Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure-mutagenicity relationships for arylamine metabolites

Article abstract of DOI:10.1016/j.bmcl.2012.10.076

The structure-activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure-mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.

Full text of DOI:10.1016/j.bmcl.2012.10.076

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7357–7362
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Heteroaryl urea inhibitors of fatty acid amide hydrolase:
Structure–mutagenicity relationships for arylamine metabolites
Mark S. Tichenor a, , John M. Keith a, William M. Jones a, Joan M. Pierce a, Jeff Merit a, Natalie Hawryluk a,
⇑
Mark Seierstad a, James A. Palmer a, Michael Webb a, Mark J. Karbarz a, Sandy J. Wilson a,
Michelle L. Wennerholm a, Filip Woestenborghs b, Dominiek Beerens b, Lin Luo a, Sean M. Brown a,
Marlies De Boeck b, Sandra R. Chaplan a, J. Guy Breitenbucher a
a Janssen Research and Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States
b Janssen Research and Development, A Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase
(FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent
modification of an active site serine with subsequent release of an aromatic amine from the urea electro-
phile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure–
mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were
identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 29 August 2012
Revised 8 October 2012
Accepted 15 October 2012
Available online 22 October 2012
Keywords:
Fatty acid amide hydrolase
FAAH
Urea
Heteroaryl amine
Mutagenic
Ames II
O
NH2
Fatty acid amide hydrolase (FAAH) is responsible for degrading
a family of fatty acid amide signaling molecules including the
endogenous cannabinoid receptor (CB1/CB2) agonist anandamide.1
Modulating the cannabinoid system by FAAH inhibition is an
attractive approach for treating a variety of conditions associated
with cannabinoid receptor function including pain, anxiety, and
depression. Elevating levels of anandamide by inhibiting its
degradation is anticipated to have an improved side effect profile
relative to exogenous cannabinoid receptor agonists such as
O
N
N
Me
H
N
H
N
O
Me
N
O
N
H
O
O
1, URB-597
2, SA-47
O
N
O
N
H
N
CF3
N
H
N
N
N
N
N
O
D
9-tetrahydrocannabinol (THC) that cause cognitive and motor
S
3, Takeda-25/JNJ-1661010
4, PF-04457845
impairment. Genetic knockout mice lacking FAAH have
anandamide levels that are elevated 15-fold relative to wild-type
animals and exhibit an analgesic phenotype but are otherwise
healthy,2 providing a rationale for the therapeutic value of FAAH
inhibition.3
The majority of FAAH modulators that have been reported in the
literature are mechanism-based inhibitors that inactivate the en-
zyme by forming a covalent bond with active site serine-241
(Fig. 1),4–7 although an increasing number of recent publications
have described compounds that inhibit FAAH by noncovalent
mechanisms.8–11 Covalent modification12 is an effective approach
O
N
N
O
N
O
Cl
N
H
N
N
O
5, JNJ-40355003
6, OL-135
Figure 1. Representative FAAH inhibitors.
for modulating FAAH activity because nearly complete enzyme
inhibition is required before anandamide levels are significantly
elevated.13,14 Phenyl carbamate inhibitors exemplified by URB-597
(1) were reported to be effective in animal models of analgesia and
anxiety.15,16 However, the clinical utility of URB-597 may be limited
⇑
Corresponding author. Tel.: +1 858 320 3512; fax: +1 858 450 2089.
E-mail address: mticheno@its.jnj.com (M.S. Tichenor).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.076

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M. S. Tichenor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7357–7362
by the high reactivity of the phenyl carbamate electrophile and the
corresponding diminished selectivity with respect to other serine
hydrolase enzymes.14 Sanofi-Aventis introduced alkyl carbamate
FAAH inhibitors (2)17 having increased chemical stability and an
improved selectivity profile relative to 1 using activity based protein
profiling.18,19 Aryl ureas such as 3 have been extensively explored in
the medicinal chemistry efforts of Takeda,20 Johnson & Johnson,21–23
Pfizer,24 and Astellas.25 Heteroaryl ureas have subsequently been
Aromatic amines are typically not harmful themselves, but they
can be susceptible to oxidation by cytochrome P450 enzymes to
reactive metabolites. In the Ames II assay, the test compounds
are preincubated with a rat liver extract (S9) to simulate metabolic
activation by liver enzymes. Several characterized pathways for
the oxidative metabolism of anilines to generate reactive species
have been described (Fig. 2).49 Carbon oxidation of the electron-
rich aromatic ring can yield electrophilic epoxides or quinones that
are capable of forming conjugates with biological macromolecules.
Alternatively, N-oxidation produces N-hydroxylamines50 that have
the potential to be reactive themselves or can act as intermediates
in the formation of other reactive species including O-acylated
hydroxylamines51 or nitrosobenzenes.41 Aryl nitrosyl functionality
plays a direct role in arylamine-induced methemoglobinemia by
mediating the oxidation of hemoglobin, and also can cause carcin-
ogenicity through covalent modification of DNA.
Quantitative structure–activity relationships (QSAR) of aro-
matic amines have been used to design computational models that
predict mutagenicity and mutagenic potency.49 An important
molecular feature that contributes to the occurrence of mutagenic-
ity in arylamines is the energy of the highest occupied molecular
orbital (HOMO),52 an indication of a molecule’s propensity for N-
oxidation. Steric hindrance surrounding the amino group can also
block the formation of reactive metabolites by reducing access of
the amine to metabolizing enzymes.53 Mutagenic potency, mea-
sured by the frequency of revertant colonies in the Ames assay,
is correlated with lipophilicity of the aniline and the number of
fused rings, which can be rationalized as an increased tendency
for planar, hydrophobic molecules to bind and intercalate DNA.
Herein, we report the synthesis and structure–activity relation-
ships for urea FAAH inhibitors having modifications to the hetero-
aryl amine fragment, and Ames II data for the corresponding
arylamine leaving groups.
disclosed by Pfizer14,26–28 (4) and Johnson & Johnson (5).29–32
A
structurally and mechanistically distinct series of ketooxazole FAAH
inhibitors exemplified by OL-135 (6) was discovered and exten-
sively characterized by Boger and co-workers at The Scripps
Research Institute.33–36 The structure of OL-135 contains an
electrophilic ketone that inhibits FAAH by trapping the active site
serine-241 in a reversible hemiketal.37–39
Our efforts to improve the pharmacological properties of aryl
urea FAAH inhibitors such as 3 have focused on identifying hetero-
aryl amine replacements for the aniline fragment to reduce the po-
tential risk associated with expelling an equivalent of aniline upon
reacting with the enzyme. Acute exposure to aniline causes methe-
moglobinemia in humans via oxidation of hemoglobin (Fe2+) to
methemoglobin (Fe3+), which does not effectively deliver oxygen
to tissues.40,41 There are many examples of arylamines that are
known to be carcinogenic,42 although reports on the carcinogenic-
ity of aniline itself are conflicting.43 Aniline replacements having a
reduced toxicity risk are required for urea FAAH inhibitors with
improved safety profiles relative to phenyl ureas.
It is desirable to identify risks such as carcinogenicity early in a
drug discovery effort,44 however the low throughput and high cost
of animal models of carcinogenicity limit their utility for triaging
compounds in the lead optimization stage. Genetic toxicology tests
are a more rapid alternative that are routinely used for predicting
the carcinogenicity of preclinical drug candidates. In general, a bat-
tery of tests is used to assess the different types of genotoxic effects
that might be associated with human disease. The widely used bac-
terial reverse mutation assay, or Ames test, detects the tendency of
a molecule or its metabolites to inflict genetic damage by measur-
ing the frequency of reverse mutations in set of Salmonella
typhimurium strains.45 Although the predictive ability of such
in vitro experiments has limitations, including an inability to de-
tect non-mutagenic mechanisms of carcinogenicity,43,46 the Ames
test is a useful selection tool for eliminating high risk molecules
before investing in more resource-intensive in vivo safety models.
The Ames II assay47,48 is a liquid fluctuation modification in micro-
plate format of the traditional Ames assay with reduced compound
requirements and higher throughput. TAMix, a mixture of TA7001–
TA7006 strains, is used for the detection of base pair substitutions
and TA98 to detect frameshift mutations.
The syntheses of non-commercially available heteroaryl amines
10–12 were completed according to Scheme 1.30 Isoxazolo[5,4-
c]pyridin-3-ylamine 10 was prepared by treating 3-chloroisonico-
tinonitrile 7 with an excess of acetohydroxamic acid under basic
conditions. SNAr addition of the hydroxamate followed by
N
Cl
Y
O
CN
NH2
a
X
Z
X
Z
Y
7, X = N, Y = Z = CH
10, X = N, Y = Z = CH
8
11
, X = Z = CH, Y = N
, X = Z = CH, Y = N
9, X = Y = CH, Z = N
12, X = Y = CH, Z = N
N
N
b
N
H2N
NH2
13
NH2
NH2
NH2
NH
N
N
N
N
b
b
ring
HO
O
O
oxidation
N
H2N
NH2
NH2 14
+
O
NH2
N
NH2
N
N
N
N
N
NH2
O
H2N
N
N
N
NH2
N
O
N
16
15
N
OH
NH
N
O
N-oxidation
17
O
R
NH
Scheme 1. Synthesis of heteroaryl amines. Reagents and conditions: (a) acetohy-
droxamic acid, K2CO3, DMF, rt to 50 °C, 20 h, 10, 41%; 11, 77%; 12, 25%; (b)
chloroacetaldehyde, H2O, NaHCO3, rt, 20 h, 13, 50%; 14, 37%; 15, 25%; 16, 30%; 17,
4%.
Figure 2. Oxidation of aniline to reactive intermediates.

M. S. Tichenor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7357–7362
7359
cyclization onto the nitrile provided the corresponding desired
product in a one-pot procedure.54,55 An analogous method was
used to prepare the isomeric heterocycles 11 and 12.
Imidazo[1,2-a]pyridin-8-ylamine (13) was synthesized by treating
2,3-diaminopyridine with chloroacetaldehyde, giving the desired
product after alkylation and cyclization to the fused imidazole.56
Pyrimidine-4,6-diamine was converted to 14 using an analogous
approach. Pyrimidine-2,4-diamine was capable of cyclizing as
distinct isomers 15 and 16 which were both isolated in comparable
yields, along with a byproduct 17 from reaction with another
equivalent of chloroacetaldehyde.
A diverse set of heteroaryl amines reacted readily with phenyl
chloroformate in the presence of pyridine to give the correspond-
ing phenyl carbamates as shown for representative examples
18a–h (Scheme 2). Some highly electron-deficient heteroaryl
amines such as benzo[d]isoxazol-3-ylamine (18l) returned only
starting material under these reaction conditions. The desired
carbamates 18i–p were prepared at elevated temperature using
phenyl chloroformate as the limiting reagent, without additional
base.
The urea FAAH inhibitors were prepared using a modular
synthesis that was readily amenable to diversification of urea
substituents (Scheme 3). The benzyl group was appended to
N-Boc piperazine by reductive amination, followed by deprotection
under acidic conditions to give intermediate 19.30 The 3-(4-chloro-
phenoxy)-benzyl piperazine was elaborated to ureas having
diverse N-heteroaryl substituents by reaction with phenyl carba-
mates. Selected ureas were assembled in a one-pot procedure by
acylating the amine with disuccinimidyl carbonate, then treating
with 19 to provide the final ureas without isolation of the interme-
diate carbamate.
The structure–activity relationships for the heteroaryl amine
fragment were defined by evaluating diverse heterocyclic substit-
uents while maintaining the optimized 3-(4-chlorophenoxy)-
benzyl-piperazine portion of the molecule constant (Table 1). All
of the compounds prepared in this series were expected to be
time-dependent inhibitors of FAAH based on their structural
similarities to characterized covalent inhibitors 3 and 4.
six-membered heteroaryl ureas were prepared, displaying low
nanomolar human FAAH inhibitory potencies that were similar
to aniline 20 under the assay conditions. 3-Aminopyridine (5)32
was the optimal six-membered heteroaryl amine in this series.
Compounds having pyridine, pyridazine, and pyrazine substituents
collectively demonstrated that nitrogen atoms were tolerated in all
ring positions. A diverse set of five-membered amino-heterocycles
(23–26) were somewhat less effective urea substituents. With the
exception of the tetrazole analog 24, the compounds are generally
several-fold more potent at human FAAH than rat FAAH.
The free arylamines in Table 1 were evaluated in the Ames II as-
say with and without (S9) metabolic activation to assess the risk of
mutagenicity for the fragment that is released after reaction with
FAAH. All unsubstituted five- and six-membered heteroaryl amines
shown in Table 1 were Ames II negative, consistent with QSAR pre-
dictions that small, electron-deficient and relatively polar hetero-
aryl amines are less susceptible to metabolic activation leading
to DNA damage. However, the 4-chloro-3-amino-oxazole found
in 26 was unexpectedly Ames II positive, despite having an elec-
tron-deficient amine substituent. Heteroaryl amines usually re-
quire metabolic activation to cause mutagenicity in the Ames II
test, but 4-chloro-3-amino-oxazole was positive with and without
metabolic activation. The molecular characteristics that determine
mutagenicity are complex, and exhaustive Ames II testing is re-
quired to detect outliers such as 4-chloro-3-amino-oxazole.
Fused bicyclic heteroaryl ureas were prepared to extend the
structure–activity relationship data to include larger amine sub-
stituents. Several bicyclic heteroaryl ureas were potent human
and rat FAAH inhibitors, including compounds having diverse spa-
cial arrangements and functionality (Table 2). Initially, the com-
pounds in Table 2 appeared to be a promising new direction for
optimization, however the bicyclic amines were much more likely
to be mutagenic than the monocyclic amines in Table 1, with six
out of the seven heteroaryl amines testing positive in the Ames II
assay. All six Ames II positive compounds in Table 2 were muta-
genic only when treated with S9 to simulate metabolic activation.
The impact of adding a fused aryl ring is apparent in the compari-
son between 3-aminopyridine (Ames II negative) in 5 versus the
more lipophilic bicyclic analog isoquinolin-4-amine (Ames II
The inhibitory potencies in Table 1 are reported as apparent IC50
values and were run under identical incubation times.57 A series of
Cl
2•HCl
O
a, b
HN
O
OPh
a or b
NBoc
HetAr NH
HetAr NH2
HN
O
18a-p
19
N
Method a: HetAr (representative examples)b
Cl
Cl
N
N
N
N
N
O
O
c
19
19
N
H
N
N
N
NH
N
N
18a
18b
18f
18c
18g
18d
18h
H
N
N
20
H
N
N
N
O
N
N
O
d, e, or f
18e
HetAr
N
H
Method b: HetAr
21−68
N
N
N
N
N
N
O
HN
HN
N
O
O
Method e: HetAr-NH2
N
Method f: HetAr-NH2
N
18i
18j
18k
18o
18l
N
N
N
N
N
N
O
O
O
N
N
Scheme 3. Preparation of heteroaryl piperazinyl ureas. Reagents and conditions:
(a) 1.5 equiv 3-(4-chlorophenoxy)benzaldehyde, 3 equiv NaB(OAc)3H, DCM, rt, 23 h
70%; (b) 4 N HCl in dioxane, DCM, rt, 23 h, 50%; c) 1.1 equiv PhNCO, 4 equiv DIPEA,
DCM, rt, 23 h, 77%; (d) 18, DIPEA, CH3CN, rt, 24 h, 50–95%; (e) HetAr-NH2,
disuccinimidyl carbonate, rt, 16 h then 19, DIPEA, 63%; (f) HetAr-NH2, disuccinim-
idyl carbonate, DMAP, rt, 16 h then 19, DIPEA, 9–53%.
N
N
18m
18n
18p
N
Scheme 2. Preparation of heteroaryl carbamates. Reagents and conditions: (a)
PhOCOCl, pyridine, CH3CN, rt, 2 h, 39–100%; (b) 0.33 equiv PhOCOCl, CH3CN, 70 °C,
20–33%.

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M. S. Tichenor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7357–7362
Table 1
Monocyclic heteroaryl ureas
O
Cl
R
N
N
O
a
a
R
Apparent hFAAH IC50 (nM)
Apparent rFAAH IC50 (nM)
HetAR-NH2 Ames II result
Neg
20
6
4
300 86
150 64
N
H
N
21
8.7 6.3
7.7 2.3
1.4 0.4
Neg
Neg
Neg
N
N
H
N
N
22
93 22
N
H
N
5
33
9
N
H
HN
N
N
23
24
25
32
5
150 80
46 9.6
Neg
Neg
Neg
N
H
HN
N
103 32
N
N
H
O
N
28
7
720 330
N
H
O
N
26
5.2 1.1
20 4.4
Pos
N
H
Cl
a
Values reported as the mean SEM of at least three independent determinations in duplicate. IC50 values were measured with a 60 min preincubation.
Table 2
Fused bicyclic heteroaryl amines
O
Cl
R
N
N
O
a
a
R
Apparent hFAAH IC50 (nM)
28 13
Apparent rFAAH IC50 (nM)
HetAR-NH2 Ames II result
Pos
27
120 50
N
H
N
O
N
N
28
5.8 1.9
25
6
Neg
N
H
N
S
29
30
1300 680
7.7 4.2
6000 2500
200 140
Pos
Pos
N
H
NH
N
O
Me
N
N
H
N
31
6.7 2.3
8.3 4.1
Pos
N
H
H
N
N
N
32
33
3.4 1.7
69 49
21
7
Pos
Pos
N
H
1100 240
N
H
N
H
a
Values reported as the mean SEM of at least three independent determinations in duplicate. IC50 values were measured with a 60 min preincubation.

M. S. Tichenor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7357–7362
7361
Table 3
Fused bicyclic heteroaryl ureas
O
Cl
R
N
N
O
a
a
R
Apparent hFAAH IC50 (nM)
Apparent rFAAH IC50 (nM)
HetAR-NH2 Ames II result
O
O
N
N
34
35
36
10
2
8.1 2.5
14 6.7
0.7 0.5
Neg
Neg
Neg
N
H
N
N
22 15
N
H
O
N
0.5 0.3
N
H
O
O
N
N
37
38
180 80
2200 700
Neg
Neg
N
H
N
13
3
22
6
N
H
N
N
39
40
46 16
>10,000
120 53
>10,000
Neg
Neg
N
N
H
N
NH
N
N
41
310 70
3500 1900
Neg
N
H
N
N
42
43
44
33 16
2600 1600
3600 2200
5.7 1.1
Pos
Neg
Neg
N
N
H
N
N
100 60
2.7 0.4
3.7 2.6
N
H
N
N
N
H
N
N
45
46
47
48
53 41
Neg
Neg
Neg
Neg
N
HN
N
N
17
2
470 100
24 11
N
N
H
N
3.0 1.2
140 70
N
N
H
N
N
2400 158
N
N
N
H
a
Values reported as the mean SEM of at least three independent determinations in duplicate. IC50 values were measured with a 60 min preincubation.
positive) in 31. This observation is consistent with previous studies
correlating increased lipophilicity with mutagenicity. Despite
having encouraging FAAH potency, the high frequency of positive
results in the Ames II assay for the bicyclic amines in Table 2
precluded these analogs from further profiling.
An additional series of bicyclic heteroaryl amines was designed
to address the high frequency of mutagenicity identified in Table 2,
based on the hypothesis that compounds having strongly electron-
deficient heteroaryl groups would be less likely to participate in
the metabolic activation that often characterizes aniline toxicity.
Benzo[d]isoxazol-3-ylamine urea 34 (Table 3) was a potent FAAH
inhibitor having an Ames II negative heteroaryl amine that served
as a starting point for further optimization. Pyridyl analogs of 34
were prepared to reduce the lipophilicity associated with the aro-
matic ring. The isoxazolo[5,4-c]pyridin-3-ylamine urea 36 was a
subnanomolar inhibitor of both human and rat FAAH, and isomers
35 and 38 were equipotent with the 34, although the location of
the nitrogen atom in isomer 37 was not well tolerated. None of
the five heteroaryl amines related to benzo[d]isoxazol-3-ylamine
scaffold were mutagenic in the Ames II assay, demonstrating that
bicyclic heteroaryl amines can be useful urea substituents
provided that they are strongly electron-deficient.
Having shown that heteroatoms are well tolerated in the urea
substituent, imidazo[1,2-a]pyridin-3-ylamine 39 was prepared as

7362
M. S. Tichenor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7357–7362
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an isosteric analog of 34 having a more polar, basic imidazole in
place of the oxazole ring. The orientation of the imidazo[1,2-a]
pyridine ring system strongly influenced the FAAH IC50. The
highest potency was generally achieved when the amino group
was located adjacent to a ring fusion (39, 44) versus in a linear
arrangement (40). Analogs having the imidazole fused to pyrimi-
dine and pyridazine rings were prepared to modify the electronic
properties of the arylamine and the fused imidazole. The urea
derived from imidazo[1,2-b]pyridazin-3-ylamine 45 was 10-fold
more potent against human FAAH than the pyridine analog 39.
Similarly, imidazopyrimidines 46–48 maintained or improved
FAAH activity relative to the corresponding imidazopyridines.
One of the five compounds having the fused imidazole ring system
(42) was positive in the Ames II assay, although two closely related
analogs 46 and 48 were not mutagenic. The more polar and elec-
tron-deficient fused pyrimidine ring may contribute to reduced
metabolic activation of 46 and 48 relative to 42.
The structure–activity relationships for a series of mechanism
based inhibitors of fatty acid amide hydrolase were explored,
focusing on modifications to the heteroaryl urea that forms a
covalent bond with FAAH. The covalent mechanism of enzyme
inhibition required evaluating each released heteroaryl amine
metabolite separately from the parent urea for its mutagenic
potential using the Ames II assay. A variety of unsubstituted five-
and six-membered heteroaryl ureas were potent inhibitors of
FAAH, and the corresponding heteroaryl amines were Ames II nega-
tive in all cases. Bicyclic heteroaryl amines were much more likely to
be mutagenic, but several analogs related to the benzo[d]isoxazol-
3-ylamine 34 and imidazo[1,2-a]pyridin-3-ylamine 39 were
potent FAAH inhibitors having Ames II negative metabolites.
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