Pyridone polycyclic fused ring system derivatives and uses thereof

Abstract

The present invention provides pyridone polycyclic fused ring system derivatives and uses thereof, specifically compounds represented by formula (VI) and pharma- ceutically acceptable salts thereof: JPEG2024502198000114.jpg4843

Description

[Technical field]

[0001] This invention claims priority to the following: CN202110024885.9, filing date: January 8, 2021; CN202110264686.5, filing date: March 11, 2021; CN202110513447.9, application date: May 11, 2021.

[0002] The present invention relates to pyridone polycyclic fused ring system derivatives and uses thereof, particularly to a compound represented by formula (VI) and pharma- ceutically acceptable salts thereof: [Background technology]

[0003] Pandemic influenza viruses, or influenza viruses (IFVs), are segmented, single-stranded, antisense RNA viruses that can cause influenza in humans and animals. Influenza viruses can cause very high morbidity and mortality, and influenza A viruses in particular can cause global pandemics, such as the 1918-1920 "Spanish Flu" (H1N1 subtype), the 1957-1958 "Asian Flu" (H2N2 subtype), the 1968-1969 "Asian Flu" (H3N2 subtype), the 1977-1978 "Hong Kong Flu" (H1N1 subtype), and the first influenza A (H1N1) outbreak in Mexico in March 2009. Influenza pandemics can cause thousands of deaths, widespread panic in society, and increase social instability.

[0004] Influenza A virus is a single-stranded negative-strand RNA virus, and its genome is divided into eight segments and encodes eight proteins. The 5' and 3' ends of the influenza virus genome segment are highly conserved, and the sequences at both ends form a complementary stem-loop structure, which plays an important role in the initiation of viral RNA replication. The size of the proteins encoded by each gene segment of the virus is different, and they play different roles in the life cycle of the influenza virus. The basic functions of some major proteins are introduced below. The HA of the influenza virus is a ligand for influenza virus to recognize the host receptor, binds to the virus-specific receptor on the cell surface, mediates the fusion of the viral outer membrane and the intracellular vesicle membrane, and releases the viral nucleocapsid into the cytoplasm. The receptor of the influenza virus has specificity, and the receptor of the influenza A virus is a sialic acid glycoprotein. The NA protein of the influenza virus can remove sialic acid on the surface of the virus particle during the replication process, which prevents the virus particle from continuing to aggregate on the surface of the host cell, thereby facilitating the release of virions and further infecting more host cells.

[0005] The role of M2 protein of influenza virus: HA protein of influenza virus binds to sialic acid, and influenza virus is endocytosed by host cells. The acidity and alkalinity of phagocytic vesicles play an important role in the uncoating of viruses, and the ion channel of M2 protein on the viral membrane can gradually reduce the pH value of phagocytic vesicles, and when the pH value drops to 5.0-6.0, there is a change in the conformation of HA2 protein, and the fusion peptide located at the amino terminus of HA2 protein shifts, thereby activating the fusion process, fusing the bilayer lipid membrane of the virus with the cell membrane, and releasing the RNP in the virus particle into the cytoplasm of the host cell. M2 protein is a transmembrane ion channel found only in influenza A virus, part of which extends to the surface of the outer membrane of the virus.

[0006] The synthesis of influenza virus proteins also utilizes the host cell translation machinery, and even viruses can pause the translation of host proteins to accelerate the synthesis of their own proteins. Unlike the polyadenylation of host cell mRNAs, which is achieved by specific adenylases, the adenylate tail of viral mRNAs is formed by the transcription of consecutive 5-7 uracils on the minus-strand vRNA. Capping of individual viral messenger RNAs (mRNAs) is performed in a similar manner: PA and PB2 proteins capture the 5' capping primer of the host pre-mRNA transcript and subsequently initiate viral mRNA synthesis, a process called "cap snatching" and mainly performed by the viral RNA-dependent RNA polymerase (RdRp), whose PA subunit has RNA endonuclease activity and is involved in the cleavage of host mRNAs. After the polyadenylation and capping processes are completed, the viral mRNA leaves the nucleus and enters the cytoplasm, where it is translated in the same way as the host cell's mRNA, and the nuclear transport of the viral vRNA fragment is mediated by the viral M1 and NS2 proteins, and when the M1 protein can interact with the vRNA and NP protein, it also interacts with the nuclear transport protein NS2, whereby the nuclear transport protein NS2 mediates the transport of M1-RNP from the nucleus to the host cell cytoplasm in the form of a nuclear protein.

[0007] Influenza generates direct costs from lost productivity and associated medical resources, as well as indirect costs from preventive measures. In the United States, influenza costs cumulatively about $10 billion per year, and future influenza pandemics are estimated to generate hundreds of billions of dollars in direct and indirect costs. The costs of prevention are also very high, with governments around the world spending billions of dollars preparing and planning for a potential H5N1 avian influenza pandemic, and prevention costs are associated with strategies for purchasing medicines and vaccines, developing disaster drills, and strengthening border controls.

[0008] Current treatment options for influenza include vaccination, chemotherapy with antiviral drugs, and chemoprevention. Vaccination against influenza is often recommended for high-risk groups such as children, the elderly, and people with asthma, diabetes, and heart disease, but vaccination does not completely prevent influenza. Vaccines against some specific influenza strains are reproduced each season, but it is impossible to cover the various strains that are actively infecting people around the world during that season. In addition, influenza viruses undergo a degree of antigenic drift, so that when multiple viruses infect a single cell, the eight individual vRNA segments in the genome mix or reassort, resulting in rapid changes in viral genetics that can cause antigenic shifts and allow the virus to infect new host species and rapidly overcome protective immunity.

[0009] Antiviral drugs can also be used to treat influenza, among which neuraminidase inhibitors such as oseltamivir (Tamiflu) have obvious effects on influenza A viruses, but clinical observations have shown that drug-resistant virus strains have emerged against this type of neuraminidase inhibitor. In the field of anti-influenza viruses, there is an urgent clinical need for anti-influenza virus drugs with novel mechanisms of action that can be used as a single agent or in combination with commercially available anti-influenza virus drugs with other mechanisms of action to prevent and treat influenza A. Among them, WO2016175224 reports RNA polymerase PA subunit inhibitors such as S-033447 and its prodrug S-033188.

[0010] [ka] Summary of the Invention

[0011] The present invention provides a compound represented by formula (VI) or a pharma- ceutically acceptable salt thereof.

[0012] [ka]

[0013] however, R7 is H and [ka] is selected from R8 is C 1-3 Alkyl and [ka] C is selected from 1-3 Alkyl and [ka] is 1, 2 or 3 R a and optionally replaced by R9 is selected from H, E1 is selected from Se, and X1 is CR 10 R 11 Selected from R 10 and R 11 C together with the atoms connected to them 3-5 Forming a cycloalkyl, Alternatively, X1 and R9 together with the atoms connected thereto may form [ka] wherein p is selected from 0 and 1, one of E1 and E2 is selected from Se, and the other is selected from S and O; R 12 are H, F, Cl, Br, I, OH, NH2, -COOH, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 alkylamino, 1-3 Alkyl, C 1-3 Alkoxy and C 1-3Alkylamino is independently one, two or three R b and optionally replaced by T1, T2, T3 and T4 are each independently selected from CH and N; q is selected from 0 and 1; t is selected from 0, 1, 2, 3, and 4; Each R a and R b are each independently selected from H, F, Cl, Br, and I; With the proviso that when T1 is selected from CH, E1 is selected from Se, and E2 is selected from O, p is selected from 1, and q is selected from 1, then each R 12 are each independently selected from OH and NH2.

[0014] In some embodiments of the present invention, each R 12 are each independently selected from F, and the other variables are as defined herein.

[0015] In some embodiments of the invention, R8 is CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, and [ka] wherein said CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and [ka] is 1, 2 or 3 R a and other variables are as defined in the present invention.

[0016] In some embodiments of the present invention, R8 is CH3, CH2CH3, CH(CH3)2, and [ka] and the other variables are as defined herein. In some embodiments of the present invention, R8 is CH3 and [ka] and the other variables are as defined herein. In some embodiments of the invention, R7 is H, [ka] and the other variables are as defined herein.

[0017] In some embodiments of the invention, R7 is H, [ka] and the other variables are as defined herein.

[0018] In some embodiments of the present invention, R7 is H and [ka] and the other variables are as defined herein.

[0019] In some embodiments of the invention, E1 is selected from Se and E2 is selected from O, with the other variables being as defined herein.

[0020] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, NH2, -COOH, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 alkylamino, 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylamino is independently one, two or three R b and other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined herein.

[0022] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined herein.

[0023] In some embodiments of the present invention, R5 is selected from F, and other variables are as defined herein.

[0024] In some embodiments of the present invention, R6 is selected from F, and other variables are as defined herein.

[0025] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined herein.

[0026] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined herein.

[0027] In some embodiments of the present invention, the structural unit [ka] teeth, [ka] and the other variables are as defined herein.

[0028] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0029] [ka]

[0030] however, R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2; R7 is H and [ka] is selected from R8 is C 1-3 Alkyl and [ka] C is selected from 1-3 Alkyl and [ka] is 1, 2 or 3 R a and optionally replaced by R9 is selected from H, E1 is selected from Se, and X1 is CR 10 R 11 Selected from R 10 and R 11 C together with the atoms connected to them 3-5 Forming a cycloalkyl, Alternatively, X1 and R9 together with the atoms connected thereto may form [ka] Forming One of E1 and E2 is selected from Se, and the other is selected from S and O; T1 is selected from CH and N; p and q are each independently selected from 0 and 1; Each R a are each independently selected from H, F, Cl, Br, and I; With the proviso that when T1 is selected from CH, E1 is selected from Se, and E2 is selected from O, p is selected from 1, and q is selected from 1, then R5 and R6 are each independently selected from OH and NH2; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0031] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0032] [ka]

[0033] however, p, q, E1, E2, T1, R5, R6 and R7 are as defined herein; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0034] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0035] [ka]

[0036] however, p, q, E1, E2, R5, R6 and R7 are as defined herein; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0037] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0038] [ka]

[0039] however, p, q, E1, E2, R5, R6 and R7 are as defined herein; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0040] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0041] [ka]

[0042] however, R1 and R2 are each independently selected from H, F, Cl, Br, I, OH, and NH2; m is selected from 0 and 1; q, R5, R6, R7 and R8 are as defined in the present invention.

[0043] In some embodiments of the present invention, R1 and R2 are each independently selected from F, and all other variables are as defined herein.

[0044] In some embodiments of the present invention, R5 and R6 are each independently selected from F, and all other variables are as defined herein.

[0045] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0046] [ka]

[0047] wherein R1, R2, R5, R6, R7 and R8 are as defined in the present invention.

[0048] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0049] [ka]

[0050] however, R1, R2, R3 and R4 are each independently selected from H, F, Cl, Br, I, OH and NH2; n and m are each independently selected from 0 and 1; In the formula (I-1) and the formula (I-2), when m is selected from 1, R1 and R2 are each independently selected from OH and NH2; q, R5, R6 and R8 are as defined in the present invention.

[0051] In some embodiments of the present invention, R1 and R2 are each independently selected from F, and all other variables are as defined herein.

[0052] In some embodiments of the present invention, R3 and R4 are each independently selected from F, and all other variables are as defined herein.

[0053] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0054] [ka]

[0055] [ka]

[0056] however, R1, R2, R3, R4, R5, R6, R7 and R8 are as defined in the present invention.

[0057] The present invention provides a compound represented by formula (V) or a pharma- ceutically acceptable salt thereof.

[0058] [ka]

[0059] however, R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2; R7 is H and [ka] is selected from R8 is C 1-3 Alkyl and [ka] C is selected from 1-3 Alkyl and [ka] is 1, 2 or 3 R a and optionally replaced by R9 is selected from H; X1 is a CR 10 R 11 Selected from R 10 and R 11 C together with the atoms connected to them 3-5 Forming a cycloalkyl, Alternatively, X1 and R9 are linked together [ka] Forming E1 is selected from S and Se; E2 is selected from O and Se, and at least one of E1 and E2 is selected from Se; T1 is selected from CH and N; p and q are each independently selected from 0 and 1; Each R aare each independently selected from H, F, Cl, Br, and I; With the proviso that when T1 is selected from CH, E1 is selected from Se, and E2 is selected from O, p is selected from 1, and q is selected from 1, then R5 and R6 are each independently selected from OH and NH2; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0060] The present invention provides a compound represented by formula (IV) or a pharma- ceutically acceptable salt thereof.

[0061] [ka]

[0062] however, T1 is selected from CH and N; E1 is selected from S and Se; E2 is selected from O and Se, and at least one of E1 and E2 is selected from Se; R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2; R7 is H and [ka] is selected from R8 is C 1-3 Alkyl and [ka] C is selected from 1-3 Alkyl and [ka] is 1, 2 or 3 R a and optionally replaced by p and q are each independently selected from 0 and 1; Each R a are each independently selected from H, F, Cl, Br, and I; With the proviso that when T1 is selected from CH, E1 is selected from Se, E2 is selected from O, p is selected from 1, and q is selected from 1, then R5 and R6 are each independently selected from OH and NH2; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0063] In some embodiments of the invention, the compound, or a pharma- ceutically acceptable salt thereof, is selected from the formula:

[0064] [ka]

[0065] however, p, q, E1, E2, R5, R6 and R7 are as defined herein; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0066] The present invention provides a compound represented by formula (III) or a pharma- ceutically acceptable salt thereof.

[0067] [ka]

[0068] however, T1 is selected from CH and N; R1 is selected from H, F, Cl, Br, I, OH and NH2; R2 is selected from H, F, Cl, Br, I, OH and NH2; m is selected from 0 and 1; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0069] The present invention provides a compound represented by formula (II) or a pharma- ceutically acceptable salt thereof.

[0070] [ka]

[0071] however, R3 is selected from H, F, Cl, Br, I, OH and NH2; R4 is selected from H, F, Cl, Br, I, OH and NH2; n is selected from 0 and 1; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0072] The present invention provides a compound represented by formula (I) or a pharma- ceutically acceptable salt thereof.

[0073] [ka]

[0074] Here, R1, R2 and m are as defined in the present invention.

[0075] The present invention provides a compound represented by formula (I) or a pharma- ceutically acceptable salt thereof.

[0076] [ka]

[0077] however, R1 is selected from H, F, Cl, Br, I, OH and NH2; R2 is selected from H, F, Cl, Br, I, OH and NH2; m is selected from 0 and 1; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either the (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

[0078] In some embodiments of the present invention, R1 is selected from F, and other variables are as defined herein.

[0079] In some embodiments of the invention, R2 is selected from F, with other variables as defined herein.

[0080] In some embodiments of the present invention, R3 is selected from F, and other variables are as defined herein.

[0081] In some embodiments of the present invention, R4 is selected from F, and other variables are as defined herein.

[0082] Further embodiments of the present invention are formed by any combination of the above variables.

[0083] The present invention provides a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0084] [ka]

[0085] [ka]

[0086] The invention further provides the use of said compound, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating a disease associated with an influenza virus. Effect of the Invention

[0087] As an RNA polymerase inhibitor, the compound of the present invention shows positive effect in the inhibition test of influenza virus replication at cell level, shows good weight protection in animal in vivo efficacy model, and has a fast recovery time.The test result of plasma protein binding rate shows that the compound of the present invention has a moderate plasma protein binding rate in plasma, and the PK result shows that the compound has good pharmacokinetic properties and good drug discovery properties. [Brief description of the drawings]

[0088] [Figure 1] 3D binding mode of S-033447 with protein (PDB ID: 6FS6). [Diagram 2] 4 shows the interactions of S-033447 with amino acids and metal ions. [Diagram 3] These are the two dihedral angles in the low-energy conformation of S-033447. [Figure 4] This is the change in energy of the two dihedral angles during rotation of S-033447. [Diagram 5] Comparison of the low-energy conformations of compound A (dark shade) and S-033447 (light shade). [Figure 6] This is the change in energy of the two dihedral angles during the rotation of compound A. [Figure 7] A comparison of the low-energy conformations of compound B (dark color) and S-033447 (light color). [Figure 8] The change in energy of the two dihedral angles during the rotation of compound B. Definition and Explanation

[0089] Unless otherwise explained, the following terms and phrases used herein shall have the following meanings: Certain terms and phrases, unless specifically defined, should not be considered indefinite or unclear but should be understood according to their ordinary meaning. When trade names are mentioned herein, it is intended to refer to the corresponding trade name or its active ingredients.

[0090] As used herein, the term "pharmacologically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals and are commensurate with a reasonable benefit / risk ratio without undue toxicity, irritation, allergic response or other problem or complication.

[0091] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention prepared from a compound having certain substituents found in the present invention and a relatively non-toxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound with a sufficient amount of a base in a pure 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 a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of an acid in a pure solution or in a suitable inert solvent. Illustrative examples of pharma- ceutically acceptable acid addition salts include inorganic and organic acid salts, as well as salts of amino acids (such as, for example, arginine) and organic acids such as glucuronic acid, such as inorganic acids 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, and the like, and organic acids including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Certain compounds of the present invention contain both basic and acidic functionalities and thus can be converted into either base or acid addition salts.

[0092] The pharma- ceutically acceptable salts of the present invention can be synthesized from the parent compound that contains an acid or base group by conventional methods. Generally, such salts are prepared by reacting the free acid or free 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 both.

[0093] The compounds of the present invention can exist in specific geometric or stereoisomeric forms. All such compounds contemplated by the present invention include cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and mixtures thereof are included within the scope of the present invention.

[0094] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, the compounds may contain tritium ( 3 H), iodine-125( 125 I), C-14( 14 C). Or, for example, deuterium can be replaced with hydrogen to form a deuterated drug, where the bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon, and compared with non-deuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of drugs. Conversion of the isotopic composition of the compounds of the present invention, whether radioactive or not, is included within the scope of the present invention.

[0095] The terms "optionally" and "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs as well as instances where the event or circumstance does not occur.

[0096] The term "substituted" means that any one or more hydrogen atoms at a particular atom are replaced with a substituent, which may include deuterium and hydrogen variants, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur in aromatic groups. The term "optionally substituted" means substituted or unsubstituted, and unless otherwise stated, the type and number of substituents are any that are chemically feasible.

[0097] When any variable (e.g., R) occurs more than one time in a composition or structure of a compound, its definition is independent at each occurrence. Thus, for example, if a group is substituted with 0-2 R, then said group may be optionally substituted with up to 2 R, and each occurrence of R is independently optional. Also, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0098] When the number of linking groups is 0, for example, -(CRR)0- means that the linking group is a single bond.

[0099] When one of the variables is a single bond, it means that the two groups to which it is linked are directly bonded; for example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0100] When the listed linking group does not indicate the direction of the link, the direction of the link is arbitrary, for example: [ka] In the formula, the linking group L is -MW-, where -MW- links ring A and ring B in the same direction as the reading order from left to right. [ka] We can also construct the following by concatenating rings A and B in the reverse order of reading from left to right: [ka] Combinations of the above linking groups, substituents and / or variables thereof are permissible only if such combinations result in stable compounds.

[0101] Unless otherwise stated, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups via chemical bonds. When the bonding mode of the chemical bond is delocalized and there is an H atom at the bondable site, the number of H atoms at the site is reduced to the corresponding valence of the group according to the number of bonded chemical bonds. The chemical bond by which the site is bonded to another group is: [ka]

[0102] For example, the straight solid bond in -OCH3 represents a bond to another group through the oxygen atom in that group, [ka] The straight-dashed bond represents a bond between the nitrogen atom of the group and another group, [ka] The wavy lines indicate that the phenyl group is bonded to another group via the 1st and 2nd carbon atoms. [ka] represents that any available bonding site of the piperidinyl can be bonded to another group via one chemical bond, and at least [ka] Even if the H atom is drawn as -N-, [ka] When one chemical bond is bonded, the H at that site decreases by one to the corresponding monovalent piperidinyl.

[0103] Unless otherwise stated, "C 1-3 The term "alkyl" is used to represent a straight or branched chain saturated hydrocarbon group of 1 to 3 carbon atoms. 1-3 Alkyl has C 1-2 and C 2-3 alkyl, which may be monovalent (e.g., methyl), divalent (e.g., methylene) or polyvalent (e.g., methine). 1-3 Illustrative examples of alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0104] Unless otherwise stated, "C 1-3 The term "alkoxy" refers to an alkyl group containing 1 to 3 carbon atoms attached to the remainder of the molecule through an oxygen atom. 1-3 Alkoxy includes C 1-2 , C 2-3 , C3 and C2 alkoxy, etc. 1-3 Illustrative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0105] Unless otherwise stated, "C 1-3 The term "alkylamino" refers to an alkyl group containing 1 to 3 carbon atoms attached to the remainder of the molecule through an amino group. 1-3 Alkylamino includes C 1-2, C3 and C2 alkylamino, etc. 1-3 Examples of alkylamino include, but are not limited to, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH3)CH2CH3, -NHCH2CH2CH3, -NHCH2(CH3)2, and the like.

[0106] Unless otherwise stated, "C 3-5 "Cycloalkyl" means a saturated cyclic hydrocarbon group of 3 to 5 carbon atoms, which is a monocyclic ring system, 3-5 Cycloalkyl has C 3-4 and C 4-5 Cycloalkyl and the like are included, which may be monovalent, divalent or polyvalent. C 3-5 Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and the like.

[0107] Unless otherwise stated, C n-n+m Or C n -C n+m includes any one specific embodiment of n to n+m carbons, for example, C 1-12 are C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , and C 12 Including any one of the ranges of n to n+m, for example, C 1-12 is C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 Similarly, n-membered to n+m-membered rings indicate that the number of atoms in the ring is n to n+m, and for example, a 3- to 12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any one of the ranges of n to n+m, and for example, a 3- to 12-membered ring includes a 3- to 6-membered ring, a 3- to 9-membered ring, a 5- to 6-membered ring, a 5- to 7-membered ring, a 6- to 7-membered ring, a 6- to 8-membered ring, and a 6- to 10-membered ring, etc.

[0108] The term "leaving group" refers to a functional group or atom that may be replaced by another functional group or atom through a substitution reaction (e.g., a nucleophilic substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, and the like; acyloxy groups such as acetoxy, trifluoroacetoxy, and the like; and the like.

[0109] The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino acid protecting groups include, but are not limited to, formyl; acyl such as alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl such as benzyl (Bn), triphenylmethyl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; silyl such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxyl protecting group" refers to a protecting group suitable for preventing side reactions of hydroxyl. Representative hydroxyl protecting groups include, but are not limited to, alkyl, such as methyl, ethyl, and tert-butyl; acyl, such as alkanoyl (e.g., acetyl); arylmethyl, such as benzyl (Bn), p-formyloxybenzyl (PMB), 9-fluorenylethyl (Fm), and diphenylmethyl (diphenylmethyl, DPM); silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and the like.

[0110] The compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0111] The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art, and when the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in the art.For example, in single crystal X-ray diffraction (SXRD), the single crystal is grown and collected diffraction intensity data by Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting relevant data, the crystal structure can be further analyzed by direct method (Shelxs97) to confirm the absolute configuration.

[0112] The following abbreviations are used in the present invention: DMAC: N,N-dimethylacetamide, PG: propylene glycol, HP-β-CD: hydroxypropyl-β-cyclodextrin, Solutol HS-15: polyethylene glycol (15)-hydroxystearate.

[0113] The solvents used in the present invention can be obtained from commercial sources. Compounds are named according to conventional naming principles in the art or using ChemDraw® software, commercially available compounds are named in the supplier's catalogue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] The present invention will be described in detail below with reference to examples, but these are not intended to limit the present invention in any adverse manner. Although the present invention has been described in detail in the present specification, specific embodiments thereof have also been disclosed, and it is obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0115] Reference example 1 [ka]

[0116] Synthesis method: [ka]

[0117] Reference example 2 [ka]

[0118] Synthesis method: [ka]

[0119] Using the anti-influenza drug S-033447 from SHIONOGI & CO., LTD. as a reference compound, the low-energy conformation of S-033447 was calculated by the Macromodel module of Schrodinger's Maestro software. In the low-energy conformation, the dihedral angle (dehidal 1) of pyridohexahydropyrimidine (hereafter referred to as the parent nucleus) was -146.6°, and the dihedral angle (dehidal 2) between the parent nucleus and 2,5-dihydrothiophene was 56.8° (see Figure 3). The conversion of S-033447 from the low-energy conformation to the active conformation of the protein binding mode (the parent nucleus rotates from -146.6° to -153.7°, and the dihedral angle between 2,5-dihydrothiophene and the parent nucleus rotates from 56.8° to 55.0°) required overcoming an energy barrier of 0.8 kcal / mol (see Figure 4). The 3D binding mode of S-033447 with protein (PDB ID: 6FS6) is shown in FIG. 1, and the interactions of S-033447 with amino acids and metal ions are shown in FIG.

[0120] Example 1 By observing the binding mode of the active conformation of S-033447 and 6FS6 protein, the lowest energy barrier conformation (low energy conformation) formed between the pyridohexahydropyrimidine parent nucleus and the benzo 2,5-dihydrothienodifluorobenzyl fragment of S-033447 while freely rotating was more consistent with the protein binding mode conformation (active conformation) in the S-033447 and 6FS6 protein cocrystal, explaining the high binding activity of S-033447 and 6FS6 protein. In general, the smaller the difference in energy barrier between the lowest energy barrier conformation (low energy conformation) of a small molecule and its binding mode conformation (active conformation) in the protein, the less energy the small molecule loses in the conversion from the low energy conformation to the binding active conformation with the protein, which means that the compound is more likely to bind to the protein and its binding activity is higher.

[0121] To lock the active conformation of S-033447 and further reduce its rotational energy barrier, the O and S in the tetrahydropyran and 2,5-dihydrothiophene fragments of S-033447 were replaced with Se by atomic substitution to obtain different cyclopentaselenium and cyclohexaselenium fragments, and the difference in energy barrier between the lowest energy barrier conformations of these Se-substituted molecules and the active conformation in the cocrystal of S-033447 and 6FS6 protein was explored.

[0122] (1) The rotational dihedral angles and rotational energy barriers of the low-energy conformations of compound A and compound B were calculated by the Macromodel module, and the results are shown in Table 1. A comparison of the low-energy conformations of compound A (dark color) and S-033447 (light color) is shown in Figure 5, the energy changes of the two dihedral angles during the rotation of compound A are shown in Figure 6, a comparison of the low-energy conformations of compound B (dark color) and S-033447 (light color) is shown in Figure 7, and the energy changes of the two dihedral angles during the rotation of compound B are shown in Figure 8.

[0123] [Table 1]

[0124] Conclusion: The low-energy conformation of compound B overlaps well with the active conformation of S-033447. The compounds of the present invention have a smaller energy difference between the lowest binding energy barrier in the 6FS6 protein structure and the energy barrier in the active conformation in the protein structure of the reference compound, so the compounds of the present invention are more likely to bind to the protein, and may show similar or better binding activity to the protein than the reference compound in actual binding to the protein.

[0125] (2) The rotational dihedral angles of the low-energy conformation of compound B were calculated by the Macromodel module, and the results are shown in Table 2.

[0126] [Table 2]

[0127] Conclusion: The low-energy conformation of compound B is essentially identical to that of S-033447.

[0128] Example 2 [ka]

[0129] Step 1: Synthesis of compound 2-2 Sodium dihydrogen phosphate (13.58 g, 113.19 mmol) was added to water (20 mL), acetonitrile (10 mL), diphenyl diselenide (1.18 g, 3.77 mmol), zinc powder (986.83 mg, 15.09 mmol) was added in batches, the reaction solution was stirred at room temperature for 1 hour, and then compound 2-1 (2 g, 7.55 mmol) was added, and the reaction solution was stirred at room temperature overnight. The reaction solution was filtered, the cake was washed with ethyl acetate (10 mL x 2), separated, the aqueous phase was extracted with ethyl acetate (10 mL x 2), the organic phase was combined, washed with saturated saline (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness. The obtained crude product was purified on a silica gel column (petroleum ether: ethyl acetate = 1: 0 to 10: 1) to obtain compound 2-2. MS m / z: 343.0 [M + H] + .

[0130] Step 2: Synthesis of compound 2-3 Compound 2-2 (1.5 g, 4.40 mmol) was added to methanol (10 mL) and water (5 mL), sodium hydroxide (527.53 mg, 13.19 mmol) was added, and the reaction solution was stirred at 60° C. for 2 hours. The reaction solution was cooled to room temperature, the reaction solution was adjusted to pH=7 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated saline (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain compound 2-3, and the crude product was used directly in the next step reaction.

[0131] Step 3: Synthesis of Compounds 2-4 Compound 2-3 (1.3 g, 3.97 mmol) was added to polyphosphoric acid (13 mL), and the reaction solution was stirred at 120° C. for 2 hours. The reaction solution was cooled to 80° C., added to stirring water (50 mL), and stirred for 5 minutes, extracted with dichloromethane (20 mL×2), and the organic phases were combined, washed with water (20 mL), washed with saturated saline (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified on a silica gel column (petroleum ether:ethyl acetate=1:0-10:1) to obtain compound 2-4. MS m / z: 310.9 [M+H]+ .

[0132] Step 4: Synthesis of Compounds 2-5 Compound 2-4 (300 mg, 970.34 μmol) was added to methanol (6 mL), then sodium borohydride (110.13 mg, 2.91 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was adjusted to pH=7 with 1N hydrochloric acid, then extracted with dichloromethane (10 mL×2), the organic phases were combined, washed with saturated saline (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified on a silica gel column (petroleum ether:ethyl acetate=10:1 to 2:1) to obtain compound 2-5.

[0133] Step 5: Synthesis of Compounds 2-7 Compound 2-6 (50 mg, 152.75 μmol) was added to ethyl acetate (1 mL), compound 2-5 (47.53 mg, 152.75 μmol) was added, then 1-propylphosphonic anhydride (388.82 mg, 611.00 μmol, 363.38 μL, 50% ethyl acetate solution) and methanesulfonic acid (58.72 mg, 611.00 μmol, 43.50 μL) were added, and the reaction solution was refluxed overnight. The reaction solution was cooled to room temperature, water (10 mL) was added, extracted with ethyl acetate (5 mL × 2), the organic phase was combined, washed with saturated saline (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified with a silica gel column (dichloromethane:methanol = 1:0 to 10:1) to obtain compound 2-7. MS m / z: 622.0 [M+H] + .

[0134] Step 6: Synthesis of Compounds 2 and 2' Compound 2-7 (10 mg, 16.12 μmol) was added to N,N-dimethylacetamide (0.5 mL), lithium chloride (3.42 mg, 80.58 μmol, 1.65 μL) was added, and the reaction solution was stirred at 80 ° C for 3 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (2 mL). The crude reaction solution was separated and purified by preparative high performance liquid chromatography (column: Xtimate C18 100 × 30 mm × 3 μm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile%: 40% to 60%, 8 min) to obtain compound 2 (retention time: 3.205 min) and compound 2' (retention time: 3.301 min).

[0135] Compound 2 (retention time: 3.205 minutes), 1 H NMR (400 MHz, deuterated methanol) δ 7.49 (d, J = 7.53 Hz, 1H), 7.23-7.32 (m, 2H), 7.16-7.22 (m, 1H), 7.07-7.15 (m, 1H), 6.85-6.97 (m, 2H), 5.85 (d, J = 7.28 Hz, 1H), 5.69 (s, 1H), 5.39 (dd, J = 2.64, 12.67 Hz, 1H), 4.73 (dd, J = 2.89, 10.16 Hz, 1H), 4.62 (br d, J = 15.56 Hz, 1H), 4.12 (d, J = 12.80 Hz, 1H), 4.07 (dd, J = 3.14, 11.17 Hz, 1H), 3.77 (dd, J = 3.01, 11.80 Hz, 1H), 3.65 (t, J = 10.54 Hz, 1H), 3.43-3.53 (m, 1H), 3.06-3.17 (m, 1H). MS m / z: 532.1 [M+H] + .

[0136] Compound 2' (retention time: 3.301 minutes) 1H NMR (400 MHz, deuterated methanol) δ 7.51 (d, J = 7.28 Hz, 1H), 7.35-7.45 (m, 2H), 7.21-7.33 (m, 2H), 6.94-7.04 (m, 1H), 6.82-6.93 (m, 1H), 6.11 (d, J = 7.53 Hz, 1H), 5.52-5.68 (m, 2H), 4.42-4.58 (m, 2H), 4.16 (d, J = 13.05 Hz, 1H), 4.06 (dd, J = 3.14, 10.92 Hz, 1H), 3.60-3.78 (m, 2H), 3.39-3.52 (m, 1H), 2.65-2.81 (m, 1H). MS m / z: 532.1 [M+H] + .

[0137] Example 3 [ka]

[0138] Step 1: Synthesis of compound 3-2 Compound 3-1 (66 g, 383.43 mmol) was dissolved in dichloromethane (460 mL), N,N-dimethylformamide (280.27 mg, 3.83 mmol, 295.02 μL) was added, and oxalyl chloride (73.00 g, 575.15 mmol, 50.35 mL) was added dropwise to the reaction solution. After the addition was completed, the reaction solution was stirred at 20 ° C for 30 minutes, and then concentrated under reduced pressure to dryness. The obtained crude product was added to dichloromethane (460 mL), and triethylamine (77.60 g, 766.87 mmol, 106.74 mL) and N,O-dimethylhydroxylamine hydrochloride (37.40 g, 383.43 mmol) were added under stirring, and the reaction solution was stirred at 20 ° C for 1 hour. Water (100 mL) was added, separated, and the aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, washed with dilute hydrochloric acid (0.2 M, 50 mL), saturated aqueous sodium bicarbonate solution (50 mL) and saturated brine (50 mL), respectively, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to give compound 3-2. 1H NMR (400 MHz, deuterated chloroform) δ 7.02-7.05 (m, 2H), 3.81 (brs, 3H), 3.49 (s, 3H), 2.28 (s, 3H).

[0139] Step 2: Synthesis of compound 3-3 Compound 3-2 (20 g, 92.94 mmol) was dissolved in tetrahydrofuran (200 mL), and methylmagnesium bromide (3 M, 37.18 mL) was added dropwise at 0° C. After the completion of the dropwise addition, the reaction solution was warmed to 20° C. and stirred for 2 hours. The reaction solution was quenched with 1 M hydrochloric acid, the pH was adjusted to 7, and extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with dilute hydrochloric acid (0.2 M, 50 mL), saturated sodium bicarbonate solution (50 mL), and saturated saline (50 mL), respectively, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain compound 3-3. 1 H NMR (400 MHz, deuterated chloroform) δ 7.47-7.50 (m, 1H), 7.03-7.07 (m, 1H), 2.60 (s, 3H), 2.47 (s, 3H).

[0140] Step 3: Synthesis of Compound 3-4 Compound 3-3 (15 g, 88.15 mmol) was dissolved in pyridine (90 mL), selenium dioxide (19.56 g, 176.31 mmol) was added, and the reaction solution was stirred at 110° C. for 12 hours. The reaction solution was cooled to room temperature, filtered, and concentrated to dryness under reduced pressure. Water (50 mL) was added to the crude product, the pH was adjusted to 4 with 1 M hydrochloric acid, and extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain compound 3-4. 1 H NMR (400 MHz, deuterated methanol) δ 7.64-7.68 (m, 1H), 7.30-7.32 (m, 1H), 2.52 (s, 3H).

[0141] Step 4: Synthesis of compounds 3-5 Compound 3-4 (15 g, 74.95 mmol) was dissolved in dichloromethane (60 mL) and methanol (60 mL), the temperature was controlled at 0-20 ° C, trimethylsilyldiazomethane (2 M, 44.97 mL) was added dropwise, the reaction solution was stirred at 20 ° C for 2 hours, and then acetic acid (3 mL) was added and stirred for 5 minutes. The reaction solution was concentrated under reduced pressure to dryness, water (50 mL) was added, and extracted with dichloromethane (50 mL × 2), the organic phase was combined, washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness. The obtained crude product was purified with a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 3-5. 1 H NMR (400 MHz, deuterated chloroform) δ 7.51-7.55 (m, 1H), 7.12-7.16 (m, 1H), 3.98 (s, 3H), 2.54 (s, 3H).

[0142] Step 5: Synthesis of Compounds 3-6 Compound 3-5 (5 g, 23.35 mmol) was dissolved in 1,2-dichloroethane (50 mL), N-bromosuccinimide (8.31 g, 46.69 mmol) and azobisisobutyronitrile (383.37 mg, 2.33 mmol) were added, and the reaction solution was stirred at 80 ° C for 12 hours. The reaction solution was cooled to room temperature, washed with saturated sodium sulfite solution (20 mL), water (20 mL) and saturated saline (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness. The obtained crude product was purified with a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-5%) to obtain compound 3-6. 1 H NMR (400 MHz, deuterated chloroform) δ 7.61-7.64 (m, 1H), 7.26-7.31 (m, 1H), 4.94 (s, 2H), 3.99 (s, 3H).

[0143] Step 6: Synthesis of compounds 3-7 Sodium dihydrogen phosphate (11.52 g, 96.05 mmol) was dissolved in water (60 mL), then acetonitrile (30 mL) was added, 3-(3-pyridyldiselenyl)pyridine (3.62 g, 11.53 mmol) was added, zinc powder (1.88 g, 28.82 mmol) was added in batches, and the reaction solution was stirred at 20 ° C for 30 min. Compound 3-6 (5.63 g, 19.21 mmol) was added, and the reaction solution was stirred at 20 ° C for 3 h. The reaction solution was filtered, and the filtrate was extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-60%) to obtain compound 3-7. MS (ESI) m / z: 373.8 [M + H] + .

[0144] Step 7: Synthesis of Compounds 3-8 Compound 3-7 (4.2 g, 11.28 mmol) was dissolved in dichloromethane (80 mL), Dess-Martin periodinane (7.18 g, 16.93 mmol) was added, and the reaction solution was stirred at 20 ° C for 12 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution and stirred for 5 minutes. Extraction was performed with dichloromethane (30 mL × 2), and the organic phases were combined, washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified with a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 3-8. MS (ESI) m / z: 371.9 [M + H] + .

[0145] Step 8: Synthesis of Compounds 3-9 Compound 3-8 (2.9 g, 7.83 mmol) was dissolved in tetrahydrofuran (16 mL), aqueous sodium hydroxide (626.67 mg, 15.67 mmol, 4 mL) was added, and the reaction solution was stirred at 20° C. for 1 h. The bulk of the tetrahydrofuran was removed by concentration under reduced pressure, the aqueous phase was adjusted to pH 6 with 1N hydrochloric acid, the solids were filtered, and the cake was drained under reduced pressure to give compound 3-9. MS(ESI) m / z: 357.9 [M+H] + .

[0146] Step 9: Synthesis of Compounds 3-10 Compound 3-9 (2.3 g, 6.46 mmol) was dissolved in dimethyl sulfoxide (23 mL), and ammonium persulfate (2.95 g, 12.91 mmol), silver nitrate (109.69 mg, 645.74 μmol) and concentrated sulfuric acid (633.34 mg, 6.46 mmol) were added, and the reaction solution was stirred at 50 ° C for 3 hours. Saturated sodium bicarbonate aqueous solution (20 mL), water (10 mL) and dichloromethane (20 mL) were added to the reaction solution, stirred for 5 minutes, filtered, the filtrate was separated, and the aqueous phase was extracted with dichloromethane (10 mL). The organic phases were combined, washed with saturated saline (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness. The obtained crude product was purified with a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 3-10. MS(ESI) m / z: 311.8 [M+H] + .

[0147] Step 10: Synthesis of Compounds 3-11 Compound 3-10 (390 mg, 1.26 mmol) was dissolved in isopropanol (8 mL), sodium borohydride (95.14 mg, 2.51 mmol) was added, and the reaction solution was stirred at 20°C for 1 hour. 1N hydrochloric acid was added to adjust the pH to 7, and the mixture was extracted with ethyl acetate (15 mL x 2). The organic phases were combined, washed with saturated saline (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified on a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 3-11. MS (ESI) m / z: 313.8 [M+H] + .

[0148] Step 11: Synthesis of Compounds 3-12 Compound 3-11 (330 mg, 1.06 mmol) was dissolved in dichloromethane (6 mL), dichlorosulfoxide (251.53 mg, 2.11 mmol, 153.37 μL) was added, and the reaction solution was stirred for 1 h at 20° C. The reaction solution was concentrated to dryness under reduced pressure to give compound 3-12, and the crude product was used directly in the next step reaction.

[0149] Step 12: Synthesis of Compounds 3-13 and 3-13' Compound 2-6 (340 mg, 1.04 mmol) was dissolved in acetonitrile (6 mL), compound 3-12 (343.41 mg, 1.04 mmol) and cesium carbonate (676.86 mg, 2.08 mmol) were added, and the reaction solution was stirred at 60° C. for 12 hours. The reaction solution was cooled to room temperature, water (5 mL) was added, and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined, washed with saturated saline (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness. The obtained crude product was purified on a silica gel column (methanol / dichloromethane, methanol ratio: 0-5%), and the obtained compounds were detected on a supercritical fluid chromatography column (analysis method: column type: Chiralpak AD-3 (50 mm × 4.6 mm, 3 μm); mobile phase: [A: carbon dioxide, B: 0.05% diethylamine / ethanol]; gradient: the concentration of mobile phase B increased from 5% to 40% in 2 min, 40% was held for 1.2 min, then 5% was held for 0.8 min), analyzed as a mixture, and chiral separation (column type: DAICEL CHIRALPAK Separation using an AD (250 mm × 30 mm, 10 μm); mobile phase: [A: carbon dioxide, B: 0.1% aqueous ammonia / ethanol]; gradient: mobile phase B was retained at 40% gave chiral isomers compound 3-13 (retention time: 1.831 min, ee = 96.1%) and compound 3-13' (retention time: 2.031 min, ee = 100%).

[0150] Step 13: Synthesis of Compound 3 Compound 3-13 (6 mg, 9.65 μmol) was added to N,N-dimethylacetamide (1 mL), lithium chloride (2.05 mg, 48.27 μmol) was added, and the reaction solution was stirred at 80 ° C for 12 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction solution was separated and purified by preparative high performance liquid chromatography (column: Phenomenex Gemini-NX C18 75 × 30 mm × 3 μm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile%: 30% to 53%, 5 min) to obtain compound 3. 1H NMR (400 MHz, deuterated methanol) δ 7.98-8.09 (m, 1H), 7.70 (dd, J = 1.51, 8.03 Hz, 1H), 7.41 (d, J = 7.53 Hz, 1H), 7.18-7.31 (m, 2H), 7.13 (dd, J = 4.52, 8.03 Hz, 1H), 5.75-5.88 (m, 2H), 5.40-5.53 (m, 1H), 4.71 (dd, J = 3.01, 10.04 Hz, 1H), 4.63 (br s, 1H), 4.17 (d, J = 12.55 Hz, 1H), 4.07 (dd, J = 3.01, 11.04 Hz, 1H), 3.77 (dd, J = 3.01, 11.54 Hz, 1H), 3.66 (t, J = 10.54 Hz, 1H), 3.48 (dt, J = 2.51, 11.80 Hz, 1H), 3.04-3.18 (m, 1H). MS(ESI) m / z: 533.1 [M+H] + .

[0151] Step 14: Synthesis of Compound 3' Compound 3-13' (5 mg, 8.05 μmol) was added to N,N-dimethylacetamide (1 mL), lithium chloride (1.71 mg, 40.23 μmol) was added, and the reaction solution was stirred at 80 ° C for 12 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction solution was separated and purified by preparative high performance liquid chromatography (column: Phenomenex Gemini-NX C18 75 × 30 mm × 3 μm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile%: 30% to 53%, 5 min) to obtain compound 3'. 1H NMR (400 MHz, deuterated methanol) δ 8.39-8.49 (m, 1H), 7.83 (dd, J = 1.51, 8.03 Hz, 1H), 7.26-7.39 (m, 2H), 6.95-7.10 (m, 1H), 6.85 (br s, 1H), 5.92 (d, J = 7.53 Hz, 1H), 5.72 (s, 1H), 5.64 (br d, J = 13.55 Hz, 1H), 4.50-4.58 (m, 1H), 4.36 (br d, J = 7.53 Hz, 1H), 4.16-4.24 (m, 1H), 4.05 (dd, J = 3.01, 11.04 Hz, 1H), 3.74 (dd, J = 3.51, 11.54 Hz, 1H), 3.65 (t, J = 10.54 Hz, 1H), 3.47 (dt, J = 2.51, 11.80 Hz, 1H), 2.79-2.91 (m, 1H). MS(ESI) m / z: 533.1 [M+H] + .

[0152] Example 4 [ka]

[0153] Compound 3 (130.00 mg, 244.65 μmol) was added to N,N-dimethylacetamide (2 mL), followed by the addition of chloromethyl methyl carbonate (45.70 mg, 366.98 μmol), potassium carbonate (67.63 mg, 489.30 μmol) and potassium iodide (40.61 mg, 244.65 μmol), and the reaction solution was stirred at 70 ° C for 3 hours. The reaction solution was cooled to room temperature, water (10 mL) was added, extracted with ethyl acetate (10 mL × 2), the organic phase was washed with saturated saline (10 mL × 4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified with a silica gel column (dichloromethane:methanol = 1:0 to 20:1) to obtain compound 4. 1H NMR (400 MHz, deuterated chloroform) δ 8.03 (dd, J=1.00, 4.52 Hz, 1H), 7.44 (dd, J=1.51, 8.03 Hz, 1H), 6.99-7.16 (m, 3H), 6.95 (dd, J=4.52, 8.03 Hz, 1H), 5.90 (d, J=6.53 Hz, 1H), 5.74-5.85 (m, 1H), 5.22-5.35 (m, 3H), 4.60 (dd, J=2.01, 13.55 Hz, 1H), 4.50 (dd, J=3.01, 10.04 Hz, 1H), 4.03 (d, J=12.55 Hz, 1H), 3.95 (dd, J=3.01, 11.04 Hz, 1H), 3.77-3.83 (m, 3H), 3.73 (dd, J=3.01, 12.05 Hz, 1H), 3.54 (t, J=10.54 Hz, 1H), 3.41 (dt, J=2.51, 11.80 Hz, 1H), 2.85-2.97 (m, 1H);MS(ESI) m / z: 621.0 [M+H] + .

[0154] The absolute configuration of compound 4 was confirmed by single crystal X-ray diffraction (SXRD) to be:

[0155] [ka]

[0156] Example 5 [ka]

[0157] Compound 2 (30.00 mg, 56.56 μmol) was added to N,N-dimethylacetamide (1 mL), followed by the addition of chloromethyl methyl carbonate (14.09 mg, 113.13 μmol), potassium carbonate (15.64 mg, 113.13 μmol) and potassium iodide (9.39 mg, 56.56 μmol), and the reaction solution was stirred at 70 ° C for 3 hours. The reaction solution was cooled to room temperature, water (3 mL) was added, extracted with ethyl acetate (3 mL × 2), the organic phase was washed with saturated saline (3 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The obtained crude product was purified on a preparative thin-layer silica gel plate (dichloromethane:methanol = 10:1) to obtain compound 5. 1 H NMR (400 MHz, deuterated methanol) δ 7.54 (d, J=7.53 Hz, 1H), 7.13-7.28 (m, 3H), 7.07-7.13 (m, 1H), 6.99-7.05 (m, 1H), 6.90-6.97 (m, 1H), 5.92 (d, J=7.78 Hz, 1H), 5.75-5.83 (m, 2H), 5.66 (s, 1H), 5.39 (dd, J=2.64, 12.67 Hz, 1H), 4.65 (dd, J=3.01, 10.04 Hz, 1H), 4.55 (dd, J=2.13, 13.43 Hz, 1H), 4.03-4.15 (m, 2H), 3.80-3.85 (m, 3H), 3.75 (dd, J=3.26, 11.54 Hz, 1H), 3.56 (t, J=10.54 Hz, 1H), 3.42 (dt, J=2.51, 11.67 Hz, 1H), 2.98-3.08 (m, 1H);MS(ESI) m / z: 620.1 [M+H] + .

[0158] Example 6 [ka]

[0159] [ka]

[0160] Step 1: Synthesis of compound 6-2 In an ice bath, a trimethylsilyldiazomethane solution (2M, 13.61mL, 27.22mmol) was added dropwise to a solution of compound 6-1 (3.35g, 13.61mmol) in methanol (8mL) and tetrahydrofuran (32mL). After the completion of the addition, the reaction solution was warmed to 20°C and stirred for 1 hour. A saturated citric acid solution (100mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100mL x 3). The organic phases were combined, washed with saturated sodium bicarbonate solution (100mL) and saturated saline (100mL) in sequence, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product of compound 6-2, which was used directly in the next step reaction.

[0161] Step 2: Synthesis of compound 6-3 Compound 6-2 (3.98 g, 15.29 mmol), tert-butyl carbazate (2.02 g, 15.29 mmol) and pyridinium p-toluenesulfonate (3.84 g, 15.29 mmol) were added to N,N-dimethylacetamide (80 mL), and the reaction solution was reacted at 60 ° C for 12 hours. The reaction solution was cooled to room temperature, water (200 mL) was added, and extracted with ethyl acetate (100 mL × 3), and the organic phases were combined, washed with water (200 mL) and saturated saline (200 mL), respectively, dried over anhydrous sodium sulfate, filtered, and spin-dried. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1 to 1: 2) to obtain compound 6-3.

[0162] Step 3: Synthesis of compound 6-4 Compound 6-3 (2.7 g, 7.21 mmol), methyl acrylate (1.24 g, 14.42 mmol, 1.30 mL), and N,N-diisopropylethylamine (2.80 g, 21.64 mmol, 3.77 mL) were dissolved in acetonitrile (35 mL), and the reaction solution was reacted at 50 ° C for 12 hours. The reaction solution was concentrated and spin-dried, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4: 1 to 1: 2, volume ratio) to obtain compound 6-4.

[0163] Step 4: Synthesis of compound 6-5 To a solution of compound 6-4 (1.6 g, 3.47 mmol) in ethyl acetate (20 mL) was added a solution of hydrogen chloride in ethyl acetate (4 M, 10 mL), and the reaction solution was stirred at 25° C. for 1 h. The reaction solution was concentrated under reduced pressure to give the crude hydrochloride salt 6-5, which was used directly in the next step reaction.

[0164] Step 5: Synthesis of compound 6-6 Compound 6-5 (1.18 g, hydrochloride salt) and potassium tert-butoxide (955.32 mg, 8.51 mmol) were added to acetonitrile (20 mL), and the reaction solution was stirred at 25 °C for 1 hour. Methanol (30 mL) was added, concentrated, and spin-dried. The crude product was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1 to 0: 1, volume ratio, then dichloromethane: methanol = 10: 1 to 0: 1) to obtain compound 6-6.

[0165] Step 6: Synthesis of Compounds 6-7 Compound 6-6 (3 g, 9.14 mmol) was dissolved in dimethyl sulfoxide (30 mL) and water (3 mL), sodium chloride (1.07 g, 18.27 mmol) was added, and the reaction solution was stirred at 90 ° C for 12 hours. The reaction solution was diluted with water (100 mL), extracted with dichloromethane (100 mL × 3), the organic phase was washed with saturated saline (100 mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to obtain compound 6-7.

[0166] Step 7: Synthesis of compounds 6-8 Compound 6-7 (0.3 g, 1.11 mmol) and diphenyl(vinyl)sulfonium trifluoromethanesulfonate (482.68 mg, 1.33 mmol) were dissolved in dimethyl sulfoxide (3.6 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (506.93 mg, 3.33 mmol) was added, and the reaction solution was stirred at 25 ° C for 1 hour. The reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL × 3), the organic phase was washed with saturated saline (30 mL × 2), dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to obtain compound 6-8. MS (ESI) m / z: 297.3 [M + H] + .

[0167] Step 8: Synthesis of Compounds 6-9 Compound 6-8 (70 mg, 236.23 μmol) and compound 2-5 (73.51 mg, 236.23 μmol) were added to ethyl acetate (2 mL), then tri-n-propylcyclophosphoric anhydride (50% ethyl acetate solution, 300.66 mg, 472.46 μmol, 280.99 μL) and methanesulfonic acid (22.70 mg, 236.23 μmol, 16.82 μL) were added, and the reaction solution was stirred at 77 ° C for 3 hours. The reaction solution was cooled to room temperature, water (10 mL) was added, extracted with ethyl acetate (5 mL × 2), the organic phase was combined, washed with saturated saline (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain the crude product. The crude product was purified by flash silica gel column (dichloromethane:methanol = 1:0 to 10:1) to obtain compound 6-9. MS(ESI)m / z: 591.1 [M+H] + .

[0168] Step 9: Synthesis of Compounds 6-9A and 6-9B Compound 6-9 was detected by supercritical fluid chromatography column (analysis method: column type: CHIRALCEL OD-3 (100 mm × 4.6 mm, 3 μm); mobile phase: [A: carbon dioxide, B: 0.05% diethylamine / ethanol]; gradient: B%: increased from 5% to 40% in 4 min, then held for 2.5 min, then 5% held for 1.5 min) and analyzed as a racemate, and was subjected to chiral separation (column type: DAICEL CHIRALCEL OD-H (25 mm × 30 mm, 5 μm); mobile phase: [A: carbon dioxide, B: 0.1% aqueous ammonia / ethanol]; gradient: B%: 40% to 40%) to give compound 6-9A (retention time: 4.024 min) and compound 6-9B (retention time: 4.447 min).

[0169] Step 10: Synthesis of compound 6 Compound 6-9A (20 mg, 33.93 μmol) was added to N,N-dimethylacetamide (1 mL), followed by lithium chloride (7.19 mg, 169.64 μmol), and the reaction solution was stirred at 80 °C for 12 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (2 mL). The crude product was separated and purified by preparative high performance liquid chromatography (column type: Xtimate C18 100 × 30 mm × 3 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 50% to 70%, 5 min) to obtain compound 6. 1 H NMR (400 MHz, deuterated methanol) δ 7.59 (d, J=7.53 Hz, 1H), 7.19-7.31 (m, 2H), 7.03-7.19 (m, 2H), 6.90 (br s, 2H), 5.86 (d, J=7.53 Hz, 1H), 5.45-5.61 (m, 2H), 4.26 (br d, J=15.06 Hz, 1H), 4.11 (d, J=12.55 Hz, 1H), 3.07 (br d, J=15.06 Hz, 1H), 1.84-1.97 (m, 1H), 1.60-1.75 (m, 1H), 0.92-1.12 (m, 2H);MS(ESI)m / z: 501.2 [M+H] + .

[0170] Step 11: Synthesis of compound 6' Compound 6-9B (20.00 mg, 33.93 μmol) was added to N,N-dimethylacetamide (1 mL), followed by lithium chloride (7.19 mg, 169.64 μmol, 3.47 μL), and the reaction solution was stirred at 80 °C for 12 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (2 mL). The crude product was separated and purified by preparative high-performance liquid chromatography (column type: Xtimate C18 100 × 30 mm × 3 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 50% to 70%, 5 min) to obtain compound 6'. 1 H NMR (400 MHz, deuterated methanol) δ 7.59 (d, J=7.53 Hz, 1H), 7.19-7.32 (m, 2H), 7.02-7.19 (m, 2H), 6.81-6.96 (m, 2H), 5.86 (d, J=7.53 Hz, 1H), 5.44-5.63 (m, 2H), 4.26 (br d, J=15.06 Hz, 1H), 4.11 (d, J=12.55 Hz, 1H), 3.07 (br d, J=15.06 Hz, 1H), 1.81-1.98 (m, 1H), 1.57-1.73 (m, 1H), 0.90-1.14 (m, 2H). MS(ESI)m / z: 501.1[M+H] + .

[0171] Biological Test Data Experimental example 1: Influenza virus cytopathic effect (CPE) experiment The 50% effective concentration (EC 50 The antiviral activity of the compounds against influenza virus (IFV) was evaluated by measuring the cytopathicity (Cytopathic) value. Cytopathic experiments are widely used to measure the protective effect of compounds against virus-infected cells to reflect the antiviral activity of the compounds.

[0172] Influenza virus CPE experiment MDCK cells were seeded into black 384-well cell culture plates at a density of 2000 cells per well, and then cultured overnight in a 37°C, 5% CO2 incubator. Compounds were diluted with an Echo555 acoustic technology non-contact nanoliter dispensing system and added to the cell wells (4-fold dilution, 8 test concentration points). Influenza virus A / PR / 8 / 34 (H1N1) strain was then added to the cell culture wells at 1–2 90% tissue culture infectious doses (TCID90) per well, and the final concentration of DMSO in the medium was 0.5%. Virus control wells (DMSO and virus added, no compound), cell control wells (DMSO added, no compound and virus), and medium control wells (medium only, no cells) were set up. Cytotoxicity measurements and antiviral activity measurements of compounds were performed in parallel, and other experimental conditions were consistent with the antiviral activity experiments, except that no virus was added. The cell plates were cultured for 5 days in a 37°C, 5% CO2 incubator. After 5 days of culture, cell viability was detected using the cell viability detection kit CCK8. The raw data were used to calculate the antiviral activity and cytotoxicity of the compounds.

[0173] The antiviral activity and cytotoxicity of a compound are expressed as the inhibition rate (%) of the compound against the cellular viral effect of the virus, respectively. The calculation formula is as follows:

[0174]

number

[0175] GraphPad Prism software was used to perform nonlinear fitting analysis of the inhibition rate and cytotoxicity of the compounds to determine the EC 50 The experimental results are shown in Table 3.

[0176] [Table 3]

[0177] Conclusion: The compounds of the present invention showed positive effects in the inhibition test of influenza virus replication at the cellular level.

[0178] Experimental Example 2: In vivo drug efficacy test Experimental objective: To evaluate the efficacy of compounds in a mouse model of influenza A virus H1N1 infection.

[0179] Experimental protocol: Mice were infected with influenza A virus A / PR / 8 / 34 (H1N1) by intranasal instillation, and treated with compounds from 48 hours after infection, orally administered twice a day for 7 consecutive days. The anti-influenza A virus H1N1 effect of the compounds in this model was evaluated by observing the body weight change and survival rate of the mice.

[0180] SPF grade BALB / c mice, 6-7 weeks, female, were used for the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to acclimate for at least 3 days before the experiment began. The day of infection was designated as experimental day 0. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg / kg, 10 mL / kg), and after the mice entered a deep anesthetized state, the mice were infected with the A / PR / 8 / 34 (H1N1) virus by intranasal instillation, with an infection volume of 50 μL. From day 2 to day 8, the test compound was orally administered twice a day at 5 mg / kg (administration volume 10 mL / kg). The first administration time was 48 hours after infection. The condition of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.

[0181] Test Results: The survival rate and weight loss rate of the animals were detected, and the weight loss rate was calculated as follows: weight loss rate = (weight on day 0 - weight on day N) / weight on day 0 × 100%. The results are shown in Table 4: Compound 5 could protect the animals with a maximum weight loss rate of 13.77% on day 7, and then began to recover, and the survival rate of the mice at the end of the experiment was 100%. Compound 4 could protect the animals with a maximum weight loss rate of 7.03% on day 3, and then began to recover, and the survival rate of the mice at the end of the experiment was 100%.

[0182] [Table 4]

[0183] Conclusion: The compounds of the present invention show excellent weight protection and rapid recovery time in animal in vivo efficacy models.

[0184] Experimental Example 3: Cytopathic Effect (CPE) Experiment of Baloxavir-Resistant A / PR / 8 / 34(H1N1)I38T Influenza Virus Strain

[0185] Purpose of the experiment: To determine the 50% effective concentration (EC 50 The objective of the present invention is to evaluate the antiviral activity of compounds against the baloxavir-resistant A / PR / 8 / 34(H1N1)I38T influenza virus strain by measuring the .DELTA.(H1N1) value.

[0186] Experimental protocol: MDCK cells were seeded into 96-well cell culture plates at a density of 15000 cells per well and cultured overnight in a 37℃, 5% CO2 incubator. The next day, compound solutions (3-fold serial dilutions, 8 concentration points, three replicate wells) and baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strain were added, and the final concentration of DMSO in the cell culture medium was 0.5%. The cells were cultured for 5 days in a 5% CO2, 37℃ incubator until the cell degeneration in the virus-infected control wells without compounds reached 80-95%. Then, CCK8 was used to detect the cell viability of each well. If the cell viability of the wells containing the compound is higher than that of the virus-infected control wells, i.e., the CPE is weakened, it indicates that the compound has an inhibitory effect on the test virus.

[0187] Test Results: The antiviral activity of a compound is expressed as the inhibitory activity (%) of the compound against the cytoviral effect of the virus. The calculation formula is as follows:

[0188] [ka]

[0189] EC 50 We used GraphPad Prism (version 5) software to perform a nonlinear fitting analysis of the inhibitory activity of the compounds and the cell viability, and the fitting method was "log(inhibitor) vs. response--Variable slope". The experimental results are shown in Table 5.

[0190] [Table 5]

[0191] Conclusion: The compounds of the present invention show positive effects in the inhibition test of baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strain replication at the cellular level.

[0192] Experimental Example 4: In vivo drug efficacy test Experimental objective: To evaluate the efficacy of compounds in a mouse model of infection with a drug-resistant strain of influenza A virus H1N1.

[0193] Experimental protocol: Mice were infected with influenza A virus baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T virus strain by intranasal instillation, and treated with compounds 2 hours before infection and orally administered twice daily for 7 consecutive days. The anti-influenza A virus H1N1 effect of the compounds in this model was evaluated by observing the body weight change and survival rate of the mice.

[0194] SPF grade BALB / c mice, 6-7 weeks, female, were used for the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to acclimate for at least 3 days before the experiment began. The day of infection was designated as experimental day 0. After the mice were deeply anesthetized by intraperitoneal injection of Zoletil 50 / xylazine hydrochloride, they were infected with the baloxavir-resistant A / PR / 8 / 34(H1N1)I38T virus strain by intranasal instillation, with an infection volume of 50 μL. From day 2 to day 8, the test compound was orally administered twice daily at 15 mg / kg or 50 mg / kg (dosage volume 10 mL / kg). The first administration time was 2 hours before infection. The condition of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.

[0195] Test Results: The survival rate and weight loss rate of the animals were detected, and the weight loss rate was calculated as follows: weight loss rate = (weight on day 0 - weight on day N) / weight on day 0 x 100%. The experimental results are shown in Table 6 below. When compound 4 was administered at a dose of 50 mg / kg, the mice showed little weight loss, and the survival rate of the mice at the end of the experiment was 100%.

[0196] [Table 6]

[0197] Conclusion: The compounds of the present invention show excellent weight protection and rapid recovery time in animal in vivo efficacy models.

[0198] Experimental Example 5 Plasma protein binding rate test of compounds Experimental objective: To evaluate the protein binding rate of the compounds of the present invention in CD-1 mice, SD rats and human plasma by equilibrium dialysis.

[0199] Test protocol: The test compound was diluted into the above five kinds of plasma with dialysis buffer to prepare samples with a final concentration of 2 μM. The samples were then added to a 96-well equilibrium dialysis device and dialyzed with phosphate buffer at 37°C for 4 hours. Warfarin was used as a control compound in the experiment. The concentrations of the test compound and warfarin in the plasma and buffer were measured by LC-MS / MS.

[0200] Experimental results: The results are shown in Table 7.

[0201] [Table 7]

[0202] Conclusion: The compounds of the present invention have moderate plasma protein binding rates in five types of plasma, which indicates that the free drug concentration ratios of the test compounds are moderate in the above five types of plasma, and have good drug discovery properties.

[0203] Experimental Example 6: Pharmacokinetic study in rats Experimental Objective: To study the plasma pharmacokinetics of the compounds of the present invention in male SD rats after a single intravenous injection and intragastric administration.

[0204] Experimental animals: male SD rats, 6–8 weeks old, weighing 200–300 g;

[0205] Experimental procedure: for injection (iv), the dose was 1mpk, the concentration was 0.50mg / mL, and the solvent was 40% DMAC + 40% PG + 20% (20% HP-β-CD + water); for oral administration (po), the dose was 10mpk, the concentration was 1mg / mL, and the solvent was 3% DMSO + 10% Solutol HS + 87% water.

[0206] Sample collection: 0.03 mL blood samples were collected from the saphenous vein of the experimental animals by puncture at each time point, and the actual blood collection time was recorded. All blood samples were added to 1.5 mL commercial EDTA-K2 anticoagulant tubes. DDV was added as a stabilizer to the plasma matrix after blood collection, where plasma:dichlorvos solution=40:1, the dichlorvos solution was a 40 mM dichlorvos solution in acetonitrile / water (1:1), and centrifuged at 3000 g for 10 min at 4°C within 30 min to aspirate the supernatant plasma, which was immediately placed on dry ice and stored in a -80°C refrigerator for LC-MS / MS analysis.

[0207] Data analysis: Plasma concentrations were treated using a noncompartmental model in Phoenix WinNonlin 6.3 pharmacokinetic software, and the linear-logarithmic trapezoidal method was used to derive the following pharmacokinetic parameters: Cl (apparent clearance rate), T 1 / 2 (time required to clear half of the compound), C max (peak concentration), AUC 0-last (integrated area under the concentration-time curve from 0 to the last sampling time) was calculated and the results are shown in Table 8.

[0208] [Table 8]

[0209] Experimental conclusion: The compounds of the present invention have relatively high oral plasma exposure and good pharmacokinetic properties.

[0210] Experimental Example 7: Pharmacokinetics study in beagle dogs Experimental Objective: To study the plasma pharmacokinetics of the compounds of the present invention in male beagle dogs after a single intravenous injection and intragastric administration.

[0211] Experimental animals: male beagle dogs, aged 6 months or older, weighing 6–12 kg;

[0212] Experimental procedure: for intravenous administration (iv), the dose was 1 mpk, the concentration was 1 mg / mL, and the solvent was 10% DMAC + 90% (20% HP-β-CD + water); for oral administration (po), the dose was 10 mpk, the concentration was 2 mg / mL, and the solvent was 3% DMSO + 10% Solutol HS + 87% water.

[0213] Sample collection: 0.8 mL blood samples were collected from the saphenous vein of the experimental animals by puncture at each time point, and the actual blood collection time was recorded. All blood samples were added to 1.5 mL commercial EDTA-K2 anticoagulant tubes. DDV was added as a stabilizer to the plasma matrix after blood collection, where plasma:dichlorvos solution=40:1, the dichlorvos solution was a 40 mM dichlorvos solution in acetonitrile / water (1:1), and centrifuged at 3000 g for 10 min at 4°C within 30 min to aspirate the supernatant plasma, which was immediately placed on dry ice and stored in a -80°C refrigerator for LC-MS / MS analysis.

[0214] Data Analysis: Plasma concentrations were treated using a noncompartmental model in Phoenix WinNonlin 6.3 pharmacokinetic software, 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 Table 9.

[0215] [Table 9]

[0216] Experimental conclusion: The compounds of the present invention have low clearance rates, long half-lives, high oral plasma exposure, and good pharmacokinetic properties.

Claims

1. A compound selected from the following formulas or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】 (however, R 5 and R 6 are each independently selected from H, F, Cl, Br, I, OH, NH 2 , —COOH, C 1-3 alkyl, C 1-3 alkoxy and C 1-3 alkylamino, wherein said C 1-3 alkyl, C 1-3 alkoxy and C 1-3 alkylamino are each independently optionally substituted by 1, 2 or 3 R b ; R ７ H and 【Chemistry 2】 is selected from R ８ is C １－３ Alkyl and 【Chemistry 3】 and C is selected from １－３ Alkyl and 【Chemistry 4】 is one, two or three R ａ and optionally replaced by p is selected from 0 and 1; E １ and E ２ one is selected from Se and the other is selected from S and O; q is selected from 0 and 1; Each R ａ and R ｂ are each independently selected from H, F, Cl, Br, and I; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in a form enriched in one enantiomer.

2. R ８ is CH ３ CH ２ CH ３ CH ２ CH ２ CH ３ CH(CH ３ ) ２ and 【Chemistry 5】 wherein said CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ and 【Chemistry 6】 is one, two or three R ａ 2. The compound of claim 1, optionally substituted by: or a pharma- ceutically acceptable salt thereof.

3. R ８ is CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ and 【Chemistry 7】 3. The compound of claim 2, selected from:

4. R ７ H, 【Chemistry 8】 2. The compound of claim 1, selected from:

5. E １ is selected from Se, E ２ or a pharma- ceutically acceptable salt thereof.

2. The compound of claim 1, wherein

6. 2. The compound of claim 1 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 9】 (however, R １ and R ２ are each independently H, F, Cl, Br, I, OH, and NH ２ is selected from m is selected from 0 and 1; q, R ５ , R ６ , R ７ and R ８ is as defined in claim 1.

7. R １ and R ２ is independently selected from F, or a pharma- ceutically acceptable salt thereof.

8. R ５ and R ６ is independently selected from F, or a pharma- ceutically acceptable salt thereof.

9. 7. The compound of claim 6 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 10】 (However, R １ , R ２ , R ５ , R ６ , R ７ and R ８ is as defined in claim 6.

10. A compound of the formula: or a pharma- ceutically acceptable salt thereof. 【Chemistry 11】

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 or a pharma- ceutically acceptable salt thereof for treating a disease associated with influenza virus.

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