Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.052

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.

Full text of DOI:10.1016/j.bmcl.2011.02.052

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2492–2496
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent, noncovalent fatty acid amide hydrolase (FAAH)
inhibitors
Darin J. Gustin a, , Zhihua Ma a, Xiaoshan Min b, Yihong Li a, Christine Hedberg a, Cris Guimaraes b,
⇑
Amy C. Porter c, Michelle Lindstrom c, Dianna Lester-Zeiner c, Guifen Xu d, Timothy J. Carlson d,
Shouhua Xiao e, Cesar Meleza e, Richard Connors a, Zhulun Wang b, Frank Kayser a
a Departments of Chemistry, Amgen Inc., South San Francisco, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Department of Structural Biology, Amgen Inc., South San Francisco, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
c Department of Neuroscience, Amgen Inc., South San Francisco, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
d Department of PKDM, Amgen Inc., South San Francisco, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
e Department of Lead Discovery, Amgen Inc., South San Francisco, 1120 Veterans Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several
spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel,
noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors.
The mechanism of action for these compounds was determined through a combination of SAR and
co-crystallography with rat FAAH.
Received 17 November 2010
Revised 10 February 2011
Accepted 14 February 2011
Available online 17 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
FAAH
FAAH inhibitor
Noncovalent
Structure based design
Fatty acid amide hydrolase is an integral membrane enzyme
that degrades the fatty acid amide family of signaling lipids,
including anandamide (N-arachidonylethanolamine, AEA). AEA
was identified as an endogenous agonist for the cannabinoid recep-
tors CB1 and CB2 in 1992.1 The anti-nociceptive benefit of stimu-
lating the CB1/CB2 receptors has long been recognized and for
some time it has been postulated that inhibiting FAAH would show
similar therapeutic benefits through increasing lifetime of AEA in
various tissues.2,3 Evidence supporting this hypothesis includes
the fact that FAAH knock-out mice show increased levels of AEA
in the brain accompanied with decreased susceptibility to neuro-
pathic and inflammatory pain.4 Likewise, inhibition of FAAH with
small molecule inhibitors (e.g., OL-1355 and URB-5976) in vivo
leads to similar increases in AEA levels and decreased sensitivities
in pain models.7 Except for the recent report by Wang,8 most FAAH
inhibitors reported to date appear to rely on covalent modification
of the active site serine 241. Herein we describe the identification
of a novel, potent and noncovalent series of FAAH inhibitors with
in vitro potencies comparable to covalent FAAH inhibitors.
As indicated in Table 1, 1a and 2 inhibited rat and human FAAH
with potencies comparable to that of OL-135 and URB-597 in our
assays.9 Subsequent studies revealed these ureas to be suicide sub-
strates.10,11 Incubation of 1b or 2 with FAAH followed by crystalli-
zation resulted in complexes where the urea carbonyl acylated
S241 with expulsion of the aniline fragment as indicated equation
1.12 The complete loss of FAAH inhibitory activity seen with amide
1d is consistent with mechanism-based inactivation suggested by
co-crystallization studies. Interestingly, simple N-methylation of
1b also resulted in a complete loss of FAAH activity (cf. 1b, 1c
and 1d, Table 1).
We were interested in developing reversible (and preferably
noncovalent) FAAH inhibitors with the expectation that they might
have a different selectivity13/safety14 profile compared to suicide
substrates like 1 or 2. We imagined that it might be possible to cre-
ate reversible FAAH inhibitors based on spirolactams or cyclic ur-
eas related to 1. Starting from a urea exemplified by 3 (Fig. 1),
we reasoned that by installing steric hindrance adjacent to the car-
bonyl group would reduce its susceptibility to nucleophilic attack.
In addition, tethering the aniline to the opposite side of the scissile
bond would prevent it from leaving the active site after initial at-
tack by S241 thus creating the possibility of reversible inhibition
through re-cyclization of the postulated enzyme-acyl intermedi-
ate. Our strategy (summarized in Fig. 1 for a spirolactam) called
Compounds 1a and 2 were identified during the course of a
high-throughput screen (HTS) of the Amgen compound collection.
⇑
Corresponding author.
E-mail address: dgustin@amgen.com (D.J. Gustin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.052

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2492–2496
2493
Table 1
results we used 4-[2-benzofuran]2-yl-pyrimidine as a key frag-
ment to evaluate additional serine-electrophile designs, that were
selected from our molecular modeling efforts.
FAAH activity of high-throughput screening hits, reference compounds and selected
SAR data
Table 2 summarizes FAAH inhibition data for a series of
4-[2-benzofuran]2-yl-pyrimidines substituted with a pyrrolidine,
piperidine or homopiperidine bearing a methyl-ketobenzimidazole
unit. As indicated, the 3-(keto-benzimidazole-3-yl)piperidine 7
displayed potent inhibition of hFAAH and moderate inhibition of
the rat enzyme. However, the pyrrolidine 6, the 4-(ketobenz-
imidazole-3-yl) piperidine 12 and the homopiperidine 13 were sig-
nificantly less potent. The potency of 7 could be improved upon by
replacing the N ꢀ 1 methyl group with an ethyl group (cf. 7 and 8).
Further improvement in potency is seen through replacement of
the ketobenzimidazole with the nitrogen bearing imidazo[4,5-
b]pyridin-2(3H)-one in 9. Examination of each enantiomer of 9 re-
vealed that only the (S) isomer 11 inhibited human and rat FAAH.
With 11 in hand we set out to re-examine the SAR of the aliphatic
binding pocket by further modifying the piperidine N-substituent.
Representative syntheses of our FAAH inhibitors are shown in
Scheme 1.17 2-Chloro-4-[2-benzofuran]2-yl-pyrimidine 14 is
available from the cross-coupling of 2,4-dichloropyrimidine and
benzofuran-2-yl-boronic acid.18 The (S)-1-(piperidin-3-yl)-1H-
benzo[d]imidazol-2(3H)-one subunit was prepared by the SNAr
reaction of 2-chloronitrobenzene and commercially available
(S)-3-amino-1-Boc-piperidine which gave 15.19 Reduction of 15
followed by treatment with 1,1-carbonyldiimidazole (CDI) and
alkylation of the ketobenzimidazole nitrogen gave 16. Removal of
the Boc protecting group provided 17.20 Finally, coupling of 17
and 14 in the presence of Hünig’s base gave the S-isomer of 8.
Alternatively, SNAr reaction of 17 with dichloro-heterocycles gave
18.21 Cross-coupling of 18 with aryl-boronic acids15 afforded
19–22 and allowed us to rapidly explore the SAR of the aryl-pyrim-
idine fragment.
H2N
O
N
O
H
N
N
R
O
O
O
OL-135
URB-597
O
O
X
N
N
N
N
O
O
H
F
N
O
S
Br
1a-d
N
2
O
N
eq.1
N
H
S241
O
N
R
FAAH
Cmpd
R
X
Rat FAAH IC50
,
l
Ma
Human FAAH IC50, l
Ma
OL-135
URB-597
—
—
—
0.025
0.003
0.010
0.003
>10
0.016
0.004
—
0.001
—
—
H
F
1a
1b
1c
1d
2
NH
NH
N-Me
CH2
—
F
F
>10
>10
—
0.036
0.005
a
Values are means of at least two experiments. All SAR data reported herein was
obtained using the assays described in Ref. 9.
Additional SAR and whole cell assay data for this series of FAAH
inhibitors is summarized in Table 3. Moving the benzofuran sub-
unit from the 4-position to the 5-position on the pyrimidine ring
(19) resulted in a complete loss of potency against rat and human
FAAH. In contrast, both alternate pyrimidine isomers (20 and 21)
were well tolerated as long as a 1,3-relationship between the
piperidine and benzofuran units was maintained.
Finally, we examined the SAR of the aryl ring attached to the
central pyrimidine (compounds 23–35, Table 3). Replacement of
the benzofuran subunit with a simple phenyl ring (cf. compounds
9 and 23) resulted in a significant loss of potency against both the
rat and human enzymes. In general, substituents in either the ortho
or meta position were unable to restore inhibitory activity, partic-
ularly on the rat enzyme. On the other hand, small para substitu-
ents were generally well tolerated with fluorine being preferred
(cf. compounds 20 to 24–28 and 23–30). Finally, changing the ben-
zofuran subunit to a 5-chloro-2-thiophene resulted in a significant
improvement in potency when combined with aza-ketobenzimi-
dazoles (cf. 11, Table 2 to 32 and 33, Table 3). Gratifyingly,
compounds 32 and 33 showed inhibition of rat and human FAAH
in both biochemical and whole cell assays with potencies compa-
rable to that observed with benchmark FAAH inhibitors.
While our compounds were originally envisioned as electro-
philes for S241 of FAAH, we were skeptical that the ketobenzimi-
dazole identified in these studies was sufficiently reactive to be
attacked by S241. Thus, we set out to determine the mechanism
of inhibition for these FAAH inhibitors in greater detail. First, we
examined the reversibility of FAAH inhibition through evaluation
of progress curves.22 In the experiment, rFAAH and a test com-
pound were incubated at a concentration 100 fold greater than
the usual assay conditions. After 1 h, the mixtures were diluted
by a factor of 100 with buffer containing anandamidoaminometh-
ylcumarin (AAMCA). The FAAH dependant hydrolysis of AAMCA
O
N
H
3
N
N
R
electrophile
design
N
O
R
O
O
N
N
N
R
HN
N
library
production
4
5a, R = H
5b, R = Me
Figure 1. FAAH inhibitors design and strategy.
for designing a serine electrophile15 that carried a substituent
to target the acyl-chain binding pocket (ABP)11 of FAAH (i.e.,
R in 4). Once suitable scaffolds were identified we intended to opti-
mize inhibitor affinity for the ABP and eventually remove the
electrophile.
Toward this end, we designed a number of bicyclic and
spirocyclic ureas and lactams as potential FAAH inhibitor electro-
philes. The designs were prioritized through their theoretical
binding to the active site of FAAH using the program Glide16 and
in-house X-ray crystallographic data.11 Selected designs were syn-
thesized and used to produce libraries. Early on in this effort, we
identified 4-[2-benzofuran]-2-yl-pyrimidine as a highly potent
piperidine nitrogen substituent. For example, spirolactam 5a
showed inhibition of rFAAH with an IC50 of 1.2
the N-methyl spirolactam 5b showed slightly improved FAAH inhi-
bition (IC50 = 0.50 M) in contrast to the complete loss of activity
seen upon N-methylating 1b, which suggested two distinct binding
modes for these two series of compounds. Encouraged by initial
lM. Interestingly,
l

2494
D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2492–2496
Table 2
was then monitored by UV. Under these conditions compounds 7,
33, and 35 were shown to be reversible inhibitors of FAAH while
URB-597 was shown to inhibit the enzyme irreversibly, as
expected.23
Rat and human FAAH SAR data for a series of 2-substituted 4-[2-benzofuran]2-yl-
pyrimidines (6–13)
N
O
Next, we were interested in replacing the ketobenzimidazole
fragment of our inhibitors with a noncarbonyl bearing heterocycle.
We reasoned that if we could replace the carbonyl while maintain-
ing comparable FAAH inhibition, it would provide evidence for a
noncovalent mechanism of action. Thus, we evaluated a number
of simple 5,6-heteroycles. We realized success with the identifica-
tion of 3-pyrrolo[2,3-b]pyridine as a replacement for the ketobenz-
imidazole (see 41–48, Table 4). The synthesis of pyrrolopyridines
41–48 is summarized in Scheme 2. Condensation of N-Boc-3-pipe-
ridinone with 36 under basic conditions yielded 37.24 Hydrogena-
tion25 and subsequent Boc removal gave 39. Finally, coupling 39
with 2-Cl-4-[2-benzothiophene]pyrimidine followed by alkylation
of the pyrrolopyridine nitrogen provided compounds 41 and 42.
Similarly, protection of 37 as its N-phenylsulfonyl-derivative, fol-
lowed by methylation provided the 2-methylpyrrolopyridine
40.26 De-protection and elaboration as described above provided
compounds 41–46. As indicated by the SAR data in Table 4, the
pyrrolopyridine-based FAAH inhibitors exhibited potencies com-
parable to the ketobenzimidazole-based inhibitors (cf. 7 and 8 with
41–48), demonstrating that the carbonyl was not required for po-
tent FAAH inhibition. Likewise, the potencies of the enantiomers of
46 (47 and 48) showed that the activity resided in the S-series, sug-
gesting a binding mode similar to that of the ketobenzimidazoles.
The X-ray co-crystal data obtained for compounds 8 and 45
bound in the rFAAH active site revealed an unexpected and unique
binding mode. As shown in Figure 2, both inhibitors adopted a
binding mode in which the pyrimidine bridges F432 and M436
thus positioning the 4-aryl substituent in the solvent exposed
portion of the membrane access channel (MAC) while the piperi-
dine was positioned in the ABP. The aryl-pyrimidine makes van
der Waals contact with several hydrophobic residues including
F432, M436, L433, W531, T488, and I491. Both the ketobenzimi-
dazole (compound 8) and pyrrolopyridine (compound 45)
fragments are perpendicular to the plane of the piperidine ring
and directed down the cytosolic port. The ketobenzimidazole and
pyrrolopyridine fragments make contact with the I238/G239 side
of the oxy-anion hole, without engaging in a covalent interaction
with S241.
R
N
6-13
Cmpd
R
Rat FAAH
Human FAAH
IC50
IC50
,
a l
M
, M
a l
N
N
6
>10
>10
N
N
Me
Me
O
7
0.78
0.03
0.01
0.009
>10
0.006
N
N
X
O
8, X = CH
9, X = N
10, (R), X = N
11, (S), X = N
0.06
0.03
>10
N
N
N
0.02
O
Me
12
13
>10
>10
>10
N
N
N
Me
O
N
0.35
N
N
Me
O
a
Values are means of at least two experiments. All SAR data reported herein was
obtained using the assays described in Ref. 9.
a
N
N
Cl
N
Cl
Cl
N
Cl
O
14
In fact, no specific polar interaction is apparent for either inhib-
itor. Overall, the binding mode displayed by both 8 and 45 is
consistent with our SAR, noncovalent inhibition, and suggested
that shape was the primary source of recognition of these inhibi-
tors by FAAH.
O
HN
N
O2N
O2N
e, f
H
N
b
c, d
N
NBoc
Me
NBoc
15
16
Me
N
In moving forward we evaluated the in vivo pharmacokinetic
(PK) parameters and CNS uptake of selected compounds
(Table 5). As shown, compounds 8, 33 and 35 exhibited low clear-
ance, displayed good absorption and reasonable mean residence
times. Unfortunately, both 8 and 33 had very limited CNS exposure.
We reasoned that the poor CNS penetration might be due in part to
the highly lipophilic nature of these compounds (c log P = 4.9–5.4).
We had already demonstrated that we could decrease lipophilicity
and improve potency by adding polarity to the ketobenzimidazole
fragment, so next we turned to the aryl-pyrimidine subunit. Based
on crystallographic data available at the time we suspected that
adding a small polar group to the pyrimidine ring of compound
21 would be well tolerated and was calculated to significantly
O
O
N
g
N
N
O
NH
N
N
17
(S)-8
h
Me
N
N
Me
N
N
O
O
O
i
het
het
N
N
X
18
19-22
Scheme 1. Representative synthesis of FAAH inhibitors. Reagents and conditions:
(a) Pd(Ph3P)4, benzofuran-2-yl boronic acid, Na2CO3, H2O, EtOH, toluene 40–66%;
(b) (S)-t-butyl 3-aminopiperidine-1-carboxylate, i-Pr2NEt, DMF 60 °C, 75%; (c) H2
(1 atm), 10% Pd/C, THF; (d) CDI, THF, 60 °C 73% from 15; (e) NaH, EtI, DMF, 0 °C, 94%;
(f) HCl, dioxane, 98%; (g) 14, i-Pr2NEt, DMF 60 °C, 72%; (h) i-Pr2NEt, THF, 25 °C; 5-
bromo-2-chloropyrimidine 46%, or 2,4-dichloropyrimidine, 73%, or 4,6-dichloropy-
rimidine, 94%; 4,6-dichloropyrimidine-2-amine, 68% or 2,6-dichloropyrazine, 8%; (i)
Pd(Ph3P)2Cl2, benzofuran-2-yl boronic acid, Na2CO3 (2 equiv), dioxane/water,
120 °C, 46–77% (for 19–21) or Pd(Ph3P)4, aryl boronic acid, 2:2:1 benzene/MeOH/
2 M Na2CO3, 85 °C, 41–52% (for 22).
reduce the compound0s lipophilicity.
A NH2 (compound 22,
c log P = 4.3) group was found to be beneficial while larger groups
were generally poorly tolerated. This modification, in combination
with a p-fluorophenyl substituent on the pyrimidine ring led to the
much less lipophilic 35 (c log P = 3.7). Compound 35 was potent
against rat and human FAAH in both our biochemical and whole cell

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2492–2496
2495
Table 3
Rat and human FAAH biochemical and whole cell assay SAR
PhSO2
R
NBoc
NBoc
S
HN
N
HN
N
N
a
b,c
N
Me
N
N
Me
N
N
36
37
38
O
O
Ar
N
N
N
e, f
NH
N
N
N
N
R
Ar
Ar
N
N
N
R
R
N
N
R1
N
HN
N
A
B
d,e,f
g,h
N
Me
R1
39, R = H
40, R = Me
41-48
O
O
N
N
N
N
N
N
N
N
Ar
Scheme 2. Synthesis of indole based FAAH inhibitors. Reagents and conditions: (a)
N-Boc-3-piperidione, NaOMe, MeOH 80 °C, 45%; (b) PhSO2Cl (2 equiv), NaH, THF
0 oC 95%; (c). LDA ꢀ78 °C, then MeI 0 °C, THF 60%; (d) KOH, MeOH, 23 °C, 95%; (e)
Pd(OH)2/C 50 PSI H2, AcOH/EtOH (6:1) 60%; (f) TFA, CH2Cl2 (1:1), 95%; (g) 2-Cl-4-[2-
benzothiophene]pyrimidine Na2CO3, DMF, 85 °C 55%; (h) R-I, NaH, DMF, 18–82%.
N
N
, R = H
, R1 = Et
F, R1 = CF3CH2
C
E
D, R = NH2
Cmpd Core Ar
Rat FAAH
IC50
RBL cells Human FAAH T84 cells
,
a l
M
EC50
IC50
,
a l
M
EC50
OL-135
URB597
0.025
0.003
0.17
0.004
0.016
0.004
3.0
0.003
19
20
21b
22
23
24
25
26
27
28
29
B
A
C
D
E
A
A
A
A
A
A
2-Benzofuran
2-Benzofuran
2-Benzofuran
2-Benzofuran
Ph
2F-Ph
3F-Ph
4F-Ph
4Cl-Ph
>10
0.30
0.57
0.20
0.22
3.0
—
—
—
—
—
—
—
—
—
—
—
>10
0.03
—
—
—
—
—
—
—
—
—
—
—
0.27
0.01
0.05
0.13
0.05
0.01
0.03
0.04
0.06
1.2
0.12
0.16
0.28
0.34
4Me-Ph
5-Cl-2-
thiophene
4F-Ph
2-Thiophene
5-Cl-2-
thiophene
5-Cl-2-
thiophene
2-
30
31
32
E
E
E
0.03
0.19
—
1.8
0.28
0.01
0.03
—
0.35
0.003
0.004
0.008
0.026
0.043
0.001
0.002
0.002
0.014
33
34
35
F
0.10
0.08
0.10
0.002
—
E
D
Benzothiophene
4F-Ph
Figure 2. Overlay of the X-ray co-crystal structures of compounds 8 (2.3 Å; PDB
0.02
code: 3QJ9) and 45 (2.1 Å; PDB code: 3QK5) with rat FAAH
making up the oxy-anion hole are shown in yellow while S241 is shown in green.
D-TM 30. Residues
a
Values are means of at least two experiments. All biochemical SAR data
reported herein was obtained using the assays described in Ref. 9.
b
Data reported for racemic compound.
Table 5
Pharmacokinetic parameters in rat for compounds 8, 33 and 35a
8
33
0.45
0.87
1.4
35
0.24
0.69
3.4
CL (L/h/Kg)b
0.40
1.5
4.4
Table 4
Vdss (L/Kg)b
Rat and human FAAH inhibition data for pyrrolopyridines
Cmax
(
l
M)^
Cmpd
41
42
R
R1
Rat FAAH IC50
,
l
Ma Human FAAH IC50 Ma
, l
MRT (h)^
5.5
3.7
5.2
F (%)
92
49
99
H
H
Me
CH2CN
H
0.12
0.009
0.009
0.005
0.011
0.002
0.003
0.002
>10
CNS uptakeb
Total concentration (
Plasma/blood/brain
0.083
0.075
0.075
0.018
0.053
0.039
l
M)
43 Me
44 Me Et
45 Me CH2CN
0.5 h
2 h
2.9/2.3/0.15
1.3/1.0/0.07
0.0016/0.001
5.4
1.0/0.41/0.05
0.17/0.05/0.02
—
2.2/0.77/0.38
0.70/0.21/0.13
0.001/0.0024
3.7
46-(rac) Me CH2CH2OH
47-(S) Me CH2CH2OH
48-(R) Me CH2CH2OH >10
Fub (plasma/brain)c
c log P
4.9
a
Permeability (nM/s)d
P-gp efflux ratiod
—
—
—
—
35
1.1
Values are means of at least two experiments. All biochemical SAR data
reported herein was obtained using the assays described in Ref. 9.
a
b
c
Values are means of at least two individual test subjects.
Following 0.5 mg/Kg dose i.v.
Determined by ultra centrifugation in rat tissues as described in Ref. 27b.
Determined in LLC-PK1 cell monolayers as described in Ref. 27a.
Following 2.0 mg/Kg dose p.o.
assays and additionally had good permeability (35 nM/s)27 and was
not a substrate for P-gp (efflux ratio = 1.1) resulting in improved
CNS penetration. Correcting the total exposure of 35 for the fraction
unbound in rat plasma and brain tissue (Table 5), one calculates a
brain/plasma ratio of 0.41 for compound 35, compared to a ratio
of <0.05 for compound 8 using the same formula.28
d
^
Overall, compounds of this series displayed good PK parameters
being of low clearance, high bioavailability and reasonable resi-
dence times. Compound 35 showed modest but reasonable CNS
penetration and shows that physico-chemical properties of this
In summary, we have identified a series of highly potent, novel,
noncovalent FAAH inhibitors.

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D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2492–2496
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series of FAAH inhibitors can be optimized to target the CNS. We
have determined these compounds to have a unique binding mode
by X-ray crystallography. We expect that these compounds will be
useful in further studying the pharmacology of FAAH inhibition.
Additionally, the novel binding mode displayed by 8 and 45 should
provide a useful starting point for the design of future FAAH
inhibitors.
13. The selectivity of several FAAH inhibitors has been described, see: (a) Johnson,
D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L. K.; Morris, M.; Beidler, D.;
Liimatta, M. B.; Smith, S. E.; Dudley, D. T.; Sadagopan, N.; Bhattachar, S. N.;
Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. F. ACS Med. Chem. Lett. 2010, 2, 91;
(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.
Neuropharmacology 2007, 52, 1095; (c) Leung, D.; Du, W.; Hardouin, C.; Cheng,
H.; Hwang, I.; Cravatt, B. F.; Boger, D. L. Bioorg. Med. Chem. Lett. 2005, 15, 1423.
14. The potential for observing idiosyncratic toxicity is generally thought to be
mitigated by avoiding covalent modifiers as drug candidates. See: Kumar, S.;
Mitra, K.; Kassahun, K.; Baillie, T. A. Adverse Drug Reactions, Handbook of
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determined by measuring AAMCA produced by UV.Whole-cell assays: RBL-2H3
or T84 cells were incubated in HBBS, 20 mM HEPES, pH 7.8, 10% glucose, 0.1%
fatty acid-free BSA and test compounds for 30 min. 1-3H AEA was added and
incubation continued 2 h before 1:1 MeOH/CHCl3 was added. The aq. layer was
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