WO2023072292A1 - Efficient antiviral compound and use thereof - Google Patents

Abstract

A compound as shown in the following formula (I-0) or a hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmacologically acceptable salt thereof and a preparation method therefor. The compound can be used for preparing a drug for preventing/treating infections caused by viruses, including influenza viruses.

Description

A kind of high-efficiency antiviral compound and its application technical field

The present invention relates to a compound with anti-influenza virus activity or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt, its preparation method and use in anti-influenza virus .

Background technique

Influenza viruses mainly include four types of influenza A virus, influenza B virus, influenza C virus and influenza D virus. Among them, influenza A virus and influenza B virus are the main human influenza viruses, and influenza A virus is one of them. The strongest, the number of people infected is the largest during the flu-prone season, and can induce severe respiratory infections, causing more than 300,000 people to die from influenza every year in the world. In China, tens of millions of people are infected with influenza virus every year, especially among infants and the elderly, the morbidity and mortality are relatively high, and it can cause pneumonia and other diseases.

Currently, the main anti-influenza drugs on the market are: Amantadine, neuraminidase inhibitor Oseltamivir or Zanamivir.

The RNA polymerase of influenza virus contains a cap-dependent endonuclease (Cap-dependent endonuclease), and inhibiting the activity of the cap-dependent endonuclease can inhibit the proliferation of the virus. This enzyme has become a promising candidate for the development of antiviral drugs. Targets, different heterocyclic compounds have been used as cap-dependent endonuclease inhibitors.

However, these compounds exhibit poor physicochemical properties, which in turn affect the relevant pharmacokinetic properties, making them unsuitable as ideal influenza therapeutics. Therefore, it is still urgent to develop new cap-dependent endonuclease inhibitors for the treatment of influenza.

Contents of the invention

Unless otherwise specified herein, the technical terms used in the present invention have basic meanings commonly understood by those skilled in the art.

The invention provides a class of compounds with anti-influenza virus effects. Most of the existing drugs play an anti-influenza virus effect by targeting neuraminidase, and the compound of the present invention plays a role in inhibiting virus replication by inhibiting the cap-dependent endonuclease in the influenza virus. Targets the earlier stage of the virus replication cycle, so it has a better effect on preventing and treating influenza. It has shown better anti-influenza virus effects in vitro or in vivo, and is expected to treat resistance to oseltamivir and avian influenza strains (H7N9, H5N1).

The compound of the present invention shows better anti-influenza virus effect in the in vivo and in vitro drug efficacy test, and also shows better in vivo exposure in the animal pharmacokinetic test. During the compound optimization process, it was unexpectedly found that: (1) after the compound of the present invention was treated, the lung virus titer of the test animal was lower, and the pathological changes in the lung tissue were milder, indicating that the anti-influenza virus activity of the compound of the present invention was significantly improved; (2) It was further found that the compounds of the present invention have good lung tissue distribution in animal experiments, and the clearance rate in the lungs is lower and the half-life is longer; (3) although the enantiomers of the compounds of the present invention have Similar antiviral activity, but only the S-configuration has higher antiviral activity in animal experiments, and the R-configuration has basically no. It overturns people's usual understanding of the differences between optical isomers in pharmacodynamic tests at the cellular level and in vivo pharmacodynamic tests in animals; (4) the ester compounds of the present invention have significant advantages over other esters in various forms, Show better and higher exposure in vivo, especially compound M19 has the best performance. These characteristics are more conducive to the anti-influenza virus effect of drugs for the treatment of influenza, which is usually transmitted through the respiratory tract, and are expected to have great clinical value and therapeutic advantages.

The present invention provides a compound represented by the following formula (I-0) or a hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt thereof:

In formula (I-0), n is 0, 1, 2, 3, 4;

_R is fluorine, chlorine, bromine, iodine, trifluoromethyl, cyano;

R _a , R _b and R _c are each independently selected from hydrogen, deuterium or methyl;

其中， Q is hydrogen,

in,

_X1 is an O atom or an S atom;

n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n2 is 0 or 1, and n1 and n2 are not 0 at the same time;

n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n4 is 0 or 1, and n3 and n4 are not 0 at the same time;

n5 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n6 is 0 or 1;

each R ₁ , R ₂ , R ₃ or R ₄ is independently selected from hydrogen or methyl;

R ₅ is the following groups substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino ;

Group A is the following groups: halogen, cyano, amino, hydroxyl, carboxyl, nitro, trifluoromethyl, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclic group, C1-C8 alkylamino group.

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts thereof are shown in formula (I-1):

The definitions of the substituents in formula (I-1) are as defined in formula (I-0).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (I-2):

The definitions of the substituents in formula (I-2) are as defined in formula (I-0).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (I-3):

The definitions of the substituents in formula (I-3) are as defined in formula (I-0).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts thereof are shown in formula (I-4):

In formula (I-4), the definitions of other substituents are as defined in formula (I-0).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (I-5):

The definitions of the substituents in formula (I-5) are as defined in formula (I-0).

In some embodiments, the compound provided by the invention or its hydrate, solvate, polymorph, isotopic derivative, pharmaceutically acceptable salt:

In formula (I), n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

n2 is 0 or 1;

_X1 is O or S;

_X2 is S or Se;

each R ₁ , R ₂ , R ₃ or R ₄ is independently hydrogen or methyl;

R ₅ is hydrogen or the following groups substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkyl Amino group;

When X ₁ is O and X ₂ is S, R ₅ is not an alkoxy group of C1-C8;

Group A is the following groups: halogen, cyano, amino, hydroxyl, carboxyl, nitro, trifluoromethyl, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclic group, C1-C8 alkylamino group.

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (II):

The definitions of the substituents in formula (II) are as defined in formula (I).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (III):

The definitions of the substituents in formula (III) are as defined in formula (I).

In some embodiments, the compounds provided by the present invention or their hydrates, solvates, polymorphs, isotopic derivatives, and pharmaceutically acceptable salts are shown in formula (IV):

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

In the embodiments of the present application, the halogen refers to fluorine, chlorine, bromine, or iodine.

In the embodiments of the present application, the heteroatom refers to N, O or S.

In the embodiments of the present application, the solvate refers to a complex formed by the interaction between the compound and a pharmaceutically acceptable solvent, and the pharmaceutically acceptable solvent includes ethanol, isopropanol, acetic acid, and ethanolamine.

In the embodiment of the present application, the C1-C8 alkyl group refers to a straight chain or branched saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms in the molecule. Including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc.

In the embodiment of the present application, the C1-C8 alkoxy group and C1-C8 alkylthio group refer to a saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms in the molecule that inserts an oxygen atom at any reasonable position or sulfur atom groups, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, 2-ethylethoxy, methylthio, ethylthio, propoxy Thio, isopropylthio, isobutylthio, etc.

In the embodiment of the present application, the C1-C8 alkylamino group refers to a saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms in the molecule, and a -NH- or _-NH2 group is inserted at any reasonable position. Groups, including monoalkylamino, dialkylamino and cycloalkylamino, including but not limited to methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, di-n-propylamine group, diisopropylamino group, etc.

In the embodiment of the present application, the C3-C8 carbocyclic group refers to a monocyclic or condensed polycyclic saturated or unsaturated cyclic hydrocarbon group containing 3 to 8 carbon atoms, including but not limited to cyclopropyl , cyclopentyl, bicyclo[3.1.0]hexyl, bicyclo[3.2.0]heptyl, cyclopentadienyl, etc.

In the embodiment of the present application, the C2-C8 heterocyclic group refers to a saturated or unsaturated cyclic group containing 2-8 carbon atoms and 1-4 heteroatoms in the molecule. Including but not limited to cycloethoxy, aziridinyl, tetrahydrothienyl, tetrahydropyrrolyl, piperidinyl, hexahydropyridazinyl, dihydropyridyl, cyclopentylsulfide, morpholinyl and the like.

In some embodiments, in formula (I-0), n is 0; in some embodiments, in formula (I-0), n is 1, 2, 3 or 4; in some specific embodiments, In formula (I-0), n is 1; in some specific embodiments, in formula (I-0), n is 2.

In some embodiments, in formula (I-0), R ₀ is fluorine, chlorine, bromine or iodine, preferably, R ₀ is fluorine or chlorine; in some embodiments, in formula (I-0), R ₀ is trifluoromethyl or cyano.

In some embodiments, in formula (I-0), R _a is hydrogen; in some embodiments, in formula (I-0), R _a is deuterium; in some embodiments, formula (I-0) In, R _a is a methyl group.

In some embodiments, _Rb and _Rc are both hydrogen; in some embodiments, _Rb and _Rc are both deuterium; in some embodiments, _Rb is hydrogen and _Rc is deuterium; in some embodiments wherein R _b and R _c are both methyl; in some embodiments, R _b is hydrogen and R _c is methyl.

一些实施方案中，式(I-0)中，Q为

一些实施方案中，式(I-0)中，Q为

In some embodiments, in formula (I-0), Q is hydrogen; In some embodiments, in formula (I-0), Q is

In some embodiments, in formula (I-0), Q is

In some embodiments, in formula (I-0), Q is

In an embodiment of the present invention, n1 and n2 are not 0 at the same time; wherein,

In some embodiments, n1 is 0; in some embodiments, n1 is 1, 2, 3, 4, 5, 6, 7, 8 or 9; in some specific embodiments, n1 is 1; in some In a specific embodiment, n1 is 2;

In some embodiments, n2 is 0; in some embodiments, n2 is 1.

In an embodiment of the present invention, n3 and n4 are not 0 at the same time; wherein,

In some embodiments, n3 is 0; in some embodiments, n3 is 1, 2, 3, 4, 5, 6, 7, 8 or 9; in some specific embodiments, n3 is 1; in some In a specific embodiment, n3 is 2;

In some embodiments, n4 is 0; in some embodiments, n4 is 1.

In some embodiments, n5 is 0; in some embodiments, n5 is 1, 2, 3, 4, 5, 6, 7, 8 or 9; in some specific embodiments, n5 is 1; in some In a specific embodiment, n5 is 2.

In some embodiments, n6 is 0; in some embodiments, n6 is 1.

In some embodiments, X _is an O atom; in some embodiments, _X is an S atom.

In an embodiment of the present invention, each R ₁ or R ₂ is independently hydrogen or methyl, which means that the carbons connected by R ₁ and R ₂ will have n1 or n3 or n5, and when n1 or n3 or n5 When greater than 1, the n1 or n3 or n5 R ₁ or R ₂ do not affect each other, and can be the same or different.

In an embodiment of the present invention, each R ₃ or R ₄ is independently hydrogen or methyl, that is, the carbon atom connected to R ₁ , R ₂ , R ₃ or R ₄ may or may not be substituted by a methyl group. replace.

In some embodiments, R ₅ is the following groups substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1 -C18 alkylamino group; preferably, R ₅ is the following group substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy; more preferably, R ₅ is a C1-C8 alkoxy group substituted or unsubstituted by one or more groups A.

In some embodiments, group A is halogen, trifluoromethyl, or cyano; in some embodiments, group A is amino, hydroxyl, carboxyl, nitro; in some embodiments, group A is Group: C1-C8 alkyl group, C1-C8 alkoxy group, C3-C8 carbocyclyl group, C2-C8 heterocyclic group, C1-C8 alkylamino group.

In an embodiment of the present invention, the formula (I-0) contains a chiral center (the place marked with * is a chiral carbon atom), and the optical isomers refer to those caused by the different configurations of the carbon atoms shown in * Optical isomers, compounds of the present invention or their intermediates can be separated by chiral separation to obtain single-configuration compounds.

In an embodiment of the present invention, the absolute configurations of S-configuration and R-configuration compounds are determined by electron circular dichroism chromatography. In some embodiments, the compounds of the invention are racemates; in some embodiments, the compounds of the invention are in the S-configuration.

In an embodiment of the present invention, the S-configuration and R-configuration compounds are tested for optical rotation (according to the Chinese Pharmacopoeia 2020 Edition-Sibu-0621 optical rotation measurement method, using methanol as a solvent). In some specific embodiments, the compounds of the present invention are racemates; in some specific embodiments, the compounds of the present invention are levorotatory.

In an embodiment of the present invention, the S-configuration compound is a levorotatory isomer.

Compounds provided by the invention include but are not limited to the following compounds:

Or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt.

Further, the pharmaceutically acceptable salts include their inorganic acid salts and organic acid salts.

In some specific embodiments, the pharmaceutically acceptable salts of the compounds include inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc., and organic acid salts include trifluoroacetate, formazan Sulfonate, tartrate, p-toluenesulfonate, etc.

The second aspect of the present invention provides a preparation route for polycyclic compounds shown in structural formula (I), as shown in the following steps:

In the above-mentioned route, the definition of each group is defined as the group in the structural formula (I), wherein L _{and L} are activating leaving groups, and the groups shown in the formula (II-3) are according to the existing literature Reported method (J.Med.Chem.52, (3), 2009, 771-778; WO2019/136112) synthesis, formula (II-1) and formula (II-2) can generate formula (II) under alkaline conditions -3), then formula (II-3) and formula compound (II-4) can generate formula (I) under basic conditions.

In an embodiment of the present invention, materials or intermediates (such as formula SM1, SM2, SM3, SM3-0 and SM5) required for the synthesis of formula (I-0) or comparative examples are carried out with reference to literature or patents, and relevant in formula SM5 The definition of substituent is as described in formula (I-0),

Formula SM1 is synthesized by referring to the synthesis method described in patent CN 110300753 A _; formula SM3 and SM5 are synthesized by referring to the synthesis method described in patent CN 113226327 A. The formula SM2 is synthesized in one step from the commercial product SM2-0 and methanesulfonyl chloride, specifically referring to the synthesis method in the patent CN 113226327 A.

The third aspect of the present invention is to provide a pharmaceutical preparation containing the above compound or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt and pharmaceutically acceptable carrier. combination. The pharmaceutically acceptable carrier includes one or a combination of fillers, binders, diluents, lubricants, preservatives, taste-masking agents or co-solvents. The pharmaceutical composition can be used for anti-influenza virus.

Further, the dosage forms of the pharmaceutical composition are tablets, capsules, powders, granules, pills, suspensions, syrups, and injections.

In the fourth aspect of the present invention, the compounds described in the present invention, including their hydrates, solvates, optical isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, or pharmaceutical compositions thereof, are used For anti-influenza virus purposes.

The compound of the present invention has stronger antiviral activity in vivo or in vitro. Studies have shown that the S-configuration product has good anti-influenza virus activity in vivo, while the R-configuration product has relatively poor activity.

In the animal drug efficacy study of the compound of the present invention, the animal lung virus titer is lower, and the pathological changes of the lung tissue are milder; in the tissue distribution study, the compound of the present invention shows good lung tissue distribution; in the pharmacokinetic study , the ester compound of the present invention shows higher in vivo exposure and shorter peak time than other ester compounds.

Stronger antiviral activity, high exposure in vivo, rapid peak concentration and high distribution in the lungs are more conducive to the drug's anti-influenza virus effect, and are expected to have great clinical value and therapeutic advantages.

The present invention provides the above-mentioned compounds, including compounds, including their hydrates, solvates, optical isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, or pharmaceutical compositions thereof in the preparation of anti-influenza virus Use in medicine.

The present invention provides a method for preventing or treating influenza virus infection, said method comprising administering a therapeutically effective amount of the above-mentioned compound, including its hydrate, solvate, optical isomer, polymorphic form, to an individual in need thereof substances, isotope derivatives, pharmaceutically acceptable salts.

Description of drawings

Figure 1 is the comparison between the theoretical electron circular dichroism spectrum predicted by quantitative calculation and the experimental ECD spectrum to determine the absolute configuration.

Fig. 2 shows the body weight changes of animals in each group in the anti-influenza virus efficacy experiment.

Fig. 3 is the change of the survival rate of animals in each group in the anti-influenza virus efficacy experiment.

Fig. 4 is the lung virus titer in the anti-influenza virus efficacy experiment.

Figure 5 shows the distribution of lung tissue.

Detailed ways

The following examples can make those skilled in the art understand the present invention more comprehensively, but do not limit the present invention in any way, the structures of all compounds are determined by MS or ¹ H-NMR, and the optical isomers of all involved single configurations , were confirmed by optical rotation test or electron circular dichroism spectrum.

Unless otherwise specified in this example, the solvents and reagents used are common commercial products. The starting materials were all purchased commercial raw materials.

Embodiment one: the synthesis of M01 and M11

Synthesis of compound 2:

At room temperature, compound 1 (100g, 793mmol, 1eq) was dissolved in DMF (1.5L), potassium carbonate (219g, 1.59mol, 2eq) was added, the temperature was cooled to 0°C in an ice-water bath, and benzyl bromide (203g, 1.19mol, 1.5eq), the dropwise addition was completed, and kept at 0°C for 30 minutes, then moved to an oil bath at 80°C for 5 hours. TLC (EA/PE=1/2, EA is ethyl acetate, PE is petroleum ether) detection raw material only has a small amount remaining, aftertreatment, system is lowered to room temperature, pours into water (3L), ethyl acetate extracts (300mlx3 ), the combined organic phases were washed with water (100mlx3), washed with saturated brine (200mlx1), dried over anhydrous sodium sulfate for 20 minutes, and the organic phase was filtered and concentrated by column chromatography (EA/PE=1/5～1/1) to obtain 157g of product. Yellow oil, yield 91.8%.

Synthesis of compound 3:

Compound 2 (100g, 115mmol, 1eq) was dissolved in bromobenzene (1L) at room temperature, added SeO ₂ (152g, 347mmol, 3eq) in an oil bath at 180°C for 16 hours, TLC (EA/PE=1/2) detected the reaction completely. The system was lowered to room temperature, adding diatomaceous earth to the Buchner funnel for suction filtration, washing with dichloromethane diatomaceous earth (100mlx3), combining the organic phase with water pump to concentrate dichloromethane, oil pump to concentrate bromobenzene to obtain crude product (100g, red oil) .

Synthesis of compound 4:

Compound 3 (100g, 434mmol, 1eq) was dissolved in DMSO (1.5L) at room temperature, cyclopropylformaldehyde (91g, 1.3mol, 3eq) was added, the temperature was cooled to 0°C in an ice-water bath, and pyrrolidine (31g, 434mmol , 1eq), keep the reaction below 15°C for 30 minutes after the dropwise addition, and move to an oil bath at 50°C for 5 hours. TLC (DCM/MeOH=20/1) detected that the reaction of raw materials was complete, the system was lowered to room temperature, poured into water (3L), extracted with ethyl acetate (250mlx3), combined organic phases were washed with water (100mlx3), washed with saturated brine (100mlx1 ), dried over anhydrous sodium sulfate for 30 minutes, and the organic phase was filtered, concentrated and purified by column chromatography (EA/PE=1/10～1/1) to obtain the product (32g, red oil, yield 24.6%). ESI-MS(+):m/z=301.1; ¹ H-NMR(CDCl ₃ ,400MHz):δ8.85(s,1H),7.69-7.67(d,1H), 7.28-7.43(m,5H) , 6.42-6.43 (d, 1H), 5.28 (s, 2H), 4.93-4.95 (d, 1H), 2.79-2.81 (d, 1H), 1.15-1.27 (m, 4H).

Synthesis of compound 5:

Compound 4 (32g, 107mmol, 1eq) was dissolved in methanol (300ml)/water (150ml) in an ice-water bath, and trifluoroacetylhydrazide (27.3g, 214mmol, 2eq) was added. The reaction was carried out at °C for 4 hours, and TLC (DCM/MeOH=20/1) detected that the reaction of the starting material was complete. Lower the reaction system to room temperature, concentrate the methanol in the system, add water (150ml), extract with ethyl acetate (100mlx3), combine the organic phases and wash with saturated brine (500mlx1), dry over anhydrous sodium sulfate for 10 minutes, and filter the organic phase Purification by concentration column chromatography (EA/PE=1/10～1/1) gave the product (14 g, yellow solid, yield 45%). ESI-MS(+):m/z=297.1; ¹ H-NMR(DMSO-D6,400MHz):δ7.90-7.92(d,1H),7.31-7.43(m,5H),7.14-7.15(s ,1H),6.28-6.30(d,1H),5.78-5.79(d,1H),5.27-5.30(d,1H),5.03-5.06(d,1H),4.09-4.10(m,1H),1.19 -1.28(m,2H),0.85-0.90(m,1H),0.61-0.64(m,1H).

Synthesis of compound 6:

Compound 5 (14g, 47.2mmol, 1eq) was dissolved in THF (150ml) and triethylamine (9.6g, 94.5mmol, 2eq) and DMAP (1.73g, 14.2mmol, 0.3eq) were added in ice-water bath. Keeping the temperature at 0°C, add acetic anhydride (9.6g, 94.5mmol, 2eq) dropwise. After the addition, keep at 0°C for 30 minutes, then move to room temperature for 2 hours. TLC (EA/PE=1/1) detected that the reaction of the starting material was complete. Pour the system into water (150ml), extract with ethyl acetate (50mlx3), combine the organic phases and wash with saturated brine (100mlx2), dry over anhydrous sodium sulfate for 10 minutes, filter and concentrate the organic phases and purify by column chromatography (EA/PE=1 /10～1/1) to obtain product 6 (15 g, yellow solid, yield 94%).

Synthesis of compound 7:

Compound 6 (15g, 44.3mmol, 1eq) was dissolved in THF (150ml) and methanol (50ml) under an ice-water bath, and sodium borohydride (3.35g, 133mmol, 3eq) was added in batches while keeping the temperature at 0°C, and kept at 0°C after the addition React for 30 minutes, move to room temperature and react for 2 hours. TLC (EA/PE=1/1) detected the disappearance of raw materials, poured the system into ammonium chloride (150ml) aqueous solution, extracted with ethyl acetate (50mlx3), combined the organic phases and washed with saturated brine (50mlx3), dried over anhydrous sodium sulfate After 10 minutes, the organic phase was filtered and concentrated to obtain a crude product (15 g, yellow solid), which was directly sent to the next step without purification.

Synthesis of compound 8:

Under ice-water bath, compound 7 (15g, 44mmol, 1eq) was dissolved in THF (150ml), DMAP (0.54g, 4.4mmol, 0.1eq), triethylamine (8.92g, 88mmol, 2eq) were added, and the temperature was kept at 0°C Boc ₂ O (19.2g, 88mmol, 2eq) was added dropwise, after the addition was completed, the reaction was maintained at 0°C for 30 minutes, then moved to room temperature for 2 hours. TLC (DCM/MeOH=20/1) detected the disappearance of the raw materials, poured the system into water (150ml), extracted with ethyl acetate (50mlx3), combined the organic phases and washed with saturated brine (50mlx3), dried over anhydrous sodium sulfate for 10 minutes, The organic phase was filtered and concentrated and purified by column chromatography (EA/PE=1/10～1/1) to obtain the product (15 g, yellow solid, yield 78%).

Synthesis of compound 9:

Compound 8 (15g, 34mmol, 1eq) was dissolved in methanol (150ml) in an ice-water bath, and potassium carbonate (4.7g, 34mmol, 1eq) was added in portions. After the addition, the mixture was kept at 0°C for 30 minutes and at room temperature for 6 hours. TLC (DCM/MeOH=20/1) detected that the reaction of raw materials was complete, poured the system into water (150ml), extracted with ethyl acetate (50mlx3), combined the organic phases and washed with saturated brine (30mlx3), dried over anhydrous sodium sulfate After 10 minutes, the organic phase was concentrated by filtration to obtain the crude product (10 g, yellow solid).

Synthesis of compound 10:

Compound 9 (10g, 25mmol, 1eq) was dissolved in DCM (100ml) under an ice-water bath, and the Dess-Martin oxidant (16g, 37.6mmol, 1.5eq) was added in batches while keeping the temperature at 0°C. After the addition, the reaction was kept at 0°C for 30 minutes , reacted at room temperature for 5 hours. TLC (DCM/MeOH=15/1) detected that the reaction of the raw materials was complete, the system was poured into an aqueous solution of sodium bicarbonate (100ml), extracted with DCM (30mlx3), and the combined organic phase was washed with saturated brine (30mlx1). Dry over anhydrous sodium sulfate for 10 minutes, filter the organic phase, concentrate and purify by column chromatography (EA/PE=1/10～1/0) to obtain 10 g of the crude product, beat the crude product with ethyl acetate to obtain 5.4 g of the pure product, and concentrate the mother liquor to obtain the crude product 4.6 g. ESI-MS (+): m/z = 397.3; ¹ H-NMR (DMSO-D6, 400MHz): δ7.84-7.86 (d, 1H), 7.28-7.46 (m, 5H), 6.30-6.32 (d ,1H),5.07-5.20(m,2H),3.85-4.20(dd,2H),1.43(s,9H),1.35-1.37(m,1H),1.18-1.28(m,2H),1.08-1.12 (m,1H).

Synthesis of Compound 12

After replacing the nitrogen in the there-necked flask, add 226ml of phenylmagnesium bromide (2.8mol/L), lower the temperature to below 10°C in an ice-water bath, add 49.9g of selenium powder in batches, control the reaction temperature not to exceed 30°C, add the selenium powder React for 2 hours after completion. Add 2 mol/L hydrochloric acid in an ice-water bath, the reaction exotherm is obvious, add EA to extract, separate the liquid, spin dry the organic phase, and obtain the product 12 as a brown oil with a strong odor. Proceed directly to the next reaction.

Synthesis of Compound 14

After replacing the nitrogen in the three-necked flask, add 81.18g of LDA (lithium diisopropylamide), drop the temperature in the dry ice-ethanol solution to -30°C, add dropwise the THF solution of 50g of compound 3,4-difluorobenzoic acid, drop During the addition process, the heat release is obvious. Control the temperature of the reaction system to be lower than -20 degrees Celsius. React for 2 hours. Add DMF. The heat release is obvious. Control the temperature of the reaction system to be lower than -20 degrees Celsius. Without temperature control, gradually transfer to room temperature and react overnight. Sampling to detect the end of the reaction. HCl was added to the reaction system, the exotherm was obvious, EA was added for extraction, the liquid was separated, and the organic phase was spin-dried to obtain 72 g of the crude product of compound 14, which was directly used in the next reaction. The obtained crude product was a yellow solid with a yield of 122% (material DMF is not spin-dried).

Synthesis of Compound 15

After nitrogen was replaced in the three-necked flask, 58.81g of compound 12, 49.6g of compound 14, 300ml of toluene, and 17.6g of camphorsulfonic acid were added, and the temperature was raised to 70°C for overnight reaction. The reaction system was lowered to room temperature, NaOH solution was added, the liquid was separated, the aqueous phase was extracted with EA, the organic phase was combined, the organic phase was washed with saturated NaCl and then spin-dried, the crude product: 123.08g, the obtained crude product was beaten with PE and suction filtered to obtain The product was 34.4g, and the product recovered from the beating filtrate was 14.83g. Compound 15 was an orange solid with a yield of 47.9%.

Synthesis of compound 16

Add 16.9g of AlCl ₃ and 250ml of toluene into the three-necked flask, cool the system in an ice-water bath, add 17.1g of tetramethyldisiloxane, stir well, add 150ml of toluene solution of compound 15 (34.4), the reaction is slightly exothermic, and AlCl ₃ Dissolve gradually, heat to 80°C, and react for 1 hour; stop the reaction, add sulfuric acid solution (16.2mL+240mL water), separate the liquid, extract the aqueous phase with EA, spin the organic phase to dry, beat the crude product with PE and filter it with suction. 22g of solid was obtained, the obtained solid was a yellow powdery solid, the theoretical amount of product: 34.68g, a total of 22g of solid product Compound 16 was obtained, and the yield: 63.4%. used directly in the next reaction.

Synthesis of compound 17:

Add 429g polyphosphoric acid into the three-neck flask, heat to 80°C, add 42g of compound 16, heat up to 120°C, the reaction system is viscous, the color changes from yellow to deep purple, react for 1 hour, take a sample, add water, after EA treatment; reduce When the reaction temperature was below 100°C, add water and stir evenly, add EA to extract, separate the organic phase, spin dry, beat the crude product with PE, filter to obtain 3.4g of the product, and separate the filtrate by column chromatography to obtain 10g of the product, compound 17 is colorless To pale yellow flocculent solid, theoretical yield: 39.69g, actual yield: 13.4g, yield: 33.76%.

Synthesis of Compound 18:

Add 2.12 g of compound 17 and 20 ml of ethanol into the three-necked flask, and stir the system. The ethanol solution of sodium borohydride (0.26g) was added dropwise in an ice-water bath, and the reaction system had a slight temperature rise. After the addition was completed, it was transferred to room temperature for reaction, and the material gradually dissolved. Treatment; add 2 mol/L hydrochloric acid until no bubbles are generated, pH=4-6, a large amount of solids are precipitated, and the solid is obtained by suction filtration, the filtrate is extracted with EA, the organic phase is separated, and the organic phase is spin-dried to obtain the product 18 as a brown solid. used directly in the next reaction.

Synthesis of Compound 19

Add 2.5g of compound 10, 2.13g of compound 18, 5.44g of compound T3P (propyl phosphoric anhydride), 1.1g of methanesulfonic acid and 30ml of EA into the single-necked bottle, heat up to reflux, react overnight, take a sample for detection, and carry out after the reaction is completed. Treatment; post-treatment: Add saturated aqueous sodium bicarbonate solution until no bubbles are released, separate liquids, extract the aqueous phase with EA, combine the organic phases, spin dry, and beat the crude product with PE to obtain product 19, which is a light brown powdery solid , Theoretical yield: 3.36g, actual yield: 2.2g, yield: 65.48%.

Synthesis of Compound M01:

Add 2.6g of compound 19, 0.94g of compound LiCl and 20ml of DMA (N,N-dimethylacetamide) into the single-necked bottle, and heat up to 100°C. The reaction solution is yellow and turbid. After 2 hours of reaction, take a sample for detection. After the reaction is completed For processing; adding saturated aqueous sodium bicarbonate solution, solids were precipitated, suction filtered, the filtrate was extracted with EA, the organic phase was spin-dried, and the crude product was beaten with PE to obtain a total of 2.28g of compound M01, the product was a brown solid powder, yield: 104 % (the yield is slightly higher than the theoretical yield, and the reason is judged to be the residual solvent DMA). used directly in the next reaction. ESI-MS (+): m/z = 501.1.

Synthesis of compound M11:

1.1 g of compound M01, 0.61 g of K ₂ CO ₃ , 0.55 g of KI and 0.55 g of chloromethyl dimethyl carbonate were added to the one-necked bottle. Add 20mL of DMA to the system, raise the temperature to 60°C, react overnight, take samples for detection, and process after the reaction is completed. Cool the reaction system to room temperature, add 2N HCl, add water, solid precipitates, filter with suction, extract the filtrate with EA, separate the organic phase, dissolve the filter cake with EA, mix with the organic phase, dry the organic phase with anhydrous sodium sulfate, After spin-dried and separated by column chromatography, 0.766 g of brown solid powder of compound M11 was obtained, yield: 58.9%. ESI-MS(+): m/z＝589.5; ¹ H-NMR (DMSO-D6, 400MHz): δ7.26-7.41(m, 5H), 7.10-7.13(m, 1H), 6.92-6.98(m ,2H),5.90-5.92(d,1H),5.74-5.86(d,1H),5.56-5.58(d,1H),5.38-5.42(m,2H),4.12-4.16(m,2H),4.01 -4.07(m,1H),3.74(s,3H),2.79(s,1H),1.73-1.75(m,1H),1.16-1.24(m,2H),0.87-0.90(m,1H),0.72 -0.74(m,1H).

Example 2: Splitting of M19

Compound M11 was prepared and isolated to obtain M19 and enantiomer M19-1, both of which were white solids. Preparation conditions: use supercritical liquid chromatography; chromatographic column: CHIRALPAK IB-N 4.6*100mm, 3μm; solvent is methanol, carrier is liquid CO ₂ ; pressure is 1500psi, flow rate is set to 2.0ml/min, column temperature is 25°C, Gradient elution. Among them, the retention time of M19 is 2.86min, and the optical rotation is measured with methanol as the solvent, and the result shows that it is a levorotatory form. The retention time of M19-1 was 2.55min, and the optical rotation was measured with methanol as the solvent, and the result showed that it was dextrorotatory. M19 and M19-1 adopt the method widely used in the world to determine the absolute configuration of chiral compounds, quantitatively calculate and predict the theoretical electron circular dichroism (ECD, usually referred to as circular dichroism) spectrum, and compare with the experimental ECD spectrum ( The comparison results are shown in Figure 1), and the experimental ECD signal is consistent with the theoretical calculation results, so that the absolute configuration is finally determined, M19 is the S configuration, and M19-1 is the R configuration.

Embodiment three: the synthesis of PX90-02

Synthesis of compound 25:

Add 2g of compound SM1, 2.54g of compound SM2, 0.6g of pyridine and 30ml of 1,4-dioxane into the reaction flask, and heat the system at 60°C for 6 hours. After the reaction, the system was concentrated to dryness, extracted with dichloromethane and water, the organic phase was concentrated to dryness, and purified by silica gel column to obtain 2.16 g of compound 25 with a yield of 59%. ESI-MS (+): m/z = 542.48.

Synthesis of compound N01:

2 g of compound 25, 20 ml of ethyl acetate, and 0.88 g of 5% palladium carbon were added to the reaction flask, and the system was filled with a hydrogen balloon and replaced with hydrogen (1 atmosphere) and stirred at room temperature for 2 hours. After filtration with celite, the system was concentrated to dryness under reduced pressure and purified by flash silica gel column to obtain 1.48 g of compound N01 with a yield of 89%, ESI-MS (+): m/z=452.78.

Synthesis of compound PX90-02-01:

1.2 g of compound N01 was dissolved in acetonitrile (15 mL), 0.6 ml of diisopropylethylamine was added, and 0.4 g of 2-bromoethanol was added. Then it was heated to 90° C., and the reaction was detected by TLC. After cooling to room temperature, evaporate to dryness under reduced pressure, add water and dichloromethane, shake, collect the organic phase, wash with 10% NaOH (10ml), water (10ml) and brine (10ml), dry over sodium sulfate, filter, reduce It was evaporated to dryness under pressure and purified by silica gel column to obtain 0.95 g of compound PX90-02-01, with a yield of 72%. [M+H] ⁺ = 496.13.

Synthesis of Compound PX90-02:

Add 0.5 g of compound PX90-02-01, 0.66 g of cesium carbonate, 33 mg of potassium iodide, 8 ml of N,N-dimethylacetamide and 20 ml of water into the reaction flask, stir the system and raise the temperature to 60 °C, add 0.2 g of chloromethyl carbonate Methyl ester in N,N-dimethylacetamide solution 5ml. The system was reacted at 55°C for 8 hours, cooled to room temperature, added ethyl acetate, washed with saturated ammonium chloride, water and saturated brine successively, separated, and the organic phase was dried with anhydrous sodium sulfate; the organic phase was concentrated and evaporated to dryness, and the residue Purified by a silica gel column to obtain 0.46 g of compound PX90-02, with a yield of 46%. [M+H] ⁺ = 584.73.

Embodiment four: the synthesis of M10

Synthesis of Compound 22:

Compound 22 was synthesized according to the synthesis reference (Pharmaceutical Research, 2005, vol.22, #3, p.390-396). 2.9 g of compound 21 in 10 ml of dichloromethane solution was cooled to 0°C, slowly added dropwise into 12 ml of dimethylamine tetrahydrofuran solution, and the system was reacted at room temperature for 24 hours. Concentrate to dryness, add dichloromethane and water, separate the organic phase, and wash the organic phase with 5% NaHCO ₃ solution 3 times. The organic phase was concentrated to dryness to obtain 1.31 g of compound 22, yield 43%. [M+H] ⁺ = 137.92.

Synthesis of compound M10:

Add 0.5 g of compound M01, 0.65 g of cesium carbonate, 33 mg of potassium iodide, 8 ml of N,N-dimethylacetamide and 20 ml of water into the reaction flask, stir the system and raise the temperature to 60°C, add 0.15 g of compound 22 in N,N-di Methylacetamide solution 5ml. The system was reacted at 55°C for 8 hours, cooled to room temperature, added ethyl acetate, washed with saturated ammonium chloride, water and saturated brine successively, separated, and the organic phase was dried with anhydrous sodium sulfate; the organic phase was concentrated and evaporated to dryness, and the residue After purification by silica gel column, 0.32 g of compound M10 was obtained, with a yield of 53%. [M+H] ⁺ = 600.73.

Embodiment five: the synthesis of M03 and M14

Synthesis of compound 23

Under nitrogen protection, 1 g of compound 17 was dissolved in 15 ml of THF, and 140 mg of lithium aluminum hydride-D4 was slowly added at 0°C, and the system was heated to 25°C for 8 hours. The system was cooled to 0°C, and water was added to quench the reaction. The system was extracted with 2N hydrochloric acid and EA. The organic phase was concentrated to dryness. Column chromatography obtained 0.58 g of compound 23 with a yield of 57%, ESI-MS (+): m/z=314.0.

Synthesis of Compound 24

Referring to the synthesis method of compound 19, a total of 0.94 g of compound 24 was synthesized, ESI-MS (+): m/z=592.1.

Synthesis of compound M03

Referring to the synthesis method of compound M01, a total of 0.51 g of compound M03 was synthesized, ESI-MS (+): m/z=502.1.

Synthesis of Compound M14

Referring to the synthesis method of compound M11, a total of 0.32 g of compound M14 was synthesized, ESI-MS (+): m/z=590.1.

Embodiment six: the synthesis of M07 and M08

Synthesis of Compound M08-1:

Under the protection of nitrogen, 1 g of compound 17 was dissolved in 15 ml of THF, and 4 ml of methyllithium reagent (1.6 M ether solution) was slowly added dropwise at -20 °C, and the system was naturally warmed to 25 °C for 8 hours. The system was cooled to 0°C, and water was added to quench the reaction. Concentrate to dryness, extract with EA and water. The organic phase was separated and concentrated to dryness. Column chromatography obtained 0.77 g of compound M08-1, with a yield of 73%, ESI-MS (+): m/z=327.1.

Synthesis of Compound M08-2

Referring to the synthesis method of compound 19, a total of 0.94 g of compound M08-2 was synthesized, ESI-MS (+): m/z=605.2.

Synthesis of Compound M07

Referring to the synthesis method of compound M01, a total of 0.51 g of compound M07 was synthesized, ESI-MS (+): m/z=515.1.

Synthesis of Compound M08

Referring to the preparation and separation method of compound M01, compound M08 was obtained. It was confirmed that M08 was a levorotatory form, and the absolute configuration was S configuration.

Embodiment seven: the synthesis of M24

Synthesis of Compound M04

Compound M01 was prepared and isolated to obtain M04 and enantiomers, all of which were white solids. Preparation conditions: use supercritical liquid chromatography; chromatographic column: CHIRALPAK IB-N 4.6*100mm, 3μm; solvent is methanol, carrier is liquid CO ₂ ; pressure is 1500psi, flow rate is set to 2.0ml/min, column temperature is 25°C, Gradient elution. Methanol was used as solvent to measure optical rotation, M04 was shown as levorotatory, and the enantiomer of M04 was shown as dextrorotary. Compared with M19 and M19-1, it can be seen that the absolute configuration of M04 is S configuration.

Synthesis of Compound M24

Under an ice-water bath, 100 mg of compound M04 was dissolved in 3 ml of DMF, and after adding 16 mg of sodium hydride (60%) and stirring for 30 minutes, 60 mg of chloromethyl tert-butyrate was added, and the reaction was slowly raised to room temperature for half an hour, and the reaction was continued for 12 hours , quenched with water, extracted 3 times with EA and water, combined the organic phases, dried with anhydrous sodium sulfate, and separated the product by chromatography. After concentration, 41.8 mg of compound M24 was obtained, with a yield of 34%. ESI-MS (+): m/z = 615.1.

Embodiment eight: the synthesis of compound M26

Synthesis of Compound M26-3

4.7ml of compound M26-1 and 3ml of pyridine were dissolved in 30ml of dichloromethane, and the system was cooled to 0°C. 3ml of compound M26-2 was slowly dropped into the above system, and the inner temperature was controlled not to be higher than 10°C. The system was reacted at room temperature for 16 hours, saturated sodium carbonate solution was added to the system for extraction, and the organic phase was concentrated to dryness under reduced pressure to obtain an oil, which was directly used in the next step.

Synthesis of Compound M26

Referring to the synthesis method of compound M11, compound M04 and compound M26-3 were reacted to obtain 48 mg of compound M26, with a yield of 42%, ESI-MS (+): m/z=633.1.

Comparative example one: the synthesis of N02

Referring to the preparation and separation method of compound M19, compound N02 was prepared and isolated from compound N01. After the optical rotation test and comparison with the optical rotation of M19, NO2 is levorotatory.

Comparative Example 2: Synthesis of N11 and N12

Referring to the synthesis method of compound M19, N11 was synthesized in one step using N01 as a material, and then compound N12 was obtained by a similar preparation and separation method of M19. After optical rotation test and comparison with M19 optical rotation, N12 is L-isomer, S configuration.

Comparative Example 3: Synthesis of Compound N13

Synthesis of Compound N13-2

Add 1 g of compound N13-1 and 60 ml of dichloromethane into the reaction flask, add 1 g of imidazole and 1.55 g of tert-butyldimethylsilyl chloride (TBSCl) into the system at room temperature, and react for 3 hours at room temperature. Concentrate to dryness, and purify through a silica gel column to obtain 0.968 g of compound N13-2. ¹ H-NMR (CDCl ₃ , 300MHz)σ:0.08(s,6H),0.42-0.58(m,4H),0.93(s,9H),2.76(brs,1H),3.44-3.60(s,2H) ,3.62-3.65(s,2H).

Synthesis of Compound N13-3

Add 0.91g of compound N13-2 and 40ml of dichloromethane into the reaction flask, add 1.96g of Dess-Martin reagent (DMP) into the system at 0°C, and keep the reaction at 0°C for 30 minutes. The system was purified by silica gel column to obtain 0.72 g of compound N13-3. ¹ H-NMR (CDCl ₃ , 300MHz)σ:0.05(s,6H),0.87(s,9H),1.12(s,4H),3.87-3.95(s,2H),8.97-9.04(s,1H) .

Synthesis of Compound N13-4

Under nitrogen protection, 1.2 g of triphenylphosphine bromide and 20 ml of anhydrous tetrahydrofuran were added to the reaction flask. The system was cooled to 0°C, and 1.5ml (2.5M) of n-butyllithium was added to react for 10min. 10 ml of compound N13-3 (0.66 g) in anhydrous tetrahydrofuran was added, and the system was reacted at room temperature for 2 hours. The reaction was quenched by adding water, the system was concentrated to dryness, extracted with dichloromethane and water, the organic phase was concentrated to dryness and purified by a silica gel column to obtain 0.53 g of compound N13-4. ¹ H-NMR (CDCl ₃ , 300MHz)σ:0.05(s,6H),0.50-0.75(m,4H),0.85-0.92(s,9H),3.62-3.66(s,2H),4.90-5.05( m,2H),5.65-

5.75 (m, 1H).

Synthesis of Compound N13-5

Add 0.52 g of compound N13-4 into the reaction flask, and then add 7.5 ml of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (1M TBAF in THF). The system reacted overnight at room temperature. The system was concentrated to dryness, extracted with dichloromethane, and purified by silica gel column to obtain 25 mg of compound N13-5. ¹ H-NMR (CDCl ₃ , 300 MHz) σ: 0.50-0.77 (m, 4H), 3.60-3.66 (s, 2H), 5.01-5.20 (m, 2H), 5.61-5.75 (m, 1H).

Synthesis of compound N13-6

Under nitrogen protection, 22mg of chloromethyl chloroformate was dissolved in anhydrous dichloromethane, 24mg of pyridine was added at 0°C, and then 15mg of compound N13-5 in dichloromethane was added dropwise. Then the system was transferred to room temperature to react for 3 hours, and the system was concentrated to dryness to obtain the crude product of compound N13-6, which was directly used in the next step without treatment.

Synthesis of compound N13

At room temperature, 30 mg of compound M04 was added to 1 ml of DMA (N,N-dimethylacetamide), and then 2 mg of potassium iodide and 16.6 mg of potassium carbonate were added. A DMA solution of compound N13-6 was added to the system, and reacted at 50° C. for 5 hours. 18.45 mg of compound N13 was obtained by reverse-phase column chromatography, with a yield of 47%, ESI-MS (+): m/z 655.1 [M+H].

According to the same method as the above examples, using commercially available compounds or intermediate compounds suitably synthesized from commercially available compounds, the following examples of compounds were synthesized:

Or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt.

Example 9: Determination of Cytopathic Extent (CPE)

MDCK cells were inoculated into 96-well culture plates and cultured at 37°C in 5% CO ₂ . During the exponential growth phase of cells, add maintenance solutions containing samples of different dilutions and positive control drugs, set 3 replicate wells for each concentration, and set normal cell control wells at the same time. After adding the sample, culture it for 72 hours, and carry out the cytotoxicity test of the sample by the CPE method. In addition, the MDCK cells were inoculated into a 96-well culture plate and cultured at 37° C. in 5% CO ₂ . Infect influenza virus (A/Hanfang/359/95(H3N2)) after 24 hours, absorb for 2 hours, discard virus solution, add maintenance solution containing samples of different dilutions and positive control drug, and set 3 replicate wells for each concentration At the same time, a cell control well and a virus control well were set up, and cultured at 5% CO ₂ at 37°C. The antiviral test of the tested samples was carried out by the CPE method, and the cytopathic degree (CPE) of each group was observed when the diseased degree (CPE) of the virus control group reached 4+. The half toxic concentration (TC ₅₀ ) of the sample to the cells and the effective concentration (EC ₅₀ ) of the drug that inhibits 50% of the cytopathic effect of the sample were calculated respectively by the Reed-Muench method, as shown in Table 1:

Table 1: Cytotoxicity and inhibitory activity against influenza virus of compounds

Sample Description: In the experiment, N01, N02, N11, and N12 are compounds disclosed in existing literature, which are used as comparative examples in the present invention.

Experiments show that the compound of the present invention has better anti-influenza virus activity. Among them, compared with the comparative example compounds N01, N02 and baloxavir, the cell activity of M01 to M08 (all of which are not esterified structures) in the compound of the present invention is significantly better; compared with the comparative example compounds N11, N12 and baloxavir Compared with esters, the compounds of the present invention also have better anti-influenza virus activity; and all compounds do not show obvious cytotoxicity.

Embodiment 10: Anti-influenza virus activity in vivo

30 BALB/c mice, female, 6-8 weeks old. Randomly divided into 6 groups: vehicle group, positive control group, test product group. The mice were inoculated with the virus (A/PR/8/34(H1N1)) by intranasal drops on the 0th day, and the inoculation dose was 1500p.f.u./mouse. From the 1st day to the 7th day, the vehicle or the positive control or the test product were continuously treated for 7 days, twice a day, the administration method was intragastric administration, the administration volume was 10mL/kg, and the first administration time was after the virus inoculation 24 hours. Animals were continuously observed from day 0 to day 14, and body weight, health and survival were recorded.

It can be seen from Figure 2 that the body weight of mice in the vehicle group, Group 3 (M19-1 low-dose group) and Group 4 (M19-1 high-dose group) began to decline significantly from the 3rd day, and then continued to decline until death Or be euthanized; the weight of the mice in the baloxavir (5mpk) group began to drop significantly from the 3rd day, and the body weight dropped to the lowest point on the 3rd day, with a maximum drop of 9.59%, and then recovered to the normal level from the 4th day; The body weight of mice in group 5 (M19 low-dose group) began to decrease significantly from the 3rd day, and the body weight dropped to the lowest point on the 6th day, with a maximum drop of 15.51%, and recovered to the normal level on the 7th day. The body weight of mice in group 6 (M19 high-dose group) began to drop significantly from the 3rd day, and the body weight dropped to the lowest point on the 3rd day, with a maximum drop of 5%, and recovered to the normal level on the 5th day.

As can be seen from Fig. 3: the mice in the vehicle group began to die on the 6th day, and all died on the 9th day, and the final survival rate was 0%; the 3rd group (M19-1 low dose group) mice The mice in the 4th group (M19-1 high-dose group) died from the 7th day, and all died on the 8th day, and the final survival rate was 0%. The survival rate was 0%; neither the baloxavir group nor the M19 high/low dose group died, and the survival rate was 100%.

The results showed that: the mice in the vehicle group showed symptoms of infection after virus inoculation, and eventually all died, the median survival period was 7.5 days, and the final survival rate was 0%; Weight loss induced by virus infection protected mice from death, showing expected in vivo anti-influenza virus efficacy. The above results meet the inclusion criteria and meet the expectations of the model, which proves that the experimental science is credible, and provides a reference and window for the evaluation of the efficacy of the test compound. Surprisingly, the test compound M19-1, which performed better in the cell activity test, failed to relieve the weight loss caused by infection in mice under the set experimental conditions, indicating that it had no anti-influenza virus efficacy in vivo. However, another test compound M19, which performed slightly worse in the cell activity test, was administered 24 hours after virus inoculation, which could alleviate the weight loss of mice caused by virus infection and protect mice from death, indicating that it had good in vivo resistance. Drug efficacy of influenza A virus.

Embodiment 11: virus titer test and histopathological examination

Twenty C57BL/6J mice (female, 8 weeks old, 18-20 g) were divided into 4 groups, 5 mice in each group. After intranasal inoculation of semi-lethal dose of A/PR/8/34 (H1N1) virus, test drug was given by intragastric administration twice a day. Lung tissue samples were collected 5 days after infection, the left lung was pathologically examined, and the right lung homogenate was tested for TCID ₅₀ . The virus titer results are shown in Figure 4.

Lung virus titer test result shows: compared with vehicle group, the lung tissue virus titer of compound M19 of the present invention, M20 and comparative example compound N12 is lower; Compared with comparative example compound N12, under the same dose, the present invention Compounds M19 and M20 had lower virus titers and showed better anti-influenza virus effects. At the same time, the results of lung histopathological examination also showed that compared with the comparative compound N12, the lung lesions of the mice treated with the compounds M19 and M20 of the present invention were milder.

Embodiment 12: Rat oral pharmacokinetic test

20 SD rats, male, 180g-220g, before the start of the experiment, the SD rats were intubated in the jugular vein and adapted for three days (free feeding and drinking, room temperature: 20-26°C; humidity: 40-70%; light illumination : dark = 12h: 12h) after the start of the experiment. The experimental animals were divided into 6 groups A/B/C/D/E/F, with 3 animals in each group. Each group was given the suspension of the test product by oral gavage respectively (the test product was respectively M04, M19, M24, M25, M26, N13, all suspended with 0.5% sodium carboxymethylcellulose), and the dosage was M19 was calculated as 2.25mg/kg, and each group was administered in equimolar amounts. Fasting starts at 5:00 p.m. the day before the administration, but water is not allowed, and the fasting is 16-17h. After 4 hours of administration, the animals were given food, and water could not be restrained during the whole process.

15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h before administration and after administration. About 0.25mL whole blood was collected through the jugular vein into sodium heparin anticoagulant tubes. Immediately after blood collection, the anticoagulant tube containing the blood sample was inverted 5 to 10 times, and temporarily stored in an ice bath. Blood samples were centrifuged at 3000 rpm for 5 minutes at 4°C within 1 hour after collection. The plasma collected by centrifugation was transferred to a new labeled centrifuge tube and temporarily stored in a -20°C refrigerator. After all samples were collected, they were handed over to the biological sample administrator and stored in a -80°C refrigerator. After the biological sample is processed, the analyte (the analyte is M04) is detected by LC-MS/MS. Use WinNonlin 7.0 to calculate the main pharmacokinetic parameters according to the non-compartmental model method. The main pharmacokinetic parameters of each group of samples after intragastric administration are shown in Table 2:

Table 2: The main pharmacokinetic parameters in rats after equimolar gavage administration of the test product

the M04 M19 M24 M25 M26 N13 T1/2(h) 5.41 4.33 4.27 5.15 4.22 4.53 Tmax(h) 2 1 1 1 1 2 Cmax(ng/ml) 4.39 34.5 22.9 5.36 19.7 2.89 AUC(h*ng/ml) 66.974 289.914 147.285 81.360 145.629 40.970

In the pharmacokinetic study of rat gavage administration, the inventor selected several different ester compounds with relatively close structures and pharmaceutical precedents for research. The results showed that: compared with M04, rats gavage After administration, the peak time (Tmax) of the compounds of the present invention is shorter, which shows that the compounds of the present invention can act rapidly; in addition, compared with M04, the plasma exposure (AUC) of the compound M19 group increased significantly, and the M24 and M26 groups The exposure also increased in varying degrees, but the exposure of M25 and M13 groups even decreased, which shows that in the pharmacokinetic experiments of rats, different ester-forming methods affect the in vivo exposure of gavage administration , presumably due to the different degrees of absorption or metabolism of different ester-forming compounds.

Embodiment 13: Mouse tissue distribution test

There were 45 DBA/1J mice (6-week-8-week-old, female), and the mice were weighed one day before the administration to calculate the dose. They were randomly divided into 3 groups, 15 rats in each group, and 1 mg/kg of M19, M20 and N12 were administered by oral gavage (N12 was used as a comparative compound in this experiment). Mice were sampled at the following 5 time points: 1 hr, 2 hrs, 4 hrs, 8 hrs and 24 hrs (3 mice per time point). Euthanize mice by _CO2 at each time point. Organs (lung, liver, kidney, spleen, and heart) were collected from euthanized mice, the surface was cleaned with normal saline, and the water was blotted dry with filter paper and weighed. Saline was ground and homogenized to obtain the homogenate of each tissue. The homogenate was stored at -80°C. Biological samples were processed and analyzed by LC-MS/MS. The results are shown in Figure 5.

As can be seen from Figure 5: within 1-4 hours after administration, the three compounds all reached the highest concentration in the above-mentioned tissues, and wherein the highest concentration was reached in about 2 hours in the lung tissue. Afterwards, the tissue concentration of compound N12 decreased rapidly, while the concentration of compounds M19 and M20 of the present invention decreased slowly, and could still be detected in lung tissue 24 hours after administration, but compound N12 was lower than the detection limit 24 hours after administration , which shows that the compounds M19 and M20 of the present invention have a low elimination rate and a long half-life in lung tissue.

The present application describes a plurality of embodiments, but the description is illustrative rather than restrictive, and there may be more embodiments and implementations within the scope of the embodiments described in the present application.

Claims (13)

A compound, as shown in formula (I-0), or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt:

In formula (I-0), n is 0, 1, 2, 3, 4; _R is fluorine, chlorine, bromine, iodine, trifluoromethyl, cyano; R _a , R _b and R _c are each independently selected from hydrogen, deuterium or methyl; Q is hydrogen,

in, _X1 is an O atom or an S atom; n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n2 is 0 or 1, and n1 and n2 are not 0 at the same time; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n4 is 0 or 1, and n3 and n4 are not 0 at the same time; n5 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n6 is 0 or 1; each R ₁ , R ₂ , R ₃ or R ₄ is independently selected from hydrogen or methyl; R ₅ is the following groups substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino ; Group A is the following groups: halogen, cyano, amino, hydroxyl, carboxyl, nitro, trifluoromethyl, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclic group, C1-C8 alkylamino group. A compound as shown in formula (I-1) or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt:

In formula (I-1), the substituents are as defined in claim 1. The compound as claimed in claim 1 or 2, as shown in formula (I-2):

The substituents in formula (I-2) are defined as defined in any one of claims 1-2. The compound as described in any one of claims 1-3, as shown in formula (I-3):

The substituents in formula (I-3) are defined as defined in any one of claims 1-3. The compound as claimed in claim 1, as shown in formula (I-4):

In formula (I-4), the substituents are as defined in claim 1. The compound as claimed in claim 1, as shown in formula (IV):

In formula (IV), the substituents are as defined in claim 1. The compound as described in any one of claims 1-5, as shown in formula (I-5):

The substituents in formula (I-5) are as defined in any one of claims 1 to 5. The compound according to any one of claims 1-7, selected from the following structures:

Or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt. A pharmaceutical composition, comprising the compound according to any one of claims 1-8 or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt and pharmaceutical acceptable carrier. The pharmaceutical composition according to claim 9, wherein the dosage form of the pharmaceutical composition is tablet, capsule, powder, granule, pill, suspension, syrup, injection. The compound described in any one of claims 1-8, including its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt, or the compound described in claim 9 The pharmaceutical composition is used for anti-influenza virus. The compound described in any one of claims 1-8, including its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt, or the compound described in claim 9 Use of the pharmaceutical composition in the preparation of anti-influenza virus drugs. A method for preventing or treating influenza virus infection, said method comprising administering a therapeutically effective amount of the compound of any one of claims 1-8, including its hydrates, solvates, optical Isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, or the pharmaceutical composition of claim 9.

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