Discovery of a potent, selective, and efficacious class of reversible α-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as analgesics

Article abstract of DOI:10.1021/jm049614v

Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen against the serine hydrolase superfamily ensuring selectivity for FAAH coupled with systematic in vivo examinations of candidate inhibitors.

Full text of DOI:10.1021/jm049614v

J. Med. Chem. 2005, 48, 1849-1856
1849
Discovery of a Potent, Selective, and Efficacious Class of Reversible
r-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase Effective as
Analgesics∇
Dale L. Boger,*,†,§ Hiroshi Miyauchi,†,§ Wu Du,†,§ Christophe Hardouin,†,§ Robert A. Fecik,†,§ Heng Cheng,†,§
Inkyu Hwang,†,§ Michael P. Hedrick,†,§ Donmienne Leung,‡,§ Orlando Acevedo,| Cristiano R. W. Guimara˜es,|
William L. Jorgensen,| and Benjamin F. Cravatt‡,§
Departments of Chemistry, Cell Biology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and Department of Chemistry, Yale University,
225 Prospect Street, New Haven, Connecticut 06520-8107
Received May 26, 2004
Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including
anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites
of action and is intimately involved in their regulation. Herein we report the discovery of a
potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in
animal models validating a new therapeutic target for pain intervention. Key to the useful
inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen
against the serine hydrolase superfamily ensuring selectivity for FAAH coupled with systematic
in vivo examinations of candidate inhibitors.
Introduction
Anandamide (1a)1 and oleamide (1b)2-4 have emerged
as the prototypical members of a class of endogenous
fatty acid amides that serve as chemical messengers.
Anandamide, the most recognizable member of the
endogenous fatty acid ethanolamides,5 binds and acti-
vates the central (CB1) and peripheral (CB2) cannab-
inoid receptors through which it is thought to exert its
biological effects. More recently, 1a was shown to
activate the vanilloid receptor (VR1) analogous to cap-
saicin and olvanil (N-vanillyoleamide) providing an
additional site of action that may contribute to its
analgesic effects and an intriguing structural-func-
tional relationship with oleamide.9,10 Anandamide, like
the cannabinoids, exhibits a range of biological proper-
ties that includes not only behavioral analgesia sup-
pressing pain neurotransmission,11 but also anxiolytic,
antiemetic, appetite enhancement, and antiproliferative
activity as well as neuroprotective effects that have
clinical implications in the treatment of sleep disorders,
anxiety, epilepsy, cachexia, cancer, and neurodegenera-
tive disorders.6-8
Figure 1. FAAH substrates.
a central role in sleep, the studies indicate the potential
of developing sleep aids that lack the side effects of
sedatives and hypnotics and the suicide-abuse potential
of such central nervous system (CNS) depressants.
Fatty acid amide hydrolase (FAAH)13-15 is an integral
membrane protein that degrades fatty acid primary
amides and ethanolamides including anandamide and
oleamide, Figure 1.16,17 Its CNS distribution indicates
that it degrades neuromodulating fatty acid amides at
their sites of action and is intimately involved in their
regulation.18 FAAH constitutes the only characterized
mammalian member of the amidase signature class of
enzymes that all bear a unique catalytic mechanism
(Ser-Ser-Lys triad).15,19-22 Significantly, FAAH knockout
mice not only proved healthy indicating no untoward
consequences attributable to the lack of enzyme, but
they also exhibited greatly augmented behavioral re-
sponses to administered anandamide23 and oleamide24
and increased endogenous brain levels of fatty acid
amides that correlated with a CB1-dependent analgesic
phenotype.23,25 As a result of its unique mammalian dis-
tribution, its selectively targetable active site and
catalytic mechanism, and the consequences of its inhibi-
tion (increased endogenous levels of anandamide and
oleamide), FAAH has emerged as a potentially exciting
Oleamide was found to accumulate in the cerebrospi-
nal fluid under conditions of sleep deprivation. In a dose-
dependent manner, it was found to induce physiological
sleep in animals where it reduced mobility, shortened
the sleep induction period, and lengthened the time
spent in slow wave sleep 2 at the expense of waken-
ing.2,12 In addition to suggesting that oleamide may play
* Corresponding author: Dale L. Boger, Department of Chemistry,
The Scripps Research Institute, 10550 North Torrey Pines Rd., La
Jolla, CA 92037. Phone: 858-784-7522. Fax: 858-784-7550. E-mail:
boger@scripps.edu.
∇ In memory of Dr. Paul Janssen, his warm friendship, and his
lasting impact on Medicinal Chemistry.
† Department of Chemistry.
‡ Department of Cell Biology.
§ The Skaggs Institute for Chemical Biology.
| Yale University.
10.1021/jm049614v CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/23/2004

1850 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Boger et al.
Scheme 1
Figure 2. Lineweaver-Burk plot of FAAH inhibition by 9f
and 11f illustrating reversible, competitive inhibition.
new therapeutic target for a range of clinical dis-
orders.26-28
Despite this interest, few FAAH inhibitors have been
disclosed.29-41 These include the discovery that the
endogenous sleep-inducing compound 2-octyl R-bro-
moacetoacetate is an effective FAAH inhibitor,29 a series
of reversible inhibitors bearing nonselective electrophilic
carbonyls30-32 (e.g., trifluoromethyl ketones), and a set
of irreversible inhibitors33-37 (sulfonyl fluorides or fluo-
rophosphonates). Recently, two classes of inhibitors
have been disclosed that promise to advance the poten-
tial of FAAH as a therapeutic target.38-41 The most
recent is a class of aryl carbamates that acylate an
active site catalytic Ser and which were shown to exhibit
anxiolytic activity in animal models.38,39 The second is
an earlier class of R-ketoheterocycle-based inhibitors
that possess extraordinary potency (Ki ) 100-200 pM)
and that act as reversible, competitive inhibitors pre-
sumably via reversible hemiketal formation with an
active site Ser.40,41
Herein we report the discovery of a class of potent,
selective, and efficacious inhibitors of FAAH that pro-
duce analgesia in animal models, providing the first
pharmacological validation of this new therapeutic
target for the treatment of pain disorders which emerged
from our continued investigations of such R-ketohetero-
cycles. Key to the useful inhibitor discovery was the
implementation of a proteomics-wide selectivity screen
against all serine hydrolases,42 ensuring selectivity for
FAAH coupled with systematic in vivo examinations of
candidate inhibitors.43
Figure 3. R-Keto oxazolopyridine FAAH inhibitors.40
obtained by the Vedejs oxazole metalation,48 aldehyde
condensation, and TBS protection of the alcohol, fol-
lowed by a selective C5-oxazole lithiation49 and subse-
quent reaction with I2 or Bu3SnCl.
Inhibition Studies. Enzyme assays were performed
at 20-23 °C with purified recombinant rat FAAH
expressed in E. coli50 (unless indicated otherwise) or
with solubilized COS-7 membrane extracts from cells
transiently transfected with human FAAH cDNA14
(where specifically indicated) in a 125 mM Tris/1 mM
EDTA/0.2% glycerol/0.02% Triton X-100/0.4 mM Hepes,
pH 9.0 buffer.29 The initial rates of hydrolysis (e10-
20% reaction) were monitored using enzyme concentra-
tions at least 3 times below the measured Ki by following
the breakdown of 14C-oleamide and Ki’s established as
described (Dixon plot).40 Lineweaver-Burk analysis
established reversible, competitive inhibition (Figure 2).
Results and Discussion
In initial studies,40 a series of candidate R-ketohet-
erocycle inhibitors were examined that incorporated the
oleyl side chain. Of these, benzoxazole 2 was selected
for detailed examination since it was amendable to
systematic exploration (Figure 3). Its modification to the
oxazolopyridine 3 with incorporation of an additional
basic nitrogen resulted in a remarkable 100-fold in-
crease in inhibitor potency. Although this increase in
potency was greatest with 3, it was not limited to a
single positional isomer and each variant on the oxa-
zolopyridine structure exhibited 50-200-fold increases
in Ki over 2. Systematic modification of the fatty acid
side chain provided the exceptionally potent FAAH
inhibitors 4-7.
Inhibitor Synthesis. The candidate inhibitors were
prepared by direct acid chloride acylation of a Zn/Cu-
metalated oxazole following the protocol of Anderson et
al.44 (Scheme 1a), selected instances of direct oxazole
lithiation and reaction with a Weinreb amide45 (Scheme
1b), or Stille coupling46 of a 5-iodo- or 5-tributylstan-
nyloxazole followed by TBS ether deprotection and
Dess-Martin periodinane oxidation (Scheme 1c).47 In
turn, the 5-iodo- or 5-tributylstannyloxazoles were

Inhibitors of Fatty Acid Amide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1851
Table 2. Modifications in the Fatty Acid Side Chaina
Table 1. Substituted R-Keto Oxazole Inhibitors of FAAH
a Measurement errors (() are provided in Supporting Informa-
tion.
substituents (9f-k), the derivatives where the H-bond
acceptor is located adjacent to the oxazole linkage site
(e.g. 2-furyl) were >10-fold more potent than the
positional isomers (e.g. 3-furyl, 9q vs 9v, and 9r vs 9w).
The Fatty Acid Chain. Enlisting the 5-(2-pyridyl)-
oxazol-2-yl heterocycle identified with 9f, well-behaved
trends were observed with modifications in the fatty
acid chain (Table 2). The greatest potency was observed
with saturated straight chain lengths of C10-C12 that
corresponds to the location of the ∆9,10 double bond of
oleamide and the ∆8,9/∆11,12 double bonds of anand-
amide. This corresponds to the location of a bend in the
inhibitor bound conformation that was identified in our
early inhibitor studies32 and confirmed in a FAAH X-ray
structure.22 The potency for 10a-n increased as the
chain length was shortened from C18 to C12 (Ki, 60 f
2 nM), leveled off at C12-C10 (Ki, 2-9 nM), and
diminished smoothly as the chain length was progres-
sively shortened (Ki, 2 f >100 000 nM). Thus, each of
first C1-C12 carbons contributes progressively to in-
hibitor binding affinity, whereas C14-C18 of the longer
inhibitors progressively reduce potency.32,40
Identical trends were observed with the incorporation
of a phenyl ring at the chain terminus. An optimal
potency was observed with the linker length of C6 (11f,
Ki 5 nM) corresponding to a C10/C12 full length chain,
although comparable potencies were observed with C5-
C9, and progressive declines in activity were seen as
the chain length was increased or decreased from C6.
Notably, the position of the phenyl π-system in 11f
corresponds to the location of the oleamide ∆9,10 double
bond or the anandamide ∆8,9/∆11,12 double bonds. Thus,
well-defined parabolic relationships were observed with
the inhibitor chain length culminating in optimal poten-
cies with 10d for the saturated straight chain inhibitors
and with 11f for the Ph(CH2)n series 11a-j.
Substituted r-Keto Oxazoles. An additional prom-
ising R-ketoheterocycle disclosed in these initial studies
was oxazole 9a (Table 1).40 We subsequently found that
4,5-disubstitution of 9a with two alkyl or phenyl sub-
stituents diminished (9b) or abolished (9c) activity, but
a single 4- or 5-phenyl substituent (9d and 9e) led to
only small reductions in potency with each exhibiting
Ki’s comparable with benzoxazole 2. Consequently, a
series of 4- and 5-substituted oleyl R-keto oxazoles
bearing each pyridine positional isomer was examined
(Table 1). Analogous to observations made with the
oxazolopyridines (e.g. 3), each exhibited 5-20-fold in-
creases in Ki over 9d or 9e. Thus, incorporation of an
additional weakly basic nitrogen proximal to the oxazole
substantially increased FAAH inhibition. Although each
isomer exhibited this increase, it was most pronounced
with 9f and 9g mirroring the pattern observed with the
oxazolopyridines.40 C5-substitution of the oxazole with
alternative six-membered heterocycles bearing two or
more weakly basic nitrogens (9l-o), one of which
overlays with 2-pyridyl nitrogen of 9f, provided potent
FAAH inhibitors with activity indistinguishable from
9f.
A revealing set of oxazoles was examined that were
substituted at C5 with a rationally chosen series of five-
membered heteroaromatics (9p-w). The inhibitor po-
tency smoothly increased as the H-bond acceptor capa-
bilities of the heteroaromatic substituent increased (N-
methylpyrrole < thiophene < furan < N-methylimidazole
< thiazole ( ) pyridine 9f) < oxazole). Notably, the 5-(2-
thiazolyl) derivative 9t proved equally potent with 9f,
while the 5-(2-oxazolyl) derivative 9u was slightly more
potent. Mirroring the pattern observed with the pyridyl

1852 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Table 3. Effect of R-Substitution
Boger et al.
Table 5. Carbonyl Role
Table 4. Heteroaromatic Modifications to 11f a
hemiketal formation with an active site Ser nucleophile.
Confirming this behavior, a series of alcohol precursors
to the R-keto oxazoles was examined and each was found
to be approximately 1000 times less potent than the
corresponding ketone (Table 5). Nonetheless, they ap-
proximate or exceed the activity of many of the early
FAAH inhibitors29-32 and many of the original R-keto-
heterocycles.40 Moreover, in the two instances examined
(22 and 24) where the hydroxyl group has been further
removed, the methylene derivatives retain much of the
activity of the corresponding alcohol. This behavior
indicates that the 5-(2-pyridyl)oxazole contributes sig-
nificantly to FAAH active site binding independent of
the electrophilic carbonyl. As such, the heterocycles
serve not only to enhance the electrophilic character of
the keto group facilitating trap of the active site Ser
nucleophile as a stable hemiketal, but they also form
substantial and selective stabilizing active site interac-
tions that contribute significantly to the binding affinity.
Notably, the R-keto oxazoles examined herein do not
exist predominantly in the hydrated (gem diol) state,
rather they are isolated as the ketones. Moreover, NMR
experiments conducted with 9f and 11f in CD3OD, 7%
D2O-acetone-d6, and 5% D2O-DMSO-d6 revealed less
than 5% hemiacetal or gem diol formation. Under
identical conditions, the oleyl trifluoromethyl ketone31,40
was completely (CD3OD) or predominately hydrated (ca.
90%, 7% D2O-acetone-d6).
Inhibition of Recombinant Human FAAH. Rat
and human FAAH are very homologous (84% sequence
identity), exhibit near identical substrate selectivities
and inhibitor sensitivities in studies disclosed to date,40
and embody an identical amidase signature sequence
suggesting the observations made with rat FAAH would
be analogous to those made with the human enzyme.
Consequently, key inhibitors in the series were exam-
ined against the human enzyme and found to exhibit
the same relative and absolute potencies (Table 6).
Selectivity Screening. Early assessments of inhibi-
tors in the R-ketoheterocycle series against candidate
competitive enzymes (phospholipase A2, ceramidase)
revealed no inhibition. In the absence of identifiable
competitive enzyme targets (FAAH constitutes the only
known mammalian amidase bearing the unusual Ser-
a Measurement errors (() are provided in Supporting Informa-
tion.
Analogous to prior observations,31,32,40 the C18 alkyne
12 (Ki 10 nM) was 2-fold more potent than the alkene
9f bearing the oleyl side chain (Ki 18 nM) which in turn
was 3-fold more potent than the saturated C18 inhibitor
10a (Ki 59 nM). Although they are not among the most
potent inhibitors in the series, the activity of alkyne 12
is notable, approaches that of 10d and 11f, and it
emerged as an especially interesting candidate inhibitor
in the selectivity screening.
r-Substitution. Consistent with past observations,41
R-methyl or R,R-dimethyl substitution of 9a resulted in
10-fold and 100-fold reductions, respectively, in activity
(Table 3).
Further Exploration of the Oxazole Heteroaro-
matic Substituent. With the emergence of 11f as a
selective FAAH inhibitor displaying efficacious in vivo
activity,43 a series of heteroaromatic substituents were
examined including those first found to be potent in the
oleyl series (cf. Table 1). Consistent with these observa-
tions, 20a bearing a C5 phenyl substituent was 30-fold
less active than 11f and each derivative incorporating
a six-membered heterocycle containing two basic nitro-
gens (one of which is placed to overlay the 2-pyridyl
nitrogen of 11f) matched (20b,d,e), or modestly ex-
ceeded (20c) the potency of 11f (Table 4). Interestingly
and like the observations made in Table 1, altering the
position of the basic nitrogen on the attached heterocycle
(20f, 3 vs 2 position) resulted in a 5-10-fold loss in
inhibitory potency. Finally, a series of five-membered
heteroaromatic substituents 20g-j similarly provided
effective FAAH inhibitors where the potency again
smoothly increased as their H-bonding capabilities
increased. Notably, both the 5-(2-thiazolyl) dertivative
20i and the 5-(2-oxazolyl) derivative 20j matched the
potency of 11f.
The Electrophilic Carbonyl. Key to the inhibitor
design is the electrophilic carbonyl and its reversible

Inhibitors of Fatty Acid Amide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1853
Table 6. Inhibition of Recombinant Human Fatty Acid Amide
SAR). Simple electrophilic carbonyl-based inhibitors
including trifluoromethyl ketones exhibit an intrinsic
selectivity that typically favors TGH by >1000-fold and
KIAA by 10-100 fold (Table 7). Despite this unfavorably
intrinsic selectivity, the identification of inhibitors selec-
tive for FAAH over KIAA proved straightforward. The
inhibitor potency for FAAH versus KIAA typically
increases as the side chain size (length) increases
thereby improving selectivity, and the affinity for KIAA
is completely or substantially disrupted with the intro-
duction of the electrophilic carbonyl heterocycle such
that the FAAH selectivity is satisfactory or superb
(>1000-fold where measurable) which we attribute
simply to the increased active site steric requirements
(size) of such inhibitors. Even more impressive given
the intrinsic >1000-fold TGH selectivity, the screening
revealed that the inhibitor potency and selectivity for
TGH decreases as the side chain size (length) increases,
and that the incorporation of a properly positioned
second weakly basic nitrogen (H-bond acceptor) into the
electrophilic carbonyl heterocycle can improve FAAH
affinity and selectivity over that of TGH. Moreover, the
selectivity of the 5-(2-pyridyl)oxazol-2-yl heterocycles
disclosed herein (FAAH > TGH > KIAA) exceeds that
of the corresponding predecessor oxazolopyridines (e.g.
9f vs 3, 11f vs 6 or 7, and 12 vs 4), which in turn exceed
and overcome the intrinsic selectivity for TGH or KIAA
observed with the simpler R-ketoheterocycles (e.g., 2 and
8) or trifluoromethyl ketone inhibitors, and useful
selectivities are achieved within simple structures,
Table 7.
Hydrolase (FAAH)
compd
Ki, µM (human)
Ki, µM (rat)
9a
9f
11f
0.045 ( 0.002
0.010 ( 0.001
0.0090 ( 0.0001
0.10 ( 0.06
0.018 ( 0.005
0.0047 ( 0.0013
Table 7. Selectivity Screening: IC50, µM (selectivity)
Ki (FAAH, µM) FAAH KIAA1363 TGH
n
CH3(CH2)7CHdCH(CH2)7COCF3
0.08
4.5
1.1 (0.25) 4.8 (1.1)
CH3(CH2)nCOCF3
6
1.2
30
10
10
1.5 (0.05) 0.002 (0.00007)
0.4 (0.04) 0.01 (0.001)
0.5 (0.05) 0.06 (0.006)
8
10
16
0.13
0.14
0.24
6.4
6.6 (1)
4.6 (0.7)
Ph(CH2)nCOCF3
0
1
5
6
7
nd
>100
10 (<0.10) 0.004 (<0.00004)
nd
>100
100 (<1)
0.8 (0.16) 0.0005 (0.0001)
0.2 (0.04) 0.001 (0.0002)
0.010 (<0.0001)
0.17
0.10
0.025
5
5
2
0.2 (0.1)
0.005 (0.002)
representative R-ketoheterocyclesa
compd Ki (FAAH) FAAH
KIAA1363
TGH
10 (1)
9 (4)
1 (25)
2
9a
3
9f
8
7
6
0.37
10
2
0.04
0.15
10
0.001
0.0003
>100 (>10)
>100 (>50)
60 (1500)
>100 (>670)
>100 (>10)
10 (10000)
20 (67000)
0.10
0.0023
0.018
0.22
0.00020
0.00028
0.0047
0.00014
0.01
>100 (>670)
0.02 (0.002)
0.001 (1)
0.003 (10)
0.6 (300)
0.5 (250)
30 (1500)
11f
4
12
0.002 >100 (>10000)
0.002
0.02
20 (10000)
>100 (>2000)
Model of Inhibitors Bound to FAAH: Key Inter-
actions. Monte Carlo (MC) simulations for the R-keto
oxazole derivatives 9f and 11f covalently bound to the
enzyme were performed. The average structure that
emerged features an extensive hydrogen-bonded net-
work between the enzyme and the pyridyl nitrogen and
oxazoyl oxygen of the inhibitors. More specifically, the
oxazoyl oxygen is hydrogen bonded to the hydroxyl
group of Ser217 of the catalytic triad, which accepts a
hydrogen bond from the protonated nitrogen of Lys142,
also from the catalytic triad. The side chain of this
residue donates hydrogen bonds to the pyridyl nitrogen
of the inhibitors, and to the hydroxyl groups of Ser218
and Thr236. An additional hydrogen bond is formed
between the pyridine nitrogen of both 9f and 11f and
the hydroxyl group of Thr236. The central role of the
pyridyl nitrogen in the network and, especially, its
interactions with Lys142 are consistent with the large
activity boost between 9d and 9f. The carbonyl group
of the third member of the catalytic triad (Ser241)
accepts hydrogen bonds from the hydroxyl group and
backbone nitrogen of Ser218. The side chain oxygen of
Ser241, covalently bound to the carbonyl group of the
inhibitors, accepts a hydrogen bond from the hydrogen
on nitrogen of Ser217. These interactions are depicted
in Figure 4a for the 9f derivative. A closer look at Figure
4a also suggests an explanation for the high activities
for 9l, 9n, 20b, 20c, 20d, and 20e. Although they would
have to pay a larger dehydration penalty due to ad-
ditional interactions between the solvent and the py-
ridazine, pyrimidine, and pyrazine rings, the second
nitrogen atoms may form additional hydrogen bonds
with the thiol groups of Cys144 and Cys269.
a Full table of results is provided in the Supporting Information.
Ser-Lys catalytic triad) and since the candidate inhibi-
tors did not effect the obvious hydrolases that are known
to act on fatty acid ester or amide substrates, a pro-
teomics-wide screen capable of globally profiling the
serine hydrolase superfamily applicable to defining the
selectivity of FAAH inhibitors was developed.42 This
permits the simultaneous assessment of all relevant
competitive enzymes including those that might be
unrecognized, lack known substrates, or are even pres-
ently unknown. Moreover, this competitive parallel
profiling of the inhibitors against all proteome serine
hydrolases requires no use of a competitive substrate,
no modification of the candidate inhibitor, and can
rapidly and quantitatively establish relative potency
and selectivity factors for each inhibitor. Thus, the IC50
values in the selectivity screen are typically higher than
the measured Ki’s, but the relative and absolute potency
and rank order determined in the assay parallels that
established by standard substrate assays.42 Only two
enzymes emerged in our screens as competitive targets
for the R-ketoheterocycles detailed herein: triacylglyc-
erol hydrolase (TGH) and an uncharacterized membrane-
associated hydrolase that lacks known substrates or
function (KIAA1363).
Summarized in Table 7 are representative results of
the selectivity screening that permitted a simultaneous
optimization of selectivity for FAAH over the two
competitive enzymes displaying distinct SAR (structure-
activity relationship) profiles conducted concurrent with
the FAAH inhibition optimization (multidimensional

1854 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Boger et al.
Figure 4. Stereoviews of a snapshot from a MC simulation illustrating the interactions between 9f and FAAH. (a) The extensive
hydrogen-bonded network between the pyridyl-substituted oxazole ring and the enzyme, and interactions for the oxyanion (similar
interactions occur for 11f). (b) Interactions of the lipid chain of 9f with hydrophobic residues in the active site.
mL, 20.6 mmol) and 15 µL of DMF. After 1 h, the solution
was allowed to warm to 25 °C and was stirred for 2.5 h.
Concentration provided the crude acid chloride that was used
directly in the next step.
Regarding the interactions for the oxyanion, the
negatively charged oxygen of both 9f and 11f is hydro-
gen bonded to the backbone nitrogens of Ile238, Gly239,
and Ser241 (Figure 4a). As for the lipid chain of the
derivatives, the longer chain of 9f is surrounded by a
number of hydrophobic residues, such as Leu192, Phe194,
Tyr335, Leu372, Ala377, Leu380, Phe381, Leu404,
Phe432, Thr488, Ile491, Val495, and Trp531 (Figure
4b), while the shorter chain of 11f makes contact with
Leu192, Leu380, Leu404, Ile491, Thr488, and Phe194.
A solution of 5-(2-pyridyl)oxazole (680 mg, 4.65 mmol) in
25 mL of anhydrous THF was treated with n-BuLi (2.2 mL,
2.5 M in hexanes, 5.5 mmol) and stirred for 35 min at -78 °C.
The mixture was allowed to warm at 0 °C and ZnCl2 (11.2 mL,
0.5 M in THF, 5.6 mmol) was added dropwise over 20 min.
After 45 min, CuI (1.05 g, 5.6 mmol) was added, and the
solution was stirred for 15 min at 0 °C. 7-Phenylheptanoyl
chloride (5.80 mmol) in anhydrous THF and added dropwise.
After 1 h, the reaction was quenched with the addition of
saturated aqueous NaHCO3 and the mixture was extracted
with EtOAc. The organic layers were combined, dried (Na2-
SO4), and concentrated. Chromatography (SiO2, 4 × 10 cm,
20% EtOAc-hexanes) afforded 11f as a yellow solid. Treat-
ment with aqueous 2 N KOH, extraction with EtOAc followed
by a wash with saturated aqueous NaCl provided 11f (1.00 g,
Conclusions
In recent studies, we showed that 11f (OL-135)
potentiates the effects of exogenously administered
anandamide, increases the endogenous levels of fatty
acid amides in the central nervous system, and produces
CB1-dependent analgesia in multiple nociceptive mod-
els, validating FAAH as an important new therapeutic
target for the management of pain.43 Key to this
development was the systematic optimization of FAAH
inhibition concurrent with a proteome-wide screening
for FAAH selectivity that distinguished the 5-(2-py-
ridyl)-2-oxazoles described herein from the predecessor
R-ketoheterocycles.
1
65%) as a pale yellow crystalline powder: mp 45-48 °C; H
NMR (500 MHz, CDCl3) δ 8.76-8.74 (m, 1H), 7.96-7.94 (m,
2H), 7.88 (td, J ) 7.8, 1.8 Hz, 1H), 7.41-7.38 (m, 1H), 7.36-
7.33 (m, 2H), 7.26-7.23 (m, 3H), 3.19 (t, 2H, J ) 7.4 Hz), 2.69
(t, 2H, J ) 7.7 Hz), 1.86 (m, 2H), 1.72 (m, 2H), 1.53-1.44 (m,
4H); 13C NMR (125 MHz, CDCl3) δ 188.3, 157.3, 153.1, 150.0,
146.2, 142.6, 137.0, 128.3 (2C), 128.1 (2C), 126.8, 125.5, 124.0,
120.3, 39.0, 35.8, 31.2, 28.9 (2C), 23.8; IR (film) νmax 3060, 3025,
2929, 2855, 1694, 1603, 1575, 1505, 1470, 1455, 1426, 1382,
1283, 1151, 1031, 990, 963, 936, 784, 741, 699 cm-1; MALDI-
FTMS m/z 335.1756 (M + H+, C21H22N2O2 requires 335.1754).
Anal. (C21H22N2O2) C, H, N.
Experimental Section
1-Oxo-1-[5-(2-pyridyl)oxazol-2-yl]-7-phenylheptane (11f).
A solution of 7-phenylheptanoic acid (1.20 g, 5.80 mmol) in 20
mL of anhydrous CH2Cl2 at 0 °C was treated with (COCl)2 (1.8
1-Hydroxy-1-[5-(2-pyridyl)oxazol-2-yl]-7-phenylhep-
tane (23). NaBH4 (3 mg, 0.08 mmol) was added to a solution

Inhibitors of Fatty Acid Amide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1855
of 11f (16 mg, 0.048 mmol) in a 1:1 mixture of MeOH and THF
(0.5 mL). After stirring at 0 °C for 30 min, the reaction was
quenched with the addition of saturated aqueous NaCl. The
mixture was concentrated and extracted with EtOAc. The
organic layers were combined, dried (Na2SO4), and concen-
trated. Chromatography (SiO2, 1 × 4 cm, 35% EtOAc-
hexanes) afforded 23 (13 mg, 0.039 mmol, 81%) as a pale
yellow oil: 1H NMR (400 MHz, CDCl3) δ 8.62 (app d, 1H, J )
4.4 Hz), 7.75 (td, 1H, J ) 7.7, 1.7 Hz), 7.64-7.62 (m, 2H), 7.28-
7.14 (m, 6H), 4.87 (app t, 1H, J ) 6.6 Hz), 3.42 (br s, 1H), 2.59
(app t, 2H, J ) 7.6 Hz), 2.05-1.93 (m, 2H), 1.64-1.33 (m, 8H);
13C NMR (125 MHz, CDCl3) δ 167.2, 152.1, 149.8, 147.1, 142.7,
136.9, 128.3 (2C), 128.2 (2C), 125.5, 125.0, 123.0, 119.3, 67.7,
35.9, 35.5, 31.4, 29.1 (2C), 25.0; IR (film) νmax 3284, 3025, 2929,
2855, 1614, 1580, 1547, 1471, 1427, 1117, 1074, 990, 950, 783,
743, 699 cm-1; MALDI-FTMS m/z 337.1911 (M + H+,
C21H24N2O2 requires 337.1916). Anal. (C21H24N2O2) C, H, N.
FAAH Inhibition. 14C-labeled oleamide was prepared from
14C-labeled oleic acid as described.2,29 The truncated rat FAAH
(rFAAH) was expressed in E. coli and purified as described.50
The purified recombinant rFAAH was used in the inhibition
assays unless otherwise indicated. The full-length human
FAAH (hFAAH) was expressed in COS-7 cells as described,14
and the lysate of hFAAH-transfected COS-7 cells was used in
the inhibition assays where explicitly indicated.
performed for 5 × 106 configurations. This was followed by 10
× 106 configurations of full equilibration and 50 × 106
configurations of averaging for each simulation. Established
procedures including Metropolis and preferential sampling53
were employed using the MCPRO 1.68 program.54 The protein
was represented with the OPLS-AA force field,55 the TIP4P
model56 was used for water, and residue-based cutoffs of 10 Å
were employed for all nonbonded solute-solute and solvent-
solute interactions.
Acknowledgment. We gratefully acknowledge the
financial support of the National Institutes of Health
(DA15648, D.L.B.; DA17259, and DA15197, B.F.C.;
GM032136, W.L.J.) and the Skaggs Institute for Chemi-
cal Biology, fellowships for R.A.F. (American Cancer
Society), and the postdoctoral sabbatical leave of H.M.
sponsored by Sumitomo Pharmaceutical.
Supporting Information Available: Full experimental
details and characterization of the FAAH inhibitors disclosed
herein. This information is available free of charge via the
Internet at http://pubs.acs.org.
References
The inhibition assays were performed as described.2,29 In
brief, the enzyme reaction was initiated by mixing 1 nM of
rFAAH (800, 500, or 200 pM rFAAH for inhibitors with Ki e
1-2 nM) with 10 µM of 14C-labeled oleamide in 500 µL of
reaction buffer (125 mM TrisCl, 1 mM EDTA, 0.2% glycerol,
0.02% Triton X-100, 0.4 mM Hepes, pH 9.0) at room temper-
ature in the presence of three different concentrations of
inhibitor. The enzyme reaction was terminated by transferring
20 µL of the reaction mixture to 500 µL of 0.1 N HCl at three
different time points. The 14C-labeled oleamide (substrate) and
oleic acid (product) were extracted with EtOAc and analyzed
by TLC as detailed.2,29 The Ki of the inhibitor was calculated
using a Dixon plot as described.40 Lineweaver-Burk analysis
was performed as described,29,40 in the presence or absence of
8 nM of 9f or 11f, respectively, confirming competitive,
reversible inhibition (see Figure 2).
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Selectivity Screening. The selectivity screening was
conducted as detailed.42
Computational Details. Cartesian coordinates for the 2.8
Å fatty acid amide hydrolase (FAAH) crystal structure com-
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1856 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
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