Industrialization Of Anti-influenza Drug Favipiravir And Research Of Oseltamivir "Me-Too" Drugs

Influenza is an acute respiratory disease caused by influenza viruses.It is highly pathogenic and widely spread with high morbidity and has become a great threat to the safety of human life and property.Highly pathogenic avian influenza has broken out continuously in recent years all around the world.H7N9 was first reported in 2013 in China and has caused over a thousand death in China with a fatality rate as high as 39%.According to the antigenic characteristics of HA and NA on its surface,influenza virus can be divided into many subtypes.Influenza viruses complete replication through the process of adsorption,introduction,uncoating,biosynthesis,assembly and release in host cells and constantly undergo antigenic drift and antigenicshift to avoid the immune system of the host.Currently,vaccination and drugs are two main therapies against influenza.Since influenza viruses mutate easily and vaccine development is hysteretic,antiviral drugs have become a major measure of dealing with influenza.Anti-influenza drugs mainly contain the following categories:(1)M2 ion channel inhibitors(amantadine,rimantadine,etc);(2)RNA polymerase inhibitors(favipiravir,etc);(3)neuraminidase inhibitors(oseltamivir,zanamivir,peramivir,etc);(4)other antiviral drugs.This paper is divided into four chapters and describes the industrialization of anti-influenza drug favipiravir and research of oseltamivir"me-too" drugs in the following two parts.Part 1:Industrialization of Anti-influenza Drug FavipiravirFavipiravir is a new antiviral drug which can selectively inhibit the RNA-dependent RNA polymerase(RdRp)of influenza viruses without affecting the DNA and RNA of the host cells.It is approved to treat new or recurrent influenza.In addition,it has been proved to be effective against many other viruses.First,this paper gave a review on the antiviral activity of favipiravir,especially its anti-influenza virus activity,and described its anti-influenza mechanism.Meanwhile,favipiravir was effective against many RNA viruses,including arenavirus,bunyavirus,yellow fever virus,alphavirus,noro virus and so on.Three synthetic routes of favipiravir were designed through bibliography retrieval and the main steps were described.These three routes took three different materiels as starting materials,namely 3-aminopyrazine-2-carboxylic acid,3-hydroxypyrazine-2-carboxylic acid and 3-aminopyrazine-2-carboxylic acid.Next,favipiravir was synthesized in accordance with the three routes in a lab scale and parts of the intermediates'structures were confirmed.Important reactions involved such as diazotization reaction,nitration reaction,hydroxyl protection and fluorine substitution were analyzed which followed by the determination of their reacting conditions.Through comparative analysis,route 1 and route 2 were found to have the following disadvantages:using expensive catalysts,large solvent consumption,and low yield which made them inappropriate for industrialization.Only one step used phosphorus oxychloride in route 3 which was difficult to operate while other steps had mild reaction conditions and the total yield of route 3 was relatively high.Therefore,route 3 could be used as industrial production route after optimization.Favipiravir was synthesized through 6 steps in route 3 which made it time-consuming and high-cost.Therefor a suitable intermediate that have been commercialized must be selected to shorten the route for industrial production.Through investigation,we found that 3,6-difluoropyrazine-2-carbonitrile was a significant chemical fragment of favipiravir and it was easy to obtain,to transport and to store.It could also be analyzed and controlled with common chemical means.Based on the reasons above,3,6-difluoropyrazine-2-carbonitrile was selected as the starting material to synthesize favipiravir,thus shortening production steps and avoiding the use of phosphorus oxychloride.Then the industrialization route of favipiravir was finally settled.Next,possible impurities in 3,6-difluoropyrazine-2-carbonitrile and their influence on the quality of favipiravir were comprehensively analyzed according to the synthetic route of 3,6-difluoropyrazine-2-carbonitrile and favipiravir.Then the quality control strategy and quality standard of the starting material were made.Through the optimization of the three critical steps:hydroxyl substitution,cyano hydrolysis,and product purification,the control standards of the critical process parameters were determined.Possible by-products were analyzed through reaction mechanism analysis and the process impurities as well as the quality standards of each intermediate were confirmed and the process was optimized.The industrialization route of favipiravir we designed had fewer steps,milder reaction conditions while it was easy to operate and the starting material was cheap and easy to obtain.The total yield was raised to 50%?60%from 24%.The purity of favipiravir was above 98%.Generally speaking,this route was suitable for industrial production.Next,based on the industrial design principles of favipiravir API required by CFDA,detailed process operation methods and process flow charts were made and the yield ranges of each step were calculated.Then the equipment used in each step was listed.Scale-up production was reasonably evaluated according to the data of small and pilot experiments.The batch size was expanded to 7.0kg.It was estimated that 3.0?3.5 kg favipiravir could be produced in each batch which could meet the demands of industrialization.Part 2:Research of Oseltamivir "Me-Too" DrugsNeuraminidase(NA),is an important active glycoprotein on the surface of virus.Its active center is composed of 18 amino acid residues.To design a compound that can combine with the amino acid residues of the NA active center is the main thought of NA inhibitor design.Now NA inhibitors mainly include the following categories:sialic acid analogs,benzoic acid analogs,cyclohexene analogs,cyclopentane analogs,pyrrolidine analogs and natural products.Currently,the listed NA inhibitors include oseltamivir,zanamivir,peramivir,laninamivir,etc.Since NA inhibitors are effective,safe and well tolerated,they have become main clinical medicines for the treatment of influenza virus infection in China.When the shape of NA near its active center changes,the binding force between NA and NA inhibitor reduces,leading to the emergence of drug resistance and the reduction of the drug effectiveness.Nowadays many viruses that are resistant to a variety of NA inhibitors have been found.The drug-resistant strain is of high infectivity and pathogenicity and it is difficult to prevent and control,hence structure optimization of the existing NA inhibitors must be conducted to search for a better NA inhibitor with high activity and selectivity.The 150-cavity near the active center of the Group-1 's NA was first reported in 2006 which offered a new way of designing NA inhibitor.Taking oseltamivir as lead compound,we did structural modification on the amino group of C-5 by introducing aliphatic or aromatic cyclic substituents or phenyl substituents with amino acid side chains.Two series:QDZT-1 and QDZT-2 which included 16 compounds were synthesized and their structures were confirmed by 1H NMR,13C NMR,and high resolution mass spectrum.The cytotoxicity tests showed that all the compounds had no significant cytotoxicity.QDZT-1-03 and QDZT-2-05 were found to have the best NA inhibitory activity against H1N1 A/Ned/378/05NA strain through CPE test and MTS test with an EC50 of 1.3?M,3.1 ?M(CPE test)and 0.387?M,1.7?M(MTS test).The NA enzyme inhibition test showed that QDZT-1-03 had a better NA inhibitory activity than QDZT-2-05 while they were all less active than their positive control oseltamivir carboxylate.The molecular docking of the two compounds indicated that the side chain on the C-5 could occupy the 150-cavity and they could combine with the active center of NA.QDZT-2-05 could form hydrogen bond inside 150-cavity which could increase the bonding power with NA.The reasons why there were differences in the inhibitory activity between the compounds were analyzed through activity tests and molecular docking,which offered a reference for the following design and synthesis of oseltamivir "me-too" drugs.Dual pro-drug has gradually become a hot area of drug research and development since it can increase drug effectiveness.The long application of antiviral drugs leads to the occurrence of cross drug resistance as well as the decrease in drug effectiveness.Therefore,dual pro-drugs are designed against different targets in different steps of the replication cycle to increase antiviral effects and to reduce side effects.It has been reported that favipiravir and oseltamivir,which was an influenza RdRp inhibitor and NA inhibitor respectively,could increase antiviral effects when they were used together.Herein we designed a synthetic route in which favipiravir and oseltamivir were linked as a dual pro-drug in one molecule by methyl formate,hoping to increase drug effects,reduce dosage and side effects.A totally new dual pro-drug QDZT-3-01 was synthesized based on the dual pro-drug principle and its structure was confirmed.It showed inhibitory activity against MDCK cells infected by H5N2 influenza virus.The results of liver microsomal metabolic stability tests and plasma microsomal metabolic stability tests showed that QDZT-3-01 was relatively stable and had potential for further research.Summary and prospect:the occurrence of drug resistance of influenza viruses has resulted in the reduction of clinically available antiviral drugs.Meanwhile there is hysteresis in the development of vaccine against new viruses,it is urgent to develop new antiviral drugs.Favipiravir is an RdRp inhibitor approved to treat new or recurrent influenza.Three feasible routes were designed through bibliography retrieval and favipiravir was synthesized in a lab scale.The industrialization route was settled after analyzing the results of the tests and the availability of the starting material as well as the production costs.Process parameters of each step were determined and the control standards of intermediates were made through progressive amplification,quality study and process optimization.The feasibility of the route had been confirmed by pilot experiments in the workshop.The design and synthesis of oseltamivir "me-too" drugs were also studied in this paper.Targeted at the 150-cavity in Group-1' NA,structure modification of oseltamivir was conducted and aliphatic or aromatic cyclic substituents or phenyl substituents with amino acid side chains were introduced at C-5.Two series:QDZT-1 and QDZT-2 were synthesized.The connection between molecular activity and the 150-cavity was further revealed through activity tests and molecular docking.A totally new compound QDZT-3-01 was synthesized based on the dual pro-drug principles which provided a new thinking for the design of NA inhibitor.