Synthesis, Identification and Anti-Cancer Activity of 1-(4-Methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane

In this study, we synthesized 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2- enyl)disulfane using sodium sulfide, 1-bromine-4-methyl-2-amylene and 1-(4-bromine-2- butylene)benzene as raw materials. The yield rate of target product was 84%. The structure of the target product was confirmed by GC-MS, 1H-NMR and elemental analysis. The results of anti-cancer activity experiments showed that 1-(4-methylpent-2-enyl)-2-(4- phenylbut-2-enyl)disulfane could significantly inhibit the proliferation, induce the apoptosis of CNE2 cells in a dose dependent manner, and could significantly enhance the activity of XIAP.


Introduction
Allicin is a natural allyl sulfide and one of the various sulfur compounds extracted from garlic, along with diallyl trisulfides, diallyl disulfides and so on [1][2][3][4][5]. Since 1980, allicin has attracted more and more attention because of its potential cancer prevention and anti-cancer effects [6]. At present, studies have shown that DADS (diallyl disulfide), a possible precursor of allicin, is a drug with broad-spectrum anti-cancer effects. It can inhibit the growth of various tumor cells, such as human colon cancer cells (HCT-15), human skin cancer cells (SK MEL-2), human gastric cancer cells, human OPEN ACCESS breast cancer cells (MCF-7, KPL-1), and so on [7][8][9][10][11][12]. Certainly, study on the bioactivity of other sulfur compounds in this area continues.
In order to investigate the apoptosis effects on the CNE2 nasopharyngeal cancer cell line induced by 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane and its molecular mechanisms, the growth inhibition and apoptosis of CNE2 cells and the protein levels of XIAP were examined by MTT assay, flow cytometry and Western blot, respectively.

Synthesis of 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane
Sodium metal sheet (12.7g, 0.55 mol) was added to DME (100 mL) with fast stirring, and then sulphur powder (16.0g, 0.50 mol) was added after the sodium metal was completely dispersed, and stirring of the mixture was continued at the room temperature for 2 h, to give the product Na 2 S 2 , which was sealed and kept in a cool place [19][20][21]. Na 2 S 2 (50 mL) was transferred into a round bottom flask (250 mL), then DME (50 mL) which contained 1-bromo-4-methyl-2-amylene (40.8 g, 0.25 mol) was added dropwise into the round bottom flask with continuous stirring, and then reacted in a 70 °C oil bath for 4 h. After that, solvent was removed by the rotary evaporator under vacuum, and a bright yellow oily matter was thus obtained. The oily matter was added into distilled water (50 mL) and dispersed with ultrasound, then extracted with ether (20 mL) five times, and extracted with chloroform three times. The combined extracts was evaporated under vacuum to remove the solvent. The product was finally purified by silica gel column chromatography, using chloroform/methanol (v/v = 99/1) as mobile phase [22].

Cell viability assay
To assess the cytotoxic effects of DADS in CNE2 cells, we used a3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) tetrazolium salt reduction assay [23]. In this assay, the MTT is used as a colorimetric substrate for measuring cell viability. When cells are injured, there is an alteration in the cellular redox activity such that cells are unable to reduce the dye [7]. Cells were seeded into 96-well plates at 1 × 10 4 cells per well 24 h before treatment. The cultures were then rinsed in phenol-free RPMI1640 medium and incubated with respective test substances in phenol-free and serum-free RPMI1640 for 24 h. In the inhibition test, cells were treated with 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2-enyl) disulfane. At the end of this time interval, 20 μL (5 mg/mL) MTT was added to each well, and after incubation at 37 °C for 4 h, the MTT solution was removed and 200 μL of dimethylsulfoxide (DMSO) was added to dissolve the crystals. The absorbance of each well was measured at 570 nm.

Flow cytometry analysis
Cells were seeded into 100 mL cell culture bottles at 12 × 10 6 cells 24 h before treatment. Then cells were treated according to the aforementioned method and incubated for 24 h. Afterwards, floating and adherent cells were collected, washed three times with PBS (pH 7.4) and fixed for 24 h with cool alcohol at 4 °C. 1 mL cell suspension (10 6 /mL) was washed three times with cooled PBS, treated with RNase for 30 min at 37 °C, stained it with PI for 30 min at 37 °C in a dark environment, and taken for flow cytometry analysis.

Western blotting
The cells were taken in the logarithmic growth phase, treated according to the aforementioned method and incubated for 24 h. After fragmentation on ice for 20 min, the lysates were centrifuged at 15,000 ×g for 10 min at 4 ºC, the protein was collected, quantitated with the BCA method, electrophoresed and isolated by the SDS-PAGE (10%) using the electrotransfer method, blocked and hybridized on the cellulose nitrate film. Then the protein expression of cells was detected using the ECL Western blotting method. The densities of protein bands were calculated using the Quantity One software.

Statistics
Data are expressed as mean ± S.D of three independent experiments and were evaluated by one-way analysis of variance (ANOVA). Significant differences were established at P < 0.05.

Conclusion
The target product 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane was characterized by GC-MS, 1 H-NMR and elemental analysis. The results of anti-cancer activity experiment showed that this compound could significantly inhibit the proliferation of CNE2 cells, induce apoptosis in a dose dependent manner, and could significantly enhance the activity of XIAP.