Preperation Of Mycophenolic Acid And Mycophenolate Mofetil As A New Immunosuppressant

In the paper, the determination of mycophenolic acid(MPA) and its morpholinoethyl ester(MMF) by HPLC , the optimization of the fermentation and purification processes of MPA were studied. It is to estabilish a comprehensive process to synthesize and prepare MMF.The chemical structure of MMF will be identified also.At first,the analytic method for MPA and MMF by HPLC was established.The chromatographic conditions were as followings.Column: Diamonsil C8(150x4.6mm, 5μm);mobile phase(MPA):acetonitrile:0.1% trifluoroacetic acid in water(50:50), mobile phase(MMF):acetonitrile:0.1% trifluoroacetic acid in water(45:55);detect wave length:249nm;flow rate:1.0mL/min;injection volumn:20μL;column temperature:room temperature. This method was linearity, accurate and sensitive.Calibration curves were linear in the range 0¹.183mg/mL for MPA and in the range 0～0.862mg/L for MMF.The strain strored in our lab was rejuvenated and purificated by natural screening,the production of MPA was stable and increased by 9%.The optimization of the fermentation process of this strain P. brevicompactum L was studied.The effects of slant medias,seed medium,carbon sources,nitrogen sources, carbon-nitrogen ratio,Fe²⁺ as well as pH, temperature, rotate speed on fermentation were studied in the shaking flasks. The optimal conditions of fermentation for production of MPA were obtained with initial pH 4.0, temperature 28℃ and culture duration for 13 days on 220r/min shaker. The optimal medium gained using uniform-design as follows: glucose 240g/L, glycine 22g/L, KH2PO₄ 6g/L. MPA production in shaking flask culture reached to 8.565 g/L which was above 89% higher than that in the basic fermentation conditions.A separation and purification process of MPA from fermentation broth was developed.MPA is found almost in the mycelium. 7%-10% diatomite was added to the fermentation broth.After being kept for 30min,it was filtrated.Recovery from the mycelium was effected by extract with 2 parts of ethanol .After reduction of theextract volume in vacuo to an aqueous concentration,MPA precipitates as a fine solid.The solid was re-dissolved completely in 2 parts of ethyl acetate. Ethyl acetate layer was treated with active carbon to remove colour.Active carbon was removeed by filtration and ethyl acetate layer was concentrated by distillation and allowed to stand at -4°C .After 24 hours large needle shaped light brown coloure crystals of MPA were found. These crystals were further dissolved in 5 parts of ethanol at 40°C and chilled to -4°C. After 24 hours the white crystals of MPA were separated by filtration and dried in vacuo. The crystals of MPA thus obtained were purity above 99%.The recovery of the whole process is about 45%.A process for making MMF by direct esterification of MPA was studied.MPA is esterified slowly in the mixture of benzene and toluene(1:1) capable of azeotropic removal of water using an excess of 2-morpholinoethanol. The product of MMF obtained was of acceptable pharmaceutical grade.UV spectrums,IR spectrums,mass spectrums,1H-NMR spectrums and 13C-NMR spectrums were applied to identify the product.The results showed that the product was the same as standard MMF sample.