Discovery and optimization of a series of liver X receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.07.048

The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a structure-based approach. A series of benzenesulfonamides was synthesized based on structural modification of a known LXR agonist and was determined to be potent dual liver X receptor (LXR α/β) ligands. Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable for pharmacological in vivo evaluation in rodents.

Full text of DOI:10.1016/j.bmcl.2012.07.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5966–5970
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a series of liver X receptor antagonists
XianYun Jiao a, , David J. Kopecky a, Ben Fisher a, Derek E. Piper b, Marc Labelle a, Sharon McKendry a,
⇑
Martin Harrison b, Stuart Jones b, Juan Jaen a, Andrew K. Shiau c, Patrick Escaron c, Jean Danao c, Anne Chai c,
Peter Coward c, Frank Kayser a
a Department of Medicinal Chemistry, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Department of Molecular Structure & Characterization, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
c Department of Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a
structure-based approach. A series of benzenesulfonamides was synthesized based on structural modifi-
cation of a known LXR agonist and was determined to be potent dual liver X receptor (LXR a/b) ligands.
Received 11 June 2012
Accepted 11 July 2012
Available online 21 July 2012
Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable
for pharmacological in vivo evaluation in rodents.
Keywords:
Ó 2012 Published by Elsevier Ltd.
LXR a/b
Antagonist
GAL4 reporter gene assay
mSREBP-1c reporter
Conversion
The liver X receptors (LXRs), LXR
a
(NR1H3) and LXRb (NR1H2),
such as sterol regulatory element-binding protein 1c (SREBP-1c)10
and the carbohydrate responsive binding protein (ChREBP).11 Thus,
limiting the potential utility of LXR agonists in a clinical setting.
One strategy to overcome this limitation centers on the use of
LXRb-selective agonists. This is based on evidence that while acti-
are members of the nuclear receptor (NR) superfamily of ligand-
regulated, DNA-binding transcription factors.1 Both LXRs bind to
their cognate response elements as heterodimers with the retinoid
X receptors (RXRs, NR2B1-3) and play important roles in lipid biol-
ogy.2a Mammalian endogenously occurring LXR ligands include the
oxysterols 24(S),25-epoxycholesterol which is mainly found in the
liver, 22(R)-hydroxycholesterol which is present in steroidogenic
tissues, and 24(S)-hydroxycholesterol which is prevalent in brain
tissue and plasma.2 Several synthetic agonists (Fig. 1) have been re-
ported and have served as valuable tools to elucidate the role of
LXRs in mammalian biology.3,4 LXR activation results in an increase
in transcription of genes involved in the catabolism (Cyp7a)5 and
transport (ABCA1, ABCG1, ABCG5, ABCG8)6 of cholesterol. In mouse
models of atherosclerosis, LXR agonists increase plasma HDL- cho-
lesterol and decrease atherosclerotic lesions,7 suggesting a poten-
tial utility in treating cardiovascular disease.8 Moreover, there is
evidence that LXR agonists are effective in suppression of inflam-
mation, play an important role in the regulation of glucose metab-
olism and might be useful for the treatment of Alzheimer’s disease
and other neurodegenerative diseases of the CNS.4,9
vation of either LXRa or LXRb provides relatively similar positive
effects on atherosclerosis, activation of LXRb is responsible for
hypertriglyceridemia.10b,12 However, the high level of similarity
in the LXRa and LXRb ligand binding pockets has limited the feasi-
bility of this approach and prevented the development of LXRb-
selective ligands. Another approach relies on the observation that
expression of certain LXR target genes is repressed in the basal
state. For example, in LXRa/b double knockout mice, although he-
patic expression of SREBP1c, FAS, SCD1, and ACC is decreased com-
pared to wild-type mice, expression of hepatic and intestinal
CF3
Cl
F3C
OH
O
O
CF3
O
N
S
However, current LXR agonists also increase plasma and hepatic
triglyceride levels in mice and hamsters, which results from LXR-
regulated induction of transcription factors involved in lipogenesis,
N
HOOC
R
R = CH3
1
R = CH2CF3 2 (T0901317)
3 (GW3965)
⇑
Corresponding author. Tel.: +1 650 244 2103; fax: +1 650 837 9369.
E-mail address: xjiao@amgen.com (XianYun Jiao).
Figure 1. Examples of LXR agonists described in the literature.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.07.048

XianYun Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5966–5970
5967
Table 1
Table 2
Binding affinities of derivatives 1, 4–14
Binding affinities of derivatives 15–28
HO
R1
CF3
CF3
R2
O
O
O
O
S
S
N
R1
N
R2
CH3
Compound R1
R2
LXRb SPA LXRb GAL4,
Compd
R1
OH
R2
LXRb SPA binding,19 IC50
(lM)
binding, IC50 lM
IC50
4.1
>30
(
l
M) (% of basal)20
1
4
5
6
7
8
9
10
11
12
13
14
CF3
CF3
CF2CF3
H
CH3
Bn
Thien-2-yl
Phenyl
Thiazol-2-yl
0.02
2.2
15
16
17
18
19
20
21
22
23
24
25
CF3
iBu
Ph
4-F-Ph
Ph
tBu
Bn
Me
Me
Me
>10
-
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
0.16
>10
>10
>10
1.5
3.6
7.5
>10
>10
>10
1.4
1.1
0.6
1.1
0.6
1.1
3.8
0.4
2.1
5.1
3.2
1.7
>10
>10
8 (28)
>10
>10
>10
>10
10
CF3CH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
Cyclohexenyl
2-Pyridyl
3-Pyridyl
1H-Imidazol-2-yl
4H-1,2,4-Triazol-3-yl
1H-Tetrazol-5-yl
3,5-Pyrimidinyl
10
26
27
28
1-Methyl-1H-imidazol-5-yl (CH3)2CHCH2
3,5-Dimethyl-isoxazol-4-yl (CH3)2CHCH2
1-Ethyl-1H-pyrazol-4-yl (CH3)2CHCH2
>10
>10
>10
ABCG5 and ABCG8 and macrophage ABCA1 and ABCG1 is either
unchanged or elevated.6a In addition, LXR
/b null mice exhibited
a
significantly reduced levels of VLDL plasma triglycerides relative
Table 3
to their wild type littermates.10a
Binding affinities of derivatives 19, 29–50
Taken together, these data suggest that an LXRa/b dual antago-
HO
nist could downregulate the SREBP-1c pathway to reduce triglycer-
ide levels in hypertriglyceridemic patients without affecting
cholesterol homeostasis. Several synthetic LXR antagonists have
been reported in the literature,13 and only one report describes a
compound which is potent in cellular assays.13d No LXR antagonist
which is suitable for pharmacological in vivo evaluation in rodents
has been reported.
CF3
O
O
S
N
R1
R2
Compound R1
R2
LXRb SPA binding, LXRb GAL4,
IC50 M) IC50 M (% of basal)
(l
l
N-Methylsulfonamide
1
and N-trifluoroethylsulfon-amide
/b agonists that have been described
in a luciferase repor-
2
(e.g., T0901317) are dual LXR
a
19
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
H
H
H
H
0.6
0.3
1.4
0.6
0.5
2.5
1.0
0.7
0.7
0.6
3.5
0.8
2.8
0.6
0.6
0.6
0.4
0.6
0.6
4.3
8 (28)
10 (34)
10 (81)
>10
previously.3a Both molecules activated LXR
a
3-CN
2,5-Cl2
3-CN
H
H
H
H
H
H
H
H
H
H
3-CN
H
3-CN
H
3-CN
3-CN
ter gene assay (GAL4) tested in HEK-293 cells.14 Furthermore a
scintillation proximity ligand binding assay (SPA) in which the trit-
urated 1 was used as a competitive binder showed that these com-
4-Cl
3-CF3
4-CF3
3-MeO
4-MeO
4-EtO
4-Me
4-Bu
>10
>10
>10
>10
>10
>10
>10
pounds displayed strong affinities for either LXR isoform3a
A
ligand-bound co-crystal structure of 2 with LXRa elucidated that
helix 12 of ligand binding domain (LBD) of LXR seals off the ligand
a
binding pocket. A hydrogen bond between His421 and the acidic
hydroxyl group of the 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP)
group is observed.15 However, we reported recently that when
one of the trifluoromethyl groups of 1 is replaced by a bulky het-
eroaromatic group, a new agonist binding mode characterized by
the lack of a hydrogen bond is observed.16 Thus, the acidity and
spatial orientation of the hydroxyl group as well as the steric envi-
ronment around the hydroxyl group influence which agonist bind-
ing mode is favored.
The approach we used for converting agonist 1 into an antago-
nist was to substitute one of the trifluoromethyl groups of 1 with a
moiety that will push helix 12 away from the binding site, prevent-
ing the ligand-bound receptor from adopting a conformation
which can undergo transcription. Similar strategies for the conver-
sion of agonists to both partial agonists as well as antagonists have
been reported previously (for e.g., the conversion of full agonist
diethylstilbestrol to antagonist 4-hydroxytamoxifen by preventing
helix 12 from adopting an agonist-bound conformation through
steric interference).17
2,4-F2
8 (0)
10 (47)
>10
2-Me, 4-NO2
3-CN
4-CN
>10
3-CH3SO2
3-CH3SO2
4-CH3SO2
4-CH3SO2
4-CF3SO2
4-PhSO2
3-COOH
4-COOH
5 (0)
7 (0)
2 (0)
2 (0)
>10
>10
>10
10 (0)
3-CN >10
H
3-CN
1.8
0.5
(Table 1, compound 4) compromises binding affinity to LXRb.
Maintaining the capability of the hydroxyl group to form a hydro-
gen bond with His435 was crucial to binding of the compounds,
thus we left the alcohol in place during further analoging efforts.
The replacement of one or both of the trifluoromethyl groups of
1 with simple alkyl groups also resulted in loss of binding
(Table 1).18 Replacement of one of the trifluoromethyl groups with
Our initial efforts focused on whether groups of different size
and shape might be attached to either the alcohol moiety or one
of the trifluoromethyl groups of 1. Not surprisingly, replacing the
acidic hydroxyl group of the HFIP moiety with a methyl ether

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XianYun Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5966–5970
Table 4
N-Alkyl modification of compound 45
HO
CF3
O
O
S
N
O
S
R
O
Compound
R
RLM%
LXRb SPA binding,
LXRb GAL4,
IC50 (lM)
rema
IC50
(
lM)
45
51
<5
85
0.6
1.0
2
9
F3C
F3C
52
42
0.5
4
53
54
55
36
>95
<5
1.2
0.5
0.3
6
2
7
56
<5
0.15
1.5
a
Compound percentage remaining after incubation in rat liver microsomes at
lM for 30 min.
1.0
Figure 3. (A) Compound 45 antagonizes compound
1 in a QuantiGene Assay
measuring endogenous SREBP-1 expression in Caco2 cells. Expression level is
normalized by expression of the housekeeping gene GAPDH. (B) Compound 45
reduces the basal expression level of SREBP-1 by approximately 50%.
However, replacement of one of the trifluoromethyl groups
with alkyne substituents (Table 2) resulted in compounds with
good binding properties. In addition, changing the N-methyl group
of the sulfonamide to a larger trifluoroethyl or isobutyl group was
mostly beneficial (Table 2). These compounds were evaluated for
antagonist activity in a GAL4 reporter gene assay using transiently
transfected HEK293 cells.20 19 showed the most robust activity,
decreasing the activation by the agonist 1 by 72%, with an IC50 of
8
lM.
Table 3 summarizes the results from a broad survey of substit-
uents around the benzenesulfonamide and phenylacetylene moi-
ety. Consistent with previous results obtained in the agonist
series, small substituents in the meta position of the benzenesul-
fonamide, such as a 3-CN group, conferred equal or better binding
properties over the unsubstituted counterparts (see e.g., 19 vs 29,
43 vs 44, 45 vs 46).3a In the phenylacetylene region, a broad range
of small substituents, such as methoxy-, cyano-, methylsulfonyl or
carboxylic acid were tolerated without significantly affecting bind-
ing, though larger, lipophilic groups were less preferred (33, 38, 47,
and 48). A 3- or 4-methylsulfonyl substituent gave compounds
with good affinity to the receptor while also showing very robust
antagonist activity in the GAL4 reporter gene assay, decreasing
the activation by the agonist 1 completely with an IC50 of 2–
Figure 2. (A) Compound 45 reduces basal activity and antagonizes the dose
response of compound 1 in a transient transfection assay in HEK-293 cells using the
mSREBP-1c promoter. (B) Compound 45 antagonizes compound 2 in a triglyceride
accumulation assay in HepG2 cells.
5 lM (43–46). When directly comparing 45 with 46, we saw no
additional benefit from the modification of the benzenesulfonyl
moiety with a 3-cyano substituent. Both p-methyl sulfonylphenyl-
a variety of aryl and heterocyclic moieties generated compounds
with generally diminished binding, consistent with our previous
observations.16

XianYun Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5966–5970
5969
Table 5
ability to block induction of endogenous SREBP-1c by the agonist
1. Consistent with data in Figure 2A, 45 lowers the basal level of
SREBP-1 transcript by 50%, and right-shifts the agonist dose re-
sponse (Fig. 3).
a,b
Pharmacokinetic parameters following iv dosing in male Sprague–Dawley rats.
Compound
CL (L/h/kg)
Vss (L/kg)
MRT (h)
AUC (lgh/L)
45
54
4.8
1.1
4.4
2.3
1.3
2.2
102
885
Unfortunately, 45 displayed a poor pharmacokinetic profile in
male Sprague–Dawley rats making it unsuitable as a tool com-
pound for in vivo proof-of-concept studies (Table 5). Metabolite
ID studies with rat liver microsomes revealed hydroxylation and
oxidative cleavage of the isobutyl group to be the major clearance
pathway. Hence we turned our attention towards the structure-
metabolism relationship of a number of N-alkyl derivatives, as
shown in Table 4. While the cyclobutylmethyl- and cyclopentylm-
ethyl groups (55 and 56) were still metabolically labile, consistent
with their possibility to undergo cytochrome p450 mediated oxi-
dative cleavage, 51 and 52 had dramatically improved microsomal
stability. This is most likely due to the reduced acidity of the
hydrogen atoms on the carbon next to the nitrogen. The cyclopro-
pylmethyl derivative 54 displayed the best balance of potency and
microsomal stability. We were pleased to see that this translated
into an improved in vivo pharmacokinetic profile with significantly
lower rat clearance of 54 (1.1 L/h/kg) compared to 45 (4.8 L/h/kg),
and about eight-fold higher AUC following i.v. dosing. (Table 5).
The oral PK profile of 54 was also significantly improved (Ta-
ble 6) displaying good oral availability and good exposure follow-
ing oral dosing at 5 mg/kg in rats (Table 6) and was suitable for
in vivo pharmacological investigations in rodents (data not
shown).
a
n = 3 animals per study.
Dosed at 0.5 mg/kg iv.
b
Table 6
Pharmacokinetic parameters following po dosing in male Sprague–Dawley ratsa,b
Compound
F (%)
AUC (lgh/L)
45
54
9
31
86
1380
a
n = 3 animals per study.
Dosed at 5 mg/kg po.
b
acetylene derivatives 45 and 46 demonstrated potent antagonism
(GAL4 IC50 = 2 M) with no evidence of cell toxicity.
l
The functional activity of 45 was further characterized in an
mSREBP reporter assay in HEK-293 cells and in a triglyceride accu-
mulation assay in HepG2 cells (Fig. 2). 45 shows antagonism on an
mSREBP-1c reporter, lowering the basal activity of the reporter and
right-shifting the agonist dose response. 45 also shows antagonism
in a triglyceride accumulation assay and at 10 lM causes a 15 fold
right shift in the agonist dose response. 45 was also tested for its
X
R1
O
X
X
S O
Cl
R-Br
b
R1
O
S
O
a
R1
O
S
NH
N
NH2
R
O
57
58
59
R2
O
F3C
OH
R2
CF3
c
d
R1
O
S
N
R1
R
O
S
N
O
R
O
60
15-56
X = Br, I; R1 = 2-Cl, 3-Cl, 3-Cyano, 2-trifluoromethoxy;
R
= cyclopropanylmethyl, isobutyl, 2,2,2-trifluoroethyl
Scheme 1. Reagents and conditions: (a) pyridine, rt; (b) NaH, DMF, rt; (c) n-BuLi / TBME, À78 °C, then ethyl trifluoroacetate; (d) n-BuLi / THF, À78 °C.
CF3
R1
O
O
S
HN
R
F
O
H2N
a
Cl
R
b
O
S
O
O
O
F3C
N
F3C
R
R1
60
62
61
Scheme 2. Reagents and conditions: (a) Et3N, acetonitrile, reflux; (b) pyridine, rt.

5970
XianYun Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5966–5970
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7604; (b) Terasaka, N.; Hiroshima, A.;
The general synthetic route to compounds with LXR antagonis-
Koieyama, T.; Ubukata, N.; Morikawa, Y.; Nakai, D.; Inaba, T. FEBS Lett. 2003,
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Zhao, C.; Karin, D.-W. J. Endocrinol. 2010, 204, 233.
tic properties is shown in Scheme 1. Sulfonylation of 57 with the
corresponding sulfonyl chloride in pyridine afforded 58 in excel-
lent yield. Aklylation of sulfonamide 58 with an appropriate alkyl
bromide or iodide in the presence of 60% sodium hydride in min-
eral oil yielded 59 which was further transformed into 60 by gen-
erating the lithium salt of 59 and quenching with ethyl
trifluoroacetate. Finally, the anion of the substituted acetylene
was generated by n-butyllithium in THF at À78 °C which was then
added to 60 to form 15–56 in good to excellent yields. The synthe-
sis of 1–14 has been described previously.18
8. (a) Tontonoz, P.; Mangelsdorf, D. J. Mol. Endocrinol. 2003, 17, 985; (b) Bennet, J.;
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nyl chloride in pyridine furnished 60.
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of the trifluoromethyl groups. 54 is the first potent cell-active
LXR antagonist reported to date and is suitable for pharmacological
in vivo evaluation in rodents.21
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Acknowledgements
We are grateful to our colleague Ji Ma for metabolite ID data
and Julio Medina and Jonathan Houze for helpful discussions.
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Binz, J. G.; Plunket, K. D.; Morgan, D. G.; Beaudet, E. J.; Whitney, K. D.; Kliewer,
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18. All data presented are for LXRb unless otherwise noted. Results for LXRa were
similar for this series.
19. SPA Assay: (scintillation proximity ligand binding assay): Binding assays were
performed with glutathione S-transferase-LXR fusion proteins and
glutathione-coated scintillation proximity assay beads. triturated compound
1 was used as the radioligand. Protein and beads were incubated for 2 h prior
4. For recent reviews, see (a) Collins, J. L. Curr. Opin. Drug Discov. Dev. 2004, 7, 692;
(b) Li, X.; Yeh, V.; Molteni, V. Expert Opin. Ther. Pat. 2010, 20, 535.
5. Chiang, J. Y.; Kimmel, R.; Stroup, D. Gene 2001, 262, 257.
to addition of test compound at concentrations ranging from 30
1 h. Radioligand (50 nM) was then added for an additional hour. Data are
expressed as IC50 values in M.
lM to 1 nM for
6. (a) Repa, J. J.; Turley, S. D.; Lobaccaro, J.-M. A.; Medina, J.; Li, L.; Lustig, K.; Shan,
B.; Heyman, R. A.; Dietschy, J. M.; Mangelsdorf, D. J. Science 2000, 289, 1524; (b)
Costet, P.; Luo, Y.; Wang, N.; Tall, A. R. J. Biol. Chem. 2000, 275, 28240; (c)
Schwartz, K.; Lawn, R. M.; Wade, D. P. Biochem. Biophys. Res. Commun. 2000,
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Mangelsdorf, D. J. J. Biol. Chem. 2002, 277, 18793; (f) Yu, L.; York, J.; von
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15565.
l
20. GAL4 assay: CBTT (Reporter gene assay) HEK-293 cells were co-transfected
with a luciferase reporter construct and DNA encoding a fusion of the DNA-
binding domain of the yeast transcription factor GAL4 and the LXR ligand-
binding domain. A CMV-b-galactosidase reporter construct was included as a
control for transfection efficiency. Compounds were dosed in the presence of
500 nM of compound 1, and treatment lasted for 20 h. Data are expressed as
IC50 values in lM, which is the molar concentration of the test compound that
affords a 50% decrease in the maximum reporter activity of compound 1.
21. Previously disclosed: Abstracts of papers, 235th ACS National Meeting, New
Orleans, LA, United States, April 6–10, 2008 (2008), MEDI-2.
7. (a) Joseph, S. B.; McKilligin, E.; Pei, L.; Watson, M. A.; Collins, A. R.; Laffitte, B. A.;
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Lusis, A. J.; Hsueh, W. A.; Law, R. E.; Collins, J. L.; Willson, T. M.; Tontonoz, P.