Compound for treating influenza and preparation method therefor

Abstract

Provided in the present invention is a compound having a new structure, or a stereoisomer, a tautomer, an isotope derivative or a pharmaceutically acceptable salt thereof. The compound of the present invention, as an RNA polymerase inhibitor, can be used for treating influenza.

Description

Compound for treating influenza and preparation method thereof Technical Field

[0001] The present application belongs to the field of medicinal chemistry, and specifically relates to a class of compounds, pharmaceutical compositions containing the same, their use as drugs for preventing and / or treating influenza, and methods for preparing the same. Background Art

[0002] Influenza viruses are RNA viruses of the family Orthomyxoviridae. Based on the antigenic and genetic characteristics of the nucleoprotein (NP) and matrix protein (M) of the virion, influenza viruses are mainly divided into three types: A, B, and C, also known as types A, B, and C. Among them, influenza A and B viruses are the main viruses that cause seasonal pandemics. Type C virus mostly exists in a scattered form, mainly invading infants and young children, generally does not cause epidemics, but can infect humans and pigs. Influenza A virus has a wide range of hosts, including wild waterfowl, pigs, etc., and there is a possibility of transmission from animals to humans, while also spreading among humans, causing a global influenza virus pandemic. The elderly, children, and people with weakened immune systems will develop severe respiratory symptoms when infected with type A and type B viruses, and in severe cases, death. Recent data show that the annual infection rate worldwide is 15%, and some patients require hospitalization.

[0003] Currently, vaccines are the most effective means of reducing influenza infection rates, but they are not effective for everyone. Globally, vaccines are effective in approximately 50-60% of the population, and nearly 60% of the elderly. Furthermore, due to vaccine design, viral mutations, and low vaccination rates in some countries, combined with peak influenza season, there is a potential for influenza pandemics.

[0004] Anti-influenza drugs are an important means of treating and preventing influenza viruses, especially for those at high risk and those with severe infections. Currently approved drugs include neuraminidase (NA) inhibitors including oseltamivir, zanamivir, peramivir, and laninamivir, which are also standard treatments; hemagglutinin (HA) inhibitors including arbidol; M2 ion channel blockers including amantadine and its derivatives; and endonuclease (PA) inhibitors including mabaloxavir.

[0005] Influenza virus proteins include the nucleocapsid protein NP and the viral RNA-dependent RNA polymerase (RdRp) complex: PA-PB1-PB2. There is one of these complexes at the end of each viral genome, where PB1 is involved in RNA replication, PB2 is involved in cap recognition, and PA is involved in nucleolytic endonucleolysis. The complex composed of the three subunits jointly completes RNA replication. Therefore, in theory, any protein of the RdRp complex PA-PB1-PB2 can inhibit influenza virus replication. There are already marketed drugs for the PB1 and PA subunits, and there is a marketed drug for the PA subunit. There is currently no marketed drug for the PB2 subunit. Therefore, it is of great significance to develop inhibitors targeting the PA subunit and / or PB2 subunit to address unmet clinical needs. Summary of the Invention

[0006] The present application provides compounds as polymerase inhibitors, their stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, which can be used to treat influenza.

[0007] In a first aspect, the present application provides a compound represented by formula (I), its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts,

[0008] Wherein, R is hydrogen, is the connection site;

[0009] R1 and R2 are both hydrogen or both deuterium;

[0010] n is selected from 0-3; preferably 0, 1, 2 or 3; more preferably 0, 1 or 2; further preferably 0 or 1;

[0011] R3 and R4 are each independently selected from H, deuterium or C 1-4 Alkyl; preferably H or C 1-3 Alkyl; more preferably H, methyl or ethyl;

[0012] R5 is independently selected at each occurrence from H, deuterium or C 1-4 Alkyl; preferably H or C 1-3 Alkyl; more preferably H, methyl or ethyl;

[0013] The condition is that when R is H or When both R5 are H, R1 and R2 are deuterium. In some embodiments, R is For the connection site.

[0014] In some embodiments, n is 0, 1, 2 or 3; more preferably 0, 1 or 2; more preferably 0 or 1.

[0015] In some embodiments, R3 and R4 are each independently selected from H or C 1-4 Alkyl; preferably H or C 1-3 Alkyl; more preferably H, methyl or ethyl; more preferably H.

[0016] In some embodiments, R5 is selected from H or C 1-3 Alkyl; more preferably H, methyl or ethyl; more preferably H.

[0017] In some embodiments, R1 and R2 are both hydrogen.

[0018] In some embodiments, R1 and R2 are both deuterium.

[0019] In some embodiments, the compound is a compound represented by formula (I-1):

[0020] R, R1, and R2 are as defined for the compound represented by formula (I).

[0021] In a second aspect, the present application provides a compound, a stereoisomer, a tautomer, an isotopic derivative, or a pharmaceutically acceptable salt thereof, wherein the compound has the following structure:

[0022] In some embodiments, the compound has the structure:

[0023] In some embodiments, the compound has the structure:

[0024] In some embodiments, the isotopic derivative is a deuterated derivative (or also known as, a deuterated form of the compound).

[0025] In a third aspect, the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the compound as described in the first aspect or the second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, and optionally further comprises a pharmaceutically acceptable carrier.

[0026] In some embodiments, the pharmaceutical composition comprises the compound as described in the first aspect or the second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers.

[0027] In some embodiments, the pharmaceutical composition is used to inhibit viral replication, comprising the compound as described in the first aspect or the second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers.

[0028] In some embodiments, the pharmaceutical composition is used to inhibit viral replication, comprising a therapeutically effective amount of the compound as described in the first aspect or the second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers.

[0029] On the other hand, the present application provides a drug combination, which includes the compound according to the first aspect or the second aspect, its stereoisomer, tautomer, isotopic derivative or pharmaceutically acceptable salt, and another treatment and / or prevention of a disease caused by a virus. In some embodiments, the additional drug includes a neuraminidase inhibitor, a polymerase acid protein inhibitor, an M2 protein inhibitor, an RNA-dependent RNA polymerase inhibitor, a PB2 Cap binding inhibitor (or PB2 inhibitor), an anti-HA antibody or an immune agent, etc. In some embodiments, the additional drug is administered simultaneously or sequentially with the compound, stereoisomer, tautomer, isotopic derivative or pharmaceutically acceptable salt described in the first aspect or the second aspect.

[0030] In some embodiments, the present application provides a drug combination comprising a compound according to the aforementioned first aspect or second aspect, a stereoisomer, a tautomer, an isotopic derivative or a pharmaceutically acceptable salt thereof, and another drug for treating and / or preventing influenza. In some embodiments, the other drugs include neuraminidase inhibitors, polymerase acid protein inhibitors, M2 protein inhibitors, RNA-dependent RNA polymerase inhibitors, PB2 Cap binding inhibitors (or PB2 inhibitors), anti-HA antibodies or immune agents, etc. In some embodiments, the other drugs are administered simultaneously or sequentially with the compound, stereoisomer, tautomer, isotopic derivative or pharmaceutically acceptable salt thereof according to the aforementioned first aspect or second aspect.

[0031] In a fourth aspect, the present application provides the use of the compound as described in the first or second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, or the pharmaceutical composition as described in the third aspect in the preparation of a drug.

[0032] In some embodiments, the drug is a polymerase inhibitor drug; preferably, the drug is an RNA polymerase inhibitor drug.

[0033] In some embodiments, the drug is a cap-dependent endonuclease inhibitor.

[0034] In some embodiments, the drug is a polymerase acid protein (PA) (or RNA polymerase PA subunit) inhibitor drug.

[0035] In some embodiments, the drug is an RNA polymerase basic protein 2 (polymerase basic protein 2, PB2) (or RNA polymerase PB2 subunit) inhibitor drug.

[0036] In some embodiments, the drug is used to treat and / or prevent a disease caused by a virus. In some embodiments, the drug is used to treat and / or prevent a disease caused by a virus having a cap-dependent endonuclease. Preferably, the drug is used to treat and / or prevent a disease caused by a virus having a polymerase acid protein (PA) (or can be called, RNA polymerase PA subunit); preferably, the disease is a disease caused by a virus of the family Orthomyxoviridae; preferably, the disease is influenza (or influenza or influenza infectious disease, referred to as influenza); further preferably, the disease is influenza A, B or C; further preferably, the disease is H1N1 influenza.

[0037] In some embodiments, the drug can be used in combination with other drugs for the treatment and / or prevention of diseases caused by viruses. In some embodiments, the other drugs include neuraminidase inhibitors, polymerase acidic protein inhibitors, M2 protein inhibitors, RNA-dependent RNA polymerase inhibitors, PB2 Cap binding inhibitors (or PB2 inhibitors), anti-HA antibodies or immune drugs, etc. In some embodiments, the other drugs are administered simultaneously or sequentially with the drug.

[0038] In some embodiments, the drug is an antiviral drug. In some embodiments, the drug is an antiviral drug against Orthomyxoviridae viruses. In some embodiments, the drug is a drug that inhibits the replication of influenza (also known as influenza or influenza epidemic) viruses; preferably, the influenza virus is influenza A virus, influenza B virus, or influenza C virus; preferably, the influenza virus is influenza A virus. Further preferably, the influenza virus is influenza H1N1 virus.

[0039] In some embodiments, the drug is an antiviral drug. In some embodiments, the drug is an antiviral drug against Orthomyxoviridae viruses. In some embodiments, the drug is a drug for treating and / or preventing influenza or an anti-influenza drug; preferably, the drug is a drug for treating and / or preventing influenza A virus, influenza B virus, or influenza C virus; preferably, the drug is a drug for treating and / or preventing influenza A virus; further preferably, the influenza A virus is an H1N1 influenza virus; further preferably, the influenza A virus is influenza A virus A / PR / 8 / 34 strain (H1N1) influenza virus.

[0040] In a fifth aspect, the present application provides a compound as described in the first or second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, or the pharmaceutical composition as described in the third aspect, which is used to inhibit influenza (or influenza or influenza infectious disease) virus replication.

[0041] In some embodiments, the compound of the first or second aspect, its stereoisomer, tautomer, isotopic derivative or pharmaceutically acceptable salt, or the pharmaceutical composition of the third aspect is used to prevent and / or treat influenza.

[0042] In a sixth aspect, the present application provides a method for inhibiting influenza virus replication, comprising administering to a subject in need thereof a therapeutically effective amount of the compound as described in the first or second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, or the pharmaceutical composition as described in the third aspect.

[0043] In some embodiments, the present application provides a method for preventing and / or treating influenza, comprising administering to a subject in need thereof a therapeutically effective amount of the compound as described in the first aspect or the second aspect, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, or the pharmaceutical composition as described in the third aspect.

[0044] In the absence of contradictions and conflicts, the technical solutions or technical features described in the fourth aspect are applicable to the fifth and sixth aspects.

[0045] In a seventh aspect, the present application provides the following compounds, their stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts:

[0046] In some embodiments, the compound is used to prepare Compound A, Compound (A-1), Compound 2, Compound (2-1), Compound 4, or Compound (4-1).

[0047] In an eighth aspect, the present application provides a method for preparing compound D.

[0048] Step 1: reducing bis-benzyl protected glycine, or bis-benzyl protected glycine methyl ester or bis-benzyl protected glycine ethyl ester with lithium deuterated aluminum to obtain intermediate B;

[0049] Step 2: Intermediate B undergoes substitution reaction with bromoacetyl dimethyl acetal to obtain intermediate C;

[0050] Step 3: Debenzylation of intermediate C to obtain intermediate D.

[0051] In a ninth aspect, the present application provides a method for preparing compound (E-1), comprising the following steps:

[0052] Using 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2 as a raw material, intermediate (E-1) was obtained through reactions such as cyclization;

[0053] In a tenth aspect, the present application provides a method for preparing compound A, comprising the following steps:

[0054] Step 1: Intermediate E reacts with 7,8-difluoro-6,11-dihydrodibenzo[b,e]thia -11-alcohol is dehydrated by condensation to obtain intermediate F;

[0055] Step 2: Debenzylation of intermediate F yields compound A.

[0056] definition

[0057] Unless otherwise indicated, the following terms used in this application have the following meanings. A particular term should not be construed as undefined or unclear unless specifically defined, but rather should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0058] 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. Such salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or separately by reacting a free base or free acid with a suitable reagent.

[0059] Unless otherwise specified, the term "isotopic derivative" means that the compounds of the present invention may exist in an isotopically labeled or enriched form, containing one or more atoms whose atomic mass or mass number is different from the atomic mass or mass number of the largest atom found in nature. Isotopes can be radioactive or non-radioactive isotopes. Isotopes commonly used as isotopic labels are: hydrogen isotopes, 2 H and 3 H; Carbon isotope: 13 C and 14 C; Chlorine isotope: 35 Cl and 37 Cl; Fluorine isotope: 18 F; Iodine isotope: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. 3 H and 13 C, because they are easy to label and detect, they are more widely used. Some heavy isotopes, such as deuterium ( 2 H), substitution can enhance metabolic stability and prolong half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques similar to those used for synthesizing non-isotope-labeled compounds.

[0060] 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 optical isomers, 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, diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0061] 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.

[0062] 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, individual 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.

[0063] The compounds of the present invention also include solvates or solvates thereof. Unless otherwise specified, the terms "solvate" and "solvate" refer to the physical association of the compounds of the present invention with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some 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 phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0064] The term "optionally" 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.

[0065] The word "comprise" or "include" and its English variations such as comprises or comprising should be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0066] The term "subject," which may also be referred to as an "individual" or "subject," refers to a cell or animal, including but not limited to mammals, such as laboratory animals or humans. An "effective amount" refers to an amount of a compound or pharmaceutical composition described herein that is sufficient to achieve the intended application, including but not limited to the treatment of a disease or the 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 status, 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.

[0067] Some compounds in the present application are optically active. The compounds in the present application may be racemates, optical isomers or mixtures thereof. The synthesis of optical isomers in the compounds in the present application may be prepared from starting materials of optical isomers or by separation of racemates.

[0068] Unless otherwise specified, the term "pharmaceutically acceptable carrier", also known as "pharmaceutically acceptable excipient" or "pharmaceutically acceptable vehicle", refers to a medium generally recognized in the field of delivering biologically active agents to animals (particularly, mammals). Pharmaceutically acceptable carriers are formulated according to many factors that are well within the scope of those of ordinary skill in the art. These include (but are not limited to) the type and nature of the active agent being formulated, the subject to whom the composition containing the agent is to be administered, the intended route of administration of the composition, and the targeted therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media, as well as various solid and semi-solid dosage forms. The preparation of the pharmaceutical compositions described herein, including but not limited to, for example, mixing the compound described in the first aspect or the second aspect, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.

[0069] The above embodiments represent exemplary embodiments of the present application, but the present application is not limited to the above embodiments. In addition, the various technical features in the above embodiments of the present application can be combined with each other to form one or more new technical solutions, which also fall within the scope of the present application as long as such new technical solutions are technically feasible.

[0070] This application has at least one or more of the following beneficial effects:

[0071] 1) The compounds of the present application have good in vitro and / or in vivo antiviral effects;

[0072] 2) Compared with the control compound, the compound of the present application has good pharmacokinetic properties, for example, low plasma clearance, and / or good oral administration performance (for example, oral exposure, oral bioavailability, etc.);

[0073] 3) Compared with the control compound, the compound of the present application has good lung tissue distribution (e.g., lung exposure, etc.);

[0074] 4) Compared with the control compound, the compound of the present application has good liver microsomal metabolic stability, especially in the liver microsomes of cynomolgus monkeys and humans;

[0075] 5) Compared with the control compound, the compound of the present application has higher hERG inhibitory activity, suggesting that it has a lower risk of cardiotoxicity;

[0076] 6) The compounds of the present application have no inhibitory effect on five CYP enzymes (>50 μM). BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1: Weight change rate graph of Test Example 8. DETAILED DESCRIPTION

[0078] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples 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 meaning 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 application method. The preferred implementation methods and materials shown in the text are for demonstration purposes only.

[0079] The structures of the compounds described herein were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or liquid chromatography (HPLC). NMR measurements were performed using a Bruker 400 MHz and / or a Varian 400 MHz instrument; LC-MS measurements were performed using an Agilent 1260 Infinity II-6120 / 6125 MSD instrument; and HPLC measurements were performed using a Waters Acquity UPLC-2 and / or a Shimadzu LC2030 and / or an Agilent 1260 Infinity II instrument.

[0080] The starting materials in the examples of the present application are known and can be purchased on the market, or can be synthesized by or according to methods known in the art.

[0081] The present application provides a method for preparing the compound. The compound can be prepared by the following steps.

[0082] Abbreviations: PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N,N-dimethylformamide; MeOH: methanol; DCM: dichloromethane; DBU: 1,8-diazobisspiro[5.4.0]undec-7-ene; DMA: N,N-dimethylacetamide.

[0083] Example 1

[0084] (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazolinone Synthesis of methyl (5-methyl-2-oxo-1,3-dioxo-4-yl)methyl) carbonate (compound 1-1):

[0085] Step 1: Synthesis of chloromethyl (5-methyl-2-oxo-2H-1,3-dioxo-4-yl) methyl carbonate:

[0086] To a solution of 4-(hydroxymethyl)-5-methyl-2H-1,3-dioxetane-2-one (1.61 g, 12.41 mmol, 1.0 eq) in dichloromethane (200 ml) was added pyridine (1.23 g). Chloromethyl chloroformate (400 mg) was then added dropwise at 0°C. The mixture was allowed to react at room temperature for 1 hour. The reaction was quenched with water, extracted with dichloromethane, and the organic phase was washed with 1N dilute hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The title compound (300 mg) was obtained by flash chromatography (silica gel, PE:EA = 5:1, volume ratio) to obtain the title compound.

[0087] Step 2: Synthesis of compound 1-1:

[0088] To a solution of baloxavir (prepared according to reference (e.g., CN112940010A)) (130 mg, 0.27 mmol, 1.0 eq) in acetonitrile (5 mL) was added the product from the first step (300 mg), potassium carbonate (224 mg), and potassium iodide (45 mg) at room temperature, and the mixture was reacted at 70°C for 3 hours. The system was then returned to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative HPLC (C18, 0.1% aqueous formic acid, acetonitrile) to obtain compound 1-1 (111 mg). LCMS (ESI) [M+H] + =670.2; 1H NMR (400MHz, DMSO-d6) δ7.47–7.35(m,2H),7.26(d,J=7.8Hz,1H),7.20–7.12(m,1H),7.09(dd,J=8.0,1.3Hz,1H),7.01(d d,J=7.8,1.1Hz,1H),6.87(td,J=7.7,1.4Hz,1H),5.78–5.70(m,3H),5.68(s,1H),5.43(dd,J=14.4,2.0Hz,1H),5.06(q,J =14.1Hz,2H),4.44(dd,J=9.9,3.0Hz,1H),4.38(dd,J=13.2,1.6Hz,1H),4.06(d,J=14.3Hz,1H),3.98(dd,J=10.8,3.0Hz ,1H),3.66(dd,J=11.5,2.8Hz,1H),3.47(t,J=10.4Hz,1H),3.26(dd,J=11.6,2.5Hz,1H),3.01–2.88(m,1H),2.17(s,3H).

[0089] Example 2

[0090] (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazolinone Synthesis of 1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2 (Compound A):

[0091] Step 1: Synthesis of dibenzylglycine methyl ester:

[0092] Glycine methyl ester hydrochloride (15 g, 119.47 mmol, 1.0 eq) was dissolved in DMF (150 mL). N,N-diisopropylethylamine (54 g) and benzyl bromide (47 g) were added under ice-cooling. The mixture was reacted at room temperature for 12 hours. The mixture was diluted with ethyl acetate, washed twice with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=20:1 to 5:1) to obtain the title compound (30 g); LCMS (ESI) [M+1] + =270.1.

[0093] Step 2: Synthesis of 2-(dibenzylamino)ethane-1,1-d2-1-ol:

[0094] Dibenzylglycine methyl ester (30 g, 111.38 mmol, 1.0 eq) was dissolved in THF (250 mL), and lithium aluminum hydride (5 g) was added under ice-cooling. The mixture was reacted at room temperature for 3 hours, and the reaction was quenched under ice-cooling. The mixture was filtered through celite, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=20:1-3:1) to obtain the title compound (24 g); LCMS (ESI) [M+1] + =244.1.

[0095] Step 3: Synthesis of N,N-dibenzyl-2-(2,2-dimethoxyethoxy)ethane-1-amine-2,2-d2:

[0096] The product from the second step (24 g, 98.63 mmol, 1.0 eq) was dissolved in DMF (250 mL). Sodium hydride (26 g) was added under ice-cooling and the mixture was allowed to react at room temperature for 1 h. 2-Bromo-1,1-dimethoxyethane (83 g) was then added and the mixture was allowed to react at room temperature for 12 h. The reaction was quenched with ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=20:1-5:1) to obtain the title compound (30 g); LCMS (ESI) [M+1] + =332.1.

[0097] Step 4: Synthesis of 2-(2,2-dimethoxyethoxy)ethane-2,2-d2-1-amine:

[0098] Under hydrogen atmosphere, the product of step 3 (32 g, 96.55 mmol, 1.0 eq) and palladium hydroxide on carbon (13 g) were dissolved in MeOH (150 mL) and reacted at room temperature for 8 hours. The mixture was filtered through celite and concentrated under reduced pressure to obtain the title compound (14 g); LCMS (ESI) [M+1] + =152.1.

[0099] Step 5: Synthesis of methyl 3-(benzyloxy)-4-oxo-4H-pyran-2-carboxylate:

[0100] 3-(Benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (20 g, 81.23 mmol, 1.0 eq) and iodomethane (21 g) were dissolved in DMF (150 mL). 1,8-diazobisspiro[5.4.0]undec-7-ene (22.3 g) was added under ice-cooling. The mixture was reacted at room temperature for 12 hours. The mixture was diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=10:1 to 1:1) to obtain the title compound (20 g); LCMS (ESI) [M+1] +=261.2.

[0101] Step 6: Synthesis of methyl 3-(benzyloxy)-1-((tert-butoxycarbonyl)amino)-4-oxo-1,4-dihydropyridine-2-carboxylate:

[0102] The product of step 5 (19 g, 73.01 mmol, 1.0 eq), (tert-butoxy)carbohydrazide (14.5 g), and pyridinium 4-methylbenzenesulfonate (57 g) were dissolved in N,N-dimethylacetamide (150 mL) and reacted at 60°C for 15 hours. The mixture was diluted with ethyl acetate, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (DCM:MeOH=20:1-10:1) to obtain the title compound (16 g); LCMS (ESI) [M+1] + =375.1.

[0103] Step 7: Synthesis of tert-butyl (3-(benzyloxy)-2-((2-(2,2-dimethoxyethoxy)ethyl-2,2-d2)carbamoyl)-4-oxopyridin-1(4H)yl)carbamate:

[0104] The product from step 6 (2 g, 5.34 mmol, 1.0 eq) and 2-(2,2-dimethoxyethoxy)ethane-2,2-d2-1-amine (1.8 g) were dissolved in THF (20 mL), and DBU (1.6 g) was added. The mixture was reacted at 60°C for 48 hours, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (DCM:MeOH = 20:1 to 10:1) to obtain the title compound (1.8 g); LCMS (ESI) [M+1] + =494.3.

[0105] Step 8: Synthesis of 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2:

[0106] The product from step 7 (16.3 g, 33.03 mmol, 1.0 eq) was dissolved in H2O (30 mL) and acetonitrile (120 mL), and then methanesulfonic acid (9.5 g) was added. The mixture was reacted at 60°C for 7 hours and concentrated under reduced pressure. The pH was adjusted to about 11 with 20% (mass fraction) aqueous sodium hydroxide solution. The mixture was extracted with ethyl acetate (200 mL) and methanol (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound (6 g). LCMS (ESI) [M+1] + =330.1.

[0107] Step 9: Synthesis of (R)-7-(benzyloxy)-12-((R)-tetrahydrofuran-2-carbonyl)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2:

[0108] The product of step 8 (4 g, 12.15 mmol, 1.0 eq) was dissolved in ethyl acetate (11.6 mL), and a solution of 1-propylphosphoric anhydride in ethyl acetate (19.3 g) was added. The temperature was raised to 60°C, and triethylamine (2.5 g) and (R)-tetrahydrofurancarboxylic acid (1.7 g) were added. The mixture was reacted at 60°C for 4.5 hours, then cooled to 0°C and stirred continuously. A portion of the ethyl acetate (about 1 / 3) was concentrated under reduced pressure, and the mixture was stirred at 0°C-10°C overnight. The title compound (1.7 g) was purified and isolated; LCMS (ESI) [M+1] + =428.1.

[0109] Step 10: Synthesis of (R)-7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2:

[0110] The product from step 9 (2.5 g, 5.85 mmol, 1.0 eq) was dissolved in ethanol (18 mL). DBU (18 mg) was added under ice-cooling and the mixture was reacted at room temperature for 2 h. Isopropyl ether was added to the reaction solution and the mixture was stirred for 1 h. The mixture was filtered and dried to obtain the title compound (1.68 g). LCMS (ESI) [M+1] + =330.1.

[0111] Step 11: (R)-7-(Benzyloxy)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thia Synthesis of tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione-3,3-d2:

[0112] The product of the tenth step (1.3 g, 3.95 mmol, 1.0 eq), 7,8-difluoro-6,11-dihydrodibenzo[b,e]thia -11-ol (1.2 g) was dissolved in ethyl acetate (2 mL), followed by the addition of a solution of 1-propylphosphoric anhydride in ethyl acetate (3.8 g) and methanesulfonic acid (759 mg), and the reaction was allowed to proceed at 70°C for 6 hours. Water was added under ice-cooling, and the mixture was extracted with ethyl acetate and a small amount of tetrahydrofuran. The organic layer was washed with water, washed with sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was added with tetrahydrofuran (8 mL), potassium carbonate (960 mg), and benzyl bromide (36 μL), and stirred at 60°C for 16 hours. 2 M hydrochloric acid was added to the reaction mixture to adjust the pH to approximately 6, and the mixture was extracted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. Purification and isolation yielded the title compound (620 mg); LCMS (ESI) [M+1] + =576.2.

[0113] Step 12: Synthesis of Compound A:

[0114] The product of step 11 (630 mg, 1.09 mmol, 1.0 eq) and lithium chloride (237 mg) were dissolved in N,N-dimethylacetamide (5 mL) and reacted at 80°C for 3 hours. 0.5 M dilute hydrochloric acid was added to the reaction solution to adjust the pH to about 6, diluted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound A (500 mg) after purification and separation; LCMS (ESI) [M+1] + =486.2.

[0115] 1 HNMR(400MHz, CDCl3)δ7.17–7.08(m,3H),7.08–6.99(m,2H),6.87–6.82(m, 1H),6.69(d,J=7.5Hz,1H),5.78(d,J=7.7Hz,1H),5.32–5.25(m,2H),4.66( d,J=13.6Hz,1H),4.59(dd,J=10.0,3.0Hz,1H),4.07(d,J=13.8Hz,1H),3.9 7(dd,J=11.1,3.0Hz,1H),3.68–3.58(m,1H),2.99(dd,J=21.4,7.7Hz,1H).

[0116] Example 3

[0117] (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazolinone Synthesis of methyl (5-methyl-2-oxo-1,3-dioxo-4-yl)methyl) carbonate (compound 2-1):

[0118] Compound A (1.02 g), potassium carbonate (821 mg), and potassium iodide (150 mg) were dissolved in acetonitrile (10 mL), reacted at 85° C. for 1.5 hours, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (DCM:MeOH=20:1-10:1) to obtain the title compound, which was further separated by preparative HPLC to obtain the title compound (155 mg); LCMS (ESI) [M+1] + =672.3[M+1]. 1 HNMR (400MHz, DMSO-d6) δ7.48–7.36(m,2H),7.26(d,J=7.8Hz,1H),7.15(t,J=7.5Hz,1H),7.09(d,J= 6.9Hz,1H),7.01(d,J=7.1Hz,1H),6.87(t,J=6.8Hz,1H),5.76–5.70(m,3H),5.68(s,1H),5.43(d,J=1 5.9Hz,1H),5.06(q,J=14.1Hz,2H),4.44(dd,J=9.9,3.0Hz,1H),4.37(d,J=13.5Hz,1H),4.06(d,J=14 .4Hz,1H),3.97(dd,J=10.7,2.9Hz,1H),3.47(t,J=10.4Hz,1H),2.93(d,J=13.6Hz,1H),2.17(s,3H).

[0119] Example 4

[0120] Synthesis of 1-((L-valine)oxy)ethyl(2S,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (Compound 3-1):

[0121] Step 1: Synthesis of ethyl (2S,3S)-3-((5-fluoro-2-(5-fluoro-1-trityl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate:

[0122] Ethyl (2S,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (860 mg, 2.01 mmol, 1.0 eq), triphenylmethane chloride (858 mg), and potassium carbonate (555 mg) were dissolved in DMF (10 mL) and reacted at room temperature for 5 hours. The reaction solution was diluted with ethyl acetate, washed twice with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=10:1 to 5:1) to obtain the title compound (1.3 g); LCMS (ESI) [M+1] + =670.5.

[0123] Step 2: Synthesis of (2S,3S)-3-((5-fluoro-2-(5-fluoro-1-trityl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid:

[0124] The product from the first step (1.3 g, 1.94 mmol, 1.0 eq) was dissolved in THF (15 mL) / MeOH (15 mL) / H2O (15 mL), and lithium hydroxide (116 mg) was added. The mixture was reacted at 45°C for 6 hours and concentrated under reduced pressure. Dilute hydrochloric acid [1 M] was added to the concentrate to adjust the pH to about 6. The product was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (DCM:MeOH = 50:1 to 10:1) to obtain the title compound (1.2 g); LCMS (ESI) [M+1] + =642.5.

[0125] Step 3: Synthesis of 1-(((tert-Butoxycarbonyl)-L-valine)oxy)ethyl(2S,3S)-3-((5-fluoro-2-(5-fluoro-1-trityl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate:

[0126] The product from the second step (250 mg, 0.39 mmol, 1.0 eq), 1-chloroethyl(tert-butyloxycarbonyl)-L-valine (327 mg), potassium carbonate (162 mg), and potassium iodide (65 mg) were dissolved in acetonitrile (8 mL), reacted at 75°C for 7 hours, and concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (PE:EA=10:1-2:1) to obtain the title compound (210 mg); LCMS (ESI) [M+1] + =886.7.

[0127] Step 4: Synthesis of compound 3-1:

[0128] The product from step 3 (180 mg, 0.2 mmol, 1 eq) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2328 mg) and triethylsilane (474 ​​mg) were added under ice-cooling. The mixture was reacted at room temperature for 2 hours, concentrated under reduced pressure, and separated by preparative HPLC (80 mg); LCMS (ESI) [M+1] + =543.4.

[0129] 1 HNMR(400MHz,DMSO-d6)δ12.27(s,1H),8.54–8.45(m,1H),8.28(s,1H),8.21–8.16(m,2H),7. 64(dd,J=14.6,7.1Hz,1H),6.82(dd,J=11.2,5.6Hz,1H),4.75(d,J=7.0Hz,1H),3.25(d,J=4.7 Hz,1H),3.12(d,J=5.2Hz,1H),2.98(dd,J=21.4,6.8Hz,1H),1.99(d,J=30.4Hz,2H),1.87–1.7 2(m,4H),1.65(s,1H),1.52(d,J=10.2Hz,3H),1.39(dd,J=7.4,5.5Hz,5H),0.86–0.72(m,6H).

[0130] Example 5

[0131] (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazolinone Synthesis of methyl carbonate (compound 4-1):

[0132] Compound A (220 mg, 0.45 mmol, 1 eq.), dimethyl chloromethyl carbonate (254 mg), potassium carbonate (188 mg), and potassium iodide (38 mg) were dissolved in acetone (3 mL) and reacted at 55°C for 16 hours. The mixture was concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography (DCM:MeOH = 20:1 to 10:1) to obtain the title compound. The title compound was further separated by preparative HPLC to obtain the title compound (190 mg); LCMS (ESI) [M+1] + =574.3; 1HNMR(400MHz,DMSO-d6)δ7.41(t,J=4.8Hz,2H),7.23(d,J=7.8Hz,1H),7.19–7.13(m,1H),7.09(d d,J=8.0,1.1Hz,1H),7.01(d,J=6.8Hz,1H),6.88–6.82(m,1H),5.78–5.70(m,3H),5.67(d,J=6.6 Hz,1H),5.46–5.38(m,1H),4.45(dd,J=9.9,3.0Hz,1H),4.39(d,J=13.5Hz,1H),4.06(d,J=14.3H z,1H),3.99(dd,J=10.8,3.0Hz,1H),3.73(s,3H),3.45(t,J=10.4Hz,1H),2.94(d,J=13.6Hz,1H).

[0133] Biological testing

[0134] Test Example 1: Pharmacokinetic evaluation after intravenous or oral administration in dogs

[0135] This study evaluated the pharmacokinetics of the present compound in healthy, adult male beagle dogs. The present compound was administered as a 5% DMSO + 10% Solutol + 85% Saline aqueous solution. Dogs were fasted overnight prior to administration and food was resumed 4 hours after administration. For intravenous (iv) administration, animals were dosed at 1.5 mg / kg; for oral (po) administration, animals were dosed at 2 mg / kg. Blood (0.5 mL) was collected from the dogs at time points of 0.083 (iv), 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 hours, collected in EDTA-K2 anticoagulant tubes, and the plasma solution was collected by centrifugation. PMSF (phenylmethylsulfonyl fluoride) was added to a final concentration of 1 mM, mixed thoroughly, and stored at -20°C or -70°C until the above-mentioned LC / MS analysis.

[0136] After testing, the compound 2-1 of the present application was mainly converted into compound A in the body, and the test results were the results of compound A.

[0137] The results showed that the compound 2-1 of the present application had good pharmacokinetic properties after administration. For example, at the same dosage (1.5 mg / kg intravenously, 2 mg / kg orally), compared with mabaloxavir, the compound 2-1 of the present application had a significantly lower T 1 / 2 The exposure time is 7.38h, which is longer than that of mabaloxavir; (2) AUC 24hThe clearance rate was 1098 hr*ng / mL, which was significantly higher than that of Mabaloxavir; (3) the clearance rate was 20.9 mL / min / kg, which was significantly lower than that of Mabaloxavir. After oral administration of compound 2-1 of the present application, (1) T 1 / 2 The oral exposure is 8.62h, which is longer than that of mabaloxavir; (2) oral exposure AUC 24h It is 834hr*ng / mL, which is significantly higher than that of mabaloxavir.

[0138] Test Example 2: Pharmacokinetic Evaluation in Mice

[0139] This study evaluated the pharmacokinetics of the compound in healthy, adult female Balb / c mice. The compound was administered as a 5% DMSO + 10% Solutol + 85% Saline solution in water. Mice were fasted overnight prior to administration and food was resumed 4 hours after administration. For intravenous (iv) administration, animals were dosed at 2 mg / kg; for oral (po) administration, animals were dosed at 10 mg / kg. Blood (0.03 mL) was collected from mice at time points: 0.083 (iv), 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 hours.

[0140] The results showed that the compound 1-1 of the present application can be converted into baloxavir in vivo, and after oral administration, it has good pharmacokinetic properties (for example, higher C max ) and good oral bioavailability (>60%).

[0141] Note: VX-787 was prepared according to references (e.g., Clark, Michael P., et al. Journal of Medicinal Chemistry. 2014, 57(15), 6668-6678.)

[0142] The test results show that the compound 3-1 of the present application can be converted into VX-787 in the body, and the test results are the results of VX-787. Test results: After oral administration, at the same dosage (2 mg / kg intravenously, 10 mg / kg orally), compared with VX-787, (1) the plasma clearance rate of the compound 3-1 of the present application after intravenous administration was 11.4 mL / min / kg, which was lower than that of VX-787; (2) the exposure amount AUC of the compound 3-1 of the present application in mice after oral administration was 11.4 mL / min / kg. 24h The oral bioavailability is 7609 hr*ng / mL, which is significantly higher than that of VX-787; (3) The oral bioavailability is 56%, which is higher than that of VX-787.

[0143] Test Example 3: Plasma and lung pharmacokinetic evaluation in mice

[0144] In order to demonstrate the drug's lung tissue distribution, a pharmacokinetic study of lung tissue distribution in mice (Balb / c) was conducted. The compound was orally administered with a 5% DMSO + 10% Solutol + 85% Saline aqueous solution. The mice were fasted overnight before administration and resumed feeding 4 hours after administration. The animals were given a dose of 10 mg / kg. Blood (0.1 mL) was collected from the mice at time points of 0.5, 1.0, 2.0, and 4.0 hours, collected in anticoagulant tubes containing EDTA-K2 anticoagulant, centrifuged to obtain plasma, and PMSF (phenylmethylsulfonyl fluoride) was added to a final concentration of 1 mM, and thoroughly mixed. After blood sample collection, the animals were euthanized by CO2 inhalation, and lung tissue samples were collected, and a homogenate containing 1 mM PMSF was added. The samples were stored at -70°C until the above-mentioned LC / MS analysis was performed.

[0145] Note: Mabaloxavir was prepared according to references (e.g., CN112940010A).

[0146] The results showed that the compound 1-1 of the present application can be converted into baloxavir in the body, and the test results are the results of baloxavir. The results showed that the compound 1-1 of the present application has good oral administration performance after administration. For example, at the same dosage, the compound 1-1 of the present application has good lung exposure AUC after oral administration (1). last The plasma exposure AUC was 6582 hr*ng / mL, which was significantly higher than that of mabaloxavir; (2) last The calculated lung tissue exposure / plasma exposure ratio was 411 hr*ng / mL, which was lower than that of mabaloxavir. (3) The calculated lung tissue exposure / plasma exposure ratio was 16, which was significantly higher than that of mabaloxavir. It can be seen that compound 1-1 can achieve higher target tissue exposure and lower peripheral and non-target tissue exposure after oral administration.

[0147] The compound 4-1 of the present application can be converted into baloxavir in the body, and the test results are the results of baloxavir. The results show that the compound 4-1 of the present application has good oral administration performance after administration. For example, at the same dosage, the compound 4-1 of the present application has good oral administration performance after oral administration. (1) Lung T max 1 hour, which is equivalent to mabaloxavir; (2) lung C max The lung exposure AUC was 5709 ng / mL, which was significantly higher than that of mabaloxavir; (3) last The plasma exposure AUC was 12752 hr*ng / mL, which was significantly higher than that of mabaloxavir; (4) lastThe calculated lung tissue exposure / plasma exposure ratio was 25.8, significantly higher than that of mabaloxavir. This indicates that compound 4-1 can achieve higher target tissue exposure and lower peripheral and non-target tissue exposure after oral administration.

[0148] Test Example 4: Pharmacokinetic evaluation of a quantitative compound in rats after intravenous or oral administration

[0149] This study evaluated the pharmacokinetics of the present compound in healthy, adult male SD rats. The present compound was administered as a 5% DMSO + 10% Solutol + 85% Saline aqueous solution. Rats were fasted overnight before administration and resumed feeding 4 hours after administration. For intravenous (iv) administration, animals were given a dose of 1 mg / kg; for oral (po) administration, animals were given a dose of 5 mg / kg. Blood (0.12 mL) was collected from rats at time points of 0.083 (iv), 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 hours, collected in anticoagulant tubes containing EDTA-K2 anticoagulant, immediately placed on wet ice, and centrifuged to collect plasma. 45 μL of plasma was immediately added to a centrifuge tube containing 5 μL of 10 mM PMSF, mixed thoroughly, and stored at -20°C or -70°C until the above-mentioned LC / MS analysis.

[0150] The results showed that: the compound 4-1 of the present application can be converted into baloxavir in vivo. (1) After oral administration, the exposure amount AUC of compound 4-1 in rats was last It is 479hr*ng / mL, which is significantly higher than that of mabaloxavir; (2) after oral administration, the half-life is 3.13hr, which is higher than that of mabaloxavir; (3) the oral bioavailability is 24.8%, which is higher than that of mabaloxavir.

[0151] Test Example 5: Pharmacokinetic evaluation of monkeys after intravenous or oral administration of a quantitative compound

[0152] This study evaluated the pharmacokinetics of the present compound in healthy, adult male cynomolgus monkeys. The present compound was administered as a 5% DMSO + 10% Solutol + 85% Saline aqueous solution. The monkeys were fasted overnight before administration and food was resumed 4 hours after administration. For intravenous (iv) administration, the animals were dosed at 1 mg / kg; for oral (po) administration, the animals were dosed at 2 mg / kg. Blood (0.5 mL) was collected from the monkeys at time points of 0.083 (iv), 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 hours, collected in EDTA-K2 anticoagulant anticoagulant tubes, immediately placed on wet ice, and centrifuged to collect plasma. 180 μL of plasma was added to a centrifuge tube containing 20 μL of 10 mM PMSF, mixed thoroughly, and stored at -20°C or -70°C until the above-mentioned LC / MS analysis.

[0153] The results showed that: after testing, the compound 4-1 of the present application can be converted into baloxavir in vivo. (1) After oral administration, the C max , which is significantly higher than that of mabaloxavir; (2) After oral administration, the T max , which is shorter than that of mabaloxavir; (3) AUC exposure in monkeys last The oral bioavailability is 48.1%, which is higher than that of mabaloxavir.

[0154] Test Example 6: Stability test of liver microsomes of different species

[0155] The metabolic stability of the compound was evaluated in different species (dog, cynomolgus monkey, and human) using the liver microsome method. Compounds at a concentration of 1 μM were incubated with 0.5 mg / mL liver microsomes at 37°C in the presence of NADPH as a cofactor for 0, 5, 15, 30, 45, and 60 minutes. The amount of compound was monitored by LC / MS / MS, and t was calculated based on the amount of compound reduction. 1 / 2 and intrinsic clearance. The experimental results are shown in Table 1.

[0156] Table 1. T values ​​of compounds in liver microsomes of different species 1 / 2 result

[0157] Test Example 7: Using patch clamp technology to detect the inhibitory effect of drugs on hERG channels

[0158] To assess the risk of QT prolongation on electrocardiogram (ECG), HEK293 cells expressing humanether-a-go-go related gene (hERG) channels were used to investigate the effects on delayed rectifier K+ current, which plays an important role in ventricular repolarization.

[0159] HEK 293 cell lines (Cat. K1236) stably expressing the hERG channel were purchased from Invitrogen. Cells were cultured in a medium containing 10% FBS, 5 μg / mL Blasticidin, and 400 μg / mL Geneticin. Prior to the assay, cells were induced with 1 μg / mL doxycycline for 48 hours. On the day of the experiment, the induced cells were resuspended and plated on coverslips before use, with approximately 5 × 10 cells per 3.5 cm cell culture dish. 5 Cells were cultured in medium without blasticin and geneticin. Compounds were diluted three-fold to five test concentrations, with a final DMSO concentration of 0.1% in the culture medium. The coverslip was removed from the cell culture dish, and the recording electrodes were placed in contact with the cells using a microelectrode manipulator under an inverted microscope. Drug administration was initiated after the hERG current recorded from the cells stabilized. Each drug concentration was allowed to act for 5 minutes (current stabilization) before the next concentration was tested, with cells treated sequentially from low to high concentrations. The experimental results are shown in Table 2.

[0160] Table-2

[0161] The test results showed that compound 4-1 had very low inhibition on hERG, suggesting that it had no potential cardiotoxicity risk or had very low cardiotoxicity risk.

[0162] Test Example 8: Evaluation of the efficacy of compounds in an influenza A virus H1N1 mouse infection model

[0163] Mice were inoculated intranasally with influenza virus (H1N1, A / PR / 8 / 34). Compounds were administered orally starting 48 hours after infection, twice daily for 7 consecutive days. The anti-influenza A (H1N1) virus activity in this model was evaluated by observing changes in mouse body weight. SPF-protected BALB / c female mice, 6-8 weeks old, were used. Experiments began after at least 3 days of acclimatization upon arrival. The day of infection was designated as day 0. One day prior, animals were randomly divided into 18 groups, with 6 mice per group. On the day of inoculation (day 0), animals were anesthetized with an intraperitoneal injection of 50 mL / kg of Zolta 50 (30 mg / kg) and 6 mg / kg of Xylazine Hydrochloride Injection II (both injection volumes were 5 mL / kg). The H1N1 (A / PR / 8 / 34) virus was then inoculated intranasally with a 50 μL inoculum. From day 2 to day 8, 5 mg / kg of the test compound was administered orally twice daily. The first dose was 48 hours after infection. The mice were observed daily and their body weights were recorded for 14 days to measure the rate of weight loss.

[0164] The results showed that compound 4-1 could achieve significant recovery of the weight of infected animals on day 9 after administration, with the weight loss rate decreasing by 2.9% compared with that before virus inoculation (Day 0), and the recovery effect was more significant than that of mabaloxavir (the weight loss rate of animals on day 9 decreased by 11.46% compared with that before virus inoculation (Day 0)) (p<0.05); further recovery of the weight of infected animals could be achieved on day 10 (the weight loss rate decreased by 0.21% compared with that before virus inoculation (Day 0)), and the recovery effect was more significant than that of mabaloxavir (the weight loss rate of animals on day 10 decreased by 8.04% compared with that before virus inoculation (Day 0)) (p<0.05); on day 11, the weight of animals in the compound 4-1 group increased compared with that on Day 0, and the weight of animals also gradually increased in the following days, which shows that compound 4-1 has good efficacy and can achieve rapid and continuous recovery of the weight of infected animals. However, the weight of animals in the mabaloxavir group did not exceed the weight before administration (Day 0) until day 13. 0). The experimental results are shown in Figure 1.

[0165] The test results showed that after viral infection, animals given compound 4-1 of the present application achieved significantly faster and better weight recovery than those given mabaloxavir at the same dose, demonstrating the compound's superior pharmacodynamic activity and rapid improvement in the animals' condition. Furthermore, compound 4-1 effectively reduced viral titers in the animals' lungs, achieving a 100% survival rate.

[0166] Test Example 9: Influenza virus cytopathic effect (CPE) and cytotoxicity experiments

[0167] The half effective concentration (EC50) of the compound was determined 50 ) values ​​were used to evaluate the antiviral activity and cytotoxic CC of the compounds against influenza virus (H1N1, A / PR / 8 / 34). 50 . Cytopathic effect assay is widely used to determine the protective effect of compounds on virus-infected cells to reflect the antiviral activity of compounds. MDCK cells were seeded into cell culture plates at a density of 15,000 cells per well and then cultured overnight in a 37°C, 5% CO2 incubator. The compounds were diluted in 3-fold increments, with 8 test concentration points, and the final DMSO concentration in the culture medium was 0.5%. Influenza virus (H1N1, A / PR / 8 / 34, MOI = 0.0067) was then added. Virus control wells (DMSO and virus added, no compound added), cell control wells (DMSO added, no compound and virus added), and culture medium control wells (only culture medium added, no cells, virus and compound added) were set up. The cell plate was cultured in a 37°C, 5% CO2 incubator for 5 days. After 5 days of culture, the cell viability was detected using the cell viability detection kit CCK8, and the raw data was used to calculate the antiviral activity and cytotoxicity of the compound. The antiviral activity of the compound is expressed by the inhibition rate (%) of the compound on the cytoviral effect caused by the virus. The calculation formula is as follows:

[0168] % inhibition rate = (average value of compound-treated wells - average value of virus control) / (average value of cell-treated wells - average value of virus control) The inhibition rate of the compound was analyzed by nonlinear fitting using GraphPad Prism software to obtain the EC value of the compound. 50 value.

[0169] The cytotoxicity of the compound is expressed by the inhibition rate (%) of the compound on cell activity. The calculation formula is as follows:

[0170] % inhibition rate = (average value of compound-treated wells - average value of culture medium control) / (average value of cell control wells - average value of culture medium control). The experimental results are shown in Table 3.

[0171] Table-3 CPE and cytotoxicity test results Note: + indicates >20nM, ++ indicates 10nM-20nM, +++ indicates 5nM-10nM, and ++++ indicates <5nM.

[0172] The test results show that the compound 4-1 of the present application and its metabolite (compound A) have less effect on cell viability, that is, less cytotoxicity, but have significantly better inhibitory activity against viral replication, indicating that the compound has better efficacy and better safety.

[0173] Test Example 10: Human CYP enzyme inhibition study

[0174] Human liver microsomes (Corning, CYP1A2, 2C9, 2C19, 2D6, 3A4-M) were removed from a -80°C freezer and thawed on ice. 20 μL of a CYP substrate mixture (phenacetin / diclofenac / smephenytoin / dextromethorphan / midazolam mixture) was added to the assay plate. 2 μL of the test compound and positive control compound were added to the corresponding wells. 158 μL of the human liver microsome working solution (0.253 mg / mL) was added to all wells of the assay plate and preheated in a 37°C water bath for 10 minutes.

[0175] Prepare NADPH cofactor solution (BONTAC, Cat. No. BT04) and add 20 μL of 10 mM NADPH cofactor to all wells. Mix and incubate in a 37°C water bath for 10 minutes. Terminate the reaction by adding 400 μL of stop solution. Centrifuge the sample at 4000 rpm for 20 minutes to precipitate the protein. Transfer 200 μL of the supernatant to 100 μL of HPLC water and shake for 10 minutes before analyzing the sample by LC / MS / MS.

[0176] Results: The IC50 values ​​of compound A of the present application against five major human CYP molecular species were all greater than 50 μM.

Claims

1. A compound of formula (I), its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, Among them, R is hydrogen, is a linking site; R1 and R2 are simultaneously hydrogen or simultaneously deuterium; n is selected from 0 - 3; preferably 0, 1, 2 or 3; more preferably 0, 1 or 2; even more preferably 0 or 1; Each occurrence of R3 and R4 is independently selected from H, deuterium or C 1-4 alkyl; preferably H or C 1-3 alkyl; more preferably H, methyl or ethyl; Each occurrence of R5 is independently selected from H, deuterium or C 1-4 alkyl; preferably H or C 1-3 alkyl; more preferably H, methyl or ethyl; Provided that when R is H or and when both R5 are H, both R1 and R2 are deuterium.

2. The compound according to claim 1, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, wherein, The compound has the following structure:

3. A compound, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, said compound having the following structure:

4. The compound according to claim 3, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, wherein, The compound has the following structure:

5. A compound, stereoisomer, tautomer, isotopic derivative or pharmaceutically acceptable salt according to any one of claims 1-4, wherein, The isotope derivative is a deuterated derivative.

6. A pharmaceutical composition comprising the compound according to any one of claims 1 - 5, its stereoisomers, tautomers, isotope derivatives or pharmaceutically acceptable salts, and optionally further comprising a pharmaceutically acceptable carrier.

7. Use of the compound according to any one of claims 1 - 5, its stereoisomers, tautomers, isotope derivatives or pharmaceutically acceptable salts, or the pharmaceutical composition according to claim 6 as a drug or in the preparation of a drug; preferably, the drug is a polymerase inhibitor drug; preferably, the drug is an RNA polymerase inhibitor drug.

8. The use according to claim 7, wherein The drug is a cap - structure - dependent endonuclease inhibitor drug; preferably, the drug is a polymerase acidic protein inhibitor drug or an RNA polymerase basic protein 2 inhibitor drug.

9. The use according to claim 7, wherein, The drug is used for the treatment and / or prevention of diseases caused by viruses; preferably, the drug is used for the treatment and / or prevention of diseases caused by viruses having a cap - structure - dependent endonuclease; preferably, the drug is used for the treatment and / or prevention of diseases caused by viruses having a polymerase acidic protein; preferably, the disease is a disease caused by an orthomyxovirus; preferably, the disease is influenza; more preferably, the disease is influenza A, B or C.

10. The use according to claim 7, wherein The drug is an antiviral drug; preferably, the drug is an anti - orthomyxovirus drug; more preferably, the drug is a drug that inhibits the replication of influenza virus; more preferably, the influenza virus is influenza A virus, influenza B virus or influenza C virus.

11. Use according to any one of claims 7-10, wherein, The drug can be used in combination with other drugs for the treatment and / or prevention of diseases caused by viruses; preferably, the other drugs include neuraminidase inhibitors, polymerase acidic protein inhibitors, M2 protein inhibitors, RNA - dependent RNA polymerase inhibitors, PB2 Cap - binding inhibitors, anti - HA antibodies or immunomodulators, etc.; more preferably, the other drugs are administered simultaneously or sequentially with the drug.

12. A drug combination comprising the compound according to any one of claims 1 - 5, its stereoisomers, tautomers, isotope derivatives or pharmaceutically acceptable salts, and another drug for the treatment and / or prevention of diseases caused by viruses; preferably, the other drug includes neuraminidase inhibitors, polymerase acidic protein inhibitors, M2 protein inhibitors, RNA - dependent RNA polymerase inhibitors, PB2 Cap - binding inhibitors, anti - HA antibodies or immunomodulators, etc.

13. A compound, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, wherein, The compound has the following structure:

14. The compound according to claim 13, its stereoisomers, tautomers, isotopic derivatives or pharmaceutically acceptable salts, wherein, The compound is used for the preparation of compound A, compound (A - 1), compound 2, compound (2 - 1), compound 4 or compound (4 - 1) according to claim 3 or 4.

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