Styrylphenylphthalimides as Novel Transrepression-Selective Liver X Receptor (LXR) Modulators

Article abstract of DOI:10.1021/acsmedchemlett.5b00170

Anti-inflammatory effects of liver X receptor (LXR) ligands are thought to be largely due to LXR-mediated transrepression, whereas side effects are caused by activation of LXR-responsive gene expression (transactivation). Therefore, selective LXR modulators that preferentially exhibit transrepression activity should exhibit anti-inflammatory properties with fewer side effects. Here, we synthesized a series of styrylphenylphthalimide analogues and evaluated their structure-activity relationships focusing on LXRs-transactivating-agonistic/antagonistic activities and transrepressional activity. Among the compounds examined, 17l showed potent LXR-transrepressional activity with high selectivity over transactivating activity and did not show characteristic side effects of LXR-transactivating agonists in cells. This representative compound, 17l, was confirmed to have LXR-dependent transrepressional activity and to bind directly to LXRβ. Compound 17l should be useful not only as a chemical tool for studying the biological functions of LXRs transrepression but also as a candidate for a safer agent to treat inflammatory diseases.

Full text of DOI:10.1021/acsmedchemlett.5b00170

Letter
pubs.acs.org/acsmedchemlett
Styrylphenylphthalimides as Novel Transrepression-Selective Liver X
Receptor (LXR) Modulators
Sayaka Nomura,† Kaori Endo-Umeda,‡ Atsushi Aoyama,† Makoto Makishima,‡ Yuichi Hashimoto,†
and Minoru Ishikawa*,†
†Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
‡Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
S
* Supporting Information
ABSTRACT: Anti-inflammatory effects of liver X receptor (LXR) ligands are thought to be largely due to LXR-mediated
transrepression, whereas side effects are caused by activation of LXR-responsive gene expression (transactivation). Therefore,
selective LXR modulators that preferentially exhibit transrepression activity should exhibit anti-inflammatory properties with
fewer side effects. Here, we synthesized a series of styrylphenylphthalimide analogues and evaluated their structure−activity
relationships focusing on LXRs-transactivating-agonistic/antagonistic activities and transrepressional activity. Among the
compounds examined, 17l showed potent LXR-transrepressional activity with high selectivity over transactivating activity and did
not show characteristic side effects of LXR-transactivating agonists in cells. This representative compound, 17l, was confirmed to
have LXR-dependent transrepressional activity and to bind directly to LXRβ. Compound 17l should be useful not only as a
chemical tool for studying the biological functions of LXRs transrepression but also as a candidate for a safer agent to treat
inflammatory diseases.
KEYWORDS: Liver X receptor, transrepression, inhibition of interleukin-6 level, styrylphenylphthalimide
syndrome.5 The ligand-mediated activation of LXRE-depend-
uclear receptors (NRs) are ligand-dependent tran-
Nscription factors that regulate multiple biological
ent gene transcription is called transactivation.
The transactivating activities of LXR ligands depend upon
their effect on recruitment of cofactors (corepressors and
coactivators), and currently known LXR ligands can be
classified into two categories with respect to transactivation.
Transactivating agonists activate expression of the target genes
mentioned above. Numerous transactivating agonists for both
LXRα/β, including T0901317 (3)6 and GW3965 (4),7 have
been reported (Figure 1).8 Although LXRs appeared to be
attractive molecular targets for medicaments, the major
drawback of transactivating agonists is upregulation of multiple
LXRE-containing target genes, including sterol regulatory
element-binding protein-1c (SREBP-1c) and fatty acid synthase
(FAS). Up-regulation of these genes, especially in liver, leads to
increased triglyceride (TG) accumulation and induces serious
hypertriglyceridemia.9 This side effect is a major barrier to
medical application of LXR-transactivating agonists.
functions, including homeostasis, metabolism, and the immune
system. Forty-eight kinds of NRs have been identified in
humans. Among them, liver X receptors (LXRs) consist of two
subtypes, LXRα and LXRβ, which both form heterodimers with
retinoid X receptor (RXR). LXRα is highly expressed in liver,
intestine, adipose tissue, and macrophages, while LXRβ is
present ubiquitously in organs and tissues.1 The physiological
ligands for LXRα/β are oxysterols, including 22(R)-hydrox-
ycholesterol (1) and 24(S),25-epoxycholesterol (2) (Figure
1).2,3 Upon binding of an agonist to the ligand-binding domain
(LBD) of LXR, the LXR/RXR heterodimer binds to LXR
response elements (LXRE) in promoter regions of specific
genes, and helix 12 (H12) in the LBD adopts a conformation
that closes the ligand binding pocket and forms a groove to
which coactivators can bind, allowing gene transcription to
occur.4 The products of LXRE-regulated genes,2 such as
ABCA1, ABCG1, ABCG5, ABCG8, ApoE, and GLUT4, are
involved in lipid metabolism, reverse cholesterol transport, and
glucose transport, so LXRs are considered to be potential drug
targets for atherosclerosis, hyperlipidemia, and metabolic
Received: April 23, 2015
Accepted: July 13, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00170
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Figure 1. Chemical structures of endogenous LXR ligands (1 and 2), synthetic LXR ligands (3 and 4), and transrepression-selective LXR ligands (5
and 6).
In contrast with transactivating agonists, transactivating
antagonists stabilize the binding of corepressors and repress
expression of the target genes. LXRs antagonists might have
therapeutic value for hepatic steatosis.10 We11,12 and other
groups10,13,14 have reported several chemical classes of LXR-
transactivational antagonists.
scaffolds should improve our understanding of the molecular
basis of LXR-mediated transrepression. In this Letter, we
present styrylphenylphthalimides as a new chemical class of
potent and transrepression-selective LXR modulators.
Styrylphenylphthalimide analogues were synthesized as
described in the Supporting Information. To identify a lead
compound, we focused on our LXR-transactivational antago-
nists since they might show transrepression in an LXR-
dependent manner and do not show LXR-agonistic activity.
Thus, we screened our antagonists and their analogues for
transrepressional activity. Cell-based transrepressional activity
was evaluated in terms of reduction of LPS-induced IL-6
expression (designated below as IL-6-inhibitory activity) in
differentiated THP-1 (human acute monocytic leukemia) cells,
which express both LXRα/β.25 LXR-transactivating agonistic
activity and antagonistic activity were measured by means of
the Gal4-human LXR reporter system. The screening identified
ortho-phenethylphenylphthalimide (7),11 which showed a 31%
reduction of IL-6 level at 10 μM (Table 1). This compound
showed no LXRα/β-transactivating agonistic activity at 30 μM,
but had weak LXRα/β-transactivational antagonistic activity.
Recent studies have revealed that LXRs also function in the
regulation of inflammation in macrophages.15−17 In vitro,
several LXR ligands inhibit the expression of proinflammatory
mediators, such as interleukin-6 (IL-6), interleukin-1β (IL-1β),
and inducible nitric oxide synthase (iNOS), in response to
bacterial infection or lipopolysaccaride (LPS) stimulation. The
promoter regions of these proinflammatory genes do not
contain LXRE, which is present in all genes showing LXR-
dependent transactivation. This gene expression-inhibitory
function of LXR, i.e., inhibition of LXRE-independent gene
transcription by an LXR ligand, is called transrepression. In
2007, Ghisletti et al. proposed that 4 showed transrepressional
activity through including SUMOylation of LXR and blocking
of corepressor clearance.18 Studies in LXR-knockout mice
indicated that transrepression is mediated by both LXRα and
LXRβ. Transrepressional activities of LXR ligands were
reported to improve skin inflammation,19 rheumatoid arthri-
tis,20 sepsis,21 and hepatic injury caused by endotoxemia22 in
animal models. Therefore, LXR ligands that show trans-
repression activity are promising candidates for treatment of
these inflammatory diseases.
Table 1. SAR of Substituted Position of Phenethylphenyl
Group
Selective LXR modulators that preferentially exhibit trans-
repression activity should exhibit anti-inflammatory properties
with fewer side effects. Although LXR ligands such as 3 and 4
exhibit both transactivating and transrepressional activities, the
first transrepression-selective LXR modulators were reported by
GlaxoSmithKline in 2008.23 Compound 5 has similar trans-
repressional activity to 3 and does not increase TG
accumulation. However, 5 showed transactivating-agonistic
activity in a cell-based reporter assay. In 2012, we reported
that 6 showed more potent transrepressional activity than 3 or
5 and lacked transactivating activity at 30 μM under our assay
conditions.24 Very few transrepression-selective LXR modu-
lators have yet been reported,23,24 and a recent Perspective
noted that “SAR of transrepression is not well understood, and
this remains an underexplored area with potential in several
disease areas.”8 Therefore, investigation of the SAR of
transrepression-selective LXR modulators having various
LXRs transactivation
LXRs
agonistic activity
EC50 (μM)
antagonistic
activity IC50 (μM)
c
transrepression
% inhibition of
IL-6 level at
10 μM
compd position
LXRα
LXRβ
LXRα
13
LXRβ
>30
b
b
7
8
9
2
3
4
31
N.A.
N.A.
(46%)
>30
(48%)
>30
(26%)
b
b
9
N.A.
N.A.
11
a
b
b
N.A.
N.A.
N.A.
>30
(20%)
a
b
c
No activity at 10 μM. No activity at 30 μM. Percent inhibition at 30
μM is given in parentheses.
B
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Table 2. SAR of Substituent Effects of Styryl Group
LXRs transactivation
agonistic activity EC50 (μM) antagonistic activity IC50 (μM)
LXRα LXRβ
d
LXRs transrepression
a
compd
bond
R
% inhibition of IL-6 level at 10 μM
LXRα
LXRβ
c
c
7
s
H
31
35
12
50
N.A.
N.A.
13
>30 (46%)
c
c
c
20b
17a
17b
17c
17d
17e
17f
17g
17h
17i
s
MeO
H
N.A.
N.A.
>30 (30%)
>30 (40%)
>30 (30%)
>30 (48%)
>30 (21%)
27
N.A.
c
c
d
d
d
d
d
d
d
d
d
N.A.
N.A.
>30 (38%)
>30 (21%)
>30 (37%)
>30 (28%)
>30 (35%)
>30 (20%)
>30 (17%)
>30 (15%)
6.8
c
c
MeO
F
N.A.
N.A.
b
c
c
N.A.
N.A.
N.A.
c
c
Br
14
27
16
27
29
N.A.
N.A.
c
c
CN
Me
CF3
n-Bu
N.A.
N.A.
c
c
N.A.
N.A.
>30 (49%)
23
c
c
N.A.
N.A.
c
c
N.A.
N.A.
>30 (38%)
6.8
c
c
t-Bu
b
16
c
N.A.
N.A.
a
d
s, single bond; d, double bond. No activity at 10 μM. No activity at 30 μM. Percent inhibition at 30 μM is given in parentheses.
Table 3. SAR of Introduction of Methoxy and Methyl Group(s)
LXRs transactivation
agonistic activity EC50 (μM) antagonistic activity IC50 (μM)
c
LXRs transrepression
compd
R
% inhibition of IL-6 level at 10 μM
LXRα
LXRβ
LXRα
LXRβ
b
b
17b
17j
4-MeO
50
N.A.
N.A.
>30 (30%)
>30 (21%)
>30 (39%)
>30 (17%)
4.3
a
b
b
2-MeO
N.A.
N.A.
N.A.
30
b
b
17k
17l
3-MeO
28
78
N.A.
N.A.
30
b
b
3,4-diMeO
3,5-diMeO
3,4,5-triMeO
N.A.
N.A.
3.3
a
b
b
17m
17n
18l
N.A.
N.A.
N.A.
13
15
b
b
15
40
13
16
N.A.
N.A.
>30 (44%)
17
>30 (38%)
21
b
b
N.A.
N.A.
b
b
20l
N.A.
N.A.
23
>30 (33%)
>30 (20%)
25
b
b
17f
4-Me
N.A.
N.A.
>30 (49%)
22
a
b
b
17o
17p
17q
17r
2-Me
N.A.
N.A.
N.A.
b
b
3-Me
10
32
N.A.
N.A.
25
>30 (20%)
b
b
b
3,4-diMe
N.A.
N.A.
>30 (41%)
>30 (43%)
N.A.
a
b
b
3,5-diMe
N.A.
N.A.
N.A.
>30 (30%)
a
b
c
No activity at 10 μM. No activity at 30 μM. Percent inhibition at 30 μM is given in parentheses.
The results of screening led us to select 7 as a novel lead
The ethylene linker moiety was next examined (Table 2). At
the same time, a methoxy group was introduced at the para-
position of the phenethylphenyl group because many types of
LXR ligands possess a methoxy group(s).11,23,26,27 However,
introduction of the methoxy group (20b) did not result in any
increase of IL-6-inhibitory activity compared with 7. Replace-
ment of the ethyl linker of methoxy analogue 20b with the
compound, and we next synthesized various analogues to clarify
the SAR. First, the substitution position of the central phenyl
group was investigated. Meta (8) and para (9) analogues11
lacked IL-6-inhibitory activity (Table 1). This result led us to fix
the site of phenethyl substitution as the ortho-position. None of
the analogues in Table 1 showed LXRs-agonistic activity.
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Table 4. IC50 Values and EC50 Values of Representative Transrepression-Selective LXR Modulators
LXRs transactivation
LXRs transrepression
agonistic activity EC50 (μM)
antagonistic activity IC50 (μM)
LXRα LXRβ
compound
inhibition of IL-6 level IC50 (μM)
LXRα
LXRβ
b
b
3
26 17
1.7 1.6
1.5 0.8
0.21 0.10
0.066 0.013
N.T.
N.T.
11 0.43
4.3 0.46
a
a
c
c
6
N.A.
N.A.
4.1 0.045
3.3 1.8
a
a
17l
N.A.
N.A.
a
b
c
No activity at 30 μM. Not tested. Data taken from ref 24.
ethylene linker (17b) improved IL-6-inhibitory activity. This
result led us to fix the linker as an ethylene group.
interspaced short palindromic repeats/CRISPR associated
proteins) technology.29 LXRβ level in differentiated THP-1
cells was decreased by transfection of LXRβ CRISPR/Cas9
knockout plasmid (Figure S1a, Supporting Information). The
IL-6 level was significantly reduced by 17l without transfection
of LXRβ CRISPR/Cas9 knockout plasmid (Figure S1b).
However, the decrease in IL-6 expression induced by 17l was
blocked by transfection of LXRβ CRISPR/Cas9 knockout
plasmid (Figure S1c). This result indicates that LXRβ
contributes to the decrease of LPS-induced IL-6 expression
by 17l, at least in part. On the other hand, ligands for other
NRs such as peroxisome proliferator-activated receptors
(PPARs) are also known to show transrepression activity.18
Therefore, selectivity of 17l over PPARs was evaluated.
Compound 17l did not show agonistic activities toward
PPARα, PPARγ, or PPARδ, respectively (Figure S2, Supporting
Information).
We next investigated whether 17l binds directly to LXR by
means of time-resolved fluorescence resonance energy transfer
(TR-FRET) assay. In theory, the binding affinity between LXR
and the coactivator peptide can be evaluated in terms of the
TR-FRET ratio in the presence of agonist 3. In our experiment,
the binding affinity was maximum when the concentration of 3
was higher than 0.1 μM. Inhibition of binding between LXR
and the coactivator by 17l was assayed in terms of decrease of
this FRET signal due to competition of 3 with 17l. Under the
assay conditions used, compound 17l inhibited the FRET signal
in a dose-dependent manner (Figure 2). This result indicates
that 17l binds directly to LXRβ LBD. IC50 value of 17l was
estimated to 1.8 μM from Figure 2.
We next explored the effect of the substituent on the
terminal benzene ring of the styryl moiety (Table 2). We
synthesized several analogues 17c−17i possessing various small
(F, Me) or large (Br, n-Bu, t-Bu) monosubstituents and with
electron-withdrawing (CN, F, Br, CF3) or electron-donating
(Me, n-Bu, t-Bu) groups. However, all the analogues prepared
showed weaker activity than 17b. This result indicates that the
methoxy group is important for transrepression activity. The
tertiary-Bu analogue 17i showed the strongest LXRs-antago-
nistic activity among the compounds listed in Table 2, but
showed only weak IL-6-inhibitory activity. This result indicated
that not all the LXRs antagonists are potent IL-6 inhibitors.
Next, we investigated the substitution pattern of methoxy
group(s) (Table 3). Compared with the case of the mono-
methoxy group, IL-6-inhibitory activity of the ortho-methoxy
analogue 17j was weaker than those of the meta (17k) and para
(17b) analogues. So, we focused on di/tri-methoxy analogues
bearing methoxy groups at the meta- and/or para-positions.
The 3,4-dimethoxy analogue 17l showed increased IL-6-
inhibitory activity (78% inhibition at 10 μM). However, the
IL-6-inhibitory activity was dramatically decreased in the cases
of 3,5-dimethoxy analogue 17m and 3,4,5-trimethoxy analogue
17n. Next, we again tried to optimize the linker moiety. We
found the E-isomer 17l was more potent than the
corresponding Z-isomer 18l or the ethyl derivative 20l.
Monomethyl isomers 17f,o,p and dimethyl isomers 17q−r
were less potent than 17l. These results again show that
methoxy group(s) are important for transrepression activity. As
for substitution positions, 3,4-diMe analogue 17q showed more
potent IL-6-inhibitory activity than 3,5-diMe analogue 17r, and
this was consistent with the results for diMeO-substituted
derivatives. Therefore, 3,4-disubstitution might match the
binding pocket of LXR.
Compounds 17l and 3 were expected to share the same
binding site because 17l showed antagonistic activity toward
agonist 3 in LXRs LBD reporter gene assay. To understand the
binding mode between 17l and LXR, 17l was computationally
docked into the cocrystal structure of LXRα LBD complexed
Compound 17l showed dose-dependent transrepressional
activity. The IC50 value was estimated to be 1.5 μM, which was
about 16-fold more potent than representative LXR agonist 3
and 1.1-fold more potent than our previous transrepression-
selective LXR ligand 6 (Table 4). Furthermore, 17l was
transrepression-selective, that is, it showed LXRα/β-antago-
nistic activity with IC50 values of 3.3 and 4.3 μM, respectively,
and did not show LXRs-agonistic activity or LXRs-inverse
agonistic activity at 30 μM.
LPS transmits inflammatory signaling through Toll-like
receptor 4 (TLR4) in the plasma membrane, and this induces
degradation of the transrepressional complex on the promoter
region of proinflammatory genes, thereby activating gene
transcription.28 So, LPS-induced IL-6 expression is also reduced
by TLR4 antagonists or TLR4-signaling inhibitors. To examine
whether the decrease of IL-6 by 17l was LXR-dependent, we
used the recently developed CRISPR/Cas (clustered regularly
Figure 2. LXR binding assay of 17l by means of TR-FRET.
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with 4 (Figure S3a, Supporting Information).30 The most
favored conformation had a free energy of binding of −11 kcal/
mol, indicating that 17l binds at the binding site of LXRα
(Figure S3b). The dimethoxyphenyl group on 17l faces toward
H12 and occupies the pocket to which the chloro-
trifluorophenyl group of 4 binds. The docking model predicts
hydrogen-bonding interaction of the carbonyl group on 17l and
the hydroxyl group on Thr302 of LXRα. According to the X-
ray crystal structures of 3 and LXRs LBD,31,32 the hydroxyl
group of 3 forms a hydrogen bond with a histidine residue
(His421 for LXRα and His435 for LXRβ) in helix 11, adjacent
to H12, and this histidine interacts with a tryptophan residue in
H12; these interactions induce proper folding of H12, affording
transcriptionally active LXRs. It is noteworthy that 17l did not
appear to form a hydrogen bond with His421. This might be
the reason why 17l showed selective transrepressional activity,
versus transactivating activity.
AUTHOR INFORMATION
Corresponding Author
*E-mail: m-ishikawa@iam.u-tokyo.ac.jp.
Funding
The work described in this Letter was partially supported by
Grants-in Aid for Scientific Research from The Ministry of
Education, Culture, Sports, Science and Technology, Japan, and
the Japan Society for the Promotion of Science, and Platform
for Drug Discovery, Informatics, and Structural Life Science.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
Cas, CRISPR-associated protein; CRISPR, clustered regularly
interspaced short palindromic repeats; fas, fatty acid synthase;
H12, helix 12; IL-1β, interleukin-1β; IL-6, interleukin-6; LBD,
ligand-binding domain; LXR, liver X receptor; LXRE, LXR
response element; NR, nuclear receptor; SREBP-1c, sterol
regulatory element-binding protein-1c; TG, triglyceride; TR-
FRET, time-resolved fluorescence resonance energy transfer
We next examined the selectivity of 17l by means of mRNA
expression analysis of ABCA1 and SREBP-1c. In THP-1 cells,
17l did not upregulate ABCA1 and SREBP-1c mRNA
expression, unlike 3 (Figure 3), indicating that it does not
REFERENCES
■
(1) Peet, D. J.; Janowski, B. A.; Mangelsdorf, D. J. The LXRs: A New
Class of Oxysterol Receptors. Curr. Opin. Genet. Dev. 1998, 8, 571−
575.
(2) Janowski, B. A.; Willy, P. J.; Devi, T. R.; Falck, J. R.; Mangelsdorf,
D. J. An Oxysterol Signalling Pathway Mediated by the Nuclear
Receptor LXR Alpha. Nature 1996, 383, 728−731.
(3) Janowski, B. A.; Grogan, M. J.; Jones, S. A.; Wisely, G. B.;
Kliewer, S. A.; Corey, E. J.; Mangelsdorf, D. J. Structural Requirements
of Ligands for the Oxysterol Liver X Receptors LXRα and LXRβ. Proc.
Natl. Acad. Sci. U. S. A. 1999, 96, 266−271.
(4) Farnegardh, M.; Bonn, T.; Sun, S.; Ljunggren, J.; Ahola, H.;
̈
̊
Figure 3. Effect of 17l on mRNA expression of (a) ABCA1 and (b)
SREBP-1c in THP-1 cells.
Wilhelmsson, A.; Gustafsson, J. Å.; Carlquist, M. The Three-
Dimensional Structure of the Liver X Receptor B Reveals a Flexible
Ligand-Binding Pocket That Can Accommodate Fundamentally
Different Ligands. J. Biol. Chem. 2003, 278, 38821−38828.
(5) Laurencikiene, J.; Ryden, M. Liver X Receptors and Fat Cell
́
Metabolism. Int. J. Obes. 2012, 36, 1494−1502.
(6) Li, L.; Liu, J.; Zhu, L.; Cutler, S.; Hasegawa, H.; Shan, B.; Medina,
J. C. Discovery and Optimization of a Novel Series of Liver X
Receptor-Alpha Agonists. Bioorg. Med. Chem. Lett. 2006, 16, 1638−
1642.
(7) Collins, J. L.; Binz, J. G.; Plunket, K. D.; Morgan, D. G.; Beaudet,
E. J.; Whitney, K. D.; Kliewer, S. A.; Willson, T. M.; Fivush, A. M.;
Watson, M. A.; et al. Identification of a Nonsteroidal Liver X Receptor
Agonist through Parallel Array Synthesis of Tertiary Amines. J. Med.
Chem. 2002, 45, 1963−1966.
(8) Tice, C. M.; Noto, P. B.; Fan, K. Y.; Zhuang, L.; Lala, D. S.;
Singh, S. B. The Medicinal Chemistry of Liver X Receptor (LXR)
Modulators. J. Med. Chem. 2014, 57, 7182−7205.
exhibit transactivating agonist activity. Therefore, 17l should
not increase blood TG and may be a safer candidate as an anti-
inflammatory medicine than LXR ligands possessing both
transactivating-agonistic activity and transrepression activity.
In summary, we have identified novel LXR antagonists with a
phenethylphenylphthalimide skeleton and investigated their
SAR, focusing on IL-6-inhibitory activity and transactivational
agonistic/antagonistic activities evaluated by means of LXR
reporter gene assay. Representative compound 17l was found
to show potent LXR transrepressional activity with high
selectivity and did not increase expression of SREBP-1c (a
side effect of LXR agonists) in cells. In addition, 17l was
confirmed to have LXR-dependent transrepressional activity by
using the CRISPR/Cas system and was shown to bind directly
to LXRβ. This compound should be useful not only as a
chemical tool for studying the biological functions of LXRs
transrepression but also as a candidate for a safer agent to treat
inflammatory diseases.
(9) Repa, J. J.; Liang, G.; Ou, J.; Bashmakov, Y.; Lobaccaro, J. M. A.;
Shimomura, I.; Shan, B.; Brown, M. S.; Goldstein, J. L.; Mangelsdorf,
D. J. Regulation of Mouse Sterol Regulatory Element-Binding Protein-
1c Gene (SREBP-1c) by Oxysterol Receptors, LXRα and LXRβ. Genes
Dev. 2000, 14, 2819−2830.
(10) Griffett, K.; Solt, L. A.; El-gendy, B. E.-D. M.; Kamenecka, T.
M.; Burris, T. P. A Liver-Selective LXR Inverse Agonist That
Suppresses Hepatic Steatosis. ACS Chem. Biol. 2013, 8, 559−567.
(11) Motoshima, K.; Noguchi-Yachide, T.; Sugita, K.; Hashimoto, Y.;
Ishikawa, M. Separation of α-Glucosidase-Inhibitory and Liver X
Receptor-Antagonistic Activities of Phenethylphenyl Phthalimide
Analogs and Generation of LXRα-Selective Antagonists. Bioorg. Med.
Chem. 2009, 17, 5001−5014.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, Figures S1−S3, experimental section, and purity of
the synthesized compounds. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acsmedchemlett.5b00170.
(12) Noguchi-Yachide, T.; Aoyama, A.; Makishima, M.; Miyachi, H.;
Hashimoto, Y. Liver X Receptor Antagonists with a Phthalimide
E
DOI: 10.1021/acsmedchemlett.5b00170
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Skeleton Derived from Thalidomide-Related Glucosidase Inhibitors.
Bioorg. Med. Chem. Lett. 2007, 17, 3957−3961.
(28) Kawai, T.; Akira, S. TLR Signaling. Cell Death Differ. 2006, 13,
816−825.
(13) Zuercher, W. J.; Buckholz, R. G.; Campobasso, N.; Collins, J. L.;
Galardi, C. M.; Gampe, R. T.; Hyatt, S. M.; Merrihew, S. L.; Moore, J.
T.; Oplinger, J. A.; et al. Discovery of Tertiary Sulfonamides as Potent
Liver X Receptor Antagonists. J. Med. Chem. 2010, 53, 3412−3416.
(14) Jiao, X.; Kopecky, D. J.; Fisher, B.; Piper, D. E.; Labelle, M.;
McKendry, S.; Harrison, M.; Jones, S.; Jaen, J.; Shiau, A. K.; et al.
Discovery and Optimization of a Series of Liver X Receptor
Antagonists. Bioorg. Med. Chem. Lett. 2012, 22, 5966−5970.
(15) Joseph, S. B.; Bradley, M. N.; Castrillo, A.; Bruhn, K. W.; Mak,
P. A.; Pei, L.; Hogenesch, J.; O’connell, R. M.; Cheng, G.; Saez, E.;
et al. LXR-Dependent Gene Expression Is Important for Macrophage
Survival and the Innate Immune Response. Cell 2004, 119, 299−309.
(16) Joseph, S. B.; Castrillo, A.; Laffitte, B. A.; Mangelsdorf, D. J.;
Tontonoz, P. Reciprocal Regulation of Inflammation and Lipid
Metabolism by Liver X Receptors. Nat. Med. 2003, 9, 213−219.
(17) Glass, C. K.; Saijo, K. Nuclear Receptor Transrepression
Pathways That Regulate Inflammation in Macrophages and T Cells.
Nat. Rev. Immunol. 2010, 10, 365−376.
(29) Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; DiCarlo, J.
E.; Norville, J. E.; Church, G. M. RNA-Guided Human Genome
Engineering via Cas9. Science 2013, 339, 823−826.
(30) Fradera, X.; Vu, D.; Nimz, O.; Skene, R.; Hosfield, D.; Wynands,
R.; Cooke, A. J.; Haunsø, A.; King, A.; Bennett, D. J.; et al. X-Ray
Structures of the LXRα LBD in Its Homodimeric Form and
Implications for Heterodimer Signaling. J. Mol. Biol. 2010, 399,
120−132.
̈
(31) Svensson, S.; Ostberg, T.; Jacobsson, M.; Norstrom, C.;
̈
́
Stefansson, K.; Hallen, D.; Johansson, I. C.; Zachrisson, K.; Ogg, D.;
Jendeberg, L. Crystal Structure of the Heterodimeric Complex of
LXRα and RXRβ Ligand-Binding Domains in a Fully Agonistic
Conformation. EMBO J. 2003, 22, 4625−4633.
(32) Williams, S.; Bledsoe, R. K.; Collins, J. L.; Boggs, S.; Lambert,
M. H.; Miller, A. B.; Moore, J.; McKee, D. D.; Moore, L.; Nichols, J.;
et al. X-Ray Crystal Structure of the Liver X Receptor Beta Ligand
Binding Domain: Regulation by a Histidine-Tryptophan Switch. J. Biol.
Chem. 2003, 278, 27138−27143.
(18) Ghisletti, S.; Huang, W.; Ogawa, S.; Pascual, G.; Lin, M.-E.;
Willson, T. M.; Rosenfeld, M. G.; Glass, C. K. Parallel SUMOylation-
Dependent Pathways Mediate Gene- and Signal-Specific Trans-
repression by LXRs and PPARgamma. Mol. Cell 2007, 25, 57−70.
(19) Fowler, A. J.; Sheu, M. Y.; Schmuth, M.; Kao, J.; Fluhr, J. W.;
Rhein, L.; Collins, J. L.; Willson, T. M.; Mangelsdorf, D. J.; Elias, P.
M.; et al. Liver X Receptor Activators Display Anti-Inflammatory
Activity in Irritant and Allergic Contact Dermatitis Models: Liver-X-
Receptor-Specific Inhibition of Inflammation and Primary Cytokine
Production. J. Invest. Dermatol. 2003, 120, 246−255.
(20) Asquith, D. L.; Miller, A. M.; Hueber, A. J.; McKinnon, H. J.;
Sattar, N.; Graham, G. J.; McInnes, I. B. Liver X Receptor Agonism
Promotes Articular Inflammation in Murine Collagen-Induced
Arthritis. Arthritis Rheum. 2009, 60, 2655−2665.
(21) Wang, Y. Y.; Ryg, U.; Dahle, M. K.; Steffensen, K. R.;
Thiemermann, C.; Chaudry, I. H.; Reinholt, F. P.; Collins, J. L.; Nebb,
H. I.; Aasen, A. O.; et al. Liver X Receptor Protects against Liver Injury
in Sepsis Caused by Rodent Cecal Ligation and Puncture. Surg. Infect.
(Larchmt). 2011, 12, 283−289.
(22) Wang, Y. Y.; Dahle, M. K.; Agren, J.; Myhre, A. E.; Reinholt, F.
P.; Foster, S. J.; Collins, J. L.; Thiemermann, C.; Aasen, A. O.; Wang, J.
E. Activation of the Liver X Receptor Protects against Hepatic Injury
in Endotoxemia by Suppressing Kupffer Cell Activation. Shock 2006,
25, 141−146.
(23) Chao, E. Y.; Caravella, J. A.; Watson, M. A.; Campobasso, N.;
Ghisletti, S.; Billin, A. N.; Galardi, C.; Wang, P.; Laffitte, B. A.;
Iannone, M. A.; et al. Structure-Guided Design of N-Phenyl Tertiary
Amines as Transrepression-Selective Liver X Receptor Modulators
with Anti-Inflammatory Activity. J. Med. Chem. 2008, 51, 5758−5765.
(24) Aoyama, A.; Endo-Umeda, K.; Kishida, K.; Ohgane, K.;
Noguchi-Yachide, T.; Aoyama, H.; Ishikawa, M.; Miyachi, H.;
Makishima, M.; Hashimoto, Y. Design, Synthesis, and Biological
Evaluation of Novel Transrepression-Selective Liver X Receptor
(LXR) Ligands with 5,11-Dihydro-5-Methyl-11-Methylene-6H-
Dibenz[b,e]azepin-6-One Skeleton. J. Med. Chem. 2012, 55, 7360−
7377.
(25) Whitney, K. D.; Watson, M. A.; Goodwin, B.; Galardi, C. M.;
Maglich, J. M.; Wilson, J. G.; Willson, T. M.; Collins, J. L.; Kliewer, S.
A. Liver X Receptor (LXR) Regulation of the LXRα Gene in Human
Macrophages. J. Biol. Chem. 2001, 276, 43509−43515.
(26) Jaye, M. C.; Krawiec, J. A.; Campobasso, N.; Smallwood, A.;
Qiu, C.; Lu, Q.; Kerrigan, J. J.; Alvaro, M. D. L. F.; Laffitte, B.; Liu, W.;
et al. Discovery of Substituted Maleimides as Liver X Receptor
Agonists and Determination of a Ligand-Bound Crystal Structure. J.
Med. Chem. 2005, 48, 5419−5422.
(27) Molteni, V.; Li, X.; Nabakka, J.; Liang, F.; Wityak, J.; Koder, A.;
Vargas, L.; Romeo, R.; Mitro, N.; Mak, P. A.; et al. N-Acylthiadiazo-
lines, a New Class of Liver X Receptor Agonists with Selectivity for
LXRbeta. J. Med. Chem. 2007, 50, 4255−4259.
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DOI: 10.1021/acsmedchemlett.5b00170
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