Intermediates Synthesize And Process Optimization Of The Anti-EBOV Antisense Oligonucleotides AVI-7537

Ebola virus(EBOV)is a kind of single-strand negative-sense RNA viruses which in the family of Filovirus,firstly found in South Sudan and Congo,District of Ebola River.The virus can be infected through contact with infected animals or humans and with very high fatality rate.It is one of the most dangerous virus in the world,its class-IV biosafety level even higher than SARS and HIV.The anti-EBOV Antisense Oligonucleotides AVI-7537 which based on the structure of Phosphorodiamidate Morpholino Oligonucleotides(PMOs)designed by Sarepta Therapeutics Inc.(used name of AVI Inc.)U.S.,Comparing 19 morpholino nucleosides and linked by Phosphorodiamidate structures.It can combine with EBOV's capsid related protein VP24 m RNA,and cover the translational start codon and downstream,influence the related protein synthesis by blocking mechanism to inhibit the process of virus infection.There are some studies show that the AVI-7537 can raise the survival rate of the primates that infected with EBOV.The structure of PMOs is modularized,can be modified through change the morpholino nucleosides' sequence and numbers in order to combine with different target m RNA sequences to realize different functions,especially stand out in the area of anti-infection and genetic diseases therapy.Therefore,the research of PMOs can also lay the theoretical and materials foundation for establish the PMOs Antisense Oligonucleotides drugs platform.This research based on related literature to study the synthesis method and the processes optimization of the intermediate structures of PMOs.Here's the details:Purpose: Based on anti-EBOV Antisense Oligonucleotides AVI-7537,explorer the synthesis method of all intermediate structures before the phosphorodiamidate morpholino oligonucleotides are formed,and ensure the intermediate structures are correct.Optimize the synthesis processes to find an efficient,convenient way to get target compounds and the processes also can be enlarged easily.Accumulate the PMOs' structure unit compounds morpholino monomers,and found the morpholino monomer compounds depot,lay the theoretical and material foundation for the further research of PMOs Antisense Oligonucleotides drugs.Methods:(1)Synthesis of morpholino monomers: Modified the ribose to morpholino and protect the key chemical group to get morpholino monomers by the initial structure of N-Benzoylcytidine,Guanosine,Adenosine and 5-Methyluridine.(2)Optimize the synthesis processes of morpholino monomers: Optimize the synthesis processes of morpholino monomers in order to get the convenient synthesis processes with low cost and high yield.(3)Synthesis and processes optimization of Phosphorodiamidate backbone structures: Synthesis and optimizate the processes of two types Phosphorodiamidate backbone linkage in AVI-7537 – N,N-dimethylphosphoramidodichloridate and N-(trifluoroacetyl piperazinyl)-N-phosphoryldichloridate.(4)Synthesis of Phosphorodiamidate Morpholino Nucleosides: Synthesize the Phosphorodiamidate Morpholino Nucleoside with two types of Phosphorodiamidate linkages and four types of morpholino monomers which can directly use for synthesize the AVI-7537 or other PMOs.There are 6 types of phosphorodiamidate morpholino nucleoside,including four types of dimethylamino-phosphorodiamidate morpholino monomer and two types of piperazinyl-phosphorodiamidate morpholino monomer.(5)Structural analysis of key compounds and foundation of morpholino monomer compounds depot: Determine the structure of key intermediate compounds in this research,accumulate the PMOs' structure unit compounds morpholino monomers to establish the morpholino monomers compounds depot,lay the theoretical and materials foundation for further research of AVI-7537 and other antisense oligonucleotides that based on structure of PMOs.Results:(1)Synthesis of morpholino monomers: The compounds N4-benzoyl-7?-hydroxy-N-trityl morpholinocytidine,N2-benzoyl-7?-hydroxy-N-trityl morpholinoguanosine,7?-hydroxy-N-trityl morpholinothymidine and N6-Benzoyl-7?-hydroxy-Ntrityl Morpholinoadenosine are synthesized by the initial structure of NBenzoylcytidine,Guanosine,5-Methyluridine,and Adenosine.7?-Hydroxy-N-trityl Morpholinothymidine,N4-Benzoyl-7?-hydroxy-N-trityl Morpholinocytidine,N2-Benzoyl-7?-hydroxyl-N-trityl Morpholinoguanosine were synthesized by the process of “base's primary amino protect(Guanosine)? synthesis of morpholine ?morpholine's secondary amino protect”.N6-Benzoyl-7?-hydroxy-N-trityl Morpholinoadenosine was synthesized by the process of “synthesis of morpholine ? secondary amino protect in morpholine ? primary amino protect in base(Adenosine)”.(2)Optimize the synthesis processes of morpholino monomers: Have optimized the two synthesis processes of four types of morpholino monomers.Comparing with the completely process that reported by literature,the yield of N4-Benzoyl-7?-hydroxy-Ntrityl Morpholinocytidine with optimized synthesis process is 60.8%,11.8% higher than reported.The yield of 7?-Hydroxy-N-trityl Morpholinothymidine is 43.2%,nearly equal with reported.N2-Benzoyl-7?-hydroxyl-N-trityl Morpholinoguanosine is a brandnew compound that not reported with yield of 26.9%.The improved process is more convenient,low cost and with good yield,also can be enlarge easily.N6-Benzoyl-7?-hydroxy-N-trityl Morpholinoadenosine with another optimized synthesis process lead to a lower yield 13.6%,also much higher than to follow the literature's operation.This result may relate to the reaction material.But the optimized synthesis process has obvious shorter reaction time,more convenient in purity operation and can be enlarge easily.(3)Synthesis and processes optimization of Phosphorodiamidate backbone linkage structures: Synthesized and optimized the process of phosphorodiamidate backbone linkage type one,N-(trifluoroacetyl piperazinyl)-N-phosphoryldichloridate,lower the dosage of some materials in synthesis,reduce the difficulty of synthesis process.Have chosen the best synthesis process to get the phosphorodiamidate backbone linkage type two,N,N-dimethylphosphoramidodichloridate.The total yield of N-(trifluoroacetyl piperazinyl)-N-phosphoryldichloridate and N,N-dimethylphosphoramidodichloridate are 40.3% and 75.4%.To avoid deterioration,the backbone linkages are not being accumulated largely.(4)Synthesis of Phosphorodiamidate Morpholino Nucleosides: Synthesized the six types of phosphorodiamidate morpholino nucleosides in AVI-7537 which can form the PMOs directly,including four types of dimethylamino-phosphorodiamidate morpholino monomers and two types of piperazinyl-phosphorodiamidate morpholino monomers.The yield of two types phosphorodiamidate morpholino nucleosides with the base thymine are highly to over 40%.The phosphorodiamidate morpholino nucleoside with the base N2-Benzoylguanine have the lowest yield 14.7%.The rest three types of phosphorodiamidate morpholino nucleosides are with yield about 30%.Because of the unstable structure,the conpounds are not been accumulated.(5)Structural determination of key compounds and foundation of morpholino monomer compounds depot: Analyzed the four types of morpholino monomers,two types of phosphorodiamidate backbone linkage and six types of phosphorodiamidate morpholino nucleosides through NMR or HRMS to ensure the molecular structure are correct.Accumulated dozens grams of the each four types morpholino monomers,lay the materials foundation and can also satisfy the requirements for the further research in AVI-7537 or others antisense oligonucleotides base on PMOs.There is no report can be found about the study of PMOs' synthesis in China.This study gives positive effects on the study of anti-EBOV drugs and the further study of antisense drugs based on structure of PMOs in China.