A new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2010.06.050

A series of oxime carbamates have been identified as potent inhibitors of fatty acid amide hydrolase (FAAH), an important regulatory enzyme of the endocannabinoid signaling system. Kinetic analysis indicates that they behave as non-competitive, reversible

Full text of DOI:10.1016/j.bmcl.2010.06.050

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4406–4411
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new group of oxime carbamates as reversible inhibitors of fatty acid
amide hydrolase
Sonia Gattinoni a, , Chiara De Simone b,c, , Sabrina Dallavalle a, Filomena Fezza b,c, Raffaella Nannei a,
Natalia Battista c, Patrizia Minetti d, Gianandrea Quattrociocchi d, Antonio Caprioli d, Franco Borsini d,
c,
Walter Cabri d, Sergio Penco d, Lucio Merlini a, , Mauro Maccarrone
*
*
a DISMA, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy
b Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università degli Studi di Roma ‘Tor Vergata’, 00133 Rome, Italy
c Dipartimento di Scienze Biomediche, Università 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
d Sigma-Tau R&D, 00040 Pomezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of oxime carbamates have been identified as potent inhibitors of fatty acid amide hydrolase
(FAAH), an important regulatory enzyme of the endocannabinoid signaling system. Kinetic analysis indi-
cates that they behave as non-competitive, reversible inhibitors, and show remarkable selectivity for
FAAH over the other components of the endocannabinoid system.
Received 7 May 2010
Revised 9 June 2010
Accepted 9 June 2010
Available online 15 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Fatty acid amide hydrolase (FAAH)
Enzyme inhibitors
Oxime carbamates
Endocannabinoids
Synthesis
Fatty acid amide hydrolase (FAAH) is an enzyme bound to intra-
cellular membranes, mainly of the endoplasmic reticulum,1 that
catalyzes the degradation of N-arachidonoylethanolamine (ananda-
mide,2 AEA), the intestinal satiety factor N-oleoylethanolamine
(OEA)3,4 the peripheral analgesic and anti-inflammatory factor
N-palmitoylethanolamine (PEA)5 and the sleep-inducing substance
oleoylamide (OA).6 AEA, other congeners termed ‘endocannabi-
noids’ and the ‘endocannabinoid-like’ compounds OEA, PEA and
OA, are a growing family of lipid signals that exert several biological
activities, including anti-cancer, anti-ischemic, anti-inflammatory,
anti-depressant, analgesic, anxiolytic, anorectic and bone-stimulant
actions (to name just a few).7 Some of the effects of endocannabi-
noids are mediated via the type-1 (CB1R) or type-2 (CB2R) cannab-
inoid receptors, while others may involve additional targets like the
transient receptor potential vanilloid-1 (TRPV1)7 and peroxisome
proliferator-activated receptors.6 Among different metabolic path-
ways that can modulate the endogenous tone of endocannabinoids,
and hence their biological actions,8 FAAH has emerged as a key
player. In fact, FAAH inactivation by either genetic ablation of the
faah gene9 or chemical inhibition10 increases the level of fatty acid
amides, and consequently their central and peripheral activities.
FAAH belongs to an unusual class of serine hydrolases, that uti-
lizes a serine–serine–lysine catalytic triad, firmly identified in 2002
when crystal structure of FAAH was reported.11 The residues of the
catalytic triad (Ser241, Ser217 and Lys142, where Ser241 is the
nucleophilic residue) interact through a network of hydrogen
bonds that facilitates proton exchange and the subsequent hydro-
lysis of the amide bond.
In the last decade these findings have boosted the search for po-
tent, selective FAAH inhibitors, as potential therapeutics for the
treatment of pain, inflammation, anxiety, depression, and other
central nervous system disorders.12,13 Important classes of FAAH
inhibitors that have been recently disclosed are
a-ketoheterocy-
cles,14,15 ureas,16 (thio)hydantoins,17 O-alkyl18 or O-aryl19 carba-
mates, and carbamoyl tetrazoles.20
A study based on the X-ray structure of FAAH bound to methoxy-
arachidonoyl-fluorophosphonate (MAFP)11 and molecularmodeling
of carbamate-based inhibitors, led Kathuria et al. to URB597 (1), a
phenol carbamate endowed with potent FAAH inhibition and
analgesic and anti-inflammatory activity.21 Mass spectral studies
* Corresponding authors. Fax: +39 02 50316801 (L.M.), fax: +39 0861 266877
(M.M.).
E-mail addresses: lucio.merlini@unimi.it (L. Merlini), mmaccarrone@unite.it
(M. Maccarrone).
These authors contributed equally to the study.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.050

S. Gattinoni et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4406–4411
4407
Treatment of the oximes with a suitable carbamoyl chloride
yielded compounds 4a, 10g–h, 10k (70–94% yield) ( Schemes 1
and 2), whereas treatment with an appropriate isocyanate gave
compounds 4b–f, 5, 6, 10a–f, 10i–j, 13a–b (50–96% yield)
(Schemes 1–3, Chart 2) (see Supplementary data for details).
Compound 6 was synthesized by addition of phenyl isocyanate
to 4-phenylcyclohexanone oxime (96%) (Chart 2).
O
R3
N R2
R1
N O
Chart 1.
In the first set of experiments the ability of all compounds to in-
hibit FAAH was tested (see Supplementary data for details). The
IC50 values of FAAH inhibition are reported in Table 1.
indicated that the SAR in this series might be driven by the ability of
the phenol group to leave the molecule.22
Firstly, a small set of biphenyl-4-yl derivatives (4a–f) was
examined. The results showed that the N,N0-dimethyl substituted
derivative (4a) did not exhibit any significant activity, differently
from what happens for the oximes reported by Sit,30 whereas the
introduction of a phenyl or a naphthyl group on the carbamate
nitrogen gave compounds with submicromolar IC50’s (4b and 4c).
Substitution of the para-biphenyl system with different rings
(4d–f, 5 and 6) was detrimental to the activity, leading to inactive
compounds, with the only exception of 4d.
CONH2
H
N
O
O
URB597
Secondly, the introduction of a biphenyl-3-yl system with a
meta distal aromatic ring in place of the para phenyl ring was ex-
plored (compds 10). The results indicated that the meta-substi-
tuted compounds were significantly more active than the
corresponding para isomers (10a vs 4b, 10b vs 4c), compound
10b being ꢀ40-fold more potent than 4c. Compound 13a, with a
polar group in the meta-position of the distal phenyl ring, showed
inhibitory activity comparable to that of the corresponding unsub-
stituted derivative 10a. Compounds 10c and 10d, possessing a
p-fluoro and p-methoxy substituted N-phenyl ring, maintained a
good inhibitory potency, while the introduction of bulky groups
on the carbamate nitrogen (10h, 10e) was again detrimental to
activity. Substitution with a piperidine ring (10g) or with a more
extended fragment (10f) restored a certain degree of inhibitory ac-
tion. The most potent compound of the series was derivative 10i
(IC50 8 nM), with a thiophene ring in place of the distal phenyl ring.
In keeping with the other results on biphenyl-4-yl derivatives, the
corresponding para isomer (4d) was found to be much less effec-
tive on FAAH activity. This preliminary SAR study clearly indicates
the preference for a ‘bent’ (compds 10 vs 4) group of two aromatic
or heteroaromatic rings, whereas a lipophilic group (piperidino,
but also phenyl, or a hydrocarbon chain) attached to the nitrogen
of the carbamate is important for activity. As for 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. This
hypothesis would indicate an interaction with the enzyme site
Other carbamates of alcohols were reported by Sanofi-Aven-
tis23–27 and carbamates of oximes in patents28,29 and in a most re-
cent paper by Bristol Myers Squibb.30 The latter publication
appeared during the preparation of this Letter, and described FAAH
inhibition by a series of oxime carbamates that differ from those
reported here. In fact, in this study we report the synthesis and
the FAAH inhibiting activity of oxime carbamates characterized
by an N-aryl or alkyl carbamic acid O-iminobiphenyl or O-imino-
heteroaryl ester moiety (Chart 1).
These compounds were designed on the hypothesis that the
carbamate of an oxime, a good leaving group, would be rapidly
hydrolysed by FAAH, and that the biphenyl group would be easily
accommodated in the enzyme active site, by analogy with the URB
class of compounds.
All the oximes (3, 9 and 12) were synthesized by reaction of the
corresponding ketones with hydroxylamine in ethanol. Oxime 9
was prepared by a Suzuki coupling between bromobenzene or
2-bromothiophene and 3-acetylbenzeneboronic acid, followed by
reaction with hydroxylamine (Scheme 2), whereas oxime 12 was
obtained by a Suzuki coupling between 3-bromobenzonitrile and
3-acetylbenzeneboronic acid, followed by hydrolysis with sodium
perborate and condensation with hydroxylamine (Scheme 3).
The configuration of the ketoximes was assigned as E (which
anyway is the preferred one in the synthesis30) on the basis of
the chemical shift of the methyl group (>2.28 ppm)31 in the 1H
NMR, and in 13C NMR (12.6 ppm), which reflects its syn relation-
ship to the OH function.32 Similar findings have been reported by
Buchanan33 and Fraser.34
R2
O
N
R3
OH
N
N
O
O
a
b
R1
R1
R1
4
3
2
4a R1 = Ph
4b R1 = Ph
R2 = Me; R3 = Me
R2 = H; R3 = Ph
R2 = H; R3 = 1-naphthyl
R2 = H; R3 = Ph
R2 = H; R3 = 1-naphthyl
R1 = Ph
4c
4d R1 = 2-thiophenyl
4e R1 = cyclohexyl
4f R1 = 2,5-dimethyl-pyrrol-1-yl R2 = H; R3 = 1-naphthyl
Scheme 1. Reagents and conditions: (a) NH2OHꢁHCl, EtOH, 2 N HCl, reflux; (b) for 4b–f: aryl isocyanate, toluene, reflux (50–96%); for 4a: N,N0-dimethylcarbamoyl chloride,
reflux, 30 min, 70%.

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S. Gattinoni et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4406–4411
R2
N
OH
O
R3
O
O
N
N
R1
R1
O
R1
(HO)2B
b
a
c
8
7
9
10
R3 = Ph
10a R1 = Ph;
10b R1 = Ph;
10c R1 = Ph;
10d R1 = Ph;
10e R1 = Ph;
10f R1 = Ph;
10g R1 = Ph;
10h R1 = Ph;
R2 = H;
R2 = H;
R2 = H;
R2 = H;
R2 = H;
R2 = H;
R3 = 1-naphthyl
R3 = 4-fluorophenyl
R3 = 4-methoxyphenyl
R3
= 5-benzo[b]thiophenyl
R3 = -(CH2)5-Ph
R2, R3 = -(CH2)5-
R2 = Ph;
R3 = -Ph
R3 = Ph
R3 = C12H25
10i R1 = 2-thiophenyl; R2 = H;
10j R1 = 2-thiophenyl; R2 = H;
10k R1 = 2-thiophenyl;
R2, R3 = -(CH2)5
-
Scheme 2. Reagents and conditions: (a) Bromobenzene or 2-bromothiophene, Pd(PPh3)4, 1 M Na2CO3, dioxane, N2, reflux, 3–8 h; (b) NH2OHꢁHCl, 2 N HCl, EtOH, reflux;
(c) aryl isocyanate, toluene, reflux (50–90%) or carbamoylchloride, reflux, 30 min (70–94%).
H
OH
O
N
N
N
H
d
a, b, c
H2N
O
N
R
O
O
NC
Br
11
13
12
13a R = H
13b R = PhNHCO
Scheme 3. Reagents and conditions: (a) ArB(OH)2, Pd(PPh3)4, 1 M Na2CO3, dioxane, N2, reflux (90%); (b) NaBO3ꢁ4H2O, H2O, dioxane, reflux (51%); (c) NH2OHꢁHCl, EtOH, N2,
HCl, reflux (64%); (d) phenyl isocyanate, toluene, reflux.
H
N
O
N
O
O
N
O
N
H
S
S
5
6
Chart 2.
analogous to that supposed by Sit et al.30 for their compounds with
the same key functional group.
confirming that URB597 is a covalent and irreversible inhibitor of
enzyme activity.35,38
We decided to further characterize FAAH inhibition by the most
potent compound, 10i. First of all, this substance was found to be
chemically stable at pH 1.5 (yielding a recovery of 69 1% after
3 h at 37 °C) and pH 8.4 (recovery of 98 1% after 6 h at 37 °C), that
mimicked stomach and intestine environment, respectively. Then,
we found that compound 10i inhibited FAAH reversibly, because
ꢀ80% of enzyme activity was recovered after 18 h dialysis of en-
Next, we ascertained by standard substrate-velocity plots that
compound 10i was a non-competitive inhibitor of FAAH with re-
spect to the natural substrate AEA (Fig. 2a). In fact, compound
10i markedly reduced the maximal velocity (Vmax) without affect-
ing the Michaelis–Menten constant (Km) of the reaction, a typical
effect of canonical non-competitive inhibitors.37 The decrease in
Vmax without effect on Km could be visualized also by Linewe-
aver–Burk analysis of double-reciprocal plots of the kinetic data
(Fig. 2b). Interestingly, also Sit et al. found that the most active
oxime carbamate of their series, compound 50, was a reversible
inhibitor of FAAH.29 However, this compound acted as a competi-
tive rather than non-competitive inhibitor, likely because of the
different structure.
zyme-inhibitor complexes in the presence of an excess (10 lM)
of inhibitor (Fig. 1a). On the contrary, under similar dialysis condi-
tions of FAAH–URB597 complexes, Karbarz et al. demonstrated
that this substance is an irreversible inhibitor of enzyme activity.35
We also performed time-dependent inhibition experiments, by
pre-incubating FAAH with compound 10i at 10 lM concentration
for different time periods, and then measuring substrate turn-
over.36 The results demonstrated that inhibition by compound
10i was not time-dependent (Fig. 1b), further suggesting that it
hits as a reversible inhibitor of FAAH. Under the same experimental
conditions, URB597 inhibited FAAH in a time-dependent manner,37
The apparent Ki of compound 10i towards FAAH, calculated
from Michaelis–Menten plots, is shown in Table 2.
Unlike direct agonists of CB1R, FAAH inhibitors do not evoke
classical effects triggered by this receptor (e.g., catalepsy, hypo-
thermia, and hyperphagia), nor are they coupled by unwanted side

S. Gattinoni et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4406–4411
4409
Table 1
Inhibition of FAAH by oxime carbamates
R3
O
N
R4
N
O
R1
R2
Compd
4a
R1
R2
R3
R4
IC50 (nM)
CH3
CH3
CH3
Ph
CH3
H
>50,000
4b
760 100
4c
4d
4e
CH3
CH3
CH3
H
H
H
401 60
200 20
>1000
Ph
S
4f
CH3
CH3
H
>1000
N
S
5
6
H
H
>1000
S
–(CH2)2–CH(Ph)–(CH2)2–
Ph
Ph
>50,000
10a
10b
10c
10d
CH3
CH3
CH3
CH3
H
H
H
H
69
10
17
28
6
1
2
3
F
MeO
10e
CH3
H
H
232 35
S
10f
10g
10h
CH3
CH3
CH3
–(CH2)5–Ph
–(CH2)5–
Ph
100 10
220 30
>50,000
Ph
H
10i
10j
10k
CH3
CH3
CH3
Ph
8
1
S
S
S
C12H25
–(CH2)5–
H
116 12
112 17
(continued on next page)

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S. Gattinoni et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4406–4411
Table 1 (continued)
Compd
R1
R2
R3
R4
H
IC50 (nM)
H NOC
2
13a
13b
CH3
Ph
84
8
PhHNOCHNOC
CH3
Ph
H
>25,000
no dialysis
dialysis
120
100
80
60
40
20
0
*
10i
CT
120
100
80
60
40
20
0
*
*
*
*
*
CT
0
5
15
30
60
Time (min)
Figure 1. (a) Residual FAAH activity pre- and post-dialysis (18 h) at 4 °C. FAAH was
pre-incubated for 20 min with compound 10i at an excess concentration of 10 M,
l
and then was dialyzed against 2 L of 20 mM Tris–HCl buffer (pH 7.0). Residual FAAH
activity was assayed as described in Supplementary data. Control values (CT) before
and after dialysis were 929 29 pmol/min per mg of protein, and 292 5 pmol/min
per mg of protein, respectively. Results are the means S.D. of three independent
experiments. (b) FAAH activity was assayed as described in Supplementary data,
after pre-incubation with 10 lM 10i at room temperature. Activity values were
Figure 2. (a) Michaelis–Menten curves of FAAH activity, determined by using
different concentrations of substrate ([3H-ethanolamine]AEA, 0–20
M range) and
inhibitor (40 and 100 nM). Results are the means S.D. of three independent
expressed as pmol/min per mg of protein, and as percentage of controls in brackets
(100% = 942 66 pmol/min per mg of protein). Results are the means S.D. of three
independent experiments. *p <0.001 versus CT.
l
experiments. Non-linear regression analysis of the kinetic data allowed to calculate
the following Km
[3H]anandamide hydrolysis: CT = 9.8 1.2 and 1938 213; 10i 40 nM = 10 1 and
779 78; 10i 100 nM = 10 and 568 57. The type of inhibition was non-
competitive, because the difference between Vmax values of control and compound
10i was statistically significant (p <0.01), whereas that between Km values was not
(p >0.05). (b) Lineweaver–Burk analysis of the same Michaelis–Menten curves
shown in panel A, further showing that the type of inhibition of compound 10i was
non-competitive. It should be recalled that in double-reciprocal plots (1/v vs 1/[S])
the intercept on the y axis corresponds to 1/Vmax, whereas that on the x axis
(lM) and Vmax (pmol/min per mg of protein) values for
effects typically due to CB1R activation. Thus, chemical inhibition
of FAAH is considered a desirable alternative to CB1R agonists for
therapeutic exploitation.39,40 Of course, it is all the more important
that the beneficial effects of increasing the endogenous tone of AEA
through FAAH inhibition are not thwarted by unwanted modula-
tion of other biological targets that recognize this endocannabi-
noid, like cannabinoid and vanilloid receptors, or the proteins
that synthesize (N-acylphosphatidylethanolamide-phospholipase
D, NAPE-PLD) and purportedly transport it (anandamide mem-
brane transporter, AMT). These proteins form the so-called ‘endo-
cannabinoid system (ECS)’, that includes other well-characterized
elements like the enzymes responsible for synthesis (diacylglyc-
erol lipase, DAGL) or degradation (monoacylglycerol lipase, MAGL)
of 2-arachidonoylglycerol.41 In this context, it is also noteworthy
that AEA can have opposite biological effects when acting at differ-
ent receptors (e.g., CBRs vs TRPV1), and a different activity com-
2
corresponds to ꢂ1/Km
.
pared to that of 2-arachidonoylglycerol.42 Therefore, it is not
surprising that interference of a drug directed towards a certain
element of the ECS with any other element of the same system
might prevent an effective therapeutic exploitation, for instance
in the treatment of cancer,39 or of neurodegenerative/neuroinflam-
matory diseases.42 Overall, the promiscuity of drugs targeted to-
wards a single element of the ECS, but able to interact with
others, has raised concerns about their true efficacy as novel ther-
Table 2
Apparent Ki values towards FAAH (nM), and inhibition (IC50, nM) of the activity of the ECS elements by compound 10i
Compd
Ki
CB1R
CB2R
TRPV1
AMT
NAPE-PLD
MAGL
DAGL
10i
38 14
>10,000
[1250]
10,000
[1250]
>10,000
[1250]
100
[12]
>10,000
[1250]
>10,000
[1250]
>10,000
[1250]
Selectivity index values are reported in square brackets.

S. Gattinoni et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4406–4411
4411
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FAAH inhibitors towards other targets of the ECS seems a pre-req-
uisite for effective drug development. For this reason, in a second
set of experiments we analysed the interaction of compound 10i
with the other well-characterized components of the ECS, in order
to calculate selectivity index values compared to FAAH (see
Supplementary data for details). The results are summarized in
Table 2.
From these data it can be concluded that 10i, besides acting as a
powerful FAAH inhibitor, also shows a good degree of selectivity
towards the other elements of ECS, leading to IC50’s ꢀ1000-fold
higher than those shown towards FAAH. Incidentally, it should
be stressed that the ability of compound 10i to inhibit the AEA
membrane transporter AMT (Table 2) could simply reflect the con-
tribution of FAAH to the transport process. In fact, hydrolysis by
FAAH drives AEA uptake by creating and maintaining a concentra-
tion gradient across the plasma membrane.40,43,44
Finally, the analysis of the interference of compound 10i with
different off-targets revealed that the IC50’s values towards 45
receptors, five ion channels and two transporters were always
>10,000 nM, with the exception of seven targets that showed
IC50’s between 1000 and 10,000 nM (Table 1 in Supplementary
data). Also these data speak in favor of a high degree of specificity
of compound 10i for FAAH over other potential unrelated targets.
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