Ebselen Derivative : Synthesis by Bingel Cyclopropanation and Enhanced Antioxidative and Neuroprotective Activity

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 ) and hydroxyl ( • OH) radicals, and the H 2 O 2 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.
C 60 -fullerenes, owing to their electronic structure as electron-poor polyolefines, are highly reactive towards various organic radicals 3 and have drawn considerable attention in the area of antioxidant research.An electron paramagnetic resonance analysis has shown that C 60 derivatives, such as carboxyfullerene, fullerenol and hexa(sulfobutyl)fullerene, have excellent capabilities to scavenge deleterious reactive oxygen species. 4In addition, neuroprotective effects of some water-soluble C 60 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 β-amyloid peptide, 5 and inhibit iron-induced oxidative stress in rat brain. 6The 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 C 60 (FC4S) and C 60 (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. 8Hexasulfobutylated C 60 (FC4S) can be employed as a free radical remover in the biological system. 9Recently, C 60 (glucosamine) 6 has been found to prevent renal I/Rinduced apoptosis formation and superoxide generation. 10ll 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. 11ecently, ebselen and its analogues were reported to have neuroprotective effect against brain ischemic insults and the glutamate-induced neurotoxicity. 12More recently, ebselen and its derivatives also confirmed to have neuroprotective effects on experimental rat spinal cord injury. 13All 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 C 60 and ebselen derivatives, it is expected that the covalent attachment of the biologically active ebselen moiety to C 60 -fullerene may lead to the formation of a new C 60 -based ebselen derivative, which could present better antioxidative and neuroprotective properties than the parent C 60 or ebselen molecules.In this article we report the synthesis of the new C 60 -based ebselen derivative 3, which contains a solubilizing ethyleneglycol chain, by Bingel cyclopropanation involving C 60 and ebselen malonate.We also report the neuroprotective activity of 3 against hydrogen peroxide-induced neuronal insults in cortical cell cultures.

Reagents and instruments
C 60 (99% purity) was prepared by the Institute of Fullerenes of Wuhan University.2-(Chloroseleno)benzoyl chloride was prepared according to the literature. 14All 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 λ-17 instrument.NMR data were recorded in CDCl 3 at room temperature on a Varian INOVA 600 NMR instrument operating at 14.09 T, observing 1 H and 13 C at 599.94 and 150.86 MHz, respectively. 1H and 13 C 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.

Primary cortical neuronal cultures
Primary cultures of rat cortical neurons were established using a modification of previous methods 17 which is briefly outlined below.Cortical tissue from 18-day fetuses of Wistar rats was dissociated and digested for 20 min at 37 °C 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 × 10 5 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 °C in a 95% air / 5% CO 2 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 H 2 O 2 -injured group, and a compound-treated group.The compound-treated group was divided into 4 subgroups according to the different chemicals employed (C 60 derivative 4, ebselen derivative 2, mixture of 4 and 2 (equimolar ratio) and C 60 -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 °C.At the time of medium change, stock solutions were further diluted and applied to the cultures.
Oxidative injury was induced by adding H 2 O 2 (final concentration 150 μmol L -1 ) to the serum-free DMEM culture medium containing 0.2% DMSO and incubating at 37 °C for 2 h. 18After 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 H 2 O 2 for other 2 h.Afterwards, the cell cultures received the same treatment as the H 2 O 2 -injured group.Control cells were not exposed to H 2 O 2 nor to the various compounds, but cultured in serum-free DMEM medium supplemented with 0.2% DMSO for 24 h.

Measurement of cell viability 19
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 °C 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 release 20
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.
The structure of 3 was confirmed by standard spectroscopic methods.Its 13 C NMR spectrum (150MHz, CDCl 3 ) displays 15 peaks between δ 139.24 and δ 145.70 owing to the sp 2 -fullerene C-atoms, indicating that compound 3 has C 2v symmetry with a [6,6]-closed structure. 22The signal at 72.37 ppm was assigned to the sp 3 -fullerene C-atoms.The six carbons of the phenyl ring in the ebselen moiety give rise to 6 peaks between δ 127.11 and δ 134.87.The carbon of the bridge is observed at 52.51 ppm.The remaining 4 signals in the δ 39.61 ~ δ 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 1 H NMR spectrum (600 MHz, CDCl 3 ) of compound 3 exhibits the aromatic hydrogens as two triplets (δ 7.65, J 7.3 Hz; δ 7.46, J 7.5 Hz) and two doublets (δ 8.06, J 7.8 Hz; δ 7.88, J 7.1 Hz).The hydrogens of the ethyleneglycol chain give rise to two triplets (δ 4.35, J 6.6 Hz; δ 4.17, J 6.3 Hz ) and two doublets between δ 3.76 and δ 3.80.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 C 60 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 dihydrofullerenes 23 and further proves that compound 3 is a [6,6]-closed methanofullerene.

Effects of C 60 derivative 4, ebselen derivative 2, mixture (4 + 2), and C 60 -based ebselen derivative 3 on cell viability and LDH release
In order to verify the enhanced neuroprotective activity of C 60 -based ebselen derivative 3, related compounds (the C 60 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).
C 60 -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 (C 60dimalonic acid, C 62 (COOH) 4 ) has cytochrome c peroxidase-like activity.They confirmed that C 62 (COOH) 4 was essential for the oxidation of reduced cytochrome c in the presence of H 2 O 2 .Hence, C 60 -fullerene should also display glutathione peroxidase-like (Gpx) activity.Besides, C 60 -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 C 60 -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 C 60 -based ebselen derivative (3), which contains an ethyleneglycol chain between the fullerene and the biologically active ebselen moiety.The C 60 -based ebselen derivative was characterized by NMR, MS, IR and UV spectroscopy analyses.In addition, it showed stronger antioxidative and neuroprotective activities than parent C 60 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 C 60 -fullerene molecules can give rise to new C 60 -based derivatives with better antioxidative ability, and this is also an effective strategy for designing and synthesizing C 60 -based antioxidants.