Exploration of a fundamental substituent effect of α-ketoheterocycle enzyme inhibitors: Potent and selective inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2008.06.084

A series of C4 substituted α-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme inhibitory potency. Thus, a plot of the Hammett σ_m versus -log K_i provided a linear correlation (R² = 0.90) with a slope of 3.37 (ρ = 3.37), that is of a magnitude that indicates that of the electron-withdrawing character of the substituent dominates its effects (a one unit change in σ_m provides a >1000-fold change in K_i).

Full text of DOI:10.1016/j.bmcl.2008.06.084

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5842–5846
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of a fundamental substituent effect of a-ketoheterocycle
enzyme inhibitors: Potent and selective inhibitors of fatty acid amide hydrolase
*
Jessica K. DeMartino, Joie Garfunkle, Dustin G. Hochstatter, Benjamin F. Cravatt, Dale L. Boger
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of C4 substituted a-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid
Received 9 May 2008
Revised 19 June 2008
Accepted 19 June 2008
Available online 28 June 2008
amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme
inhibitory potency. Thus, a plot of the Hammett
with a slope of 3.37 (
r
m versus Àlog Ki provided a linear correlation (R2 = 0.90)
q
= 3.37), that is of a magnitude that indicates that of the electron-withdrawing
character of the substituent dominates its effects (a one unit change in rm provides a >1000-fold change
in Ki).
Keyword:
Ó 2008 Elsevier Ltd. All rights reserved.
Fatty acid amide hydrolase
Fatty acid amide hydrolase (FAAH)1,2 is the enzyme that serves
to hydrolyze endogenous lipid amides3,4 including anandamide
(1a)5 and oleamide (1b),6 Fig. 1. Its distribution is consistent with
its role in degrading and regulating such signaling fatty acid
amides at their sites of action.3 Although it is a member of the ami-
dase signature family of serine hydrolases, for which there are a
number of prokaryotic enzymes, it is currently the only character-
ized mammalian enzyme bearing the family’s unusual Ser–Ser–Lys
catalytic triad.7,8
O
OH
N
H
O
1a, Anandamide
1b, Oleamide
NH2
Fatty Acid Amide Hydrolase
(FAAH)
O
Due to the therapeutic potential of inhibiting FAAH9 especially
for the treatment of pain,10 inflammation,11 or sleep disorders,12
there has been increasing interest in the development of selective
and potent inhibitors of the enzyme.9 Early studies shortly follow-
ing the initial discovery and characterization of FAAH led to the
demonstration that the endogenous sleep-inducing molecule 2-oc-
OH
O
Arachidonic Acid
Oleic Acid
OH
Figure 1. FAAH Substrates.
tyl
a
-bromoacetoacetate is an effective FAAH inhibitor,13 the dis-
but also members of this class have been shown to be efficacious
analgesics in vivo.28
In the course of these latter studies, we disclosed a fundamental
substituent effect in which a well-defined correlation between the
closure of a series of nonselective, reversible inhibitors bearing
an electrophilic ketone (e.g., trifluoromethyl ketone-based inhibi-
tors),14,15 and the reports of a set of irreversible inhibitors16 (e.g.,
fluorophosphonates and sulfonyl fluorides). To date, two classes
of inhibitors have been disclosed that provide opportunities for
the development of inhibitors with therapeutic potential. One class
is the reactive aryl carbamates and ureas17–24 that irreversibly
electronic character of a para substituent (Hammett
rp) and the
inhibitor potency (ÀlogKi) was observed.27 Thus, the inhibitor po-
tency was found to smoothly increase as the electron-withdrawing
character of the substituent increased, and the magnitude of the ef-
acylate a FAAH active site serine.25 A second class is the
a-ketohet-
fect was remarkably large (
change in p leads to nearly a 1000-fold increase in Ki. Presumably
q
= 2.7–3.0)26,27 indicating that a unit
erocycle-based inhibitors26–29 that bind to FAAH via reversible
hemiketal formation with an active site serine. Many of these latter
competitive inhibitors are not only potent and extraordinarily
selective for FAAH versus other mammalian serine hydrolases,
r
this reflects the electronic effect of the substituent on activating
the electrophilic carbonyl toward nucleophilic attack by the FAAH
active site catalytic Ser. Herein, we further generalize this funda-
mental effect to include meta substituents on the
ketoheterocycle.
a-
Whereas the former para substituents are directly conjugated
with the electrophilic carbonyl, the meta substituents would exert
* Corresponding author.
E-mail address: boger@scripps.edu (D.L. Boger).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.084

J. K. DeMartino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5842–5846
5843
their effects through their inductive electron-withdrawing proper-
ties. Moreover and although intuitive expectations might suggest
that such a non-conjugated substituent effect might be small, a
comparison of Hammett rp and rm constants suggests that the
magnitude of the effects may be surprisingly similar and, in some
instances, even enhanced (e.g., F, Cl, Br, and I).
4p using LiOH and methanolic ammonia, respectively. Carboxylic
acid 4o was also coupled with methylamine and dimethylamine
to give the substituted carboxamides 4q and 4r. Carboxamide 4p
was dehydrated with TFAA and pyridine to provide nitrile 4s. The
trifluoromethyl derivative 3t was prepared from iodide 3d using
the method developed by Chen et al.32 and iodide 3d also served
as the precursor to methyl ether 3u.33 In each case, deprotection
of the TBS ether followed by Dess–Martin periodinane oxidation34
The candidate inhibitors bearing a varied C4 oxazole substitu-
ent were accessed from the readily available 2,26 enlisting its
in situ conversion to the isomeric 4-bromooxazole via a halogen
dance rearrangement, Scheme 1.30 Thus, C4-lithiation of 2 followed
by its in situ rearrangement to the more stable 5-lithio-4-bromo-
oxazole31 and its quench with water provided 3b, the requisite pre-
cursor for the series of derivatives to be examined. These were
accessed by metallation of 3b with n-BuLi or t-BuLi followed by
reaction with appropriate electrophiles (NCS, I2, CH3I, (MeS)2,
DMF, CH3CONMe2, CF3CO2Et, NCCO2Me). Similarly, the C4 pyridine
(3k, 3l, and 3m) and phenyl (3n) derivatives were prepared from
bromide 3b using a Stille coupling reaction with the respective
pyridyl or phenyl tributylstannanes. Many of these derivatives
served as precursors to additional candidate inhibitors bearing fur-
ther modified C4 substituents. Methyl ester 4j was directly con-
verted to its corresponding carboxylic acid 4o and carboxamide
of the liberated alcohol yielded the corresponding
a-ketooxazole
(4b–u).
The FAAH inhibition derived from the examination of the series
of inhibitors and the correlation derived from a plot of Àlog Ki ver-
sus rm (q
= 3.37, R2 = 0.90 excluding 4e, 4m, 4p, 4u) are provided
in Fig. 2. The magnitude of the effect defined by the correlation
is large (
with rp
q
q
= 3.37) and satisfyingly comparable to that observed
(
= 3.0)27 where a unit change in rm leads to an increase
in the Ki of more than 1000-fold. As such and although the substit-
uents may engage in additional, more subtle interactions at the en-
zyme active site, the magnitude of the electronic effect indicates
that it dominates the behavior of the FAAH inhibitors.
Importantly, this allows one to quantitatively predict a Ki from
the correlation based on the Hammett substituent constant (
rp and
rm) or use the measured Ki to draw inferences about active site
binding that might not be known a priori. As an example of the lat-
ter, we can confidently establish that 4o binds the FAAH active site
R
R
as its deprotonated carboxylate (
rm = 0.02) versus carboxylic acid
N
N
1. Bu4NF
(rm = 0.35) from the measured Ki, and this conclusion is reasonable
—
given the pH of the enzyme assay conditions (pH = 9.0).26 More
subtly, we were able to establish that aldehyde 4g (and trifluoro-
methyl ketone 4i) exists in protic solution as a gem diol (at C4,
not C2; 1H and 13C NMR, data in Supporting Information) and that
it inhibits FAAH with a potency at a level more consistent with this
C4 substituent gem diol versus carbonyl active site binding.
Although the latter C(OH)2CF3 gem diol most likely suffers signifi-
cant destabilizing steric interactions at the enzyme active site com-
2. Dess Martin
6
Ph
O
OTBS
3b- j
6
Ph
O
4b- j
O
R= Cl, I, Me, SCH3, CHO, CH3CO, CF3CO, CO2CH3
1. BuLi 2. Electrophile
Br
N
N
1. LDA
2. H2O
75%
6
Br
Ph
O
6
Ph
OTBS
O
3b
OTBS
2
Pd(PPh3)4
Bu3SnAr
R
N
Ar
Ar
N
N
1. Bu4NF
O
O
—
6
2. Dess Martin
6
Ph
Ph
Ph
O
O
O
OTBS
cmpd
R
Ki, nM
cmpd
R
Ki, nM
4k-n
3k-n
4a
4b
4c
4d
4e
4f
H
48
4k
4l
2-Pyr
3-pyr
4-pyr
Ph
1.9
18
Ar = 2-pyr, 3-pyr, 4-pyr, Ph
Br
Cl
3.0
4.0
6.5
520
29
R
N
4m
4n
4o
4p
4q
4r
H3CO2C
1.6
65
N
I
LiOH
CH3
SCH3
CHO
COCH3
CF3CO
CO2H
CONH2
53
1.6
6
Ph
6
O
O
O
O
4g
4h
4i
55
2.0
470
CONHMe 1.8
CONMe2 35
CN
CF3
OMe
4j
4q
, R = CONHMe
MeNH2
4s
4t
0.5
3.7
740
4o
EDCI
, R = CO2H
NH3
CO2CH3 3.4
4j
Me2NH
4r
, R = CONMe2
4u
H2NOC
NC
N
N
TFAA, pyr
6
6
Ph
Ph
6
O
Ph
O
O
O
4p
4s
I
F3C
N
N
FSO2CF2CO2Me
CuI
O
6
Ph
O
3t
OTBS
OTBS
3d
I
MeO
CuI, N,N-
N
N
dimethylglycine
6
Ph
O
6
Ph
OTBS
MeOH, Cs2CO3
O
3u
OTBS
3d
Scheme 1.
Figure 2. Rat FAAH inhibition (Ki, nM) and plot of rm versus Àlog Ki.

5844
J. K. DeMartino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5842–5846
parable to that of a t-butyl substituent, the measured Ki of the hy-
drated aldehyde (CH(OH)2) is of a magnitude that suggests it may
provide a good estimate of the rm for this substituent (0.02 for
CH(OH)2 vs 0.35 for CHO). That is, the correlation between rm
and Ki is sufficiently dependable that deviations from the expecta-
tions can be utilized to establish features of active site binding not
a priori known.
FAAH
FAAH
KIAA1363
TGH
Cmpd
Ki, nM IC50, μM IC50, μM
IC50, μM
N
O
O
4a
(H)
48
2.5
20 (8)
0.02 (0.008)
In this correlation, there are several inhibitors (4m, 4k, 4p, and
4q) that deviate productively from expectations being more potent
than predicted. All four could benefit from additional H-bonding at
the active site that may increase affinity beyond that expected.
Based on their relative Ki’s, the 4-pyridyl derivative 4m and, to a
lesser extent, the 2-pyridyl derivative 4k may interact with H-bond
donors including the mobile catalytic Lys142 at the FAAH active
site26 where such a potential H-bond may be regarded not only
as a conventional H-bond stabilizing interaction, but also as a par-
tial protonation of the pyridyl nitrogen enhancing its electron-
withdrawing properties. Similarly, the primary carboxamide 4p
and, to a lesser extent, the secondary carboxamide 4q productively
deviate from correlation expectations, whereas the tertiary carbox-
amide 4r falls below extrapolated35 expectations. Attractive expla-
nations for this behavior include questions on the accuracy of the
R
N
O
O
4t (CF3)
4s (CN)
3.7
0.6
1.9
0.4
10 (25)
0.06 (0.15)
0.03 (1.5)
0.02
0.03
0.14
0.5
30 (1500)
4k
>100 (>3000) 10 (330)
(2-pyr)
>100 (>700)
>100 (>200)
0.7 (5)
2.5 (5)
4m (4-pyr) 1.7
4o (CO2H) 53
N
R
O
O
5a
(CF3)
>100 (>1400)
0.2 (3)
0.8
0.4
4.7
30
0.07
carboxamide
rm, a productive H-bonding interaction of RCONHR
5b (CN)
>100 (>14000) 0.02 (3)
>100 (>50000) 0.6 (300)
0.007
0.002
0.9
at the FAAH active site for 4p and 4q (but not 4r) that further in-
crease affinity, and/or destabilizing steric interactions that emerge
only with the tertiary amide 4r. Two substituents (–Me, –OMe) dis-
play substantial nonproductive deviations from the correlation.
Although we do not yet have attractive explanations for their
behavior, both represent electron-donating and electron-rich sub-
stituents whose activity is predicted to be among the poorest.
Thus, while additional substituent features can and will modulate
the binding affinity of the candidate inhibitors (e.g., H-bonding,
hydrophobic, or steric interactions), the magnitude of the elec-
5c (2-pyr)
5d
(CO2H)
>100 (>100)
>100 (>100)
Figure 3. Selectivity screening, IC50, l
M (selectivity).
KIAA1363 selectivity (>25-fold selective vs eightfold for 4a), but
not always as substantially as the 5-substituted oxazoles (>100-
fold) where such inhibitors typically fail to inhibit KIAA1363. Sim-
ilarly, the addition of a 4-substituent converts the TGH selective
inhibitor 4a (>100-fold selective for TGH vs FAAH) into inhibitors
that are modestly selective for FAAH (up to fivefold selective).
The exception to this is 4k which like OL-135 (5c) was found to
be >300-fold selective for FAAH versus TGH. This enhancement in
FAAH selectivity, while remarkable (typically >100-fold, but
>40,000 for 4k), is generally not as great as that observed in the
5-substituted oxazole series.
One additional feature of this study merits highlighting. In ear-
lier studies,26 we found that either a 4-substituent or 5-substituent
on the oxazole can be utilized to enhance FAAH inhibitor potency,
but candidate inhibitors bearing both were significantly less active.
Although not extensively investigated, analogous observations
were made in the course of these studies.39 This suggests that
the two classes of oxazole-based inhibitors may bind at the FAAH
active site in a manner that places the substituent in a comparable
location. This simply requires a flipped orientation of the oxazole
at the active site reversing the location of the N and O of the het-
erocycle (Fig. 4). Consistent with this suggestion, related inhibi-
tors26 bearing a C5 substituent have been shown to exhibit a
significant sensitivity to steric hindrance surrounding the analo-
gous oxazole C4 center (potency: N > O > CH) indicating that there
may be ample room for one, but not two such substituents. Provoc-
atively and while this flipped orientation of the oxazole between
the two series has little impact on the FAAH inhibitor potency, it
tronic effect of the substituent (q = 3.37) indicates that the latter
will typically dominate, especially for small and simple
substituents.
Finally, the oxazole substituents in such inhibitors not only
influence the FAAH inhibitor potency, but they can have an equally
remarkable impact on the FAAH inhibition selectivity.26 Although
there are no other characterized mammalian members of the ser-
ine hydrolase family that bear the amidase signature sequence
and its unusual Ser–Ser–Lys catalytic triad and no resulting close
family of enzymes against which to counter-screen the candidate
inhibitors, a close collaboration with Professor Cravatt led to the
implementation of a proteome-wide assay capable of simulta-
neously interrogating all mammalian serine hydrolases applicable
to assessing the selectivity of reversible enzyme inhibitors.36 This
assay, which requires no modification of the inhibitor, no purified
protein for conventional substrate assay, no knowledge of candi-
date off-site targets or even the function or substrate of the en-
zymes, can globally detect, identify, and quantitate all potential
competitive enzyme targets in the human proteome for such inhib-
itors.37 To date, two enzymes have emerged at potential competi-
tive targets for inhibitors in this class: triacylglycerol hydrolase
(TGH) and a previously uncharacterized membrane-associated
hydrolase that lacked known substrates or function at the time
(KIAA1363), but has since been characterized by Cravatt and
coworkers.38 Enlisting this proteome selectivity assay, we have
been able to simultaneously optimize inhibitors for both FAAH po-
tency and selectivity, identifying key features of candidate inhibi-
tors that can increase binding at the FAAH active site while
simultaneously disrupting KIAA1363 and TGH affinity. This multi-
dimensional SAR optimization is highlighted beautifully with the
inhibitors 4t, 4s, 4k, 4m, and 4o with the results summarized in
Fig. 3. Like observations made with the 5-substituted oxazoles,26
the addition of a 4-substituent to 4a enhances the FAAH versus
O
O
O
N
O
N
vs
O
N
O
Figure 4. Binding models.

J. K. DeMartino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5842–5846
5845
15. Patterson, J. E.; Ollmann, I. R.; Cravatt, B. F.; Boger, D. L.; Wong, C.-H.; Lerner, R.
A. J. Am. Chem. Soc. 1996, 118, 5938; Boger, D. L.; Sato, H.; Lerner, A. E.; Austin,
B. J.; Patterson, J. E.; Patricelli, M. P.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 1999,
9, 265.
may have a more significant impact on the FAAH selectivity which
often, but not always, appears to erode with the 4-substituted ser-
ies disclosed herein.
16. Deutsch, D. G.; Omeir, R.; Arreaza, G.; Salehani, D.; Prestwich, G. D.; Huang, Z.;
Howlett, A. Biochem. Pharmacol. 1997, 53, 255; Deutsch, D. G.; Lin, S.; Hill, W. A.
G.; Morse, K. L.; Salehani, D.; Arreaza, G.; Omeir, R. L.; Makriyannis, A. Biochem.
Biophys. Res. Commun. 1997, 231, 217; Edgemond, W. S.; Greenberg, M. J.;
McGinley, P. J.; Muthians, S.; Campbell, W. B.; Hillard, C. J. J. Pharmacol. Exp.
Ther. 1998, 286, 184.
17. Kathuria, S.; Gaetani, S.; Fegley, D.; Valino, F.; Duranti, A.; Tontini, A.; Mor, M.;
Tarzia, G.; La Rana, G.; Calignano, A.; Giustino, A.; Tattoli, M.; Palmery, M.;
Cuomo, V.; Piomelli, D. Nat. Med. 2003, 9, 76; Jayamanne, A.; Greenwood, R.;
Mitchell, V. A.; Aslan, S.; Piomelli, D.; Vaughan, C. W. Br. J. Pharmacol. 2006, 147,
281; Mor, M.; Rivara, S.; Lodola, A.; Plazzi, P. V.; Tarzia, G.; Duranti, A.; Tontini,
A.; Piersanti, G.; Kathuria, S.; Piomelli, D. J. Med. Chem. 2004, 47, 4998; Tarzia,
G.; Duranti, A.; Tontini, A.; Piersanti, G.; Mor, M.; Rivara, S.; Plazzi, P. V.; Park,
C.; Kathuria, S.; Piomelli, D. J. Med. Chem. 2003, 46, 2352; Tarzia, G.; Duranti, A.;
Gatti, G.; Piersanti, G.; Tontini, A.; Rivara, S.; Lodola, A.; Plazzi, P. V.; Mor, M.;
Kathuria, S.; Piomelli, D. Chem. Med. Chem. 2006, 1, 130.
18. Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.; Stevenson, T.;
Lund, E. T.; Nugent, R. A.; Normanbhoy, T.; Alexander, J. P.; Cravatt, B. F.
Biochemistry 2007, 46, 13019.
19. Abouab–Dellah, A.; Burnier, P.; Hoornaert, C.; Jeunesse, J.; Puech, F. WO 2004/
099176.; Abouab–Dellah, A.; Almario, G. A.; Froissant, J.; Hoornaert, C. WO
2005/077898.; Abouab–Dellah, A.; Almario, G. A.; Hoornaert, C.; Li, A. T. WO
2005/070910.; Abouab–Dellah, A.; Almario, G. A.; Hoornaert, C.; Li, A. T. WO
2007/027141.
20. Sit, S. -Y.; Xie, K.; Deng, H. WO2003/06589. Sit, S. -Y.; Xie, K. WO 2002/087569.;
Sit, S. Y.; Conway, C.; Bertekap, R.; Xie, K.; Bourin, C.; Burris, K.; Deng, H. Bioorg.
Med. Chem. Lett. 2007, 17, 3287.
21. Apodaca, R.; Breitenbucher, J. G.; Pattabiraman, K.; Seierstad, M.; Xiao, W. US
2006/0173184.; Apodaca, R.; Breitenbucher, J. G.; Pattabiraman, K.; Seierstad,
M.; Xiao, W. US 2007/004741.
22. Matsumoto, T.; Kori, M.; Miyazaki, J.; Kiyota, Y. WO 2006054652. Matsumoto,
T.; Kori, M.; Kouno, M. WO 2007020888.
23. Ishii, T.; Sugane, T.; Maeda, J.; Narazaki, F.; Kakefuda, A.; Sato, K.; Takahashi, T.;
Kanayama, T.; Saitoh, C.; Suzuki, J.; Kanai, C. WO 2006/088075.
A series of C4-substituted
a-ketooxazoles were examined as
inhibitors of the serine hydrolase fatty acid amide hydrolase in ef-
forts that further define and generalize a fundamental substituent
effect on enzyme inhibitory potency. A plot of the Hammett rm
versus ÀlogKi provided a linear correlation (R2 = 0.90) with a slope
of 3.37 (q = 3.37), that is of a magnitude that indicates that the
electron-withdrawing character of the substituent dominates its
effects (a one unit change in rm provides a >1000-fold change in
Ki). Moreover, this meta substituent effect is comparable, essen-
tially identical, to that we previously defined for para substituents
(q
= 2.7–3.0, R2 = 0.91–0.97)26,29 confirming both its generality and
magnitude independent of the site of substitution. Importantly, the
correlation provides a useful and predictive design principle for en-
zyme inhibitors and is of a sufficient accuracy that subtleties of ac-
tive site binding that are not known a priori may be established
from a measured Ki. These observations may prove useful not only
to extend to other enzyme classes, but also have provided herein
an additional and useful class of potent and selective FAAH
inhibitors.
Acknowledgments
We gratefully acknowledge the financial support of the National
Institutes of Health (DA15648), and the Skaggs Institute for Chem-
ical Biology. We are especially grateful to Professor B.F. Cravatt for
the supply of rat and recombinant human FAAH and for stimulat-
ing collaborations.
24. Moore, S. A.; Nomikos, G. G.; Dickason–Chesterfield, A. K.; Sohober, D. A.;
Schaus, J. M.; Ying, B. P.; Xu, Y. C.; Phebus, L.; Simmons, R. M.; Li, D.; Iyengar, S.;
Felder, C. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17852; Alexander, J. P.;
Cravatt, B. F. J. Am. Chem. Soc. 2006, 128, 9699.
Appendix Supplementary. data
25. Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179.
26. Boger, D. L.; Sato, H.; Lerner, A. E.; Hedrick, M. P.; Fecik, R. A.; Miyauchi, H.;
Wilkie, G. D.; Austin, B. J.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 5044; Boger, D. L.; Miyauchi, H.; Hedrick, M. P. Bioorg. Med.
Chem. Lett. 2001, 11, 1517; Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.;
Fecik, R. A.; Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.;
Guimaráes, C. R. W.; Jorgensen, W. L.; Cravatt, B. F. J. Med. Chem. 2005, 48, 1849;
Guimaráes, C. R. W.; Boger, D. L.; Jorgensen, W. L. J. Am. Chem. Soc. 2005, 127,
17377; Leung, D.; Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Cravatt, B. F.;
Boger, D. L. Bioorg. Med. Chem. Lett. 2005, 15, 1423; Romero, F. A.; Du, W.;
Hwang, I.; Rayl, T. J.; Kimball, F. S.; Leung, D.; Hoover, H. S.; Apodaca, R. L.;
Breitenbucher, B. J.; Cravatt, B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 1058;
Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.; Hwang, I.; Cravatt,
B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 3359; Kimball, F. S.; Romero, F. A.;
Ezzili, C.; Garfunkle, J.; Rayl, T. J.; Hochstatter, D. G.; Hwang, I.; Boger, D. L. J.
Med. Chem. 2008, 51, 937; Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.;
Full experimental details on the preparation and characteriza-
tion of the inhibitors, the FAAH inhibition assay, and FAAH assay
measurement errors are provided. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2008.06.084.
References
1. Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner, R. A.; Gilula, N. B.
Nature 1996, 384, 83.
2. Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2238.
3. Patricelli, M. P.; Cravatt, B. F. Vit. Hormones 2001, 62, 95.
4. Boger, D. L.; Fecik, R. A.; Patterson, J. E.; Miyauchi, H.; Patricelli, M. P.; Cravatt, B.
F. Bioorg. Med. Chem. Lett. 2000, 10, 2613.
5. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.;
Gibson, D.; Mandlebaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 258,
1946.
6. Cravatt, B. F.; Prospero-Garcia, O.; Suizdak, F.; Gilula, N. B.; Henriksen, S. J.;
Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506; Cravatt, B. F.; Boger, D. L.;
Lerner, R. A. J. Am. Chem. Soc. 1996, 118, 580; Boger, D. L.; Henriksen, S. J.;
Cravatt, B. F. Curr. Pharm. Des. 1998, 4, 303.
7. Patricelli, M. P.; Cravatt, B. F. J. Biol. Chem. 2000, 275, 19177.
8. Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793.
9. Cravatt, B. F.; Lichtman, A. H. Curr. Opin. Chem. Biol. 2003, 7, 469; Lambert, D.
M.; Fowler, C. J. J. Med. Chem. 2005, 48, 5059.
10. Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.; Giang, D. K.; Martin,
B. R.; Lichtman, A. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9371; Lichtman, A. H.;
Shelton, C. C.; Advani, T.; Cravatt, B. F. Pain 2004, 109, 319; Cravatt, B. F.;
Saghatelian, A.; Hawkins, E. G.; Clement, A. B.; Bracey, M. H.; Lichtman, A. H.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10821.
Hwang, I.; Boger, D. L. J. Med. Chem. 2008, 51, in press.; For a review of
a-
ketoheterocycle enzyme inhibitors, see: Maryanoff, B. E.; Costanzo, M. J. Bioorg.
Med. Chem. 2008, 16, 1562.
27. Romero, F. A.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006, 68, 14004.
Although we are unaware of reports that enlist Hammett rp or rm values on
oxazoles, studies on heterocycles including furan and thiophene exhibit near
perfect Hammett rp or rm correlations with those established for phenyl as we
have used them, see:Freeman, F. J. Chem. Edu. 1970, 47, 140.
28. Lichtman, A. H.; Leung, D.; Shelton, C. C.; Saghatelian, A.; Hardouin, C.; Boger,
D. L.; Cravatt, B. F. J. Pharmacol. Exp. Ther. 2004, 311, 441; Chang, L.; Luo, L.;
Palmer, J. A.; Sutton, S.; Wilson, S. J.; Barbier, A. J.; Breitenbucher, J. G.; Chaplan,
S. R.; Webb, M. Br. J. Pharmacol. 2006, 148, 102.
29. For additional studies, see: Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Boger, D.
L. Bioorg. Med. Chem. Lett. 2005, 15, 103; Muccioli, G. G.; Fazio, N.; Scriba, G. K.
E.; Poppitz, W.; Cannata, F.; Poupaert, J. H.; Wouters, J.; Lambert, D. M. J. Med.
Chem. 2006, 49, 417; Saario, S. M.; Poso, A.; Juvonen, R. O.; Jarvinen, T.; Salo–
Ahen, O. M. H. J. Med. Chem. 2006, 49, 4650; Myllymaki, M. J.; Saario, S. M.;
Kataja, A. O.; Castillo–Melendez, J. A.; Navalainen, T.; Juvonen, R. O.; Jarvinen,
T.; Koskinen, A. M. P. J. Med. Chem. 2007, 50, 4236.
11. Karsak, M.; Gaffal, E.; Date, R.; Wang–Eckhardt, L.; Rehnelt, J.; Petrosino, S.;
Starowicz, K.; Steuder, R.; Schlicker, E.; Cravatt, B. F.; Mechoulam, R.; Buettner,
R.; Werner, S.; Di Marzo, V.; Tueting, T.; Zimmer, A. Science 2007, 316, 1494.
12. Huitrón–Reséndiz, S.; Sanchez–Alavez, M.; Wills, D. N.; Cravatt, B. F.;
Henriksen, S. J. Sleep 2004, 27, 857.
13. Patricelli, M. P.; Patterson, J. P.; Boger, D. L.; Cravatt, B. F. Bioorg. Med. Chem.
Lett. 1998, 8, 613.
14. Koutek, B.; Prestwich, G. D.; Howlett, A. C.; Chin, S. A.; Salehani, D.; Akhavan,
N.; Deutsch, D. G. J. Biol. Chem. 1994, 269, 22937.
30. Stanetty, P.; Spina, M.; Mihovilovic, M. D. Synlett 2005, 9, 1433.
31. Schnürch, M.; Spina, M.; Khan, A. F.; Mihovilovic, M. D.; Stanetty, P. Chem. Soc.
Rev. 2007, 36, 1046.
32. Chen, Q.-Y.; Wu, S.-W. J. Chem. Soc., Chem. Commun. 1989, 705; Qing, F.-L.; Fan,
J.; Sun, H.-B.; Yue, X.-J. J. Chem. Soc., Perkin Trans. 1 1997, 3053.
33. Hui, Z.; Ma, D.; Cao, W. Synlett 2007, 2, 243.
34. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
35. We could not locate a rm for –CONMe2, but would expect it to approach that
reported for –CONH2 (0.28) or –CONHMe (0.35).

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J. K. DeMartino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5842–5846
36. Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotech 2003, 21, 687.
37. Kidd, D.; Liu, Y.; Cravatt, B. F. Biochemistry 2001, 40, 4005; Liu, Y.; Patricelli, M.
P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694.
38. Chiang, K. P.; Niessen, S.; Saghatelian, A.; Cravatt, B. F. Chem. Biol. 2006, 13,
1041.
39. For example, the 4,5-dibromo derivative, 1-(4,5-dibromooxazol-2-yl)-7-
phenylheptan-1-one, exhibited Ki of 47 nM. This is 16-fold less potent
a
than the 5-bromo derivative (Ki = 3 nM), 16-fold less potent than 4b, and
>100-fold less active than estimates based on the combined substituent
effects.