Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2008.07.081

A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are

Full text of DOI:10.1016/j.bmcl.2008.07.081

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4838–4843
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Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase
à
*
John M. Keith , Richard Apodaca , Wei Xiao, Mark Seierstad, Kanaka Pattabiraman , Jiejun Wu,
Michael Webb, Mark J. Karbarz, Sean Brown, Sandy Wilson, Brian Scott, Chui-Se Tham, Lin Luo,
James Palmer, Michelle Wennerholm, Sandra Chaplan, J. Guy Breitenbucher
Johnson & Johnson Pharmaceutical Research and Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The
molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a
covalent bond with Ser241. SAR and PK properties are presented.
Received 19 May 2008
Revised 18 July 2008
Accepted 21 July 2008
Available online 25 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
FAAH
Fatty acid amide hydrolase
Anandamide
Time dependant inhibition
Urea
Piperazine
Thiadiazole
Lipid
CB1
CB2
Me
The endogenous cannabinoid system consists of two well-de-
fined G protein-coupled receptors, CB1 (1990)1 and CB2, a family
of lipid mediators, and several degradative enzymes which termi-
nate the actions of these mediators by hydrolysis. Anandamide
(AEA)2 (Fig. 1) was the first endogenous substance discovered with
activity at cannabinoid receptors, at both of which it is a weak par-
tial agonist. Although exogenous substances active at the cannab-
inoid receptors (of which the prototype is the plant-derived
O
OH
HO
H
N
H
H
Me
Me
Me
O
Me
Δ9-THC
Anandamide (AEA)
O
O
HO
substance
D
9-THC, Fig. 1) have analgesic activity, their known side
N
H
H2N
Me
effects (including dysphoria, memory deficit and abuse liability)
limit their therapeutic utility. Nevertheless, this observation does
suggest that manipulating the endogenous system may produce
analgesic benefit. In addition, the observation that endocannabi-
noid synthesis is selectively up-regulated in active neural path-
Me
N
-Palmitoylethanolamide
Oleamide
Figure 1. D9ÀTHC and substrates of FAAH.
ways also suggests that this strategy may have
selectivity and lower side effect profile than global activation of
cannabinoid receptors via exogenous agonists.
a greater
Endogenous levels of AEA are normally very low, as it is rapidly
metabolized by the enzyme Fatty Acid Amide Hydrolase (FAAH) to
give ethanolamine and arachidonic acid.3 FAAH also breaks down
several other lipids including: oleoyl ethanolamide (OEA), palmi-
toyl ethanolamide (PEA), O-arachidonylethanolamine (virod-
amine), oleamide, and N-arachidonyldopamine.4
* Corresponding author. Tel.: +1 858 784 3275.
E-mail address: jkeith@prdus.jnj.com (J.M. Keith).
Present address: Metamolecular, LLC, PO Box 13091, La Jolla, CA 92039-3091,
USA.
Present address: Amgen, 1120 Veterans Boulevard, S. San Francisco, CA 94080,
USA.
Although 2-AG is a full agonist at both CB1 and CB2 its primary
route of breakdown is through the action of monoacylglycerol
lipase (MAGL).5 Of the other FAAH substrates, PEA is known to
have anti-inflammatory properties and to exert analgesic effects
through a non-cannabinoid pathway.6 OEA is a known anorectic
à
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.081

J.M. Keith et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4838–4843
4839
lipid that has effects on satiety.7 Oleamide appears to be involved
in sleep induction.8 Less is known about the pharmacology of the
other FAAH substrates.
FAAH knockout mice have elevated levels of AEA, PEA and OEA
in the brain, suggesting that degradation by FAAH is the primary
metabolic fate of these lipids.9 The knockout mice are viable and
healthy, but display attenuated responses in several pain assays,
supporting the suggestion that a FAAH inhibitor would be an effec-
tive analgesic.
The amidine salts were treated with perchloromethyl mercaptan
in strongly alkaline solution for 30 min at low temperature and
then given a typical workup.17 The crude products were treated
with piperazine and stirred overnight. Treatment of the resultant
piperazines with aryl isocyanates gave ureas in good yields (Table
1). Alternatively, the piperazines could be treated with phenyl
carbamates of heteroaryl amines. The phenyl carbamates were
prepared by treating an excess of the heteroaryl amine with phenyl
chloroformate, either at room temperature or at reflux (Scheme 2).
The yields of the phenyl carbamates varied considerably (Table 2).
The data presented in Table 1 indicate very tight SAR18 on both
of the pendant aryl rings. The phenyl ring attached to the urea
nitrogen appears to be best if either unsubstituted (1) or with a
chloro (10) or a fluoro (13) group in the 2-position. Other substit-
uents yielded weakly active compounds.
A number of groups have reported the preparation and testing
of FAAH inhibitors (Fig. 2). Several series of highly potent
a-keto
heterocycles were described by Boger et al.10 The ketoheterocycle
OL-135 is thought to form a reversible tetrahedral intermediate
derived from the interaction of the ketone to give a hemiketal.
Piomelli et al.11 disclosed carbamate-based inhibitors as did Sanofi.12
The carbamate compounds are relatively potent and form a cova-
lent bond with Ser 241 within the active site of FAAH.13 URB597
and SA-47 also form a carbamate with the FAAH enzyme with
the concomitant loss of a phenolic or alcoholic fragment, respec-
tively. Both OL-13514 and URB59715 have been reported to be
efficacious in the treatment of pain in various animal models with-
out the motor impairment associated with direct CB1 agonism.
We discovered the potent FAAH inhibitor (1) (Fig. 3) in an HTS
screen of our chemical library.16 Analogs of (1) could be prepared
in three steps from an aryl amidine hydrochloride (Scheme 1).
Despite the tight SAR observed for substituted phenyl groups on
the urea nitrogen, many heteroaryl replacements for phenyl gave
moderate to highly potent compounds (Table 2). In general, 6-
membered ring heterocycles were more potent than 5-membered
ring analogs. The most potent compound prepared, however, was
Table 1
SAR on pendant aryl groups
O
R
O
N
H
N
O
O
N
N
N
H2N
O
N
H
Ar
O
S
N
N
Compound
R
Ar
Apparent rFAAH
Apparent hFAAH
OL-135
Ki = 4.7 nM
URB597
IC50 = 48 nM
a
a
IC50 (nM)b
IC50 (nM)b
1
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
34 6.5
33 2.1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4-MeO
3-MeO
2-MeO
4-Me
3-Me
2-Me
4-Cl
3-Cl
2-Cl
4-F
84 15
280 145
47 9.8
152 15
1860 856
173 18
1300 430
Me
1270 452
148 21
1666 408
350 19
163 22
18.3 5.0
91 43
152 97
6.2 1.5
8330 1080
>10000
>10000
>10000
>10000
350 21
117 35
248 58
N
193
8
O
N
H
N
2000
0
347 36
257 15
31.7 4.7
96 36
143 93
19 2.1
5000 3000
>10000
10000
>10000
>10000
1066 82
833 204
>10000
>10000
69 26
Me
O
N
H
O
SA-47
3-F
2-F
Figure 2. Known FAAH inhibitors.
4-OCF3
4-SCF3
4-NO2
2-CF3
2-OCF3
2-SMe
H
H
H
H
H
O
Ph
N
H
N
(1) JNJ-1661010
rFAAH IC50 = 34 6.5 nM
hFAAH IC50 = 33 2.1 nM
4-F-Ph
4-Cl-Ph
4-Me-Ph
3-Me-Ph
4-MeO-Ph
N
N
Ph
147
8
S
N
122 36
300 131
>10000
Figure 3. Initial HTS hit: JNJ-1661010. Values measured at 20 min (N P 3).
Range is the standard error of the mean. A reference for this ubiquitous method of
reporting error ranges may be excessive.
a
IC50s were determined with a 20 min preincubation with the enzyme.
All values are with N P 3.
b
HN
NH
a, b
c
N
N
Ar
H2N
Ar
S
N
O
O
HetAr
N
H
N
O
a
b
N
H
N
N
HetAr
N
HetAr NH2
R
N
H
OAr
Ph
N
N
S
Ar
N
S
N
Scheme 2. Reagents and conditions: (a) 1/3 equiv ClCO2Ph, THF or ACN, rt—reflux;
(b) 0.9 equiv 1-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine, DMSO, microwave
100 °C, 30 min or 50 °C 18 h.
Scheme 1. Reagents and conditions: (a) CCl3SCl, 6 N NaOH, CH2Cl2, 0 °C, 30 min; (b)
piperazine, rt, 18 h, ꢀ50% for two steps; (c) R-PhNCO, CH2Cl2, 80%-quant. yield.

4840
J.M. Keith et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4838–4843
Table 2
Heteroaryl ureas
a
a
Compound
Yield (%) step (a)
—
Yield (%) step (b)
46c
HetAr
Apparent rFAAH IC50 (nM)b
Apparent hFAAH IC50 (nM)b
25
26 2.5
26.7 2.2
9.8 1.5
N
N
26
80
97
8.7 2.7
N
N
27
28
26
32
68
98
57 20
266 60
N
6.3 0.41
10.3 1.5
N
N
N
29
30
10
4
53
18
28.8
7
13 3.4
253 33
MeO
N
N
188 35
CN
N
N
31
32
33
71
18
14
97
14
24
54
9
50 15
2667 408
23.0 9.9
Cl
N
N
1317 419
10.8 4.4
N
N
N
S
N
N
34
38
85
1.35 0.79
2.02 0.48
35
36
37
38
57
78
11
30
37
N
125 23
638 140
113 17
>10000
1167 204
1037 105
320 12
N
H
Ph
Ph
80
N
N
H
N
S
Quant.
82
N
S
>10000
Me
O
N
39
40
24
74
84
57
25.7 1.5
41 5.6
70 2.9
320 13
N
HN
N
N
Range is the standard error of the mean. A reference for this ubiquitous method of reporting error ranges may be excessive.
a
IC50s were measured with 10 min preincubation.
Values are with N P 3.
Prepared from the isocyanate.
b
c
the bicyclic benzothiadiazole derivative (34). Interestingly, despite
the mechanism of FAAH inhibition, having more electron deficient
heteroaryl substituents, and thus better leaving groups, on the urea
does not translate to more potent/rapid inhibition of the enzyme.
This suggests that interactions between the heteroaryl amine and
the enzyme may be an important factor in determining the rate
(and therefore apparent IC50) of inhibition of the FAAH enzyme.
Under standard conditions, (1) has an IC50 of 12 nM at human
FAAH (60 min assay with no preincubation). To determine the ef-
fect of preincubation time on apparent IC50, the assay was short-

J.M. Keith et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4838–4843
4841
N
S
N
N
H
N
N
+
FAAH
O
JNJ-1661010 (1)
MW = 365
FAAH
FAAH
N
S
H
N
O
N
N
O
N
O
O
Figure 4. Apparent IC50 of (1) with differing preincubation times.
MW = FAAH + 119
Not observed
MW = FAAH + 273
ened to 10 min and (1) was preincubated with enzyme for different
times (Fig. 4). The apparent IC50 for (1) at human FAAH was re-
duced from 70 to 15 nM after a 40 min preincubation. A time
dependence of apparent IC50 values for enzyme inhibition is usu-
ally observed when a compound is either a slow on tight binding
ligand, an irreversible inhibitor, or a mechanism-based substrate.
To determine the reversibility of (1), we used a dialysis ap-
proach to examine the mechanism of FAAH inhibition. Test com-
pounds (at IC80 concentrations) were incubated with membranes
of recombinant cells expressing rat FAAH, and dialyzed against
1 L of PBS buffer at 4 or 22 °C. After 18 h dialysis, the contents
of the dialysis cassette were recovered and assayed for FAAH
activity (Fig. 5). The activity of enzyme preincubated with OL-
135 was fully recovered19 after dialysis at both temperatures,
consistent with a full reversibility of inhibition. FAAH preincu-
bated with URB597 failed to recover activity after 18 h of dialysis
at either temperature, consistent with the formation of an irre-
versible covalent bond, as expected for this carbamate. Enzyme
incubated with (1) remained substantially inhibited after dialysis
at 4 °C. However, at 22 °C, significant activity was recovered, sug-
gesting a temperature-dependent release of the compound from
the active site. The recovery of enzyme activity after 18 h dialysis
at 22 °C, albeit not complete, rules out irreversible inhibition of
FAAH due to (1).
Figure 6. Results of LC-ESI-MS analysis of partially purified rat FAAH incubated
with (1).
ingly, the measured MW increased by 274 Da to that of FAAH pro-
tein itself, suggesting a covalent modification of the enzyme with
the piperazinyl fragment and concomitant loss of aniline (Fig. 6).
This increase in mass, along with a return of FAAH activity in the
dialysis experiments, suggests that (1) must be a covalent, mecha-
nism-based substrate inhibitor.
To confirm the formation of a covalently linked piperazinyl
fragment, we incubated partially purified soluble rat FAAH lacking
the transmembrane domain with (1) tritiated on the piperazinyl
group. Ten micrograms of this preparation was incubated with
400 nM 3H (1) for 60 min, and then unbound ligand was separated
from bound ligand by gel filtration. Enzyme activity of an unla-
beled preparation run on a parallel column confirmed co-elution
A
To distinguish between a tightly bound non-covalent ligand and
a mechanism-based substrate inhibitor, compound (1) was incu-
bated (twofold molar excess of FAAH) with partially purified rat
FAAH enzyme for 3 h at 22 °C and examined by LC-ESI-MS. Accord-
B
70
C
60
50
40
30
20
10
0
inside
outside
Disposition of recovered radiolabel
Figure 7. (A) Separation of radiolabeled FAAH after incubation with 3H (1); (B)
autoradiograph of sds gel of column fractions 4-8 after denaturation (grey, labeling
carried out in excess unlabeled (1); (C) dialysis of FAAH 3H (1) complex.
Figure 5. Residual FAAH activity pre- and post-dialysis (18 h) for JNJ-1661010, OL-
135 (reversible) and URB597 (irreversible) at 4 and 22 °C (N P 3).

4842
J.M. Keith et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4838–4843
of the labeled protein peak with enzyme activity (Fig. 7A). The radi-
olabeled preparation was then denatured in 8 M urea and sub-
jected to SDS gel electrophoresis and autoradiography. A single
radiolabeled band (Fig. 7B) was found, suggesting that the complex
between enzyme and the piperazinyl fragment was covalent and
stable to protein denaturation. The peak radiolabeled fractions
were subjected to dialysis as described, and after 18 h at room
temperature, about 35% of the total radioactivity was recovered
in the outer compartment, confirming that the bond formed be-
tween the enzyme and piperazinyl fragment is reversible (Fig. 7c).
A published crystal structure of rFAAH bound to MAFP20 reveals
the presence of a long lipophilic tunnel and a relatively large
hydrophilic area containing Ser241 and other polar residues. As it
was not immediately clear how compounds such as (1) bound to
the active site, we docked (1) in two ways21: with the phenyl urea
portion of the molecule in the hydrophobic tunnel and the phenyl-
thiadiazole in the hydrophilic pocket (Fig. 8); and, with the phenyl-
thiadiazole in the hydrophobic tunnel and the phenylurea in the
hydrophilic pocket (Fig. 9). Molecular modeling suggested, on the
basis of size, that both configurations of (1) within the enzyme ac-
tive site were possible, however several factors suggested binding
as shown in Figure 9. First, the crystal structure with FAAH bound
to MAFP showed the long lipophilic tail of MAFP in the hydropho-
bic pocket, and the phenylthiadiazole is lipophilic. Second, the loss
of aniline is consistent with the urea portion of the molecule in the
more solvent accessible hydrophilic pocket, and would be consis-
tent with work published by others.22 Finally, the observed SAR
for this class of molecules is consistent with the phenythiadiazole
being in the hydrophobic pocket.
Figure 10. Overlay of 3- and 4-substituted phenyl compounds (5) and (6).
Substitution was not tolerated in the 4-position of the phenyl
pendant to the thiadiazole by the human enzyme, but 3-substitu-
tion gave compounds equipotent to (1). A rationale for the preced-
ing observation can be found in Figure 10, which shows an overlay
of compounds (5) and (6) docked within the active site of rFAAH.
The 4-methyl group of (5) is proposed to be in a sterically crowded
space whereas the methyl group in (6) resides within a small lipo-
philic pocket.
We assessed the possibility of mechanism-based non-specificity
by assaying liver carboxylesterases. In an assay measuring changes
in the rate of ester (4-nitrophenyl-acetate) hydrolysis in rat liver
microsomes,23 (1) did not measurably inhibit ester hydrolysis.
OL-135 and URB597 significantly decreased the rate of ester hydro-
lysis (Fig. 11). These data suggest that (1) is not a general inhibitor
of esterases, whereas URB597 decreases liver esterase activity by
40%.
Liver Esterase Assay
JNJ-1661010
OL-135
URB597
Figure 8. Compound (1) docked with the phenyl urea portion of the molecule in the
hydrophobic tunnel and the phenylthiadiazole in the hydrophilic pocket.
0
10 20 30 40 50 60 70 80 90 100
% Relative Activity
Figure 11. Inhibition of liver esterases by various FAAH inhibitors (10 lM with
30 min preincubation). DMSO control showed no inhibition.
Table 3
Summary of pharmacokinetic data for JNJ-1661010
P.O.
I.P.
I.V.
1
Dose (mg/kg)
Cmax M)
Tmax (min)
Plasma T1/2 (min)
Mean retention Time (min)
Cl (L/min)
Volume of distribution (L)
F (%)
10
10
(l
0.301 ( 57%)
110 ( 103%)
264 ( 66%)
382 ( 95%)
0.116 ( 18)
45
1.58 ( 7%)
35 ( 66%)
94 ( 5%)
135 ( 7%)
0.032 ( 10%)
4.6
3.06 ( 38%)
—
34 ( 13%)
49 ( 19%)
0.006 ( 18%)
0.57
5
20
—
Figure 9. Compound (1) docked with the phenylthiadiazole in the hydrophobic
tunnel and the phenylurea in the hydrophilic pocket.
Vehicle is 1:4:15 pharmasolve: cremaphore: 5% dextrose in water.

J.M. Keith et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4838–4843
4843
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Rats dosed with 20 mg/kg i.p. (1) had a plasma Cmax of 26.9 lM
at the Tmax of 0.75 h and a Cmax in the brain of 6.04 lM at the Tmax
of 2 h (Table 3). Compound (1) had a blood–brain barrier coeffi-
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O.; Monaghan, E. P.; Parrott, J. A.; Putman, D. CNS Drug Rev. 2006, 12, 21.
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cient of 0.042.24
We examined the in vivo properties of (1) by measuring both ex
vivo inhibition of brain FAAH and the elevation of AEA after dosing
the compound at 20 mg/kg i.p. Brain FAAH was profoundly inhib-
ited by a single 20 mg/kg dose of (1) for an extended period (Fig.
12). Even after 24 h, FAAH activity had recovered only to 25% of un-
treated values. In parallel, rats that had been dosed with 1 (20 mg/
kg, i.p.) showed elevated levels of AEA in brain tissue. Four hours
post dosing of (1), rat brain AEA levels increased by up to a factor
of 1.4 thus suggesting in vivo inhibition of FAAH. Additional in vivo
studies will be reported elsewhere.
19. We consistently see an increase in FAAH activity post dialysis with reversible
inhibitors, but have no concrete explanation for this phenomenon. All
experiments (at each dialysis temperature) are compared to their dialyzed
vehicle control under the same conditions, which sets the 100% value.
20. Bracey, M. H.; Hanson, M. A.; Stevens, R. C.; Cravatt, B. F. PCT Int. Appl.,
2004044169, 2004.
21. Ligands were docked by hand or using Glide (Schrödinger, LLC, New York, NY,
2007) into a single FAAH unit (PDB ID: 1mt5). Minimization of the covalently
bound ligand was performed with MacroModel (Schrödinger). Images were
created with PyMOL (DeLano WL: The PyMOL Molecular Graphics System 2002
DeLano Scientific, Palo Alto, CA, USA. http://www.pymol.org).
22. 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.; Cravatt, B. F.
Biochemistry 2007, 46, 13019.
23. Zhang, D.; Saraf, A.; Kolasa, T.; Bhatia, P.; Zheng, G. Z.; Patel, M.; Lannoye, G. S.;
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24. The BBB coefficient is defined as the Log (AUCbrain)/(AUCplasma).
25. For the related system, see Ref. 22. Apodaca, R.; Breitenbucher, J. G.;
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In conclusion, we have described a series of aryl piperazinyl
urea FAAH inhibitors with characteristics consistent with them
being mechanism-based substrates of the enzyme. While this work
was in progress, a report describing the mechanism of action of a
closely related series of molecules (first disclosed in one of our pat-
ent applications)25 appeared.
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