Discovery of potent dual PPARα agonists/CB1 ligands

Article abstract of DOI:10.1021/ml200091q

This letter describes the synthesis and in vitro and in vivo evaluation of dual ligands targeting the cannabinoid and peroxisome proliferator-activated receptors (PPAR). These compounds were obtained from fusing the pharmacophores of fibrates and the diar

Full text of DOI:10.1021/ml200091q

LETTER
pubs.acs.org/acsmedchemlett
Discovery of Potent Dual PPARα Agonists/CB1 Ligands
||
Ruth Pꢀerez-F^ernꢀandez,† Nieves Fresno^,† Manuel Macías-Gon^zꢀalez,‡ Josꢀe Elguero,† Juan Decara,§,
ꢀ
Rocío Girꢀon, Ana Rodríguez-Alvarez, María Isabel Martín,
||
Fernando Rodríguez de Fonseca,§, and Pilar Goya*,†
†Instituto de Química Mꢀedica, IQM-CSIC, Juan de la Cierva 3, 28006, Madrid, Spain
‡Servicio de Endocrinología Nutriciꢀon, Hospital Virgen de la Victoria (Fundaciꢀon IMABIS), Mꢀalaga,
CIBER Fisiopatología de la Obesidad y Nutriciꢀon, CB06/03, Instituto de Salud Carlos III, Spain
§Fundaciꢀon Hospital Carlos Haya, Avda. Carlos Haya 82, 29010, Mꢀalaga, Spain
CIBER OBN (Centro de Investigaciꢀon Biomꢀedica en Red de la Fisiopatología de la Obesidad y Nutriciꢀon), Instituto de Salud Carlos III,
Ministerio de Ciencia e Innovaciꢀon, 28029 Madrid, Spain
^Departamento de Farmacología y Nutriciꢀon, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Avda. Atenas S/N,
28922 Alcorcꢀon, Madrid, Spain
S Supporting Information
b
ABSTRACT: This letter describes the synthesis and in vitro and
in vivo evaluation of dual ligands targeting the cannabinoid and
peroxisome proliferator-activated receptors (PPAR). These com-
pounds were obtained from fusing the pharmacophores of fibrates
and the diarylpyrazole rimonabant, a cannabinoid receptor an-
tagonist. They are the first examples of dual compounds with
nanomolar affinity for both PPARα and cannabinoid receptors.
Besides, lead compound 2 proved to be CB1 selective. Unexpect-
edly, the phenol intermediates tested were equipotent (compound 1
as compared to 2) or even more potent (compound 3 as compared with 4). This discovery opens the way to design new dual
ligands.
KEYWORDS: Dual ligands, PPARα, cannabinoids, rimonabant, fibrates, neuroprotective
eroxisome proliferator-activated receptors (PPARs) belong
to the superfamily of nuclear receptors involved in the
The endocannabinoid system comprising the endocannabi-
noids, cannabinoid receptors, and the proteins involved in
regulation mechanisms, including the anandamide transporter
(ANT), fatty acid amide hydrolase (FAAH), and monoacylgly-
cerol lipase (MAGL), is a relevant therapeutic target.5,6 Among
the synthetic ligands of these receptors, the cannabinoid receptor
antagonist rimonabant proved effective in reducing weight gain
and plasma lipids, although side effects banned it as a marketed
drug.7
PPARs are sensors of fatty acid levels. There is increasing
evidence suggesting that endocannabinoids or related com-
pounds may activate PPARs. One of the endogenous ligands
that has nanomolar affinity for the PPARα receptor is OEA.8 The
first study that established the interactions between cannabinoids
and PPARs was published in 2002,9 in which the endocannabi-
noid 2-AG proved to increase the transcriptional activity of
PPARα. Additional significance comes from the fact that both
cannabinoid antagonists and PPARα agonists may be useful as
therapeutic agents for neuroprotective treatment.10,11
P
regulation of glucose and lipid metabolism, as well as in
adipogenesis, feeding, and other processes such as inflammation
and neuroprotection. Therefore, they are interesting targets in
medicinal chemistry.1 Among the three isoforms, PPARα is
a relevant target for pharmaceutical development, oleoylethanol-
amide (OEA) being its endogenous ligand.2 PPARα agonists
(e.g., fenofibrate, clofibrate) are capable of lowering triglycerides
and raising high-density lipoprotein levels. The limitations of
currently approved fibrates are their poor selectivity, weak affinity
to rodent and human cloned receptors, and the fact that they
require high micromolar concentrations to be effective. Recent
advances in this field have led to the discovery of the so-called
second generation of PPARα agonists such as BMS-687453,
which showed affinity in the nanomolar range.3 On the other
hand, the therapeutic properties of cannabinoids including plant-
derived compounds as well as synthetic derivatives are due to the
interactions with the cannabinoid receptors CB1 and CB2. The
identification of these G-protein-coupled receptors4 responsible
for many biochemical processes prompted the discovery of
anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) as
endogenous ligands.
Received: April 6, 2011
Accepted: September 16, 2011
Published: September 16, 2011
r
2011 American Chemical Society
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LETTER
Figure 1. Structures of dual PPARα-cannabinoid compounds 1ꢀ4.
Cannabinoids (endogenous, phytoderived, and synthetic) do
not bind to or activate PPARs to the same extent as available
synthetic PPAR ligands; therefore, they may work successfully as
partial agonists of PPARs, reducing the potential carcinogenic
effects of PPAR agonists. For this reason, cannabinoid actions at
PPARs would provide an alternative mechanism for cannabi-
noids as therapeutic agents.
Scheme 1. Synthesis of Compounds 1ꢀ4
It has been reported that combining cannabinoids with other
compounds (other cannabinoids or PPAR agonists) increases
their therapeutic potential. For example, the combination of OEA
(PPARα agonist) with rimonabant (CB1 antagonist) resulted in
more effective suppression of feeding and increased weight loss.12
Another example can be found in the combination of AEA (CB1)
and GW7647 (PPARα agonist) leading to synergistic effects on
reducing pain behavior.13
We have previously reported oleylethanolamide analogues as
PPARα activators14 and cannabinoid antagonist derivatives
containing a 1,2,4-triazole motif.15 We now report molecules
incorporating cannabinoid and PPAR activities into a single
structure using the strategy known as “designed multiple ligands”
(DML).16 Hence, we decided to integrate in one molecule the
pharmacophore of the fibrates, fenofibrate (PPARα agonist), and
a structural motif of rimonabant, a proven CB1 antagonist/
inverse agonist (Figure 1).
The procedure employed in our laboratory for the synthesis of
compounds 1ꢀ4 is depicted in Scheme 1. The synthesis began
with the carboxylate derivative 5, accessible through a two-step
synthesis reported in the literature.17 The trimethyl aluminum-
mediated amide formation between compound 5 and 3-amino-
phenol was completed in 3 min and 30 s at 125 ꢀC under
microwave irradiation yielding compound 1. This methodology
avoided the traditional ester hydrolysis, acid activation to obtain
in three steps the amide derivative, apart from reducing the
reaction time from 18 h to 3 min using a microwave reactor.
Deprotonation of compound 1, followed by treatment with ethyl
2-bromo-2-methylpropionate gave the target compound 2. A
similar synthetic route using 4-aminophenol instead of 3-amino-
phenol was followed for the synthesis of phenol derivative 3 and
the fibratelike 4. Deprotonation of compound 3 and nucleophilic
substitution of the corresponding bromo derivative yielded
compound 4.
a (i) 3-Aminophenol, Al(CH3)3 in 2.0 M heptane, THF, 125 ꢀC, 3 min 30 s.
(ii) Ethyl 2-bromo-2-methylpropionate, K2CO3, CH3CN, 90 ꢀC, 18 h. (iii)
4-Aminophenol, Al(CH3)3 in 2.0 M heptane, THF, 125 ꢀC, 3 min 30 s. (iv)
Ethyl 2-bromo-2-methylpropionate, K2CO3, CH3CN, 90 ꢀC, 18 h.
The compounds reported in this study were first evaluated for
basal and ligand-induced activity of PPARα (Figure 2).
Luciferase reporter gene assays were performed with extracts
from MCF-7 cells that were transiently transfected with a
reporter construct containing four copies of the human CPTI
DR1-type RE (for PPARs) and the indicated expression vectors
for PPARα, RXRα, and NCoR (CoR). Cells were treated for 16 h
with a 10 μM concentration of different compounds: DMSO
(solvent), the reference PPARα agonist WY14643, the endogen-
ous PPARα, agonist OEA, and compounds 1 and 2 (Figure 2a).
MD simulation of luciferase activity was normalized to the basal
activity of PPARαꢀRXRαꢀSRC1 in the presence of the solvent
(DMSO). Details on the methodology have been previously
published.14 Compounds 1 and 2 displayed agonistic activity at
the PPARα receptor at high nanomolar concentrations (Table 1).
They were similar in potency to fibrate WY14643, although
compound 2 was slightly more potent than compound 1 but 4ꢀ
5-fold less potent than the natural ligand oleylethanolamide.
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Table 2. CB1 Receptor Affinitya
compd
CB1 Ki (nM)
rimonabant
7.3 ( 0.97
45.6 ( 8.6
52 ( 22
WIN 55,212-2
1
2
3
4
65 ( 17
4.6 ( 0.96
109 ( 50
a Data are mean values of n = 3 separate experiments and are expressed
as Ki (nM) for CB1 and binding.
reference compound rimonabant. Compound 4 was the least
potent of the series. The diarylpyrazole scaffold used for design-
ing the compounds and the lack of potency at the cannabinoid
CB2 receptor (data not shown) are usually associated with
selective cannabinoid CB1 receptors antagonist activity.
The biological activity of compounds 2 (fibratelike) and 3 (p-
phenol) was evaluated carrying out pharmacological experiments
in the isolated mouse vas deferens (MVD).18 It is a commonly
used tissue to study and characterize cannabinoid effects as
agonists or antagonists.19 Compound 2 was chosen as the best
dual compound of the series and compound 3 because of having
a binding constant (4.6 nM) similar to rimonabant (7.3 nM).
The effect of the compounds was always compared with that of
reference drugs: arachidonyl-2-chloroethylamide (ACEA) and
AM251 as cannabinoid CB1 selective agonist and antagonist,
respectively.
Figure 2. Basal and ligand-induced activity of PPARα. Columns
represent the mean of at least three experiments, and bars indicate
standard deviations. Two-tailed, paired Student's test was performed,
and P values were calculated in reference to the respective solvent
control (*P < 0.01, **P < 0.001, or ‡P < 0.01) to compare every ligand in
the absence or presence of NCoR. (a) After 16 h of incubation. (b) After
6 h of incubation.
The MVD is a nerve-smooth muscle preparation that serves as
a highly sensitive and quantitative functional in vitro bioassay for
cannabinoid receptor agonists. Additionally, it is commonly used
as a bioassay for competitive surmountable cannabinoid receptor
antagonists and also provides a means for distinguishing neutral
cannabinoid antagonists from inverse agonists. The bioassay of
cannabinoid receptor agonists relies on the ability of these
ligands to produce concentration-related decreases in the ampli-
tude of electrically evoked contractions of the vas deferens by
acting on prejunctional neuronal receptors and inhibiting the
release of the contractile neurotransmitters such as noradrenaline
and ATP. The bioassay of competitive surmountable cannabi-
noid receptor antagonists examines the ability of these com-
pounds to produce dextral shifts in cannabinoid receptor
agonist log concentrationꢀresponse curves in electrically
stimulated tissues.20ꢀ22
Data obtained from compounds 2 and 3 at the tested
concentrations (10ꢀ7ꢀ1.8 ꢁ 10ꢀ5 M) did not reveal any effect
either on the nonstimulated basal recording or on the electrically
induced contractile response on MVD (the % of inhibition of the
electrically evoked contractions was <10% and not different from
that induced by the vehicle), so it could be established that they
both lack a direct (activation or blockade) or indirect (induction
or inhibition of neurotransmitter release) intrinsic activity on
a large number of well-known receptors that play a role in the
basal tone or on the electrically induced contraction in this
tissue (adrenergic, purinergic, cannabinoid, δ, μ, and k opioid
receptors).
Table 1. Pharmacological Properties of Compounds 1ꢀ4a
PPARα activation
EC50 (nM)b
PPARα activation
EC50 (nM)c
compd
OEA
152 ( 31
650 ( 134
989 ( 175
743 ( 109
ND
185 ( 64
542 ( 98
WY14643
1
2
3
4
887 ( 185
856 ( 166
971 ( 180
1480 ( 253
ND
a EC50 values calculated in the presence of the different compounds by
GraphPad Prism 4. Results are the means ( SEMs of three experiments.
ND, no data. b Sixteen hours of incubation. c Six hours of incubation.
To assess the stability of the compounds in the assay, two
different experiments changing the incubation time were per-
formed. Cells were treated for 6 h with a 10 μM concentration of
different compounds: DMSO (solvent), WY14643, OEA, and
compounds 1ꢀ4 (Figure 2b). These results were consistent with
the previous ones after 16 h of incubation, and the phenol
derivatives 1 and 3 showed agonistic activity at the PPARα
receptor at high nanomolar concentrations. Compound 2 was
the more potent of the series, whereas its homologue 4 with a
para substitution at the benzene ring was less potent with an
EC50 of 1.5 μM.
The compounds were screened for cannabinoid activity in
radioligand binding assays (Table 2). All compounds were found
to displace [3H]-CP55940 binding from cannabinoid CB1
receptors at nanomolar concentrations. Ki values of compounds
1 and 2 were very similar to the reference agonist WIN 55,212-2,
while compound 3 with a Ki of 4.6 nM was more potent than the
An interesting finding is that compounds 2 and 3 (at the two
tested concentrations 10ꢀ6 and 2 ꢁ 10ꢀ6 M) significantly
attenuate the inhibition induced by the selective CB1 receptor
agonist ACEA (Figure 3) as also occurred with the reference
compound AM251. These data suggest that both behave as CB1
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LETTER
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic experimental details,
b
analytical data, and biological assay protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +34915622900. Fax: +34915644853. E-mail: pgoya@iqm.
csic.es.
Figure 3. Functional activity of compounds 2 and 3 on MVD. Lines
show the mean % ( SEM (n = 6ꢀ8) inhibition of the electrically
induced contraction of the MVD induced by the addition of increasing
concentrations of ACEA in control tissues or in tissues incubated with 2,
3, or AM251. Statistically significant difference: *p < 0.05, 2 (10ꢀ6 M) +
ACEA vs ACEA; #p < 0.05 and ##p < 0.01, 2 (2 ꢁ 10ꢀ6 M) + ACEA vs
< 0.01 and @@@p < 0.001, 3 (2 ꢁ 10ꢀ6 M) + ACEA vs ACEA; and &p <
0.05 and &&p < 0.01, AM251 (10ꢀ6 M) + ACEA vs ACEA (two-way
ANOVA test, Bonferroni's posthoc test).
Funding Sources
The present work has been supported by grants SAF2009-12422-
C02-02, RTA (RED Trastornos Adictivos RD06/001/0014), the
European Union's seventh Framework Programme (Health-F2-
2008-223713, Reprobesity), and the following grants from the
Spanish Ministry of Science and Innovation: SAF2010-20521,
National Institute of Health 'Carlos III' Red de Trastornos
Adictivos EU-ERDF (RD06/0001/0000), and CIBER-OBN
EU-ERDF (CB06/03/1008). M.M.-G. is supported by the
Research Stabilization Program of the Instituto de Salud Carlos
III (CES 10/004).
ACEA; $$p < 0.01 and $$$p < 0.001, 3 (10ꢀ6 M) + ACEA vs ACEA; @@
p
receptor antagonists, compound 2 being as effective as AM251,
whereas compound 3 is significantly more effective than AM251.
From the shape of the curve, it could be suggested that it behaves
like a competitive antagonist, although more work is required to
confirm this possibility.
Regarding CB2 antagonism experiments, compound 2 failed
in modifying the inhibitory effect of the selective CB2 receptor
agonist JWH-015 (data not shown), so it could be suggested
that it is a selective CB1 antagonist; on the other hand, phenol
derivative 3 was able to inhibit the effect of JWH-015
(Supporting Information), although this effect was lower than
that observed on CB1 receptor, suggesting a relatively selec-
tive CB1 effect.
Despite the affinity for both cannabinoid CB1 receptors and
PPARα receptors, the compounds did not display activity as
feeding suppressants in vivo food deprived rats when adminis-
tered intraperitoneally (Supporting Information). The lack of
acute effects on feeding of these acyl derivatives may be
attributable to several reasons. First, the in vitro profile of the
compounds may not be manifested in vivo due to poor pharma-
cokinetics. Second, the compounds may affect non-CB1, non-
PPARα-mediated signaling mechanisms that control feeding.
For instance, OEA is a feeding suppressant that acts not only at
the PPARα receptor but also on the orphan receptor GPR119 to
inhibit feeding.23 Third, the lack of acute effects does not exclude
the potential induction of changes in energy expenditure when
these compounds are given chronically. All of these aspects will
be addressed in future studies.
In summary, we have prepared and evaluated four com-
pounds, three of which showed nanomolar affinity for both the
PPARα and the CB1 receptors. The middle-low potency at the
PPARα receptor similar to fibrates will probably lead to a
hypolipemiant and neuroprotective profile, while the high affinity
for the CB1 receptor may help to modulate metabolism. The
intermediates phenol derivatives, 1 and 3, were found to be
unexpectedly potent, offering new possibilities in the search for
dual PPARα agonists/CB1 ligands. Therefore, the four synthe-
sized compounds can be considered interesting leads in the
search for a new class of dual ligands capable of modulating
metabolism with potential neuroprotective activities.
’ ACKNOWLEDGMENT
We thank Prof. J. Fernꢀandez Ruiz for generous use of the
equipment to perform the binding studies and M. Gꢀomez Ca~nas
for her technical assistance.
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