Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas

Article abstract of DOI:10.1016/j.bmcl.2009.05.097

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Full text of DOI:10.1016/j.bmcl.2009.05.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4241–4244
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral
azetidine ureas
a,
a
a
a
a
Terance Hart a, , Alba T. Macias , Karen Benwell , Teresa Brooks , Jalanie D’Alessandro , Pawel Dokurno ,
Geraint Francis a, Ben Gibbons b, Timothy Haymes b, Guy Kennett b, Sean Lightowler b, Howard Mansell b,
Natalia Matassova a, Anil Misra b, Anthony Padfield b, Rachel Parsons a, Robert Pratt b, Alan Robertson a,
*
*
Steven Walls a, Melanie Wong a, Stephen Roughley a,
*
a Vernalis (R&D) Ltd, Granta Park, Cambridge CB21 6GB, UK
b Vernalis (R&D) Ltd, Oakdene Court, 613 Reading Road, Winnersh RG41 5UA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase
(FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH
and also hCB1.
Received 15 April 2009
Revised 22 May 2009
Accepted 23 May 2009
Available online 29 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
FAAH, fatty acid amide hydrolase
Azetidine
Urea
Inhibitor
Fatty Acid Amide Hydrolase (FAAH) is the serine hydrolase
that is thought to be responsible for the hydrolysis of ananda-
mide (AEA), an endogenous agonist of the cannabinoid receptor
1 (CB1).1 Neither FAAH knockout mice, nor rodents treated with
FAAH inhibitors, elicit behaviours associated with cannabinoid-
like central side effects. Furthermore, FAAH knockout mice are
viable, showing increased brain levels of AEA with attenuated re-
sponses in a variety of inflammatory and neuropathic pain mod-
inhibitors tend to form time-dependent, covalent adducts by
reaction of the catalytic Ser-241 residue within the FAAH
enzyme.
Irreversible inhibitors need to be selective and, based upon
their serine hydrolase selectivity profiles in Activity-Based Pro-
tein Profiling (ABPP) assays,5 most of the ureas and carbamates
shown in Figure 1, apart from URB597, are highly selective as
both rat and human FAAH inhibitors. Because of the substrate
and agonist connection for AEA between FAAH and CB1, we
decided to screen our hCB1 antagonist collection of substituted
ureas for rFAAH activity, and discovered that the racemic,
azetidine VER-24052, was a reasonably potent inhibitor of re-
combinant rat FAAH (Table 1). This appears to be the first
els.2
A separation of the different CB1-mediated effects is
supported by the observation that cannabinoid-like side effects
may be mediated by 2-arachidonoylglycerol (2-AG) and not
AEA.3 Thus, orally bioavailable FAAH inhibitors may be new
treatments not just for acute and chronic pain, but also for anx-
iety and depression. Both SanofiAventis (SSR411298 (Phase II))
and Pfizer (PF-4457845 (Phase I)) have recently advanced FAAH
inhibitors into the clinic, primarily for these respective indica-
tions. A variety of FAAH inhibitors, including keto-heterocycles
(e.g., OL-135), carbamates (e.g., URB597 and SA47), and ureas
(e.g., PF-750, PF-622 and JNJ-1661010) (Fig. 1) have now been
reported.4 To date all these carbamate and urea-containing FAAH
example of FAAH inhibitor that contains
a tetrasubstituted
urea. However, VER-24052 was originally designed as a potent
CB1 antagonist and since this activity would negate much of
the benefits from inhibiting FAAH, we decided to characterize
biologically both enantiomers of VER-24052 before embarking
on a lead optimisation programme. We separated the enantio-
mers of VER-24052 (Fig. 1), as shown in Scheme 1, using chiral
preparative HPLC (AD-H: iso-Hexane:EtOH (70:30)) to produce
two enantiomers of unknown stereochemistry, designated,
VER-156084 (>99% ee) and VER-156085 (>99% ee), respec-
tively.11 Re-profiling against FAAH and human CB1 and CB2
* Corresponding authors. Tel.: +44 0 1223 895426 (T.H.).
E-mail addresses: t.hart@vernalis.com (T. Hart), a.macias@vernalis.com (A.T.
Macias), s.roughley@vernalis.com (S. Roughley).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.097

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T. Hart et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4241–4244
O
Conversion of chloronicotinoyl chloride 1 to the Weinreb
O
N
amide, 2, followed by Grignard reagent addition and NaBH4
reduction furnished the racemic alcohol 5 (Scheme 1). Reaction
of 5 with 1-benzhydrylazetidin-3-ol (PTSA, PhMe, reflux, Dean-
Stark), followed by deprotection under standard conditions (1-
chloroethyl chloroformate, followed by MeOH) gave the racemic
azetidine ether, 6, as the hydrochloride salt. Resolution of the
azetidine 6 was achieved using the same chiral HPLC system
as previously used on VER-24052, while re-crystallisation from
NH
OH
Ph
O
OL-135
IC50 230 nM
N
Anandamide (AEA)
O
N
O
2NH
O
N
N
NH O
NH
URB597
IC50 109 nM
O
MeCN of the D-dibenzoyl tartrate salt (mpt 140 °C) of the enan-
N
H
O
SA-47
tiomer, 7, gave crystals suitable for X-ray crystallography, and
confirmed the absolute stereochemistry of chiral amine, 7,
(Fig. 2).11
O
O
N
NH
Ph
N
NH
Ph
N
Examples of low energy conformers for VER-156084 and VER-
156085 are shown in Figure 2. These molecular geometries were
obtained from a conformational sampling study and optimized
N
PF-750
IC50 16.2 nM
PF-622
IC50 33 nM
using B3LYP/6-311G in Jaguar.7 A large number of conformers
**
O
N
Cl
O
were first produced by molecular mechanics with MOE’s sto-
chastic method.8 Low energy conformers were then used as
the initial structures for relaxed dihedral scans of the pyridyl
ring using quantum mechanics. VER-156084 and VER-156085
have populations of stable conformers that differ in the spatial
arrangement of the substituents in the aromatic rings and results
in the lower activity of the R isomer. Ring rotations are hindered
by repulsion between the 2-Cl and the ether lone pairs or the
electron cloud of the adjacent ring giving a degree of rigidity
to the molecule. The crystal of 7, a fragment of VER-156084,
underscored the preference of the 2-Cl to point away from the
lone pairs of the ether oxygen. Common to both isomers is the
puckering of the urea nitrogens and an out of plane twist be-
tween the saturated rings. The twist away from the carbonyl
plane in the azetidine (h–60°) is greater than that of the piperi-
dine (hꢀ40°).
N
NH
Ph
N
N
N
N
N
S
O
Cl
JNJ-1661010
IC50 33 nM
VER-24052
Figure 1. Substrates and inhibitors of FAAH.
receptors showed that both the rat and human FAAH activity
was due to VER-156084, whereas the hCB1 and hCB2 activity
resided mainly with its enantiomer, VER-156085 (Table 1).
VER-156084 also showed a time-dependent inhibition of rat
FAAH which is consistent with other urea inhibitors, indicating
that it also probably inhibits FAAH by reaction with the Ser-
241 residue of the active site. However, most surprisingly to
us, and perhaps unknown in this area, VER-156084 was signif-
icantly much weaker as an inhibitor of human FAAH (hFAAH)
than rat FAAH (rFAAH).
To understand better how VER-156084 fits into the hFAAH cat-
alytic pocket, we decided to determine unambiguously the abso-
lute configuration of VER-156084. However, because neither
VER-156084 nor VER-156085 provided crystals of sufficient quality
for detailed X-ray analysis, we needed to separate the amine enan-
tiomers of the precursor racemic azetidine, 6, and then convert
each of these separately to VER-156084 and VER-156085, respec-
tively.6 During this process we showed that neither the intermedi-
ates nor the products had racemised by chiral HPLC. Repeating
both the rat and human FAAH bioassays in tandem with chiral
HPLC monitoring allowed us to connect the chiral amine precursor,
7 to the end product, 8 (VER-156084) and, hence, amine 9 to 10
(VER-156085).
VER-156084 is less potent in hFAAH than rFAAH by ꢀ7-fold.
Species selectivity has been reported previously for FAAH inhib-
itors, with PF-750 being more potent in human than rat FAAH
by ꢀ8-fold,9 while several other inhibitors, including JNJ-
1661010,4 are equipotent. Mileni and co-workers studied the
structural basis for this selectivity by crystallizing the adduct
of PF-750 bound to a humanized rat protein, in which six rat
residues in the arachidonoyl binding channel (ACB) were mu-
tated to human residues. Figure 3 highlights the differences be-
tween the rat (1MT5.pdb)10 and hybrid (2VYA.pdb)9 crystals
and their effect on the binding site shape. Flexible alignment
of VER-156084 with known inhibitors (Fig. 2C) shows the over-
lap between the pyridyl ring in VER-156084 and the quinoline
ring in PF-750. We postulate that the aromatic ring in VER-
156084 occupies a similar site in the protein as the pyridyl ring
of PF-750 (Fig. 3). This would place VER-156084 in close con-
tact with residues that differ between rat and human FAAH.
The species selectivity observed for VER-156084 supports the
Table 1
Rat FAAH, human FAAH inhibition and CB receptor binding
a
a
Compds
Rat FAAH inhibition IC50 (nM)
Human FAAH inhibition IC50 (nM)
hCB1 binding
hCB2 binding
Kib (nM)
Kib (nM)
t = 0 h
t = 1 h
t = 3 h
t = 0 h
t = 1 h
t = 3 h
URB597
OL-135
VER-24052
VER-156084
VER-156085
1148
191
1205
465
78
119
291
140
63
22
306
NT
1031
>10,000
112
188
78
NT
>2000
>10,000
NT
531
>10,000
<10
560
<10
NT
7190
10900
>10,000
>10,000
>10,000
a
Apparent IC50 determined with rat or human FAAH by pre-incubation of compound with FAAH for time period indicated, followed by addition of arachidonylami-
nomethylcoumarin amide (AAMCA) probe.
b
Ki determined by radioligand displacement binding assay with 3H-SR141716A (hCB1) or 3H-CP55940 (hCB2).

T. Hart et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4241–4244
4243
N
Cl
N
Cl
N
Cl
N
Cl
N
Cl
N
N
Cl
Cl
O
O
OH
a
O
b
e
c
d
NH
O
O
N
Ph
Ph
O
Cl
Cl
Cl
Cl
1
2
3
4
6
5
N
Cl
N
Cl
Cl
g
O
O
N
N
NH
VER-156084
O
[a]D +16° (c 0.6 MeOH-d4)
Cl
N
Cl
Cl
f
g
(S)-urea
(S)-amine
f
O
6
8
7
N
N
O
Cl
O
N
Cl
O
N
g
11
VER-24052
N
N
NH
VER-156085
O
Cl
Cl
(R)-urea
(R)-amine
10
9
Scheme 1. Reagents and conditions: (a) MeNHOMeÁHCl, Et3N, DCM, 10 °C, 2 h, 83%; (b) 4-chlorophenylmagnesium bromide, THF, 0 °C ?rt, 19 h, 100%; (c) NaBH4, MeOH,
1.5 h, 89%; (d) 1-benzhydrylazetidin-3-ol, PTSAÁH2O, PhMe, Dean-Stark conditions, 3.5 h, 35%; (e) (i) 1-chloroethyl chloroformate, DCM, rt, 2 h; (ii) MeOH, rt, 24 h, 85%; (f)
chiral HPLC; (g) (i) triphosgene, pyridine DCM, rt; (ii) piperidine, DCM, 50%.
Figure 2. (A) X-ray crystal structure of 7, dibenzoyl tartrate salt has been removed for clarity. (B) Ab initio optimized structures of VER-156084 (S) and VER-156085 (R). (C)
Flexible alignment of VER-156084 (grey carbons) with inhibitors JNJ-1661010 (green carbons) and PF-750 (brown carbons).
placement of this ligand in a site where there are mutations be-
tween rat and human. Leu192 in rFAAH is replaced in hFAAH
and high microsomal turnover. The surprisingly lower inhibitory
potency of these molecules for human over rat FAAH also repre-
sents a key drug design and development issue regarding species
selectivity for workers in this area.
In conclusion, we have identified a new series of tetra substi-
tuted urea-based FAAH inhibitors and believe that this is also the
first example of two enantiomers having different activity versus
both human and rat FAAH, which can best be explained by the nat-
ure of the residues in the ACB channel. In future publications we
shall describe how we used this modeling information to design
highly selective, potent, orally active hFAAH inhibitors with excel-
lent in vivo activity.
by a Phe which can form an aromatic CH–p interaction with
the ligands. rFAAH Ile491 is replaced in hFAAH by a smaller
Val residue. Both mutations have an impact on the binding cav-
ity shape and volume, which should affect bulky rigid ligands
like VER-156084. Additional protein–ligand interactions and a
better fit could explain the enhanced binding of VER-156084
to hFAAH.
Further investigative profiling of VER-156084 showed that it
suffered from off-target serine hydrolase activity, as shown by
ABPP, was prone to high plasma protein binding across species

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T. Hart et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4241–4244
Acknowledgements
The authors thank Dr. J. E. Davies, Cambridge University Chem-
ical Laboratory, UK, for his help with the X-ray studies.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.097.
References and notes
1. Ahn, K.; McKinney, M. K.; Cravatt, B. F. Chem. Rev. 2008, 108, 1687.
2. Lichtman, A. H.; Shelton, C. C.; Advani, T.; Cravatt, B. F. Pain 2004, 109, 319.
3. Long, J. Z.; Li, W.; Booker, L.; Burston, J. J.; Kinsey, S. G.; Schlosburg, J. E.; Pavon,
F. P.; Serrano, A. M.; Selley, D. E.; Parsons, L.; Lichtman, AH.; Cravatt, B. F. Nat.
Chem. Biol. 2009, 5, 37.
4. (a) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.; Cheng, H.;
Hwang, I.; Hedrick, M. P.; Acevedo, O.; Guimaraes, C. R. W.; Jorgensen, W. L.;
Cravatt, B. F. J. Med. Chem. 2005, 48, 1849; (b) Keith, J. M.; Apodaca, R.; Xiao, W.;
Seierstad, M.; Pattabiraman, K.; Wu, J.; Webb, M.; Karbarz, M. J.; Brown, S.;
Wilson, S. Bioorg. Med. Chem. Lett. 2008, 18, 4838; (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.; Cravatt, B. F. Biochemistry 2007, 46,
13019.
5. (a) 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; (b) Cravatt, B. F.; Wright, A. T.; Kozarich, J.
W. Annu. Rev. Biochem. 2008, 77, 383; (c) Hoover, H. S.; Blankman, J. L.; Niessen,
S.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 2008, 18, 5838.
6. Both precursor amines 7 and 9 were inactive in both rat and human FAAH
assays.
7. Jaguar, version 7.5, Schrödinger, LLC, New York, NY, 2008.
8. MOE; Chemical Computing Group: 1010 Sherbrooke St. W, Suite 910, Montreal,
Quebec, Canada H3A 2R7. http://www.chemcomp.com.
9. Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.; Liimatta, M.; Pabst, B.;
Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt, B. F.; Ahn, K.; Stevens, R.
C. PNAS 2008, 105, 12820.
10. Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793.
11. (a) See Supplementary data.; (b) The crystal structure of 7 has been deposited
at the Cambridge Crystallographic Data Centre and allocated the deposition
number CCDC 731592.
Figure 3. Binding sites of rFAAH (A, 1MT5) and hybrid r/hFAAH (B, 2VYA). Residues
shown include three that differ between both species and a conserved flexible
phenylalanine.