The Synthesis Of Beclomethasone Dipropionate By Chemical Reaction And Microbial Biotransformation

Beclomethasone dipropionate (BD), namely 9α-chloro-11β,17α,21-trihydroxy-16β-methyl-pregna-1,4-diene-3,20-dione-17,21-dipropionate, is a new generation of synthetical glucocorticoid, which has a remarkable local anti-inflammation effect. It can also induce vasoconstriction, and is widely used in the treatment of asthma, skin inflammation and allergic rhinitis. Since BD selectively acts on histiocytes of the respiratory organ, and affects specific procedure of the transcription of the target gene, its aerosol dosage is much more effective and economical than the peroral dosage. At present, lots of domestic companies are engaged in the production of BD aerosols. Unfortunately, only a small proportion of raw materials are produced in China. Therefore, it is greatly beneficial to design a convenient, cost-effective and green synthetic route for the production of BD with high yield.An integrated synthetic route for BD was developed by the combination of chemical reaction and microbial biotransformation in this dissertation. Epoxyprogesterone was used as the initial substrate. After two steps of biotransformation, namely C₁₁-hydroxylation and C_1,2-dehydrogenation, as well as five steps of chemical synthesis, namely C_16β-methylation together with C_17α-, C₂₁-hydroxylation, C₂₁-propionic esterification, C_9,11-dehydrogenation, C₁₇-propionic esterification and C_9α-chlorination as well as C_11β-hydroxylation, BD was obtained with the content of 99.7%, the total yield of 30%.Firstly, by UV-γ ray radiation, a high-producing mutant, Absidia coerulea AB04-12, was obtained for the microbial biotransformation of C₁₁-hydroxylation. The feed mode and fermentation conditions were optimized. The effects of methanol, ethanol, 1, 2-propanediol and N, N-dimethylformamide on conversion was compared, and 1, 2-propanediol was found as the optimum solvent in solvent-suspending feed mode. A novel water-suspending feed which was an ultrasonic-vibrated mixture containing 0.84 μm-diameter substrate, water, β-cyclodextrin, tween 80 and a little ethanol was also proposed. Under this feed mode, the conversion was increased to 70% at 10 g/L feed concentration. Multi-layer feed-forward neural network (MFNN) and particle swarm optimization (PSO) were used in the optimization of the medium components and some additives in the water-suspending feed mode, and the optimized components were: 15.58g/L glucose, 27.02g/L corn steep liquor, 3.47g/L yeast extract, 10.72g/L magnesium sulfate, 0.62g/L ammonium sulfate, 0.68g/L β-cyclodextrin, 10.00g/L tween 80, and 0.04g/L penicillin. With the optimal conditions and feed mode, the conversion was 87.5% in shaking flasks culture at 10g/L feed concentration and maintained 86.6% in the 3.7 L fermentor at 20g/L feed concentration, indicating a great potential in large-scale production. The extracellular 11-hydroxylation was preliminarily studied, however, lots of byproducts were obtained and low activity of hydroxylase was measured.Secondly, in the following C_1,2-dehydrogenation reaction of 11-hydroxide by Arthrobacter simplex, MFNN and PSO were also used for the optimization of themedium, and the optimized components were: glucose 6.89g/L, corn steep liquor 9.48g/L, peptone 1.74g/L, potassium dihydrogen phosphate 2.69g/L. The influences of reaction time, batches, treatment mode and concentration of feed on conversion were investigated. The results indicated that that feeding once at 28h was convenient and less risky of contamination. Whereas for batches biotransformation, feeding twice at 26h and 32h respectively, would induce the dehydrogenase, leading to a higher conversion than that of feeding once. Ethanol was found to be the optimum solvent in solvent-suspending feed mode. The conversion was further up to 91% at 25 g/L feed concentration under water-suspending feed mode. The C_1,2-dehydrogenation by encapsulated Arthrobacter simplex in the sodium cellulose sulphate (NaCS) /poly-dimethyl-diallyl-ammonium chloride (PDMDAAC) microcapsules was preliminarily investigated. The bacteria cells were firstly incubated to fulfill the requirement of feeding, and then encapsulated into NaCS/PDMDAAC microcapsules. After 4h activation in medium, the immobilized Arthrobacter simplex cells were used for the following bioconversion. On the contrary the conversion was higher under solvent-suspending feed mode than that under water-suspending feed mode, which was different from free Arthrobacter simplex bioconversion. At low feed concentration, the conversion could be as high as 90%.The conversion of substrate in organic/aqueous two-phase microbial biotransformation by NaCS/PDMDAAC encapsulated cells was better than that of free cells, since the microcapsules could reduce the influences of organic solvents on the growth of Arthrobacter simplex.Thirdly, trimethylchlorosilane and methylmagnesium bromide as Grignard reagent were used to afford reaction, then m-chloroperbenzoic acid was added. The dihydroxylation was carried out by the rearrangement of the intermediate. The 16β-methyl, 17α-hydroxyl and 21-hydroxyl were continuously introduced to the compound with high steric selectivity to shorten reaction steps and reduce byproducts. The product was obtained with the yield of 81%. Then, propionic anhydride was used for 21-propionic esterification. The reaction was easy to be achieved with a high yield of 87%. And then, elimination reaction was carried out by the use of a mixture of triethylamine and vinyl chloroformate to make C_9,11-double bond formed. The C_11,12-double bond byproduct, which was quite common in the conventional process, would not be observed in this process. Product with high purity was obtained. The content of isomerous byproducts, content of product after recrystallization and yield were 2%, 98% and 84%, respectively. And, the inactive C_17α-hydroxyl was propionic esterified with the catalysis of p-toluene sulfonic acid, and the yield was up to 85%. Finally, the Cnp-hydroxy-C_9α-chloride, namely BD, was synthesized with N-chlorobutanimide (NCS) under the protection of nitrogen, and the yield was 90%.