Indazole-based Liver X Receptor (LXR) modulators with maintained atherosclerotic lesion reduction activity but diminished stimulation of hepatic triglyceride synthesis

Article abstract of DOI:10.1021/jm800799q

A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRα than on LXRβ. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.

Full text of DOI:10.1021/jm800799q

J. Med. Chem. 2008, 51, 7161–7168
7161
Indazole-Based Liver X Receptor (LXR) Modulators with Maintained Atherosclerotic Lesion
Reduction Activity but Diminished Stimulation of Hepatic Triglyceride Synthesis|
Jay Wrobel,*,† Robert Steffan,† S. Marc Bowen,† Ronald Magolda,† Edward Matelan,† Rayomand Unwalla,† Michael Basso,‡
Valerie Clerin,‡ Stephen J. Gardell,‡ Ponnal Nambi,‡ Elaine Quinet,‡ Jason I. Reminick,3 George P. Vlasuk,‡ Shuguang Wang,‡
Irene Feingold,§ Christine Huselton,§ Tomas Bonn,# Mathias Farnegardh,# Tomas Hansson,# Annika Goos Nilsson,#
Anna Wilhelmsson,# Edouard Zamaratski,# and Mark J. Evans‡
Wyeth Pharmaceuticals, 500 Arcola Road, CollegeVille, PennsylVania 19426, and Karo Bio AB, Huddinge, Sweden
ReceiVed July 2, 2008
A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These
compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly
weaker with respect to potency and efficacy on LXRR than on LXRꢀ. Lead compounds in this series 12
(WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full
agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol
efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR
knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13
showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses
in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In
contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to
increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results
suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.
Cardiovascular risk is increased in patients with dyslipidemia,
diabetes, and the metabolic syndrome. In spite of the wide
availability and use of effective lipid-lowering and glucose-lowering
therapies, cardiovascular disease remains the leading cause of
morbidity and mortality in the industrialized world. Liver X
receptors (LXRsa) belong to a transcription factor subfamily that
consists of LXRR (expressed in adipose, intestine, liver, kidney,
and
macrophages)
and
LXRꢀ
(expressed
ubiqui-
tously).1-4 Oxysterols are endogenous ligands for LXR. Ligand
binding results in exchange of corepressors and coactivators
associated with LXR-RXR heterodimers that are bound to DNA
response elements. LXR-mediated gene expression has been shown
to induce reverse cholesterol transport (RCT)-cholesterol efflux
from macrophages via ApoE and ATP-binding cassettes (ABC
transporters ABCA1, and ABCG1), and to enhance bile acid
production in mice. In mice, the loss of LXR expression increases
atherosclerosis,5 while conversely, treatment of mice with an LXR
agonist reduces atherosclerosis development.6 An LXR agonist may
offer potential benefits on lipid metabolism, improving glucose
metabolism and vascular inflammation and thereby reducing the
Figure 1. Known LXR agonists and the indazole lead.
risk of cardiovascular disease. Specifically, LXR agonists may
increase insulin sensitivity, suppress gluconeogenic genes, and
antagonize the expression of inflammatory genes (e.g., iNOS, IL-
1B, IL-6, and COX2), as well as enhance reverse cholesterol
transport, all contributing to the possibility of reduced morbidity
and mortality in patients with established atherosclerosis. Known
LXR modulators such as 1 (TO901317)7 and 2 (GW3965)6,8
(Figure 1) up-regulate LXR target genes in vivo and retard the
advancement of atherosclerotic processes in mouse models of
atherosclerosis. However, these compounds also induce hypertrig-
lyceridemia by LXRR induction of genes in the liver involved in
fatty acid biosynthesis, including sterol regulatory binding element
protein 1c (SREBP-1c) and fatty acid synthase (FAS).7 The
pharmacological challenge, therefore, is to identify LXR modulators
that selectively activate desirable LXR target genes.
|
Dedicated to the memory of Dr. Ronald Magolda.
* To whom the correspondence should be addressed. Phone: 484-865-
2480. Fax: 484-865-9399. E-mail: wrobelj@wyeth.com.
†
Chemical and Screening Sciences, Wyeth Pharmaceuticals.
Cardiovascular and Metabolic Diseases, Wyeth Pharmaceuticals.
Translational Medicine, Wyeth Pharmaceuticals.
Drug Metabolism, Wyeth Pharmaceuticals.
‡
3
§
#
Karo Bio AB.
a Abbreviations: LXR, liver X receptor; ABCA1/G1, adenosine triph-
osphate binding cassette, A1/G1; HepG2, human hepatocellular liver
carcinoma cell line; THP-1, human acute monocytic leukemia cell line;
LDLR, low density lipoprotein receptor; TG, triglycerides; RXR, retinoic
acid X receptor; RCT, reverse cholesterol transport; SREBP-1c, sterol
regulatory binding element 1c; FAS, fatty acid synthase; LBD, ligand
binding domain; PPARs (R, γ, or δ), peroxisome proliferator-activated
receptor R, γ, or δ; FXR, farnesoid X receptor; PXR, pregnane X receptor;
TR, thyroid receptor; cyp7A1, cytochrome P450 7A1 hydroxylase; CETP,
cholesterol ester transfer protein; DMF, dimethylforamide, EtOAc, ethyl
acetate.
Previous drug design efforts to improve the pharmaceutical
properties of an early quinoline lead series led to the preparation
potent phenylacetic acid substituted quinolines represented by
3 (WAY-254011)9 and 4.9,10 Both compounds significantly
10.1021/jm800799q CCC: $40.75
2008 American Chemical Society
Published on Web 10/31/2008

7162 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Table 1. LXR Activity of Indazoles of General Structure 10a
Wrobel et al.
compd
Y
R1
R2
LXRꢀ bindingb
LXRR bindingb
Gal4 LXRꢀc
Gal4 LXRRc
1
9
13
n ) 3
100
n ) 7
10
n ) 15
8
n ) 3
279
n ) 5
78
0.17 (100)
n ) 177
0.31 (77)
n ) 5
0.09 (63)
n ) 3
0.09 (70)
n ) 3
3.26 (53)
0.14 (100)
n ) 177
0.66 (58)
n ) 5
0.24 (90)
n ) 3
0.16 (82)
n ) 3
4.27 (31)
n ) 1
1.81 (58)
n ) 1
6.66 (53)
n ) 12
6.10 (35)
n ) 13
5.02 (32)
n ) 3
8.40 (43)
12.77 (35)
13.28 (64)
14.40 (45)
15.21 (36)
13.44 (50)
15.83 (5)
NT
n ) 5
12
n ) 7
2
n ) 15
2
n ) 3
41
2
3
4
5
H
F
F
F
F
H
H
n ) 5
10
11
12
13
14
Me
Cl
F
Me
F
0.99 (75)
24
n ) 8
33
n ) 9
32
n ) 8
49
58
38
30
40
38
48
1853
n ) 1
179
n ) 8
248
n ) 9
297
n ) 8
317
401
260
252
379
304
465
3101
n ) 1
3.67 (73)
n ) 12
4.15 (64)
n ) 13
2.89 (54)
n ) 3
5.72 (66)
9.05 (74)
9.80 (104)
11.22 (77)
10.2 (67)
7.44 (68)
15.46 (38)
NT
H
Cl
H
15
16
17
18
19
20
21
22
F
F
H
H
F
Cl
Cl
F
CF3
H
F
Cl
Cl
F
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
CF3
a Results are given as the mean of two independent experiments unless otherwise indicated. b IC50 in nM. The standard deviations for these assays were
typically (30% of the mean or less. c EC50 in µM (% efficacy). The standard deviations for these assays were typically (50% of the mean or less. The %
of efficacy is relative to that of 1. NT ) not tested.
Scheme 1a
reduced lesion size in an atherosclerosis mouse model (8-week
LDLR -/- mouse model) at 10 mg/kg.8,11,12 However, 3 also
caused substantial liver and plasma triglyceride (TG) increases
in hamster (vide infra). These compounds were potent full
agonists for both LXR isoforms (relative to a standard LXR
modulator 1). The growing body of evidence supports the
hypothesis that the lipogenic effects in liver are primarily LXRR
mediated.13,14 Furthermore, both isoforms play a role in mac-
rophage RCT processes and atherosclerosis but the LXRꢀ
isoform alone may be sufficient for these processes.13,15,16 Thus,
a more desired profile for a compound would be one that
minimizes the agonist activity due to the LXRR isoform and
maximizes the LXRꢀ agonist activity.16 With this premise our
medicinal chemistry efforts focused on finding compounds with
reduced LXRR agonist activity.
In this regard, our indazole-based series (lead candidate 5)
a Reaction conditions: (a) (COCl)2, 95%; (b) NH(OMe)Me ·HCl, K2CO3,
showed promise as a series to explore for optimization.
Compound 5 had an IC50 value of 41 nM for binding to LXRꢀ
and was a partial agonist on LXRR according to our transac-
tivation assay (Table 1). This compound is structurally analogous
to quinoline series members such as 39 and can be viewed as
collapse of the 6/6 quinoline ring system to a 6/5 indazole
system. However, compound 5 was rapidly metabolized in
mouse, rat, and human microsomes and subsequent PK studies
in mouse revealed that the compound had high clearance, low
drug levels, and poor oral bioavailability. Metabolic identifica-
tion studies indicated that the primary metabolite was oxidative
metabolism on the phenyl rings. Thus, the optimization of
ADME and PK parameters of compound 5 became a primary
area of focus. Herein, we describe analogues of 5 with improved
metabolic stability and their subsequent LXR activity in vitro
and in a hamster lipid and mouse atherosclerosis model.17,18
toluene, water, 100%; (c) p-fluoro- or p-chlorophenylmagnesium bromide,
THF, 0 °C to room temp; (d) NH2NH2 ·H2O, THF, 55-65 °C, 8 h, 77%;
(e) compound 9, DMF, 120 °C, 60-90%.
ethylbenzoic acid was converted to the Weinreb amide under
standard means and reacted with p-fluoro- or p-chlorophenylmag-
nesium bromide to afford the 2-fluorobenzophenones 7. Reaction
of 7 with hydrazine led to indazoles 8. Heating 8 with the appropriate
benzyl bromide 9 in DMF with the exclusion of a base led to the N2
alkylated indazoles 10 with no or very little N1 alkylation. The NMR
spectra for the N2 analogues displayed a typical pattern distinct from
the N1 substituted regioisomers. In the case of compound 14 the proton
at position 4 displays a doublet at δ 7.85, whereas in the regioisomer
substituted at N-1 the same proton displays a doublet at δ 8.39. A 1H
NMR NOESY spectrum for compound 14 was obtained confirming
N2 substitution (see Supporting Information). Further evidence of N2
substitution is confirmed in the X-ray crystal structure of compound
12 (Figure 3).
Chemistry
The synthesis of the indazole analogues related to 5 is shown
in Scheme 1. The commercially available 2-fluoro-3-trifluorom-

LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7163
Figure 2. Path toward metabolically stable analogues of 5.
Figure 4. X-ray crystal structure of hLXRꢀ complexed with 12
(colored cyan) overlaid with published X-ray structure of compound 2
(colored magenta). Only key residues within the binding site are shown
from 2.
Table 2. Cellular Activity of Selected Compoundsa
compd
ABCA1b
C-effluxc
SREBP1cd
lipid accume
1
44 (100)
n ) 203
434 (178)
n ) 4
84 (155)
n ) 8
541 (100)
n ) 3
551 (89)
n ) 4
3 (100)
n ) 3
61 (100)
n ) 14
137 (100)
n ) 73
2002 (45)
n ) 3
223 (85)
n ) 3
1911 (35)
n ) 3
998 (38)
2
3
31 (84)
210 (137)
6 (88)
n ) 4
1275 (140)
n ) 5
549 (132)
120 (96)
2114 (58)
1222 (72)
12
13
Figure 3. X-ray crystal structure of 12 bound to LXRꢀ. Only key
residues involved in ligand interaction are shown for clarity. Hydrogen
bonds to key residues are shown as yellow dotted lines.
a EC50 in nM (% efficacy). Results are given as the mean of two
independent experiments unless otherwise indicated. The standard deviations
for these assays were typically (50% of the mean or less. The % of efficacy
is relative to that of 1. b In differentiated THP-1 cells. c In differentiated
THP-1 cells loaded with ac-LDL and labeled with [3H]cholesterol represent-
ing atherosclerotic foam cells. d In HepG2 cells. e Lipid accumulation is
measured in HepG2 cells.
Results and Discussion
The LXR binding activities of compounds were evaluated
(Table 1) using recombinant human LXRR or LXRꢀ ligand
binding domains (LBDs) with [3H]1 as a tracer. Huh-7 human
hepatoma cell based Gal4 LXRR or LXRꢀ transactivation assays
were used to assess the agonist potency and efficacy. Efficacy
in this assay is relative to 1, which is defined as 100%. SAR
studies on the indazole series revealed that small lipophilic
substituents on the 2-benzyl moiety enhanced potency. For
example, compound 11 (Figure 2) with 2′, 4′-dimethyl groups
on the 2-benzyl moiety had a LXRꢀ binding IC50 of 10 nM.
Like 5, this compound showed rapid in vitro metabolism and
poor PK parameters in mice [high clearance (Clp), low exposure
(AUC), and low oral bioavailability (% F)]. However, oxidative
metabolism was now shunted toward the methyl groups of the
2-(2′, 4′-dimethyl)benzyl substituent although the p-fluorine
substituent of 11 impeded oxidative metabolism at the 3-phenyl
ring. Replacement of the dimethyl groups of 11 with halogens
led to compounds (12-22) with increased metabolic stability
and improved PK parameters.
Compounds 12-16, like compound 11, contain a p-fluoro
substituent on the indazole 3-phenyl ring (i.e. Y ) F). However,
the benzyl moiety of 12-16 contained chloro, fluoro, or
hydrogen in place of the dimethyl groups of 11. These
compounds were slightly weaker binders on both LXRꢀ (IC50
values of of 24-58 nM) and LXRR (IC50 values of 179-401
nM) compared to 11, and this translated to weaker transacti-
vation potencies, although the efficacy on LXRꢀ or LXRR was
similar to that for 11. The position or type (Cl or F) of the
halogen (R1 or R2) did not have much effect on the binding or
transactivation potencies of the compound with the exception
of a p-chloro group (R2 ) Cl), which resulted in a slightly
weaker potency compound (16). Congeners with a 3-(p-
chloro)phenyl group (17-20) had similar binding potencies
compared to the 3-(p-fluoro)phenyl containing compounds
(12-16); however, they were less potent agonists. The analogues
with trifluoromethyl groups (21 and 22) showed reduced potency
relative to compounds with chloro or fluoro groups. Compound
22 was an exceptionally weak binder, most likely because of
steric and electronic effects within the LBD.
An X-ray structure of 12 bound to the LXRꢀ ligand-binding
domain was also obtained (Figure 3).19 There are many
similarities to previously published structure of 3.9 Ligand
recognition was achieved by multiple interactions that line the
pocket, the N1 indazole nitrogen formed a H-bond interaction
with the Nꢀ(epsilon) of His435, while the 7-trifluoromethyl
group, being in proximity, made attractive electrostatic interac-
tions; i.e., d(N-F) ) 2.9 Å with this residue. The N2-benzyl
group was enclosed in a hydrophobic pocket surrounded by three
Phe residues (271, 340, and 349). This structure provides a
plausible explanation for the weak potency of compound 22.
In compound 22 the p-fluoro moiety of the 2-benzyl group of
12 is replaced by the more bulky and electronegative trifluoro-
methyl moiety. This would place the CF3 group of 22 near the
π-cloud of Phe340 residue and lead to an unfavorable electro-
static stacking interaction.
Interestingly, an overlay of X-ray structure of 12 with
published structure of a larger LXR ligand, i.e., 220 (Figure 4)19
revealed important differences in their interactions with the
receptor. Although both ligands induced an agonist conformation
of helix H12, the binding pocket of 2 was considerably larger
and different from that of 12. The carboxylic acid group of 2

7164 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Table 3. PK Parameters for Compounds 5 and 13 in C57 Male Micea
Wrobel et al.
compd
iv t1/2 (h)
Cl ((mL/min)/kg)
Vss (L/kg)
po t1/2 (h)
Cmax (ng/mL)
Tmax (h)
AUC(0-∞) (h·ng/mL)
F (%)
5
13
1.9
7.9
81
57
5.2
30.3
2.2
5.8
124
633
0.25
0.5
321
3101
16
106
a Single dose PK study done in male C57 mice (weight 20-35 g). For iv, compound was given at 1 mg/kg in 2% ethanol, 40% PEG200 in saline. For
po, compound was given at 10 mg/kg in 0.5% methylcellulose, 2% Tween-80 in water. Dose volume for iv and po studies was 5 mL/kg.
Figure 5. Male LDLR -/- mice were fed a Western diet (gray bars, n ) 28) or diet supplemented to deliver 10 mg/kg compound 2 (n ) 28),
10 mg/kg compound 3 (n ) 20), or 15 mg/kg compound 13 (n ) 9). After 8 weeks, plasma total cholesterol (TC) and total triglyceride (TG) were
determined using a Roche 911 clinical chemistry analyzer. Values are the mean ( SEM. Aortic lesion area was determined by en face analysis of
Oil Red O stained aortas. Lesion size in each individual animal is shown (filled circles) along with the group mean (lines): (/) p < 0.05 vs control
animals by ANOVA.
was able to extend out to the ꢀ-hairpin loop and make additional
interactions with residues from this region, i.e., Arg319 and the
NH backbone of Leu330 residue. By reaching into this cavity,
compound 2 was further able to stabilize this flexible region
over compound 12. Additionally, the diphenylethyl chain of 2
induced small shifts within the side chain residues lining the
hydrophobic pocket, i.e., phe271, phe340, and phe349 in order
to accommodate this larger group. Taken together, these
observed differences in the binding pocket might explain why
compound 2 has increased binding and transactivation potency
over compound 12.
Compounds 12 (WAY-252623) and 13 (WAY-214950) were
among the most potent and stable of the compounds 12-22
showing reduced LXRR agonism, and so these two compounds
were further profiled in gene regulation and functional assays
to better assess their ability to promote the desired reverse
cholesterol transport activity (ABCA1 expression in THP-1 cells
and cholesterol efflux in macrophage-like foam cells) in relation
to undesired TG synthesis activities (SREBP1c expression and
lipid accumulation in HepG2 liver cells). The data are shown
in Table 2. Literature compounds 1 and 3, which are potent
full agonists for LXRR and LXRꢀ in the transactivation assays,
showed little differentiation between cellular reverse cholesterol
transport in macrophage cells versus cellular TG synthesis in
HepG2 cells. In contrast, treatment of cells with either 12 or
13 increased ABCA1 gene expression in THP-1 cells with
similar full efficacy, albeit with lower potency than 1 or 3.
However, 12 or 13 were partial agonists and had weaker potency
for increasing SREBP1c mRNA in liver cells compared to 1 or
3. An LXR modulator having this profile may have less liability
with respect to TG synthesis versus promoting reverse choles-
terol transport.
Compounds 12 and 13 did not display agonist activity against
PPARs (R, γ, or δ) and FXR and were >40-fold (for 12) and
>65 fold (for 13) less potent, based on binding IC50 values over
PXR, RXR, GR, MR, AR, PR, ER, and TR over LXRꢀ (see
Supporting Information for details).
Compound 13 showed acceptable PK parameters in C57 mice
(Table 3). Following an iv dose, the half-life was long, the
clearance was moderate, and the volume of distribution was
high. Following an oral dose, the terminal half-life was long
and the oral bioavailablity was high. This was in contrast to
the des-halogen early indazole lead 5, which had high clearance,
low oral bioavailability, and much lower exposure and Cmax
relative to 13, thus proving that halogenation protects the
molecule from rapid metabolism.
Synthetic LXR agonists such as compound 2 significantly
reduce atherosclerotic lesion size in LDLR -/- mice. To
compare the in vivo activity of selected compounds, male LDLR
-/- mice were fed a Western diet or Western diet supplemented
with 2, 3, or 13 for a period of 8 weeks.21 Compound 2 reduced
total cholesterol levels by 17%, had no effect on plasma
triglyceride level, and reduced lesion size by 40% (Figure 5),
nearly identical to published results of 18% total cholesterol
reduction, no TG effect, and 34% lesion reduction.6 Compound
3 produced very similar results, lowering plasma total cholesterol
by 20%, having no effect on plasma triglyceride levels, and
reducing lesion size by 38%. Compound 13 produced a
nonsignificant 10% decrease in total cholesterol, had no effect
on plasma triglyceride levels, and reduced lesion size by 28%.
Compound treatment did not affect animal weights. There was
no significant difference between the magnitude of lesion
reduction by 2, 3, or 13. Thus, all three LXR agonists similarly
reduced lesion size without producing any detrimental plasma
lipid effects.
The gene expression data were borne out in cellular functional
assays. Compounds 12 and 13 stimulated [3H] cholesterol efflux
in THP-1 foam cells in a concentration-dependent manner. The
compounds were as efficacious as 1 or 3 although not as potent
in this assay. On the other hand, 12 or 13 showed relatively
poor efficacy (and potency) relative to the full agonists 1 and 3
with respect to TG accumulation in HepG2 cells and had
reduced potency for SREBP-1c induction. The differential
activities on liver cells and macrophages are a favorable profile
for an LXR modulator.
In addition, the effects of compound 13 on LXR responsive
genes were monitored in several tissues. In the duodenum
ABCA1 gene expression was up-regulated (2.3 ( 0.14)-fold
by 13 and (6.9 ( 0.08)-fold by compound 2. In the liver,
CYP7A1, which is regulated by LXR only in mice, was
increased (4.0 ( 0.10)-fold by compound 13 and (1.9 ( 0.14)-
fold by 2.
Although LXR agonists such as 2 favorably reduced athero-
sclerotic lesion size in mice, in species such as the hamster and

LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7165
Figure 6. Male Golden Syrian hamsters were treated by oral gavage with 1 mL of 0.5% methylcellulose/2% polysorbate (Tween) 80 vehicle (open
bars), 3 mg/kg compound 3 in vehicle (gray bars), or 100 mg/kg compound 13 in vehicle (black bars) for 7 days. On the final day, food was
removed at time of final dosing. Two hours later the animals were euthanized. RNA was prepared from duodenum and liver and quantified by real
time PCR. All gene expression values were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, with the expression
seen in vehicle treated animals defined as 1.0 for each gene. Plasma total cholesterol and triglyceride were determined using a Roche 912 clinical
chemistry analyzer: (/) p < 0.05 vs vehicle control.
primates, compound 2 elevated plasma triglyceride as well as
plasma total and LDL cholesterol but lowered HDL choles-
terol.22 The basis for this effect may be due to LXR induction
of cholesterol ester transfer protein (CETP) expression in these
species. To determine whether compounds 3 and 13 produced
similar lipid effects, hamsters fed a standard chow diet were
treated with 3 mg/kg compound 3 or 100 mg/kg compound 13.
These doses were chosen to produce equivalent induction of
ABCA1 and ABCG1 expression in both the duodenum and the
liver (Figure 6). Both compounds 3 and 13 produced equivalent
increases in plasma total cholesterol levels, and both compounds
produced identical increases in hepatic CETP expression. In
contrast, while compound 3 increased plasma triglyceride levels
by 25%, compound 13 had no effect on plasma triglyceride
levels.23 Neither compound showed a hepatic TG increase at
these doses. Hepatic expression of fatty acid synthase (FAS),
the rate limiting enzyme for triglyceride synthesis in the liver,
was induced 4-fold by compound 3 but was not induced by
compound 13. Together, these results suggest that LXRR partial
agonist compound 13 has a reduced propensity to elevate plasma
triglycerides compared to other synthetic LXR agonists in the
hamster, potentially due to a selective gene regulation profile
in the liver.
transcription in hamster relative to the potent LXR agonist 3
when both compounds were administered at doses that generate
comparable ABC transporter gene activity. Thus, our premise
of minimizing LXRR agonist efficacy while maintaining LXRꢀ
efficacy was shown to provide compounds that retain antiath-
erosclerosis benefits of LXR agonists while reducing the
unwanted lipid effects of these agonists.
Experimental Section
General. Solvents and chemicals were purchased from EM
Sciences, VWR, Oakwood, and Aldrich Chemical Co. and used
without further purification. MS results were obtained on an Agilent
MS. High-resolution mass spectra were obtained on a Waters
LC-TOFMS instrument and were measured to within 5 ppm of
calculated values. 1H NMR spectra were obtained on a Varian (400
MHz) instrument. NMR data are given as δ values (ppm) using
tetramethylsilane as an internal standard (δ ) 0 ppm).
2-(2,4-Dimethylbenzyl)-3-(4-fluorophenyl)-7-(trifluoromethyl)-
2H-indazole (11). Step 1: Preparation of (2-Fluoro-3-
(trifluoromethyl)phenyl)(4-fluorophenyl)methanone. A solution
of 2-fluoro-3-trifluoromethylbenzoic acid (10 g, 65 mmol) and
oxalyl chloride (5.7 mL, 65 mmol) in 100 mL of dry CH2Cl2 was
treated with a few drops of DMF. The solution was stirred at
ambient temperature for 1 h. In one portion was added N,O-
dimethylhydroxylamine hydrochloride (16.8 g, 174 mmol) followed
by 16 mL of pyridine dropwise. The mixture was stirred for 48 h
at ambient temperature. The mixture was partitioned with H2O. The
organic phase was washed with 2 N HCl, brine and was dried with
Na2SO4. Concentration of the organic phase resulted in 13.3 g of
the intermediate 2-fluoro-N-methoxy-N-methyl-3-(trifluoromethyl)-
benzamide as a clear oil.
Conclusion
Indazoles 5-21 were partial agonists in transactivation assays,
particularly on LXRR relative to full agonists 1-4. Despite their
partial agonism in transactivation assays, compounds 12 and
13 showed full agonism (relative to 1) in THP-1 cells with
respect to increasing ABCA1 gene expression and on cholesterol
efflux in THP-1 foam cells compared to 1-3. These in vitro
LXR activities of 13, although lower in potency than 1 or 3,
were sufficient to translate to in vivo activities, since the
compound showed reduction of aortic arch lesion progression
and stimulation of LXR genes in the duodenum and liver in
LDLR -/- mice. Compounds 12 and 13 also showed less
potential for triglyceride elevation over full agonists such as 1
and 3. The compounds were partial agonists in HepG2 cells
with respect to up-regulation of SREBP1-c, a gene involved in
liver TG synthesis, and were less efficient in promoting lipid
accumulation in this cell type. This translated to an inability of
13 to increase plasma triglyceride levels or liver FAS gene
To a stirred solution of 2-fluoro-N-methoxy-N-methyl-3-(trif-
luoromethyl)benzamide (12.8 g, 65 mmol) in 150 mL of THF was
added 4-fluorophenylmagnesium bromide (2 N in diethyl ether, 32.5
mL, 65 mmol), and the mixture was heated at 50 °C overnight.
The reaction mixture was partitioned with EtOAc and H2O. The
organic phase was dried (Na2SO4) and concentrated in vacuo. The
residue was purified by flash chromatography (silica gel, hexane/
1
CH2Cl2, 3:1) to give the title compound (9.78 g, 52% yield). H
NMR (DMSO-d6, 400 MHz) δ 7.43 (t, 2 H, J ) 8.83 Hz), 7.61 (t,
1 H, J ) 7.81 Hz), 7.82 (m, 3 H), 8.06 (t, 1 H, J ) 7.04).
Step 2: Preparation of 3-(4-Fluorophenyl)-7-(trifluoro-
methyl)-1H-indazole. A solution of (2-fluoro-3-(trifluorometh-
yl)phenyl)(4-fluorophenyl)methanone (6.58 g, 23 mmol), hydrazine
hydrate (7.36 mL, 230 mmol), and dimethylaminopyridine (2.8 g,

7166 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Wrobel et al.
23 mmol) in 20 mL of pyridine was heated at 110 °C overnight.
The reaction mixture was partitioned with EtOAc and H2O. The
organic phase was dried (Na2SO4) and concentrated in vacuo to
give 8.0 g of crude yellow solid. The crude product was dissolve
in CH2Cl2 and passed through a plug of silica gel. The plug was
washed with additional CH2Cl2. The combined filtrate was con-
centrated in vacuo to give 5.18 g of product as a white solid (yield
) 80%). 1H NMR (DMSO-d6, 400 MHz) δ 7.39 (t, 3 H, J ) 8.84
Hz), 7.82 (d, 1 H, J ) 7.3 Hz), 8.04 (m, 2 H), 8.37 (d, 1 H, J )
8.2 Hz), 13.81 (s, 1H); LC/MS 281.22 [M + H]+.
Step 3: Preparation of 2-(2,4-Dimethylbenzyl)-3-(4-
fluorophenyl)-7-(trifluoromethyl)-2H-indazole. A solution of
3-(4-fluorophenyl)-7-(trifluoromethyl)-1H-indazole (0.28 g, 1 mmol)
and 2,4-dimethylbenzylmethane sulfonate (0.321 g, 1.5 mmol) in
DMF was heated at 120 °C overnight. The reaction mixture was
partitioned with EtOAc and H2O. The organic phase was washed
with brine and concentrated in vacuo. The crude residue was
purified by flash chromatography (CH2Cl2) to give 0.117 g product
as a white solid (yield ) 29%). 1H NMR (DMSO-d6, 400 MHz) δ
2.17 (s, 3H), 2.19 (s, 3H), 5.67 (s, 2 H), 6.33 (s, 1H, J ) 7.93 Hz),
6.83 (d, 1 H, J ) 7.79 Hz), 6.97 (s, 1H), 7.23 (t, 1H, J ) 7.8 Hz),
7.42 (m, 2H), 7.60 (m, 2H), 7.74 (d, 1H, J ) 7.01 Hz), 7.83 (d,
1H, J ) 8.45); MS (ESI) m/z 399 [M + H]+. HRMS: calcd for
C23H18F4N2 + H+, 399.1479; found (ESI, [M + H]+ obsd),
399.1481. Anal. (C23H18F4N2) C, H, N. HPLC purity: 100%.
HRMS: calcd for C21H13F5N2 + H+, 389.1076; found (ESI, [M +
H]+ obsd), 389.1072. Anal. (C21H13F5N2) C, H, N.
2-(4-Chlorobenzyl)-3-(4-fluorophenyl)-7-(trifluoromethyl)-2H-
indazole (16). Reaction was performed according to the procedure
of compound 12 from 3-(4-fluorophenyl)-7-(trifluoromethyl)-1H-
indazole (0.15 g, 0.54 mmol) and 4-chlorobenzyl bromide (0.124
1
g, 0.6 mmol) to give 0.060 g of product (yield ) 27%). H NMR
(DMSO-d6, 400 MHz) δ 5.73 (s, 2H), 7.01 (d, 2H, J ) 8.44 Hz),
7.23 (t, 1H, J ) 7.80 Hz), 7.34 (d, 2H, J ) 8.58 Hz), 7.43 (m,
2H), 7.61 (m, 2H), 7.75 (d, 1H, J ) 7.01 Hz), 7.82 (d, 1H, J )
8.45 Hz); MS (ESI) m/z 405 [M + H]+. HRMS: calcd for
C21H13F4N2 + Na+, 427.0592; found (ESI, [M + Na]+ obsd),
427.0596. Anal. (C21H13F4N2) C, H, N. HPLC purity: 100%.
2-(4-Chloro-2-fluorobenzyl)-3-(4-chlorophenyl)-7-(trifluoro-
methyl)-2H-indazole (17). Step 1: Preparation of 3-(4-fluoro-
phenyl)-7-(trifluoromethyl)-1H-indazole. A solution of (2-fluoro-
3-(trifluoromethyl)phenyl)(4-chlorophenyl)methanone (10.5 g, 34.7
mmol, prepared as described above for 4-fluoroanalogue intermedi-
ate of 12), hydrazine hydrate (11 mL, 347 mmol), and dimethy-
laminopyridine (4.27 g, 35 mmol) in 20 mL of pyridine was heated
at 110 °C overnight. The reaction mixture was partitioned with
EtOAc and H2O. The organic phase was dried (Na2SO4) and
concentrated in vacuo to give 8.73 g of crude yellow solid. The
crude product was triturated with petroleum ether to give 7.5 g of
product as a white solid (yield ) 73%). 1H NMR (DMSO-d6, 400
MHz) δ 7.40 (t, 1H, J ) 7.92 Hz), 7.62 (d, 2H, J ) 8.44 Hz),
7.83(d, 1 H, J ) 7.41 Hz), 8.04 (d, 2 H, J ) 8.44 Hz), 8.39 (d, 1
H, J ) 8.31 Hz), 13.88 (s, 1H); LC/MS 297.02/299.10 [M + H]+.
Step 2: Preparation of 2-(4-Chloro-2-fluorobenzyl)-3-(4-
chlorophenyl)-7-(trifluoromethyl)-2H-indazole. A solution of 3-(4-
chlorophenyl)-7-(trifluoromethyl)-1H-indazole (0.15 g, 0.506 mmol)
and 2-chloro-4-fluorobenzyl bromide (0.134 g, 0.6 mmol) in DMF
was heated at 120 °C overnight. The reaction mixture was
partitioned with EtOAc and H2O. The organic phase was washed
with brine and concentrated in vacuo. The crude residue was
purified by preparative HPLC (silica gel, Biotage 12M, solvent
hexane/CH2Cl2, 1:1, flow rate 10 mL/min) to give 0.102 g of
product as a white solid (yield ) 46%). 1H NMR (DMSO-d6, 400
MHz) δ 5.77 (s, 2 H), 7.01 (t, 1H, J ) 8.18 Hz), 7.23 (m, 2H),
7.40 (dd, 1H), 7.66 (m, 4H), 7.755 (dd, 1H), 7.83 (d, 1H, J ) 8.45);
MS (ES) m/z 439.1 [M + H]+. HRMS: calcd for C21H12Cl2F4N2
+ H+, 439.0386; found (ESI, [M + H]+ obsd), 439.0390. Anal.
(C21H12Cl2F4N2) C, H, N. HPLC purity: 100%.
2-(2-Chloro-4-fluorobenzyl)-3-(4-fluorophenyl)-7-(trifluoro-
methyl)-2H-indazole (12). A solution of 3-(4-fluorophenyl)-7-
(trifluoromethyl)-1H-indazole (0.15 g, 0.54 mmol) and 2-chloro-
4-fluorobenzyl bromide (0.134 g, 0.6 mmol) in DMF was heated
at 120 °C overnight. The reaction mixture was partitioned with
EtOAc and H2O. The organic phase was washed with brine and
concentrated in vacuo. The crude residue was purified by preparative
HPLC over silica gel (Biotage 12M; hexane/methyl tert-butyl ether
gradient, 5% methyl tert-butyl ether to 95% over 15 min at 10 mL/
min) to give 0.106 g of product as a white solid (yield ) 46%, mp
1
93 °C). H NMR (DMSO-d6, 400 MHz) δ 5.77 (s, 2H), 6.92 (m,
1H), 7.14 (dt, 1H), 7.24 (t, 1H, J ) 7.8 Hz), 7.43 (m, 3H), 7.65
(m, 2H), 7.76 (d, 1H, J ) 7.01 Hz), 7.83 (d, 1H, J ) 8.45 Hz);
MS (ESI) m/z 423 [M + H]+. HRMS: calcd for C21H12ClF5N2 +
H+, 423.06819; found (ESI-FTMS, [M + H]1+ 423.0683. Anal.
(C21H12ClF5N2) C, H, N. HPLC purity 100%.
2-(2-Fluorobenzyl)-3-(4-fluorophenyl)-7-(trifluoromethyl)-2H-
indazole (13). Reaction was run according to the procedure of
compound 12 from 3-(4-fluorophenyl)-7-(trifluoromethyl)-1H-in-
dazole (0.15 g, 0.54 mmol) and 2- fluorobenzyl bromide (0.113 g,
0.6 mmol) to give 0.103 g of product (yield ) 49%, mp 89-90
°C). 1H NMR (DMSO-d6, 400 MHz) δ 5.77 (s, 2H), 6.93 (dt, 1H),
7.08-7.17 (m, 2H), 7.23 (t, 1H, J ) 7.67 Hz), 7.31 (m, 1H), 7.43
(m, 2H), 7.63 (m, 2H), (dd, 1H), 7.83 (d, 1H, J ) 8.44 Hz); MS
(ESI) m/z 389 [M + H]+. HRMS: calcd for C21H13F5N2 + H+,
389.1072; found (ESI, [M + H]+ obsd), 389.1081. Anal.
(C21H13F5N2) C, H, N. HPLC purity: 100.0%.
2-(2-Chloro-4-fluorobenzyl)-3-(4-chlorophenyl)-7-(trifluoro-
methyl)-2H-indazole (18). Reaction was performed according to
the procedure of compound 17 from 3-(4-chlorophenyl)-7-(triflu-
oromethyl)-1H-indazole (0.15 g, 0.506 mmol) and 2-chloro-4-
fluorobenzyl bromide (0.124 g, 0.6 mmol) to give 0.140 g of product
1
(yield ) 63%). H NMR (DMSO-d6, 400 MHz) δ 5.79 (s, 2 H),
6.92 (m, 1H), 7.16 (dt, 1 H), 7.24 (dt,, 1H), 7.43 (dd, 1H), 7.64
(m, 4H), 7.765 (dd, 1H), 7.836 (d, 1H, J ) 8.44); MS (ES) m/z
439.1[M + H]+. HRMS: calcd for C21H12Cl2F4N2 + H+, 439.0386;
found (ESI, [M + H]+ obsd), 439.0389. Anal. (C21H12Cl2F4N2) C,
H, N. HPLC purity: 98.9%.
2-(2-Chlorobenzyl)-3-(4-fluorophenyl)-7-(trifluoromethyl)-2H-
indazole (14). Reaction run according to the procedure of compound
12 from 3-(4-fluorophenyl)-7-(trifluoromethyl)-1H-indazole (0.15
g, 0.54 mmol) and 2- chlorobenzylbromide (0.124 g, 0.6 mmol) to
give 0.081 g product (yield ) 37%). 1H NMR (DMSO-d6, 400
MHz) δ 5.80 (s, 2H), 6.765 (dd, 1H), 7.25 (dt, 1H), 7.31 (dt, 1H),
7.42 (m, 2H), 7.63 (m, 2H), 7.77 (dd, 1H), 7.84 (d, 1H, J ) 8.44
Hz); MS (ESI) m/z 405 [M + H]+; HRMS: calcd for C21H13ClF4N2
+ H+, 405.0776; found (ESI, [M + H]+ obsd), 405.0782. Anal.
(C21H13ClF4N2) C, H, N. HPLC purity: 100.0%.
2-(2-Chlorobenzyl)-3-(4-chlorophenyl)-7-(trifluoromethyl)-2H-
indazole (19). Reaction was performed according to the procedure
of compound 17 from 3-(4-chlorophenyl)-7-(trifluoromethyl)-1H-
indazole (0.15 g, 0.50 mmol) and 2-chlorobenzyl bromide (0.133
g, 0.65 mmol) to give 0.056 g of product (yield ) 27%). 1H NMR
(DMSO-d6, 400 MHz) δ 5.76 (s, 2 H), 6.77 (dd, 1H), 7.26 (m,
2H), 7.4 (dd, 1H), 7.64 (m, 4H), 7.7 (dd, 1H), 7.87 (d, 1H, J )
8.45); MS (ES) m/z 421.1 [M + H]+. HRMS: calcd for
C21H13Cl2F3N2 + H+, 421.0483; found (ESI, [M + H]+ obsd),
421.0481. Anal. (C21H13Cl2F3N2) C, H, N. HPLC purity: 100%.
3-(4-Chlorophenyl)-2-(2-fluorobenzyl)-7-(trifluoromethyl)-2H-
indazole (20). Reaction was performed according to the procedure
of compound 17 from 3-(4-chlorophenyl)-7-(trifluoromethyl)-1H-
indazole (0.15 g, 0.50 mmol) and 2-fluorobenzyl bromide (0.123
g, 0.65 mmol) to give 0.043 g of product (yield ) 19%). 1H NMR
(DMSO-d6, 400 MHz) δ 5.79 (s, 2 H), 6.94 (dt, 1H), 7.12 (m, 2H),
7.24 (dt, 1H), 7.31 (m, 1H), 7.645 (m, 4H), 7.75 (d, 1H, J ) 7.01
2-(4-Fluorobenzyl)-3-(4-fluorophenyl)-7-(trifluoromethyl)-2H-
indazole (15). Reaction was performed according to the procedure
of compound 12 from 3-(4-fluorophenyl)-7-(trifluoromethyl)-1H-
indazole (0.15 g, 0.54 mmol) and 4- fluorobenzyl bromide (0.113
1
g, 0.6 mmol) to give 0.085 g of product (yield ) 40%). H NMR
(DMSO-d6, 400 MHz) δ 5.72 (s, 2H), 7.03-7.14 (m, 4H), 7.23 (t,
1H, J ) 7.67), 7.44 (m, 2H), 7.61 (m, 2H), 7.74 (d, 1H, J ) 7.01
Hz), 7.81 (d, 1H, J ) 8.44 Hz); MS (ESI) m/z 389 [M + H]+.

LXR Modulators
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7167
Hz), 7.83 (d, 1H, J ) 8.45); MS (ES) m/z 405.1 [M + H]+. HRMS:
calcd for C21H13ClF4N2 + H+, 405.0779; found (ESI, [M + H]+
obsd), 405.0776. Anal. (C21H13ClF4N2) C, H, N. HPLC purity:
100%.
compared to a glycerol standard curve, and converted into a triolein
mg/mL equivalent. In parallel with samples, 400 µg of triolein was
added to control liver extracts and processed to determine recovery
efficiency.
In Vitro Assays. LXRR and LXRꢀ binding assays (Table 1),
Gal4 transactivation assays (Table 1), ABCA1 gene regulation in
THP-1 cells (Table 2), C-efflux (Table 2), and SREBP1c in HepG2
cells (Table 2) were performed as described previously.9 NHR
cross-reactivity assays and data are outlined in Supporting Informa-
tion.
3-(4-Chlorophenyl)-7-(trifluoromethyl)-2-[2-(trifluoromethyl)-
benzyl]-2H-indazole (21). Reaction was performed according to the
procedure of compound 17 from 3-(4-chlorophenyl)-7-(trifluoro-
methyl)-1H-indazole (0.15 g, 0.50 mmol) and 2-(trifluoromethyl-
)benzyl bromide (0.155 g, 0.65 mmol) to give 0.054 g of product
1
(yield ) 24%). H NMR (DMSO-d6, 400 MHz) δ 5.92 (s, 2 H),
TG Accumulation in HepG2 Cells. Results for compounds in
6.62 (d, 1H, J ) 7.79 Hz), 7.28 (t, 1 H, J ) 7.8 Hz), 7.49 (t, 1H,
J ) 7.66 Hz), 7.61 (m, 4H), 7.75 (d, 1H, J ) 7.27 Hz), 7.80 (d,
1H, J ) 7.01 Hz), 7.89 (d, 1H, J ) 8.44); MS (ES) m/z 455.1 [M
+ H]+. HRMS: calcd for C22H13ClF6N2 + H+, 455.0751; found
(ESI, [M + H]+ obsd), 455.0744. Anal. (C22H13ClF6N2) C, H, N.
HPLC purity: 100%.
Table 2 were obtained using methods previously described.24
LDL KO Mouse Inhibition of Lesion Progression Model.
Eight-week old male LDLRKO were fed atherogenic diet or diet
supplemented to deliver 2, 3, or 13 for 8 weeks. Aortas were
obtained and analyzed using methods previously described.9
X-ray Crystal Structure of 12 with LXRꢀ LBD. Results were
obtained using methods as previously described.9
3-(4-Chlorophenyl)-7-(trifluoromethyl)-2-[4-(trifluoromethyl)-
benzyl]-2H-indazole (22). Reaction was performed according to the
procedure of compound 17 from 3-(4-chlorophenyl)-7-(trifluorometh-
yl)-1H-indazole (0.15 g, 0.50 mmol) and 4-(trifluoromethyl)benzyl
bromide (0.155 g, 0.65 mmol) to give 0.043 g of product (yield )
19%). 1H NMR (DMSO-d6, 400 MHz) δ 5.86 (s, 2 H), 7.21 (d, 2H,
J ) 8.05 Hz), 7.28 (dt, 1 H), 7.63 (m, 5H), 7.78 (d, 1H, J ) 7.02 Hz),
7.86 (d, 1H, J ) 8.31); MS (ES) m/z 455.1. HRMS: calcd for
C22H13ClF6N2 + H+, 455.0744; found (ESI, [M + H]+ obsd),
455.0744. Anal. (C22H13ClF6N2) C, H, N. HPLC purity: 100%.
2-Benzyl-3-phenyl-7-(trifluoromethyl)-2H-indazole (5). 5 was
prepared identically to the 4-fluoro (e.g., 11) analogues starting from
Acknowledgment. We thank the Wyeth Discovery Analyti-
cal Chemistry Department for the analytical data, and Liang
Chen, Anita Halpern, Qiang Liu, Johnny Sandberg, and Dawn
Savio for biological assay data, and Dr. Baihua Hu for
resynthesis of compound 13. We also thank Dr. Magid Abou-
Gharbia for his support of this work.
Supporting Information Available: Additional analytical data
for compounds 5 and 11-21 and cross-reactivity data for com-
pounds 12 and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
(2-fluoro-3-(trifluoromethyl)phenyl)(phenyl)methanone. H NMR
(DMSO-d6, 400 MHz) δ 5.75 (s, 2H), 7.0 (dd, 2H), 7.26 (m, 4H),
7.58 (m, 5H), 7.75 (d, 1H, J ) 7.05 Hz), 7.84 (d, 1H, J ) 8.46).
HRMS: calcd for C21H15F3N2 + H+, 353.1260; found (ESI, [M +
H]+ obsd), 353.1267. Anal. (C21H15F3N2) C. HPLC purity: 100%.
Seven Day Hamster Model. The 5-6 week old (90-100 g)
male Golden Syrian hamsters were obtained from Harlan (India-
napolis IN). Hamsters were randomized by body weight and group
and housed two per cage in plastic cages with ad libitum access to
water and diet (Purina Rodent Chow 5001). Animals were allowed
to acclimate for 1 week under standard conditions with a 12 h/12
h light/dark cycle (lights on at 0600 h) and with temperature
controlled at 22 ( 2 °C. All experimental procedures were
performed according to protocols approved by the Wyeth Institu-
tional Animal Care and Use Committee (Collegeville, PA). Animals
were divided into four groups (n ) 6 per group) and were dosed
once daily by oral gavage with vehicle (0.5% methylcellulose/2.0%
Tween-80) or vehicle containing compound at a concentration
designed to deliver a target dose of either 3 (mg/kg)/day for 3 or
100 (mg/kg)/day for 13. On the final day, food was removed at
time of final dosing. Two hours later the animals were euthanized.
RNA was prepared from duodenum and liver and quantified by
real time PCR. All gene expression values were normalized for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression,
with the expression seen in vehicle treated animals defined as 1.0
for each gene. Whole blood was collected into serum separator
tubes, and serum was isolated and analyzed for total cholesterol,
triglycerides, AST, and ALT using enzymatic methods. Plasma total
cholesterol and triglyceride were determined using a Roche 912
clinical chemistry analyzer.
For hepatic triglyceride analysis, 200 mg of frozen liver was
homogenized in 10 mL of 2:1 chloroform/methanol, stored under
nitrogen, and shaken overnight. Then 4 mL of water was added
and the samples were centrifuged at 500g for 20 min. The organic
phase was filtered through cheesecloth and centrifuged at 500g for
10 min, and a portion (0.5 mL) was evaporated under nitrogen.
The samples were resuspended in 2.5 mL of 30 mM pipes, pH 7.0,
60 mM MgCl2, 0.1% NP-40 by sonication, stored under nitrogen,
and shaken overnight. Then 0.2 mL of resuspended sample was
treated with 10 µg of lipase (Pseudomonas sp., Sigma) at 37 °C
for 1 h. An amount of 80 µL of sample was transferred to a clear
96-well plate and incubated with 160 µL of Free glycerol reagent
(Sigma) for 10 min. The absorbance at 540 nm was determined,
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