Discovery of a new binding mode for a series of liver X receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2012.02.028

Structural modification of a series of dual LXRα/β agonists led to the identification of a new class of LXRβ partial agonists. An X-ray co-crystal structure shows that a representative member of this series, pyrrole 5, binds to LXRβ with a reversed orient

Full text of DOI:10.1016/j.bmcl.2012.02.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2407–2410
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Bioorganic & Medicinal Chemistry Letters
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Discovery of a new binding mode for a series of liver X receptor agonists
David J. Kopecky a, , Xian Yun Jiao a, Ben Fisher a, Sharon McKendry a, Marc Labelle a, Derek E. Piper b,
⇑
Peter Coward c, Andrew K. Shiau c, Patrick Escaron c, Jean Danao c, Anne Chai c, Juan Jaen a, 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:
Structural modification of a series of dual LXRa/b agonists led to the identification of a new class of LXRb
partial agonists. An X-ray co-crystal structure shows that a representative member of this series, pyrrole
5, binds to LXRb with a reversed orientation compared to 1.
Received 18 January 2012
Revised 8 February 2012
Accepted 10 February 2012
Available online 20 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
LXR
Agonist
Nuclear receptor
SAR
Pyrrole
Liver X receptors (LXRs) are nuclear receptors that function as
ligand-activated transcription factors and are present in two highly
similar isoforms, LXRa (NR1H3) and LXRb (NR1H2). Heterodimer-
A series of dual LXRab agonists, which include N-methylsulf-
onamide 1 and N-trifluoroethylsulfonamide 2 (also known as
T0901317), was identified at Tularik Inc. (now Amgen) and was de-
scribed previously.5 These compounds exhibit potent affinities for
ization of LXRs with retinoid X receptors (RXRs) is required to
achieve DNA binding. LXRs have been implicated in cholesterol
homeostasis by regulating ABCA1, a key gene involved in managing
cellular HDL cholesterol transport.1 LXRs also play a critical role in
fatty acid metabolism and lipid biosynthesis by regulating sterol
regulatory element-binding protein-1c (SREBP-1c) production;
SREBP-1c is a transcription factor which regulates the expression
of a number of key genes implicated in lipogenesis including fatty
acid synthase (FAS) and stearoyl CoA desaturases (SCDs).2
both LXR
say (SPA) using tritium-labeled 1 as the competitive binder
(Table 1).5 Compounds 1 and 2 were also shown to activate LXR
in a luciferase reporter gene assay performed in HEK293 cells.6 An
X-ray co-crystal structure of 2 bound to the LXR has been re-
a and LXRb in a scintillation proximity ligand binding as-
a
a
ported and establishes the presence of a strong hydrogen bond be-
tween His421 and the acidic hydroxyl group of the 1,1,1,3,3,3-
hexafluoroisopropanol moiety of 2.7 Further, helix 12 of the LBD
Several reports on the viability and lipid profile of LXR
a
/b null
of LXRa effectively seals off the exposed face of the ligand binding
mice raised our interest in developing a dual-LXR /b antagonist
a
pocket.
for the treatment of hypertriglyceridemia, a risk factor for athero-
sclerotic cardiovascular disease.3 Two observations in particular
were noteworthy. First, LXRa/b null mice exhibited significantly
Table 1
reduced levels of VLDL plasma triglycerides relative to wild type.3b
Second, basal expression of ABCA1 was elevated and intestinal
Binding affinities of early LXR agonists 1–2
absorption of cholesterol was unaffected in LXR
a
/b null mice.1a
/b dual
Taken together, these data suggest that a potent LXR
a
antagonist could down-regulate the SREBP-1c pathway to reduce
triglyceride levels in hypertriglyceridemic patients without affect-
ing cholesterol homeostasis. To date, several reports of synthetic
LXR antagonists have been documented in the literature.4
Compound
R
SPA binding, IC50 (lM)
LXR
a
LXRb
1
2
CH3
CH2CF3
0.04
0.1
0.02
0.1
⇑
Corresponding author.
E-mail address: dkopecky@amgen.com (D.J. Kopecky).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.028

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D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2407–2410
We postulated that replacement of one of the trifluoromethyl
ether analog 4 relative to 3. On the other hand, N-methoxyethyl-
pyrrole 5 unexpectedly becomes a more potent binder upon
O-alkylation (compound 6) or deoxygenation (compound 7). A
similar trend is observed for N-ethoxyethylpyrrole 8 and its corre-
sponding deoxygenated analog 9.
Pyrroles 3–9 were synthesized from commercially available
2-(trifluoroacetyl)pyrrole (10) (Scheme 1). N-alkylation of 10 with
an appropriate bromide followed by a tert-butyllithium-mediated
condensation with N-(4-bromophenyl)-N-methylbenzenesulfona-
groups of 1 with a bulky aromatic moiety might force helix 12 to
point away from the binding site and thus prevent the ligand-
bound receptor from adopting a transcriptionally active conforma-
tion. There is precedent for this type of agonist to antagonist con-
version in the nuclear receptor field. For example, the estrogen
receptor (ER) agonist diethylstilbestrol was converted into the
antagonist 4-hydroxytamoxifen by the attachment of a suitable
bulky side chain to the agonist core structure. This structural
change prevented helix 12 from adopting an agonist-bound confor-
mation upon 4-hydroxytamoxifen binding.8
In our case, the replacement of one of the trifluoromethyl
groups of compound 1 with a variety of aryl and heterocyclic moi-
eties failed to generate compounds displaying appreciable levels of
LXR antagonism.9 During the course of this study, however, a sur-
prising SAR trend for a series of racemic N-substituted pyrrole
derivatives was identified (Table 2). As expected, removal of the
hydrogen bond donating capability of N-benzylpyrrole 3 signifi-
cantly reduced both LXR
a and LXRb binding affinities for methyl
Table 2
Binding affinities of pyrrole derivatives 3–9
Compound
R1
R2
SPA binding, IC50 (lM)
LXR
a
LXRb
3
4
5
6
7
8
9
OH
OCH3
OH
OCH3
H
Bn
Bn
0.8
>10
1.5
0.4
0.2
0.5
0.2
0.8
>10
1.8
0.9
0.8
1.5
0.7
CH2CH2OCH3
CH2CH2OCH3
CH2CH2OCH3
CH2CH2OCH2CH3
CH2CH2OCH2CH3
OH
H
Figure 1. (A) Compounds 1 and 5 increase transcriptional activation by LXRb in a
cell-based reporter gene assay. HEK-293 cells were transfected with a plasmid
containing a gene encoding a fusion of the GAL4 DNA-binding domain and the LXRb
ligand-binding domain, a luciferase reporter gene, and a b-galactosidase gene to
control for transfection efficiency. (B) Compounds
2 and 5 reduce the basal
interaction of LXRb and the nuclear receptor co-repressor NCoR in a homogeneous
Scheme 1. Reagents and conditions: (a) NaH, RBr, DMF, rt (for R = Bn) or 60 °C, 75–
80%; (b) (i) t-BuLi, N-(4-bromophenyl)-N-methylbenzene-sulfonamide, Et2O,
À100 °C, 10 min; (ii) add ketone, Et2O, À100 °C to rt, 65–70%; (c) NaH, MeI, DMF,
45–60%; (d) BF3ÁOEt2, Et3SiH, CH2Cl2, 33–34%.
time-resolved fluorescence (HTRF) assay. The emission intensity ratio has been
expressed as
a percentage relative to the control (DMSO) emission ratio. (C)
Compounds 2 and 5 increase endogenous ABCA1 gene expression in Caco-2 cells.
Expression level is normalized by expression of the housekeeping gene GAPDH.

D. J. Kopecky et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2407–2410
2409
and hydroxyalkyl chains (compounds 11–19, Fig. 2).11 Also, a series
of analogs 20–31 with modified aryl-heteroaryl linkers was made
(Fig. 3).12 Unfortunately, no significant improvements in binding
were observed for either set of derivatives.
Co-crystal structures of compounds 1 and 5 bound to LXRb have
been determined and provide a rational explanation for these
unanticipated results (Fig. 4). For compound 1, the usual agonist
binding conformation is observed, including a strong hydrogen
bond between the hydroxyl group of 1 and His435 (analogous to
His421 in LXRa). On the other hand, compound 5 adopts a novel
reversed agonist binding mode in the ligand binding site of LXRb,
presumably due to the large steric size of the functionalized pyr-
role. In this structure, the sulfonamide region of the molecule
points toward helix 12, and no hydrogen bond interaction is pres-
ent between 5 and the protein. The lack of a hydrogen bond be-
tween compound 5 and the ligand binding site in this reversed
agonist binding mode could account for the reduced LXRb binding
affinity of 5 relative to 1. This weakened interaction with helix 12
might also explain why compound 5 behaves as a partial agonist in
comparison to 1. Note that while compound 5 is racemic, only the
(S)-epimer of 5 is observed in the co-crystal structure.
Figure 2. Pyrrole N-alkyl and N-hydroxyalkyl derivatives.
In conclusion, an attempt to transform dual-LXRa/b agonists 1
and 2 into an antagonists via structural modification of the
1,1,1,3,3,3-hexafluoroisopropanol moiety led to the unexpected
discovery of a new series of LXR agonists. An X-ray co-crystal struc-
ture of 5 bound to LXRb displayed a novel, reversed agonist binding
mode characterized by the lack of a hydrogen bond anchoring the
substrate into the binding site. This new binding mode accommo-
dates for the bulky substituted pyrrole side chain of 5 without sig-
nificant reorientation of helix 12 relative to its position when
bound to 1.
Figure 3. Pyrrole derivatives with modified central linkers.
mide10 generated alcohols 3, 5, and 8. From these alcohol interme-
diates, methyl ether formation was accomplished with methyl io-
dide to afford
4 and 6. Alternatively, deoxygenation with
triethylsilane in the presence of boron trifluoride etherate led to
7 and 9.
References and notes
We were initially surprised that despite the presence of a bulky
pyrrole side chain, several compounds in this series behave as par-
tial agonists. For example, compound 5 exhibits approximately 33%
of the maximal response of 1 in an LXRb cell-based reporter gene
assay (Fig. 1).6 Compounds 6 and 8 also act as partial agonists,
while 3 is a very weak antagonist (data not shown). Moreover, 5
displays partial agonism (as compared to 2) in a biochemical assay
measuring LXRb co-repressor recruitment, and in a cell-based as-
say measuring endogenous expression of ABCA1 (Fig. 1).
Further structural modification of LXR agonist 5 was examined
in an effort to identify pyrrole-based agonists with enhanced bind-
ing affinities and cellular potencies. To this end, the pyrrole nitro-
gen was functionalized with a number of flexible, non-bulky alkyl
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Figure 4. X-ray co-crystal structure of full agonist 1 bound to LXRb (left picture, resolution = 2.45 Å, pdb code 4DK7) shows that a key hydrogen bond interaction exists
between 1 with His435. The X-ray co-crystal structure of partial agonist 5 bound to LXRb (right picture, resolution = 2.75 Å, pdb code 4DK8) shows that 5 binds in a ‘flipped’
orientation with no hydrogen bond interactions. The electron density map indicates that only the (S)-epimer of 5 is present. In both cases, Helix 12 adopts a closed
conformation that seals off the ligand binding site.

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11. Compounds 11–19 were synthesized from 2-(trifluoroacetyl)pyrrole (10) by a
similar route to that described in Scheme 1.
12. Compounds 20–24 and 27–31 were synthesized from pyrrole-2-
carboxaldehyde, while compounds 25–26 were made from 2-acetylpyrrole.