6α-甲基强的松龙生产工艺开发
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摘要
6α-甲基强的松龙及其衍生物是一类高效的抗免疫应激反应的肾上腺类皮质激素类固醇,广泛地应用于脏器移植、抗免疫应激反应的临床之中,亦可用于急性肾上腺皮质功能不全及手术后休克等,它还是治疗非典最重要的药物之一。目前国内该药物的生产能力与生产技术水平还比较低,无法满足市场的需求,导致其售价高达25000元/kg左右,从而阻碍了它的推广与应用。因此开发一条低成本、高收率、可操作性强、环保的6α-甲基强的松龙合成路线具有极其重大的经济效益。
本文结合化学合成法及微生物转化法开发了一条6α-甲基强的松龙的合成工艺路线。以醋酸可的松为原料,首先在不必保护酮簇的基础上,通过烯醇醚化,曼尼希反应在C_6位上引入次甲基;然后在酸性条件下将次甲基氢化、经过构型转换合成6α-甲基,解决了合成路线中最为关键也是最困难的一步;再通过简单节杆菌催化6α-甲基醋酸可的松C_(1,2)位脱氢生成6α-甲基醋酸强的松,这是本工艺中比较有特色之处;最后通过保护C_3,C_(20)-酮基,选择性还原C_(11)-酮为C_(11)β-羟基,生成6α-甲基强的松龙,获得较高的收率和质量,通过高效液相色谱、红外光谱及核磁共振谱分析检测合成物质确为6α-甲基强的松龙,目标产物收率为36.7%,纯度达到97.8%。该合成过程具有位置专一性强、收率高的特点,并能缩短反应步骤,避免了易燃、易爆试剂的使用,还可避免化学反应所带来的环境污染问题,对甾体药物的制备具有重要意义。
同时,对简单节杆菌催化6α-甲基醋酸可的松C_(1,2)位脱氢转化过程进行了研究,对脱氢转化工艺条件进行了探索,并采用遗传算法对简单节杆菌发酵培养基进行了优化,得到脱氢转化过程较优的反应条件及培养基组成,并进一步将脱氢转化过程放大到3.7L发酵罐中,得到底物的转化曲线和菌体的生长曲线,并维持较高的甾体底物转化率。
在前述脱氢转化过程研究的基础上,深入研究了简单节杆菌催化6α-甲基醋酸可的松C_(1,2)脱氢转化生成6α-甲基醋酸强的松的动力学,考察了菌体浓度、底物浓度及产物浓度等各种因素对脱氢转化过程的影响,并建立了该脱氢反应的动力学模型,经线性化回归拟合出该模型的动力学参数,理论结果与实验数据得到很好的吻合。
6α-Methylprednisolone and its derivatives which have high anti-immunity activity belong to the adrenal cortex hormone. They are widely used in the replant of viscera, the anti-immunity reaction and the shock after surgery. 6α-Methylprednisolone was also one of the most important clinical pharmaceuticals for SARS (Severe Acute Respiratory Syndrome) due to its special effect on the anti-immunity system. At present the productivity and technology of 6α-Methylprednisolone synthesis is still in the low level in domestic market and can not satisfy the demanding requirement. As a result the price of 6α-Methylprednisolone is up to 25000 RMB/kg which blocks the wide application of 6α-Methylprednisolone. So it is quite urgent to explore a novel synthesis technique for 6a-Methylprednisolone with high yield.An integrated synthesis route for 6a-Methylprednisolone was developed which combined the chemical reaction and microbial bioconversion. The 6α-Methylprednisolone was prepared by a series of steps beginning with Cortisone acetate including the synthesis of 3-enol ether, the mannich reaction, the synthesis of 6α-methyl, the 1-en-dehydrogenation and the transformation of 11 β-hydroxy steroid. The product which was obtained was proved to be 6α-Methylprednisolone by HPLC, FTIR and NMR. The yield and purity of 6a-Methylprednisolone is 36.7% and 97.8% respectively. The synthesis of 6a-Methylprednisolone has high position specificity and high conversion which can be applied into the preparation of other important steroids.Meanwhile, the 1 -en-dehydrogenation of 6a-methyl cortisone by Arthrobacter simplex was investigated. The transformation technique was explored and the optimization method of genetic algorithm was used to obtain the compositions of the fermentation medium. Furthermore, the dehydrogenation transformation was carried out in the 3.7L fermentor which resulted in high conversion rate.Based upon the experimental results, the kinetic characteristics of the 1-en-dehydrogenation of 6a-methyl cortisone by Arthrobacter simplex were investigated. The effects of concentrations of biomass, substrate and product on the conversion process of dehydrogenation were considered. As a result, a noncompetitive inhibitory model was proposed to describe the dehydrogenation process, and the parameters of the kinetic equation were calculated.
本文结合化学合成法及微生物转化法开发了一条6α-甲基强的松龙的合成工艺路线。以醋酸可的松为原料,首先在不必保护酮簇的基础上,通过烯醇醚化,曼尼希反应在C_6位上引入次甲基;然后在酸性条件下将次甲基氢化、经过构型转换合成6α-甲基,解决了合成路线中最为关键也是最困难的一步;再通过简单节杆菌催化6α-甲基醋酸可的松C_(1,2)位脱氢生成6α-甲基醋酸强的松,这是本工艺中比较有特色之处;最后通过保护C_3,C_(20)-酮基,选择性还原C_(11)-酮为C_(11)β-羟基,生成6α-甲基强的松龙,获得较高的收率和质量,通过高效液相色谱、红外光谱及核磁共振谱分析检测合成物质确为6α-甲基强的松龙,目标产物收率为36.7%,纯度达到97.8%。该合成过程具有位置专一性强、收率高的特点,并能缩短反应步骤,避免了易燃、易爆试剂的使用,还可避免化学反应所带来的环境污染问题,对甾体药物的制备具有重要意义。
同时,对简单节杆菌催化6α-甲基醋酸可的松C_(1,2)位脱氢转化过程进行了研究,对脱氢转化工艺条件进行了探索,并采用遗传算法对简单节杆菌发酵培养基进行了优化,得到脱氢转化过程较优的反应条件及培养基组成,并进一步将脱氢转化过程放大到3.7L发酵罐中,得到底物的转化曲线和菌体的生长曲线,并维持较高的甾体底物转化率。
在前述脱氢转化过程研究的基础上,深入研究了简单节杆菌催化6α-甲基醋酸可的松C_(1,2)脱氢转化生成6α-甲基醋酸强的松的动力学,考察了菌体浓度、底物浓度及产物浓度等各种因素对脱氢转化过程的影响,并建立了该脱氢反应的动力学模型,经线性化回归拟合出该模型的动力学参数,理论结果与实验数据得到很好的吻合。
6α-Methylprednisolone and its derivatives which have high anti-immunity activity belong to the adrenal cortex hormone. They are widely used in the replant of viscera, the anti-immunity reaction and the shock after surgery. 6α-Methylprednisolone was also one of the most important clinical pharmaceuticals for SARS (Severe Acute Respiratory Syndrome) due to its special effect on the anti-immunity system. At present the productivity and technology of 6α-Methylprednisolone synthesis is still in the low level in domestic market and can not satisfy the demanding requirement. As a result the price of 6α-Methylprednisolone is up to 25000 RMB/kg which blocks the wide application of 6α-Methylprednisolone. So it is quite urgent to explore a novel synthesis technique for 6a-Methylprednisolone with high yield.An integrated synthesis route for 6a-Methylprednisolone was developed which combined the chemical reaction and microbial bioconversion. The 6α-Methylprednisolone was prepared by a series of steps beginning with Cortisone acetate including the synthesis of 3-enol ether, the mannich reaction, the synthesis of 6α-methyl, the 1-en-dehydrogenation and the transformation of 11 β-hydroxy steroid. The product which was obtained was proved to be 6α-Methylprednisolone by HPLC, FTIR and NMR. The yield and purity of 6a-Methylprednisolone is 36.7% and 97.8% respectively. The synthesis of 6a-Methylprednisolone has high position specificity and high conversion which can be applied into the preparation of other important steroids.Meanwhile, the 1 -en-dehydrogenation of 6a-methyl cortisone by Arthrobacter simplex was investigated. The transformation technique was explored and the optimization method of genetic algorithm was used to obtain the compositions of the fermentation medium. Furthermore, the dehydrogenation transformation was carried out in the 3.7L fermentor which resulted in high conversion rate.Based upon the experimental results, the kinetic characteristics of the 1-en-dehydrogenation of 6a-methyl cortisone by Arthrobacter simplex were investigated. The effects of concentrations of biomass, substrate and product on the conversion process of dehydrogenation were considered. As a result, a noncompetitive inhibitory model was proposed to describe the dehydrogenation process, and the parameters of the kinetic equation were calculated.
引文
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