Synthetic Of Stable Isotope Labeled β-Agonists

With development of economic globalization, cross-border and cross-regional of food flowed more frequently, and a great variety of food safety issues exposed many shortcomings, they made a great threat on people’s lives and health. Residuing in food of β-agonists not only brings harm to human beings, but also obstructs the development of the society. Using stable isotope reagents as an internal standard reagent,and dilution mass spectrometry with isotope to detect residual β-agonists, can greatly improve the accuracy of detection methods.Based on the requirements of the detection of the internal standard, we synthesized butylamine -D9, tulobuterol-D9. During the experiments,we used the natural abundance of raw materials to make the preliminary tests and optimize the reaction conditions. Based on the improvement of each step of experimental exploration,we did the total synthesis. Then we did the isotopically labeled experiments, and characterized the products with high performance liquid chromatography analysis methods, mass spectrometry, nuclear magnetic resonance spectroscopy, nuclear magnetic resonance spectra of nitrogen, IR.(1) The process route synthesis of tert-butylamine -D9:deuterated methanol, deuterated acetone were reagent to synthesize tert-butanol -D9(abundance of 99.0%), then we used tert-butyl formamide method to synthesis of tert-butylamine -D9. In Synthesis of tert-butyl-methylamine, the order of addition has a great influence on the yield of the reaction. Based on the experimental optimization, we got the best order of addition:acetic acid sodium cyanide, then concentrated sulfuric acid was slowly added dropwise to the mixture, again tert-butanol was added dropwise to the mixture. The purity of Tert-butylamine -D9 was 99.8%, the isotopic abundance was 97.7% atom% D determined.by mass spectrometry.(2) 3,5-dichloro-Aminoacetophenone was bromided, aminated and reductived to synthesize clenbuterol with tert-butylamine as labeling precursor. Amimation step was optimized by uniform design, then we obtained optimum synthesis results with control A2 feeding amount, the optimal reaction conditions:reaction time 4h, the reaction temperature is 63℃, the molar ratio of tertiary amine and A2 of 2:1, chloroform (solvent) used 60mL. Reduction steps investigated influences of solvent on the yield by single factor, the experiment results from a single solvent,, the yield of methanol as the reaction solvent was the highest. Reduction with the amount of water added then the reduction has been significantly increase yield reached 65.9%, So this experiment choosed methanol-water mixed as the solvent.Based on the consuming of T-butylamine, the total yield of clenbuterol of was 31.2%. Confirming the structure by HPLC, MS,’NMR and other means of instrumental analysis and chemical purity was hinger was 98.7%.(3) Aminoacetophenone was bromided by NBS and CuBr2, aminated and reductived to synthesize brombuterol with tert-butylamine as labeling precursor. NBS bromination step investigated solvent and temperature, we ultimately selected the DMF as solvent, the temperature is 70 ℃, reaction time was 4h by experiments. Based on the consuming of T-butylamine, the total yield of brombuterol was 27.3%. Confirming the structure by HPLC, MS,1NMR and other means of instrumental analysis and chemical purity was 98.5%.(4) O-chloroacetophenone was bromided by CuBr2, aminated and reductived to synthesize tulobuterol-D9 with tert-butylamine -D9 as labeling precursor. Amination step was optimized by uniform design, then we obtained optimum synthesis results with control C2 feeding amount, the optimal reaction conditions:reaction time is 5h, the reaction temperature is 59 ℃, the molar ratio of 2-tert-butylamine and C2 is 2:1, chloroform (solvent) used 25mL. Reduction steps investigated influences of sodium borohydride on reaction yield, based on the experimental results when the molar ratio of sodium borohydride and C3 is 2:1, the yield best results. Based on the consuming of T-butylamine -D9, the total yield of brombuterol-D9was 17.4%. Confirming the structure by HPLC, MS,1NMR and other means of instrumental analysis and chemical purity was 98.0%, isotopic abundances was 97.0 atom%D.(5) 1-(two-3,5-(N, N-dimethylamino carbamoyloxy) phenyl)-2-bromo-ethanone was aminated by t-butylamine, and reducted by sodium borohydride to get Bambuterol. The yield was 20.8% of the amination step, amination product liquid purity of over 99%, mp 165-169 ℃. The yield of reduction step was 21.0%, the liquid purity of Bambuterol was 98%, mp 222～225 ℃.(6) 3,5-dihydroxyacetophenone was bromide by copper bromide, esterificated by chloride. Yield of the first two steps were 57%,72.8%, purity of the liquid phase were 99%, 99%.