Synthesis and evaluation of selenoflavones that have potential neuroprotective effects

Article abstract of DOI:10.3987/COM-14-13112

We synthesized selenoflavones and evaluated their physicochemical properties and antioxidant effects. When oxygen was substituted with selenium, the compounds exhibited improved polarity and lipophilicity, implying that this change could lead to better BBB penetration. Selenoflavones revealed more potent antioxidant activity in our in-vitro assay. This suggests that selenoflavones would be more druggable than flavones and have a better potential as a neuroprotective agent.

Full text of DOI:10.3987/COM-14-13112

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HETEROCYCLES, Vol. 89, No. 12, 2014
HETEROCYCLES, Vol. 89, No. 12, 2014, pp. 2794 - 2805. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 20th October, 2014, Accepted, 21st November, 2014, Published online, 12th December, 2014
DOI: 10.3987/COM-14-13112
SYNTHESIS AND EVALUATION OF SELENOFLAVONES THAT HAVE
POTENTIAL NEUROPROTECTIVE EFFECTS
Yong-Sung Choi,1,2 Yoon-Jung Kim,2 Ju Yeun Lee,2 Jinu Lee,2 and Jin-Hyun
Jeong2,*
1
College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro,
2
Dongdaemun-gu, Seoul 130-701, Republic of Korea. College of Pharmacy,
Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85
Songdogwahak-ro, Yeonsu-gu, Incheon 406-840, Republic of Korea. E-Mail:
organicjeong@yonsei.ac.kr
Abstract – We synthesized selenoflavones and evaluated their physicochemical
properties and antioxidant effects. When oxygen was substituted with selenium,
the compounds exhibited improved polarity and lipophilicity, implying that this
change could lead to better BBB penetration. Selenoflavones revealed more
potent antioxidant activity in our in-vitro assay. This suggests that selenoflavones
would be more druggable than flavones and have a better potential as a
neuroprotective agent.
Flavonoids are phenolic compounds, which are mainly found in fruits, herbs, and grains.1,2 In recent years,
flavonoids have increasingly attracted scientific and public attention, due to their beneficial effects
against oxidative stress, inflammation, and certain cancers.3-5 Up to date, several synthetic and biological
studies have been done to evaluate flavonoids as a potential neuroprotective drug.6-8 However, many
hydroxyl- and alkoxy- groups of flavonoids make them strongly polar. The high polarity makes it difficult
to pass them through membranes. We hypothesized that polarity and lipophilicity can be improved by
substituting selenium for oxygen atom in flavones(1).
It has been known that selenium, as a trace element in the human body, exists in a form of selenoproteins.
Glutathione peroxidase (GPx) is one of the selenoproteins that exhibit antioxidant function.9,10
Furthermore, synthetic GPx mimetic compounds have been developed. Those synthetic organoselenium
compounds show more potent antioxidant effects.11-13

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Based on these previous studies, we hypothesized that selenoflavonoids can pass through blood-brain
barrier (BBB) more easily and have a synergetic antioxidant effect. To test this hypothesis, our group
focused on synthesizing selenoflavones (2) as a potential neuroprotective agent.
O
Se
O
R
R
O
flavones (1)
2
selenoflavones ( )
Figure 1. Structure of flavones and selenoflavones
Synthetic pathway to generate selenoflavones is illustrated in Scheme 1. 3-Phenylpropioloyl chloride was
prepared from 3-phenylpropiolic acid and used without purifications.14 Bromobenzenes and
3-phenylpropioloyl chloride were conducted at Friedel-Crafts condition which formed 3. Then compound
3 was treated with sodium hydrogen selenide (NaHSe), in-situ generated from selenium powder and
sodium borohydride (NaBH4) to afford the selenoflavone (2a, 2b).15
Scheme 1. Reagents and conditions : (a) COCl2, DMF(cat.) CH2Cl2, rt, 1 h;
(b) AlCl3, CH2Cl2, 0 °C, 2 h; (c) Se, NaBH4, DMF, 100 °C, 5 h

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Treatment of 5,6,7-trihydoxyflavone (baicalein, 1g) and 5,7-dihydoxyflavone (chrysin, 1f) with dimethyl
sulfate generated corresponding methylated flavones (1a, 1b) (Scheme 2).
Scheme 2. Reagents and conditions : (a) Me2SO4, K2CO3, acetone, reflux, overnight
Further modification of compounds (1a-1c, 2a-2e) by boron tribromide (BBr3) at low temperature
resulted in selective demethylation to get compounds (1c-1e, 2c-2g) (Scheme 3). All the prepared
flavones (1a-1f) and selenoflavones (2a-2f) are listed in Table 1.
R3
R2
M
O
R3
R2
M
O
a
R1
R1
1
2
3
1a
1b
1c
2a
2b
2c
2d
2e
1c
1d
1e
2c
2d
2e
2f
: M = O, R = OMe, R = OMe, R = OMe
: M = O, R1 = OH, R2 = OMe, R3 = OMe
: M = O, R1 = OH, R2 = H, R3 = OMe
: M = O, R1 = OH, R2 = OH, R3 = OMe
1
2
: M = O, R = OMe, R = H,
R3 = OMe
1
: M = O, R = OH, R2 = OMe, R3 = OMe
1
2
3
1
2
3
: M = Se, R = OMe, R = OMe, R = OMe
: M = Se, R = OH, R = OMe, R = OMe
1
2
1
2
: M = Se, R = OMe, R = H,
R3 = OMe
: M = Se, R = OH, R = H,
R3 = OMe
: M = Se, R = OH, R = OH, R3 = OMe
: M = Se, R1 = OH, R2 = H R3 = OH
: M = Se, R = OH, R = OH, R3 = OH
1
1
2
: M = Se, R = OH, R2 = OMe, R3 = OMe
1
: M = Se, R = OH, R2 = H
R3 = OMe
1
1
2
: M = Se, R = OH, R2 = OH, R3 = OMe
2g
Scheme 3. Reagents and conditions : (a) BBr3, CH2Cl2, -40 °C to rt, 2 h
To confirm the difference between flavones and selenoflavones, topological polar surface area (tPSA) and
calculated partition coefficient (ClogP) were generated by Cambridgesoft ChemBioDraw software (Table
2) and retention time of compounds in reversed phase HPLC was measured (Table 3).
Polar surface area (PSA) of a molecule is the total surface of polar atoms. It is one of the druglikeness
factors showing the polarity of molecules. Molecules with a PSA of less than 140 Å2 were predicted to
have good cell membrane permeability.16 And a PSA less than 90 Å2 is usually needed to penetrate the
BBB.17 Calculated tPSA was decreased by changing oxygen to selenium. Therefore, selenoflavones were
expected to move through BBB more easily than flavones.

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Table 1. Structure-analytical data of flavone and selenoflavone derivatives
R3
R2
M
O
R1
Compound
M
O
O
O
O
R1
OMe
OMe
OH
OH
OH
R2
OMe
H
OMe
H
OH
H
R3
OMe
OMe
OMe
OMe
OMe
OH
1a
1b
1c
1d
1e
1f
O
O
OH
O
OH
OH
OMe
H
OMe
H
OH
H
OH
OH
1g
2a
2b
2c
2d
2e
2f
Se
Se
Se
Se
Se
Se
Se
OMe
OMe
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OH
OH
OH
OH
2g
Table 2. Calculated physicochemical properties of flavones and selenoflavones
Physicochemical properties
Physicochemical properties
Flavone
Selenoflavone
ClogPa
2.88
3.62
3.52
4.15
3.33
3.56
3.00
ClogPa
3.37
4.11
4.01
4.64
3.82
4.05
3.49
tPSAa (Å2)
54.0
tPSAa (Å2)
44.8
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
44.8
65.0
55.8
76.0
66.8
87.0
35.5
55.8
46.5
66.8
57.5
77.8
1g
2g
(a) Generated by Cambridgesoft ChemBioDraw software
Table 3. Retention time of flavones and selenoflavones in reversed phase HPLC
Retention time in HPLC (min)
Retention time in HPLC (min)
Flavone
Selenoflavone
Method Aa
1.421
Method Bb
1.862
Method Aa
1.562
Method Bb
2.230
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
1.515
2.069
1.912
3.285
1.556
2.201
1.776
2.801
1.943
3.248
2.375
4.549
1.316
1.641
1.451
1.986
1.458
1.964
1.626
2.457
1.265
1.527
1.399
1.860
1g
2g
(a) MeOH:H2O = 90:10, flow 1 mL/min; (b) MeOH:H2O = 80:20, flow 1 mL /min

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LogP is the concentration ratio of a compound between 1-octanol and water. The logP is known as a
measure of lipophilicity. Compounds with high logP value are lipophilic which are well distributed to
hydrophobic compartment like lipid bilayer of the membrane. Selenoflavones exhibited larger ClogP
value which represents to be more lipophilic than flavones.
For confirming the accuracy of the calculated ClogP value of compounds, it was plotted against the
retention time. Because retention time of compounds is also affected by its partition coefficient, LogP can
be determined by correlating retention time of compounds with known logP value of similar
compounds.18,19 Thus, we inferred that correlation between ClogP and retention time might signify the
accuracy of the calculated value. As shown in Figure 2, the slope of Method A was approximately linear
with correlation coefficient r = 0.8763. Method B also represented a high correlation between these two
factors (r = 0.8769). It means that calculated ClogP is trustworthy and increased ClogP value of
compounds reveals actual increment of lipophilicity.
Figure 2. Relationship of the retention time and the calculated ClogP value
To compare the antioxidant activity of flavones and selenoflavones, we treated neuro2a cells with these
compounds and quantified reactive oxygen species (ROS) in cells by DCFDA, a ROS-detecting
fluorescence dye.20 Decrease in cellular ROS by a chemical represents antioxidant activity. As shown in
Figure 3, compounds 1e, 2e, 1g, and 2g showed higher antioxidant activity compared to that of other
compounds. However, these compounds have larger tPSA and smaller ClogP values than others. The
results of antioxidant assay were not be correlated by only one factor. The factors could include the
oxidation potential, polarity, and lipophilicity of compounds. There is no consistent difference between
antioxidant activity of flavones and selenoflavones. However, we did not deduce from this observation

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2799
that selenium has no influence to antioxidant effect. It should be necessary to do additional research to
ensure the results. Instead, we suspected that antioxidant effect could be in discord with prooxidant effect.
The next experiment which showed in Figure 4 could be explained the possible difference between
antioxidant effect and prooxidant effect.
Figure 3. Antioxidant activity of flavones and selenoflavones at 10 M
Figure 4. Dose-response experiments with 1e and 2e
We performed the dose-response experiments with 2e which presented best antioxidant activity among
selenoflavones at 10 M and corresponding flavone (1e). Compound 2e exhibited antioxidant effect up to

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90% at 100M. In contrast, 1e showed a sharp decline in its antioxidant effect at 100M. Depending on
the condition, some substances can behave as either antioxidants or prooxidants.21,22 Concentration is one
of the important conditions.23 At high concentration, 1e might act not as an antioxidant but as a
prooxidant that induces oxidative stress. From these results, 2e is expected to be more potent and safer
antioxidant than 1e.
In summary, we have synthesized selenoflavones and evaluated their physicochemical properties and
in-vitro antioxidant effects. As we hypothesized, substitution of selenium for oxygen atom in flavone led
to improved physicochemical and biological properties. Therefore, selenoflavoids are anticipated to
penetrate BBB better and be more druggable as a neuroprotective drug.
EXPERIMENTAL
General Information
CH2Cl2 used for the reaction was LC grade reagent and redistilled from calcium hydride. All other
reagents were used as received without purification. Reactions were monitored by thin-layer
chromatography on 0.25 mm silica plate (F-254) visualizing with UV light (254 nm) and KMnO4 solution.
Flash chromatography was performed using silica gel (230-400 mesh) with hexanes and EtOAc as eluent.
1H and 13C NMR spectra were recorded on Agilent 400 MHz NMR spectrometer. Chemical shifts are
reported in parts per million (δ), and coupling constants (J) are expressed in Hertz. HR-MS were
measured with electrospray ionization (ESI) and Q-TOF mass analyzer. Physicochemical properties were
generated by CS ChemProp. Retention time of compounds was measured with Agilent 1200 series HPLC
and Agilent ZORBAX Eclipse Plus C18 (4.6 x 100 mm, 3.5 µm) column. Cellular fluorescence was
measured with multi-plate reader (Tecan, Männedorf, Switzerland) at 490nm/520nm.
Procedure for the Formation of 3-Phenylpropioloyl Chloride
Oxalyl chloride (1.2 mmol) and DMF (cat.) were added to a solution of 3-phenylpropiolic acid (1.0
mmol) in CH2Cl2 (10 mL) and the mixture was stirred for 1 h at rt. After the excess reagent and solvent
were distilled off, the residue was dried in vacuo. 3-Phenylpropioloyl chloride was used without further
purification.
General Procedure for the Friedel-Crafts Acylation
Aluminum chloride powder (2.0 mmol) was suspended in CH2Cl2 (10 mL) at 0 °C. 3-Phenylpropioloyl
chloride (1.2 mmol) in CH2Cl2 (5 mL) was added and the mixture was stirred for 30 min. Then a solution
of bromobenzene (1.0 mmol) in CH2Cl2 (5 mL) was added dropwise and stirred for another 2 h. After the
addition of water (20 mL), the organic layer was separated and the aqueous layer was extracted with

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CH2Cl2 (3 x 30 mL). The combined organic extracts were filtered through celite pad, washed with water
(3 x 100 mL) and brine (100 mL), dried (MgSO4), and concentrated. The resulting residue was purified
by flash chromatography to give (3a, 3b) in 85-92% yields.
1
1-(6-Bromo-2,3,4-trimethoxyphenyl)-3-phenylprop-2-yn-1-one (3a) H NMR (400 MHz, CDCl3) δ
7.60 (d, J = 7.1 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.38 (t, J = 7.4 Hz, 2H), 6.91 (s, 1H), 3.98 (s, 3H), 3.90
(s, 3H), 3.88 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 177.5, 155.5, 152.5, 141.7, 133.1, 130.8, 128.6,
120.2, 113.1, 112.3, 110.0, 92.9, 89.2, 62.1, 60.9, 56.4; HR-ESI-MS: m/z 375.0212 [M+H]+ (calcd for
+
C18H16BrO4 , 375.0226)
1
1-(2-Bromo-4,6-dimethoxyphenyl)-3-phenylprop-2-yn-1-one (3b) H NMR (400 MHz, CDCl3) δ 7.58
(d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.3 Hz, 1H), 7.37 (t, J = 7.3 Hz, 2H), 6.74 (s, 1H), 6.45 (s, 1H), 3.84 (d, J
13
= 0.7 Hz, 6H); C NMR (101 MHz, CDCl3) δ 177.9, 162.1, 159.2, 133.2, 130.7, 128.5, 124.0, 120.7,
+
120.4, 109.8, 98.4, 92.3, 89.3, 56.2, 55.8; HR-ESI-MS: m/z 345.0119 [M+H]+ (calcd for C17H14BrO3 ,
345.0121)
General Procedure for the Formation of Selenoflavones
To the stirred solution of NaHSe (1.2 mmol) which was prepared from selenium powder (1.2 mmol) and
NaBH4 (1.2 mmol) in DMF (10 mL) at 100 °C for 1 h, a solution of (3a, 3b) (1.0 mmol) in DMF (10 mL)
was slowly added and the mixture was stirred for 5 h under these conditions. After the addition of water
(10 mL), the mixture was filtered to remove remained selenium. The filtrate was extracted with CH2Cl2 (3
x 10 mL). The combined organic extracts were washed with water (5 x 20 mL) and brine (20 mL), dried
(MgSO4), and concentrated. The resulting residue was purified by flash chromatography to give (2a, 2b)
in 56-62% yields.
5,6,7-Trimethoxyselenoflavone (2a) 1H NMR (400 MHz, CDCl3) δ 7.64 – 7.56 (m, 2H), 7.50 – 7.43 (m,
3H), 7.23 (s, 1H), 6.94 (s, 1H), 4.01 – 3.89 (m, 9H); 13C NMR (101 MHz, CDCl3) δ 182.8, 156.6, 156.2,
149.1, 143.0, 137.6, 134.1, 130.5, 129.2, 126.8, 126.8, 120.8, 105.7, 62.0, 61.4, 56.2; HR-ESI-MS: m/z
377.0275 [M+H]+ (calcd for C18H17O4Se+, 377.0287)
1
5,7-Dimethoxyselenoflavone (2b) H NMR (400 MHz, CDCl3) δ 7.65 – 7.55 (m, 2H), 7.50 – 7.41 (m,
3H), 7.21 (s, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.51 (d, J = 2.2 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 183.1, 163.7, 162.1, 147.9, 141.4, 137.5, 130.4, 129.2, 127.8, 126.8, 116.4, 102.9,
99.3, 56.3, 55.7; HR-ESI-MS: m/z 347.0207 [M+H]+ (calcd for C17H15O3Se+, 347.0181)

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General Procedure for the Methylation of Flavones
Dimethyl sulfate (3.5mmol) and K2CO3 (4.5 mmol) were added to a solution of (1g, 1f) (1.0 mmol) in
acetone (10 mL) and the mixture was stirred for overnight at reflux condition. After the excess solvent
was distilled off, the residue was diluted with EtOAc (10 mL) and water (10 mL). Then the organic layer
was separated and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic
extracts were washed with water (2 x 20 mL) and brine (20 mL), dried (MgSO4), and concentrated. The
resulting residue was purified by flash chromatography to give (1a, 1b) in 92-95% yields.
1
5,6,7-Trimethoxyflavone (1a) H NMR (400 MHz, CDCl3) δ 7.88 (s, 2H), 7.52 (s, 3H), 6.83 (s, 1H),
6.68 (s, 1H), 4.16 – 3.72 (m, 9H); 13C NMR (101 MHz, CDCl3) δ 177.2, 161.1, 157.7, 154.5, 152.5, 140.3,
131.5, 131.3, 129.0, 125.9, 112.9, 108.4, 96.3, 62.2, 61.6, 56.3; HR-ESI-MS: m/z 313.1071 [M+H]+
+
(calcd for C18H17O5 , 313.1071)
1
5,7-Dimethoxyflavone (1b) H NMR (400 MHz, CDCl3) δ 7.90 – 7.84 (m, 2H), 7.54 – 7.48 (m, 3H),
6.68 (s, 1H), 6.58 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H).; 13C NMR (101
MHz, CDCl3) δ 177.6, 164.1, 160.9, 160.6, 159.9, 131.5, 131.2, 128.9, 125.9, 109.3, 109.1, 96.2, 92.8,
+
56.4, 55.8; HR-ESI-MS: m/z 283.0961 [M+H]+ (calcd for C17H15O4 , 283.0965)
General Procedure for the Demethylation of Flavones and Selenoflavones
1M BBr3 in CH2Cl2 (1 mL, 1 mmol) was added dropwise to a solution of (1a-1c, 2a-2e) (1.0 mmol) in
CH2Cl2 (10 mL) at -40 °C. Then temperature was slowly raised to rt during 2 h. After the addition of
water (10 mL), the organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3 x 10
mL). The combined organic extracts were washed with water (3 x 20 mL) and brine (20 mL), dried
(MgSO4), and concentrated. The resulting residue was purified by flash chromatography to give (1c-1e,
2c-2g) in 71-89% yields.
5-Hydroxy-6,7-dimethoxyflavone (1c) 1H NMR (400 MHz, CDCl3) δ 12.70 (s, 1H), 7.95 – 7.84 (m, 2H),
7.63 – 7.49 (m, 3H), 6.69 (s, 1H), 6.58 (s, 1H), 3.98 (s, 3H), 3.94 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
182.7, 163.9, 158.9, 153.3, 153.0, 132.6, 131.9, 131.3, 129.1, 126.3, 112.5, 105.6, 90.6, 60.9, 56.3;
+
HR-ESI-MS: m/z 299.0917 [M+H]+ (calcd for C17H15O5 , 299.0914)
1
5-Hydroxy-7-methoxyflavone (1d) H NMR (400 MHz, CDCl3) δ 12.72 (s, 1H), 7.88 (dd, J = 7.9, 1.6
Hz, 2H), 7.58 – 7.49 (m, 3H), 6.66 (s, 1H), 6.50 (d, J = 2.2 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 3.88 (s,

HETEROCYCLES, Vol. 89, No. 12, 2014
2803
3H); 13C NMR (101 MHz, CDCl3) δ 182.5, 165.6, 164.0, 162.2, 157.8, 131.8, 131.3, 129.1, 126.3, 105.8,
+
105.7, 98.2, 92.7, 55.8; HR-ESI-MS: m/z 269.0807 [M+H]+ (calcd for C16H13O4 , 269.0808)
5,6-Dihydroxy-7-methoxyflavone (1e) 1H NMR (400 MHz, CDCl3) δ 12.51 (s, 1H), 7.90 (d, J = 6.8 Hz,
13
2H), 7.59 – 7.48 (m, 3H), 6.70 (s, 1H), 6.63 (s, 1H), 4.02 (s, 3H); C NMR (101 MHz, cdcl3) δ 182.7,
164.1, 152.9, 150.7, 145.6, 138.3, 131.8, 131.4, 129.6, 129.1, 126.3, 105.5, 90.5, 56.5; HR-ESI-MS: m/z
+
285.0753 [M+H]+ (calcd for C16H13O5 , 285.0757)
5-Hydroxy-6,7-dimethoxyselenoflavone (2c) 1H NMR (400 MHz, CDCl3) δ 7.64 – 7.59 (m, 2H), 7.54 –
7.48 (m, 3H), 7.23 (s, 1H), 6.76 (s, 1H), 3.98 (s, 3H), 3.94 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 187.8,
158.9, 157.2, 155.5, 137.4, 135.3, 134.7, 131.0, 129.4, 126.8, 124.4, 112.5, 100.6, 60.7, 56.3;
HR-ESI-MS: m/z 363.0120 [M+H]+ (calcd for C17H15O4Se+, 363.0130)
5-Hydroxy-7-methoxyselenoflavone (2d) 1H NMR (400 MHz, CDCl3) δ 7.61 – 7.57 (m, 2H), 7.49 (d, J
13
= 7.0 Hz, 3H), 7.19 (s, 1H), 6.71 (d, J = 2.3 Hz, 1H), 6.47 (d, J = 2.3 Hz, 1H), 3.86 (s, 3H); C NMR
(101 MHz, CDCl3) δ 187.4, 167.2, 163.7, 155.0, 140.1, 137.4, 131.0, 129.4, 126.7, 124.6, 111.1, 104.3,
100.7, 55.6; HR-ESI-MS: m/z 333.0054 [M+H]+ (calcd for C16H13O3Se+, 333.0024)
5,6-Dihydroxy-7-methoxyselenoflavone (2e) 1H NMR (400 MHz, CDCl3) δ 7.65 – 7.59 (m, 2H), 7.54 –
7.48 (m, 3H), 7.24 (s, 1H), 6.78 (s, 1H), 5.65 (s, 1H), 4.01 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 187.6,
156.5, 151.6, 151.2, 137.6, 132.3, 131.0, 129.9, 129.4, 126.8, 124.2, 111.7, 100.6, 56.4; HR-ESI-MS: m/z
348.9979 [M+H]+ (calcd for C16H13O4Se+, 348.9974)
1
5,7-Dihydroxyselenoflavone (2f) H NMR (400 MHz, CD3OD) δ 7.66 – 7.56 (m, 2H), 7.48 (d, J = 3.9
Hz, 3H), 7.04 (s, 1H), 6.57 (d, J = 1.6 Hz, 1H), 6.14 (d, J = 1.6 Hz, 1H); 13C NMR (101 MHz, CD3OD) δ
186.1, 169.5, 166.6, 154.0, 140.1, 137.6, 130.5, 129.0, 126.3, 123.6, 107.8, 107.5, 103.6; HR-ESI-MS:
m/z 318.9888 [M+H]+ (calcd for C15H11O3Se+, 318.9868)
1
5,6,7-Trihydroxyselenoflavone (2g) H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 6.7 Hz, 2H), 7.51 –
7.39 (m, 3H), 7.08 (s, 1H), 6.75 (s, 1H), 3.27 (s, 2H); 13C NMR (101 MHz, CD3OD) δ 188.8, 157.7, 154.1,
152.8, 139.0, 133.3, 132.0, 130.8, 130.5, 127.7, 124.5, 111.4, 106.1; HR-ESI-MS: m/z 334.9810 [M+H]+
(calcd for C15H11O4Se+, 334.9817)
Antioxidant Assay

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To measure the antioxidant activities of flavones and selenoflavones, we quantified reactive oxygen
species (ROS) in cells treated with the compounds. Dichlorofluorescin diacetate (DCFDA) is
cell-permeable, but once it enters cells, it is deacetylated by cellular esterases and cannot exit cells. Since
non-fluorescent dichlorofluorescin is oxidized by intracellular ROS to a fluorescent dye,
dichlorofluorescein (DCF), if a compound reduces the fluorescence of cells loaded with DCFDA, it has
antioxidant activity. Neuro2a cells were plated in 96-well plates and were grown to 80% confluence.
Media were changed to new media containing each compound at the indicated concentration 1 h before
loading of DCFDA at 10 μM for 20 min. After further incubation with 10 μM H2O2, cellular fluorescence
was measured with multi-plate reader (Tecan, Männedorf, Switzerland) at 490nm/520nm. Antioxidant
activity of a compound was measured as decrease in fluorescence compared to DMSO treated group and
expressed as % ± SD.
ACKNOWLEDGEMENTS
This work was supported by grants from Yonsei University Global Specialization Project of 2013, and
Ukseung Chemical Co., Ltd.
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