Use of nucleoside analogues or combination preparations containing nucleoside analogues in antiviral treatment

Abstract

The present invention relates to the use of nucleoside analogs in antiviral treatment. Specifically, the present invention relates to the use of nucleoside analogs and their pharmaceutical compositions as (a) inhibitors for inhibiting the replication of coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviruses, filoviruses, and / or porcine epidemic diarrhea virus (PEDV); and / or (b) medicaments for treating and / or preventing and alleviating related diseases caused by infections of coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviruses, filoviruses, and / or porcine epidemic diarrhea virus (PEDV). The nucleoside analogs of the present invention can treat and / or prevent and alleviate related diseases such as respiratory infections and pneumonia (COVID-19) caused by the infection of the 2019 novel coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the use of nucleoside analogues or combination preparations containing nucleoside analogues in antiviral treatment. Background Art

[0002] Among acute infectious diseases, the vast majority are viral infectious diseases, which have a high incidence and a high mortality rate. Viruses are a type of pathogenic microorganism that is extremely tiny, lacks the ability of independent metabolism, and exists in a parasitic manner. There are a wide variety of viruses. Currently, many viruses with high infectivity and high pathogenicity to humans have been discovered. These viruses often cause local or even global infectious disease outbreaks, posing a great threat to human society, such as influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, Ebola virus, etc. Some viruses can also infect animals, causing various diseases ranging from mild to severe. At the same time, animals have become the sources of these viruses, making it impossible for humans to guard against them.

[0003] Coronaviruses belong to the order Nidovirales, the family Coronaviridae, and the genus Coronavirus. They are a large class of single-stranded positive-sense RNA viruses that widely exist in nature and can cause diseases in the respiratory, digestive, and nervous systems of humans and animals. According to the phylogenetic tree, coronaviruses can be divided into four genera: α, β, γ, and δ. Among them, β-coronaviruses can be further divided into four independent subgroups: A, B, C, and D.

[0004] As of April 2020, the novel coronavirus (SARS-CoV-2) has caused more than 100,000 deaths, resulting in another very serious global public health event after the Spanish flu in 1918. SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) all belong to the β-coronavirus genus. Currently, these three viruses have become the coronaviruses with the strongest pathogenic ability to humans.

[0005] Currently, there are no effective vaccines and antiviral drugs for the severe pneumonia caused by the SARS-CoV-2 coronavirus. These infectious diseases have seriously affected people's lives and health. It is urgent to develop antiviral drugs with good effects. Developing low-toxic and highly effective antiviral drugs against the SARS-CoV-2 coronavirus to meet the clinical needs of SARS-CoV-2 coronavirus-infected patients at home and abroad has great social significance.

[0006] Coronaviruses can also infect a variety of mammals, including bats, pigs, dogs, cats, mice, cattle, horses, camels, etc. Most of these viruses belong to the genera α and β. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that can cause acute intestinal infectious diseases in pigs. Pigs of all ages can be infected and develop the disease. Among them, suckling pigs and newborn piglets are the most severely affected, and the breeding industry often suffers heavy losses due to the outbreak of PEDV.

[0007] Respiratory virus infections are the most common and widespread type of viral infectious diseases clinically, causing a large number of deaths globally every year. In addition to coronaviruses, influenza viruses, respiratory syncytial viruses, parainfluenza viruses, etc. can also cause respiratory infections and lead to pneumonia, which are important killers threatening human life and health.

[0008] In summary, there is an urgent need in this field to develop inhibitors that can inhibit virus replication for the treatment of related diseases caused by virus infections. Summary of the Invention

[0009] The object of the present invention is to provide an active ingredient that can effectively inhibit virus replication and its new use in related diseases caused by virus infections.

[0010] Specifically, the present invention provides the use of the nucleoside analogues shown in Formula I and their compositions in antiviral (such as coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviridae viruses, filoviridae viruses and / or porcine epidemic diarrhea virus), especially in the treatment of novel coronavirus (SARS-CoV-2).

[0011] In the first aspect of the present invention, there is provided a compound shown in Formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof or a prodrug thereof:

[0012]

[0013] In the formula,

[0014] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, amino, C 1-6 alkyl-substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyl-substituted amino, halo C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 2-6 alkynyl, C 3-6 cycloalkyl, halo C 3-6 cycloalkyl, carbamoyl, hydroxymethyl, cyanomethyl (-CH2 CN), amidino, guanidino, ureido, thiocyanato (-SCN), cyanato (-OCN);

[0015] R 2 selected from hydrogen, halogen, OR 3 , cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0016] R 3 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl, C 1-6 alkylamino C 1-6 alkanoyl, C 1-6 alkoxy C 1-6 alkyl, α - amino acid, the carboxyl group of which is linked to the hydroxyl group on the furan ring by an ester bond;

[0017] R 4 selected from hydrogen, deuterium, halogen, azido, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azido C 1-6 alkyl, cyano C 1-6 alkyl, hydroxy C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 1-6 alkanoyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyanato, cyanato;

[0018] R 5 selected from R 3 ,

[0019] R 6 selected from amino, hydroxy, halogen, cyano, cyanato, thiocyanato, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;

[0020] R 7Selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;

[0021] R 8 Selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl-substituted carbamoyl, C 1-6 alkoxy amide group, C 1-6 alkoxycarbonyl, hydroxy, hydroxy C 1-6 alkyl, amino, C 1-6 alkanoyl-substituted amino, C 1-6 alkyl-substituted amino, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0022] X is selected from -CH 2 -, -CD 2 -, -CHD-;

[0023] R 9 Selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0024] R 10 Selected from C 1-18 alkyl, methylene C 6-20 aryl;

[0025] R 11 Selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0026] R 12 Selected from C 1-20 alkyl;

[0027] Each M is independently selected from hydrogen, metal, -NH 4 , or protonated organic amine;

[0028] In another preferred example, each position represented as deuterium (D) has a deuterium enrichment of at least 50%; preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 80%; more preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 90%; most preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 95%.

[0029] In another preferred embodiment, the 5- to 15-membered heteroaryl contains 1, 2, 3 or 4 heteroatoms selected from N, O and S.

[0030] In another preferred embodiment, the metal is selected from the group consisting of alkali metals, alkaline earth metals, or combinations thereof.

[0031] In another preferred embodiment, in the compound of formula (I),

[0032] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, methyl, chloromethyl, fluoromethyl, vinyl, ethynyl, cyclopropyl, carbamoyl, hydroxymethyl, methoxy, formyl, amidino; and / or

[0033] R 2 is selected from halogen, cyano, amino, formyl, OR 3 ; and / or

[0034] R 3 is selected from hydrogen, C 1-20 alkyl acyl, α-amino acid, wherein the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond; preferably, the α-amino acid is selected from the group consisting of alanine, valine, isoleucine, tryptophan, phenylalanine; and / or

[0035] R 4 is selected from hydrogen, deuterium, halogen, azido, cyano, methyl, chloromethyl, fluoromethyl, difluoromethyl, vinyl, ethynyl, cyclopropyl, hydroxymethyl, azidomethyl (-CH 2 N 3 );), formyl, acetyl, formamido, acetamido; and / or

[0036] R 5 is selected from R 3 , and / or

[0037] R 6 is selected from amino, hydroxyl, halogen, cyano, methylamino (-NH 2 CH 3 ), NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ; and / or

[0038] R 7 is selected from hydrogen, deuterium, halogen, amino; and / or

[0039] R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, N-methylcarbamoyl (CH 3NHCO-), methyl, ethyl, ethynyl, methoxycarbonyl, ethoxycarbonyl, hydroxy, hydroxymethyl, hydroxyethyl, methoxy, ethoxy, formyl, acetyl, formylamino, acetylamino, methoxycarbonylamino (CH 3 OCONH 2 -), ethoxycarbonylamino (C 2 H 5 OCONH 2 -), methoxycarbonyloxy (CH 3 OCOO-), ethoxycarbonyloxy (C 2 H 5 OCOO-); and / or

[0040] X is selected from -CH 2 -, -CD 2 -, -CHD-; and / or

[0041] R 9 is selected from C 6-20 aryl, 5- to 15-membered heteroaryl; and / or

[0042] R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl; and / or

[0043] R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5- to 15-membered heteroaryl; and / or

[0044] R 12 is selected from C 1-20 alkyl; and / or

[0045] M is independently selected from hydrogen, zinc, magnesium, calcium, sodium, potassium, NH 4 , protonated trimethylamine, protonated triethylamine, protonated tri-n-butylamine.

[0046] In another preferred embodiment, the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and X are each independently the specific groups corresponding to each of the compounds (such as any one of compounds A1 to A221) in the examples.

[0047] In another preferred embodiment, the compound of formula (I) is any one of compounds A1 to A221 having the following structure, or a combination thereof:

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of: compound A1, A9, A10, A11, A12, A49, A50, A51, A52, A53, A69, A70, A71, A72, A74, A75, A76, A77, A84, A87, A102, A106, A107, A108, A109, A124, A131, A138, A140, A144, A146, A147, A151, A164, A171, A173, A174, A180, A181, A188, A196, A198, A209, A212, A213, A214, A215, A216, A221 or a combination thereof.

[0061] In a second aspect of the present invention, there is provided the use of an active ingredient or a preparation containing the active ingredient, wherein the active ingredient is a compound represented by formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof or a prodrug thereof:

[0062]

[0063] wherein,

[0064] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, amino, C 1-6 alkyl-substituted amino, C1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyl-substituted amino, halo C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 2-6 alkynyl, C 3-6 cycloalkyl, halo C 3-6 cycloalkyl, carbamoyl, hydroxymethyl, cyanomethyl (-CH 2 CN), amidino, guanidino, ureido, thiocyanato (-SCN), cyanato (-OCN);

[0065] R 2 is selected from hydrogen, halogen, OR 3 , cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0066] R 3 is selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl, C 1-6 alkylamino C 1-6 alkanoyl, C 1-6 alkoxy C 1-6 alkyl, α-amino acid, the carboxyl group of which is linked to the hydroxyl group on the furan ring by an ester bond;

[0067] R 4 is selected from hydrogen, deuterium, halogen, azido, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azido C 1-6 alkyl, cyano C 1-6 alkyl, hydroxy C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 1-6 alkanoyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyanato, cyanato;

[0068] R 5 is selected from R 3 ,

[0069] R 6 is selected from amino, hydroxy, halogen, cyano, cyanoxy, cyanothio, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;

[0070] R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;

[0071] R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl-substituted carbamoyl, C 1-6 alkoxycarbonylamino, C 1-6 alkoxycarbonyl, hydroxy, hydroxyC 1-6 alkyl, amino, C 1-6 alkanoyl-substituted amino, C 1-6 alkyl-substituted amino, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0072] X is selected from -CH 2 -, -CD 2 -, -CHD-;

[0073] R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15-membered heteroaryl;

[0074] R 10 is selected from C 1-18 alkyl, methyleneC 6-20 aryl;

[0075] R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15-membered heteroaryl;

[0076] R 12 is selected from C 1-20 alkyl;

[0077] M is independently selected from hydrogen, metal, -NH4 or a protonated organic amine;

[0078] Furthermore, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting virus replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by virus infection.

[0079] In another preferred example, each position represented as deuterium (D) has a deuterium enrichment of at least 50%; preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 80%; more preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 90%; most preferably, each position represented as deuterium (D) has a deuterium enrichment of at least 95%.

[0080] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting coronavirus replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by coronavirus infection.

[0081] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting respiratory syncytial virus (RSV) replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by respiratory syncytial virus (RSV) infection.

[0082] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting influenza virus replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by influenza virus infection.

[0083] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting flavivirus replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by flavivirus infection.

[0084] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting filovirus replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by filovirus infection.

[0085] In another preferred example, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting porcine epidemic diarrhea virus (PEDV) replication; and / or (b) a medicament for treating and / or preventing and alleviating related diseases caused by porcine epidemic diarrhea virus (PEDV) infection.

[0086] In another preferred embodiment, the active ingredient or the preparation containing the active ingredient is used for preparing (a) an inhibitor for inhibiting the replication of the 2019 novel coronavirus (SARS-Cov-2); and / or (b) a drug for treating and / or preventing and alleviating related diseases caused by the infection of the 2019 novel coronavirus (SARS-CoV-2).

[0087] In another preferred embodiment, the virus is selected from:

[0088] (1) Coronaviruses infecting humans: Severe acute respiratory syndrome coronavirus (SARS-CoV), 2019 novel coronavirus (2019-nCoV or SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV)

[0089] (2) Coronaviruses causing common colds: The coronaviruses causing common colds are preferably selected from the following group: Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1;

[0090] (3) Human respiratory syncytial virus (RSV);

[0091] (4) Human influenza viruses: Influenza A virus, Influenza B virus, Influenza C virus;

[0092] (5) Viruses of the Flaviviridae family: Hepatitis C virus (HCV), Dengue virus (DENV), Zika virus;

[0093] (6) Viruses of the Filoviridae family: Marburg virus (MBV), Ebola virus (EBV);

[0094] (7) Coronaviruses infecting other mammals: Porcine epidemic diarrhea virus (PEDV).

[0095] In another preferred embodiment, the related diseases caused by the virus are selected from the following group:

[0096] (D1) Common colds, high-risk symptom infections, respiratory tract infections, pneumonia and its complications caused by human coronavirus infections;

[0097] (D2)Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and their complications caused by human respiratory syncytial virus (RSV);

[0098] (D3)Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and their complications caused by human influenza virus;

[0099] (D4)Chronic hepatitis C and its complications caused by hepatitis C virus (HCV);

[0100] (D5)Dengue fever and its complications caused by dengue virus (DENV);

[0101] (D6)Infection and its complications caused by Zika virus;

[0102] (D7)Hemorrhagic fever and its complications caused by Marburg virus (MBV) and Ebola virus (EBV);

[0103] (D8)Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2;

[0104] (D9)Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV);

[0105] (D10)Any combination of the above diseases.

[0106] In another preferred example, the related diseases caused by the 2019 novel coronavirus infection are selected from the group consisting of: respiratory tract infection, pneumonia and their complications, or a combination thereof.

[0107] In another preferred example, the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.

[0108] In another preferred example, the active ingredient is a nucleoside analogue selected from the group consisting of, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a prodrug thereof:

[0109] Compound A1, A5, A6, A8, A9, A10, A11, A12, A13, A14, A28, A30, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A49, A50, A51, A52, A53, A54, A55, A57, A58, A63, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A84, A86, A87, A88, A89, A91, A95, A97, A99, A101, A102, A105, A106, A107, A108, A109, A110, A111, A113, A114, A115, A116, A117, A118, A119, A120, A121, A122, A123, A124, A125, A126, A127, A128, A129, A130, A131, A132, A133, A134, A135, A136, A137, A138, A139, A140, A141, A142, A143, A144, A145, A146, A147, A148, A149, A150, A151, A152, A153, A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, A164, A165, A166, A167, A168, A169, A170, A171, A172, A173, A174, A175, A176, A177, A178, A179, A180, A181, A182, A183, A184, A185, A186, A187, A188, A189, A190, A191, A192, A193, A194, A195, A196, A197, A198, A199, A200, A201, A202, A203, A204, A205, A206, A207, A208, A209, A210, A211, A212, A213, A214, A215, A216, A217, A218, A219, A220, A221 or a combination thereof.

[0110] In another preferred example, the compound of formula (I) is selected from the group consisting of: compound A1, A9, A10, A11, A12, A49, A50, A51, A52, A53, A69, A70, A71, A72, A74, A75, A76, A77, A84, A87, A102, A106, A107, A108, A109, A124, A131, A138, A140, A144, A146, A147, A151, A164, A171, A173, A174, A180, A181, A188, A196, A198, A209, A212, A213, A214, A215, A216, A221 or a combination thereof.

[0111] In another preferred example, the preparation (or the pharmaceutical composition containing the compound) may further contain other antiviral drugs.

[0112] In another preferred example, the other antiviral drugs further include additional components selected from the group consisting of:

[0113] Remdesivir (also known as GS-5734), Favipiravir, Galidesivir, GS-441524, NHC (EIDD-1931), EIDD-2801, GC-376, Lopinavir, Ritonavir, Nelfinavir; Chloroquine, Hydroxychloroquine, Cyclosporine, Carrimycin, Baicalin, Baicalein, Forsythoside, Chlorogenic Acid, Emodin, Mycophenolic Acid, Mycophenolate Mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, Baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV (Respi )、MEDI-557、A-60444 (RSV-604)、MDT-637、BMS-433771、or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0114] In another preferred embodiment, the pharmaceutical composition containing the compound further comprises administering a therapeutically effective amount of at least one other therapeutic agent selected from the group consisting of corticosteroids, anti-inflammatory signal transduction regulators, β2-adrenergic receptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs for treating viral infections; or a combination thereof.

[0115] In another preferred embodiment, the formulation is a pharmaceutical composition.

[0116] In another preferred embodiment, the preparation (or pharmaceutical composition) includes: oral preparations and non-oral preparations.

[0117] In another preferred embodiment, the preparation includes: powders, granules, capsules, injections, inhalants, tinctures, oral liquids, tablets, lozenges, or dripping pills.

[0118] In the third aspect of the present invention, a pharmaceutical composition is provided, and the pharmaceutical composition contains:

[0119] (a1) A first active ingredient, which is a compound represented by formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof or a prodrug thereof:

[0120]

[0121] In the formula,

[0122] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, amino, C 1-6 alkyl-substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyl-substituted amino, halo C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 2-6 alkynyl, C 3-6 cycloalkyl, halo C 3-6 cycloalkyl, carbamoyl, hydroxymethyl, cyanomethyl (-CH 2 CN), amidino, guanidino, ureido, thiocyanato (-SCN), cyanato (-OCN);

[0123] R 2 is selected from hydrogen, halogen, OR 3 , cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0124] R 3 is selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl, C 1-6 alkylamino C 1-6 alkanoyl, C 1-6 alkoxy C 1-6 alkyl, an α-amino acid, and the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond;

[0125] R 4 selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azido C 1-6 alkyl, cyano C 1-6 alkyl, hydroxy C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 1-6 alkanoyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, cyanothio, cyanato;

[0126] R 5 selected from R 3 ,

[0127] R 6 selected from amino, hydroxy, halogen, cyano, cyanato, cyanothio, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;

[0128] R 7 selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;

[0129] R 8 selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl-substituted carbamoyl, C 1-6 alkoxycarbonylamide, C 1-6 alkoxycarbonyl, hydroxy, hydroxy C 1-6 alkyl, amino, C 1-6 alkanoyl-substituted amino, C 1-6 alkyl-substituted amino, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0130] X is selected from CH 2 , CD 2, -CHD-;

[0131] R 9 Selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0132] R 10 Selected from C 1-18 alkyl, methylene C 6-20 aryl;

[0133] R 11 Selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0134] R 12 Selected from C 1-20 alkyl;

[0135] M are each independently selected from hydrogen, metal, -NH 4 or protonated organic amine;

[0136] and (b) a pharmaceutically acceptable carrier.

[0137] In another preferred embodiment, the composition further comprises (a2) a second active ingredient;

[0138] Among them, the second active ingredient is an antiviral drug, which is selected from the following group: interferon, RNA-dependent RNA polymerase inhibitors (such as Remdesivir (Remdesivir or GS-5734), Favipiravir, Galidesivir, GS-441524, NHC (EIDD-1931), EIDD-2801), 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, Nelfinavir; Chloroquine, Hydroxychloroquine, Cyclosporine, Carrimycin, Baicalin, Baicalein, Forsythoside, Chlorogenic acid, Emodin, Mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, Baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV (Respi ), MEDI-557, A-60444 (RSV-604), MDT-637, BMS-433771, or a pharmaceutically acceptable salt thereof, or a combination thereof;

[0139] And / or the second active ingredient is selected from the group consisting of: bronchodilators and corticosteroids for treating respiratory tract infections, wherein the corticosteroids include dexamethasone, dexamethasone sodium phosphate, fluorometholone, fluorometholone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinonide, fluocinonide, flunisolide, fluocortin-21-butylate, flumethasone, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide; or a pharmaceutically acceptable salt thereof.

[0140] And / or the second active ingredient is selected from the group consisting of: Zinc, Fingolimod, Vitamin C, Olmesartan Medoxomil, valsartan, Losartan, Thalidomide, glycyrrhizic acid, Artemisinin, dihydroartemisinin, Artesunate, Artemisone, Azithromycin, Escin, Naproxen, or a combination thereof.

[0141] In another preferred embodiment, in the formula (I) compound,

[0142] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, methyl, chloromethyl, fluoromethyl, vinyl, ethynyl, cyclopropyl, carbamoyl, hydroxymethyl, methoxy, formyl, amidino;

[0143] R 2 is selected from halogen, cyano, amino, formyl, OR 3 ;

[0144] R 3 is selected from hydrogen, C 1-20 alkyl acyl, α-amino acid, wherein the carboxyl group of the α-amino acid is linked to the hydroxyl group on the furan ring by an ester bond; preferably, the α-amino acid is selected from alanine, valine, isoleucine, tryptophan, phenylalanine;

[0145] R 4 is selected from hydrogen, deuterium, halogen, azido, cyano, methyl, chloromethyl, fluoromethyl, difluoromethyl, vinyl, ethynyl, cyclopropyl, hydroxymethyl, azidomethyl (-CH 2 N3 ), formyl, acetyl, formamido, acetamido;

[0146] R 5 selected from R 3 ,

[0147] R 6 selected from amino, hydroxy, halogen, cyano, methylamino (-NH 2 CH 3 ), NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;

[0148] R 7 selected from hydrogen, deuterium, halogen, amino;

[0149] R 8 selected from hydrogen, deuterium, halogen, cyano, carbamoyl, N-methylcarbamoyl (CH 3 NHCO-), methyl, ethyl, ethynyl, methoxycarbonyl, ethoxycarbonyl, hydroxy, hydroxymethyl, hydroxyethyl, methoxy, ethoxy, formyl, acetyl, formylamino, acetamido, methoxycarbonylamino (CH 3 OCONH 2 -), ethoxycarbonylamino (C 2 H 5 OCONH 2 -), methoxycarbonyloxy (CH 3 OCOO-), ethoxycarbonyloxy (C 2 H 5 OCOO-);

[0150] X is selected from CH 2 , CD 2 , -CHD-;

[0151] R 9 selected from C 6-20 aryl, 5- to 15-membered heteroaryl;

[0152] R 10 selected from C 1-18 alkyl, methylene C 6-20 aryl;

[0153] R 11 selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5- to 15-membered heteroaryl;

[0154] R 12 selected from C1-20 alkyl; and / or

[0155] M each independently selects hydrogen, zinc, magnesium, calcium, sodium, potassium, NH 4 , protonated trimethylamine, protonated triethylamine, protonated tri-n-butylamine.

[0156] In another preferred example, the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.

[0157] In another preferred example, the compound of formula (I) is selected from the group consisting of: compound A1, A5, A6, A8, A9, A10, A11, A12, A13, A14, A28, A30, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A49, A50, A51, A52, A53, A54, A55, A57, A58, A63, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A84, A86, A87, A88, A89, A91, A95, A97, A99, A101, A102, A105, A106, A107, A108, A109, A110, A111, A113, A114, A115, A116, A117, A118, A119, A120, A121, A122, A123, A124, A125, A126, A127, A128, A129, A130, A131, A132, A133, A134, A135, A136, A137, A138, A139, A140, A141, A142, A143, A144, A145, A146, A147, A148, A149, A150, A151, A152, A153, A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, A164, A165, A166, A167, A168, A169, A170, A171, A172, A173, A174, A175, A176, A177, A178, A179, A180, A181, A182, A183, A184, A185, A186, A187, A188, A189, A190, A191, A192, A193, A194, A195, A196, A197, A198, A199, A200, A201, A202, A203, A204, A205, A206, A207, A208, A209, A210, A211, A212, A213, A214, A215, A216, A217, A218, A219, A220, A221 or a combination thereof.

[0158] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of: compound A1, A9, A10, A11, A12, A49, A50, A51, A52, A53, A69, A70, A71, A72, A74, A75, A76, A77, A84, A87, A102, A106, A107, A108, A109, A124, A131, A138, A140, A144, A146, A147, A151, A164, A171, A173, A174, A180, A181, A188, A196, A198, A209, A212, A213, A214, A215, A216, A221 or a combination thereof.

[0159] In another preferred embodiment, the second active ingredient is selected from the group consisting of: (Y1) RNA replicase inhibitors (such as Remdesivir (Remdesivir or GS-5734), Favipiravir, Galidesivir, GS-441524, NHC, EIDD-2801); (Y2) Lopinavir; (Y3) Ritonavir; (Y4) Favipiravir; (Y5) Chloroquine, Hydroxychloroquine, or a pharmaceutically acceptable salt thereof (such as Chloroquine Phosphate), (Y6) Nelfinavir; (Y7) any combination of the above Y1 - Y6.

[0160] In another preferred embodiment, the pharmaceutical composition is used to inhibit the replication of coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviruses, filoviruses, and / or porcine epidemic diarrhea virus (PEDV).

[0161] In another preferred embodiment, the drug is used to inhibit the replication of the 2019 novel coronavirus (SARS-CoV-2).

[0162] In a fourth aspect of the present invention, there is provided the use of the pharmaceutical composition described in the third aspect of the present invention, which is used to prepare (a) an agent for inhibiting coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviruses, filoviruses, and / or porcine epidemic diarrhea virus (PEDV); and / or (b) a drug for treating and / or preventing, alleviating related diseases caused by infection with coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviruses, filoviruses, and / or porcine epidemic diarrhea virus (PEDV).

[0163] In another preferred example, it is used to prepare (a) an inhibitor that inhibits the replication of the novel coronavirus 2019 (SARS-CoV-2); and / or (b) a drug for treating and / or preventing and alleviating related diseases caused by the infection of the novel coronavirus 2019 (SARS-CoV-2).

[0164] In the fifth aspect of the present invention, a method for inhibiting virus replication is provided, including the steps of:

[0165] Contacting a first active ingredient or a preparation containing the first active ingredient with a virus, thereby inhibiting the replication of the virus;

[0166] Wherein, the first active ingredient is a compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate of a crystal thereof or a solvate thereof or a prodrug thereof:

[0167]

[0168] In the formula,

[0169] R 1 is selected from hydrogen, deuterium, halogen, cyano, azido, amino, C 1-6 alkyl-substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkanoyl-substituted amino, halo C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 2-6 alkynyl, C 3-6 cycloalkyl, halo C 3-6 cycloalkyl, carbamoyl, hydroxymethyl, cyanomethyl (-CH 2 CN), amidino, guanidino, ureido, thiocyanato (-SCN), cyanato (-OCN);

[0170] R 2 is selected from hydrogen, halogen, OR 3 、cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl;

[0171] R 3 is selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl, C 1-6 alkylamino C 1-6 alkanoyl, C 1-6 alkoxy C 1-6An alkyl group and an α-amino acid, wherein the carboxyl group of the α-amino acid is connected to the hydroxyl group on the furan ring by an ester bond. Preferably, the α-amino acid is selected from alanine, valine, isoleucine, tryptophan, and phenylalanine;

[0172] R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azido C 1-6 alkyl, cyano C 1-6 alkyl, hydroxy C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 1-6 alkanoyl, C 2-6 alkenyloxy, C 2-6 alkynyloxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, cyanothio, cyanato;

[0173] R 5 is selected from R 3 ,

[0174] R 6 is selected from amino, hydroxy, halogen, cyano, cyanato, cyanothio, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;

[0175] R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;

[0176] R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl-substituted carbamoyl, C 1-6 alkoxyamide, C 1-6 alkoxycarbonyl, hydroxy, hydroxy C 1-6 alkyl, amino, C 1-6 alkanoyl-substituted amino, C 1-6 alkyl-substituted amino, C 1-6 alkyl, C 2-6 C 1-6Alkoxy, alkenyl, C 2-6 alkynyl;

[0177] X is selected from CH 2 , CD 2 , -CHD-;

[0178] R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0179] R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;

[0180] R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;

[0181] R 12 is selected from C 1-20 alkyl;

[0182] M are each independently selected from hydrogen, metal, NH 4 or protonated organic amine;

[0183] In another preferred example, the virus is selected from the group consisting of coronavirus, influenza virus, respiratory syncytial virus, flavivirus, filovirus, porcine epidemic diarrhea virus, or a combination thereof.

[0184] In another preferred example, the virus is 2019 novel coronavirus (SARS-CoV-2).

[0185] In another preferred example, the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.

[0186] In another preferred example, the compound of formula (I) is selected from the group consisting of: compound A1, A5, A6, A8, A9, A10, A11, A12, A13, A14, A28, A30, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A49, A50, A51, A52, A53, A54, A55, A57, A58, A63, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A84, A86, A87, A88, A89, A91, A95, A97, A99, A101, A102, A105, A106, A107, A108, A109, A110, A111, A113, A114, A115, A116, A117, A118, A119, A120, A121, A122, A123, A124, A125, A126, A127, A128, A129, A130, A131, A132, A133, A134, A135, A136, A137, A138, A139, A140, A141, A142, A143, A144, A145, A146, A147, A148, A149, A150, A151, A152, A153, A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, A164, A165, A166, A167, A168, A169, A170, A171, A172, A173, A174, A175, A176, A177, A178, A179, A180, A181, A182, A183, A184, A185, A186, A187, A188, A189, A190, A191, A192, A193, A194, A195, A196, A197, A198, A199, A200, A201, A202, A203, A204, A205, A206, A207, A208, A209, A210, A211, A212, A213, A214, A215, A216, A217, A218, A219, A220, A221 or a combination thereof.

[0187] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of: compound A1, A9, A10, A11, A12, A49, A50, A51, A52, A53, A69, A70, A71, A72, A74, A75, A76, A77, A84, A87, A102, A106, A107, A108, A109, A124, A131, A138, A140, A144, A146, A147, A151, A164, A171, A173, A174, A180, A181, A188, A196, A198, A209, A212, A213, A214, A215, A216, A221 or a combination thereof.

[0188] In another preferred embodiment, the method is an in vitro method.

[0189] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0190] In the sixth aspect of the present invention, there is provided a method for (a) inhibiting virus replication and / or (b) treating and / or preventing and / or alleviating related diseases caused by virus infection, comprising the step of administering to a subject in need a safe and effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof or a prodrug thereof:

[0191]

[0192] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and X are as defined in the first aspect of the present invention.

[0193] In another preferred embodiment, the subject is a mammal, such as a human.

[0194] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0195] None. DETAILED DESCRIPTION OF THE INVENTION

[0196] Through extensive and in-depth research and a large number of screenings, the present inventor has unexpectedly developed a class of active ingredients that can effectively inhibit virus replication for the first time. Experiments have shown that the active ingredients of the present invention can efficiently inhibit the replication and viability of various viruses such as the 2019 novel coronavirus (SARS-CoV-2), and thus can be used to inhibit the replication of coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviridae viruses, filoviridae viruses, and / or porcine epidemic diarrhea virus. On this basis, the present invention has been completed.

[0197] Specifically, the present invention discloses the use of nucleoside analogs represented by formula (I) and their compositions in antiviral treatment, such as in the treatment of coronaviruses, influenza viruses, respiratory syncytial viruses, flaviviridae viruses, filoviridae viruses, and / or porcine epidemic diarrhea virus (PEDV). The nucleoside analogs represented by formula (I) have excellent inhibitory effects on the replication of viruses such as SARS-CoV-2 and have good prospects for clinical application.

[0198] The term

[0199] As used herein, the terms "active compounds of the present invention", "active ingredients of the present invention", "nucleoside analogs of the present invention", and "active compounds for inhibiting coronavirus replication of the present invention" are used interchangeably and refer to nucleoside analogs having excellent activity in inhibiting coronavirus replication, including the compounds represented by formula I, or their pharmaceutically acceptable salts, or their solvates, or their prodrugs, or combinations thereof.

[0200] As used herein, "the preparation of the present invention" refers to a preparation containing the active compound of the present invention.

[0201] As used herein, the term "comprising" or its variants such as "including" or "including having" etc. are understood to include the stated elements or components, without excluding other elements or other components.

[0202] As used herein, the terms "novel coronavirus", "2019-nCoV", or "SARS-CoV-2" are used interchangeably. The 2019 novel coronavirus is the 7th known coronavirus that infects humans and causes COVID-19, which is one of the serious infectious diseases threatening global human health.

[0203] As used herein, "halogen" generally refers to fluorine, chlorine, bromine, and iodine; preferably fluorine, chlorine, or bromine; more preferably fluorine or chlorine.

[0204] As used herein, the term "C n -C m " is used interchangeably with C n-m and refers to having n to m carbon atoms.

[0205] As used herein, the term "C 1 -C 6 alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, isohexyl, 3-methylpentyl, or n-hexyl, etc., and is preferably methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or tert-butyl.

[0206] Halogenated C 1 -C 6 alkyl refers to a straight-chain or branched-chain saturated hydrocarbon group containing 1 to 6 carbon atoms in which one or more hydrogen atoms are replaced by one or more identical or different halogen atoms, such as trifluoromethyl, fluoromethyl, difluoromethyl, chloromethyl, bromomethyl, dichlorofluoromethyl, chloroethyl, bromopropyl, 2-chlorobutyl, or pentafluoroethyl, etc.;

[0207] C 1 -C 6 alkoxy refers to a straight-chain or branched-chain alkoxy group containing 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, isohexyloxy, 3-methylpentyloxy, or n-hexyloxy, etc., and is preferably methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy; C1-C6 alkanoyl refers to a straight-chain or branched-chain alkanoyl group containing 1 to 6 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, tert-butyryl, or hexanoyl, etc.

[0208] C 1-6 alkyl-substituted amino refers to an amino group in which one or more hydrogen atoms are replaced by one or more C 1 -C 6 alkyl groups, such as -NHCH 3 、-N(CH 3 ) 2 etc.

[0209] C 1-6 alkanoyl-substituted amino refers to an amino group in which one or more hydrogen atoms are replaced by one or more C 1 -C 6 alkyl groups, such as -NHCOCH 3 、-NHCOCH 2 CH 3 etc.

[0210] C 2 -C 6Alkenyl refers to a straight-chain or branched-chain unsaturated hydrocarbon group containing 1-3 double bonds and 2-6 carbon atoms, including both cis and trans configurations. For example, vinyl, 1-propenyl, 2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-butadienyl, 1,3-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 3,3-dimethyl-1-propenyl, 2-ethyl-1-propenyl, etc.

[0211] C 2 -C 6 Alkynyl refers to a straight-chain or branched-chain alkynyl group containing 2-6 carbon atoms. For example, ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 2-pentynyl, or 2-hexynyl, etc.

[0212] C 2 -C 6 Alkenyloxy refers to a straight-chain or branched-chain alkenyloxy group containing 1-3 double bonds and 2-6 carbon atoms, such as vinyloxy, 1-propenyloxy, 1-methyl-1-propenyloxy, 2-methyl-1-propenyloxy, 1-pentenyloxy, 1,3-pentadienyloxy, or 2-pentenyloxy, etc.

[0213] C 2 -C 6 Alkynyloxy refers to a straight-chain or branched-chain alkynyloxy group containing 2-6 carbon atoms. For example, ethynyloxy, 2-propynyloxy, 2-butynyloxy, 3-butynyloxy, 1-methyl-2-propynyloxy, 2-pentynyloxy, or 2-hexynyloxy, etc.

[0214] Amino C 1-20 Alkanoyl refers to the connection of one carbon atom of a straight-chain or branched-chain alkanoyl group containing 1-20 carbon atoms to an amino group, such as -COCH 2 NH 2 、-COCH 2 CH 2 NH 2 etc.

[0215] C 1-6 Alkylamino C 1-6 Alkanoyl refers to C 1-6 The nitrogen atom of an alkyl-substituted amino group is connected to a non-carbonyl carbon atom of a straight-chain or branched-chain alkanoyl group containing 1-6 carbon atoms, such as -COCH 2 NHCH 3 、-COCH 2 CH 2 NHCH 2 CH3 etc.

[0216] C 1-6 alkoxy C 1-6 The alkyl group refers to the oxygen atom of a straight-chain or branched alkoxy group containing 1 to 6 carbon atoms being connected to the carbon atom of C 1-6 -CH 2 OCH 3 -CH 2 CH 2 OCH 2 CH 3 etc.

[0217] amino C 1-6 The alkyl group refers to a carbon atom of a straight-chain or branched alkyl group containing 1 to 6 carbon atoms being connected to the amino group, such as -CH 2 NH 2 -CH 2 CH 2 NH 2 -CH(NH 2 )CH 3 -CH 2 CH 2 CH 2 NH 2 or -CH 2 CH 2 CH 2 CH 2 NH 2 etc.

[0218] C 1-6 The C-alkyl-substituted carbamoyl group refers to the hydrogen atom on the carbamoyl group being substituted by 1 or 2 identical or different C 1 -C 6 alkyl groups, such as -CONHMe, -CONHEt, -CON(Me)Et, -CONEt 2 or -CONMe 2 etc.

[0219] hydroxy C 1 -C 6 The alkyl group refers to a carbon atom of a straight-chain or branched alkyl group containing 1 to 6 carbon atoms being connected to the hydroxyl group, such as -CH 2 OH, -CH 2 CH 2 OH, -CH(OH)CH 3 -CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2OH or -CH 2 CH(CH 3 )CH 2 OH etc.

[0220] C 1-6 Alkoxy amide group means that the oxygen atom of a straight-chain or branched-chain alkoxy group containing 1-6 carbon atoms is connected to the carbonyl group of the amide group, such as -NHCOOCH 3 , -NHCOOCH 2 CH 3 etc.

[0221] C 1-6 Alkoxy carbonyl means that the oxygen atom of a straight-chain or branched-chain alkoxy group containing 1-6 carbon atoms is connected to the carbonyl group, such as -COOCH 3 , -COOCH 2 CH 3 etc.

[0222] Coronavirus

[0223] Coronavirus (Coronavirus, CoV) belongs to the order Nidovirales, family Coronaviridae, and is an enveloped positive-strand RNA virus. Its subfamilies include four genera: α, β, δ, and γ.

[0224] Among the currently known human-infecting coronaviruses, HCoV-229E and HCoV-NL63 belong to the genus α coronavirus, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV, and SARS-CoV-2 are all β coronavirus. SARS-CoV-2 is also known as 2019-nCoV.

[0225] The highly pathogenic coronaviruses "SARS" (SARS-CoV) and "Middle East Respiratory Syndrome" (MERS-CoV) that broke out in 2003 and 2012 respectively both belong to the genus β coronavirus. The novel coronavirus (SARS-CoV-2) that broke out at the end of 2019 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and also belongs to the genus β coronavirus.

[0226] The genome of this type of virus is a single-stranded positive-strand RNA, which is one of the RNA viruses with the largest genome, encoding proteins including replicase, spike protein, envelope protein, membrane protein, and nucleocapsid protein, etc. In the initial stage of virus replication, the genome is translated into two peptide chains thousands of amino acids long, namely precursor polyprotein, and then the precursor protein is cleaved by protease to generate non-structural proteins (such as RNA polymerase and helicase), structural proteins (such as spike protein), and accessory proteins.

[0227] Influenza virus

[0228] Influenza virus, abbreviated as flu virus, common influenza viruses are divided into types A (H1N1), B, C, and D. Influenza viruses can cause infections and diseases in various animals such as humans, birds, pigs, horses, and bats, and are the pathogens of human influenza, avian influenza, swine influenza, equine influenza and other human and animal diseases.

[0229] Clinical symptoms caused by influenza viruses include acute high fever, general pain, significant fatigue and respiratory symptoms. Human influenza is mainly caused by influenza A virus and influenza B virus. Influenza A virus often undergoes antigenic variation and can be further divided into subtypes such as H1N1, H3N2, H5N1, H7N9, etc.

[0230] Respiratory syncytial virus

[0231] Respiratory syncytial virus (RSV, also known as syncytial virus, belonging to the Paramyxoviridae family) is the most common pathogen causing viral pneumonia in children and can cause interstitial pneumonia.

[0232] RSV is similar to parainfluenza virus. The virus particles are about 150nm in size, slightly smaller than parainfluenza virus, and it is an RNA virus.

[0233] Flaviviridae viruses

[0234] Flaviviridae viruses are a class of RNA viruses that mainly infect mammals, including three virus genera: Flavivirus, Pestivirus, and Hepacivirus. Dengue virus (DENV) and Zika virus belong to the Flavivirus genus and are transmitted by mosquitoes. Dengue virus infection can cause obvious fever and pain symptoms, and severe dengue symptoms also include headache, nausea, vomiting, confusion, and even shock, etc. Zika virus infection symptoms are similar to dengue fever and are generally mild. Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is the pathogen of chronic hepatitis C, which can lead to cirrhosis and liver cancer.

[0235] Filoviridae viruses

[0236] The Filoviridae family currently contains three genera, namely Ebolavirus, Marburgvirus, and Cuevavirus. Marburg virus and Ebola virus can both cause severe hemorrhagic fever. After human infection, there will be high fever and bleeding symptoms, which will further lead to shock and organ failure in patients until death.

[0237] Porcine epidemic diarrhea virus (PEDV)

[0238] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Coronavirus in the family Coronaviridae. Porcine epidemic diarrhea is an acute intestinal infectious disease of piglets and fattening pigs caused by the PEDV virus.

[0239] After oral and nasal infection with the PEDV virus, it directly enters the small intestine. The replication of the PEDV virus can occur in the cytoplasm of the epithelial cells of the villi in the small intestine and colon. PEDV can cause diarrhea, which belongs to osmotic diarrhea. Severe diarrhea causes dehydration, which is the main cause of death in diseased pigs.

[0240] The active compounds and active ingredients of the present invention

[0241] In the present invention, there is provided an active ingredient that can effectively inhibit the replication of coronaviruses, influenza viruses, respiratory syncytial viruses, and / or porcine epidemic diarrhea virus (PEDV), especially the novel coronavirus 2019 (SARS-CoV-2).

[0242] In the present invention, the active ingredient is selected from the nucleoside analogs represented by formula (I) described in the first aspect of the present invention, or pharmaceutically acceptable salts thereof, or prodrugs thereof, or combinations thereof, or crystals thereof, or solvates thereof.

[0243] The structural formula of the preferred nucleoside analog is the compound prepared in the examples or its pharmaceutically acceptable salt, or its prodrug, or a combination thereof, or a crystal thereof, or a solvate thereof, especially any one of the compounds A1 to A221, or a combination thereof.

[0244] In another preferred example, the compound of formula (I) is selected from the group consisting of: Compound A1, A5, A6, A8, A9, A10, A11, A12, A13, A14, A28, A30, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A49, A50, A51, A52, A53, A54, A55, A57, A58, A63, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A84, A86, A87, A88, A89, A91, A95, A97, A99, A101, A102, A105, A106, A107, A108, A109, A110, A111, A113, A114, A115, A116, A117, A118, A119, A120, A121, A122, A123, A124, A125, A126, A127, A128, A129, A130, A131, A132, A133, A134, A135, A136, A137, A138, A139, A140, A141, A142, A143, A144, A145, A146, A147, A148, A149, A150, A151, A152, A153, A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, A164, A165, A166, A167, A168, A169, A170, A171, A172, A173, A174, A175, A176, A177, A178, A179, A180, A181, A182, A183, A184, A185, A186, A187, A188, A189, A190, A191, A192, A193, A194, A195, A196, A197, A198, A199, A200, A201, A202, A203, A204, A205, A206, A207, A208, A209, A210, A211, A212, A213, A214, A215, A216, A217, A218, A219, A220, A221 or a combination thereof.

[0245] Tests show that the active ingredient of the present invention can effectively inhibit the replication of the novel coronavirus 2019 (SARS-CoV-2), thereby preventing, treating, and / or alleviating SARS-CoV-2-related diseases.

[0246] As used herein, the terms "active compound of the present invention" and "active compound for inhibiting virus replication of the present invention" are used interchangeably and refer to a compound having excellent activity for inhibiting virus replication, including a nucleoside analogue of formula I or a pharmaceutically acceptable salt thereof, or a crystal thereof, or a solvate thereof.

[0247] It should be understood that the active ingredient of the present invention includes a compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate thereof, or a prodrug thereof. It should be understood that the active ingredient of the present invention also includes forms such as crystal forms of the active compound of the present invention, amorphous compounds, and deuterated compounds.

[0248] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the active compound of the present invention with an inorganic acid or an organic acid. For example, a conventional non-toxic salt can be prepared by reacting the active compound of the present invention with an inorganic acid or an organic acid. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc. The organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or a sodium salt, zinc salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by reacting the active compound of the present invention with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid to form an ester and then reacting with an inorganic base; or a corresponding inorganic acid salt formed by reacting the active compound of the present invention with lysine, arginine, ornithine to form an ester and then reacting with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid or a corresponding organic acid salt formed by reacting with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid; or a salt formed by reacting 1-4 phosphate groups in the molecule of the active compound of the present invention with 1-4 molecules of organic amine, including but not limited to trimethylamine monophosphate salt, triethylamine monophosphate salt, tributylamine monophosphate salt, trimethylamine triphosphate salt, triethylamine triphosphate salt, tributylamine triphosphate salt.

[0249] In addition, the active ingredient of the present invention is also particularly suitable for combination with other antiviral drugs or anti-coronavirus drugs. Representative other antiviral drugs or anti-coronavirus drugs include (but are not limited to): interferons, RNA-dependent RNA polymerase inhibitors (such as Remdesivir (GS-5734), favipiravir, Galidesivir, GS-441524, NHC, EIDD-2801); 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, Nelfinavir; Chloroquine (Sigma-C6628), hydroxychloroquine, cyclosporine, Carrimycin, baicalin, baicalein, forsythoside, chlorogenic acid, emodin, mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, nafamostat, nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, baricitinib, Ruxolitinib, Dasatinib, Saquinavir, Beclabuvir, Simeprevir, Palivizumab, Motavizumab, RSV-IGIV (Respi ), MEDI-557, A-60444 (RSV-604), MDT-637, BMS-433771 or a pharmaceutically acceptable salt thereof, or a combination thereof. The interferons include one or more of interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-1a, and interferon β-1b.

[0250] Additional active therapeutic agents for treating respiratory symptoms and sequelae of infection can also be used in combination with the compounds of formula I. The other agents are preferably administered orally or by direct inhalation. For example, other preferred additional therapeutic agents for treating respiratory tract infections in combination with the compounds of formula I include, but are not limited to, bronchodilators and corticosteroids.

[0251] Glucocorticoids, first introduced as an asthma therapy in 1950 (Carryer, Journal of Allergy, 21, 282-287, 1950), remain the most effective and consistent therapy for the disease, but their mechanism of action is not fully understood (Morris, J. Allergy Clin. Immunol., 75(1 Pt) 1-13, 1985). Unfortunately, oral glucocorticoid therapy is associated with profound undesirable side effects such as centripetal obesity, hypertension, glaucoma, glucose intolerance, accelerated cataract formation, bone mineral loss, and psychological effects, all of which limit their use as long-term therapeutic agents (Goodman and Gilman, 10th Edition, 2001). A solution to the systemic side effects is to deliver the steroid drug directly to the site of inflammation. Inhaled corticosteroids (ICS) have been developed to mitigate the severe side effects of oral steroids. Non-limiting examples of corticosteroids that can be used in combination with the compounds of formula I are dexamethasone, dexamethasone sodium phosphate, fluorometholone, fluorometholone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinonide, flucetonide, flunisolide, fluocortin-21-butylate, flumethasone, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide; or a pharmaceutically acceptable salt thereof.

[0252] Other anti-inflammatory agents that act through the anti-inflammatory cascade mechanism can also be used as additional therapeutic agents in combination with the compounds of formula I for the treatment of viral respiratory infections. Application of "anti-inflammatory signal transduction modulators" (referred to herein as AISTM) such as phosphodiesterase inhibitors (e.g., specific for PDE-4, PDE-5 or PDE-7), transcription factor inhibitors (e.g., blocking NFκB through IKK inhibition) or kinase inhibitors (e.g., blocking P38MAP, JNK, PI3K, EGFR or Syk) is a logical way to cut off inflammation because these small molecules target a limited number of common intracellular pathways - those signal transduction pathways that are key points of anti-inflammatory therapeutic intervention (see, reviewed by P.J. Barnes, 2006). These non-limiting additional therapeutic agents include: 5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl)-amide (P38Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluoromethoxy-benzamide (PDE-4 inhibitor roflumilast); 4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenyl-ethyl]-pyridine (PDE-4 inhibitor CDP-840); N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8-[(methylsulfonyl)amino]-1-dibenzofuran-carboxamide (PDE-4 inhibitor omalizumab); N-(3,5-dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide (PDE-4 inhibitor AWD12-281); 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl)-amide (PDE-4 inhibitor Sch351591); 4-[5-(4-fluorophenyl)-2-(4-methylsulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine (P38 inhibitor SB-203850); 4-[4-(4-fluoro-phenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (2-diethyl-ethyl ester prodrug of cilomilast, PDE-4 inhibitor); (3-chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (gefitinib, EGFR inhibitor); and 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (imatinib, EGFR inhibitor).

[0253] A combination of an inhaled β2-adrenergic receptor agonist bronchodilator such as formoterol, salbutamol or salmeterol and a compound of formula I is a suitable but non-limiting combination that can also be used for the treatment of viral respiratory infections.

[0254] Combinations of inhaled β2-adrenergic receptor agonist bronchodilators such as formoterol or salmeterol with an ICS are also used for the treatment of both bronchial stenosis and inflammation. Combinations comprising such ICS and β2-adrenergic receptor agonist combinations together with a compound of formula I are also suitable but non-limiting combinations for the treatment of viral respiratory infections.

[0255] Anticholinergic agents have potential utility for the treatment or prevention of pulmonary bronchial stenosis and may thus be used as additional therapeutic agents in combination with compounds of formula I for the treatment of viral respiratory infections.These anticholinergic agents include, but are not limited to, antagonists of muscarinic receptors (especially the M3 subtype) that have shown therapeutic efficacy in humans for the control of cholinergic properties in COPD (Witek, 1999); 1-{4-hydroxy-1-[3,3,3-tris-(4-fluorophenyl)-propanoyl]-pyrrolidine-2-carbonyl}-pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl)-amide; 3-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-8-isopropyl-8-methyl-8-azonia-bicyclo[3.2.1]octane (ipratropium-N,N-diethylglycine ester or salt); 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester (solifenacin); 2-hydroxymethyl-4-methylsulfinyl-2-phenyl-butanoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester (revatropate); 2-{1-[2-(2,3-dihydro-benzofuran-5-yl)-ethyl]-pyrrolidin-3-yl}-2,2-diphenyl-acetamide (darifenacin); 4-azepan-1-yl-2,2-diphenyl-butyramide (buzepide); 7-[3-(2-diethylamino-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane (oxitropium-N,N-diethylglycine ester or salt); 7-[2-(2-diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-9,9-dimethyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane (tiotropium-N,N-diethylglycine ester or salt); dimethylamino-acetic acid 2-(3-diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester (tolterodine-N,N-dimethylglycine ester or salt); 3-[4,4-bis-(4-fluorophenyl)-2-oxo-imidazolin-1-yl]-1-methyl-1-(2-oxo-2-pyridin-2-yl-ethyl)-pyrrolidinium; 1-[1-(3-fluorobenzyl)-piperidin-4-yl]-4,4-bis-(4-fluorophenyl)-imidazolidin-2-one; 1-cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1-phenyl-prop-2-yn-1-ol; 3-[2-(2-diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-1-(3-phenoxypropyl)-1-azonia-bicyclo[2.2.2]octane (aclidinium-N,N-diethylglycine ester or salt); or (2-diethylamino-acetoxy)-di-thiophen-2-yl-acetic acid 1-methyl-1-(2-phenoxyethyl)-piperidin-4-yl ester.

[0256] In addition, since SARS-CoV-2 infection can cause acute lung injury, the active ingredient of the present invention is also particularly suitable for combination with drugs having the effect of improving acute lung injury. Representative drugs include, but are not limited to, Zinc, Fingolimod, Vitamin C, Olmesartan Medoxomil, valsartan, Losartan, Thalidomide, glycyrrhizic acid, Artemisinin, dihydroartemisinin, Artesunate, Artemisone, Azithromycin, Escin, Naproxen. The Zinc includes, but is not limited to, zinc sulfate, zinc glycyrrhizinate, zinc gluconate.

[0257] The active ingredient of the present invention can inhibit the infectivity of novel coronaviruses such as SARS-CoV-2. Therefore, when the active ingredient of the present invention is therapeutically administered or given, the infection of the 2019 novel coronavirus (SARS-CoV-2) can be inhibited, thereby achieving an antiviral effect.

[0258] The compound of formula I can also be combined with a mucolytic to treat both the infection and the symptoms of respiratory tract infection. A non-limiting example of the mucolytic is ambroxol. Similarly, the compound of formula I can be combined with an expectorant to treat both the infection and the symptoms of respiratory tract infection. A non-limiting example of the expectorant is guaifenesin.

[0259] Nebulized hypertonic saline is used to improve the immediate and long-term clearance of the small airways in patients with lung diseases (Kuzik, J. Pediatrics 2007, 266). The compound of formula I can also be combined with nebulized hypertonic saline, especially when paramyxovirus infection is complicated with bronchiolitis. The combination of the compound of formula I and hypertonic saline can also include any of the additional agents discussed above. In a preferred aspect, about 3% nebulized hypertonic saline is used.

[0260] It is also possible to combine any compound of the present invention with one or more additional active therapeutic agents in a unit dosage form for simultaneous or sequential administration to a patient. The combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations.

[0261] The co - administration of the compounds of the present invention with one or more other active therapeutic agents generally means the administration, simultaneously or sequentially, of the compounds of the present invention and one or more other active therapeutic agents such that a therapeutically effective amount of the compounds of the present invention and one or more other active therapeutic agents are both present in the patient's body.

[0262] Co - administration includes administering a unit dose of the compounds of the present invention before or after administering a unit dose of one or more other active therapeutic agents. For example, the compounds of the present invention are administered within seconds, minutes, or hours of administering one or more other active therapeutic agents. For example, a unit dose of the compounds of the present invention can be administered first, followed by a unit dose of one or more other active therapeutic agents within seconds or minutes. Or, a unit dose of one or more other therapeutic agents can be administered first, followed by a unit dose of the compounds of the present invention within seconds or minutes. In some cases, it may be necessary to administer a unit dose of the compounds of the present invention first, and then administer a unit dose of one or more other active therapeutic agents after a period of hours (e.g., 1 - 12 hours). In other cases, it may be necessary to administer a unit dose of one or more other active therapeutic agents first, and then administer a unit dose of the compounds of the present invention after a period of hours (e.g., 1 - 12 hours).

[0263] Combination therapy can provide "synergistic effects" and "synergism", that is, the effect obtained when the active ingredients are used together is greater than the sum of the effects obtained when the compounds are used separately. Synergism can be obtained when the active ingredients: (1) are co - formulated and administered or delivered simultaneously in the form of a combination preparation; (2) are administered or delivered alternately as separate preparations or in parallel; or (3) are delivered by some other administration regimen. When delivered by alternate therapy, synergism can be obtained when the compounds are administered or delivered sequentially, for example, as separate tablets, pills, or capsules, or by different injections from separate syringes. Generally, during alternate therapy, an effective dose of each active ingredient is administered sequentially, that is, continuously, while in combination therapy, an effective dose of two or more active ingredients is administered together. A synergistic antiviral effect means an antiviral effect greater than the predicted pure additive effect of the individual compounds in the combination.

[0264] In yet another embodiment, a method of inhibiting viral RNA polymerase in a cell is provided, which comprises contacting a virus - infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., an ester) thereof, thereby inhibiting the viral RNA polymerase.

[0265] In yet another embodiment, a method of inhibiting viral RNA polymerase in a cell is provided, which comprises contacting a virus-infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., ester) thereof, and at least one additional active therapeutic agent, thereby inhibiting the viral RNA polymerase.

[0266] In yet another embodiment, a method of inhibiting viral RNA polymerase in a cell is provided, which comprises contacting a virus-infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., ester) thereof, and at least one selected additional active therapeutic agent.

[0267] In yet another embodiment, a method of treating a respiratory virus infection in a human or other mammal is provided, which comprises administering to the human or other mammal a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., ester) thereof.

[0268] In yet another embodiment, a method of treating a respiratory virus infection in a human or other mammal is provided, which comprises administering to the human or other mammal a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., ester) thereof, and at least one additional active therapeutic agent, thereby inhibiting the respiratory viral RNA polymerase.

[0269] In yet another embodiment, a method of treating a respiratory virus infection in a human or other mammal is provided, which comprises administering to the human or other mammal a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or prodrug (e.g., ester) thereof, and at least one additional active therapeutic agent.

[0270] Pharmaceutical compositions and uses

[0271] The present invention also provides the use of a mixture of one or more of the active compounds for inhibiting virus replication of the present invention, or a pharmaceutically acceptable salt thereof, or a prodrug thereof as an active ingredient in the preparation of a medicament for treating and / or preventing, alleviating respiratory infections, pneumonia and other related diseases caused by virus infections such as coronavirus, influenza virus, respiratory syncytial virus and / or porcine epidemic diarrhea virus (PEDV), especially the 2019 novel coronavirus.

[0272] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99 wt%, and a preferred ratio is that the active compound of the present invention as an active ingredient accounts for 0.1 wt% to 90 wt% or 1 wt% to 50 wt% of the total weight, and the remaining part is a pharmaceutically acceptable carrier, diluent or solution or salt solution.

[0273] When needed, one or more pharmaceutically acceptable carriers may also be added to the medicament of the present invention. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0274] The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, etc., and can be present in suitable solid or liquid carriers or diluents and suitable sterilized appliances for injection or infusion.

[0275] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to the conventional preparation methods in the pharmaceutical field. The unit dosage of its formulation usually contains 0.05 - 1000 mg of the active compound of the present invention. Preferably, the unit dosage of the formulation contains 1 mg - 500 mg of the active compound of the present invention.

[0276] The compounds and pharmaceutical compositions of the present invention can be clinically used in mammals, including humans and animals, and can be administered through routes such as oral, nasal, skin, lung, or gastrointestinal tract, etc. The most preferred is oral administration. The most preferred daily dose is 0.01 - 400 mg / kg body weight, taken once, or 0.01 - 200 mg / kg body weight taken in divided doses. Regardless of the administration method, the optimal dose for an individual should be determined according to the specific treatment. Usually, it starts with a small dose and gradually increases the dose until the most suitable dose is found.

[0277] The medicament or inhibitor of the present invention can be administered in various different ways. For example, it can be introduced into the body through injection, spraying, nasal dropping, eye dropping, penetration, absorption, physical or chemical mediated methods such as intramuscular, intradermal, subcutaneous, intravenous, mucosal tissues; or it can be mixed or encapsulated with other substances and then introduced into the body.

[0278] Typically, the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0279] The pharmaceutical dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including O / W type, W / O type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion and a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol (powder aerosol), a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0280] The active ingredient of the present invention can be made into an ordinary preparation, or can also be made into a sustained-release preparation, a controlled-release preparation, a targeted preparation and various particulate drug delivery systems.

[0281] In order to make the active ingredient of the present invention into a tablet, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and glidant can be talc powder, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0282] The tablet can also be further made into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a bilayer tablet and a multilayer tablet.

[0283] In order to make the dosage unit into a capsule, the active ingredient of the present invention as the effective ingredient can be mixed with a diluent and a glidant, and the mixture is directly placed into a hard capsule or a soft capsule. The effective ingredient can also be first made into granules or pellets with a diluent, a binder and a disintegrant, and then placed into a hard capsule or a soft capsule. The varieties of each diluent, binder, wetting agent, disintegrant and glidant used for preparing the tablet of the present invention can also be used for preparing the capsule of the present invention.

[0284] To prepare the active ingredient of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and appropriate solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing a freeze-dried powder injection, mannitol, glucose, etc. can also be added as a support agent.

[0285] In addition, if necessary, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparation.

[0286] The active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0287] When there is a synergistic effect between the active ingredient of the present invention and other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0288] The main advantages of the present invention include:

[0289] (a) The active compound of the present invention can efficiently inhibit the replication of viruses such as SARS-CoV-2, human respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV OC43), porcine epidemic diarrhea virus (PEDV), Zika virus, dengue virus (DENV), etc., with a small EC 50 value and having a broad-spectrum antiviral characteristic.

[0290] (b) The active compound of the present invention has low toxicity and side effects (cytotoxicity CC 50 > 10 μM) and good drug-forming properties. This indicates that the nucleoside analog of the present invention has good medicinal prospects in the treatment of viral infectious diseases, especially in the field of anti-COVID-19.

[0291] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the reagent manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0292] Preparation Example 1: Synthesis of Compound A1

[0293]

[0294] Compound 1-1 was synthesized according to the reported method in the literature (Nature. 2016, 531, 381-385). Compound 1-1 (1.5 g, 2.67 mmol) was added to acetonitrile (30 mL), and then 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor, 1.13 g, 3.2 mmol, 1.2 eq) and sodium bicarbonate (0.67 g, 8.0 mmol, 3 eq) were added. After the addition was complete, the reaction was carried out at room temperature for 3-4 hours, and TLC showed that the reaction was complete. The reaction solution was added to water (120 mL), extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and separated by silica gel column chromatography to obtain compound 1-2, 0.43 g of off-white solid, with a yield of 28%. 1 H NMR(500MHz,DMSO-d 6 )δ8.12(brs,1H),7.87(s,1H),7.50–7.20(m,16H),6.59(s,1H),4.92(d,J=11.7Hz,1H),4.85–4.78(m,2H),4.57–4.46(m,4H),4.42–4.35(m,1H),4.12(t,J=5.7Hz,1H),3.73(dd,J=11.2,3.2Hz,1H),3.60(dd,J=11.2,4.3Hz,1H).

[0295] Compound 1-2 (0.25 g, 0.43 mmol) was added to dichloromethane (10 mL), the temperature was cooled to -60 °C, and 1M boron tribromide dichloromethane solution (1.72 mL, 1.72 mmol, 4.0 eq) was added dropwise. After the addition was complete, the temperature was naturally raised to -40 °C, and the reaction was stirred at this temperature for 2 hours. Then TLC monitored that the reaction was complete. The temperature was cooled to -60 °C, and methanol (0.2 mL) and triethylamine (0.52 g, 5.16 mmol, 12 eq) were added dropwise in sequence. After the addition was complete, the solvent was evaporated to obtain a yellow solid. The solid was added to methanol (2 mL) and ethyl acetate (10 mL), stirred and filtered to remove the white insoluble matter. The filtrate was concentrated and separated by preparative plate to obtain compound A1, 49 mg of off-white solid, with a yield of 37%. 1 H NMR(500MHz,DMSO-d 6)δ8.11(brs,1H),7.88(s,1H),7.41(brs,1H),6.80(s,1H),6.23(d,J=6.1Hz,1H),5.20(d,J=5.7Hz,1H),4.93(t,J=5.7Hz,1H),4.54(t,J=5.5Hz,1H),4.08–4.01(m,1H),3.96–3.90(m,1H),3.71–3.64(m,1H),3.55–3.48(m,1H). MS m / z=310.1[M+1] + 。

[0296] Preparation Example 2: Synthesis of Compound A2

[0297]

[0298] Compound 2-1 was synthesized according to the reported method in the literature (Nature. 2016, 531, 381-385). Compound 2-1 (291 mg, 1.0 mmol) was added to N,N-dimethylformamide (5 mL), and N-chlorosuccinimide (245 mg, 1.1 mmol, 1.1 eq) and trifluoroacetic acid (24 mg, 0.2 mmol, 0.2 eq) were added. The reaction was carried out at 50 °C for 1 hour, and TLC showed that the reaction was complete. The reaction solution was added to a mixed solution of sodium sulfite and sodium carbonate, and filtered to obtain Compound A2, 185 mg of white solid, with a yield of 57%. 1 H NMR(500MHz,DMSO-d 6 )δ8.26(brs,1H),7.95(s,1H),7.09(brs,1H),6.98(s,1H),6.23(d,J=5.7Hz,1H),5.18(d,J=5.6Hz,1H),4.92(t,J=5.6Hz,1H),4.52(t,J=5.0Hz,1H),4.06–4.00(m,1H),3.96–3.88(m,1H),3.69–3.61(m,1H),3.54–3.45(m,1H). MS m / z=326.0[M+1] + 。

[0299] Preparation Example 3: Synthesis of Compound A3

[0300]

[0301] Compound 2-1 (291 mg, 1.0 mmol) was added to N,N-dimethylformamide (5 mL). N-Iodosuccinimide (245 mg, 1.1 mmol, 1.1 eq) and trifluoroacetic acid (24 mg, 0.2 mmol) were added. The reaction was carried out at 50 °C for 1 hour. TLC showed that the reaction was complete. The reaction solution was added to a mixed solution of sodium sulfite and sodium carbonate, and filtered to obtain compound A3, 200 mg of white solid, with a yield of 48%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.98 (s, 1H), 7.12 (s, 1H), 6.21 (brs, 1H), 5.20 (brs, 1H), 4.91 (s, 1H), 4.52 (d, J = 4.6 Hz, 1H), 4.11–3.99 (m, 1H), 3.97–3.85 (m, 1H), 3.64 (d, J = 11.3 Hz, 1H), 3.49 (d, J = 10.9 Hz, 1H). MS m / z = 418.0 [M+1] + .

[0302] Preparation Example 4: Synthesis of Compound A4

[0303]

[0304] Compound 1-1 (350 mg, 0.62 mmol) was added to 80% aqueous acetic acid solution (10 mL). The temperature was lowered to 0 °C, and sodium nitrite (856 mg, 12.4 mmol, 20 eq) was added in portions. After addition, it was kept warm for 5 minutes, stirred at room temperature for 30 minutes, then gradually heated to 90 °C and kept warm and stirred for 4 hours. TLC showed that the raw materials were completely converted. (5 mL) of water and (15 mL) of toluene were added, and the organic layer was separated. The aqueous layer was extracted with toluene again. The organic phases were combined, and the combined organic phase was washed successively with water, aqueous sodium bicarbonate solution and brine, dried, concentrated, and separated by silica gel column chromatography to obtain compound 4-1, 345 mg of pale yellow foamy solid.

[0305] The product 4-1 obtained in the previous step was dissolved in dichloromethane (3 mL), protected by nitrogen, cooled to -35 °C, and a dichloromethane solution of 1 M boron trichloride (2.1 mL, 2.1 mmol) was added dropwise. The addition was completed in about 10 minutes, and the mixture was kept warm for 15 minutes. Methanol (0.3 mL) was slowly added dropwise. After the addition was completed, the mixture was kept warm for 20 minutes. The reaction solution was concentrated, n-heptane (5 mL) was added, and the mixture was stirred at room temperature for 1 hour, filtered, and the filter cake was washed with a small amount of n-heptane to obtain 255 mg of a yellow solid. This solid was refluxed in methanol (0.75 mL) and water (0.1 mL) for 30 minutes, activated carbon (12 mg) was added, filtered, the filter residue was washed with a small amount of methanol, the filtrate was evaporated to dryness, a small amount of ethyl acetate was added, and the mixture was slurried and filtered to obtain 85 mg of compound A4 as a pale yellow solid, with a yield of 44%. 1 H NMR(500MHz,DMSO-d 6 )δ11.91(d,J=4.1Hz,1H),7.98(d,J=4.1Hz,1H),6.91(d,J=4.4Hz,1H),6.81(d,J=4.4Hz,1H),6.14(brs,1H),5.22(brs,1H),4.55(d,J=5.1Hz,1H),4.06–4.03(m,1H),3.95(t,J=5.4Hz,1H),3.62(dd,J=12.2,3.4Hz,1H),3.49(dd,J=12.2,4.6Hz,1H). MS m / z=293.0[M+1] + ,m / z=291.0[M-1] - 。

[0306] Preparation Example 5: Synthesis of Compound A9

[0307]

[0308] Compound 1-1 (561 mg, 1.0 mmol) was dissolved in N,N-dimethylformamide (5 mL), and iodine (508 mg, 2 mmol, 2 eq) was added in portions. The reaction was carried out overnight at room temperature, and TLC showed that there was remaining raw material. The reaction solution was added to a mixed solution of sodium sulfite and sodium carbonate, extracted with ethyl acetate, the organic phase was separated, and the organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain 400 mg of compound 9-1 as a white solid, with a yield of 58%. 1 H NMR(500MHz,DMSO-d 6)δ 7.98 (s, 1H), 7.40–7.22 (m, 15H), 6.86 (s, 1H), 4.91 (d, J=11.7 Hz, 1H), 4.84–4.78 (m, 2H), 4.50 (q, J=12.0 Hz, 4H), 4.41–4.35 (m, 1H), 4.12–4.08 (m, 1H), 3.72 (dd, J=11.2, 2.8 Hz, 1H), 3.59 (dd, J=11.2, 4.1 Hz, 1H).

[0309] Compound 9-1 (69 mg, 0.1 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). Under an ice bath, trimethylchlorosilane (24 mg, 0.22 mmol, 2.2 eq) was added. After stirring for 10 minutes, the temperature was lowered to -10 °C, and then a 2-methyltetrahydrofuran solution of 3.0 M methylmagnesium bromide (74 μL, 0.22 mmol, 2.2 eq) was added dropwise. After addition, the mixture was stirred for 30 minutes. The temperature was lowered to -20 °C, and a 1.3 M solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (0.1 mL, 0.13 mmol, 1.3 eq) was added, and stirring was continued for 1 hour. Heavy water (0.2 mL) was added to the reaction system. After stirring for 15 minutes, the reaction solution was added to saturated ammonium chloride solution, and extracted with ethyl acetate. The organic layer was separated, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain compound 9-2, 28 mg of white solid, yield 50%. 1 H NMR (500 MHz, Methanol-d 4 )δ 7.65 (s, 1H), 7.34–7.16 (m, 15H), 6.84 (s, 1H), 4.95 (d, J=5.1 Hz, 1H), 4.74 (q, J=11.9 Hz, 2H), 4.57 (d, J=11.9 Hz, 1H), 4.53–4.43 (m, 4H), 4.11 (t, J=5.2 Hz, 1H), 3.73 (dd, J=10.9, 4.1 Hz, 1H), 3.62 (dd, J=10.9, 4.5 Hz, 1H).

[0310] Compound 9-2 (90 mg, 0.16 mmol) was dissolved in dichloromethane (10 mL). At -60 °C, a 1.0 M solution of boron trichloride in dichloromethane (0.56 mL, 0.56 mmol) was added dropwise. After addition, the mixture was stirred at -40 °C for 1 hour. TLC showed that the reaction was complete. Methanol (0.1 mL) was added to the reaction solution, and then triethylamine was added until the pH of the reaction solution was 7-8. The reaction solution was concentrated and separated by silica gel column chromatography to obtain compound A9, 15 mg of off-white solid, yield 50%. 1 H NMR (500 MHz, DMSO-d 6)δ8.03–7.78(m,3H),6.87(s,1H),6.09(d,J=6.3Hz,1H),5.19(d,J=5.2Hz,1H),4.91(t,J=5.7Hz,1H),4.64(t,J=5.7Hz,1H),4.09–4.02(m,1H),3.99–3.92(m,1H),3.68–3.59(m,1H),3.55–3.47(m,1H). MS m / z=293.0[M+1] + 。

[0311] Preparation Example 6: Synthesis of Compound A9

[0312]

[0313] Compound 9-1 (69 mg, 0.1 mmol) was added to a mixed solution of dry tetrahydrofuran (5 mL) and heavy water (1 mL). After the solution was concentrated to dryness, dry tetrahydrofuran (5 mL) and heavy water (1 mL) were added again. Then, [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (8 mg, 0.01 mmol) and tetramethylethylenediamine (3 mg, 0.02 mmol) were added successively. After addition, the mixture was stirred for 10 minutes, and then sodium borodeuteride (21 mg, 0.5 mmol) was added in portions. After 2 hours, TLC showed that the raw material had completely reacted. The reaction solution was added to water, extracted with ethyl acetate, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain Compound 9-2 with a deuteration rate of not less than 97%, 30 mg of white solid, and a yield of 53%.

[0314] Referring to the synthesis method in Reference Example 5, Compound 9-2 (90 mg, 0.16 mmol) was deprotected to obtain Compound A9, 15 mg of off-white solid, and a yield of 50%. 1 H NMR(500MHz,DMSO-d 6 )δ8.01–7.79(m,3H),6.88(s,1H),6.09(d,J=6.4Hz,1H),5.19(d,J=5.3Hz,1H),4.91(t,J=5.8Hz,1H),4.65(t,J=5.7Hz,1H),4.08–4.04(m,1H),3.98–3.93(m,1H),3.67–3.61(m,1H),3.54–3.48(m,1H). 13 C NMR(126MHz,DMSO-d 6)δ 156.08, 148.32, 124.35, 117.82, 116.94, 111.16, 85.92, 79.04, 74.72, 70.56, 61.43. The deuteration rate is not less than 97%, MS m / z = 293.0 [M+1] + 。

[0315] Preparation Example 7: Synthesis of Compound A10

[0316]

[0317] Compound 2-1 (0.5 g, 1.72 mmol) was added to acetone (10 mL), and 2,2-dimethoxypropane (0.89 g, 8.6 mmol, 5 eq) and p-toluenesulfonic acid monohydrate (0.59 g, 3.1 mmol, 1.8 eq) were added successively. After addition, the mixture was stirred at 45 °C for 2 hours, and the reaction was monitored by TLC to be complete. The reaction solution was added to saturated sodium bicarbonate aqueous solution (20 mL), extracted with ethyl acetate, the organic layer was separated, dried, and the solvent was evaporated to obtain Compound 10-1, 0.33 g of white solid, with a yield of 58%.

[0318] Compound 10-1 (0.25 g, 0.75 mmol) was added to acetonitrile / water (V / V = 1 / 1, 15 mL), and TEMPO (0.047 g, 0.3 mmol, 0.4 eq), iodobenzene diacetate (1.06 g, 3.3 mmol, 4.4 eq), and sodium bicarbonate (0.25 g, 3 mmol, 4.0 eq) were added successively. After addition, the mixture was stirred at room temperature for about 5 hours, and TLC showed the reaction was complete. The reaction solution was added to 0.5 M sodium hydroxide solution (20 mL), extracted twice with ethyl acetate (20 mL × 2), the organic phase was discarded, the aqueous layer was adjusted to pH 4-5 with dilute hydrochloric acid (2 M), extracted with ethyl acetate (50 mL × 2), the organic layers were combined, dried, and concentrated to obtain Compound 10-2, 0.23 g of white solid, with a yield of 89%.

[0319] The product 10-2 (0.18 g, 0.52 mmol) obtained in the previous step was added to tetrahydrofuran and methanol (8 mL, 1:1). At room temperature, 2 M trimethylsilyldiazomethane n-hexane solution (1.2 mL, 2.4 mmol, 4.5 eq) was added dropwise. After addition, the reaction was carried out at room temperature, and the reaction was complete after 1 hour. Acetic acid was added dropwise to the reaction solution until no more bubbles were produced, the solvent was evaporated, and separation was carried out on a preparative plate to obtain Compound 10-3, 0.11 g of white solid, with a yield of 59%. 1 H NMR (500 MHz, DMSO-d 6)δ8.06–7.88(m,3H),6.92(d,J=4.6Hz,1H),6.89(d,J=4.6Hz,1H),5.52(d,J=6.1Hz,1H),5.37(dd,J=6.1,2.2Hz,1H),4.92(d,J=2.2Hz,1H),3.40(s,3H),1.62(s,3H),1.42(s,3H).

[0320] Compound 10-3 (0.10 g, 0.28 mmol) was added to anhydrous tetrahydrofuran (4 mL) and deuterated methanol (1 mL). Under ice bath conditions, sodium borodeuteride (0.047 g, 1.12 mmol, 4 eq) was added. After addition, the reaction was carried out at room temperature. After 1 hour, TLC showed that the reaction was complete. Dilute hydrochloric acid was added dropwise to the reaction solution until no more bubbles were generated. The reaction solution was concentrated and separated by preparative thin layer chromatography to obtain compound 10-4, 0.06 g of white solid, with a yield of 64%.

[0321] The product 10-4 (60 mg, 0.18 mmol) from the previous step was added to tetrahydrofuran (2 mL), and concentrated hydrochloric acid (0.4 mL) was added dropwise. The reaction was carried out at 40 °C until the reaction was complete. Sodium bicarbonate solution was added to the reaction solution until the pH was neutral. The mixture was extracted with tetrahydrofuran, and the organic phase was separated. After drying, the organic phase was evaporated to dryness to obtain an off-white solid. The obtained solid was added to ethyl acetate and stirred for 0.5 hour, then filtered and dried to obtain compound A10, 34 mg of white solid, with a yield of 64%. 1 H NMR(500MHz,DMSO-d 6 )δ8.03–7.82(m,3H),6.92(d,J=4.5Hz,1H),6.89(d,J=4.6Hz,1H),6.10(d,J=6.3Hz,1H),5.20(d,J=5.2Hz,1H),4.89(s,1H),4.66(t,J=5.7Hz,1H),4.06(d,J=5.4Hz,1H),4.00–3.93(m,1H). 13 CNMR(126MHz,DMSO-d 6 )δ156.10,148.35,124.35,117.81,117.01,111.26,101.27,85.80,79.04,74.70,70.54. The deuteration rate was 99%, MS m / z=294.0[M+1] + .

[0322] Preparation Example 8: Synthesis of Compound A11

[0323]

[0324] Compound A9 (1.17 g, 4.0 mmol, deuterium incorporation rate not less than 97%) was added to acetone (20 mL). Then, 2,2-dimethoxypropane (2.08 g, 20.0 mmol, 5 eq) and p-toluenesulfonic acid monohydrate (1.37 g, 7.2 mmol, 1.8 eq) were added successively. After addition, the mixture was stirred at 45 °C for 2 hours, and a large amount of solid appeared. The reaction was monitored by TLC and was found to be complete. The reaction solution was added to saturated aqueous sodium bicarbonate, and the mixture was extracted with ethyl acetate. The organic layer was separated, dried, and the solvent was evaporated to obtain Compound 11-1 as a white solid (0.86 g, yield 65%).

[0325] Compound 11-1 (0.225 g, 0.68 mmol) was added to acetonitrile / water (V / V = 1 / 1, 10 mL). Then, TEMPO (0.02 g, 0.14 mmol, 0.2 eq), iodobenzene diacetate (0.66 g, 2.04 mmol, 3.0 eq), and sodium bicarbonate (0.17 g, 2.04 mmol, 3.0 eq) were added successively. After addition, the mixture was stirred at room temperature for 4 - 5 hours. The reaction was monitored by TLC and was found to be complete. The reaction solution was added to an aqueous KOH solution (0.5 M, 20 mL), and the aqueous layer was extracted with ethyl acetate (20 mL × 3). The aqueous layer was separated. The pH of the aqueous layer was adjusted to 4 - 5 with dilute hydrochloric acid (2 M), and the mixture was extracted with ethyl acetate (20 mL × 3). The organic layers were combined, dried, and concentrated to obtain the crude product of 11-2, which was directly used in the next step of the reaction.

[0326] The crude product of Compound 11-2 (0.68 mmol, calculated as 100% yield) was added to tetrahydrofuran / methanol (V / V = 1 / 1, 8 mL). A hexane solution of trimethylsilyldiazomethane (2 M, 1.36 mL, 4.0 eq) was added dropwise at room temperature. After addition, the reaction was carried out at room temperature and was complete after 30 minutes. Acetic acid was added dropwise to the reaction solution until no more bubbles were generated. The reaction solution was evaporated to dryness, ethyl acetate (20 mL) was added, and the mixture was washed with saturated aqueous sodium bicarbonate (10 mL × 2). The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Compound 11-3 as a white solid (0.06 g, overall yield of two steps 24%).

[0327] Compound 11-3 (0.06 g, 0.17 mmol) was added to deuterated methanol / tetrahydrofuran (V / V = 1 / 5, 6 mL). NaBD was added under ice-bath conditions 4(0.028 g, 0.68 mmol, 4 eq). After addition, the reaction was carried out at room temperature and was complete after 1 - 2 hours. The reaction solution was added to a mixture of ethyl acetate / water (20 mL / 20 mL), and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (20 mL) once more. The ethyl acetate layers were combined and evaporated to dryness. The obtained product was added to methanol (10 mL), and dilute hydrochloric acid (1 M) was added dropwise to adjust the pH of the solution to 2 - 3. After stirring at room temperature for 1 - 2 hours, the pH was adjusted to neutral with saturated aqueous sodium bicarbonate solution. A mixture of ethyl acetate / water (20 mL / 20 mL) was added, and the organic phase was separated. The aqueous layer was extracted with ethyl acetate (20 mL) once more. The organic layers were combined, dried, concentrated, and separated by silica gel preparative plate to obtain compound 11 - 4, 0.05 g of white solid, with a yield of 89%.

[0328] 11 - 4 (0.05 g, 0.15 mmol) was added to tetrahydrofuran (1.5 mL), and concentrated hydrochloric acid (0.3 mL) was added dropwise under an ice bath. After addition, the reaction was stirred at room temperature for 2 - 3 hours, and the reaction was monitored by TLC to be complete. Under an ice bath, the pH of the reaction solution was adjusted to neutral with 1 M aqueous NaOH solution. After evaporating tetrahydrofuran, filtration was carried out. The filter cake was slurried successively with distilled water (10 mL) and ethyl acetate (10 mL), filtered, and dried to obtain compound A11, 0.03 g of white solid, with a yield of 68%. 1 H NMR (500 MHz, DMSO - d 6 ) δ 8.04–7.76 (m, 3H), 6.89 (s, 1H), 6.07 (d, J = 6.3 Hz, 1H), 5.18 (d, J = 5.2 Hz, 1H), 4.87 (s, 1H), 4.67 (t, J = 5.8 Hz, 1H), 4.07 (d, J = 5.3 Hz, 1H), 4.01–3.93 (m, 1H). m / z = 295.0 [M + 1] + . The deuteration rates at the two sites were not less than 97% respectively.

[0329] Preparation Example 9: Synthesis of Compound A12

[0330]

[0331] Compound 10-1 (662 mg, 2.0 mmol) was added to acetonitrile (15 mL), and sodium bicarbonate (336 mg, 4 mmol, 2 eq) and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) (850 mg, 2.4 mmol, 1.2 eq) were added. The mixture was stirred at room temperature for 24 h, and TLC showed that some starting materials remained. Ethyl acetate (50 mL) and water (20 mL) were added to the reaction mixture. The organic phase was separated, dried, concentrated, and purified by silica gel column chromatography to obtain compound 12-1, 140 mg of off-white solid, with a yield of 20%.

[0332] Compound 12-1 (140 mg, 0.4 mmol) was added to a mixed solution of acetonitrile and water (6 mL, 1:1). At room temperature, iodobenzene diacetate (515 mg, 1.6 mmol, 4 eq), sodium bicarbonate (135 mg, 1.6 mmol, 4 eq), and TEMPO (25 mg, 0.16 mmol, 0.4 eq) were added successively. After about 3 h, the reaction was complete. An aqueous sodium hydroxide solution (10 mL, 224 mg, 10 eq) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phase was discarded. 2M dilute hydrochloric acid was added to the aqueous phase to adjust the pH to 4-5, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and the solvent was evaporated to obtain compound 12-2, 116 mg of off-white solid, with a yield of 80%.

[0333] Compound 12-2 (116 mg, 0.32 mmol) was added to a mixed solution of tetrahydrofuran and methanol (4 mL, 1:1). A 2M hexane solution of trimethylsilyldiazomethane (0.48 mL, 0.96 mmol, 3 eq) was added slowly. Monitored by TLC, if starting materials remained, an appropriate amount of trimethylsilyldiazomethane could be added until the reaction was complete. Acetic acid was added dropwise to the reaction mixture until no more bubbles were produced. The solvent was evaporated, and the residue was purified by silica gel column chromatography to obtain compound 12-3, 105 mg of white solid, with a yield of 87%.

[0334] The product 12-3 (105 mg, 0.28 mmol) from the previous step was added to anhydrous tetrahydrofuran (2 mL), and deuterated methanol (0.5 mL) was added. At room temperature, sodium borodeuteride (35 mg, 0.84 mmol, 3 eq) was added in portions within 1 h, and stirring was continued for 5 h. TLC showed that the starting materials had reacted completely. Ethyl acetate (0.5 mL) was added to the reaction mixture, and the reaction mixture was concentrated. The residue was separated by preparative TLC to obtain compound 12-4, 80 mg of white foamy solid, with a yield of 82%.

[0335] The product 12-4 (80 mg, 0.23 mmol) from the previous step was added to tetrahydrofuran (3 mL), and concentrated hydrochloric acid (0.5 mL) was added dropwise. The reaction was carried out at 40 °C until the reaction was complete. An insoluble substance precipitated in the reaction solution, which was filtered to obtain 55 mg of a white solid. The solid was added to water (1.5 mL), sodium carbonate (16 mg) was added, and the mixture was stirred for 0.5 h, filtered, and dried to obtain 44 mg of compound A12 as a white solid, with a yield of 62%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.10 (brs, 1H), 7.88 (s, 1H), 7.41 (brs, 1H), 6.80 (s, 1H), 6.23 (brs, 1H), 5.20 (brs, 1H), 4.89 (s, 1H), 4.54 (d, J = 4.9 Hz, 1H), 4.04 (d, J = 6.3 Hz, 1H), 3.94 (t, J = 5.5 Hz, 1H). The deuteration rate was 98%, MS m / z = 312.0 [M+1] + 。

[0336] Preparation Example 10: Synthesis of Compound A35

[0337]

[0338] Compound 35-1 was synthesized according to the method reported in the literature (WO2015069939). Hydroxylamine hydrochloride (547 mg, 7.87 mmol, 30 eq) was added to water (3.2 mL), and 10% sodium hydroxide solution was added dropwise slowly to adjust the pH value of the system to 6.0. Then compound 35-1 (69 mg, 0.26 mmol, 1 eq) was added, and the mixture was stirred overnight at 40 °C under nitrogen protection. TLC showed that the reaction was complete. The reaction solution was separated by a reverse-phase column to obtain 52 mg of off-white solid A35, with a yield of 71%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 10.61 (d, J = 4.0 Hz, 1H), 10.06 (s, 1H), 7.39 (d, J = 4.0 Hz, 1H), 6.38–6.33 (m, 2H), 4.96 (d, J = 6.6 Hz, 1H), 4.93 (d, J = 6.3 Hz, 1H), 4.86 (d, J = 5.1 Hz, 1H), 4.74–4.68 (m, 1H), 4.15 (q, J = 6.1 Hz, 1H), 3.91 (q, J = 4.9 Hz, 1H), 3.74 (q, J = 4.5 Hz, 1H), 3.55–3.49 (m, 1H), 3.47–3.43 (m, 1H). MS m / z = 283.0 [M+1] + 。

[0339] Preparation Example 11: Synthesis of Compounds A49 and A124

[0340]

[0341] Compound 2-1 (2.2 g, 7.6 mmol) was added to N,N-dimethylformamide (20 mL), imidazole (3.1 g, 45.6 mmol, 6 eq) was added, and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (2.9 g, 9.5 mmol, 1.2 eq) was added dropwise under an ice bath. After the addition was complete, the reaction was carried out at room temperature and the reaction was complete after 4 hours. The reaction solution was added to water (120 mL), extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and separated by silica gel column chromatography to obtain compound 49-1, 3.2 g of white solid, with a yield of 80%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.99–7.82 (m, 3H), 6.88 (d, J = 4.5 Hz, 1H), 6.79 (d, J = 4.4 Hz, 1H), 6.45 (d, J = 5.7 Hz, 1H), 4.56 (t, J = 5.0 Hz, 1H), 4.22–4.09 (m, 3H), 3.95–3.86 (m, 1H), 1.09–0.76 (m, 28H).

[0342] Compound 49-1 (1.2 g, 2.2 mmol) was added to toluene (20 mL), N,N-dimethylformamide dimethyl acetal (0.52 g, 4.4 mmol, 2 eq) was added, and the reaction was carried out at 50 °C. The reaction was complete after about 3 h. The solvent was removed under reduced pressure, ethyl acetate (60 mL) and water (20 mL) were added, the organic phase was separated, washed with saturated sodium chloride, dried, and evaporated to dryness to obtain compound 49-2, 1.2 g of white solid, with a yield of 92%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.96 (s, 1H), 8.16 (s, 1H), 6.91 (d, J = 4.5 Hz, 1H), 6.81 (d, J = 4.4 Hz, 1H), 6.49 (d, J = 5.8 Hz, 1H), 4.59 (t, J = 5.1 Hz, 1H), 4.26–4.11 (m, 3H), 3.93 (dd, J = 13.3, 2.4 Hz, 1H), 3.26 (s, 3H), 3.20 (s, 3H), 1.09–0.87 (m, 28H).

[0343]

[0344] Compound 49-2 (294 mg, 0.5 mmol) was added to dichloromethane (6 mL). At room temperature, triethylamine (101 mg, 1 mmol, 2 eq), DMAP (12 mg, 0.1 mmol, 0.2 eq), and isobutyryl chloride (85 mg, 0.8 mmol, 1.6 eq) were added successively. The reaction was carried out overnight, and TLC showed that the reaction was complete. Saturated aqueous sodium bicarbonate solution and dichloromethane were added to the reaction solution. The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain compound 49-3, 240 mg of white foamy solid, with a yield of 72%.

[0345] The product 49-3 (240 mg, 0.36 mmol) obtained in the previous step was added to tetrahydrofuran (5 mL). A tetrahydrofuran solution of 1 M tetrabutylammonium fluoride (0.72 mL, 0.72 mmol) was added. After 1 hour, the reaction was complete. Water (10 mL) was added to the reaction solution, and it was extracted with ethyl acetate. The organic layer was separated, dried, and evaporated to dryness to obtain an oily substance. This oily substance was added to tetrahydrofuran (6 mL), water (0.5 mL), and trifluoroacetic acid (205 mg, 1.8 mmol) were added, and it was stirred at room temperature overnight. Aqueous sodium bicarbonate solution was added to the reaction solution, and it was extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and separated by silica gel column chromatography to obtain compounds A49 and A124, with the proportion of A124 being greater than 95%. A124 was recrystallized from isopropyl acetate to obtain 80 mg of white solid, with a two-step yield of 61%. The obtained compound A124's 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.09–7.84 (m, 3H), 6.94 (d, J = 4.6 Hz, 1H), 6.90 (d, J = 4.6 Hz, 1H), 6.43 (d, J = 6.5 Hz, 1H), 5.22 (dd, J = 5.7, 3.3 Hz, 1H), 5.07 (t, J = 5.8 Hz, 1H), 5.01 (t, J = 6.1 Hz, 1H), 4.31–4.25 (m, 1H), 3.66–3.52 (m, 2H), 2.68–2.58 (m, 1H), 1.19 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H). MS m / z = 362.0 [M+1] + 。

[0346] Preparation Example 12: Synthesis of Compound A50

[0347]

[0348] Compound 10-1 (0.664 g, 2.0 mmol) was added to toluene (10 mL), and N,N-dimethylformamide dimethyl acetal (0.47 g, 4.0 mmol, 2.0 eq) was added. After addition, the reaction was carried out at 60 °C for 2 - 3 hours, and the reaction was monitored by TLC until completion. The solvent was evaporated to obtain Compound 50-1, 0.65 g of yellow solid, with a yield of 84%.

[0349] The product 50-1 (0.2 g, 0.52 mmol) from the previous step was added to dichloromethane (10 mL), and triethylamine (0.1 g, 1.0 mmol, 2 eq), DMAP (0.012 g, 0.10 mmol, 0.2 eq), and isobutyryl chloride (0.083 g, 0.78 mmol, 1.5 eq) were added in sequence. After addition, the mixture was stirred at room temperature for 1 - 2 hours, and the reaction was monitored by TLC until completion. The reaction solution was added to 1 M dilute hydrochloric acid (10 mL), and extracted with dichloromethane. The organic layer was separated, washed with saturated sodium bicarbonate (20 mL), dried and concentrated to obtain Compound 50-2, 0.2 g of off-white solid, with a yield of 84%.

[0350] Compound 50-2 (0.1 g, 0.22 mmol) was added to tetrahydrofuran (3 mL), and concentrated hydrochloric acid (0.6 mL) was added dropwise under an ice bath. After addition, the reaction was stirred at room temperature for 3 hours, and TLC showed a small amount of raw material remaining. The reaction solution was added to saturated aqueous sodium bicarbonate (20 mL), extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and purified by preparative thin-layer chromatography to obtain Compound A50, 13 mg of white solid, with a yield of 16%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.00–7.80 (m, 3H), 6.92 (d, J = 4.6 Hz, 1H), 6.82 (d, J = 4.5 Hz, 1H), 6.33 (d, J = 6.0 Hz, 1H), 5.39 (d, J = 5.8 Hz, 1H), 4.70 (t, J = 5.5 Hz, 1H), 4.32 (dd, J = 12.0, 2.9 Hz, 1H), 4.27–4.21 (m, 1H), 4.18 (dd, J = 12.0, 5.3 Hz, 1H), 3.99–3.94 (m, 1H), 2.57–2.52 (m, 1H), 1.07 (d, J = 2.4 Hz, 3H), 1.06 (d, J = 2.4 Hz, 3H). MS m / z = 362.0 [M+1] + 。

[0351] Preparation Example 13: Synthesis of Compound A51 and A212

[0352]

[0353] Compound 49-2 (260 mg, 0.44 mmol) and Boc-L-valine (115 mg, 0.53 mmol, 1.2 eq) were added to dichloromethane (10 mL). Subsequently, HOBT (89 mg, 0.66 mmol, 1.5 eq), EDCI (169 mg, 0.88 mmol, 2 eq) and DMAP (214 mg, 1.76 mmol, 4 eq) were added successively, and the mixture was stirred at room temperature overnight. Dichloromethane (20 mL) and water (20 mL) were added to the reaction mixture, and it was stirred for 5 minutes. The organic phase was separated, washed with saturated sodium chloride, dried, concentrated, and separated by silica gel column chromatography to obtain compound 51-1, 260 mg of white solid, with a yield of 75%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.98 (s, 1H), 8.15 (s, 1H), 7.23 (d, J = 9.0 Hz, 1H), 6.96 (d, J = 4.5 Hz, 1H), 6.85 (d, J = 4.6 Hz, 1H), 5.90 (d, J = 5.0 Hz, 1H), 4.57 (dd, J = 9.2, 4.9 Hz, 1H), 4.27–4.17 (m, 3H), 3.95 (dd, J = 13.8, 2.8 Hz, 1H), 3.27 (s, 3H), 3.21 (s, 3H), 2.30–2.19 (m, 1H), 1.45–1.34 (m, 9H), 1.10–0.86 (m, 34H).

[0354] Compound 51-1 (180 mg, 0.23 mmol) was added to tetrahydrofuran (4 mL). At room temperature, a tetrahydrofuran solution of 1 M tetrabutylammonium fluoride (0.46 mL, 0.46 mmol) was added. After 1 hour, the reaction was complete. Water (10 mL) was added to the reaction mixture, and it was extracted with ethyl acetate. The organic phase was separated, dried and evaporated to dryness to obtain an oil. The oil was dissolved in tetrahydrofuran (6 mL), water (0.5 mL) and trifluoroacetic acid (131 mg, 1.15 mmol, 5 eq) were added, and the mixture was stirred at room temperature overnight. An aqueous sodium bicarbonate solution was added to the reaction mixture, and it was extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and separated by silica gel column chromatography to obtain compounds 51-2a and 51-2b, with the proportion of 51-2b being greater than 90%. Further recrystallization gave pure 51-2b, 70 mg of white solid, with a two-step yield of 62%. The obtained compound 51-2b's 1 H NMR (500 MHz, DMSO-d 6)δ8.08–7.87(m,3H),7.06(d,J=8.6Hz,1H),6.94(d,J=4.5Hz,1H),6.91(d,J=4.6Hz,1H),6.50(d,J=6.7Hz,1H),5.19–5.14(m,1H),5.10–5.05(m,1H),5.03–4.98(m,1H),4.26–4.20(m,1H),4.10(dd,J=8.7,5.5Hz,1H),3.66–3.53(m,2H),2.31–2.22(m,1H),1.45–1.35(m,9H),0.93(d,J=6.8Hz,3H),0.90(d,J=6.8Hz,3H).

[0355] Compound 51-2b (42 mg, 0.086 mmol) was added to a saturated hydrogen chloride / methanol solution (4 mL), stirred at 35 °C, and TLC showed complete reaction after 2 hours. The reaction solution was concentrated, methyl tert-butyl ether was added, stirred and filtered to obtain Compound A212, which was the dihydrochloride salt, 45 mg of white solid, yield 81%. 1 H NMR(500MHz,Methanol-d 4 )δ8.17(s,1H),7.50(d,J=4.8Hz,1H),7.21(d,J=4.8Hz,1H),5.52(dd,J=5.8,3.2Hz,1H),5.19(d,J=5.8Hz,1H),4.54–4.48(m,1H),4.16–4.10(m,1H),3.83(d,J=3.7Hz,2H),2.58–2.48(m,1H),1.18(d,J=4.2Hz,3H),1.17(d,J=4.2Hz,3H). MS m / z=391.1[M+1] + . If the mixture 51-2a and 51-2b obtained in the previous step (51-2a accounted for 5-10%) was directly deprotected, a mixture of A51 and A212 could be obtained, and the content of A51 was about 10%.

[0356] Preparation Example 14: Synthesis of Compound A52

[0357]

[0358] Compound 2-1 (291 mg, 1.0 mmol) was added to pyridine (8 mL). Pyridine was removed by evaporation under reduced pressure, and the operation was repeated once. Then pyridine (8 mL) was added again. At room temperature, N,N-dimethylformamide dimethyl acetal (477 mg, 4.0 mmol) was added. Under nitrogen protection, the reaction was carried out overnight at room temperature. The reaction solution was concentrated to obtain compound 52-1, an oily substance, which was directly used in the next step without separation.

[0359] Boc-L-valine (304 mg, 1.4 mmol), 1-hydroxybenzotriazole (203 mg, 1.5 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (422 mg, 2.2 mmol) were added to dichloromethane (10 mL). After stirring at room temperature for 15 minutes, a dichloromethane solution (1 mL) of 52-1 obtained in the previous step and 4-dimethylaminopyridine (684 mg, 5.4 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, methanol was added, and then concentrated again to obtain an oily substance. Water was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product of compound 52-2, an oily substance, which was directly used in the next step.

[0360] Compound 52-2 was dissolved in acetonitrile (10 mL), 85% hydrazine hydrate (236 mg, 4.0 mmol) was added, and the reaction was carried out at room temperature for 3 hours. Then the reaction solution was added to water, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain compound 52-3, 104 mg of white solid, and the total yield of the three steps was 21%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.98–7.83 (m, 3H), 7.13 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 4.5 Hz, 1H), 6.82 (d, J = 4.5 Hz, 1H), 6.33 (d, J = 6.0 Hz, 1H), 5.38 (d, J = 5.9 Hz, 1H), 4.69 (t, J = 5.4 Hz, 1H), 4.33–4.28 (m, 1H), 4.27–4.20 (m, 2H), 3.94–3.90 (m, 1H), 3.89–3.85 (m, 1H), 1.98–1.91 (m, 1H), 1.37 (s, 9H), 0.82 (t, J = 6.5 Hz, 6H).

[0361] Compound 52-3 (104 mg, 0.21 mmol) was added to a methanol solution saturated with hydrogen chloride (5 mL), and the mixture was stirred at 36 °C. After 1 hour, the reaction was complete. The reaction solution was concentrated, then isopropyl ether was added, and a solid precipitated. The solid was filtered to obtain Compound A52 as the dihydrochloride, 80 mg of white solid, with a yield of 82%. 1 H NMR (500 MHz, Methanol-d 4 ) δ 8.15 (s, 1H), 7.49 (d, J = 4.8 Hz, 1H), 7.12 (d, J = 4.8 Hz, 1H), 4.74 (d, J = 5.2 Hz, 1H), 4.62 (dd, J = 12.1, 7.4 Hz, 1H), 4.54 (dd, J = 12.1, 2.8 Hz, 1H), 4.45 (td, J = 7.5, 2.7 Hz, 1H), 4.05–4.00 (m, 2H), 2.35–2.27 (m, 1H), 1.08 (d, J = 1.9 Hz, 3H), 1.07 (d, J = 1.8 Hz, 3H). MS m / z = 391.1 [M+1] + 。

[0362] Preparation Example 15: Synthesis of Compound A53

[0363]

[0364] Compound 49-1 (150 mg, 0.28 mmol) was dissolved in dichloromethane (2 mL), and pyridine (265 mg, 3.35 mmol, 12 eq) was added. Under an ice bath, trimethylchlorosilane (93 mg, 0.86 mmol) was added, and after stirring for 30 minutes, TLC showed that the raw material was completely converted. n-Amyl chloroformate (120 mg, 0.8 mmol) was added to the reaction solution, and the ice bath was maintained while stirring continued for 2 hours. TLC detected that the reaction was complete. Dichloromethane and water were added to the reaction solution, and the organic layer was separated. The organic layer was washed successively with dilute hydrochloric acid and brine, dried and concentrated to obtain the crude product of 53-1, 200 mg of colorless oil. This oil was added to tetrahydrofuran (2 mL), and a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.2 mL, 0.2 mmol) was added dropwise at room temperature. After addition, the mixture was stirred at room temperature for 50 minutes, and TLC showed that the reaction was complete. The reaction solution was concentrated and purified by silica gel column chromatography to obtain Compound A53, 55 mg of white solid, with a two-step yield of 48%. 1 H NMR (600 MHz, DMSO-d 6)δ10.88(s,1H),8.37(s,1H),7.30(d,J=4.8Hz,1H),7.11(d,J=4.8Hz,1H),6.21(d,J=6.2Hz,1H),5.23(d,J=5.5Hz,1H),4.90(t,J=5.6Hz,1H),4.61(t,J=5.7Hz,1H),4.18(t,J=6.7Hz,2H),4.10–4.04(m,1H),3.95(q,J=5.5Hz,1H),3.68–3.61(m,1H),3.54–3.47(m,1H),1.71–1.61(p,J=6.8Hz,2H),1.39–1.29(m,4H),0.89(t,J=7.0Hz,3H). MS m / z=406.0[M+1] + 。

[0365] Preparation Example 16: Synthesis of Compounds A69 and A144

[0366]

[0367] Compound A9 (879 mg, 3.01 mmol, deuterium incorporation rate not less than 97%) was added to N,N-dimethylformamide (15 mL), imidazole (819 mg, 12.03 mmol, 4 eq) was added, and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.32 g, 4.21 mmol, 1.4 eq) was added dropwise under an ice bath. After the addition was complete, the reaction was carried out at room temperature and the reaction was complete after 1 hour. The reaction solution was added to water, extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and slurried with petroleum ether to obtain Compound 69-1, 1.29 g of white solid, with a yield of 80%. 1 H NMR(600MHz,DMSO-d 6 )δ8.04–7.78(m,3H),6.79(s,1H),6.46(d,J=5.7Hz,1H),4.56(t,J=5.1Hz,1H),4.24–4.08(m,3H),3.91(d,J=12.0Hz,1H),1.08–0.86(m,28H).

[0368] Compound 69-1 (1.10 g, 2.07 mmol) was added to toluene (20 mL), N,N-dimethylformamide dimethyl acetal (370 mg, 3.11 mmol, 1.5 eq) was added, and the reaction was carried out at 45 °C and the reaction was complete in about 30 minutes. The solvent was removed under reduced pressure, ethyl acetate (60 mL) and water (20 mL) were added, the organic phase was separated, washed with saturated sodium chloride, dried, and evaporated to dryness to obtain Compound 69-2, 1.12 g of white solid, with a yield of 92%.

[0369] Compound 69-2 (614 mg, 1.04 mmol) was added to dichloromethane (10 mL). At room temperature, triethylamine (210 mg, 2.08 mmol, 2 eq), isobutyryl chloride (166 mg, 1.56 mmol, 1.5 eq) and DMAP (127 mg, 1.04 mmol, 1 eq) were added successively. The mixture was stirred at room temperature and the reaction was completed in about 1 hour. Saturated aqueous sodium bicarbonate and dichloromethane were added to the reaction solution. The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain compound 69-3, 494 mg of white foamy solid, with a yield of 72%.

[0370] Compound 69-3 (350 mg, 0.53 mmol) was added to acetonitrile (8 mL). 85% hydrazine hydrate (125 mg, 2.12 mmol, 4 eq) was added. The mixture was stirred at room temperature and the reaction was completed in about 30 minutes. The reaction solution was added to water and extracted with ethyl acetate. The organic layer was separated. The organic phase was washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride respectively, dried, and evaporated to dryness to obtain compound 69-4, 289 mg of white solid, with a yield of 90%.

[0371] Compound 69-4 (289 mg, 0.48 mmol) was added to tetrahydrofuran (10 mL). A tetrahydrofuran solution of 1 M tetrabutylammonium fluoride (0.48 mL, 0.48 mmol, 1 eq) was added. The mixture was stirred at room temperature and the reaction was completed in about 30 minutes. The reaction solution was added to water and extracted with isopropyl acetate. The organic layer was separated. The organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride respectively, dried, and evaporated to dryness to obtain compounds A69 and A144, with the proportion of A144 being greater than 95%. It was slurried with a mixed solution of n-heptane / isopropanol (1:1), filtered and dried to obtain pure compound A144, 121 mg of white solid, with a yield of 70%. The obtained compound A144 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.06–7.85 (m, 3H), 6.88 (s, 1H), 6.41 (d, J = 6.5 Hz, 1H), 5.21 (dd, J = 5.7, 3.3 Hz, 1H), 5.08–5.04 (m, 1H), 4.99 (t, J = 6.0 Hz, 1H), 4.26 (q, J = 3.7 Hz, 1H), 3.64–3.52 (m, 2H), 2.67–2.58 (m, 1H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6)δ175.92,156.13,148.45,123.24,117.50,117.27,111.60,84.59,78.30,73.14,72.69,61.25,33.81,19.18,19.08. MS m / z=363.0[M+1] + .

[0372] Preparation Example 17: Synthesis of Compound A70

[0373]

[0374] Compound A9 (145 mg, 0.5 mmol, deuterium incorporation rate not less than 97%) was added to pyridine (5 mL). Pyridine was removed by evaporation under reduced pressure, and the operation was repeated once. Then pyridine (5 mL) was added again. At room temperature, N,N-dimethylformamide dimethyl acetal (240 mg, 2.0 mmol) was added, and the reaction was carried out overnight under nitrogen protection at room temperature. The reaction solution was concentrated to obtain Intermediate 70-1, an oily substance, which was directly used in the next step without separation.

[0375] The Intermediate 70-1 obtained in the previous step was dissolved in pyridine (5 mL). 4-Dimethylaminopyridine (6 mg, 0.05 mmol) and isobutyryl chloride (80 mg, 0.75 mmol) were added successively. After 3 hours, methanol was added, and the solvent was removed by evaporation to obtain Intermediate 70-2, an oily substance, which was directly used in the next step without separation.

[0376] The Intermediate 70-2 obtained in the previous step was added to acetonitrile (5 mL). Hydrazine hydrate (176 mg, 3.0 mmol) was added, and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was separated, and the organic layer was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain Compound A70, 80 mg of white solid, with a yield of 44%. 1 H NMR(600MHz,DMSO-d 6 )δ8.01–7.79(m,3H),6.80(s,1H),6.32(d,J=6.0Hz,1H),5.38(d,J=5.8Hz,1H),4.69(t,J=5.4Hz,1H),4.30(dd,J=12.1,2.9Hz,1H),4.25–4.21(m,1H),4.17(dd,J=12.1,5.3Hz,1H),3.95(q,J=5.9Hz,1H),2.54–2.51(m,1H),1.06(d,J=2.9Hz,3H),1.05(d,J=2.9Hz,3H). 13 C NMR(126MHz,DMSO-d6 ) δ 176.36, 156.06, 148.40, 123.99, 117.40, 117.00, 110.61, 81.78, 79.50, 74.49, 70.65, 63.40, 33.63, 19.19, 19.11. MS m / z = 363.0 [M+1] + 。

[0377] Preparation Example 18: Synthesis of Compounds A71 and A213

[0378]

[0379] Compound 69-2 (160 mg, 0.27 mmol) was dissolved in dichloromethane (10 mL), and under ice bath, Boc-L-Val (82 mg, 0.38 mmol), 1-hydroxybenzotriazole (55 mg, 0.41 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (113 mg, 0.59 mmol) and 4-dimethylaminopyridine (132 mg, 1.08 mmol) were added successively. After addition, the reaction was carried out at room temperature overnight. The reaction solution was concentrated, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain Compound 71-1, 158 mg of white solid, with a yield of 74%.

[0380] Compound 71-1 (158 mg, 0.20 mmol) was dissolved in acetonitrile (10 mL). At room temperature, 85% hydrazine hydrate (71 mg, 1.20 mmol) was added. After stirring for 1 hour, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was separated, and the organic phase was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a white solid. The obtained white solid was added to tetrahydrofuran (8 mL). Under ice bath, acetic acid (14 mg, 0.24 mmol) and 1M tetrabutylammonium fluoride in tetrahydrofuran solution (0.24 mL, 0.24 mmol) were added. After addition, the mixture was stirred at room temperature. After 2 hours, TLC showed that the reaction was complete. The reaction solution was concentrated, water was added, and the mixture was extracted with isopropyl acetate. The organic phase was separated, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and then concentrated. The obtained product was slurried with petroleum ether, filtered and dried to obtain Compounds 71-2a and 71-2b, with the proportion of 71-2b being greater than 90%. Further recrystallization gave pure 71-2b, 60 mg of white solid, and the two-step yield was 61%. The obtained Compound 71-2b's 1 1H NMR (500 MHz, DMSO-d 6)δ8.07–7.86(m,3H),7.06(d,J=8.6Hz,1H),6.91(s,1H),6.50(d,J=6.7Hz,1H),5.17(dd,J=5.8,3.6Hz,1H),5.07(t,J=5.8Hz,1H),5.01(t,J=6.2Hz,1H),4.27–4.21(m,1H),4.10(dd,J=8.6,5.4Hz,1H),3.66–3.53(m,2H),2.31–2.23(m,1H),1.45–1.36(m,9H),0.93(d,J=6.9Hz,3H),0.90(d,J=6.9Hz,3H).

[0381] Compound 71-2b (60 mg, 0.12 mmol) was added to a saturated hydrogen chloride / methanol solution (5 mL), stirred at 35 °C, and after 2 hours, TLC showed that the reaction was complete. The reaction solution was concentrated and triturated with methyl tert-butyl ether to obtain Compound A213 as the dihydrochloride, a white solid (45 mg, yield 81%). 1 H NMR (500 MHz, Methanol-d 4 )δ8.17(s,1H),7.21(s,1H),5.55–5.50(m,1H),5.19(d,J=5.8Hz,1H),4.53–4.48(m,1H),4.12(d,J=4.4Hz,1H),3.83(d,J=3.7Hz,2H),2.58–2.49(m,1H),1.18(d,J=4.3Hz,3H),1.17(d,J=4.3Hz,3H). 13 C NMR (126 MHz, Methanol-d 4 )δ167.48,148.78,135.20,128.58,115.31,113.58,113.19,84.12,77.64,74.29,73.56,60.15,57.64,28.91,16.63,16.32. MS m / z=392.1[M+1] + . If the mixture 71-2a and 71-2b obtained in the previous step was directly deprotected, a mixture of A71 and A213 could be obtained, and the content of A71 was about 10%.

[0382] Preparation Example 19: Synthesis of Compound A72

[0383]

[0384] Compound A9 (350 mg, 1.2 mmol) was added to pyridine (10 mL). Pyridine was removed by evaporation under reduced pressure, and the operation was repeated once. Then pyridine (10 mL) was added again. At room temperature, N,N-dimethylformamide dimethyl acetal (596 mg, 5.0 mmol) was added. Under nitrogen protection, the reaction was carried out overnight at room temperature. The reaction solution was concentrated to obtain intermediate 70-1, an oily substance, which was directly used in the next step without separation.

[0385] Boc-L-valine (369 mg, 1.7 mmol), 1-hydroxybenzotriazole (243 mg, 1.8 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (498 mg, 2.6 mmol) were added to dichloromethane (15 mL). After stirring at room temperature for 15 minutes, a dichloromethane solution (2 mL) of 70-1 obtained in the previous step and 4-dimethylaminopyridine (733 mg, 6.0 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, methanol was added, and it was concentrated again to obtain an oily substance. Water was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, and the organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain crude intermediate 72-1, an oily substance, which was directly used in the next step without separation.

[0386] Intermediate 72-1 was dissolved in acetonitrile (10 mL), 85% hydrazine hydrate (283 mg, 4.8 mmol) was added, and the reaction was carried out at room temperature for 3 hours. Then the reaction solution was added to water, and the mixture was extracted with ethyl acetate. The organic phase was separated, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain compound 72-2, 195 mg of white solid, and the total yield of three steps was 33%. 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.01–7.80 (m, 3H), 7.14 (d, J = 8.2 Hz, 1H), 6.82 (s, 1H), 6.33 (d, J = 6.0 Hz, 1H), 5.38 (d, J = 5.9 Hz, 1H), 4.69 (t, J = 5.4 Hz, 1H), 4.34–4.28 (m, 1H), 4.26–4.21 (m, 2H), 3.95–3.90 (m, 1H), 3.89–3.85 (m, 1H), 1.99–1.92 (m, 1H), 1.40–1.28 (m, 9H), 0.81 (t, J = 7.1 Hz, 6H).

[0387] Compound 72-2 (100 mg, 0.2 mmol) was added to a methanol solution saturated with hydrogen chloride (5 mL), and the mixture was stirred at 36 °C. After 1 hour, the reaction was complete. The reaction solution was concentrated, then isopropyl ether was added, a solid was precipitated, and it was filtered to obtain compound A72, which was a hydrochloride salt, 74 mg of white solid, and the yield was 80%.1 1H NMR (600 MHz, Methanol-d 4 ) δ 8.15 (s, 1H), 7.12 (s, 1H), 4.75 (d, J = 5.2 Hz, 1H), 4.62 (dd, J = 12.1, 7.4 Hz, 1H), 4.54 (dd, J = 12.1, 2.8 Hz, 1H), 4.45 (td, J = 7.5, 2.8 Hz, 1H), 4.05–4.01 (m, 2H), 2.34–2.28 (m, 1H), 1.08 (d, J = 2.4 Hz, 3H), 1.07 (d, J = 2.3 Hz, 3H). 13 13C NMR (151 MHz, Methanol-d 4 ) δ 168.15, 148.61, 135.04, 129.77, 114.70, 113.28, 112.57, 80.77, 79.53, 74.42, 70.17, 64.81, 57.56, 29.19, 16.48, 16.32. MS m / z = 392.0 [M+1] + .

[0388] Preparation Example 20: Synthesis of Compounds A74 and A164

[0389]

[0390] Referring to the synthesis method of reference compound A144, using A10 (176 mg, 0.6 mmol, deuterium substitution rate 99%) as the raw material, compounds A74 and A164 were obtained through five-step reactions. Further recrystallization yielded 139 mg of pure A164 as a white solid, with an overall yield of 64%. The 1 1H NMR (600 MHz, DMSO-d 6 ) δ 8.07–7.83 (m, 3H), 6.92 (d, J = 4.6 Hz, 1H), 6.88 (d, J = 4.6 Hz, 1H), 6.41 (d, J = 6.6 Hz, 1H), 5.21 (dd, J = 5.7, 3.3 Hz, 1H), 5.02 (s, 1H), 5.01–4.97 (m, 1H), 4.25 (d, J = 3.2 Hz, 1H), 2.66–2.58 (m, 1H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 6.9 Hz, 3H). MS m / z = 364.0 [M+1] + .

[0391] Preparation Example 21: Synthesis of Compound A75

[0392]

[0393] Referring to the synthesis method of reference compound A70, using A10 (176 mg, 0.6 mmol) as the raw material, compound A75 was obtained through three-step reaction, 109 mg of white solid, with an overall yield of 50%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.03–7.82 (m, 3H), 6.92 (d, J = 4.5 Hz, 1H), 6.82 (d, J = 4.5 Hz, 1H), 6.33 (d, J = 6.0 Hz, 1H), 5.39 (d, J = 5.8 Hz, 1H), 4.71 (t, J = 5.5 Hz, 1H), 4.24 (d, J = 6.6 Hz, 1H), 3.97 (q, J = 5.8 Hz, 1H), 2.57–2.53 (m, 1H), 1.07 (d, J = 2.5 Hz, 3H), 1.06 (d, J = 2.5 Hz, 3H). MS m / z = 364.0 [M+1] + .

[0394] Preparation Example 22: Synthesis of Compounds A76 and A214

[0395]

[0396] Referring to the synthesis method of reference compound A213, using 74-2 (295 mg, 0.5 mmol) as the raw material, compound A214 was obtained through three-step reaction, as the dihydrochloride salt, 97 mg of white solid, with an overall yield of 42%. 1 H NMR (500 MHz, Methanol-d 4 ) δ 8.17 (s, 1H), 7.49 (d, J = 4.8 Hz, 1H), 7.21 (d, J = 4.9 Hz, 1H), 5.53 (dd, J = 5.8, 3.1 Hz, 1H), 5.19 (d, J = 5.8 Hz, 1H), 4.50 (d, J = 3.1 Hz, 1H), 4.13 (d, J = 4.5 Hz, 1H), 2.58–2.50 (m, 1H), 1.18 (d, J = 4.6 Hz, 3H), 1.17 (d, J = 4.6 Hz, 3H). MS m / z = 393.0 [M+1] + . If the products 76-2a and 76-2b obtained in the second step are directly deprotected, a mixture of A76 and A214 can be obtained, and the content of A76 is about 10%.

[0397] Preparation Example 23: Synthesis of Compound A77

[0398]

[0399] Referring to the synthesis method of reference compound A72, using A10 (1.0 mmol, 293 mg) as the raw material, compound A77 was obtained through four steps of reaction. It is the dihydrochloride salt, a white solid of 107 mg, with an overall yield of 23%. MS m / z = 393.0 [M+1] + 。

[0400] Preparation Example 24: Synthesis of Compound A84

[0401]

[0402] Compound A1 (49 mg, 0.16 mmol) was added to acetone (3 mL), and 2,2-dimethoxypropane (83 mg, 0.8 mmol, 5 eq) and p-toluenesulfonic acid monohydrate (30 mg, 0.16 mmol, 1 eq) were added successively. After addition, the temperature was raised to 40 °C and the reaction was stirred for 2 hours. The reaction was monitored by TLC until completion. The reaction solution was added to saturated sodium bicarbonate aqueous solution (20 mL), extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and separated by preparative thin layer chromatography to obtain compound 84-1, a white solid of 27 mg, with a yield of 48%.

[0403] Compound 84-1 (27 mg, 0.077 mmol) was added to anhydrous tetrahydrofuran (3 mL). Under ice bath, a 3M methylmagnesium bromide solution in methyltetrahydrofuran (0.05 ml, 0.15 mmol, 2 eq) was added dropwise. After addition, it was stirred for 5 minutes, and then a solution of compound B (57 mg, 0.12 mmol, 1.5 eq) in anhydrous tetrahydrofuran (1 mL) was added dropwise. After addition, the reaction was carried out at room temperature for 2 hours. The reaction was monitored by TLC until completion. The reaction solution was added to saturated ammonium chloride solution (20 mL), extracted with ethyl acetate, the organic layer was separated, dried, and concentrated to obtain compound 84-2, an oil, which was directly used for the next step of reaction.

[0404] The product of the previous step was added to tetrahydrofuran (2 mL), and concentrated hydrochloric acid (0.4 mL) was added dropwise under ice bath. After addition, the reaction was stirred at room temperature until completion. The reaction solution was added to saturated sodium bicarbonate aqueous solution (20 mL), extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and separated by preparative thin layer chromatography to obtain compound A84, a white solid of 10 mg, with a two-step yield of 21%. 1 H NMR (500 MHz, DMSO-d 6)δ8.12(brs,1H),7.88(s,1H),7.42(brs,1H),7.36(t,J=7.7Hz,2H),7.24–7.14(m,3H),6.73(s,1H),6.45(d,J=6.0Hz,1H),6.12–6.02(m,1H),5.39(d,J=6.0Hz,1H),4.56(t,J=5.2Hz,1H),4.31–4.19(m,2H),4.16–4.06(m,1H),4.01–3.77(m,4H),1.45–1.38(m,1H),1.28–1.20(m,7H),0.80(t,J=7.4Hz,6H). 1 H NMR(500MHz,Methanol-d 4 )δ7.82(s,1H),7.34(t,J=7.9Hz,2H),7.27–7.16(m,3H),6.72(s,1H),4.73(d,J=5.2Hz,1H),4.46–4.35(m,2H),4.33–4.27(m,1H),4.15(t,J=5.7Hz,1H),4.06(dd,J=11.0,5.8Hz,1H),4.01–3.90(m,2H),1.53–1.45(m,1H),1.38–1.30(m,7H),0.89(t,J=7.5Hz,6H).

[0405] Preparation Example 25: Synthesis of Compound A102

[0406]

[0407] Compound A1 (62 mg, 0.2 mmol) and 1,8-bis(dimethylamino)naphthalene (56 mg, 0.26 mmol, 1.3 eq) were added to trimethyl phosphate (3 mL), and the mixture was stirred in an ice bath. Phosphorus oxychloride (153 mg, 1.0 mmol, 5 eq) was slowly added. After about 3 hours, TLC showed that the raw materials had basically reacted completely. A solution of tributylammonium pyrophosphate (549 mg, 1.0 mmol, 5 eq) in DMF (3 mL) was added to the above system, and then tri-n-butylamine (222 mg, 1.2 mmol, 6 eq) was added. After stirring in an ice bath for 10 minutes, the reaction solution was allowed to warm to room temperature naturally. Monitored by TLC until the product no longer increased. 0.5 M aqueous triethylammonium bicarbonate solution was added to the reaction solution to adjust the pH to 7.5, and deionized water (5 mL) was added. The mixture was extracted with dichloromethane and ethyl acetate respectively, and the organic phase was discarded. The aqueous phase was separated by gel column chromatography to obtain the crude product of A102. The crude product was further purified by reverse-phase column chromatography to obtain A102 as the diethylamine salt, a foamy solid, 44 mg, with a yield of 29%.1 H NMR (500 MHz, D 2 O) δ 7.88 (s, 1H), 6.87 (s, 1H), 4.76 (d, J = 5.2 Hz, 1H), 4.45–4.41 (m, 1H), 4.38 (t, J = 4.8 Hz, 1H), 4.17–4.10 (m, 2H), 3.12 (q, J = 7.3 Hz, 12H), 1.19 (t, J = 7.3 Hz, 18H). 31 P NMR (202 MHz, D 2 O) δ -10.89 (d), -11.46 (d), -23.19 (t). MS m / z = 548.0 [M - 1] - .

[0408] Preparation Example 26: Synthesis of Compound A106

[0409]

[0410] Referring to the synthesis method of Compound A102, using A9 (58 mg, 0.2 mmol, deuterium substitution rate not less than 97%) as the raw material, the product A106 was obtained as the triethylamine salt, 55 mg of foamy solid, with a yield of 33%. 1 H NMR (500 MHz, D 2 O) δ 7.92 (s, 1H), 7.02 (s, 1H), 4.92 (d, J = 4.4 Hz, 1H), 4.50–4.42 (m, 2H), 4.16–4.10 (m, 1H), 4.09–4.01 (m, 1H), 3.11 (q, J = 7.3 Hz, 18H), 1.19 (t, J = 7.2 Hz, 27H). 31 P NMR (202 MHz, D 2 O) δ -10.90 (d), -11.52 (d), -23.29 (t). MS m / z = 531.0 [M - 1] - .

[0411] Preparation Example 27: Synthesis of Compound A107

[0412]

[0413] Referring to the synthesis method of Compound A102, using A10 (59 mg, 0.2 mmol, deuterium substitution rate 99%) as the raw material, the product A107 was obtained as the tetraethylamine salt, 54 mg of foamy solid, with a yield of 29%. 1 H NMR (500 MHz, D 2O) δ 7.97 (s, 1H), 7.17 (d, J = 4.9 Hz, 1H), 7.07 (d, J = 4.8 Hz, 1H), 4.87 (d, J = 5.0 Hz, 1H), 4.48–4.40 (m, 2H), 3.12 (q, J = 7.3 Hz, 24H), 1.20 (t, J = 7.3 Hz, 36H). 31 P NMR (202 MHz, D 2 O) δ -10.92 (d), -11.45 (d), -23.29 (t). MS m / z = 532.0 [M - 1] - .

[0414] Preparation Example 28: Synthesis of Compound A109

[0415]

[0416] Compound 2 - 1 (145 mg, 0.5 mmol) was added to trimethyl phosphate (2 mL). Under an ice bath, phosphorus oxychloride (153 mg, 1 mmol, 2 eq) was added, and the mixture was stirred for 3 - 4 hours. Monitored by TLC, there was still a large amount of starting material remaining. After adding additional phosphorus oxychloride (80 mg), stirring was continued under the ice bath for 2 hours. An acetonitrile suspension (2 mL) of tri - n - butylammonium pyrophosphate (1.1 g, 2 mmol, 4 eq) and tri - n - butylamine (741 mg, 4 mmol, 8 eq) were added to the reaction solution, and stirring was continued for 2 hours. 1 M aqueous triethylammonium bicarbonate solution (8 mL) was added to the reaction solution, followed by distilled water (8 mL), and the mixture was stirred. Extracted with ethyl acetate, the organic phase was discarded, the aqueous phase was freeze - dried, and separated by preparative liquid chromatography to obtain Compound A109 as the tri - n - butylamine salt, 55 mg of white solid, with a yield of 10%. 1 H NMR (400 MHz, D 2 O) δ 7.85 (s, 1H), 7.00–6.91 (m, 2H), 4.89–4.83 (m, 1H), 4.43–4.36 (m, 2H), 4.13–4.04 (m, 1H), 4.03–3.94 (m, 1H), 3.04–2.93 (m, 18H), 1.60–1.47 (m, 18H), 1.29–1.17 (m, 18H), 0.79 (t, J = 7.4 Hz, 27H). MS m / z = 530.0 [M - 1] - .

[0417] Preparation Example 29: Synthesis of Compound A112

[0418]

[0419] Compound 2-1 (291 mg, 1.0 mmol) was added to tetrahydrofuran (6 mL), concentrated hydrochloric acid (3 mL) was added, and the mixture was stirred at 45 °C. The reaction was monitored by TLC until completion. The solvent was evaporated, and the residue was separated by silica gel column chromatography to obtain 168 mg of compound A112 as a white solid in a yield of 54%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.77 (s, 1H), 7.72 (brs, 2H), 7.49 (s, 1H), 7.44 (s, 1H), 6.83 (d, J = 4.5 Hz, 1H), 6.62 (d, J = 4.5 Hz, 1H), 5.58 (s, 1H), 4.93 (s, 1H), 4.85 (d, J = 4.4 Hz, 1H), 4.68 (s, 1H), 4.08–3.98 (m, 2H), 3.52 (d, J = 12.0 Hz, 1H), 3.34–3.29 (m, 1H). MS m / z = 310.1 [M+1] + 。

[0420] Preparation Example 30: Synthesis of Compounds A173 and A188

[0421]

[0422] Referring to the synthetic method of reference compound A144, using A11 (294 mg, 1.0 mmol) as the starting material, compounds A173 and A188 were obtained through five-step reactions. Further recrystallization gave 91 mg of pure A188 as a white solid with a total yield of 25%. The :1 H NMR (600 MHz, DMSO-d 6 ) δ 8.07–7.83 (m, 3H), 6.88 (s, 1H), 6.41 (d, J = 6.6 Hz, 1H), 5.21 (dd, J = 5.7, 3.3 Hz, 1H), 5.02 (s, 1H), 5.01–4.97 (m, 1H), 4.25 (d, J = 3.2 Hz, 1H), 2.66–2.58 (m, 1H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 6.9 Hz, 3H). MS m / z = 365.0 [M+1] + 。

[0423] Preparation Example 31: Synthesis of Compound A180

[0424]

[0425] Referring to the synthetic method of reference compound A70, using A11 (147 mg, 0.5 mmol) as the raw material, compound A180 was obtained through three-step reactions, 80 mg of white solid, with an overall yield of 44%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.03–7.83 (m, 3H), 6.82 (s, 1H), 6.32 (d, J = 6.1 Hz, 1H), 5.39 (d, J = 5.8 Hz, 1H), 4.71 (t, J = 5.5 Hz, 1H), 4.23 (d, J = 6.6 Hz, 1H), 3.97 (q, J = 5.8 Hz, 1H), 2.57–2.53 (m, 1H), 1.10–1.01 (m, 6H). MS m / z = 365.0 [M+1] + 。

[0426] Preparation Example 32: Synthesis of Compounds A174 and A215

[0427]

[0428] Referring to the synthetic method of reference compound A213, using 173-2 (296 mg, 0.5 mmol) as the raw material, compound A215 was obtained through three-step reactions, as the dihydrochloride salt, 93 mg of white solid, with a yield of 40%. 1 H NMR (500 MHz, Methanol-d 4 ) δ 8.17 (s, 1H), 7.21 (s, 1H), 5.53 (dd, J = 5.8, 3.1 Hz, 1H), 5.19 (d, J = 5.8 Hz, 1H), 4.50 (d, J = 3.1 Hz, 1H), 4.13 (d, J = 4.5 Hz, 1H), 2.58–2.50 (m, 1H), 1.18 (d, J = 4.6 Hz, 3H), 1.17 (d, J = 4.6 Hz, 3H). MS m / z = 394.0 [M+1] + 。If the products 174-2a and 174-2b obtained in the second step are directly deprotected, a mixture of A174 and A215 can be obtained, with the content of A174 being approximately 10%.

[0429] Preparation Example 33: Synthesis of Compound A181

[0430]

[0431] Referring to the synthetic method of reference compound A72, using A11 (147 mg, 0.5 mmol) as the raw material, compound A181 was obtained through four-step reactions, as the dihydrochloride salt, 75 mg of white solid, with a yield of 32%. 11H NMR (600 MHz, Methanol-d 4 ) δ 8.15 (s, 1H), 7.12 (s, 1H), 4.75 (d, J = 5.2 Hz, 1H), 4.62 (dd, J = 12.1, 7.4 Hz, 1H), 4.54 (dd, J = 12.1, 2.8 Hz, 1H), 4.45 (td, J = 7.5, 2.8 Hz, 1H), 2.35–2.28 (m, 1H), 1.08 (d, J = 2.4 Hz, 3H), 1.07 (d, J = 2.3 Hz, 3H). MS m / z = 394.0 [M+1] + .

[0432] Preparation Example 34: Synthesis of Compound A198

[0433]

[0434] Referring to the synthesis method of Compound A102, using A11 (59 mg, 0.2 mmol) as the raw material, the product A198 was obtained as the triethylamine salt, 59 mg of foamy solid, with a yield of 35%. 1 1H NMR (500 MHz, D 2 2O) δ 7.90 (s, 1H), 7.00 (s, 1H), 4.84 (d, J = 4.6 Hz, 1H), 4.42–4.38 (m, 2H), 3.07 (q, J = 7.3 Hz, 18H), 1.15 (t, J = 7.3 Hz, 27H). 31 31P NMR (202 MHz, D 2 2O) δ -10.93 (d), -11.46 (d), -23.31 (t). MS m / z = 533.0 [M-1] - .

[0435] Preparation Example 35: Synthesis of Compound A131

[0436]

[0437] Compound 2-1 (58 mg, 0.2 mmol) was added to N,N-dimethylformamide (3 mL), and N,N-dimethylformamide dimethyl acetal (160 mg, 1.34 mmol, 6.7 eq) was added. After the addition, the reaction was carried out at room temperature and was complete after 1 h. Methanol was added to the reaction solution, concentrated, and slurried with isopropanol and toluene to obtain Compound 131-1, 59 mg of white solid, with a yield of 85%.

[0438] Compound 131-1 (59 mg, 0.17 mmol) was added to dichloromethane (5 mL). At room temperature, triethylamine (138 mg, 1.36 mmol, 8 eq), DMAP (62 mg, 0.51 mmol, 3 eq) and isobutyryl chloride (72 mg, 0.68 mmol, 4 eq) were successively added, and the reaction was carried out overnight at room temperature. The reaction solution was concentrated, ethyl acetate (30 mL) and water (10 mL) were added, the organic phase was separated, and the organic phase was washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride respectively, dried, evaporated to dryness, and Compound 131-2 was obtained as a white solid, 85 mg, with a yield of 90%.

[0439] Compound 131-2 (85 mg, 0.15 mmol) was added to acetonitrile (10 mL), hydrazine hydrate (30 mg, 0.60 mmol, 4 eq) was added, and after completion of the addition, the reaction was complete in about 30 minutes. The reaction solution was added to water, extracted with ethyl acetate, the organic layer was separated, and the organic phase was washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride respectively, dried, evaporated to dryness, and separated by silica gel column chromatography to obtain Compound A131 as a white solid, 70 mg, with a yield of 93%. 1 H NMR (600 MHz, DMSO-d 6 ) δ 8.04 (br, 1H), 7.96 (br, 1H), 7.93 (s, 1H), 6.93 (d, J = 4.6 Hz, 1H), 6.75 (d, J = 4.6 Hz, 1H), 6.08 (d, J = 5.7 Hz, 1H), 5.44 (dd, J = 5.7, 3.7 Hz, 1H), 4.63 (q, J = 3.7 Hz, 1H), 4.33 (dd, J = 12.4, 3.3 Hz, 1H), 4.28 (dd, J = 12.4, 4.1 Hz, 1H), 2.66–2.57 (m, 2H), 2.50–2.46 (m, 1H), 1.17 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 6.9 Hz, 3H), 1.12–1.09 (m, 6H), 1.05 (d, J = 7.0 Hz, 3H), 1.02 (d, J = 7.0 Hz, 3H). MS m / z = 502.0 [M+1] + 。

[0440] Preparation Example 36: Synthesis of Compound A151

[0441]

[0442] Referring to the synthesis method of Compound A131, using A9 (58 mg, 0.2 mmol) as the raw material, through three-step reaction, Compound A151 was obtained as a white solid, 69 mg, with an overall yield of 68%. 1 H NMR (600 MHz, DMSO-d6 ) δ 8.04 (broad, 1H), 7.96 (broad, 1H), 7.93 (singlet, 1H), 6.75 (singlet, 1H), 6.08 (doublet, J = 5.7 Hz, 1H), 5.44 (doublet of doublets, J = 5.7, 3.7 Hz, 1H), 4.63 (quartet, J = 3.7 Hz, 1H), 4.33 (doublet of doublets, J = 12.4, 3.3 Hz, 1H), 4.28 (doublet of doublets, J = 12.4, 4.1 Hz, 1H), 2.66–2.57 (multiplet, 2H), 2.49–2.46 (multiplet, 1H), 1.17 (doublet, J = 7.0 Hz, 3H), 1.15 (doublet, J = 6.9 Hz, 3H), 1.12–1.09 (multiplet, 6H), 1.05 (doublet, J = 7.0 Hz, 3H), 1.02 (doublet, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 175.53, 174.90, 174.13, 155.58, 148.12, 120.98, 117.17, 115.44, 110.30, 81.25, 75.81, 72.05, 70.30, 62.46, 33.20, 33.16, 33.09, 18.55, 18.46, 18.40, 18.35, 18.33, 18.18. MS m / z = 503.0 [M+1] + .

[0443] Preparation Example 37: Synthesis of Compound A171

[0444]

[0445] Referring to the synthesis method of Compound A131, using A10 (59 mg, 0.2 mmol) as the starting material, through three-step reaction, Compound A171 was obtained as a white solid (70 mg), with an overall yield of 69%. MS m / z = 504.0 [M+1] + .

[0446] Preparation Example 38: Synthesis of Compound A196

[0447]

[0448] Referring to the synthesis method of Compound A131, using A11 (59 mg, 0.2 mmol) as the starting material, through three-step reaction, Compound A196 was obtained as a white solid (59 mg), with an overall yield of 58%. MS m / z = 505.0 [M+1] + .

[0449] Preparation Example 39: Synthesis of Compound A209

[0450]

[0451] Compound 1-1 (0.5 g, 0.89 mmol) was added to DMF (9 ml), and NBS (0.16 g, 0.89 mmol, 1.0 eq) was added. The mixture was stirred at room temperature for 3 - 4 hours, and the reaction was monitored by TLC until completion. Saturated sodium thiosulfate (10 mL) solution was added to the reaction mixture, stirred, extracted with ethyl acetate, and the layers were separated. The aqueous layer was extracted with ethyl acetate (20 mL) again. The ethyl acetate layers were combined, dried, concentrated, and separated by column chromatography to obtain compound 209-1, 0.5 g of white solid, with a yield of 87%.

[0452] Compound 209-1 (0.15 g, 0.23 mmol) was added to N,N-dimethylacetamide (5 mL), and Zn (2 mg, 0.031 mmol, 0.13 eq), Zn(CN) 2 (0.06 g, 0.51 mmol, 2.2 eq), NiCl 2 (dppf) (0.03 g, 0.043 mmol, 0.2 eq) and Pd 2 (dba) 3 (0.02 g, 0.02 mmol, 0.1 eq) were added successively. After addition, the reaction was carried out at 140 °C overnight, and TLC showed that the reaction was complete. The reaction mixture was poured into ethyl acetate / water (20 mL / 20 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (20 mL) again. The ethyl acetate layers were combined, dried, concentrated, and separated by preparative TLC to obtain compound 209-2, 0.1 g of white solid, with a yield of 74%.

[0453] Compound 209-2 (0.1 g, 0.17 mmol) was added to dichloromethane (3 mL). At -30 °C, 1 M boron trichloride dichloromethane solution (0.7 mL, 4.1 eq) was added dropwise. After addition, the mixture was stirred at the same temperature for 1 - 2 hours, and the reaction was monitored by TLC until completion. Methanol (0.5 mL) and triethylamine (0.3 mL) were added dropwise to the reaction mixture successively. The reaction mixture was evaporated to dryness and separated by preparative TLC to obtain compound A209, 15 mg of white solid, with a yield of 28%. 1 H NMR (500 MHz, DMSO-d 6)δ8.23(s,1H),7.50(s,1H),6.30(d,J=6.1Hz,1H),5.22(d,J=5.9Hz,1H),4.93(t,J=5.7Hz,1H),4.55(t,J=5.5Hz,1H),4.11–4.03(m,1H),3.94(q,J=5.8Hz,1H),3.73–3.65(m,1H),3.57–3.49(m,1H).MS m / z=317.1[M+1] + 。

[0454] Preparation Example 40: Synthesis of Compound A87

[0455]

[0456] Compound 11-1 (0.05 g, 0.15 mmol) was added to tetrahydrofuran (3 mL). Under an ice bath, a 2-methyltetrahydrofuran solution of 3 M methylmagnesium bromide (0.1 mL, 0.3 mmol) was added dropwise. After the addition, the mixture was stirred for 5 minutes. Then, a tetrahydrofuran solution (1 mL) of Compound B (0.1 g, 0.23 mmol) was added dropwise. After the addition, the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The reaction mixture was added to saturated ammonium chloride solution (10 mL), and extracted with ethyl acetate. The organic layer was separated, dried, and concentrated to obtain the crude product of Compound 87-1, which was directly used in the next step of the reaction.

[0457] The product obtained in the previous step was added to tetrahydrofuran (2 mL). Under an ice bath, concentrated hydrochloric acid (0.4 mL) was added dropwise. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was added to saturated sodium bicarbonate solution (10 mL), and extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Compound A87, 0.03 g of white solid, and the overall yield of two steps was 33%. 1 H NMR(500MHz,Methanol-d 4 )δ7.89(s,1H),7.36–7.30(m,2H),7.24–7.15(m,3H),6.94(s,1H),4.82(d,J=5.4Hz,1H),4.46–4.37(m,2H),4.35–4.28(m,1H),4.20(t,J=5.6Hz,1H),4.05(dd,J=10.9,5.8Hz,1H),3.98–3.88(m,2H),1.51–1.44(m,1H),1.37–1.30(m,7H),0.88(t,J=7.5Hz,6H).MS m / z=604.1[M+1] + 。

[0458] Preparation Example 41: Synthesis of Compound A138

[0459]

[0460] Referring to the synthesis method of reference compound A70, using A9 (147 mg, 0.5 mmol) as the raw material, compound A138 was obtained through three-step reaction. 65 mg of white solid was obtained, with an overall yield of 37%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.99–7.83 (m, 3H), 6.80 (s, 1H), 6.32 (d, J = 6.1 Hz, 1H), 5.38 (d, J = 5.8 Hz, 1H), 4.71–4.65 (m, 1H), 4.33 (dd, J = 12.0, 2.8 Hz, 1H), 4.25–4.19 (m, 1H), 4.15 (dd, J = 12.0, 5.5 Hz, 1H), 3.97–3.90 (m, 1H), 2.30 (q, J = 7.5 Hz, 2H), 1.00 (t, J = 7.5 Hz, 3H). MS m / z = 349.2 [M+1] + 。

[0461] Preparation Example 42: Synthesis of Compound A140

[0462]

[0463] Referring to the synthesis method of A70, using A9 (147 mg, 0.5 mmol) as the raw material, compound A140 was obtained through three-step reaction. 77 mg of white solid was obtained, with an overall yield of 41%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.03–7.80 (m, 3H), 6.80 (s, 1H), 6.32 (d, J = 6.1 Hz, 1H), 5.37 (d, J = 5.7 Hz, 1H), 4.70 (t, J = 5.3 Hz, 1H), 4.30–4.22 (m, 2H), 4.21–4.14 (m, 1H), 4.01–3.94 (m, 1H), 1.09 (s, 9H). MS m / z = 377.2 [M+1] + 。

[0464] Preparation Example 43: Synthesis of Compound A146

[0465]

[0466] Compound A70 (0.18 g, 0.5 mmol) was added to acetic acid (3 mL), and trimethyl orthoisobutyrate (0.37 g, 2.5 mmol) was added. The mixture was stirred at 50 °C until the reaction was complete. The solvent was evaporated to obtain the unstable intermediate 146-1. This intermediate was dissolved in tetrahydrofuran (6 mL), 1 M dilute hydrochloric acid (0.5 mL) was added, and the mixture was stirred at room temperature. The reaction was complete in about 1 hour. Saturated aqueous sodium bicarbonate was added to the reaction solution until the pH was neutral. The mixture was extracted with ethyl acetate. The organic phase was dried and evaporated to dryness to obtain the crude product of compound A146. This crude product was recrystallized with isopropanol / water to obtain 0.18 g of white solid with a yield of 82%. 1 H NMR(600MHz,DMSO-d 6 )δ8.05–7.83(m,3H),6.85(s,1H),6.56(d,J=6.5Hz,1H),5.16(dd,J=5.6,4.1Hz,1H),5.09(t,J=6.1Hz,1H),4.46(q,J=4.3Hz,1H),4.27(dd,J=12.2,4.0Hz,1H),4.23(dd,J=12.2,4.8Hz,1H),2.67–2.58(m,1H),2.55–2.49(m,1H),1.16(d,J=7.0Hz,3H),1.15(d,J=7.0Hz,3H),1.05(d,J=7.0Hz,3H),1.03(d,J=7.2Hz,3H). MS m / z=433.2[M+1] + 。

[0467] Preparation Example 44: Synthesis of Compound A147

[0468]

[0469] Compound 151-1 (1.51 g, 4.34 mmol) was added to N,N-dimethylformamide (15 mL). Under an ice bath, tert-butyldiphenylchlorosilane (2.39 g, 8.69 mmol) and imidazole (1.18 g, 17.37 mmol) were added. The mixture was stirred at room temperature for 5 hours. Water and ethyl acetate were added to the reaction solution, and the organic phase was separated. The organic phase was washed successively with dilute hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain compound 147-1, 2.05 g of foamy solid.

[0470] Compound 147-1 (2.05 g, 3.50 mmol) was added to dichloromethane (30 mL). At room temperature, triethylamine (1.24 g, 12.26 mmol), DMAP (0.21 g, 1.75 mmol) and isobutyric anhydride (1.39 g, 8.76 mmol) were added. Under nitrogen protection, the reaction was carried out at room temperature for 1 hour. After the reaction solution was concentrated, water and ethyl acetate were added. The organic phase was separated, and the organic phase was washed with dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution and saturated brine respectively, dried over anhydrous sodium sulfate, concentrated to obtain the crude product of compound 147-2, an oily substance. This intermediate was directly used in the next step without separation.

[0471] The product obtained in the previous step was added to tetrahydrofuran (30 mL). At room temperature, acetic acid (0.11 g, 1.75 mmol) and a tetrahydrofuran solution of 1M tetrabutylammonium fluoride (3.5 mL, 3.5 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Water and ethyl acetate were added to the reaction solution. The organic phase was separated, and the organic phase was washed with saturated sodium bicarbonate and saturated brine respectively, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product of compound 147-3, an oily substance.

[0472] The crude product of 147-3 obtained in the previous step was added to ethanol (20 mL). At room temperature, acetic acid (4.2 g, 70 mmol) was added. Under nitrogen protection, the mixture was stirred at 50 °C overnight. After the reaction solution was concentrated, water and ethyl acetate were added. The organic phase was separated, and the organic phase was washed with saturated sodium bicarbonate and saturated brine respectively, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to obtain compound A147, 0.85 g of white solid, and the overall yield of three steps was 56%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.15–7.89 (m, 3H), 6.80 (s, 1H), 6.01 (d, J = 5.7 Hz, 1H), 5.44 (dd, J = 5.7, 3.1 Hz, 1H), 5.18 (dd, J = 6.1, 5.2 Hz, 1H), 4.41 (q, J = 3.4 Hz, 1H), 3.71–3.60 (m, 2H), 2.69–2.54 (m, 2H), 1.19 (d, J = 7.0 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.11 (d, J = 2.4 Hz, 3H), 1.09 (d, J = 2.4 Hz, 3H). MS m / z = 433.2 [M+1] + 。

[0473] Preparation Example 45: Synthesis of Compound A216

[0474]

[0475] Compound 1-1 (0.2 g, 0.36 mmol) was added to dichloromethane (2 mL). At -78 °C, a toluene solution of 1.2 M diisobutylaluminum hydride (0.9 mL, 1.08 mmol) was slowly added dropwise. After the addition, the mixture was stirred at the same temperature for 3 - 4 hours. TLC showed that the reaction was complete. Ethyl acetate (3 mL) was added dropwise to the reaction solution. After the addition, the temperature was allowed to rise to room temperature naturally. 20% aqueous solution of potassium sodium tartrate (5 mL) was added, and the mixture was stirred at room temperature overnight. The reaction solution was added to water (15 mL), and the mixture was extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and Compound 216-1 was obtained.

[0476] Compound 216-1 (0.6 g, 1.06 mmol) was added to a mixture of ethanol (10 mL) and dichloromethane (10 mL). Under an ice bath, NaBH 4 (0.1 g, 2.6 mmol) was added portionwise. After the addition, the mixture was stirred at room temperature for 2 - 3 hours. TLC showed that the reaction was complete. Glacial acetic acid was added dropwise to the reaction solution until no more bubbles were generated. Water (30 mL) was added, and the mixture was extracted with ethyl acetate. The organic layer was separated, washed with saturated sodium bicarbonate, dried, concentrated, and separated by silica gel column chromatography to obtain Compound 216-2, 0.48 g of white solid, with a yield of 80%.

[0477] Compound 216-2 (0.48 g, 0.85 mmol), formic acid (2 mL), and 10% palladium on carbon (0.06 g) were successively added to methanol (6 mL). At room temperature, hydrogen was introduced, and the mixture was stirred at atmospheric pressure for 16 hours. The mixture was filtered, concentrated, and separated by reverse-phase column chromatography to obtain Compound A216, 0.05 g of white solid, with a yield of 20%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 7.80 (s, 1H), 7.61 (s, 2H), 6.81 (d, J = 4.4 Hz, 1H), 6.73 (d, J = 4.4 Hz, 1H), 5.18 (d, J = 4.7 Hz, 1H), 4.89 (d, J = 6.9 Hz, 1H), 4.79 (t, J = 5.8 Hz, 1H), 4.51–4.38 (m, 2H), 4.16 (dd, J = 11.6, 6.7 Hz, 1H), 3.99 (dd, J = 11.6, 4.7 Hz, 1H), 3.94–3.83 (m, 1H), 3.78–3.69 (m, 1H), 3.70–3.59 (m, 1H), 3.56–3.47 (m, 1H). MS m / z = 297.1 [M+1] + .

[0478] Preparation Example 46: Synthesis of Compound A28

[0479]

[0480] Under an ice bath, a 0.5 M solution of cyclopropylmagnesium bromide in tetrahydrofuran (18 mL, 9 mmol) was added dropwise to Compound 1-1 (0.5 g, 0.9 mmol). After the addition was complete, the mixture was stirred at room temperature overnight. Under an ice bath, saturated ammonium chloride solution (10 mL) was added dropwise to the reaction mixture, followed by water (50 mL). The mixture was extracted with ethyl acetate, and the organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Compound 28-1 as a white solid (0.2 g, yield 37%).

[0481] Compound 28-1 (0.2 g, 0.34 mmol) was added to dichloromethane (4 mL). Under an ice bath, methanesulfonic acid (0.1 g, 1.04 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 10 - 12 hours, and the reaction was monitored by TLC until completion. Saturated sodium bicarbonate solution was added to the reaction mixture until the pH was neutral, followed by water (15 mL). The mixture was extracted with ethyl acetate, and the organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Compound 28-2 as a white solid (0.1 g, yield 51.0%).

[0482] Compound 28-2 (0.24 g, 0.42 mmol), formic acid (1 mL), and 10% palladium on carbon (0.03 g) were successively added to methanol (3 mL). Under room temperature, hydrogen gas was introduced, and the mixture was stirred at atmospheric pressure for 16 hours. The reaction was monitored by TLC until completion. The mixture was filtered, concentrated, and purified by silica gel column chromatography to obtain Compound A28 as a white solid (13 mg, yield 10%). 1 H NMR (500 MHz, Methanol-d 4 ) δ 7.78 (s, 1H), 6.85 (d, J = 4.5 Hz, 1H), 6.79 (d, J = 4.5 Hz, 1H), 4.78 (d, J = 5.1 Hz, 1H), 4.01–3.95 (m, 1H), 3.95–3.89 (m, 1H), 3.81 (dd, J = 11.9, 2.8 Hz, 1H), 3.68 (dd, J = 11.8, 5.5 Hz, 1H), 2.02–1.92 (m, 1H), 0.68–0.52 (m, 2H), 0.28–0.19 (m, 1H), 0.19–0.08 (m, 1H). MS m / z = 307.1 [M+1] + 。

[0483] Preparation Example 47: Synthesis of Compound A218

[0484]

[0485] Under an ice bath, 1.0 M isopropenylmagnesium bromide in tetrahydrofuran solution (40 mL, 40 mmol) was added dropwise to Compound 1-1 (2.2 g, 3.98 mmol). After the addition was complete, the mixture was stirred at room temperature overnight. To the reaction mixture, saturated aqueous ammonium chloride solution (10 mL) was slowly added dropwise. After the addition was complete, water (40 mL) was added, and the mixture was extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Intermediate 218-1, 1.6 g of white solid, with a yield of 68%.

[0486] Intermediate 218-1 (1.6 g, 2.69 mmol) was added to dichloromethane (30 mL). Under an ice bath, methanesulfonic acid (0.77 g, 8.01 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was monitored by TLC to completion. Under an ice bath, saturated sodium bicarbonate solution was added dropwise to the reaction mixture until the pH was neutral. Then, it was added to water (40 mL), and the mixture was extracted with dichloromethane. The organic layer was separated, dried, concentrated, and purified by silica gel column chromatography to obtain Intermediate 218-2, 0.7 g of yellow oil, with a yield of 45%.

[0487] Referring to the method of debenzylation in Reference Example 5, Intermediate 218-2 (0.7 g, 1.21 mmol) was reacted with 1.0 M BCl 3 dichloromethane solution (6.0 mL, 6.0 mmol). The product was separated by column chromatography to obtain Compound A218, 0.025 g of white solid. 1 H NMR (600 MHz, DMSO-d 6 ) δ 7.74 (s, 1H), 7.55 (s, 2H), 6.82 (d, J = 4.5 Hz, 1H), 6.58 (d, J = 4.4 Hz, 1H), 5.03 (s, 1H), 4.83–4.73 (m, 3H), 4.72–4.64 (m, 2H), 4.05–3.94 (m, 1H), 3.84–3.76 (m, 1H), 3.70–3.60 (m, 1H), 3.51–3.42 (m, 1H), 1.59 (s, 3H). m / z = 307.2 [M+1] + .

[0488] Preparation Example 48: Synthesis of Compound A219

[0489]

[0490] Compound 1-1 (0.56 g, 1.0 mmol) was added to tetrahydrofuran (5 mL). Under nitrogen protection and in an ice bath, a 3 M solution of methylmagnesium bromide in tetrahydrofuran (1.7 mL, 5.1 mmol) was slowly added dropwise. After addition, the mixture was stirred at 60 °C for 2 - 3 hours. The reaction was monitored by TLC and found to be complete. In an ice bath, saturated aqueous ammonium chloride solution (1.0 mL) was added dropwise, followed by water (10 mL). The mixture was extracted with ethyl acetate, and the organic layer was separated, dried, concentrated, and purified by column chromatography to obtain 219-1, a white solid (0.38 g, yield 66%).

[0491] Referring to the method for debenzylation in Reference Example 5, intermediate 219-1 (0.2 g, 0.345 mmol) was reacted with a 1.0 M solution of BCl 3 in dichloromethane (1.7 mL, 1.7 mmol). The product was separated by silica gel column chromatography to obtain compound A219, a white solid (0.05 g, yield 47%). 1 H NMR (600 MHz, DMSO-d 6 ) δ 7.99–7.43 (m, 3H), 6.79 (s, 1H), 6.62 (s, 1H), 5.49 (s, 1H), 5.10–4.93 (m, 1H), 4.83–4.67 (m, 1H), 4.60–4.40 (m, 1H), 4.19–3.96 (m, 2H), 3.59–3.44 (m, 1H), 3.27–3.16 (m, 1H), 2.30 (s, 3H). MS m / z = 309.2 [M+1] + .

[0492] Preparation Example 49: Synthesis of Compound A221

[0493]

[0494] Compound 49-2 (0.6 g, 1 mmol) was added to DMF (10 mL). In an ice bath, methyl iodide (0.28 g, 2.0 mmol) and 60% sodium hydride (0.014 g, 2.0 mmol) were added successively. After stirring for 15 minutes, TLC showed that the reaction was complete. The reaction mixture was added to saturated aqueous ammonium chloride solution (15 mL), and the mixture was extracted with ethyl acetate. The organic layer was separated, dried, concentrated, and evaporated to dryness to obtain compound 221-1. This intermediate was used directly in the next step without purification.

[0495] The 221-1 obtained in the previous step was added to acetonitrile (2 mL), 85% hydrazine hydrate (0.24 g, 4.0 mmol) was added, and the mixture was stirred at room temperature for 1 hour. TLC showed that the reaction was complete. The reaction solution was added to water (15 mL), extracted with ethyl acetate, the organic layer was separated, dried, concentrated, and separated by column chromatography to obtain compound 221-2, 0.2 g of white solid, and the yield of two steps was 37%.

[0496] Compound 221-2 (0.2 g, 0.36 mmol) was added to tetrahydrofuran (2 mL), 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.4 mL, 0.4 mmol) was added, and the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The reaction solution was concentrated and separated by preparative plate to obtain compound A221, 0.045 g of white solid, and the yield was 41%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.07–7.78 (m, 3H), 6.87 (s, 1H), 5.30 (d, J = 5.4 Hz, 1H), 4.94 (t, J = 5.8 Hz, 1H), 4.37 (d, J = 4.9 Hz, 1H), 4.11 (q, J = 5.4 Hz, 1H), 4.08–4.02 (m, 1H), 3.71–3.62 (m, 1H), 3.55 (s, 3H), 3.54–3.48 (m, 1H). MS m / z = 306.0 [M+1] + 。

[0497] Preparation Example 50: Synthesis of Nucleoside Analogs

[0498] Referring to the methods of Preparation Examples 1-49, with the difference that different starting compounds were used, compounds A5, A6, A8, A13, A14, A28, A30, A36, A37, A54, A55, A57, A58, A63, A73, A78, A79, A80, A81, A86, A88, A89, A91, A95, A97, A99, A101, A105, A113, A114, A115, A116, A117, A118, A119, A120, A121, A122, A123, A125, A126, A127, A128, A129, A130, A132, A133, A134, A135, A136, A137, A139, A141, A142, A143, A145, A148, A149, A150, A152, A153, A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, A165, A166, A167, A168, A169, A170, A172, A175, A176, A177, A179, A182, A183, A184, A185, A186, A187, A189, A190, A191, A192, A193, A194, A195, A197, A199, A200, A201, A202, A203, A204, A205, A206, A207, A208, A210, A211 were prepared.

[0499] Test Example 1: Study on the inhibitory effect of compounds on the replication of novel coronavirus

[0500] The inhibitory activity of the compounds of the present invention against the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was determined: Vero E6 cells were purchased from ATCC, and the SARS-CoV-2 virus was obtained from the National Center for Microbial Culture Collection of the National Virus Resource Bank. Vero E6 cells were cultured overnight in a 48-well cell culture dish at a density of 5×10 4 cells / well, pretreated with different concentrations of the compounds of the present invention for 1 hour, then the virus (multiplicity of infection MOI = 0.05) was added to infect the cells for 1 hour, and then the virus-compound mixture was removed, and the cells were further cultured with fresh medium containing the compounds of the present invention. At 24 h post-infection (p.i.), the cell supernatant was collected and lysed in lysis buffer, and the viral copy number in the cell supernatant was quantitatively evaluated by quantitative real-time RT-PCR (qRT-PCR).

[0501] The results showed that multiple compounds significantly inhibited the replication of SARS-CoV-2 virus at a concentration of 10 μM or 5 μM. Compared with the control group without drug treatment, the inhibition rate of the replication of the novel coronavirus was > 99%. The EC 50 values of some compounds were at a low micromolar level, and their antiviral activities were significantly better than that of the control compound remdesivir.

[0502] The EC 50 and inhibition rate data of some preferred compounds are listed in Table 1.

[0503] Table 1. Inhibitory activity against the replication of 2019 novel coronavirus (SARS-CoV-2)

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510] Note: " / " indicates not determined.

[0511] Test Example 2: Determination of the half-toxic concentration of the compound

[0512] In this example, the half-toxic concentration (CC 50 ) of the compounds of the present invention (each compound in Table 1) against Vero E6 cells was determined by analyzing with a CCK8 kit.

[0513] The results showed that the test compounds had no cytotoxic effect on Vero E6 cells at the highest concentration of 10 μM, which indicated that the CC 50 of the compounds of the present invention was much greater than 10 μM.

[0514] Test Example 3: Study on the inhibitory effect of the compound on the RNA-dependent RNA polymerase (RdRp) of the novel coronavirus

[0515] The method reported in the reference (Virus Genes, 2015, 50: 498-504) was used to determine the inhibitory activity of the compounds of the present invention against the RNA-dependent RNA polymerase (RdRp) of the 2019 novel coronavirus (SARS-CoV-2) by fluorescence method: After co-incubating the COVID-19 RdRP solution, substrate, compound solution, buffer, etc. for 1 hour, a fluorescent DNA-binding dye was added. After 10 minutes, data collection was carried out to calculate the inhibitory activity of the compound against RdRp.

[0516] The results showed that some compounds had significant inhibitory activity against RdRp, as shown in Table 2.

[0517] Table 2. Inhibitory activity of compounds against the RdRp of the 2019 novel coronavirus

[0518]

[0519]

[0520] A: < 1 μM; B: 1 μM - 10 μM; C: > 10 μM; " / " indicates not determined.

[0521] Test Example 4: Study on the inhibitory effect of compounds on the replication of other viruses

[0522] The inhibitory activities of the compounds against the replication of respiratory syncytial virus (RSV), human coronavirus OC43, influenza A virus, and Zika virus were tested using the cytopathic effect (CPE) experiment.

[0523] Cytopathic effect (CPE) experiment

[0524] The experimental cells were inoculated into a 96-well cell culture plate at a certain cell density and cultured overnight in an incubator at 5% CO 2 , 37 °C. The next day, the compounds and viruses were added. Depending on the virus to be tested, the cells were cultured in an incubator at 5% CO 2 , 33 °C or 37 °C for 3 - 7 days until the cytopathic effect of the virus-infected control wells without compounds reached 80 - 95%. Then, the cell viability of each well was detected using CellTiter-Glo or CCK-8. If the cell viability of the wells containing the compounds was higher than that of the virus-infected control wells, that is, the CPE was weakened, it indicated that the compounds had an inhibitory effect on the tested viruses. The method of the cytotoxicity experiment was the same as the corresponding antiviral experiment method, but without virus infection.

[0525] The antiviral activity and cytotoxicity of the compounds were respectively represented by the inhibition rate (%) of the cytopathic effect of the virus on the cells and the cell viability rate (%). The calculation formulas are as follows:

[0526] Inhibition rate (%) = (OD value of test well - average value of virus control) / (average value of cell control - average value of virus control) × 100;

[0527] Cell viability (%) = (OD value of test well - average value of medium control) / (average value of cell control - average value of medium control) × 100;

[0528] EC 50 and CC 50 values were calculated using Prism software, and the inhibition curve fitting method was "log(inhibitor) vs. response--Variable slope".

[0529] Dengue virus plaque reduction assay

[0530] Vero cells were seeded into 6-well cell culture plates at a density of 600,000 cells per well and cultured overnight in a 5% CO 2 2 incubator at 37°C. The next day, compounds and virus (40 - 50 PFU / well) were added. The cells were cultured in a 5% CO 2 2 incubator at 37°C for 2 hours, then the supernatant was aspirated, and low melting point agarose culture medium containing the corresponding concentration of the compound was added. The cells were cultured in a 5% CO 2 2 incubator at 33°C or 37°C for 6 - 7 days until obvious virus plaques could be observed in the virus infection control wells without the compound under the microscope. The cells were fixed with 4% paraformaldehyde and stained with crystal violet. The number of plaques in each well was calculated.

[0531] The cytotoxicity assay was performed in parallel with the antiviral assay. Vero cells were seeded into 96-well cell culture plates at a density of 20,000 cells per well and cultured overnight in a 5% CO 2 2 incubator at 37°C. The next day, compounds (1 - 5 concentration points, single point) were added. The cells were cultured in a 5% CO 2 2 incubator at 33°C or 37°C for 6 - 7 days. Then the cell viability of each well was detected using CCK-8.

[0532] The antiviral activity (EC 50 or inhibition rate) and cytotoxicity (CC 50 ) of the compounds were calculated as described above.

[0533] Anti-porcine epidemic diarrhea virus (PEDV) activity assay

[0534] Vero cells were digested and passaged, and the cell density was adjusted to 1×10 5 / mL with cell growth medium, and then inoculated into 96-well plates, 100 μL / well, and placed in an incubator at 37°C and 5% CO 2Incubate in an incubator for 24 h; Take out the 96-well plate, discard the culture medium in the wells, wash three times with 1×PBS, and after draining, add a mixture of the compound (10 concentration points) and the virus (0.01 MOI per well) to each well. Set 8 replicate wells for each concentration, and incubate in a 37°C, 5% CO 2 incubator, while setting up virus control and cell control at the same time. After 36 h, collect the cell samples, and use fluorescence quantitative PCR to measure the changes in virus content in different treatment groups, and calculate the EC of the compound 50 .

[0535] The results showed that multiple compounds had significant activities against respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV OC43), porcine epidemic diarrhea virus (PEDV), Zika virus, and dengue virus (DENV). Compared with GS-441524, the deuterated derivatives had stronger antiviral effects, and the EC of some selected compounds 50 are listed in Tables 3 and 4.

[0536] Table 3, Replication inhibition activities against respiratory syncytial virus (RSV) and human coronavirus OC43 (HCoV OC43)

[0537]

[0538]

[0539]

[0540]

[0541] Note: " / " indicates not measured.

[0542] Table 4, Replication inhibition activities against porcine epidemic diarrhea virus (PEDV), Zika virus, and dengue virus (DENV)

[0543]

[0544]

[0545]

[0546] Note: " / " indicates not measured.

[0547] Test Example 5: Pharmacokinetic evaluation in mice

[0548] Experimental method:

[0549] Fifteen male CD-1 mice were randomly divided into 5 groups, with 3 mice in each group. They were fasted for 12 h before the experiment and allowed free access to water; they were fed uniformly 2 h after drug administration. Four of the groups were intragastrically administered GS-441524, A9, A10, and A11 at 50 mg / kg, and approximately 30 μL of blood was collected from the femoral vein at 0.25, 0.5, 1, 2, 4, 8, and 24 h after drug administration and placed in a heparin anticoagulant tube. Immediately, 20 μL of whole blood was accurately aspirated into a centrifuge tube pre-added with 2 μL of PhosSTOP and 7 μL of DTNB (0.5 M), mixed well, centrifuged at 4 °C, and then 10 μL of plasma was taken and placed in a centrifuge tube pre-added with 100 μL of precipitant (methanol:acetonitrile, 1:1, v / v), mixed well, centrifuged at 4 °C, stored temporarily in dry ice for transportation, and frozen at -80 °C for later measurement. The remaining 1 group was intravenously injected with GS-441524 at 25 mg / kg, and blood was collected from the femoral vein at 5 min, 0.25, 0.5, 1, 2, 4, 8, and 24 h after drug administration and placed in a heparin anticoagulant tube, and the treatment method was the same as above.

[0550] The LC-MS-MS method was used to determine the concentrations of GS-441524, A9, A10, and A11 in plasma. 20 μL of the supernatant was mixed with 20 μL of deionized water and then injected for analysis. After the sample was separated by a Waters HSS T3 (2.1 * 50 mm, 1.8 μm) chromatographic column, an electrospray ionization source was used, and the multi-reaction monitoring mode was used for detection under positive ion conditions. The linear range of the samples was all: 10 - 30000 ng / mL.

[0551] Table 5, Pharmacokinetic parameters of compounds GS-441524, A9, A10, and A11 in mice

[0552]

[0553]

[0554] Test Example 6: Pharmacokinetic evaluation in rats

[0555] Experimental method:

[0556] Eighteen male SD rats were randomly divided into 6 groups, with 3 rats in each group. They were fasted for 12 h before the experiment (the intravenous experimental group was not fasted) and allowed free access to water; they were fed uniformly 4 h after drug administration. Three of the groups were intragastrically administered A9, A146, and A151 at 10 mg / kg, and the remaining 3 groups were intravenously injected with A9, A146, and A151 at 2 mg / kg. The drug solvent was DMSO / EtOH / PEG300 / 0.9% NaCl (5 / 5 / 40 / 50, v / v / v / v).

[0557] Blood samples (0.2 mL) were collected from the jugular vein at 5 min (only for intravenous administration), 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h after dosing, placed in EDTA-K2 tubes, centrifuged at 11,000 rpm for 5 min to separate plasma, and frozen at -70 °C for later measurement. The operations were performed in an ice-water bath. The concentration of A9 in plasma was determined by LC-MS-MS method, and the pharmacokinetic parameters were calculated.

[0558] Table 6. Pharmacokinetic parameters of compounds A9, A146, and A151 in rats

[0559]

[0560] Test Example 7: Tissue distribution test

[0561] Experimental method:

[0562] Sixteen male CD-1 mice were randomly divided into 4 groups, with 4 mice in each group. The mice were fasted for 12 h before the experiment and allowed free access to water; they were fed uniformly 2 h after dosing. The 4 groups of mice were respectively administered A151 (dissolved in DMSO-ethanol-PEG300-saline with a volume ratio of 5 / 5 / 40 / 50) at a dose of 200 mg / kg by gavage. At 1 h, 2 h, 4 h, and 8 h after dosing, the mice were anesthetized, and blood was collected by cardiac puncture. Liver, kidney, heart, and lung tissue samples were collected, and the distribution of A9 in each tissue was determined by HPLC-MS / MS method.

[0563] Table 7. Tissue distribution of metabolite A9 after oral administration of A151 (200 mg / Kg) in mice

[0564]

[0565] Discussion

[0566] The compound of formula I of the present invention has significant anti-SARS-CoV-2 activity. In Vero E6 cells infected with the virus, representative compounds A9 (Example 6), A10 (Example 7), A124 (Example 11), A50 (Example 12), A212 (Example 13), A144 (Example 16), A213 (Example 18), A164 (Example 20), A214 (Example 22), A151 (Example 36) can significantly inhibit virus replication, and the EC 50 values are 0.44 μM, 0.43 μM, 0.25 μM, 0.23 μM, 0.11 μM, 0.24 μM, 0.10 μM, 0.27 μM, 0.11 μM, and 0.31 μM respectively, which are significantly better than the control compound remdesivir (EC 50= 2.0 μM). In addition, compounds such as A11, A12, A52, A70, A72, A75, A77, A131, A171, A180, A181, A188, A196, A215 can significantly inhibit virus replication at a concentration of 5 μM, and the inhibition rate is above 98%, which is significantly higher than the inhibition rate of remdesivir on the virus at this concentration (65%).

[0567] A102, A106, A107, A198 can significantly inhibit the activity of novel coronavirus RdRp, indicating that this type of compound exerts antiviral effects by acting on RdRp.

[0568] The compound of formula I of the present invention also has significant inhibitory effects on other viruses, including respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV OC43), porcine epidemic diarrhea virus (PEDV), Zika virus, and dengue virus (DENV). It is worth noting that the antiviral effect of the deuterated compound is more significant.

[0569] The mouse PK test shows that after oral administration of the deuterated compounds A9 and A11, the drug exposure in plasma is higher than that of GS-441524, which are 1.3 times and 1.2 times the exposure of GS-441524, respectively; the oral bioavailability of A9 in rats is 21.3%, and the oral bioavailabilities of compounds A146 and A151 are 48.5% and 56.7% respectively, which are greatly improved compared with A9.

[0570] The tissue distribution test in mice shows that after oral administration of A151 for 1 hour, the metabolite A9 has the highest concentration in the liver, kidney, lung, and heart, and is evenly distributed, without liver and kidney accumulation.

[0571] From the above data, it can be speculated that the compounds of the present invention have good application prospects for antiviral, especially against novel coronavirus (SARS-CoV-2).

[0572] All the documents mentioned in the present invention are cited in this application for reference as if each document is cited separately for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Wherein, R 1 is a cyano group; R 2 is OR 31 ; R 3 、R 31 and R 5 one of which is selected from C 1-20 alkanoyl, amino C 1-20 alkanoyl; and R 3 、R 31 and R 5 the remaining two of which are selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl; R 4 selected from hydrogen, deuterium, and halogen; R 6 is an amino group; R 7 is hydrogen; R 8 selected from hydrogen, deuterium, a halogen; and X is selected from -CH 2 -, -CD 2 -, -CHD-; And the compound of formula I is not:

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that, R 3 selected from C 1-20 alkanoyl, amino C 1-20 alkanoyl; R 31 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl; and R 5 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that, R 3 is C 1-20 alkanoyl.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that, R 3 、R 31 and R 5 One selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α - aminoisovaleryl, 2 - ethylbutyryl, 3,3 - dimethylbutyryl; and R 3 、R 31 and R 5 The remaining two of R 3 31 and R 5 are selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that, R 3 selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α - aminoisovaleryl, 2 - ethylbutyryl, 3,3 - dimethylbutyryl; R 31 selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl; and R 5 Selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

6. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, Characterized in that, R 8 is deuterium.

7. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of (a) an inhibitor for inhibiting virus replication; and / or (b) a medicament for treating, preventing or alleviating virus infection or related diseases caused by virus infection; Wherein, R 1 is cyano; R 2 is OR 31 ; R 3 , R 31 and R 5 One of the following is selected from C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; R 3 , R 31 and R 5 The other two are selected from hydrogen, C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; R 4 selected from hydrogen, deuterium, and halogen; R 6 is an amino group; R 7 is hydrogen; R 8 selected from hydrogen, deuterium, a halogen; and X is selected from -CH 2 -, -CD 2 -, -CHD-; And the virus is selected from: (1) Human-infecting coronaviruses; (2) Human respiratory syncytial virus (RSV); (3) Flaviviridae viruses: which are selected from dengue virus (DENV), Zika virus; (4) Porcine epidemic diarrhea virus (PEDV).

8. The use according to claim 7, Characterized in that, R 3 selected from C 1-20 alkanoyl, amino C 1-20 alkanoyl; R 31 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl; and R 5 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl.

9. The use according to claim 7, Characterized in that, R 3 is C 1-20 alkanoyl.

10. The use according to claim 7, Characterized in that, R 3 , R 31 and R 5 one of which is selected from the group consisting of formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarboxyl, α-aminoisovaleryl, 2-ethylbutyryl, 3,3-dimethylbutyryl; and R 3 , R 31 and R 5 The remaining two of R 3 , R 31 and R 5 are selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

11. The use according to claim 7, Characterized in that, R 3 selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, 3,3-dimethylbutyryl; R 31 selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl; and R 5 Selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

12. The use according to claim 7, Characterized in that, R 8 is deuterium.

13. The use according to any one of claims 7 to 12, Characterized in that, The virus is selected from: Severe acute respiratory syndrome coronavirus (SARS-CoV), 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1.

14. The use according to any one of claims 7 to 12, Characterized in that, The related diseases caused by the virus are selected from the following group: (D1) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human coronavirus infection; (D2) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human respiratory syncytial virus (RSV) infection; (D3)Dengue fever and its complications caused by dengue virus (DENV); (D4)Infections and their complications caused by Zika virus (Zika); (D5)Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV); (D6)Any combination of the above diseases.

15. The use according to any one of claims 7 to 12, characterized in that the related diseases caused by the virus are: Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2.

16. A pharmaceutical composition, characterized in that the pharmaceutical composition contains: (a1) A compound of formula I or a pharmaceutically acceptable salt thereof: In the formula, R 1 is a cyano group; R 2 is OR 31 ; R 3 , R 31 and R 5 One of the following is selected from C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; and R 3 , R 31 and R 5 The other two are selected from hydrogen, C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; R 4 selected from hydrogen, deuterium, and halogen; R 6 is an amino group; R 7 is hydrogen; R 8 selected from hydrogen, deuterium, a halogen; and X is selected from CH 2 , CD 2 , -CHD-; and (b) a pharmaceutically acceptable carrier.

17. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that R 3 selected from C 1-20 alkanoyl, amino C 1-20 alkanoyl; R 31 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl; and R 5 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl.

18. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that R 3 is C 1-20 alkanoyl.

19. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that R 3 、R 31 and R 5 One selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, 3,3-dimethylbutyryl; and R 3 、R 31 and R 5 The remaining two of R are selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

20. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that R 3 selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, 3,3-dimethylbutyryl; R 31 selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl; and R 5 Selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

21. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that R 8 is deuterium.

22. The use of a pharmaceutical composition according to any one of claims 16 - 21, characterized in that for the preparation of (a) an inhibitor for inhibiting virus production; and / or (b) a drug for treating, preventing, or alleviating virus infection or related diseases caused by virus infection; wherein the virus is selected from: (1) Coronaviruses that infect humans; (2) Human respiratory syncytial virus (RSV); (3) Flaviviridae viruses: selected from dengue virus (DENV), Zika virus (Zika); (4) Porcine epidemic diarrhea virus (PEDV).

23. The use according to any one of claims 16 - 21, characterized in that the virus is selected from: Severe acute respiratory syndrome coronavirus (SARS-CoV), 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1.

24. The use according to any one of claims 16 - 21, It is characterized in that, the related diseases caused by the virus are selected from the following group: (D1) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human coronavirus infection; (D2) Common cold, high-risk symptom infection, respiratory tract infection, pneumonia and its complications caused by human respiratory syncytial virus (RSV) infection; (D3) Dengue fever and its complications caused by dengue virus (DENV); (D4) Infection and its complications caused by Zika virus; (D5) Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV); (D6) Any combination of the above diseases.

25. The use according to any one of claims 16-21, it is characterized in that, the related disease caused by the virus is: novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) caused by SARS-CoV-2.

26. An in vitro non-therapeutic method for inhibiting virus replication, it is characterized in that, comprising the steps of: contacting a compound of formula I or a pharmaceutically acceptable salt thereof or a preparation containing the compound of formula I or a pharmaceutically acceptable salt thereof with a virus, thereby inhibiting the replication of the virus; wherein, R 1 is a cyano group; R 2 is OR 31 ; R 3 , R 31 and R 5 One of the following is selected from C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; and R 3 , R 31 and R 5 The other two are selected from hydrogen, C 1-20 Alkanoyl, amino C 1-20 Alkanoyl; R 4 selected from hydrogen, deuterium, and halogen; R 6 is an amino group; R 7 is hydrogen; R 8 selected from hydrogen, deuterium, a halogen; and X is selected from CH 2 , CD 2 , -CHD-; wherein, the virus is selected from: (1) Coronaviruses that infect humans; (2) Human respiratory syncytial virus (RSV); (3) Flaviviridae viruses: which are selected from dengue virus (DENV), Zika virus; (4) Porcine epidemic diarrhea virus (PEDV).

27. The method according to claim 26, it is characterized in that, R 3 selected from C 1-20 alkanoyl, amino C 1-20 alkanoyl; R 31 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl; and R 5 selected from hydrogen, C 1-20 alkanoyl, amino C 1-20 alkanoyl.

28. The method according to claim 26, it is characterized in that, R 3 is C 1-20 alkanoyl.

29. The method according to claim 26, it is characterized in that, R 3 、R 31 and R 5 One selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, 3,3-dimethylbutyryl; and R 3 、R 31 and R 5 The remaining two of R 3 31 5 are selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

30. The method according to claim 26, it is characterized in that, R 3 selected from formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α - aminoisovaleryl, 2 - ethylbutyryl, 3,3 - dimethylbutyryl; R 31 selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl; and R 5 selected from hydrogen, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl, cyclopropanecarbonyl, α-aminoisovaleryl, 2-ethylbutyryl, and 3,3-dimethylbutyryl.

31. The method according to claim 26, it is characterized in that, R 8 is deuterium.

32. The method according to any one of claims 26-31, it is characterized in that, the virus is selected from: Severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV), Human coronavirus OC43 (Human coronavirus OC43), Human coronavirus 229E (Human coronavirus 229E), Human coronavirus NL63 (Human coronavirus NL63), Human coronavirus HKUl (Human coronavirus HKUl).

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