because 
 of the therapeutic potential of inhibiting fatty acid amide 
 hydrolase ( faah ) for the treatment of pain , inflammatory , or sleep disorders , there is a continuing interest in the development 
 of selective inhibitors of the enzyme .
the distribution of faah is consistent with its role in regulating 
 signaling fatty acid amides including anandamide ( 1a ) and oleamide ( 1b ) at their sites of action ( figure 1 ) .
although 
 faah is a member of the amidase signature family of serine hydrolases 
 for which there are a number of prokaryotic enzymes , it is the only 
 well - characterized mammalian enzyme bearing the family s unusual 
 ser  ser  lys catalytic triad .
early studies following the initial identification of the 
 enzyme 
 led to the disclosure of a series of substrate - inspired inhibitors 
 that were used to characterize the enzyme as a serine hydrolase .
subsequent studies disclosed several classes of inhibitors that 
 provide opportunities for the development of inhibitors with therapeutic 
 potential .
these include the reactive aryl carbamates and ureas that irreversibly carbamylate the faah active site catalytic serine .
a second , and one of the earliest classes , is 
 the -ketoheterocycle - based inhibitors that bind to faah by reversible hemiketal formation with the active 
 site catalytic serine . many of these reversible , competitive inhibitors 
 have been shown to be selective for faah versus other mammalian serine 
 hydrolases as well as efficacious analgesics in vivo . in these studies , 2 ( ol-135 ) emerged as a potent ( ki = 4.7 nm ) and selective ( > 60300 fold ) prototypical faah inhibitor that induces analgesia 
 and increases endogenous anandamide levels .
it lacks significant off - site target activity , does not bind cannabinoid 
 ( cb1 or cb2 ) or vanilloid ( trp ) receptors , and does not significantly 
 inhibit common p450 metabolism enzymes or the human ether - a - go - go 
 related gene product ( herg ) .
the analgesic effects of 2 are observed without the respiratory depression or chronic dosing 
 desensitization characteristic of opioid administration or the increased 
 feeding and decreased motor control characteristic of cannabinoid 
 ( cb ) agonist administration .
it possesses 
 a relatively short duration of in vivo activity relative to irreversible 
 inhibitors , although further conformational constraints in the c2 
 acyl chain of 2 have provided inhibitors that are not 
 only orally active but also exhibit extended durations of in vivo 
 activity .
activity 
 relationship ( sar ) studies on 2 exploring substitution 
 of the central oxazole , the c2 acyl side chain , and the central heterocycle , the x - ray characterization of inhibitor - bound complexes defined 
 key features that impact inhibitor affinity and selectivity .
these include not only the ser241 hemiketal formation with the inhibitor 
 electrophilic carbonyl and its interaction with the enzyme oxyanion 
 hole but also an unusual ser217-mediated oh h - bond 
 to the activating heterocycle and the key anchoring interaction of 
 the terminal phenyl group of the c2 acyl chain . the structural studies 
 also revealed that cys269 is located adjacent to c5 of the inhibitor 
 pyridine substituent , which in turn is engaged in a series of intricate 
 interactions in the enzyme cytosolic port .
herein , we report results of a systematic study of candidate inhibitors 
 containing modifications at the pyridyl c5-position of 2 and related inhibitors that in principle could covalently trap this 
 proximal cys269 to provide inhibitors that alkylate or cross - link 
 the faah active site . in turn
, this could be expected to enhance their 
 potency , potentially enhance their selectivity , and extend their in 
 vivo duration of action ( figure 2 ) .
herein , 
 we detail the systematic inhibitor modifications that led to the discovery 
 and characterization of such inhibitors and the unexpected trends that the additional strategically placed 
 electrophiles display .
the series 1 analogues 
 ( 322 ) were accessed from 5-(tributylstannyl)oxazole 1e(36 ) by stille coupling with the appropriate 2-chloro- or 2-bromopyridine 
 ( scheme 1 ) .
this was followed typically by 
 tbs ether deprotection 
 ( bu4nf ) and oxidation of the liberated alcohol with dess 
martin 
 periodinane ( dmp ) to provide the corresponding 
 -ketoheterocycles : 3 , 7 , 9 , 14 , and 1822 .
the 
 remaining inhibitors were accessed by further modification of the 
 pyridyl c5 substituent ( scheme 1 ) .
the second series , in which the pyridine of 2 is replaced 
 with an alkyl linker to the pendant electrophile , was accessed by 
 sonogashira coupling of 5-bromooxazole 1f(43 ) with the appropriate alkyne ( scheme 2 ) .
the alkyne intermediate was reduced to the corresponding 
 alkane with h2 and palladium on carbon or palladium hydroxide . 

this was followed by tbs ether deprotection ( bu4nf ) and 
 oxidation of the liberated alcohol with dess 
martin periodinane 
 ( dmp ) to yield the series 2 c5-substituted oxazoles : 23 and 28
. further elaboration of the terminal electrophile 
 ( r group ) yielded the remaining compounds : 2427 and 2932 .
the initial characterization of the 
 candidate inhibitors and their comparison with 2 was 
 conducted using purified recombinant rat faah ( rfaah ) expressed in escherichia coli(55 ) at 
 2023 c as previously disclosed .
the initial rates of hydrolysis ( > 1020% reaction ) were 
 monitored 
 using enzyme concentrations below the initially measured ki values by following the breakdown of c - oleamide , 
 and ki values were established as previously 
 described ( dixon plot ) .
series 1 was developed directly on the basis of 2 ( ki = 4.7 nm ) , placing a potential thiol - capturing 
 electrophile at the 5-position of the pyridine ring ( 58 , 11 , 12 , 14 , and 1622 ) .
thioesters 5 and 16 were expected to be the most straightforward 
 traps for the cys269 thiol by thioester exchange . without preincubation 
 of the inhibitors with the enzyme , these inhibitors along with their 
 precursors ( 322 ) were tested for 
 binding and inhibition of rfaah ( figure 3 ) . 

all display potencies similar to 2 , exhibiting ki values in the low nanomolar range . in series 
 2 ,
the pyridine ring was replaced by an alkyl chain of appropriate 
 length capped with the thiol - engaging moiety .
as modeled , this flexible 
 linker is able to reach through the cytosolic pocket and place the 
 potentially reactive electrophile proximal to cys269 . like the series 
 1 inhibitors and without preincubation with the enzyme , all series 
 2 inhibitors exhibited effective faah inhibition with potencies that 
 approach or match that of 2 ( figure 3 ) . because the cys269 
 alkylation was expected to be slow relative to the rapid hemiketal 
 formation , the time - dependent inhibition of faah was examined .
this 
 was accomplished by preincubation of the inhibitors with recombinant 
 rfaah for a period of 16 h. as previously observed , reversible , 
 competitive inhibitor 2 does not display time - dependent 
 inhibition of faah , and its ki value remains 
 unchanged with the enzyme 
in contrast , a select 
 subset of inhibitors ( 11 , 14 , 17 , and 2022 ) in series 1 exhibited 
 significant increases in potency , displaying 220-fold improvements 
 in ki over the same time period , consistent 
 with slow irreversible inhibition of faah .
surprisingly , thioesters 5 or 16 did not exhibit this time - dependent increase 
 in enzyme inhibition potency .
similarly , chloride 12 was 
 found to be relatively nonpotent and insensitive to preincubation 
 with the enzyme , whereas the corresponding bromide 11 was initially more potent and exhibited the most pronounced time - dependent 
 increase in potency of all inhibitors .
both nitrile 7 and its imidate 8 , where the candidate electrophile 
 is attached directly to the pyridyl ring , did not display time - dependent 
 increases in potency , whereas both the homologated nitrile 14 and its imidate 17 , where a methylene spacer separates 
 the electrophile and pyridyl ring , did exhibit increases in potency 
 with the enzyme 
inhibitor preincubation . of the series of inhibitors 
 that might be expected to serve as michael acceptors for a thiol conjugate 
 addition ( 1822 ) , including the ,-unsaturated 
 ester 18 and nitrile 19 , only those bearing 
 the weaker activating substituents ( 2022 vs 18 and 19 ) that would be expected to 
 react slower and to be intrinsically less reversible displayed the 
 exceptionally potent and time - dependent faah inhibition improvements . 
 notably and throughout this series , it was not the anticipated electrophiles 
 that exhibited the time - dependent inhibition of faah characteristic 
 of a slow irreversible inhibitor , but rather it was a less - well - recognized 
 alternative ( 14 and 17 vs 16 , 11 vs 12 , and 2022 vs 1819 ) .
finally , no 
 inhibitor in series 2 that bears the flexible linker to the second 
 electrophile displayed the time - dependent increases in potency , indicating 
 that the conformationally restricted placement of the second electrophile 
 is important to observation of the targeted alkylation .
for the inhibitors 
 that displayed time - dependent increases in inhibitor potency , enzyme 
 activity did not recover after this time period and is indicative 
 of irreversible enzyme inhibition .
the compounds 
 that demonstrated time - dependent improvements in potency were further 
 investigated by lineweaver 
-ketoheterocycle inhibitors including 2 were shown to display well - behaved competitive , reversible inhibition 
 kinetics . despite expectations but consistent with the lack of time - dependent 
 faah inhibition , lineweaver  burk kinetic analysis of thioesters 5 , 16 , and 25 after 3 h preincubation 
 with the enzyme confirmed that they also behave as reversible , competitive 
 inhibitors , analogous to 2 and related -ketoheterocycle 
 inhibitors ( figure 5 ) .
thus , despite the expectations 
 of a facile transthioesterification with cys269 , the thioesters exhibit 
 enzyme inhibition characteristic of reversible inhibitors , suggesting 
 that reaction with cys269 does not occur .
significantly , thioesters 5 and 25 were 
 recovered unchanged from the assay buffer ( 6 h ) and from enzymatic 
 assays ( 5 ) , indicating that they are not undergoing chemical 
 hydrolysis or transient enzyme adduct formation and subsequent hydrolysis 
 under the conditions of the assay .
burk kinetic analysis 
 of 5 , 16 , and 25 demonstrate 
 reversible , competitive 
 inhibition .
in contrast , the inhibitors 
 that demonstrated a time - dependent 
 increase in inhibitor potency also exhibited noncompetitive inhibition 
 of faah when preincubated with the enzyme for 3 h prior to lineweaver 
this is expected 
 of irreversible enzyme inhibition and consistent with cys269 alkylation 
 or addition to the pendant electrophile . in the case of 11 , this entails cys269 thiol nucleophilic displacement of the benzylic 
 bromide to provide the corresponding thioether , and its structure 
 has been confirmed by x - ray analysis of the inhibitor bound to faah .
the noncompetitive enzyme inhibition presumably 
 entails thiol nucleophilic addition to the electrophile to provide 
 the cys269-linked thioimidate for 14 and 17 , and it presumably involves an apparent irreversible thiol conjugate 
 addition to 2022 .
interestingly , 
 both the ,-unsaturated nitrile 19 and ester 18 , which do not exhibit time - dependent increases in inhibitor 
 activity or the potent ki values consistent 
 with irreversible inhibition , displayed mixed kinetics , exhibiting 
 competitive inhibition at low inhibitor concentrations and noncompetitive 
 inhibition at high concentrations .
presumably , this indicates that 
 the thiol conjugate addition products derived from 18 and 19 are either formed less effectively or , more 
 likely , that they may be sufficiently reversible at 23 c to 
 less effectively trap cys269 as an apparent irreversible inhibitor 
 of the enzyme .
burk analysis demonstrates noncompetitive 
 faah 
 inhibition for ( a ) 11 , ( b ) 14 , ( c ) 17 , ( d ) 20 , ( e ) 21 , and ( f ) 22
. dialysis dilution ( 4 
 c , 18 h , 370-fold ) of the faah - inhibitor mixture following 3 
 h of preincubation with 2 restored full enzyme activity , 
 consistent with its reversible enzyme inhibition , whereas the mixtures 
 containing 11 , 14 , and 1722 remained relatively unchanged , failing to restore faah 
 activity , indicative of irreversible enzyme inhibition under the conditions 
 monitored ( 4 c , ph 9 , figure 7 ) .
it is 
 notable that 14 and 17 ( not shown ) , which 
 presumably form a cys269 thioimidate adduct , do not appear to be even 
 slowly reversible under these conditions .
similarly , 2022 displayed irreversible inhibition of faah , 
 consistent with their time - dependent , noncompetitive enzyme inhibition . 

interestingly , dialysis dilution at 4 c also did not restore 
 enzyme activity with both the ,-unsaturated nitrile 19 and ester 18 , which do not exhibit time - dependent 
 faah inhibition and displayed concentration - dependent mixed competitive / noncompetitive 
 kinetics in the lineweaver 
this 
 suggests that their inhibition of faah following the 3 h incubation 
 ( 22 c ) is not reversible at 0 c .
unfortunately , the reversibility 
 of 18 and 19 at 23 c could not be 
 established because of the instability of faah at 23 c over 
 the dialysis time frame .
faah mixtures 
 illustrates 
 reversible inhibition by 2 and establishes irreversible 
 faah inhibition by 11 , 14 , and 1722 .
after 3 h preincubation of purified recombinant 
 rat faah with compounds at concentrations that result in inhibition 
 of ca .
80% enzyme activity ( 22 c ; 3 h ; 100 nm , 2 ; 80 nm , 11 and 14 ; 100 nm , 18 and 19 ; 150 nm , 20 ; and 80 nm , 21 and 22 ) , and following measurement of residual enzyme 
 activity , dialysis dilution ( 4 c , 18 h , 370-fold dilution ) of 
 the mixtures resulted in nearly full recovery of enzyme activity for 2 but little or no recovery of enzyme activity for 11 , 14 , and 1722 under 
 the conditions monitored ( 4 c , ph 9 ) ; conducted in triplicate 
 and reported as the percent enzyme inhibition  sd .
the selectivity 
 of the time - dependent , 
 irreversible faah inhibitors 17 and 2022 were examined along with 11 and 14 that were recently disclosed using 
 activity - based protein profiling ( abpp ) of the serine hydrolases .
abpp methods permit the testing of serine hydrolases 
 in their native state and eliminate the need for their recombinant 
 expression , purification , and the development of specific substrate 
 assays .
because inhibitors are screened against many enzymes in the 
 proteome in parallel , both relative potency and selectivity can be 
 simultaneously evaluated .
previous studies have shown that the -ketoheterocycle 
 class of inhibitors are selective for faah , although four enzymes 
 have emerged as potential competitive targets : triacylglycerol hydrolase 
 ( tgh ) ,  hydrolase containing domain 6 ( abhd6 ) , monoacylglycerol 
 lipase ( magl ) , and the membrane - associated hydrolase kiaa1363 .
each 
 inhibitor was tested for its effects on the fluorophosphonate ( fp)-rhodamine 
 probe labeling of serine hydrolases in the mouse brain ( contains kiaa1363 , 
 magl , and abhd6 ) and heart membrane ( contains tgh ) proteome at concentrations 
 ranging from 10 nm to 100 m .
the selectivity assessments were 
 conducted following 6 h inhibitor incubation with the proteomes and 
 all inhibitors showed superb selectivity for faah over kiaa1363 and 
 abhd6 ( > 10-fold ) , excellent selectivity over magl ( > 200-fold ) , 
 and good selectivity over tgh ( figure 8) .
abpp selectivity 
 screen in mouse brain membrane proteome ( 1 mg / ml ) 
 with fp - rhodamine ( 100 nm ) ,
inhibitor 
 preincubation with the proteome was conducted for 6 h. in initial efforts 
 to screen for in vivo inhibition of faah and its subsequent pharmacological 
 effects , the set of inhibitors displaying the time - dependent , irreversible 
 faah inhibition ( 11 , 14 , 17 , and 2022 ) were examined alongside 
 of 2 for their ability to increase the endogenous levels 
 of a series of lipid amide signaling molecules that are substrates 
 for faah in both the brain ( cns effect ) and liver ( peripheral effect , 
 not shown ) .
thus , the effects of the inhibitors on the endogenous 
 levels of the faah substrates anandamide ( aea ) , oleoyl ethanolamide 
 ( oea ) , and palmitoyl ethanolamide ( pea ) were measured .
notably , it 
 is the increase in endogenous levels of anandamide and its subsequent 
 action at cannabinoid ( cb1 and cb2 ) receptors that are thought to 
 be responsible for the analgesic and anti - inflammatory effects of 
 faah inhibitors .
administration of inhibitor in three mice per time point for 
 an initial screen ( 30 mg / kg ) .
significantly , increases in endogenous 
 levels of anandamide in the brain requires > 90% inhibition of faah 
 for in vivo enzyme inhibition . with the 
 exception of imidate 17 , which matched the increased 
 anandamide levels observed with 2 after 3 h , each of 
 the additional inhibitors proved to be roughly equivalent ( 11 , 14 , and 20 > 21 and 22 ) , increasing anandamide levels approximately 2-fold over 
 that of 2 and approximately 3-fold over vehicle treatment 
 ( figure 9 ) .
, 
 30 mg / kg , n = 3 ) . with pea and oea , which show significant enhancements in 
 endogenous 
 levels with partial enzyme inhibition and
are less sensitive to the 
 extent of faah inhibition , all of the inhibitors that displayed time - dependent , 
 irreversible faah inhibition matched or exceeded the activity of 2 , producing elevations of 312-fold over vehicle . 
 of these
as a result , more detailed dose- 
 and time - dependent studies of 11 and 14 were 
 conducted as reported elesewhere .
the 
 results of these studies revealed that they cause accumulation of 
 all three lipid amides in the brain with peak levels achieved within 
 1.53 h , that these elevations exceed those achieved with the 
 reversible inhibitor 2 , that these elevations are maintained 
 > 6 h ( vs 23 h for 2 ) , consistent with irreversible 
 enzyme inhibition , and that they exhibit long acting in vivo activity 
 in a mouse model of neuropathic pain .
the design , synthesis , and characterization 
 of -ketoheterocycles 
 that additionally target the remote cys269 nucleophile found in the 
 cytosolic port of faah provided inhibitors 
 that slowly react with the enzyme nucleophile , effectively providing 
 time - dependent , irreversible inhibitors of the enzyme that maintain 
 or enhance their selectivity for faah over other serine hydrolases . 

the electrophiles capable of targeting cys269 were incorporated as 
 a c5 substituent on the pyridyl group of the 5-(pyrid-2-yl ) oxazole 
 of 2 and ranged from the reactive benzylic bromide 11 to the otherwise benign nitrile 14 .
the irreversible 
 inhibitors of faah displayed an expected sensitivity to the position 
 of the electrophile introduction , but those that were successful exhibited 
 surprising trends in apparent reactivity toward cys269 that would 
 not be easily predicted .
a preliminary in vivo characterization of 
 the identified irreversible faah inhibitors confirmed their ability 
 to raise endogenous brain levels of the enzyme substrates , including 
 anandamide , in mice to a greater extent ( > 2-fold ) and for a longer 
 duration ( > 6 h ) than the reversible -ketoheterocycles on 
 which 
 they are based .
two of these ( 11 and 14 ) 
 were characterized in greater detail , as reported elsewhere , along 
 with their long acting in vivo efficacy in a mouse model of neuropathic 
 pain .
c - labeled oleamide was 
 prepared from c - labeled oleic acid as described .
the truncated rat faah ( rfaah ) was expressed 
 in e. coli and purified as described , and the purified recombinant rfaah was used 
 in the inhibition and reversibility assays unless otherwise indicated . 

the purity of each tested compound ( > 95% ) was determined on an 
 agilent 
 1100 lc / ms instrument using a zorbax sb - c18 column ( 3.5 mm , 4.6 mm 
  50 mm , with a flow rate of 0.75 ml / min and detection at 220 
 and 253 nm ) with a 1098% acetonitrile / water/0.1% formic acid 
 gradient ( two different gradients ) .
the enzyme reaction 
 was initiated by mixing 1 nm rfaah ( 800 , 500 , or 200 pm rfaah for 
 inhibitors with ki 
12 
 nm ) with 20 m c - labeled oleamide in 500 l 
 of reaction buffer ( 125 mm triscl , 1 mm edta , 0.2% glycerol , 0.02% 
 triton x-100 , and 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 l of the reaction 
 mixture to 500 l of 0.1 n hcl at three different time points . 

the c - labeled oleamide ( substrate ) and oleic acid ( product ) 
 were extracted with etoac and analyzed by tlc as detailed .
burk kinetic 
 analysis was performed as described , confirming 
 competitive , reversible inhibition for 5 , 16 , and 25 and noncompetitive inhibition for 11 , 14 , 17 , and 2022 ( figures 5 and 6 ) .
the reversibility 
 of faah inhibition by 2 , 11 , 14 , and 1722 was assessed by dialysis 
 dilution using purified recombinant rfaah .
the enzyme was placed in 
 15 ml of faah assay buffer ( 125 mm tris , 1 mm edta , 0.2% glycerol , 
 0.02% triton x-100 , and 0.4 mm hepes , ph 9.0 ) .
the dialysis experiment 
 was performed in the predialysis mix at or near the apparent ic80 .
the final assay inhibitor concentrations used were 100 
 nm , 2 , 18 , and 19 ; 80 nm , 11 , 14 , 21 , and 22 ; 
 and 150 nm , 20 .
samples were preincubated with the enzyme 
 for 3 h at room temperature ( 22 c ) before 300 l was removed 
 and assayed in triplicate in a faah activity assay .
the remaining 
 sample ( 2.7 ml ) was injected into a dialysis cassette employing a 
 10  000 mw cutoff membrane .
the mixture was dialyzed against 
 1 l of pbs at 4 c on a stir plate for 18 h. the postdialysis 
 faah activity was assessed by assaying 300 l samples taken 
 from the dialysis cassettes in triplicate .
faah activity is expressed 
 as a percentage of vehicle - treated faah ( dmso alone ) and is shown 
 in figure 7 .
mouse tissues were dounce - homogenized 
 in pbs buffer ( ph 8.0 ) , and 
 membrane proteomes were isolated by centrifugation at 4 c ( 100  000 g , 45 min ) , washed , resuspended in pbs buffer , and adjusted 
 to a protein concentration of 1 mg / ml . proteomes were preincubated 
 with inhibitors ( 10100  000 nm , dmso stocks ) for 6 h 
 and then treated with fp - rhodamine ( 100 nm , dmso stock ) at room temperature 
 for 10 min .
reactions were quenched with sds - page loading buffer , 
 subjected to sds - page , and visualized in - gel using a flatbed fluorescence 
 scanner ( mirabio ) .
labeled proteins were quantified by measuring integrated 
 band intensities ( normalized for volume ) ; control samples ( dmso alone ) 
 were considered to have 100% activity .
inhibitors 
 were prepared as a saline  emulphor emulsion for intraperitoneal 
 ( i.p . )
administration by vortexing , sonicating , and gently heating 
 neat compound directly in an 18:1:1 v / v / v solution of saline / ethanol / emulphor . 

male c57bl/6j mice ( < 6 months old , 2028 g ) were administered 
 inhibitors in saline  emulphor emulsion or an 18:1:1 v / v / v saline / emulphor / ethanol 
 vehicle i.p . at a volume of 10 l / g weight .
after the indicated 
 amount of time ( 1 , 3 , or 6 h ) , mice ( n = 3 for each 
 compound at each time point ) 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 guidelines of the institutional animal care and use committee 
 of the scripps research institute .
tissue was weighed and 
 subsequently 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-aea , 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 l ) , and 10 l of 
 this resolubilized lipid was injected onto an agilent g6410b qqq instrument . 

lc separation was achieved with a gemini reverse - phase c18 column 
 ( 5 m , 4.6 mm  50 mm , phenomonex ) together with a precolumn 
 ( c18 , 3.5 m , 2 mm  20 mm ) . mobile phase
a was composed 
 of 95:5 v / v h2o / meoh , and mobile phase b was composed of 
 65:35:5 v / v / v
. 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 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 ) .
the following 
 ms parameters were used to measure the indicated metabolites in positive 
 mode ( precursor ion , product ion , collision energy in v ) : aea ( 348 , 
 62 , 11 ) , oea ( 326 , 62 , 11 ) , pea ( 300 , 62 , 11 ) , d4-aea ( 352 , 66 , 11 ) , and d4-pea 
 ( 304 , 62 , 11 ) . the capillary was set to 4 kv , the ionization source 
 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 
 ( n = 3 for each inhibitor at each time point ) .
 a series 
 of -ketooxazoles incorporating electrophiles at 
 the c5 position of the pyridyl ring of 2 ( ol-135 ) and 
 related compounds were prepared and examined as inhibitors of fatty 
 acid amide hydrolase ( faah ) that additionally target the cytosolic 
 port cys269 . from this series , a subset of the candidate inhibitors 
 exhibited time - dependent faah inhibition and noncompetitive irreversible 
 inactivation of the enzyme , consistent with the targeted cys269 covalent 
 alkylation or addition , and maintained or enhanced the intrinsic selectivity 
 for faah versus other serine hydrolases . 
 a preliminary in vivo assessment 
 demonstrates that these inhibitors raise endogenous brain levels of 
 anandamide and other faah substrates upon intraperitoneal ( i.p . ) 
 administration 
 to mice , with peak levels achieved within 1.53 h , and that 
 the elevations of the signaling lipids were maintained > 6 h , indicating 
 that the inhibitors effectively reach and remain active in the brain , 
 inhibiting faah for a sustained period . 