Pyridone multiple fusion ring derivatives and their uses

Abstract

Provides a pyridone multiple fusion ring derivative and its uses, specifically a compound represented by formula (VI) and a pharmaceutically acceptable salt thereof.

Description

Technology Field

[0001] The present invention claims the following priority.

[0002] CN 202110024885.9, Filing Date: January 8, 2021;

[0003] CN 202110264686.5, Filing Date: March 11, 2021;

[0004] CN 202110513447.9, Filing Date: May 11, 2021.

[0005] The present invention relates to pyridone multiple fused cyclic derivatives and uses thereof, specifically to a compound represented by formula (VI) and a pharmaceutically acceptable salt thereof. Background Technology

[0006] The influenza virus (IFV), also known as the epidemic cold virus, is a segmented single-stranded antisense RNA virus capable of causing influenza in humans and animals. Influenza viruses can cause very high morbidity and mortality rates; in particular, Type A influenza viruses can cause global pandemics such as the "Spanish Influenza" (H1N1 subtype) of 1918–1920, the "Asian Influenza" (H2N2 subtype) of 1957–1958, the "Asian Influenza" (H3N2 subtype) of 1968–1969, the "Hong Kong Influenza" (H1N1 subtype) of 1977–1978, and the Type A H1N1 influenza that first emerged in Mexico in March 2009. Large-scale influenza outbreaks have resulted in thousands of deaths, causing immense social panic and amplifying social anxiety.

[0007] Influenza A virus is a single-negative RNA virus, and its genome is divided into eight fragments encoding eight proteins. The 5' and 3' ends of the influenza virus genome fragments are highly conserved; the sequences of these two ends form a complementary stem-loop structure, which plays a crucial role in initiating viral RNA replication. The proteins encoded by each viral gene fragment differ in size and play different roles in the influenza virus life cycle; first, the basic functions of several key proteins are introduced as follows. The HA of the influenza virus acts as a ligand that recognizes the host receptor; it binds to the virus's specific receptor on the cell surface and mediates the fusion of the viral envelope and the intracellular body membrane, thereby releasing the viral nucleocapsid to enter the cytoplasm. The influenza virus receptor is specific, and the receptor for Influenza A virus is a sialoglycan protein. The NA protein of the influenza virus can remove sialic acid from the surface of viral particles during replication, preventing the virus particles from continuing to aggregate on the surface of host cells; this facilitates the release of virions and leads to the infection of more host cells.

[0008] Role of the M2 protein of the influenza virus: The HA protein of the influenza virus binds to sialic acid, causing the influenza virus to undergo endocytosis by the host cell. The acidity and alkalinity of the phagocytic vesicle play a crucial role in the shedding of the virus. The ion channel of the M2 protein located in the viral membrane can gradually decrease the pH value of the phagocytic vesicle; when the pH drops to between 5.0 and 6.0, it induces a structural change in the HA2 protein and moves the fusion peptide located at the amino terminus of the HA2 protein, thereby activating the fusion process, which fuses the viral bilipid membrane with the cell membrane and releases RNPs from within the viral particle into the host cytoplasm. The M2 protein is a transmembrane ion channel found only in type A influenza virus, with a portion of it extending to the surface of the viral outer membrane.

[0009] The synthesis of influenza virus proteins also utilizes the translation mechanisms of host cells, and the virus can even accelerate the synthesis of its own proteins by halting the translation of host proteins. Unlike the polyadenylation of host cell mRNA, which is completed by specific adenylases, the adenylate tail of viral mRNA is formed by the transcription of 5 to 7 consecutive uracils from the negative strand of vRNA. The capping of each viral messenger RNA (mRNA) is completed in a similar manner. PA and PB2 proteins capture the 5' cap primer of the host pre-mRNA transcript to initiate the synthesis of viral mRNA; this process is called "cap snatching" and is primarily completed by the viral RNA-dependent RNA polymerase (RdRp), the PA subunit of which possesses RNA endonuclease activity and serves to cleave the host mRNA. When the polyadenylation and capping processes are completed, the viral mRNA exits the nucleus, enters the cytoplasm, and is translated like the host cell's mRNA; the nuclear export of viral vRNA fragments is mediated by the viral M1 protein and NS2 protein, and when the M1 protein can interact with vRNA and NP proteins, it can also simultaneously interact with the nuclear export protein NS2; from this, the nuclear export protein NS2 mediates the M1-RNP to exit the nucleus in the form of a nuclear protein and enter the cytoplasm of the host cell.

[0010] Influenza generates direct costs resulting from the loss of productivity and related medical resources, as well as indirect costs associated with preventive measures. In the United States, influenza causes a cumulative annual loss of approximately $10 billion, and future influenza pandemics are estimated to result in hundreds of billions of dollars in direct and indirect costs. Prevention costs are also very high, and governments around the world have already spent billions of dollars on preparedness and planning for a potential H5N1 avian influenza pandemic, the associated costs and procurement of drugs and vaccines, the development of disaster drills, and related strategies to improve border controls.

[0011] Current treatment options for influenza include vaccination and chemotherapy and chemoprevention using antiviral drugs. For example, anti-influenza vaccination is often recommended for high-risk groups, such as children, the elderly, or people with asthma, diabetes, or heart disease; however, receiving the vaccine does not guarantee complete prevention of the flu. While vaccines for certain specific influenza strains are manufactured seasonally, it is impossible to cover all types of viral variants in people who are voluntarily infected worldwide during that season. Furthermore, because influenza viruses exhibit a certain degree of antigenic drift, when one or more viruses infect a single cell, eight individual vRNA fragments within the genome are mixed or rearranged; these rapid changes in induced viral genetics cause antigenic shifts, allowing the virus to infect a new host species and rapidly overcome protective immunity.

[0012] Antiviral drugs can also be used to treat influenza; while neuraminidase inhibitors such as oseltamivir (Tamiflu) are significantly effective against influenza A virus, clinical observations have revealed the emergence of viral strains resistant to this type of neuraminidase inhibitor. In the field of anti-influenza, there is an urgent need for anti-influenza drugs with novel mechanisms of action that can either support the clinical use of a single drug for the treatment of influenza A or be used for the prevention and treatment of influenza A in combination with other commercially available anti-influenza drugs with different mechanisms of action. Here, WO2016175224 reported RNA polymerase PA subunit inhibitors such as S-033447 and its prodrug, S-033188.

[0013] Prodrug:

[0014] The present invention provides a compound represented by formula (VI) or a pharmaceutically acceptable salt thereof, and

[0015] ,

[0016] In the above formula,

[0017] R7 is H and Selected from;

[0018] R8 is C 1-3 alkyl and Selected from, and the above C 1-3 alkyl and is 1, 2, or 3 R a Arbitrarily substituted by;

[0019] 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 atoms jointly connected to it.3-5 Forming a cycloalkyl;

[0020] Or, X1 and R9 together with the atoms connected thereto It forms, 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;

[0021] R 12 is H, F, Cl, Br, I, OH, NH2, -COOH, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Selected from alkylaminos, and C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Each alkylamino has independently 1, 2, or 3 R groups b Arbitrarily substituted by;

[0022] T1, T2, T3, and T4 are each independently selected from CH and N;

[0023] q is selected from 0 and 1;

[0024] t is selected from 0, 1, 2, 3, and 4;

[0025] Each R a and R b Each is independently selected from H, F, Cl, Br, and I;

[0026] The condition is that if T1 is selected from CH, and 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 Each is independently selected from OH and NH2.

[0027] In some aspects of the present invention, each R 12 Each is independently selected from F, and other variables are as defined in the present invention.

[0028] In some aspects of the present invention, the R8 is CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and Selected from, and the above CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and is 1, 2, or 3 R a It is arbitrarily substituted by, and other variables are as defined in the present invention.

[0029] In some aspects of the present invention, the R8 is CH3, CH2CH3, CH(CH3)2 and Selected from, and other variables as defined in the present invention.

[0030] In some aspects of the present invention, the R8 is CH3 and Selected from, and other variables as defined in the present invention.

[0031] In some aspects of the present invention, the R7 is H, , , and Selected from, and other variables as defined in the present invention.

[0032] In some aspects of the present invention, the R7 is H, and Selected from, and other variables as defined in the present invention.

[0033] In some aspects of the present invention, the R7 is H and Selected from, and other variables as defined in the present invention.

[0034] In some aspects of the present invention, E1 is selected from Se, E2 is selected from O, and other variables are as defined in the present invention.

[0035] In some aspects of the present invention, the structural unit Is Selected from, where R5 and R6 are each independently H, F, Cl, Br, I, OH, NH2, -COOH, C1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Selected from alkylaminos, and C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Each alkylamino has independently 1, 2, or 3 R groups b It is arbitrarily substituted by, and other variables are as defined in the present invention.

[0036] In some aspects of the present invention, the structural unit Is , , and Selected from, and other variables as defined in the present invention.

[0037] In some aspects of the present invention, the structural unit Is Selected from, and other variables as defined in the present invention.

[0038] In some aspects of the present invention, R5 is selected from F, and other variables are as defined in the present invention.

[0039] In some embodiments of the present invention, R6 is selected from F, and other variables are as defined in the present invention.

[0040] In some aspects of the present invention, the structural unit Is , , and Selected from, and other variables as defined in the present invention.

[0041] In some aspects of the present invention, the structural unit Is , , and Selected from, and other variables as defined in the present invention.

[0042] In some aspects of the present invention, the structural unit Is Selected from, and other variables as defined in the present invention.

[0043] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0044]

[0045] In the above formula,

[0046] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2;

[0047] R7 is H and Selected from;

[0048] R8 is C 1-3 alkyl and Selected from, and the above C 1-3 alkyl and is 1, 2, or 3 R a Arbitrarily substituted by;

[0049] 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 atoms jointly connected to it. 3-5 Forming a cycloalkyl;

[0050] Or, X1 and R9 together with the atoms connected thereto Forms,

[0051] One of E1 and E2 is selected from Se, and the other is selected from S and O;

[0052] T1 is selected from CH and N;

[0053] p and q are each independently selected from 0 and 1;

[0054] Each R a Each is independently selected from H, F, Cl, Br, and I;

[0055] The condition is that when T1 is selected from CH, and E1 is selected from Se, and E2 is selected from O, p is selected from 1, and q is selected from 1, R5 and R6 are independently selected from OH and NH2, respectively;

[0056] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0057] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0058] and ,

[0059] In the above formula,

[0060] p, q, E1, E2, T1, R5, R6, and R7 are as defined in the present invention;

[0061] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0062] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0063] and ,

[0064] In the above formula,

[0065] p, q, E1, E2, R5, R6, and R7 are as defined in the present invention;

[0066] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0067] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0068] and ,

[0069] In the above formula,

[0070] p, q, E1, E2, R5, R6, and R7 are as defined in the present invention;

[0071] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0072] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0073]

[0074] and ,

[0075] In the above formula,

[0076] R1 and R2 are each independently selected from H, F, Cl, Br, I, OH, and NH2;

[0077] m is selected from 0 and 1;

[0078] q, R5, R6, R7, and R8 are as defined in the present invention.

[0079] In some aspects of the present invention, R1 and R2 are each independently selected from F, and other variables are as defined in the present invention.

[0080] In some aspects of the present invention, R5 and R6 are each independently selected from F, and other variables are as defined in the present invention.

[0081] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0082]

[0083] and ,

[0084] In the above formula, R1, R2, R5, R6, R7, and R8 are as defined in the present invention.

[0085] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0086]

[0087]

[0088] and ,

[0089] In the above formula,

[0090] R1, R2, R3, and R4 are each independently selected from H, F, Cl, Br, I, OH, and NH2;

[0091] n and m are each independently selected from 0 and 1;

[0092] The condition is that, in equations (I-1) and (I-2), when m is selected from 1, R1 and R2 are each independently selected from OH and NH2;

[0093] q, R5, R6, and R8 are as defined in the present invention.

[0094] In some aspects of the present invention, R1 and R2 are each independently selected from F, and other variables are as defined in the present invention.

[0095] In some embodiments of the present invention, R3 and R4 are each independently selected from F, and other variables are as defined in the present invention.

[0096] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0097]

[0098]

[0099]

[0100]

[0101] and ,

[0102] In the above formula,

[0103] R1, R2, R3, R4, R5, R6, R7 and R8 are as defined in the present invention.

[0104] The present invention provides a compound represented by formula (V) or a pharmaceutically acceptable salt thereof, and

[0105]

[0106] In the above formula,

[0107] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2;

[0108] R7 is H and Selected from;

[0109] R8 is C 1-3alkyl and Selected from, and the above C 1-3 alkyl and is 1, 2, or 3 R a Arbitrarily substituted by;

[0110] R9 is selected from H;

[0111] X1 is CR 10 R 11 Selected from, R 10 and R 11 C together with atoms jointly connected to it. 3-5 Forming a cycloalkyl;

[0112] Or, X1 and R9 are connected together Forming;

[0113] E1 is selected from S and Se;

[0114] E2 is selected from O and Se, and at least one of E1 and E2 is selected from Se;

[0115] T1 is selected from CH and N;

[0116] p and q are each independently selected from 0 and 1;

[0117] Each R a Each is independently selected from H, F, Cl, Br, and I;

[0118] The condition is that when T1 is selected from CH, and E1 is selected from Se, and E2 is selected from O, p is selected from 1, and q is selected from 1, R5 and R6 are independently selected from OH and NH2, respectively;

[0119] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0120] The present invention provides a compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, and

[0121]

[0122] In the above formula,

[0123] T1 is selected from CH and N;

[0124] E1 is selected from S and Se;

[0125] E2 is selected from O and Se, and at least one of E1 and E2 is selected from Se;

[0126] R5 and R6 are each independently selected from H, F, Cl, Br, I, OH, and NH2;

[0127] R7 is H and Selected from;

[0128] R8 is C 1-3 alkyl and Selected from, and the above C 1-3 alkyl and is 1, 2, or 3 R a Arbitrarily substituted by;

[0129] p and q are each independently selected from 0 and 1;

[0130] Each R a Each is independently selected from H, F, Cl, Br, and I;

[0131] The condition is that when T1 is selected from CH, and E1 is selected from Se, E2 is selected from O, p is selected from 1, and q is selected from 1, R5 and R6 are independently selected from OH and NH2, respectively;

[0132] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0133] In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formula, and

[0134]

[0135] In the above formula,

[0136] p, q, E1, E2, R5, R6, and R7 are as defined in the present invention;

[0137] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0138] The present invention provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof, and

[0139]

[0140] In the above formula,

[0141] T1 is selected from CH and N;

[0142] R1 is selected from H, F, Cl, Br, I, OH, and NH2;

[0143] R2 is selected from H, F, Cl, Br, I, OH, and NH2;

[0144] m is selected from 0 and 1;

[0145] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0146] The present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, and

[0147]

[0148] In the above formula,

[0149] R3 is selected from H, F, Cl, Br, I, OH, and NH2;

[0150] R4 is selected from H, F, Cl, Br, I, OH, and NH2;

[0151] n is selected from 0 and 1;

[0152] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0153] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and

[0154]

[0155] In the above formula, R1, R2 and m are as defined in the present invention.

[0156] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and

[0157]

[0158] In the above formula,

[0159] R1 is selected from H, F, Cl, Br, I, OH, and NH2;

[0160] R2 is selected from H, F, Cl, Br, I, OH, and NH2;

[0161] m is selected from 0 and 1;

[0162] The carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer.

[0163] In some embodiments of the present invention, R1 is selected from F, and other variables are as defined in the present invention.

[0164] In some embodiments of the present invention, R2 is selected from F, and other variables are as defined in the present invention.

[0165] In some embodiments of the present invention, R3 is selected from F, and other variables are as defined in the present invention.

[0166] In some aspects of the present invention, R4 is selected from F, and other variables are as defined in the present invention.

[0167] Another aspect of the present invention is obtained by arbitrarily combining the above variables.

[0168] The present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof.

[0169] and .

[0170] The present invention further provides the use of the compound or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating influenza virus-related diseases.

[0171] Effects of the invention

[0172] The compound of the present invention is an RNA polymerase inhibitor and exhibits a positive effect in tests inhibiting the replication of influenza virus at the cellular level, exhibits excellent body weight protection in animal in vivo efficacy models, has a fast recovery time, and plasma protein binding rate test results indicate that the compound of the present invention has an intermediate plasma protein binding rate in plasma, and PK results indicate that it has excellent pharmacokinetic properties and excellent drug manufacturing properties. Brief explanation of the drawing

[0173] Figure 1 shows the 3D binding mode of S-033447 and protein (PDB ID: 6FS6). Figure 2 shows the interaction between S-033447 and amino acids and metal ions. Figure 3 is the dihedral angle of the S-033447 low-energy form. Figure 4 shows the change in energy of the two dihedral angles of S-033447 during the rotation process. Figure 5 shows a compound A This is a comparison of the low-energy forms of (dark color) and S-033447 (bright color). Fig. 6 shows a compound during the rotation process A It is the change in energy of the two dihedral angles. Figure 7 shows a compound B This is a comparison of the low-energy forms of (dark color) and S-033447 (bright color). Fig. 8 shows a compound during the rotation process B It is the change in energy of the two dihedral angles. Specific details for implementing the invention

[0174] Related definitions

[0175] Unless otherwise specified, the following terms and phrases used in this text have the following meanings. No specific term or phrase shall be considered indeterminate or unclear unless specifically defined otherwise, and shall be understood in its ordinary meaning. Where a product name appears in this text, it indicates the corresponding product or its active ingredient.

[0176] As used herein, the term “pharmaceuticalally acceptable” means that, within the scope of reliable medical judgment, such compounds, materials, compositions and / or formulations are suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, hypersensitivity reactions or other problems or complications, and have a reasonable benefit / risk ratio.

[0177] The term “pharmaceutically acceptable salt” refers to a salt of a compound of the present invention, prepared with a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base. If the compound of the present invention contains a relatively acidic functional group, a base addition salt may be obtained by contacting such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. If the compound of the present invention contains a relatively basic functional group, an acid addition salt may be obtained by contacting such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic salts, said inorganic acids including, for example, hydrochloric acid, hydrobromide, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodide, phosphite, etc.; and includes organic acid salts, wherein the organic acid includes, 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, and methanesulfonic acid; further include salts of amino acids (e.g., arginine, etc.) and salts of organic acids such as glucuronic acid. Some specific compounds of the present invention may include basic and acidic functional groups and may be converted into any base or acid addition salt.

[0178] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods with a parent compound containing an acid group or a base. In general, the method of preparing such salts is to react such compounds in the form of a free acid or base with a chemically weighed suitable base or acid in water or an organic solvent or a mixture of both.

[0179] The compounds of the present invention may exist in specific geometric or stereoisomer forms. These compounds anticipated in the present invention include cis and trans isomers, (-)- and (+)- enantiomers, such as mixtures rich in enantiomers or diastereomers, ( R )- and ( S )-enhanced mirror image isomer, diastereomer, ( D )-isomer, ( L )-isomers, and mixtures of racemic mixtures and other mixtures, all of which fall within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and mixtures thereof fall within the scope of the present invention.

[0180] The compound of the present invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, tritium ( 3 H), iodine-125( 125 I) or C-14( 14 Compounds can be labeled with radioactive isotopes such as C). As another example, deuterated drugs can be formed by replacing hydrogen with deuterium; since the bond composed of deuterium and carbon is stronger than the bond composed of ordinary hydrogen and carbon, deuterated drugs have advantages over non-deuterated drugs, such as reduced side effects, increased drug stability, enhanced drug efficacy, and extended biological half-life. All transformations composed of isotopes of the compounds of the present invention, whether radioactive or not, fall within the scope of the present invention.

[0181] The terms “arbitrarily” or “optional” refer to the fact that an event or situation may appear in the description below, including cases where the event or situation occurs and cases where the event or situation does not occur, but does not necessarily appear.

[0182] The term “substituted” refers to the substitution of any one or more hydrogen atoms of a specific atom by a substituent, and the substituent may include variants of deuterium and hydrogen in which the valence of the specific atom is normal and the compound after substitution is stable. If the substituent is a ketone group (i.e., =O), it means that two hydrogen atoms are substituted. Ketone substitution does not occur in aromatic groups. The term “arbitrarily substituted” means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary in terms of chemical feasibility.

[0183] If any variable (e.g., R) appears one or more times in the composition or structure of a compound, the definition in each case is independent. Thus, for example, if one radical is substituted by 0 to 2 Rs, said radical may optionally be substituted by 2 or fewer Rs, and R in each case is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations produce a stable compound.

[0184] When the number of a single connector is 0, as in -(CRR)0-, the connector represents a single connection.

[0185] If one of the variables is selected from a single bond, it indicates that the two radicals are directly connected; for example, if L in ALZ is a single bond, the structure is substantially AZ.

[0186] If the connection direction of the listed connectors is not specified, the connection direction is arbitrary, for example, In this case, connector L is -MW-, where -MW- connects loop A and loop B in the same direction as the left-to-right reading order. It can form, and by connecting ring A and ring B from left to right in the opposite direction to the reading order It can form. The combination of the linker, substituent, and / or variant thereof is permitted only if such combination produces a stable compound.

[0187] Unless otherwise specified, if any radical has one or more connectable sites, any one or more sites of said radical may be connected to other radicals through chemical bonds. The form of the chemical bond connection is amorphous, and if H atoms are present in the connectable sites, upon chemical bond connection, the number of H atoms in the site is correspondingly reduced according to the number of connected chemical bonds, transforming into a radical of the corresponding valence. The chemical bond connecting said site to another radical is a straight solid line bond ( ), straight dotted line combination( ), or wavy line ( It can be represented as ). For example, the straight solid bond of -OCH3 indicates that it is connected to another radical through the oxygen atom in the radical; The straight dotted line connection indicates that it is connected to another radical through both ends of the nitrogen atom in the radical; The wavy line indicates that it is connected to another radical through the carbon atoms of the first and second sites of the phenyl radical; This means that any connectable site of the piperidinyl group can be connected to another radical through a single chemical bond, and at least , , , It includes four connection types, and even if an H atom is drawn on -N- is still It includes a radical of the linked form, and when only one chemical bond is connected, the H at the above site is accordingly reduced by one to become the corresponding monovalent piperidinyl group.

[0188] Unless otherwise specified, the term “C1-3 “Alkyl group” represents a saturated hydrocarbon group composed of 1 to 3 carbon atoms in a straight or branched chain. The above C 1-3 The alkyl group is C 1-2 and C 2-3 It includes alkyl groups, etc.; it may be monovalent (e.g., methyl group), divalent (e.g., methylene group), or polyvalent (e.g., methine group). C 1-3 Examples of alkyl groups include methyl (Me), ethyl (Et), and propyl groups (e.g., n - Includes, but is not limited to, propyl groups and isopropyl groups) etc.

[0189] Unless otherwise specified, the term “C 1-3 "Alkoxy group" represents an alkyl group comprising 1 to 3 carbon atoms connected to the rest of the molecule through one oxygen atom. The above C 1-3 The alkoxy group is C 1-2 , C 2-3 Includes C3 and C2 alkoxy groups, etc. C 1-3 Examples of alkoxy groups include, but are not limited to, methoxy groups, ethoxy groups, and propoxy groups (including n-propoxy groups and isopropoxy groups).

[0190] Unless otherwise specified, the term “C 1-3 “Alkylamino group” refers to an alkyl group comprising 1 to 3 carbon atoms connected to the rest of the molecule through an amino group. The above C 1-3 The alkylamino group is C 1-2 , includes C3 and C2 alkylamino groups, etc. C 1-3 Examples of alkylamino groups include, but are not limited to, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH3)CH2CH3, -NHCH2CH2CH3, -NHCH2(CH3)2.

[0191] Unless otherwise specified, the term “C 3-5 The “cycloalkyl group” represents a saturated cyclic hydrocarbon group composed of 3 to 5 carbon atoms, which is a monocyclic system, and the above C3-5 The cycloalkyl group is C 3-4 and C 4-5 It includes cycloalkyl groups, etc.; it may be monovalent, divalent, or polyvalent. C 3-5 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl groups, cyclobutyl groups, and cyclopentyl groups.

[0192] Unless otherwise specified, C n-n+m or C n -C n+m It includes any one specific case of n to n+m carbons, for example, C 1-12 C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 It includes, and also includes any one range from 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 Includes the like; likewise, n to n+m members indicate that the number of atoms in the ring is n to n+m, for example, 3 to 12 members include 3 members, 4 members, 5 members, 6 members, 7 members, 8 members, 9 members, 10 members, 11 members, and 12 members, and also includes any one of the ranges from n to n+m, for example, 3 to 12 members include 3 to 6 members, 3 to 9 members, 5 to 6 members, 5 to 7 members, 6 to 7 members, 6 to 8 members, and 6 to 10 members, etc.

[0193] The term “leaving group” refers to a functional group or atom that has been substituted by another functional group or atom through a substitution reaction (e.g., nucleophilic substitution). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, and iodine; sulfonate groups such as methanesulfonate, tosylate, p-bromobenzenesulfonate, and p-toluenesulfonate; and acyloxy groups such as acetoxy groups and trifluoroacetoxy groups.

[0194] The term “protecting group” includes, but is not limited to, “amino protecting group,” “hydroxyl protecting group,” or “mercapto protecting group.” The term “amino protecting group” refers to a protecting group suitable for preventing side reactions at the nitrogen position of an amino group. Representative amino protecting groups include the formyl group; acyl groups such as, e.g., alkanoyl groups (e.g., acetyl group, trichloroacetyl group, or triplefluoroacetyl group); alkoxycarbonyl groups such as the tert-butoxycarbonyl group (Boc); arylmethoxycarbonyl groups such as the benzyloxycarbonyl group (Cbz) and the 9-fluorenylmethoxycarbonyl group (Fmoc); and arylmethyl groups such as the benzyl group (Bn), triphenylmethyl group (Tr), and 1,1-bis-(4'-methoxyphenyl)methyl group. 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 groups such as methyl, ethyl, and tert-butyl groups; acyl groups such as alkanoyl groups (e.g., acetyl groups); aryl methyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (diphenylmethyl, DPM); and silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS).

[0195] The compounds of the present invention may be prepared by various synthesis methods known to those skilled in the art, and include specific embodiments exemplified below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitution methods known to those skilled in the art, and include but are not limited to embodiments of the present invention as preferred embodiments.

[0196] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art, and where the present invention relates to the absolute configuration of the compound, said absolute configuration can be confirmed by conventional technical means in the art. For example, the absolute configuration can be confirmed by single-crystal X-ray diffraction (SXRD) by collecting diffraction intensity data by collecting a cultured single crystal with a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: φ / ω, collecting the relevant data, and then analyzing the crystal structure using the direct method (Shelxs97).

[0197] The present invention uses the following abbreviations: DMAC represents N,N-dimethylacetamide, PG represents propylene glycol, HP-β-CD represents hydroxypropyl-β-cyclodextrin, and Solutol HS-15 represents polyethylene glycol (15)-hydroxystearate.

[0198] All solvents used in this invention are commercially available. Compounds are named using the conventional naming conventions of the art or ChemDraw® software, and for commercially available compounds, the supplier's listing name is used.

[0199] The present invention is described in detail through the following examples, but this does not imply any adverse limitations to the invention. Although the present specification has described the invention in detail and disclosed specific embodiments thereof, it will be obvious to a person skilled in the art that various changes and improvements can be made to specific embodiments of the invention without departing from the gist and scope of the invention.

[0200] Reference Example 1

[0201]

[0202] Synthesis method:

[0203]

[0204]

[0205] Reference Example 2

[0206]

[0207] Synthesis method:

[0208]

[0209]

[0210] Using SHIONOGI & CO., LTD.'s anti-influenza drug S-033447 as a reference compound, Schr The low-energy form of S-033447 was calculated using the Macromodel module of Dinger's Maestro software. In the low-energy form, the dihedral angle (dehidal 1) of the pyridohexahydropyrimidine (hereinafter referred to as the parent nucleus) is -146.6°, and the dihedral angle (dehidal 2) between the parent nucleus and 2,5-dihydrothiophene is 56.8° (see Fig. 3). To convert from its low-energy form to its protein binding mode active form (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°), S-033447 must overcome an energy barrier of 0.8 kcal / mol (see Fig. 4). The 3D binding mode of S-033447 and protein (PDB ID: 6FS6) is as shown in Fig. 1, and the interaction between S-033447 and amino acids and metal ions is as shown in Fig. 2.

[0211] Example 1

[0212] By observing the active structural binding modes of S-033447 and 6FS6 proteins, it was discovered that the lowest energy barrier form (low-energy form) formed between the pyridohexahydropyrimidine matrix nuclei while the benzo 2,5-dihydrothienodifluorobenzyl fragment of S-033447 rotates freely corresponds relatively to the protein binding mode form (active form) in the co-crystallization of S-033447 and 6FS6 proteins, which explains the high binding activity of S-033447 and 6FS6 proteins. In general, the smaller the energy barrier difference between the lowest energy barrier form (low-energy form) of a small molecule and its binding mode form (active form) in the corresponding protein, the less energy is lost when the small molecule is converted from the low-energy form to the binding active form of the corresponding protein, meaning that the compound binds more easily to the protein and its binding activity increases.

[0213] To fix the active form of S-033447 and further lower its rotational energy barrier, O and S of the tetrahydropyran fragment and 2,5-dihydrothiophene fragment of S-033447 were replaced with Se through atomic substitution to obtain different cyclopentaselenium fragments and cyclohexaselenium fragments, and the energy barrier difference between the lowest energy barrier form of these Se-substituted molecules and the active form in S-033447 and 6FS6 protein co-crystals was explored.

[0214] (1) Macromodel modulo compound A and compounds B The rotational dihedral angle and rotational energy barrier of the low-energy form were calculated, and the results are shown in Table 1. Compound A A comparison of the low-energy forms of (dark color) and S-033447 (light color) is shown in Fig. 5, and the compound during the rotation process A The change in energy of the two dihedral angles is as shown in Fig. 6, and the compound B A comparison of the low-energy forms of (dark color) and S-033447 (light color) is shown in Fig. 7, and the compound during the rotation process B The change in energy of the two dihedral angles is as shown in Fig. 8.

[0215]

[0216] Note: Dihedral angle 1 is the dihedral angle of pyridohexahydropyrimidine, dihedral angle 2 is the dihedral angle of pyridohexahydropyrimidine and 2,5-dihydrothiophene, and ΔE is the energy barrier required to convert from the low-energy form to the protein binding mode active form of S-033447 (dihedral angle 1 is -153.7° and dihedral angle 2 is 55.0°).

[0217] Conclusion: Compound BThe low-energy form of the compound of the present invention overlapped excellently with the active form of S-033447. Since the lowest energy barrier for binding of the compound of the present invention to the 6FS6 protein structure is relatively small compared to the energy barrier of the active form of the reference compound in the corresponding protein structure, the compound of the present invention could bind more easily to the corresponding protein and exhibit binding activity similar to or better than that of the reference compound in actual binding with the corresponding protein.

[0218] (2) Macromodel modulo compound B The dihedral angle of the low-energy form rotation was calculated, and the results are shown in Table 2.

[0219]

[0220] Note: Dihedral angle 1 is the dihedral angle of pyridohexahydropyrimidine, and dihedral angle 2 is the dihedral angle of pyridohexahydropyrimidine and 2,5-dihydroselenothiophene.

[0221] Conclusion: Compound B The low-energy form of was basically identical to the low-energy form of S-033447.

[0222] Example 2

[0223]

[0224]

[0225]

[0226] Step 1: Compound 2-2 Synthesis of

[0227] Sodium dihydrogen phosphate (13.58 g, 113.19 mmol) was added to water (20 mL), acetonitrile (10 mL) and diphenyl diselenide (1.18 g, 3.77 mmol) were added, zinc powder (986.83 mg, 15.09 mmol) was added in a batch, and the reaction solution was stirred at room temperature for 1 hour, and then the compound 2-1(2g, 7.55mmol) was added, and the reaction solution was stirred overnight at room temperature. The reaction solution was filtered, the cake was washed with ethyl acetate (10mL × 2), separated, the aqueous phase was extracted with ethyl acetate (10mL × 2), the organic phase was combined, washed with saturated saline (20mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried. The obtained crude product was purified using a silica gel column (petroleum ether:ethyl acetate = 1:0 to 10:1) to obtain a compound 2-2 was obtained. MS m / z: 343.0 [M+H] + .

[0228] Step 2: Compound 2-3 Synthesis of

[0229] compound 2-2 (1.5g, 4.40mmol) was added to methanol (10mL) and water (5mL), sodium hydroxide (527.53mg, 13.19mmol) was added, and the reaction solution was stirred at 60°C for 2 hours. The reaction solution was cooled to room temperature, the pH of the reaction solution was adjusted to 7 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phase was combined, washed with saturated saline (10mL × 2), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried to obtain the compound. 2-3 A crude product was obtained and used directly in the next step.

[0230] Step 3: Compound 2-4 Synthesis of

[0231] compound 2-3(1.3g, 3.97mmol) was added to polyphosphoric acid (13mL), and the reaction solution was stirred at 120°C for 2 hours. The reaction solution was cooled to 80°C, added to water (50mL) under stirring, stirred continuously for 5 minutes, extracted with dichloromethane (20mL × 2), the organic phase was combined, washed with water (20mL), washed with saturated saline (20mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried. The obtained crude product was purified using a silica gel column (petroleum ether:ethyl acetate = 1:0 to 10:1) to obtain a compound 2-4 was obtained. MS m / z: 310.9 [M+H] + .

[0232] Step 4: Compound 2-5 Synthesis of

[0233] compound 2-4 300 mg (970.34 μmol) was added to methanol (6 mL), followed by the addition of sodium borohydride (110.13 mg, 2.91 mmol), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was adjusted to pH=7 with 1N hydrochloric acid, extracted with dichloromethane (10 mL × 2), the organic phase was combined, washed with saturated saline (10 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried. The obtained crude product was purified using a silica gel column (petroleum ether:ethyl acetate = 10:1 to 2:1) to obtain a compound 2-5 obtained.

[0234] Step 5: Compound 2-7 Synthesis of

[0235] compound 2-6 (50 mg, 152.75 μmol) was added to ethyl acetate (1 mL), and the compound 2-5After adding (47.53 mg, 152.75 μmol), 1-propyl phosphate 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 with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried. The obtained crude product was purified using a silica gel column (dichloromethane:methanol = 1:0 to 10:1) to obtain the compound 2-7 was obtained. MS m / z: 622.0 [M+H] + .

[0236] Step 6: Compound 2 and 2' Synthesis of

[0237] 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 reaction solution of the crude product was separated by preparative high-performance liquid chromatography and purified (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 the compound 2 (Retention time 3.205 min) and compound 2' (Retention time 3.301 minutes) was obtained.

[0238] compound 2 (Duration of stay 3.205 minutes), 1 ¹H NMR (400 MHz, deuterium 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] + .

[0239] compound 2' (Duration of stay 3,301 minutes) 1 ¹H NMR (400 MHz, deuterium 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] + .

[0240] Example 3

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Step 1: Compound 3-2 Synthesis of

[0247] compound 3-1 (66g, 383.43mmol) was dissolved in dichloromethane (460mL), N,N-dimethylformamide (280.27mg, 3.83mmol, 295.02μL) was added, oxalyl chloride (73.00g, 575.15mmol, 50.35mL) was added dropwise to the reaction solution, and after the addition was completed, the reaction solution was stirred at 20℃ for 30 minutes, and then concentrated under reduced pressure until dry. Dichloromethane (460 mL) was added to the obtained crude product, 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, the liquids were 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 sodium bicarbonate aqueous solution (50 mL), and saturated saline solution (50 mL), respectively, dried with anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure until dry to obtain the compound 3-2 obtained. 1 ¹H NMR (400 MHz, deuteriumized chloroform) δ 7.02-7.05 (m, 2H), 3.81 (brs, 3H), 3.49 (s, 3H), 2.28 (s, 3H).

[0248] Step 2: Compound 3-3 Synthesis of

[0249] compound3-2 (20g, 92.94mmol) was dissolved in tetrahydrofuran (200mL), and methyl magnesium bromide (3M, 37.18mL) was added dropwise at 0℃. After the addition was complete, the reaction solution was heated to 20℃ and stirred for 2 hours. The reaction solution was quenched with 1M hydrochloric acid, the pH was adjusted to 7, extracted with ethyl acetate (100mL × 2), and the organic phases were combined. The mixtures were washed with dilute hydrochloric acid (0.2M, 50mL), saturated sodium bicarbonate solution (50mL), and saturated saline solution (50mL), respectively, dried with anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure until dry to obtain the compound. 3-3 obtained. 1 ¹H NMR (400 MHz, deuteriumized chloroform) δ 7.47-7.50 (m, 1H), 7.03-7.07 (m, 1H), 2.60 (s, 3H), 2.47 (s, 3H).

[0250] Step 3: Compound 3-4 Synthesis of

[0251] compound 3-3 (15g, 88.15mmol) was dissolved in pyridine (90mL), selenium dioxide (19.56g, 176.31mmol) was added, and the reaction solution was stirred at 110°C for 12 hours. The reaction solution was cooled to room temperature, filtered, concentrated under reduced pressure, and dried. Water (50mL) was added to the crude product, the pH was adjusted to 4 with 1M hydrochloric acid, extracted with ethyl acetate (50mL × 3), the organic phase was combined, washed with saturated saline (50mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry to obtain the compound. 3-4 obtained. 1 ¹H NMR (400 MHz, deuteriumized methanol) δ 7.64–7.68 (m, 1H), 7.30–7.32 (m, 1H), 2.52 (s, 3H).

[0252] Step 4: Compound 3-5 Synthesis of

[0253] compound 3-4 (15g, 74.95mmol) was dissolved in dichloromethane (60mL) and methanol (60mL), the temperature was controlled to 0 to 20°C, trimethylsilyldiazomethane (2M, 44.97mL) was added dropwise, and the reaction solution was stirred at 20°C for 2 hours, then acetic acid (3mL) was added and stirred for 5 minutes. The reaction solution was concentrated under reduced pressure until dry, water (50mL) was added, extracted with dichloromethane (50mL × 2), the organic phase was combined, washed with saturated saline (30mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 20%) to obtain a compound 3-5 obtained. 1 ¹H NMR (400 MHz, deuteriumized chloroform) δ 7.51–7.55 (m, 1H), 7.12–7.16 (m, 1H), 3.98 (s, 3H), 2.54 (s, 3H).

[0254] Step 5: Compound 3-6 Synthesis of

[0255] 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 solution (20 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 5%) to obtain the compound 3-6 obtained. 1¹H NMR (400 MHz, deuteriumized chloroform) δ 7.61–7.64 (m, 1H), 7.26–7.31 (m, 1H), 4.94 (s, 2H), 3.99 (s, 3H).

[0256] Step 6: Compound 3-7 Synthesis of

[0257] 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 a batch, and the reaction solution was stirred at 20°C for 30 minutes. Compound 3-6 (5.63 g, 19.21 mmol) was added, and the reaction solution was stirred at 20°C for 3 hours. The reaction solution was filtered, and the filtrate was extracted with ethyl acetate (30 mL × 2). The organic phase was combined, washed with saturated saline (30 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 60%) to obtain the compound 3-7 was obtained. MS(ESI) m / z: 373.8 [M+H] + .

[0258] Step 7: Compound 3-8 Synthesis of

[0259] compound 3-7(4.2 g, 11.28 mmol) was dissolved in dichloromethane (80 mL), dess-martin periodionan (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), the organic phase was combined, washed with saturated saline (30 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 50%) to obtain the compound 3-8 was obtained. MS(ESI) m / z: 371.9 [M+H] + .

[0260] Step 8: Compound 3-9 Synthesis of

[0261] compound 3-8 (2.9 g, 7.83 mmol) was dissolved in tetrahydrofuran (16 mL), an aqueous sodium hydroxide solution (626.67 mg, 15.67 mmol, 4 mL) was added, and the mixture was stirred at 20°C for 1 hour. Most of the tetrahydrofuran was removed by concentration under reduced pressure, the pH of the aqueous phase was adjusted to 6 with 1N hydrochloric acid, the solid was filtered, and the cake was suction-dried under reduced pressure to obtain the compound. 3-9 was obtained. MS(ESI) m / z: 357.9 [M+H] + .

[0262] Step 9: Compound 3-10 Synthesis of

[0263] compound 3-9(2.3g, 6.46mmol) was dissolved in dimethyl sulfoxide (23mL), ammonium persulfate (2.95g, 12.91mmol), silver nitrate (109.69mg, 645.74μmol), and concentrated sulfuric acid (633.34mg, 6.46mmol), respectively, and the reaction solution was stirred at 50°C for 3 hours. Saturated sodium bicarbonate aqueous solution (20mL), water (10mL), and dichloromethane (20mL), respectively, were added to the reaction solution, stirred for 5 minutes, filtered, the filtrate was separated, and the aqueous phase was extracted with dichloromethane (10mL). The organic phase was combined, washed with saturated saline solution (30mL × 3), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product is purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 50%) to obtain a compound 3-10 Obtained. MS(ESI) m / z: 311.8 [M+H] + .

[0264] Step 10: Compound 3-11 Synthesis of

[0265] 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. The pH was adjusted to 7 with 1N hydrochloric acid, extracted with ethyl acetate (15 mL × 2), the organic phase was combined, washed with saturated saline (10 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0 to 50%) to obtain the compound 3-11 was obtained. MS(ESI) m / z: 313.8 [M+H] + .

[0266] Step 11: Compound 3-12 Synthesis of

[0267] compound 3-11 (330 mg, 1.06 mmol) was dissolved in dichloromethane (6 mL), thionyl chloride (251.53 mg, 2.11 mmol, 153.37 μL) was added, and the reaction solution was stirred at 20°C for 1 hour. The reaction solution was concentrated under reduced pressure until dry to obtain the compound 3-12 A crude product was obtained and used directly in the next step of the reaction.

[0268] Step 12: Compound 3-13 and 3-13' Synthesis of

[0269] compound 2-6 Dissolve (340 mg, 1.04 mmol) in acetonitrile (6 mL), and the 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, extracted with ethyl acetate (5 mL × 3), the organic phase was combined, washed with saturated saline (5 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product is purified using a silica gel column (methanol / dichloromethane, methanol ratio: 0 to 5%), and the resulting compound is detected using 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: mobile phase B concentration increases from 5% to 40% within 2 minutes, is maintained at 40% for 1.2 minutes, and then maintained at 5% for 0.8 minutes) to be analyzed as a mixture, and separated using chiral separation (column type: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: [A: carbon dioxide, B: 0.1% ammonia water / ethanol]; gradient: mobile phase B is maintained at 40%) to determine the chiral isomer compounds 3-13(Retention time 1.831 min, ee=96.1%) and compound 3-13' (Retention time 2.031 min, ee=100%) was obtained.

[0270] Step 13: Compound 3 Synthesis of

[0271] 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 reaction solution of the crude product was separated by preparative high-performance liquid chromatography and purified (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 the compound 3 obtained. 1 ¹H NMR (400 MHz, deuteride 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] + .

[0272] Step 14: Compound 3' Synthesis of

[0273] 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 reaction solution of the crude product was separated by preparative high-performance liquid chromatography and purified (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 the compound 3' obtained. 1 ¹H NMR (400 MHz, deuterium 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] + .

[0274] Example 4

[0275]

[0276]

[0277] compound 3 (130.00 mg, 244.65 μmol) was added to N,N-dimethylacetamide (2 mL), followed by the addition of methyl chloromethyl 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, and the mixture was extracted with ethyl acetate (10 mL × 2). The organic phase was washed with saturated saline solution (10 mL × 4), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a silica gel column (dichloromethane:methanol = 1:0 to 20:1) to obtain the compound 4 obtained. 1¹H NMR (400 MHz, deuteriumized 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] + .

[0278] compound 4 The absolute configuration is determined by single-crystal X-ray diffraction (SXRD). It was confirmed that it is.

[0279] Example 5

[0280]

[0281]

[0282] compound 230.00 mg (56.56 μmol) was added to N,N-dimethylacetamide (1 mL), followed by the addition of methyl chloromethyl 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, and the mixture was extracted with ethyl acetate (3 mL × 2). The organic phase was washed with saturated saline solution (3 mL × 3), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until dry. The obtained crude product was purified using a thin-layer silica gel plate for preparative extraction (dichloromethane:methanol = 10:1) to obtain the compound 5 obtained. 1 ¹H NMR (400 MHz, deuteride 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] + .

[0283] Example 6

[0284]

[0285]

[0286]

[0287]

[0288] Step 1: Compound 6-2 Synthesis of

[0289] Under an ice bath, compound 6-1 A trimethylsilanediazomethane solution (2M, 13.61 mL, 27.22 mmol) was added dropwise to a solution of methanol (8 mL) and tetrahydrofuran (32 mL) (3.35 g, 13.61 mmol). After the addition was complete, the reaction solution was heated to 20°C and stirred for 1 hour. A saturated citric acid solution (100 mL) was added to the reaction solution, extracted with ethyl acetate (100 mL × 3), the organic phase was combined, washed sequentially with saturated sodium bicarbonate (100 mL) and saturated saline solution (100 mL), dried with anhydrous sodium sulfate, filtered, and rotary dried to obtain a crude compound product. 6-2 was obtained and used directly in the next step of the reaction.

[0290] Step 2: Compound 6-3 Synthesis of

[0291] 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, extracted with ethyl acetate (100 mL × 3), the organic phase was combined, washed with water (200 mL) and saturated saline (200 mL) respectively, dried with anhydrous sodium sulfate, filtered, and rotary dried. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:2) to obtain the compound 6-3 obtained.

[0292] Step 3: Compound6-4 Synthesis of

[0293] compound 6-3 (2.7g, 7.21mmol), methyl acrylate (1.24g, 14.42mmol, 1.30mL), and N,N-diisopropylethylamine (2.80g, 21.64mmol, 3.77mL) were dissolved in acetonitrile (35mL), and the reaction solution was reacted at 50°C for 12 hours. The reaction solution was concentrated and rotary dried, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:2, by volume ratio) to obtain the compound 6-4 obtained.

[0294] Step 4: Compound 6-5 Synthesis of

[0295] compound 6-4 10 mL of ethyl acetate hydrochloride solution (4 M) was added to 20 mL of ethyl acetate solution (1.6 g, 3.47 mmol), and the reaction solution was stirred at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure to obtain a crude product in the form of a hydrochloride salt. 6-5 was obtained and used directly in the next step of the reaction.

[0296] Step 5: Compound 6-6 Synthesis of

[0297] compound 6-5 (1.18 g, hydrochloride) 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 rotary dried. The crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 0:1 by volume, followed by dichloromethane:methanol = 10:1 to 0:1) to obtain the compound 6-6 obtained.

[0298] Step 6: Compound 6-7 Synthesis of

[0299] compound6-6 (3g, 9.14mmol) was dissolved in dimethyl sulfoxide (30mL) and water (3mL), sodium chloride (1.07g, 18.27mmol) was added, and the reaction solution was stirred at 90°C for 12 hours. The reaction solution was diluted with water (100mL), extracted with dichloromethane (100mL × 3), the organic phase was washed with saturated saline (100mL × 2), dried with anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to obtain a compound 6-7 obtained.

[0300] Step 7: Compound 6-8 Synthesis of

[0301] 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]undek-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 with anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to obtain the compound 6-8 Obtained. MS (ESI) m / z: 297.3 [M+H] + .

[0302] Step 8: Compound 6-9 Synthesis of

[0303] compound 6-8 (70 mg, 236.23 μmol) and compound 2-5(73.51 mg, 236.23 μmol) was added to ethyl acetate (2 mL), then tri-n-propyl cyclophosphate 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 with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and dried to obtain a crude product. The crude product is purified using a flash silica gel column (dichloromethane:methanol = 1:0 to 10:1) to obtain a compound 6-9 was obtained. MS(ESI)m / z: 591.1 [M+H] + .

[0304] Step 9: Compound 6-9A and 6-9B Synthesis of.

[0305] compound 6-9 It is analyzed as a racemic compound by detection using a 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% within 4 minutes, then maintained for 2.5 minutes; then maintained at 5% for 1.5 minutes), and the compound is analyzed by chiral separation (column type: DAICEL CHIRALCEL OD-H (25 mm × 30 mm, 5 μm); mobile phase: [A: carbon dioxide, B: 0.1% ammonia water / ethanol]; gradient: B%: 40% to 40%). 6-9A (Retention time 4.024 min) and compound 6-9B (Duration time 4.447 minutes) was obtained.

[0306] Step 10: Compound 6 Synthesis of

[0307] compound 6-9A After adding (20 mg, 33.93 μmol) to N,N-dimethylacetamide (1 mL), lithium chloride (7.19 mg, 169.64 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 (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 the compound 6 obtained. 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] + .

[0308] Step 11: Compound 6' Synthesis of

[0309] compound 6-9BAfter adding (20.00 mg, 33.93 μmol) to N,N-dimethylacetamide (1 mL), lithium chloride (7.19 mg, 169.64 μmol, 3.47 μL) 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 (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 the compound 6' obtained. 1 ¹H NMR (400 MHz, deuteride 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] + .

[0310] Biological test data

[0311] Experimental Example 1: Influenza Virus Cellular Degeneration (CPE) Experiment

[0312] The antiviral activity of a compound against the influenza virus (IFV) is at half the effective concentration (EC₀) of the compound. 50 The value was evaluated by measuring it. Cell denaturation tests are widely used to measure the protective effect of a compound on virus-infected cells to reflect the antiviral activity of the compound.

[0313] Influenza virus CPE experiment

[0314] MDCK cells were seeded into black 384-well cell culture plates at a cell density of 2,000 cells per well and cultured overnight in an incubator at 37°C with 5% CO2. The compound was diluted using an Echo555 non-contact nano-upgraded sonic pipetting system and added to the cell wells (diluted 4-fold, 8 test concentration points). Influenza virus A / PR / 8 / 34 (H1N1) strains were added to the cell culture plates at 1 to 2 90% tissue culture infection doses (TCID90) per well, and the final concentration of DMSO in the medium was 0.5%. Virus control wells (DMSO and virus added, but no compound added), cell control wells (DMSO added, but no compound or virus added), and medium control wells (medium only, no cells) were established. The cytotoxicity and antiviral activity of the compound were measured in parallel, and other experimental conditions were consistent with those of the antiviral activity experiment, except that no virus was added. Cell plates were cultured in an incubator at 37°C and 5% CO2 for 5 days. After 5 days of culture, cell activity was detected using the CCK8 cell activity detection kit. The raw data were used to calculate the antiviral activity and cytotoxicity of the compound.

[0315] The antiviral activity and cytotoxicity of the compounds were expressed as the inhibition rate (%) of the compounds against cytoviral effects induced by viruses, respectively. The calculation formulas are as follows:

[0316]

[0317] Non-linear fitting analysis was performed on the inhibition rate and cytotoxicity of the compound using GraphPad Prism software to determine the EC of the compound 50 Values ​​were obtained. The experimental results are shown in Table 3.

[0318]

[0319] Conclusion: The compound of the present invention showed a positive effect in experiments inhibiting the replication of the influenza virus at the cellular level.

[0320] Experimental Example 2: Study of In vivo drug efficacy

[0321] Experimental Objective: To evaluate the efficacy of the compound in a mouse model of influenza A virus H1N1 infection.

[0322] Experimental plan: Mice were infected with influenza A virus A / PR / 8 / 34 (H1N1) by nasal instillation, and treatment with the compound was started 48 hours after infection and administered orally twice a day for 7 consecutive days. The anti-influenza A virus H1N1 effect of the compound in the model was evaluated by observing changes in body weight and survival rates of the mice.

[0323] For the experiment, female SPF-grade BALB / c mice aged 6 to 7 weeks were selected. The mice were acclimatized for at least 3 days after arriving at the BSL-2 animal room before the experiment began. 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); once the animals entered a state of deep anesthesia, they were infected with the A / PR / 8 / 34 (H1N1) virus via nasal infusion, with an infection volume of 50 μL. From Day 2 to Day 8, the test compound was administered orally twice daily at a dose of 5 mg / kg (administration volume 10 mL / kg). The first administration was given 48 hours after infection. The condition of the mice was monitored daily, and their body weight and survival rate were recorded. On Day 14, all surviving animals were euthanized.

[0324] Experimental Results:

[0325] The survival rate and body weight loss rate of animals are detected, and the body weight loss rate is calculated as: (body weight on day 0 - body weight on day N) / body weight on day 0 × 100%. The results are as shown in Table 4, regarding the compounds. 5It can protect the animals to achieve a maximum body weight reduction of 13.77% on day 7, after which recovery begins and the survival rate of mice is 100% until the end of the experiment; compound 4 The maximum rate of animal body weight loss was 7.03% on the third day, after which recovery began, and the survival rate of the mice was 100% until the end of the experiment.

[0326]

[0327] Conclusion: The compound of the present invention was shown to have excellent body weight protection and a rapid recovery time in an animal in vivo drug efficacy model.

[0328] Experimental Example 3: Cytogenetic Evolution (CPE) Test on Baloxavir-Resistant A / PR / 8 / 34 (H1N1) I38T Influenza Virus Strain

[0329] Experimental Objective: Half effective concentration of a compound (EC 50 We intend to evaluate the antiviral activity of the compound against the Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T influenza virus strain by measuring the value.

[0330] Experimental Plan: MDCK cells were seeded into 96-well cell culture plates at a cell density of 15,000 cells per well and cultured overnight in an incubator at 37°C and 5% CO2. The next day, the compound solution (three-fold serial dilution, 8 concentration points, triple wells) and the 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%. Cells in virus-infected control wells without the compound were cultured in an incubator at 5% CO2 and 37°C for 5 days until cytodenaturation reached 80 to 95%. Subsequently, cell activity in each well was detected using CCK8. If the cell activity in the wells containing the compound was higher than that in the virus-infected control wells—that is, if the CPE was weakened—it indicated that the compound had an inhibitory effect against the measured virus.

[0331] Experimental Results:

[0332] The antiviral activity of the compound was expressed as the inhibitory activity (%) of the compound against the cytoviral effect caused by the virus. The calculation formula is as shown below.

[0333]

[0334] EC 50 Non-linear fitting analysis was performed on the inhibitory activity of the compound and cell viability using GraphPad Prism (version 5) software, and the fitting method was “log(inhibitor) vs. response -- Variable slope”. The experimental results are shown in Table 5.

[0335]

[0336] Conclusion: The compound of the present invention showed a positive effect in experiments inhibiting the replication of baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strains at the cellular level.

[0337] Experimental Example 4: Study of In vivo drug efficacy

[0338] Experimental Objective: To evaluate the efficacy of the compound in a mouse infection model of H1N1 influenza virus resistant strains.

[0339] Experimental method: Mice were infected with the baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T strain of influenza A virus by nasal instillation, and treatment with the compound was started 2 hours before infection and administered orally twice daily for 7 consecutive days. Changes in body weight and survival rates of the mice were observed to evaluate the anti-influenza A virus H1N1 effect of the compound in the model.

[0340] For the experiment, female SPF-grade BALB / c mice aged 6 to 7 weeks were selected. The mice were acclimatized for at least 3 days after arriving at the BSL-2 animal room before the experiment began. The day of infection was set as Day 0 of the experiment. Mice were deeply anesthetized by intraperitoneal injection of Zoletil 50 / xylazine hydrochloride, and then infected with the baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T virus strain via nasal infusion, with an infection volume of 50 μL. From Day 2 to Day 8, the test compound was administered orally twice daily at a dose of 15 mg / kg or 50 mg / kg (administration volume 10 mL / kg). The first administration was given 2 hours prior to infection. The condition of the mice was monitored daily, and their body weight and survival rate were recorded. On Day 14, all surviving animals were euthanized.

[0341] Experimental Results:

[0342] The survival rate and body weight loss rate of animals are detected, and the body weight loss rate is calculated as: (Weight on Day 0 - Weight on Day N) / Weight on Day 0 × 100%. The experimental results are shown in Table 6 below. Compound 4 When administered at a dose of 50 mg / kg, the mice's body weight hardly decreased, and the survival rate of the mice was 100% until the end of the experiment.

[0343]

[0344] Conclusion: The compound of the present invention was shown to have excellent body weight protection and a rapid recovery time in an animal in vivo drug efficacy model.

[0345] Experimental Example 5: Test of Plasma Protein Binding Rate of Compound

[0346] Experimental Objective: To evaluate the protein binding rate of the compound of the present invention in CD-1 mice, SD rats, and human plasma using an equilibrium dialysis method.

[0347] Experimental Plan: The test compound was diluted in each of the five types of plasma above with dialysis buffer to prepare samples with a final concentration of 2 μM. The samples were then added to a 96-well equilibrium dialysis apparatus and dialyzed with phosphate buffer at 37°C for 4 hours. Warfarin was used as the control compound in the experiment. The concentrations of the test compound and warfarin in the plasma and buffer were measured by the LC-MS / MS method.

[0348] Experimental Results: The results are as shown in Table 7.

[0349]

[0350] Note: H represents human, R represents rat, M represents mouse, D represents dog, and C represents cynomolgus monkey.

[0351] Conclusion: The compound of the present invention has an intermediate plasma protein binding rate in all five types of plasma, which exemplifies that the ratio of the free-state drug concentration of the test compound in the five types of plasma is appropriate and has excellent drug manufacturing characteristics.

[0352] Experimental Example 6: Rat Pharmacokinetic Study Experiment

[0353] Experimental Objective: To investigate the plasma pharmacokinetics in male SD rats after single intravenous injection and gastric tube administration of the compound of the present invention.

[0354] Experimental animals: male SD rats weighing 200 to 300 g, aged 6 to 8 weeks;

[0355] Experimental procedure: administered by injection (iv), with a dosage of 1 mpk and a concentration of 0.50 mg / mL, and the solvent was 40% DMSO + 40% PG + 20% (20% HP-β-CD + water); administered orally (po), with a dosage of 10 mpk and a concentration of 1 mg / mL, and the solvent was 3% DMSO + 10% solutol HS + 87% water.

[0356] Sample Collection: 0.03 mL of blood samples were collected from experimental animals via saphenous venipuncture at each time point, and the actual time of collection was recorded. All blood samples were placed in commercial EDTA-K2 anticoagulant tubes with a standard size of 1.5 mL. After collecting the blood samples, DDV was added as a stabilizer to the plasma matrix, wherein the plasma:dichlorvos solution ratio was 40:1, and the dichlorvos solution was a 1:1 acetonitrile / water solution of 40 mM dichlorvos. Within 30 minutes, the sample was centrifuged at 4°C and 3000 g for 10 minutes to aspirate the supernatant plasma, rapidly placed on dry ice, and stored in a refrigerator at -80°C for use in LC-MS / MS analysis.

[0357] Data Analysis: Plasma concentrations were processed using the non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetics software, and pharmacokinetic parameters Cl (apparent elimination rate) and T were analyzed using the linear logarithmic trapezoidal method. 1 / 2 (Time required to remove half of the compound), C max (Peak concentration), AUC 0-last (0 - concentration integration area within the last sampling time) was calculated, and the results are shown in Table 8.

[0358]

[0359] Experimental Conclusion: The compound of the present invention had a relatively high oral plasma exposure and excellent pharmacokinetic properties.

[0360] Experimental Example 7: Pharmacokinetic Study of Beagle Dogs

[0361] Experimental Objective: To investigate the plasma pharmacokinetics in male Beagle dogs after single intravenous injection and gastric tube administration of the compound of the present invention.

[0362] Experimental animals: Male Beagle dogs ≥6 months old weighing 6 to 12 kg;

[0363] Experimental procedure: administered by injection (iv) with a dosage of 1 mpk, a concentration of 1 mg / mL, and a solvent of 10% DMSO + 90% (20% HP-β-CD + water); administered orally (po) with a dosage of 10 mpk, a concentration of 2 mg / mL, and a solvent of 3% DMSO + 10% solutol HS + 87% water.

[0364] Sample Collection: 0.8 mL of blood samples were collected from experimental animals via saphenous venipuncture at each time point, and the actual time of collection was recorded. All blood samples were placed in commercial EDTA-K2 anticoagulant tubes with a standard size of 1.5 mL. After collecting the blood samples, DDV was added as a stabilizer to the plasma matrix, where the plasma:dichlorvos solution ratio was 40:1 and the dichlorvos solution was a 1:1 acetonitrile / water solution of 40 mM dichlorvos. Within 30 minutes, the sample was centrifuged at 4°C and 3000 g for 10 minutes to aspirate the supernatant plasma, rapidly placed on dry ice, and stored in a refrigerator at -80°C for use in LC-MS / MS analysis.

[0365] Data Analysis: Plasma concentrations were processed using the non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetics software, and pharmacokinetic parameter Cl was analyzed using the linear logarithmic trapezoidal method. , T 1 / 2 , C max , AUC 0-last ...was calculated, and the results are as shown in Table 9.

[0366]

[0367] Experimental Conclusion: The compound of the present invention had a low elimination rate, a long half-life, a high plasma exposure after oral administration, and excellent pharmacokinetic properties.

Claims

Claim 1 A compound represented by formula (IV-A) or a pharmaceutically acceptable salt thereof, or a compound represented by formula (VIA) or a pharmaceutically acceptable salt thereof: and ,in the above formula, R5 and R6 are F; R7 is H and Selected from; R8 is CH3 and Selected from; p is 1, one of E1 and E2 is selected from Se, and the other is selected from S and O; q is 1; and the carbon atom with "*" is a chiral carbon atom and exists in a form rich in (R) or (S) single enantiomer or one enantiomer. Claim 2 A compound or a pharmaceutically acceptable salt thereof, wherein E1 is Se and E2 is O, in claim 1. Claim 3 In claim 1, a compound or a pharmaceutically acceptable salt thereof selected from the following formula: and In the above formula, R1 and R2 are F; m is 1; and q, R5, R6, R7 and R8 are as defined in the first term. Claim 4 In paragraph 3, a compound or a pharmaceutically acceptable salt thereof selected from the following formula: and In the above formula, R1, R2, R5, R6, R7, and R8 are as defined in Paragraph 3. Claim 5 A compound or a pharmaceutically acceptable salt thereof selected from the following formula: and . Claim 6 A pharmaceutical composition for treating influenza virus-related diseases, comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete

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