Methyl substitution of a rexinoid agonist improves potency and reveals site of lipid toxicity

Article abstract of DOI:10.1021/jm5004792

(2E,4E,6Z,8E)-8-(3′,4′-Dihydro-1′(2′H) -naphthalen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD.

Full text of DOI:10.1021/jm5004792

Article
pubs.acs.org/jmc
Methyl Substitution of a Rexinoid Agonist Improves Potency and
Reveals Site of Lipid Toxicity
Venkatram R. Atigadda,† Gang Xia,† Anil Desphande,† LeeAnn J. Boerma,‡ Susan Lobo-Ruppert,§
Clinton J. Grubbs,§ Craig D. Smith,∥ Wayne J. Brouillette,*,† and Donald D. Muccio*,†
†
‡
§
∥
Departments of Chemistry, Biochemistry and Molecular Genetics, Medicine, and Vision Sciences, University of Alabama at
Birmingham, Birmingham, Alabama 35294, United States
S
* Supporting Information
ABSTRACT: (2E,4E,6Z,8E)-8-(3′,4′-Dihydro-1′(2′H)-naphthalen-1′-ylidene)-
3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays
substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an
analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid
biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced
potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with
methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The
syntheses and biological evaluations of these new analogues are reported here
along with the X-ray crystal structures of each homologue bound to the ligand
binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is
a more potent agonist, but only the 7-methyl-9cUAB30 caused severe
hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new
rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had
methyl groups that interacted with helix 7 residues of the LBD.
9cUAB30. These conformational changes and interactions were
also present in the 9cUAB30 homologue with a methyl group
at the 4-position of the tetralone ring.9 This homologue of
9cUAB30, 4-methyl-9cUAB30, is more potent than 9cUAB30,
but it enhances lipid biosynthesis to an extent exhibited by
bexarotene rather than its parent compound. When the gene
arrays were analyzed from rat livers obtained from treatment of
bexarotene, 4-methyl-9cUAB30, and 9cUAB30, both bexar-
otene and 4-methyl-9cUAB30 induce signaling through the
RXR:LXR heterodimers and the levels of mRNA of key
enzymes involved in lipogenesis increase.10 In contrast, the
levels of these key lipogenic liver enzymes of 9cUAB30 treated
rats are nearly identical to those from untreated rats, suggesting
that this rexinoid is not an agonist in liver tissues (tissue
selective).
1. INTRODUCTION
Bexarotene (Targretin) is a rexinoid that selectively activates
signaling of retinoid X receptors (RXRs) over signaling through
retinoic acid receptors (RARs). Bexarotene is the first clinically
approved rexinoid, and it is used to treat refractory cutaneous
T-cell lymphoma (CTCL).1 Human phase 1 trials reveal that
bexarotene is better tolerated than the RAR agonist, all-trans-
retinoic acid (ATRA), and the RAR and RXR pan-agonist, 9-cis-
retinoic acid (9cRA).2,3 The dose-limiting toxicity of oral
bexarotene treatment is hyperlipidemia (both elevated serum
triglycerides (TGs) and serum total cholesterol). CTCL
patients treated with bexarotene often need to be administered
lipid-lowering drugs to control side effects, and if still
unmanaged, these patients are removed from bexarotene
treatment until lipid levels return to normal ranges.
As mentioned earlier, most of the potent rexinoids including
bexarotene and 9cRA are associated with severe hyperlipidemia,
which limits their clinical utility for chronic administration. So
far it is not clear what structural features of the rexinoids
contribute to the increase in lipids. Do the potent rexinoids
associated with hyperlipidemia interact differently with the
RXR protein than rexinoids which do not induce hyper-
lipidemia? Our laboratory is engaged in the rational design of
rexinoids with significant potency for preventing cancer in the
healthy population. Toward this end, we focus in this study on
understanding the structural features that contribute to and
9cUAB30 is a rexinoid containing a tetralone ring rather than
the tetramethyltetrahydronapthelene ring of bexarotene or the
trimethylcyclohexenyl ring of 9cRA (Figure 1).4,5 9cUAB30,
bexarotene, and 9cRA each reduce proliferation, enhance
apoptosis in mammary tumors, and efficiently prevent
mammary cancers in rodent models.6 We reported 9cRA,
9cUAB30, and bexarotene each bind to hRXRα-LBD in an L-
shaped geometry, and binding of these agonists causes a nearly
identical set of conformational changes on the ligand binding
domain of human RXRα (hRXRα-LBD) to recruit a coactivator
peptide to the surface of the domain.7,8 There were small
differences in these structures localized to the ligand binding
pocket. Both 9cRA and bexarotene occupy more volume in this
pocket, and each interacts with helices 3 and 7 more than
Received: March 26, 2014
Published: May 6, 2014
© 2014 American Chemical Society
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Figure 1. Structures of 9-cis-retinoic acid, bexarotene (Targretin), 9-cis-UAB30, and four methyl homologues of 9-cis-UAB30 (1−4). The position of
the methyl group substitution on 9-cis-UAB30 uses the tetralone ring numbering scheme.
Scheme 1. Synthesis of Four 9cUAB30 Homologs with Methyl Substitutions at the 5-, 6-, 7-, or 8-Position of the Tetralone Ring
enhance the potency of our parent rexinoid, 9cUAB30, from
those structural features that induce hyperlipidemia. Molecular
modeling of rexinoids bound to the ligand binding domain of
RXR is a common and useful approach in rexinoid design. But
we were surprised to find that our low-energy docked structure
of 4-methyl-9cUAB30 in the retinoid binding site was quite
different from what was found from the actual crystal structures
we reported recently.9 Thus, in this study we synthesized a
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Table 1. Summary of Biological Data for 9cRA, 9cUAB30, and Its Analogues 1−4
RXRα binding,
Kd (nM)
RXRα activation,
EC50 (nM)
KLF4 inhibition,
EC50 (nM)
serum triglyceride,
increase (%)
proliferation index,
decrease (%)
retinoid treatment
a
a
9cRA
14
33
18
15
8
3
5
3
2
120 30
820 70
720 20
100 30
160 10
110 13
400 90
440 150
110 50
>1000
326
56
a
a
9cUAB30
63
65
1
31
80
79
82
29
2
51
3
2
642
59
4
10
25
26
1
5
3
620 50
880
b
b
b
b
b
b
(R)-4-Me-9cUAB30
120
40
5
19
4
420
16
b
b
b
b
bexarotene
6
ND
456
56
a
b
Data reported in Grubbs et al.6 for 150 mg/kg diet for 9cRA and 200 mg/kg diet for 9cUAB30. Data reported in Deshpande et al.9 for 150 mg/kg
diet for bexarotene and 200 mg/kg diet for (R)-4-methyl-9cUAB30.
Table 2. Summary of ITC Measurements of GRIP-1 to hRXRα-LBD:UAB Rexinoid Complexes
rexinoid (37 °C)
Kd (μM)
ΔH (kcal/mol)
−TΔS (kcal/mol)
ΔG (kcal/mol)
n
9cRA
1.56 0.09
1.88 0.12
1.89 0.13
2.24 0.14
1.75 0.08
1.86 0.09
2.33 0.18
1.55 0.06
−12.3 0.12
−13.2 0.16
−13.0 0.18
−12.6 0.17
−13.4 0.11
−13.4 0.13
−13.0 0.22
−12.6 0.11
4.1
5.1
4.9
4.7
5.2
5.2
5.0
4.3
−8.2
−8.1
−8.1
−8.0
−8.2
−8.2
−8.0
−8.2
0.98
0.96
0.96
0.99
0.96
0.96
0.95
0.97
9cUAB30
bexarotene
1
2
3
4
4-Me-9cUAB30
series of methylated analogues of 9cUAB30 and obtained
crystal structures for each member of the series. These new
analogues (1−4) possess a single methyl group at the 5-, 6-, 7-,
or 8-position of the tetralone ring, which probes the space
surrounding the tetralone of 9cUAB30 in a systematic manner
(Figure 1). When we compare the structures of methylated
9cUAB30 homologues that induce hyperlipidemia to those we
published for bexarotene, we find that rexinoids that
significantly induce hyperlipidemia contain one or more methyl
groups that interact with the side chains of amino acid residues
(F346 and V349) on helix 7 of the LBD of RXR.
2.2. Binding and Transactivation of RXR. 9cRA is a
potent pan-agonist for both RARs and RXRs.14 By use of
fluorescence quenching, the 9cRA:hRXRα-LBD homodimer
complex had a dissociation constant (Kd) of 14 nM (Table 1).7
This was consistent with a previously reported result.15 By use
of this method, the Kd for the 9cUAB30:hRXRα-LBD
homodimer complex was determined to be 33
5 nM,
which was about 2-fold weaker than 9cRA binding. These
fluorescence titrations were also performed for the compounds
1−4. Each methyl analogue quenched more than 90% of the
fluorescence signal at 337 nm when the ratio of the protein to
the rexinoids reached 1:1. The fluorescence quenching data
were fit to a single-site binding model, and the Kd values (Table
1) indicated that each methyl analogue of 9cUAB30 bound to
the hRXRα-LBD with similar magnitude as 9cRA. Rexinoids
1−4 had a 2- to 4-fold stronger binding affinity to hRXRα-LBD
than 9cUAB30.
Previous studies have shown that 9cUAB30 is a RXR-
selective agonist in two cell lines while 9cRA is a potent pan-
agonist.5,16 In RK3E cells, the EC50 value for 9cUAB30
activating RXRα transcription was about 6-fold weaker than for
9cRA activation and it was a full agonist (Table 1). Using 9cRA
as a positive control, 9cUAB30 did not activate RARα-mediated
transcription at a high concentration (<1% at 10−6 M). Using
these receptor reporter assays in RK3E cells, we evaluated the
capability of methyl-substituted 9cUAB30 analogues (1−4) for
activating RXRα. The EC50 values for RXRα activation by 1
and 4 were similar to 9cUAB30; however, the EC50 values of 2
and 4 were much lower than that for 9cUAB30 and similar to
that found for 9cRA (Table 1). When RARα activation was
examined, rexinoids 1−3 did not activate transcription relative
to controls (less than 1%); however, rexinoid 4 induced 20%
activation of RAR-mediated transcription only at the highest
dose tested (10−6 M).
2. RESULTS AND DISCUSSION
2.1. Chemistry. Scheme 1 summarizes the methods
employed to prepare the new retinoids 1−4. The α-tetralones
with methyl substituents (5−8) were synthesized using
previously reported methods.11−13 The synthesis of retinoids
1−4 began with a Reformatsky reaction between methyl
substituted α-tetralones 5−8 and ethyl 4-bromo-3-methyl-2-
butenoate in 1,4-dioxane to provide the 9Z-acids 9−12 in 60−
86% yield. The carboxyl groups in compounds 9−12 were then
reduced in the presence of LiAlH4 to produce the desired 9Z-
alcohols 13−16 in quantitative yield. The alcohol functional
groups in compounds 13−16 were next oxidized using IBX to
provide the desired 9Z-aldehydes 17−20 containing minor
amounts of all E-aldehyde (5−7%). Pure 9Z-isomers (17−20)
were isolated in 68−80% yields following column chromatog-
raphy. Olefination of aldehydes 17−20 in the presence of
triethyl phosphonosenecioate under Horner−Emmons con-
ditions provided the esters 21−25 as an 85:15 mixture of the
9Z-isomer to the 9Z,13Z-isomer. The isomers were readily
separated by flash silica column chromatography, and the
desired 9Z-isomers were obtained in 70−85%. Subsequent
hydrolysis of the 9Z-esters 21−25 under basic conditions
provided the final compounds 1−4 in 70−85% yields after
recrystallization.
We used isothermal titration calorimetry (ITC) to measure
GRIP-1 binding to hRXRα-LBD homodimers containing
9cUAB30 or its methylated analogues. All measurements
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were performed at 37 °C. When 9cRA, 9cUAB30, or
bexarotene was bound to hRXRα-LBD homodimers, GRIP-1
occupied both coactivator binding sites on the monomers
(stoichiometry nearly 1:1 for GRIP-1/hRXRα-LBD monomer
subunit). For rexinoids 1−4, the stoichiometries for GRIP-1
binding were all near 1:1. This indicates that these homologues
of 9cUAB30 can recruit coactivators to both binding sites on
the surface of the LBD, and it is consistent with their agonist
properties. The free energy of GRIP-1 binding to the hRXRα-
LBD complexes containing rexinoids 1−4 was similar in
proliferation index correlates well with their capacity to prevent
estrogen receptor-positive mammary cancers.6 We evaluated
each of these analogues 1−4 in antiproliferative assays using
rats with established mammary cancers induced by the chemical
carcinogen N-methylnitrosourea. As displayed in Table 1, for
rexinoids 1−3 the proliferation index decreased by 79−82%
when rats containing existing mammary cancers were fed for 7
days a diet containing 200 mg/kg 1, 2, or 3. The decrease in
proliferation index was only 29% in rat tumors treated with oral
dosing of 4 (Table 1).
2.5. Crystal Structures of hRXRα-LBD Homodimers
Bound with Methyl Homologues. Previously we deter-
mined the X-ray crystal structures of hRXRα-LBD homodimers
bound to the pan-agonist 9cRA with the coactivator peptide
GRIP-1 (686KHKILHRLLQDSS698).7 We also used this
coactivator peptide when we compared the structures of
bexarotene to 9cUAB30 bound to hRXRα-LBD homodimer8 or
when we compared the structures of (R)-4-methyl-9cUAB30
and (S)-4-methyl-9cUAB30 to that of 9cUAB30.9 Here we
report four additional crystal structures of hRXRα-LBD
homodimers containing rexinoid 1 (PDB code 4PP5), 2
(PDB code 4PP3), 3 (PDB code 4PPJ), or 4 (PDB code
4POH) in the presence of coactivator peptide GRIP-1. Each
crystal structure belonged to the P4(3)2(1)2 space group, and
each unit cell contained two monomers with GRIP-1 bound to
each monomer. The summary of the X-ray crystallography and
refinement statistics for the structures are provided in the
Supporting Information.
magnitude (−8.1
0.1 kcal/mol). The negative free energy
change for GRIP-1 binding was driven strongly by a large
negative enthalpy change (−13.0 0.4 kcal/mol) regardless of
which homologue of 9cUAB30 was present in the LBP.
Likewise, binding was opposed by entropy even though the
coactivator peptide contains the ILxxLL hydrophobic motif
(Table 2). This thermodynamic signature for GRIP-1 binding
to homodimers was very similar for GRIP-1 binding to
homodimers containing two other rexinoids (9cUAB30 or
bexarotene) or the pan-agonist 9cRA in its LBP (Table 2).7
2.3. Inhibition of Oncogenic Transformation. Previ-
ously we showed that the pan-agonist 9cRA and two RXR-
selective agonists, 9cUAB30 and bexarotene, inhibited trans-
formation of rat kidney epithelial (RK3E) cells infected with
the oncogene KLF4.16 9cUAB30 was 3-fold less effective than
9cRA in this assay (Table 1). We demonstrated that 4-methyl-
9cUAB30 had equal potency to 9cRA in blocking oncogenesis.
Most of the potency of this racemic rexinoid was present in the
(S)-enantiomer.9 Here, the potencies of the 9cUAB30 methyl
analogues 1−4 in blocking transformation of oncogenes were
compared to the activity of 9cUAB30. Relative to DMSO, 2 was
the most potent methyl homologue for the inhibition of KLF4-
ER mediated transformation. Homologue 1 had potency similar
to 9cUAB30, while 4 was 2-fold less active than the parent
rexinoid (Table 1). The inhibition of transformation correlated
well with reduction of squamous cell carcinoma in the skin in
transgenic mice models.16 These studies indicate that 2 has the
most promising potential for preventing squamous cell
carcinoma of the skin.
The 3D fold of the hRXRα-LBD homodimer was nearly
identical regardless which 9cUAB30 homologue was bound.
The backbone atoms of each structure were overlaid with the
structure of hRXRα-LBD bound to 9cUAB30 and GRIP-1
complex (4K4J). The rmsd values for this overlay of 229
backbone residues were 0.130, 0.139, 0.105, and 0.128 for
homodimers bound to 1, 2, 3, and 4, respectively (Figure 2).
2.4. In Vivo Triglyceride Levels and Inhibition of
Proliferation of Rat Mammary Cancers. The elevation of
serum triglyceride (TG) is a dose limiting toxicity of many
rexinoids like bexarotene.3 In contrast the tissue-selective
9cUAB30 did not increase serum TG in chronic administration
in preclinical evaluation in rodents and dogs or as a single dose
in humans.17,18 In order to study if methyl homologues of
9cUAB30 had a similar effect, we measured serum TG in rats
(216 days of age) fed a diet of rexinoids 1−4 containing 200
mg/kg diet. As displayed in Table 1, rexinoids 1, 2, and 4
increased serum TG only slightly (30−60%), but rexinoid 3
raised TG levels by 642% over controls. There was a dose
response to the TG increase; rats fed a diet containing only 100
mg rexinoid/kg diet increased serum TG by 206%. No dose
response was observed for rexinoids 1, 2, 4 or the parent
rexinoid 9cUAB30. There was no change in the body weights of
the rats for all treated groups in comparison to the controls.
Liver weights normalized to body weight also showed no
variation from the control rats.
Figure 2. Overlay of X-ray crystal structures of hRXRα-LBD bound to
9cUAB30 (green), 1 (yellow), 2 (blue), 3 (magenta), and 4 (cyan).
The coactivator peptide GRIP-1 is displayed in red, and the ligand
binding pocket of hRXRα-LBD is highlighted in brown-gray mesh.
We established that four conformational changes occur in the
layer between the rexinoid binding site and the coactivator
binding site.7,8 Each of these conformational changes was
present in the structures containing the methyl homologues
studied here. These allow helix 12 of the hRXRα-LBD to form
the coactivator binding site. Together with helices 3 and 4
residues, the ILxxLL motif of GRIP-1 bound to the hydro-
phobic pocket on the surface of the receptor and GRIP-1 was
held by charge clamps. These interactions were identical for the
structures reported here and those reported previously.7 The
Proliferation of estrogen receptor-positive mammary cancers
is decreased using estrogen receptor antagonists or agents that
block estrogen biosynthesis. Potent rexinoid agonists also
decrease proliferation as we demonstrated for bexarotene,
9cUAB30, and 4-methyl-UAB30.7−9 The decrease in BRDU
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only exception occurred when rexinoid 2 was bound. In this
structure, the side chain of E456 (helix 12) extended
completely into the solvent and the charge clamp was not
formed with H687 on GRIP-1.
orientation of the tetralone ring in compounds 1−4 and
9cUAB30 were in distinct contrast to those of (S)-4-methyl-
9cUAB30 (C8−C9 = −56.6°) and (R)-4-methyl-9cUAB30
(C8−C9 = −64.4°), where the tetralone ring was flipped by
nearly 180° in the ligand binding pocket relative to 9cUAB30
and the other methyl homologues, 1−4.
In the bound structure of 9cUAB30, the cyclohexenyl ring
adopted a half-chair conformation with the C3 methylene of
the tetralone ring pointed to the same side as C19, and the C2
methylene was on the opposite side. The dihedral angle of C2−
C3 in 9cUAB30 was 51.9°. This orientation reduced the steric
effect between the C19 methyl group on C9 and the methylene
group on C2 on the tetralone ring. The conformations of the
tetralone ring for 1−4 were different from those of 9cUAB30.
Both the C2 and C3 atoms in rexinoids 1−4 were in a twisted
envelope conformation (Figure 3B). The C2−C3 dihedral
angles for rexinoids 1−4 were −43.0°, −41.8°, −44.3°, and
−9.8°, respectively. C2 and C3 were on the same side of the
tetralone ring plane and pointed in the opposite direction of
C19 in order to avoid any unfavorable steric interactions with
C19.
2.6. Ligand Binding Pocket of hRXRα-LBD Bound to
Methyl Homologues. The ligand binding pocket is defined
by residues from four helices (helices 3, 5, 7, and 11) with a few
residues from the β sheet. The ligand binding pocket is buried
near the center of the ligand binding domain and away from the
coactivator binding site. Rexinoids 1, 2, 3, and 4 adopted an L-
shaped conformation inside the ligand binding pocket (LBP) of
hRXRα-LBD, and these conformations were similar to the
twisted conformations of 9cRA, 9cUAB30, 4-methyl-UAB30
enantiomers or bexarotene in the LBP.7,8 Rexinoids 1−4
contain a twisted C8−C9 torsional angle, and these rexinoids
also adopt an L-shape in the LBP similar to 9cUAB30 (Figure
3A and Figure 3B). The C8−C9 dihedral angles of 1−3 were
2.7. Ligand Protein Contacts between Rexinoids and
hRXRα-LBD. In the crystal structures of hRXRα-LBD:UAB
rexinoid/GRIP-1 complexes, 9cUAB30 and its methyl ana-
logues were completely buried in the LBP of hRXRα-LBD.
Rexinoid 1 had a similar orientation in the LBP as 9cUAB30.
The methyl group at the 5-position in 1 resulted in 30% more
contact area with V342 and I345 on helix 7 than 9cUAB30
(Figure 3C). The carboxylate group in rexinoid 1 was also
closer to the β sheet than 9cUAB30. For 2, the methyl group at
the 6-position of the tetralone ring made more contacts with
V342 and F346 on helix 7 than observed for 9cUAB30. The
phenyl ring of F346 rotated by 37° (along the Cβ) to avoid
unfavorable steric interaction with the methyl group on C6
(Figure 3D). Rexinoid 3 contains a methyl group at the 7-
position on the tetralone ring. This methyl group interacted
with F346 and V349 (Figure 3E). The position of rexinoid 4 in
the LBP was much different from those of rexinoids 1−3 and
the parent compound 9cUAB30. Rexinoid 4 was shifted by 1.2
Å toward helices 3 and 5 (Figure 3F). This movement led to an
increased surface area contact with helix 3 (10%) and helix 5
(17%) and a decreased surface area contact with helix 7 (19%)
relative to 9cUAB30. The methyl group at the 8 position in 4
interacted strongly with I268 of helix 3, a residue with which
9cUAB30 had very little contact.
2.8. Comparison of Structural Results and Biological
Activities. In our previous publications, we examined the
structures of 9cUAB30, 4-methyl-9cUAB30 enantiomers,
bexarotene, and 9cRA bound to hRXRα-LBD homodimers
with GRIP-1.7−9 The crystal structure of bexarotene revealed
that the C23/C24 methyl groups pointed toward helix 7
(Figure 4A). These methyl groups were tightly positioned
between V349 and F346 (3.7−4.6 Å), and they made strong
van der Waals contacts to each of these residues. In the crystal
structure of 9cUAB30 bound to this protein domain (Figure
4B), these interactions were missing. Only minor van der Waals
interactions occurred between the methylene groups of the
tetralone and F346/V349. In the crystal structures of (R)- and
(S)-4-methyl-9cUAB30 bound to the RXR LBD, we found that
the methyl group at the 4-position on the tetralone of each
enantiomer occupied a similar space in the LBP as the C23/
C24 methyl groups of bexarotene (Figure 4C). Using the
bound structure of 9cUAB30 as a guide, we expected the 4-
Figure 3. (A) Overlay of UAB rexinoids in the ligand binding pocket
of hRXRα-LBD: 9cUAB30 (green), 1 (yellow), 2 (blue), 3 (magenta),
and 4 (cyan). (B) Side view of this overlay showing the common L-
shaped twist in the rexinoid structure and the tetralone ring pucker.
(C−F). Electron density maps (2Fo − Fc) for the ligand binding
pocket containing 1 (C), 2 (D), 3 (E), and 4 (F).
121.6°, 123.5°, 124.7°, respectively. These torsional angles were
close to the torsional angle found when 9cUAB30 occupied the
LBP (121.4°). The twisted conformation of the rexinoids
reduced steric effects between the C2 and C3 methylene groups
of the tetralone ring and the methyl group at C9 (C19). The
C8−C9 dihedral angle on rexinoid 4 (102°) was noticeably
smaller than the angle measured for 9cUAB30 and the other
methyl homologues. This decrease in dihedral angle was due to
interactions between the methyl group on the 8-position of the
tetralone ring and H8 of the tetraene chain. The observed
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Figure 4. Interactions between the rexinoid rings with helix 7 residues, F346 and V347, with van der Waals surfaces of rexinoid and protein residues
shown as dots: bexarotene (Targretin) (A), 9cUAB30 (B), 4-methyl-UAB30 (C), and 7-methyl-UAB30, 3 (D).
methyl group to be oriented toward helix 11 residues. However,
the conformation of the tetralone ring of 4-methyl-9cUAB30
enantiomers changed because of a nearly 180° rotation around
the C8−C9 bond. The conformational change in the backbone
of the polyene chain reoriented the tetralone to optimize van
der Waals interactions between the methyl group at the 4-
position of the tetralone and helix 7 residues (3.5−4.4 Å). It is
well-established that bexarotene and 4-methyl-9cUAB30
enantiomers raise serum triglycerides significantly when fed
to rodents, whereas 9cUAB30 displays minimal effect on
triglyceride levels.9 In a recent publication, genomic studies of
liver tissues from animals treated with bexarotene and racemic
4-methyl-UAB30 demonstrated that the signaling pathways for
lipogenesis were enhanced by rexinoid activation of RXR−LXR
heterodimers.10 In contrast to these rexinoids, liver tissues from
9cUAB30-treated rats did not affect these lipogenic pathways.10
Prior studies suggest that rexinoids with structural features
that interact with helix 7 residues may cause enhanced
lipogenesis. To test this hypothesis, the structures of several
9cUAB30 homologues were analyzed bound to RXR LBD
containing the GRIP-1 coactivator peptide. These homologues
position methyl groups systematically around the tetralone ring.
Homologue 3 is a potent agonist that contains a methyl group
at the 7-position of the tetralone ring (Table 1). The crystal
structure of 3 bound to this protein domain revealed an L-
shaped conformation that was similar to 9cUAB30 (Figure 3),
which allowed for the methyl group on position 7 to be
directed toward helix 7 residues F346/V349 (Figure 4D). In a
similar fashion to bexarotene and 4-methyl-UAB30, the 7-
methyl group of 3 made strong contact with V349 (3.88 Å) and
F346 (3.70 Å). The serum TG levels of rodents dosed with 3 at
200 mg/kg diet for 7 days were increased by over 600% in a
dose-dependent manner, further reinforcing the argument that
the strong lipophilic interactions between the rexinoid and
these helix 7 residues of RXR lead to signaling that enhances
lipogenesis. Methyl homologues 1, 2, and 4 did not significantly
induce lipogenesis, even though they were potent RXR agonists
(Table 1). The methyl group at the 6-position of the tetralone
ring of homologue 2 was pointed toward the N-terminal end of
helix 7, but it made no direct van der Waals contact with F346/
V349. A small conformational change occurred in this crystal
structure, and the phenyl ring of F346 rotated away from the
rexinoid by about 34° (data not shown). For methyl
homologues 1 and 4, which have methyl groups at the 5- and
8-positions of the tetralone ring, there were essentially no
contacts with helix 7 residues (data not shown). Both
homologues 1 and 4 bound strongly and activated RXR,
without significantly affecting serum triglyceride levels. It
should be noted that homologue 4 was shifted in the LBP
toward helices 5 and 11 to prevent the steric crowding between
the methyl group and I268 on helix 3.
3. CONCLUSIONS
In this study we demonstrate that methyl substitution of
9cUAB30 improved the potency relative to the parent RXRα
agonist, 9cUAB30, by nearly 10-fold. This is consistent with the
increased potency we observed for 4-methyl-9cUAB30
enantiomers and how methyl substitution often affects the
potency of other drugs.9,19 Rexinoid 2 displays efficacy in all
biological evaluations of cancer prevention and therapy while
not influencing signaling that elevates serum triglyceride levels.
These results warrant further evaluation of rexinoid 2 for
chemoprevention. This study further improved our under-
standing of the rexinoid−protein interactions and more
importantly shed light on the interactions between rexinoid
and helix 7 residues that may contribute to lipogenesis. Taken
together, subtle changes in lipophilic interactions in this region
of the rexinoid binding pocket may account for the undesired
lipogenesis by enhancing signaling of RXR−LXR heterodimers,
which control many lipogenic genes.
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Journal of Medicinal Chemistry
4. EXPERIMENTAL SECTION
Article
(2Z,4E)-4-[7′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenoic Acid (11). A Zn−Cu complex was
generated as described earlier, from Zn dust (12.59 g, 192.0 mmol)
and copper(II) acetate (1.30 g) in glacial acetic acid (40 mL). This
complex in dry dioxane (50 mL) was treated with a solution of 7-
methyltetralone 7 (8.80 g, 55.0 mmol) and ethyl 4-bromo-3-
methylbut-2-enoate (22.8 g, 110 mmol) in dry dioxane (50 mL).
Following the usual workup provided 11 (10.2 g, 77.0% yield): mp
127−129 °C [ether/hexanes (1:2)]; UV λmax 309 (ε = 11 637); IR
(KBr) 2944 (−OH), 1665 (CO), 1623 (CC) cm−1; MS m/z
1H and 13C NMR spectra were recorded on Bruker ARX 300 and DRX
400 spectrometers. IR spectra were recorded using a 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 and ionized by 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 was
prepared by the reaction of ethyl 3,3-dimethylacrylate with N-
bromosuccinimide.20 Triethyl phosphonosenecioate was prepared via
the Arbusov reaction.21 Purity of the all the compounds were
determined to be ≥95% which was determined by combustion analysis
performed by Atlantic Microlabs, Inc. (Norcross, GA).
243.42 (MH+); H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 7.08 (s,
1
1H), 6.98 (m, 2H), 5.78 (s, 1H), 2.75 (t, J = 6.2 Hz, 2H), 2.56−2.51
(m, 2H), 2.29 (s, 3H), 2.10 (s, 3H), 1.85−1.77 (m, 2H); 13C NMR
(75 MHz, CDCl3) δ 171.73, 156.24, 140.08, 135.64, 135.57, 135.37,
129.11, 128.91, 125.41, 121.95, 118.09, 29.93, 28.85, 25.93, 23.63,
21.39.
(2Z,4E)-4-[8′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenoic Acid (12). A Zn−Cu complex was
generated as described earlier, from Zn dust (9.15 g, 140.0 mmol) and
copper(II) acetate (1.0 g) in glacial acetic acid (30 mL). This complex
in dry dioxane (40 mL) was treated with a solution of 8-
methyltetralone 8 (6.40 g, 40.0 mmol) and ethyl 4-bromo-3-
methylbut-2-enoate (16.6 g, 80.0 mmol) in dry dioxane (40 mL).
Following the usual workup provided 12 (6.00 g, 62.0% yield): mp
158−160 °C [ether/hexanes (1:2)]; UV λmax 287 (ε = 16 226); IR
(KBr) 2944 (OH), 1676 (CO), 1654 (CC) cm−1; MS m/z
(2Z,4E)-4-[5′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenoic Acid (9). A suspension of Zn dust
(12.60 g, 192.0 mmol) and copper(II) acetate (1.30 g) in glacial acetic
acid (40 mL) was stirred under nitrogen for 1 h in a 250 mL round-
bottomed flask. The mixture was diluted with anhydrous ether (50
mL) and vacuum-filtered using a sintered glass funnel under air. The
Zn−Cu complex was then washed successively with anhydrous ether
(3 × 20 mL) and anhydrous benzene (3 × 20 mL). This complex was
dried under vacuum for 1 h at room temperature and transferred to a
250 mL, flame-dried, three-neck flask fitted with a condenser, addition
funnel, and nitrogen inlet. Anhydrous dioxane (50 mL) was transferred
to the flask, and this suspension was heated to 90 °C in an oil bath.
This reaction mixture was then treated dropwise with a solution of 5-
methyltetralone (5) (8.80 g, 55.0 mmol) and ethyl 4-bromo-3-
methylbut-2-enoate (22.8 g, 110 mmol) in dry dioxane (50 mL).
Vigorous bubbling was noticed during the addition process, and the
reaction mixture was stirred at reflux for 8 h and then cooled to room
temperature. Water (50 mL) and 2 N HCl (50 mL) were added. The
mixture was diluted with ether (100 mL) and allowed to stir for 10
min. The mixture was filtered, and the acidic layer was separated. The
organic layer was washed with water (2 × 50 mL) and then with 1 N
NaOH (3 × 40 mL). The combined basic layers were cooled in an ice
bath, and the pH of the solution was adjusted to ∼2 with HCl (2 N).
The product was extracted into ether (3 × 40 mL), and the combined
organic layers were washed with water (40 mL), brine (40 mL), dried
(Na2SO4), and evaporated under vacuum to give a semisolid that was
crystallized from ether/hexanes to obtain pure 9 (9.98 g, 75.0% yield):
mp 153−155 °C [ether/hexanes (1:2)]; UV λmax 301 nm (ε = 10
729); IR (KBr) 2938 (−OH), 1669 (CO), 1607 (CC) cm−1; MS
243.45 (MH+); H NMR (300 MHz, CDCl3) δ 7.07 (d, J = 4.8 Hz,
1
2H), 6.93 (t, J = 4.5 Hz, 1H), 6.52 (s, 1H), 5.78 (s, 1H), 2.59 (t, J =
6.2 Hz, 2H), 2.51−2.48 (m, 2H), 2.46 (s, 3H), 2.09 (s, 3H), 1.79−1.70
(m, 2H); 13C NMR (75 MHz, CDCl3) δ 171.62, 155.73, 140.69,
139.24, 138.28, 135.03, 129.47, 127.76, 126.81, 125.14, 118.01, 30.10,
27.96, 26.14, 22.63, 21.44.
(2Z,4E)-4-[5′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenol (13). To a flame-dried two-neck round-
bottomed flask fitted with a nitrogen inlet and addition funnel were
added acid 9 (7.26 g, 30.0 mmol) and anhydrous ether (100 mL). The
flask was cooled to 0 °C in an ice bath, and the reaction mixture was
treated with 1 M LiAlH4/ether (39 mL, 39.0 mmol) dropwise. The
mixture was stirred for 2 h at 0 °C, cooled to −80 °C in a dry ice/
acetone bath, and slowly quenched with methanol (5 mL) followed by
1 N HCl (40 mL). The reaction mixture was allowed to come to room
temperature and extracted with ether (3 × 75 mL). The combined
ether layers were washed with water (40 mL), brine (40 mL), dried
(Na2SO4), and concentrated under vacuum to give the alcohol 13: mp
79−81 °C [ether/hexanes (1:2)]; UV λmax 267.5 (ε = 15 390); IR
(KBr) 3307 (−OH), 1656 (CC), 1624 (CC) cm−1; MS m/z
211.51 (MH+ − H2O); 1H NMR (300 MHz, CDCl3) δ 7.50−7.40 (m,
1H), 7.15−7.03 (m, 2H), 6.32 (s, 1H), 5.55 (t, J = 6.6 Hz, 1H), 4.06
(d, J = 6.7 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 2.32 (m, 2H), 2.23 (s,
3H), 1.91−1.80 (m, 2H), 1.86 (s 3H), 1.40 (brs, 1H); 13C NMR (75
MHz, CDCl3) δ 138.64, 136.74, 136.45, 136.02, 135.95, 129.19,
126.36, 125.72, 122.51, 122.06, 61.08, 27.66, 27.30, 24.25, 23.86,
19.94.
(2Z,4E)-4-[6′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenol (14). This preparation employed the
acid 10 (6.05 g, 25.0 mmol) in anhydrous ether (75 mL) and 1 M
LiAlH4/ether (32.5 mL, 33.0 mmol). After the workup described
above was obtained the alcohol 14 (5.36 g, 94.0% yield) as an oil: UV
λmax 268 (ε = 10 465); IR (neat) 3340 (−OH), 1610 (CC),cm−1;
MS m/z 211.55 (MH+ − H2O); 1H NMR (400 MHz, CDCl3) δ 7.49
(d, J = 8.1 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.93 (s, 1H), 6.35 (s,
1H), 5.55 (t, J = 6.4 Hz, 1H), 4.06 (d, J = 6.7 Hz, 2H), 2.80 (t, J = 6.3
Hz, 2H), 2.35 (m, 2H), 2.31 (s, 3H), 1.87 (s, 3H), 1.85−1.78 (m, 2H),
1.55 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 139.52, 139.42,
139.34, 138.56, 134.86, 131.92, 129.11, 128.30, 126.26, 123.23, 63.15,
32.37, 30.27, 26.25, 25.89, 23.22.
1
m/z 243.45 (MH+); H NMR (300 MHz, CDCl3) δ 7.51−7.47 (m,
1H), 7.07 (d, J = 4.5 Hz, 2H), 7.02 (s, 1H), 5.79 (s, 1H), 2.69 (t, J =
6.6 Hz, 2H), 2.52−2.48 (m, 2H), 2.23 (s, 3H), 2.11 (s, 3H), 1.93−1.84
(m, 2H); 13C NMR (75 MHz, CDCl3) δ 171.73, 156.23, 141.04,
136.50, 136.25, 129.58, 125.86, 123.17, 122.12, 118.08, 28.33, 27.15,
25.99, 23.46, 19.91.
(2Z,4E)-4-[6′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenoic Acid (10). A Zn−Cu complex was
generated as described earlier, from Zn dust (11.4 g, 175.0 mmol) and
copper(II) acetate (1.15 g) in glacial acetic acid (40 mL). This
complex in dry dioxane (50 mL) was treated with a solution of 6-
methyltetralone 6 (8.00 g, 50.0 mmol) and ethyl 4-bromo-3-
methylbut-2-enoate (20.7 g, 100 mmol) in dry dioxane (50 mL).
Following the usual workup provided 10 (8.35 g, 69.0% yield): mp
149−151 °C [ether/hexanes (1:2)]; UV λmax 308 (ε = 13 491); IR
(KBr) 2941 (−OH), 1681 (CO), 1621 (CC) cm−1; MS m/z
1
243.60 (MH+); H NMR (300 MHz, CDCl3) δ 7.56 (d, J = 8.1 Hz,
1H), 7.16 (s, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.91 (s, 1H), 5.76 (s, 1H),
2.76 (t, J = 6.3 Hz, 2H), 2.60−2.55 (m, 2H), 2.30 (s, 3H), 2.13 (s,
3H), 1.86−1.78 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 171.48,
156.19, 140.32, 138.33, 137.85, 133.17, 129.75, 127.37, 125.05, 121.34,
117.93, 30.37, 29.00, 25.96, 23.54, 21.28.
(2Z,4E)-4-[7′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenol (15). This preparation employed the
acid 11 (8.00 g, 34.0 mmol) in anhydrous ether (100 mL) and 1 M
LiAlH4/ether (43 mL, 43.0 mmol). After workup as described earlier
5376
dx.doi.org/10.1021/jm5004792 | J. Med. Chem. 2014, 57, 5370−5380

Journal of Medicinal Chemistry
Article
was obtained the alcohol 15 (7.08 g, 94% yield) as an oil: UV λmax 264
(ε = 12 516); IR (neat) 3332 (−OH), 1607 (CC),cm−1; MS m/z
(2Z,4E)-4-[8′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenal (20). This preparation employed the
alcohol 16 (4.50 g, 20.0 mmol) and IBX (22.1 g, 79.0 mmol)
suspended in acetone (80 mL). After the workup described earlier and
chromatographic purification was obtained the pure 9(Z)-aldehyde 20
as an oil (3.57 g, 80% yield): UV λmax 296 (ε = 7665); IR (neat) 1674
(CO), 1609 (CC) cm−1; MS m/z 227.56 (MH+); 1H NMR (300
MHz, CDCl3) δ 9.82 (d, J = 8.2 Hz, 1H), 7.15−7.09 (m, 2H), 7.03−
6.95 (m, 1H), 6.15 (s, 1H), 6.03 (dt, J = 1.0 and 8.0 Hz, 1H), 2.65 (t, J
= 6.5 Hz, 2H), 2.48 (s, 3H), 2.51−2.42 (m, 2H), 2.06 (s 3H), 1.84−
1.75 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 193.14, 159.25, 141.58,
140.52, 137.43, 134.47, 129.53, 129.30, 127.35, 126.45, 125.54, 29.97,
28.00, 25.38, 22.93, 21.59.
1
211.32 (MH+ − H2O); H NMR (300 MHz, CDCl3) δ 7.41 (s, 1H),
7.00 (m, 2H), 6.36 (s, 1H), 5.55 (t, J = 6.6 Hz, 1H), 4.06 (d, J = 6.7
Hz, 2H), 2.79 (t, J = 6.3 Hz, 2H), 2.35 (m, 2H), 2.32 (s, 3H), 1.86 (s
3H), 1.86−1.76 (m, 2H), 1.57 (brs, 1H); 13C NMR (75 MHz, CDCl3)
δ 137.61, 136.41, 135.45, 135.37, 134.67, 129.28, 128.48, 126.38,
124.73, 121.93, 61.05, 29.95, 28.19, 24.17, 23.97, 21.37.
(2Z,4E)-4-[8′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenol (16). This preparation employed the
acid 12 (5.56 g, 23.0 mmol) in anhydrous ether (75 mL) and 1 M
LiAlH4/ether (30 mL, 30.0 mmol). After the workup described earlier
was obtained the alcohol 16 (5.03 g, 96.0% yield) as an oil: IR (neat)
3332 (−OH), 1607 (CC) cm−1; MS m/z 229.55 (MH+); 1H NMR
(300 MHz, CDCl3) δ 7.08 (m, 2H), 6.95 (m, 1H), 5.93 (s, 1H), 5.57
(t, J = 6.7 Hz, 1H), 4.15 (d, J = 6.6 Hz, 2H), 2.62 (t, J = 6.4 Hz, 2H),
2.45 (s, 3H), 2.33 (m, 2H), 1.85 (s 3H), 1.80−1.71 (m, 2H), 1.55 (brs,
1H); 13C NMR (75 MHz, CDCl3) δ 140.36, 138.26, 136.88, 136.30,
134.42, 129.32, 128.25, 126.53, 126.50, 125.28, 61.32, 30.18, 27.38,
24.21, 23.03, 21.73.
(2Z,4E)-4-[5′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenal (17). A single-neck round bottomed
flask fitted with a reflux condenser was charged with o-iodoxybenzoic
acid (IBX) (28.0 g, 100 mmol) and acetone (100 mL) and warmed to
50−55 °C in an oil bath. A solution of alcohol 13 (5.70 g, 25.0 mmol)
in acetone (25 mL) was added all at once to the reaction mixture. The
mixture was then allowed to stir at 50−55 °C for 1.5 h. It should be
noted that IBX at temperatures greater than 200 °C has been
demonstrated to be explosive. We have not experienced any
problematic incidents with IBX used in these reactions. The reaction
mixture was cooled to 0 °C in an ice bath, diluted with ether (50 mL),
and filtered through a sintered glass funnel. The filtrate (solids retained
on the funnel) was washed with ether (2 × 75 mL), and the combined
organic layers were concentrated under vacuum to furnish the crude
product 17 (5.5 g). This was purified by flash column chromatography
using 10% ether in hexane to give pure 9Z-aldehyde 17 as an oil (4.2 g,
75% yield): UV λmax 303 (ε = 9288); IR (neat) 1669 (CO), 1607
(CC) cm−1; MS m/z 227.48 (MH+); 1H NMR (300 MHz, CDCl3)
δ 9.63 (d, J = 8.2 Hz, 1H), 7.50−7.45 (m, 1H), 7.15−7.10 (m, 2H),
6.52 (s, 1H), 6.01 (dt, J = 1.1 and 8.2 Hz, 1H), 2.73 (t, J = 6.4 Hz,
2H), 2.46 (m, 2H), 2.43 (s, 3H), 2.09 (s 3H), 1.94−1.86 (m, 2H); 13C
NMR (75 MHz, CDCl3) δ 193.37, 159.54, 143.22, 137.01, 136.58,
135.15, 130.15, 129.13, 125.91, 122.73, 120.20, 28.06, 27.19, 25.47,
23.86, 19.88.
(2E,4E,6Z,8E)-Ethyl 8-[5′-Methyl-3′,4′-dihydro-1′(2′H)-naph-
thalen-1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoate (21). To a
flame-dried, three-neck 250 mL round-bottomed flask fitted with a
nitrogen inlet, addition funnel, and rubber septum was added NaH
(60% suspension in mineral oil, 0.850 g, 21.0 mmol). Dry THF (15
mL, distilled over Na/benzophenone) was added to the flask followed
by a solution of triethyl phosphonosenecioate (5.60 g, 21.0 mmol) in
dry THF (15 mL). The resulting solution was stirred for 15 min, and
then freshly distilled HMPA (5 mL) was added under a nitrogen
atmosphere. The flask was covered with aluminum foil and stirred for
15 min. A solution of aldehyde 17 (4.00 g, 18.0 mmol) in dry THF
(20 mL) was added dropwise through the addition funnel, and the
mixture was then stirred at room temperature for 2.5 h. The reaction
mixture was quenched with water (25 mL) and extracted with ether (2
× 75 mL). The combined ether layers were washed with brine (60
mL), dried over Na2SO4, and concentrated under vacuum to provide
the crude product as an oil. The product was purified by column
chromatography (n-hexane/ether, 9/1) to give 4.46 g of 21 as a 85:15
mixture of (9Z)/(9Z,13Z) (78% combined yield). Separation of these
isomers was achieved by column chromatography using n-hexane/
benzene (1:1) to obtain pure (9Z)-21 (3.57 g, 60.0% yield) as a yellow
oil: UV λmax 334.6 (ε = 28 387); IR (neat) 1706 (CO), 1610 (C
1
C) cm−1; MS m/z 337.71 (MH+); H NMR (300 MHz, CDCl3) δ
7.54−7.48 (m, 1H), 7.15−7.04 (m, 2H), 6.64 (dd, J = 4.3 Hz & 11.0
Hz, 1H), 6.42 (s, 1H), 6.22 (d, J = 15.5 Hz, 1H), 6.11 (d, J = 11.0 Hz,
1H), 5.74 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 2.72 (t, J = 6.4 Hz, 2H),
2.40−2.32 (m, 2H), 2.25 (s, 3H), 2.21 (s 3H), 1.97 (s, 3H), 1.87 (m,
2H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 167.39,
153.17, 140.79, 139.33, 136.85, 136.25, 134.12, 133.18, 129.40, 128.53,
127.48, 125.81, 122.78, 122.59, 118.52, 59.80, 28.25, 27.36, 24.97,
24.00, 19.97, 14.56, 14.07.
(2E,4E,6Z,8E)-Ethyl 8-[6′-Methyl-3′,4′-dihydro-1′(2′H)-naph-
thalen-1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoate (22). This
preparation employed a suspension of NaH (60% suspension in
mineral oil, 0.479 g, 12.0 mmol) in anhydrous THF (15 mL), a
solution of phosphonate ester (3.17 g, 12.0 mol) in anhydrous THF
(10 mL), HMPA (4 mL), and a solution of aldehyde 18 (2.26 g, 10.0
mmol) in anhydrous THF (15 mL). After the workup described earlier
and chromatographic purification was obtained the ester (2.42 g,
72.0% yield (9Z + 9Z,13Z)) as an oil. Separation of these isomers was
achieved by column chromatography using n-hexane/benzene (1:1) to
obtain pure (9Z)-22 (1.95 g, 58.0% yield): UV λmax 334 (ε = 21 112);
IR (neat) 1706 (CO), 1610 (CC) cm−1; MS m/z 337.81 (MH+);
1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.2 Hz, 1H), 7.01 (d, J =
(2Z,4E)-4-[6′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenal (18). This preparation employed the
alcohol 14 (4.50 g, 20.0 mmol) and IBX (22.1 g, 79.0 mmol)
suspended in acetone (80 mL). After the workup described earlier and
chromatographic purification was obtained the pure 9(Z)-aldehyde 18
as an oil (3.03 g, 68.0% yield): UV λmax 307 (ε = 8052); IR (neat)
1674 (CO), 1609 (CC) cm−1; MS m/z 227.59 (MH+); 1H NMR
(400 MHz, CDCl3) δ 9.63 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 8.1 Hz,
1H), 7.03 (d, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.53 (s, 1H), 6.00 (dt, J =
1.1 and 8.2 Hz, 1H), 2.82 (t, J = 6.3 Hz, 2H), 2.49 (m, 2H), 2.32 (s,
3H), 2.09 (s 3H), 1.87−1.81 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ 195.48, 161.71, 144.00, 140.56, 140.22, 134.00, 132.12, 131.07,
129.31, 126.52, 121.43, 32.19, 30.74, 27.49, 25.93, 23.28.
(2Z,4E)-4-[7′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-1′-yli-
dene]-3-methyl-2-butenal (19). This preparation employed the
alcohol 15 (6.16 g, 27.0 mmol) and IBX (30.8 g, 110 mol) suspended
in acetone (100 mL). After the workup described earlier and
chromatographic purification was obtained the pure 9(Z)-aldehyde
19 as an oil (4.4 g, 72% yield): UV λmax 304 (ε = 6830); IR (neat)
1672 (CO), 1610 (CC) cm−1; MS m/z 227.46 (MH+); 1H NMR
(300 MHz, CDCl3) δ 9.64 (d, J = 8.2 Hz, 1H), 7.45 (s, 1H), 7.10−
6.98 (m, 2H), 6.56 (s, 1H), 6.00 (dt, J = 1.1 Hz & 8.2 Hz, 1H), 2.82 (t,
J = 6.3 Hz, 2H), 2.48 (m, 2H), 2.35 (s, 3H), 2.09 (s 3H), 1.88−1.80
(m, 2H); 13C NMR (75 MHz, CDCl3) δ 193.11, 159.43, 141.90,
135.53, 135.18, 134.35, 129.37, 129.36, 128.97, 124.84, 119.96, 29.65,
28.51, 25.27, 23.89, 21.22.
8.1 Hz, 1H), 6.92 (s, 1H), 6.64 (dd, J = 4.2 and 11.1 Hz, 1H), 6.43 (s,
1H), 6.22 (d, J = 15.3 Hz, 1H), 6.10 (d, J = 11.0 Hz, 1H), 5.74 (s, 1H),
4.15 (q, J = 7.1 Hz, 2H), 2.81 (t, J = 6.3 Hz, 2H), 2.40−2.37 (m, 2H),
2.32 (s, 3H), 2.22 (s 3H), 1.97 (s, 3H), 1.84−1.78 (m, 2H), 1.28 (t, J
= 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 167.34, 153.16,
140.80, 138.05, 137.78, 137.57, 134.03, 133.18, 133.04, 129.95, 127.46,
127.16, 124.50, 121.69, 118.46, 59.76, 30.39, 28.85, 24.90, 23.99,
21.22, 14.52, 14.02.
(2E,4E,6Z,8E)-Ethyl 8-[7′-Methyl-3′,4′-dihydro-1′(2′H)-naph-
thalen-1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoate (23). This
preparation employed a suspension of NaH (60% suspension in
mineral oil, 0.878 mg, 22.0 mmol) in anhydrous THF (15 mL), a
solution of phosphonate ester (5.81 g, 22.0 mol) in anhydrous THF
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(15 mL), HMPA (5 mL), and a solution of aldehyde 19 (4.07 g, 18.0
mmol) in anhydrous THF (20 mL). After the workup described earlier
and chromatographic purification was obtained the ester (4.23 g,
70.0% yield (9Z + 9Z,13Z)) as an oil. Separation of these isomers was
achieved by column chromatography using n-hexane/benzene (1:1) to
obtain pure (9Z)-23 (3.69 g, 61.0% yield): UV λmax 333 (ε = 25 568);
IR (neat) 1705 (CO), 1601 (CC) cm−1; MS m/z 337.67
(d, J = 11.0 Hz, 1H), 5.76 (s, 1H), 2.81 (t, J = 6.1 Hz, 2H), 2.40−2.35
(m, 2H), 2.32 (s, 3H), 2.16 (s 3H), 1.98 (s, 3H), 1.84−1.78 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 172.93, 155.82, 141.69, 138.27,
137.87, 137.69, 134.17, 133.87, 133.04, 130.01, 127.43, 127.20, 124.56,
121.70, 117.73, 30.41, 28.89, 24.99, 24.03, 21.27, 14.25.
(2E,4E,6Z,8E)-8-[7′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-
1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoic Acid (3). This prep-
aration utilized 9Z-ester 23 (3.00 g, 9.00 mmol) suspended in
methanol (100 mL) and a solution of 2 N KOH (45 mL, 90.0 mmol)
in water (45 mL). After the workup described earlier was obtained the
acid 3 (2.63 g, 95.0% yield) as a yellow solid, which was crystallized
from ether/n-hexane to furnish pure (9Z)-3 (1.91 g, 69.0% yield): mp
179−181 °C [ether/hexanes (1:1)]; UV λmax 323 (ε = 31 955); IR
(KBr) 2930 (−OH), 1679 (CO), 1592 (CC) cm−1; MS m/z
1
(MH+); H NMR (300 MHz, CDCl3) δ 7.47 (s, 1H), 7.02 (s, 2H),
6.63 (dd, J = 4.3 Hz & 11.0 Hz, 1H), 6.45 (s, 1H), 6.23 (d, J = 15.3
Hz, 1H), 6.11 (d, J = 11.0 Hz, 1H), 5.75 (s, 1H), 4.15 (q, J = 7.1 Hz,
2H), 2.81 (t, J = 6.3 Hz, 2H), 2.40−2.32 (m, 2H), 2.35 (s, 3H), 2.22 (s
3H), 1.97 (s, 3H), 1.84−1.77 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 167.25, 153.05, 140.64, 138.24, 135.53,
135.48, 134.90, 134.11, 133.08, 129.29, 128.64, 127.48, 124.96, 122.39,
118.50, 59.70, 29.97, 28.76, 24.82, 24.08, 21.36, 14.49, 14.00.
1
309.71 (MH+); H NMR (300 MHz, CDCl3) δ 7.47 (s, 1H), 7.02 (s,
2H), 6.68 (dd, J = 4.3 and 11.0 Hz, 1H), 6.46 (s, 1H), 6.26 (d, J = 15.4
Hz, 1H), 6.12 (d, J = 11.0 Hz, 1H), 5.77 (s, 1H), 2.81 (t, J = 6.3 Hz,
2H), 2.40−2.35 (m, 2H), 2.35 (s, 3H), 2.23 (s 3H), 1.98 (s, 3H),
1.85−1.77 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 172.74, 155.78,
141.62, 138.49, 135.62, 135.59, 135.05, 134.13, 133.97, 129.39, 128.76,
127.46, 125.06, 122.45, 117.74, 30.03, 28.83, 24.96, 24.15, 21.44,
14.28.
(2E,4E,6Z,8E)-8-[8′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-
1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoic Acid (4). This prep-
aration utilized 9Z-ester 24 (2.02 g, 6.0 mmol) suspended in methanol
(75 mL) and a solution of 2 N KOH (30 mL, 60.0 mmol) in water (30
mL). After the workup described earlier was obtained the acid 4 (1.79
g, 97.0% yield) as a yellow solid, which was crystallized from ether/n-
hexane to furnish pure (9Z)-4 (1.40 g, 76.0% yield): mp 180−182 °C
[ether/hexanes (1:1)]; UV λmax 316.67 (ε = 26 077); IR (KBr) 2949
(−OH), 1675 (CO), 1593 (CC) cm−1; MS m/z 309.7 (MH+);
1H NMR (400 MHz, CDCl3) δ 7.20−7.05 (m, 2H), 6.98 (d, J = 6.5
Hz, 1H), 6.82 (dd, J = 4.3 and 11.0 Hz, 1H), 6.28 (d, J = 15.4 Hz, 1H),
6.13 (d, J = 10.8 Hz, 1H), 6.05 (s, 1H), 5.78 (s, 1H), 2.63 (t, J = 6.4
Hz, 2H), 2.50 (s, 3H), 2.40−2.35 (m, 2H), 2.24 (s 3H), 1.97 (s, 3H),
1.80−1.73 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 172.44, 155.64,
141.47, 140.66, 138.48, 137.76, 134.55, 134.11, 133.89, 129.44, 128.66,
127.67, 126.76, 125.35, 117.79, 30.21, 27.96, 24.79, 22.98, 21.75,
14.12.
4.2. Binding Affinity, Transient Transfection, and Luciferase
Reporter Assays. The binding affinity of 1−4 to hRXRα-LBD
homodimer was measured using a fluorescence quenching method.22
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.22
Transient transfection and luciferase reporter assays were performed
using a previously reported protocol.16 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 after
transfection, rexinoid was added to the culture medium. At 48 h after
transfection, reporter activity was determined using the dual-luciferase
reporter assay (Promega). The EC50 of the luciferase assay was
determined using a dose response model with two duplicates at four
different concentrations (1, 10, 100, 1000 nM).
4.3. Inhibition of Oncogenic Transformation Assay. BOSC23
ecotropic packaging cells at 80−90% confluence were transfected with
30 μg of KLF4-ER, ErB2 or pBpuro (control) plasmid DNA. Virus was
collected and filtered at 24 and 48 h after transfection and frozen in
liquid nitrogen. Following viral titer, RK3E cells with 30% confluence
were infected with virus in the presence of 10 μg/mL Polybrene. After
a 15 h infection, virus was removed and RK3E was supplemented with
DMEM. Cells infected with KLF4-ER were given DMEM
supplemented with DMSO (control), 9cRA, 9cUAB30, 1, 2, 3, and
4 every other day in duplicate at three different concentrations. Three
(2E,4E,6Z,8E)-Ethyl 8-[8′-Methyl-3′,4′-dihydro-1′(2′H)-naph-
thalen-1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoate (24). This
preparation employed a suspension of NaH (60% suspension in
mineral oil, 0.560 g, 14.0 mmol) in anhydrous THF (15 mL), a
solution of phosphonate ester (3.70 g, 14.0 mmol) in anhydrous THF
(10 mL), HMPA (4 mL), and a solution of aldehyde 20 (2.71 g, 12.0
mmol) in anhydrous THF (15 mL). After the workup described earlier
and chromatographic purification was obtained the ester (3.14 g,
78.0% yield (9Z + 9Z,13Z)) as an oil. Separation of these isomers was
achieved by column chromatography using n-hexane/benzene (1:1) to
obtain pure (9Z)-24 (2.58 g, 62.0% yield): UV λmax 328 (ε = 27 316);
IR (neat) 1708 (CO), 1603 (CC) cm−1; MS m/z 337.84 (MH+);
1H NMR (400 MHz, CDCl3) δ 7.20−7.06 (m, 2H), 6.92 (s, 1H), 6.78
(dd, J = 4.3 and 11.1 Hz, 1H), 6.22 (d, J = 15.4 Hz, 1H), 6.12 (d, J =
11.2 Hz, 1H), 6.05 (s, 1H), 5.76 (s,1H), 4.16 (q, J = 7.1 Hz, 2H), 2.63
(t, J = 6.5 Hz, 2H), 2.50 (s, 3H), 2.40−2.30 (m, 2H), 2.24 (s 3H), 1.96
(s, 3H), 1.80−1.73 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 167.36, 153.00, 140.63, 138.52, 137.55, 134.53,
134.08, 133.17, 129.42, 128.71, 127.10, 126.71, 125.32, 118.69, 59.83,
30.21, 27.92, 24.73, 22.96, 21.74, 14.56, 13.92.
(2E,4E,6Z,8E)-8-[5′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-
1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoic Acid (1). The 9Z-
ester 21 (3.02 g, 9.00 mmol) was suspended in methanol (100 mL)
and warmed to about 70 °C in an oil bath. An aqueous solution of 2.0
N KOH (45 mL, 90.0 mmol) (prepared with distilled and degassed
water) was added to the above suspension and stirred under reflux for
1.5 h. Then the reaction mixture was cooled in an ice bath, diluted
with ice-cold water (50 mL), and acidified to pH 1−2 with ice-cold 2
N HCl. The resulting precipitate was filtered, redissolved in ether (100
mL), washed with H2O (2 × 40 mL), brine (40 mL), dried over
Na2SO4, and concentrated under vacuum to furnish the final acid 1
(2.64 g, 96.0% yield) as a yellow solid, which was crystallized from
ether/n-hexanes (1:1) to furnish pure (9Z)-1 (2.05 g, 74.0% yield):
mp 189−191 °C [ether/hexanes (1:1)]; UV λmax 324.6 (ε = 28 747);
IR (KBr) 2936 (−OH), 1669 (CO), 1592 (CC) cm−1; MS m/z
309.57 (MH+); 1H NMR (300 MHz, CDCl3) δ 11.2 (brs, 1H), 7.55−
7.45 (m, 1H), 7.15−7.05 (m, 2H), 6.68 (dd, J = 4.3 and 11.1 Hz, 1H),
6.42 (s, 1H), 6.29 (d, J = 9.7 Hz, 1H), 6.12 (d, J = 11.1 Hz, 1H), 5.76
(s, 1H), 2.72 (t, J = 6.4 Hz, 2H), 2.40−2.32 (m, 2H), 2.25 (s, 3H),
2.21 (s 3H), 1.98 (s, 3H), 1.92−1.82 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ 172.73, 155.78, 141.64, 139.52, 136.88, 136.30, 136.22,
134.13, 133.93, 129.46, 127.44, 125.83, 122.80, 122.55, 117.71, 28.27,
27.36, 25.03, 24.00, 19.98, 14.26.
(2E,4E,6Z,8E)-8-[6′-Methyl-3′,4′-dihydro-1′(2′H)-naphthalen-
1′-ylidene]-3,7-dimethyl-2,4,6-octatrienoic Acid (2). This prep-
aration utilized 9Z-ester 22 (1.80 g, 5.40 mmol) suspended in
methanol (70 mL) and a solution of 2 N KOH (27 mL, 54.0 mmol) in
water (27 mL). After the workup described earlier was obtained the
acid 2 (1.58 g, 96.0% yield) as a yellow solid, which was crystallized
from ether/n-hexane to furnish pure (9Z)-2 (1.16 g, 70.0% yield): mp
173−174 °C [ether/hexanes (1:1)]; UV λmax 326 (ε = 30 749); IR
(KBr) 2935 (−OH), 1677 (CO), 1594 (CC) cm−1; MS m/z
1
309.70 (MH+); H NMR (400 MHz, CDCl3) δ 11.77 (brs, 1H), 7.56
(d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.00 Hz, 1H), 6.95 (s, 1H), 6.68 (dd, J
= 3.8 Hz & 11.2 Hz, 1H), 6.43 (s, 1H), 6.25 (d, J = 15.3 Hz, 1H), 6.11
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weeks after infection, transformed foci were fixed, stained, counted,
and analyzed against DMSO control.
CA089019 (D.D.M.) and the Komen Foundation (Grant
BCTR 20000690) (D.D.M.).
4.4. In Vivo Triglyceride Levels, Antiproliferative, and
Apoptosis Assays. 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 TG, antiproliferative, and apoptosis 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 N-
methylnitrosourea (75 mg/kg BW). The animals were fed a Teklad
diet according to previous reports.6 The retinoids tested were mixed
into the diet according to the protocols reported previously and fed for
7 days.6 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.23 The infinity triglycerides
assay kit was purchased from Thermo DMA. For evaluation of
antiproliferative index, animals were injected with bromodeoxyuridine
(BRDU) 2 h prior to killing using CO2. Cancers were removed and
fixed with 10% formalin. Cell proliferation and cell apoptosis were
performed as previously reported.6 The proliferation/apoptotic indices
were calculated as the ratio of proliferation/apoptosis of treated
animals to vehicle fed animals.
4.5. Protein Purification, Crystallization, and X-ray Crystal-
lography. The hRXRα-LBD (T223-T462) was overexpressed in
Escherichia coli and purified using AKTA purifier system.7 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.7 Diffraction data of crystals were collected
at synchrotron source of Advanced Proton Source (APS) at Argonne
National Laboratory or on a Rigaku IV+ diffractometer at UAB. The
collected diffraction data were processed using the program D*Trek.24
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.25 A complete summary of data for these structures is
given in the Supporting Information. Interactions between ligand/
peptide and hRXRα-LBD were analyzed using a program Ligand-
Protein Contacts (LPC)/Contacts of Structural Units (CSU).26 All
structures were prepared using PyMol (The PyMOL Molecular
ABBREVIATIONS USED
■
RXR, retinoid X receptor; RAR, retinoic acid receptor; RA,
retinoic acid; LBD, ligand binding domain; IBX, o-iodox-
ybenzoic acid; RKE, rat kidney epithelial; SCC, squamous cell
carcinoma; TG, triglyceride; GRIP, 1-glucocorticoid receptor
interacting peptidie 1; LBP, ligand binding pocket; THF,
tetrahydrofuran; HMPA, hexamethylphosphoramide; TLC,
thin layer chromatography; BRDU, bromodeoxyuridine
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̈
ASSOCIATED CONTENT
■
S
* Supporting Information
Tables containing the crystallographic data collection, refine-
ment statistics, and elemental analysis data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*W.J.B.: phone, 1-205-934-8288; 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.
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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. Matthew B. Renfrow. This work was supported by grants
from the National Institutes of Health (NIH) Grant 2 P50
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