Synthesis and biological evaluation of new potential inhibitors of N-acylethanolamine hydrolyzing acid amidase

Article abstract of DOI:10.1016/j.bmcl.2009.11.134

N-Acylethanolamines, including N-palmitoyl-ethanolamine (PEA), are hydrolyzed to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). Recently, N-acylethanolamine-hydrolyzing acid amidase (NAAA) was identified as being able to specifically hydrolyze PEA. In order to find selective and effective inhibitors of this enzyme, we synthesized and screened several amides, retroamides, esters, retroesters and carbamates of palmitic acid (1-21) and esters with C15 and C17 alkyl chains (22-27). Cyclopentylhexadecanoate (13) exhibited the highest inhibitory activity on NAAA (IC₅₀ = 10.0 μM), without inhibiting FAAH up to 50 μM. Compound 13 may become a useful template to design new NAAA inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.11.134

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1210–1213
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of new potential inhibitors
of N-acylethanolamine hydrolyzing acid amidase
Carmela Saturnino a, , Stefania Petrosino a,b, , Alessia Ligresti b, Chiara Palladino a,
Giovanni De Martino a, Tiziana Bisogno b, Vincenzo Di Marzo b,
*
a Department of Pharmaceutical Sciences, University of Salerno, Via Ponte don Melillo 84084, Fisciano (SA), Italy
b Endocannabinoid Research Group, Institute of Biomolecular Chemistry-CNR-Via Campi Flegrei 34, 80078, Pozzuoli (NA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Acylethanolamines, including N-palmitoyl-ethanolamine (PEA), are hydrolyzed to the corresponding
fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). Recently, N-acylethanolamine-
hydrolyzing acid amidase (NAAA) was identified as being able to specifically hydrolyze PEA. In order
to find selective and effective inhibitors of this enzyme, we synthesized and screened several amides, ret-
roamides, esters, retroesters and carbamates of palmitic acid (1–21) and esters with C15 and C17 alkyl
chains (22–27). Cyclopentylhexadecanoate (13) exhibited the highest inhibitory activity on NAAA
Received 2 October 2009
Revised 26 November 2009
Accepted 30 November 2009
Available online 4 December 2009
Keywords:
(IC50 = 10.0
l
M), without inhibiting FAAH up to 50
l
M. Compound 13 may become a useful template
N-Acylethanolamines
Palmitoylethanolamide
Inflammation
Degradation
Enzyme
to design new NAAA inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Endocannabinoid
Vanilloid
NAAA
N-Palmitoyl-ethanolamine (PEA) is an endogenous lipid pro-
duced by most mammalian cells1,2 well known for its anti-inflam-
matory and analgesic properties.3–5 It is biosynthesized from a
phospholipid precursor, N-palmitoyl-phosphatidyl-ethanolamine,
through the catalytic action of N-acyl-phosphatidyl-ethanol-
amine-selective phospholipase D (NAPE–PLD).6 Its inactivation to
palmitic acid and ethanolamine is catalyzed by fatty acid amide
hydrolase (FAAH)7 and, more selectively, also by N-acylethanol-
amine-hydrolyzing acid amidase (NAAA).8 The molecular mecha-
nism of action of PEA is still controversial and several hypotheses
have been put forward to explain its anti-inflammatory and anal-
gesic effects. These include: (1) an Autacoid Local Inflammation
Antagonism (ALIA) mechanism through which PEA acts by down-
regulating mast-cell degranulation;9 (2) the direct stimulation of
an as-yet uncharacterized cannabinoid CB2 receptor-like tar-
get;3,10,11 (3) an ‘entourage effect’,12–14 through which PEA would
enhance the anti- inflammatory and anti-nociceptive effects ex-
erted by another fatty acid ethanolamide, anandamide (AEA),
which is often produced together with PEA, and activates cannab-
inoid CB1 and CB2 receptors or the transient receptor potential
vanilloid receptor type 1 (TRPV1) channel;15–18 (4) the direct acti-
vation of the nuclear peroxisome proliferator-activated receptor-
(PPAR- ), which clearly mediates many of the anti-inflammatory
a
a
effects of this compound;19 and (5) the direct activation of the or-
phan receptor G-protein coupling, GPR55.20 Whatever its mecha-
nism of action, based on its pharmacological properties, and on
the fact that its tissue concentrations are altered during several
pathological conditions,5,21 PEA has been proposed to act as a
protective endogenous mediator produced ‘on demand’ during
inflammatory and neurodegenerative conditions to counteract
inflammation, neuronal damage and pain. However, the lack of
pharmacological tools able to selectively modulate its tissue levels
(such as specific inhibitors of its biosynthesis or degradation), has
negated so far the conclusive demonstration of such protective
functions for endogenous PEA. Indeed, at the present time, there
is almost no report of NAAA inhibitors. The screening of different
esters, retroesters and retroamides of palmitic acid22 allowed to
identify three compounds that inhibited NAAA: N-(3-hydroxy-pro-
pionyl)pentadecanamide, cyclohexylhexadecanoate (15) and hexa-
decyl propionate (Fig. 1). These compounds were selective for
NAAA, with no inhibitory activity on FAAH,22 and exhibited IC50
values of 32, 19, and 54 l
M, respectively.23 Tsuboi et al. reported
two additional compounds, N-pentadecylbenzamide and N-penta-
decylcyclohexanecarboxamide (28) (Fig. 1), which exhibited
* Corresponding author. Tel.: +39 081 8675093; fax: +39 081 8041770.
E-mail address: vdimarzo@icmib.na.cnr.it (V.D. Marzo).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.134

C. Saturnino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1210–1213
1211
O
H
N
Equation 1
OH
1
O
H N-R
O
2
1
CH (CH ) -COCl
CH (CH ) -CONH-R
3 2 14
3
3
2 14
Toluene, Na CO
N-(3-hydroxy-propionyl)pentadecanamide
Cyclohexyhexadecanoate (15)
2
(1-9)
Equation 2
O
O
H
N
TEA, DCM
2
CH (CH ) -NH
CH (CH ) -NHCO-R
3 2 15
3
2 15
2
2
O
R -COCl
(10-12)
Hexadecylpropionate
N-pentadecylbenzamide
Equation 3
DCC, DMAP
H
N
CH (CH ) -COOH
CH (CH ) -COO-R
3 2 n
3
2 n
3
DCM, R -OH
3
6
O
n=14 (13-16) with R
n=13 (22-24) with R
n=15 (25-27) with R
6
or R -OH
N-pentadecylcyclohexanecarboxamide(28)
6
Figure 1. Chemical structures of the previously reported esters, retroesters and
retroamides of palmitic acid and NAAA inhibitors.
Equation 4
4
-COCl
R
4
CH (CH ) -CH OH
CH (CH ) -CH -OCO-R
3
2 14
2
3
2 14
2
DCM, TEA
stronger inhibitory activity, with IC50 values of 8.3 lM and 4.5 lM,
(17-19)
respectively.24 Compound 28 is, therefore, the most efficacious and
selective inhibitor of NAAA reported so far, and its mechanism of
action is reversible and non-competitive.24
Equation 5
5
NCO-R
5
CH (CH ) -CH OH
CH (CH ) -CH -OCONH-R
3 2 14 2
3
2 14
2
The aim of the present work was to gain new insights into the
structure–activity relationships of NAAA inhibition, and possibly
to obtain new NAAA inhibitors able to increase the endogenous
levels of PEA, to be used as pharmacological tools to corroborate
the suggested role of this compound in various pathological condi-
tions.21 With this purpose, we established a specific enzymatic as-
say of NAAA activity, using human embryonic kidney (HEK)-293
cells in which the cDNA encoding the human enzyme was stably
overexpressed (HEK-NAAA cells). NAAA activity was measured as
the capability of membranes from these cells to hydrolyze
Toluene
(20-21)
Scheme 1. Chemical synthesis of compounds (1–27).
[
14C]PEA to palmitic acid and [1,2-14C]ethanolamine, as quantified
using a liquid scintillation analyzer. In parallel, we designed and
synthesized several new PEA analogues, to be tested as potential
NAAA inhibitors in this assay. We carried out the synthesis of
new amides (1–9), retroamides (10–12), esters (13–16), retroesters
(17–19) and carbamates (20–21) of palmitic acid, and of esters
with C15 and C17 alkyl chains (22–24 and 25–27, respectively)
(Scheme 1).
In agreement with the findings previously obtained in various
cells and mammalian tissues,23,25 we found that significantly high-
er NAAA activity is found at pH 5 in the membranes (submitted to
two cycles of freezing and thawing) of HEK-NAAA cells, whereas no
appreciable activity was detected in HEK293 wild-type (HEK-WT)
cells (data not shown).26 Consequently, we concluded that HEK-
NAAA cell membrane fractions could be used for our subsequent
experiments. To validate the assay, we next tested the most potent
selective NAAA inhibitor identified so far, compound 28, and found
that the inhibition was concentration-dependent with an IC50
Figure 2. Concentration-dependent inhibition of human recombinant NAAA by 13.
Mean values S.D. are shown. N = 3 separate determinations were carried out for
these experiments.
inserting an aromatic ring also considerably reduced or even
erased the inhibitory activity. In fact, 15 and 4-methoxyphenyl
hexadecanoate (14) exhibited, respectively, 18% inhibition at the
maximal concentration tested and no activity (Table 1).
All other compounds were significantly less active or inactive,
value under our conditions (17.0 1.4
lM) higher than that previ-
with IC50 values always higher than 50 lM (Table 1). In particular,
ously reported by Tsuboi et al., possibly because these authors used
a different source of enzyme from another species (the rat lung) to
measure NAAA activity.24 The screening of the new analogues syn-
thesized here allowed to identify a compound more active than 28,
in which the amide bond and the cyclohexyl group were changed
to an ester bond and a cyclopentyl group, respectively.
among the amide derivatives, only the insertion of a p-nitrophenyl
substituent (1) introduced some NAAA inhibitory activity (15%
inhibition at the maximal concentration tested), whereas all other
amide derivatives were significantly inactive. We also observed
that all the ester derivatives with an alkyl chain length of C15 or
C17 exhibited a modest activity or were inactive, thus suggesting
that the length of alkyl chain also influences the potency of these
compounds (Table 1).
The new compound, cyclopentylhexadecanoate (13), inhibited
NAAA by 85% at 50
lM. The inhibition was concentration-depen-
dent and the IC50 was calculated as 10.0 2.1
l
M (Fig. 2). It should
Compound 13 exhibited no inhibitory activity on FAAH, nor any
binding activity at CB1 and CB2 receptors, or any functional activity
be noted that among the ester derivatives, the nature of the cycle
inserted was important to observe an inhibitor activity. In fact,
although much less than 13, cyclobutylhexadecanoate (16) also
inhibited NAAA activity to some extent (41% inhibition at the
maximal concentration tested). Increasing the steric hindrance or
at TRPV1 receptors, at concentrations as high as 50 lM (data not
shown).26 The type of inhibition of NAAA by 13 was examined by
incubating different concentrations of PEA with HEK-NAAA mem-
branes in the presence or absence of 20 lM 13. As indicated by

1212
C. Saturnino et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1210–1213
Table 1
Table 1 (continued)
Inhibition of NAAA by new analogues of palmitic acid
R5
Compound
R1
Maximal inhibition
of NAAA at 50 lM
21
OCH3
Inactive
CH3(CH2)14–CONH–R1
Compound
n
R6
Maximal inhibition
1
NO2
15%
of NAAA at 50
lM
CH3(CH2)n–COO–R6
2
OCH3
Inactive
Inactive
22
13
25%
3
4
H2CH2C
OCH3
OCH3
23
24
25
13
13
15
28%
29%
H2CH2C
Inactive
Inactive
OCH3
26
27
15
15
Inactive
Inactive
5
6
N
N
N
N
Cl
F
Inactive
Inactive
Data are means of three separate determinations and are expressed as IC50
(
lM) or,
when this was higher than 10
concentration.
l
M, as maximum inhibition observed at a 50
lM
N
N
CH2
7
8
Inactive
Inactive
Lineweaver–Burk plots (not shown), the inhibition appeared to be
of a competitive nature, since, in the presence of 13, the apparent
Km value for PEA was significantly enhanced from 118 2.0
N
N
lM to
198 1.0 M, with no significant change in the Vmax value (11.1
l
3.6 nmol minÀ1 mg proteinsÀ1 and 14.1 3.4 nmol minÀ1 mg pro-
teinsÀ1, respectively). Finally, 13 also appeared to inhibit NAAA
in intact HEK-NAAA cells since it selectively increased the levels
of PEA (as determined by isotope-dilution liquid chromatogra-
phy-mass spectrometry)27,28 in these cells from 2.39 0.21 pmol/
mg lipid to 3.96 0.03 pmol/mg lipid, without influencing the lev-
els of the other N-acylethanolamines, AEA and OEA, nor of the
other endocannabinoid, 2-arachidonoylglycerol.
9
Inactive
N
CH3(CH2)15–NHCO–R2
R2
10
11
8%
4%
NO2
In summary, 13 is a selective and competitive inhibitor of NAAA
with potency, under our assay conditions using a human recombi-
nant enzyme, nearly two-fold higher than that of the previously
described 28. Compound 13 inhibits NAAA both in the cell-free
preparations and intact cells, and will be useful both as a template
to design new and more potent NAAA inhibitors and as a pharma-
cological tool to investigate the consequences of pharmacological
elevation of endogenous PEA levels in cells and tissues.
12
OCH3
Inactive
CH3(CH2)14–COO–R3
R3
13
14
85%
OCH3
Inactive
Acknowledgments
This work was partly supported by Epitech Group S.r.l. The
authors are grateful to Marco Allarà for technical assistance.
15
16
18%
41%
Supplementary data
CH3(CH2)14–CH2–OCO–R4
R4
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.134.
17
18
19
17%
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NO2
26%
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