WO2021213288A1 - 核苷类似物或含有核苷类似物的组合制剂在抗病毒中的应用 - Google Patents

核苷类似物或含有核苷类似物的组合制剂在抗病毒中的应用 Download PDF

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WO2021213288A1
WO2021213288A1 PCT/CN2021/087928 CN2021087928W WO2021213288A1 WO 2021213288 A1 WO2021213288 A1 WO 2021213288A1 CN 2021087928 W CN2021087928 W CN 2021087928W WO 2021213288 A1 WO2021213288 A1 WO 2021213288A1
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alkyl
virus
amino
compound
hydrogen
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PCT/CN2021/087928
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English (en)
French (fr)
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谢元超
肖庚富
何洋
张磊砢
蒋华良
沈敬山
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中国科学院上海药物研究所
中国科学院武汉病毒研究所
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Priority to IL297410A priority Critical patent/IL297410A/en
Priority to CN202180002969.0A priority patent/CN114096543B/zh
Priority to US17/996,430 priority patent/US11919923B2/en
Priority to JP2022564346A priority patent/JP2023526179A/ja
Priority to BR112022021226A priority patent/BR112022021226A2/pt
Priority to KR1020227032612A priority patent/KR20220143919A/ko
Priority to AU2021260618A priority patent/AU2021260618B2/en
Priority to CA3171091A priority patent/CA3171091A1/en
Application filed by 中国科学院上海药物研究所, 中国科学院武汉病毒研究所 filed Critical 中国科学院上海药物研究所
Priority to EP21792567.6A priority patent/EP4141007A1/en
Priority to CN202211058832.XA priority patent/CN115448924A/zh
Priority to MX2022013270A priority patent/MX2022013270A/es
Publication of WO2021213288A1 publication Critical patent/WO2021213288A1/zh
Priority to CONC2022/0015415A priority patent/CO2022015415A2/es
Priority to US18/519,408 priority patent/US20240140975A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/06—Heterocyclic radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/005—Sugars; Derivatives thereof; Nucleosides; Nucleotides; Nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of medicine, in particular to the application of nucleoside analogues or combined preparations containing nucleoside analogues in anti-virus.
  • viruses are pathogenic microorganisms that are extremely small, lack independent metabolism, and exist in a parasitic manner. There are many types of viruses. At present, many viruses that are highly contagious and highly pathogenic to humans have been discovered. These viruses often cause local or even global outbreaks of infectious diseases, which are extremely harmful to human society, such as influenza viruses. , Respiratory syncytial virus (RSV), parainfluenza virus, atypical pneumonia (SARS) virus, Middle East respiratory syndrome (MERS) virus, Ebola virus, etc. Some viruses can also infect animals and cause various mild to severe diseases. At the same time, animals have become the source of infection of these viruses, and humans cannot prevent them.
  • RSV Respiratory syncytial virus
  • SARS atypical pneumonia
  • MERS Middle East respiratory syndrome
  • Ebola virus etc.
  • Coronaviruses belong to the order Nested Virus, Coronaviridae, and Coronavirus. They are a large class of single-stranded positive-stranded RNA viruses that are widespread in nature and can cause diseases of the respiratory tract, digestive tract and nervous system of humans and animals. According to the phylogenetic tree, coronaviruses can be divided into four genera: ⁇ , ⁇ , ⁇ , and ⁇ . Among them, ⁇ genus coronaviruses can be divided into four independent subgroups A, B, C, and D.
  • SARS-CoV-2 SARS-CoV
  • SARS-CoV SARS-CoV
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • Coronavirus can also infect a variety of mammals, including bats, pigs, dogs, cats, mice, cattle, horses, and camels. Most of these viruses belong to the genus ⁇ and ⁇ .
  • Porcine Epidemic Diarrhea Virus (PEDV) is a type of coronavirus that can cause acute intestinal infectious diseases in pigs. Pigs of all ages can be infected. Among them, suckling pigs and newborn piglets suffer the most. The outbreak of PEDV suffered heavy losses.
  • Respiratory viral infections are the most common and most widespread type of viral infectious diseases in clinical practice, causing a large number of deaths in the world every year.
  • influenza virus influenza virus
  • respiratory syncytial virus respiratory syncytial virus
  • parainfluenza virus etc.
  • respiratory infections and cause pneumonia which is an important killer that threatens human life and health.
  • the purpose 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 infection.
  • the present invention provides the nucleoside analogues represented by formula I and their compositions in anti-virus (such as coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or pig Epidemic diarrhea virus), especially the use of anti-new coronavirus (SARS-CoV-2).
  • anti-virus such as coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or pig Epidemic diarrhea virus
  • SARS-CoV-2 anti-new coronavirus
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, amino, C 1-6 alkyl substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C Amino substituted by 1-6 alkanoyl, halogenated 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, thiocyano (-SCN ), cyanooxy (-OCN);
  • 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;
  • 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 amino acid, wherein the carboxyl group of the ⁇ -amino acid is connected to the hydroxyl group on the furan ring by an ester bond;
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azide 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 alkyne Oxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyano , Cyanooxy;
  • R 5 is selected from R 3 ,
  • R 6 is selected from amino, hydroxyl, halogen, cyano, cyanooxy , thiocyano, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;
  • R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl substituted carbamoyl, C 1-6 alkoxy amide, C 1-6 alkoxycarbonyl, hydroxyl, hydroxyl 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;
  • X is selected from -CH 2 -, -CD 2 -, -CHD-;
  • R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 12 is selected from C 1-20 alkyl
  • M is each independently selected from hydrogen, metal, -NH 4 , or protonated organic amine
  • each position denoted as deuterium (D) has a deuterium enrichment of at least 50%; preferably, each position denoted as deuterium (D) has a deuterium enrichment of at least 80%; more preferably Specifically, each position denoted as deuterium (D) has a deuterium enrichment of at least 90%; most preferably, each position denoted as deuterium (D) has a deuterium enrichment of at least 95%.
  • the 5-15 membered heteroaryl group contains 1, 2, 3 or 4 heteroatoms selected from N, O and S.
  • the metal is selected from the following group: alkali metals, alkaline earth metals, or combinations thereof.
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, methyl, chloromethyl, fluoromethyl, vinyl, ethynyl, cyclopropyl, carbamoyl, hydroxymethyl, methoxy, methyl Acyl, amidino; and/or
  • R 2 is selected from halogen, cyano, amino, formyl, OR 3 ;
  • R 3 is selected from hydrogen, C 1-20 alkyl acyl group, ⁇ -amino acid, and the carboxyl group of the ⁇ -amino acid is connected to the hydroxyl group on the furan ring by an ester bond; preferably, the ⁇ -amino acid is selected from the following group: C Acid, valine, isoleucine, tryptophan, phenylalanine; and/or
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, methyl, chloromethyl, fluoromethyl, difluoromethyl, vinyl, ethynyl, cyclopropyl, hydroxymethyl, azidomethyl (-CH 2 N 3 ), formyl, acetyl, formamido, acetamido; and/or
  • R 5 is selected from R 3 , and / or
  • R 6 is selected from amino, hydroxyl, halogen, cyano, methylamino (-NH 2 CH 3 ), NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ; and/or
  • R 7 is selected from hydrogen, deuterium, halogen, amino; and/or
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, N-methylcarbamoyl (CH 3 NHCO-), methyl, ethyl, ethynyl, methoxycarbonyl, ethoxycarbonyl, hydroxyl, 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-); and/or
  • X is selected from -CH 2 -, -CD 2 -, -CHD-;
  • R 9 is selected from C 6-20 aryl, 5-15 membered heteroaryl.
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl; and/or
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl; and/or
  • R 12 is selected from C 1-20 alkyl
  • M is each independently selected from hydrogen, zinc, magnesium, calcium, sodium, potassium, NH 4 , protonated trimethylamine, protonated triethylamine, and protonated tri-n-butylamine.
  • the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and X are each independently the compound in the embodiment (such as in A1 to A221 Any compound) corresponding to the specific group.
  • the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof, having the following structures:
  • the compound of formula (I) is selected from the following group:
  • the compound of formula (I) is selected from the group consisting of compounds 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.
  • an active ingredient or a preparation containing the active ingredient the active ingredient being a compound represented by formula I or a pharmaceutically acceptable salt or crystal hydrate thereof Or its solvates or prodrugs:
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, amino, C 1-6 alkyl substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C Amino substituted by 1-6 alkanoyl, halogenated 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, thiocyano (-SCN ), cyanooxy (-OCN);
  • 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;
  • 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 amino acid, wherein the carboxyl group of the ⁇ -amino acid is connected to the hydroxyl group on the furan ring by an ester bond;
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azide 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 alkyne Oxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyano , Cyanooxy;
  • R 5 is selected from R 3 ,
  • R 6 is selected from amino, hydroxyl, halogen, cyano, cyanooxy , thiocyano, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;
  • R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl substituted carbamoyl, C 1-6 alkoxy amide, C 1-6 alkoxycarbonyl, hydroxyl, hydroxyl 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;
  • X is selected from -CH 2 -, -CD 2 -, -CHD-;
  • R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 12 is selected from C 1-20 alkyl
  • M is each independently selected from hydrogen, metal, -NH 4 or protonated organic amines
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors that inhibit viral replication; and/or (b) treat and/or prevent and alleviate related diseases caused by viral infections. drug.
  • each position denoted as deuterium (D) has a deuterium enrichment of at least 50%; preferably, each position denoted as deuterium (D) has a deuterium enrichment of at least 80%; more preferably Specifically, each position denoted as deuterium (D) has a deuterium enrichment of at least 90%; most preferably, each position denoted as deuterium (D) has a deuterium enrichment of at least 95%.
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors for inhibiting the replication of coronavirus; and/or (b) treating and/or preventing and alleviating coronavirus Drugs for related diseases caused by viral infections.
  • the active ingredient or a preparation containing the active ingredient is used to prepare (a) an inhibitor for inhibiting the replication of respiratory syncytial virus (RSV); and/or (b) treatment and/or Drugs to prevent and alleviate related diseases caused by respiratory syncytial virus (RSV) infection.
  • RSV respiratory syncytial virus
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors for inhibiting influenza virus replication; and/or (b) treating and/or preventing and alleviating influenza Drugs for related diseases caused by viral infections.
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors for inhibiting the replication of Flaviviridae viruses; and/or (b) treatment and/or prevention and alleviation Drugs for related diseases caused by infections of flaviviridae viruses.
  • the active ingredient or a preparation containing the active ingredient is used to prepare (a) inhibitors for inhibiting the replication of viruses of the filoviridae family; and/or (b) treatment and/or prevention, Drugs for alleviating related diseases caused by filoviridae virus infection.
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors of porcine epidemic diarrhea virus (PEDV) replication; and/or (b) treatment and/ Or prevent and alleviate related diseases caused by porcine epidemic diarrhea virus (PEDV) infection.
  • PDV porcine epidemic diarrhea virus
  • the active ingredient or a preparation containing the active ingredient is used to prepare (a) an inhibitor for inhibiting the replication of the 2019 novel coronavirus (SARS-Cov-2); and/or (b ) Drugs for the treatment and/or prevention and alleviation of related diseases caused by the 2019 novel coronavirus (SARS-CoV-2) infection.
  • SARS-Cov-2 2019 novel coronavirus
  • SARS-CoV-2 2019 novel coronavirus
  • the virus is selected from:
  • Coronavirus that infects humans severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome, SARS-CoV), 2019 new coronavirus (2019-nCoV or SARS-CoV-2), Middle East Respiratory Syndrome Sign the coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus, MERS-CoV)
  • coronavirus that causes the common cold is preferably selected from the following group: Human Coronavirus OC43 (Human Coronavirus OC43), Human Coronavirus 229E (Human Coronavirus 229E), Human Coronavirus NL63 (Human coronavirus NL63), human coronavirus HKUl (Human coronavirus HKUl);
  • Human influenza virus influenza A virus, influenza B virus, and influenza C virus;
  • Flaviviridae virus hepatitis C virus (HCV), dengue fever virus (DENV), Zika virus (Zika);
  • Filovirus family viruses Marburg virus (MBV), Ebola virus (EBV);
  • Coronavirus that infects other mammals Porcine Epidemic Diarrhea Virus (PEDV).
  • the related disease caused by the virus is selected from the following group:
  • D2 Common cold, high-risk symptom infection, respiratory infection, pneumonia and its complications caused by human respiratory syncytial virus (RSV) infection;
  • RSV human respiratory syncytial virus
  • D5 Dengue fever and its complications caused by dengue fever virus (DENV);
  • D9 Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV);
  • the related disease caused by the 2019 novel coronavirus infection is selected from the following group: respiratory tract infection, pneumonia and its complications, or a combination thereof.
  • the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.
  • the active ingredient is a nucleoside analog or a pharmaceutically acceptable salt or a crystalline hydrate or a solvate or a prodrug thereof selected from the following group:
  • the compound of formula (I) is selected from the group consisting of compounds 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.
  • the preparation (or pharmaceutical composition containing the compound) may also contain other antiviral drugs.
  • the other antiviral drugs also include additional components selected from the following group:
  • Remdesivir (Remdesivir or GS-5734), Favipiravir, Galidesivir, GS-441524, NHC (EIDD-1931), EID-2801, GC-376, Lopinavir (Lopinavir), Ritonavir, Nelfinavir; Chloroquine, hydroxychloroquine, cyclosporine, Carrimycin, baicalin, baicalin (baicalein), forsythoside, chlorogenic acid, emodin, mycophenolic acid, mycophenolate mofetil, naphthoquine, Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitrazole Nitazoxanide, Darunavir, Arbidol, Camostat, Niclosamide, Baricitinib, Rucotinib (Ruxolitinib), Dasatinib, Saquinavir, Beclabuvir, Simeprevir
  • the pharmaceutical composition containing the compound further includes administering a therapeutically effective amount of at least one other therapeutic agent selected from the group consisting of corticosteroids, anti-inflammatory signal transduction modulators, ⁇ 2-adrenergic receptors Body agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline and other drugs used to treat viral infections; or combinations thereof.
  • at least one other therapeutic agent selected from the group consisting of corticosteroids, anti-inflammatory signal transduction modulators, ⁇ 2-adrenergic receptors Body agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline and other drugs used to treat viral infections; or combinations thereof.
  • the formulation is a pharmaceutical composition.
  • the formulation includes: oral formulations and non-oral formulations.
  • the preparations include powders, granules, capsules, injections, inhalants, tinctures, oral liquids, tablets, lozenges, or dripping pills.
  • a pharmaceutical composition which contains:
  • the first active ingredient, said first active ingredient is a compound represented by formula I or a pharmaceutically acceptable salt or crystalline hydrate or solvate or prodrug thereof:
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, amino, C 1-6 alkyl substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C Amino substituted by 1-6 alkanoyl, halogenated 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, thiocyano (-SCN ), cyanooxy (-OCN);
  • 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;
  • 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 amino acid, wherein the carboxyl group of the ⁇ -amino acid is connected to the hydroxyl group on the furan ring by an ester bond;
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azide 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 alkyne Oxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyano , Cyanooxy;
  • R 5 is selected from R 3 ,
  • R 6 is selected from amino, hydroxyl, halogen, cyano, cyanooxy , thiocyano, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;
  • R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl substituted carbamoyl, C 1-6 alkoxy amide, C 1-6 alkoxycarbonyl, hydroxyl, hydroxyl 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;
  • X is selected from CH 2 , CD 2 , -CHD-;
  • R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 12 is selected from C 1-20 alkyl
  • M is each independently selected from hydrogen, metal, -NH 4 or protonated organic amines
  • composition further contains (a2) a second active ingredient
  • the second active ingredient is an antiviral drug, which is selected from the following group: interferon, RNA-dependent RNA polymerase inhibitor (such as Remdesivir (Remdesivir or GS-5734), favipiravir ( favipiravir), Galidesivir, GS-441524, NHC (EIDD-1931), EIDD-2801), 3CL protease inhibitors (such as GC-376), Lopinavir (Lopinavir), Ritonavir (Ritonavir), Nefe Nelfinavir; Chloroquine, hydroxychloroquine, cyclosporine, Carrimycin, baicalin, baicalein, forsythiaside ( forsythoside, chlorogenic acid, emodin, mycophenolic acid, Mycophenolate mofetil, Naphthoquine, Ciclesonide, Lime Bavirin (Ribavirin), penciclovir (Penciclovir), lefluor,
  • the second active ingredient is selected from the group consisting of bronchodilators and corticosteroids for the treatment of respiratory tract infections
  • the corticosteroids include dexamethasone, dexamethasone sodium phosphate, fluorometholone, and fluoroacetate Milong, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate , Methylprednisolone, fluocinolone, flucinonide, flunisolide, fluocortin-21-butylate (fluocortin-21-butylate), flumethasone, flumethasone pivalate, budesonide, halobenium propionate Tasso, mometasone furoate, fluticasone propionate, cicleson
  • the second active ingredient is selected from the following group: Zinc, Fingolimod, Vitamin C, Olmesartan Medoxomil, Valsartan , Losartan, Thalidomide, Glycyrrhizic Acid, Artemisinin, Dihydroartemisinin, Artesunate, Artemisinone (Artemisone), Azithromycin, Escin, Naproxen, or a combination thereof.
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, methyl, chloromethyl, fluoromethyl, vinyl, ethynyl, cyclopropyl, carbamoyl, hydroxymethyl, methoxy, methyl Acyl, amidino;
  • R 2 is selected from halogen, cyano, amino, formyl, OR 3 ;
  • R 3 is selected from hydrogen, C 1-20 alkyl acyl, ⁇ -amino acid, and the carboxyl group of the ⁇ -amino acid is connected with the hydroxyl group on the furan ring by an ester bond; preferably, the ⁇ -amino acid is selected from alanine, Valine, isoleucine, tryptophan, phenylalanine;
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, methyl, chloromethyl, fluoromethyl, difluoromethyl, vinyl, ethynyl, cyclopropyl, hydroxymethyl, azidomethyl (-CH 2 N 3 ), formyl, acetyl, formamido, acetamido;
  • R 5 is selected from R 3 ,
  • R 6 is selected from amino, hydroxyl, halogen, cyano, methylamino (-NH 2 CH 3 ), NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;
  • R 7 is selected from hydrogen, deuterium, halogen, and amino
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, N-methylcarbamoyl (CH 3 NHCO-), methyl, ethyl, ethynyl, methoxycarbonyl, ethoxycarbonyl, hydroxyl, 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-);
  • X is selected from CH 2 , CD 2 , -CHD-;
  • R 9 is selected from C 6-20 aryl and 5-15 membered heteroaryl
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 12 is selected from C 1-20 alkyl
  • M independently selects hydrogen, zinc, magnesium, calcium, sodium, potassium, NH 4 , protonated trimethylamine, protonated triethylamine, and protonated tri-n-butylamine.
  • the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.
  • the compound of formula (I) is selected from the group consisting of compounds 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, A
  • the compound of formula (I) is selected from the group consisting of compounds 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.
  • the second active ingredient is selected from the following group: (Y1) RNA replicase inhibitor (such as Remdesivir (Remdesivir or GS-5734), favipiravir, Galidesivir , GS-441524, NHC, EIDD-2801); (Y2) Lopinavir (Lopinavir); (Y3) Ritonavir (Ritonavir); (Y4) Fapilavir; (Y5) Chloroquine, Hydroxychloroquine, or a pharmaceutically acceptable salt thereof (such as chloroquine phosphate), (Y6) Nelfinavir; (Y7) any combination of the above Y1 to Y6.
  • RNA replicase inhibitor such as Remdesivir (Remdesivir or GS-5734), favipiravir, Galidesivir , GS-441524, NHC, EIDD-2801
  • Y2 Lopinavir (Lopinavir);
  • the pharmaceutical composition is used to inhibit the replication of coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or porcine epidemic diarrhea virus (PEDV).
  • coronavirus influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or porcine epidemic diarrhea virus (PEDV).
  • PEDV porcine epidemic diarrhea virus
  • the drug is used to inhibit the replication of the 2019 novel coronavirus (SARS-CoV-2).
  • SARS-CoV-2 2019 novel coronavirus
  • the use of the pharmaceutical composition according to the third aspect of the present invention is provided, which is used to prepare (a) inhibit coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, silk Virus and/or Porcine Epidemic Diarrhea Virus (PEDV); and/or (b) Treatment and/or prevention and alleviation of coronaviruses, influenza viruses, respiratory syncytial virus, flaviviridae viruses, filoviridae Drugs for related diseases caused by virus and/or porcine epidemic diarrhea virus (PEDV) infection.
  • PEDV Porcine Epidemic Diarrhea Virus
  • it is used for the preparation of (a) inhibitors for inhibiting the replication of 2019 new coronavirus (SARS-CoV-2); and/or (b) treatment and/or prevention and alleviation of 2019 new coronavirus (SARS-CoV-2) -CoV-2) Drugs for related diseases caused by infection.
  • SARS-CoV-2 2019 new coronavirus
  • SARS-CoV-2 2019 new coronavirus
  • SARS-CoV-2) -CoV-2 2019 new coronavirus
  • a method for inhibiting virus replication which includes the steps:
  • the first active ingredient is a compound of formula I or a pharmaceutically acceptable salt or crystalline hydrate or solvate or prodrug thereof:
  • R 1 is selected from hydrogen, deuterium, halogen, cyano, azide, amino, C 1-6 alkyl substituted amino, C 1-6 acyl, C 1-6 alkyl, C 1-6 alkoxy, C Amino substituted by 1-6 alkanoyl, halogenated 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, thiocyano (-SCN ), cyanooxy (-OCN);
  • 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;
  • 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 amino acid, the carboxyl group of the ⁇ -amino acid is connected to the hydroxyl group on the furan ring by an ester bond.
  • the ⁇ -amino acid is selected from the group consisting of alanine, valine, isoleucine, tryptophan, and phenylalanine acid;
  • R 4 is selected from hydrogen, deuterium, halogen, azide, cyano, C 1-6 alkyl, halo C 1-6 alkyl, azide 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 alkyne Oxy, C 1-6 alkoxy C 1-6 alkyl, amino C 1-6 alkyl, C 1-6 alkylamino C 1-6 alkyl, amidino, guanidino, ureido, thiocyano , Cyanooxy;
  • R 5 is selected from R 3 ,
  • R 6 is selected from amino, hydroxyl, halogen, cyano, cyanooxy , thiocyano, C 1-6 alkoxy, C 1-6 alkylamino, NHOH, NHCOR 12 , NHOCOR 12 , NHCOOR 12 ;
  • R 7 is selected from hydrogen, deuterium, halogen, amino, methyl, NHCOR 12 , NHCOOR 12 ;
  • R 8 is selected from hydrogen, deuterium, halogen, cyano, carbamoyl, C 1-6 alkyl substituted carbamoyl, C 1-6 alkoxy amide, C 1-6 alkoxycarbonyl, hydroxyl, hydroxyl 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 1-6 alkoxy, alkenyl , C 2-6 alkynyl;
  • X is selected from CH 2 , CD 2 , -CHD-;
  • R 9 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 10 is selected from C 1-18 alkyl, methylene C 6-20 aryl;
  • R 11 is selected from C 1-6 alkyl, C 3-6 cycloalkyl, C 6-20 aryl, 5-15 membered heteroaryl;
  • R 12 is selected from C 1-20 alkyl
  • M is each independently selected from hydrogen, metal, NH 4 or protonated organic amines
  • the virus is selected from the following group: coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus, porcine epidemic diarrhea virus, or a combination thereof.
  • the virus is 2019 novel coronavirus (SARS-CoV-2).
  • the compound of formula (I) is any one of compounds A1 to A221, or a combination thereof.
  • the compound of formula (I) is selected from the group consisting of compounds 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, A
  • the compound of formula (I) is selected from the group consisting of compounds 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.
  • the method is an in vitro method.
  • the method is non-therapeutic and non-diagnostic.
  • a method for (a) inhibiting virus replication and/or (b) treating and/or preventing and alleviating related diseases caused by virus infection including the steps of: administering safe and effective administration to a subject in need An amount of the compound represented by formula I or its pharmaceutically acceptable salt or its crystalline hydrate or its solvate or its prodrug:
  • 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.
  • the subject is a mammal, such as a human.
  • Figure 1 shows that in the embodiment of the present invention, a virus control group, A151-S oral 50mg/kg group, A151-S oral 100mg/kg, remdesivir oral 50mg/kg group, and remdesivir intraperitoneal injection 50mg/kg In kg group, virus RNA copies 2 days after administration.
  • Figure 2 shows that in the embodiment of the present invention, the virus control group, A151-S oral 50mg/kg group, A151-S oral 100mg/kg, remdesivir oral 50mg/kg group, and remdesivir intraperitoneal injection 50mg/kg are set. kg group, virus RNA copy status 5 days after administration.
  • Fig. 3 shows the virus RNA copy situation of the virus control group and the A151-S intraperitoneal injection 100 mg/kg group in the embodiment of the present invention 5 days after administration.
  • the inventors unexpectedly developed a class of active ingredients that can effectively inhibit virus replication for the first time.
  • the active ingredients of the present invention can effectively inhibit the replication and viability of various viruses such as the 2019 novel coronavirus (SARS-CoV-2), and therefore can be used to inhibit coronavirus, influenza virus, respiratory syncytial virus, and flaviviridae. Replication of viruses, filoviridae and/or porcine epidemic diarrhea virus.
  • SARS-CoV-2 2019 novel coronavirus
  • the present invention has been completed on this basis.
  • nucleoside analogs represented by formula (I) discloses the use of nucleoside analogs represented by formula (I) and their compositions in anti-virus, such as anti-coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, silk
  • anti-virus such as anti-coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, silk
  • PDV Porcine Epidemic Diarrhea Virus
  • the nucleoside analogue represented by the formula (I) has an excellent inhibitory effect on the replication of viruses such as SARS-CoV-2, and has a good clinical application prospect.
  • the active compound of the present invention As used herein, “the active compound of the present invention”, “the active ingredient of the present invention”, “the nucleoside analog of the present invention” and “the active compound of the present invention for inhibiting the replication of coronavirus” are used interchangeably and refer to having excellent Nucleoside analogs that inhibit the replication of coronaviruses include the compound represented by formula I, or a pharmaceutically acceptable salt, or a solvate thereof, or a prodrug, or a combination thereof.
  • the formulation of the present invention refers to a formulation containing the active compound of the present invention.
  • new coronavirus As used herein, the terms “new coronavirus”, “2019-nCoV” or “SARS-CoV-2” are used interchangeably.
  • the 2019 new coronavirus is the seventh type of coronavirus that is known to infect humans and causes new coronary pneumonia. (COVID-19) is one of the serious infectious diseases that threaten human health around the world.
  • halogen generally refers to fluorine, chlorine, bromine and iodine; preferably fluorine, chlorine or bromine; more preferably fluorine or chlorine.
  • C n -C m and C nm are used interchangeably and refer to having n to m carbon atoms.
  • C 1 -C 6 alkyl refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl Group, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, isohexyl, 3-methylpentyl or n-hexyl, etc., preferably methyl Group, ethyl, n-propyl, isopropyl, butyl, isobutyl or tert-butyl.
  • Halogenated C 1 -C 6 alkyl refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms whose 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.;
  • C 1 -C 6 alkoxy refers to a straight or branched chain alkoxy containing 1-6 carbon atoms, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy , Isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentyloxy, isohexyloxy, 3-methylpentoxy or n-hexyloxy, etc., preferably Methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy;
  • C1-C6 alkanoyl refers to direct or Branched alkanoyl groups, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, tert-butyryl or hexanoyl, etc.
  • a C 1-6 alkyl substituted amino group means that the hydrogen atom of the amino group is substituted with one or more C 1 -C 6 alkyl groups, such as -NHCH 3 , -N(CH 3 ) 2 and the like.
  • a C 1-6 alkanoyl substituted amino group means that the hydrogen atom of the amino group is substituted with one or more C 1 -C 6 alkyl groups, such as -NHCOCH 3 , -NHCOCH 2 CH 3 and the like.
  • C 2 -C 6 alkenyl refers to a straight or branched 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-butanyl Alkenyl, 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 or 2-ethyl-1 -Allyl and so on.
  • C 2 -C 6 alkynyl refers to a straight or branched 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.
  • C 2 -C 6 alkenyloxy group refers to a straight or branched chain alkenyloxy group containing 1-3 double bonds and 2-6 carbon atoms, such as vinyloxy, 1-propenoxy, 1-methyl 1-propenyloxy, 2-methyl-1-propenyloxy, 1-pentenyloxy, 1,3-pentadienyloxy or 2-pentenyloxy, etc.
  • C 2 -C 6 alkynyloxy refers to a straight or branched alkynyloxy group containing 2-6 carbon atoms, for example, ethynyloxy, 2-propynyloxy, 2-butynyloxy, 3-butyne Oxy, 1-methyl-2-propynyloxy, 2-pentynyloxy or 2-hexynyloxy, etc.
  • Amino C 1-20 alkanoyl group refers to a linear or branched alkanoyl group containing 1-20 carbon atoms connected to one carbon atom with an amino group, such as -COCH 2 NH 2 , -COCH 2 CH 2 NH 2 and the like.
  • C 1-6 Alkylamino C 1-6 Alkanoyl means that the nitrogen atom of the C 1-6 alkyl substituted amino group is connected to the non-carbonyl carbon atom of a straight or branched chain alkanoyl group containing 1-6 carbon atoms, such as- COCH 2 NHCH 3 , -COCH 2 CH 2 NHCH 2 CH 3 and so on.
  • C 1-6 alkoxy C 1-6 alkyl refers to the oxygen atom of a straight or branched chain alkoxy containing 1-6 carbon atoms and the carbon atom of the C 1-6 alkyl group, such as -CH 2 OCH 3 , -CH 2 CH 2 OCH 2 CH 3 and so on.
  • Amino C 1-6 alkyl refers to one carbon atom of a straight or branched chain alkyl containing 1-6 carbon atoms connected to an 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 and so on.
  • the C 1-6 alkyl substituted carbamoyl group means that the hydrogen atom on the carbamoyl group is substituted by one or two identical or different C 1 -C 6 alkyl groups, such as -CONHMe, -CONHEt, -CON(Me ) Et, -CONEt 2 or -CONMe 2 etc.
  • Hydroxy C 1 -C 6 alkyl refers to a straight or branched chain alkyl group containing 1-6 carbon atoms with one carbon atom 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 2 OH, or -CH 2 CH(CH 3 )CH 2 OH, etc.
  • the C 1-6 alkoxy amide group refers to a straight or branched chain alkoxy group containing 1-6 carbon atoms whose oxygen atom is connected to the carbonyl group of the amide group, such as -NHCOOCH 3 , -NHCOOCH 2 CH 3 and the like.
  • the C 1-6 alkoxycarbonyl group has a linear or branched chain alkoxy group containing 1-6 carbon atoms.
  • the oxygen atom of the alkoxy group is connected to the carbonyl group, such as -COOCH 3 , -COOCH 2 CH 3 and the like.
  • Coronavirus belongs to the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), which is an enveloped positive-stranded RNA virus, and its subfamily includes four genera of ⁇ , ⁇ , ⁇ and ⁇ .
  • HCoV-229E and HCoV-NL63 belong to the ⁇ genus coronavirus
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all ⁇ genus coronavirus.
  • Virus. SARS-CoV-2 is also called 2019-nCoV.
  • the new 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 it also belongs to the beta coronavirus.
  • the genome of this type of virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses in the genome.
  • the codes include replicase, spike protein, envelope protein, envelope protein, and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to produce non-structural proteins (such as RNA polymerase and unwinding). Enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • Influenza virus is abbreviated as influenza virus. Common influenza viruses are divided into types A (A), B (B), C (C) and D (D). Influenza viruses can cause infection and disease in many animals such as humans, poultry, pigs, horses, and bats, and are the pathogens of human and animal diseases such as human influenza, avian influenza, swine influenza, and equine influenza.
  • influenza viruses 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 viruses often undergo antigenic mutations and can be further divided into H1N1, H3N2, H5N1, H7N9 and other subtypes.
  • Respiratory syncytial virus (RSV, abbreviated as syncytial virus, also belongs to the Paramyxoviridae) is the most common pathogen that causes viral pneumonia in children and can cause interstitial pneumonia.
  • RSV is similar to the parainfluenza virus.
  • the size of the virus particle is about 150nm, which is slightly smaller than the parainfluenza virus. It is an RNA virus.
  • Flaviviridae viruses are a class of RNA viruses that mainly infect mammals, including 3 virus genera, flavivirus, pestivirus and hepacivirus.
  • Dengue fever virus (DENV) and Zika virus (Zika) belong to the flavivirus genus and are transmitted by mosquito vectors. Dengue virus infection can cause obvious fever and pain symptoms. Severe dengue fever symptoms can also manifest as headache, nausea, vomiting, unconsciousness, and even shock.
  • Zika virus (Zika) infection is similar to dengue fever, and is generally mild.
  • Hepatitis C virus (HCV) belongs to the genus of hepatitis C virus and is the causative agent of chronic hepatitis C, which can cause liver cirrhosis and liver cancer.
  • the Filovirus family currently contains three genera, namely Ebolavirus, Marburg virus, and Cuevavirus. Both Marburg virus and Ebola virus can cause severe hemorrhagic fever. Humans will experience high fever and bleeding symptoms after infection, which can further lead to shock, organ failure, and death.
  • PEDV Porcine Epidemic Diarrhea Virus
  • Porcine Epidemic Diarrhea Virus belongs to the genus Coronavirus of the Coronavirus family. Porcine epidemic diarrhea is an acute intestinal infection of piglets and fattening pigs caused by PEDV virus.
  • PEDV virus enters the small intestine directly after being infected through the mouth and nose.
  • the replication of PEDV virus can be carried out in the cytoplasm of villi epithelial cells in the small intestine and colon.
  • PEDV can cause diarrhea, which is osmotic diarrhea. Severe diarrhea causes dehydration and is the main cause of death in sick pigs.
  • an active ingredient that can effectively inhibit the replication of coronavirus, influenza virus, respiratory syncytial virus and/or porcine epidemic diarrhea virus (PEDV), especially 2019 novel coronavirus (SARS-CoV-2) .
  • PEDV porcine epidemic diarrhea virus
  • SARS-CoV-2 2019 novel coronavirus
  • the active ingredient is selected from the nucleoside analog represented by formula (I) described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a prodrug, or a combination thereof, or Crystals, or solvates thereof.
  • nucleoside analogues are the compounds prepared in the examples or their pharmaceutically acceptable salts, or their prodrugs, or their combinations, or their crystals, or their solvates, especially in compounds A1 to A221 Any compound, or combination thereof.
  • the compound of formula (I) is selected from the group consisting of compounds 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, A
  • Tests have shown that the active ingredients of the present invention can effectively inhibit the replication of the 2019 novel coronavirus (SARS-CoV-2), thereby preventing, treating and/or alleviating SARS-CoV-2 related diseases.
  • SARS-CoV-2 2019 novel coronavirus
  • the active compound of the present invention and “the active compound of the present invention for inhibiting viral replication” are used interchangeably, and refer to compounds having excellent activity for inhibiting viral replication, including formula I nucleoside analogues or their pharmacological agents.
  • the active ingredient of the present invention includes a compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate, or prodrug thereof. It should be understood that the active ingredient of the present invention also includes the crystal form, amorphous compound, and deuterated compound of the active compound of the present invention.
  • the "pharmaceutically acceptable salt” refers to the reaction of the active compound of the present invention with an inorganic acid or an organic acid to form a conventional non-toxic salt.
  • conventional non-toxic salts can be prepared by reacting the active compound of the present invention with inorganic or organic acids.
  • the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, and phosphoric acid.
  • Acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic 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-aminobenzenesulfonate Acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the active compound of the present invention is combined with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic
  • the active ingredients of the present invention are particularly suitable for combined use with other antiviral drugs or anti-coronavirus drugs.
  • Representative other antiviral drugs or anti-coronavirus drugs include (but are not limited to): interferon, RNA-dependent RNA polymerase inhibitors (such as Remdesivir (Remdesivir or GS-5734), favipiravir (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 (Ribavirin), Pen
  • Additional active therapeutic agents for the treatment of respiratory symptoms and sequelae of infection can also be used in combination with the compound of formula I.
  • Other agents are preferably administered orally or by direct inhalation.
  • other preferred additional therapeutic agents for the treatment of respiratory infections in combination with compounds of formula I include, but are not limited to, bronchodilators and corticosteroids.
  • Glucocorticoids originally introduced as asthma treatments in 1950 (Carryer, Journal of Allergy, 21, 282-287, 1950), remain the most effective and consistently effective treatments for the disease, but they are not yet fully understood The mechanism of action (Morris, J. Allergy Clin. Immunol., 75(1Pt) 1-13, 1985).
  • oral glucocorticoid therapy is associated with far-reaching undesirable side effects, such as central obesity, hypertension, glaucoma, glucose intolerance, accelerated cataract formation, bone mineral loss, and psychological effects. All these limit their use as long-term therapeutics (Goodman and Gilman, 10th edition, 2001).
  • the solution to systemic side effects is to deliver steroid drugs directly to the site of inflammation.
  • corticosteroids Inhaled corticosteroids (ICS) have been developed to alleviate the serious side effects of oral steroids.
  • corticosteroids that can be used in combination with the compound of formula I are dexamethasone, dexamethasone sodium phosphate, flumetholone, flumetholone acetate, loteprednol, loteprednol etabonate, hydrocortisone Pine, prednisolone, fludrocortisone, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone, flucinonide, flunisolide, fluoride Fluocortin-21-butylate, flumethasone, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide ; Or a pharmaceutical
  • anti-inflammatory signal transduction modulators such as phosphodiesterase inhibitors (such as PDE-4, PDE-5 or PDE-7 specific), transcription factor inhibitors (such as by IKK inhibition (blocking NF ⁇ B) or kinase inhibitors (such as blocking P38MAP, JNK, PI3K, EGFR, or Syk) is a logical way to cut inflammation, because these small molecules target a limited number of common intracellular pathways-yes Those signal transduction pathways that are key points of anti-inflammatory therapeutic intervention (see, reviewed by PJ Barnes, 2006).
  • 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-pyridyl)-4-(difluoromethoxy)-8-[(methylsulfonyl)amino]-1-dibenzofuran Amide (PDE-4 inhibitor Omisite); N-(3,5-Dichloro-pyridin-4-y
  • Combinations of bronchodilators including inhaled ⁇ 2-adrenergic receptor agonists such as formoterol, salbutamol or salmeterol and compounds of formula I are also suitable but non-limiting combinations that can be used to treat viral infections of the respiratory tract.
  • Inhaled ⁇ 2-adrenergic receptor agonist bronchodilators such as formoterol or salmeterol in combination with ICS are also used to treat both bronchial stenosis and inflammation.
  • Combinations comprising these ICS and ⁇ 2-adrenergic receptor agonists together with compounds of formula I are also suitable but non-limiting combinations that can be used to treat viral infections of the respiratory tract.
  • anticholinergic drugs For the treatment or prevention of pulmonary bronchial stenosis, anticholinergic drugs have potential uses and therefore can be used as additional therapeutic agents in combination with the compound of formula I for the treatment of viral respiratory infections.
  • anticholinergic drugs include, but are not limited to, muscarinic receptor (especially M3 subtype) antagonists that have shown therapeutic efficacy in the control of cholinergic properties in COPD in humans (Witek, 1999) ; 1- ⁇ 4-hydroxy-1-[3,3,3-tris-(4-fluoro-phenyl)-propionyl]-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-nitrogen cation (azonia)-bicyclo[3.2.1]octan
  • Nonane Teotropium bromide-N,N-diethylglycinate or salt
  • dimethylamino-acetic acid 2-(3-diisopropylamino-1-phenyl-propyl) -4-methyl-phenyl ester (tolterodine-N,N-dimethylglycinate or salt)
  • the active ingredient of the present invention is particularly suitable for combined use with drugs that have the effect of improving acute lung injury.
  • Representative drugs include but are not limited to zinc (Zinc), Fingolimod (Fingolimod), Vitamin C (Vitamin C), Olmesartan Medoxomil, Valsartan, Losartan , Thalidomide, glycyrrhizic acid, Artemisinin, dihydroartemisinin, Artesunate, Artemisone, Azithromycin ), Escin (Escin), Naproxen (Naproxen).
  • the zinc (Zinc) includes, but is not limited to, zinc sulfate, zinc glycyrrhizinate, and zinc gluconate.
  • the active ingredient of the present invention can inhibit the infectious activity of new coronaviruses such as SARS-CoV-2. Therefore, when the active ingredient of the present invention is administered or administered therapeutically, the infection of the 2019 novel coronavirus (SARS-CoV-2) can be inhibited, and the antiviral effect can be achieved.
  • new coronaviruses such as SARS-CoV-2. Therefore, when the active ingredient of the present invention is administered or administered therapeutically, the infection of the 2019 novel coronavirus (SARS-CoV-2) can be inhibited, and the antiviral effect can be achieved.
  • Compounds of formula I can also be combined with mucolytics to treat both infections and symptoms of respiratory infections.
  • a non-limiting example of a mucolytic drug is ambroxol.
  • compounds of formula I can be combined with expectorants to treat both infections and symptoms of respiratory infections.
  • a non-limiting example of an expectorant is guaifenesin.
  • Nebulized hypertonic saline is used to improve the immediate and long-term clearance of small airways in patients with lung disease (Kuzik, J. Pediatrics 2007, 266).
  • the compound of formula I can also be combined with nebulized hypertonic saline, especially when the Paramyxoviridae virus infection is complicated by bronchiolitis.
  • the combination of a compound of formula I and hypertonic saline may also include any of the additional agents discussed above. In a preferred aspect, about 3% nebulized hypertonic saline is used.
  • 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 applied sequentially, the combination can be applied in two or more applications.
  • the co-administration of the compound of the present invention and one or more additional active therapeutic agents generally refers to simultaneous or sequential administration of the compound of the present invention and one or more additional active therapeutic agents such that a therapeutically effective amount of the compound of the present invention and one Or multiple additional active therapeutic agents are all present in the patient.
  • Co-administration includes administration of a unit dose of a compound of the invention before or after administration of a unit dose of one or more additional active therapeutic agents, for example, within seconds, minutes, or hours of administration of one or more additional active therapeutic agents Inside, the compound of the present invention is administered.
  • a unit dose of a compound of the invention can be administered first, followed by a unit dose of one or more additional active therapeutic agents within seconds or minutes.
  • a unit dose of one or more other therapeutic agents may be administered first, followed by a unit dose of the compound of the invention within seconds or minutes.
  • a unit dose of a compound of the invention it may be necessary to first administer a unit dose of a compound of the invention, and after a period of several hours (e.g., 1-12 hours), administer a unit dose of one or more additional active therapeutic agents. In other cases, it may be necessary to first administer a unit dose of one or more additional active therapeutic agents, and after a period of several hours (e.g., 1-12 hours), administer a unit dose of the compound of the invention.
  • Combination therapy can provide "synergism” 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.
  • Synergistic effects can be obtained when the active ingredients: (1) are co-formulated and administered or delivered simultaneously in the form of a combined preparation; (2) are alternately administered or delivered in parallel as separate preparations; or (3) are delivered by some other administration schedule.
  • alternation therapy when the compounds are administered or delivered sequentially, for example, in separate tablets, pills, or capsules, or different injections through separate syringes, a synergistic effect can be obtained.
  • an effective dose of each active ingredient is administered sequentially, that is, consecutively, while in combination therapy, effective doses of two or more active ingredients are administered together.
  • a synergistic antiviral effect means an antiviral effect that is greater than the predicted pure additive effect of the individual compounds in the combination.
  • a method for inhibiting viral RNA polymerase in a cell comprises contacting a virus-infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate and/or precursor thereof.
  • Drugs e.g., esters
  • a method for inhibiting viral RNA polymerase in a cell comprises contacting a virus-infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate and/or precursor thereof.
  • a drug e.g., ester
  • at least one additional active therapeutic agent thereby inhibiting viral RNA polymerase.
  • a method for inhibiting viral RNA polymerase in a cell comprises contacting a virus-infected cell with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate and/or precursor thereof.
  • a drug e.g., an ester
  • a method for treating respiratory viral infections in humans or other mammals which comprises administering to said human or other mammals a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, Solvates and/or prodrugs (e.g., esters).
  • a method for treating respiratory viral infections in humans or other mammals which comprises administering to said human or other mammals a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, Solvates and/or prodrugs (e.g., esters), and at least one additional active therapeutic agent, thereby inhibiting respiratory viral RNA polymerase.
  • a method of treating respiratory viral infections in humans or other mammals which comprises administering to said human or other mammals a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, Solvates and/or prodrugs (e.g., esters), and at least one additional active therapeutic agent.
  • the present invention also provides the active compound for inhibiting viral replication of the present invention, or a pharmaceutically acceptable salt thereof, or a mixture of one or more of its prodrugs as an active ingredient, which is used in the preparation of treatment and/or prevention, alleviation Use in medicine for respiratory infections, pneumonia and other related diseases caused by infections of viruses such as coronavirus, influenza virus, respiratory syncytial virus and/or porcine epidemic diarrhea virus (PEDV) (especially 2019 new coronavirus).
  • viruses such as coronavirus, influenza virus, respiratory syncytial virus and/or porcine epidemic diarrhea virus (PEDV) (especially 2019 new coronavirus).
  • PEDV epidemic diarrhea virus
  • the pharmaceutical composition provided by the present invention preferably contains the active ingredient in a weight ratio of 0.001-99wt%, and the preferred ratio is that the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight,
  • the remaining part is a pharmaceutically acceptable carrier, diluent or solution or salt solution.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or diluents. Neutralization is suitable for sterilization equipment for injection or drip infusion.
  • Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field.
  • the unit measurement of the preparation formula usually contains 0.05-1000 mg of the active compound of the present invention.
  • the unit measurement of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.
  • the compounds and pharmaceutical compositions of the present invention can be used clinically on mammals, including humans and animals, and can be administered via oral, nose, skin, lung, or gastrointestinal tract. Most preferred is oral administration.
  • the most preferred daily dose is 0.01-400 mg/kg body weight, taken in one time, or 0.01-200 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dose for an individual should depend on the specific treatment. Usually, start with a small dose and gradually increase the dose until the most suitable dose is found.
  • the drugs or inhibitors of the present invention can be administered in various ways, for example, can be introduced into the body by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemically mediated methods such as muscle, intradermal, subcutaneous, and intravenous methods. , Mucosal tissue; or mixed or wrapped by other substances into the body.
  • the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous injection, nasal cavity, oral mucosa, Eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O/W type, W/O type and double emulsion), suspensions, injections (including water injections, powder injections and infusions), eye drops
  • the solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the active ingredients of the present invention can be made into ordinary preparations, and can also be made into slow-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • 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 syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • the disintegrant can be dry starch, microcrystalline cellulose
  • the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule.
  • the active ingredient can also be made into granules or pellets with diluents, binders, and disintegrants, and then placed in hard or soft capsules.
  • the various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.
  • solubilizing agent or co-solvent can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • the pH adjusting agent can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.
  • the osmotic pressure adjusting agent can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • mannitol, glucose, etc. can also be added as proppants.
  • coloring agents if necessary, coloring agents, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations.
  • the active ingredient or composition of the present invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs.
  • the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the active compound of the present invention can effectively inhibit SARS-CoV-2, human respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV OC43), porcine epidemic diarrhea virus (PEDV), Zika virus (Zika)
  • RSV human respiratory syncytial virus
  • HoV OC43 human coronavirus OC43
  • PDV porcine epidemic diarrhea virus
  • Zika Zika virus
  • the replication of viruses such as dengue fever virus (DENV) has a low EC 50 value and has broad-spectrum antiviral characteristics.
  • the active compound of the present invention has low toxic and side effects (cytotoxicity CC 50 > 10 ⁇ M) and good drug-making properties. This suggests that the nucleoside analogs of the present invention have a good medicinal prospect in the treatment of viral infectious diseases, especially in the field of anti-new coronary pneumonia.
  • Compound 1-1 was synthesized by reference report method (Nature.2016,531,381-385).
  • Compound 1-1 (1.5g, 2.67mmol) was added to acetonitrile (30mL), followed by the addition of 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis (tetra Fluoroborate (Selectfluor, 1.13g, 3.2mmol, 1.2eq), sodium bicarbonate (0.67g, 8.0mmol, 3eq), after the addition, react at room temperature for 3-4 hours, TLC shows that the reaction is complete.
  • 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%.
  • Compound 2-1 was synthesized according to the method reported 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 ( 24mg, 0.2mmol, 0.2eq), reacted at 50°C for 1 hour, TLC showed that the reaction was complete. The reaction solution was added to the mixed solution of sodium sulfite and sodium carbonate, and compound A2 was filtered to obtain 185 mg of white solid, with a yield of 57%.
  • the product 4-1 obtained in the previous step was dissolved in dichloromethane (3mL), protected by nitrogen, and the temperature was reduced to -35°C.
  • 1M boron trichloride in dichloromethane solution (2.1mL, 2.1mmol) was added dropwise for 10 minutes After adding left and right, keep for 15 minutes, slowly add methanol (0.3mL) dropwise, after addition, keep for 20 minutes, concentrate the reaction solution, add n-heptane (5mL), stir at room temperature for 1 hour, filter, filter cake with a small amount of n-heptane After washing with alkane, 255 mg of yellow solid was obtained.
  • 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%. , White solid 30mg, yield 53%.
  • reaction solution was added to 0.5M sodium hydroxide solution (20mL), ethyl acetate (20mL ⁇ 2) was extracted twice, the organic phase was discarded, the aqueous layer was adjusted to pH 4-5 with dilute hydrochloric acid (2M), ethyl acetate (50 mL ⁇ 2) extraction, combined organic layers, dried, and concentrated to obtain compound 10-2, 0.23 g of white solid, with a yield of 89%.
  • the product 10-4 (60mg, 0.18mmol) of the previous step was added to tetrahydrofuran (2mL), concentrated hydrochloric acid (0.4mL) was added dropwise, and the reaction was carried out at 40°C until the reaction was complete.
  • the obtained solid was added to ethyl acetate, stirred for 0.5 hour, filtered, and dried to obtain compound A10, 34 mg of white solid, with a yield of 64%.
  • the aqueous layer was adjusted to pH 4-5 with dilute hydrochloric acid (2M), extracted with ethyl acetate (20 mL ⁇ 3), and the organic layers were combined, dried, and concentrated to obtain 11-2 crude product, which was directly thrown into the step reaction.
  • 2M dilute hydrochloric acid
  • the obtained product was added to methanol (10 mL), diluted hydrochloric acid (1M) was added dropwise to make the pH of the solution 2-3, and after stirring at room temperature for 1-2 hours, the pH was adjusted to neutral with a saturated aqueous sodium bicarbonate solution.
  • a mixture of ethyl acetate/water (20mL/20mL) was added, the organic phase was separated, and the aqueous layer was extracted once more with ethyl acetate (20mL).
  • the organic layers were combined, dried, concentrated, and separated on a silica gel preparation plate to obtain compound 11-4, 0.05 g of white solid, with a yield of 89%.
  • the product 12-4 (80mg, 0.23mmol) of the previous step was added to tetrahydrofuran (3mL), concentrated hydrochloric acid (0.5mL) was added dropwise, and the reaction was carried out at 40°C until the reaction was complete. The insoluble matter precipitated in the reaction solution and filtered to obtain 55 mg of white solid. The solid was added to water (1.5 mL), sodium carbonate (16 mg) was added, stirred for 0.5 hours, filtered, and dried to obtain compound A12 and white solid 44 mg. The rate is 62%.
  • the product 49-3 (240 mg, 0.36 mmol) obtained in the previous step was added to tetrahydrofuran (5 mL), and a 1M tetrabutylammonium fluoride solution in tetrahydrofuran (0.72 mL, 0.72 mmol) was added. After 1 hour, the reaction was complete. Water (10 mL) was added to the reaction solution, extracted with ethyl acetate, the organic layer was separated, and the organic phase was dried and evaporated to dryness to obtain an oily substance.
  • the product 50-1 (0.2g, 0.52mmol) from the previous step was added to dichloromethane (10mL), followed by triethylamine (0.1g, 1.0mmol, 2eq), DMAP (0.012g, 0.10mmol, 0.2eq) , Isobutyryl chloride (0.083g, 0.78mmol, 1.5eq), after the addition, stir at room temperature for 1-2 hours, TLC monitors that the reaction is complete.
  • the reaction solution was added to 1M dilute hydrochloric acid (10 mL), extracted with dichloromethane, and the organic phase was separated. The organic phase was washed with saturated sodium bicarbonate (20 mL), dried and concentrated to obtain compound 50-2, 0.2 g of off-white solid , The yield is 84%.
  • 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 at room temperature overnight. Water was added to the reaction solution, extracted with ethyl acetate, and the organic phase was separated. 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 a white solid. The rate is 44%.
  • the white solid obtained was added to tetrahydrofuran (8mL), under ice bath, acetic acid (14mg, 0.24mmol) and 1M tetrabutylammonium fluoride in tetrahydrofuran solution (0.24mL, 0.24mmol) were added, after addition, stirred at room temperature . After 2 hours, TLC showed that the reaction was complete. The reaction solution was concentrated, water was added, and isopropyl acetate was extracted. The organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated.
  • compound A181 was obtained through four steps of reaction as dihydrochloride salt, 75 mg of white solid, and a yield of 32%.
  • reaction solution was poured into ethyl acetate/water (20 mL/20 mL), the layers were separated, and the aqueous layer was extracted once more with ethyl acetate (20 mL).
  • the ethyl acetate layers were combined, dried, concentrated, and separated by a preparation plate to obtain compound 209-2, 0.1 g of white solid, with a yield of 74%.
  • the product obtained in the previous step was added to tetrahydrofuran (30 mL).
  • a solution of acetic acid (0.11 g, 1.75 mmol) and 1M tetrabutylammonium fluoride (3.5 mL, 3.5 mmol) in tetrahydrofuran was added, and stirred at room temperature for 2 hours.
  • Water and ethyl acetate were added to the reaction solution to separate the organic phase.
  • the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain crude compound 147-3 as an oily substance.
  • 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 column chromatography was separated to obtain compound 221-2, a white solid 0.2 g, with a two-step yield of 37%.
  • the crude compound 36-3 was added to dichloromethane (10mL), 2,4,6-trimethylpyridine (194mg, 1.6mmol), silver nitrate (272mg, 1.6mmol), triphenylchloromethane (374mg, 1.2 mmol), react at room temperature for 2 hours, add methanol, concentrate, add ethyl acetate, wash with saturated copper sulfate and saturated sodium chloride respectively, dry with anhydrous sodium sulfate, and concentrate to obtain crude compound 36-4;
  • Test Example 1 Study on the inhibitory effect of the compound on the replication of the new coronavirus
  • Vero E6 cells were purchased from ATCC, and the SARS-CoV-2 virus was derived from the National Virus Resource Bank Microbial Species Collection. The Vero E6 cells were cultured overnight in a 48-well cell culture dish with a density of 5 ⁇ 10 4 cells/well. The cells were pre-treated with different concentrations of the compound of the present invention for 1 hour, and then virus was added (the MOI of infection was 0.05) to infect After 1 hour, the virus compound mixture was taken out, and the cells were further cultured in a fresh medium containing the compound of the present invention. At 24h pi, the cell supernatant was collected and lysed in lysis buffer, and the virus copy number in the cell supernatant was quantitatively evaluated by quantitative real-time RT-PCR (qRT-PCR).
  • qRT-PCR quantitative real-time RT-PCR
  • Test Example 2 Determination of the half-toxic concentration of the compound
  • the CCK8 kit analysis was used to determine the half-toxic concentration (CC 50 ) of the compounds of the present invention (each compound in Table 1) on Vero E6 cells.
  • test compound has no cytotoxic effect on Vero E6 cells at the highest concentration of 10 ⁇ M, which suggests that the CC 50 of the compound of the present invention is far greater than 10 ⁇ M.
  • Test Example 3 Study on the inhibitory effect of the compound on the novel coronavirus RNA-dependent RNA polymerase (RdRp)
  • Reference report method (Virus Genes, 2015, 50:498-504), using the fluorescence method to determine the inhibitory activity of the compounds of the present invention against the 2019 novel coronavirus (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp): new coronavirus After the RdRP solution, substrate, compound solution, buffer, etc. were incubated for 1 hour, fluorescent DNA binding dye was added. After 10 minutes, data was collected and the compound's inhibitory activity on RdRp was calculated.
  • SARS-CoV-2 2019 novel coronavirus
  • RdRp RNA-dependent RNA polymerase
  • Test Example 4 Study on the inhibitory effect of the compound on the replication of other viruses
  • CPE cytopathic effect
  • the experimental cells were seeded into a 96-well cell culture plate at a certain cell density , and cultured overnight in a 5% CO 2 , 37°C incubator. The compound and virus were added the next day. Depending on the virus to be tested, the cells are cultured in an incubator under 5% CO 2 , 33°C or 37°C for 3-7 days, until the cytopathic changes in the control wells with virus-free virus infection reach 80-95%. Then use CellTiter-Glo or CCK-8 to detect cell viability in each well. If the cell viability of the compound-containing well is higher than that of the virus-infected control well, that is, the CPE is weakened, it indicates that the compound has an inhibitory effect on the tested virus.
  • the cytotoxicity test method is the same as the corresponding antiviral test method, but there is no virus infection.
  • the antiviral activity and cytotoxicity of the compound are represented by the inhibitory rate (%) and cell viability (%) of the compound on the cellular viral effect caused by the virus, respectively. Calculated as follows:
  • Inhibition rate (%) (reading value of test well-average value of virus control)/(average value of cell control-average value of virus control) ⁇ 100;
  • Cell viability (%) (reading value of test well-average value of medium control)/(average value of cell control-average value of medium control) ⁇ 100;
  • the EC 50 and CC 50 values are calculated by Prism software, and the inhibition curve fitting method is "log(inhibitor) vs.response--Variable slope".
  • Vero cells were seeded into a 6-well cell culture plate at a density of 600,000 cells per well , and cultured overnight in a 5% CO 2 , 37°C incubator. Compound and virus (40-50 PFU/well) were added the next day. The cells were cultured in an incubator under the conditions of 5% CO 2 and 37°C for 2 hours, and then the supernatant was aspirated, and a low melting point agarose culture medium containing the corresponding concentration of the compound was added. The cells are cultured in an incubator under 5% CO 2 , 33° C. or 37° C. for 6-7 days, until obvious virus plaques can be observed in the virus-infected control wells without compound under the microscope. Cells were fixed with 4% paraformaldehyde and stained with crystal violet. Calculate the number of plaques in each hole.
  • the cytotoxicity experiment was carried out in parallel with the antiviral experiment. Vero cells were seeded into a 96-well cell culture plate at a density of 20,000 cells per well , and cultured overnight in a 5% CO 2 , 37°C incubator. The compound was added the next day (1-5 concentration points, single point). The cells are cultured in an incubator for 6-7 days under the conditions of 5% CO 2, 33°C or 37°C. Then use CCK-8 to detect cell viability in each well.
  • the Vero cells were digested and passaged, the cell density was adjusted to 1 ⁇ 10 5 /mL with cell growth solution, and the cells were seeded in a 96-well plate, 100 ⁇ L/well, placed in a 37 °C, 5% CO 2 incubator for 24 hours; take out the 96 wells Plate, discard the medium in the well, wash three times with 1 ⁇ PBS, add a mixture of compound (10 concentration points) and virus (0.01 MOI per well) to each well after spin-drying, set 8 replicate wells for each concentration, Incubate in a 37°C, 5% CO 2 incubator, and set virus control and cell control at the same time. After 36 hours, cell samples were collected, and the changes in virus content in different treatment groups were determined by fluorescence quantitative PCR, and the EC 50 of the compound was calculated.
  • CD-1 mice Fifteen male CD-1 mice were randomly divided into 5 groups, 3 in each group, fasted for 12 hours before the experiment and free drinking; 2 hours after the administration, they ate uniformly.
  • Four groups were given 50mg/kg of GS-441524, A9, A10 and A11 by intragastric administration, and approximately 30 ⁇ L of blood was taken from the femoral vein at 0.25, 0.5, 1, 2, 4, 8, 24 h after administration, and placed in heparin. Anticoagulation tube.
  • the remaining 1 group was given intravenous injection of 25mg/kg of GS-441524, and blood was taken from the femoral vein at 5 min, 0.25, 0.5, 1, 2, 4, 8, 24 h after administration, and placed in a heparin anticoagulation tube for treatment
  • the method is the same as above.
  • the concentration of GS-441524, A9, A10 and A11 in plasma was determined by LC-MS-MS method. Take 20 ⁇ L of supernatant and 20 ⁇ L of deionized water and mix well and sample for analysis. After the sample is separated by a Waters HSS T3 (2.1*50 mm, 1.8 ⁇ m) chromatographic column, the electrospray ionization source is used, and the multi-reaction monitoring mode is used for detection under positive ion conditions. The linear range of the sample is 10-30000ng/mL.
  • Eighteen male SD rats were randomly divided into 6 groups, 3 in each group, fasted for 12 hours before the experiment (the intravenous experimental group was not fasted), and they were free to drink; 4 hours after the administration, they ate uniformly.
  • Three groups were given 10mg/kg of A9, A146 and A151 by gavage, and the remaining three groups were given 2mg/kg of A9, A146 and A151 by intravenous injection.
  • the administration vehicle is DMSO/EtOH/PEG300/0.9% NaCl (5/5/40/50, v/v/v/v).
  • mice 16 male CD-1 mice were randomly divided into 4 groups, 4 mice in each group, fasted for 12 hours before the experiment, and free drinking water; 2 hours after the administration, they ate uniformly.
  • the 4 groups of mice were given 200 mg/kg of A151 (dissolved in DMSO-enthanol-PEG300-saline, volume ratio is 5/5/40/50). Anesthesia was performed, 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.
  • Test Example 8 In vivo efficacy test of anti-SARS-CoV-2
  • mice Eight-week-old Balb/c mice were divided into 5 groups, virus control group (vehicle: 40% PEG400 + 10% HS15 + 50% ultrapure water), A151 hydrobromide (A151-S) oral group (50 mg /Kg, 100mg/Kg), Radixivir oral group (50mg/kg), Radixivir intraperitoneal injection group (50mg/kg). Animals in each group were transduced with adenovirus with human ACE2 gene, 5 days after transduction, they were infected with new coronavirus, and 1 hour after infection, they were given the vehicle, A151-S and the control drug Remdesivir by gavage or intraperitoneal injection, respectively. It is administered once a day.
  • virus control group vehicle: 40% PEG400 + 10% HS15 + 50% ultrapure water
  • A151 hydrobromide (A151-S) oral group 50 mg /Kg, 100mg/Kg
  • Radixivir oral group 50mg/kg
  • mice were sacrificed on the 2nd and 5th days after infection, and the lung tissues of the mice were taken to detect the copy number of viral RNA in the lung tissues of the mice.
  • the virus RNA copies of 2 days after administration and 5 days after administration are shown in Fig. 1 and Fig. 2, respectively.
  • test results show that oral administration of compound A151-S at 50 mg/kg and 100 mg/kg can effectively reduce the amount of viral RNA in mouse lung tissue, with a certain dose-dependence.
  • the oral antiviral effect of A151-S was better than that of the remdesivir oral group at a dose of 50mg/Kg, and the oral antiviral effect of A151-S was better than that of remdesivir intraperitoneal injection after 5 days of administration. Group.
  • mice Eight-week-old Balb/c mice were divided into 2 groups, virus control group (vehicle: 5% DMSO + 5% Solutol HS15 + 90% Saline) and A151-S intraperitoneal injection group (100 mg/Kg).
  • virus control group vehicle: 5% DMSO + 5% Solutol HS15 + 90% Saline
  • A151-S intraperitoneal injection group 100 mg/Kg.
  • Two groups of animals were transduced with human-derived adenoviruses carrying the ACE2 gene. They were given A151-S the day before infection with the new coronavirus, and A151-S was given by intraperitoneal injection once a day from the day to the 4th day after infection.
  • the control group received the same treatment.
  • the mice were sacrificed on the 4th day after infection, and the lung tissues of the mice were taken to detect the copy number of virus RNA in the lung tissues of the mice. The result is shown in Figure 3.
  • test results show that compound A151-S can significantly reduce the amount of viral RNA in the lung tissue of mice by intraperitoneal injection, and the viral load is reduced by more than 30 times compared with the control group.
  • the compound of formula I of the present invention has significant anti-SARS-CoV-2 activity.
  • A11, A12, A52, A70, A72, A75, A77, A131, A171, A180, A181, A188, A196, A215 and other compounds can significantly inhibit virus replication at a concentration of 5 ⁇ M, and the inhibition rate is above 98%, which is significantly higher The inhibitory rate of Yuredcivir on the virus at this concentration (65%).
  • A102, A106, A107, and A198 can significantly inhibit the activity of new coronavirus RdRp, indicating that these compounds exert antiviral effects by acting on RdRp.
  • the compound of formula I of the present invention is also effective against other viruses, including respiratory syncytial virus (RSV), human coronavirus OC43 (HCoV OC43), porcine epidemic diarrhea virus (PEDV), Zika virus (Zika), and dengue fever virus (DENV).
  • RSV respiratory syncytial virus
  • HoV OC43 human coronavirus OC43
  • PDV porcine epidemic diarrhea virus
  • Zika Zika virus
  • DENV dengue fever virus
  • the mouse PK test showed that after oral administration of deuterated compounds A9 and A11, the drug exposure in plasma was higher than that of GS-441524, which was 1.3 times and 1.2 times the exposure of GS-441524; the oral bioavailability of A9 in rats With a degree of 21.3%, the oral bioavailability of compounds A146 and A151 were 48.5% and 56.7%, respectively, which was greatly improved compared with A9.
  • the tissue distribution test in mice showed that after 1 hour of oral administration of A151, the metabolite A9 had the highest concentration in the liver, kidney, lung, and heart, and the distribution was relatively uniform, and there was no accumulation of liver and kidney.
  • the A151 hydrobromide (A151-S) can be administered orally once a day in an adenovirus-transduced mouse model expressing the human ACE2 of the new coronavirus receptor, which dose-dependently reduces the viral load in the lungs of mice And at the same dose (50mg/Kg), A151-S has better anti-coronavirus effect than Redcivir.
  • the above data shows that A151-S has significant anti-SARS-COV-2 effects and has oral advantages. From the above data, it can be inferred that the compound of the present invention has good anti-virus, especially anti-new coronavirus (SARS-CoV-2) application prospects.

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Abstract

本发明涉及核苷类似物在抗病毒中的应用。具体而言，本发明涉及核苷类似物及其药物组合物作为(a)抑制冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)复制的抑制剂；和/或(b)治疗和/或预防、缓解由冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)感染引起的相关疾病的药物中的用途。本发明的核苷类似物可治疗和/或预防、缓解由2019新型冠状病毒感染引起的呼吸道感染、肺炎(COVID-19)等相关疾病。

Description

核苷类似物或含有核苷类似物的组合制剂在抗病毒中的应用 技术领域
本发明涉及医药领域，具体地涉及核苷类似物或含核苷类似物的组合制剂在抗病毒中的应用。
背景技术
在急性传染病中，绝大部分都是病毒性传染病，病毒性传染病的发病率高，死亡率也很高。病毒一类个体极其微小、缺乏独立代谢能力、以寄生方式存在的病原微生物。病毒的种类繁多，目前，已发现了许多对人类具有高传染性和高致病性的病毒，这些病毒常造成地方性甚至全球性传染病暴发，对人类社会有极大的危害，如流感病毒、呼吸道合胞病毒(RSV)、副流感病毒、非典型性肺炎(SARS)病毒、中东呼吸综合征(MERS)病毒、埃博拉病毒等。一些病毒也可感染动物，引起各种轻到重度疾病，同时，动物也成为这些病毒的传染源，令人类对其防不胜防。
冠状病毒属于套式病毒目、冠状病毒科、冠状病毒属，是自然界广泛存在的一大类单股正链的RNA病毒，可引起人和动物呼吸道、消化道和神经系统疾病。根据系统发育树，冠状病毒可分为四个属：α、β、γ、δ，其中β属冠状病毒又可分为四个独立的亚群A、B、C和D群。
截止2020年4月，新型冠状病毒(SARS-CoV-2)已导致死亡人数超过10万，造成了自1918年西班牙大流感后，又一次非常严重的全球公共卫生事件。SARS-CoV-2、非典型性肺炎病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)同属于β属冠状病毒，目前，这三种病毒已成为对人类致病能力最强的冠状病毒。
目前，针对SARS-CoV-2冠状病毒导致的严重肺炎疾病尚无特效的疫苗和抗病毒药物。这些感染性疾病严重影响了人们的生命健康，研发效果好的抗病毒药物迫在眉睫。针对SARS-CoV-2冠状病毒开发出低毒高效的抗病毒药物，以满足国内外SARS-CoV-2冠状病毒感染患者的临床需求，具有重大的社会意义。
冠状病毒也可感染多种哺乳动物，包括蝙蝠、猪、犬、猫、鼠、牛、马、骆驼等，这些病毒多为α、β属。猪流行性腹泻病毒(PEDV)是一种能引起猪急性肠道传染病的冠状病毒，各种年龄的猪都能感染发病，其中，尤以哺乳猪、新生仔猪受害最为严重，养殖业常因PEDV的爆发而蒙受重大损失。
呼吸道病毒感染是临床上最常见的、影响最广的一类病毒感染性疾病，每年都会造成全球大量人口死亡。除冠状病毒外，流感病毒、呼吸道合胞病毒、副流感病毒等也可引起呼吸道感染，导致肺炎，是威胁人类生命健康的重要杀手。
综上所述，本领域迫切需要开发针抑制病毒复制的抑制剂以用于治疗由病毒感染引起的相关疾病。
发明内容
本发明的目的是提供一种可有效抑制病毒复制的活性成分及其在病毒感染引起的相关疾病中的新用途。
具体地，本发明提供了式I所示的核苷类似物及其组合物在抗病毒(如冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒)，尤其是抗新型冠状病毒(SARS-CoV-2)中的用途。
在本发明第一方面，提供了一种式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
Figure PCTCN2021087928-appb-000001
式中，
R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
R 5选自R 3、
Figure PCTCN2021087928-appb-000002
Figure PCTCN2021087928-appb-000003
R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
X选自-CH 2-、-CD 2-、-CHD-；
R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 10选自C 1-18烷基、亚甲基C 6-20芳基；
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 12选自C 1-20烷基；
M各自独立地选自氢、金属、-NH 4、或质子化的有机胺；
在另一优选例中，表示为氘(D)的各个位置具有至少50％的氘富集度；优选地，表示为氘(D)的各个位置具有至少80％的氘富集度；更优选地，表示为氘(D)的各个位置具有至少90％的氘富集度；最优选地，表示为氘(D)的各个位置具有至少95％的氘富集度。
在另一优选例中，所述的5-15元杂芳基含有1、2、3或4个选自N、O和S的杂原子。
在另一优选例中，所述的金属选自下组：碱金属、碱土金属、或其组合。
在另一优选例中，所述式(I)化合物式中，
R 1选自氢、氘、卤素、氰基、叠氮、甲基、氯甲基、氟甲基、乙烯基、乙炔基、环丙基、氨甲酰基、羟甲基、甲氧基、甲酰基、脒基；和/或
R 2选自卤素、氰基、氨基、甲酰基、OR 3；和/或
R 3选自氢、C 1-20烷基酰基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；优选地，所述α-氨基酸选自下组：丙氨酸、缬氨酸、异亮氨酸、色氨酸、苯丙氨酸；和/或
R 4选自氢、氘、卤素、叠氮、氰基、甲基、氯甲基、氟甲基、二氟甲基、乙烯基、乙炔基、环丙基、羟甲基、叠氮甲基(-CH 2N 3)、甲酰基、乙酰基、甲酰胺基、乙酰胺基；和/或
R 5选自R 3、
Figure PCTCN2021087928-appb-000004
Figure PCTCN2021087928-appb-000005
和/或
R 6选自氨基、羟基、卤素、氰基、甲基氨基(-NH 2CH 3)、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；和/或
R 7选自氢、氘、卤素、氨基；和/或
R 8选自氢、氘、卤素、氰基、氨甲酰基、N-甲基氨基甲酰基(CH 3NHCO-)、甲基、乙基、乙炔基、甲氧羰基、乙氧羰基、羟基、羟甲基、羟乙基、甲氧基、乙氧基、甲酰基、乙酰基、甲酰氨基、乙酰氨基、甲氧羰基氨基(CH 3OCONH 2-)、乙氧羰基氨基(C 2H 5OCONH 2-)、甲氧羰基氧基(CH 3OCOO-)、乙氧羰基氧基(C 2H 5OCOO-)；和/或
X选自-CH 2-、-CD 2-、-CHD-；和/或
R 9选自C 6-20芳基、5-15元杂芳基；和/或
R 10选自C 1-18烷基、亚甲基C 6-20芳基；和/或
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；和/或
R 12选自C 1-20烷基；和/或
M各自独立地选自氢、锌、镁、钙、钠、钾、NH 4、质子化的三甲胺、质子化的三乙胺、质子化的三正丁胺。
在另一优选例中，所述的R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和X各自独立地为实施例中各化合物(如A1至A221中任一化合物)中所对应的具体基团。
在另一优选例中，所述式(I)化合物为具有如下结构的化合物A1至A221中任一化合物、或其组合：
Figure PCTCN2021087928-appb-000006
Figure PCTCN2021087928-appb-000007
Figure PCTCN2021087928-appb-000008
Figure PCTCN2021087928-appb-000009
Figure PCTCN2021087928-appb-000010
Figure PCTCN2021087928-appb-000011
在另一优选例中，所述的式(I)化合物选自下组：
Figure PCTCN2021087928-appb-000012
Figure PCTCN2021087928-appb-000013
Figure PCTCN2021087928-appb-000014
Figure PCTCN2021087928-appb-000015
Figure PCTCN2021087928-appb-000016
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在本发明的第二方面，提供了一种活性成分或含所述活性成分的制剂的用途，所述的活性成分为式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
Figure PCTCN2021087928-appb-000017
式中，
R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
R 5选自R 3、
Figure PCTCN2021087928-appb-000018
Figure PCTCN2021087928-appb-000019
R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
X选自-CH 2-、-CD 2-、-CHD-；
R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 10选自C 1-18烷基、亚甲基C 6-20芳基；
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 12选自C 1-20烷基；
M各自独立地选自氢、金属、-NH 4或质子化的有机胺；
并且，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由病毒感染引起的相关疾病的药物。
在另一优选例中，表示为氘(D)的各个位置具有至少50％的氘富集度；优选地，表示为氘(D)的各个位置具有至少80％的氘富集度；更优选地，表示为氘(D)的各个位置具有至少90％的氘富集度；最优选地，表示为氘(D)的各个位置具有至少95％的氘富集度。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制冠状病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由冠状病毒感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制呼吸道合胞病毒(RSV)复制的抑制剂；和/或(b)治疗和/或预防、缓解由呼吸道合胞病毒(RSV)感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制流感病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由流感病毒感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制黄病毒科病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由黄病毒科病毒感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制丝状病毒科病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由丝状病毒科病毒感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制猪流行性腹泻病毒(PEDV)复制的抑制剂；和/或(b)治疗和/或预防、缓解由猪流行性腹泻病毒(PEDV)感染引起的相关疾病的药物。
在另一优选例中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制2019新型冠状病(SARS-Cov-2)毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由2019新型冠状病(SARS-CoV-2)感染引起的相关疾病的药物。
在另一优选例中，所述的病毒选自：
(1)感染人的冠状病毒：重症急性呼吸综合征冠状病毒SARS-CoV(Severe acute respiratory syndrome coronavirus，SARS-CoV)、2019新型冠状病毒(2019-nCoV或SARS-CoV-2)、中东呼吸综合征冠状病毒MERS-CoV(Middle East respiratory syndrome coronavirus，MERS-CoV)
(2)致普通感冒的冠状病毒：所述的致普通感冒的冠状病毒优选选自下组：人冠状病毒OC43(Human coronavirus OC43)、人冠状病毒229 E(Human coronavirus  229E)、人冠状病毒NL63(Human coronavirus NL63)、人冠状病毒HKUl(Human coronavirus HKUl)；
(3)人呼吸道合胞病毒(RSV)；
(4)人流感病毒：甲型流感病毒、乙型流感病毒、丙型流感病毒；
(5)黄病毒科病毒：丙型肝炎病毒(HCV)、登革热病毒(DENV)、寨卡病毒(Zika)；
(6)丝状病毒科病毒：马尔堡病毒(MBV)、埃博拉病毒(EBV)；
(7)感染其他哺乳动物的冠状病毒：猪流行性腹泻病毒(PEDV)。
在另一优选例中，所述病毒引起的相关疾病选自下组：
(D1)人冠状病毒感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
(D2)人呼吸道合胞病毒(RSV)感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
(D3)人流感病毒感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
(D4)丙型肝炎病毒(HCV)引起的慢性丙型肝炎及其并发症；
(D5)登革热病毒(DENV)引起的登革热及其并发症；
(D6)寨卡病毒(Zika)引起的感染及其并发症；
(D7)马尔堡病毒(MBV)、埃博拉病毒(EBV)引起的出血热及其并发症；
(D8)SARS-CoV-2引起的新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)；
(D9)猪流行性腹泻病毒(PEDV)引起的猪流行性腹泻；
(D10)上述疾病的任意组合。
在另一优选例中，所述由2019新型冠状病毒感染引起的相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。
在另一优选例中，所述式(I)化合物为化合物A1至A221中任一化合物、或其组合。
在另一优选例中，所述的活性成分为选自下组的核苷类似物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
化合物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或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在另一优选例中，所述的制剂(或含所述化合物的药物组合物)还可以含有其他抗病毒药。
在另一优选例中，所述的其他抗病毒药物还包括选自下组的额外组分：
Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524、NHC(EIDD-1931)、EIDD-2801、GC-376、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine)、羟氯喹(hydroxychloroquine)、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、连翘脂苷(forsythoside)、绿原酸(chlorogenic acid)、大黄素(emodin)、霉酚酸(mycophenolic acid)、霉酚酸酯(Mycophenolate mofetil)、萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、帕利珠单抗、莫维珠单抗(Motavizumab)、RSV-IGIV
Figure PCTCN2021087928-appb-000020
MEDI-557、A-60444(RSV-604)、MDT-637、BMS-433771、或其药学上可接受的盐、或其组合。
在另一优选例中，含所述化合物的药物组合物还包括施用治疗有效量的选自下组的至少一种其它治疗剂：皮质类固醇、抗炎信号转导调节剂、β2-肾上腺素受体激动剂支气管扩张药、抗胆碱能药、黏液溶解药、高渗盐水和用于治疗病毒感染的其它药物；或其组合。
在另一优选例中，所述的制剂为药物组合物。
在另一优选例中，所述的制剂(或药物组合物)包括：口服制剂和非口服制剂。
在另一优选例中，所述的制剂包括：粉剂、颗粒剂、胶囊剂、注射剂、吸入剂、 酊剂、口服液、片剂、含片、或滴丸。
在本发明的第三方面。提供了一种药物组合物，所述的药物组合物含有：
(a1)第一活性成分，所述的第一活性成分为式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
Figure PCTCN2021087928-appb-000021
式中，
R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
R 5选自R 3、
Figure PCTCN2021087928-appb-000022
Figure PCTCN2021087928-appb-000023
R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
X选自CH 2，CD 2、-CHD-；
R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 10选自C 1-18烷基、亚甲基C 6-20芳基；
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 12选自C 1-20烷基；
M各自独立地选自氢、金属、-NH 4或质子化的有机胺；
以及(b)药学上可接受的载体。
在另一优选例中，所述的组合物还含有(a2)第二活性成分；
其中，所述的第二活性成分为抗病毒药物，其选自下组：干扰素、RNA依赖的RNA聚合酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524、NHC(EIDD-1931)、EIDD-2801)、3CL蛋白酶抑制剂(如GC-376)、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine)、羟氯喹(hydroxychloroquine)、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、连翘脂苷(forsythoside)、绿原酸(chlorogenic acid)、大黄素(emodin)、霉酚酸(mycophenolic acid)、霉酚酸酯(Mycophenolate mofetil)、萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、帕利珠单抗、莫维珠单抗(Motavizumab)、RSV-IGIV
Figure PCTCN2021087928-appb-000024
MEDI-557、A-60444(RSV-604)、MDT-637、BMS-433771、或其药学上可接受的盐、或其组合；
和/或所述的第二活性成分选自下组：用于治疗呼吸道感染的支气管扩张药和皮质类固醇，其中所述皮质类固醇包括地塞米松、地塞米松磷酸钠、氟米龙、醋酸氟米龙、氯替泼诺、依碳酸氯替泼诺、氢化可的松、泼尼松龙、氟氢可的松、曲安西龙、曲安奈德、倍他米松、双丙酸倍氯美松、甲泼尼龙、氟轻松、氟西奈德、氟尼缩松、氟可丁-21-丁酯(fluocortin-21-butylate)、氟米松、新戊酸氟米松、布地奈德、丙酸卤贝他索、糠酸莫米他松、丙酸氟替卡松、环索奈德；或其药学上可接受的盐。
和/或所述的第二活性成分选自下组：锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)、或其组合。
在另一优选例中，所述式(I)化合物式中，
R 1选自氢、氘、卤素、氰基、叠氮、甲基、氯甲基、氟甲基、乙烯基、乙炔基、 环丙基、氨甲酰基、羟甲基、甲氧基、甲酰基、脒基；
R 2选自卤素、氰基、氨基、甲酰基、OR 3；
R 3选自氢、C 1-20烷基酰基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；优选地，所述α-氨基酸选自丙氨酸、缬氨酸、异亮氨酸、色氨酸、苯丙氨酸；
R 4选自氢、氘、卤素、叠氮、氰基、甲基、氯甲基、氟甲基、二氟甲基、乙烯基、乙炔基、环丙基、羟甲基、叠氮甲基(-CH 2N 3)、甲酰基、乙酰基、甲酰胺基、乙酰胺基；
R 5选自R 3、
Figure PCTCN2021087928-appb-000025
Figure PCTCN2021087928-appb-000026
R 6选自氨基、羟基、卤素、氰基、甲基氨基(-NH 2CH 3)、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
R 7选自氢、氘、卤素、氨基；
R 8选自氢、氘、卤素、氰基、氨甲酰基、N-甲基氨基甲酰基(CH 3NHCO-)、甲基、乙基、乙炔基、甲氧羰基、乙氧羰基、羟基、羟甲基、羟乙基、甲氧基、乙氧基、甲酰基、乙酰基、甲酰氨基、乙酰氨基、甲氧羰基氨基(CH 3OCONH 2-)、乙氧羰基氨基(C 2H 5OCONH 2-)、甲氧羰基氧基(CH 3OCOO-)、乙氧羰基氧基(C 2H 5OCOO-)；
X选自CH 2，CD 2、-CHD-；
R 9选自C 6-20芳基、5-15元杂芳基；
R 10选自C 1-18烷基、亚甲基C 6-20芳基；
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 12选自C 1-20烷基；和/或
M各自独立地选择氢、锌、镁、钙、钠、钾、NH 4、质子化的三甲胺、质子化的三乙胺、质子化的三正丁胺。
在另一优选例中，所述式(I)化合物为化合物A1至A221中任一化合物、或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在另一优选例中，所述的第二活性成分选自下组：(Y1)RNA复制酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524、NHC、EIDD-2801)；(Y2)洛匹那韦(Lopinavir)；(Y3)利托那韦(Ritonavir)；(Y4)法匹拉韦；(Y5)氯喹(Chloroquine)、羟氯喹(hydroxychloroquine)、或其药学上可接受的盐(如磷酸氯喹)、(Y6)奈非那韦(Nelfinavir)；(Y7)上述Y1～Y6的任意组合。
在另一优选例中，所述的药物组合物用于抑制冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)复制。
在另一优选例中，所述药物用于抑制2019新型冠状病毒(SARS-CoV-2)复制。
在本发明的第四方面，提供了本发明第三方面所述的药物组合物的用途，它被用于制备(a)抑制冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)；和/或(b)治疗和/或预防、缓解由冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)感染引起的相关疾病的药物。
在另一优选例中，用于制备(a)抑制2019新型冠状病毒(SARS-CoV-2)复制的抑制剂；和/或(b)治疗和/或预防、缓解由2019新型冠状病毒(SARS-CoV-2)感染引起的相关疾病的药物。
在本发明第五方面，提供了一种抑制病毒复制的方法，包括步骤：
将第一活性成分或含所述第一活性成分的制剂与病毒接触，从而抑制所述病毒的复制；
其中，所述的第一活性成分为式I化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
Figure PCTCN2021087928-appb-000027
式中，
R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连，优选地，所述α-氨基酸选自丙氨酸、缬氨酸、异亮氨酸、色氨酸、苯丙氨酸；
R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
R 5选自R 3、
Figure PCTCN2021087928-appb-000028
Figure PCTCN2021087928-appb-000029
R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基，C 2-6C 1-6烷氧基、烯基、C 2-6炔基；
X选自CH 2，CD 2、-CHD-；
R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 10选自C 1-18烷基、亚甲基C 6-20芳基；
R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
R 12选自C 1-20烷基；
M各自独立地选自氢、金属、NH 4或质子化的有机胺；
在另一优选例中，所述的病毒选自下组：冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒、猪流行性腹泻病毒、或其组合。
在另一优选例中，所述的病毒为2019新型冠状病毒(SARS-CoV-2)。
在另一优选例中，所述式(I)化合物为化合物A1至A221中任一化合物、或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
在另一优选例中，所述方法是体外方法。
在另一优选例中，所述方法是非治疗性和非诊断性的。
在本发明第六方面，提供了一种(a)抑制病毒复制和/或(b)治疗和/或预防、缓解由病毒感染引起的相关疾病的方法，包括步骤：给需要的对象施用安全有效量的式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
Figure PCTCN2021087928-appb-000030
式中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和X如本发明第一方面中所定义。
在另一优选例中，所述的对象为哺乳动物，如人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明实施例中，设置病毒对照组、A151-S口服50mg/kg组、A151-S口服100mg/kg、瑞德西韦口服50mg/kg组、瑞德西韦腹腔注射50mg/kg组，给药2天后的病毒RNA拷贝情况。图2显示了本发明实施例中，设置病毒对照组、A151-S口服50mg/kg组、A151-S口服100mg/kg、瑞德西韦口服50mg/kg组、瑞德西韦腹腔注射50mg/kg组，给药5天后的病毒RNA拷贝情况。
图3显示了本发明实施例中，设置病毒对照组、A151-S腹腔注射100mg/kg组给药5天后的病毒RNA拷贝情况。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次意外地开发了一类可有效抑制病毒复制的活性成分。实验表明，本发明的活性成分可高效地抑制2019新型冠状病毒(SARS-CoV-2)等多种病毒的复制和活力，因此可用于抑制冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒的复制。在此基础上完成了本发明。
具体地，本发明揭示了具有式(I)所示的核苷类似物及其组合物在抗病毒中的用途，例如在抗冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)中的用途。式(I)所示的核苷类似物对SARS-CoV-2等病毒的复制具有优良的抑制作用，具有良好的临床应用前景。
术语
如本文所用，“本发明的活性化合物”、“本发明的活性成分”、“本发明的核苷类似物”“本发明的抑制冠状病毒复制的活性化合物”可互换使用，指具有优异的抑制冠状病毒复制的活性的核苷类似物，包括式I所示的化合物，或其药学上可接受的盐、或其溶剂合物、或其前药、或其组合。
如本文所用，“本发明的制剂”指含有本发明活性化合物的制剂。
如本文所用，术语“包括”或其变换形式如“包含”或“包括有”等等，被理解为包括所述的元件或组成部分，而并未排除其它元件或其它组成部分。
如本文所用，术语“新型冠状病毒”、“2019-nCoV”或“SARS-CoV-2”可互换使用，该2019新型冠状病毒是已知感染人的第7种冠状病毒，并且造成新冠肺炎(COVID-19)，是威胁全球人类健康的严重传染性疾病之一。
如本文所用，“卤素”通常是指氟、氯、溴及碘；优选为氟、氯或溴；更优选为氟或氯。
如本文所用，术语“C n-C m”与C n-m可互换使用，指具有n至m个碳原子。
如本文所用，术语“C 1-C 6烷基”指含有1-6个碳原子的直链或支链的饱和烃基，例如，甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1-乙基丙基、异戊基、新戊基、异己基、3-甲基戊基或正己基等，优选为甲基、乙基、正丙基、异丙基、丁基、异丁基或叔丁基。
卤代C 1-C 6烷基是指含有1-6个碳原子的直链或支链的饱和烃基的氢原子被1个或多个相同或不同的卤原子取代，例如三氟甲基、氟甲基、二氟甲基、氯甲基、溴甲基、二氯氟甲基、氯乙基、溴丙基、2-氯丁基或五氟乙基等；
C 1-C 6烷氧基指含有1-6个碳原子的直链或支链烷氧基，例如，甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、仲丁氧基、正戊氧基、异戊氧基、新戊氧基、异己氧基、3-甲基戊氧基或正己氧基等，优选为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基或叔丁氧基；C1-C6烷酰基指含有1-6个碳原子的直连或支链烷酰基，如甲酰基、乙酰基、丙酰基、丁酰基、异丁酰基、戊酰基、叔丁酰基或己酰基等。
C 1-6烷基取代的氨基是指氨基氢原子被一个或多个C 1-C 6烷基取代，例如-NHCH 3、-N(CH 3) 2等。
C 1-6烷酰基取代的氨基是指氨基氢原子被一个或多个C 1-C 6烷基取代，例如-NHCOCH 3、-NHCOCH 2CH 3等。
C 2-C 6烯基指含有1-3个双键和2-6个碳原子的直链或支链的不饱和烃基，既包括顺式构型也包括反式构型，例如，乙烯基、1-丙烯基、2-丙烯基、1-甲基-1-丙烯基、2-甲基-1-丙烯基、2-甲基-2-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、4-戊烯基、1,3-丁二烯基、1,3-戊二烯基、1-己烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、3,3-二甲基-1-丙烯基或2-乙基-1-丙烯基等。
C 2-C 6炔基指含有2-6个碳原子的直链或支链炔基，例如，乙炔基、2-丙炔基、2-丁炔基、3-丁炔基、1-甲基-2-丙炔基、2-戊炔基、2-戊炔基或2-己炔基等。
C 2-C 6烯氧基是指含有1-3个双键和2-6个碳原子的直链或支链的链烯氧基，如乙烯氧基、1-丙烯氧基、1-甲基-1-丙烯氧基、2-甲基-1-丙烯氧基、1-戊烯氧基、 1,3-戊二烯氧基或2-戊烯氧基等。
C 2-C 6炔氧基指含有2-6个碳原子的直链或支链炔氧基，例如，乙炔氧基、2-丙炔氧基、2-丁炔氧基、3-丁炔氧基、1-甲基-2-丙炔氧基、2-戊炔氧基或2-己炔氧基等。
氨基C 1-20烷酰基指与含有1-20个碳原子的直链或支链烷酰基的一个碳原子与氨基连接，如-COCH 2NH 2、-COCH 2CH 2NH 2等。
C 1-6烷氨基C 1-6烷酰基指C 1-6烷基取代的氨基的氮原子与含有1-6个碳原子的直链或支链烷酰基的非羰基碳原子连接，如-COCH 2NHCH 3、-COCH 2CH 2NHCH 2CH 3等。
C 1-6烷氧基C 1-6烷基指与含有1-6个碳原子的直链或支链烷氧基的氧原子与C 1-6烷基的碳原子连接，如-CH 2OCH 3、-CH 2CH 2OCH 2CH 3等。
氨基C 1-6烷基指与含有1-6个碳原子的直链或支链烷基的一个碳原子与氨基连接，如-CH 2NH 2、-CH 2CH 2NH 2、-CH(NH 2)CH 3、-CH 2CH 2CH 2NH 2或-CH 2CH 2CH 2CH 2NH 2等。
C 1-6烷基取代的氨基甲酰基是指氨基甲酰基上的氢原子被1个或2个相同或不同的C 1-C 6烷基取代，例如-CONHMe、-CONHEt、-CON(Me)Et、-CONEt 2或-CONMe 2等。
羟基C 1-C 6烷基指含有1-6个碳原子的直链或支链烷基的一个碳原子与羟基连接，如-CH 2OH、-CH 2CH 2OH、-CH(OH)CH 3、-CH 2CH 2CH 2OH、-CH 2CH 2CH 2CH 2OH或-CH 2CH(CH 3)CH 2OH等。
C 1-6烷氧基酰胺基指含有1-6个碳原子的直链或支链烷氧基的氧原子与酰胺基的羰基连接，例如-NHCOOCH 3、-NHCOOCH 2CH 3等。
C 1-6烷氧羰基含有1-6个碳原子的直链或支链烷氧基的氧原子与羰基连接，例如-COOCH 3、-COOCH 2CH 3等。
冠状病毒
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。SARS-CoV-2也被称为2019-nCoV。
2003年和2012年分别爆发的高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白 (如刺突蛋白)及辅助蛋白。
流感病毒
流行性感冒病毒简称流感病毒,常见的流感病毒分为甲(A)、乙(B)、丙(C)和丁(D)型。流感病毒可引起人、禽、猪、马、蝙蝠等多种动物感染和发病，是人流感、禽流感、猪流感、马流感等人与动物疫病的病原。
流感病毒引起的临床症状包括急性高热、全身疼痛、显著乏力和呼吸道症状。人流感主要是甲型流感病毒和乙型流感病毒引起的。甲型流感病毒经常发生抗原变异，可以进一步分为H1N1、H3N2、H5N1、H7N9等亚型。
呼吸道合胞病毒
呼吸道合胞病毒(RSV，简称合胞病毒，也属副粘病毒科)是引起小儿病毒性肺炎最常见的病原，可引起间质性肺炎。
RSV与副流感病毒类似，病毒颗粒大小约为150nm，较副流感病毒稍小，为RNA病毒。
黄病毒科病毒
黄病毒科病毒是一类主要感染哺乳类动物的RNA病毒，包括3个病毒属，黄病毒属(flavivirus)、瘟病毒属(pestivirus)和丙型肝炎病毒属(hepacivirus)。登革热病毒(DENV)和寨卡病毒(Zika)属于黄病毒属，由蚊媒传播。登革病毒感染可引起明显的发热和疼痛症状，重度的登革热症状还表现为头痛，恶心，呕吐，意识不清，甚至休克等。寨卡病毒(Zika)感染后症状与登革热相似，一般较轻。丙肝病毒(HCV)属于丙型肝炎病毒属，是慢性丙型肝炎的病原体，可导致肝硬化、肝癌。
丝状病毒科病毒
丝状病毒科目前包含三个属，分别为埃博拉病毒属、马尔堡病毒属、奎瓦病毒属。马尔堡病毒和埃博拉病毒都可以引起严重的出血热，人感染后会出现高烧和出血症状，进一步会导致患者休克、器官衰竭，直至死亡。
猪流行性腹泻病毒(PEDV)
猪流行腹泻病毒(PEDV),属于冠状病毒科冠状病毒属。猪流行性腹泻是由PEDV病毒引起的仔猪和育肥猪的一种急性肠道传染病。
PEDV病毒经口和鼻感染后，直接进入小肠。PEDV病毒的复制可在小肠和结肠绒毛上皮细胞浆中进行。PEDV可导致腹泻，属于渗透性腹泻。严重腹泻引起脱水，是导致病猪死亡的主要原因。
本发明的活性化合物和活性成分
在本发明中，提供了一种可有效抑制冠状病毒、流感病毒、呼吸道合胞病毒和/或猪流行性腹泻病毒(PEDV)尤其是2019新型冠状病毒(SARS-CoV-2)复制的活性成分。
在本发明中，活性成分选自本发明第一方面中所述的式(I)所示的核苷类似物、或其药学上可接受的盐、或其前药、或其组合、或其晶体、或其溶剂化物。
优选的核苷类似物的结构式为实施例中所制备的化合物或其药学上可接受的盐、或其前药、或其组合、或其晶体、或其溶剂化物，尤其是化合物A1至A221中任一化合物、或其组合。
在另一优选例中，所述的式(I)化合物选自下组：化合物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或其组合。
试验表明，本发明的活性成分可有效地抑制2019新型冠状病毒(SARS-CoV-2)的复制，从而预防、治疗和/或缓解SARS-CoV-2相关疾病。
如本文所用，“本发明的活性化合物”、“本发明的抑制病毒复制的活性化合物”可互换使用，指具有优异的抑制病毒复制的活性的化合物，包括式I核苷类似物或其药学上可接受的盐、或其晶体、或其溶剂化物。
应理解，本发明的活性成分包括式I化合物、或其药学上可接受的盐、对映异构体、非对映异构体或外消旋体、或其前药。应理解，本发明的活性成分还包括本发明的活性化合物的晶型、无定形化合物、以及氘代化合物等形式。
所述“药学上可接受的盐”为本发明的活性化合物与无机酸或有机酸反应形成常规的无毒盐。例如，常规的无毒盐可通过本发明的活性化合物与无机酸或有机酸反应制得，所述无机酸包括盐酸、氢溴酸、硫酸、硝酸、胺基磺酸和磷酸等，所述有机酸包括柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙 酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者本发明的活性化合物与丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、天冬氨酸或谷氨酸形成酯后再与无机碱形成的钠盐、锌盐、钾盐、钙盐、铝盐或铵盐；或者本发明的活性化合物与赖氨酸、精氨酸、鸟氨酸形成酯后再与盐酸、氢溴酸、氢氟酸、硫酸、硝酸或磷酸形成的对应的无机酸盐或与甲酸、乙酸、苦味酸、甲磺酸或乙磺酸形成的对应的有机酸盐；或者本发明的活性化合物分子中的1-4个磷酸基与1-4分子有机胺形成盐，包括但不限于单磷酸三甲胺盐、单磷酸三乙胺盐、单磷酸三正丁胺盐、三磷酸三甲胺盐、三磷酸三乙胺盐、三磷酸三正丁胺盐。
此外，本发明的活性成分还特别适合与其他抗病毒药物或抗冠状病毒药物联用。代表性的其他的抗病毒药物或抗冠状病毒药物包括(但并不限于)：干扰素、RNA依赖的RNA聚合酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524、NHC、EIDD-2801)；3CL蛋白酶抑制剂(如GC-376)、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、连翘脂苷(forsythoside)、绿原酸(chlorogenic acid)、大黄素(emodin)、霉酚酸(mycophenolic acid)、霉酚酸酯(Mycophenolate mofetil)、萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、帕利珠单抗、莫维珠单抗(Motavizumab)、RSV-IGIV
Figure PCTCN2021087928-appb-000031
MEDI-557、A-60444(RSV-604)、MDT-637、BMS-433771或其药学上可接受的盐、或其组合。所述干扰素包括干扰素α-2a、干扰素α-2b、干扰素α-n1、干扰素α-n3、干扰素β-1a、干扰素β-1b中的一种或多种。
用于治疗呼吸症状和感染后遗症的另外活性治疗剂也可以与式I化合物组合使用。其它药剂优选地口服或通过直接吸入来施用。例如，与式I化合物组合用于治疗呼吸道感染的其它优选的另外治疗剂包括但不限于支气管扩张药和皮质类固醇。
糖皮质激素类，最初在1950年作为哮喘疗法被介绍(Carryer,Journal of Allergy,21，282-287,1950)，对该疾病保持是最有效的且一贯有效的疗法，但还未完全理解它们的作用机制(Morris,J.Allergy Clin.Immunol.,75(1Pt)1-13,1985)。不幸地，口服糖皮质激素疗法与深远的不期望副作 用相关联，副作用例如向心性肥胖症、高血压、青光眼、葡萄糖耐受不良、白内障形成加速、骨密度损失(bone mineral loss)和心理影响，所有这些限制它们用作长期治疗剂(Goodman和Gilman,第10版，2001)。对全身副作用的解决方案是将类固醇药物直接递送至炎症部位。已经开发被吸入的皮质类固醇(ICS)来减轻口服类固醇的严重副作用。可以与式I化合物组合使用的皮质类固醇的非限制性实例是地塞米松、地塞米松磷酸钠、氟米龙、醋酸氟米龙、氯替泼诺、依碳酸氯替泼诺、氢化可的松、泼尼松龙、氟氢可的松、曲安西龙、曲安奈德、倍他米松、双丙酸倍氯美松、甲泼尼龙、氟轻松、氟西奈德、氟尼缩松、氟可丁-21-丁酯(fluocortin-21-butylate)、氟米松、新戊酸氟米松、布地奈德、丙酸卤贝他索、糠酸莫米他松、丙酸氟替卡松、环索奈德；或其药学上可接受的盐。
通过抗炎级联机制起作用的其它抗炎剂还可用作与式I化合物组合用于治疗病毒性呼吸道感染的另外的治疗剂。应用“抗炎信号转导调节剂”(在本文中被称为AISTM)比如磷酸二酯酶抑制剂(例如PDE-4、PDE-5或PDE-7特异的)、转录因子抑制剂(例如通过IKK抑制阻断NFκB)或激酶抑制剂(例如阻断P38MAP、JNK、PI3K、EGFR或Syk)，是切断炎症的合乎逻辑的方法，因为这些小分子的目标是有限数量的常见细胞内路径-是抗炎治疗干预的关键点的那些信号转导路径(参见，由P.J.Barnes,2006综述的)。这些非限制性的另外的治疗剂包括：5-(2,4-二氟-苯氧基)-1-异丁基-1H-吲唑-6-甲酸(2-二甲基氨基-乙基)-酰胺(P38Map激酶抑制剂ARRY-797)；3-环丙基甲氧基-N-(3,5-二氯-吡啶-4-基)-4-二氟甲氧基-苯甲酰胺(PDE-4抑制剂罗氟司特)；4-[2-(3-环戊氧基-4-甲氧基苯基)-2-苯基-乙基]-吡啶(PDE-4抑制剂CDP-840)；N-(3,5-二氯-4-吡啶基)-4-(二氟甲氧基)-8-[(甲基磺酰基)氨基]-1-二苯并呋喃甲酰胺(PDE-4抑制剂奥米斯特)；N-(3,5-二氯-吡啶-4-基)-2-[1-(4-氟苄基)-5-羟基-1H-吲哚-3-基]-2-氧代-乙酰胺(PDE-4抑制剂AWD12-281)；8-甲氧基-2-三氟甲基-喹啉-5-甲酸(3,5-二氯-1-氧基-吡啶-4-基)-酰胺(PDE-4抑制剂Sch351591)；4-[5-(4-氟苯基)-2-(4-甲亚磺酰基-苯基)-1H-咪唑-4-基]-吡啶(P38抑制剂SB-203850)；4-[4-(4-氟-苯基)-1-(3-苯基-丙基)-5-吡啶-4-基-1H-咪唑-2-基]-丁-3-炔-1-醇(P38抑制剂RWJ-67657)；4-氰基-4-(3-环戊氧基-4-甲氧基-苯基)-环己烷甲酸2-二乙基氨基-乙酯(西洛司特的2-二乙基-乙酯前药、PDE-4抑制剂)；(3-氯-4-氟苯基)-[7-甲氧基-6-(3-吗啉-4-基-丙氧基)-喹唑啉-4-基]-胺(吉非替尼，EGFR抑制剂)；和4-(4-甲基-哌嗪-1-基甲基)-N-[4-甲基-3-(4-吡啶-3-基-嘧啶-2-基氨基)-苯基]-苯甲酰胺(伊马替尼，EGFR抑制剂)。
包括吸入的β2-肾上腺素受体激动剂支气管扩张药例如福莫特罗、沙丁胺醇或沙美特罗和式I化合物的组合还是可用于治疗呼吸道病毒性感染的合适的但非限制性的组合。
吸入的β2-肾上腺素受体激动剂支气管扩张药例如福莫特罗或沙美特罗与ICS的组合还用于治疗支气管狭窄和炎症两者。包括这些ICS和β2-肾上腺素受体激 动剂组合连同式I化合物的组合还是可用于治疗呼吸道病毒性感染的合适的但非限制性的组合。
对于治疗或预防肺支气管狭窄，抗胆碱能药具有潜在的用途且因此可用作与式I化合物组合用于治疗病毒性呼吸道感染的另外的治疗剂。这些抗胆碱能药包括但不限于，已经在人中对于COPD中的胆碱能特性的控制显示出治疗功效的毒蕈碱性受体(特别是M3亚型)拮抗剂(Witek,1999)；1-{4-羟基-1-[3,3,3-三-(4-氟-苯基)-丙酰基]-吡咯烷-2-羰基}-吡咯烷-2-甲酸(1-甲基-哌啶-4-基甲基)-酰胺；3-[3-(2-二乙基氨基-乙酰氧基)-2-苯基-丙酰氧基]-8-异丙基-8-甲基-8-氮阳离子(azonia)-双环[3.2.1]辛烷(异丙托品-N,N-二乙基甘氨酸酯或盐)；1-环己基-3,4-二氢-1H-异喹啉-2-甲酸1-氮杂-双环[2.2.2]辛-3-基酯(索利那新)；2-羟基甲基-4-甲亚磺酰基-2-苯基-丁酸1-氮杂-双环[2.2.2]辛-3-基酯(瑞伐托酯)；2-{1-[2-(2,3-二氢-苯并呋喃-5-基)-乙基]-吡咯烷-3-基}-2,2-二苯基-乙酰胺(达非那新)；4-氮杂环庚烷-1-基-2,2-二苯基-丁酰胺(甲碘布卓(Buzepide))；7-[3-(2-二乙基氨基-乙酰氧基)-2-苯基-丙酰氧基]-9-乙基-9-甲基-3-氧杂-9-氮阳离子-三环[3.3.1.02,4]壬烷(氧托品-N,N-二乙基甘氨酸酯或盐)；7-[2-(2-二乙基氨基-乙酰氧基)-2,2-二-噻吩-2-基-乙酰氧基]-9,9-二甲基-3-氧杂-9-氮阳离子-三环[3.3.1.02,4]壬烷(噻托溴铵-N,N-二乙基甘氨酸酯或盐)；二甲基氨基-乙酸2-(3-二异丙基氨基-1-苯基-丙基)-4-甲基-苯基酯(托特罗定-N,N-二甲基甘氨酸酯或盐)；3-[4,4-双-(4-氟-苯基)-2-氧代-咪唑啉-1-基]-1-甲基-1-(2-氧代-2-吡啶-2-基-乙基)-吡咯烷鎓；1-[1-(3-氟-苄基)-哌啶-4-基]-4,4-双-(4-氟-苯基)-咪唑烷-2-酮；1-环辛基-3-(3-甲氧基-1-氮杂-双环[2.2.2]辛-3-基)-1-苯基-丙-2-炔-1-醇；3-[2-(2-二乙基氨基-乙酰氧基)-2,2-二-噻吩-2-基-乙酰氧基]-1-(3-苯氧基-丙基)-1-氮阳离子-双环[2.2.2]辛烷(阿地溴铵-N,N-二乙基甘氨酸酯或盐)；或(2-二乙基氨基-乙酰氧基)-二-噻吩-2-基-乙酸1-甲基-1-(2-苯氧基-乙基)-哌啶-4-基酯。
此外，由于SARS-CoV-2)感染可引起急性肺损伤，本发明的活性成分还特别适合与具有改善急性肺损伤作用的药物联用。代表性的药物包括但不限于锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)。所述锌(Zinc)包括但不限于硫酸锌、甘草酸锌、葡萄糖酸锌。
本发明的活性成分可抑制SARS-CoV-2等新型冠状病毒的感染活性。因此，当在治疗上施用或给予本发明的的活性成分时，可抑制2019新型冠状病毒(SARS-CoV-2)的感染，进而达到抗病毒作用。
式I化合物还可以与黏液溶解药组合治疗感染和呼吸道感染症状两者。黏液溶解药的非限制性实例是氨溴索。同样地，式I化合物可以与祛痰药组合治疗感染和呼吸道感染症状两者。祛痰药的非限制性实例是愈创甘油醚。
雾化高渗盐水用于改进肺病患者中的小气道的即时和长期清除(Kuzik,J.Pediatrics2007,266)。式I化合物还可以与雾化高渗盐水组合，特别是当副黏病毒科病毒感染并发细支气管炎时。式I化合物与高渗盐水的组合还可以包括上面所讨论的任意另外的药剂。在优选的方面，使用约3％雾化高渗盐水。
还可能使本发明的任意化合物与一种或多种另外活性治疗剂相组合在单位剂型中，用于同时或依次施用给患者。组合治疗可以作为同时或依次方案来施用。当依次施用时，可以在两次或更多次施用中施用该组合。
本发明化合物与一种或多种另外活性治疗剂的共同施用一般是指，同时或依次施用本发明的化合物与一种或多种另外活性治疗剂，使得治疗有效量的本发明化合物和一种或多种另外活性治疗剂均存在于患者体内。
共同施用包括在施用单位剂量的一种或多种另外活性治疗剂之前或之后施用单位剂量的本发明的化合物，例如，在施用一种或多种另外活性治疗剂的数秒、数分钟或数小时内，施用本发明的化合物。例如，可以首先施用单位剂量的本发明的化合物，随后在数秒或数分钟内，施用单位剂量的一种或多种另外活性治疗剂。或者，可以首先施用单位剂量的一种或多种其它治疗剂，随后在数秒或数分钟内，施用单位剂量的本发明的化合物。在有些情况下，可能需要首先施用单位剂量的本发明的化合物，在数小时时期(例如1-12小时)后，施用单位剂量的一种或多种另外活性治疗剂。在其它情况下，可能需要首先施用单位剂量的一种或多种另外活性治疗剂，在数小时时期(例如1-12小时)后，施用单位剂量的本发明的化合物。
组合治疗可提供“增效作用”和“协同作用”，即，当活性成分一起使用时获得的效果大于分开使用化合物所得效果之和。当活性成分：(1)被共同配制并以组合制剂形式同时施用或递送；(2)作为单独的制剂交替施用或平行递送；或(3)通过一些其它施用方案递送时，可获得协同作用。当以交替治疗递送时，当化合物依次施用或递送时，例如以单独的片剂、丸剂或胶囊剂，或通过单独注射器的不同注射，可获得协同作用。通常，在交替治疗期间，有效剂量的每种活性成分被依次施用，即连续地施用，而在组合治疗中，有效剂量的两种或多种活性成分被一起施用。协同的抗病毒作用表示，大于所述组合中的单个化合物的预测的纯累加效应的抗病毒作用。
在又一个实施方案中，提供了抑制细胞中的病毒RNA聚合酶的方法，其包括使受到病毒感染的细胞接触有效量的式I化合物或其药学上可接受的盐、溶剂化物和/或前药(例如，酯)，由此抑制病毒RNA聚合酶。
在又一个实施方案中，提供了抑制细胞中的病毒RNA聚合酶的方法，其包括使受到病毒感染的细胞接触有效量的式I化合物或其药学上可接受的盐、溶剂化物 和/或前药(例如，酯)，和至少一种另外活性治疗剂，由此抑制病毒RNA聚合酶。
在又一个实施方案中，提供了抑制细胞中的病毒RNA聚合酶的方法，其包括使受到病毒感染的细胞接触有效量的式I化合物或其药学上可接受的盐、溶剂化物和/或前药(例如，酯)，和选择的至少一种另外活性治疗剂。
在又一个实施方案中，提供了治疗人或其他哺乳动物中的呼吸道病毒感染的方法，其包括对所述人或其他哺乳动物施用治疗有效量的式I化合物或其药学上可接受的盐、溶剂化物和/或前药(例如，酯)。
在又一个实施方案中，提供了治疗人或其他哺乳动物中的呼吸道病毒感染的方法，其包括对所述人或其他哺乳动物施用治疗有效量的式I化合物或其药学上可接受的盐、溶剂化物和/或前药(例如，酯)，和至少一种另外活性治疗剂，由此抑制呼吸道病毒RNA聚合酶。
在又一个实施方案中，提供了治疗人或其他哺乳动物中的呼吸道病毒感染的方法，其包括对所述人或其他哺乳动物施用治疗有效量的式I化合物或其药学上可接受的盐、溶剂化物和/或前药(例如，酯)，和至少一种另外活性治疗剂。
药物组合物和应用
本发明还提供了以本发明的抑制病毒复制的活性化合物、或其药学上可接受的盐、或其前药的一种或多种的混合物为有效成分，在制备治疗和/或预防、缓解由冠状病毒、流感病毒、呼吸道合胞病毒和/或猪流行性腹泻病毒(PEDV)等病毒(尤其是2019新型冠状病毒)感染引起的呼吸道感染、肺炎等相关疾病的药物中的用途。
本发明所提供的药物组合物优选含有重量比为0.001-99wt％的活性成份，优选的比例是本发明的活性化合物作为活性成分占总重量的0.1wt％～90wt％或1wt％～50wt％，其余部分为药学可接受的载体、稀释液或溶液或盐溶液。
需要的时候，在本发明药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
本发明所提供的化合物和药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中和适宜的用于注射或滴注的消毒器具中。
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。其制剂配方的单位计量中通常包含0.05-1000mg本发明的活性化合物，优选地，制剂配方的单位计量中包含1mg-500mg本发明的活性化合物。
本发明的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服。最优选日剂量为0.01-400mg/kg体重，一次性服用，或0.01-200mg/kg体重分次服用。不管用何 种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
本发明的药物或抑制剂可通过各种不同方式施用，例如可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹导入机体。
典型地，本发明活性成分或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括O/W型、W/O型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明活性成分可以被制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明活性成分被制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；黏合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明活性成分与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明的胶囊剂。
为将本发明活性成分制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、PH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；PH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、 葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其他添加剂。
本发明的活性成分或组合物可单独服用，或与其他治疗药物或对症药物合并使用。
当本发明的活性成分与其他治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明的主要优点包括：
(a)本发明活性化合物可高效地抑制SARS-CoV-2、人呼吸道合胞病毒(RSV)、人冠状病毒OC43(HCoV OC43)、猪流行性腹泻病毒(PEDV)、寨卡病毒(Zika)、登革热病毒(DENV)等病毒的复制，EC 50值小,并且具有广谱抗病毒特点。
(b)本发明活性化合物的毒副作用低(细胞毒性CC 50>10μM)，成药性好。这提示着本发明的核苷类似物在治疗病毒感染性疾病，尤其是抗新冠肺炎领域有很好的药用前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照试剂制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
制备实施例1：化合物A1的合成
Figure PCTCN2021087928-appb-000032
化合物1-1参考文献报道方法合成(Nature.2016,531,381-385)。将化合物1-1(1.5g，2.67mmol)加入到乙腈(30mL)中，随后加入1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷二(四氟硼酸)盐(Selectfluor，1.13g，3.2mmol，1.2eq)，碳酸氢钠(0.67g，8.0mmol，3eq)，加毕，室温反应3-4小时，TLC显示反应完全。将反应液加入到水(120mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物1-2，类白色固体0.43g，收率28％。 1H 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)。
化合物1-2(0.25g，0.43mmol)加入到二氯甲烷(10mL)中，降温至-60℃，滴加1M三溴化硼二氯甲烷溶液(1.72mL，1.72mmol，4.0eq)，加毕，自然升温至-40℃，在该温度下继续搅拌2小时后，TLC监测反应完全。降温至-60℃，依次滴加甲醇(0.2mL)和三乙胺(0.52g，5.16mmol，12eq)，加毕，蒸除溶剂，得到黄色固体。固体加入到甲醇(2mL)和乙酸乙酯(10mL)中，搅拌后过滤，除去白色不溶物，滤液浓缩，制备板分离，得化合物A1，类白色固体49mg，收率37％。 1H 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] +。
制备实施例2：化合物A2的合成
Figure PCTCN2021087928-appb-000033
化合物2-1参照文献报道方法合成(Nature.2016,531,381-385)。化合物2-1(291mg，1.0mmol)加入到N,N-二甲基甲酰胺(5mL)中，加入N-氯代丁二酰亚胺(245mg,1.1mmol，1.1eq)和三氟乙酸(24mg，0.2mmol，0.2eq)，50℃下反应1小时，TLC显示反应完全。将反应液加入亚硫酸钠和碳酸钠的混合溶液中，过滤得化合物A2，白色固体185mg，收率57％。 1H 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] +。
制备实施例3：化合物A3的合成
Figure PCTCN2021087928-appb-000034
化合物2-1(291mg，1.0mmol)加入到N,N-二甲基甲酰胺(5mL)中，加入 N-碘代丁二酰亚胺(245mg，1.1mmol，1.1eq)和三氟乙酸(24mg，0.2mmol)，50℃下反应1小时，TLC显示反应完全。将反应液加入亚硫酸钠和碳酸钠的混合溶液中，过滤得化合物A3，白色固体200mg，收率48％。 1H NMR(500MHz,DMSO-d 6)δ7.98(s,1H),7.12(s,1H),6.21(brs,1H),5.20(brs,1H),4.91(s,1H),4.52(d,J＝4.6Hz,1H),4.11–3.99(m,1H),3.97–3.85(m,1H),3.64(d,J＝11.3Hz,1H),3.49(d,J＝10.9Hz,1H)。MS m/z＝418.0[M+1] +。
制备实施例4：化合物A4的合成
Figure PCTCN2021087928-appb-000035
化合物1-1(350mg，0.62mmol)加入到80％乙酸水溶液(10mL)中，降温至0℃，分批多次加入亚硝酸钠(856mg，12.4mmol，20eq)，加毕，保温5分钟，室温下搅拌30分钟后，逐渐升温至90℃，保温搅拌4小时，TLC显示原料转化完毕。加入(5mL)水和(15mL)甲苯，分出有机层，水层用甲苯再萃取一次，合并有机相，有机相依次用水、碳酸氢钠水溶液和食盐水洗涤，干燥，浓缩，硅胶柱层析分离得化合物4-1，淡黄色泡沫状固体345mg。
将上步得到的产物4-1溶于二氯甲烷中(3mL)，氮气保护，降温至-35℃，滴加1M三氯化硼的二氯甲烷溶液(2.1mL，2.1mmol)，10分钟左右加毕，保温15分钟，缓慢滴加入甲醇(0.3mL)，加毕，保温20分钟，浓缩反应液，加入正庚烷(5mL)，室温下搅拌1小时，过滤，滤饼用少量正庚烷洗，得黄色固体255mg。该固体于甲醇(0.75mL)和水(0.1mL)中回流30分钟，加入活性炭(12mg)，过滤，滤渣用少量甲醇洗，滤液蒸干，加入少量乙酸乙酯，打浆，过滤，得化合物A4，淡黄色固体85mg，收率44％。 1H 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] -。
制备实施例5：化合物A9的合成
Figure PCTCN2021087928-appb-000036
化合物1-1(561mg，1.0mmol)溶于N,N-二甲基甲酰胺(5mL)中，分批加入碘(508mg，2mmol，2eq)，室温反应过夜，TLC显示有原料剩余。将反应液加入到亚硫酸钠和碳酸钠的混合溶液中，乙酸乙酯萃取，分出有机相，有机相依次用水和饱和食盐水洗涤，无水硫酸钠干燥，硅胶柱层析分离，得化合物9-1，白色固体400mg，收率58％。 1H NMR(500MHz,DMSO-d 6)δ7.98(s,1H),7.40–7.22(m,15H),6.86(s,1H),4.91(d,J＝11.7Hz,1H),4.84–4.78(m,2H),4.50(q,J＝12.0Hz,4H),4.41–4.35(m,1H),4.12–4.08(m,1H),3.72(dd,J＝11.2,2.8Hz,1H),3.59(dd,J＝11.2,4.1Hz,1H)。
化合物9-1(69mg，0.1mmol)溶于无水四氢呋喃(5mL)中，冰浴下，加入三甲基氯硅烷(24mg，0.22mmol，2.2eq)，搅拌10分钟后，降温至-10℃，随后滴加3.0M甲基溴化镁的2-甲基四氢呋喃溶液(74uL，0.22mmol，2.2eq)，加毕，搅拌30分钟。降温至-20℃，加入1.3M异丙基氯化镁氯化锂四氢呋喃溶液(0.1mL，0.13mmol，1.3eq)，继续搅拌1小时。向反应体系中加入重水(0.2mL)，搅拌15分钟后将反应液加入到饱和氯化铵溶液中，乙酸乙酯萃取，分出有机相，有机相依次用水和饱和食盐水洗涤，无水硫酸钠干燥，硅胶柱层析分离，得化合物9-2，白色固体28mg，收率50％。 1H NMR(500MHz,Methanol-d 4)δ7.65(s,1H),7.34–7.16(m,15H),6.84(s,1H),4.95(d,J＝5.1Hz,1H),4.74(q,J＝11.9Hz,2H),4.57(d,J＝11.9Hz,1H),4.53–4.43(m,4H),4.11(t,J＝5.2Hz,1H),3.73(dd,J＝10.9,4.1Hz,1H),3.62(dd,J＝10.9,4.5Hz,1H)。
化合物9-2(90mg，0.16mmol)溶于二氯甲烷(10mL)中，-60℃下，滴加1.0M三氯化硼二氯甲烷溶液(0.56mL，0.56mmol)，加毕，在-40℃搅拌1小时，TLC显示反应完全。向反应液中加入甲醇(0.1mL)，随后加入三乙胺至反应液pH为7-8，将反应液浓缩，硅胶柱层析分离，得化合物A9，类白色固体15mg，收率50％。 1H NMR(500MHz,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] +。
制备实施例6：化合物A9的合成
Figure PCTCN2021087928-appb-000037
化合物9-1(69mg，0.1mmol)加入到干燥的四氢呋喃(5mL)和重水(1mL)的混合溶液中，将溶液浓缩干后，再加入干燥的四氢呋喃(5mL)和重水(1mL)，然后依次加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(8mg，0.01mmol)和四甲基乙二胺(3mg，0.02mmol)，加完后搅拌10分钟，然后分批加入硼氘化钠(21mg，0.5mmol)，2小时后TLC显示原料反应完全。将反应液加入水中，乙酸乙酯萃取，分出有机相，有机相用饱和食盐水洗涤，无水硫酸钠干燥，硅胶柱层析分离，得化合物9-2，氘代率不低于97％，白色固体30mg，收率53％。
参考实施例5中的合成方法，化合物9-2(90mg，0.16mmol)脱除保护基后得化合物A9，类白色固体15mg，收率50％。 1H 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)。 13C NMR(126MHz,DMSO-d 6)δ156.08,148.32,124.35,117.82,116.94,111.16,85.92,79.04,74.72,70.56,61.43。氘代率不低于97％，MS m/z＝293.0[M+1] +。
制备实施例7：化合物A10的合成
Figure PCTCN2021087928-appb-000038
化合物2-1(0.5g，1.72mmol)加入到丙酮(10mL)中，依次加入2,2-二甲氧基丙烷(0.89g，8.6mmol，5eq)和对甲苯磺酸一水合物(0.59g，3.1mmol，1.8eq)，加毕，45℃下搅拌2小时，TLC监测反应完全。将反应液加入到饱和碳酸氢钠水溶液(20mL)中，乙酸乙酯萃取，分出有机层，干燥，蒸除溶剂，得化合物10-1，白色固体0.33g，收率58％。
化合物10-1(0.25g，0.75mmol)加入到乙腈/水(V/V＝1/1，15mL)中，依次加入TEMPO(0.047g，0.3mmol，0.4eq)、二乙酸碘苯(1.06g，3.3mmol，4.4eq)、碳酸氢钠(0.25g，3mmol，4.0eq)，加毕，室温搅拌5小时左右，TLC显示反应完全。将反应液加入到0.5M氢氧化钠溶液(20mL)中，乙酸乙酯(20mL×2)萃取两次，舍弃有机相，水层用稀盐酸(2M)调节pH至4-5，乙酸乙酯(50mL×2)萃取，合并有机层，干燥，浓缩，得化合物10-2，白色固体0.23 g，收率89％。
将上步得到的产物10-2(0.18g，0.52mmol)加入到四氢呋喃和甲醇(8mL，1:1)中，室温下，滴加2M三甲基硅基重氮甲烷正己烷溶液(1.2mL，2.4mmol，4.5eq)，加毕，室温反应，1小时后反应完全。向反应液滴加醋酸直至不再产生气泡，蒸除溶剂，制备板分离，得化合物10-3，白色固体0.11g，收率59％。 1H NMR(500MHz,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)。
化合物10-3(0.10g，0.28mmol)加入到无水四氢呋喃(4mL)和氘代甲醇(1mL)中，冰浴条件下，加入硼氘化钠(0.047g，1.12mmol，4eq)，加毕，室温反应，1小时后TLC显示反应完全。向反应液滴加稀盐酸直至不再产生气泡，浓缩反应液，制备板分离，得化合物10-4，白色固体0.06g，收率64％。
将上步产物10-4(60mg，0.18mmol)加入到四氢呋喃(2mL)中，滴加浓盐酸(0.4mL)，40℃下反应，直至反应完全。向反应液中加入碳酸氢钠溶液至pH为中性，四氢呋喃萃取，分出有机相，有机相干燥后蒸干，得类白色固体。所得固体加入到乙酸乙酯中，搅拌0.5小时，过滤，烘干，得化合物A10，白色固体34mg，收率64％。 1H 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)。 13C NMR(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。氘代率为99％，MS m/z＝294.0[M+1] +。
制备实施例8：化合物A11的合成
Figure PCTCN2021087928-appb-000039
化合物A9(1.17g，4.0mmol，氘代率不低于97％)加入到丙酮(20mL)中，依次加入2,2-二甲氧基丙烷(2.08g，20.0mmol，5eq)和对甲苯磺酸一水合物(1.37g，7.2mmol，1.8eq)，加毕，45℃下搅拌2小时，出现大 量固体，TLC监测反应完全。将反应液加入到饱和碳酸氢钠水溶液中，乙酸乙酯萃取，分出有机层，干燥，蒸除溶剂，得化合物11-1，白色固体0.86g，收率65％。
化合物11-1(0.225g，0.68mmol)加入到乙腈/水(V/V＝1/1，10mL)中，依次加入TEMPO(0.02g，0.14mmol，0.2eq)、二乙酸碘苯(0.66g，2.04mmol，3.0eq)、碳酸氢钠(0.17g，2.04mmol，3.0eq)，加毕，室温搅拌4-5小时，TLC监测反应完全。将反应液加入到KOH水溶液(0.5M，20mL)中，乙酸乙酯(20mL×3)萃取水层，分出水层。水层用稀盐酸(2M)调节pH至4-5，乙酸乙酯(20mL×3)萃取，合并有机层，干燥，浓缩得11-2粗品，直接投下步反应。
化合物11-2粗品(0.68mmol，按100％产率)加入到四氢呋喃/甲醇(V/V＝1/1，8mL)中，室温滴加三甲基硅基重氮甲烷正己烷溶液(2M，1.36mL，4.0eq)，加毕，室温反应，30分钟后反应完全。向反应液滴加醋酸直至不再产生气泡。蒸干反应液，加入乙酸乙酯(20mL)，饱和碳酸氢钠水溶液(10mL×2)洗涤，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物11-3，白色固体0.06g，两步反应收率24％。
化合物11-3(0.06g，0.17mmol)加入到氘代甲醇/四氢呋喃(V/V＝1/5，6mL)中，冰浴条件下加入NaBD 4(0.028g，0.68mmol，4eq)，加毕，室温反应，1-2小时后反应完全。将反应液加入到乙酸乙酯/水(20mL/20mL)混合液中，分出有机层，水层再用乙酸乙酯(20mL)萃取一次。合并乙酸乙酯层，并蒸干。将得到的产物加入到甲醇(10mL)中，滴加稀盐酸(1M)，使溶液pH为2-3，室温搅拌1-2小时后，饱和碳酸氢钠水溶液调节pH至中性。加入乙酸乙酯/水(20mL/20mL)混合液，分出有机相，水层再用乙酸乙酯(20mL)萃取一次。合并有机层，干燥，浓缩，硅胶制备板分离，得到化合物11-4，白色固体0.05g，收率89％。
11-4(0.05g，0.15mmol)加入到四氢呋喃(1.5mL)中，冰浴下滴加浓盐酸(0.3mL)，加毕，室温搅拌反应2-3小时，TLC监测反应完全。冰浴下，反应液用1M的NaOH水溶液调节pH至中性，蒸除四氢呋喃后，过滤，滤饼依次使用蒸馏水(10mL)、乙酸乙酯(10mL)打浆，过滤，烘干，得化合物A11，白色固体0.03g，收率68％。 1H NMR(500MHz,DMSO-d 6)δ8.04–7.76(m,3H),6.89(s,1H),6.07(d,J＝6.3Hz,1H),5.18(d,J＝5.2Hz,1H),4.87(s,1H),4.67(t,J＝5.8Hz,1H),4.07(d,J＝5.3Hz,1H),4.01–3.93(m,1H)。m/z＝295.0[M+1] +。两个位点的氘代率分别不低于97％。
制备实施例9：化合物A12的合成
Figure PCTCN2021087928-appb-000040
化合物10-1(662mg，2.0mmol)加入到乙腈(15mL)中，加入碳酸氢钠(336mg，4mmol，2eq)和1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷二(四氟硼酸)盐(850mg，2.4mmol，1.2eq)，室温下搅拌24小时，TLC显示有部分原料剩余。向反应液中加入乙酸乙酯(50mL)和水(20mL)，分出有机相，干燥，浓缩，硅胶柱层析分离，得化合物12-1，类白色固体140mg，收率20％。
化合物12-1(140mg，0.4mmol)加入到乙腈和水的混合溶液中(6mL，1:1)，室温下，依次加入二乙酸碘苯(515mg，1.6mmol，4eq)、碳酸氢钠(135mg，1.6mmol，4eq)和TEMPO(25mg，0.16mmol，0.4eq)，3小时左右，反应完全。向反应液中加入氢氧化钠水溶液(10mL，224mg，10eq)，乙酸乙酯萃取2次，舍弃有机相。向水相中加入2M稀盐酸，使其pH至4-5，乙酸乙酯萃取3次，合并有机相，干燥，蒸除溶剂，得化合物12-2，类白色固体116mg，收率80％。
将化合物12-2(116mg，0.32mmol)加入到四氢呋喃和甲醇(4mL，1:1)混合溶液中，缓慢加入2M三甲基硅烷化重氮甲烷正己烷溶液(0.48mL，0.96mmol，3eq)，TLC监测，若原料仍剩余，可再补加适量三甲基硅烷化重氮甲烷，直至反应完全。向反应液滴加醋酸至不再产生气泡，蒸除溶剂，硅胶柱层析分离，得化合物12-3，白色固体105mg，收率87％。
将上步产物12-3(105mg，0.28mmol)加入到无水四氢呋喃(2mL)中，加入氘代甲醇(0.5mL)，室温下，分批次加入硼氘化钠(35mg，0.84mmol，3eq)，1小时内加完，继续搅拌5小时，TLC显示原料反应完全。向反应液中加入乙酸乙酯(0.5mL)，浓缩反应液，制备板分离，得化合物12-4，白色泡沫状固体80mg，收率82％。
将上步产物12-4(80mg，0.23mmol)加入到四氢呋喃(3mL)中，滴加浓盐酸(0.5mL)，40℃反应，直至反应完全。反应液中析出不溶物，过滤，得白色固体55mg，将固体加入到水(1.5mL)中，加入碳酸钠(16mg)，搅拌0.5小时，过滤，烘干，得化合物A12，白色固体44mg，收率62％。 1H NMR(500MHz,DMSO-d 6)δ8.10(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＝4.9Hz,1H),4.04(d,J＝6.3Hz,1H),3.94(t,J＝5.5Hz,1H)。氘代率为98％，MS m/z＝312.0[M+1] +。
制备实施例10：化合物A35的合成
Figure PCTCN2021087928-appb-000041
化合物35-1参照文献(WO2015069939)报道方法合成。盐酸羟胺(547mg，7.87mmol，30eq)加入到水(3.2mL)中，缓慢滴加10％氢氧化钠溶液，使体系pH值为6.0，然后加入化合物35-1(69mg，0.26mmol，1eq)，氮气保护，40℃下搅拌过夜，TLC显示反应完全。反应液用反相柱分离，得类白色固体A35，52mg，收率71％。 1H NMR(500MHz,DMSO-d 6)δ10.61(d,J＝4.0Hz,1H),10.06(s,1H),7.39(d,J＝4.0Hz,1H),6.38–6.33(m,2H),4.96(d,J＝6.6Hz,1H),4.93(d,J＝6.3Hz,1H),4.86(d,J＝5.1Hz,1H),4.74–4.68(m,1H),4.15(q,J＝6.1Hz,1H),3.91(q,J＝4.9Hz,1H),3.74(q,J＝4.5Hz,1H),3.55–3.49(m,1H),3.47–3.43(m,1H)。MS m/z＝283.0[M+1] +。
制备实施例11：化合物A49、A124的合成
Figure PCTCN2021087928-appb-000042
化合物2-1(2.2g，7.6mmol)加入到N,N-二甲基甲酰胺(20mL)中，加入咪唑(3.1g，45.6mmol，6eq)，冰浴下，滴加1,3-二氯-1,1,3,3-四异丙基二硅氧烷(2.9g，9.5mmol，1.2eq)，加毕，室温反应，4小时后反应完全。将反应液加入到水(120mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物49-1，白色固体3.2g，收率80％。 1H NMR(500MHz,DMSO-d 6)δ7.99–7.82(m,3H),6.88(d,J＝4.5Hz,1H),6.79(d,J＝4.4Hz,1H),6.45(d,J＝5.7Hz,1H),4.56(t,J＝5.0Hz,1H),4.22–4.09(m,3H),3.95–3.86(m,1H),1.09–0.76(m,28H)。
化合物49-1(1.2g，2.2mmol)加入到甲苯(20mL)中，加入N,N-二甲基甲酰胺二甲基缩醛(0.52g，4.4mmol，2eq)，50℃反应。3h左右反应完全。减压蒸除溶剂，加入乙酸乙酯(60mL)和水(20mL)，分出有机相，饱和氯化钠 洗涤，干燥后，蒸干，得化合物49-2，白色固体1.2g，收率92％。 1H NMR(500MHz,DMSO-d 6)δ8.96(s,1H),8.16(s,1H),6.91(d,J＝4.5Hz,1H),6.81(d,J＝4.4Hz,1H),6.49(d,J＝5.8Hz,1H),4.59(t,J＝5.1Hz,1H),4.26–4.11(m,3H),3.93(dd,J＝13.3,2.4Hz,1H),3.26(s,3H),3.20(s,3H),1.09–0.87(m,28H)。
Figure PCTCN2021087928-appb-000043
化合物49-2(294mg，0.5mmol)加入到二氯甲烷(6mL)中，室温下，依次加入三乙胺(101mg，1mmol，2eq)、DMAP(12mg，0.1mmol，0.2eq)和异丁酰氯(85mg，0.8mmol，1.6eq)，反应过夜，TLC显示反应完全。向反应液中加入饱和碳酸氢钠水溶液和二氯甲烷，分出有机层，有机相干燥，浓缩，硅胶柱层析纯化，得化合物49-3，白色泡沫状固体240mg，收率72％。
将上步得到的产物49-3(240mg，0.36mmol)加入到四氢呋喃(5mL)中，加入1M四丁基氟化铵的四氢呋喃溶液(0.72mL，0.72mmol)，1小时后，反应完全。向反应液中加入水(10mL)，乙酸乙酯萃取，分出有机层，有机相干燥后蒸干，得油状物。该油状物加入到四氢呋喃(6mL)中，加入水(0.5mL)和三氟乙酸(205mg，1.8mmol)，室温搅拌过夜。向反应液中加入碳酸氢钠水溶液，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物A49和A124，A124占比大于95％，醋酸异丙酯重结晶得A124，白色固体80mg，两步收率61％。所得化合物A124的 1H NMR(500MHz,DMSO-d 6)δ8.09–7.84(m,3H),6.94(d,J＝4.6Hz,1H),6.90(d,J＝4.6Hz,1H),6.43(d,J＝6.5Hz,1H),5.22(dd,J＝5.7,3.3Hz,1H),5.07(t,J＝5.8Hz,1H),5.01(t,J＝6.1Hz,1H),4.31–4.25(m,1H),3.66–3.52(m,2H),2.68–2.58(m,1H),1.19(d,J＝7.0Hz,3H),1.17(d,J＝7.0Hz,3H)。MS m/z＝362.0[M+1] +。
制备实施例12：化合物A50的合成
Figure PCTCN2021087928-appb-000044
化合物10-1(0.664g，2.0mmol)加入到甲苯(10mL)中，加入N，N-二甲基甲酰胺二甲基缩醛(0.47g，4.0mmol，2.0eq)，加毕，60℃下反应2-3 小时，TLC监测反应完全。蒸除溶剂，得化合物50-1，黄色固体0.65g，收率84％。
将上步产物50-1(0.2g，0.52mmol)加入到二氯甲烷(10mL)中，依次加入三乙胺(0.1g，1.0mmol，2eq)、DMAP(0.012g，0.10mmol，0.2eq)，异丁酰氯(0.083g，0.78mmol，1.5eq)，加毕，室温搅拌1-2小时，TLC监测反应完全。将反应液加入到1M稀盐酸(10mL)中，二氯甲烷萃取，分出有机相，有机相用饱和碳酸氢钠(20mL)洗涤，干燥后浓缩，得化合物50-2，类白色固体0.2g，收率84％。
化合物50-2(0.1g，0.22mmol)加入到四氢呋喃(3mL)中，冰浴下滴加浓盐酸(0.6mL)，加毕，室温搅拌反应3小时，TLC显示原料有小量剩余。将反应液加入到饱和碳酸氢钠水溶液(20mL)中，乙酸乙酯萃取，分出有机相，有机相干燥，浓缩，制备板分离得化合物A50，白色固体13mg，收率16％。 1H NMR(500MHz,DMSO-d 6)δ8.00–7.80(m,3H),6.92(d,J＝4.6Hz,1H),6.82(d,J＝4.5Hz,1H),6.33(d,J＝6.0Hz,1H),5.39(d,J＝5.8Hz,1H),4.70(t,J＝5.5Hz,1H),4.32(dd,J＝12.0,2.9Hz,1H),4.27–4.21(m,1H),4.18(dd,J＝12.0,5.3Hz,1H),3.99–3.94(m,1H),2.57–2.52(m,1H),1.07(d,J＝2.4Hz,3H),1.06(d,J＝2.4Hz,3H)。MS m/z＝362.0[M+1] +。
制备实施例13：化合物A51、A212的合成
Figure PCTCN2021087928-appb-000045
化合物49-2(260mg，0.44mmol)和Boc-L-缬氨酸(115mg，0.53mmol，1.2eq)加入到二氯甲烷(10mL)中，依次加入HOBT(89mg，0.66mmol，1.5eq)、EDCI(169mg，0.88mmol，2eq)和DMAP(214mg，1.76mmol，4eq)，室温搅拌过夜。向反应液中加入二氯甲烷(20mL)和水(20mL)，搅拌5分钟，分出有机相，饱和氯化钠洗涤，干燥，浓缩，硅胶柱层析分离，得化合物51-1，白色固体260mg，收率75％。 1H NMR(500MHz,DMSO-d 6)δ8.98(s,1H),8.15(s,1H),7.23(d,J＝9.0Hz,1H),6.96(d,J＝4.5Hz,1H),6.85(d,J＝4.6Hz,1H),5.90(d,J＝5.0Hz,1H),4.57(dd,J＝9.2,4.9Hz, 1H),4.27–4.17(m,3H),3.95(dd,J＝13.8,2.8Hz,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)。
化合物51-1(180mg，0.23mmol)加入到四氢呋喃(4mL)中，室温下，加入1M四丁基氟化铵的四氢呋喃溶液(0.46mL，0.46mmol)，1小时后，反应完全。向反应液中加入水(10mL)，乙酸乙酯萃取，分出有机相，有机相干燥后蒸干，得到油状物。该油状物溶于四氢呋喃(6mL)中，加入水(0.5mL)和三氟乙酸(131mg，1.15mmol，5eq)，室温搅拌过夜。向反应液中加入碳酸氢钠水溶液，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物51-2a和51-2b，51-2b占比大于90％，进一步重结晶得到51-2b纯品，白色固体70mg，两步收率62％。所得化合物51-2b的 1H NMR(500MHz,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)。
化合物51-2b(42mg，0.086mmol)加入到饱和氯化氢/甲醇溶液(4mL)中，35℃下搅拌，2小时后TLC显示反应完全。将反应液浓缩，加入甲基叔丁基醚，搅拌后过滤，得化合物A212，为二盐酸盐，白色固体45mg，收率81％。 1H 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] +。若将上步得到的混合物51-2a和51-2b(51-2a占比5-10％)直接进行脱保护，可得到A51和A212混合物，A51含量约为10％。
制备实施例14：化合物A52的合成
Figure PCTCN2021087928-appb-000046
化合物2-1(291mg，1.0mmol)加入到吡啶(8mL)中，减压蒸除吡啶，重复操作一次，然后再加入吡啶(8mL)，室温下，加入N,N-二甲基甲酰胺二甲缩醛(477mg,4.0mmol)，氮气保护，室温反应过夜，将反应液浓缩得化合物52-1，油状物，不经分离直接用于下步反应。
将Boc-L-缬氨酸(304mg，1.4mmol)、1-羟基苯并三唑(203mg，1.5mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(422mg，2.2mmol)加入到二氯甲烷(10mL)中，室温搅拌15分钟后，加入上步得到的52-1的二氯甲烷溶液(1mL)和4-二甲氨基吡啶(684mg，5.4mmol)，室温搅拌过夜。将反应液浓缩后加入甲醇，再次浓缩，得油状物，加入水，乙酸乙酯萃取，分出有机相，有机相依次用水和饱和食盐水洗涤，无水硫酸钠干燥，浓缩得化合物52-2粗品，油状物，直接用于下步反应。
化合物52-2溶于乙腈(10mL)中，加入85％水合肼(236mg，4.0mmol)，室温反应3小时后，将反应液加入到水中，乙酸乙酯萃取，分出有机相，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物52-3，白色固体104mg，三步总收率21％。 1H NMR(500MHz,DMSO-d 6)δ7.98–7.83(m,3H),7.13(d,J＝8.1Hz,1H),6.91(d,J＝4.5Hz,1H),6.82(d,J＝4.5Hz,1H),6.33(d,J＝6.0Hz,1H),5.38(d,J＝5.9Hz,1H),4.69(t,J＝5.4Hz,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＝6.5Hz,6H)。
化合物52-3(104mg，0.21mmol)加入到饱和氯化氢的甲醇溶液(5mL)中，36℃下搅拌，1小时后反应完全。将反应液浓缩，然后加入异丙醚，析出固体，过滤，得化合物A52，为二盐酸盐，白色固体80mg，收率82％。 1H NMR(500MHz,Methanol-d 4)δ8.15(s,1H),7.49(d,J＝4.8Hz,1H),7.12(d,J＝4.8Hz,1H),4.74(d,J＝5.2Hz,1H),4.62(dd,J＝12.1,7.4Hz,1H),4.54(dd,J＝12.1,2.8Hz,1H),4.45(td,J＝7.5,2.7Hz,1H),4.05–4.00(m,2H),2.35–2.27(m,1H),1.08(d,J＝1.9Hz, 3H),1.07(d,J＝1.8Hz,3H)。MS m/z＝391.1[M+1] +。
制备实施例15：化合物A53的合成
Figure PCTCN2021087928-appb-000047
化合物49-1(150mg，0.28mmol)，溶于二氯甲烷(2mL)中，加入吡啶(265mg，3.35mmol，12eq)，冰浴下，加入三甲基氯硅烷(93mg，0.86mmol)，搅拌30分钟后，TLC显示原料转化完毕。向反应液中再加入氯甲酸正戊酯(120mg，0.8mmol)，保持冰浴,继续搅拌2小时，TLC检测反应完全。向反应液中加入二氯甲烷和水，分出有机层，有机层依次用稀盐酸洗和食盐水洗涤，干燥后浓缩，得53-1粗品，无色油状物200mg。该油状物加入到四氢呋喃(2mL)中，室温下滴加入1M四丁基氟化铵四氢呋喃溶液(0.2mL，0.2mmol)，加毕，室温下搅拌50分钟，TLC显示反应完全。反应液浓缩，硅胶柱层析纯化，得化合物A53，白色固体55mg，两步收率48％。 1H NMR(600MHz,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] +。
制备实施例16：化合物A69、A144的合成
Figure PCTCN2021087928-appb-000048
化合物A9(879mg，3.01mmol，氘代率不低于97％)加入到N，N-二甲基甲酰胺(15mL)中，加入咪唑(819mg，12.03mmol，4eq)，冰浴下，滴加1,3二氯-1,1,3,3-四异丙基二硅氧烷(1.32g，4.21mmol，1.4eq)，加毕，室温反应，1小时后反应完全。将反应液加入到水中，乙酸乙酯萃取，分 出有机层，干燥，浓缩，用石油醚打浆，得化合物69-1，白色固体1.29g，收率80％。 1H 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)。
化合物69-1(1.10g，2.07mmol)加入到甲苯(20mL)中，加入N,N-二甲基甲酰胺二甲基缩醛(370mg，3.11mmol，1.5eq)，45℃反应，30分钟左右反应完全。减压蒸除溶剂，加入乙酸乙酯(60mL)和水(20mL)，分出有机相，饱和氯化钠洗涤，干燥后，蒸干，得化合物69-2，白色固体1.12g，收率92％。
化合物69-2(614mg，1.04mmol)加入到二氯甲烷(10mL)中，室温下，依次加入三乙胺(210mg，2.08mmol，2eq)、异丁酰氯(166mg，1.56mmol，1.5eq)和DMAP(127mg，1.04mmol，1eq)，室温搅拌，1小时左右反应完全。向反应液中加入饱和碳酸氢钠水溶液和二氯甲烷，分出有机层，干燥，浓缩，硅胶柱层析纯化，得化合物69-3，白色泡沫状固体494mg，收率72％。
化合物69-3(350mg，0.53mmol)加入到乙腈(8mL)中，加入85％水合肼(125mg，2.12mmol，4eq)，室温搅拌，30分钟左右反应完全。将反应液加入到水中，乙酸乙酯萃取，分出有机层，有机相分别用稀盐酸、饱和碳酸氢钠和饱和氯化钠洗涤，干燥后，蒸干，得化合物69-4，白色固体289mg，收率90％。
化合物69-4(289mg，0.48mmol)加入到四氢呋喃(10mL)中，加入1M四丁基氟化铵的四氢呋喃溶液(0.48mL，0.48mmol，1eq)，室温搅拌，30分钟左右反应完全。将反应液加入到水中，乙酸异丙酯萃取，分出有机层，有机相分别用饱和碳酸氢钠和饱和氯化钠洗涤，干燥后，蒸干，得到化合物A69和A144，A144占比大于95％，用正庚烷/异丙醇(1:1)混合溶液打浆，过滤、干燥，得化合物A144纯品，白色固体121mg，收率70％。所得化合物A144的 1H NMR(600MHz,DMSO-d 6)δ8.06–7.85(m,3H),6.88(s,1H),6.41(d,J＝6.5Hz,1H),5.21(dd,J＝5.7,3.3Hz,1H),5.08–5.04(m,1H),4.99(t,J＝6.0Hz,1H),4.26(q,J＝3.7Hz,1H),3.64–3.52(m,2H),2.67–2.58(m,1H),1.17(d,J＝7.0Hz,3H),1.15(d,J＝7.0Hz,3H)。 13C NMR(126MHz,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] +。
制备实施例17：化合物A70的合成
Figure PCTCN2021087928-appb-000049
化合物A9(145mg，0.5mmol，氘代率不低于97％)加入到吡啶(5mL)中，减压蒸除吡啶，重复操作一次，然后再加入吡啶(5mL)，室温下，加入N,N-二甲基甲酰胺二甲缩醛(240mg，2.0mmol)，氮气保护，室温反应过夜。将反应液浓缩得中间体70-1，油状物，该中间体不经分离直接用于下步反应。
将上步得到的中间体70-1溶于吡啶(5mL)中，依次加入4-二甲氨基吡啶(6mg，0.05mmol)和异丁酰氯(80mg，0.75mmol)，3小时后，加入甲醇，蒸除溶剂，得中间体70-2，油状物，该中间体不经分离直接用于下步反应。
将上步得到的中间体70-2加入到乙腈(5mL)中，加入水合肼(176mg，3.0mmol)，室温搅拌过夜。向反应液中加入水，乙酸乙酯萃取，分出有机相，有机层依次用水和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物A70，白色固体80mg，收率44％。 1H 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)。 13C NMR(126MHz,DMSO-d 6)δ176.36,156.06,148.40,123.99,117.40,117.00,110.61,81.78,79.50,74.49,70.65,63.40,33.63,19.19,19.11。MS m/z＝363.0[M+1] +。
制备实施例18：化合物A71、A213的合成
Figure PCTCN2021087928-appb-000050
化合物69-2(160mg，0.27mmol)溶于二氯甲烷(10mL)中，冰浴，依次加入Boc-L-Val(82mg，0.38mmol)、1-羟基苯并三唑(55mg，0.41mmol)、 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(113mg，0.59mmol)和4-二甲氨基吡啶(132mg，1.08mmol)，加完后室温反应过夜。将反应液浓缩，加入水，乙酸乙酯萃取，有机层依次用稀盐酸、饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物71-1，白色固体158mg，收率74％。
化合物71-1(158mg，0.20mmol)溶于乙腈(10mL)中，室温下加入85％水合肼(71mg，1.20mmol)，搅拌1小时后，向反应液中加入水，乙酸乙酯萃取，分出有机相，有机相依次用稀盐酸、饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，得白色固体。将得到的白色固体加入到四氢呋喃(8mL)中，冰浴下，加入醋酸(14mg，0.24mmol)和1M四丁基氟化铵的四氢呋喃溶液(0.24mL，0.24mmol)，加完后，室温搅拌。2小时后，TLC显示反应完全。将反应液浓缩，加入水，醋酸异丙酯萃取，分出有机相，有机相用饱和食盐水洗涤，无水硫酸钠干燥后浓缩，所得产物用石油醚打浆，过滤、干燥，得化合物71-2a和71-2b，71-2b占比大于90％，进一步重结晶得71-2b纯品，白色固体60mg，两步收率61％。所得化合物71-2b的 1H NMR(500MHz,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)。
化合物71-2b(60mg，0.12mmol)加入饱和氯化氢/甲醇溶液(5mL)中，35℃下搅拌，2小时后TLC显示反应完全。将反应液浓缩，甲基叔丁基醚打浆，得化合物A213，为二盐酸盐，白色固体45mg，收率81％。 1H NMR(500MHz,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)。 13C NMR(126MHz,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] +。若将上步得到的混合物71-2a和71-2b直接脱保护，可得到A71和A213混合物，A71含量约为10％。
制备实施例19：化合物A72的合成
Figure PCTCN2021087928-appb-000051
化合物A9(350mg，1.2mmol)加入到吡啶(10mL)中，减压蒸除吡啶，重复操作一次，然后再加入吡啶(10mL)，室温下，加入N,N-二甲基甲酰胺二甲缩醛(596mg，5.0mmol)，氮气保护，室温反应过夜，将反应液浓缩得中间体70-1，油状物，该中间体不经分离直接用于下步反应。
将Boc-L-缬氨酸(369mg，1.7mmol)、1-羟基苯并三唑(243mg，1.8mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(498mg，2.6mmol)加入到二氯甲烷(15mL)中，室温搅拌15分钟后，加入上步得到的70-1的二氯甲烷溶液(2mL)和4-二甲氨基吡啶(733mg，6.0mmol)，室温搅拌过夜。将反应液浓缩后加入甲醇，再次浓缩，得油状物，加入水，乙酸乙酯萃取，分出有机相，有机相依次用水和饱和食盐水洗涤，无水硫酸钠干燥，浓缩得中间体72-1粗品，油状物，该中间体不经分离直接用于下步反应。
中间体72-1溶于乙腈(10mL)中，加入85％水合肼(283mg，4.8mmol)，室温反应3小时后，将反应液加入水中，乙酸乙酯萃取，分出有机相，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物72-2，白色固体195mg，三步总收率33％。 1H NMR(600MHz,DMSO-d 6)δ8.01–7.80(m,3H),7.14(d,J＝8.2Hz,1H),6.82(s,1H),6.33(d,J＝6.0Hz,1H),5.38(d,J＝5.9Hz,1H),4.69(t,J＝5.4Hz,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＝7.1Hz,6H)。
化合物72-2(100mg，0.2mmol)加入到饱和氯化氢的甲醇溶液(5mL)中，36℃下搅拌，1小时后反应完全。将反应液浓缩，然后加入异丙醚，析出固体，过滤，得化合物A72，为盐酸盐，白色固体74mg，收率80％。 1H NMR(600MHz,Methanol-d 4)δ8.15(s,1H),7.12(s,1H),4.75(d,J＝5.2Hz,1H),4.62(dd,J＝12.1,7.4Hz,1H),4.54(dd,J＝12.1,2.8Hz,1H),4.45(td,J＝7.5,2.8Hz,1H),4.05–4.01(m,2H),2.34–2.28(m,1H),1.08(d,J＝2.4Hz,3H),1.07(d,J＝2.3Hz,3H)。 13C NMR(151MHz,Methanol-d 4)δ168.15,148.61,135.04,129.77,114.70,113.28, 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] +。
制备实施例20：化合物A74、A164的合成
Figure PCTCN2021087928-appb-000052
参考化合物A144的合成方法，以A10(176mg，0.6mmol，氘代率99％)为原料，经五步反应得到化合物A74和A164，进一步重结晶得到A164纯品，白色固体139mg，总收率64％。所得化合物A164的 1H NMR(600MHz,DMSO-d 6)δ8.07–7.83(m,3H),6.92(d,J＝4.6Hz,1H),6.88(d,J＝4.6Hz,1H),6.41(d,J＝6.6Hz,1H),5.21(dd,J＝5.7,3.3Hz,1H),5.02(s,1H),5.01–4.97(m,1H),4.25(d,J＝3.2Hz,1H),2.66–2.58(m,1H),1.17(d,J＝7.0Hz,3H),1.15(d,J＝6.9Hz,3H)。MS m/z＝364.0[M+1] +。
制备实施例21：化合物A75的合成
Figure PCTCN2021087928-appb-000053
参考化合物A70的合成方法，以A10(176mg，0.6mmol)为原料，经三步反应得到化合物A75，白色固体109mg，总收率50％。 1H NMR(500MHz,DMSO-d 6)δ8.03–7.82(m,3H),6.92(d,J＝4.5Hz,1H),6.82(d,J＝4.5Hz,1H),6.33(d,J＝6.0Hz,1H),5.39(d,J＝5.8Hz,1H),4.71(t,J＝5.5Hz,1H),4.24(d,J＝6.6Hz,1H),3.97(q,J＝5.8Hz,1H),2.57–2.53(m,1H),1.07(d,J＝2.5Hz,3H),1.06(d,J＝2.5Hz,3H)。MS m/z＝364.0[M+1] +。
制备实施例22：化合物A76、A214的合成
Figure PCTCN2021087928-appb-000054
参考化合物A213的合成方法，以74-2(295mg，0.5mmol)为原料，经三步反应得到化合物A214，为二盐酸盐，白色固体97mg，总收率42％。 1H NMR(500MHz,Methanol-d 4)δ8.17(s,1H),7.49(d,J＝4.8Hz,1H),7.21(d,J＝4.9Hz,1H),5.53(dd,J＝5.8,3.1Hz,1H),5.19(d,J＝5.8Hz,1H),4.50(d,J＝3.1Hz,1H),4.13(d,J＝4.5Hz,1H),2.58–2.50(m,1H),1.18(d,J＝4.6Hz,3H),1.17(d,J＝4.6Hz,3H)。MS m/z＝393.0[M+1] +。若第二步得到的产物76-2a和76-2b直接脱保护，可得到A76和A214混合物，A76含量约为10％。
制备实施例23：化合物A77的合成
Figure PCTCN2021087928-appb-000055
参考化合物A72的合成方法，以A10(1.0mmol，293mg)为原料，经四步反应得到化合物A77，为二盐酸盐，白色固体107mg，总收率23％。MS m/z＝393.0[M+1] +。
制备实施例24：化合物A84的合成
Figure PCTCN2021087928-appb-000056
将化合物A1(49mg，0.16mmol)加入到丙酮(3mL)中，依次加入2,2-二甲氧基丙烷(83mg，0.8mmol，5eq)和对甲苯磺酸一水合物(30mg，0.16mmol，1eq)，加毕，升温至40℃，搅拌反应2小时，TLC监测反应完全。将反应液加入到饱和碳酸氢钠水溶液(20mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，制备板分离，得化合物84-1，白色固体27mg，收率48％。
化合物84-1(27mg，0.077mmol)加入到无水四氢呋喃(3mL)中，冰浴下，滴加3M甲基溴化镁的甲基四氢呋喃溶液(0.05ml，0.15mmol，2eq)，加毕，搅拌5分钟后，滴加化合物B(57mg，0.12mmol，1.5eq)的无水四氢呋喃溶液(1mL)，滴毕，室温反应2小时，TLC监测反应完全。将反应液加入到饱和氯化铵溶液(20mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，得化合物84-2，油状物，直接进行下一步反应。
将上一步产物加入到四氢呋喃(2mL)中，冰浴下滴加浓盐酸(0.4mL)，加毕，室温搅拌反应直至反应完全。将反应液加入到饱和碳酸氢钠水溶液(20mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，制备板分离，得化合物A84，白色固体10mg，两步收率21％。 1H NMR(500MHz,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)。 1H 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)。
制备实施例25：化合物A102的合成
Figure PCTCN2021087928-appb-000057
化合物A1(62mg，0.2mmol)和1,8-双(二甲基氨基)萘(56mg，0.26mmol，1.3eq)加入到磷酸三甲酯(3mL)中，冰浴下搅拌，缓慢加入三氯氧磷(153mg，1.0mmol，5eq)，3小时左右，TLC显示原料基本反应完全。将三丁基焦磷酸铵(549mg，1.0mmol，5eq)的DMF(3mL)溶液加入到上述体系，随后加入三正丁胺(222mg，1.2mmol，6eq)，冰浴下搅拌10分钟后，反应液自然升温。TLC监测，直至产物不再增多。向反应液中加入0.5M三乙基碳酸氢铵水溶液，使pH为7.5，加入去离子水(5mL)，分别用二氯甲烷和乙酸乙酯萃取，舍弃有机相，水相用凝胶柱分离得到A102粗品，该粗品进一步用反相柱纯化，得到A102，为二三乙胺盐，泡沫状固体，44mg，收率29％。 1H NMR(500MHz,D 2O)δ7.88(s,1H),6.87(s,1H),4.76(d,J＝5.2Hz,1H),4.45–4.41(m,1H),4.38(t,J＝4.8Hz,1H),4.17–4.10(m,2H),3.12(q,J＝7.3Hz,12H),1.19(t,J＝7.3Hz,18H)。 31P NMR(202MHz,D 2O)δ-10.89(d),-11.46(d),-23.19(t)。MS m/z＝548.0[M-1] -。
制备实施例26：化合物A106的合成
Figure PCTCN2021087928-appb-000058
参考化合物A102的合成方法，以A9(58mg，0.2mmol，氘代率不低于97％)为原料，得产物A106，为三三乙胺盐，泡沫状固体55mg，收率33％。 1H NMR(500MHz,D 2O)δ7.92(s,1H),7.02(s,1H),4.92(d,J＝4.4Hz,1H),4.50–4.42(m,2H),4.16–4.10(m,1H),4.09–4.01(m,1H),3.11(q,J＝7.3Hz,18H),1.19(t,J＝7.2Hz,27H)。 31P NMR(202MHz,D 2O)δ-10.90(d),-11.52(d),-23.29(t)。MS m/z＝531.0[M-1] -。
制备实施例27：化合物A107的合成
Figure PCTCN2021087928-appb-000059
参考化合物A102的合成方法，以A10(59mg，0.2mmol，氘代率99％)为原料，得产物A107，为四三乙胺盐，泡沫状固体54mg，收率29％。 1H NMR(500MHz,D 2O)δ7.97(s,1H),7.17(d,J＝4.9Hz,1H),7.07(d,J＝4.8Hz,1H),4.87(d,J＝5.0Hz,1H),4.48–4.40(m,2H),3.12(q,J＝7.3Hz,24H),1.20(t,J＝7.3Hz,36H)。 31P NMR(202MHz,D 2O)δ-10.92(d),-11.45(d),-23.29(t)。MS m/z＝532.0[M-1] -。
制备实施例28：化合物A109的合成
Figure PCTCN2021087928-appb-000060
化合物2-1(145mg，0.5mmol)加入到磷酸三甲酯(2mL)中，冰浴下，加入三氯氧磷(153mg，1mmol，2eq)，搅拌3-4小时，TLC监测，仍有较多原料剩余，补加三氯氧磷(80mg)后，继续在冰浴下搅拌2小时。向反应液中加入三正丁焦磷酸铵(1.1g，2mmol，4eq)的乙腈悬浮液(2mL)和三正丁胺(741mg，4mmol，8eq)，继续搅拌2小时。向反应液中加入1M三乙基碳酸氢铵水溶液(8mL)，再加入蒸馏水(8mL)，搅拌。乙酸乙酯萃取，舍弃有机相，水相冻干，制备液相分离，得化合物A109，为三三正丁胺盐，白色固体55mg，收率10％。 1H NMR(400MHz,D 2O)δ7.85(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＝7.4Hz,27H)。MS m/z＝530.0[M-1] -。
制备实施例29：化合物A112的合成
Figure PCTCN2021087928-appb-000061
化合物2-1(291mg，1.0mmol)加入到四氢呋喃(6mL)中，加入浓盐酸(3mL)，45℃下搅拌，TLC监测直至反应完全。蒸除溶剂，硅胶柱层析分离，得化合物A112，白色固体168mg，收率54％。 1H NMR(500MHz,DMSO-d 6)δ7.77(s,1H),7.72(brs,2H),7.49(s,1H),7.44(s,1H),6.83(d,J＝4.5Hz,1H),6.62(d,J＝4.5Hz,1H),5.58(s,1H),4.93(s,1H),4.85(d,J＝4.4Hz,1H),4.68(s,1H),4.08–3.98(m,2H),3.52(d,J＝12.0Hz,1H),3.34–3.29(m,1H)。MS m/z＝310.1[M+1] +。
制备实施例30：化合物A173、A188的合成
Figure PCTCN2021087928-appb-000062
参考化合物A144的合成方法，以A11(294mg，1.0mmol)为原料，经五步反应得到化合物A173和A188，进一步重结晶得到A188纯品，白色固体91mg，总收率25％。所得化合物A188的 :1H NMR(600MHz,DMSO-d 6)δ8.07–7.83(m,3H),6.88(s,1H),6.41(d,J＝6.6Hz,1H),5.21(dd,J＝5.7,3.3Hz,1H),5.02(s,1H),5.01–4.97(m,1H),4.25(d,J＝3.2Hz,1H),2.66–2.58(m,1H),1.17(d,J＝7.0Hz,3H),1.15(d,J＝6.9Hz,3H)。MS m/z＝365.0[M+1] +。
制备实施例31：化合物A180的合成
Figure PCTCN2021087928-appb-000063
参考化合物A70的合成方法，以A11(147mg，0.5mmol)为原料，经三步反应得到化合物A180，白色固体80mg，总收率44％。 1H NMR(500MHz,DMSO-d 6)δ8.03–7.83(m,3H),6.82(s,1H),6.32(d,J＝6.1Hz,1H),5.39(d,J＝5.8Hz,1H),4.71(t,J＝5.5Hz,1H),4.23(d,J＝6.6Hz,1H),3.97(q,J＝5.8Hz,1H),2.57–2.53(m,1H),1.10–1.01(m,6H)。MS m/z＝365.0[M+1] +。
制备实施例32：化合物A174、A215的合成
Figure PCTCN2021087928-appb-000064
参考化合物A213的合成方法，以173-2(296mg，0.5mmol)为原料，经三步反应得到化合物A215，为二盐酸盐，白色固体93mg，收率40％。 1H NMR(500MHz,Methanol-d 4)δ8.17(s,1H),7.21(s,1H),5.53(dd,J＝5.8,3.1Hz,1H),5.19(d,J＝5.8Hz,1H),4.50(d,J＝3.1Hz,1H),4.13(d,J＝4.5Hz,1H),2.58–2.50(m,1H),1.18(d,J＝4.6Hz,3H),1.17(d,J＝4.6Hz,3H)。MS m/z＝394.0[M+1] +。若第二步得到的产物174-2a和174-2b直接脱保护，可得到A174和A215混合物，A174含量约为10％。
制备实施例33：化合物A181的合成
Figure PCTCN2021087928-appb-000065
参考化合物A72的合成方法，以A11(147mg，0.5mmol)为原料，经四步反应得到化合物A181，为二盐酸盐，白色固体75mg，收率32％。 1H NMR(600MHz,Methanol-d 4)δ8.15(s,1H),7.12(s,1H),4.75(d,J＝5.2Hz,1H),4.62(dd,J＝12.1,7.4Hz,1H),4.54(dd,J＝12.1,2.8Hz,1H),4.45(td,J＝7.5,2.8Hz,1H),2.35–2.28(m,1H),1.08(d,J＝2.4Hz,3H),1.07(d,J＝2.3Hz,3H)。MS m/z＝394.0[M+1] +。
制备实施例34：化合物A198的合成
Figure PCTCN2021087928-appb-000066
参考化合物A102的合成方法，以A11(59mg，0.2mmol)为原料，得产物A198，为三三乙胺盐，泡沫状固体59mg，收率35％。 1H NMR(500MHz,D 2O)δ7.90(s,1H),7.00(s,1H),4.84(d,J＝4.6Hz,1H),4.42–4.38(m,2H),3.07(q,J＝7.3Hz,18H),1.15(t,J＝7.3Hz,27H)。 31P NMR(202MHz,D 2O)δ-10.93(d),-11.46(d),-23.31(t)。MS m/z＝533.0[M-1] -。
制备实施例35：化合物A131的合成
Figure PCTCN2021087928-appb-000067
化合物2-1(58mg，0.2mmol)加入到N,N-二甲基甲酰胺(3mL)中，加入N,N-二甲基甲酰胺二甲基缩醛(160mg，1.34mmol，6.7eq)，加毕，室温反应，1h后反应完全。向反应液中加入甲醇，浓缩，用异丙醇和甲苯打浆，得化合物131-1，白色固体59mg，收率85％。
化合物131-1(59mg，0.17mmol)加入到二氯甲烷(5mL)中，室温下，依次加入三乙胺(138mg，1.36mmol，8eq)、DMAP(62mg，0.51mmol，3eq)和异丁酰氯(72mg，0.68mmol，4eq)，室温反应过夜。将反应液浓缩，加入乙酸乙酯(30mL)和水(10mL)，分出有机相，有机相分别用稀盐酸、饱和碳酸氢钠和饱和氯化钠洗涤，干燥后，蒸干，得化合物131-2，白色固体85mg，收率90％。
化合物131-2(85mg，0.15mmol)加入到乙腈(10mL)中，加入水合肼(30mg，0.60mmol，4eq)，加毕，30分钟左右反应完全。将反应液加入到水中，乙酸乙酯萃取，分出有机层，有机相分别用稀盐酸、饱和碳酸氢钠和饱和氯化钠洗涤，干燥后，蒸干，硅胶柱层析分离，得化合物A131，白色固体70mg，收率93％。 1H NMR(600MHz,DMSO-d 6)δ8.04(br,1H),7.96(br,1H),7.93(s,1H),6.93(d,J＝4.6Hz,1H),6.75(d,J＝4.6Hz,1H),6.08(d,J＝5.7Hz,1H),5.44(dd,J＝5.7,3.7Hz,1H),4.63(q,J＝3.7Hz,1H),4.33(dd,J＝12.4,3.3Hz,1H),4.28(dd,J＝12.4,4.1Hz,1H),2.66–2.57(m,2H),2.50–2.46(m,1H),1.17(d,J＝7.0Hz,3H),1.15(d,J＝6.9Hz,3H),1.12–1.09(m,6H),1.05(d,J＝ 7.0Hz,3H),1.02(d,J＝7.0Hz,3H)。MS m/z＝502.0[M+1] +。
制备实施例36：化合物A151的合成
Figure PCTCN2021087928-appb-000068
参考化合物A131的合成方法，以A9(58mg，0.2mmol)为原料，经三步反应，得化合物A151，白色固体69mg，总收率68％。 1H NMR(600MHz,DMSO-d 6)δ8.04(br,1H),7.96(br,1H),7.93(s,1H),6.75(s,1H),6.08(d,J＝5.7Hz,1H),5.44(dd,J＝5.7,3.7Hz,1H),4.63(q,J＝3.7Hz,1H),4.33(dd,J＝12.4,3.3Hz,1H),4.28(dd,J＝12.4,4.1Hz,1H),2.66–2.57(m,2H),2.49–2.46(m,1H),1.17(d,J＝7.0Hz,3H),1.15(d,J＝6.9Hz,3H),1.12–1.09(m,6H),1.05(d,J＝7.0Hz,3H),1.02(d,J＝7.0Hz,3H)。 13C NMR(126MHz,DMSO-d 6)δ175.53,174.90,174.13,155.58,148.12,120.98,117.17,115.44,110.30,81.25,75.81,72.05,70.30,62.46,33.20,33.16,33.09,18.55,18.46,18.40,18.35,18.33,18.18。MS m/z＝503.0[M+1] +。
制备实施例37：化合物A171的合成
Figure PCTCN2021087928-appb-000069
参考化合物A131的合成方法，以A10(59mg，0.2mmol)为原料，经三步反应，得化合物A171，白色固体70mg，总收率69％。MS m/z＝504.0[M+1] +。
制备实施例38：化合物A196的合成
Figure PCTCN2021087928-appb-000070
参考化合物A131的合成方法，以A11(59mg，0.2mmol)为原料，经三步反应，得化合物A196，白色固体59mg，总收率58％。MS m/z＝505.0[M+1] +。
制备实施例39：化合物A209的合成
Figure PCTCN2021087928-appb-000071
化合物1-1(0.5g，0.89mmol)加入到DMF(9ml)中，加入NBS(0.16g，0.89mmol，1.0eq)，室温搅拌3-4小时，TLC监测反应完全。向反应液中加入饱和硫代硫酸钠(10mL)溶液，搅拌，乙酸乙酯萃取，分层，水层再用乙酸乙酯(20mL)萃取一次。合并乙酸乙酯层，干燥，浓缩，柱层析分离得化合物209-1，白色固体0.5g，收率87％。
化合物209-1(0.15g，0.23mmol)加入到N,N-二甲基乙酰胺(5mL)中，依次加入Zn(2mg，0.031mmol，0.13eq)，Zn(CN) 2(0.06g，0.51mmol，2.2eq)，NiCl 2(dppf)(0.03g，0.043mmol，0.2eq)和Pd 2(dba) 3(0.02g，0.02mmol，0.1eq)，加毕，140℃反应过夜，TLC显示反应完全。将反应液倒入乙酸乙酯/水(20mL/20mL)中，分层，水层再用乙酸乙酯(20mL)萃取一次。合并乙酸乙酯层，干燥，浓缩，制备板分离，得化合物209-2，白色固体0.1g，收率74％。
化合物209-2(0.1g，0.17mmol)加入到二氯甲烷(3mL)中，-30℃下，滴加1M三氯化硼二氯甲烷溶液(0.7mL，4.1eq)，滴毕，保温搅拌1-2小时，TLC监测反应完全。向反应液依次滴加甲醇(0.5mL)，三乙胺(0.3mL)，蒸干反应液，制备板分离，得到化合物A209，白色固体15mg，收率28％。 1H NMR(500MHz,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] +。
制备实施例40：化合物A87的合成
Figure PCTCN2021087928-appb-000072
化合物11-1(0.05g，0.15mmol)加入到四氢呋喃(3mL)中，冰浴下，滴加3M甲基溴化镁的2-甲基四氢呋喃溶液(0.1mL，0.3mmol)，加毕，搅拌5分钟，滴加化合物B(0.1g，0.23mmol)的四氢呋喃溶液(1mL)，滴毕，室温搅拌2小时后，TLC显示反应完全。将反应液加入到饱和氯化铵溶液(10mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩得化合物87-1粗品，直接投下步反应。
将上步得到的产物加入到四氢呋喃(2mL)中，冰浴下，滴加浓盐酸(0.4mL)，加毕，室温搅拌直至反应完全。将反应液加入到饱和碳酸氢钠溶液(10mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物A87，白色固体0.03g，两步收率33％。 1H 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] +。
制备实施例41：化合物A138的合成
Figure PCTCN2021087928-appb-000073
参考化合物A70的合成方法，以A9(147mg，0.5mmol)为原料，经三步反应得到化合物A138，白色固体65mg，总收率37％。 1H NMR(400MHz,DMSO-d 6)δ7.99–7.83(m,3H),6.80(s,1H),6.32(d,J＝6.1Hz,1H),5.38(d,J＝5.8Hz,1H),4.71–4.65(m,1H),4.33(dd,J＝12.0,2.8Hz,1H),4.25–4.19(m,1H),4.15(dd,J＝12.0,5.5Hz,1H),3.97–3.90(m,1H),2.30(q,J＝7.5Hz,2H),1.00(t,J＝7.5Hz,3H)。MS m/z＝ 349.2[M+1] +。
制备实施例42：化合物A140的合成
Figure PCTCN2021087928-appb-000074
参考A70的合成方法，以A9(147mg，0.5mmol)为原料，经三步反应得到化合物A140，白色固体77mg，总收率41％。 1H NMR(500MHz,DMSO-d 6)δ8.03–7.80(m,3H),6.80(s,1H),6.32(d,J＝6.1Hz,1H),5.37(d,J＝5.7Hz,1H),4.70(t,J＝5.3Hz,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] +。
制备实施例43：化合物A146的合成
Figure PCTCN2021087928-appb-000075
化合物A70(0.18g，0.5mmol)加入到醋酸(3mL)中，加入原异丁酸三甲酯(0.37g，2.5mmol)，50℃下搅拌，直至反应完全。蒸除溶剂，得到不稳定中间体146-1。将该中间体溶于四氢呋喃(6mL)中，加入1M稀盐酸(0.5mL)，室温搅拌，1小时左右反应完全。向反应液中加入饱和碳酸氢钠水溶液，至pH为中性，乙酸乙酯萃取，有机相干燥后蒸干，得化合物A146粗品。该粗品用异丙醇/水重结晶，得白色固体0.18g，收率82％。 1H 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] +。
制备实施例44：化合物A147的合成
Figure PCTCN2021087928-appb-000076
化合物151-1(1.51g，4.34mmol)加入到N,N-二甲基甲酰胺(15mL)中，冰浴下，加入叔丁基二苯基氯硅烷(2.39g，8.69mmol)和咪唑(1.18g，17.37mmol)，室温下搅拌5小时。向反应液中加入水和乙酸乙酯，分出有机相，有机相依次用稀盐酸、饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物147-1，泡沫状固体2.05g。
化合物147-1(2.05g，3.50mmol)加入到二氯甲烷(30mL)中，室温下，加入三乙胺(1.24g，12.26mmol)、DMAP(0.21g，1.75mmol)和异丁酸酐(1.39g，8.76mmol)，氮气保护，室温反应1小时。反应液浓缩后，加入水、乙酸乙酯，分出有机相，有机相分别用稀盐酸、饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，得化合物147-2粗品，油状物，该中间体不经分离直接用于下步反应。
将上步得到的产物加入到四氢呋喃(30mL)中，室温下，加入乙酸(0.11g，1.75mmol)和1M四丁基氟化铵(3.5mL，3.5mmol)的四氢呋喃溶液，室温搅拌2小时。向反应液中加入水和乙酸乙酯，分出有机相，有机相分别用饱和碳酸氢钠和饱和食盐水洗涤，无水硫酸钠干燥，浓缩得化合物147-3粗品，油状物。
将上步得到的147-3粗品加入到乙醇(20mL)中，室温下，加入乙酸(4.2g，70mmol)，氮气保护，50℃搅拌过夜。将反应液浓缩后加入水和乙酸乙酯，分出有机相，有机相分别用饱和碳酸氢钠和饱和食盐水洗涤，无水硫酸钠干燥，浓缩，硅胶柱层析分离，得化合物A147，白色固体0.85g，三步收率56％。 1H NMR(500MHz,DMSO-d 6)δ8.15–7.89(m,3H),6.80(s,1H),6.01(d,J＝5.7Hz,1H),5.44(dd,J＝5.7,3.1Hz,1H),5.18(dd,J＝6.1,5.2Hz,1H),4.41(q,J＝3.4Hz,1H),3.71–3.60(m,2H),2.69–2.54(m,2H),1.19(d,J＝7.0Hz,3H),1.16(d,J＝7.0Hz,3H),1.11(d,J＝2.4Hz,3H),1.09(d,J＝2.4Hz,3H)。MS m/z＝433.2[M+1] +。
制备实施例45：化合物A216的合成
Figure PCTCN2021087928-appb-000077
化合物1-1(0.2g，0.36mmol)加入到二氯甲烷(2mL)中,零下78℃下，缓慢滴加1.2M二异丁基氢化铝(0.9mL，1.08mmol)的甲苯溶液，加毕，保温搅拌3-4小时，TLC显示反应完全。向反应液滴加乙酸乙酯(3mL)，加毕，自然升温至室温，加入20％酒石酸钠钾水溶液(5mL)，室温搅拌过夜。将反应液加入到水(15mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，得到化合物216-1。
化合物216-1(0.6g，1.06mmol)加入到乙醇(10mL)和二氯甲烷(10mL)的混合液中，冰浴下，分批加入NaBH 4(0.1g，2.6mmol)，加毕，室温搅拌2-3小时，TLC显示反应完全。向反应液中滴加冰醋酸至不再产生气泡，加入水(30mL)，乙酸乙酯萃取，分出有机层，饱和碳酸氢钠洗涤，干燥，浓缩，硅胶柱层析分离，得化合物216-2，白色固体0.48g，收率80％。
化合物216-2(0.48g,0.85mmol)、甲酸(2mL)和10％钯碳(0.06g)依次加入到甲醇(6mL)中,室温下，通入氢气，常压搅拌16小时。过滤，浓缩，反相柱分离，得到化合物A216，白色固体0.05g，收率20％。 1H NMR(500MHz,DMSO-d 6)δ7.80(s,1H),7.61(s,2H),6.81(d,J＝4.4Hz,1H),6.73(d,J＝4.4Hz,1H),5.18(d,J＝4.7Hz,1H),4.89(d,J＝6.9Hz,1H),4.79(t,J＝5.8Hz,1H),4.51–4.38(m,2H),4.16(dd,J＝11.6,6.7Hz,1H),3.99(dd,J＝11.6,4.7Hz,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] +。
制备实施例46：化合物A28的合成
Figure PCTCN2021087928-appb-000078
冰浴下，向化合物1-1(0.5g，0.9mmol)中滴加0.5M环丙基溴化镁四氢呋喃溶液(18mL，9mmol)，加毕，室温搅拌过夜。冰浴下，向反应液中滴加饱和氯化铵溶液(10mL)，加入水(50mL)，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物28-1，白色固体0.2g，收率37％。
化合物28-1(0.2g，0.34mmol)加入到二氯甲烷(4mL)中,冰浴下，滴加甲磺酸(0.1g，1.04mmol)，加毕，室温搅拌10-12小时，TLC监测反 应完全。向反应液中加入饱和碳酸氢钠溶液至pH为中性，加入水(15mL)，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得化合物28-2，白色固体0.1g，收率51.0％。
化合物28-2(0.24g,0.42mmol)、甲酸(1mL)和10％钯碳(0.03g)依次加入到甲醇(3mL)中,室温下，通入氢气，常压搅拌16小时，TLC监测反应完全。过滤，浓缩，硅胶柱层析分离，得化合物A28，白色固体13mg，收率10％。 1H NMR(500MHz,Methanol-d 4)δ7.78(s,1H),6.85(d,J＝4.5Hz,1H),6.79(d,J＝4.5Hz,1H),4.78(d,J＝5.1Hz,1H),4.01–3.95(m,1H),3.95–3.89(m,1H),3.81(dd,J＝11.9,2.8Hz,1H),3.68(dd,J＝11.8,5.5Hz,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] +。
制备实施例47：化合物A218的合成
Figure PCTCN2021087928-appb-000079
冰浴下，向化合物1-1(2.2g，3.98mmol)中滴加1.0M异丙烯基溴化镁四氢呋喃溶液(40mL，40mmol)，加毕，室温搅拌过夜。向反应液中，缓慢滴加饱和氯化铵水溶液(10mL)，加毕，加入水(40mL)，乙酸乙酯萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得中间体218-1，白色固体1.6g，收率68％。
中间体218-1(1.6g，2.69mmol)加入到二氯甲烷(30mL)中，冰浴下，缓慢滴加甲磺酸(0.77g，8.01mmol)，加毕，室温搅拌过夜。TLC监测反应完全，冰浴下，向反应液中滴加饱和碳酸氢钠溶液，至pH为中性，加入到水(40mL)，二氯甲烷萃取，分出有机层，干燥，浓缩，硅胶柱层析分离，得中间体218-2，黄色油状物0.7g，收率45％。
参考实施例5中脱苄基的方法，中间体218-2(0.7g，1.21mmol)与1.0M BCl 3二氯甲烷溶液(6.0mL,6.0mol)反应，产物经柱层析分离，得化合物A218,白色固体0.025g。 1H NMR(600MHz,DMSO-d 6)δ7.74(s,1H),7.55(s,2H),6.82(d,J＝4.5Hz,1H),6.58(d,J＝4.4Hz,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] +。
制备实施例48：化合物A219的合成
Figure PCTCN2021087928-appb-000080
化合物1-1(0.56g，1.0mmol)加入到四氢呋喃(5mL)中，氮气保护，冰浴下，缓慢滴加3M甲基溴化镁四氢呋喃溶液(1.7mL，5.1mmol)，加毕，60℃下搅拌2-3小时。TLC监测反应完全，冰浴下，滴加饱和氯化铵水溶液(1.0mL)，加入水(10mL)，乙酸乙酯萃取，分出有机层，干燥，浓缩，柱层析分离得219-1，白色固体0.38g，收率66％。
参考实施例5中脱苄基的方法，中间体219-1(0.2g，0.345mmol)与1.0M BCl 3二氯甲烷溶液(1.7mL,1.7mmol)反应，产物经硅胶柱层析分离，得化合物A219，白色固体0.05g，收率47％。 1H NMR(600MHz,DMSO-d 6)δ7.99–7.43(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] +。
制备实施例49：化合物A221的合成
Figure PCTCN2021087928-appb-000081
化合物49-2(0.6g，1mmol)加入到DMF(10mL)中，冰浴下，依次加入碘甲烷(0.28g，2.0mmol)和60％氢化钠(0.014g，2.0mmol)，搅拌15分钟后，TLC显示反应完全。将反应液加入到饱和氯化铵水溶液(15mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，蒸干，得化合物221-1，该中间体不经纯化直接进行下一步反应。
将上步得到的221-1加入到乙腈(2mL)中，加入85％水合肼(0.24g，4.0mmol)，室温搅拌1小时，TLC显示反应完全。将反应液加入到水(15mL)中，乙酸乙酯萃取，分出有机层，干燥，浓缩，柱层析分离得化合物221-2，白色固体0.2g，两步收率37％。
化合物221-2(0.2g，0.36mmol)加入到四氢呋喃(2mL)中，加入1M四丁基氟化铵的四氢呋喃溶液(0.4mL，0.4mmol)，室温搅拌2小时，TLC显示反应完全。将反应液浓缩，制备板分离，得化合物A221，白色固体0.045g，收率41％。 1H NMR(500MHz,DMSO-d 6)δ8.07–7.78(m,3H),6.87(s,1H), 5.30(d,J＝5.4Hz,1H),4.94(t,J＝5.8Hz,1H),4.37(d,J＝4.9Hz,1H),4.11(q,J＝5.4Hz,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] +。
制备实施例50：化合物A36的合成
Figure PCTCN2021087928-appb-000082
化合物35-1(8.4g，31.5mmol)溶于吡啶(85mL)中，加入三苯基磷(33.1g，126.2mmol)，氮气保护升温至30℃，40℃以下分批加入碘(32.0g，126.2mmol)，搅拌1h，TLC显示反应完毕，加入饱和硫代硫酸钠溶液，硅胶柱层析分离(DCM:MeOH＝100:1-10:1)，得化合物36-1，8.5g，收率72％。
化合物36-1(3.0g，8.0mmol)溶于乙腈(60mL)中，加入DBU(2.4g，16.0mmol)，40℃搅拌3h，TLC显示反应完毕，硅胶柱层析分离(DCM:MeOH＝50:1-10:1)，得到化合物36-2，600mg，收率30％。
化合物36-2(200mg，0.8mmol)加入THF(5mL)中，加入DBU(731mg，4.8mmol)，TBSCl(543mg，3.6mmol)，室温搅拌2h，TLC显示反应完毕，加入甲醇(1mL)，饱和柠檬酸水溶液(4mL)，搅拌，乙酸乙酯萃取，分别用饱和食盐水和饱和碳酸氢钠溶液洗涤，无水硫酸钠干燥，浓缩，得化合物36-3粗品。
化合物36-3粗品加入二氯甲烷(10mL)中，加入2,4,6-三甲基吡啶(194mg，1.6mmol)，硝酸银(272mg，1.6mmol)，三苯基氯甲烷(374mg，1.2mmol)，室温反应2h后，加入甲醇，浓缩，加入乙酸乙酯，分别用饱和硫酸铜和饱和氯化钠洗涤，无水硫酸钠干燥，浓缩，得化合物36-4粗品；
化合物36-4粗品加入THF(5mL)中，加入四丁基氟化铵水合物(508mg，1.6mmol)的四氢呋喃溶液，反应1h，补加四丁基氟化铵水合物(508mg，1.6mmol)的四氢呋喃溶液反应，继续反应1h，加入水，乙酸乙酯萃取，浓缩，硅胶柱层析分离，得到化合物36-5，白色固体84mg，三步收率22％；
化合物36-5(70mg，0.14mmol)加入乙腈(2mL)中，冰盐浴冷却到-5℃，加入三乙胺三氟化氢(35mg，0.21mmol)，碘代丁二酰亚胺(36mg，0.15 mmol)，保温反应2小时，升到室温反应2h，加入饱和碳酸氢钠和饱和硫代硫酸钠的混合溶液，乙酸乙酯萃取，饱和氯化钠洗涤，无水硫酸钠干燥，浓缩，得化合物36-6粗品；
化合物36-6粗品加入二氯甲烷(5mL)中，依次加入吡啶(114mg，1.4mmol)，异丁酸酐(162mg，1.0mmol)和DMAP(5mg)，室温反应2小时，加入甲醇，浓缩，加入乙酸乙酯，分别用饱和硫酸铜溶液和饱和氯化钠溶液洗涤，浓缩，硅胶柱层析分离(PE：EA＝20：1-10：1)，得到化合物36-7，淡黄色固体28mg，收率20％。
化合物36-7(28mg，0.036mmol)加入DMF(2mL)中，加入18-冠-6(26mg)和异丁酸钠(35mg，0.32mmol)，110℃反应18h，加入水，乙酸乙酯萃取，饱和氯化钠洗涤，硅胶柱层析分离(PE：EA＝20：1-10：1)，得到化合物36-8，淡黄色固体20mg。
化合物36-8(100mg，0.14mmol)加入乙酸(3mL)和水(1mL)中，加热到85℃，反应10mins，原料消失，加入饱和碳酸氢钠溶液，乙酸乙酯萃取，浓缩，硅胶柱层析分离(DCM:MeOH＝100:1-50:1)，得到化合物A36，淡黄色固体50mg。MS m/z＝495.2[M+1] +。
制备实施例51：核苷类似物的合成
参照制备实施例1－50的方法，不同点在于采用不同的原料化合物，从而制得化合物A5、A6、A8、A13、A14、A28、A30、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。
测试实施例1：化合物对新型冠状病毒复制的抑制作用研究
测定本发明化合物对2019新型冠状病毒(SARS-CoV-2)复制抑制活性：Vero E6细胞购买自ATCC，SARS-CoV-2病毒来源于国家病毒资源库微生物菌毒种保藏中心。将Vero E6细胞在密度为5×10 4细胞/孔的48孔细胞培养皿中培养过夜，用不同浓度的本发明化合物预先处理细胞1小时，然后加入病毒(感染复数MOI为0.05)使其感染1小时，然后取出病毒化合物混合物，用含本发明化合物的新鲜培养基进一步培养 细胞。在24h p.i.时，收集细胞上清液并在裂解缓冲液中裂解，通过定量实时RT-PCR(qRT-PCR)对细胞上清液中的病毒拷贝数进行定量评估。
结果表明，在10μM或5μM浓度下多个化合物显著抑制SARS-CoV-2病毒复制，与药物未处理的对照组比较，对新型冠状病毒复制的抑制率＞99％，部分化合物的EC 50值在低微摩尔水平，其抗病毒活性显著优于对照化合物瑞德西韦(remdesivir)。
部分优选化合物的EC 50和抑制率数据列于表1。
表⒈对2019新型冠状病毒(SARS-CoV-2)复制抑制活性
Figure PCTCN2021087928-appb-000083
Figure PCTCN2021087928-appb-000084
Figure PCTCN2021087928-appb-000085
Figure PCTCN2021087928-appb-000086
Figure PCTCN2021087928-appb-000087
注：“/”表示未测定。
测试实施例2：化合物的半数毒性浓度测定
在本实施例中，通过CCK8试剂盒分析确定本发明化合物(表1中的各化合物)对Vero E6细胞的半数毒性浓度(CC 50)。
结果显示，测试化合物在最高浓度10μM下对Vero E6细胞无细胞毒作用，这提示本发明化合物CC 50远大于10μM。
测试实施例3：化合物对新型冠状病毒RNA依赖的RNA聚合酶(RdRp)抑制作用研究
参考文献报道方法(Virus Genes,2015,50:498-504)，利用荧光法测定本发明化合物对2019新型冠状病毒(SARS-CoV-2)RNA依赖的RNA聚合酶(RdRp)抑制活性：新冠病毒RdRP溶液、底物、化合物溶液、缓冲液等共孵育1小时后，加入荧光DNA结合染料，10分钟后，进行数据采集，计算化合物对RdRp的抑制活性。
结果显示，部分化合物对RdRp具有显著抑制活性，见表2。
表2.化合物对2019新型冠状病毒RdRp的抑制活性
Figure PCTCN2021087928-appb-000088
A：<1μM；B:1μM～10μM；C：>1μM；“/”表示未测定。
测试实施例4：化合物对其它病毒复制的抑制作用研究
利用细胞病变效应(CPE)实验测试化合物对呼吸道合胞病毒(RSV)，人冠状病毒OC43，A型流感病毒，寨卡病毒(Zika)复制的抑制活性。
细胞病变效应(CPE)实验
将实验细胞以一定的细胞密度接种到96孔细胞培养板中，于5％CO 2、37℃培养箱中培养过夜。第二天加入化合物和病毒。依所测病毒不同而异，细胞在 5％CO 2、33℃或37℃条件下于培养箱中培养3-7天，直至无化合物的病毒感染对照孔内细胞病变达80-95％。然后用CellTiter-Glo或CCK-8检测每孔细胞活力。如含化合物孔的细胞活力较病毒感染对照孔高，即CPE减弱，则表明化合物对所测病毒有抑制作用。细胞毒性实验方法与相应的抗病毒实验方法相同，但无病毒感染。
化合物的抗病毒活性和细胞毒性分别由化合物对病毒引起的细胞病毒效应的抑制率(％)和细胞活率(％)表示。计算公式如下：
抑制率(％)＝(测试孔读值-病毒对照平均值)/(细胞对照平均值-病毒对照平均值)×100；
细胞活率(％)＝(测试孔读值-培养基对照平均值)/(细胞对照平均值-培养基对照平均值)×100；
EC 50和CC 50值通过Prism软件计算，抑制曲线拟合方法为“log(inhibitor)vs.response--Variable slope”。
登革热病毒空斑减少实验
将Vero细胞以每孔600,000个细胞的密度接种到6孔细胞培养板中、于5％CO 2、37℃培养箱中培养过夜。第二天加入化合物和病毒(40-50PFU/孔)。细胞在5％CO 2、37℃条件下于培养箱中培养2个小时，随后吸去上清，加入含对应浓度化合物的低融点琼脂糖培养液。细胞在5％CO 2、33℃或37℃条件下于培养箱中培养6-7天，直至显微镜下无化合物的病毒感染对照孔内可观察到明显的病毒空斑。使用4％多聚甲醛固定细胞，使用结晶紫染色。计算每孔内空斑数量。
细胞毒性实验与抗病毒实验平行进行。将Vero细胞以每孔20,000个细胞的密度接种到96孔细胞培养板中、于5％CO 2、37℃培养箱中培养过夜。第二天加入化合物(1-5个浓度点、单点)。细胞在5％CO 2、33℃或37℃条件下于培养箱中培养6-7天。然后用CCK-8检测每孔细胞活力。
化合物的抗病毒活性(EC 50或抑制率)和细胞毒性(CC 50)的计算方法同上。
抗猪流行性腹泻病毒(PEDV)活性实验
将Vero细胞进行消化传代，用细胞生长液调整细胞密度为1×10 5/mL，并接种于96孔板，100μL/孔，放置于37℃、5％CO 2培养箱培养24h；取出96孔板，弃去孔中培养基，用1×PBS清洗三次，甩干后每孔加入化合物(10个浓度点)和病毒(每孔0.01MOI)的混合液，每个浓度设置8个重复孔，于37℃、5％CO 2培养箱培养，同时设置病毒对照与细胞对照。36h后，收集细胞样品，荧光定量PCR测定不同处理组病毒含量变化，计算化合物的EC 50。
结果表明，多个化合物具有显著的抗呼吸道合胞病毒(RSV)、人冠状病毒OC43(HCoV OC43)、猪流行性腹泻病毒(PEDV)、寨卡病毒(Zika)、登革热病毒(DENV)活性，与GS-441524相比，氘代衍生物具有更强的抗病毒效果，部分优选化合物 的EC 50列于表3和表4。
表3，对呼吸道合胞病毒(RSV)、人冠状病毒OC43(HCoV OC43)复制抑制活性
Figure PCTCN2021087928-appb-000089
Figure PCTCN2021087928-appb-000090
Figure PCTCN2021087928-appb-000091
注：“/”表示未测定。
表4，对猪流行性腹泻病毒(PEDV)、寨卡病毒(Zika)、登革热病毒(DENV)复制抑制活性
Figure PCTCN2021087928-appb-000092
Figure PCTCN2021087928-appb-000093
Figure PCTCN2021087928-appb-000094
注：“/”表示未测定。
测试实施例5：小鼠体内的药代动力学评价
实验方法：
15只雄性CD-1小鼠，随机分成5组，每组3只，实验前禁食12h，自由饮水；给药后2h统一进食。其中4组分别灌胃给予50mg/kg的GS-441524、A9、A10和A11，并于给药后0.25，0.5，1，2，4，8，24h经股静脉取血约30μL，置于肝素抗凝管中。立即准确吸取20μL全血于预先加好2μL PhosSTOP和7μL DTNB(0.5M)的离心管中，混匀，4℃离心后,取10μL血浆于预先加好100μL沉淀剂(甲醇：乙腈，1:1，v/v)的离心管中,混匀，4℃离心后置于干冰中暂存及运输，冻存于-80℃待测。剩余1组分别静脉注射给予25mg/kg的GS-441524，并于给药后5min，0.25，0.5，1，2，4，8，24h经股静脉取血，置于肝素抗凝管中，处理方法同上。
采用LC-MS-MS法测定血浆中GS-441524、A9、A10和A11的浓度。取20μL上清液与20μL去离子水混匀后进样分析。样品经Waters HSS T3(2.1*50 mm，1.8μm)色谱柱分离后，采用电喷雾电离源，在正离子条件下采用多反应监测模式检测。样品的线性范围均为：10-30000ng/mL。
表5，化合物GS-441524，A9，A10，A11小鼠体内的药动学参数
Figure PCTCN2021087928-appb-000095
测试实施例6：大鼠体内的药代动力学评价
实验方法：
18只雄性SD大鼠，随机分成6组，每组3只，实验前禁食12h(静脉实验组不禁食)，自由饮水；给药后4h统一进食。其中3组分别灌胃给予10mg/kg的A9、A146和A151，剩余3组分别静脉注射给予2mg/kg的A9、A146和A151。给药溶媒为DMSO/EtOH/PEG300/0.9％NaCl(5/5/40/50,v/v/v/v)。
于给药后5min(仅静脉)、0.25，0.5，1.0，2.0，4.0，6.0，8.0和24h经颈静脉取血0.2mL，置EDTA-K2试管中，11000rpm离心5min，分离血浆，于–70℃冰箱中冷冻待测。冰水浴操作。采用LC-MS-MS法测定血浆中A9的浓度，计算药动学参数。
表6，化合物A9，A146，A151大鼠体内的药动学参数
Figure PCTCN2021087928-appb-000096
Figure PCTCN2021087928-appb-000097
测试实施例7：组织分布试验
实验方法：
16只雄性CD-1小鼠，随机分成4组，每组4只，实验前禁食12h，自由饮水；给药后2h统一进食。4组小鼠分别灌胃给予200mg/kg的A151(DMSO-enthanol-PEG300-saline溶解，体积比为5/5/40/50)，在给药1h，2h，4h，和8h后，小鼠进行麻醉，心脏穿刺取血，采集肝、肾、心、肺组织样本,采用HPLC-MS/MS法测定各组织中A9的分布情况。
表7，小鼠口服A151(200mg/Kg)后，代谢产物A9的组织分布
Figure PCTCN2021087928-appb-000098
测试实施例8：抗SARS-CoV-2体内药效试验
实验8.1：
8周龄的Balb/c小鼠，分为5组，病毒对照组(溶媒：40％PEG400+10％HS15+50％超纯水)、A151氢溴酸盐(A151-S)口服组(50mg/Kg，100mg/Kg)、瑞德西韦口服组(50mg/kg)、瑞德西韦腹腔注射组(50mg/kg)。各组动物转导带人源ACE2基因的腺病毒，转导5天后感染新冠病毒，感染1小时后，采用灌胃或腹腔注射的方式分别给予溶媒、A151-S和对照药瑞德西韦，每天给药一次。分别于感染后第2天和第5天处死小鼠，取小鼠肺组织，检测小鼠肺组织中病毒RNA拷贝数。给药2天和给药5天后的病毒RNA拷贝情况分别如图1和图2所示。
试验结果显示，化合物A151-S口服50mg/kg和100mg/kg，均能够有效降低小鼠肺组织中的病毒RNA量，具有一定的剂量依赖性。在给药2天后，在50mg/Kg剂量下，A151-S口服抗病毒效果优于瑞德西韦口服组，在给药5天 后，A151-S口服抗病毒效果优于瑞德西韦腹腔注射组。
实验8.2：
8周龄的Balb/c小鼠，分为2组，病毒对照组(溶媒：5％DMSO+5％SolutolHS15+90％Saline)和A151-S腹腔注射给药组(100mg/Kg)。两组动物转导带人源ACE2基因的腺病毒，感染新冠病毒前一天给予A151-S，并于感染后当天到第4天每天一次腹腔注射给予A151-S，对照组进行同样处理。于感染后第4天处死小鼠，取小鼠肺组织，检测小鼠肺组织中病毒RNA拷贝数。结果如图3所示。
试验结果显示，化合物A151-S通过腹腔注射，能够显著降低小鼠肺组织中的病毒RNA量，与对照组相比，病毒载量下降大于30倍。
讨论
本发明的式I化合物具有显著的抗SARS-CoV-2活性。在病毒感染的Vero E6细胞中，代表性化合物A9(实施例6)、A10(实施例7)、A124(实施例11)、A50(实施例12)、A212(实施例13)、A144(实施例16)、A213(实施例18)、A164(实施例20)、A214(实施例22)、A151(实施例36)能显著抑制病毒复制，EC 50分别为0.44μM、0.43μM、0.25μM、0.23μM、0.11μM、0.24μM、0.10μM、0.27μM、0.11μM和0.31μM，显著优于对照化合物瑞德西韦(remdesivir，EC 50＝2.0μM)。此外，A11、A12、A52、A70、A72、A75、A77、A131、A171、A180、A181、A188、A196、A215等化合物在5μM浓度下能显著抑制病毒复制，抑制率在98％以上，显著高于瑞德西韦在该浓度下对病毒的抑制率(65％)。
A102、A106、A107、A198能够显著抑制新型冠状病毒RdRp活性，表明该类化合物通过作用于RdRp发挥抗病毒作用。
本发明的式I化合物对其它病毒，包括呼吸道合胞病毒(RSV)、人冠状病毒OC43(HCoV OC43)、猪流行性腹泻病毒(PEDV)、寨卡病毒(Zika)、登革热病毒(DENV)也具有显著的抑制作用。值得注意的是，氘代化合物的抗病毒效果更加显著。
小鼠PK试验显示，氘代化合物A9、A11口服后，血浆中药物的暴露量较GS-441524高，分别是GS-441524暴露量的1.3倍和1.2倍；A9在大鼠上的口服生物利用度为21.3％，化合物A146和A151的口服生物利用度分别为48.5％和56.7％，与A9相比，大幅度提高。
小鼠体内组织分布试验显示，A151口服1小时后，代谢产物A9在肝、肾、肺、心脏中浓度最高，并且分布较均匀，无肝、肾蓄积情况。
A151的氢溴酸盐(A151-S)在表达新冠病毒受体人源ACE2的腺病毒转导 小鼠模型上，可通过一天一次口服给药，剂量依赖地降低小鼠肺部的病毒载量，并且在同样剂量下(50mg/Kg),A151-S抗新冠病毒的效果优于瑞德西韦。以上数据表明，A151-S抗SARS-COV-2作用显著，并且具有口服优势。从以上数据可推测，本发明的化合物具有很好的抗病毒，尤其是抗新型冠状病毒(SARS-CoV-2)的应用前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
    Figure PCTCN2021087928-appb-100001
    式中，
    R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
    R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
    R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
    R 5选自R 3、
    Figure PCTCN2021087928-appb-100002
    Figure PCTCN2021087928-appb-100003
    R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
    R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
    R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    X选自-CH 2-、-CD 2-、-CHD-；
    R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 10选自C 1-18烷基、亚甲基C 6-20芳基；
    R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 12选自C 1-20烷基；
    M各自独立地选自氢、金属、-NH 4、或质子化的有机胺。
  2. 一种活性成分或含所述活性成分的制剂的用途，其特征在于，所述的活性成分为式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
    Figure PCTCN2021087928-appb-100004
    式中，
    R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
    R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
    R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
    R 5选自R 3、
    Figure PCTCN2021087928-appb-100005
    Figure PCTCN2021087928-appb-100006
    R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
    R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
    R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    X选自-CH 2-、-CD 2-、-CHD-；
    R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 10选自C 1-18烷基、亚甲基C 6-20芳基；
    R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 12选自C 1-20烷基；
    M各自独立地选自氢、金属、-NH 4、或质子化的有机胺；
    并且，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制病毒复制的抑制剂；和/或(b)治疗和/或预防、缓解由病毒感染引起的相关疾病的药物。
  3. 如权利要求2所述的用途，其特征在于，所述的病毒选自：
    (1)感染人的冠状病毒：重症急性呼吸综合征冠状病毒SARS-CoV(Severe acute respiratory syndrome coronavirus，SARS-CoV)、2019新型冠状病毒(2019-nCoV或SARS-CoV-2)、中东呼吸综合征冠状病毒MERS-CoV(Middle East respiratory syndrome coronavirus，MERS-CoV)；
    (2)致普通感冒的冠状病毒：所述的致普通感冒的冠状病毒优选选自下组：人冠状病毒OC43(Human coronavirus OC43)、人冠状病毒229 E(Human coronavirus229E)、人冠状病毒NL63(Human coronavirus NL63)、人冠状病毒HKUl(Human coronavirus HKUl)；
    (3)人呼吸道合胞病毒(RSV)；
    (4)人流感病毒：甲型流感病毒、乙型流感病毒、丙型流感病毒；
    (5)黄病毒科病毒：丙型肝炎病毒(HCV)、登革热病毒(DENV)、寨卡病毒(Zika)；
    (6)丝状病毒科病毒：马尔堡病毒(MBV)、埃博拉病毒(EBV)；
    (7)感染其他哺乳动物的冠状病毒：猪流行性腹泻病毒(PEDV)。
  4. 如权利要求2所述的用途，其特征在于，所述病毒引起的相关疾病选自下组：
    (D1)人冠状病毒感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
    (D2)人呼吸道合胞病毒(RSV)感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
    (D3)人流感病毒感染引起的普通感冒、高危症状感染、呼吸道感染、肺炎及其并发症；
    (D4)丙型肝炎病毒(HCV)引起的慢性丙型肝炎及其并发症；
    (D5)登革热病毒(DENV)引起的登革热及其并发症；
    (D6)寨卡病毒(Zika)引起的感染及其并发症；
    (D7)马尔堡病毒(MBV)、埃博拉病毒(EBV)引起的出血热及其并发症；
    (D8)SARS-CoV-2引起的新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)；
    (D9)猪流行性腹泻病毒(PEDV)引起的猪流行性腹泻；
    (D10)上述疾病的任意组合。
  5. 如权利要求2所述的用途，其特征在于，所述式(I)化合物为具有如下结构的化合物A1至A221中任一化合物、或其组合：
    Figure PCTCN2021087928-appb-100007
    Figure PCTCN2021087928-appb-100008
    Figure PCTCN2021087928-appb-100009
    Figure PCTCN2021087928-appb-100010
    Figure PCTCN2021087928-appb-100011
    Figure PCTCN2021087928-appb-100012
    更佳地，所述的活性成分为选自下组的核苷类似物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药、化合物：
    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或其组合。
  6. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (a1)第一活性成分，所述的第一活性成分为式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
    Figure PCTCN2021087928-appb-100013
    式中，
    R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
    R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连；
    R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
    R 5选自R 3、
    Figure PCTCN2021087928-appb-100014
    Figure PCTCN2021087928-appb-100015
    R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
    R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
    R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    X选自CH 2，CD 2、-CHD-；
    R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 10选自C 1-18烷基、亚甲基C 6-20芳基；
    R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 12选自C 1-20烷基；
    M各自独立地选自氢、金属、NH 4或质子化的有机胺；
    以及(b)药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述式(I)化合物为化合物A1至A221中任一化合物、或其组合。
  8. 一种权利要求6所述的药物组合物的用途，其特征在于，用于制备(a)抑制冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)；和/或(b)治疗和/或预防、缓解由冠状病毒、流感病毒、呼吸道合胞病毒、黄病毒科病毒、丝状病毒科病毒和/或猪流行性腹泻病毒(PEDV)感染引起的相关疾病的药物。
  9. 一种抑制病毒复制的方法，其特征在于，包括步骤：
    将第一活性成分或含所述第一活性成分的制剂与病毒接触，从而抑制所述病毒的复制；
    其中，所述的第一活性成分为式I化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
    Figure PCTCN2021087928-appb-100016
    式中，
    R 1选自氢、氘、卤素、氰基、叠氮、氨基、C 1-6烷基取代的氨基、C 1-6酰基、C 1-6烷基、C 1-6烷氧基、C 1-6烷酰基取代的氨基、卤代C 1-6烷基、C 2-6烯基、C 2-6烯氧基、C 2-6炔氧基、C 2-6炔基、C 3-6环烷基、卤代C 3-6环烷基、氨甲酰基、羟甲基、氰基甲基(-CH 2CN)、脒基、胍基、脲基、氰硫基(-SCN)、氰氧基(-OCN)；
    R 2选自氢、卤素、OR 3、氰基、C 1-6烷基、C 1-6烷氧基、C 2-6烯基、C 2-6炔基；
    R 3选自氢、C 1-20烷酰基、氨基C 1-20烷酰基、C 1-6烷氨基C 1-6烷酰基、C 1-6烷氧基C 1-6烷基、α-氨基酸，所述α-氨基酸的羧基与呋喃环上的羟基以酯键相连，优选地，所述α-氨基酸选自丙氨酸、缬氨酸、异亮氨酸、色氨酸、苯丙氨酸；
    R 4选自氢、氘、卤素、叠氮、氰基、C 1-6烷基、卤代C 1-6烷基、叠氮C 1-6烷基、氰基C 1-6烷基、羟基C 1-6烷基、C 2-6烯基、C 2-6炔基、C 3-6环烷基、C 1-6烷酰基、C 2-6烯氧基、C 2-6炔氧基、C 1-6烷氧基C 1-6烷基、氨基C 1-6烷基、C 1-6烷氨基C 1-6烷基、脒基、胍基、脲基、氰硫基、氰氧基；
    R 5选自R 3、
    Figure PCTCN2021087928-appb-100017
    Figure PCTCN2021087928-appb-100018
    R 6选自氨基、羟基、卤素、氰基、氰氧基、氰硫基、C 1-6烷氧基、C 1-6烷氨基、NHOH、NHCOR 12、NHOCOR 12、NHCOOR 12；
    R 7选自氢、氘、卤素、氨基、甲基、NHCOR 12、NHCOOR 12；
    R 8选自氢、氘、卤素、氰基、氨甲酰基、C 1-6烷基取代的氨基甲酰基、C 1-6烷氧基酰胺基、C 1-6烷氧羰基、羟基、羟基C 1-6烷基、氨基、C 1-6烷酰基取代的氨基、C 1-6烷基取代的氨基、C 1-6烷基，C 2-6 C 1-6烷氧基、烯基、C 2-6炔基；
    X选自CH 2，CD 2、-CHD-；
    R 9选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 10选自C 1-18烷基、亚甲基C 6-20芳基；
    R 11选自C 1-6烷基、C 3-6环烷基、C 6-20芳基、5-15元杂芳基；
    R 12选自C 1-20烷基；
    M各自独立地选自氢、金属、NH 4或质子化的有机胺。
  10. 一种(a)抑制病毒复制和/或(b)治疗和/或预防、缓解由病毒感染引起的相关疾病的方法，其特征在于，包括步骤：给需要的对象施用安全有效量的式I所示的化合物或其药学上可接受的盐或其结晶水合物或其溶剂化物或其前药：
    Figure PCTCN2021087928-appb-100019
    式中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和X如权利要求1中所定义。
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