Synthesis and in vitro evaluation of novel liver X receptor agonists based on naphthoquinone derivatives

Article abstract of DOI:10.3390/molecules24234316

We aimed to synthesize novel liver X receptor (LXR) agonists with potent agonist activity and subtype selectivity. Our synthetic scheme started with naphthoquinone derivatives, such as menadione and 2,3-dichloro-1,4-naphthoquinone. We introduced different

Full text of DOI:10.3390/molecules24234316

molecules
Article
Synthesis and In Vitro Evaluation of Novel Liver X
Receptor Agonists Based on
Naphthoquinone Derivatives
Tatsuma Nishioka 1,†, Kaori Endo-Umeda 2,†, Yuki Ito 1, Akane Shimoda 1, Atsuko Takeuchi 3,
Chisato Tode 3, Yoshihisa Hirota 4,5 , Naomi Osakabe 5,6, Makoto Makishima 2
and Yoshitomo Suhara 1,5,
*
1
Laboratory of Organic Synthesis and Medicinal Chemistry, Department of Bioscience and Engineering,
College of Systems Engineering and Science, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku,
Saitama 337-8570, Japan; mf15065@shibaura-it.ac.jp (T.N.); mf17010@shibaura-it.ac.jp (Y.I.);
mf17036@shibaura-it.ac.jp (A.S.)
2
3
4
Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine,
30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan; umeda.kaori@nihon-u.ac.jp (K.E.-U.);
makishima.makoto@nihon-u.ac.jp (M.M.)
Instrumental Analysis Center, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi,
Higashinada-ku, Kobe 658-8558, Japan; takeuchi@kobepharma-u.ac.jp (A.T.);
c-tode@kobepharma-u.ac.jp (C.T.)
Laboratory of Biochemistry, Department of Bioscience and Engineering,
College of Systems Engineering and Science, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku,
Saitama 337-8570, Japan; hirotay@sic.shibaura-it.ac.jp
Bio-Intelligence for well-being Association, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan;
nao-osa@sic.shibaura-it.ac.jp
5
6
Food and Nutrition Laboratory, Department of Bioscience and Engineering,
College of Systems Engineering and Science, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku,
Saitama 337-8570, Japan
*
†
Correspondence: suhara@sic.shibaura-it.ac.jp; Tel.: +81-48-720-6043
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 11 November 2019; Accepted: 24 November 2019; Published: 26 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We aimed to synthesize novel liver X receptor (LXR) agonists with potent agonist activity
and subtype selectivity. Our synthetic scheme started with naphthoquinone derivatives, such as
menadione and 2,3-dichloro-1,4-naphthoquinone. We introduced different substituents into the
naphthoquinone structures, including aniline, piperidine, pyrrolidine, and morpholine, in one or two
steps, and thus, we produced 14 target compounds. All 14 synthetic ligands were tested to determine
whether they mediated LXR-mediated transcriptional activity. We investigated the transcriptional
activity of each compound with two types of receptors, LXR
α
and LXR
β. Among all 14 compounds,
two showed weak LXR -agonist activity, and two others exhibited potent LXR
β
α
-agonist activity.
We also performed docking studies to obtain a better understanding of the modes of compound
binding to LXR at the atomic level. In conclusion, we successfully synthesized naphthoquinone
derivatives that act as LXRα/β agonists and selective LXRα agonists.
Keywords: liver X receptor (LXR); naphthoquinone; α-selective; agonist; transcriptional activity
1. Introduction
Liver X receptors (LXRs) are members of the nuclear receptor superfamily [
1]. LXRs include two
subtypes, LXR and LXR , which have different tissue distributions. LXR is highly expressed in liver,
α
β
α
Molecules 2019, 24, 4316; doi:10.3390/molecules24234316
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4316
2 of 11
intestine, adipose tissue, and macrophages; in contrast, LXR
β
is expressed ubiquitously in organs and
tissues. These receptors are ligand-activated transcription factors involved in regulating cholesterol
and lipid metabolism. An LXR agonist might be useful in preventing and treating atherosclerosis,
because an agonist could promote the production of high density lipoprotein (HDL), which activates
the cholesterol reverse transport system by elevating expression of the ATP-binding cassette transporter
A1 and G1 genes [
and decrease amyloid
LXR agonists have both desirable and undesirable pharmacological effects, but ligands that act specially
on LXR or LXR may be applicable as therapeutic agents. A recent study reported that some LXR
selective agonists have been found in natural products or had been chemically synthesized [
LXR regulates human cholesteryl ester transfer protein expression, which plays an important role in
reverse cholesterol transport, both in vitro and in transgenic mice. On the other hand, LXR -selective
2,
3]. Additionally, LXR agonists were shown to increase apolipoprotein E expression
β
protein accumulation thought to cause Alzheimer’s disease [ ]. Thus,
4,5
α
β
α/β
6–11].
α
β
agonists may be useful as therapeutic agents for arteriosclerosis and Alzheimer’s disease [7,10,11].
Many previous studies have described synthetic LXR agonists, including T0901317 and GW3965,
which are known potent LXR agonists (Figure 1). However, most LXR agonists have not shown
subtype selectivity [6]. As aforementioned, an LXR agonist with selectivity for an LXR subtype could
be a useful biologically active compound. Therefore, we aimed to synthesize novel LXR agonists that
possessed potent agonistic activity as well as subtype selectivity.
Figure 1. Chemical structures of T0901317 (left) and GW3965 (right).
We focused on synthetic compounds which have naphthoquinone moiety since they showed
various biological activities. For example, some naphthoquinone derivatives have previously
been described as antitumor agents [12
naphthoquinone moiety were reported [14
derivatives as active motifs for our compounds. We anticipated that synthetic compounds bearing the
naphthoquinone skeleton could apply for LXR agonists and the selectivity for LXR and LXR would
,13]. Recently, several types of LXR agonists that bore a
,15]. We employed the chemical structure of those known
α
β
be controlled by substituents. Our design included the introduction of a nitrogen atom or a sulfur atom
into the molecule, with the expectation that it would improve the interaction between the compounds
and the receptor proteins. We also synthesized compounds with either a fluorine or trifluoromethyl
group which is an electron-withdrawing functional group, like that incorporated into T0901317, in
addition to piperidine, pyrrolidine, morpholine, aniline, and phenylsulfide moieties. At the same time,
we also synthesized compounds with a methyl group, which is an electron-donating functional group,
as comparators. We demonstrated that these newly synthesized LXR agonists displayed potent agonist
activity, compared to known LXR agonists.
2. Results
2.1. Synthesis of LXR Ligands
We synthesized the compounds according to the methods shown in Schemes 1 and 2. Compounds
1
and
we introduced an aniline derivative in the presence of cerium (III) chloride heptahydrate, according
to a previously reported method [12 16 18]. On the other hand, compounds 14 were obtained
2 were obtained in high yield, with menadione (15) as a starting material (Scheme 1). Briefly,
,
–
3–
in a 2-step synthesis method (Scheme 2). In the first step, one molecule of the aniline derivative
was introduced into 2,3-dichloro-1,4-naphthoquinone (18) in the presence of cerium (III) chloride

Molecules 2019, 24, 4316
3 of 11
heptahydrate, in aqueous conditions, to produce the monochloro derivatives, 20
–
22, in a 62–99% yield.
The intermediates, 20 and 21, were described previously [19]. In the second step, we reacted the
intermediates with a nucleophilic reagent, such as excess cycloalkylamine or benzenethiol, which led
to the desired compounds:
11 12 with morpholine; and 13
compounds that were candidate LXR agonists (The data refer to Supplementary Materials).
3–5, with aniline derivatives;
6–8 with piperidine; 9–10 with pyrrolidine;
–
–14 with phenylsulfide, in 41–86% yields. Thus, we prepared 14 different
Scheme 1. One-step synthesis of candidate liver X receptor (LXR) agonists,
menadione (15).
1 and 2, based on
Scheme 2. Two-step synthesis of candidate LXR agonists,
naphthoquinone (18).
3–14, based on 2,3-dichloro-1,4-
2.2. Transcriptional Activity of LXR Ligand
To evaluate the agonist activity of the synthesized compounds, we tested whether they induced
transcriptional activity mediated by LXR and LXR with a reporter gene assay. We used T0901317
(1.0
10−7 M) as a positive control for this assay. Briefly, HEK293 cells were plated at a density
that corresponded to 70–80% confluence (1
104 cells per each well) in a 96-well plate, 24 h
prior to transfection. Then, cells were co-transfected with an expression plasmid that carried one
of two nuclear receptors, under the control of the ctyomegalovirus promoter (pCMX-hLXR or
pCMX-hLXR ), a reporter plasmid (rCYP7A-DR4 3-tk-LUC), and a CMX- -galactosidase vector,
α
β
×
×
α
β
×
β
as an indicator of expression efficiency. Transfections were performed according to the calcium
phosphate co-precipitation method. After 24 h, transfected cells were treated with test compounds
(
3.0 × 10−6
M) or dimethyl sulfoxide (DMSO) for 16 h. Treated cells were assayed for luciferase reporter
activity in a luminometer. The luciferase activity measured in each sample was normalized with
respect to the level of β-galactosidase activity (Figure 2) [6].

Molecules 2019, 24, 4316
4 of 11
Figure 2. Human LXR
with compounds 14 induced reporter activity through activation of (
LXR . T: T0901317: positive control (assigned 100%). HEK293 cells were treated with 3.0
each compound or 1.0
10−7 M T0901317. Negative control cells (-) were treated with DMSO alone.
α
/
β
reporter gene assays in human embryonic kidney (HEK)293 cells. Treatment
) human LXR and ( ) human
10−6 M of
1
–
A
α
B
β
×
×
Data are the means of three independent experiments; error bars indicate the SD. Significant differences
from the control group (-) are indicated as follows: *** p > 0.001, * p > 0.05 (Dunnett’s t-test).
Our compounds did not show the potency of T0901317; however, the substituents on the
compounds affected the transcriptional activities of LXR
compared to control, were observed with compounds
which carried the trifluoromethyl group, exhibited the most potent induction of LXR
all our compounds. However, compounds 10, and 13 showed no significant difference
with induction of LXR activity (Figure 2A). On the other hand, weak inductions of LXR activity were
observed only with compounds and . Other compounds had no significant difference compared
to control group (Figure 2B). Based on these findings, compounds and had agonist effects on
both LXR and LXR . Furthermore, from the comparison between LXR and LXR , the activities
of compounds 11 12, and 14 suggested that these agonists were candidates with selective LXR
α
and LXR
11 12, and 14. In particular,
activity, among
β
. Significant LXR
α
activities,
6,
7,
8,
,
8
and 14
,
α
1, 2, 3, 4, 5, 9,
α
β
6
8
6
8
α
β
α
β
7
,
,
α
binding. In particular, 14 showed the highest LXRαselective activity in this assay.
3. Discussion
To better understand the binding modes of compounds to LXRs at the atomic level (based on the
results shown in Figure 2), we performed molecular docking studies on compounds with LXR (PDB
ID: 1UHL [20]) using the docking program of the MOE suite (see Computational Details) as shown
in Figure 3. We calculated the binding affinity by replacing T-0901317, originally bound to LXR
with compounds using the “dock” mode. We selected compounds and 14, because they had the
highest agonistic activity for LXR , and and 13 randomly picked up from 10, and 13
because they showed no agonist activity for LXR . When compounds and 14 were in their most
stable binding conformations (it means the lowest binding energy was exhibited), their aromatic rings
faced to the Phe315 (sky blue) of LXR , which facilitated the formation of a stacking interaction
(Figure 3B,C). On the other hand, when compounds and 13 were in their most stable binding
α
α
,
8
α
3
1,
2
,
3,
4
,
5,
9,
,
α
8
α
π-π
3
conformations, they faced the opposite direction, and the aromatic rings did not interact with Phe315
(Figure 3D,E). Previous studies on LXR also suggested that residues His435 and Trp457 (PDB ID: 5HJP)
functioned as an activation switch, and the interaction between the ligand and His435 was particularly
important [
Trp443 (corresponding to His435 and Trp457 in 5HJP, respectively) in the docking simulation with
1UHL. In the most stable structure of and 14 bound to LXR , we obtained a conformation similar
7]. Therefore, we performed docking simulations to analyze interactions with His421 and
8
α
to T0901317, with the (CF3)2OH moiety facing His421 (purple) and Trp443 (yellow) (Figure 3A–C).
These similarities were suggested to show agonistic activity. However, the docking simulations could
not clarify the differential effects of these compounds on LXRα and LXRβ. This observation was
consistent with results reported in a previous study. Those authors speculated that ligand selectivity for
LXRα vs. LXRβ arises from different post-binding conformational changes or differential coactivator
recruitment, rather than from different binding preferences [6].

Molecules 2019, 24, 4316
5 of 11
Figure 3. Simulations of test compounds docking to LXR
white) are shown interacting with amino acid residues of LXR
in 1UHL; ( ) compound and ( ) compound 14 exhibited the most potent activity among all 14 test
compounds. Compounds and 14 exhibited stacking interactions with Phe315 (sky blue), and those
α
(PDB ID: 1UHL). The test compounds (center,
α
(red). ( ) T0901317 originally contained
A
B
8
C
8
π–π
(CF3)2OH moiety faced to His421 (purple) and Trp443 (yellow), similar to T0901317; (
D) Compound 3
and (E) compound 13 showed no agonist activity.
4. Materials and Methods
4.1. Synthetic Method of Compounds
4.1.1. General Experimental Procedures
1H and 13C-NMR spectra were recorded at 400 and 100 MHz in CDCl3 or CD3OD,
with tetramethylsilane used as an internal standard. High-resolution mass spectra were measured in
the time of flight (TOF) mass mode.
4.1.2. Preparation of 2-[(4-Fluorophenyl)amino]-3-methylnaphthalene-1,4-dione (1)
Menadione (15) (172 mg, 1.00 mol), 4-fluoroaniline (16) (194
µ
L, 2.00 mmol), and CeCl4
•
7H2O
(19 mg, 50.0
µ
mol) were dissolved in 15 mL of ethanol, and then heated to reflux at 90 ◦C overnight.

Molecules 2019, 24, 4316
6 of 11
The reaction mixture was poured into ice-water and extracted with CH2Cl2 (100 mL
organic layer was washed with water (100 mL) and brine (100 mL) and dried over MgSO4 to concentrate
×
3). The combined
the product. The residue was purified with silica gel column chromatography (n-hexane/AcOEt = 6:1)
1
to afford compound
1
(163 mg, 58%) as an orange powder: H-NMR (500 MHz, CDCl3)
δ
1.72 (3H, s),
13.6,
6.97–7.05 (4H, m), 7.33 (1H, s), 7.64–7.72 (2H, m), 8.06–8.12 (2H, m); 13C-NMR (125 MHz, CDCl3)
115.5, 115.7, 118.2, 124.2, 124.3, 126.2, 126.3, 132.4, 134.4, 158.3, 160.7, 182.4, 184.4; HRMS ([M + H]+) m/z
δ
was calculated for C17H12FNO2 282.0930; Found: 282.0925.
4.1.3. Preparation of 2-Methyl-3-[(4-methylphenyl)amino]naphthalene-1,4-dione (2)
Similar to the synthesis of
obtained from 15 (344 mg, 2.00 mmol), p-toluidine (17) (430 mg, 4.00 mmol), and CeCl4
100 mol) in ethanol (30 mL), was purified with silica gel column chromatography (n-hexane/AcOEt =
6:1), which gave compound
(3H, s), 2.37 (3H, s), 6.88–6.90 (2H, d), 7.11–7.13 (2H, d), 7.37 (1H, s), 7.60–7.72 (2H, m), 8.04–8.11 (2H,
1
from 15 and 4-fluoroaniline (16), the crude product 2, which was
•
7H2O (38 mg,
µ
1
2
(229 mg, 82%) as a purple powder: H-NMR (400 MHz, CDCl3) δ 1.73
m); 13C-NMR (100 MHz, CDCl3)
δ 13.7, 16.5, 20.8, 117.8, 122.6, 126.0, 126.1, 126.2, 126.5, 129.3, 132.2,
133.5, 133.6, 134.0, 134.3, 135.6, 137.1, 182.6, 184.4, 184.9, 185.5; HRMS ([M + H]+) m/z calculated for
C18H16NO2 278.1181; Found: 278.1177.
4.1.4. Preparation of 2-chloro-3-[(4-fluorophenyl)amino]naphthalene-1,4-dione (20)
2,3-Dichloro-1,4-naphthoquinone (18) (909 mg, 4.00 mmol), 16 (767
(75 mg, 200 mol) were dissolved in 15 mL of ethanol, and stirred overnight. The reaction mixture
was poured into ice-water and extracted with CH2Cl2 (100 mL 3). The combined organic layer was
µL, 8.00 mmol), and CeCl4
µ
×
washed with water (100 mL) and brine (100 mL), and dried over MgSO4 to concentrate the product.
The residue was purified with silica gel column chromatography (n-hexane/AcOEt = 3:1) to afford 20
(1.23 g, 88%) as a dark red powder.
4.1.5. Preparation of 2-Chloro-3-[(4-methylphenyl)amino]naphthalene-1,4-dione (21)
Similar to the synthesis of 20 from 16 and 18, the crude product 21, which was obtained from 18
(681 mg, 3.00 mmol), 17 (645 mg, 6.00 mmol), and CeCl4 7H2O (57 mg, 150 µmol) in ethanol (15 mL),
•
was purified with silica gel column chromatography (n-hexane/AcOEt = 3:1), which gave compound
21 (890 mg, 99%) as a purple powder.
4.1.6. Preparation of 2-Chloro-3-{[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]amino}
naphthalene-1,4-dione (22)
Similar to the synthesis of 20 from 16 and 18, the crude product 22, which was obtained from 18
(341 mg, 1.50 mmol), 2-(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (19) (441 mg, 1.70 mmol),
and CeCl4
•
7H2O (28 mg, 75
µ
mol) in ethanol (15 mL), was purified with silica gel chromatography
1
(n-hexane/AcOEt = 3:1), which gave compound 22 (416 mg, 62%) as a red powder: H-NMR (400 MHz,
CD3OD)
δ 7.19 (dt, 2H, J = 8.8, 2.6 Hz), 7.68 (d, 2H, J = 8.4 Hz), 7.79 (m, 2H), 8.11 (dd, 2H, J = 8.8,
1.2 Hz); 13C-NMR (CD3OD, 100 MHz):
δ 115.0, 121.8, 123.3, 124.6, 126.3, 126.5, 126.8, 127.4, 130.4, 132.3,
133.0, 134.5, 140.2, 142.9, 177.8, 180.0; HRMS ([M + H]+) m/z calculated for C19H11ClF6NO3 450.0332;
Found: 450.0332.
4.1.7. Preparation of 2-[(4-Fluorophenyl)amino]-3-(piperidin-1-yl)naphthalene-1,4-dione (3)
To a solution of compound 20 (302 mg, 1.00 mmol) in toluene (25 mL), we added 16 (144
1.50 mmol), sodium butoxide (144 mg, 1.50 mmol), palladium chloride (188 mg, 230 mol),
and 1,10-bis(diphenylphosphino)ferrocene (128 mg, 230
mol); the mixture was stirred at 100 C
for 6 h under Ar. The reaction mixture was poured into ice-water and extracted with CH2Cl2 (100 mL
3). The combined organic layer was washed with water (100 mL) and brine (100 mL), and dried over
MgSO4 to concentrate the product. The residue was purified with silica gel column chromatography
µL,
µ
◦
µ
×

Molecules 2019, 24, 4316
7 of 11
1
(n-hexane/AcOEt = 4:1) to afford
δ
3 (204 mg, 54%), as a navy-blue powder: H-NMR (400 MHz, CDCl3)
6.31 (4H, m), 6.64-6.66 (4H, m), 7.19 (2H, d), 7.69 (2H, m), 8.07–8.11 (2H, m); 13C-NMR (100 MHz,
CDCl3)
δ 114.1, 114.3, 121.8, 121.9, 123.5, 126.4, 131.4, 133.1, 133.1,133.7, 157.3, 159.7, 181.3; HRMS ([M +
H]+) m/z calculated for C22H15N2O2F2 377.1102; Found: 377.1102
4.1.8. Preparation of 2-[(4-Fluorophenyl)amino]-3-(p-tolylamino)naphthalene-1,4-dione (4)
Similar to the synthesis of
(298 mg, 1.00 mmol), 16 (144 L, 1.50 mmol), sodium butoxide (144 mg, 1.50 mmol), palladium chloride
(188 mg, 230 mol) in toluene (25 mL),
mol), and 1,10-bis(diphenylphosphino)ferrocene (128 mg, 230
3 from 20 and 16, the crude product 4, which was obtained from 21
µ
µ
µ
was purified with silica gel chromatography (n-hexane/AcOEt = 4:1), which gave compound
4
(229 mg,
1
61%) as a navy-blue solid: H-NMR (400 MHz, CDCl3)
δ 2.18 (3H, s), 6.24–6.28 (4H, m), 6.58–6.75 (4H,
m), 7.25 (2H, d), 7.64–7.66 (2H, m), 8.06–8.08 (2H, m); 13C-NMR (100 MHz, CDCl3)
δ 20.8, 113.9, 114.1,
120.6, 121.6, 121.7, 126.3, 126.3, 128.0, 133.5, 133.6, 157.2, 159.6, 181.3, 181.5; HRMS ([M + Na]+) m/z
calculated for C23H17N2O2FNa 395.1172; Found: 395.1172.
4.1.9. Preparation of 2,3-Bis[(4-methylphenyl)amino]naphthalene-1,4-dione (5)
Similar to the synthesis of
21 (298 mg, 1.00 mmol), 17 (161 mg, 1.50 mmol), sodium butoxide (144 mg, 1.50 mmol), palladium
chloride (188 mg, 230 mol) in toluene
mol), and 1,10-bis(diphenylphosphino)ferrocene (128 mg, 230
3 from 20 and 16, the crude product 5, which was obtained from
µ
µ
(25 mL), was purified with silica gel chromatography (n-hexane/AcOEt = 4:1), which gave compound
1
5
(175 mg, 47%) as a navy-blue powder: H-NMR (400 MHz, CDCl3)
δ
2.17 (6H, s), 6.23–6.25 (4H, m),
6.69–6.71 (4H, m), 7.14–7.25 (2H, d), 7.62–7.65 (2H, m), 8.05–8.08 (2H, m); 13C-NMR (100 MHz, CDCl3):
20.7, 120.3, 123.6, 126.1, 127.8, 131.3, 131.5, 133.3, 134.8, 181.3; HRMS ([M + Na]+) m/z calculated for
δ
C24H20N2O2Na 391.1422; Found: 391.1422.
4.1.10. Preparation of 2[(4-Fluorophenyl)amino]-3-(piperidin-1-yl)naphthalene-1,4-dione (6)
Compound 20 (302 mg, 1.00 mol) was dissolved in piperidine (5 mL), and heated to reflux at
125 ◦C for 1 day. After cooling to room temperature, the mixture was poured into water and extracted
with ethyl acetate (100 mL
brine (100 mL) and dried over MgSO4 to concentrate the product. The residue was purified with silica
gel column chromatography (n-hexane/AcOEt = 5:1) to afford (154 mg, 44%) as a navy-blue powder.
1H-NMR (400 MHz, CDCl3)
1.21–1.59 (6H, m), 3.06–3.07 (4H, m), 6.78–6.83 (2H, m), 6.91–6.96 (2H,
m), 7.10 (1H, br s), 7.55–7.65 (2H, m), 7.96–8.00 (2H, m); 13C-NMR (100 MHz, CDCl3):
24.2, 26.1, 49.9,
×
3). The combined organic layer was washed with water (100 mL) and
6
δ
δ
114.8, 115.0, 121.7, 121.8, 125.6, 126.4, 130.7, 132.7, 133.67, 133.71, 157.4, 159.8, 182.0, 182.3; HRMS (M +
H+) m/z calculated for C21H20O2N2F 351.1509. Found: 351.1508.
4.1.11. Preparation of 2-(Piperidin-1-yl)-3-[(4-methylphenyl)amino]naphthalene-1,4-dione (7)
Similar to the synthesis of
6 from 20 and piperidine, the crude product 7, which was obtained
from 21 (298 mg, 1.00 mmol) and piperidine (5 mL), was purified with silica gel chromatography
1
(n-hexane/AcOEt = 5:1), which gave compound
(400 MHz, CDCl3)
7
(271 mg, 76%) as a navy-blue powder: H-NMR
δ
1.26–1.38 (6H, m), 2.32 (3H, s), 3.08–3.11 (4H, m), 6.76–6.78 (2H, m), 7.04–7.06 (2H,
m), 7.11(1H, br s), 7.58–7.63 (2H, m), 7.97–8.01 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 21.0, 24.3, 26.1,
49.8, 120.3, 125.6, 126.3, 128.8, 130.8, 131.9, 132.6, 132.9, 133.6, 133.7, 137.3, 182.0, 182.4. HRMS (M + H+)
m/z calculated for C22H23O2N2 347.1760. Found: 351.1750.
4.1.12. Preparation of 2-{[4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)phenyl]amino}-3-(piperidin-
1-yl)naphthalene- 1,4-dione (8)
Similar to the synthesis of
6 from 20 and piperidine, the crude product 8, which was obtained
from 22 (225 mg, 0.50 mmol) and piperidine (5 mL), was purified with silica gel chromatography
1
(n-hexane/AcOEt = 5:1), which gave compound
8
(157 mg, 63%) as a yellow solid: H-NMR (400 MHz,

Molecules 2019, 24, 4316
8 of 11
CDCl3)
δ 1.26–1.35 (7H, m), 3.13–3.16 (4H, m), 6.88–6.90 (2H, m), 7.16 (1H, br s), 7.54–7.72 (4H, m),
8.01–8.03 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 24.1, 25.9, 49.9, 119.6, 122.4, 125.7, 126.6, 126.7, 128.8,
130.7, 132.7, 133.0, 133.6, 135.4, 141.4, 182.2, 182.3; HRMS (M + H+) m/z calculated for C24H21O2N2F6
499.1456. Found: 499.1456.
4.1.13. Preparation of 2-[(4-Fluorophenyl)amino]-3-(pyrrolidin-1-yl)naphthalene-1,4-dione (9)
Compound 20 (302 mg, 1.00 mol) was dissolved in pyrrolidine (5 mL), and heated to reflux at
107 ◦C for 6 h. After cooling to room temperature, the mixture was poured into water and extracted
with ethyl acetate (100 mL
brine (100 mL) and dried over MgSO4 to concentrate the product. The residue was purified with silica
gel column chromatography (n-hexane/AcOEt = 5:1) to afford (248 mg, 73%) as a navy-blue powder.
1H-NMR (400 MHz, CDCl3)
1.62-1.63 (4H, m), 3.44-3.48 (4H, m), 6.58 (2H, m), 6.88–6.90 (3H, m),
×
3). The combined organic layer was washed with water (100 mL) and
9
δ
7.60 (2H, m), 7.96–8.01 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 25.4, 51.1, 115.1, 115.3, 117.2, 117.3,
123.4, 125.5, 126.1, 131.5, 132.7, 133.3, 156.1, 158.4, 180.5, 183.3; HRMS ([M + H]+) m/z calculated for
C20H18N2O2F 377.1352; Found: 337.1351.
4.1.14. Preparation of 2-(Pyrrolidin-1-yl)-3-[(4-methylphenyl)amino]naphthalene-1,4-dione (10)
Similar to the synthesis of 9 from 20 and pyrrolidine, the crude product 10, which was obtained
from 21 (298 mg, 1.00 mmol) and pyrrolidine (5 mL), was purified with silica gel chromatography
1
(n-hexane/AcOEt = 5:1), which gave compound 10 (255 mg, 76%) as a navy-blue powder: H-NMR
(400 MHz, CDCl3)
δ
1.61–1.63 (4H, m), 2.17 (3H, s), 3.46–3.49 (4H, m), 6.52–6.55 (2H, m), 6.81 (1H, s),
20.6, 25.4, 50.9,
6.98–7.00 (2H, m), 7.59–7.60 (2H, m), 7.97–8.00 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ
116.1, 123.5, 125.3, 125.9, 129.1, 129.4, 131.5, 131.9, 132.5, 133.1, 136.7, 139.9, 180.4, 183.3; HRMS ([M +
H]+) m/z calculated for C21H21N2O2 333.1603; Found: 333.1597.
4.1.15. Preparation of 2-[(4-Fluorophenyl)amino]-3-morpholinonaphthalene-1,4-dione (11)
Compound 20 (302 mg, 1.00 mol) was dissolved in morpholine (5 mL), and heated to reflux at
140 ◦C for 2 days. After cooling to room temperature, the mixture was poured into water and extracted
with ethyl acetate (100 mL
brine (100 mL) and dried over MgSO4 to concentrate the product. The residue was purified with silica
×
3). The combined organic layer was washed with water (100 mL) and
gel column chromatography (n-hexane/AcOEt = 5:1) to afford 11 (196 mg, 55%) as a black powder.
1H-NMR (400 MHz, CDCl3)
δ
1H-NMR (400 MHz, CDCl3):
δ
3.15–3.18 (4H, m), 3.32–3.44 (4H, m),
6.87–6.90 (2H, m), 6.98–7.02 (2H, m), 7.27 (1H, br s), 7.62–7.69 (2H, m), 8.01–8.03 (2H, m); 13C-NMR
(100 MHz, CDCl3):
δ 48.9, 66.8, 115.0, 115.2, 122.7, 122.8, 125.8, 126.5, 130.4, 131.3, 132.8, 134.1, 157.8,
160.2, 182.0, 182.5; HRMS (M + H+) m/z calculated for C20H18O3N2F 353.1301. Found: 353.1299.
4.1.16. Preparation of 2-Morpholino-3-[(4-methylphenyl)amino]naphthalene-1,4-dione (12)
Similar to the synthesis of 11 from 20 and morpholine, the crude product 12, which was obtained
from 21 (298 mg, 1.00 mmol) and morpholine (5 mL), was purified with silica gel chromatography
1
(n-hexane/AcOEt = 5:1), which gave compound 12 (145 mg, 41%) as a black powder: H-NMR (400
MHz, CDCl3):
δ 2.34 (3H, s), 3.16–3.18 (4H, m), 3.31–3.33 (4H, m), 6.81–6.83 (2H, m), 7.09–7.11 (2H, m),
7.21 (1H, s), 7.601–7.68 (2H, m), 8.00–8.03 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 21.0, 48.9, 66.8, 121.5,
125.8, 126.4, 128.9, 130.5, 131.1, 132.3, 132.6, 132.9, 133.0, 134.0, 136.3, 181.9, 182.7; HRMS (M + H+) m/z
calculated for C21H21O3N2 349.1552. Found: 349.1548.
4.1.17. Preparation of 2-[(4-Fluorophenyl)amino]-3-(phenylthio)naphthalene-1,4-dione (13)
To a stirred solution of thiophenol (45 µL, 0.32 mmol), triethylamine (45 µL, 0.32 mmol), potassium
hydroxide (107 mg, 1.9 mmol), and a 0.5% SDS solution (5 mL), we added compound 20 (97 mg,
0.32 mmol) and heated to 60 ◦C for 14 h. After cooling to room temperature, the mixture was neutralized

Molecules 2019, 24, 4316
9 of 11
with 5% hydrochloric acid solution. Water was added to the mixture, and the solution was extracted
with dichloromethane (100 mL 3). The combined organic layer was dried over MgSO4 to concentrate
×
the product. The residue was purified with silica gel column chromatography (n-hexane/AcOEt = 15:1)
1
to afford 13 (75 mg, 62%) as a red-purple powder: H-NMR (400 MHz, CDCl3):
δ 6.62–6.65 (2H, m),
6.71–6.75 (2H, m), 6.81–6.85 (2H, m), 7.00–7.10 (3H, m), 7.67–7.78 (2H, m), 7.91 (1H, br s), 8.10–8.19 (2H,
m); 13C-NMR (100 MHz, CDCl3):
δ 114.4, 114.6, 124.8, 125.9, 126.9, 127.2, 127.9, 128.5, 133.0, 135.0, 142.6,
158.9, 161.3, 180.8, 181.3; HRMS (M + H+) m/z calculated for C22H15O2NFS 376.0808. Found: 376.0808.
4.1.18. Preparation of 2-{[4-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)phenyl]amino}-3
-(phenylthio)naphthalene-1,4-dione (14)
Thiophenol (33 µL, 0.32 mmol), trimethylamine (45 µL, 0.32 mmol), and compound 22 (144 mg,
0.32 mmol) were dissolved in methanol (50 mL) and heated at 70 ◦C for 24 h. After cooling to room
temperature, water was added to the mixture and the solution was extracted with dichloromethane
(100 mL
×
3). The combined organic layer was dried over MgSO4 to concentrate the product. The residue
was purified with silica gel column chromatography (n-hexane/AcOEt = 10:1) to afford 14 (144 mg,
86%) as a red-brown powder: H-NMR (400 MHz, CDCl3):
1
δ 3.59 (1H, br s), 6.53–6.55 (2H, m), 6.59–6.61
(2H, m), 6.90–6.94 (2H, m), 6.98–7.03 (1H, m), 7.45–7.47 (2H, d, J = 8.0 Hz), 7.71–7.81 (2H, m), 7.95 (1H,
br s), 8.14–8.22 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 115.1, 121.8, 124.5, 125.8, 126.3, 127.0, 127.2,
128.3, 128.7, 130.4, 131.2, 133.3, 135.0, 136.9, 139.6, 180.6, 181.8; HRMS (M + H+) m/z calculated for
C25H16O3NF6S 524.0755. Found: 524.0756.
4.2. Cell Culture Conditions
Human embryonic kidney (HEK) 293 cells were cultured in DMEM containing 5% FBS and a
penicillin/streptomycin mixture, at 37 ◦C, in a humidified atmosphere of 5% CO2 in air.
4.3. Transient Transfection Assays
HEK293 cells were plated at a density corresponding to 70–80% confluence (1
each well) in a 96-well plate, 24 h prior to transfection. Cells were co-transfected with 15 ng of an
expression plasmid that carried one of two nuclear receptors (pCMX-hLXR ), 50 ng of reporter
plasmid (rCYP7A-DR4 3-tk-LUC), and 10 ng of pCMX- -galactosidase expression vector. Transfection
×
104 cells per
α/β
×
β
was performed according to the calcium phosphate co-precipitation method. After 24 h, transfected
cells were treated with test compounds or DMSO for 16 h. Agonist activity was measured in response
to each test compound (3.0
luciferase activity in a luminometer. The luciferase activity of each sample was normalized to the level
of -galactosidase activity. Each transfection was carried out twice in triplicate. Error bars represent
×
10−6 M) or T0901317 (1.0
×
10−7 M). Treated cells were assayed for
β
SD. Statistics were performed with the Dunnett’s t-test.
4.4. Computational Details
Calculations were performed on a Dell Precision T3500 workstation. Conformational analysis of
T0901317 and compounds 3, 8, 13, 14 were conducted using Molecule Operating Environment (MOE,
2019.01; Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada)
and Amber10: EHT molecular mechanics force field [21]. Docking of the minimized energy structure
of the compounds into the crystal structure of LXRα in complex with T0901317 and compounds were
carried out with the docking program of the MOE suite.
4.5. Molecular Docking Experiment
The drug-bound LXRα structure (PDB code: 1UHL, Resolution: 2.9 A) was obtained from Protein
Data Bank (PDB) [20]. The cocrystalized structure was prepared using MOE 2019.01 for correcting
structural issues (such as break bond, miss loop, etc.), adding hydrogen, and calculating partial charge.
The 2D structure of the compounds were downloaded from the CS ChemDraw with mol file format

Molecules 2019, 24, 4316
10 of 11
and converted to 3D in MOE through energy minimization. MOE-Docking was used for docking
simulation of the compounds and predicting the binding affinity with the LXR protein structure.
α
The original drug-binding pocket was chosen as the active site for docking. Site Finder in MOE was
also used to identify the potential binding pockets and analyze the conserved pocket residues. Classical
triangle matching was chosen as placement method, and the number of placement poses was set
to 100. The output docking poses were evaluated by the London dG score and top 30 poses were
chosen. Then, the rigid receptor method was employed in the refinement step. The number of the
final output docking poses was set to 20, followed by minimizing using Amber10: EHT force field in
MOE. The GBVI/WSA dG score was used to estimate free energy of binding of the compounds with
the LXRα. The binding mode was analyzed in MOE after the refinement minimization [22,23].
5. Conclusions
In summary, we prepared a series of compounds based on naphthoquinone derivatives that
displayed selective binding to LXRα. In future studies, we will examine the skeletons of these
compounds or the structural modifications that afford the best selectivity for LXRα vs. LXRβ.
Supplementary Materials: The following are available online. Figure S1: 1H and 13C NMR chart of 1–14.
Author Contributions: T.N. synthesized compounds and analyzed the data. K.E.-U. conceived and designed
the research and edited the manuscript. Y.I., A.S., A.T., C.T., Y.H., and N.O. analyzed the data. T.N. and K.E.-U.
contributed materials and analysis tools. M.M. contributed supervision. Y.S. designed the experiments and wrote
the original draft of the manuscript.
Funding: This work was supported by SIT Project for Research Grant (Linked with KAKENHI) in FY2013.
Acknowledgments: We thank Yoshirou Kimura, MOLSIS Inc. for technical advice about MOE.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Schultz, J.R.; Tu, H.; Luk, A.; Repa, J.J.; Medina, J.C.; Li, L.; Schwendner, S.; Wang, S.; Thoolen, M.;
Mangelsdorf, D.J.; et al. Role of LXRs in control of lipogenesis. Genes Dev. 2000, 14, 2831–2838. [CrossRef]
Venkateswaran, A.; Laffitte, B.A.; Joseph, S.B.; Mak, P.A.; Wilpitz, D.C.; Edwards, P.A.; Tontonoz, P. Control
of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc. Natl. Acad. Sci. USA 2000, 97,
12097–12102. [CrossRef]
2.
3.
4.
5.
6.
7.
Kennedy, M.A.; Venkateswaran, A.; Tarr, P.T.; Xenarios, I.; Kudoh, J.; Shimizu, N.; Edwards, P.A.
Characterization of the human ABCG1 gene: Liver X receptor activates an internal promoter that produces a
novel transcript encoding an alternative form of the protein. J. Biol. Chem. 2001, 276, 39438–39447. [CrossRef]
Cramer, P.E.; Cirrito, J.R.; Wesson, D.W.; Lee, C.Y.; Karlo, J.C.; Zinn, A.E.; Casali, B.T.; Restivo, J.L.;
Goebel, W.D.; James, M.J.; et al. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in
AD mouse models. Science 2012, 335, 1503–1506. [CrossRef] [PubMed]
Namjoshi, D.R.; Martin, G.; Donkin, J.; Wilkinson, A.; Stukas, S.; Fan, J.; Carr, M.; Tabarestani, S.; Wuerth, K.;
Hancock, R.E.; et al. The liver X receptor agonist GW3965 improves recovery from mild repetitive traumatic
brain injury in mice partly through apolipoprotein E. PLoS ONE 2013, 8, e53529. [CrossRef] [PubMed]
Nomura, S.; Endo-Umeda, K.; Makishima, M.; Hashimoto, Y.; Ishikawa, M. Development of
tetrachlorophthalimides as liver X receptor
[CrossRef] [PubMed]
β (LXRβ)-selective agonists. ChemMedChem 2016, 11, 2347–2360.
Stachel, S.J.; Zerbinatti, C.; Rudd, M.T.; Cosden, M.; Suon, S.; Nanda, K.K.; Wessner, K.; DiMuzio, J.;
Maxwell, J.; Wu, Z.; et al. Identification and in vivo evaluation of liver X receptor -selective agonists for the
β
potential treatment of Alzheimer’s disease. J. Med. Chem. 2016, 59, 3489–3498. [CrossRef] [PubMed]
Song, C.; Hiipalla, R.A.; Liao, S. Selective activation of liver X receptor alpha by 6alpha-hydroxy bile acids
and analogs. Steroids 2000, 65, 423–427. [CrossRef]
Honzumi, S.; Shima, A.; Hiroshima, A.; Koieyama, T.; Ubukata, N.; Terasaka, N. LXRalpha regulates human
CETP expression in vitro and in transgenic mice. Atherosclerosis 2010, 212, 139–145. [CrossRef] [PubMed]
8.
9.

Molecules 2019, 24, 4316
11 of 11
10. Kick, E.K.; Busch, B.B.; Martin, R.; Stevens, W.C.; Bollu, V.; Xie, Y.; Boren, B.C.; Nyman, M.C.; Nanao, M.H.;
Nguyen, L.; et al. Discovery of Highly Potent Liver X Receptor
1207–1212. [CrossRef] [PubMed]
β Agonists. ACS Med. Chem. Lett. 2016, 7,
11. Swahn, B.M.; Macsari, I.; Viklund, J.; Öhberg, L.; Sjödin, J.; Neelissen, J.; Lindquist, J. Liver X receptor
agonists with selectivity for LXRbeta; N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides. Bioorg. Med.
Chem. Lett. 2009, 19, 2009–2012. [CrossRef] [PubMed]
12. Benites, J.; Valderrama, J.A.; Bettega, K.; Pedrosa, R.C.; Calderon, P.B.; Verrax, J. Biological evaluation
of donor-acceptor aminonaphthoquinones as antitumor agents. Eur. J. Med. Chem. 2010, 45, 6052–6057.
[CrossRef] [PubMed]
13. Kongkathip, B.; Akkarasamiyo, S.; Hasitapan, K.; Sittikul, P.; Boonyalai, N.; Kongkathip, N. Synthesis of
novel naphthoquinone aliphatic amides and esters and their anticancer evaluation. Eur. J. Med. Chem. 2013
60, 271–284. [CrossRef] [PubMed]
,
14. Granot, Y.; Bittner, S. Pyrrolidino-1,4-naphthoquinone Deriviatives and Their Use for Treating Malignancies
and Cardiovascular Diseases. U.S. Patent 8513437B2, 20 August 2013.
15. Matsuda, T.; Miura, T.; Watanabe, Y. LXR Agonist. JP Patent 2007284367A, 1 November 2007.
16. Tandon, V.K.; Maurya, H.K. ‘On water’: Unprecedented nucleophilic substitution and addition reactions
with 1, 4-quinones in aqueous suspension. Tetrahedron Lett. 2009, 50, 5896–5902. [CrossRef]
17. Tandon, V.K.; Maurya, H.K.; Verma, M.K.; Kumar, R.; Shukla, P.K. ‘On water’ assisted synthesis and
biological evaluation of nitrogen and sulfur containing hetero-1, 4-naphthoquinones as potent antifungal
and antibacterial agents. Eur. J. Med. Chem. 2010, 45, 2418–2426. [CrossRef] [PubMed]
18. Wang, X.L.; Zheng, X.F.; Wang, L.; Reiner, J.; Xie, W.L.; Chang, J.B. [1,1’-bis(diphe-nylphosphino)ferrocene]
dichloropalladium/ 1,1’-bis(di-phenylphosphino) ferrocene catalyzed synthesis of 2,3-diamino-
1,4-naphthoquinones. Synthesis 2007, 7, 989–998.
19. Satheshkumar, A.; Ganesh, K.; Elango, K.P. Charge transfer facilitated direct electrophilic substitution in
phenylaminonaphthoquinones: Experimental, theoretical and electrochemical studies. N. J. Chem. 2014, 38,
993–1003. [CrossRef]
20. Svensson, S.; Ostberg, T.; Jacobsson, M.; Norström, C.; Stefansson, K.; Hallén, D.; Johansson, I.C.;
Zachrisson, K.; Ogg, D.; Jendeberg, L. Crystal structure of the heterodimeric complex of LXRalpha and
RXRbeta ligand-binding domains in a fully agonistic conformation. EMBO J. 2003, 22, 4625–4633. [CrossRef]
[PubMed]
21. Molecular Operating Environment; Chemical Computing Group Inc.: Montreal, QC, Canada, 2019.
22. Corbeil, C.R.; Willams, C.I.; Labute, P. Variability in docking success rates due to dataset preparation.
J. Comput. Aided Mol. Des. 2012, 26, 775–786. [CrossRef] [PubMed]
23. Zhang, W.; Liu, M.; Yang, L.; Huang, F.; Lan, Y.; Li, H.; Wu, H.; Zhang, B.; Shi, H.; Wu, X. P-glycoprotein
Inhibitor Tariquidar Potentiates Efficacy of Astragaloside IV in Experimental Autoimmune Encephalomyelitis
Mice. Molecules 2019, 24, 561. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).