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CONCISE ARTICLE

Discovery of potent, non-carbonyl inhibitors of fatty acid amide hydrolase

(FAAH)
Sumithra Gowlugari, Jeff DeFalco, Margaret T. Nguyen, Carl Kaub, Candace Chi, Matthew A. J. Duncton,
Daniel E. Emerling, Michael G. Kelly, John Kincaid and Fabien Vincent*
Received 4th June 2012, Accepted 19th July 2012
DOI: 10.1039/c2md20146a
Fatty acid amide hydrolase (FAAH) inhibition is a promising target for the treatment of pain, anxiety
and depression. The vast majority of FAAH inhibitors contain an electrophilic moiety and are known
to react covalently with the enzyme. Herein we present the discovery of potent inhibitors, such as
RN-450 29, which are based upon a novel tetrahydropyridopyridine scaffold lacking an obvious
electrophilic site, and which appear to inhibit FAAH in a reversible and non-covalent manner.

Introduction
The endocannabinoid system comprises two G-protein coupled
receptors, CB1 and CB2, their endogenous ligands, with anandamide (AEA 1) and 2-arachidonoyl glycerol (2-AG) being the
most thoroughly characterized, and the enzymes controlling the
levels of these signaling lipids through hydrolysis, fatty acid
amide hydrolase (FAAH) for AEA, and monoacylglycerol lipase
(MAGL) for 2-AG.1 Isolated and characterized in 1996, FAAH
has become the focus of intense research efforts, both within the
academic community and the pharmaceutical industry. The
latter interest has arisen from FAAH inhibition promising to
harness at least some of the known therapeutic benefits of
cannabinoids and CB1/CB2 agonism (e.g. analgesia, anti-emesis,
anxiolysis and anti-inflammation), while displaying none of the
equally well known psychotropic side-effects.2,3
FAAH possesses a highly nucleophilic serine, S241, which
together with K142 and S217, forms part of an unusual catalytic
triad within the active site of the enzyme. The vast majority of
known FAAH inhibitors take advantage of the powerful reactivity of this catalytic serine, and contain electrophilic moieties
(i.e., activated ketone, carbamate and urea) which form a covalent bond with the enzyme.4,5 For example, OL-135 2 benefits
from covalent and reversible hemi-ketal formation with S241,
while URB-597 3 reacts in an essentially irreversible manner with
the same residue.5
Early covalent inhibitors (e.g. 25) have been used to
convincingly document the potential therapeutic effects of
FAAH inhibition in animal models of human disease.510 More

Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate

Drive, South San Francisco, CA 94080, USA. E-mail:
fabien_vincent_us@yahoo.com
Electronic supplementary information (ESI) available: Assay protocols
for FAAH inhibition and biochemical evaluation. Synthetic details for
preparation of compounds 1530. See DOI: 10.1039/c2md20146a
Present address: Pfizer, Groton, CT, USA.

1258 | Med. Chem. Commun., 2012, 3, 12581263

recently, several compounds, notably PF-4457845, and

SSR-411298 have entered clinical development for pain and
depression.11 While covalent modification provides undisputable
advantages in terms of active site occupancy, clinical candidates
and drugs bearing reactive moieties are also over-represented in
cases of idiosyncratic toxicity, potentially leading to failure in
clinical trials or withdrawal from the marketplace.12 As robust
in vivo efficacy has been observed for reversible, competitive
inhibitors that benefit from covalent hemiketal formation (e.g.
OL-135 2), it may also be possible to develop non-covalent,
reversible FAAH inhibitors.13 While some compounds have been
reported to behave in a manner consistent with a reversible
mechanism of inhibition, to the best of our knowledge only a
limited number of these molecules, such as compound 6, have
been thoroughly characterized as reversible and non-covalent
inhibitors of FAAH.14 In this paper, we present the discovery of
tetrahydropyridopyridine-based inhibitors of FAAH and characterize the mechanism of action for a representative probe
compound, RN-450. The data indicate that tetrahydropyridopyridines such as RN-450 inhibit FAAH in a reversible and
non-covalent manner (Fig. 1).15

Results and discussion

High-throughput screen and preliminary structureactivity
studies
An initial screen of our compound library was conducted against
hFAAH using microsomal protein preparations and a fluorescent assay readout.16 Amongst the different hit series, tetrahydropyridopyrimidine derivatives such as compound 7 were
conspicuous, due to the lack of an obvious electrophilic moiety.
Consequently, this scaffold was prioritized for exploratory
structureactivity studies aimed at producing a tool compound
suitable for detailed mechanistic studies. As tetrahydropyridopyrimidines are a relatively well-known substructure within
This journal is The Royal Society of Chemistry 2012

Fig. 1 Anandamide and representative inhibitors of FAAH.

medicinal chemistry programmes,17 the corresponding tetrahydropyridopyridine scaffold (e.g. 15) was investigated first.
The synthesis of the tetrahydropyridopyridine core and of
representative compounds with substituents at the 1-position of
the tetrahydropyridopyridine ring are shown in Scheme 1. The
results from our biological testing indicated that the FAAH
enzyme was very sensitive to changes at this position (Table 1).
For example, changing from quinolin-3-yl in compound 15 to
quinolin-6-yl (compound 16) resulted in a greater than five-fold
reduction in potency. Additionally, removing the benzenoid ring
in compound 15, to give rise to pyridine analogues such as 18 and
19, resulted in a dramatic decrease in FAAH inhibition. A similar
dramatic decrease in potency was also observed when the
secondary nitrogen in compound 15 was alkylated with a methyl
group to give compound 20, thus showing the importance of an
anilino NH to the activity of compound 15.
We then proceeded to examine replacement of the benzyl
group in lead structure 15. Thus, sulfonamides 2226, and alkyl
derivatives 2730 were prepared by reaction of amine 21 with
sulfonyl chlorides and alkylation respectively (Scheme 2). Some
structureactivity relationships for sulfonamide and alkyl
analogues are shown in Tables 2 and 3 respectively. Amide and
urea derivatives of amine 21 were also prepared, but the resulting
analogues displayed poor activity (not shown).
As shown in Tables 13, discrepancies in potency between
human and rat FAAH were apparent for numerous compounds.
This phenomenon has been noted previously for a diverse range
of inhibitors.18,19 For example, discrepancies amongst literature
urea-based inhibitors such as PF-750 4 were demonstrated to

originate from differences in up to six amino acid residues

between the two species.18 Accordingly, on the basis of its
potency against both rat and human FAAH, we chose
compound 29 (RN-450) as a tool compound with which to
investigate the mechanism of action of inhibitors based on a
tetrahydropyridopyridine scaffold.
Biochemical investigations with RN-450
A variety of biochemical approaches were employed to characterize the interaction of RN-450 29 with hFAAH.20 First, its
mechanism of inhibition was investigated using enzyme kinetics
(Fig. 2A). The activity pattern observed in the presence of
increasing concentrations of inhibitor (increased KM, stable
Vmax) is characteristic of a reversible and competitive inhibitor
(Ki 14.3 nM, 95% CI: 9.818.8 nM). Predictably, URB-597 3
displayed a reduced Vmax under similar conditions, an apparent
non-competitive pattern also observed with irreversible enzyme
inhibitors.20 Next, the effect of pre-incubation of compounds
with hFAAH was evaluated (Fig. 2B). As expected, the IC50
value of carbamate URB-597 3 shifted significantly (365 fold)
when a 3 h pre-incubation with hFAAH was introduced. Such a
lengthy pre-incubation, however, had no effect on the potency of
RN-450 29 (1.7 fold shift), a result consistent with it being a
reversible FAAH inhibitor. Dilution was the third biochemical
approach employed to study the mechanism of RN-450
(Fig. 2C).20 Following an incubation of hFAAH with IC90
concentrations of select inhibitors, the reaction mixture was
diluted 300 fold and the recovered enzymatic activity was then

Scheme 1 Synthesis of tetrahydropyridopyridines. Reagents and conditions: (a) EtI, CH2Cl2, rt, 86%. (b) NaCN, K2CO3, 50  C. (c) DMF.DMA,
pyrrolidine, 130  C. (d) 48% aq. HBr, EtOH, reflux, 40%. (e) BnBr, K2CO3, EtOH, H2O, reflux, then (f) NaBH4, 0  C, 32% over 2 steps. (g) POCl3,
reflux, 80%, (h) R1NH2, Pd2(dba)3, NaOtBu, tBuOH, 120  C. (i) MeI, CH2Cl2.

This journal is The Royal Society of Chemistry 2012

Med. Chem. Commun., 2012, 3, 12581263 | 1259

Table 1 Tetrahydropyridopyridine analogues as FAAH inhibitorsa

R2

hFAAH
IC50 (nM)a

rFAAH
IC50 (nM)a

15

76  5.6

446  72

16

479

1486

17

>10 000

ndb

18

2124

>10 000

Compound

19

20

R1

CH3

>10 000

>10 000

Table 2 SAR for tetrahydropyridopyridine sulfonamides

hFAAH
IC50 (nM)a

rFAAH
IC50 (nM)a

22

190

939

23

93  17

638

24

45

614

25

45

969

26

21  6

249

Compound

R1

nd

nd

a
Values are presented means  sem of independent experiments, except
when only an n 1 was obtained; URB-597 displayed an average IC50 of
3 nM under the conditions used. b nd: not determined.

measured. Enzyme treated with carbamate URB-597 3 and urea

PF-750 4 did not show any measurable activity recovery, thus
confirming the irreversibility of their inhibition. On the other
hand, enzyme treated with oleyl trifluoromethyl ketone (OlCF3),
a covalent yet reversible inhibitor of hFAAH, recovered a
significant amount of activity (35%). Finally, treatment with

a
Values are presented means  sem of independent experiments, except
when only an n 1 was obtained; URB-597 displayed an average IC50 of
3 nM under the conditions used.

RN-450 29 led to 65% enzymatic activity recovery, demonstrating the reversible nature of its inhibition of hFAAH.
As mass spectrometry has been used to document the covalent
adduction of PF-750 4 to rFAAH,5 we used this same approach
to study RN-450 29 (Fig. 3). As expected, enzyme incubated with
PF-750 4 displayed a modified 213243 peptide, with a molecular
mass consistent with the carbamoylation of catalytic serine S241.
On the other hand, no such modification was detected using

Scheme 2 Preparation of sulfonamide and alkyl derivatives. Reagents and conditions: (a) H2, Pd/C, EtOH, rt, 18 h, (b) R0 SO2Cl, CH2Cl2, DIPEA, (c)
CsCO3, NMP, RX.

1260 | Med. Chem. Commun., 2012, 3, 12581263

This journal is The Royal Society of Chemistry 2012

Fig. 2 Modification of rFAAH with PF-750 4 and RN-450 29. Biochemical studies on the mechanism of action of RN-450 29: (A) Enzyme kinetic
analysis of hFAAH inhibition by URB-597 3 (upper panel) and RN-450 29 (lower panel); (B) Time dependency of hFAAH inhibition by URB-597 3 and
RN-450 29; (C) Dilution experiment with hFAAH for URB-597 3, PF-750 4, RN-450 29 and O1CF3 recovered activity after dilution shown.

Fig. 3 Modification of rFAAH with PF-750 4 and RN-450 29. Mass spectrometry analysis of peptide 213243 from rFAAH protein treated with (A)
RN-450 29, (B) DMSO (control) and (C) PF-750 4.

This journal is The Royal Society of Chemistry 2012

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Table 3 SAR for alkylated tetrahydropyridopyridines

hFAAH
IC50 (nM)a

rFAAH
IC50 (nM)a

15

76  5.6

446  72

27

28  6

449  98

28

2.6  0.8

606  64

29

13  3

25  11

30

>10 000

ndb

Compound

a
Values are presented means  sem of independent experiments, except
when only an n 1 was obtained; URB-597 displayed an average IC50 of
3 nM under the conditions used. b nd: not determined.

RN-450 29. Similar results were observed following the use of an

alternative method, 2D electrophoresis, to detect covalent bond
formation (data not shown).

Conclusions
In summary, a novel class of FAAH inhibitors based on a
tetrahydropyridopyridine scaffold lacking reactive electrophilic
moieties was identified. Compound 29 (RN-450) displayed low
nM potency against hFAAH, and after extensive biochemical
characterization was found to be a reversible, non covalent,
inhibitor of the FAAH enzyme. Compounds such as 29, by virtue
of their structural novelty and unique mechanism of inhibition,
represent attractive tools with which to study FAAH and the
endocannabinoid pathway.

Notes and references

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This journal is The Royal Society of Chemistry 2012

S. Gowlugari, D. J. R. OMahony, M. T. Nguyen, D. E. Emerling,

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