Reversible competitive α-ketoheterocycle inhibitors of fatty acid amide hydrolase containing additional conformational constraints in the acyl side chain: Orally active, long-acting analgesics

ARTICLE

pubs.acs.org/jmc

Reversible Competitive r-Ketoheterocycle Inhibitors of Fatty

Acid Amide Hydrolase Containing Additional Conformational

Constraints in the Acyl Side Chain: Orally Active, Long-Acting

Analgesics

Cyrine Ezzili,^†Mauro Mileni,^‡Nicholas McGlinchey,^§Jonathan Z. Long,^||Steven G. Kinsey,^{^}

Dustin G. Hochstatter,^†Raymond C. Stevens,^‡,#Aron H. Lichtman,^{^}Benjamin F. Cravatt,^||,#

Edward J. Bilsky,^§and Dale L. Boger*^,†,#

^†Departments of Chemistry, ^‡Molecular Biology, Chemical Physiology, and ^#The Skaggs Institute for Chemical Biology,

The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States

^§Department of Pharmacology, College of Osteopathic Medicine, University of New England, Biddeford, Maine 04005, United States

^{^}Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, United States

S Supporting Information

b

ABSTRACT: A series of R-ketooxazoles containing conformational constraints in the C2

acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase

(FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs

R enantiomer), and their potency is comparable or improved relative to 2, indicating that the

conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure

of the R-ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding

details, conﬁrmed that the (S)-enantiomer is the bound active inhibitor, shed light on the

origin of the enantiomeric selectivity, and conﬁrmed that the catalytic Ser241 is covalently

bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo

characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain

anandamide levels following either intraperitoneal (ip) or oral (po) administration indicative

of eﬀective in vivo FAAH inhibition. Signiﬁcantly, the oral administration of 12 caused dramatic accumulation of anandamide in the

brain, with peak levels achieved between 1.5 and 3 h, and these elevations were maintained over 9 h. Additional studies of these two

representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the

demonstration that 12 administered orally signiﬁcantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained

periods consistent with its long-acting eﬀects in raising the endogenous concentration of anandamide.

’ INTRODUCTION

ketone (e.g., triﬂuoromethyl ketone-based inhibitors)^28ꢀ31and

irreversible inhibitors^32ꢀ37(e.g., ﬂuorophosphonates and sulfonyl

ﬂuorides) that were used to characterize the enzyme as a serine

hydrolase. Subsequent studies have deﬁned two major classes of

inhibitors that provide opportunities for the development of

inhibitors with greater therapeutic potential. One class is the

reactive aryl carbamates and ureas^38ꢀ50that irreversibly acylate a

FAAH active site serine⁴⁹and that have been shown to exhibit

Fatty acid amide hydrolase (FAAH)^1,2serves as the catabolic

regulator of several endogenous lipid amides^3ꢀ6including ana-

ndamide (1a)^7ꢀ10and oleamide (1b),^11ꢀ13Figure 1. Its dis-

tribution is consistent with its role in hydrolyzing and regulating

such signaling fatty acid amides⁶at their sites of action.³Although

it is a member of the amidase signature family of serine hydro-

lases for which there are a number of prokaryotic enzymes, it is

the only well-characterized mammalian enzyme bearing the

family’s unusual SerꢀSerꢀLys catalytic triad.^14ꢀ17

anxiolytic activity³⁸and produce antinociceptive eﬀects.³⁹

A

second class is the R-ketoheterocycle-based inhibitors^51ꢀ60that

bindto FAAH by reversible hemiketalformation with an active site

serine. Many of these latter reversible competitive inhibitors have

been shown to be potent and selective for FAAH versus other

mammalian serine hydrolases, and members of thisclass have been

shown to be eﬃcacious in preclinical animal models of pain.⁶¹

Due to the therapeutic potential of inhibiting FAAH^18ꢀ20for

the treatment of pain,^21ꢀ23inﬂammation,²⁴or sleep disorders,^13,25

there has been growing interest in the development of selective

inhibitors of the enzyme.²⁶Early studies following the initial

characterization of the enzyme led to the discovery that the

endogenous sleep-inducing molecule 2-octyl R-bromoacetoace-

tate is an eﬀective FAAH inhibitor²⁷and the disclosure of a series

of nonselective reversible inhibitors bearing an electrophilic

Received: December 16, 2010

Published: March 23, 2011

r

2011 American Chemical Society

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Figure 1. Substrates of FAAH.

In these latter studies, 2 (OL-135)⁵³emerged as an important

lead inhibitor for further study. It is a potent (K_i= 4.7 nM)⁵³and

selective (>60ꢀ300-fold)³¹FAAH inhibitor that induces analge-

sia and increases endogenous anandamide levels.⁶¹It exhibits

antinociceptive and anti-inﬂammatory activity in a range of

preclinical animal models that include the tail ﬂick assay, hot

plate assay, formalin test of noxious chemical pain (ﬁrst and

second phase), the mild thermal injury (MTI) model of periph-

eral pain, the spinal nerve ligation (SNL) and chronic constriction

injury (CCI) models of neuropathic pain, and an inﬂammatory

model of pruritus with eﬃcacies that match or exceed those of

morphine (1ꢀ3 mg/kg in MTI/SNL), ibuprofen (100 mg/kg in

MTI), or gabapentin (500 mg/kg in SNL) and at administered

doses (10ꢀ20 mg/kg, ip) that approach or are below those of

such common pain or anti-inﬂammatory medications.⁶¹The

compound lacks signiﬁcant oﬀsite target activity (Cerep assay

proﬁling), does not bind cannabinoid (CB₁or CB₂) or vanilloid

receptors, and does not signiﬁcantly inhibit common P450

metabolism enzymes or the human ether-a-go-go related gene

(hERG). The observed antinociceptive eﬀects are not asso-

ciated with concomitant respiratory depression or tolerance/

dependence characteristic of opioid administration, nor with

increased feeding or decreased locomotor coordination fre-

quently observed with CB agonists.⁶¹Signiﬁcantly, 2 does not

produce antinociception in FAAH knockout mice,⁶¹establish-

ing that FAAH is the relevant target responsible for the

observed in vivo eﬀects.

Consequently, we have conducted a series of systematic

structureꢀactivity relationship (SAR) studies on 2 exploring

the 4- and 5-position of the central oxazole,^53,55,56,60the C2 acyl

side chain,^53,57,58and the central heterocycle,^54,59each of which

was found to independently impact inhibitor potency or selec-

tivity, Figure 2.⁶²Herein, we report results of studies examining

candidate inhibitors containing further conformational con-

straints in the C2 acyl side chain of 2 and related inhibitors,

the X-ray crystal structure characterization of a prototypical

inhibitor in the series bound to the enzyme, and preliminary

in vivo characterization of two representative inhibitors in this

series.

Figure 2. Progression of the inhibitor series.

additional conformational restriction in the C2 acyl side chain is

shown in Scheme 1. The inhibitors contain a tetrahydronaphtha-

lene or indane central to the C2 acyl chain, a newly introduced

chiral center adjacent to the electrophilic carbonyl, and pendant

terminal aryl groups that were varied in our studies. Expectations

are that the introduction of further conformational restraints in the

C2 acyl chain would improve oral bioavailability of the candidate

inhibitors, potentially maintain or enhance FAAH inhibitory

activity and selectivity, and sterically further reduce potential

competitive metabolic or oﬀ-target reactivity of the electrophilic

carbonyl.

Introduction of a methyl ester R to the ketone of the

commercially available 6-methoxytetralone or 6-methoxyinda-

none proceeded as reported using dimethylcarbonate and NaH,

and was followed by hydrogenation removal of the cyclic ketone

using H₂and Pd/C.⁶³Simultaneous deprotection of the aryl

methyl ether and the methyl ester with aqueous HBr in HOAc

yielded the phenolic carboxylic acid. Esteriﬁcation of the car-

boxylic acid using H₂SO₄and MeOH aﬀorded the advanced

phenol intermediates^63,64on which the varied aryl substituent

was added. A Suzuki coupling with phenylboronic acid via the

corresponding triﬂate intermediate, a Mitsunobu alkylation⁶⁵of

the phenol with benzyl alcohol and Ph₃P-diethyl azodicarbox-

ylate (DEAD), and a modiﬁed Ullmann reaction⁶⁶of the phenol

with phenylboronic acid yielded the corresponding 6-phenyl,

6-benzyloxy, and 6-phenoxy-1,2,3,4-tetrahydronapthalenes and

indanes, respectively. Reduction of the methyl ester to the

primary alcohol using LiAlH₄followed by oxidation with

DessꢀMartin periodinane⁶⁷gave the corresponding alde-

hyde. Vedejs oxazole metalation⁶⁸and condensation with

the various C2 side chain aldehydes was followed by tert-

butyldimethylsilyl (TBS) ether protection of the resulting

alcohols. Selective C5-oxazole lithiation⁶⁹of these inter-

mediates followed by treatment with Bu₃SnCl aﬀorded the

corresponding C5 tributylstannanes. Stille coupling⁷⁰of the

’ CHEMISTRY

The general method for the synthesis of the oxazole-based

inhibitors bearing a C5 aryl substituent and containing the

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Scheme 1

Scheme 2

Figure 3. Prior constraints in the C2 acyl side chain and impact of the

terminal aryl substituent.⁵⁷

stannane intermediates with pyridine halides produced the

C5-substituted oxazoles, which could be readily converted to

the corresponding ketones by TBS ether deprotection

(Bu₄NF) and oxidation of the liberated alcohol with

DessꢀMartin periodinane. These candidate inhibitors were

separated into their two enantiomers by resolution on a

semipreparative Chiracel OD or AD column. The candidate

inhibitors containing a methyl ester were then converted to

their corresponding carboxylic acid using (CH₃)₃SnOH.⁷¹

This reagent and the conditions employed resulted in minimal

racemization of the chiral center whereas the conventional use

of LiOH (1 equiv, THF/H₂O 3:2, 25 ꢀC) resulted in more

extensive racemization.

The synthesis of candidate inhibitors that bear a nonaro-

matic oxazole C5-substituent is summarized in Scheme 2.

Following oxazole C5-lithiation, treatment with Mander’s

reagent (NCCO₂Me) provided the corresponding C5-substi-

tuted oxazoles bearing a methoxycarbonyl group in good

conversions. In each case, deprotection of the TBS ether

followed by DessꢀMartin periodinane oxidation of the liber-

ated alcohol yielded the corresponding R-ketooxazole. The

methyl esters were also converted to the corresponding

carboxamides by treatment with NH₃ꢀCH₃OH and the

carboxamides were dehydrated with triﬂuoroacetic anhydride

(TFAA) and pyridine to provide the C5 nitriles that were

converted to the R-ketooxazoles as well. These derivatives

were separated into their two enantiomers by resolution on a

semipreparative Chiracel OD or AD column.

’ ENZYME ASSAY

Enzyme assays were performed at 20ꢀ23 ꢀC with puriﬁed

recombinant rat FAAH expressed in Escherichia coli⁷²or 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 (e10ꢀ20% reaction) were monitored using

enzyme concentrations (typically 1 nM) at least three times

below the measured K_iby following the breakdown of ¹⁴C-

oleamide, and K_ivalues (standard deviations are provided in the

Supporting Information tables) were established as described

(Dixon plot).²⁹LineweaverꢀBurk analysis of 12 and 14 estab-

lished that they behave as reversible, competitive inhibitors

analogous to 2⁵³and related inhibitors (see Experimental

Section).

’ RESULTS AND DISCUSSION

In early work, some of the most potent inhibitors that emerged

contained conformational constraints in the ﬂexible C2 acyl side

chain of 2 removing many of its rotatable bonds and introducing

additional π-unsaturation. This portion of the inhibitors binds in

a hydrophobic channel of the FAAH active site reserved for the

unsaturated lipid chain of the fatty acid amide substrates.

Additional restriction in this C2 acyl side chain may contribute

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Figure 4. FAAH inhibitors with 1,2,3,4-tetrahydronaphthalene C2 acyl side chain, K_i(nM).

to an enhancement in binding aﬃnity while simultaneously

improving the drug-like characteristics of the candidate inhibi-

tors. Three improved C2 side chains previously identiﬁed⁵⁷

(Figure 3) were now incorporated into the candidate inhibitors

that contain a 1,2,3,4-tetrahydronaphthalene or indane core and

were combined with representative or optimized C5 oxazole

substituents that were found to impact inhibitor potency, selec-

tivity, and physical properties.

Scheme 3

Tetrahydronaphthalene Series. The series examined in-

clude the 6-phenyl, 6-phenoxy, and 6-benzyloxy-1,2,3,4-tetrahy-

dronaphthalene C2 acyl side chains combined with a set of

representative oxazole C5-substituents. For each derivative, the

racemic mixture and the pure enantiomers were prepared and

examined, but only the results of the examination of the

individual enantiomers are reported in Figure 4. In each instance,

it was the slower eluting second enantiomer obtained from

chromatographic resolution (Chiralcel OD or AD) that was

found to be more potent, and as detailed later, it was established

to be the (S)-enantiomer. Thus, the inhibitors displayed a

consistently more potent activity for the assigned (S)-enantio-

mer that was of a magnitude and range (10ꢀ400-fold, avg = 70-

fold) that suggests the observed activity for the less active (R)-

enantiomer is not distinguishable from that potentially derived

from contaminant (S)-enantiomer in the assayed samples de-

rived directly from the chromatographic resolutions. At this level

of enantiomeric purity achieved by the chromatographic resolu-

tions, we can confidently state that the reported activity of the

active (S)-enantiomers is accurate and unaffected by any trace

amounts of contaminate (R)-enantiomer (g98% ee), whereas

that of the less interesting (less active) or inactive (R)-enantio-

mer is not easy to distinguish from the potential presence of

contaminant (S)-enantiomer. Moreover and from these and later

studies (see Scheme 3), we can establish that these samples do

not significantly epimerize under the conditions of the FAAH

assay (pH = 9). For each oxazole C5-substituent, the potency of

the (S)-enantiomers of the C2 acyl side chain aryl substituents

consistently followed the order phenoxy > benzyloxy > phenyl

indicating that the added conformational constraints in the

C2 acyl linking chain have subtly reordered this aryl

substituent preference (compare Figure 3). Here the distinctions

between a phenoxy and benzyloxy substituent are small (typically

1.4ꢀ4-fold), whereas the differences with the less active and

more rigid phenyl substituent are larger and more easily distin-

guished. Interestingly and unlike observations made with 2 itself,

the impact of the oxazole C5-substituent on the activity in each

series is much more modest, although it is most significant in the

less active biaryl series. However, the trends for the oxazole C5-

substituent, but not their magnitude, are maintained in these

series, and most significant is the enhanced potency observed

with inhibitors that lack the C5 substituent. This suggests that

beneficial enhancements in binding affinity in this series are

gained by the C2 acyl side conformational restriction and the

added hydrophobic interactions of the tetrahydronaphthalene.

In order to unambiguously establish the absolute stereochem-

ical assignment for the active enantiomer, an inhibitor in the

6-benzyloxy-1,2,3,4-tetrahydronaphthalene series was prepared

with an iodo substituent at the oxazole C5 position following the

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Figure 5. FAAH inhibitors with indane C2 acyl side chain, K_i(nM).

general procedure described earlier, Scheme 3. The racemic

mixture and the pure enantiomers were tested for FAAH

inhibition, and one enantiomer was found to be ca. 200-fold

more active. Its structure and absolute stereochemistry were

established with an X-ray crystal structure determination⁷³

indicating that the most potent enantiomer is the (S)-enantio-

mer, Scheme 3. With 21, the activity of the less potent (R)-

enantiomer cannot be distinguished from potential contaminate

(S)-enantiomer (0.5%) in the assay sample, and this comparison

indicates that the active (S)-enantiomers may be g200-fold

more active than the corresponding (R)-enantiomers.

Additional Derivatives. A set of additional derivatives in the

6-phenoxy-1,2,3,4-tetrahydronaphthalene series were examined

that incorporate a 1,3,4-oxadiazole as the central activating

heterocycle. In earlier studies, comprehensive systematic changes

in the central activating heterocycle of 2 were examined and

found to significantly influence the inhibitor activity^54,59with the

1,3,4-oxadiazole derivatives providing extraordinarily potent in-

hibitors. Accordingly, the preparation of a representative small

series of 1,3,4-oxadiazoles containing the 6-phenoxy-1,2,3,4-

tetrahydronaphthalene side chain was conducted and is illu-

strated in Scheme 4. Reaction of the C2 side chain aldehyde with

KCN afforded the corresponding cyanohydrin and was followed

by conversion of the nitrile to the methyl ester. TBS protection of

the alcohol followed by saponification of the methyl ester yielded

the carboxylic acid, which was condensed with a series of

hydrazides in a reaction promoted by 1-[3-(dimethylamino)

propyl]-3-ethylcarbodiimide hydrochloride (EDCI) to provide

the diacyl hydrazide intermediates. These intermediates were

cyclized to the corresponding 1,3,4-oxadiazoles upon treatment

with p-toluenesulfonyl chloride (TsCl) and Et₃N. The desired R-

keto-1,3,4-oxadiazoles were obtained after TBS ether deprotec-

tion (Bu₄NF or TASF) and oxidation of the liberated alcohol

with DessꢀMartin periodinane. These derivatives were sepa-

rated into their two enantiomers by resolution on a semipre-

parative Chiracel OD column.

Finally and for comparison purposes, a series of derivatives

were prepared and examined that lack an aryl substituent on the

acyl side chain 1,2,3,4-tetrahydronaphthalene or indane core.

Their synthesis entailed Vedejs C2-lithiation of oxazole followed

by condensation of the corresponding aldehyde and TBS protec-

tion of the resulting alcohol. Selective oxazole C5-lithiation (n-

BuLi) followed by treatment with Bu₃SnCl aﬀorded the corre-

sponding tributylstannane intermediates. Subsequent Stille cou-

pling with 2-bromopyridine produced the C5-substituted

oxazoles, which were converted to the corresponding ketones

by TBS ether deprotection (Bu₄NF) and oxidation of the

liberated alcohols using DessꢀMartin periodinane, Scheme 5.

The 1,2,3,4-tetrahydronaphthalene derivatives were separated

into their two enantiomers by resolution on a semipreparative

Chiracel OD column. The indane derivatives are meso com-

pounds, and no resolution is required.

Indane Series. An analogous, but smaller series of related

inhibitors containing the 5-phenyl, 5-phenoxy, and 5-benzylox-

yindane acyl chains were examined, Figure 5.

Thisseries exhibited an analogous enantiomeric selectivity with

the tentatively assigned (S)-enantiomers being on average 60-fold

(10ꢀ320-fold, avg = 60-fold) more potent than the correspond-

ing (R)-enantiomers, and they approached or matched the

potency of the corresponding inhibitors bearing the 1,2,3,4-

tetrahydronaphthalene C2 acyl chain core. Although the number

of comparisons is more limited in this series, the expected

signiﬁcant improvement in potency with the introduction of the

oxazole C5 pyridyl substitutent (avg 9-fold enhancement) is

observed, and there appears to be less of a distinction between

choices of acyl side chain aryl substituent in this series. Interest-

ingly, and again with the caveat that the number of comparisons is

limited, this latter observation results from small improvements in

the potency of the inhibitors bearing the acyl chain C5 phenyl and

benzyloxy substituents, whereas those bearing the C5 phenoxy

substituent appear to be reduced relative to the corresponding

inhibitors bearing a 1,2,3,4-tetrahydronaphthalene C2 acyl chain

core. Since all are potent inhibitors of FAAH, this suggests that

there are minor reorientation diﬀerences in the binding of the C2

acyl side chains in the two series (tetrahydronaphthalene vs

indane) that may subtly impact the inhibitor aﬃnity.

The Electrophilic Carbonyl. In several instances, the diaster-

eomeric mixture of racemic alcohols used as the penultimate

precursors to the R-ketoheterocycles 15, 19, 30, and 32 were also

assessed for FAAH inhibition, and all were found to be inactive

(K_i> 10 μM) confirming the importance of the electrophilic

carbonyl.

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Figure 6. Additional FAAH inhibitors, K_i(nM).

Scheme 4

Scheme 5

The results of the examination of these derivatives are

summarized in Figure 6. The incorporation of the activating

1,3,4-oxadiazole heterocycle in the 6-phenoxy-1,2,3,4-tetra-

hydronaphthalene series further enhanced the activity of the

candidate inhibitors providing extraordinarily potent inhibi-

tors. This was most evident with 34, lacking a 1,3,4-oxadiazole

C5-substituent, which exhibited a K_iof 500 pM (0.5 nM) for

the more potent (S)-enantiomer. Analogous to prior

observations,^54,59this represents a 4ꢀ5-fold improvement in

potency relative to the corresponding oxazole 9. In this series,

the measured diﬀerences between the (S)- and (R)-enantio-

mers proved modest, indicating that a well-precedented⁷⁴

enhanced racemization of the 1,3,4-oxadiazole most likely

occurs under the conditions (pH 9) and time course of the

assay because of the stronger electron-withdrawing properties

of the activating heterocycle.

conformational constraints (K_i= 200 nM, Figure 3) indicating

that their introduction may subtly, but not seriously, aﬀect

their active site aﬃnity. More interestingly, the enantiomer

distinctions with 37 were modest (5.1 vs 12 μM, ca. 2-fold). As

revealed in the X-ray structure of 12 bound to FAAH, it is the

spatial relationship of the dominant anchoring C6-phenoxy

substituent of 12 with the chiral center that imposes the

enantiomeric selectivity observed in the tetrahydrona-

phthalene or indane series. Additionally, the introduction of

the oxazole C5 pyridyl substituent increased the potency

7ꢀ12-fold compared with the unsubstituted counterparts in

this series (38 vs 37 and 40 vs 39), analogous to prior observa-

tions,⁵³suggesting that the hydrogen-bond capability of the

weakly basic pyridyl substitutent is responsible for their

enhanced aﬃnities.

Inhibition of Recombinant Human FAAH. Rat and human

FAAH are very homologous (82% sequence identity),²exhibit

near identical substrate selectivity and inhibitor sensitivity in

our studies with the R-ketoheterocycles disclosed to date, and

embody an identical amidase signature sequence, suggesting

that the observations made with rat FAAH (rFAAH) would

be analogous to those made with human FAAH (hFAAH).

For the candidate inhibitors without an acyl side chain aryl

substituent, a substantial loss in potency (from nanomolar to

micromolar range) is observed, highlighting the importance of its

anchoring interaction within the enzyme active site, Figure 6.

Notably, 38 (2-fold, K_i= 430 nM) and 40 (5-fold, K_i= 1.1 μM)

approach the activity of the FAAH inhibitor lacking the

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Figure 7. Inhibition of human versus rat FAAH (hFAAH vs rFAAH),

K_i(nM).

Figure 9. Two views of the superposition of the FAAHꢀ12 (in yellow/

orange) complex with FAAHꢀ2 complex⁷⁶(in green). The rearrange-

ment of Phe192 and Met495 residues are shown.

summarized in Table S1 (Supporting Information). Compound

12 was found covalently attached to the catalytic Ser241 residue

through its electrophilic carbonyl bound as a deprotonated

hemiketal mimicking the enzymatic tetrahedral intermediate, a

hallmark of the R-ketoheterocycle class of FAAH inhibitors.^76,77

The electron density of the bound inhibitor and its excellent

resolution provide an unambiguous depiction of the chiral

center of 12 confirming that it is the (S)-enantiomer, Figure 8.

A structural overlay of the X-ray crystal structure of 2, whose

interactions with FAAH have been described in detail,⁷⁶and 12 is

provided in Figure 9. The terminal phenyl ring of the acyl side

chain of 12 is positioned in the same plane and only slightly

shifted (0.6 Å) from the analogous group of 2, Figure 9. This

phenyl group serves as a key anchoring interaction with FAAH,

which adopts an active site conformation that leads to a broa-

dened and open membrane access channel with truncation of

the acyl chain-binding pocket.⁷⁶Beautifully, the R, β, and γ

carbons of the intervening flexible hydrophobic linker in the

acyl side chain of 2, which adopt a gauche conformation, nearly

superimpose with C2ꢀC4 of the tetrahydronaphthalene core of

12, Figure 9.

Figure 8. (top) View of 12 in the binding pocket of FAAH and its

interactions. (bottom) An enlarged view of the chiral center within the

tetrahydronaphthalene is shown. Electron density at 1.5σ contour is

shown with white meshes.

Consequently, the active (S)-enantiomers of two representa-

tive inhibitors, 12 and 14, were examined with hFAAH, and

the results are summarized in Figure 7. As observed with 2, no

significant distinction was observed for 12, whereas 14 ex-

hibited a modest 4-fold reduction in activity against the human

enzyme.

X-ray Structure of the Active Enantiomer of Inhibitor 12

Bound to FAAH. The X-ray structure of the active enantiomer of

the R-ketooxazole 12 bound to h/rFAAH⁷⁵was solved at 1.9 Å

resolution, and the data processing and refinement statistics are

The backbone of the enzyme bound to 12 did not exhibit any

major conformational changes relative to previously published

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Figure 10. Initial screen for the eﬀects of FAAH inhibitors on brain and liver lipid levels following in vivo inhibitor treatment; n = 1 per group.

structures, although two residues lining the active site undergo

rearrangement. The most signiﬁcant change is the relocation

of the side chain of the Phe192 residue that rotates and shifts

away from the compound acyl chain due to the size of the

tetrahydronaphthalene core of 12, Figure 9. This reorganization

of the lining of the hydrophobic binding pocket, also observed in

other structures,⁷⁷conﬁrms that this residue is highly ﬂexible and

is likely a major adapter residue when binding diﬀerent substrates

and inhibitors. As a consequence of this rearrangement, the

inhibitor oxazole C5-pyridyl substituent is pushed toward Ile238

and Leu278. Despite this, the pyridyl nitrogen remains hydro-

gen-bonded to an ordered cytosolic port water molecule that in

turn is hydrogen-bonded to Thr236, a feature that is conserved in

all related structures analyzed to date.^76,77Interestingly, the space

generated by the shift of the pyridine is compensated by the

appearance of a new water molecule in the cytosolic port that had

not appeared in earlier structures. It sits above the aromatic ring

of the pyridine at a distance of 3.6 Å, and is coordinated, perhaps

polarized, by the backbone amide of Cys269, previously identi-

ﬁed as a key residue forming part of an anion binding site,

Figure 8.⁷⁷The activating oxazole and its attached pyridine

substituent are bound in the cytosolic port, adopting bound

orientations and a biaxial twist (18ꢀ) analogous to those found

with 2 (15ꢀ) and related inhibitors. However, the unusual Ser217

OH hydrogen bond to the π-system of activating oxazole

observed with 2 is now replaced with a hydrogen bond directly

from Ser217 OH to the oxazole nitrogen in a fashion observed for

histidine in serine proteases,⁷⁸Figure 9. This change is not

derived from a signiﬁcant relocation of the Ser217 residue, but

rather is a result of the reorientation of activating heterocycle

(oxazole) and the displacement of its attached C5-pyridyl

substituent. These oﬀsetting changes, which still provide inhibi-

tors more potent than 2, may account for the reason that the

introduction of the pyridine substituent did not improve aﬃnity

in this series (12 vs 9 and 18 vs 15) although it still enhances their

selectivity for FAAH.³¹An additional change unique to this

structure is the rearrangement of the distal rotamer of Met495,

which now points toward the inhibitor and sits over the acyl chain

linker aromatic ring at a distance of 4.4 Å, Figure 9.

modeling⁷⁹studies were performed and used to calculate the

relative energies involved in the binding of the two enantiomers.

Covalent docking (ICM, Molsoft Inc.) of 12 with Ser241 and

Monte Carlo simulations for sampling bound conformations

were used to compare the two enantiomers of 12. As empirically

predicted from the crystal structure analysis and established in

the enzymatic assays, the (R)-enantiomer binding is destabilized

relative to the (S)-enantiomer suﬀering from a large penalty in

the van der Waals term (steric clash). Analysis of the contribu-

tions of single amino acids to the total binding energy established

that this coincides predominately with a van der Waals repulsion

with Ser193. When bound with the same orientation as (S)-12

and in order to maintain phenoxy binding at the terminus of the

truncated acyl chain binding pocket, (R)-12 suﬀers a destabiliz-

ing steric clash with Ser193 proximal to the chiral center

(Figure 8, bottom). If (R)-12 is ﬂipped 180ꢀ to avoid this steric

interaction and the tetrahydronaphthalene core is arranged in a

disposition similar to (S)-12 (chiral C2ꢀH down as found in

Figure 8, bottom), the terminal phenoxy group is no longer

positioned to bind in this key region of the acyl chain binding

pocket. Thus, it is not surprising that while the two enantiomers

of 37 and 38 that lack the phenoxy group bind FAAH with

comparable, albeit weak aﬃnities, the two enantiomers of 12 and

related inhibitors exhibit much more pronounced diﬀerences.

Preliminary In Vivo Characterization. In initial efforts to

establish in vivo inhibition of FAAH and the potential pharma-

cological effects, a select set of the conformationally restricted

inhibitors (12, 14, 27, and 29) were examined alongside 2 for

their ability to increase the endogenous levels of a series of lipid

amide signaling molecules in the brain (CNS effect) and liver

(peripheral effect). This includes monitoring the effects of the

inhibitors on the endogenous levels of the FAAH substrates

anandamide (N-arachidonoyl ethanolamine), N-oleoyl ethanol-

amine (OEA), and N-palmitoyl ethanolamine (PEA), as well as

the key lipids 2-arachidonoylglycerol (2-AG) and arachidonic

acid that are not endogenous substrates for FAAH. Notably, it is

the increase in endogenous levels of anandamide and its sub-

sequent action at CB₁and CB₂receptors that are thought to be

responsible for the antinociceptive and anti-inflammatory effects

of FAAH inhibitors, although both PEA and 2-AG are also

known to exhibit anti-inflammatory and CB receptor-mediated

In order to establish the origin of the inhibitor enantiomer

selectivity extrapolated from the crystal structure, molecular

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Figure 11. Brain lipid amide levels following oral versus intraperitoneal dosing.

Figure 12. Dose- (panels a and b, analysis performed 1 h post-treatment) and time-dependent (panels c and d at 50 mg/kg 12) impact on brain lipid

amide levels following oral dosing of 12. AEA = anandamide, PEA = N-palmitoyl ethanolamine, OEA = N-oleoyl ethanolamine.

antinociceptive effects, respectively. Pharmacological effects

were initially established 1 h after intraperitoneal (ip) adminis-

tration of 30 mg/kg inhibitor in a single mouse for the initial

screen, and the results are summarized in Figure 10. The

inhibitors 12 and 27 increased the endogenous levels of key

lipid amides thought to be responsible for antinociceptive effects

(anandamide, PEA) without impacting the endogenous levels of

2-AG, consistent with selective FAAH inhibition in vivo. They

also had a minimal impact on reducing the endogenous levels of

arachidonic acid, the hydrolysis product of anandamide and a key

proinflammatory fatty acid.⁸⁰The effects of 12 (and presumably

27) were observed both in the brain (CNS) and liver

(peripheral) matching or exceeding the effects of 2, whereas

the impact of the more polar inhibitor 14 (and presumably 29)

was principally seen peripherally (liver) with more muted effects

in the CNS (brain).

Given the results of this initial screen, both 12 and 14 were

examined side-by-side not only with ip (30 mg/kg) but also with

oral (po, 50 mg/kg) dosing with three mice per group to provide

the results presented in Figure 11. Signiﬁcantly, the oral dosing

matched and even improved on the results observed with ip

administration.

Given the oral activity established in the PD model with 12, a

follow up dose- and time-dependent study of its eﬀects on the

endogenous brain levels of anandamide, PEA, and OEA was

conducted alongside 2 and the irreversible FAAH inhibitor 41

(PF-3845).^42cIn the ﬁrst of these studies, each comparison

standard (2 and 41) was administered orally at 50 mg/kg, 12

was administered orally at 10, 25, and 50 mg/kg, and the resulting

impact on the endogenous brain levels of fatty acid amides was

measured at a single time point (1 h). Compound 12 increased

the levels of anandamide (12ꢀ13-fold at 25ꢀ50 mg/kg) in a

dose-dependent manner more eﬃcaciously and more potently

than 2 (11-fold at 50 mg/kg) but not as eﬀectively as the

irreversible FAAH inhibitor 41, Figure 12a. In contrast, the

increases in the brain levels of PEA and OEA observed with 12

at 1 h were substantial (5.5ꢀ7.8-fold) and essentially the same at

all three administered doses. These increases exceeded the eﬀects

observed with 2 at 50 mg/kg and matched the eﬀects of the

irreversible inhibitor 41 administered at 50 mg/kg, Figure 12b.

These data are consistent with reports showing that partial

blockade of FAAH can cause elevations in PEA and OEA but

that >90% FAAH blockade is required to elevate anandamide

levels.^42cIn the second of the studies, 12 was administered to

mice (50 mg/kg, po) and the animals were sacriﬁced at various

time points up to 9 h postadministration. Brains from these mice

were analyzed for levels of anandamide and the additional FAAH

substrates PEA and OEA. Administration of 12 caused dramatic

accumulations of all three N-acylethanolamines in brain, with

peak levels of anandamide achieved between 1.5 and 3 h

(Figure 12c,d). Remarkably, the elevations in these lipids were

maintained over the 9 h time course, similar to the time course

reported for the irreversible FAAH inhibitor 41 and signiﬁcantly

longer than that reported for the irreversible carbamate inhibitor

URB597.^42cThese time course data suggest that the reversible

inhibitor 12 remains in the brain after its initial oral dosing at

suﬃciently high concentrations to completely inhibit FAAH

(>90%) for a prolonged period.

An additional feature of the R-ketoheterocycle FAAH inhibi-

tors that we have not disclosed previously is that they rapidly

establish an equilibirum mixture of active ketone and reduced

alcohol in vivo. Although this is likely a general feature of the

entire class of R-ketoheterocycles that have been examined as

enzyme inhibitors,⁸¹we are not aware of its discussion elsewhere.

Consequently, triaging screens for compound development using

rat or human liver microsomes (rlm and hlm) may often

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Figure 13. Antinociception in the tail ﬂick assay (52 ꢀC) following iv administration of 14, 2, and morphine.

Figure 14. FAAH inhibition by 12 signiﬁcantly attenuated neuropathic pain for up to 9 h. Male C57BL/6 mice were subjected to chronic constriction

injury (CCI) of the sciatic nerve and tested 10 days later for (A) mechanical allodynia, as measured with von Frey ﬁlaments, and (B) acetone-induced

cold allodynia. Inhibitor 12 (50 mg/kg, po) signiﬁcantly attenuated CCI-induced mechanical allodynia, as well as cold allodynia, in paws ipsilateral to

CCI surgery, but had no eﬀect in paws contralateral to CCI surgery. Circles, vehicle treatment; triangles, 12 treatment; open shapes, control paws; ﬁlled

shapes, CCI paws. Data expressed as mean ( SEM (n = 9ꢀ10). / p < 0.05; // p < 0.01 vs vehicle.

misleadingly suggest rapid metabolic reduction that is not repre-

sentative of the true metabolic fate of such candidate inhibitors

(e.g., rlm and hlm t_1/2for 2 and 18 are 2ꢀ4 min and 12ꢀ15 min,

respectively). Rather, 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 inhibitors being

determined by other features of the molecule. For 2, this

steady-state equilibrium is established within 15 min and was

found to be 1/3 (ketone/alcohol), independent of the means of

administration.⁸²For 12, we also measured the compound levels

in the brain following its oral administration (50 mg/kg) and

could detect both the parent ketone and the reduced alcohol in

inhibitor-treated but not vehicle-treated mice. Therelative ratio of

ketone/alcohol was 3ꢀ3.5/1 at early stages following compound

administration (0.5ꢀ1 h) and slowly equilibrated to 1ꢀ1.5/1 at

the longer times following administration (1.5ꢀ9 h) where both

the ratio and brain levels persisted. Although preliminary, the

results seem to indicate that the added conformational constraints

in the C2 acyl chain and the increased steric hindrance surround-

ing the electrophilic ketone both slow the rate of equilibration and

improve the ketone/alcohol ratio in vivo.

Concurrent with these studies, the two prototypical inhibitors

12 (not shown) and 14 were examined for antinociceptive

activity in a preclinical model of acute thermal pain (tail ﬂick at

52 ꢀC) in mice alongside 2 and morphine (iv administration).

The results of the doseꢀresponse studies for 14, the more

soluble of the two inhibitors, are summarized in Figure 13

alongside those of morphine. Compound 14 produced the same

maximal eﬀect (eﬃcacy) observed with morphine requiring only

a 2-fold higher dose, reached its maximal eﬀect at 30ꢀ60 min,

and exhibited a duration of eﬀect matching that of morphine

(150 min).

In an important extension of the studies and following the

observation of the long-acting in vivo eﬀects of 12 on endogen-

ous anandamide levels following oral administration, mice were

subjected to chronic constriction injury (CCI) and tested 10 days

later for signs of neuropathic pain. Inhibitor 12 administered

orally (50 mg/kg) signiﬁcantly attenuated mechanical allodynia

[F(1,51) = 17.1; p < 0.001; Figure 14A] and cold allodynia

[F(1ꢀ51) = 26.4; p < 0.0001; Figure 14B], in paws ipsilateral to

CCI surgery. In the control paws of the same mice, 12 had no

eﬀect on mechanical (p = 0.16) or cold allodynia (p = 0.48),

indicating a lack of sedative eﬀects. Signiﬁcantly, the eﬀects of 12

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following its oral dosing were sustained, lasting >6 h in the

mechanical allodynia and >9 h in the cold allodynia, consistent

with its long-acting eﬀects in raising the endogenous concentra-

tion of anandamide (see Figure 12c).

Finally and signiﬁcantly, inhibitor 12 was also examined for

cannabimimetic side eﬀects following oral administration at a

dose that provided analgesic eﬀects (50 mg/kg). Like 2 and

earlier FAAH inhibitors in this class,⁶¹12 did not produce

catalepsy, hypothermia, or hypomotility (data not shown) in-

dicating that it does not produce THC-like eﬀects characteristic

of a classical CB receptor agonist.

min before a solution of 6-phenoxy-1,2,3,4-tetrahydronaphthalene-2-car-

boxaldehyde (870 mg, 3.44 mmol) in THF (20 mL) was added. The

reaction mixture was stirred at ꢀ78 ꢀC for 2 h before being warmed to

room temperature. A 5% HOAcꢀEtOH solution (50 mL) was added, and

this mixture was stirred at room temperature for 12 h. The solvent was

removed under reduced pressure, and the residue was dissolved in EtOAc

and washed with H₂O, saturated aqueous NaHCO₃and saturated aqueous

NaCl before the organic layer was dried over MgSO₄and the solvent was

removed under reduced pressure. Flash chromatography (SiO₂, 40%

EtOAcꢀhexanes) afforded oxazol-2-yl(6-phenoxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)methanol (740 mg, 67%) as colorless oil: ¹H NMR

(CDCl₃, 600 MHz) δ 7.65 (s, 1H), 7.32ꢀ7.31 (m, 2H), 7.10ꢀ6.98 (m,

5H), 6.78ꢀ6.75 (m, 2H), 4.78ꢀ4.74 (m, 1H), 2.88ꢀ2.78 (m, 4H),

2.61ꢀ2.59 (m, 0.5H), 2.34 (m, 0.5H), 2.14ꢀ2.12 (m, 0.5H), 1.80ꢀ1.77

(m, 0.5H), 1.62ꢀ1.51 (m, 1H); ¹³C NMR (CDCl₃, 150 MHz) δ 165.3,

157.5, 154.8, 138.9, 137.9, 137.7, 130.5, 130.3, 130.2, 130.1, 129.5 (2C),

126.5, 122.8, 122.7, 118.86 (2C), 118.82, 118.49, 118.42, 116.78, 116.73,

71.2, 71.0, 39.8, 39.7, 30.8, 30.2, 29.6, 28.9, 28.8, 25.1, 24.4.

A solution of oxazol-2-yl(6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-

yl)methanol (400 mg, 1.24 mmol), TBSCl (450 mg, 2.98 mmol), and

imidazole (421 mg, 6.2 mmol) in DMF (20 mL) was stirred at room

temperature for 16 h before it was diluted with EtOAc and washed with

H₂O and saturated aqueous NaCl. The organic layer was dried over

MgSO₄, and the solvent was removed under reduced pressure. Flash

chromatography (SiO₂, 10% EtOAcꢀhexanes) yielded 2-((tert-butyldi-

methylsilyloxy)(6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)

oxazole (459 mg, 85%) as a thick colorless oil: ¹H NMR (CDCl₃, 500

MHz) δ 7.65 (s, 1H), 7.31 (t, 2H, J = 8.5 Hz), 7.12 (d, 1H, J = 8.0 Hz),

7.01ꢀ6.97 (m, 4H), 6.81ꢀ6.75 (m, 2H), 4.80 (d, 0.5H, J = 7.0 Hz), 4.74

(d, 0.5H, J = 7.0 Hz), 2.99ꢀ2.73 (m, 3H), 2.55ꢀ2.51 (m, 1H),

2.39ꢀ2.25 (m, 1H), 1.74ꢀ1.71 (m, 1H), 1.53ꢀ1.49 (m, 1H), 0.96 (s,

4.5H), 0.93 (s, 4.5H), ꢀ0.03 (s, 1.5H), ꢀ0.04 (s, 1.5H), ꢀ0.05 (s,

1.5H), ꢀ0.06 (s, 1.5H); ¹³C NMR (CDCl₃, 125 MHz) δ 164.4, 164.3,

157.6, 157.5, 154.7, 154.6, 138.47, 138.41, 138.0, 137.7, 130.7, 130.4,

130.3, 130.1 (2C), 129.4, 126.7, 122.6, 122.5, 118.8, 118.3, 118.2, 116.7,

116.6, 72.2, 72.1, 40.3, 30.67, 30.62, 28.8, 28.7, 25.6 (3C), 25.1, 24.8,

18.0, ꢀ5.3, ꢀ5.41, ꢀ5.44, ꢀ5.6.

’ CONCLUSIONS

A series of R-ketooxazoles containing conformational con-

straints in the C2 acyl side chain of 2 were prepared and examined

as inhibitors of FAAH. Members of this new series exhibited

comparable or improved enzyme inhibition potency relative to 2,

indicating that additional conformational restriction in the C2

acyl side chain is achievable and beneﬁcial. A cocrystal X-ray

structure of the prototypical R-ketoheterocycle 12 bound to a

humanized variant of rat FAAH conﬁrmed that the (S)-enantio-

mer is the bound active inhibitor, shed light on the structural

origin of the enantiomeric selectivity, and conﬁrmed that the

active site catalytic Ser241 is covalently bound to the electrophilic

carbonyl, mimicking the enzymatic tetrahedral intermediate.

Preliminary in vivo characterization of the prototypical inhibitors

12 and 14 in mice was reported demonstrating that they raise

endogenous anandamide levels with either intraperitoneal or oral

administration, and that the oral adminstration of 12 caused

sustained accumulation of three major N-acylethanolamines

(anandamide, OEA, and PEA) in the brain with peak levels of

anandamide achieved between 1.5 and 3 h with elevations that

were maintained over the 9 h time course of the examination. This

duration of action with oral administration of 12 proved similar to

that reported for the irreversible urea FAAH inhibitor 41 and

signiﬁcantly longer than that reported for the irreversible carba-

mate inhibitor URB597, suggesting that the reversible inhibitor

12 remains in the brain after its initial oral dosing at suﬃciently

high concentrations to completely inhibit FAAH (>90%) for a

prolonged period. Preliminary in vivo studies with the two

representative members of the series (12 and 14) indicate that

they exhibit robust analgesic activity in mouse models of thermal

hyperalgesia and neuropathic pain including the demonstration

that oral administration of 12 (50 mg/kg) signiﬁcantly attenuated

both mechanical (>6 h) and cold (>9 h) allodynia for sustained

periods consistent with its long-acting eﬀects in raising the

endogenous concentration of anandamide.

A solution of 2-((tert-butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetra-

hydronaphthalen-2-yl)methyl)oxazole (459 mg, 1.05 mmol) in anhy-

drous THF(15 mL) wascooledto ꢀ78 ꢀC before it was treated with 2.16

M n-BuLi (0.6 mL, 1.15 mmol) dropwise. The reaction mixture was

stirred at ꢀ78 ꢀC for 2 h and treated with Bu₃SnCl (0.6 mL, 2.1 mmol)

and stirred for 5 min. The solution was warmed to room temperature,

diluted with EtOAc, and washed with saturated aqueous NaCl. The

organic layer was dried over MgSO₄, and the solvent was removed under

reduced pressure. Flash chromatography (SiO₂, 10% EtOAcꢀhexanes)

yielded 2-((tert-butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)methyl)-5-(tributylstannyl)oxazole (500 mg, 78%) as

a thick colorless oil: ¹H NMR (CDCl₃, 500 MHz) δ 7.30 (t, 2H, J = 7.5

Hz), 7.12 (d, 1H, J = 6.0 Hz), 7.06 (t, 2H, J = 7.0 Hz), 6.97 (m, 2H), 6.77

(dd, 2H, J = 2.5, 8.5 Hz), 4.80 (d, 0.5H, J = 7.0 Hz), 4.75 (d, 0.5H, J = 7.0

Hz), 2.96ꢀ2.92 (m, 2H), 2.82ꢀ2.70 (m, 2H), 2.52ꢀ2.22 (m, 2H),

1.58ꢀ1.46 (m, 8H), 1.36ꢀ1.30 (m, 6H), 1.15ꢀ1.11 (m, 5H), 0.94ꢀ0.90

(s, 18H), 0.08 (s, 1.5H), 0.06 (s, 1.5H), ꢀ0.11 (s, 1.5H), ꢀ0.12 (s,

1.5H); ¹³C NMR (CDCl₃, 125 MHz) δ 168.5, 168.4, 157.8, 154.9, 154.8,

154.7, 154.6, 138.2, 138.0, 137.1, 131.2, 130.8, 130.4, 130.2, 129.5 (2C),

122.67, 122.64, 118.9, 118.4, 118.3, 116.8, 116.6, 72.5, 72.3, 40.69, 40.64,

30.85, 30.81, 29.3, 29.2 (3C), 29.1, 29.0, 28.98, 28.90, 28.8, 28.5, 27.6,

27.4, 27.3, 27.2 (3C), 27.1, 27.0, 26.8, 25.7, 25.2, 25.0, 18.1, 13.69, 13.60

(3C), 11.6, 10.7, 10.2 (3C), 9.98, ꢀ5.3, ꢀ5.4, ꢀ5.61, ꢀ5.62.

’ EXPERIMENTAL SECTION

General Experimental. The purity of each inhibitor (>95%) was

determined on an Agilent 1100 LC/MS instrument on a ZORBAX SB-

C18, 3.5 mm, 4.6 mm ꢁ 50 mm, with a flow rate of 0.75 mL/min and

detection at 220 and 254 nm, with a 10ꢀ98% acetonitrile/water/0.1%

formic acid gradient and a 50ꢀ98% acetonitrile/water/0.1% formic acid

gradient (see Table S2 in Supporting Information).

(6-Phenoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(5-(pyridin-

2-yl)oxazol-2-yl)methanone (12). A solution of oxazole (0.226 mL,

3.44mmol) in anhydrousTHF (20 mL) wastreatedwithBH₃THF (1 M,

2-((tert-Butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetrahydronaph-

thalen-2-yl)methyl)-5-(tributylstannyl)oxazole (5.0 g, 6.89 mmol),

Pd(PPh₃)₄(800 mg, 0.689 mmol), and 2-bromopyridine (0.9 mL,

8.96 mmol) were dissolved in anhydrous 1,4-dioxane (30 mL), and the

3

3.74 mL, 3.74 mmol), and the solution was stirred at room temperature for

1 h before being cooled to ꢀ78 ꢀC and treated with 2.16 M n-BuLi (2 mL,

4.47 mmol) dropwise. The reaction mixture was stirred at ꢀ78 ꢀC for 40

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mixture was warmed at reﬂux for 16 h under Ar. The mixture was

diluted with EtOAc, washed with saturated aqueous NaCl, and dried

over Na₂SO₄. Flash chromatography (SiO₂, 20% EtOAcꢀhexanes)

yielded 2-((tert-butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)methyl)-5-(pyridin-2-yl)oxazole (1.49 g, 42%; typi-

cally 42ꢀ61%) as a colorless oil: ¹H NMR (CDCl₃, 500 MHz) δ 8.64

(d, 1H, J = 4.5 Hz), 7.78ꢀ7.76 (m, 1H), 7.71ꢀ7.67 (m, 2H), 7.30 (t,

2H, J = 7.5 Hz), 7.24ꢀ7.22 (m, 1.5H), 7.07ꢀ7.04 (m, 1.5H),

6.98ꢀ6.95 (m, 2H), 6.78ꢀ7.72 (m, 2H), 4.81 (d, 0.5H, J = 7.0 Hz),

4.75 (d, 0.5H, J = 7.0 Hz), 2.96ꢀ2.73 (m, 2H), 2.58ꢀ2.55 (m, 1H),

2.39ꢀ2.34 (m, 1H), 2.26ꢀ2.20 (m, 1H), 1.81ꢀ1.77 (m, 1H),

1.58ꢀ1.53 (m, 1H), 0.90 (s, 9H), 0.11 (s, 1.5H), 0.09 (s, 1.5H),

ꢀ0.05 (s, 1.5H), ꢀ0.04 (s, 1.5H); ¹³C NMR (CDCl₃, 125 MHz) δ

157.7, 157.6, 154.8, 154.7, 149.6, 138.1, 137.9, 137.18, 137.14, 132.1,

130.8, 130.5, 130.4 (2C), 130.3, 129.5, 128.5 (2C), 125.5, 125.4, 122.8,

122.78, 122.73, 119.1, 118.9, 118.4, 118.3, 116.8, 116.7, 72.5, 72.4, 40.5,

30.9, 30.5, 29.0, 28.9, 25.7 (3C), 25.3, 24.9, 18.2, ꢀ5.0, ꢀ5.23, ꢀ5.26.

2-((tert-Butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetrahydronaph-

thalen-2-yl)methyl)-5-(pyridin-2-yl)oxazole (1.49 g, 2.90 mmol) was

dissolved in THF (30 mL) and treated with Bu₄NF (1 M in THF, 4 mL,

3.48 mmol) and the solution was 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, and the solvent

was removed under reduced pressure. Flash chromatography (SiO₂,

50ꢀ100% EtOAcꢀhexanes) yielded (6-phenoxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)(5-(pyridin-2-yl)oxazol-2-yl)methanol (740 mg, 64%)

as a yellow oil: ¹H NMR (CDCl₃, 600 MHz) δ 8.63 (d, 1H, J = 4.2 Hz),

7.78 (t, 1H, J = 7.8 Hz), 7.71ꢀ7.65 (m, 2H), 7.30 (t, 2H, J = 7.2 Hz),

7.27ꢀ7.25 (m, 2H), 7.07ꢀ6.96 (m, 3H), 6.77ꢀ6.73 (m, 2H), 4.87 (d,

0.5H, J = 7.0 Hz), 4.82 (d, 0.5H, J = 7.0 Hz), 2.86ꢀ2.68 (m, 4H),

2.45ꢀ2.42 (m, 1H), 2.17ꢀ2.15 (m, 1H), 1.92ꢀ1.89 (m, 1H),

1.66ꢀ1.61 (m, 1H); ¹³C NMR (CDCl₃, 150 MHz) δ 157.6, 154.8,

149.5, 146.7, 137.9, 137.7, 137.3, 130.5, 130.4, 130.3, 130.2, 129.6 (2C),

125.37, 125.34, 123.1, 122.8, 119.4, 118.9, 118.8, 118.4 (2C), 116.85,

116.81, 71.5, 71.3, 39.9, 39.8, 30.9, 30.0, 29.6, 28.98, 28.94, 25.3, 24.3.

(6-Phenoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(5-(pyridin-2-yl)-

oxazol-2-yl)methanol (740 mg, 1.85 mmol) was dissolved in CH₂Cl₂

(40 mL), and DessꢀMartin periodinane (1.0 g, 2.22 mmol) was added.

The mixture was stirred at room temperature for 2 h, and the reaction

mixture was evaporated in vacuo. Flash chromatography (SiO₂, 20%

EtOAcꢀhexanes) yielded (6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(5-(pyridin-2-yl)oxazol-2-yl)methanone (12, 650 mg, 88%) as a

5-yl)picolinate (2.88 g, 73%) as a colorless oil: ¹H NMR (CDCl₃, 500

MHz) δ 8.01 (dd, 1H, J = 4.5, 7.0 Hz), 7.99ꢀ7.97 (m, 1H), 7.89ꢀ7.85

(m, 1H), 7.80ꢀ7.78 (m, 1H), 7.65ꢀ7.59 (m, 1H), 7.25ꢀ7.22 (m, 2H),

7.01ꢀ6.97 (m, 1H), 6.92ꢀ6.90 (m, 2H), 6.73ꢀ6.66 (m, 2H), 4.80 (d,

0.5H, J = 7.0 Hz), 4.77 (d, 0.5H, J = 7.0 Hz), 3.96 (s, 1.5H), 3.93 (s,

1.5H), 2.91ꢀ2.87 (m, 1H), 2.78ꢀ2.76 (m, 3H), 2.73ꢀ2.71 (m, 1H),

2.38ꢀ2.33(m, 0.5H), 2.23ꢀ2.20(m, 0.5H), 1.62ꢀ1.52(m, 1H), 0.90 (s,

9H), 0.11 (s, 1.5H), 0.09 (s, 1.5H), ꢀ0.05 (s, 1.5H), ꢀ0.04 (s, 1.5H);

¹³C NMR (CDCl₃, 125 MHz) δ 165.0, 164.9, 164.8, 164.0, 157.47,

157.40, 154.6, 154.5, 149.9, 149.8, 148.4, 148.0, 147.38, 147.35, 141.8,

138.9, 137.8, 137.6, 131.8, 131.7, 131.5, 130.5, 130.2, 130.1, 130.0, 129.3

(2C), 128.3, 128.23, 126.20, 126.1, 123.8, 123.7, 122.56, 122.52, 121.8

(2C), 118.7, 118.2, 118.1, 72.3, 72.1, 52.8, 52.6, 40.2, 30.7, 30.3, 28.7,

28.6, 27.6, 26.5, 25.5 (3C), 25.1, 24.6, 17.9, 17.3, 13.3, ꢀ5.2, ꢀ5.40,

ꢀ5.44.

Methyl 6-(2-((tert-butyldimethylsilyloxy)(6-phenoxy-1,2,3,4-tetrahy-

dronaphthalen-2-yl)methyl)oxazol-5-yl)picolinate (2.88 g, 5.04 mmol)

was dissolved in THF (50 mL) and treated with Bu₄NF (1 M in THF,

6 mL, 6.05 mmol), and the solution was 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, and the solvent was

removed under reduced pressure. Flash chromatography (SiO₂,

50ꢀ100% EtOAcꢀhexanes) yielded methyl 6-(2-(hydroxy(6-phenoxy-

1,2,3,4-tetrahydronaphthalen-2-yl)methyl)oxazol-5-yl)picolinate (2.0 g,

86%) as a yellow oil: ¹H NMR (CDCl₃, 400 MHz) δ 8.20 (dd, 1H, J =

1.2, 7.6 Hz), 8.05 (t, 1H, J = 8.0 Hz), 7.98ꢀ7.96 (m, 2H), 7.48 (t, 2H, J =

7.2 Hz), 7.25ꢀ7.12 (m, 4H), 6.95ꢀ6.90 (m, 2H), 5.06 (d, 0.5H, J = 6.8

Hz), 5.01 (d, 0.5H, J = 6.8 Hz), 4.18 (s, 3H), 3.08ꢀ2.95 (m, 3H),

2.84ꢀ2.81 (m, 1H), 2.65ꢀ2.61 (m, 1H), 2.38ꢀ2.03 (m, 1H), 1.81ꢀ1.45

(m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 165.8, 165.7, 165.1, 157.4,

154.77, 154.74, 150.1, 148.0, 147.1, 137.8, 137.6, 130.4, 130.3, 130.2,

130.1, 129.49 (2C), 129.47, 125.9, 123.9, 122.6, 122.2, 118.79, 117.74,

118.33, 118.30, 116.7, 116.6, 71.2, 71.0, 64.2, 52.8, 39.69, 39.65, 30.9,

30.1, 28.8, 25.2, 24.4, 18.9, 17.4, 13.4.

Methyl 6-(2-(hydroxy(6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-

yl)methyl)oxazol-5-yl)picolinate (2.0 g, 4.38 mmol) was dissolved in

CH₂Cl₂(60 mL) and DessꢀMartin periodinane (2.7 g, 6.25 mmol) was

added. The mixture was stirred at room temperature for 2 h before the

reaction mixture was evaporated in vacuo. Flash chromatography (SiO₂,

30% EtOAcꢀhexanes) yielded methyl 6-(2-(6-phenoxy-1,2,3,4-tetrahy-

dronaphthalene-2-carbonyl)oxazol-5-yl)picolinate (13, 1.67 g, 70%) as a

white solid: ¹H NMR (CDCl₃, 500 MHz) δ 8.09 (dd, 1H, J = 1.0, 8.0

Hz), 8.03 (s, 1H), 8.01 (dd, 1H, J = 1.5, 8.0 Hz), 7.95 (t, 1H, J = 7.5 Hz),

7.29 (t, 2H, J = 7.5 Hz), 7.06 (t, 2H, J = 7.5 Hz), 6.98ꢀ6.96 (m, 2H),

6.79ꢀ6.77 (m, 2H), 4.01 (s, 3H), 3.91ꢀ3.86 (m, 1H), 3.08 (d, 2H, J =

8.0 Hz), 2.93ꢀ2.87 (m, 2H), 2.31ꢀ2.27 (m, 1H), 1.94ꢀ1.89 (m, 1H);

¹³C NMR (CDCl₃, 125 MHz) δ 190.3, 164.9, 157.4, 156.8, 154.9, 152.3,

148.4, 146.3, 138.1, 137.1, 130.0, 129.6 (2C), 129.5, 127.8, 125.0, 123.1,

122.7, 118.8, 118.4 (2C), 116.8, 52.9, 43.4, 30.4, 28.6, 25.6; HRMS-ESI-

TOF m/z 455.1617 ([M þ H]^þ, C₂₇H₂₂N₂O₅requires 455.1601).

The enantiomers were separated using a semipreparative chiral

phase HPLC column (Daicel ChiraCel OD, 10 μm, 2 cm ꢁ 25 cm,

40% EtOHꢀhexanes, 7 mL/min, R = 1.19). (S)-13: [R]²³_Dꢀ0.7 (c 0.8,

THF). (R)-13: [R]²³_Dþ0.5 (c 0.8, THF).

1

yellow oil: H NMR (CDCl₃, 600 MHz) δ 8.68 (d, 1H, J = 4.2 Hz),

7.93 (s, 1H), 7.90ꢀ7.83 (m, 2H), 7.34ꢀ7.31 (m, 3H), 7.19ꢀ7.14 (m,

4H), 6.88ꢀ6.78 (m, 2H), 3.92ꢀ3.90 (m, 1H), 3.10ꢀ2.90 (m, 4H),

2.32ꢀ2.30 (m, 1H), 1.95ꢀ1.93 (m, 1H); ¹³C NMR (CDCl₃, 150 MHz)

δ 190.5, 157.5, 156.8, 155.1, 153.3, 150.0, 146.1, 137.2, 137.0, 130.2,

129.7, 129.6 (2C), 127.0, 124.2, 122.9, 120.4, 118.9, 118.5 (2C), 116.9,

43.5, 30.6, 28.8, 25.7; HRMS-ESI-TOF m/z 397.1551 ([M þ H]^þ,

C₂₅H₂₀N₂O₃requires 397.1547). The enantiomers were separated

using a semipreparative chiral phase HPLC column (Daicel ChiraCel

OD, 10 μm, 2 cm ꢁ 25 cm, 10% EtOHꢀhexanes, 7 mL/min, R = 1.35).

(S)-12: [R]²³_Dꢀ2.0 (c 0.1, THF). (R)-12: [R]²³_Dþ1.8 (c 0.1, THF).

Methyl 6-(2-(6-Phenoxy-1,2,3,4-tetrahydronaphthalene-

2-carbonyl)oxazol-5-yl)picolinate (13). 2-((tert-Butyldimethylsi-

lyloxy)(6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)-5-(tribu-

tylstannyl)oxazole (5.0 g, 6.89 mmol), Pd(PPh₃)₄(800 mg, 0.68 mmol),

and methyl 6-bromopicolinate (2.0 g, 8.96 mmol) were dissolved in

anhydrous 1,4-dioxane (30 mL), and the mixture was warmed at

reflux for 16 h under Ar. The reaction mixture was diluted with EtOAc,

washed with saturated aqueous NaCl, and dried over Na₂SO₄, and the

solvent was removed under reduced pressure. Flash chromatography

(SiO₂, 30% EtOAcꢀhexanes) yielded methyl 6-(2-((tert-butyldimethyl-

silyloxy)(6-phenoxy-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)oxazol-

6-(2-(6-Phenoxy-1,2,3,4-tetrahydronaphthalene-2-carbo-

nyl)oxazol-5-yl)picolinic Acid (14). Each pure enantiomer (S)-13

and (R)-13 (0.010 mmol) was dissolved in 1,2-dichloroethane, and after

addition of trimethyltin hydroxide (3 equiv), the mixture was warmed at

70 ꢀC for 16 h. The mixture was concentrated in vacuo and diluted with

EtOAc, and the organic layer was washed with aqueous 0.01 N KHSO₄

and saturated aqueous NaCl and dried over Na₂SO₄. Evaporation in

vacuo yielded the crude acid, which was purified by flash chromatogra-

phy (SiO₂, 5% HOAcꢀEtOAc) to yield 6-(2-(6-phenoxy-1,2,3,4-tetra-

hydronaphthalene-2-carbonyl)oxazol-5-yl)picolinic acid (14, 70%) as a

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yellow solid: ¹H NMR (CDCl₃þ 0.1% TFA, 600 MHz) δ 8.34 (d, 1H,

J = 6.0 Hz), 8.22ꢀ8.19 (m, 2H), 7.98 (s, 1H), 7.36 (t, 2H, J = 8.0 Hz),

7.13ꢀ7.10 (m, 2H), 7.03 (d, 2H, J = 7.8 Hz), 6.85ꢀ6.78 (m, 2H),

3.85ꢀ3.84 (m, 1H), 3.13ꢀ3.09 (m, 2H), 2.96ꢀ2.90 (m, 2H),

2.34ꢀ2.31 (m, 1H), 1.97ꢀ1.94 (m, 1H); ¹³C NMR (CDCl₃þ 0.1%

TFA, 150 MHz) δ 191.0, 157.2, 156.8, 155.3, 151.2, 145.0, 140.5, 136.8,

130.2, 129.7 (2C), 128.9, 127.9, 125.8, 125.2, 123.2, 118.9 (2C), 118.6,

117.1, 43.9, 30.2, 28.5, 25.7; HRMS-ESI-TOF m/z 441.1451 ([M þ

H]^þ, C₂₆H₂₀N₂O₅requires 441.1445). (S)-14: [R]²³ꢀ5.5 (c 0.7,

D

THF). (R)-14: [R]²³_Dþ5.4 (c 0.6, THF).

FAAH Inhibition. ¹⁴C-labeled oleamide was prepared from ¹⁴C-

labeled oleic acid as described.¹³The truncated rat FAAH (rFAAH) was

expressed in E. coli and purified as described.⁷¹The 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,²and the lysate of hFAAH-transfected COS-7 cells was used in

the inhibition assays where explicitly indicated. The inhibition assays

were performed as described.¹³In brief, the enzyme reaction was

initiated by mixing 1 nM rFAAH (800, 500, or 200 pM rFAAH for

inhibitors with K_ie 1ꢀ2 nM) with 20 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.¹³The K_iof the inhibitor was

calculated using a Dixon plot as described (standard deviations are

provided in the Supporting Information tables). LineweaverꢀBurk

analysis was performed as described⁵³confirming competitive, reversible

inhibition for 12 and 14, Figure 15.

In Vivo Pharmacodynamic Studies with Inhibitors. Inhibi-

tors were prepared as a salineꢀemulphor emulsion for ip administration

by vortexing, sonicating, and gently heating neat compound directly into

an 18:1:1 v/v/v solution of saline/ethanol/emulphor or as a homo-

geneous PEG solution for po administration by vortexing, sonicating,

and gently heating neat compound directly into PEG300 (Fluka). Male

C57Bl/6J mice (<6 months old, 20ꢀ28 g) were administered inhibitors

in salineꢀemulphor emulsion or an 18:1:1 v/v/v saline/emulphor/

ethanol vehicle ip at a volume of 10 μL/g weight, or alternatively

inhibitors in PEG300 or a PEG300 vehicle po at a volume of 4 μL/g

weight. After the indicated amount of time, mice were anesthetized with

isofluorane and killed by decapitation. Total brains (∼400 mg) and a

portion of the liver (∼100 mg) were removed and flash frozen in liquid

N₂. Animal experiments were conducted in accordance with the guide-

lines of the Institutional Animal Care and Use Committee of The

Scripps Research Institute.

Measurement of Brain Lipids. Tissue was weighed and subse-

quently Dounce homogenized in 2:1:1 v/v/v CHCl₃/MeOH/Tris, pH

8.0 (8 mL), containing standards for lipids (50 pmol of d₄-PEA, 2 pmol

of d₄-anandamide, 0.5 nmol of d₅-2-AG, and 10 nmol of pentadecanoic

acid). The mixture was vortexed and then centrifuged (1400 ꢁ g,

10 min). The organic layer was removed, dried under a stream of N₂, and

resolubilized in 2:1 v/v CHCl₃/MeOH (120 μL), and 10 μL of this

resolubilized lipid was injected onto an Agilent G6410B QQQ instru-

ment. LC separation was achieved with a Gemini reverse-phase C18

column (5 μm, 4.6 mm ꢁ 50 mm, Phenomonex) together with a

precolumn (C18, 3.5 μm, 2 mm ꢁ 20 mm). Mobile phase A was

composed of a 95:5 v/v H₂O/MeOH, and mobile phase B was

composed of a 65:35:5 v/v/v i-PrOH/MeOH/H₂O. Formic acid

(0.1%) or ammonium hydroxide (0.1%) was included to assist in ion

formation in positive and negative ionization mode, respecitvely. The

flow rate for each run started at 0.1 mL/min with 0% B. At 5 min,

the solvent was immediately changed to 60% B with a flow rate of

Figure 15. LineweaverꢀBurk analysis of 12 and 14 illustrating rever-

sible, competitive inhibition of FAAH.

0.4 mL/min and increased linearly to 100% B over 10 min. This was

followed by an isocratic gradient of 100% B for 5 min at 0.5 mL/min

before equilibrating for 3 min at 0% B at 0.5 mL/min (23 min total per

sample). MS analysis was performed with an electrospray ionization

(ESI) source. The following MS parameters were used to measure the

indicated metabolites in positive mode (precursor ion, product ion,

collision energy in V): anandamide (348, 62, 11), OEA (326, 62, 11),

PEA (300, 62, 11), d₄-anandamide (352, 66, 11), d₄-PEA (304, 62, 11),

2-AG (379, 287, 8), and d₅-2-AG (384, 287, 8). For negative polarity, the

analysis was performed in MS2 scan mode from 100ꢀ1000 m/z. The

capillary was set to 4 kV, the fragmentor was set to 100 V, and the delta

EMV was set to 0. Lipids were quantified by measuring the area under

the peak in comparison to the standards.

Mouse Tail Flick Assay. Male CD-1 (25ꢀ35 g, Charles River)

mice were housed in groups of five in Plexiglas chambers with food and

water available ad libitum. All animals were maintained on a 12 h light/

dark cycle (lights on at 7:00 a.m.) in a temperature- and humidity-

controlled animal colony. All tail-flick experiments were performed

under an approved University of New England animal protocol in

accordance with institutional guidelines and in accordance with the

Guide for the Care and Use of Laboratory Animals as adopted and

promulgated by the National Institutes of Health. Efficacy of the test

compound was assessed using the 52 ꢀC warm water tail-flick test. The

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latency to the first sign of a rapid tail-flick was taken as the behavioral

end point. Each mouse was first tested for baseline latency by immersing

its tail in the water and recording the time to response. Mice not

responding within 5 s were excluded from further testing. Mice were

then administered the test compound and tested for antinociception at

various time points afterward. Antinociception was calculated by the

following formula: % antinociception = 100 ꢁ (test latency ꢀ control

latency)/(10 ꢀ control latency). A maximum score was assigned

(100%) to animals not responding within 10 s to avoid tissue damage.

Chronic Constriction Injury (CCI). Surgery was performed as

described previously.⁸³Briefly, the right hind leg of male C57BL/6 mice

was shaved and swabbed with betadine solution and ethanol. Posterior

to the femur, an incision was made and the sciatic nerve was visualized

and isolated, following muscle separation. The nerve was ligated twice

with 5-0 (1.0 metric) black silk braided suture (Surgical Specialties

Corporation, Reading, PA). The surrounding muscle and skin were then

sutured with 6-0 nylon. Mice were recovered in a heated cage and

observed for approximately 2 h before being returned to the vivarium.

Anesthesia was maintained by constant inhalation of 1.5% isoflurane. In

addition, mice were administered acetaminophen (2.4 mg/mL in

drinking water) from 24 h before surgery through 48 h post surgery.

Allodynia Assays. Allodynia was initially tested 10 days after

surgery. Male C57BL/6 mice were habituated to the test apparatus for

2 h on the 2 days prior to testing. On the test day, the mice were brought

into the test room, weighed, and allowed to acclimate for at least 1 h

before the start of the experiment. Mice were administered inhibitor 12

(50 mg/kg) in PEG300 (4 μL/g body weight) or vehicle (po), then

placed in ventilated polycarbonate cylinders on a mesh table. Mechanical

(von Frey) and cold (acetone-induced) allodynia were tested at 1, 3, 6,

and 9 h post-drug administration. Testing was carried out by a separate

observer who was blinded to treatment conditions. Mechanical allodynia

was assessed with von Frey filaments (North Coast Medical, Morgan

Hill, CA), using the “upꢀdown” method.^61d,83Each hind paw was

stimulated 5 times per filament (0.16ꢀ6.0 g), starting with the 0.6 g

filament and increasing weight. Paw clutching or lifting in response to

three or more stimulations was coded as a positive response. Once a

positive response was detected, sequentially lighter-weight filaments

were used to assess paw withdrawal threshold. Approximately 30 min

after completing the von Frey test, cold allodynia was tested by

propelling 10 μL of acetone (Fisher Bioscience) via air burst, from a

200 μL pipet (Rainin Instruments, Oakland, CA) onto the plantar

surface of each hind paw. Total time lifting or clutching each paw was

recorded, with a maximum time of 20 s.⁸⁴

of 100 K. The cocrystal structure of FAAH bound to 12 was solved at

1.90 Å resolution (Table S1, Supporting Information). Data processing

was performed using the XDS software package, and the structure was

solved by molecular replacement (Phaser, CCP4 package⁸⁵) using the

coordinates from a previous h/rFAAH structure (PDB code 2WJ1) as a

search model and refined using programs Phenix suite, coot, Refmac5,

and BUSTER.⁸⁶Chemical parameters for the inhibitors were calculated

by the Dundee PRODRG Web server.⁸⁷For the last steps of refinement,

TLS (Translation/Libration/Screw) parametrization has been applied

by dividing each monomer in eight partitions (16 partitions in the

crystallographic asymmetric unit).

Molecular Modeling and Binding Energy Evaluation. The

program ICM-Pro (Molsoft, L.L.C.), employing a Monte Carlo mini-

mization algorithm for energy stochastic optimization of ligands, has

been previously adopted for covalent docking simulation and binding

energy calculation of FAAH inhibitors. Here, the coordinates of h/

rFAAH crystal structure with bound 12 (PDB 3OJ8) were used for the

simulation of both the (R)- and (S)-enantiomers (X and Y), and binding

energies were calculated. The energy functions included the following

ICM terms: van der Waals (vw) and 1ꢀ4 van der Waals, hydrogen

bonding (hb), electrostatics (el), entropic free energy, and constant

surface tension (sf). Estimation of the electrostatic energy was accurately

calculated with analytical molecular surface as dielectric boundary.⁸⁸

After addition of hydrogens, application of global energy minimization,

and assignment of partial charges, the inhibitor was removed from the

model and each enantiomer was created in silico and manually con-

jugated to the γ-oxygen of the catalytic Ser241. The docking of (S)-12

was, as expected, fully superimposable to the experimentally (X-ray)

determined structure.

’ ASSOCIATED CONTENT

S

Supporting Information. Full experimental details and

b

characterization of the candidate inhibitors, FAAH assay mea-

surement errors, purities of the FAAH inhibitors disclosed, table

of data processing and reﬁnement statstics for the FAAH-12

X-ray structure. This material is available free of charge via the

Internet at http://pubs.acs.org.

Accession Codes

Pdb deposition code: faah-12 (3oj8).

’ AUTHOR INFORMATION

Data Analyses. Behavioral data were analyzed using a two-way mixed

factorial analysis of variance (ANOVA) for each paw, with drug

treatment as the between subjects measure and time as the within

subjects measure. Follow-up comparisons were made using the Bonfer-

roni test. All animals were included in the analyses. Differences between

groups were considered statistically significant at p < 0.05.

Corresponding Author

*Phone: 858-784-7522. Fax: 858-784-7550. E-mail: boger@

scripps.edu.

’ ACKNOWLEDGMENT

FAAH Production, Crystallization, and Crystal Structure

Determination. The N-terminal transmembrane-deleted humanized

We gratefully acknowledge the ﬁnancial support of the

National Institutes of Health (Grants DA015648, D.L.B.;

DA009789 and DA017259, B.F.C.; DA017259, R.C.S.;

NS052727-01A2, E.J.B.; DA007027, DA009789, and DA017259,

A.H.L.) and thank Raj K. Chadha for the X-ray crystal

structure of 21.

version of FAAH (amino acids 32ꢀ579) was expressed in E. coli and

purified as previously described,⁷⁵using 0.08% n-undecyl-β-D-maltoside

in the ion exchange and size exclusion chromatography steps of the

purification. Samples of pure protein were concentrated up to 35 mg/

mL and supplemented with 13% xylitol and 2% benzyldimethyl(2-

dodecyloxyethyl)ammonium chloride (Aldrich). Large crystals of h/

rFAAH were obtained using a reservoir buffer (ratio 1:1) containing

100 mM MES, pH 5.5, 100 mM KCl, 30% PEG400, and 8% poly-

propylene glycol-P400 by sitting drop vapor diffusion at 14 ꢀC in 96-well

plates (Innovaplate SD-2, Innovadyne Technologies, Inc.). The crystals

were frozen directly in liquid nitrogen and complete data sets were

collected from a single crystal at the Stanford Synchrotron Radiation

Laboratory (SSRL, Menlo Park, CA) on beamline 11-1 at a temperature

’ ABBREVIATIONS

2-AG, 2-arachidonylglycerol; CB, cannabinoid; CCI, chronic

constriction injury; DEAD, diethyl azodicarboxylate; EDCI,

1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlor-

ide; FAAH, fatty acid amide hydrolase; hERG, human ether-a-

go-go related gene; MTI, mild thermal injury; hlm, human liver

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microsomes; OEA, N-oleoyl ethanolamine; PEA, N-palmitoyl

ethanolamine; rlm, rat liver microsomes; SNL, spinal nerve liga-

tion; TBS, tert-butyldimethylsilyl; TFAA, triﬂuoroacetic anhy-

dride; TGH, triacylglycerol hydrolase; TsCl, p-toluenesulfonyl

chloride

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Reversible competitive α-ketoheterocycle inhibitors of fatty acid amide hydrolase containing additional conformational constraints in the acyl side chain: Orally active, long-acting analgesics

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