Discovery of a 2-hydroxyacetophenone derivative as an outstanding linker to enhance potency and β-selectivity of liver X receptor agonist

Article abstract of DOI:10.1016/j.bmc.2016.05.048

Our research found that the 2-hydroxyacetophenone derivative is an outstanding linker between the 1,1-bistrifluoromethylcarbinol moiety and the imidazolidine-2,4-dione moiety to enhance the potency and β-selectivity of liver X receptor (LXR) agonist in ou

Full text of DOI:10.1016/j.bmc.2016.05.048

Accepted Manuscript
Discovery of a 2-hydroxyacetophenone derivative as an outstanding linker to
enhance potency and β-selectivity of liver X receptor agonist
Minoru Koura, Yuki Yamaguchi, Sayaka Kurobuchi, Hisashi Sumida, Yuichiro
Watanabe, Takashi Enomoto, Takayuki Matsuda, Ayumu Okuda, Tomoaki
Koshizawa, Yuki Matsumoto, Kimiyuki Shibuya
PII:
S0968-0896(16)30379-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.048
BMC 13032
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2016
20 May 2016
21 May 2016
Please cite this article as: Koura, M., Yamaguchi, Y., Kurobuchi, S., Sumida, H., Watanabe, Y., Enomoto, T.,
Matsuda, T., Okuda, A., Koshizawa, T., Matsumoto, Y., Shibuya, K., Discovery of a 2-hydroxyacetophenone
derivative as an outstanding linker to enhance potency and β-selectivity of liver X receptor agonist, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.048
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Discovery of a 2-hydroxyacetophenone derivative as an outstanding linker to
enhance potency and β-selectivity of liver X receptor agonist
Minoru Koura, Yuki Yamaguchi, Sayaka Kurobuchi, Hisashi Sumida, Yuichiro Watanabe, Takashi
Enomoto, Takayuki Matsuda, Ayumu Okuda, Tomoaki Koshizawa, Yuki Matsumoto and Kimiyuki
Shibuya∗
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Co., Ltd, 2-17-43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
ARTICLE INFO
ABSTRACT
Our research found that the 2-hydroxyacetophenone derivative is an outstanding linker between
the 1,1-bistrifluoromethylcarbinol moiety and the imidazolidine-2,4-dione moiety to enhance the
Article history:
Received
Received in revised form
Accepted
Available online
potency and β-selectivity of liver X receptor (LXR) agonist in our head-to-tail molecular design.
The incorporation of this linker is 20-fold more potent than our previous compound (2) for
LXRβ agonistic activity (EC50) in a GAL-4 luciferase assay. Furthermore, we also identified 5-
[5-(1-methylethoxy)pyridyl-2-yl]-5-methylimidazoline-2,4-dione (54), which lowers the
lipophilicity of 2-hydroxyacetophenone derivative. We revealed that a combination of our newly
developed linker and hydantoin (54) plays a pivotal role in improving the potency and
−)
( -56 increases high-density lipoprotein
selectivity of LXRβ. The optically separated
Keywords:
cholesterol levels without elevating plasma triglyceride levels and results in a decrease of the
lipid accumulation area in the aortic arch in a high-fat- and cholesterol-fed low-density
Liver X Receptor (LXR) β-selective
2-hydroxyacetophenone
ABCA1
−)
lipoprotein receptor knock-out mice. In this manuscript, we report that ( -56 is a highly potent
and β-selective LXR agonist for use in the treatment of atherosclerosis.
HDL-C
Anti-atherosclerosis
2016 Elsevier Ltd. All rights reserved.
Liver X receptors (LXRs) are ligand-activated transcription
factors involved in cholesterol metabolism, glucose homeostasis,
1. Introduction
inflammation and lipogenesis.3,4 LXRα (known as NR1H3) is the
dominant subtype in the liver, small intestine and in
macrophages, whereas LXRβ (known as NR1H2) is distributed
ubiquitously. LXR activation is known to regulate the expression
of the ATP-binding cassette transporter A1 (ABCA1), ABCG1
and ABCG8, cholesterol metabolism, increasing high-density
lipoprotein cholesterol (HDL-C) levels and cholesterol efflux.5-11
The agonistic actions of LXR would be expected to have an anti-
atherogenic effect in peripheral blood vessels. However,
elevations in the plasma and hepatic TG occurred.12,13 The
activation LXRα induces the expression of the sterol regulatory
element-binding protein-1c (SREBP-1c), and leads to the
observed lipogenesis.12,13 To suppress the undesirable effect of
increased lipogenesis in an LXR agonist, our efforts were
focused on differentiating between the two LXR subtypes.14-15
Although we have demonstrated the pharmacologically useful
effects of 1 and 2, there remain pharmacokinetic (PK) issues with
oral administration at higher doses. We could not detect the
plasma drug concentrations of 1 at doses of 30 and 100 mg/kg
due to the high clearance (CLs) of hamster hepatic microsomes
In our drug discovery program, we recently reported the 2-
oxochromene molecule 1 and the 1,1-bistrifluoromethylcarbinol
molecule 2 as liver X receptor (LXR) β-selective agonists and
demonstrated their potential pharmacological uses.1,2 Both
compounds have anti-atherogenic and hypolipidemic effects that
do not elevate triglycerides (TG) in the F1B hamster. These
compounds have a common feature in their molecular design, a
butylene linker between their head moieties (2-oxochromene and
1,1-bistrifluoromethylcarbinol) and their tail
(imidazolidine-2,4-diones), as shown in Figure 1.
moieties
Figure 1. Our discovered LXRβ agonists
———
∗ Corresponding author. Tel.: +81-423916211; fax: +81-423950312; e-mail: k-sibuya@kowa.co.jp

presence
of
imidazole
to
produce
4-((tert-
(500 µL/min/mg protein). Similarly, we monitored the lower
plasma drug concentration of 2 and the higher plasma
concentration of its metabolite, despite the improvement in the
CL of hamster hepatic microsome (49 µL/min/mg protein) after
oral administration at a dose of 100 mg/kg.2 We identified the
major metabolite of 2 as the carboxylate derivative 3 with the
oxidative cleavage of the C-N bond between the butylene unit
and the imidazolidine-2,4-dione unit as shown in Figure 2.
Therefore, our purpose in this study is to modify the chemical
structure of 2 and optimize the metabolic stability, potency and
selectivity of LXRβ. In this manuscript, we report our
optimization process and the discovery of a highly potent and β-
selective LXR agonist for the treatment of atherosclerosis.
butyldiphenylsilyl)oxy)but-2-yn-1-ol (12), which was followed
by a Mitsunobu reaction16 of 8 to produce 13. Next, we carried
out the deprotection of 13 by tetra-n-butylammonium fluoride
(TBAF) to release the alcohol 14, which reacted with a
combination of triphenylphosphine (PPh3) and carbon
tetrabromide (CBr4) to release the propalgyl bromide 15. The
alkylation of 15 with 5 in the presence of K2CO3 gave the product
16. Finally, we deprotected the MOM ether of 16 under acidic
conditions to yield the desired compound 17.
Scheme 2. Reagents and conditions: (a) TBDPSCl, imidazole, DMF, rt,
overnight, 29%; (b) DEAD, PPh3, THF, rt, overnight, 55%; (c) TBAF, THF,
1 h, 90%; (d) CBr4, PPh3, CH2Cl2, rt, 1 h, 87%; (e) K2CO3, DMF, rt,
overnight, 77%; (f) HCl/EtOAc, rt, 1 h, 88%.
Figure 2. Plan for the improvement of metabolic stability, potency (EC50)
and selectivity for LXRβ through the modification of the structure of 2.
2.2 Evaluation of the unsaturated alkyl linker derivatives
To evaluate the impact of the unsaturated alkyl linker on function,
we conducted a GAL4-LXR luciferase assay on 10a, 10b and 17.
Interestingly, the 2-cis-butene derivative 10a lost LXR activity
while the 2-trans-butene derivative 10b has slightly improved
potency (EC50 for LXRβ = 0.80 µΜ) and selectivity (EC50 ratio
2. Results and discussion
2.1 Synthesis of the unsaturated alkyl linker derivatives
First, we focused on the linker between the 1,1-bistrifluoro-
methylcarbinol moiety (head) and the imidazolidine-2,4-dione
moiety (tail), which could be a metabolic target (first pass effect)
in the liver. We then investigated how much the agonistic
activities change after modifying the structure of a butylene unit.
We prepared cis and trans-2-butene 10a-b as depicted in Scheme
for LXRα/LXRβ = 1.5) compared with
2
(EC50 for
LXRβ = 1.15 µΜ, EC50 ratio for LXRα/LXRβ = 1.0).
Furthermore, the butyne derivative 17 is more potent than 10b.
As can be seen, even the small modification of the linker
influences the activity and the selectivity towards LXRβ. Our
results are summarized and shown in Table 1.
1.
The
alkylation
of
4-(1,1,1,3,3,3-hexafluoro-2-
(methoxymethoxy)propan-2-yl)-2,6-di-n-propylphenol (5)2 with
cis-1,4-dichloro-2-butene (6a) and trans-1,4-dibromo-2-butene
(6b) under basic conditions produced the allylhalides 7a and 7b,
followed by alkylation with 5-(4-(1-methylethoxy)phenyl)-5-
methylimidazolidine-2,4-dione (8)2 under basic conditions to
produce 9a and 9b. Finally, under acidic conditions, the
methoxymethyl (MOM) ethers of 9a-b were deprotected to yield
the desired compounds in 3 steps of 10a and 10b at yields of
71% and 14%, respectively.
Table 1. LXR activity of the 1,1-bistrifluoromethylcarbinol derivatives
containing various linkers 4 (2, 10a, 10b, 17)a
Compound
linker
LXRα
EC50b (%)c
1.12 (26)
LXRβ
ClogP
EC50 α/β
EC50b (%)c
1.15 (146)
OMOM
OMOM
F3C
F3C
F3C
F3C
O
2
7.63
7.58
7.58
7.33
b
a
O
0.97
-
HN
+
+
X
X
NH
OH
X
O
O
6a
6b
cis: X = Cl
trans: X = Br
10a
10b
17
ia (0)
ia (1)
5
7a (cis, X = Cl)
8
7b (trans, X = Br)
OH
OMOM
F3C
F3C
F3C
F3C
O
O
1.23 (40)
0.86 (27)
0.80 (182)
0.61 (149)
c
O
O
1.54
1.41
N
N
NH
NH
O
O
O
O
9a
9b
10a
10b
(cis)
(trans)
(cis)
(trans)
ia = inactive at 10 µM.
Scheme 1. Reagents and conditions: (a) cis-1,4-dichloro-2-butene (6a) or
trans-1,4-dibromo-2-butene (6b), K2CO3, DMF, rt, overnight, 42-89%; (b)
K2CO3, DMF, rt, overnight, 21-73%; (c) HCl/EtOAc, rt, 1 h, 80%.
a The GAL4-LXR luciferase assay was performed at a maximum dose of 10
µM. The results are given as the mean of two independent experiments.
b EC50 data are reported in µM.
c The Emax (%) is defined as the percentage ratio between maximum fold
induction for the test compound and fold induction for T0901317 at 10 µM in
the same experiment.
We then prepared the but-2-yne derivative 17, as depicted in
Scheme 2. We performed the protection of but-2-yne-1,4-diol
(11) with tert-butyl(chloro)diphenylsilane (TBDPSCl) in the

These results led us to conclude that the orientation and the
distance of the linker between the head and tail moieties plays a
pivotal role in the agonistic actions of LXRs. We next replaced
the unsaturated alkyl with an aromatic part as a rigid linker to fix
the orientation of the head and tail moieties.
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane to produce the
stylene 40a-c, at yields of 99%, 64% and 75%, respectively. We
performed the procedure described in Scheme 4 (step b) to give
the phenethyl alcohol 41a-c. The bromination of 41a failed
because of its chemical instability; thus, we conducted the
tosylation of 41a with TsCl and pyridine to produce the tosylate
42a in 50% yield. We obtained the bromides 41b-c, which
resulted in lower yields than those produced by Scheme 2 (step
d). We prepared the desired compounds 44a-c via the cleavage of
the MOM ether 43a-c, which was obtained from 42a and 42b-c
by alkylation of 8 with overall yields of 17%, 4% and 3% with 8,
9 and 10 steps, respectively. The desired compound 44d was
prepared by the hydrogenolysis of 44c.
Finally, we also produced the acetophenone derivatives 49a-e as
depicted in Scheme 6. A Grignard reaction of 24b with MeMgBr
produced the 2-alcohol 45, followed by oxidation with MnO2 to
give the acetophenone 46. A Stille coupling reaction21 of 38a-c
with tributyl(1-ethoxyvinyl)stannane yielded the acetophenone
derivatives 47a-c. The direct tosylation of the methyl group of 46
and 47a-c was performed by treating with ((hydroxy)(tosyloxy)-
iodo)benzene (PhI(OH)OTs)22 to produce the tosylate 48a-d at
yields of 24%, 82%, 37% and 18%, respectively. The connection
between 48a-d and 8 was carried out under basic conditions in
the same manner (Scheme 5, step h) to produce 49a-d. The
hydrogenolysis of 49d yielded the desired phenol 49e.
2.3 Synthesis of the aromatic linker derivatives
Considering the increasing lipophilicity of the newly created
molecule after incorporation of an aromatic substituent into the
linker part, we used the mono-propyl phenol 182 as a starting
material instead of 5. To examine the impact of the orientation of
the linker between the head and tail moieties, we introduced the
benzyl part as a rigid linker and prepared the 22 derivative in a
retro-synthetic plan. The diaryl ether formation between 18 and
19 produced 20, then alkylation of 20 with hydantoin 8 produced
21 and finally, the deprotection of MOM ether 21 gave the
desired molecule 22 as shown in Figure 3.
2.4 Evaluation of the aromatic linker derivatives
The results of the LXR transactivation assay are summarized in
Table 2. The benzyl derivatives 28a-b and the phenethyl
derivatives 34a-b have moderate potency (EC50) toward LXRβ.
The para-substitution system has the tendency to express higher
subtype selectivity than the meta-substitution system (selectivity
for LXRα EC50 / LXRβ EC50; 28b = 5.5 vs 28a = 3.9; 34b = 5.3
vs 34a = 1.0). Based on 34b, which has good potency and
selectivity (EC50 for LXRβ = 1.88 µM, Emax = 223%), we
conducted further structural modifications and introduced
substituents at the 3-position of the phenethyl 34b. The methyl
group in 44a significantly enhanced the Emax value of LXRβ
(EC50 = 0.70 µM, Emax = 615%), but has the higher ClogP value
(8.12). To address this issue, we incorporated a polar substituent
group into the benzene ring. Interestingly, the methoxy and the
hydroxyl groups increased the agonistic activities of LXRβ (44b;
Figure 3. Outline of our retro-synthetic plan.
First, we prepared the benzyl derivatives 28a-b as depicted in
Scheme 3. Copper-promoted arylation17 of 18 with 3- or 4-
formylphenylboronic acid (23a or 23b) produced 3- or 4-
phenoxybenzaldehyde (24a or 24b), followed by a reduction with
NaBH4 gave the benzyl alcohol 25a-b product. The bromination
of 25a-b using a combination of PPh3 and CBr4 produced the
benzyl bromide 26a-b. The resultant 26a-b reacted with the
hydantoin 8 in the presence of K2CO3 to produce the bis-aryl
ether 27a-b. Finally, the MOM ether of 27a-b was deprotected
under acidic conditions to yield the desired compound 28a-b.
Second, to introduce the phenethyl into the linker, we prepared
the phenethyl derivatives 34a-b as depicted in Scheme 4.
Similarly, we usedthe copper-promoted arylation between 18 and
3- or 4-vinylphenylboronic acid (29a or 29b) to produce the
stylene 30a-b. Hydroboration of 30a-b with borane-THF
complex, followed by oxidation with NaBO3-4H2O, resulted in
the phenethyl alcohol 31a-b. We performed the reaction process
depicted in Scheme 2 (step d-f) to obtain the desired compound
34a-b.
Third, to introduce a substituent at the 2-potision of the
phenethyl moiety and examine the effects of the substituents, we
prepared the 2-substituted phenethyl derivatives 44a-d as
depicted in Scheme 5. The phenol 35 was reacted with methyl
iodide and benzyl bromide to give the phenyl ethers 36b and 36c,
respectively. The substitution of 4-fluoro-2-substituted-1-
nitrobenzene (36a R= Me, 36b R = OMe, 36c R = OBn) with 18
was carried out under basic conditions to give the 4-aryloxy-2-
substituted-1-nitrobenzene 37a-c, at yields of 71%, 91% and 96%,
respectively. Hydrogenation of 37a-b with Pd/C under a
hydrogen atmosphere gave the aniline 38a-b. In contrast, to
avoid deprotection of the benzyl ether, the reduction of 37c was
performed with iron powder under acid conditions to produce the
aniline 38c. We used a Sandmeyer reaction18,19 of 38a-c in the
presence of KI to produce the iodobenzene 39a-c. We also
conducted a Suzuki--Miyaura coupling reaction20 of 39a-c with
EC50 = 1.66 µM, Emax = 534%, 44d; EC50 = 2.87 µM, Emax
=
284%) and slightly lowered its lipophilicity (ClogP = 7.50 and
7.24). Having obtained the products 28a-44d, we turned our
attention to the benzylic position because it can be metabolically
labile. Introduction of the hydroxyl group at the benzylic position
of 34b (yielding 5023) results in slightly lower potency (EC50 for
LXRβ = 2.33 µM, Emax = 176%). Next, the conversion of the
alcohol 50 into the ketone 49a remarkably improves potency and
lipophilicity (EC50 for LXRβ = 0.12 µM, Emax = 236%, ClogP =
6.54), but has only moderate LXRα agonistic activity (EC50 for
LXRα = 0.36 µM, Emax = 46%). Successively, we examined
whether substitution at the 2-position of the acetophenone could
ameliorate the subtype selectivities of 44b and 44d. We obtained
satisfactory results of LXRβ activity (49b : 49c : 49e; EC50
=
0.064 : 0.36 : 0.11 µM; Emax = 264 : 306 : 348%), but could not
suppress LXRα activity (49b : 49c : 49e; EC50 = 0.40 : 3.74 :
0.34 µM; Emax = 71 : 38 : 55%). Accordingly, we noted the lower
lipophilicity of 49e and the fixed 2-hydroxyacetophenone linker,
and then explored how to modify the structure of 5-(4-(1-
methylethoxy)phenyl)-5-methylimidazolidine-2,4-dione (8). To
this end, we incorporated a nitrogen atom into the benzene ring
of 8.

Scheme 3. Reagents and conditions: (a) Cu(OAc)2, pyridine, MS4A, CH2Cl2, rt, overnight, 68-75%; (b) NaBH4, MeOH, 0 °C, 1 h, 99%; (c) CBr4, PPh3, CH2Cl2,
rt, 0.5-2 h, 18-94%; (d) K2CO3, DMF, rt, overnight, 21-73%; (e) HCl/EtOAc, rt, 1 h, 80%.
Scheme 4. Reagents and conditions: (a) Cu(OAc)2, pyridine, MS4A, CH2Cl2, rt, overnight, 68-75%; (b) BH3-THF, THF, rt, 1 h then NaBO3-4H2O, H2O, rt,
overnight, 46-85%; (c) CBr4, PPh3, CH2Cl2, rt, 0.5-2 h, 18-94%; (d) K2CO3, DMF, rt, overnight, 21-73%; (e) HCl/EtOAc, rt, 1 h, 80%.
Scheme 5. Reagents and conditions: (a) MeI or BnBr, K2CO3, DMF, 60 °C, 1 h, 99%; (b) K2CO3, DMF, 80 °C, 2 h, 71-96%; (c) i) H2, Pd/C, MeOH, rt, 1 h, 92%;
or Fe, aq. AcOH, rt, 4 h, 99%; (d) NaNO2, p-TsOH-H2O, KI, MeCN, rt, overnight, 30-67%; (e) 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane, Pd(PPh3)4,
Na2CO3, DMF-H2O, microwave, 80 °C, 20 min, 64-99%; (f) BH3-THF, THF, rt, 1 h then NaBO3-4H2O, H2O, rt, overnight, 62-73%; (g) i) TsCl, pyridine,
CH2Cl2, 40 °C, 2 h, 50% (42a) or ii) CBr4, PPh3, CH2Cl2, rt, 0.5-2 h, 10-31% (42b, 42c); (h) K2CO3, DMF, rt, overnight, 21-73%; (i) HCl/EtOAc, rt, 1 h, 99%.
OMOM
OMOM
OH
OMOM
O
F3C
F3C
F3C
F3C
F3C
F3C
CHO
a
b
O
O
O
24b (
p-)
45
46
O
N
OH
O
OMOM
O
OH
O
O
F3C
F3C
F3C
F3C
F3C
F3C
39a: R1 = Me
OTs
NH
d
c
e
: R1 = OMe
39b
: R1 = OBn
8
+
R1
R1
O
O
R1
O
O
39c
R1 = H
R1 = H,
: R1 = Me
48a:
48b:
48c:
49a:
49b:
49c:
47a
47b
R1 = CH3
R1 = CH3,
R1 = OCH3,
: R1 = OMe
R1 = OCH3
48d: R1 = OBn
47c: R1 = OBn
49d: R1 = OBn,
49e: R1 = OH,
f
Scheme 6. Reagents and conditions: (a) MeMgBr, THF, 0 °C, 0.5 h then rt, 1 h; (b) MnO2, CH2Cl2, rt, overnight, 77% for 2 steps; (c) (CH2CH(OEt))SnBu3,
Pd(PPh3)4, PhMe, reflux, 3 h, then 1 N HCl, rt, overnight, 50-80%; (d) PhI(OH)OTs, CH3CN, reflux, 6 h, 18-82%; (e) K2CO3, DMF, rt, overnight, 21-73%; (f)
H2, Pd/C, MeOH, rt, 3 h, 88%.

Table 2. LXR activity of the 1,1-bistrifluoromethylcarbinol derivatives containing various linkers 28-50a
Compound
linker
LXRα
EC50b (%)c
1.96 (53)
LXRβ
EC50b (%)c
0.50 (429)
ClogP
EC50
α/β
28a
28b
34a
34b
44a
44b
44d
50
7.35
7.35
7.63
7.63
8.12
7.50
7.24
6.81
3.92
>5.52
1.01
>5.32
4.89
6.02
3.48
ia (0)
1.81 (22)
2.89 (23)
ia (1)
2.85 (126)
1.88 (223)
0.70 (615)
1.66 (534)
2.87 (284)
2.33 (176)
3.14 (53)
ia (0)
ia (1)
ia (5)
4.29
3.00
O
49a
49b
49c
49e
0.36 (46)
0.40 (71)
3.74 (38)
0.34 (55)
0.12 (236)
0.064 (264)
0.36 (306)
0.11 (348)
6.54
7.02
6.41
6.15
6.25
10.4
3.09
ia = inactive at 10 µM.
a The GAL4-LXR luciferase assay was performed at a maximum dose of 10 µM. The results are given as the mean of two independent experiments.
b EC50 data are reported in µM.
c The Emax (%) is defined as the percentage ratio between maximum fold induction for the test compound and fold induction for T0901317 at 10 µM in the
same experiment.
2.5 Synthesis of the pyridylhydantoin derivative 56
We prepared the pyridylhydantoin 54 from 5-hydroxy-picolinonitrile (51) as depicted in Scheme 7. We performed the alkylation of
51 with 2-iodopropane to yield 5-isopropoxy-picolinonitrile (52) at 59%. Upon exposure of 52 to MeMgBr, followed by the work-
up produced the ketone 53. Finally, we conducted a Bucherer-Bergs reaction24-27 of 53 using NaCN and (NH4)2CO3 in aq. EtOH at
70 °C to produce 54 at 85% yield.
Scheme 7. Reagents and conditions: (a) i-PrI, K2CO3, DMF, rt, 6 h, 59%; (b)
MeMgBr, THF, 0 °C, 1 h, then HCl aq., 1 h, 91%; (c) NaCN, (NH4)2CO3, aq. EtOH, 70°C, 85%.

We performed the same procedure described in Scheme 6 (step e-f) to give the desired compound 56, as depicted in Scheme 8.
Scheme 8. Reagents and conditions: (a) K2CO3, DMF, rt, overnight, 21%; (b) H2, Pd/C, MeOH, rt, 3 h, 80%.
2.6 Evaluation of the pyridylhydantoin derivative 56
Interestingly, the pyridylhydantoin derivative 56 remarkably improved the potency and selectivity of LXRβ (EC50 for LXRβ =
0.058 µM, Emax = 329%, selectivity EC50 ratio for LXRα/LXRβ = 5.69, ClogP = 5.23) as shown in the dose-response curves of
T0901317, 2, 49e and 56 in Gal4-LXRα and LXRβ luciferase assays (Figure 4).
Figure 4. Dose-response curves of T0901317, ( )-2, ( )-49e and ( )-56
Successively, we evaluated an in vitro liver CL of 56 compared to the prototype compounds 2 and 49e. To our delight, the metabolic
stability of 56 has an improved value in all species examined as shown in Table 3.
Table 3. hepatic CLint (µL/min/mg protein) of each animal obtained using an in vitro assay.*
Compound
Mouse
84
Hamster
206
Human
2
71
12
7
49e
56
76
27
24
11
*The hepatic CLint values of compounds 2, 49e and 56 were assessed using hepatic microsomes from each animal (mouse, hamster and human).28
We concluded that 56 would be a well-balanced LXRβ agonist and warrants further evaluation.
2.7. Synthesis and evaluation of both enantiomers of 56
We conducted the chiral separation of ( )-54 by optical resolution using a CHIRALPAK AY-H column and obtained both
enantiomers of (+)-54 and (−)-54.29 Having prepared (+)-56 and (−)-56 in the same manner in Scheme 8 (steps a-b), we evaluated
−)
the compounds in Gal4-LXRα and LXRβ luciferase assays. We found that ( -56 is the desired LXRβ agonist (EC50 for LXRβ; (
−)
-
56: 0.007 µM, EC50 α/β selectivity: 28.5), while (+)-56 has almost no LXRα agonistic activity and has significantly decreased
LXR β agonistic activity (EC50 for LXRβ; (+)-56: 2.9 µM) as shown in Figure 5.

Figure 5. Dose-response curves of (̶−)-56 and (+)-56
To examine systemic PK, we measured the drug concentration of (−)-56 after oral administration in mice at a dose of 10 mg/kg
−)
using PEG400 as a delivery vehicle. Our results show that the plasma drug concentration levels of ( -56 have nearly equivalent
levels of 2 at a dose of 100 mg/kg (Table 4).30
−)
Table 4 PK profile of ( )-2 and ( -56
Compound Dose (mg/kg)
Cmax (ng/mL)
AUC (h*ng/mL)
( )-2
−)
100
10
406
503
3419
3027
(
-56
Considering the enhanced potency of (−)-56, we then conducted an in vivo test in another established animal model. We changed
animal model from high-fat- and cholesterol-fed F1B hamsters to high-fat and cholesterol-fed low-density lipoprotein (LDL)
receptor knock-out mice, which is one of the most widely used and promising animal models for atherosclerosis.31,32 We evaluated
33
−)
(
-56 and T0901317 in this mouse model. At a dose of 10 mg/kg, T0901317 significantly decreased the area of lipid
accumulation in the aortic arch (40% relative to the control) with a decrease of LDL-C, but significantly elevated the plasma TG
level (465%) and decreased HDL-C (47%). In contrast, even at doses of 1 and 3 mg/kg, (−)-56 remarkably decreased the area of
lipid accumulation (62 and 59%, respectively) and increased in HDL-C (115 and 116%, respectively) without altering LDL-C. From
−)
these results, we speculate that the increase in HDL-C by ( -56 substantially contributes the cholesterol efflux from atherogenic
lesions.34 On the other hand, the anti-atherosclerotic effect of T0901317 could be attributed to a significant lowering of LDL-C
(55%) as shown in Table 5.
34
−)
Table 5. In vivo study of T0901317 and ( -56
Compound Dose
TC
HDL-C
(%)
LDL-C
(%)
Plasma
TG
Area of
lipid
accumul
(mg/k
g)
(%)
(%)
ation
(%)
T0901317
−)
10
1
96
47*
55*
108
97
465*
152
40*
62*
59*
(
-56
111
102
115*
116*
3
166*
*p < 0.05; Statistical analysis was conducted using Dunnett’s test. The % value is calculated relative to control.
3
Conclusion
In conclusion, we discovered a 2-hydroxyacetophenone derivative that has an outstanding linker between the 1,1-
−)
bistrifluoromethylcarbinol moiety and imidazolidine-2,4-dione moiety in our head-to-tail molecular design. Our novel ( -56
compound increased HDL-C without significantly affecting TG levels and leaded to a decrease in the lipid accumulation area of the
aortic arch in high-fat and cholesterol-fed LDL receptor knock-out mice. However, the identification of the absolute configuration
−)
of ( -56 and its efficient synthesis requires further study. Further development of this compound is currently underway in our
laboratory.
4 Experimental Section
4.1. Chemistry
4.1.1. General comments

Commercially available reagents and solvents were used without further purification. Thin layer chromatography (TLC) analyses
were performed on silica gel 60 F254 plates (Merck). 1H NMR and 13C NMR spectra were obtained on a JEOL JNM-LA 400 MHz
spectrometer using CDCl3 or CD3OD as the solvent with tetramethylsilane as the internal standard. Infrared (IR) spectra were
recorded on a Thermo Nicolet 370 FT-IR (ATR) spectrometer. Mass spectra were obtained on a JEOL MS-BU20 mass
spectrometer. Elemental analyses (C, H, N) were performed using a Yanaco MT-5 instrument. Melting points were determined in
open glass capillaries on a Buchi B-545 melting point apparatus. Optical rotations were measured on a JASCO P2200 polarimeter
operating at the sodium D line at room temperature. The chiral HPLC analyses were performed on a Shimazu LC-2010A HT liquid
chromatograph.
4.1.2. Representative synthetic procedure of (−)-56
2-Benzyloxy-4-fluoro-1-nitrobenzene (36c):
To a stirred suspension of 4-fluoro-2-hydroxy-1-nitrobenzene (35) (5.0 g, 31.8 mmol) and K2CO3 (5.28 g, 38.2 mmol) in N, N-
dimethylformamide (DMF) (50 mL) was added benzyl bromide (5.99 g, 35.0 mmol) at room temperature. The reaction mixture was
stirred at 60 °C for 1 h. After the reaction was completed, the reaction mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc = 5/1) to give the title compound (9.47 g, 99%) as a pale yellow crystal; 1H NMR (400
MHz, CDCl3) δ 5.23 (2H, s), 6.74 (1H, ddd, J = 2.4, 7.3, 9.0 Hz), 6.83 (1H, dd, J = 2.4, 10.2 Hz), 7.33-7.47 (5H, m), 7.97 (1H, dd, J
= 6.1, 9.0 Hz); MS (EI) m/z 247 [M]+; Anal. Calcd for C13H10FNO3: C, 63.16; H, 4.08; N, 5.67. Found: C, 63.23; H, 4.11; N, 5.61.
2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-(methoxy-methoxy)propan-2-yl)-2-n-propylphenoxy)-1-nitrobenzene (37c):
To a stirred suspension of 4-(1,1,1,3,3,3-hexafluoro-2-(methoxy-methoxy)propan-2-yl)-2-n-propylphenol (18) (5.30 g, 15.3 mmol)
and K2CO3 (2.64 g, 19.1 mmol) in DMF (25 mL) was added 36c (3.15 g, 12.7 mmol) at room temperature. The reaction mixture
was stirred at 80 °C for 3 h. After the reaction was completed, the reaction mixture was diluted with water and extracted with
EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane/acetone = 10/1) to give the title compound (8.31 g, 99%) as a pale yellow crystal; 1H NMR
(400 MHz, CDCl3) δ 0.89 (3H, t, J = 7.1 Hz), 1.55 (2H, qt, J = 7.1, 7.8 Hz), 2.50 (2H, t, J = 7.8 Hz), 3.58 (3H, s), 4.89 (2H, s), 5.18
(2H, s), 6.50 (1H, dd, J = 2.2, 8.6 Hz), 6.58 (1H, d, J = 2.2 Hz), 6.92 (1H, d, J = 8.8 Hz), 7.31-7.39 (5H, m), 7.46 (1H, d, J = 8.8
Hz), 7.54 (1H, s), 7.97 (1H, d, J = 8.6 Hz); MS (EI) m/z 573 [M]+; Anal. Calcd for C27H25F6NO6: C, 56.55; H, 4.39; N, 2.44. Found:
C, 56.57; H, 4.33; N, 2.42.
2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-(methoxy-methoxy)propan-2-yl)-2-propylphenoxy)aniline (38c):
To a stirred solution of 37c (8.31 g, 12.7 mmol) in acetic acid (64 mL) and water (4.0 mL) was added iron powder (10.7 g, 191
mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 h. After completing the reaction, 1 N NaOH
aq. was added to the reaction mixture. The resulting mixture was filtered through a pad of Celite and rinsed with EtOAc. The
reaction mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo
to give the title compound (8.49 g, 99%) as a yellow amorphous; 1H NMR (400 MHz, CDCl3) δ 0.97 (3H, t, J = 7.3 Hz), 1.68 (2H,
qt, J = 7.3, 7.3 Hz), 2.70 (2H, t, J = 7.3 Hz), 3.55 (3H, s), 4.84 (2H, s), 5.04 (2H, s), 6.50 (1H, dd, J = 2.4, 8.3 Hz), 6.61 (1H, d, J =
2.4 Hz), 6.67 (1H, d, J = 8.8 Hz), 6.72 (1H, d, J = 8.3 Hz), 7.33-7.40 (7H, m); MS (EI) m/z 543 [M]+; Anal. Calcd for C27H27F6NO4:
C, 59.67; H, 5.01; N, 2.58. Found: C, 59.66; H, 4.87; N, 2.65.
2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-(methoxy-methoxy)propan-2-yl)-2-n-propylphenoxy)-1-iodobenzene (39c):
To a stirred suspension of 38c (8.49 g, 12.7 mmol) and p-toluene sulfonic acid monohydrate (7.27 g, 38.2 mmol) in CH3CN (64
mL) was added the solution (water 8.5 mL) of a mixture of NaNO2 (1.76 g, 25.5 mmol) and KI (5.28 g, 31.8 mmol) at 0 °C. The
reaction mixture was stirred at 0 °C for 10 min, and stirred at room temperature overnight. To the reaction mixture was added
saturated Na2S2O3 aq. and saturated NaHCO3 aq. The aqueous layer was extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-
hexane/EtOAc = 10/1) to give the title compound (4.86 g, 58%) as a yellow crystal; 1H NMR (400 MHz, CDCl3) δ 0.92 (3H, t, J =
7.3 Hz), 1.61 (2H, qt, J = 7.3, 7.6 Hz), 2.60 (2H, t, J = 7.6 Hz), 3.56 (3H, s), 4.86 (2H, s), 5.11 (2H, s), 6.36 (1H, dd, J = 2.4, 8.6
Hz), 6.52 (1H, d, J = 2.4 Hz), 6.78 (1H, d, J = 8.9 Hz), 7.31-7.43 (6H, m), 7.47 (1H, s), 7.70 (1H, d, J = 8.6 Hz); MS (EI) m/z 654
[M]+; Anal. Calcd for C27H25F6IO4: C, 49.56; H, 3.85. Found: C, 49.74; H, 3.79.
1-(2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-(methoxy-methoxy)propan-2-yl)-2-propylphenoxy)phenyl)ethan-1-one (47c):
To a stirred solution of 39c (852 mg, 1.3 mmol) in toluene (13 mL) was added ethoxyvinyltributyl tin (2.35 g, 6.5 mmol) and
tetrakis(triphenylphosphine)palladium (0) (Pd(PPh3)4) (300 mg, 0.26 mmol) under an argon atmosphere. The reaction mixture was
refluxed for 1 h under an argon atmosphere. The reaction mixture was diluted with 5% HCl at 0 °C, and then stirred overnight at
room temperature. The reaction mixture was filtered through a pad of Celite and rinsed with EtOAc. The aqueous layer was
extracted with EtOAc. The organic layer was washed with saturated NaHCO3 aq. and brine, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (n-hexane/EtOAc = 10/1) to give the title compound (348 mg, 47%) as a yellow

oil; 1H NMR (400 MHz, CDCl3) δ 0.93 (3H, t, J = 7.3 Hz), 1.61 (2H, qt, J = 7.3, 7.8 Hz), 2.59 (2H, t, J = 7.8 Hz), 2.62 (3H, s), 3.60
(3H, s), 4.90 (2H, s), 5.13 (2H, s), 6.54 (1H, dd, J = 2.4, 8.6 Hz), 6.60 (1H, d, J = 2.4 Hz), 6.93 (1H, d, J = 8.9 Hz), 7.34-7.45 (6H,
m), 7.53 (1H, s), 7.83 (1H, d, J = 8.6 Hz); MS (EI) m/z 570 [M]+; Anal. Calcd for C29H28F6O5: C, 61.05; H, 4.95. Found: C, 60.91;
H, 4.94.
2-(2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-propan-2-yl)-2-n-propylphenoxy)phenyl)-2-oxoethyl
benzenesulfonate (48d):
4-methyl
To a stirred solution of 47c (70 mg, 0.12 mmol) in CH3CN (0.65 mL) was added ((hydroxy)(tosyloxy)iodo)benzene (PhI(OH)OTs)
(96 mg, 0.25 mmol). The reaction mixture was refluxed for 6 h and then concentrated in vacuo. The residue was diluted with water
at 0 °C. The aqueous layer was extracted with EtOAc. The organic layer was washed with brine, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (n-hexane/EtOAc = 3/1) to give the title compound (80 mg, 93%) as a
yellow oil; 1H NMR (400 MHz, CDCl3) δ 0.89 (3H, t, J = 7.3 Hz), 1.57 (2H, qt, J = 7.3, 7.3 Hz), 2.43 (3H, s), 2.52 (2H, t, J = 7.3
Hz), 5.07 (2H, s), 5.14 (2H, s), 6.50-6.54 (2H, m), 6.94 (1H, d, J = 8.6 Hz), 7.24-7.43 (7H, m), 7.54 (1H, d, J = 8.6 Hz), 7.62 (1H,
s), 7.67 (2H, d, J = 8.6 Hz), 7.92 (1H, d, J = 9.2 Hz); MS (EI) m/z 696 [M]+.
5-(1-Methylethoxy)picolinonitrile (52):
To a stirred suspension of 5-hydroxypicolinonitrile (51) (616 mg, 5.13 mmol) and K2CO3 (1.42 g, 10.3 mmol) in DMF (3.0 mL)
was added 2-iodopropane (1.31 g, 7.7 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 5.5 h. To the
reaction mixture was added water (3.0 mL). The aqueous layer was extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-
hexane/EtOAc = 10/1) to give the title compound (491 mg, 59%) as a yellow oil; 1H NMR (400 MHz, CDCl3) δ 1.40 (6H, d, J = 6.2
Hz), 4.66 (1H, sept, J = 6.2 Hz), 7.19 (1H, dd, J = 2.7, 8.6 Hz), 7.62 (1H, d, J = 8.6 Hz), 8.32 (1H, d, J = 2.7 Hz); MS (EI) m/z 162
[M]+.
1-(5-(1-Methylethoxy)pyridin-2-yl)ethan-1-one (53):
To a stirred solution of 52 (491 mg, 3.0 mmol) in THF (20 mL) was added methylmagnesium bromide (MeMgBr) (0.97 M in THF
solution, 9.3 mL, 9.1 mmol) at 0 °C under an argon atmosphere. The reaction mixture was stirred 0 °C for 2 h. To the reaction
mixture was added 1 N HCl (10 mL). Then, the reaction mixture was neutralized with saturated NaHCO3 aq. at 0 °C. The aqueous
layer was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography (n-hexane/EtOAc = 10/1) to give the title compound (418 mg, 77%) as a
yellow oil; 1H NMR (400 MHz, CDCl3) δ 1.40 (6H, d, J = 5.9 Hz), 2.68 (3H, s), 4.68 (1H, sept, J = 5.9 Hz), 7.22 (1H, dd, J = 3.0,
8.6 Hz), 8.03 (1H, d, J = 8.6 Hz), 8.28 (1H, d, J = 3.0 Hz); MS (EI) m/z 179 [M]+.
Racemic-5-[5-(1-methylethoxy)pyridin-2-yl]-5-methyl- imidazolidine-2,4-dione (( )-54):
To a stirred solution of 53 (417 mg, 2.3 mmol) in EtOH (2.3 mL) was added NaCN (1.02 g, 20.9 mmol), (NH4)2CO3 (4.03 g, 41.9
mmol) and water (2.3 mL) at room temperature. The reaction mixture was irradiated using a microwave reactor (Biotage® Initiator)
at 100 °C for 1 h. The reaction mixture was concentrated to remove EtOH. The resulting precipitate was filtered off and washed
with water. The solid was recrystallized from a mixture solvent of CHCl3 and MeOH (20/1) to give the title compound (504 mg,
87%) as a brown amorphous; 1H NMR (400 MHz, CDCl3) δ 1.33 (6H, d, J = 6.2 Hz), 1.79 (3H, s), 4.67 (1H, sept, J = 6.2 Hz), 7.36
(1H, dd, J = 2.7, 8.9 Hz), 7.46 (1H, d, J = 8.9 Hz), 8.18 (1H, d, J = 2.7 Hz); MS (EI) m/z 249 [M]+.
The hydrochloric acid salt of the ( )-54 was prepared by adding methanolic HCl to a solution of ( )-54 in MeOH. A sample was
recrystallized from MeOH to yield colorless crystals for analysis.
Hydrochloric acid salt of ( )-54; mp 222-225 °C; IR (KBr): 3133, 2984, 1752, 1540, 1240, 935 cm-1;1H NMR (400 MHz, CD3OD)
δ 1.42 (6H, d, J = 5.6 Hz), 1.94 (3H, s), 4.90 (1H, sept, J = 5.6 Hz), 8.12 (1H, d, J = 9.6 Hz), 8.21 (1H, dd, J = 2.8, 9.6 Hz), 8.41
(1H, d, J = 2.8 Hz); 13C NMR (100 MHz, CD3OD) δ 21.7 (2C), 24.4, 64.9, 74.6, 127.0, 132.5, 133.6, 145.3, 157.9, 158.4, 175.2; MS
(EI) m/z 239 [M+]; Anal. Calcd for C12H16ClN3O3: C, 50.44; H, 5.64; N, 14.71. Found: C, 50.39; H, 5.68; N, 14.53.
Resolution of ( )-hydantoin 54 by HPLC
−)
( )-Hydantoin 54 was optically separated by HPLC on a CHIRALPAK AY-H column. Each elution was concentrated to give ( -54
and (+)-54 as a colorless amorphous compound. The chiral HPLC conditions to separate ( )-hydantoin are described below.
Analytical chiral HPLC conditions
Column: CHIRALPAK AY-H, 5 µm, 0.46 × 150 mm; Mobile phase: n-hexane/i-PrOH = 70/30; Flow rate: 1.0 mL/min; Column
−)
temperature: 40 °C; Wavelength: 264 nm; Retention time: ( -form 4.55 min/(+)-form 5.81 min.
Preparative chiral HPLC conditions
Column: CHIRALPAK AY-H, 5 µm, 20 × 250 mm; Mobile phase: n-hexane/i-PrOH = 70/30; Flow rate: 10 mL/min; Column
−)
temperature: 40 °C; Wavelength: 264 nm; Retention time: ( -form 6.05 min/(+)-form 12.1 min.

Hydrochloric acid salt of (+)-54; mp 222-225 °C; MS (EI) m/z 249 [M+]; Anal. Calcd for C12H16ClN3O3: C, 50.44; H, 5.64; N,
14.71. Found: C, 50.34; H, 5.60; N, 14.92; [α]2D0 = +26.5 (c = 1.0, MeOH); Optical purity: >99% ee. The IR, 1H NMR, and 13C NMR
spectra of (+)-54 are identical to those of ( )-54
+
−)
Hydrochloric acid salt of ( -54; mp 222-224 °C; MS (EI) m/z 249 [M ]; Anal. Calcd for C12H16ClN3O3: C, 50.44; H, 5.64; N,
20
1
−26.6 (c = 1.0, MeOH);
14.71. Found: C, 50.36; H, 5.60; N, 14.93; [α]D =
Optical purity: >99% ee. The IR, H NMR and 13C NMR
spectra of (−)-54 are identical to those of ( )-54.
Racemic-3-(2-(2-(Benzyloxy)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-propan-2-yl)-2-propylphenoxy)phenyl)-2-oxoethyl)-5-(5-
(1-methylethoxy)pyridin-2-yl)-5-methylimidazolidine-2,4-dione (55):
To a stirred suspension of 54 (20.4 mg, 81.8 µmol) and K2CO3 (22.7 mg, 164 µmol) in DMF (160 µL) was added 48d (28 mg, 40.9
µmol) at room temperature. The reaction mixture was stirred at room temperature overnight. To the reaction mixture was added
water. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 2/1) to give the title compound (6.5 mg,
21%) as a colorless oil; 1H NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 7.6 Hz), 1.36 (6H, d, J = 6.5 Hz), 1.54 (2H, qt, J = 7.6, 7.8
Hz), 1.87 (3H, s), 2.49 (2H, t, J = 7.8 Hz), 4.58 (1H, sept, J = 6.5 Hz), 4.87 (2H, s), 5.13 (2H, s), 6.29 (1H, brs), 6.48 (1H, d, J = 2.4
Hz), 6.53 (1H, dd, J = 2.4, 8.9 Hz), 6.92 (1H, d, J = 8.9 Hz), 7.20 (1H, dd, J = 2.7, 8.9 Hz), 7.29-7.36 (5H, m), 7.52 (1H, d, J = 8.9
Hz), 7.61 (1H, s), 7.64 (1H, d, J = 8.9 Hz), 7.97 (1H, d, J = 8.9 Hz), 8.21 (1H, d, J = 2.7 Hz); MS (EI) m/z 772 [M]+.
Racemic-3-(2-(4-(4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan- 2-yl)-2-propylphenoxy)-2-hydroxyphenyl)-2-oxoethyl)-5-(5-(1-
methylethoxy)pyridin-2-yl)-5-methylimidazolidine-2,4-dione (( )-56):
To a stirred solution of 55 (3.7 mg, 4.8 µmol) in MeOH (500 µL) was added palladium carbon (1.0 mg). The reaction mixture was
stirred for 3 h under a hydrogen atmosphere. The reaction mixture was filtered through a pad of Celite and rinsed with MeOH. The
filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 2/1) to give
the title compound (2.6 mg, 80%) as a colorless amorphous; IR (film) 3130, 2985, 1740, 1545, 1240, 934 cm-1; 1H NMR (400 MHz,
CD3OD) δ 0.90 (3H, t, J = 7.2 Hz), 1.33 (6H, d, J = 6.0 Hz), 1.61 (2H, qt, J = 7.2, 7.6 Hz), 1.90 (3H, s), 2.59 (2H, t, J = 7.6 Hz),
4.68 (1H, sept, J = 6.0 Hz), 4.98 (2H, s), 6.34 (1H, d, J = 2.4 Hz), 6.55 (1H, dd, J = 2.4, 9.2 Hz), 7.12 (1H, d, J = 8.8 Hz), 7.39 (1H,
dd, J = 2.8, 8.8 Hz), 7.60 (1H, d, J = 8.8 Hz), 7.64 (1H, dd, J = 2.8, 8.8 Hz), 7.71 (1H, d, J = 2.8 Hz), 7.95 (1H, d, J = 9.2 Hz), 8.20
(1H, d, J = 2.8 Hz); 13C NMR (100 MHz, CD3OD) δ 14.0, 22.1 (2C), 23.6, 24.3, 33.2, 45.8, 66.5, 71.9, 78.3 (sept, J = 29.8 Hz),
105.2, 110.0, 115.2, 122.2, 122.5, 124.2, 124.5 (2C, q, J = 285.7 Hz), 127.7, 129.7, 131.2, 133.4, 136.1, 139.6, 150.0, 155.0, 155.5,
158.1, 165.1, 166.1, 177.3, 195.9; MS (EI) m/z 683 [M]+.
−)
(
-3-(2-(4-(4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-2-propylphenoxy)-2-hydroxyphenyl)-2-oxoethyl)-5-(5-(1-
methylethoxy)pyridin-2-yl)-5-methylimidazolidine-2,4-dione ((−)-56):
−)
The preparation of ( -56 was performed using (+)-54 in the same procedure as that for racemic ( )-56.
20
+
−56.5 (c
MS (EI) m/z 683 [M ]; Anal. Calcd for C32H31N3O7F6: C, 56.22; H, 4.57; N, 6.15. Found: C, 56.07; H, 4.61; N, 6.08; [α]D =
= 1.0, CHCl3). The IR, 1H NMR and 13C NMR spectra of ( -56 were identical to those of ( )-56.
−)
(+)-3-(2-(4-(4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-2-propylphenoxy)-2-hydroxyphenyl)-2-oxoethyl)-5-(5-(1-
methylethoxy)pyridin-2-yl)-5-methylimidazolidine-2,4-dione ((+)-56):
The preparation of (+)-56 was performed using (−)-54 in the same procedure as that for racemic ( )-56.
MS (EI) m/z 683 [M+]; Anal. Calcd for C32H31N3O7F6: C, 56.22; H, 4.57; N, 6.15. Found: C, 56.06; H, 4.63; N, 6.09; [α]20 = +56.1 (c
D
= 1.0, CHCl3). The IR, 1H NMR and 13C NMR spectra of (+)-56 were identical to those of ( )-56.
4.2. Pharmacology
4.2.1. In vitro study
The CHO K-1 cells stably transfected with LXRs/GAL4 fused protein expression vectors and the GAL4 –responsive reporter
vector (pG5luc, Promega, WI) were seeded at 20,000 cells/well on a 96-well plate in HAM-F12 medium containing 10% bovine
fetal serum, 100 units/mL of penicillin G, and 100 µg/mL of streptomycin sulfate and incubated in a wet atmosphere with 5% CO2
at 37 °C. After 24 h, media with various concentrations of the test compound (0.01 µM, 0.1 µM, 1 µM and 10 µM) was added,
and the cells incubated for an additional 24 h. We then used Bright-Glo (Promega) as a substrate and measured the luminescence
intensity with a luminometer LB960 (Berthold Technologies) to find the effect of the test compound on the activation of luciferase
transcription via LXRα- or LXRβ-LBD.
4.2.2. In vivo study

Low-density lipoprotein (LDL) receptor knock-out (male, 10 weeks old) mice (n = 60) (Charles River Japan, Inc., Kanagawa,
Japan) were used for animal experiments. The animal room was controlled at 23 3 °C and relative humidity of 50 20%. Animals
were fed a CE-2 chow diet (CLEA Japan Inc., Tokyo, Japan), followed by supplementation with western diet (TD.88137, Harlan
Laboratories, WI, USA) for 10 weeks.
−)
During fat loading, a suspension of ( -56 in 0.5% methyl cellulose (MC) was administered orally at 1 or 3 mg/kg/day (n = 15 for
each dose group). As a comparative agent, a suspension of T0901317 in 0.5% MC was administered orally at 10 mg/kg/day (n=15
for each dose group). The control group (n = 15) received an aqueous solution of 0.5% MC instead of T0901317. Blood samples
were collected to determine plasma lipid levels using a commercially available kit (total cholesterol: Cholesterol E-Test Wako;
Wako Pure Chemical Industries, Osaka, Japan, triglyceride: Triglyceride E-Test Wako; Wako Pure Chemical Industries, Osaka,
Japan). Plasma lipoprotein profiles were analyzed by CLiP method using a Shimadzu HPLC system (LC-20A) and Superose 6
column (10 mm×300 mm, GE Healthcare, UK). Briefly, 15 µL of plasma was diluted 10-fold in PBS containing 1 mM EDTA.
Diluted plasma (20 µL) was separated by the column at 0.5 mL/min with PBS containing 1 mM EDTA and kept at 40 °C for
simultaneous determination of cholesterol contents in the eluents.
For analyses of atherosclerotic lesions, the animals were anesthetized by intraperitoneal injection of pentobarbital sodium (50
mg/kg) followed by vascular perfusion for 5 min with saline containing 4% paraformaldehyde at a perfusion pressure of 120
mmH2O.
The heart was fixed with 4% paraformaldehyde for 24 hrs. Tissues were embedded in OCT compound (Tissue-Tek) and snap
frozen in liquid N2. Cryosections of 10 µm thickness were cut from the end of the aortic sinus. Sections were stained with Oil-Red
O for lipid and counterstained with hematoxylin. A positive area was quantified at 600 µm distally using an image analysis system
(SP500F; Olympus, Tokyo, Japan).
4.3. Pharmacokinetics
The oral doses of 2 at 100 mg/kg and (−)-56 at 10 mg/kg were formulated in PEG 400. A solution of 2 and (−)-56 in PEG 400 were
orally administered to a CE-2 chow diet-fed Golden Syrian hamsters. Blood samples (heparin plasma) were collected from a
−)
forearm vein 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 h after administration. The drug concentrations of 2 and ( -56 in the supernatant was
measured by HPLC-LC-MS/MS.
Supplementary Data
Supplementary data (general preparation procedure and the analytical data of all synthetic compounds) associated with this article
can be found.
Acknowledgments
We thank Dr. S. Tanabe, managing director of the Tokyo New Drug Research Laboratories, Kowa Co., Ltd, for his support.
References and notes
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14. Since preparing the manuscript, some reports on orally efficient LXRβ agonists have been published. Therefore, we add the following references
(References 14 and 15):
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B.; Meng, S.; Hardy, A.; Howard, A.; Krosky, P.; Guo, J.; Lipinski, K.; Kandpal, G.; Bukhtiyarov, Y.; Zhao, Y.; Lala, D.; Orden, R. V.; Zhou, J.;
Chen, G.; Wu, Z.; McKeever, B. M.; McGeehan, G. M.; Gregg, R. E.; Claremon, D. A.; Singh, S. B. J. Med. Chem. 2016, 59, 3264-3271.

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23. The compound 50 was obtained by the reduction of 49a with NaBH4 as described in detail in Supplementary Data.
24. Bergs, H. Ger. Pat. 566,094, 1929.
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28. The hepatic CLint values in Table 3 are the intrinsic clearances of the compounds (1 µmol/L) incubated with liver microsomes (0.5 mg protein/mL).
−)
29. The absolute configuration of ( -54 has not been decided.
30. See: in the Supplementary Data of reference 1-(b). In vivo test, we used a solution of (−)-56 in 0.5%MC when orally administered to mice.
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34. In an in vivo study of LDL receptor knock-out mice, we measured the gene expression of ABCA1 mRNA in blood and SREBP-1c in the liver as
shown in the table 6. As reported, both LXR agonists directly increase an expression of ABCA1 in peripheral blood. T0901317 at a dose of 10 mg/kg
−)
significantly increased SREBP-1c mRNA expression in the liver (2.9-fold relative to the control) leading to TG elevation. In contrast, ( -56 at a
dose of 1 mg/kg did not affect SREBP-1c mRNA expression in the liver, but significantly elevated SREBP-1c mRNA expression at a dose of 3
mg/kg.
Table 6 The upregulation of ABCA1 mRNA and SREBP-1c
mRNA in in vivo study with T0901317 and (−)-56
Compound Dose
ABCA1
in blood
SREBP-1c
in liver
(mg/kg)
(fold)
2.5*
2.1*
3.0*
(fold)
2.9*
1.2
T0901317
−)
10
(
-56
1
3
1.5*
*p < 0.05; Statistical analysis was conducted using Dunnett’s test. The fold value was calculated relative to control.
35. A reviewer recommended that the authors map the correlation between the CLogP and the lipophilic ligand efficiency (LLE) values. This correlation
analysis would help improve the understanding of the lipophilic binding contribution.
*LLE = pEC50-CLogP

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Discovery of a 2-hydroxyacetophenone derivative
as an outstanding linker to enhance potency
and β-selectivity of liver X receptor agonist
Leave this area blank for abstract info.
Minoru Koura, Yuki Yamaguchi, Sayaka Kurobuchi, Hisashi Sumida, Yuichiro Watanabe, Takashi Enomoto,
Takayuki Matsuda, Ayumu Okuda, Tomoaki Koshizawa, Yuki Matsumoto and Kimiyuki Shibuya∗
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Co., Ltd, 2-17-43, Noguchicho,
Higashimurayama, Tokyo 189-0022, Japan
a