WO2021147235A1 - 法匹拉韦在治疗冠状病毒感染方面的应用 - Google Patents

法匹拉韦在治疗冠状病毒感染方面的应用 Download PDF

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WO2021147235A1
WO2021147235A1 PCT/CN2020/097395 CN2020097395W WO2021147235A1 WO 2021147235 A1 WO2021147235 A1 WO 2021147235A1 CN 2020097395 W CN2020097395 W CN 2020097395W WO 2021147235 A1 WO2021147235 A1 WO 2021147235A1
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pharmaceutical composition
pharmaceutically acceptable
sars
coronavirus
preparation
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PCT/CN2020/097395
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English (en)
French (fr)
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钟武
曹瑞源
曹诚
高婷
肖庚富
胡志红
王曼丽
张磊砢
李松
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2021147235A1 publication Critical patent/WO2021147235A1/zh

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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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

Definitions

  • This application relates to the compound Favipiravir represented by the following formula I, its geometric isomers, its pharmaceutically acceptable salts and/or both solvates and/or both hydrates, and pharmaceutical compositions containing the above-mentioned compounds ,
  • coronavirus especially SARS coronavirus (SARS-CoV) and SARS-CoV-2 infection.
  • Fapilavir (compound of formula I), chemically named 6-fluoro-3-hydroxypyrazine-2-carboxamide, is a nucleoside analog drug and a viral RNA polymerase inhibitor. This type of drug is a broad-spectrum antiviral drug, which has been approved as an anti-influenza drug in Japan.
  • Fapilavir is against members of the filoviridae, Bunyaviridae, arenaviridae, togaviridae and other virulent RNAviridae, as well as Orthomyxoviridae, Paramyxoviridae, Picornaviridae, and Flaviviridae Other members of the RNA virus family have good inhibitory effects in vitro and in vivo, but the activity of coronaviruses has not been reported.
  • Favipiravir can be converted into a triphosphate active form after entering the cell, and it is incorporated into the RNA chain during the transcription and replication of the viral RNA, and non-specifically terminates the extension of the viral RNA chain, thereby achieving antiviral effects.
  • T-705 can effectively inhibit the virus titer in the supernatant after virus infection on the vero E6 cell line, and the half inhibitory concentration (IC50) for EBOV Zaire is 67 ⁇ mol/L.
  • IC50 half inhibitory concentration for EBOV Zaire is 67 ⁇ mol/L.
  • the in vivo pharmacodynamic study using the IFNAR-/-C57BL/6 mouse model deficient in type I interferon receptors showed that at the challenge dose of 100% death in the placebo group, the dose was 300 mg/kg/d The dose was administered from the 6th day to the 13th day, and the protection rate was 100%.
  • the body weight, alanine aminotransferase, aspartate aminotransferase and viremia were also significantly improved.
  • the 2019 Novel Coronavirus (2019-nCoV) is a new strain of coronavirus that has never been found in humans before.
  • ICTV International Commission for Classification of Viruses
  • SARS-CoV- 2 severe acute respiratory syndrome coronavirus 2
  • WHO World Health Organization
  • the symptoms of SARS-CoV-2 infection are mainly pneumonia. According to the severity of the disease, it can be divided into simple infection, mild pneumonia, severe pneumonia, acute respiratory distress syndrome, sepsis, septic shock and so on.
  • Patients with simple infections may have non-specific symptoms, such as fever, cough, sore throat, nasal congestion, fatigue, headache, muscle pain or discomfort, the elderly and immunosuppressed people may have atypical symptoms.
  • Patients with mild pneumonia mainly cough, dyspnea + shortness of breath. Severe pneumonia can be seen in adolescents, adults or children. The main symptoms are increased respiratory rate, severe respiratory failure or dyspnea, central cyanosis, lethargy, unconsciousness or convulsions, gas pumping, etc.
  • the lung images of acute respiratory distress syndrome are bilateral ground-glass shadows, but they cannot be completely explained by effusion, lobar exudation, atelectasis, or pulmonary block shadows. Pulmonary edema is the main symptom. Patients with sepsis often have fatal organ dysfunction, and septic shock is the most critical patient with a higher probability of death.
  • the purpose of this application is to discover drugs with antiviral activity against coronaviruses, especially SARS-CoV (SARS-CoV) and SARS-CoV-2, which can be used for related diseases such as simple infections such as fever, cough and sore throat, etc. , Pneumonia, acute or severe acute respiratory infections, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock.
  • SARS-CoV SARS-CoV
  • SARS-CoV-2 SARS-CoV-2
  • the pharmaceutically acceptable salt of the compound represented by formula I of the present application includes its inorganic or organic acid salts, and inorganic or organic base salts.
  • the present application relates to all forms of the foregoing salts, including but not limited to :Sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrogen salt, hydrogen syrup, nitrate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, ethyl Acid salt, propionate, butyrate, oxalate, trimethyl acetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate , Fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and paxine Acid salts and so on.
  • the compound represented by formula I can inhibit the replication of coronavirus on cells and reduce the nucleic acid load of coronavirus in cell culture.
  • the compound represented by formula I can reduce the viral nucleic acid load level of SARS-CoV-2 infected cells at a micromolar concentration
  • the compound represented by formula I has a significant protective effect on mice infected with SARS coronavirus (SARS-CoV).
  • This application relates to the preparation of compounds represented by formula I, their geometric isomers, their pharmaceutically acceptable salts and/or their solvates and/or their hydrates for the treatment of coronaviruses, especially SARS coronavirus (SARS- CoV) and diseases or infections caused by SARS-CoV-2 (including but not limited to respiratory diseases (such as simple infections such as fever, cough and sore throat, etc.), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), low Oxygen respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.))),
  • This application also relates to the use of the compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates in the preparation of drugs as coronavirus inhibitors.
  • This application also relates to the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt and/or its solvate and/or its hydrate in preparation for inhibiting coronavirus in cells (such as mammalian cells). ) In the replication or reproduction of medicines.
  • This application also relates to a pharmaceutical composition, which comprises the compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or solvates and/or hydrates thereof,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to the pharmaceutical composition
  • the pharmaceutical composition comprising the compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates or the said formula I
  • the shown compound, its geometric isomer or its pharmaceutically acceptable salt and/or its solvate and/or its hydrate are prepared for the treatment of respiratory diseases but not limited to respiratory diseases (including simple infections such as fever) , Cough and sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc. )
  • respiratory diseases including simple infections such as fever) , Cough and sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.
  • respiratory diseases including simple infections such as fever) , Cough and sore throat, pneumonia, acute respiratory infection, severe
  • This application also relates to a method for treating and/or preventing disease in a mammal in need or a method for inhibiting the replication or reproduction of coronavirus in a mammal in need, the method comprising administering treatment and/or prevention to a mammal in need / Or a preventively effective amount of the pharmaceutical composition or the pharmaceutical composition comprising the compound represented by formula I, its geometrical isomer or its pharmaceutically acceptable salt and/or its solvate and/or its hydrate.
  • the diseases caused by the coronavirus include but are not limited to respiratory diseases (e.g., simple infections such as fever, cough, and sore throat).
  • respiratory diseases e.g., simple infections such as fever, cough, and sore throat.
  • Etc. pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.).
  • This application also relates to the preparation of a pharmaceutical composition for the treatment of diseases or infections caused by coronaviruses, especially SARS coronavirus (SARS-CoV) and SARS-CoV-2 (e.g., respiratory diseases (e.g., simple infections such as fever, cough and Sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.))
  • respiratory diseases e.g., simple infections such as fever, cough and Sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.
  • SARS severe acute respiratory syndrome
  • the pharmaceutical composition comprises the compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to the use of a pharmaceutical composition in the preparation of a drug as a coronavirus inhibitor, wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or Solvates and/or hydrates,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to the use of a pharmaceutical composition in the preparation of a medicament for inhibiting the replication or reproduction of coronaviruses in cells (such as mammalian cells), wherein the pharmaceutical composition comprises the compound represented by formula I and its geometric isomers , Its acceptable salt and/or its solvate and/or its hydrate,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to the compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates, which are used for the treatment of coronaviruses, especially SARS coronavirus (SARS -CoV) and diseases or infections caused by SARS-CoV-2 (including but not limited to respiratory diseases (such as simple infections such as fever, cough and sore throat, etc.), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), Hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.)).
  • This application also relates to the compound represented by Formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates, which are used as coronavirus inhibitors.
  • This application also relates to the compound represented by Formula I, its geometric isomers, its pharmaceutically acceptable salts and/or its solvates and/or its hydrates, which are used to inhibit the presence of coronavirus in cells (such as mammalian cells). ) To copy or reproduce.
  • This application also relates to a pharmaceutical composition for the treatment of diseases or infections caused by coronaviruses, especially SARS coronaviruses (SARS-CoV) and SARS-CoV-2 (e.g., respiratory diseases (e.g., simple infections such as fever, cough and Sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection (SARI), hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.)),
  • the pharmaceutical composition comprises the compound represented by formula I, its geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to a pharmaceutical composition for use as a coronavirus inhibitor, wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, its pharmaceutically acceptable salts and/or solvates and/ Or its hydrate,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to a pharmaceutical composition for inhibiting the replication or reproduction of coronaviruses in cells (such as mammalian cells), wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, and its pharmaceutically acceptable compounds. Accepted salts and/or solvates and/or hydrates thereof,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • the coronavirus described in this application is SARS coronavirus (SARS-CoV) or SARS-CoV-2.
  • the coronavirus described in this application is SARS-CoV-2.
  • the disease caused by the coronavirus described in this application is a disease caused by SARS-CoV-2, namely COVID-19.
  • the disease caused by the coronavirus described in this application is atypical pneumonia caused by SARS coronavirus (SARS-CoV).
  • the mammals described in this application include bovines, equines, ovines, swines, canines, felines, rodents, primates, such as humans. , Cat, dog or pig.
  • 2019-nCoV 2019-nCoV
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the term "therapeutically effective amount” or “prophylactically effective amount” refers to an amount that is sufficient to treat or prevent the patient's disease but is low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of reasonable medical judgment.
  • the therapeutically effective amount of the compound will depend on the specific compound selected (for example, considering the potency, effectiveness and half-life of the compound), the route of administration selected, the disease to be treated, the severity of the disease to be treated, and the patient's condition to be treated. Factors such as age, size, weight, and physical disease, the medical history of the patient being treated, the duration of treatment, the nature of concurrent therapy, the desired therapeutic effect and other factors have changed, but they can still be routinely determined by those skilled in the art.
  • the specific dosage and method of use of the compound represented by formula I, its geometric isomers or its pharmaceutically acceptable salts and/or its solvates and/or its hydrates for different patients are determined by many Factors include the patient’s age, weight, gender, natural health status, nutritional status, active strength of the drug, taking time, metabolic rate, severity of the disease, and the subjective judgment of the physician.
  • the preferred dosage here is between 0.001-1000 mg/kg body weight/day.
  • the pharmaceutical composition described in this application can be prepared into various forms according to different administration routes.
  • the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular , Intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or medication with the aid of an explanted reservoir.
  • oral, intraperitoneal or intravenous administration is preferred.
  • the compound represented by formula I can generally be prepared in the form of suppositories, which can be used to form a suppository. It is prepared by mixing with a suitable non-irritating excipient.
  • the excipient presents a solid state at room temperature, but melts at the rectal temperature to release the drug.
  • excipients include cocoa butter, beeswax and polyethylene glycol.
  • the compound represented by formula I When applied locally to the eye, the compound represented by formula I, its geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof can be formulated into a micronized suspension or solution
  • the carrier used is isotonic sterile saline with a certain pH, which may or may not be added with preservatives such as chlorinated benzyl alkanolate.
  • the compound can also be made into an ointment form such as petrolatum.
  • the compound represented by formula I, its geometric isomers or its pharmaceutically acceptable salts and/or its solvates and/or its hydrates can be made into appropriate ointments, lotions or creams A dosage form in which the active ingredient is suspended or dissolved in one or more carriers.
  • the carriers that can be used for the ointment here include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; the carriers that can be used for lotions or creams include but are not limited to: Mineral oil, sorbitan monostearate, Tween 60, cetyl ester wax, hexadecenyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the compound represented by formula I When administered locally in the lower intestinal tract, the compound represented by formula I, its geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof can be prepared into rectal suppository formulations as described above Or a suitable enema preparation form, and a topical transdermal patch can also be used.
  • the compound represented by formula I can also be administered in the form of sterile injection preparations, including sterile injection water or oil suspensions Solution, or sterile injection solution.
  • usable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution.
  • sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.
  • Figure 1 Shows that Favipiravir effectively reduces the viral nucleic acid load on SARS-CoV-2 infected vero E6 cells.
  • (a) shows that favipiravir can inhibit the viral RNA load on the cells 48h after the cells are infected with SARS-CoV-2, and the inhibitory activity is dose-dependent.
  • the ordinate is the copy number of viral RNA in the sample, and the abscissa is the drug concentration;
  • (b) shows that favipiravir treated the tested cells at the test concentration for 48 hours, and no cytotoxicity was observed.
  • the ordinate is the percentage of cell viability relative to the negative control group (cells only, no drug is added), and the abscissa is the drug concentration.
  • FIG. 1 Shows that Favipiravir effectively protects SARS coronavirus-infected mice from death.
  • the mice were given intraperitoneal Fabiravir injection once a day 4 hours after challenge and the following time.
  • the control group was given the same volume of solvent injection. It can be seen that all the control groups died, while the mice in the Fabiravir treatment group survived. The survival rate is dose-dependent.
  • Example 1 Favipiravir reduces the viral nucleic acid load of SARS-CoV-2 infected cells
  • Vero E6 cells purchased from ATCC, article number 1586
  • 2% cell maintenance solution formula: FBS (purchased from Gibco company, article number 16000044)
  • MEM purchased from Gibco, article number 10370021
  • SARS-CoV-2 2019-nCoV
  • SARS-CoV-2019BetaCoV/Wuhan/WIV04/2019 strain was diluted from the Wuhan Institute of Virology, CAS provided to the appropriate concentration and added 24 per well plates containing viral manipulation amount 100TCID 50.
  • favipiravir purchased from Selleck Chemicals, catalog number S7975
  • favipiravir purchased from Selleck Chemicals, catalog number S7975
  • the cell control group only added 2% cell maintenance solution without any test drug.
  • the reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, article number RR047Q) produced by TaKaRa was used for RNA reverse transcription.
  • 1gDNA removal Collect RNA samples of each experimental group, and take 1 ⁇ g for reverse transcription. First, add 2 ⁇ l 5 ⁇ gDNA Eraser Buffer to the RNA of each experimental group, fill up the reaction system to 10 ⁇ l with RNase Free water, mix well, and remove the g DNA that may exist in the sample in a water bath at 42°C for 2 minutes;
  • Fluorescence quantitative PCR was used to detect the number of copies per milliliter of the original virus solution.
  • RBD-qR CTCAAGTGTCTGTGGATCACG
  • Cycle parameters 95°C for 15 seconds, 54°C for 15 seconds, 72°C for 30 seconds, a total of 40 cycles.
  • MEM medium purchased from Gibco, product number 10370021
  • FBS Gibco company, product number 10370021
  • Cell viability (%) (A (drug treatment group) -A (blank control) )/(A (negative control) -A (blank control) ) ⁇ 100%
  • test compound can effectively inhibit the replication of the SARS-CoV-2 virus genome in the infection supernatant at the concentrations of 100 ⁇ M, 33 ⁇ M, 11.1 ⁇ M and 3.7 ⁇ M (Table 1 and Figure 1)
  • Example 2 Experiment of favipiravir protecting SARS coronavirus-infected mice from death
  • mice (provided by the Shanghai Pasteur Institute of the Chinese Academy of Sciences) 3-4 weeks old and weighing 9-13 g were randomly divided into 4 groups, namely the virus control group, the high-dose administration group, the middle-dose administration group and the low-dose administration group. Dosage group, 10 per group, ear studs mark.
  • CMC-Na as a solvent to dissolve Fapiravir.
  • the challenge was by intraperitoneal injection.
  • Each mouse was intraperitoneally injected with SARS coronavirus (provided by the Academy of Military Medicine) at a challenge dose of 1*10 6 PFU, and the virus was diluted with normal saline to the required dose. 4h, 24h, 48h, 72h, 96h, 120h, 144h after the challenge, the doses are high dose 200mg/kg, medium dose 100mg/kg and low dose 50mg/kg.
  • the virus control group is given the same 0.5% CMC-Na solvent by volume. Weigh and record at fixed points every day. At the same time, the death of mice is recorded, and the survival curve is drawn, as shown in Figure 2.
  • mice The results of in vivo experiments show that the terminal survival rate of mice is in a dose-dependent relationship with the dose (Table 3 and Figure 2), showing that the treatment of the test compound fapilavir can effectively protect mice from SARS coronavirus Death caused by infection.

Abstract

本申请涉及式I所示法匹拉韦类化合物，及其药学上可接受的盐和/或其溶剂化物和/或其水合物，及含有此化合物的药物组合物，用于治疗冠状病毒的感染。

Description

法匹拉韦在治疗冠状病毒感染方面的应用
本申请是以CN申请号为202010070142.0，申请日为2020年1月21日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请涉及下面式I所示化合物法匹拉韦，其几何异构体、其药学上可接受的盐和/或两者溶剂化物和/或两者水合物，及含上述化合物的药物组合物，用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2感染方面的用途。
Figure PCTCN2020097395-appb-000001
背景技术
法匹拉韦(式I化合物),化学名为6-氟-3-羟基吡嗪-2-甲酰胺，是一种核苷类似物的药物，是一种病毒RNA聚合酶抑制剂。该类药物是一种广谱抗病毒药物，目前已在日本被批准作为抗流感药物上市。
法匹拉韦对丝状病毒科、布尼亚病毒科、沙粒病毒科、披膜病毒科等烈性RNA病毒科成员以及正黏病毒科、副黏病毒科、小RNA病毒科、黄病毒科等其他RNA病毒科成员在体外及体内均具有良好的抑制效果，但是对冠状病毒的活性未见报道。法匹拉韦在进入细胞后能够被转化为三磷酸活性形式，在病毒RNA的转录与复制时掺入RNA链，非特异性终止病毒RNA链的延伸，进而实现抗病毒作用。
有研究报道，T-705在vero E6细胞系上能够有效抑制病毒感染后上清中的病毒滴度，对EBOV扎伊尔型的半数抑制浓度(IC50)为67μmol/L。利用I型干扰素受体缺陷的IFNAR-/-C57BL/6小鼠模型进行体内药效学研究结果显示，在安慰剂组100％死亡的攻毒剂量下，以300mg/kg/d的给药剂量从第6天开始给药至第13天，保护率为100％，与此同时，体质量、谷丙转氨酶、谷草转氨酶和病毒血症等参数也有明显改善。英国防卫科学与技术实验室的相关研究结果显示，法匹拉韦在1.95g/L的高浓度时对Vero C1008细胞无细胞毒性，当药物浓度在62.5mg/L以上时，可以完全抑制EBOV扎伊尔型对细胞的CPE作用。在I型、II型干扰素同时缺陷的A129小鼠模型中，以安慰剂组100％死亡的剂量对小鼠进行攻毒，攻毒之 后1h立即以300mg/kg/d(2次/d，每次150mg/kg)的给药剂量进行灌胃给药，连续给药14d，能够保护100％的小鼠免于死亡，且给药组的小鼠体质量具有显著改善。
2019新型冠状病毒(2019-nCoV)是以前从未在人类中发现的冠状病毒新毒株。2020年2月11日，国际病毒分类委员会(ICTV)宣布，2019新型冠状病毒(2019-nCoV)的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)。同日，世界卫生组织(WHO)宣布，由这一病毒导致的疾病的正式名称为COVID-19。SARS-CoV-2感染的症状主要以肺炎为主，依据病情的轻重程度可分为单纯性感染、轻症肺炎、重症肺炎、急性呼吸窘迫综合症、脓毒症、脓毒症休克等。单纯性感染的患者可能有非特异性症状，例如发热、咳嗽、咽痛、鼻塞、乏力、头痛、肌肉疼痛或不适，老年人和免疫抑制者可能会出现非典型症状。轻症肺炎的患者主要以咳嗽、呼吸困难+呼吸急促为主。重症肺炎可见于青少年、成人或儿童，主要症状为呼吸频率增加，严重的呼吸衰竭或呼吸困难，中心型发绀、嗜睡、意识不清或惊厥、抽气等。急性呼吸窘迫综合症的肺部影像为双侧磨玻璃影，但不能完全由积液、大叶渗出或者肺不张或者肺部块影解释，以肺水肿为主要症状。脓毒症患者往往有致命的器官功能障碍，脓毒性休克是最为危重的患者，死亡可能性较高。
目前，针对新型冠状病毒感染，临床上以支持治疗为主，无特异抗病毒药物可用。
申请内容
本申请目的是发现对冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2有抗病毒活性的药物，可用于其感染引起相关疾病如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等的救治。本申请通过创造性的研究发现式I所示化合物法匹拉韦，具有抑制SARS冠状病毒(SARS-CoV)和SARS-CoV-2复制方面的功能，在治疗SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病方面具有很好的潜在的治疗效果。
本申请提供式I所示的化合物，其几何异构体，其药学上可接受的盐和/或其溶剂化物和/或其水合物:
Figure PCTCN2020097395-appb-000002
在某些实施方案中，本申请式I所示化合物的药学上可接受的盐包括其无机或有机酸盐，以及无机或有机碱盐，本申请涉及上述盐的所有形式，其中包括但不限于：钠盐、钾盐、钙盐、锂盐、葡甲胺盐、盐酸盐，氢澳酸盐，氢腆酸盐，硝酸盐，硫酸盐，硫酸氢盐，磷酸盐，磷酸氢盐，乙酸盐，丙酸盐，丁酸盐，草酸盐，三甲基乙酸盐，己二酸盐，藻酸盐，乳酸盐，柠檬酸盐，酒石酸盐，琥珀酸盐，马来酸盐，富马酸盐，苦味酸盐，天冬氨酸盐，葡糖酸盐，苯甲酸盐，甲磺酸盐，乙磺酸盐，苯磺酸盐，对甲苯磺酸盐和双羟萘酸盐等。
式I所示化合物可在细胞上抑制冠状病毒复制，减少细胞培养物中冠状病毒核酸载量。
本申请的发明人在经过创造性的发明和研究后，发现了式I化合物的一些新作用特点：
第一、式I所示化合物可以在微摩尔级浓度下降低SARS-CoV-2感染的细胞病毒核酸载量水平；
第二、式I所示化合物对SARS冠状病毒(SARS-CoV)感染的小鼠具有显著的保护作用。
本申请涉及式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))的药物中的用途，
Figure PCTCN2020097395-appb-000003
本申请还涉及式I所示的化合物，其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备作为冠状病毒抑制剂的药物中的用途。
本申请还涉及式I所示的化合物，其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途。
本申请还涉及一种药物组合物，其包含所述的式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药 物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及所述包含所述的式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物或所述式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备用于治疗呼吸系统疾病但不限于呼吸系统疾病(包括单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等)的药物中的用途。
本申请还涉及一种在有需要的哺乳动物中治疗和/或预防疾病的方法或者在有需要的哺乳动物中抑制冠状病毒复制或繁殖的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的所述包含所述的式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物或所述的式I所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，其中所述的疾病包括冠状病毒引起的疾病。
在某些实施方案中，所述的冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等)。
本申请还涉及药物组合物在制备用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))的药物中的用途，其中所述药物组合物包含所述的式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物
Figure PCTCN2020097395-appb-000004
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及药物组合物在制备作为冠状病毒抑制剂的药物中的用途，其中所述 药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020097395-appb-000005
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及药物组合物在制备用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药盐上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020097395-appb-000006
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，其用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))。
本申请还涉及式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，其用作冠状病毒抑制剂。
本申请还涉及式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，其用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖。
本申请还涉及药物组合物，其用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(例如单纯性感染如发热、咳嗽 和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))，其中所述药物组合物包含所述的式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物
Figure PCTCN2020097395-appb-000007
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及药物组合物，其用作冠状病毒抑制剂，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020097395-appb-000008
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
本申请还涉及药物组合物，其用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020097395-appb-000009
优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
在某些实施方案中，本申请所述的冠状病毒为SARS冠状病毒(SARS-CoV)或SARS-CoV-2。
在某些实施方案中，本申请所述的冠状病毒为SARS-CoV-2。
在某些实施方案中，本申请所述冠状病毒引起的疾病为SARS-CoV-2引起的疾病，即COVID-19。
在某些实施方案中，本申请所述冠状病毒引起的疾病为SARS冠状病毒(SARS-CoV)引起的非典型肺炎。
在某些实施方案中，本申请所述哺乳动物包括牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，例如是人、猫、狗或猪。
本申请中，所用术语“2019新型冠状病毒(2019-nCoV)”的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)。
本申请中，所用术语“2019新型冠状病毒(2019-nCoV)引起的疾病”的正式名称为COVID-19。
本申请中，术语“治疗有效量”或“预防有效量”是指在合理的医学判断范围内，足以治疗或预防患者疾病但足够低地避免严重副作用(在合理的利益/风险比)的量。化合物的治疗有效量将根据所选择的具体化合物(例如考虑化合物的效力、有效性和半衰期)、所选择的给药途径、所治疗的疾病、所治疗的疾病的严重性、所治疗的患者的年龄、大小、体重和身体疾病、所治疗的患者的医疗史、治疗持续时间、并行疗法的性质、所需的治疗效果等因素发生变化，但仍可以由本领域技术人员常规确定。
另外需要指出，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物针对不同患者的特定使用剂量和使用方法决定于诸多因素，包括患者的年龄，体重，性别，自然健康状况，营养状况，药物的活性强度，服用时间，代谢速率，病症的严重程度以及诊治医师的主观判断。这里优选使用剂量介于0.001-1000mg/kg体重/天。
本申请所述药物组合物可以根据不同给药途径而制备成各种形式。
根据本申请，所述的药物组合物可以以下面的任意方式施用：口服、喷雾吸入、直肠用药、鼻腔用药、颊部用药、阴道用药、局部用药、非肠道用药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内和颅内注射或输入、或借助一种外植储器用药。其中优选口服、腹膜内或静脉内用药方式。
当口服用药时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物可制成任意口服可接受的制剂形式，包括但不限于片剂、胶囊、水溶液或水悬浮液。其中，片剂一般使用的载体包括乳糖和玉米淀粉，另外也可加入润滑剂如硬质酸镁。胶囊制剂一般使用的稀释剂包括乳糖和干燥玉米淀粉。水悬浮液制剂则通 常是将活性成分与适宜的乳化剂和悬浮剂混合使用。如果需要，以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当直肠用药时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物一般可制成栓剂的形式，其通过将药物与一种适宜的非刺激性赋形剂混合而制得。该赋形剂在室温下呈现固体状态，而在直肠温度下熔化释出药物。该类赋形剂包括可可脂、蜂蜡和聚乙二醇。
当局部用药时，特别是治疗局部外敷容易达到的患面或器官，如眼睛、皮肤或下肠道神经性疾病时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物可根据不同的患面或器官制成不同的局部用药制剂形式，具体说明如下：
当眼部局部施用时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物可配制成一种微粉化悬浮液或溶液的制剂形式，所使用载体为等渗的一定pH的无菌盐水，其中可加入也可不加防腐剂如氯化苄基烷醇盐。此外对于眼用，也可将化合物制成膏剂形式如凡士林膏。
当皮肤局部施用时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物可制成适当的软膏、洗剂或霜剂制剂形式，其中活性成分悬浮或溶解于一种或多种载体中。这里软膏即可使用的载体包括但不限于：矿物油、液体凡士林、白凡士林、丙二醇、聚氧化乙烯、聚氧化丙烯、乳化蜡和水；洗剂或霜剂可使用的载体包括但不限于：矿物油、脱水山梨糖醇单硬脂酸酯、吐温60、十六烷酯蜡、十六碳烯芳醇、2-辛基十二烷醇、苄醇和水。
当下肠道局部施用时，所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物可制成如上所述的直肠栓剂制剂或适宜的灌肠制剂形式，另外也可使用局部透皮贴剂。
所述式I所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物还可以无菌注射制剂形式用药，包括无菌注射水或油悬浮液，或无菌注射溶液。其中，可使用的载体和溶剂包括水，林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
上述各种剂型的药物均可以按照药学领域的常规方法制备。
附图说明
图1.显示法匹拉韦有效降低SARS-CoV-2感染的vero E6细胞上病毒核酸载量。其中(a)显示法匹拉韦在细胞感染SARS-CoV-2 48h后能够抑制细胞上的病毒RNA载量，且抑制活性呈剂量依赖性。(a)中纵坐标为样品中病毒RNA的拷贝数，横坐标为药物浓度；(b)显示法匹拉韦在受试浓度下对受试细胞处理48h，观察不到细胞毒性。(b)中纵坐标为相对于阴性对照组(只有细胞，未加药物)的细胞活力百分比，横坐标为药物浓度。
图2.显示法匹拉韦有效保护SARS冠状病毒感染小鼠免于死亡。在小鼠攻毒后4h及接下来的时间每天给予一次的腹腔法匹拉韦注射，对照组给予同等体积的溶剂注射，可见对照组全部死亡，而法匹拉韦治疗组小鼠存活，且存活率呈剂量依赖关系。
具体实施方式
下面的实施例是本申请说明性优选实施方案，对本申请不构成任何限制。
实施例1：法匹拉韦降低SARS-CoV-2感染的细胞病毒核酸载量实验
(1)药物处理感染病毒的细胞
将Vero E6细胞(购自ATCC，货号1586)接种至24孔板，培养24h；然后进行病毒感染，具体的，用2％细胞维持液(配方为：将FBS(购自Gibco公司，货号16000044)按照2％的体积比加入MEM(购自Gibco公司，货号10370021)，即为2％细胞维持液)将SARS-CoV-2(2019-nCoV)病毒(nCoV-2019BetaCoV/Wuhan/WIV04/2019株，由中国科学院武汉病毒研究所提供)稀释成相应浓度，然后加入24孔板中使每孔含有病毒量为100TCID 50。接下来再用2％细胞维持液将法匹拉韦(购自Selleck Chemicals，货号S7975)分别稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别为100μM、33μM、11μM、3.7μM、1.23μM、0.41μM、0.14μM，然后放37℃、5％CO 2孵箱继续培养48h，细胞对照组只加不含有任何受试药物的2％细胞维持液。
(2)RNA提取
RNA提取试剂盒购自Qiagen公司，货号74106。下述RNA提取步骤中所涉及的耗材(离心柱、无RNA酶的2ml收集管等)及试剂(RLT、RW1、RPE、无RNA酶水等)均为试剂盒的组成部分。下述提取步骤均为试剂盒说明书所推荐的步骤。
1)取受试培养板的上清液100μL，加入无核酸酶EP管中，然后每孔加入350μL Buffer RLT，用移液枪吹吸混匀使其充分裂解后，离心取上清；
2)向1)中所得上清液加入等体积的70％乙醇，混匀；
3)将上述2)中所得混合液转入无RNA酶的离心柱中，12000rpm离心15s，弃废液；
4)加入700μL Buffer RW1，12000rpm离心15s清洗离心柱，弃废液；
5)加入500μL Buffer RPE，12000rpm离心15s清洗离心柱，弃废液；
6)加入500μL Buffer RPE，12000rpm离心2min清洗离心柱，弃废液；
7)换新的无RNA酶的2ml收集管，12000rpm离心1min，干燥离心柱，然后离心柱整体转移至步骤8)的1.5ml收集管中；
8)换上新的1.5ml收集管，放入步骤7)中干燥后的离心柱，并向离心柱中加入30μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA，加入RNA酶抑制剂(购自NEB公司，货号M0314L)，用Nano Drop(购自Thermo scientific，型号Nano Drop One)检测各RNA浓度。
(3)RNA反转录
实验采用TaKaRa公司生产的反转录试剂盒(PrimeScript TM RT reagent Kit with gDNA Eraser，货号RR047Q)进行RNA反转录，步骤如下。
①gDNA去除：收集各实验组RNA样品，分别取1μg进行反转录。首先，向各实验组RNA中加入2μl 5×gDNA Eraser Buffer，用RNase Free水补足反应体系至10μl，充分混匀，42℃水浴2min去除样品中可能存在的g DNA；
②逆转录：向①所得样品中加入适量的酶和引物Mix及反应缓冲液，用RNase Free水补足体积至20μl，37℃水浴反应15min，之后投入85℃水中5sec，既可转录得到cDNA。
(4)Real-time PCR
采用荧光定量PCR检测原病毒液每毫升所含拷贝数。
采用TB Green Premix(Takara，Cat#RR820A)混好反应体系，在StepOne Plus Real-time PCR仪(品牌：ABI)进行扩增反应和读数。计算原病毒液每毫升所含拷贝数。步骤如下：
①首先建立标准品：将质粒pMT-RBD(质粒由中国科学院武汉病毒研究所提供)稀释成5×10 8copies/μL,5×10 7copies/μL,5×10 6copies/μL,5×10 5copies/μL,5×10 4copies/μL,5×10 3copies/μL,5×10 2copies/μL。取2μL标准品或cDNA模板用于qPCR反应。
②实验过程中所用引物序列如下(均为5’-3’方向表示)：
RBD-qF:CAATGGTTTAACAGGCACAGG
RBD-qR:CTCAAGTGTCTGTGGATCACG
③反应程序如下：
预变性：95℃5分钟；
循环参数：95℃15秒，54℃15秒，72℃30秒，共40个循环。
(5)药物对细胞毒性测试
药物对细胞毒性的检测利用CCK-8试剂盒(Beoytime)测定。具体步骤如下：
①96孔板中接种1×10 4个Vero E6(ATCC)细胞，37℃培养8小时。
②将药物用DMSO稀释到合适的母液浓度，再用含2％FBS(购自Gibco公司，货号16000044)的MEM培养基(购自Gibco公司，货号10370021)稀释到与药物处理同样的浓度，弃96孔板中原培养基，取100μL含药物的MEM培养基加入到细胞中，每个浓度做三个复孔。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和空白对照(不含细胞，加DMSO和培养基)。加药完毕，细胞37℃培养48小时。
③向待测孔中加入20μL CCK-8溶液(Beoytime)，轻轻混匀，不要产生气泡，37℃继续培养2小时。在酶标仪(购自Molecular Devices公司，型号SpectraMax M5)上读取OD 450，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (空白对照))/(A (阴性对照)-A (空白对照))×100％
其中A为酶标仪读数。
(6)实验结果
病毒增殖抑制实验的结果显示，受试化合物在100μM，33μM，11.1μM以及3.7μM的浓度下，均能够有效抑制感染上清中SARS-CoV-2病毒基因组的复制(表1和图1)
表1.受试化合物(法匹拉韦)的抗病毒实验
Figure PCTCN2020097395-appb-000010
细胞毒性结果显示，在所有受试浓度下，受试化合物(法匹拉韦)的处理均未改变细胞活力，即受试化合物在所有浓度下对细胞均无毒性作用(表2和图1)。
表2.受试化合物(法匹拉韦)的细胞毒性实验
Figure PCTCN2020097395-appb-000011
实施例2：法匹拉韦保护SARS冠状病毒感染小鼠免于死亡的实验
(1)小鼠分组与标记
将3-4周龄、体重9-13g的A129小鼠(中国科学院上海巴斯德研究所提供)随机分为4组，即病毒对照组、高剂量给药组、中剂量给药组和低剂量给药组，每组10只，耳钉标记。
(2)药物配置
利用0.5％CMC-Na作为溶剂溶解法匹拉韦。先精确称量药物，加入适量0.5％CMCNa溶液，涡旋和超声交替处理15min，直至样品呈颗粒均匀的悬浊液，然后用0.5％CMC-Na进行2倍的稀释，按照200mg/kg、100mg/kg以及50mg/kg的给药剂量稀释好。4℃保存备用。
(3)小鼠攻毒、给药及数据采集
攻毒采用腹腔注射方式，每只小鼠按照1*10 6PFU的攻毒剂量进行腹腔注射SARS冠状病毒(由军事医学研究院提供)，病毒用生理盐水稀释至所需剂量。攻毒后4h、24h、48h、72h、96h、120h、144h各腹腔给药一次，给药剂量为高剂量200mg/kg、中计量100mg/kg以及低剂量50mg/kg，病毒对照组给与相同体积的0.5％CMC-Na溶剂。每日定点称重并记录，同时记录小鼠死亡情况，绘制生存曲线，如图2所示。
(4)实验结果
体内实验结果显示，小鼠的终末生存率和给药剂量呈量效依赖性关系(表3和图2)，显示受试化合物法匹拉韦的处理能够有效保护小鼠免于SARS冠状病毒感染所致的死亡。
表3 受试化合物(法匹拉韦)的体内抗病毒实验
Figure PCTCN2020097395-appb-000012

Claims (24)

  1. 式I所示的化合物，其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备用于治疗冠状病毒引起的疾病或感染(包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))的药物中的用途，
    Figure PCTCN2020097395-appb-100001
  2. 药物组合物在制备用于治疗冠状病毒引起的疾病或感染(例如呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100002
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  3. 药物组合物在制备作为冠状病毒抑制剂的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100003
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药 物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  4. 药物组合物在制备用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药盐上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100004
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  5. 式I所示的化合物，其几何异构体、其药物学上可接受的盐和/或其溶剂化物和/或其水合物在制备作为冠状病毒抑制剂的药物中的用途
    Figure PCTCN2020097395-appb-100005
  6. 式I所示的化合物，其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物在制备用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
    Figure PCTCN2020097395-appb-100006
  7. 权利要求1-6任一项所述的用途，其中所述的冠状病毒为SARS冠状病毒(SARS-CoV)或SARS-CoV-2。
  8. 权利要求1-6任一项所述的用途，其中所述的冠状病毒为SARS-CoV-2。
  9. 权利要求1或2所述的用途，其中所述冠状病毒引起的疾病为COVID-19或SARS冠状病毒(SARS-CoV)引起的非典型肺炎。
  10. 权利要求4或6的用途，其中所述哺乳动物包括牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，例如是人、猫、狗或猪。
  11. 式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100007
    其用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))。
  12. 式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100008
    其用作冠状病毒抑制剂。
  13. 式I所示的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100009
    其用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖。
  14. 药物组合物，其用于治疗冠状病毒特别是SARS冠状病毒(SARS-CoV)和SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等))，其中所述药物组合物包含所述的式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100010
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  15. 药物组合物，其用作冠状病毒抑制剂，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100011
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  16. 药物组合物，其用于抑制冠状病毒在细胞(例如哺乳动物细胞)中复制或繁殖，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020097395-appb-100012
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  17. 一种在有需要的哺乳动物中治疗和/或预防疾病的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的所述包含所述的式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，
    Figure PCTCN2020097395-appb-100013
    或所述的式I所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物 和/或其水合物，其中所述的疾病包括冠状病毒引起的疾病。
  18. 一种在有需要的哺乳动物中抑制冠状病毒复制或繁殖的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的所述包含所述的式Ⅰ所示化合物、其几何异构体或其药学上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，
    Figure PCTCN2020097395-appb-100014
    或所述的式I所示化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，其中所述的疾病包括冠状病毒引起的疾病。
  19. 权利要求11-13任一项所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求14-16任一项所述的药物组合物，或权利要求17或18所述的方法，其中所述的冠状病毒为SARS冠状病毒(SARS-CoV)或SARS-CoV-2。
  20. 权利要求11-13任一项所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求14-16任一项所述的药物组合物，或权利要求17或18所述的方法，其中所述的冠状病毒为SARS-CoV-2。
  21. 权利要求11所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求14所述的药物组合物，或权利要求17所述的方法，其中所述冠状病毒引起的疾病为SARS-CoV-2引起的疾病，即COVID-19。
  22. 权利要求11所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求14所述的药物组合物，或权利要求17所述的方法，其中所述冠状病毒引起的疾病为SARS冠状病毒(SARS-CoV)引起的非典型肺炎。
  23. 权利要求11所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求14所述的药物组合物，或权利要求17所述的方法，其中所述的冠状病毒引起的疾病包括但不限于呼吸系统疾病(例如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染、严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克、重症急性呼吸综合征(SARS)等)。
  24. 权利要求13所述的化合物、其几何异构体、其药学上可接受的盐和/或其溶剂化物和/或其水合物，权利要求16所述的药物组合物，或权利要求18所述的方法，其中所述哺乳动物包括牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、 啮齿类动物、灵长类动物，例如是人、猫、狗或猪。
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