Fluorescent retinoid X receptor ligands for fluorescence polarization assay

Article abstract of DOI:10.1016/j.bmcl.2010.07.011

Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino] nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.

Full text of DOI:10.1016/j.bmcl.2010.07.011

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5143–5146
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorescent retinoid X receptor ligands for fluorescence polarization assay
Shoya Yamada a, Fuminori Ohsawa a,b, Shuji Fujii a, Ryosuke Shinozaki a, Makoto Makishima c,
Hirotaka Naitou d, Shuichi Enomoto a,b, Akihiro Tai e, Hiroki Kakuta a,
*
a Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Okayama 700-8530, Japan
b Multiple Molecular Imaging Research Laboratory, RIKEN Center for Molecular Imaging Science, Minatojima-minamimachi 6-7-3, Chuo-ku, Kobe, Hyogo 650-0047, Japan
c Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
d Graduate School of Nutritional and Environmental Sciences and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
e Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and
type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X
receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or
binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable
for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization
(FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands
by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected
the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-
yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of
RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should
be a lead compound for use in high-throughput assay systems for screening RXR ligands.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 23 May 2010
Revised 25 June 2010
Accepted 6 July 2010
Available online 8 July 2010
Keywords:
Retinoid X receptor
Fluorescence polarization
Molecular design
Carbostyril
Retinoid X receptors (RXRs) are nuclear receptors that func-
tion as homodimers or heterodimers with other nuclear receptors,
such as peroxisome proliferator-activated receptors (PPARs), liver
X receptors (LXRs), farnesoid X receptor (FXR), retinoic acid recep-
tors (RARs), thyroid hormone receptors (TRs), and so on.1,2 In RXR-
heterodimers, RXRs are involved in stabilization of the binding of
the heterodimer partner to DNA.2 RXR agonists can stabilize the
heterodimeric structures and sometimes potentiate the agonistic
activity of the heterodimer partners. For example, an RAR agonist
at a low concentration which does not induce cell differentiation
by itself may induce cell differentiation upon synergistic activation
of RAR/RXR with an RXR agonist.3 Moreover, PPARs, which are target
molecules for treatment of metabolic syndrome and type 2 diabetes,
and LXRs, whichare involvedin lipid and glucosemetabolism, canbe
activated synergistically by RXR agonists in combination with PPAR
or LXR agonists, respectively, and also can be activated by RXR ago-
nists alone (permissive mechanism) to promote transcription of
downstream genes.4 Thus, RXRs are attractive targets for the treat-
ment of metabolic syndrome and type 2 diabetes.5,6
philic RXR agonists, NEt-3IP (5), and NEt-3IB (6).12–14 Interestingly,
3 and 4 have different characteristics in activation of permissive
heterodimers PPAR/RXR and LXR/RXR,15 although we have ob-
tained similar results with carboxy bioisosteric derivatives.16 In
other words, RXR agonists can show a structure-dependent activa-
tion profile towards RXR-heterodimers, and thus are attractive
candidates for application as RXR-heterodimer modulators.
For the screening of RXR ligands, reporter gene assays based on
gene transactivation or binding assays using radioisotopes are gen-
erally used.17 Though the former approach is useful for the verifi-
cation of RXR-agonistic or antagonistic activities, it takes several
days. On the other hand, although the latter approach offers high
sensitivity, the use of radioisotopes is inconvenient. We considered
that safe, simple, and inexpensive high-throughput RXR ligand
screening might be attained by using fluorescence polarization
(FP) assay systems. However, no fluorescent RXR ligand has yet
been reported. Here we describe the molecular design, synthesis,
and characterization of fluorescent RXR ligands suitable for fluo-
rescence polarization-based screening of RXR ligands.
Known RXR agonists include 9-cis retinoic acid (1), an endoge-
nous ligand of RXRs, and LGD1069 (2), which is approved in the
USA for treatment of cutaneous T cell lymphoma, HX630 (3),
PA024 (4), and so on (Fig. 1).2,3,7–11 We have also created less lipo-
Fluorescent ligands for FP assay are generally created by intro-
duction of a fluorophore, such as fluorescein or rhodamine, into an
existing ligand via a spacer.18–20 However, X-ray structure analysis
of RXRs indicates that there is insufficient space at the ligand-bind-
ing site for the introduction of a fluorophore or a spacer (e.g.,
1FBY). Thus, we considered that a part of the RXR ligand would
have to be converted to a fluorophore. Focusing on RXR agonist
* Corresponding author. Tel./fax: +81 (0)86 251 7963.
E-mail address: kakuta@pharm.okayama-u.ac.jp (H. Kakuta).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.011

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S. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5143–5146
Figure 1. Chemical structures of known RXR agonists.
Figure 2. Design strategy of fluorescent RXR ligands.
Scheme 1. Reagents and conditions: (a) CH3COCH2CO2Et, water, microwave, 150 °C, 46%. (b) CF3COCH2CO2Et, p-TsOH, EtOH, reflux, 42%. (c) RI (R = Me, Et), NaH, DMF or i-
BuBr, NaH, KI, DMF, rt 22–72%. (d) 6-Chloronicotinic acid methyl ester, p-TsOH, dioxane, DMSO, reflux, 20–87%. (e) EtI, NaH, DMF, rt 13–67%. (f) NaOHaq, MeOH, 60 or 65 °C,
19–97%.

S. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5143–5146
5145
400
8b
8c
8d
2
8d (1 μM)
100
75
50
25
0
8d (1 μM) + 2 (1 μM)
350
300
250
200
10-10
10-9
10-8
10-7
10-6
10-5
0.01
0.1
1
Concentration (M)
Concentration of RXRα (mg/mL)
Figure 3. Co-transfection data (RXRa) for synthetic compounds and known RXR
agonist LGD1069 (2) in COS-1 cells. Data are the average (n = 3) SEM.
Figure 5. Fluorescence polarization values upon addition of RXR
HEPES buffer. Data are the average (n = 3) SEM.
a
to 8d (1
lM) in
Table 1
with corresponding alkyl halides and sodium hydride in dry DMF
to afford anilino derivatives 11. After coupling reaction of the ob-
tained anilino derivatives and methyl 6-chloronicotinate with a
catalytic amount of p-tosylic acid in dioxane and DMSO, N-alkyl-
ation at the linking group and deprotection of the carboxylic group
afforded the target molecules 8.
RXR-agonistic activity of 8a–d, as an indicator of RXR binding
activity, was evaluated by reporter gene assay. While 8a showed
no RXR-agonistic activity (data not shown), 8b–d show RXR-ago-
Fluorescence properties of 8b–d (10
l
M) in PBS
R
N
Et
N
O
N
CO2H
CF3
Compound
R
Exa (nm)
Emb (nm)
Intensityc (arb. units)
nistic activity with EC50 values of about 1 lM, although they are
less potent than 2 (Fig. 3).
8b
8c
8d
Me
Et
i-Bu
372
368
368
494
496
493
5.60
7.90
14.5
Next, the fluorescence properties of 8b–d were evaluated (Table
1). Each compound showed a fluorescence excitation maximum at
around 370 nm, and emission at around 490 nm. As the N-alkyl
chain at the lactam ring was extended, the relative fluorescence
intensity tended to increase. Thus, we selected 8d as the most po-
tent RXR agonist, with the highest fluorescence intensity, and eval-
uated its FP characteristics according to the procedure reported by
Peterson et al.19 Figure 4a shows the FP of 8d in glycerol solution, a
viscous liquid, that impedes solute motion. As the ratio of glycerol
in the solution was increased, increasing FP of 8d was observed.
The fluorescence intensity of 8d increased in proportion to the con-
centration of 8d, while the FP value remained constant, at a fixed
ratio of glycerol (Fig. 4b).
a
b
c
Excitation maximum wavelength.
Emission maximum wavelength.
Fluorescent intensity shown by arbitrary units.
7, in which the structure of the so-called lipophilic domain resem-
bles a fluorescent carbostyril structure, we designed 8, which pos-
sesses a carbostyril structure as its lipophilic domain in the general
RXR structure (Fig. 2).21
The synthetic scheme is shown in Scheme 1, starting from m-
phenylenediamine. 7-Amino-4-methyl-1H-quinolin-2-one (10a:
R = methyl) was synthesized by the use of microwave irradiation
at 150 W and 150 °C for 80 min,22 while 7-amino-4-trifluoro-
methyl-1H-quinolin-2-one (10b: R = trifluoromethyl) was pre-
pared by Knorr condensation with ethyl acetoacetate or ethyl
1,1,1-trifluoroacetoacetate and a catalytic amount of p-tosylic acid
in ethanol.23 N-Alkylation at the lactam ring of 10 was performed
In order to evaluate the binding of 8d to RXR, we added RXR
a
protein to a solution of 1 M 8d in PBS buffer (Fig. 5). With increas-
l
ing RXR concentration, the FP value of 8d was increased, indicating
that 8d was bound to RXR. Moreover, addition of 2 to the solution
decreased the FP value, indicating that binding of 8d is competitive
b
a
500
450
400
500
Intensity
Polarization
450
400
350
320
350
320
0
2
4
6
8
10
0
20
40
60
80
100
Concentration of 8d (μM)
% Glycerol in PBS
Figure 4. Fluorescence polarization characteristics of 8d. (a) Fluorescence polarization calibration curve of 8d (1
values as a function of the concentration of 8d in PBS. Data are the average (n = 3) SEM.
lM) in glycerol/PBS buffer. (b) Intensity and polarization

5146
S. Yamada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5143–5146
4. Mukherjee, R.; Davies, P. J.; Crombie, D. L.; Bischoff, E. D.; Cesario, R. M.; Jow, L.;
Hamann, L. G.; Boehm, M. F.; Mondon, C. E.; Nadzan, A. M.; Paterniti, J. R., Jr.;
Heyman, R. A. Nature 1997, 386, 407.
with that of 2, and therefore presumably occurs at the same site in
RXR.
We designed, synthesized, and characterized fluorescent RXR
agonists that were expected to be suitable for high-throughput
FP screening of RXR ligands. One of them, 6-[ethyl(1-isobutyl-2-
oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic
acid 8d, was concluded to be suitable for this purpose. Further
work is planned to increase the RXR-agonistic activity and fluores-
cence intensity of 8d. The strategy presented here is expected to be
useful for creation and development of other fluorescent RXR ago-
nists for RXR ligand screening systems.
5. Pinaire, J. A.; Reifel-Miller, A. PPAR Res. 2007, 2007, 604.
6. Leibowitz, M. D.; Ardecky, R. J.; Boehm, M. F.; Broderick, C. L.; Carfagna, M. A.;
Crombie, D. L.; D’Arrigo, J.; Etgen, G. J.; Faul, M. M.; Grese, T. A.; Havel, H.; Hein,
N. I.; Heyman, R. A.; Jolley, D.; Klausing, K.; Liu, S.; Mais, D. E.; Mapes, C. M.;
Marschke, K. B.; Michellys, P. Endocrinology 2006, 147, 1044.
7. Heyman, R. A.; Mangelsdorf, D. J.; Dyck, J. A.; Stein, R. B.; Eichele, G.; Evans, R.
M.; Thaller, C. Cell 1992, 68, 397.
8. Boehm, M. F.; Zhang, L.; Badea, B. A.; White, S. K.; Mais, D. E.; Berger, E.; Suto, C.
M.; Goldman, M. E.; Heyman, R. A. J. Med. Chem. 1994, 37, 2930.
9. Gottardis, M. M.; Bischoff, E. D.; Shirley, M. A.; Wagoner, M. A.; Lamph, W. W.;
Heyman, R. A. Cancer Res. 1996, 56, 5566.
10. Rizvi, N. A.; Marshall, J. L.; Dahut, W.; Ness, E.; Truglia, J. A.; Loewen, G.; Gill, G.
M.; Ulm, E. H.; Geiser, R.; Jaunakais, D.; Hawkins, M. J. Clin. Cancer Res. 1999, 5,
1658.
Acknowledgments
11. Cohen, M. H.; Hirschfeld, S.; Honig, S. F.; Ibrahim, A.; Johnson, J. R.; O’leary, J. J.;
White, R. M.; Williams, G. A.; Pazdur, R. Oncologist 2001, 6, 4.
12. Takamatsu, K.; Takano, A.; Yakushiji, N.; Morishita, K.; Matsuura, N.;
Makishima, M.; Ali, H. I.; Akeho, E.; Tai, A.; Sasaki, K.; Kakuta, H.
ChemMedChem 2008, 3, 454.
13. Takamatsu, K.; Takano, A.; Yakushiji, N.; Morohashi, K.; Morishita, K.;
Matsuura, N.; Makishima, M.; Tai, A.; Sasaki, K.; Kakuta, H. ChemMedChem
2008, 3, 780.
The authors are grateful to the SC-NMR Laboratory of Okayama
University for the NMR experiments. This work was partially sup-
ported by a Grant-in Aid for Scientific Research on Priority Areas
from the Ministry of Education, Science, Culture and Sports of Ja-
pan. The authors are also grateful to Mr. Ryosuke Fukai, Ms. Mariko
Nakayama, and Mr. Kohei Kawata for helpful discussions during
the preparation of this manuscript.
14. Morishita, K.; Yakushiji, N.; Ohsawa, F.; Takamatsu, K.; Matsuura, N.;
Makishima, M.; Kawahata, M.; Yamaguchi, K.; Tai, A.; Sasaki, K.; Kakuta, H.
Bioorg. Med. Chem. Lett. 2009, 19, 1001.
15. Nishimaki-Mogami, T.; Tamehiro, N.; Sato, Y.; Okuhira, K.; Sai, K.; Kagechika,
H.; Shudo, K.; Abe-Dohmae, S.; Yokoyama, S.; Ohno, Y.; Inoue, K.; Sawada, J.
Biochem. Pharmacol. 2008, 76, 1006.
Supplementary data
16. Fujii, S.; Ohsawa, F.; Yamada, S.; Shinozaki, R.; Fukai, R.; Makishima, M.;
Enomoto, S.; Tai, A.; Kakuta, H. Bioorg. Med. Chem. Lett. 2010, 20, 5139.
17. Boehm, M. F.; McClurg, M. R.; Pathirana, C.; Mangelsdorf, D.; White, S. K.;
Hebert, J.; Winn, D.; Goldman, M. E.; Heyman, R. A. J. Med. Chem. 1994, 37, 408.
18. Parker, G. J.; Law, T. L.; Lenoch, F. J.; Bolger, R. E. J. Biomol. Screen. 2000, 5, 77.
19. DeGrazia, M. J.; Thompson, J.; Heuvel, J. P. V.; Peterson, B. R. Bioorg. Med. Chem.
2003, 11, 432.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.011.
References and notes
20. Han, K.; Kim, J. H.; Kim, K.; Kim, E. E.; Seo, J.; Yang, E. G. Anal. Biochem. 2010,
398, 185.
21. Saroja, G.; Sankaran, N. B.; Samanta, A. Chem. Phys. Lett. 1996, 249, 392.
22. Lee, H.; Cao, H.; Rana, T. M. J. Comb. Chem. 2005, 7, 279.
23. Oeveren, A.; Pio, B. A.; Tegley, C. M.; Higuchi, R. I.; Wu, M.; Jones, T. K.;
Marschke, K. B.; Negro-Vilar, A.; Zhi, L. Bioorg. Med. Chem. Lett. 2007, 17, 1523.
1. Rowe; Eager, N. S. C.; Brickell, P. M. Development 1991, 111, 771.
2. Umemiya, H.; Kagechika, H.; Fukasawa, H.; Kawachi, E.; Ebisawa, M.;
Hashimoto, Y.; Eisenmann, G.; Erb, C.; Pornon, A.; Chambon, P.; Gronemeyer,
H.; Shudo, K. Biochem. Biophys. Res. Commun. 1997, 233, 121.
3. Ohta, K.; Kawachi, E.; Inoue, N.; Fukasawa, H.; Hashimoto, Y.; Itai, A.;
Kagechika, H. Chem. Pharm. Bull. 2000, 48, 1504.