Nucleoside prodrug and use thereof

Abstract

The present invention relates to a compound represented by formula I and a pharmaceutically acceptable salt or stereoisomer thereof. The compound is a nucleoside prodrug with a completely new structure. The activity and bioavailability of the nucleoside prodrug are significantly improved, thereby laying a completely new material foundation for the drug development of diseases caused by infection with viruses.

Description

Nucleoside prodrugs and their uses Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to nucleoside prodrugs, pharmaceutical compositions comprising said nucleoside prodrugs, and the use of said nucleoside prodrugs or pharmaceutical compositions. Background Technology

[0002] Nucleoside analogues possess excellent antiviral activity. For example, compound GS-441524 has shown good efficacy in treating coronavirus-induced diseases in mammals, including humans and cats (Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment, Medical Chemicstry Letters, Victoria C. et al.; Development and validation of a UHPLC MS / MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2 / COVID-19 and Ebola virus disease, J Antimicrob Chemother, Valeria Avataneo et al.). Researchers have already designed several drugs or prodrugs based on GS-441524 for the treatment of humans or other mammals (Potency and pharmacokinetics of GS-441524 derivatives against SARS-CoV-2, Bioorg. Med. Chem. 46(2021) 116364). However, there are few reports on oral prodrugs of GS-441524 available in the prior art, and even fewer oral drugs available for the treatment of animals.

[0003] Compared to methods such as injection, oral administration offers advantages such as convenience and reduced pain. However, because oral administration involves absorption through the digestive tract, the environment it comes into contact with is more complex. Therefore, designing an effective, orally administered prodrug of GS-441524 remains a pressing technical problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a GS-441524 prodrug, which has the advantages of being orally administered, rapidly absorbed, and having improved bioavailability.

[0005] Another object of the present invention is to provide a pharmaceutical composition comprising the prodrug.

[0006] Other objects of the present invention are to provide the use of the prodrug and pharmaceutical composition in the preparation of medicaments for treating, inhibiting or preventing diseases caused by viral infections, and methods of treating diseases caused by viral infections using the prodrug or pharmaceutical composition.

[0007] In a first aspect, the present invention provides compounds of Formula I, pharmaceutically acceptable salts or stereoisomers thereof.

[0008] In the formula,

[0009] R1, R2, and R3 are each independently selected from: H, C(=O)C(=X)R4, C(=O)R5, R6, and R1, R2, and R3 are not all H at the same time;

[0010] X is selected from O or S;

[0011] R4 is selected from: substituted or unsubstituted C 5-8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C-aryl groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S 1-10 alkyl;

[0012] R5 is selected from: substituted or unsubstituted 5-14 membered cyclic alkyl groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S; substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 1-10 Alkoxy;

[0013] Preferably, R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted 5-14 membered spirocyclic groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted 5-14 membered fused ring groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 1-10 Alkoxy;

[0014] More preferably, the cyclic group is any one of the following groups:

[0015] R6 is selected from: substituted or unsubstituted phosphoryl groups;

[0016] or,

[0017] R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively.

[0018] In a preferred embodiment, X is O.

[0019] In a preferred embodiment, R4 is substituted or unsubstituted C. 5-8 Aryl.

[0020] In a preferred embodiment, R4 is a substituted or unsubstituted (preferably unsubstituted) phenyl group.

[0021] In a preferred embodiment, R5 is a substituted or unsubstituted 5-12 membered bridging ring containing 0, 1, 2, or 3 (preferably 0) heteroatoms independently selected from N, O, or S, and a substituted or unsubstituted C 1-10 alkyl.

[0022] In a preferred embodiment, R5 is a substituted or unsubstituted 5-12 membered bridging ring containing 0, 1, 2, or 3 (preferably 0) heteroatoms independently selected from N, O, or S, and a substituted or unsubstituted C 3-4 alkyl.

[0023] In a preferred embodiment, R5 is a substituted or unsubstituted 5-12-membered bridged ring group containing 0, 1, 2, or 3 (preferably 0) heteroatoms independently selected from N, O, or S, i-Pr, i-Bu, or

[0024] In a preferred embodiment, R5 is a 7-10 membered bridging ring group, substituted or unsubstituted, containing 0, 1, 2, or 3 (preferably 0) heteroatoms independently selected from N, O, or S, i-Pr, i-Bu, or

[0025] In a preferred embodiment, R5 is a 5-12 bridging ring group, substituted or unsubstituted, containing 0, 1, 2 or 3 (preferably 0) heteroatoms independently selected from N, O or S.

[0026] In a specific embodiment, R5 is a 7-10 bridging ring group, substituted or unsubstituted, containing 0, 1, 2 or 3 (preferably 0) heteroatoms independently selected from N, O or S.

[0027] In a preferred embodiment, R5 is adamantyl, bicyclo[2.2.2]octyl, or bicyclo[2.2.1]heptyl.

[0028] In a preferred embodiment, the substituted or unsubstituted phosphoryl group is as shown in P(=O)(OR7)(OR8), wherein R7 and R8 are each independently selected from: H, substituted or unsubstituted C. 1-6Alkyl, C 1-3 Alkylene OC(=O)OR9, where R9 is a substituted or unsubstituted C. 1-6 Alkyl groups; or, R7 and R8 together with the oxygen atoms attached to them form substituted or unsubstituted 5-12 membered rings.

[0029] In a preferred embodiment, the substituted or unsubstituted phosphoryl group, such as P(=O)(OR7)(OR8), R7 and R8, together with the oxygen atom attached to them, form a substituted or unsubstituted 5-12 membered ring or a C ring. 1-3 Alkylene OC(=O)OR9, where R9 is a substituted or unsubstituted C. 1-6 alkyl.

[0030] In a preferred embodiment, R7 and R8, together with the oxygen atom attached to them, form a substituted or unsubstituted 6-membered ring or a C1 alkylene ring OC(=O)OR9, wherein R9 is a substituted or unsubstituted C1 alkylene ring. 1-6 alkyl.

[0031] In a preferred embodiment, the substituted or unsubstituted phosphoryl group is as follows:

[0032] In a specific implementation, R2 and R3 form a substituted or unsubstituted 5-12 membered ring containing 0-2 carbonyl groups.

[0033] In a preferred embodiment, where R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12-membered rings containing 0-2 carbonyl groups, the formed 5-12-membered rings may have one or more (preferably two) substituents, which may be substituted or unsubstituted carbonyl groups. 1-10 Alkyl, substituted or unsubstituted C 1-10 alkylthio, substituted or unsubstituted C 1-10 Alkylamino, substituted or unsubstituted C 1-10 Alkoxy groups; or, the substituents on the 5-12 membered cyclic groups may further form substituted or unsubstituted C atoms with the carbon atoms attached to them. 4-8 A carbon ring, substituted or unsubstituted, containing one or two (preferably one) heteroatoms independently selected from N, O or S. 4-8 Heterocyclic rings, substituted or unsubstituted 5-14 fused-ring bridges, or substituted or unsubstituted C 5-12 Spiral ring.

[0034] In a specific embodiment, R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings containing 0-2 carbonyl groups, as shown below:

[0035] In a specific implementation, R1, R2 and R3 are each independently selected from: H, C(=O)C(=X)R4, C(=O)R5, R6, and R1, R2 and R3 are not all H at the same time;

[0036] X is selected from O;

[0037] R4 is selected from: substituted or unsubstituted C 5-8 Aryl;

[0038] R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S;

[0039] or,

[0040] R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively.

[0041] R6 is selected from either substituted or unsubstituted phosphoryl groups.

[0042] In a specific implementation, R1, R2, and R3 are each independently selected from: H, C(=O)C(=X)R4, and C(=O)R5; and R1, R2, and R3 are not all H at the same time;

[0043] X is selected from O;

[0044] R4 is selected from: substituted or unsubstituted C 5-8 Aryl;

[0045] R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S;

[0046] or,

[0047] R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively.

[0048] In a preferred embodiment, "substituted" means that the group modified by the term has one or more substituents, which are selected from: halogens, hydroxyl groups, nitro groups, C... 1-6 Alkyl, C 1-6 Alkoxy, amino, C 5-8 aryl, C containing one or two heteroatoms selected from N, O or S 5-8 Mixed aromatic compounds.

[0049] In a preferred embodiment, the "carbon ring" includes, but is not limited to, C. 4-8 cycloalkyl, C 5-8 Aryl.

[0050] In a preferred embodiment, the "heterocyclic ring" includes, but is not limited to, C.4-8 Heterocyclic alkyl, C 5-8 Mixed aromatic compounds.

[0051] In a specific embodiment, the compound represented by Formula I is selected from any one of the following compounds:

[0052] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0053] In a preferred embodiment, the pharmaceutical composition is administered by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulation or inhalation.

[0054] In a preferred embodiment, the dosage form of the pharmaceutical preparation includes, but is not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, etc.

[0055] In a preferred embodiment, the pharmaceutical composition is used to treat, inhibit, or prevent diseases caused by viral infection.

[0056] In a preferred embodiment, the virus comprises one or more of the following:

[0057] (1) Coronaviruses, including but not limited to zoonotic viruses such as novel coronavirus, SARS coronavirus, Middle East Respiratory Syndrome coronavirus, human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1 that infect humans, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine type D coronavirus that infect pigs, feline infectious peritonitis virus, feline enteric coronavirus, canine enteritis coronavirus, canine respiratory coronavirus, and pan-trophic canine coronavirus that infect pets, and chicken infectious bronchitis virus, duck coronavirus, goose coronavirus, pigeon coronavirus, wild bird coronavirus, and turkey coronavirus that infect birds;

[0058] (2) Flaviviridae viruses, including but not limited to hepatitis C virus and dengue virus that infect humans, zoonotic viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, Casanova forest disease virus, bovine viral diarrhea mucosal disease virus that infects cattle, Tembusu virus that infects poultry, and classical swine fever virus that infects pigs.

[0059] (3) Filoviridae viruses, including but not limited to zoonotic viruses such as Marburg virus and Ebola virus;

[0060] (4) Arenaviridae viruses, including but not limited to zoonotic viral prototypes such as lymphocytic choroid plexus meningitis virus, Lassa virus, Luyo virus, Mobara virus, Mopeya virus and Eipy virus infecting animals.

[0061] (5) Viruses of the Paramyxoviridae family, including but not limited to human parainfluenza virus, mumps virus, measles virus, human respiratory syncytial virus, human metapneumovirus that infects humans, canine distemper virus that infects pets, rinderpest virus, peste des petits ruminants virus, swine rubella virus, bovine respiratory syncytial virus that infects animals, Newcastle disease virus, avian metapneumovirus, avian parainfluenza virus that infects birds, and canine distemper virus and canine parainfluenza virus that infect dogs.

[0062] (6) Orthomyxoviridae viruses, including but not limited to influenza A virus, influenza B virus, influenza C virus that infect humans, zoonotic influenza A virus, and Sogoto virus;

[0063] (7) Bunyaviridae, including but not limited to zoonotic viruses such as neo Bunyavira virus, Hantavirus, Rift Valley fever virus, Tuscan virus, Seoul virus, Dobrava-Belgrade virus, Pumara virus, and Andean virus.

[0064] (8) Rhabdoviridae viruses, including but not limited to zoonotic viruses such as rabies virus, vesicular stomatitis virus, and equine infectious anemia virus that infects horses.

[0065] (9) Viruses of the Clostridium Viraeidae family, including but not limited to Chikungunya virus and rubella virus that infect humans, zoonotic viruses such as Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus.

[0066] (10) Arteritis Viridae, including but not limited to equine arteritis virus and porcine reproductive and respiratory syndrome virus;

[0067] (11) Rotaviruses, including but not limited to zoonotic rotaviruses A, B and C, rotaviruses D, F and G infecting birds, and rotaviruses E and H infecting pigs.

[0068] (12) Microribonucleic acid viruses, including but not limited to poliovirus, Coxsackievirus, echovirus, rhinovirus that infect humans, zoonotic viruses such as encephalomyocarditis virus and foot-and-mouth disease virus, avian myelitis virus and avian Sicinivirus that infect birds, and swine Seneca virus type A, swine Seneca Valley virus and swine parvovirus that infect pigs.

[0069] (13) Disegmental RNAviridae viruses, including but not limited to infectious bursal disease virus that infects birds and infectious pancreatic necrosis virus that infects aquatic animals.

[0070] (14) Reoviridae viruses, including but not limited to human reovirus and Banervirus that infect humans, zoonotic viruses such as rotavirus, bovine reovirus and epidemic hemorrhagic disease virus, bluetongue virus, African horse sickness virus and swine reovirus that infect animals, avian reovirus that infects birds and aquatic reovirus that infects aquatic animals.

[0071] (15) Parvoviruses, including but not limited to parvovirus B19 and human bocavirus that infect humans, canine parvovirus and feline panleukopenia virus that infect pets, bovine and porcine cell viruses that infect animals, avian parvovirus that infects birds, and pyrenoidosis viruses that infect arthropods.

[0072] (16) Caliciviridae viruses, including but not limited to zoonotic viruses such as norovirus and Sapporo virus, feline calicivirus that infects pets, and swine vesicular herpesvirus and rabbit hemorrhagic fever virus that infect animals.

[0073] In a preferred embodiment, the disease caused by the viral infection is selected from:

[0074] (1) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human coronavirus infection;

[0075] (2) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human respiratory syncytial virus infection;

[0076] (3) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human influenza virus infection;

[0077] (4) Chronic hepatitis C caused by hepatitis C virus and its complications;

[0078] (5) Dengue fever caused by dengue virus and its complications;

[0079] (6) Infections and complications caused by Zika virus;

[0080] (7) Hemorrhagic fever and its complications caused by Marburg virus and Ebola virus;

[0081] (8) Lassa hemorrhagic fever caused by Lassa virus;

[0082] (9) Novel coronavirus pneumonia caused by SARS-CoV-2;

[0083] (10) Feline infectious peritonitis or feline enteritis caused by feline coronavirus infection, or porcine epidemic diarrhea caused by porcine epidemic diarrhea virus infection; and

[0084] (11) Any combination of the above diseases.

[0085] In a third aspect, the present invention provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for treating, inhibiting or preventing diseases caused by viral infection.

[0086] In a specific implementation, the virus includes one or more of the following:

[0087] (1) Coronaviruses, including but not limited to zoonotic viruses such as novel coronavirus, SARS coronavirus, Middle East Respiratory Syndrome coronavirus, human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1 that infect humans, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine type D coronavirus that infect pigs, feline infectious peritonitis virus, feline enteric coronavirus, canine enteritis coronavirus, canine respiratory coronavirus, and pan-trophic canine coronavirus that infect pets, and chicken infectious bronchitis virus, duck coronavirus, goose coronavirus, pigeon coronavirus, wild bird coronavirus, and turkey coronavirus that infect birds;

[0088] (2) Flaviviridae viruses, including but not limited to hepatitis C virus and dengue virus that infect humans, zoonotic viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, Casanova forest disease virus, bovine viral diarrhea mucosal disease virus that infects cattle, Tembusu virus that infects poultry, and classical swine fever virus that infects pigs.

[0089] (3) Filoviridae viruses, including but not limited to zoonotic viruses such as Marburg virus and Ebola virus;

[0090] (4) Arenaviridae viruses, including but not limited to zoonotic viral prototypes such as lymphocytic choroid plexus meningitis virus, Lassa virus, Luyo virus, Mobara virus, Mopeya virus and Eipy virus infecting animals.

[0091] (5) Viruses of the Paramyxoviridae family, including but not limited to human parainfluenza virus, mumps virus, measles virus, human respiratory syncytial virus, human metapneumovirus that infects humans, canine distemper virus that infects pets, rinderpest virus, peste des petits ruminants virus, swine rubella virus, bovine respiratory syncytial virus that infects animals, Newcastle disease virus, avian metapneumovirus, avian parainfluenza virus that infects birds, and canine distemper virus and canine parainfluenza virus that infect dogs.

[0092] (6) Orthomyxoviridae viruses, including but not limited to influenza A virus, influenza B virus, influenza C virus that infect humans, zoonotic influenza A virus, and Sogoto virus;

[0093] (7) Bunyaviridae, including but not limited to zoonotic viruses such as neo Bunyavira virus, Hantavirus, Rift Valley fever virus, Tuscan virus, Seoul virus, Dobrava-Belgrade virus, Pumara virus, and Andean virus.

[0094] (8) Rhabdoviridae viruses, including but not limited to zoonotic viruses such as rabies virus, vesicular stomatitis virus, and equine infectious anemia virus that infects horses.

[0095] (9) Viruses of the Clostridium Viraeidae family, including but not limited to Chikungunya virus and rubella virus that infect humans, zoonotic viruses such as Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus.

[0096] (10) Arteritis Viridae, including but not limited to equine arteritis virus and porcine reproductive and respiratory syndrome virus;

[0097] (11) Rotaviruses, including but not limited to zoonotic rotaviruses A, B and C, rotaviruses D, F and G infecting birds, and rotaviruses E and H infecting pigs.

[0098] (12) Microribonucleic acid viruses, including but not limited to poliovirus, Coxsackievirus, echovirus, rhinovirus that infect humans, zoonotic viruses such as encephalomyocarditis virus and foot-and-mouth disease virus, avian myelitis virus and avian Sicinivirus that infect birds, and swine Seneca virus type A, swine Seneca Valley virus and swine parvovirus that infect pigs.

[0099] (13) Disegmental RNAviridae viruses, including but not limited to infectious bursal disease virus that infects birds and infectious pancreatic necrosis virus that infects aquatic animals.

[0100] (14) Reoviridae viruses, including but not limited to human reovirus and Banervirus that infect humans, zoonotic viruses such as rotavirus, bovine reovirus and epidemic hemorrhagic disease virus, bluetongue virus, African horse sickness virus and swine reovirus that infect animals, avian reovirus that infects birds and aquatic reovirus that infects aquatic animals.

[0101] (15) Parvoviruses, including but not limited to parvovirus B19 and human bocavirus that infect humans, canine parvovirus and feline panleukopenia virus that infect pets, bovine and porcine cell viruses that infect animals, avian parvovirus that infects birds, and pyrenoidosis viruses that infect arthropods.

[0102] (16) Caliciviridae viruses, including but not limited to zoonotic viruses such as norovirus and Sapporo virus, feline calicivirus that infects pets, and swine vesicular herpesvirus and rabbit hemorrhagic fever virus that infect animals.

[0103] In a preferred embodiment, the disease caused by the viral infection is selected from:

[0104] (1) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human coronavirus infection;

[0105] (2) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human respiratory syncytial virus infection;

[0106] (3) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human influenza virus infection;

[0107] (4) Chronic hepatitis C caused by hepatitis C virus and its complications;

[0108] (5) Dengue fever caused by dengue virus and its complications;

[0109] (6) Infections and complications caused by Zika virus;

[0110] (7) Hemorrhagic fever and its complications caused by Marburg virus and Ebola virus;

[0111] (8) Lassa hemorrhagic fever caused by Lassa virus;

[0112] (9) Novel coronavirus pneumonia caused by SARS-CoV-2;

[0113] (10) Feline infectious peritonitis or feline enteritis caused by feline coronavirus infection, or porcine epidemic diarrhea caused by porcine epidemic diarrhea virus infection; and

[0114] (11) Any combination of the above diseases.

[0115] In a fourth aspect, the present invention provides the compound described in the first aspect or a pharmaceutically acceptable salt or stereoisomer thereof for the treatment, inhibition or prevention of diseases caused by viral infection.

[0116] In a preferred embodiment, the virus comprises one or more of the following:

[0117] (1) Coronaviruses, including but not limited to zoonotic viruses such as novel coronavirus, SARS coronavirus, Middle East Respiratory Syndrome coronavirus, human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1 that infect humans, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine type D coronavirus that infect pigs, feline infectious peritonitis virus, feline enteric coronavirus, canine enteritis coronavirus, canine respiratory coronavirus, and pan-trophic canine coronavirus that infect pets, and chicken infectious bronchitis virus, duck coronavirus, goose coronavirus, pigeon coronavirus, wild bird coronavirus, and turkey coronavirus that infect birds;

[0118] (2) Flaviviridae viruses, including but not limited to hepatitis C virus and dengue virus that infect humans, zoonotic viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, Casanova forest disease virus, bovine viral diarrhea mucosal disease virus that infects cattle, Tembusu virus that infects poultry, and classical swine fever virus that infects pigs.

[0119] (3) Filoviridae viruses, including but not limited to zoonotic viruses such as Marburg virus and Ebola virus;

[0120] (4) Arenaviridae viruses, including but not limited to zoonotic viral prototypes such as lymphocytic choroid plexus meningitis virus, Lassa virus, Luyo virus, Mobara virus, Mopeya virus and Eipy virus infecting animals.

[0121] (5) Viruses of the Paramyxoviridae family, including but not limited to human parainfluenza virus, mumps virus, measles virus, human respiratory syncytial virus, human metapneumovirus that infects humans, canine distemper virus that infects pets, rinderpest virus, peste des petits ruminants virus, swine rubella virus, bovine respiratory syncytial virus that infects animals, Newcastle disease virus, avian metapneumovirus, avian parainfluenza virus that infects birds, and canine distemper virus and canine parainfluenza virus that infect dogs.

[0122] (6) Orthomyxoviridae viruses, including but not limited to influenza A virus, influenza B virus, influenza C virus that infect humans, zoonotic influenza A virus, and Sogoto virus;

[0123] (7) Bunyaviridae, including but not limited to zoonotic viruses such as neo Bunyavira virus, Hantavirus, Rift Valley fever virus, Tuscan virus, Seoul virus, Dobrava-Belgrade virus, Pumara virus, and Andean virus.

[0124] (8) Rhabdoviridae viruses, including but not limited to zoonotic viruses such as rabies virus, vesicular stomatitis virus, and equine infectious anemia virus that infects horses.

[0125] (9) Viruses of the Clostridium Viraeidae family, including but not limited to Chikungunya virus and rubella virus that infect humans, zoonotic viruses such as Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus.

[0126] (10) Arteritis Viridae, including but not limited to equine arteritis virus and porcine reproductive and respiratory syndrome virus;

[0127] (11) Rotaviruses, including but not limited to zoonotic rotaviruses A, B and C, rotaviruses D, F and G infecting birds, and rotaviruses E and H infecting pigs.

[0128] (12) Microribonucleic acid viruses, including but not limited to poliovirus, Coxsackievirus, echovirus, rhinovirus that infect humans, zoonotic viruses such as encephalomyocarditis virus and foot-and-mouth disease virus, avian myelitis virus and avian Sicinivirus that infect birds, and swine Seneca virus type A, swine Seneca Valley virus and swine parvovirus that infect pigs.

[0129] (13) Disegmental RNAviridae viruses, including but not limited to infectious bursal disease virus that infects birds and infectious pancreatic necrosis virus that infects aquatic animals.

[0130] (14) Reoviridae viruses, including but not limited to human reovirus and Banervirus that infect humans, zoonotic viruses such as rotavirus, bovine reovirus and epidemic hemorrhagic disease virus, bluetongue virus, African horse sickness virus and swine reovirus that infect animals, avian reovirus that infects birds and aquatic reovirus that infects aquatic animals.

[0131] (15) Parvoviruses, including but not limited to parvovirus B19 and human bocavirus that infect humans, canine parvovirus and feline panleukopenia virus that infect pets, bovine and porcine cell viruses that infect animals, avian parvovirus that infects birds, and pyrenoidosis viruses that infect arthropods.

[0132] (16) Caliciviridae viruses, including but not limited to zoonotic viruses such as norovirus and Sapporo virus, feline calicivirus that infects pets, and swine vesicular herpesvirus and rabbit hemorrhagic fever virus that infect animals.

[0133] In a preferred embodiment, the disease caused by the viral infection is selected from:

[0134] (1) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human coronavirus infection;

[0135] (2) Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human respiratory syncytial virus infection;

[0136] (3) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human influenza virus infection;

[0137] (4) Chronic hepatitis C caused by hepatitis C virus and its complications;

[0138] (5) Dengue fever caused by dengue virus and its complications;

[0139] (6) Infections and complications caused by Zika virus;

[0140] (7) Hemorrhagic fever and its complications caused by Marburg virus and Ebola virus;

[0141] (8) Lassa hemorrhagic fever caused by Lassa virus;

[0142] (9) Novel coronavirus pneumonia caused by SARS-CoV-2;

[0143] (10) Feline infectious peritonitis or feline enteritis caused by feline coronavirus infection, or porcine epidemic diarrhea caused by porcine epidemic diarrhea virus infection; and

[0144] (11) Any combination of the above diseases.

[0145] In a fifth aspect, the present invention provides a treatment method comprising the step of administering a therapeutically effective amount of the compound of the first aspect or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of the second aspect, to a subject in need of such treatment.

[0146] In a preferred embodiment, the subject is an animal.

[0147] In a preferred embodiment, the animal is a mammal.

[0148] In a preferred embodiment, the mammal is a human, cat, dog, pig, rat, or mink.

[0149] In a preferred embodiment, the mammal is a cat.

[0150] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0151] Through extensive and in-depth research, the inventors unexpectedly discovered a series of novel GS-441524 prodrug compounds. These prodrugs exhibit improved activity and bioavailability, enabling their oral use in the treatment of viral infections in humans and animals. This invention was completed based on these findings.

[0152] Terminology Definition

[0153] The terms used herein have the meanings conventionally understood by those skilled in the art. For clarity of understanding, some terms used herein are defined as follows.

[0154] As used herein, the term "substituent" or "substituent group" refers to an atom or group of atoms that substitutes for a hydrogen atom in a molecule. The term "substituted" means that a particular molecule has one or more substituents.

[0155] The term "alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group containing, for example, 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, and tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl. When a number appears as a subscript after the symbol "C", the subscript more specifically defines the number of carbon atoms that a particular group may contain. For example, "C 1-6 "Alkyl" refers to straight-chain and branched alkyl groups having one to six carbon atoms. C 1-6 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and pentyl.

[0156] The term "heteroatoms" refers to oxygen (O), sulfur (S), and nitrogen (N).

[0157] The term "halogenated" or "halogen" refers to F, Cl, Br, and I.

[0158] The term "aromatic ring" or "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic ring system of a hydrocarbon. The aryl group may optionally be substituted by one or more substituents. In one embodiment, 0, 1, 2, 3, 4, 5, or 6 atoms of each ring of the aryl group may be substituted by substituents. Examples of aryl groups include phenyl, naphthyl, anthraceneyl, fluorenyl, indene, azulel, and so on.

[0159] The term "aromatic heterocycle" or "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups and 9- or 10-membered bicyclic groups, wherein the group has at least one heteroatom (O, S, or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2, or 3 heteroatoms independently selected from O, S, and / or N. Each ring in the heteroatom-containing heteroaryl group may contain 1 or 2 oxygen or sulfur atoms and / or 1-4 nitrogen atoms, provided that the total number of heteroatoms in each ring is 4 or less and each ring has at least one carbon atom. The heteroaryl group may be attached to any available nitrogen or carbon atom in any ring. The heteroaryl ring system may be unsubstituted or may contain one or more substituents.

[0160] The term "alkoxy" refers to an -O-alkyl group. An alkoxy group may optionally be substituted with one or more substituents. Similarly, the term "alkathio" refers to an -S-alkyl group.

[0161] The term "phosphoryl group" refers to P(=O)(OR) a (OR) b The group shown in the figure, wherein R a and R b Each can be independently H, substituted or unsubstituted C 1-6 Alkyl, C 1-3 Alkylene OC (=O) OR c , where R c Is it substituted or unsubstituted C? 1-6 Alkyl; or, R a and R b They form substituted or unsubstituted 5-12 membered rings together with the oxygen atoms attached to them.

[0162] The term "cyclic group" refers to a cyclic aliphatic hydrocarbon group, that is, an aliphatic hydrocarbon group containing one or more rings. Cyclic groups may contain, for example, 3-20, preferably 5-14 carbon atoms.

[0163] The term "bridged ring group" or "bridged ring system" refers to a class of polycyclic aliphatic hydrocarbons in which two carbon rings share two or more carbon atoms. Bridged ring groups can be divided into bicyclic bridged rings and polycyclic bridged rings. Bicyclic bridged rings are composed of two aliphatic rings sharing two or more carbon atoms; polycyclic bridged ring hydrocarbons are composed of three or more rings sharing two or more carbon atoms.

[0164] The term "spirocyclic" refers to a system in which a molecule has two rings that share a single carbon atom.

[0165] The term "fused ring" refers to a fused ring system formed by two or more carbon rings (such as cyclohexane) sharing adjacent atoms.

[0166] In this invention, the cyclic group may specifically be one of the following groups:

[0167] The term "pharmaceutically acceptable" means a compound, substance, composition, and / or dosage form that, within reasonable pharmaceutical judgment, is suitable for contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0168] The compounds of the present invention or their pharmaceutically acceptable salts

[0169] The inventors designed and synthesized a series of prodrugs for GS-441524. These prodrugs have increased oral bioavailability, thereby enabling oral therapy.

[0170] In specific embodiments, the present invention provides a compound of Formula I, a pharmaceutically acceptable salt or stereoisomer thereof.

[0171] In the formula, R1, R2, and R3 are as described above.

[0172] The compounds of this invention can form salts, and therefore these salts are also within the scope of this application. Those skilled in the art will understand that salts formed by the compounds of this invention refer to acidic and / or basic salts formed by the compounds with inorganic and / or organic acids and bases. Examples of pharmaceutically acceptable salts of the compounds of this invention include, but are not limited to, inorganic and organic acid salts, such as hydrochlorides, hydrobromic acids, sulfates, citrates, lactates, tartrates, maleates, fumarates, mandelates, and oxalates; and inorganic and organic base salts formed with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, aminobutanetriol), and N-methylglucosamine.

[0173] The pharmaceutical composition of the present invention

[0174] Given that the compounds of the present invention are pharmaceutically active prodrug compounds, those skilled in the art will understand that the present invention also provides a pharmaceutical composition. The pharmaceutical composition comprises a therapeutically effective amount of the compounds of the present invention and a pharmaceutically acceptable excipient.

[0175] The pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for various routes of administration, including but not limited to those formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical administration. A dosage is an amount of medicine that effectively improves or eliminates one or more symptoms. For the treatment of a specific disease, an effective amount is an amount sufficient to improve or alleviate, in some way, the symptoms associated with the disease. Such a dosage may be administered as a single dose or may be administered according to an effective treatment regimen. A dosage may cure the disease, but administration is usually intended to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. The dosage of the medicine will be determined based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0176] The pharmaceutical formulations of this invention can be administered to any animal, particularly mammals, provided they can obtain the therapeutic effects of the compounds of this invention. Humans are the most important of these mammals.

[0177] The pharmaceutical formulations of the present invention can be manufactured using known methods. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolving, or freeze-drying processes. When manufacturing oral formulations, solid excipients and active compounds can be combined, and the mixture can be selectively ground. If desired or necessary, appropriate excipients can be added, and the granular mixture can be processed to obtain tablets or tablet cores.

[0178] Suitable excipients, especially fillers, include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added. Adjuvants, especially flow conditioners and lubricants, include silica, talc, stearates such as calcium magnesium stearate, stearic acid, or polyethylene glycol. If necessary, a suitable coating that resists gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures. To prepare a coating resistant to gastric juice, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of the tablet or lozenge core, for example, to identify or characterize combinations of active ingredient dosages.

[0179] Based on the teachings of this invention, those skilled in the art can prepare the compounds of this invention into various dosage forms and determine the dosage, administration method, and administration time according to the actual situation of the patient to be treated, including but not limited to age, gender, disease severity, and previous treatment history. Furthermore, based on the teachings of this invention, those skilled in the art can understand that the compounds of this invention are compounds with therapeutic activity; in other words, the compounds of this invention have the potential to be used as drugs. Therefore, those skilled in the art can qualitatively and quantitatively detect various characteristics of the compounds of this invention as drugs using conventional techniques, including but not limited to therapeutic activity, toxicity, bioavailability, and drug-likeness. Performing these tests is obvious to those skilled in the art and requires no inventive effort.

[0180] use

[0181] The compounds or pharmaceutical compositions of the present invention have therapeutic, inhibitory, or preventive effects against diseases caused by viral infections. Therefore, based on the content of the present invention, those skilled in the art can prepare the compounds or pharmaceutical compositions of the present invention into drugs for treating, inhibiting, or preventing diseases caused by viral infections. In specific embodiments, the virus may be (1) a coronavirus, including but not limited to zoonotic viruses such as novel coronavirus, SARS coronavirus, Middle East Respiratory Syndrome coronavirus, human coronaviruses OC43, 229E, NL63, and HKU1 infecting humans, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine type D coronavirus infecting pigs, feline infectious peritonitis virus, feline enteric coronavirus, canine enteritis coronavirus, canine respiratory coronavirus, and pantropic canine coronavirus infecting pets, and chicken infectious bronchitis virus and duck infectious bronchitis virus infecting poultry. Coronaviruses, goose coronaviruses, pigeon coronaviruses, wildfowl coronaviruses, turkey coronaviruses; (2) Flaviviridae viruses, including but not limited to hepatitis C virus and dengue virus that infect humans, zoonotic viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, Casanorr forest disease virus, bovine viral diarrhea mucosal disease virus that infects cattle, Tembusu virus that infects birds, and classical swine fever virus that infects pigs; (3) Filoviridae viruses, including but not limited to zoonotic viruses such as Marburg virus and Ebola virus; (4) Arenaviridae viruses, The viruses of the Arenaviridae family include, but are not limited to, zoonotic viral prototypes such as lymphocytic choriomeningitis virus, Lassa virus, Luyo virus, and Mobara virus; and veterinary viruses such as Mopeya virus and Eipy virus; (5) Paramyxoviridae viruses, including but not limited to, human parainfluenza virus, mumps virus, measles virus, human respiratory syncytial virus, and human metapneumovirus infecting humans; canine distemper virus infecting pets; rinderpest virus, peste des petits ruminants virus, swine rubella virus, and bovine respiratory syncytial virus infecting veterinarians; Newcastle disease virus, avian metapneumovirus, and avian parainfluenza virus infecting poultry; and canine distemper virus and canine parainfluenza virus infecting dogs. (6) Orthomyxoviridae viruses, including but not limited to influenza A virus, influenza B virus, and influenza C virus that infect humans, and zoonotic influenza A virus and Sogoto virus; (7) Bunyaviridae viruses, including but not limited to zoonotic viruses such as neo-Buniavira virus, Hantavirus, Rift Valley fever virus, Tuscan virus, Seoul virus, Dobrava-Belgrade virus, Pumara virus, and Andean virus; (8) Rhabdoviridae viruses, including but not limited to zoonotic viruses such as rabies virus, vesicular stomatitis virus, and equine infectious anemia virus that infects horses;(9) Viruses of the Clostridium Viridae family, including but not limited to chikungunya virus and rubella virus that infect humans, and zoonotic viruses such as eastern equine encephalitis virus, western equine encephalitis virus, and Venezuelan equine encephalitis virus; (10) Viruses of the Arteritis Viridae family, including but not limited to equine arteritis virus and porcine reproductive and respiratory syndrome virus; (11) Viruses of the Rotavirus family, including but not limited to zoonotic viruses such as group A rotavirus, group B rotavirus, and group C rotavirus. Rotaviruses of groups D, F, and G infecting birds, and rotaviruses of groups E and H infecting pigs; (12) MicroRNA viruses, including but not limited to poliovirus, Coxsackievirus, echovirus, rhinovirus, zoonotic viruses such as encephalomyocarditis virus and foot-and-mouth disease virus, avian myelitis virus and avian Sicinivirus infecting birds, and swine Seneca virus, swine Seneca Valley virus, and swine parvovirus infecting pigs; (13) Disegmental RNAviridae viruses, including but not limited to infectious bursal disease virus infecting birds and infectious pancreatic necrosis virus infecting aquatic animals; (14) Reoviridae viruses, including but not limited to human reovirus and banner virus infecting humans, zoonotic viruses such as rotavirus, bovine reovirus, and epidemic hemorrhagic disease virus, bluetongue virus, African horse sickness virus, and swine reovirus infecting animals, avian reovirus infecting birds, and aquatic animals infecting aquatic animals. (15) Parvoviruses, including but not limited to parvovirus B19 and human bocavirus, canine parvovirus and feline panleukopenia virus infecting pets, bovine and porcine cell viruses infecting animals, avian parvovirus infecting birds, and cystoviruses infecting arthropods; (16) Caliciviridae viruses, including but not limited to zoonotic viruses such as norovirus and Sapporo virus, feline calicivirus infecting pets, and swine vesicular herpesvirus and rabbit hemorrhagic fever virus infecting animals.

[0182] Accordingly, the diseases caused by the viral infection may be: (1) common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human coronavirus infection; (2) common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human respiratory syncytial virus infection; (3) common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human influenza virus infection; (4) chronic hepatitis C and its complications caused by hepatitis C virus; (5) dengue fever and its complications caused by dengue virus; (6) infection and its complications caused by Zika virus; (7) hemorrhagic fever and its complications caused by Marburg virus and Ebola virus; (8) Lassa hemorrhagic fever caused by Lassa virus; (9) novel coronavirus pneumonia caused by SARS-CoV-2; (10) feline infectious peritonitis or feline enteritis caused by feline coronavirus infection, or swine epidemic diarrhea caused by swine epidemic diarrhea virus infection; etc.

[0183] Advantages of this invention:

[0184] 1. This invention provides a series of novel nucleoside prodrugs;

[0185] 2. The nucleoside prodrug of the present invention has improved activity and bioavailability, etc.

[0186] 3. The nucleoside prodrug of the present invention lays a completely new material basis for the development of drugs for diseases caused by viral infections.

[0187] The technical solution of the present invention is further described below with reference to specific implementation examples. However, the following implementation examples do not constitute a limitation on the present invention. All application methods based on the principles and technical means of the present invention are within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0188] Example 1. Synthesis of Compound 1

[0189] Step 1: Preparation of Compounds 1-2

[0190] Compound 1-1 (5.8 g, 15.22 mmol) was dissolved in acetone (30 mL), and 2,2-dimethoxypropane (9.5 g, 91.31 mmol) and 98% sulfuric acid (2.54 g, 25.87 mmol) were added. The mixture was stirred at 45 °C for 30 min. TLC analysis showed that the reaction proceeds were completely reacted. The reaction solution was evaporated to dryness to remove acetone, diluted with EA (20 mL), washed once with saturated sodium bicarbonate (30 mL), and twice with saturated brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / anhydrous methanol = 15:1) to give a white solid compound 1-2 (6 g, yield: 92.30%).

[0191] Step 2: Preparation of compounds 1-3

[0192] Compounds 1-2 (500 mg, 1.51 mmol) were dissolved in DCM (5 mL), followed by the addition of 2-oxo-2-phenylacetic acid (226.8 mg, 1.51 mmol) and 4-dimethylaminopyridine (26.7 mg, 0.151 mmol). The mixture was stirred at room temperature for 10 min, then N,N'-dicyclohexylcarbodiimide (342 mg, 1.66 mmol) was added, and the mixture was stirred overnight at room temperature. TLC analysis showed that the reaction proceeds were completely reacted. The reaction mixture was filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / anhydrous methanol = 20:1) to give a pink solid compound 1-3 (330 mg, yield: 47.21%).

[0193] Step 3: Preparation of Compound 1

[0194] Compounds 1-3 (330 mg, 0.71 mmol) were dissolved in tetrahydrofuran (4 mL), and concentrated hydrochloric acid (0.8 mL) was added. The reaction was carried out at room temperature for 3 hours. TLC analysis showed that the reaction proceeds were complete. The reaction solution was adjusted to alkalinity with saturated sodium bicarbonate and extracted twice with ethyl acetate (10 mL). The organic phases were combined, washed twice with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / anhydrous methanol = 10:1) to give compound 1 (250 mg, yield: 83.06%) as a white solid.

[0195] 1H NMR (400MHz, DMSO-d6) δ7.98–7.94(m,2H),7.87(s,3H),7.82–7.76(m,1H),7.61–7.55(m,2H),6.83(d,J=4.5Hz,1H),6.78(d,J=4.5Hz,1H),6. 33(d,J=6.0Hz,1H),5.46(d,J=5.7Hz,1H),4.76–4.66(m,2H),4.60(dd,J=12.1,5.9Hz,1H),4.38(td,J=6.1,3.1Hz,1H),4.04(q,J=5.7Hz,1H)

[0196] Example 2. Synthesis of Compound 2

[0197] The synthesis of compound 1 was performed using adamantane-1-carboxylic acid as the starting material, and the relevant synthetic steps were the same as those for compound 1.

[0198] 1 H NMR (400MHz, DMSO-d6) δ7.93(s,3H),6.92(d,J=4.5Hz,1H),6.82(d,J=4.5Hz,1H),6.35(d,J=5.9Hz,1H),5.36(d,J=6.0Hz,1H),4.7 0(dd,J=5.9,4.8Hz,1H),4.31–4.22(m,2H),4.18–4.11(m,1H),3.99(td,J=6.2,4.7Hz,1H),1.96–1.90(m,3H),1.75–1.58(m,12H).

[0199] Example 3. Synthesis of Compound 3

[0200] Step 1: Preparation of Compound 3

[0201] Compound 1 (200 mg, 0.47 mmol) was dissolved in acetonitrile (2 mL), and N,N'-carbonyldiimidazole (152 mg, 0.94 mmol) was added. The reaction was carried out at room temperature for 1 hour. TLC analysis showed that the reaction proceeded completely. The reaction solution was prep-HPLC (acetonitrile / water = 1:4) to obtain a white solid compound 3 (100 mg, yield: 47.17%).

[0202] 1H NMR (400MHz, DMSO-d6) δ8.10–7.96(m,2H),7.94–7.89(m,2H),7.86(s,1H),7.81–7.73(m,1H),7.60–7.51(m,2H),6.91(s,2H),5.97( d,J=7.7Hz,1H),5.59(dd,J=7.7,3.9Hz,1H),4.99(dt,J=5.5,3.8Hz,1H),4.76(dd,J=12.1,3.7Hz,1H),4.62(dd,J=12.2,5.5Hz,1H).

[0203] Example 4. Synthesis of Compound 4

[0204] Step 1: Preparation of Compound 4-1

[0205] Compound 1-2 (7.7 g, 23.2 mmol) was dissolved in dichloromethane (80 mL), followed by the sequential addition of isovaleric acid (2.85 g, 27.9 mmol) and DMAP (2.6 g, 23.2 mmol), and the mixture was stirred for 5 min. DCC (5.74 g, 27.9 mmol) was then added, and the reaction was carried out at room temperature for 2 h. TLC showed that intermediate compound 1-2 had completely reacted. Silica gel was added to prepare precipitate, and the mixture was purified by column chromatography (eluting PE:EA = 2:1) to give a white solid compound 4-1 (9.5 g, 98% yield).

[0206] Step 2: Preparation of compound 4-2

[0207] Compound 4-1 (7.4 g, 17.8 mmol) was dissolved in tetrahydrofuran (75 mL), and 37% HCl (15 mL) was added dropwise. The reaction was carried out at room temperature for 3 h. TLC showed that compound 4-1 reacted completely. The pH of the system was adjusted to about 8 with saturated NaHCO3, and the mixture was extracted twice with ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and concentrated to give crude white solid compound 4-2 (5.3 g, yield 79%), which was directly used in the next step of the reaction.

[0208] Step 3: Preparation of compound 4-3

[0209] The crude compound 4-2 (2.3 g) was dissolved in dichloromethane (30 mL), and then Boc anhydride (4.0 g) and triethylamine (0.93 g) were added sequentially. The mixture was reacted at room temperature for 1 h. The reaction was confirmed by TLC and the target molecular weight was found by LCMS. The mixture was concentrated and purified by column chromatography (elution PE:EA = 1:50 → PE:EA = 1:1) to give 2 g of white solid compound 4-3 (2 g, yield 56.7%).

[0210] Step 4: Preparation of compound 4-4

[0211] Compound 4-3 (500 mg, 0.868 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of 1,2-cyclohexanedicarboxylic acid (300 mg, 2.08 mmol) and DMAP (290 mg, 2.4 mmol). The mixture was stirred for 5 min, and then DCC (538 mg, 2.61 mmol) was added. The reaction was allowed to proceed overnight at room temperature. The target product was formed. Silica gel was added to prepare precipitate, and the mixture was purified by column chromatography to give a white solid compound 4-4 (300 mg, yield: 50.5%).

[0212] Step 5: Preparation of Compound 4

[0213] Compound 4-4 (300 mg, 0.438 mmol) was dissolved in DCM (10 mL), and trifluoroacetic acid (4 mL) was added dropwise. The reaction was carried out at room temperature for 2 h. TLC showed that the reaction was complete. Part of the trifluoroacetic acid was concentrated under reduced pressure, and 15 mL of dichloromethane was added. The pH was adjusted to about 9 with triethylamine, and the mixture was concentrated to dryness. The solution was then sent to HPLC to prepare a white solid compound 4 (72 mg, yield: 33.9%).

[0214] 1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),7.26(d,J=4.8Hz,1H),6.85(d,J=4.8H z,1H),4.77(d,J=3.6Hz,1H),4.66(p,J=4.3,3.9Hz,2H),4.46(dd,J=12.3,3.6Hz ,1H),4.39–4.28(m,2H),3.68–3.57(m,2H),3.10(d,J=3.8Hz,1H),2.95–2.83(m, 2H),2.56–2.47(m,2H),2.15–2.12(m,1H),2.11–2.01(m,2H),0.98–0.87(m,6H).

[0215] Example 5. Synthesis of Compound 5

[0216] 1,2-cyclohexanedicarboxylic acid was used as the starting material, and the relevant synthesis steps are the same as those for the synthesis of compound 4.

[0217] 1H-NMR (400MHz, CDCl3): δ8.63 (s, 1H), 7.22 (d, 1H, J = 4.83), 6.78 (d, 1H, J = 4 .86),4.65(m,2H),4.45(dd,1H,J=12.30,3.61),4.36(dd,1H,J=5.70,3.74) ,4.30(dd,1H,J=12.32,4.50),3.22(q,2H,J=3.28,1.94),2.13(m,2H),2.04 -2.02(m,5H),2.98(d,1H,J=2.98),1.30(m,3H),0.93(dd,6H,J=6.57,1.74)

[0218] Example 6. Synthesis of Compound 6

[0219] Step 1: Preparation of Compound 6

[0220] Compound 4-2 (100 mg, 0.26 mmol) was dissolved in DCM (5 mL), and benzoylformic acid (120 mg, 0.80 mmol), DMAP (32 mg, 0.26 mmol), and DCC (120 mg, 0.58 mmol) were added sequentially. The mixture was reacted at room temperature for 5 min. The reaction was confirmed to be complete by TLC (MeOH:DCM = 1:20). The reaction solution was concentrated, and the crude product was prepared and purified (C18, water, acetonitrile) to give compound 6 (56 mg, yield: 33%) as a white solid.

[0221] 1 H NMR (400MHz, DMSO-d6) δ8.28–8.12(m,2H),8.05–7.95(m,5H),7.80–7.74(m,2H),7.55(q,J=7.9H z,4H),7.04(dd,J=4.6,2.9,1.6Hz,1H),6.99(dd,J=4.6,1.3Hz,1H),6.67(d,J=5.8Hz,1H),5.99 (dd,J=5.9,4.5Hz,1H),4.86(q,J=4.2Hz,1H),4.49(dd,J=12.4,3.4Hz,1H),4.41(dd,J=12.4,4. 7Hz, 1H), 2.19 (dd, J=7.1, 5.7Hz, 2H), 1.94 (dd, J=13.6, 6.8Hz, 1H), 0.87 (dd, J=6.7, 1.3Hz, 6H).

[0222] Example 7. Synthesis of Compound 7

[0223] Step 1: Preparation of Compound 7-1

[0224] Compound 4-2 (300 mg, 0.80 mmol) was dissolved in DMF (6 mL), and DMF-DMA (638 mg, 5.35 mmol) was added. The reaction was carried out at 60 °C for 1 hour, and the reaction was monitored by TLC (MeOH:DCM = 1:15). The reaction proceeds were completely reacted. The reaction solution was extracted with EA (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by rapid column chromatography (eluting MeOH:DCM = 0-5%) to give a white solid compound 7-1 (265 mg, yield: 77.2%).

[0225] Step 2: Preparation of compound 7-2

[0226] Compound 7-1 (160 mg, 0.37 mmol) was dissolved in DCM (6 mL), and TEMED (432 mg, 3.72 mmol) and DMAP (45 mg, 0.37 mmol) were added sequentially. Then, adamantyl chloride (890 mg, 4.4 mmol) was added in five batches under a N2 atmosphere. The reaction was carried out at room temperature for 6 days. TLC monitoring (MeOH:DCM = 1:40) showed that there was still a starting material remaining. The reaction solution was extracted with DCM (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by rapid column chromatography (eluting PE:EA = 0-60%) to give a white solid compound 7-2 (140 mg, yield: 49.9%).

[0227] Step 3: Preparation of Compound 7

[0228] Compound 7-2 (100 mg, 0.13 mmol) was dissolved in acetonitrile (6 mL), and 50% hydrazine hydrate (0.5 mL) was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored by TLC (MeOH:DCM = 1:30), indicating that the starting material reacted completely. The reaction solution was purified by reverse-phase preparative separation (C18, water, acetonitrile) to give a white solid compound 7 (76 mg, yield: 81.9%).

[0229] 1H NMR (400MHz, DMSO-d6): δ7.93(s,3H),6.94(d,J=4.6Hz,1H),6.77(d,J=4.6Hz,1H) ,6.08(d,J=5.6Hz,1H),5.37(dd,J=5.6,4.0Hz,1H),4.55(q,J=4.0Hz,1H),4.39–4. 24(m,2H),2.20–2.09(m,2H),1.99(dp,J=6.6,3.1Hz,6H),1.91(d,J=2.8Hz,6H),1 .85(d,J=3.0Hz,6H),1.73–1.62(m,10H),1.24(s,3H),0.84(dd,J=6.6,1.6Hz,6H).

[0230] Example 8. Synthesis of Compound 8

[0231] Phthalic acid was used as the starting material, and the relevant synthesis steps are the same as those for the synthesis of compound 4.

[0232] 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.09(dd,J=5.5,3.0Hz,2H),7.93(dd,J=5.5,3.1Hz,2H),7.24(d,J=4.8H z,1H),6.92(d,J=4.7Hz,1H),4.71–4.64(m,2H),4.52–4.24(m,4H),2.19–1.99(m,4H),0.92(dd,J=6.6,1.7Hz,6H).

[0233] Example 9. Synthesis of Compound 9

[0234] Compound 2 was used as the starting material, and the relevant synthesis steps were the same as those for the synthesis of compound 3.

[0235] 1 H NMR (400MHz, DMSO-d6) δ7.98(s,3H),6.97(d,J=4.6Hz,1H),6.89(d,J=4.6Hz,1H),5.96(d,J=7.4Hz,1H),5.5 1(dd,J=7.4,3.1Hz,1H),4.90(q,J=3.7Hz,1H),4.32–4.22(m,2H),1.87(p,J=3.1Hz,3H),1.68–1.51(m,12H).

[0236] Example 10. Synthesis of Compound 10

[0237] Compound 2 (200 mg, 0.44 mmol) was dissolved in DCM (100 mL), and isobutyric acid (85 mg, 0.97 mmol), DMAP (118 mg, 0.97 mmol), and DCC (4.5 g, 21.75 mmol) were added sequentially. The mixture was reacted at room temperature for 1 hour, and the reaction was detected by TLC (MeOH:DCM = 1:15). A small amount of the starting material remained. The mixture was filtered and concentrated, and purified by reverse-phase separation (C18, water, acetonitrile) to give a white solid compound 10 (100 mg, yield: 37.8%).

[0238] 1 H NMR (400MHz, DMSO-d6): δ7.91(s,3H),6.95(d,J=4.5Hz,1H),6.75(d,J=4.6Hz,1H),6.10(d,J=5.6Hz,1H),5.44(dd,J=5.6,3.8Hz,1H),4 .65(q,J=3.5Hz,1H),4.27(d,J=3.4Hz,2H),2.62(dp,J=13.9,7.0Hz,2H),1.91(q,J=3.2Hz,3H),1.74–1.56(m,12H),1.21–1.09(m,12H).

[0239] Example 11. Synthesis of Compound 11

[0240] Compound 4-2 (150 mg, 0.40 mmol) was dissolved in a mixed solvent of DCM (2 mL) and THF (0.4 mL), and tetrahydropyranone (40 mg, 0.40 mmol) and BF3 in Et2O (0.53 mL, 0.53 mmol) were added. The reaction was carried out at room temperature for 12 hours. TLC (MeOH:DCM = 1:15) showed that the starting material was completely reacted. The system was added to a saturated NaHCO3 solution (10 mL), and the aqueous phase was extracted once with DCM (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by reverse-phase preparative separation (C18, water, acetonitrile) to give a white solid compound 11 (53 mg, yield: 29.0%).

[0241] 1H NMR (400MHz, DMSO-d6) δ7.97(s,3H),6.92(d,J=4.6Hz,1H),6.86(d,J=4.6Hz,1H),5.54(d,J=6.3Hz,1H),5.01(dd,J=6.4,2.8Hz,1H),4.63–4.58( m,1H),4.23(dd,J=12.0,4.1Hz,1H),4.10(dd,J=12.0,5.9Hz,1H),3.79– 3.56(m,4H),2.13–1.96(m,4H),1.90–1.74(m,3H),0.83(d,J=6.6Hz,6H).

[0242] Example 12. Synthesis of Compound 12

[0243] Adamantane was used as the starting material, and the relevant synthesis steps are the same as those for the synthesis of compound 11.

[0244] 1 H NMR (400MHz, DMSO-d6) δ7.95(s,1H),7.86(s,2H),6.93(d,J=4.6Hz,1H),6.87(d,J= 4.6Hz,1H),5.54(d,J=6.4Hz,1H),4.97(dd,J=6.5,3.4Hz,1H),4.51(dt,J=6.0,4.0 Hz,1H),4.23(dd,J=12.0,4.4Hz,1H),4.09(dd,J=12.0,6.1Hz,1H),2.21(s,1H),2. 15–2.04(m,3H),2.03–1.85(m,5H),1.83–1.64(m,8H),0.86(dd,J=6.6,0.9Hz,6H).

[0245] Example 13. Synthesis of Compound 13

[0246] Spiro[3.3]heptane-2-one was used as the starting material, and the relevant synthesis steps were described in the synthesis of compound 11.

[0247] 1H NMR (400MHz, DMSO-d6) δ7.94(s,1H),7.86(s,2H),6.92(d,J=4.5Hz,1H),6.83(d,J=4.6H z,1H),5.41(d,J=6.2Hz,1H),4.87(dd,J=6.2,3.1Hz,1H),4.54(m,1H),4.20(dd,J=12.0 ,4.3Hz,1H),4.08(dd,J=12.0,5.9Hz,1H),2.68(dd,J=13.0,2.4Hz,1H),2.58–2.53(m,1 H),2.39–2.30(m,2H),2.14–2.00(m,6H),1.92–1.76(m,3H),0.84(dd,J=6.6,1.0Hz,6H).

[0248] Example 14. Synthesis of Compound 14

[0249] Step 1: Preparation of Compound 14-1

[0250] Compound 1-2 (500 mg, 1.5 mmol, 1.0 eq.) was added to a 100 mL reaction flask. Bicyclo[2.2.2]octane-1-carboxylic acid (279 mg, 1.8 mmol, 1.2 eq.) was dissolved in DCM (10 mL). DMAP (180 mg, 1.5 mmol, 1.0 eq.) was added, followed by DCC (370 mg, 1.8 mmol, 1.2 eq.). The reaction was allowed to proceed overnight at room temperature. The reaction was carried out by TLC with a ratio of MeOH:DCM of 1:15. The reaction proceeded completely. The sample was purified by rapid column chromatography (200-300 mesh, EA:DCM = 42%) to obtain the title compound 14-1 (580 mg, yield: 82.7%) as a white solid.

[0251] LCMS(ESI)[M+1] + =468.0.

[0252] Step 2: Preparation of Compound 14

[0253] Add 14-1 (580 mg, 1.24 mmol, 1.0 eq.) dissolved in tetrahydrofuran (15 mL) to a 100 mL reaction flask, add concentrated hydrochloric acid (3 mL), and stir overnight at room temperature. The reaction proceeds completely under TLC with MeOH:DCM = 1:15. Add the mixture to 100 mL of the solution, adjust the pH to 8-9 with saturated Na2CO3 solution, extract the aqueous phase twice with EA (100 mL), combine the organic phases, dry over anhydrous sodium sulfate, and purify using rapid column chromatography (200-300 mesh silica gel, MeOH:DCM = 0-12%) to obtain the title compound 14 (55 mg, yield: 10.4%) as a white solid.

[0254] LCMS(ESI)[M+1] + =428.

[0255] 1 H NMR (400MHz, DMSO-d6) δ7.93 (s, 3H), 6.93 (d, J = 4.5Hz, 1H), 6.82 (d, J = 4.5Hz, 1H), 4.72 (d, J = 4. 8Hz,1H),4.32-4.20(m,2H),4.16-4.09(m,1H),3.98(dd,J=6.7,4.8Hz,1H),1.64-1.40(m,13H)

[0256] HPLC: (210nm) 97.4%.

[0257] Example 15. Synthesis of Compound 15

[0258] Step 1: Preparation of Compound 15-1

[0259] Add 1-2 (252 mg, 1.8 mmol, 1.2 eq.) to a 100 mL reaction flask, dissolve in DCM (10 mL), add DMAP (180 mg, 1.5 mmol, 1.0 eq.), and finally add DCC (370 mg, 1.8 mmol, 1.2 eq.). React overnight at room temperature. TLC MeOH:DCM = 1:15. The starting material reacted completely. Separate and purify by rapid column chromatography (200-300 mesh, EA:DCM = 40%) to give the title compound 15-1 (633 mg, yield: 93.2%) as a white solid.

[0260] LCMS(ESI)[M+1] + =454.0.

[0261] Step 2: Preparation of Compound 15

[0262] Add 15-1 (633 mg, 1.39 mmol, 1.0 eq) dissolved in tetrahydrofuran (10 mL) to a 100 mL reaction flask, add concentrated hydrochloric acid (2 mL), and stir overnight at room temperature. The reaction proceeds completely under TLC with MeOH:DCM = 1:15. Add the mixture to 100 mL of the solution, adjust the pH to 8-9 with saturated Na2CO3 solution, extract the aqueous phase twice with EA (100 mL), combine the organic phases, dry over anhydrous sodium sulfate, and purify using rapid column chromatography (200-300 mesh silica gel, MeOH:DCM = 0-12%) to obtain the title compound 15 (57 mg, yield: 9.9%) as a white solid.

[0263] LCMS(ESI)[M+1] + =414.

[0264] 1 H NMR (400MHz, DMSO-d6) δ7.93(d,J=2.1Hz,3H),6.92(d,J=4.5Hz,1H),6.82(d,J=4.5Hz,1H),4.69(dd,J=4.9,2.8Hz,1H),4.35–4.26(m,1H),4.20(dt,J =16.3,5.2Hz,2H),4.00–3.91(m,1H),2.81–2.71(m,1H),2.39(d,J=4.4Hz, 1H),2.20(s,1H),1.68–1.54(m,1H),1.49–1.19(m,5H),1.18–1.05(m,2H).

[0265] HPLC: (210nm) 98.1%.

[0266] Example 16. Synthesis of Compound 15

[0267] Step 1: Preparation of Compound 16-1

[0268] Dissolve compound 1-2 (978 mg, 2.95 mmol) in DCM (10 mL), add 3-methyl-2-oxobutyric acid (343 mg, 2.95 mmol), add 4-dimethylaminopyridine (36 mg, 0.295 mmol), stir at room temperature for 10 min, add N,N'-dicyclohexylcarbodiimide (670 mg, 3.35 mmol), stir at room temperature overnight. TLC analysis showed that the starting material was not completely reacted and was directly post-processed. The reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane / anhydrous methanol = 20:1) to give a white solid, title compound 16-1 (790 mg, yield: 65.8%).

[0269] LC-MS:m / z[M+H] + =430.2

[0270] Step 2: Preparation of Compound 16

[0271] 16-1 (250 mg, 0.58 mmol) was dissolved in DCM (5 mL), and TFA (2.5 mL) was added. The reaction was carried out at room temperature for 4 hours. TFA (2.5 mL) was added again, and the reaction was carried out at room temperature for 1 hour. TLC analysis showed that the starting material was not completely reacted. The mixture was directly concentrated to remove DMC and TFA. The solution was dissolved in DCM (2 mL), and the liquid was loaded for silica gel column chromatography (methanol / dichloromethane = 4%). Multiple column chromatography cycles yielded a white solid, title compound 16 (55 mg, yield: 23%).

[0272] LC-MS:m / z[M+H] + =390.1

[0273] 1 H NMR (400MHz, DMSO-d6) δ8.16(brs,2H),7.97(s,1H),7.00(d,J=4.6Hz,1H),6.86(d,J=4.6Hz,1H),4.71(d,J=4.9Hz,2H ), 4.53(dd,J=12.0,2.7Hz,1H),4.43–4.30(m,2H),4.03–3.99(m,1H),3.99–3.15(m,1H),1.04(dd,J=6.9,2.8Hz,6H).

[0274] Assay of the virus inhibitory activity of the compounds of this invention:

[0275] healthy CRFK cells were divided into 10 4 Cells were seeded at a density of cells / well in 96-well plates and incubated in a CO2 incubator for 24 hours. After the cells grew into a monolayer, 100 μL of test compounds diluted to different concentrations were added, with three replicates for each compound. Cell controls and virus controls were also included. Cells were pretreated with the drug for 2 hours, then washed three times with sterile DMEM, and inoculated with FIPV-Luci virus at 50 TCID50 / 100 μL / well. After 20 hours, the inhibitory effect was detected using a Nano-Glo luciferase assay system (Promega), and the half-maximal effective concentration (EC50) of the compound against the virus was calculated using GraphPadprism 7 software. 50 )value.

[0276] Table 1. In vitro inhibitory activity of the compounds of the present invention against feline infectious peritonitis virus (FIPV). Note: EC 50Less than or equal to 1µM: +; Less than 1µM and greater than or equal to 0.5µM: ++; Less than 0.5µM and greater than or equal to 0.1µM: +++; Less than 0.1µM: ++++

[0277] As can be seen from Table 1, the compounds of the present invention, especially 1, 2, 3, 5, 6, 8, 9 and 10, can effectively inhibit the replication of feline infectious peritonitis virus (FIPV) in vitro.

[0278] FCV test steps description

[0279] healthy F81 cells were divided into groups of 1.5 x 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 24-well plates and incubated in a CO2 incubator. After 24 hours of culture and complete cell adhesion, the medium was replaced with medium containing 2% FBS and inoculated with FCV virus. Two hours after infection, the compound was diluted to the corresponding working concentration with medium containing 2% FBS, and each compound was performed in triplicate. After 24 hours of drug treatment, cells were collected, RNA was extracted and reverse-engineered into cDNA, and viral load was quantified by qPCR to calculate the inhibition rate. The half-maximal effective concentration (EC50) of the compound against the virus was then calculated using GraphPadprism 7 software. 50 )value.

[0280] Table 2. In vitro inhibitory activity of the compounds of the present invention against feline calicivirus (FCV). Note: EC 50 ≥10µM: +; <10µM ≥5µM: ++; <5µM ≥1µM: +++; <1µM: ++++

[0281] As can be seen from Table 2, the compounds of the present invention, especially 2, 7, 9, 17, 18, and 21, can effectively inhibit the replication of feline calicivirus (FCV) in vitro.

[0282] RSV Test Procedure Description

[0283] healthy Hep2 cells were divided into 10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated in a CO2 incubator. After 24 hours of culture and complete cell adhesion, the medium was replaced with medium containing 2% FBS, and the test compound was diluted to the specified working concentration. Each compound was tested in triplicate. Two hours after drug treatment, the cells were infected with RSV-A2 strain. Forty-eight hours after infection, the supernatant was collected, RNA was extracted, and the RSV viral load in the supernatant was quantified using the TaqMan probe assay to calculate the inhibition rate. The half-maximal effective concentration (EC50) of the compound against the virus was then calculated using GraphPadprism 7 software. 50 )value.

[0284] Table 3. In vitro inhibitory activity of the compounds of the present invention against human respiratory syncytial virus (RSV). Note: EC50 ≥ 0.5µm: +; < 0.5µm ≥ 0.3µm: ++; < 0.3µm: +++

[0285] As can be seen from Table 3, the compounds of the present invention, especially 5, 6, 9, 10, 16, 19, 20, 21 and 22, can effectively inhibit the replication of human respiratory syncytial virus (RSV) in vitro.

[0286] In vitro cytotoxicity assay of the compounds of this invention

[0287] Healthy CRFK cell suspensions were seeded into 96-well plates (100 μL / well), and test compounds diluted to different concentrations were added. Cell viability was measured using a microplate reader after 20 hours. The concentrations of compounds associated with 50% cytotoxicity (CC) were then calculated using GraphPadprism 7 software. 50 )value.

[0288] Table 4. Cytotoxicity of the compounds of the present invention on CRFK cells Note: CC 50 500µM or greater is represented by "+"; less than 500µM but greater than or equal to 100µM is represented by "++"; less than 100µM is represented by "+++".

[0289] As can be seen from Table 4, the compounds of the present invention exhibit low cytotoxicity in CRFK cells.

[0290] Pharmacokinetic evaluation in rats

[0291] Male SD rats were fasted before the experiment (except for the intravenous group, which was allowed free access to water), and were fed uniformly 2 hours after drug administration. Compound 2 was administered intravenously at a dose of 5.0 mg / kg (n=3) and orally at a dose of 30.0 mg / kg (n=3). Compounds 3, 5, 9, 10, and GS-441524 were administered intravenously at a dose of 2 mg / kg (n=3) and orally at a dose of 10 mg / kg (n=3). The drug solution was 5% DMSO + 5% enthanol + 40% PEG300 + 50% saline. At different time points after drug administration, 0.2 mL of blood was collected via the jugular vein, placed in heparin sodium anticoagulant tubes, gently mixed, centrifuged at 2000g for 10 min, and the plasma was separated and frozen at –70℃ for analysis. The concentration of nucleoside (GS-441524) in the plasma was determined by LC MS-MS, and pharmacokinetic parameters were calculated. The results are shown in Table 3.

[0292] Table 5: Pharmacokinetic parameters of nucleoside (GS-441524) in rats after single intravenous and single oral administration of compounds 2, 3, 5, 9, 10 and GS-441524.

[0293] As shown in Table 5, the oral bioavailability of the nucleoside metabolites of compounds 2, 3, 5, 9, and 10 in rats after gavage administration was higher than that of GS-441524. Compound 2, in particular, had an oral bioavailability of 86.6%.

[0294] Example 17. Membrane permeability experiment of compound 2 and GS-441524 in Caco-2 cells.

[0295] Remove the Transwell plates from the incubator. Rinse the cell membranes twice with preheated transport buffer and incubate at 37°C for 30 minutes. First, add 210 μL of the dosing end solution to each well of the upper chamber (top), then immediately remove 10 μL of the dosing end solution and add it to a new 96-well plate. Mix this with 90 μL of transport buffer and 300 μL of quenching solution (acetonitrile, containing 5 ng / mL verapamil and 50 ng / mL glibenclamide). This sample is used as the initial dosing end sample TA0 (A→B). Add 1300 μL of the receiving end solution to each well of the lower chamber (base end) and determine the transport rate of compound 2 and GS-441524 from the top to the base end; then reverse the process to test the transport rate from the base end to the top. After centrifuging all samples, aspirate the supernatant to a new 96-well plate and mix with water at a certain ratio before LC-MS / MS analysis. Each experimental sample was tested in duplicate. The results are shown in Table 4.

[0296] Table 6: Results of membrane permeability experiments of compound 2 and GS-441524 on Caco-2 cells Notes: a: Internal measured data; b: Literature data

[0297] As shown in Table 6, compound 2 can be classified as having high membrane permeability and good absorption; it is also not a substrate for efflux receptors (such as PGP). Compared with GS-441524, it has significant advantages, and it can be predicted that the oral absorption of the prodrug molecule compound 2 will be superior to that of GS-441524.

[0298] Evaluation of rat tissue distribution

[0299] The active metabolites of prodrugs directly reflect drug efficacy, and optimizing their pharmacokinetics and tissue distribution is the goal of oral prodrug design. Therefore, in male SD rats, the distribution of the active metabolites of compounds 3, 5, 9, and 10 was studied. Male SD rats were randomly grouped according to body weight. The target compounds were dissolved in 5% DMSO + 5% enthanol + 40% PEG300 + 50% saline to prepare a solution. The administration route was oral, with a single dose of 10 mg / kg. Rats were fasted overnight before administration and fed 4 hours after administration. Plasma and tissues were collected 8 hours later. The tissues were cleaned with physiological saline in an ice bath and dried. They were placed in labeled sample tubes, weighed, and then homogenized immediately with 4 times the volume of homogenate. After homogenization, protein precipitation was performed on-site by a bioanalyst. After vortexing, the concentration of nucleoside (GS-441524) in the plasma was immediately determined by LC MS-MS. The results are shown in Table 5.

[0300] Table 7: Tissue distribution of compounds 3, 5, 9 and 10 in rats via gavage (results in nucleoside GS-441524).

[0301] As shown in Table 7, when compounds 3, 5, 9, and 10 were administered by gavage, their nucleoside metabolites were significantly enriched in the tissues of rats, especially compounds 5 and 9, which showed the most significant enrichment in the intestines compared to plasma concentrations (compound 5 was 215 times higher and compound 9 was 40 times higher).

[0302] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The pharmaceutically acceptable salt or stereoisomer of the compound shown in Formula I, In the formula, R1, R2, and R3 are each independently selected from: H, C(=O)C(=X)R4, C(=O)R5, R6, and R1, R2, and R3 are not all H at the same time; X is selected from O or S; R4 is selected from: substituted or unsubstituted C 5-8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C-aryl groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S 1-10 alkyl; R5 is selected from: substituted or unsubstituted 5-14 membered cyclic alkyl groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S; substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 1-10 Alkoxy; Preferably, R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted 5-14 membered spirocyclic groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted 5-14 membered fused ring groups containing 0, 1, 2 or 3 heteroatoms independently selected from N, O or S; substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 1-10 Alkoxy; More preferably, the cyclic group is any one of the following groups: R6 is selected from: substituted or unsubstituted phosphoryl groups; or, R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively.

2. The compound of claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that, R5 is a 7-10 bridging ring group, substituted or unsubstituted, containing 0, 1, 2 or 3 (preferably 0) heteroatoms independently selected from N, O or S.

3. The compound of claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that, R2 and R3 form substituted or unsubstituted 5-12 membered rings containing 0-2 carbonyl groups.

4. The compound of claim 3, its pharmaceutically acceptable salt or stereoisomer, characterized in that, R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings containing 0-2 carbonyl groups, as shown below:

5. The compound of claim 1, its pharmaceutically acceptable salt or stereoisomer, characterized in that, R1, R2, and R3 are each independently selected from: H, C(=O)C(=X)R4, C(=O)R5, R6, and R1, R2, and R3 are not all H at the same time; X is selected from O; R4 is selected from: substituted or unsubstituted C 5-8 Aryl; R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S; or, R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively. R6 is selected from either substituted or unsubstituted phosphoryl groups.

6. The compound of claim 5, its pharmaceutically acceptable salt or stereoisomer, characterized in that, R1, R2, and R3 are each independently selected from: H, C(=O)C(=X)R4, C(=O)R5; and R1, R2, and R3 are not all H at the same time; X is selected from O; R4 is selected from: substituted or unsubstituted C 5-8 Aryl; R5 is selected from: substituted or unsubstituted 5-14 membered bridged ring groups containing 0, 1, 2 or 3 independent heteroatoms selected from N, O or S; or, R2 and R3, together with the oxygen atoms attached to them, form substituted or unsubstituted 5-12 membered rings, respectively.

7. The compound, its pharmaceutically acceptable salt, or stereoisomer as described in any one of claims 1-6, characterized in that, The compound represented by Formula I is selected from any one of the following compounds; 8. A pharmaceutical composition comprising the compound of any one of claims 1-7 or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient.

9. Use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for treating, inhibiting or preventing diseases caused by viral infection.

10. The use as described in claim 9, characterized in that, The virus includes one or more of the following: (1) Coronaviruses, including but not limited to zoonotic viruses such as novel coronavirus, SARS coronavirus, Middle East Respiratory Syndrome coronavirus, human coronavirus OC43, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1 that infect humans, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine type D coronavirus that infect pigs, feline infectious peritonitis virus, feline enteric coronavirus, canine enteritis coronavirus, canine respiratory coronavirus, and pan-trophic canine coronavirus that infect pets, and chicken infectious bronchitis virus, duck coronavirus, goose coronavirus, pigeon coronavirus, wild bird coronavirus, and turkey coronavirus that infect birds; (2) Flaviviridae viruses, including but not limited to hepatitis C virus and dengue virus that infect humans, zoonotic viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, Casanova forest disease virus, bovine viral diarrhea mucosal disease virus that infects cattle, Tembusu virus that infects poultry, and classical swine fever virus that infects pigs. (3) Filoviridae viruses, including but not limited to zoonotic viruses such as Marburg virus and Ebola virus; (4) Arenaviridae viruses, including but not limited to zoonotic viral prototypes such as lymphocytic choroid plexus meningitis virus, Lassa virus, Luyo virus, Mobara virus, Mopeya virus and Eipy virus infecting animals. (5) Viruses of the Paramyxoviridae family, including but not limited to human parainfluenza virus, mumps virus, measles virus, human respiratory syncytial virus, human metapneumovirus that infects humans, canine distemper virus that infects pets, rinderpest virus, peste des petits ruminants virus, swine rubella virus, bovine respiratory syncytial virus that infects animals, Newcastle disease virus, avian metapneumovirus, avian parainfluenza virus that infects birds, and canine distemper virus and canine parainfluenza virus that infect dogs. (6) Orthomyxoviridae viruses, including but not limited to influenza A virus, influenza B virus, influenza C virus that infect humans, zoonotic influenza A virus, and Sogoto virus; (7) Bunyaviridae, including but not limited to zoonotic viruses such as neo Bunyavira virus, Hantavirus, Rift Valley fever virus, Tuscan virus, Seoul virus, Dobrava-Belgrade virus, Pumara virus, and Andean virus. (8) Rhabdoviridae viruses, including but not limited to zoonotic viruses such as rabies virus, vesicular stomatitis virus, and equine infectious anemia virus that infects horses. (9) Viruses of the Clostridium Viraeidae family, including but not limited to Chikungunya virus and rubella virus that infect humans, zoonotic viruses such as Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, and Venezuelan Equine Encephalitis Virus. (10) Arteritis Viridae, including but not limited to equine arteritis virus and porcine reproductive and respiratory syndrome virus; (11) Rotaviruses, including but not limited to zoonotic rotaviruses A, B and C, rotaviruses D, F and G infecting birds, and rotaviruses E and H infecting pigs. (12) Microribonucleic acid viruses, including but not limited to poliovirus, Coxsackievirus, echovirus, rhinovirus that infect humans, zoonotic viruses such as encephalomyocarditis virus and foot-and-mouth disease virus, avian myelitis virus and avian Sicinivirus that infect birds, and swine Seneca virus type A, swine Seneca Valley virus and swine parvovirus that infect pigs. (13) Disegmental RNAviridae viruses, including but not limited to infectious bursal disease virus that infects birds and infectious pancreatic necrosis virus that infects aquatic animals. (14) Reoviridae viruses, including but not limited to human reovirus and Banervirus that infect humans, zoonotic viruses such as rotavirus, bovine reovirus and epidemic hemorrhagic disease virus, bluetongue virus, African horse sickness virus and swine reovirus that infect animals, avian reovirus that infects birds and aquatic reovirus that infects aquatic animals. (15) Parvoviruses, including but not limited to parvovirus B19 and human bocavirus that infect humans, canine parvovirus and feline panleukopenia virus that infect pets, bovine and porcine cell viruses that infect animals, avian parvovirus that infects birds, and pyrenoidosis viruses that infect arthropods. (16) Caliciviridae viruses, including but not limited to zoonotic viruses such as norovirus and Sapporo virus, feline calicivirus that infects pets, and swine vesicular herpesvirus and rabbit hemorrhagic fever virus that infect animals.

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