C60-based ebselen derivative: synthesis by bingel cyclopropanation and enhanced antioxidative and neuroprotective activity

Abstracts

C60-based ebselen derivative 3 was synthesized through Bingel cyclopropanation of C60 with the ebselen malonate 2. Compound 3 was obtained in 42% yield (based on consumed C60) in a three-step synthesis starting from 2-(chloroseleno)benzoyl chloride and 2-(2aminoethoxy)ethanol. Its structure was confirmed by ¹H NMR, 13C NMR, IR, UV and FAB-MS spectroscopy analyses. In order to verify the enhanced antioxidative and neuroprotective activity of 3, a C60 derivative (4), an ebselen derivative (2), and their mixture (4 plus 2 in equimolar ratio) were employed to treat cortical neuronal cells, following the same procedure used with 3 and at the same final concentration (30 µmol L-1). Cell viabilities of the four treated groups were estimated by LDH (lactic dehydrogenase) leakage and MTT (3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) assays. Results showed that the antioxidative and protective activities of C60-based ebselen derivative 3 against H2O2-mediated neuronal injury (MTT(OD) 0.364 ± 0.028; LDH release (UL-1) 4.66 ± 0.28) were significantly higher than those of C60 derivative 4 (MTT(OD) 0.324 ± 0.025; LDH release (UL-1) 5.39 ± 0.17), ebselen derivative 2 (MTT(OD) 0.294 ± 0.021; LDH release (UL-1) 5.71 ± 0.27), and the mixture of 4 and 2 (MTT(OD) 0.310 ± 0.018; LDH release (UL-1) 5.54 ± 0.39). These findings demonstrated that the combination of two molecular units with similar biological activities (C60 and ebselen) may be a desirable way of obtaining new and more biologically effective C60-based compounds.

fullerene; ebselen derivative; Bingel cyclopropanation; neuroprotective activity; cell viability

Um derivado de ebselen baseado em C60, 3, foi sintetizado pela reação de ciclopropanação de Bingel envolvendo C60 e manolato de ebselen, 2. O produto foi sintetizado em três etapas, partindo de cloreto de 2-cloro-selenobenzoila e 2-(2-aminoetoxietanol), com rendimento de 42% (baseado no consumo de C60). O composto 3 foi caracterizado estruturalmente por análises espectroscópicas de RMN de ¹H e de 13C, IV, UV e FAB-MS. Com o objetivo de verificar um possível aumento na atividade antioxidante e neuroprotetora do derivado 3, um derivado de C60 (4), um derivado de ebselen (2) e sua mistura (4 + 2 em razão equimolar) foram selecionados para o tratamento de células neurais pelo mesmo procedimento adotado para 3 e na mesma concentração final (30 µmol L-1). As viabilidades celulares dos quatro grupos de tratamento foram estimadas por ensaios de LDH (desidrogenase láctica) e MTT (brometo de 3-[4,5dimetil-tiazol-2-il]-2,5-difenil-tetrazólio). Os resultados mostraram que a atividade antioxidante e neuroprotetora de 3 frente à lesão neural mediada por H2O2 (MTT(OD) 0,364 ± 0,028; LDH liberado (UL-1) 4,66 ± 0,28) foi significativamente mais alta que a do derivado de C60 4 (MTT(OD) 0,324 ± 0,025; LDH liberado (UL-1) 5,39 ± 0,17), do derivado de ebselen 2 (MTT(OD) 0,294 ± 0,021; LDH liberado (UL-1) 5,71 ± 0,27), e da sua mistura (4 + 2) (MTT(OD) 0,310 ± 0,018; LDH liberado (UL-1) 5,54 ± 0,39). Esses resultados mostram que a combinação de dois componentes moleculares com atividades biológicas similares (C60 e ebselen) pode ser uma forma desejável de preparação de novos compostos biologicamente efetivos derivados de C60.

ARTICLE

C₆₀-based ebselen derivative: synthesis by bingel cyclopropanation and enhanced antioxidative and neuroprotective activity

Xufeng Liu^I,II; Wenchao Guan^I,*; Wengshan Ke^III

^IMinistry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Hubei University, Wuhan 430062, P. R. China

^IIDepartment of Chemistry, HuaZhong University of Science and Technology, Wuhan 430074, P. R. China

^IIICollege of Life Science, Hubei University, Wuhan 430062, P. R. China

ABSTRACT

C₆₀-based ebselen derivative 3 was synthesized through Bingel cyclopropanation of C₆₀ with the ebselen malonate 2. Compound 3 was obtained in 42% yield (based on consumed C₆₀) in a three-step synthesis starting from 2-(chloroseleno)benzoyl chloride and 2-(2aminoethoxy)ethanol. Its structure was confirmed by ¹H NMR, ¹³C NMR, IR, UV and FAB-MS spectroscopy analyses. In order to verify the enhanced antioxidative and neuroprotective activity of 3, a C₆₀ derivative (4), an ebselen derivative (2), and their mixture (4 plus 2 in equimolar ratio) were employed to treat cortical neuronal cells, following the same procedure used with 3 and at the same final concentration (30 µmol L^-1). Cell viabilities of the four treated groups were estimated by LDH (lactic dehydrogenase) leakage and MTT (3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) assays. Results showed that the antioxidative and protective activities of C₆₀-based ebselen derivative 3 against H₂O₂-mediated neuronal injury (MTT(OD) 0.364 ± 0.028; LDH release (UL^-1) 4.66 ± 0.28) were significantly higher than those of C₆₀ derivative 4 (MTT(OD) 0.324 ± 0.025; LDH release (UL^-1) 5.39 ± 0.17), ebselen derivative 2 (MTT(OD) 0.294 ± 0.021; LDH release (UL^-1) 5.71 ± 0.27), and the mixture of 4 and 2 (MTT(OD) 0.310 ± 0.018; LDH release (UL^-1) 5.54 ± 0.39). These findings demonstrated that the combination of two molecular units with similar biological activities (C₆₀ and ebselen) may be a desirable way of obtaining new and more biologically effective C₆₀-based compounds.

Keywords: fullerene, ebselen derivative, Bingel cyclopropanation, neuroprotective activity, cell viability

RESUMO

Um derivado de ebselen baseado em C₆₀, 3, foi sintetizado pela reação de ciclopropanação de Bingel envolvendo C₆₀ e manolato de ebselen, 2. O produto foi sintetizado em três etapas, partindo de cloreto de 2-cloro-selenobenzoila e 2-(2-aminoetoxietanol), com rendimento de 42% (baseado no consumo de C₆₀). O composto 3 foi caracterizado estruturalmente por análises espectroscópicas de RMN de ¹H e de ¹³C, IV, UV e FAB-MS. Com o objetivo de verificar um possível aumento na atividade antioxidante e neuroprotetora do derivado 3, um derivado de C₆₀ (4), um derivado de ebselen (2) e sua mistura (4 + 2 em razão equimolar) foram selecionados para o tratamento de células neurais pelo mesmo procedimento adotado para 3 e na mesma concentração final (30 µmol L^-1). As viabilidades celulares dos quatro grupos de tratamento foram estimadas por ensaios de LDH (desidrogenase láctica) e MTT (brometo de 3-[4,5dimetil-tiazol-2-il]-2,5-difenil-tetrazólio). Os resultados mostraram que a atividade antioxidante e neuroprotetora de 3 frente à lesão neural mediada por H₂O₂ (MTT(OD) 0,364 ± 0,028; LDH liberado (UL^-1) 4,66 ± 0,28) foi significativamente mais alta que a do derivado de C₆₀ 4 (MTT(OD) 0,324 ± 0,025; LDH liberado (UL^-1) 5,39 ± 0,17), do derivado de ebselen 2 (MTT(OD) 0,294 ± 0,021; LDH liberado (UL^-1) 5,71 ± 0,27), e da sua mistura (4 + 2) (MTT(OD) 0,310 ± 0,018; LDH liberado (UL^-1) 5,54 ± 0,39). Esses resultados mostram que a combinação de dois componentes moleculares com atividades biológicas similares (C₆₀ e ebselen) pode ser uma forma desejável de preparação de novos compostos biologicamente efetivos derivados de C₆₀.

Introduction

The investigation of modern medical sciences has suggested that free radicals are associated with a number of neurodegenerative diseases,¹ such as Parkinson and Huntington's diseases and Alzheimer's dementia. Some reactive oxygen species (ROS), including superoxide (O₂^–•) and hydroxyl (^•OH) radicals, and the H₂O₂ molecule, are believed to be major contributors to oxidative damage in neurodegenerative diseases. Reactive oxygen species, produced during oxidative stress, may cause different kinds of biological damage, such as lipid peroxidation, protein oxidation and protein cross-linking, and lead to neuronal cell death.² In the search for a link between active oxygen species and neurodegenerative conditions, the development of chemopreventive agents with antioxidative activities could help preventing neurological injury caused by the ROS.

C₆₀-fullerenes, owing to their electronic structure as electron-poor polyolefines, are highly reactive towards various organic radicals³ and have drawn considerable attention in the area of antioxidant research. An electron paramagnetic resonance analysis has shown that C₆₀ derivatives, such as carboxyfullerene, fullerenol and hexa(sulfobutyl)fullerene, have excellent capabilities to scavenge deleterious reactive oxygen species.⁴ In addition, neuroprotective effects of some water-soluble C₆₀ derivatives on oxidative injuries have also been extensively studied in several cell cultures and animal models of CNS (central nervous system) damage. For example, carboxyfullerenes prevent apoptotic injury of cultured cortical neurons evoked by N-methyl-D-aspartate and b-amyloid peptide,⁵ and inhibit iron-induced oxidative stress in rat brain.⁶ The same carboxyfullerenes were also reportedly able to inhibit cerebellar granule cell apoptosis, possibly by reducing the generation of ROS.⁷ Other fullerene derivatives, such as fullerenol, hexasulfobutylated C₆₀ (FC4S) and C₆₀(glucosamine)₆, also showed neuroprotective activity related to their antioxidative property. Fullerenol demonstrated to be a powerful scavenger for the free radicals induced by I/R (ischemia/reperfusion) injury of the small intestine.⁸ Hexasulfobutylated C₆₀ (FC4S) can be employed as a free radical remover in the biological system.⁹ Recently, C₆₀(glucosamine)₆ has been found to prevent renal I/Rinduced apoptosis formation and superoxide generation.¹⁰ All these results reveal that fullerene and its derivatives possess antioxidative activity and may be useful as neuroprotective agents in several acute or chronic neurodegenerative diseases.

On the other hand, ebselen and its analogues seem to possess glutathione (GSH) peroxidase-like activity, and may act as antioxidants and free radical scavengers.¹¹ Recently, ebselen and its analogues were reported to have neuroprotective effect against brain ischemic insults and the glutamate-induced neurotoxicity.¹² More recently, ebselen and its derivatives also confirmed to have neuroprotective effects on experimental rat spinal cord injury.¹³ All these biological effects shown by ebselen and its analogues are obviously related to their antioxidative properties and free radical scavenge activities.

In view of the above mentioned antioxidative and neuroprotective properties of both C₆₀ and ebselen derivatives, it is expected that the covalent attachment of the biologically active ebselen moiety to C₆₀-fullerene may lead to the formation of a new C₆₀-based ebselen derivative, which could present better antioxidative and neuroprotective properties than the parent C₆₀ or ebselen molecules. In this article we report the synthesis of the new C₆₀-based ebselen derivative 3, which contains a solubilizing ethyleneglycol chain, by Bingel cyclopropanation involving C₆₀ and ebselen malonate. We also report the neuroprotective activity of 3 against hydrogen peroxide-induced neuronal insults in cortical cell cultures.

Experimental

Reagents and instruments

C₆₀ (99% purity) was prepared by the Institute of Fullerenes of Wuhan University. 2-(Chloroseleno)benzoyl chloride was prepared according to the literature.¹⁴ All solvents and reagents were dried prior to use by standard procedures and other commercially available compounds were used as received. Melting points were determined with a Digital Melting Point Apparatus (WRS-IB). Column chromatography was performed on silica Gel (200-300 mesh). IR spectra (KBr pellets) were obtained on a Spectrum One Perkin Elmer spectrometer. UV spectra were recorded on a Perkin-Elmer l-17 instrument. NMR data were recorded in CDCl₃ at room temperature on a Varian INOVA 600 NMR instrument operating at 14.09 T, observing ¹H and ¹³C at 599.94 and 150.86 MHz, respectively. ¹H and ¹³C NMR chemical shifts are given in ppm relative to TMS as internal standard at 0.00 ppm. Mass spectra were recorded on a JMS AXS05H instrument using 3-nitrobenzyl alcohol (m-NBA) as a matrix.

Wister rats of 18-day pregnancy were provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology (China). D-Hanks medium, trypsinase and DMEM (Dulbecco's Modification of Eagle's Medium) were purchased from Gibco Co. (USA). Fetal calf serum (FBS) and cytarabine hydrochloride were from Tianjin Biochemical Factory (China). Poly-L-lysine, DMSO, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma Chemical Co. (USA). C₆₀ derivative 4 (Figure 1) was prepared according to the literature.¹⁵ LDH (lactic dehydrogenase) assay kit was obtained from Beijing Chemical Kit Company (China). The optical density of each well was measured at 550 nm on a BIO-RAD550 (USA) enzyme-linked analyzer.

Synthesis of 2-(2-(2-hydroxyethoxy)ethyl)benzo[d][1,2] selenazol-3(2H)-one (1)

Details of the procedure are described in reference 16. Reaction of diethylene glycolamine (1.456 g, 13.86 mmol) with 2-(chloroseleno)benzoyl chloride gave ebselen derivative 1 (2.5 g, 63%). White needles, mp 116.7~117.5 ºC; IR n _max/cm^-1: 3630 (O-H), 3048 (C-H_Ar), 1610, 1573 (amide C=O), 1283 (amide C-N), 1170 (C-OC); ¹H NMR (CDCl₃, 600 MHz) d 8.00 (dd, J 7.6, 1.5 Hz, 1 H, ArH), 7.86 (dd, J 7.1, 1.4 Hz, 1 H, ArH), 7.63 (ddd, J 7.3, 7.3, 1.7 Hz, 1 H, ArH), 7.43 (ddd, J 7.3, 7.3, 1.7 Hz,1 H, ArH), 4.92 (br, s, 1 H, OH), 4.10 (t, J 6.2 Hz, 2 H, NCH₂), 3.76 (t, J 6.0 Hz, 2 H, ethylene glycol aliphatic hydrogens), 3.65 (t, J 6.0 Hz, 2 H, ethylene glycol aliphatic hydrogens), 3.54 (t, J 6.2Hz, 2 H, ethylene glycol aliphatic hydrogens); ¹³C NMR (CDCl₃, 150MHz) d 166.55 (amide C=O), 134.33, 132.54, 132.08, 129.20, 127.85, 127.00 (aromatic C), 72.26 (OCH₂CH₂OH), 69.23 (NCH₂CH₂O), 61.52 (OCH₂CH₂OH), 39.20 (NCH₂CH₂O).

Preparation of ebselen malonate 2

A solution of malonyl dichloride (0.37 mL, 3.87 mmol) in dry CHCl₃ (50 mL) was added dropwise to a stirred solution of ebselen derivative 1 (2.44 g, 8.51 mmol) and pyridine (0.84 mL, 10.2 mmol) at 0 ºC in dry CHCl₃ (150 mL) and the resulting mixture was allowed to warm slowly to room temperature. After stirring overnight, the mixture was washed with aqueous NaHCO₃ (5%), dried over MgSO₄ and evaporated in vacuum. The residue was chromatographed on silica gel, and elution with ethyl acetate/petroleum ether (3:1) gave ebselen malonate 2 (183.5 mg, 74%) as a pale yellow oil. TLC R_f = 0.48 (ethyl acetate/petroleum ether 4:1); IR n _max/cm^-1: 3045 (C-H_Ar), 1750, 1735 (ester C=O), 1613, 1568 (amide C=O), 1330, 1276 (amide C-N), 1160, 1170 (C-O-C); ¹H NMR (CDCl₃, 600 MHz) d8.04 (dd, J 7.8, 1.5 Hz, 2 H, ArH), 7.86 (dd, J 7.2, 1.5 Hz, 2 H, ArH), 7.66 (ddd, J 7.3, 7.3, 1.8 Hz, 2 H, ArH), 7.45 (ddd, J 7.5, 7.5, 1.8 Hz, 2 H, ArH), 4.22 (t, J 7.0 Hz, 4 H, COOCH₂), 4.08 (t, J 6.0 Hz, 4 H, NCH₂), 3.74 (t, J 6.0 Hz, 4 H, ethylene glycol aliphatic hydrogens), 3.70 (t, J 7.2 Hz, 4 H, ethylene glycol aliphatic hydrogens), 3.30 (s, malonic CH₂); ¹³C NMR (CDCl₃, 150 MHz) d 168.32 (ester C=O), 167.85 (amide C=O), 134.85, 132.43, 132.12, 129.16, 127.70, 127.02 (aromatic C), 70.73 (OCH₂CH₂O), 69.46 (NCH₂CH₂O), 65.32 (OCH₂CH₂O), 41.27 (malonic CH₂), 39.53 (NCH₂CH₂O).

Bingel cyclopropanation of C₆₀ with ebselen malonate 2 to give 3

To a solution of C₆₀ (150 mg, 0.208 mmol), CBr₄ (103.5 mg, 0.312 mmol), and ebselen malonate 2 (159.5 mg, 0.249 mmol) in 200 mL of toluene, 63.33 mg of DBU (0.416 mmol) was added. The reaction mixture was stirred under N₂ at room temperature for 10 h. The solvent was distilled, and the residue was chromatographed on silica gel, and eluting first with toluene (to remove unreacted C₆₀), then with toluene/CHCl₃ (8:1) provided the fullereneebselen mono-adduct 3 (95.23 mg, 42%, based on consumed C₆₀) as a dark brown solid. TLC R_f = 0.51 toluene/chloroform 8:1); UV-Vis l _max/nm (CHCl₃) 433, 332, 228.5; IR n _max/cm^-1: 3052 (C-H_Ar), 1756, 1740 (ester C=O), 1610, 1570 (amide C=O), 1433 (C₆₀), 1334, 1272 (amide C-N), 1185 (C₆₀), 1158, 1174 (C-O-C), 528 (C₆₀); ¹H NMR (CDCl₃, 600MHz) d8.06 (dd, J 7.8, 1.5 Hz, 2 H, ArH), 7.88 (dd, J 7.1, 1.5 Hz, 2 H, ArH), 7.65 (ddd, J 7.3, 7.3, 1.8 Hz, 2 H, ArH), 7.46 (ddd, J 7.5, 7.5, 1.8 Hz, 2 H, ArH), 4.35 (t, J 6.6 Hz, 4 H, COOCH₂), 4.17 (t, J 6.3 Hz, 4 H, NCH₂), 3.80 (t, J 7.5 Hz, 4 H, ethylene glycol aliphatic hydrogens), 3.76 (t, J 7.8 Hz, 4 H, ethylene glycol aliphatic hydrogens); ¹³C NMR (CDCl₃, 150 MHz) d 168.80 (ester C=O), 167.88 (amide C=O), 145.70, 145.66, 145.53, 145.46, 145.16, 144.92, 144.86, 144.14, 143.34, 143.26, 143.24, 142.46, 142.17, 141.20, 139.24 (15 C₆₀-sp²-carbons), 134.87 (aromatic C), 132.44, 132.14, 129.13, 127.73, 127.11 (aromatic C), 72.37 (C₆₀-sp³carbons), 70.80 (OCH₂CH₂O), 69.80 (NCH₂CH₂O), 65.71 (OCH₂CH₂O), 52.51 (malono bridge), 39.61 (NCH₂CH₂O); FAB-MS [(+), m-NBA] m/z 1361 [(M+H)⁺, 13%], 720 [(M+H-ebselen malonate)⁺, 100].

Primary cortical neuronal cultures

Primary cultures of rat cortical neurons were established using a modification of previous methods¹⁷ which is briefly outlined below. Cortical tissue from 18-day fetuses of Wistar rats was dissociated and digested for 20 min at 37 ºC in D-Hanks medium with 0.125% trypsinase. Trypsinase was then removed and the dissociated neurons were washed with DMEM / 10% fetal calf serum (FCS) and filtrated through a 200-order stainless steel sieve. After centrifugation at 4000 g for 10 min, the cells were gently resuspended in DMEM / 10% FCS and vaccinated to poly-L-lysine (0.01%) coated 96-well plates at the density of 1 × 10⁵ cells per well. All cells were incubated in DMEM culture medium containing 10% fetal calf serum (FCS), penicillin (100 U mL^-1) and streptomycin (100 µg mL^-1) at 37 ºC in a 95% air / 5% CO₂ humidified culture box. On the third day of vaccination, cytarabine (final concentration 10 µmol L^-1) was added to the culture medium to prevent proliferation of non-neuronal cells.

On the 10^th day of culture, the neural cells were divided at random into a control group, an H₂O₂-injured group, and a compound-treated group. The compound-treated group was divided into 4 subgroups according to the different chemicals employed (C₆₀ derivative 4, ebselen derivative 2, mixture of 4 and 2 (equimolar ratio) and C₆₀-based ebselen derivative 3) (Figure 1).

Culture treatments

Stock solutions (10 mmol L^-1) of compounds 4, 2, mixture (4 + 2) and 3 prepared in DMSO and diluted with sterile water were kept at 4 ºC. At the time of medium change, stock solutions were further diluted and applied to the cultures.

Oxidative injury was induced by adding H₂O₂ (final concentration 150 µmol L^-1) to the serum-free DMEM culture medium containing 0.2% DMSO and incubating at 37 ºC for 2 h.¹⁸ After that treatment, the culture medium was replaced with fresh serum-free DMEM and the cells were incubated for 24 h in culture box. For the four compound-treated groups, cortical neuronal cultures were pretreated with compound 4, 2, mixture (4 + 2), or 3 (final concentration: 30 µmol L^-1) for 2 h and then exposed to 150 µmol L^-1 H₂O₂ for other 2 h. Afterwards, the cell cultures received the same treatment as the H₂O₂-injured group. Control cells were not exposed to H₂O₂ nor to the various compounds, but cultured in serum-free DMEM medium supplemented with 0.2% DMSO for 24 h.

Measurement of cell viability¹⁹

Cell viability was determined by measuring the mitochondrial dehydrogenase activity (MDHA) through the reduction of MTT. After the above described procedure, an MTT solution (final concentration 0.5 mg mL^-1) was added to each well, and the cells were incubated at 37 ºC for 4 h. After removal of the medium, formazan crystals, produced by MDHA in viable cells, were dissolved in dimethyl sulfoxide (200 µL). The optical density of each well was measured at 550 nm using an enzyme-linked analyzer.

Determination of LDH release²⁰

Cell damage was evaluated by measuring the release of lactate dehydrogenase (LDH) into the culture medium. LDH contents in the extracellular medium were determined using the LDH assay kit according to the kit instructions.

Results and Discussion

Synthesis and spectroscopic properties of C₆₀-based Ebselen derivative 3

The synthesis of 3 is achieved after a three-step process as outlined in Scheme 1. Reaction of 2-(chloroseleno)benzoyl chloride with slight excess 2-(2-aminoethoxy)ethanol (1.1 equiv.) in dry CH₃CN afforded ebselen derivative 1, which is N-substituted with an ethylene glycol chain, in 63% yield. The ebselen derivative 1 was esterified with malonyl dichloride in 2.2:1 molar ratio in dry CHCl₃ in the presence of pyridine (1.2 equiv. to 1). After purification by flash chromatography (silica gel, eluent: ethyl acetate/petroleum ether 3:1), ebselen malonate 2 was obtained in 74% yield. Finally, Bingel cyclopropanation²¹ of C₆₀ (toluene, room temperature, 10 h) with the ebselen malonate 2 (1.2 equiv. to C₆₀) in the presence of 1,8-diaza-bicyclo[5.4.0]undec-7ene (DBU, 2.0 equiv. to C₆₀) and CBr₄ (1.5 equiv. to C₆₀) gave rise to the fullerene-ebselen mono-adduct 3 in a 42% yield (based on consumed C₆₀). Mono-adduct 3 was isolated by column chromatography on silica-gel, eluted with pure toluene, followed by toluene/CHCl₃ (8:1, v/v).

The structure of 3 was confirmed by standard spectroscopic methods. Its ¹³C NMR spectrum (150MHz, CDCl₃) displays 15 peaks between d139.24 and d 145.70 owing to the sp²-fullerene C-atoms, indicating that compound 3 has C_2v symmetry with a [6,6]-closed structure.²² The signal at 72.37 ppm was assigned to the sp³-fullerene C-atoms. The six carbons of the phenyl ring in the ebselen moiety give rise to 6 peaks between d127.11 and d134.87. The carbon of the bridge is observed at 52.51 ppm. The remaining 4 signals in the d 39.61 ~ d 70.80 region are assignable to the aliphatic carbons of the ethyleneglycol chain. The carbons of the carboxylate and amide groups are detected at 168.80 and 167.88 ppm, respectively. The ¹H NMR spectrum (600 MHz, CDCl₃) of compound 3 exhibits the aromatic hydrogens as two triplets (d 7.65, J 7.3 Hz; d 7.46, J 7.5 Hz) and two doublets (d8.06, J 7.8 Hz; d 7.88, J 7.1 Hz). The hydrogens of the ethyleneglycol chain give rise to two triplets (d4.35, J 6.6 Hz; d 4.17, J 6.3 Hz ) and two doublets between d3.76 and d3.80. The FTIR spectrum of compound 3 shows strong carbonyl absorptions at 1756, 1740 cm^-1 (ester) and 1610, 1570 cm^-1 (imide), and three characteristic absorptions of the C₆₀ skeleton at 1433, 1185, and 528 cm^-1. The FAB-MS analysis of compound 3 gives the molecular ion peak at m/z 1361 (M+H)⁺ and a relevant fragment at m/z 720 (M+H-ebselen malonate)⁺. The UV-Vis spectrum of compound 3 contains a typical medium intensity absorption band at around 430 nm, which is characteristic of most dihydrofullerenes²³ and further proves that compound 3 is a [6,6]-closed methanofullerene.

Effects of C₆₀ derivative 4, ebselen derivative 2, mixture (4 + 2), and C₆₀-based ebselen derivative 3 on cell viability and LDH release

In order to verify the enhanced neuroprotective activity of C₆₀-based ebselen derivative 3, related compounds (the C₆₀ derivative 4, the ebselen derivative 2, and a mixture of 4 and 2) were also used to treat cortical neuronal cells according to the same procedures employed for 3 (Figure 1).

Cell viability and cell damage were estimated by MTT and LDH leakage assays, respectively. Results of LDH release and MTT(OD) measurements are shown in Figure 2. Compared with the control group, treatment of cortical culture with 150 µmol L^-1 H₂O₂ for 2 h caused a decrease of MTT(OD) from 0.458 ± 0.024 to 0.215 ± 0.024 (P < 0.01) and an increase of LDH content in the extra-cellular media from 3.69 ± 0.17 to 8.02 ± 0.27 (P < 0.01), indicating H₂O₂-induced neuronal injury. Cytotoxicity tests demonstrated that compounds 4, 2 and 3, as well as the mixture 4 + 2, at a concentration up to 30 µmol L^-1, have no toxic effect on cortical neurons. Therefore, 30 µmol L^-1 was selected to detect their protective effects on H₂O₂-mediated neuronal injury. As expected, they were effectively able to attenuate the H₂O₂-mediated increase of LDH release and decrease of MTT(OD) value (Figure 2). MTT(OD) was elevated from 0.215 ± 0.024 (H₂O₂ alone) to 0.324 ± 0.025 (4), 0.294 ± 0.021 (2), 0.310 ± 0.018 (4 + 2), 0.364 ± 0.028 (3) (P < 0.01) (Figure 2a), and LDH release was reduced from 8.02 ± 0.27 (H₂O₂ alone) to 5.39 ± 0.17 (4), 5.71 ± 0.27 (2), 5.54 ± 0.39 (4 + 2) and 4.66 ± 0.28 (3) (P < 0.01) (Figure 2b).

Cell viability in compound 3-treated group (MTT(OD) 0.364 ± 0.028) was higher than those obtained for compound 4 (0.324 ± 0.025), compound 2 (0.294 ± 0.021), or mixture (4 + 2) (0.310 ± 0.018). LDH release (3: 4.66 ± 0.28 UL^-1), in its turn, was consistently lower: compound 4 (5.39 ± 0.17), compound 2 (5.71± 0.27), or mixture (4 + 2) (5.54 ± 0.39). These results indicated that the antioxidative and protective activities of C₆₀-based ebselen derivative 3 against H₂O₂-mediated neuronal injury are significantly higher than those shown by C₆₀ derivative 4, ebselen derivative 2, and their mixture (4 + 2). Hence, the covalent incorporation of fullerene and ebselen units into a new molecule is of significance for enhancing antioxidative activity.

C₆₀-fullerene is a redox-active compound, since it has low LUMO and high HOMO levels. Okuda et al.²⁴ have observed that a water-soluble fullerene derivative (C₆₀dimalonic acid, C₆₂(COOH)₄) has cytochrome c peroxidase-like activity. They confirmed that C₆₂(COOH)₄ was essential for the oxidation of reduced cytochrome c in the presence of H₂O₂. Hence, C₆₀-fullerene should also display glutathione peroxidase-like (Gpx) activity. Besides, C₆₀-fullerene possesses an excellent antioxidative property derived from its polyolefine bonds, which are highly reactive towards deleterious reactive oxygen species. On the other hand, ebselen has excellent Gpx activites and limited radical scavenging ability. Therefore, we think that the enhanced neuroprotective activity of C₆₀-based ebselen derivative 3 can reasonably be attributed to the improvement of both its free radical scavenging and Gpxlike activities.

Conclusions

The present work describes the synthesis of a C₆₀-based ebselen derivative (3), which contains an ethyleneglycol chain between the fullerene and the biologically active ebselen moiety. The C₆₀-based ebselen derivative was characterized by NMR, MS, IR and UV spectroscopy analyses. In addition, it showed stronger antioxidative and neuroprotective activities than parent C₆₀ derivative 4, ebselen derivative 2, and a mixture of 4 and 2. From this encouraging result, it can be concluded that the selection and incorporation of appropriate antioxidant components into C₆₀-fullerene molecules can give rise to new C₆₀-based derivatives with better antioxidative ability, and this is also an effective strategy for designing and synthesizing C₆₀-based antioxidants.

Acknowledgment

This work was supported by the National Science Foundation of China (No. 20474020)

Supplementary Information

Relevant spectral data, including NMR and mass spectra, are available free of charge at http://jbcs.sbq.org.br, as a PDF file.

References

1. Beyer, C. E.; Steketee, J. D.; Saphire, D.; BioChem. Pharmacol.1998, 56, 1265.

2. Stadtman, E. R.; Berlett, B. S.; Chem. Res. Toxicol. 1997, 10, 485; Moore, T. M.; Khimenko, P. L.; Taylor, A. E.; Chin. J. Physiol. 1996, 9, 65.

3. Krusic, P. J.; Wasserman, E.; Ketzer, P. N.; Morton, J. R.; Preston, K. F.; Science 1991, 254, 1183.

4. Cheng, F.; Yang, X.; Zhu, H.; Sun, J.; Liu, Y.; J. Phys. Chem. Solids 2000, 61, 1145; Zhu, Y. S.; Sun, D. Y.; Liu, G. Z.; Chem. J. Chin. Univ. 1996, 17, 1127; Chiang, L. Y.; Lu, F. J.; Lin, J. T.; J. Chem. Soc., Chem. Commun. 1995, 1283; Chueh, S. C.; Lai, M.K.; Lee, M. S.; Chiang, L. Y.; Ho, T. I.; Chen, S. C.; Transplant. Proceed. 1999, 31, 1976.

5. Dugan, L. L.; Turetsky, D. M.; Du, C.; Lobner, D.; Wheeler, M.; Almli, C. R.; Shen, C. K.-F.; Luh, T.-Y.; Choi, D. W.; Lin, T. S.; Proc. Natl. Acad. Sci. USA 1997, 94, 9434; Dugan, L. L.; Gabrielsen, J. K.; Yu, S. P.; Lin, T-S.; Choi, D. W.; Neurobiol. Disease 1996, 3, 129.

6. Lin, A. M. Y.; Chyi, B. Y.; Wang, S. D.; Yu, H. H.; Kanakamma, P. P.; Luh, T. Y.; Chou, C. K.; Ho, L. T.; J. NeuroChem. 1999, 72, 1634.

7. Bisaglia, M.; Natalini, B.; Pellicciari, R.; Straface, E.; Malorni, W.; Monti, D.; Franceschi, C.; Schettini, G.; J. NeuroChem. 2000, 74, 1197.

8. Lai, H-Sh.; Chen, W-J.; Chiang, L-Y.; World J. Surg. 2000, 24,450.

9. Lee, Y. T.; Chiang, L. Y.; Chen, W. J.; Hsu, H. C.; PSEBM 2000, 224, 69; Huang, S. S.; Tsai, Sh. K.; Chin, Ch. L.; Chiang, L. Y.; Hsieh, H. M.; Teng, C. M.; Free Radic. Biol. Med. 2001, 30, 643.

10. Chien, Ch. T.; Chen, Ch-F.; Hsu, Su-M.; Chiang, L. Y.; Lai, MK; Fullerene Science and Technology 2001, 9, 77.

11. Muller, A.; Cadenas, E.; Graf, P.; Sies, H.; BioChem. Pharmacol. 1984, 33, 3235; Ren, X. J.; Yang, L.; Liu, J.; Su, D.; You, D.; Liu, Ch.; Zhang, K.; Luo, G.; Arch. BioChem. Biophys. 2001, 387, 250.

12. Porciuncula, L. O.; Rocha, J. B. T.; Boeck, C. R.; Vendite, D.; Souza, D. O.; Neurosci. Lett. 2001, 299, 217; Graham, I. H.; Masayasu, D. I.; Macrae, H.; Free Radic. Biol. Med. 2003, 34, 56.

13. Kalayci, M.; Coskun, O.; Cagavi, F.; Kanter, M.; Armutcu, F.; Gul, S.; NeuroChem. Res.2005, 30, 403.

14. Schewe, T.; Gen. Pharmac. 1995, 26, 1153.

15. Guldi, D. M.; Tian, Y.; Fendler, J. H.; Hungerbuehler, H.; Asmus, K-D.; J. Phys. Chem. 1995, 99, 17673.

16. Osajda, M.; Mlochowski, J.; Tetrahedron 2002, 58, 7531.

17. Choi, D. W.; Mauluccigedde, M.; Kriegstein, A. R.; J. Neurosci.1987, 7, 357; Schena, M.; Shalon, D.; Dais, R. W.; Science 1995, 270, 467.

18. Hansen, M. B.; Nielsen, S. E.; Berg, K.; J. Immunol. Methods 1989, 119, 203; Yoshizumi, M.; Kogame, T.; Suzaki, Y., Fuijita, Y.; Br. J. Pharmacol. 2002, 136, 1023.

19. Mosmann, T.; J. Immunol. Methods 1983, 65, 55; Behl, C.; Davis, J. B.; Lesley, R.; Schubert, D.; Cell 1994, 77, 817.

20. Koh, J. Y.; Choi, D. W.; J. Neurosci. Methods 1987, 20, 83; Hartnett, K. A.; Stout, A. K.; Rajdev, S.; Rosenberg, P. A.; Reynolds, I. J.; Aizenman, E.; J. NeuroChem. 1997, 68, 1836.

21. Camps, X.; Hirsch, A.; J. Chem. Soc., Perkin Trans. 1 1997, 1595.

22. Diederich, F.; Isaacs, L.; Philp, D.; Chem. Soc. Rev. 1994, 243.

23. Isaacs, L.; Wehrsig, A.; Diederich, F.; Helv. Chim. Acta 1993, 76, 1231.

24. Okuda, K.; Mashino, T.; Hirobe, M.; Bioorg. Med. Chem. Lett. 1996, 5, 539.

Received: October 8, 2006

Web Release Date: October 31, 2007

Supplementary information

Publication Dates

• Publication in this collection
  08 Jan 2008
• Date of issue
  2007

History

• Received
  08 Oct 2006
• Accepted
  31 Oct 2007