Conformationally Defined Rexinoids and Their Efficacy in the Prevention of Mammary Cancers

Article abstract of DOI:10.1021/acs.jmedchem.5b00829

(2E,4E,6Z,8Z)-8-(3′,4′-Dihydro-1′(2H)-naphthalen-1′-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid (UAB30) is currently undergoing clinical evaluation as a novel cancer prevention agent. In efforts to develop even more highly potent rexinoids that prevent breast cancer without toxicity, we further explore here the structure-activity relationship of two separate classes of rexinoids. UAB30 belongs to the class II rexinoids and possesses a 9Z-tetraenoic acid chain bonded to a tetralone ring, whereas the class I rexinoids contain the same 9Z-tetraenoic acid chain bonded to a disubstituted cyclohexenyl ring. Among the 12 class I and class II rexinoids evaluated, the class I rexinoid 11 is most effective in preventing breast cancers in an in vivo rat model alone or in combination with tamoxifen. Rexinoid 11 also reduces the size of established tumors and exhibits a therapeutic effect. However, 11 induces hypertriglyceridemia at its effective dose. On the other hand rexinoid 10 does not increase triglyceride levels while being effective in the in vivo chemoprevention assay. X-ray studies of four rexinoids bound to the ligand binding domain of the retinoid X receptor reveal key structural aspects that enhance potency as well as those that enhance the synthesis of lipids.

Full text of DOI:10.1021/acs.jmedchem.5b00829

Article
pubs.acs.org/jmc
Conformationally Defined Rexinoids and Their Efficacy in the
Prevention of Mammary Cancers
Venkatram R. Atigadda,† Gang Xia,† Anil Deshpande,† Lizhi Wu,‡ Natalia Kedishvili,‡ Craig D. Smith,§
Helen Krontiras,∥ Kirby I. Bland,∥ Clinton J. Grubbs,∥ Wayne J. Brouillette,*,† and Donald D. Muccio*,†
†
‡
§
∥
Departments of Chemistry, Biochemistry and Molecular Genetics, Vision Sciences, and Surgery, University of Alabama at
Birmingham, Birmingham, Alabama 35294, United States
S
* Supporting Information
ABSTRACT: (2E,4E,6Z,8Z)-8-(3′,4′-Dihydro-1′(2H)-naph-
thalen-1′-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid
(UAB30) is currently undergoing clinical evaluation as a
novel cancer prevention agent. In efforts to develop even more
highly potent rexinoids that prevent breast cancer without
toxicity, we further explore here the structure−activity
relationship of two separate classes of rexinoids. UAB30
belongs to the class II rexinoids and possesses a 9Z-tetraenoic acid chain bonded to a tetralone ring, whereas the class I rexinoids
contain the same 9Z-tetraenoic acid chain bonded to a disubstituted cyclohexenyl ring. Among the 12 class I and class II
rexinoids evaluated, the class I rexinoid 11 is most effective in preventing breast cancers in an in vivo rat model alone or in
combination with tamoxifen. Rexinoid 11 also reduces the size of established tumors and exhibits a therapeutic effect. However,
11 induces hypertriglyceridemia at its effective dose. On the other hand rexinoid 10 does not increase triglyceride levels while
being effective in the in vivo chemoprevention assay. X-ray studies of four rexinoids bound to the ligand binding domain of the
retinoid X receptor reveal key structural aspects that enhance potency as well as those that enhance the synthesis of lipids.
between the ligands and the hydrophobic residues that line the
ligand binding pocket of hRXRα-LBD. Oral dosing of two
methyl homologues of UAB30, 1 and 4, substantially increases
the synthesis of triglycerides in the liver and accumulation in
serum when orally administered to rodents. The enhanced levels
of triglycerides observed in the serum after oral administration of
1 or 4 are similar to those found when bexarotene is
administered. In contrast to these methyl homologues, the
methyl substitution of UAB30 at other positions of the tetralone
produces rexinoids 2, 3, and 5 that are more potent agonists than
UAB30 without stimulating accumulation of triglycerides in the
serum.11 The X-ray crystallography structure of 1 or 4 bound to
the hRXRα-LBD revealed that the methyl groups on the
tetralone ring interacts strongly with helix 7 residues, Phe346 and
Val349; whereas the methyl groups of the tetralone rings of 2, 3,
and 5 did not interact with these residues. Furthermore, two
methyl groups of the bexarotene tetrahydronaphthalene occupy
the same space in the binding pocket as the methyl groups of 1
and 4. Prior studies suggest that RXR potency can be separated
from lipogenic toxicity by avoiding these interactions.9,11 In this
study we prepare each monomethyl homologue of UAB30 in
multigram levels and evaluate them for their capability to prevent
mammary cancers in rats initiated by the potent carcinogen, 1-
methyl-1-nitrosourea (MNU). This study also explores the ring-
expanded homologue of UAB30, 6, which contains a
benzosuberone ring bonded to a 9Z-tetraenoic acid side chain.
1. INTRODUCTION
Rexinoid agonists target exclusively the retinoid X receptors
(RXRs) over agonists for the retinoic acid receptors (RARs).
Bexarotene (Targretin) is the only clinically used rexinoid
approved by the FDA (Figure 1). UAB30 is a rexinoid that is
derived from the structure of 9-cis-retinoic acid, except it contains
a tetralone ring connected to a 9Z-tetraenoic acid side chain.
Recently the X-ray structures of each of these rexinoids were
determined bound to the ligand binding domain of the human
retinoid X receptor (hRXRα-LBD). Each rexinoid adopts an L-
shaped conformation, which is characteristic of the way rexinoid
agonists or the pan-agonist 9-cis-retinoic acid binds to the
receptor.1,2 The binding of the rexinoid agonists cause similar,
but not identical, conformational and dynamical changes to the
ligand binding domain enabling the recruitment of an
amphipathic coactivator peptide containing the LLxxLL motif.
Each rexinoid reduces proliferation and enhances apoptosis in
mammary tumors and efficiently prevents mammary cancers in
rodent models.3−5 Hyperlipidemia is the dose-limiting toxicity of
bexarotene in humans.6,7 Genomic, proteomic, and metabolo-
mics studies establish that oral dosing of bexarotene induces
triglyceride synthesis in rat livers by hyperstimulating tran-
scription of genes under the control of the RXR:LXR
heterodimer, but oral dosing of UAB30 does not.8,9
To improve upon the potency of UAB30, a series of UAB30
homologues were generated with a single methyl group added to
the carbon positions of the tetralone ring.10,11 The monomethyl
substituted UAB30 homologues 1−5 are more potent than
UAB30, likely because of the increased lipophilic interactions
Received: May 29, 2015
© XXXX American Chemical Society
A
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Figure 1. (Top) Structures of bexarotene and 9-cis-retinoic acid. Structures of class II rexinoids: UAB30, five methyl homologues of UAB30 1−5, and a
benzosuberone homologue of UAB30, 6. (Bottom) Structure of class I rexinoid: UAB8 and six more highly substituted analogs of UAB8 (7−12).
Scheme 1. Synthesis of New Class I UAB Rexinoids (9−12) with Different Substituents at R1
The larger ring of 6 is expected to interact better with the ligand
binding pocket residues (particularly helix 11 residues) while
providing minimal interaction to the helix 7 residues that are
thought to induce triglyceride biosynthesis.
B
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Table 1. Summary of Biological Data for UAB Rexinoids, Bexarotene, and 9-cis-Retinoic Acid
cd
,
reduction in cancer, 200
mg/kg diet (unless
otherwise noted)
rexinoid or pan-agonist RXRα binding, Kd (nM) RXRα activation, EC50 (nM) increase in serum triglyceride, 200 mg/kg diet (%) number (%) weight (%)
b
b
b
b
b
b
b
a
a
b
a
9-cis-RA
14
26
33
25
18
15
8
3
3
5
5
3
120 30
326
456
65
70
63
78
0
90
76
76
72
0
b
b
bexarotene
40
3
b
a
a
UAB30
820 70
63
b
b
b
1
120
5
560
b
b
b
b
b
2
720 20
100 30
160 10
620 50
31
b
3
2
51
10
>10
51
0
b
4
2
642
61, 100 mg
b
b
5
10
9
1
59
0
6
3
30
50
5
5
175
289
30
43
33
32
21
86
64
96
27↑
7
34 10
b
b
8
125 25
215 30
6
15
9
14
35
9
3
4
23
19
22
5
5
5
341
71
69
62
99
10
11
12
3
430
b
b
150 10
80 15
76
1
a
b
Data reported by Grubbs et al.3,11 using 150 mg/kg diet of bexarotene, 60 mg/kg diet of 9cRA, and 200 mg/kg diet for 9cUAB30. Data reported in
Atigadda et al. and Deshpande et al.10,11 for 150 mg/kg diet for Targretin and 200 mg/kg diet for (R)-4-Me-UAB30. The percent reduction is
c
cancer is determined as [(number of cancers in rats fed control diet − number of cancers in rats fed control diet plus test rexinoid)/(number cancers
d
in rats fed control diet)] × 100. Methods are described refs 5 and 8.
To continue to explore the relationship between potency and
lipid toxicity, class I UAB rexinoids are revisited. Class I rexinoids
contain a disubstituted cyclohexenyl ring bonded to the 9Z-
tetraenoic acid side chain rather than the substituted tetralone
ring (Figure 1). Previously, we produced UAB8, which contains
an ethyl group at R1 and isopropyl group at R2 of the
cyclohexenyl ring.12,13 The 9-cis-isomer of UAB8 displayed
RXR-selectivity with potency that approaches that of the pan-
agonist 9-cis-retinoic acid.12,13 Using structural information on
the ligand binding pocket of RXR containing UAB30, methyl
homologues of UAB30, bexarotene, and 9-cis-retinoic acid, we
predicted that analogs of UAB8 containing larger alkyl groups at
R1 and R2 can be introduced into the generic structure of class I
rexinoids (Figure 1).
In the second part of this study, we prepare a series of class I
rexinoids 7−12 to understand how steric bulk at the cyclo-
hexenyl ring affects agonist potency and activity as a preventive
agent. We identify the class I rexinoid 11 (R1 = isopropyl group;
R2 = isopentyl group) which is substantially more potent than 9-
cis-retinoic acid and even exceeds the potency of bexarotene. We
determine X-ray crystal structures of hRXRα-LBD bound to 9,
10, or 11 to provide information on the key interactions in the
ligand binding pocket that controls potency. As with class II
rexinoids, the newly designed class I rexinoids, 7−12, are
synthesized at multigram levels for evaluation in 90-day
chemoprevention assays for mammary cancer prevention. This
affords us a comparison of the efficacies of each class of rexinoids
as potential drugs to prevent mammary cancer.
synthesized using methods that were described previously for
UAB8; the detailed experimental procedures are also provided in
the Supporting Information. Scheme 1 summarizes the syntheses
of the retinoids 9−12. The starting ketones (13−16) with the
appropriate substituents at R1 and R2 were synthesized using
previously reported methods, and the detailed experimental
procedures for these precursors are provided in the Supporting
Information.12,13 The synthesis of retinoids 9−12 began with a
Reformatsky reaction between the ketones 13−16 and ethyl 4-
bromo-3-methyl-2-butenoate (1:1 mixture of isomers) in 1,4-
dioxane to provide the 9Z-acids 17−20 in 70−85% yield. The
carboxyl groups in compounds 17−20 were then reduced in the
presence of LiAlH4 to produce the desired 9Z-alcohols 21−24 in
quantitative yield. The alcohol functional groups in compounds
21−24 were next oxidized using IBX14 (rather than MnO2) to
provide the desired 9Z-aldehydes 25−28 containing minor
amounts of all E-aldehyde (5−7%). Pure 9Z-isomers (25−28)
were isolated in 65−80% yields following column chromatog-
raphy. Olefination of aldehydes 25−28 in the presence of triethyl
phosphonosenecioate (1:1 mixture of isomers) under Horner−
Emmons conditions provided the esters 29−32 as an 85:15
mixture of the 9Z-isomer to the 9Z,13Z-isomer. Since the two
isomers were not separable by column chromatography,
hydrolysis of the esters in compounds 29−32 was performed
on the mixture of 9Z and 9Z,13Z-isomers under basic conditions
to provide the acids 9−12. The desired pure 9Z-isomers 9−12
were obtained after recrystallization in 60−65% yields.
2.2. Binding and Transactivation of Rexinoids to RXR.
In our previous publications we determined the dissociation
constants (Kd) for retinoid-receptor complexes by using a
fluorescence quenching method.1,10,11 By use of this method, the
Kd values for the dissociation of either 9-cis-retinoic acid or
UAB30 from its complex with the hRXRα-LBD homodimer
were determined to be 14 and 33 nM, respectively (Table 1).1
Fluorescence titrations of rexinoids 1−5 demonstrated each
methyl homologue was a better binder to hRXRα-LBD than
UAB30 (Table 1). By use of the same method, 6−12 were
evaluated for binding to hRXRα-LBD. Rexinoids 6, 7, and 9−11
2. RESULTS AND DISCUSSION
2.1. Chemistry. Rexinoids 1−5 were synthesized at the 30 g
levels using the previously reported methods to generate smaller
quantities.10,11 Rexinoid 1 was synthesized using commercially
available racemic 4-methyltetralone. Rexinoid 6 was synthesized
utilizing the same methodology starting from benzosuberone
rather than tetralones. The detailed experimental procedure is
provided in the Supporting Information. Rexinoids 7 and 8 were
C
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Table 2. Summary of ITC Measurements of GRIP-1 Binding to hRXRα-LBD:Rexinoid Complexes at 25 °C
rexinoid (25 °C)
Kd (μM)
ΔH (kcal/mol)
−TΔS (kcal/mol)
ΔG (kcal/mol)
n
9-cis-retinoic acid
1.81 0.06
1.33 0.12
1.59 0.04
1.21 0.03
0.47 0.02
−9.2 0.03
−8.9 0.03
−9.5 0.02
−9.4 0.04
−7.6 0.05
0.61 0.06
0.52 0.12
0.84 0.04
0.36 0.03
0.92 0.05
−8.5 0.03
−8.4 0.03
−8.6 0.12
−8.5 0.06
−7.2 0.05
0.92
1.05
0.96
0.93
0.96
UAB30
bexarotene
11
12
quenched more than 90% of the protein fluorescence signal at
337 nm when the ratio of the protein to the rexinoids reached
1:1. Each of these rexinoids was a better binder to hRXRα-LBD
than UAB30. Rexinoids 8 or 12 were the exception; they were 5-
fold weaker binders than UAB30 to hRXRα-LBD.
For the RXR full agonists 9-cis-retinoic acid, UAB30, several
methyl analogs of UAB30 or bexarotene, the thermodynamic
parameters determined from ITC titration of the coactivator
peptide GRIP-1 into hRXRα-LBD homodimers saturated with
agonist were similar regardless of structure of the rexinoid agonist
in the ligand binding pocket.4,5,7 In this study, we compared the
thermodynamics of GRIP-1 binding to hRXRα-LBD homo-
dimers containing the full and potent agonist, 11, to that of the
partial agonist, 12. This comparison was undertaken to decide if
the partial agonist, 12, was capable of recruiting coactivator
peptide to the surface of the RXR receptor like other structurally
different full agonists. Isothermal titration calorimetry (ITC)
measurements were performed at 25 °C (Table 2). When 9-cis-
retinoic acid, UAB30, or bexarotene was bound to hRXRα-LBD
homodimers, GRIP-1 bound well to each coactivator binding site
on the monomers (stoichiometry of nearly 1 for GRIP-
1:hRXRα-LBD monomer subunit). For 11, the stoichiometry
for GRIP-1 binding was also 1. The binding affinities, binding
enthalpies, entropies, or free energies of GRIP-1 to the full
agonist bound complexes were nearly identical to each other
(Table 2). This indicates that hRXRα-LBD homodimers
containing 11 adopt a similar surface on the LBD to recruit
coactivators as found in RXR homodimers containing other
potent agonists. The thermodynamic signature of coactivator
binding is a necessary but not sufficient property of a potent
agonist. The binding of GRIP-1 to hRXRα-LBD homodimers
containing 12 was different from those found from all other ITC
studies. The binding affinity of GRIP was 3-fold less as was
reflected in the 1 kcal/mol lower free energy of binding (−7.2
kcal/mol). The negative free energy change for GRIP-1 binding
to the receptor complex bound with 12 was still present, but the
enthalpy change was substantially smaller in magnitude than
observed for the full agonists studied (−7.6 kcal/mol versus −9.4
kcal/mol). The change in enthalpy may be a thermodynamic
signature for GRIP-1 binding to homodimers containing full
agonists versus partial agonists, and it may be used as a rapid
screen for partial agonists of the RXR receptor.
Binding affinity to hRXRα-LBD is the simplest evaluation of
the potential potency of the rexinoid. To evaluate the efficacy of
rexinoids as agonists, the HEK293 cell line was transiently
transfected with a Gal4 reporter construct containing the
hRXRα-LBD. The enhancement of gene transcription mediated
by agonist was measured using a luciferase reporter system. This
cell line and reporter were recently used to compare the activities
of UAB30, 9-cis-retinoic acid and bexarotene as well as
comparing the agonist activities of different methyl homologues
of UAB30.10,11 Selective data from the previous studies are
included in Table 1 so that the potencies of class II rexinoids can
be compared to class I rexinoids and bexarotene. In this cell line
bexarotene is 3-fold more potent as a RXR agonist (EC50 ≈ 40
nM) than 9-cis-retinoic acid (EC50 ≈ 120 nM). UAB30 is a 20-
fold weaker agonist in this cell line than bexarotene, even though
it was a potent agonist (EC50 = 118 nM) in the CV-1 cell line
used previously.4 Apparently, UAB30 does not recruit
endogenous coactivator proteins in the HEK293 cell line as
well as those found in CV-1 cells (coactivator types and
abundance vary according to cell type). Given the demonstrated
tissue-selective effects of this rexinoid, it is not suprising to find its
potency changing with different in vitro evaluation conditions.
Relative to these data, 6 was evaluated next. Rexinoid 6 is a
closely related homologue of UAB30 containing an extra
methylene group in the benzosuberone ring relative to the
tetralone ring. Consistent with its potent binding to hRXRα-
LBD, 6 was also a potent activator of RXR-mediated tran-
scription, similar to that of bexarotene (Table 1). Rexinoid 7 is
built on the cyclohexenyl ring scaffold (Figure 1). The EC50 value
of 7 was similar to that of bexarotene. When the steric size of the
R1 substituent was increased to a phenyl group, the potency of 8
was substantially lost relative to 7. The R2 substituent of class I
rexinoids was increased to an isopentyl group to give 9. The
potency of 9 was 2-fold better than that of 7. The potencies of
rexinoids 10 (R1 = cyclopropyl) and 11 (R1 = isopropyl) were
similar to the potency of 9 (R1 = ethyl). When the size of the R1
was increased to a phenyl group, the potency of 12 was much
poorer (EC50 ≈ 80 nM) than those of 9−11. Furthermore, the
response at the 1000 nM dose was only 30% of that of 12,
suggesting that 12 is a partial agonist. This loss of agonist
properties for 12 (R1 = phenyl; R2 = isopentyl) relative to 11 (R1
= isopropyl; R2 = isopentyl) was similar to the loss of activity of 8
relative to 7. The EC50 value of 12 was 10-fold less than those of
9−11. Rexinoid 12 was a partial agonist in this cell line inducing
only 20% of the signal of 9-cis-retinoic acid at 1000 nM. When
RARα activation was examined, rexinoids 1−6 and 12 did not
activate transcription relative to controls (less than 5%) at 10−6
M; rexinoids 5, 9−11 induced 20% activation of RARα-mediated
transcription at 10−6 M.
2.3. Crystal Structures of hRXRα-LBD Homodimers
Bound with Rexinoids 6, 9, 10, or 11. The X-ray crystal
structures of hRXRα-LBD homodimers bound to the pan-
agonist 9-cis-retinoic acid or RXR-selective agonists, bexarotene,
UAB30, and its methyl analogs with coactivator peptide GRIP-1
(
686KHKILHRLLQDSS698) were valuable to understanding
structural factors that influenced biological activity and
toxicity.1,2,10 Here crystal strutures of hRXRα-LBD homodimers
containing the coactivator peptide GRIP-1 with one of four
potent agonists (6, 9, 10, and 11) were obtained and compared.
Crystals of the homodimer containing the partial agonist 8 or 12
were not successfully obtained after repeated tries using similar
conditions of crystallization to those of full agonists. In our
laboratory, 13 crystals of hRXRα-LBD homodimers each
containing a potent rexinoid agonist and GRIP-1 were generated
using similar crystallization conditions, suggesting that these
conditions are suited to generate crystals of homodimers bound
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to full RXR agonists but not for the crystallization of homodimers
bound to partial agonists. Each crystal structure of hRXRα-LBD
homodimers containing GRIP-1 and either agonist 6 or 9−11
belonged to the P4(3)2(1)2 space group. The unit cell contained
two monomers of LBD with GRIP-1 bound to each monomer.
The summary of the X-ray crystallography and refinement
statistics for the structures is listed in a table provided in the
Supporting Information.
The 3D fold of the hRXRα-LBD homodimer was nearly
identical to previously reported structures.2 The backbone atoms
of each structure were overlaid with the structure of hRXRα-LBD
bound to UAB30 and GRIP-1 complex (4K4J). The rmsd values
for this overlay of 229 backbone residues were 0.177, 0.211,
0.161, and 0.166 Å for homodimers bound to 6, 9, 10, and 11,
respectively (Figure 2). Four conformational changes occurred
Figure 2. Overlay of X-ray crystal structures of hRXRα-LBD bound to
UAB30 (PDB code 4K4J), 6 (PDB code 4RFW), 9 (PDB code 4RMC),
10 (PDB code 4RMD), 11 (PDB code 4RME). The coactivator peptide
GRIP-1 is displayed in red, and the ligand binding pocket of hRXRα-
LBD is highlighted in gray mesh.
Figure 3. (A) Overlay of UAB30 (green, PDB code 4K4J) and class II
UAB rexinoid, 6 (red, PDB code 4RFW) in the ligand binding pocket
(gray). (B) Overlay of UAB30 (green, PDB code 4K4J) and class I UAB
rexinoids, 9 (blue, PDB code 4RMC), 10 (yellow, PDB code 4RMD),
and 11 (purple, PDB code 4RME) in the ligand binding pocket (gray).
in the layer between the rexinoid binding site and the coactivator
binding site when GRIP-1 binds to holo-protein complex.1,2,11
These conformational changes allow helix 12 of the hRXRα-LBD
to form and stabilize the coactivator binding site. Each of these
conformational changes were observed in the structures of
hRXRα-LBD containing 6, 9, 10, or 11. Together with helices 3
and 4 residues, the ILxxLL motif of GRIP-1 bound to the
hydrophobic pocket on the surface of the receptor and GRIP-1
was held by two charge clamps involving Glu453 of helix 12 and
Lys284 of helix 3. The ITC study of the GRIP-1 binding to
hRXRα-LBD with 11 is consistent with the structural data
presented here. GRIP-1 binding to hRXRα-LBD containing the
partial agonist 12 produced a different thermodynamic signature
(weaker binding affinity and smaller negative enthalpy change)
than observed when a full agonist, 11, is bound. The change in
thermodynamics of GRIP-1 binding to LBD complexes with 11
versus 12 supports the notion that the surface of the LBD
complex when partial agonist 12 is bound is different from the
surface when the full agonist 11 is bound.
2.4. Ligand Binding Pocket of hRXRα-LBD Containing
Rexinoid 6, 9, 10, or 11. Rexinoids 6, 9, 10, and 11 were well-
defined by their electron density maps in the LBP of hRXRα-
LBD. Each rexinoid adopted a nonplanar L-shape conformation
that filled the ligand binding pocket of the LBD (Figure 3). The
dihedral angles of C7−C8−C9−C10 of 6, 9, 10, and 11 were
106.0°, 111.5°, 120.1°, and 100.9°, respectively. The dihedral
angle of C7−C8−C9−C10 of 6 was twisted less than that of
UAB30 (121.4°). Even with this change, the structures of 6 and
UAB30 overlaid well (Figure 3A). The benzosuberone ring of 6
occupied the same space as the tetralone ring of UAB30, except it
was slightly less planar as expected for the seven-membered ring
of the benzosuberone relative to the six-membered ring of the
tetralone. The structures of 9, 10, and 11 occupied the ligand
binding pocket in a very similar manner to each other, and these
structures overlaid well with that of UAB30 (Figure 3B). The
polyene chains of these rexinoids were nearly identical, and the
substituted cyclohexenyl rings of each rexinoid were oriented in
the same space as the tetralone ring of UAB30. The cyclohexenyl
ring of 11 is tilted toward helix 11 residues more than rings of 9
and 10. The cyclohexenyl rings of 9 and 10 were in an envelope
conformation in which the C1′ and C2′ atoms are all on the
opposite side of C19. The cyclohexenyl ring of 11 adopted a half-
chair conformation with C1′ on the same side as C19 and with
C2′ on the opposite side of C19. The extra space in the electron
density map of the rexinoids at R1 was limited. The phenyl group
at R1 of 12 could not occupy this space without disrupting the 3D
fold of the protein, which provides a structural explanation for its
partial agonist properties.
For structures studied in our laboratory, the ligand binding
pocket is small when UAB30 is bound to hRXRα-LBD (442 Å3).
The ligand binding pocket of the RXR domain containing 6 was
slightly larger (464 Å3) than found for UAB30. The size of this
pocket increased to 545 Å3 to accommodate 9, 10, and 11. Even
though the size of the ligand binding pocket changed, the
rexinoids each occupied 75% of available space. To examine
changes in the geometry of the protein residues that surrounded
the ligand, we provide electron density maps of each rexinoid
focusing on the ring region of the ligand binding pocket (Figure
4A). Even though 6, 9, 10, and 11 made more contacts with the
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Figure 4. (A) Electron density maps (2Fo − Fc) for the ligand binding pocket containing 6 (PDB code 4RFW). (B) Electron density maps (2Fo − Fc) for
the ligand binding pocket containing 9 (PDB code 4RMC). (C) Electron density maps (2Fo − Fc) for the ligand binding pocket containing 10 (PDB
code 4RMD). (D) Electron density maps (2Fo − Fc) for the ligand binding pocket containing 11 (PDB code 4RME).
protein residues surrounding them in the ligand binding pockets
of their complexes with hRXRα-LBD than found for the smaller
UAB30, the protein residues that interact with these rexinoids
were nearly identical to each other and to UAB30 (Figure 4A).
For 6 relative to UAB30, the change in total contact surface area
was only 25 Å2. The polyene chain of 6 increased its contact to
residues on helices 3 and 5 slightly more than found for UAB30.
For 9, 10, and 11, the total contact surface areas of the rexinoid to
hRXRα-LBD were each about 100 Å2 larger than those observed
for 9cUAB30. In particular 9, 10, and 11 made more contacts
with the protein residues of helices 3, 5, and 7 and the β sheet
than either 9-cis-retinoic acid or UAB30 did. When helix 11
residues were examined, 9 and 10 made fewer contacts with these
residues than either 9-cis-retinoic acid or UAB30, while the total
contact surface area of 11 with helix 11 residues was similar to
those found in UAB30. The ethyl, cyclopropyl, and isopropyl
groups at R1 of 9, 10, and 11 interacted with residues on helix 7.
The isopropyl group of 11 had one methyl group pointed to
Val342 of helix 7 and the other methyl group pointed to Phe439
and His436 of helix 11. The cyclopropyl group of 10 exhibited
similar interactions with helix 7 and helix 11 residues, but the
methylene groups of the cyclopropyl group provided less surface
area for interaction than the methyl groups of the isopropyl
group. The ethyl group of 9 pointed toward Val342 of helix 7.
In our prior publication we demonstrated how a single methyl
substitution at carbon 7 of the tetralone ring of UAB30 (rexinoid
4) substantially enhanced the agonist potency of this methyl
derivative over its parent compound, but this enhanced potency
was at the expense of substantially enhancing lipid biosynthesis.11
For class II rexinoid 4, the methyl group at carbon 7 interacted
strongly with Phe346 and Val349 of helix 7. The methyl groups
on the reduced naphthalene ring of bexarotene interacted with
same residues on helix 7. The class II rexinoid 6 is situated in the
ligand binding pocket in a nearly identical manner to UAB30
(Figure 3A contains an overlay of the two rexinoids). The
benzosuberone of 6 did not make van der Vaals contacts with
Phe346 and Val349 of helix 7 (Figure 4A). The ring of 6 was only
slightly larger than the ring of UAB30 and did not contain methyl
groups that protrude toward the helix 7 residues. In contrast to 6,
rexinoids 9, 10, and 11 contained an isopentyl group at R2 of the
cyclohexenyl ring. When these structures were overlaid with
those of UAB30, the isopentyl group extended well beyond the
space occupied by the tetralone ring of UAB30 (Figure 3B) and
pointed toward helix 7 (Figure 4B−D). As shown in the 2Fo − Fc
plots, the methyl groups in isopentyl substituent of 9, 10, and 11
made substantial van der Waals contact with both Phe346 and
Val349 of helix 7 (Figure 4B−D). Additionally an isopentyl
methyl group interacted well with Phe313 of helix 5. This
interaction was not present in 6 or UAB30; in these structures
the methine at carbon-8 of the tetralone ring of UAB30 was just
outside of van der Waals contact to Phe313. In the published
structure of bexarotene, the methyl groups of the tetrahydro-
naphthalene ring interacted strongly with Phe313.2 Rexinoids 9,
10, and 11 are as potent as or more potent than bexarotene as a
RXR agonist in the in vitro transient transfection assays, which is
consistent with the enhanced contacts between its ring R-groups
and helix 7 (Table 1).
2.5. In Vivo Triglyceride Levels and Effectiveness for
the Prevention of MNU-Initiated Mammary Cancer.
Elevated serum triglyceride levels were observed in humans
orally administered bexarotene or 9-cis-retinoic acid. The
triglyceride levels measured in humans were similar to those
found in rats given these drugs.7 A 7-day in vivo screen was used
to evaluate if oral dosing of other rexinoids (6−12) increases
serum triglyceride levels. Serum triglycerides were measured in
rats fed each rexinoid at a dose of 200 mg rexinoid/kg diet for 7
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days. As displayed in Table 1, class I rexinoids 8, 10, and 12 did
not significantly increase triglycerides over control rodents. The
modest increase in serum triglyceride levels measured for either 8
or 12 is consistent with their status as a partial RXR agonists.
However, rexinoid 10 is a potent full agonist, yet triglyceride
levels were low. Three class II rexinoids (2, 3, and 5) are other
examples of potent agonists that did not increase serum
triglycerides significantly above normal. Rexinoid 6 is a full
agonist with similar potency to 10; it elevated serum TG levels to
175% which is higher than those of UAB30, 2, 3, 5, or 10 but well
below levels of bexarotene, 9-cis-retinoic acid, 1, and 4. Rexinoids
7, 9, and 11 are even more potent agonists than the class II
rexinoids, and their administration raised triglycerides levels by
280−640% over controls, which are serum levels achieved when
9-cis-retinoic acid, bexarotene, 1, or 3 is administered. Compared
to the large increase seen with the 200 mg/kg dosing, a 100 mg/
kg diet dose of 9 or 11 resulted in a smaller increase in serum
triglyceride levels (about 1/2 the value observed for 200 mg/kg
diet dosing). The structures of 9 and 11 contained methyl groups
that interact strongly with helix 7 residues. These interactions
were similar to those observed for 1, 4, or bexarotene.10,11 Thus,
each rexinoid that contained these structural features increased
lipid biosynthesis and accumulation in serum. Unexpectedly, 10
with an isopentyl group at R2, which interacted with helix 7
residues, did not substantially raise serum TG levels. As discussed
previously, there were subtle differences in structure of 10 versus
11 in the ligand binding pocket. The cyclopropyl group at R1 of
10 apparently influenced agonist activity as well as mitigated lipid
biosynthesis.
The NCI Division of Cancer Prevention uses the MNU-
initiated mammary model to evaluate the potential of new
compounds for the prevention of ER-positive breast cancers in
humans. Recent gene array studies revealed that aromatase
inhibitors modulated gene products in the MNU-chemically
initiated model for mammary cancer in rats that were remarkably
similar to how these drugs modulated gene products in humans
with ER-positive breast cancers.15 Like aromatase inhibitors,
rexinoid agonists prevented chemically initiated mammary
cancers in this rat model. We demonstrated that UAB30 or
racemic 1 was as effective as 9-cis-retinoic and bexarotene in
preventing cancers in the MNU model (Table 1).1,2,10 In this
study, we evaluated the efficacy of both class I and class II
rexinoids in the same 3-month model that informs on the
preventive effects of new drugs in humans. The effectiveness of
class I and class II rexinoids reported in Table 1 are reported at a
single dose of 200 mg/kg diet, which was used for the evaluation
of UAB30 (except for 4 as discussed later). After 3 months of
treatment at 200 mg/kg diet after carcinogen administration, 11
was clearly the most effective rexinoid; it was even more effective
than bexarotene. Rexinoid 11 prevented essentially all mammary
cancers at this dose without overt toxicity (except elevated
lipids). Rexinoids 9 and 10 were also effective in preventing 60−
70% of mammary cancers. Rexinoid 7 had intermediate
effectiveness and reduced only 43% of cancers, which is less
than the more potent class I rexinoids, 9, 10, or 11. Rexinoids 8
and 12, two partial agonists, exhibited no effectiveness in the
prevention of mammary cancers. The effectiveness of cancer
prevention clearly correlated with agonist potency for class I
rexinoids.
particularly toxic to rats when evaluated in the MNU-initiated
prevention model using a 200 mg/kg diet dose. After a month of
chronic treatment at this dose, rats displayed clinical signs of
hypervitaminosis A toxicity (e.g., reduction of body weight gain,
skin reddening, and mucus membrane dryness). The dose was
lowered to 100 mg/kg diet after toxicity was observed, and the
study was continued to duration without further clinical
observation of vitamin A toxicity. Essentially no tumors appeared
at the termination of the study in the treated group. To further
evaluate the effectiveness of this rexinoid, 4 was evaluated again
in this prevention model using a 100 mg/kg diet dose for the
entire 3 months of study. Rexinoid 4 was 61% effective at the
lower dose (Table 1). Serum triglycerides were elevated by 350%
in rat serum when fed the 100 mg/kg diet. The UAB30 methyl
homologues 2, 3, and 5 and rexinoid 6 were not effective (or
poorly effective) at the 200 mg/kg diet dose. The low
effectiveness of 2, 3, 5, or 6 as preventative agents was surprising.
For class I rexinoids the effectiveness as an RXR agonist
correlated well with its effectiveness in cancer prevention, but
this correlation was not observed for 2, 3, 5, or 6 (Table 1). To
evaluate metabolism, the serum levels were measured after the
study. The serum levels of 1, 2, 3, 5, and 6 were well below 1 μM,
which were much lower than observed for UAB30 (2 μM). The
serum levels of every class I rexinoids were 3 μM or greater (3−6
μM). The low serum levels of 2, 3, and 5 suggest that oxidative
metabolism (e.g., benzylic oxidation of methyl groups) may play
a role in the reduced potency of these rexinoids. Of those
rexinoids examined in our laboratory, only the pan-agonist 9-cis-
retinoic acid prevents mammary cancers in this rodent model
when serum levels are low (<1 μM). Every other active rexinoid
in this mammary model exhibited higher serum levels in excess of
1 μM.
The preventive effects of 11 encouraged us to continue its
evaluation as a putative preventive agent for mammary cancer.
We undertook a study using lower doses alone and in
combination with the selective estrogen receptor modulator
(SERM) tamoxifen. When 11 was administered at 50 mg/kg diet,
cancer incidence was 26% alone (decrease in tumor size was
37%). With a 0.4 mg/dg diet dose of tamoxifen, the cancer
incidence in this group of rats was 10% (decrease in tumor size
was 3%). When rats were fed the low-dose 11 and tamoxifen
together, the cancer incidence was 53% after 120 days (decrease
in tumor size was 68%), which suggested an additive or synergic
effect of preventive actions of the SERM and rexinoid. As
importantly, the lower dose of 11 only increased serum TG by
about 65% (substantially lower than a 200 kg/diet dose). The
lower dose is more viable for use as a potential preventive agent
in the clinic, since lipid toxicity would be less of a problem for
long-term administration. These studies were similar to those
published for 9-cis-retinoic acid, UAB30, or bexarotene using a
lower dose.3,8
2.6. Inhibition of Growth of MNU-Initiated Mammary
Tumors. SERMs are administered to humans with ER-positive
mammary cancers in an adjuvant or neoadjuvant setting in the
clinic. Since rexinoids reduce proliferation in existing MNU-
initiated mammary cancers, we next investigated if rexinoid
treatment would slow the growth of existing mammary tumors.
To investigate the effectiveness of rexinoids for therapy of ER-
positive mammary cancers, rats (N = 9) with small MNU-
initiated tumors of about 200 mm2 were treated with 200 mg/kg
diet of UAB30 or with 11. The size of mammary tumors in
control animals rapidly grew. The rate of tumor growth was
different for each animal, which is consistent with chemically
Among class II rexinoids, only two methyl homologues of
UAB30 were as effective or more effective than UAB30. Rexinoid
1 was slightly more effective than UAB30, consistent with its
enhanced agonist potency. Rexinoid 4 was effective but
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induced tumors containing different genomic instabilities. To
account for slightly different sizes of tumors at day 0, the data
were normalized to the size of the tumor at day 0 (just prior to
rexinoid administration). The change in the size of a tumor with
time is reported as a percent change relative to the size of the
tumor at day 0. After 7 days of rexinoid treatment, the average
tumor size of the untreated group nearly doubled. After 14 days,
the tumors from control group rats were on average 4-fold larger
(Figure 5). At this time point one rat was removed from the study
aromatase inhibitors are similar to those observed in humans
treated with the same class of drug.15 Aromatase inhibitors block
cell proliferation by downregulation of numerous genes (e.g.,
cyclins). Rexinoids (bexarotene or UAB30) display significant
capability to prevent MNU-initiated mammary cancers; they
significantly decrease cell proliferation in MNU-initiated cancers
as well as increase apoptosis.3,5,6,8 As reviewed by den Holannder
et al.,16 the RAR:RXR heterodimer is an important target of
rexinoids. Retinoic acid, which is synthesized by oxidative
metabolism of vitamin A, binds to RAR:RXR heterodimers,
slows tumor cell proliferation, induces cell differentiation, and
enhances apoptosis.17 RXR agonists are known to enhance
signaling of retinoic acid or other RAR agonists through the
permissive RAR:RXR heterodimer. Bexarotene induces RXR and
RAR expression in normal mammary epithelial cells and
mammary cancers (T47D).18 Bexarotene also stimulates the
expression of insulin-like growth factor binding protein 6 and
reduces cell proliferation and increases apoptosis.18 Rexinoids
have an added advantage of stimulating additional signaling
pathways that control cell proliferation besides those pathways
mediated by the RAR:RXR heterodimers. In vitro studies suggest
rexinoids also enhance PPARγ:RXR signaling, which kill cancer
cells. Studies by Bonofiglio et al. demonstrate profound effects of
low doses of RXR and PPARγ agonists in inducing apoptosis in
human breast cancer cells but not in normal breast epithelial
cells.19 This suggests that rexinoids may in fact be preventing
mammary cancer by working with endogenous PPARγ agonists.
Other recent work suggests PPARγ agonists and rexinoids work
synergistically to block inflammatory signaling in cancer stem
cells that surround and support growth of breast tumors.20
Rexinoids (e.g., bexarotene) may also prevent mammary cancer
development by suppressing the expression of COX-2 in normal
and premalignant mammary epithelial cells.21 We are still unsure
if the profound effects that rexinoids have in the prevention of
cancer are due to slowing growth and inducing cell death in
microscopic disease (transformed tissue) or if these effects are
due to preventing transformed epithelial cells from progressing
from normal phenotypes to frank cancers. It is quite possible
each rexinoid has different modes of action, even though they
bind with high affinity to the same RXR receptor.
As the answer to this question emerges, we focus here on the
clinical utility of a limited set of class I and II rexinoids. We search
for a nontoxic rexinoid with clinical potential greater than that of
UAB30, which is in clinical development for breast cancer
prevention. Among class II rexinoids, we examined methyl
homologues of UAB30, 1−5, and a ring expanded rexinoid, 6.
Even though rexinoids 1−6 were substantially more potent than
the parent RXRα agonist, UAB30, only two class II rexinoids, 1
and 4, were more effective than UAB30, and each of these
rexinoids produced substantial triglyceride accumulation in
serum (a hallmark of lipid toxicity). Since cancer prevention in
the high-risk population requires chronic dosing for extended
periods (or even lifetimes), toxicity is a real concern in drug
development. Among class II rexinoids, UAB30 is the clear
compromise between reasonable effectiveness and low toxicity.
We also examined rexinoids built from our original design of class
I rexinoids. To date, UAB8 was the most potent homologue
among this class reported.12 Our recent structures of hRXRα-
LBD containing bound class II rexinoids and bexarotene revealed
that larger groups could be accommodated in the binding pocket
of the LBD.1,2,10,11 The most potent class I rexinoid contained an
isopentyl group at R2 and smaller alkyl groups (cyclopropyl or
isopropyl) at R1. Rexinoid 11 was substantially more potent than
Figure 5. Treatment of small existing MNU-initiated rat mammary
cancers with UAB30 (red) or class I rexinoid, 11 (blue), at 200 mg/kg
diet versus controls (black). The size of the mammary cancers was
normalized to 100% at day zero of treatment (N = 9). The change of the
size of the cancer for controls or treated rats were measured once a week
for 4 weeks and averaged at each time point. The average tumor size (y-
axis) was determined by the ratio of the average size at the indicated day
to the average size at day zero. Nine animals (n = 9) survived in each
treated group, and five animals (n = 5) survived in the control group
after 28 days.
to prevent death from a large necrotic tumor. At later time points,
especially rapid tumor growth resulted in removal of three
additional rats from the study in the control group to avoid death.
The average size of the tumors was smaller at time points of 21
and 28 days because of the smaller average size of the remaining
tumors in control rats (since these are chemically initiated
spontaneous tumors with different genetic mutations, tumors
grow at different rates). In contrast to tumors from rats fed only
control diets, the average tumor size of rats fed 11 mainly
decreased after 28 days of treatment (Figure 5). In one rat, the
tumor completely disappeared at day 24; in four other animals
the tumor size significantly decreased after 28 days of treatment
with 11 (30−55% decrease in size). In only one treated rat, the
tumor growth was similar to that in the control rats. These data
demonstrate that treatment with 11 is capable of regressing
tumor growth for many cancer genotypes. The preclinical studies
suggest 11 could be used as an adjuvant or neoadjuvant agent of
estrogen-positive tumors in addition to its use to prevent new
cancers. A similar effect was observed for bexarotene treated
MNU-mammary cancers.8 UAB30 was also studied, since this is
the most potent and least toxic rexinoid we have developed. The
average size of tumors from rats treated with UAB30 increased
slightly relative to day 0 (40% increase in the average size of
tumors between day 0 and day 14), but tumors from control rats
were 400% larger during the same time period (Figure 5).
3. CONCLUSIONS
The MNU-initiated mammary model is used currently by the
NCI to identify new preventive breast cancer drugs. Recent
studies demonstrate that the genomic changes observed in
MNU-initiated mammary cancers from rats treated with
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ketone 13 (47.0 g, 242 mmol) and ethyl 4-bromo-3-methylbut-2-enoate
(120 g, 579.43 mmol) in dry dioxane (200 mL). Vigorous bubbling was
noticed during the addition process, and the reaction mixture was stirred
at reflux for 3 h and then cooled to temperature. Water (100 mL) and 2
N HCl (250 mL) were added. The mixture was diluted with ether (500
mL) and allowed to stir for 15 min. The mixture was filtered, and the
acidic layer was separated. The organic layer was washed successively
with water (2 × 100 mL), 1 N NaOH (3 × 150 mL). The basic wash was
cooled in an ice bath, acidified with HCl (2 N) to pH 1−2, and washed
with ether (2 × 200 mL). The combined organic layers were washed
with water (2 × 50 mL), brine (50 mL), dried (Na2SO4), and evaporated
under vacuum to provide a semisolid. This was crystallized from
hexanes/ether, filtered, and dried to give 41 g (62%) of pure 17: mp 71−
73 °C; MS m/z 277 (M + 1); 1H NMR (300 MHz, CDCl3) δ 6.6 (s, 1H),
5.7 (s, 1H), 2.3−2.2 (m, 4H), 2.2−2.1 (m, 4H), 2.1 (s, 3H), 1.7−1.6 (m,
2H), 1.6−1.5 (m, 1H), 1.4−1.3 (m, 2H), 1.0 (t, 3H), 0.9 (d, 6H); 13C
NMR (CDCl3) δ 171.1, 157.2, 141.7, 141.2, 132.0, 120.2, 116.9, 38.7,
30.2, 28.8, 28.7, 27.7, 25.9, 25.8, 22.9, 22.5, 12.8.
(2Z)-4-(2′-(3-Methyl)butyl-3′-cyclopropyl-2′-cyclohexen-1′-
ylidene)-3-methyl-2-butenoic Acid (18). Mp 82−84 °C; MS m/z
289 (M + 1); 1H NMR (300 MHz, CDCl3) δ 12.0−10.0 (br, 1H), 6.6 (s,
1H), 5.7 (s, 1H), 3.0−2.9 (m, 1H), 2.3−2.2 (m, 4H), 2.1 (s, 3H), 2.1−
1.9 (m, 2H), 1.7−1.5 (m, 3H), 1.3−1.2 (m, 2H), 1.0 (d, 6H), 0.9 (d,
6H); 13C NMR (CDCl3) δ 171.9, 157.5, 141.6, 139.4, 134.4, 120.3,
117.3, 38.5, 29.1, 28.9, 26.5, 26.3, 25.9, 23.1, 22.9, 14.7, 5.3.
(2Z)-4-(2′-(3-Methyl)butyl-3′-isopropyl-2′-cyclohexen-1′-yli-
dene)-3-methyl-2-butenoic Acid (19). Mp 100−102 °C; MS m/z
291 (M + 1); 1H NMR (300 MHz, CDCl3) δ 6.6 (s, 1H), 5.7 (s, 1H),
2.96−2.9 (m, 1H), 2.4−2.2 (m, 4H), 2.1 (s, 3H), 2.1−2.0 (m, 2H) 1.6−
1.5 (m, 3H), 1.4−1.3 (m, 2H), 1.0 (d, 6H), 0.9 (d, 6H); 13C NMR
(CDCl3) δ 171.8, 157.7, 145.5, 141.8, 131.6, 120.9, 117.4, 39.2, 30.8,
29.3, 29.2, 26.2, 26.0, 24.7, 23.4, 22.9, 21.0.
bexarotene, and it exhibited an extremely high effectiveness to
prevent mammary cancer. The structural studies revealed in this
study demonstrated the isopentyl group at R2 filled the ligand
binding pocket optimally. Rexinoid 11 also demonstrated
therapeutic potential at doses well below its cytotoxic effects.
The increase in potency of 11 came at a cost. Rexinoid 11
exhibited enhanced triglyceride biosynthesis and accumulation in
serum levels similar to bexarotene or 9-cis-retinoic acid. The
structural studies provided here were consistent with our prior
studies that revealed interactions between the rexinoid and helix
7 residues as a putative “hot-spot” for inducing serum triglyceride
synthesis. As proposed in other studies, we believe this occurs by
enhancing signaling of RXR:LXR heterodimers in the liver.9 To
lessen toxicity, we also revealed in this study that rexinoid 11
effectively reduced mammary cancers when used at a lower dose
and placed in combination with the SERM tamoxifen. The low-
dose 11 administration with SERM does not substantially elevate
serum triglyceride levels, which is a clinical concern when
administering a preventive agent chronically to the high-risk but
disease free population.
A surprising find of this study occurred when we changed the
isopropyl group at R1 of the cyclohexenyl ring to a cyclopropyl
group. The effectiveness of 10 in cancer prevention was less than
11, but levels of serum triglycerides were substantially mitigated.
While 10 is clearly less potent than 11, 10 displays similar
effectiveness to that of UAB30. On the basis of the results of
these studies, UAB30, 10 alone, or 11 in combinations with
SERMs appears to offer the most promise for translational
development in the prevention of human mammary cancer.
(2Z)-4-(2′-(3-Methyl)butyl-3′-phenyl-2′-cyclohexen-1′-yli-
dene)-3-methyl-2-butenoic Acid (20). Mp 136−138 °C; MS m/z
325 (M + 1); 1H NMR (300 MHz, CDCl3) δ 7.4−7.3 (m, 2H), 7.3−7.2
(m, 1H), 7.2−7.1 (m, 2H), 6.7 (s, 1H), 5.7 (s, 1H), 2.44 (t, 2H), 2.37 (t,
2H), 2.2−2.1 (m, 2H), 2.1 (s, 3H), 1.8−1.7 (m, 2H), 1.4−1.2 (m, 3H),
0.7 (d, 6H); 13C NMR (CDCl3) δ 171.9, 157.2, 145.0, 141.1, 140.2,
135.0, 128.5, 128.1, 126.7, 122.7, 117.9, 39.2, 34.2, 28.8, 27.7, 26.9, 26.2,
23.5, 22.7.
4. EXPERIMENTAL SECTION
1H and 13C NMR spectra were recorded on Bruker ARX 300 or DRX
400 spectrometer. IR spectra were recorded using an ABB Bomem FTIR
spectrometer. UV−vis spectra were recorded on a Varian (Carry 100
Conc) spectrophotometer in methanol (Aldrich, spectrograde). Mass
spectra were taken on a Hewlett-Packard 1100 LC−MS instrument
using electrospray ionization (ESI). Melting points were recorded on an
Electrothermal melting point apparatus and are uncorrected. All
reactions, unless otherwise mentioned, were monitored by thin layer
chromatography (TLC) on 0.25 mm silica gel plates (60F-254, E. Merck
or Silicycle). Flash chromatography was performed using Silicycle silica
gel (40−63 μm). Reactions and purifications were conducted with
nitrogen-saturated solvents and under subdued lighting. Ethyl 4-bromo-
3-methylbut-2-enoate (1:1 mixture of isomers) was prepared by the
reaction of ethyl 3,3-dimethylacrylate with N-bromosuccinimide.
Triethyl phosphonosenecioate (1:1 mixture of isomers) was prepared
via the Arbusov reaction.22,23 Triethyl phosphonosenecioate was
prepared by using the procedure described in ref 21 except that we
used a roughly 1:1 mixture of ethyl 4-bromo-3-methylbut-2-enoate as
the starting material. Purity of all the compounds was ≥95%, which was
determined by combustion analysis performed by Atlantic Microlabs,
Inc. (Norcross, GA).
General Procedure for the Reduction of C15 Acids. By use of
this general procedure, all the alcohols were prepared.
(2Z)-4-(2′-(3-Methyl)butyl-3′-ethyl-2′-cyclohexen-1′-yli-
dene)-3-methyl-2-butenol (21). To a flame-dried three-neck round-
bottomed flask fitted with a nitrogen inlet and addition funnel were
added acid 17 (40 g, 145 mmol) and anhydrous ether (800 mL). The
flask was cooled to 0 °C in an ice bath, and the reaction mixture was
treated with 1 M LiAlH4/ether (200 mL, 202 mmol) dropwise. The
reaction mixture was stirred for an additional 1 h at 0 °C, cooled to −78
°C in dry ice/acetone bath, and slowly quenched with methanol (100
mL) followed by 10% H2SO4 (200 mL). The reaction mixture was
allowed to come to room temperature and extracted with ether (3 × 200
mL). The combined ether layers were washed with water (100 mL),
brine (2 × 100 mL), dried (Na2SO4), and concentrated under vacuum
to give the alcohol 21 (38 g,100%) as a colorless oil, which was used in
the next reaction without further purification. MS m/z 263 (M + 1); 1H
NMR (300 MHz, CDCl3) δ 5.8 (s, 1H), 5.5−5.4 (m, 1H), 4.0 (d, 2H),
2.3−2.2 (m, 2H), 2.1−2.0 (m, 6H), 1.8 (s, 3H), 1.7−1.5 (m, 3H), 1.3−
1.2 (m, 2H), 1.0 (t, 3H), 0.9 (d, 6H); 13C NMR (CDCl3) δ 139.3, 138.1,
137.4, 131.1, 125.3, 119.7, 61.2, 38.9, 30.2, 28.8, 27.9, 27.3, 25.5, 24.2,
23.2, 22.6, 12.9.
Reformatsky Reaction for Preparing C15 Acids. By use of this
general procedure, all C15 acids were prepared.
(2Z)-4-(2′-(3-Methyl)butyl-3′-ethyl-2′-cyclohexen-1′-yli-
dene)-3-methyl-2-butenoic Acid (17). A suspension of Zn dust (42
g) in 10% HCl (150 mL) was stirred under nitrogen for 10 h in a 1 L,
three-neck round bottomed flask. The aqueous layer was decanted, and
the zinc was washed successively with distilled water (3 × 150 mL),
anhydrous acetone (3 × 150 mL), and anhydrous ether (3 × 150 mL).
After removal of the residual ether, the zinc dust was heated strongly
with a Bunsen burner flame for about a minute under vacuum. The
clumps of zinc were then carefully broken up with a stirring rod. The
cooled zinc was suspended in anhydrous dioxane (200 mL), and the
stirred suspension was heated to 125 °C in an oil bath. This reaction
mixture was then treated dropwise over a period of 1 h with a solution of
(2Z)-4-(2′-(3-Methyl)butyl-3′-cyclopropyl-2′-cyclohexen-1′-
ylidene)-3-methyl-2-butenol (22). MS m/z 275 (M + 1); 1H NMR
(300 MHz, CDCl3) δ 5.8 (s, 1H), 5.5−5.4 (m, 1H), 4.0 (d, 2H), 2.5−2.4
(m, 2H), 2.1−2.0 (m, 2H), 1.8 (s, 3H), 1.8−1.7 (m, 3H), 1.6−1.5 (m,
3H), 1.4−1.3 (m, 2H), 0.9 (d, 6H), 0.7−0.6 (m, 2H), 0.6−0.5 (m, 2H).
(2Z)-4-(2′-(3-Methyl)butyl-3′-isopropyl-2′-cyclohexen-1′-yli-
1
dene)-3-methyl-2-butenol (23). MS m/z 277 (M + 1); H NMR
(300 MHz, CDCl3) δ 5.8 (s, 1H), 5.5−5.4 (m, 1H), 4.0 (d, 2H), 2.9−2.8
I
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(m, 1H), 2.3−2.2 (m, 2H), 2.1−2.0 (m, 4H), 1.8 (s, 3H), 1.6−1.5 (m,
3H), 1.3−1.2 (m, 2H), 1.0 (d, 6H), 0.9 (d, 6H).
MHz, CDCl3) δ 6.6 (dd, 1H), 6.2 (d, 1H), 6.02 (d, 1H), 5.9 (s, 1H), 5.7
(s, 1H), 4.1 (q, 2H), 2.3−2.2 (m, 2H), 2.2 (s, 3H), 2.2−2.1 (m, 6H), 1.9
(s, 3H), 1.7−1.5 (m, 3H), 1.4−1.3 (m, 2H), 1.3 (t, 3H), 1.0 (t, 3H), 0.9
(d, 6H); 13C NMR (CDCl3) δ 167.3, 153.2, 142.0, 140.0, 138.9, 133.4,
133.1, 131.5, 126.7, 120.1, 118.0, 59.5, 39.0, 30.3, 28.7, 28.4, 27.4, 25.6,
24.8, 23.4, 22.6, 14.4, 13.7, 12.9.
(2E,4E,6Z)- and (2Z,4E,6Z)-Ethyl 8-(2′-(3-Methyl)butyl-3′-
cyclopropyl-2′-cyclohexen-1′-ylidene)-3,7-dimethyl-2,4,6-oc-
tatrienoate (30). MS m/z 383 (M + 1); 1H NMR (300 MHz, CDCl3)
δ 6.6 (dd, 1H), 6.18 (d, 1H), 6.02 (d, 1H), 5.9 (s, 1H), 5.7 (s, 1H), 4.1
(q, 2H), 2.5−2.4 (m, 2H), 2.2 (s, 3H), 2.1 (t, 2H), 1.9 (s, 3H), 1.8−1.7
(m, 3H), 1.7−1.5 (m, 3H), 1.4−1.3 (m, 2H), 1.3 (t, 3H), 0.9 (d, 6H),
0.7−0.6 (m, 2H), 0.6−0.5 (m, 2H); 13C NMR (CDCl3) δ 167.6, 153.5,
142.4, 139.3, 137.7, 133.8, 133.5, 127.1, 120.0, 118.4, 59.9, 38.9, 29.1,
28.6, 26.3, 26.1, 25.2, 23.4, 23.0, 14.8, 14.6, 14.2, 5.2
(2E,4E,6Z)- and (2Z,4E,6Z)-Ethyl 8-(2′-(3-Methyl)butyl-3′-iso-
propyl-2′-cyclohexen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrie-
noate (31). MS m/z 385 (M + 1); 1H NMR (300 MHz, CDCl3) δ 6.6
(dd, 1H), 6.2 (d, 1H), 6.02 (d, 1H), 5.9 (s, 1H), 5.7 (s, 1H), 4.1 (q, 2H),
3.0−2.9 (m, 1H), 2.3−2.2 (m, 2H), 2.2 (s, 3H), 2.15 (t, 3H), 2.0 (t, 3H),
1.9 (s, 3H), 1.6−1.5 (m, 3H), 1.3−1.2 (m, 5H), 1.0 (d, 6H), 0.9 (d, 6H);
13C NMR (CDCl3) δ 167.6, 153.5, 143.9, 142.4, 139.5, 133.7, 133.5,
(2Z)-4-(2′-(3-Methyl)butyl-3′-phenyl-2′-cyclohexen-1′-yli-
1
dene)-3-methyl-2-butenol (24). MS m/z 311 (M + 1); H NMR
(300 MHz, CDCl3) δ 7.4−7.3 (m, 2H), 7.3−7.2 (m, 1H), 7.2−7.1 (m,
2H), 5.9 (s, 1H), 5.6−5.5 (m, 1H), 4.0 (d, 2H), 2.4−2.3 (m, 2H), 2.3−
2.2 (m, 2H), 2.2−2.1 (m, 2H), 1.8 (s, 3H), 1.8−1.7 (m, 3H), 1.3−1.2
(m, 2H), 0.7 (d, 6H).
General Procedure for the Oxidation of Alcohols. By use of this
general procedure, all the aldehydes were prepared.
(2Z)-4-(2′-(3-Methyl)butyl-3′-ethyl-2′-cyclohexen-1′-yli-
dene)-3-methyl-2-butenal (25). A three-neck, round-bottomed flask
fitted with a reflux condenser was charged with o-iodoxybenzoic acid
(IBX) (125 g, 446 mmol) and acetone (1000 mL) and warmed to 50−
55 °C. A solution of crude alcohol 21 (38 g, 145.0 mmol) in acetone
(600 mL) was added all at once to the reaction mixture. The reaction
was then allowed to stir at 50−55 °C for 1.5 h under subdued light. The
reaction mixture was cooled to 0 °C in an ice bath, diluted with ether
(500 mL), and filtered through a sintered glass funnel. The filtrate was
washed with ether (2 × 500 mL) and the combined organic layers were
concentrated under vacuum (rotary evaporator water bath temperature
kept at <35 °C) to give a crude oil. This was purified by flash column
chromatography (silica gel, 1:6 ether/hexanes, all column solvents
purged with nitrogen) to give pure 9Z-aldehyde 25 (27 g (73%) and 0.5
g of all-E 25. MS m/z 261 (M + 1); 1H NMR (300 MHz, CDCl3) δ 9.5
(d, 1H), 6.0 (s, 1H), 5.95−5.9 (m, 1H), 2.3−2.2 (m, 4H), 2.2−2.1 (m,
4H), 2.0 (s, 3H), 1.7−1.5 (m, 3H), 1.3−1.2 (m, 2H), 1.0 (t, 3H), 0.9 (d,
6H).
130.9, 127.1, 120.6, 118.4, 59.9, 39.6, 30.7, 29.1, 29.0, 25.8, 25.2, 24.8,
23.8, 23.0, 21.2, 14.7, 14.2
(2E,4E,6Z)- and (2Z,4E,6Z)-Ethyl 8-(2′-(3-Methyl)butyl-3′-
phenyl-2′-cyclohexen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrie-
noate (32). MS m/z 419 (M + 1); 1H NMR (300 MHz, CDCl3) δ 7.4−
7.3 (m, 2H), 7.3−7.2 (m, 1H), 7.2−7.1 (m, 2H), 6.7 (dd, 1H), 6.2 (d,
1H), 6.08 (d, 1H), 6.0 (s, 1H), 5.7 (s, 1H), 4.18 (q, 2H), 2.4 (t, 2H), 2.3
(s, 3H), 2.3−2.2 (m, 2H), 2.2−2.1 (m, 2H), 1.95 (s, 3H), 1.8−1.7 (m,
2H), 1.4−1.2 (m, 3H), 1.2 (t, 3H), 0.7 (d, 6H); 13C NMR (CDCl3) δ
167.6, 153.4, 144.9, 141.8, 140.0, 138.5, 134.4, 133.9, 133.5, 128.5, 128.2,
127.5, 126.7, 122.6, 118.6, 60.0, 39.6, 34.3, 28.8, 28.6, 27.5, 25.1, 23.9,
22.8, 14.8, 14.2
(2Z)-4-(2′-(3-Methyl)butyl-3′-cyclopropyl-2′-cyclohexen-1′-
ylidene)-3-methyl-2-butenal (26). MS m/z 273 (M + 1); 1H NMR
(300 MHz, CDCl3) δ 9.5 (d, 1H), 6.0 (s, 1H), 5.9 (d, 1H), 2.5−2.4 (m,
2H), 2.2−2.1 (m, 2H), 2.0 (s, 3H), 1.8−1.7 (m, 3H), 1.7−1.5 (m, 3H),
1.4−1.3 (m, 2H), 0.9 (d, 6H), 0.8−0.7 (m, 2H), 0.7−0.6 (m, 2H); 13C
NMR (CDCl3) δ 193.6, 161.1, 142.5, 140.0, 132.8, 128.5, 117.7, 38.3,
28.7, 28.2, 25.9, 25.7, 25.5, 22.9, 22.6, 14.3, 5.0.
General Procedure for Hydrolysis of the Esters. By use of this
general procedure, all other C20 acids were prepared.
(2Z)-4-(2′-(3-Methyl)butyl-3′-isopropyl-2′-cyclohexen-1′-yli-
(2E,4E,6Z)-8-(2′-(3-Methyl)butyl-3′-ethyl-2′-cyclohexen-1′-
ylidene)-3,7-dimethyl-2,4,6-octatrienoic Acid (9). The ester 29
(85:15 mixture of (9Z):(9Z,13Z)-29) (30.0 g, 81.0 mmol) was
suspended in methanol (1300 mL) and warmed to about 70 °C. An
aqueous solution of KOH (2.5 N, 325 mL) was added to the above
solution and stirred under reflux for 1 h. Then the reaction mixture was
cooled in an ice bath, diluted with ice cold water (500 mL), and acidified
slowly to pH 2−3 with ice cold 1 N HCl. The resulting yellow precipitate
was filtered and washed with ice-cold water. The wet precipitate was
dissolved in ether (1000 mL), washed with brine (2 × 200 mL), dried
(Na2SO4), and concentrated under vacuum to about 100 mL volume.
The mixture was diluted with hexanes (200 mL) and cooled in the
freezer for 18 h. The resulting yellow crystalline solid was filtered,
washed with ice-cold hexanes, and dried to give 16.5 g (59.5%) of pure 9
as single 9Z isomer. Mp 119−120 °C; MS m/z 342 (M + 1); UV λmax
318 nm (ϵ 25 550); 1H NMR (300 MHz, CDCl3) δ 6.6 (dd, 1H), 6.2 (d,
1H), 6.04 (d, 1H), 5.9 (s, 1H), 5.7 (s, 1H), 2.3−2.2 (m, 2H), 2.2 (s, 3H),
2.2−2.1 (m, 6H), 1.9 (s, 3H), 1.6−1.5 (m, 3H), 1.4−1.3 (m, 2H), 1.0 (t,
3H), 0.9 (d, 6H); 13C NMR (CDCl3) δ 172.7, 155.8, 142.9, 140.2, 139.1,
134.4, 132.9, 131.4, 126.7, 120.1, 117.1, 39.0, 30.3, 28.7, 28.4, 27.4, 25.6,
24.9, 23.4, 22.6, 13.9, 12.9.
(2E,4E,6Z)-8-(2′-(3-Methyl)butyl-3′-cyclopropyl-2′-cyclohex-
en-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic Acid (10). Mp
160−162 °C; MS m/z 355 (M + 1); UV λmax 326 nm (ϵ 22 300); 1H
NMR (300 MHz, CDCl3) δ 6.65 (dd, 1H), 6.2 (d, 1H), 6.04 (d, 1H), 5.9
(s, 1H), 5.7 (s, 1H), 2.5−2.4 (m, 2H), 2.2 (s, 3H), 2.15 (t, 2H), 1.9 (s,
3H), 1.8−1.7 (m, 3H), 1.7−1.5 (m, 3H), 1.4−1.3 (m, 2H), 0.9 (d, 6H),
0.7−0.6 (m, 2H), 0.6−0.5 (m2H); 13C NMR (CDCl3) δ 173.2, 156.2,
143.3, 139.5, 137.8, 134.8, 133.8, 133.3, 127.1, 120.0, 117.6, 38.9, 29.1,
28.7, 26.4, 26.1, 25.3, 23.4, 23.1, 14.6, 14.4, 5.2
1
dene)-3-methyl-2-butenal (27). MS m/z 275 (M + 1); H NMR
(300 MHz, CDCl3) δ 9.5 (d, 1H), 6.0 (s, 1H), 5.9 (d, 1H), 3.0−2.9 (m,
1H), 2.3−2.2 (m, 4H), 2.1 (t, 2H), 2.0 (s, 3H), 1.6−1.5 (m, 3H), 1.3−
1.2 (m, 2H), 1.00 (d, 6H), 0.9 (d, 6H); 13C NMR (CDCl3) δ 193.9,
161.5, 146.3, 142.9, 130.4, 128.9, 118.7, 39.4, 30.8, 29.1, 29.0, 25.7, 25.6,
24.8, 23.6, 22.9, 21.1
(2Z)-4-(2′-(3-Methyl)butyl-3′-phenyl-2′-cyclohexen-1′-yli-
1
dene)-3-methyl-2-butenal (28). MS m/z 309 (M + 1); H NMR
(300 MHz, CDCl3) δ 9.6 (d, 1H), 7.4−7.3 (m, 2H), 7.3−7.2 (m, 1H),
7.2−7.1 (m, 2H), 6.1 (s, 1H), 6.0 (d, 1H), 2.4−2.3 (m, 4H), 2.2−2.1 (m,
2H), 2.0 (s, 3H), 1.8−1.7 (m, 2H), 1.4−1.2 (m, 3H), 0.7 (d, 6H); 13C
NMR (CDCl3) δ 193.8, 160.9, 144.4, 142.2, 141.9, 133.8, 129.2, 128.6,
127.9, 127.0, 120.6, 39.4, 34.3, 28.8, 28.5, 27.4, 25.7, 23.7, 22.7.
Horner−Emmons Reaction. By use of this general procedure, all
the C20 esters were prepared.
(2E,4E,6Z)- and (2Z,4E,6Z)-Ethyl 8-(2′-(3-Methyl)butyl-3′-
ethyl-2′-cyclohexen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrie-
noate (29). To a flame-dried, 2 L, three-neck round-bottomed flask
fitted with a nitrogen inlet, addition funnel, and rubber septum was
added NaH (60% suspension in mineral oil, 4.96 g, 124.0 mmol). Dry
THF (600 mL, distilled over Na/benzophenone) was added to the flask
followed by addition of a solution of freshly distilled triethyl
phophonosenecioate (33 g, 124.0 mmol). The resulting solution was
stirred for 15 min, and then freshly distilled HMPA (87 mL) was added
under a nitrogen atmosphere. The flask was covered with aluminum foil
and stirred for 15 min. A solution of aldehyde 25 (21.5 g, 82.7 mmol) in
dry THF (250 mL) was added dropwise through the addition funnel,
and the mixture was then stirred for an additional 1.5 h. The reaction
mixture was quenched with water (200 mL) and extracted with ether (3
× 400 mL). The combined ether layers were washed with brine (2 × 250
mL), dried (Na2SO4), and concentrated under vacuum to provide the
crude product as an oil. The product was purified by chromatography
(silica gel; hexanes/ether 8:1) to give 30.0 g of 29 (98%) as an oil (85:15
(2E,4E,6Z)-8-(2′-(3-Methyl)butyl-3′-isopropyl-2′-cyclohexen-
1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic Acid (11). Mp 169−
170 °C; MS m/z 356 (M + 1); UV λmax 328 nm (ϵ 25 900); 1H NMR
(300 MHz, CDCl3) δ 6.6 (dd, 1H), 6.2 (d, 1H), 6.04 (d, 1H), 5.9 (s,
1H), 5.7 (s, 1H), 3.0−2.9 (m, 1H), 2.3−2.2 (m, 2H), 2.2 (s, 3H), 2.1 (t,
1
mixture of (9Z):(9Z,13Z)-29). MS m/z 371 (M + 1); H NMR (300
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2H), 2.0 (t, 2H), 1.9 (s, 3H), 1.6−1.5 (m, 3H), 1.3−1.2 (m, 2H), 1.0 (d,
6H), 0.9 (d, 6H); 13C NMR (CDCl3) δ 173.2, 156.2, 144.1, 143.4, 139.8,
134.7, 133.3, 130.9, 127.1, 120.6, 117.6, 39.6, 30.7, 29.1, 29.0, 25.8, 25.3,
24.8, 23.8, 23.0, 21.2, 14.4
coli and purified using AKTA purifier system.1 The hRXRα-LBD
homodimers were isolated from a gel filtration chromatography. The
protein was mixed with a 4-fold excess of UAB rexinoids and then 5-fold
excess of GRIP-1 coactivator peptide. The ternary complexes were
crystallized using vapor diffusion technique in hanging drops.1
Diffraction data of crystals were collected at synchrotron source of
Advanced Proton Source (APS) at Argonne National Laboratory. The
collected diffraction data were processed using the program HKL2000.
The structures were solved using the molecular replacement method
with a high-resolution hRXRα-LBD structure with its ligand deleted as a
search model (PDB code 3OAP). The structures were refined using
CNS software.27 A complete summary of data for these structures is
given in a Supporting Information table. Interactions between ligand/
peptide and hRXRα-LBD were analyzed using a program Ligand-
Protein Contacts (LPC)/Contacts of Structural Units (CSU).28 All
structures were prepared using PyMol (The PyMOL Molecular Graphics
(2E,4E,6Z)-8-(2′-(3-Methyl)butyl-3′-phenyl-2′-cyclohexen-1′-
ylidene)-3,7-dimethyl-2,4,6-octatrienoic Acid (12). Mp 168−169
°C; MS m/z 391 (M + 1); UV λmax 328 nm (ϵ 26 300); 1H NMR (300
MHz, CDCl3) δ 7.4−7.3 (m, 2H), 7.3−7.2 (m, 1H), 7.2−7.1 (m, 2H),
6.7 (dd, 1H), 6.24 (d, 1H), 6.1 (d, 1H), 6.0 (s, 1H), 5.8 (s, 1H), 2.4 (t,
2H), 2.3 (s, 3H), 2.3−2.2 (m, 2H), 2.2−2.1 (m, 2H), 1.97 (s, 3H), 1.8−
1.7 (m, 2H), 1.4−1.3 (m, 3H), 0.7 (d, 6H); 13C NMR (CDCl3) δ 173.1,
156.1, 144.9, 142.7, 140.1, 138.7, 134.5, 134.4, 133.7, 128.5, 128.2, 127.5,
126.8, 122.5, 117.8, 39.6, 34.3, 28.8, 28.6, 27.6, 25.2, 23.9, 22.8, 14.5.
Binding Affinity, Transient Transfection, and Luciferase
Reporter Assays. The binding affinity of 1−12 to hRXRα-LBD
homodimer was measured using a fluorescence quenching method.24
hRXRα-LBD homodimers (0.5 μM) were excited at 280 nm, and the
protein fluorescence was measured at 337 nm with a Cary Eclipse
fluorescence spectrophotometer (Varian, Palo Alto, CA). The binding
association constant Ka was calculated by using a nonlinear least-squares
regression to fit the raw data.24
Transient transfection and luciferase reporter assays were performed
using a previously reported protocol.25 At 24 h prior to transfection,
human embryonic kidney (HEK) 293 cells were plated at 2 × 105 cells
per well in six-well plates. Transfection mixtures included 0.2 μg of the
Gal4 reporter plasmid pGL4.31[luc2P/Gal4UAS/Hygro] (Promega),
0.5 μg of pCMXGal4-hRARα or pCMX-Gal4-hRXRα expression vector,
and 0.01 μg of Renilla luciferase reporter plasmid, pRL-TK. TransIT-
LT1 transfection reagent (Mirus) was used. At 24 h post-transfection,
rexinoid was added to the culture medium. At 48 h post-transfection,
reporter activity was determined using the dual luciferase reporter assay
(Promega). The EC50 of the hRXRα luciferase assay was determined
using a dose response model with triplicates at four different
concentrations (1, 10, 100, 1000 nM). A single 1000 nM dose of
rexinoid was used to evaluate the percentage of hRARα activation using
the same concentration of 9-cis-retinoic acid as the positive 100%
control.
Isothermal Titration Calorimetry of GRIP-1 Binding to holo-
hRXRα-LBD. A Microcal VP-Isothermal titration calorimeter (Micro-
cal, Piscataway, NJ) was used to measure binding of the GRIP-1
coactivator peptide to holo-hRXRα-LBD homodimers as described
previously.1 Each titration experiment consisted of 30 injections of 8 μL
of GRIP-1 peptide (0.04−0.12 mM) into the sample cell containing 1.34
mL of hRXRα-LBD homodimers (0.05 mM) in 10 mM Tris buffer (pH
8.0) containing 50 mM NaCl, 0.5 mM EDTA, and 2 mM DTT.
Rexinoids 11 or 12 were dissolved in DMSO and added at a ratio of 2:1
(retinoid:protein). Both retinoid solution and hRXRα-LBD solution
were degassed at least 15 min before the rexinoids were added to the
protein solution. The ITC data were processed using the ORIGIN 7
software. The titration curves were fit to a single site binding model by a
nonlinear least-squares method. The ITC experiments were performed
at 37 °C.
In Vivo Triglyceride Levels Assay. All animal studies were
performed in accordance with the University of Alabama at Birmingham
guidelines as defined by the Institutional Animal Care and Use
Committee (IACUC-121008309). The triglyceride assays were
conducted on female Sprague-Dawley rats bearing small mammary
cancers. Mammary cancers were induced in 50-day-old female Sprague-
Dawley rats by iv injection of the chemical carcinogen methylnitrosour-
ea (75 mg/kg BW). The animals were fed a Teklad diet according to
previous reports.3 The retinoids tested were mixed into the diet
according to the protocols reported previously and fed for 7 days.3 For
the evaluation of the compounds on serum triglycerides, blood was
collected from the inferior vena cava at the time of sacrifice of the
animals. The blood was kept at 5 °C during centrifugation (3800 rpm for
15 min). Serum was immediately collected and frozen at −85 °C until
analyzed for triglycerides.26 The infinity triglycerides assay kit was
purchased from Thermo DMA.
System, version 0.99; Schrodinger, LLC: New York.).
̈
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00829.
Synthesis of compounds 6−8 and intermediates 13−16,
tables containing the crystallographic data, refinement
statistics, and elemental analysis data (PDF)
Molecular formula strings (XLSX)
Accession Codes
PDB codes: compound 6, 4RFW; compound 9, 4RMC;
compound 10, 4RMD; compound 11, 4RME.
AUTHOR INFORMATION
Corresponding Authors
■
*W.J.B.: phone, 1-205-934-8288; fax, 1-205-934-2543; e-mail,
wbrou@uab.edu.
*D.D.M.: phone, 1-205-934-8285; fax, 1-205-934-2543; e-mail,
muccio@uab.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Ellen Li (Washington University in St. Louis, MO)
for providing the protein expression vector. We appreciate
critical reading and helpful comments provided by Dr. Debasish
Chattopadhyay. This work was supported by the National
Institutes of Health (NIH) Grant 2 P50 CA089019, the Komen
Foundation (Grant BCTR 20000690), and Grant RO1AA12153.
ABBREVIATIONS USED
■
RXR, retinoid X receptor; RAR, retinoic acid receptor; RA,
retinoic acid; LBD, ligand binding domain; IBX, o-iodoxybenzoic
acid; HEK293, human embryonic kidney cell; TG, triglycerides;
GRIP-1, glucocorticoid receptor interacting peptide 1; LBP,
ligand binding pocket; MNU-1, methyl-1-nitrosourea; ER,
estrogen receptor; SERM, selective estrogen receptor modu-
lator; THF, tetrahydrofuran; HMPA, hexamethylphosphorami-
date; TLC, thin layer chromatography
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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DOI: 10.1021/acs.jmedchem.5b00829
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