Synthesis And Quality Control Of Tropane Intermediates

Objective:Compounds 2β-carbomethoxy-3β-（4-chlorophenyl）tropane（β-CCT）and 2β-carbomethoxy-3β-（4-fluorophenyl）tropane（β-CFT）are key intermediates for the synthesis of corresponding clinical dopamine transporter（DAT）imaging agents,such as^99mTc-TRODAT-1,[¹⁸F]FECNT,[¹¹C]CFT.The research and quality control of intermediates are important in the preparation of radio-labelling precursors（Active Pharmaceutical Ingredients,APIs）.It is significance to stabilize the preparation process and provide important information for the quality research of APIs.In this study,we synthesized and finished quality control of the two tropane intermediates.Methods:Firstly,anhydroecgonine methyl ester was obtained from ecgonine hydrochloride by dehydration and esterification.Compoundsβ-CCT andβ-CFT were achieved by Michael addition of corresponding Grignard reagent to anhydroecgonine methyl ester,respectively.Their corresponding related substances（2α-carbomethoxy-3β-（4-chlorophenyl）tropane,α-CCT;2α-carbomethoxy-3β-（4-fluorophenyl）-tropane,α-CFT）were also separated.The corresponding hydrolysate compound（2β-carboxyl-3β-（4-chlorophenyl）tropane,β-CXCT;2β-carboxyl-3β-（4-fluorophenyl）tropaneβ-CXFT）were obtained by hydrolysis ofβ-CCT andβ-CFT in aqueous dioxane,respectively.The structures of all compounds were verified by NMR,MS and UV.Properties of physical and chemical properties including melting range/melting point and specific rotation.Method parameters of high-performance liquid chromatography（HPLC）ofβ-CCT andβ-CFT were validated for specificity,linearity,precision,accuracy,robustness,respectively.The established chromatographic methods were used for the content analysis ofβ-CCT andβ-CFT,respectively.Results:1.The key intermediatesβ-CCT,β-CFT and their corresponding related substances were prepared and confirmed in our laboratory.The structures were verified by ¹H NMR,¹³C NMR and UV.The specific rotation ofβ-CCT and its related substancesα-CCT andβ-CXCT was-19.8,+12.56 and-96.86,respectively.The specific rotation ofβ-CFT and its related substancesα-CFT andβ-CXFT was-22.29,+7.49 and-92.48,respectively.The melting range ofβ-CCT and its related substanceα-CCT was 112.9～113.4℃,76.3～77.1℃.The melting range ofβ-CFT and its related substanceα-CFT was 91.2～92.5℃,55.9～56.6℃.The melting point ofβ-CXCT andβ-CXFT was over 250℃.2.The HPLC separation ofβ-CCT and its related substances and method validation were carried out on a reverse-phase phenomenex^TMGemini C₁₈column（150×4.6 mm,particle size 5μm 118（?））with the mobile phase consisting of methanol,water and TFA（30/70/0.1,v/v/v）.The temperature of the column temperature box was set to 30℃and the flow rate of mobile phase for isocratic elution was 1.0 m L/min.The detection wavelength was set to 220 nm to monitor analytes.The total completed analysis time was 30 minutes and the injection volume was 10μL for analytes.The HPLC analysis conditions ofβ-CFT and its related substances were following:the stationary phase,flow rate,column temperature and flow rate were the same asβ-CCT.The mobile phase was methanol,water and TFA（22/78/0.1,v/v/v）.The detection wavelength was set to205nm.3.Correlation coefficient within the corresponding concentration range of key intermediates and their corresponding related substances were greater than 0.9995.The recovery was 98.00～102.00%.The relative standard deviations（RSD%）of intra-day precision and intra-day precision were less than 2.0%.Forced degradation experiments displayed thatβ-CCT andβ-CFT were stable in thermal and photolytic degradation conditions,and were unstable in alkaline and hydrogen peroxide environment.β-CCT was also stable in acid environment butβ-CFT was not.The peaks of the key intermediates and their corresponding related substances were not significantly affected by any of the small modifications in chromatographic conditions.Both the sample solution and the mobile phase were stable at room temperature for at least 72 hours.4.The developed and validated HPLC method was used for the content analysis of samples.The content ofβ-CCT was 99.16～99.75%and the content ofβ-CFT was98.84～99.91%.Conclusion:Compoundβ-CCT,β-CFT and their corresponding related substances have been synthesized in our laboratory.A rapid and accurate HPLC method was developed and validated for the analysis ofβ-CCT andβ-CFT,respectively.A key technology for analysis of tropane derivatives was developed.A preliminary analysis method was established for tropane intermediates and valuable information was provided for tropane drugs research.