Discovery of a potent retinoid X receptor antagonist structurally closely related to RXR agonist NEt-3IB

Article abstract of DOI:10.1021/ml200197e

We discovered a potent retinoid X receptor (RXR) antagonist, 6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(E)-styrylphenyl)amino]nicotinic acid (13e), that is structurally closely related to the RXR full agonist 6-[N-ethyl-N-(3-isobutoxy-4-isopropylphenyl)amin

Full text of DOI:10.1021/ml200197e

LETTER
pubs.acs.org/acsmedchemlett
Discovery of a Potent Retinoid X Receptor Antagonist Structurally
Closely Related to RXR Agonist NEt-3IB
Mariko Nakayama,† Shoya Yamada,† Fuminori Ohsawa,† Yui Ohta,† Kohei Kawata,† Makoto Makishima,‡
and Hiroki Kakuta*,†
†Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
1-1-1, Tsushima-Naka, Okayama 700-8530, Japan
‡Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho,
Itabashi-ku, Tokyo 173-8610, Japan
S Supporting Information
b
ABSTRACT: We discovered a potent retinoid X receptor (RXR) antagonist,
6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(E)-styrylphenyl)amino]nicotinic
acid (13e), that is structurally closely related to the RXR full agonist 6-[N-
ethyl-N-(3-isobutoxy-4-isopropylphenyl)amino]nicotinic acid (NEt-3IB) (4).
Compound 13e was synthesized via a simple route from 11, a methyl ester
precursor of 4. Because 11 possesses high electrophilic reactivity because of
the amino and alkoxy groups, it was readily transformed to 12 by iodization,
and the iodine atom of 12 was converted to a CÀC or CÀN bond by means
of palladium-catalyzed reaction to afford 13. Transcriptional activation assay
revealed that 13g (in which the iodine atom was replaced with an amino
group) is a weak RXR agonist, while 13d (a phenyl group), 13e (a styryl
group), and 13f (an anilino group) are RXR antagonists. Among them, 13e
was found to be more potent than the known RXR antagonist PA452 (9).
KEYWORDS: Synthetic route, nuclear receptors, agonists, antagonists,
retinoids, RXR
etinoid X receptors (RXRs) are nuclear receptors that
control gene expression in response to ligand binding, and
Figure 1 shows the chemical structures of known RXR agonists
1À74,16À20 and RXR antagonists 8À10.15,21À23 Although several
agonist/antagonist pairs, such as compounds 5 (agonist)18 and 8
(antagonist),21 6 (agonist)19 and PA452 (9; antagonist),22 and 7
(agonist)23 and 10 (antagonist),15 each possess similar skeletons,
the starting materials for their synthesis differ from each other. If
RXR agonists and antagonists could be synthesized from com-
mon starting materials, thiswould facilitate systematic RXR ligand
production and be both convenient and environmentally friendly.
RXR ligands generally contain common structural features,
having a hydrophobic domain such as 1,1,4,4-tetramethyltetralin,
an acidic domain such as an aromatic carboxyl group, and a linker
domain connecting the two.2,3 StructureÀactivity relationship
studies have shown that RXR agonists can be changed to antago-
nists by introducing an alkyl side chain into the ortho position of the
hydrophobic domain with respect to the linker.2,3,13,24 We have
R
they function as homodimers or heterodimers with other
nuclear receptors.1 Because of their biological activity, RXRs
are interesting targets for drug discovery.2,3 For example,
Targretin (bexarotene) (1),4 an RXR full agonist, has already
been launched for the treatment of cutaneous T cell lymphoma
(CTCL) in the United States.5 In addition, peroxisome prolif-
erator-activated receptors (PPARs) and liver X receptors (LXRs)
function as RXR heterodimers to control lipid/sugar metabo-
lism, and because these heterodimers can be activated even by
RXR agonists alone (permissive mechanism),6 RXR agonists are
considered to be candidate drugs for the treatment of metabolic
syndrome.7,8 However, currently known RXR agonists induce
hepatomegaly,9 hyperlipidemia,10 and hypothyroidism,11 and
other studies have aimed at finding RXR agonists without these
side effects.12 Nevertheless, RXR antagonists have been re-
ported to be effective against diabetes13 and allergic diseases14
in animal models and are also useful for analyzing various
biological phenomena involving RXRs.15
Received: August 12, 2011
Accepted: September 27, 2011
Published: September 27, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of RXR agonists 1À7 and RXR antagonists 8À10.
Scheme 1a
a Reagents and conditions. (a) I2, Ag2SO4, MeOH. (b) (i) 2 N NaOH, THF, MeOH; (ii) 2 N HCl. (c) TMS acetylene, PdCl2(PPh3)2, CuI,
triethylamine, THF. (d) K2CO3, MeOH. (e) (i) 2 N NaOH, MeOH; (ii) 2 N HCl. (f) H2, PdÀC, MeOH. (g) Phenylboronic acid, PdÀC, Na2CO3,
EtOH. (h) Styrene, Pd(OAc)2, tris(o-tolyl)phosphine, triethylamine, acetonitrile. (i) Aniline, Pd(OAc)2, (()-BINAP, Cs2CO3, toluene. (j)
Benzophenoneimine, Pd(OAc)2, (()-BINAP, Cs2CO3, toluene. (k) 2 N HCl, THF.
reported RXR full agonists NEt-3IP (3) and NEt-3IB (4), which
contain a phenyl group.17 Because these compounds have an amino
group at the linker domain and an alkoxy group at the meta position
(position-3 carbon) of the hydrophobic ring with respect to the
amino group, the position-6 carbon (C-6) is electron-rich and
reactive to electrophilic reagents. Therefore, we considered that it
should be possible to introduce an iodine atom at C-6. On the basis
of this idea, we designed a new method to synthesize RXR ligands,
not only agonists but also antagonists, via iodine derivative 12
obtained from precursor 11 (previously used in the synthesis of
potent RXR agonist 4), by substitution of the iodine atom with the
aid of palladium catalyst.
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Figure 2. Dose-dependent RXR agonistic activities of compounds
13aÀg against RXRα. The relative transactivation activity is based on
the luciferase activity of 1 μM 1, taken as 1.0.
Scheme 1 shows the synthetic scheme using 12 as a common
intermediate. Compound 12 was synthesized by iodinating
methyl ester 11 with silver sulfate. Hydrolysis of the methyl
ester of 12 gave 13a. Coupling reaction of 12 and TMS-acetylene
with palladium catalyst afforded intermediate 14,25 and depro-
tection of the TMS and methyl ester moieties gave 13b. The
acetylene moiety of 13b was catalytically reduced to afford 13c.
Moreover, 12 was reacted with phenylboronic acid by SuzukiÀ
Miyaura coupling,26 styrene by Heck reaction,27 and aniline or
benzophenoneimine by Buckwald coupling reaction,28 followed
by deprotection to give 13dÀg. The trans structure of 13e was
supported by the 1H NMR coupling constant between the ethylene
protons.29
The compounds obtained were assessed by reporter gene
assay using COS-1 cells. Compound 13g shows RXR full
agonistic activity, although it was less potent than 4 (Figure 2).
Compounds 13d, 13e, and 13f, which showed no RXR agonistic
activity, were examined for RXR antagonistic activity. Figure 3a
shows doseÀresponse curves of NEt-TMN (2), a RXR full
agonist,16 in the absence and in the presence of these compounds
or 9, an RXR antagonist, at 1 μM. The presence of 9 shifted the
response curve of 2 to a higher concentration (to the right),
showing that 9 acts as an RXR antagonist. Although 13d did not
shift the doseÀresponse curve, compounds 13e and 13f did do
so, in the same way as did 9. Among them, compound 13e,
possessing a stilbene structure, shifted the doseÀresponse curve
of 2 more strongly than 9, indicating that 13e is a more potent
RXR antagonist than 9. Thus, to assess the RXR-antagonistic
activity of 13e in detail, the pA2 value (common logarithm of the
concentration of an antagonist that doubles the EC50 value of an
agonist; used as an antagonistic activity index) was obtained from
Schild plots.30 Figure 3bÀd shows the doseÀresponse curves of 2
or 4 in the absence or presence of each concentration of 9 or 13e,
respectively. The Schild plot using 2 indicated that the pA2 values of
9 and 13e were 7.11 and 8.23, respectively, indicating that 13e is 1
order of magnitude more potent than 9. The pA2 value of 13e
against 4 was 8.26 (see the Supporting Information, S17).
Figure 3. (a) Dose-dependent RXRα agonistic activities of NEt-TMN (2),
an RXR full agonist, in the absence or presence of 9 or 13dÀf. (b) Dose-
dependent RXRα agonistic activities of 2 in the absence or presence of
compound 9. (c) Dose-dependent RXRα agonistic activities of 2 in the
absence or presence of compound 13e. (d) Dose-dependentRXRαagonistic
activities of 4 in the absence or presence of compound 13e. The transactiva-
tion activity is based on the luciferase activity of 1 μM 1, taken as 1.0.
To examine the reason why 13e shows potent RXR antag-
onistic activity, we performed a docking study using AutoDock
4.0 (Figure 4aÀc) (see the Supporting Information, S19).31
First, we were interested in the driving force that causes 13e to
induce the antagonistic form of helix 12 (H12). Bourguet et al.
suggested that RXR-full antagonist 10 induces the antagonistic
form as a consequence of steric hindrance between leucine 451
(Leu451) in H12 of the ligand binding pocket of RXRα and the
alkoxy side chain of 10, based on the superposition of 10 with the
X-ray structure of RXR-partial agonist UVI3002 in the ligand-binding
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Figure 4. (a) Antagonist 13e (yellow) modeled into the ligand-binding pocket of RXRa (PDB code: 2P1V)32 and superposed onto the agonist 4. The
white stick molecule is a RXR-partial agonist UVI3002, which binds in the ligand-binding domain of RXRα (2P1V). The asterisk indicates a steric clash
between the side chain of 13e and Leu451 in helix 12 (H12). (b) Antagonist 13e (yellow) modeled into the ligand-binding pocket of RXRα (PDB code:
3A9E).33 The white stick molecule is RXR antagonist LG100754, which binds in the ligand-binding domain of RXRα (3A9E). (c) An enlarged view of
the boxed region in b.
pocket (PDB code: 2P1V).32 Thus, according to their method,
we superposed the structure of 13e on the most stable docked
structure of 4 in the ligand binding pocket and examined its
interaction with RXRα (Figure 4a). The result reveals that the
styryl moiety of 13e lies closer to Leu451 in H12 than does the
alkoxy group of UVI3002 and thus indicates that 13e exerts its
antagonistic effect through the classical mechanism of antagonist
action. Next, to examine the binding structure of 13e in the
antagonistic form of the RXR-ligand binding pocket, docking
simulations using an X-ray structure of the RXR antagonistic
form (PDB code: 2P1V)33 were performed (Figure 4b,c). The
docking energies of 13e and 13f, which are less potent than 13e,
were calculated to be À11.60 and À11.07 kcal/mol, respectively,
indicating that 13e affords a more stable complex than 13f.
In summary, we have discovered a potent RXR antagonist,
6-[N-ethyl-N-(5-isobutoxy-4-isopropyl-2-(E)-styrylphenyl)-
amino]nicotinic acid (13e), that is structurally closely related
to the RXR full agonist 6-[N-ethyl-N-(3-isobutoxy-4-isopropyl-
phenyl)amino]nicotinic acid (NEt-3IB) (4). A series of com-
pounds 13 were synthesized from 11, a methyl ester precursor of
4, as a key intermediate, thus providing a new methodology to
synthesize RXR agonists and antagonists easily via a common
precursor. RXR agonistic and antagonistic activity assay revealed
that compound 13e is a more potent RXR antagonist than the
known antagonist 9. This synthetic route provides a convenient
approach for RXR ligand synthesis. Compound 13e is expected to
be a useful tool for analyzing biological phenomena involving RXRs.
’ AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +81(0)86-251-7963. E-mail: kakuta@pharm.okayama-u.
ac.jp.
Funding Sources
We thank the Ministry of Education, Science, Culture and Sports
of Japan, JST A-STEP research project, and Takeda Science
Foundation for financial support.
’ ACKNOWLEDGMENT
We thank Dr. Kagechika (School of Biomedical Science, Tokyo
Medical and Dental University) for kindly providing PA452. We
are also grateful to Professor Yoshio Naomoto and Dr. Takuya
Fukazawa (Department of General Surgery, Kawasaki Medical
School) for preparing plasmids.
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