Synthesis and evaluation of neuroprotective selenoflavanones

Article abstract of DOI:10.3390/ijms161226188

The physicochemical properties and antioxidant activity of a molecule could be improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones showed improved physicochemical properties, suggestive of the ability to pass through the blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse model were significantly reduced by the selenoflavanone treatments.

Full text of DOI:10.3390/ijms161226188

Article
Synthesis and Evaluation of Neuroprotective
Selenoflavanones
Yong-Sung Choi 1, Dong-Myung Kim 2, Yoon-Jung Kim 1, Sai Yang 1, Kyung-Tae Lee 3,
Jong Hoon Ryu 3 and Jin-Hyun Jeong 1,
*
Received: 1 November 2015; Accepted: 4 December 2015; Published: 10 December 2015
Academic Editor: Katalin Prokai-Tatrai
1
College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro,
Yeonsu-gu, Incheon 21983, Korea; uriys2@gmail.com (Y.-S.C.); sunshine333@naver.com (Y.-J.K.);
saiyang871001@gmail.com (S.Y.)
Office of Research Affairs, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03720, Korea;
2
dongmk@yonsei.ac.kr
3
College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02453, Korea;
ktlee@khu.ac.kr (K.-T.L.); jhryu63@khu.ac.kr (J.-H.R.)
*
Correspondence: organicjeong@yonsei.ac.kr; Tel.: +82-32-749-4509; Fax: +82-32-749-4105
Abstract: The physicochemical properties and antioxidant activity of a molecule could be
improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized
selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones
showed improved physicochemical properties, suggestive of the ability to pass through the
blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and
did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse
model were significantly reduced by the selenoflavanone treatments.
Keywords: selenoflavanone; flavanone; selenium; neuroprotect; antioxidant; blood-brain barrier (BBB)
1. Introduction
Flavonoids, a group of phenolic compounds with diverse chemical structures and characteristics,
occur naturally in the fruits, seeds, flowers, and bark of plants [1,2]. To date, several studies have
demonstrated the anti-oxidant, anti-inflammation, and anti-cancer activities of flavonoids [3–5].
Reactive oxygen species (ROS), such as hydrogen peroxide, are generated by cerebral ischemia
conditions, such as stroke [6,7]. These ROS induce cell apoptosis, resulting in damage to brain
cells [8–10]. Therefore, an antioxidant compound that scavenges ROS could play an important
role in the treatment of neurodegenerative disease. The neuroprotective effects of antioxidants
have been well studied [11,12]. Recently, the flavonoids were highlighted as potentially promising
neuroprotective agents [13–15], but their high polarity limits their ability to penetrate the blood-brain
barrier (BBB).
Our group recently reported that the substitution of an oxygen atom in flavone with selenium
led to improvement in the physicochemical properties and biological activity of molecules [16]. These
selenoflavones had lower polarity (tPSA) and higher lipophilicity (ClogP) than their corresponding
flavones, and, consequently, were anticipated to better penetrate the BBB. Moreover, they showed
improved in vitro antioxidant activity with respect to scavenging hydrogen peroxide. Therefore, we
expected the selenoflavonoids to be more druggable and more potent neuroprotective agents.
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Int. J. Mol. Sci. 2015, 16, 29574–29582
2. Results and Discussion
Based on previous studies, we decided to synthesize some additional selenoflavanoids
and evaluate their in vivo neuroprotective activity. We designed and synthetized two simple
selenoflavanones (1a and 1b). The corresponding flavanones (2a and 2b) were also synthesized to
allow for a comparison of their neuroprotective activities. The structures of the compounds utilized
in this study are indicated in Figure 1.
Selenoflavanone 1
Flavanone 2
Figure 1. The structures of selenoflavanones and flavanones.
2.1. Synthesis of the Selenoflavanones and Flavanones
The synthetic pathway of the Selenium flavanones 1 is described in Scheme 1. Compound 4 was
conducted from bromobenzene 3 and cinnamoyl chloride under AlCl3 conditions [17]. To introduce
a selenium atom into the heterocycle, compound 4 was treated with t-BuLi, followed by elemental
selenium [18]. The selenoflavanones 1 were obtained using a two-step synthesis resulting in 76%–80%
overall yields.
Scheme 1. Reagents and conditions: (a) cinnamoyl chloride, AlCl3, CH2Cl2, 0 ˝C, 2 h; (b) t-BuLi, Se,
THF, ´78 ˝C to room temperature, 75 min.
We successfully synthesized the corresponding flavanones 2, as shown in Scheme 2. Acylation
of the phenol compound 5 with cinnamoyl chloride was carried out under BF3-Et2O conditions [19].
Treatment of compound 6 with NaOAc led to intramolecular cyclization to form the flavones 2 [20]
in 72%–75% two-step yields.
Scheme 2. Reagents and conditions: (a) cinnamoyl chloride, BF3-Et2O, reflux, 15 min; (b) NaOAc,
EtOH, H2O (cat.), reflux, 6 h.
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2.2. Physicochemical Properties of the Selenoflavanones and Flavanones
First, we evaluated the physicochemical properties of the selenoflavanones and flavanones.
Polar surface area (PSA) is one of the drug-like factors demonstrating the polarity of molecules.
The partition coefficient (logP) is known to be a measure of lipophilicity. The topological polar
surface area (tPSA) and calculated partition coefficient (ClogP) were generated using Cambridgesoft
ChemBioDraw software (Waltham, MA, USA) (Table 1). The selenoflavanones had lower tPSA and
higher ClogP values than their corresponding flavanones. By substituting oxygen with selenium, the
polarity and lipophilicity were improved, likely allowing the compounds to more easily pass through
the BBB [21,22].
Table 1. Calculated physicochemical properties of selenoflavanones and flavanones.
Physicochemical Properties
Physicochemical Properties
Selenoflavanone
Flavanone
ClogP a
ClogP a
tPSA a (Å2)
tPSA a (Å2)
1a
1b
26.3
35.5
a
4.20
3.88
2a
2b
35.5
44.8
3.50
3.11
Generated by Cambridgesoft ChemBioDraw software.
2.3. Biological Activities of the Selenoflavanones and Flavanones
2.3.1. Antioxidant Effect and Cytotoxicity of the Selenoflavanones and Flavanones
To confirm the antioxidant effect and cytotoxicity of the compounds, cell viability in the presence
of hydrogen peroxide (100 µM) was measured using an MTT assay (tetrazolium dye colorimetric
assay) [23] (Figure 2). Treatment with hydrogen peroxide (H2O2) caused oxidative stress and
resulted in the death of 60% of SH-SY5Y cells. H2O2 induced cell death could be reduced by
antioxidant–reactive oxygen species scavenger. All of the selenoflavanone (1a, 1b) and flavanone (2a)
treatment groups showed fully recovered cell viability at all concentrations, except for compound 1b
´6
(10 M). Based on these observations, we believe that flavanone 2a and the selenoflavanones have
antioxidant activity, and that selenoflavanone 1b behaves as a pro-oxidant at high concentrations,
similar to some flavonoids, which show antioxidant or pro-oxidant effects depending on their
concentration [8,24]. The selenoflavanones did not result in cytotoxicity at low concentrations.
Consequently, we believe that they could be developed into neuroprotective drugs.
Figure 2. Changes in cell viability due to treatment with compounds. *** p < 0.001 compared with the
###
control group.
p < 0.001 compared with the H2O2-only treated group.
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2.3.2. Neuroprotective Activity of the Selenoflavanones and Flavanones
Next, we studied the in vivo neuroprotective activity of the compounds. A transient ischemia
mouse model [25] was used to confirm the actual changes in infarction volume resulting from
treatment with the compounds (Figure 3). Considerable and reproducible focal infarction and
hemispheric bulging were induced all over the cortical and subcortical structures when the subjects
were treated under middle cerebral artery occlusion (MCAO) for 90 min followed by 22.5 h of
reperfusion condition. An extensive infarct was observed in the control group animals, as shown
in Figure 3 (the total infarction volume was 177.58 ˘ 6.69 mm3). In comparison with the control
group, the anterior and medial striatum, and large areas of the cortex were remained unaffected
in the selenoflavanone-treated group. In other words, selenoflavanone treatment significantly
reduced the total infarction volumes in the ipsilateral hemisphere of the ischemia-reperfusion
mice. Total infarct volumes at doses of 40 mg/kg were reduced by approximately 45% or
41% by either 7-methoxyselenoflavanone (1a) or 6,7-dimethoxyselenoflavanone (1b) treatment,
respectively. The selenoflavanones showed better protective effects than the flavanones. Moreover,
treatment with MCI-186 (positive control, 5 mg/kg, intravenous (IV)) reduced the infarct volume
by 45%. Selenoflavanones (1a, 1b) showed more potent neuroprotective activity than corresponding
flavanones (2a, 2b).
Figure 3. Changes in infarction volume due to treatment with compounds. MCI-185: positive
control (5 mg/kg, iv); 1a: 7-methoxylselenoflavanone; 2a: 6,7-dimethoxyselenoflavanones, 2a:
7-methoxyflavanone; 2b: 6,7-dimethoxyflavanone; IP: intraperitoneal; *** p < 0.001 compared with
#
the control group. p < 0.05, ### p < 0.001 compared with the flavanone treated group.
Blood concentration and brain level of the compounds were not monitored. Nevertheless,
changes in physicochemical properties suggested that selenium substitution improved bioavailability
and/or BBB penetration of the compound. Flavanones also showed similar in vitro antioxidant
activity, but it had more polar and hydrophilic characters than corresponding selenoflavanones. As
a result, 1a and 1b proved to have more potential to penetrate membranes in gastrointestinal or
brain while 2a and 2b not. Antioxidant or pro-oxidant activity of compounds can be switched by
concentration and other conditions [8,24]. Although oxidation potential of selenoflavonoids has not
been identified yet, molecular behavior between antioxidant and pro-oxidant might be affected by
the substituted atom. We postulate that the results of the neuroprotective activity assay were affected
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by changes in both the physicochemical properties and oxidation-potential of the compounds as a
result of the selenium substitution.
3. Experimental Section
3.1. Chemistry
3.1.1. General Information
CH2Cl2 and THF used for the reaction were Liquid Chromatography grade reagents and were
redistilled from calcium hydride and sodium benzophenone ketyl, respectively. All other reagents
were obtained commercially and used as received without additional purification. Reactions were
monitored by thin-layer chromatography on 0.25 mm silica plates (F-254) visualized with ultraviolet
(UV) light (254 nm) and KMnO4 solution. Flash chromatography was performed using silica gel
(230–400 mesh) with hexanes and EtOAc as eluents. 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded on Varian Gemini 200 MHz or Agilent 400 MHz NMR spectrometers.
Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are expressed
in Hertz (Hz).
3.1.2. General Procedure for the Acylation of Bromobenzenes
˝
Aluminum chloride powder (2.0 mmol) was suspended in CH2Cl2 (10 mL) at 0 C. Cinnamoyl
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 CH2Cl2 (3 ˆ 30 mL). The combined organic extracts were filtered through a celite pad,
washed with water (3 ˆ 100 mL) and brine (100 mL), dried (MgSO4), and concentrated. The resulting
residue was purified by flash chromatography to give (4a, 4b) in 85%–87% yields.
1
(E)-1-(2-Bromo-4-methoxyphenyl)-3-phenylprop-2-en-1-one (4a) H NMR (400 MHz, CDCl3) δ
7.60–7.55 (m, 2H), 7.54–7.45 (m, 2H), 7.43–7.36 (m, 3H), 7.22–7.14 (m, 2H), 6.92 (dd, J = 8.6, 2.4 Hz,
1H), 3.86 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 193.4, 161.7, 145.4, 134.8, 133.5, 131.3, 130.8, 129.1,
128.6, 126.2, 121.2, 119.1, 113.4, 55.9.
(E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-phenylprop-2-en-1-one (4b) 1H NMR (400 MHz,
CDCl3) δ 7.65–7.58 (m, 2H), 7.55 (d, J = 16.0 Hz, 1H), 7.46–7.37 (m, 3H), 7.22 (d, J = 15.9 Hz, 1H),
7.08 (s, 1H), 7.04 (s, 1H), 3.94 (s, 3H), 3.90 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 193.3, 151.2, 148.3,
145.2, 134.6, 133.2, 130.7, 129.0, 128.6, 125.9, 115.9, 112.4, 111.2, 56.3, 56.2.
3.1.3. General Procedure for the Formation of Selenoflavanones
˝
To a THF (10 mL) solution of (4a, 4b) at ´78 C, 1.7 M t-BuLi solution in pentane was slowly
˝
added over a 10 min period. The reaction mixture was stirred at 0 C for 30 min, and selenium
powder (0.34 mmol) was added in two portions. The reaction mixture was kept at 0 C for 5 min and
˝
then at room temperature for 30 min. After the addition of HCl (1 M, 10 mL), the organic material
was extracted with Et2O (3 ˆ 10 mL). The combined organic extracts were dried over MgSO4 and
concentrated. The residue was dissolved in absolute EtOH (10 mL) and stirred vigorously in the
presence of NaOH (15 mg) for 30 min. The solution was concentrated, and the resulting residue was
purified by flash chromatography to give (1a, 1b) in 88%–92% yields.
1
7-Methoxyselenoflavanone (1a) H NMR (200MHz, CDCl3) δ 7.16 (m, 3H), 7.05 (m, 1H), 6.92
(d, J = 8.8 Hz, 1H), 6.71 (dd, J = 11.0, 2.4 Hz, 1H), 3.8 (s, 3H), 3.42 (m, 2H), 3.05 (dd, J = 16.1, 5.3 Hz,
1H); 13C NMR (50 MHz, CDCl3) δ 202.9, 161.4, 141.6, 134.1, 130.8, 129.6, 127.7, 126.3, 118.8, 113.1, 55.8,
52.2, 43.8.
6,7-Dimethoxyselenoflavanone (1b) 1H NMR (200MHz, CDCl3) δ 7.14 (m, 3H), 7.02 (m, 2H), 6.95
(s, 1H), 6.12 (s, 1H), 3.89 (s, 3H), 3.61 (s, 3H), 3.46 (m, 2H), 2.98 (dd, J = 15.6, 5.0 Hz, 1H); 13C NMR
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(50 MHz, CDCl3) δ 204.3, 150.6, 147.8, 141.3, 134.2, 129.7, 127.5, 126.2, 115.3, 111.8, 109.4, 56.2, 55.8,
52.7, 43.9.
3.1.4. General Procedure for the Acylation of Phenols
An appropriate phenol compound (1 mmol) and cinnamoyl chloride (1.07 mmol) were dissolved
in a BF3-Et2O complex (5 mL) and heated to reflux. After a 15 min reaction, the reaction mixture was
quenched with an excess of water. Then, the organic layer was separated and the aqueous layer
was extracted with EtOAc (2 ˆ 10 mL). The combined organic extracts were washed with water
(2 ˆ 10 mL) and brine (10 mL), dried (MgSO4), and concentrated. The resulting residue was purified
by flash chromatography to give (6a, 6b) in 84%–90% yields.
(E)-1-(2-Hydroxy-4-methoxyphenyl)-3-phenylprop-2-en-1-one (6a) 1H NMR (400 MHz, CDCl3)
δ 13.44 (s, 1H), 7.90 (d, J = 15.5 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.68–7.64 (m, 2H), 7.59 (d, J = 15.5 Hz,
1H), 7.46–7.40 (m, 3H), 6.55–6.44 (m, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 191.9, 166.7,
166.2, 144.4, 134.8, 131.2, 130.7, 129.0, 128.5, 120.3, 114.1, 107.8, 101.1, 55.6.
(E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-phenylprop-2-en-1-one (6b) 1H NMR (400 MHz,
CDCl3) δ 8.20 (d, J = 15.2 Hz, 1H), 7.70 (d, J = 7.2 Hz, 2H), 7.58–7.45 (m, 3H), 7.39 (d, J = 15.2 Hz,
1H), 7.04 (s, 1H), 6.40 (s, 1H), 5.30 (s, 1H), 3.98 (s, 3H), 3.92 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
180.8, 165.2, 163.7, 150.8, 144.7, 133.9, 132.7, 129.8, 129.3, 115.7, 109.5, 106.5, 101.8, 57.0, 56.5.
3.1.5. General Procedure for the Formation of Flavanones
To a solution of (6a, 6b) (0.1 mmol) and sodium acetate (50 mg) in EtOH (5 mL), 1 drop of water
was added and the mixture was heated to reflux for 6 h. The reaction mixture was poured into cold
water and extracted with EtOAc (2 ˆ 10 mL). The combined organic extracts were washed with water
(2 ˆ 10 mL) and brine (10 mL), dried (MgSO4), and concentrated. The resulting residue was purified
by flash chromatography to give (2a, 2b) in 80%–89% yields.
7-Methoxyflavanone (2a) 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.8 Hz, 1H), 7.52–7.36 (m, 5H),
6.63 (dd, J = 8.8, 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 5.48 (dd, J = 13.3, 2.8 Hz, 1H), 3.84 (s, 3H), 3.05
(dd, J = 16.9, 13.3 Hz, 1H), 2.84 (dd, J = 16.9, 2.9 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 190.6, 166.2,
163.5, 138.8, 128.8, 128.7, 126.1, 114.8, 110.3, 100.9, 80.0, 55.6, 44.3.
6,7-Dimethoxyflavanone (2b) 1H NMR (400 MHz, CDCl3) δ 7.54–7.37 (m, 5H), 7.33 (s, 1H), 6.54
(s, 1H), 5.47 (dd, J = 13.5, 2.9 Hz, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.05 (dd, J = 16.9, 13.5 Hz, 1H), 2.83
(dd, J = 16.9, 3.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 190.7, 158.0, 156.3, 144.7, 138.8, 128.9, 128.8,
126.2, 113.2, 106.6, 100.3, 80.3, 56.3, 56.2, 44.2.
3.2. In Vitro Cell Viability
3.2.1. Cell Culture
SH-SY5Y cells (human neuroblastoma) were obtained from the Korean cell line bank (KCLB,
Seoul, Korea). Cells were cultured in minimum essential medium (MEM) supplemented with
10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin sulfate
˝
(100 µg/mL) (Life Technologies, Grand Island, NY, USA). Cells were cultured at 37 C in an
atmosphere of 5% CO2.
3.2.2. MTT Assay
SH-SY5Y cells (1 ˆ 104) were seeded in wells containing 100 µL of Roswell Park Memorial
Institute (RPMI) medium supplemented with 10% FBS in a 96-well plate. Cells were pretreated
´8
with various concentrations (10´6–10 M) of the test compounds for 1 h, followed by exposure to
100 µM of H2O2 for another 3 h. To produce oxidative stress, H2O2 was freshly prepared from a 30%
stock solution prior to each experiment. The control cells were added to the same medium without
H2O2 or test compounds. Then media were carefully changed to new media (H2O2 free). After 48 h
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incubation, 50 µL of MTT (5 mg/mL stock solution) was added and the plates were incubated for an
additional 4 h. The medium was discarded and the formazan blue, which was formed in the cells,
was dissolved with 100 µL DMSO. The optical density was measured at 540 nm. Data were expressed
as mean ˘ S.E.M. Statistical analyses were performed using one-way analysis of variance (ANOVA)
###
followed by the Newman-Keuls test. *** p < 0.001 compared with the control group.
p < 0.001
compared with the H2O2-only treated group
3.3. In Vivo Neuroprotective Activity
3.3.1. Transient Ischemia Model
All in vivo experimental sections were carried out in accordance with the Principles of Laboratory
Animal Care (National Institutes of Health (NIH) publication (NIH: Bethesda, MD, USA), #85-23,
revised 1985) and the Animal Care and Use guidelines of Kyung Hee University, Korea. All test
subjects were adult male imprinting control region (ICR) mice weighing between 24–32 g at the time
of surgery. Anesthesia of the animals was conducted in a chamber with a mixture of N2O and O2
(70:30) containing 2.5% isoflurane, and were continued by inhalation of 1.5% isoflurane. Intraluminal
filament method [25] with the modifications described previously [26] was used to induce middle
cerebral artery occlusion (MCAO). The procedures were as in the following. A mid-line incision was
made on the ventral surface of the neck, and the right common carotid arteries and external carotid
artery were isolated and ligated with an 8.0 silk suture. A polyamide monofilament (Ethilon, Johnson
and Johnson Intl., Zaventum, Belgium) coated into a round tip with silicone resin (Xantopren, Byer
Dental, Kiel, Germany) was introduced into the intracranial internal carotid artery through an incision
in the common carotid artery. The filament was then carefully advanced approximately 10 mm distal
to the carotid bifurcation, beyond the origin of the middle cerebral artery. After 90 min of MCAO,
mice were re-anesthetized and the occluding filament was gently withdrawn back into the common
carotid artery to allow reperfusion to take place. Sham-operated mice underwent the same surgical
procedure, except that the filament was not advanced far enough to occlude the middle cerebral
˝
artery. Body temperature was maintained at 37 ˘ 0.5 C throughout the surgery using a heating
pad (Biomed S.L., Balearic Islands, Spain). Heart rate, arterial blood oxygen saturation of arterial
hemoglobin and ECG were monitored throughout the procedure (SurgiVet, Norwell, MA, USA).
3.3.2. Drug Administration
Mice subjected to MCAO were randomly assigned to receive either drug or vehicle treatments. Each
drug (40 mg/kg), dissolved in 6% Cremophor with 0.5% DMSO, was introduced intraperitoneally
at 30 min after MCAO. MCI-186 (3-Methyl-1-phenyl-2-pyrazolin-5-one, Sigma-Aldrich Co. Natick,
MA, USA) was used as a positive control. MCI-186 is a free radical scavenger that has been used to
reduce the neuronal damage following acute brain ischemia and subsequent cerebral infarction [27].
It is also called Edaravone and has marteked in Japan by the brand name Radicut. To the mice in the
MCI-186 treatment group, 5 mg/kg of MCI-186 dissolved in saline was injected intravenously using
the same time schedule. The vehicle treatment group was subjected to the identical experimental
protocol, except that they received the same volume/weight of vehicle only.
3.3.3. Morphometric Measurement of Infarct Volume and Edema
2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure the cerebral infarction
area. TTC is a reliable marker to highlight mitochondrial function, as well as stable indicator of
ischemic areas for up to 3 days after ischemia [28,29]. Animals were decapitated after 22.5 h of
reperfusion and their brains were carefully taken out. The brain slices were prepared along the
coronal plane with a brain slicer for mice (World Precision Instruments, Inc., Sarasota, FL, USA) at
2 mm intervals, starting 1 mm from the frontal pole. TTC staining of the brain slices were performed
˝
by 60 min of incubation in a 2% solution of TTC at 37 C followed by fixed-immersion with 10%
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neutral-buffered formalin. Computerized image analysis system (TDI Scope Eye 3.0, Samkyung Intl.
Co., Seoul, Korea) was used to determine the infarction area (mm2) in each section. The total lesion
volume (mm3) was calculated by summing the infarct area in each section (seven slices in all) and
multiplying this by the distance between sections. Both contralateral and ipsilateral hemisphere areas
were measured, and the differences between the two areas were used to calculate the edema volume.
TTC and neutral-buffered formalin were purchased from the Sigma-Aldrich Co. (Natick, MA, USA).
The significance of the differences between the total infarction volumes of the control (n = 10),
MCI-186 (5 mg/kg, IV, n = 8), the selenoflavanone (40 mg/kg, IP, n = 8), and the flavanone (40 mg/kg,
IP, n = 8) treated groups was determined by one way ANOVA followed by Student-Newman-Keuls
#
test. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group. p < 0.05, ## p < 0.01,
###
p < 0.001 compared with the flavanone treated group.
4. Conclusions
We synthesized selenoflavanones and flavanones using two steps, and evaluated their
physicochemical properties and biological activities. The selenoflavanones showed lower polarity
and higher lipophilicity than the corresponding flavanones, which suggests that they would be able to
more easily penetrate the BBB. In our in vitro assay, hydrogen peroxide-induced cell death decreased
with selenoflavanone treatment. Consequently, we confirmed the antioxidant effect and safety of
the selenoflavanones. The total infarction volumes in the transient ischemia mouse model were
significantly reduced by selenoflavanone treatment. Furthermore, the selenoflavanones resulted in
more potent neuroprotective activity than the flavanones. Based on these observations, we anticipate
that selenium substitution could be an effective approach for developing a neuroprotective agent.
Acknowledgments: This work was supported by grants from Yonsei University Global Specialization Project of
2014 and the Yonsei University Research Fund of 2015.
Author Contributions: Yong-Sung Choi, Dong-Myung Kim, and Jin-Hyun Jeong conceive the study and
designed the research; Yong-Sung Choi, Dong-Myung Kim, Yoon-Jung Kim and Sai Yang performed
experiments; Kyung-Tae Lee, Jong Hoon Ryu and Jin-Hyun Jeong discussed the results; Yong-Sung Choi wrote
the paper; Jin-Hyun Jeong supervised the project. All authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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