Preparation Of High Fatty Acid Analogue K And Its Resistance Reversal Activity

Objective: To optimize the preparation of analogue K that had been synthesized and found to show anti-tumor activities in vitro and vivo. To determine the physical and chemical constants of compound K as a basis for further animal experiments. To study the resistance reversal effect of K and the possible mechanism.Methods: The synthesis scale of K was expanded by one step reaction, K was purified by dry column flash chromatography and recrystallization method. The melting point, boiling point, dissociation and solubility constant of compound K were measured with method from the Chinese Pharmacopoeia. By MTT colorimetric assay,the reversal effect of compounds K on resistant cells line were detected. Flow cytometry was used to analyze the accumulation effect of Rho123. The activity of GST on cells effected by analogue K were detected.Results: The synthesis scale of K was expanded to 10 times, K was obtained with dry column flash chromatography and recrystallization, the yield of K was 65.5%, the purity of K was 99.45%. The melting point of K was 82-84℃, the boiling point of K was 112℃. The solubility of K was 0.219, 810.724, 708.666 and 2215.844μg/ml in water, ethanol, petroleum ether and ethyl acetate. The calculated pKa of K was 7.16±0.24, the pKa of K was 6.98 by A-pH curves. The concentration of K on the two resistant cell with non-toxic effect was 200μg/ml. Reversal of K was obvious on the BEL-7404/ADM and TCA8113/ADM cells. The relative reversal efficiency of K was 58.37% and 89.56%. The rho123 accumulation in the resistant cells was determined, the experimental results showed that the resistant cells within K had no effect on the accumulation of Rho123. The activity Glutathione-S transferase was studied. The experimental results showed that the Glutathione-S transferase activity of the BEL-7404/ADM (246.327.16) and TCA8113/ADM (222.122.85) cells compared with the Glutathione-S transferase activity of the BEL-7404 (90.820.04) and TCA8113 (94.118.68) improved significantly. After compound K was added, the enzyme activity of the BEL-7404/ADM cell activity was reduced to 117.6±5.23.The enzyme activity of the TCA8113/ADM cell activity also significantly reduced to 103.83±2.32, showed that compound K had a significant effect on the resistant cells of glutathione-S transferase activity.Conclusion: Compound K can be purified by dry column flash chromatography and recrystallization. The physical and chemical constants of K can be determined. The results show that K has fat-soluble and insoluble in water. It shows that K does not result in inhibition of resistant cells, but with resistance reversal effect. Mechanism of K is independent of the role of P-gp, maybe reduce cellular glutathione-S transferase vitality and reduce its role in detoxification of chemotherapeutic drugs.