WO2022105669A1 - Se-se-containing macrocyclic compound - Google Patents

Abstract

Se-containing macrocyclic compounds and application thereof. Specifically disclosed are a compound as shown in formula (II) and a pharmaceutically acceptable salt thereof.

Description

Se-containing macrocycles

The present invention claims the following priority:

CN202011288166.X, filing date Nov. 17, 2020.

technical field

The present invention relates to a class of Se-containing macrocyclic compounds and applications thereof, in particular to compounds represented by formula (II) and pharmaceutically acceptable salts thereof.

Background technique

Influenza virus, namely influenza virus (IFV), is a segmented single-stranded antisense RNA virus that can cause influenza in humans and animals. The synthesis of influenza virus proteins utilizes the host cell translation mechanism, and even the virus can suspend the translation of host proteins and speed up the synthesis of its own proteins. The polyadenylation of host cell mRNA is completed by a specific adenylase, unlike the adenylate tail of viral mRNA, which is formed by the transcription of 5-7 consecutive uracils on the negative-strand vRNA. of. The capping of viral individual messenger RNAs (mRNAs) is accomplished in a similar manner: PA and PB2 proteins grab the 5' capping primer of the host pre-mRNA transcript and in turn initiate viral mRNA synthesis, a process known as "cap" snatching". After the polyadenylation process and capping process are completed, the mRNA of the virus exits the nucleus, enters the cytoplasm, and is translated like the mRNA of the host cell. The nuclear export of the viral vRNA fragment is mediated by the M1 and NS2 proteins of the virus. As a result, when M1 protein can interact with vRNA and NP protein, it also interacts with nuclear export protein NS2; thus, nuclear export protein NS2 mediates the export of M1-RNP as a nuclear protein into the cytoplasm of host cells.

Current flu treatment options include vaccination and chemotherapy and chemoprophylaxis with antiviral drugs. Influenza vaccine against influenza is often recommended for high-risk groups, such as children and the elderly, or people with asthma, diabetes, or heart disease, but even vaccination does not completely prevent the flu. Vaccines for some specific influenza strains are recreated each season, but it is impossible to cover the various strains that actively infect people globally during that season. In addition, because influenza viruses undergo a certain degree of antigenic drift, if more than one virus infects a single cell, the 8 separate vRNA segments in the genome mix or reassort, resulting in rapid changes in viral genetics that can produce The antigenic shift enables the virus to infect new host species and rapidly overcome protective immunity. Wherein WO2016175224 reported the following compounds and their prodrugs:

In the field of anti-influenza virus, anti-influenza virus drugs with a new mechanism of action are urgently needed in clinical practice.

SUMMARY OF THE INVENTION

The present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

in,

R ₁ and R ₂ are each independently selected from H and C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _a ;

Alternatively, R ₁ and R ₂ are attached to the carbon atom to which they are attached to form a C _3-5 cycloalkyl or 3-5 membered heterocycloalkyl;

R ₃ is selected from H and

each R ₄ is independently selected from H, F, Cl, Br, I, OH and NH ₂ ;

R ₅ is selected from C _1-3 alkyl and

The C _1-3 alkyl and

each independently optionally substituted with 1, ₂ or 3 R;

each R _a is independently selected from F, Cl, Br, I and OH;

each R _b is independently selected from F, Cl, Br, I and OH;

n is selected from 0, 1, 2, 3 and 4;

The "3-5 membered heterocycloalkyl" contains 1 or 2 heteroatoms independently selected from O, S, N and NH.

In some aspects of the present invention, the above R ₁ and R ₂ are selected from H, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ and R ₂ are attached to the carbon atoms to which they are attached to form a cyclopropyl, cyclobutyl, oxetanyl or azetidinyl group, other variables being as defined in the present invention definition.

_In some embodiments _of the present invention, the above R1 and R2 are attached to the carbon atom to which they are attached to form a cyclobutyl group, other variables are as defined herein.

In some aspects of the invention, the above R ₅ is selected from CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

The CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

Optionally substituted with 1, 2 or 3 R _b , other variables are as defined in the present invention.

In some aspects of the invention, the above R ₅ is selected from CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

Other variables are as defined in the present invention.

In some aspects of the present invention, above-mentioned R ₃ is selected from H,

Other variables are as defined in the present invention.

In some aspects of the invention, _R above is selected from H and

Other variables are as defined in the present invention.

In some aspects of the present invention, each of the above R ₄ is independently selected from F, and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned structural units

selected from

Other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned structural units

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

wherein, R ₁ , R ₂ and R ₃ are as defined in the present invention;

The carbon atoms with "*" are chiral carbon atoms, which exist as (R) or (S) single enantiomer or enriched in one enantiomer.

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

in,

R ₁ and R ₂ are each independently selected from H;

Alternatively, R ₁ and R ₂ are attached to the carbon atom to which they are attached to form a C _3-5 cycloalkyl or 3-5 membered heterocycloalkyl;

The "3-5 membered heterocycloalkyl" contains 1 or 2 heteroatoms independently selected from O and NH.

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

wherein, R ₁ and R ₂ are as defined in the present invention;

The carbon atoms with "*" are chiral carbon atoms, which exist as (R) or (S) single enantiomer or enriched in one enantiomer.

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

wherein, R ₁ and R ₂ are as defined in the present invention;

The carbon atoms with "*" are chiral carbon atoms, which exist as (R) or (S) single enantiomer or enriched in one enantiomer.

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

wherein, R ₁ and R ₂ are as defined in the present invention.

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in,

R ₁ and R ₂ are selected from H;

Alternatively, R ₁ and R ₂ are attached to the carbon atoms to which they are attached to form C _3-5 cycloalkylcyclobutyl or 3-5 membered heterocycloalkyl;

The "3-5 membered heterocycloalkyl" contains 1 or 2 heteroatoms independently selected from O and NH.

There are also some solutions of the present invention, which are obtained by any combination of the above variables.

The present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof

In some aspects of the invention, the above-mentioned compound, or a pharmaceutically acceptable salt thereof, is selected from,

In some aspects of the present invention, the above-mentioned compounds or their pharmaceutically acceptable salts are used in the preparation of medicines for the treatment of influenza virus-related diseases.

The present invention also provides a method for testing the stability of liver microsomes:

Experimental purpose: To test the stability of compound liver microsomes

experimental method:

1. Preparation of test compounds and working solutions

Dilute 5 microliters of test article stock solution (10 mM DMSO solution) with 495 microliters of acetonitrile (intermediate working solution concentration: 100 μM, 99% acetonitrile).

2 materials

NADPH powder: NADPH·4Na, β-nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt, supplier: Chem-Impex International, Cat.No.00616.

HLM: Human liver microsomes, supplier: Corning, Cat No. 452117, Lot No. 38295.

3. Preparation of working solution for NADPH regeneration system

An appropriate amount of NADPH powder was weighed and diluted into a 10 mM magnesium chloride solution (concentration of working solution: 10 mM; final concentration of reaction system: 1 mM).

4. Preparation of Liver Microsomes

The liver microsome solution was diluted to 1 mg/mL with 100 mM potassium phosphate buffer.

5. Quenching Solution Preparation

Cold (4°C) acetonitrile containing 200 ng/mL tolbutamide and 200 ng/mL labetalol as internal standard (IS) was used as a quench solution.

6. Test procedure

1) Preheat empty "incubation" plates T60 and NCF60 for 10 minutes;

2) Dilute liver microsomes to 0.56 mg/mL with 100 mM phosphate buffer;

3) Transfer 445 μL of microsomal working solution (0.56 mg/mL) into pre-warmed “incubation” plates T60 and NCF60, then pre-incubate “incubation” plates T60 and NCF60 for 10 minutes at 37°C with constant shaking. Transfer 54 μL of liver microsomes to a blank plate, then add 6 μL of NAPDH cofactor to the blank plate, and then add 180 μL of quenching solution to the blank plate;

4) Add 5 μL of test compound in dimethyl sulfoxide (100 μM) to the “incubation” plate (T60 and NCF60) containing microsomes and mix thoroughly 3 times;

5) For NCF60 plate, add 50 μL of buffer and mix well 3 times. Start the timer; the plate will be shaken at 37°C for 60 minutes;

6) In "quenched" plate T0, add 180 μL of quenching solution and 6 μL of NAPDH cofactor. Make sure to cool the plate to prevent evaporation;

7) For T60 plates, mix thoroughly 3 times and immediately transfer 54 μL of the mixture to the "quench" plate at the 0 minute time point. 44 μL of NAPDH cofactor was then added to the plate (T60). Start the timer; the plate will be shaken at 37°C for 60 minutes;

8) At 5, 15, 30, 45 and 60 min, 180 μL of quench solution was added to the “quenched” plate, mixed once, and 60 μL of the sample was continuously transferred from the T60 plate to the “quenched” plate at each time point;

9) For NCF60: Mix once and transfer 60 μL of sample from the NCF60 dish to the "quench" plate containing the quench solution at the 60 minute time point;

10) Shake all sampling plates for 10 minutes, then centrifuge at 4000rpm for 20 minutes at 4°C;

11) Transfer 80 μL of the supernatant into 240 μL of high performance liquid chromatograph water, and stir with a plate shaker for 10 minutes;

12) Seal and shake each bioassay plate for 10 minutes prior to LC-MS/MS analysis.

technical effect

The compound of the present invention shows a positive effect in the test of inhibiting the replication of influenza virus at the cellular level, and has excellent pharmacokinetic properties, excellent metabolic stability and good liver microsome stability, and exhibits excellent in vivo pharmacodynamic model in animals weight protection.

Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.

In addition to salt forms, the compounds provided herein also exist in prodrug forms. Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the present invention. Furthermore, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise indicated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.

Unless otherwise specified, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a stereocenter, using a straight solid key

and straight dashed keys

Indicate the relative configuration of the stereocenter, with a wavy line

Represents a solid wedge key

or wedge-dotted key

or with wavy lines

Represents a straight solid key

or straight dashed key

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, and the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

The terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. .

The term "substituted" means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically achievable basis.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may optionally be substituted with up to two Rs, with independent options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, when a group has one or more attachable sites, any one or more sites in the group can be linked to other groups by chemical bonds. When the connection method of the chemical bond is not located, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of the connected chemical bond. the group. The chemical bond connecting the site to other groups can be represented by straight solid line bonds

straight dotted key

or wavy lines

express. For example, a straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in this group;

The straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;

The wavy line in the phenyl group indicates that it is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;

Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These 4 connection methods, even if the H atom is drawn on -N-, but

still includes

The group in this connection method is only when one chemical bond is connected, the H at the site will be correspondingly reduced by one to become the corresponding monovalent piperidinyl group.

Unless otherwise specified, when there is a double bond structure in the compound, such as carbon-carbon double bond, carbon-nitrogen double bond and nitrogen-nitrogen double bond, and each atom on the double bond has two different substituents attached (including nitrogen atom) In the double bond of the compound, a lone pair of electrons on the nitrogen atom is regarded as a substituent for its connection), if the atom on the double bond in the compound and its substituent are

represents the (Z) isomer, (E) isomer or a mixture of both isomers of the compound.

Unless otherwise specified, when there is a double bond structure in the compound, such as carbon-carbon double bond, carbon-nitrogen double bond and nitrogen-nitrogen double bond, and each atom on the double bond has two different substituents attached (including nitrogen atom) In the double bond, a lone pair of electrons on the nitrogen atom is regarded as a substituent for its connection), if a wavy line is used between the atom on the double bond and its substituent in the compound

If connected, it means the (Z) isomer, (E) isomer or a mixture of both isomers of the compound. For example, the following formula (A) indicates that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2) The following formula (B) indicates that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or exists in two forms of formula (B-1) and formula (B-2) exists as a mixture of isomers. The following formula (C) represents that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of two isomers of formula (C-1) and formula (C-2) exists in the form of a mixture.

Unless otherwise indicated, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in one enantiomer" refer to one of the isomers or pairs The enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).

Unless otherwise specified, the term "C _1-3 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (eg methyl), divalent (eg methylene) or multivalent (eg methine) . Examples of _C1-3 alkyl groups include, but are not limited to, _methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, "C _3-5 cycloalkyl" means a saturated cyclic hydrocarbon group consisting of 3 to 5 carbon atoms, which is a monocyclic ring system, said C _3-5 cycloalkyl including C _{3 -4} and C _4-5 cycloalkyl, etc.; it may be monovalent, divalent or polyvalent. Examples of _C3-5 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and the like.

Unless otherwise specified, the term "3-5 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated monocyclic group consisting of 3 to 5 ring atoms, 1, 2, 3 or 4 ring atoms, respectively are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, wherein the nitrogen atom is optionally quaternized and the nitrogen atom is optionally oxidized (ie, NO). Furthermore, with respect to the "3-5 membered heterocycloalkyl", a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule. The 3-5 membered heterocycloalkyl includes 4-5 membered, 4 membered, and 5 membered heterocycloalkyl and the like. Examples of 3-5 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.) or tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.) and the like.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, a nucleophilic substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.

The present invention adopts the following abbreviations: DMAC: N,N-dimethylacetamide, PG: propylene glycol, HP-β-CD: hydroxypropyl-β-cyclodextrin.

The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffractometry (SXRD), using Bruker D8 venture diffractometer to collect the diffraction intensity data of the cultivated single crystal, the light source is CuKα radiation, scanning mode: φ/ω scanning, after collecting the relevant data, further adopt the direct method (Shelxs97) analysis of the crystal structure, the absolute configuration can be confirmed.

Compounds are named according to conventional nomenclature in the art or are used

Software naming, commercially available compounds use supplier catalog names.

Detailed ways

The present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention. will be obvious.

Reference Example 1: Synthesis of Fragment BB-1

synthetic route:

Step 1: Synthesis of Compound BB-1-2

Metal magnesium (8.4g, 345.61mmol) and iodine (432.40mg, 1.70mmol) were added to the three-necked flask, the reaction flask was heated to fill the entire reaction flask with iodine vapor, and methanol (60mL) and toluene (200mL) were added to the reaction flask , then warmed to 80°C until the magnesium turnings were dissolved. The reaction solution was distilled under reduced pressure to remove methanol, toluene (200 mL) was added, and then BB-1-1 (30 g, 170.36 mmol) was added, and the reaction solution was stirred at 110° C. for 12 hours. The reaction solution was quenched by adding hydrochloric acid solution (10%, 200 mL), and extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, the crude product obtained was stirred with petroleum ether (100 mL) for 10 minutes, filtered, and the filter cake was dried under reduced pressure to obtain BB-1 -2 was used directly in the next reaction.

Step 2: Synthesis of Compound BB-1-3

Under nitrogen protection, tetramethylethylenediamine (37.06 g, 318.93 mmol) was dissolved in tetrahydrofuran (200 mL), sec-butyllithium (1.4 M, 227.81 mL, 318.93 mmol) was added dropwise at -60 °C, then A solution of BB-1-2 (20 g, 106.31 mmol) in tetrahydrofuran (200 mL) was slowly added dropwise, and the reaction solution was stirred at -60° C. for 2 hours. Iodomethane (61.38 g, 432.44 mmol, 26.92 mL) was added, the reaction solution was slowly heated to 20° C., and stirring was continued for 2 hours. Water (200 mL) and aqueous sodium hydroxide solution (1N, 100 mL) were added to the reaction solution respectively, extracted with methyl tert-butyl ether (200 mL×2), the organic phases were combined, and 1N aqueous sodium hydroxide solution (100 mL×2) was used for extraction. Wash, combine the alkaline aqueous phases extracted twice, adjust the pH to 3-4 with 6N hydrochloric acid, extract with ethyl acetate (100 mL×3), combine the organic phases, wash with saturated brine (100 mL×2), anhydrous Dry over sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain crude product BB-1-3, which is directly used in the next reaction.

Step 3: Synthesis of Compound BB-1-4

Under an ice bath, BB-1-3 (5 g, 24.73 mmol) was dissolved in dichloromethane (50 mL), oxalyl chloride (3.77 g, 29.68 mmol, 2.60 mL) and a catalytic amount of N,N-dichloromethane were added dropwise Methylformamide (90.39 mg, 1.24 mmol), the reaction solution was stirred at 20°C for 2 hours, the reaction solution was concentrated under reduced pressure, the obtained crude product was dissolved in ethanol (50 mL), and then stirred at 20°C for 1 hour. The reaction solution was concentrated to dryness under reduced pressure. The obtained crude product was purified by silica gel column (petroleum ether:ethyl acetate=100:1 to 50:1) to obtain compound BB-1-4. ¹ HNMR (400MHz, deuterated chloroform) δppm 6.23-6.58 (m, 1H), 4.39 (q, J=7.06Hz, 2H), 3.79 (s, 3H), 2.25 (d, J=2.4Hz, 3H) , 1.23 (t, J=7.20Hz, 3H).

Step 4: Synthesis of Compound BB-1-5

BB-1-4 (3.5 g, 15.20 mmol) was dissolved in carbon tetrachloride (50 mL), azobisisobutyronitrile (124.83 mg, 760.18 μmol) and N-bromosuccinimide (2.98 g) were added , 16.72 mmol), the reaction solution was stirred at 80 °C overnight. The reaction solution was diluted with water (50 mL), and extracted with dichloromethane (100 mL×2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product BB-1-5, which was directly used in the next reaction. ¹ HNMR (400MHz, deuterated chloroform) δppm 6.74-6.80 (m, 1H), 4.51 (s, 2H), 4.41-4.46 (m, 2H), 3.82 (s, 3H), 1.39-1.46 (m, 3H) .

Step 5: Synthesis of Compound BB-1-6

BB-1-5 (4.2 g, 10.87 mmol, 80% purity) was dissolved in a mixed solvent of acetonitrile (60 mL) and water (20 mL), zinc powder (1.42 g, 21.74 mmol), sodium dihydrogen phosphate (6.52 mL) were added. g, 54.35 mmol), diphenyl diselenide (2.04 g, 6.52 mmol), the reaction solution was stirred at 25°C for 2 hours. The reaction solution was diluted with water (50 mL), and extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (petroleum ether:ethyl acetate=50:1 to 20:1) to obtain BB-1-6. ¹ H NMR (400MHz, deuterated dimethyl sulfoxide) δppm 7.46-7.49 (m, 2H), 7.30-7.31 (m, 3H), 7.18-7.29 (m, 1H), 4.17-4.27 (m, 2H), 4.14 (s, 2H), 3.77 (s, 3H), 1.25 (q, J=7.20 Hz, 3H).

Step 6: Synthesis of Compound BB-1-7

BB-1-6 (3.6 g, 9.34 mmol) was dissolved in ethanol (50 mL), sodium hydroxide aqueous solution (2 M, 25.05 mL, 50.1 mmol) was added, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure, diluted with water (20 mL), adjusted to pH=2-3 with 1N hydrochloric acid, extracted with ethyl acetate (50 mL×3), combined with organic phases, washed with saturated brine (100 mL), and washed with anhydrous sulfuric acid. Dry over sodium, filter, and concentrate the filtrate to obtain crude BB-1-7, which is directly used in the next reaction. MS (ESI) m/z: 357.0 (MH) ⁺ .

Step 7: Synthesis of Compound BB-1-8

BB-1-7 (0.05 g, 139.97 μmol) was dissolved in 1,2-dichlorobenzene (1 mL), polyphosphoric acid (1 mL) was added, and the reaction solution was stirred at 130° C. for 2 hours. The reaction solution was poured into ice water (10 mL), and extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by preparative thin layer chromatography (petroleum ether:ethyl acetate=10:1) to obtain BB-1-8. ¹ HNMR (400MHz, deuterated chloroform) δppm 7.89-7.91(m,1H), 7.30-7.36(m,3H), 6.60-6.65(m,1H), 4.07(s,2H), 3.74(s,3H) .

Step 8: Synthesis of Compound BB-1-9

Under ice bath, BB-1-8 (0.05g, 147.41μmol) was dissolved in dichloromethane (1mL), and a solution of boron tribromide (92.32mg, 368.52μmol) in dichloromethane (1mL) was added dropwise, The reaction solution was stirred at 25°C for 2 hours. The reaction solution was poured into ice water (10 mL) to quench, and extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product BB-1-9, which was directly used in the next reaction. ¹ HNMR (400MHz, deuterated chloroform) δppm 12.79(s, 1H), 8.07-8.10(m, 1H), 7.62-7.65(m, 1H), 7.30-7.40(m, 1H), 6.72-6.76(m, 1H), 4.11 (s, 2H).

Step 9: Synthesis of Compound BB-1-10

Under an ice bath, BB-1-9 (0.38 g, 1.17 mmol) was dissolved in acetonitrile (6 mL), potassium carbonate (484.55 mg, 3.51 mmol) and allyl bromide (424.14 mg, 3.51 mmol) were added, and the reaction solution Stir at 50°C for 12 hours. The reaction solution was diluted with water (10 mL), and extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to crude BB-1-10, which was directly used in the next reaction. MS (ESI) m/z: 367.0 (M+H) ⁺ .

Step 10: Synthesis of Compound BB-1-11

Under ice bath, BB-1-10 (0.05 g, 136.90 μmol) was dissolved in a mixed solvent of dichloromethane (1 mL) and methanol (0.5 mL), and sodium borohydride (15.54 mg, 410.70 μmol) was added to react. The solution was stirred at 25°C for 12 hours, additional sodium borohydride (15.54 mg, 410.70 μmol) was added, and the reaction solution was further stirred at 25°C for 2 hours. The reaction solution was quenched with saturated aqueous ammonium chloride solution (10 mL), and then extracted with dichloromethane (20 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by column silica gel column (petroleum ether:ethyl acetate=20:1 to 10:1) to obtain BB-1-11. ¹ HNMR (400MHz, deuterated chloroform) δppm 7.49-7.51 (m, 1H), 7.10-7.23 (m, 3H), 6.60-6.63 (m, 1H), 6.28-6.30 (m, 1H), 6.05-6.10 ( m, 1H), 5.38-5.46 (m, 2H), 4.74-4.77 (m, 1H), 4.15-4.51 (m, 3H).

Step 11: Synthesis of Compound BB-1

Under ice bath, BB-1-11 (0.05g, 136.15μmol) was dissolved in dichloromethane (1mL), thionyl chloride (32.40mg, 272.30μmol) was added, and the reaction solution was heated to 25°C and continued to stir for 1 Hour. The reaction solution was concentrated under reduced pressure to obtain crude product BB-1, which was directly used in the next reaction.

Example 1

Step 1: Synthesis of Compounds 1-2

Under an ice bath, 1-1 (3 g, 8.32 mmol) was added to dichloromethane (30 mL), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate ( 3.80g, 9.99mmol) and N,N-diisopropylethylamine (2.15g, 16.65mmol, 2.90mL), the reaction solution was reacted at 25 ° C for 0.5 hour, and allylamine (0.85g, 14.89mmol) was added to the reaction solution , the reaction was continued at 25°C for 12 hours. Water (30 mL) was added to the reaction solution, and the solution was extracted with dichloromethane (50 mL×3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=50:1 to 20:1) to obtain compound 1-2. MS (ESI) m/z: 399.9 (M+H) ⁺ .

Step 2: Synthesis of Compounds 1-3

Compound 1-2 (0.9 g, 2.25 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (6.16 g, 54.03 mmol, 4 mL) was added, and the reaction solution was stirred at 25° C. for 3 hours. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of crude product 1-3, which was directly used in the next reaction.

Step 3: Synthesis of Compounds 1-4

The trifluoroacetate salt of compound 1-3 (0.9 g, crude product) was dissolved in ethanol (5 mL), sodium hydroxide aqueous solution (1 M, 2.61 mL) and formalin solution (353.39 mg, 4.35 mmol, 324.21 μL, 37%) were added purity), the reaction solution was stirred at 25°C for 20 minutes. The reaction solution was extracted with dichloromethane (30 mL×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=50:1 to 20:1) to obtain compound 1-4. MS (ESI) m/z: 312.0 (M+H) ⁺ .

Step 4: Synthesis of Compounds 1-5

Under ice bath, compound 1-4 (0.16 g, 513.92 μmol) was dissolved in N,N-dimethylformamide (3 mL), sodium hydrogen (61.66 mg, 1.54 mmol, 60% purity) was added, and the reaction was The solution was stirred at 0°C for 0.5 hours, a solution of BB-1 (0.23 g, 596.33 μmol) in N,N-dimethylformamide (2 mL) was added to the reaction solution, and the reaction solution was further stirred at 25°C for 1 hour. The reaction solution was poured into ice water (10 mL) to quench, extracted with ethyl acetate (30 mL×2), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=100:1 to 50:1) to obtain compound 1-5. MS (ESI) m/z: 662.1 (M+H) ⁺ .

Step 5: Synthesis of Compounds 1-6

Compound 1-5 (60 mg, 90.83 μmol) was dissolved in dichloroethane (8 mL), and [1,3-bis(2,4,6-trimethylphenyl)imidazolin-2-yl]- Dichloro-[(2-isopropoxyphenyl)methylene]ruthenium (11.38 mg, 18.17 μmol), and the reaction solution was stirred at 80° C. for 3 hours. The reaction solution was concentrated under reduced pressure. The obtained crude product was purified twice by preparative thin layer chromatography (dichloromethane:methanol=10:1) to obtain compound 1-6. MS (ESI) m/z: 634.1 (M+H) ⁺ .

Step 6: Synthesis of Compounds 1-6A, 1-6B, 1-6C and 1-6D

Compounds 1-6 were detected by supercritical fluid chromatography (column model: Chiralcel OJ-3 (100mm*4.6mm, 3μm); mobile phase: [A: carbon dioxide, B: 0.05% diethylamine/ethanol]; gradient: 5% ~40%(B%), 4min; 40%(B%), 2.5min, 5%(B%) 1.5min) is a mixture of 4 peaks, and the mixture is subjected to supercritical fluid chromatography (column model: DAICEL CHIRALCEL OJ- H (250mm*30mm, 5μm); mobile phase: A: carbon dioxide, B: [0.1% ammonia water/ethanol]; gradient: B%: 35%-35%) separated to obtain 4 isomers: 1-6A (retained time 3.300min, chiral purity=90.2%), 1-6B (retention time 3.459min, chiral purity=75.0%), 1-6C (retention time 3.781min, chiral purity=97.0%) and 1-6D ( Retention time 4.146 min, chiral purity=99.0%).

Step 7: Synthesis of Compound 1A

Compound 1-6A (20 mg, 31.62 μmol) was added to N,N-dimethylacetamide (0.5 mL), lithium chloride (13.40 mg, 316.20 μmol) was added, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was separated and purified by preparative high performance liquid phase (column model: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile]; gradient: B%: 38% -68%) to obtain compound 1A. ¹ H NMR (400MHz, deuterated methanol) δ 7.72 (d, J=7.53Hz, 1H), 7.22 (d, J=8.03Hz, 1H), 7.04-7.18 (m, 2H), 6.83-6.94 (m ,2H),6.29-6.42(m,1H),5.93-6.03(m,2H),5.60(dd,J=2.76,12.55Hz,1H),5.54(s,1H),5.22(d,J=14.05 Hz,1H),5.01(dd,J＝5.14,14.43Hz,1H),4.75-4.80(m,1H),4.54(d,J＝13.80Hz,1H),4.39(br t,J＝10.04Hz, 1H), 4.13 (d, J=12.55Hz, 1H), 3.16 (dd, J=7.65, 14.18Hz, 1H). MS (ESI) m/z: 544.0 (M+H) ⁺ .

Step 8: Synthesis of Compound 1B

Compound 1-6B (8 mg, 12.65 μmol) was added to N,N-dimethylacetamide (0.5 mL), lithium chloride (5.36 mg, 126.48 μmol) was added, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was separated and purified by preparative high performance liquid phase (column model: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile]; gradient: B%: 38% -68%) to obtain compound 1B. ¹ H NMR (400MHz, deuterated methanol) δ 7.61 (d, J=7.53Hz, 1H), 7.23-7.30 (m, 1H), 7.04-7.19 (m, 2H), 6.83-6.93 (m, 1H) ,6.69(d,J＝6.78Hz,1H),6.47-6.56(m,1H),6.26-6.39(m,2H),6.00-6.08(m,1H),5.64(dd,J＝2.89,12.92Hz ,1H),5.09(d,J＝13.55Hz,1H),4.75-4.80(m,1H),4.51-4.60(m,2H),4.41(dd,J＝7.53,13.80Hz,1H),4.01( d, J=12.80Hz, 1H), 3.77 (dd, J=8.53, 13.55Hz, 1H). MS (ESI) m/z: 544.0 (M+H) ⁺ .

Step 9: Synthesis of Compound 1C

Compound 1-6C (8 mg, 12.65 μmol) was added to N,N-dimethylacetamide (0.5 mL), then lithium chloride (5.36 mg, 126.48 μmol) was added, and the reaction solution was stirred at 80° C. for 12 hours . The reaction solution was separated and purified by preparative high performance liquid phase (column model: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile]; gradient: B%: 38% -68%) to obtain compound 1C. ¹ H NMR (400MHz, deuterated methanol) δ 7.63 (d, J=7.28Hz, 1H), 7.22-7.28 (m, 1H), 7.07-7.18 (m, 2H), 6.87 (t, J=6.90Hz) ,1H),6.70(d,J＝6.78Hz,1H),6.47-6.57(m,1H),6.27-6.40(m,2H),6.07(d,J＝7.53Hz,1H),5.65(dd, J=2.89, 12.92Hz, 1H), 5.09 (d, J=13.55Hz, 1H), 4.75-4.80 (m, 1H), 4.51-4.63 (m, 2H), 4.42 (dd, J=7.53, 13.80Hz) , 1H), 4.02 (d, J=12.80Hz, 1H), 3.78 (dd, J=8.78, 13.80Hz, 1H). MS (ESI) m/z: 544.0 (M+H) ⁺ .

Step 10: Synthesis of Compound 1D

Compound 1-6D (20 mg, 31.62 μmol) was added to N,N-dimethylacetamide (0.5 mL), lithium chloride (13.40 mg, 316.20 μmol) was added, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was separated and purified by preparative high performance liquid phase (column model: Boston Green ODS 150*30mm*5μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile]; gradient: B%: 38% -68%) to obtain compound 1D. ¹ H NMR (400MHz, deuterated methanol) δ 7.73 (d, J=7.53Hz, 1H), 7.22 (d, J=7.78Hz, 1H), 7.05-7.18 (m, 2H), 6.85-6.95 (m ,2H),6.35(td,J＝7.72,15.18Hz,1H),5.93-6.04(m,2H),5.60(dd,J＝2.51,12.80Hz,1H),5.54(s,1H),5.22( d, J＝14.05 Hz, 1H), 5.01 (dd, J＝4.77, 14.81 Hz, 1H), 4.75-4.80 (m, 1H), 4.54 (d, J＝13.80 Hz, 1H), 4.39 (br t, J=10.16Hz, 1H), 4.13 (d, J=12.55Hz, 1H), 3.16 (dd, J=7.65, 14.43Hz, 1H). MS (ESI) m/z: 544.0 (M+H) ⁺ .

Example 2

Step 1: Synthesis of Compound 2-2

Under an ice bath, compound 2-1 (10 g, 27.75 mmol) was dissolved in N,N-dimethylformamide (100 mL), and benzotriazole-N,N,N',N'-tetramethylene Biurea hexafluorophosphate (12.66g, 33.30mmol) and N,N-diisopropylethylamine (17.93g, 138.75mmol, 24.17mL) were added to the reaction solution, and the reaction solution was reacted at 25°C for 0.5 hours, and then 1-Vinylcyclobutylamine hydrochloride (4.45 g, 33.30 mmol) was added to the reaction solution, and the reaction solution was continued to react at 25° C. for 12 hours. The reaction solution was diluted with water (100 mL), and extracted with dichloromethane (100 mL×3). The organic phases were combined and washed with saturated brine (400 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=50:1 to 20:1) to obtain compound 2-2. MS (ESI) m/z: 440.1 (M+H) ⁺ .

Step 2: Synthesis of Compounds 2-3

Compound 2-2 (4 g, 9.10 mmol) was dissolved in dichloromethane 40 mL), trifluoroacetic acid (11.20 g, 98.20 mmol, 7.27 mL) was added, and the reaction solution was stirred at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of crude product 2-3, which was directly used in the next reaction. MS (ESI) m/z: 340.1 (M+H) ⁺ .

Step 3: Synthesis of Compounds 2-4

The trifluoroacetate salt of compound 2-3 (4 g, crude product) was dissolved in ethanol (40 mL), sodium hydroxide aqueous solution (3M, 3.53 mL) and formalin solution (859.22 mg, 10.59 mmol, 788.27 μL, 37% purity) were added ), the reaction solution was stirred at 25°C for 1 hour. The reaction solution was extracted with dichloromethane (50 mL×3), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=50:1 to 20:1) to obtain compound 2-4. MS (ESI) m/z: 352.1 (M+H) ⁺ .

Step 4: Synthesis of Compounds 2-5

Under ice bath, compound 2-4 (0.8 g, 2.28 mmol) was dissolved in acetonitrile (15 mL), potassium carbonate (943.92 mg, 6.83 mmol) and BB-1 (1.05 g, 2.73 mmol) were added, and the reaction solution was Stirring was continued for 12 hours at 25°C. The reaction solution was added to water (50 mL) to quench, extracted with ethyl acetate (50 mL×2), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=100:1 to 50:1) to obtain compound 2-5. MS (ESI) m/z: 702.1 (M+H) ⁺ .

Step 5: Synthesis of Compounds 2-6

Compound 2-5 (0.47 g, 670.82 μmol) was dissolved in dichloroethane (240 mL), and [1,3-bis(2,4,6-trimethylphenyl)imidazolin-2-yl] was added -Dichloro-[(2-isopropoxyphenyl)methylene]ruthenium (84.07 mg, 134.16 μmol), the reaction solution was stirred at 80° C. for 12 hours. Sulfur silica gel (500 mg) was added to the reaction solution, stirred for 1 hour, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (dichloromethane:methanol=100:1 to 50:1) to obtain compound 2-6. . MS (ESI) m/z: 674.1 (M+H) ⁺ .

Step 6: Synthesis of Compounds 2-6A and 2-6B

Compound 2-6 was detected by supercritical fluid chromatography (column model: Chiralcel OJ-3 (100mm*4.6mm, 3μm); mobile phase: [A: carbon dioxide, B: 0.05% diethylamine/ethanol]; gradient: 5% ~40%(B%), 4min; 40%(B%), 2.5min, 5%(B%) 1.5min) is a mixture of 2 peaks, and the mixture is subjected to supercritical fluid chromatography (column model: DAICEL CHIRALCEL OJ- H (250mm*30mm, 5μm), mobile phase: [A: carbon dioxide, B: 0.1% ammonia water/ethanol]; gradient: B%: 25%-25%) separated to obtain 2 isomers: compound 2-6A ( Retention time 3.295 min, chiral purity=100%) and compound 2-6B (retention time 3.931 min, chiral purity=99.3%).

Step 7: Synthesis of Compound 2A

Compound 2-6A (90 mg, 133.81 μmol) was added to dichloromethane (2 mL), magnesium chloride (63.70 mg, 669.07 μmol) was added, and the reaction solution was stirred at 25° C. for 12 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the obtained crude product was separated and purified by preparative high-performance liquid phase (column model: Welch Xtimate C18 100*40mm*3 μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile) ]; gradient: B%: 58%-88%) to obtain compound 2A. ¹ H NMR (400MHz, deuterated methanol) δ 7.78 (d, J=7.53Hz, 1H), 7.14-7.25 (m, 2H), 7.06 (t, J=6.90Hz, 1H), 6.85-6.94 (m ,1H),6.78-6.84(m,1H),6.48(br d,J＝15.81Hz,1H),6.23-6.33(m,1H),5.93(d,J＝7.53Hz,1H),5.67(dd ,J=2.38,12.92Hz,1H),5.55(s,1H),5.17(d,J=13.80Hz,1H),4.75-4.81(m,1H),4.62(br dd,J=7.03,11.04Hz ,1H),4.19(d,J＝13.80Hz,1H),4.11(d,J＝12.55Hz,1H),2.93-3.08(m,1H),2.53-2.64(m,1H),2.17-2.27( m, 1H), 2.07-2.16 (m, 1H), 1.81-1.91 (m, 1H), 1.69-1.80 (m, 1H). MS (ESI) m/z: 584.0 (M+H) ⁺ .

Step 8: Synthesis of Compound 2B

Compound 2-6B (90 mg, 133.81 μmol) was added to dichloromethane (1 mL), lithium chloride (127.41 mg, 1.34 mmol) was added, and the reaction solution was stirred at 25° C. for 12 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the obtained crude product was separated and purified by preparative high-performance liquid phase (column model: Welch Xtimate C18 100*40mm*3 μm; mobile phase: [A: water (0.075% trifluoroacetic acid)-B: acetonitrile) ]; gradient: B%: 45%-75%) to obtain compound 2B. ¹ H NMR (400MHz, deuterated methanol) δ 7.80 (br d, J=7.28Hz, 1H), 7.14-7.30 (m, 2H), 7.06 (br t, J=7.53Hz, 1H), 6.76-6.95 (m,2H),6.48(br d,J=15.81Hz,1H),6.23-6.37(m,1H),5.97(d,J=7.53Hz,1H),5.66(br d,J=14.56Hz, 1H), 5.56(s, 1H), 5.18(br d, J=13.80Hz, 1H), 4.78(br d, J=8.28Hz, 1H), 4.58-4.67(m, 1H), 4.18-4.24(m ,1H),4.11(br d,J＝12.55Hz,1H),2.95-3.08(m,1H),2.54-2.65(m,1H),2.09-2.25(m,2H),1.73-1.88(m, 2H). MS (ESI) m/z: 584.0 (M+H) ⁺ .

Example 3

Step 1: Synthesis of Compound 3

Compound 2B (180.00 mg, 309.04 μmol) was added to acetone (4 mL), then chloromethyl methyl carbonate (150.83 mg, 618.07 μmol) and cesium carbonate (503.45 mg, 1.55 mmol) were added, and the reaction solution was stirred at room temperature 1 hour. The reaction solution was concentrated to dryness under reduced pressure, and the obtained crude product was purified by silica gel column (dichloromethane:methanol=100:1 to 50:1) to obtain compound 3. ¹ H NMR (400MHz, deuterated dimethyl sulfoxide) δ 7.84 (br d, J=7.78Hz, 1H), 7.13-7.25 (m, 2H), 7.01-7.11 (m, 1H), 6.85-6.98 (m,2H),6.50(br d,J＝15.81Hz,1H),6.23-6.39(m,1H),5.99(br d,J＝7.53Hz,1H),5.63-5.81(m,3H), 5.53(s, 1H), 5.10(br d, J=13.80Hz, 1H), 4.71-4.80(m, 1H), 4.54-4.66(m, 1H), 4.10(br dd, J=9.66, 12.92Hz, 2H), 3.85(s, 3H), 2.79(q, J=10.54Hz, 1H), 2.57(br s, 1H), 2.07-2.20(m, 2H), 1.68-1.92(m, 2H); MS( ESI) m/z: 672.2 [M+H] ⁺ .

biological test data

Experimental Example 1: Influenza Virus Cytopathic (CPE) Experiment

The antiviral activity of the compounds against Influenza virus (IFV) was evaluated by determining the half effective concentration (EC ₅₀ ) value of the compounds. Cytopathic assays are widely used to measure the protective effect of compounds on virus-infected cells to reflect the antiviral activity of compounds.

Influenza virus CPE experiment

MDCK cells were seeded in black 384-well cell culture plates at a density of 2000 cells per well, and then placed in a 37°C, 5% CO ₂ incubator overnight. Compounds were diluted by Echo555 non-contact nanoliter sonic pipetting system and added to cell wells (4-fold dilution, 8 test concentration points). Influenza virus A/PR/8/34 (H1N1) strain was then added to cell culture wells at a 1-2 90% tissue culture infectious dose (TCID90) per well at a final concentration of 0.5% DMSO in the medium. Virus control wells (DMSO and virus added, no compound added), cell control wells (DMSO added, no compound and virus added) and medium control wells (medium only, no cells) were set up. The cytotoxicity assay and the antiviral activity assay of the compounds were performed in parallel, except that no virus was added, the other experimental conditions were the same as the antiviral activity experiments. Cell plates were placed in a 37°C, 5% _CO2 incubator for 5 days. After 5 days of culture, the cell viability was detected using the cell viability detection kit CCK8. Raw data were used for compound antiviral activity and cytotoxicity calculations.

The antiviral activity and cytotoxicity of the compounds were expressed by the inhibition rates (%) of the compounds against virus-induced cellular viral effects, respectively. Calculated as follows:

The inhibition rate and cytotoxicity of the compounds were analyzed by nonlinear fitting using GraphPad Prism software, and the EC ₅₀ values of the compounds were obtained. The experimental results are shown in Table 1.

Table 1 Inhibitory activity of compounds against influenza virus A/PR/8/34 (H1N1)

compound	EC50 (nM)
1C	7.5
1D	2.9
2B	1.9

Conclusion: The compounds of the present invention exhibited positive effects in the assay of inhibiting influenza virus replication at the cellular level.

Experimental Example 2: Pharmacokinetic Research Test in Mice

Experimental purpose: To investigate the plasma pharmacokinetics in male BALB/c mice after single intravenous injection and intragastric administration of the compounds of the present invention.

Experimental animals: male BALB/c mice, 7-9 weeks old, weighing 17-23 grams;

Experimental procedure: injection administration (i.v.): the dose is 1 mpk, the concentration is 0.50 mg/mL, and the vehicle is 40% DMAC+40% PG+20% (20% HP-β-CD in water); oral administration (po ): The dose is 10 mpk, the concentration is 1 mg/mL, and the vehicle is 3% DMSO+10% solutol HS+87% water.

Sample collection: After collecting blood from saphenous vein puncture, add dichlorvos to the plasma matrix as a stabilizer (plasma: dichlorvos solution = 40:1, dichlorvos solution is 40mM dichlorvos in acetonitrile: water = 1:1 solution), within half an hour, The supernatant plasma was collected by centrifugation at 3000g at 4°C for 10 minutes, quickly placed in dry ice, and stored in a -80°C refrigerator for LC-MS/MS analysis.

Data analysis: The non-compartmental model of the Phoenix WinNonlin 6.3 pharmacokinetic software was used to process the plasma concentration, and the linear logarithmic trapezoidal method was used to calculate the pharmacokinetic parameters Cl, T _1/2 , C _max , AUC _0-last , the results are shown in the table 2.

Table 2 PK results of compounds of the present invention

compound	Cl(mL/Kg/min)	T 1/2 (h)	Cmax (nM)	AUC 0-last (nM h)
Compound 2B (i.v.)	20	3.6	/	1386
Compound 3(po)	/	4.2	681	4379

Note: Cl: apparent clearance; T _1/2 : time required to clear half of the compound; C _max : peak concentration; AUC _0-last : 0-concentration integrated area within the last sampling time.

Experimental conclusion: The results of the mouse pharmacokinetic study show that the compound of the present invention has a low clearance rate and a high oral plasma exposure of its prodrug, and has good pharmacokinetic properties.

Experimental Example 3: Pharmacokinetic Research Test in Rats

Experimental purpose: To investigate the plasma pharmacokinetics in male SD rats after single intravenous injection and intragastric administration of the compounds of the present invention.

Experimental animals: male SD rats, 6-8 weeks old, weighing 200-300 grams;

Experimental procedure: injection administration (iv), the dose is 1mpk, the concentration is 0.50 mg/mL, the vehicle is 40% DMAC+40% PG+20% (20% HP-β-CD+H ₂ O); oral administration (po) at a dose of 10 mpk at a concentration of 1 mg/mL in a vehicle of 3% DMSO + 10% solutol HS + 87% _H2O ).

Sample collection: 0.03 mL of blood samples were collected from saphenous vein puncture of experimental animals at each time point, and the actual blood collection time was recorded. All blood samples were added into 1.5mL commercial EDTA-K2 anticoagulant tubes. After the blood sample was collected, DDV was added to the plasma matrix as a stabilizer (plasma: dichlorvos solution (40mM, acetonitrile: water = 1:1) was 40:1), and within half an hour, centrifuge at 4°C and 3000g for 10 minutes to absorb the supernatant. Plasma was immediately stored in dry ice and stored in a -80°C freezer for LC-MS/MS analysis.

Data analysis: The non-compartmental model of the Phoenix WinNonlin 6.3 pharmacokinetic software was used to process the plasma concentration, and the linear logarithmic trapezoidal method was used to calculate the pharmacokinetic parameters Cl, T _1/2 , C _max , AUC _0-last , the results are shown in the table 3.

Table 3 PK results of compounds of the present invention

compound	Cl(mL/Kg/min)	T 1/2 (h)	Cmax (nM)	AUC 0-last (nM h)
Compound 2B (i.v.)	12.9	5.7	/	1948
Compound 3(po)	/	4.3	490	4089

Note: Cl: apparent clearance rate; T _1/2 : time required to clear half of the compound; C _max : peak concentration; AUC _0-last : 0-concentration integrated area within the last sampling time.

Experimental conclusion: The results of pharmacokinetic study in rats show that the compound of the present invention has low clearance rate, high oral plasma exposure of its prodrug and good pharmacokinetic properties.

Experimental Example 4: In vivo efficacy study

Experimental purpose: To evaluate the efficacy of the compounds of the present invention in the influenza A virus H1N1 mouse infection model

Experimental protocol: Mice were infected with influenza A virus A/PR/8/34 (H1N1) by intranasal instillation, and were treated with compounds starting 48 hours after infection and administered orally for 7 consecutive days, twice a day. The anti-influenza A virus H1N1 effect of the compound in this model was evaluated by observing the changes in body weight and survival rate of mice.

BALB/c mice of SPF grade, 6-7 weeks old, female were selected for the experiment. The mice were acclimated for at least 3 days after arriving in the BSL-2 animal room to start the experiment. The day of infection was set as day 0 of the experiment. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (75 mg/kg, 10 mL/kg). After the animals entered deep anesthesia, they were infected with A/PR/8/34 (H1N1) virus by intranasal infusion, and the infection volume was 50 μL. From day 2 to day 8, the test compound was orally administered at 5 mg/kg (administration volume 10 mL/kg) twice a day. The first dose was 48 hours after infection. The state of the mice was observed every day, and the body weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.

Experimental results:

The animal survival rate and body weight loss rate were detected, and the calculation formula was as follows: body weight loss rate on day N=(body weight on day N-body weight on day 0)/body weight on day 0*100%. The results are shown in Table 4. Compound 3 can achieve a maximum weight loss rate of -9.22% in protected animals on the 4th day, and then begins to recover, and the survival rate of mice is 100% at the end of the experiment. The maximum weight loss rate of the animals in the vehicle group was over -35% on the 8th day, and all the animals died on the 9th day.

Table 4 Results of animal survival rate and weight loss rate

compound	Maximum weight loss rate (day N)	Survival rate (percentage)
vehicle group	Over -35% (Day 8)	0%
Compound 3	-9.22% (Day 4)	100%

Conclusion: The compounds of the present invention show excellent body weight protection and early recovery time in an animal pharmacodynamic model.

Claims (16)

1. A compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

   

   in,

   R ₁ and R ₂ are each independently selected from H and C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _a ;

   Alternatively, R ₁ and R ₂ are attached to the carbon atom to which they are attached to form a C _3-5 cycloalkyl or 3-5 membered heterocycloalkyl;

   R ₃ is selected from H and

   

   each R ₄ is independently selected from H, F, Cl, Br, I, OH and NH ₂ ;

   R ₅ is selected from C _1-3 alkyl and

   

   The C _1-3 alkyl and

   

   each independently optionally substituted with 1, ₂ or 3 R;

   each R _a is independently selected from F, Cl, Br, I and OH;

   each R _b is independently selected from F, Cl, Br, I and OH;

   n is selected from 0, 1, 2, 3 and 4;

   The "3-5 membered heterocycloalkyl" contains 1 or 2 heteroatoms independently selected from O, S, N and NH.
2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₁ and R ₂ are each independently selected from H.
3. The _compound of claim ₁ , or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are attached to the carbon atom to which they are attached to form a cyclopropyl, cyclobutyl, oxetanyl or azetidine alkyl.
4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₅ is selected from CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

   

   The CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

   

   Optionally substituted with 1, 2 or 3 R _b .
5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein R ₅ is selected from CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ and

   
6. The compound according to claim 1 or 5, or a pharmaceutically acceptable salt thereof, wherein _R is selected from H and

   
7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each R ₄ is independently selected from F.
8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

   
9. The compound according to claim 7 or 8 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

   
10. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, which is selected from,

    

    in,

    R ₁ , R ₂ and R ₃ are as defined in any one of claims 1 to 9;

    The carbon atoms with "*" are chiral carbon atoms, which exist as (R) or (S) single enantiomer or enriched in one enantiomer.
11. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from,

    

    in,

    R ₁ and R ₂ are each independently selected from H;

    Alternatively, R ₁ and R ₂ are attached to the carbon atom to which they are attached to form a C _3-5 cycloalkyl or 3-5 membered heterocycloalkyl;

    The "3-5 membered heterocycloalkyl" contains 1 or 2 heteroatoms independently selected from O and NH.
12. The compound according to claim 10 or 11, or a pharmaceutically acceptable salt thereof, selected from,

    

    in,

    R ₁ and R ₂ are as defined in claim 10 or 11;

    The carbon atoms with "*" are chiral carbon atoms and exist as (R) or (S) single enantiomer or enriched in one enantiomer.
13. The compound according to claim 10, or a pharmaceutically acceptable salt thereof, selected from,

    

    in,

    R ₁ and R ₂ are as defined in claim 10;

    The carbon atoms with "*" are chiral carbon atoms, which exist as (R) or (S) single enantiomer or enriched in one enantiomer.
14. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, selected from,

    

    

    in,

    R ₁ and R ₂ are as defined in claim 13 .
15. A compound of the formula or a pharmaceutically acceptable salt thereof,

    
16. The compound according to claim 15, or a pharmaceutically acceptable salt thereof, selected from,