162.3,
169.0, 169.3, 171.9. IR (cm-1): υ = 2930, 1805, 1736, 1720, 1454,
1381, 1313, 1213. HRMS: C25H27NO6Na: calculated:
460.1736, found: 460.1722.
1-(3-Phenylpropanoyl)-(3R,4R)-3-[1(R)-(4-phenylbutanoylo-
xy)-ethyl]-4-(acetoxy)-azetidin-2-one (13b). Yield: 70% (153
mg from 0.49 mmol of 11a). [R]D = -17.7 (c = 2.6). Rf =
0.48 (cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 474.2
((M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.41 (d, 3H,
J = 6.5 Hz), 1.94 (m, 2H), 2.14 (s, 3H), 2.36 (t, 2H, J = 7.3 Hz),
2.65 (t, 2H, J = 7.6 Hz), 2.93-3.08 (m, 4H), 3.32 (dd, 1H, J =
1.6 Hz, J = 5.7 Hz), 5.32 (m, 1H), 6.53 (d, 1H J = 1.6 Hz),
7.12-7.39 (m, 10H). 13C NMR (75 MHz, CDCl3): δ = 18.1,
20.7, 26.4, 29.7, 33.5, 35.0, 38.2, 62.6, 65.5, 74.6, 126.0, 126.4,
128.4 (2C), 128.5, 128.5, 139.8, 141.2, 162.3, 168.9, 169.2,
172.2. IR (cm-1): υ = 2932, 1803, 1742, 1720, 1454, 1381,
1313, 1213, 1188. HRMS: C26H29NO6Na: calculated:
474.1893, found: 474.1893.
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-(3-phenylpropanoyloxy)-
ethyl]-azetidin-2-one (16a). Yield: 79% (64 mg from 0.21 mmol
of 12a). [R]D = -17.1 (c = 2.7). Rf = 0.41 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 402.1 ((M þ Na)þ). 1H NMR (300
MHz, CDCl3): δ = 1.29 (d, 3H, J = 6.4 Hz), 2.61 (t, 2H, J = 7.7
Hz), 2.93 (t, 2H, J = 7.7 Hz), 2.96-3.08 (m, 4H), 3.31 (m, 1H),
3.41 (dd, 1H, J = 3.6 Hz, J = 7.7 Hz), 3.56 (dd, 1H, J = 6.6 Hz,
J = 7.7 Hz), 5.21 (m, 1H), 7.09-7.60 (m, 10H). 13C NMR (75
MHz, CDCl3): δ = 18.3, 30.1, 30.9, 35.8, 38.3, 40.1, 53.6,
67.6, 126.4, 126.5, 128.3, 128.56, 128.62 (2C), 140.1, 140.2,
164.3, 170.2, 171.9. IR (cm-1): υ = 2931-3028, 1786, 1735,
1701, 1454-1497, 1383, 1315, 1238, 1132-1161. HRMS:
C23H25NO4Na: calculated: 402.1681, found: 402.1675.
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-(4-phenylbutanoyloxy)-et-
hyl]-azetidin-2-one (16b). Yield: 75% (79 mg from 0.27 mmol of
12a). [R]D = -9.6 (c = 3.4). Rf = 0.44 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 416.2 ((M þ Na)þ), 808.7 ((2 M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.37 (d, 3H, J = 6.4
Hz), 1.95 (m, 2H), 2.32 (t, 2H, J = 7.5 Hz), 2.66 (t, 2H, J = 7.6
Hz), 2.90-3.11 (m, 4H), 3.41 (m, 1H), 3.32 (dd, 1H, J = 3.6 Hz,
J = 7.7 Hz), 3.68 (dd, 1H, J = 6.8, Hz, J = 7.7 Hz), 5.28 (m, 1H),
7.12-7.42 (m, 10H). 13C NMR(75 MHz, CDCl3): δ = 18.4, 26.6,
30.1, 33.7, 35.1, 38.3, 40.0, 53.6, 67.3, 126.2, 126.4, 128.5 (2C),
128.6 (2C), 140.2, 141.2, 164.4, 170.2, 172.4. IR (cm-1): υ =
2858-3086, 1784, 1732, 1697, 1454-1497, 1381, 1313, 1238,
1132-1190. HRMS: C24H27NO4Na: calculated: 416.1838,
found: 416.1827.
1-(4-Phenylbutanoyl)-(3R,4R)-3-[1(R)-(3-phenylpropanoylo-
xy)-ethyl]-4-(acetoxy)-azetidin-2-one (14a). Yield: 88% (90 mg
from 0.23 mmol of 11b). [R]D = -17.1 (c = 6.2). Rf = 0.42
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 473.9 ((M þ
Na)þ), 925.3 ((2 M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ =
1.36 (d, 3H, J = 6.5 Hz), 2.00 (m, 2H), 2.12 (s, 3H), 2.57-2.77
(m, 6H), 2.93 (t, 2H, J = 7.7 Hz), 3.28 (dd, 1H, J = 1.7 Hz, J =
5.9 Hz), 5.31 (m, 1H), 6.46 (d, 1H, J = 1.7 Hz), 7.14-7.34 (m,
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-(3-phenylpropanoyloxy)-et-
hyl]-azetidin-2-one (17a). Yield: 89% (71 mg from 0.20 mmol of
12b). [R]D = -9.9 (c = 4.9). Rf = 0.40 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 394.0 ((M þ H)þ), 416.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.31 (d, 3H, J = 6.4
Hz), 2.00 (m, 2H), 2.56-2.83 (m, 6H), 2.93 (t, 2H, J = 7.6 Hz),
3.33 (m, 1H), 3.41 (dd, 1H, J = 3.6, Hz, J = 7.7 Hz), 3.56 (dd,
1H, J = 6.7, Hz, J = 7.7 Hz), 5.24 (m, 1H), 7.01-7.59 (m, 10H).

7064 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Feledziak et al.
13C NMR(75MHz, CDCl3):δ = 18.3, 25.6, 30.9, 35.1, 35.8, 36.0,
40.0, 53.5, 67.6, 126.1, 126.5, 128.3, 128.4, 128.5, 128.6, 140.1,
141.3, 164.3, 170.8, 171.8. IR (cm-1): υ = 2935-3026, 1786,
1736, 1701, 1454-1497, 1383, 1313, 1250, 1132-1190. HRMS:
C24H27NO4Na: calculated: 416.1838, found: 416.1821.
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-(4-phenylbutanoyloxy)-eth-
yl]-azetidin-2-one (17b). Yield: 87% (68 mg from 0.19 mmol of
12b). [R]D = -3.3 (c = 4.8). Rf = 0.41 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 408.0 ((M þ H)þ), 430.1 ((M þ
Na)þ). 1H NMR (500 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.4
Hz), 1.87-2.03 (m, 4H), 2.29 (t, 2H, J = 7.5 Hz), 2.56-2.83 (m,
6H), 3.38 (m, 1H), 3.52 (dd, 1H, J = 3.7 Hz, J = 7.7 Hz), 3.64
(dd, 1H, J = 6.7 Hz, J = 7.7 Hz), 5.28 (m, 1H), 7.12-7.32 (m,
10H). 13C NMR (125 MHz, CDCl3): δ = 18.4, 25.7, 26.6, 33.7,
35.1, 35.2, 36.1, 40.0, 53.6, 67.3, 126.1, 126.2, 128.5, 128.55,
128.57 (2C), 141.2, 141.4, 164.4, 170.9, 172.4. IR (cm-1): υ =
2934, 1786, 1734, 1701, 1454-1497, 1383, 1312, 1246,
1130-1159. HRMS: C25H29NO4Na: calculated: 430.1994,
found: 430.1985.
40.0, 53.6, 67.3, 115.6, 126.2, 128.6 (2C), 137.7, 141.2, 164.5,
171.1, 172.4. IR (cm-1): υ = 2934-2976, 1786, 1734, 1701,
1454, 1381, 1313, 1250, 1132-1190. HRMS: C21H27NO4Na:
calculated: 380.1838, found: 380.1827.
General Procedure for Esterification with Carboxylic Acid
(16c-d, 17c-d, 18d, 19d and 19f, 20d and 23). To a stirred
solution of alcohol precursor, DCC (1.1 equiv) and DMAP
(cat.) in dry dichloromethane (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 a
vacuum. After purification by flash chromatography
(dichloromethane/ethyl acetate), white solids (13d, 14d, 16d,
17d, 18d, 19d, 20d, and 23) or colorless oils (16c, 17c, and 19f)
were obtained.
1-(3-Phenylpropanoyl)-(3R,4R)-3-[1(R)-(biphenylacetyloxy)-
ethyl]-4-(acetoxy)-azetidin-2-one (13d). Yield: 67% (35 mg from
0.16 mmol of 11a). Mp: 98.0-103.0 ꢀC. [R]D = -15.1 (c = 2.3).
Rf = 0.28 (cyclohexane/ethyl acetate: 5/2). MS (ESI): m/z:
522.2 ((M þ Na)þ), 1020.9 ((2 M þ Na)þ). 1H NMR (300 MHz,
CDCl3): δ = 1.42 (d, 3H, J = 6.5 Hz), 2.14 (s, 3H), 2.76-2.94
(m, 4H), 3.29 (dd, 1H, J = 1.7 Hz, J = 5.6 Hz), 3.64 (d, 1H, J =
17.1 Hz, AB system), 3.70 (d, 1H, J = 17.1 Hz, AB system), 5.33
(m, 1H), 6.46 (d, 1H, J = 1.7 Hz), 7.13-7.66 (m, 14H). 13C
NMR (75 MHz, CDCl3): δ = 18.3, 20.9, 29.8, 38.3, 41.0, 62.8,
66.2, 74.8, 126.5, 127.2, 127.4 (2C), 128.6, 128.6, 128.9, 129.8,
132.6, 140.0, 140.2, 140.7, 162.2, 169.1, 169.2, 170.5. IR (cm-1):
υ = 2931-3029, 1803, 1740, 1718, 1454-1489, 1381, 1313,
1213. HRMS: C30H29NO6Na: calculated: 522.1893, found:
522.1899.
1-(4-Phenylbutanoyl)-(3R,4R)-3-[1(R)-(biphenylacetyloxy)-
ethyl]-4-(acetoxy)-azetidin-2-one (14d). Yield: 77% (62 mg
from 0.16 mmol of 11b). Mp: 87.5-89.0 ꢀC. [R]D = -8.2
(c = 4.7). Rf = 0.32 (cyclohexane/ethyl acetate: 5/2). MS
(ESI): m/z: 536.1 ((M þ Na)þ), 1048.6 ((2 M þ Na)þ). 1H
NMR (300 MHz, CDCl3): δ = 1.43 (d, 3H, J = 6.5 Hz),
1.85-2.00 (m, 2H), 2.12 (s, 3H), 2.48-2.73 (m, 4H), 3.28 (dd,
1H, J = 1.7 Hz, J = 5.6 Hz), 3.64 (d, 1H, J = 15.5 Hz, AB
system), 3.70 (d, 1H, J = 15.5 Hz, AB system), 5.35 (m, 1H),
6.46 (d, 1H, J = 1.7 Hz), 7.15-7.70 (m, 14H). 13C NMR (75
MHz, CDCl3): δ = 18.3, 20.9, 25.2, 35.0, 36.0, 41.0, 62.7, 66.2,
74.7, 126.1, 127.1, 127.4 (2C), 128.5 (2C), 128.9, 129.7, 132.6,
140.2, 140.7, 141.2, 162.2, 169.1, 169.8, 170.4. IR (cm-1): υ =
2854-3082, 1803, 1740, 1717, 1452-1489, 1381, 1312, 1213.
HRMS: C31H31NO6Na: calculated: 536.2049, found:
536.2062.
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-(5-phenylpentanoyloxy)-
ethyl]-azetidin-2-one (16c). Yield: 59% (48 mg from 0.20 mmol
of 12a). [R]D = -9.9 (c = 1.8). Rf = 0.44 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 430.1 ((M þ Na)þ). 1H NMR (300
MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.5 Hz), 1.58-1.76 (m,
4H), 2.32 (m, 2H), 2.63 (m, 2H), 2.96-3.09 (m, 4H), 3.38 (m,
1H), 3.52 (dd, 1H, J = 3.6 Hz, J = 7.7 Hz), 3.66 (dd, 1H, J =
6.5 Hz, J = 7.7 Hz), 5.25 (m, 1H), 7.14-7.40 (m, 10H). 13C
NMR (75 MHz, CDCl3): δ = 18.4, 24.6, 30.2, 30.9, 34.3, 35.6,
38.3, 40.1, 53.6, 67.3, 125.9, 126.4, 128.5 (2C), 128.6 (2C), 140.2,
142.0, 164.4, 170.3, 172.5. IR (cm-1): υ = 2932-3026, 1785,
1734, 1701, 1454-1497, 1387, 1315, 1238-1255, 1132-1175.
HRMS: C25H29NO4Na: calculated: 430.1994, found:
430.1990.
1-(5-Phenylpentanoyl)-(3S)-3-[1(R)-(4-phenylbutanoyloxy)-eth-
yl]-azetidin-2-one (18b). Yield: 63% (34 mg from 0.13 mmol of
12c). [R]D = -2.2 (c = 1.8). Rf = 0.43 (cyclohexane/ethyl acetate:
1
5/3). MS (ESI): m/z: 444.1 ((M þ Na)þ). H NMR (300 MHz,
CDCl3): δ = 1.35 (d, 3H, J = 6.3 Hz), 1.57-1.79 (m, 4H), 1.91
(m, 2H), 2.29 (t, 2H, J = 7.5 Hz), 2.54-2.79 (m, 6H), 3.39 (m,
1H), 3.53 (dd, 1H, J = 3.6 Hz, J = 7.5 Hz), 3.65 (dd, 1H, J = 6.8
Hz, J = 7.5 Hz), 5.28 (m, 1H), 7.08-7.38 (m, 10H). 13C NMR (75
MHz, CDCl3): δ = 18.4, 23.7, 26.6, 30.9, 33.7, 35.1, 35.6, 36.5,
39.9, 53.6, 67.3, 125.9, 126.2, 128.4, 128.5, 128.6 (2C), 141.2, 142.1,
164.5, 171.1, 172.4. IR (cm-1): υ = 2858-3026, 1786, 1734, 1699,
1452-1497, 1381, 1313, 1242, 1132-1192. HRMS: C26H31NO4-
Na: calculated: 444.2151, found: 444.2152.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-(4-phenylbutanoyloxy)-ethyl]-az-
etidin-2-one (19b). Yield: 84% (89 mg from 0.31 mmol of 12d).
[R]D = -1.3 (c = 3.5). Rf = 0.46 (cyclohexane/ethyl acetate: 5/3).
MS (ESI): m/z: 344.0 ((M þ H)þ), 366.1 ((M þ Na)þ). 1H NMR
(300 MHz, CDCl3): δ = 1.34 (d, 3H, J = 6.4 Hz), 1.92 (m, 2H),
2.30 (t, 2H, J = 7.6 Hz), 2.39 (m, 2H), 2.63 (t, 2H, J = 7.6 Hz),
2.77 (t, 2H, J = 7.4 Hz), 3.40 (m, 1H), 3.53 (dd, 1H, J = 3.7 Hz,
J = 7.7 Hz), 3.66 (dd, 1H, J = 6.6 Hz, J = 7.7 Hz), 4.96-5.09 (m,
2H), 5.28(m, 1H), 5.81 (m,1H), 7.07-7.36 (m,5H). 13CNMR (75
MHz, CDCl3): δ = 18.4, 26.5, 27.9, 33.7, 35.0, 35.8, 39.9, 53.6,
67.2, 115.9, 126.1, 128.5 (2C), 136.4, 141.2, 164. 4, 170.3, 172.4. IR
(cm-1): υ = 2864-3026, 1786, 1734, 1703, 1454, 1381, 1313,
1238, 1132-1191. HRMS: C20H25NO4Na: calculated:
366.1681, found: 366.1685.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-(pent-4-enoyloxy)-ethyl]-azeti-
din-2-one (19e). Yield: 88% (500 mg from 2.03 mmol of 12d).
[R]D = -0.25 (c = 4.9). Rf = 0.63 (cyclohexane/ethyl acetate:
5/3). MS (ESI): m/z: 280.0 ((M þ H)þ), 302.1 ((M þ Na)þ). 1H
NMR (500 MHz, CDCl3): δ = 1.34 (d, 3H, J = 6.5 Hz), 2.37
(m, 6H), 2.78 (m, 2H), 3.39 (m, 1H), 3.52 (dd, 1H, J = 3.7 Hz,
J = 7.8 Hz), 3.65 (dd, 1H, J = 6.5 Hz, J = 7.8 Hz), 4.99 (m,
2H), 5.03 (m, 1H), 5.06 (m, 1H), 5.26 (m, 1H), 5.77 (ddt, 1H,
J = 5.9 Hz, J = 10.2 Hz, J = 16.2 Hz), 5.81 (ddt, 1H, J = 6.5
Hz, J = 10.2 Hz, J = 16.8 Hz). 13C NMR (125 MHz, CDCl3):
δ = 18.2, 27.8, 28.7, 33.4, 35.7, 39.8, 53.4, 67.2, 115.6, 115.7,
136.1, 136.2, 164.2, 170.2, 171.7. IR (cm-1): υ = 2927-2979,
1785, 1738, 1702, 1320. HRMS: C15H21NO4Na: calculated:
302.1368, found: 302.1358.
1-(Hexa-5-enoyl)-(3S)-3-[1(R)-(4-phenylbutanoyloxy)-ethyl]-
azetidin-2-one (20b). Yield: 77% (20 mg from 0.07 mmol of 12e).
[R]D = -2.6 (c = 1.0). Rf = 0.43 (cyclohexane/ethyl acetate:
5/3). MS (ESI): m/z: 358.0 ((M þ H)þ), 380.1 ((M þ Na)þ). 1H
NMR (300 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.4 Hz), 1.74
(m, 2H), 1.92 (m, 2H), 2.10 (m, 2H), 2.29 (t, 2H, J = 7.5 Hz),
2.56-2.74 (m, 4H), 3.39 (m, 1H), 3.53 (dd, 1H, J = 3.7 Hz, J =
7.7 Hz), 3.66 (dd, 1H, J = 6.6 Hz, J = 7.7 Hz), 4.93-5.02 (m,
2H), 5.28 (m, 1H), 5.77 (m, 1H), 7.10-7.36 (m, 5H). 13C NMR
(75 MHz, CDCl3): δ = 18.4, 23.2, 26.6, 33.1, 33.7, 35.1, 35.9,
1-(3-Phenylpropanoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-ethyl]-
azetidin-2-one (16d). Yield: 93% (84 mg from 0.20 mmol of 12a).
Mp: 61.5-62.0 ꢀC. [R]D = -18.2 (c = 3.7). Rf = 0.38
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 464.2 ((M þ
Na)þ), 904.8 ((2 M þ Na)þ). 1H NMR (300 MHz, CDCl3): δ =
1.29 (d, 3H, J = 6.4 Hz), 2.77-2.96 (m, 4H), 3.31 (m, 1H), 3.42
(dd, 1H, J = 3.7 Hz, J = 7.7 Hz), 3.54 (dd, 1H, J = 7.2 Hz, J =
7.7Hz), 3.58 (s, 2H), 5.22 (m, 1H), 7.09-7.60 (m, 14H). 13C NMR
(75 MHz, CDCl3): δ = 18.5, 30.2, 38.4, 39.9, 41.3, 53.7, 67.9,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7065
126.5, 127.3, 127.5 (2C), 128.7 (2C), 129.0, 129.7, 132.8, 140.3,
140.4, 140. 7, 164.3, 170.3, 170.5. IR (cm-1): υ = 2906-3058,
1786, 1734, 1701, 1454-1489, 1387, 1315, 1251, 1132-1157.
HRMS: C28H27NO4Na: calculated: 464.1838, found: 464.
1845.
1701, 1381, 1315, 1238, 1134-1168. HRMS: C16H23NO4Na:
calculated: 316.1525, found: 316.1515.
1-(Hexa-5-enoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-ethyl]-aze-
tidin-2-one(20d). Yield: 66% (39mg from 0.06mmol of 12e). Mp:
44.2-45.1 ꢀC. [R]D = -11.7 (c = 1.1). Rf = 0.43 (cyclohexane/
ethyl acetate: 5/3). MS (ESI): m/z: 428.1 ((M þ Na)þ). 1H NMR
(300 MHz, CDCl3): δ = 1.36 (d, 3H, J = 6.4 Hz), 1.71 (m, 2H),
2.06 (m, 2H), 2.61 (m, 2H), 3.39 (m, 1H), 3.49 (dd, 1H, J = 3.7
Hz, J = 7.7 Hz), 3.61 (dd, 1H, J = 7.2 Hz, J = 7.7 Hz), 3.64
(s, 2H), 4.90-5.06 (m, 2H), 5.30(m, 1H), 5.74(m, 1H), 7.23-7.65
(m, 9H). 13C NMR (75 MHz, CDCl3): δ = 18.4, 23.2, 33.0, 35.9,
39.8, 41.2, 53.5, 67.8, 115.5, 127.1, 127.4 (2C), 128.9, 129.6, 132.7,
137.7, 140.3, 140.6, 164.3, 170.5, 171.0. IR (cm-1): υ =
2934-2976, 1786, 1736, 1701, 1450-1489, 1389, 1315, 1252,
1132-1194. HRMS: C25H27NO4Na: calculated: 428.1838,
found: 428.1825.
1-(Benzyloxycarbonyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-eth-
yl]-azetidin-2-one (23). Yield: 83% (75 mg from 0.20 mmol of
22). Mp: 90.8-91.6 ꢀC. [R]D = -19.8 (c = 0.6). Rf = 0.37
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 466.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.3
Hz), 3.39 (m, 1H), 3.51 (dd, 1H, J = 3.6 Hz, J = 7.0 Hz),
3.60-3.70 (m, 3H), 5.19 (s, 2H), 5.29 (m, 1H), 7.26-7.67 (m,
14H). 13C NMR (75 MHz, CDCl3): δ = 18.4, 41.2, 41.4, 54.5,
68.0, 68.2, 127.2 (2C), 128.4 (2C), 128.7, 128.8, 128.9, 129.7,
132.7, 135.0, 140.2, 140.8, 148.9, 163.6, 170.6. IR (cm-1):
2920-3059, 1813, 1772, 1730, 1456-1489, 1389, 1329, 1128.
HRMS: C27H25NO5Na: calculated: 466.1630, found:
466.1609.
1-(Benzyloxycarbonyl)-(3S)-3-[1(R)-(tert-butyldimethylsilyloxy)-
ethyl]-azetidin-2-one (21). To a stirred solution of lithium hexam-
ethyldisilazide (436 μL, 0.44 mmol) in tetrahydrofurane (2 mL)
at -78 ꢀC was added 8 (100 mg, 0.44 mmol) in tetrahydrofurane
(2 mL) under argon atmosphere. The mixture was stirred for 30 min
at -78 ꢀC; then benzyl chloroformate (75 μL, 0.52 mmol) was
added. After being stirred for 1 h, at low temperature, the solution
was allowed to warm up and was stirred for 1 h at 20 ꢀC. After
dilution in dichloromethane, the organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated under a
vacuum. Purification by flash chromatography (cyclohexane/ethyl
acetate) gave 21 as a white solid. Yield: 99% (157 mg from 0.44
mmol of 8). Mp: 44.6-46.0 ꢀC. Rf = 0.46 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 386.1 ((M þ Na)þ). 1H NMR (300
MHz, CDCl3): δ = 0.03 (s, 3H), 0.06 (s, 3H), 0.81 (s, 9H), 1.17 (d,
3H, J = 6.3 Hz), 3.22 (m, 1H), 3.58 (dd, 1H, J = 3.5 Hz, J = 6.5
Hz), 3.73 (dd, 1H, J = 6.4 Hz, J = 6.5 Hz), 4.29 (m, 1H), 5.25 (s,
2H), 7.27-7.48 (m, 5H). 13C NMR (75 MHz, CDCl3): δ = -5.1,
-4.1, 17.9, 22.3, 25.7, 39.8, 57.3, 64.8, 68.0, 128.4, 128.5, 128.7,
135.4, 149.3, 165.8. IR (cm-1): υ = 2854-2924, 1801, 1726, 1464,
1387, 1323-1339, 1259. HRMS: C19H29NO4SiNa: calculated:
386.1764, found: 386.1776.
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-(5-phenylpentanoyloxy)-
ethyl]-azetidin-2-one (17c). Yield: 93% (76 mg from 0.19 mmol
of 12b). [R]20/D = -2.3 (c = 4.0). Rf = 0.41 (cyclohexane/ethyl
acetate: 5/3). MS (ESI): m/z: 421.9 ((M þ H)þ), 444.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.4
Hz), 1.56-1.73 (m, 4H), 2.01 (m, 2H), 2.31 (m, 2H), 2.62 (m,
2H), 2.66-2.84 (m, 4H), 3.38 (m, 1H), 3.51 (dd, 1H, J = 3.7
Hz, J = 7.7 Hz), 3.63 (dd, 1H, J = 6.6 Hz, J = 7.7 Hz), 5.27
(m, 1H), 7.08-7.45 (m, 10H). 13C NMR (75 MHz, CDCl3):
δ = 18.4, 24.6, 25.7, 30.8, 34.2, 35.1, 35.5, 36.0, 39.9, 53.5,
67.2, 125.9, 126.1, 128.4 (2C), 128.5 (2C), 141.3, 142.0, 164.4,
170.8, 172.5. IR (cm-1): υ = 2856-3026, 1786, 1734, 1701,
1454, 1383, 1313, 1250, 1130. HRMS: C26H31NO4Na: cal-
culated: 444.2151, found: 444.2141.
1-(4-Phenylbutanoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-ethyl]-
azetidin-2-one (17d). Yield: 83% (72 mg from 0.19 mmol of 12b).
Mp: 96.0-96.5 ꢀC. [R]D = -14.8 (c = 4.7). Rf = 0.40 (cyclo-
hexane/ethyl acetate: 5/3). MS (ESI): m/z: 456.0 ((M þ H)þ),
478.1 ((M þ Na)þ). 1H NMR (500 MHz, CDCl3): δ = 1.37 (d,
3H, J = 6.4 Hz), 1.95 (m, 2H), 2.57-2.76 (m, 4H), 3.37 (m, 1H)
3.48 (dd, 1H, J = 3.7 Hz, J = 7.7 Hz), 3.59 (dd, 1H, J = 6.6 Hz,
J = 7.7 Hz), 3.65 (s, 2H), 5.31 (m, 1H), 6.98-7.76 (m, 14H). 13
C
NMR (125 MHz, CDCl3): δ = 18.4, 25.5, 35.1, 36.0, 39.7, 41.1,
53.5, 67.8, 126.1, 127.1, 127.38, 127.43, 128.4, 128.5, 128.9, 129.6,
132.6, 140.2, 140.6, 141.3, 164.2, 170.4, 170.7. IR (cm-1): υ =
2936-3028, 1786, 1736, 1697, 1452-1489, 1389, 1313, 1248,
1132-1155. HRMS: C29H29NO4Na: calculated: 478.1994,
found: 478.1994.
1-(5-Phenylpentanoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-eth-
yl]-azetidin-2-one (18d). Yield: 66% (39 mg from 0.12 mmol of
12c). Mp: 70.5-71.3 ꢀC. [R]D = -1.1 (c = 7.0). Rf = 0.44
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 492.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.36 (d, 3H, J = 6.4
Hz), 1.52-1.73 (m, 4H), 2.50-2.75 (m, 4H), 3.38 (m, 1H), 3.48
(dd, 1H, J = 3.6 Hz, J = 7.7 Hz), 3.56-3.65 (m, 3H), 5.31 (m,
1H), 7.07-7.64 (m, 14H). 13C NMR (75 MHz, CDCl3): δ =
18.4, 23.7, 30.8, 35.6, 36.4, 39.7, 41.2, 53.5, 67.8, 125.8, 127.1,
127.4, 127.5, 128.4, 128.5, 128.9, 129.6, 132.7, 140.2, 140.6,
142.1, 164.3, 170.4, 170.9. IR (cm-1): υ = 2854-3028, 1786,
1736, 1699, 1452-1489, 1389, 1315, 1246, 1132-1159. HRMS:
C30H31NO4Na: calculated: 492.2151, found: 492.2133.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-(biphenylacetyloxy)-ethyl]-aze-
tidin-2-one (19d). Yield: 68% (70 mg from 0.26 mmol of 12d).
Mp: 92.5-93.0 ꢀC. [R]D = -9.8 (c = 2.5). Rf = 0.44
(cyclohexane/ethyl acetate: 5/3). MS (ESI): m/z: 414.1 ((M þ
Na)þ). 1H NMR (300 MHz, CDCl3): δ = 1.36 (d, 3H, J = 6.4
Hz), 2.36 (m, 2H), 2.70 (m, 2H), 3.39 (m, 1H), 3.48 (dd, 1H, J =
3.7 Hz, J = 7.7 Hz), 3.61 ((dd, 1H, J = 6.6 Hz, J = 7.7 Hz), 3.64
(s, 2H), 5.00 (m, 2H), 5.30 (m, 1H), 5.79 (m, 1H), 7.27-7.70 (m,
9H). 13C NMR (75 MHz, CDCl3): δ = 18.4, 27.9, 35.8, 39.8,
41.2, 53.6, 67.8, 115.8, 127.1, 127.4, 128.9 (2C), 129.6, 132.7,
136.4, 140.2, 140.6, 164.3, 170.3, 170.4. IR (cm-1): υ = 2916,
1788, 1734, 1701, 1488, 1387, 1315, 1238-1259. HRMS:
C24H25NO4Na: calculated: 414.1681, found: 414.1692.
1-(Pent-4-enoyl)-(3S)-3-[1(R)-(hexa-5-enoyloxy)-ethyl]-azeti-
din-2-one (19f). Yield: 84% (65 mg from 0.26 mmol of 12d).
[R]D = -0.5 (c = 4.0). Rf = 0.41 (cyclohexane/ethyl acetate:
5/3). MS (ESI): m/z: 316.1 ((M þ Na)þ). 1H NMR (300 MHz,
CDCl3): δ = 1.32 (d, 3H, J = 6.4 Hz), 1.66 (m, 2H), 2.04 (m,
2H), 2.26 (t, 2H, J = 7.5 Hz), 2.38 (m, 2H), 2.76 (t, 2H, J = 7.5
Hz), 3.39 (m, 1H), 3.51 (dd, 1H, J = 3.6 Hz, J = 7.7 Hz), 3.64
(dd, 1H, J = 6.8 Hz, J = 7.7 Hz), 4.85-5.12 (m, 4H), 5.22 (m,
1H), 5.76 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 18.4, 24.1,
28.0, 33.0, 33.6, 35.9, 39.9, 53.6, 67.2, 115.7, 115.9, 136.4, 137.5,
164.5, 170.4, 172.5. IR (cm-1): υ = 2935-2978, 1788, 1736,
(3S)-3-[1(R)-(Biphenylacetyloxy)-ethyl]-azetidin-2-one (24).
To a stirred solution of 23 (56 mg, 0.13 mmol) in ethyl acetate
(2.5 mL) and ethanol (3 mL) was added 10% Pd/C (5.6 mg).
After being stirred under hydrogen atmosphere (P = 1 atm),
during 1 h at room temperature, the mixture was filtered
through a short pad of Celite and concentrated under a vacuum.
24 was obtained without further purification as a white solid.
Yield: 96% (38 mg from 0.13 mmol of 23). Mp: 119.5-120.8 ꢀC.
[R]D = -27.2 (c = 2.0). Rf = 0.06 (cyclohexane/ethyl acetate: 5/
1
3). MS (ESI): m/z: 309.8 ((M þ H)þ), 332.0 ((M þ Na)þ). H
NMR (300 MHz, CDCl3): δ = 1.37 (d, 3H, J = 6.3 Hz), 3.14
(dd, 1H, J = 2.3 Hz, J = 5.5 Hz), 3.32 (dd, 1H, J = 5.4 Hz, J =
5.5 Hz), 3.35-3.43 (m, 1H), 3.65 (s, 2H), 5.25 (m, 1H), 5.96 (br s,
1H), 7.36 (m, 3H), 7.44 (m, 2H), 7.56 (m, 4H). 13C NMR (75
MHz, CDCl3): δ = 18.5, 39.3, 41.2, 56.5, 69.1, 127.1, 127.3,
127.4, 128.9, 129.7, 133.0, 140.1, 140.7, 167.7, 170.8. IR (cm-1):
υ = 3248, 2922-2978, 1755, 1732, 1489, 1250, 1136-
1155. HRMS: C19H19NO3Na: calculated: 332.1263, found:
332.1249.

7066 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Feledziak et al.
In Vitro Assays for Human FAAH. Tubes containing the
enzyme47 (10 mM Tris-HCl, 1 mM EDTA, 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 con-
centration, 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 buffer only were used as
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.
tubes containing buffer only were used as control for chemical
hydrolysis (blank) and this value was systematically subtracted.
Docking Studies. Docking of the inhibitors into the active site
of FAAH was performed using the GOLD program. GOLD is
based on a genetic algorithm, performing docking of flexible
ligands into proteins with partial flexibility in the neighborhood
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: protein-ligand hydrogen bond energy,
protein-ligand van der Walls energy, ligand internal van der
Waals energy, and ligand torsional strain energy. The figures
were produced using PyMOL57 and Ligplot.58
In Vitro Assays for Human MGL Activity. Tubes containing
purified enzyme46 (10 mM Tris-HCl, 1 mM EDTA, 0.1% (w/v)
BSA, pH 8.0, 165 μL), test compounds in DMSO or DMSO
alone for controls (10 μL) and [3H]-2-OG (50 000 dpm, 2 μM
final concentration, 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) fol-
lowed by vigorous mixing. Phases were separated by centrifuga-
tion at 850g, and aliquots (200 μL) of the upper methanol/buffer
phase were counted for radioactivity by liquid scintillation
counting. In all experiments, tubes containing buffer only were
used as control for chemical hydrolysis (blank) and this value
was systematically subtracted.
Preincubation Studies. Tubes containing enzyme (10 mM
Tris-HCl, 1 mM EDTA, 0.1% (w/v) BSA, pH 7.4, 165 μL)
and test compounds in DMSO or DMSO alone (10 μL) were
preincubated 90, 45, 15, and 0 min at room temperature prior to
addition of [3H]-AEA (50 000 dpm, 2 μM final concentration,
25 μL). 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 centri-
fugation at 850g, and aliquots (200 μL) of the upper methanol/
buffer phase were counted for radioactivity by liquid scintilla-
tion counting. In all experiments, tubes containing buffer only
were used as control for chemical hydrolysis (blank) and this
value was systematically subtracted.
Reversibility Studies. In a total volume of 15 μL, human
FAAH (27.5 μg) and inhibitors (or DMSO for controls) at
concentrations allowing inhibition of the enzyme before dilution
and no inhibition after the 100-fold dilution were preincubated
during 1 h at room temperature. The mixtures were then diluted
100-fold by adding assay buffer. Immediately after, an aliquot
(165 μL) was taken and [3H]-AEA (50 000 dpm, 2 μM final
concentration, 25 μL) was added. Two samples were taken at 30
and 90 min after the dilution too. Each aliquots were incubated
at 37 ꢀC for 30 min and reactions were stopped by rapidly
placing the tubes in ice and adding 400 μL of ice-cold chloro-
form/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 radio-
activity by liquid scintillation counting. In all experiments, tubes
containing buffer only were used as control for chemical hydro-
lysis (blank) and this value was systematically subtracted.
Determination of Inhibitor Interactions with hFAAH. Tubes
containing enzyme (10 mM Tris-HCl, 1 mM EDTA, 0.1% (w/v)
BSA, pH 7.4, 165 μL; except for 150 μM of AEA, 159.5 μL and
250 μM of AEA, 139.5 μL) and test compounds in DMSO or
DMSO alone (10 μL) were incubated at 37 ꢀC with increasing
concentrations of [3H]-AEA (50,000 dpm, 1, 2, 5, 10, 15, 20, 30,
75, 150, and 250 μM final concentration, 25 μL; except for
150 μM, 30.5 μL and 250 μM, 50.5 μL). 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 850 g, and aliquots
(200 μL) of the upper methanol/buffer phase were counted for
radioactivity by liquid scintillation counting. In all experiments,
ꢀ
Acknowledgment. The UCL (Universite catholique de
Louvain) and the F. R. S.-FNRS (Fonds de la Recherche
Scientifique, Belgium) are gratefully aknowledged for finan-
cial support of this work (FRFC grant, nꢀ2.4.654.06 F). J.M.-B.
is senior research associate of the Belgian F. R. S.-FNRS,
C.M. is a scientific research worker associate of the Belgian
F. R. S.-FNRS. The authors wish to warmly thank Kaneka
corporation (Japan), in particular Dr. Claudio Salvagnini, for
the donation of the starting azetidinone 7 and Guillaume
Menneson for technical assistance.
Supporting Information Available: Synthesis of compound 8,
pI50 and standard deviation of each tested compound, repre-
sentative “dose-response” curves, docking showing aminoa-
cids involved in hydrophobic contacts and Ramachadran plot of
the modeled human FAAH. This material is available free of
charge via the Internet at http://pubs.acs.org.
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