RXR partial agonist produced by side chain repositioning of alkoxy RXR full agonist retains antitype 2 diabetes activity without the adverse effects

Article abstract of DOI:10.1021/jm501863r

We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid: 5, EC₅₀ = 143 nM, E_max = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: 8b, EC₅₀ = 169 nM, E_max = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: 7b, EC₅₀ = 19 nM). NEt-4IB (8b) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to 5.

Full text of DOI:10.1021/jm501863r

Article
pubs.acs.org/jmc
RXR Partial Agonist Produced by Side Chain Repositioning of Alkoxy
RXR Full Agonist Retains Antitype 2 Diabetes Activity without the
Adverse Effects
Kohei Kawata,† Ken-ichi Morishita,† Mariko Nakayama,† Shoya Yamada,†,‡ Toshiki Kobayashi,†
Yuki Furusawa,† Sakae Arimoto-Kobayashi,† Toshitaka Oohashi,§ Makoto Makishima,∥ Hirotaka Naitou,⊥
Erika Ishitsubo,# Hiroaki Tokiwa,#,∇ Akihiro Tai,○ 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
‡Research Fellowship Division, Japan Society for the Promotion of Science, Sumitomo-Ichibancho FS Bldg., 8 Ichibancho,
Chiyoda-ku, Tokyo102-8472, Japan
§Division of Medical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan
∥Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho,
Itabashi-ku, Tokyo 173-8610, Japan
⊥Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
#Department of Chemistry, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
∇Research Center for Smart Molecules, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
○Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka-cho, Shobara, Hiroshima 727-0023,
Japan
S
* Supporting Information
ABSTRACT: We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-
1H-benzotriazole-5-carboxylic acid: 5, EC50 = 143 nM, Emax = 75%), which showed a potent glucose-lowering effect without
causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the
glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated
differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB
(6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: 8b, EC50 = 169 nM, Emax = 55%), can be obtained simply by
repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist
NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: 7b, EC50 = 19 nM). NEt-4IB (8b) showed
antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to 5.
treat such diseases with drugs targeting specific receptors or
enzymes associated with each individual disease. In contrast,
INTRODUCTION
■
The pathogenesis of many diseases associated with a modern
lifestyle, such as cancer, circulatory disease, type 2 diabetes, and
Alzheimer’s disease, often appears to involve impaired homeo-
stasis of major body systems. Western medicine generally aims to
Received: October 5, 2014
Published: December 8, 2014
© 2014 American Chemical Society
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Figure 1. Chemical structures of RXR ligands 1, 4−8, PPARγ agonist 2, and LXR agonist 3.
traditional Chinese medicine or Japanese Kampo medicine, which
is a Japanese development of traditional Chinese medicine, aims
to treat such diseases by normalizing or improving patients’
overall physical condition. Our aim is to develop a westernized
Kampo medicine1 by synthesizing small molecules that target
receptors or enzymes but focusing on targets that play critical
roles in whole homeostatic systems and regulatory networks.
With this goal in mind, we have focused on retinoid X receptors
(RXRs), which are key regulators of sugar/lipid metabolism.
RXRs are nuclear receptors that control gene transcription
dependently upon ligand binding.2 RXRs act as homodimers or
as heterodimers with peroxisome proliferator-activated receptors
(PPARs), liver X receptors (LXRs), and other receptors.3−5
PPARs are involved in glucose/lipid metabolism, and the PPARγ
agonist pioglitazone (2) (Figure 1) is used for the treatment of
type 2 diabetes.6 LXRs control glucose/cholesterol metabo-
lism,7,8 and the LXR agonist T0901317 (3) (Figure 1) is reported
to elevate HDL cholesterol and to decrease very low density
lipoprotein (VLDL), low density lipoprotein (LDL) cholesterol,
and arteriosclerosis lesion area in LDL receptor knockout
(LDLR KO) mice fed a high-fat diet.9 Heterodimers of RXRs
with PPARs and LXRs are known to be activated synergistically
by RXR agonists in the presence of agonists of the heterodimer
partners.10,11 Moreover, interestingly, these heterodimers can be
activated by RXR agonists alone even in the absence of any
heterodimer partner agonist (so-called permissive effect).12,13
Thus, targeting RXRs is a reasonable strategy to modulate glucose/
lipid metabolism, which is deeply related to diseases associated
with a modern lifestyle, via the synergistic or permissive effect
of RXRs.
agonists, including bexarotene (1) and NEt-TMN (4),19,20 cause
various side effects including elevation of blood triglyceride,21,22
weight gain,23 hepatomegaly,23,24 and hypothyroidism.25 Thus,
we are interested in finding ways to eliminate the side effects of
RXR agonists, by analogy with the idea of reviving abandoned
drugs by eliminating or avoiding their side effects.1
The adverse effects mentioned above are all associated with
RXR full agonists, which maximally activate RXRs. We con-
sidered that moderate activation of RXR might be enough to
normalize the body systems, and so we focused on RXR partial
agonists whose maximum activation of RXR is lower than that
of RXR full agonists or whose transcriptional efficacy is restricted
to a particular subset of genes. Although several RXR partial
agonists have been reported,26−28 only CBt-PMN (5) (Figure 1)
has been evaluated in vivo.29 This compound showed a potent
glucose-lowering effect, together with improvements of insulin
secretion and glucose tolerance in type 2 diabetes model mice,
without inducing the serious adverse effects caused by RXR full
agonists. We also discussed the mechanism of the RXR partial
agonistic activity of 5 and reported that 6 is an RXR partial
agonist having similar Emax (67 2%) and lower EC50 (15
0 nM) to those of 5 (Emax = 75 4%, EC50 = 143 2 nM).30
However, the level of RXR activating efficacy required to achieve
a glucose-lowering effect remains unclear. In addition, the
beneficial or adverse effects of RXR agonists may be related to
not only RXR activation, but also RXR heterodimer activation,
because RXR agonists may differentially activate RXR hetero-
dimers.31,32 Thus, in this work, we created a novel RXR partial
agonist having a distinct chemical structure from 5 and whose
activating efficacy toward RXR is weaker than that of 5 and we
evaluated its beneficial effects and side effects in mice and rats.
RXR agonists are well-known to be effective in the treat-
ment of various diseases.14 For example, RXR full agonist
bexarotene (LGD1069: 1) (Figure 1) is used for the treatment of
cutaneous T cell lymphoma (CTCL) in the United States15 and
is also effective against type 2 diabetes, metabolic syndrome,16
Alzheimer’s disease,17 and Parkinson’s disease.18 However, RXR
RESULTS AND DISCUSSION
■
To create new RXR partial agonists, we focused on NEt-4IP (8a),
which was previously found during the process of developing
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a
Scheme 1
a
(a) HNO3, ZnCl2, EtOAc, ultrasonication, 48%; (b) i-PrBr, K2CO3, KI, DMF, 80%; (c) (1) H2, Pd/C, MeOH, (2) 6-chloronicotinic acid,
MeSO3H, dioxane, (3) H2SO4, MeOH, 73%; (d) Et-I, NaH, DMF, 75%; (e) AlCl3, CH2Cl2, 97%; (f) R-X, K2CO3, KI, DMF, 16−94%; (g) (1) 2 N
NaOH, MeOH, (2) 2 N HCl, 12%−93%.
Table 1. Transactivation Activity of Compounds 8a−k toward RXRα in COS-1 Cells
a,b
RXRα
efficacy (%)
10 μM
c
c
c
compd
R
1 μM
EC50 (nM)
22
293 29
169
Emax (%)
lowest conc for Emax (μM)
1
100
85
6
3
3
0
6
2
3
3
1
1
2
2
2
100
3.8 0.9
4.6 1.5
8a
8b
8c
8d
8e
8f
i-Pr
72
1
0
6
6
2
3
2
1
1
3
2
83
54
64
83
84
75
58
51
75
49
44
83
55
62
83
84
73
57
52
73
45
42
2
2
2
3
3
3
4
1
1
1
2
i-Bu
53
52
78
77
53
37
28
52
15
20
3
4.0 1.5
c-Pr-CH2
CH2CMe-CH2
Me2CCH-CH2
n-Bu
324 62
218 55
268 32
567 57
772 80
918 53
14.7 7.7
12.9 10.1
8.4 5.9
20.0 0.0
12.7 8.7
28.7 4.3
24.3 4.3
4.0 5.8
8g
8h
8i
n-Pen
n-Hex
Ph-CH2
540
4
8j
Ph-(CH2)2
Ph-(CH2)3
1130 60
1030 80
8k
10.0 5.0
a
COS-1 cells were transfected with three kinds of vectors consisting of RXRα, a luciferase reporter gene under the control of the appropriate RXR
b
response element (CRBPII-tk-Luc), and secreted alkaline phosphatase (SEAP) gene as a background. The relative transactivation activity is based
on the luciferase activity of 1 μM 1 (RXR full agonist) taken as 100%. All values represent the mean value of at least three separate experiments with
c
triplicate determinations. Each value was determined from full dose−response curves.
RXR full agonists NEt-3IP (7a) and NEt-3IB (7b), because 8a
showed a lower RXR activation efficacy than either 7a or 7b.33
Additionally, because changing the isopropoxy group of 7a to
various other alkoxy groups afforded ligands with distinct RXR
heterodimer activation properties,30 we applied this approach to
the isopropoxy group of 8a.
Various alkoxy derivatives were synthesized according to
Scheme 1. 2-Isopropylphenol (12) was nitrated in the presence of
zinc chloride under ultrasonication to give 13, and the hydroxyl
group was converted to an isopropoxy group to afford 14.
Reduction of the nitro group was performed, and the coupling
reaction of the resulting amine and 6-chloronicotinic acid in the
presence of mesyl acid as a catalyst gave 15. Next, 16 was obtained
by N-ethylation of the linking nitrogen, and deprotection of the
isopropoxy group of 16 with aluminum chloride afforded com-
mon intermediate 17. Reaction of 17 with various alkyl halides
and hydrolysis of the methyl ester gave the target molecules 8a−j.
RXR-activating efficacy of the compounds obtained was
evaluated using a luciferase transcription assay in COS-1 cells.
The data are shown in Table 1 as relative values with respect to
100% activation by RXRfull agonist 1 at 1 μM. All the compounds
synthesized showed all reduced Emax values compared to 1.
Because we wished to know whether compounds whose RXR
activation efficacy is lower than that of 5 show pharmacological
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Figure 2. Evaluation of transactivation activity toward RXRα and cofactor recruitment. (a) Relative transactivation activity of CBt-PMN (5: closed black
circles), CBTF-PMN (6: closed purplish-red triangles), NEt-3IB (7b: closed green triangles), and 8b (closed blue squares) toward RXRα. Blue broken
lines and dotted lines show the activity of 8b toward RXRβ and RXRγ, respectively. COS-1 cells were transfected with three kinds of vectors consisting of
an RXR receptor subtype, a luciferase reporter gene under the control of the appropriate RXR response element (CRBPII-tk-Luc), and secreted alkaline
phosphatase (SEAP) gene as a background. The results for 5 and 6 are taken from ref 30. (b) Relative transactivation activity of 5 with 1 μM 1 (open
black circles), 6 with 1 μM 1 (open purplish-red triangles), or 8b with 1 μM 1 (open blue squares) toward RXRα. Luciferase activity of 1 at 1 μM was
defined as 1. Data shown are the average (n = 3) SEM. The results for 5 and 6 were taken from ref 30. (c,d) Changes in fluorescence polarization of
each fluorescein-labeled cofactor with RXR in the presence or absence of each test compound. (c) 5 nM fluorescein-labeled coactivator peptide D22. (d)
Fluorescein-labeled corepressor peptide SMRT-ID2 (5 nM). Fluorescence polarization values, expressed in mP, are the average (n = 3) SEM. The
data were analyzed by one-way ANOVA followed by Bonferroni test. *, p < 0.05; **, p < 0.01 vs cofactor peptide with RXR. Results for D22, D22 + RXR,
1, and PA452 were taken from ref 30 because these experiments were performed at the same time.
effects or not, we focused on compound 8b (NEt-4IB), whose
Emax value was lowest of all and EC50 value was lower than 8h
(Table 1).
was clearly increased, while that of RXR full agonist 1 or 7b was
not increased and that of 8b showed a nonsignificant increase.
Taken together, these results indicate that compound 8b
recruited both CoA and CoR and support the conclusion that
8b is an RXR partial agonist.
We found that 8b at 10 μM did not activate any RAR subtype
(Figure 3a). We also examined heterodimer activation by 8b.
Although 8b did not activate PPARγ or LXRα in the absence
of RXRα (Figure 3c,e), it induced activation of LXRα/RXRα
or PPARγ/RXRα heterodimers generated by cotransfection of
RXRα with PPARγ or LXRα, i.e., permissive activation occurred
(Figure 3d,f). In addition, 8b also showed RXRα partial agonistic
activity with HepG2 instead of COS-1 as host cells (Supporting
Information, Figure S1a). On the other hand, PPARγ/RXRα
activation by 8b was similar to that by 5, and LXRα/RXRα
activation by 8b was much lower than that by 5 or 7b
(Supporting Information, Figure S1b,c).
Next, to understand the mechanism of the RXR partial
agonistic activity of 8b, we performed docking simulation using
AutoDock4.2 (Figure 4).40 Compounds 5 and 6 are proposed to
induce RXR partial activation not by direct interference with
helix 12 but via electrostatic repulsion with Asn306 in helix 4.30
Our simulation predicted that the isobutoxy group at the 4′
position on the hydrophilic phenyl group of 8b is located close to
His435 in helix 11 of RXR. Steric interference between the
isobutoxy group of 8b and His435 is thought to influence helix 12
indirectly, which might produce a conformation that allows RXRs
to bind either CoA or CoR to produce RXR partial activation.
Evaluation of dose-dependency toward RXR revealed that
compound 8b showed 50−60% efficacy toward not only RXRα
but also RXRβ and RXRγ (Figure 2a). To confirm the RXR
partial antagonist character of 5, 6, and 8b, they were each
examined in combination with RXR full agonist 1 at 1 μM
(Figure 2b). Like 5 and 6, 8b showed partial inhibition of the
activity of 1 in proportion to the concentration of 8b, confirming
that 8b is an RXR partial agonist.
RXRs bind to a corepressor (CoR), which inhibits DNA
transcription, in the absence of agonists. Binding of RXRs to
agonists induces conformational change of helix 12 of RXRs and
promotes dissociation of CoR and association of a coactivator
(CoA), leading to induction of DNA transcription of target
genes.34 That is, RXR ligands that favor a stable RXR−CoA
complex are RXR full agonists, while RXR ligands that favor
a stable RXR−CoR complex are RXR full antagonists. RXR
partial agonists are thought to induce an equilibrium of the two
complexes. These phenomena can be detected by fluorescence
polarization (FP) measurements using fluorescence-labeled
cofactor peptides.30 Thus, cofactor behavior was examined by
FP assay in the presence of an RXR full agonist, an RXR full
antagonist, an RXR partial agonist, or 8b (Figure 2c,d). The FP
value of 8b for CoA was much higher than that of RXR antagonist
PA452,35 although lower than that of RXR full agonist 1 or 7b
(Figure 2c). On the other hand, the FP value of PA452 for CoR
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Figure 3. Relative transactivation activity of 5, 7b, and 8b toward RARα, RARβ, RARγ, PPARγ, PPARγ/RXRα, LXRα, and LXRα/RXRα using COS-1
cells. (a) Relative transactivation activity of 8b toward RARα (open blue circles), RARβ (open blue triangles), and RARγ (open blue squares). COS-1
cells were transfected with three kinds of vectors: each RAR receptor subtype, a luciferase reporter gene under the control of the appropriate RAR
response element (tk-TREPII-Luc), and secreted alkaline phosphatase (SEAP) gene as a background. Luciferase activity of Am80 (RARα/β dual
agonist) or ATRA (RAR pan agonist) at 1 μM was defined as 1 for RARα and RARβ or for RARγ, respectively. Data shown are the average (n = 3)
SEM. (b) Chemical structures of Am80 (references36,37), all-trans retinoic acid, TIPP-703 (ref 38) (PPAR pan agonist), and carba-T0901317 (ref 39)
(LXR pan agonist). (c−f) Relative transactivation activity of 5 (closed black circles), 7b (closed green triangles), and 8b (closed blue squares) toward
LXRα, LXRα/RXRα, PPARγ, and PPARγ/RXRα. (c,d) Relative transactivation activity of 2 (closed orange diamonds) toward PPARγ and PPARγ/
RXRα. COS-1 cells were transfected with four kinds of vectors, consisting of RXRα, a partner receptor (PPARγ or LXRα), the partner response element
(tk-PPREx3-Luc for PPARγ or tk-rBARx3-Luc for LXRα), and secreted alkaline phosphatase (SEAP) gene as a background. Luciferase activity of TIPP-
703 (PPAR pan agonist) or carba-T0901317 (LXR pan agonist) at 1 μM was defined as 1 for LXRα or LXRα/RXRα, PPARγ, or PPARγ/RXRα,
respectively. Data shown are the average (n = 3) SEM. The data for 5 was taken from ref 29.
The results suggest that 5 and 8b induce partial activation through
different mechanisms.
the higher blood concentration of 8b after oral administration
compared with 7b. However, because oral administration of each
compound at 30 mg/kg gave a sufficient blood concentration
to obtain Emax in each case, adverse effects were evaluated at this
dosage.
Compounds 7b and 8b were repeatedly administered orally
to mice or rats at 30 mg/kg/day, and weight change and other
parameters were evaluated. Figure 5b,c,d and Table 2 show
weight change, liver weight, blood triglyceride, and other blood
parameters before and after consecutive administration for 7 days
in mice. The changes of liver weight and blood triglyceride in the
group treated with 8b were similar to those in the case of the
vehicle control, whereas those in the group treated with 7b
were significantly increased (Figure 5c,d and Table 2); however,
there was no significant difference in weight change between the
groups given 7b and 8b (Figure 5b). We previously found that
In a preliminary in vivo study of 8b, the blood concentration
after oral administration was evaluated and compared with that
of RXR full agonist NEt-3IB (7b), a regioisomer of 8b. Single
oral administration of NEt-4IB (8b) at 30 mg/kg gave a blood
concentration of about 13 μM 8b at 1 h, which is higher than the
concentration producing Emax (Figure 5a). The blood concen-
tration after oral administration of 7b at 30 mg/kg reached about
0.75 μM at 1 h, which again is higher than the concentration
producing Emax. The reason why NEt-3IB (7b) and NEt-4IB
(8b) gave significantly different blood concentrations was
examined by using positron emission tomography (PET) tracers,
and the results will be reported elsewhere.42 Briefly, although
many factors are known to affect absorption, we found that higher
intestinal absorbability and lower biliary excretion contribute to
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Figure 4. Proposed docking structures of each RXR agonist in the RXRα LBP obtained with AutoDock4.2 (ref 40). (a) Crystal structure of RXRα LBD
in complex with 1 (pdb code: 4K6I) (ref 41). (b−e) Docking models obtained with AutoDock4.2. a, c, d, and e were modeled with 5, 6, 7b, and 8b,
respectively. Electronic and steric repulsions were indicated by yellow and white curves, respectively.
RXR full agonists 4, 7a, and 7b similarly activate RXR but
differently activate PPAR/RXR and LXR/RXR,30 and 7b
induced lower degrees of hepatomegaly and blood triglyceride
elevation than 4 and 7a, even though all of them showed similar
blood glucose-lowering action on repeated administration.20
Because the liver weight and blood triglyceride increases induced
by 7b after consecutive administration for 7 days in mice were
smaller than those caused by RXR full agonists 4 and 7a,20 8b
clearly shows reduced side effects compared to the full agonists.
Among other blood parameters examined, only blood urea
nitrogen (BUN) was elevated by 8b (Table 2). The drug
exposures in the single-dose pharmacokinetic studies of 7b or 8b
would not necessarily reflect those in the multiday accumulated
dosing studies, but nevertheless, these results at least indicate that
8b shows reduced side effects compared to 7b. Further studies,
including evaluation of drug exposures and dose-dependency
studies, would be needed to evaluate the efficacy in detail,
because many factors, such as cascade effects, total exposure, and
peak-to-trough ratio can influence agonist effects. And, although
further investigation is required, a PET tracer experiment in mice
revealed that excretion of 8b is not renal, but biliary,42 so that
renal function should not be an issue. In rats administered 8b
orally at 30 mg/kg/day for consecutive 28 days, food intake,
water intake, and body weight change were similar to those of
the vehicle control group (Supporting Information, Figure S2).
The RXR full agonist 7b produced hepatomegaly, whereas RXR
partial agonist 8b did not (Supporting Information, Table S1).
Although blood triglyceride was elevated in females by 8b, the
increase was less than that caused by 7b. Other blood parameters
were not impaired by 8b but were by 7b (Supporting
Information, Table S2). In short, 8b did not induce significant
weight gain, hepatomegaly, or TG elevation even after prolonged
administration. These results strongly suggest that the partial
agonist character of 8b is the reason for the reduced incidence of
adverse effects. Nevertheless, we cannot completely rule out the
possibilities that the adverse effects of 7b are due in part to
off-target activities or are caused in part by its metabolites, and
further studies are desirable to confirm the critical role of partial
antagonism.
Because 8b had no significant adverse effect upon repeated
oral administration at 30 mg/kg/day, we next examined its
antitype 2 diabetes effect in an animal model because not only
RXR partial agonists 5 and 6 but also RXR full agonist 7b show a
glucose-lowering effect in KK-Ay type 2 diabetes model mice.29,43
The insulin sensitizer pioglitazone (2) was used as a positive
control. Repeated oral administration of 2 at 10 mg/kg/day
reduced water intake similarly to 8b (Figure 6b) and caused
significant weight gain (Figure 6c) and hepatomegaly (Table 3),
but did not reduce glucose, in accordance with the findings
of Arakawa et al.44 On the other hand, 8b at the same dosage
showed a marked glucose-lowering effect (Figure 6a−d, Table 3)
and a tendency for improvement of OGTT (Figure 6e) without
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CONCLUSION
■
Compound 8b is an RXR partial agonist with a maximum RXR
efficacy of 55% in luciferase reporter gene assay and shows an
antitype 2 diabetes effect without the serious side effects caused
by full agonists in our animal models. Because RXR full agonists
are reported to show therapeutic effects against not only type 2
diabetes but also Alzheimer’s disease and Parkinson’s disease
in animal models, our findings also suggest that RXR partial
agonists may be candidate therapeutic agents for those diseases
too. The mechanism of the activity of 8b appears to be distinct
from those of other partial agonists involving steric interaction
with His435 in helix 11 of RXR. This mechanism may open up a
new approach to the design of RXR partial agonists as candidate
drugs.
EXPERIMENTAL SECTION
■
Chemistry. General. Flash column chromatography was performed
using Kanto Chemical Silica Gel 60 (spherical) 40−50 μm. Melting
points were determined with a Yanagimoto hot-stage melting point
1
apparatus and are uncorrected. H NMR spectra were recorded on a
Figure 5. Comparison between compounds 7b and 8b in male ICR
mice. (a) Plasma concentrations of 7b (closed green triangles) and 8b
(closed blue squares) (single oral dose of 30 mg/kg). The horizontal
scale is elapsed time in hours, and the vertical scale is normalized by the
micromolar concentration. Data shown are the average (n = 4−7)
SEM. (b−d) Evaluation of adverse effects of repeated oral
administration of vehicle (open), 7b (green), and 8b (blue) at
30 mg/kg/day to male ICR mice for 7 consecutive days. (b) Time
course of body weight change for 7 days. Effects of compounds on (c)
liver weight/body weight and (d) serum triglyceride. Data shown are the
average (n = 8) SEM. The data were analyzed by one-way ANOVA
followed by Bonferroni test. **: p < 0.01 vs vehicle.
JEOL JMN-AL-300 (300 MHz), Varian VXR-300 (300 MHz), or Varian
VXR-500 (500 MHz) spectrometer. 13C NMR spectra were recorded
on a Varian VXR-400 (100 MHz) spectrometer. The purity of all tested
compounds was >95%, as confirmed by combustion analysis.
Combustion analysis was carried out with a Yanagimoto MT-5 CHN
recorder elemental analyzer, and results were within 0.4% of the
theoretical values. FAB-MS was carried out with a VG70-SE.
4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-
ethenyl]benzoic Acid (1). This compound was prepared according to
ref 45.
NEt-3IB (7b) and NEt-4IP (8a). These compounds were prepared
according to ref 33.
2-Isopropyl-4-nitrophenol (10). Conc HNO3 (3.4 mL, 45 mmol)
was added to a solution of 2-isopropylphenol (6.1 mL, 45 mmol) in
EtOAc (200 mL) in an ice bath. The reaction mixture was placed in an
ultrasonic reactor, ZnCl2 (6.1 g, 45 mmol) was added, and the whole
was stirred for 0.8 h. The reaction mixture was washed with H2O (2 ×
100 mL) and brine (100 mL) and dried over MgSO4. The solvent was
evaporated under reduced pressure. The residue was purified by flash
column chromatography (EtOAc:n-hexane = 1:5) to yield 10 (4.9 g,
60%) as a brown solid. H NMR (300 MHz, CDCl3) δ: 8.13 (d, J =
2.8 Hz, 1H), 8.00 (dd, J = 9.0, 2.8 Hz, 1H), 6.83 (d, J = 9.0 Hz, 1H), 6.04
(s, 1H), 3.26 (sep, J = 7.0 Hz, 1H), 1.30 (d, J = 7.0 Hz, 6H).
Isopropoxy-2-isopropyl-4-nitrobenzene (11). 2-Bromopropane
(990 μL, 11 mmol), K2CO3 (2.6 g, 19 mmol), and KI (800 mg,
4.8 mmol) were added to a solution of 10 (1.7 g, 9.6 mmol) in dry DMF
(4.8 mL). The reaction mixture was stirred at 70 °C under an Ar
atmosphere for 3.0 h, then poured into 2 N HCl (70 mL) and extracted
significant weight gain or hepatomegaly. Because this dosage is
lower than that used to evaluate adverse effects, it appears that
this compound produces antitype 2 diabetes effects without
the adverse effects produced by RXR full agonists. RT-PCR
examination of changes in the expression levels of genes
associated with glucose/lipid metabolism in the liver of KK-Ay
mice (Figure 6f) showed that 8b increased Gck similarly to RXR
partial agonist 5,29 although the difference from the vehicle
control was not significant. Thus, it is possible that one of the
mechanisms of the glucose-lowering effect of 5 is induction of
glycolysis via Gck. In Ames tests using five strains, 8b did not
exhibit mutagenic properties (Supporting Information, Table S3).
These results indicate that 8b is a promising candidate drug for
the treatment of type 2 diabetes.
1
Table 2. Serum Parameters of Male ICR Mice after Oral Administration of Vehicle, 7b, or 8b at 30 mg/kg/day for 7 Consecutive
a
Days
c
vehicle
7b
8b
reference value
AST (U/I)
47.6 4.9
16.6 0.87
5.38 0.38
327 28
77.5 22
30.9 4.7**
5.88 0.23
594 43**
ND
48.4 4.0
23.4 1.2
6.00 0.33
367 32
ND
80.5 67
45 16
ALT (U/I)
γ-GTP (U/I)
ALP (U/I)
b
CRE (mg/dL)
BUN (mg/dL)
TG (mg/dL)
TCHO (mg/dL)
GLU (mg/dL)
ND
23.6 1.6
197 33
126 9.3
91.1 8.2
25.7 1.3
321 22**
182 23**
216 25**
30.7 2.9**
199 22
160 8.1
129 8.7
14
3
192 66
164 27
164 27
a
Data shown are the average (n = 8) SEM. The data were analyzed by one-way ANOVA followed by Bonferroni test. Significant differences:
b
c
**p < 0.01 vs vehicle. ND means not detected. Taken from the Clinical Laboratory Parameters for Crl:CD1(ICR) Mice (50−70 day old) by
Charles River. Analyzing equipment: Alfa Wassermann Ace Alera.
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Figure 6. Evaluation of antitype 2 diabetes effects of repeated oral administration of pioglitazone (2: closed orange diamonds) or 8b (closed blue
squares) at 10 mg/kg/day to male KK-Ay mice for 14 consecutive days. Open black circles indicate vehicle treatment. Time course of (a) food intake, (b)
water intake, (c) body weight change, and (d) blood glucose levels. Data shown are the average (n = 4−6) SEM. The data were analyzed by one-way
ANOVA followed by Bonferroni test. Significant differences: * (for 2), † (for 8b) p < 0.05; ** (for 2), †† (for 8b) p < 0.01, vs vehicle, respectively. (e)
Oral glucose tolerance test (OGTT) results of male KK-Ay mice treated with vehicle (open black circles), pioglitazone (2) (closed orange diamonds), or
8b (closed blue squares) at 10 mg/kg/day for 14 consecutive days (n = 4−5). (f) Fold changes in mRNA expression of Irs1 (i), Irs2 (ii), Slc2a1 (iii),
Slc2a2 (iv), G6pc (v), Pck (vi), Gck (vii), Scd1 (viii), Fasn (ix), Srebp1c (x), and ApoE (xi) in liver tissue of male KK-Ay mice treated with vehicle, 2, or 8b
at 10 mg/kg/day for 14 consecutive days (n = 3−4). The gray, orange, and blue bars indicate vehicle, 2, and 8b treatment, respectively. Data shown are
the average SEM. The data were analyzed by one-way ANOVA followed by Bonferroni test. Significant differences: *p < 0.05 or **p < 0.01 vs vehicle.
Data for vehicle and pioglitazone (2) were taken from ref 30 because these experiments were performed at the same time.
Table 3. Liver Weight and Serum Parameters of Male KK-Ay
purified by flash column chromatography (EtOAc:n-hexane = 1:15) to
yield 11 (1.7 g, 80%) as an orange oil. 1H NMR (300 MHz, CDCl3) δ:
Mice after Oral Administration of Vehicle, Pioglitazone (2),
a
8.13 (d, J = 2.5 Hz, 1H), 8.07 (dd, J = 9.0, 3.0 Hz, 1H), 6.86 (d, J =
9.0 Hz, 1H), 4.70 (sep, J = 6.0 Hz, 1H), 3.31 (sep, J = 7.0 Hz, 1H), 1.40
or 8b at 10 mg/kg/day for 14 Consecutive Days
(d, J = 6.0 Hz, 6H), 1.24 (d, J = 7.0 Hz, 6H).
vehicle
2
8b
Methyl 6-[(4-Isopropoxy-3-isopropylphenyl)amino]pyridine-3-
carboxylate (12). Activated 10% Pd−C (catalytic amount) was added
to a solution of 11 (4.7 g, 21 mmol) in MeOH (30 mL). The reaction
mixture was stirred under an H2 atmosphere at room temperature for
5.5 h, then filtered through Celite, and the Celite cake was washed
with EtOAc. The combined filtrate and washing were evaporated
under reduced pressure. 6-Chloronicotinic acid (3.7 g, 23 mmol) and
MeSO3H (1.5 mL, 23 mmol) were added to a solution of the residue in
dry dioxane (30 mL). This mixture was stirred at 120 °C under an Ar
atmosphere for 17 h and then evaporated under reduced pressure. Conc
H2SO4 (2.0 mL) was added to a solution of the residue in dry MeOH
(20 mL). The reaction mixture was stirred at 80 °C under an Ar
atmosphere for 5.7 h, evaporated under reduced pressure, neutralized
with 2 N NaOH, and then extracted with EtOAc (3 × 80 mL). The
organic layer was collected, washed with H2O (2 × 100 mL) and brine
(100 mL), and dried over MgSO4. The solvent was evaporated under
reduced pressure. The residue was purified by flash column chromato-
graphy (EtOAc:n-hexane = 1:8 to 1:5) to yield 12 (5.6 g, 73%) as a
liver weight/body
weight (%)
3.78 0.096
4.95 0.33**
3.68 0.18
AST (U/I)
49.6 2.3
17.6 0.93
5.80 0.58
358 43
45.3 8.4
21.3 1.9*
7.33 2.3
396 44
ND
41.8 1.9
17.0 0.58
5.25 0.63
397 40
ND
ALT (U/I)
γ-GTP (U/I)
ALP (U/I)
b
CRE (mg/dL)
BUN (mg/dL)
TG (mg/dL)
TCHO (mg/dL)
ND
21.9 1.1
122 11
153 11
22.2 1.3
124 28
147 3.8
21.6 1.8
101 12
146 6.5
a
Data shown are the average (n = 3−5)
SEM. The data were
analyzed by one-way ANOVA followed by Bonferroni test. Significant
b
differences: **p < 0.01 vs vehicle. ND means “not detected”.
with EtOAc (3 × 80 mL). The organic layer was collected, washed
with H2O (2 × 80 mL) and brine (80 mL), and dried over MgSO4.
The solvent was evaporated under reduced pressure. The residue was
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brown solid. 1H NMR (300 MHz, CDCl3) δ: 8.79 (d, J = 2.0 Hz, 1H),
8.00 (dd, J = 9.0, 2.0 Hz, 1H), 7.12−7.09 (m, 2H), 6.90 (s, 1H), 6.85 (d,
J = 9.0 Hz, 1H), 6.64 (d, J = 9.0 Hz, 1H), 4.54 (sep, J = 6.0 Hz, 1H), 3.88
(s, 3H), 3.33 (sep, J = 7.0 Hz, 1H), 1.36 (d, J = 6.0 Hz, 6H), 1.20 (d, J =
7.0 Hz, 6H).
Methyl 6-[Ethyl-(4-isopropoxy-3-isopropylphenyl)amino]-
pyridine-3-carboxylate (13). Compound 12 (1.2 g, 3.6 mmol) was
added to a suspension of NaH (200 mg, 5.0 mmol, 60% dispersion in
oil) in dry DMF (6.0 mL) at rt under an Ar atmosphere. The mixture
was stirred for 5 min, then iodoethane (400 μL, 5.0 mmol) was added,
and stirring was continued for 0.3 h. The reaction mixture was poured
onto ice and extracted with EtOAc (3 × 40 mL). The organic layer
was collected, washed with H2O (2 × 50 mL) and brine (30 mL),
and dried over MgSO4. The solvent was evaporated under reduced
pressure. The residue was purified by flash column chromatography
(EtOAc:n-hexane = 1:5 to 1:1) to yield 13 (960 mg, 75%) as a brown oil.
1H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 2.5 Hz, 1H), 7.77 (dd, J =
9.0, 2.5 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.5 Hz, 1H),
6.88 (d, J = 8.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 4.58 (sep, J = 6.0 Hz,
1H), 4.00 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.32 (sep, J = 7.0 Hz, 1H),
1.38 (d, J = 6.0 Hz, 6H), 1.22 (t, J = 7.0 Hz, 3H), 1.19 (d, J = 7.0 Hz, 6H).
Methyl 6-[Ethyl-(4-hydroxy-3-isopropylphenyl)amino]pyridine-3-
carboxylate (14). AlCl3 (1.2 g, 9.0 mmol) was added to a solution of 13
(960 mg, 2.7 mmol) in dry CH2Cl2 (14 mL). The reaction mixture
was stirred at rt under an Ar atmosphere for 3.5 h, then poured into
H2O (100 mL) and extracted with EtOAc (3 × 50 mL). The organic
layer was collected, washed with H2O (2 × 50 mL) and brine (50 mL),
and dried over MgSO4. The solvent was evaporated under reduced
pressure. The residue was purified by flash column chromatography
(EtOAc:n-hexane = 1:4) to yield 14 (820 mg, 97%) as a pale-yellow oil.
1H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 2.5 Hz, 1H), 7.78 (dd, J =
9.0, 2.5 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.90 (dd, J = 8.5, 2.5 Hz, 1H),
6.82 (d, J = 8.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 5.06 (s, 1H), 4.00 (q,
J = 7.0 Hz, 2H), 3.86 (s, 3H), 3.22 (sep, J = 7.0 Hz, 1H), 1.25 (d, J =
7.0 Hz, 6H), 1.24 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ:
166.89, 160.79, 152.44, 151.05, 137.44, 136.78, 136.15, 126.34, 116.45,
113.96, 107.35, 51.68, 45.48, 27.10, 22.46, 12.94. FAB-MS m/z:
315 [M + H]+.
Methyl 6-[Ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-
3-carboxylate (15b). 1-Bromo-2-methylpropane (370 μL, 3.4 mmol),
K2CO3 (240 mg, 1.7 mmol), and KI (catalytic amount) were added to a
solution of 14 (310 mg, 1.0 mmol) in dry DMF (10 mL). The reaction
mixture was stirred at 60 °C under an Ar atmosphere for 18 h, then
poured into H2O (100 mL) and extracted with EtOAc (3 × 30 mL). The
organic layer was collected, washed with H2O (2 × 50 mL) and brine
(30 mL), and dried over MgSO4. The solvent was evaporated under
reduced pressure. The residue was purified by flash column
chromatography (EtOAc:n-hexane = 1:6) to yield 15b (300 mg, 81%)
as a pale-yellow oil. 1H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 2.5 Hz,
1H), 7.77 (dd, J = 9.0, 2.5 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 6.97 (dd, J =
8.5, 2.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.14 (d, J = 9.0 Hz, 1H), 4.00
(q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.77 (sep, J = 6.0 Hz,1H), 3.36 (sep, J =
7.0 Hz, 1H), 2.22−2.08 (m, 1H), 1.22 (d, J = 7.0 Hz, 6H), 1.22 (t, J = 7.0
Hz, 3H), 1.08 (d, J = 6.5 Hz, 6H).
Methyl 6-[Ethyl-(4-cyclopropylmethoxy-3-isopropylphenyl)-
amino]pyridine-3-carboxylate (15c). (Bromomethyl)cyclopropane
(100 μL, 1.1 mmol), K2CO3 (190 mg, 1.4 mmol), and KI (60 mg,
0.35 mmol) were added to a solution of 14 (220 mg, 0.69 mmol) in dry
DMF (3.0 mL). The reaction mixture was stirred at 60 °C under an Ar
atmosphere for 4.0 h, then poured into H2O (20 mL), acidified with 2 N
HCl, and extracted with EtOAc (3 × 30 mL). The organic layer was
collected, washed with H2O (2 × 30 mL) and brine (30 mL), and dried
over MgSO4. The solvent was evaporated under reduced pressure. The
residue was purified by flash column chromatography (EtOAc:n-hexane =
1:6) to yield 15c (240 mg, 94%) as a pale-yellow oil. 1H NMR (300 MHz,
CDCl3) δ: 8.82 (dd, J = 2.5, 0.5 Hz, 1H), 7.76 (dd, J = 9.0, 2.5 Hz, 1H),
7.02 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.5 Hz, 1H), 6.84 (d, J = 8.5 Hz,
1H), 6.14 (d, J = 9.0, 0.5 Hz, 1H), 4.00 (q, J = 7.0 Hz, 2H), 3.86 (d, J =
7.5 Hz, 2H), 3.85 (s, 3H), 3.37 (sep, J = 7.0 Hz, 1H), 1.23−1.20 (m, 5H),
0.67−0.62 (m, 2H), 0.40−0.38 (m, 2H).
Methyl 6-{Ethyl-[3-isopropyl-4-(2-methylallyloxy)phenyl]amino}-
pyridine-3-carboxylate (15d). 3-Bromo-2-methylpropene (100 μL,
1.0 mmol), K2CO3 (69 mg, 0.50 mmol), and KI (catalytic amount) were
added to a solution of 14 (80 mg, 0.25 mmol) in dry DMF (3.0 mL).
The reaction mixture was stirred at 60 °C under an Ar atmosphere for
4.0 h, poured into H2O (60 mL), acidified with 2 N HCl, and then
extracted with EtOAc (3 × 20 mL). The organic layer was collected,
washed with H2O (40 mL) and brine (40 mL), and dried over MgSO4.
The solvent was evaporated under reduced pressure. The residue was
purified by flash column chromatography (EtOAc:n-hexane = 1:6) to
1
yield 15d (73 mg, 78%) as a pale-yellow oil. H NMR (300 MHz,
CDCl3) δ: 8.83 (dd, J = 2.5, 0.5 Hz, 1H), 7.77 (dd, J = 9.0, 2.5 Hz, 1H),
7.03 (d, J = 2.5 Hz, 1H), 6.97 (dd, J = 9.0, 2.5 Hz, 1H), 6.88 (d, J =
8.5 Hz, 1H), 6.14 (dd, J = 9.0, 0.5 Hz, 1H), 5.15 (s, 1H), 5.02 (s, 1H),
4.47 (s, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.39 (sep, J = 7.0 Hz,
1H), 1.88 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.22 (d, J = 7.0 Hz, 6H).
Methyl 6-{Ethyl-[3-isopropyl-4-(3-methyl-but-2-enyloxy)phenyl]-
amino}pyridine-3-carboxylate (15e). 1-Bromo-3-methyl-2-butene
(210 μL, 1.8 mmol), K2CO3 (330 mg, 2.4 mmol), and KI (catalytic
amount) were added to a solution of 14 (190 mg, 0.60 mmol) in dry
DMF (20 mL). The reaction mixture was stirred at 120 °C under an Ar
atmosphere for 21 h, then poured into H2O (120 mL) and extracted
with EtOAc (3 × 50 mL). The organic layer was collected, washed
with H2O (2 × 50 mL) and brine (50 mL), and dried over MgSO4.
The solvent was evaporated under reduced pressure. The residue was
purified by flash column chromatography (EtOAc:n-hexane = 1:12) to
yield 15e (62 mg, 27%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
δ: 8.83 (dd, J = 2.5, 0.5 Hz, 1H), 7.78 (dd, J = 9.0, 2.5 Hz, 1H), 7.02 (d,
J = 2.5 Hz, 1H), 6.97 (dd, J = 8.5, 2.5 Hz, 1H), 6.89 (d, J = 8.5 Hz,
1H), 6.16 (dd, J = 9.0, 0.5 Hz, 1H), 5.52 (t, J = 6.5 Hz, 1H), 4.55 (d, J =
6.5 Hz, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.35 (sep, J = 7.0 Hz,
1H), 1.82 (s, 3H), 1.76 (s, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.20 (d, J =
7.0 Hz, 6H).
Methyl 6-[(4-Butoxy-3-isopropylphenyl)ethylamino]pyridine-3-
carboxylate (15f). 1-Bromobutane (69 μL, 0.64 mmol), K2CO3 (88 mg,
0.64 mmol), and KI (catalytic amount) were added to a solution of 14
(100 mg, 0.32 mmol) in dry DMF (3.0 mL). The reaction mixture was
stirred at 60 °C under an Ar atmosphere for 3.0 h, poured into H2O
(60 mL), acidified with 2 N HCl, and then extracted with EtOAc
(3 × 20 mL). The organic layer was washedwith water (40 mL) and brine
(40 mL) and dried over MgSO4. The solution was evaporated under
reduced pressure. The residue was purified by flash column chromato-
graphy (EtOAc:n-hexane = 1:6) to yield 15f (69 mg, 59%) as a yellow oil.
1H NMR (300 MHz, CDCl3) δ: 8.84 (d, 1H, J = 2.0 Hz), 7.78 (dd, J = 9.0,
2.5 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.5, 2.5 Hz, 1H), 6.89
(d, J = 9.0 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 4.01 (q, J = 7.0 Hz, 2H), 4.01
(t, J = 6.0 Hz, 2H), 3.85 (s, 3H), 3.34 (sep, J = 7.0 Hz, 1H), 1.87−1.78 (m,
2H), 1.62−1.51 (m, 2H), 1.22 (t, J = 7.0 Hz, 3H), 1.21 (d, J = 7.0 Hz,
6H), 1.01 (t, J = 7.5 Hz, 3H).
Methyl 6-[Ethyl-(3-isopropyl-4-pentyloxyphenyl)amino]pyridine-
3-carboxylate (15g). n-Amyl bromide (82 μL, 0.66 mmol), K2CO3
(91 mg, 0.66 mmol), and KI (catalytic amount) were added to a solution
of 14 (104 mg, 0.33 mmol) in dry DMF (3.0 mL). The reaction mixture
was stirred at 60 °C under an Ar atmosphere for 6.5 h, poured into
H2O (60 mL), acidified with 2 N HCl, and then extracted with EtOAc
(3 × 20 mL). The organic layer was washed with water (40 mL) and
brine (40 mL) and dried over MgSO4. The solution was evaporated
under reduced pressure. The residue was purified by flash column
chromatography (EtOAc:n-hexane = 1:6) to yield 15g (76 mg, 60%) as a
yellow oil. 1H NMR (300 MHz, CDCl3) δ: 8.84 (d, J = 2.5 Hz, 1H), 7.78
(dd, J = 9.0, 2.5 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.5, 2.5
Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 4.01 (q, J =
7.0 Hz, 2H), 4.00 (t, J = 7.0 Hz, 2H), 3.86 (s, 3H), 3.34 (sep, J = 7.0 Hz,
1H), 1.89−1.80 (m, 2H), 1.55−1.41 (m, 4H), 1.22 (t, J = 6.5 Hz, 3H),
1.21 (d, J = 6.5 Hz, 6H), 1.01 (t, J = 7.0 Hz, 3H).
Methyl 6-[Ethyl-(4-hexyloxy-3-isopropylphenyl)amino]pyridine-3-
carboxylate (15h). 1-Iodohexane (97 μL, 0.66 mmol), K2CO3 (91 mg,
0.66 mmol), and KI (catalytic amount) were added to a solution of 14
(105 mg, 0.33 mmol) in dry DMF (3.0 mL). The reaction mixture
was stirred at 60 °C under an Ar atmosphere for 3.7 h, poured into H2O
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(60 mL), acidified with 2 N HCl, and then extracted with EtOAc
(3 × 20 mL). The organic layer was washed with water (40 mL) and
brine (40 mL) and dried over MgSO4. The solution was evaporated
under reduced pressure. The residue was purified by flash column
chromatography (EtOAc:n-hexane = 1:6) to yield 15h (110 mg, 82%) as
a yellow oil. 1H NMR (300 MHz, CDCl3) δ: 8.84 (d, J = 2.0 Hz, 1H),
7.78 (dd, J = 9.0, 2.5 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.5,
2.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 4.01 (q,
J = 6.5 Hz, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.85 (s, 3H), 3.34 (sep, J = 7.0
Hz, 1H), 1.88−1.79 (m, 2H), 1.57−1.52 (m, 2H), 1.40−1.35 (m, 4H),
1.22 (t, J = 7.0 Hz, 3H), 1.22 (d, J = 6.5 Hz, 6H), 0.92 (t, J = 7.0 Hz, 3H).
Methyl 6-[(4-Benzyloxy-3-isopropylphenyl)ethylamino]pyridine-
3-carboxylate (15i). Benzyl bromide (48 μL, 0.40 mmol), K2CO3
(69 mg, 0.40 mmol), and KI (catalytic amount) were added to a solution
of 14 (106 mg, 0.34 mmol) in dry DMF (3.0 mL). The reaction mixture
was stirred at 60 °C under an Ar atmosphere for 2.5 h, then poured
into H2O (60 mL) and extracted with EtOAc (3 × 20 mL). The organic
layer was washed with water (2 × 40 mL) and brine (30 mL) and
dried over MgSO4. The solution was evaporated under reduced
pressure. The residue was purified by flash column chromatography
(EtOAc:n-hexane = 1:6) to yield 15i (76 mg, 55%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 2.5 Hz, 1H), 7.78 (dd, J =
9.0, 2.5 Hz, 1H), 7.48−7.35 (m, 5H), 7.06−6.97 (m, 3H), 6.16 (d, J = 9.0
Hz, 1H), 5.12 (s, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.42 (sep,
J = 7.0 Hz, 1H), 1.23 (d, J = 7.0 Hz, 6H), 1.22 (t, J = 7.0 Hz, 3H).
Methyl 6-[Ethyl-(3-isopropyl-4-phenethyloxyphenyl)lamino]-
pyridine-3-carboxylate (15j). (2-Bromoethyl)benzene (190 μL,
1.4 mmol), K2CO3 (170 mg, 1.2 mmol), and KI (catalytic amount)
were added to a solution of 14 (220 mg, 0.70 mmol) in dry DMF
(4.0 mL). The reaction mixture was stirred at 80 °C under an Ar
atmosphere for 16 h, then poured into H2O (100 mL) and extracted
with EtOAc (3 × 30 mL). The organic layer was washed with water
(2 × 40 mL) and brine (30 mL) and dried over MgSO4. The solution
was evaporated under reduced pressure. The residue was purified by
flash column chromatography (EtOAc:n-hexane = 1:9 to 1:3) to yield
15j (46 mg, 16%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ: 8.82
(d, J = 2.0 Hz, 1H), 7.76 (dd, J = 9.0, 2.0 Hz, 1H), 7.33−7.32 (m, 5H),
7.00 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.5 Hz, 1H), 6.86 (d, J =
8.5 Hz, 1H), 6.13 (d, J = 9.0 Hz, 1H), 4.21 (t, J = 6.5 Hz, 2H), 4.00 (q, J =
7.0 Hz, 2H), 3.85 (s, 3H), 3.30 (sep, J = 7.0 Hz, 1H), 3.15 (t, J = 6.5 Hz,
2H), 1.21 (t, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 6H).
Methyl 6-{Ethyl-[(3-isopropyl-4-phenylpropoxy)phenyl]amino}-
pyridine-3-carboxylate (15k). 3-Phenylpropyl bromide (54 μL,
0.36 mmol), K2CO3 (83 mg, 0.60 mmol), and KI (17 mg, 0.10 mmol)
were added to a solution of 14 (75 mg, 0.24 mmol) in dryDMF (3.0 mL).
The reaction mixture was stirred at 90 °C under an Ar atmosphere for
14 h, then poured into H2O (100 mL) and extracted with EtOAc (3 ×
30 mL). The organic layer was washed with water (2 × 40 mL) and brine
(30 mL) and dried over MgSO4. The solution was evaporated under
reduced pressure. The residue was purified by flash column chromato-
graphy (EtOAc:n-hexane = 1:6) to yield 15k (76 mg, 73%) as a white oil.
1H NMR (300 MHz, CDCl3) δ: 8.83 (d, J = 2.5 Hz, 1H), 7.78 (d, J = 9.0,
2.5 Hz, 1H), 7.35−7.22 (m, 5H), 7.03 (d, J = 2.5 Hz, 1H), 6.96 (dd, J =
8.5, 2.5 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.15 (d, J = 9.0 Hz, 1H), 4.01 (t,
J = 6.0 Hz, 2H), 4.01 (q, J = 7.5 Hz, 2H), 3.85 (s, 3H), 3.37 (sep, J = 7.0
Hz, 1H), 2.87 (t, J = 7.0 Hz, 2H), 2.22−2.12 (m, 2H), 1.24 (d, J = 7.0 Hz,
6H), 1.22 (t, J = 7.5 Hz, 3H).
6-[Ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-car-
boxylic Acid (8b). To a solution of 15b (300 mg, 0.81 mmol) in MeOH
(10 mL) were added 2 N NaOH (4.0 mL) and THF (3.0 mL). The
reaction mixture was stirred at 60 °C for 2.0 h and then evaporated
under reduced pressure. The residue was neutralized with 2 N HCl
and extracted with EtOAc (3 × 20 mL). The organic layer was washed
with water (2 × 40 mL) and brine (30 mL) and dried over MgSO4. The
solution was evaporated under reduced pressure, and the residue was
recrystallized from MeOH to yield 8b (140 mg, 49%) as white needles;
mp 162.0−164.0 °C. IR (KBr) cm−1: 1677 (CO). 1H NMR (300 MHz,
CDCl3) δ: 8.90 (d, J = 2.5 Hz, 1H), 7.81 (dd, J = 9.0, 2.5 Hz, 1H),
7.03 (d, J = 2.5 Hz, 1H), 6.98 (dd, J = 8.5, 2.5 Hz, 1H), 6.88 (d, J =
8.5 Hz, 1H), 6.16 (d, J = 9.0 Hz, 1H), 4.00 (q, J = 7.0 Hz, 2H),
3.77 (d, J = 6.0 Hz, 2H), 3.49 (s, 1H), 3.36 (sep, J = 7.0 Hz, 1H), 1.23 (t,
J = 7.0 Hz, 3H), 1.22 (d, J = 7.0 Hz, 6H), 1.08 (d, J = 6.5 Hz, 6H).
FAB-MS m/z: 357 [M + H]+. Anal. Calcd for C21H28N2O3: C, 70.76;
H, 7.92; N, 7.86. Found: C, 70.49; H, 7.64; N, 7.73.
6-[Ethyl-(4-cyclopropylmethoxy-3-isopropylphenyl)amino]-
pyridine-3-carboxylic Acid (8c). To a solution of 15c (130 mg,
0.35 mmol) in MeOH (8.0 mL) were added 2 N NaOH (5.0 mL) and
THF (2.0 mL). The reaction mixture was stirred at 60 °C for 1.5 h and
then evaporated under reduced pressure. The residue was poured into
satd NH4Cl and extracted with EtOAc (3 × 30 mL). The organic layer
was washed with water (2 × 40 mL) and brine (40 mL) and dried over
MgSO4. The solution was evaporated under reduced pressure, and the
residue was recrystallized from MeOH to yield 8c (120 mg, 93%) as
white needles; mp 194.0−196.0 °C. 1H NMR (300 MHz, DMSO-d6) δ:
8.65 (dd, J = 2.5, 0.8 Hz, 1H), 7.76 (dd, J = 9.0, 2.5 Hz, 1H), 7.05−7.01
(m, 3H), 6.13 (d, J = 9.0, 0.8 Hz, 1H), 3.94 (q, J = 7.0 Hz, 2H), 3.88 (q,
J = 7.0 Hz, 2H), 1.14 (d, J = 7.0 Hz, 6H), 1.12 (t, J = 7.0 Hz, 3H), 0.62−
0.56 (m, 2H), 0.38−0.33 (m, 2H). FAB-MS m/z: 355 [M + H]+. Anal.
Calcd for C21H26N2O3: C, 71.16; H, 7.39; N, 7.90. Found: C, 70.99; H,
7.25; N, 7.81.
6-{Ethyl-[3-isopropyl-4-(2-methylallyloxy)phenyl]amino}pyridine-
3-carboxylic Acid (8d). To a solution of 15d (73 mg, 0.20 mmol) in
MeOH (3.0 mL) was added 2 N NaOH (3.0 mL). The reaction mixture
was stirred at 60 °C for 2.0 h and then evaporated under reduced
pressure. The residue was neutralized with 2 N HCl and extracted with
EtOAc (3 × 30 mL). The organic layer was washed with water (40 mL)
and brine (40 mL) and dried over MgSO4. The solution was evaporated
under reduced pressure, and the residue was recrystallized from MeOH
to yield 8d (26 mg, 37%) as white needles; mp 162.0−163.5 °C. IR
(KBr) cm−1: 1677 (CO). 1H NMR (300 MHz, CDCl3) δ: 8.91 (d, J =
2.0 Hz, 1H), 7.82 (dd, J = 9.0, 2.5 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 6.99
(dd, J = 8.5, 2.5 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.17 (d, J = 9.0 Hz,
1H), 5.15 (s, 1H), 5.02 (s, 1H), 4.47 (s, 2H), 4.03 (q, J = 7.0 Hz, 2H),
3.39 (sep, J = 7.0 Hz, 1H), 1.88 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H), 1.23 (d,
J = 7.0 Hz, 6H). FAB-MS m/z: 355 [M + H]+. Anal. Calcd for
C21H26N2O3: C, 71.16; H, 7.39; N, 7.90. Found: C, 71.54; H, 7.52;
N, 7.95.
6-{Ethyl-[3-isopropyl-4-(3-methyl-but-2-enyloxy)phenyl]amino}-
pyridine-3-carboxylic Acid (8e). To a solution of 15e (62 mg,
0.16 mmol) in MeOH (3.0 mL) were added 2 N NaOH (3.0 mL)
and THF (2.0 mL). The reaction mixture was stirred at 60 °C for 0.75 h
and then evaporated under reduced pressure. The residue was poured
into satd NH4Cl (40 mL) and extracted with EtOAc (3 × 30 mL). The
organic layer was washed with water (2 × 30 mL) and brine (30 mL)
and dried over MgSO4. The solution was evaporated under reduced
pressure, and the residue was recrystallized from MeOH to yield 8e
(7.2 mg, 12%) as white needles; mp 139.5−141.0 °C. IR (KBr) cm−1:
1671 (CO). 1H NMR (300 MHz, DMSO-d6) δ: 8.66 (d, J = 2.5 Hz, 1H),
7.76 (dd, J = 9.0, 2.5 Hz, 1H), 7.06−7.05 (m, 3H), 6.14 (d, J = 9.0 Hz,
1H), 5.48 (t, J = 6.5 Hz, 1H), 4.58 (d, J = 6.5 Hz, 2H), 3.94 (q, J = 7.0 Hz,
2H), 1.78 (s, 3H), 1.73 (s, 3H), 1.15 (d, J = 7.0 Hz, 6H), 1.12 (t, J = 7.0
Hz, 3H). FAB-MS m/z: 369 [M + H]+. Anal. Calcd for C22H28N2O3: C,
71.71; H, 7.66; N, 7.60. Found: C, 71.77; H, 7.62; N, 7.57.
6-[(4-Butoxy-3-isopropylphenyl)ethylamino]pyridine-3-carbox-
ylic Acid (8f). To a solution of 15f (69 mg, 0.19 mmol) in MeOH
(3.0 mL) was added 2 N NaOH (3.0 mL). The reaction mixture was
stirred at 60 °C for 4.0 h and then evaporated under reduced pressure.
The residue was neutralized with 2 N HCl and extracted with EtOAc
(3 × 20 mL). The organic layer was washed with water (40 mL) and
brine (40 mL) and dried over MgSO4. The solution was evaporated
under reduced pressure, and the residue was recrystallized from MeOH
to yield 8f (29 mg, 44%) as white needles; mp 161.0−163.5 °C. IR
(KBr) cm−1: 1613 (CO). 1H NMR (300 MHz, DMSO-d6) δ: 8.66 (d, J =
2.0 Hz, 1H), 7.76 (dd, J = 9.0, 2.5 Hz, 1H), 7.09−7.00 (m, 3H), 6.13 (d,
J = 9.0 Hz, 1H), 4.01 (t, J = 6.0 Hz, 2H), 3.93 (q, J = 7.0 Hz, 2H), 3.24
(sep, J = 7.0 Hz, 1H), 1.79−1.70 (m, 2H), 1.55−1.43 (m, 2H), 1.16 (d,
J = 7.0 Hz, 6H), 1.11 (t, J = 7.0 Hz, 3H), 0.95 (t, J = 7.5 Hz, 3H). FAB-
MS m/z: 357 [M + H]+. Anal. Calcd for C21H28N2O3: C, 70.76; H, 7.92;
N, 7.86. Found: C, 70.70; H, 7.73; N, 7.86.
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6-[Ethyl-(3-isopropyl-4-pentyloxyphenyl)amino]pyridine-3-car-
boxylic Acid (8g). To a solution of 15g (76 mg, 0.20 mmol) in MeOH
(6.0 mL) were added 2 N NaOH (3.0 mL) and THF (1.5 mL). The
reaction mixture was stirred at 60 °C for 3.0 h and then evaporated
under reduced pressure. The residue was neutralized with 2 N HCl and
extracted with EtOAc (3 × 20 mL). The organic layer was washed with
water (40 mL) and brine (40 mL) and dried over MgSO4. The solution
was evaporated under reduced pressure and the residue was recrystal-
lized from MeOH to yield 8g (47 mg, 64%) as pale-yellow needles; mp
The reaction mixture was stirred at 60 °C for 2.0 h and then evaporated
under reduced pressure. The residue was poured into satd NH4Cl and
extracted with EtOAc (3 × 30 mL). The organic layer was washed
with water (2 × 30 mL) and brine (30 mL) and dried over MgSO4.
The solution was evaporated under reduced pressure and recrystallized
from CH2Cl2/n-hexane to yield 8k (56 mg, 75%) as colorless plates; mp
1
171.5−176.0 °C. IR (KBr) cm−1: 1660 (CO). H NMR (300 MHz,
DMSO-d6) δ: 8.65 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 9.0, 2.5 Hz, 1H),
7.33−7.17 (m, 5H), 7.05−7.01 (m, 3H), 6.13 (d, J = 9.0 Hz, 1H), 4.01
(t, J = 6.0 Hz, 2H), 3.94 (q, J = 7.0 Hz, 2H), 2.81 (t, J = 8.0 Hz, 2H),
2.13−2.04 (m, 2H), 1.20 (d, J = 7.0 Hz, 6H), 1.13 (t, J = 7.0 Hz, 3H).
FAB-MS m/z: 419 [M + H]+. Anal. Calcd for C26H30N2O3: C, 74.61; H,
7.22; N, 6.69. Found: C, 74.70; H, 7.23; N, 6.69.
Luciferase Reporter Gene Assay. Culture of COS-1 and HepG2
Cells. COS-1 and HepG2 cells were maintained in Dulbecco’s Modified
Eagle’s Medium supplemented with 10% FBS, 0.2% NaHCO3, 4 mM
L-glutamine, penicillin (50000 U/L), and streptomycin (50000 μg/L) in
a humidified atmosphere of 5% CO2 in air at 37 °C.
1
154.5−156.0 °C. IR (KBr) cm−1: 1691 (CO). H NMR (300 MHz,
DMSO-d6) δ: 8.67 (d, J = 2.5 Hz, 1H), 7.77 (dd, J = 9.0, 2.5 Hz, 1H),
7.10−7.01 (m, 3H), 6.14 (d, J = 9.0 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H),
3.94 (q, J = 7.0 Hz, 2H), 3.26 (sep, J = 7.0 Hz, 1H), 1.82−1.73 (m, 2H),
1.49−1.37 (m, 4H), 1.17 (d, J = 6.5 Hz, 6H), 1.12 (t, J = 7.0 Hz, 3H),
0.92 (t, J = 7.0 Hz, 3H). FAB-MS m/z: 371 [M + H]+. Anal. Calcd
for C22H30N2O3: C, 71.32; H, 8.16; N, 7.56. Found: C, 71.16; H, 7.98;
N, 7.29.
6-[Ethyl-(4-hexyloxy-3-isopropylphenyl)amino]pyridine-3-car-
boxylic Acid (8h). To a solution of 15h (109 mg, 0.27 mmol) in MeOH
(6.0 mL) were added 2 N NaOH (3.0 mL) and THF (1.5 mL). The
reaction mixture was stirred at 60 °C for 2.3 h and then evaporated
under reduced pressure. The residue was neutralized with 2 N HCl
and extracted with EtOAc (3 × 20 mL). The organic layer was washed
with water (40 mL) and brine (40 mL) and dried over MgSO4. The
solution was evaporated under reduced pressure, and the residue was
recrystallized from MeOH to yield 8h (64 mg, 61%) as white needles;
mp 167.0−169.0 °C. IR (KBr) cm−1: 1654 (CO). 1H NMR (300 MHz,
DMSO-d6) δ: 8.67 (d, J = 2.5 Hz, 1H), 7.77 (dd, J = 9.0, 2.5 Hz, 1H),
7.10−7.01 (m, 3H), 6.14 (d, J = 9.0 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H),
3.94 (q, J = 7.0 Hz, 2H), 3.26 (sep, J = 7.0 Hz, 1H), 1.81−1.72 (m, 2H),
1.50−1.44 (m, 2H), 1.37−1.30 (m, 4H), 1.17 (d, J = 7.0 Hz, 6H), 1.12
(t, J = 7.0 Hz, 3H), 0.89 (t, J = 7.0 Hz, 3H). FAB-MS m/z: 385 [M + H]+.
Anal. Calcd for C23H32N2O3: C, 71.84; H, 8.39; N, 7.29. Found: C,
71.63; H, 8.38; N, 7.37.
6-[(4-Benzyloxy-3-isopropylphenyl)ethylamino]pyridine-3-car-
boxylic Acid (8i). To a solution of 15i (76 mg, 0.19 mmol) in MeOH
(8.0 mL) were added 2 N NaOH (3.0 mL) and THF (2.0 mL). The
reaction mixture was stirred at 60 °C for 2.0 h and then evaporated
under reduced pressure. The residue was neutralized with 2 N HCl
and extracted with EtOAc (3 × 20 mL). The organic layer was washed
with water (2 × 40 mL) and brine (30 mL) and dried over MgSO4. The
solution was evaporated under reduced pressure and the residue was
recrystallized from MeOH to yield 8i (38 mg, 52%) as colorless cubes;
mp 183.0−184.5 °C. IR (KBr) cm−1: 1674 (CO). 1H NMR (300 MHz,
DMSO-d6) δ: 12.36 (br s, 1H), 8.65 (d, J = 2.5 Hz, 1H), 7.76 (dd, J = 9.0,
2.5 Hz, 1H), 7.51−7.35 (m, 5H), 7.16−7.04 (m, 3H), 6.14 (d, J = 9.0 Hz,
1H), 5.15(s, 2H), 3.95 (q, J = 7.0 Hz, 2H), 1.19 (d, J = 7.0 Hz, 6H), 1.13
(t, J = 7.0 Hz, 3H). FAB-MS m/z: 391 [M + H]+. Anal. Calcd for
C24H26N2O3: C, 73.82; H, 6.71; N, 7.17. Found: C, 73.58; H, 6.93;
N, 7.14.
6-[Ethyl-(3-isopropyl-4-phenethyloxyphenyl)lamino]pyridine-3-
carboxylic Acid (8j). To a solution of 15j (46 mg, 0.11 mmol) in MeOH
(6.0 mL) were added 2 N NaOH (2.0 mL) and THF (2.0 mL). The
reaction mixture was stirred at 60 °C for 4.3 h and then evaporated
under reduced pressure. The residue was neutralized with 2 N HCl
and extracted with EtOAc (3 × 20 mL). The organic layer was washed
with water (2 × 30 mL) and brine (30 mL) and dried over MgSO4. The
solution was evaporated under reduced pressure, and the residue was
purified by flash column chromatography (EtOAc:n-hexane = 1:4), then
was recrystallized from MeOH to yield 8j (25 mg, 58%) as colorless
cubes; mp 167.5−169.0 °C. IR (KBr) cm−1: 1693 (CO). 1H NMR (300
MHz, DMSO-d6) δ: 8.63 (d, J = 2.5 Hz, 1H), 7.72 (dd, J = 9.0, 2.5 Hz,
1H), 7.36−7.22 (m, 5H), 7.03−6.99 (m, 3H) 6.10 (d, J = 9.0, 2.5 Hz,
1Hx), 4.23 (t, J = 6.5 Hz, 2H), 3.93 (q, J = 7.0 Hz, 2H), 3.19 (sep, J =
7.0 Hz, 1H), 3.08 (t, J = 6.5 Hz, 2H), 1.12 (t, J = 7.0 Hz, 3H), 1.09 (d, J =
7.0 Hz, 6H). FAB-MS m/z: 405 [M + H]+. Anal. Calcd for C25H28N2O3:
C, 74.23; H, 6.98; N, 6.93. Found: C, 74.33; H, 6.88; N, 7.06.
6-{Ethyl-[(3-isopropyl-4-phenylpropoxy)phenyl]amino}pyridine-
3-carboxylic Acid (8k). To a solution of 15k (76 mg, 0.18 mmol) in
MeOH (7.0 mL) were added 2 N NaOH (2.0 mL) and THF (3.0 mL).
Luciferase Reporter Gene Assay. Luciferase reporter gene assays
were performed using COS-1 or HepG2 cells transfected with three
kinds of vectors: each receptor subtype, a luciferase reporter gene under
the control of the appropriate nuclear receptor response element, and
secreted alkaline phosphatase (SEAP) gene as a background. CRBPII-tk-
Luc, tk-TREPII-Luc, tk-PPRE×3-Luc, and tk-rBAR×3-Luc reporters were
used as RXR, RAR, PPAR, and LXR response elements, respectively. The
amounts of each receptor subtype and response element were 1.0 and
4.0 μg, respectively. Transfection was performed with Effectene transfec-
tion reagent (QIAGEN)according to thesupplier’s protocol. Inthe case of
heterodimer assay, RXRα (0.5μg), each partner receptor (PPAR or LXRα,
0.5 μg), and the partner response element (4.0 μg) were transfected into
COS-1 or HepG2 cells as described above. Test compound solutions were
added to the suspension of transfected cells, which were seeded at about
2.0 × 104 cells/well in 96-well white plates (DMSO concentration 1%).
After incubation in a humidified atmosphere of 5% CO2 at 37 °C for 18 h,
25 μL of the medium was used for analyzing SEAP activities, and the
remaining cells were used for luciferase reporter gene assays with a
Steady-Glo luciferase assay system (Promega) according to the supplier’s
protocol. The luciferase activities were normalized using SEAP activities.
The assays were carried out in triplicate three times.
Production and Purification of RXRα Protein. This experiment was
performed at the same time as the work reported in ref 30. Produc-
tion and purification of recombinant RXRα protein were done using
the GATEWAY technology.46,47 Destination vectors were generated
by insertion of human RXRα DNA (Ultimate Human ORF Clone,
Invitrogen)48 into a pDEST17 vector (Invitrogen) and were trans-
formed into Escherichia coli BL21-AI cells (Invitrogen) by means of LR
reaction. These cells were used as expression clones. The expression
clones were cultured in LB medium containing 100 μg/mL ampicillin
at 37 °C with shaking until the OD600 reached 0.6 to 1.0 and then diluted
to OD 0.1 at 600 nm. After addition of 0.2% L-arabinose and 0.1%
glucose to the culture during the exponential phase of growth (OD 0.4 at
600 nm), cells were further cultured for 2.0 h and then harvested.
The cell pellets were resuspended in Lysis buffer (50 mM Tris-HCl,
pH 7.5, 0.15 M NaCl, 0.1% SDS, 1.0% Triton X-100, 1.0 mM PMSF)
at 4 °C. The RXR protein was purified using a His GraviTrap
column (GE Healthcare)49 to give 2.2 mg/L pure protein in the culture
medium.
Fluorescence Polarization Assay. This experiment was performed at
the same time as the work reported in ref 30. Fluorescein-labeled
cofactor peptides were purchased from Invitrogen. Assays were
performed in 96-well half-area black plates (Greiner) in a final volume
of 40 μL. All reagents were diluted in phosphate buffer (50 mM sodium
phosphate pH 7.2, 154 mM NaCl, 1.0 mM dithiothreitol, 1.0 mM
EDTA, 0.01% NP40), and the final DMSO concentration in the assay
mixtures was adjusted to 1%. The mixtures containing fluorescein-
labeled cofactor peptide, RXRα, and various RXR ligands in phosphate
buffer were incubated for 1.0 h at 25 °C. The fluorescence polarization of
the mixtures was measured at an excitation wavelength of 485 nm and an
emission wavelength of 535 nm. Fluorescence polarization measure-
ments were made with a TECAN Polarion. Fluorescence polarization is
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Journal of Medicinal Chemistry
Article
the ratio of the difference between the intensities of parallel and
perpendicularly polarized fluorescent light to the total light intensity.
Molecular Docking. The crystal structure of human RXRα−ligand
binding domain (pdb code: 4K6I)41 was retrieved from the Brookhaven
Protein Data Bank: http://www.rcsb.org/pdb/Welcome.do. The
After arrival of the animals, all were group-housed and acclimated to the
colony for 1 or 2 days before the experiment. They were housed with
four mice per cage, with free access to water and chow pellets in a
light (12 h on, 8:00 a.m./12 h off, 8:00 p.m.), temperature (23 1 °C),
and relative humidity (50
20%) controlled environment. Before
structure was also refined by Refmac5 of the CCP4 suite (Rfactor
=
experiments, mice were assigned to experimental groups so as to
minimize the variance between groups based on the measured weight
(four per cage (17 × 33 × 15 cm3)). Body weight was measured at
approximately 10:00 a.m. every day for 7 days before dosing. Mice
were administered orally with a solution of test compound at a dose of
30 mg/kg or with the vehicle (1% ethanol and 0.5% CMC in distilled
water) in a volume of 10 mL/kg of animal at approximately 10:00 a.m.
every day for 7 days. On the final day of dosing, animals were fasted from
5:00 p.m. and given water ad libitum. On the next day, at approximately
10:00 a.m., animals were weighed and anesthetized with diethyl ether.
Blood and liver were removed immediately, and the liver was weighed.
Approximately 1 mL of blood in an Eppendorf sample tube was
centrifuged to afford a serum sample. Blood samples were centrifuged at
1900g for 5 min at rt.
0.207, Rfree = 0.284).50 Hydrogen atoms were automatically added at
pH 7.0 by the protonate 3D program in the MOE 2010.10 package
(Molecular Operating Environment, Chemical Computing Group Inc.,
Montreal, QC, Canada). All hydrogen atomic coordinates were
optimized by the conjugate gradient method with the AMBER99 force
field under the Born solvent model. N- and C-termini were ionized
−NH3+ and −COO−, respectively. The 3D structures of ligands used for
the docking study were constructed by using Spartan (Wavefunction,
Inc.). After semiempirical PM3 calculations, ab initio calculations at
the level of HF/6-31G* were performed to find the lowest energy
conformers. The AutoDock4.2 molecular docking program40 was
employed by using a genetic algorithm with local search (GALS).
One hundred individual GA runs, 150 chromosomes, a crossover ratio of
0.80, a rate of gene mutation of 0.02, and an elitism ratio of 0.10 were used
for each ligand. The grid box was created with dimensions of 40 × 40 ×
40 Å3, which encloses the original ligand. Molfeat (FiatLux Co., Tokyo,
Japan) was used for molecular modeling.
Observation of Side Effects after Once-Daily Oral Administration
at 30 mg/kg for 28 Consecutive Days in SD Rats. Four-week-old male
and female SD rats were purchased from Charles River Laboratories
Japan, Inc.. This experiment was conducted in accordance with the
Guidelines for Animal Experiments at Okayama University Advanced
Science Research Center, and all procedures were approved by the
Animal Research Control Committee of Okayama University. After
arrival of the rats, all were group-housed and acclimated to the colony for
6 (male) or 7 (female) days before the experiment. They were housed
with two rats per cage, with free access to water and chow pellets in a
light (12 h on, 8:00 a.m./12 h off, 8:00 p.m.), temperature (23 1 °C),
In Vivo Assays. Measurement of Serum Concentration of Test
Compounds after Oral Administration at 30 mg/kg to Mice. Six-
week-old male ICR mice were purchased from Charles River
Laboratories Japan, Inc.. This experiment was conducted in accordance
with the Guidelines for Animal Experiments at Okayama University
Advanced Science Research Center, and all procedures were approved
by the Animal Research Control Committee of Okayama University.
Groups of six-week-old ICR male mice (n = 5−8 in each) were treated
with suspensions of test compound at 30 mg/kg (1% ethanol and 0.5%
CMC in distilled water) in a volume of 10 mL/kg of body weight by oral
administration. At the indicated times (0, 0.5, 1.0, 3.0, and 6.0 h after
compound administration), 0.6−1 mL of blood was taken from the
inferior vena cava under diethyl ether anesthesia. Each blood sample was
centrifuged at 1900g for 5 min at rt. To 100 μL of the resulting plasma
were added 100 μL of ice-cold 5.0 mM ammonium acetate solution
(adjusted with acetic acid to pH 5.0) and 1.0 mL of ice-cold EtOAc. The
resulting mixture was vortexed for 30 s, kept at room temperature for
10 min, and centrifuged at 1900g for 30 s at room temperature. An
800 μL aliquot of the EtOAc phase was removed and concentrated to
dryness in a centrifugal evaporator. To the resulting residue was added
100 μL of HPLC eluent solution (for 7b, 33.3 mM ammonium acetate
(adjusted with acetic acid to pH 5.0) with methanol (85:15, v/v); for 8b,
25 mM ammonium acetate (adjusted with acetic acid to pH 5.0)
with methanol (80:20, v/v), respectively). Each solution was directly
subjected to HPLC analysis, and the concentration of each compound
was determined from the peak area of the sample with reference to a
calibration plot obtained with the authentic compound.
HPLC Conditions. The HPLC system used in this study was a
Shimadzu liquid chromatographic system (Kyoto, Japan) consisting of
an SCL-10A system controller, LC-10AD pump, SPD-10AV UV−vis
spectrophotometric detector, SIL-10AD autoinjector, CTO-10A column
oven, DGU-14A degasser, and C-R7A Chromatopac. The samples (each
20 μL) were injected using a refrigerated autosampler kept at 10 °C. The
chromatographic analyses were carriedout on an Inertsil ODS-3 (4.6 mm
i.d. × 250 mm, 5 μm, GL Sciences, Tokyo, Japan) kept at 40 °C, using the
appropriate HPLC eluent solution (for 7b, 33.3 mM ammonium acetate
(adjusted with acetic acid to pH 5.0) with methanol (85:15, v/v); for 8b,
25 mM ammonium acetate (adjusted with acetic acid to pH 5.0) with
methanol (80:20, v/v), respectively) as a mobile phase. The flow rate was
0.7 mL/min and the absorbance was monitored at 280 nm.
and relative humidity (50
20%) controlled environment. Before
experiments, rats were assigned to experimental groups so as to
minimize the variance between groups based on the measured weight
(two per cage (25.0 × 41.5 × 19.0 cm3)). Body weight, food intake, and
water intake were measured at approximately 10:00 a.m. every day for
28 days before dosing. Rats were administered orally with a solution of
test compound at a dose of 30 mg/kg or with the vehicle (1% ethanol
and 0.5% CMC in distilled water) in a volume of 5 mL/kg of animal at
approximately 10:00 a.m. every day for 28 days. On the final day of
dosing, animals were fasted from 5:00 p.m. and given water ad libitum.
On the next day, at approximately 10:00 a.m., animals were weighed
and anesthetized with isoflurane. Blood, liver, brain, kidney, spleen, and
testis (male only) were removed immediately. The liver, brain, kidney,
spleen, and testis were weighed and frozen with liquid nitrogen.
Approximately 10 mL of blood in a centrifuge tube was centrifuged
at 2000g for 10 min at 4 °C to obtain a serum sample. Measurements
of blood parameters were performed according to the method
described below.
Evaluation of Blood Glucose-Lowering Activity in KK-Ay Mice. This
experiment was performed at the same time as the work reported in
ref 30. Type 2 diabetic KK-Ay mice, in which the Ay allele at the agouti
locus had been transferred to the inbred KK strain by repetitive
backcrossing, were used as the congenic strain. The introduction of the
Ay allele caused DM and massive hereditary obesity. Four-week-old male
KK-Ay mice were purchased from CLEA Japan Inc. The KK-Ay mice
were allowed free access to solid food and tap water. This experiment
was conducted in accordance with the Guidelines for Animal
Experiments at Okayama University Advanced Science Research
Center, and all procedures were approved by the Animal Research
Control Committee of Okayama University. After arrival of the animals,
all were group-housed and acclimated to the colony for 6 weeks before
the experiment. Before the experiment, they were housed with one
mouse per cage, with free access to water and chow pellets in a light
(12 h on, 8:00 a.m./12 h off, 8:00 p.m.), temperature (23 1 °C), and
relative humidity (50 20%) controlled environment. Before experi-
ments, mice were assigned to experimental groups so as to minimize the
variance between groups based on the blood glucose level (one per cage
(17 × 33 × 15 cm3)). Body weight, food intake, and water intake were
measured at approximately 10:00 a.m. every day for 14 days before
dosing. Mice were administered orally with a solution of test compound
Observation of Side Effects after Once-Daily Oral Administration
at 30 mg/kg for 7 Consecutive Days in Male ICR Mice. Six−seven-
week-old male ICR mice were purchased from Charles River
Laboratories Japan, Inc.. This experiment was conducted in accordance
with the Guidelines for Animal Experiments at Okayama University
Advanced Science Research Center, and all procedures were approved
by the Animal Research Control Committee of Okayama University.
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Journal of Medicinal Chemistry
Article
at a dose of 10 mg/kg or with the vehicle (1% ethanol and 0.5% CMC in
distilled water) in a volume of 10 mL/kg of animal at approximately
10:00 a.m. every day for 14 days. At day 15, an oral glucose tolerance test
(OGTT) was performed, and animals were fasted from 5:00 p.m. and
given water ad libitum. On the next day, at approximately 10:00 a.m.,
animals were weighed and anesthetized with diethyl ether. Blood was
removed immediately and centrifuged in an Eppendorf sample tube to
obtain serum. Each blood sample was centrifuged at 1900g for 5 min
at room temperature. Samples for measurements of fed blood glucose
level were taken from the tail vein of the mice, and glucose was measured
by using the glucose oxidase method (Medisafe-Mini, TERUMO,
Tokyo, Japan).
Measurements of Blood Parameters. Aspartate aminotransferase
(AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase
(γ-GTP), alkaline phosphatase (ALP), creatinine (CRE), blood urea
nitrogen (BUN), triglyceride (TG), total cholesterol (TCHO), and
glucose (GLU) were measured by using a Fuji Dry Chem system (Dry
Chem 4000 V, Fuji Medical Co., Tokyo, Japan). White blood cells
(WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT),
mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), mean corpuscular hemoglobin concentration (MCHC), and
platelets (PLT) were measured by using a pocH-100i (Sysmex).
Oral Glucose Tolerance test (OGTT). This experiment was
performed at the same time as the work reported in refs 29 and 30.
KK-Ay mice treated with each compound for 14 days were fasted for
17 h, and orally given glucose solution (100 mg glucose in 1.0 mL
distilled water) at a dose of 1 g/kg body weight. At 0, 15, 30, 45, 60, 90,
and 120 min after the glucose loading, blood glucose level was measured
as described above.
is to use a fully adequate protocol including all strains with and without
metabolic activation, a suitable dose range that fulfills criteria for top
dose selection, and appropriate positive and negative controls. The
recommended set of bacterial strains includes those that detect base
substitution and frameshift mutations were Salmonella typhimurium
TA98, Salmonella typhimurium TA100, Salmonella typhimurium
TA1535, either Salmonella typhimurium TA1537 or TA97 or TA97a,
and either Salmonella typhimurium TA102 or Escherichia coli WP2 uvrA
or Escherichia coli WP2 uvrA (pKM101). For this experiment, five strains
were selected (Salmonella typhimurium TA98, Salmonella typhimurium
TA100, Salmonella typhimurium TA1535, Salmonella typhimurium
TA97, and Salmonella typhimurium TA102). As the maximum dose
level recommended is 5000 μg/plate (or 5 μL/plate for liquid test
substance) when not limited by solubility or cytotoxicity, 5000 μg/plate
(0.1 mL of 50 mg/mL solution) was selected as the top dose. All assays
were performed with plate-incorporation methods52 and with and
without metabolic activation (hereafter referred as +S9 and −S9,
respectively). Assays were performed in duplicate. All experiments were
carried out in accordance with the Safety Guidelines of Okayama
University and the Japanese Government Management Law for Toxic
Chemicals (no. 303).
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR data for compound 14; in vitro and in vivo data are
described (PDF, CSV). This material is available free of charge
via the Internet at http://pubs.acs.org.
RNA Preparation and Quantitative Real-Time RT-PCR. This
experiment was performed according to ref 29. Fifty mg of liver tissue
from KK-Ay mice described above was resected and mechanically
homogenized with a Polytron PT 10/35 (Kinematica Inc., Littau-Luzern,
Switzerland) in 0.5 mL of Trizol reagent (Invitrogen, Carlsbad, CA).
Total RNA was extracted as previously described (S4). Quantitative real-
time RT-PCR analysis was performed using a LightCycler rapid thermal
cycler system (Roche Applied Science, Mannheim, Germany) following
the protocol previously reported (S5). Two μg of total RNA was reverse-
transcribed by random hexamer priming using ReverTra Ace (Toyobo,
Osaka, Japan). The PCR mixture consisted of 1× SYBR Green PCR
Master Mix (Toyobo), which includes DNA polymerase, SYBR Green I
Dye, dNTPs, PCR buffer, and 10 pmol forward and reverse primers and
cDNA of samples in a total volume of 20 μL. The amplification of a
housekeeping gene, Rps18, was used to normalize the efficiency of cDNA
synthesis and the amount of RNA applied. PCR was performed with
initial denaturation at 94 °C for 30 s, followed by amplification for
40 cycles, each cycle consisting of denaturation at 95 °C for 5.0 s,
annealing at 60 °C for 15 s, and polymerization at 72 °C for 15 s. Primers
used for this study are listed in Supporting Information, Table 3.
Ames Test. Salmonella enterica subspecies I, serovar Typhimurium
(Salmonella typhimurium) strain TA97 [hisO1242, hisD6610 DuvrB
gal bio chl1005 rfa1001/pKM101], TA98 [hisD3052 DuvrB gal bio
chl1005 rfa1001/pKM101], TA100 [hisG46 DuvrB gal bio chl1005
rfa1001/pKM101], TA102 [hisD(G)8476 DuvrB gal bio chl1005
rfa1001/pKM101 pAQ1], and TA1535 [hisG46 DuvrB gal bio chl1005
rfa1001] were gifts from Dr. B. N. Ames of the University of California,
Berkeley.51 The supernatant fraction of rat liver homogenate (S9) was
prepared from Sprague−Dawley rats (male, 6 weeks old) that had been
induced with phenobarbital and 5,6-benzoflavone. Positive controls,
2-aminoanthracene (2AA), 6-chloro-9-[3-(2-chloroethylamino)-
propylamino]-2-methoxyacridine 2HCl (ICR191), methylnitrosourea
(MNU), 2-nitrofluorene (2NF), N-nitrosomorpholine (NMOR),
4-nitroquinoline-1-oxide (4NQO), and other reagents were purchased
from commercial sources. Compound 8b and positive controls except
MNU and NMOR were dissolved in dimethyl sulfoxide. MNU and
NMOR were dissolved in sterilized distilled water.
AUTHOR INFORMATION
Corresponding Author
*Phone and Fax: +81-(0)86-251-7963. E-mail: kakuta-h@cc.
okayama-u.ac.jp.
■
Author Contributions
H.K. conceived and designed the project. K.K. and K.M.
synthesized compounds. S.Y. and K.M. performed reporter
gene assays. M.M. prepared plasmids. H.N. prepared RXR
proteins. S.Y. performed cofactor recruitment assays. S.Y., E.I.,
and H.T. performed docking simulation. K.K., M.N., T.K., Y.F.,
and H.K. performed in vivo experiments. A.T. and H.K.
performed HPLC analysis. PCR was performed by T.K. and
T.O. Ames test was performed by S.A. The manuscript was
written by K.K., A.T., and H.K.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Miyachi (Okayama University) for kindly
providing TIPP-703 and carba-T0901317. We also thank Dr.
Kagechika (Tokyo Medical and Dental University) for kindly
providing PA452. The authors are also grateful to Prof. Yoshio
Naomoto, Dr. Takuya Fukazawa (Kawasaki Medical School),
and Dr. Kaori Endo-Umeda (Nihon University) for preparing
plasmids. We are grateful to the Division of Instrumental
Analysis, Okayama University, for the NMR and the mass
measurements. This work was supported by Health and Labour
Science research grants for Research on Seeds for Publicly
Essential Drugs and Medical Devices (grant no. 23080401 to
H.K. and T.O.) from the Ministry of Health, Labour, and Welfare
of Japan, the subsidy to promote science and technology in the
prefectures where nuclear power plants and other power plants
are located (to H.K.), and a Grant-in-aid for Scientific Research
<KAKENHI> (C) (grant no. 25460157 to H.T.) from the
Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan. H.K. acknowledges Okayama University
Genotoxicity assays were performed in accordance with ICH
harmonized tripartite guideline guidance on genotoxicity testing and
data interpretation for pharmaceuticals intended for human use S2(R1)
Current Step 4 version dated 9 November 2011.51 The recommendation
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Article
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■
RXR, retinoid X receptor; PPAR, peroxisome proliferator-
activated receptor; LXR, liver X receptor; Emax, maximum
efficacy; EC50, half-maximal (50%) effective concentration; Asn,
asparagine; His, histamine; SEM, standard error of the mean;
CMC, carboxymethyl cellulose; AST, aspartate aminotransfer-
ase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl trans-
peptidase; ALP, alkaline phosphatase; CRE, creatinine; BUN,
blood urea nitrogen; GLU, glucose; Rps18, ribosomal protein
S18; Irs, insulin receptor substrate; Slc2a, solute carrier family 2
(facilitated glucose transporter); G6pc, glucose-6-phosphatase,
catalytic subunit; Pck, phosphoenolpyruvate carboxykinase; Gck,
glucokinase; Scd1, stearoyl-CoA desaturase-1; Fasn, fatty acid
synthase; Srebp1c, sterol regulatory element binding protein-1c;
ApoE, apolipoprotein E
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