Synthesis Of Ataluren,Related Substances And Ataluren Pharmacokinetic Studies

Ataluren?also known as PTC124,trade name Translarna?,3-??5-?2-fluorophenyl??-3-?1,2,4-oxadiazolyl??-benzoic acid is a small molecule oral drug developed by the United States PTC company,which can treat nonsense mutations in Duchenne muscular dystrophy?Duchenne muscular dystrophy and Becker muscular dystrophy?and cystic fibrosis.Ataluren induces to ignore the termination codons,continue reading normal genetic information,and complete functional protein synthesis.It plays an important role in forcing the body ignoring gene mutations and producing normal proteins.Part?Synthesis of AtalurenObjective:The aim is to establish synthetic methods of Ataluren.Methods:Using 3-cyanobenzoicacid?2?as the starting material,methyl-3-?N-hydroxycarbamimidoyl?-benzoate?4?was prepared through esterification and addition.Condensation of?4?and 2-fluorobenzoyl chloride provided methyl-3-?5-?2-fluorophenyl?-1,2,4-oxadiazol-3-yl?-benzoate?5?producted.Final,hydrolysis gave Ataluren?1?.The structure of intermediates and Ataluren were confirmed by mass spectrometry,nuclear magnetic resonance and melting point.Results:Ataluren was successfully synthesized and the structure was confirmed by mass spectrometry,nuclear magnetic resonance and melting point.The synthetic route was optimized and the yield was improved.Conclusions:In this study,using 3-cyanobenzoicacid as the stating material,the target compound Ataluren was synthesized in 4 steps.This synthetic route is concise and the reaction condition is mild.The purity of Ataluren is high enough to be used as a pharmaceutical ingredient.Part ? HPLC study on chemical purity and related substances of AtalurenObjective:To establish a effective strategy in determination of Ataluren content by HPLC,and to understand the possible degradation pathways and degradation products of the drug.Methods:The chromatographic separation was carried out on SunFire^TMM C18?150 mm×4.6 mm,5?m?.The mobile phase consisted of acetonitrile and0.1%formic acid solution?50?50?,flow rate:0.8 mL/min,Injection volume10?L,Column temperature:room temperature.Results:In the above chromatographic conditions,the relevant impurities produced by high humidity,high heat,acid,alkali,light,and oxidation damage are effectively separated from Ataluren main peak.Ataluren linear equation is Y=55.237X+285.82,?r²=0.9992??5?g/mL²50?g/mL?.The minimum detection limit of 1?g/mL.The test solution was stable at room temperature for 24 hours.The same test solution after repeated injection 6 times,the peak area RSD values are less than 1.5%,good precision.The average recovery was99.30%and the RSD values were less than 1.5%.Conclusions:The HPLC method is simple,sensitive,specific and accurate to analyze related substances of Ataluren.It can be used for the inspection of relevant substances of Ataluren during manufacturing and storage.Part ? The study of Ataluren pharmacokinetics in ratsObjective:To establish a strategy in determination of Ataluren concentration in rat plasma by HPLC/MS to study pharmacokinetics of Ataluren in rats.Methods:1 HPLC conditions:The chromatographic separation was carried out on SunFire^TM C18?150 mm×4.6 mm×5?m?;the mobile phase consisted of acetonitrile and 0.1%formic acid solution with a gradient elution,elution program is as follows,10%-35%A?0-15 min?,35%-60%A?15-30 min?,60%-80%A?30-45 min?,85%-95%A?45-60 min?;Flow rate:0.6 mL/min.Injection volume:10?L.2 MS conditions:The mass spectrometer was operated in the positive ion electrospray mode,Monitoring mode:Multi-reaction monitoring.Ion spray voltage?IS?:5500 V;turbo spray temperature,650?;nebulizer gas?GS1?:25Psi;heater gas?GS2?:60 psi;curtain gas?CUR?:65 psi;The monitored ion pair m/z 285/123,desorption voltage?DP?:44 V,collision energy?CE?:23eV.The monitoring of the internal standard ion pair m/z 254/157,dissociation voltage?DP?:53 V,collision energy?CE?:22 eV.3 Sample collection and pretreatment:Six healthy male Wister rats?250±10 g?were used in this study.After a single gavage administration of Ataluren?30mg/kg?,blood samples were collected at 0.25?0.5?1?2?3?4?6?8?10?12 and 24 h.All the samples were precipitated with acetonitrile.Using internal standard method for quantitative.4 Pharmacokinetic Analysis:Based on the results of the assay,Drug concentration-time curve was obtained based on the results of this assay.The pharmacokinetic parameters were calculated using a non-compartmental model.Results:1 Methodological validation:The Ataluren curve was Y=1.67416X-0.1000109?r²=0.9851?,and the linear range was 50¹000 ng/mL for Ataluren.The RSD values of intra-day are 3.3%,11.8%and 12.0%,inter-day are 2.3%,7.5%and 7.8%respectively;The accuracy values are 0.6%,1.1%,-1.1%.The recoveries of quality control samples at above three concentrations for Ataluren were 94.7%,96.8%and 93.7%.RSD were 8.2%,9.4%and7.9%,respectively;matrix effects of quality control samples were98.7%,96.8%,98.7%.2 Pharmacokinetics:AUC_0-t:14.98±2.37?g/L·h,AUC_0-?:15.94±3.15?g/L·h,C_max:3.14±0.33?g/L,T_max:3h,t_1/2:5.86±0.17h,Cl:1.935±0.32L/h/kg,V:13.97±0.49L/kg.Conclusions:The LC-MS/MS method has a good performance in terms of sensitivity,accuracy and precision.It is suitable to determine the plasma concentration of the Ataluren in rats.Part ? The study on metabolites in rats after gavage administration of AtalurenObjective:To establish a universal and effective strategy in finding and indentifying metabolites of Ataluren in vivo by UPLC/qTOF-MS,which was used to study the metabolites in rat plasma.Methods:1 UPLC conditions:Analysis was performed on a UPLC/q TOF-MS.The chromatographic separation was carried out on SunFire^TM C18?150 mm×4.6 mm×5?m?;The mobile phase consisted of acetonitrile and0.1%formicic acid solution with a gradient elution,elution program is as follows,10%-35%A?0-15 min?,35%-60%A?15-30 min?,60%-80%A?30-45min?,85%-95%A?45-60 min?;flow rate:0.6 mL/min;injection volume:5?L;sampler temperature:room temperature.2 qTOF-MS conditions:qTOF-MS using ESI source,The high-resolution mass spectrometer was operated in the positive ion electrospray mode,full scan acquisition,A single run was 60 min.ion spray voltage?IS?:5500 V;turbo spray temperature,650?;nebulizer gas?GS1?:65 Psi;heater gas?GS2?:60 psi;curtain gas?CUR?:35 psi;DP:44 V;CE:23 eV;Each three-pin correction with CDS calibration fluid.3 Sample collection and pretreatment:Six healthy male Wister rats?250±10 g?were used.After a single gavage administration of Ataluren?30mg/kg?,blood samples were collected at 0.25,0.5,1,2,3,4,6,8,10,12and 24 h.All the samples were precipitated with acetonitrile.4 Analytical strategy:Triple TOF 5600 mass spectrometer was used to analyze all biological samples,while Metabolite 1.5 software was applied to screen non target metabolites and search for potential metabolites.Results:Six main metabolites were identified in the plasma samples.Their respective metabolic pathways were analyzed through their secondary ion mass spectroscopy.Conclusions:The main metabolites of Ataluren in the plasma sample were successfully screened and indentified by UPLC/qTOF-MS,which will be helpful for better understanding of Ataluren metabolism in vivo.