Syntheses of diacyltanshinol derivatives and their suppressive effects on macrophage foam cell formation by reducing oxidized LDL uptake

Article abstract of DOI:10.1016/j.bioorg.2013.11.001

A series of diacyltanshinol derivatives were synthesized by esterifying the corresponding o-hydroquinones of tanshinones. The suppressive effects of the synthesized compounds on oxidized low-density lipoprotein (oxLDL) uptake and oxLDL-induced macrophage-derived foam cell formation were evaluated. Our results indicated that the nicotinate derivatives 1a and 2a, modified from tanshinone IIA and cryptotanshinone, showed stronger suppressive activity on oxLDL uptake and the resultant foam cell formation relative to tanshinone IIA. Western Blot analysis indicated that derivatives 1a and 2a could dose-dependently inhibit the expression of oxLDL-induced LOX-1, implying that the suppressive effects of 1a and 2a on oxLDL uptake and foam cell formation could be at least partially attributed to the inhibition of LOX-1 expression in macrophages.

Full text of DOI:10.1016/j.bioorg.2013.11.001

Bioorganic Chemistry 52 (2014) 24–30
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Syntheses of diacyltanshinol derivatives and their suppressive effects
on macrophage foam cell formation by reducing oxidized LDL uptake
Xi Cheng a,1, Da-Li Zhang a,1, Xiao-Bing Li a, Jian-Tao Ye a, Lei Shi b, Zhi-Shu Huang a,
Lian-Quan Gu a, Lin-Kun An a,
⇑
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b Department of Pharmacy, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 August 2013
Available online 15 November 2013
A series of diacyltanshinol derivatives were synthesized by esterifying the corresponding o-hydroqui-
nones of tanshinones. The suppressive effects of the synthesized compounds on oxidized low-density
lipoprotein (oxLDL) uptake and oxLDL-induced macrophage-derived foam cell formation were evaluated.
Our results indicated that the nicotinate derivatives 1a and 2a, modified from tanshinone IIA and cryp-
totanshinone, showed stronger suppressive activity on oxLDL uptake and the resultant foam cell forma-
tion relative to tanshinone IIA. Western Blot analysis indicated that derivatives 1a and 2a could dose-
dependently inhibit the expression of oxLDL-induced LOX-1, implying that the suppressive effects of
1a and 2a on oxLDL uptake and foam cell formation could be at least partially attributed to the inhibition
of LOX-1 expression in macrophages.
Keywords:
Atherosclerosis
Diacyltanshinol derivative
Foam cell formation
Lipid uptake
Tanshinone
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
Asian countries for the prevention and management of cardiovas-
cular diseases including atherosclerosis [8]. Tanshinone IIA is one
Atherosclerosis, the leading cause of morbidity and mortality
[1], is a systemic cardiovascular disease characterized by the accu-
mulation of lipids, extracellular matrix, and various inflammatory
cells in the subendothelium, resulting in the narrowing of the ves-
sel lumen [2]. In the early stages of atherosclerosis, the uptake of
modified low-density lipoprotein (LDL), such as oxidized LDL
(oxLDL), by scavenger receptors (SRs) causes the lipoproteins accu-
mulation in macrophages, resulting in the formation of foam cell
[3–5]. It has been suggested that the down-regulation of SRs
expression and activity may inhibit the macrophage-derived foam
cell formation, and plays an important role in the prevention and
treatment of atherogenesis [6,7].
of the major tanshinones that have been intensively investigated
in recent years. It was recently reported that tanshinone IIA could
down-regulate the expression of SR lectin-like oxLDL receptor-1
(LOX-1) at both the mRNA and protein level [9], and thereby sup-
press oxLDL uptake and prevent atherogenesis in several animal
models [10–12]. However, the efficacy of tanshinone IIA is limited
by its present poor oral bioavailability due to the relatively high
lipophilic properties (the absolute oral bioavailability of tanshi-
none IIA is only about 3% in rats) [8]. To improve the bioavailability
of tanshinones, the chemical modification of tanshinones has at-
tracted much attention in the past decade. Tian et al. found that
the acetylated product of the corresponding o-hydroquinone of
tanshinone IIA showed improved aqueous solubility and activity
[13]. However, in our screening study, the acetylated product
showed lower suppressive activity against oxLDL uptake than the
parent compound tanshinone IIA.
Tanshinones are major lipophilic pharmacologically active com-
pounds isolated from the Salvia miltiorrhiza Bunge, a well-known
traditional Chinese medicinal herb that has been widely used in
Nicotinic acid is clinically used to regulate lipid metabolism for
more than 50 years [14]. Nicotinic acid can decrease LDL-choles-
terol, total cholesterol and lipoprotein levels, and increase HDL-
cholesterol in plasma [14–18]. We designed to acylate the o-hydro-
quinones of tanshinones with nicotinic anhydride derivatives to
obtain the potential protective agents against atherosclerosis. Here,
we report the syntheses of the acylated derivatives, named as diacyl-
tanshinol derivatives, and their suppressive effects on oxLDL uptake
in macrophages and formation of macrophage-derived foam cells.
Abbreviations: LDL, low-density lipoprotein; oxLDL, oxidized low-density
lipoprotein; HDL, high-density lipoprotein; SRs, scavenger receptors; LOX-1,
lectin-like oxidized low-density lipoprotein receptor 1; BSA, Bovine Serum Albu-
min; DiI, l,l0-dioctadecyl-3,3,30,30-tetramethyl-indocarbocyanine perchlorate; DAPI,
40,6-diamidino-2-phenylindole; DMAP, 4-dimethylaminopyridine.
⇑
Corresponding author. Fax: +86 20 39943052.
E-mail address: lssalk@mail.sysu.edu.cn (L.-K. An).
1
These authors contributed equally to this paper.
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.11.001

X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
25
suppressive effect. The derivatives from tanshinone IIA generally
showed more potency than that from cryptotanshinone, as well as
the substituent of fluorine atom on the pyridinyl ring did not im-
prove their potency. Among them, compounds 1a and 2a showed
stronger activity. To confirm their suppressive effects, the effects
in various concentrations of compounds 1a and 2a were studied.
As shown in Fig. 1, lipid accumulation in RAW264.7 macrophages
was observed and the cells displayed typical foamy appearances
after incubation with oxLDL for 24 h. After treatment with com-
pounds 1, 1a and 2a for 12 h, lipid accumulation and foam cell for-
mation was attenuated dose-dependently.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, Tanshinone IIA (1) was Pd/C catalytically
hydrogenated into the corresponding o-hydroquinone intermedi-
ate, which was subsequently acylated with excess nicotinic anhy-
dride to give the colorless solid 1a in 70%. HRMS indicated that
compound 1a had a formula C31H26N2O5. In the 1H NMR spectrum,
the three signals at 8.17 (d) ppm, 7.63 (d) ppm and 7.52 (q) ppm
and the six signals between 3.6 ppm and 1.36 ppm indicated the
presence of tanshinone IIA skeleton. 13C NMR spectrum and the
colorless appearance implied the disappearance of o-quinone
structure. 1H NMR and 13C NMR spectra indicated that compound
1a contained two nicotinoyl groups. All spectra of compound 1a
proved its 1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]
furan-10,11-diyl dinicotinate structure.
To assess the effect of pyridinyl fragment and tanshinone scaf-
fold on the activity, nicotinic anhydride analogues and tanshinone
analogues, including tanshinone IIA, cryptotanshinone (2) and
tanshinone I (3), were used as materials to give the target
compounds 1a–d, 2a–d and 3a (Scheme 1), respectively. Since
compound 3 and 3a showed high cytotoxicity against macrophage
cells, only one compound, 3a, derived from tanshinone I was
synthesized. All diacyltanshinol derivatives were characterized by
HRMS, 1H NMR and 13C NMR spectra.
To further confirm and quantify the effects of compounds on lipid
accumulation and foam cell formation, DiI-oxLDL uptake assays of
compounds 1a and 2a were conducted. The cells were pretreated
with compounds followed by incubation with DiI-oxLDL
(20 lg/ml) for 6 h. Cellular uptake of DiI-oxLDL was detected with
confocal microscopy, and quantified by flow cytometry. As shown
in Fig. 2, pretreatment with compounds dose-dependently inhibited
the uptake of DiI-oxLDL in RAW264.7 macrophages, and their
suppressive effects were significantly higher than that of compound
1
at 10
lM concentration. Compounds 1a and 2a (10 lM)
suppressed DiI-oxLDL uptake by 91% and 87%, respectively.
LOX-1 is one of the crucial types of SRs involved in the uptake of
modified LDL into macrophages [19]. Furthermore, the up regula-
tion of LOX-1 has been demonstrated to be closely associated with
the macrophage-derived foam cell formation and subsequent
development of atherosclerosis. Fig. 3 showed that, similar to
compound 1 [9], compounds 1a and 2a could markedly reduce
oxLDL-induced LOX-1 protein expression dose-dependently.
Although their suppressive effects on LOX-1 protein expression
were similar to that of tanshinone IIA, compounds 1a and 2a
showed significantly stronger suppressive activity than tanshinone
IIA in oxLDL uptake, implying that their suppression effects on
LOX-1 expression might partially contribute to the decrease in
oxLDL uptake.
2.2. Biological activities
The diacyltanshinol derivatives at 5 lM concentration were
pre-screened for their suppressive activity on oxLDL-induced mac-
rophage-derived foam cell formation by the oil red O staining assay
(Fig. S1, Supporting Information). The results indicated that the lipid
accumulationwas attenuated by pretreatment with compounds 1a–
1e. Although cryptotanshinone (2) showed no effects, its derivatives
2a–2d could suppress lipid accumulation to some content, implying
that it might be the diacyltanshinol pharmacophore to exert the
To evaluate the cytotoxicity of diacyltanshinol derivatives and
their protection on the oxLDL-induced cytotoxicity in RAW264.7
Scheme 1. The syntheses of diacyltanshinol derivatives.

26
X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
Fig. 1. Effects on oxLDL-induced lipid accumulation and foam cell formation in RAW264.7 macrophages. The macrophages were pre-incubated with indicated compounds for
12 h, followed with the treatment of oxLDL (50 g/ml) for 24 h. The lipid laden macrophages (foam cells) were stained with oil red O. Blank: normal cells (without compound
l
and oxLDL treatment). Control: foam cells (only with oxLDL treatment).
macrophages, MTT assay was performed. The results indicated that
LCMS-IT-TOF mass spectrometer. The purities of the tested com-
pounds were more than 95%, analyzed by High Performance Liquid
Chromatography (HPLC) on SHIMADZU LC-20A equipped with a
the IC50 values of the compounds 1a–d and 2a–d (6.25–50
were more than 50 M after incubation for 48 h. At the concentra-
tion of 10 M, compounds 1, 1a and 2a showed low cytotoxicity
lM)
l
l
reverse-phase Waters Sunfire C18 column, 5
lm, 4.6 ꢀ 250 mm.
with the percentage of viable cells >84% by pretreatment for 12 h
(Fig. 4). The oxLDL could significantly reduce the viability of
RAW264.7 macrophages by 70%. However, compounds 1, 1a and
2a could increase the macrophage cell viability up to 55%, 43%
and 60%, respectively. This result indicated that compounds 1, 1a
and 2a had protective effect against oxLDL-induced cytotoxicity
in RAW264.7 macrophages.
4.1.1. Synthesis of nicotinic anhydride derivatives
The anhydride derivatives were prepared according to the
reported method with slight modification [21]. To a 500 ml
three-necked round-bottomed flask, nicotinic acid derivative
(0.081 mol) and new distilled toluene (275 ml) were added. To
remove traces of moisture introduced with the nicotinic acid
derivative, the mixture was heated until about 75 ml of toluene
had distilled. The mixture was cooled to 0–5 °C with an ice-water
bath, and added with new distilled triethylamine (8.65 g,
0.086 mol). To the resulting clear solution, a solution of thionyl
chloride (0.043 mol) in toluene (200 ml) was added dropwise over
a period to make sure the temperature of the reaction mixture was
lower than 7 °C. White precipitate appeared along with the addi-
tion. After the addition of thionyl chloride solution, the resulting
suspension was stirred at room temperature for 45 min, and then
heated to about 60 °C. The precipitate was removed by filtration
under slightly reduced pressure while hot. The precipitate was
washed with warm toluene (60 °C, 25 ml ꢀ 3). The combined tolu-
ene solution was concentrated by reduced pressure. The resulting
residue was simmered with new distilled warm toluene (75 ml).
The precipitate was removed with filtration. The filtrate was al-
lowed to stand for 3 h at room temperature. The white crystalline
product was collected with filtration and washed with cold new
distilled toluene (4 ml ꢀ 2). The dry anhydride product was used
immediately.
3. Conclusion
In summary, we synthesized a series of diacyltanshinol deriva-
tives, which potently suppress oxLDL uptake and oxLDL-induced
macrophage-derived foam cell formation to some content. Among
these compounds, 1a and 2a showed higher efficacy than tanshi-
none IIA. Our experiments also revealed that their effects might
be partially attributed to the inhibition of LOX-1 expression in
macrophages, underscoring their potential protective effects
against atherosclerosis. Pharmacological evaluation of the anti-
atherosclerotic potential and underlying mechanisms of these
compounds are underway in our laboratory.
4. Experimental section
4.1. Chemistry
All solvents were obtained from commercial suppliers and used
without further purification unless mentioned. Niacin analogues
were purchased from Aladdin Industrial Inc. Pd/C (10%) was pur-
chased from Acros Organics. Tanshinone IIA (1), Cryptotanshinone
(2) and Tanshinone I (3) were isolated from the Chinese medicinal
herb, S. miltiorrhiza Bunge by our lab [20]. Their purities analyzed
by HPLC are 95% (1), 96% (2) and 96% (3), respectively. Silica gel col-
umn chromatography was carried out with silica gel (200–
300 mesh). Melting points were measured in open capillary tubes
on a SRS-OptiMelt automated melting point instrument without
correction. Nuclear magnetic resonance spectra were recorded on
Bruker AVANCE III 400 MHz spectrometer using tetramethylsilane
as the internal standard. Mass spectra were analyzed on an Agilent
LC-MS 6120 (Quadrupole LC-MS) mass spectrometer. High-resolu-
4.1.2. General syntheses of diacyltanshinol derivatives
To a solution of tanshinone (0.60 g) in ethyl acetate (150 ml),
palladium charcoal (10%, 0.12 g) was added. The mixture was stir-
red at room temperature for 10 min in a hydrogen atmosphere.
And then, a solution of excess anhydride (5 equivalents) and DMAP
(0.80 mg, 6.6 mmol) in DMF (20 ml) was dropwise added into the
reaction solution. The mixture solution was stirred and refluxed
for 4 h. The reaction solution was filtered. The filtrate was washed
with water (50 ml ꢀ 4), saturated aqueous NaHCO3 (50 ml) and
water (50 ml), respectively, and dried over anhydride Na2SO4. Sol-
vent was evaporated under vacuum. The residue was purified by
silica gel column chromatography using ethyl acetate/petroleum
ether (1:1–1:9) mixture as eluent.
tion mass spectra (HRMS) were analyzed on
a SHIMADZU

X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
27
Fig. 2. (A) Effects of compounds 1, 1a and 2a on DiI-oxLDL uptake by RAW264.7 macrophages. The cells were serum starved for 12 h and pre-incubated with indicated
compounds for 12 h, and then treated with DiI-oxLDL for additional 6 h. Subsequently, cells were fixed and analyzed by confocal microscopy. Blank: cells incubated without
DiI-oxLDL. Control: cells incubated with DiI-oxLDL and vehicle (0.1% DMSO). (B) Quantitation of DiI-oxLDL uptake by flow cytometry analysis from panel A.
4.1.2.1. 1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,
11-diyl dinicotinate (1a). Colorless solid, 0.72 g, 70% yield.
Mp = 189.6–193.0 °C. 1H NMR (400 MHz, CDCl3) d 9.34 (s, 1H),
9.27 (s, 1H), 8.78–8.75 (m, 2H), 8.40 (d, J = 7.6 Hz, 1H), 8.33 (d,
J = 8.0 Hz, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.52
(q, J = 1.2 Hz, 1H), 7.39 (dd, J = 7.6, 4.8 Hz, 1H), 7.34 (dd, J = 8.0,
4.8 Hz, 1H), 3.59–3.50 (m, 1H), 2.95–2.80 (m, 1H), 2.18 (d,
J = 1.2 Hz, 3H), 1.75–1.70 (m, 2H), 1.66–1.60 (m, 2H), 1.36 (s, 6H).
13C NMR (100 MHz, CDCl3) d 163.7, 163.3, 154.2, 154.1, 151.2,
151.1, 150.6, 144.6, 141.9, 137.7, 137.5, 134.8, 134.6, 131.5, 126.8,
125.0, 124.4, 124.3, 123.7, 123.6, 119.6, 118.5, 117.5, 115.5, 38.1,
34.9, 31.9, 30.1, 20.0, 9.0. ESI-MS m/z: 507.3 [M+H]+. HRMS (ESI)
m/z: calcd for C31H27N2O5 507.1914 [M+H]+; found: 507.1933.
Mp = 184.5–187.1 °C. 1H NMR (400 MHz, CDCl3)
d 8.79 (d,
J = 5.6 Hz, 2H), 8.74 (d, J = 5.6 Hz, 2H), 8.18 (d, J = 8.8 Hz, 1H),
7.93 (dd, J = 4.4, 1.6 Hz, 2H), 7.86 (dd, J = 4.4, 1.6 Hz, 2H), 7.64 (d,
J = 8.8 Hz, 1H), 7.52 (q, J = 1.2 Hz, 1H), 3.59–3.46 (m, 1H), 2.86–
2.76 (m, 1H), 2.17 (d, J = 1.2 Hz, 3H), 1.74–1.68 (m, 2H), 1.65–
1.60 (m, 2H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) d 163.7,
163.3, 150.99, 150.96, 150.7, 144.8, 142.1, 136.1, 135.5, 131.4,
127.0, 124.2, 123.0, 122.9, 119.7, 118.6, 117.4, 115.5, 38.1, 35.0,
31.9, 30.2, 20.0, 9.0. ESI-MS m/z: 507.2 [M+H]+; HRMS (ESI) m/z:
calcd for C31H27N2O5 507.1914 [M+H]+; found: 507.1924.
4.1.2.3. 1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,
11-diyl bis(2-fluoronicotinate) (1c). Colorless solid, 0.72 g, 65%
yield. Mp = 130.2–132.4 °C. 1H NMR (400 MHz, CDCl3) d 8.54
(ddd, J = 9.4, 7.6, 2.0 Hz, 1H), 8.48 (ddd, J = 9.4, 7.6, 2.0 Hz, 1H),
8.43–8.39 (m, 2H), 8.16 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H),
4.1.2.2. 1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,
11-diyl diisonicotinate (1b). Colorless solid, 0.60 g, 58% yield.

28
X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
13C NMR (100 MHz, CDCl3) d 162.6 (d, JC4—F = 2.1 Hz), 162.3 (d,
J4C—F = 2.1 Hz), 159.1 (d, JC1—F = 258.5 Hz), 159.0 (d, J1C—F = 257.1 Hz),
150.7, 147.0 (d, J4C—F = 4.3 Hz), 146.8 (d, JC4—F = 4.2 Hz), 144.9, 143.4
(d, J2C—F = 22.9 Hz), 143.3 (d, JC2—F = 22.9 Hz), 142.1, 134.6, 134.4,
131.3, 127.0, 126.2 (d, JC3—F = 3.7 Hz), 125.6 (d, J3C—F = 3.7 Hz), 124.1
(d, J2C—F = 17.5 Hz), 123.9 (d, JC2—F = 19.6 Hz), 123.8, 119.6, 118.6,
117.3, 115.4, 38.1, 34.9, 31.9, 30.2, 20.0, 9.1.ESI-MS m/z: 543.2
[M+H]+. HRMS (ESI) m/z: calcd for C31H25F2N2O5 543.1726
[M+H]+; found: 543.1751.
4.1.2.5. 1,6,6-trimethyl-1,2,6,7,8,9-hexahydrophenanthro[1,2-b]
furan-10,11-diyl dinicotinate (2a). Colorless solid, 0.79 g, 77% yield.
Mp = 165.4–167.3 °C. 1H NMR (400 MHz, CDCl3) d 9.31 (s, br, 1H),
9.21 (d, J = 1.2 Hz, 1H), 8.76 (d, J = 3.6 Hz, 1H), 8.73 (d, J = 3.6 Hz,
1H), 8.37 (s, br, 1H), 8.28 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.8 Hz,
1H), 7.48 (d, J = 8.8 Hz, 1H), 7.37 (dd, J = 7.4, 5.0 Hz, 1H), 7.31 (dd,
J = 8.0, 4.8 Hz, 1H), 4.92 (t, J = 9.0 Hz, 1H), 4.36 (dd, J = 8.8,
6.4 Hz), 3.92–3.72 (m, 1H), 3.52–3.40 (m, 1H), 2.86–2.69 (m, 1H),
1.72–1.66 (m, 2H), 1.64–1.58 (m, 2H), 1.33–1.26 (m, 9H). 13C
NMR (100 MHz, CDCl3) d 163.8, 162.5, 155.2, 154.1, 153.9, 151.1,
151.0, 145.3 137.7, 137.5, 132.0, 130.6, 127.3, 125.7, 125.1, 124.5,
123.6, 123.5, 119.9, 118.3, 118.2, 79.6, 38.1, 37.0, 34.9, 31.72,
31.68, 30.0, 19.9, 19.1. ESI-MS m/z: 509.2 [M+H]+. HRMS (ESI)
m/z: calcd for C31H29N2O5 509.2071 [M+H]+; found: 509.2094.
Fig. 3. Effects of compounds 1, 1a and 2a on LOX-1 protein expression. Blk.: normal
cells (without compound and oxLDL treatment). Con.: cells treated with oxLDL.
4.1.2.6. 1,6,6-trimethyl-1,2,6,7,8,9-hexahydrophenanthro[1,2-b]
furan-10,11-diyl diisonicotinate (2b). Colorless solid, 0.72 g, 70%
yield. Mp = 145.6–148.1 °C. 1H NMR (400 MHz, CDCl3) d 8.77 (d,
J = 5.6 Hz, 2H), 8.71 (d, J = 6.0 Hz, 2H), 7.89 (s, br, 2H), 7.84–7.80
(m, 3H), 7.48 (d, J = 8.8 Hz, 1H), 4.93 (t, J = 8.8 Hz, 1H), 4.36 (dd,
J = 8.8, 6.4 Hz, 1H), 3.90–3.72 (m, 1H), 3.52–3.38 (m, 1H), 2.79–
2.69 (m, 1H), 1.72–1.59 (m, 7H), 1.33 (s, 6H). 13C NMR (100 MHz,
CDCl3) d 163.8, 162.5, 155.3, 151.0, 150.9, 145.5, 137.5, 136.1,
135.5, 131.8, 130.9, 130.6, 128.9, 127.2, 125.9, 123.0, 122.8,
119.9, 118.4, 118.0, 79.6, 38.1, 37.0, 34.9, 31.72, 31.67, 30.00,
19.89, 19.03. ESI-MS m/z: 509.2 [M+H]+; HRMS (ESI) m/z: calcd
for C31H29N2O5 509.2071 [M+H]+; found: 509.2082.
4.1.2.7. 1,6,6-trimethyl-1,2,6,7,8,9-hexahydrophenanthro[1,2-b]
furan-10,11-diyl bis(2-fluoronicotinate) (2c). Colorless solid, 0.78 g,
70% yield. Mp = 125.7–127.2 °C. 1H NMR (400 MHz, CDCl3) d
8.53–8.49 (m, 1H), 8.46–8.36 (m, 3H), 7.81 (d, J = 8.8 Hz, 1H),
7.47 (d, J = 9.2 Hz, 1H), 7.34–7.27 (m, 2H), 4.92 (t, J = 8.8 Hz, 1H),
4.36 (dd, J = 8.6, 6.2 Hz, 1H), 3.92–3.76 (m, 1H), 3.60–3.36 (m,
1H), 2.91–2.69 (m, 1H), 1.74–1.67 (m, 2H), 1.64–1.58 (m, 2H),
Fig. 4. Effects of compounds 1, 1a and 2a on RAW264.7 macrophage cell viability in
the absence or presence of oxLDL. In the absence of oxLDL, cells were serum starved
for 12 h and pre-incubated with indicated compounds for 12 h. In the presence of
oxLDL, cells were serum starved for 12 h and pre-incubated with compounds for
12 h, and then treated with oxLDL (50
measured by MTT assay.
lg/ml) for 24 h. The cell viability was
1.33 (s, br, 9H). 13C NMR (100 MHz, CDCl3)
d 161.7 (d,
7.51 (q, J = 1.2 Hz, 1H), 7.35–7.28 (m, 2H), 3.64–3.46 (m, 1H), 2.98–
2.74 (m, 1H), 2.21 (d, J = 1.2 Hz, 1H), 1.76–1.70 (m, 2H), 1.67–1.62
(m, 2H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) d 162.7 (d,
J1C—F = 249.4 Hz), 162.2 (d, JC1—F = 249.3 Hz), 161.6 (d, J3C—F = 8.5 Hz),
161.3 (d, J3C—F = 8.5 Hz), 152.7 (d, JC3—F = 15.3 Hz), 152.6 (d,
J3C—F = 15.3 Hz), 150.6, 144.6, 143.7, 143.5, 142.0, 134.7, 134.4,
131.5, 126.9, 124.2, 121.82 (d, J3C—F = 4.3 Hz), 121.78 (d,
J3C—F = 4.4 Hz), 119.6, 118.5, 117.4, 115.5, 112.8 (d, J2C—F = 24.3 Hz),
112.2 (d, J2C—F = 24.8 Hz), 38.1, 34.9, 31.9, 29.9, 20.0, 8.9. ESI-MS
m/z: 543.2 [M+H]+. HRMS (ESI) m/z: calcd for C31H25F2N2O5
543.1726 [M+H]+; found: 543.1726.
J1C—F = 249.6 Hz), 161.5 (d, JC1—F = 249.0 Hz), 161.7 (d, J3C—F = 8.7 Hz),
160.3 (d, J3C—F = 10.6 Hz), 155.3, 152.6 (d, JC3—F = 15.5 Hz), 152.5 (d,
J3C—F = 15.4 Hz), 145.3, 143.6, 143.4, 137.4, 131.9, 130.6, 127.3,
125.8, 121.7 (d, JC3—F = 5.5 Hz), 121.6 (d, JC3—F = 5.6 Hz), 119.9,
118.4, 118.1, 112.9 (d, J2C—F = 24.2 Hz), 112.4 (d, J2C—F = 25.1 Hz),
79.6, 38.2, 37.1, 34.9, 31.8, 31.7, 29.7, 19.9, 19.0. ESI-MS m/z:
545.2 [M+H]+. HRMS (ESI) m/z: calcd for C31H27F2N2O5 545.1883
[M+H]+; found: 545.1897.
4.1.2.8. 1,6,6-trimethyl-1,2,6,7,8,9-hexahydrophenanthro[1,2-b]
furan-10,11-diyl bis(5-fluoronicotinate) (2d). Colorless solid, 0.87 g,
78% yield. Mp = 148.6–150.8 °C. 1H NMR (400 MHz, CDCl3) d 9.14
(s, br, 1H), 9.04 (s, br, 1H), 8.67 (d, J = 2.8 Hz, 1H), 8.64 (d,
J = 2.8 Hz, 1H), 8.11–8.03 (m, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.83 (d,
J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 4.94 (t, J = 9.0 Hz, 1H), 4.37
(dd, J = 8.4, 6.4 Hz, 1H), 3.94–3.77 (m, 1H), 3.49–3.37 (m, 1H),
2.84–2.68 (m, 1H), 1.73–1.67 (m, 2H), 1.64–1.58 (m, 2H), 1.33 (s,
br, 9H). 13C NMR (100 MHz, CDCl3) d 162.8 (d, J4C—F = 4.8 Hz),
161.5 (d, J4C—F = 1.4 Hz), 159.0 (d, JC1—F = 258.8 Hz), 158.9 (d,
J1C—F = 258.7 Hz), 155.4, 146.9 (d, JC4—F = 4.2 Hz), 146.8 (d,
4.1.2.4. 1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,
11-diyl bis(5-fluoronicotinate) (1d). Colorless solid, 0.83 g, 75%
yield. Mp = 171.4–172.7 °C. 1H NMR (400 MHz, CDCl3) d 9.10 (s,
1H), 9.03 (s, 1H), 8.60 (d, J = 2.8 Hz, 1H), 8.59 (d,J = 2.8 Hz, 1H),
8.10 (d, J = 8.8 Hz, 1H), 8.02 (ddd, J = 8.4, 2.8, 1.6 Hz, 1H), 7.97
(ddd, J = 8.4, 2.8, 1.6 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.45 (q,
J = 1.2 Hz, 1H), 3.52–3.39 (m, 1H), 2.81–2.71 (m, 1H), 2.10 (d,
J = 0.8 Hz, 3H), 1.68–1.61 (m, 2H), 1.58–1.55 (m, 2H), 1.29 (s, 6H).

X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
29
J4C—F = 4.1 Hz), 145.6, 143.24 (d, JC2—F = 23.2 Hz), 143.15 (d,
J2C—F = 23.1 Hz), 137.4, 131.7, 130.5, 127.2, 126.2 (d, J3C—F = 2.6 Hz),
125.9, 125.7 (d, J3C—F = 4.7 Hz), 124.0 (d, JC2—F = 19.3 Hz), 123.8 (d,
J2C—F = 19.7 Hz), 119.9, 118.4, 118.0, 79.6, 38.1, 37.0, 34.9, 31.73,
31.68, 30.1, 19.9, 19.1. ESI-MS m/z: 545.2 [M+H]+. HRMS (ESI)
m/z: calcd for C31H27F2N2O5 545.1883 [M+H]+; found: 545.1895.
4.2.4. Preparation and uptake of DiI-oxLDL
LDL was labeled with fluorescent probe, l,l0-dioctadecyl-
3,3,30,30-tetramethyl-indocarbocyanine perchlorate (DiI) according
to the reported method with minor modification [25]. Briefly, LDL
was incubated overnight at 37 °C under nitrogen and light protec-
tion with 50 lL of DiI (3 mg/ml in DMSO) for each milligram of LDL
protein. Unbound dye was removed by ultracentrifugation at
46,000 rpm for 5.5 h at 4 °C. For the preparation of DiI-oxLDL,
DiI-LDL (0.9 mg/ml) was incubated with 10 lM CuSO4 (final con-
4.1.2.9. 1,6-dimethylphenanthro[1,2-b]furan-10,11-diyl dinicotinate
(3a). Colorless solid, 0.76 g, 72% yield. Mp = 179.3–180.2 °C. 1H
centration) at 37 °C in dark for 24 h. The DiI-oxLDL distributed in
the middle layer was re-isolated and subjected to dialysis against
PBS buffer containing 0.24 mM EDTA. The protein content of
DiI-oxLDL was determined using BCA Protein Assay Kit (Thermo).
To assess the cellular DiI-oxLDL uptake, RAW264.7 macrophage
cells were seeded overnight on confocal dishes (Corning, NY) at a
density of 5 ꢀ 105 cells/ml, then were serum starved for 12 h and
pre-incubated with the tested compounds or control (0.1% DMSO)
NMR (400 MHz, CDCl3)
d 9.45 (d, J = 1.6 Hz, 1H), 9.32(d,
J = 1.6 Hz, 1H), 8.90 (d, J = 8.8 Hz, 1H), 8.83 (dd, J = 5.0, 1.4 Hz,
1H), 8.79 (dd, J = 4.8, 1.6 Hz, 1H), 8.48 (dt, J = 8.0, 2.0 Hz, 1H),
8.39 (dt, J = 8.0, 2.0 Hz, 1H), 8.37 (d, J = 9.2 Hz, 1H), 8.17 (d,
J = 9.2 Hz, 1H), 7.63 (q, J = 1.2 Hz, 1H), 7.44–7.36 (m, 3H), 7.32
(dd, J = 8.4, 7.2 Hz, 1H), 2.79 (s, 3H), 2.26 (d, J = 1.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) d 163.5, 163.4, 154.4, 151.3, 151.2, 150.6,
142.8, 137.7, 137.6, 135.8, 135.3, 134.8, 131.6, 129.3, 128.0,
126.8, 124.9, 124.5, 124.4, 123.75, 123.69, 123.66, 121.1, 119.3,
118.8, 118.6, 115.6, 20.5, 9.1. ESI-MS m/z: 489.2 [M+H]+. HRMS
(ESI) m/z: calcd for C30H21N2O5 489.1445 [M+H]+; found: 489.1466.
for 12 h, and then loaded with DiI-oxLDL (20 lg/ml) for another 6 h
at 37 °C. At the end of incubation period, cells were washed once
with PBS buffer (with 2 mg/ml BSA) and twice with PBS buffer,
then were fixed in phosphate buffered 10% formalin for 10 min.
Thereafter, for nucleus staining, the cells were incubated for
4.2. Biological testing
20 min with 10 lg/ml DAPI (Sigma) and washed 3 times with
PBS buffer. The cells were then placed for confocal microscopy with
a X 100 oil immersion objective using a 546 nm filter set (LSM 710,
ZEISS, Germany), and quantitatively analyzed by flow cytometry.
4.2.1. Cell culture and MTT assay
RAW 264.7 cells were cultured in 96-well plates (Costar, Corn-
ing, NY) at a density of 5 ꢀ 104 cells/ml in RPMI 1640 medium
(with 100 IU/ml of penicillin G, 100 lg/ml streptomycin, 2 mM
4.2.5. Western Blot analysis
L-glutamine, and 10% (v/v) heat-inactivated fetal bovine serum),
incubated in a humidified atmosphere of 5% CO2 in incubator at
37 °C. To evaluate the cytotoxicity, cells were exposed to the
synthesized compounds and measured using the MTT assay [22].
RAW264.7 macrophage cells were pretreated with compounds
for 6 h before stimulation with oxLDL (50
lg/ml) for 24 h. Cells
were lysed in lysis buffer containing PMSF (Sigma), and 40
lg pro-
teins were loaded and separated on a 10% SDS–PAGE, then trans-
ferred to PVDF membrane as described [23]. The membrane was
probed with a rabbit anti-mouse LOX-1 antibody (1:1000, Biovi-
sion), followed by incubation with an HRP-conjugated anti-rabbit
secondary antibody (1:3000, Cell Signaling Technology). Immuno-
complexes were detected by ECL method (Pierce). GAPDH was
used to normalize the protein loading.
4.2.2. LDL isolation, modification, and labeling
The use of human plasma (from Guangzhou General Hospital of
Guangzhou Military Command) conformed to the principle out-
lined in the Declaration of Helsinki. Human native-LDL (d = 1.019
to 1.063 g/ml) was prepared from the plasma of normolipidemic
volunteers by sequential ultracentrifugation [23]. LDL was exten-
sively dialyzed for 24 h at 4 °C in Tris buffer (20 mM Tris,
Acknowledgments
150 mM NaCl, 300
for 24 h to generate oxLDL followed by dialysis against PBS buffer
(5 mM Tris, 50 nM NaCl) with 10 M EDTA and then PBS buffer to
lM EDTA). LDL was oxidized with 10 lM CuSO4
This work was financially supported by Natural Science Foun-
dation of China (No. 81373257), Guangdong Provincial Science
and Technology Projects (No. 2012B031800114) and Fundamental
Research Funds for the Central Universities.
l
remove EDTA. The quality of LDL and oxLDL were monitored by
agarose gel electrophoresis. The relative electrophoretic mobility
of oxLDL was 2.5–2.7 relative to native-LDL. The protein content
of lipoprotein was determined by BCA Protein Assay Kit (Thermo)
using BSA as a standard. All lipoproteins were filter sterilized,
stored at 4 °C in dark and used within 3 weeks.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.
11.001.
4.2.3. Treatment of compounds and oil red O staining
The RAW264.7 cells were seeded on 6-well tissue culture plates
at a density of 5 ꢀ 105 cells/ml for 18 h, and then starved in RPMI
1640 medium without FBS for 12 h. The cells were pretreated with
indicated compounds for 12 h in RPMI 1640 medium without FBS
References
before treatment with oxLDL (50 lg/ml) for 24 h, while the cells in
[1] D. Lloyd-Jones, R.J. Adams, T.M. Brown, M. Carnethon, S. Dai, G. De Simone, T.B.
Ferguson, E. Ford, K. Furie, C. Gillespie, et al., Circulation 121 (2010) 948–954.
[2] J. Lusis, Nature 407 (2000) 233–241.
control group was pretreated with 0.1% DMSO for 12 h. Foam cell
formation was revealed by oil red O staining according to the re-
ported method [24]. Briefly, cells were fixed in phosphate buffered
10% formalin for 10 min and washed once with PBS buffer. After
flushing with 60% isopropanol for 30 s at room temperature, the
cells were stained with 0.5% oil red O (Sigma) in 60% isopropanol
for 20 min (37 °C, in darkness) and washed once with 60%
isopropanol for 5 s. Photomicrographs were taken by phase
contrast microscopy with a X 40 objective (CKX41, Olympus,
Japan).
[3] K.J. Moore, I. Tabas, Cell 145 (2011) 341–355.
[4] N. Shibata, C.K. Glass, Circ. J. 74 (2010) 2045–2051.
[5] M.S. Brown, J.L. Goldstein, Annu. Rev. Biochem. 52 (1983) 223–261.
[6] M.J. Chen, T. Masaki, T. Sawamura, Pharmacol. Therapeut. 95 (2002) 89–100.
[7] D.Y. Li, H.J. Chen, J.L. Mehta, Cardiovasc. Res. 52 (2001) 130–135.
[8] S. Gao, Z.P. Liu, H. Li, P.J. Little, P.Q. Liu, S.W. Xu, Atherosclerosis 220 (2012) 3–
10.
[9] S.W. Xu, Z.P. Liu, Y. Huang, K. Le, F.T. Tang, H.Q. Huang, S. Ogura, P.J. Little, X.Y.
Shen, P.Q. Liu, Transl. Res. 160 (2012) 114–124.
[10] W.Y. Chen, F.T. Tang, B.L. Xie, S.R. Chen, H.Q. Huang, P.Q. Liu, Eur. J. Pharmacol.
674 (2012) 359–364.

30
X. Cheng et al. / Bioorganic Chemistry 52 (2014) 24–30
[11] F.T. Tang, Y.A. Cao, T.Q. Wang, L.J. Wang, J.A. Guo, X.S. Zhou, S.W. Xu, W.H. Liu,
P.Q. Liu, H.Q. Huang, Eur. J. Pharmacol. 650 (2011) 275–284.
[12] S.W. Xu, P.J. Little, T. Lan, Y. Huang, K. Le, X.Q. Wu, X.Y. Shen, H.Q. Huang, Y. Cai,
F.T. Tang, H. Wang, P.Q. Liu, Arch. Biochem. Biophys. 515 (2011) 72–79.
[13] H.L. Tian, T. Yu, N.N. Xu, C. Feng, L.Y. Zhou, H.W. Luo, D.C. Chang, X.F. Le, K.Q.
Luo, Cancer Lett. 297 (2010) 18–30.
[14] R. Altschul, A. Hoffer, J.D. Stephen, Arch. Biochem. Biophys. 54 (1955) 558–559.
[15] H.E. Carlson, A.S. Brickman, G.F. Bottazzo, N. Engl, J. Med. 296 (1977) 140–144.
[16] W.B. Parsons Jr., J.H. Flinn, Arch. Intern. Med. 103 (1959) 783–790.
[17] J. Shepherd, C.J. Packard, J.R. Patsch, A.M. Gotto Jr., O.D. Taunton, J. Clin. Invest.
63 (1979) 858–867.
[19] S.W. Xu, S. Ogura, J.W. Chen, P.J. Little, J. Moss, P.Q. Liu, Cell. Mol. Life Sci. 70
(2013) 2859–2872.
[20] L.K. An, X.Z. Bu, H.Q. Wu, X.D. Guo, L. Ma, L.Q. Gu, Tetrahedron 58 (2002)
10315–10321.
[21] H. Rinderknecht, M. Gutenstein, Org. Synth. 47 (1967) 89.
[22] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.
[23] S.W. Xu, J.J. Fu, J.W. Chen, P.X. Xiao, T. Lan, K. Le, F. Cheng, L. He, X.Y. Shen, H.Q.
Huang, P.Q. Liu, Cytotechnology 61 (2009) 65–72.
[24] S.W. Xu, Y. Huang, Y. Xie, T.A. Lan, K. Le, J.W. Chen, S.R. Chen, S. Gao, X.Z. Xu,
X.Y. Shen, H.Q. Huang, P.Q. Liu, Cytotechnology 62 (2010) 473–481.
[25] T.W. Lian, L.S. Wang, Y.H. Lo, I.J. Huang, M.J. Wu, Biochim. Biophys. Acta 1781
(2008) 601–609.
[18] L. Berglund, R. Ramakrishnan, Arterioscler. Thromb. Vasc. 24 (2004)
2219–2226.