CN117924325A - Anti-influenza virus derivative and application thereof - Google Patents

Abstract

The invention discloses a high-efficiency antiviral derivative and application thereof; the derivative is a compound shown in the following formula (I) or a hydrate, a solvate, an optical isomer, a polymorph, an isotope derivative and a pharmaceutically acceptable salt thereof, and a preparation method of the derivative is also disclosed. The compound of the invention can be used for preparing medicines for preventing/treating influenza viruses.

Description

Anti-influenza virus derivative and application thereof

The application relates to a split application of an anti-influenza virus derivative and application thereof, wherein the application date is 2023, 08, 07, the application number is 202310984260.6.

Technical Field

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

Background

Influenza viruses mainly include four types of influenza a virus, influenza b virus, influenza c virus and influenza d virus.

The drugs currently on the global market are four types of neuraminidase inhibitors, hemagglutinin inhibitors, RNA polymerase inhibitors and M2 ion channel blockers. M2 ion channel blockers include amantadine and rimantadine, which are resistant to influenza A virus but are not recommended by the current guidelines for influenza virus strains. The hemagglutinin inhibitor Abidol does not enter the mainstream medicine market, and has limited clinical application data in China. Neuraminidase inhibitors include oseltamivir, zanami Weila nimesulide and peramivir, oseltamivir being currently the dominant drugs in the market. However, neuraminidase inhibitors have the problem of drug resistance.

The influenza virus genome is small and the synthesis of the desired protein depends on the translation system of the host cell. Thus, the messenger RNA (mRNA) of influenza virus needs to possess both a 5 'CAP (CAP) structure and a 3' -poly (A) tail structure that are recognized by the host cell translation system. Wherein the 5 'cap structure is "preempted" from the 5' end of the host cell pre-mRNA by cleavage of the endoenzyme activity of the PA subunit in the influenza RNA polymerase complex. This manner, known as "CAP-snatching," which captures host mRNA for transcription of viral self mRNA, is necessary for transcription initiation by influenza viruses. RNA polymerase of influenza virus contains Cap-dependent endonuclease (Cap-DEPENDENT ENDONUCLEASE), and inhibition of the activity of the Cap-dependent endonuclease can inhibit proliferation of viruses. The enzyme has now become a promising target for the development of antiviral drugs, many companies turn their eyes towards cap-dependent endonucleases, and different heterocyclic compounds have been used as cap-dependent endonuclease inhibitors. However, even the cap-dependent endonuclease inhibitor baluo Sha Wei currently on the market, there are reports of drug resistance, and in addition, poor physicochemical properties are exhibited: such as low solubility, low bioavailability, etc., there is still a need to develop new generation of cap-dependent endonuclease inhibitors.

After careful study, the cyclopropyl-containing compounds are found to have high-efficiency broad-spectrum anti-influenza virus effects, and are expected to be developed into anti-influenza virus drugs; further structural optimization, the molecules with higher activity are discovered, and the drug has better drug-forming property. The compound plays a role in inhibiting virus replication by inhibiting cap-dependent endonuclease in influenza virus, and targets the earlier stage of the virus replication cycle, so that the compound has better effects of preventing and treating influenza.

Disclosure of Invention

Unless specifically stated otherwise herein, the terms used herein are all the basic meanings commonly understood by those skilled in the art.

The invention provides a compound with an anti-influenza virus effect, a preparation method and application thereof.

The invention provides a compound shown in the following formula (I) or hydrate, solvate, optical isomer, polymorph, isotope derivative and pharmaceutically acceptable salt thereof:

in formula (I), R _a is selected from hydrogen, deuterium, methyl or deuterated methyl;

r _b and R _c are each independently selected from hydrogen, deuterium, C1-C3 alkyl, deuterated C1-C3 alkyl, or R _b、R_c together with the carbon atom to which they are attached form cyclopropyl or deuterated cyclopropyl;

y ₁、Y₂、Y₃ and Y ₄ are each independently CH or N, and at least one of them is N;

X is Se or S;

R is hydrogen, Wherein,

X ₁ is an O atom or an S atom;

n1 is 0,1 or 2;

each R ₁ or R ₂ is independently selected from hydrogen, deuterium, methyl, or deuterated methyl;

R ₃ is selected from the following groups with or without substitution of one or more hydrogen atoms with deuterium: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino;

R ₄ and R ₅ are each independently a hydroxyl group, the following groups with or without deuterium substitution of one or more hydrogen atoms: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino, C3-C8 cycloalkoxy, C3-C8 heterocycloalkoxy, C6-C10 aryloxy, C7-C12 aralkyloxy; or R ₄ and R ₅ together with the phosphorus atom to which they are attached, e.g A 5-7 membered ring of (2); wherein R ₆,R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃ and R ₁₄ are each independently hydrogen or C1-C3 alkyl, or R ₆ and R ₇、R₈ and R ₉、R₁₁ and R ₁₂、R₁₂ and R ₁₃ each together with the attached carbon atom form an aromatic ring, and R ₄ and R ₅ together with the attached phosphorus atom form a 5-7 membered ring and one or more of the hydrogen atoms in the ring substituents may be substituted or unsubstituted with deuterium.

In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (II-1) and/or formula (II-2):

The definition of the substituent in the formula (II-1) and/or the formula (II-2) is defined as in the formula (I).

In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (III-1) and/or formula (III-2):

the definition of the substituent in the formula (III-1) and/or the formula (III-2) is defined as in the formula (I).

In some embodiments, the compounds provided herein, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, are represented by formula (IV-1) and/or formula (IV-2):

the definition of the substituent in the formula (IV-1) and/or the formula (IV-2) is defined as in the formula (I).

In an embodiment 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 ethanol, isopropanol, acetic acid and ethanolamine.

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

In the embodiments of the present application, the alkoxy group of C1-C8 and the alkylthio group of C1-C8 refer to a group in which a saturated aliphatic hydrocarbon group having 1-8 carbon atoms is inserted into an oxygen atom or a sulfur atom at any reasonable position, and include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, 2-ethylethoxy, methylthio, ethylthio, propylthio, isopropylthio, isobutylthio and the like.

In the embodiment of the application, the C1-C8 alkylamino refers to a group in which a saturated aliphatic hydrocarbon group containing 1-8 carbon atoms is inserted into an-NH-or-NH ₂ group at any reasonable position, and the saturated aliphatic hydrocarbon group comprises mono-, di-and naphthene amino groups, including but not limited to methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, di-n-propylamino, diisopropylamino and the like.

In the embodiment of the application, the C1-C3 alkyl refers to alkane containing 1-3 carbon atoms in the molecule, and the method comprises the following steps: methyl, ethyl, propyl, isopropyl, cyclopropyl.

In embodiments of the present application, the C3-C8 cycloalkoxy group refers to a monocyclic or fused polycyclic saturated or unsaturated cyclic hydrocarbyloxy group having 3 to 8 carbon atoms, including, but not limited to, cyclopropyloxy, cyclopentyloxy, bicyclo [3.1.0] hexyloxy, bicyclo [3.2.0] heptyloxy, and the like.

In an embodiment of the present application, the C3-C8 heterocycloalkyl group refers to a group in which a C3-C8 heterocycloalkyl group is attached to oxygen, and the C3-C8 heterocycloalkyl group refers to a saturated or unsaturated cyclic group having 3 to 8 carbon atoms and 1 to 4 hetero atoms in the molecule; the C3-C8 heterocycloalkyl group includes, but is not limited to, aziridinyl, tetrahydrothienyl, tetrahydropyrrolyl, piperidinyl, hexahydropyridazinyl, dihydropyridinyl, cyclopentylsulfanyl, morpholinyl, and the like.

In an embodiment of the present application, the aryloxy group of C6-C10 refers to a group having an aromatic ring composed of 6 to 10 carbon atoms bonded to an oxygen atom, including but not limited to phenoxy, naphthoxy.

In an embodiment of the present application, the C7-C12 aralkyloxy group refers to a group containing an aralkyl group of 7 to 12 carbon atoms attached to an oxygen atom, and includes, but is not limited to, benzyloxy, phenethyloxy and the like.

In embodiments of the application, the substitution of one or more hydrogen atoms with deuterium means that a hydrogen atom at any reasonable position in the group may be substituted with a deuterium atom.

In the embodiment of the application, the deuterated C1-C8 alkyl refers to any hydrogen atom in the C1-C8 alkyl which can be substituted by deuterium, and the deuterated hydrogen atom can be one or a plurality of deuterated hydrogen atoms, and can be on the same carbon atom or different carbon atoms. The deuterated C1-C8 alkyl group comprises, but is not limited to, deuterated methyl, deuterated ethyl, deuterated propyl, deuterated isopropyl, deuterated butyl, deuterated isobutyl, deuterated pentyl, deuterated hexyl and deuterated heptyl.

In the embodiment of the application, the substitution of one or more hydrogen atoms by deuterium for a C1-C8 alkoxy group means that any hydrogen atom in the C1-C8 alkoxy group may be substituted by deuterium, and the deuterated hydrogen atom may be one or more, or may be on the same carbon atom or may be on different carbon atoms. The alkoxy groups in which one or more hydrogen atoms are substituted with deuterium for C1-C8 include, but are not limited to, deuterated methoxy, deuterated ethoxy, deuterated propoxy, deuterated isopropoxy, deuterated butoxy, deuterated isobutoxy, deuterated pentyloxy, deuterated hexyloxy, deuterated heptyloxy.

In some embodiments, R _a is hydrogen; in some embodiments, R _a is deuterium; in some embodiments, R _a is methyl; in some embodiments, R _a is deuterated methyl.

In some embodiments, R _b and R _c are both hydrogen; in some embodiments, R _b and R _c are both deuterium; in some embodiments, R _b is hydrogen and R _c is deuterium;

In some embodiments, R _b and R _c are each independently C1-C3 alkyl; in some specific embodiments, R _b and R _c are both methyl;

In some embodiments, R _b and R _c are each independently deuterated C1-C3 alkyl; in some specific embodiments, R _b and R _c are both deuterated methyl groups;

In some embodiments, R _b is hydrogen and R _c is C1-C3 alkyl; in some specific embodiments, R _b is hydrogen and R _c is methyl;

in some embodiments, R _b is hydrogen and R _c is deuterated C1-C3 alkyl; in some specific embodiments, R _b is hydrogen and R _c is deuterated methyl.

In some embodiments, R _b is deuterium, R _c is C1-C3 alkyl; in some specific embodiments, R _b is deuterium and R _c is methyl;

In some embodiments, R _b is deuterium, R _c is deuterated C1-C3 alkyl; in some specific embodiments, R _b is deuterium and R _c is deuterated methyl;

in some embodiments, R _b is C1-C3 alkyl, and R _c is deuterated C1-C3 alkyl;

In some embodiments, R _b and R _c together with the attached carbon form cyclopropyl; in some embodiments, R _b and R _c together with the attached carbon form a deuterated cyclopropyl group.

In an embodiment of the invention, Y ₁、Y₂、Y₃ and Y ₄ are each independently CH or N, and at least one of them is N;

In some specific embodiments, Y ₁ is N, Y ₂、Y₃ and Y ₄ are each independently CH or N; in some specific embodiments, Y ₁ is N, Y ₂、Y₃ and Y ₄ are each independently CH;

In some specific embodiments, Y ₂ is N, Y ₁、Y₃ and Y ₄ are each independently CH or N; in some specific embodiments, Y ₃ is N, Y ₁、Y₂ and Y ₄ are each independently CH or N; in some specific embodiments, Y ₄ is N, and Y ₁、Y₂ and Y ₃ are each independently CH or N.

In some embodiments, X is Se; in some embodiments, X is S.

In some embodiments, R is hydrogen; in some embodiments, R isIn some embodiments, R is/>

In some embodiments, n1 is 0; in some embodiments, n1 is 1; in some embodiments, n1 is 2.

In some embodiments, X ₁ is an O atom; in some embodiments, X ₁ is an S atom.

In an embodiment of the invention, each R ₁ or R ₂ is independently hydrogen, deuterium, methyl or deuterated methyl; in some specific embodiments, R ₁ or R ₂ are both methyl; in some specific embodiments, R ₁ is methyl and R ₂ is hydrogen; in some specific embodiments, R ₁ or R ₂ are both hydrogen; in some specific embodiments, R ₁ or R ₂ are each deuterated methyl groups, including 1 or 2 or 3 hydrogens in the methyl groups substituted with deuterium; in some specific embodiments, R ₁ is methyl and R ₂ is deuterium; in some specific embodiments, R ₁ or R ₂ are both deuterium; in some specific embodiments, R ₁ is hydrogen and R ₂ is deuterated methyl.

In some embodiments, R ₃ is selected from the following groups: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkylamino; preferably, R ₃ is selected from the following groups: C1-C8 alkyl, C1-C8 alkoxy; more preferably, R ₃ is C1-C8 alkoxy;

In some embodiments, R ₃ is selected from the following groups substituted with deuterium for one or more hydrogen atoms: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkylamino; preferably, R ₃ is selected from the following groups substituted with deuterium for one or more hydrogen atoms: C1-C8 alkyl, C1-C8 alkoxy; more preferably, R ₃ is an alkoxy group in which one or more hydrogen atoms are replaced by deuterium in C1-C8.

In some specific embodiments, n1 is 1, X ₁ is an O atom, R ₁ and R ₂ are both hydrogen, R ₃ is C1-C8 alkoxy or C1-C8 alkyl; in some specific embodiments, n1 is 1, X ₁ is an O atom, R ₁ and R ₂ are deuterium, R ₃ is C1-C8 alkoxy or C1-C8 alkyl;

In some specific embodiments, n1 is 1, X ₁ is an O atom, R ₁ and R ₂ are both hydrogen, R ₃ is deuterated C1-C8 alkoxy or deuterated C1-C8 alkyl; in some specific embodiments, n1 is 1, X ₁ is an O atom, R ₁ and R ₂ are both deuterium, R ₃ is deuterated C1-C8 alkoxy or deuterated C1-C8 alkyl;

In some specific embodiments, n1 is 0, X ₁ is an O atom, R ₃ is a C1-C8 alkoxy group or a C1-C8 alkyl group;

In some specific embodiments, n1 is 1, X ₁ is an O atom, R ₁ is hydrogen, R ₂ is deuterated methyl, R ₃ is deuterated C1-C8 alkoxy or deuterated C1-C8 alkyl.

In some embodiments, R ₄ and R ₅ are both hydroxy; in some embodiments, R ₄ and R ₅ are the following groups: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino, C3-C8 cycloalkoxy, C3-C8 heterocycloalkoxy, C6-C10 aryloxy, C7-C12 aralkyloxy; in some embodiments, R ₄ and R ₅ are the following groups with one or more hydrogen atoms replaced with deuterium: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino, C3-C8 cycloalkoxy, C3-C8 heterocycloalkoxy, C6-C10 aryloxy, C7-C12 aralkyloxy;

In some embodiments, R ₄ and R ₅ together with the phosphorus atom to which they are attached form, e.g A 5-7 membered ring of (2); wherein R ₆、R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃ and R ₁₄ are each independently hydrogen or C1-C3 alkyl, or R ₆ and R ₇、R₈ and R ₉、R₁₁ and R ₁₂、R₁₂ and R ₁₃ together with the carbon atom to which they are attached form an aromatic ring;

In some embodiments, R ₄ and R ₅ together with the phosphorus atom to which they are attached form, e.g A 5-7 membered ring of (2); wherein one or more of the hydrogen atoms in the 5-7 membered ring and the ring substituents, which R ₄ and R ₅ together with the phosphorus atom to which they are attached, may be substituted with deuterium.

In some specific embodiments, n1 is 1, x ₁ is an O atom, R ₁ and R ₂ are both hydrogen, and R ₄ and R ₅ are both hydroxy; in some specific embodiments, n1 is 1, x ₁ is an O atom, R ₁ and R ₂ are both deuterium, and R ₄ and R ₅ are both hydroxyl; in some specific embodiments, n1 is 1, x ₁ is an O atom, R ₁ is hydrogen, R ₂ are both deuterated methyl groups, and R ₄ and R ₅ are both hydroxyl groups; in some specific embodiments, n1 is 0, x ₁ is an O atom, and R ₄ and R ₅ are both hydroxyl.

In some embodiments, when R is hydrogen, the pharmaceutically acceptable salts of the compounds may be alkali metal salts, alkaline earth metal salts, amine salts, and amino acid salts; preferably, the pharmaceutically acceptable salts include: sodium salt, potassium salt, magnesium salt, zinc salt, amine salt, basic amino acid salt, and the like.

In some embodiments, where R ₄ and R ₅ are both hydroxy, the pharmaceutically acceptable salts of the compounds may be alkali metal salts, alkaline earth metal salts, amine salts, and amino acid salts; preferably, the pharmaceutically acceptable salts include: sodium salt, potassium salt, magnesium salt, zinc salt, amine salt, basic amino acid salt, and the like.

The salt related in the invention is obtained by adopting a conventional salification method.

In an embodiment of the invention, the pharmaceutically acceptable salt is structurally verified by nuclear magnetism, mass spectrometry, atomic absorption spectrometry, elemental analysis, melting point detection and other means.

In an embodiment of the invention, the compounds of formula (I) comprise two chiral centers, and the compounds of the invention or intermediates thereof may be isolated by chiral separation to give compounds of single configuration.

In some specific embodiments, the compounds of the present invention are optical isomers of a single configuration as shown in formula (I).

In an embodiment of the invention, the optical isomers of a single configuration are subjected to absolute configuration determination by electron circular dichroism.

In an embodiment of the invention, racemates and optical isomers of a single configuration are subjected to optical rotation testing according to the optical rotation assay of "chinese pharmacopoeia" 2020 edition-four-0621.

The compounds provided by the present invention include, but are not limited to, the following:

Or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof.

The materials N158-1 and N158-3 required by the synthesis of the compounds are completely synthesized according to the method in the document W0202214834A 1; materials N158-2 and N158-4 are synthesized by referring to the synthesis method of N158-1 and N158-3,

The comparative compounds synthesized by the synthetic methods of the references, patents and examples of the present invention were used in the relevant biological tests when the detected purity was > 98%.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising the above compound or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include one or a combination of several fillers, binders, diluents, lubricants, preservatives, taste masking agents or co-solvents.

Further, the dosage form of the pharmaceutical composition is a tablet, a capsule, a powder, a granule, a pill, a suspension, a syrup, an injection or an inhalation.

In a third aspect of the invention, the present invention provides the use of the above compounds, including hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for combating influenza virus.

The invention provides application of the compound, including hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt or pharmaceutical composition thereof in preparing anti-influenza virus drugs.

The present invention provides a method for preventing or treating influenza virus infection comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described above, including hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof.

The compound has stronger antiviral activity in vivo or in vitro, and has better safety. The compound is effective on various influenza virus strains including avian influenza virus strains, and is also effective on oseltamivir-resistant influenza viruses and other drug-resistant strains;

The compound has stronger capability of entering cells, is known to be a cap-dependent endonuclease inhibitor, needs to enter cells to play a role in the link of virus replication, and is more beneficial to inhibiting the virus replication;

the compound has good lung tissue distribution in animal experiments, and is more beneficial to exerting the anti-influenza virus effect;

The compounds of the present invention are less vascular-irritating when administered by injection, and studies have also shown that they are acceptably irritating to intravenous injection and are expected to be useful for intravenous administration in patients with severe influenza.

In an in vivo pharmacodynamic test of a mouse model of the compound, the lung virus titer is lower, and the lesions of lung tissues are lighter; the method has outstanding advantages in the aspects of weight protection and survival protection, and shows the characteristic of rapid recovery after virus infection.

Drawings

FIG. 1 shows the rate of change of body weight of mice model of anti-influenza drug effect.

Detailed Description

The following examples will allow one skilled in the art to more fully understand the invention, but are not intended to limit the invention in any way, the structure of all compounds being determined by MS or ¹ H-NMR, all optical isomers of the single configuration involved being configuration determined by optical rotation testing or electron circular dichroism.

In this example, unless otherwise specified, the solvents and reagents used were all commercially available. The starting materials were all commercially available.

Embodiment one: 9a Synthesis

Synthesis of Compound 3a

5G of Compound 1a, 2.95g of Compound 2a and 7ml of triethylamine were added to a mixed solvent of 40ml of DMF (N, N-dimethylformamide) and 40ml of toluene, and the system was heated to 130℃for reaction for 8 hours. Adding water and stirring to precipitate solid. The system was filtered to give 7.03g of compound 3a as a white solid. The reaction mixture is directly used for the next reaction without separation.

Synthesis of Compound 4a

5.84G of the compound 2-bromo-1, 1-dimethoxyethane and 5g of the compound 3a were added to 40ml of DCM, and 3.9g of potassium tert-butoxide were slowly added to the system at room temperature. After the addition, the system was heated to 40℃and reacted for 5 hours. Cooling the system to room temperature, adding water, stirring for 15min, concentrating to dryness, purifying with silica gel column to obtain 5.2g of compound 4a, and obtaining the yield 74％;¹H NMR(CDCl₃,400MHz)：δ7.83-7.88(m,2H),7.68-7.70(m,2H),4.46(t,1H),3.81(s,2H),3.55(d,2H),3.31(s,6H),0.47-0.95(m,4H).

Synthesis of Compound 5a

5G of compound 4a are added to 15ml of ethanol and 15ml of water and the system is heated to 60 ℃. 1.5g of aqueous hydrazine hydrate (80%) was added and the reaction was continued at 60℃for 5 hours. The system was concentrated to dryness, cooled to room temperature, and 40ml of DCM and 40ml of 1N aqueous NaOH were added. The organic phase was separated and the aqueous phase was extracted 2 more times with DCM and the organic phases combined. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to give 2.75g of crude compound 5a which was used directly in the next step without further isolation.

Synthesis of Compound 7a

5G of Compound 6a was added to 10ml of DMA (N, N-dimethylacetamide), 1.62g of sodium hydride (60%) and 3.2g of methyl iodide were added. The system was stirred at 25℃for 12h, 100ml of water were added and stirred for 0.5h, and extracted with EA. The organic phases were combined and washed successively with 0.5N diluted hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to give 4.65g of an oil. To this oil was added 30ml of DMA, 3.54g of Boc hydrazine and 13.47g of pyridine p-toluenesulfonate. The system was heated to 60℃and reacted for 18 hours. After the completion of the reaction, the system was extracted with water and EA, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness. Purification by silica gel column gave 4.68g of compound 7a in 61.65% yield; the product was a yellow oil which turned to a yellow solid upon standing, ESI-MS (+): m/z= 375.2.

Synthesis of Compound 8a

4.5G of Compound 7a are added to 30ml of ethanol, and 24ml of 1N NaOH solution are added. The system was reacted at 60℃for 15 hours and the pH was adjusted with dilute hydrochloric acid. The system was extracted with DCM (dichloromethane), the organic phases combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to dryness to give an off-white solid. The resulting off-white solid was added to DCM and 2.53g of Compound 5a, 4ml of triethylamine and 6.85g of HATU (2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) were added at 25 ℃. The system was stirred at 25 ℃ for 11h, diluted with water and extracted with DCM. The organic phases were combined and washed with saturated brine. Drying by anhydrous sodium sulfate, and passing through a silica gel column to obtain 4.1g of compound 8a, wherein the yield is 66%; the product was a white solid, ESI-MS (+): m/z=518.3.

Synthesis of Compound 9a

To 3g of Compound 8a, 20ml of acetonitrile and 4ml of water were added, the system was heated to 60℃and 1.7g of methanesulfonic acid was added dropwise, and the reaction was continued at that temperature for 5 hours. After the reaction, the system is cooled to room temperature, and sodium bicarbonate aqueous solution is added to adjust the pH to be slightly alkaline. The system was concentrated, extracted with DCM and water, the organic phase separated, dried over anhydrous sodium sulfate and concentrated to dryness, and purified by silica gel column to give compound 9a as a white solid 1.21g in 59% yield, ESI-MS (+): m/z= 354.2.

Embodiment two: synthesis of N158-3 and N158-1

Synthesis of compound y 0:

10g of 3-iodopyridine compound and 100ml of dimethyl sulfoxide are sequentially added into a reaction bottle, 7.90g of selenium powder, 194.02mg of copper oxide and 5.47g of potassium hydroxide are respectively added, and the reaction solution is stirred for 2 hours at 120 ℃. The reaction mixture was cooled to 25 ℃, saturated aqueous ammonium chloride (100 mL), water (50 mL) and dichloromethane (100 mL) were added to the reaction mixture, stirred for 5 minutes, filtered, the filtrate was separated, and the aqueous phase was extracted with dichloromethane (50 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product obtained was purified by a silica gel column to give 2.5g of compound y0 in a yield of 16.32% and a purity of 67% as yellow oil.

Synthesis of compound y 2:

To the reaction flask, 20g of the compound y1 and N, N-dimethylformamide (200 mL) were added, 23.55g of 1-hydroxybenzotriazole, 76.18g of N, N-diisopropylethylamine and 33.33g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, after the addition was completed, the reaction solution was stirred at 25℃for 30 minutes, and then 11.90g of N, O-dimethylhydroxylamine hydrochloride was added to the reaction solution, and the reaction solution was stirred at 25℃for 12 hours. Water (100 mL) was added and the aqueous phase was extracted with 100mL (50 mL. Times.2) of methylene chloride. The combined organic phases were washed with dilute hydrochloric acid (0.2M, 50 mL), saturated aqueous sodium bicarbonate (50 mL) and saturated brine (50 mL), respectively, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give 25g of Compound y2 as a yellow oil ,¹H NMR(400MHz,CDCl₃)δ＝6.99-6.92(m,2H),3.41(brs,3H),3.25(brs,3H),2.20(d,J＝2.0Hz,3H).

Synthesis of compound y 3:

10g of compound y2 was dissolved in 200mL of tetrahydrofuran, and methylmagnesium bromide (3M, 18.59 mL) was added dropwise at 0℃and the reaction mixture was stirred for 2 hours after the completion of the addition at 25 ℃. The reaction solution was quenched with 1M hydrochloric acid to adjust pH to 7, extracted with ethyl acetate, and the organic phases were combined, washed with dilute hydrochloric acid, saturated sodium bicarbonate solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give 14.6g of compound y3 as yellow oil.

Synthesis of compound y 4:

7.3g of compound y3 was dissolved in 73mL of pyridine, 9.52g of selenium dioxide was added, and the reaction solution was stirred at 110℃for 12 hours. The reaction solution was cooled to room temperature, filtered, and concentrated to dryness under reduced pressure. Water was added to the crude product, pH was adjusted to 4 with 1M hydrochloric acid, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give 15.2g of Compound y4 as a yellow oil.

Synthesis of compound y 5:

7.6g of compound y4 was dissolved in methylene chloride (76 mL) and methanol (76 mL), the temperature was controlled at 0-20℃and trimethylsilyl diazomethane (2M, 22.78 mL) was added dropwise, and the reaction mixture was stirred at 20℃for 2 hours, followed by addition of acetic acid (4 mL) and stirring for 5 minutes. The reaction solution was concentrated to dryness under reduced pressure, water was added, the extracts were extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product obtained was purified by means of a silica gel column to give 12.8g of compound y5 as a yellow solid.

Synthesis of compound y 6:

5g of compound y5 was dissolved in 1, 2-dichloroethane (50 mL), 8.31g of N-bromosuccinimide and 383.37mg of azobisisobutyronitrile were added, and the reaction solution was stirred at 80℃for 12 hours. The reaction solution was cooled to room temperature, washed with a saturated sodium sulfite solution, water and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product obtained is purified by a silica gel column. Repeating 5 batches gave 33.5g of compound y6 in 97.9% yield as a yellow solid .¹H NMR(400MHz,CDCl₃)δ＝7.55(ddd,J＝1.9,4.8,8.8Hz,1H),7.25-7.13(m,1H),4.85(d,J＝2.0Hz,2H),3.91(s,3H).

Synthesis of compound y 7:

10.24g of sodium dihydrogen phosphate was dissolved in 100mL of water, then 50mL of acetonitrile was added, 4.80g of compound y0 (purity: 67%) was added, 1.67g of zinc powder was added in portions, and the reaction solution was stirred at 25℃for 30 minutes. 5g of compound y6 are added and the reaction mixture is stirred for a further 3 hours at 25 ℃. The reaction mixture was filtered, and the filtrate was extracted with 30mL of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product is purified by a silica gel column, and 5 batches are combined and treated to obtain 17g of yellow solid of the compound y7 ,¹H NMR(400MHz,CDCl₃)δ＝8.54(brs,1H),8.46(brd,J＝3.9Hz,1H),7.75(brd,J＝7.7Hz,1H),7.55-7.47(m,1H),7.16-7.02(m,2H),4.39(d,J＝1.7Hz,2H),3.90(s,3H).

Synthesis of compound y 8:

16g of compound y7 were dissolved in 160mL of methylene chloride, 27.35g of dess-martin periodate was added, and the reaction mixture was stirred at 20℃for 12 hours. 200mL of saturated sodium sulfite solution was added and stirred for 5 minutes. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product obtained was purified by a silica gel column to give 14.3g of compound y8 in 89.8% yield as a yellow solid.

Synthesis of compound y 9:

14g of compound y8 was dissolved in 140mL of tetrahydrofuran, and an aqueous solution of sodium hydroxide (4M, 18.91 mL) was added thereto, and the reaction mixture was stirred at 25℃for 1 hour. The aqueous phase was adjusted to pH 6 with 1N hydrochloric acid, the solid filtered and the filter cake was dried under reduced pressure to give 13.1g of compound y9 in 97.2% yield as a white solid.

Synthesis of Compound N158-3:

4g of compound y9 was dissolved in 40mL of dimethyl sulfoxide, 5.13g of ammonium persulfate, 839.39mg of silver nitrate and 1.10g of concentrated sulfuric acid were sequentially added, and the reaction solution was stirred at 50℃for 3 hours. To the reaction solution, a saturated aqueous sodium hydrogencarbonate solution and methylene chloride were added, followed by stirring for 5 minutes, filtration, separation of the filtrate and extraction of the aqueous phase with methylene chloride. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purifying the crude product by silica gel column to obtain white solid, combining 3 batches to obtain 1.5g of compound N158-3, and obtaining the yield 14.0％,¹H NMR(400MHz,CDCl₃)δ＝8.61(dd,J＝1.1,4.4Hz,1H),7.80(dd,J＝1.3,8.0Hz,1H),7.37(ddd,J＝1.6,4.9,8.6Hz,1H),7.22-7.19(m,1H),7.12-7.01(m,1H),4.11(s,2H).

Synthesis of Compound N158-1:

1.3g of Compound N158-1 was dissolved in 13mL of isopropanol, 316.67mg of sodium borohydride was added, and the reaction was stirred at 20℃for 1 hour. The pH was adjusted to 7 by addition of 1N hydrochloric acid, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product obtained was purified by column chromatography on silica gel to give 1.1g of compound N158-1 in 84.0% yield as white solid ,ESI-MS(+):m/z＝313.9.¹H NMR(400MHz,DMSO-d6)δ＝8.31(brd,J＝4.2Hz,1H),7.71(brd,J＝7.7Hz,1H),7.32-7.24(m,2H),7.18(brdd,J＝4.7,7.6Hz,1H),6.29(brd,J＝4.9Hz,1H),6.17(brd,J＝4.5Hz,1H),4.81(brd,J＝12.6Hz,1H),4.46(brd,J＝12.0Hz,1H). example three: synthesis of N158-4 and N158-2

Synthesis of compound N3:

1g of compound N2 and 10ml of tetrahydrofuran are added into a reaction bottle, the system is cooled to 0 ℃, 0.36g of sodium hydride (60%) and 2.64g of compound y6 are sequentially added into the system, and the system is reacted for 30min at 60 ℃. The system was extracted with dilute hydrochloric acid and ethyl acetate, the organic phase was washed with aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, and separated by column chromatography to give compound N3 at about 1.51g in 52% yield, ESI-MS (+): m/z=324.3.

Synthesis of compound N4:

To the reaction flask, 1.5g of Compound N3 and 15ml of tetrahydrofuran were added, and the system was stirred at room temperature, followed by addition of 2.3ml of aqueous sodium hydroxide solution (4M) and stirring at 25℃for 3 hours. The system was concentrated under reduced pressure to remove most of the tetrahydrofuran, pH was adjusted to 6 by addition of 1N hydrochloric acid, the solid was separated out, and filtered off and dried to give 1.32g of Compound N4, ESI-MS (-): m/z=308.3.

Synthesis of Compound N158-4:

1.15g of Compound N4 was dissolved in 10ml of DMSO (dimethyl sulfoxide), 1.7g of ammonium persulfate, 63mg of silver nitrate and 370mg of concentrated sulfuric acid were sequentially added, and the reaction solution was heated to 50℃to react for 4 hours. To the reaction solution were added 20ml of saturated aqueous sodium bicarbonate solution, 10ml of water and 30ml of DCM, respectively, the system was stirred for 5 minutes, filtered, the filtrate was separated, the aqueous phase was extracted again with DCM, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated to dryness, and purified by column chromatography to give 440mg of compound N158-4 in a yield of 45% and ESI-MS (+): m/z=264.2.

Synthesis of Compound N158-2:

0.4g of Compound N158-4 was dissolved in 5ml of isopropanol, 0.12g of sodium borohydride was added, and the reaction solution was stirred at 20℃for 1 hour. The pH was adjusted to 7 by addition of 1N hydrochloric acid, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give 0.31g of Compound N158-2 in 77% yield, ESI-MS (+): m/z= 266.3.

Embodiment four: synthesis of DSC1581 and DSC1583

Synthesis of Compound DSC 1581-1:

To a single-necked flask, 0.5g of Compound 9a, 0.44g of Compound N158-1, 1.13g of Compound T3P (propylphosphoric anhydride, 50% EA solution), 0.28g of methanesulfonic acid and 10ml of EA were added, and the mixture was heated to reflux, reacted overnight, sampled and inspected, and treated after the reaction was completed; saturated aqueous sodium bicarbonate was added until no bubbles evolved, the solution was separated, the aqueous phase extracted with EA, the organic phases combined, dried by spinning and purified by column on silica gel. Then preparing and separating by a chiral column to obtain 0.37g of compound DSC1581-1; ESI-MS (+): m/z= 649.2.

Synthesis of compound DSC 1581:

0.28g of compound DSC1581-1, 0.12g of compound LiCl (lithium chloride) and 5ml of DMA (N, N-dimethylacetamide) are added into a single-port bottle, the temperature is raised to 100 ℃, the reaction solution is yellow and turbid, the reaction is carried out for 2 hours, sampling detection is carried out, and the treatment is carried out after the reaction is completed; saturated aqueous sodium bicarbonate was added, the solid precipitated, suction filtered, the filtrate extracted with EA, the organic phase was spin-dried and purified by a silica gel column to give 0.19g of compound DSC1581 in 79% yield, ESI-MS (+): m/z= 558.6.

Synthesis of compound DSC 1583:

To a single vial was added 0.15g of compound DSC1581, 74mg of K ₂CO₃, 5mg of KI and 40mg of chloromethyl dimethyl carbonate. 5mL of DMA is added into the system, the temperature is raised to 60 ℃, the reaction is carried out overnight, sampling detection is carried out, and the treatment is carried out after the reaction is completed. Cooling the reaction system to room temperature, adding 2N HCl, adding water, separating out solids, adding EA and water for extraction, separating out an organic phase, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain 0.1g of compound DSC1583, wherein the yield is: 58%. ESI-MS (+): m/z=646.6.

Fifth embodiment: synthesis of DSC1581-1

Under nitrogen, the flask was charged with 20mg of N158-1, 31mg of thionyl chloride and 2ml of DCM. The system is heated to 40 ℃, the reaction is carried out for 2 hours, the TLC detection reaction is complete, and the crude t1 product is obtained after concentration to dryness and is directly used for the next reaction.

Under the protection of nitrogen, 20mg of compound 9a, crude product of the previous step t1, 36mg of cesium carbonate and 2ml of acetonitrile are added into a reaction bottle, and the mixture is reacted overnight at 60 ℃. Water and EA were added to extract, and the organic phase was separated, dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column. Then 8.06mg of compound DSC1581-1 is prepared and separated by chiral column, yield: 22%. ESI-MS (+): m/z= 649.2.

Example six: synthesis of DSC1582 and DSC1584

Synthesis of compound DSC 1582:

The compound DSC1582 is synthesized according to the above steps by the synthesis method of the reference compound DSC1581, ESI-MS (+);

Synthesis of compound DSC 1584:

To a single vial was added 50mg of compound DSC1582, 63.8mg of Cs ₂CO₃ (cesium carbonate), 1.6mg of KI (potassium iodide), 22mg of diethyl chloromethyl phosphate and 2mL of DMA. The temperature of the system is raised to 50 ℃, the reaction is carried out for 2.5h, and the reaction is completed. The reaction system was cooled to room temperature, extracted with EA and water, the organic phase was separated, dried over anhydrous sodium sulfate, and spin-dried, and separated by column chromatography to give 39.1mg of a solid of compound DSC1584, yield: 59%. ESI-MS (+): m/z= 677.6.

Embodiment seven: synthesis of Compounds DSC1821 and DSC1825

Synthesis of Compound DSC1821-2

Under the protection of nitrogen, 1.3g of compound N158-3 is dissolved in 20ml of THF, 175mg of lithium aluminum hydride-D4 is slowly added at 0 ℃, and the system is heated to 25 ℃ to react for 8 hours. The system is cooled to 0 ℃, and water is added for quenching reaction. The system was extracted with 2N hydrochloric acid and EA. The organic phase was concentrated to dryness. Column chromatography gave compound DSC1821-2 in a total of 0.87g, 66% yield, ESI-MS (+): m/z=315.0.

Synthesis of Compound DSC1821-1

Referring to the synthesis method of the compound DSC1581-1, the compound DSC1821-1 is synthesized from the compound 9a and the compound DSC1821-2, and the total amount of the compound DSC1821-1 is 0.2g, and the yield is 36%; ESI-MS (+): m/z=650.1.

Synthesis of Compound DSC1821

Referring to a synthesis method of the compound DSC1581, 75mg of compound DSC1821 is obtained through synthesis, and the yield is 73%; ESI-MS (+): m/z= 560.1.

Synthesis of Compound DSC1825

With reference to the synthesis method of the compound DSC1583, 0.42mg of compound DSC1825 is synthesized, and the yield is 67%; ESI-MS (+): m/z= 648.1.

Example eight: synthesis of Compounds DSC1823 and DSC1828

Synthesis of Compound DSC 1823-2:

under the protection of nitrogen, 0.7g of compound N158-3 is dissolved in 15ml of THF, 2.8ml of methyl lithium reagent (1.6M diethyl ether solution) is slowly added dropwise at the temperature of minus 20 ℃, and the system is naturally heated to the temperature of 25 ℃ for reaction for 8 hours. The system is cooled to 0 ℃, and water is added for quenching reaction. Concentrating to dryness, and extracting with EA and water. The organic phase was separated and concentrated to dryness. Column chromatography gave compound DSDC, 1823-2 in a total amount of 0.52g, 71% yield, ESI-MS (+): m/z=328.0.

Synthesis of Compound DSC1823-1

Referring to the synthesis method of the compound DSC1581-1, the compound DSC1823-1 is synthesized from the compound 9a and the compound DSC1823-2, and the total amount of the compound DSC1823-1 is 0.31g, and the yield is 25%; ESI-MS (+): m/z= 663.1.

Synthesis of Compound DSC1823

With reference to the synthesis method of the compound DSC1581, 0.17g of compound DSC1823 is synthesized, and the yield is 69%; ESI-MS (+): m/z= 573.1.

Synthesis of Compound DSC1828

Referring to a synthesis method of the compound DSC1583, 33mg of compound DSC1828 is obtained through synthesis, and the yield is 57%; ESI-MS (+): m/z=661.1.

Example nine: synthesis of DSC1585

Synthesis of Compound DSC 1585-1:

to the reaction flask were added 0.7g of compound DSC1581, 0.37g of t-butyl chloromethyl phosphate and 1ml of triethylamine, 10ml of DMF (N, N-dimethylformamide) was added, and the system was reacted at 45℃for 8 hours. The system was water-added to crystallize and filter to give about 0.45g of crude compound DSC1585-1 in 46% yield, ESI-MS (+): m/z= 781.2. The product was used in the next step without further purification.

Synthesis of compound DSC 1585:

Under the protection of nitrogen, 0.36g of compound DSC1585-1 and 5ml of anhydrous dichloromethane are added into a reaction bottle, and 1ml of trifluoroacetic acid is added dropwise under stirring at room temperature of the system, and stirring is carried out at 25 ℃ for 3 hours. The system was concentrated to dryness and isopropanol and water were added to crystallize to give 0.23g of compound DSC1585 in 74% yield, ESI-MS (+): m/z=669.1, ESI-MS (-): m/z= 667.2.

Example ten: synthesis of compound DSC 1826:

Synthesis of Compound DSC1826-1

0.9G of deuterated methanol, 4.5ml of triethylamine and 30ml of dichloromethane are added into a reaction bottle, and after the temperature of the system is reduced to 0 ℃, 3g of chloromethyl chloroformate is slowly dripped. After the dripping, the system naturally rises to room temperature to react for 3 hours. The reaction mixture was quenched with water, extracted with dichloromethane, and the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to dryness to give 2.3g of a liquid of the compound DSC1826-1 in 79% yield.

Synthesis of Compound DSC1826

With reference to the synthesis of compound DSC1583, 47mg of compound DSC1826 was synthesized using compounds DSC1581 and DSC1826-1 as starting materials in 52% yield with ESI-MS (+): m/z=650.1.

The following examples were synthesized in the same manner as in the above examples, using commercially available compounds or intermediate compounds appropriately synthesized from the commercially available compounds:

example eleven: synthesis of Compounds DSC15815 and DSC18212

Synthesis of Compound DSC15815

0.9G of compound DSC1585 is added into 10ml of absolute ethyl alcohol, the system is heated to 30 ℃, 108mg of sodium hydroxide is added, stirring is carried out for 2 hours, cooling is carried out to-10 ℃, filtering is carried out, and the obtained product is dried under reduced pressure, thus obtaining DSC15815, the yield is 93%, and the content is 99.5%. The obtained product is confirmed by element analysis: actual measurement value: na,6.55%; se,11.13%; theoretical value: na,6.46%; se,11.10%.

Synthesis of Compound DSC18212

0.6G of compound DSC15815 is dissolved in 25ml of deionized water, 185mg of deionized water (5 ml) solution of zinc acetate dihydrate is added, the mixture is stirred for 2 hours at room temperature, the mixture is filtered, and the obtained product is dried under reduced pressure to obtain DSC18212, the yield is 88%, and the purity is 99.1%. The obtained product is confirmed by element analysis: actual measurement value: se,10.98%; zn,9.07%; theoretical value: se,10.81%; zn,8.95%.

Embodiment twelve: synthesis of Compound DSC18213

0.7G of compound DSC1581 is added into 10ml of absolute ethyl alcohol, the system is heated to 50 ℃, 50mg of sodium hydroxide is added, stirring is carried out for 2 hours, cooling is carried out to 0 ℃, filtering is carried out, and the obtained product is dried under reduced pressure, thus obtaining DSC18213, the yield is 93%, and the content is 99.2%. The obtained product is confirmed by element analysis: actual measurement value: na,3.81%; se,13.44%; theoretical value: na,3.97%; se,13.63%.

Referring to the synthesis of the metal salts in the present invention, the following example compounds were obtained:

Comparative example one: synthesis of M41 and M42

Compounds M41 and M42 were synthesized by the method described in reference WO2019141179A 1;

Compounds M46 and M47 were synthesized by the method described in reference patent WO2022148434A 1;

reference compounds DSC1581 and DSC1583 were synthesized as comparative compounds M39, M40.

Comparative example two: synthesis of M43, M44 and M45

Comparative example compounds M43, M44 and M45 were synthesized by the synthesis method of reference compound DSC 1585.

Embodiment thirteen: anti-influenza virus cell Activity assay

MDCK cells were inoculated into 96-well plates and incubated in a 5% co ₂, 37 ℃ incubator. During the cell exponential growth phase, cell culture maintaining liquid containing different dilutions of sample and positive reference medicine is added, and 3 compound wells are set in each concentration, and normal cell reference wells are set simultaneously. After the sample was added, the sample was cultured for 72 hours, and the cytotoxicity test of the sample was performed by the CPE method. MDCK cells were also inoculated into 96-well plates and incubated in a 5% co ₂, 37 ℃ incubator. After 24 hours of culture, influenza virus (A/han-defenses/359/95 (H3N 2)) is infected, virus is adsorbed for 2 hours, virus liquid is discarded, cell culture maintaining liquid containing samples with different dilutions and positive control drugs is added, 3 compound holes are arranged at each concentration, meanwhile, cell control holes and virus control holes are arranged, and the mixture is placed in a culture box with 5% CO ₂ and at 37 ℃ for culture. Antiviral test of the tested sample is carried out by CPE method, and when the pathological change degree (CPE) of the virus control group reaches 4+, the cytopathic change degree (CPE) of each group is observed. The half toxic concentration of the sample on cells (TC ₅₀) and the drug effective concentration (EC ₅₀) to inhibit 50% of the cytopathic effect were calculated using the Reed-Muench method, respectively, as shown in table 1:

table 1: cytotoxicity of the compound and inhibitory Activity against influenza Virus

Experiments show that the compound has better anti-influenza virus activity. The compounds DSC1581, DSC1582 and DSC1821 to DSC1824 of the present invention have significantly better antiviral activity and higher safety than the comparative compounds M41, M46 and baluo Sha Wei. The prodrug compounds DSC1583 to DSC15811, DSC1825 to DSC18211 of the present invention also have better antiviral activity and higher safety than the comparative example compounds M42, M47 and baluo Sha Weizhi.

Fourteen examples: vascular irritation test for intravenous administration

21 SPF-grade Japanese white rabbits with weights of 2.0-2.2 kg are male. Rabbits were purchased from Liaoning long life biosciences Inc. The common grade environment has the environment temperature of 20-22 ℃ and the relative humidity of 40-70 percent. During the test, the rabbits can freely ingest conventional rabbit breeding feed and drink water. The rabbit breeding feed is purchased from Liaoning long-life biotechnology limited company, and comprises the main components of soybean meal, corn, flour, wheat bran, alfalfa, vitamins and minerals. The nutrient components are as follows: the crude protein is more than or equal to 14%, the crude fat is more than or equal to 3% and the crude fiber is 10% -15%. Rabbits were kept adaptively for 4 days prior to the test.

The experiment adopts the self-control of the left ear and the right ear of the same rabbit, the intravenous drip of the tested medicine at the left ear edge and the intravenous drip of the normal saline with equal volume dose at the right ear edge. The administration concentration is 0.2mg/mL, the administration dosage is 5mL/kg, and the administration time is 2.8min.

15 Rabbits were randomly balanced into 5 groups, group 1, group 2, group 3, group 4 and group 5, respectively. Each group was given in a single dose. Group 1 ear edge intravenous drip DSC1585 solution, group 2 ear edge intravenous drip DSC18212 solution, group 3 ear edge intravenous drip M43 solution, group 4 ear edge intravenous drip M44 solution, group 5 ear edge intravenous drip M45 solution, wherein M43, M44 and M45 structures are synthesized according to the synthesis method of the patent, the structures are as follows:

Sample preparation and use: each compound was precisely weighed and dissolved in physiological saline to prepare a physiological saline solution having a concentration of 0.2 mg/ml. All samples were prepared the day before first use, stored in a sealed condition at 2-8 ℃, and after each use the solution was returned to room temperature, filtered through a 0.22 micron polyethersulfone microporous filter membrane and administered intravenously, and the process was performed as aseptically as possible.

The rabbits were observed for general status before, during, after and 24, 48 and 72 hours after administration, and the injection sites were visually observed and recorded by photographing. Rabbits were euthanized after the end of the observation period, ear tissues were collected, and HE stained for pathology examination.

Visual observation of

Visual observations were scored according to table 2 (vascular stimulation response score) and table 3 (vascular stimulation grade grading criteria), while general status, behavior, signs, etc. of animals were observed and recorded. Visual inspection scoring results are shown in Table 4, and visual inspection grade determination results of vascular irritation are shown in Table 5.

TABLE 2 vascular stimulation response score

Lesion extent	Score value
		No obvious reaction	0
Mild hyperemia	1
		Light to moderate congestion and swelling	2
Moderate to severe congestion, swelling, and sagging ear	3
		Moderate to severe congestion, swelling, ear drop, and light to moderate necrosis	4
Moderate to severe congestion, swelling, ear drop, and severe extensive necrosis	5

TABLE 3 blood vessel stimulation level grading criteria

Score value	Stimulation level
		0-0.4	Without any means for
0.5-1.4	Slight
		1.5-2.4	Mild condition
2.5-3.4	Moderate degree
		3.5-4.4	Severe severity of
>4.5	Is very serious

Table 4: visual inspection of vascular irritation scoring results

Table 5: visual assessment of vascular irritation

	Group 1	Group 2	Group 3	Group 4	Group 5
						After administration of the drug	Slight	Slight	Mild condition	Mild condition	Mild condition
Recovery period	Without any means for	Without any means for	Slight	Slight	Slight

Histopathological evaluation

Pathological material selection: the rabbit ear hair is carefully removed before the sample is obtained, so that the skin is prevented from being damaged. All animals were left ear cut with 3 partial ear pieces: the distal end of the ear is not injected with a site (labeled A1, not mechanically stimulated); 1.5cm from the needle insertion site (labeled as A2, observe the response caused by needling, bleeding and drug stimulation); 3cm from the injection site (labeled A3, the ear is not physically stimulated, only the drug directly stimulates the vessel-induced response); similarly, the control selected group 1 animals had their right ears cut out 3 partial ears: the distal uninjected site of the ear (labeled B1, not mechanically stimulated); 1.5cm from the needle insertion site (labeled B2, observe the response caused by needling, bleeding and drug stimulation); 3cm from the injection site (labeled B3, ear was not physically stimulated, but only the response from direct stimulation of the vessel with saline). Each portion was cut into 0.5cm length of ear pieces.

HE staining performs pathological examination, details pathological changes at the examination site, and analyzes and judges vascular irritation. The main observation is as follows: whether the epidermis of the skin is swollen, broken and bleeding; vascular endothelial cell elastic tissue and adventitia have lesions and vascular ruptures; intravascular thrombus, and the area of the lumen cross-section occupied; perivascular soft tissue lesions. The results of the major pathological changes are summarized in table 6 below:

Table 6: vascular irritation test histopathological examination

It can be seen that during the dosing period, group 2 rabbits did not struggle, group 1 rabbits struggle slightly, and group 3, group 4 and group 5 rabbits all appeared to struggle significantly; the rabbits in each group have local reddening and swelling of blood vessels with different degrees after administration, but compared with the rabbits in the group 3, the rabbits in the group 4 and the rabbits in the group 2 have obviously lighter symptoms and lower macroscopic scores; histopathological results showed that none of the groups showed necrosis of vascular endothelial or extravascular tissue nor inflammatory cell infiltration, but rabbits of group 3, group 4 and group 5 had more pronounced pathological changes, relative to the milder changes of group 1 and group 2; in the convalescence period, the vascular stimulation symptoms of the rabbits in each group are gradually recovered, and the stimulation scores of the rabbits in the group 1 and the group 2 are obviously lower than those of the rabbits in the group 3, the group 4 and the group 5 from the visual observation scores in the convalescence period. Histopathological results showed that none of the rabbits showed necrosis of vascular endothelial or extravascular tissue nor inflammatory cell infiltration. However, it is also evident that rabbits of group 3, group 4 and group 5 have more pronounced pathological changes, and that the changes of group 1 and group 2 are relatively milder.

Based on visual inspection, histopathological examination results and the state of animals during administration, it was comprehensively judged that the compound of the present invention had a milder vascular irritation at the time of intravenous drip administration.

Example fifteen: drug concentration in Peripheral Blood Mononuclear Cells (PBMC)

About 100mg of compound DSC1581, comparative example compound M46 (compound 2 in WO2022148434A 1) and comparative example compound M41 were weighed respectively, 100ml of 30% sodium sulfobutyl- β -cyclodextrin solution was added, and the mixture was placed on a magnetic stirrer to be stirred and dissolved, and the solution was colorless, clear and transparent. The pH was adjusted to 3.50 using 0.2mol/L dilute hydrochloric acid. The vials were filled with 20ml penicillin bottles, each about 4ml. And (3) dissolving the filled sample, half adding a plug, and freeze-drying to obtain DSC1581 freeze-dried powder, M46 freeze-dried powder and M41 freeze-dried powder, wherein each bottle contains 4mg of active ingredient (API). Before use, the freeze-dried powder is dissolved by adding normal saline to prepare a solution with the concentration of 0.2mg/mL, and the solution is filtered by a polyethersulfone microporous filter membrane with the concentration of 0.22 micrometers and then is administrated.

After 12 cynomolgus monkeys are adaptively bred, the cynomolgus monkeys are randomly divided into three groups according to body weight and sex, and 4 cynomolgus monkeys in each group are respectively half male and female. Pre-dose weighing, group 1 intravenous infusion test sample DSC1581, group 2 intravenous infusion test sample M46, group 3 intravenous infusion test sample M41. The administration dosage is 1mg/kg, the administration volume is 5ml/kg, and the intravenous infusion time is 20min. Blood sample collection time points were pre-dose (0 h), 1h, 6h, 10h, 12h, 14h, 18h, 24h, 30h, 36h, 48h, and 72h after start of dose. Blood was collected via femoral vein (2 mL whole blood/time point), K ₂ EDTA was anticoagulated, and placed on ice after collection. Blood sample centrifugation was performed at 20.+ -. 2 ℃ and PBMCs were isolated within 2 hours after whole blood collection. After treatment of the PBMC samples, test compounds were detected using the LC-MS/MS method.

The results showed that AUC _0-t for DSC1581 group was 36.16h ng/1000000PBMC, AUC _0-t for m46 group was 30.75h ng/1000000PBMC, AUC _0-t for m41 group was 27.54h ng/1000000PBMC; AUC _0-∞ for DSC1581 group was 36.84h ng/1000000PBMC, AUC _0-∞ for m46 group was 31.79h ng/1000000PBMC, AUC _0-∞ for m41 group was 28.13h ng/1000000PBMC; DSC1581 group t _1/2 was 14.34h, M46 group t _1/2 was 11.68h, and M41 group t _1/2 was 12.72h. The exposure of DSC1581 was increased by 17.6% and 31.3% compared to M46 and M41, respectively, and the half-life was longer, indicating a greater ability of DSC1581 to enter cells.

Example sixteen: in vivo antiviral assay

Female BALB/c mice of 6-8 weeks of age, without specific pathogen grade, purchased from Shanghai Ji Hui laboratory animal feeding limited, after 3 days of adaptive feeding, 30 were selected and randomly divided into 5 groups: group A, group B, group C, group D and group E, 6 each; the solvent is sodium carboxymethylcellulose (CMC-Na) solution with concentration of 0.5%, and the positive control agent and the test sample are all suspended with CMC-Na solution with concentration of 0.5% for administration. Group A is vehicle group, and 0.5% sodium carboxymethylcellulose (CMC-Na) solution is given; group B administration of a suspension of balofluo Sha Weizhi; group C administration of DSC1583 suspension; group D administers a suspension of M47; group E administration of a suspension of M42;

Mice were vaccinated nasally on day 0 (Influenza Virus, a/PR/8/34 (H1N 1)) at 900p.f.u./mouse. The treatment with solvent or test sample was continued for 7 days from day 1 to day 7, 2 times daily, with a dosing regimen of gastric lavage and a dosing volume of 10mL/kg, and the doses of each of the other groups except the solvent group were 5mg/kg, with the first dosing time being 24 hours after virus inoculation. Animals were observed continuously from day 0 to day 14 and weight, health and survival were recorded. The rate of change of body weight is shown in figure 1.

The test results show that: the animals in the solvent group show infection symptoms after virus inoculation, the weight is rapidly and obviously reduced, and finally all animals die, and the survival rate is 0%; the control medicine baluo Sha Weizhi (5 mpk) can relieve the weight loss of mice caused by virus infection (the maximum reduction is-2.29%) under the set experimental conditions, protect the mice from death, and the final survival rate is 100%, thus the expected in vivo anti-influenza virus drug effect is shown; the tested compound M42 (5 mpk) can relieve the weight loss (the maximum reduction amplitude is-3.49%) of mice caused by virus infection under the set experimental conditions, protect the mice from death, and the final survival rate is 100%; the tested compound M47 (5 mpk) can relieve weight loss of mice caused by virus infection (maximum reduction of-11.94%) under set experimental conditions, protect the mice from death, and the final survival rate is 100%, and the tested compounds M42 and M47 show expected in vivo anti-influenza virus drug effects; the tested compound DSC1583 (5 mpk) can obviously relieve the weight loss (the maximum reduction amplitude is-0.41%) of mice caused by virus infection under the set experimental conditions, protect the mice from death, and has the final survival rate of 100 percent, thus showing the expected anti-influenza virus efficacy in vivo.

From the weight recovery time analysis, the weight of animals in DSC1583 group was not substantially reduced, the weight of animals in Ballon Sha Weizu and M42 group was recovered at about day 8, and the weight of animals in M47 group was recovered at day 11.

From the above analysis, it can be seen that although the animal survival rates of baluo Sha Weizu, M42, M47 and DSC1583 were all 100%, DSC1583 showed outstanding advantages in terms of weight change and weight recovery.

The present application has been described in terms of several embodiments, but the description is intended to be illustrative and not limiting and many more embodiments and implementations are possible within the scope of the described embodiments.

Claims (9)

1. A compound represented by formula (I), or an optical isomer, an isotopic derivative, a pharmaceutically acceptable salt thereof:

in formula (I), R _a is selected from hydrogen, deuterium, methyl or deuterated methyl;

R _b and R _c are each independently selected from hydrogen, deuterium, C1-C3 alkyl, deuterated C1-C3 alkyl, or R _b、R_c together with the carbon atom to which they are attached form cyclopropyl or deuterated cyclopropyl;

y ₁、Y₂、Y₃ and Y ₄ are each independently CH or N, and at least one of them is N;

X is Se or S;

R is hydrogen, And when X is Se, R is not/>Wherein,

X ₁ is an O atom or an S atom;

n1 is 0,1 or 2;

each R ₁ or R ₂ is independently selected from hydrogen, deuterium, methyl, or deuterated methyl;

R ₃ is selected from the following groups with or without substitution of one or more hydrogen atoms with deuterium: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino;

R ₄ and R ₅ are each independently a hydroxyl group, the following groups with or without deuterium substitution of one or more hydrogen atoms: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino, C3-C8 cycloalkoxy, C3-C8 heterocycloalkoxy, C6-C10 aryloxy, C7-C12 aralkyloxy; or R ₄ and R ₅ together with the phosphorus atom to which they are attached, e.g A 5-7 membered ring of (2); wherein R ₆,R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃ and R ₁₄ are each independently hydrogen or C1-C3 alkyl, or R ₆ and R ₇、R₈ and R ₉、R₁₁ and R ₁₂、R₁₂ and R ₁₃ each together with the attached carbon atom form an aromatic ring, and R ₄ and R ₅ together with the attached phosphorus atom form a 5-7 membered ring and one or more of the hydrogen atoms in the ring substituents may be substituted or unsubstituted with deuterium.

2. A compound represented by formula (ii-1) and/or formula (ii-2), or an optical isomer, an isotopic derivative, a pharmaceutically acceptable salt thereof:

The substituents of the formula (II-1) and/or of the formula (II-2) are as defined in the formula (I) of claim 1.

3. A compound represented by formula (iii-1) and/or formula (iii-2), or an optical isomer, an isotopic derivative, a pharmaceutically acceptable salt thereof:

The substituents of the formula (III-1) and/or of the formula (III-2) are as defined in the formula (I) of claim 1.

4. A compound of formula (iv-2), or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof:

the substituents in formula (IV-2) are as defined in claim 1 for formula (I).

5. The compound of any one of claims 1-4, wherein when R ₄ and R ₅ are selected from hydroxy, the pharmaceutically acceptable salts thereof include sodium, potassium, magnesium, zinc, amine, basic amino acid salts, and the like.

6. A compound according to any one of claims 1 to 5 selected from the following structures:

7. a pharmaceutical composition comprising a compound of any one of claims 1-6, or an optical isomer, an isotopic derivative, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier thereof.

8. The pharmaceutical composition of claim 7, in the form of a tablet, capsule, powder, granule, pill, suspension, syrup, injection or inhalant formulation.

9. Use of a compound according to any one of claims 1 to 6, or an optical isomer, an isotopic derivative, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7 or 8 for the preparation of an anti-influenza virus medicament.