Study On The Preparation And Analysis Of Impurities In Tedizolid Phosphate API

Tedizolid phosphate was developed by Cubist pharmaceutical company and was approved by the FDA in June 2014.The chemical name of tedizolid phosphate is(R)-3-(4-(2-(2-methyltetrazol-5-yl)pyridin-5-yl)-3-fluorophenyl)-5-Hydroxymethyloxazolidin-2-one dihydrogen phosphate and its trade name is Sivextro.Its mechanism of action is similar to that of other oxazolidinone antibiotics.By binding to the 23S ribosomal RNA(rRNA)of the 50S subunit,it prevents the formation of the 70S initiation complex,thereby inhibiting protein synthesis.Acute bacterial skin and skin structure infections caused by gram-positive bacteria can be treated by two drug delivery routes:oral and injection.The most common side effects confirmed in clinical trials are nausea,headache,diarrhea,vomiting and dizziness.Tedizolid phosphate,as the second marketed oxazolidinone drug,has broad prospect of application.It is often seen in researches on its antibacterial activity,clinical efficacy,safety and drug resistance.There are few researches on the synthesis and determination of its related substances.In the process of drug development,research on impurities is also an important content.This dissertation takes tedizolid phosphate API as the research object,analyzes the impurities produced in its synthetic route.It uses directional synthesis method to prepare five impurities of tedizolid phosphate API and uses preparative high performance liquid chromatography to purify them.The structure of these five impurities was identified.Using these five impurities as reference substances,an HPLC method for the determination of related substances of tedizolid phosphate API was established and the methodology was verified,laying the foundation for the quality control of Tedizolid phosphate API.The research content is as follows:1 Literature researchThe ABSSSI of the indications for tedizolid phosphate is summarized in terms of definition,symptoms,pathogens and current treatment methods.Summarize methods for preparation and analysis by liquid chromatography of API impurities to provide a theoretical basis for the research on impurities of tedizolid phosphate API.A comprehensive analysis and summary of the pharmaceutical research progress of tedizolid phosphate in recent years such as synthesis process research,impurity research,in vitro activity research and pharmacokinetics,safety,tolerability research was made to lay the foundation for the quality research on tedizolid phosphate API.2 Research on preparation method of impurities in tedizolid phosphate APIUsing(5R)-3-(4-bromo-3-fluorophenyl)-5-hydroxymethyloxazolidin-2-one and pinacol biborate in[1,1’-bis(diphenyl)Phosphine)ferrocene]dichloride palladium dichloromethane complex catalyzed to obtain borate ester compound(5R)-3-(3-fluoro-4-(4,4,5,5-tetramethyl-1),3,2-Dioxaborin-2-yl)phenyl)-5-hydroxymethyloxazol idin-2-one followed by 5-bromo-2-(2-methyl-2H-tetrazole-5-yl)pyridine undergoes Suzuki coupling to obtain tedizolid.It is then reacted with phosphorus oxychloride to form tedizolid phosphate.According to the synthesis process of tedizolid phosphate,multiple reaction by-products are produced during its preparation.Tedizolid reacts with phosphorus oxychloride and triethylamine to form the intermediate state((R)-3-(3-fluoro-4-(6-(2-methyl)-2H-tetrazol-5-yl)pyridyl-3-yl)phenyl)-2-oxazolidin-5-yl)methyl dichlorophosphate may continue to react with tedizolid and hydrolyze to form impurities B0201.Tedizolid phosphate may hydrolyze to produce impurity A01.Chloride ion replaces phospholipid to produce impurity A020501.N on the pyridine ring of tedizolid phosphate is easily oxidized into impurities C01 in the air under high temperature conditions.Tedizolid phosphate will hydrolyze and open the ring to generate impurity C02 under alkaline conditions.The five impurities are prepared by a directional synthesis method.(5R)-3-(3-fluoro-4-(dihydroxyboron)phenyl)-5-hydroxymethyloxazolidin-2-one and 5-bromo-2-(2-methyl-2H-tetrazol-5-yl)pyridine occurs Suzuki coupling to obtain impurity A01.Tedizolid is substituted with thionyl chloride to form impurity A020501.Tedizolid produces impurity B0201 through condensation reaction.Tedizolid phosphate can be oxidized into impurity C01 with 30%hydrogen peroxide.Tedizolid phosphate is ring-opened in sodium hydroxide solution and then acidified with hydrochloric acid to obtain impurity C02.Separate and purify five impurities by preparative high performance liquid chromatography[Column:YMC ODS-AQ(50×250 mm,10 μm);Detection wavelength 300 nm;Flow rate 60 mL/min;Mobile phase A:20 mM ammonium formate solution adjusted to pH 4.5 with formic acid;Mobile phase B:acetonitrile;linear gradient elution].Collect fractions according to the peak type.Use HPLC[column:Sepax BR-C18(4.6×250 mm,5 μm);detection wavelength 300 nm;flow rate 1.0 mL/min;column temperature 30℃;Mobile phase A:10 mM ammonium formate solution adjusted to pH 4.5 with formic acid;Mobile phase B:acetonitrile;linear gradient elution:0-40 min,10%B-90%B,40-50 min.90%B,50-52 min,90%-10%B,52-60 min,10%B]to detect the purity of the fractions.The fractions with a purity greater than 95 percent are then desalted[Column:YMC ODS-AQ(50×250 mm,10 μm);detection wavelength 300 nm;flow rate 60 mL/min;Mobile phase A:purified water;Mobile phase B:acetonitrile;linear gradient elution:0-30 min,10%B,30-31 min,10%B-90%B,31-51 min.90%B].Use HPLC detects the purity of the fractions.The fractions with a purity greater than 95 percent are concentrated and dried under reduced pressure at 30℃ to obtain impurity reference substances.The structures of the five impurities were confirmed by HRMS and 1H NMR.The molecular formula and molecular weight measured in the high-resolution mass spectrometry ES+combined with 1H NMR and the research background of tedizolid phosphate can confirm that the impurities are consistent with the corresponding structure.The impurities were calibrated from the aspects of character and HPLC purity.The character is visually inspected and the purity of impurity is determined by the HPLC method(area normalization method calculation).As a result,the impurities meet the limit requirements of the reference substance.3 Research on analysis method of impurities in tedizolid phosphate APIThe related substances of tedizolid phosphate API were determined by HPLC.The established chromatographic condition for related substances were:Column:Sepax BR-C18(4.6×250 mm,5 μm);detection wavelength 300 nm;flow rate 1.0 mL/min;column temperature 30℃;Mobile phase A:10 mM ammonium formate solution adjusted to pH 4.5 with formic acid;Mobile phase B:acetonitrile;linear gradient elution:0-40 min,10%B-90%B,40-50 min,90%B,50-52 min,90%-10%B,52-60 min,10%B;injection volume:10 μL.Perform methodological verifications such as the selection of detection wavelength,system applicability,specificity,durability,detection limit and quantification limit,repeatability,stability and linearity.The results showed that:It is more suitable to detect at the wavelength of 300nm and impurity C02 was calculated by self-control method with correction factor at this wavelength.Tedizolid phosphate was well separated from each impurity.Good separation can be achieved between the main degradation products and tedizolid phosphate that appear under forced degradation(acid,alkali,oxidation,high temperature,strong light)conditions.The test solution and the reference solution of tedizolid phosphate are stable within 24 hours at room temperature.Tedizolid phosphate and each impurity has a good linear relationship within the range of 0.02%～0.20%(relative to the concentration of the test solution).Repeatability,durability and system applicability are all good.Three batches of self-made raw materials were tested.The result showed that the detected content of each impurity was less than 0.10%and the maximum detected content of total impurities was 0.32%,which met the requirements.