Study On The Synthesis Of Several Ursodeoxycholic Acid Process Impurities And Lithocholic Acid Intermediates

Primary biliary cirrhosis（PBC）is a chronic cholestatic liver disease.Ursodeoxycholic acid is the only drug approved by the FDA for the treatment of PBC.The domestic and foreign market has reached 2.5 billion yuan and is still growing rapidly.At present,there is no industrialized production of ursodeoxycholic acid using plant-derived BA as raw material in China.This is due to the difficulty in purifying the raw material BA,the long overall reaction route,the harsh reaction conditions of some parts and the separation and characterization of impurities in the reaction process have not been completed,etc.Lithocholic acid not only has a variety of biological activities,but also can be used as a raw material to prepare a variety of biologically active substances.The reported synthesis method of lithocholic acid intermediate 3-oxo-lithocholic acid ester has disadvantages such as time-consuming and difficult separation of by-products.Therefore,it has important application value to study the impurity generated in the process of BA synthesis of ursodeoxycholic acid and the synthesis process of lithocholic acid intermediate 3-oxo-lithocholic acid ester.The specific work of this paper is as follows:（1）Through literature research,a total of 9 reaction routes were identified,such as selective oxidation reaction,reduction reaction,Mitsunobu reaction,etc.,23 kinds of ursodeoxycholic acid process impurities and related intermediates were synthesized,and completed structural characterization of impurities and their intermediates.Among them,Mitsunobu reaction,reduction reaction and hydroxyl selective protection reaction can be applied to the synthesis of other cholic acid system impurities and obeticholic acid,so the reaction conditions are systematically optimized.The reported method for synthesizing 3β-ursodeoxycholic acid by the Mitsunobu reaction is by reacting methyl ursodeoxycholate with formic acid,triphenylphosphine,and diethyl azodicarboxylate in benzene or toluene solution with a yield of 50～60%.One of the disadvantages of this method is the use of toxic benzene or toluene as a solvent,and the second is that the yield is low.In order to overcome these deficiencies,after a comprehensive investigation of the reaction selectivity,operational safety and cost,p-nitrobenzoic acid was selected instead of formic acid,and dried THF was used as the solvent.The optimal conditions are that the molar ratio of p-nitrobenzoic acid,triphenylphosphine,diethyl azodicarboxylate and methyl ursodeoxycholate is 1.8:1.8:1.8:1.0,with THF solvent,sample addition and stirring at 0°C Then,the temperature was slowly raised to 35°C for 12 h,and the yield was 72%;after investigating the carbonyl reduction reaction,the large sterically hindered lithium tri-tert-butoxyaluminum hydride was selected as the reducing agent,and the optimal condition is that the molar ratio of 3β-hydroxy-7-methyl ketolithocholic acid to lithium tri-tert-butoxyaluminum hydride was 1.0:2.0.The reaction was stirred at 0°C for 4 h in THF solution,and the yield was 91%.The selectivity of the hydroxyl protection reaction was comprehensively investigated,after the cost,selecting trimethylacetyl is the protecting group of the C-3 position hydroxyl,and the optimum condition is that the mol ratio of methyl chenodeoxycholate and trimethylacetyl chloride is 1.0:1.8,taking toluene as a solvent,pyridine As a base,the reaction was carried out at 70°C for 4 h with a yield of 94%.（2）The synthesis process of lithocholic acid intermediate 3-oxo-lithocholic acid methyl ester was studied.The by-product 3-oxo-5α-lithocholic acid methyl ester is similar in polarity to the target product 3-oxo-lithocholic acid methyl ester,which makes its separation difficult.In the past reports,high-activity Pd/C was often used as the catalyst,but the content of by-products was relatively high,about 6-11%.This paper explores the preparation of methyl 3-oxo-lithocholic acid by catalytic hydrogenation of nano-Pd-Cu,a bimetallic material with high activity,high stability and unique structure,as a catalyst,and the reaction conditions are systematically investigated.The optimal condition is to add 6 mg of catalyst,26 mg of triethylamine,and 0.1 g of raw materials to 5 ml of mixed solution（Me OH/DCM=4/1,v/v）,and react at 10°C for 9 h under the atmosphere of hydrogen,the yield was 92%,and only1～2%of the by-products in the 5α-H configuration were in the reaction product.The used nano-Pd-Cu can still be reused after being recovered and dried.The structures of the compounds synthesized in the above studies were confirmed by means of ¹H NMR,¹³C NMR,MS and melting point determination.