Optimization of the central heterocycle of α-ketoheterocycle inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1021/jm800136b

The synthesis and evaluation of a refined series of α- ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity. 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well-defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes.

Full text of DOI:10.1021/jm800136b

4392
J. Med. Chem. 2008, 51, 4392–4403
Articles
Optimization of the Central Heterocycle of r-Ketoheterocycle Inhibitors of Fatty Acid
Amide Hydrolase
Joie Garfunkle, Cyrine Ezzili, Thomas J. Rayl, Dustin G. Hochstatter, Inkyu Hwang, and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
ReceiVed February 7, 2008
The synthesis and evaluation of a refined series of R-ketoheterocycles based on the oxazole 2 (OL-135)
incorporating systematic changes in the central heterocycle bearing a key set of added substituents are
described. The nature of the central heterocycle, even within the systematic and minor perturbations explored
herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles,
the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles >
1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom
at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to
a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying
well-defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the
inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity
and simultaneous disruption of binding affinity for competitive off-target enzymes.
Introduction
Fatty acid amide hydrolase (FAAHa)1,2 is the enzyme that
serves to hydrolyze endogenous lipid amides3–6 including
anandamide (1a)7–10 and oleamide (1b),11–13 Figure 1. Its
distribution is consistent with its role in degrading and regulating
such neuromodulating and signaling fatty acid amides at their
sites of action.3 Although it is a member of the amidase signature
family of serine hydrolases, for which there are a number of
prokaryotic enzymes, it is currently the only characterized
mammalian enzyme bearing the family’s unusual Ser-Ser-Lys
catalytic triad.1,2,14–17
Because of the therapeutic potential of inhibiting FAAH,3,18–20
especially for the treatment of pain,21–23 inflammatory,24 or sleep
disorders,13,25 there has been an increasing interest in the
development of selective and potent inhibitors of the enzyme.26
Figure 1. Substrates of fatty acid amide hydrolase (FAAH).
Early studies shortly following the initial characterization of
the enzyme led to the discovery that the endogenous sleep-
inducing molecule 2-octyl R-bromoacetoacetate is an effective
of inhibitors that irreversibly and covalently modify the target
enzyme. A second class is the R-ketoheterocycle-based
inhibitors51–59 that bind to FAAH via reversible hemiketal
formation with an active site serine. Many of these competitive
inhibitors are not only potent and extraordinarily selective for
FAAH versus other mammalian serine hydrolases, but members
of this class have been shown to be efficacious analgesics in
vivo.58,59
FAAH inhibitor,27 the disclosure of a series of nonselective
reversible inhibitors bearing an electrophilic ketone (e.g.,
trifluoromethyl ketone-based inhibitors),28–31 and the reports of
a set of irreversible inhibitors32–37 (e.g., fluorophosphonates and
sulfonyl fluorides). To date, only two classes of inhibitors have
been disclosed that provide opportunities for the development
of inhibitors with therapeutic potential. One class is the reactive
aryl carbamates and ureas38–50 that irreversibly acylate a FAAH
active site serine49 and that have been shown to exhibit
anxiolytic activity38 and produce analgesic effects.39 To date
and with some exceptions, the selectivity of such inhibitors has
often been low,31,42,48–50 further complicating the development
In these studies, 253 emerged as an important lead inhibitor
for further study (Figure 2). It has been shown that 2 is a potent
(Ki ) 4.7 nM)53 and selective (g100-300 fold)31 FAAH
inhibitor that induces analgesia and increases endogenous
anandamide levels.53,59 It has been shown to exhibit analgesic
activity in the tail flick assay, hot plate assay, formalin test of
noxious chemical pain (1st and second phase), the mild thermal
injury (MTI) model of peripheral pain, and the spinal nerve
ligation (SNL) model of neuropathic pain with efficacies that
match or exceed those of morphine (@1-3 mg/kg in MTI/SNL),
* To whom correspondence should be addressed. Phone: 858-784-7522.
Fax: 858-784-7550. E-mail: boger@scripps.edu.
a Abbreviations: FAAH, fatty acid amide hydrolase, TGH, triacylglycerol
hydrolase.
10.1021/jm800136b CCC: $40.75
2008 American Chemical Society
Published on Web 07/16/2008

Central Heterocycle of R-Ketoheterocycle Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4393
Scheme 1
Figure 2. Progression of the inhibitor series.
ibuprofen (@100 mg/kg in MTI), or gabapentin (@500 mg/kg
in SNL) and at administered doses (10-20 mg/kg, ip) that
approach or exceed those of such common pain medications.59
It has been shown to lack significant offsite target activity (Cerep
assay profiling), does not bind cannabinoid (CB1 or CB2) or
vanilloid (TRP) receptors and does not significantly inhibit
common P450 metabolism enzymes (3A4, 2C9, 2D6) or the
human ether-a-go-go related gene (hERG). Significantly, the
inhibitor was ineffective at promoting analgesia in FAAH
knockout mice,58,59 verifying that FAAH is the only relevant
target responsible for the in vivo analgesic effects of 2.
Moreover, the analgesic effects were observed without the
respiratory depression or chronic dosing desensitization char-
acteristic of opioid administration59 or the increased feeding and
decreased mobility and motor control characteristic of a can-
nabinoid (CB1) agonist administration.59
Scheme 2
accomplished via a metal-halogen exchange of the correspond-
ing alkylbromide to give the R-ketooxadiazole or R-ketothia-
diazole.
Consequently, we conducted a series of systematic structure-
activity relationship (SAR) studies on 2 independently targeting
the 5-position of the central oxazole (aryl and nonaromatic
substituents)53,55,56 and the C2 acyl side chain,53,57,58 both of
which have provided extraordinarily potent and selective FAAH
inhibitors (Figure 2).60 Herein, we report results of systematic
studies examining candidate inhibitors exploring and further
optimizing the central heterocycle of the R-ketoheterocycle
within the structure of 2 along with results of the proteome-
wide selectivity screening31 of the resulting candidate inhibitors.
Synthesis of the 1,2,4-triazole series (14) in an analogous
fashion was unsuccessful in which the side chain nucleophilic
addition to the corresponding methyl ester failed to afford
product. Therefore, an alternative approach was utilized to obtain
the desired 1,2,4-triazoles (Scheme 2). 7-Phenylheptanal was
converted to the silyl-protected cyanohydrin upon treatment with
potassium cyanide, TBSCl, and catalytic ZnI2.61 The corre-
sponding amidrazone62 was formed by in situ generation of
sodium hydrazine followed by dropwise addition of the cyano-
hydrin, which was treated with an aryl(R) acid chloride.
Surprisingly, the cyclization afforded mainly the 1,3,4-oxadia-
zole product along with a small amount of the desired 1,2,4-
triazole product. Although this offered an additional route to
the 1,3,4-oxadiazoles, it required modification for 1,2,4-triazole
formation. The alternative was a two-step condensation and
oxidation pathway via reaction of the amidrazone with aryl(R)
aldehydes to give a stable imine, which undergoes oxidative
cyclization to the triazole upon treatment with DDQ.63 The
desired 1,2,4-triazoles 14 were obtained after TBS deprotection
(Bu4NF) and oxidation of the liberated alcohol with Dess-Martin
periodinane64 (Scheme 2).
Chemistry
The preparation of the 1,3,4-thiadiazole (12), 1,3,4-oxadiazole
(8), and 1,2,4-oxadiazole (9) inhibitors is illustrated in Scheme
1. A one-pot procedure was utilized to prepare the C-5 aryl
substituted 1,3,4-thiadiazole, 1,3,4-oxadiazole, and 1,2,4-oxa-
diazole methyl esters. In the case of the 1,3,4-thiadiazole and
1,3,4-oxadiazole, the corresponding aryl(R) methyl ester was
converted to the hydrazide with hydrazine monohydrate, whereas
the aryl(R) nitrile was transformed to the N-hydroxycarbamide
with hydroxylamine for the 1,2,4-oxadiazole, all of which were
subsequently treated with methyl oxalyl chloride in the pres-
ence of Et3N to give a diacyl hydrazide intermediate. These
were cyclized upon treatment with Lawesson’s reagent to give
the desired 1,3,4-thiadiazole or with p-toluenesulfonyl chloride
(TsCl) for the 1,3,4-oxadiazole and 1,2,4-oxadiazole. Subsequent
addition of the requisite side chain (R1) to the methyl ester was
The TBS-protected cyanohydrin was also a key intermediate
for the preparation of two additional inhibitor series, the isomeric
1,2,4-oxadiazoles (10) and the tetrazoles (15). For the 1,2,4-
oxadiazoles, the cyanohydrin was converted to the N-hydroxy-
carbamide upon treatment with hydroxylamine. Its treatment
with acid chlorides in the presence of Et3N to give the diacyl

4394 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Garfunkle et al.
Scheme 3
Scheme 6
Scheme 4
philicity of the intermediate for subsequent side chain addition,
which was accomplished via metal-halogen exchange of the
corresponding alkylbromide. The alcohol precursor was oxidized
with Dess-Martin periodinane64 to give the 1,2,-diazines 13
(Scheme 6).
Enzyme Assay
Enzyme assays were performed at 20-23 °C with purified
recombinant rat FAAH expressed in Escherichia coli71 (unless
indicated otherwise) or with solubilized COS-7 membrane
extracts from cells transiently transfected with human FAAH
cDNA2 (where specifically indicated) in a buffer of 125 mM
Tris/1 mM EDTA/0.2% glycerol/0.02% Triton X-100/0.4 mM
Hepes, pH 9.0. The initial rates of hydrolysis (e10-20%
reaction) were monitored using enzyme concentrations (typically
1 nM) at least 3 times below the measured Ki by following the
breakdown of 14C-oleamide, and Ki values (standard deviations
are provided in the Supporting Information tables) were
established as described (Dixon plot). Lineweaver-Burk analy-
sis previously established reversible, competitive inhibition for
2 and related inhibitors.53
Scheme 5
Results and Discussion
In the course of early studies,51 a range of oleyl R-ketohet-
erocycles were explored in which the benzoxazolyl51 and
oxazolyl53 derivatives were found to be among the most potent
and were chosen for further examination. Their examination,
which included the elaboration to the exceptionally potent
oxazolopyridines51 or 5-(2-pyridyl)oxazoles53 including 2,
defined important features of the heterocycle substituents and
the C2-aryl side chain that enhance inhibitor potency or inhibitor
selectivity.51–58 With this knowledge of the generalized SAR
surrounding these two classes of R-ketoheterocycles and with
the benefit of insightful modeling and computational studies72a
indicating that the active site hydrogen bonding and steric
interactions favor a 5-membered versus 6-membered hetero-
cycle, we reexamined the central heterocycle, extending the
series to include systems not yet explored.
Oleyl-Based Inhibitors. Even before a systematic examina-
tion of the central heterocycle of 2 was undertaken, a small
series of additional substituted heterocycles bearing an oleyl
acyl side chain were examined beyond those originally dis-
closed.53 This included the candidate 1,3,4-oxadiazole and 1,3,4-
thiadiazole inhibitors 4a-4e and 5a-5c, respectively, bearing
C5 substituents that probe a well-defined trend reflecting the
hydrogen bond acceptor capabilities of the substituent (Figure
3). This provided inhibitors that followed the identical substitu-
ent trends (Ki for 2-pyr < 2-furyl < 2-thienyl < H) and that
exhibited potencies that exceeded (4a-4e, 1,3,4-oxadiazoles,
2-10 fold) or roughly matched (5a-5c, 1,3,4-thiadiazoles) those
hydrazide intermediates followed by dehydration with p-
toluenesulfonyl chloride (TsCl) at elevated temperatures gave
the desired 1,2,4-oxadiazoles 10 after TBS deprotection (Bu4NF)
and oxidation of the liberated alcohol with Dess-Martin
periodinane64 (Scheme 3).
The tetrazoles (15) were obtained by treatment of the
cyanohydrin with sodium azide.65 Reprotection of the alcohol
with TBSCl followed by regioselective N-arylation using a
copper-catalyzed Ullmann condensation66 with iodopyridine or
with mixed-aryl hypervalent iodonium salts67 afforded N2-
substituted tetrazoles, which were converted to the corresponding
products upon TBS deprotection (Bu4NF) and oxidation of the
liberated alcohol with Dess-Martin periodinane64 (Scheme 4).
The synthesis of the 1,3-thiazole inhibitors (11) entailed a
selective C2-lithiation of thiazole followed by condensation with
7-phenylheptanal. TBS protection of the resulting alcohol
followed by selective C5-lithiation (t-BuLi)68 and stannylation
or iodination and subsequent Stille coupling69 produced the
substituted thiazoles, which were converted to the corresponding
ketones by TBS deprotection (Bu4NF) and oxidation of the
liberated alcohol with Dess-Martin periodinane64 (Scheme 5).
Synthesis of the 1,2-diazine inhibitors (13) began with Stille
coupling of a known chloro-pyridazine70 and a series of
2-(tributylstannyl)arenes or 2-aryl boronic esters to afford the
series of 6-aryl substituted pyridazine-3-carboxylates. The
corresponding methyl ester was converted to the aldehyde by a
reduction-oxidation process in order to increase the electro-

Central Heterocycle of R-Ketoheterocycle Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4395
cycles, this matrix examination confidently permits the relative
extrapolation of the extensive SAR surrounding 2 and the
oxazole-based inhibitors onto each of the new R-ketohetero-
cycles (Figure 5).
Thus, the substituent impact within each heterocycle series
was found to follow the well-defined potency order of 2-pyr
>2-furanyl >2-thienyl > phenyl, reflecting their relative
hydrogen bonding ability53 and for which the 2-pyr derivatives
were 10-20 fold more active than the corresponding phenyl
derivative. Moreover, the unsubstituted derivative (R ) H) was
always less potent than the corresponding 2-pyr derivative but
more potent than the corresponding phenyl derivative displaying
a Ki that approached or exceeded those of the 2-furanyl or
2-thienyl derivative. Typically, the 2-pyr-6-CO2Me derivative
was less potent than the corresponding 2-pyr derivative and the
corresponding 2-pyr-6-CO2H derivative was 2-10 fold less
potent than its ester. However, the results with these latter
carboxylic acid derivatives represent Ki’s measured at pH 9
under conditions where they are fully ionized destabilizing their
active site binding. These Ki’s improve when measured at pH
8 and 7.4 (physiological pH); for example, oxazole 7g was
examined and displayed an increase in inhibitory potency with
decreasing pH (7g: pH ) 9, Ki ) 20 nM; pH ) 8, Ki ) 14.5
nM; pH ) 7.4, Ki ) 10.3 nM). These overall substituent trends
are analogous to those defined with the oxazole-based inhibitors
(7 series)53–58 that includes 2.
The trends for the heterocycles themselves proved even more
interesting. The thiazole-based inhibitors 11 proved significantly
less active than the oxazole-based inhibitors 7, and replacing
the position 4 CH of the oxazole or thiazole with a heteroatom
uniformly and substantially improved the potency. The most
potent of these were the 1,3,4-oxadiazoles 8 and 1,2,4-
oxadiazoles 9, which were typically 10-70 fold more active
than the corresponding oxazoles, followed closely by the
isomeric 1,2,4-oxadiazoles 10. Perhaps the most dramatic change
in potency occurred between the thiazole 11 and 1,3,4-
thiadiazole 12 series, where incorporation of the position 4
nitrogen improved potencies 30-600 fold. As such, the 1,3,4-
thiadiazoles 12 typically exceed the potency of the correspond-
ing oxazoles 7, approaching the activity of the 1,2,4-oxadiazoles
10, albeit still being less potent than the isomeric 1,2,4-
oxadiazoles 9 or 1,3,4-oxadiazoles 8. Notably, this position 4
heteroatom effect (N > O > CH) is consistent with its role in
reducing a destabilizing steric interaction at the active site as
well as lowering the torsional energy penalty for coplanar
binding of the heterocycle and its aryl C5 substituent.72 Given
that this latter effect is not contributing to the differences
observed with the heterocycles when R ) H, the result suggests
that it is the former steric effect or an as yet unrecognized effect
that dominates the differences in binding affinity. Such unrec-
ognized effects could include the introduction of a stabilizing
hydrogen bond acceptor site (N > O . CH), heteroatom effects
leading to increases in the heterocycle pKb and intrinsic
hydrogen bonding capabilities or even simply productive
increases in the intrinsic electron-withdrawing character of the
central heterocycle.
Figure 3. Oleyl-based inhibitors.
Figure 4. Activity of the isomeric oxazole.
of the corresponding oxazole (3a-3e).53 Thus, the introduction
of an additional heteroatom into the central heterocycle had a
beneficial impact that in the case of the 1,3,4-oxadiazole could
be attributed to a reduced destabilizing steric interaction in the
active site (position 4 N vs CH) and a lower torsional energy
penalty for near coplanar binding of the central heterocycle and
its C5 substituent.72a These, and related studies,54 indicated that
systematic modifications to the central oxazole of 2 would be
productive.
Phenhexyl-Based Inhibitors. The first of the inhibitors
examined was the oxazole isomer 6. Simply switching the
location of the oxazole nitrogen atom within 6 resulted in a
>4000-fold loss of FAAH inhibition (Figure 4). This result was
anticipated based on the reduced electrophilicity of such ketones,
and additional members of this class were not examined.
Much more interesting was the behavior observed in the
systematic examination of alternative 5-membered heterocycles
bearing two or more heteroatoms. In these comparisons, a
constant series of representative C5 substituents was examined
that permitted an assessment of not only the central heterocycle
trends but also served to generalize the substituent trends defined
with the oxazole series.53,56 In principle and because the
substituent trends remain analogous across each of the hetero-
Interestingly, the isosteric replacement of sulfur in the 1,3,4-
thiadiazoles 12 with CHdCH, providing the corresponding 1,2-
diazines 13, afforded effective but significantly less potent
inhibitors (typically 5-50 fold). Nonetheless, such inhibitors
were still more effective than the corresponding triazoles 14.
The 1,2,4-triazoles 14 proved inactive, but this behavior
represents the destabilizing binding of the deprotonated acidic

4396 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Garfunkle et al.
Figure 5. Inhibitors containing alternative central heterocycles, Ki (nM).
heterocycle under the pH 9 assay conditions resulting from the
C2 acyl substitution (Figure 6).
One of the most interesting series to emerge from these
studies was the tetrazoles 15. In addition to being the most
electron-deficient of the heterocycles examined, position 4
(oxazole numbering) incorporates a N (N > O > CH), and all
three accessible sites on the heterocycle constitute potential
hydrogen bond acceptor sites (N). Consistent with these features,
the tetrazoles 15 proved substantially more active than the
oxazoles 7, comparable in potency with the 1,2,4-oxadiazoles
10, albeit not quite as potent as the isomeric 1,2,4-oxadiazoles
9 or 1,3,4-oxadiazoles 8. Clearly, there are subtle and unrec-
ognized effects that account for the small differences in binding
affinity between the most potent classes of heterocycles (8, 9
> 10, 12, 15) that in turn are easily distinguished from the less
active series (13 > 11 > 14). These latter studies with the
tetrazoles seems to suggest that the most potent activity within
the 5-membered heterocycles is observed with those that
incorporate a hydrogen bonding acceptor N and O (vs N and
N) bracketing the electrophilic carbonyl attachment site. This
may account for the subtly more potent activity of the 1,3,4-
oxadiazoles 8 and 1,2,4-oxadiazoles 9 (N and O) relative to
the isomeric 1,2,4-oxadiazoles 10 and the comparable tetrazoles
15 (N and N). Each incorporate a heteroatom at the oxazole
position 4 that enhance their activity relative to the oxazole
series, and the results with the tetrazole series 15 suggest the
subtle differences in the 7-15 series has more to do with
the location of the oxygen atom than a preference for an added
nitrogen versus oxygen at the oxazole position 4. The exception
to the behavior in the tetrazole series 15 is the unsubstituted
derivative 15a. This unsubstituted and acidic central heterocycle,
like the 1,2,4-triazole series 14, is deprotonated under the pH 9
Figure 6. Effect of N-methylation on inhibitor potency of 1,2,4-
triazoles and tetrazoles.
assay conditions, destabilizing active site binding or its activation
of the reversible hemiketal formation with the otherwise

Central Heterocycle of R-Ketoheterocycle Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4397
Scheme 7
Figure 7. Methyl ester substituted R-ketoheterocycle inhibitors of
FAAH.
Scheme 8
Figure 8. Additional C6 substituted 2-pyridyl substituents.
R-ketoheterocycles, we sought to establish that analogous
modifications or substitutions in the C2 acyl side chain would
be possible. Thus, a small preliminary series of key 1,3,4-
oxadiazoles bearing the conformationally restricted biphenyl-
ethyl side chain was prepared (Scheme 7) and examined (Figure
9). Analogous to trends observed in the oxazole-based series,58
the replacement of the phenhexyl side chain with the biphe-
nylethyl side chain maintained or further enhanced the extraor-
dinary potency of the candidate inhibitors (e.g., Ki of 27 ) 300
pM). Such studies continue and will be disclosed in due course.
The Electrophilic Carbonyl. A select set of the candidate
inhibitors were also examined that bear a secondary alcohol or
a methylene in place of the ketone. The former were often
prepared en route to the R-ketoheterocycles and simply entailed
examination of this alcohol intermediate. The candidate 1,3,4-
oxadiazole inhibitors bearing a methylene were prepared by
simple dehydrative ring closure of the corresponding diacyl-
hydrazide as the key step (Scheme 8). Consistent with a
mechanism of reversible Ser addition to the electrophilic
carbonyl forming a hemiketal at the enzyme active site, the
corresponding alcohol and methylene inhibitors were found to
electrophilic carbonyl. However, simple methylation of 15a
rendered the isomeric 2- and 1-methyltetrazole derivatives 19a
and 19b, respectively, incapable of this deprotonation and which
now displayed FAAH inhibition at levels consistent with this
series (Figure 6). Notably, the isomer assignments for 16-19
were made by HMBC (long-range proton-carbon correlation)
NMR analysis.
An additional smaller series was examined in which a methyl
ester substituent was placed directly on the heterocycle (Figure
7). Such candidate FAAH inhibitors in the oxazole-based series
typically display activity reflecting the strength of the conjugated
electron-withdrawing substituent55,58 but lack the intrinsic
enzyme selectivity observed with 2 and related inhibitors.58 With
the exception of the acidic triazole 14h, which was inactive
presumably because of its deprotonation under the assay
conditions, this substitution with 8h, 10h, and 12h provided
potent FAAH inhibitors (Ki < 10 nM). However and unlike
the behavior of the corresponding oxazole,56 in each case the
activity approached but did not exceed that of the unsubstituted
heterocycles (8a, 10a, and 12a).
A final series of inhibitors incorporating a substituted pyridine
substituent on the central heterocycle was examined and
constituted potential synthetic intermediates en route to 8f-13f
(Figure 8). Each displayed activity that approached the unsub-
stituted pyridine derivative (b series in Figure 5) or, in the case
of 24 and 26, the corresponding methyl esters 9f and 13f.
Biphenylethyl-Based Inhibitors. After having established
that several central heterocycle replacements further improve
on the potency of the oxazole-based inhibitors and that its trends
in the substituent effects carry forward onto these additional
Figure 9. Biphenylethyl-based inhibitors.

4398 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Garfunkle et al.
Scheme 9
Figure 11. ꢀ-Keto-1,3,4-oxadiazoles.
be g10000-fold less active than the corresponding ketone,
Figure 10. Although not investigated in detail for the inhibitors
disclosed herein, even this level of reduced inhibition (0.01%
activity) is most likely attributable to contaminant ketone in
the samples of the alcohol and methylene compounds that can
arise from air oxidation upon storage or even while undergoing
the assay.56 Important in these comparisons is the fact that the
ketone is essential to the potent activity of the inhibitors and
that reduction to an alcohol or removal altogether leads to g104
reductions in activity.
The results of an additional and unique series that were
prepared (Scheme 9) and examined are summarized in Figure
11. These constitute ketone inhibitors with a methylene inserted
between the reactive carbonyl and heterocycle in efforts to
establish whether the inductive electron-withdrawing properties
of the electron-deficient heterocycle would be sufficient to
activate the carbonyl for active site hemiketal formation. Unlike
the potent activity of the R-ketoheterocycles, these ꢀ-ketohet-
Figure 12. Inhibition of recombinant human fatty acid amide
hydrolase.
erocycles and their corresponding alcohols were found to be
inactive against FAAH.
Inhibition of Recombinant Human FAAH. Rat and human
FAAH are very homologous (82% sequence identity),2 exhibit
near identical substrate selectivity and inhibitor sensitivity in
our studies disclosed to date 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
examined against the human enzyme and were found to exhibit
the same relative and absolute potencies consistent with previous
observations (Figure 12).
Selectivity. Early assessments of R-ketoheterocycle inhibitors
of FAAH against possible competitive enzymes (e.g., phospho-
lipase A2, ceramidase) revealed no inhibition. Consequently, a
method for proteome-wide screening capable of globally profil-
ing all mammalian serine hydrolases was developed,31,73 and
studies have shown that the R-ketoheterocycle class of inhibitors
can be exquisitely selective for FAAH. However, two enzymes
did emerge as potential competitive targets: triacylglycerol
hydrolase (TGH) and a previously uncharacterized membrane-
associated hydrolase that lacked known substrates or function
(KIAA1363).74 In this screen, IC50 values are typically higher
than the measured Ki values, but the relative potency, the
magnitude of binding affinity differences, and the rank order
binding determined in the assay parallel those established by
standard substrate assays.
Summarized in Figure 13 are the results of the selectivity
screening of selected candidate inhibitors. In general, the
inhibitors were selective for FAAH over TGH and KIAA1363.
The trends within the series examined are very clear and mirror
the observations disclosed in our preceding studies.53,54,56–58
Thus, the inhibitors bearing the unsubstituted heterocycles
(7-13a) were selective for FAAH over KIAA1363 but were
typically more selective for TGH over FAAH (1-125-fold).
The potent inhibitors incorporating even the small methyl ester
substituent (7-12h series) on the heterocycle were found to be
selective for FAAH versus KIAA1363 (>100-fold) and now
Figure 10. Effect of the electrophilic carbonyl.

Central Heterocycle of R-Ketoheterocycle Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4399
Figure 13. Activity based protein profiling of R-ketoheterocycles.
modestly selective for FAAH versus TGH (2-200-fold). In line
with prior observations, the addition of a 2-pyridyl substituent
(7-15b series) increased not only the FAAH potency but also
FAAH selectivity such that the most potent inhibitors failed to
inhibit KIAA1363 (>104-fold selective) and are now typically
4-300-fold selective for FAAH versus TGH. Addition of a C6-
carboxylic acid to the 2-pyridyl substituent (7-15g series)
further enhanced this intrinsic selectivity such that the resulting
inhibitors are typically no longer viable competitive inhibitors
of KIAA1363 or TGH (Figures 13 and 14). Notably, many of
the central heterocycle features and added substituents that were
found to increase FAAH potency were also found to enhance
FAAH selectivity by simultaneously disrupting KIAA1363 and
TGH affinity.
potent (Ki ) 300 pM) inhibitors of fatty acid amide hydrolase.
The nature of the central heterocycle, even with the modest and
systematic perturbations explored herein, significantly influenced
the inhibitor activity that can be defined as 1,3,4-oxadiazoles
(50) and 1,2,4-oxadiazoles 9 (50) > tetrazoles (5), the isomeric
1,2,4-oxadiazoles 10 (7), 1,3,4-thiadiazoles (2-30) > oxazoles
including 2 (rel activity ) 1) > 1,2-diazines (0.3) > thiazoles
(0.06) > 1,3,4-triazoles (-). Most evident in the observations
is that the introduction of an additional position 4 heteroatom
(oxazole numbering) substantially increases the activity (N >
O > CH) that may be attributed in part to a reduced destabilizing
steric interaction at the FAAH active site. Additionally the
position 1 and 3 requisite heteroatoms exhibit a N,O > N,N >
N,S preference that is reflected in the observed heterocycle
trends. Within each heterocycle series, the impact of an added
substituent was systematically explored and was found to follow
well defined trends first observed with 2. Exemplifying these
effects, additional aryl substituents (e.g., 2-pyr or 2-pyr-6-CO2H)
placed on the central heterocycle increase FAAH potency and
Conclusions
Herein, we report the synthesis and evaluation of a key series
of R-ketoheterocycles based on 2 incorporating systematic
changes in the central heterocycle that provided extraordinarily

4400 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Garfunkle et al.
significantly modify physical properties (e.g., solubility) in a
manner that may impact PK and PD behavior of the inhibitors.
Just as significantly, the nature of the substituent substantially
impacts the selectivity of the FAAH inhibitors, and these trends
proved general across the range of inhibitors examined to
date.53–58 This is especially evident with the TGH selectivity
for the unsubstituted heterocycles (7-13a series, 1-100-fold
selective for TGH vs FAAH), which can be improved by the
independent choice of the C2 side chain (compare 2)58 as well
as the heterocycle substituent (compare 7-13a vs 7-15b and
7-15g). A combination of these independent features which
simultaneously improve FAAH potency and disrupt TGH or
KIAA1363 binding provide exquisitely selective and potent
FAAH inhibitors.
Figure 14. Activity based protein profiling of FAAH inhibitors 10b,
10f, 10g, 10a with FP-Rh in brain and heart membrane proteome.
Enzyme targets such as FAAH, KIAA1363, and TGH are highlighted.
0.006 mmol) in DMF (200 µL) was purged with Ar and sealed.
The reaction mixture was warmed at 100 °C for 18 h before it was
cooled to room temperature, diluted with EtOAc, and washed with
H2O, 9:1 NH4OH:saturated aqueous NH4Cl, and saturated aqueous
NaCl. The organic layer was dried over Na2SO4, and the solvent
was removed under reduced pressure to afford the crude material
that was purified by flash chromatography (SiO2, 1.5 × 15 cm,
5-10% acetone-hexanes) to afford 2-(5-(1-(tert-butyldimethylsi-
lyloxy)-7-phenylheptyl)-2H-tetrazol-2-yl)-pyridine (4.0 mg, 21%)
Experimental Section
7-Phenyl-1-(2-(pyridin-2-yl)-2H-tetrazol-5-yl)-heptan-1-one
(15b). A solution of 7-phenylheptanal (1.93 g, 0.01 mol) in MeCN
(64 mL) was treated with KCN (2.65 g, 0.04 mol), ZnI2 (77.7 mg,
0.0002 mol), and TBSCl (3.06 g, 0.02 mol) under Ar at room
temperature. The reaction mixture was stirred vigorously and the
progress monitored by TLC (SiO2, 5% EtOAc-hexanes). After
72 h, the solvent was removed in vacuo and the residue resuspended
in Et2O. The salts were removed by filtration and rinsed thoroughly
with Et2O. The filtrate was washed with water, dried over Na2SO4,
and concentrated in vacuo to give a yellow oil that was purified by
flash chromatography (SiO2, 4 × 25 cm, 1% EtOAc-hexanes) to
afford 2-(tert-butyldimethylsilyloxy)-8-phenyloctanitrile (2.6 g,
1
as a colorless oil. H NMR (CDCl3, 400 MHz): δ 8.69 (dd, 1H, J
) 0.9, 4.8 Hz), 8.16 (d, 1H, J ) 7.5 Hz), 7.98 (dt, 1H, J ) 1.8, 7.9
Hz), 7.48 (m, 1H), 7.26 (m, 2H), 7.16 (m, 3H), 5.16 (dd, 1H, J )
5.9, 7.4 Hz), 2.58 (t, 2H, J ) 7.7 Hz), 1.99 (m, 2H), 1.60 (m, 2H),
1.34 (m, 6H), 0.88 (s, 9H), 0.10 (s, 3H), -0.02 (s, 3H). 13C NMR
(CDCl3, 150 MHz): δ 169.7, 149.6, 149.0, 142.9, 139.5, 128.5,
128.4, 125.7, 125.0, 115.2, 67.4, 37.5, 36.1, 31.5, 29.3, 25.9 (3C),
25.4, 18.4, -4.1, -4.8. HRMS-ESI-TOF m/z 452.2823 ([M + H]+,
C25H37N5OSi requires 452.2840).
1
80%) as a colorless oil. H NMR (CDCl3, 500 MHz): δ 7.30 (m,
2H), 7.20 (m, 3H), 4.43 (t, 1H, J ) 6.4 Hz), 2.63 (t, 2H, J ) 7.7
Hz), 1.80 (m, 2H), 1.65 (m, 2H), 1.49 (m, 2H), 1.39 (m, 4H), 0.94
(s, 9H), 0.21 (s, 3H), 0.16 (s, 3H). 13C NMR (CDCl3, 125 MHz):
δ 142.7, 128.5, 128.4, 125.7, 120.2, 62.0, 36.4, 36.0, 31.4, 29.1,
28.9, 25.6, 24.6, 18.2 (3C), -5.0, -5.2. HRMS-ESI-TOF m/z
354.2221 ([M + Na]+, C20H33NOSi requires 354.2223).
2-(5-(1-(tert-Butyldimethylsilyloxy)-7-phenylheptyl)-2H-tetrazol-
2-yl)-pyridine (4.4 mg, 0.009 mmol) was dissolved in THF (122
µL), treated with Bu4NF (1 M in THF, 0.013 mL, 0.013 mmol),
and stirred at room temperature for 2 h under Ar. The reaction
mixture was diluted with EtOAc, washed with saturated aqueous
NaCl, and dried over Na2SO4. Evaporation in vacuo yielded the
crude alcohol that was purified by flash chromatography (SiO2, 0.5
× 4 cm, 20-50% EtOAc-hexanes) to afford 7-phenyl-1-(2-
(pyridin-2-yl)-2H-tetrazol-5-yl)-heptan-1-ol (2.7 mg, 79%) as a
white solid. 1H NMR (CDCl3, 600 MHz): δ 8.69 (dd, 1H, J ) 1.1,
4.7 Hz), 8.18 (d, 1H, J ) 8.1 Hz), 8.00 (m, 1H), 7.50 (ddd, 1H, J
) 0.7, 4.8, 7.4 Hz), 7.26 (m, 2H), 7.16 (m, 3H), 5.16 (m, 1H),
2.59 (t, 2H, J ) 7.7 Hz), 2.48 (d, 1H (-OH), J ) 6.1 Hz), 2.07
(m, 2H), 1.61 (m, 2H), 1.52 (m, 2H), 1.40 (m, 4H). 13C NMR
(CDCl3, 150 MHz): δ 169.5, 149.6, 148.8, 142.9, 139.6, 128.5,
128.4, 125.7, 125.3, 115.3, 67.0, 36.7, 36.1, 31.5, 29.3 (2C), 25.2.
HRMS-ESI-TOF m/z 338.1966 ([M + H]+, C19H23N5O requires
338.1975).
A sample of 2-(tert-butyldimethylsilyloxy)-8-phenyloctanitrile
(335 mg, 1.01 mmol) was dissolved in a mixture of 2-propanol
(1.4 mL):water (2.9 mL). NaN3 (197 mg, 3.04 mmol) and ZnBr
(250 mg, 1.11 mmol) were added to the reaction mixture as solids,
which was subsequently warmed at 100 °C for 90 h. Upon
disappearance of starting material, the solution was cooled to room
temperature and diluted with EtOAc. Then 2 N HCl was added to
the reaction mixture, which was stirred until all solids dissolved.
The organic layer was isolated, and the aqueous layer was washed
several times with EtOAc. The combined organic phases were dried
over Na2SO4, and the solvent was removed under reduced pressure
to afford 7-phenyl-1-(2H-tetrazol-5-yl)-heptan-1-ol as a colorless
1
oil that was used without further purification (270 mg, quant). H
NMR (CDCl3, 600 MHz): δ 7.25 (m, 2H), 7.15 (m, 3H), 5.26 (m,
1H), 2.57 (t, 2H, J ) 7.7 Hz), 2.02 (m, 1H), 1.90 (m, 1H), 1.59
(m, 2H), 1.44 (m, 2H), 1.34 (m, 4H). HRMS-ESI-TOF m/z 261.1707
([M + H]+, C14H20N4O requires 261.1710).
7-Phenyl-1-(2-(pyridin-2-yl)-2H-tetrazol-5-yl)-heptan-1-ol (2.7
mg, 0.007 mmol) was dissolved in CH2Cl2 (0.24 mL), and
Dess-Martin periodinane (4.5 mg, 0.011 mmol) was added. The
mixture was stirred at room temperature for 2 h before the reaction
mixture was reduced to half-volume, and this mixture was directly
loaded onto silica gel and purified by flash chromatography (SiO2,
0.5 × 4 cm, 10-30% EtOAc-hexanes) to afford 7-phenyl-1-(2-
(pyridin-2-yl)-2H-tetrazol-5-yl)-heptan-1-one (15b, 2.7 mg, 99%)
A solution of 7-phenyl-1-(2H-tetrazol-5-yl)-heptan-1-ol (36 mg,
0.14 mmol), TBSCl (63 mg, 0.42 mmol), and imidazole (28 mg,
0.42 mmol) in DMF (0.7 mL) was stirred at room temperature for
72 h before it was diluted with EtOAc and washed with H2O and
saturated aqueous NaCl. The organic layer was dried over Na2SO4,
and the solvent was removed under reduced pressure to afford the
crude material that was purified by flash chromatography (SiO2,
1.5 × 15 cm, 10% acetone-hexanes) to afford 5-(1-(tert-butyldim-
ethylsilyloxy)-7-phenylheptyl)-2H-tetrazole (39 mg, 75%) as a
1
as a white solid. H NMR (CDCl3, 600 MHz): δ 8.74 (m, 1H),
8.23 (m, 1H), 8.05 (dt, 1H, J ) 1.6, 7.9 Hz), 7.57 (dd, 1H, J )
4.8, 7.5 Hz), 7.27 (t, 2H, J ) 7.6 Hz), 7.17 (m, 3H), 3.26 (t, 2H,
J ) 7.4 Hz), 2.61 (t, 2H, J ) 7.7 Hz), 1.83 (m, 2H), 1.64 (m, 2H),
1.43 (m, 4H). 13C NMR (CDCl3, 150 MHz): δ 191.5, 162.5, 149.9,
148.6, 142.8, 139.8, 128.5, 128.4, 126.0, 125.8, 115.9, 41.0, 36.0,
31.4, 29.1, 29.0, 23.5. HRMS-ESI-TOF m/z 336.1813 ([M + H]+,
C19H21N5O requires 336.1819). Purity 99%.
1
colorless oil. H NMR (CDCl3, 400 MHz): δ 7.26 (dd, 2H, J )
6.3, 8.4 Hz), 7.16 (m, 3H), 5.25 (t, 1H, J ) 5.8 Hz), 2.57 (t, 2H,
J ) 7.7 Hz), 1.85 (m, 2H), 1.58 (m, 2H), 1.31 (m, 6H), 0.90 (s,
9H), 0.13 (s, 3H), 0.01 (s, 3H).
In a gas-tight vessel, a solution of 5-(1-(tert-butyldimethylsily-
loxy)-7-phenylheptyl)-2H-tetrazole (16 mg, 0.043 mmol), 2-io-
dopyridine (7 µL, 0.064 mmol), CuI (1 mg, 0.004 mmol), K2CO3
(12 mg, 0.085 mmol), and N,N-dimethylethylene diamine (1 µL,
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.71 The purified

Central Heterocycle of R-Ketoheterocycle Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4401
phospholipids and Their Derivatives. Prog. Lipid Res. 1990, 29, 1–
43.
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,2 and the lysate of hFAAH-
transfected COS-7 cells was used in the inhibition assays where
explicitly indicated.
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The inhibition assays were performed as described.13 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 temperature 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.13 The Ki of the inhibitor
was calculated using a Dixon plot as described (standard deviations
are provided in the Supporting Information tables). Lineweaver-Burk
analysis was performed as described confirming competitive,
reversible inhibition.53
Selectivity Screening. The selectivity screening was conducted
as detailed.31
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (DA15648) and the
Skaggs Institute for Chemical Biology. We wish to acknowledge
Dr. Wu Du and his preparation of 4b, 4e, 8b, 8c, 8e (ref 54),
Dr. Robert Fecik for the preparation of 4a and 5a, and Dr. F.
Anthony Romero for the preparation of 6. We are especially
grateful to Professor B. F. Cravatt for the supply of rat and
recombinant human FAAH and to H. S. Hoover in the Cravatt
laboratories for the training and guidance in conducting the
selectivity assay. J.G. is a Skaggs and ARCS Fellow.
Supporting Information Available: Full experimental details
and characterization of the candidate inhibitors, FAAH assay
measurement errors, and purities of the FAAH inhibitors disclosed.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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