Studies On The Purification Of Natural Product Rapamycinand Synthesis Of Its Derivative Everolimus

Rapamycin is a kind of natural product with multiple effects and a new type of highly effective immunosuppressant drugs. However, rapamycin has poor aqueous solubility and chemical stability because of the structure of macrolide, precluding its clinical development greatly. Therefore, It has been of great significance developing new derivatives with the same or even better biological activities and more excellent pharmacological properties.Recently, the biological activities of anti-proliferation, anti-tumor, neuroprotective and anti-aging have been found when structural modification of rapamycin. Rapamycin and its derivatives attracted a great attention because of its multiple effects, highly effective,distinct mode of action, novel macrocyclic structure and lower toxicity. Especially its excellent anti-tumor activity has been hot research topics in recent years. This dissertation is divided into two parts.Part I, Purification of the crude rapamycin extract from fermentation broth: We optimized the separation conditions by using silica gel column chromatography, and then recrystallized, qualified rapamycin. Furthermore, as a starting material of the derivativeeverolimus, we have established quality control methods of rapamycin from appearance,water content, sulphated ash, quality content by HPLC and other aspects, so as to ensure the stability of the synthesis process.Part II, A study on synthesis of everolimus which is one of rapamycin derivatives:Firstly, through aeeessing relevant literature, we have optimized a method that selective synthesis of 28-TMS-rapamycin when experimented the synthetic routes reported before, it has provided an effective method to synthesis of rapamycin 40-derivatives and has practical application. Summary the four methods synthesis of everolimus reported, we found that the synthetic technology is low yield, high cost, poor operability and not suitable for industrial production. So as to develop a new process for the industrial production, we try to use various active esters reacting with rapamycin, and found that the ethylene glycol triflates with group protected has high activity and stability. By changing the other hydroxyl protecitive group of ethylene glycol, we optimized a kind of acetal protecting groups that using 2-tetrahydropyranyl instead of silyl-ethers protecitive group, the forming active ester2-[(tetrahydro-2H- pyran-2-yl)oxy] ethyl triflate had better selectivity, simplified workup and improved the yield. The active ester reacted with rapamycin under organic base, the HPLC purity of reaction was 93.0% after optimization, the intermediate crude removal of the acetal protecting group to give product everolimus under the condition of 0.5N sulfuric acid, 0℃, and anhydrous tetrahydrofuran as the reaction solvent, the HPLC purity of crude product was 80.0%. The crude everolimus was separated by high pressure preparation system, after normal phase and reverse phase separation, we got the acceptable product more than 99.0% purity and less than 0.3% single impurity. The product was characterized by measuring melting point, LC/MS, 1HNMR and 13 CNMR. The total yield of the new synthetic technology got to 66.3%. Lastly, we have synthesized, analyzed the related substances of everolimus and achieved an ideal result.The new synthetic technology had mild reaction conditions, higher yield, lower cost,simple workup, workable, and industrial applications.