Enol carbamates as inhibitors of fatty acid amide hydrolase (FAAH) endowed with high selectivity for FAAH over the other targets of the endocannabinoid system

Article abstract of DOI:10.1002/cmdc.200900472

FAAH inhibitors: This study identifies a new class of enol carbamates as potent reversibleinhibitors of fatty acid amide hydrolase (FAAH), endowed with high selectivity towards this target over other components of endocannabinoid system.

Full text of DOI:10.1002/cmdc.200900472

DOI: 10.1002/cmdc.200900472
Enol Carbamates as Inhibitors of Fatty Acid Amide Hydrolase (FAAH)
Endowed with High Selectivity for FAAH over the Other Targets of the
Endocannabinoid System
Sonia Gattinoni,[a] Chiara De Simone,[b, c] Sabrina Dallavalle,[a] Filomena Fezza,[b, c] Raffaella Nannei,[a]
Daniele Amadio,[b, c] Patrizia Minetti,[d] Gianandrea Quattrociocchi,[d] Antonio Caprioli,[d] Franco Borsini,[d]
Walter Cabri,[d] Sergio Penco,[d] Lucio Merlini,*[a] and Mauro Maccarrone*[c]
Fatty acid amide hydrolase (FAAH) is an enzyme bound to in-
tracellular membranes, mainly of the endoplasmic reticulum,[1]
which regulates the cellular level and activity of fatty acid
amides.[2] These substances belong to the endogenous canna-
binoids (“endocannabinoids”), a family of lipid signals that
exert several biological activities, including anticancer, anti-is-
chemic, anti-inflammatory, antidepressant, analgesic, anxiolytic,
anorectic and bone-stimulant actions, to name just a few.[3] En-
docannabinoids include the neurotransmitter N-arachidonoyl-
ethanolamine (anandamide, AEA),[4] the anti-inflammatory
factor N-palmitoylethanolamine,[5] the sleep-inducing com-
pound oleoylamide,[6] and the satiating signal N-oleoylethanol-
amine.[7] Some of the effects of endocannabinoids are mediat-
ed via the type-1 (CB1R) or type-2 (CB2R) cannabinoid recep-
tors, while others may involve additional targets like the transi-
ent receptor potential vanilloid-1 (TRPV1)[3] and peroxisome
proliferator-activated receptors (PPARs).[8]
therapeutic approach for the treatment of cancer, eating disor-
ders, inflammation, pain, anxiety, depression, sleep disturban-
ces, and other central nervous system (CNS) disorders.[12,13]
Of course, it is also important that the beneficial effects of
increasing the endogenous level of AEA through FAAH inhibi-
tion are not offset by unwanted modulation of other biological
targets that recognize this lipid, such as the cannabinoid and
vanilloid receptors, or the proteins that synthesize N-acylphos-
phatidylethanolamide-phospholipase D (NAPE-PLD) and alleg-
edly transport it (anandamide membrane transporter, AMT).
These proteins form the endocannabinoid system (ECS), which
includes other well-characterized elements, such as the en-
zymes responsible for synthesis (diacylglycerol lipase, DAGL) or
degradation (monoacylglycerol lipase, MAGL) of 2-arachido-
noylglycerol.[14] It is also noteworthy that AEA can have contra-
ry biological effects when acting at different receptors (e.g.,
CBRs vs TRPV1), and a different activity compared to that of 2-
arachidonoylglycerol.[15] Therefore, it is not surprising that in-
teraction of a drug, targeted towards a particular ECS enzyme,
with other ECS components could lead to reduced therapeutic
efficacy, for example in the treatment of cancer,[14] or of neuro-
degenerative/inflammatory diseases.[15] For instance, the thera-
peutic advantage of a popular AMT inhibitor AM404 has been
reconsidered when it was shown to also activate TRPV1 recep-
tors.[16] Similarly, the TRPV1 agonist olvanil has been shown to
inhibit AMT,[17] and the putative AMT blocker LY2183240 is
known to also inhibit FAAH and MAGL.[18] Overall, the promis-
cuity of such drugs, intended to target a single ECS compo-
nent but able to interact with others, has raised concerns
about their true efficacy as therapeutics. On this basis, a careful
evaluation of the selectivity of inhibitors for FAAH over other
targets of the ECS seems a prerequisite for effective drug
development.
Among different metabolic pathways that can modulate the
endogenous levels of endocannabinoids, and hence their bio-
logical actions,[9] FAAH has emerged as a key player. In fact,
FAAH inactivation by either genetic ablation of the faah
gene[10] or chemical inhibition[11] increases the level of fatty
acid amides, and consequently their central and peripheral ac-
tivities. However, unlike direct agonists of CB1R, FAAH inhibi-
tors do not cause classical effects triggered by this receptor
(e.g., catalepsy, hypothermia, and hyperphagia), nor are they
accompanied by undesirable side effects typically due to CB1R
activation, like impairment of cognition and motor control.
Therefore, chemical inhibition of FAAH is considered a useful
[a] Dr. S. Gattinoni,+ Dr. S. Dallavalle, Dr. R. Nannei, Prof. L. Merlini
DISMA, Universitꢀ degli Studi di Milano, Via Celoria 2, 20133 Milano (Italy)
Fax: (+39)02-50316801
E-mail: lucio.merlini@unimi.it
[b] Dr. C. De Simone,+ Dr. F. Fezza, Dr. D. Amadio
FAAH belongs to an unusual class of serine hydrolases that
utilizes a serine–serine–lysine catalytic triad,[2] and therefore
potent electrophiles, such as fluorophosphonates and fluoro-
sulfonates, are important tools for evaluating the biochemistry
of FAAH.[19] Starting from activated ketones designed as puta-
tive active-site traps, and including chloroketones, a-keto-
amides, a-ketoesters, trifluoromethylketones and diazoke-
tones,[12] past development of FAAH inhibitors has led to the
recent discovery of several classes of a-ketoheterocycles as
potent and highly selective blockers of FAAH, compared to
other serine hydrolases.[13,20] However, the systematic study of
specificity towards FAAH over all the other well-defined ECS el-
ements has been investigated only rarely (e.g., in the case of
Dipartimento di Medicina Sperimentale e Scienze Biochimiche
Universitꢀ degli Studi di Roma “Tor Vergata”, 00133 Rome (Italy)
[c] Dr. C. De Simone,+ Dr. F. Fezza, Dr. D. Amadio, Prof. Dr. M. Maccarrone
Dipartimento di Scienze Biomediche, Universitꢀ degli Studi di Teramo
Piazza A. Moro 45, 64100 Teramo, Italy, & Centro Europeo di Ricerca sul
Cervello (CERC)/IRCCS Fondazione Santa Lucia, 00143 Rome (Italy)
Fax: (+39)0861-266877
E-mail: mmaccarrone@unite.it
[d] Dr. P. Minetti, Dr. G. Quattrociocchi, Dr. A. Caprioli, Dr. F. Borsini,
Dr. W. Cabri, Dr. S. Penco
Sigma-Tau R&D, 00040 Pomezia (Italy)
[+] These authors contributed equally to the study.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900472.
ChemMedChem 2010, 5, 357 – 360
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
357

MED
OL-135 only with respect to CB1R, CB2R and TRPV1).[21] Anoth-
er class of FAAH inhibitors currently under investigation are
the carbamate-based compounds.[12] Modification of the initial
hit compounds and molecular modeling based on the X-ray
co-crystal structure of methoxy-arachidonoyl-fluorophospho-
nate (MAFP) bound to FAAH[22] led Kathuria and co-workers to
develop URB597, an O-aryl carbamate endowed with potent
FAAH inhibition, with analgesic
and anti-inflammatory activity.[23]
Mass spectral studies indicated
that the structure–activity relation-
Scheme 1. Synthesis of compounds 1. Reagents and conditions: a) NaH,
DMSO, RT, 1 h, 18–57%; b) ClCON(R2 R1), DMSO, RT, 3 h, 18–57%; c) R4B(OH)2,
NaHCO3, Pd(tetrakis), MeOH, reflux, 7 h, Ar, or MW, 1408C, 30 min (15–66%).
ships (SAR) within the carbamate-
based series might be driven by
the ability of the phenol group to
leave the molecule.[24]
Table 1. Inhibition of FAAH by compounds 1.
Other carbamates have been reported by Sanofi–Aventi-
s[25–29]and Bristol–Myers Squibb.[30,31] More recently, aryl ureas
were found to be remarkably selective FAAH inhibitors.[32–34] In
this context, it should be stressed that both carbamates and
aryl ureas covalently bind to FAAH, and generally act as irrever-
sible inhibitors. However, it has been suggested that reversible
inhibitors might offer advantages over irreversible blockers as
lead compounds for drug design.[35] However, only a few rever-
sible FAAH inhibitors have been described in the literature, like
the a-ketoheterocycle OL-135,[36] and the covalent but slowly
reversible piperazine urea JNJ-1661010 developed by Johnson
& Johnson.[33]
Compd
R1
R2
R3
R4
IC50 [nm]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
CH3
(CH2)5
(CH2)2O(CH2)2
CH3
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
3930Æ500
9Æ1
292Æ30
4600Æ920
40Æ5
CH3
Ph
(CH2)5
(CH2)5
(CH2)5
2-thienyl
3’-NH2COPh
2’-NH2COPh
116Æ12
167Æ17
1743Æ180
133Æ14
13Æ3
CH3
CH3
CH3
Ph
H
H
H
H
(CH2)5
(CH2)5
Herein, we report the synthesis of a new series of enol car-
bamates (1), the most potent of which appear to reversibly in-
hibit FAAH. These novel com-
1j
1k
3’-CNPh
Ph
340Æ20
pounds were designed based on
the hypothesis that FAAH would
wished to ascertain whether changes in the position of the
substituents could affect the mechanism-of-action or the selec-
tivity of the compound. The position of the phenyl group with
respect to the enol carbamate group differs in compounds 1b
(para, R4) and 1i (meta, R3), while the substitution of the carba-
mate nitrogen differs in compounds 1h (dimethyl) and 1i
(cyclic amine structure). Initial SAR showed that dimethyl sub-
stitution of the carbamate nitrogen (compounds 1a and 1h)
leads to weaker activity than analogues with a bulkier (e.g., pi-
peridine 1b,e–g, i, j) but not aromatic (1k) cyclic amines. In
addition, the presence of substituents on the distal phenyl ring
can modulate the activity (1j vs 1i and 1 f,g vs 1b).
catalyze the hydrolysis of the car-
bamate group to release an enol,
which would immediately convert
to the tautomeric keto form, thus shifting the equilibrium to-
wards the products of hydrolysis. To the best of our knowl-
edge, there are no other examples of use of this class of com-
pounds in medicinal chemistry.
Compounds 1 were prepared by reacting the appropriate
ketone with a strong base, such as NaH, usually in dimethyl-
sulfoxide, followed by treatment with the corresponding carba-
moyl chloride (route A, Scheme 1),[38] or by reaction of a bro-
mophenylvinylcarbamate with an arylboronic acid via
Suzuki–Miyaura reaction (route B, Scheme 1).
a
Compounds 1b, 1h, 1i, 1j were found to inhibit FAAH re-
versibly. At least 80% of enzyme activity was recovered after
dialysis of the enzyme–inhibitor complexes for 18 h (see Sup-
porting Information figure S1). In contrast, dialysis of FAAH–
URB597 complexes under similar conditions has shown that
this agent is an irreversible inhibitor of the enzyme,[39] due to
carbamoylation of Ser241 in the active site.[40] Furthermore,
nonlinear regression analysis of the Michaelis–Menten curves
demonstrated that all of the selected compounds were non-
competitive inhibitors of FAAH with respect to the natural sub-
strate AEA (see Supporting Information figure S2A). In fact,
Compounds 1 were evaluated for their ability to inhibit
FAAH. The IC50 values were calculated as detailed in the Sup-
porting Information, and are summarized in Table 1. In addi-
tion, we investigated the effect of the selected FAAH inhibitors
towards other components of the ECS; the assays were carried
out as previously described,[37] (for details see Supporting Infor-
mation).
Two compounds (1b, 1j) were found to be potent inhibitors
of FAAH, with IC50 values in the low nanomolar range (Table 1).
Therefore, these inhibitors were selected for further evaluation
along with the less potent FAAH inhibitors 1h and 1i, selected
because of their structural similarities with compound 1b; we
compounds 1 markedly reduced the maximal velocity (Vmax
)
without affecting the Michaelis–Menten constant (Km), a typical
effect of canonical noncompetitive inhibitors.[40] The same phe-
358
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 357 – 360

nomenon can also be visualized by Lineweaver–Burk analysis
of double-reciprocal plots of the kinetic data (see Supporting
Information figure S2B).
The apparent Ki values of compounds 1b, 1h, 1i, 1j to-
wards FAAH are shown in Table 2. In a second set of experi-
ments, we analyzed the interaction of compounds 1b, 1h, 1i,
Noncompetitive
inhibition
seems in keeping with the differ-
ences in structures between
compounds 1 and AEA. In fact, it
should be recalled that by defini-
tion, a noncompetitive inhibitor
binds reversibly and equally well
to the enzyme alone and to the
enzyme–substrate complex, and
therefore it does not affect (nor
is affected by) the binding of the
substrate at the active site.[41] On
the other hand, compounds 1b,
1h, 1i, 1j might lead to carbon-
ylation of Ser241, that is the al-
ternative mode of FAAH inhibi-
tion by carbamate blockers,[40]
Table 2. Apparent Ki values towards FAAH, and inhibition (IC50) of the activity of the ECS elements by selected
compounds 1. Selectivity index values are reported in square brackets.
Compd
Ki [nm]
IC50 [nm]
CB1R
CB2R
TRPV1
AMT
NAPE-PLD
MAGL
DAGL
1b
1h
1i
54Æ4
2259Æ356
274Æ93
46Æ6
>10000
[1111]
>10000
[6]
>10000
[75]
>10000
[769]
>10000
[1111]
>10000
[6]
10000
[75]
>10000
[1111]
>10000
[6]
>10000
[75]
>10000
[769]
100
[111]
> 10000
[6]
10000
[75]
100
>10000
[1111]
>10000
[6]
>10000
[75]
>10000
[769]
>10000
[1111]
>10000
[6]
>10000
[75]
>10000
[769]
>10000
[1111]
>10000
[6]
>10000
[75]
>10000
[769]
1j
>1000
[769]
[77]
Details of the methods used to perform dialysis, to determine the type of inhibition, and to calculate the Ki
values and selectivity index values are reported in the Supporting Information.
thus forming hydrolysable (and hence reversible) intermedi-
ates. The actual mechanism by which our compounds operate
can be ascertained using mass spectrometry as described by
Cravatt et al.,[40] an experiment that is planned for the future.
Another unanswered question is the binding mode of our
compounds to FAAH. Their structural similarity with URB597
would suggest the occupation of the cytosolic access (CA)
channel by the biphenylyl moiety, with the N-terminal moiety
positioned in the acyl-chain-binding (ACB) channel of the
enzyme.[42] In the absence of a crystal structure, further SAR
data and molecular modeling studies are needed to support
this binding mode or suggest alternative. In addition, it has re-
cently been demonstrated that compound JNJ-1661010 inhib-
its FAAH by carbamoylation followed by hydrolysis, thus acting
as a covalent but slowly reversible blocker.[33] Therefore, the
possibility that compounds 1b, 1h, 1i, 1j also inhibit FAAH
through reversible carbamoylation can not be ruled out. On a
general note, it seems noteworthy that the ability of carba-
mate compounds to inhibit FAAH in a noncompetitive manner
is not unprecedented,[43] and that clinically used carbamate
drugs (i.e., rivastigmine) are known to act through a covalent
carbamoylation of acetylcholinesterase,[44] that leads to a rever-
sible and noncompetitive inhibition.[45,46] In any case, com-
pounds 1b, 1h, 1i, 1j appear to be the first reversible enol
carbamate inhibitors of FAAH described to date.
1j with the other targets of the ECS, in order to calculate selec-
tivity index values compared with FAAH (see Table 2). From
these results it can be concluded that compounds 1b and 1j,
in addition to yielding potent FAAH inhibition, also show a
high degree of selectivity towards FAAH over any other ECS
target, with IC50 values ~1000-fold higher than those shown
towards FAAH. Incidentally, it should be stressed that the abili-
ty of compounds 1b and 1j to inhibit the AEA membrane
transporter AMT (Table 2) could simply reflect the contribution
of FAAH to the transport process. In fact, hydrolysis by FAAH
drives AEA uptake by creating and maintaining a concentration
gradient across the plasma membrane.[2,13,18]
Overall, compound 1b showed the best combination of in-
hibitory potency towards FAAH and selectivity for the target
over the other ECS components. Therefore, further experi-
ments were performed to confirm the stability of 1b at 378C
at pH 1.5 and pH 8.4, mimicking the stomach and intestine en-
vironments, respectively (Supporting Information table S2).[48]
Moreover, compound 1b was not at all hydrolyzed by esteras-
es from mouse or rat plasma after 1 h of incubation at 378C,
and was only slightly (~20%) degraded by esterases from
human plasma (Supporting Information table 2). In addition,
compound 1b was subjected to in vitro pharmacology experi-
ments in order to ascertain its possible interference with differ-
ent off-targets (i.e., 45 receptors, 5 ion channels and 2 neuro-
transmitter transporters). The data, shown in the Supporting
Information (table S3), demonstrate that compound 1b had no
effect on any of the off-targets tested, yielding IC50 values
>10000 nm in all cases. Finally, oral administration of com-
pound 1b to mice (at 30 mgkgÀ1) produced anxiolytic-like
effects in the elevated plus maze model, where an increase in
time spent in open arms was observed, in a same manner as
the reference compound, diazepam (Supporting Information,
table S2). These in vivo data support the pharmacological effi-
cacy of enol carbamates as promising anxiolytic therapeu-
tics.[49,50]
To further support this conclusion, we performed time-de-
pendent inhibition experiments. The most potent compounds
(1b and 1j) were preincubated with FAAH for different time
periods and the substrate turnover was measured.[47] The re-
sults show that inhibition by either compound was not time-
dependent (Supporting Information table S1), confirming that
1b and 1j are indeed reversible inhibitors of FAAH. URB597
was also tested under the same experimental conditions and
shown to inhibit FAAH in a time-dependent manner, which is
in accordance with a previous report.[47] This result corrobo-
rates the hypothesis that URB597 acts as a covalent and irre-
versible inhibitor of the enzyme.[39,40]
ChemMedChem 2010, 5, 357 – 360
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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In conclusion, a new class of enol carbamates have been
identified as potent reversible inhibitors of FAAH endowed
with high selectivity towards FAAH over the other targets of
the endocannabinoid system. Notably, our systematic investi-
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Experimental Section
Representative and general experimental procedures, compound
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and details of the biochemical assays and the associated data are
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Keywords: endocannabinoids · enol carbamates · FAAH ·
hydrolases · inhibitors
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