HK40101381B - Anti-influenza virus derivative and use thereof - Google Patents

Description

An anti-influenza virus derivative and its uses

Technical Field

This invention relates to compounds with anti-influenza virus activity, or their hydrates, solvates, optical isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, methods of preparation thereof, and their use in anti-influenza virus activities.

Background Technology

Influenza viruses mainly include four types: influenza A, influenza B, influenza C, and influenza D. The main antiviral drugs on the market are amantadine, neuraminidase inhibitors oseltamivir and zanamivir. However, resistance to these compounds has emerged in all of them to date.

Influenza virus RNA polymerase contains a cap-dependent endonuclease; inhibiting the activity of this endonuclease can inhibit viral replication. This enzyme has become a promising target for antiviral drug development, and many companies are focusing on cap-dependent endonucleases. Various heterocyclic compounds have already been used as cap-dependent endonuclease inhibitors. Even with the currently marketed cap-dependent endonuclease inhibitor baloxavir, drug resistance has been reported. Furthermore, these compounds often exhibit poor physicochemical properties, such as low solubility and low bioavailability. Therefore, it remains necessary to develop a new generation of cap-dependent endonuclease inhibitors.

After detailed research, we discovered a class of selenized cyclopropyl-containing compounds that exhibit highly effective and broad-spectrum anti-influenza virus effects. Further research shows that these compounds have a specific distribution, making them promising candidates for development into anti-influenza virus drugs. The compounds of this invention inhibit viral replication by suppressing cap-dependent endonucleases in the influenza virus. By targeting an earlier stage of the viral replication cycle, they offer better preventative and therapeutic effects against influenza.

Summary of the Invention

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

This invention provides a class of compounds with anti-influenza virus activity, their preparation methods, and uses.

This invention provides a hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt thereof, as shown in formula (I-0).

In formula (I-0), _Ra is selected from hydrogen, deuterium, methyl, or deuterated methyl;

X is S or Se, and when X is S, the formula (I-0) contains at least one deuterium atom;

_Rb and _Rc are each independently selected from hydrogen, deuterium, C1-C3 alkyl, deuterated C1-C3 alkyl, or _Rb , _Rc and the carbon atom attached thereto form a cyclopropyl or deuterated cyclopropyl group.

R is hydrogen, where,

_X1 is an O atom or an S atom;

n1 is 0, 1, or 2;

Each _R1 or _R2 is independently selected from hydrogen, deuterium, methyl, or deuterated methyl;

R ₃ is selected from the following groups whose one or more hydrogen atoms are substituted with deuterium or not: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine;

_R4 and _R5 are each independently a hydroxyl group, or one or more hydrogen atoms substituted with or not substituted with deuterium, of the following groups: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine, C6-C20 arylalkoxy; or _R4 and _R5 together form a 5-7 membered ring with the attached phosphorus atom; wherein _R6 , _R7 , _R8 , _R9 , _R10 , _{R11, R12, R13 and R14 are} each independently hydrogen or C1-C3 alkyl, or _R6 and _R7 , R8 and _R9 , _R11 and _R12 , _R12 and _R13 each together form an aromatic ring with the _{attached carbon atom, and R4 and} R _5. One or more of the hydrogen atoms in the 5-7 membered ring formed by the phosphorus atom and the substituents on the ring may be substituted with deuterium or not substituted.

In some embodiments, the present invention provides a compound or its hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt represented by formula (I):

In formula (I), _Ra is selected from hydrogen, deuterium, and methyl;

_Rb and _Rc are each independently selected from hydrogen, deuterium, C1-C3 alkyl groups, or _Rb , _Rc and the carbon atom attached to them together form a cyclopropyl group;

R is hydrogen, where,

_X1 is an O atom or an S atom;

n1 is 0, 1, or 2;

Each _R1 or _R2 is independently selected from hydrogen or methyl;

R ₃ is selected from the following groups: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, and C1-C8 alkylamine;

_R4 and _R5 are each independently a hydroxyl group or one of the following groups: C1-C8 alkoxy, C1-C8 alkylthio, or C1-C8 alkylamine.

In some embodiments, the present invention provides a compound or its hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt represented by formula (I):

In formula (I), _Ra is selected from hydrogen, deuterium, methyl, or deuterated methyl;

_Rb and _Rc are each independently selected from hydrogen, deuterium, C1-C3 alkyl, deuterated C1-C3 alkyl, or _Rb , _Rc and the carbon atom attached to them together form cyclopropyl or deuterated cyclopropyl.

R is hydrogen, where,

_X1 is an O atom or an S atom;

n1 is 0, 1, or 2;

Each _R1 or _R2 is independently selected from hydrogen, deuterium, methyl, or deuterated methyl;

R ₃ is selected from the following groups whose one or more hydrogen atoms are substituted with deuterium or not: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine;

_R4 and _R5 are each independently a hydroxyl group, or one or more hydrogen atoms substituted with or not substituted with deuterium, of the following groups: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine, C6-C20 arylalkoxy; or _R4 and _R5 together form a 5-7 membered ring with the attached phosphorus atom; wherein _R6 , _R7 , _R8 , _R9 , _R10 , _{R11, R12, R13 and R14 are} each independently hydrogen or C1-C3 alkyl, or _R6 and _R7 , R8 and _R9 , _R11 and _R12 , _R12 and _R13 each together form an aromatic ring with the _{attached carbon atom, and R4 and} R _5. One or more of the hydrogen atoms in the 5-7 membered ring formed by the phosphorus atom and the substituents on the ring may be substituted with deuterium or not substituted.

In some embodiments, the compound provided by the present invention, or its hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt, is shown in formula (II):

The substituents in formula (II) are defined as defined in formula (I-0) and/or formula (I).

In some embodiments, the compound provided by the present invention, or its hydrate, solvate, optical isomer, polymorph, isotope derivative, or pharmaceutically acceptable salt, is shown in formula (Ⅲ):

The substituents in formula (Ⅲ) are defined as defined in formula (I-0) and/or formula (I).

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

In the embodiments of this 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. This includes, but is not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc.

In the embodiments of this application, the C1-C8 alkoxy and C1-C8 alkylthio groups refer to groups in which oxygen or sulfur atoms are inserted at any reasonable position into a saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms in the molecule, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, 2-ethylethoxy, methylthio, ethylthio, propylthio, isopropylthio, isobutylthio, etc.

In the embodiments of this application, the C1-C8 alkylamine group refers to a group in which a saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms is inserted with a -NH- or -NH ₂ group at any reasonable position, including monoalkylamine, dialkylamine and cycloalkylamine, including but not limited to methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, etc.

In the embodiments of this application, the C1-C3 alkyl refers to an alkane containing 1 to 3 carbon atoms in the molecule, including: methyl, ethyl, propyl, isopropyl, and cyclopropyl.

In the embodiments of this application, the C6-C20 arylalkoxy group refers to an aryl group with 6-20 carbon atoms bonded to an oxygen atom, including but not limited to benzyloxy, phenethoxy, and naphthoxy.

In the embodiments of this application, deuteration refers to the replacement of a hydrogen atom at any reasonable position in the molecule by a deuterium atom.

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

In some embodiments, _{both Rb} and _Rc are hydrogen; in some embodiments, _{both Rb} and _Rc are deuterium; in some embodiments, _Rb is hydrogen and _Rc is deuterium.

In some embodiments, _Rb and _Rc are each independently a C1-C3 alkyl group; in some embodiments, _Rb is hydrogen and _Rc is a C1-C3 alkyl group; in some embodiments, _Rb is deuterium and _Rc is a C1-C3 alkyl group.

In some embodiments, _Rb and _Rc are each independently a deuterated C1-C3 alkyl group; in some embodiments, _Rb is hydrogen and _Rc is a deuterated C1-C3 alkyl group; in some embodiments, _Rb is deuterium and _Rc is a deuterated C1-C3 alkyl group.

In some embodiments, _Rb is a C1-C3 alkyl group, and _Rc is a deuterated C1-C3 alkyl group;

In some embodiments, _Rb , _Rc , and the carbon atom attached thereto together form a cyclopropyl group; in other embodiments, _Rb , _Rc , and the carbon atom attached thereto together form a deuterated cyclopropyl group.

In some implementations, X is S, in which case formula (I-0) contains at least one deuterium atom;

In some implementations, X is Se.

In some embodiments, R is hydrogen; in some embodiments, R is...

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

In some implementations, _X1 is an O atom; in other implementations, _X1 is an S atom.

In some embodiments, _R1 and _R2 are both hydrogen; in some specific embodiments, _R1 or _R2 are both deuterium; in some embodiments, _R1 and _R2 are both methyl; in some embodiments, _R1 and _R2 are both deuterated methyl.

In some specific embodiments, _R1 is methyl and _R2 is hydrogen; in some specific embodiments, _R1 is deuterated methyl and _R2 is hydrogen.

In some specific implementation schemes, _R1 is deuterated methyl and _R2 is deuterium.

In some embodiments, _R3 is selected from the following groups: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkylamine; preferably, _R3 is selected from the following groups: C1-C8 alkyl, C1-C8 alkoxy; more preferably, _R3 is a C1-C8 alkoxy.

In some embodiments, _R3 is selected from one or more hydrogen atoms substituted with deuterium of the following groups: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, and C1-C18 alkylamine; preferably, _R3 is selected from one or more hydrogen atoms substituted with deuterium of the following groups: C1-C8 alkyl and C1-C8 alkoxy; more preferably, _R3 is a C1-C8 alkoxy group with one or more hydrogen atoms substituted with deuterium.

In some specific implementations, n1 is 1, _X1 is an O atom, _R1 and _R2 are both hydrogen atoms, and _R3 is a C1-C8 alkoxy or C1-C8 alkyl group;

In some specific implementations, n1 is 1, _X1 is an O atom, _R1 and _R2 are both hydrogen atoms, and _R3 is a C1-C8 alkoxy group in which one or more hydrogen atoms are replaced by deuterium, or a C1-C8 alkyl group in which one or more hydrogen atoms are replaced by deuterium.

In some specific implementations, n1 is 0, _X1 is an O atom, and _R3 is a C1-C8 alkoxy or C1-C8 alkyl group;

In some specific implementations, n1 is 0, _X1 is an O atom, _R3 is a C1-C8 alkoxy group with one or more hydrogen atoms replaced by deuterium, or a C1-C8 alkyl group with one or more hydrogen atoms replaced by deuterium.

In some embodiments, _R4 and _R5 are both hydroxyl groups; in some embodiments, _R4 and _R5 are the following groups: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine; in some embodiments, _R4 and _R5 are C6-C20 arylalkoxy, preferably, _R4 and _R5 are benzyloxy.

In some embodiments, _R4 and _R5 are the following groups in which one or more hydrogen atoms are substituted with deuterium: C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamine;

In some embodiments, _R4 and _R5 together form a 5-7 membered ring with the attached phosphorus atom; wherein _R6 , _R7 , _R8 , _R9 , _R10 , _R11 , _R12 , _R13 and _R14 are each independently hydrogen or C1-C3 alkyl, or _R6 and _R7 , _R8 and _R9 , _R11 and _R12 , _R12 and _R13 each together form an aromatic ring with the attached carbon atom;

In some embodiments, _R4 and _R5 together form a 5-7 membered ring with the attached phosphorus atom, and one or more of the hydrogen atoms in the 5-7 membered ring formed by _R4 and _R5 together with the attached phosphorus atom and the substituents on the ring can be substituted with deuterium.

In some specific implementations, n1 is 1, _X1 is an O atom, _R1 and _R2 are both hydrogen atoms, and _R4 and _R5 are both hydroxyl atoms; in some specific implementations, n1 is 0, _X1 is an O atom, and _R4 and _R5 are both hydroxyl atoms.

In some embodiments, when R is hydrogen, the pharmaceutically acceptable salt of the compound can be an alkali metal salt, an alkaline earth metal salt, an amine salt, or an amino acid salt; preferably, the pharmaceutically acceptable salt includes: sodium salt, potassium salt, magnesium salt, zinc salt, amine salt, basic amino acid salt, etc.

In some embodiments, when _{both R4} and _R5 are hydroxyl groups, the pharmaceutically acceptable salt of the compound can be an alkali metal salt, an alkaline earth metal salt, an amine salt, or an amino acid salt; preferably, the pharmaceutically acceptable salt includes: sodium salt, potassium salt, magnesium salt, zinc salt, amine salt, basic amino acid salt, etc.

In an embodiment of the present invention, the pharmaceutically acceptable salt is obtained using conventional salt-forming methods.

In an embodiment of the present invention, the pharmaceutically acceptable salt is structurally confirmed using methods such as nuclear magnetic resonance, mass spectrometry, atomic absorption spectroscopy, elemental analysis, and melting point detection.

In embodiments of the present invention, formula (I-0) or/and formula (I) contain two chiral centers, and the compounds of the present invention or their intermediates can be obtained as single-configuration compounds by chiral separation.

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

In an embodiment of the present invention, the absolute configuration of a single-configuration optical isomer is determined by electronic circular dichroism spectroscopy.

In an embodiment of the present invention, the optical rotation of the racemic mixture and the single-configuration optical isomer is tested according to the optical rotation determination method of Chinese Pharmacopoeia 2020 Edition-Part IV-0621.

The comparative compounds synthesized in this invention with reference to the synthesis methods in the literature, patents and embodiments of this invention, and whose purity is all >98%, are used in relevant biological experiments.

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

Or its hydrates, solvates, optical isomers, polymorphs, isotope derivatives, or pharmaceutically acceptable salts.

The metal salts of the compounds provided by this invention include, but are not limited to, the following compounds:

Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound or its hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more combinations of fillers, binders, diluents, lubricants, preservatives, flavor masking agents, or solubilizers. This pharmaceutical composition can be used against influenza viruses.

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

In a third aspect, the present invention provides the use of the above-mentioned compounds, including their hydrates, solvates, optical isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, or pharmaceutical compositions thereof, for use against influenza viruses.

The present invention provides the use of the above-mentioned compounds, including compounds such as their hydrates, solvates, optical isomers, polymorphs, isotope derivatives, pharmaceutically acceptable salts, or pharmaceutical compositions thereof, in the preparation of medicaments for the treatment of influenza viruses.

The present invention provides a method for preventing or treating influenza virus infection, the method comprising administering a therapeutically effective amount of the above-mentioned compound, including its hydrates, solvates, optical isomers, polymorphs, isotope derivatives, and pharmaceutically acceptable salts, to an individual in need.

The compounds of this invention exhibit stronger antiviral activity both in vivo and in vitro, demonstrating a better safety margin.

The compounds of this invention are effective against a variety of influenza virus strains, including avian influenza virus strains, and are also effective against oseltamivir-resistant influenza viruses and other drug-resistant strains, and have superior activity compared to other similar structures; in particular, they show superior activity in inhibiting influenza B.

The compounds of this invention show significantly enhanced in vivo anti-influenza virus activity, lower viral titers in the lungs, and histopathological results also show that the lesions in the lung tissue are milder after treatment with the compounds of this invention.

Further research revealed that the compound of this invention exhibits good lung tissue distribution in animal experiments, consistent with its results in in vivo anti-influenza virus experiments, showing low viral titers in the lungs and milder lung tissue lesions. This is more conducive to exerting its anti-influenza virus effect and is expected to have significant clinical value and therapeutic advantages.

Pharmacokinetic studies of the compounds of this invention have shown that they have a shorter average absorption time, which indicates that they can be absorbed more quickly after oral administration, and thus exert their effects more quickly.

Attached Figure Description

Figure 1 shows the viral titer in lung tissue of a mouse model of influenza virus in a drug efficacy test.

Detailed Implementation

The following examples are intended to enable those skilled in the art to fully understand the present invention, but do not limit the invention in any way. The structures of all compounds were determined by MS or ^1H -NMR, and the optical isomers of all single configurations involved were determined by optical rotation testing or electronic circular dichroism spectroscopy.

Unless otherwise specified, all solvents and reagents used in this embodiment are commercially available products. All starting materials are commercially purchased raw materials.

Example 1: Synthesis of 9a

Synthesis of compound 3a

5 g of compound 1a, 2.95 g of compound 2a, and 7 ml of triethylamine were added to a mixed solvent of 40 ml of DMF (N,N-dimethylformamide) and 40 ml of toluene. The system was heated to 130 °C and reacted for 8 hours. Water was added and the mixture was stirred to precipitate a solid. The system was filtered to obtain 7.03 g of a white solid of compound 3a. No separation was required, and it was used directly in the next reaction.

Synthesis of compound 4a

5.84 g of compound 2-bromo-1,1-dimethoxyethane and 5 g of compound 3a were added to 40 mL of DCM. 3.9 g of potassium tert-butoxide was slowly added to the system at room temperature. After the addition was complete, the system was heated to 40 °C and reacted for 5 hours. The system was cooled to room temperature, water was added, and the mixture was stirred for 15 min. The mixture was separated, the organic phase was concentrated to dryness, and purified by silica gel column chromatography to give 5.2 g of compound 4a, yield 74%. ^¹H NMR ( _CDCl₃ , 400 MHz): δ 7.83–7.88 (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

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

Synthesis of compound 7a

5 g of compound 6a was added to 10 ml of DMA, along with 1.62 g of sodium hydride (60%) and 3.2 g of iodomethane. The system was stirred at 25 °C for 12 h, then 100 ml of water was added and stirred for 0.5 h. Extraction was performed using EA. The combined organic phases were washed successively with 0.5 N dilute hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 4.65 g of an oil. 30 ml of DMA, 3.54 g of Boc hydrazine, and 13.47 g of pyridine p-toluenesulfonate were added to this oil. The system was heated to 60 °C and reacted for 18 h. After the reaction, the system was extracted with water and EA, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness. Purification by silica gel column chromatography yielded 4.68 g of compound 7a, with a yield of 61.65%; the product was a yellow oil, ESI-MS (+): m/z = 375.2.

Synthesis of compound 8a

4.5 g of compound 7a was added to 30 mL of ethanol, followed by 24 mL of 1 N NaOH solution. The reaction mixture was incubated at 60 °C for 15 hours, and the pH was adjusted with dilute hydrochloric acid. The mixture was extracted with dichloromethane (DCM), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a grayish-white solid. The grayish-white solid was added to DCM, and 2.53 g of compound 5a, 4 mL of triethylamine, and 6.85 g of HATU were added at 25 °C. The mixture was stirred at 25 °C for 11 hours, diluted with water, and extracted with DCM. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give 4.1 g of compound 8a (66% yield); the product was a white solid, and ESI-MS (+): m/z = 518.3.

Synthesis of compound 9a

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

Example 2: Synthesis of 21a

Synthesis of compound 15a

After purging the three-necked flask with nitrogen, 226 ml of phenyl magnesium bromide (2.8 mol/L) was added. The mixture was cooled to below 10°C in an ice-water bath. 49.9 g of selenium powder was added in batches, controlling the reaction temperature to not exceed 30°C. After the selenium powder was added, the reaction was allowed to proceed for 2 hours. Then, 2 mol/L hydrochloric acid was added in an ice-water bath. The reaction was exothermic. Extraction was performed with EA, and the mixture was separated. The organic phase was evaporated to dryness, yielding product 15a, a brown oily substance with a strong odor. The next reaction was then carried out directly.

Synthesis of compound 17a

After purging the three-necked flask with nitrogen, 81.18 g of LDA (lithium diisopropylamino) was added. The mixture was cooled to -30°C in a dry ice-ethanol solution. 50 g of a THF solution of compound 3,4-difluorobenzoic acid was added dropwise. The addition was exothermic, and the reaction temperature was controlled below -20°C. The reaction was allowed to proceed for 2 hours. DMF was then added, again exothermic, and the reaction temperature was controlled below -20°C. No further temperature control was needed; the mixture was gradually brought to room temperature and allowed to react overnight. A sample was taken to confirm the reaction was complete. HCl was added to the reaction system, resulting in exothermic reaction. Extraction with EA was performed, followed by separation. The organic phase was evaporated to dryness, yielding 72 g of crude compound 17a. This product was used directly in the next reaction step. The crude product was a yellow solid with a yield of 122% (the material contained unevaporated DMF).

Synthesis of compound 18a

After purging the three-necked flask with nitrogen, 58.81 g of compound 15a, 49.6 g of compound 17a, 300 ml of toluene, and 17.6 g of camphor sulfonic acid were added. The mixture was heated to 70°C and reacted overnight. The reaction system was cooled to room temperature, and NaOH solution was added. The mixture was separated into liquid and aqueous phases. The aqueous phase was extracted with EA, and the organic phases were combined. The organic phase was washed with saturated NaCl and evaporated to dryness. The crude product was 123.08 g. The crude product was pulped with PE and filtered to obtain 34.4 g of product. 14.83 g of product was recovered from the pulping filtrate. Compound 18a was an orange-yellow solid, with a yield of 47.9%.

Synthesis of compound 19a

In a three-necked flask, 16.9 g of _AlCl3 and 250 ml of toluene were added. The system was cooled in an ice-water bath, and 17.1 g of tetramethyldisiloxane was added. The mixture was stirred until homogeneous, and then 150 ml of a toluene solution of compound 18a (34.4 g) was added. The reaction was slightly exothermic, _{and AlCl3} gradually dissolved. The mixture was heated to 80 °C and reacted for 1 hour. The reaction was stopped, and sulfuric acid solution (16.2 mL + 240 mL water) was added. The mixture was separated, and the aqueous phase was extracted with EA. The organic phase was evaporated to dryness. The crude product was pulped with PE and filtered to obtain 22 g of a yellow powdery solid of 19a. Theoretical yield: 34.68 g, yield: 63.4%. This product was used directly in the next reaction.

Synthesis of compound 20a:

429g of polyphosphoric acid was added to a three-necked flask and heated to 80℃. 42g of compound 19a was added, and the temperature was raised to 120℃. The reaction system became viscous, and the color changed from yellow to dark purple. After reacting for 1 hour, a sample was taken, water was added, and the mixture was treated with EA. The reaction temperature was lowered to below 100℃, water was added and stirred until homogeneous, EA was added for extraction, the organic phase was separated, and the mixture was evaporated to dryness. The crude product was pulped with PE and filtered to obtain 3.4g of product. The filtrate was separated by column chromatography to obtain 10g of product. Compound 20a was a colorless to pale yellow flocculent solid. Theoretical yield: 39.69g, actual yield: 13.4g, yield: ^33.76 %. NMR (DMSO-D6, 400MHz): δ7.95-8.09(d.1H), 7.54-7.67(d,1H), 731-7.52(m,3H), 7.20-7.29(m,1H), 4.21-4.27(s,2H).

Synthesis of compound 21a:

Add 2.12 g of compound 20a and 20 ml of ethanol to a three-necked flask and stir the system. Add an ethanol solution of sodium borohydride (0.26 g) dropwise under an ice-water bath. The reaction system exhibits a slight temperature rise. After the addition is complete, the reaction is allowed to proceed to room temperature. The materials gradually dissolve. After the reaction solution is clear, a sample is taken for analysis. After the reaction is complete, 2 mol/L hydrochloric acid is added until no more bubbles are produced and the pH is 4–6. A large amount of solid precipitates out. The solid is obtained by filtration. The filtrate is extracted with EA to separate the organic phase. The organic phase is evaporated to dryness. The solids are combined to obtain product 21a, a brown solid, which is directly used in the next reaction step.

Example 3: Synthesis of DS1491 and DS1494

Synthesis of compound 22a:

0.8 g of compound 9a, 0.77 g of compound 21a, 1.8 g of compound _T3P (propylphosphonic anhydride, 50% EA solution), 0.33 g of methanesulfonic acid, and 10 ml of EA were added to a single-necked flask. The mixture was heated to reflux and reacted overnight. Samples were taken for analysis. After the reaction was complete, the following steps were performed: Post-treatment: Saturated sodium bicarbonate aqueous solution was added until no more bubbles were released. The mixture was separated into liquid and aqueous phases, extracted with EA, and the organic phases were combined, evaporated to dryness, and purified by silica gel column chromatography. Then, 0.52 g of compound 22a was obtained by chiral column chromatography, yield: 37%.

Synthesis of compound DS1491:

0.5 g of compound 22a, 0.17 g of compound LiCl, and 5 ml of DMA (N,N-dimethylacetamide) were added to a single-necked flask. The temperature was raised to 100 °C, and the reaction solution became yellow and turbid. The reaction was carried out for 2 hours, and samples were taken for analysis. After the reaction was completed, the solution was processed. Saturated sodium bicarbonate aqueous solution was added, and a solid precipitated out. The solution was filtered, and the filtrate was extracted with EA. The organic phase was evaporated to dryness and purified by silica gel column chromatography to obtain 0.34 g of compound DS1491, with a yield of 77%. ES I-MS (+): m/z = 558.1.

Synthesis of compound DS1494:

0.3 g of compound DS1491, 150 _mg of _K₂CO₃ , 10 mg of KI, and 110 mg of dimethyl chloromethyl carbonate were added to a single-necked flask. 5 mL of DMA was added to the system, and the mixture was heated to 60 °C and reacted overnight. Samples were taken for analysis, and the reaction was processed after completion. The reaction system was cooled to room temperature, 2N HCl was added, and water was added. A solid precipitated, which was extracted with EA and water. The organic phase was separated, dried over anhydrous sodium sulfate, and then evaporated to dryness. After separation by column chromatography, 0.21 g of compound DS1494 was obtained, with a yield of 61%. ES I-MS (+): m/z = 646.1.

Example 4: Synthesis of compounds DS1492 and DS1495

Synthesis of compound 1b

Under nitrogen protection, 1 g of compound 20a was dissolved in 15 mL of THF. 140 mg of lithium aluminum hydride-D4 was slowly added at 0 °C, and the mixture was heated to 25 °C and reacted for 8 hours. The mixture was then cooled to 0 °C and the reaction was quenched with water. The mixture was extracted with 2N hydrochloric acid and EA. The organic phase was concentrated to dryness, and column chromatography yielded 0.58 g of compound 1b (57% yield). ES I-MS (+): m/z = 314.0.

Synthesis of compound 2b

Following the synthetic method of compound 22a, compounds 9a and 1b were synthesized to obtain 0.36 g of compound 2b in total, with a yield of 35%; ES I-MS (+): m/z = 649.2.

Synthesis of compound DS1492

Following the synthetic method of compound DS1491, 0.21 g of compound DS1492 was synthesized, with a yield of 795%; ES I-MS (+): m/z = 559.1.

Synthesis of compound DS1495

Following the synthetic method of compound DS1494, 0.13 g of compound DS1495 was synthesized, with a yield of 63%; ES I-MS (+): m/z = 647.1.

Example 5: Synthesis of compounds DS1493 and DS1496

Synthesis of compound 1c:

Under nitrogen protection, 1 g of compound 20a was dissolved in 15 mL of THF. 4 mL of methyllithium reagent (1.6 M diethyl ether solution) was slowly added dropwise at -20 °C. The system was allowed to naturally warm to 25 °C and reacted for 8 hours. The system was then cooled to 0 °C, and the reaction was quenched with water. The mixture was concentrated to dryness and extracted with EA and water. The organic phase was separated and concentrated to dryness. Column chromatography yielded 0.77 g of compound 1c, a yield of 73%, ES I-MS (+): m/z = 327.1.

Synthesis of compound 2c

Following the synthetic method of compound 22a, compounds 9a and 1c were synthesized to obtain 0.33 g of compound 2c in total, with a yield of 23%; ES I-MS (+): m/z = 662.1.

Synthesis of compound DS1493

Following the synthetic method of compound DS1491, 0.19 g of compound DS1493 was synthesized, with a yield of 73%; ES I-MS (+): m/z = 571.1.

Synthesis of compound DS1496

Following the synthetic method of compound DS1494, 95 mg of compound DS1496 was synthesized, with a yield of 55%; ES I-MS (+): m/z = 660.1.

Example 6: Synthesis of DS1497

Synthesis of compound 1d:

1 g of compound DS1491, 0.7 g of tert-butyl chloromethyl phosphate, and 1 ml of triethylamine were added to a reaction flask, followed by 10 ml of DMF (N,N-dimethylformamide). The reaction mixture was incubated at 45 °C for 8 hours. After crystallization and filtration, approximately 0.59 g of crude compound 1d was obtained, with a yield of 42%. ES I-MS (+): m/z = 779.5. The product was used directly in the next step without further purification.

Synthesis of compound DS1497:

Under nitrogen protection, 0.5 g of compound 1d and 5 ml of anhydrous dichloromethane were added to a reaction flask. While stirring at room temperature, 1 ml of trifluoroacetic acid was added dropwise, and the mixture was stirred at 25°C for 3 hours. The system was concentrated to dryness, and isopropanol and water were added to crystallize, yielding 0.29 g of compound DS1497, with a yield of 69%. ES I-MS (+): m/z = 668.1, ES I-MS (-): m/z = 666.2.

Example 7: Synthesis of compound DS1831:

70 mg of compound DS1491, 82 _mg of _Cs₂CO₃ , 2.1 mg of KI, 30 mg of compound diethyl chloromethyl phosphate, and 10 mL of DMA were added to a single-necked flask. The system was heated to 50 °C and reacted for 2.5 h until the reaction was complete. The reaction system was cooled to room temperature and extracted with EA and water to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to obtain 55.45 mg of compound DS1831, with a yield of 61% and ESI-MS (+): m/z = 724.1.

Example 8: Synthesis of compound DS1833:

Synthesis of compound DS1833-1

1.1 g of deuterated methanol, 5.2 ml of triethylamine, and 30 ml of dichloromethane were added to a reaction flask. After the system was cooled to 0 °C, 4 g of chloromethyl chloroformate was slowly added dropwise. After the addition was complete, the system was allowed to naturally rise to room temperature and react for 3 hours. The reaction was quenched with water, extracted with dichloromethane, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to give 3.28 g of liquid DS1833-1, with a yield of 83%.

Synthesis of compound DS1833

Following the synthetic method of compound DS1494, 78 mg of compound DS1833 was synthesized using compounds DS1491 and DS1833-1 as raw materials, with a yield of 55% and ES I-MS (+): m/z = 649.1.

Example 9: Synthesis of compound DS14914:

1 g of compound DS1497 was added to 10 ml of anhydrous ethanol. The system was heated to 30 °C, and 120 mg of sodium hydroxide was added. The mixture was stirred for 2 hours, cooled to -10 °C, and filtered. The resulting product was dried under reduced pressure to obtain DS14914, with a yield of 90% and a purity of 99.7%. ES I-MS (+): m/z = 734.1 (M+Na). Elemental analysis: Measured values: Na, 6.33%; Se, 11.02%; Theoretical values: Na, 6.47%; Se, 11.12%.

Example 10: Synthesis of compound DS14915:

0.6 g of compound DS14914 was dissolved in 25 ml of deionized water, and 185 mg of zinc acetate dihydrate in 5 ml of deionized water was added. The mixture was stirred at room temperature for 2 hours, filtered, and the resulting product was dried under reduced pressure to obtain DS14915, with a yield of 84% and a purity of 99.4%. ES I-MS (+): m/z = 729.9 (M+H). Elemental analysis: Measured values: Se, 10.66%; Zn, 8.81%; Theoretical values: Se, 10.82%; Zn, 8.96%.

Example 11: Synthesis of compound DS1836:

1 g of compound DS1491 was added to 10 ml of anhydrous ethanol. The system was heated to 50 °C, and 100 mg of potassium hydroxide was added. The mixture was stirred for 2 hours, cooled to 0 °C, and filtered. The resulting product was dried under reduced pressure to obtain DS1836, with a yield of 95% and a purity of 99.1%. ES I-MS (+): m/z = 617.9 (M+Na). Elemental analysis: Measured values: K, 6.65%; Se, 13.15%; Theoretical values: K, 6.58%; Se, 13.28%.

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

Referring to the synthesis methods of metal salts in this invention and the literature, the following compound examples were obtained:

Comparative Example 1: Synthesis of M41 and M42

The two pairs of proportioned compounds M41 and M42 were synthesized using the same synthetic methods as compounds DS1491 and DS1494.

Compound M43 was synthesized exactly according to the synthesis method in the literature (CN113226327B).

Example 12: Anti-influenza virus cell activity experiment

MDCK cells were seeded in 96-well plates and cultured in a 5% _CO2 incubator at 37°C. During the exponential growth phase, cell culture maintenance medium containing different dilutions of the sample and a positive control drug was added, with three replicates for each concentration, and normal cell control wells were also included. After 72 hours of culture following sample addition, the cytotoxicity of the samples was tested using the CPE method. Separately, MDCK cells were seeded in 96-well plates and cultured in a 5% _CO2 incubator at 37°C. After 24 hours of culture, the plates were infected with influenza virus (A/Hanfang/359/95(H3N2)). After 2 hours of virus adsorption, the virus solution was discarded, and cell culture maintenance medium containing different dilutions of the sample and a positive control drug was added, with three replicates for each concentration, and cell control wells and virus control wells were also included. The plates were then cultured in a 5% _CO2 incubator at 37°C. The antiviral assay of the test samples was performed using the CPE method. The degree of cytopathic effect (CPE) in each group was observed when the cytopathic effect (CPE) in the virus control group reached 4+. The Reed-Muench method was used to calculate the half-maximal toxic concentration (TC ₅₀ ) of the sample to cells and the effective drug concentration (EC ₅₀ ) that inhibited 50% cytopathic effect, as shown in Table 1.

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

Note: In Table 1, IAV represents the A/Hanfang/359/95(H3N2) virus strain.

Experiments show that the compounds of this invention possess excellent in vitro anti-influenza virus activity and a large therapeutic index (TC ₅₀ /EC ₅₀ ). Compared with comparative compounds M41 and baloxavir, compounds DS1491 to DS1493 (active ingredients) of this invention exhibit significantly superior in vitro anti-influenza virus activity and a larger therapeutic index. Compared with comparative compounds M42 and baloxavir ester, the prodrug compounds in this invention also possess superior in vitro anti-influenza virus activity and a larger therapeutic index.

Example 13: Activity against other influenza viruses

The antiviral efficacy of the compound against five influenza A virus (IAV) strains—A/WSN/33 (H1N1), A/PR/8/34 (H1N1)(PR8), A/Weiss/43 (H1N1) oseltamivir-resistant strain, and influenza B virus strains—B/Beijing-Haidian/1386/2013 (BV) and B/Massachusetts/2/2012 (BY-3)—was determined using the following method. The specific experimental method was as follows: MDCK cells were seeded into 24-well plates, with 1 × ^10⁵ cells per well. After seeding, the cells were incubated in a 5% _CO₂ incubator at 37°C. After 24 hours of cell culture, serially diluted test compounds were added to each well. One hour after compound addition, the cells were infected with different strains of influenza virus. Normal cell control wells and virus control wells were also included. The antiviral assay was performed on the test samples using the CPE method. The degree of cytopathic effect (CPE) in each group was observed when the CPE in the viral control group reached 4+. The _EC50 of each sample was calculated using the Reed-Muench method. The experiment was repeated three times. The results are shown in Table 2.

Table 2: Inhibitory activity against other influenza viruses

Note: A/WSN/33 = A/WSN/33 (H1N1); PR8 = A/PuertoRico/8/1934 (H1N1); OSE-R = A/Weiss/43 (H1N1) oseltamivir-resistant strain; HD = B/Beijing-Haidian/1386/2013 (BV); BY-3 = BY/Massachusetts/02/2012

Experiments show that the compounds of this invention have good activity against oseltamivir-resistant strains and influenza B virus. Compared with the comparative compound M42, the activity of the compounds of this invention is significantly improved, and even more significantly higher than that of Ribavirin; the inhibitory activity against influenza B virus of this invention is also significantly better than that of the comparative compound M43.

Example 14: Viral titer test and histopathological examination

Fifteen female C57BL/6J mice (8 weeks old, 18-20g) were randomly divided into five groups (Group A, Group B, Group C, Group D, and Group E), with three mice in each group. After intranasal inoculation with a median lethal dose of A/PR/8/34 (H1N1) virus, groups A, B, and C were administered the test drug (Group A: DS1494; Group B: DS1833; Group C: M42, all administered via gavage in 1% methylcellulose solution, at a dose of 2 mg/kg) respectively. Group D was administered the same volume of 1% methylcellulose solution orally via gavage. Group E received an intraperitoneal injection of the test drug DS1835 (1.4 mg/kg, dissolved in physiological saline). All groups received the drug once daily. Lung tissue samples were collected 5 days post-infection. Pathological examination of the left lung was performed, and _TCID50 determination was performed on the right lung homogenate. The viral titer results are shown in Figure 1.

The results of the lung virus titer test showed that the lung tissue virus titers of the other groups were lower than those of the solvent group; and compared with the comparative compound M42, the mouse lung tissue virus titers of the compounds DS1494 and DS1833 of the present invention were lower at the same dose, showing better anti-influenza virus effects; DS1835, administered intraperitoneally at a relatively lower dose, showed the lowest lung tissue virus titer.

Pathological examination of lung tissue showed that: the lung tissue structure of group D (model) mice was significantly damaged, with blurred alveolar structure, localized consolidation, and epithelial cell degeneration and necrosis; the bronchial structure of the lung tissue of group E (intraperitoneal injection of DS1835) mice was clear, and the epithelial cells were intact and neatly arranged; the bronchial structure of the lung tissue of group A (gavage with DS1494), group B (gavage with DS1833), and group C (gavage with M42) mice was clear, the epithelial cells were neatly arranged, and a small number of inflammatory cells infiltrated; a small number of epithelial cells were necrotic in the lung tissue of group C mice; compared with the comparative compound M42, the lung lesions of mice treated with the compound of the present invention were milder, and the compound of the present invention can improve the pathological damage of lung tissue caused by influenza virus.

The above results demonstrate that the compound of this invention has a better anti-influenza virus effect.

Example 15: Mouse Tissue Distribution Assay

Eighteen female DBA/1J mice (6-8 weeks old) were used. Mice were weighed the day before administration to calculate the dosage. They were randomly divided into two groups of nine mice each, and administered DS1494 and M42 (both 2 mg/kg, suspended in 1% methylcellulose solution) orally by gavage. Samples were taken from the mice at three time points: 2 hours, 4 hours, and 8 hours (3 mice at each time point). Mice were euthanized by _CO2 at each time point. Organs (lung, liver, kidney, spleen, and heart) were collected from the euthanized mice. After washing away blood with physiological saline, the organs were blotted dry with filter paper, weighed, and placed in glass homogenization tubes. Three times the volume of physiological saline was added for homogenization to obtain tissue homogenates. The homogenates were stored at -80°C. After processing, the biological samples were analyzed by LC-MS/MS (DS1494 group was analyzed for DS1491, M42 group was analyzed for M41).

The results showed that two hours after administration, both compounds reached their highest concentrations in the aforementioned tissues. In lung tissue, the concentration of DS1491 in the DS1494 group was higher than that of M41 in the M42 group. Furthermore, at 4 and 8 hours after administration, the rate of decrease in DS1491 concentration in the DS1494 group was significantly slower than that of M41 concentration in the M42 group; by 8 hours after administration, the concentration of DS1491 in the DS1494 group was approximately four times that of M41 in the M42 group.

Example 16: A Comparative Study of Crab-Eating Macaques

Twenty-four cynomolgus macaques (average grade, half male and half female), aged 3-6 years and weighing 2.5-4 kg, were randomly divided into eight groups: A1, B1, C1, D1, A2, B2, C2, and D2. Groups A1, B1, C1, and D1 received oral administration (po), with fasting but water allowed overnight before administration. Groups A2, B2, C2, and D2 received intravenous administration (iv), with fasting but water allowed overnight before administration.

Oral samples were prepared into suspensions using 0.5% CMC-Na aqueous solution, with a dosage of 2 mg/kg; group A1 received M42, group B1 received DS1494, group C1 received DS1833, and group D1 received DS1837. Intravenous samples were prepared into solutions using a mixed solvent (5% DMSO + 35% PEG400 + 60% water for injection), with a dosage of 0.5 mg/kg; group A2 received M42, group B2 received DS1494, group C2 received DS1833, and group D2 received DS1837.

For the oral gavage group, blood samples were collected via femoral vein before administration (0h), and at 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, and 24h after administration. For the intravenous injection group, blood samples were collected via femoral vein before administration (0h), and at 0.033h, 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, and 24h after administration. Approximately 1.5mL of blood collected each time was placed in a tube containing heparin anticoagulant and centrifuged at 2–8°C for 10 minutes. The target analytes in the plasma were detected by LC/MS/MS (compound M41 was detected in the M42 and DS1837 groups, and compound DS1491 was detected in the DS1833 and DS1494 groups). Pharmacokinetic parameters were calculated using DAS based on the blood drug concentration data at different time points, and the results are shown in Table 3.

Table 3: Comparison Parameters of Crab-Eating Mammals

PK parameters unit M42 DS1494 DS1833 DS1837 Tmax* h 3 2 2 3 MAT h 6.45 5.11 4.58 4.96

*Data for oral gavage.

The results showed that the compounds of this invention had shorter peak time (Tmax) and mean absorption time (MAT) (MAT = MRT(po) - MRT(iv)), indicating rapid absorption after administration compared to the control group M42. Other compounds in this invention also exhibited this characteristic.

This application describes several embodiments, but these descriptions are exemplary and not restrictive. There are many more embodiments and implementations that can be found within the scope of the embodiments described in this application.

Claims (7)

1. A compound, as shown in formula (I), or an isotopic derivative thereof, or a pharmaceutically acceptable salt thereof: In formula (I), _Ra is selected from hydrogen, deuterium, and methyl; Both _Rb and _Rc are hydrogen; R is hydrogen, where, _X1 represents an O atom; n1 is 1; Both _R1 and _R2 are hydrogen; R ₃ is selected from the following groups: C1-C8 alkoxy groups, in which one or more hydrogen atoms are substituted with or not substituted with deuterium. 2. A compound, as shown in formula (II), or an isotopic derivative thereof, or a pharmaceutically acceptable salt thereof: The substituents in formula (II) are defined as defined in claim 1. 3. The compound according to any one of claims 1-2, selected from the following structures: Or its isotope derivatives or pharmaceutically acceptable salts. 4. The compound according to any one of claims 1-2, selected from the following structures: Or its isotope derivatives. 5. A pharmaceutical composition comprising the compound or isotopic derivative thereof as described in any one of claims 1-3, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier. 6. The pharmaceutical composition of claim 5, wherein the dosage form of the pharmaceutical composition is a tablet, capsule, powder, granule, pill, suspension, syrup, injection, or inhalation preparation. 7. Use of the compound of any one of claims 1-3, or its isotopic derivatives, pharmaceutically acceptable salts, or the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for treating influenza virus.