α-Ketoheterocycle inhibitors of fatty acid amide hydrolase: Exploration of conformational constraints in the acyl side chain

Article abstract of DOI:10.1016/j.bmc.2014.03.013

A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH.

Full text of DOI:10.1016/j.bmc.2014.03.013

Bioorganic & Medicinal Chemistry 22 (2014) 2763–2770
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
a
-Ketoheterocycle inhibitors of fatty acid amide hydrolase:
Exploration of conformational constraints in the acyl side chain
⇑
Katharine K. Duncan, Katerina Otrubova, Dale L. Boger
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of
a-ketooxazoles containing heteroatoms embedded within conformational constraints in the
Received 4 March 2014
Accepted 8 March 2014
Available online 18 March 2014
C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydro-
lase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint
is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the
analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candi-
date inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively
inhibit FAAH.
Keywords:
Fatty acid amide hydrolase
a-Ketoheterocycles
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
two generalized classes of inhibitors with promising therapeutic
potential have been identified. One class is the reactive aryl carba-
Fatty acid amide hydrolase (FAAH)1,2 hydrolyzes and terminates
the signaling of several endogenous lipid amides3–6 including the
endocannabinoid anandamide (1a)7–9 and the sleep-inducing lipid
oleamide (1b)10–12 (Fig. 1). The distribution of FAAH in the central
nervous system is consistent with its role in regulating these neu-
romodulating fatty acid amides6 at their sites of action.3 FAAH is a
member of amidase signature family of serine hydrolases. While
there are a number of prokaryotic enzymes in this family, FAAH
is the only well-characterized mammalian enzyme bearing the
family’s diagnostic Ser–Ser–Lys catalytic triad.13–16
mates and ureas37–49 that irreversibly acylate a FAAH active site
serine. The second class consists of the
a-ketoheterocycle-based
inhibitors that bind to an active site serine in FAAH by reversible
hemiketal formation.51–53 Many members of this class have been
shown to be potent FAAH inhibitors and selective for FAAH over
other mammalian serine hydrolases and several have been shown
to be efficacious in vivo.54
Within this class, 2 (OL-135)55 emerged as an important lead
inhibitor for further development.55 This compound is a potent
(Ki = 4.7 nM)55 and selective (>60–300 fold)30 FAAH inhibitor that
exhibits antinociceptive and anti-inflammatory activity and
There has been wide-spread interest17 in the development of
selective inhibitors of FAAH given their therapeutic potential18–20
for the treatment of pain,21–23 inflammation,24 or sleep disor-
ders.13,25 FAAH inhibition potentiates an activated pathway,
increasing the endogenous levels of a released lipid signaling mol-
ecule only at its site of action, thus providing a temporal and spa-
tial pharmacological control not available with classical cell
surface receptor agonists. Following the initial characterization of
the enzyme, the endogenous sleep-inducing molecule 2-octyl
O
OH
N
H
Anandamide (1a)
O
NH2
Oleamide (1b)
a
-bromoacetoacetate26 was found to be a potent, reversible FAAH
inhibitor (Ki = 0.8 M). Subsequent work led to the disclosure of
Fatty Acid Amide
l
Hydrolase (FAAH)
O
nonselective, substrate-inspired, reversible inhibitors with an elec-
trophilic ketone (e.g., trifluoromethyl ketone-based inhibitors)27–30
and irreversible inhibitors (e.g., fluorophosphonates and sulfonyl
fluorides).31–36 Since that time and with notable exceptions,50
OH
Arachidonic Acid (1c)
O
OH
Oleic Acid (1d)
⇑
Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784 7550.
E-mail address: boger@scripps.edu (D.L. Boger).
Figure 1. Substrates of fatty acid amide hydrolase.
http://dx.doi.org/10.1016/j.bmc.2014.03.013
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2764
K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
increases levels of anandamide in vivo.54 No antinociception was
observed in FAAH knockout mice, indicating FAAH is the target
responsible for the in vivo effects.54 Significantly, 2 does not bind
to cannabinoid (CB1 and CB2) or vanilloid receptors and does not
inhibit the human ether-a-go-go related gene (hERG) or common
P450 metabolism enzymes (3A4, 2C9, 2D6). Further, the antinoci-
ceptive effects are not associated with the side effects observed
with opioid administration (respiratory depression and dosing
desensitization) or cannabinoid agonists (increased feeding and
decreased motor control).54
Because of its therapeutic potential, we have conducted a series
of systematic structure–activity relationship (SAR) studies explor-
ing the 4- and 5-position of the central oxazole,55,57,58,62 the C2 acyl
side chain,55,59,60 and the central heterocycle56,61 (Fig. 2). Each of
these sites were found to independently impact inhibitor potency
or selectivity.62
with conformationally restricted C2 acyl side chains. To probe
the tolerance for additional modifications, two series of analogues
were designed and prepared: one with an oxygen atom
a to the
electrophilic carbonyl (X = O, the 6-phenoxychroman-2-yl-2-keto-
oxazoles) and a second with an oxygen atom b to the carbonyl
(Y = O, the 7-arylchroman-3-yl-2-ketooxazoles). The former
change was expected to substantially increase the electrophilic
character of the activated ketone by virtue of the inductive elec-
tron-withdrawing
a-alkoxy substituent (X = O). Potentially, this
could improve FAAH inhibitor potency provided the change does
not competitively promote gem-diol formation, an attribute not
observed with 2 or 3 and related compounds that would compete
with active site adduct formation. An analogous, but dampened ef-
fect might be expected of the introduction of an ether oxygen b to
the activated ketone (Y = O). However, the impact of the added
heteroatom on the intrinsic FAAH active site binding through
introduction of either destabilizing electrostatic or stabilizing
H-bonding interactions with the proximal active site residues
was difficult to a priori predict.
In the most recent of these studies, we disclosed63,64 a series of
inhibitors including 3 with added conformational constraints in
the flexible C2 acyl side chain of 2 (Fig. 2). This latest series showed
improved or comparable enzyme inhibition potency relative to 2,
indicating that removing many of the rotatable bonds in the C2
acyl side chain can simultaneously enhance target binding affinity
and improve the drug-like characteristics of the inhibitor. Although
many may be under the impression such inhibitors bearing elec-
trophilic carbonyls may be metabolically labile, we have found that
they are subject to competitive reduction/reoxidation metabolism
that sets up a steady-state equilibrium between the two states
(ketone/alcohol) with the true in vivo fate of the candidate inhibi-
tors being determined by other features of the molecule.63 More-
over, the added conformational constraints in the C2 acyl chain
and the increased steric hindrance surrounding the electrophilic
ketone both slow the rate of equilibration and improve the ke-
tone/alcohol ratio in vivo.63 As a result, the lead 1,2,3,4-tetrahydro-
naphthalene analogue 3 exhibited robust, long acting analgesic
activity when administered orally in mouse models of thermal
hyperalgesia and neuropathic pain in preliminary in vivo studies.63
Herein, we report our continued exploration of such inhibitors
2. Chemistry
The synthesis of the 6-phenoxychroman-2-yl-2-ketooxazole
series of analogues is described in Scheme 1. The carboxylic acid
4 was prepared in five steps from commercially available 4-phen-
oxyphenol according to literature procedure.65 The carboxylic acid
was converted to the corresponding aldehyde 6 through reduction
of the Weinreb amide 5 (96%), accessed by coupling 4 with N,O-
dimethylhydroxyamine (67%).
Figure 2. Progression of the inhibitor series.
Scheme 1.

K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
2765
Vedejs oxazole metalation66 and condensation with aldehyde 6
yielded the secondary alcohol 7 (70%). The secondary alcohol was
protected as its TBS ether to give 8 (95%). Selective C5-lithiation67
and quench with Bu3SnCl generated the tributylstannane interme-
diate 9 (72%). Stille coupling68 with either 2-bromopyridine (76%)
or methyl 6-bromopicolinate (80%) produced the C5-aryl oxazoles
10 and 11, respectively. Subsequent TBS ether deprotection
(Bu4NF) provided 12 and 13, and oxidation with Dess–Martin peri-
odinane69 provided the desired
a-ketooxazoles 14 and 15. Because
ketones 14 and 15 were too polar for facile resolution by chiral
HPLC, the chromatographic enantiomer separation was conducted
at an earlier stage of the synthesis. Specifically, the TBS ethers 10
and 11 were separated by flash chromatography (SiO2) into their
two diastereomers and each diastereomer was resolved by chiral
HPLC to provide the two enantiomers (ChiralPak AD for 10 and
ChiralCel OD for 11). This enantiopure material was then carried
through the deprotection and oxidation steps described above to
generate each single enantiomer of the ketone. The unsubstituted
parent
a-ketooxazole 16 was generated by the Dess–Martin peri-
odinane oxidation of the alcohol 7 (71%), followed by resolution
on a ChiralPak AD HPLC column.
The synthesis of the 7-arylchroman-3-yl-2-ketooxazole series
of analogues began with commercially available 4-benzyloxy-2-
hydroxybenzaldehyde, which was transformed into the tert-butyl
ester 17 in two steps according to a literature procedure.70 One
of three side chains (R1 = Ph, OPh, or OBn) was then added to the
chromane core by reaction of the free phenol of 17 (Scheme 2).
For the 7-phenyl series (R1 = Ph), the phenol 17 was converted to
the corresponding triflate intermediate 18 (91%) and coupled with
phenylboronic acid under Suzuki conditions to provide 19 (88%).
Alkylation of the phenol with BnBr (cat. Bu4NI, 2 equiv K2CO3) pro-
vided 20 (93%) for accessing the 7-benzyloxy series (R1 = OBn).
Alternatively, 17 was subjected to a modified Ullmann reaction68
with phenylboronic acid to produce 21 (63%) needed to prepare
the 7-phenoxy series (R1 = OPh). For 19–21, the tert-butyl esters
were reduced to the corresponding alcohols 22–24 with LiAlH4
and subsequently oxidized with Dess–Martin periodinane69 to pro-
vide the corresponding aldehydes 25–27.
With the aldehydes in hand, the synthesis of the a-ketooxazole
inhibitorsproceeded as describedfor thechroman-2-yl-2-ketooxaz-
ole series. Vedejs C2 metalation66 of oxazole was followed by
condensation with the aldehydes to yield the secondary alcohols.
The alcohols were TBS protected before selective C5-lithiation67 of
the oxazole and quench with Bu3SnCl to generate the tributylstann-
ane intermediates. Stille coupling71 with either 2-bromopyridine or
methyl 6-bromopicolinate generated the C5-aryl oxazoles.
Subsequent TBS ether deprotection (Bu4NF) and oxidation using
Dess–Martin periodinane69 provided the desired
a-ketooxazoles.
For the 7-benzyloxy and 7-phenyl derivatives, each separated
diastereomer of the corresponding TBS ethers were chromatograph-
ically resolved into the two enantiomers and independently carried
Scheme 2.
forward to the enantiomerically pure
a-ketooxazoles. Only the
7-phenoxy derivatives could be resolved into its enantiomers as
the final ketone on a ChiralPak AD column. The parent a-ketooxaz-
4. Results and discussion
oles were generated by the Dess–Martin periodinane oxidation69
of the secondary alcohols.
Previous studies revealed that the C2 acyl side chain of 2 and re-
lated inhibitors bind in a hydrophobic channel of the FAAH active
site reserved for the unsaturated lipid chain of the fatty acid amide
3. Enzyme assay
substrates. Both the hydrophobic character and
p-saturation of
Enzyme assays were performed at 20–23 °C with purified recom-
binant rat FAAH (rFAAH) expressed in Escherichia coli as previously
described.72 The initial rates of hydrolysis were monitored using en-
zyme concentrations (typically 1 nM) at least three times below the
measured Ki by following the breakdown of 14C-oleamide, and the Ki
values were established as described (Dixon plot).28
these inhibitors mimic the nature, location, and unsaturation
(e.g., the oleyl D9,10 double bond) of the fatty acid chain of the
endogenous substrates. Removing many of the rotatable bonds
and introducing additional p-saturation led to a series of inhibitors
that exhibited enhanced binding affinity and drug-like proper-
ties.63 In early work59 exploring conformational constraints in

2766
K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
the C2 acyl side chain, three terminal aryl substituents displayed
improved potency relative to following the trend of
the assay (pH = 9.1), the sample was dissolved in DMSO and placed
in the assay buffer without enzyme. The enantiopurity of the sam-
ple was monitored at 4-min intervals for 30 min, the time course of
the assay. For the parent inhibitor 16 and the C5-pyridine substi-
tuted inhibitor 14, the total change in ee was only À6.8% and
À4.4%, respectively. This suggests that the 2 to 3-fold differences
in the inhibitor enantiomer potencies is reflective of their activity
and not significantly impacted by competitive epimerization in
the assay although that cannot be conclusively ruled out. To fur-
ther characterize the potential for racemization at this center, we
monitored the enantiopurity of the sample after dissolution in a
variety of solvents. In this case, only the presence of base (5%
Et3N in EtOAc) led to significant racemization within the first hour
of study, and continued exposure to these conditions led to com-
plete epimerization within 48 h (Fig. 4).
2
phenyl > benzyloxy > phenoxy (Fig. 2). With the later 1,2,3,4-tetra-
hydronaphthalene series exemplified by 3, a subtle reordering of
aryl substituent preference was observed, following the trend
phenoxy > benzyloxy > phenyl.63 Previous work also established
that the aryl C5 oxazole substituents exhibit well-defined potency
trends (2-Pyr > 3-Pyr > 4-Pyr > Ph) that correlate with the location
and H-bond acceptor capabilities of the weakly basic heterocycle
substituent.58 The addition of electron-withdrawing and water-
solubilizing substituents onto the pyridine ring (6-CO2Me or 6-
CO2H) weakly modulated the potency of these inhibitors, improved
water solubility, and did not appear to impact CNS penetration.58
These optimized C5 oxazole substituents and the phenoxy aryl
substituent were combined with the candidate C2 or C3 chromane
cores. With the C3 chromane series, all three improved side chains
(R1 = Ph, OBn, OPh) were synthesized and tested.
It was previously observed that 2 and related
a-ketooxazoles
preferentially exist in solution in their ketone form and do not
adopt a hydrated (gem diol) state. Similarly, 3 showed no detect-
able hydrate or hemiketal formation in CD3OD and 7% D2O–
DMSO-d6. In contrast and consistent with an expected enhanced
electrophilic character, significant hemiketal formation was ob-
4.1. 6-Phenoxychroman-2-yl-2-ketooxazole series
For each derivative, the racemic mixture and the pure enantio-
mers were prepared and tested in the in vitro enzyme inhibition
assay. The results (Ki) for this series are reported in Figure 3.
In each case, it was the faster eluting enantiomer obtained from
the chiral phase chromatographic resolution that was the more po-
tent. Unlike derivatives of 2 itself but like the observations made
with 3, there was no improvement in potency upon introduction
of the C5-pyridine substituent (14 vs 16), rather there was an anal-
ogous 2-fold reduction in activity. Analogous to the behavior of 2,
which experienced a 13-fold loss in activity with the introduction
served for the parent
a-ketooxazole 16 (38%), 14 (34%), and 15
(45%) in CD3OD. These three compounds also displayed slow,
time-dependent hydrate formation (7% D2O–DMSO-d6) with no
gem diol detected initially, but with a significant amount observed
at 24 h: 16 (13%), 14 (25%), 15 (27%). It is conceivable that this
competitive hydrate formation contributes to the less effective
FAAH inhibition by 14–16 relative to 3 and 2.
4.2. 7-Arylchroman-3-yl-2-ketooxazole series
of an
to an 8-fold loss in activity (3 vs 14). Thus, the intrinsic electron-
withdrawing effect of an -oxygen atom that would be expected
a a-oxygen to 3 led
-oxygen atom,59 the introduction of the
For each derivative, the racemic mixture and the pure enantio-
mers were prepared and tested in the in vitro enzyme inhibition
assay. The results (Ki) for this series are reported in Figure 5.
The most interesting C5-pyridyloxazole series (45–47) were
found to be 2- to 4-fold less potent than 3 against FAAH, indicating
that the introduction of the b-oxygen atom only slightly reduced
the inhibitor activity. This is in sharp contrast to the analogous im-
pact within the structure of 2, where the introduction of b-oxygen
atom lead to a 20-fold loss in activity.59 With the exception of 48,
the difference in potency between the two enantiomers (average of
1.5-fold) was less than that observed for the 6-phenoxychroman-
2-yl-2-ketooxazole series and distinct from the 1,2,3,4-tetrahydro-
naphthalene series including 3. Unlike in the C2 chromane series
(Fig. 3), the installation of the pyridine substituent at C5 provided
a roughly equipotent (e.g., 46) or slightly more potent ((S)-45)
inhibitor than the unsubstituted parent oxazoles.
a
to enhance the electrophilic reactivity of the activated ketone does
not productively improve the FAAH inhibition. The distinguishing
feature of the series was that the activity of the slower eluting enan-
tiomer was only 2–4-fold less potent than the faster eluting more
potent enantiomer. This is in contrast to the 1,2,3,4-tetrahydro-
naphthalene series, where the more potent (S) enantiomer was an
average of 70-fold more potent than the less active (R) enantiomer.
One might suspect that the proton on the stereogenic center
adjacent to the ketone could be sufficiently activated such that epi-
merization of this center might take place during synthetic proce-
dures or under the conditions of the enzyme inhibition assay. The
enantiopurity of each sample was measured before the sample was
tested in vitro. In each case, the enantiomeric excess (ee) of the
sample was P95%, indicating little erosion in enantiopurity during
the deprotection, oxidation reaction, and purification. To establish
whether the compounds were epimerizing under the conditions of
In order to unambiguously establish the absolute stereochemis-
try of each enantiomer, the inhibitor 54 in the series with an iodo
substituent at the C5 position of the oxazole was prepared and re-
solved (ChiralPak AD, 20% iPrOH/hexane,
a = 1.18, Scheme 3). A
single-crystal X-ray crystal structure determination73 conducted
on a heavy atom derivative established that the slower eluting
enantiomer from the chiral phase HPLC separation and the less
Figure 3. FAAH inhibition, Ki (nM).
Figure 4. Change in enantiomeric excess for 14 in solution.

K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
2767
Figure 5. FAAH inhibition, Ki (nM).
Figure 6. Change in enantiopurity of 45 in solution.
inhibitor enantiomers although that cannot be conclusively ruled
out. To further explore the potential for racemization, the enantio-
purity of 45 was monitored after exposure to a variety of solvents.
Only exposure to 5% Et3N in EtOAc led to any significant racemiza-
tion within the first hour of study (Fig. 6). For all the inhibitors in
this series, no detectable hydrate (gem diol) or hemiketal formation
was observed in CD3OD and 7% D2O–DMSO-d6.
Interestingly, there appears little preference for the (R) or (S)
configuration of the inhibitors although there are subtle trends
that track with the C7 aryl substituent. The C7-phenoxy (R-enan-
tiomer) and C7-phenyl (S-enantiomer) series appear to switch
the modest enantioselectivity (45 vs 47), whereas the 7-benzyloxy
series exhibit no selectivity (46). Although this may appear unu-
sual at first glance, it may simply reflect a 180° flip of the chromane
core in the active site to preserve the key anchoring interactions of
the C2 acyl chain terminal phenyl group. In our earlier 1,2,3,4-tet-
rahydronaphthalene series, the X-ray structure of 3 bound to the
enzyme revealed that the enantiomeric selectivity is similarly im-
posed by the spatial relationship of the anchoring C6-phenoxy sub-
stituent relative to the chiral center adjacent to the electrophilic
carbonyl.63,74,75
Most interesting of these unusual observations is the potential
that the racemic inhibitors like 46 may serve as potent FAAH inhib-
itors just as effective as either enantiomer precluding the need for
resolution, that they approach the activity of 2 and 3 (Ki = 4.7 and
4.4 nM vs 17 nM for 46), and that they may be expected to main-
tain the attributes of the conformationally-constrained inhibitor
3 (orally active, long acting FAAH inhibitor).
Scheme 3.
potent enantiomer possesses the (S)-configuration. The absolute
configuration of the additional inhibitors 45, 46, 47, and 48 were
tentatively assigned based on their analogous chromatographic
mobility on the ChiralPak AD column with the slower eluting enan-
tiomer assigned the (S)-configuration. Confirming this assignment
for 47 and consistent with the additional tentative assignments,
Stille coupling of the faster eluting (R)-enantiomer of 54 with 2-
tributylstannylpyridine provided (R)-47 (faster eluting enantio-
mer). Interestingly, the two enantiomers of 54 were only 1.6-fold
different in potency in inhibiting FAAH.
To determine if the results in Figure 5 could be attributed to epi-
merization of the stereocenter during the enzyme inhibition assay,
the enantiopurity of the samples were monitored by chiral HPLC
analysis over the course of a 30 min exposure to the assay buffer
(pH 9.1). For the 5-pyridyloxazole inhibitors, the loss of enantiopu-
rity was modest and followed the trend (greatest to least) of 7-phe-
nyl 45 (À7.7%), 7-benzyloxy 46 (À4.8%), and 7-phenoxy 47 (À1.7%).
Similarly, the unsubstituted parent oxazoles were only modestly
susceptible to epimerization under the assay conditions: 7-phenyl
49 (À9.5%) and 7-benzyloxy 50 (À6.6%). Thus, it seems unlikely that
the results in Figure 5 reflect sufficient racemization under the as-
say conditions to account for the near equivalent activities of the
4.3. Inhibitor selectivity
One inhibitor from each series was examined for FAAH selectiv-
ity in an activity-based protein profile (ABPP) assay for serine
hydrolases76 conducted using the mouse brain proteome. Previous

2768
K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
against FAAH relative to 2 and 3 despite the inductive electron-
(a)
withdrawing effect of the heteroatom insertion, and maintained
their exquisite selectivity for FAAH. Unlike the observations made
with 3, both enantiomers of the candidate FAAH inhibitors proved
to be effective. Representative of these observations, 46 was found
be only 4-fold less active than either 2 or 3, and the two enantio-
mers proved to be equally active (Ki = 18 and 17 nM).
< FAAH
<KIAA1363
6. Experimental section
6.1. Inhibitors
<ABHD6
<MAGL
Full experimental for the synthesis, characterization, and purity
of the candidate inhibitors is provided in Supporting information.
The purity of each tested compound (>95%) was determined on
an Agilent 1100 LC/MS instrument using a ZORBAX SB-C18 column
(3.5 mm, 4.6 mm  50 mm, with a flow rate of 0.75 mL/min and
detection at 220 and 253 nm) with a 10–98% acetonitrile/water/
0.1% formic acid gradient and 50–98% acetonitrile/water/0.1% for-
mic acid gradient.
(b)
6.2. FAAH inhibition
14C-labeled oleamide was prepared from 14C-labeled oleic acid
as described.13 The truncated rat FAAH (rFAAH) was expressed in
E. coli and purified as described.72 The inhibition assays were per-
formed as described.13 In brief, the enzyme reaction was initiated
< FAAH
<KIAA1363
<ABHD6
<MAGL
by mixing 1 nM rFAAH with 20
500 L reaction buffer (125 mM TrisCl, 1 mM EDTA, 0.2% glycerol,
l
M of 14C-labeled oleamide in
l
0.02% Triton X-100, 0.4 mM Hepes, pH 9.0) at room temperature
in the presence of three different concentrations of the inhibitor.
The enzyme reaction was terminated by transferring 20
l
L of the
reaction mixture to 500
lL of 0.1 N HCl at three different time
points. The 14C-labeled oleamide (substrate) and oleic acid (prod-
uct) were extracted with EtOAc and analyzed by TLC as detailed.13
The Ki of the inhibitor was calculated using a Dixon plot as
described.13
The purity of each inhibitor (>95%) was determined on an
Agilent 1100 LC/MS instrument on
a
ZORBAX SB-C18,
3.5 mm  50 mm, with a flow rate of 0.75 mL/min and detection
at 220 and 254 nm, with a 10–98% acetonitrile/water/0.1% formic
acid gradient and a 50–98% acetonitrile/water/0.1% formic acid
gradient (see Supporting information).
Figure 7. ABPP screen of (a) 14 and (b) 47 in mouse brain proteome (1 mg/mL) with
FP-rhodamine (100 nM). Inhibitors were preincubated with proteome for 10 min
prior to 10 min treatment with probe.
6.3. Preparations of mouse tissue proteomes
studies30 have shown that the C5-pyridyl
exquisitely selective for FAAH over other proteome-wide serine
hydrolases, although other derivatives display offsite activity
against the membrane associated hydrolase KIAA1363, monoacyl-
a-ketooxazoles are
Tissues were Dounce-homogenized in PBS, pH 7.5, followed by
a low-speed spin (1400g, 5 min) to remove debris. The supernatant
was then subjected to centrifugation (64,000g, 45 min) to provide
the cytosolic fraction in the supernatant and membrane fraction
as a pellet. The pellet was washed and resuspended in PBS buffer
by sonication. Total protein concentration in each fraction was
determined using a protein assay kit (Bio-Rad).
glycerol lipase (MAGL), and/or a,b-hydrolase containing domain 6
(ABHD6). Both enantiomers of the inhibitors 14 and 47 were tested
for their effects on the fluorophosphonate (FP)-rhodamine probe
labeling of serine hydrolases in the mouse brain proteome at con-
centrations ranging from 10 nM to 100 lM (Fig. 7). Each inhibitor
6.4. ABPP studies
showed superb selectivity for FAAH over all other serine hydro-
lases in the mouse brain proteome including KIAA1363, MAGL,
and ABHD6 (>200-fold).
Tissue proteomes, diluted to 1 mg/mL in PBS, were preincu-
bated with inhibitors (10–10,000 nM, DMSO stocks) for 10 min
and then treated with rhodamine-tagged fluorophosphonate (FP-
rhodamine 100 nM, DMSO stock) at 25 °C for 10 min. Reactions
were quenched with SDS–PAGE loading buffer, subjected to SDS–
PAGE, and visualized in-gel using a flatbed fluorescence scanner
(MiraBio). Labeled proteins were quantified by measuring inte-
grated band intensities (normalized for volume); control samples
(DMSO alone) were considered 100% activity and inhibitor-treated
5. Conclusions
Herein, we report the synthesis and evaluation of a series of
a-ketooxazoles as candidate FAAH inhibitors based on 3, incorpo-
rating an oxygen heteroatom into a conformationally constrained
C2 acyl side. This modification led to slightly diminished potency

K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
2769
33. Deutsch, D. G.; Lin, S.; Hill, W. A. G.; Morse, K. L.; Salehani, D.; Arreaza, G.;
Omeir, R. L.; Makriyannis, A. Biochem. Biophys. Res. Commun. 1997, 231, 217.
34. Edgemond, W. S.; Greenberg, M. J.; McGinley, P. J.; Muthians, S.; Campbell, W.
B.; Hillard, C. J. J. Pharmacol. Exp. Ther. 1998, 286, 184.
35. Fernando, S. R.; Pertwee, R. G. Br. J. Pharmacol. 1997, 121, 1716.
36. Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Boger, D. L. Bioorg. Med. Chem. Lett.
2005, 15, 103.
37. Kathuria, S.; Gaetani, S.; Fegley, D.; Valino, F.; Duranti, A.; Tontini, A.; Mor, M.;
Tarzia, G.; La Rana, G.; Calignano, A.; Giustino, A.; Tattoli, M.; Palmery, M.;
Cuomo, V.; Piomelli, D. Nat. Med. 2003, 9, 76.
38. Jayamanne, A.; Greenwood, R.; Mitchell, V. A.; Aslan, S.; Piomelli, D.; Vaughan,
C. W. Br. J. Pharmacol. 2006, 147, 281.
samples were expressed as a percentage of remaining activity. IC50
values were determined from dose–response curves from three tri-
als at each inhibitor concentration using Prism software
(GraphPad).
Acknowledgments
We gratefully acknowledge the financial support of the National
Institutes of Health (Grant DA015648, D.L.B.). We thank Raj K. Cha-
dha for the X-ray crystal structure of (S)-54 and B.F. Cravatt for the
supply of FAAH used in the enzymatic assays.
39. Biancalani, C.; Giovanni, M. P.; Pieretti, S.; Cesari, N.; Graziano, A.; Vergeli, C.;
Cilibrizzi, A.; Di Gianuario, A.; Colucci, M.; Mangano, G.; Garrone, B.; Polezani,
L.; Dal Piaz, V. J. Med. Chem. 2009, 52, 7397.
40. Clapper, J. R.; Vacondio, F.; King, A. R.; Duranti, A.; Tontini, A.; Silva, C.;
Sanchini, S.; Tarzia, G.; Mor, M.; Piomelli, D. ChemMedChem 2009, 4, 1505.
41. (a) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.; Stevenson,
T.; Lund, E. T.; Nugent, R. A.; Normanbhoy, T.; Alexander, J. P.; Cravatt, B. F.
Biochemistry 2007, 46, 13019; (b) Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.;
Long, J. Z.; McKinney, M. K.; Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith,
S. E.; Lazerwith, S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney,
S.; Van Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Chem. Biol. 2009, 16, 411; (c)
Johnson, D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L. K.; Morris, M.;
Beidler, D.; Liimatta, M. B.; Smith, S. E.; Dudley, D. T.; Sadogopan, N.;
Bhattachar, S. N.; Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. F.; Ahn, K. ACS
Med. Chem. Lett. 2011, 2, 91.
Supplementary data
Supplementary data (full experimental procedures, character-
ization and purities of the candidate inhibitors, and enzyme inhibi-
tion measurement standard deviations for Figures 3, 5 and 7 and
Scheme 3) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2014.03.013.
References and notes
42. Meyers, J. M.; Long, A. S.; Pelc, J. M.; Wang, L. J.; Bowen, J. S.; Schweitzer, A. B.;
Wilcox, C. M.; McDonald, J.; Smith, E. S.; Foltin, S.; Rumsey, J.; Yang, S. Y.;
Walker, C. M.; Kamtekar, S.; Beidler, F.; Torarensen, A. Bioorg. Med. Chem. Lett.
2011, 21, 6545.
1. Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner, R. A.; Gilula, N. B.
Nature 1996, 384, 83.
2. Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2238.
3. Patricelli, M. P.; Cravatt, B. F. Vitam. Horm. 2001, 62, 95.
4. Egertova, M.; Cravatt, B. F.; Elphick, M. R. Neuroscience 2003, 119, 481.
5. Boger, D. L.; Fecik, R. A.; Patterson, J. E.; Miyauchi, H.; Patricelli, M. P.; Cravatt, B.
F. Bioorg. Med. Chem. Lett. 2000, 10, 2613.
6. Ezzili, C.; Otrubova, K.; Boger, D. L. Bioorg. Med. Chem. Lett. 2010, 20, 5959.
7. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.;
Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 258,
1946.
8. Martin, B. R.; Mechoulam, R.; Razdan, R. K. Life Sci. 1999, 65, 573.
9. Schmid, H. H. O.; Schmid, P. C.; Natarajan, V. Prog. Lipid Res. 1990, 29, 1.
10. Boger, D. L.; Henriksen, S. J.; Cravatt, B. F. Curr. Pharm. Des. 1998, 4, 303.
11. Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580.
12. (a) Cravatt, B. F.; Prospero-Garcia, O.; Suizdak, G.; Gilula, N. B.; Henriksen, S. J.;
Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506; (b) Lerner, R. A.; Siuzdak, G.;
Prospero-Garcia, O.; Henriksen, S. J.; Boger, D. L.; Cravatt, B. F. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 9505.
13. (a) Patricelli, M. P.; Cravatt, B. F. Biochemistry 1999, 38, 14125; (b) Patricelli, M.
P.; Cravatt, B. F. J. Biol. Chem. 2000, 275, 19177; (c) Patricelli, M. P.; Lovato, M.
A.; Cravatt, B. F. Biochemistry 1999, 38, 9804.
14. McKinney, M. K.; Cravatt, B. F. J. Biol. Chem. 2003, 278, 37393.
15. McKinney, M. K.; Cravatt, B. F. Annu. Rev. Biochem. 2005, 74, 411.
16. Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793.
17. Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674.
18. Clement, A. B.; Hawkins, E. G.; Lichtman, A. H.; Cravatt, B. F. J. Neurosci. 2003,
23, 3916.
19. (a) Cravatt, B. F.; Lichtman, A. H. Curr. Opin. Chem. Biol. 2003, 7, 469; (b) Ahn, K.;
McKinney, M. K.; Cravatt, B. F. Chem. Rev. 2008, 108, 1687; (c) Ahn, K.; Johnson,
D. S.; Cravatt, B. F. Exp. Opin. Drug Disc. 2009, 4, 763.
20. Lambert, D. M.; Fowler, C. J. J. Med. Chem. 2005, 48, 5059.
21. Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.; Giang, D. K.; Martin,
B. R.; Lichtman, A. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9371.
22. Lichtman, A. H.; Shelton, C. C.; Advani, T.; Cravatt, B. F. Pain 2004, 109, 319.
23. Cravatt, B. F.; Saghatelian, A.; Hawkins, E. G.; Clement, A. B.; Bracey, M. H.;
Lichtman, A. H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10821.
24. Karsak, M.; Gaffal, E.; Date, R.; Wang-Eckhardt, L.; Rehnelt, J.; Petrosino, S.;
Starowicz, K.; Steuder, R.; Schlicker, E.; Cravatt, B. F.; Mechoulam, R.; Buettner,
R.; Werner, S.; Di Marzo, V.; Tueting, T.; Zimmer, A. Science 2007, 316, 1494.
25. (a) Huitrón-Reséndiz, S.; Gombart, L.; Cravatt, B. F.; Henriksen, S. J. Exp. Neurol.
2001, 172, 235; (b) Huitrón-Reséndiz, S.; Sanchez-Alavez, M.; Wills, D. N.;
Cravatt, B. F.; Henriksen, S. J. Sleep 2004, 27, 857.
26. Patricelli, M. P.; Patterson, J. P.; Boger, D. L.; Cravatt, B. F. Bioorg. Med. Chem.
Lett. 1998, 8, 613.
27. Koutek, B.; Prestwich, G. D.; Howlett, A. C.; Chin, S. A.; Salehani, D.; Akhavan,
N.; Deutsch, D. G. J. Biol. Chem. 1994, 269, 22937.
28. Patterson, J. E.; Ollmann, I. R.; Cravatt, B. F.; Boger, D. L.; Wong, C.-H.; Lerner, R.
A. J. Am. Chem. Soc. 1996, 118, 5938.
29. Boger, D. L.; Sato, H.; Lerner, A. E.; Austin, B. J.; Patterson, J. E.; Patricelli, M. P.;
Cravatt, B. F. Bioorg. Med. Chem. Lett. 1999, 9, 265.
43. (a) Sit, S. Y.; Conway, C.; Bertekap, R.; Xie, K.; Bourin, C.; Burris, K.; Deng, H.
Bioorg. Med. Chem. Lett. 2007, 17, 3287; (b) Sit, S. Y.; Conway, C. M.; Xie, K.;
Bertekap, R.; Bourin, C.; Burris, K. D. Bioorg. Med. Chem. Lett. 2010, 20, 1272.
44. (a) Keith, J. M.; Apocada, R.; Xiao, W.; Seierstad, M.; Pattabiraman, K.; Wu, J.;
Webb, M.; Karbarz, M. J.; Brown, S.; Wilson, S.; Scott, B.; Tham, C.-S.; Luo, L.;
Palmer, J.; Wennerholm, M.; Chaplan, S.; Breitenbucher, J. G. Bioorg. Med. Chem.
Lett. 2008, 18, 4838; (b) Keith, J. M.; Apodaca, R.; Tichenor, M.; Xiao, W.; Jones,
W.; Pierce, J.; Seierstad, M.; Palmer, J.; Webb, M.; Karbarz, M.; Scott, B.; Wilson,
S.; Luo, L.; Wennerholm, M.; Chang, L.; Brown, S.; Rizzolio, M.; Rynberg, R.;
Chaplan, S.; Breitenbucher, J. G. ACS Med. Chem. Lett. 2012, 3, 823.
45. Kono, M.; Matsumoto, T.; Kawamura, T.; Nishimura, A.; Kiyota, Y.; Oki, H.;
Miyazaki, J.; Igaki, S.; Bahnke, C. A.; Shimojo, M.; Kori, M. Bioorg. Med. Chem.
2013, 21, 28.
46. Moore, S. A.; Nomikos, G. G.; Dickason-Chesterfield, A. K.; Sohober, D. A.;
Schaus, J. M.; Ying, B. P.; Xu, Y. C.; Phebus, L.; Simmons, R. M.; Li, D.; Iyengar, S.;
Felder, C. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17852.
47. Alexander, J. P.; Cravatt, B. F. J. Am. Chem. Soc. 2006, 128, 9699.
48. Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179.
49. (a) Minkkila, A.; Myllymaki, M. J.; Saario, S. M.; Castillo-Melendez, J. A.;
Koskinen, A. M. P.; Fowler, C. J.; Leppanen, J.; Nevalainene, T. Eur. J. Med. Chem.
2009, 44, 2294.
50. For additional studies, see: (a) Wang, X.; Sarris, K.; Kage, K.; Zhang, D.; Brown,
S. P.; Kolassa, T.; Surowy, C.; El Kouhen, O. F.; Muchmore, S. W.; Brioni, J. D.;
Stewart, A. O. J. Med. Chem. 2009, 52, 170; (b) Min, X.; Thibault, S. T.; Porter, A.
C.; Gustin, D. J.; Carlson, T. J.; Xu, H.; Lindstrom, M.; Xu, G.; Uyeda, C.; Ma, Z.; Li,
Y.; Kayser, F.; Walker, N. P. C.; Wang, Z. Proc. Natl. Acad. Sci. 2011, 108, 7379; (c)
Tian, G.; Pachetto, K. A.; Gharahdaghi, F.; Gordon, E.; Wilkens, D. E.; Luo, X.;
Scott, C. W. Biochemistry 2011, 50, 6867.
51. Otrubova, K.; Boger, D. L. ACS Chem. Neurosci. 2012, 3, 340.
52. Boger, D. L.; Sato, H.; Lerner, A. E.; Hedrick, M. P.; Fecik, R. A.; Miyauchi, H.;
Wilkie, G. D.; Austin, B. J.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 5044.
53. Boger, D. L.; Miyauchi, H.; Hedrick, M. P. Bioorg. Med. Chem. Lett. 2001, 11, 1517.
54. (a) Lichtman, A. H.; Leung, D.; Shelton, C. C.; Saghatelian, A.; Hardouin, C.;
Boger, D. L.; Cravatt, B. F. J. Pharmacol. Exp. Ther. 2004, 311, 441; (b) Schlosburg,
J. E.; Boger, D. L.; Cravatt, B. F.; Lichtman, A. H. J. Pharmacol. Exp. Ther. 2009, 329,
314; (c) Kinsey, S. G.; Long, J. Z.; O’Neal, S. T.; Abdulla, R. A.; Poklis, J. L.; Boger,
D. L.; Cravatt, B. F.; Lichtman, A. H. J. Pharmacol. Exp. Ther. 2009, 330, 902; (d)
Booker, L.; Kinsey, S. G.; Abdullah, R. A.; Blankman, J. L.; Long, J. Z.; Boger, D. L.;
Cravatt, B. F.; Lichtman, A. H. Br. J. Pharmacol. 2012, 165, 2485.
55. Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.; Cheng, H.; Hwang,
I.; Hedrick, M. P.; Leung, D.; Acevedo, O.; Guimaráes, C. R. W.; Jorgensen, W. L.;
Cravatt, B. F. J. Med. Chem. 2005, 48, 1849.
56. Leung, D.; Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Cravatt, B. F.; Boger, D. L.
Bioorg. Med. Chem. Lett. 2005, 15, 1423.
57. Romero, F. A.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006, 68, 14004.
58. Romero, F. A.; Du, W.; Hwang, I.; Rayl, T. J.; Kimball, F. S.; Leung, D.; Hoover, H.
S.; Apodaca, R. L.; Breitenbucher, B. J.; Cravatt, B. F.; Boger, D. L. J. Med. Chem.
2007, 50, 1058.
59. Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.; Hwang, I.; Cravatt,
B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 3359.
60. Kimball, F. S.; Romero, F. A.; Ezzili, C.; Garfunkle, J.; Rayl, T. J.; Hochstatter, D.
G.; Hwang, I.; Boger, D. L. J. Med. Chem. 2008, 51, 937.
61. Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang, I.; Boger, D. L. J.
Med. Chem. 2008, 51, 4393.
30. Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotechnol. 2003, 21, 687.
31. De Petrocellis, L.; Melck, D.; Ueda, N.; Maurelli, S.; Kurahashi, Y.; Yamamoto, S.;
Marino, G.; Di Marzo, V. Biochem. Biophys. Res. Commun. 1997, 231, 82.
32. Deutsch, D. G.; Omeir, R.; Arreaza, G.; Salehani, D.; Prestwich, G. D.; Huang, Z.;
Howlett, A. Biochem. Pharmacol. 1997, 53, 255.

2770
K. K. Duncan et al. / Bioorg. Med. Chem. 22 (2014) 2763–2770
62. DeMartino, J. K.; Garfunkle, J.; Hochstatter, D. G.; Cravatt, B. F.; Boger, D. L.
Bioorg. Med. Chem. Lett. 2008, 18, 5842.
63. Ezzili, C.; Mileni, M.; McGlinchey, N.; Long, J. Z.; Kinsey, S. G.; Hochstatter, D.
G.; Stevens, R. C.; Lichtman, A. H.; Cravatt, B. F.; Bilsky, E. J.; Boger, D. L. J. Med.
Chem. 2011, 54, 2805.
64. Otrubova, K.; Brown, M.; McCormick, M. S.; Han, G. W.; O’Neil, S. T.; Cravatt, B.
F.; Stevens, R. C.; Lichtman, A. H.; Boger, D. L. J. Am. Chem. Soc. 2013, 135, 6289.
65. Witiak, D. T.; Heilman, W. P.; Sankarappa, S. K.; Cavestri, R. C.; Newman, H. A. I.
J. Med. Chem. 1975, 19, 934.
66. Vedejs, E.; Monahan, S. D. J. Org. Chem. 1996, 61, 5192.
67. Hari, Y.; Obika, S.; Sakaki, M.; Morio, K.; Yamagata, Y.; Imanishi, T. Tetrahedron
2002, 58, 3051.
71. Farina, V.; Krishnamurthy, V.; Scott, W. J. Org. React. 1997, 50, 1.
72. Patricelli, M. P.; Lashuel, H. A.; Giang, D. K.; Kelly, J. W.; Cravatt, B. F.
Biochemistry 1998, 37, 15177.
73. The structure and absolute configuration of (S)-54 was established with single-
crystal X-ray structure determination conducted on a colorless needle grown
from EtOAc/CH2Cl2 (CCDC 975738).
74. (a) Mileni, M.; Garfunkle, J.; DeMartino, J. K.; Cravatt, B. F.; Boger, D. L.; Stevens,
R. C. J. Am. Chem. Soc. 2009, 131, 10497; (b) Mileni, M.; Garfunkle, J.; Kimball, F.
S.; Cravatt, B. F.; Stevens, R. C.; Boger, D. L. J. Med. Chem. 2010, 53, 230; (c)
Mileni, M.; Garfunkle, J.; Ezzili, C.; Cravatt, B. F.; Stevens, R. C.; Boger, D. L. J. Am.
Chem. Soc. 2011, 133, 4092.
75. Guimaráes, C. R. W.; Boger, D. L.; Jorgensen, W. L. J. Am. Chem. Soc. 2005, 127,
17377.
76. (a) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279; (b) Liu, Y. S.; Patricelli,
M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694.
68. Ley, S. V.; Thomas, A. W. Angew. Chem., Int. Ed. 2003, 42, 5400.
69. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
70. Choi, Y.-M.; Shin, H.; Ilankumaran, P.; Kim, H.-W. Patent WO 2005/021540,
2005.