N-acylthiadiazolines, a new class of liver X receptor agonists with selectivity for LXRβ

Article abstract of DOI:10.1021/jm070453f

We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRβ subtype with selectivity over LXRα. LXRβ selectivity was confirmed using macrophages derived from LXR mutant mice. Despite its selectivity and modest potency, the compou

Full text of DOI:10.1021/jm070453f

J. Med. Chem. 2007, 50, 4255-4259
4255
N-Acylthiadiazolines, a New Class of Liver X Receptor Agonists with Selectivity for LXRâ
Valentina Molteni,*,† Xiaolin Li,† Juliet Nabakka,† Fang Liang,† John Wityak,† Alan Koder,† Leo Vargas,† Russell Romeo,†
Nico Mitro,†,¶ Puiying A. Mak,† H. Martin Seidel,† Jennifer A. Haslam,† Donald Chow,† Tove Tuntland,† Tracy A. Spalding,†
Ansgar Brock,† Michelle Bradley,‡ Antonio Castrillo,‡ Peter Tontonoz,‡ and Enrique Saez*,†,¶
Genomics Institute of the NoVartis Research Foundation, 10675 John Jay Hopkins DriVe, San Diego, California 92121, Department of Cell
Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, and Howard Hughes Medical Institute
and Department of Pathology and Laboratory Medicine, UniVersity of California, Los Angeles, California 90095
ReceiVed April 17, 2007
We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRâ subtype with selectivity
over LXRR. LXRâ selectivity was confirmed using macrophages derived from LXR mutant mice. Despite
its selectivity and modest potency, the compound can induce APO-AI-dependent cholesterol efflux from
macrophages with full efficacy. Our results indicate that it is possible to achieve significant LXRâ selectivity
in a small molecule while maintaining functional LXR activity.
Chart 1
Introduction
The liver X receptors (LXR)a R and â (NR1H3, NR1H2)
are ligand-activated transcription factors of the nuclear receptor
superfamily that control gene expression linked to cholesterol,
lipid, and glucose homeostasis.1,2 The endogenous ligands for
LXRs are primarily oxysterols, but several synthetic ligands have
been described that have facilitated the study of LXR function.3-10
In particular, the dual LXRR/â agonists 1 (GW3965)6 and 1a
(T0901317;7 Chart 1) have been widely utilized as nonsteroidal
chemical tools to explore the biology of the LXRs. Use of these
agonists has shown the potential of the LXRs as drug targets
for cardiovascular disease, diabetes, inflammation, and neuro-
degenerative disease.11-16 However, development of LXR
ligands as therapeutic agents has been hampered because dual
LXR agonists induce a detrimental increase in hepatic triglyc-
eride production. Because LXRR is the dominant subtype in
the liver, where LXRâ is expressed at very low levels, it is
thought that an LXRâ-selective agonist may retain efficacy
without deleteriously increasing hepatic lipogenesis.17-19 Hence,
the identification of LXRâ-selective agonists may be of
significant therapeutic value.
The crystal structures of the LXRs reveal that all the residues
in the ligand binding domain (LBD) that form the ligand binding
pocket (LBP) are conserved between the two subtypes, hinting
that isolation of subtype-selective LXR ligands may be a
challenging endeavor.20-22 However, LXR transcriptional activ-
ity in vivo is the result of a complex interplay between
recruitment of co-activators and release of co-repressors, the
nature of which cannot be anticipated simply by the in vitro
binding properties of a compound to the LBD. Thus, while LXR
subtype selectivity measured as a separation in binding potency
(EC50) may be difficult to attain given the degree of similarity
between the two ligand binding pockets, it is conceivable that
functional selectivity measured as a separation in the degree of
transcriptional activation induced (i.e., efficacy) may be achiev-
able, even if it cannot be described at a molecular level.
Results and Discussion
To identify novel LXR modulators, a cell-based HTS using
a chimeric GAL-hLXRâ LBD construct was run. A few N-acyl-
thiadiaziolines with high micromolar activity that appeared
selective for LXRâ over LXRR under the conditions used were
identified. These molecules activated LXRâ but failed to activate
LXRR in transfection assays. Through a round of optimization
of the HTS hits, a more potent LXRâ-selective N-acylthiadia-
zoline 2 was synthesized (GAL-hLXRâ assay EC50 ) 1.29 µM,
% efficacy ) 55; no activity on GAL-hLXRR). This thiadia-
zolidine was inactive in cell-based assays for a panel of nuclear
receptors (RXR, FXR, PXR, PPARs, GR, VDR) both in agonist
and in antagonist mode (10 µM top dose, not shown). To explore
the molecular basis of the apparent selectivity of this compound,
we profiled it on cell-free LXR binding assays (Table 1). In
scintillation proximity assays (SPA) measuring total binding, 2
bound to LXRâ with 30-fold greater potency than to LXRR,
and to a more complete extent (100% vs 52% occupancy).
Similarly, in FRET-based coactivator recruitment assays mea-
suring agonist activity, 2 stimulated co-activator recruitment to
the LXRâ LBD with 10-fold greater potency than to the hLXRR
LBD and with greater efficacy (100% vs 53% efficacy).
To evaluate the ability of compound 2 to stimulate in vivo
expression of LXR target genes, it was profiled on the natural
receptors in cell-based assays. Compound 2 retained LXRâ
selectivity at the level of efficacy separation (Table 1), stimulat-
ing LXRâ activity with nearly full effectiveness (82%), while
* To whom correspondence should be addressed. Telephone: 858-332-
4736 (V.M.); 858-784-7305 (E.S.). Fax: 858-812-1648 (V.M.); 858-784-
7333 (E.S.). E-mail: vmolteni@gnf.org (V.M.); esaez@scripps.edu (E.S.).
† Genomics Institute of the Novartis Research Foundation.
¶ The Scripps Research Institute.
‡ University of California, Los Angeles.
a Abbreviations: LXR, liver X receptor; RXR, retinoid X receptor; FXR,
farnesoid X receptor; PXR, pregnane X receptor; PPAR, peroxisome-
proliferator activated receptor; GR, glucocorticoid receptor; VDR, vitamin
D receptor; ABCA1, ATP-binding cassette transporter A1; FAS, fatty acid
synthase; FRET, fluorescence resonance energy transfer; SPA, scintillation
proximity assay.
10.1021/jm070453f CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/01/2007

4256 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Table 1. Biological Properties of Racemate 2 and Its Enantiomersa
Brief Articles
1
2
3
4
assay
racemate
R
S
IC50 SPA-LXRR (%E)
IC50 SPA-LXRâ (%E)
IC50 FRET-LXRR (%E)
IC50 FRET-LXRâ (%E)
EC50 hLXRR (%E)
0.121 (100) 9.8 (52)
0.023 (100) 0.3 (100) 0.066 (100) NA
0.097 (100) 2.3 (53) 1.6 (64) NA
0.027 (100) 0.25 (100) 0.067 (94) NA
0.066 (100) 0.608 (19) 0.346 (41) NA
0.004 (100) 0.098 (82) 0.063 (98) NA
5 (80)
NA
EC50 hLXRâ (%E)
ABCA1 induction (1 µM)
ABCA1 induction (10 µM ) 100%
100%
34%
59%
39%
60%
0%
0%
a NA ) no activity: no significant efficacy (<15%) detected at any
concentration. Reporter gene assays were performed in quadruplicate with
a top dose of 10 µM. EC50 data is reported in µM. Efficacy of compounds
is presented as percent efficacy (%E) relative to 1 and defined as the
percentage ratio between maximum fold induction for the test compound
and maximum fold induction for 1 in the same experiment. FRET assays
were performed in duplicate with a top dose of 20 µM; EC50 data is reported
in µM and efficacy of compounds is reported as percent efficacy (%E)
relative to 1, defined as the percentage ratio between maximum coactivator
recruitment for the test compound and maximum coactivator recruitment
for 1 in the same experiment. SPA assays were run in displacement mode
using [3H]-1a and a top dose of 30 µM test compound; efficacy represents
maximum displacement relative to 1. ABCA1 induction in human THP-1
cells was measured using qRT-PCR.
only marginally activating LXRR (19%). As may be anticipated
based on this profile, compound 2 increased expression of an
endogenous LXR target gene (ABCA1) in the human macroph-
age cell line THP-1 that expresses similar levels of the two LXR
subtypes23 with significant efficacy relative to the dual agonist
1 (34% at 1 µM and 59% at 10 µM). In contrast, in liver-derived
HepG2 cells that contain primarily LXRR, compound 2 induced
expression of the LXR target gene FAS with considerably
reduced efficacy relative to 1 (10% at 1 µM and 33% at 10
µM), as may be expected of an LXRâ-selective ligand.29
Furthermore, when profiled in human primary hepatocytes, 2
failed to induce FAS expression, while 1 and 1a elicited robust
increases in FAS message levels (Figure 1a).
Figure 1. (a) Induction of endogenous FAS expression in primary
human hepatocytes. Cells were treated with 1 µM of the indicated com-
pounds, and gene expression was analyzed by Taqman-based qRT-
PCR. (b) Dose response in FRET-based coactivator recruitment assay.
Note that there are several concentrations at which 3 activates LXRâ
but not LXRR.
Scheme 1. General Synthesis of Thiadiazolinesa
Compound 2, synthesized as per Scheme 1, was obtained as
a racemic mixture.24,25 Separation by chiral HPLC afforded the
two enantiomers 3 and 4. The S absolute configuration of 4
was established by X-ray crystallography. In cell-based and
biochemical assays, compound 4 was found to be inactive on
both LXR subtypes, while compound 3 was active (Figure 1b
and Table 1). Compound 3 contributed to the functional activity
of the racemic mixture, stimulating endogenous ABCA1 expres-
sion to the same degree as 2, while 4 did not alter ABCA1
expression in THP-1 cells (Table 1). The cellular behavior of
the two enantiomers correlated with their in vitro LXR-binding
properties: compound 4 failed to bind either LXR, while 3
bound LXRâ with greater potency and efficacy compared to
the racemic mixture 2 (Table 1).
To determine if the subtype selectivity of 2 was physiologi-
cally relevant, we measured ABCA1 expression in primary
macrophages derived from LXRR- and â-null mice treated with
1 or 2 (Figure 2a). The dual agonist 1 induced ABCA1 in
LXRR- and LXRâ-null macrophages to a similar level. In
contrast, mixture 2 induced higher levels of ABCA1 in LXRR-
null than in LXRâ-null macrophages, illustrating that the
compound is functionally LXRâ-selective at the concentrations
tested. Mixture 2 also failed to induce ABCA1 in LXRR/â
double null cells, corroborating its dependence on LXR for
activity (not shown). Interestingly, when tested for its ability
to promote APO-AI-dependent cholesterol efflux, mixture 2 and
its active enantiomer 3 both induced cholesterol efflux in a dose-
dependent manner, consistent with their ability to induce
ABCA1 expression (Figure 2b). Notably, the efficacy of 3 in
a Reagents and conditions: (i) t-BuOCONHNH2, EDC, DMAP, DMF/
THF; (ii) Lawesson’s reagent, THF, MW 80 °C; (iii) HCl (4 N), 1,4-dioxane;
(iv) (1) R2PhCHO, DIEA, CH2Cl2; (2) R3PhCOCl, DIEA.
this assay is similar to that of 1, despite its subtype selectivity
and lower potency and efficacy.
To explore the chemical basis of LXR activity and subtype
selectivity, we performed SAR analysis around lead compound
2 using a FRET-based coactivator recruitment assay to guide
our survey (Table 2). Substitutions around the phenyl group A
(Scheme 1) appeared limited to halogens and small lipophilic
substituents in the 3- and 4- position (5-7), while 4-substitutions
such as MeO, CN, SONH2, Et2N, t-Bu, and CF3 yielded inactive
mixtures (data not shown). Substitution of ring B with only one
methoxy group in the 2-position (as in 8) yielded a 10-fold more
potent mixture for LXRâ but less selective than the parent
mixture 2. Mixture 9 with only one methoxy group in the
3-position showed complete selectivity but a 10-fold decrease
in potency and a reduction in efficacy, suggesting that the
2-MeO substituent may be sufficient for activity and that the
3-MeO substituent, while not necessary for activity, may be
beneficial for selectivity. Addition of a 4-fluoro substitution to
the monomethoxy phenyl compounds yielded a 50-fold increase
in LXRâ potency but ablated subtype selectivity, as in 10.
2-Trifluoromethoxy substitution, as in 11, abolished LXR
activity while substitution with 2-OCHF2 still provided active
mixtures (12). Tying the two oxygens in a cycle as in the
difluoromethylenedioxy phenyl derivative 13 yielded active but

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4257
nonselective mixtures. Compounds where the 2-methoxy phenyl
ring was exchanged with a 2-methoxy pyridine ring were found
to retain activity with improved efficacy but showed less subtype
selectivity, as exemplified by compound 14.
Replacement of the hydrogen atom on the stereogenic center
of the thiadiazoline ring (C5) with an alkyl group caused almost
complete loss of activity, as in compound 15. Phenyl ring C
tolerated a variety of substitutions. In general, substitution of
the phenyl ring with at least one halogen atom in different
positions and in combination with other groups yielded active
mixtures with varying degrees of potency and efficacy that were
invariably LXRâ-selective in terms of efficacy separation, as
seen in compounds 17-27. Other phenyl substitutions such as
3-MeO, 3-F, 4-MeO, 4-OCF3, 4-NO2, and 4-NH2 were detri-
mental to activity (not shown).
Compounds were also tested in a qRT-PCR assay measuring
upregulation of endogenous ABCA1 expression in THP-1 cells
to verify the cellular properties of newly synthesized molecules
(Table 2). As expected, the compounds that showed the highest
level of efficacy were the less LXRâ-selective mixtures 10 and
14. Among the more selective mixtures, 5 elicited expression
of ABCA1 comparable to mixture 2 despite being slightly more
potent in the FRET assay. In contrast, mixture 25 showed a
comparable level of activity in the FRET assay, but demon-
strated a higher degree of efficacy in terms of ABCA1
expression when compared to mixture 2.
Figure 2. (a) Induction of ABCA1 expression in primary mouse
macrophages. Genotype of cells is shown above bars. Two concentra-
tions of 1 and 2 were used. Note the increased basal expression in
LXRâ null cells. (b) Apo AI-dependent cholesterol efflux from primary
mouse macrophages.
Unfortunately, despite adequate in vivo PK,30 when dosed
in mice, both mixtures 2 and 5 failed to elicit induction of
ABCA1 and SREBP-1c in liver or other tissues. One likely
reason may be that mixtures 2 and 5, as well as most of the
Table 2. SAR Analysisa
ABCA1
induction (%)
FRET LXRR
EC50 (%E)
FRET LXRâ
EC50 (%E)
cmpd
R1
R2
R3
1 µM
10 µM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15g
16
17
18
19
20
21
22
23
24
0.097 (100)
2.3 (42)
1.6 (64)
no fit
1.4 (62)
1.4 (52)
no fit
0.18 (80)
no fit
0.07 (92)
no fit
1.9 (76)
3.9 (71)
0.4 (71)
no fit
no fit
no fit
0.027 (100)
0.3 (99)
0.067 (94)
no fit
0.076 (98)
0.2 (98)
>10 (51)
0.035 (110)
3.7 (60)
0.006 (100)
3.4 (31)
0.15 (90)
0.8 (74)
0.21 (93)
4 (50)
No Fit
100
34
39
0
42
24
18
100
59
60
0
53
34
46
54
ND
59e
23
ND
61
4-Cl
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2-MeO
3-MeO
2-MeO-4-F
2-OCF3
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
2,4,6-tri-F
H
4-Clb
4-Clc
4-F
4-Me
3,4-di-Cl
4-Cl
4-Cl
4-F
4-Cl
4-F
4-Cl
4-F
4-F
4-Cl
4-Cl
4-Cl
4-F
4-F
3,4-di-F
4-F
57
NDd
99
ND
26
35
100
ND
ND
ND
23
35
30
27
21
2-OCHF2
2,3-difluoromethylenedioxy
f
79
2-MeO
ND
ND
ND
40
42
48
47
37
36
32
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
2-F
7.7(48)
2,4-di-F
2,3,6-tri-F
2,4,5-tri-F
3-F
3,5-di-F
3-Cl
6.7 (50)
no fit
no fit
1.5 (30)
2.1 (40)
no fit
0.98 (80)
0.97 (74)
0.6 (40)
0.56 (83)
0.47 (95)
0.25 (82)
0.14 (90)
4-F
4-F
13
21
2-Me,
2.4 (46)
5-F
2,6-di-F,
4-MeO
2,6-di-F,
4-Br
2,4,5-tri-F,
3-OMe
25
26
27
4-F
4-F
4-F
2,3-di-MeO
2,3-di-MeO
2,3-di-MeO
0.9 (47)
0.9 (43)
0.7 (64)
0.17 (100)
0.16 (92)
81
42e
42
ND
30
0.081 (100)
59
a No fit ) no significant efficacy (<15%) detected at any concentration. For FRET assays, data is reported in µM, assay performed in duplicate, variance
< 15%. Efficacy of compounds is reported as percent efficacy (%E) relative to 1 and defined as the percentage ratio between maximum coactivator recruitment
for the test compound and maximum coactivator recruitment for 1 in the same experiment. ABCA1 induction in human THP-1 cells was measured using
qRT-PCR. b R-enantiomer. c S-enantiomer. d ND ) not determined. e Drop-off in activity is due to toxicity. f 2-Methoxy-3-pyridyl. g Methyl-substituted
compound on thiadiazoline ring C5.

4258 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Brief Articles
transfected using FuGENE 6 (Roche). After 24 h, ligands were
added as 11-point dose-response curves starting at 10 µM. After
an additional 16 h, luciferase activity was read.
compounds shown, were found to be highly protein-bound
(>99.9%). This was reflected by a dramatic decrease in their
ability to induce ABCA1 in THP-1 cells when the assay was
run using a higher concentration of serum (20% FBS, not
shown). This liability limits the utility of these molecules in
animal models of disease; further optimization of this class of
thiadiazolines is required to increase plasma-free fraction and
establish their therapeutic potential in vivo.
Fluorescence-Resonance Energy Transfer Assay: FRET assay
was performed in 384-well plates in a 20 µL final volume. A mix
of 20 nM His-tagged hLXR, 300 nM biotinylated SRC-1 peptide
(Biotin-CPSSHSSLTERHKILHRLLQEGSPSC-OH), Europium-
labeled anti-His antibody (Perkin-Elmer), and allophycocyanin-
labeled streptavidin (Prozyme) was prepared. Ligands were tested
in 12-point dose-response curves starting at 20 µM. Plates were
incubated for 1 h at 37 °C and FRET was read on an AnalystGT
(Molecular Devices).
Scintillation Proximity Assay: SPA was performed as in
Janowski et al.27 Scintillant-filled beads (Amersham) were diluted
in SPA buffer (10 mM K2HPO4, 10 mM KH2PO4, 2 mM EDTA,
50 mM NaCl, 1 mM DTT, 2 mM CHAPS, and 10% glycerol) to
a final concentration of 5 mg/mL. The assay was performed in 384-
well plates (Packard) in 20 µL containing beads (0.2 mg per well)
and His-tagged hLXR LBD (200 ng per well). The amount of
protein did not deplete ligand concentration. Displacement assays
used 30 nM of [3H]-1a (Amersham). Ligands were tested in 12-
point dose-response curves starting at 20 µM. Plates were shaken
for 3 h at room temperature and read in a Packard Topcount at 1
min per well. Wells devoid of competitor represented 100% binding.
Nonspecific binding was measured by leaving LXR protein out of
the SPA reaction.
Conclusions
In summary, we have described the first examples of LXRâ
agonists with separation of efficacy from LXRR. Functional
selectivity was confirmed using macrophages derived from
LXR-null mice: compounds showed significantly higher ex-
pression of ABCA1 in LXRR-null macrophages than in LXRâ-
null macrophages. In spite of their selectivity and modest
potency, these compounds can induce APO-AI-dependent
cholesterol efflux with similar efficacy to that of the potent dual
LXR agonist 1. Our results indicate that it is possible to achieve
significant LXRâ selectivity in a small molecule while main-
taining functional LXR activity in a relevant setting.
Experimental Section
General Procedure for the Preparation of Benzothiohy-
drazides Hydrochloride Salt. To a solution of a benzoic acid (35.7
mmol) in 72 mL of a mixture of DMF and THF (1:1) were added
tert-butyl carbazate (37.5 mmol), EDC (39.3 mmol), and N,N-
dimethylaminopyridine (0.54 mmol). After 10 min, the mixture
became homogeneous and stirring was continued for 3 h until the
reaction was complete by TLC and LC/MS. The reaction mixture
was poured into ice. Upon addition of diethylether, the organic layer
was separated. The organic layer was washed with sodium bisulfite,
satd sodium bicarbonate, and satd sodium chloride solution, dried
over magnesium sulfate, and concentrated to yield the benzoylhy-
drazinecarboxylic acid tert-butyl ester. To a mixture of the
benzoylhydrazinecarboxylic acid tert-butyl ester (11.1 mmol) in
10 mL of dry THF, Lawesson’s reagent (11.6 mmol) was added,
and the mixture was heated in the microwave oven at 80 °C for 20
min. The reaction mixture was concentrated and purified by
automated column chromatography using hexanes/EtOAc. The
thiobenzoyl hydrazinecarboxylic acid tert-butyl ester obtained (18.5
mmol) was added to HCl (4 N) in 1,4-dioxane (185 mmol). The
mixture was stirred at room temperature for 1 h. Hexanes were
added to further precipitate the product, which was filtered off,
yielding the benzothiohydrazide.
General Procedure for the Preparation of N-Acyl Thiadia-
zolines. To a heterogeneous mixture of a benzothiohydrazide
hydrochloride salt (0.107 mmol) in CH2Cl2 (1 mL) an aldehyde
(0.128 mmol) and DIEA (0.128 mmol) were added. After 10 min,
the mixture became homogeneous and the reaction was complete
by TLC and LCMS to give the 2,5-substituted-2,3-dihydro-[1,3,4]-
thiadiazole, which was used in the next step without evaporation
of the solvent. To the solution was added DIEA (0.16 mmol) and
2-fluorobenzoyl chloride (0.16 mmol), and the reaction mixture was
stirred for 12 h at room temperature. After evaporation of the
solvent, the residue was purified by automated chromatography
(hexane/EtOAc) or preparative LC/MS (20 to 100% MeCN/H2O).
Biological Assays. Transcriptional Assays: Expression con-
structs for chimeric GAL-hLXR LBDs were generated by fusing
the hLXRR and â LBDs to the DBD of GAL4 and inserting them
into pcDNA3 (Invitrogen) as previously described.5 Assays were
validated using known ligands. The pTK-2xLXRE-luciferase
reporter and pcDNA3 expression plasmids for full-length human
and mouse LXRR/â have been described.5,26 Cells were cultured
in DMEM with 10% FBS. For transcriptional assays, cells were
plated in 384-well plates in DMEM with 10% fetal bovine
lipoprotein-deficient serum (FBLDS, Intracel) in the presence of
7.5 µM lovastatin and 100 µM mevalonic acid. HepG2 cells were
Dose-response and competition curves were generated by
nonlinear regression analysis using GraphPad Prism, and the
apparent equilibrium association/dissociation constants (Kd and Ki
values) were determined using the method described by DeBlasi
and colleagues.28
Cholesterol Efflux: Primary peritoneal macrophages were plated
at 50% confluence in 24-well plates. On day 2, the cells were
washed and incubated for 24 h in medium A supplemented with
an ACAT inhibitor (58-035; 2 µg/mL) and [3H]cholesterol (1.0
µCi/mL). Compounds were added to the cells as indicated. On day
3, the cells were washed twice with PBS and then incubated for
2-4 h in fresh medium, devoid of radiolabeled cholesterol, but
containing the indicated compounds. The cells were rewashed
before addition of 1.0 mL medium B (DMEM containing 0.2%
BSA) in the absence or presence of apoAI (15 µg/mL). After 4 h
incubation, the medium was removed and centrifuged at 14 000 ×
g for 10 min, and the radioactivity was determined by liquid
scintillation counting. The cells were dissolved in isopropanol, and
an aliquot was used to determine total cell-associated radioactivity.
The apoAI-dependent efflux of radioactive cholesterol from the cells
into the medium was determined as a percentage of the total
radioactivity in the cells and medium for each condition. Each efflux
assay was performed in triplicate.
Gene Expression Analysis: Human THP-1 cells were grown
in propagation media (10% defined FBS, 2 mM L-glutamine, 10
mM HEPES, 1.0 mM sodium pyruvate, 4.5 g /L glucose, 1.5 g/L
bicarbonate, and 0.05 mM 2-mercaptoethanol in RPMI 1640). On
day 1, 125 000 cells/well were plated in propagation media plus
40 ng/mL PMA on a 48-well dish. On day 2, media was replaced
with fresh assay media (same as propagation media but with 2%
FBLDS as the serum supplement) and compounds were added 6 h
later (1 or 10 µM in DMSO). Cells were then incubated for 24 h
before being harvested, and RNA was isolated using the RNeasy
kit (Qiagen) with DNaseI option. RNA was analyzed by TaqMan
qRT-PCR using the one-step Superscript III platinum reagent
(Invitrogen). Samples were run in triplicate as multiplexed reactions
with an internal control (36B4). Probe and primer sequences for
ABCA1 and FAS are available on request. Fold induction induced
by compound was calculated in reference to DMSO. Primary
peritoneal macrophages were elicited from LXR mutant mice by
IP injection of 4% thioglycollate (Difco) and subsequent lavage.12
Cells were washed extensively and then plated for 24 h. Adherent
cells were treated in all subsequent steps in identical fashion to the
THP-1 cells described above, except no PMA was used to prompt

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4259
(14) Laffitte, B. A.; Chao, L. C.; Li, J.; Walczak, R.; Hummasti, S.; Joseph,
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and 5 in mice (2.5 mg/mL in PEG300/Tween 80 [4:1]). Intravenously,
mixture 2 dosed at 5 mpk showed AUC ) 428.24 min‚ug/mL, Cmax
) 20.4 µM, Cl ) 12.23 (mL/min/kg), Vss ) 1.65 (L/kg). Mixture 5
dosed at 5 mpk showed AUC ) 2143.69 min‚ug/mL, Cmax ) 70.9
µM, Cl ) 2.26 (mL/min/kg), Vss ) 0.44 (L/kg). Orally, mixture 2
dosed at 20 mpk demonstrated an AUC ) 475 min‚ug/mL and Cmax
of 4.9 µM; bioavailability was 28% and t1/2 was 6.7 hours. Mixture
5 dosed at 20 mpk showed an AUC ) 1005 min‚ug/mL, Cmax of 6.9
µM but poorer bioavailability (11%); t1/2 was 4.5 hours.
differentiation. Primary human hepatocytes were cultured and
analyzed as described in reference 14.
Animal Experiments: The 10-week-old male C57BL/6 mice
were maintained on a standard chow diet. Mice were gavaged daily
with vehicle, 1 (50 mpk), or test compound (20-200 mpk) for 3
days before sacrifice. Mice were fasted overnight prior to the last
dose of compound and sacrificed 3 h later. RNA was isolated from
tissues and gene expression analyzed using TaqMan-based qRT-
PCR. All experiments were approved by the Institutional Animal
Care and Use Committee of The Genomics Institute of the Novartis
Research Foundation.
Acknowledgment. We thank Glen Spraggon for resolution
of the crystal structure of 4, Van Nguyen-Tran, Cara Cuc T.
Ngo, and David Huang for animal studies and Patricia McNeeley
for technical help.
Supporting Information Available: Spectral and purity data
for compounds 2-27. This material is available free of charge via
the Internet at http://pubs.acs.org
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