Picolinamide derivative and application thereof

Abstract

The invention discloses a picolinamide derivative and application thereof. The derivative is a compound as shown in the following formula (I) or a solvate, a polymorphic substance, an isotope derivative, a tautomer, a stereoisomer and a pharmaceutically acceptable salt thereof, wherein the definition of each substituent is shown in the specification. In addition, the invention also discloses a preparation method of the derivative. The compound provided by the invention can be used for preparing drugs for treating and / or preventing influenza virus infection. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to pyridine amide derivatives or their solvates, polymorphs, isotope derivatives, tautomers, stereoisomers and pharmaceutically acceptable salts, as well as preparation methods and uses thereof in preparing drugs for treating and / or preventing influenza virus infection. Background Art

[0002] Anti-influenza drugs mainly act on viral proteins or different steps of viral replication, such as viral RNA polymerase, neuraminidase (NA), hemagglutinin, M2 ion channel, viral nucleoprotein (RNP) or sialic acid receptors. In addition, inhibitors acting on host cell targets include protease and vesicle proton ATP inhibitors, but the efficacy and potential toxic side effects of such drugs still need further evaluation.

[0003] Due to the shortcomings of influenza drugs such as M2 ion channel inhibitors and neuraminidase inhibitors, such as low efficacy, narrow time window and / or easy emergence of drug resistance, recent anti-influenza drug research and development has focused on viral RNA polymerase. Influenza virus RNA polymerase is composed of three subunits: PA, PB1 and PB2, which work together to complete the "three steps" of influenza virus RNA replication. First, the PB2 subunit specifically binds to the cap structure of the host cell mRNA precursor; then the PA subunit cuts and hijacks the mRNA precursor cap structure to the viral RNA at 10-15 nucleotides downstream of the cap structure through the action of an endonuclease; finally, the PB1 subunit replicates the viral RNA through the activity of the replicase. In recent years, RNA polymerase inhibitors have also developed a certain degree of drug resistance.

[0004] Influenza virus nucleoprotein (NP) is wrapped around viral RNA to form viral ribonucleoprotein (vRNP), which mediates many key steps in the viral life cycle, including genome replication, and remains highly conserved in influenza viruses. Therefore, NP protein has been considered as a target for anti-influenza virus drugs.

[0005] Naproxen is a type of nonsteroidal anti-inflammatory drug (NSAID). In addition to its anti-inflammatory effect, it can also block the replication of influenza virus by binding to NP protein and blocking NP nuclear export. Phase IIb / III clinical trials have shown that clarithromycin-naproxen-oseltamivir combination therapy reduced the mortality of hospitalized patients with H3N2 influenza infection at 30 days and 90 days, and shortened the length of hospital stay. In another study of hospitalized children with influenza, the clarithromycin-naproxen-oseltamivir combination therapy group required a shorter time to defervescence than the oseltamivir monotherapy group, and the influenza virus titer decreased faster. Since naproxen is already on the market and widely used in clinical practice, the time required to develop it as a clinical anti-influenza drug may be shorter than that required for other new anti-influenza drugs.

[0006] In addition, preclinical studies have found that FA-6005 can inhibit the activity of viral RNP and block its intracellular transport, and can interfere with the viral life cycle through multiple mechanisms, which has been verified in cell and animal models and needs to be further evaluated in clinical trials.

[0007] Most of the anti-influenza drugs currently on the market need to be used within 48 hours of the onset of symptoms, and they have all developed varying degrees of drug-resistant mutations. There is still hope to develop multi-target anti-influenza drugs that can take effect quickly and prolong the duration of drug action, thereby reducing drug resistance and side effects. Summary of the invention

[0008] Unless otherwise specified herein, the professional terms used in the present invention have the basic meanings generally understood by those skilled in the art.

[0009] The present invention provides a class of compounds capable of treating and / or preventing influenza virus infection, and a preparation method and use thereof.

[0010] The present invention provides a compound as shown in formula (I), or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt:

[0011]

[0012] In formula (I), R1 is selected from hydrogen, deuterium or fluorine;

[0013] R2 is selected from: fluorine, C1-C3 alkyl substituted by one or more deuterium atoms, C1-C3 alkyl substituted by one or more fluorine atoms;

[0014] R3, R4, R5 and R6 are each independently hydrogen or deuterium;

[0015] R7 and R8 are each independently selected from hydrogen, or the following groups which are substituted or unsubstituted by one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl;

[0016] The group A is selected from the following groups in which deuterium or one or more hydrogens are substituted or unsubstituted by deuterium: C1-C6 alkoxy, C3-C6 cycloalkoxy;

[0017] X1 and / or X2 are each independently selected from: absent, hydrogen, When R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, any one of X1 and / or X2 is not hydrogen; wherein Ra is selected from the following groups which are substituted or unsubstituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl; Rb and Rc are each independently selected from hydroxyl, or the following groups which are substituted or unsubstituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl, or Rb and Rc together with the oxygen atom and the phosphorus atom to which they are connected form a 5-7 membered ring;

[0018] “--” indicates bonding or non-bonding, and the “--” shown has one and only one bonding. When “--” indicates bonding, the two connected atoms form a double bond, and X1 or X2 connected to the double bond does not exist;

[0019] In particular, formula (I) shall not be

[0020] In some preferred embodiments, the compounds of formula (I-1) provided by the present invention, or their solvates, polymorphs, isotopic derivatives, tautomers, stereoisomers and pharmaceutically acceptable salts, when R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, X1 and / or X2 shall not be simultaneously selected from: hydrogen or not present.

[0021] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, tautomer, pharmaceutically acceptable salt is as shown in formula (II):

[0022]

[0023] The substituents in formula (II) are as defined in formula (I).

[0024] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (III):

[0025]

[0026] The substituents in formula (III) are as defined in formula (I).

[0027] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (IV):

[0028]

[0029] The substituents in formula (IV) are as defined in formula (I).

[0030] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (V):

[0031]

[0032] The substituents in formula (V) are as defined in formula (I).

[0033] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (VI):

[0034]

[0035] The substituents in formula (VI) are as defined in formula (I).

[0036] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (VII):

[0037]

[0038] The substituents in formula (VII) are as defined in formula (I).

[0039] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (VIII):

[0040]

[0041] The substituents in formula (VIII) are as defined in formula (I).

[0042] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (II-1):

[0043]

[0044] In formula (II-1), R1 is selected from hydrogen or deuterium;

[0045] R2 is selected from: trifluoromethyl, methyl, deuterated methyl;

[0046] R3, R4, R5 and R6 are each independently hydrogen or deuterium;

[0047] R7 is selected from: hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;

[0048] R8 is selected from: hydrogen, or the following groups which may be substituted or unsubstituted by one or more groups A: C1-C6 alkyl;

[0049] The group A is selected from: deuterium, C1-C6 alkoxy, C3-C6 cycloalkoxy;

[0050] X1 and / or X2 are each independently selected from: absent, hydrogen, When R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, X1 and / or X2 shall not be simultaneously selected from: hydrogen or not exist; wherein, Ra is selected from the following groups substituted or not substituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl; Rb and Rc are each independently selected from hydroxyl, or the following groups substituted or not substituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl, or Rb and Rc together with the oxygen atom and phosphorus atom to which they are connected form a 5-7 membered ring;

[0051] “--” indicates bonding or non-bonding, and the “--” shown has one and only one bonding. When “--” indicates bonding, the two connected atoms form a double bond, and X1 or X2 connected to the double bond does not exist.

[0052] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (III-1):

[0053]

[0054] In formula (III-1), R1 is selected from hydrogen or deuterium;

[0055] R2 is selected from: trifluoromethyl, methyl, deuterated methyl;

[0056] R3, R4, R5 and R6 are each independently hydrogen or deuterium; and at least one of R1, R2, R3, R4, R5 and R6 is deuterium;

[0057] R7 is selected from: hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;

[0058] R8 is selected from: hydrogen, or the following groups which may be substituted or unsubstituted by one or more groups A: C1-C6 alkyl;

[0059] Group A is selected from: C1-C6 alkoxy, C3-C6 cycloalkoxy.

[0060] In some embodiments, the compound provided by the present invention or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt are as shown in formula (IV-1):

[0061]

[0062] In formula (IV-1), R1 is selected from hydrogen or deuterium;

[0063] R2 is selected from: trifluoromethyl, methyl, deuterated methyl;

[0064] R3, R4, R5 and R6 are each independently hydrogen or deuterium;

[0065] R7 is selected from: hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;

[0066] R8 is selected from: hydrogen, or the following groups which may be substituted or unsubstituted by one or more groups A: C1-C6 alkyl;

[0067] The group A is selected from: deuterium, C1-C6 alkoxy, C3-C6 cycloalkoxy;

[0068] X1 is selected from: Wherein, Ra is selected from the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl; Rb and Rc are each independently selected from hydroxyl, or the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl, or Rb and Rc together with the oxygen atom and the phosphorus atom to which they are connected form a 5-7 membered ring.

[0069] In some embodiments, the compounds provided by the present invention or their solvates, polymorphs, isotopic derivatives, tautomers, stereoisomers and pharmaceutically acceptable salts are as shown in formula (IV-2):

[0070]

[0071] In formula (IV-2), R1 is selected from hydrogen or deuterium;

[0072] R2 is selected from: trifluoromethyl, methyl, deuterated methyl;

[0073] R3, R4, R5 and R6 are each independently hydrogen or deuterium;

[0074] R7 is selected from: hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl;

[0075] R8 is selected from: hydrogen, or the following groups which may be substituted or unsubstituted by one or more groups A: C1-C6 alkyl;

[0076] The group A is selected from: deuterium, C1-C6 alkoxy, C3-C6 cycloalkoxy;

[0077] X2 is selected from: Wherein, Ra is selected from the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl; Rb and Rc are each independently selected from hydroxyl, or the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl, or Rb and Rc together with the oxygen atom and the phosphorus atom to which they are connected form a 5-7 membered ring.

[0078] In some embodiments, R1 is hydrogen; in some embodiments, R1 is deuterium; in some embodiments, R1 is fluorine.

[0079] In some embodiments, R2 is fluorine; in some embodiments, R2 is a C1-C3 alkyl group in which one or more hydrogen atoms are replaced by a deuterium atom; in some embodiments, R2 is a C1-C3 alkyl group in which one or more hydrogen atoms are replaced by a fluorine atom.

[0080] In some embodiments, R3, R4, R5 and R6 are all hydrogen; in some embodiments, R3, R4, R5 and R6 are all deuterium; in some embodiments, R3 and R4 are hydrogen, and R5 and R6 are all deuterium; in some embodiments, R3 and R4 are deuterium, and R5 and R6 are all hydrogen.

[0081] In some embodiments, R7 is selected from hydrogen, and R8 is selected from the following groups which are substituted or unsubstituted with one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl;

[0082] In some embodiments, R7 is selected from methyl, and R8 is selected from the following groups which are substituted or unsubstituted by one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl;

[0083] In some embodiments, R8 is selected from hydrogen, and R7 is selected from the following groups which are substituted or unsubstituted with one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl;

[0084] In some embodiments, R7 and R8 are both selected from the following groups which are substituted or unsubstituted by one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl.

[0085] In some embodiments, group A is selected from deuterium;

[0086] In some embodiments, group A is selected from C1-C6 alkoxy and C3-C6 cycloalkoxy; preferably, group A is selected from C1-C3 alkoxy and cyclopropyloxy.

[0087] In some embodiments, group A is selected from the following groups in which one or more hydrogen is replaced by deuterium: C1-C6 alkoxy, C3-C6 cycloalkoxy; preferably, group A is selected from the following groups in which one or more hydrogen is replaced by deuterium: C1-C3 alkoxy; more preferably, group A is selected from -OCD3, -OCD2CD3, -OCD(CD3)2.

[0088] In some embodiments, R7 is selected from methyl, and R8 is C1-C6 alkyl substituted with C1-C6 alkoxy; preferably, R7 is selected from methyl, and R8 is C1-C3 alkyl substituted with C1-C3 alkoxy;

[0089] In some embodiments, R7 is selected from methyl, and R8 is C1-C6 alkyl substituted with -OCD3.

[0090] In the embodiments of the present application, "--" indicates bonding or non-bonding; in the embodiments of the present invention, the two "--" shown in formula (I) have one and only one bonding, that is, when one of the "--" indicates bonding, the two connected atoms form a double bond, and the other "--" indicates non-bonding, and X1 or X2 connected to the double-bonded atom does not exist.

[0091] In the embodiments of the present application, X1 and / or X2 are each independently selected from: absent, hydrogen, When R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, X1 and X2 cannot be simultaneously selected from: hydrogen or not exist.

[0092] In some embodiments, when at least one of R1, R2, R3, R4, R5 and R6 is a deuterium atom, X1 is hydrogen and X2 is absent;

[0093] In some embodiments, when at least one of R1, R2, R3, R4, R5 and R6 is a deuterium atom, X2 is hydrogen and X1 is absent;

[0094] In some embodiments, when at least one of R1, R2, R3, R4, R5 and R6 is a deuterium atom, X1 is absent and X2 is selected from:

[0095] In some embodiments, when at least one of R1, R2, R3, R4, R5 and R6 is a deuterium atom, X2 is absent and X1 is selected from:

[0096] In some embodiments, regardless of whether R1, R2, R3, R4, R5 and R6 contain deuterium atoms, X1 is absent, and X2 is selected from:

[0097] In some embodiments, whether or not R1, R2, R3, R4, R5 and R6 contain deuterium atoms, X2 is absent, and X1 is selected from:

[0098] In some embodiments, Ra is selected from the following groups: C1-C6 alkyl, C3-C6 cycloalkyl; in some embodiments, Ra is selected from the following groups in which one or more hydrogen atoms are replaced by deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl.

[0099] In some embodiments, Rb and Rc are both hydroxyl groups;

[0100] In some embodiments, Rb is hydroxyl, and Rc is selected from the following groups which are substituted or unsubstituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl;

[0101] In some embodiments, Rb and Rc are each independently selected from the following groups: C1-C6 alkyl, C3-C6 cycloalkyl;

[0102] In some embodiments, Rb and Rc are each independently selected from the following groups substituted with one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl;

[0103] In some embodiments, Rb and Rc together with the oxygen atom and phosphorus atom to which they are attached form a 5-7 membered ring.

[0104] In the embodiments of the present application, the "one or more" includes "one", "all" or "any one from one to all". For example, "one or more hydrogen atoms of a C1-C3 alkyl group are replaced by deuterium atoms", which means that all hydrogen atoms involved in the C1-C3 alkyl group can be replaced by deuterium, specifically, 1, 2, 3, 4, 5, 6 or 7 hydrogen atoms are replaced by deuterium.

[0105] In the embodiments of the present application, the “at least one” mentioned includes “one”, “all” or “any one from one to all”.

[0106] In the embodiments of the present application, the solvate refers to a complex formed by the interaction of a compound with a pharmaceutically acceptable solvent, and the pharmaceutically acceptable solvent includes common solvents such as water, ethanol, isopropanol, acetic acid, and ethanolamine.

[0107] In the embodiments of the present application, the tautomers refer to structural isomers with different energies that can be mutually converted by low energy barriers. If tautomerism is possible (such as in solution), the chemical equilibrium of tautomers can be reached. For example, proton tautomers include mutual conversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0108] In the embodiments of the present application, the pharmaceutically acceptable salt refers to the organic salt and inorganic salt of the compound of the present invention. Pharmaceutically acceptable salts are well known to us in the art, as described in the literature (SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19). Pharmaceutically acceptable salts formed by non-toxic acids, including but not limited to inorganic acid salts (hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates) formed without amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates), or other methods such as ion exchange methods described in books and documents to obtain these salts.

[0109] In an embodiment of the present application, when one or both of Rb and Rc are hydroxyl groups, the pharmaceutically acceptable salt is a salt of phosphoric acid with an alkali metal, an alkaline earth metal, zinc, iron, an organic amine, or a basic amino acid.

[0110] In the embodiments of the present application, the C1-C6 alkyl group refers to a straight-chain or branched saturated aliphatic hydrocarbon group containing 1 to 6 carbon atoms in the molecule, including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc.

[0111] In the embodiments of the present application, the C1-C6 alkoxy group refers to a group in which an oxygen atom is inserted in any reasonable position of a saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms in the molecule, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, 2-ethylethoxy, etc.

[0112] In the embodiments of the present application, the C3-C6 cycloalkyl refers to a cycloalkane containing 3 to 6 carbon atoms in the molecule, including: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2-methyl-cyclopropyl.

[0113] In the embodiments of the present application, the deuterated refers to that a hydrogen atom at any reasonable position in a molecule is replaced by a deuterium atom.

[0114] In the embodiments of the present application, the compounds provided by the present invention include but are not limited to compounds having the following structures or their solvates, polymorphs, isotopic derivatives, tautomers, stereoisomers and pharmaceutically acceptable salts:

[0115]

[0116]

[0117] In an embodiment of the present invention, the material It was synthesized according to the method described in the literature (WO_2023002443_A1), and other starting materials were purchased commercially or obtained according to the method described in the literature.

[0118] In an embodiment of the present invention, the position where X1 and X2 are connected in the structures shown in formula (III) and formula (IV), and (VI) and formula (VII) is 1 H NMR confirmed.

[0119] Another aspect of the present invention is to provide a pharmaceutical composition comprising the above-mentioned compound or its solvate, polymorph, isotopic derivative, tautomer, stereoisomer and pharmaceutically acceptable salt and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or a combination of fillers, binders, diluents, lubricants, preservatives, taste masking agents or cosolvents.

[0120] Furthermore, the pharmaceutical composition is in the form of tablets, capsules, powders, granules, pills, suspensions, syrups, injections or aerosol inhalers.

[0121] In the third aspect of the present invention, the present invention provides the above-mentioned compound, including its solvate, polymorph, isotope derivative, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition thereof, for treating and / or preventing influenza virus infection.

[0122] The present invention provides a method for treating and / or preventing influenza virus infection, which comprises administering a therapeutically effective amount of the above-mentioned compound, including its solvate, polymorph, isotope derivative, tautomer, and pharmaceutically acceptable salt, to an individual in need thereof.

[0123] The compounds of the present invention show better effects of treating and preventing influenza virus infection both in vivo and in vitro; in the efficacy test of the influenza model in mice, compared with compound A1, the mice in the group administered with the compounds of the present invention had a lower weight loss and a faster recovery speed; 72 hours after infection with the influenza virus, at a lower dose, there was still a significant effect of inhibiting the influenza virus;

[0124] The compounds of the present invention have significantly enhanced resistance to drug resistance;

[0125] The compounds of the present invention have little effect on mitochondrial membrane potential;

[0126] The compound of the present invention exhibits good bioavailability, rapid absorption, decreased clearance rate, and prolonged half-life in animal in vivo pharmacokinetic studies, and is less affected by gender and food after administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Attached Figure 1 :Effects of compounds on mitochondrial membrane potential in HepG2 cells. DETAILED DESCRIPTION

[0128] The following examples can enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The structures of all compounds are determined by MS or 1 All the single-configuration optical isomers involved were determined by optical rotation test or electronic circular dichroism spectroscopy.

[0129] Unless otherwise specified, the solvents and reagents used in this example are all common commercial products, and the starting materials are all purchased commercial raw materials or simply synthesized from purchased commercial raw materials.

[0130] Example 1: Synthesis of Compound A1

[0131]

[0132] Synthesis of compound a2

[0133] Under nitrogen protection, 50 ml of tetrahydrofuran (THF) was added to the reaction flask, the system was cooled to 0°C, 1.68 g of NaH (60w%) and 7.55 g of 2-methylallyl alcohol were added in sequence, the system was stirred for 10 min, 6 g of compound a1 ((S)-2-((4-bromophenoxy)methyl)oxirane) was added, and the system was heated to 65°C for 3 hours. After cooling, methanol and water were added to quench, concentrated to dryness, extracted with ethyl acetate (EA) and water, the organic phase was separated, concentrated to dryness, and purified by silica gel column to obtain 4.26 g of compound a2, with a yield of 54%, ESI-MS (+): m / z = 301.2, 303.2.

[0134] Synthesis of compound a3

[0135] Under nitrogen protection, 4 g of compound a2 and 20 ml of dichloromethane (DCM) were added to the reaction flask, and 3.9 g of m-chloroperbenzoic acid (m-CPBA, 77 w%) was added at room temperature. The system continued to react at room temperature for 30 minutes, and sodium thiosulfate aqueous solution was added to quench the reaction. Ether was added to the system for extraction, and the organic phase was washed with sodium thiosulfate aqueous solution, saturated sodium bicarbonate solution, and saturated brine in sequence and concentrated to dryness to obtain compound a3, ESI-MS (+): m / z = 317.2.

[0136] Synthesis of compound a4

[0137] Under nitrogen protection, 2 g of compound a3, 100 ml of DCM, and 1 ml of trifluoroacetic acid (TFA) were added to the reaction flask. The system was reacted at room temperature for 3 hours. Saturated brine was added to the system, the organic phase was separated, the aqueous phase was extracted twice with DCM, the organic phases were combined for 3 times, dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column to obtain 1.74 g of compound a4, with a yield of 87%, ESI-MS (+): m / z = 317.2.

[0138] Synthesis of compounds a5, a6 and a7

[0139] Under nitrogen protection, 1.5 g of compound a4, 50 ml of THF and 380 mg of NaH (60w%) were added to the reaction flask. After the system was stirred at room temperature for 10 minutes, 3.35 g of iodomethane was added, and the system was stirred at room temperature for 3 hours. After cooling, methanol and water were added to quench, and the mixture was concentrated to dryness, extracted with ethyl acetate (EA) and water, and the organic phase was separated and concentrated to dryness. The mixture was purified by silica gel column to obtain 1.38 g of compound a5 with a yield of 88%. Compounds a6 and a7 were obtained by preparative separation, ESI-MS (+): m / z = 331.2, and the configurations of a6 and a7 were determined by electronic circular dichroism.

[0140] Synthesis of compound a8

[0141] Under nitrogen protection, 0.6 g of compound a6, 690 mg of bis(pinacolato)diboron, 100 mg of Pd(dppf)Cl2, 533 mg of potassium acetate and 40 ml of 1,4-dioxane were added to the reaction flask, and the system was heated to 85°C for 12 hours. The mixture was extracted with water and EA, and the organic phase was washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain 363 mg of compound a8, with a yield of 53%, ESI-MS (+): m / z = 379.3.

[0142] Synthesis of Compound A1

[0143] Under nitrogen protection, 0.2 g of compound a8, 0.126 g of compound b1, 16 mg of Pd(dppf)Cl2, 430 mg of cesium carbonate and 25 ml of ethylene glycol dimethyl ether were added to the reaction bottle, and the system was heated under reflux for 48 h. The mixture was extracted with water and EA, and the organic phase was washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, concentrated to dryness, and purified on a silica gel column to obtain compound A1, ESI-MS (+): m / z = 457.5.

[0144] Example 2: Synthesis of Compound V1

[0145]

[0146] Synthesis of compound c2

[0147] Compound c2 was obtained by completely referring to the synthesis method in the literature (Cho; Beale; Graff; Mocek; Nakagawa; Omura; Floss Journal of the American Chemical Society, 1993, vol. 115, #26 p. 12296-12304).

[0148] Synthesis of compound c3

[0149] Under nitrogen protection, 1.57g of compound b2 ((R)-epichlorohydrin) and 1ml of water were added to the reaction flask, and the system was cooled to -5°C to obtain system 1. 2g of compound c2, 42mg of benzyltrimethylammonium chloride, 0.45g of sodium hydroxide and water were added to another reaction flask, and the system was stirred at room temperature until homogeneous to obtain system 2. System 2 was slowly added dropwise to the coolant of system 1, and the reaction was continued at -5°C for 5 hours. The temperature was naturally raised to room temperature, and water and EA were added for extraction. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain compound c3, ESI-MS (+): m / z = 233.1.

[0150] Synthesis of compounds C4, C5, C6 and C7

[0151] The synthesis method of compound a2 was completely referred to, compound c3 was used instead of compound a1, and the reaction with 2-methylallyl alcohol gave compound c4, ESI-MS (+): m / z=305.1.

[0152] According to the synthetic method of compound a3, compound c4 was reacted with m-chloroperbenzoic acid to obtain compound c5, ESI-MS (+): m / z = 321.2.

[0153] With complete reference to the synthesis method of compound a4, compound c5 was intramolecularly cyclized under acidic conditions to obtain compound c6, which was then separated and prepared to obtain compound c7, ESI-MS (+): m / z = 321.2.

[0154] Synthesis of compound c8

[0155] According to the synthetic method of compound a5, compound c7 was reacted with iodomethane to obtain compound c8, ESI-MS (+): m / z = 335.3.

[0156] Synthesis of compound V1

[0157] With complete reference to the synthesis method of compounds a8 and A1, compound c8 was catalytically boronized and reacted with compound b1 to obtain compound V1, ESI-MS (+): m / z = 461.4.

[0158] Example 3: Synthesis of Compound V3

[0159]

[0160] Synthesis of compound d1

[0161] Referring to the synthesis method of compound a1, compound c2 was used instead of 4-bromophenol to react with compound (S)-epichlorohydrin to obtain compound d1 ((S)-2-((4-bromo-2,3,5,6-tetradeuterium-phenoxy)methyl)oxirane).

[0162] Synthesis of compound d2

[0163] Under nitrogen protection, 10 ml of tetrahydrofuran (THF) was added to the reaction flask, the system was cooled to 0°C, 0.28 g of NaH (60w%) and 1 g of allyl alcohol were added in sequence, the system was stirred for 10 min, 1 g of compound d1 ((S)-2-((4-bromo-2,3,5,6-tetradeuterium-phenoxy)methyl)oxirane) was added, and the system was heated to 65°C for 3 hours. After cooling, methanol and water were added to quench, concentrated to dryness, extracted with ethyl acetate (EA) and water, the organic phase was separated, concentrated to dryness, and purified by silica gel column to obtain 0.74 g of compound d2, with a yield of 59%, ESI-MS (+): m / z = 291.2.

[0164] Synthesis of compound d3

[0165] Under nitrogen protection, 0.7 g of compound d2 and 10 ml of dichloromethane (DCM) were added to the reaction flask, and 0.7 g of m-chloroperbenzoic acid (m-CPBA, 77w%) was added at room temperature. The system continued to react at room temperature for 30 minutes, and sodium thiosulfate aqueous solution was added to quench the reaction. Ether was added to the system for extraction, and the organic phase was washed with sodium thiosulfate aqueous solution, saturated sodium bicarbonate solution, and saturated brine in sequence and concentrated to dryness to obtain compound d3, ESI-MS (+): m / z = 307.2, and the next step was directly carried out without purification.

[0166] Synthesis of compound d4

[0167] Under nitrogen protection, 0.5 g of compound d3, 20 ml of DCM and 0.25 ml of trifluoroacetic acid (TFA) were added to the reaction flask. The system was reacted at room temperature for 3 hours. Saturated brine was added to the system, the organic phase was separated, the aqueous phase was extracted twice with DCM, the organic phases were combined for 3 times, dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column to obtain 0.4 g of compound d4, with a yield of 79%, ESI-MS (+): m / z = 307.2.

[0168] Synthesis of compounds d5 and d6

[0169] Under nitrogen protection, 0.3 g of compound d4, 10 ml of anhydrous THF and 100 mg of NaH (60w%) were added to the reaction flask, the system was stirred at room temperature for 10 minutes, 0.7 g of iodomethane was added, the system was stirred at room temperature for 3 hours, methanol and water were added to quench after cooling, concentrated to dryness, extracted with ethyl acetate (EA) and water, the organic phase was separated, concentrated to dryness, and purified by silica gel column to obtain 0.28 g of compound d5, with a yield of 90%. Compound d6 was obtained by chiral column separation, with a yield of 44%, ESI-MS (+): m / z = 321.1.

[0170] Synthesis of compounds d7 and V3

[0171] Referring to the synthesis method of compound a8, compound d7 was obtained by replacing a6 with compound d6, ESI-MS (+): m / z = 369.4.

[0172] Referring to the synthesis method of compound A1, compound d7 was used instead of a8 to obtain compound V3, ESI-MS (+): m / z = 447.3.

[0173] Example 4: Synthesis of Compound V2

[0174]

[0175] Compound V2 was synthesized by referring to the synthesis method of compounds A1 and V1 using compound d1 as the starting material, ESI-MS (+): m / z=461.4.

[0176] Example 5: Synthesis of Compound V4

[0177]

[0178] Synthesis of compound e2

[0179] Compound e1 was obtained by referring to the synthetic method of compound c7;

[0180] Under nitrogen protection, 1.4 g of compound e1 and 15 ml of DCM were added to the reaction flask, the system was cooled to 0°C, 1.85 g of DMP (Dess-Martin reagent) was added, the system was heated to room temperature for reaction, and TLC was monitored until the reaction was complete. 20 ml of 2M NaOH aqueous solution was added to the system for quenching, and DCM was added for extraction 3 times. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain compound e2, ESI-MS (+): m / z = 319.2, which was used directly in the next step without further purification.

[0181] Synthesis of compound e3

[0182] Under nitrogen protection, 0.95g of PPh3MeBr and 15ml of anhydrous THF were added to the reaction flask, and the system was cooled to 0°C. 1.1ml of n-BuLi (2.5M in n-hexane) was slowly added dropwise, and the system was reacted at 0°C for 30 minutes, then cooled to -78°C, and 0.7g of compound e2 in THF (15ml) solution was added dropwise. After reacting for 30 minutes, the temperature was slowly raised to room temperature, and the system was monitored by TLC until the reaction was complete. The system was quenched with saturated NH4Cl solution, extracted with water and EA, and the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and purified by silica gel column to obtain 0.46g of compound e3, with a yield of 66%, ESI-MS (+): m / z = 317.2.

[0183] Synthesis of compound e4

[0184] Under nitrogen protection, 0.42 g of compound e3, 5 ml of THF and 2.2 ml of borane dimethyl sulfide complex (2M THF solution) were added to the reaction flask in sequence, and the system was stirred at room temperature for 2 hours. 5 ml of water and 2 g of sodium perborate tetrahydrate were added, and the system was stirred at room temperature for 2 hours. The system was filtered and concentrated to dryness, and purified by silica gel column to obtain 0.27 g of compound e4, with a yield of 62%, ESI-MS (+): m / z = 335.2.

[0185] Synthesis of compound e5

[0186] Under nitrogen protection, 0.22 g of compound e4, 10 ml of THF and 52 mg of NaH (60w%) were added to the reaction bottle. After the system was stirred at room temperature for 10 minutes, 0.47 g of iodomethane was added, and the system was stirred at room temperature for 3 hours. After cooling, methanol and water were added to quench, and the mixture was concentrated to dryness. The mixture was extracted with ethyl acetate (EA) and water, and the organic phase was separated and concentrated to dryness. The mixture was purified by silica gel column to obtain 0.19 g of compound e5 with a yield of 83%. ESI-MS (+): m / z = 348.2.

[0187] Synthesis of compounds e6 and V4

[0188] Referring to the synthesis method of compound a8, compound e6 was obtained by replacing compound a6 with compound e5, ESI-MS (+): m / z = 397.4.

[0189] Referring to the synthesis method of compound A1, compound e6 was used instead of a8 to obtain compound V4, ESI-MS (+): m / z = 475.5.

[0190] Example 6: Synthesis of Compounds VT-3 and VT-4

[0191]

[0192] Under nitrogen protection, 180 mg of compound A1, 154 mg of cesium carbonate, 74 mg of ethyl chloromethyl carbonate and 10 ml of DMF (N,N-dimethylformamide) were added to the reaction flask in sequence, and the system was reacted at 25°C for 24 hours, extracted with ethyl acetate and water, and the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column and then purified by preparative liquid phase to obtain 77 mg of compound VT-3 (yield 36%) and 58 mg of compound VT-4 (yield 27%), ESI-MS (+): m / z = 545.5.

[0193] Example 7: Synthesis of Compounds VT-11 and VT-12

[0194]

[0195] Under nitrogen protection, 200 mg of compound V2, 170 mg of cesium carbonate, 74 mg of chloromethyl isobutyrate and 10 ml of DMF (N,N,-dimethylformamide) were added to the reaction flask in sequence, and the system was reacted at 25°C for 24 hours. The mixture was extracted with ethyl acetate and water, and the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column and preparative liquid phase to obtain 100 mg of compound VT-11 (yield 41%) and 80 mg of compound VT-12 (yield 33%), ESI-MS (+): m / z = 561.5.

[0196] Example 8: Synthesis of Compound VT-23

[0197]

[0198] Synthesis of Deuterated Ethyl Chloromethyl Carbonate (D5)

[0199] 3.96g of deuterated ethanol (D5), 13.5ml of triethylamine and 70ml of dichloromethane were added to the reaction flask. After the system was cooled to 0°C, 10g of chloromethyl chloroformate was slowly dripped in. After the dripping, the system was naturally warmed to room temperature and reacted for 3 hours. Water was added to quench the reaction, and dichloromethane was extracted to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to obtain 8.7g of liquid of the compound deuterated ethyl chloromethyl carbonate (D5), with a yield of 78%.

[0200] Synthesis of Compound VT-23

[0201] Referring to the synthesis method of compound VT-3, compound A1 and compound deuterated ethyl chloromethyl carbonate (D5) were used as raw materials to synthesize 52 mg of compound VT-23 with a yield of 35%. ESI-MS (+): m / z = 564.5.

[0202] Example 9: Synthesis of Compound VT-24

[0203]

[0204] Synthesis of compound VT-24-01

[0205] Referring to the synthesis method of compound VT-3, compound A1 and di-tert-butyl chloromethyl phosphate were used as raw materials to synthesize 320 mg of compound VT-24-01 with a yield of 35%. ESI-MS (+): m / z = 679.5.

[0206] Synthesis of Compound VT-24

[0207] Under nitrogen protection, 300 mg of compound VT-24-01 and 30 ml of DCM were added to the reaction flask, and 1 ml of trifluoroacetic acid was added dropwise. The system was reacted at 25°C for 5 hours, concentrated to dryness under reduced pressure, and purified by preparative liquid phase to obtain 110 mg of compound VT-24, with a yield of 44%, ESI-MS (-): m / z = 565.2.

[0208] Example 10: Synthesis of Compounds V7, VT-26 and VT27

[0209]

[0210] The synthesis of compound f7 was based on compound b1. It was synthesized by the method described in the document (WO_2023002443_A1). The details are as follows:

[0211] Synthesis of compound f3

[0212] Under nitrogen protection, 5 g of f1 (ethoxyethylene) and 50 ml of DCM were added to the reaction bottle, the system was cooled to -10°C, 8.25 g of f2 (deuterated acetic anhydride-d6) and 0.5 g of DMAP (dimethylaminopyridine) were added in sequence, the system was heated to 25°C and reacted for 24 hours, concentrated to dryness, and purified by silica gel column to obtain compound f3.

[0213] Synthesis of compound f5

[0214] At room temperature, 5 g of compound f3, 5.81 g of sodium ethoxide, 6.72 g of compound f4 and 50 ml of ethanol were added to the reaction flask in sequence, and the system was heated to 80°C for 18 hours. The system was quenched with water, extracted with ethyl acetate, and the organic phase was separated, dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column to obtain compound f5, ESI-MS (-): m / z = 185.2.

[0215] Synthesis of compound f6

[0216] Under nitrogen protection, 2 g of compound f5 and 20 ml of DMF were added to the reaction flask, and 2.32 g of compound NBS (N-bromosuccinimide) was added to the system at room temperature, and the temperature was maintained for 1 hour. Water and EA were added to the system for extraction, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and purified by silica gel column to obtain 2.09 g of compound f6, with a yield of 73%, ESI-MS (-): m / z = 263.1.

[0217] Synthesis of compound f7

[0218] 1 g of compound f6 and 30 ml of ammonia methanol solution (7 M) were added to the high pressure reactor, and the system was heated to 60°C for 24 hours. After the reaction, the system was concentrated to dryness and purified by silica gel column to obtain 0.7 g of compound f7, with a yield of 78%, ESI-MS (-): m / z = 234.1.

[0219] Synthesis of compound V7

[0220] Referring to the synthesis method of compound A1, compound f7 and compound a8 were reacted to obtain compound V7, ESI-MS (+): m / z = 406.3.

[0221] Synthesis of compounds VT-26 and VT-27

[0222] Referring to the synthesis method of compound VT-3 and compound VT-4, compound V7 was reacted with methyl chloromethyl carbonate to obtain compounds VT-26 and VT-27 by preparative separation, ESI-MS (+): m / z = 494.5.

[0223] The following example compounds were synthesized by the same method as in the above examples using commercially available compounds or intermediate compounds appropriately synthesized from commercially available compounds:

[0224]

[0225]

[0226] Comparative Example 1: Synthesis of Compound A2

[0227] Compound A2 was synthesized by referring to the synthesis method of compound A1.

[0228] Example 10: Anti-influenza virus cell activity experiment

[0229] MDCK cells were inoculated into 96-well culture plates and cultured in a 5% CO2, 37°C incubator. After 24 hours of culture, the cells were infected with influenza virus (A / Hanfang / 359 / 95 (H3N2)). After 2 hours of virus adsorption, the virus solution was discarded and cell culture maintenance medium containing samples of different dilutions was added. Three replicate wells were set for each concentration. Cell control wells and virus control wells were also set up and cultured in a 5% CO2, 37°C incubator. The antiviral test of the test samples was carried out using the CPE method. When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect (CPE) of each group was observed. The effective drug concentration (EC) of the samples that inhibited 50% of the cytopathic effect was calculated using the Reed-Muench method. 50 ), as shown in Table 1:

[0230] Table 1: Inhibitory activity of compounds against influenza virus

[0231] Compound <![CDATA[EC 50 / nM]]> Compound <![CDATA[EC 50 / nM]]> Compound <![CDATA[EC 50 / nM]]> Compound <![CDATA[EC 50 / nM]]> V1 2.60±0.24 V2 2.29±0.18 V3 2.73±0.21 V4 2.43±0.25 V5 2.82±0.31 V6 2.33±0.17 V7 2.38±0.37 V8 2.82±0.33 VT-1 5.52±0.38 VT-2 5.83±0.36 VT-3 9.23±0.83 VT-4 8.72±0.73 VT-5 6.12±0.42 VT-6 5.77±0.46 VT-7 8.78±0.71 VT-8 5.95±0.45 VT-9 9.05±0.78 VT-10 9.09±0.81 VT-11 6.25±0.43 VT-12 6.18±0.47 VT-13 8.88±0.98 VT-14 9.49±0.84 VT-15 9.08±0.79 VT-16 8.81±0.92 VT-17 9.03±0.82 VT-18 8.66±0.73 VT-19 9.14±0.97 VT-20 8.83±0.75 VT-21 9.15±0.80 VT-22 9.21±0.95 VT-23 8.74±0.89 VT-24 8.79±0.83 VT-25 8.92±0.95 VT-26 5.69±0.38 VT-27 6.03±0.53 VT-28 5.66±0.52 VT-29 6.03±0.56 A1 6.92±0.47 A2 7.32±0.55

[0232] The experiment shows that the compounds of the present invention have good in vitro anti-influenza virus activity. Compared with comparative example compounds A1 and A2, the in vitro anti-influenza virus activity of compounds V1 to V8 of the present invention is significantly better; compounds VT-1 to VT-29 of the present invention also have anti-H3N2 activity at the nanomolar level.

[0233] Example 11: Drug resistance induction experiment

[0234] MDCK was inoculated into 12-well plates and cultured until more than 90% confluence. PR8 virus was inoculated into MDCK cells at MOI = 0.01 and adsorbed at 4°C for 90 min. 1 nM of compound V4, V7 and comparative example A1 were immediately added and co-cultured for 3 days. The supernatant was collected, diluted 10 times and inoculated into MDCK cells with more than 90% confluence and continued to be co-cultured with 1 nM concentration of drug for 3 days. Continuously passaged to the 15th generation. The total RNA of the virus was extracted, its PA gene sequence was determined, and the mutation sites and the generations at which they appeared were analyzed.

[0235] The results showed that the mutations caused by compounds V4 and V7 were far less than those caused by A1, which indicated that compounds V4 and V7 had stronger resistance to drug resistance.

[0236] Example 12: Effect on mitochondrial membrane potential of HepG2 cells

[0237] The experiment set up 4 groups, each group had 2 subgroups (cell subgroup and non-cell subgroup, the difference was whether cells were added), and the specific groupings were: control group (normal well, normal control well), positive drug group (positive drug well, positive drug control well), sample group 1 (sample 1 well, sample 1 control well), sample group 2 (sample 2 well, sample 2 control well).

[0238] HepG2 cells (density 1×10 5 cells / mL, inoculation volume of 100 μL / well) were inoculated on a 96-well transparent bottom side black board (normal well, positive drug well, sample 1 well, sample 2 well); 100 μL / well complete culture medium (MEM+10% FBS+1% Non Essential AminoAcids (NEAA)+1% Sodium Pyruvate (NaP)+1% P / S, without cells) was added to the normal control well, positive drug control well, sample 1 control well, and sample 2 control well, respectively; and the cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0239] The test sample was dissolved in DMSO, diluted with 1×Supplemented DilutionBuffer solution, and sterilized through a 0.22μm filter membrane. The supernatant was discarded from the 96-well plate, and 100μL of sample V2 solution (50μM) diluted with 1×Supplemented DilutionBuffer solution was added to the sample 1 well and the sample 1 control well respectively; 100μL of sample A2 solution (50μM) diluted with 1×Supplemented DilutionBuffer solution was added to the sample 2 well and the sample 2 control well respectively, and 100μL of 1×Supplemented DilutionBuffer solution was added to the other wells (positive drug wells, positive drug control wells, normal control wells, and blank control wells), and culture was continued overnight.

[0240] About 20 hours after adding samples V2 and A2, add 11μL of 1mM FCCP to each positive well and positive control well (positive FCCP is diluted to 1mM using 1×Supplemented DilutionBuffer solution). About 23 hours after adding samples, dissolve JC-1 in DMSO, then add 1×Dilution buffer to dilute to 40μM and store at 37℃ for later use. 10-30 minutes before adding samples (24 hours of co-culture of samples is the end point of the experiment), add the JC-1 solution prepared in the previous step, 100μL per well, and continue incubation until the end point of the experiment.

[0241] Discard the supernatant and wash each well twice with 1× Dilution buffer or 1× Dilution buffer to dilute different concentrations of samples / positive. The last wash solution remains in the well and continue the microplate reader detection.

[0242] When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is a monomer and can produce green fluorescence. The fluorescence values ​​were detected by microplate reader, and the following values ​​were detected: ①Excitation wavelength: 535±17.5nm (polymer only); Emission wavelength: 590±17.5nm (polymer only); ②Excitation wavelength: 475±20nm (polymer and monomer); Emission wavelength: 530±15nm (polymer and monomer).

[0243] The fluorescence values ​​of each well were tested at wavelengths of 530nm and 590nm. The difference between the fluorescence value of the cell subgroup and the fluorescence value of the non-cell subgroup in each group was recorded as Em530 or Em590. The ratio of Em530 and Em590 was calculated for each group, recorded as Ratio (Em530 / 590), to evaluate the effect of the compound on mitochondrial membrane potential. Specific results are shown in the attached Figure 1 .

[0244] From the attached Figure 1 It can be seen that compared with the control group, the positive drug FCCP group can significantly reduce the mitochondrial membrane potential of HepG2 cells; the test results of the test sample V2 group were not statistically different from those of the control group; the test results of the comparative compound A2 group were statistically different from those of the control group (P<0.05), and there was also a statistical difference compared with the V2 group (P<0.05). This shows that under the conditions of this experiment, the test sample V2 had no significant effect on the mitochondrial membrane potential of HepG2 cells at a concentration of 50μM, and the comparative compound A2 had a certain effect on the mitochondrial membrane potential of HepG2 cells at a concentration of 50μM.

[0245] Example 13: Rat PK study

[0246] 18 SD rats, male, 180g-220g, before the experiment, the SD rats were cannulated in the jugular vein, and the experiment was started after three days of adaptation (free access to food and water, room temperature: 20-26℃; humidity: 40-70%; light illumination: dark = 12h: 12h). The experimental animals were divided into 6 groups A / B / C / D / E / F, with 3 rats in each group. Each group was given a suspension of the test article by oral gavage (the test articles were A1, VT-1, VT-3, VT-7, VT-16 and VT-26, all suspended with 0.5% sodium carboxymethyl cellulose), the dosage was 2mg / kg based on A1, and each group was given equimolar administration. Fasting but not water was started at 5 pm the day before administration, and fasting for 16-17h. The animals were fed 4h after administration, and water was not allowed during the whole process.

[0247] 0.5h before administration (0h) and 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h after administration. About 0.25mL of whole blood was collected from the jugular vein in a sodium heparin anticoagulation tube, mixed thoroughly, and placed in an ice bath. Within 1 hour after blood sample collection, centrifuge at 3000rpm for 5 minutes at 4℃. Take plasma and transfer it to a labeled centrifuge tube and temporarily store it in a -20℃ refrigerator. After all samples are collected, hand it over to the biological sample manager and store it in a -80℃ refrigerator. After processing, the biological samples were detected by LC-MS / MS (VT-1 group detected compound V2; A1 group, VT-3 group, VT-7 group and VT-16 group detected compound A1; VT-26 group detected compound V7). WinNonlin 7.0 was used to calculate the main pharmacokinetic parameters according to the non-compartmental model method.

[0248] The results of pharmacokinetic studies of rats administered orally showed that, compared with compound A1, the compound of the present invention had significant enterohepatic circulation in the pharmacokinetic study in rats, and exhibited good bioavailability, rapid absorption, decreased clearance rate, and prolonged half-life.

[0249] Example 13: Pharmacokinetic study of oral administration in monkeys

[0250] Cynomolgus monkeys, ordinary grade, 12, half male and half female, aged 3-6 years old, weighing 2.5-4.5kg, were randomly divided into 2 groups according to body weight, half male and half female, marked as group A and group B. Group A was gavaged with compound VT-3, and group B was gavaged with compound A1. Compounds VT-3 and A1 were prepared with mixed solutions (5% dimethyl sulfoxide: 50% polyethylene glycol 400: 45% 0.2g / ml hydroxypropyl β-cyclodextrin), and the dosage was 1mg / kg. The experiment was divided into two cycles. The first cycle was fasting administration. Each group fasted for 12h to 13h before administration, but could not drink water. Then, a single oral gavage was administered. A meal was taken 4h after administration, and water was not allowed during the whole experiment. The second cycle was postprandial administration. Each group did not fast before and after administration, but could not drink water. A single oral gavage was administered immediately after breakfast. The interval between the two cycles was 5 days.

[0251] In each test cycle, about 1.5 mL of blood was collected from the femoral vein within 0.5 h (0 h) before administration, and 0.25 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h, and 24 h after administration; the blood collected each time was added to a tube containing heparin anticoagulant placed in an ice bath, and centrifuged at 2-8 ° C for 10 minutes to obtain plasma. The concentration of the target analyte in plasma was determined by LC / MS / MS (compound A1 was detected in both VT-3 and A1 groups), and the main pharmacokinetic parameters were calculated using DAS, and the C concentrations of male and female monkeys in the fasting VT-3 and A1 groups were calculated respectively. maxand AUC (0-t) The ratio of fasting and postprandial C max and AUC (0-t) The effect of gender and food on the main pharmacokinetic parameters was evaluated (Table 2).

[0252] Table 2: Gender differences and food effects of oral gavage in cynomolgus monkeys

[0253]

[0254] The results showed that in the fasting state, the Cmax or AUC of male monkeys after VT-3 administration was significantly lower than that of A1 administration group. (0-t) and Cmax or AUC in female monkeys (0-t) The ratio is closer to 1, which indicates that the compound VT-3 of the present invention is less affected by gender after administration. In addition, after administration to male monkeys, the difference between fasting and postprandial in the VT-3 administration group is much smaller than that in the A1 administration group, and female animals also show a similar trend, which also indicates that the compound VT-3 of the present invention is not easily affected by food.

[0255] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A compound as represented by formula (I), or a solvate, polymorph, isotope derivative, tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: In formula (I), R1 is selected from hydrogen, deuterium or fluorine; R2 is selected from: fluorine, C1-C3 alkyl substituted by one or more deuterium atoms, C1-C3 alkyl substituted by one or more fluorine atoms; R3, R4, R5 and R6 are each independently hydrogen or deuterium; R7 and R8 are each independently selected from hydrogen, or the following groups which are substituted or unsubstituted by one or more groups A: C1-C6 alkyl, C3-C6 cycloalkyl; The group A is selected from the following groups in which deuterium or one or more hydrogens are substituted or unsubstituted by deuterium: C1-C6 alkoxy, C3-C6 cycloalkoxy; X1 and / or X2 are each independently selected from: absent, hydrogen, When R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, any one of X1 and / or X2 is not hydrogen; in, Ra is selected from the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl; Rb and Rc are each independently selected from hydroxyl, or the following groups which are substituted or unsubstituted by one or more deuterium atoms: C1-C6 alkyl, C3-C6 cycloalkyl, or Rb and Rc together with the oxygen atom and the phosphorus atom to which they are connected form a 5-7 membered ring; "--" indicates bonding or non-bonding, and the "--" shown has one and only one bonding. When "--" indicates bonding, the two atoms connected form a double bond, and X1 or X2 connected to the double bond does not exist; In particular, formula (I) shall not be 2. The compound of claim 1, or its solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt, characterized in that: When R1, R3, R4, R5 and R6 are all hydrogen and R2 is a C1-C3 alkyl group substituted with one or more fluorine atoms, X1 and / or X2 must not be simultaneously selected from: hydrogen or not exist.

3. A compound represented by formula (II-1), or a solvate, polymorph, isotope derivative, tautomer, stereoisomer, or pharmaceutically acceptable salt thereof: In formula (II-1), R1 is selected from hydrogen or deuterium; R2 is selected from: trifluoromethyl, methyl, deuterated methyl; R3, R4, R5 and R6 are each independently hydrogen or deuterium; R7 is selected from: hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl; R8 is selected from hydrogen, or the following groups which may be substituted or unsubstituted by one or more groups A: C1-C6 alkyl; The group A is selected from deuterium, C1-C6 alkoxy, C3-C6 cycloalkoxy; The other substituents in formula (II-1) are as defined in claim 2.

4. A compound represented by formula (III-1), or a solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt thereof: In formula (III-1), R1 is selected from hydrogen or deuterium; R2 is selected from: trifluoromethyl, methyl, deuterated methyl; R3, R4, R5 and R6 are each independently hydrogen or deuterium; and at least one of R1, R2, R3, R4, R5 and R6 is deuterium; In formula (III-1), other substituents are as defined in claim 3.

5. A compound represented by formula (IV-1), or a solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt thereof: In formula (IV-1), X1 is selected from: The other substituents are as defined in claim 3.

6. A compound represented by formula (IV-2), or a solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt thereof: In formula (IV-2), X2 is selected from: The other substituents are as defined in claim 3.

7. The compound according to any one of claims 1 to 6, selected from the following structures: or a solvate, polymorph, isotope derivative, tautomer, stereoisomer and pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 7 or its solvate, polymorph, isotopic derivative, tautomer, stereoisomer, pharmaceutically acceptable salt and pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, which is in the form of tablets, capsules, powders, granules, pills, suspensions, syrups, injections or aerosol inhalants.

10. A compound according to any one of claims 1 to 7, including its solvate, polymorph, isotope derivative, tautomer, pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 8 or 9, for preparing a medicament for treating and / or preventing influenza virus infection.

11. A method for treating and / or preventing influenza virus infection, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 7, including a solvate, polymorph, isotopic derivative, tautomer, pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8 or 9.

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