Dual RXR agonists and RAR antagonists based on the stilbene retinoid scaffold

Article abstract of DOI:10.1021/ml400521f

Arotinoids containing a C5,C8-diphenylnaphthalene-2-yl ring linked to a (C3-halogenated) benzoic acid via an ethenyl connector (but not the corresponding naphthamides), which are prepared by Horner-Wadsworth-Emmons reaction of naphthaldehydes and benzylphosphonates, display the rather unusual property of being RXR agonists (15-fold induction of the RXR reporter cell line was achieved at 3- to 10-fold lower concentration than 9-cis-retinoic acid) and RAR antagonists as shown by transient transactivation studies. The binding of such bulky ligands suggests that the RXR ligand-binding domain is endowed with some degree of structural elasticity.

Full text of DOI:10.1021/ml400521f

Letter
pubs.acs.org/acsmedchemlett
Dual RXR Agonists and RAR Antagonists Based on the Stilbene
Retinoid Scaffold
†
‡
†
†
†
́
́
Claudio Martínez, Michele Lieb, Susana Alvarez, Fatima Rodríguez-Barrios, Rosana Alvarez,
́
Harshal Khanwalkar,‡ Hinrich Gronemeyer,*,‡ and Angel R. de Lera*,†
†Departamento de Química Organ
Biomedica de Vigo (IBIV), 36310 Vigo, Spain
‡Equipe Labellisee
Ligue Contre le Cancer, Department of Functional Genomics and Cancer, Institut de Gen
Moleculaire et Cellulaire (IGBMC)/CNRS/INSERM/UdS, BP 10142, 67404 Illkirch, Cedex, C. U. de Strasbourg, France
́ ́
ica, Facultade de Química, CINBIO, Universidade de Vigo, and Instituto de Investigacion
́
́
́ ́
etique et de Biologie
́
S
* Supporting Information
ABSTRACT: Arotinoids containing a C5,C8-diphenylnaph-
thalene-2-yl ring linked to a (C3-halogenated) benzoic acid via
an ethenyl connector (but not the corresponding naphtha-
mides), which are prepared by Horner−Wadsworth−Emmons
reaction of naphthaldehydes and benzylphosphonates, display
the rather unusual property of being RXR agonists (15-fold
induction of the RXR reporter cell line was achieved at 3- to
10-fold lower concentration than 9-cis-retinoic acid) and RAR
antagonists as shown by transient transactivation studies. The
binding of such bulky ligands suggests that the RXR ligand-
binding domain is endowed with some degree of structural
elasticity.
KEYWORDS: Retinoid receptor subtypes, agonists, antagonists, arotinoids, transactivation, molecular modeling
ll-trans-retinoic acid 1, its isomer 9-cis-retinoic acid 2, and
Ligands that act as agonists of RXR and antagonists of RAR
A
other natural retinoids are important signaling molecules
are interesting chemical probes to help understand the signaling
options of the RAR/RXR and other heterodimers.25 We wish
to report a new retinoid scaffold that displays this unusual
property, as it acts in transactivation assays as a potent RXR
agonist and RAR antagonist.
that act in the embryo1 and throughout adult life.2−4 At the
genomic level, retinoids bind the retinoid receptors (RARs,
subtypes RARα,β,γ)5 and retinoic X receptors (RXRs, subtypes
RXRα,β,γ)6 to regulate many important biological activities.
The binding of these native retinoids and also of synthetic
analogues to their receptors affect cell growth, proliferation,
apoptosis, development, and homeostasis.2,7−9 For the direct
regulation of gene transcription, the functional unit is a RAR−
RXR heterodimeric structure, which provides a binding
interface with other recruited protein complexes that sense
the ligand-modulated activation status of the receptor(s) and
bind to DNA regulatory elements in the promoter regions of
target genes to control transcription.10,11 Within the series of
heterodimeric complexes with RXR as a partner, RAR/RXR
heterodimers are nonpermissive since their functional activa-
tion requires agonist binding to RAR, but not to RXR, in a
phenomenon called RXR subordination.12,13
The structure of the new retinoid receptor modulator is
inspired in the parent RAR pan-agonist TTNPB (3).26−30
Numerous modifications of the basic scaffold have been
systematically carried out in order to correlate structure and
functional consequences of retinoid ligand modifications on
receptor activation.17−19 Substitution at the naphthalene C8
position afford in general ligands with RAR antagonist/inverse
agonist activities,28−30 and this is modulated31 by synergy with
halogen atoms at the C3 position32 of the benzoic acid
terminus. In these structures, the LBP volume around the
naphthalene C5 position is filled with a hydrophobic gem-
dimethyl group. Manual docking experiments carried out with
RARα suggested that further extension of the substituents was
possible, and this increase in bulk could impact on H12
positioning and thus endow the ligand with antagonist
properties. Moreover, for synthetic purposes the naphthalene
core was considered as a more suitable starting material than
With the exception of 9-cis-retinoic acid (2) and a few other
flexible analogues, which can bind both RXRs and RARs,14−16
most of the more than 2500 synthetic retinoids so far reported
show selectivity for either of them.17−19 This is due to the
different architecture of the ligand binding pockets (LBPs) in
RAR and RXR. Whereas the former is an I-shaped pocket that
binds elongated ligands,20−22 the latter is L-shaped,23,24 and
RXR ligands (so-called rexinoids) must adopt a bent
conformation to effectively bind the receptor.
Received: December 18, 2013
Accepted: March 19, 2014
Published: March 19, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
the dihydroderivative due to the easy functionalization with
halogen atoms using SEAr reactions, and these halogens could
in turn be replaced with aryl groups employing Pd-catalyzed
cross-coupling reactions. Both an olefin and an amide
connector (compounds 10 and 11, Scheme 1) were included
in the design, as they are present in many of the well-
characterized modulators in this series of compounds.17−19,31
DIBAL at −78 °C followed by oxidation of 19 provided
aldehyde 20. Reaction of the phosphonate-stabilized anions,
obtained by treatment of 21a,b,d with n-BuLi in THF in the
presence of DMPU at −78 °C with aldehyde 20 produced the
E-isomers (3JH−H between 16 and 18 Hz for the vinyl protons
1
in the H NMR spectra) of stilbenoids 22a,b,d in good yield.
These esters were used in the next saponification step without
further purification, due to their ease of isomerization to the Z
isomers, and furnished the final arotinoids 11a,b,d (67−73%).
To evaluate the effects of the described retinoids on RARα,
RARβ, RARγ, and RXRβ-mediated transactivation, a reporter
assay with genetically engineered HeLa cell lines17,28 was used.
A single RXR reporter cell line (expressing RXRβ LBD) is
assumed to reveal the ligand responsiveness as a readout for all
three RXRs, given the identity of the amino acids constituting
the ligand-binding pockets of the RXR subtypes.6,7 The
reporter cell lines are engineered to express a fusion protein,
comprising the ligand-binding domain of the corresponding
receptor and the DNA-binding domain of the yeast GAL4
transcription factor. In addition, the cells contain a stably
integrated luciferase reporter gene, which is controlled by five
Gal4 response elements in front of a β-globin promoter; this is
termed (17m)5-βG-Luc.12,42 This reporter system is largely
insensitive to endogenous receptors, which cannot recognize
the GAL4-binding site. The transcriptional activity of the
various compounds was compared with that of the pan-RAR
agonists 3 and 2 (Figure 1) as positive controls for RAR and
a
Scheme 1
a
Reagents and reaction conditions: a. Ipy2BF4, TfOH, CH2Cl2, 25 °C,
20 h, 71%. b. Pd(PPh3)4, PhB(OH)2, Na2CO3, 1:1 THF/H2O,
microwaves, 110 °C, 5 min, 91%. c. 2 M KOH, MeOH, 50 °C, 3 h,
then H3O+ (15, 99%; 10a, 72%; 10b, 51%; 10c, 43%; 11a, 89%; 11b,
87%; 11d, 81%). d. EDCI, HOBt, NH3, DMF, 25 °C, 3 h, 60%. e. CuI,
(1S,2S)-cyclohexane-1,2-diamine, K2CO3, dioxane, 140 °C, 17 h (18a,
74%; 18b, 75%; 18c, 64%). f. DIBAL, THF, −78 °C, 4 h, then H3O+,
78%. g. MnO2, CH2Cl2, 25 °C, 3 h, 84%. h. 21a−d, n-BuLi, THF,
DMPU, −78 °C (22a, 67%; 22b, 70%; 22d, 73%).
Using excess bis-(pyridine)iodonium(I) tetrafluoroborate
(Ipy2BF4)33 and triflic acid, the diiodo derivative 13 was
obtained from methyl 2-naphthoate 12 by substitution at both
activated positions (C5 and C8, Scheme 1). Cross-coupling of
the diiodide 13 with phenylboronic acid [Pd(PPh3)4, Na2CO3,
60 °C, 2 h]34 afforded compound 14. The preparation of N-
arylamides was based on the Cu(I)-diamine-catalyzed amida-
tion of the 4-halo- or 3,4-dihaloderivatives (using, in this case,
the more reactive iodides at the C4 position) with the
corresponding naphthamides.35,36 Saponification of 14 was
followed by primary amide formation using EDCI and HOBt in
Figure 1. Natural RAR ligands (1 and 2), parent arotinoid pan-agonist
TTNPB (3), and dihydronaphthalene analogues substituted at C8″
(4−6) and C5″ (7−9).
RXR activation, respectively. In the antagonist assays the
positive controls are challenged with increasing amounts of
putative antagonists, resulting in decreased transactivation of
the reporter. Note that depending on their receptor binding
affinities weak agonists can appear in this in vivo competition
assay as antagonists, albeit they retain at high concentration
their weak agonist activity. The transcriptional data of the
naphthamide and stilbene arotinoids with the RAR subtypes is
presented in Figures 2 (agonist activity) and 3 (antagonist
activity).
37
MeOH in the presence of NH3 to furnish the disubstituted
naphthamide 16. Using CuI as catalyst, (1S,2S)-cyclohexane-
1,2-diamine as ligand, and K3PO4 in dioxane,38−41 the
condensation of 16 with halogenated benzoates 17 produced
the corresponding amides 18 after CAr-N bond formation at
the more reactive position in good yield (64−75%). The
sequence was completed with the saponification of the esters
using KOH in MeOH at 60 °C, which proceeded in high yield
except for the 3-chloro analogue (10c; 43%).
The stilbenoids 22 were prepared by Horner−Wadsworth−
Emmons condensation of naphthaldehyde 20 and the
corresponding benzylphosphonates 21a,b,d (X = H, F, Br) as
previously described.31 Reduction of naphthoate 14 with
Naphthamides showed no (RARα) to modest activities
(RARβ and RARγ at 10 μM), and only 10a was able to
considerably activate RARβ at very high concentrations (>1
μM) to levels obtained with 0.1 μM 3 (Figure 2). There is
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ACS Medicinal Chemistry Letters
Letter
some residual activation of RARγ at such high concentrations,
indicating that 10a can bind to RARγ but may not induce a
highly active conformation. This likely explains why this ligand
acts at the same time as the weak antagonist of RARγ (Figures
2 and 3, left panels). In the case of the stilbenes, however,
strong activation of RXRβ was observed, while these
compounds were unable to activate RAR subtypes even at 1
μM concentration (Figure 2, right panels). In particular, 11a
and less pronounced 11b showed a dose−response curve
resembling that of 2 but shifted by about 2- and 10-fold,
respectively, to higher concentrations (“right shift”); specifi-
cally, a 15-fold induction of the RXR reporter cell line was
achieved at 51 nM 11b, 125 nM 11a, and 875 nM 11d,
compared to 541 nM obtained with 3. Moreover, competition
studies between 3 and the synthesized arotinoids in this series
revealed strong antagonism of the RAR subtypes, particularly of
11a and analogue 11b (with a fluorine atom at C3) and to a
lesser extent of 11d (with a bromine at C3) (Figure 3, right
panels).
The dual modulatory activity of compounds 11a and 11b led
us to explore the structural basis of their behavior as RXR
agonist and RAR antagonist, which is surprising as their
structure contains a trans-olefin as a connector. Since the
structure of RARα bound to antagonist BMS195,614
(Supporting Information) at 2.50 Å resolution (PDB code:
1dkf)24 is known, the model of RARα-antagonist-11a complex
was constructed. The structure of human RARβ complexed
with 3 (PDB code: 1xap)22 provided the template for the highly
homologous 210−216 region of RARα, which is severely
disordered in the antagonist-bound crystal structure of RARα.
Ligand 11a was positioned in the active site on the basis of the
canonical positions revealed by the crystal struc-
tures.20−22,32,43,44 Preferred docking sites for functional groups
were evaluated with the program GRID,45 which assisted in the
selection of binding modes.31 The resulting model showed the
two phenyl moieties at C5 and C8 positions running almost
perpendicular to the ethenylbenzoic acid buried in a
predominantly hydrophobic pocket (Figure 4). Unrestrained
molecular dynamics simulations31 showed a notably stable
behavior reflecting that the overall architecture of the protein
was preserved for the whole length of the simulation, including
the ionic bridge anchor. The evolution of the root-mean-square
deviation (rmsd) of 11a with respect to the initial structure
shows rmsd lower than 0.5 Å (see Supporting Information) and
maintains the ionic bridge of the carboxylate to Arg276 (Figure
4).
Figure 2. In vitro dose−response luciferase reporter assays revealing
the RAR subtype agonist activities of the retinoids described in this
study relative to agonists 3 and 2 (Figure 1). For details on the assay
system, see the Supporting Information. The data are derived from at
least three independent experiments; the standard deviations are
indicated.
Similar docking experiments with RXRα failed to provide
stable solutions without severe distortion of the protein-binding
pocket, and no binding poses could be found due to the bulky
phenyl extensions protruding from both sides of the ligand
naphthyl core, in particular the C5 phenyl group interacting
with H11 (see Supporting Information). Extended pockets in
the LBD of nuclear receptors to allow binding of bulky agonists
have been noted by X-ray crystalography of ligand-bound
estrogen receptor α (ERα).46 This precedent suggests that the
RXR ligand-binding domain is also endowed with some degree
of structural elasticity to allow binding of bulky stilbenes such
as 11a or 11b.
To summarize, diphenylsubstituted (E)-4-(2-(naphthalen-2-
yl)vinyl)benzoic acids and the corresponding 4-(2-
naphthamido)benzoic acids with the same substitution pattern
have been prepared and evaluated as retinoid receptor
modulators. Transactivation studies in this series revealed
Figure 3. In vitro dose−response luciferase reporter assays revealing
the RAR subtype antagonist activities of the retinoids synthesized in
this study. The agonists 3 (10 or 100 nM) and 2 (100 nM) are
challenged with increasing amounts of the various retinoids, and
luciferase activity is determined. The decreased activity relative to 3 or
2 reveals antagonism. For further details on the assay system, see the
Supporting Information. The data are derived from at least three
independent experiments; the standard deviations are indicated.
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Ipy, bis-(pyridine)iodonium(I); LBP, ligand binding pocket;
PDB, protein data bank
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ASSOCIATED CONTENT
* Supporting Information
Experimental and computational details. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Authors
*(A.R.d.L.)Tel: +34-986-812316. Fax +34-986-818622. E-mail:
qolera@uvigo.es.
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The authors declare no competing financial interest.
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■
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Galicia (INBIOMED-FEDER ″Unha maneira de facer Euro-
pa″), EU FP7 (BIOCAPS 316265/REGPOT-2012-2013.1), the
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and Antagonists of Retinoid Receptors. In Methods in Enzymology;
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