WO2022012624A1 - 2,4,5-三取代的1,2,4-三唑酮在抗病毒中的应用 - Google Patents

2,4,5-三取代的1,2,4-三唑酮在抗病毒中的应用 Download PDF

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WO2022012624A1
WO2022012624A1 PCT/CN2021/106486 CN2021106486W WO2022012624A1 WO 2022012624 A1 WO2022012624 A1 WO 2022012624A1 CN 2021106486 W CN2021106486 W CN 2021106486W WO 2022012624 A1 WO2022012624 A1 WO 2022012624A1
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virus
cov
tautomer
sars
compound
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French (fr)
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钟武
周辛波
曹瑞源
肖典
王曼丽
樊士勇
胡志红
李松
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Academy of Military Medical Sciences AMMS of PLA
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Priority to US17/615,287 priority Critical patent/US20220193039A1/en
Priority to JP2023503228A priority patent/JP7594084B2/ja
Priority to EP21841781.4A priority patent/EP4183394A4/en
Priority to US17/552,609 priority patent/US20220105077A1/en
Publication of WO2022012624A1 publication Critical patent/WO2022012624A1/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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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 application is based on the CN application number 202010692337.9 and the filing date is July 17, 2020, and claims its priority.
  • the disclosure of the CN application is hereby incorporated into the present application as a whole.
  • the present application relates to the field of chemical medicines, in particular to the application of 2,4,5-trisubstituted 1,2,4-triazolone compounds in antiviral.
  • the compound can effectively inhibit the infection of various viruses, especially, the compound BAY2402234 has a high-efficiency inhibitory effect on coronaviruses, influenza viruses, enteroviruses, Zika virus, Bunya virus and the like.
  • Viruses are non-cellular organisms that contain only one nucleic acid (DNA or RNA) and must be parasitic in living cells and reproduce by means of replication. It can invade the body through a variety of routes and proliferate in susceptible host cells.
  • DNA or RNA nucleic acid
  • Virus infection in humans can cause varying degrees of damage to the human body.
  • diseases caused by some viruses are almost fatal, such as HIV, Ebola virus, rabies virus, etc.; other viruses such as hepatitis A, hepatitis B and other 5 kinds of hepatitis caused by hepatitis virus, Coxsackie virus, influenza virus, etc.
  • causesd by viral myocarditis, etc. people will lose the labor force or even be disabled for life.
  • other viral diseases also pose serious threats to human health, such as dengue and Zika viruses in the Flaviviridae family, bunyaviruses in the Buniaviridae family, and enteroviruses in the Picornaviridae family.
  • coronavirus is an enveloped non-segmented single-stranded positive-stranded RNA virus with a wide range of animal hosts. SARS-CoV and MERS-CoV derived from zoonotic diseases can cause death in humans.
  • the 2,4,5-trisubstituted 1,2,4-triazolone compound (the compound of formula I below) is a new type of antitumor drug developed by Bayer Company in Germany. It exerts antitumor effect by inhibiting tumor cell proliferation and inducing differentiation. It has great potential for clinical treatment of bone marrow malignant tumors.
  • BAY 2402234 showed good tumor-inhibiting activity in vivo and in vitro, and is currently undergoing a phase I clinical trial study for myeloid tumors.
  • a compound of formula I or an N-oxide, tautomer, geometric isomer, solvate, hydrate, pharmaceutically acceptable salt, or such tautomer
  • the use of the salt of the isomer or N-oxide in the preparation of a medicament, wherein the medicament is used for the prevention and/or treatment of a virus-related disease, and the structure of the compound of formula I is as follows:
  • R 1 represents a group selected from:
  • Monocyclic heteroaryl selected from oxazol-2-yl, pyrazol-3-yl, pyrazol-5-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidine- 2-yl, pyrimidin-4-yl, quinolin-5-yl, indazol-5-yl, and which are optionally substituted once or twice, each substituent independently selected from methyl and methoxy,
  • R 2 represents a hydrogen atom or a fluorine or chlorine atom
  • R 3 represents a group selected from the group consisting of:
  • propyl 2-methylpropyl, 3-pentyl, cyclopropylmethyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclohexyl, difluoromethyl, trifluoromethyl yl, 1,1-difluoroethyl, prop-2-en-1-yl, 2-methyl-prop-1-en-1-yl, N,N-dimethylaminoethyl and phenyl,
  • R 4 represents a group selected from the group consisting of:
  • R 5 represents a chlorine atom or a group selected from:
  • the compound is a compound of formula II,
  • the virus is an RNA virus.
  • the virus is a Coronaviridae, Orthomyxoviridae, Flaviviridae, Bunyaviridae, Picornaviridae, Arenavirus, Filoviridae, or Western Equine encephalitis virus.
  • the virus is a Coronaviridae virus.
  • the Coronaviridae virus is selected from the group consisting of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.
  • the virus is SARS-CoV-2.
  • the virus is an Orthomyxoviridae virus.
  • the virus is an influenza virus.
  • the Orthomyxoviridae virus is an influenza virus.
  • influenza virus is an influenza A virus (eg, an H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, or H10N8 subtype), an influenza B virus, or an influenza C virus.
  • influenza A virus eg, an H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, or H10N8 subtype
  • influenza B virus eg, an influenza B virus, or an influenza C virus.
  • the virus is a Flaviviridae virus.
  • the Flaviviridae virus is selected from the group consisting of Zika virus, Dengue virus, West Nile virus, Yellow fever virus, and HCV virus.
  • the virus is a Bunyaviridae virus.
  • the Bunyaviridae virus is a Bunya virus or a sand fly virus.
  • the virus is a Picornaviridae virus.
  • the picornavirus is an enterovirus or a foot-and-mouth disease virus.
  • the virus is a Filoviridae virus.
  • the Filoviridae virus is selected from the group consisting of Ebola virus, Marburg virus, and Kuva virus.
  • the medicament is for the prevention and/or treatment of SARS-CoV, MERS-CoV, SARS-CoV-2, influenza virus, Zika virus, dengue virus, bunya virus or enterovirus disease caused.
  • the medicament is used to prevent and/or treat diseases caused by SARS-CoV, MERS-CoV or SARS-CoV-2, such as SARS, MERS or COVID-19.
  • BAY 2402234 has high antiviral activity against coronaviruses, especially SARS-CoV-2, and can be used for related diseases caused by its infection, such as simple infections such as fever, cough and sore throat, pneumonia, acute or severe acute respiratory infections, low Treatment of oxygen respiratory failure and acute respiratory distress syndrome, sepsis and septic shock.
  • the inventor found through creative research that BAY 2402234 has the function of inhibiting the replication of SARS-CoV-2, and has a good therapeutic effect in the treatment of diseases caused by SARS-CoV-2. Therefore, in some particularly preferred embodiments, the medicament is used to prevent and/or treat diseases caused by SARS-CoV-2, such as COVID-19.
  • the medicament is for human use.
  • the medicament is for veterinary use.
  • the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the compound acts as the only pharmaceutically active ingredient.
  • the compounds may be used in combination with other pharmaceutically active ingredients.
  • the compound is in the same dosage unit as the other pharmaceutically active ingredient.
  • the compound and the other pharmaceutically active ingredients are in separate formulation units.
  • the compound is administered concurrently, separately or sequentially with the other pharmaceutically active ingredient.
  • the other pharmaceutical active ingredient is an antiviral drug, eg, amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir Wei, famciclovir, foscarnet, lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil, efavirenz, nevirapine, saquinavir, oseltamivir, zaquinavir Namivir, ribavirin or interferon, etc.
  • an antiviral drug eg, amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir Wei, famciclovir, foscarnet, lamivudine, zidovudine, emtricita
  • the drug is a solid or liquid formulation. In some embodiments, the medicament is a tablet, injection or spray. In some embodiments, the drug is a tablet or injection.
  • this document also relates to a compound as defined in the first aspect, or an N-oxide, tautomer, geometric isomer, solvate, hydrate, pharmaceutically acceptable salt, Or the use of the tautomer or salt of the N-oxide in the preparation of a medicament for inhibiting the replication or reproduction of a virus in a cell (eg mammalian cell).
  • a cell eg mammalian cell
  • the mammal is a human, dog or pig.
  • the medicament is for human or veterinary use.
  • the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the compound is used as the sole pharmaceutically active ingredient or in combination with other pharmaceutically active ingredients (eg, the drug is a combination formulation).
  • the other pharmaceutical active ingredient is an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz, Nevirapine, Saquinavir, Oseltamivir, Zana One or more of mivir, ribavirin, and interferon.
  • an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz,
  • the drug is a solid or liquid formulation.
  • the medicament is a tablet, injection or spray; preferably a tablet or injection.
  • this document also relates to a compound as defined in the first aspect, or an N-oxide, tautomer, geometric isomer, solvate, hydrate, pharmaceutically acceptable salt, Or the use of the tautomer or N-oxide salt in the preparation of a virus inhibitor.
  • the inhibitor is used to inhibit virus replication or reproduction in cells (eg, mammalian cells).
  • the host is a mammal.
  • the mammal is a human, dog or pig.
  • this paper also relates to a method of preventing and/or treating a virus-related disease, comprising administering to a subject in need thereof an effective amount of a compound as defined in the first aspect, or an N- The steps of oxides, tautomers, geometric isomers, solvates, hydrates, pharmaceutically acceptable salts, or salts of said tautomers or N-oxides.
  • the subject is a mammal, such as a human.
  • the salts of N-oxides are administered alone, or in combination with other pharmaceutically active ingredients, eg simultaneously, separately or sequentially.
  • the other pharmaceutical active ingredient is an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz, Nevirapine, Saquinavir, Oseltamivir, Zana One or more of mivir, ribavirin, and interferon.
  • an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz,
  • this paper also relates to a method of inhibiting the replication or propagation of a virus in a cell (eg a mammalian cell), comprising administering to a subject or cell an effective amount of a compound as defined in the first aspect, or The steps of its N-oxide, tautomer, geometric isomer, solvate, hydrate, pharmaceutically acceptable salt, or salt of said tautomer or N-oxide.
  • a cell eg a mammalian cell
  • the subject is a mammal, such as a human.
  • the mammalian cell is a human-derived cell.
  • the salts of N-oxides are administered alone, or in combination with other pharmaceutically active ingredients, eg simultaneously, separately or sequentially.
  • the other pharmaceutical active ingredient is an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz, Nevirapine, Saquinavir, Oseltamivir, Zana One or more of mivir, ribavirin, and interferon.
  • an antiviral drug such as amantadine, rimantadine, enfuviride, maraviroc, acyclovir, ganciclovir, valacyclovir , Famciclovir, Foscarnet, Lamivudine, Zidovudine, Emtricitabine, Tenofovir, Adefovir dipivoxil, Efavirenz,
  • viruses and diseases of any of the second to fifth aspects herein are as defined in the first aspect.
  • the virus is a Coronaviridae virus, such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or SARS-CoV-2, especially SARS- CoV-2.
  • a Coronaviridae virus such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or SARS-CoV-2, especially SARS- CoV-2.
  • the disease is a disease caused by SARS-CoV-2, such as simple infection (eg, fever, cough and/or sore throat), pneumonia, acute or severe acute respiratory infection, hypoxic respiratory failure, Acute respiratory distress syndrome, sepsis or septic shock, especially COVID-19.
  • simple infection eg, fever, cough and/or sore throat
  • pneumonia acute or severe acute respiratory infection
  • hypoxic respiratory failure e.g., hypoxic respiratory failure
  • Acute respiratory distress syndrome e.g., sepsis or septic shock, especially COVID-19.
  • the term "pharmaceutically acceptable salts” includes inorganic or organic acid salts of compounds of formula I, as well as inorganic or organic base salts such as sodium, potassium, calcium, lithium, meglumine, Hydrochloride, Hydrogenate, Hydrogenate, Nitrate, Sulfate, Hydrogensulfate, Phosphate, Hydrogenphosphate, Acetate, Propionate, Butyrate, Oxalate, Trimethyl Acetate, Adipate, Alginate, Lactate, Citrate, Tartrate, Succinate, Maleate, Fumarate, Picrate, Aspartate, Glucose acid salt, benzoate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or pamoate, etc.
  • inorganic or organic base salts such as sodium, potassium, calcium, lithium, meglumine, Hydrochloride, Hydrogenate, Hydrogenate, Nitrate, Sulfate, Hydrogens
  • geometric isomer refers to the existence of different spatial arrangements in molecules with double bonds or cyclic structures due to the hindered free rotation of atoms or groups of atoms connected to the double bonds or rings in the molecule.
  • the resulting stereoisomers such as cis/trans isomers.
  • the compounds of formula I described herein may exist in the form of solvates, preferably hydrates, comprising polar solvents, in particular water, methanol or ethanol, as structural elements of the crystal lattice of the compounds.
  • polar solvents in particular water, methanol or ethanol
  • the amount of polar solvent, especially water may be present in stoichiometric or non-stoichiometric ratios. It is to be understood that any solvate of a compound of formula I for use in the treatment of a disease or infection described herein, although likely to provide different properties (including pharmacokinetic properties), once absorbed into a subject, Compounds of formula I will be obtained such that the use of compounds of formula I encompass the use of any solvates of compounds of formula I, respectively.
  • Nitrogen-containing heterocycles are capable of forming N-oxides since nitrogen requires available lone pairs of electrons to oxidize to oxides; Nitrogen-containing heterocycles. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides.
  • N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include the use of peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl Hydrogen peroxides such as t-butyl hydroperoxide, sodium perborate and dioxiranes such as dimethyldioxirane are used to oxidize heterocycles and tertiary amines.
  • peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA)
  • hydrogen peroxide alkyl Hydrogen peroxides such as t-butyl hydroperoxide
  • sodium perborate and dioxiranes such as dimethyldioxirane
  • the compounds of formula I may exist as mixtures of two or more structurally distinct forms in rapid equilibrium (often referred to as tautomers).
  • Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers Wait. It is to be understood that the scope of this application covers all such in any ratio (eg 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %) of isomers or mixtures thereof.
  • the term "therapeutically effective amount” or “prophylactically effective amount” refers to an amount sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment.
  • a therapeutically effective amount of a compound will depend on the particular compound selected (eg, taking into account the potency, effectiveness and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, the severity of the patient being treated. Factors such as age, size, weight and physical ailment, medical history of the patient being treated, duration of treatment, nature of concurrent therapy, desired therapeutic effect, and the like vary, but can still be routinely determined by those skilled in the art.
  • the specific dosage and use method of the compound represented by the formula I, or its geometric isomer, pharmaceutically acceptable salt, solvate or hydrate for different patients depends on many factors, including the age of the patient. , weight, gender, natural health status, nutritional status, active strength of the drug, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. It is preferred here to use doses between 0.001 and 1000 mg/kg body weight/day.
  • Figure 1 shows the effect of BAY 2402234 on viral nucleic acid load on vero E6 cells infected with SARS-CoV-2; in which (a) shows that BAY 2402234 can inhibit the viral RNA load on cells 24h after cell infection with SARS-CoV-2 , and the inhibitory activity was dose-dependent.
  • the left ordinate is the percentage inhibition rate calculated based on the copy number of viral RNA in the sample (corresponding to the red dot and its fitting line in the figure), and the right ordinate is the percentage toxicity calculated based on cell viability (corresponding to the figure).
  • (b) shows that BAY 2402234 can inhibit the viral RNA load on cells 48h after cell infection with SARS-CoV-2, and the inhibitory activity is dose-dependent sex.
  • the left ordinate is the percentage inhibition rate calculated based on the copy number of viral RNA in the sample (corresponding to the red dot and its fitting line in the figure), and the right ordinate is the percentage toxicity calculated based on cell viability (corresponding to the figure).
  • the blue squares and their fitted lines in ), and the abscissa is the drug concentration.
  • Figure 2 shows the evaluation of BAY 2402234 cytotoxicity and in vitro anti-influenza virus activity.
  • Example 1 BAY 2402234 reduces the viral nucleic acid load of SARS-CoV-2 infected cells
  • Vero E6 cells purchased from ATCC, Cat. No. 1586 were seeded into 24-well plates, cultured for 24 h and then virus-infected. Specifically, use 2% cell maintenance solution (recipe: FBS (purchased from Gibco, Item No. 16000044) is added to MEM (purchased from Gibco, Item No.
  • RNA extraction kit was purchased from Qiagen, Cat. No. 74106.
  • the consumables spin column, 2ml collection tube without RNase, etc.
  • reagents RLT, RW1, RPE, RNase-free water, etc.
  • RNA concentration was detected by Nano Drop (purchased from Thermo scientific, model Nano Drop One).
  • reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, product number RR047Q) produced by TaKaRa company was used for RNA reverse transcription, and the steps were as follows.
  • 1gDNA removal Collect RNA samples from each experimental group, and take 1 ⁇ g of them for reverse transcription. First, add 2 ⁇ l of 5 ⁇ gDNA Eraser Buffer to the RNA of each experimental group, make up the reaction system to 10 ⁇ l with RNase Free water, mix well, and remove the gDNA that may exist in the sample in a water bath at 42°C for 2 minutes;
  • Fluorescence quantitative PCR was used to detect the copy number per milliliter of the original virus solution.
  • the reaction system was mixed with TB Green Premix (Takara, Cat#RR820A), and the amplification reaction and reading were carried out on the StepOne Plus Real-time PCR machine (brand: ABI). Calculate the number of copies per milliliter of the original virus solution. Proceed as follows:
  • RBD-qF CAATGGTTTAACAGGCACAGG
  • RBD-qR CTCAAGTGTCTGTGGATCACG
  • Cycling parameters 95°C for 15 seconds, 54°C for 15 seconds, and 72°C for 30 seconds, a total of 40 cycles.
  • Inhibition rate (%) (RNA copy number in drug treatment group)/(RNA copy number in cell infection group) ⁇ 100%
  • Cell viability (%) (A (drug treatment group) -A (blank control) )/(A (negative control) -A (blank control) ) ⁇ 100%
  • the results of the virus proliferation inhibition experiment showed that the test compounds at the concentrations of 10 ⁇ M, 3.3 ⁇ M, 1.1 ⁇ M, 0.3 ⁇ M, 0.1 ⁇ M and 0.03 ⁇ M could effectively inhibit the replication of the SARS-CoV-2 virus genome in the infection supernatant ( figure 1)
  • the compound BAY 2402234 has a significant inhibitory effect on the SARS-CoV-2 virus isolate nCoV-2019BetaCoV/Wuhan/WIV04/2019, the EC 50 values after virus infection 24h and 48h are 0.0070 ⁇ M, 0.0027 ⁇ M, respectively, and the CC 50 value is 198.8 ⁇ M, the corresponding therapeutic indices SI were 28400 and 73629.63, respectively.
  • the cells were washed 3 times with PBS (purchased from Gibco, product number 10010049), and 100 ⁇ L of D/F-12 medium (purchased from Sigma company, product number T1426) containing 2 ⁇ g/mL TPCK trypsin (purchased from Sigma company, product number T1426) was added. Gibco, Catalog No. 11330032). Then the stock solution of BAY 2402234 (purchased from MCE Company, Cat. No.
  • HY-112645) at a concentration of 50 mM was serially diluted with the above D/F-12 medium to 0.444 ⁇ M, 0.148 ⁇ M, 0.049 ⁇ M, 0.016 ⁇ M, 0.005 ⁇ M, 1.829 nM , 0.609nM, 0.203nM, take 50 ⁇ L and add to the cell culture plate.
  • influenza virus strain A/PR/8 preserved by the Academy of Military Medical Sciences
  • A/California/07/2009 preserved by the Academy of Military Medical Sciences
  • A/Hongkong/08/1968 purchased from ATCC, item number: VR- 1679
  • A/Zhenxing1109/2010 preserved by the Academy of Military Medical Sciences
  • B/Lee/40 purchased from ATCC, Cat. No.: VR-1535
  • D/F-12 medium containing 2 ⁇ g/mL TPCK trypsin. According to the corresponding concentration, 50 ⁇ L was added to a 96-well plate so that each well contained 100 TCID 50 of virus.
  • the final drug concentration is 0.25 times of the pretreatment concentration, that is, 0.111 ⁇ M as the initial concentration, 3 times dilution, the final drug concentration is: 0.111 ⁇ M, 0.037 ⁇ M, 0.012 ⁇ M, 0.004 ⁇ M, 0.0014 ⁇ M, 0.457nM, 0.152 nM, 0.051 nM.
  • Negative controls DMSO and medium without drug added to cell wells
  • positive controls DMSO, medium and virus added to cell wells without drug addition
  • the cells were washed 3 times with PBS (purchased from Gibco, product number 10010049), and 150 ⁇ L of D/F-12 medium (purchased from Sigma company, product number T1426) containing 2ug/mL TPCK trypsin (purchased from Sigma company, product number T1426) was added. Gibco, Catalog No. 11330032). Then, the stock solution of BAY 2402234 (purchased from MCE Company, Cat. No.
  • HY-112645) at a concentration of 50 mM was serially diluted with the above D/F-12 medium to 4 ⁇ M, 1.333 ⁇ M, 0.444 ⁇ M, 0.148 ⁇ M, 0.049 ⁇ M, 0.016 ⁇ M, 0.005 ⁇ M, 1.829 nM, 0.609 nM, 0.203 nM, 50 ⁇ L was added to the cell culture plate.
  • the final drug concentration was 0.25 times of the pretreatment concentration, that is, 1 ⁇ M was used as the initial concentration and 3-fold dilution.
  • the final drug concentrations were: 1 ⁇ M, 0.333 ⁇ M, 0.111 ⁇ M, 0.037 ⁇ M, 0.012 ⁇ M, 0.004 ⁇ M, 0.0014 ⁇ M, 0.457nM, 0.152nM, 0.051nM. Note that a negative control was set (DMSO and medium were added to the wells, but no drug was added). Incubate at 37°C for 72 hours.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • Buffer and substrate of chemiluminescent cell viability detection reagent were mixed in the dark to prepare a working solution.
  • 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken for 7 min with an orbital shaker to induce cell lysis.
  • a microplate reader purchased from Molecular Devices, model SpectraMax M5
  • the plate reading program is the CellTiter-Glo preset program, and calculate the cytotoxicity:
  • Cytotoxicity (%) (A (negative control) -A (drug treatment group) )/A (negative control) ⁇ 100%
  • test compound BAY 2402234 inhibited influenza virus strains A/PR/8, A/California/07/2009, A/Hongkong/08/1968, A/Zhenxing1109/2010 and B/Lee/40.
  • EC 50 were 0.01 ⁇ 0.0003 ⁇ M, 0.03 ⁇ 0.009 ⁇ M, 0.007 ⁇ 0.003 ⁇ M, 0.04 ⁇ 0.01 ⁇ M and 0.03 ⁇ 0.02 ⁇ M (as shown in Table 1);
  • the cytotoxicity test results showed that the CC50 values of the test compounds on MDCK cells was 0.27 ⁇ 0.09 ⁇ M (as in Table 1).
  • the corresponding therapeutic indices were 27, 9, 38.57, 6.75 and 9, respectively.
  • the compound BAY 2402234 has a relatively broad-spectrum (H1N1, H1N1-oseltamivir-resistant strain, H3N2, type B) inhibitory effect on influenza virus.
  • DMSO and medium, but no drug added to cell wells were set up.
  • DMSO, medium, and virus, but no drug added to cell wells were set up.
  • Cultured at 37°C BHK cells need to be cultured for 9 days, and Vero cells need to be cultured for 7 days.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • the working solution was mixed with PBS (purchased from Gibco, Cat. No. 10010049) at a ratio of 4:6. After the cell culture plate was discarded, 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken by an orbital shaker for 5 min to induce cell lysis.
  • the final drug concentration is 0.25 times of the pretreatment concentration, that is, with 200nM as the initial concentration and 3-fold dilution, the final drug concentrations are: 200nM, 66.67nM, 22.22nM, 7.41nM, 2.47nM, 0.82nM, 0.27nM, 0.09nM. Note that a negative control was set (DMSO and medium were added to the cell wells, but no drug was added). Cultured at 37°C, BHK cells need to be cultured for 9 days, and Vero cells need to be cultured for 7 days.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • the working solution was mixed with PBS (purchased from Gibco, Cat. No. 10010049) at a ratio of 4:6. After the cell culture plate was discarded, 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken by an orbital shaker for 5 min to induce cell lysis.
  • Cytotoxicity (%) (A (negative control) -A (drug treatment group) )/A (negative control) ⁇ 100%
  • test compound BAY 2402234 has an inhibitory effect on Zika virus.
  • Huh7 cells preserved by the Military Medical Research Institute
  • the cell culture medium is DMEM medium (purchased from Gibco, Item No. 16000044) containing 10% FBS Gibco Company, Cat. No. 11995065), placed in a CO 2 incubator, and incubated at 37°C for 24 hours.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • the working solution was mixed with PBS (purchased from Gibco, Cat. No. 10010049) at a ratio of 4:6. After the cell culture plate was discarded, 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken by an orbital shaker for 5 min to induce cell lysis.
  • Huh7 cells preserved by the Military Medical Research Institute
  • the cell culture medium is DMEM medium (purchased from Gibco, Item No. 16000044) containing 10% FBS Gibco Company, Cat. No. 11995065), placed in a CO 2 incubator, and incubated at 37°C for 24 hours.
  • the final drug concentration is 0.25 times of the pretreatment concentration, that is, with 200nM as the initial concentration and 3-fold dilution, the final drug concentrations are: 200nM, 66.67nM, 22.22nM, 7.41nM, 2.47nM, 0.82nM, 0.27nM, 0.09nM. Note that a negative control was set (DMSO and medium were added to the cell wells, but no drug was added). Incubate at 37°C for 6 days.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • the working solution was mixed with PBS (purchased from Gibco, Cat. No. 10010049) at a ratio of 4:6. After the cell culture plate was discarded, 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken by an orbital shaker for 5 min to induce cell lysis.
  • Cytotoxicity (%) (A (negative control) -A (drug treatment group) )/A (negative control) ⁇ 100%
  • test compound BAY 2402234 has an inhibitory effect on bunya virus.
  • 1Vero cells purchased from ATCC, Item No.: CCL-81 were seeded in a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and the cell culture medium was DMEM containing 2% FBS (purchased from Gibco, Item No. 16000044). base (purchased from Gibco, product number 11995065), placed in a CO 2 incubator, and cultured at 37°C for 24 hours.
  • HY-112645) at a concentration of 50 mM was serially diluted with the above DMEM medium to 40 ⁇ M, 13.33 ⁇ M, 4.44 ⁇ M, 1.48 ⁇ M, 0.49 ⁇ M, 0.16 ⁇ M, 0.05 ⁇ M, 18.29 ⁇ M nM, 6.09nM, 2.03nM, 50 ⁇ L was added to the cell culture plate.
  • 50 ⁇ L of enterovirus CB3 (preserved by the Military Medical Research Institute) diluted with DMEM medium containing 2% FBS was added to the cells, so that each well contained 100 TCID 50 of virus.
  • the final drug concentration was 0.25 times the pretreatment concentration, that is, 10 ⁇ M was used as the initial concentration and 3-fold dilution was performed.
  • the final drug concentrations were: 10 ⁇ M, 3.33 ⁇ M, 1.11 ⁇ M, 0.37 ⁇ M, 0.12 ⁇ M, 0.04 ⁇ M, 13.72 nM, 4.57nM, 1.52nM, 0.51nM. Note that a negative control (DMSO and medium, but no drug added to cell wells) and a positive control (DMSO, medium, and virus, but no drug added to cell wells) were set up. Incubate at 37°C for 5 days.
  • 1Vero cells purchased from ATCC, Item No.: CCL-81 were seeded in a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and the cell culture medium was DMEM containing 2% FBS (purchased from Gibco, Item No. 16000044). base (purchased from Gibco, product number 11995065), placed in a CO 2 incubator, and cultured at 37°C for 24 hours.
  • HY-112645) at a concentration of 50 mM was serially diluted with the above DMEM medium to 40 ⁇ M, 13.33 ⁇ M, 4.44 ⁇ M, 1.48 ⁇ M, 0.49 ⁇ M, 0.16 ⁇ M, 0.05 ⁇ M, 18.29 ⁇ M nM, 6.09nM, 2.03nM, 50 ⁇ L was added to the cell culture plate.
  • the final drug concentration was 0.25 times the pretreatment concentration, that is, 10 ⁇ M was used as the initial concentration and 3-fold dilution was performed.
  • the final drug concentrations were: 10 ⁇ M, 3.33 ⁇ M, 1.11 ⁇ M, 0.37 ⁇ M, 0.12 ⁇ M, 0.04 ⁇ M, 13.72 nM, 4.57nM, 1.52nM, 0.51nM. Note that a negative control was set (DMSO and medium were added to the cell wells, but no drug was added). Incubate at 37°C for 5 days.
  • chemiluminescent cell viability detection reagent purchased from Promega, product number G7573
  • Buffer and substrate of chemiluminescent cell viability detection reagent were mixed in the dark to prepare a working solution.
  • 100 ⁇ l of detection reagent was added to each well, and the 96-well plate was shaken by an orbital shaker for 5 min to induce cell lysis.
  • the signal was stabilized in the dark for 3 minutes, use a microplate reader (purchased from Molecular Devices, model SpectraMax M5) to determine the chemiluminescence unit, and the plate reading program was the CellTiter-Glo preset program, and the cytotoxicity was calculated:
  • Cytotoxicity (%) (A (negative control) -A (drug treatment group) )/A (negative control) ⁇ 100%
  • test compound BAY 2402234 has an inhibitory effect on enteroviruses.

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Abstract

涉及2,4,5-三取代的1,2,4-三唑酮在抗病毒中的应用，具体涉及式I化合物在制备药物中的用途，其中所述药物用于预防和/或治疗与病毒有关的疾病。体外实验显示所述化合物能高效抑制多种病毒的感染，并且在一些试验中显示出较低的细胞毒性，

Description

2,4,5-三取代的1,2,4-三唑酮在抗病毒中的应用
本申请是以CN申请号为202010692337.9，申请日为2020年7月17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
本申请涉及化学药物领域，具体涉及2,4,5-三取代的1,2,4-三唑酮化合物在抗病毒中的应用。体外实验显示所述化合物能高效抑制各种病毒的感染，尤其地，化合物BAY2402234对冠状病毒科病毒、流感病毒、肠道病毒、寨卡病毒、布尼亚病毒等具有高效抑制作用。
背景技术
病毒是只含一种核酸(DNA或RNA)，须在活细胞内寄生并以复制方式增殖的非细胞型生物。其可通过多种途径侵入机体，并在易感的宿主细胞中进行增殖。
病毒感染人类可对人体产生轻重不一的损伤。例如，一些病毒引发的疾病几乎是致命的，如艾滋病毒、埃博拉病毒、狂犬病毒等等；另一些病毒如甲肝、乙肝等5种肝炎病毒引发的肝炎，柯萨奇病毒、流感病毒等引发的病毒性心肌炎等等会使人丧失劳动力甚至终生残疾。另外，其他病毒性疾病也给人类健康带来了严重的威胁，例如黄病毒科登革病毒和寨卡病毒、布尼亚病毒科的布尼亚病毒、小RNA病毒科的肠道病毒等。
其中，冠状病毒(Coronavirus)是有包膜的不分节段的单股正链RNA病毒，具有广泛的动物宿主。来源于动物传染病的SARS冠状病毒和MERS冠状病毒可引起人类的死亡。
2020年2月11日，国际病毒分类委员会(ICTV)命名了一种新型冠状病毒--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。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。研发能够有效抑制以上病毒尤其是能够抑制SARS-CoV-2的药品仍是十分紧迫和必要的。
2,4,5-三取代的1,2,4-三唑酮化合物(下述式I化合物)为德国Bayer公司研发的 一种新型抗肿瘤药物，通过抑制肿瘤细胞增殖和诱导分化来发挥抗癌作用，对骨髓恶性肿瘤有较大的临床治疗潜力。其中，BAY 2402234表现出较好的体内和体外抑制肿瘤的活性，目前正在进行适应症为骨髓性肿瘤的一期临床试验研究。
Figure PCTCN2021106486-appb-000001
发明内容
发明人在研究中发现BAY 2402234能够有效抑制多种病毒，并且该化合物在一些试验中显示出较低的细胞毒性，为有效预防和/或治疗病毒引发的疾病或症状提供了新的途径和选择。
在第一个方面，本文提供式I化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐在制备药物中的用途，其中所述药物用于预防和/或治疗与病毒有关的疾病，式I化合物结构如下所示：
Figure PCTCN2021106486-appb-000002
其中:
R 1代表选自以下的基团：
3-戊基、2,2-二甲基丙基、4-庚基、4-氟苯基环丙基、环戊基、环己基、环庚基、环戊基甲基、环己基甲基、1-环己基乙基、1-羟基丙-2-基、2-羟基丙基、1-羟基丁-2-基、1-氰基丁-2-基、1-苯基丁-2-基、1-氨基-2-丙基、1-氨基-2-丁基、1-氨基-1-氧代丁-2-基、茚满-2-基、5-至6-元杂环烷基，其选自四氢呋喃-3-基、四氢-2H-吡喃-4-基和哌啶-4-基，并且其 任选被甲基取代一次或两次，
苯基，其任选被取代一次、两次或三次，每个取代基独立地选自氟原子或氯原子或选自以下的基团：甲基、乙基、丙基、异丙基、二氟甲基、三氟甲基、甲氧基、-O-C(＝O)-1,1-二甲基乙基、羟基、-C(＝O)OCH 3、-C(＝O)NH-环丙基、氨基、甲基氨基、氨基甲基、-S-CH 3、-S(＝O) 2CH 3和-S(＝O)(NH)CH 3，和，
单环杂芳基，其选自噁唑-2-基、吡唑-3-基、吡唑-5-基、吡啶-2-基、吡啶-3-基、吡啶-4-基、嘧啶-2-基、嘧啶-4-基、喹啉-5-基、吲唑-5-基，并且其任选被取代一次或两次，每个取代基独立地选自甲基和甲氧基，
R 2代表氢原子或氟或氯原子，
R 3代表选自以下的基团：
丙基、2-甲基丙基、3-戊基、环丙基甲基、环丙基、环丙基甲基，环丁基、环戊基、环己基、二氟甲基、三氟甲基、1,1-二氟乙基、丙-2-烯-1-基、2-甲基-丙-1-烯-1-基，N,N-二甲基氨基乙基和苯基，
R 4代表选自以下的基团：
甲基、乙基、丙基、异丙基、2-丁基、丙-2-烯-1-基、环丙基甲基、苄基、环丙基、环丁基、环戊基和2-羟基乙基，
R 5代表氯原子或选自以下的基团：
甲基、乙基、丙基、异丙基、2-丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、三氟甲基、羟基甲基、1-羟基乙基、2-羟基丙-2-基、1-氯乙基、1-羟基-2,2,2-三氟乙基、1-甲氧基乙基、甲氧基、异丙氧基、甲基硫基、氨基甲基、(甲基氨基)甲基、(二甲基氨基)甲基、1-氨基乙基、2-氨基乙基、甲基氨基和乙基(甲基)氨基、-C(＝O)OH、-C(＝O)OCH 3、-C(＝O)NH 2、-C(＝O)NHCH 3、-C(＝O)NH环丙基、-C(＝O)N(CH 3) 2和-S(＝O)(＝NH)CH 3
在一些实施方案中，所述化合物为式II化合物，
Figure PCTCN2021106486-appb-000003
在一些实施方案中，所述病毒为RNA病毒。
在一些实施方案中，所述病毒为冠状病毒科病毒、正粘病毒科病毒、黄病毒科病毒、布尼亚病毒科病毒、小RNA病毒科病毒、沙粒病毒、丝状病毒科病毒或西方马脑炎病毒。
在一些实施方案中，所述病毒为冠状病毒科病毒。
在一些实施方案中，所述冠状病毒科病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2。
在一些实施方案中，所述病毒为SARS-CoV-2。
在一些实施方案中，所述病毒为正粘病毒科病毒。
在一些实施方案中，所述病毒为流感病毒。
在一些实施方案中，所述正粘病毒科病毒为流感病毒。
在一些实施方案中，所述流感病毒为甲型流感病毒(例如H1N1、H5N1、H7N1、H7N2、H7N3、H7N7、H7N9、H9N2或H10N8亚型)、乙型流感病毒或丙型流感病毒。
在一些实施方案中，所述病毒为黄病毒科病毒。
在一些实施方案中，所述黄病毒科病毒选自寨卡病毒、登革病毒、西尼罗病毒、黄热病病毒和HCV病毒。
在一些实施方案中，所述病毒为布尼亚病毒科病毒。
在一些实施方案中，所述布尼亚病毒科病毒为布尼亚病毒或白蛉病毒。
在一些实施方案中，所述病毒为小RNA病毒科病毒。
在一些实施方案中，所述小RNA病毒为肠道病毒或口蹄疫病毒。
在一些实施方案中，所述病毒为丝状病毒科病毒。
在一些实施方案中，所述丝状病毒科病毒选自埃博拉病毒、马尔堡病毒和库瓦病毒。
在一些实施方案中，所述药物用于预防和/或治疗由SARS-CoV、MERS-CoV、SARS-CoV-2、流感病毒、寨卡病毒、登革病毒、布尼亚病毒或肠道病毒引起的疾病。在一些优选的实施方案中，所述药物用于预防和/或治疗由SARS-CoV、MERS-CoV或SARS-CoV-2引起的疾病，例如SARS、MERS或COVID-19。
BAY 2402234对冠状病毒特别是SARS-CoV-2有很高的抗病毒活性，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。发明人通过创造性的研究发现BAY 2402234具有抑制SARS-CoV-2复制方面的功能，在治疗SARS-CoV-2引起的疾病方面具有很好的治疗效果。因此，在一些特别优选的实施方案中，所述药物用于预防和/或治疗由SARS-CoV-2引起的疾病，例如COVID-19。
在一些实施方案中，所述药物为人用。
在一些实施方案中，所述药物为兽用。
在一些实施方案中，所述药物中还包含药学上可接受的载体或辅料。
在一些实施方案中，所述化合物作为唯一的药物活性成分。
在一些实施方案中，所述化合物可与其它药物活性成分联用。在一些实施方案中，所述化合物与其它药物活性成分在同一制剂单元中。在一些实施方案中，所述化合物与其它药物活性成分在不同的制剂单元中。在一些实施方案中，所述化合物与其它药物活性成分同时、分别或依次给药。在一些实施方案中，所述其它药物活性成分为抗病毒药物，例如，金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、奥司他韦、扎那米韦、利巴韦林或干扰素等。
在一些实施方案中，所述药物为固体制剂或液体制剂。在一些实施方案中，所述药物为片剂、注射剂或喷剂。在一些实施方案中，所述药物为片剂或注射剂。
在第二个方面，本文还涉及如第一方面所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐在制备药物中的用途，所述药物用于抑制病毒在细胞(例如哺乳动物细胞)中复制或繁殖。
在一些实施方案中，所述哺乳动物是人，狗或猪。
在一些实施方案中，所述药物为人用或兽用。
在一些实施方案中，所述药物中还包含药学上可接受的载体或辅料。
在一些实施方案中，所述化合物作为唯一的药物活性成分或与其它药物活性成分联用(例如，所述药物为复方制剂)。
在一些实施方案中，所述其它药物活性成分为抗病毒药物，例如金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、 奥司他韦、扎那米韦、利巴韦林和干扰素中的一种或多种。
在一些实施方案中，所述药物为固体制剂或液体制剂。
在一些实施方案中，所述药物为片剂、注射剂或喷剂；优选为片剂或注射剂。
在第三个方面，本文还涉及如第一方面所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐在制备病毒抑制剂中的用途。
在一些实施方案中，所述抑制剂用于抑制病毒在细胞(例如哺乳动物细胞)中复制或繁殖。
在一些实施方案中，所述宿主为哺乳动物。
在一些实施方案中，所述哺乳动物是人，狗或猪。
在第四个方面，本文还涉及一种预防和/或治疗与病毒有关的疾病的方法，其包括向有此需要的受试者施用有效量的第一方面所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐的步骤。
在一些实施方案中，所述受试者为哺乳动物，例如人。
在一些实施方案中，所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐单独施用，或与其它药物活性成分联用，例如同时、分别或依次给药。
在一些实施方案中，所述其它药物活性成分为抗病毒药物，例如金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、奥司他韦、扎那米韦、利巴韦林和干扰素中的一种或多种。
在第五个方面，本文还涉及一种抑制病毒在细胞(例如哺乳动物细胞)中复制或繁殖的方法，其包括向受试者或细胞施用有效量的第一方面中所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐的步骤。
在一些实施方案中，所述受试者为哺乳动物，例如人。
在一些实施方案中，所述哺乳动物细胞为来自人的细胞。
在一些实施方案中，所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐单独施用，或与其它药物活性成分联用，例如同时、分别或依次给药。
在一些实施方案中，所述其它药物活性成分为抗病毒药物，例如金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、奥司他韦、扎那米韦、利巴韦林和干扰素中的一种或多种。
本文第二方面至第五任一方面所述病毒以及疾病如第一方面所定义。
在一些实施方案中，所述病毒为冠状病毒科病毒，例如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或SARS-CoV-2，特别是SARS-CoV-2。
在一些实施方案中，所述疾病为SARS-CoV-2引起的疾病，例如单纯性感染(如发热、咳嗽和/或咽痛)、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症或脓毒性休克，特别是COVID-19。
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
在本文中，所用术语“药学上可接受的盐”包括式I化合物的无机或有机酸盐，以及无机或有机碱盐，例如钠盐、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，对甲苯磺酸盐或双羟萘酸盐等。
在本文中，术语“几何异构体”是指在有双键或环状结构的分子中，由于分子中与双键或环相连接的原子或原子团的自由旋转受阻碍，存在不同的空间排列而产生的立体异构体，例如顺式/反式异构体。
本文中所述式I化合物可以溶剂合物(优选水合物)的形式存在，其包含作为所述化合物晶格的结构要素的极性溶剂，特别是水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。应理解的是，在治疗本申请所述的疾病或感染中使用的式I化合物的任何溶剂合物尽管可能提供不同的性质(包括药代动力学性质)，但是一旦吸收至受试者中，会得到式I化合物，使得式I化合物的使用分别涵盖式I化合物的任何溶剂合物的使用。
本领域技术人员应当理解，由于氮需要可用的孤对电子来氧化成氧化物，因此并非所有的含氮杂环都能够形成N-氧化物；本领域技术人员会识别能够形成N-氧化物的含氮杂环。本领域技术人员还会认识到叔胺能够形成N-氧化物。用于制备杂环和叔胺的N-氧化物的合成方法是本领域技术人员熟知的，包括用过氧酸如过氧乙酸和间氯过氧苯甲酸(MCPBA)、过氧化氢、烷基过氧化氢如叔丁基过氧化氢、过硼酸钠和双环氧乙烷(dioxirane)如二甲基双环氧乙烷来氧化杂环和叔胺。这些用于制备N-氧化物的方法已在文献中得到广泛描述和综述，参见例如：T.L.Gilchrist,Comprehensive Organic Synthesis,vol.7,pp748-750；A.R.Katritzky和A.J.Boulton,Eds.,Academic Press；以及G.W.H.Cheeseman和E.S.G.Werstiuk，Advances in Heterocyclic Chemistry,vol.22,pp 390-392,A.R.Katritzky和A.J.Boulton,Eds.,Academic Press。
所述式I化合物可以两种或更多种处于快速平衡的结构不同的形式的混合物(通常称作互变异构体)存在。互变异构体的代表性实例包括酮-烯醇互变异构体、苯酚-酮互变异构体、亚硝基-肟互变异构体、亚胺-烯胺互变异构体等。要理解，本申请的范围涵盖所有这样的以任意比例(例如60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的异构体或其混合物。
在本文中，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、或其几何异构体、药学上可接受的盐、溶剂化物或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。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本文示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1显示BAY 2402234对SARS-CoV-2感染的vero E6细胞上病毒核酸载量的影响；其中(a)显示BAY 2402234在细胞感染SARS-CoV-2 24h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。左侧纵坐标为依据样品中病毒RNA的拷贝数计算出的百分比抑制率(对应图中的红色圆点及其拟合线)，右侧纵坐标为依据细胞活力计算出的百分比毒性(对应图中的蓝色方块及其拟合线)，横坐标为药物浓度；(b)显示BAY 2402234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。左侧纵坐标为依据样品中病毒RNA的拷贝数计算出的百分比抑制率(对应图中的红色圆点及其拟合线)，右侧纵坐标为依据细胞活力计算出的百分比毒性(对应图中的蓝色方块及其拟合线)，横坐标为药物浓度。
图2显示BAY 2402234细胞毒性及体外抗流感病毒活性评价。
具体实施方式
下面将结合实施例中的附图，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 BAY 2402234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保存)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BAY 2402234(购自MCE公司，货号HY-112645)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5秒，即可转录得到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保存)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。
抑制率(％)＝(药物处理组RNA拷贝数)/(细胞感染组RNA拷贝数)×100％
(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10μM，3.3μM，1.1μM，0.3μM，0.1μM 以及0.03μM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(图1)
细胞毒性结果显示，受试化合物BAY 2402234在10μM以下的浓度未改变细胞活力，即受试化合物在所有浓度下对细胞均无毒性作用(图1)。
(7)结论
化合物BAY 2402234对SARS-CoV-2病毒分离株nCoV-2019BetaCoV/Wuhan/WIV04/2019具有显著的抑制作用，在病毒感染24h、48h后的EC 50值分别为0.0070μM，0.0027μM，CC 50值为198.8μM，相应的治疗指数SI分别为28400和73629.63。
实施例2 BAY 2402234体外抗流感病毒活性评价以及安全性评估
(1)药物抗流感病毒活性评价
①将MDCK细胞(购自ATCC，货号：CCL-34)按照1.5×10 4细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②实验前，用PBS(购自Gibco公司，货号10010049)将细胞洗涤3遍，加入100μL含2μg/mL TPCK胰酶(购自Sigma公司，货号T1426)的D/F-12培养基(购自Gibco公司，货号11330032)。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述D/F-12培养基系列稀释至0.444μM，0.148μM，0.049μM，0.016μM，0.005μM，1.829nM，0.609nM，0.203nM，取50μL加入到细胞培养板中。最后，将流感病毒株A/PR/8(军事医学研究院保存)，A/California/07/2009(军事医学研究院保存)，A/Hongkong/08/1968(购自ATCC，货号：VR-1679)，A/Zhenxing1109/2010(军事医学研究院保存)，B/Lee/40(购自ATCC，货号：VR-1535)用含2μg/mL TPCK胰酶的D/F-12培养基稀释成相应浓度，取50μL加入96孔板中使每孔含有病毒量为100TCID 50。最终药物终浓度为预处理浓度的0.25倍，即以0.111μM为初始浓度，3倍倍比稀释，药物终浓度为：0.111μM，0.037μM，0.012μM，0.004μM，0.0014μM，0.457nM，0.152nM，0.051nM。设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和阳性对照(细胞孔中加DMSO、培养基和病毒，而不加药物)。37℃培养72小时。
③将
Figure PCTCN2021106486-appb-000004
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS按4:6比例混合。细胞培养 板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡7min，以诱导细胞裂解。避光稳定信号5min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (阳性对照))/(A (阴性对照)-A (阳性对照))×100％
其中A为酶标仪读数。
(2)药物对细胞毒性测试
①将MDCK细胞(购自ATCC，货号：CCL-34)按照1.5×10 4细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②实验前，用PBS(购自Gibco公司，货号10010049)将细胞洗涤3遍，加入150μL含2ug/mL TPCK胰酶(购自Sigma公司，货号T1426)的D/F-12培养基(购自Gibco公司，货号11330032)。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述D/F-12培养基系列稀释至4μM，1.333μM，0.444μM，0.148μM，0.049μM，0.016μM，0.005μM，1.829nM，0.609nM，0.203nM，取50μL加入到细胞培养板中。最终药物终浓度为预处理浓度的0.25倍，即以1μM为初始浓度，3倍倍比稀释，药物终浓度为：1μM，0.333μM，0.111μM，0.037μM，0.012μM，0.004μM，0.0014μM，0.457nM，0.152nM，0.051nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)。37℃培养72小时。
③将
Figure PCTCN2021106486-appb-000005
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡7min，以诱导细胞裂解。避光稳定信号5min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞毒性：
细胞毒性(％)＝(A (阴性对照)-A (药物处理组))/A (阴性对照)×100％
其中A为酶标仪读数。
(3)实验结果
体外抗病毒实验结果显示，受试化合物BAY 2402234抑制流感病毒株A/PR/8，A/California/07/2009，A/Hongkong/08/1968，A/Zhenxing1109/2010和B/Lee/40的EC 50分别为0.01±0.0003μM，0.03±0.009μM，0.007±0.003μM，0.04±0.01μM和0.03±0.02μM(如表1)；细胞毒性实验结果显示，受试化合物在MDCK细胞上的CC50值为0.27±0.09μM(如 表1)。相应的治疗指数分别为27,9,38.57,6.75和9。
(4)结论
化合物BAY 2402234对流感病毒具有较为广谱(H1N1，H1N1-奥司他韦耐药株，H3N2，B型)的抑制效果。
表1 BAY 2402234抗流感病毒的半数最大有效浓度(EC 50)及安全性(CC 50)
Figure PCTCN2021106486-appb-000006
实施例3 BAY 2402234体外抗寨卡病毒活性评价以及安全性评估
(1)药物对寨卡病毒的活性评价
①将BHK细胞(军事医学研究院保存)按照5×10 3细胞/孔的浓度或Vero细胞(军事医学研究院保存)按照1×10 4细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②弃96孔板中原培养基，取100μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)加入到细胞中。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至800nM，266.67nM，88.89nM，29.63nM，9.88nM，3.29nM，1.10nM，0.37nM，取50μL加入到细胞培养板中。最后细胞中加入50μL用含2％FBS的DMEM培养基稀释好的寨卡病毒SZ-SMGC-01株(军事医学研究院保存)，使每孔含有病毒量为100TCID 50。最终药物终浓度为预处理浓度的0.25倍，即以200nM为初始浓度，3倍倍比稀释，药物终浓度为：200nM，66.67nM，22.22nM，7.41nM，2.47nM，0.82nM，0.27nM，0.09nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和阳性对照(细胞孔中加DMSO、培养基和病毒，而不加药物)。37℃培养，BHK细胞需培养9天，Vero细胞需培养7天。
③将
Figure PCTCN2021106486-appb-000007
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS(购自Gibco公司，货号10010049)按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振 荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号2min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (阳性对照))/(A (阴性对照)-A (阳性对照))×100％
其中A为酶标仪读数。
(2)药物对细胞毒性测试
①将BHK细胞(军事医学研究院保存)按照5×10 3细胞/孔的浓度或Vero细胞(军事医学研究院保存)按照1×10 4细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②弃96孔板中原培养基，取150μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)加入到细胞中。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至800nM，266.67nM，88.89nM，29.63nM，9.88nM，3.29nM，1.10nM，0.37nM，取50μL加入到细胞培养板中。最终药物终浓度为预处理浓度的0.25倍，即以200nM为初始浓度，3倍倍比稀释，药物终浓度为：200nM，66.67nM，22.22nM，7.41nM，2.47nM，0.82nM，0.27nM，0.09nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)。37℃培养，BHK细胞需培养9天，Vero细胞需培养7天。
③将
Figure PCTCN2021106486-appb-000008
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS(购自Gibco公司，货号10010049)按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号2min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞毒性：
细胞毒性(％)＝(A (阴性对照)-A (药物处理组))/A (阴性对照)×100％
其中A为酶标仪读数。
(3)实验结果
初步实验结果显示，受试化合物BAY 2402234对寨卡病毒具有抑制作用。
实施例4 BAY 2402234体外抗布尼亚病毒活性评价以及安全性评估
(1)药物对布尼亚病毒的活性评价
①将Huh7细胞(军事医学研究院保存)按照5×10 3细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②弃96孔板中原培养基，取100μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)加入到细胞中。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至800nM，266.67nM，88.89nM，29.63nM，9.88nM，3.29nM，1.10nM，0.37nM，取50μL加入到细胞培养板中。最后细胞中加入50μL用含2％FBS的DMEM培养基稀释好的布尼亚病毒(本实验室分离：首都医科大学北京地坛医院患者血清)，使每孔含有病毒量为100TCID 50。最终药物终浓度为预处理浓度的0.25倍，即以200nM为初始浓度，3倍倍比稀释，药物终浓度为：200nM，66.67nM，22.22nM，7.41nM，2.47nM，0.82nM，0.27nM，0.09nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和阳性对照(细胞孔中加DMSO、培养基和病毒，而不加药物)。37℃培养6天。
③将
Figure PCTCN2021106486-appb-000009
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS(购自Gibco公司，货号10010049)按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号2min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (阳性对照))/(A (阴性对照)-A (阳性对照))×100％
其中A为酶标仪读数。
(2)药物对细胞毒性测试
①将Huh7细胞(军事医学研究院保存)按照5×10 3细胞/孔的浓度接种96孔板，细胞培养基为含10％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②弃96孔板中原培养基，取150μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)加入到细胞中。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至800nM，266.67nM，88.89nM，29.63nM，9.88nM，3.29nM，1.10nM，0.37nM， 取50μL加入到细胞培养板中。最终药物终浓度为预处理浓度的0.25倍，即以200nM为初始浓度，3倍倍比稀释，药物终浓度为：200nM，66.67nM，22.22nM，7.41nM，2.47nM，0.82nM，0.27nM，0.09nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)。37℃培养6天。
③将
Figure PCTCN2021106486-appb-000010
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS(购自Gibco公司，货号10010049)按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号2min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞毒性：
细胞毒性(％)＝(A (阴性对照)-A (药物处理组))/A (阴性对照)×100％
其中A为酶标仪读数。
(3)实验结果
初步实验结果显示，受试化合物BAY 2402234对布尼亚病毒具有抑制作用。
实施例5 BAY 2402234体外抗肠道病毒活性评价以及安全性评估
(1)药物抗肠道病毒活性评价
①将Vero细胞(购自ATCC，货号：CCL-81)按照1×10 4细胞/孔的浓度接种96孔板，细胞培养基为含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②实验前，用PBS(购自Gibco公司，货号10010049)将细胞洗涤3遍，加入100μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至40μM，13.33μM，4.44μM，1.48μM，0.49μM，0.16μM，0.05μM，18.29nM，6.09nM，2.03nM，取50μL加入到细胞培养板中。最后细胞中加入50μL用含2％FBS的DMEM培养基稀释好的肠道病毒CB3(军事医学研究院保存)，使每孔含有病毒量为100TCID 50。最终药物终浓度为预处理浓度的0.25倍，即以10μM为初始浓度，3倍倍比稀释，药物终浓度为：10μM，3.33μM，1.11μM，0.37μM，0.12μM，0.04μM，13.72nM，4.57nM，1.52nM，0.51nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和阳性对照(细胞孔中加DMSO、培养基和病毒，而 不加药物)。37℃培养5天。
③将
Figure PCTCN2021106486-appb-000011
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号3min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (阳性对照))/(A (阴性对照)-A (阳性对照))×100％
其中A为酶标仪读数。
(2)药物对细胞毒性测试
①将Vero细胞(购自ATCC，货号：CCL-81)按照1×10 4细胞/孔的浓度接种96孔板，细胞培养基为含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)，置入CO 2孵箱，37℃培养24小时。
②实验前，用PBS(购自Gibco公司，货号10010049)将细胞洗涤3遍，加入150μL含2％FBS(购自Gibco公司，货号16000044)的DMEM培养基(购自Gibco公司，货号11995065)。然后将浓度为50mM的BAY 2402234(购自MCE公司，货号HY-112645)母液，用上述DMEM培养基系列稀释至40μM，13.33μM，4.44μM，1.48μM，0.49μM，0.16μM，0.05μM，18.29nM，6.09nM，2.03nM，取50μL加入到细胞培养板中。最终药物终浓度为预处理浓度的0.25倍，即以10μM为初始浓度，3倍倍比稀释，药物终浓度为：10μM，3.33μM，1.11μM，0.37μM，0.12μM，0.04μM，13.72nM，4.57nM，1.52nM，0.51nM。注意设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)。37℃培养5天。
③将
Figure PCTCN2021106486-appb-000012
化学发光细胞活力检测试剂(购自Promega公司，货号G7573)的Buffer和底物避光混合，配制为工作液。将工作液与PBS按4:6比例混合。细胞培养板弃液后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡5min，以诱导细胞裂解。避光稳定信号3min后，使用酶标仪(购自Molecular Devices公司，型号SpectraMax M5)测定化学发光单位，读板程序为CellTiter-Glo预设程序，计算细胞毒性：
细胞毒性(％)＝(A (阴性对照)-A (药物处理组))/A (阴性对照)×100％
其中A为酶标仪读数。
(3)实验结果
初步实验结果显示，受试化合物BAY 2402234对对肠道病毒具有抑制作用。
Figure PCTCN2021106486-appb-000013
除本文中描述的那些外，根据前述描述，本发明的各种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (18)

  1. 式I化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐在制备药物中的用途，其中所述药物用于预防和/或治疗与病毒有关的疾病，式I化合物结构如下所示：
    Figure PCTCN2021106486-appb-100001
    其中:
    R 1代表选自以下的基团：
    3-戊基、2,2-二甲基丙基、4-庚基、4-氟苯基环丙基、环戊基、环己基、环庚基、环戊基甲基、环己基甲基、1-环己基乙基、1-羟基丙-2-基、2-羟基丙基、1-羟基丁-2-基、1-氰基丁-2-基、1-苯基丁-2-基、1-氨基-2-丙基、1-氨基-2-丁基、1-氨基-1-氧代丁-2-基、茚满-2-基、5-至6-元杂环烷基，其选自四氢呋喃-3-基、四氢-2H-吡喃-4-基和哌啶-4-基，并且其任选被甲基取代一次或两次，
    苯基，其任选被取代一次、两次或三次，每个取代基独立地选自氟原子或氯原子或选自以下的基团：甲基、乙基、丙基、异丙基、二氟甲基、三氟甲基、甲氧基、-O-C(＝O)-1,1-二甲基乙基、羟基、-C(＝O)OCH 3、-C(＝O)NH-环丙基、氨基、甲基氨基、氨基甲基、-S-CH 3、-S(＝O) 2CH 3和-S(＝O)(NH)CH 3，和，
    单环杂芳基，其选自噁唑-2-基、吡唑-3-基、吡唑-5-基、吡啶-2-基、吡啶-3-基、吡啶-4-基、嘧啶-2-基、嘧啶-4-基、喹啉-5-基、吲唑-5-基，并且其任选被取代一次或两次，每个取代基独立地选自甲基和甲氧基，
    R 2代表氢原子或氟或氯原子，
    R 3代表选自以下的基团：
    丙基、2-甲基丙基、3-戊基、环丙基甲基、环丙基、环丙基甲基，环丁基、环戊基、环己基、二氟甲基、三氟甲基、1,1-二氟乙基、丙-2-烯-1-基、2-甲基-丙-1-烯-1-基，N,N- 二甲基氨基乙基和苯基，
    R 4代表选自以下的基团：
    甲基、乙基、丙基、异丙基、2-丁基、丙-2-烯-1-基、环丙基甲基、苄基、环丙基、环丁基、环戊基和2-羟基乙基，
    R 5代表氯原子或选自以下的基团：
    甲基、乙基、丙基、异丙基、2-丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、三氟甲基、羟基甲基、1-羟基乙基、2-羟基丙-2-基、1-氯乙基、1-羟基-2,2,2-三氟乙基、1-甲氧基乙基、甲氧基、异丙氧基、甲基硫基、氨基甲基、(甲基氨基)甲基、(二甲基氨基)甲基、1-氨基乙基、2-氨基乙基、甲基氨基和乙基(甲基)氨基、-C(＝O)OH、-C(＝O)OCH 3、-C(＝O)NH 2、-C(＝O)NHCH 3、-C(＝O)NH环丙基、-C(＝O)N(CH 3) 2和-S(＝O)(＝NH)CH 3。
  2. 权利要求1所述的用途，其中所述化合物为式II化合物，
    Figure PCTCN2021106486-appb-100002
  3. 权利要求1或2中所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐在制备药物中的用途，其中所述药物用于抑制病毒在细胞(例如哺乳动物细胞)中复制或繁殖。
  4. 权利1-3任一项所述的用途，其中所述病毒为RNA病毒。
  5. 权利要求1-4任一项所述的用途，其中所述病毒为冠状病毒科病毒、正粘病毒科病毒、黄病毒科病毒、布尼亚病毒科病毒、小RNA病毒科病毒、沙粒病毒、丝状病毒科病毒或西方马脑炎病毒；
    优选地，所述冠状病毒科病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2；
    优选地，所述正粘病毒科病毒为流感病毒，例如甲型流感病毒、乙型流感病毒或丙型流感病毒；
    优选地，所述黄病毒科病毒选自寨卡病毒、登革病毒、西尼罗病毒、黄热病病毒 和HCV病毒；
    优选地，所述布尼亚病毒科病毒为布尼亚病毒或白蛉病毒；
    优选地，所述小RNA病毒为肠道病毒或口蹄疫病毒；
    优选地，所述丝状病毒科病毒选自埃博拉病毒、马尔堡病毒和库瓦病毒。
  6. 权利1-5任一项所述的用途，其中所述药物用于预防和/或治疗由SARS-CoV、MERS-CoV、SARS-CoV-2、流感病毒、寨卡病毒、登革病毒、布尼亚病毒或肠道病毒引起的疾病；
    优选地，所述药物用于预防和/或治疗由SARS-CoV、MERS-CoV或SARS-CoV-2引起的疾病；
    优选地，所述药物用于预防和/或治疗由SARS-CoV-2引起的疾病，例如单纯性感染(如发热、咳嗽和/或咽痛)、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症或脓毒性休克；
    优选地，所述药物用于预防和/或治疗COVID-19。
  7. 权利要求1-6任一项所述的用途，其中所述药物为人用或兽用。
  8. 权利要求1-7任一项所述的用途，其中所述药物中还包含药学上可接受的载体或辅料。
  9. 权利要求1-8任一项所述的用途，其中所述化合物作为唯一的药物活性成分或与其它药物活性成分联用(例如，所述药物为复方制剂)；
    优选地，所述其它药物活性成分为抗病毒药物，例如金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、奥司他韦、扎那米韦、利巴韦林和干扰素中的一种或多种。
  10. 权利要求1-9任一项的用途，其中所述药物为固体制剂或液体制剂；
    优选地，所述药物为片剂、注射剂或喷剂；优选为片剂或注射剂。
  11. 一种预防和/或治疗与病毒有关的疾病的方法，其包括向有此需要的受试者施用有效量的权利要求1或2中所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐的步骤。
  12. 一种抑制病毒在细胞(例如哺乳动物细胞)中复制或繁殖的方法，其包括向受试者或细胞施用有效量的权利要求1或2中所定义的化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化 物的盐的步骤。
  13. 权利要求11或12所述的方法，其中所述病毒为RNA病毒。
  14. 权利要求11-13任一项所述的方法，其中所述病毒为冠状病毒科病毒、正粘病毒科病毒、黄病毒科病毒、布尼亚病毒科病毒、小RNA病毒科病毒、沙粒病毒、丝状病毒科病毒或西方马脑炎病毒；
    优选地，所述冠状病毒科病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2；
    优选地，所述正粘病毒科病毒为流感病毒，例如甲型流感病毒、乙型流感病毒或丙型流感病毒；
    优选地，所述黄病毒科病毒选自寨卡病毒、登革病毒、西尼罗病毒、黄热病病毒和HCV病毒；
    优选地，所述布尼亚病毒科病毒为布尼亚病毒或白蛉病毒；
    优选地，所述小RNA病毒为肠道病毒或口蹄疫病毒；
    优选地，所述丝状病毒科病毒选自埃博拉病毒、马尔堡病毒和库瓦病毒。
  15. 权利11-14任一项所述的方法，其中所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐用于预防和/或治疗由SARS-CoV、MERS-CoV、SARS-CoV-2、流感病毒、寨卡病毒、登革病毒、布尼亚病毒或肠道病毒引起的疾病；
    优选地，其中所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐用于预防和/或治疗由SARS-CoV、MERS-CoV或SARS-CoV-2引起的疾病；
    优选地，其中所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐用于预防和/或治疗由SARS-CoV-2引起的疾病，例如单纯性感染(如发热、咳嗽和/或咽痛)、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭、急性呼吸窘迫综合征、脓毒症或脓毒性休克；
    优选地，其中所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐用于预防和/或治疗COVID-19。
  16. 权利要求11-15任一项所述的方法，其中所述受试者为哺乳动物，例如人。
  17. 权利要求12所述的方法，其中所述哺乳动物细胞为来自人的细胞。
  18. 权利要求11-17任一项所述的方法，其中所述化合物、或其N-氧化物、互变异构体、几何异构体、溶剂合物、水合物、药学上可接受的盐、或所述互变异构体或N-氧化物的盐单独施用，或与其它药物活性成分联用，例如同时、分别或依次给药；
    优选地，所述其它药物活性成分为抗病毒药物，例如金刚烷胺、金刚乙胺、恩夫韦地、马拉韦罗、阿昔洛韦、更昔洛韦、伐昔洛韦、泛昔洛韦、膦甲酸钠、拉米夫定、齐多夫定、恩曲他滨、替诺福韦、阿德福韦酯、依法韦仑、奈韦拉平、沙奎那韦、奥司他韦、扎那米韦、利巴韦林和干扰素中的一种或多种。
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