Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain

Article abstract of DOI:10.1016/j.bmcl.2011.08.048

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.

Full text of DOI:10.1016/j.bmcl.2011.08.048

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6545–6553
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH).
Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally
efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain
⇑
Marvin J. Meyers , Scott A. Long, Matthew J. Pelc, Jane L. Wang, Scott J. Bowen, Barbara A. Schweitzer,
Mark V. Wilcox, Joseph McDonald, Sarah E. Smith, Susan Foltin, Jeanne Rumsey, Young-Sun Yang,
⇑
Mark C. Walker, Satwik Kamtekar, David Beidler, Atli Thorarensen
Pfizer Global Research & Development, St. Louis Laboratories, 700 Chesterfield Parkway West, Chesterfield, MO 63017, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 19 August 2011
Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degra-
dation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has
been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spi-
rocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using
an iterative design and optimization strategy, lead compounds were identified with a remarkable reduc-
tion in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltet-
razole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and
pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to
move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remark-
able potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was
advanced as a clinical candidate.
Keywords:
Fatty acid amide hydrolase
FAAH
Pain
Irreversible inhibitor
Endocannabinoid
Ó 2011 Elsevier Ltd. All rights reserved.
Fatty acid amide hydrolase (FAAH) is an integral membrane ser-
ine hydrolase responsible for the degradation of fatty acid amide
signaling molecules including the endocannabinoid anandamide
(AEA).1 It has been shown that inhibition of FAAH leads to elevated
levels of AEA and analgesic effects in rodent models of pain with-
out evidence of side effects commonly seen with cannabinoid
receptor agonists, suggesting that inhibition of FAAH may result
in a new class of analgesic agents.2 In our previous report, we de-
scribed our campaign to replace the methylenepiperidine core of
our lead inhibitor of fatty acid amide hydrolase (FAAH), the clinical
candidate PF-04457845 (1).3 Compound 1 is a covalent inhibitor
with exquisite potency and selectivity for FAAH, is orally effica-
cious at 0.1 mpk in a rat model of pain and is being evaluated in
human clinical trials.4
In the previous report,3 we described the successful identifica-
tion of two new series of FAAH inhibitors with novel cores,
7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, repre-
sented by lead compounds 2a–b and 4, respectively. These
compounds have modest potency for FAAH (kinact/Ki = 1570–
3040 MÀ1 sÀ1) but were found to lack efficacy at modest doses in
the rat CFA model for pain (10 mpk, ip). Our medicinal chemistry
optimization program aimed to reduce MW while improving FAAH
k
inact/Ki potency to values greater than 2500 MÀ1 sÀ1, minimizing
in vivo clearance (CL <15), and improving oral in vivo efficacy to
1 mpk or less in rodent models of pain. Herein, we report our
successful identification of a series of preclinical candidates, includ-
ing clinical candidate PF-04862853 (15p).
Having identified two optimal spirocyclic cores, we outlined a
parallel medicinal chemistry optimization strategy relying heavily
on design of small, iterative compound libraries (ꢀ50 analogs/li-
brary) to optimize both the lipophilic tail group and the heteroar-
omatic head group (Chart 1). The aim of the compound libraries
was to increase compound potency for FAAH while maintaining
enzyme selectivity and CNS drug-like properties (e.g., MW <450,
HBD <2, PSA <90 Å3, c log P 2–5).5 This strategy would enable the
rapid identification of high quality compounds likely to have the
physiochemical properties needed to advance into rodent efficacy
models and into the clinic.
Abbreviations: FAAH, fatty acid amide hydrolase; AEA, anandamide; ABPP,
activity-based protein profiling; CFA, complete Freund’s adjuvant; MED, minimum
efficacious dose; SAR, structure–activity relationship; CNS, central nervous system;
PK, pharmacokinetic.
⇑
Corresponding authors at present address: Center for World Health and
Medicine at Saint Louis University, 1100 S. Grand Blvd., Saint Louis, MO 63104,
United States. Tel.: +1 314 977 5197; fax: +1 314 977 5220 (M.J.M.).
E-mail addresses: mmeyers8@slu.edu (M.J. Meyers), atli.thorarensen@pfizer.com
(A. Thorarensen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.048

6546
M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
O
F3C
N
N
N
H
N
N
O
1
PF-04457845 ( )
Core Replacement
(Reference 3)
O
O
R
2a R =
2b R =
N
N
N
Optimization
Head
Head
H
∗
∗
N
O
N
N
H
N
O
N
Tail
Tail
F3C
F3C
3
O
O
N
N
Optimization
N
N
H
N
N
H
O
O
O
N
4
5
Chart 1. Medicinal chemistry optimization strategy.
Boc
Boc
Boc
N
N
b or c
N
a
HO
HO
BnO
X
X
X
O
X = CH2 or CH2O
8
7
6
O
Boc
Ar
N
N
N
e,f
d
H
HetArO
O
X
HetArO
X
Ar
N
O
10H
9
11
Scheme 1. Library synthesis of terminal heteroaryl tail analogs. Reagents and conditions:6 (a) 3-benzyloxyphenylmagnesium bromide, 2-MeTHF, 0 °C; (b) Raney Ni, EtOH,
reflux; (c) (i) Et3SiH, TFA, BF3OEt2, DCM, 0 °C; (ii) Boc2O, TEA, DCM; (iii) H2, 10% Pd/C, MeOH, 45 psi (36–38% from 6); (d) heteroaryl chloride, cesium carbonate, DMF, 90 °C; (e)
4 N HCl/dioxane, DCM; (f) 10, DIEA, CH3CN, room temp.
Initially, the tail group was optimized while keeping the urea
head group fixed as either 3-pyridazinyl or 3,4-dimethylisoxazol-
5-yl. Libraries designed to replace the terminal heteroaryl group
were prepared from the advanced intermediate phenol 8 as shown
in Scheme 1.6 Ketone intermediate 6 was treated with 3-benzyl-
oxyphenylmagnesium bromide to give protected alcohol 7. The
alcohol was reduced during benzyl deprotection with Raney Ni to
give phenol 8. Alternatively, alcohol 7 was reduced with triethylsi-
lane under strong acidic conditions. This method required repro-
tection of the amine with Boc anhydride and removal of the
benzyl group. Phenol 8 was then treated with an array of hetero-
aryl chlorides in the presence of cesium carbonate to give the crude
displacement products. After concentration, treatment with HCl
gave the Boc deprotected products which were concentrated, trea-
ted with phenyl aryl carbamates 10, and purified by reverse phase
HPLC to give the final biaryl ether products 11.
In an aim to reduce molecular weight to produce more CNS
drug-like compounds,5 the biaryl ether group was replaced with
smaller substituted phenyl moieties using a modified Suzuki cou-
pling strategy (Scheme 2).6 Ketone 6 was reduced with sodium
O
Boc
Boc
N
N
HetAr
a,b
c
d,e
N
N
H
6
X
X
Br
Ar
X
Ar
12
X = CH2 or CH2O
13
14
Scheme 2. Library synthesis of terminal truncated aryl tail analogs. Reagents and conditions:6 (a) NaBH4, MeOH, 0 °C (86%); (b) PPh3, CBr4, THF (42–51%); (c) ArB(OH)2, NiI2,
LiHMDS, trans-1,2-aminohexanol, isopropanol; (d) 4 N HCl/dioxane, DCM; (e) 10, DIEA, CH3CN, room temp.

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
6547
Figure 1. 7-Azaspiro[3.5]nonane tail group SAR for different tail/head group combinations. (A) Biaryl ether/pyridazine. (B) Biaryl ether/dimethylisoxazole. (C) Truncated
phenyl/pyridazine. (D) Truncated phenyl/dimethylisoxazole.
Figure 2. 1-Oxa-8-azaspiro[4.5]decane tail group SAR for different tail/head group combinations. (A) Biaryl ether/pyridazine. (B) Biaryl ether/dimethylisoxazole.
(C) Truncated phenyl/pyridazine. (D) Truncated phenyl/dimethylisoxazole.

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M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
borohydride to give the alcohol which was readily converted to
bromide 12 by treatment with triphenylphosphine and carbon
tetrabromide. Bromide 12 was the key intermediate used to pre-
pare libraries of truncated aryl tail groups using aryl boronic acids
and the Ni-catalyzed Suzuki coupling methodology developed by
Fu and co-workers.7 Intermediate crude coupling products 13 were
treated with HCl to remove the Boc protecting group, concentrated,
exposed to heteroaryl phenyl carbamates 10 under mild basic con-
ditions, and purified by reverse phase HPLC to give the final prod-
ucts 14.
Analysis of hFAAH activity as a function of the tail group substi-
tution revealed some interesting trends. Representative com-
pounds from the 7-azaspiro[3.5]nonane series illustrate these
trends as shown in Figure 1. Replacement of the terminal trifluoro-
methyl pyridyl group with other heterocycles resulted in only mar-
ginal gains in hFAAH potency with the 3-pyridazinyl head group
being preferred over the 3,4-dimethylisoxazol-5-yl head group
(Fig. 1A and B). Surprisingly, this trend was only observed for the
biaryl ether tail group. Remarkably, truncation of the biaryl ether
to a phenyl ring with small lipophilic substituents such as Cl, F,
Me, OMe, CF3, or OCF3 gave compounds with a reduced molecular
weight near 400 and significantly improved hFAAH activity
(Fig. 1D; Table 1). This effect was observed only when combined
with the 3,4-dimethylisoxazol-5-yl head group whereas the direct
comparators in this class with the 3-pyridazinyl head group were
only weakly active (Fig. 1C). As a result, subsequent libraries were
designed focusing on optimization of the truncated aryl tail group
with small lipophilic groups and the 3,4-dimethylisoxazol-5-yl
head group (Fig. 1D).
Similar trends were observed for the 1-oxa-8-azaspiro[4.5]dec-
ane series ( Fig. 2A–D). For this series, however, higher levels
of FAAH potency were more readily achieved with the biaryl
ethers/pyridazine tail/head group combination than with the
truncated/dimethylisoxazole tail/head group combination. Like
the 7-azaspiro[3.5]nonane series, subsequent libraries focused
on optimization of the truncated aryl tail group with small
lipophilic groups and the 3,4-dimethylisoxazol-5-yl head group
(Fig. 2D). Other tail group derivatives were also explored but are
outside the scope of this manuscript and will be reported
elsewhere.
Specific representative examples truncated aryl tail analogs
from the 7-azaspiro[3.5]nonane series (15a–p) and the 1-oxa-8-
azaspiro[4.5]decane series (16a–j) are shown in Table 1. For the
7-azaspiro[3.5]nonane series, the simple phenyl analog (15a) was
potent for human FAAH (hFAAH), but did not have sufficient po-
tency against the rat FAAH (rFAAH) necessary for advancement
into efficacy studies. Similarly, methoxyphenyl analogs (15b–d)
were equipotent for hFAAH but insufficient for rFAAH. Interest-
ingly, the corresponding trifluoromethoxyphenyl analogs (15e–g)
had equipotency for hFAAH and rFAAH but greater sensitivity to
the substitution pattern (i.e., 3-OCF3 15f retained hFAAH and
rFAAH potency but 2-OCF3 15e and 4-OCF3 15g were four to eight-
fold less potent). A similar hFAAH: rFAAH potency trend was ob-
served for trifluoromethyl (15h–j) versus non-halogenated
methyl (15k), and ethyl (15l) analogs. 3-Chloro and 4-chlorophenyl
analogs (15m–n) also demonstrated superior hFAAH and rFAAH
potency, and the combination of 3,4-dihalogenation resulted in po-
tent analogs such as 15o–p.
was observed for lead compound 4,3 both enantiomers retained
activity similar to the racemate (16f).
Having established small lipophilic groups as optimal replace-
ments for the biaryl ether in the 7-azaspiro[3.5]nonane series,
we turned our focus towards optimization of the heteroaromatic
urea leaving group. For this, we selected a few optimal tail groups
(e.g., 3-trifluoromethoxyphenyl) and prepared the requisite amine
(e.g., 17;Scheme 3).6 Amines such as 17 could be converted directly
to the desired ureas 18 as described above or first converted to
nitrophenyl carbamate 19 which was then reacted with an array
of heteroaromatic amines to give a broader collection of ureas 18
after purification by reverse phase HPLC. Targeting access to FAAH
in the CNS compartment, we designed libraries of heterocyclic ur-
eas wherein no H-bond donors were added to the final products
(i.e., the requisite primary amino heterocycles did not contain
additional H-bond donors).
Using this approach, more than 70 ureas were prepared on the
3-trifluormethyoxyphenyl 7-azaspiro[3.5]nonane scaffold. A plot
of hFAAH activity as a function of heteroaromatic group reveals a
rather remarkable sensitivity to the makeup of the heterocyclic
leaving group (Fig. 3). Out of 72 examples, only four examples
had FAAH kinact/Ki potency values greater than 2500 MÀ1 sÀ1
.
Specific examples are shown in Table 2. The most potent heteroar-
omatic groups identified were 1-methyltetrazole and 1-ethyltet-
razole with roughly five- and two-fold potency enhancements
over 3,4-dimethylisoxazol-5-yl (entries 1–2 and 5). Remarkably,
the 2-methyltetrazole isomer was ꢀ100-fold less active than the
1-methyl isomer (entry 3) and no FAAH activity was detected for
the corresponding methyltriazole derivative (entry 4). A similar
level of sensitivity was observed for the isoxazol-5-yl series. The
3-ethyl-4-methylisoxazol-5-yl analog lost only twofold FAAH
potency relative to the dimethyl isomer (entries 5 and 6) while
4-ethyl-3-methyl isomer was nearly 10-fold less potent (entry 7).
Deletion of the 4-methyl group (entry 8) or reversal of the O and
N atoms (entry 9) resulted in more than 20-fold potency reduc-
tions. The only other heterocycle identified with retention of FAAH
potency was 5-methyl-1,3,4-oxadiazol-2-yl (entry 10).
Given the superior potency observed with 1-methyltetrazole
ureas (e.g., 18a), several of the optimal truncated tail templates
(e.g., 3-CF3O, 3-CF3, 3-Cl and 3-Cl,4-F) from both the 7-azaspi-
ro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane series were com-
bined with 5-amino-1-methyltetrazole using the nitrophenyl
carbamate synthetic methodology shown in Scheme 3. Representa-
tive compounds obtained are shown in Table 3. Examples from the
7-azaspiro[3.5]nonane series (18a, 20–21) have exceptional po-
tency for hFAAH and sufficient potency for rFAAH. The correspond-
ing 1-oxa-8-azaspiro[4.5]decane analogs (22–24) are also quite
potent, albeit at a two- to fourfold reduction.
Compounds with suitable FAAH potency (FAAH
kinact/Ki
>2500 MÀ1 sÀ1) were resynthesized6 profiled for selectivity versus
the serine hydrolase super family of enzymes (>200 human en-
zymes, including FAAH), as previously described.8 The compounds
were assayed at 100 lM in a functional proteomic screen based
on competitive activity-based protein profiling (ABPP) in human
brain membrane and soluble liver proteomes using a rhodamine-
tagged fluorophosphonate ABPP probe (ActivX screen). Sufficiently
potent compounds that demonstrated selectivity for FAAH in
the ActivX, dofetilide and CYP inhibition counter screens were
Truncated aryl tail analogs from the 1-oxa-8-azaspiro[4.5]dec-
ane series (16a–j) were generally two- to five-fold less potent that
the corresponding analogs from the 7-azaspiro[3.5]nonane series
(e.g., compare 16a–15a, 16b–15f, and 16h–15p). General SAR
trends noted above for the substitution pattern on the aryl group
was consistent for both series (e.g., 16a–h) with small lipophilic
groups being preferred substituents in the 3-position. In contrast,
small polar groups such as cyano were not tolerated (16i–j). As
advanced to a screen of oral efficacy in the rat Complete
Fruend’s Adjuvant (CFA) pain model (Fig. 4 and Table 4).9,10 This
experiment was a key decision making assay which enabled us
to determine both pharmacokinetic (PK) parameters and in vivo
efficacy; importantly, this assay afforded the ability to move
quickly from compound synthesis to selection of preclinical
candidates. In practice, compounds were initially screened at an
oral fixed dose of 3 mpk. Three h post dose, the FAAH inhibition

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
6549
Table 1
Representative truncated compounds from tail group optimization
N
O
O
O
N
N
N
H
O
N
N
H
Ar
Ar
O
15a-r
16a-m
a
Compd
Ar
hFAAH kinact/Kia (MÀ1 sÀ1
)
rFAAH kinact/Ki (MÀ1 sÀ1
)
MW
c log P
2b
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
3-((5-(Trifluoromethyl)pyridin-2-yl)oxy)phenyl
Phenyl
2-Methoxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
2-(Trifluoromethoxy)phenyl
3-(Trifluoromethoxy)phenyl
4-(Trifluoromethoxy)phenyl
2-(Trifluoromethyl)phenyl
3-(Trifluoromethyl)phenyl
4-(Trifluoromethyl)phenyl
3-Methylphenyl
1760
2080
2640
2190
1760
839
2830
353
1140
3060
245
2870
5670
3940
2030
4550
4190
461
1800
162
1330
481
9460
679
265
741
585
564
3860
229
500
339
369
369
369
423
423
423
407
407
407
353
367
373
373
407
391
355
439
439
423
423
389
389
389
423
407
380
380
4.6
3.0
2.5
2.9
2.9
3.6
4.0
4.0
3.9
3.9
3.9
3.5
4.0
3.7
3.7
4.3
3.8
1.4
2.5
2.5
2.3
2.3
2.1
2.1
2.1
2.7
2.3
0.9
0.9
3690
348
1620
1340
3560
2070
3470
5820
315
3110
461
2680
714
2670
1560
3830
4360
3950
999
3-Ethylphenyl
3-Chlorophenyl
4-Chlorophenyl
3,4-Dichlorophenyl
3-Chloro-4-fluorophenyl
Phenyl
3-(Trifluoromethoxy)phenyl
4-(Trifluoromethoxy)phenyl
3-(Trifluoromethyl)phenyl
4-(Trifluoromethyl)phenyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
3,4-Dichlorophenyl
3-Chloro-4-fluorophenyl
3-Cyanophenyl
15p
( )-16a
( )-16b
( )-16c
( )-16d
( )-16e
( )-16f
16f-ent1
16f-ent2
( )-16g
( )-16h
( )-16i
( )-16j
2040
2390
2590
2430
1580
243
4-Cyanophenyl
43.2
26.1
a
Each kinact/Ki value corresponds to an average of at least two independent determinations.
O
HetAr
NH
a
N
N
H
F3CO
O
F3C
18
17
c
b
NO2
O
N
O
F3CO
19
Scheme 3. Preparation of urea libraries. Reagents and conditions:6 (a) 10, DIEA, CH3CN, room temp; (b) 4-nitrophenyl chloroformate, dioxane, satd sodium bicarbonate
(92%); (c) H2N-HetAr, NaH, DMA.
in the brain as well as compound concentration in the brain and
plasma were determined. Additionally, to facilitate estimation of
rat clearance values, compound plasma concentrations at 24 h
were determined.
With the ultimate goal of choosing the ‘best’ compound suitable
for QD dosing with good safety margins, a few key factors were
emphasized during the screening process. First, it was necessary
to have sufficient brain exposure relative to potency to achieve
>95% FAAH inhibition; this defined the minimum efficacious dose
(MED). It was also desirable for a compound to have a low effica-
cious concentration (Ceff ꢀ Cmin), resulting a low dose, and a shal-
low Cmax/Cmin profile in plasma. In general, rat clearance values
of <15 mL/min/kg, coupled with FAAH potency
kinact/Ki
>2500 MÀ1 sÀ1, was optimal for a low dose QD profile. More de-
tailed evaluation of the dose response relationships for several
compounds in the CFA model, enabled us to develop the correla-
tion between in vitro potency, exposure and MED.11,12 As shown
in Figure 4, it was determined that, in general, a [brain]/Ki >0.2

6550
M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
Figure 3. Urea group SAR.
Table 2
Urea heteroaromatic group SAR
O
HetAr
N
N
H
O
F3C
Entry
1
Compd
HetAr
hFAAH kinact/Kia (MÀ1 sÀ1
)
rFAAH kinact/Kia (MÀ1 sÀ1
)
MW
410
c log P
N
N
N
18a
13,200
4570
3.7
N
∗
N
N
N
2
18b
6940
3420
424
4.2
N
∗
N
N
N
3
4
18c
18d
165
<10
152
—
410
409
2.6
3.3
N
∗
N
N
N
∗
N
O
∗
5
6
15f
2830
1650
3860
1660
423
437
4.0
4.5
N
N
O
18e
∗
O
7
18f
338
—
437
4.5
∗
N
O
O
8
9
18g
18h
113
64
—
409
423
4.4
4.0
∗
N
34.2
∗

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
6551
Table 2 (continued)
Entry
Compd
HetAr
hFAAH kinact/Kia (MÀ1 sÀ1
)
rFAAH kinact/Kia (MÀ1 sÀ1
)
MW
410
c log P
O
10
18i
1530
2460
3.3
N
N
∗
a
Each kinact/Ki value corresponds to an average of at least two independent determinations.
Table 3
1-Methyltetrazole tail group SAR
N
N
O
N
O
N
N
N
N
N
N
H
N
N
N
H
Ar
Ar
O
A
B
Compd
Ar
Series
hFAAH kinact/Kia (MÀ1 sÀ1
)
rFAAH kinact/Kia (MÀ1 sÀ1
)
MW
c log P
18a
20
21
22-ent1
23-ent1
23-ent2
24-ent1
3-(Trifluoromethoxy)phenyl
3-(Trifluoromethyl)phenyl
3-Chloro-4-fluorophenyl
3-(Trifluoromethoxy)phenyl
3-(Trifluoromethyl)phenyl
3-(Trifluoromethyl)phenyl
3-Chlorophenyl
A
A
A
B
B
B
B
13,200
12,400
16,300
7110
4600
3760
4570
5750
5940
4560
2160
2110
1970
410
394
379
426
410
410
377
3.7
3.5
3.5
2.1
2.0
2.0
1.8
5600
a
Each kinact/Ki value corresponds to an average of at least two independent determinations.
was required for efficacy. Importantly, this single experiment pro-
vided information on target inhibition at the site of action ([brain]/
Ki), PK profile and their relationship to efficacy.
advancement to more resource and time intensive studies. The most
promising compounds were selected for a more complete analysis
including a dose-response study in the CFA model and IV/PO rat
PK. This enabled a more definitive projection of human PK and Ceff
using multiple data points and the generation of an EC95 based upon
a simple Emax model relating %FAAH inhibition in the brain to com-
pound plasma exposure. Importantly, for the compounds which we
conducted this more rigorous analysis, there was good agreement
with the data estimated from the screening model (Table 4).
On the basis of the in vivo efficacy/PK screen and subsequent
dose response efficacy experiments, compound 15p was selected
as a candidate for human clinical trials. This compound has calcu-
lated properties consistent with that for known CNS drugs
(MW = 391, c log P = 3.8, tPSA = 58 Å3) and clean selectivity profiles
in CYP, hERG, Ames, micronucleous, and CEREP assays (data not
shown). Furthermore, characterization in the ActivX selectivity
screen, 15p was found to inhibit only FAAH at concentrations up
For further prioritization purposes, human PK parameters were
estimated from this single point efficacy/PK experiment utilizing
allometric scaling from the estimated rat PK.13 Rat clearance was
estimated from the 3 h and 24 h time points. We assumed that both
the absorption constant (Ka) and volume of distribution were rela-
tively invariant across similar chemical space. We took a conserva-
tive approach to human dose setting and assumed that efficacy
required 24 h coverage of the Ceff derived from the CFA studies
(i.e., Cmin >Ceff). This exposure was likely higher than that required
at 24 h to maintain efficacy. The predicted human dose and pharma-
cokinetic profile, including the estimated Cmax and Cmax/Cmin ratio,
proved to be useful tools for prioritization of compounds before
to 100 lM. In rats, 15p has a half-life of 7.1 h (CL = 8.5 mL/min/
Table 4
In vivo efficacy data for representative compounds
Compd hFAAH
rFAAH
CFA
3 mpk
PO
Predicted
CFA MED
(mpk)
Actual CFA
MED
(mpk)
k
inact/Kia
k
inact/Kia
(MÀ1 sÀ1
)
(MÀ1 sÀ1
)
15f
15l
15m
15p
18a
20
2830
5670
3940
3860
1340
3560
5820
4570
5750
7110
Active
Inactive N/A
Active
Active
Active
Active
0.5
1
N/A
1
0.3
0.1
1
0.8
0.1
0.04
0.2
Active
4190
13,200
12,400
ent1
22-
0.3
4560
2160
0.1
23-
ent1
4600
Active
0.1
Figure 4. The correlation between efficacy and brain/Ki across several compound
classes.3,4 Colored by efficacy in the CFA assay: green = active, red = inactive; and
shaped by dose: N = single dose PO, d = single dose IP, j = dose response PO. Single
point studies were 3 mpk, 10 mpk or 25 mpk.
1.0
a
Each kinact/Ki value corresponds to an average of at least two independent
determinations.

6552
M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
B. Residual FAAH Activity
A. Rat CFA Efficacy
25
20
15
10
5
* p< 0.05 ANOVA (Dunnetts)
20000
15000
10000
5000
0
30000
20000
10000
0
Brain
Leukocyte
*
*
*
*
0
C. Compound Levels
D. Anandamide (AEA) Levels
5000
4000
3000
2000
1000
0
1000
800
600
400
200
0
20
15
10
5
1.5
1.0
0.5
0.0
Brain
Plasma
Brain
Plasma
0
All data 4 Hours Post dose
Figure 5. Effect of compound 15p in the CFA model of inflammatory pain: relationship between efficacy, PK, and modulation of target and mechanism biomarkers. All
samples were taken 4 h post dose. All PK/biomarker samples and behavioural assessments were taken from the same animals. (A) Paw withdrawal threshold assessment via
von Frey filaments. N = 8 animals/group. ⁄Significance with respect to vehicle (p <0.05, ANOVA/Dunnetts). (B) Residual FAAH activity from brain tissue or leukocytes were
measured in a FAAH assay with 3H-anandamide as substrate. (C and D) Compound and AEA levels were determined by LC/MS. (B, C and D) n = 4 animals/group.
kg, Vdss = 3.4 L/kg) and 53% bioavailability. Similarly, in dogs, 15p
has a half-life of 7.8 h (CL = 11.1 mL/min/kg, Vdss = 5.0 L/kg) and
33% bioavailability. In the rat CFA model, 15p has a MED of
0.3 mpk (Fig. 5A). At 0.3 mpk, residual FAAH activity is completely
inhibited in both leukocytes and brain isolates, corresponding to
resolutions, S. Yang for 2D NMR determinations, and T. K. Noma-
nbhoy at ActivX for ABPP proteome profiling. Finally, we would like
to thank Dr. Robert O. Hughes for his critical evaluation of this
manuscript.
0.459
l
M plasma concentration of 15p (Fig. 5B and C). As expected,
References and notes
levels of the FAAH substrate AEA were elevated in a dose-depen-
dent manner (Fig. 5D), consistent with the observed efficacy and
residual FAAH activity data.
1. Ahn, K.; McKinney, M. K.; Cravatt, B. F. Chem. Rev. 2008, 108, 1687.
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6. (a) For detailed experimental procedures for the preparation of 7-
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Meyers, M. J.; Schweitzer, B. A.; Pelc, M.; Wang, J. L.; Long, S. A.;
Thorarensen, A. WO2010/049841, 2010.; (b) For detailed experimental
procedures for the preparation of 1-oxa-8-azaspiro[4.5]decane compounds,
see: Long, S. A.; Wang, J. L.; Meyers, M. J.; Pelc, M.; Schweitzer, B. A.;
Thorarensen, A. WO2010/058318, 2010.
In summary, we have optimized the spirocyclic 7-azaspi-
ro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane cores for FAAH po-
tency, identifying leads with significantly reduced molecular
weights and favorable CNS drug-like properties. Additionally, 3,4-
dimethylisoxazole and 1-methyltetrazole were identified as supe-
rior urea moieties for inhibition of FAAH. Once suitable low molec-
ular weight tail groups and urea heterocycles were identified, the
most promising compounds were advanced using the ActivX selec-
tivity panel and CFA efficacy assay to select preclinical candidates.
On the basis of the remarkable potency, selectivity, pharmacoki-
netic properties and in vivo efficacy, 15p was advanced as a clinical
candidate.
7. (a) Gonzalez-Bobes, F.; Fu, G. C. J. Am. Chem. Soc. 2006, 128, 5360; (b) Note: This
chemistry only worked on small scale; when a larger quantity of a specific
compound was desired, a sequence similar to Scheme 1 was utilized.
8. (a) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2008, 77, 383;
(b) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
Acknowledgments
We would like to thank Professor Benjamin F. Cravatt for his ad-
vice and extensive discussions on this project, J. T. Collins for chiral

M. J. Meyers et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6545–6553
6553
14694; (c) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.;
Stevenson, T.; Lund, E. T.; Nugent, R. A.; Nomanbhoy, T. K.; Alexander, J. P.;
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the MED was then predicted using the above knowledge.
relationship was assumed for all compounds allowing calculation of these
values. MED dose calculation is the simplified equation: (CFA-SP dose/
A
linear dose
9. Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.;
Weerpana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith, S.; Stiff, C.;
Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Becelaere, K. V.; Stevens,
R. C.; Cravatt, B. F. Chem. Biol. 2009, 16, 411.
([brain]SP/Ki)) = (MED
rFAAH = 4900 MÀ1 sÀ1
dose = 3 mpk;
dose/0.2).
[brain]SP = 2.44
MED-pred = 3 mpk/4.52 ⁄ 0.2 = 0.12 mpk;
Example
M;
calculation
[brain]/Ki = 4.52;
for
15p:
CFA-SP
actual
;
l
MED = 0.3 mpk]. Plasma exposure at MED: [plasma]MED = ([plasma]SP/CFA-SP
10. The Pfizer Institutional Animal Care and Use Committee reviewed and
approved the animal use in these studies. The animal care and use program
is fully accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care, International.
dose) ⁄ MED dose pred.
12. Maurer, T. S.; DeBartolo, D. B.; Tess, D. A.; Scott, D. O. Drug Metab. Dispos. 2005,
33, 175.
13. Hosea, N. A.; Collard, W. T.; Cole, S.; Maurer, T. S.; Fang, R. X.; Jones, H.; Kakar, S.
11. Calculation of [brain]/Ki was estimated by: [brain] ⁄ (kinact/Ki)/kinact
.
This
M.; Nakai, Y.; Smith, B. J.; Webster, R.; Beaumont, K. J. Clin. Pharmacol. 2009, 49,
513.
required the assumption that the kinact was constant and we utilized a value
for kinact = 0.00265 sÀ1. The MED and subsequently the [plasma] exposure at