Determinants of retinoid X receptor transcriptional antagonism

Article abstract of DOI:10.1021/jm030651g

The synthesis and bioactivity of the retinoid X receptor (RXR) antagonist 4-[(3′-n-butyl-5′,6′,7′,8′-tetrahydro-5′, 5′,8′,8′-tetramethyl-2′-naphthalenyl)(cyclopropylidene) methyl]benzoic acid and several heteroatom-substituted analogues are described. Lig

Full text of DOI:10.1021/jm030651g

4360
J . Med. Chem. 2004, 47, 4360-4372
Articles
Deter m in a n ts of Retin oid X Recep tor Tr a n scr ip tion a l An ta gon ism
Claudio N. Cavasotto,| Gang Liu,† Sharon Y. J ames,† Peter D. Hobbs,‡ Valerie J . Peterson,#
Ananyo A. Bhattacharya,† Siva K. Kolluri,† Xiao-kun Zhang,† Mark Leid,# Ruben Abagyan,§
Robert C. Liddington,† and Marcia I. Dawson*,†
Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La J olla, California 92037, Molsoft L.L.C.,
3366 North Torrey Pines Court, Suite 300, La J olla, California 92037, Retinoid Program, SRI International,
333 Ravenswood Avenue, Menlo Park, California 94025, College of Pharmacy, Oregon State University,
Corvallis, Oregon 97331, and The Scripps Research Institute, 10550 North Torrey Pines Road, La J olla, California 92037
Received December 31, 2003
The synthesis and bioactivity of the retinoid X receptor (RXR) antagonist 4-[(3′-n-butyl-5′,6′,7′,8′-
tetrahydro-5′,5′,8′,8′-tetramethyl-2′-naphthalenyl)(cyclopropylidene)methyl]benzoic acid and
several heteroatom-substituted analogues are described. Ligand design was based on the scaffold
of the 3′-methyl RXR-selective agonist analogue and reports that 3′-n-propyl and longer n-alkyl
groups conferred RXR antagonism. The transcriptional antagonism of the 3′-n-butyl analogue
was demonstrated by its blockade of retinoic acid receptor (RAR) â expression induced by the
RXRR/peroxisome proliferator-activated receptor (PPAR) γ heterodimer complexed with an
RXRR agonist plus the PPARγ agonist ciglitazone and the inhibition of 9-cis-RA-induced
coactivator SRC-1a recruitment to RXRR. Receptor-ligand docking studies using full-atom
flexible ligand and flexible receptor suggested that binding of the antagonist to the RXRR
antagonist conformation was favored because the salt bridge that formed between the retinoid
carboxylate and the RXRR helix H5 arginine-321 was far stronger than that formed on its
binding to the agonist conformation. The antagonist also blocked activation of RAR subtypes
R and â by 9-cis-RA but not that of RARγ.
In tr od u ction
lography on RXRR-agonist complexes reveals that the
terminus of the RXRR helix H12 is more mobile.4-7
These differences are highlighted by the structure of the
RXRR/peroxisome proliferator activated receptor (PPAR)
γ heterodimer complex with both RXR agonist 9-cis-RA
(1 in Figure 1) and PPARγ transcriptional agonist
rosiglitazone5 and by the transcriptional activation
activity of the RXRR/RARR heterodimer complex with
both RXR transcriptional agonist SR112378 (2) and RAR
antagonist AGN192870 (14).6 This inherent flexibility
in the RXRR helix H12 permits the functional variability
exhibited by RXR as the heterodimeric partner of many
other members of the nuclear receptor family.7
The retinoid nuclear receptors function as transcrip-
tion factors that regulate such cell processes as morpho-
genesis, proliferation, and differentiation.1 This homolo-
gous retinoid receptor subfamily has two classes, namely,
the retinoic acid receptors (RARs) and retinoid X recep-
tors (RXRs). Each class consists of three subtypes (R, â,
and γ). In vivo, these receptors typically function as
heterodimers that bind to specific DNA sequences
termed response elements (REs) and undergo confor-
mational changes on binding retinoid transcriptional
agonists to release corepressors and recruit coactivators.
Binding by the latter facilitates the interactions neces-
sary to engage the multiprotein machinery for gene
transcription. X-ray crystallographic studies indicate
that transcriptional agonist binding induces major shifts
in the RAR ligand-binding domain (LBD) helices H3,
H11, and H12 so that helix H3 and the activation
function-2 (AF-2) region of helix H12 form a cleft with
helix H4 on the RAR surface to which a coactivator
binds.2,3 In contrast, RAR transcriptional antagonists
are unable to effect these same changes. Crystal-
Synthetic receptor-selective retinoids facilitate mecha-
nistic studies9-12 because the natural retinoids lack
specificity with trans-RA (15) activating all the RAR
subtypes, 1 activating both RAR and RXR subtypes,1
and both interconverting by isomerization.9 RAR class
and subtype-selective transcriptional agonists and an-
tagonists have been reported,9-12 as have RXR class-
selective retinoids (rexinoids).8,9,11-18 The extensive
homology of the RXR subtype ligand-binding pocket
(LBP) residues has as yet precluded the identification
of RXR subtype-selective retinoids. To facilitate mecha-
nistic studies, we undertook the identification of RXR-
selective transcriptional antagonists, as have other
groups.19-22 Previously, the homology between the
residues surrounding the RAR and RXR LBPs1 led to
our successful exploitation of the mirrored structural
* To whom correspondence should be addressed. Telephone: 1-858-
646-3165. Fax: 1-858-646-3197. E-mail: mdawson@burnham.org.
| Molsoft L.L.C.
† The Burnham Institute.
‡ SRI International.
#
Oregon State University.
§ The Scripps Research Institute.
10.1021/jm030651g CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/29/2004

Retinoid X Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4361
reported to bind to the RXR subtypes but not to bind to
the RAR subtypes or to activate the RAR or RXR
subtypes.16 Because this activity profile suggested that
8 was an RXR transcriptional antagonist, we synthe-
sized its 3′-n-butyl analogue (9) as a potential antagonist
for probing RXR-selective retinoid (rexinoid) signaling
pathways. Unfortunately, because 9 activated RXRR on
the (TREpal1)2-tk-chloramphenicol acetyl transferase
(CAT) reporter construct (Table 1), further design and
synthesis were necessary. Our first objective in the
present studies was to discern whether the addition of
hydrophobic substituents at the 2-position of the ethenyl
bridge of 9 would produce an RXR antagonist as a
similar strategy did for the diazepinyl-bridged rexinoids
reported by Kagechika and co-workers.19 The validity
of such an approach has since been supported by their
recent report on the conversion of a series of potent 1,3-
pyrimidine-5-carboxylic acid-terminated, MeN-bridged
rexinoid agonists to antagonists by replacing the 3′-
methyl groups on their 5′,6′,7′,8′-tetrahydro-2′-naph-
thalenyl rings with 3′-n-pentoxy and n-hexyloxy groups.20
Klaus and co-workers first reported the use of a 3′-n-
alkoxy (n-heptyloxy) substituent to confer retinoid
antagonist activity in RARR-selective Ro41-5253 (16).26
We substantiated the utility of this substitution strategy
in the related RAR antagonist 17.27
In the construction of 3, the potent RXR-selective
agonist 4-[(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-
2′-naphthalenyl)(cyclopropylidene)methyl]benzoic acid
(10)14 was used as the scaffold and the 3′-n-butyl group
of 9 was used to introduce antagonist activity. The
synthesis of 3, which is illustrated in Scheme 1, was
also based on that of 10. Serendipitously for the first
phase of this work, the 6-(n-butyl)tetrahydronaphtha-
lene 19 was available as a byproduct from the lithiation
of the 6-bromotetrahydronaphthalene 18 using an n-
butyllithium solution evidently containing appreciable
unreacted n-butyl halide. Subsequently, 19 was directly
prepared in much higher yield by lithiation of 18
followed by alkylation with excess n-butyl bromide. The
overall yield for the five-step synthesis of 3 from 18 was
28%. Substitution of methyl(triphenyl)phosphonium
bromide in the fourth step of Scheme 1 afforded 9.
This series was extended with heteroatom-substituted
analogues 4-7 for the following reasons. Replacing the
3′-alkyl group on the TTN ring with an alkoxy group
has been shown to facilitate the synthesis of rexinoid
analogues.16,20 Pyridine and pyrimidinecarboxylic acid
termini were reported to confer high-binding affinities
to RXRs having an MeN or cyclopropyl C bridge.16,20 In
addition, according to the Lipinski rule of five,28 the
introduction of an H-bond acceptor group should im-
prove druglike properties. Thus, the 3′-n-propoxy and
3′-n-butoxy-5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-
naphthalenyl (TTN) benzoic acid analogues 4 and 5 of
F igu r e 1. Structures of RAR and RXR panagonist 1, rexinoid
agonists 2 and 8-13, RXR antagonist 3 and analogues 4-7,
RAR antagonists 14 and 17, RAR agonist trans-RA (15), and
RARR-selective antagonist 16.
similarities of their selective ligands to guide the design
of RXR agonists based on that of RARγ agonists.9,13,14
We then extended this strategy to RXR antagonist
design from the structures of RAR antagonists. Here,
we report one such RXR antagonist, 4-[(3′-n-butyl-
5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-2′-naphtha-
lenyl)(cyclopropylidene)methyl]benzoic acid (3), and four
heteroatom-substituted analogues, 4-7.
To understand the structural determinants that
govern receptor binding by 3, we undertook computa-
tional studies on RAR and RXR ligand-binding domain
(LBD)-retinoid complexes using state-of-the-art, full-
atom flexible ligand-flexible receptor docking, a tech-
nique that we previously used successfully on G-protein-
coupled seven-transmembrane receptors23 and protein
kinases.24 The present studies suggested that the pref-
erence of retinoid 3 for the antagonist-bound conforma-
tion of the RXRR LBD25 was not due to steric clashes
with residues in the LBP of the agonist-bound confor-
mation but to the greater strength of the salt bridge
between the carboxylate group of 3 and the guanidinium
group of arginine (R)-321 in helix H5 in the LBP of the
antagonist-bound conformation. These studies provide
evidence that ligand binding to these mobile retinoid
receptors may be more complex than that anticipated
from using rigid crystallographic structures as models.
Resu lts
Earlier, retinoid 8, which has a 3′-n-propyl group
ortho to the ethenyl carbon joining its aryl rings, was
Ta ble 1. Effects of Retinoids 3 and 9 on Retinoid Receptor Activity Induced by 1 on the TREpal Retinoid Response Elementa
relative activation (%)
RXRR concn (M)
RARR concn (M)
RARâ concn (M)
RARγ concn (M)
10-7
10-6
10-5
10-7
10-6
10-5
10-7
10-6
10-5
10-7
10-6
10-5
3
9
115 ( 5
114 ( 4
88 ( 4
130 ( 6
48 ( 4
150 ( 11
82 ( 3
75 ( 3
45 ( 3
30 ( 2
22 ( 1
20 ( 1
98 ( 4
94 ( 3
88 ( 3
102 ( 4
65 ( 3
84 ( 5
95 ( 4
89 ( 5
99 ( 3
94 ( 4
91 ( 5
110 ( 6
a
Receptor activities were determined using the (TREpal)2-tk-CAT reporter construct in cotransfected CV-1 cells as described11 and
expressed relative to that of 1 × 10-7 M 1 as 100%.

4362 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Cavasotto et al.
Sch em e 1a
a
(a) n-BuLi, THF; 20-35 °C, n-BuBr; (b) (COCl)2, PhH, reflux; (c) AlCl3, CH2Cl2, 0 °C to reflux; (d) [cyclopropylmethyl(Ph)3PBr,
KN(SiMe3)2, PhMe], [Me(OCH2CH2)2]3N, 100 °C; (e) aqueous KOH, EtOH, 80-90 °C, H3O+; (f) 5-carbomethoxypyridinecarboxylic acid
(25), SOCl2, DMF, benzene, reflux; (g) 21 or 26, CH2Cl2, AlCl3; (h) (28 and 31) n-PrBr, K2CO3, acetone, reflux; (i) (29 and 32) n-BuBr,
K2CO3, acetone, reflux; (j) aqueous NaOH, MeOH, reflux; dilute HCl.
3, as well as their 5-pyridinecarboxylic acid analogues
6 and 7, were readily prepared from the tetrahydro-
tetramethylnaphthol 24 and the benzoyl chloride 21 and
the 2-pyridylcarbonyl chloride 26 by routes that were
very similar to that used for the preparation of 3
(Scheme 1). However, in the cases of 4-7, Fries rear-
rangements were used to introduce the aroyl groups of
acyl chlorides 21 and 26 at the least hindered position
adjacent to the OH group on 24 to produce 27 and 30,
respectively. Alkylation of the hydroxyl groups of 27 and
30, Wittig olefination of the diaryl ketone group of the
resulting aryl ethers 28, 29, 31, and 32, and methyl
ester hydrolysis of the Wittig reaction products 33-36
then produced 4-7 in overall yields of 67%, 66%, 24%,
and 25%, respectively, for the five steps starting from
24.
F igu r e 2. Binding affinity of 3 and 10 to the RXRR LBD.
Competitive radioligand binding assays were performed as
Rexin oid 3 bin d s to RXRr. Competitive binding
studies using recombinant histidine-tagged human (h)
RXRR LBD and [11,12-3H2]9-cis-RA indicated that the
relative binding affinities as measured by IC50 values
were 21 nM for 10, 29 nM for 1, and 0.5 µM for 3 (Figure
2). Thus, under these conditions, 3 was a more than 20-
fold weaker competitive binder to RXRR than the
agonist 10 from which it was derived. Binding affinity
assays using a second recombinant hRXRR LBD sample
indicated that heteroatom substitution on the scaffold
of 3 improved binding affinity to RXRR. Relative IC50
values for 4-7 were 120, 270, 6, and 10 nM, respec-
tively, compared to 45 nM for 1. Thus, on the basis of
comparing the IC50 values of 4 with 5 and of 6 with 7,
CH2 homologation of the 3′-propoxy group decreased
binding affinity by about half. In contrast, the pyridine
rings of 6 and 7 enhanced affinity at least an order of
magnitude over that of 4 and 5 having benzene rings
at the same position.
described in Methods. Binding was conducted in duplicate.
Differences were 10% or less. The data represent the relative
percentage of bound cpm compared to cpm bound in the
absence of added ligand.
RXRr a ctiva tion by 9-cis-RA (1) or r exin oid 2 is
a n ta gon ized by 3. The retinoid receptor transcrip-
tional activation activity of 3 was assessed in classical
cotransfection assays. Transcriptional activation in
CV-1 cells using cotransfected vectors for one of the
retinoid receptors and a retinoid-responsive chloram-
phenicol acetyl transferase (CAT) reporter construct (the
(TREpal)2-tk-CAT,1 RAR-specific cellular retinol-binding
protein (CRBP)-I-tk-CAT,1 or RXR-specific CRBP-II-tk-
CAT8) showed that 1 µM 3 was not able to activate
RARR, â, or γ or RXRR (Table 1). The TREpal is a
palindromic response element that is activated by either
RAR or RXR-agonist complexes. However, a 1 log

Retinoid X Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4363
Ta ble 2. Effects of Retinoid 3 on Retinoid Receptor Activity Induced by 9-cis-RA (1) on the CRBP-II and trans-RA (15) on the
CRBP-Ia
relative activation (%)
RXRR concn (M)
RARR concn (M)
RARâ concn (M)
RARγ concn (M)
0
10-7
10-6
0
10-7
10-6
0
10-7
10-6
0
10-7
10-6
3
100
98 ( 6
34 ( 2
100
52 ( 4
10 ( 2
100
80 ( 4
59 ( 1
100
94 ( 8
108 ( 6
a
RXRR and RAR subtype activities were determined using the CRBP-II-tk-CAT and CRBP-I-tk-CAT reporter constructs, respectively,
in cotransfected CV-1 cells treated with 1 × 10-7 M 9-cis-RA and 1 × 10-7 M trans-RA, respectively, in the absence or presence of the
indicated concentrations of 3 as described121 and expressed relative to that of 1 × 10-7 M 9-cis-RA as 100% for RXRR activation and 1 ×
10-7 M trans-RA as 100% for the RAR subtype activations.
excess of 3 was able to reduce 0.1 µM 1-induced RXRR
activation of the CRBP-II by over 66% (Table 2) and that
induced by 0.1 µM rexinoid agonist 2 by 80%. Interest-
ingly, antagonism by 3 was weaker on the (TREpal)2-
tk-CAT reporter because a 2 log excess of 3 was required
to decrease 1-induced RXRR activation by 50%. Thus,
while having no intrinsic RAR subtype or RXRR agonist
activity, 3 was able to successfully block the activation
F igu r e 3. Retinoid 3 inhibits RARâ induction by rexinoid
of RARR and RARâ by trans-RA (15) or 1 and that of
agonist 2 and the PPARγ ligand ciglitazone. Calu-6 cells were
RXRR by 1. However, 3 had only minimal effects on the
treated for 24 h with 1.0 µM trans-RA (15), 2, or 3, or with 10
activation of RARγ by 15 or 1 (Table 2).
µM ciglitazone alone, or with 2 plus ciglitazone in the absence
In contrast to the inhibitory effects of 3 on 1-induced
RXRR activation, at 0.1, 1.0, and 10 µM 9 enhanced the
activation of RXRR by 10 nM 1 on the (TREpal)2-tk-CAT
reporter in a concentration-dependent manner (114%,
130%, and 150%, respectively) (Table 1). At 1.0 and 10
µM, 9 reduced RARR activation by 10 nM 1 to 30% and
20%, respectively. The inhibitory effects of 10 µM 9 on
RARâ and RARγ activation were considerably smaller
(reduction from 92% to 82% and none, respectively). On
the basis of these results, 9 functioned as an RXRR
transcriptional agonist and an RARR antagonist.
Act iva t ion of t h e R ARâ2 r esp on se elem en t
(â2RARE) by RXRr/P P ARγ h eter od im er liga n d s is
block ed by a n ta gon ist 3. RARâ is considered to
function as a tumor suppressor gene for several reasons.
The loss of RARâ expression in many cancer cell lines
and in tumor biopsy specimens has been found to
correlate with their insensitivity to growth inhibition
by retinoid agonists.29,30 Restoration of RARâ expression
by activating the â2RARE response element in the
RARâ2 gene promoter with transfected RARR in the
presence of a retinoid agonist or by transfection of
RARâ2 also restored the sensitivity of several cancer cell
lines to growth inhibition by retinoids.31,32 Recently, we
observed that the combination of rexinoid transcrip-
tional agonist 2 and the PPARγ agonist ciglitazone
cooperatively transactivated the transfected â2RARE
reporter construct in both retinoid-resistant MDA-MB-
231 and retinoid-sensitive ZR-75-1 breast cancer cells.33
Antagonist 3 was able to block the induction of RARâ
protein expression in the Calu-6 lung cancer cell line
by the combination of rexinoid 2 and ciglitazone (Figure
3).
or presence of 3. Cell lysates were prepared, and RARâ protein
was assessed by Western analysis.
F igu r e 4. Antagonist 3 prevents coactivator recruitment to
RXRR. (A) GST-pulldown experiments using GST-RXRR LBD
or GST and [35S]methionine-labeled, full-length SRC-1a (SRC-
1) in the presence of vehicle (0.1% v/v ethanol, lanes 2 and 8)
or RXR ligand as indicated. These experiments were conducted
as previously described34 with ligands at a final concentration
of 1.0 µM. (B) GST-pulldown experiments as described in (A)
using 1.0 µM 1 in the absence (lanes 2-7) or presence (lanes
8-13) of 5.0 µM 3. The input lane (lane 2 of parts A and B)
corresponds to 15% of the [35S]methionine-labeled SRC-1a used
in the pulldown reaction. The autoradiographs are representa-
tive of three independent experiments.
demonstrated in the glutathione-S-transferase (GST)
pulldown experiments using the recombinant mouse
RXRR LBD and 35S-labeled SRC-1a that are shown in
Figure 4. RXRR agonists 1 and 10-12 strongly pro-
moted SRC-1a binding to the GST-RXRR LBD in vitro
(Figure 4A, lanes 3-6). This result is consistent with
the transcriptional activation properties of these ligands.
In contrast, 3 did not promote SRC-1a interaction with
the GST-RXRR LBD (Figure 4A, lane 7). The findings
that 3 and 10-12 did not promote the interaction of
SRC-1a with GST alone indicate the specificity of the
interaction between the GST-RXRR LBD and SRC-1a
(Figure 4A, lanes 9-13). This result and the demonstra-
tion that the binding of 3 and that of labeled 9-cis-RA
to the RXRR LBD were mutually exclusive (Figure 2)
suggested that 3 exerts RXRR antagonistic activity in
Coa ctiva tor r ecr u itm en t to RXRr is in h ibited by
a n ta gon ist 3. Retinoid transcriptional agonists induce
conformational changes in the retinoid receptor LBD
that permit coactivator binding, whereas antagonists do
not. Unlike RXR agonist 1 and RXR-selective agonists
10-12,13,14 antagonist 3 was not able to induce the
recruitment of the steroid receptor coactivator (SRC)-
1a34 to the RXRR LBD coactivator site in vitro but did
retard the recruitment of SRC-1a induced by 1, as was

4364 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Cavasotto et al.
conformation, which were comparable to that of 1.
Superposing the docked configurations of 10 and 13
showed that their scaffold orientations were conserved
(Figure 6A). Both 10 and 13 made similar contacts with
the LBP surface, although those of 10 covered about
20% more of the surface. Both the higher contact surface
and the slightly stronger electrostatic interaction be-
tween the carboxylate group of 10 and the guanidinium
group of the RXRR LBD helix H5 arginine-316 (1FM6
numbering)35 suggested why 10 had the lower IC50 value
in competitive binding to RXRR (21 nM for 10 compared
to 44 nM for 13) and a 2-fold lower AC50 value for
activating RXRR on the (TREpal)2-tk-CAT than 13.14
The docked conformation of 3 made considerable
contacts with LBP residues in the RXRR LBD antago-
nist conformation. Its carboxylate group made a strong
salt bridge with arginine-321 in helix H5 (1DKF num-
bering).2 Surprisingly and despite our experimental
studies demonstrating that 3 functioned as an RXRR
antagonist (Table 1 and Figures 3 and 4), 3 was also
able to dock to the RXRR LBD agonist conformation
(Figure 6B). We next undertook docking experiments
to assess whether the structural determinants for
binding to the RXRR LBD antagonist conformation2
were preferred by 3. From the conformations we had
generated by grid docking, we performed cycles of global
energy stochastic optimization using both flexible ligand
and flexible LBP side chains and then minimized the
energy of resultant complexes. This full-atom flexible
ligand-flexible receptor method revealed that in the
agonist LBP conformation 3 superposed well with the
native ligand 1 (Figure 6B). In this docking simulation,
the two water molecules that were found in the vicinity
of the ligand (RXRR LBD-135 PDB structure 1FBY)
were conserved and were not affected by the benzoate
group of 3. One water molecule continued to hydrogen-
bond to leucine-309, the carboxylate of 3, and the second
water molecule remained hydrogen-bonded to glutamine-
275.
F igu r e 5. Structures of RXR antagonists 37, 41, and 43; RXR
transcriptional agonists 38, 40, and 42; and RARR-selective
antagonist 39.
vitro. This possibility was tested directly in GST-
pulldown assays, the results of which are shown in
Figure 4B. When 3 was absent, 1 promoted receptor-
SRC-1a interaction in a concentration-dependent man-
ner (Figure 4B, lanes 3-7). In the presence of 3 at 5.0
µM, a concentration at which receptor-coactivator
interaction did not occur (compare lanes 1 and 8 of
Figure 4B), the efficacy with which 1 induced recruit-
ment of SRC-1a to the RXRR LBD was clearly reduced
(compare lanes 3-7 to lanes 9-13 in Figure 4B).
However, because the 3-mediated antagonism of 1-in-
duced recruitment of SRC-1a to the RXRR LBD was
surmounted by increasing the concentration of 1 (com-
pare lanes 7 and 13 in Figure 4B, which correspond to
1.0 µM 1), 3 functioned as a competitive antagonist and
not as a rexinoid agonist. Thus, binding by 3 to the
RXRR LBD did not induce a conformation that could
recruit a coactivator protein to the RXRR AF-2 site.
Considered together, these in vitro studies demon-
strated that 3 bound directly to the RXRR LBD but in
a manner distinct from that of 1.
Com p u ta tion a l Stu d ies. To understand how 3
functioned as a rexinoid antagonist in binding to the
RXRR LBD, its docking conformation was compared to
those of rexinoid agonists 10 and 13,13 which have
CdC(CH2)2 and C(CH2)2 diaryl bridges, respectively.
Docking studies were performed using the X-ray crys-
tallographic structures of the RXRR LBDs complexed
to 1 and 9-cis-oleic acid (37 in Figure 5) to represent
the agonist35 (Protein Data Bank (PDB) entry 1FM6)
and antagonist2 (PDB 1DKF) conformations of the holo-
RXRR LBD-retinoid complex, respectively. Water mol-
ecules in the vicinity of the ligand were taken from the
PDB 1DKF structure for the antagonist conformation
and the PDB 1FBY structure for the agonist conforma-
tion because no water molecules in the vicinity of the
ligand were reported for the PDB 1FM6 structure.
Rapid grid dockings with flexible ligands followed by
global energy stochastic optimizations using the full-
atom representations of the receptors and flexible
ligands36 were performed. Rexinoid agonists 10 and 13
had very good docking scores to the RXRR LBD agonist
In the LBP antagonist conformation the TTN ring of
3 was rotated by 180° from that in the agonist confor-
mation so that instead of the 3′-n-butyl group of 3
pointing to helix H11 in the LBP as it had in the agonist
conformation, this group now pointed to helix H7. As a
result, the benzoate group of 3 was nearly orthogonal
(90°) to that in the agonist conformation and its car-
boxylate formed a strong salt bridge with the RXRR
helix H5 arginine. During the course of this docking
simulation, the water molecule that had originally
hydrogen-bonded to the carbonyl oxygen of leucine-314
and the oleic acid (37) carboxylate in the LBP antagonist
conformation was displaced from its position by the
benzoate moiety of 3 and moved to become hydrogen-
bonded to arginine-321, the hydroxyl hydrogen of serine-
317, and the carboxylate of 3 (Figure 6C). Our calcula-
tions indicated that the salt bridge made by 3 in the
antagonist conformation was much stronger than that
in the agonist conformation. The binding affinity of 3
to the antagonist conformation was further strength-
ened by the reciprocal rearrangement of the LBP side
chains that produced additional and closer hydrophobic
contacts between 3 and the LBP surface than those
produced in the agonist conformation (Figure 6D). Thus,
these studies suggested that the binding of 3 to the

Retinoid X Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4365
F igu r e 6. (A) Superposition of 10 (aqua) and 13 (dark-gray) in the human (h)RXRR ligand-binding pocket (LBP) after global
optimization of side chains (carbon, gray; nitrogen, blue; oxygen, red; sulfur, yellow). (B) Superposition of complexes of 3 in the
RXRR LBP in its agonist- (light-magenta) and antagonist-bound (green) conformations with that of 1 found in the agonist
conformation. Some helices are indicated. The helix H5 arginine (R) 316 (agonist)/321 (antagonist) side chain conformations forming
salt bridges with the ligand carboxylate conformations are also displayed. Retinoid 3 in the antagonist-bound conformation makes
the stronger salt bridge with the RXRR LBD R321 and so functions as a competitive antagonist. The arginine nitrogens are in
blue. (C) Hydrogen-bonding network of the water molecule making contacts with 3 (green) in the antagonist bound conformation
of the RXRR LBP. The hydrogen bonds of the water contacting serine-317, arginine-321, and the carboxylate group of 3 are
displayed in blue. This water as originally found hydrogen-bonded to leucine-314 and the 37 carboxylate (PDB 1DKF) is shown
with its oxygen colored magenta. Atom color code: nitrogen, blue; oxygen, red; hydrogen, black. (D) Space-filling representation
of 3 in the agonist-bound conformation of the hRARγ LBP indicating that 3 fits well (color coding of the pocket surface is green
for hydrophobic regions, blue for hydrogen-bond donor, and red for hydrogen-bond acceptor). Helices H3, H5, H10, and H11 are
indicated. (E) Superposition of 3 (green), 9 (purple), and trans-RA (15) (black) in the RARγ LBP shows the weaker electrostatic
interaction for the carboxylate groups of rexinoids 3 and 9 with the helix H5 R278 side chain compared to that of trans-RA (15).
R278 carbons are displayed in gray and nitrogens are in blue; ligand oxygens are in red. (F) Superposed conformations of 3-7 as
found in docked to the RXRR LBD antagonist conformation. Structure color code: 3, green; 4, purple; 5,; orange 6, aqua; and 7,
yellow.
RXRR LBD antagonist conformation was energetically
favored. We also performed flexible ligand-flexible
receptor docking of analogues 4-7 to the antagonist
conformation. As shown in Figure 6F, the high degree
of overlapping of their resultant conformations with that
of 3 suggested that the binding conformation of this
series of ligands to the antagonist form of the RXRR
LBD was conserved. Identical results were obtained
performing the simulations in the absence of the water
molecules.
water molecules reported to be in the vicinity of trans-
RA (15) suggested that the lack of RARγ transcriptional
activation activities for 3 and 9 was not due to any of
their atoms clashing with those of RARγ LBP residues
(Figure 6E). In fact, 3 and 9 fit very well in the LBP
agonist conformation without any significant side chain
clashes. Superposing the docked conformations of 3 and
9 with that of trans-RA (15)3 (PDB entry 2LBD)
revealed that electrostatic interactions between their
retinoid carboxylates and the RARγ helix H5 arginine-
278 were much weaker than those of trans-RA (15)
(Figure 6E). As a result, the free energies36 calculated
for binding of 3 and 9 to the agonist form of the RARγ
Docking of 3 and 9 into the RARγ LBD agonist
conformation using the structure of the human (h)RARγ
LBD-trans-RA (15) complex3 (PDB 2LBD) and the two

4366 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Cavasotto et al.
LBD were about 4.5 kcal/mol higher than that for trans-
RA (15). The same results were obtained when water
molecules were omitted in the simulation. Thus, our
computational studies agreed with experimental results
on the lack of transcriptional activation of RARγ by 3
and 9.
AGN192870 (9) complex also recruited a coactivator
(TIF-II).6 In the RXRR LBD-1 monomer complex, the
movement of helices H3 and H11 permitted stabilization
of the hydrogen bonds between the N-terminus of helix
H12 and helix H3 residues but not coactivator-binding
site formation.6 In contrast and unlike the binding of
agonists to RARs, the agonists 1, 2, and docosahexaenoic
acid (38 in Figure 5) on binding to RXRR did not
stabilize the RXRR helix H12 position through direct
contacts.7 In the RXRRF318A LBD mutant-oleic acid
(37)/RARR LBD-RARR-selective antagonist BMS190614
(39) complex,2 the H12 helices of both RXRR and RARR
adopted comparable canonical antagonist conforma-
tions.2 In this case, the RXRR helix H12 leucine-456,
methionine-459, and leucine-460 side chains occupied
the coactivator-binding site by mimicking the three
leucines of the coactivator-binding motif. These studies
indicate that the position of the RXR helix H12 varies
considerably and is not stabilized by contacts with the
ligand.
Moras and co-workers35 reported that on docking the
RXR agonist HX600 (40) into the LBP of the RXRR
LBD-1 complex the position of helix H12 was stabilized
by van der Waals contacts with helices H5 and H11. In
contrast, docking of the nitro-substituted analogue
HX531 (41),19 which functioned as an antagonist, pro-
duced steric clashes between the nitro group of 41 and
helices H5 and H11. Docking of 41 to the RXRR LBD
antagonist conformation2 suggested to them that ad-
ditional conformational adaptations of ligand or protein
were required to ensure an acceptable fit.35
In the structure (1H9U)37 for the RXRâ LBD ho-
modimer complex with the potent rexinoid agonist 2-[1-
(3′-methyl-5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-2′-
naphthalenyl)cyclopropyl]-5-pyridinecarboxylic acid
(LG100268, 42),17 helix H12 by not contacting the
receptor surface was found to have an apo, rather than
a holo agonist, orientation. Love and co-workers specu-
lated that had helix H12 assumed the classical agonist
conformation on binding 42 and a coactivator, the 3′-
methyl group on its TTN ring would have interacted
with the RXRR leucine-451 to stabilize the helix H12
agonist conformation and coactivator interaction. They
hypothesized that a longer 3′-group, such as the 3′-n-
propoxy group of the RXR homodimer antagonist
LG10075422 (43), would have destabilized the helix H12
coactivator-binding site position through a similar
interaction with helix H12.
Discu ssion
The presence of clashes between a ligand and the side
chains in the receptor LBP has been considered to be a
prime determinant to its ability to bind. Our present
docking studies suggest another paradigm in which the
strength of the salt bridge formed between the retinoid
carboxylate group and the guanidinium group of the
conserved arginine on the RAR or RXR LBD helix H5
plays a major role in determining whether a retinoid
functions as a transcriptional agonist or antagonist.
Thus, 3 behaved as a transcriptional antagonist because
the LBP side chain rearrangements that occurred on
the binding of 3 to the RXRR LBP antagonist conforma-
tion produced a stronger salt bridge and more and closer
hydrophobic contacts than those that occurred on its
binding to the agonist conformation. The subtle differ-
ence in the energies of the resulting complexes would
have shifted the equilibrium to favor the binding of 3
to the LBP antagonist conformation. Similarly, the
stronger salt bridge between the carboxylate of rexinoid
10 and arginine-316 of the RXRR LBD agonist confor-
mation and the resulting higher number of van der
Waals contacts between 10 and the LBP surface would
explain the higher binding affinity and transcriptional
activation activity of 10 compared to 13.14 The results
as to binding preferences to receptor agonist or antago-
nist conformations by these ligands remained the same
regardless of whether the presence of water molecules
in the vicinity of the salt bridge were included in the
docking simulations.
The LBDs of the RARs and RXRs are flexible. X-ray
crystallographic studies of the apo (nonliganded) and
holo (liganded) RARγ and RXRR LBDs indicated that
ligand binding produced significant and mutually co-
operative conformational changes that influenced the
position of their H12 helices. Thus, binding by a
transcriptional agonist caused the N-terminus of the
LBD helix H3 to tilt more than 10 Å and helix H11 to
rotate 180° about its axis so that its hydrophobic side
chains moved from the LBP to provide space for the
ligand to bind. To accommodate the tilting of helix H3,
helix H2 of RXRR unwound to increase the length of
the loop between helices H1 and H335 and the loop
joining helices H1 and H3 of RARγ straightened out.
In agonist-bound RARs and RXRs, these changes al-
lowed helix H12 to move to cap the LBP. In RARs, the
position of the helix H12 was stabilized by direct
interactions with the hydrophobic terminus of the ligand
so that its AF-2 sequence formed the coactivator-binding
site with helices H3 and H4.35
Our docking studies do not support the latter premise
but support the results of RXRR crystallographic studies
by suggesting another binding paradigm. Docking re-
sults indicated that steric clashes of 3 with LBP residues
of either helix H12 or other helices did not prevent
docking to agonist conformations of the RXRR and RARγ
LBDs. Instead, the binding of 3 to the agonist confor-
mations of RXRR and RARγ was observed to be ener-
getically disfavored. Antagonist 3 docked in the RXRR
LBP antagonist conformation without significant steric
clashes by adopting another configuration that en-
hanced the strength of its salt bridge to helix H5.
Moreover, although docking of 3 to the RARγ LBD
agonist conformation permitted salt bridge formation,
the strength of this salt bridge was so much weaker
than that formed by trans-RA (15) that efficient com-
In RXRR, the position of the helix H12 C-terminus
depended on the ligand bound, the dimeric partner, and
whether a coactivator was present. Thus, in the RXRR
LBD-1/PPARγ LBD-rosiglitazone-SRC-1 coactivator
peptide complex, both the PPARγ and RXRR H12 AF-2
sequences formed coactivator-binding sites.5 The RXRR
LBD-RXR
agonist
2/RARR
LBD-antagonist

Retinoid X Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4367
IR (CHCl3) 1745 cm-1; 1H NMR δ 0.84 (t, J ) 7.1 Hz, 3H, 4′′-
CH3), 1.20 (s, 6H, CH3), 1.30 (m, 2H, 3′′-CH2), 1.32 (s, 6H, CH3),
1.50 (m, 2H, 2′′-CH2), 1.70 (s, 4H, 6′,7′-CH2), 2.66 (t, J ) 7.8
Hz, 2H, 1′′-CH2), 3.96 (s, 3H, OCH3), 7.20 and 7.22 (2 s, 2H,
1′,4′-NapH), 7.86 (dd, J ) 2.0, 6.6 Hz, 2H, 3,5-ArH), 8.11 ppm
(dd, J ) 1.9, 6.6 Hz, 2H, 2,6-ArH); MALDI-FTMS (HRMS)
calcd C27H34NaO3 (MNa+) 429.2400, found 429.2412.
petitive binding was not possible. These results suggest
that ligand alignment in addition to steric clashing must
be considered when conducting docking studies to
retinoid receptors. Because a protein in solution exists
in dynamic equilibrium with its environment and
ligand, it is reasonable to assume that equilibration
would favor the lowest-energy conformations of the
receptor-ligand complex. Thus, in the case of the
binding of 3 to RXRR, the antagonist conformation
would predominate.
Meth yl 4-[(3′-n -Bu tyl-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m et h yl-2′-n a p h t h a len yl)(cyclop r op ylid en e)m et h yl]-
ben zoa te (23). To a solution of cyclopropylmethyl(triphenyl)-
phosphonium bromide (0.84 g, 2.2 mmol) dissolved in anhydrous
toluene (2.0 mL) was added 0.5 M potassium bis(trimethylsi-
lyl)amide (2.0 mmol) in toluene (4.0 mL) under argon at room
temperature. Stirring was continued for 15 min before 22 (0.41
g, 1.0 mmol) and tris(2-methoxyethoxyethyl)amine (65 mg, 0.2
mmol) in toluene (1.5 mL) were added. The reaction mixture
was heated in a 100 °C oil bath for 2 h to give a dark
suspension, which was cooled, poured into aqueous NaHCO3,
and extracted (EtOAc). Drying and concentration gave a
colorless oil, which was chromatographed (20-30% CH2Cl2/
hexanes) to give 213 mg of three byproducts, then 173 mg of
23 (40%) as a white gum, followed by 144 mg (approximately
33%) of predominantly 23. TLC (30% CH2Cl2/hexanes) Rf )
Meth od s
Ch em istr y. Ma ter ia ls. [11,12-3H2]9-cis-retinoic acid ([3H]9-
cis-RA, 43 Ci/mmol) was purchased (Amersham), and 1,38
SR11237 (2),8 SR11173 (10),14 SR11345 (11),13 and SR11346
(12)14 were synthesized as we described previously.
Gen er a l. Unless otherwise mentioned, during workup
procedures organic layers were washed with water and
saturated brine, dried (anhydrous Na2SO4), filtered, and
concentrated at reduced pressure. Standard column chroma-
tography employed silica gel (Merck 60), as did flash chroma-
tography (Merck, grade 9385, 230-400 mesh). Experimental
procedures were not optimized and were typically conducted
only once. Melting points were determined in sealed capillaries
using a Mel-Temp II apparatus and are uncorrected. Fourier
transform IR spectra were obtained on powdered samples,
unless otherwise specified, using an FT-IR Mason satellite
1
0.48; IR (CHCl3) 1734, 1663 cm-1; H NMR δ 0.73 (t, J ) 7.2
Hz, 3H, 4′′-CH3), 1.14 (m, 2H, 3′′-CH2), 1.25 (s, 6H, CH3), 1.30
(m, 2H, 2′′-CH2), 1.33 (s, 6H, CH3), 1.62 (m, 4H, CdC(CH2)2),
1.71 (s, 4H, 6′,7′-CH2), 2.25 (t, J ) 7.2 Hz, 2H, 1′′-CH2), 3.91
(s, 3H, OCH3), 7.05 (s, 1H, 4′-NapH), 7.12 (s, 1H, 1′-NapH),
7.48 (d, J ) 8.0 Hz, 2H, 3,5-ArH), 7.95 ppm (d, J ) 8.4 Hz,
2H, 2,6-ArH); MALDI-FTMS (HRMS) calcd C30H39O2 (MH+)
431.2944, found 431.2939.
1
spectrophotometer. H NMR spectra were recorded on a 300
MHz Varian Unity Inova spectrometer, and shift values are
expressed in ppm (δ) relative to Me4Si as the internal
standard. Unless mentioned otherwise, compounds were dis-
4-[(3′-n -Bu tyl-5′,6′,7′,8′-tetr ah ydr o-5′,5′,8′,8′-tetr am eth yl-
2′-n a p h th a len yl)(cyclop r op ylid en e)m eth yl]ben zoic Acid
(3). Ester 23 (144 mg, 0.32 mmol) in EtOH (3.0 mL) and 20%
KOH in water (1.0 mL) was heated at 80-90 °C for 2.0 h under
argon, cooled to room temperature, and concentrated. The
residue was acidified (1 N H2SO4), washed repeatedly (water),
dried, and diluted (EtOAc/CH2Cl2) to give a cloudy suspension,
which was eluted through a silica gel pad (1.5 cm × 20 cm)
using 5% MeOH/CH2Cl2 (150 mL), then 0.25% HOAc/5%
MeOH/CH2Cl2 to give 142 mg (100%) of 3 as a pale-yellow
solid: mp 207-209 °C; TLC (50% EtOAc/hexane) Rf ) 0.72;
2
solved in HCCl3. MALDI-FTMS high-resolution mass spectra
were run on an IonSpec Ultima instrument at The Scripps
Research Institute (La J olla, CA). Electrospray mass spec-
trometry was performed on an ABI EPI-3000 instrument.
6-n -Bu tyl-1,2,3,4-tetr a h yd r o-1,1,4,4-tetr a m eth yln a p h -
th a len e (19). To a stirred solution of 6-bromo-1,2,3,4-tetrahy-
dro-1,1,4,4-tetramethylnaphthalene13,39 (18) (1.34 g, 5.0 mmol)
in THF (5 mL) under argon and cooled in a water bath was
added a solution (3.0 mL) of 2.5 M n-BuLi (25 mmol) in
hexanes dropwise to maintain the temperature between 20-
35 °C. Stirring was continued for 1 h at room temperature.
The reaction mixture was then cooled to -78 °C, and n-C4H9-
Br (0.685 g, 5.0 mmol) in THF (2.0 mL) was added dropwise.
This mixture was stirred at room temperature for 0.5 h before
quenching with water and dilution with 1 N HCl and hexanes
(100 mL). The organic layer was washed (brine, 3×), dried,
and concentrated to give a pale-yellow liquid; TLC (hexanes)
indicated one product (19, Rf ) 0.66). Chromatography gave
1
IR (CHCl3) 2961, 1608 cm-1; H NMR (C2HCl3/MeOH-2H4) δ
0.73 (t, J ) 7.3 Hz, 3H, 4′′-CH3), 1.15 (m, 2H, 3′′-CH2), 1.26 (s,
6H, CH3), 1.30 (m, 2H, 2′′-CH2), 1.33 (s, 6H, CH3), 1.63 (m,
4H, CdC(CH2)2), 1.71 (s, 4H, 6′,7′-CH2), 2.25 (t, J ) 7.0 Hz,
2H, 1′′-CH2), 7.05 (s, 1H, 4′-NapH), 7.13 (s, 1H, 1′-NapH), 7.52
(d, J ) 8.5 Hz, 2H, 3,5-ArH), 8.02 ppm (d, J ) 8.5 Hz, 2H,
2,6-ArH); MALDI-FTMS (HRMS) calcd C29H36O2 (M+) 416.2715,
found 416.2715.
4-[1-(3′-n -Bu t yl-5′,6′,7′,8′-t e t r a h yd r o-5′,5′,8′,8′-t e t r a -
m eth yl-2′-n a p h th a len yl)eth en yl]ben zoic Acid (9). To a
solution of methyl(triphenyl)phosphonium bromide (0.79 g, 2.2
mmol) dissolved in anhydrous toluene (2.0 mL) was added 0.5
M potassium bis(trimethylsilyl)amide (2.0 mmol) in toluene
(4.0 mL) under argon at room temperature. Stirring was
continued for 15 min before 22 (0.41 g, 1.0 mmol) and tris(2-
methoxyethoxyethyl)amine (65 mg, 0.2 mmol) in toluene (1.5
mL) were added. The reaction mixture was heated in a 100
°C oil bath for 2 h, then worked up and chromatographed as
in the synthesis of ester 23 to give 148 mg (36%) of the ester
of 9 as white crystals. TLC (30% CH2Cl2/hexanes) Rf ) 0.42;
1H NMR δ 0.76 (t, J ) 7.3 Hz, 3H, 4′′-CH3), 1.17 (m, 2H, 3′′-
CH2), 1.28 (s, 6H, CH3), 1.31 (m, 2H, 2′′-CH2), 1.32 (s, 6H, CH3),
1.71 (s, 4H, 6′,7′-CH2), 2.25 (t, J ) 8.0 Hz, 2H, 1′′-CH2), 3.91
(s, 3H, OCH3), 5.31 (d, J ) 1.4 Hz, 1H, CdCH), 5.81 (d, J )
1.4 Hz, 1H, CdCH), 7.09 and 7.10 (2 s, 2H, 1′,4′-NapH), 7.35
(dd, J ) 1.5, 8.2 Hz, 2H, 3,5-ArH), 7.95 ppm (dd, J ) 1.5, 8.1
Hz, 2H, 2,6-ArH).
1.01 g (83%) of 19 as a colorless liquid. IR (CHCl3) 1466 cm-1
;
1H NMR δ 0.94 (t, J ) 7.2 Hz, 3H, 4′-CH3), 1.28 (s, 12H, CH3),
1.38 (m, 2H, 3′-CH2), 1.58 (m, 2H, 2′-CH2), 1.67 (s, 4H, 2,3-
CH2), 2.55 (d, J ) 7.8 Hz, 2H, 1′-CH2), 6.95 (d, J ) 7.8 Hz,
1H, 7-ArH), 7.09 (s, 1H, 5-ArH), 7.20 ppm (d, J ) 7.8 Hz, 1H,
8-ArH).
Meth yl 4-(3′-n -Bu tyl-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m eth yl-2′-n a p h th a len ylca r bon yl)ben zoa te (22). To a
suspension of 630 mg (3.5 mmol) of 4-carbomethoxybenzoic
acid (20) in benzene (15 mL) was added oxalyl chloride (2.0
mL, 23 mmol). The mixture was heated at reflux for 2 h, cooled,
and concentrated to give 4-carbomethoxybenzoyl chloride (21)
as a white powder, which was used without further purification
as follows. The powder was dissolved in CH2Cl2 (20 mL), and
19 (587 mg, 2.4 mmol) was added. This solution was cooled to
0 °C in an ice bath before AlCl3 (1.06 g, 8 mmol) was added
over a period of 15 min. After being stirred for 15 min at 0 °C,
the mixture was heated at reflux for 0.5 h, poured into ice/
water, and extracted with CH2Cl2 (100 mL). The combined
organic layers were washed with water and brine, dried, and
concentrated to give a yellow solid. Chromatography (2%
EtOAc/hexane) afforded 820 mg (85%) of 22 as a white
powder: mp 100-102 °C; TLC (5% EtOAc/hexane) Rf ) 0.54;
The methyl ester of 9 (47 mg, 0.116 mmol) in EtOH (1.5
mL) and 20% aqueous KOH (0.5 mL) was heated at 80-90 °C
for 2.0 h under argon, then cooled, concentrated, and diluted
with 1.0 N H2SO4 to give a white solid that was thoroughly
washed with water, then 10% EtOAc/hexanes. The resultant

4368 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Cavasotto et al.
solid was dissolved in 5% MeOH/CHCl3, filtered, and concen-
trated to give after drying 42 mg (93%) of 9 as a white solid:
mp 260-262 °C; TLC (50% EtOAc/hexanes) Rf ) 0.70; IR
(CHCl3) 3416, 1663 cm-1; 1H NMR (C2HCl3/MeOH-2H4) δ 0.71
(t, J ) 7.2 Hz, 3H, 4′′-CH3), 1.12 (q, J ) 7.7 Hz, 2H, 3′′-CH2),
1.23 (s, 6H, CH3), 1.27 (s, 6H, CH3), 1.30 (m, 2H, 2′′-CH2), 1.66
(s, 4H, 6′,7′-CH2), 2.22 (t, J ) 7.5 Hz, 2H, 1′′-CH2), 5.21 (d, J
) 1.4 Hz, 1H, CdCH), 5.72 (d, J ) 1.4 Hz, 1H, CdCH), 7.05
(s, 2H, 1′,4′-NapH), 7.25 (d, J ) 7.2 Hz, 2H, 3,5-ArH), 7.86
ppm (d, J ) 8.7 Hz, 2H, 2,6-ArH); MALDI-FTMS (HRMS) calcd
1H, 4′-NapH), 7.43 (s, 1H, 1′-NapH), 7.81 (dd, J ) 2.1, 6.6 Hz,
2H, 3,5-ArH), 8.07 ppm (dd, J ) 1.8, 8.1 Hz, 2H, 2,6-ArH);
MALDI-FTMS (HRMS) calcd C27H35O4 (MH+) 423.2530, found
423.2512.
Meth yl 2-(3′-P r op oxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m eth yl-2′-n a p h th a len ylca r bon yl)-5-p yr id in eca r boxy-
la te (31). White powder; mp 135-137 °C; TLC (10% EtOAc/
hexane) Rf ) 0.28; IR (CHCl3) 1728 cm-1; 1H NMR δ 0.59 (t, J
) 7.5 Hz, 3H, 3′′-CH3), 1.26 (m, 2H, 2′′-CH2), 1.30 (s, 12H, CH3),
1.69 (s, 4H, 6′,7′-H), 3.72 (t, J ) 6.6 Hz, 2H, 1′′-CH2), 3.98 (s,
3H, OCH3), 6.79 (s, 1H, 4′-NapH), 7.66 (s, 1H, 1′-NapH), 7.77
(d, J ) 8.1 Hz, 1H, 3-PyH), 8.42 (dd, J ) 2.4, 7.8 Hz, 1H,
4-PyH), 9.21 ppm (d, J ) 2.1 Hz, 1H, 6-PyH); MALDI-FTMS
(HRMS) calcd C25H32NO4 (MH+) 410.2326, found 410.2317.
Meth yl 2-(3′-Bu toxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m eth yl-2′-n a p h th a len ylca r bon yl)-5-p yr id in eca r boxy-
la te (32). White powder; mp 115-117 °C; TLC (10% EtOAc/
hexane) Rf ) 0.28; IR (CHCl3) 1733 cm-1; 1H NMR δ 0.70 (t, J
) 7.2 Hz, 3H, 4′′-CH3), 0.93 (m, 2H, 3′′-CH2), 1.26 (m, 2H, 2′′-
CH2), 1.30 (s, 12H, CH3), 1.69 (s, 4H, 6′,7′-H), 3.75 (t, J ) 6.3
Hz, 2H, 1′′-CH2), 3.98 (s, 3H, OCH3), 6.78 (s, 1H, 4′-NapH),
7.66 (s, 1H, 1′-NapH), 7.86 (d, J ) 8.1 Hz, 1H, 3-PyH), 8.43
(dd, J ) 1.8, 8.4 Hz, 1H, 4-PyH), 9.21 ppm (d, J ) 1.8 Hz, 1H,
C
27H35O2 (MH+) 391.2631, found 391.2616.
Tetr a h yd r on a p h th a len e 24, P yr id in eca r boxyla te 25,
a n d Ben zoa te 27. 5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-
naphthol (24),22 5-carbomethoxypyridine-2-carboxylic acid (25),40
and methyl 4-(5′,6′,7′,8′-tetrahydro-3′-hydroxy-5′,5′,8′,8′-tet-
ramethyl-2′-naphthalenylcarbonyl)benzoate (27)17 were syn-
1
thesized according to the literature and had H NMR spectra
identical to those reported.
Meth yl 2-(3′-Hyd r oxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m eth yl-2′-n a p h th a len ylca r bon yl)p yr id in e-5-ca r boxy-
la te (30). To a suspension of 5-carbomethoxypyridine-2-
carboxylic acid (25) (543 mg, 3.0 mmol) in benzene (5 mL) was
added oxalyl chloride (1.0 mL, 11.5 mmol) and DMF (2 drops).
This mixture was heated at reflux for 2 h, cooled, and
concentrated to give 5-carbomethoxypyridine-2-carbonyl chlo-
ride (26) as a white powder, which was used without further
purification in the next step.
6-PyH); MALDI-FTMS (HRMS) calcd
424.2482, found 424.2461.
C
26H34NO4 (MH+)
Gen er a l P r oced u r e for In tr od u cin g th e (Cyclop r op yli-
d en e)m eth yl Gr ou p in to 28, 29, 31, a n d 32. To a solution
of cyclopropylmethyl(triphenyl)phosphonium bromide (192 mg,
0.5 mmol) dissolved in anhydrous toluene (1.0 mL) was added
0.5 M potassium bis(trimethylsilyl)amide (0.5 mmol) in toluene
(1.0 mL) under argon at room temperature. Stirring was
continued for 1 h before diaryl ketone 28, 29, 31, or 32 (0.2
mmol) and tris(2-methoxyethoxyethyl)amine (13 mg, 0.4 mmol)
in toluene (0.5 mL) were added. The reaction mixture was
stirred at room temperature for 1 h, then heated in a 100 °C
oil bath for 2 h to give a dark suspension, which after cooling
was poured into aqueous NaHCO3 and extracted (EtOAc).
Drying and concentration gave an oil, which was chromato-
graphed (20-30% CH2Cl2/hexanes) to give the methyl ester
33 (87%), 34 (89%), 35 (62%), or 36 (67%), respectively.
Meth yl 4-[(3′-n -P r op oxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-
tetr a m eth yl-2′-n a p h th a len yl)(cyclop r op ylid en e)m eth yl]-
ben zoa te (33). White gum; TLC (4% EtOAc/hexane) Rf ) 0.57;
IR (CHCl3) 1723 cm-1; 1H NMR δ 0.60 (t, J ) 7.5 Hz, 3H, 3′′-
CH3), 1.26 (s, 6H, CH3), 1.30 (m, 4H, 2′′-CH2, cyclopropyl H),
1.31 (s, 6H, CH3), 1.54 (t, J ) 7.8 Hz, 2H, cyclopropyl H), 1.70
(s, 4H, 6′,7′-H), 3.70 (t, J ) 6.3 Hz, 2H, 1′′-CH2), 3.90 (s, 3H,
OCH3), 6.77 (s, 1H, 4′-NapH), 7.19 (s, 1H, 1′-NapH), 7.50 (d, J
) 8.4 Hz, 2H, 3,5-ArH), 7.94 ppm (d, J ) 8.4 Hz, 2H, 2,6-ArH).
Meth yl 4-[(3′-n -Bu toxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-
tetr a m eth yl-2′-n a p h th a len yl)(cyclop r op ylid en e)m eth yl]-
ben zoa te (34). White gum; TLC (4% EtOAc/hexane) Rf ) 0.58;
IR (CHCl3) 1725 cm-1; 1H NMR δ 0.70 (t, J ) 7.5 Hz, 3H, 4′′-
CH3), 0.97 (m, 2H, 3′′-CH2), 1.26 (s, 6H, CH3), 1.28 (m, 4H,
2′′-CH2, cyclopropyl H), 1.31 (s, 6H, CH3), 1.57 (t, J ) 7.8 Hz,
2H, cyclopropyl H), 1.70 (s, 4H, 6′,7′-H), 3.73 (t, J ) 6.3 Hz,
2H, 1′′-CH2), 3.90 (s, 3H, OCH3), 6.76 (s, 1H, 4′-NapH), 7.20
(s, 1H, 1′-NapH), 7.48 (d, J ) 8.4 Hz, 2H, 3,5-ArH), 7.93 ppm
(d, J ) 8.7 Hz, 2H, 2,6-ArH).
Meth yl 2-[(3′-n -P r op oxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-
tetr a m eth yl-2′-n a p h th a len yl)(cyclop r op ylid en e)m eth yl]-
5-p yr id in eca r boxyla te (35). White gum; TLC (10% EtOAc/
hexane) Rf ) 0.41; IR (CHCl3) 1723 cm-1; 1H NMR δ 0.59 (t, J
) 7.5 Hz, 3H, 3′′-CH3), 1.26 (m, 2H, 2′′-CH2), 1.28 (s, 6H, CH3),
1.30 (s, 6H, CH3), 1.40 (m, 2H, cyclopropyl H), 1.61 (m, 2H,
cyclopropyl H), 1.69 (s, 4H, 6′,7′-H), 3.68 (t, J ) 6.6 Hz, 2H,
1′′-CH2), 3.94 (s, 3H, OCH3), 6.75 (s, 1H, 4′-NapH), 7.34 (s,
1H, 1′-NapH), 7.58 (d, J ) 8.4 Hz, 1H, 3-PyH), 8.20 (dd, J )
2.4, 8.1 Hz, 1H, 4-PyH), 9.16 ppm (d, J ) 2.1 Hz, 1H, 6-PyH).
Meth yl 2-[(3′-n -Bu toxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-
tetr a m eth yl-2′-n a p h th a len yl)(cyclop r op ylid en e)m eth yl]-
5-p yr id in eca r boxyla te (36). White gum; TLC (10% EtOAc/
hexane) Rf ) 0.42; IR (CHCl3) 1726 cm-1; 1H NMR δ 0.59 (t, J
) 7.5 Hz, 3H, 4′′-CH3), 0.94 (m, 2H, 3′′-CH2), 1.20 (m, 2H, 2′′-
The acyl chloride 26 dissolved in CH2Cl2 (20 mL) was added
to a mixture of 24 (408 mg, 2.0 mmol) and AlCl3 (1.33 g, 10
mmol) with cooling in an ice bath. After being stirred for 15
min at 0 °C, the reaction mixture was heated at reflux for 1 h.
An additional portion of AlCl3 (399 mg, 3.0 mmol) was added,
and heating at reflux was continued for 0.5 h. The mixture
was then cooled to room temperature, poured into ice/water,
and extracted with CH2Cl2 (100 mL). The organic layers were
washed (water and brine), dried, and concentrated to give a
yellow solid, which on chromatography (5% EtOAc/hexane)
afforded 380 mg (52%) of 30 as a golden powder: mp 162-
164 °C; TLC (10% EtOAc/hexane) Rf ) 0.32; IR (CHCl3) 1730
1
cm-1; H NMR δ 1.16 (s, 6H, CH3), 1.31 (s, 6H, CH3), 1.68 (s,
4H, 6′,7′-H), 4.02 (s, 3H, OCH3), 6.99 (s, 1H, 4′-NapH), 7.97
(d, J ) 8.1 Hz, 1H, 3-PyH), 8.00 (s, 1H, 1′-NapH), 8.51 (dd, J
) 2.1, 8.1 Hz, 1H, 4-PyH), 9.32 ppm (d, J ) 1.8 Hz, 1H, 6-PyH).
ESI-TOF (HRMS) calcd C22H26NO4 (MH+) 368.1862, found
368.1851.
Gen er a l Meth od for th e Syn th esis of th e n -P r op yl a n d
n -Bu tyl Eth er s of Tetr a h yd r on a p h th ols 27 a n d 30. To a
solution of 27 or 30 (0.5 mmol) and n-propyl or n-butyl bromide
(1.0 mmol) in acetone (10 mL) was added K2CO3 (4.0 mmol).
The resulting suspension was heated at reflux for 20-24 h,
at which time TLC showed that 27 or 30 had disappeared,
then concentrated, and diluted with CH2Cl2 and water (20 mL
each). The organic layer was washed (brine), dried, and
concentrated. Chromatography (5% EtOAc/hexane) gave the
n-propyl ether 28 (92%) or n-butyl ether 29 (93%) from 27 or
the n-propyl ether 31 (84%) or n-butyl ether 32 (82%) from
30.
Meth yl 4-(3′-n -P r op oxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-
tetr am eth yl-2′-n aph th alen ylcar bon yl)ben zoate (28). White
powder; mp 117-119 °C; TLC (4% EtOAc/hexane) Rf ) 0.42;
IR (CHCl3) 1728 cm-1; 1H NMR δ 0.64 (t, J ) 7.5 Hz, 3H, 3′′-
CH3), 1.27 (s, 6H, CH3), 1.32 (s, 6H, CH3), 1.39 (m, 2H, 2′′-
CH2), 1.71 (s, 4H, 6′,7′-H), 3.78 (t, J ) 6.2 Hz, 2H, 1′′-CH2),
3.95 (s, 3H, OCH3), 6.82 (s, 1H, 4′-NapH), 7.43 (s, 1H,
1′-NapH), 7.82 (d, J ) 8.1 Hz, 2H, 3,5-ArH), 8.07 ppm (d, J )
8.1 Hz, 2H, 2,6-ArH); MALDI-FTMS (HRMS) calcd C26H33O4
(MH+) 409.2373, found 409.2359.
Meth yl 4-(3′-n -Bu toxy-5′,6′,7′,8′-tetr a h yd r o-5′,5′,8′,8′-tet-
r a m eth yl-2′-n a p h th a len ylca r bon yl)ben zoa te (29). White
powder; mp 96-97 °C; TLC (4% EtOAc/hexane) Rf ) 0.43; IR
(CHCl3) 1728 cm-1 1H NMR δ 0.64 (t, J ) 7.5 Hz, 3H, 4′′-
;
CH3), 1.00 (m, 2H, 3′′-CH2), 1.27 (s, 6H, CH3), 1.31 (m, 2H,
2′′-CH2), 1.32 (s, 6H, CH3), 1.70 (d, J ) 1.2 Hz, 4H, 6′,7′-H),
3.81 (t, J ) 6.6 Hz, 2H, 1′′-CH2), 3.95 (s, 3H, OCH3), 6.81 (s,

Retinoid X Receptor
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4369
CH2), 1.28 (s, 6H, CH3), 1.30 (s, 6H, CH3), 1.41 (m, 2H,
cyclopropyl H), 1.57 (m, 2H, cyclopropyl H), 1.69 (s, 4H, 6′,7′-
H), 3.68 (t, J ) 6.6 Hz, 2H, 1′′-CH2), 3.94 (s, 3H, OCH3), 6.74
(s, 1H, 4′-NapH), 7.35 (s, 1H, 1′-NapH), 7.57 (d, J ) 8.4 Hz,
1H, 3-PyH), 8.21 (dd, J ) 2.1, 8.4 Hz, 1H, 4-PyH), 9.15 ppm
(d, J ) 2.4 Hz, 1H, 6-PyH).
Gen er a l P r oced u r e for th e Hyd r olysis of Meth yl
Ester s 33-36. Each ester (0.1 mmol) in MeOH (2.0 mL) and
10% aqueous NaOH (0.5 mL) was heated at 80-90 °C for 2.0
h under argon, cooled to room temperature, then diluted with
EtOAc or CH2Cl2 (20 mL). The mixture was acidified (0.5 N
HCl) and washed repeatedly (water and brine), then dried and
concentrated to give the benzoic acid 4 (99%) or 5 (94%) or
the pyridinecarboxylic acid 6 (89%) or 7 (88%).
resulting structure in the internal coordinate space of the LBP
that was within a 4.0 Å vicinity using the ICM method.36,41,42
One water molecule in the vicinity of 9-cis-oleic acid (37) was
kept in the simulations. The agonist-bound hRARγ LBD
conformation was based on the X-ray structure reported for
the trans-RA (15) bound complex3 (PDB entry 2LBD, 2.0 Å
resolution). Two water molecules in the vicinity of the native
ligand carboxylate were kept in these simulations. As imple-
mented in the ICM program, the PDB structures were adjusted
by adding hydrogens and missing heavy atoms, assigning
partial charges, and then energy-minimizing.
En er gy Eva lu a tion a n d Op tim iza tion . According to the
ICM method, the molecular system was described using
internal coordinates as variables. Energy calculations were
based on the ECEPP/343 force field with a distance-dependent
dielectric constant. The biased probability Monte Carlo41,44
(BPMC) was used to optimize global energy by iterative cycles
of a random conformational change of the free variables
according to a predefined continuous probability distribu-
tion,41,44 the local energy minimization of analytical dif-
ferentiable terms, a complete energy evaluation including
nondifferentiable terms such us entropy41 and solvation en-
ergy, and the acceptance or rejection of the total energy on
the basis of the Metropolis criterion.45 The nonpolar contribu-
tion to the solvation energy in the implicit solvation model used
was assumed to be proportional to the solvent-accessible
surface, and the electrostatic contribution to solvation was
determined from the Poisson equation using the boundary
element algorithm.46
4-[(3′-n -P r op oxy-5′,6′,7′,8′-t et r a h yd r o-5′,5′,8′,8′-t et r a -
m e t h yl-2′-n a p h t h a le n yl)(cyclop r op ylid e n e )m e t h yl]-
ben zoic Acid (4). White powder; mp 226-228 °C; TLC (20%
EtOAc/hexane) Rf ) 0.34; IR (CHCl3) 3405, 1728, 1689 cm-1
;
1H NMR δ 0.60 (t, J ) 7.5 Hz, 3H, 3′′-CH3), 1.26 (s, 6H, CH3),
1.29 (m, 4H, 2′′-CH2, cyclopropyl H), 1.32 (s, 6H, CH3), 1.58 (t,
J ) 7.8 Hz, 2H, cyclopropyl H), 1.70 (s, 4H, 6′,7′-H), 3.71 (t, J
) 6.3 Hz, 2H, 1′′-CH2), 6.77 (s, 1H, 4′-NapH), 7.20 (s, 1H, 1′-
NapH), 7.52 (d, J ) 8.7 Hz, 2H, 3,5-ArH), 8.00 ppm (d, J )
8.1 Hz, 2H, 2,6-ArH). ESI-TOF (HRMS) calcd C28H35O3 (MH+)
419.2581, found 419.2572.
4-[1-(3′-n -Bu t oxy-5′,6′,7′,8′-t et r a h yd r o-5′,5′,8′,8′-t et r a -
m e t h yl-2′-n a p h t h a le n yl)(cyclop r op ylid e n e )m e t h yl]-
ben zoic Acid (5). White powder; mp 220-222 °C; TLC (20%
EtOAc/hexane) Rf ) 0.34; IR (CHCl3) 3400, 1685 cm-1 1H
;
NMR δ 0.60 (t, J ) 7.5 Hz, 3H, 4′′-CH3), 0.97 (m, 2H, 3′′-CH2),
1.27 (s, 6H, CH3), 1.29 (m, 4H, 2′′-CH2, cyclopropyl H), 1.32
(s, 6H, CH3), 1.58 (t, J ) 9.0 Hz, 2H, cyclopropyl H), 1.70 (s,
4H, 6′,7′-H), 3.74 (t, J ) 6.0 Hz, 2H, 1′′-CH2), 6.77 (s, 1H, 4′-
NapH), 7.21 (s, 1H, 1′-NapH), 7.52 (d, J ) 8.1 Hz, 2H, 3,5-
ArH), 8.00 ppm (d, J ) 7.8 Hz, 2H, 2,6-ArH). ESI-TOF (HRMS)
calcd C29H37O3 (MH+) 433.2737, found 433.2724.
2-[(3′-n -P r op oxy-5′,6′,7′,8′-t et r a h yd r o-5′,5′,8′,8′-t et r a -
m e t h yl-2′-n a p h t h a le n yl)(cyclop r op ylid e n e )m e t h yl]-
5-p yr id in eca r boxylic Acid (6). White powder; mp 126-128
°C; TLC (50% EtOAc/hexane) Rf ) 0.25; IR (CHCl3) 3405, 1722
cm-1; 1H NMR δ 0.59 (t, J ) 7.5 Hz, 3H, 3′′-CH3), 1.24 (m, 2H,
2′′-CH2), 1.28 (s, 6H, CH3), 1.30 (s, 6H, CH3), 1.40 (m, 2H,
cyclopropyl H), 1.60 (m, 2H, cyclopropyl H), 1.69 (s, 4H, 6′,7′-
H), 3.68 (t, J ) 6.6 Hz, 2H, 1′′-CH2), 6.75 (s, 1H, 4′-NapH),
7.34 (s, 1H, 1′-NapH), 7.59 (d, J ) 8.4 Hz, 1H, 3-PyH), 8.25
(dd, J ) 1.8, 8.7 Hz, 1H, 4-PyH), 9.22 ppm (s, 1H, 6-PyH);
MALDI-FTMS (HRMS) calcd C27H34NO3 (MH+) 420.2533,
found 420.2528.
Liga n d -Recep tor Dock in g. In the flexible-ligand-rigid-
receptor docking, the receptor was represented by six potential
energy maps, namely, electrostatic, hydrogen bond, hydropho-
bic, and three van der Waals. The flexible ligand in the
receptor field was subjected to global optimization47 so that
both the intramolecular ligand energy and the ligand-receptor
interaction energy were optimized during the calculation. Each
docked compound was assigned a score according to its fit in
the LBP that also accounted for desolvation and hydrophobic
effects and entropy loss, which occurred on docking.24,47,48
Further structural refinement through flexible-ligand-flex-
ible-receptor docking was achieved through cycles of global
energy stochastic optimization47,49 of the flexible ligand and
flexible side chains within 6.0 Å of the ligand, followed by
energy minimization of the complex, including backbone
relaxations, which were important for relieving any residual
van der Waals clashes between ligand and receptor. For the
energy minimization of the complex, heavy atoms were
tethered using quadratic restraints,42 and the weight of the
tether function was decreased after each minimization as
follows: 50, 20, 10, 5, 1, and 0 kcal/mol.
2-[(3′-n -Bu t oxy-5′,6′,7′,8′-t e t r a h yd r o-5′,5′,8′,8′-t e t r a -
m e t h yl-2′-n a p h t h a le n yl)(cyclop r op ylid e n yl)m e t h yl]-
5-p yr id in eca r boxylic Acid (7). White powder; mp 120-122
°C; TLC (50% EtOAc/hexane) Rf ) 0.25; IR (CHCl3) 3400, 1728
cm-1; 1H NMR δ 0.59 (t, J ) 7.5 Hz, 3H, 4′′-CH3), 0.92 (m, 2H,
3′′-CH2), 1.21 (m, 2H, 2′′-CH2), 1.28 (s, 6H, CH3), 1.30 (s, 6H,
CH3), 1.40 (m, 2H, cyclopropyl H), 1.60 (m, 2H, cyclopropyl
H), 1.68 (s, 4H, 6′,7′-H), 3.72 (t, J ) 6.0 Hz, 2H, 1′′-CH2), 6.74
(s, 1H, 4′-NapH), 7.35 (s, 1H, 1′-NapH), 7.61 (d, J ) 8.1 Hz,
1H, 3-PyH), 8.27 (d, J ) 7.8 Hz, 1H, 4-PyH), 9.23 ppm (s, 1H,
Bin d in g-En er gy Ca lcu la tion s. The binding free energy
calculation implemented in ICM50 included an electrostatic
term for Coulombic interactions and partial charge desolvation,
a
hydrophobic term for variation of the water/nonwater
interface upon ligand binding, and an entropy term for loss of
torsional entropy upon binding. A constant term for loss of
translational/rotational entropy and change in entropy from
variations in the concentration of free molecules was also
included. No van der Waals term was used because it was
considered too sensitive to small geometrical errors, whereas
the average van der Waals interaction was included in the
hydrophobic term because it was proportional to the surface
interaction. No clashes between the ligand and the LBP were
assumed in these calculations.
Biology. RXRr LBD Exp r ession . A reported procedure35
was modified. The cDNA sequence coding for the hRXRR LBD
(amino acids 223-462) was amplified using the polymerase
chain reaction (PCR) on forward 5′-AGTCCATATGACCAG-
CAGCGCCAACGAG-3′ and reverse 5′-GCCGCTCGAGCT-
AAGTCATTTGGTGCGG-3′ primers. The PCR product was
purified (Gene Clean II kit, Q-Biogene) and then cloned into
the vector pET15b (Novagen) using the NdeI and XhoI (New
England Biolabs) restriction sites. After sequence conforma-
tion, the vector bearing the sequence for an N-terminus
6-PyH); MALDI-FTMS (HRMS) cald
434.2690, found 434.2670.
C
28H36NO3 (MH+)
Recep tor Mod el Con str u ction . The agonist-bound hu-
man (h) RXRR LBD model was based on the X-ray crystal
structure of the LBD bound to 1 at 2.1 Å resolution5 (PDB
entry 1FM6, chain A), which we selected on the basis of its
high resolution and structural integrity. Because no water
molecules were observed in the vicinity of the ligand in PDB
1FM6, the positions of water molecules in the vicinity of 1 were
taken from those found in the LBP of the RXRR LBD-9-cis-
RA complex in the PDB entry 1FBY structure.35 The antagonist-
bound hRXRR LBD model was derived from the crystal
structure of the mouse RXRR LBD-F318A mutant bound to
9-cis-oleic acid (37) at 2.5 Å resolution2 (PDB entry 1DKF,
chain A) by virtually mutating the alanine at 318 back to the
wild-type phenylalanine and then energy-minimizing the

4370 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Cavasotto et al.
hexahistidine tag was transformed into E. coli BL21 (DE3)
(Novagen) for protein overexpression, for which a representa-
tive procedure is described. Cells were grown in TB medium
containing bactotryptone, yeast extract, and glycerol in potas-
sium phosphate (KP) buffer at 37 °C to an OD600 of 0.4-0.6,
induced for 1-2 h with 0.25 mM 1-thio-â-D-galactopyranoside
(Invitrogen), and then harvested by centrifugation. The cell
pellet from 833 mL of culture was resuspended in 30 mL of
buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH
7.9), then stored at -80 °C until required. Upon thawing, the
suspension was made 1 mM in 4-(2-aminoethyl)benzenesulfo-
nylfluoride hydrochloride (Calbiochem) and sonicated on ice.
The lysate was centrifuged (39000g for 30 min) to remove
debris and purified on a 5-mL HiTrap Ni(II)-chelating column
(Pharmacia Biotech) by sequentially washing (30 mL of 60 mM
imidazole in 20 mM Tris-HCl, pH 7.9, containing 500 mM
NaCl) and then eluting (30 mL of 100 mM EDTA in the same
buffer). The His tag from the fusion protein was removed by
proteolysis using thrombin. The hRXRR LBD (residues 223-
462 plus the glycine-serine-histidine-methionine (GSHM)
sequence remaining after cleavage of the His tag) was thor-
oughly dialyzed against 20 mM KP buffer, pH 7.9, containing
200 mM NaCl and analyzed by sodium dodecyl sulfate-
polyacrylamide (SDS-PAGE) gel electrophoresis. A typical
yield was 40 mg of RXRR LBD. Electrospray MS (GSHM +
hRXRR LBD residues 223-462) calcd 27235.5, found 27234.8.
The thrombin cleavage step was omitted for the protein used
in competition binding assays.
fetal calf serum (FCS), 100 units/mL of penicillin, and 100 µg/
mL of streptomycin. For transfection assays, cells were seeded
at 1.0 × 105 cells/mL in 24-well plates for 16-24 h before
transfection. Cells were then transfected using the calcium
chloride precipitation method54 with (TREpal)2-tk-CAT or
CRBP-I-tk-CAT (200 µg) alone or together with an RAR
subtype vector (100 µg) or with CRBP-II-tk-CAT (200 µg) alone
or together with RXRR (20 µg). In addition, cells were also
transfected with â-galactosidase (â-gal) expression vector (pCH
110, Amersham Biosciences) and carrier DNA (pBluescript,
Stratagene) to a final concentration of 1000 µg/well. At 20 h
after transfection, the medium was changed to DMEM con-
taining 5% charcoal-stripped FCS, and cells were treated for
24 h with one or more of the retinoids of interest. Chloram-
phenicol acetyl transferase (CAT) activity was expressed
relative to â-galactosidase activity to normalize for transfection
efficiency.
Wester n An a lysis. Cell cultures were harvested and lysed
in lysis buffer (50 mM Tris-HCl, pH 8.0, and 150 mM NaCl,
with 0.1% Triton X-100, 0.25% sodium deoxycholate, 1 mM
ethylenediaminetetraacetic acid, 1 mM phenylmethanesulfonyl
fluoride, 1 µg/mL of aprotinin, 1 µg/mL of leupeptin, and 1 mM
sodium orthovanadate (all from Sigma)). Equivalent protein
extracts from each sample were separated on 8% SDS-PAGE
gels. Protein was quantitated by a total protein assay (Bio-
Rad). Proteins were transferred onto nitrocellulose membranes
(Trans-Blot, Bio-Rad). Nitrocellulose membranes were pre-
blocked with 5% nonfat milk powder in phosphate-buffered
saline (PBS) containing 0.05% Tween 20 detergent for 1 h at
room temperature. Following PBS/Tween washes, preblocked
membranes were incubated with 1 µg/mL equivalent of anti-
rabbit RARâ polyclonal antibody (Santa Cruz, CA). RARâ
proteins were detected by horseradish peroxidase conjugated
secondary antibodies to antirabbit immunoglobulins (Amer-
sham Pharmacia) after a 1 h incubation at room temperature,
and specific bands were visualized by enhanced chemilumi-
nescence (ECL, Amersham Pharmacia). Equivalent loading of
samples was determined by reprobing each nitrocellulose
membrane with a mouse monoclonal antibody recognizing
â-actin (Sigma).
Hexahistidine-tagged mouse RXRR lacking the AB domain
(mRXRR∆AB) for the GST-pulldown experiments was ex-
pressed and purified as previously described.51 Constructs
corresponding to domains D and E (LBD) of human RXRR
fused to GST (GST-hRXRR LBD) and full-length human SRC-
1a in pSG5 were kind gifts from Dr. David Heery (University
of Leicester, England). GST-RXRR LBD was expressed in E.
coli BL21(DE3)pLysS and purified by glutathione affinity
chromatography using standard techniques.
Liga n d Bin d in g. hRXRR LBD was expressed in E. coli and
purified as a polyhistidine-tagged fusion protein for use in
competition binding assays.52 Briefly, His-tagged RXR (1.0 µg)
was incubated in binding buffer (0.15 M KCl in 10 mM Tris-
HCl, pH 7.4, containing 0.5% 3-[(3-cholamidopropyl)dimethyl-
ammonio]-1-propane sulfonate (CHAPS detergent, Roche Di-
agnostics) and 8% glycerol; 300 µL) with 1 nM [11,12-3H2]9-
cis-RA (44 Ci/mmol, NEN) in the absence or presence of
increasing concentrations of nonlabeled 1 or retinoid for 16-
18 h at 4 °C. Next, yttrium silicate copper His-tag beads (500
µg, Amersham Pharmacia Biotech) were added, and incubation
with shaking was continued for 1 h at room temperature. The
His-tagged beads were washed (3 × 1 mL of binding buffer)
to separate receptor-bound from nonbound label, then sus-
pended (500 µL of buffer) and transferred for scintillation
counting (3.5 mL of EcoLume liquid scintillation fluid, ICN;
Beckman Coulter LS 3801 counter). Nonspecific [3H]9-cis-RA-
binding determined in the presence of 1.0 µM nonlabeled 1
was typically less than 10% of the total bound radiolabel.
Experiments were performed in duplicate, and specific binding
was calculated as the average of the percentage of the total
bound cpm remaining (cpm/(total bound cpm) × 100). Com-
petitive binding using purified mRXRR∆AB and [11,12-3H2]9-
cis-RA was conducted essentially as previously described.51 At
1.0 µM, 1 displaced 80% ( 5% of the label from mRXRR∆AB
and 3 displaced 83% ( 5%.
Ack n ow led gm en t. We thank Dr. David Heery
(University of Leicester) for the kind gifts of full-length
SRC-1a and GST-RXRR LBD. These studies were sup-
ported by NIH Grants P01 CA51993 (M.I.D., M.L., and
X.Z.) and P30 ES00210 (M.L.).
Su p p or tin g In for m a tion Ava ila ble: Figure 7, in which
the information given in Tables 1 and 2 has been expanded to
other concentrations of 3 and 9 and to 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Mangelsdorf, D. J .; Umesono, K.; Evans, R. M. The retinoid
receptors. In The Retinoids: Biology, Chemistry, and Medicine;
Sporn, M. B., Roberts, A. B., Goodman, D. S., Eds.; Raven
Press: New York, 1994; pp 319-349.
(2) Bourguet, W.; Vivat, V.; Wurtz, J . M.; Chambon, P.; Gronemeyer,
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