Ferrocenyl-appended aurone and flavone: Which possesses higher inhibitory effects on DNA oxidation and radicals?

Article abstract of DOI:10.1021/tx500405b

The aim of the present work was to compare the antioxidative effect of the ferrocenyl-appended aurone with that of ferrocenyl-appended flavone; therefore, nine aurones together with the flavone-type analogues were synthesized by using chalcone as the reactant. The radical-scavenging property was evaluated by reacting with the 2,2β-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+·), 2,2β-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively. The cytotoxicity was estimated by inhibiting 2,2β-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. It was found that the introduction of the ferrocenyl group remarkably increased the radical-scavenging activities of aurone and flavone. Especially, the ferrocenyl group in flavones can quench radicals even in the absence of the phenolic hydroxyl group, while ferrocenyl-appended aurones can efficiently protect DNA against AAPH-induced oxidation. Therefore, the antioxidative effect was generated by the ferrocenyl group and enhanced by the electron-donating group attaching to the para-position of the ferrocenyl group. Introducing the ferrocenyl group into natural compounds may be a useful strategy for increasing the antioxidative effectiveness.

Full text of DOI:10.1021/tx500405b

Article
pubs.acs.org/crt
Ferrocenyl-Appended Aurone and Flavone: Which Possesses Higher
Inhibitory Effects on DNA Oxidation and Radicals?
Jia-Feng Chen and Zai-Qun Liu*
Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road, Changchun 130021, China
S
* Supporting Information
ABSTRACT: The aim of the present work was to compare the
antioxidative effect of the ferrocenyl-appended aurone with that of
ferrocenyl-appended flavone; therefore, nine aurones together with the
flavone-type analogues were synthesized by using chalcone as the
reactant. The radical-scavenging property was evaluated by reacting
with the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic
radical (ABTS+·), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and
galvinoxyl radical, respectively. The cytotoxicity was estimated by
inhibiting 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-in-
duced oxidation of DNA. It was found that the introduction of the
ferrocenyl group remarkably increased the radical-scavenging activities
of aurone and flavone. Especially, the ferrocenyl group in flavones can
quench radicals even in the absence of the phenolic hydroxyl group,
while ferrocenyl-appended aurones can efficiently protect DNA against AAPH-induced oxidation. Therefore, the antioxidative
effect was generated by the ferrocenyl group and enhanced by the electron-donating group attaching to the para-position of the
ferrocenyl group. Introducing the ferrocenyl group into natural compounds may be a useful strategy for increasing the
antioxidative effectiveness.
flavones are prepared in order to clarify the aforementioned
problems. So, aurones and flavones used herein contain the
same functional groups including hydroxyl, methoxyl, bromide,
and nitro groups attached to ring A.
INTRODUCTION
■
Aurone, an isomer of flavone, contains double aromatic rings
(as rings A and B) and a furan-3(2H)-one (as ring C) instead of
the pyran-4-one in flavone (see Scheme 1).1 As diet nutrients in
Medicago species,2 flavone and aurone exhibit widely biological
activities3 and significant chemosystematics.4 The natural
distribution5 and the pharmacological applications6 including
the cytotoxicity,7 the probe for β-amyloid plaques,8 and anti-
inflammatory9 and anticancer effects10 attract much research
attention. The quantitative study on the structure−activity
relationship (QSAR) reveals that the hydroxyl groups attaching
to ring B in flavone play the main role in generating
bioactivities.11 Meanwhile, efforts also contributed to introduce
some active groups into scaffolds of aurone and flavone for
increasing the cytotoxicity.12 For example, the methoxyl group
at ring A may enhance the cytotoxicity of the hydroxyl group
(at ring B) on K562 human leukemia cells.13 Recently, it was
found that the cytotoxicity of aurone toward breast cancer cells
can be increased by a ferrocenyl group as ring B.14 Meanwhile,
the bioactivities of ferrocene-involved molecules attract much
research attention,15 and some protocols are applied to
synthesize the ferrocenyl-appended aurones and flavones, in
which chalcones act as the reactants in the presence of some
catalysts.16 However, how the ferrocenyl group (as ring B)
influences the antioxidative effects of aurone and flavone
remains unclear, and the antioxidative effects of the ferrocenyl-
appended aurone and flavone need to be compared. Therefore,
as shown in Scheme 1, 18 ferrocenyl-appended aurones and
The antioxidative effect can be estimated in many
experimental systems,17 in which the oxidative damage of
DNA and radical-scavenging property are usually applied
because the radical-induced oxidation of DNA is regarded as
the pathogenesis for many fatal diseases.18 Presented here is a
comparison of the abilities of ferrocenyl-appended aurones and
flavones to quench the 2,2′-azinobis(3-ethylbenzothiazoline-6-
sulfonate) radical (ABTS+·), 2,2′-diphenyl-1-picrylhydrazyl
radical (DPPH), and the galvinoxyl radical. In addition, the
cytotoxicity of ferrocenyl-appended aurones and flavones on
2,2′-azobis(2-amidinopropane hydrochloride) (AAPH, R-N
N-R; R = -CMe2C(=NH) NH2))-induced oxidation of DNA is
investigated.
MATERIALS AND METHODS
■
Materials and Instrumentation. Diammonium salt of 2,2′-
azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS salt), DPPH, and
the galvinoxyl radical were purchased from Fluka Chemie GmbH,
Buchs, Switzerland. AAPH and the naked DNA sodium salt were
purchased from Acros Organics, Geel, Belgium. Other agents were of
Special Issue: Chemical Toxicology in China
Received: October 4, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/tx500405b
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Scheme 1. Synthetic Routines and Structures of Ferrocenyl-Appended Aurones and Flavones
analytical grade and used directly. The structures of the synthetic
compounds were identified by 1H and 13C NMR (Varian Mercury 400
NMR spectrometer).
Selective Protection of the Hydroxyl Group in Dihydrox-
yacetophenone. To a dry acetone solution (50 mL) of 2,4-
dihydroxyacetophenone or 2,5-dihydroxyacetophenone (3.5 g),
anhydrous K2CO3 (10 g) was added and stirred for 10 min.
CH3OCH2Cl (2.5 mL) was added and heated at 30 °C for 12 h.
After the mixture was cooled to room temperature, the precipitate was
filtered and washed with acetone. The acetone solution was dried over
Na2SO4. After acetone was evaporated, the residue was purified on a
flash column chromatograph (CH2Cl2 as the eluent) to yield 2-
B
DOI: 10.1021/tx500405b
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hydroxyl-4-(methoxymethoxy)acetophenone (72%) or 2-hydroxy-5-
(methoxymethoxy)acetophenone (61%).
(Z)-2-Benzylidene-benzofuran-3-one, 1. Rf = 0.63 (ethyl
acetate/petroleum ether = 1:3, v/v), 1.97 g of white solid, yield
88%. m.p.: 111−113 °C. 1H NMR (400 MHz, CDCl3) δ: 7.93 (d, J =
7.2 Hz, 2H), 7.81 (d, J = 7.6 Hz, 1H), 7.66 (td, J = 7.6 Hz, J = 1.6 Hz,
1H), 7.48−7.44 (m, 2H), 7.42−7.38 (m, 1H), 7.34 (d, J = 7.6 Hz,
1H), 7.22 (t, J = 7.2 Hz, 1H), 6.90 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ: 184.8, 166.2, 146.9, 136. 9, 132.3, 131.5, 129.9, 128.9,
124.7, 123.5, 121.6, 113.1, 112.9. HRMS (ESI): m/z calcd for
C15H10O2Na+, 245.0573; found, 245.0548.19
Synthesis of Chalcones. To a stirred ethanol solution (50 mL) of
corresponding 2-hydroxyacetophenone (0.01 mol) and benzaldehyde
or ferrocenecarboxaldehyde (0.012 mol), NaOH (3.0 g) was added
and stirred at room temperature overnight. The mixture was cooled to
0 °C and acidified with HCl aqueous solution (30%, v/v) (pH < 7) to
afford the precipitate. The mixture was extracted with CH2Cl2, which
was then dried over Mg2SO4. After CH2Cl2 was evaporated, the
residue was recrystallized by ethanol to give the corresponding
chalcones.
Synthesis of Phenyl-Substituted Aurones (1 and 3) and
Flavones (2 and 4). To a pyridine solution (50 mL) of 2-
hydroxychalcone or 2-hydroxy-5-methoxymethoxychalcone (0.01
mol), 0.015 mol Hg(OAc)2 was added and stirred at 110 °C for 1.5
h. The cooled mixture was poured into ice-cold water and acidified
with HCl aqueous solution (30%, v/v). The produced precipitate was
extracted with CH2Cl2, which was dried over Mg2SO4. After CH2Cl2
was evaporated, the residue was recrystallized by ethanol to give 1 (or
5-methoxymethoxy-aurone). The deprotection of methoxyl for 5-
methoxymethoxyaurone was carried out in 100 mL of methanol
(containing 15 mL of 10% (v/v) HCl aqueous solution) under
refluxing for 30 min. After the mixture was cooled to room
temperature, a yellow precipitate was formed and recrystallized by
ethanol to give 3.
2-Phenyl-chromen-4-one, 2. Rf = 0.40 (ethyl acetate/petroleum
ether = 1:3, v/v), 1.57 g of white solid, yield 70%. m.p.: 92−95 °C. 1H
NMR (400 MHz, CDCl3) δ: 8.25 (d, J = 8.0 Hz, 1H), 7.96−7.94 (m,
2H), 7.71 (t, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.51−7.50 (m,
3H), 7.45−7.42 (m, 1H), 6.86 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ: 178.5, 164.1, 156.4, 134.1, 131.9, 131.6, 129.1, 126.5, 125.8, 125.5,
+
123.5, 118.1, 107.2. HRMS (ESI) [M + H]+: m/z calcd for C15H11O2 ,
223.0754; found, 223.0761.20
(Z)-5-Hydroxy-2-benzylidene-benzofuran-3-one, 3. Rf = 0.72
(ethyl acetate/petroleum ether = 1:1, v/v), 1.27 g of yellow solid, yield
1
53%. m.p.: 260−264 °C. H NMR (400 MHz, DMSO-d6) δ: 9.80
(s,1H), 7.98 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.2 Hz, 2H), 7.46 (d, J =
6.8 Hz, 1H), 7.44−7.40 (m, 1H), 7.23 (dd, J = 6.0, 2.0 Hz, 1H), 7.04
(d, J = 2.4 Hz, 1H), 6.89 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ:
184.0, 159.3, 153.9, 147.1, 132.0, 131.3, 129.9, 129.0, 125.8, 121.2,
113.9, 111.7, 107.8. MS (ESI) [M + H]+: m/z 239.21
The 2-hydroxychalcone or 2-hydroxy-5-methoxymethoxychalcone
(0.002 mol) and CuI (0.5 mmol) were dissolved in 20 mL of N,N-
dimethylacetamide (DMA) and heated at 130 °C for 16 h. After the
solvent was evaporated, the crude product was purified on a column
chromatograph with ethyl acetate and petroleum ether (1:3, v/v) as
the eluent to afford 2 (or 6-methoxymethoxyflavone). The methoxyl
group in 6-methoxymethoxyflavone was removed to afford 4 (see the
synthesis of 3).
6-Hydroxy-2-phenyl-chromen-4-one, 4. Rf = 0.45 (ethyl
acetate/petroleum ether = 1:1, v/v), 1.32 g of white solid, yield
55%. m.p.: 250−252 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.01 (s,
1H), 8.08 (d, J = 6.8 Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.60−7.58 (m,
3H), 7.33 (s, 1H), 7.27 (d, J = 8.8 Hz, 1H), 6.96 (s, 1H). 13C NMR
(100 MHz, DMSO-d6) δ: 177.0, 162.2, 154.9, 149.4, 131.6, 131.4,
129.1, 126.2, 124.2, 123.1, 119.8, 107.5, 105.9. MS (ESI) [M + H]+:
m/z 239.22
(Z)-2-Ferrocenylidene-benzofuran-3-one, 5. Rf = 0.75 (ethyl
Synthesis of Ferrocenyl-Appended Aurones (5, 7, 9, 11, 13,
15, and 17). Ferrocenyl-appended chalcone (0.01 mol) was dissolved
in 30 mL of pyridine, and Hg(OAc)2 (2.5 equiv) was added and stirred
for 10 min at room temperature. Then, the mixture was heated at 80
°C for 2 h and poured into water (cooling to 0 °C). The pH value of
the mixture was adjusted to <3 by adding HCl aqueous solution (30%,
v/v). The mixture was extracted by CH2Cl2 and washed by water. The
organic phase was dried over MgSO4, filtered, and evaporated. The
residue was purified on a silica gel column with CH2Cl2 being the
eluent to give 5, 7, 9, 15, and 17. The methoxyl group in the
corresponding products was removed to give 11 and 13.
acetate/petroleum ether = 1:3, v/v), 2.31 g of violet solid, yield 70%.
1
m.p.: 131−133 °C. H NMR (400 MHz, CDCl3) δ: 7.81 (d, J = 7.6
Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.19 (t, J =
7.6 Hz, 1H), 6.90 (s, 1H), 4.88 (s, 2H), 4.56 (s, 2H), 4.19 (s, 5H). 13C
NMR (100 MHz, CDCl3) δ: 183.0, 165.4, 146.0, 136.1, 124.5, 123.1,
122.7, 116.5, 113.0, 75.1, 71.8, 71.5, 70.0. HRMS (ESI): m/z calcd for
C19H14FeO2Na+, 353.02409; found, 353.02354.16
2-Ferrocenyl-chromen-4-one, 6. Rf = 0.56 (ethyl acetate/
petroleum ether = 1:3, v/v), 52 mg of red solid, yield 52%. m.p.:
1
112−115 °C. H NMR (400 MHz, CDCl3) δ: 8.22 (d, J = 8.0 Hz,
Synthesis of Ferrocenyl-Appended Flavones (6, 8, 10, 12,
14, 16, and 18). Two steps were carried out for preparing ferrocenyl-
appended flavones from the corresponding aurones. The first step was
a ring-opening reaction for generating ferrocenyl-ynones. The
ferrocenyl-appended aurone (5, 7, 9, 11, 13, 15, and 17) (300 mg)
was dissolved in 25 mL of tetrahydrofuran (THF) and cooled to −78
°C. Then, lithium diisopropylamide (LDA, 2.5 equiv, 2 M in THF)
was added and stirred at −78 °C for 1 h. After the temperature of the
solution returned to ambient temperature, the solution was poured
into 12 M HCl aqueous solution, extracted with CH2Cl2, and washed
with water. The organic phase was dried over MgSO4 and then
evaporated. The residue was purified on a silica gel column with
petroleum ether and dichloromethane (1:1, v/v) being the eluent to
give the ferrocenyl-ynone. The second step was the annulation of the
ferrocenyl-ynone to form ferrocenyl-appended flavones. A ferrocenyl-
ynone (100 mg) was dissolved in 30 mL of C2H5OH, and NaOC2H5
(1.5 g) was added and stirred at room temperature for 6−24 h. After
the reaction was completed, the mixture was poured into 12 M HCl
aqueous solution, extracted with CH2Cl2, and washed with water. The
organic phase was dried over MgSO4 and then evaporated. The
residue was purified on a silica gel column with CH2Cl2 being the
eluent to give 6, 8, 10, 16, and 18. The methoxyl group in the
corresponding products was removed to give 12 and 14. The NMR
data of all the synthetic compounds and mass spectra (MS) data of
new compounds (from high resolution mass spectra) given below. The
MS data of the reported compounds were cited from references.
1H), 7.68 (t, J = 7.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.6
Hz, 1H), 6.50 (s, 1H), 4.89 (s, 2H), 4.54 (s, 2H), 4.18 (s, 5H). 13C
NMR (100 MHz, CDCl3) δ: 177.5, 168.4, 156.3, 133.4, 125.7, 125.0,
124.1, 117.8, 105.8, 75.0, 71.5, 70.3, 67.6. HRMS (ESI): m/z calcd for
C19H14FeO2Na+, 353.02409; found, 353.02354.16
(Z)-6-Methoxy-2-ferrocenylidene-benzofuran-3-one, 7. Rf =
0.60 (ethyl acetate/petroleum ether = 1:3, v/v), 2.35 g of violet solid,
1
yield 65%. m.p.: 152−155 °C. H NMR (400 MHz, CDCl3) δ: 7.71
(d, J = 9.2 Hz, 1H), 6.81 (s, 1H), 6.75−6.74 (m, 2H), 4.84 (s, 2H),
4.53 (s, 2H), 4.18 (s, 2H), 3.93 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 181.4, 167.8, 166.9, 146.9, 125.6, 115.8, 114.8, 111.9, 96.6, 75.2,
+
71.4, 71.2, 69.8, 56.0. HRMS (ESI): m/z calcd for C20H16FeO3 ,
360.04489; found, 360.04434.14
7-Methoxy-2-ferrocenyl-chromen-4-one, 8. Rf = 0.32 (ethyl
acetate: petroleum ether = 1:3, v/v), 45 mg of red solid, yield 45%.
1
m.p.: 145−147 °C. H NMR (400 MHz, CDCl3) δ: 7.59 (d, J = 3.2
Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.28−7.25 (m, 2H), 6.47 (s, 1H),
4.87 (s, 2H), 4.53 (s, 2H), 4.18 (s, 5H), 3.91 (s, 3H). 13C NMR (100
MHz, CDCl3) δ: 167.9, 156.9, 151.1, 123.2, 119.1, 105.3, 105.0, 75.2,
71.3, 70.2, 67.4, 56.0. HRMS (ESI) [M + H]+: m/z calcd for
23
+
C20H17FeO3 , 361.0520; found, 361.0527.
(Z)-5-Methoxy-2-ferrocenylidene-benzofuran-3-one, 9. Rf =
0.62 (ethyl acetate/petroleum ether = 1:3, v/v), 2.24 g of violet solid,
yield 62%. m.p.: 139−144 °C. 1H NMR (400 MHz, CDCl3) δ: 7.27−
7.22 (m, 3H), 6.91 (s, 1H), 4.89 (s, 2H), 4.58 (s, 2H), 4.21 (s, 5H),
3.87 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 183.0, 160.5, 155.9,
C
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146.8, 125.5, 122.7, 116.5, 113.7, 105.1, 77.0, 75.2, 71.8, 71.5, 70.0,
56.0. HRMS (ESI): m/z calcd for C20H16FeO3Na+, 383.03466; found,
383.03411.16
Hz, 1H), 8.39 (dd, J = 7.6 Hz, J = 2.8 Hz, 1H), 7.43 (d, J = 9.2 Hz,
1H), 6.94 (s, 1H), 4.91 (s, 2H), 4.73 (s, 2H), 4.26 (s, 5H). 13C NMR
(100 MHz, DMSO-d6) δ: 195.5, 172.6, 162.1, 140.6, 128.1, 124.7,
123.1, 113.9, 99.6, 73.0, 70.3, 68.8, 65.8. HRMS (ESI) [M + H]+: m/z
calcd for C19H13FeNO4, 376.0272; found, 376.0297.
6-Methoxy-2-ferrocenyl-chromen-4-one, 10. Rf = 0.36 (ethyl
acetate/petroleum ether = 1:3, v/v), 43 mg of red solid, yield 43%.
ABTS+•, DPPH, and Galvinoxyl Radical Test. ABTS salt (4.39
mg) was mixed with K2S2O8 (0.76 mg) in 2.0 mL of water and kept for
20 h to form ABTS+•. Then, 100 mL of ethanol was added to make the
1
m.p.: 139−141 °C. H NMR (400 MHz, CDCl3) δ: 7.60 (d, J = 3.2
Hz, 1H), 7.45 (d, J = 9.2 Hz, 1H), 7.30−7.27 (m, 1H), 6.55 (s, 1H),
4.89 (s, 2H), 4.54 (s, 2H), 4.18 (s, 5H), 3.92 (s, 3H). 13C NMR (100
MHz, CDCl3) δ: 177.3, 167.9, 156.9, 151.1, 124.8, 123.2, 119.1, 105.2,
105.1, 75.2, 71.3, 70.2, 67.4, 56.0. HRMS (ESI): m/z calcd for
C20H16FeO3Na+, 383.03466; found, 383.03411.16
+•
absorbance around 1.20 at 734 nm (εABTS = 1.6 × 104 M−1 cm−1).
DPPH and the galvinoxyl radical were dissolved in 50 mL of ethanol to
make the absorbance of DPPH around 1.20 at 517 nm (εDPPH = 1.09
× 104 M−1 cm−1) and the absorbance of the galvinoxyl radical around
1.50 at 428 nm (εgalvinoxy = 1.4 × 105 M−1 cm−1). The dimethyl
sulfoxide (DMSO) solutions of 1−18 (0.1 mL) were added to 1.9 mL
of the aforementioned radical solutions. The decay of the absorbance
of the radical was recorded at room temperature (25 °C) with a certain
time interval.
AAPH-Induced Oxidation of the DNA Test. The sodium salt of
DNA and AAPH were dissolved in phosphate buffered solution (PBS:
8.1 mM Na2HPO4, 1.9 mM NaH2PO4, and 10.0 μM EDTA). Then,
AAPH, DNA, and a synthetic compound (dissolved in DMSO as the
stock solution) were mixed, and the final concentrations of AAPH and
DNA were 40 mM and 2.24 mg/mL, respectively. The mixture was
dispatched into test tubes (each one containing 2.0 mL) and incubated
at 37 °C for initiating the DNA oxidation. Three test tubes were taken
out at a certain period and cooled immediately. Then, 1.0 mL of
thiobarbituric acid (TBA) solution (1.00 g of TBA and 0.40 g of
NaOH dissolved in 100 mL of PBS) and 1.0 mL of 3.0%
trichloroacetic acid aqueous solution were added and heated in
boiling water for 15 min. After the test tubes were cooled to room
temperature, 1.5 mL of n-butanol was added and shaken vigorously to
extract thiobarbituric acid reactive substance (TBARS). The
absorbance of TBARS was measured at 535 nm and plotted vs the
reaction period.
(Z)-6-Hydroxy-2-ferrocenylidene-benzofuran-3-one, 11. Rf =
0.55 (ethyl acetate/petroleum ether = 1:1, v/v), 1.90 g of violet solid,
1
yield 55%. m.p.: 280−290 °C. H NMR (400 MHz, DMSO-d6) δ:
11.06 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 2.0 Hz, 1H), 6.71−
6.68 (m, 2H), 4.89 (s, 2H), 4.57 (s, 2H), 4.19 (s, 5H). 13C NMR (100
MHz, DMSO-d6) δ: 179.8, 167.2, 165.9, 146.2, 125.5, 113.7, 113.3,
112.7, 98.51, 75.0, 71.2, 70.9, 69.6. HRMS (ESI) [M + H]+: m/z calcd
for C19H14FeO3, 347.0371; found, 347.0393.
7-Hydroxy-2-ferrocenyl-chromen-4-one, 12. Rf = 0.29 (ethyl
acetate/petroleum ether = 1:1, v/v), 29 mg of red solid, yield 29%.
m.p.: 174−180 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.71 (s, 1H),
7.84 (d, J = 8.4 Hz, 1H), 6.91−6.88 (m, 2H), 6.48 (s, 1H), 5.05 (s,
2H), 4.59 (s, 2H), 4.20 (s, 5H). 13C NMR (100 MHz, DMSO-d6) δ:
175.5, 166.8, 162.3, 157.5, 126.5, 116.4, 114.5, 104.7, 102.4, 75.0, 71.1,
70.0, 67.3. HRMS (ESI) [M + H]+: m/z calcd for C19H14FeO3,
347.0371; found, 347.0393.
(Z)-5-Hydroxy-2-ferrocenylidene-benzofuran-3-one, 13. Rf =
0.71 (ethyl acetate/petroleum ether = 1:1, v/v), 1.73 g of violet solid,
yield 50%. m.p.: 245−252 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.70
(s, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.00 (s,
1H), 6.82 (s, 1H), 4.92 (s, 2H), 4.62 (s, 2H), 4.21 (s, 5H). 13C NMR
(100 MHz, DMSO-d6) δ: 181.8, 158.6, 153.6, 146.1, 125.0, 122.2,
115.4, 113.8, 107.4, 75.0, 71.6, 71.2, 69.7. HRMS (ESI) [M + H]+: m/
z calcd for C19H14FeO3, 347.0371; found, 347.0394.
Statistical Analysis. All of the data were the average value from at
least three independent measurements with the experimental error
within 10%. The equations were analyzed by one-way ANOVA in
Origin 6.0 professional software, and p < 0.001 indicated a significance
difference.
6-Hydroxy-2-ferrocenyl-chromen-4-one, 14. Rf = 0.39 (ethyl
acetate/petroleum ether = 1:1, v/v), 20 mg of red solid, yield 20%.
1
m.p.: 160−164 °C. H NMR (400 MHz, DMSO-d6) δ: 9.95 (s, 1H),
7.55 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.22 (d, J = 7.2 Hz, 1H), 6.55
(s, 1H), 5.06 (s, 2H), 4.60 (s, 2H), 4.20 (s, 5H). 13C NMR (100 MHz,
DMSO-d6) δ: 176.4, 167.7, 155.1, 149.9, 125.0, 122.9, 119.9, 108.2,
104.8, 75.5, 71.7, 70.5, 67.9. HRMS (ESI) [M + H]+: m/z calcd for
C19H14FeO3, 347.0371; found, 347.0398.
RESULTS AND DISCUSSION
■
Synthesis. As shown in Scheme 1, nine aurones and the
corresponding flavones are synthesized by using the same
chalcone. It is worth noting that the design of the synthetic
methods for aurones and flavones is a challengeable work. For
example, the five-membered ring in aurone is originally derived
from o-bromoacetophenone under basic conditions, and then,
the formed benzofuranone takes part in aldol condensation
with benzaldehydes in the presence of KOH.24 Recently, the
cyclization of the five-membered ring in aurone was combined
with the formation of chalcone by benzophenone and 2,6-
dihydroxyacetophenone within a one-pot operation.25 Mean-
while, applications of some catalysts including AuCl,19
trifluoromethanesulfonic acid,26 and 4-dimethylaminopyri-
dine27 simplify the cyclization of o-alkynoylphenol to form
aurones or flavones.
(Z)-5-Bromo-2-ferrocenylidene-benzofuran-3-one, 15. Rf =
0.73 (ethyl acetate/petroleum ether = 1:3, v/v), 2.79 g of violet solid,
yield 68%. m.p.: 188−191 °C. 1H NMR (400 MHz, CDCl3) δ: 7.93 (s,
1H), 7.72 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 6.93 (s, 1H),
4.88 (s, 2H), 4.60 (s, 2H), 4.21 (s, 5H). 13C NMR (100 MHz, CDCl3)
δ: 181.2, 163.9, 145.9, 138.5, 127.1, 124.4, 117.9, 115.9, 114.7, 74.7,
+
72.2, 71.7, 70.0. HRMS (ESI): m/z calcd for C19H13BrFeO2 ,
430.93460 and 432.93256; found, 430.93406 and 432.93201.16
6-Bromo-2-ferrocenyl-chromen-4-one, 16. Rf = 0.54 (ethyl
acetate/petroleum ether = 1:3, v/v), 40 mg of red solid, yield 40%.
m.p.: 177−179 °C. 1H NMR (400 MHz, CDCl3) δ: 8.34 (s, 1H), 7.75
(d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 6.47 (s, 1H), 4.87 (s,
2H), 4.56 (s, 2H), 4.18 (s, 5H). 13C NMR (100 MHz, CDCl3) δ:
176.0, 168.7, 155.0, 136.2, 128.4, 125.6, 119.7, 118.4, 105.7, 74.5, 71.7,
+
70.3, 67.5. HRMS (ESI): m/z calcd for C19H13BrFeO2 , 430.93460
and 432.93256; found, 430.93406 and 432.93201.16
(Z)-5-Nitro-2-ferrocenylidene-benzofuran-3-one, 17. Rf
In order to compare the influence of the ferrocenyl and
phenyl group on the antioxidant effects of aurone and flavone,
as shown in Scheme 1, aurone (1) and flavone (2) are prepared
by using the chalcone as the reactant and Hg(OAc)216 or CuI20
as the catalyst. Owing to the susceptibility to copper ion,
ferrocenyl-appended chalcone cannot be annulated by using
CuI as the catalyst. Meanwhile, the cyclyzation temperature for
the ferrocenyl-appended chalcone decreases from 110 °C (for
the phenyl-related chalcone) to 80 °C, followed by a ring-
opening reaction of the ferrocenyl-appended aurone to form
ferrocenyl-ynone in the presence of lithium diisopropylamide
=
0.65 (ethyl acetate/petroleum ether = 1:3, v/v), 2.28 g of black
1
solid, yield 60%. m.p.: 194−197 °C. H NMR (400 MHz, CDCl3) δ:
8.71 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.05
(s, 1H), 4.95 (s, 2H), 4.71 (s, 2H), 4.27 (s, 5H). 13C NMR (100 MHz,
CDCl3) δ: 180.6, 167.4, 146.2, 144.0, 130.8, 123.4, 121.0, 120.3, 113.8,
74.4, 73.1, 72.1, 70.5. HRMS (ESI) [M + H]+: m/z calcd for
C19H13FeNO4, 376.0272; found, 376.0299.
6-Nitro-2-ferrocenyl-chromen-4-one, 18. Rf = 0.46 (ethyl
acetate/petroleum ether = 1:3, v/v), 37 mg of red solid, yield 37%.
m.p.: 180−183 °C. 1H NMR (400 MHz, DMSO-d6) δ: 8.66 (d, J = 2.8
D
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(LDA). Then, the ferrocenyl-ynone is annulated to generate the
corresponding flavone under basic conditions. As a result, a
two-step operation converts the ferrocenyl-appended aurones
into flavones. Moreover, because of the sensitivity of the
hydroxyl group toward Hg(OAc)2, the hydroxyl group in
acetophenone is etherized by CH3OCH2Cl, which can be
removed under acidic conditions. Interestingly, the hydroxyl
group at the ortho-position of acetophenone cannot react with
CH3OCH2Cl because of an intramolecular hydrogen bond
formed between −OH and CO.
Table 1. Rate Constants (k) for Aurones and Flavones in
Scavenging ABTS+•, DPPH, and the Galvinoxyl Radical, and
the Stoichiometric Factor (n) in Protecting DNA against
a
AAPH-Induced Oxidation
rate constant (k) in scavenging
radicals ( × 103 M−1·s−1
)
compd
number
galvinoxyl
radical
stoichiometric factor
ABTS+• DPPH
(n)
1
2
Radical-Scavenging Properties. ABTS+•, DPPH (stable
N-centered radicals at room temperature), and the galvinoxyl
radical (stable O-centered radicals at room temperature) are
usually applied to estimate the ability of an antioxidant to
quench radicals.28 Reactions of antioxidants with the
aforementioned radicals are regarded as chemical measurement
of oxygen radical absorbance capacity (ORAC) and as the
antioxidative potential of the whole molecule. Especially,
reactions of antioxidants and these radicals can be expressed
by chemical kinetics, in which the reaction rate (r) is proved to
follow a second-order reaction. As an example, eq 1 expresses
the correlation of the reaction rate with the concentration of
DPPH.29
3
0.83
0.28
3.29( 0.16)
2.79 ( 0.14)
7.12 ( 0.36)
6.22 ( 0.31)
8.50 ( 0.43)
7.39 ( 0.37)
10.79 ( 0.54)
8.47 ( 0.42)
16.06 ( 0.80)
16.56 ( 0.82)
19.39 ( 0.97)
19.59 ( 0.98)
7.46 ( 0.37)
6.48 ( 0.32)
2.91 ( 0.15)
2.65 ( 0.13)
4
0.043
5
8.03
4.00
4.04
4.37
4.26
4.45
2.63
6
9.30
7
15.03
17.44
16.13
19.37
29.61
35.29
34.54
38.13
8.89
8
9
10
11
12
13
14
15
16
17
18
Trolox
0.23
0.61
2.45
1.89
0.57
2.42
4.04
1.57
2.41
0.353
11.49
4.63
d[DPPH]
−
= r = k[DPPH][antioxidant]
(1)
dt
4.98
Equation 1 is also available when the reaction time is 0 min. If
[DPPH]t=0 and [antioxidant]t=0 refer to concentrations of
DPPH and antioxidant at the beginning of the reaction, the
kinetic eq 1 can be converted into eq 2, in which the rate
constant (k) is calculated when the reaction rate at t = 0 min
(r0) is measured.30
29.2
1.70
a
The initial concentrations of every compound are listed in the
Supporting Information, and in the stoichiometric factor (n), Ri = Rg =
1.4 × 10−6 [AAPH] s−1 = 3.36 μM·min−1 when 40 mM AAPH is
employed; thus, n = coefficient × 3.36 μM·min−1.
effect on scavenging DPPH. However, a hydroxyl group
attached to aurone and flavone (as in 11, 13, 12, and 14)
increases the ability to quench ABTS+• even higher than that of
Trolox. However, the intramolecular interaction between
hydroxyl and ferrocenyl groups makes ferrocenyl-appended
aurone (11 and 13) inactive toward DPPH. The high k value of
13 in scavenging the galvinoxyl radical (2.45 mM−1 s−1, higher
than that of Trolox, 1.70 mM−1 s−1) indicates that the hydroxyl
group at the 5-position in aurone is more active than that at the
6-position. Moreover, the high k values of 12 and 14 in
scavenging all the radicals demonstrate that ferrocenyl-
appended flavones exhibit relatively high activities in quenching
radicals, especially, with a hydroxyl group attached to the 6-
position.
As shown in Scheme 2, the rate constant (k) of every
ferrocenyl-appended aurone or flavone in scavenging ABTS+• is
higher than that in scavenging DPPH or the galvinoxyl radical,
revealing that ferrocenyl-appended aurone and flavone are
more active in reducing radicals (such as ABTS+•) than
donating their electron or hydrogen atom to N- or O-centered
radicals (such as DPPH and galvinoxyl radical). ABTS+• is
generated by the oxidation of ABTS salt, and the redox couples
of ABTS•−/ABTS2− and ABTS•−/HABTS− are 0.68 and 0.81 V
(vs NHE), respectively. The second wave of the oxidative
potential (E1/2, associated with ABTS0/ABTS•−) is 1.09 V.31
The high values of E1/2 of ABTS in every state imply that
ABTS+• readily reacts with the phenolic hydroxyl group. As
reported in a previous work,14 E1/2 of ferrocenyl-appended
aurone (5) is 0.144 V (vs the redox of ferrocene cation/
ferrocene), and a methoxyl group attached to the 5-position (as
9) does not markedly change E1/2 (0.147 V). However, the
r0 = k[DPPH]t=0[antioxidant]t=0
(2)
The process for calculating the rate constant (k) is listed in
Table S1 (Supporting Information), and final results are
collected in Table 1 (Trolox as the reference compound) and
schematized in Scheme 2.
Aurone (1) and flavone (2) are not active toward any radical
because they do not contain a phenolic hydroxyl group. A
hydroxyl group attached to the 5- or 6-position of aurone or
flavone (as in 3 and 4) is active toward ABTS+•, indicating that
the phenolic hydroxyl group plays the major role in trapping
radicals. Especially, 4 can even donate its hydrogen atom from
the hydroxyl group to the galvinoxyl radical, implying that the
6-hydroxyl group in flavone (also called the para-position of
ring B) exhibits higher activity toward the O-centered radical
than the 5-hydroxyl group in aurone. By comparing 1 and 5, as
well as 2 and 6, it can be found that a ferrocenyl group as ring B
leads to a similar radical-scavenging property for aurone and
flavone, demonstrating that the ferrocenyl group can provide
the electron for quenching ABTS+• and DPPH and acts as a
novel radical-scavenging feature for aurone and flavone. The k
values of 7, 8, 9, and 10 in quenching ABTS+• (15−19 mM−1
s−1) are higher than that of 5 and 6 (8−9 mM−1 s−1), revealing
that methoxyl as an electron-donating group (EDG) is
beneficial to the ferrocenyl group (as ring B) in quenching
ABTS+•. However, their k values in quenching DPPH do not
vary markedly compared with those of 5 and 6 (both ∼4 mM−1
s−1), implying that the EDG cannot affect the ability of the
ferrocenyl group in quenching the N-centered radical. Mean-
while, an electron-withdrawing group (EWG) such as bromide
and the nitro group in 15, 17, and 18 does not exhibit an active
E
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Scheme 2. Comparison of the Radical-Scavenging Abilities of Aurones and Flavones
same group (CH3O−) attached to the 6-position (as 7)
decreases E1/2 to 0.118 V. The published result has
demonstrated that the introduction of the ferrocenyl group
actually decreases E1/2 and makes ferrocenyl-appended aurone
and flavone readily quench ABTS+•. However, the reaction of
an antioxidant with DPPH or galvinoxyl radical is owing to the
hydrogen atom or electron in the phenolic hydroxyl group
being transferred to the N- or O-centered radical.28 Thus,
different radicals are employed to react with aurones and
flavones in order to reveal the abilities toward different single-
electron centers. As shown in Scheme 2, higher k values in
quenching ABTS+• indicate higher abilities of aurones and
flavones to reduce the radical, while lower k values in
quenching DPPH and the galvinoxyl radical imply that these
compounds cannot efficiently provide a hydrogen atom or an
electron for the N- or O-centered radical. So, the different k
values of the same compound reveal the different abilities of the
compound toward different single-electron centers. The radical-
scavenging properties of ferrocenyl-appended aurones and
flavones motivate us to explore their abilities to protect DNA
against radical-induced oxidation.
inhibition concentrations (IC50).14 The IC50 values of 7 and 9
in inhibiting human fetal lung cells are 3.5 and 1.0 μM,
respectively, and 4.1 and 1.1 μM in inhibiting breast carcinoma
cells, respectively. Although the lower IC50 implies the potential
application of ferrocenyl-appended aurone in anticancer drugs,
the data obtained from cell lines cannot reveal the details of the
chemical reaction between biological materials and antiox-
idants. A research work published two decades ago reveals the
prooxidation of α-tocopherol in low-density lipoprotein (LDL)
by using a chemical kinetic method.32 The rate constant (r) and
stoichiometric factor (n) chemically express the reaction
between the α-tocopherol radical and LDL. Therefore, the
application of chemical kinetics on a biological system can
display reactions among biomaterials. The aim of the present
work is to employ the chemical kinetics in DNA oxidation
because the oxidation of DNA induced by 2,2′-azobis(2-
amidinopropane hydrochloride) (AAPH) is an in vitro
experimental system for DNA undergoing oxidation.33 The
products from DNA oxidation can be measured after reacting
with thiobarbituric acid (TBA) because the absorbance of the
thiobarbituric acid reactive species (TBARS) can be detected at
535 nm.34 As shown in Figure S4 (Supporting Information),
the increase of the absorbance in the control experiment
Inhibiting DNA Oxidation. Cytotoxicities of some
ferrocenyl-appended aurones have been expressed by the half-
F
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Scheme 3. Relationship between the n Values and the Structures of Aurones and Flavones
indicates that several TBARS are produced during the longer
reaction time. A higher absorbance value at 535 nm is observed
in the case of the added ferrocenyl-related compounds. This
phenomenon has been found in our previous report on the
antioxidant effect of ferrocenyl-appended ailanthoidol in the
same experimental system.35 The λmax of ferrocenyl-appended
compound is around 340 nm, and the absorbance at 535 nm is
very low. However, the addition of AAPH can also lead to the
decrease of the absorbance of ferrocenyl-appended compound
at 535 nm, indicating that the ferrocenyl-appended compound
can react with AAPH directly, and thus protect DNA against
AAPH-induced oxidation.35 The inhibition period (tinh) can be
measured by the time point from the beginning of the reaction
to the cross-point of tangents (for the decrease and increase
periods in the absorbance line), and the lower absorbance of
ferrocenyl-appended compounds at 535 nm cannot influence
the measurement of tinh. Additions of 1 and 2 (or Trolox, data
not shown) cannot affect the increase of the absorbance of
TBARS, but positions of the absorbance lines are lower than
that of the control experiment, revealing that 1 and 2 are able to
retard DNA oxidation. However, the addition of other
compounds can actually hinder the increase of the absorbance
for a period and then recover as the control experiment. Figure
S5 (Supporting Information) outlines the correlation of tinh
with the concentration of the tested compounds, and the
corresponding quantitative equations are in Table S4
(Supporting Information). In view of chemical kinetics, the
relationship between tinh and the concentration of an
antioxidant is expressed by eq 3, in which n (stoichiometric
factor) is a quantitative parameter for the radical-propagation
terminated by one molecule of the antioxidant, while Ri stands
for the radical-initiation rate mediated by AAPH.32
(Supporting Information) are equal to n/Ri. The sodium salt of
DNA and AAPH are both water-soluble compounds, and
radicals generated from AAPH attack DNA at the aqueous
phase. The radical-generation rate from the decomposition of
AAPH (Rg = (1.4 0.2) × 10−6 [AAPH] s−1)32 is assumed to
be equal to Ri. Hence, the n value is the product of the
coefficient of equations in Table S4 (Supporting Information)
and Ri, and listed in Table 1. A higher n value implies that the
compound possesses high cytotoxicity in AAPH-induced
oxidation of DNA, and correlations of structures of
ferrocenyl-appended aurones and flavones with n values are
schematized in Scheme 3.
The phenyl-appended aurone (1) and flavone (2) cannot
generate tinh in AAPH-induced oxidation of DNA, and thus, no
n values are obtained. The n values of ferrocenyl-appended
aurone (5) and flavone (6) are 7.12 and 6.22, respectively,
indicating that the introduction of the ferrocenyl group into
scaffolds of aurone and flavone generates the cytotoxicity for
aurone and flavone. Moreover, the higher n values of 5 and 6
(7.12 and 6.22, respectively) than those of 3 (3.29) and 4
(2.79) indicate that the ferrocenyl group is even more active
than the phenolic hydroxyl group in phenyl-appended aurone
and flavone. We have reported that the ferrocenyl group in
ailanthoidol is able to react with AAPH directly and protects
DNA against AAPH-induced oxidation.35 Thus, the cytotoxicity
of ferrocenyl-appended aurone and flavone is due to the
ferrocenyl group directly scavenging the radical. This has been
proved by the fact that 5 and 6 (without the phenolic hydroxyl
group) can quench ABTS+• and DPPH. Meanwhile, the
antioxidant effect of ferrocenyl-appended aurone is higher than
that of ferrocenyl-appended flavone. This rule can also be found
in ferrocenyl-appended aurones and flavones with other
substituents. The bromide cannot affect the inhibitory effects
of ferrocenyl-appended aurone and flavone because n values of
16 (6.48) and 15 (7.46) are the same as those of 6 and 5, but
the nitro group as an EWG decreases n values of 18 and 17 to
tinh = (n/Ri)[antioxidant]
(3)
Equations in Table S4 (Supporting Information) are equivalent
to eq 3, and the coefficients of the equations in Table S4
G
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2.65 and 2.91, respectively. On the contrary, the methoxyl
group as an EDG can markedly increase the cytotoxicities of
ferrocenyl-appended aurone and flavone since n values of 8
(7.39), 10 (8.47), 7 (8.50), and 9 (10.79) are much higher than
those of 5 and 6. In particular, when the methoxyl group
attaches to the 5-position of aurone or the 6-position of flavone,
the cytotoxicities of the corresponding aurone and flavone are
higher than that of the methoxyl group at the 6- or 7- position,
again demonstrating that the EDG at the para-position is
beneficial for the antioxidant effect. Furthermore, introducing
the hydroxyl group into the ferrocenyl-appended aurone and
flavone increases n values of 11 (16.06), 12 (16.56), 13
(19.39), and 14 (19.59) to the zenith among compounds used
herein. The benefit of the para-hydroxyl group for the
antioxidant effect is still available for 13 and 14, in which the
ferrocenyl in combination with the hydroxyl group in both
aurone and flavone can efficiently protect DNA against radical-
induced oxidation. To sum up, the n value is not a simple
phenomenon but a quantitative parameter for revealing the
antioxidative effectiveness. The measurement of the parameter
is derived from various concentrations of antioxidant employed,
and the initiation rate (Ri) for the DNA oxidation is also taken
into consideration. So, n is not a concentration- and initiation-
rate-dependent value. As pointed out in the aforementioned
discussion, the comparison of the n values quantitatively
displays the influence of the substituents on the antioxidative
effectiveness.
ABBREVIATIONS
■
AAPH, 2,2′-azobis(2-amidinopropane hydrochloride; ABTS+·,
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radi-
cal; DPPH, 2,2′-diphenyl-1-picrylhydrazyl radical; EDG,
electron-donating group; EWG, electron-withdrawing group;
k, rate constant; n, stoichiometric factor; tinh, inhibition period;
TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive
species
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CONCLUSIONS
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ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra of the synthetic compounds, the decay of the
concentration of radicals together with the calculation of the
rate constant (k) in quenching radicals, and the TBARS in the
case of AAPH-induced oxidation of DNA together with the
calculation of n values. This material is available free of charge
via the Internet at http://pubs.acs.org.
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(2012) In vitro inhibitory properties of ferrocene-substituted chalcones
and aurones on bacterial and human cell cultures. Dalton Trans. 41,
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flavonols. Organometallics 30, 5424−5432.
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Evaluation of antioxidants: Scope, limitations and relevance of assays.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: zaiqun-liu@jlu.edu.cn.
Funding
Financial support from Jilin Provincial Science and Technology
Department, China, is gratefully acknowledged
(20130206075GX).
Notes
The authors declare no competing financial interest.
H
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Chemical Research in Toxicology
Article
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DOI: 10.1021/tx500405b
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX