Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties

Article abstract of DOI:10.1016/j.bmcl.2016.02.061

Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using representative FAAH crystal structures revealed that compounds containing

Full text of DOI:10.1016/j.bmcl.2016.02.061

Accepted Manuscript
Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly rever-
sible FAAH inhibitors with analgesic properties
Stephen O. Pember, Galo L. Mejia, Theodore J. Price, Robert J. Pasteris
PII:
S0960-894X(16)30183-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.061
BMCL 23615
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
15 December 2015
18 February 2016
19 February 2016
Please cite this article as: Pember, S.O., Mejia, G.L., Price, T.J., Pasteris, R.J., Piperidinyl thiazole isoxazolines: A
new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.061
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Title Page
Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with
analgesic properties.
Stephen O. Pembera, Galo L. Mejiab, Theodore J. Priceb, and Robert J. Pasterisa
a) E.I. Du Pont de Nemours and Company, Stine Haskell Research Center, 1094 Elkton Rd., Newark, DE
19711, USA. steve.o.pember@dupont.com, and robert.j.pasteris@dupont.com
b) School of Behavioral and Brain Sciences University of Texas at Dallas, 800 W. Campbell Rd.,
Richardson, TX, 75080, and University of Arizona Department of Pharmacology, 1501 N. Campbell Ave.,
Tucson, AZ, 85724, USA. theodore.price@utdallas.edu and glm140230@utdallas.edu
Corresponding author: steve.o.pember@dupont.com (302-451-0037)
1

Abstract Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a
principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using
representative FAAH crystal structures revealed that compounds containing a novel piperdinyl thiazole
isoxazoline core fit within the ligand binding domains. New potential FAAH inhibitors were designed
and synthesized incorporating urea, carbamate, alkyldione and thiourea reactive centers as potential
pharmacophores. A small library of candidate compounds (75) was then screened against human FAAH
leading to the identification of new carbamate and urea based inhibitors (Ki = pM and nM respectively).
Representative carbamate and urea based chemotypes displayed slow, time dependent inhibition
kinetics leading to enzyme inactivation which was slowly reversible. However, evidence indicated that
features of the mechanism of inactivation differ between the two pharmacophore types. Selected
compounds were also evaluated for analgesic activity in the mouse-tail flick test.
Keywords: Fatty Acid Amide Hydrolase
Inhibition
Inactivation
Analgesia
2

Fatty acid amide hydrolase (FAAH) is a membrane anchored protein that has an important role
in regulating the activity of the G-protein coupled receptors (GPCRs) CB1 and CB2 via regulation of
metabolism of one of their endogenous ligands, anadamide1. CB1 receptors are expressed throughout
the central nervous system, and act presynaptically to regulate neurotransmitter release. Fatty acid
amides such as anandamide (AEA, arachidonyl ethanolamide), oleamide, and palmitoyl ethanolamide
are endogenous agonists (endocannabinoids) of CB1 that have been implicated in a number of
physiological effects including endogenous analgesia, feeding behavior and sleep induction2. FAAH
catalyzes the deacylating hydrolysis of these lipid amide agonists thereby down regulating the levels of
the cognate ligands. By blocking hydrolysis of these agonists, in particular AEA, through inhibition of
FAAH, CB1-mediated effects can be increased and prolonged leading to physiological effects such as
analgesia3. FAAH inhibition is an emerging target for analgesia and a variety of FAAH inhibitors have
shown efficacy in pre-clinical chronic pain models through a mechanism of action that includes sites in
the central and peripheral nervous systems3, 4. Despite this promising pre-clinical data, a recent trial in
humans failed to show efficacy for osteoarthritis pain5.
FAAH is a member of the amidase enzyme family whose catalytic mechanism depends on a SER-
SER-LYS catalytic triad for activity6, 7. The enzymatic mechanism involves nucleophilic attack of SER241
on the carbonyl of fatty acid amide substrates. With AEA, ethanolamine is eliminated leaving an acyl
enzyme arachidonyl intermediate. The enzyme is then deacylated by hydrolysis of the covalent
intermediate with water thereby releasing arachidonic acid. This completes the catalytic cycle restoring
active enzyme. A variety of potent inhibitors of FAAH have been developed as potential drugs based on
exploiting these features of the reaction mechanism that have led to both irreversible and reversible
type inhibitors8. Electrophilic urea and carbamate pharmacophores have been used in a number of
scaffolds. Compounds such as URB5979, and aryl ureas from Johnson and Johnson, Takeda, and Pfizer10-
15 (Fig 1) display potent binding properties leading to enzyme inactivation through covalent modification
of the active site SER241. Co-crystallographic studies with inhibitors and mammalian FAAH have helped
3

map critical structural features of the enzyme16, 17. The cytoplasmic port (CP) domain, a hydrophilic
region adjacent to the catalytic site, interacts with the polar head groups of substrates and inhibitors.
Also, adjacent to the catalytic domain are a hydrophobic acyl binding pocket (ABP), and membrane
access channel (MAC) that bind the extended lipid chains of substrates and hydrophobic scaffolds of
inhibitors. We now report on new acyl urea and carbamate inhibitors of FAAH with high in vitro
potency, and their evaluation for analgesia in mice.
New bioactive chemotypes can be derived from primary scaffolds by addition or linking of
secondary heterocycles guided by compatibility with structural or mechanistic features of target
proteins. The aryl urea FAAH inhibitors reported by both Takeda and Johnson and Johnson contain
piperzine thiazole and thiadiazole scaffolds which are similar to the piperdinyl thiazole central core of
Oxathiapiprolin (Fig 1), an antifungal agent discovered by DuPont that is used for control of plant
diseases caused by oomycetes.18 Although there are structural similarities to FAAH inhibitors,
Oxathiapiprolin
O
N
S
O
N
N
N
N
N
N
H
N
H
PF-750
JNJ-1661010/Takeda
O
NH2
N
O
N
O
O
O
N
OL-135
URB597
Figure 1 – Oxathiapiprolin fungicide; Piperdine and piperzine aryl urea FAAH inhibitors (PF-750, JNJ-1661010); α-ketooxazole
and carbamate FAAH inhibitors (OL-135, URB597).
4

the mechanism of action of Oxathiapiprolin is not thought to involve a homologue of FAAH.19 This is due
in part to the presence of amidase signature domains in Phytophthora infestans (a target organism)
which appear to be restricted to the glutamyl t-RNA amidotransferase protein family. In addition,
Oxathiapiprolin differs significantly from FAAH chemotypes since it has an atypical substitution off the
piperdine ring and extended aryl isoxazoline substitution from the thiazole ring. We hypothesized that
because of the extended ABP and adjacent MAC regions in FAAH that further extension from the
thiazole might be well tolerated, and that appropriate substitution of piperdinyl ring with electrophilic
substituents might yield efficacious FAAH inhibitors.
Crystallographic results with FAAH and inhibitory ligands have revealed a dynamic relation
between the ABP and CP domains, and alterations in the flexible ABP and MAC regions depending on
chemotype and mechanism of inhibition.17, 20 For example, in the crystallographic structure 2WJ2 (PDB
code) containing α-ketoheterocycle inhibitor OL-135, the conformation of the ABP is associated with the
MAC in the “open” state.17 OL-135 forms a covalent tetrahedral transition state mimic with SER241 that
is also fully reversible. Alternatively, crystallographic structure 2WAP (PDB code) was determined from
FAAH covalently inactivated with an aryl urea (PF-3845) that yields a carbamoylated SER241.4 2WAP
depicts an endpoint inactivation with reorganized ABP and MAC domains viewed as a “closed” state.
Docking experiments with the aryl carbamate 1a and aryl urea 2c (Table 1) containing the piperdinyl
thiazole isoxazoline core yielded estimated binding constants in the low nM range when docked to
either 2WAP or 2WJ2. Fig 2 depicts a view of compound 1a docked to 2WAP. In this view compound 1a
extends deeply into the ABP domain in an extended linear conformation. In addition to hydrophobic
accommodation in the ABP domain additional interactions appear to contribute to the binding affinity:
The terminal aryl ring is positioned close to PHE381 showing an edge facing π-π interaction. The
thiazole ring is near MET495 showing possible H-bonding with the ring nitrogen. PHE192 is positioned
close to the piperdine ring with a C-H Ar interaction from the 3-position methylene. The carbamate aryl
ring is positioned close to MET191 with apparent stabilization through a C-H Ar interaction. Docking of
5

the aryl urea 2c to structure 2WAP was nearly identical to compound 1a. A key feature for both
compound 1a and 2c is the nearly covalent bond distance (2.0Å) between SER241 and the carbonyl
thereby supporting the likelihood of covalent enzyme modification by these inhibitors.
Figure 2 –Docking of compound 1a (green carbon backbone) to FAAH structure 2WAP.
Docking of 1a and 2c to 2WJ2, representative of the “open” conformation of the MAC and
associated ABP domains repositions the aryl carbamate and urea portions of the molecules extending
further into the CP domain. The extended aryl isoxazoline portions of the compounds were
accommodated in the ABP and MAC domain similarly to bound OL-135. In these experiments the aryl
ring of the carbamate (1a) was positioned very close to the S of CYS269 (3.6Å) with the ring hydrogens
capable of interacting favorably with the sulfur lone pairs. An S-H Ar interaction appeared also be
possible, but would be highly dependent on specific orientation.21 In contrast, the urea aryl ring of 2c
was positioned close to the backbone amide of CYS269 forming a potential N-H Ar interaction, however
it was farther away from the CYS269 thiol (6.5Å) relative to the carbamate aryl ring of 1a which can
perhaps be attributed to the non-rotation of the urea amide bond in 2c.
As a result of the favorable modeling results with respect to estimated binding affinity and
active site orientation, targeted synthesis then yielded a variety of analogues containing aryl carbamate
6

and ureas, as well as alkyl carbamates, aryl thioureas, isothioureas, and hydrazone electrophilic
substituents.
Table 1
X2
X1
A
Z
Compound
X1
X2
R
Apparent IC50 (nM) Ki (pM) estimate
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
CH
CH
CH
CH
CH
CH
H
0.07
0.14
0.03
0.08
0.36
0.06
42
0.04
35
0.41
2000
0.6
6.3
12.8
2.5
7.2
33.6
5.3
3962
3.4
3302
38.3
188,680
56.3
18.5
2735
INDh
INDh
56,603
20,943
17,924
36,037
924
2358
147
8.1
18.5
INDh
14.7
18.5
4-CN
3-CN
4-Cl
4-CF3
2-CL
CH 3,5-CH3
CH 2-CH3
CH 2,5-CH3
CH 3-CL
CH 2,6-CH3
CH
CH
CH
CH
3-CH3
4-CH3
H
1m
0.2
29
1n (notea)
1o (noteb)
H
0.02
0.01
600
222
190
382
9.8
1p
CH 4-OCH3
2a
NH CH
NH CH
NH CH
NH CH
NH CH
NH CH
O
O
O
O
O
O
2-CH3
H
2b (notec)
2c
H
2d (noted)
NAg
H
2e (notee)
2f (notef)
H
H
25
3a
3b
3c
3d
3e
3f
N
N
N
N
N
N
1.56
0.09
0.2
0.01
0.16
0.2
4-CN
3-CN
4-CL
4-CF3
4-NO2
X1
X1
N
O
a) fragment Z =
b) fragment A =
c) fragment A =
X1
X1
X1
d) fragment A =
e) fragment A =
f) fragment A =
IC50 values were determined as described in Experimental Procedures. Reactions were measured over 40
min, but because inhibition was time dependent, only initial linear rates were used (0 to about 25min).
A small library of about 75 compounds was then screened in vitro for inhibition of FAAH. Out of
this initial pool, active compounds with the highest level of activity resided in either aryl urea or aryl
carbamate substituted compounds (region A, figure in Table 1). Interestingly, in the urea series when
7

region A was a thiazole or an acyl ester, IC50 values were still observed below 1 M (Table 1). Within the
urea series when region A was substituted with the weakly basic diazine heterocycles (pyrimidine,
pyridazine, 2e and 2f respectively) a large increase in binding affinity was observed relative to simple
phenyl substitution (2c). This trend is qualitatively similar to the effects of nitrogen addition within the
central activating oxazole of the α-ketoheterocycle FAAH inhibitors.17 As listed in Table 1, better binding
affinity was observed with aryl carbamates relative to acyl ureas.
Additional analogues were also prepared around piperzine ring substitution as used in other
FAAH inhibitor chemotypes. Attention was given to the aryl carbamates because of better intrinsic
binding affinity and enzyme inactivation potency relative to the aryl urea series. As listed in Table 1,
many of the piperdine and piperzine aryl carbamates have very high binding affinity with observed IC50
values and Ki estimates reaching the pM range. The IC50 values determined for several standard
inhibitors under our assay conditions (0.24 nM URB597 and 0.4 nM OL-135, Fig 1) were generally lower
than those reported in the literature (3 nM URB597 and 0.3, 4.7 nM OL-135).9, 22, 23 This can likely be
attributed to the low concentration of active enzyme used in the fluorometric assay (8 pM). For several
compounds the IC50 values (within experimental error) reached a limiting value equivalent to [Et]/2
indicating that a Ki value could not be estimated under the conditions of the experiments. Nevertheless,
the relative values for the majority of compounds in the analogue sets are comparable for structure
activity purposes. Among the piperdinyl analogues listed in Table 1, di-substitution of the phenyl
carbamate ring significantly decreased relative binding affinity. In particular, the 2, 6-dimethyl
substitution (1k) decreased binding affinity greater than 4 orders of magnitude relative to the
unsubstituted analogue (1a). Mono-substitution in the 2, 3, or 4 positions on the phenyl carbamate ring
in the piperdine series was relatively well tolerated without much shift in binding affinity observed
among analogues with the exception being the 3 substituted CL and CH3 analogues. Within the
piperzine series substitution in the 4 position of the phenyl ring (3b, 3d, 3e) showed similar inhibition
values compared to identically substituted piperdine analogues (1b, 1d, 1e). Interestingly, when the
8

phenyl ring is unsubstituted (3a), or substituted in the 3 position (3c) binding affinity decreased relative
to the corresponding piperdine analogue.
Representative aryl carbamates and ureas were examined in more detail to explore the
mechanism of inhibition. Both the aryl carbamates and ureas examined showed time dependent loss of
enzyme activity consistent with inactivation of the enzyme as observed with other carbamate and urea
chemotypes. This response is consistent with several possible mechanisms of inhibition. Reversible,
slow-tight binding inhibition, reversible or irreversible mechanism-based inhibition, or irreversible
modification of active site residues due to reactive propinquity can yield time dependent inhibition
kinetics. A kinact for representative compounds was determined using a 2 step model; an initial binding
equilibrium followed by a first order inactivation event, and competitive with substrate binding and
catalysis. The individual ki’ values determined at different concentrations of inhibitor were used to
estimate a kinact. Determination of ki’ values for compound 1l are shown in Fig 3A. A representative fit of
the data to determine kinact is depicted in Fig 3B for the aryl urea 2b (Table 1). Table 2 summarizes the
inactivation kinetics for representative ureas and carbamates. Based on comparison of the second order
inactivation constants the ureas were less effective than the carbamates, and directly related to the
A
B
0.0024
0.002
0.0016
0.0012
0.0008
0.0004
0
1800
1600
1400
1200
1000
800
600
400
200
0
0.0001 0.001
0.01
0.1
1
10
0
500
1000
1500
2000
2500
Inhibitor concentration (uM)
Time (sec)
Figure 3 – A, Time dependent loss of FAAH activity in the presence of increasing concentrations of compound 1l. Baseline (open
circles), 10nM (dark circles), 1 nM (open squares), 0.1 nM (dark squares), and the uninhibited reaction (open triangles). The
solid lines represent the computer fit of the data using the apparent inactivation constant (ki’) determined at each
concentration of 1l. B, computer fit of the apparent ki’ values for compound 2b to determine Kinact as described in the methods
section.
9

lower binding affinity for the enzyme. The kinact values generally correlated with the electrophilicity of
the activating group on the carbamate or urea. For example, in the urea series, benzyl (2c) is > thiazole
(2b) is > acyl ester (2d). Taken together with the docking experiments, the results for the urea and
carbamates are consistent with inactivation of the enzyme through covalent enzyme modification of
SER241 as observed with other FAAH chemotypes.13, 14
Table 2
Compound
kinact (sec-1)
.0004
Ki (nM)
.007
kinact/Ki (M-1sec-1)
1a
6.1 x 107
1b
1l
.0012
.0014
.0043
.0025
.0015
.013
.056
18
9.2 x 107
2.5 x 107
2.4 x 105
1.2 x 105
4.2 x 105
2c
2b
2d
21
36
The Kinact and in vitro efficacies of the selected acyl ureas and carbamates were determined as described in the methods.
To examine the reversibility of inactivation by the aryl carbamates and ureas, FAAH samples that
had been completely inactivated by compounds 1a, 2c, 2d, and JNJ-1661010 were extensively dialyzed
at 4oC to determine if enzymatic activity could be regained. Activity was sampled at 24, 48, and 144 hrs.
The results are shown in Fig 4. At 24 hrs the ureas, 2c and 2d showed some recovery of activity relative
to the others. Progressive recovery of activity for 2c and 2d treated enzyme was observed over the time
course reaching about 60% recovery of activity. FAAH inactivation by the ureas appears to be
predominately reversible. Interestingly, the aryl carbamate (1a) inactivated enzyme also showed some
recover of activity (30%) at 144hrs, and was similar to recovery observed for JNJ-1661010. The extent of
recovered activity for JNJ-1661010 inactivated enzyme was similar to that previously reported at 4oC.13
Control, untreated enzyme maintained about 80% activity over the duration of the experiment. These
results support an inactivation mechanism through reversible intermediates. Covalent modification of
the enzyme by 1a would be consistent with the very slow partial recovery of activity as observed with
the acylating inhibitor, JNJ-1661010.
10

1
0.8
0.6
0.4
0.2
0
Control
a
b
c
d
Figure 4 – Recovery of FAAH activity (relative to controls) through exhaustive dialysis after inactivation by various compounds
(a) compound 1a; (b) JNJ-1661010; (c) compound 2c; (d) compound 2d as described in the methods. Bars: light gray, 24 hrs;
hash marks, 48 hrs; dark gray, 144 hrs; and black, range of control activity over the time course. Structures are given in Table 1
and Fig 1.
Both the representative aryl carbamates and ureas displayed slow, time dependent inactivation,
and slow reversibility. This is characteristic of either a mechanism-based inactivating substrate, or
formation of an inhibitory transition-state type mimic. The former type would suggest hydrolysis of the
inhibitor forming a very slowly released acylated enzyme intermediate with initial release of either
phenol or aniline. To investigate this, single turnover (inactivation) experiments with enzyme and added
inhibitor were analyzed by LC/APCI MS for the presence of hydrolytic products in the incubation
mixtures using compounds 2c and 1a respectively. Mixtures were analyzed for parent inhibitor, aniline
(2c) or phenol (1a), and evidence of the carbamic acid piperdinyl thiazole isoxazoline core. For
compound 1a, phenol and parent inhibitor were detectable in the reaction mix. However for compound
2c, parent inhibitor, aniline, and evidence of the carbamic acid core were observed (Table 3). The
product ion corresponding to m/z = 359.4 is consistent with the sodium adduct of the free piperdinyl
11

scaffold resulting from instability of the carbamic acid. Only parent inhibitor was detected in control
incubations containing all components except enzyme for both 2c and 1a.
Table 3
Y (+H)
W (+OH)
Compound
Parent
434.5 [M+H]+ 113.1 [M+H3O]+
433.5 [M+H]+ 94.1 [M+H]+
Y
W(-CHO2)
1a
2c
Not Found
359.4 [M+2Na]+
m/z values for the parent and product ions of the aryl carbamate (1a) and urea (2c). HPLC/MS conditions are
described in the methods. Under the conditions of the experiment phenol was detected primarily as an adduct
(water), although the parent [M+H] + = 95.1 was present in trace amounts. The product ion m/z=359.4 was also detected
in control extraction of incubation buffers containing only the piperdinyl scaffold. For 1a X is O; for 2c X is N.
FAAH is a serine hydrolase, but as a member of the amidase enzyme family contains a catalytic
triad that is unusual, and not found in other typical serine hydrolases such as proteases and esterases16.
However, some features of the reaction mechanism appear similar, such as the use of an activated
serine nucleophile, with some potent FAAH inhibitor chemotypes showing some degree of non-
selectivity inhibiting unrelated serine hydrolases24. In preliminary tests, to probe FAAH specificity, we
examined the ability of selected compounds to inhibit mammalian pancreatic esterase and elastase
activities. All compounds tested showed at most, only slight inhibition of pancreatic elastase at the
highest concentration tested (10 μM). IC50 values for 2b, 2c, 1f, 1d, 1h, and 1a were >10, 5.6, 0.87,
0.47, 0.84, and 2.4 uM respectively for inhibition of liver esterase. These values were orders of
12

magnitude less potent compared to FAAH inhibition. These initial results indicated promising specificity
for FAAH inhibition by these compounds.
We then sought to determine in vivo efficacy of these compounds using the mouse tail-flick test.
Previous studies have shown that FAAH blockade produces a short-lasting analgesia in this assay as
reflected by an increase latency to respond to noxious heat stimulation of the tail24. Since the
bioavailablity properties for our aryl urea, and aryl carbamate inhibitor types were unknown,
compounds were selected from both classes for in vivo evaluation. The 2 most potent aryl ureas (2e, 2f),
and 4 of the more potent aryl carbamates (1a, 1b, 1o, and 1p) were screened at 50 mg/kg
intraperitoneal (IP) dosing (Fig 5). Compounds 1a, 1b, 2e and 2f displayed significant analgesic effects at
this dose and were tested at further doses to establish a dose-response relationship. Compounds 1o
and 1p did not produce analgesia (Fig 5C, D). Lower doses of compound 1a, 2e and 2f failed to produce
significant analgesia, however, 1b produced clear dose-dependent effects down to 5 mg/kg. Summary
data for all tested compounds over the entire time course of testing is shown in Fig 5G.
The aryl isoxazoline carbamate and urea FAAH inhibitors reported here are among the most
potent in vitro FAAH inhibitors identified. Contributions to the high binding affinities implicated through
docking experiments are the extended interactions of the aryl isoxazoline moiety within the ABP
domain, and interactions of both the aryl carbamate and urea portions of the chemotypes with key
residues in the CP domain of FAAH. The SAR relationships are supported by the modeling results, for
example, the bulky spiro-tetralin substitution on the isoxazoline (1n) ring showed a significant decrease
in binding affinity relative to other extended aryl isoxazoline analogues. This is consistent with steric
constraints imposed by the tetralin relative to the extended aryl isoxazoline within the ABP, and
interaction with residues such as PHE192 and MET495. The aryl carbamate series showed higher
binding affinity to FAAH than the aryl urea series where the common piperdinyl backbone is shared
suggesting differences in interaction with the CP domain likely also occur between the two chemotypes.
13

Figure 5 - in vivo efficacies of novel FAAH inhibitors (Table1) in the mouse tail flick test. A-F shows tail flick latencies in the 52 C
tail flick test following intraperitoneal injection of doses of novel FAAH inhibitors G shows summary data for all compounds
where compound 1b shows the best in vivo potency. * p < 0.05, ** p < 0.01, *** p < 0.001 two way ANOVA with Bonferroni
posthoc test.
14

This was supported through experimental modeling (2WJ2) where favorable interactions with CYS269
appeared diminished with aryl urea 2c relative to the aryl carbamate 1a. Additionally, the greater
electrophilicity of the aryl carbamates relative to the aryl ureas would lead to a greater interaction
potential with SER241, and is likely reflected in the higher binding affinity of the aryl carbamates.
Previous structural observations have identified chemotype dependent conformational
differences in critical active site residues that are coupled to corresponding structural changes in the
ABP region20. This indicates a dynamic relationship between the ABP and CP domains. This is observed in
crystallographic structures that show dynamic progress through the reaction states of the enzyme with
reorientation of critical binding domains. The docked view of 1a (Fig 2) depicts the covalent approach of
SER241 (Oγ) to the carbonyl in the “closed” conformation of the MAC and associated ABP domains, and
is consistent with an expected inactivation mechanism leading to covalent modification of SER241, likely
through carbamoylation. This was supported experimentally by detection of phenol as the resulting
elimination product, and the absence of an extractable carbamic piperdinyl core in the reaction mixture.
The absence of the latter indicates the formation of a non-dissociable carbamoylated intermediate
supported by the view in Fig 2. Compound 1a displayed time-dependent inactivation, and very slow
partial reversibility. The data suggests 1a is a mechanism based inhibitory substrate similar to JNJ-
1661010.
The data also indicates that the distribution and nature of inactivating intermediates formed
during inactivation of FAAH by 2c is distinct from that of 1a. This is based on the recovery of both the
aniline leaving group, and the piperdinyl thiazole isoxazoline core as hydrolysis products. While this
does not eliminate the presence of a stable carbamoylated intermediate (SER241), it nevertheless
indicates that a significant proportion of the inactivating intermediate is dissociable. This is also
supported by significant reversibility of inactivated enzyme by 2c upon dialysis. The nature of FAAH
inhibition by 2c may therefore be through the reversible formation of a quaternary transition state
mimic resulting from attack of SER241 Oγ on the urea carbonyl, similar to OL-135 inactivation. The
15

ability to recover both hydrolysis products of 2c is consistent with this, and would likely result from
partitioning of the quaternary intermediate; collapsing to either parent, carbamoylated intermediate, or
hydrolysis products during aqueous denaturation of the complex. The observed differences in
inactivation of FAAH by 1a or 2c can likely be attributed to the greater intrinsic susceptibility of aryl
carbamates to nucleophilic attack relative to aryl ureas.
The inactivation rates (kinact) determined for the three carbamate and urea analogues are slower
than those reported for other chemotypes such as carbamate URB597, and the biarylether urea
inhibitor PF-3845 (0.0033 sec-1 each)4. This may be due to the relative intrinsic susceptibility differences
among the individual electrophilic substituents. Among the aryl carbamates tested the greater kinact for
1b over 1a is consistent with greater susceptibility of the carbonyl toward nucleophilic attack because of
the greater electron withdrawing character of the 4-CN (σ = +.66) on the phenyl ring of 1b. However, if
ring substitution were solely dominant, then the kinact for 1l would be expected to be similar to that for
1a since only modest effects from the 3-CH3 (σ= -.069) on the aryl leaving group would be expected,
however the kinact for 1l is about 2 fold higher than 1a. From the docked example of 1a (Fig 2) it is
evident that the terminal aryl ring of the carbamate potentially interacts significantly with MET191 in
the oxyanion pocket. The greater kinact for 1l may therefore result from greater delocalization through a
C-H Ar type interaction due to the electron donating effect of the 3-CH3 substitution.
We chose a subset of compounds for in vivo testing in mice. Inhibition of FAAH results in
increased AEA levels in the periphery and in the central nervous system2, 6. One result of this increased
AEA concentration is analgesia via the activation of CB1 receptors. Recent evidence suggests that at
least part of this effect is mediated by a peripheral mechanism26-28. We observed strong, dose-
dependent analgesia in the tail flick test with 1b whereas several other active compounds only produced
significant effects at 50 mg/kg. While the disposition of 1b in vivo is not currently known, the strong in
vitro and in vivo potency for this compound make it an excellent starting point for further optimization
based on this scaffold.
16

Experimental Procedures
Materials – JNJ-1661010, N-succinyl-ala-ala-ala-ρ-nitroanilide, 4-nitrophenyl butyrate, amino-4-methyl
coumarin, porcine liver esterase, porcine pancreatic elastase, and the computer program GraFit (ver 3,
Erithacus software) were from Sigma Aldrich. Sephacryl™ S100 was obtained from GE Healthcare.
Expression vector pMAL-c4x, E. coli T7 Express cells, and restriction enzymes were from New England
BioLabs. EDTA-free protease inhibitor cocktail was from Roche Life sciences. The human FAAH gene
was purchased from Open BioSystems, GE Healthcare. Microplates were from Costar. Enzyme assays
using chromogenic or fluorogenic substrates were measured on either a SpectraMax ™ Plus or
SpectraMax ™ Gemini plate reader (Molecular Devices, Inc.), respectively. URB597 was from Asinex, Inc.
NMR measurements were made on either a Varian INOVA 400 MHz system equipped with Nalorac
indirect detection probe or a Brucker 500 MHz Avance III spectrometer using a 5mm 1H, X BBO probe.
Mass analysis supporting chemical synthesis was performed using a Waters Alliance e2695 liquid
chromatography system coupled to a SQ detector 2 (single quad) mass detector. Enzymatic reaction
products were analyzed using an Agilent 1100 liquid chromatography system coupled to a Thermo LTQ
mass spectrometer. Slide-A-Lyzer® mini dialysis units were from Thermo Scientific. Melting points were
determined using a Thomas Hoover apparatus. All IC50 and enzyme kinetic data were fit with the
computer program GraFit. All other chemicals, reagents, and materials were of reagent grade or better.
Compound Preparation – The synthetic chemistry used to prepare the compounds described in this
article has been previously described in patent WO201107220729. As an example, phenyl 4-[4-(4, 5-
dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]-1-piperidinecarboxylate (Compound 1a, Table A) can be
prepared in the following series of steps:
Preparation
of
1,1-dimethylethyl
4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]-1-
piperidinecarboxylate.
To mixture of 1,1-dimethylethyl 4-(4-formyl-2-thiazolyl)-1-
a
17

piperidinecarboxylate (1.0 g, 3.4 mmol) in ethanol (5 mL) was added an aqueous solution of
hydroxylamine (50 wt. %, 0.25 mL, 4.0 mmol). The reaction mixture was heated at 60 °C for 1 h, during
which time the reaction mixture became homogeneous. The resulting reaction solution was cooled to
room temperature and diluted with tetrahydrofuran (10 mL). Styrene (0.57 mL, 5 mmol) was added to
the reaction mixture, followed by a portion wise addition of Clorox® (aqueous sodium hypochlorite
solution) (10.5 mL) over 3 h. The reaction mixture was stirred overnight at room temperature and then
filtered. The solid collected by filtration was washed with water and diethyl ether and then air dried to
give the title compound as a white powder (610 mg). The filtrate was diluted with saturated aqueous
sodium bicarbonate solution and extracted with diethyl ether. The extract was dried (MgSO4) and
concentrated under reduced pressure to give more of the title compound as yellow oil (850 mg). The oil
was diluted with diethyl ether (4 mL) and upon standing provided the title compound as a white solid
(233 mg). 1H NMR (CDCl3) δ 1.47 (s, 9H), 1.7 (m, 2H), 2.1 (m, 2H), 2.85 (m, 2H), 3.2 (m, 1H), 3.45 (m,
1H), 3.84 (m, 1H) 4.2 (br s, 2H), 5.75 (m, 1H), 7.25-7.40 (m, 5H), 7.61 (s, 1H).
Preparation of 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]piperidine. To a solution of 1,1-
dimethylethyl 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]-1-piperidinecarboxylate
(0.815 g,
1.97 mmol) in dichloromethane (50 mL) was added a solution of hydrogen chloride in diethyl ether (2 M,
10 mL, 20 mmol). The reaction mixture was stirred at room temperature for 1 h to give a gummy
precipitate. Methanol was added to dissolve the precipitate, and the reaction mixture was stirred for an
additional 1 h. The reaction mixture was concentrated under reduced pressure and partitioned
between ethyl acetate and saturated aqueous sodium bicarbonate. The organic layer was dried
(MgSO4) and concentrated to give the title compound as a clear oil (0.31 g), which solidified on
standing. 1H NMR (CDCl3) δ 1.65 (br s, 1 H), 1.7 (m, 2H), 2.1 (m, 2H), 2.75 (m, 2H), 3.1-3.25 (m, 3H), 3.41
(m, 1H), 3.83 (m, 1H), 5.75 (m, 1H), 7.25-7.40 (m, 5H), 7.60 (s, 1H).
18

Preparation of phenyl 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]-1- piperidinecarboxylate.
To a solution of 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2-thiazolyl]piperidine (3.3 g, 10 mmol) and
triethylamine (2 mL, 14 mmol) in dichloromethane (40 mL) cooled to -5o C, was added a solution of
phenyl chloroformate (1.6 g, 10 mmol) in dichloromethane (10 mL) dropwise over 5 minutes. The
reaction mixture was stirred at -5o C for 30 minutes and then allowed to warm to room temperature.
After 2 h, the mixture was washed with 1 N hydrochloric acid and brine, dried (MgSO4) and
concentrated under reduced pressure to give the title compound as white foam (4.3 g). A 1 g sample
was crystallized from ethanol (20 mL) to give a white powder (0.81 g) melting at 123-125o C. 1H NMR
(CDCl3) δ 1.85 (m, 2H), 2.20 (m, 2H), 2.95-3.22 (m, 2H), 3.30 (m, 1H), 3.45 (m, 1H), 3.85 (m, 1H), 4.30-
4.50 (m, 2H), 5.75 (m, 1H), 7.15 (m, 2H), 7.22 (m, 1H), 7.25-7.42 (m, 7H), 7.63 (s, 1H); m/z AP+ (M+1) =
434.
Ligand Docking - Ligand docking to the crystal structures FAAH was explored using AutoDock 4.2.6 and
AutoDock Tools 1.5.6.30 Python Molecular viewer was used for graphics.31 Ligand and protein files were
prepared using standard AutoDock protocols. Default values for number of ligand rotatable bonds and
torsion angles were used which included assignment of urea amide bonds as non-rotatable. Medium
level exhaustiveness was used in docking conformation searches.
FAAH Expression and Purification – recombinant human FAAH was expressed in truncated form, in
which the transmembrane (TM) portion of the enzyme was removed from the N-terminal (amino acids
1-33), and then heterologously expressed as a MBP (maltose-binding protein) fusion protein in E. coli
(MBP-ΔTM-FAAH) similar to previously described methods32. The region of the gene corresponding to
amino acids 34 to 579 was cloned into pMAL-c4x (New England BioLabs, Inc.) using EcoR1 and Sal1
restriction sites. E. coli T7 Express cells, containing the FAAH constructs, were used for expression of
protein by induction with IPTG (isopropyl-β-D-thiogalactopyranoside) (100 μM) overnight at room
19

temperature in Lennox Broth with 0.2% glucose. After harvest, the cells were resuspended in 20 mM
Hepes buffer (pH 7.4) containing 200 mM NaCl, 2mM DTT (dithiothreitol), and protease inhibitor
cocktail. The cell suspension was lysed by sonication, and the cell debris removed by centrifugation.
The soluble extract was adjusted to 2.5 mg/mL protein, and the FAAH fusion protein (~105 kDa) loaded
onto a 5 mL column of amylose affinity resin. The enzyme was eluted using 15 mM maltose as per
manufacturer's (New England BioLabs, Inc.) instruction. Fractions containing FAAH were concentrated
and further purified using Sephacryl™ S100 (HIPrep™ 26/60, GE Healthcare, Inc.) chromatography.
Fractions enriched in FAAH were pooled, concentrated, and made 10% in glycerol then stored at -80oC
until use. Stock samples used for single turnover (inactivation) experiments were stored without
glycerol. All column chromatography steps used the Hepes buffer described above. Protein purity was
about 90% determined by SDS-PAGE.
FAAH Assay and Compound Evaluation – Enzyme activity was measured using the fluorogenic
substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) as described in the literature33. Briefly, the
assay buffer consisted of 125 mM Tris-CL, 1 mM EDTA, and 0.1% BSA (pH 8.0). D-AMC was used at final
concentration of 5 μM in all assays. Under these conditions specific activity of usual enzyme
preparations were about 4000 to 6000 RFU min-1mg-1, although some preparations reached 15,000
RFU/mg-1/min-1. Reactions were carried out in black 96-well microplates using a SpectraMax Gemini™
o
(Molecular Devices, Inc.) fluorescence plate reader in a reaction volume of 200 µL per well at 37 C.
Reaction rates were monitored at an emission wavelength of 430 nm using an excitation wavelength of
351 nm over 30 to 40 minutes. Stock experimental compounds were dissolved in DMSO, and initially
evaluated at a single diluted test concentration of 2 μM. Reactions were initiated by the addition of
enzyme using 3 ug protein per well. Compounds inhibiting the reaction ≥ 90% were subsequently
retested in microplates using serial dilution of compounds to determine IC50 values. The final
concentrations of DMSO in the assay were 1% or lower.
20

Enzyme Inhibition and Kinetic Analysis – IC50 values were determined by computer fit of the dose
response data using initial rates over a 9 dose range. Approximate inhibition constants (Ki values) were
determined for experimental compounds using the relationship34, IC50 = Ki (1+[S]/Km) + [Et]/2. A Km value
of 0.52 µM was used for D-AMC as reported in the literature33. To determine inactivation constants
(kinact) for specific compounds the apparent first order rates of inactivation (ki′) of the enzyme were
determined at several different concentrations of inhibitor by computer fit to the relationship, Fo=F∞(1-
e-ki′t) where Fo is initial observed fluorescence and F∞ is the maximum final observed fluorescence. The
apparent inactivation constants at each corresponding inhibitor concentration were then used to
determine the kinact and the inhibition constant Ki from the relationship; ki´ = ([I]/Ki) kinact/ (1 + [I]/Ki +
[S]/Km) through solution (fit) of the resulting simultaneous equations. To assess the reversibility of aryl
carbamate and urea enzyme inactivation, 50 μL (158 μg protein) of stock enzyme was mixed with 50 μL
of assay buffer (described above), and incubated in the presence of excess inhibitor for 2 hr at 25oC.
Control enzyme sample was treated identically, but without inhibitor. The samples were then assayed
to insure complete inactivation of treated enzyme versus the control. Following inactivation, control
and treated enzyme samples were dialyzed against multiple 1L changes (3) of 20mM Hepes buffer
(pH7.4) containing 200mM NaCL, and 0.5 mM DTT (dithiothreitol) over a 144 hr period at 4oC. At various
intervals during dialysis, samples were assayed as described above. To assess product release from aryl
carbamate and urea inhibitors as a result of turnover during enzyme inactivation, the enzyme (50uL; 180
ug protein, high specific activity preparation, 15,730 RFU min-1mg-1) was first inactivated with inhibitor
at 35oC as described above. The final concentrations of compound 1a and 2c (Table 1) were 200 nM and
10 uM respectively. The enzyme solutions (100uL) were then extracted with 100 uL of Hexane: Ethyl
acetate (60:40) by vigorously vortexing the mixture (2x20 sec). Samples were centrifuged to separate
the phases, the organic layer removed, and evaporated under reduced pressure at room temperature.
100uL of ACN: H20 (1:1) was added to resuspend the sample. Analysis was by LC MS. Sample
separation was on an ACE 3 (50x4.6mm) C18 column equilibrated in H20 (0.1% FA): ACN (0.1% FA)
21

[90:10]. A linear gradient (0.8 mL/min) from 1 to 10 min reaching H20 (0.1% FA): ACN (0.1% FA) [5:95]
was used to elute analytes. MS analysis was made using an APCI source (vaporizer temperature 450oC)
on a Thermo LTQ mass spectrometer. Detection was in positive polarity. The concentration of active
enzyme used in assays was determined by stoichiometric titration of the enzyme with compound 1a
(table 1). Stock enzyme (10 uL) was mixed with varying concentrations of inhibitor in assay buffer (10uL),
and incubated at 25oC for 20 min to reach an endpoint. The enzyme samples were then assayed as
above. A plot of the fraction of active enzyme remaining versus [I] was used to estimate active enzyme
concentration.
Evaluation of FAAH Inhibitor Selectivity - The specificity of FAAH inhibition relative to other
mechanistically similar enzymes, such as porcine liver esterase and porcine pancreatic elastase, was also
explored for selected compounds. Both enzymes and substrates were obtained from commercial
sources, and assayed in microplate format. N-succinyl-ala-ala-ala-ρ-nitroanilide was used as a substrate
for pancreatic elastase, and 4-nitrophenyl butyrate was used as a substrate for measuring liver esterase
activity. Enzyme activity was measured by following the release of ρ-nitroaniline and ρ-nitrophenol at
400 nm from the respective chromogenic substrates. The assay reaction mixture contained enzyme,
100uM substrate, 0.125 M TrisCl, and 0.2 mM EDTA, pH 8.0 in a total volume of 200 uL. Reactions were
started by the addition of substrate. Control reactions give linear reaction rates (20 to 50 mOD/min)
over at least 5 min.
Animal experiments - Male ICR mice (18-22g) were purchased from Harlan. Animals were housed in a
climate-controlled room on a 12-12hr light/dark cycle where food and water were available ad libitum.
All experiments were performed under an approved protocol in accordance with the policies and
recommendations from IASP, NIH and IACUC of the University of Arizona. Stock solutions of compounds
were made in 100% dimethyl sulfoxide (DMSO). All compounds were diluted to final doses in Ringer's
solution for injection. Final concentrations of DMSO in solutions used for injection in all tests were less
than 10%. For intraperitoneal injections in mice the mouse was restrained and its head was held down
22

at an angle to allow the abdominal contents to move away from the injection site. The needle was
placed parallel to the linea alba and inserted at a 30-45 degree angle in one of the two lower quadrants
of the abdomen. Proper placement of the needle was verified prior to injection by withdrawing the
syringe plunger. A lack of intestinal or bladder content in the syringe confirmed a good placement of the
needle. The injection was then administered. A volume of 0.1 mL was used. In all experiments observers
and lab personnel conducting the injections were blinded to the experimental conditions.
Tail Flick: Analgesia was measured using the tail immersion method35. The mice were restrained with the
tail extending out. The distal portion of the tail (2–3 cm) was immersed in a water bath thermostatically
controlled at 52°C ± .5. The tail withdrawal reaction time (in seconds) was initially recorded as the tail
flick latency before drug administration and then recorded at 10, 20, 30, 45, and 60 minutes after the
administration of the test compound. A cutoff latency of 10 seconds was maintained to prevent tissue
damage. Percent maximum possible effect (%MPE) was calculated as %MPE = [(Test latency-Baseline
latency)/ (Cutoff latency-Baseline latency)]*100. All statistical analysis was done using Graph Pad Prism
for Mac OS X Version 6.0c. Comparisons between groups were made by two-way anova with
Bonferroni’s multiple comparison tests. At least 6 mice were used for each compound at each dose.
Data are presented as mean  SEM in all cases.
23

Abbreviations: FAAH, fatty acid amide hydrolase; AEA, arachidonyl ethanolamide; CB1, cannabinoid
receptor type 1; CB2 cannabinoid receptor type 2; CP, cytoplasmic port; ABP, acyl binding pocket; MAC,
membrane access channel; D-AMC, decanoyl 7-amino-4-methyl coumarin; Ar, aromatic, and SE,
standard error.
Acknowledgements: The authors would like to thank Drs. Pat Confalone, William Barnett, Stephen
Gutteridge, and Scott Dax, for their interest and support of the project, and DuPont crop protection
management for allowing exploration of the research area. We also thank Dr. John Andreassi for helpful
discussion. We also acknowledge the skilled technical assistance of Daniel Rhoades and Boris
Klyashchitsky. Special thanks to Dr. Seetha Krishnan (Syngene International Ltd, Bangalore, India) for
molecular biology support and Dr. Satheesh Anand (DuPont) for preliminary assessment of analgesic
activity. Work in the Price lab was supported by NIH grant GM102575.
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Figure Captions:
Figure 1 – Oxathiapiprolin fungicide; Piperdine and piperzine aryl urea FAAH inhibitors (PF-750, JNJ-
1661010); α-ketooxazole and carbamate FAAH inhibitors (OL-135, URB597).
Figure 2 – Docking of 1a (green carbon backbone) to FAAH structure 2WAP.
Figure 3 – A, Time dependent loss of FAAH activity in the presence of increasing concentrations of
compound 1l. Baseline (open circles), 10nM (dark circles), 1 nM (open squares), 0.1 nM (dark squares),
and the uninhibited reaction (open triangles). The solid lines represent the computer fit of the data
using the apparent inactivation constant (ki’) determined at each concentration of 1l. B, computer fit of
the apparent ki’ values for compound 2b to determine Kinact as described in the methods section.
Figure 4 – Recovery of FAAH activity (relative to controls) through exhaustive dialysis after inactivation
by various compounds (a) compound 1a; (b) JNJ-1661010; (c) compound 2c; (d) compound 2d as
described in the methods. Bars: light gray, 24 hrs; hash marks, 48 hrs; dark gray, 144 hrs; and black,
range of control activity over the time course. Structures are given in Table 1 and Fig 1.
Figure 5 - in vivo efficacies of novel FAAH inhibitors (Table 1) in the mouse tail flick test. A-F shows tail
flick latencies in the 52 C tail flick test following intraperitoneal injection of doses of novel FAAH
inhibitors. G shows summary data for all compounds where compound 1b shows the best in vivo
potency. * p < 0.05, ** p < 0.01, *** p < 0.001 two way ANOVA with Bonferroni posthoc test.
28

Graphical Abstract
IA
IB
phenol
†EIB
E + IA or B
EI
EI′
+ CH2O2
E +
very slow
†EIA
aniline
29