WO2025195347A1 - Polycyclic pyridone derivative and use thereof - Google Patents

Abstract

The present invention provides a polycyclic pyridone derivative, a composition containing same, and a use thereof. The compound of the present invention has an inhibitory effect on influenza viruses, and can be used for treating cap-dependent endonuclease-mediated diseases.

Description

Polycyclic pyridone derivatives and uses thereof Technical Field

The present invention relates to the field of medical technology, and in particular to a class of polycyclic pyridone derivatives, compositions and uses thereof.

Background Art

Influenza is a highly contagious respiratory disease caused by the influenza virus. Among them, influenza A virus has the widest host range, infecting both birds and mammals, making it highly likely to cause a worldwide pandemic. Currently, vaccination is generally effective in preventing influenza infection, but the vaccine is only partially effective, with an overall efficacy of approximately 60%. Furthermore, since it typically takes about six months between antigen identification and vaccine production, the virus may mutate during this period, thereby reducing the effectiveness of the vaccine. Regarding anti-influenza drugs, there are two types of FDA-approved anti-influenza drugs: influenza neuraminidase protein inhibitors (oseltamivir, zanamivir, peramivir, and nanamivir) and M2 proton channel blockers, the amantadine class of drugs (amantadine and rimantadine). However, there are concerns about the emergence of drug-resistant strains, side effects, and the global prevalence of novel influenza viruses with high pathogenicity or lethality, leading to the development of anti-influenza drugs with novel mechanisms.

Cap-dependent endonucleases, enzymes derived from influenza viruses, are considered suitable targets for anti-influenza drugs because they are essential for viral proliferation and possess virus-specific enzymatic activity not possessed by the host. The influenza virus cap-dependent endonuclease is an endonuclease activity that generates a fragment containing 9 to 13 bases of the cap structure (the bases of the cap structure are not included in the above base count) with the host mRNA precursor as a substrate. This fragment functions as a primer for the viral RNA polymerase, used for the synthesis of mRNA encoding viral proteins. In other words, substances that inhibit cap-dependent endonucleases are believed to inhibit viral protein synthesis by inhibiting viral mRNA synthesis, thereby inhibiting viral proliferation. Mabaloxavir, a drug already marketed in multiple countries, is an innovative cap-dependent endonuclease inhibitor. With a single oral dose, it can halt viral shedding within about 24 hours, shorten the infectious period, and quickly relieve flu symptoms such as fever and body aches. Based on the fact that cap-dependent nuclease drugs have been proven to have strong anti-influenza virus effects, it is still necessary to develop new drugs with richer types and more outstanding effects.

Summary of the Invention

The present invention provides a novel polycyclic pyridone derivative, a pharmaceutical composition thereof and use thereof in resisting influenza virus.

In a first aspect, the present invention provides a compound as shown in Formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof:

wherein R ₁ , R ₂ , R ₃ and R ₄ are all hydrogen or all deuterium, R ₅ is absent or is an oxo group, and R ₆ is hydrogen or

In a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ and R ₄ are deuterium, R ₅ is absent or is oxo, and R ₆ is hydrogen or

In a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are hydrogen, R ₅ is oxo, and R ₆ is hydrogen or

In a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are deuterium, R ₅ is an oxo group, the configuration of the sulfur atom is S or R, and R ₆ is hydrogen or

In a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ , and R ₄ are hydrogen, R ₅ is an oxo group, the configuration of the sulfur atom is S or R, and R ₆ is hydrogen or

The compound of the present invention is selected from:

and its tautomers, stereoisomers or pharmaceutically acceptable salts.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, and optionally, a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides the use of a compound represented by Formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for treating and/or preventing diseases or conditions mediated by cap-dependent nucleases.

The present invention also provides the use of the compound represented by Formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the second aspect of the present invention in the preparation of drugs for treating and/or preventing diseases or conditions caused by viral infections.

In a fourth aspect, the present invention provides a method for treating and/or preventing diseases or conditions mediated by cap-dependent nucleases, comprising administering to a patient a therapeutically effective dose of a compound of formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the second aspect of the present invention.

The present invention also provides a method for treating and/or preventing a disease or condition caused by a viral infection, comprising administering to a patient a therapeutically effective dose of a compound shown in Formula I, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the second aspect of the present invention. Preferably, the viral infection of the present invention is an influenza virus infection. Further, the influenza virus is selected from type A, type B, type C, or avian influenza (H5N1, H7N9); further, the disease of the present invention is selected from cold-like symptoms accompanied by fever, chills, headache, muscle pain, general fatigue, etc., or respiratory inflammation with sore throat, runny nose, nasal congestion, cough, and sputum; gastrointestinal symptoms of abdominal pain, vomiting, and diarrhea, and then accompanied by complications of acute encephalopathy, secondary pneumonia infection, or a combination thereof.

The present invention also provides the use of the compound represented by Formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the second aspect of the present invention as a drug or in the preparation of a drug.

Preferably, the drug is a drug for treating and/or preventing a disease or condition mediated by a cap-dependent endonuclease. Preferably, the disease or condition is an influenza infectious disease. Preferably, the influenza infectious disease is caused by an influenza virus selected from influenza A, B, C or avian influenza (H5N1, H7N9). Preferably, the influenza infectious disease is accompanied by one or more of the following symptoms: cold-like symptoms such as fever, chills, headache, muscle pain, general fatigue, or respiratory inflammation such as sore throat, runny nose, nasal congestion, cough, and sputum; gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea. Preferably, it is further accompanied by complications of acute encephalopathy, secondary pneumonia infection, or a combination thereof.

definition

Unless otherwise specified, D in the present invention represents deuterium ( ² H).

Unless otherwise specified, the term "oxo" refers to a group in which two hydrogen atoms at the same substitution position are replaced by the same oxygen atom to form a double bond.

Unless otherwise specified, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissues, particularly human tissues, without excessive toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. For example, pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or separately by reacting the free base or free acid with a suitable reagent.

Unless otherwise specified, pharmaceutically acceptable salts of the compounds of the present invention also include "solvates" thereof. The terms "solvate" and "solvate" refer to the physical association of a salt of a compound of the present invention with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In certain cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to be separated. The solvent molecules in the solvate may exist in a regular and/or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

Unless otherwise specified, pharmaceutically acceptable salts of the compounds of the present invention also include "hydrates" thereof. The term "hydrate" refers to a substance formed by water molecules binding to cations or anions in the compound by coordinate bonds or covalent bonds, or refers to a substance formed by water ions not directly binding to cations or anions but existing in a certain proportion at a certain position in the solid crystal lattice.

Unless otherwise specified, the compounds of the present invention also include their "prodrugs," which refers to drugs that are converted into the parent drug in vivo. Prodrugs are generally useful because they can improve certain, undesirable physical or biological properties. Physical properties are often related to solubility (excessive or insufficient lipid or water solubility) or stability, while problematic biological properties include rapid metabolism or poor bioavailability, which may themselves be related to physicochemical properties. For example, they may be bioavailable by oral administration, whereas the parent drug is not. Prodrugs also have improved solubility in pharmaceutical compositions compared to the parent drug. An example, but not limited to, of a prodrug is any compound of the present invention administered as an ester ("prodrug") to facilitate transport across cell membranes, where water solubility is detrimental to mobility, but once inside the cell, water solubility is beneficial, which is then metabolically hydrolyzed to the carboxylic acid, the active entity. Another example of a prodrug is a short peptide (polyamino acid) conjugated to an acid group, where the peptide is metabolized to reveal the active moiety.

Unless otherwise specified, the term "stereoisomer" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans) isomers, atropisomers, etc. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and/or fractional crystallization.

Unless otherwise specified, the term "geometric (cis/trans) isomers" may contain carbon-carbon double bonds or carbon-nitrogen double bonds in the E or Z configuration, wherein the term "E" represents higher-order substituents on opposite sides of the carbon-carbon or carbon-nitrogen double bond and the term "Z" represents higher-order substituents on the same side of the carbon-carbon or carbon-nitrogen double bond (determined using the Cahn-Ingold Prelog priority rules). The compounds of the present invention may also exist as mixtures of "E" and "Z" isomers.

Unless otherwise specified, the term "tautomer" refers to structural isomers of different energies that are interconvertible through a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions that occur via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur via reorganization of some of the bonding electrons.

Unless otherwise indicated, the structural formulas described herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformers)): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, and (Z) and (E) conformers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformers) are within the scope of the present invention.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

An "effective amount," also referred to as a "therapeutically effective amount," refers to an amount of a compound or pharmaceutical composition described herein sufficient to achieve the intended application, including but not limited to treatment of a disease or alleviation of symptoms. In some embodiments, for example, the amount is a dose that can induce a specific response in cells, or a dose that can exert a therapeutic effect on a disease in a model animal. The specific amount will vary depending on, for example, the specific compound selected, the type of subject and their age/existing health condition or risk of health condition, the dosing regimen followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system used to carry it.

A "pharmaceutically acceptable carrier," also known as a "pharmaceutically acceptable excipient" or "pharmaceutically acceptable vehicle," refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid excipient, solvent, or encapsulating material, that is used to transport the active ingredient from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other components of the formulation and harmless to the patient. The preparation of the pharmaceutical compositions described herein includes, but is not limited to, for example, mixing the compound of Formula I or a pharmaceutically acceptable salt thereof described in the first aspect with a pharmaceutically acceptable carrier.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail one by one.

The beneficial effects of the present invention are:

This invention designs a class of novel compounds, offering a new direction for the development of drugs known as cap-dependent endonuclease inhibitors. Experimental studies have shown that these compounds exhibit potent inhibitory effects against influenza virus, good PK properties, and/or oral absorption, making them promising compounds for the treatment of cap-dependent endonuclease-mediated diseases.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials shown herein are for demonstration purposes only.

The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and/or liquid chromatography-mass spectrometry (LC-MS) and/or liquid chromatography (HPLC). The NMR spectrometer used is a Bruker Advance 600, the LC-MS spectrometer is a Waters ACQUITY UPLC I-Class Xevo TQ-S, and the HPLC spectrometer is a Waters e2695.

The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

Preparation Example 1: Preparation of Intermediate 1-8

Method 1: Toluene (36 mL), compound 1-1 (5.5 g, 29.6 mmol), deuterated thiophenol (8.2 g, 35.4 mmol), and D-camphorsulfonic acid (2.32 g, 10.0 mmol) were added to a round-bottom flask and stirred at 60°C for 4 hours, then cooled to 5°C. 2 mol/L sodium hydroxide solution (20 mL) was added and stirred until the mixture returned to room temperature. Toluene (20 mL) was added for extraction and phase separation. The organic phase was washed once with 2 mol/L sodium hydroxide (20 mL) and once with water (20 mL), and then concentrated under reduced pressure to obtain compound 1-3 (9.2 g). MS m/z: 284.1 [M+H].

Aluminum chloride (11.1 g, 82.9 mmol) and toluene (50 mL) were added to a reaction flask, stirred and cooled to 0°C. A solution of 1,1,3,3-tetramethyldisiloxane (11.1 g, 82.8 mmol) in toluene (20 mL) was added dropwise. After the addition was completed, the temperature was raised to 25°C and the toluene solution of compound 1-3 was slowly added. The mixture was stirred at 25°C for 2.5 hours. 15% aqueous sulfuric acid solution (70 mL) was added for extraction and phase separation. The organic phase was washed twice with water (40 mL) and concentrated under reduced pressure. n-heptane (80 mL) was added and the mixture was slurried at 0°C. The mixture was filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 1-4 (14.5 g). MS m/z: 286.1 [M+H].

Polyphosphoric acid (59 g) was added to the reaction flask, stirred and heated to 80°C, compound 1-4 (12 g) was added, the temperature was raised to 120°C and stirred for reaction for 3 hours, the temperature was lowered to 80°C, water (30 mL) was slowly added, the temperature was lowered to 30°C, water (120 mL) and ethyl acetate (120 mL) were added to extract the phases, the organic phase was washed with 10% sodium bicarbonate aqueous solution (35 mL), and the organic phase was concentrated under reduced pressure. n-heptane (60 mL) was added and the mixture was slurried at room temperature, filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 1-5 (10.2 g). MS m/z: 267.1 [M+H].

Isopropanol (40 mL), water (4.5 mL), and compound 1-5 (9.2 g, 17.2 mmol) were added to a reaction flask, stirred, and heated to 40°C. Sodium borohydride (0.47 g, 12.4 mmol) was slowly added to a 0.5% aqueous sodium hydroxide solution (3.6 mL) to prepare a suspension. The suspension was slowly added to the reaction system, stirred at 40°C for 1.5 hours, cooled to room temperature, and water (65 mL) was added, followed by a 4% aqueous sulfuric acid solution (15 mL). The temperature was lowered to 5°C, filtered, and the filter cake was rinsed with water (20 mL) and dried to obtain compound 1-6 (8.7 g). MS m/z: 269.1 [M+H].

Ethyl acetate (16 mL), cyclohexane (4 mL), compound 1-6 (3.0 g, 11.2 mmol) and compound 1-7 (4.5 g, 13.4 mmol) were added to the reaction flask and stirred at room temperature. A 50% ethyl acetate solution of propylphosphonic anhydride (7.8 g, 12.3 mmol) was added, followed by methanesulfonic acid (1.5 g, 16.1 mmol). The mixture was heated to 60°C and stirred for 24 hours. After cooling to room temperature, tetrahydrofuran (20 mL) and water (12 mL) were added, followed by slow addition of 24% aqueous sodium hydroxide solution (12 mL) for extraction and separation. The organic phase was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to give compound 1-8 (5.2 g). MS m/z: 572.2 [M+H].

Method 2: Toluene (150 mL), compound 1-1 (15 g), deuterated thiophenol (10 g), and D-camphorsulfonic acid (2.8 g) were added to a round-bottom flask, stirred at 60°C for 4 hours, and then cooled to 5°C. 2 mol/L sodium hydroxide aqueous solution (30 mL) was added, stirred and returned to room temperature, and toluene (30 mL) was added for phase separation. The organic phase was washed once with 2 mol/L sodium hydroxide aqueous solution (30 mL) and once with water (30 mL). The organic phase was collected, concentrated under reduced pressure, and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to obtain compound 1-3 (19.7 g). MS m/z: 284.1 [M+H].

Aluminum chloride (23.7 g) and toluene (100 mL) were added to a reaction flask, stirred and cooled to 0°C. A solution of 1,1,3,3-tetramethyldisiloxane (23.9 g) in toluene (40 mL) was added dropwise. After the addition was completed, the temperature was raised to 25°C and a solution of compound 1-3 (18 g) in toluene (60 mL) was slowly added. The mixture was stirred at 25°C for 2.5 hours. 15% aqueous sulfuric acid solution (140 mL) was added for extraction and phase separation. The organic phase was washed twice with water (80 mL) and concentrated under reduced pressure. n-heptane (80 mL) was added and the mixture was slurried at 0°C. The mixture was filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 1-4 (14 g). MS m/z: 286.1 [M+H].

Polyphosphoric acid (60 g) and sulfolane (120 g) were added to the reaction flask, stirred and heated to 80°C, compound 1-4 (12 g) was added, the temperature was raised to 120°C and stirred for reaction for 3 hours, the temperature was lowered to 80°C, water (30 mL) was slowly added, the temperature was lowered to 30°C, water (120 mL) and ethyl acetate (120 mL) were added for phase separation, the organic phase was washed with 10% aqueous sodium bicarbonate solution (35 mL), and the organic phase was concentrated under reduced pressure. n-heptane (60 mL) was added and the mixture was slurried at room temperature, filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 1-5 (10.6 g). MS m/z: 267.1 [M+H].

Isopropanol (40 mL), water (4.5 mL), and compound 1-5 (9.2 g) were added to a reaction flask, stirred and heated to 40°C. Sodium borohydride (0.47 g) was slowly added to a 0.5% aqueous sodium hydroxide solution (3.6 mL) to prepare a suspension. The suspension was slowly added to the reaction system, stirred at 40°C for 1.5 hours, cooled to room temperature, added with water (65 mL), and then added with a 4% aqueous sulfuric acid solution (15 mL). The temperature was cooled to 5°C, filtered, and the filter cake was rinsed with water (20 mL) and dried to obtain compound 1-6 (8.7 g). MS m/z: 269.1 [M+H].

Ethyl acetate (16 mL), cyclohexane (4 mL), compound 1-6 (4.0 g) and compound 1-7 (7.35 g) were added to the reaction flask and stirred at room temperature. A 50% ethyl acetate solution (19 g) of propylphosphoric anhydride was added, followed by methanesulfonic acid (2.9 g). The mixture was heated to 60°C and stirred for 24 hours. After cooling to room temperature, tetrahydrofuran (20 mL) and water (12 mL) were added, followed by slow addition of 24% aqueous sodium hydroxide solution (12 mL) for extraction and separation. The organic phase was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to give compound 1-8 (7.2 g). MS m/z: 572.2 [M+H].

Preparation Example 2: Preparation of Intermediate 1-9

To the reaction flask, dichloromethane (20 mL), compound 1-8 (2.0 g), S-1,1'-bi-2-naphthol (0.2 g), tetraisopropyl titanate (0.2 g), and water (0.14 g) were added and stirred at room temperature for 2 hours. A 70% aqueous solution of tert-butyl peroxide (0.6 g) was added dropwise, maintaining the temperature at 40°C. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to afford compound 1-9 (1.6 g). MS m/z: 588.2 [M+H].

Preparation Example 3: Preparation of Intermediate 1-10

To the reaction flask, dichloromethane (20 mL), compound 1-8 (2.0 g), R-1,1'-bi-2-naphthol (0.2 g), tetraisopropyl titanate (0.2 g), and water (0.14 g) were added and stirred at room temperature for 2 hours. A 70% aqueous solution of tert-butyl peroxide (0.6 g) was added dropwise, maintaining the temperature at 40°C. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to afford compound 1-10 (1.6 g). MS m/z: 588.2 [M+H].

Preparation Example 4: Preparation of Intermediate 2-6

Method 1: Toluene (36 mL), compound 1-1 (5.0 g), thiophenol (7.2 g), and D-camphorsulfonic acid (2.1 g) were added to a round-bottom flask and stirred at 60°C for 4 hours. The temperature was then lowered to 5°C. 2 mol/L sodium hydroxide solution (18 mL) was added and the mixture was stirred and returned to room temperature. Toluene (20 mL) was added for extraction and phase separation. The organic phase was washed once with 2 mol/L sodium hydroxide (20 mL) and once with water (20 mL). The mixture was concentrated under reduced pressure to obtain compound 2-1 (7.8 g). MS m/z: 279.0 [M+H].

Aluminum chloride (10.0 g) and toluene (50 mL) were added to a reaction flask, stirred and cooled to 0°C. A solution of 1,1,3,3-tetramethyldisiloxane (10.0 g) in toluene (20 mL) was added dropwise. After the addition was completed, the temperature was raised to 25°C and the toluene solution of compound 2-1 was slowly added. The mixture was stirred at 25°C for 2.5 hours. 15% aqueous sulfuric acid solution (70 mL) was added to extract the phases. The organic phase was washed twice with water (40 mL) and concentrated under reduced pressure. n-heptane (80 mL) was added and the mixture was slurried at 0°C. The mixture was filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 2-2 (13.0 g). MS m/z: 281.0 [M+H].

Polyphosphoric acid (45 g) was added to the reaction flask, stirred and heated to 80°C, compound 2-2 (9.0 g) was added, the temperature was raised to 120°C and stirred for 3 hours, the temperature was lowered to 80°C, water (25 mL) was slowly added, the temperature was lowered to 30°C, water (90 mL) and ethyl acetate (90 mL) were added for phase separation, the organic phase was washed with 10% aqueous sodium bicarbonate solution (30 mL), and the organic phase was concentrated under reduced pressure. n-heptane (50 mL) was added and the mixture was slurried at room temperature, filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 2-3 (7.5 g). MS m/z: 263.0 [M+H].

Isopropanol (25 mL), water (3 mL), and compound 2-3 (6.0 g) were added to a reaction flask, stirred, and heated to 40°C. Sodium borohydride (0.3 g) was slowly added to a 0.5% aqueous sodium hydroxide solution (2.0 mL) to prepare a suspension. The suspension was slowly added to the reaction system, stirred at 40°C for 1.5 hours, cooled to room temperature, and water (45 mL) was added, followed by a 4% aqueous sulfuric acid solution (10 mL). The temperature was lowered to 5°C, filtered, and the filter cake was rinsed with water (15 mL) and dried to obtain compound 2-4 (10.0 g). MS m/z: 265.0 [M+H].

Ethyl acetate (10 mL), cyclohexane (2 mL), compound 2-4 (2.0 g) and compound 1-7 (3.0 g) were added to the reaction flask and stirred at room temperature. A 50% ethyl acetate solution of propylphosphoric anhydride (5.1 g) was added, followed by methanesulfonic acid (1.0 g). The mixture was heated to 60°C and stirred for 24 hours. After cooling to room temperature, tetrahydrofuran (15 mL) and water (10 mL) were added, followed by slow addition of 24% aqueous sodium hydroxide solution (10 mL) for extraction and separation. The organic phase was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to give compound 2-5 (3.5 g). MS m/z: 568.2 [M+H].

To the reaction flask, dichloromethane (20 mL), compound 2-5 (2.0 g), S-1,1'-bi-2-naphthol (0.2 g), tetraisopropyl titanate (0.2 g), and water (0.14 g) were added and stirred at room temperature for 2 hours. A 70% aqueous solution of tert-butyl peroxide (0.6 g) was added dropwise, maintaining the temperature at 40°C. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to afford compound 2-6 (1.6 g). MS m/z: 584.2 [M+H].

Method 2: Toluene (100 mL), compound 1-1 (15.0 g), thiophenol (21.6 g), and D-camphorsulfonic acid (6.3 g) were added to a round-bottom flask and stirred at 60°C for 4 hours, then cooled to 5°C. 2 mol/L sodium hydroxide solution (54 mL) was added, and the mixture was stirred and returned to room temperature. Toluene (60 mL) was added for extraction and phase separation. The organic phase was washed once with 2 mol/L sodium hydroxide (60 mL) and once with water (60 mL). After concentration under reduced pressure, compound 2-1 (20.4 g) was obtained. MS m/z: 279.0 [M+H].

Aluminum chloride (30.0 g) and toluene (150 mL) were added to a reaction flask, stirred and cooled to 0°C. A solution of 1,1,3,3-tetramethyldisiloxane (30.0 g) in toluene (60 mL) was added dropwise. After the addition was completed, the temperature was raised to 25°C. A solution of compound 2-1 (22.8 g) in toluene (90 mL) was slowly added. The mixture was stirred at 25°C for 2.5 hours. 15% aqueous sulfuric acid solution (210 mL) was added for extraction and phase separation. The organic phase was washed twice with water (120 mL) and concentrated under reduced pressure. n-heptane (240 mL) was added and the mixture was slurried at 0°C. The mixture was filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 2-2 (18.0 g). MS m/z: 281.0 [M+H].

Polyphosphoric acid (45 g) and sulfolane (70 mL) were added to the reaction flask, stirred and heated to 80°C, compound 2-2 (9.0 g) was added, the temperature was raised to 120°C and stirred for 3 hours, the temperature was lowered to 80°C, water (25 mL) was slowly added, the temperature was lowered to 30°C, water (90 mL) and ethyl acetate (90 mL) were added for phase separation, the organic phase was washed with 10% aqueous sodium bicarbonate solution (30 mL), and the organic phase was concentrated under reduced pressure. n-heptane (50 mL) was added and the mixture was slurried at room temperature, filtered, the filter cake was rinsed with n-heptane, and dried to obtain compound 2-3 (7.5 g). MS m/z: 263.0 [M+H].

Isopropanol (25 mL), water (3 mL), and compound 2-3 (6.0 g) were added to a reaction flask, stirred, and heated to 40°C. Sodium borohydride (0.3 g) was slowly added to a 0.5% aqueous sodium hydroxide solution (2.0 mL) to prepare a suspension. The suspension was slowly added to the reaction system, stirred at 40°C for 1.5 hours, cooled to room temperature, and water (45 mL) was added, followed by a 4% aqueous sulfuric acid solution (10 mL). The temperature was cooled to 5°C, filtered, and the filter cake was rinsed with water (15 mL) and dried to obtain compound 2-4 (4.0 g). MS m/z: 265.0 [M+H].

Ethyl acetate (10 mL), cyclohexane (2 mL), compound 2-4 (2.4 g) and compound 1-7 (3.0 g) were added to the reaction flask and stirred at room temperature. A 50% ethyl acetate solution of propylphosphonic anhydride (7.7 g) was added, followed by methanesulfonic acid (1.0 g). The mixture was heated to 60°C and stirred for 24 hours. After cooling to room temperature, tetrahydrofuran (15 mL) and water (10 mL) were added, followed by slow addition of 24% aqueous sodium hydroxide solution (10 mL) for extraction and separation. The organic phase was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to give compound 2-5 (4.1 g). MS m/z: 568.2 [M+H].

To the reaction flask, dichloromethane (20 mL), compound 2-5 (2.0 g), S-1,1'-bi-2-naphthol (0.2 g), tetraisopropyl titanate (0.2 g), and water (0.14 g) were added and stirred at room temperature for 2 hours. A 70% aqueous solution of tert-butyl peroxide (0.6 g) was added dropwise, maintaining the temperature at 40°C. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to afford compound 2-6 (1.6 g). MS m/z: 584.2 [M+H].

Preparation Example 5, Preparation of Intermediate 2-7

To the reaction flask, dichloromethane (20 mL), compound 2-5 (2.0 g), R-1,1'-bi-2-naphthol (0.2 g), tetraisopropyl titanate (0.2 g), and water (0.14 g) were added and stirred at room temperature for 2 hours. A 70% aqueous solution of tert-butyl peroxide (0.6 g) was added dropwise, maintaining the temperature at 40°C. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to afford compound 2-7 (1.8 g). MS m/z: 584.2 [M+H].

Example 1: Preparation of Compound 6

N-methylpyrrolidone (5 mL), intermediate compound 1-8 (1 g), and lithium chloride (0.57 g) were added to a reaction flask. The temperature was raised to 75°C, and methanesulfonic acid (70 mg) was added. The reaction was maintained at 75°C for 20 h. The temperature was lowered to 45°C, and acetonitrile (1.5 mL) and water 1 (10 mL) were added. The mixture was stirred at 45°C for 1 hour, then lowered to 15°C, stirred at this temperature for 1 hour, and filtered. The filter cake was slurried with isopropanol (10 mL) at room temperature for 2 hours, filtered, rinsed with isopropanol (5 mL), and dried to yield compound 6 (0.7 g). MS m/z: 488.1 [M+H]. ¹ H NMR (600MHz, CDCl ₃ ) δ11.52(s,1H),7.10(dd,J＝16.8,8.5Hz,1H),7.06–6.97(m,2H),5.77(d,J＝7.7Hz,1H),5.29 -2.27(m,2H),4.67(d,J＝13.5Hz,1H),4.58(dd,J＝10.0,2.8Hz,1H),4.07(d,J＝13.9Hz,1H),3.96(dd,J＝11.1,2. 8Hz,1H),3.81(dd,J＝12.0,3.2Hz,1H),3.62(t,J＝10.6Hz,1H),3.47(td,J＝11.8,2.1Hz,1H),3.06–2.95(m,1H).

Example 2-5: The following compounds of Example 2-5 were prepared by referring to the preparation method of Example 1

Example 6: Preparation of Compound 1

Tetrahydrofuran (5 mL), N,N-dimethylacetamide (0.5 mL), compound 6 (0.5 g), anhydrous potassium carbonate (0.19 g), potassium iodide (70 mg), isopropyl alcohol (30 mL), and purified water (15 mL) were added to the reaction flask and heated to 60°C with stirring. Chloromethyl methyl carbonate (0.22 g) was then added. The mixture was reacted at 60°C for 10 hours. The mixture was cooled to room temperature, glacial acetic acid (0.1 g) was added, and the mixture was stirred for 15 minutes. Water (2.5 mL) was added and stirred for 30 minutes. The mixture was extracted and separated. The organic phase was concentrated and separated by column chromatography using a mixed solvent of ethyl acetate and n-hexane to obtain compound 1 (0.47 g). MS m/z: 576.2 [M+H]. ¹ H NMR (600 MHz, CDCl ₃ )δ7.14(d,J＝7.6Hz,1H),7.10(dd,J＝16.8,8.4Hz,1H),7.01(dd,J＝8.2,4.3Hz,1H),5.93(d,J＝ 7.7Hz,1H),5.90(s,2H),5.33(s,1H),5.27(d,J＝14.3Hz,1H),4.64(d,J＝13.2Hz,1H),4.52(dd, J＝9.9,2.6Hz,1H),4.07(d,J＝13.9Hz,1H),3.94(dd,J＝10.9,2.4Hz,1H),3.86(s,3H),3.76(dd ,J＝11.8,2.9Hz,1H),3.56(t,J＝10.5Hz,1H),3.44(dd,J＝11.7,10.0Hz,1H),2.98–2.90(m,1H).

Example 7-35: The following compound of Example 7-35 was prepared by referring to the preparation method of Example 6

Biological testing

The following test examples further illustrate the present invention, but these examples are not intended to limit the scope of the present invention.

Test Example 1: Pharmacokinetics experiment in rats

1. Purpose:

The pharmacokinetic characteristics of the compounds of the present invention after intravenous and oral administration were investigated.

2. Reagents and experimental animals:

Reagents: dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG400), methylcellulose (MC).

Experimental animals: SD rats, male.

3. Comparative Pharmacokinetic Experimental Methods in Rats

(1) Drug preparation

The control compound (mabaloxavir) and the compounds 1-5 and 11-35 of the present invention were prepared respectively using the following formulations.

Intravenous administration: Dosage: 1 mg/kg, volume: 5 ml/kg, concentration: 0.2 mg/ml; 5% DMSO + 10% PEG400 + 85% aqueous solution;

Oral administration: Dosage: 5 mg/kg, Dosage volume: 10 ml/kg, Dosage concentration: 0.5 mg/ml; 0.5% methylcellulose (MC) suspension.

(2) Dosage regimen

Healthy adult SD rats (3 animals per group) were fasted overnight (with free access to water) and then administered with the drug via tail vein injection (iv 1 mg/kg) or oral gavage (po 5 mg/kg). In the iv group, 0.2 mL of blood was collected from the jugular vein in EDTA-K2 anticoagulant tubes at 5 min, 15 min, 0.5, 1, 2, 4, 8, and 24 h after administration. The blood samples were placed on wet ice and centrifuged within 1 h (4°C, 3500 g, 5 min) to separate the plasma. The plasma was placed in a labeled 1.5 mL EP tube and stored in a -80°C ultra-low temperature freezer for testing. In the po. group, 0.2 mL of blood was collected from the jugular vein before administration and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. The treatment method was the same as that of the iv administration group. The concentration of the unchanged drug in plasma was determined by LC/MS/MS, and the blood concentration-time curve was drawn. The main pharmacokinetic parameters were calculated using WinNonlin 7.2 software.

Exemplary test results are shown in the table below:

Table 1 Pharmacokinetic results in rats

The experimental results show that the compounds of the present invention have good PK properties and/or oral absorption properties.

Test Example 2: Detection of virus inhibition activity (EC50) and cytotoxicity (CC50) of compounds 1

Cell plating: MDCK cells were seeded at a certain density in a 96-well plate, and then the cells were cultured in a 37°C, 5% CO2 incubator overnight.

Compound Treatment: Test compounds (control compound baloxavir and compounds 6-10 of the present invention) were serially diluted with DMSO to a final DMSO concentration of 0.5% and added to the cell culture plates. CC50 determinations were performed using a 3-fold dilution from 50 μM for 10 concentrations. EC50 determinations were performed using a 3-fold dilution from 100 nM for 10 concentrations.

Virus Inoculation: Add diluted influenza virus (A/WSN/33 (H1N1) or A/PR/8/34 (H1N1)) to the antiviral activity test wells; add only the compound, without virus, to the cytotoxicity test wells. Incubate the cells in a 37°C, 5% CO2 incubator for 3-5 days until the cytopathic effect (CPE) rate in the virus control wells (no compound) reaches 80-95%.

Cell viability assay: Cell viability assay reagent (CCK-8 assay reagent) was added to each well. After culturing the cells in a 37°C, 5% CO2 incubator for 3-4 hours, the signal value was detected by a microplate reader. The raw data was used to calculate the anti-influenza virus activity and cytotoxicity of the compound.

GraphPad Prism software was used for analysis to obtain the compound dose-response curve and its EC50 and CC50 values, and the therapeutic index was calculated (therapeutic index = CC50/EC50).

The experimental results show that the compound of the present invention has good influenza virus inhibitory activity and low cytotoxicity.

Test Example 3: Detection of Virus Inhibitory Activity (EC50) and Cytotoxicity (CC50) of Compounds 2

1. Purpose:

The in vitro anti-influenza virus activity of the compounds of the present invention was investigated using an indirect immunofluorescence virus quantitative assay (FFA) method.

2. Key reagents, strains and cells:

Key reagents: Influenza Anti-HA Serum (strain-specific serum), Alexa Fluor 488 donkey anti-sheep IgG

Strain: Type A (H1N1) A/Victoria/2570/2019 & A/Victoria/4897/2022

Type A (H3N2)A/Cambodia/E0826360/2020&A/Darwin/9/2021

Cell plating: MDCK cells

3. Detection method:

Cell preparation: Observe the cell status and ensure the confluence is 85% to 95% and the viability is ≥90%. Digest the cells and inoculate them into 96-well cell culture plates at a concentration of 2.5 to 3.0 × 10 ⁵ cells/mL, 100 μL/well, and culture in a 37°C CO 2 incubator for approximately 24 hours.

Virus dilution: Take the virus solution and dilute it with MEM medium according to the virus titer of each strain to 8000 PFU/mL as the virus working solution. Place it on ice or at 2-8°C until ready for use.

Compound Dilution: Test compounds (control compound 1: baloxavir, control compound 2: Example compound 1A in CN108440564A, and compounds of the present invention) were serially diluted with DMSO to a final DMSO concentration of 0.5% and added to the cell culture plate. EC50 determination dilution factor: 20 concentrations, starting from 100 nM, with 3-fold dilutions.

Compound Dosing and Virus Inoculation: Remove the pre-seeded cell plate from the 37°C CO2 incubator, spin dry the culture supernatant, add 100 μL/well MEM medium, and wash twice. Spin dry any remaining liquid. According to the designed plate layout, pipette the compound serial dilutions and compound blank dilutions at a rate of 50 μL/well onto the antiviral assay plate using a multichannel pipette. Except for wells CC (cell control), add 50 μL of the corresponding virus working solution to each well. Gently shake the plate to mix thoroughly. Incubate the plate in a 37°C, 5% _CO2 incubator for 18–24 hours.

GraphPad Prism software was used to analyze the compound dose-response curve and its EC50 value.

Table 2 In vitro anti-influenza virus activity of compounds

The experimental results show that the compound of the present invention is significantly superior to the control compound and has good influenza virus inhibitory activity.

Test Example 4: Pharmacokinetics experiment in mice

1. Purpose:

The distribution levels of the compounds of the present invention in the lungs and plasma were investigated after oral administration to mice.

2. Reagents and experimental animals:

Reagents: methylcellulose (MC);

Experimental animals: Balb/c mice, male.

3. Comparative Pharmacokinetic Experimental Methods in Mice

(1) Drug preparation

Oral administration: The dosage is 10 mg/kg, the administration volume is 10 ml/kg, and 0.5% methylcellulose (MC) is used to prepare a uniform suspension with a dosage concentration of 1.0 mg/ml.

(2) Dosage regimen

6-8 week old Balb/c mice (3 animals at each time point) were fasted overnight (with free access to water) and then orally administered with 10 mg/kg of compound (such as compound 1 of the present invention); blood samples were collected at 0.5, 1, 2, 4, and 8 hours after the drug administration for each compound group, and the mice were immediately killed and lung tissue was collected. The blood samples were collected into test tubes with stabilizers added in advance, mixed by inversion, and centrifuged within 30 minutes (4°C, 3000g, 10 minutes). The plasma was separated and frozen for testing. After the lung tissue was collected, it was immediately placed in an ice water bath, weighed within 20 minutes, and glacial acetonitrile was quickly added according to 1g:3mL. The homogenate was completed within 1 hour for testing. The metabolite compounds (such as compound 6 of the present invention) in the plasma were determined by LC/MS/MS method, and the results are as follows:

The experimental results show that the exposure levels of the compound of the present invention in mouse plasma and lungs, as well as the ratio of lung blood exposure, are both good.

Experimental Example 5: Pharmacokinetic Experiment in Cynomolgus Monkeys

1. Purpose:

The pharmacokinetic characteristics of the compounds of the present invention were investigated after intravenous and oral administration to cynomolgus monkeys.

2. Reagents and experimental animals:

Reagents: dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG400), methylcellulose (MC).

Experimental animals: Cynomolgus monkeys, male.

3. Comparative Pharmacokinetic Experimental Methods in Cynomolgus Monkeys

(1) Drug preparation

Intravenous administration: Dosage: 1 mg/kg, dosing volume: 2 ml/kg, dosing concentration: 0.5 mg/ml; 5% DMSO + 30% PEG400 + 65% aqueous solution;

Oral administration: Dosage: 10 mg/kg, administration volume: 2 ml/kg, administration concentration: 0.5 mg/ml; 0.5% methylcellulose (MC) suspension.

(2) Dosage regimen

Healthy eluted cynomolgus monkeys (n=4 per oral group and n=2 per intravenous group) were fasted overnight (with free access to water) and then administered with either tail vein injection (1 mg/kg) or oral administration (1 mg/kg). Blood samples (0.3 mL) were collected from all four limb veins in the iv group at 2, 5, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h after administration. The samples were placed in EDTA-K2 anticoagulant tubes (pre-added with 5 μL PMSF solution (300 mM in methanol)). The tubes were gently shaken several times, placed in an ice-water bath, and centrifuged within 30 min (3000 g, 4°C, 10 min). Plasma was separated and frozen at -70°C as soon as possible for analysis. Blood samples were collected from the po group before administration and at 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h after administration and processed as described for the intravenous group. The metabolites in plasma (such as compound 6, the metabolite of the invention compound) were determined by LC/MS/MS, and the blood drug concentration-time curve was drawn. The main pharmacokinetic parameters were calculated using WinNonlin 8.0 software.

The experimental results show that the compound of the present invention has good PK properties and oral absorption properties after oral administration.

Test Example 6: Stability test of compound in human liver S9 in vitro

Test samples: 1 μM baloxavir and compound 1 of the present invention

Reagents: Phosphate buffer: 200 mM, 200 μL, final concentration 100 mM; Ultrapure water: 138 μL, _MgCl2 solution: 50 mM, 40 μL, final concentration 5 mM; S9 component: 20 mg/mL, 20 μL, final concentration 1 mg/mL

Procedure: Add 2 μL of a 200 μM test compound or control compound solution to the above reagent system. Verapamil serves as a positive control, and the final concentration of the test and control compounds is 1 μM. After 0, 5, 15, 30, 45, and 60 minutes, the reaction solution is mixed on a vortex mixer for 10 seconds. Then, 50 μL is transferred to 400 μL of acetonitrile, sealed, and placed on ice. Mixing is continued for 10 minutes. After centrifugation at 3,220 g for 50 minutes at 4°C, mabaloxavir and compound 1 concentrations are analyzed by LC-MS/MS. The measured concentrations are used to calculate the percentage remaining at each time point using Microsoft Excel. The slopes are then used to calculate the half-life (T _1/2) and in vitro intrinsic clearance (CL _{int )} .

Table 3 In vitro stability results of compound human liver S9

The experimental results show that the compound of the present invention is metabolized faster in human liver S9 than Yangshen, and is more likely to release the original drug molecule.

Test Example 7: Stability test of compound in human intestinal S9 in vitro

Test samples: 1 μM baloxavir and compound 1 of the present invention

Reagents: Phosphate buffer: 200 mM, 200 μL, final concentration 100 mM; Ultrapure water: 138 μL, _MgCl2 solution: 50 mM, 40 μL, final concentration 5 mM; S9 component: 20 mg/mL, 20 μL, final concentration 1 mg/mL

Procedure: Add 2 μL of a 200 μM test compound or control compound solution to the above reagent system. Verapamil serves as a positive control, and the final concentration of the test and control compounds is 1 μM. After 0, 5, 15, 30, 45, and 60 minutes, the reaction solution is mixed on a vortex mixer for 10 seconds. Then, 50 μL is transferred to 400 μL of acetonitrile, sealed, and placed on ice. Mixing is continued for 10 minutes. After centrifugation at 3,220 g for 50 minutes at 4°C, mabaloxavir and compound 1 concentrations are analyzed by LC-MS/MS. The measured concentrations are used to calculate the percentage remaining at each time point using Microsoft Excel. The slopes are then used to calculate the half-life (T _1/2) and in vitro intrinsic clearance (CL _{int )} .

Table 4 In vitro stability results of compound human intestinal S9

The experimental results show that the compounds of the present invention are metabolized faster in human intestinal S9 than the Yangshen compounds and are more likely to release the original drug molecules.

Test Example 8: Stability test of compound in human plasma

Test samples: Yangshenmabaloxavir and compound 1 of the present invention

Procedure: 50 μL of a 400 ng/mL solution of test compound or control compound was added to human plasma. The final concentration of the test compound and control compound was 20 ng/mL. After incubation at 37°C for 0, 5, 15, 30, and 60 minutes, the stabilized plasma was pooled and 30 μL was transferred to 270 μL of acetonitrile. After mixing, the mixture was rapidly centrifuged at 10,000 g for 5 minutes at 4°C. The concentrations of mabaloxavir and compound 1 were analyzed by LC-MS/MS. The measured concentrations were used to calculate the percentage remaining at each time point using Microsoft Excel.

Table 5 In vitro stability results of compounds in human plasma

Experimental results show that the compound of the present invention is metabolized faster in human plasma than the Yangshen compound and is more likely to release the original drug molecules.

Claims (10)

The compound represented by formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof:
wherein R ₁ , R ₂ , R ₃ and R ₄ are all hydrogen or all deuterium, R ₅ is absent or is an oxo group, and R ₆ is hydrogen or The compound according to claim 1, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, characterized in that R ₁ , R ₂ , R ₃ and R ₄ are deuterium, R ₅ is absent or is an oxo group, and R ₆ is hydrogen or The compound according to claim 1, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, characterized in that R ₁ , R ₂ , R ₃ and R ₄ are hydrogen, R ₅ is oxo, and R ₆ is hydrogen or The compound according to claim 1, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, characterized in that R ₁ , R ₂ , R ₃ and R ₄ are deuterium, R ₅ is an oxo group, the configuration of the sulfur atom is S or R, and R ₆ is hydrogen or The compound according to claim 1, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, characterized in that R ₁ , R ₂ , R ₃ and R ₄ are hydrogen, R ₅ is an oxo group, the configuration of the sulfur atom is S or R, and R ₆ is hydrogen or A compound selected from the group consisting of:
and its tautomers, stereoisomers or pharmaceutically acceptable salts. A pharmaceutical composition, characterized in that it comprises the compound according to any one of claims 1 to 6, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, and optionally further comprises a pharmaceutically acceptable carrier. Use of the compound according to any one of claims 1 to 6, its tautomer, stereoisomer or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and/or preventing a disease or condition mediated by a cap-dependent endonuclease. Use of the compound according to any one of claims 1 to 6, its tautomer, stereoisomer or pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and/or preventing a disease or condition caused by viral infection. The use according to claim 9 is characterized in that the viral infection is influenza virus infection; further, the influenza virus is selected from type A, type B, type C or avian influenza (H5N1, H7N9); further, the symptoms are selected from cold-like symptoms accompanied by fever, chills, headache, muscle pain, general fatigue, etc., or respiratory tract inflammation with sore throat, runny nose, nasal congestion, cough, and sputum; gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea, and further accompanied by complications of acute encephalopathy, secondary pneumonia infection, or a combination thereof.