Synthesis and biological evaluation of isoflavone amide derivatives with antihyperlipidemic and preadipocyte antiproliferative activities

Article abstract of DOI:10.1016/j.bmc.2015.06.032

A series of isoflavone amides were designed with isoflavone in place of the scaffold of 2-arylbenzoxazole as cholesterol ester transfer protein (CETP) inhibitors. Twelve new compounds were synthesized, and their inhibitory activities of CETP and preadipocyte proliferation were assayed. The hypolipidemic potency of the most effective compound HY-2c was further tested in vivo by hamster. The results indicate that HY-2c exhibited favorable antihyperlipidemic and preadipocyte antiproliferative activities.

Full text of DOI:10.1016/j.bmc.2015.06.032

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of isoflavone amide derivatives
with antihyperlipidemic and preadipocyte antiproliferative activities
Wenbin Wang a,b, Yi He a,b, Pei Xu a,b, Qidong You a,b, Hong Xiao c, , Hua Xiang a,b,
⇑
⇑
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
Nanjing Brain Hospital Affiliated to Nanjing Medical University, 264 Guangzhou Road, Nanjing 210029, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isoflavone amides were designed with isoflavone in place of the scaffold of 2-arylbenzoxazole
as cholesterol ester transfer protein (CETP) inhibitors. Twelve new compounds were synthesized, and
their inhibitory activities of CETP and preadipocyte proliferation were assayed. The hypolipidemic
potency of the most effective compound HY-2c was further tested in vivo by hamster. The results indicate
that HY-2c exhibited favorable antihyperlipidemic and preadipocyte antiproliferative activities.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 2 March 2015
Revised 6 June 2015
Accepted 8 June 2015
Available online xxxx
Keywords:
CETP
Isoflavone
Hypolipidemic
1
. Introduction
Cardiovascular diseases (CVD’s) are among the major causes of
on novel therapeutic agents aiming to effectively elevating HDL-C
levels are needed.
CETP is a plasma protein that naturally transfers cholesterol
from high-density lipoproteins to very low-density lipoproteins
morbidity and mortality globally today.1 Dyslipidemia is consid-
ered a major risk factor in the progression of cardiovascular dis-
eases. Epidemiological studies have established that both high
low-density-lipoprotein cholesterol (LDL-C) level and low high-
density-lipoprotein cholesterol (HDL-C) level are contributors to
the progression of atherosclerosis and the underlying chronic dis-
6
(VLDLs) or low-density lipoproteins (LDLs). CETP has an additional
role in the uptake of HDL-C by human adipocytes. Thus, inhibition
of CETP may raise the concentration of HDL-C and lower plasma
7
LDL-C levels thereby reduce the risk of CVD.
Obesity can cause various health problems including diabetes,
cardiovascular diseases and dyslipidemia. Increased production
of CETP in obese status could directly result in reduced HDL-C
levels. In obese patients, plasmatic CETP exhibits stronger bioactiv-
2
,3
order of CVD.
Current clinical strategies of lipid regulation for the treatment
of atherosclerosis have primarily focused on lowering circulating
levels of LDL-C via the use of agents such as statins. However,
CVD risk remains for some patients, and low HDL-C level could
8
ity due to its higher concentration. Adipose tissue which secretes
CETP, may be an important source of plasmatic CETP in humans.9
Preadipocytes are the initial formation of adipose depots and pos-
sess the ability to undergo complete differentiation into mature
adipocytes. Thus, the inhibition of preadipocytes proliferation is
4
contribute to the residual risk. HDL promotes reverse cholesterol
transport (RCT), a process by which cholesterol is transferred from
peripheral tissues and cells to the liver for excretion. Thus, elevat-
ing the concentration of HDL-C appears to be an attractive strategy
of reducing atherosclerosis risk for patients with low HDL-C level.
To date, two classes of HDL-C elevating drugs, fibrates and niacins,
are currently used in clinical applications. However, they still go
beyond the clinical requirement with the limited efficacy of
fibrates and the gastric intolerance of niacins.5 Therefore, studies
1
0
an early target to reduce adipose tissue mass. Therefore, inhibi-
tion of preadipocyte proliferation could reduce the activity of
plasma CETP and treat dyslipidemia indirectly. The preadipocyte
proliferation could be reduced by (1) antagonizing the actions of
a gene whose activation is essential for cell growth; (2) limiting
substrate availability for growth; (3) causing growth arrest; or
1
1
(
4) being cytotoxic.
Soy isoflavones, such as genistein, daidzein and formononetin
Fig. 1) which are found in soybean and soybean-derived products,
are a major source of phytoestrogens in human diet. They have a
similar scaffold as estrogen, which enables them to bind estrogen
(
⇑
Corresponding authors. Tel./fax: +86 025 83271096 (H.X.).
E-mail addresses: xiaohong63xx@163.com (H. Xiao), 1020030692@cpu.edu.cn
(
H. Xiang).
http://dx.doi.org/10.1016/j.bmc.2015.06.032
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
W. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
OH
OH
2.2. Biological evaluation
OH
O
O
O
The commercially available CETP Inhibitor Drug Screening Kit
BioVision, USA) was used to assay CETP inhibitory efficacy of
(
HO
HO
O
our compounds according to the manufacturer’s protocol. The
screening kit uses a donor molecule containing a fluorescent self-
quenched neutral lipid (NBD-labeled Cholesterol in HDL) that is
transferred to an acceptor molecule (LDL) in the presence of
CETP (rabbit serum). CETP-mediated transfer of the fluorescent
neutral lipid to the acceptor molecule results in an increase in flu-
orescence (ExEm = 465/535 nm). CETP Inhibitors will inhibit the
lipid transfer and therefore decrease fluorescence intensity.
Anacetrapib, which was developed by Merck and now in phase
III clinical trial, was selected as positive control drug. The assay
results are shown in Figure 2. The result showed that some com-
pounds with an acetyl group at 7-OH of the isoflavone moiety,
including HY-2c, HY-2d and HY-2f, had better activity than the
corresponding unesterified one. While other acetylized compounds
such as HY-2a, HY-2b and HY-2e had no activity. Methyl substitu-
tion at the 2, 3, or 4 positions of ring B would lead to reduction of
Genistein
Daidzein
O
O
O
O
N
O
NH
HO
O
Formononetin
Compound 1
Figure 1. Structures of some isoflavone compounds and compound 1.
receptor (ER), and act as estrogen agonists. It has been shown that
genistein has anti-3T3-L1 preadipocyte proliferation activity.
Studies in humans and rodents support the hypothesis that soy iso-
flavones may be beneficial to the prevention of obesity and dyslipi-
demia.
Compound 1 (Fig. 1) was identified as a CETP inhibitor after
screening using the BODIPY-CE fluorescence assay by Bristol-
Myers Squibb (BMS). Its IC50 value of CETP inhibition in human
whole plasma assay is 10 M. Further efforts to optimize the struc-
tures of 2-arylbenzoxazole class of CETP inhibitors were made
1
2
3
activities. 2-OCH substitution led to a loss of activity, while HY-2c
with no substitution at ring B displayed the best activity. The inhi-
bition rate of HY-2c and HY-2f was over 70% at the concentration
1
3–15
of 100
as 1.52
bition in seven concentration gradients (Fig. 5).
T3-L1 cells were selected to assess the effects of the com-
l
M. Then the IC50 value of the two compounds was obtained
l
M and 8.42 M respectively by testing the activity of inhi-
l
1
6
3
l
pounds in this study on preadipocyte proliferation using the stan-
dard MTT assay. Oleoyl Formononetin, previously reported by our
through the substitution of the benzoxazole moiety and modifica-
tion of the
2
2
-alkoxyamide side chain by Merck.1
7–21
lab, was selected as the positive control. HY-2c and HY-2f exhib-
ited satisfactory inhibition efficacy (Fig. 3). The mechanisms medi-
ating such inhibition are not clear, but this effect is not due to a
cytotoxic activity, since HY-2c was also tested on other cell types
and it was found to have no significant effect on cell viability.
Figure 4 showed that HY-2c had no proliferation inhibition effect
on HepG2 cells.
a
The 2-arylben-
zoxazole moiety of compound 1 shows a structural similarity to
the molecular framework of cholesterol.16 Isoflavone is similar to
1
7-b-estradiol in chemical structure and it is a common parent
nucleus for scaffold hopping of steroids. Thus, isoflavone may
mimic cholesterol better than 2-arylbenzoxazole moiety. In addi-
tion, isoflavones can also inhibit preadipocyte proliferation as
mentioned above. In this paper, by replacing the 2-arylbenzoxazole
with isoflavone scaffold 12 isoflavone amide derivatives were syn-
thesized and they were anticipated to have CETP inhibition and
preadipocyte antiproliferative activities.
We further investigated the hypolipidemic activities of HY-2c
in vivo using male Syrian hamster dyslipidemia model induced
by high lipid diet. The results were presented in Figure 6. After oral
administration of HY-2c for 28 days, LDL-C was reduced by 44.6%
and HDL-C was increased by 66%. This result suggested that HY-
2
c had a good lipid regulating potency.
2
2
. Results and discussion
3
. Conclusions
.1. Chemical synthesis
In order to maintain the hypolipidemic activities, we changed
Synthesis of these isoflavone amide derivatives is outlined in
the 2-arylbenzoxazole scaffold into isoflavone and synthesized 12
isoflavone amide derivatives. Two of them, HY-2c and HY-2f,
embodied remarkable CETP inhibition in vitro and preadipocyte
antiproliferation activities. Furthermore, HY-2c exerted remark-
able lipid regulating activity in vivo.
Scheme 1. The isoflavone scaffold and the side chains were synthe-
sized respectively.
Synthesis of the side chains was performed by a facile two-step
procedure. The phenols (2a–f) reacted with ethyl chloroacetate (3)
to give the corresponding ethers (4a–f). Then the acids (5a–f) were
obtained through the hydrolysis of the ethers. The acyl chloride
intermediates (6a–f) were synthesized by treating 5a–f with thio-
nyl chloride.
4. Experimental
0
As a key intermediate, 7-hydroxy-4 -nitroisoflavone (9) was
4.1. Chemical synthesis
prepared with resorcinol (8) and 4-nitrobenzoic acid (7) as starting
materials by Friedel–Crafts Acylation using boron trifluoride ether-
ate as solvent and catalyst. The intermediate was then directly
4.1.1. Materials
Most chemicals and solvents were of analytical grade and, when
necessary, were purified and dried by standard methods. Reactions
were monitored by thin-layer chromatography (TLC) using pre-
coated silica gel plates (silica gel GF/UV 254), and spots were visu-
alized under UV light (254 nm). Melting points were determined
on a Mel-TEMP II melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded on a Nicolet Impact 410 instru-
treated with Vilsmeier reagent to give the isoflavone scaffold.
0
7
-Hydroxy-4 -aminoisoflavone (10) was prepared from the reduc-
0
tion of 7-hydroxy-4 -nitroisoflavone (9).
Compounds HY-1a–HY-1f were obtained by condensation of
0
acyl chloride intermediates (6a–f) with 7-Hydroxy-4 -aminoisofla-
vone (10). Acetylation of HY-1a–HY-1f with acetyl chloride yielded
HY-2a–HY-2f.
1
ment (KBr pellet). H NMR spectra were recorded with a Bruker
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W. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
a
O
COOCH CH
b
OH
2
3
+
Cl
COOCH CH
R
R
2
3
2a~2f
3
4a~4f
O
COOH
c
O
COCl
R
R
R
H
N
B
5
a~5f
6
a~6f
g
O
O
O
NH2
O
O
A
HO
HY-1a ~ HY-1f
h
1
0
HO
O
f
R
COOH
+
H
N
B
OH
NO2
1
d
O
O
O
O
O
2 e
A
OH
HO
O
9
AcO
NO2
7
HY-2a ~ HY-2f
8
Ra=4-CH
3
Rb=3-CH
3
Rc=-H Rd=
3 3
Re=2-OCH Rf=2-CH
Scheme 1. Reagents and conditions: (a) K
2
CO
3
, KI, DMF, 75 °C, 12 h; (b) 7.2% KOH, EtOH, rt, 1.5 h; (c) SOCl
2
, THF, DMF, 50 °C, 5 h; (d) BF
3
–Et
2
O, 85 °C, 1.5 h; (e) PCl
5
, DMF,
2
5 °C, 2 h; (f) Fe, H
2
O, NH
4
Cl, EtOH, reflux, 4 h; (g) THF, pyridine, 50 °C, 3 h; (h) CH
3
COCl, THF, pyridine, reflux, 2 h.
1
00
*
90
80
70
60
50
40
30
20
10
0
*
1.4
*
1.2
*
*
*
1
0.8
0.6
0
0
.4
.2
0
compound number
Figure 2. CETP inhibition activity of the synthetic compounds (100
lM) with
control
HY-2c
⁄
Anacetrapib (100
l
M) as positive control. P 6 0.05 versus control.
compound number
Figure 4. The MTT assay result of HY-2c (100 lM) on HepG2 cells. Cells were
treated with HY-2c for 48 h. The control group, absence of HY-2c. The HY-2c group
was not significantly different from control (p>0.05).
1
20
00
*
*
1
80
60
40
20
0
(
EI) or a Mariner Mass Spectrum (ESI), or a LC/MSD TOF HR-MS
Spectrum. Chemical shifts (d) were expressed in parts per million
relative to tetramethylsilane, which was used as an internal stan-
dard, coupling constants (J) were in hertz (Hz), and the signals
were designated as follows: s, singlet; d, doublet; t, triplet; m, mul-
tiplet; br s, broad singlet.
*
*
*
4
.1.2. 7-Hydroxy-3-(4-nitrophenyl)-4H-benzopyran-4-one (9)
A mixture of 4-nitrophenylacetic acid (7; 1.0 g, 6.0 mmol),
compound number
Figure 3. The antiproliferation effect of the synthetic compounds (100
preadipocytes by MTT assay. Cells were treated with the synthetic compounds for
l
M) on
resorcinol (8; 0.72 g, 6.5 mmol) and BF
at 85 °C for 1.5 h with stirring, then cooled down to 10 °C and
0 ml DMF was added slowly. In another flask, 10 ml DMF was
added and cooled to 10 °C, PCl (2.0 g, 9 mmol) was added in
3 2
/Et O (10 ml) was heated
4
8 h. Values represent % of the control (the control group, absence of synthetic
1
⁄
compounds). P 6 0.05 versus control group.
5
batches, afterwards the mixture was heated to 55 °C and stirred
for 0.5 h, after that cooled to 10 °C. Then the mixture was added
by droplet into the first flask and stirred at 25 °C for 2 h. Then
Avance 500 MHz spectrometer at 300 K, using TMS as an internal
standard. MS spectra were recorded on a Shimadzu GC–MS 2010
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4
W. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Then the mixture was refluxed for 4 h. The hot solution was fil-
tered, and the filtrate was concentrated under reduced pressure
1
to give 10 (2 g, 0.75 mmol, 75%) as a white solid. H NMR
(
5
DMSO, 300 MHz): d 8.14 (s, 1H, H-2), 8.05 (d, 1H, J = 8.8 Hz, H-
0
0
), 7.46 (d, 2H, J = 8. 8 Hz, H-2 , H-6 ), 6.99 (dd, 1H, J = 8.8, 2.4 Hz,
0
0
H-6), 6.90 (d, 1H, J = 2.4 Hz, H-8), 6.88 (2H, J = 8.8 Hz, H-3 , H-5 );
MS (ESI): m/z = 254 [M+H] .
+
4
.1.4. General procedure for the synthesis of 5a–f
A mixture of phenols (0.3 mmol), acetone (10 ml), dimethylfor-
mamide (10 ml), ethyl chloroacetate (2.5 ml, 0.26 mmol), potas-
sium carbonate (2 g) and potassium iodide (0.15 g) was heated at
7
5 °C for 12 h. Afterwards, the solid was filtered and the solvent
of the filtrate was removed in vacuo. The colorless liquid (4a–f)
obtained was dissolved in ethanol (20 ml). A solution of potassium
hydroxide (7.2%, 5 ml) was added and stirred at room temperature
for 1.5 h. The mixture was poured into hydrochloric acid (1 mol/L)
and a white precipitate was formed. Purification by recrystalliza-
tion from methanol afforded the corresponding aryloxyl acid
(a) HY-2c
(5a–f).
4
.1.4.1. 4-Methyl phenyloxyl acid (5a). Compound (5a) was
1
obtained as
a
white solid. Yield: 80.7%.
H NMR (DMSO,
3
00 MHz): d 7.12 (m, 2H, H-3, H-5), 6.90 (m, 2H, H-2, H-6), 4.62
(
[
s, 2H, –OCH
MꢀH] .
2 3
CO–), 2.22 (s, 3H, Ar-CH ); MS (ESI): m/z = 165
ꢀ
4
.1.4.2. 3-Methyl phenyloxyl acid (5b). Compound (5b) was
obtained as a white solid. Yield: 83%. d 7.17 (m, 1H, H-5), 6.60-
6
.84 (m, 3H, H-2, H-4, H-6), 4.66 (s, 2H, –OCH
2
CO–), 2.28 (s, 3H,
ꢀ
Ar-CH
3
); MS (ESI): m/z = 165 [MꢀH] .
(
b) HY-2f
4
.1.4.3. Phenyloxyl acid (5c). Compound (5c) was obtained as a
Figure 5. CETP inhibition activity of HY-2c and HY-2f in seven concentration
white solid. Yield: 84.6%. d 7.35 (m, 2H, H-3, H-5), 6.84-7.05 (m,
gradients.
3
H, H-2, H-4, H-6), 4.69 (s, 2H, –OCH
2
CO–); MS (ESI): m/z = 151
ꢀ
[
MꢀH] .
5
4
4
3
3
2
2
1
1
00
50
00
50
00
50
00
50
00
4
.1.4.4. 1-Naphthyloxyl phenyloxyl acid (5d). Compound (5d)
white solid. Yield: 76.2%. 7.80–7.91
(m, 3H, H-4, H-5, H-8), 7.49 (m, 1H, H-7), 7.39 (m, 1H, H-6), 7.33
was obtained as
a
d
*
(m, 1H, H-1), 7.28 (m, 1H, H-3), 4.88 (s, 2H, –OCH
(ESI): m/z = 201 [MꢀH] .
2
CO–); MS
+
control
HY-2c
*
4
.1.4.5. 2-Methoxy phenyloxyl acid (5e). Compound (5e) was
obtained as a white solid. Yield: 82.4%. d 6.84–7.02 (m, 4H, Ar-5),
*
4
.70 (s, 2H, –OCH
2
CO–), 3.85 (s, 3H, –OCH
3
); MS (ESI): m/z = 181
ꢀ
[MꢀH] .
50
4
.1.4.6. 2-Methyl phenyloxyl acid (5f). Compound (5f) was
0
obtained as a white solid. Yield: 81.3%. d 7.14 (m, 1H, H-3),
TC
TG
HDL-C
LDL-C
6
.81–6.89 (m, 3H, H-4, H-5, H-6), 4.73 (s, 2H, –OCH
2
CO–), 2.26
Figure 6. The biochemistry analysis of hamster treated with HY-2c for 4 weeks.
ꢀ
(
s, 3H, Ar-CH
3
); MS (ESI): m/z = 165 [MꢀH] .
(
ig). Control: high-fat diet-fed group untreated with HY-2c; HY-2c: high-fat diet-
⁄
fed hamster treated with HY-2c (50 mg/kg/d). P 6 0.05, HY-2c group versus
4
.1.5. General procedure for the synthesis of HY-1a–HY-1f
control.
A mixture of aryloxyl acid (5a–f) (10 mmol), thionyl chloride
the reaction mixture was poured into heated dilute hydrochloric
acid (0.5 mol/L). The product was filtered, and purified by recrys-
tallization from methanol to yield 9 (0.36 g, 1.26 mmol, 21%) as a
(15 mmol), tetrahydrofuran (50 ml) and dimethylformamide
(1 ml) was stirred at 50 °C for 5 h. Then tetrahydrofuran and thio-
nyl chloride were removed in vacuo. Yellow liquid (6a–f) obtained
and pyridine (2.5 ml) were then added to a solution of 10 (10 mmol
in 25 ml tetrahydrofuran). The solution was stirred at 50 °C for 3 h.
When the reaction was completed, the solution was poured into
1
white solid. H NMR (DMSO, 300 MHz): d 8.48 (s, 1H, H-2), 8.28
0
0
(
2
(
2H, J = 8.8 Hz, H-3 , H-5 ), 8.07 (d, 1H, J = 8.8 Hz, H-5), 7.96 (d,
0
0
H, J = 8. 8 Hz, H-2 , H-6 ), 7.05 (dd, 1H, J = 8.8, 2.4 Hz, H-6), 6.97
d, 1H, J = 2.4 Hz, H-8); MS (ESI): m/z = 282 [MꢀH] .
ꢀ
hydrochloric acid (1 mol/L) and extracted with CH Cl2
2
(
20 ml ꢁ 3). The combined organic phase was washed with brine
4
.1.3. 7-Hydroxy-3-(4-aminophenyl)-4H-benzopyran-4-one (10)
To a solution of 9 (3 g, 10 mmol) in 30 ml ethanol, iron powder
(20 ml ꢁ 3), dried over Na SO , concentrated to afford yellow solid.
2
4
Purification by silica gel column chromatography (PE:EA = 15:1)
(
6 g, 100 mmol) and NH
4
Cl (1 g in 3 ml water) solution were added.
yielded the isoflavone amide derivatives HY-1a–HY-1f.
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W. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
4
.1.5.1. 7-Hydroxy-3-[4-(4-methylphenyloxyacethy)phenyl]-4H-
into aqueous HCl (1 mmol/L) to give the white solid. The mixture
was filtered, dried and the product was obtained.
benzopyran-4-one (HY-1a). Compound HY-1a was obtained as a
white solid. Yield: 56.8%. Mp 170–172 °C; IR (cm ): 3382, 3127,
ꢀ1
1
2
(
1
365, 2324, 1655, 1588, 1509, 1400, 1260, 1236; H NMR
DMSO, 300 MHz): d 10.80 (s, 1H, NH), 10.11 (s, 1H, OH), 8.37 (s,
H, H-2), 7.96 (d, 1H, J = 8.91 Hz, H-5), 7.56 (m, 4H, Ar-H), 7.12
4.1.6.1. 7-Acetoxy-3-[4-(4-methylphenyloxyacethy)phenyl]-4H-
benzopyran-4-one (HY-2a). Compound HY-2a was obtained as a
ꢀ
1
white solid. Yield: 85%. Mp 165–167 °C; IR (cm ): 3119, 2909,
0
0
(
d, 2H, J = 8.58 Hz, H-2 , H-6 ), 6.90 (m, 4H, Ar-H), 4.62 (s, 2H, –
OCH CO–), 2.22 (s, 3H, Ar-CH ); HRMS (EI) calcd for C24
02.1336, found 402.1330.
2585, 1738, 1608, 1593, 1510, 1430, 1400, 1379, 1339, 1302,
1
2
3
H19NO
5
1254; H NMR (DMSO, 300 MHz): d 8.37 (s, 1H, H-2), 7.96 (d, 1H,
0
4
J = 8.91 Hz, H-5), 7.56 (m, 4H, Ar-H), 7.12 (d, 2H, J = 8.58 Hz, H-2 ,
0
H-6 ), 6.90 (m, 4H, Ar-H), 4.62 (s, 2H, –OCH
2
CO–), 3.43 (s, 3H, –
4
.1.5.2. 7-Hydroxy-3-[4-(3-methylphenyloxyacethy)phenyl]-4H-
COCH
3
), 2.22 (s, 3H, Ar-CH
3
–); HRMS (EI) calcd for C24H19NO
5
benzopyran-4-one (HY-1b). Compound HY-1b was obtained as a
444.1442, found 444.1441.
ꢀ1
white solid. Yield: 37.5%. Mp 168–170 °C; IR (cm ): 3394, 3100,
2
3
2
363, 2326, 1686, 1587, 1522, 1265, 1171; 1H NMR (DMSO,
00 MHz): d 10.79 (s, 1H, NH), 10.12 (s, 1H, OH), 8.38 (s, 1H, H-
),7.97 (d, 1H, J = 8.91 Hz, H-5), 7.57 (m, 4H, Ar-H), 6.96 (t, 2H,
4.1.6.2. 7-Acetoxy-3-[4-(3-methylphenyloxyacethy)phenyl]-4H-
benzopyran-4-one (HY-2b). Compound HY-2b was obtained as a
white solid. Yield: 87.2%. Mp 160–163 °C; IR (cm ): 3391, 3126,
1767, 1682, 1635, 1618, 1533, 1441, 1221, 1178; H NMR
(DMSO, 300 MHz): d 10.14 (s, 1H, NH) 8.54 (s, 1H, H-2), 8.18 (d,
ꢀ1
0
0
1
J = 8.48 Hz, H-2 , H-6 ), 6.85 (m, 4H, Ar-H), 4.68 (s, 2H, –OCH
, 2.28 (s, 3H, Ar-CH ); HRMS (EI) calcd for C24 402.1336,
found 402.1336.
2
CO–
)
3
H19NO
5
0
0
1H, J = 8.91 Hz, H-5), 7.59 (m, 5H, Ar-H), 7.56 (m, 2H, H-2 , H-6 ),
6
.90 (m, 4H, Ar-H), 4.69 (s, 2H, –OCH
2
CO–), 2.29 (s, 3H, –COCH
3
),
2
.26 (s, 3H, Ar-CH -); HRMS (EI) calcd for C24H19NO
3
5
444.1442,
4
.1.5.3. 7-Hydroxy-3-(4-phenyloxyacethyphenyl)-4H-benzopy-
ran-4-one (HY-1c). Compound HY-1c was obtained as a white
found 444.1450.
ꢀ1
solid. Yield: 46.8%. Mp 166–168 °C; IR (cm ): 3125, 2578, 2365,
1
4.1.6.3. 7-Acetoxy-3-(4-phenyloxyacethyphenyl)-4H-benzopy-
ran-4-one (HY-2c). Compound HY-2c was obtained as a white
solid. Yield: 86.3%. Mp 159–160 °C; IR (cm ): 3131, 2359, 2330,
1783, 1639, 1618, 1513, 1440, 1399, 1272, 1248, 1234, 1151; 1
NMR (CDCl 300 MHz): 8.43 (s, 1H, H-2), 8.35 (m, 1H,
J = 8.91 Hz, H-5), 7.71 (m, 1H, Ar-H), 7.61 (m, 4H, Ar-H), 7.23 (d,
2
348, 1734, 1705, 1670, 1585, 1497, 1437, 836, 756, 685;
NMR (CDCl , 300 MHz): d 10.85 (s, 1H, NH), 10.22 (s, 1H, OH),
.43 (s, 1H, H-2),8.34 (d, 1H, J = 8.81 Hz, H-5), 7.57 (m, 4H, Ar-H),
H
3
ꢀ1
8
6
2
3
0
0
H
.96 (t, 2H, J = 8.48 Hz, H-2 , H-6 ), 6.85 (m, 4H, Ar-H), 4.68 (s,
3
,
d
H, –OCH
2 5
CO–); HRMS (EI) calcd for C24H19NO 388.1179, found
88.1176.
0
0
3
H, J = 8.58 Hz, H-2 , H-6 ,Ar-H), 6.88 (m, 3H, Ar-H), 4.65 (s, 2H, –
OCH CO–), 2.38 (s, 3H, –COCH ); HRMS (EI) calcd for C24
30.1285, found 430.1286.
2
3
H19NO
5
4
.1.5.4. 7-Hydroxy-3-(4-naphthyloxyacethyphenyl)-4H-benzopy
4
ran-4-one (HY-1d). Compound HY-1d was obtained as a white
solid. Yield: 50.8%. Mp 166–168 °C;IR (cm ): 3403, 3133, 2353,
ꢀ1
1
4.1.6.4. 7-Acetoxy-3-(4-naphthyloxyacethyphenyl)-4H-benzop
yran-4-one (HY-2d). Compound HY-2d was obtained as a white
solid. Yield: 88.7%. Mp 149–151 °C; IR (cm ): 3125, 2571, 1728,
2
342, 1682, 1622, 1577, 1533, 1508, 1398, 1266, 1240, 1108;
NMR (CDCl , 300 MHz): d 10.80 (s, 1H, NH), 10.30 (s, 1H, OH),
.34 (m, 2H, H-2 Ar-H),7.88 (m, 2H, H-5 Ar-H), 7.64 (m, 8H, Ar-
H
3
ꢀ1
8
1
0
0
1601, 1399, 1266, 1224, 1155, 1100, 817, 521; H NMR (DMSO,
H), 6.95 (d, 2H, J = 8.28 Hz, H-2 , H-6 ), 6.88 (s, 1H, Ar-H), 4.90 (s,
3
00 MHz): d 10.34 (s, 1H, NH), 8.54 (s, 1H, H-2), 8.17 (d, 1H,
2
H, –OCH
2 5
CO–); HRMS (EI) calcd for C24H19NO 438.1336, found
0
0
J = 8.34 Hz, H-5), 7.77 (d, 2H, Ar-H), 7.53 (m, 9H, H-2 , H-6 , Ar-
H), 6.95 (m, 1H, Ar-H), 4.62 (s, 2H, –OCH CO–), 2.34 (s, 3H, –
COCH ); HRMS (EI) calcd for C24 480.1436, found 480.1440.
4
38.1332.
2
3
H19NO
5
4
4
.1.5.5. 7-Hydroxy-3-[4-(2-methyloxyphenyloxyacethy)phenyl]-
H-benzopyran-4-one (HY-1e). Compound HY-1e was obtained
ꢀ
1
4.1.6.5. 7-Acetoxy-3-[4-(2-methyloxyphenyloxyacethy)phenyl]-
4H-benzopyran-4-one (HY-2e). Compound HY-2e was obtained
as a white solid. Yield: 84.4%. Mp 164–167 °C; IR (cm ): 3132,
1
as a white solid. Yield: 58.3%. Mp 168–170 °C; IR (cm ): 3385,
1
3
133, 2355, 2334, 1697, 1532, 1504, 1400, 1263, 1219, 1126;
NMR (DMSO, 300 MHz): 9.09 (s, 1H, OH), 8.19 (d, 1H,
J = 8.91 Hz, H-5), 7.95 (s, 1H, H-2), 7.63 (m, 4H, Ar-H), 7.02 (m,
H
ꢀ1
d
1
628, 1601, 1548, 1506, 1262; H NMR (DMSO, 300 MHz): d
0
0
10.20 (s, 1H, NH), 8.36 (s, 1H, H-2), 7.97 (d, 1H, J = 8.81 Hz, H-5),
5
H, J = 8.48 Hz, H-2 , H-6 ), 6.86 (s, 1H, Ar-H), 4.70 (s, 2H, –
OCH CO–), 3.97 (s, 3H, Ar-OCH ); HRMS (EI) calcd for C24
18.1285, found 418.1298.
0
0
7
.60 (m, 4H, Ar-H), 6.95 (m, 6H, Ar-H, H-2 , H-6 ), 4.70 (s, 2H, –
OCH CO–), 3.81 (s, 3H, -OCH ), 2.22 (s, 3H, COCH ); HRMS (EI)
calcd for C24 460.1391, found 460.1397.
2
3
H19NO
5
2
3
3
4
H19NO
5
4
.1.5.6. 7-Hydroxy-3-[4-(2-methylphenyloxyacethy)phenyl]-4H-
4
.1.6.6. 7-Acetoxy-3-[4-(2-methylphenyloxyacethy)phenyl]-4H-
benzopyran-4-one (HY-1f). Compound HY-1f was obtained as a
white solid. Yield: 40%. Mp 168–170 °C; IR (cm ): 3397, 3126,
benzopyran-4-one (HY-2f). Compound HY-2f was obtained as a
ꢀ1
ꢀ1
white solid. Yield: 85.8%. Mp 169–160 °C; IR (cm ): 3126, 2579,
1
2
355, 2334, 1623, 1590, 1529, 1493, 1455, 1400, 1243, 1183;
NMR (DMSO, 300 MHz): d 10.80 (s, 1H, NH), 10.13 (s, 1H, OH),
.38 (s, 1H, H-2), 7.96 (d, 1H, J = 8.91 Hz, H-5), 7.60 (m, 4H, Ar-
H
481, 1739, 1704, 1637, 1602, 1497, 1458, 1423, 1268, 758; 1
2
H
NMR (DMSO, 300 MHz): d 10.16 (s, 1H, NH), 8.55 (s, 1H, H-2),
8
0
8
1
2
C
.18 (d, 1H, J = 8.73 Hz, H-5), 7.59 (m, 5H, Ar-H, H-2 ), 7.12 (d,
H, J = 8.58 Hz, H-6 ), 6.95 (m, 4H, Ar-H), 4.74 (s, 2H, –OCH
.34 (s, 3H, –COCH
444.1442, found 444.1439.
0
0
H), 6.16 (t, 2H, J = 8.48 Hz, H-2 , H-6 ), 6.89 (m, 4H, Ar-H), 4.73 (s,
0
2
CO–),
2
H, –OCH
2
3
CO–), 2.26 (s, 3H, Ar-CH ); HRMS (EI) calcd for
), 2.26 (s, 3H, Ar-CH ); HRMS (EI) calcd for
3 3
C
H19NO 402.1336, found 402.1342.
24 5
24 5
H19NO
4
.1.6. General procedure for the synthesis of HY-2a–HY-2f
4.2. 3T3-L1 cell and HepG2 cells proliferation assay by MTT
To a solution of HY-1a–HY-1f (1 mmol in 10 ml tetrahydrofu-
ran), pyridine (0.3 ml) and acetyl chloride (0.3 ml) were added
and stirred at reflux for 2 h. Then the reaction mixture was poured
3T3-L1 preadipocytes and HepG2 cells (5000 cells/well) were
seeded in 96-well plates. After two days’ conventional culture,
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.032

6
W. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
the cells were incubated with Dulbecco’s Modified Eagle’s medium
DMEM, Gibco) containing the selected compounds (100 mol/L)
Supplementary data
(
l
for 48 h. The culture solution containing 0.1% (V/V) ethanol (96%)
was given to the control group. Twenty microliters of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
solution (5 mg/mL) was placed in each well for 4 h at 37 °C.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.032.
References and notes
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1
.
Jorgensen, T.; Capewell, S.; Prescott, E.; Allender, S.; Sans, S.; Zdrojewski, T.; De
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to extract the MTT formazan, and followed by agitation on a plate
shaker for 15 min. The optical density (OD) was then measured on
the multi-well enzyme-linked immunosorbent assay automatic
spectrometer reader at 540 nm. The inhibition ratio was calculated
through formula 1:
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4
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Inhibition ratio ¼ ð1 ꢀ ODexperimental group=ODblank groupÞ ꢁ 100%
ð1Þ
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4
.3. CETP inhibitory assay
3
l
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was as background and that contains rabbit serum but no testing
compounds as the positive control. Donor molecule (10 l), accep-
tor molecule (10 l) and CETP assay buffer (20 l) were mixed and
(
100
l
l
2
1
39, 369.
1
1
1
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using a fluorometer (ExEm = 465/535 nm). The inhibition ratio
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4
.4. In vivo study in hamsters
Male Syrian golden hamsters (4 weeks old; Vital River
1
7. Smith, C. J.; Ali, A.; Chen, L.; Hammond, M. L.; Anderson, M. S.; Chen, Y.;
Eveland, S. S.; Guo, Q.; Hyland, S. A.; Milot, D. P.; Sparrow, C. P.; Wright, S. D.;
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cated to control and treated groups (n = 5). Hamsters were placed
on a high-fat diet for a month, blood samples were drawn from
orbit. After the establishment of the hyperlipidemia model, com-
pound HY-2c was suspended in olive oil and administered at
18. Hunt, J. A.; Gonzalez, S.; Kallashi, F.; Hammond, M. L.; Pivnichny, J. V.; Tong, X.;
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1019.
5
0 mg/kg/day for 28 days to the treated group, and vehicle to the
19. Kallashi, F.; Kim, D.; Kowalchick, J.; Park, Y. J.; Hunt, J. A.; Ali, A.; Smith, C. J.;
Hammond, M. L.; Pivnichny, J. V.; Tong, X.; Xu, S. S.; Anderson, M. S.; Chen, Y.;
Eveland, S. S.; Guo, Q.; Hyland, S. A.; Milot, D. P.; Cumiskey, A. M.; Latham, M.;
Peterson, L. B.; Rosa, R.; Sparrow, C. P.; Wright, S. D.; Sinclair, P. J. Bioorg. Med.
Chem. Lett. 2011, 21, 558.
control group. The general condition was observed daily and body
weight was measured. Again blood samples were drawn from orbit
and the serum lipid levels were measured.
2
0. Sweis, R. F.; Hunt, J. A.; Kallashi, F.; Hammond, M. L.; Chen, Y.; Eveland, S. S.;
Guo, Q.; Hyland, S. A.; Milot, D. P.; Cumiskey, A. M.; Latham, M.; Rosa, R.;
Peterson, L.; Sparrow, C. P.; Wright, S. D.; Anderson, M. S.; Sinclair, P. J. Bioorg.
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Acknowledgments
2
1. Sweis, R. F.; Hunt, J. A.; Sinclair, P. J.; Chen, Y.; Eveland, S. S.; Guo, Q.; Hyland, S.
A.; Milot, D. P.; Cumiskey, A. M.; Latham, M.; Rosa, R.; Peterson, L.; Sparrow, C.
P.; Anderson, M. S. Bioorg. Med. Chem. Lett. 2011, 21, 2597.
This work was supported by grants from the Twelfth Five-Year
Plan Major Project of Candidate Drugs (Ministry of National
Science and Technology, 2012ZX09103101041) and the
Fundamental Research Funds for the Central Universities.
22. Xiang, H.; Zhao, W.; Xiao, H.; Qian, L.; Yao, Y.; Li, X. B.; Liao, Q. J. Bioorg. Med.
Chem. 2010, 18, 3036.
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.032