Synthesis And Optimization Of The Linker Mc-Val-Cit-PABC-PNP For Antibody-Drug Conjugate

Objective:Brentuximab vedotin（Adcetris）is an Antibody-drug conjugate（ADC）developed by Seattle Genetics and Takeda.Its structure is composed of antibody c AC10specific for the antitumor antigen CD30,cleavable dipeptide linker Mc-Val-Cit-PABC-PNP,and small molecule toxic drug Monomethyl auristatin E（MMAE）.It is mainly used for the treatment of Hodgkin’s lymphoma and systemic anaplastic large cell lymphoma.Since Adcetris is still under patent protection（expires in July 2023）,only the expensive original drug is currently sold in China.Therefore,this project focuses on the study of the linker of Adcetris,and obtains a stable,high-yield,green chemistry requirements,and suitable for industrial production of the synthesis process through design and experiments.It can provide a reference for the development of subsequent Adcetris generic drugs and ADC with the same linker to achieve the goal of reducing production costs.Methods:First,a better synthetic route was selected by comparing and analyzing the existing synthetic processes of the linker Mc-Val-Cit-PABC-PNP.The route uses N-[（9H-Fluoren-9-ylmethoxy）carbonyl]-L-valyl-2,5-dioxo-1-pyrrolidinyl ester（Fmoc-Val-OSu）as the starting material,and first undergoes an ammonolysis reaction with citrulline;under the action of N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline（EEDQ）,amidation reaction was carried out with p-aminobenzyl alcohol to connect a spacer group that can be spontaneously eliminated;then diethylamine was used as the organic base to remove the Fmoc protecting group on valine;the exposed N-terminal was subjected to ammonolysis reaction with N-Succinimidyl 6-maleimido hexanoate（Mc-OSu）;the target compound was obtained by transesterification with bis（p-nitrophenyl）carbonate.The purity and yield of the products were improved by optimizing the reaction time,purification of intermediates,dosage ratio,use of organic bases,reagents used in the synthesis of the target compounds and purification methods in single-factor tests,and the structures of each intermediate and end product were confirmed by ¹H NMR,¹³C NMR,IR and HRMS.Then,the amount of starting material was increased to 100 g,and the optimized synthesis process was verified by scaling-up test.Subsequently,the HPLC conditions and methods selected for the determination of the linker Mc-Val-Cit-PABC-PNP were validated by methodological investigation.Finally,the reaction of the obtained linker with the MMAE model compound was performed to determine the conditions for the coupling reaction of the linker Mc-Val-Cit-PABC-PNP with small molecule toxic drugs.Results:The structure of the end product obtained by ¹H NMR,¹³C NMR,IR and HRMS methods was confirmed as the linker Mc-Val-Cit-PABC-PNP in small pilot and scale-up tests,with a content of 99.2%.When the feeding scale was 100 g,the total yield of the synthesis process was stable up to 28.3%.The method conditions for the determination of content by HPLC were as follows:C₁₈ chromatographic column;mobile phase:V（acetonitrile）:V（water）=50:50;the injection volume was 20μL;the column temperature was 30℃;the detection wavelength was 254 nm.The coupling conditions of the linker and small molecule toxic drug were determined as follows:n（Mc-Val-Cit-PABC-PNP）:n（HOBt）:n（small molecule toxic drug）:n（N,N-diisopropylethylamine）=1.0:1.0:1.1:2.0,and the reaction was carried out overnight at room temperature.Conclusion:The optimized synthesis process of the linker Mc-Val-Cit-PABC-PNP avoids the use of hazardous chemicals and highly toxic substances,and is suitable for industrial production because of its mild conditions and easy operation compared with the original route,high purity and stable yield of the product.After the methodological investigation,the chromatographic conditions for the determination of the content of the linker Mc-Val-Cit-PABC-PNP by HPLC had a good linear relationship,and the recovery rate,precision and repeatability experiments all met the requirements of 2020 edition of“Chinese Pharmacopoeia”for the determination of content by HPLC.The product structure obtained by the coupling experiment of the linker Mc-Val-Cit-PABC-PNP and MMAE model compound was confirmed as the expected compound,and the coupling condition of the linker with small molecule toxic drug was feasible.The experimental results can provide a favorable basis for the study of Adcetris generic drugs,and promote the synthesis and development of ADC with same linker.