Structure-guided design of N-phenyl tertiary amines as transrepression- selective liver X receptor modulators with anti-inflammatory activity

Article abstract of DOI:10.1021/jm800612u

A cocrystal structure of T1317 (3) bound to hLXRβ was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 (20) as a high-affinity LXRβ li

Full text of DOI:10.1021/jm800612u

5758
J. Med. Chem. 2008, 51, 5758–5765
Structure-Guided Design of N-Phenyl Tertiary Amines as Transrepression-Selective Liver X
Receptor Modulators with Anti-Inflammatory Activity
Esther Y. Chao,† Justin A. Caravella, Mike A. Watson, Nino Campobasso,† Serena Ghisletti,‡ Andrew N. Billin,†
Cristin Galardi,† Ping Wang,† Bryan A. Laffitte, Marie A. Iannone,† Bryan J. Goodwin,† Jason A. Nichols,† Derek J. Parks,†
Eugene Stewart,† Robert W. Wiethe,† Shawn P. Williams,† Angela Smallwood,† Kenneth H. Pearce,† Christopher K. Glass,‡
Timothy M. Willson,† William J. Zuercher,† and Jon L. Collins*,†
GlaxoSmithKline Research and DeVelopment, Research Triangle Park, North Carolina 27709, and Departments of Cellular and Molecular
Medicine and Medicine, School of Medicine, UniVersity of California, La Jolla, San Diego, California 92093
ReceiVed May 22, 2008
A cocrystal structure of T1317 (3) bound to hLXRꢀ was utilized in the design of a series of substituted
N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator
GSK9772 (20) as a high-affinity LXRꢀ ligand (IC50 ) 30 nM) that shows separation of anti-inflammatory
and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the
structurally related analog 19 bound to LXRꢀ reveals regions within the receptor that can affect receptor
modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold
selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of
lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with
improved therapeutic indexes.
Chart 1. Structures of 24(S),25-Epoxycholesterol 1, 2, and 3
Introduction
Liver X receptors (LXR)a R and ꢀ are ligand-activated
transcription factors that belong to the nuclear receptor super-
family of hormone receptors. The identification of oxysterols,
which is exemplified by 24(S),25-epoxycholesterol (1),1,2 as
natural LXR ligands as well as 2 (GW3965)3 and 3 (T1317)4
as synthetic, nonsteroidal LXR agonists facilitated the discovery
of biological signaling pathways that are regulated by LXRs.5-8
The LXRs regulate the expression of key target genes that are
involved in cholesterol metabolism and transport (abca1, abcg1,
cyp7a), glucose metabolism (pepck, g6p), and inflammation (IL-
6, inos, cox2). These effects are manifested in vivo because 2
and 3 show beneficial effects in animal models of antiathero-
sclerosis, diabetes, inflammation, and neurodegenerative dis-
eases. However, the beneficial effects of LXR agonists are
compromised by the LXR-dependent increase in the expression
of genes that control hepatic lipogenesis including srebp1c and
fas. As a direct consequence, the chronic treatment of rodents
with first-generation LXR agonists 2 and 3 leads to the
accumulation of hepatic triglycerides and, with 2, the elevation
of low-density lipoprotein (LDL) particles in primates.4,9 There
is a clear need for LXR modulators that show the beneficial
effects on cholesterol transport and inflammation but do not
induce hepatic lipogenesis.
The crystal structures of 1-3 bound to the LXRs reveal large
plastic ligand-binding pockets (LBPs) that vary in size and shape
depending on the nature of the ligand.10 The cumulative data
from these structural studies suggest that the mechanism of
receptor activation of the different ligands involves a conserved
ligand-guided interaction between His435 on helix 10/11 and a
Trp457 on the activation function-2 (AF-2) helix. This interaction
stabilizes the position of the AF-2 helix against the LBP, which
leads to a transcriptionally active receptor. Three key observa-
tions were noted in the cocrystal structure of 3 bound to LXRꢀ.
First, the acidic bistrifluoromethyl alcohol in 3 was involved in
a strong hydrogen-bond interaction with His435, which suggests
that this interaction is critical for ligand binding and receptor
activation. Second, the sulfonyl moiety was not involved in an
interaction with residues that line the LBP, which suggests that
the replacement of this group with a methylene would not
dramatically reduce the binding affinity. Third, syn and anti
conformations around the tertiary sulfonamide bond were
observed in different unit cells of the cocrystal structure, which
* To whom correspondence should be addressed. Address: Five Moore
Drive, North-1510, Research Triangle Park, NC 27709. Phone: 919-483-
5694. Fax: 919-315-0430. E-mail: Jon.L.Collins@gsk.com.
†
GlaxoSmithKline Research and Development.
Departments of Cellular and Molecular Medicine and Medicine,
‡
University of California.
a Abbreviations: LXR, liver X receptor; abca1/g1, adenosine triphosphate
binding cassette a/g; cyp7a, cytochrome P450 7a hydroxylase; pepck,
phosphoenolpyruvate carboxykinase; g6p, glucose-6-phosphate; IL-6, in-
terleukin-6; inos, inducible nitric oxide synthase; cox2, cyclooxygenase 2;
srebp1c, sterol regulatory element binding protein-1c; fas, fatty acid
synthase; LBP, ligand-binding pocket; LBD, ligand-binding domain; NPTA,
N-phenyl tertiary amine; DMAP, 4-dimethylaminopyridine; Et3N, triethyl-
amine; NaHB(OAc)3, sodium triacetoxyborohydride; LAH, lithium alumi-
num hydride; DMF, N,N-dimethylformamide; AF-2, activation function-2;
SUMO, small ubiquitin-like modifier; SRC-1, steroid receptor coactivator-
1; NCoR, nuclear receptor repressor; ChIP, chromatin immunoprecipitation.
10.1021/jm800612u CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

Transrepression-SelectiVe LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5759
Table 1. LXR Modulator Profile of N-Phenyl Tertiary Aminesa,b
Scheme 1a
binding
LXRR LXRꢀ IL-6 ELISA TG
IC50 IC50 IC50 RE RE
235 1.0
functional
cmpd R1
R2
2
3
30
20
1.0
75
30
100 1.0
1.0
a Reagents: (i) R1C(O)OC(O)R1, Et3N, DMAP, DCM; (ii) LAH, Et2O;
(iii) R2CHO, NaBH(OAc)3, AcOH, DCE; or (iv) R2CH2X, DMF, 140°C,
microwave.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Me Ph
n-Bu Ph
Bn Ph
n-Bu 3-HOCH2CH2-Ph
n-Bu 2-pyridyl
n-Bu 4-pyridyl
n-Bu 2-HO-Ph
n-Bu 3-HO-Ph
n-Bu 4-HO-Ph
n-Bu 2-HO-3-F-Ph
n-Bu 2-HO-5-CF3O-Ph
n-Bu 2-HO-3,5-Cl2-Ph
n-Bu 2-HO-5-Cl-Ph
n-Bu 3-Cl-4-HO-Ph
310
170
315
115
120
90
180
160
955
615
190
890
220
560
170
40
175
15
30
25
NT
25
NT
5
20
20
35
35
NT
60
NT
NT
1.0
NT
1.0
1.0
1.0
1.0
1.0
NT
1.0
NT
>0.5
0.8
>0.5
1.8
1.1
1.1
0.6
0.5
NT
1.9
indicates that this region of the pocket was quite plastic. These
three observations led us to explore modifications of 3 that
would perturb the receptor conformation and potentially lead
to dissociated functional activities.
40
30
740
120
165
290
115
50
45
25
30
30
1.0
Chemistry
1290 <0.5 ia
Commercially available 2-(4-aminophenyl)-1,1,1,3,3,3-hexaflu-
oropropan-2-ol was converted to an amide following treatment
with an acyl anhydride, Et3N, and DMAP (Scheme 1) and was
reduced with LAH to provide an intermediate secondary aniline.
Subjection of the product aniline to NaBH(OAc)3 and AcOH
in the presence of aldehydes gave rise to substituted N-phenyl
tertiary amines (NPTAs) in good overall yield and purity.
Alternatively, the intermediate secondary aniline was alkylated
with benzyl halides in DMF while heating in a microwave. Both
procedures proved to be very efficient for the preparation of
the targeted N-phenyl tertiary amines. Compound 6 was prepared
according to the published procedure.11
55
25
10
15
15
10
15
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.6
<0.2
0.4
<0.1
0.7
0.8
n-Bu 3-Cl-4-HO-5-MeO-Ph 775
n-Bu 3-Cl-4,5-(OMe)2-Ph
n-Bu 3-,5-Cl2-4-HO-Ph
n-Bu 3,5-Cl2-Ph
165
180
195
170
n-Bu 3,5-Cl2-4-MeO-Ph
30
a IC50 data are in nM and are expressed as the mean of g3 replicates
(variation <25%). RE is relative efficacy compared with 2, 3, or both. NT
is not tested. b TG RE data were determined by dose-response testing up to
10 µM test concentration and are expressed relative to 2, 3, or both (g3
replicates).
and lipogenic activities in IL-6 ELISA and triglyceride ac-
cumulation assays, respectively.14 Compounds 4-12 were
profiled in these functional assays to determine whether
substituted NPTAs would show a differential profile relative to
3. Analogs containing an unsubstituted phenyl (compound 5),
pyridine (compounds 8 and 9), or hydroxyethyl ether (7) did
not show a significant separation of anti-inflammatory and
lipogenic activity. In contrast, NPTAs 10 and 11, which
contained a phenol substituent, consistently showed anti-
inflammatory activity that was comparable to that of 3 and only
partial (50-60%) induction of triglyceride accumulation.
In an attempt to explore the SAR of LXR modulation and to
reduce lipogenic activity further, a second set of analogs was
prepared wherein the N-butyl group was maintained and
variations were made to the benzyl group. Screening of the
analogs in competitive binding assays provided several high-
affinity LXRꢀ ligands that contained phenols and phenolic ethers
(compounds 13-20). The preference for o- and m-phenols over
p-phenols for derivatives 10-12 was reversed with more highly
substituted analogs 13-20; p-phenols showed a higher affinity
for the latter. Functional profiling of 13-20 in anti-inflammatory
and triglyceride accumulation assays revealed that LXRꢀ
binding affinity generally correlated with the inhibition of IL-6
secretion but not with lipogenic potential. With the exception
of 13, the majority of these NPTA analogs showed reduced
lipogenic potential relative to that of 3. Of these, 20 dose-
dependently inhibited IL-6 secretion (IC50 ) 15 nM) but did
not induce triglyceride accumulation in a human liver cell line
(Table 1, Figure 1A,B). Interestingly, analogs that lacked a
phenol group (21) or contained a methyl ether (22) showed
increased lipogenic potential compared with 20, which suggests
that the phenol is critical for receptor modulation. To address
whether metabolism could explain the differential profile with
Results and Discussion
The targeting of the classical nuclear receptor ligand-AF-2
helix interaction has proven to be an effective strategy for the
conversion of full agonists to partial agonists and antagonists.12,13
On the basis of this, our initial efforts focused on determining
whether one of the bistrifluoromethyl groups in 3 could be
replaced with hydrophobic groups of different size, shape, and
directionality. Simple replacement of one of the bis-trifluoro-
methyl groups with small substituted or unsubstituted aliphatic,
aryl, or alkyne groups led to only weak partial LXR agonism
(data not shown), which reinforced our hypothesis that the acidic
bistrifluoromethyl alcohol-His435-Trp457 triad would be re-
quired for high-affinity binding to LXR. These results prompted
us to design and synthesize analogs that retain the bistrifluo-
romethyl benzyl alcohol pharmacophore of 3 yet contain a
replacement of the tertiary sulfonamide moiety with substituted
N-phenyl tertiary amines.
The cocrystal structure of 3 bound to LXRꢀ suggested that
the trifluoroethyl group could be replaced with small hydro-
phobic groups. The syn conformer of 3 occupied a hydrophobic
pocket; in contrast, the anti conformer occupied a polar region
within the LBP. An initial set of compounds was prepared from
small alkyl anhydrides and aryl/heteroaryl aldehydes. Screening
in LXRR and LXRꢀ competitive binding assays provided
several potent dual LXRR/ꢀ ligands (Table 1, compounds
4-12). Whereas an N-butyl group was the preferred N-alkyl
substituent, the benzenesulfonyl moiety could be replaced by
various benzyl, substituted-benzyl, and heteroaryl (pyridine,
furan, thiophene) groups, suggesting that this region of the LBP
could accommodate a variety of N-benzyl substitutions. We
previously reported that LXR agonists show anti-inflammatory

5760 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chao et al.
Figure 1. Effects of 3 (2) and 20 (b) on (A) LPS-stimulated IL-6 secretion in THP-1 cells and (B) triglyceride accumulation in HuH7 cells. (C)
Effects of vehicle (open bars) and 20 (1 µM, solid bars) on LPS-induced IL-6 secretion in peritoneal macrophages that were isolated from wild-type
vs LXRR/ꢀ knockout mice. (D) Induction of srebp1c (open bars) and fas (solid bars) mRNA expression in HepG2 cells by 2 (200 nM) alone or
in the presence of 20 (500 nM). Data are expressed as fold expression over vehicle-treated cells. The data are the average of G3 replicates.
20, we determined the percentage of parent compound that
remained in HuH7 cells under conditions that were used for
the triglyceride accumulation assay. After being dosed at 1 µM
and incubated for 24 h, compounds 3 and 20 showed 65 and
40% of parent compound remaining, respectively, which sug-
gests that the unique profiles were not due to differential
metabolism. Therefore, 20 (GSK9772) is a high-affinity LXR
modulator that dissociates anti-inflammatory signaling from
lipogenic signaling.
Compound 20 was characterized in nuclear receptor selectiv-
ity and functional assays to support our hypothesis further that
the dissociated activities were LXR-dependent (Figure 1). With
the exception of full-length human PXR (EC50 ) 250 nM), 20
was 100-fold selective when screened in a panel of nuclear
receptor selectivity assays including AR, GR, PR, MR, ERR/ꢀ,
FXR, and PPARR/γ/δ (data not shown). The dissociated profile
with 20 would not be due to PXR cross reactivity because PXR
agonists do not affect IL-6 secretion or triglyceride accumula-
tion. Importantly, the ability of 20 to repress IL-6 secretion from
peritoneal macrophages of wild-type mice was completely
abolished in macrophages that were derived from LXR-null mice
(Figure 1C). Moreover, compound 20 effectively antagonized
the LXR agonist-induced expression of the lipogenic genes
srebp1c and fas in HuH7 cells (Figure 1D). The cumulative
data from these experiments confirm that the dissociation of
anti-inflammatory signaling from lipogenic signaling by 20 is
indeed LXR-dependent.
observed when the compounds were screened in cell-free
fluorescent resonance energy transfer (FRET) assays that used
peptide fragments from the steroid receptor coactivator-1 (SRC-
1) and nuclear corepressor (NCoR).3 Moreover, the screening
of 3, 19, and 20 in cell-free multiplexed peptide interaction
assays that used a panel of 90 coactivator and corepressor
peptide fragments (21- to 26-mer) did not differentiate the three
compounds (Figure 2A).15,16 In contrast, significant differences
between 3 and 20 were observed upon the screening in cellular
mammalian two-hybrid (M2H) assays that used longer peptide
fragments of SRC-1 (190 amino acids) and NCoR (61 amino
acids) (Figure 2B). Whereas 3 effectively recruited SRC-1 and
effectively displaced NCoR, LXR modulator 20 was signifi-
cantly less potent at recruiting SRC-1 and did not displace NCoR
from the LXR ligand-binding domain (LBD), which suggests
that 20 binds and stabilizes the LXR in an NCoR-associated,
repressed state.
In an attempt to explore the structural basis for this finding,
we solved the cocrystal structure of 19 bound to mLXRR to
2.35 Å resolution (Figure 3). The binding orientation was very
similar to that of the anti conformer of the LXR agonist 3 with
the bistrifluoromethyl alcohol directed toward AF-2. Consistent
with cell-free cofactor recruitment data, the positions of the AF-2
helix in both cocrystal structures were nearly identical. A
difference in the structure of 19 was the presence of a hydrogen
bond between the 4-methoxy group of 19 and Ser262 of LXR.
It is interesting that relatively minor changes in this region of
the ligand can significantly alter the effect on triglyceride
accumulation. (See the results for ligands 17-22 in Table 1.)
For example, compound 20 contains a 4-phenol that also would
be expected to hydrogen bond with Ser262. In contrast, com-
pound 21, which lacks a 4-phenol, shows much higher triglyc-
eride accumulation than does 20. The substituents in this region
do, however, appear to affect the conformation of the loop
between helices 5 and 6. Whereas no firm crystal structure-
The design of NPTAs as LXR modulators that are centered
around the bistrifluoromethyl alcohol moiety is a key pharma-
cophore for high-affinity binding to the LXRs. Because of the
proximity of this functional group to the cofactor-AF-2
interface, compounds 3, 19, and 20 were profiled in cell-free
and cellular cofactor-LXRR/ꢀ interaction assays to probe
whether differential cofactor peptide recruitment could explain
the dissociated activities. No significant differences were

Transrepression-SelectiVe LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5761
Figure 2. (A) Profiling of 3, 19, and 20 in cell-free multiplexed peptide
interaction assays by the use of a panel of 90 coactivator and corepressor
peptide fragments that ranged from 21 to 26 amino acids. Data are
expressed as log fluorescence units (peptide + compound/peptide basal)
(n ) 4). Nearly identical profiles were observed for the three LXR
ligands. (B) Screening of 3 and 20 (5 µM) in cellular mammalian two-
hybrid assays that used fragments of SRC-1 (190 amino acids) and
NCoR (61 amino acids) revealed significant differences between 3 and
anti-inflammatory LXR modulator 20.
Figure 4. (A) Transrepression of LPS-induced (black bar) inos
promoter activation in RAW264.7 cells with 2 (1 µM, hatched bar)
and 20 (1 µM, gray bar) in the absence and presence of LXRR and
LXRꢀ. (B) ChIP analysis using antibody against NCoR (vs IgG control)
of LPS-treated primary macrophages in the absence and presence of
20 (1 µM). Immunoprecipitated DNA was analyzed by real-time PCR
using primers that were specific for the inos promoter. (C) LXRꢀ is
SUMOylated by SUMO2 and SUMO3 upon treatment with 2 (1 µM)
and 20 (1 µM) in HeLa cells. Whole cell lysates were immunoblotted
for FLAG tag.
of the repression of gene expression, designated transrepression,
has been poorly understood. Recent literature data has demon-
stated that the LXRs transrepress LPS-induced proinflammatory
gene expression (IL-6, inos) by the ligand-dependent SUMOy-
lation of lysine residues that are contained in the LBD, which
results in the targeting of SUMO-LXR to promoters of
inflammatory genes, the inhibition of NCoR clearance from the
promoters, and the repression of proinflammatory gene
expression.17,18 To test whether N-phenyl tertiary amines
transpress via a similar mechanism, 20 was evaluated in LPS-
treated reporter gene transrepression, SUMOylation, and chro-
matin immunoprecipitation (ChIP) assays (Figure 4). Whereas
2 and 20 did not show an effect in the iNOS promoter-driven
reporter gene assay in the absence of LXRR or LXRꢀ, both
compounds showed comparable repression of LPS-stimulated
inos expression in the presence of both LXRR and LXRꢀ
(Figure 4A). Morever, upon treatment with LPS, both com-
pounds inhibited the clearance of NCoR from the inos promoter
and induced the SUMOylation of LXRꢀ but not of LXRR (data
Figure 3. Crystal structure of 3 (green, gauche conformer) bound to
hLXRꢀ (magenta). The AF-2 helix is highlighted in pink. The cocrystal
structure of 19 bound to mLXRR is superimposed and shown in orange.
A hydrogen-bond interaction between the bistrifluoromethyl alcohol
of 19 and His419 of helix 10/11 is similar to that seen in the complex
with 3. There is also a hydrogen bond between Ser262 and the 4-methoxy
group of 19. The conformation of the loop, particularly Phe313, between
helices 5 and 6 of the receptor (lower right) appears to be dramatically
influenced by the substituents on the phenyl ring of 19.
functional-activity relationship could be gleaned from the 19-
LXR cocrystal structure, the crystallographic data coupled with
the M2H data suggest that regions of the receptor that are distal
from the coactivator peptide-binding site can modulate core-
pressor interaction and LXR functional activity.
Mechanism of Anti-inflammatory versus Lipogenic
Activities. The identification of 20 provided a chemical tool to
explore the mechanistic basis for the differential effects in anti-
inflammatory versus lipogenic assays. The mechanistic basis

5762 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chao et al.
(4:1 hexanes/EtOAc) provided 85 mg (63%) of an intermediate
secondary amine. A solution of 60 mg (0.17 mmol) of the
intermediate in 600 µL of glacial acetic acid was treated with excess
paraformaldehyde followed by 53 mg (0.85 µmol) of NaCNBH3.
After being stirred at room temperature for 15 h, the reaction was
filtered through silica gel with EtOAc as the eluent, and the filtrate
was concentrated in vacuo. Purification by preparative TLC (SiO2,
1000 µm) that used hexanes/EtOAc (4:1) provided 30 mg (50%)
of 4: 1H NMR (CDCl3, δ): 3.07 (s, 3H), 4.56 (s, 2H), 6.76 (d, J )
8.8 Hz, 2H), 7.18-7.29 (m, 3H), 7.33 (t, J ) 7.4 Hz, 2H), 7.49 (d,
J ) 8.8 Hz, 2H). MS (ES) m/e: 364 (M + 1). >99% pure by HPLC
(Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O/CH3CN
(85:15) with 0.1% HCOOH to 100% CH3CN over 4 min, flow rate
) 1.2 mL/min). tR ) 4.61 min. HRMS (ES+) m/e: (M + 1) calcd
for C17H17F6N1O, 364.1136; found, 364.1131.
not shown) upon analysis in Western blot and NCoR ChIP
assays (Figure 4B,C), respectively, which demonstrates that 20
shows anti-inflammatory activity via a mechanism that is similar
to that of the first-generation LXR ligand 2.
First-generation LXR ligands increase the expression of target
genes through a complex series of events: designated transac-
tivation, which includes ligand binding, heterodimerization with
RXR, DNA binding, coactivator recruitment, and assembly of
cellular transcriptional machinery. The ability of 3 and 20 to
transactivate target genes differentially was determined by
profiling in reporter gene functional assays. Consistent with the
relatively weak recruitment of the coactivator SRC-1 (Figure
2B) and the minimal induction of the lipogenic genes srebp1c
and fas (Figure 1D), 20 showed very weak (EC50 ) 3-5 uM)
transactivation potential when it was tested in cell-based
LXRR- and LXRꢀ-GAL4 reporter gene expression assays and
only weakly induced the reverse cholesterol transport pathway
via ABCA1 (data not shown).3 Overall, the cumulative data
suggest that the mechanism of anti-inflammatory activity versus
lipogenic activity with 20 results from the greater than 10-fold
selectivity for the transrepression of anti-inflammatory target
genes versus the transactivation of lipogenic genes.
2-{4-[Butyl(pyridin-2-ylmethyl)amino]phenyl}-1,1,1,3,3,3-hexaflu-
oropropan-2-ol (8). To a solution of 2-(bromomethyl)pyridine (0.06
g, 0.32 mmol) in 0.2 mL DMF were added 2-[4-(butylamino)phe-
nyl]-1,1,1,3,3,3-hexafluoropropan-2-ol (0.05 g, 0.16 mmol) and
K2CO3 (0.06 g, 0.48 mmol). The mixture was heated in a microwave
at 140 °C for 15 min and was then evaporated to dryness. We
purified the residue on a prep Agilent HPLC system with a
Phenomenex Luna 5 µm C18 (150 × 21 mm) column by using
30-100% CH3CN/H2O that contained 0.1% TFA over 10 min to
1
give compound 8: H NMR (CD3OD, δ): 1.01 (t, 3H), 1.45 (m,
2H), 1.71 (m, 2H), 3.61 (t, 2H), 4.98 (s, 2H), 6.79 (d, J ) 9.1 Hz,
2H), 7.54 (d, J ) 8.8 Hz, 2H), 7.86 (t, 2H), 8.42 (t, 1H), 8.72 (d,
J ) 5.5 Hz, 1H). MS (APCI) m/e: 407 (M + 1). >99% pure by
HPLC (Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O
(with 0.1% HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to
100% MeOH (with 0.075% HCOOH) over 5 min, flow rate ) 1.0
mL/min, 210-400 nm). tR ) 3.13 min. HRMS (ES+) m/e: (M +
1) calcd for C19H21F6N2O, 407.1558; found, 407.1553.
2-{4-[Benzyl(butyl)amino]phenyl}-1,1,1,3,3,3-hexafluoropropan-
2-ol (5). Step 1: To a solution of 2-(4-aminophenyl)-1,1,1,3,3,3-
hexafluoropropan-2-ol (10 g, 0.04 mol) in 170 mL of DCM were
added butyric anhydride (12 mL, 0.07 mol), triethylamine (14 mL,
0.09 mol), and catalytic DMAP. The mixture was heated to 40 °C
for 18 h and was then evaporated to dryness. The residue was
dissolved in ethyl acetate, treated with 10% K2CO3, and stirred at
25 °C for 2 h. A solid precipitated and was collected by filtration.
The crude amide/ester was treated with 1 M LAH (95 mL, 0.095
mmol) in Et2O and was then stirred at 25 °C for 4 h. The reaction
was treated with H2O (20 mL) at 0 °C, followed by 15% NaOH
(50 mL) and was then filtered through celite. The mixture was
extracted with EtOAc, and the organic layers were dried (MgSO4),
filtered, and evaporated to dryness to provide 9.5 g (75%) of 2-[4-
(butylamino)phenyl]-1,1,1,3,3,3-hexafluoropropan-2-ol. 1H NMR
(CDCl3, δ): 0.98 (t, 3H), 1.45 (m, 2H), 1.63 (m, 2H), 3.15 (t, 2H),
6.63 (dd, J ) 1.9, 9.0 Hz, 2H), 7.45 (d, J ) 8.7 Hz, 2H).
Conclusions
The 3-LXRꢀ cocrystal structure led to the design and
synthesis of a series of N-phenyl tertiary amines. Profiling in
binding and functional assays led to the discovery of 20 as an
LXR modulator that shows potent anti-inflammatory activity
but does not induce the expression of lipogenic genes or hepatic
triglyceride accumulation. Cofactor profiling revealed key
differences between first-generation LXR agonist 3 and 20 by
the use of M2H profiling assays with more than 60 amino acid
fragments of SRC-1 and NCoR, which suggests that 20 stabilizes
LXR in a basal, repressed state. An NPTA-LXR cocrystal
structure revealed unexpected regions within the LXR ligand-
pocket that affect receptor conformation and functional activity.
Importantly, in vitro receptor profiling with 15- to 21-mer
peptides was not able to differentiate the phenotype of receptor
modulation distal to the AF-2 helix. Our results define the
limitations of short coactivator and corepressor peptide profiling
for the characterization and optimization of NR modulators.
Whereas compounds that directly affect AF-2 can be binned
by the use of these technologies,16,19 we now demonstrate that
compounds that modulate receptor function distal to AF-2
require large coactivator and corepressor fragments to distin-
guish their pharmacology. Mechanistic studies showed that 20,
like first-generation LXR agonist 2, effectively suppressed the
expression of proinflammatory target genes via a SUMOylation-
dependent mechanism, which resulted in a greater than 10-fold
selectivity for transrepression versus transactivation. LXR
modulator 20 will serve as a valuable chemical tool for exploring
nuclear receptor transrepression and will provide an opportunity
for the future discovery of LXR modulators with improved
therapeutic indexes.
Step 2: To a solution of benzaldehyde (1.7 g, 15.9 mmol) in
DCE (30 mL) were added glacial AcOH (1 mL), 2-[4-(butylami-
no)phenyl]-1,1,1,3,3,3-hexafluoropropan-2-ol (1 g, 3.2 mmol), and
NaHB(OAc)3 (4 g, 16 mmol). The mixture was stirred at 25 °C for
18 h and was then evaporated to dryness. The residue was purified
on a prep Agilent HPLC system with a Phenomenex Luna 5 µm
C18 (150 × 21 mm2) column and was eluted with 30-100%
CH3CN/H2O that contained 0.1% TFA over 10 min to give
1
compound 5. H NMR (CD3OD, δ): 0.97 (t, 3H), 1.41 (m, 2H),
1.65 (m, 2H), 3.49 (t, 2H), 4.62 (s, 2H), 6.76 (d, J ) 9.0 Hz, 2H),
7.21-7.33 (m, 5H), 7.48 (d, J ) 8.8 Hz, 2H). MS (APCI) m/e:
406 (M + 1). >99% pure by HPLC (Waters symmetry shield, RPq
3.5 µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH (with
0.075% HCOOH) (50:50) to 100% MeOH (with 0.075% HCOOH)
over 5 min, flow rate ) 0.8 mL/min, 210-400 nm). tR ) 4.27
min. HRMS (ES+) m/e: (M + 1) calcd for C20H22F6NO (M + 1);
found, 406.1600.
Experimental Section
1,1,1,3,3,3-Hexafluoro-2-{4-[methyl(phenylmethyl)amino]phenyl}-
2-propanol (4). A solution of 2-(4-aminophenyl)-1,1,1,3,3,3-
hexafluoro-2-propanol (101 mg, 0.39 mmol) in 0.8 mL of MeOH/
trimethyl orthoformate (1:1) at 25 °C was treated with benzaldehyde
(40 µL, 0.39 mmol). After being stirred overnight, the reaction was
treated with solid NaBH4 in small portions until TLC indicated
that the intermediate imine was consumed. The reaction was filtered
through a pad of silica gel with EtOAc as the eluent, and the filtrate
was concentrated in vacuo. Purification by SiO2 chromatography
Compounds Prepared in an Analogous Fashion. 2-(4-{Butyl[3-
(2-hydroxyethoxy)benzyl]amino}phenyl)-1,1,1,3,3,3-hexafluoropro-
1
pan-2-ol (7). H NMR (CD3OD, δ): 0.98 (t, 3H), 1.42 (m, 2H),
1.66 (m, 2H), 3.48 (t, 2H), 3.84 (t, 2H), 3.98 (t, 3H), 4.58 (s,

Transrepression-SelectiVe LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5763
2H), 6.72 (d, J ) 9.0 Hz, 2H), 6.81 (s, 1H), 6.82 (d, J ) 6.9 Hz,
2H), 7.23 (t, 1H), 7.45 (d, J ) 8.7 Hz, 2H). MS (APCI) m/e: 466
(M + 1). >97% pure by HPLC (Waters symmetry shield, RPq 3.5
µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH (with 0.075%
HCOOH) (50:50) to 100% MeOH (with 0.075% HCOOH) over 5
min, flow rate ) 0.8 mL/min, 210-400 nm). tR ) 4.26 min. HRMS
(ES+) m/e: (M + 1) calcd for C20H22F6NO, 466.1817; found,
466.1820.
2-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
nyl}amino)methyl]-4,6-dichlorophenol (15). H NMR (CD3OD, δ):
1
0.90 (t, 3H), 1.41 (m, 2H), 1.68 (m, 2H), 3.48 (t, 2H), 4.57 (s,
2H), 6.70 (d, J ) 9.0 Hz, 2H), 6.86 (d, J ) 2.3 Hz, 1H), 7.25 (d,
J ) 2.5 Hz, 1H), 7.49 (d, J ) 8.7 Hz, 2H). MS (APCI) m/e: 490
(M + 1). >96% pure by HPLC (Waters symmetry shield, RPq 3.5
µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH (with 0.075%
HCOOH) (50:50) to 100% MeOH (with 0.075% HCOOH) over 5
min, flow rate ) 0.8 mL/min, 210-400 nm). tR ) 4.89 min. HRMS
(ES+) m/e: (M + 1) calcd for C20H20Cl2F6NO2, 490.0775; found,
490.0770.
2-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phen-
yl}amino)methyl]-4-chlorophenol (16). 1H NMR (CD3OD, δ): 0.99
(t, 3H), 1.43 (m, 2H), 1.66 (m, 2H), 3.52 (t, 2H), 4.56 (s, 2H),
6.77-6.9 0 (m, 4H), 7.06 (dd, J ) 2.6, 8.6 Hz, 1H), 7.54 (d, J )
8.9 Hz, 2H). MS (ES) m/e: 456 (M + 1). >99% pure by HPLC
(Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with
0.1% HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100%
MeOH (with 0.075% HCOOH) over 3.5 min, flow rate ) 1.5 mL/
min, 210-400 nm). tR ) 2.96 min. HRMS (ES+) m/e: (M + 1)
calcd for C20H20ClF6NO2, 456.1165; found, 456.1160.
4-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phen-
yl}amino)methyl]-2-chlorophenol (17). 1H NMR (CD3OD, δ): 0.96
(t, 3H), 1.39 (m, 2H), 1.61 (m, 2H), 3.50 (t, 2H), 4.53 (s, 2H),
6.83-6.99 (m, 4H), 7.13 (d, J ) 1.9 Hz, 1H), 7.55 (d, J ) 8.7 Hz,
2H). MS (ES) m/e: 456 (M + 1). >97% pure by HPLC (Waters
symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1%
HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100% MeOH
(with 0.075% HCOOH) over 3.5 min, flow rate ) 1.5 mL/min,
210-400 nm). tR ) 2.82 min. HRMS (ES+) m/e: (M + 1) calcd
for C20H20ClF6NO2, 456.1165; found, 456.1160.
4-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phen-
yl}amino)methyl]-2-chloro-6-methoxyphenol (18). 1H NMR (CD3OD,
δ): 0.96 (t, 3H), 1.41 (m, 2H), 1.61 (m, 2H), 3.51 (t, 2H), 3.77 (s,
3H), 4.53 (s, 2H), 6.69 (d, J ) 1.7 Hz, 1H), 6.76 (d, J ) 1.6 Hz,
1H), 6.89 (d, J ) 9.0 Hz, 2H), 7.56 (d, J ) 8.7 Hz, 2H). MS (ES)
m/e: 486 (M + 1). >97% pure by HPLC (Waters symmetry shield,
RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH
(with 0.075% HCOOH) (50:50) to 100% MeOH (with 0.075%
HCOOH) over 3.5 min, flow rate ) 1.5 mL/min, 210-400 nm). tR
) 2.81 min. HRMS (ES+) m/e: (M + 1) calcd for C21H23ClF6NO3,
486.1271; found, 486.1265.
2-[4-(Butyl{[3-chloro-4,5-bis(methyloxy)phenyl]methyl}amino)phen-
yl]-1,1,1,3,3,3-hexafluoro-2-propanol (19). 1H NMR (CD3OD, δ): 0.98
(t, J ) 7.2 Hz, 3H), 1.30-1.50 (m, 2H), 1.55-1.75 (m, 2H), 3.50
(t, J ) 7.7 Hz, 2H), 3.78 (s, 3H), 3.79 (s, 3H), 4.56 (s, 2H), 6.82
(dd, J ) 6.3, 1.7 Hz, 4H), 7.54 (d, J ) 8.8 Hz, 2H). MS (ES) m/e
) 500 (M + 1). >99% pure by HPLC (Waters symmetry shield,
RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH
(with 0.075% HCOOH) (50:50) to 100% MeOH (with 0.075%
HCOOH) over 3 min, flow rate ) 1.5 mL/min, 210-400 nm). tR
) 2.89 min. HRMS (ES+) m/e: (M + 1) calcd for C22H25ClF6NO3,
500.1427; found, 500.1421.
4-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]ph-
enyl}amino)methyl]-2,6-dichlorophenol (20). 1H NMR (CD3OD, δ):
0.98 (t, 3H), 1.41 (m, 2H), 1.64 (m, 2H), 3.46 (t, 2H), 4.50 (s,
2H), 6.75 (d, J ) 9.1 Hz, 2H), 7.12 (s, 2H), 7.50 (d, J ) 8.9 Hz,2H).
MS (APCI) m/e: 490 (M + 1). >95% pure by HPLC (Waters
symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1%
HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100% MeOH
(with 0.075% HCOOH) over 5 min, flow rate ) 0.8 mL/min,
210-400 nm). tR ) 4.58 min. HRMS (ES+) m/e: (M + 1) calcd
for C20H20Cl2F6NO2, 490.0775; found, 490.0770.
2-{4-[Butyl(pyridin-4-ylmethyl)amino]phenyl}-1,1,1,3,3,3-hexaflu-
1
oropropan-2-ol (9). H NMR (CD3OD, δ): 1.01 (t, 3H), 1.46 (m,
2H), 1.70 (m, 2H), 3.59 (t, 2H), 4.94 (s, 2H), 6.72 (d, J ) 9.2 Hz,
2H), 7.50 (d, J ) 8.7 Hz, 2H), 7.90 (d, J ) 4.8 Hz, 2H), 8.74 (br
s, 2H). MS (APCI) m/e: 407 (M + 1). >99% pure by HPLC
(Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with
0.1% HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100%
MeOH (with 0.075% HCOOH) over 5 min, flow rate ) 0.8 mL/
min, 210-400 nm). tR ) 2.66 min. HRMS (ES+) m/e: (M + 1)
calcd for C19H21F6N2O, 407.1558; found, 407.1553.
2-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
1
nyl}amino)methyl]phenol (10). H NMR (CDCl3, δ): 0.91 (t, 3H),
1.32 (m, 2H), 1.56 (m, 2H), 3.31 (t, 2H), 4.47 (s, 2H), 6.87 (m,
2H), 7.03 (d, J ) 8.9 Hz, 2H), 7.18 (m, 2H), 7.59 (d, J ) 8.7 Hz,
2H). MS (APCI) m/e: 422 (M + 1). >98% pure by HPLC (Waters
symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1%
HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100% MeOH
(with 0.075% HCOOH) over 5 min, flow rate ) 0.8 mL/min,
210-400 nm). tR ) 4.34 min. HRMS (ES+) m/e: (M + 1) calcd
for C20H21F6NO2, 422.1555; found, 422.1549.
3-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
nyl}amino)methyl]phenol (11). 1H NMR (CD3OD, δ): 0.98 (t, 3H),
1.41 (m, 2H), 1.67 (m, 2H), 3.45 (t, 2H), 4.54 (s, 2H), 6.65-6.73
(m, 5H), 7.14 (m, 1H), 7.45 (d, J ) 8.9 Hz, 2H). MS (APCI) m/e:
422 (M + 1). >99% pure by HPLC (Waters symmetry shield, RPq
3.5 µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH (with
0.075% HCOOH) (50:50) to 100% MeOH (with 0.075% HCOOH)
over 5 min, flow rate ) 0.8 mL/min, 210-400 nm). tR ) 4.27
min. HRMS (ES+) m/e: (M + 1) calcd for C20H21F6NO2, 422.1555;
found, 422.1549.
4-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
nyl}amino)methyl]phenol (12). 1H NMR (CD3OD, δ): 0.89 (t, 3H),
1.25 (m, 2H), 1.51 (m, 2H), 3.08 (t, 2H), 3.88 (s, 2H), 6.73 (d, J
) 8.4 Hz, 2H), 6.84 (d, J ) 8.7 Hz, 1H), 6.99 (d, J ) 8.4 Hz, 2H),
7.43 (s, 1H), 7.52 (d, J ) 8.6 Hz, 1H). MS (ES) m/e: 422 (M + 1).
>96% pure by HPLC (Waters symmetry shield, RPq 3.5 µm, 2.1
× 30 mm2, H2O (with 0.1% HCOOH)/MeOH (with 0.075%
HCOOH) (50:50) to 100% MeOH (with 0.075% HCOOH) over
3.5 min, flow rate ) 1.5 mL/min, 210-400 nm). tR ) 2.56 min.
HRMS (ES+) m/e: (M + 1) calcd for C20H21F6NO2, 422.1555;
found, 422.1549.
2-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
nyl}amino)methyl]-6-fluorophenol (13). 1H NMR (CD3OD, δ): 0.98
(t, 3H), 1.43 (m, 2H), 1.67 (m, 2H), 3.50 (t, 2H), 4.61 (s, 2H),
6.78 (m, 4H), 6.96 (m, 1H), 7.48 (d, J ) 8.8 Hz, 2H). MS (APCI)
m/e: 440 (M + 1). >96% pure by HPLC (Waters symmetry shield,
RPq 3.5 µm, 2.1 × 30 mm2, H2O (with 0.1% HCOOH)/MeOH
(with 0.075% HCOOH) (50:50) to 100% MeOH (with 0.075%
HCOOH) over 5 min, flow rate ) 0.8 mL/min, 210-400 nm). tR
) 4.37 min. HRMS (ES+) m/e: (M + 1) calcd for C20H21F7NO2,
440.1461; found, 440.1455.
2-[(Butyl{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phe-
nyl}amino)methyl]-4-(trifluoromethoxy)phenol (14). 1H NMR
(CD3OD, δ): 0.98 (t, 3H), 1.42 (m, 2H), 1.67 (m, 2H), 3.50 (t,
2H), 4.57 (s, 2H), 6.74-6.88 (m, 4H), 6.98 (dd, J ) 2.2, 8.6 Hz,
1H), 7.50 (d, J ) 8.7 Hz, 2H). MS (ES) m/e: 506 (M + 1). >99%
pure by HPLC (Waters symmetry shield, RPq 3.5 µm, 2.1 × 30
mm2, H2O (with 0.1% HCOOH)/MeOH (with 0.075% HCOOH)
(50:50) to 100% MeOH (with 0.075% HCOOH) over 3.5 min, flow
rate ) 3.5 mL/min, 210-400 nm). tR ) 3.03 min. HRMS (ES+)
m/e: (M + 1) calcd for C21H21F9NO3, 506.1378; found, 506.1372.
2-{4-[Butyl(3,5-dichlorobenzyl)amino]phenyl}-1,1,1,3,3,3-hexaflu-
oropropan-2-ol (21). 1H NMR (CD3OD, δ): 0.98 (t, 3H), 1.42 (m,
2H), 1.66 (m, 2H), 2.51 (s, 3H), 3.48 (t, 2H), 4.55 (s, 2H), 6.71 (d,
J ) 9.1 Hz, 2H), 7.17 (d, J ) 1.5 Hz, 2H), 7.30 (s, 1H), 7.49 (d,
J ) 8.9 Hz, 2H). MS (APCI) m/e: 474 (M + 1). >99% pure by
HPLC (Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O/
CH3CN (with 0.1% HCOOH) (85:15) to 100% CH3CN over 3.5

5764 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chao et al.
min, flow rate ) 1.0 mL/min). tR ) 4.45 min. HRMS (ES+) m/e:
(M + 1) calcd for C20H20Cl2F6NO, 474.0826; found, 474.0821.
dated, charcoal- stripped FBS. The final concentration of DMSO
was 0.1%. Cells were incubated for 24 h in the presence of drug,
after which the media was sampled and assayed for alkaline
phosphatase activity, and the cells were assayed for R-galac-
tosidase activity to normalize for transfection efficiency. Plates
were read on a Thermomax plate reader (Molecular Devices) at
405 nm.
2-{4-[Butyl(3,5-dichloro-4-methoxybenzyl)amino]phenyl}-
1,1,1,3,3,3-hexafluoropropan-2-ol (22). 1H NMR (CD3OD, δ): 0.98
(t, 3H), 1.41 (m, 2H), 1.64 (m, 2H), 3.48 (t, 2H), 3.85 (s, 3H),
4.55 (s, 2H), 6.74 (d, J ) 9.1 Hz, 2H), 7.21 (s, 2H), 7.52 (d, J )
8.8 Hz, 2H). MS (ES) m/e: 504 (M + 1). >99% pure by HPLC
(Waters symmetry shield, RPq 3.5 µm, 2.1 × 30 mm2, H2O (with
0.1% HCOOH)/MeOH (with 0.075% HCOOH) (50:50) to 100%
MeOH (with 0.075% HCOOH) over 5 min, flow rate ) 1.5 mL/
min, 210-400 nm). tR ) 3.06 min. HRMS (ES+) m/e: (M + 1)
calcd for C21H22Cl2F6NO2, 504.0932; found, 504.0926.
LXRr and LXRꢀ Binding Assays. Human biotinylated LXRR
and LXRꢀ LBDs20 were incubated at 50 and 25 nM, respectively,
with 0.25 mg/mL streptavidin-coupled LEADseeker imaging
beads (GE Healthcare) in assay buffer (50 mM MOPS (pH 7.5),
50 mM NaF, 0.05 mM CHAPS, 0.1 mg/mL FAF-BSAA) for
60 min at 25 °C. After being incubated, the receptor-bead slurry
was pelleted by centrifugation at 1200g. The supernatant was
discarded, and the beads were resuspended in the original volume
of assay buffer that contained freshly added 10 mM DTT with
gentle mixing. D-Biotin (Pierce Chemical) was added to the
resuspended receptor bead solution at 200 uM, and the mixture
was allowed to incubate at room temperature for 60 min. After
the incubation, [N-methyl-3H]-GW043821 was added to the
receptor/bead mix, and the mixture was mixed gently. The
receptor/bead/radio-ligand mixture (25 µL) was added to each
well of an assay plate that containined 0.5 µL of the test
compound. The final test compound concentrations were between
300 pM and 20 µM. Plates were incubated at room temperature
for 3 h and were then imaged on a ViewLux 1430 ultraHTS
microplate imager (Perkin-Elmer).
Coactivator and Corepressor Panel Peptide Scanning. The
reagents for the FRET-based cell-free peptide scanning assays
were prepared as described in ref 20 with minor modifications.
Europium-labeled streptavidin (12 µL, 10 nM) was added to each
well of a 384-well plate that contained 1 µL of 500 nM
biotinylated test peptide and was allowed to equilibrate for 30
min at room temperature. At the same time, each test compound
at 10 µM was allowed to incubate for 30 min with the biotin-
blocked LXR APC solution. The LXR APC test compound
mixture (12 µL) was added to a plate that contained the test
peptide coupled europium-streptavidin complex, was allowed
to equilibrate for 1 h, and was then imaged on a ViewLux 1430
ultraHTS microplate imager (Perkin-Elmer) in time-resolved
mode. The final assay volume was 25 µL. The values were
normalized by log (compound activity/basal activity).
Mammalian Two-Hybrid (M2H) Assay. CV-1 cells were
cultured in DME high-glucose media that was supplemented with
10% FBS and 2 mM glutamine in a humidified incubator (5%
CO2 in air) at 37 °C. 3 days Three days prior to plating for
transfection, cells were harvested with 0.25% trypsin/2 mM
EDTA in phenol red-free Dulbecco’s phosphate-buffered saline
(without calcium or magnesium) and were collected in phenol
red-free DMEM-F12/15 mM HEPES medium that was supple-
mented with 10% charcoal/dextran-treated fetal bovine serum
and 2 mM L-glutamine (experimental medium). After 3 days in
culture, cells were again harvested and were suspended in
experimental medium. The cells were seeded at 2.0 × 104 cells
per well in a 96-well plate and were returned to the incubator
for 24 h. Cells were then transfected by the use of Lipofectamine
(Life Technologies) according to the manufacturer’s instructions.
The total amount of DNA that was transfected into each well
was 80 ng. Transfection mixtures contained 15 ng of VP16-
LXRꢀ plasmid, 8 ng of SPAP reporter, 25 ng of pCH110-R-gal
control plasmid, 24 ng of pBluescript II KS+ (Clonetech), and
8 ng of either coactivator or corepressor plasmid. Cells were
transfected for 16 h, the medium was aspirated, and 100 µL of
ligand was added. We prepared appropriate ligand concentrations
from DMSO stock solutions by using phenol red-free DMEM/
F-12 that was supplemented with 10% heat-inactivated, delipi-
Transrepression, ChIP, and SUMOylation Assays. Experi-
mental procedures were performed according to ref 17.
Supporting Information Available: 1H NMR spectra for
compounds 4-5 and 7-22 as well as experimental procedures
for the crystallography research described in this manuscript.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) (a) Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver, T. A.;
Blanchard, D. E.; Spencer, T. A.; Willson, T. M. Activation of the
nuclear receptor LXR by oxysterols defines a new hormone response
pathway. J. Biol. Chem. 1997, 272, 3137–3140.
(2) Spencer, T. A.; Gayen, A. K.; Phirwa, S.; Nelson, J. A.; Taylor, F. R.;
Kandutsch, A. A.; Erickson, S. K. 24(S),25-Epoxycholesterol. Evidence
consistent with a role in the regulation of hepatic cholesterogenesis.
J. Biol. Chem. 1985, 260, 13391–13394.
(3) Collins, J. L.; Fivush, A. M.; Watson, M. A.; Galardi, C. M.; Lewis,
M. C.; Moore, L. B.; Parks, D. J.; Wilson, J. G.; Tippin, T. K.; Binz,
J. G.; Plunket, K. D.; Morgan, D. G.; Beaudet, E. J.; Whitney, K. D.;
Kliewer, S. A.; Willson, T. M. Identification of a nonsteroidal liver X
receptor agonist through parallel array synthesis of tertiary amines.
J. Med. Chem. 2002, 45, 1963–1966.
(4) Schultz, J. R.; Tu, H.; Luk, A.; Repa, J. J.; Medina, J. C.; Li, L.;
Schwendner, S.; Wang, S.; Thoolen, M.; Mangelsdorf, D. J.; Lustig,
K. D.; Shan, B. Role of LXRs in control of lipogenesis. Genes DeV.
2000, 14, 2831–2838.
(5) Rader, D. J. Liver X receptor and farnesoid X receptor as therapeutic
targets. Am. J. Cardiol 2007, 100, 15N–19N.
(6) Zelcer, N.; Tontonoz, P. Liver X receptors as integrators of metabolic
and inflammatory signaling. J. Clin. InVest. 2006, 116, 607–614.
(7) Collins, J. L. Therapeutic opportunities for liver X receptor modulators.
Curr. Opin. Drug DiscoV. DeVel. 2004, 7, 692–702.
(8) (a) Jaye, M. LXR agonists for the treatment of atherosclerosis. Curr.
Opin. InVest. Drugs 2003, 4, 1053–1058. (b) Lund, E. G.; Menke,
J. G.; Sparrow, C. P. Liver X receptor agonists as potential therapeutic
agents for dyslipidemia and atherosclerosis. Arterioscler., Thromb.,
Vasc. Biol. 2003, 237, 1169–1177.
(9) Groot, P. H. E.; Pearce, N. J.; Yates, J. W.; Stocker, C.; Sauermelch,
C.; Doe, C. P.; Willette, R. N.; Olzinski, A.; Peters, T.; d’Epagnier,
D.; Morasco, O.; Krawiec, J. A.; Webb, C. L.; Aravindhan, K.;
Jucker, B.; Burgert, M.; Ma, C.; Marino, J. P.; Collins, J. L.;
Macphee, C. H.; Thompson, S. K.; Jaye, M. Synthetic LXR agonists
increase LDL in CETP species. J. Lipid Res. 2005, 46, 2182–2191.
(10) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert,
M. H.; Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols,
J.; Parks, D.; Watson, M.; Wisely, B.; Willson, T. M. X-ray crystal
structure of the liver X receptor ꢀ ligand binding domain: regulation
by a histidine-tryptophan switch. J. Biol. Chem. 2003, 278, 27138–
27143.
(11) Masciadri, R.; Kamer; Matthias, N. N regioselective Friedel-Crafts
alkylation of anilines and amino-substituted heteroarenes with
hexafluoroacetone sesquihydrate. Eur. J. Org. Chem. 2003, 21, 4286–
4291.
(12) Resche-Rigon, M.; Gronemeyer, H. Therapeutic potential of selective
modulators of nuclear receptor action. Curr. Opin. Chem. Biol. 1998,
2, 501–507.
(13) de Lera, A. R.; Bourguet, W.; Altucci, L.; Gronemeyer, H. Design of
selective nuclear receptor modulators: RAR and RXR as a case study.
Nat. ReV. Drug DiscoVery 2007, 6, 811–820.
(14) Jaye, M.; Krawiec, J. A.; Campobasso, N.; Smallwood, A.; Qiu, C.;
Lu, Q.; Kerrigan, J. J.; De Los Frailes Alvaro, M.; Laffitte, B.; Liu,
W.-S.; Marino, J. P., Jr.; Meyer, C. R.; Nichols, J. A.; Parks, D. J.;
Perez, P.; Sarov-Blat, L.; Seepersaud, S. D.; Steplewski, K. M.;
Thompson, S. K.; Wang, P.; Watson, M. A.; Webb, C. L.; Haigh, D.;
Caravella, J. A.; Macphee, C. H.; Willson, T. M.; Collins, J. L.
Discovery of substituted maleimides as liver X receptor agonists and
determination of a ligand-bound crystal structure. J. Med. Chem. 2005,
48, 5419–5422.
(15) Iannone, M. A.; Consler, T. G.; Pearce, K. H.; Stimmel, J. B.; Parks,
D. J.; Gray, J. G. Multiplexed molecular interactions of nuclear

Transrepression-SelectiVe LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5765
receptors using fluorescent microspheres. Cytometry 2001, 44, 326–
337.
(16) Iannone, M. A.; Simmons, C. A.; Kadwell, S. H.; Svoboda, D. L.;
Vanderwall, D. E.; Deng, S.-J.; Consler, T. G.; Shearin, J.; Gray,
J. G.; Pearce, K. H. Correlation between in vitro peptide binding
profiles and cellular activities for estrogen receptor-modulating
compounds. Mol. Endocrinol. 2004, 18, 1064–1081.
(17) Ghisletti, S.; Huang, W.; Ogawa, S.; Pascual, G.; Lin, M.-E.;
Willson, T. M.; Rosenfeld, M. G.; Glass, C. K. Parallel SUMOy-
lation-dependent pathways mediate gene- and signal-specific trans-
repression by LXRs and PPARγ. Mol. Cell 2007, 25, 57–70.
(18) Treuter, E.; Gustafsson, J.-A. Wrestling rules in transrepression:
as easy as SUMO-1, -2, -3. Mol. Cell 2007, 25, 178–180.
(19) Trump, R. P.; Cobb, J. E.; Shearer, B. G.; Lambert, M. H.; Nolte,
R. T.; Willson, T. M.; Buckholz, R. G.; Zhao, S. M.; Leesnitzer,
L. M.; Iannone, M. A.; Pearce, K. H.; Billin, A. N.; Hoekstra, W. J.
Co-crystal structure guided array synthesis of PPARγ inverse
agonists. Bioorg. Med. Chem. Lett. 2007, 17, 3916–3920.
(20) Spencer, T. A.; Li, D.; Russel, J. S.; Collins, J. L.; Bledsoe, R. K.;
Consler, T. G.; Moore, L. B.; Galardi, C. M.; McKee, D. D.; Moore,
J. T.; Watson, M. A.; Parks, D. J.; Lambert, M. H.; Willson, T. M.
Pharmacophore analysis of the nuclear oxysterol receptor LXRR.
J. Med. Chem. 2001, 44, 886–897.
(21) Noble, S. M.; Carnahan, V. E.; Moore, L. B.; Luntz, T.; Wang, H.;
Ittoop, O. R.; Stimmel, J. B.; Davis-Searles, P. R.; Watkins, R. E.;
Wisely, G. B.; LeCluyse, E.; Tripathy, A.; McDonnell, D. P.; Redinbo,
M. R. Human PXR forms a tryptophan zipper-mediated homodimer.
Biochemistry 2006, 45, 8579–8589.
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