Design and discovery of 2-oxochromene derivatives as liver X receptor β-selective agonists

Article abstract of DOI:10.1016/j.bmcl.2015.01.047

In an attempt to molecularly design liver X receptor (LXR) β-selective agonists, we discovered that the combination of the 2-oxochromene moiety (head) and the imidazoline-2,4-dione moiety (tail) plays an important role in the expression potency and select

Full text of DOI:10.1016/j.bmcl.2015.01.047

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and discovery of 2-oxochromene derivatives as liver X
receptor b-selective agonists
Takayuki Matsuda, Ayumu Okuda, Yuichiro Watanabe, Tohru Miura, Hidefumi Ozawa, Ayako Tosaka,
⇑
Koichi Yamazaki, Yuki Yamaguchi, Sayaka Kurobuchi, Minoru Koura, Kimiyuki Shibuya
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Co., Ltd, 2-17-43, Noguchicho, Higashimurayam, Tokyo 189-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to molecularly design liver X receptor (LXR) b-selective agonists, we discovered that the
combination of the 2-oxochromene moiety (head) and the imidazoline-2,4-dione moiety (tail) plays an
important role in the expression potency and selectivity toward LXRb. We synthesized a series of 2-oxo-
chromene derivatives and identified 43 as a LXRb-selective agonist that increased the HDL-C level with-
out significantly elevating the TG level and resulted in a decreased lipid-accumulation area in the aortic
arch in a high-fat-and-cholesterol-fed Bio F1B hamster. In this manuscript, we report the design, synthe-
sis and pharmacology of these 2-oxochromene derivatives.
Received 7 November 2014
Revised 9 January 2015
Accepted 21 January 2015
Available online xxxx
Keywords:
Liver X receptor
LXRb-selective
HDL-C
Ó 2015 Elsevier Ltd. All rights reserved.
2-Oxochromene
Anti-atherosclerosis
Atherosclerotic cardiovascular disease (CVD), which includes
myocardial infarction and ischemic stroke, is a major cause of mor-
bidity and mortality in developed countries.1 CVD is characterized
by the accumulation of low-density lipoprotein (LDL) particles in
the arterial wall, which leads to the formation of cholesterol-laden
foam cells. Therefore, lipid-lowering drugs, such as statins, are
widely used to lower the circulating LDL as therapeutics for dysli-
pidemia, and thus far, these drugs have contributed to the suppres-
sion of such diseases.2 However, this therapy does not
satisfactorily reduce cardiovascular events.3 Therefore, we have
dedicated our efforts to developing a novel drug for the treatment
of atherosclerosis.
transport (RCT) plays an important role in regulating LXRs in clin-
ical trials.
The synthetic agonists T0901317 and GW3965 have been
reported as valuable tools for LXR studies (Fig. 1).6,7 In murine
and hamster models of atherosclerosis, LXR agonists increase the
plasma HDL-C levels and decrease atherosclerotic lesions.8 These
findings indicate the potential utility of LXR agonists for the treat-
ment of cardiovascular disease. However, significant side effects,
such as elevations in the plasma and hepatic triglyceride (TG) lev-
els, were observed as a result of lipogenesis due to the induction of
the sterol regulatory element-binding protein-1c (SREBP-1c) by
LXRa activation in the liver.
Liver X receptors (LXRs) are ligand-activated transcription fac-
tors involved in cholesterol metabolism, glucose homeostasis,
To develop an LXR agonist as an anti-atherosclerotic agent, we
should eliminate this undesirable side effect, which relies on
differentiating between these two LXR subtypes and discovering
inflammation and lipogenesis.4 LXR
a (known as NR1H3) is the
dominant subtype in the liver, small intestine, and macrophage,
whereas LXRb (known as NR1H2) is distributed ubiquitously. LXR
activation is known to regulate the expression levels of ATP-bind-
ing cassette transporter A1 (ABCA1), ABCG1 and G8 and cholesterol
metabolism, increasing the high-density lipoprotein particle cho-
lesterol (HDL-C) level and providing cholesterol efflux. These
actions induce an anti-atherogenic effect in the peripheral blood
vessels and can yield a synergistic effect when in combination with
statin therapy.5 We hypothesized that this reverse cholesterol
region-1
region-1
OH
CF3
Cl
N
F3C
F3C
region-2
CO2H
O
O
S
O
N
F3C
GW3965 (2)
T0901317 (1)
LXRα EC50 (Emax): 0.49 μM (100%)
LXR EC50 (Emax): 0.41 M (100%)
LXRα EC50 (Emax): 1.3 μM (60%)
LXRβ EC50 (Emax): 0.42 μM (62%)
β
μ
⇑
Corresponding author. Tel.: +81 42 391 6211; fax: +81 42 395 0312.
E-mail address: k-sibuya@kowa.co.jp (K. Shibuya).
Figure 1. Structure of the LXR agonists from Tularik and GlaxoSmithKline.
http://dx.doi.org/10.1016/j.bmcl.2015.01.047
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Matsuda, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.047

2
T. Matsuda et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
LXRb-selective agonists.9 Considering the high homology of the
LXR and LXRb ligand-binding domains (LBD; 77% amino acid iden-
region-1
CF3
region-2
R1
CF3
a
R2
Y
tity), it appears difficult to create an LXRb-selective agonist. In our
drug-discovery program, we first compared the differences in the
profiles for T0901317 and GW3965. The former agonist has no
X
N
O
O
O
O
O
N
O
hybridization
X = NR3, S
Y = O, H2
6
5
selectivity between LXR
a and LXRb, whereas the latter shows
LXRα EC50 (Emax): 6.1 μM (16%)
LXRβ EC50 (Emax): 3.2 μM (19%)
three-fold increased selectivity for LXRb.10 Through X-ray analysis,
we then found that the hydroxyl group of 1,1-bistrifluoromethyl-
carbinol (region-1) in T0901317 and 2-chloro-3-trifluorophenyl
group (region-1) in GW3965 can interact with His435 in the LXR
ligand-binding pocket.11 In contrast, the interaction of the phenyl
acetic acid (region-2) in GW3965 with Arg319, Ser242 and Leu330
could also be observed. These results led us to hypothesize that
the structural unit of region-2 may play an important role in activat-
ing LXRb. Our molecular design was based on the combination of
head-to-tail structures (corresponding to region-1 and region-2).
To explore the head core structure, we performed a high-through-
put screening of our chemical library for the assay of the up-regula-
tion of ABCA1 mRNA in a THP-1 human macrophage cell line and of
SREBP-1c mRNA in a HepG2 cell line. After the screening, com-
pounds 3, 4 and 5 were identified as hit compounds with a common
2-oxochromene core structure and a lipophilic substituent at the 4-
position. As shown in Table 1, 3 and 5 showed moderate induction of
ABCA1 mRNA compared with that of SREBP-1c mRNA, whereas 4
showed lower induction. The reference compound GW3965 showed
greater potency for ABCA1 up-regulation than 5.
region-1
CF3
region-1
region-2
region-2
F3C
R1
Y
R2
3
O
N
X
N
O
O
O
O
O
N
7
S
O
X = NR3, S
Y = O, H2
O
7
Merck's compound
LXRα EC50 (Emax): 1.3 μM (105%)
LXRβ EC50 (Emax): 0.69 μM (102%)
Figure 2. Drug design of the LXR agonists.
a
b
HO
OH
O
d
O
HO
OH
8
9
10
CF3
O
c
HO
OH
O
OH
11
12
Based on the structure of 5, we speculated that the trifluoro-
methyl group or the carbonyl oxygen can interact with the
His435-Trp457 activation switch11 to express LXR agonistic action
and that another lipophilic substituent at the 8- or 10-position may
be located in the highly hydrophobic LXR ligand-binding pocket.
However, the analysis of the tetracyclic structure of 5 revealed a
highly planar structure, indicating possible intercalation of the
DNA chain.12 Therefore, we disconnected the C–N bond of the
quinoline ring of 5 to dismantle the tetracyclic system. We then
referred to the Merck compound13 as the head-to-tail design to
denote the arrangement of the 3-trifluoromethyl group and the
7-n-propyl group on the benzisoxazole moiety (head) and the thi-
azolidine-2,4-dione moiety (tail). We also incorporated a similar
head-and-tail combination into our molecular design presented
in Figure 2 with an optimal linker. Based on this concept, we syn-
thesized two types of bis-n-propyl-2-oxochromen 6 and mono-n-
Scheme 1. Reagents and conditions: (a) allyl chloride, K2CO3, DMF, 70 °C, 24 h, 85%;
(b) N,N-dimethylaniline, 200 °C, 10 h, 57%; (c) H2, Pd/C, MeOH, rt, 18 h, 98%; (d)
ethyl 4,4,4-trifluoro-3-oxobutanoate, ZnCl2, 110 °C, 18 h, 59%.
nated to yield 11. Finally, 11 was condensed with ethyl 4,4,4-tri-
fluoro-3-oxobutanoate to furnish 12.
7-Hydroxy-8-n-propyl-4-(trifluoromethyl)-2H-chromen-2-one
(16) was prepared as depicted in Scheme 2. Lithiation of 1,3-dime-
thoxybenzene (13) with n-BuLi followed by alkylation with n-pro-
pyl iodide gave 1,3-dimethoxy-2-n-propylbenzene (14). After
demethylation of 14 with BBr3, the 2-n-propylbenzene-1,3-diol
(15) obtained was condensed with ethyl 4,4,4-trifluoro-3-oxobut-
anoate to furnish 16.
Imidazolidine-2,4-dione 1814 was prepared for a Bucherer–
Bergs reaction15 of ketone 17 using NaCN and (NH4)2CO3, as
depicted in Scheme 3.
propyl-2-oxochromen
relationships.
7 and studied their structure–activity
The preparation of 2-oxochromene derivatives 6 and 7 is
depicted in Scheme 4.16 6,8-Bis-n-propyl-2-oxochromene 12 and
8-n-propyl-2-oxochromene 16 were reacted with 1,4-dibromobu-
tane to obtain the alkylbromide intermediates, which were then
reacted with imidazolidine-2,4-dione, thiazolidine-2,4-dione, suc-
cinimide or oxazolidin-2-one to produce 6 and 7, respectively.
The activities of the series of 2-oxochromene derivatives 6 and
7-Hydroxy-6,8-di-n-propyl-4-(trifluoromethyl)-2H-chromen-2-
one (12) was prepared as depicted in Scheme 1. 1,3-Diallyloxyben-
zene (9) was obtained by the bis-allylation of resorcinol (8). A Cla-
isen rearrangement reaction of 9 was performed to yield 2,4-
diallylbenzene-1,3-diol (10), which was subsequently hydroge-
7 were evaluated through GAL4-LXRa
/b luciferase assays.17 We
Table 1
Structure and cellular activity of the hit compoundsa
investigated which atoms X and Y are favorable for compound 6.
The results are summarized in Table 2. Thiazolidine-2,4-dione 19
and imidazolidine-2,4-dione 20 showed moderate activities (LXRb
CF3
CF3
O
4
10
EC50 1.4
l
M, 1.3
l
M; LXRb Emax 45%, 47%) and exhibited seven- and
O
O
N
O
O
N
O
N
2
8
3
4
5
CF3
Compound
ABCA1b
SREBP-1cb
ABCA1/SREBP-1c
a
b
c
MeO
OMe
MeO
OMe
3
4
5
5.0
1.3
9.1
15
1.4
3.3
3.9
4.9
3.6
0.40
2.3
3.1
HO
OH
O
O
OH
13
14
15
16
GW3965
Scheme 2. Reagents and conditions: (a) n-BuLi, n-PrI, THF, rt, overnight, 45%; (b)
BBr3, CH2Cl2, À70 °C, 1 h to rt, 2 h, 76%; (c) ethyl 4,4,4-trifluoro-3-oxobutanoate,
ZnCl2, 110 °C, 18 h, 77%.
a
The assays were conducted at 5
The value is the ratio of the activation relative to that of the control (DMSO).
l
M.
b
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T. Matsuda et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
Table 3
O
R1
R2
a
LXR activity of the 2-oxochromene derivatives in the series 6 (20, 24–39) and 7 (40–
HN
R1 R2
47)a
NH
O
CF3
O
17
18
CF3
O
Y
Y
R1
R2
X
R1
R2
X
Scheme 3. Reagents and conditions: (a) NaCN, (NH4)2CO3, EtOH aq, 100 °C, 50–85%
N
N
O
O
O
O
O
O
6 (20, 24-39)
(Y = O)
7 (40-47) (Y = O)
CF3
Y
R1
a,b
Compound
X
R1
R2
LXR
a
LXRb
R2
12
16
N
b
b
EC50 (%)c
EC50 (%)c
O
O
O
O
O
X
O
20
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
NMe
NMe
NMe
NH
NMe
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
NMe
NH
NH
NH
NH
NH
NH
H
H
Me
H
H
2.6 (10)
1.7 (16)
11 (21)
1.5 (5)
ia (0)
ia (0)
ia (0)
ia (0)
ia (0)
ia (0)
ia (0)
nd (2)
ia (0)
ia (0)
ia (0)
ia (0)
ia (0)
nd (4)
2.4 (7)
ia (0)
nd (2)
ia (0)
nd (4)
ia (0)
ia (0)
1.3 (47)
1.3 (52)
4.7 (70)
1.3 (14)
ia (0)
5.3 (6)
2.4 (9)
ia (0)
ia (0)
0.96 (8)
ia (0)
1.2 (32)
ia (0)
nd (1)
8.0 (7)
ia (0)
6
7
Me
Me
Me
Ph
Ph
CF3
O
Y
R1
R2
a,b
N
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
X
O
4-MePh
2-MeOPh
3-MeOPh
4-MeOPh
3,4-diMeOPh
3,4-OCH2OPh
4-Me2NPh
4-CF3Ph
4-NO2Ph
4-HO2CPh
4-HOPh
Scheme 4. Reagents and conditions: (a) 1,4-dibromobutane, K2CO3, DMF, rt,
overnight, 80–96%; (b) amide or imide, K2CO3, DMF, rt, overnight, 51–99%.
Table 2
LXR activity of 2-oxochromene derivatives 6a
CF3
R1
Y
ia (0)
R2
H
Me
Ph
1.3 (26)
1.02 (51)
2.0 (8)
1.4 (31)
3.0 (9)
1.1 (39)
ia (0)
N
X
O
O
O
O
6 (R1 = R2 = H)
4-MeOPh
3,4-diMeOPh
3,4-OCH2OPh
4-HO2CPh
4-HOPh
b
b
Compound
X
Y
LXR
a
EC50 (%)c
LXRb EC50 (%)c
19
20
21
22
23
S
O
O
O
H2
H2
2.6 (6)
2.6 (10)
nd (2)
nd (1)
ia (0)
1.4 (45)
1.3 (47)
1.4 (22)
2.2 (5)
ia (0)
nd (2)
NMe
CH2
O
nd = not determined.
ia = inactive at 10 M.
l
a
CH2
The GAL4-LXR luciferase assay was performed with a maximal dose of 30 lM.
The results are provided as the means of two independent experiments.
nd = not determined.
ia = inactive at 10 M.
b
EC50 data are reported in
The% efficacy is defined as the percentage ratio between the maximal fold
l
M.
l
c
a
The GAL4-LXR luciferase assay was performed with a maximal dose of 30 lM.
induction for the test compound and the fold induction for T0901317 at 10
the same experiment.18
lM from
The results are provided as the means of two independent experiments.
b
EC50 data are reported in
The % efficacy is defined as the percentage ratio between the maximal fold
lM.
c
induction for the test compound and the fold induction for T0901317 at 10
the same experiment.18
lM from
the 4-dimethylaminophenyl group 35 was inactive. The analysis
of the electron-withdrawing groups showed that both the 4-tri-
fluoromethyl and 4-nitro groups (36–37) were weak and that the
4-carboxylic acid group was inactive (38). Considering the lipophil-
icity of the head moiety (e.g., ClogP of 34 shows 7.57), we removed
the n-propyl group at either position 6 or 8 from the 6,8-bis-n-pro-
pyl-2-oxochromenes and tested them. As a result, the 8-n-propyl-
2-oxochromene derivatives not only maintained their activities but
also improved their EC50 values (41 vs 25) and relatively decreased
five-fold selectivities for LXRb (Emax ratio), respectively. In contrast,
succinimide 21, oxazolidin-2-one 22 and pyrrolidin-2-one 23 pre-
sented significantly decreased and even negligible activities.
Both thiazolidine-2,4-dione and imidazolidine-2,4-dione were
identified as effective tails with LXRb agonistic action. In this study,
we selected 20 as a lead compound that could be varied for further
development of diversity. We then examined the effect of the sub-
stituent on the imidazolidine-2,4-dione ring in the series of 6 and
7, as shown in Table 3. Interestingly, the more substituted 25
showed a tendency for improved activity (25 > 24 > 20 (Emax)),
whereas 5-methyl-5-phenyl-substituted 27 lost activity. In the 5-
phenyl-substituted 27 and 28, the N-methyl-substituted 27 com-
their LXR
a activities. Molecule 43 showed prominent selectivity
with maintained potency, similarly to 32. Consequently, the hydro-
gen-bond-acceptor groups (4-MeO-43, 3,4-diMeO-44, and 3,4-
OCH2O-45) were superior to the hydrogen-bond-donor groups
(4-HO2C-46 and 4-HO-47).
To confirm our initial concept of LXRb-selective agonists, we
assessed 43 despite its weak potency (Emax 31%) toward LXRb
pletely lost both LXRa and LXRb activities, but the NH-free deriva-
and lack of LXRa activity (Emax 2%), as shown in Figure 3.
tive 28 maintained weak activity and moderate selectivity. The
results obtained for 1-NH-5-phenylimidazolidine-2,4-dione 28
prompted us to focus on the substituent of the phenyl group. To
investigate the influence of the substituents on the benzene ring,
we prepared and tested a variety of compounds (29–39). The anal-
ysis of the electron-donating groups (29–35) revealed the 4-meth-
ylphenyl group 29 prevailed over the 2-methoxy, 3-methoxy, 3,4-
dimethoxy groups (30–31, 33). 4-Methoxy and 3,4-methylenedi-
oxy-phenyl groups (32, 34) were the same or more than 29, and
We examined 43 through an in vivo test using a high-fat-and-
cholesterol-fed Bio F1B hamster. The F1B hamster was suitable for
the evaluation of the lipid-accumulation area in the aortic arch in
the hyperlipidemia model.19 T0901317 at doses of 3 and 10 mg/
kg decreased the lipid-accumulation area in the aortic arch (53%
and 52% relative to the control, respectively) but significantly ele-
vated the plasma TG (206% and 243%, respectively). In contrast, our
LXRb-selective agonist 43 was orally administered to the hamster
Please cite this article in press as: Matsuda, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.047

4
T. Matsuda et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 3. LXR
a
b activity of T0901317 and 43 determined through an in vitro GAL4-LXR luciferase assay.
Table 4
(c) Tice, C. M.; Noto, P. B.; Fan, K. Y.; Zhuang, L.; Lala, D. S.; Singh, S. B. J. Med.
Chem. 2014, 57, 7182.
In vivo study of the LXR agonists22
5. Giannarelli, C.; Cimmino, G.; Connolly, T. M.; Ibanez, B.; GarciaRuiz, J. M.;
Alique, M.; UroojZafar, M.; Fuster, V.; Feuerstein, G.; Badimon, J. J. Eur. Heart J.
2011
6. Terasaka, N.; Hiroshima, A.; Koieyama, T.; Ubukata, N.; Morikawa, Y.; Nakai, D.;
Inaba, T. FEBS Lett. 2003, 536, 6.
7. Joseph, S. B.; McKilligin, E.; Pei, L.; Watson, M. A.; Collins, A. R.; Laffitte, B. A.;
Chen, M.; Noh, G.; Goodman, J.; Hagger, G. N.; Tran, J.; Tippin, T. K.; Wang, X.;
Lusis, A. J.; Hsueh, W. A.; Law, R. E.; Collins, J. L.; Willson, T. M.; Tontonoz, P.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7604.
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P. Nat. Rev. Mol. Cell. Biol. 2012, 13, 213; (c) Fievet, C.; Staels, B. Biochem.
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23, 1169; (g) Calkin, A. C.; Tontonoz, P. Arterioscler. Thromb. Vasc. Biol. 2010, 30,
1513.
Compound Dose
TC
HDL-C Plasma TG Area of lipid accumulation
(mg/kg) (%)
(%)
(%)
in the aortic arch (%)
T0901317
3
10
123 67*
127* 59*
115 96
101 96
206*
243*
136
131
96
53*
52*
119
102
56*
43
30
100
300
98
113*
a The % value was calculated relative to the control.
*
p <0.05; the statistical analysis was conducted using Dunnett’s test.
at three-fold higher doses (30, 100 and 300 mg/kg) due to its poor
PK profile.20 Molecule 43 at a dose of 300 mg/kg increased the
HDL-C (113%) level and significantly decreased the lipid-accumula-
tion area in the aortic arch (56%) without elevating the plasma TG
(96%),21 as shown in Table 4.
9. (a) Schultz, J. R.; Tu, H.; Luk, A.; Repa, J. J.; Medina, J. C.; Li, L.; Schwendner, S.;
Wang, S.; Thoolen, M.; Mangelsdorf, D. J.; Lustig, K. D.; Shan, B. Genes Dev. 2000,
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Gustafsson, J.-A.; Basso, M. D.; Nambi, P. Mol. Pharmacol. 2006, 70, 1340.
In conclusion, a combination of the 2-oxochromene moiety
(head) and the imidazolidine-2,4-dione moiety (tail) in the molec-
ular design played a pivotal role in LXR activity and b selectivity.
The novel LXRb-selective agonist 43 increased the HDL-C level
without significant TG elevation and resulted in a decrease in the
lipid-accumulation area in the aortic arch in a high-fat-and-choles-
terol-fed Bio F1B hamster. Although we have successfully proven
our initial concept, it remains possible to further improve the
potency, selectivity and pharmacokinetics of our agonist, and fur-
ther developments are in progress.
10. In in vitro GAL4-LXR
a
l
b luciferase assay, LXR
M (60%) and LXRb EC50 (%); 0.42 l
a
b activity of GW3965 showed
LXR EC50 (Emax); 1.3
a
M (62%).
11. (a) Svensson, S.; Ostberg, T.; Jacobsson, M.; Norstrom, C.; Stefansson, K.; Hallen,
D.; Johansson, I. C.; Zachrisson, K.; Ogg, D.; Jendeberg, L. EMBO J. 2003, 22,
4625; (b) Hoerer, S.; Schmid, A.; Heckel, A.; Budzinski, R. M.; Nar, H. J. Mol. Biol.
2003, 334, 853; (c) 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.; Parks, D.;
Watson, M.; Wisely, B.; Willson, T. M. J. Biol. Chem. 2003, 278, 27138; (d)
Farnegardh, M.; Bonn, T.; Sun, S.; Ljunggren, J.; Ahola, H.; Wilhelmsson, A.;
Gustafsson, J. A.; Carlquist, M. J. Biol. Chem. 2003, 278, 38821.
12. Igarashi, J.; Sunagawa, M. Bioorg. Med. Chem. Lett. 1995, 23, 2923.
13. Int. Patent Appl. WO2004011448, 2004. In the Merck patent documentation,
only concrete biological data (% cholesterol efflux) for two compounds were
disclosed. We prepared the corresponding Merck compounds and evaluated
them through our luciferase assay using
structure.
a tail moiety as the reference
Acknowledgment
14. 1,5,5-Trimethylimidazolidine-2,4-dione (CAS NO. 6851-81-6) is commercially
available (Tokyo Chemical Industry Co., Ltd).
15. (a) Bergs, H. Ger. Pat. 1929, 566, 094; (b) Bucherer, H. T.; Fischbeck, H. T. J. Prakt.
Chem. 1934, 140, 69; (c) Bucherer, H. T.; Steiner, W. T. J. Prakt. Chem. 1934, 140,
291; (d) Ware, E. Chem. Rev. 1950, 46, 403.
We thank Dr. S. Tanabe, Director of Tokyo New Drug Research
Laboratories, Kowa Co., Ltd, for his support.
16. Int. Patent Appl. WO2009107387, 2009.
17. LXRa/GAL4 or LXRb/GAL4 hybrid and GAL4-responsive reporter vector pG5luc-
Supplementary data
stable-expression CHOK-1 cells were seeded at 20,000 cells/well on a 96-well
plate in HAM-F12 medium containing 10% immobilized bovine fetal serum,
Supplementary data (pharmacological experimental details,
pharmacokinetic experimental details, general preparation proce-
dure and analytical data of all synthetic compounds) associated
with this article can be found, in the online version, at http://dx.
doi.org/10.1016/j.bmcl.2015.01.047.
100 units/ml of penicillin G, and 100
incubated under a wet atmosphere with 5% CO2 at 37 °C. After 24 h, medium
with the test compound at various concentrations (0.01 M, 0.1 M, 1 M,
10 M, and 30 M) was added, and the cells were incubated for an additional
lg/ml of streptomycin sulfate and
l
l
l
l
l
24 hours. Using Bright-Glo (Promega) as a luciferase assay substrate and
measuring the luminescence intensity with a luminometer LB960 (Berthold
Technologies), the effect of the test compound on the activation of luciferase
References and notes
transcription via the LXRa- or LXRb-LBD was measured.
18. The luciferase activity results are shown in Tables 1–3 as activity values (%eff)
at the respective concentration of the test compound relative to the T0901317
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luminescence intensity of 100 at 10 lM.
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19. Srivastava reported that the hyperlipidemic Bio F1B hamster has been proven
to be an adequate atherosclerosis model for the evaluation of lipid-modulating
agents Srivastava, R. A. K. Atherosclerosis 2011, 214, 86; However, the above
experimental condition requires 21 weeks for the evaluation. To reduce the
experimental period to 10 weeks, we conducted our established protocol as
described in the following paper: Ikenoy, M.; Yoshinaka, Y.; Kobayashi, H.;
Kawamine, K.; Shibuya, K.; Sato, F.; Sawanobori, K.; Watanabe, T.; Miyazaki, A.
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Please cite this article in press as: Matsuda, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.047

T. Matsuda et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
20. The PK profiles of 43 were measured as follows. The CLs (
l
L/min/mg protein)
21. A reviewer suggested the possibility that the insignificant effect of 43 on
plasma TG could be attributed to LXRb partial agonism and/or poor liver
exposure (site of TG synthesis). At this stage, the cause, with the exception of
poor liver exposure, has not been clarified.
22. Interestingly, Srivastava reported that T0901317 did not alter the LDL-C level
but increased the TG level by three-fold and the HDL-C level by 50% under the
fed condition. Our pharmacological experimental details are described in the
Supplementary data.
of human, mouse and hamster hepatic microsomes were 393, 397 and 500,
respectively. After a solution of 43 in PEG400 was orally administered to
hamsters at doses of 30 and 100 mg/kg, the drug concentration of 43 in plasma
could not be detected because it is easily metabolized. Even at a dose of
300 mg/kg, 43 showed a low concentration in plasma for up to 2 h after
administration and disappeared 6 h after administration, as shown in the drug
concentration curve obtained for
Supplementary data.
a dose of 300 mg/kg presented in the
Please cite this article in press as: Matsuda, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.047