Benzothiophene piperazine and piperidine urea inhibitors of fatty acid amide hydrolase (FAAH)

Article abstract of DOI:10.1016/j.bmcl.2009.03.080

The synthesis and structure-activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzyme's active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freund's adjuvant (CFA) model of inflammatory pain.

Full text of DOI:10.1016/j.bmcl.2009.03.080

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2865–2869
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzothiophene piperazine and piperidine urea inhibitors of fatty acid
amide hydrolase (FAAH)
Douglas S. Johnson a,b, , Kay Ahn a,b, Suzanne Kesten a, Scott E. Lazerwith a, Yuntao Song a, Mark Morris a,
*
Lorraine Fay a, Tracy Gregory a,b, Cory Stiff a,b, James B. Dunbar Jr. a, Marya Liimatta a,c, David Beidler a,d
,
Sarah Smith a,d, Tyzoon K. Nomanbhoy e, Benjamin F. Cravatt f
a Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
b Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
c Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, USA
d Pfizer Global Research and Development, 700 Chesterfield Parkway, Chesterfield, MO 63017, USA
e ActivX Biosciences, 11025 North Torrey Pines Road, La Jolla, CA 92037, USA
f Department of Chemical Physiology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North 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:
The synthesis and structure–activity relationships (SAR) of a series of benzothiophene piperazine and
piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying
the enzyme’s active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these
urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Sev-
eral compounds showed in vivo activity in a rat complete Freund’s adjuvant (CFA) model of inflammatory
pain.
Received 24 February 2009
Revised 16 March 2009
Accepted 20 March 2009
Available online 24 March 2009
Keywords:
FAAH inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
Fatty acid amide hydrolase
Urea
Activity-based protein profiling
Covalent inhibitor
O
Fatty acid amide hydrolase (FAAH) is an integral membrane en-
zyme that degrades the fatty acid amide family of signaling lipids,
including the endocannabinoid anandamide.1 Genetic1g or phar-
macological1f inactivation of FAAH leads to elevated endogenous
levels of fatty acid amides and a range of behavioral effects includ-
ing analgesic and anti-inflammatory phenotypes in rodents.
Importantly, these behavioral phenotypes occur in the absence of
alterations in motility, weight gain, or body temperature that are
typically observed with direct cannabinoid receptor 1 (CB1) ago-
nists, indicating that FAAH may represent an attractive therapeutic
target for the treatment of inflammatory pain and related condi-
tions.2 Several classes of FAAH inhibitors have been reported
including electrophilic ketones (e.g., OL-1353), carbamates (e.g.,
URB5974 and SA-475) and, more recently, piperidine/piperazine ur-
eas (e.g., PF-7506 and Takeda-25/JNJ-16610107) (Fig. 1). We re-
cently reported that PF-750 inhibits FAAH by covalently
modifying the enzyme’s active site serine nucleophile and is com-
pletely selective for FAAH relative to other mammalian serine
hydrolases.6a In this Letter, we describe the synthesis and struc-
N
O
N
N
OL-135
O
N
H
O
Me
N
O
NHMe
N
H
O
URB597
O
O
NH2
SA-47
O
O
N
N
N
H
N
N
H
N
S
N
N
PF-750
JNJ-1661010/Takeda-25
Figure 1. Structures of FAAH inhibitors.
ture–activity relationships (SAR) of a series of benzothiophene
piperazine and piperidine urea FAAH inhibitors.
A series of piperazine phenyl ureas, exemplified by 1, was dis-
covered by high throughput screening of the Pfizer chemical file
* Corresponding author.
E-mail address: doug.johnson@pfizer.com (D.S. Johnson).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.080

2866
D. S. Johnson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2865–2869
F
F
O
O
F
a
Boc
O
O
Boc
N
N
H
N
N
H
N
b
N
N
N
S
N
S
S
7
1
PF-622
O
OH
N
9
10
Figure 2. Structures of HTS lead (1) and PF-622.
F
F
O
N
c, d
N
H
e
NH
against human FAAH (Fig. 2). We prepared piperazine phenyl urea
intermediate 2 and performed a series of reductive aminations
with various aromatic aldehydes (Scheme 1, Eqs. 1 and 2). This al-
lowed us to easily vary the aldehyde component and resulted in
the identification of several bicyclic cores including PF-6226a and
a series of benzothiophenes represented by 4h which is the subject
of this manuscript (Scheme 1, Eq. 2).
S
S
13
11
Boc
Me
N
N
N
MeO
OCN
O
12
8
Scheme 2. Synthesis of piperidine urea 13: (a) n-BuLi, THF, À78 °C; then add 8, 70%,
(b) NaBH4, EtOH, quant; (c) TsOH, toluene, quant; (d) H2(g), 20% Pd/C, MeOH, 87%;
(e) 12, CH2Cl2, 60%.
We then explored the benzothiophene lead 4h in more detail.
Therefore, the benzothiophene piperazine intermediate 5 was pre-
pared according to equation 3 and reacted with various commer-
cially available alkyl and aryl isocyanates or phenyl carbamates
of heterocyclic amines8 to give the corresponding piperazine ureas
4 and 6 (Scheme 1).
Schemes 2 and 3 outline the synthesis of representative piperi-
dine urea FAAH inhibitors utilized in the current studies. Lithiation
of benzothiophene 7 followed by reaction with Weinreb amide 8
provided ketone 9 in good yield.9 Ketone 9 was reduced with so-
dium borohydride to give alcohol 10 which underwent elimination
when treated with p-toluene sulfonic acid (TsOH). The resulting
double bond was hydrogenated to give piperidine 11 which was
treated with isocyanate 12 as described previously to give the de-
sired urea 13.8
The linker analogs 15–18 were synthesized according to
Scheme 3. Deprotection of Boc-piperidine 14 with TFA followed
by reaction with isocyanate 12 afforded urea 15 with a ketone lin-
ker. Wittig olefination of ketone 15 provided compound 16 with a
methylene substituted linker. Hydrogenation of the double bond
gave 17 with a methyl substituted linker as the racemate and the
enantiomers were separated by chiral chromatography. Further-
more, ketone 15 was reduced to give racemic 18 with a hydroxy
substituted linker.
O
N
Boc
N
N
H
N
N
c
a, b
S
S
S
O
O
15
14
f
O
O
N
N
N
N
N
H
d
H
S
R
16
R = Me: 17-rac
e
17-E1
17-E2
R = Me:
R =OH: 18-rac
and
Scheme 3. Synthesis of linker analogs 15–18: (a) TFA, CH2Cl2, 99%; (b) 12, CH2Cl2,
85%; (c) Ph3PCH3Br, n-BuLi, THF, À20 °C to rt, 97%; d) H2(g), Pd/C, MeOH, 76%; (e)
chiral chromatography (CHIRALCELÒ OD); (f) NaBH4, EtOH, quant.
to what has been observed for N-alkyl urea analogs of URB597.4a In
contrast, we found that aryl ureas such as phenyl urea 4h were po-
tent FAAH inhibitors. Next, we investigated a series of heteroaro-
matic ureas (4i–l, 6a) in part to avoid the generation of aniline
upon covalent binding to FAAH. The 3-aminopyridyl group (6a)
was preferred over the 2-aminopyridyl (4i) and 3-aminopyridazi-
nyl (4j) groups in the benzothiophene series. The SAR of the het-
erocyclic group was highly sensitive. For example, isoxazole 4k
retained potency, while the potency was lost with methylthiazole
4l, which is consistent with observations made by Keith et al. in a
related thiadiazolopiperazinyl urea series.7b Boger and co-workers
As previously reported, these piperidine/piperazine ureas inhi-
bit FAAH by covalent carbamylation of the catalytic Ser241 nucle-
ophile.6a,b Therefore, the potency of these benzothiophene
piperidine/piperazine urea inhibitors were determined by the sec-
ond order rate constants kinact/Ki values using an enzyme-coupled
FAAH assay as described previously.6b,10 Unlike IC50 values, kinact
/
Ki values do not change with various preincubation times and have
been described as the best measure of potency for irreversible
inhibitors.11
Table 1 shows the SAR of the right hand portion of the urea. The
alkyl ureas (4a–g) had very low potency for FAAH, which is similar
also observed that subtle changes in the central heterocycle of a-
ketoheterocycle FAAH inhibitors greatly influence inhibitor activ-
O
O
NH
a, b
Ph
Ph
RCHO
c
(eq 1)
(eq 2)
N
2
N
H
N
N
H
N
Boc
HN
R
N
3
O
O
Ph
Ph
O
c
N
N
H
N
N
H
CHO
+
S
HN
N
S
4h
2
R
d
e or f
CHO
NH
N
N
H
(eq 3)
S
N
N
S
S
5
4 and 6
Scheme 1. Synthesis of piperazine ureas 4 and 6: (a) PhNCO, CH2Cl2, 92%; (b) TFA, CH2Cl2, 80–99% (c) NaBH(OAc)3, DCE, 50–95%; (d) N-Boc-piperazine, NaBH(OAc)3, CH2Cl2;
then TFA, CH2Cl2, 64%; (e) RNCO (R = alkyl, Ph, 3-pyr; ie. 4d–g, 4h, 6a), CH2Cl2, 50–90%; (f) RNHCO2Ph (R = heterocycle, ie. 4j–1, 6), DMSO, 60 °C, 50–80%.

D. S. Johnson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2865–2869
2867
Table 1
Table 3
Human and rat FAAH potency (kinact/Ki values)15 for compounds 4a–l, 6a showing SAR
Human and rat FAAH potency (kinact/Ki values)15 for compounds 13, 19–39 showing
of the right hand side of the urea
SAR of substituted benzothiophenes
R3
R2
O
R
R5
O
R1
N
N
H
N
R4
N
N
H
N
S
X
S
R
h kinact/Ki (MÀ1 sÀ1
)
r kinact/Ki (MÀ1 sÀ1
)
R1
H
6a Me
20 Me
21 Et
22 Et
6b Me
23 Cl
R2 R3
R4
R5
X
h kinact/Ki (MÀ1 sÀ1
)
r kinact/Ki (MÀ1 sÀ1
)
4a
4b
4c
4d
4e
4f
4g
4h
4i
Butyl
44
42
<10
70
<10
<10
<10
1918
1002
2401
924
13
19
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
N
N
C
N
C
695
2401
5129
1402
1668
1481
900
174
CH2CH2OH
CH2C(O)NHMe
iPr
Cyclohexyl
tBu
Bn
Ph
2-pyr
3-pyr
<10
<10
<10
<10
<10
<10
762
635
1083
191
1083
2613
774
1013
390
N
N
133
24 Me
25 Me
26 Me
F
F
F
H
H
H
H
H
H
H
H
Me
N
C
N
3321
4147
1980
1203
1547
758
6a
4j
27 Me
13 Me
28 Me
29 Me
30 Me
31 Me
32 Me
33 Et
H
H
H
H
H
H
H
H
H
H
H
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
C
N
C
N
N
N
N
C
3134
4474
2635
3024
2822
1389
1381
1808
2292
1871
1169
779
729
557
482
1133
219
500
617
1141
260
183
N
N
4k
4l
1559
<10
1433
<10
Me
Me
Cl
CF3
OMe
F
F
F
Me
N
O
S
N
34 Et
35 Me
36 Me
H
Me
Me
N
N
ity.3c Also it is interesting to note that isoxazole 4k exhibited equal
potency for hFAAH and rFAAH, while phenyl urea 4h and pyrida-
zine urea 4j showed higher potency for hFAAH than rFAAH.
Next, we investigated the effect of substituents at the 6-position
of the 3-amino pyridine (Table 2). The compounds with 6-methoxy
(6g), 6-amino (6i) and 6-NHAc (6n) substituents are roughly equi-
potent to 6a, while the 6-Cl (6d) and 6-NHMe (6j) groups result in
compounds that are about twofold less potent and the potency is
further diminished by the 6-NHEt (6l) substituent (fourfold less
potent than 6a). Furthermore, 6-CF3 (6c) and 6-NEt2 (6m) substit-
uents are detrimental to activity. Taken together with the results in
Table 1, it is clear that the nature of the aromatic amine portion of
the urea is critical for activity, but the potency does not appear to
correlate with the leaving group ability. There appears to be a
steric limitation at the 6-position as hFAAH potency generally
37 Me
38 Me
39 Cl
H
H
H
H
H
H
OMe
OEt
Me
H
H
Me
N
N
N
73
14
73
113
<10
14
Subsequent SAR studies focused on optimization of the benzo-
thiophene substitution while maintaining the optimal 3-amino-
pyridyl urea (Table 3). It was found that incorporating an alkyl
group at the 3-position of the benzothiophene (R1) led to improved
potency. For example, compound 6a was 3–4-fold more potent
than the unsubstituted benzothiophene 19. In most cases for
hFAAH, the piperidine analogs were slightly more potent than
the corresponding piperazine analogs (i.e., 20 > 6a), which is most
likely a result of more favorable Van der Waals interactions of the
piperidine with the acyl chain binding channel of FAAH.1b,6b Sub-
stitution was tolerated at the 4- and 5-position of the benzothio-
phene (24–36), but no significant improvement in potency was
realized. Conversely, moving the substituent (R4) to the 6-position
of the benzothiophene impaired potency (37–39), which is pre-
dicted to introduce steric clashes with the FAAH protein (Fig. 5).
As shown in Table 4, the potency is maintained when a ketone
or methylene is incorporated within the linker (15 and 16, Scheme
3) whereas the reduced compounds 17 and 18 with methyl and hy-
droxyl substituents lose much of their activity. The sp2 hybridiza-
tion of the ketone and methylene linker compounds 15 and 16
imposes a conformational constraint that appears to help position
the benzothiophene in a more favorable binding orientation. The
SAR in this linker region is sensitive as further evidenced by the
>10-fold difference in potency for the enantiomers of 17.
decreases with the larger substituents (i.e.,
h kinact/Ki for
NH2 > NHMe > NMe2 > NEt2). Interestingly, several 6-amino com-
pounds (6j–m) actually show a preference for rFAAH over hFAAH.
Table 2
Human and rat FAAH potency (kinact/Ki values)15 for compounds 6a–6n showing SAR
of substituted pyridyl ureas
R
O
N
N
N
H
N
S
R
h kinact/Ki (MÀ1 sÀ1
)
r kinact/Ki (MÀ1 sÀ1
)
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
Me
CF3
CI
Br
Ph
OMe
O-(3-Pyr)
NH2
NHMe
NMe2
NHEt
NEt2
NHAc
2401
1481
69
1238
645
631
2931
605
1871
1108
892
597
90
1083
390
<10
242
157
1215
1060
101
1067
1513
1811
1891
227
Table 4
Human and rat FAAH potency (kinact/Ki values)15 for compounds 15–18 showing SAR
of the piperidine linker
h kinact/Ki (MÀ1 sÀ1
)
r kinact/Ki (MÀ1 sÀ1
)
15
16
17-rac
17-E1
17-E2
18-rac
3601
5441
114
34
617
200
1226
991
48
<10
110
79
6j
6k
61
6m
6n
2011
1719

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D. S. Johnson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2865–2869
fully conjugated urea
"activated" urea
FAAH binding
N
H
S
S
H
N
20
δ+
NH2
Lys142
δ−
O
H
O
H
- 3-aminopyridine
Ser241
Ser217
O
N
OH
NH2
O
Ser241 Ser217
Lys142
S
Figure 3. Proposed mechanism of covalent inhibition of FAAH by piperidine ureas. The catalytic triad Ser-Ser-Lys is shown.
We have proposed that the urea may be activated upon binding
in the active site of FAAH by undergoing a conformational change
that diminishes conjugation of the nitrogen lone pair with the car-
bonyl, which would activate the urea toward nucleophile attack as
shown in Figure 3.6a It appears that a cyclic secondary amine such
as piperazine or piperidine is critical for activation of the urea as
ureas prepared from primary amines are ineffective.4a
Compound 13 (Fig. 4) and compound 38 (Fig. 5) were mod-
eled,12 using Sybyl 8.0, into the ‘humanized’ rat FAAH (h/rFAAH)
crystal structure (2VYA).6b Compound 13 fit within the binding site
in almost identical fashion to that of PF-750 with only very slight
movement of the surrounding residue atoms, in essence, occupying
virtually identical space. The green surface depicting the binding
site volume and the space-filled representations were generated
from the compound 13-h/rFAAH model and are virtually identical
to those generated from the PF-750-h/rFAAH crystal structure. In
contrast, when the model of compound 38-h/rFAAH was mini-
mized, there was a significant shift in the position of the side chain
of Met436. As shown in Figure 5, the ethoxy portion of compound
38 extends beyond the green surface generated from the com-
pound 13 model illustrating the increased size of the ligand and
the subsequent displacement (i.e., push) of the Met436 side chain.
This steric interaction with Met436 could explain the decreased
potency of compound 38.
Figure 4. Model of compound 13 covalently attached to Ser241 of h/rFAAH using
coordinates from the PF-750-h/rFAAH crystal structure.6b Overlay with PF-750
shown in blue.
To assess the selectivity of the benzothiophene piperidine/
piperazine urea series, compounds 6b, 6g (PF-465), and 13 (PF-
946) were profiled at 100 lM by competitive activity-based pro-
tein profiling (ABPP)13 in a number of different proteomes derived
from mouse and human sources (Fig. 6). Competitive ABPP pro-
vides a global view of the proteome-wide selectivity of enzyme
inhibitors as described previously.13b,c In the case of FAAH inhibi-
tors, we used a reporter-tagged fluorophosphonate (FP) ABPP pro-
be,13c which serves as a general activity-based profiling tool for the
Figure 5. Model of compound 38 covalently attached to Ser241 of h/rFAAH using
coordinates from the PF-750-h/rFAAH crystal structure.6b Overlay with PF-750
shown in blue.
A. Mouse Liver
Cytosol
B. Mouse Kidney
Cytosol
C. Human Liver
Cytosol
D. Human Heart
Cytosol
E. Human Brain
Membrane
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
MW
80-
66-
FAAH
45-
42-
Figure 6. Selectivity profiling of FAAH inhibitors. Gel images of proteomes labeled with FP-rhodamine in the absence or presence of FAAH inhibitors (Lane 1, buffer; lane 2, 6b
at 100 M; lane 3, 6g at 100 M; lane 4, 13 at 100 M; lane 5, URB597 at 100 M; lane 6, URB597 at 10 M). Inhibited bands are shown with arrows. (A) soluble mouse liver
l
l
l
l
l
proteome, (B) soluble mouse kidney proteome, (C) soluble human liver proteome, (D) soluble human heart proteome, and (E) human brain membrane proteome. Fluorescent
gel signals shown in grayscale.

D. S. Johnson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2865–2869
2869
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3
10
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[Compound 6g] (mg/kg, i.p.)
Figure 7. Anti-hyperalgesic effects of compound 6g (3–30 mg/kg, ip) in the CFA
model of inflammatory pain. Compound 6g produces a dose-dependent reduction
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was profiled at 10 and 100 lM. Gel images of soluble proteomes of
mouse liver, mouse kidney, human liver, and human heart and the
membrane proteome of human brain are shown in Figure 6. Con-
sistent with previous reports,6a,14 multiple off-targets were ob-
served in peripheral tissues for URB597 even at 10 lM,
particularly amongst FP-labeled proteins migrating between 55
and 65 kDa. It is noteworthy that URB597, which has been shown
to be highly selective for serine hydrolases in mouse brain proteo-
mes, inhibits at least one additional hydrolase migrating at 55–
60 kDa in the human brain membrane proteome. In contrast, no
off-targets were observed for compounds 6b, 6g, and 13 even when
tested at 100 lM (Fig. 6). Under the assay conditions, compounds
6b, 6g, 13, and URB597 completely inhibited FAAH from both
mouse and human tissues.
10. Ahn, K. Patent WO 2006/085196, 2006.
Next we selected compound 6g (PF-465) to assess its in vivo
efficacy in a rat model of inflammatory pain. Subcutaneous injec-
tion of complete Freund’s adjuvant (CFA) into the plantar surface
of the hind paw produced a significant decrease in mechanical
paw weight threshold (PWT) at 5 days post injection (Fig. 7). Com-
pound 6g dosed at 3, 10 and 30 mg/kg (ip) caused a dose-depen-
11. Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis, 2nd ed.; Wiley-VCH: New York, 2000. pp 318–349.
12. All the hydrogens were added to the protein and each compound was
covalently attached to the sidechain oxygen of Ser241. The protein/ligand
structures were then minimized using the Sybyl forcefield with the backbone
atoms held constant; charges were not used. These minimized structures were
then compared to the PF-750-h/rFAAH crystal structure.
13. (a) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2008, 77, 383;
(b) Kidd, D.; Liu, Y.; Cravatt, B. F. Biochemistry 2001, 40, 4005; (c) Leung, D.;
Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotechnol. 2003, 21, 687; (d)
Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Proteomics 2001, 1,
1067; (e) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 14694.
14. (a) Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179; (b) Zhang, D.; Saraf,
A.; Kolasa, T.; Bhatia, P.; Zheng, G. Z.; Patel, M.; Lannoye, G. S.; Richardson, P.;
Stewart, A.; Rogers, J. C.; Brioni, J. D.; Surowy, C. S. Neuropharmacol. 2007, 52,
1095.
15. The hFAAH (amino acids 32–579) and rFAAH (amino acids 30–579) constructs
were generated as the N-terminal transmembrane-deleted truncated forms
with N-terminal His6 tags, and were expressed in E. coli and purified as
previously described.6b Both hFAAH and rFAAH enzymes used in the present
study had purity greater than 95% based on SDS–PAGE visualized by Coomassie
blue staining. The GDH-coupled FAAH assay was performed for determination
of potencies (kinact/Ki values) of inhibitors in 384-well microplates with a final
dent inhibition of mechanical allodynia with
a minimum
effective dose (MED) of 10 mg/kg. At doses of 10 and 30 mg/kg,
compound 6g inhibited pain responses to an equivalent degree
as the nonsteroidal anti-inflammatory drug naproxen (10 mg/kg,
ip).
In conclusion, we have described a series of benzothiophene
piperazine/piperidine urea FAAH inhibitors that covalently modify
the enzyme’s active site serine nucleophile. Activity-based protein
profiling revealed that these urea inhibitors are highly selective for
FAAH relative to other mammalian serine hydrolases. Furthermore,
in vivo activity was demonstrated in a rat CFA model of inflamma-
tory pain. Additional SAR development of this class of piperazine/
piperidine urea FAAH inhibitors will be reported in due course.
volume of 50 lL. The details on the assay and derivations of the overall
potency, kinact/Ki value, have been described previously.6b In the 384-well
format assay, the kinact/Ki values were generally calculated from the slope,
kinact/[Ki (1 + [S]/Km)], which is obtained from the kobs versus [I] linear lines. The
kinact/Ki values are averages of at least two independent experiments.
References and notes
1. (a) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner, R. A.; Gilula, N.
B. Nature 1996, 384, 83; (b) Bracey, M. A.; Hanson, M. A.; Masuda, K. R.; Stevens,