THE SYNTHESIS OF A NOVEL BIBENZOPYRAN RELIED ON ELECTROOXYDATION OF MALLOAPELTA B BY CYCLIC VOLTAMMETRY

A novel bibenzopyran named bimalloapelta ( 1) was synthesized relying on electrooxydation of malloapelta B by cyclic voltammetry in acetonitril adding LiClO 4 0.1 M as supporting electrolyte. Its structure determination based on extensive NMR studies and ESI mass spectral measurements. The new compound showed significant cytotoxicity againsts two cancer cell lines as human hepatocellular carcinoma (Hep -2; IC50 : 0.46 μg/m) and rhabdosarcom a (RD; IC50: 0.33 μg/m). These results were the same that of malloapelta B (Hep -2, IC50: 0.49 μg/ml and KB , IC50: 0.54 μg/ml).


INTRODUCTION
As part of an ongoing program to discover new anti-cancer agents from nature resources, we have reported previously the isolation and the structural elucidation of malloapelta B, a new cytotoxic compound from Mallotus apelta [1].To seak malloapelta B's derivatives, which have stronger cytotoxic activities than that of malloapelta B, we have synthesized a series of its derivatives based on organic synthesis and electroorganic reactions.
Currently, electroorganic synthetic method has been numerous using to synthesize organic compounds.The advantages of electroorganic reactions are that they react quickly and very selectivity [2].Malloapelta B, a new cytotoxic component and new inhibitor againsts NF-B activation from Mallotus apelta is an organic agent having some double bonds [1].Therefore, it can be also used as an electroorganic active agent.That means malloapelta B can be oxydated or reduced by electroorganic methods.Among them is the cyclic voltammetric method, which is an efficient synthetic way.In our experiment we chose the voltage located in oxydation area.The voltammograms of base solvent as well as research solvent were taken by electroorganic workstation system IM6 from Zahner Elektrik (Germany) in the voltage range from 1 to 1.5 V at a scan rate of 150 mV/s.This report deals with the electroorganic synthesis of bimalloapelta (1), the structural detemination and the evaluation of its cytotoxic activity by in vitro assay.

General experimental procedures
The IR spectra were obtained on a Hitachi 270 -30 type spectrometer using KBr discs.The Electron Spray Ionization Mass (ESI) spectrum was obtained using a AGILENT 1100 LC-MSD trap spectrometer.The 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer using TMS as the internal standard.Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 -230 mesh and 230 -400 mesh, Merck).Thin layer chromatography (TLC) was performed on DC-Alufolien Kiesegel 60 F254 (Merck).

Synthesis
Electroorganic cell: Using Electroorganic cell (100 ml) in three electrodes compartment with a platine net as counter electrode (CE), a platine plate of 1cm 2 as working electrode (WE), and a Ag/AgCl electrode as reference electrode (RE).
200 mg Malloapelta B agent (12 g/l) was added to base electrolyte.

Cyclic voltammetry:
The current-potential curves (Fig. 3) obtained by the application of a triangular impulse of potential was shown in Fig. 2. Investigations of electrosynthesis by cyclic voltammetry are usually carried out by choosing the voltage spans and sweep rates.The voltammograms were taken using electroorganic workstation system IM6 from Zahner Elektrik unit (Germany) in the voltage range from 1 to 1.5 V at a scan rate of 150 mV/s (2000 cycles).

Purification
After finishing reaction, the reactive solvent was evaporated in vacum to get 250 mg extract, which was then chromatographed on a silica gel column ( 20 × L 500 mm) eluted with hexaneacetone (4 : 1) as the eluent to give 1 (180 mg) as white powder.

Cytotoxicity
The cytotoxic activities of compounds 1 and malloapelta B were assayed on Hep-2 (human hepatocellular carcinoma) and RD (rhabdosarcoma) cells by SRB method [4 -6].In brief, the cell lines were stored in the liquid N 2 , then were cultured in Dulbeco's Modified Eagle Medium (DMEM) suplemented with 7 -10% Fecal Bovine Serum (FBS) for test.Cells were typically grown to 60% -70% confluence, the medium was then changed and the cells were used for test procedures one day later.In each case, 96-well tissue culture plates were used.Test samples (4-10 mg) were initially dissolved in 1 ml of 100% DMSO, then diluted 10 fold with H 2 O. Serial dilutions were performed using 10% aqueous DMSO as the solvent, and 10 µl were added to each well.Control groups were also added in which 10 µl of 10% DMSO and 10 µl of 0,01 mM Elipticine in DMSO were added to each well as negative and positive control in turn.After the plates were prepared, cell were removed from the tissue culture flasks by treatment with Tripsin 0.05%, enumerated, and diluted with fresh media.The quantities of cells (in 190 µl of media) were added to the 96-well plates (KB: 3 × 10 4 ; Hep-G2 and FL: 4 × 10 4 ), and incubation were perform for three days at 37 0 C in a CO 2 incubator with the plates capped in the nornal fashion.
After the incubation period, cell were fixed to the plastic substratum by the addition of 50 µl of cold 50% aqueous Trichloroacetic acid (TAC).The plate were incubated at 4 o C for 1 h, washed with tap water (4×), and air-dried.Cells then were stained by the addition of 0.4% Suforhodamine B (w/v) dissolved in 1% AcOH (30 min).Free sulforhodamine B solution were then removed by washing with 1% aqueous AcOH (4×).The plates were air-dried, and the bound dye was solubilized by the addition of 10mM unbuffered Tris base, pH 10.The plates were placed on a shake for 5 min, and the absorption was determined at 515 nm using an ELISA plate reader.

RESULTS AND DISCUSSION
As known about the electroorganic reactions that cation radicals, carboniums or uncharged radicals are formed at the anode, and anion radiacals, carbanions, or uncharged radicals are formed at the cathode [2]. Figure 3 showed the cyclic voltammograms of the solution with and without malloapelta B in acetonitril plus LiClO 4 (0.1 M) as supporting electrolyte.There was a clear appearence of the oxidation peak of malloapelta B when the potential ranged from 1 to 1.5 V versus Ag/AgCl.Some derivatives of malloapelta B were formed during oxidation process.The main product among them was found.The electroorganic reaction could be: An electroorganic oxidation mechanism of malloapelta B was proposed as shown in Scheme 1.
Firstly, malloapelta B transfered one electron to convert into corresponding cation radical, which was further oxidated by LiClO 4 , (LiClO 4 is much oxidative than CH 3 CN) then converted into 1.By chromatography on silica gel, compound 1 was obtained as white powder from the reactive solution.The 1 H-NMR spectrum of 1 inhibited one singlet of two methyl groups at δ 1.33, one doublet of doublet of the other methyl group at δ 1.87 (J = 7.0, 1.5 Hz), one singlet at δ 6,09 was assigned to H-6, a doublet of doublet at δ 6.30 (J = 15.5, 1.5 Hz) and a doublet of quartet at 6.55 (J = 15.5, 7.0 Hz) were assigned to two proton H-14 and H-15, respectively at trans configuration.Two other singlets at 3.81 and 3.78 were assigned to two methoxyl groups.A methine proton resonanced at higher field (δ 2.33, d, J = 6.5 Hz) coupled with the other methine proton bearing to oxygen atom at δ 4.89 (d, J = 6.5 Hz).The 13 C-NMR, DEPT 135 o and DEPT 90 o spectra of 1 confirmed the present of 17 carbon, including 3 methyl, 2 methoxyl, 5 methine and 7 quartenary carbon groups.Comparing the NMR of 1 with that of malloapelta B suggested the quite difference between them at C-3 and C-4 of the benzopyran ring.The double bond at C- The coupling constant between protons H-3 and H-4 (J 3-4 = 6.5 Hz) confirmed that they are all axial configuration (Silverstein, 1998).Accordingly, the structure of 1 was determined as shown in Fig. 4, which named bimalloapelta.Compounds 1 and malloapelta B were assayed on Hep-2 (human hepatocellular carcinoma) and RD (rhabdosarcoma) cells by SRB method.As a result, 1 also inhibited strongly cytotoxic activity on both tested cancer cell lines Hep-2 and RD with the IC 50 values of 0.46 µg/ml and 0.33 µg/ml, respectively.Comparing these results with those of malloapelta B (Hep-2, IC 50 : 0.49 µg/ml and KB, IC 50 : 0.54 µg/ml) indicated that IC 50 values of both compounds were the same and that the oxydation of double bond at C-3/C-4 did not affect to its cytotoxic activity.

Fig. 2 :Fig. 1 :
Fig. 2: Variation of applied potential with time in cyclic voltammetry shows the maximum (E max )and minimum (E min ) potentials.The sweep rate |dE/dt| = v.