Optimization of α-ketooxazole inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1021/jm701210y

A series of α-ketooxazoles containing conformational constraints in the flexible C2 acyl side chain of 2 (OL-135) and representative oxazole C5 substituents were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH). Exceptionally potent and selective FAAH inhibitors emerged from the series (e.g., 6, K_i = 200 and 260 pM for rat and rhFAAH). With simple and small C5 oxazole substituents, each series bearing a biphenylethyl, phenoxyphenethyl, or (phenoxymethyl)phenethyl C2 side chain was found to follow a well-defined linear relationship between -log K_i and Hammett σ_p of a magnitude (ρ = 2.7-3.0) that indicates that the substituent electronic effect dominates, confirming its fundamental importance to the series and further establishing its predictive value. Just as significantly, the nature of the C5 oxazole substituent substantially impacts the selectivity of the inhibitors whereas the effect of the C2 acyl chain was more subtle but still significant even in the small series examined. Combination of these independent features, which display generalized trends across a range of inhibitor series, simultaneously improves FAAH potency and selectivity and can provide exquisitely selective and potent FAAH inhibitors.

Full text of DOI:10.1021/jm701210y

J. Med. Chem. 2008, 51, 937–947
937
Optimization of r-Ketooxazole Inhibitors of Fatty Acid Amide Hydrolase
F. Scott Kimball, F. Anthony Romero, Cyrine Ezzili, Joie Garfunkle, Thomas J. Rayl, Dustin G. Hochstatter, Inkyu Hwang, and
Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
ReceiVed September 25, 2007
A series of R-ketooxazoles containing conformational constraints in the flexible C2 acyl side chain of 2
(OL-135) and representative oxazole C5 substituents were prepared and examined as inhibitors of fatty acid
amide hydrolase (FAAH). Exceptionally potent and selective FAAH inhibitors emerged from the series
(e.g., 6, Ki ) 200 and 260 pM for rat and rhFAAH). With simple and small C5 oxazole substituents, each
series bearing a biphenylethyl, phenoxyphenethyl, or (phenoxymethyl)phenethyl C2 side chain was found
to follow a well-defined linear relationship between -log Ki and Hammett σp of a magnitude (F ) 2.7–3.0)
that indicates that the substituent electronic effect dominates, confirming its fundamental importance to the
series and further establishing its predictive value. Just as significantly, the nature of the C5 oxazole substituent
substantially impacts the selectivity of the inhibitors whereas the effect of the C2 acyl chain was more
subtle but still significant even in the small series examined. Combination of these independent features,
which display generalized trends across a range of inhibitor series, simultaneously improves FAAH potency
and selectivity and can provide exquisitely selective and potent FAAH inhibitors.
Fatty acid amide hydrolase (FAAHa)1,2 is an enzyme that
serves as the catabolic regulator3,4 of several endogenous lipid
amides5,6 including anandamide (1a)7–10 and oleamide (1b)11–13
(Figure 1). Its distribution is consistent with its role of regulating
(degrading) such neuromodulating and signaling fatty acid
amides at their sites of action.3 Although it is a member of the
amidase signature family of serine hydrolases, for which there
are a number of prokaryotic enzymes, it is currently the only
characterized mammalian enzyme bearing the family’s unusual
Ser-Ser-Lys catalytic triad.1,2,14–17
Because of the therapeutic potential of inhibiting FAAH3,17–20
especially for the treatment of pain,21–23 inflammatory,24 or sleep
disorders13,25 and with the emerging concerns surrounding
alternative targets (e.g., COX-2 inhibitors), there has been an
increasing interest in the development of potent inhibitors of
this enzyme.26 Early studies led to the discovery that the
Figure 1. Substrates of fatty acid amide hydrolase (FAAH).
endogenous sleep-inducing molecule 2-octyl R-bromoacetoac-
cycle-based inhibitors51–59 that bind to FAAH via reversible
etate is an effective FAAH inhibitor27 and to the disclosure of
hemiketal formation with an active site serine. Not only are
a series of nonselective reversible inhibitors bearing an elec-
these competitive inhibitors potent and extraordinarily selective
trophilic ketone (e.g., trifluoromethyl ketone based inhibitors),28–31
for FAAH versus other mammalian serine hydrolases, but
as well as a set of irreversible inhibitors32–37 (e.g., fluorophos-
members of this class have been shown to be efficacious
phonates and sulfonyl fluorides). To date, only two classes of
analgesics in vivo.58,59
inhibitors have been disclosed that provide significant op-
In these preceding studies, 253 emerged as an important lead
portunities for the development of inhibitors with therapeutic
inhibitor for further study (Figure 2). It has been shown that 2
potential. One class is the reactive aryl carbamates and ureas38–50
is a potent (Ki ) 4.7 nM)53 and selective (>100- to 300-fold)31
that irreversibly acylate a FAAH active site serine49 and that
FAAH inhibitor that induces analgesia by raising endogenous
have been shown to exhibit anxiolytic activity38 and produce
anandamide levels.53,58,59 It has been shown to exhibit analgesic
analgesic effects.39 To date and where examined, the selectivity
activity in the tail flick assay,58 hot plate assay,58 formalin test
of such inhibitors has often been low,31,48–50 further complicating
of noxious chemical pain (first and second phases),58 the mild
the development of inhibitors that irreversibly and covalently
thermal injury (MTI) model of peripheral pain,59 and the spinal
modify the target enzyme. A second class is the R-ketohetero-
nerve ligation (SNL) model of neuropathic pain59 with efficacies
that match or exceed those of morphine (at 1–3 mg/kg in MTI/
* To whom correspondence should be addressed. Phone: 858-784-7522.
SNL), ibuprofen (at 100 mg/kg in MTI), or gabapentin (at 500
Fax: 858-784-7550. E-mail: boger@scripps.edu.
a Abbreviations: CB1, cannabinoid 1 receptor; CB2, cannabinoid 2
receptor; EDTA, ethylenediaminetetraacetic acid; EDCI, 1-ethyl-3-(3′-
dimethylaminopropyl)carbodiimide hydrochloride; FAAH, fatty acid amide
hydrolase; MTI, mild thermal injury; SAR, structure–activity relationship;
SNL, spinal nerve ligation; TGH, triacylglycerol hydrolase; TRP, vanilloid
receptors.
mg/kg in SNL) and at administered doses (10–20 mg/kg, ip)
that approach or exceed those of such common pain medica-
tions.59 It was shown to lack significant off-site target activity
(Cerep assay profiling), does not bind cannabinoid (CB1 or CB2)
or vanilloid (TRP) receptors, and does not significantly inhibit
10.1021/jm701210y CCC: $40.75
2008 American Chemical Society
Published on Web 02/05/2008

938 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Kimball et al.
Scheme 1
Scheme 2
Figure 2. Progression of the inhibitor series.
common P450 metabolism enzymes (3A4, 2C9, 2D6) or the
human ether-a-go-go related gene (hERG). Significantly, the
inhibitor was ineffective at promoting analgesia in FAAH
knockout mice,58,59 verifying that FAAH is the only relevant
target responsible for the in vivo analgesic effects of 2.
Moreover, the analgesic effects were observed without the
respiratory depression or chronic dosing desensitization char-
acteristic of opioid administration59 or the increased feeding and
decreased mobility and motor control characteristic of a can-
nabinoid (CB1) agonist administration.58,59
Consequently, we have conducted systematic structure–ac-
tivity relationship (SAR) studies on 2 independently targeting
the 5-position of the central oxazole (aryl and nonaromatic
substituents)53,55,56 and the C2 acyl side chain,53,57 both of which
have provided extraordinarily potent and selective FAAH
inhibitors (Figure 2).60 Herein, we report results of studies
examining candidate inhibitors formed by combining the
optimized or most representative C5 oxazole substituents with
optimized C2 acyl side chains along with results of the
proteome-wide selectivity screening31 of the resulting candidate
inhibitors.
Chemistry
in good conversions. In each case, deprotection of the TBS ether
followed by Dess-Martin periodinane oxidation of the liberated
alcohol yielded the corresponding R-ketooxazoles. Many such
substitution products served as intermediates en route to
additional key inhibitors. The C5 carboxylic acids were directly
converted to corresponding amides (RNH2 or R2NH, EDCI, and
HOAt) as well as to their corresponding methyl esters by
treatment with TMSCHN2. In turn, the esters were converted
to the carboxamides by treatment with methanolic ammonia and
the carboxamides were dehydrated with TFAA and pyridine to
provide the C5 nitriles. Similarly, the C5 iodides were trans-
formed (FSO2CF2CO2CH3, CuI) to candidate inhibitors bearing
a C5 trifluoromethyl substituent.65,66
The general method for the synthesis of the C5 aryl inhibitors
containing a conformationally restricted C2 acyl side chain is
shown in Scheme 1. Vedejs oxazole metalation61 and condensa-
tion with the appropriate side chain aldehyde were followed by
TBS protection of the resulting alcohol. Selective C5 oxazole
lithiation62 of these intermediates followed by treatment with
Bu3SnCl afforded the corresponding stannanes. Stille coupling63
of the stannanes with the arylpyridine halides produced the
substituted oxazoles, which could be readily converted to the
corresponding ketones via TBS deprotection (Bu4NF) and
oxidation of the liberated alcohol with Dess-Martin periodi-
nane.64
The synthesis of the candidate inhibitors that bear a nonaro-
matic C5 oxazole substituent is summarized in Scheme 2.
Following the analogous C5 oxazole lithiation, treatment with
various electrophiles (CO2(g), CF3CON(CH3)2, I2, dimethyl
disulfide) provided the corresponding C5 substituted oxazoles
Enzyme Assay
Enzyme assays were performed at 20–23 °C with purified
recombinant rat FAAH expressed in Escherichia coli67 (unless

R-Ketooxazole Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 939
Figure 4. Plot of -log Ki (µM) versus σp for biphenyl series.
Figure 3. Effect of nonaromatic substituents.
indicated otherwise) or with solubilized COS-7 membrane
extracts from cells transiently transfected with human FAAH
cDNA2 (where specifically indicated) in a buffer of 125 mM
Tris/1 mM EDTA/0.2% glycerol/0.02% Triton X-100/0.4 mM
Hepes, pH 9.0. The initial rates of hydrolysis (<10–20%
reaction) were monitored using enzyme concentrations (typically
1 nM) at least 3 times below the measured Ki by following the
breakdown of 14C-oleamide, and Ki values (standard deviations
are provided in the Supporting Information tables) were
established as described (Dixon plot). Lineweaver–Burk analysis
previously established reversible, competitive inhibition for 2
and related inhibitors.53
Results and Discussion
Many of the most potent inhibitors that emerged from
previous SAR studies of 2 contained conformational constraints
in the flexible C2 acyl side chain 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. Consequently, both their hydrophobic character and
π-unsaturation mimic the nature and location of the fatty acid
chain and its unsaturation (arachidonyl ∆5,6, ∆8,9, and ∆11,12
double bonds or oleyl ∆9,10 double bond) and the added
conformational restriction may contribute to their enhanced
binding affinity while simultaneously improving their drug-like
characteristics. Three such improved C2 side chains identified
in these studies were now incorporated into candidate inhibitors
that contain representative or optimized C5 oxazole aryl and
nonaryl substituents that impact FAAH potency, selectivity, and
physical properties (e.g., solubility).
Biphenyl Series. The series most extensively examined
contained the C2 biphenylethyl side chain. A set of candidate
inhibitors bearing representative small nonaryl C5 oxazole
substituents were examined and found to follow a well-defined
correlation55 between the electronic character of the substituent
and the inhibitor potency (Figure 3). Thus, the inhibitor potency
was found to smoothly increase as the electron-withdrawing
character of the substituent increased. As observed in an earlier
study,55 the magnitude of the effect is remarkably large and a
plot of the substituent Hammett σp constant versus -log Ki
provided a F value of 2.70 (slope ) F, R2 ) 0.97), indicating
that a unit change in σp leads to an increase in the Ki of nearly
1000-fold (Figure 4). As such and although such substituents
may engage in additional more subtle interactions at the enzyme
active site (H-bonding or hydrophobic interactions), the mag-
nitude of electronic effect indicates that the substituent electronic
character dominates the behavior of this series of relatively small
substituents. Presumably this reflects the electronic effect of the
Figure 5. Effect of pyridyl derivatives.
substituent on activating the electrophilic carbonyl toward
nucleophilic attack by the FAAH active site catalytic Ser.
A series of aryl C5 oxazole substituents were also examined
including a key set of substituted 2-pyridyl derivatives (Figure
5). Preceding studies have shown that such substituents exhibit
well defined potency trends (2-Pyr > 3-Pyr > 4-Pyr > Ph) that
correlate with the location and H-bond acceptor capabilities of
the weakly basic heterocycle substituent. Although this has been
discussed in detail elsewhere,53 the potency of such 2-Pyr
substituents exceeds that predicted by their intrinsic electron-
withdrawing properties further benefiting from a key H-bonding
interaction at the enzyme active site.56,68 Consistent with prior
SAR observations with 2,53 the inhibitor potencies followed the
order 14 > 24 > 27 (2-Pyr > 3-Pyr > 4-Pyr), suggesting that
the extensive heterocycle series explored with 251,53,56 would
provide analogous, albeit more potent, inhibitors in this biphe-
nylethyl series with 14 being among the most potent. Similarly,
the addition of electron-withdrawing and water-solubilizing
substituents weakly modulates the potency of such inhibitors
(15–23 vs 14) and many (e.g., -CO2H and 2-tetrazole) further

940 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Kimball et al.
measured Ki about active site binding that may not be known a
-
priori (e.g., -CO2H vs -CO2 binding).55,56
Additional Derivatives. A small series of additional deriva-
tives in the biphenylethyl series were also examined which
incorporate a basic amine at the terminus of the C2 acyl side
chain. These derivatives, which contain substituents that impact
the physical properties of compounds (solubility) and might be
expected to alter the PK properties, were prepared by late stage
reductive amination incorporation of the tertiary amine as shown
in Scheme 3. Interestingly and although not investigated in
detail, conducting the reductive amination on the intermediate
aldehyde containing the R-ketooxazole resulted only in reductive
amination of the aldehyde and not the R-ketooxazole. Moreover
and perhaps more surprisingly, the reactive R-ketooxazole was
not even reduced to the corresponding alcohol when the reaction
was conducted with NaBH(OAc)3, whereas it was competitively
reduced if NaCNBH3 was employed.
Figure 6. Conformational restriction in the C2 side chain.
The results of the examination of these derivatives are
summarized in Figure 10. Despite the incorporation of a polar,
weakly basic amine in the acyl side chain that occupies the
active site hydrophobic channel, the candidate inhibitors proved
to be effective FAAH inhibitors that typically displayed activity
that approached that of the parent biphenylethyl derivative.
Moreover, albeit in a small series, the activity smoothly parallels
the relative hydrophobic character of the derivatives (77 > 78
> 79 > 80, 76) with 75, 77, 78, 83, and 84 most effectively
approaching the activity of the corresponding parent derivatives
3, 15, and 17. In contrast, the much more basic and polar
N-methylpiperizine derivative 81 was much less active. Thus,
while polar functionality is poorly tolerated in the portion of
the enzyme hydrophobic channel that binds C1-C7 of the fatty
acid amide substrates,17 it is much more readily accommodated
at the far end of this channel.
Similarly, a small series of derivatives were examined in
which the second aryl ring in the C2 acyl side chain was
replaced with one containing a secondary, basic amine (Scheme
4). Although those that contained the free amine (87 and 89)
were relatively poor inhibitors of FAAH, the precursors in which
the free amine was protected as a carbamate (Boc derivative)
proved to be effective FAAH inhibitors (Figure 11).
enhance the intrinsic FAAH selectivity. Several such inhibitors
exhibit subnanomolar Ki values, and those of the carboxylic
acids (17, 19, 26, and 29) are artificially underestimated with
an in vitro enzymatic assay conducted at pH 9 where they are
fully deprotonated.
4-Phenoxyphenethyl and 4-(Phenoxymethyl)phenethyl Se-
ries. In the preceding SAR study of 2 and beyond the
biphenylethyl derivative 14,56 the 4-phenoxyphenethyl and
4-(phenoxymethyl)phenethyl C2 acyl side chains represented
the least effective and next most effective members of the
conformationally restricted series examined (Figure 6).
All were more effective than the parent 2 itself (Ki ) 0.0047
µM) approaching the potency of 14 indicating that all benefit
from the conformational restriction and incorporation of two
phenyl rings in the C2 acyl side chain. Consequently, we
examined the candidate inhibitors incorporating the 4-phenoxy-
phenethyl and 4-(phenoxymethyl)phenethyl side chains with a
somewhat smaller series of the same representative or optimized
C5 oxazole aryl and nonaryl substituents. The results of their
examination are presented in Figure 7 alongside the results of
the corresponding phenhexyl series,56 of which 2 is a member,
and the biphenylethyl series disclosed herein. With a few notable
exceptions, the phenhexyl inhibitor was less potent than each
of the corresponding inhibitors in the biphenylethyl and (phe-
noxymethyl)phenethyl series and comparable in potency with
the phenoxyphenethyl series. Thus, the potency for any given
C5 substituent typically followed the order of 4-biphenylethyl
> 4-(phenoxymethyl)phenethyl > phenhexyl > phenoxyphen-
ethyl. However, the activity of each such inhibitor is so good
that the potency distinctions between members of the new
classes are subtle especially with data collected over a 1–2 year
period. Nonetheless, for the most interesting inhibitors that bear
a pyridyl (14 > 68 > 54 > 2) or substituted pyridyl C5 oxazole
substituent that modulates the solubility properties of the
inhibitors (74 > 23 > 44), the trends are readily discernible.
Like observations made with 2 (F ) 3.0, R2 ) 0.91)55 and
the biphenylethyl series (Figure 4), a plot of the Hammett σp
constant versus -log Ki for the small C5 oxazole substituents
provided well defined correlations (R2 ) 0.95 and 0.93) with F
values of 2.99 and 2.67 (Figures 8 and 9). Thus, a consistent
magnitude (F ) 3.0–2.7) of the substituent effect was observed
for all four series, suggesting that the correlation represents one
of a fundamental nature with predictive value. Importantly, this
allows one to quantitatively predict a Ki from an existing
relationship in a given C2 acyl series, to qualitatively estimate
or extrapolate to members of a new C2 series if a single member
has been examined, and to confidently draw inferences from a
Electrophilic Carbonyl. A select set of the candidate
inhibitors were also examined that bear a secondary alcohol or
a methylene in place of the ketone. The former were prepared
en route to the R-ketoheterocycles and simply entailed examina-
tion of this alcohol intermediate. The candidate inhibitors
bearing a methylene were prepared by enlisting an alkylative
substitution of the Vedejs metalated oxazole as a key step
(Scheme 5). Consistent with a mechanism of reversible Ser
addition to the electrophilic carbonyl forming a hemiketal at
the enzyme active site, the corresponding alcohol and methylene
inhibitors were found to be >1000-fold less active than the
corresponding ketone (Figure 12). Although not investigated
in detail for the inhibitors disclosed herein, even this level of
reduced inhibition (0.1% activity) is most likely attributable to
contaminant ketone in the samples of the alcohol and methylene
compounds that can arise from air oxidation upon storage or
even while undergoing assay.56 Important in these comparisons
is the fact that the ketone is essential to the potent activity of
the inhibitors and that their reduction to an alcohol or removal
altogether leads to >103 reductions in activity.
Inhibition of Recombinant Human FAAH. Rat and human
FAAH are very homologous (84% sequence identity),2 exhibit
near identical substrate selectivities and inhibitor sensitivities
in studies disclosed to date, and embody an identical amidase

R-Ketooxazole Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 941
Figure 7. FAAH inhibitors with conformationally restricted C2 side chains.
Figure 9. Plot of -log Ki (µM) versus σp for 4-(phenoxymethyl)phen-
ethyl series.
Figure 8. Plot of -log Ki (µM) versus σp for 4-phenoxyphenethyl
series.
generally are exquisitely selective for FAAH. However, two
enzymes did emerge as potential competitive targets: triacyl-
glycerol hydrolase (TGH) and an uncharacterized membrane-
associated hydrolase that lacked known substrates or function
(KIAA1363).70 In this screen, IC50 values are typically higher
than the measured Ki values, but the relative potency, the
magnitude of binding affinity differences, and the rank order
binding determined in the assay parallel those established by
standard substrate assays.
signature sequence, suggesting the observations made with rat
FAAH would be analogous to those made with the human
enzyme. Consequently, key inhibitors in the series were
examined against the human enzyme and, consistent with
previous observations, were found to exhibit the same relative
and absolute potencies (Figure 13).
Selectivity. Early assessments of R-ketoheterocycle inhibitors
of FAAH against possible competitive enzymes (e.g., phospho-
lipase A2, ceramidase) revealed no inhibition. Consequently, a
method for proteome-wide screening capable of globally profil-
ing all mammalian serine hydrolases was developed,31,69 and
studies have shown that the R-ketoheterocycle class of inhibitors
Summarized in Figure 14 are the results of the selectivity
screening of selected candidate inhibitors. In general, the
inhibitors were very selective for FAAH over TGH and

942 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Kimball et al.
Scheme 3
Scheme 4
oxazole (3, 30, 45, and 59) were selective for FAAH over
KIAA1363 but more selective for TGH over FAAH (1- to 125-
fold). The potent inhibitors incorporating the small C5 nitrile
(6, 33, 48, and 62) were found to be sufficiently selective for
FAAH versus KIAA1363 (>103- to 104-fold) but only modestly
selective for FAAH versus TGH (3- to 50-fold). Here, the subtle
impact of the nature of the C2 acyl side is also apparent with
the series following the selectivity order of (phenoxymeth-
yl)phenethyl > biphenylethyl > phenoxyphenethyl ) phen-
hexyl. In line with prior observations, the addition of a 2-pyridyl
C5 substituent (2, 14, 54, and 68) increases not only the FAAH
potency but also FAAH selectivity such that they no longer
inhibit KIAA1363 (104-fold selective) and are typically >100-
fold selective for FAAH versus TGH. The corresponding
tetrazolyl substituted 2-pyridyl derivatives 23, 44, and 74
KIAA1363. The trends within the series examined are very clear
and mirror the observations disclosed in our preceding
studies.53,54,56–58 Thus, the inhibitors bearing an unsubstituted
Figure 10. Effect of tertiary amine derivatives.
Figure 11. Effect of secondary amine derivatives.

R-Ketooxazole Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 943
Scheme 5
Figure 13. Inhibition of recombinant human fatty acid amide
hydrolase.
exhibited essentially identical selectivity trends exhibited by 2,
33, and 62, whereas the addition of a carboxylic acid to the
2-pyridyl C5 substituent (-CO2H: 17, 40, 56, and 70) further
enhances this intrinsic selectivity such that the resulting inhibi-
tors are no longer viable competitive inhibitors of either
KIAA1363 or TGH. In fact, the selectivity impact of the addition
of a weakly acidic C5 oxazole substituent is so large that the
candidate inhibitors bearing only a C5 carboxylic acid (7, 34,
44, and 63) were found to be surprisingly selective for FAAH
(typically >100-fold). Superimposed on this impact of the
oxazole C5 substituent is the impact of the C2 acyl side chain.
In general, the increased size and conformational restraints of
the biphenylethyl, phenoxyphenethyl, and (phenoxymethyl)phen-
ethyl side chains matched or enhanced the intrinsic selectivity
of the phenhexyl series that includes 2 typically following the
order of (phenoxymethyl)phenethyl > biphenylethyl > phe-
noxyphenethyl > phenhexyl (Figure 15).
Conclusions
Herein, we report the synthesis and evaluation of a key series
of R-ketooxazoles based on 2 incorporating conformationally
constrained C2 acyl side chains and optimized or representative
C5 oxazole substituents that provided extraordinarily potent (Ki
) 200 pM) inhibitors of fatty acid amide hydrolase. Those
Figure 14. Selectivity screening.
Figure 15. Selectivity trends.
incorporating small C5 oxazole substituents were found to
follow a well-defined linear relationship between -log Ki and
Hammett σp of predictive value and that is of a magnitude (F
) 2.7–3.3) that indicates that the substituent electronic properties
dominate its inhibitor effects. Presumably this reflects the
substituent electron-withdrawing effect on enhancing the reac-
tivity of the electrophilic carbonyl. Additional C5 aryl substit-
Figure 12. Effect of the electrophilic carbonyl.

944 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Kimball et al.
HOAc/EtOH 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 NaHCO3, and saturated
aqueous NaCl before the organic layer was dried over MgSO4 and
the solvent was removed under reduced pressure. Flash chroma-
tography (SiO2, 10-40% EtOAc/hexanes) afforded 3-(biphenyl-
4-yl)-1-(oxazol-2-yl)propan-1-ol (3.32 g, 82%) as a colorless oil:
1H NMR (CDCl3, 600 MHz) δ 7.62 (s, 1H), 7.57 (d, 2H, J ) 7.2
Hz), 7.51 (d, 2H, J ) 7.8 Hz), 7.43 (t, 2H, J ) 6.6 Hz), 7.34–7.28
(m, 4H), 7.10 (s, 1H), 4.90–4.88 (m, 1H), 4.34 (s, 1H), 2.85–2.82
(m, 2H), 2.30–2.25 (m, 2H); 13C NMR (CDCl3, 150 MHz) δ 167.1,
141.9, 141.0, 140.0, 139.9, 129.8, 129.6, 128.1, 128.0, 127.9, 127.1,
67.6, 37.7, 31.7.
uents (e.g., 2-Pyr or 2-PyrCO2H) similarly increase FAAH
potency, enhance FAAH selectivity, and significantly modify
physical properties (e.g., solubility) in a manner that may impact
PK and PD behavior of the inhibitors. Just as significantly, the
nature of the C5 oxazole substituent substantially impacts the
selectivity of the FAAH inhibitors whereas the effect of the C2
acyl substituent was more subtle but still significant in the small
series examined. These trends proved to be general across a
range of inhibitors examined to date.53–58 This is especially
evident with the TGH selectivity for the unsubstituted oxazole
30 (125-fold selective for TGH vs FAAH), which can be
improved by the independent choice of the C2 side chain
(compare 59) as well as the C5 oxazole substituent (compare
33, 34, 2, 40, and 44). Combination of these independent
features that simultaneously improve FAAH potency and disrupt
TGH or KIAA1363 binding can provide exquisitely selective
and potent FAAH inhibitors.
A solution of 3-(biphenyl-4-yl)-1-(oxazol-2-yl)propan-1-ol (5.70
g, 20.4 mmol), TBSCl (4.62 g, 30.7 mmol), and imidazole (2.09 g,
30.7 mmol) in DMF (50 mL) was stirred at room temperature for
72 h before it was diluted with ether 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, 2–10% EtOAc/hexanes) yielded 2-(3-
(biphenyl-4-yl)-1-(tert-butyldimethylsilyloxy)propyl)oxazole (7.54
Experimental Section
Methyl 6-(2-(3-(Biphenyl-4-yl)propanoyl)oxazol-5-yl)picoli-
nate (15). A solution of triethyl phosphonoacetate (13.73 mL, 1.25
equiv) in 100 mL of anhydrous THF at -78 °C was treated with
n-BuLi (2.5 M in hexanes, 28.5 mL, 1.5 equiv). After the mixture
was stirred for 45 min, 4-biphenylcarboxaldehyde (10 g, 1 equiv)
in 80 mL of anhydrous THF was added dropwise. The reaction
mixture was allowed to warm to 25 °C and was stirred overnight.
The reaction was quenched with the addition of saturated aqueous
NH4Cl and extracted with EtOAc. The organic layer was dried over
Na2SO4, filtered, and concentrated to yield the crude R,ꢀ-unsaturated
ester. The R,ꢀ-unsaturated ester (13.85 g) was dissolved in EtOH
(250 mL), and 10% Pd/C (1.17 g, 0.2 equiv) was added. The
solution was purged with H2 and stirred for 16 h. The mixture was
filtered through a Celite pad, and the solution was concentrated.
Column chromatography (SiO2, 5–20% EtOAc/hexanes) afforded
ethyl 3-(biphenyl-4-yl)propanoate as a clear oil (9.60 g, 69%): 1H
NMR (CDCl3, 500 MHz) δ 7.71 (d, 2H, J ) 7.0 Hz), 7.66 (d, 2H,
J ) 8.0 Hz), 7.55 (t, 2H, J ) 7.5 Hz), 7.47–7.46 (m, 3H), 4.28 (q,
2H, J ) 5.6 Hz), 3.13 (t, 2H, J ) 8.0 Hz), 2.79 (t, 2H, J ) 7.0
Hz), 1.38 (t, 3H, J ) 7.5 Hz); 13C NMR (CDCl3, 125 MHz) δ
173.3, 141.4, 140.2, 139.7, 129.2, 129.2, 127.7, 127.6, 127.5, 60.9,
36.4, 31.1, 14.7.
Ethyl 3-(biphenyl-4-yl)propanoate (9.27 g, 36.45 mmol) in
anhydrous CH2Cl2 (150 mL) was cooled to -78 °C, and DIBAL-H
(1 M in hexanes, 47.4 mL, 47.4 mmol) was added dropwise. The
reaction mixture was stirred for 2 h at -78 °C before methyl
formate (2.25 mL, 36.45 mmol) was added dropwise to quench
the reaction. The reaction mixture was warmed to 0 °C, saturated
aqueous NH4Cl (20 mL) and saturated aqueous Na+K+ tartrate (25
mL) were added, and the reaction mixture was stirred vigorously
overnight. The aqueous phase was separated and extracted with
CH2Cl2, and the combined organic layers were washed with
saturated aqueous NaHCO3 and saturated aqueous NaCl and dried
over Na2SO4. Evaporation in vacuo yielded the crude aldehyde,
which was purifed by flash chromatography (SiO2, 2–10% EtOAc/
hexanes) to yield 3-(biphenyl-4-yl)propanal as a white solid (6.83
g, 89%): 1H NMR (CDCl3, 500 MHz) δ 9.94 (s, 1H), 7.66 (d, 2H,
J ) 7.0 Hz), 7.62 (d, 2H, J ) 8.0 Hz), 7.52 (t, 3H, J ) 7.5 Hz),
7.43 (t, 1H, J ) 7.5 Hz), 7.36 (d, 2H, J ) 8.0 Hz), 3.10 (t, 2H, J
) 7.5 Hz), 2.91 (t, 2H, J ) 7.5 Hz); 13C NMR (CDCl3, 125 MHz)
δ 201.9, 141.3, 139.9, 139.8, 129.2, 129.2, 127.8, 127.6, 127.4,
45.7, 28.2.
1
g, 94%) as a thick colorless oil: H NMR (CDCl3, 600 MHz) δ
7.63 (s, 1H), 7.60 (d, 2H, J ) 7.8 Hz), 7.53 (d, 2H, J ) 7.8 Hz),
7.44 (t, 2H, J ) 7.8 Hz), 7.34 (t, 1H, J ) 7.8 Hz), 7.28 (d, 2H, J
) 8.4 Hz), 7.10 (s, 1H), 4.92 (t, 1H, J ) 6.0 Hz), 2.86–2.81 (m,
1H), 2.74–2.69 (m, 1H), 2.34–2.19 (m, 2H), 0.93 (s, 9H), 0.11 (s,
3H), -0.04 (s, 3H); 13C NMR (CDCl3, 150 MHz) δ 165.9, 142.0,
141.4, 139.8, 139.5, 129.7, 129.6, 128.0, 127.9, 127.9, 127.8, 68.8,
38.9, 32.0, 26.7, 19.1, -4.2, -4.3.
A solution of 2-(3-(biphenyl-4-yl)-1-(tert-butyldimethylsilyloxy)
propyl)oxazole (842 mg, 2.14 mmol) in THF (20 mL) was cooled
to -78 °C before it was treated with 1.6 M t-BuLi (1.74 mL, 2.78
mmol) dropwise. The reaction mixture was stirred at -40 °C for
2 h, cooled to -78 °C, treated with a solution of Bu3SnCl (1.15
mL, 3.53 mmol) in THF (5 mL), 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, 0–2% EtOAc/hexanes) yielded 2-(3-(bi-
phenyl-4-yl)-1-(tert-butyldimethylsilyloxy)propyl)-5-(tributylstan-
nyl)oxazole (1.09 mg, 90%) as a thick colorless oil: 1H NMR
(CDCl3, 600 MHz) δ 7.58 (d, 2H, J ) 7.8 Hz), 7.51 (d, 2H, J )
7.8 Hz), 7.43 (t, 2H, J ) 7.4 Hz), 7.44 (t, 2H, J ) 7.8 Hz), 7.32
(t, 1H, J ) 7.2 Hz), 7.27 (d, 2H, J ) 7.8 Hz), 7.10 (s, 1H), 4.92
(t, 1H, J ) 6.6 Hz), 2.84–2.79 (m, 1H), 2.72–2.67 (m, 1H),
2.30–2.18 (m, 2H), 1.58–1.55 (m, 6H), 1.36–1.32 (m, 6H),
1.13–1.10 (m, 6H), 0.90 (s, 18H), 0.08 (s, 3H), -0.09 (s, 3H); 13
C
NMR (CDCl3, 150 MHz) δ 169.9, 155.8, 142.0, 141.7, 139.7, 138.1,
129.7, 129.6, 128.0, 127.9, 127.9, 68.9, 39.0, 32.0, 29.8, 28.0, 26.6,
19.1, 14.5, 11.1, -4.2, -4.3.
2-(3-(Biphenyl-4-yl)-1-(tert-butyldimethylsilyloxy)propyl)-5-
(tributylstannyl)oxazole (191 mg, 0.337 mmol), Pd(PPh3)4 (39 mg,
0.034 mmol), and methyl 6-chloropicolinate (116 mg, 0.674 mmol)
were dissolved in anhydrous 1,4-dioxane (18 mL), and the mixture
was warmed to reflux for 24 h under Ar. The mixture was diluted
with EtOAc, washed with saturated aqueous NaCl, and dried over
Na2SO4. Evaporation in vacuo yielded the crude coupling product.
Flash chromatography (SiO2, 10–20% EtOAc/hexanes) yielded
methyl 6-(2-(3-(biphenyl-4-yl)-1-(tert-butyldimethylsilyloxy)pro-
pyl)oxazol-5-yl)picolinate as a yellow oil (145 mg, 81%): 1H NMR
(CDCl3, 500 MHz) δ 8.09 (d, 1H, J ) 7.5 Hz), 7.95 (t, 1H, J )
7.5 Hz), 7.88–7.86 (m, 2H), 7.61 (d, 2H, J ) 7.8 Hz), 7.57 (d, 2H,
J ) 7.8 Hz), 7.47 (t, 2H, J ) 7.8 Hz), 7.39–7.34 (m, 3H), 5.04 (t,
1H, J ) 6.0 Hz), 4.08 (s, 3H), 2.98–2.92 (m, 1H), 2.86–2.80 (m,
1H), 2.47–2.33 (m, 2H), 1.00 (s, 9H), 0.20 (s, 3H), 0.07 (s, 3H);
13C NMR (CDCl3, 125 MHz) δ 166.0, 165.8, 150.5, 148.7, 148.0,
141.4, 140.8, 139.3, 138.4, 129.3, 129.1, 128.5, 127.6, 127.5, 127.4,
126.9, 122.5, 68.5, 53.3, 38.4, 31.5, 26.2, 18.7, -4.5, -4.6.
Methyl 6-(2-(3-(biphenyl-4-yl)-1-(tert-butyldimethylsilyloxy)pro-
pyl)oxazol-5-yl)picolinate (139 mg, 0.263 mmol) was dissolved in
Oxazole (1.0 g, 14.5 mmol) in anhydrous THF (100 mL) was
treated with BH3 ·THF (1 M, 15.9 mL, 15.9 mmol), and the solution
was stirred at room temperature for 1 h before being cooled to
-78 °C and treated with 1.5 M t-BuLi (12.6 mL, 18.9 mmol)
dropwise. The reaction mixture was stirred at -78 °C for 40 min
before a solution of 3-(biphenyl-4-yl)propanal (3.04 g, 14.5 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%

R-Ketooxazole Inhibitors of FAAH
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 945
THF (5 mL), treated with Bu4NF (1 M in THF, 0.316 mL, 0.316
mmol), and stirred at room temperature for 2 h under Ar. The
reaction mixture was diluted with EtOAc, washed with saturated
aqueous NaCl, and dried over Na2SO4. Evaporation in vacuo yielded
the crude alcohol, which was filtered through a short silica gel pad.
The silica gel pad was washed with 10% EtOAc/hexanes followed
by 60% EtOAc/hexanes to afford the alcohol, which required no
further purification. The alcohol was dissolved in CH2Cl2 (12 mL),
and Dess-Martin periodinane (167 mg, 0.395 mmol) was added.
The mixture was stirred at room temperature for 2 h before silica
gel was added, and the reaction mixture was evaporated in vacuo
to afford the crude ketone absorbed on silica gel. Flash chroma-
tography (SiO2, 10–40% EtOAc/hexanes) yielded methyl 6-(2-(3-
(biphenyl-4-yl)propanoyl)oxazol-5-yl)picolinate (15) as a white
solid (37 mg, 75%): 1H NMR (CDCl3, 600 MHz) δ 8.10 (d, 1H, J
) 7.5 Hz), 8.01–8.00 (m, 2H), 7.95 (t, 1H, J ) 7.5 Hz), 7.56 (d,
2H, J ) 7.8 Hz), 7.52 (d, 2H, J ) 7.8 Hz), 7.41 (t, 2H, J ) 7.8
Hz), 7.34–7.30 (m, 3H), 4.02 (s, 3H), 3.50 (t, 2H, J ) 7.2 Hz),
3.15 (t, 2H, J ) 7.2 Hz); 13C NMR (CDCl3, 150 MHz) δ 188.2,
166.0, 158.2, 153.3, 149.4, 147.4, 141.8, 140.3, 140.1, 139.2, 129.8,
129.6, 128.9, 128.1, 128.0, 127.9, 126.1, 124.2, 54.0, 41.5, 30.1;
HR ESI-TOF m/z 413.1491 (M + H+, C25H21N2O4, requires
413.1496).
and the Skaggs Institute for Chemical Biology and the post-
doctoral fellowship support for F.A.R. (American Cancer
Society). We are especially grateful to Professor B. F. Cravatt
for the supply of rat and recombinant human FAAH and to H. S.
Hoover in the Cravatt laboratories for the training and guidance
in conducting the selectivity assay. J.G. is a Skaggs and ARCS
Fellow.
Supporting Information Available: Full experimental details
and characterization of the candidate inhibitors, FAAH assay
measurement errors, and purities of the FAAH inhibitors. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
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6-(2-(3-(Biphenyl-4-yl)propanoyl)oxazol-5-yl)picolinic Acid
(17). Methyl 6-(2-(3-(biphenyl-4-yl)propanoyl)oxazol-5-yl)picoli-
nate (15) (15 mg, 0.036 mmol) was dissolved in a mixture of 3:2
THF/H2O (3 mL), and LiOH (3 mg, 0.1 mmol) was added. The
reaction mixture was stirred for 2 h at room temperature before
the mixture was made acidic with the addition of aqueous 1 N HCl.
The solution was diluted with EtOAc, and the organic layer was
separated from the aqueous layer. The aqueous layer was extracted
with EtOAc. The combined organic extracts were washed with
saturated aqueous NaCl and dried over Na2SO4. Evaporation in
vacuo yielded the crude acid, which was purified by flash
chromatography (SiO2, 0–2% AcOH/EtOAc) to yield 6-(2-(3-
(biphenyl-4-yl)propanoyl)oxazol-5-yl)picolinic acid (17) as a white
1
solid (10 mg, 71%): H NMR (THF-d8, 600 MHz) δ 8.12–8.06
(m, 4H), 7.60 (d, 2H, J ) 7.8 Hz), 7.55 (d, 2H, J ) 7.8 Hz),
7.40–7.36 (m, 4H), 7.28 (t, 1H, J ) 7.8 Hz), 3.47 (t, 2H, J ) 7.2
Hz), 3.10 (t, 2H, J ) 7.2 Hz); 13C NMR (THF-d8, 150 MHz) δ
187.0, 165.4, 158.6, 153.3, 149.8, 146.9, 141.8, 141.0, 139.8, 139.5,
129.6, 129.3, 128.4, 127.6, 127.4, 125.2, 123.4, 41.2, 29.9; HR ESI-
TOF m/z 399.1334 (M + H+, C24H19N2O4, requires 399.1339).
FAAH Inhibition. 14C-Labeled oleamide was prepared from
14C-labeled oleic acid as previously described.13 The truncated rat
FAAH (rFAAH) was expressed in E. coli and purified as previously
described.67 The purified recombinant rFAAH was used in the
inhibition assays unless otherwise indicated. The full-length human
FAAH (hFAAH) was expressed in COS-7 cells as previously
described,2 and the lysate of hFAAH-transfected COS-7 cells was
used in the inhibition assays where explicitly indicated.
The inhibition assays were performed as previously described.13
In brief, the enzyme reaction was initiated by mixing 1 nM rFAAH
(800, 500, or 200 pM rFAAH for inhibitors with Ki e 1–2 nM)
with 10 µM 14C-labeled oleamide in 500 µL of reaction buffer (125
mM Tris-Cl, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100,
0.4 mM Hepes, pH 9.0) at room temperature in the presence of
three different concentrations of inhibitor. The enzyme reaction was
terminated by transferring 20 µL of the reaction mixture to 500
µL of 0.1 N HCl at three different time points. The 14C-labeled
oleamide (substrate) and oleic acid (product) were extracted with
EtOAc and analyzed by TLC as detailed.13 The Ki of the inhibitor
was calculated using a Dixon plot as described (standard deviations
are provided in the Supporting Information tables). Lineweaver–
Burk analysis was performed as described, confirming competitive
and reversible inhibition.53
Selectivity Screening. The selectivity screening was conducted
as previously detailed.31
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (Grant DA15648)

946 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Kimball et al.
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