Methyl-substituted conformationally constrained rexinoid agonists for the retinoid X receptors demonstrate improved efficacy for cancer therapy and prevention

Article abstract of DOI:10.1016/j.bmc.2013.11.039

(2E,4E,6Z,8Z)-8-(3′,4′-Dihydro-1′(2H)-naphthalen- 1′-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid, 9cUAB30, is a selective rexinoid for the retinoid X nuclear receptors (RXR). 9cUAB30 displays substantial chemopreventive capacity with little toxicity and is being translated to the clinic as a novel cancer prevention agent. To improve on the potency of 9cUAB30, we synthesized 4-methyl analogs of 9cUAB30, which introduced chirality at the 4-position of the tetralone ring. The syntheses and biological evaluations of the racemic homolog and enantiomers are reported. We demonstrate that the S-enantiomer is the most potent and least toxic even though these enantiomers bind in a similar conformation in the ligand binding domain of RXR.

Full text of DOI:10.1016/j.bmc.2013.11.039

Bioorganic & Medicinal Chemistry 22 (2014) 178–185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Methyl-substituted conformationally constrained rexinoid agonists
for the retinoid X receptors demonstrate improved efficacy for
cancer therapy and prevention
Anil Desphande a, Gang Xia a, LeeAnn J. Boerma b, Kimberly K. Vines a, Venkatram R. Atigadda a,
Susan Lobo-Ruppert c, Clinton J. Grubbs d, Fariba L. Moeinpour d, Craig D. Smith e, Konstantin Christov f,
a,
Wayne J. Brouillette a, , Donald D. Muccio
⇑
⇑
a Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA
b Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
c Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA
d Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA
e Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA
f Department of Surgical Oncology, University of Illinois, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
(2E,4E,6Z,8Z)-8-(30,40-Dihydro-10(2H)-naphthalen-10-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid, 9cUAB30,
is a selective rexinoid for the retinoid X nuclear receptors (RXR). 9cUAB30 displays substantial chemopreventive
capacity with little toxicity and is being translated to the clinic as a novel cancer prevention agent. To improve on
the potency of 9cUAB30, we synthesized 4-methyl analogs of 9cUAB30, which introduced chirality at the 4-posi-
tion of the tetralone ring. The syntheses and biological evaluations of the racemic homolog and enantiomers are
reported. We demonstrate that the S-enantiomer is the most potent and least toxic even though these enantio-
mers bind in a similar conformation in the ligand binding domain of RXR.
Article history:
Received 3 September 2013
Revised 11 November 2013
Accepted 20 November 2013
Available online 1 December 2013
Keywords:
Conformationally constrained retinoids
Rexinoids
Ó 2013 Elsevier Ltd. All rights reserved.
Cancer prevention
Ligand–protein crystal structures
1. Introduction
designed to selectively enhance signaling through RXRs indepen-
dent of RAR-signaling.2 Selective RXR agonists (rexinoids) are
Nuclear receptor (NR) proteins are ligand-inducible transcrip-
tion factors that up-regulate target genes. NR signaling is tightly
effective in cancer therapy and prevention, and they are less toxic
than RAR agonists since they do not hyperactivate RAR signaling.
Targretin (bexarotene) was the first FDA-approved rexinoid, and
it is clinically used to treat refractory cutaneous T-cell lymphoma.3
Phase I trials revealed that Targretin was better tolerated than
all-trans-RA or 9cRA in humans.4,5 Hyperlipidemia was the dose-
limiting toxicity of oral Targretin treatment. To reduce toxicity, we
pursued the design of potent rexinoids that were tissue-selective.
Tissue-selective rexinoids, which do not exhibit dose-limiting lipid
toxicities, could be administered chronically for cancer prevention.
9cUAB30 was designed by substituting a tetralone ring for the trim-
ethylcyclohexenyl ring of 9cRA (Fig. 1).6,7 9cUAB30 and Targretin
each reduced proliferation, enhanced apoptosis in mammary
tumors, and efficiently prevented mammary cancers in rodent mod-
els.8 However, unlike Targretin, 9cUAB30 did not induce hyperlipid-
emia, providing a clinical advantage.9 To improve upon the potency
of 9cUAB30, we have designed and synthesized a series of methyl
derivatives of 9cUAB30. In the X-ray crystal structure of
regulated in
a ligand and coactivator/corepressor-dependent
mechanism. Vitamin A acids (all-trans-RA; 9-cis-RA) bind to two
classes of NR proteins, retinoic acid receptors (RARs) and retinoid
X receptors (RXRs), and activate transcription by recruitment of
coactivator proteins.1 Many classes of RXR agonists have been
Abbreviations: RXR, retinoid X receptor; RAR, retinoic acid receptor; RA, retinoic
acid; LBD, ligand binding domain; IBX, 2-iodoxybenzoic acid; RKE, rat kidney
epithelial; SCC, squamous cell carcinoma; TG, triglycerides; LBP, ligand binding
pocket; THF, tetrahydrofuran; HMPA, hexamethylphosphoramide; TLC, thin layer
chromatography; DMEM, Dulbecco’s modified Eagle medium; ER, estrogen recep-
tor; GRIP-1, glucocorticoid receptor interacting protein-1; KLF4, Kruppel-like factor
4; LXRs, liver X receptors; NR, nuclear receptor; PDB, protein data bank; RMSD, root
mean square deviation.
⇑
Corresponding authors. Tel.: +1 205 9348288; fax: +1 205 9342543 (W.J.B.);
tel.: +1 205 9348285 (D.D.M.).
E-mail addresses: wbrou@uab.edu (W.J. Brouillette), muccio@uab.edu
(D.D. Muccio).
hRXRa-LBD homodimers bound to 9cUAB30 with the coactivator
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.039

A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
179
3
19
2
16
17
4
9
10
1
9
10
5
O
8
6
18
7
CO2H
Tetralone
20
CO2H
9-cis-RA
9-cis-UAB30 (9cUAB30)
Me
3
4
2
1
5
O
Me
CO2H
8
6
7
-1
Targretin
CO2H
Tetralone
(R)-1
(S)-1
Figure 1. The structures of 9cRA, 9cUAB30, Targretin and 4-methyl-9cUAB30, and numbering schemes for the tetralone ring of 9cUAB30 and 4-methyl-9cUAB30.
peptide GRIP-1, we revealed that the binding pocket contained en-
ough space to accommodate a small lipophilic group on the
tetralone ring.10 Methyl substitution has proven to be very effective
in improving activity through lipophilic interactions and/or from
energetic gains realized by burial of the methyl group in the hydro-
phobic pockets of the protein.11 Racemic 1 (( )-1) possesses a
methyl group at the 4-position of the tetralone ring, and it is also
known as 4-methyl-9c-UAB30 in the literature (Fig. 1). ( )-1 has
been evaluated in cancer prevention studies. It has been compared
directly to its parent rexinoid, 9cUAB30, and to other rexinoids
including Targretin.8 ( )-1 reduced cancer cell proliferation more
than 9cUAB30 and prevented formation of mammary cancers
in vivo better than 9cUAB30. However, this single methyl substitu-
tion substantially enhanced lipogenesis. The serum triglycerides
from dosing of ( )-1 were elevated to the levels displayed by
Targretin dosing.8 Recently, gene expression in liver tissues from
rats dosed with 9cUAB30, Targretin, and ( )-1 were compared.9 This
study demonstrated that Targretin and ( )-1 stimulated lipogenesis
using signaling pathways in the liver involving RXR heterodimers
with the liver X receptors (LXRs), but 9cUAB30 had little effect on
these lipogenic pathways.
4-bromo-3-methyl-2-butenoate in 1,4-dioxane under Reformatsky
reaction conditions to give lactones 3. The intermediate lactones
were treated with NaOMe/methanol to generate the 9Z-acids
( )-4, (R)-4, and (S)-4 in 60–86% yield. The carboxyl groups in com-
pounds( )-4, (R)-4, and (S)-4 were subjected to reductive conditions
in the presence of LiAlH4 to produce the desired 9Z-alcohols ( )-5,
(R)-5 and (S)-5 in quantitative yield. The alcohol functional groups
in compounds ( )-5, (R)-5 and (S)-5 were then oxidized in the pres-
ence of IBX to provide the desired 9Z-aldehydes ( )-6, (R)-6 and (S)-6
containing minor amounts of all E-aldehyde (5–7%). Pure 9Z isomers
( )-6, (R)-6 and (S)-6 were isolated in 68–80% yields following
column chromatography. Olefination of aldehydes ( )-6, (R)-6 and
(S)-6 in the presence of triethyl phosphonosenecioate under Horn-
er–Emmons conditions provided the esters ( )-7, (R)-7 and (S)-7 as
an 85:15 mixture of (9Z,13E) and (9Z,13Z)-isomers. The isomers
were separated by flash silica column chromatography to obtain
the desired (9Z,13E) isomers in 70–85% yield. Subsequent hydrolysis
of (9Z,13E)-esters ( )-7, (R)-7 and (S)-7 under basic conditions pro-
vided the final compounds ( )-1, (R)-1 and (S)-1 in 70–85% yields
after recrystallization.
In this manuscript, we prepare the R/S-enantiomers of 1. We
compare how each R/S-enantiomer activates the RXR receptor, pre-
vents oncogenesis, and reduces proliferation of established mam-
mary cancers relative to 9cUAB30. We also examine if lipid
biosynthesis is induced by each enantiomer. Further, we report
the X-ray crystal structures of each enantiomer bound to the li-
2.2. Binding and transactivation of the RXRs
9cRA is a potent pan-agonist for both RAR and RXR.16 Using
fluorescence quenching, the 9cRA:hRXRa-LBD homodimer com-
plex has a dissociation constant (Kd) of 14 nM (Table 1).10 This
was consistent with a previously reported result.17 Using this
gand-binding domain (LBD) of human RXR
a
in the presence of
method, the Kd for the 9cUAB30:hRXRa-LBD homodimer complex
the coactivator peptide, GRIP-1. We compare these crystal struc-
tures to those we obtained for 9cRA, 9cUAB30 and Targretin in
the hopes of revealing how this single methyl substitution im-
proves potency and controls lipid biosynthesis.10,12,13
was determined to be 33 5 nM, which was about 2-fold weaker
than 9cRA binding. Targretin exhibited a Kd in between that of
9cRA and 9cUAB30 (Table 1). These fluorescence titrations were
also performed for the compounds ( )-1, (R)-1 and (S)-1. Methyl
substitution of 9cUAB30 at the 4-position lowered Kd values to
13 4 nM [(S)-1)] and 25 5 1 nM [(R)-1)] (Table 1).
2. Results and discussion
2.1. Chemistry
Previous studies have shown that 9cUAB30 is an RXR-selective
agonist in two cell lines while 9cRA is a potent pan-agonist.7,18 In
RK3E cells, the EC50 value for 9cUAB30 activating RXRa transcrip-
tion is about 6-fold weaker than for 9cRA activation (Table 1).
Using 9cRA as a positive control, 9cUAB30 did not activate
Scheme 1 summarizes the methods employed to prepare the new
retinoids. Enantiomerically pure tetralones (R)-2 and (S)-2 were
synthesized utilizing a previously reported procedure.14,15 The syn-
theses of compounds ( )-1, (R)-1 and (S)-1 began with the conden-
sation between ( )-4-methyl-1-tetralone (2) or enantiomerically
RARa-mediated transcription at a high concentration (<1% at
10ꢀ6 M). Using these receptor reporter assays in RK3E cells, we
evaluated the methyl-substituted 9cUAB30 analogs. The EC50 val-
ues for RXRa activation by (R)-1 and (S)-1 were much lower than
pure
(R)-
or
(S)-4-methyl-1-tetralone
and
ethyl
the EC50 for 9cUAB30. They were similar to those found for 9cRA

180
A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
Me
CO2Et
Me
Me
Br
NaOMe
O
Zn/Cu(II)OAc
THF
O
CO2H
O
_
+
2
_
+
4
(R)-2
_
+
3
(R)-4
(
S
)-2
(R)-3
( )-4
S
(S)-3
Me
Me
O
CO2Et
LiAlH4
(EtO)2P
IBX
CH2OH
CHO
NaH/THF
_
+
6
_
+
5
(R)-6
(R)-5
( )-6
(S)-5
S
Me
Me
KOH
MeOH/H2O
_
+
_
1
+
7
CO2H
CO2Et
(R)-7
(R)-1
(S)-7
(S)-1
Scheme 1.
Table 1
Summary of biological data for ( )-1 and enantiomers
Retinoid
treatment
RXR
a
RXR
a
KLF4
Serum
Proliferation
Apoptotic
indexd
bindinga
activationb
inhibitionc
triglycerided
indexd
Kd
(nM)
EC50
(nM)
EC50
(nM)
Increase
(%)
Decrease
(%)
Increase
(%)
9cRA
14
26
33
23
25
13
3
3
5
5
5
4
120
40
820 70
110
120
120
3
6
110 13
NDe
326%d
456%d
65%d
370%
420%
175%
56%d
89%d
65%d
89%
16%
89%
67%d
483%d
33%d
300%
150%
300%
Targretin
9cUAB30
( )-1
(R)-1
(S)-1
400 90
5
5
6
NDe
19
27
4
1
a
b
c
The dissociation constant Kd for retinoids and RXR
The EC50 values for RXR
transcriptional activation in RK3E cells was measured using a Luciferase assay as reported by Jiang et al.18
The EC50 value for retinoid inhibition of KLF4 transformation in RK3E cells was described by Jiang et al.18 and Boerma et al.10
a
LBD was measured using fluorescence quenching method as reported by Xia et al.12
a
The serum triglyceride levels, proliferation index, and apoptotic index were measured in female rats with MNU-initiated mammary tumors as reported by Grubbs et al.8
d
Data for 9cRA, Targretin, and 9cUAB30 were taken from Grubbs et al.8
e
Not determined.
(Table 1). When RAR
a
activation was examined, (R)-1 and (S)-1
for (R)-1 and (S)-1, respectively (Table 1). These data indicate
(R)-1 and (S)-1 may be more effective in preventing SCC than
9cUAB30.
each activated transcription by only 17% at the high dose
(10ꢀ6 M). Enantiomers (R)-1 and (S)-1 exhibited increased potency
but reduced selectivity for the RXR receptors over the parent rexi-
noid 9cUAB30.
2.4. In vivo triglyceride levels and inhibition of proliferation in
mammary cancers
2.3. Inhibition of oncogenic transformation
The elevation of serum triglycerides (TG) is the dose-limiting
toxicity of rexinoids like Targretin.5 9cUAB30 exhibited no increase
in serum TG in its preclinical evaluation in rats and dogs or in hu-
man trials.19,20 We previously reported that serum TG increased by
566% over controls when rats were fed 200 mg/kg diet of ( )-1 for
7 days.8 Each enantiomer of 1 was evaluated in a similar manner
and compared to ( )-1. The study conducted here used older rats
(216 days of age versus 50 days). As displayed in Table 1, both
(R)-1 and (S)-1 increased serum TG; however TG levels due to
(R)-1 dosing were more than 2-fold higher than for (S)-1. The body
Previously we have shown the pan-agonist 9cRA and two
RXR-selective agonists, 9cUAB30 and Targretin, inhibited transfor-
mation of rat kidney epithelial (RK3E) cells infected with the onco-
gene KLF4.10,18 9cUAB30 was 3.6-fold less effective than 9cRA in
this assay (Table 1). The inhibition of transformation correlated
well with reduction of squamous cell carcinoma (SCC) in trans-
genic mice models.18 Here, the 4-methyl derivatives of 9cUAB30
were compared. Relative to DMSO controls, the EC50 values for
inhibition of KLF4-ER mediated transformation are 19 and 27 nM

A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
181
Table 2
weights of the rats increased by about 3% for all treated groups, but
Crystallographic data-collection and refinement statistics for hRXR
UAB30: GRIP-1 complexes
a-LBD:4Me
the increase was slightly larger for rats fed (R)-1. Liver weights
were normalized to body weight. The livers from rats fed (R)-1
were larger than those fed (S)-1 (4.2 0.1 g/100 g BW for (R)-1;
3.8 0.1 g/100 g BW for (S)-1; 3.3 g/100 g BW for controls). Each
enantiomer of 1 induced hyperlipidemia and hepatomegaly to a
different extent.
Data set
PDB ID
(R)-1
4M8H
(S)-1
4M8E
Unit cell parameters (Å, °)
a = 65.92, b = 65.92,
c = 111.65
a = 66.28, b = 66.28,
c = 111.01
a
= 90, b = 90,
P43212
29.30–2.20 (2.28–2.20) 26.15–2.40 (2.49–2.40)
c
= 90
a
= 90, b = 90,
c = 90
Previously we demonstrated that ( )-1 was very potent as an
antiproliferative agent, which correlated well with its capacity to
prevent estrogen receptor-positive mammary cancers.8 We evalu-
ated each enantiomer of 1 in the same antiproliferative assays. Using
mammary cancers of about 200 mm2 that have been chemically in-
duced with N-methyl-nitrosourea in rats, the animals were treated
orally for 7 days and cancers removed. As displayed in Table 1, the
proliferation index decreased by 90% relative to controls when rats
containing existing mammary cancers were fed a diet containing
200 mg/kg of either ( )-1 or (S)-1. The decrease in proliferation
index was only 16% in rat tumors treated with oral dosing of (R)-1
(Table 1). We reported that the apoptotic index increased for mam-
mary tumors treated with potent chemopreventive rexinoids.8 For
each treated group, the apoptotic index increased, but the increase
observed for ( )-1 or for (S)-1 was twice that of (R)-1 (Table 1).
Taken together these data clearly demonstrate that (S)-1 is the ma-
jor contributor toward the potency of ( )-1 and (S)-1 is also the
enantiomer that is least likely to induce lipid toxicity.
Space group
Resolution (last shell) (Å)
P43212
No. of total/unique reflections 34,390/12,013
103,513/10,066
0.076 (0.314)
10.28 (10.03)
98.7 (99.3)
Rmerge (last shell) (%)
Redundancy (last shell)
Completeness (last shell) (%)
I/sigma (last shell)
0.078 (0.218)
2.86 (2.66)
91.8 (94.5)
8.7 (4.3)
17.0 (6.5)
Refinement
No. of residues
No. of protein atoms
No. of water molecules
No. of ligand atoms
Rcryst (%)
224
1785
130
23
22.81
26.52
224
1785
138
23
20.85
24.68
Rfree (%)
R.M.S. deviation from ideality
Bond length (Å)
Bond angles (°)
0.0081
1.115
0.0074
1.091
Ramachandran plot
Most favored regions (%)
Additional allowed regions (%)
Generously allowed regions (%)
Average B factor (Å2)
Non-hydrogen protein atoms
(Å2)
94.06
3.65
2.28
29.22
28.74
93.61
4.57
1.83
40.06
39.75
2.5. Crystal structures of hRXRa-LBD homodimers bound with
(R)-1 or (S)-1
Non-hydrogen ligand atoms (Å2) 31.67
Water molecules (Å2)
36.00
44.75
37.92
We have determined the X-ray crystal structures of hRXRa-LBD
homodimers bound to 9cRA (3OAP), 9cUAB30 (pdb ID: 4K4J) and Tar-
gretin (pdb ID: 4K6I) with the coactivator peptide GRIP-1
(
686KHKILHRLLQDSS698).10,12 Here we report two crystal structures
of hRXRa-LBD containing either (R)-1 or (S)-1 in the presence of
GRIP-1 (4M8H and 4M8E). The summary of the X-ray crystallography
and refinement statistics for the two structures are listed in Table 2.
The structures were highly similar to previously reported struc-
ture of hRXR
ture of hRXR
a
-LBD.10,12 Each structure was overlaid with the struc-
a-LBD bound to 9cUAB30 and GRIP-1. RMSD values
were 0.137 and 0.126 for (R)-1 and (S)-1 (Fig. 2). The active confor-
mation was present in each structure, which had helix 12 folded
back to the core of the hRXRa-LBD to form the coactivator binding
site along with helix 3 and helix 4. In each structure, the ILxxLL
motif of GRIP-1 bound to the hydrophobic pocket and the charge
clamps identified previously were present.10
2.6. Ligand binding pocket of hRXRa-LBD
The ligand binding pocket is defined by helices 3, 5, 7, 11 and the
beta sheet. Interactions between coactivator binding site and ligand
binding pocket were present in each structure of hRXRa-LBD
homodimers containing (R)-1 or (S)-1. 9cRA, 9cUAB30 and Targre-
tin adopted an L-shaped conformation inside the ligand binding
pocket (LBP) of hRXRa
-LBD.10 For 9cUAB30, the C8-C9 torsional an-
gle was 121.4°. The twisted conformation reduced steric effect be-
tween C2 and C3 methylene groups of the tetralone ring and the
methyl group at C9 (C19). Both (R)-1 and (S)-1 also adopted an
Figure 2. Overlay of X-ray crystal structures of hRXRa-LBD bound to 9cUAB30
(green), (R)-1 (yellow) and (S)-1 (Blue). GRIP-1 is highlighted in red.
L-shaped conformation inside the LBP of hRXRa-LBD (Figure 3).
However, when the polyene chain of (R)-1 or (S)-1 were overlaid
with that of 9cUAB30, the tetralone ring was in a different orienta-
tion. The C8-C9 dihedral angle was ꢀ64.4° for (R)-1 and was ꢀ56.6°
for (S)-1. In the 9cUAB30 structure, the benzyl ring of the tetralone
was pointed to helix 7 (V342, I345, F346, V349) (Figure 3B). In the
structures containing (R)-1 and (S)-1, this part of the tetralone rings
were pointed toward helix 11 residues (H435, L436 and F439)
(Fig. 3C and D).
The tetralone ring of each rexinoid adopted an envelope confor-
mation for 9cUAB30, (R)-1 and (S)-1. For each rexinoid, C3 of the
tetralone was out of plane relative to the benzyl ring. C2 and C3
were very close to the phenyl ring of F313 on helix 5, but there
were little interactions between the tetralone ring and F313 in
the 9cUAB30 structure. The surface area contact between (R)-1

182
A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
Figure 3. Conformation of rexinoids in the LBP of holo-hRXR
a-LBD (A) Overlay of 9cUAB30 (green), (R)-1 (yellow) and (S)-1 (magenta) in the ligand binding pocket. Electron
density maps (2Fo ꢀ Fc) for the ligand binding pocket of hRXR
a
-LBD homodimers containing (B) 9cUAB30; (C) (R)-1 and (D) (S)-1. (E) Overlay of 9cUAB30 (green), (R)-1
(yellow) and (S)-1 (magenta) in the ligand binding pocket from the point of view of the tetralone ring with C2 and C3 in front. (F) Puckered conformations of the 4-methyl
tetralone ring in (R)-1 and (S)-1.
or (S)-1 and helix 5 residues increased relative to that of 9cUAB30.
This increase was more apparent for (R)-1 (20% increase) than (S)-1
(7% increase). The difference in surface area contact to helix 5 res-
idues for the enantiomers was due to the 0.8 Å translational shift of
(R)-1 relative to (S)-1 in the ligand binding pocket (Fig. 3A). The
surface area contact between (R)-1 and (S)-1 and helix 7 residues
also increased relative to those found for 9cUAB30. This increase
in helix 5 contacts occurred mostly for (S)-1 (26% increase) relative
to (R)-1 (14% increase). There were fewer interactions between he-
lix 11 residues and (R)-1 (18% decrease) and (S)-1 (9% decrease)
than observed for 9cUAB30.
In the structures of (R)-1 and (S)-1, the 4-methyl groups were
each in a pseudo-equatorial position of the puckered tetralone ring.
The 4-methyl group carbons were about 3.5 Å from the phenyl ring
of F346. The tetralone rings of (R)-1 and (S)-1 were essentially mir-
ror images of each other with the 4-methyl group the same side of
the plane as C3 (Fig. 3E and F). The puckered tetralone was also ob-
served in the structure of 9cUAB30 bound to this LBD and in crystal
structures without the protein (unpublished data).10
9cUAB30, by nearly 8-fold. This is consistent with observations of
how methyl substitution affects the potency for other drugs.11
Methyl substitution also improved inhibition of KLF4-ER mediated
transformation by more than 10-fold when compared with
9cUAB30. While potency was increased, ( )-1 and its enantiomers
were less selective than 9cUAB30. ( )-1 was more potent than
9cUAB30 in receptor activation and cancer chemoprevention.
However, ( )-1 also demonstrated a profound and undesired in-
crease in lipogenesis, which reduces its value as a chemopreven-
tive agent for cancer. But since ( )-1 and its S enantiomer are
more potent than 9cUAB30 and as potent as Targretin, these rexi-
noids may be candidates for cancer therapy. When each enantio-
mer was evaluated for potency and toxicity, we demonstrate
here that (S)-1 significantly reduced mammary cancer cell prolifer-
ation in vivo suggesting that this enantiomer contributes most to
the demonstrated chemopreventive capacity of ( )-1. Further,
(S)-1 displayed significantly lower TG biosynthesis and it should
be less toxic than (R)-1 or Targretin. The separation of potency
using chiral rexinoids was also observed by the Chandraratna
group for another class of RXR agonists.13 The X-ray crystal struc-
tures revealed that both enantiomers had similar, twisted struc-
3. Conclusion
tures in the LBP of hRXRa-LBD. However, the tetralone ring
regions of (S)-1 and (R)-1 were oriented differently from the tetra-
lone ring of 9cUAB30 in the chiral ligand binding pocket. The
In this study we demonstrate that methyl substitution of
9cUAB30 improved the potency of the selective RXR agonist,

A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
183
differences observed in the structures of (R)-1 and (S)-1 with the
surrounding residues on the LBD, as well as differences in the phar-
macokinetic parameters (transport, distribution, and metabolism),
may contribute to the way these enantiomers produce differential
potency and toxicity.
anhydrous methanol (15 mL). To this Na (0.506 g, 23.0 mmol)
pieces were added slowly at room temperature followed by the
addition of d-lactone (S)-3 (4.0 g, 16.5 mmol) in anhydrous metha-
nol (10 mL). The reaction mixture was stirred at reflux for 1.5 h,
cooled to room temperature, and the solvent was removed on a ro-
tary evaporator under vacuum. The resulting crude oil was diluted
with ice-cold water (100 mL), acidified with HCl (5 N) to pH 1–2
and washed with ether (2 ꢁ 50 mL). The combined organic layers
were washed with water (25 mL), brine (25 mL), dried (Na2SO4)
and evaporated under vacuum to give the acid (S)-4 (4.0 g) in
quantitative yield; mp 98–99 °C [ether/hexanes (1:2)]; UV kmax
4. Experimental section
Detailed experimental procedures for the synthesis of (S)-1 are
described here and these same procedures were followed to syn-
thesize (R)-1 and ( )-1. 1H and 13C NMR spectra were recorded
on Bruker ARX 300 or DRX 400 spectrometers. IR spectra were re-
corded using a Bomem FTIR spectrometer. UV–vis spectra were re-
corded on a Varian (Cary 100) spectrophotometer in methanol
(Aldrich, spectrograde). Mass spectra were measured on a Hew-
lett-Packard 1100 LC–MS instrument using electrospray ionization
(ESI). All optical rotations were recorded at room temperature
(25 °C) using Jasco P-1030 polarimeter. Melting points were re-
corded on an Electrothermal melting point apparatus and were
uncorrected. Combustion analyses were performed by Atlantic
Microlabs, Inc. (Norcross, GA). All reactions, unless otherwise men-
tioned, 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
309 nm (
e = 12242); IR (KBr): 2937 (OH), 1677 (C@O), 1606
(C@C) cmꢀ1; [
a]
ꢀ44.5° (c 0.42, MeOH); MS m/z 243 (MH+); 1H
D
NMR (300 MHz, CDCl3) d 7.63 (d, J = 7.5 Hz, 1H), 7.26–7.14 (m,
4H), 5.79 (t, J = 1.3 Hz, 1H), 2.97–2.89 (m, 1H), 2.71–2.64 (m, 1H),
2.57–2.49 (m, 1H), 2.15 (s, 3H), 1.99–1.91 (m, 1H), 1.65–1.53 (m,
1H), 1.29 (d, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 171.14,
156.14, 143.24, 140.53, 135.63, 128.17, 127.84, 126.38, 125.24,
122.37, 118.08, 33.22, 30.97, 25,92, 25.91, 21.82; Anal. Calcd for
C16H18O2: C, 79.31; H, 7.49. Found: C, 79.22; H, 7.47.
4.1.3. (2Z,4E)-4-[(4R)-Methyl-3,4-dihydro-1(2H)-naphthalen-1-
ylidene]-3-methyl-2-butenoic acid [(R)-4]
A solution of (R)-4-methyl tetralone [(R)-2] (3.50 g, 22.0 mmol)
and bromoester (9.06 g, 44.0 mmol) was employed as described for
(S)-4. [a]D +44.7° (c 0.64, MeOH). All other spectral data were iden-
tical to its enantiomer.
(40–63 lm). 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. Triethyl
phosphonosenecioate was prepared via the Arbusov reaction.
4.1.4. (2Z,4E)-4-[(4S)-Methyl-3,4-dihydro-1(2H)-naphthalen-1-
ylidene]-3-methyl-2-butenol [(S)-5]
To a flame-dried two-neck round-bottomed flask fitted with a
nitrogen inlet and addition funnel was added acid (S)-4 (4.00 g,
16.5 mmol) and anhydrous ether (50 mL). The flask was cooled to
0 °C in an ice bath and the reaction mixture was treated with
1 M LiAlH4/ether (22 mL, 22.0 mmol) dropwise. The reaction mix-
ture was stirred for 2 h at 0 °C, cooled to ꢀ80 °C in a dry ice/ace-
tone bath, and slowly quenched with methanol (2 mL) followed
by 1 N HCl (40 mL). The reaction mixture was allowed to come to
room temperature and extracted with ether (2 ꢁ 75 mL). The com-
bined ether layers were washed with water (40 mL), brine (40 mL),
dried (Na2SO4) and concentrated under vacuum to give the alcohol
(S)-5 (3.6 g, 96%); mp 42–48 °C [ether/hexanes (1:2)]; UV kmax
4.1. General procedure for the preparation of compounds
4.1.1. (9S)-7,8-Benzo-4,9-dimethyl-1-oxaspiro[5,5]undec-3-ene-
2-one [(S)-3]
A suspension of Zn dust (5.76 g, 88.0 mmol) and copper(II) ace-
tate (0.580 g) in glacial acetic acid (25 mL) was stirred under nitro-
gen for 1 h in a 250 mL, round-bottomed flask. The mixture was
diluted with anhydrous ether (50 mL), vacuum-filtered, and the
Zn–Cu complex was washed successively with anhydrous ether
(3 ꢁ 20 mL) and anhydrous benzene (3 ꢁ 20 mL). This complex
was dried under vacuum for 1 h and transferred to a 250 mL,
flame-dried, three-neck flask fitted with a condenser, addition fun-
nel and nitrogen inlet. Anhydrous THF (40 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 (S)-4-methyl tetralone [(S)-2] (4.00 g, 25.0 mmol) and ethyl
4-bromo-3-methylbut-2-enoate (10.4 g, 50.0 mmol) in dry THF
(40 mL). Vigorous bubbling was noticed during the addition pro-
cess and the reaction mixture was stirred at reflux for 4 h and then
cooled to room temperature. Water (40 mL) and 2 N HCl (40 mL)
were added. The mixture was diluted with ether (100 mL) and al-
lowed to stir for 10 min. The mixture was filtered and the acidic
layer was separated. The organic layer was washed successively
with water (50 mL), 1 N NaOH (2 ꢁ 40 mL), water (40 mL) and
brine (40 mL). The organic layer was dried (Na2SO4) and concen-
trated under vacuum to result in thick oil. The unreacted tetralone
and the other volatile materials were removed under high vacuum
distillation to give d-lactone (S)-3 (4.1 g, 68% yield, mixture of iso-
mers) as a cream colored solid, which was used in the next reaction
without further purification.
265 nm (e a]
= 19165); IR (KBr): 3347 (OH), 1605 (C@C) cmꢀ1; [
D
ꢀ31.6° (c 1.55, MeOH); MS m/z 229 (MH+); 1H NMR (300 MHz,
CDCl3) d 7.57 (d, J = 7.3 Hz, 1H), 7.23–7.04 (m, 3H), 6.34 (s, 1H),
5.56 (t, J = 6.7 Hz, 1H), 4.07 (d, J = 6.7 Hz, 2H), 2.96 (sex,
J = 6.7 Hz, 1H), 2.43–2.37 (m, 1H), 2.33–2.28 (m, 1H),1.95–1.82
(m, 1H), 1.87 (s, 3H), 1.80 (brs, 1H), 1.62–1.56 (m, 1H), 1.30 (d,
J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 142.57, 138.00,
136.50, 135.40, 128.23, 127.76, 126.44, 126.17, 124.50, 122.31,
61.13, 33.23, 31.50, 25.20, 24.20, 22.31; Anal. Calcd for C16H20O:
C, 84.16; H, 8.83. Found: C, 84.06; H, 9.17.
4.1.5. (2Z,4E)-4-[(4R)-Methyl-3,4-dihydro-1(2H)-naphthalen-1-
ylidene]-3-methyl-2-butenol [(R)-5]
A solution of acid (R)-4 (4.3 g, 7.7 mmol) was employed as de-
scribed for the preparation of (S)-5. [a] +32.7° (c 0.81, MeOH).
D
All other spectral data were identical to its enantiomer.
4.1.6. (2Z,4E)-4-[(40S)-Methyl-30,40-dihydro-10(20H)-naphthalen-
10-ylidene]-3-methyl-2-butenal [(S)-6]
A single-neck, round-bottomed flask fitted with a reflux con-
denser was charged with o-iodoxybenzoic acid (IBX) (21.0 g,
75.0 mmol) and acetone (75 mL) and warmed to 50-55 °C in an
oil bath. A solution of alcohol (S)-5 (3.53 g, 15.0 mmol) in acetone
(25 mL) was added all at once to the reaction mixture. The reaction
4.1.2. (2Z,4E)-4-[(4S)-Methyl-3,4-dihydro-1(2H)-naphthalen-1-
ylidene]-3-methyl-2-butenoic acid [(S)-4]
A dry, 100 mL, two-neck round-bottomed flask fitted with a re-
flux condenser under nitrogen atmosphere was charged with

184
A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
was then allowed to stir at 50–55 °C for 1.5 h. 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 was washed
with ether (2 ꢁ 50 mL) and the combined organic layers were con-
centrated under vacuum to furnish the crude product (S)-6 (2.5 g,
74% yield). This was purified by flash column chromatography
using 15% ether in hexane to give pure 9Z-aldehyde (S)-6 (2.1 g,
4.1.10. (2E,4E,6Z,8E)-8-[(40S)Methyl-30,40-dihydro-10(20H)-
naphthalen-10-ylidene]-3,7-dimethyl-2,4,6-octatrienoic Acid
[(S)-1]
The (9Z)-ester (S)-7 (1.68 g, 5.00 mmol) was suspended in
methanol (75 mL) and warmed to about 70 °C in an oil bath. An
aqueous solution of 1.25 N KOH (40 mL, 50.0 mmol) (prepared
with distilled and degassed water) was added to the above suspen-
sion and stirred under reflux for 1 h. Then the reaction mixture was
cooled in an ice bath, diluted with ice-cold water (50 mL) and acid-
ified to pH 1–2 with ice-cold 2 N HCl. The resulting precipitate was
extracted with ether (2 ꢁ 75 mL), washed with brine (40 mL), dried
over Na2SO4 and concentrated under vacuum to furnish the final
acid (S)-1 (1.45 g, 95.0% yield) as a yellow solid, which was crystal-
lized from ether/n-hexanes (1:1) to furnish pure (9Z)-isomer of
(S)-1 (0.955, 62.0% yield); mp 166–167 °C [ether/hexanes (1:1)];
62% yield); UV kmax 306 nm (e = 8937); IR (neat): 1673 (C@O),
1611 (C@C) cmꢀ1; [
a
]
D
ꢀ51.7° (c 0.86, MeOH); MS z/e 227 (MH+);
1H NMR (300 MHz, CDCl3) d 9.66 (d, J = 8.3 Hz, 1H), 7.61 (d,
J = 7.8 Hz, 1H), 7.35–7.15 (m, 3H), 6.54 (s, 1H), 6.01 (dt, J = 1.1 Hz
& 8.2 Hz, 1H), 3.03–2.93 (m,1H), 2.62–2.53 (m, 1H), 2.48–2.44
(m, 1H), 2.09 (s, 3H), 2.06–1.92 (m, 1H), 1.64–1.59 (m, 1H), 1.31
(d, J = 7.0, 3H); 13C NMR (75 MHz, CDCl3) d 193.40, 159.58,
143.17, 142.46, 134.47, 129.14, 128.78, 128.46, 126.33, 124.71,
120.43, 33.09, 31.48, 25.71, 25.43, 22.22. Anal. Calcd for C16H18O:
C, 84.92; H, 8.02. Found: C, 84.71; H, 8.27.
UV kmax 331 nm (
e
= 28616); IR (KBr) 2937 (–OH), 1667 (C@O),
1590 (C@C) cmꢀ1; [
a]
ꢀ70.5° (c 0.72, MeOH); MS m/z 309.41
D
(MH+); 1H NMR (300 MHz, CDCl3) d 10.9 (br s, 1H), 7.63 (d,
J = 7.2 Hz, 1H), 7.26–7.17 (m, 3H), 6.70 (dd, J = 4.3 & 11.1 Hz, 1H),
6.46 (s, 1H), 6.26 (d, J = 15.4 Hz, 1H), 6.13 (d, J = 11.1 Hz, 1H),
5.77 (s, 1H), 2.97 (m, 1H), 2.54–2.43 (m, 1H), 2.40–2.31 (m, 1H),
2.23 (s, 3H), 1.99 (s, 3H), 2.00–1.90 (m, 1H), 1.64–1.54 (m, 1H),
1.30 (d, J = 7.00 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 172.50,
155.74, 142.90, 141.54, 38.71, 135.54, 134.01, 128.28, 127.99,
127.61, 126.26, 124.75, 122.75, 117.74, 33.27, 31.53, 25.78, 24.93,
22.23, 14.22; Anal. Calcd for C21H24O2: C, 81.78; H, 7.84. Found:
C, 81.92; H, 7.74.
4.1.7. (2Z,4E)-4-[(40R)-Methyl-30,40-dihydro-10(20H)-naphthalen-
10-ylidene]-3-methyl-2-butenal [(R)-6]
A solution of alcohol [(R)-5] (4.0 g, 17.5 mmol) was employed.
[a] +52.1° (c 0.84, MeOH). All other spectral data were identical
D
to its enantiomer.
4.1.8. (2E,4E,6Z,8E)-Ethyl-8-[(40S)methyl-30,40-dihydro-10(20H)-
naphthalen-10-ylidene]-3,7-dimethyl-2,4,6-octatrienoate [(S)-7]
To a flame-dried, 250 mL, three-neck round-bottomed flask fit-
ted with a nitrogen inlet, addition funnel and rubber septum was
added NaH (60% suspension in mineral oil, 0.440 g, 11.0 mmol).
Dry THF (25 mL, distilled over Na/benzophenone) was added to
the flask followed by a solution of triethyl phosphonosenecioate
(2.90 g, 11.0 mmol) in dry THF (25 mL). The resulting solution
was stirred for 15 min and then freshly distilled HMPA (7.5 mL)
was added under a nitrogen atmosphere. The flask was covered
with aluminum foil and stirred for 15 min. A solution of aldehyde
(S)-6 (2.03 g, 9.00 mmol) in dry THF (25 mL) was added dropwise
through the addition funnel, and the mixture was then stirred 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 2.35 g of (S)-7 as a 85:15 mixture of (9Z): (9Z,13Z)-7S (78%
combined yield). Separation of these isomers was achieved by
column chromatography using n-hexane/benzene (1:1) to obtain
the pure (9Z)-isomer of (S)-7 (1.8 g, 61% yield) as a yellow oil; UV
4.1.11. (2E,4E,6Z,8E)-8-[(40R)Methyl-30,40-dihydro-10(20H)-
naphthalen-10-ylidene]-3,7-dimethyl-2,4,6-octatrienoic acid
[(R)-1]
A solution of ester (R)-7 (1.4 g, 4.2 mmol) was employed. [a]
+70.6° (c 1.1, MeOH). All other spectral data were identical to its
D
enantiomer.
4.2. Binding affinity and transient transfection and luciferase
reporter assays
The binding affinities of compounds ( )-1, (R)-1 and (S)-1 to
hRXR
a
-LBD homodimer were measured using a fluorescence
quenching method.21 hRXR
a
-LBD homodimers (1 M) were ex-
l
cited at 280 nM and the protein fluorescence was measured at
337 nM with Cary Eclipse fluorescence spectrophotometer (Varian,
Palo Alto, CA). The binding dissociation constant Kd was calculated
by using a nonlinear least square regression to fit a single-site
binding model to the raw data.21
Transient transfection and luciferase reporter assays were per-
formed using a previously reported protocol.18 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 in-
kmax 331 nm (e ;
= 30079); IR (Neat) 1708 (C@O), 1603 (C@C) cmꢀ1
[
a
]
ꢀ61.2° (c 1.1, MeOH); MS m/z 337.65 (MH+); 1H NMR
D
(300 MHz, CDCl3) d 7.63 (d, J = 7.2 Hz, 1H), 7.26–7.17 (m, 3H),
6.65 (dd, J = 4.3 Hz
& 11.1 Hz, 1H), 6.45 (s, 1H), 6.24 (d,
cluded 0.2
Hygro] (Promega), 0.5
4-hRXR expression vector, and 0.01
lg of the Gal4 reporter plasmid pGL4.31[luc2P/Gal4UAS/
J = 15.4 Hz, 1H), 6.13 (d, J = 11.1 Hz, 1H), 5.75 (s, 1H), 4.15 (q,
J = 7.1 Hz, 2H), 3.01–2.91 (m, 1H), 2.55–2.45 (m, 1H), 2.40–2.30
(m, 1H), 2.23 (s, 3H), 1.98 (s, 3H), 2.00–1.90 (m, 1H), 1.65–1.50
(m, 1H), 1.31 (d, J = 7.0 Hz, 3H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR
lg
of pCMXGal4-hRAR
a or pCMX-Gal
a
lg of Renilla luciferase repor-
ter plasmid, pRL-TK. TransIT-LT1 Transfection Reagent (Mirus) was
used. At 24 h post-transfection, retinoid was added to the culture
medium. At 48 h post-transfection, reporter activity was deter-
mined using the Dual- Luciferase Reporter Assay (Promega). The
EC50 values from the luciferase assay were determined using a dose
response model containing 2 duplicates at 4 different concentra-
tions (1 nM, 10 nM, 100 nM, 1000 nM).
(75 MHz, CDCl3)
d 167.39, 153.11, 142.86, 140.69, 138.52,
135.57, 134.21, 133.06, 128.25, 127.93, 127.67, 126.24, 124.73,
122.78, 118.61, 59.83, 33.27, 31.53, 25.76, 24.87, 22.22, 14.56,
14.03; Anal. Calcd for C23H28O2: C, 82.10; H, 8.39. Found: C,
82.39; H, 8.63.
4.1.9. (2E,4E,6Z,8E)-Ethyl-8-[(40R)methyl-30,40-dihydro-10(20H)-
naphthalen-10-ylidene]-3,7-dimethyl-2,4,6-octatrienoate [(R)-7]
A solution of aldehyde (R)-6 (2.00 g, 8.85 mmol) was employed.
4.3. Inhibition of oncogenic transformation assay
BOSC23 ecotropic packaging cells at 80–90% confluence were
[a] +60.4° (c 1.1, MeOH). All other spectral data were identical to
D
transfected with 30
lg of KLF4-ER, ErB2 or pBpuro (control) plas-
its enantiomer.
mid DNA. Virus was collected and filtered at 24 h and 48 h post

A. Desphande et al. / Bioorg. Med. Chem. 22 (2014) 178–185
185
transfection and frozen in liquid nitrogen. Following viral titer,
RK3E cells with 30% confluence were infected with virus in the
(CSU).26 All structures were prepared using PyMol (The PYMOL Molec-
ular Graphics System, Version 0.99. Schrödinger, LLC).
presence of 10 lg/mL Polybrene. After 15 h infection, virus was re-
moved and RK3E were supplemented with DMEM. To cells infected
with KLF4-ER were added DMEM supplemented with DMSO (con-
trol), 9cRA, 9cUAB30, ( )-1, (R)-1 and (S)-1 every other day in
duplicate at 3 different concentrations. Three weeks after post-
infection, transformed foci were fixed, stained, counted and ana-
lyzed relative to DMSO control.
Acknowledgements
We thank Dr. Ellen Li (Washington University in St. Louis) for
providing the protein expression vector. We appreciate critical
reading and helpful comments provided by Dr. Matthew B Ren-
frow. We also appreciate the help from Dr. Bingdong Sha in the
refinement of the X-ray crystal structures. This work was sup-
ported by grants from the National Institutes of Health (NIH) 2
P50 CA089019 (D.D.M.) and the Komen Foundation (BCTR
20000690) (D.D.M.).
4.4. In vivo triglyceride levels assay and antiproliferative and
apoptosis assays
The triglyceride, antiproliferative and apoptosis assays were
conducted on female Spraque Dawley rats having small mammary
tumors. The animals were fed a Teklad diet according to previous
reports.8 The retinoids tested were mixed into the diet according
to the previously published protocols and fed for 7 days.8 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 fro-
zen at ꢀ85 °C until it was analyzed for triglycerides.22 The infinity
triglycerides assay kit was purchased from Thermo DMA. For eval-
uation of antiproliferative index, animals were injected with
bromodeoxyuridine (BRDU) 2 h prior to euthanasia using CO2. Can-
cers were removed and fixed with 10% formalin. Cell proliferation
and cell apoptosis were performed as previously reported.8 The
proliferation/apoptotic indices were calculated as the ratio of pro-
liferation/apoptosis of treated animals to vehicle fed animals.
References and notes
1. Mangelsdorf, D. J.; Ong, E. S.; Dyck, J. A.; Evans, R. M. Nature 1990, 345, 224.
2. Lehmann, J. M.; Jong, L.; Fanjul, A.; Cameron, J. F.; Lu, X. P.; Haefner, P.; Dawson,
M. I.; Pfahl, M. Science 1992, 258, 1944.
3. Boehm, M. F.; Zhang, L.; Badea, B. A.; White, S. K.; Mais, D. E.; Berger, E.; Suto, C.
M.; Goldman, M. E.; Heyman, R. A. J. Med. Chem. 1994, 37, 2930.
4. Duvic, M.; Martin, A. G.; Kim, Y.; Olsen, E.; Wood, G. S.; Crowley, C. A.; Yocum,
R. C. Arch. Dermatol. 2001, 137, 581.
5. Miller, V. A.; Benedetti, F. M.; Rigas, J. R.; Verret, A. L.; Pfister, D. G.; Straus, D.;
Kris, M. G.; Crisp, M.; Heyman, R.; Loewen, G. R.; Truglia, J. A.; Warrell, R. P., Jr. J.
Clin. Oncol. 1997, 15, 790.
6. Atigadda, V. R.; Vines, K. K.; Grubbs, C. J.; Hill, D. L.; Beenken, S. L.; Bland, K. I.;
Brouillette, W. J.; Muccio, D. D. J. Med. Chem. 2003, 46, 3766.
7. Muccio, D. D.; Brouillette, W. J.; Breitman, T. R.; Taimi, M.; Emanuel, P. D.;
Zhang, X.; Chen, G.; Sani, B. P.; Venepally, P.; Reddy, L.; Alam, M.; Simpson-
Herren, L.; Hill, D. L. J. Med. Chem. 1998, 41, 1679.
8. Grubbs, C. J.; Lubet, R. A.; Atigadda, V. R.; Christov, K.; Deshpande, A. M.; Tirmal,
V.; Xia, G.; Bland, K. I.; Eto, I.; Brouillette, W. J.; Muccio, D. D. Carcinogenesis
2006, 27, 1232.
9. Vedell, P. T.; Lu, Y.; Grubbs, C. J.; Yin, Y.; Jiang, H.; Bland, K. I.; Muccio, D. D.;
Cvetkovic, D.; You, M.; Lubet, R. Mol. Pharmacol. 2013, 83, 698.
10. Boerma, L. J.; Xia, G.; Qui, C.; Cox, B. D.; Chalmers, M. J.; Smith, C. D.; Lobo-
Ruppert, S.; Griffin, P.; Muccio, D. D.; Renfrow, M. B. J. Biol. Chem. 2013 http://
dx.doi.org/10.1074/jbc.M113.476861.
4.5. Protein purification, crystallization and X-ray
crystallography
11. Leung, C. S.; Leung, S. S.; Tirado-Rives, J.; Jorgensen, W. L. J. Med. Chem. 2012,
55, 4489.
12. Xia, G.; Boerma, L. J.; Cox, B. D.; Qiu, C.; Kang, S.; Smith, C. D.; Renfrow, M. B.;
Muccio, D. D. Biochemistry 2011, 50, 93.
13. Vuligonda, V.; Thacher, S. M.; Chandraratna, R. A. J. Med. Chem. 2001, 44, 2298.
14. Barry, J.; Kagan, H. B.; Snatzke, G. Tetrahedron 1971, 27, 4737.
15. Premasagar, V.; Palaniswamy, V. A.; Eisenbraun, E. J. J. Org. Chem. 1981, 46,
2974.
16. Allegretto, E. A.; McClurg, M. R.; Lazarchik, S. B.; Clemm, D. L.; Kerner, S. A.;
Elgort, M. G.; Boehm, M. F.; White, S. K.; Pike, J. W.; Heyman, R. A. J. Biol. Chem.
1993, 268, 26625.
17. Bourguet, W.; Ruff, M.; Chambon, P.; Gronemeyer, H.; Moras, D. Nature 1995,
375, 377.
18. Jiang, W.; Deng, W.; Bailey, S. K.; Nail, C. D.; Frost, A. R.; Brouillette, W. J.;
Muccio, D. D.; Grubbs, C. J.; Ruppert, J. M.; Lobo-Ruppert, S. M. Cancer Biol. Ther.
2009, 8, 289.
19. Lindeblad, M.; Kapetanovic, I. M.; Kabirov, K. K.; Dinger, N.; Mankovskaya, I.;
Morrisey, R.; Martin-Jimenez, T.; Lyubimov, A. Drug Chem. Toxicol. 2011, 34,
300.
20. Kolesar, J. M.; Hoel, R.; Pomplun, M.; Havighurst, T.; Stublaski, J.; Wollmer, B.;
Krontiras, H.; Brouillette, W.; Muccio, D.; Kim, K.; Grubbs, C. J.; Bailey, H. E.
Cancer Prev. Res. (Phila) 2010, 3, 1565.
21. Cheng, L.; Norris, A. W.; Tate, B. F.; Rosenberger, M.; Grippo, J. F.; Li, E. J. Biol.
Chem. 1994, 269, 18662.
22. Hartge, P.; Hoover, R.; Lesher, L. P.; McGowan, L. JAMA 1983, 250, 1844.
23. Pflugrath, J. W. Acta Crystallogr. D Biol. Crystallogr. 1999, 55, 1718.
24. Schuttelkopf, A. W.; van Aalten, D. M. Acta Crystallogr. D Biol. Crystallogr. 2004,
60, 1355.
25. Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-
Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr. D 1998, 54, 905.
26. Sobolev, V.; Sorokine, A.; Prilusky, J.; Abola, E. E.; Edelman, M. Bioinformatics
1999, 15, 327.
The hRXR
a
-LBD (T223–T462) was overexpressed in Escherichia coli
-LBD homo-
and purified using AKTA purifier system.12 The hRXR
a
dimers were isolated from Ni-chelating column followed by size
exclusion column. The protein was mixed with 4-fold excess of
UAB rexinoids and then 5-fold excess of GRIP-1 coactivator peptide
(
686KHKILHRLLQDSS698). The ternary complexes were crystallized
using vapor diffusion technique in hanging drops.12 Crystals with
suitable diffraction size appeared in 2 weeks with conditions of
2–10% PEG4000, 4–12% Glycerol and 0.1 M Bis–Tris, pH 7.0. Crystals
were flash frozen in liquid nitrogen using a reservoir cryoprotectant
solution containing glycerol. The crystals were placed in stepwise
increasing concentration of glycerol (10.0%, 12.5%, 15%, 17.5%,
20.0%, 22.5%, 25.0%) in order to avoid damage to the crystals due
to the sharp increase in glycerol concentrations. Diffraction data
of crystals were collected on a Rigaku IV+ diffractometer with a cop-
per rotating anode and processed using the program D⁄Trek.23 The
structures were solved using the molecular replacement method
with a high-resolution hRXRa-LBD structure with its ligand deleted
as a search model (pdb ID: 3OAP). The ligand files and their associ-
ated topology and parameter files were created by PRODRG.24 The
structures were refined using CNS software.25 A complete summary
of data for these structures was listed in Table 2. Interactions be-
tween ligand/peptide and hRXR
gram ligand–protein contacts (LPC)/Contacts of Structural Units
a-LBD were analyzed using a pro-