Chalcone derivatives enhance ATP-binding cassette transporters A1 in human THP-1 macrophages

Article abstract of DOI:10.3390/molecules23071620

Atherosclerosis is a process of imbalanced lipid metabolism in the vascular walls. The underlying pathology mainly involves the deposition of oxidized lipids in the endothelium and the accumulation of cholesterol in macrophages. Macrophages export excessive cholesterol (cholesterol efflux) through ATP-binding cassette transporter A1 (ABCA1) to counter the progression of atherosclerosis. We synthesized novel chalcone derivatives and assessed their effects and the underlying mechanisms on ABCA1 expression in macrophages. Human THP-1 macrophages were treated with synthetic chalcone derivatives for 24 h. In Western blot and flow cytometry analyses, a chalcone derivative, (E)-1-(3,4-diisopropoxyphenyl)-3-(4-isopropoxy-3-methoxyphenyl)prop- 2-en-1-one (1m), was observed to significantly enhance ABCA1 protein expression in THP-1 cells (10 μM, 24 h). Levels of mRNA of ABCA1 and liver X receptor alpha (LXRα) were quantified using a real-time quantitative polymerase chain reaction technique and were found to be significantly increased after treatment with the novel chalcone derivative 1m. Several microRNAs, including miR155, miR758, miR10b, miR145, miR33, and miR106b, which functionally inhibit ABCA1 expression were suppressed after treatment with 1m. Collectively, 1m increases ABCA1 expression in human THP-1 macrophages. The mechanisms involve the activation of the LXRα-ABCA1 pathway and suppression of certain microRNAs that regulate ABCA1 expression.

Full text of DOI:10.3390/molecules23071620

molecules
Article
Chalcone Derivatives Enhance ATP-Binding Cassette
Transporters A1 in Human THP-1 Macrophages
I-Jou Teng 1,†, Min-Chien Tsai , Shao-Fu Shih , Bi-Feng Tsuei , Hsin Chang ,
2,†
3
4
5
6
3
5,‡
7, ,‡
Yi-Ping Chuang , Chin-Sheng Lin , Ching-Yuh Chern
and Sy-Jou Chen *
1
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 11490, Taiwan;
topy900703@gmail.com
2
3
4
5
6
7
Department of Physiology and Biophysics, Graduate Institute of Physiology, National Defense Medical
Center, Taipei 11490, Taiwan; mctsaisyy@gmail.com
Division of Cardiology, Department of Medicine, Tri-Service General Hospital, National Defense Medical
Center, Taipei 11490, Taiwan; evarei080@gmail.com (S.-F.S.); littlelincs@gmail.com (C.-S.L.)
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan;
befun1214@gmail.com
Department of Applied Chemistry, National Chiayi University, Chiayi City 60004, Taiwan;
s1060317@mail.ncyu.edu.tw (H.C.); cychern@mail.ncyu.edu.tw (C.-Y.C.)
Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center,
Taipei 11490, Taiwan; ypchuang@mail.ndmctsgh.edu.tw
Department of Emergency Medicine, Tri-Service General Hospital, National Defense Medical Center,
Taipei 11490, Taiwan; syjou.chen@gmail.com
*
†
‡
Correspondence: syjou.chen@gmail.com; Tel.: +886-2-8792-3311
These authors contributed equally to this work.
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 22 May 2018; Accepted: 29 June 2018; Published: 3 July 2018
Abstract: Atherosclerosis is a process of imbalanced lipid metabolism in the vascular walls.
The underlying pathology mainly involves the deposition of oxidized lipids in the endothelium
and the accumulation of cholesterol in macrophages. Macrophages export excessive cholesterol
(cholesterol efflux) through ATP-binding cassette transporter A1 (ABCA1) to counter the progression
of atherosclerosis. We synthesized novel chalcone derivatives and assessed their effects and the
underlying mechanisms on ABCA1 expression in macrophages. Human THP-1 macrophages were
treated with synthetic chalcone derivatives for 24 h. In Western blot and flow cytometry analyses,
a chalcone derivative, (E)-1-(3,4-diisopropoxyphenyl)-3-(4-isopropoxy-3-methoxyphenyl)prop- 2-en-1-one
(1m), was observed to significantly enhance ABCA1 protein expression in THP-1 cells (10
µM, 24 h).
Levels of mRNA of ABCA1 and liver X receptor alpha (LXR ) were quantified using a real-time
α
quantitative polymerase chain reaction technique and were found to be significantly increased after
treatment with the novel chalcone derivative 1m. Several microRNAs, including miR155, miR758,
miR10b, miR145, miR33, and miR106b, which functionally inhibit ABCA1 expression were suppressed
after treatment with 1m. Collectively, 1m increases ABCA1 expression in human THP-1 macrophages.
The mechanisms involve the activation of the LXRα-ABCA1 pathway and suppression of certain
microRNAs that regulate ABCA1 expression.
Keywords: atherosclerosis; cholesterol efflux; ATP-binding cassette transporters A1; chalcone derivative
1
. Introduction
Atherosclerosis is the leading cause of cardiovascular diseases including myocardial infarction,
stroke, unstable angina, and sudden cardiac death worldwide [
1
]. The underlying pathology
Molecules 2018, 23, 1620; doi:10.3390/molecules23071620
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1620
2 of 16
has been considered a chronic proinflammatory consequence of excess deposition of oxidized
low-density lipoprotein (oxLDL) in arterial walls [ ]. The formation of foam cells, namely lipid laden
2,3
macrophages, in the vascular intima plays a critical role in the initiation and progression of endothelial
dysfunction in atherosclerosis [4–6].
Reverse cholesterol transport, a process by which cholesterol is transferred from peripheral
tissues and cells to the liver for biliary secretion, contributes to protection from atherosclerosis.
Excess oxLDL is considered to be counterbalanced by cholesterol efflux mediated mainly by reverse
cholesterol transporters (RCT) [7]. ATP-binding cassette transporter A1 (ABCA1), a major RCT,
exports excess cholesterol from cells to apolipoprotein A-I and mediates the synthesis of high-density
lipoprotein (HDL) cholesterol. Human epidemiology studies have shown an inverse relationship
between HDL cholesterol and cardiovascular events [8–10]. Moreover, cholesterol efflux capacity is
inversely associated with the incidence of cardiovascular events demonstrated in a population-based
cohort [11].
Chalcone (1,3-diphenyl-2-propen-1-one) is a phenolic compound that is widely biosynthesized
in plants [12]. Epidemiological studies have suggested that increased intake of polyphenols from
fruits and vegetables reduces the risk of cardiovascular disease. Naturally occurring chalcones as
well as synthetic chalcone analogues have indicated numerous pharmaceutical effects, including
anti-inflammatory, antioxidant, antiapoptosis, antiparasite, and antitumor activities [13–19]. Because of
privileged structural and chemical properties, chalcones are excellent at modifying new drug
designs and development [12]. However, the antiatherosclerotic effects of chalcones remain poorly
0
investigated [20]. A study on 2-hydroxy-4 -methoxychalcone, a synthetic chalcone derivative, reported
that this derivative significantly inhibited the oxLDL-induced proliferation of human aortic smooth
muscle cells [21]. Synthetic indole–chalcone fibrates exhibited in vitro antioxidant and in vivo
antidyslipidemic effects in mice [22]. Although few studies have started to focus on its potential
in atherosclerosis prevention, no studies have investigated its effects on regulating ABCA1.
Therefore, the present study investigated the effects of chalcone on the expression of ABCA1 in
macrophages. We synthesized several chalcone derivatives and identified a novel chalcone derivative
that significantly increased the expression of ABCA1. The mechanism works by enhancing the liver X
receptor alpha (LXRα) signaling pathway and inhibiting several microRNAs (miRNA) that regulate
the expression of ABCA1.
2
. Results
2
.1. Chemistry
For this study, we synthesized 20 chalcones 1a
were obtained using a procedure similar to the one described previously in References [23
Intramolecular hydrogen bonding such as that observed in 1a is known to prevent Aldol reactions.
Therefore, O-isoproxyl-acetophenones 3a were used as the starting materials for our synthesis.
The preparation of O-isoproxyl- acetophenones 3a was straightforward. Acetophenone 2a were
protected with isopropyl bromide and potassium carbonate in dimethylformamide at around 95%
yield. The isolated products 3a were then reacted with appropriate benzaldehydes with 5N KOH to
offer the intermediates 4a . The O-isopropyl ether was removed quantitatively with BCl to afford
the target chalcones 1a
–
t
(Figure 1). These Aldol intermediates
–
27].
–g
–
g
–g
–g
–g
–g
3
–
g
(Scheme 1), and each compound displayed > 95% purity based on the proton
1
nuclear magnetic resonance ( H-NMR) results [23].

Molecules 2018, 23, 1620
3 of 16
Figure 1. List of chalcone derivatives 1a–t.
Scheme 1. Chalcone derivatives 1a–g.

Molecules 2018, 23, 1620
4 of 16
The starting materials 2h
–
m
used in this study are new and have not been reported before.
We were the first group to synthesize the target compounds 1h
–
m. In the present study, we employed
the same methodology to synthesize the new compounds 1h
–
m
and the known compounds 1o–t
(Scheme 2).
Scheme 2. Chalcone derivatives 1h–t.
2
.2. Screening for ABCA1 Expression in Human THP-1 Macrophages Treated with Chalcone Derivatives
Excessive cholesterol accumulation in macrophages aggravates foam cell formation. ABCA1 is
one of the most crucial RCTs facilitating the removal of intracellular cholesterol in macrophages.
Subsequently, flow cytometry was used to screen chalcon derivatives that potentially enhance
ABCA1 expression in human THP-1 macrophages. THP-1 macrophages were treated with synthetic
chalcone derived compounds at 10
µM for 24 h. As shown in Figure 2A, ABCA1 expression
significantly increased in the THP-1 macrophages treated with 1h and 1m. The results were further
confirmed through Western blot analysis, which showed enhanced ABCA1 protein expression in the
THP-1 macrophages treated with 1h and 1m compared with the vehicle (Figure 2B). To determine
whether these chalcone derivatives affect cell viability, cell cytotocixity was analyzed using an MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay in the presence of various
chalcone compounds at 10 M for 24 h. The results of the MTT assay showed that most chalcone
µ
analogues significantly suppressed cell viability, except for 1m (Figure 2C). Therefore, 1m was
selected for subsequent studies. These results indicated that 1m is not cytotoxic to human
THP-1 cells and that 1m increases ABCA1 expression without inducing cell death. Furthermore,
to determine the different dose effects on ABCA1 expression, THP-1 cells were treated with indicated
concentrations (1, 5, and 10 µM) of 1m for 24 h. The results showed that 1m significantly and
concentration-dependently increased the expression of ABCA1 (Figure 2D).

Molecules 2018, 23, 1620
5 of 16
Figure 2. Effects of chalcone derivatives on the expression of ABCA1 in human THP-1 macrophages.
) Human THP-1 macrophages were treated with control ( ), vehicle (V; 0.1% DMSO) and various
chalcone derivatives at a concentration of 10 M for 24 h; cells were then washed and stained with
ABCA1 antibody. The expression of cell surface ABCA1 was analyzed using flow cytometry. ( ) Cell
lysates were collected after treatment with the selected chalcones 1h and 1m at 10 M for 24 h.
The expression of ABCA1 and Actin in THP-1 macrophages was determined by Western blot. ( ) THP-1
macrophages were incubated with vehicle (0.1% DMSO) or chalcone compounds (10 M) for 24 h.
(
A
C
µ
B
µ
C
µ
Cells were then incubated with MTT for six hours. Cell viability was measured by the amount
of dissolved reduced MTT crystals using an enzyme-linked immunosorbent assay reader; 1m was
nontoxic to THP-1 macrophages and was selected for subsequent studies. (
treated with the indicated concentrations of 1m. The expression of ABCA1 protein was determined
by Western blot. Data are shown by representative bands and presented as the mean SEM of three
D) THP-1 macrophages were
±
independent experiments in triplicate after densitometric analysis. (** p < 0.01, *** p < 0.001 vs. control).
2
.3. Effects of Chalcone 1m on the Expression of Transcription Factors LXRα and PPARγ
Liver X receptors (LXRs) are sterol-activated transcriptional factors. LXR
genes that regulate RCTs, and it is critical for the regulation of peroxisome proliferator-activated
receptor gamma expression (PPAR ) and several downstream genes such as chemokine (C–C motif)
ligand 2 (CCL2). To investigate whether LXR is involved in 1m-induced ABCA1 expression at the
transcriptional level, several mRNAs were assessed. As shown in Figure 3A, treatment with 1m for
4 h increased levels of mRNA of LXR , ABCA1, and ATP-binding cassette transporter G1(ABCG1)
and decreased CCL2 mRNA expression in THP-1 macrophages in a concentration-dependent manner
5 and 10 M). Subsequently, we investigated the protein expression of LXR and PPAR . THP-1
macrophages were incubated with 10 M of 1m for 2, 4, and 6 h. The protein expression of
LXR significantly increased at 6 h, but that of PPAR did not (Figure 3B). Treatment of 1m also
concentration-dependently enhanced LXR protein expression at 6 h, but it did not alter the expression
α targets promoter
γ
α
2
α
(
µ
α
γ
µ
α
γ
α

Molecules 2018, 23, 1620
6 of 16
of PPAR
γ
(Figure 3C). These results suggest that LXR
α
is involved in 1m-induced ABCA1 expression
in THP-1 macrophages.
Figure 3. Protein and mRNA expression of transcriptional factor LXR
macrophages treated with chalcone 1m ) THP-1 macrophages were treated with indicated
concentrations (1, 5, and 10 M) of 1m for 24 h. Expression of mRNA of LXR , ABCA1, ABCG1,
and CCL2 was determined using qRT-PCR analysis. The relative expressions of mRNA are shown.
B) THP-1 macrophages were treated with 10 M of 1m for the time indicated (2, 4, and 6 h),
or (C) treated with indicated doses (1, 5, and 10 M) for 6 h. Cell lysates were collected and protein
expression of LXR , PPAR , and Actin were determined by Western blot. Data are shown by
representative bands and presented as the mean SEM of three independent experiments in triplicate
α was enhanced in THP-1
.
(A
µ
α
(
µ
µ
α
γ
±
after densitometric analysis. (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
2
.4. Effects of Chalcone 1m on the Expression of MicroRNAs that Regulate ABCA1.
A pool of miRNAs is known to target several genes that are critical for the regulation of cholesterol
metabolism. Specific candidates such as miR10b, miR33, miR106b, miR144, miR145, miR155, miR206,
and miR758, which have been demonstrated to directly and indirectly inhibit the expression or function
of ABCA1, were selected [28
qRT-PCR) (Figure 4) revealed that treatment with 1m for 24 h reduced the expression of miR155,
miR758, miR10b, miR145, miR33, and miR106b, but did not affect the expression of miR144 and miR206.
–35]. The results of real-time quantitative polymerase chain reaction
(

Molecules 2018, 23, 1620
7 of 16
Figure 4. MicroRNAs were suppressed in THP-1 macrophages treated with chalcone 1m. THP-1
macrophages were incubated with a vehicle control (V) and 10 M of 1m for 24 h and were lysed
µ
for measuring the indicated microRNAs. The levels of specific microRNA (miR144, miR33, miR106b,
miR155, miR758, miR145, miR10b, and miR206) were measured using qRT-PCR. Data are shown by the
relative expressions of miRNA. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. vehicle control).
3
. Discussion
Chalcone-based derivatives exert various biological functions beneficial to the cardiovascular
system. However, the antiatherosclerotic effects have been rarely evaluated until recently.
We synthesized several novel chalcone derivatives and determined that 1m can significantly enhance
ABCA1 expression in THP-1 macrophages; 1m significantly increased ABCA1 protein and mRNA
expression in THP-1 macrophages. The mechanisms may be partly mediated by the LXRα-ABCA1
pathway and the inhibition of several microRNAs that regulate ABCA1 expression (Figure 5).
This novel synthetic chalcone derivative that targets ABCA1 critical in foam cell formation can be
beneficial to the prevention of atherosclerosis.
Figure 5. Potential mechanisms of the chalcone derivative (1m) in regulation of ABCA1 expression
in macrophage.

Molecules 2018, 23, 1620
8 of 16
For a newly developed drug or chemical, it is generally considered that less than 10
concentration of a drug in plasma is accepted because higher than 10
necessary enzymes [36]. In the study by Peluso MR [37], Xanthohumol (chalcone) at 20
to test its anti-inflammatory effects on THP-1 cells, suggesting that 20 M of Xanthohumol exerts no
µM (1–10)
µM of a drug might inhibit
µM was used
µ
cytotoxicity effect on THP-1 cells. Moreover, in the study of in vitro structure-toxicity relationship of
chalcones in human hepatic stellate cells, nineteen chalcones were analyzed for their structure related
toxicity. Seventeen out of 19 studied chalcones showed an IC50 > 50
all (18/19) studied chalcones showed decreased cell viability at 50
µ
M by the MTT assay, and nearly
µ
M [38]. Accordingly, we chose the
concentration of 10 µM as a reasonable initial screening concentration in the current study.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that control
transcriptional signals involved in processes of lipid metabolism, including fatty acid oxidation,
fat cell development, and lipoprotein metabolism [39]. PPARγ activation can decrease the rate of
cholesterol esterification by reducing acetyl-coenzyme acetyltransferase 1 mRNA levels, and controls
cholesterol efflux in human macrophages. Furthermore, the activation of PPAR
its anti-inflammatory properties. Liu et al. [21] studied a synthetic 2-hydroxy-4 -methoxychalcone in
human aortic smooth muscle cells and demonstrated that it significantly inhibited the proliferation
γ
0
reflects the nature of
of oxLDL–induced aortic smooth muscle cells. The effects manifest through the activation of PPAR
inhibition of p44/42 mitogen-activated protein kinase, and the progression of the cell cycle. However,
did not alter the expression of PPAR , but enhanced the expression of LXR in THP-1 macrophages
in the present study. Previous studies have shown that PPARs cooperate with LXRs to enhance
transcription of the ABCA1 gene and that the activation is in an LXR -dependent manner, suggesting
that LXR play a crucial role in the regulation of ABCA1 [40]. In this study, we demonstrated that
treatment with 1m in THP-1 macrophages significantly increase LXR but not PPAR expression,
indicating that LXR instead of PPAR is the main target of this pharmacological action. The results are
γ,
1
m
γ
α
α
α
α
γ
α
γ
also in consistence with some reports, suggesting that the activation of LXRα and ABCA1 expression
does not need pre-activation of PPARγ [41,42]
Liver X receptors (LXRs) are nuclear receptors, which have pivotal roles in regulating cholesterol
homeostasis [43]. These receptors are ligand-dependent and control the expression of genes involved in
the uptake, transport, efflux, and excretion of cholesterol in a tissue-dependent manner [44]. Treatment
with LXRα activator increases expression of ABCA1 mRNA and promotes cholesterol efflux to apoA-I
in macrophages [45]. LXRs have been identified as inflammatory regulators that suppress genes
involved in inflammation, such as the C–C motif ligand 2 (CCL2) gene; furthermore, they can exert
antiatherosclerotic effects independent of cholesterol efflux pathways [46
member of the CC chemokine family and is capable of suppressing HDL cholesterol internalization and
cholesterol efflux [49 50]. Our results show that 1m selectively upregulates LXR and its downstream
suppression gene, but not PPAR , which suggests that 1m might specifically upregulate ABCA1 at the
–48]. C–C motif ligand 2 is a
,
γ
transcriptional level through the LXR-ABCA1 pathway.
MicroRNAs (miRNAs) are short, noncoding RNAs (18–25 nucleotides) that are incorporated
0
into the RNA-induced silencing complex where it binds to the 3 -untranslated region of its target
genes, promoting translational repression and mRNA degradation [51]. Studies have shown that
ABCA1 expression is tightly regulated at the posttranscriptional level by miRNAs, including HDL
biogenesis, cholesterol efflux, cholesterol uptake in the liver, and bile acid synthesis and secretion [52].
Numerous miRNAs have been shown to regulate ABCA1 expression. For example, antagonists
of miR-33 increase hepatic ABCA1 expression and elevated plasma HDL cholesterol levels [28].
Previous studies suggested that several microRNAs, including miR-145, miR-106b, miR-206, miR-155,
miR-144, miR-758, miR-10b, and miR-33 are involved in manipulation of ABCA1 expression [28–35].
We found that treatment with 1m reduced the expression of miR-155, miR-758, miR-10b, miR-145,
miR-33, and miR-106b, but did not affect the expression of miR-144 and miR-206, which suggests that
1
m may participate in regulation of ABCA1 expression at a posttranscriptional level.

Molecules 2018, 23, 1620
9 of 16
Various biological activities of chalcone analogues have been identified. Specific characteristics
that are beneficial for protecting against atherosclerosis include anti-inflammatory, antioxidizing,
and antiplatelet properties. The present study proposed a novel synthetic chalcone compound
and demonstrated its superior effects on increasing the expression of ABCA1, a critical cholesterol
regulator in macrophages. Although we found that treatment with 1m significantly increased ABCA1
expression, the effect of 1m on cholesterol efflux has not been determined, a limitation in the present
study. Further in vitro and in vivo cholesterol efflux studies are warranted to investigate its effect on
atherosclerotic cardiovascular disease.
4
. Materials and Methods
4
.1. Synthesis of Chalcone Analogues
Proton (300 MHz) and carbon (75 MHz) NMR spectra were recorded on a Varian Mercury-300
NMR spectrometer (Agilent, Santa Clara, CA, USA). Chemical shifts were reported on the
δ scale
as parts per million (ppm) downfield from tetramethylsilane (TMS), which was used as an internal
reference. Mass spectra were measured using a VG Analytical Model 70–250 s Mass Spectrometer
(Varian, Palo Alto, CA, USA). All reagents were used as obtained commercially.
1
(
E)-1-(2-hydroxy-phenyl)-3-phenyl-propenone (1a) [24]. H-NMR (CDCl ):
δ 12.80 (1H, s, –OH), 7.93 (1H,
3
d, J = 15.6 Hz), 7.93 (1H, d, J = 8.0 Hz), 7.66 (1H, d, J = 15.6 Hz), 7.68–7.65 (1H, m), 7.55–7.42 (5H, m),
7
.03 (1H, dd, J = 8.0 Hz, 1.0 Hz), 6.95 (1H, td, J = 7.2 Hz, 1.0 Hz); 13C-NMR (CDCl3):
δ
193.8, 163.5,
1
45.4, 136.4, 134.5, 130.9, 129.6, 129.0, 128.6, 120.0, 119.9, 118.8, 118.6.
1
(
(
(
E)-1-(4-hydroxy-phenyl)-3-phenyl-propenone (1b) [24]. H-NMR (CDCl ):
δ
8.00 (2H, d, J = 8.8 Hz), 7.80
3
1H, d, J = 15.6 Hz), 7.63 (2H, dd, J = 2.6 Hz, 2.0 Hz), 7.53 (1H, d, J = 15.6 Hz), 7.42–7.40 (3H, m), 6.93
2H, d, J = 8.8 Hz), 2.10–1.90 (1H, br s, –OH); C-NMR (CDCl3):
31.0, 129.8, 129.4, 123.0, 116.2.
13
δ 188.4, 162.9, 143.8, 136.4, 132.0, 131.2,
1
1
(
E)-1-(3-hydroxy-phenyl)-3-phenyl-propenone (1c) [24]. H-NMR (CDCl ):
δ
7.83 (1H, d, J = 15.6 Hz),
3
7
4
.66–7.58 (3H, m), 7.51 (1H, d, J = 15.6 Hz), 7.45–7.34 (5H, m), 7.14 (1H, ddd, J = 8.0 Hz, 2.4 Hz, 1.2 Hz),
.8–4.2 (1H, br s, –OH); 13C-NMR (CDCl3):
δ
190.9, 156.5, 145.5, 139.4, 134.7, 130.7, 129.9, 129.0, 128.6,
1
21.9, 120.9, 120.5, 115.2.
1
(
E)-1-(2,6-Dihydroxy-4-methoxy-phenyl)-3-phenyl-propenone (1d) [25]. H-NMR (d -acetone):
δ
12.07 (2H,
6
br s, –OH), 8.25 (1H, d, J = 15.6 Hz), 7.73 (1H, d, J = 15.6 Hz), 7.71–7.66 (2H, m), 7.46–7.40 (3H, m), 6.04
(
2H, s), 3.81 (3H, s); 13C-NMR (d -acetone):
δ
193.4, 167.2, 165.4, 142.9, 136.4, 130.9, 129.8, 129.1, 128.3,
6
1
06.2, 94.6, 55.8.
1
(
E)-1-(2-hydroxy-4-methoxy-phenyl)-3-phenyl-propenone (1e) [53]. H-NMR (CDCl ):
δ
13.48 (1H, br s,
3
–
OH), 8.10 (1H, d, J = 15.6 Hz), 6.45–7.94 (8H, m), 6.41 (1H, d, J = 15.6 Hz), 3.86 (3H, s).
1
(
E)-1-(3-hydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (1f) [54]. H NMR (d -acetone):
δ
9.02 (1H,
br s, –OH), 8.76 (1H, br s, –OH), 7.73 (1H, d, J = 15.8 Hz), 7.60 (1H, d, J = 15.8 Hz,), 7.72–7.52 (4H, m),
.36 (1H, t, J = 8.0 Hz), 7.09 (1H, ddd, J = 8.8 Hz, 2.4 Hz, 1.0 Hz), 6.91 (2H, d, J = 8.8 Hz); 13C-NMR
6
7
(d6-acetone): δ 189.7, 160.8, 158.5, 145.0, 140.9, 131.5, 130.5, 127.6, 120.5, 119.8, 116.7, 115.5, 113.8.
1
(
E)-1-(3-hydroxyphenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one (1g) [25]. H NMR(d -acetone):
δ 8.90 (1H,
6
br s, –OH), 8.77 (1H, br s, –OH), 7.77 (2H, s, A ), 7.57 (1H, d, J = 7.6 Hz), 7.53 (1H, t, J = 2.2 Hz), 7.37
2
(
1H, t, J = 7.8 Hz), 7.31–7.22 (3H, m), 7.11 (1H, ddd, J = 7.6 Hz, 2.6 Hz, 1.0 Hz), 6.96–6.90 (1H, m).
1
3
C-NMR (d -acetone):
δ
189.9, 158.7, 158.6, 144.8, 140.5, 137.3, 130.8, 130.6, 122.9, 120.9, 120.8, 120.6,
6
1
18.5, 115.8, 115.6.
1
(
E)-1-(4-fluorophenyl)-3-(4-isopropoxyphenyl)prop-2-en-1-one (1h). H-NMR (CDCl , 300 MHz):
δ
8.07–8.02
3
(
2H, m), 7.76 (1H, d, J = 15.6 Hz), 7.35 (1H, d, J = 15.6 Hz), 7.23–7.14 (4H, m), 6.91 (2H, d, J = 8.4 Hz),

Molecules 2018, 23, 1620
10 of 16
+
4
.63 (1H, septa, J = 6.3 Hz), 1.41 (6H, d, J = 6.3 Hz) ; EI-MS m/z (relative intensity%): 284 (M , 49), 241
(
100), 225 (11), 147 (10), 123 (9); HRMS Calcd for C H FO : 284.1213, Found: 284.1213.
18
17
2
1
(
E)-3-(4-isopropoxyphenyl)-1-phenylprop-2-en-1-one (1i). H-NMR (CDCl ):
δ 8.01 (2H, dd, J = 8.1 Hz,
3
1
.5 Hz), 7.79 (1H, d, J = 15.6 Hz), 7.59–7.53 (3H, m), 7.50 (2H, t, J = 8.4 Hz), 7.42 (1H, d, J = 15.6 Hz), 6.94
(
2
2H, d, J = 8.4 Hz), 4.69 (1H, septa, J = 6.3 Hz), 1.43 (6H, d, J = 6.3 Hz); EI-MS m/z (relative intensity%):
+
66 (M , 64), 223 (100), 147 (16); HRMS Calcd for C H O : 266.1307, Found: 266.1315.
18
18
2
1
(
E)-3-(4-Fluoro-phenyl)-1-(4-isopropoxy-3-methoxy-phenyl)-propenone (1j). H-NMR(CDCl ):
δ
7.77(1H, d,
J = 15.6 Hz), 7.67–7.61(3H, m), 7.49 (1H, d, J = 15.6 Hz), 7.14–7.08 (2H, td, J = 8.7, 2.1 Hz), 6.93 (1H, d,
J = 8.4 Hz), 4.69 (1H, septa, J = 6.0 Hz), 3.95 (3H, s), 1.43 (6H, d, J = 6.0 Hz). 13C-NMR(CDCl3):
188.2,
65.4, 162.1, 151.8, 150.0, 142.4, 131.3, 130.2, 130.1, 122.8, 121.3, 116.1, 115.8, 112.6, 111.3, 71.1, 56.0, 21.8;
3
δ
1
+
EI-MS m/z (relative intensity%): 314(M , 70), 272(98), 271(100), 241(55), 124(87), 123(82), 95(75); HRMS
Calcd for C H FO :314.1318 Found: 314.1315.
18
17
2
1
(
E)-3-(4-isopropoxy-3-methoxyphenyl)-1-phenylprop-2-en-1-one (1k). H-NMR(CDCl ):
δ
8.01 (2H, dd,
J = 8.1 Hz, 1.5 Hz), 7.76 (1H, d, J = 15.6 Hz), 7.58 (1H, td, J = 7.2 Hz, 1.5 Hz), 7.50 (2H, td, J = 7.5 Hz,
.5 Hz), 7.38 (1H, d, J = 15.6 Hz), 7.21(1H, dd, J = 8.4 Hz, 2.1 Hz), 7.17 (1H, d, J = 2.1 Hz), 6.91(1H, d,
J = 8.4 Hz), 4.62 (1H, septa, J = 6.0 Hz), 3.92 (3H, s), 1.41 (6H, d, J = 6.0 Hz); 13C-NMR (CDCl3):
190.2,
50.1, 149.8, 144.8, 138.3, 132.3, 128.3, 128.2, 127.6, 122.8, 119.7, 114.4, 110.9, 71.0, 55.8, 21.8; HRMS
Calcd for C H O : 296.1412, Found: 296.1410.
3
1
δ
1
19
20
3
1
(
E)-3-(4-isopropoxy-3-methoxyphenyl)-1-p-tolylprop-2-en-1-one (1l). H-NMR(CDCl ): δ 7.93 (2H, d,
3
J = 8.1 Hz), 7.75 (1H, d, J = 15.6 Hz), 7.38 (1H, d, J = 15.6 Hz), 7.30 (2H, d, J = 8.1 Hz), 7.20 (1H,
dd, J = 8.4, 1.8 Hz), 7.16 (1H, d, J = 1.8 Hz), 6.91(1H, d, J = 8.4 Hz), 4.62 (1H, septa, J = 6.0 Hz), 3.92 (3H,
s), 2.44 (3H, s), 1.41 (6H, d, J = 6.0 Hz); 13C-NMR (CDCl3):
δ
189.9, 150.2, 149.8, 144.5, 143.2, 135.8, 129.1,
+
1
1
28.5, 127.8, 122.8, 119.8, 114.4, 110.9, 71.2, 55.9, 21.9, 21.5; EI-MS m/z (relative intensity%): 310(M ,
00), 268(96), 253(98), 145(53), 119(92), 91(93); HRMS Calcd for C H O :310.1569, Found: 310.1563.
20
22
3
1
(
E)-1-(3,4-diisopropoxyphenyl)-3-(4-isopropoxy-3-methoxyphenyl)prop-2-en-1-one (1m). H-NMR (CDCl ):
3
δ
7.74 (1H, d, J = 15.6 Hz), 7.65 (1H, d, J = 8.4 Hz), 7.64 (1H, s), 7.38 (1H, d, J = 15.6 Hz), 7.20 (1H, d,
J = 8.4 Hz), 7.16 (1H, s), 6.95 (1H, d, J = 8.0 Hz), 6.90 (1H, d, J = 8.4 Hz), 4.62 (2H, septa, J = 5.6 Hz),
.45 (1H, septa, J = 6.4 Hz), 3.92 (3H, s), 1.40 (3H, d, J = 5.6 Hz), 1.39 (3H, d, J = 6.0 Hz), 1.36 (3H, d,
4
1
3
J = 6.0 Hz); C-NMR(75 MHz):
δ 188.9, 153.6, 150.2, 149.7, 148.5, 144.1, 131.6, 128.0, 123.4, 122.7, 119.7,
1
18.1, 115.0, 114.5, 110.9, 72.6, 71.7, 71.3, 56.1, 22.2, 22.1, 22.0; HRMS Calcd for C H O : 412.2250,
25
32
5
Found: 412.2252.
1
(
E)-3-(4-fluorophenyl)-1-phenylprop-2-en-1-one (1n) [55]. H-NMR (CDCl ): δ 8.02 (2H, d, J = 7.3 Hz), 7.77
3
(1H, d, J = 15.6 Hz), 7.66–7.56 (3H, m), 7.49–7.42 (3H, m), 7.10 (2H, t, J = 8.7 Hz).
1
(
E)-4-(3-Oxo-3-phenylprop-1-en-1-yl)benzonitrile (1o) [55]. H-NMR (CDCl ):
δ
8.01 (2H, d, J = 7.4 Hz),
3
7
.78–7.70 (3H, m), 7.68 (2H, d, J = 8.6 Hz), 7.64–7.57 (2H, m), 7.51 (2H, t, J = 8.6 Hz).
1
(
E)-1-Phenyl-3-(p-tolyl)prop-2-en-1-one (1p) [56]. H-NMR (CDCl ):
δ
3
8.07–7.99 (2H, m), 7.80 (1H, d,
3
1
J = 15.6 Hz), 7.61–7.46 (6H, m), 7.24 (2H, t, J = 7.6 Hz), 2.40 (3H, s); C-NMR (CDCl3):
δ 190.6, 144.9,
1
41.0, 138.3, 132.6, 132.1, 129.7, 128.5, 128.4, 128.4, 121.0, 21.49.
1
(
E)-1,3-Bis(4-nitrophenyl)prop-2-en-1-one (1q) [57]. H-NMR (CDCl ):
δ
7.97–7.98 (2H, m), 7.53–7.52 (2H,
3
m), 8.01–8.03 (2H, m), 8.17 (2H, m), 6.88 (1H, d, J = 15.6 Hz), 6.37 (1H, d, J = 15.6 Hz).
1
(
E)-1-(4-Nitrophenyl)-3-phenylprop-2-en-1-one (1r) [58]. H-NMR (CDCl ):
δ
8.34 (2H, d, J = 8.4 Hz), 8.15
3
(
2H, d, J = 8.4 Hz), 7.85 (1H, d, J = 15.6 Hz), 7.69–7.63 (2H, m), 7.49 (1H, d, J = 15.6 Hz), 7.47–7.43 (3H,
13
m); C-NMR (CDCl ): δ 189.0, 150.0, 146.8, 143.0, 134.2, 131.2, 129.4, 129.1, 128.7, 123.8, 121.2.
3

Molecules 2018, 23, 1620
11 of 16
1
(
E)-1-(4-Methoxyphenyl)-3-phenylprop-2-en-1-one (1s) [55]. H-NMR (CDCl ):
δ
8.03 (2H, d, J = 8.8 Hz),
3
7.80 (1H, d, J = 15.6 Hz), 7.66–7.63 (2H, m), 7.56 (1H, d, J = 15.6 Hz), 7.44–7.40 (3H, m), 6.95 (2H, d,
J = 8.8 Hz), 3.86 (3H, s).
1
(
E)-1-(4-Fluorophenyl)-3-phenylprop-2-en-1-one (1t) [56]. H-NMR (CDCl ):
δ
8.06 (2H, dd, J = 8.7, 5.5 Hz),
3
7
.82 (1H, d, J = 15.6 Hz), 7.63–7.61 (2H, m), 7.51 (1H, d, J = 15.6 Hz), 7.46–7.37 (3H, m), 7.17–7.13 (2H,
13
m); C-NMR (CDCl3):
δ 188.7, 165.5 (d, JC–F = 252.9 Hz), 144.9, 134.7, 134.4 (d, JC–F = 2.9 Hz), 131.0 (d,
JC–F = 9.2 Hz), 130.5, 128.9, 128.4, 121.5, 115.7 (d, JC–F = 21.7).
4
.2. Cell Culture
Human monocytic THP-1 cell line was originally obtained from the Bioresource Collection
and Research Center (BCRC, Taiwan, catalog #60430). The cell line was cultured in RPMI 1640
medium (Corning, Corning, NY, USA, catalog #10-040-CMR) with 10% fetal bovine serum (FBS)
(catalog #10437-028), MEM Non-Essential Amino Acids Solution (NEAA) (gibco, Grand Island,
NY, USA, catalog #11140-050), sodium pyruvate (gibco, catalog #11360-070), penicillin (100 U/mL),
◦
and streptomycin (100
µ
6
g/mL) in a humidified incubator with 5% CO at 37 C. For each experiment,
2
equal amount (1
×
10 /mL) of THP-1 cells were incubated with PMA (100 ng/mL) for 3 days to
differentiate into macrophages adherent to dishes. For each analysis, cells were incubated in starvation
medium containing 0.1% FBS, MEM NEAA, penicillin, and streptomycin.
4
.3. Cell Viability Assay
The possible cytotoxic effect induced by chalcone compounds was measured by
-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. THP-1 monocytes were
3
seeded into 96-well plates containing 100
µL of culture medium with PMA to differentiate into
macrophages. After incubating THP-1 macrophages for 3 days, they were treated with a series of
chalcone compounds (10
compounds, 25
h. MTT was then replaced with 100
MTT crystals was measured using an enzyme-linked immunosorbent assay reader.
µ
M) in 100
µ
L of starvation medium for 24 h. After treatment with target
◦
µ
M of MTT (5 mg/mL) was added to each well; the cells were incubated at 37 C for
6
µL of dimethyl sulfoxide, and the amount of dissolved reduced
4
.4. Flow Cytometry
5
THP-1 cells were treated with PMA in 12-well culture plates (5
×
10 cells/well) for 3 days to
differentiate, and subsequently, THP-1 macrophages were treated with various chalcone compounds
for 24 h. The harvested cells were digested by 0.5 M EDTA and washed with phosphate-buffered
saline. After washing, cells were fixed with 0.5% paraformaldehyde/PBS and stained with ABCA1
◦
antibody (abcam, catalog #ab18180, 1:250) for 1 h at 4 C. The cells were then stained using the second
antibody with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG for 40 min in a dark place
◦
at 4 C. The expression of ABCA1 was analyzed using FACSCalibur (BD Biosciences, Franklin Lakes,
NJ, USA).
4
.5. Western Blot
At first, the cells were lysed using RIPA lysis buffer (Millipore, Burlington, MA, USA, catalog
20-188) and protease and phosphatase inhibitor (Thermo, Waltham, MA, USA, catalog #78442). Whole
#
cell lysates were harvested through centrifugation and collection of the supernatants. After calculating
the protein concentration using Bradford reagent (Bio-Rad, Hercules, CA, USA) with BSA as a standard,
equal amounts of protein sample were added to 5X loading buffer (iNtTRON Biotechnology, catalog
#IBS-BS002). The protein samples were resolved on 8–12% SDS-PAGE gels and transferred onto
membranes using a wet transfer system. Membranes were blocked in 5% (wt/vol) BSA (Affymetrix,
Santa Clara, CA, USA, catalog #9048-46-8) in TBS-T (50 mM Tris/HCL, pH 7.6, 150 mM NaCl and 0.1%
(vol/vol) Tween-20) for 1 h at room temperature. Subsequently, the membranes were incubated with

Molecules 2018, 23, 1620
12 of 16
primary antibodies that included those for ABCA1 (abcam, catalog #ab18180, 1:5000), LXR
α
(Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, USA, catalog #sc-1202, 1:1000), PPARγ (Santa Cruz, catalog
#sc-7196, 1:1000) and Actin (Thermo, catalog #MA5-15739, 1:10000) diluted in 2.5% (wt/vol) BSA in
TBS-T and then with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies
diluted in 2.5% (wt/vol) BSA in TBS-T. Immunodetection of relative intensities was performed using
ImageQuant LAS 4000 and quantified using ImageJ.
4
.6. Quantification of mRNA
6
All RNA samples were isolated from a 6-cm-dish of fresh human THP-1 macrophage (2
×
10 cells
per dish) using RNeasy kit (QIAGEN, Hilden, Germany, catalog #74106) according to manufacturer
instructions. The concentration and purity were evaluated by Nanodrop spectrophotometer. 1000 ng
RNA were reverse transcripted into cDNA using an iScript cDNA synthesis kit (BIO-RAD, catalog
#
170-8891) following the manufacturer protocol. KAPA SYBR FAST kit (catalog #kk4609) were used
in qRT-PCR and 20 ng cDNA with 200 nM primers were applied in each reaction. qRT-PCR reaction
◦
was carried out by Roche LC480 2X. The cycling program consists of an initial activation at 95 C
◦
◦
◦
for 3 min, followed by 40 cycles of 95 C for 10 s, 60 C for 20 s and 72 C for 1 s. Immediately
◦
◦
after amplification, melt-curve analysis was performed from 65 C to 97 C. The quantification of
mRNA was performed following MIQE guidelines [59]. The calibration curve for a primer set was
generated from a series of dilution of the cDNA template. Each primer efficiency was then calculated
−
1/slope
(
E = 10
)C. t(Ttahrgeet)relative quantification of target mRNA was calculated based on the equation
Etarget∆
(
RQ = E
) and GAPDH was served as the reference gene. For all genes determined
∆
Ct(re f erence)
re f erence
in our study, single peak was shown in melting curve which indicated the PCR amplification was
specific. There was no Cq value of NTC (no template control) detected in all primer sets. The detailed
information regarding primer sequences, efficiencies, and calibration curves was provided in the
supplementary information (Supplementary Table S1, Figure S1).
4
.7. Quantification of MicroRNA
6
All miRNA samples were isolated from a 6-cm-dish of human THP-1 macrophage (2 × 10 cells
per dish) using miRNeasy kit (QIAGEN, catalog #1038703) following manufacturer instructions.
The concentration and purity were evaluated by Nanodrop spectrophotometer. cDNA was then
synthesized from 1000 ng miRNA using miScript II RT kit with HiFlex Buffer (QIAGEN, catalog
#218161). Each cDNA synthesis was then diluted by RNase-free water (20 ng cDNA in each well)
and mixed with miScript SYBR Green PCR kit (QIAGEN, catalog #218073). qRT-PCR was optimized
◦
by Roche LC480 2X. The three-step cycling program was settled as an initial activation at 95 C for
1
◦ ◦ ◦
5 min, followed by 40 cycles of 95 C for 15 s, 55 C for 30 s and 70 C for 30 s. The calibration
curve for a primer set was generated from a series of dilution of the cDNA template. Each primer
efficiency was then calculated to adjust corresponding PCR results. The expression of miRNA was
analyzed by relative quantification with comparative CT method and normalized to RNU6-2 as the
universal invariant reference primer (∆Cq), followed by a comparison of vehicle sample (∆∆Cq).
The detailed primer information was provided in the supplementary information. (Supplementary
Table S2, Figure S1).
4
.8. Statistical Analysis
Data were expressed as mean
were analyzed using a Student’s unpaired t test or one-way ANOVA; p < 0.05 was considered
±
SEM for at least three independent experiments. Statistical data
statistically significant.

Molecules 2018, 23, 1620
13 of 16
5
. Conclusions
Privileged structures and chemical properties make certain chalcone derivatives potential for
modifying new drug designs and developments for treating atherosclerotic diseases associated with
lipid dysmetabolism. With their unique and multiplex properties, certain chalcone derivatives are
promising antiatherosclerotic agents that warrant further investigation.
Supplementary Materials: The following are available online: Table S1: The sequences and efficiencies of mRNAs
for real-time quantitative PCR assay; Table S2: The sequences and efficiencies of miRNAs for real-time quantitative
PCR assay; Figure S1: Standard curves of the significant primers.
Author Contributions: I.-J.T., M.-C.T., S.-F.S., and B.-F.T. carried out the cell experiments and analyzed the data.
H.C. synthesize all the chalcone derivatives. M.-C.T., C.-Y.C., Y.-P.C., C.-S.L. gave discussion and helped in editing
the manuscript. C.-S.L., C.-Y.C., and S.-J.C. were principal investigators of this project and I.-J.T., S.-F.S., and S.-J.C.
drafted this manuscript.
Funding: This work is supported by grants from Ministry of National Defense-Medical Affairs Bureau
(
MAB-105-088 to SJ Chen); Tri-Service General Hospital (TSGH-C105-057 to SJ Chen); Teh-Tzer Study Group
for Human Medical Research Foundation (B1041005 to SJ Chen); Taiwan Ministry of Science and Technology
MOST-106-2320-B-016-010 to MC Tsai).
(
Acknowledgments: The authors thank Wan-Lin Wu and Meng-Lin Chiang for their valuable comments.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
References
1.
2.
3.
4.
5.
6.
7.
Weber, C.; Noels, H. Atherosclerosis: Current pathogenesis and therapeutic options. Nat. Med. 2011, 17,
410–1422. [CrossRef] [PubMed]
Shapiro, M.D.; Fazio, S. From lipids to inflammation: New approaches to reducing atherosclerotic risk.
Circ. Res. 2016, 118, 732–749. [CrossRef] [PubMed]
1
Sorci-Thomas, M.G.; Thomas, M.J. Microdomains, inflammation, and atherosclerosis. Circ. Res. 2016, 118,
6
79–691. [CrossRef] [PubMed]
Moore, K.J.; Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145, 341–355. [CrossRef]
PubMed]
Hansson, G.K.; Hermansson, A. The immune system in atherosclerosis. Nat. Immunol. 2011, 12, 204–212.
CrossRef] [PubMed]
[
[
Tabas, I.; Garcia-Cardena, G.; Owens, G.K. Recent insights into the cellular biology of atherosclerosis.
J. Cell. Biol. 2015, 209, 13–22. [CrossRef] [PubMed]
Tang, C.; Houston, B.A.; Storey, C.; LeBoeuf, R.C. Both STAT3 activation and cholesterol efflux contribute to
the anti-inflammatory effect of apoA-I/ABCA1 interaction in macrophages. J. Lipid. Res. 2016, 57, 848–857.
[CrossRef] [PubMed]
8
9
1
1
1
.
Barter, P.; Gotto, A.M.; LaRosa, J.C.; Maroni, J.; Szarek, M.; Grundy, S.M.; Kastelein, J.J.; Bittner, V.;
Fruchart, J.C. Treating to new targets, I. HDL cholesterol, very low levels of LDL cholesterol,
and cardiovascular events. N. Engl. J. Med. 2007, 357, 1301–1310. [CrossRef] [PubMed]
Khera, A.V.; Cuchel, M.; de la Llera-Moya, M.; Rodrigues, A.; Burke, M.F.; Jafri, K.; French, B.C.; Phillips, J.A.;
Mucksavage, M.L.; Wilensky, R.L.; et al. Cholesterol efflux capacity, high-density lipoprotein function,
and atherosclerosis. N. Engl. J. Med. 2011, 364, 127–135. [CrossRef] [PubMed]
.
0. Saleheen, D.; Scott, R.; Javad, S.; Zhao, W.; Rodrigues, A.; Picataggi, A.; Lukmanova, D.; Mucksavage, M.L.;
Luben, R.; Billheimer, J.; et al. Association of HDL cholesterol efflux capacity with incident coronary heart
disease events: A prospective case-control study. Lancet. Diabetes. Endocrinol. 2015, 3, 507–513. [CrossRef]
1. Rohatgi, A.; Khera, A.; Berry, J.D.; Givens, E.G.; Ayers, C.R.; Wedin, K.E.; Neeland, I.J.; Yuhanna, I.S.;
Rader, D.R.; de Lemos, J.A.; et al. HDL cholesterol efflux capacity and incident cardiovascular events.
N. Engl. J. Med. 2014, 371, 2383–2393. [CrossRef] [PubMed]
2. Gomes, M.N.; Muratov, E.N.; Pereira, M.; Peixoto, J.C.; Rosseto, L.P.; Cravo, P.V.L.; Andrade, C.H.; Neves, B.J.
Chalcone derivatives: Promising starting points for drug design. Molecules 2017, 22, 1210. [CrossRef]
[PubMed]

Molecules 2018, 23, 1620
14 of 16
1
3. Kontogiorgis, C.; Mantzanidou, M.; Hadjipavlou-Litina, D. Chalcones and their potential role in
inflammation. Mini. Rev. Med. Chem. 2008, 8, 1224–1242. [CrossRef] [PubMed]
1
4. Kamal, A.; Ramakrishna, G.; Raju, P.; Viswanath, A.; Ramaiah, M.J.; Balakishan, G.; Pal-Bhadra, M. Synthesis
and anti-cancer activity of chalcone linked imidazolones. Bioorg. Med. Chem. Lett. 2010, 20, 4865–4869.
[CrossRef] [PubMed]
1
5. Kumar, V.; Kumar, S.; Hassan, M.; Wu, H.; Thimmulappa, R.K.; Kumar, A.; Sharma, S.K.; Parmar, V.S.;
Biswal, S.; Malhotra, S.V. Novel chalcone derivatives as potent Nrf2 activators in mice and human lung
epithelial cells. J. Med. Chem. 2011, 54, 4147–4159. [CrossRef] [PubMed]
1
1
1
6. Bukhari, S.N.; Jasamai, M.; Jantan, I. Synthesis and biological evaluation of chalcone derivatives (mini
review). Mini. Rev. Med. Chem. 2012, 12, 1394–1403. [PubMed]
7. Bukhari, S.N.; Jantan, I.; Jasamai, M. Anti-inflammatory trends of 1, 3-diphenyl-2-propen-1-one derivatives.
Mini. Rev. Med. Chem. 2013, 13, 87–94. [CrossRef] [PubMed]
8. Wu, J.Z.; Cheng, C.C.; Shen, L.L.; Wang, Z.K.; Wu, S.B.; Li, W.L.; Chen, S.H.; Zhou, R.P.; Qiu, P.H. Synthetic
chalcones with potent antioxidant ability on H( )O( )-induced apoptosis in PC12 cells. Int. J. Mol. Sci. 2014
,
2
2
1
5, 18525–18539. [CrossRef] [PubMed]
1
9. Wu, J.; Li, J.; Cai, Y.; Pan, Y.; Ye, F.; Zhang, Y.; Zhao, Y.; Yang, S.; Li, X.; Liang, G. Evaluation and discovery
of novel synthetic chalcone derivatives as anti-inflammatory agents. J. Med. Chem. 2011, 54, 8110–8123.
[CrossRef] [PubMed]
2
2
0. Singh, P.; Anand, A.; Kumar, V. Recent developments in biological activities of chalcones: A mini review.
Eur. J. Med. Chem. 2014, 85, 758–777. [CrossRef] [PubMed]
0
1. Liu, C.S.; Chang, C.C.; Du, Y.C.; Chang, F.R.; Wu, Y.C.; Chang, W.C.; Hsieh, T.J. 2-hydroxy-4 -
methoxychalcone inhibits proliferation and inflammation of human aortic smooth muscle cells by increasing
the expression of peroxisome proliferator-activated receptor gamma. J. Cardiovasc. Pharmacol. 2012, 59,
3
39–351. [CrossRef] [PubMed]
2
2
2
2. Sashidhara, K.V.; Dodda, R.P.; Sonkar, R.; Palnati, G.R.; Bhatia, G. Design and synthesis of novel
indole-chalcone fibrates as lipid lowering agents. Eur. J. Med. Chem. 2014, 81, 499–509. [CrossRef]
[PubMed]
3. Chen, Y.H.; Wang, W.H.; Wang, Y.H.; Lin, Z.Y.; Wen, C.C.; Chern, C.Y. Evaluation of the anti-inflammatory
effect of chalcone and chalcone analogues in a zebrafish model. Molecules 2013, 18, 2052–2060. [CrossRef]
[PubMed]
4. Lee, Y.T.; Fong, T.H.; Chen, H.M.; Chang, C.Y.; Wang, Y.H.; Chern, C.Y.; Chen, Y.H. Toxicity assessments of
chalcone and some synthetic chalcone analogues in a zebrafish model. Molecules 2014, 19, 641–650. [CrossRef]
[PubMed]
2
5. Chen, Y.H.; Chang, C.Y.; Chang, C.F.; Chen, P.C.; Lee, Y.T.; Chern, C.Y.; Tsai, J.N. Pro-angiogenic effects of
chalcone derivatives in zebrafish embryos in vivo. Molecules 2015, 20, 12512–12524. [CrossRef] [PubMed]
6. Karki, R.; Thapa, P.; Kang, M.J.; Jeong, T.C.; Nam, J.M.; Kim, H.L.; Na, Y.; Cho, W.J.; Kwon, Y.; Lee, E.S.
Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study of
hydroxylated 2,4-diphenyl-6-aryl pyridines. Bioorg. Med. Chem. 2010, 18, 3066–3077. [CrossRef] [PubMed]
7. Qian, Y.P.; Shang, Y.J.; Teng, Q.F.; Chang, J.; Fan, G.J.; Wei, X.; Li, R.R.; Li, H.P.; Yao, X.J.; Dai, F.; et al.
Hydroxychalcones as potent antioxidants: Structure-activity relationship analysis and mechanism
considerations. Food Chem. 2011, 126, 241–248. [CrossRef]
2
2
2
2
3
3
8. Rayner, K.J.; Suarez, Y.; Davalos, A.; Parathath, S.; Fitzgerald, M.L.; Tamehiro, N.; Fisher, E.A.; Moore, K.J.;
Fernandez-Hernando, C. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 2010, 328,
1
570–1573. [CrossRef] [PubMed]
9. Sala, F.; Aranda, J.F.; Rotllan, N.; Ramirez, C.M.; Aryal, B.; Elia, L.; Condorelli, G.; Catapano, A.L.;
Fernandez-Hernando, C.; Norata, G.D. MiR-143/145 deficiency attenuates the progression of atherosclerosis
in Ldlr-/-mice. Thromb. Haemost. 2014, 112, 796–802. [CrossRef] [PubMed]
0. Kim, J.; Yoon, H.; Ramirez, C.M.; Lee, S.M.; Hoe, H.S.; Fernandez-Hernando, C.; Kim, J. MiR-106b impairs
cholesterol efflux and increases Abeta levels by repressing ABCA1 expression. Exp. Neurol. 2012, 235,
4
76–483. [CrossRef] [PubMed]
1. Vinod, M.; Chennamsetty, I.; Colin, S.; Belloy, L.; De Paoli, F.; Schaider, H.; Graier, W.F.; Frank, S.; Kratky, D.;
Staels, B.; et al. MiR-206 controls LXRalpha expression and promotes LXR-mediated cholesterol efflux in
macrophages. Biochim. Biophys. Acta. 2014, 1841, 827–835. [CrossRef] [PubMed]

Molecules 2018, 23, 1620
15 of 16
3
3
3
2. Tian, F.J.; An, L.N.; Wang, G.K.; Zhu, J.Q.; Li, Q.; Zhang, Y.Y.; Zeng, A.; Zou, J.; Zhu, R.F.; Han, X.S.; et al.
Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis. Cardiovasc. Res.
014, 103, 100–110. [CrossRef] [PubMed]
3. Ramirez, C.M.; Rotllan, N.; Vlassov, A.V.; Davalos, A.; Li, M.; Goedeke, L.; Aranda, J.F.; Cirera-Salinas, D.;
Araldi, E.; Salerno, A.; et al. Control of cholesterol metabolism and plasma high-density lipoprotein levels by
microRNA-144. Circ. Res. 2013, 112, 1592–1601. [CrossRef] [PubMed]
4. Ramirez, C.M.; Davalos, A.; Goedeke, L.; Salerno, A.G.; Warrier, N.; Cirera-Salinas, D.; Suarez, Y.;
Fernandez-Hernando, C. MicroRNA-758 regulates cholesterol efflux through posttranscriptional repression
of ATP-binding cassette transporter A1. Arterioscler. Thromb. Vasc. Biol. 2011, 31, 2707–2714. [CrossRef]
2
[PubMed]
3
5. Wang, D.; Xia, M.; Yan, X.; Li, D.; Wang, L.; Xu, Y.; Jin, T.; Ling, W. Gut microbiota metabolism of anthocyanin
promotes reverse cholesterol transport in mice via repressing miRNA-10b. Circ. Res. 2012, 111, 967–981.
[
CrossRef] [PubMed]
6. Hughes, J.P.; Rees, S.; Kalindjian, S.B.; Philpott, K.L. Principles of early drug discovery. Br. J. Pharmacol. 2011
62, 1239–1249. [CrossRef] [PubMed]
7. Peluso, M.R.; Miranda, C.L.; Hobbs, D.J.; Proteau, R.R.; Stevens, J.F. Xanthohumol and related prenylated
flavonoids inhibit inflammatory cytokine production in LPS-activated THP-1 monocytes: Structure-activity
relationships and in silico binding to myeloid differentiation protein-2 (MD-2). Planta. Med. 2010, 76,
3
3
,
1
1
536–1543. [CrossRef] [PubMed]
3
8. Zenger, K.; Dutta, S.; Wolff, H.; Genton, M.G.; Kraus, B. In vitro structure-toxicity relationship of chalcones
in human hepatic stellate cells. Toxicology 2015, 336, 26–33. [CrossRef] [PubMed]
3
9. Ricote, M.; Valledor, A.F.; Glass, C.K. Decoding transcriptional programs regulated by PPARs and LXRs
in the macrophage: Effects on lipid homeostasis, inflammation, and atherosclerosis. Arterioscler. Thromb.
Vasc. Biol. 2004, 24, 230–239. [CrossRef] [PubMed]
4
4
4
4
0. Ogata, M.; Tsujita, M.; Hossain, M.A.; Akita, N.; Gonzalez, F.J.; Staels, B.; Suzuki, S.; Fukutomi, T.; Kimura, G.;
Yokoyama, S. On the mechanism for PPAR agonists to enhance ABCA1 gene expression. Atherosclerosis 2009
05, 413–419. [CrossRef] [PubMed]
,
2
1. Trogan, E.; Feig, J.E.; Dogan, S.; Rothblat, G.H.; Angeli, V.; Tacke, F.; Randolph, G.J.; Fisher, E.A. Gene
expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression
in ApoE-deficient mice. Proc. Natl. Acad. Sci. USA 2006, 103, 3781–3786. [CrossRef] [PubMed]
2. Babaev, V.R.; Yancey, P.G.; Ryzhov, S.V.; Kon, V.; Breyer, M.D.; Magnuson, M.A.; Fazio, S.; Linton, M.F.
Conditional knockout of macrophage PPARgamma increases atherosclerosis in C57BL/6 and low-density
lipoprotein receptor-deficient mice. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 1647–1653. [CrossRef] [PubMed]
3. Bensinger, S.J.; Bradley, M.N.; Joseph, S.B.; Zelcer, N.; Janssen, E.M.; Hausner, M.A.; Shih, R.; Parks, J.S.;
Edwards, P.A.; Jamieson, B.D.; et al. LXR signaling couples sterol metabolism to proliferation in the acquired
immune response. Cell 2008, 134, 97–111. [CrossRef] [PubMed]
4
4. Hong, C.; Tontonoz, P. Liver X receptors in lipid metabolism: Opportunities for drug discovery. Nat. Rev.
Drug Discov. 2014, 13, 433–444. [CrossRef] [PubMed]
4
5. 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,
1
2097–12102. [CrossRef] [PubMed]
4
6. Joseph, S.B.; Castrillo, A.; Laffitte, B.A.; Mangelsdorf, D.J.; Tontonoz, P. Reciprocal regulation of inflammation
and lipid metabolism by liver X. receptors. Nat. Med. 2003, 9, 213–219. [CrossRef] [PubMed]
4
7. Kappus, M.S.; Murphy, A.J.; Abramowicz, S.; Ntonga, V.; Welch, C.L.; Tall, A.R.; Westerterp, M. Activation of
−
/−
liver X receptor decreases atherosclerosis in Ldlr
mice in the absence of ATP-binding cassette transporters
A1 and G1 in myeloid cells. Arterioscler. Thromb. Vasc. Biol. 2014, 34, 279–284. [CrossRef] [PubMed]
8. Bensinger, S.J.; Tontonoz, P. Integration of metabolism and inflammation by lipid-activated nuclear receptors.
Nature 2008, 454, 470–477. [CrossRef] [PubMed]
4
4
9. Bursill, C.A.; Castro, M.L.; Beattie, D.T.; Nakhla, S.; van der Vorst, E.; Heather, A.K.; Barter, P.J.;
Rye, K.A. High-density lipoproteins suppress chemokines and chemokine receptors in vitro and in vivo
.
Arterioscler. Thromb. Vasc. Biol. 2010, 30, 1773–1778. [CrossRef] [PubMed]

Molecules 2018, 23, 1620
16 of 16
5
0. Sun, R.L.; Huang, C.X.; Bao, J.L.; Jiang, J.Y.; Zhang, B.; Zhou, S.X.; Cai, W.B.; Wang, H.; Wang, J.F.; Zhang, Y.L.
CC-chemokine ligand 2 (CCL2) suppresses high density lipoprotein (HDL) internalization and cholesterol
efflux via CC-chemokine receptor 2 (CCR2) induction and p42/44 mitogen-activated protein kinase (MAPK)
activation in human endothelial cells. J. Biol. Chem. 2016, 291, 19532–19544. [CrossRef] [PubMed]
1. Canfran-Duque, A.; Lin, C.S.; Goedeke, L.; Suarez, Y.; Fernandez-Hernando, C. Micro-RNAs and high-density
lipoprotein metabolism. Arterioscler. Thromb. Vasc. Biol. 2016, 36, 1076–1084. [CrossRef] [PubMed]
5
5
5
5
2. Rottiers, V.; Naar, A.M. MicroRNAs in metabolism and metabolic disorders. Nat. Rev. Mol. Cell. Biol. 2012
3, 239–250. [CrossRef] [PubMed]
3. Ohkatsu, Y.; Satoh, T. Antioxidant and photo-antioxidant activities of chalcone derivatives. J. Jpn. Petrol. Inst.
008, 51, 298–308. [CrossRef]
,
1
2
4. Karki, R.; Thapa, P.; Yoo, H.Y.; Kadayat, T.M.; Park, P.H.; Na, Y.; Lee, E.; Jeon, K.H.; Cho, W.J.; Choi, H.; et al.
Dihydroxylated 2,4,6-triphenyl pyridines: Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity,
and structure-activity relationship study. Eur. J. Med. Chem. 2012, 49, 219–228. [CrossRef] [PubMed]
5. Guo, T.; Jiang, Q.; Yu, L.; Yu, Z. Synthesis of chalcones via domino dehydrochlorination/Pd(OAc)2-catalyzed
Heck reaction. Chin. J. Catal. 2015, 36, 78–85. [CrossRef]
6. Zhou, Y.; Li, Z.; Yang, X.; Chen, X.; Li, M.; Chen, T.; Yin, S.F. Phosphorous acid promoted
hydration–condensation of aromatic alkynes with aldehydes affording chalcones in an oil/water two-phase
system. Synthesis 2016, 48, 231–237. [CrossRef]
5
5
5
5
5
7. Singh, U.P.; Pathak, M.; Dubey, V.; Bhat, H.R.; Gahtori, P.; Singh, R.K. Design, synthesis, antibacterial activity,
and molecular docking studies of novel hybrid 1,3-thiazine-1,3,5-triazine derivatives as potential bacterial
translation inhibitor. Chem. Biol. Drug Des. 2012, 80, 572–583. [CrossRef] [PubMed]
8. Silva, W.A.; Andrade, C.K.Z.; Napolitano, H.B.; Vencato, I.; Lariucci, C.; de Castro, M.R.C.; Camargo, A.J.
Biological and structure-activity evaluation of chalcone derivatives against bacteria and fungi. J. Braz.
Chem. Soc. 2013, 24, 133–144. [CrossRef]
9. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.;
Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time
PCR experiments. Clin. Chem. 2009, 55, 611–622. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2018 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/).