Aryl piperazinyl ureas as inhibitors of fatty acid amide hydrolase (FAAH) in rat, dog, and primate

Article abstract of DOI:10.1021/ml300186g

A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatt

Full text of DOI:10.1021/ml300186g

Letter
pubs.acs.org/acsmedchemlett
Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase
(FAAH) in Rat, Dog, and Primate
John M. Keith,* Rich Apodaca, Mark Tichenor, Wei Xiao, William Jones, Joan Pierce, Mark Seierstad,
James Palmer, Michael Webb, Mark Karbarz, Brian Scott, Sandy Wilson, Lin Luo, Michelle Wennerholm,
Leon Chang, Sean Brown, Michele Rizzolio, Raymond Rynberg, Sandra Chaplan,
and J. Guy Breitenbucher
Janssen Pharmaceutical Research and Development, LLC, 3210 Merryfield Row, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty
acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit
FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels
of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl
ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels
of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a
significant role in regulating plasma levels of fatty acid ethanolamides in primates.
KEYWORDS: FAAH, urea, ethanolamides, FAAH-2, enzyme, anandamide
annabis sativa has been used medicinally for the treatment
of pain and other conditions for thousands of years,1 but
(PEA), N-oleoylethanolamide (OEA), oleamide (OA), 2-
arachidonyl glyceryl ether (noladin), O-arachidonylethanol-
amine (virodamine), and N-arachidonyldopamine.11,12 PEA is
known to have anti-inflammatory properties and exert analgesic
effects through a noncannabinoid pathway.13,14 OEA appears to
be involved in regulating satiety,15 whereas the similar OA is an
important contributor to sleep induction.16 The pharmacology
of the other FAAH substrates is less well understood.
FAAH appears to be the primary enzyme responsible for the
metabolism of AEA, PEA, and OEA as the levels of these fatty
acid amides (FAAs) are all greatly elevated in FAAH knockout
mice.8 These knockout mice are healthy but are phenotypically
less sensitive to pain, thus supporting the notion that
pharmacologically induced FAAH inhibition may afford
analgesia. More recently, another fatty acid amide metabolizing
enzyme (FAAH-2) was discovered and cloned.17 FAAH-2 is
not expressed in rats or dogs but is present in monkeys and
humans. While the preferred substrates of FAAH-2 are primary
amides, its expression in primates raises the question of
whether or not a selective FAAH inhibitor would be able to
elevate the levels of FAAs in primates significantly.
C
it was not until the discovery and cloning of two cannabinoid
receptors CB12 and CB23 in the early 1990s that a rationale for
the analgesic effects of cannabis preparations became possible.
The principal active ingredient of cannabis, Δ9-THC, is not an
ideal drug due to numerous adverse side effects (hypothermia,
catalepsy, hyperphagia, delayed gastric emptying, dizziness, and
diminished motor coordination and memory) associated with
its use.4 The later discovery of anandamide (AEA)5 (Figure 1),
Numerous groups have reported the preparation and
pharmacological testing of FAAH inhibitors (Figure 2). Boger's
group described several series of highly potent α-keto
heterocycles.18,19 Piomelli20 disclosed carbamate-based inhib-
itors as did Sanofi.21 Pfizer,22−24 Takeda,25 and Johnson &
Johnson26 have disclosed phenyl and heteroaryl urea inhibitors
of FAAH. All of these compounds are quite potent and form
covalent bonds with Ser241 within the FAAH active site. OL-
Figure 1. Δ9-THC and substrates of FAAH.
an agonist of CB1,6−8 and fatty acid amide hydrolase
(FAAH),10 the enzyme that hydrolyzes AEA, opened the
possibility of engaging the cannabinoid system without the use
of direct exogenous cannabinoid agonists.
Unfortunately, AEA is also a nonideal drug substance
because it has poor physical properties and is rapidly (T1/2
on the order of a few minutes)9 metabolized by FAAH to give
ethanolamine and arachidonic acid.10 FAAH also breaks down
several other substances, including N-palmitoylethanolamide
Received: July 6, 2012
Accepted: August 22, 2012
Published: August 22, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Preparation of Anilinyl Piperazinyl Ureas:
a
Variation of Benzyl Substituents
a
Reagents and conditions: (a) 1.1 mol equiv PhNCO, methylene
chloride, 4 h, rt, 93%. (b) 4 N HCl in 1,4-dioxane, methylene chloride,
40 h, rt, 96%. (c) 1.5 mol equiv aldehyde (R defined in Table 1), 9 mol
equiv diisopropylethylamine, 2.5 mol equiv MP-BH(OAc)3, THF, rt,
24 h, 17−66%.
in reasonable yield. A selection of compounds prepared in this
manner is shown in Table 1. Because these aryl ureas are
known to be covalent inhibitors of FAAH, their activities are
reported as apparent IC50 values after 1 h of incubation with the
enzyme.22
Figure 2. Some known FAAH inhibitors.
135 forms a reversible tetrahedral hemiketal intermediate
derived from the attack of Ser241 onto the ketone.27,28
URB59729 and the carbamates reported by Sanofi carbamylate
the active site Ser241 of the FAAH enzyme with the
concomitant loss of a phenolic or alcoholic fragment,
respectively.30 The ureas also form a carbamate with the
FAAH enzyme but with the loss of an aniline or heteroaryl
amine rather than an alcohol.31,32 OL-135,33 URB597,34 JNJ-
1661010,26 and PF-0445784535 have been found to be
efficacious in the treatment of pain in various animal models
without the motor impairment associated with direct CB1
agonism. Encouragingly, at least two compounds have been
evaluated in the clinic. Pfizer's PF-04457845 was evaluated as
an analgesic (phase II) and Sanofi-Aventis' SSR411298 for
major depression (phase II).36 While no data have been posted
for SSR411298, the study was completed in 2010. PF-
04457845, on the other hand, was found to be ineffective in
people for the reduction of OA pain in the knee.37
Table 1. SAR of the Pendant Benzyl Group
a
IC50 (nM)
entry
R
hFAAH
>10000
rFAAH
>10000
3
4
H
2-Cl
4670 1100
840 150
128 60
>10000
5600 860
2670 2000
>10000
5
3-Cl
6
4-Cl
7
3-F
2470 650
2770 780
6670 2300
4670 1800
350 90
8
4-F
>10000
9
3-CN
4-CN
3-CF3
4-CF3
3-OMe
4-OMe
3-OPh
4-OPh
10000
0
10
11
12
13
14
15
16
17
18
19
20
21
7670 1800
830 200
260 70
>10000
Our internal HTS efforts identified a series of potent FAAH
inhibitors from a purchased combinatorial library (Figure 3).
50 19
1530 220
490 200
74 14
>10000
6670 800
>10000
2830 1400
3-(4-chlorophenoxy)
4-(4-chlorophenoxy)
3-(4-methylphenoxy)
3-(4-methoxy-phenoxy)
3-(4-t-butyl-phenoxy)
6
4
300 90
4000 1000
640 120
133 11
75 16
62 28
11 2.4
12 0.7
46 12
Figure 3. From an impure HTS hit to benzyl piperazine ureas.
a
These are apparent IC50 values with values obtained after a 60 min
preincubation with the enzyme; n ≥ 3 standard error.
Resynthesis of an example showed that FAAH inhibition came
not from the presumed structure but from an unknown
impurity. An analysis of the likely synthesis and side products
suggested the possibility of a benzyl piperidine urea
contaminant. A combination of synthesis and cheminformatics
analyses of our corporate collection identified benzyl piperazine
urea (1). Compound (1) was a potent FAAH inhibitor, and its
modular structure suggested the possibility of rapidly
optimizing this chemotype to yield more potent analogues.
We elected to first optimize the pendent benzyl substituent,
keeping the phenyl urea constant. Treatment of 1-Boc
piperazine with phenyl isocyanate and then removing the Boc
group with 4.0 N HCl in dioxane gave (2), a common
intermediate that allowed for rapid diversification of the benzyl
group (Scheme 1).23 Treatment of (2) with a benzaldehyde,
diisopropylethylamine, and resin supported triacetoxyborohy-
dride [MP-BH(OAc)3] in THF gave the desired final products
From Table 1, it is apparent that substitution is required on
the benzyl group for there to be some affinity for the FAAH
enzyme. Generally, substituents in the 3- and 4-positions of the
benzyl group were better accommodated than those in the 2-
position. Polar substituents, such as nitriles, were less preferable
than lipophilic groups, which is consistent with the lipophilic
nature of the enzyme binding pocket. When the lipophilic
groups are small (Cl, CF3, and OMe), there is a larger boost in
potency when they are in the 4-position. However, as the
lipophilic groups get larger, it becomes clear that the enzyme
can better accommodate extra bulk in the 3-position of the
benzyl group, thus giving rise to potent FAAH inhibitors (17
and 19−21).
Recognizing that anilines have a propensity to be
genotoxic38−40 and that a covalent FAAH inhibitor would
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ACS Medicinal Chemistry Letters
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release an equivalent of aniline upon inactivation of the
enzyme, we began to examine heteroaromatic amines as
possible substitutes. As 17 was both one of the most potent
analogues made and had the least obvious metabolic liabilities,
we elected to keep the 3-(4-chlorophenoxy)benzyl substituent
constant while exploring aniline replacements. Heteroaryl ureas
were not synthesized from isocyanates but rather from their
corresponding phenyl carbamates, which can be prepared in a
single step (Scheme 2). Consistent with previous reports,32 the
3-pyridyl analogue (23, JNJ-40355003) was the most potent
inhibitor of both h- and rFAAH.
the extent of >20 mg/mL in deionized water. Concentrated
samples of JNJ-40355003 gave rise to highly acidic solutions,
which, if intended for iv delivery, were titrated to pH 4.5 with
aqueous NaOH.
Rats dosed orally with JNJ-40355003 showed a dose
proportional increase in compound concentrations both in
the plasma and in the brain with a brain to plasma ratio of 2.62
(Figure 3 in the Supporting Information).43 A time−course
study of FAAH inhibition and FAA elevation was conducted
(Figure 4 in the Supporting Information) using JNJ-40355003
at a dose of 3 mg/kg (po). Consistent with observations by
others,20,22 an almost complete blockade of the FAAH enzyme
is required to elevate AEA, with peak concentrations observed
approximately 4 h (Cp of JNJ-40355003 ≈ 800 nM) postdosing.
PEA and OEA concentrations peaked approximately 6 h (Cp of
JNJ-40355003 ≈ 600 nM) postdosing. PEA and OEA increased
more readily and to a much greater degree than AEA, which is
consistent with AEA being a better substrate for the enzyme
than the other two ethanolamides.44 By 12 h postdose (Cp of
JNJ-40355003 ≈ 200 nM), levels of the FAAs are decreasing
even though FAAH is still 90% inhibited. By 24 h (Cp of JNJ-
40355003 ≈ 15 nM), AEA, PEA, and OEA levels had returned
to their basal levels, and by 48 h, there was complete recovery
of enzymatic activity.
We then conducted a dose−response (0.1−60 mg/kg) study
of FAA elevation with JNJ-40355003. FAA levels were
measured at the 4 h time point postdose (Figure 5 in the
Supporting Information) in both the brain and the plasma.
Maximum FAA elevations in the brain were observed at the 10
mg/kg dose (approximate fold increase: AEA, 1.75; PEA, 3.7;
OEA, 3.25; FAA levels plateau at higher doses), whereas
maximal plasma concentrations were achieved at 3 mg/kg
(approximate fold increase: AEA, 3; PEA, 2.25; and OEA, 1.9).
The behavior of JNJ-40355003 in the dog was somewhat
surprising. The PK properties of JNJ-40355003 in beagle dogs
were reasonable (Cl = 2.3 L/h/kg, Vz = 1.4 L/kg, Vss = 1.24 L/
kg, and F = 39%), but the degree to which the individual FAAs
were elevated was quite unexpected (Figure 6 in the Supporting
Information). Whereas in rats FAAs typically achieve a 2−4-
fold increase over basal levels, the peak concentrations of all
three FAAs measured in dogs were >30-fold over basal levels.
AEA and OEA concentrations peaked 2−3 h postdosing,
whereas PEA levels peaked in the 6−8 h time frame. These
dramatic elevations of FAAs in dogs were not associated with
any adverse behavioral findings.
a
Scheme 2. Preparation of Heteroaryl Piperazinyl Ureas
a
Reagents and conditions (note: HetAr as defined in Table 2 as a
subset of Ar): (a) 0.9 mol equiv ClCO2Ph, 1.1 mol equiv pyridine,
CH3CN, 0 °C−rt, 2 h, 80−81%. b) 1/3 mol equiv ClCO2Ph, CH3CN,
rt, 31 h, 90%. (c) 1 mol equiv 1-Boc-piperazine or (40), 1−1.2 mol
equiv heteroaryl phenyl carbamate, 2 mol equiv Et3N, 1,2-dichloro-
ethane or DMSO, 18−24 h, rt, 34%-quant. (d) 2 M HCl, MeOH, 18 h,
rt, 75% or methylene chloride/trifluoroacetic acid, quant. (e) 1.2−2
mol equiv 3-(4-chlorophenoxy)benzaldehyde, 2−4 mol equiv MP-
BH(OAc)3, 6.0 mol equiv Et3N, THF, rt, 18−48 h, 36−82%.
Table 2. Heteroaryl Urea SAR
a
IC50 (nM)
entry
Ar
hFAAH
rFAAH
17
22
23
24
25
phenyl
6
4
300 90
520 80
b
4-pyridyl
3-pyridyl
2-pyridyl
4-pyrimidyl
15 6.1
1.4 0.41
12 2.5
41 17
33
9
470 180
1500 620
a
These are apparent IC50 values with values obtained after a 60 min
b
preincubation with the enzyme; n ≥ 3 standard error. N = 2.
Unlike humans and monkeys, neither the rat nor the dog
expresses functional FAAH-2 enzyme. The degree to which
enzymatically active FAAH-2 in primates would offset any
benefits of inhibition of FAAH is, perhaps, an open question.
PF-04457845 is reported to be completely selective for FAAH,
at least among serine hydrolases,24 but no specific mention of
selectivity over FAAH-2 was published. However, a closely
related molecule, PF-3845, has been found to have an IC50 >10
μM against this FAAH isoform.32,45 As JNJ-40355003 also lacks
FAAH-2 inhibitory activity (>10 μM), we were interested in
determining the effect that this compound would have on FAA
levels in a species expressing FAAH-2. To this end, we
measured FAA levels in cynomolgous monkeys dosed with JNJ-
40355003.
We modeled JNJ-40355003 in the crystal structure of
humanized rFAAH31,41 with the carbonyl bound covalently to
Ser241, forming a tetrahedral hemiketal-like intermediate
(Figure 1 in the Supporting Information). The benzyl
substituent is in an environment very similar to that of the
reported crystal structure, making a key face-to-edge π-
interaction with Phe192. Before collapse of the tetrahedral
intermediate and expulsion of 3-aminopyridine, there is a
possibility of hydrogen bond interactions between the urea N−
H and the carbonyl of Met191 and the side chain of Thr236
(alternatively, rotation of the aryl group could expose the
nitrogen to water molecules in this cavity).
JNJ-40355003 had good PK properties in the monkey (Cl =
1.52 L/h/kg, Vz = 2.18 L/kg, Vss = 1.89 L/kg, and F = 62%),
and more importantly, plasma levels for all three FAAs
examined were elevated (300−400% for AEA, 150−200%
We elected to profile JNJ-40355003 in greater detail. JNJ-
40355003 has excellent physical properties42 and is readily
formulated for both po and iv dosing. JNJ-40355003 has two
basic nitrogens, and the bis-HCl salt 1.5 hydrate is soluble to
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OEA, and 150−175% PEA; Figure 7 in the Supporting
Information). The degree of AEA elevation was greater than
that observed in the rat but less than in the dog. The peak levels
of all three FAAs were reached 4 h after iv dosing. As PF-
04457845 raised plasma levels of the FAAs in human subjects,
it may be reasonable to postulate that dual inhibition of FAAH
and FAAH-2 is not necessary to achieve elevated levels of
FAAH's primary substrates in primates. Because it is not known
what FAA levels could be achieved if both FAAH and FAAH-2
were inhibited,10,46 we cannot rigorously conclude that
inhibition of FAAH-2 would have no impact on the efficacy
of a FAAH inhibitor. However, it is reassuring that FAA levels
can be influenced by the selective inhibition of FAAH.
In conclusion, we have described a series of substituted
phenyl and heteroaryl urea FAAH inhibitors. The highly potent
FAAH inhibitor JNJ-40355003 was found to have good physical
and PK properties and elevated the plasma and brain
concentrations of AEA, PEA, and OEA in rats after oral
dosing. Similarly, plasma levels of these three lipids were found
to be elevated in dogs and monkeys orally dosed with JNJ-
40355003. The elevation of FAAs in the monkey strongly
suggests that FAAH-2 is not contributing greatly to the
breakdown of fatty acid ethanolamides in the plasma; thus, its
inhibition is unlikely to be required for there to be a
pharmacological benefit from blockade of the FAAH enzyme
in humans. Additional studies involving urea inhibitors of
FAAH will be reported in due course.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures, annotated 1H NMR, and MS
data for the described compounds as well as graphical
representations of in vivo data for JNJ-40355003 can be
found in the supporting information. This material is available
free of charge via the Internet at http://pubs.acs.org.
(17) Wei, B. Q.; Mikkelsen, T. S.; McKinney, M. K.; Lander, E. S.;
Cravatt, B. F. A second fatty acid amide hydrolase with variable
distribution among placental mammals. J. Biol. Chem. 2006, 281,
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Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.;
Guimaraes, C. R.; Jorgensen, W. L.; Cravatt, B. F. Discovery of a
potent, selective, and efficacious class of reversible α-ketoheterocycle
inhibitors of fatty acid amide hydrolase effective as analgesics. J. Med.
Chem. 2005, 48, 1849−1856.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 858 784-3275. E-mail: jkeith@its.jnj.com.
Notes
The authors declare no competing financial interest.
(19) Romero, F. A.; Du, W.; Hwang, I.; Rayl, T. J.; Kimball, F. S.;
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(41) PDB ID: 2wap.
(42) Physical properties: pKa1 (piperazine) = 6.08; pKa2 (pyridine) =
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(resultant pH = 1.5); mp of bis-HCl salt 1.5 hydrate = 219 °C (onset);
suitable formulations 30% SBE β-CDEX (83 mg/mL, resultant pH =
1.94), 20% SBE β-CDEX (62.6 mg/mL, resultant pH = 1.67.)
(43) Other rat PK parameters: Cl = 0.69 L/h/kg, Vz = 8.3 L/kg, Vss
= 3.2 L/kg, and F = 53−69%.
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(31) PF-750: Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.;
Liimatta, M.; Pabst, B.; Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.;
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(32) PF-3845: Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long,
J. Z.; McKinney, M. K.; Weerapana, E.; Sadagopan, N.; Liimatta, M.;
Smith, S. E.; Lazerwith, S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.;
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(35) Unfortunately, many compounds that inhibit FAAH-2 are not
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(36) Data from clinicaltrials.gov as of 6/21/11.
(37) Huggins, J. P.; Smart, T. S.; Langman, S.; Taylor, L.; Young, T.
An efficient randomised, placebo-controlled clinical trial with the
irreversible fatty acid amide hydrolase-1 inhibitor PF-04457845, which
modulates endocannabinoids but fails to induce effective analgesia in
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