Modification at the lipophilic domain of RXR agonists differentially influences activation of RXR heterodimers

Article abstract of DOI:10.1021/ml100184k

RXR permissive heterodimers are reported to be activated differently depending upon the chemical structure of RXR agonists, but the relationship of agonist structure to differential heterodimer activation has not been explored in detail. In this study, we

Full text of DOI:10.1021/ml100184k

pubs.acs.org/acsmedchemlett
Modification at the Lipophilic Domain of RXR Agonists
Differentially Influences Activation of RXR Heterodimers
Fuminori Ohsawa,†,‡ Ken-ichi Morishita,† Shoya Yamada,† Makoto Makishima,§ and
Hiroki Kakuta*,†
†Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, 1-1-1, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan, ‡Multiple Molecular Imaging Research Laboratory, RIKEN
Center for Molecular Imaging Science, Minatojima-minamimachi 6-7-3, Chuo-ku, Kobe, Hyogo 650-0047, Japan, and
§Division of Biochemistry, Nihon University School of Medicine, Itabashi-Ku, Tokyo 173-8610, Japan
ABSTRACT RXR permissive heterodimers are reported to be activated differently
depending upon the chemical structure of RXR agonists, but the relationship of
agonist structure to differential heterodimer activation has not been explored in
detail. In this study, we performed systematic conversion of the alkoxy side chain of
5a (6-[ethyl-(3-isopropoxy-4-isopropylphenyl)amino]nicotinic acid, NEt-3IP) and
evaluated the RXR-, PPAR/RXR-, and LXR/RXR-agonist activities of the products.
The cyclopropylmethoxy analogue (5c) showed similar RXR- and LXR/RXR-
agonistic activities to the benzyloxy analogue (5i) and n-propoxy analogue (5k)
but exhibited more potent PPAR/RXR-agonistic activity than 5i or 5k. Differential
modulation of RXR heterodimer-activating ability by conversion of the alkoxy
group located in the lipophilic domain of the RXR-agonist common structure is
expected be a useful approach in the design of new RXR agonists for the treatment
of hyperlipidemia or type 2 diabetes.
KEYWORDS RXR agonists, permissive heterodimers, PPAR, LXR, heterodimer
activation, reporter gene assay
etinoid X receptors (RXRs) are nuclear receptors that
act as ligand-dependent transcription factors, func-
tioning as homodimers or as heterodimers with
disorders associated with cutaneous T-cell lymphoma
(CTCL).13-15 Moreover, several RXR agonists, including 1,
are under investigation for the treatment of metabolic
syndrome.16 However, repeated administration of RXR ago-
nists can elevate blood triglycerides (TGs) and induce hypo-
thyroidism.17-19 However, recently, PA024 (2) and HX630
(3) were reported to show differential transcriptional activa-
tion activities,20 raising the possibility that RXR agonists
without the above side effects may be obtainable by means
of appropriate structural modification.
The general chemical structure of RXR agonists can be
divided into a lipophilic domain based on 1,1,4,4-tetra-
methyltetralin structure, an acidic domain comprising ben-
zoic acid or nicotinic acid, and a linking domain connecting
the other two domains. So far, to create RXR agonists without
the side effects described above and to examine the influ-
ence of the carboxylic acid moiety in the acidic domain on
RXR-heterodimer activation, we have synthesized com-
pounds whose acidic domains contain carboxylic acid
bioisosters, such as a 5-tetrazolyl or hydroxamic acid group,
and evaluated their RXR-, PPAR/RXR-, and LXR/RXR-agonist
R
peroxisome proliferator-activated receptors (PPARs), liver X
receptors (LXRs), farnesoid X receptors (FXRs), and retinoic
acid receptors (RARs).1-3 Among RXR heterodimers, PPARγ/
RXR is known to be a target of thiazolidinediones (TZDs), which
are used for the treatment of insulin resistance,4 and LXR/RXR
is reported to be involved in glucose/lipid metabolism.5,6
Although these heterodimers can be activated by PPAR ago-
nists or LXR agonists, respectively, synergistic activation with
RXR agonists7 and activation by RXR agonists alone8 are also
known to occur. The RXR-heterodimer partners, which can be
activated by RXR agonists alone, are called permissive hetero-
dimer partners.8 Because the permissive mechanism is
thought to be relevant to several RXR heterodimers associated
with glucose/lipid metabolism, RXR agonists are considered to
be candidate therapeutic agents for the treatment of chronic
disorders such as metabolic syndrome. In addition, because
PPARγ or LXRR may influence insulin resistance in type 2
diabetes and autoimmune disease, including rheumatism
(RA),9-12 modulation of RXR heterodimers containing PPARγ
or LXRR with RXR agonists may be a promising approach for
the treatment of these diseases.
Received Date: August 5, 2010
LGD1069 (1) (Targretin; Figure 1), an RXR agonist, is used
clinically in the United States for the treatment of skin
Accepted Date: August 24, 2010
Published on Web Date: August 27, 2010
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Scheme 1a
Figure 1. Chemical structures of known RXR ligands 1-5.
activities.21 We found that structural modification at this
location does not influence RXR-heterodimer activation, and
the RXR-heterodimer-agonist activities correspond well with
the RXR-agonistactivities.21 On the other hand, to create less
lipophilic RXR agonists, NEt-3IP (5a) and NEt-3IB (5b)
(Figure 1), which possess an alkoxy group in the lipophilic
domain, were synthesized.22 Because known RXR agonists
have a 1,1,4,4-tetramethyltetralin structure, structural con-
version of this domain is of interest from the viewpoint of not
only RXR-agonistic activity but also RXR-heterodimer acti-
vation. Therefore, we converted the isopropoxy group of 5a
to a hydroxyl group by treatment with aluminum chloride
and alkylated the resulting intermediate. The RXR- and RXR-
heterodimer-agonist activities of the products were exam-
ined. We found that while 1, 4a, and 5b show similarly
potent RXR-agonistic activities, their patterns of PPAR/RXR-
and LXR/RXR-agonist activities were different. Compound
5c (cyclopropylmethoxy) showed similar RXR- and LXR/
RXR-agonist activities to 5i (n-pentyloxy group) and 5k
(benzyloxy), but it was a significantly more potent activator
of PPAR/RXR, as compared with 5i and 5k. In this letter, we
report the patterns of RXR- and RXR-heterodimer-agonist
activities of modified RXR agonists bearing an alkoxy group
in the lipophilic domain.
aReagents and conditions: (a) AlCl3, CH2Cl2. (b) Alkyl halide, K2CO3, KI,
DMF. (c) NaOH(aq), MeOH, THF.
Table 1. Cotransfection Data for 1, 4a, and 5a-m in COS-1 Cells
Various alkoxy derivatives were obtained from 6 as shown
in Scheme 1. Deisopropylation of intermediate 6, the synth-
esis of which has already been reported,22 was performed by
using aluminum chloride in methylene chloride at room
temperature. Compound 7 thus obtained was alkylated with
various alkyl halides in the presence of potassium carbonate
and potassium iodide in DMF. Then, de-esterification under
alkaline conditions afforded the desired products 5.
The transactivation assay of the compounds was per-
formed by a reporter gene assay in COS-1 cells. The results
for RXRR are shown in Table 1. Because 5b showed signifi-
cant RXR-agonistic activity, cyclopropylmethyl derivative 5c
and compounds 5d and 5e, which possess a double bond,
were evaluated. Although their RXR-agonistic activity was
reduced, each compound showed an EC50 value of approxi-
mately 100 nM. Compound 5e bearing a 1,1,1-trifluoroethyl
group showed similar RXR-agonistic activity to the com-
pounds possessing a double bond, so we next focused on
aAll values represent the standard error of the mean value of at least
three separate experiments with triplicate determinations. bEC50 values
were determined from full dose-response curves ranging from 10-8 to
10-5 M in COS-1 cells. c The luciferase activity of 1 at 1 μM was defined
as 100%.
compounds possessing a linear alkyl chain on the alkoxy
group. Interestingly, elongation of the alkyl chain to n-propyl,
n-butyl, or n-pentyl increased the RXR-agonistic activity.
Even n-hexyl derivative 5j showed an EC50 value of about
300 nM, indicating that there is a cavity around the so-called
lipophilic moiety of RXR agonists in the ligand-binding domain
of RXR. Thus, the RXR-agonistic activity of compounds bearing
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DOI: 10.1021/ml100184k ACS Med. Chem. Lett. 2010, 1, 521–525
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a phenyl group was evaluated. All of the compounds examined
showed an EC50 value of several hundred nanomolar.
Although the activity was slightly reduced, these results in-
dicate that the cavity of RXR that contacts the 3-alkoxy group
of our RXR agonists is of sufficient size to accommodate a
phenyl ring.
Because compounds 5a-m possessing various alkoxy side
chains showed rather similar RXR-agonistic activity despite
their structural differences, we performed a reporter gene
assay with PPAR, LXR, PPAR/RXR, and LXR/RXR, anticipating
RXR-permissive/synergistic action. Figure 2 shows the dose-
dependent plots of selected compounds for RXRR. Com-
pounds 1, 4a, and 5b (open circles, triangles, and squares)
all showed extremely similar RXR-agonistic activities, as did
5c, 5i, and 5k (closed circles, triangles, and squares).
First, we took the three compounds in the former group
and examined their agonistic activities with PPARγ, LXRR,
PPAR/RXR, and LXR/RXR (Figure 3). The reason why PPARγ
and LXRR were selected from among the various possible
RXR-heterodimer partners is that these receptors have
been well studied, and their modulation is associated with
improved insulin resistance, improved glucose control,
anti-inflammatory activity, and improved autoimmune
regulation.5,9,11,23-26 These compounds did not activate
PPARγ but slightly activated LXR. Every compound activated
each RXR heterodimer, but interestingly, 1 was not active at a
low concentration, differently from 4a and 5b. These results
are consistent with a previous report20 showing that the
pattern of heterodimeric activation is dependent on the
chemical structure of RXR agonists. In the concentration
range from 10-7 to 10-6 M, 4a and 5b showed similar RXR-
agonistic activity (Figure 2), but the activity toward PPAR/
RXR was weaker (Figure 3), indicating that alteration of the
lipophilic domain of RXR agonists can differentially modu-
late the RXR-heterodimer-activating ability.
Next, a similar study was performed on compounds 5c, 5i,
and 5k, belonging to the latter group shown in Figure 2.
Although LXR/RXR heterodimer was activated similarly by
each compound, the PPAR/RXR heterodimer was more
potently activated by 5c than by the other two compounds
(Figure 4). Because these compounds differ only in the
alkoxy side chain, it appears that conversion of the alkoxy
group in the lipophilic domain of RXR agonists can differen-
tially influence the heterodimer-activating ability.
Activation of LXR is reported to improve disordered
glucose metabolism in type 2 diabetes.5 However, excessive
activation of LXR induces SREBP-1c expression, resulting in
elevation of blood TGs.27 To avoid side effects of RXR
agonists, including TG elevation, moderate activation of
LXR/RXR appears to be desirable. Similarly, although activa-
tion of PPARγ improves insulin resistance, inflammation,
and rheumatoid arthritis,4,10-12,23-26 excessive activation of
PPARγ can also cause edema and obesity.28 Therefore, RXR
Figure 2. Results of RXRR reporter gene assay for 1 (open circle),
4a (open triangle), 5b (open square), 5c (closed circle), 5i (closed
triangle), and 5k (closed square).
Figure 3. Relative transactivation data of 1 (open circle), 4a (open triangle), and 5b (open square) toward PPARγ, PPARγ/RXRR, LXRR, and
LXRR/RXRR.
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Figure 4. Relative transactivation data of 5c (closed circle), 5i (closed triangle), and 5k (closed square) toward PPARγ, PPARγ/RXRR, LXRR,
and LXRR/RXRR.
agonists that activate these heterodimers moderately with-
out causing side effects are attractive candidates for clinical
application.
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