Pyridinone polycyclic derivative and use thereof

Abstract

Provided are pyridinone polycyclic derivative and application thereof, and specifically disclosed are compounds represented by formula (VI) and pharmaceutically acceptable salts thereof.

Description

[0001] This invention claims the following priority:

[0002] CN 202110024885.9, application date January 8, 2021;

[0003] CN 202110264686.5, application date March 11, 2021;

[0004] CN 202110513447.9, application date May 11, 2021. Technical Field

[0005] This invention relates to a class of pyridone polycyclic derivatives and their applications, specifically to compounds of formula (VI) and their pharmaceutically acceptable salts. Background Technology

[0006] Influenza virus (IFV) is a segmented, single-stranded, antisense RNA virus that causes influenza in humans and animals. Influenza viruses can cause very high morbidity and mortality rates, and type A influenza viruses, in particular, can cause global pandemics.

[0007] Influenza A virus is a single-stranded negative-sense RNA virus with a genome divided into eight segments, encoding eight proteins. The 5' and 3' ends of the influenza virus genome segments are highly conserved; these two ends are complementary, forming a stalk-loop structure, which plays a crucial role in initiating viral RNA replication. The proteins encoded by the different viral gene segments vary in size and play different roles in the influenza virus's life cycle. The basic functions of several key proteins are introduced below. The influenza virus HA (hypoacetic acid) ligand is responsible for recognizing the host receptor. It binds to the virus-specific receptor on the cell surface, mediating the fusion of the viral outer membrane with the intracellular cytosome membrane, releasing the viral nucleocapsid into the cytoplasm. The influenza virus receptor is specific; the receptor for influenza A virus is a sialic acid glycoprotein. During replication, the influenza virus NA protein removes sialic acid from the surface of the viral particle, preventing the viral particle from accumulating on the host cell surface, thus facilitating the release of viruses and further infecting more host cells.

[0008] The role of the M2 protein in influenza virus: The HA protein of influenza virus binds to sialic acid, allowing the virus to be endocytosed by the host cell. The pH of the phagocytic vesicle plays a crucial role in viral uncoating. The ion channel of the M2 protein on the viral membrane can gradually lower the pH of the phagocytic vesicle. When the pH drops to 5.0-6.0, it causes a conformational change in the HA2 protein, resulting in the translocation of the fusion peptide at the amino terminus of the HA2 protein. This activates the fusion process, leading to the fusion of the viral lipid bilayer with the cell membrane and the release of RNPs from the viral particle into the host cytoplasm. The M2 protein is a transmembrane ion channel found only in type A influenza virus, and a portion of it extends to the surface of the viral outer membrane.

[0009] The synthesis of influenza virus proteins also utilizes the host cell's translation mechanism; in fact, the virus can even pause the translation of host proteins to accelerate its own protein synthesis. Unlike host cell mRNA polyadenylation, which is accomplished by a specific adenylate esterase, the adenylate tail of viral mRNA is formed by transcription of 5-7 consecutive uracils on the negative-stranded vRNA. Capping of various viral messenger RNAs (mRNAs) is accomplished in a similar manner: PA and PB2 proteins seize the 5' capping primer of the host pre-mRNA transcript, thereby initiating viral mRNA synthesis. This process, known as "cap snatching," is primarily accomplished by the viral RNA-dependent RNA polymerase (RdRp), whose PA subunit possesses RNA endonuclease activity, responsible for cleaving host mRNA. After completing the polyadenylation and capping processes, the viral mRNA exits the nucleus, enters the cytoplasm, and is translated like the mRNA of the host cell. The nuclear export of the viral vRNA fragment is mediated by the viral M1 and NS2 proteins. The M1 protein can interact with the vRNA and NP protein, and at the same time, it also interacts with the nuclear export protein NS2. Thus, the nuclear export protein NS2 mediates the M1-RNP to exit the nucleus and enter the cytoplasm of the host cell in the form of a nuclear protein.

[0010] Current influenza treatment options include vaccination and chemotherapy with antiviral drugs. Influenza vaccines are frequently recommended for high-risk groups, such as children and the elderly, or those with asthma, diabetes, or heart disease; however, even vaccination does not completely prevent influenza. Vaccines for specific influenza strains are re-prepared each season, but it is impossible to cover all the viral strains actively infecting humans globally during that season. Furthermore, due to antigenic drift, if more than one virus infects a single cell, the eight individual vRNA segments in the genome mix or reassort, resulting in rapid changes in viral genetics that can produce antigenic shifts, enabling the virus to infect new host species and quickly overcome protective immunity.

[0011] Antiviral drugs can also be used to treat influenza. Neuraminidase inhibitors, such as oseltamivir (Tamiflu), are effective against influenza A viruses. However, clinical observations have revealed resistant viral strains to these inhibitors. In the field of antiviral therapy, there is an urgent clinical need for antiviral drugs with novel mechanisms of action to support monotherapy for influenza A, or to be used in combination with other marketed antiviral drugs with different mechanisms of action for the prevention and treatment of influenza A. WO2016175224 reports RNA polymerase PA subunit inhibitors, such as S-033447 and its prodrug S-033188. Prodrug: . Summary of the Invention

[0012] This invention provides compounds of formula (VI) or pharmaceutically acceptable salts thereof. , in, R7 is selected from H and ; R8 is selected from C 1-3 Alkyl and The C 1-3 Alkyl and Choose 1, 2, or 3 Rs a replace; R9 is selected from H, E1 is selected from Se, and X1 is selected from CR. 10 R 11 R 10 and R 11 Together with the atoms they are bonded to, they form C 3-5 cycloalkyl; Alternatively, X1 and R9 together with the atoms they are connected to form p is selected from 0 and 1, and E1 and E2 are selected from Se and S and O respectively; R 12 Selected from H, F, Cl, Br, I, OH, NH2, -COOH, C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylamino, the C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylamino groups are independently and optionally bounded by 1, 2, or 3 R groups. b replace; 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 Ra and R b They are each independently selected from H, F, Cl, Br, and I; The condition is 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, each R 12 They are selected independently from OH and NH2, respectively.

[0013] In some embodiments of the present invention, the respective R 12 Each variable is independently selected from F, and other variables are as defined in this invention.

[0014] In some embodiments of the present invention, R8 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, and The CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and Choose 1, 2, or 3 Rs a Replacement, other variables as defined in this invention.

[0015] In some embodiments of the present invention, R8 is selected from CH3, CH2CH3, CH(CH3)2, and Other variables are as defined in this invention.

[0016] In some embodiments of the present invention, R8 is selected from CH3 and Other variables are as defined in this invention.

[0017] In some embodiments of the present invention, R7 is selected from H, , , and Other variables are as defined in this invention.

[0018] In some embodiments of the present invention, R7 is selected from H, and Other variables are as defined in this invention.

[0019] In some embodiments of the present invention, R7 is selected from H and Other variables are as defined in this invention.

[0020] In some embodiments of the present invention, E1 is selected from Se, E2 is selected from O, and other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, the structural unit Selected from R5 and R6 are independently selected from H, F, Cl, Br, I, OH, NH2, -COOH, and C, respectively. 1-3 Alkyl, C1-3 Alkoxy and C 1-3 Alkylamino, the C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkylamino groups are independently and optionally bounded by 1, 2, or 3 R groups. b Replacement, other variables as defined in this invention.

[0022] In some embodiments of the present invention, the structural unit Selected from , , and Other variables are as defined in this invention.

[0023] In some embodiments of the present invention, the structural unit Selected from Other variables are as defined in this invention.

[0024] In some embodiments of the present invention, R5 is selected from F, and other variables are as defined in the present invention.

[0025] In some embodiments of the present invention, R6 is selected from F, and other variables are as defined in the present invention.

[0026] In some embodiments of the present invention, the structural unit Selected from , , and Other variables are as defined in this invention.

[0027] In some embodiments of the present invention, the structural unit Selected from , , and Other variables are as defined in this invention.

[0028] In some embodiments of the present invention, the structural unit Selected from Other variables are as defined in this invention.

[0029] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from...

[0030] in, R5 and R6 are independently selected from H, F, Cl, Br, I, OH and NH2, respectively; R7 is selected from H and ; R8 is selected from C1-3 Alkyl and The C 1-3 Alkyl and Choose 1, 2, or 3 Rs a replace; R9 is selected from H, E1 is selected from Se, and X1 is selected from CR. 10 R 11 R 10 and R 11 Together with the atoms they are bonded to, they form C 3-5 cycloalkyl; Alternatively, X1 and R9 together with the atoms they are connected to form , E1 and E2, one of which is selected from Se, and the other is selected from S and O; T1 is selected from CH and N; p and q are independently selected from 0 and 1, respectively; Each R a They are each independently selected from H, F, Cl, Br, and I; The condition is 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, R5 and R6 are independently selected from OH and NH2, respectively. bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0031] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, p, q, E1, E2, T1, R5, R6 and R7 are as defined in this invention; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0032] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, p, q, E1, E2, R5, R6 and R7 are as defined in this invention; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0033] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, p, q, E1, E2, R5, R6 and R7 are as defined in this invention; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0034] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, R1 and R2 are independently selected from H, F, Cl, Br, I, OH and NH2, respectively; m is selected from 0 and 1; q, R5, R6, R7 and R8 are as defined in this invention.

[0035] In some embodiments of the present invention, R1 and R2 are each independently selected from F, and other variables are as defined in the present invention.

[0036] In some embodiments of the present invention, R5 and R6 are each independently selected from F, and other variables are as defined in the present invention.

[0037] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , R1, R2, R5, R6, R7 and R8 are as defined in this invention.

[0038] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, R1, R2, R3, and R4 are independently selected from H, F, Cl, Br, I, OH, and NH2, respectively; n and m are independently selected from 0 and 1, respectively; The condition is that, in equations (I-1) and (I-2), when m is selected from 1, R1 and R2 are independently selected from OH and NH2, respectively; q, R5, R6 and R8 are as defined in this invention.

[0039] In some embodiments of the present invention, R1 and R2 are each independently selected from F, and other variables are as defined in the present invention.

[0040] 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.

[0041] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... and , in, R1, R2, R3, R4, R5, R6, R7 and R8 are as defined in this invention.

[0042] This invention provides compounds of formula (V) or pharmaceutically acceptable salts thereof.

[0043] in, R5 and R6 are independently selected from H, F, Cl, Br, I, OH and NH2, respectively; R7 is selected from H and ; R8 is selected from C1-3 Alkyl and The C 1-3 Alkyl and Choose 1, 2, or 3 Rs a replace; R9 is selected from H; X1 is selected from CR 10 R 11 R 10 and R 11 Together with the atoms they are bonded to, they form C 3-5 cycloalkyl; Alternatively, X1 and R9 can be connected together to form... ; 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 independently selected from 0 and 1, respectively; Each R a They are each independently selected from H, F, Cl, Br, and I; The condition is 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, R5 and R6 are independently selected from OH and NH2, respectively. bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0044] This invention provides compounds of formula (IV) or pharmaceutically acceptable salts thereof. , in, 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 independently selected from H, F, Cl, Br, I, OH and NH2, respectively; R7 is selected from H and ; R8 is selected from C 1-3 Alkyl and The C 1-3 Alkyl and Choose 1, 2, or 3 Rs a replace; p and q are independently selected from 0 and 1, respectively; Each R a They are each independently selected from H, F, Cl, Br, and I; 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. bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0045] In some embodiments of the invention, the compound or a pharmaceutically acceptable salt thereof is selected from... , in, p, q, E1, E2, R5, R6 and R7 are as defined in this invention; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0046] This invention provides compounds of formula (III) or pharmaceutically acceptable salts thereof.

[0047] in, 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; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0048] This invention provides compounds of formula (II) or pharmaceutically acceptable salts thereof.

[0049] in, 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; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0050] This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0051] Wherein, R1, R2 and m are as defined in this invention.

[0052] This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0053] in, 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; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer."

[0054] In some embodiments of the present invention, R1 is selected from F, and other variables are as defined in the present invention.

[0055] In some embodiments of the present invention, R2 is selected from F, and other variables are as defined in the present invention.

[0056] In some embodiments of the present invention, R3 is selected from F, and other variables are as defined in the present invention.

[0057] In some embodiments of the present invention, R4 is selected from F, and other variables are as defined in the present invention.

[0058] Some solutions in this invention are derived from arbitrary combinations of the aforementioned variables.

[0059] This invention provides compounds of the following formula or pharmaceutically acceptable salts thereof. and .

[0060] The present invention also provides the use of the said compound or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating diseases related to influenza virus.

[0061] Technical effect

[0062] The compound of this invention, as an RNA polymerase inhibitor, showed a positive effect in the inhibition of influenza virus replication at the cellular level. It also demonstrated excellent weight protection and early recovery in an in vivo animal pharmacodynamic model. Plasma protein binding rate test results showed that the compound of this invention has a moderate plasma protein binding rate. PK results showed that it has good pharmacokinetic properties and good drug-like properties. Attached Figure Description

[0063] Figure 1 3D binding mode of S-033447 to protein (PDB ID: 6FS6).

[0064] Figure 2 The interaction between S-033447 and amino acids and metal ions is shown.

[0065] Figure 3 S-033447 low-energy conformation with two dihedral angles.

[0066] Figure 4 The energy changes of the two dihedral angles of S-033447 during rotation.

[0067] Figure 5 Comparison of the low-energy conformations of compound A (dark) and S-033447 (light).

[0068] Figure 6 The energy changes of the two dihedral angles of compound A during rotation.

[0069] Figure 7 Comparison of the low-energy conformations of compound B (dark) and S-033447 (light).

[0070] Figure 8 The energy changes of the two dihedral angles of compound B during rotation.

[0071] Related definitions

[0072] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0073] The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0074] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this invention, with a specific substituent. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds 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. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0075] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0076] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention envisions all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, ( R )- and( S - Enantiomers, diastereomers, ( D )-Isomer, ( L (Isomers, racemic mixtures thereof, and other mixtures, such as mixtures enriched with enantiomers or diastereomers, are all within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.)

[0077] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), Iodine-125 ( 125I) or C-14 14 C). For example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0078] The terms “optional” or “optionally” refer to events or conditions that may occur but are not required to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0079] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the resulting compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, unless otherwise specified, and the type and number of substituents can be arbitrary on a chemically feasible basis.

[0080] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0081] When the number of a linking group is 0, such as -(CRR)0-, it indicates that the linking group is a single bond.

[0082] When one of the variables is selected as a single bond, it means that the two groups it connects to are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0083] When the listed linking groups do not specify their linking direction, the linking direction is arbitrary, for example, The linker group L is -MW-. In this case, -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form a ring. Alternatively, rings A and B can be connected in the opposite direction to the left-to-right reading order to form a ring. The combination of the linking group, substituents, and / or their variants is permitted only if such a combination produces a stable compound.

[0084] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds (…). Straight dashed key ( ), or wavy lines ( () indicates that the oxygen atom in the group is bonded to another group. For example, a straight solid line bond in -OCH3 indicates that the oxygen atom in the group is bonded to another group. The straight dashed bond in the diagram indicates that the group is connected to other groups through both ends of the nitrogen atom in the group; The wavy lines in the text indicate that the phenyl group is connected to other groups through the carbon atoms at positions 1 and 2 of the phenyl group. This indicates that any connectable site on the piperidinyl group can be linked to other groups via a single chemical bond, including at least... , , , Even if H atoms are drawn on -N- in these four connection methods, Still includes In this type of linkage, when a chemical bond is attached, the number of hydrogen atoms at that site is reduced by one, resulting in a monovalent piperidinyl group.

[0085] Unless otherwise specified, the term "C" 1-3 "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl groups, etc.; they can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). C 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), and propyl (including...). n -propyl and isopropyl), etc.

[0086] Unless otherwise specified, the term "C" 1-3 "Alkoxy" refers to alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule by an oxygen atom. The C 1-3 Alkoxy groups include C 1-2 C 2-3 C3 and C2 alkoxy groups, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.

[0087] Unless otherwise specified, the term "C" 1-3 "Alkylamino" refers to alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule via an amino group. The C 1-3 Alkylamino groups include C 1-2 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, etc.

[0088] Unless otherwise specified, "C 3-5 "Cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 3 to 5 carbon atoms, which is a monocyclic system. 3-5 Cycloalkyl groups include C 3-4 and C 4-5 Cycloalkyl groups, etc.; they can be monovalent, divalent, or polyvalent. C 3-5 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, etc.

[0089] Unless otherwise specified, C n-n+m Or C n -C n+m This includes any specific case with n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 C 11 and C 12 It also includes any range from n to n+m, such as C 1-12 Including 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 represent the number of atoms in the ring from n to n+m. For example, 3-12-membered rings include 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, and 12-membered rings, and also any range from n to n+m. For example, 3-12-membered rings include 3-6-membered, 3-9-membered, 5-6-membered, 5-7-membered, 6-7-membered, 6-8-membered, and 6-10-membered rings, etc.

[0090] The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (such as a nucleophilic substitution reaction). For example, representative leaving groups include trifluoromethanesulfonates; chlorine, bromine, and iodine; sulfonate groups, such as methanesulfonates, toluenesulfonates, p-bromobenzenesulfonates, p-toluenesulfonates, etc.; acyloxy groups, such as acetoxy groups, trifluoroacetoxy groups, etc.

[0091] The term "protecting group" includes, but is not limited to, "amino protecting group," "hydroxy protecting group," or "thiol protecting group." The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the nitrogen position of an amino group. Representative amino 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), etc. The term "hydroxyl protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxyl protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanolyl groups (e.g., acetyl); arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (diphenylmethyl, DPM); silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc.

[0092] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0093] The structures of the compounds of this invention can be confirmed using conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, that absolute configuration can be confirmed using conventional techniques in the art. For example, single-crystal X-ray diffraction (SXRD) is used, where the cultured single crystal is used to collect diffraction intensity data using a Bruker D8 venture diffractometer with CuKα radiation as the light source. The scanning method is as follows: After collecting relevant data by ω scanning, the crystal structure can be further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0094] The present invention uses the following abbreviations: DMAC: N,N-dimethylacetamide, PG: propylene glycol, HP-β-CD: hydroxypropyl-β-cyclodextrin, and Solutol HS-15 represents polyethylene glycol (15)-hydroxystearate.

[0095] The solvents used in this invention are commercially available. Compounds are named according to conventional naming principles in the art or using ChemDraw® software; commercially available compounds are named using supplier catalog names. Detailed Implementation

[0096] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0097] Reference Example 1

[0098] Synthesis method:

[0099] See Example 2

[0100] Synthesis method:

[0101] Using Shionogi's anti-influenza drug S-033447 as a reference compound, the low-energy conformation of S-033447 was calculated using the Macromodel module of Schrödinger's Maestro software. In the low-energy conformation, the dihedral angle (dehidal 1) of 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 Appendix). Figure 3 The transformation of S-033447 from its low-energy conformation to its protein-binding active conformation (rotation of the parent nucleus from -146.6° to -153.7°, and rotation of the dihedral angle between 2,5-dihydrothiophene and the parent nucleus from 56.8° to 55.0°) requires overcoming an energy barrier of 0.8 kcal / mol (see Appendix). Figure 4 The 3D binding mode of S-033447 to the protein (PDB ID: 6FS6) is shown in the appendix. Figure 1 The interactions between S-033447 and amino acids and metal ions are shown in the appendix. Figure 2 .

[0102] Example 1

[0103] By observing the binding modes of the active conformations of S-033447 and 6FS6 proteins, we found that the lowest energy barrier conformation (low-energy conformation) formed between the benzo-2,5-dihydrothiophene-difluorobenzyl fragment of S-033447 and the pyridohexahydropyrimidine core during free rotation closely matches the protein binding mode conformation (active conformation) in the cocrystal of S-033447 and 6FS6 proteins. This explains the high binding activity of S-033447 and 6FS6 proteins. Generally, the smaller the energy barrier difference 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 when transforming from the low-energy conformation to the active conformation that binds to the protein. This makes it easier for the compound to bind to the protein, resulting in higher binding activity.

[0104] To lock the active conformation of S-033447 and further reduce its rotational energy barrier, we replaced the O and S of the tetrahydropyran and 2,5-dihydrothiophene fragments of S-033447 with Se atoms, obtaining different cycloselenopentane and cycloselenohexane fragments. We then explored the energy barrier difference between the lowest energy barrier conformation of these Se-substituted molecules and the active conformation in the cocrystal of S-033447 and 6FS6 proteins.

[0105] (1) The low-energy conformational rotation dihedral angles and rotational energy barriers of compounds A and B were calculated using the Macromodel module, and the results are shown in Table 1. A comparison of the low-energy conformations of compound A (dark) and S-033447 (light) is shown in the appendix. Figure 5 The energy changes of the two dihedral angles of compound A during rotation are shown in the appendix. Figure 6 A comparison of the low-energy conformations of compound B (dark) and S-033447 (light) is shown in the appendix. Figure 7 The energy changes of the two dihedral angles of compound B during rotation are shown in the appendix. Figure 8 .

[0106] Table 1. Rotation dihedral angles and rotation barriers of the low-energy conformations of the compounds of this invention.

[0107] Note: Dihedral 1 is the dihedral angle of pyridohexahydropyrimidine, Dihedral 2 is the dihedral angle of pyridohexahydropyrimidine and 2,5-dihydrothiophene, and ΔE is the energy barrier required to transform from the low-energy conformation to the protein-binding active conformation of S-033447 (Dihedral 1 is -153.7°, Dihedral 2 is 55.0°).

[0108] Conclusion: The low-energy conformation of compound B overlaps well with the active conformation of S-033447. The lowest binding energy barrier of the compound of the present invention in the 6FS6 protein structure has a small energy difference with that of the reference compound in the active conformation of the same protein structure. Therefore, the compound of the present invention binds more readily to this protein and may exhibit similar or better binding activity to the reference compound in actual binding.

[0109] (2) The low-energy conformational rotation dihedral angle of compound B was calculated using the Macromodel module, and the results are shown in Table 2.

[0110] Table 2. Low-energy conformational dihedral angles of the compounds of this invention

[0111] Note: Dihedral 1 is the dihedral angle of pyridohexahydropyrimidine, and Dihedral 2 is the dihedral angle of pyridohexahydropyrimidine and 2,5-dihydroselenothiophene.

[0112] Conclusion: The low-energy conformation of compound B is basically consistent with that of S-033447.

[0113] Example 2

[0114] Step 1: Synthesis of Compound 2-2

[0115] Sodium dihydrogen phosphate (13.58 g, 113.19 mmol) was added to water (20 mL), followed by acetonitrile (10 mL), diphenyldiselenoether (1.18 g, 3.77 mmol), and zinc powder (986.83 mg, 15.09 mmol) in portions. The reaction mixture was stirred at room temperature for 1 hour, then compound 2-1 (2 g, 7.55 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate (10 mL × 2). The mixture was separated, and the aqueous phase was extracted with ethyl acetate (10 mL × 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give compound 2-2. MS m / z: 343.0 [M+H] + .

[0116] Step 2: Synthesis of compounds 2-3

[0117] Compound 2-2 (1.5 g, 4.40 mmol) was added to methanol (10 mL) and water (5 mL), and sodium hydroxide (527.53 mg, 13.19 mmol) was added. The reaction mixture was stirred at 60 °C for 2 hours. The reaction mixture was cooled to room temperature, and the pH was adjusted to 7 with 1 N hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2-3. The crude product was used directly in the next step.

[0118] Step 3: Synthesis of compounds 2-4

[0119] Compounds 2-3 (1.3 g, 3.97 mmol) were added to polyphosphoric acid (13 mL), and the reaction mixture was stirred at 120 °C for 2 hours. The reaction mixture was cooled to 80 °C and added to water (50 mL) under stirring. Stirring was continued for 5 minutes. Extraction was performed with dichloromethane (20 mL × 2). The organic phases were combined, washed with water (20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give compounds 2-4. MS m / z: 310.9 [M+H] + .

[0120] Step 4: Synthesis of compounds 2-5

[0121] Compound 2-4 (300 mg, 970.34 µmol) was added to methanol (6 mL), followed by sodium borohydride (110.13 mg, 2.91 mmol). The reaction mixture was stirred at room temperature for 1 hour. The pH of the reaction mixture was adjusted to 7 with 1N hydrochloric acid, and then extracted with dichloromethane (10 mL × 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 2:1) to give compound 2-5.

[0122] Step 5: Synthesis of compounds 2-7

[0123] Compounds 2-6 (50 mg, 152.75 µmol) were added to ethyl acetate (1 mL), followed by compound 2-5 (47.53 mg, 152.75 µmol), 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). The reaction mixture was refluxed overnight. The reaction mixture was cooled to room temperature, and water (10 mL) was added. Extraction was performed with ethyl acetate (5 mL × 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to give compound 2-7. MS m / z: 622.0 [M+H] + .

[0124] Step 6: Synthesis of compounds 2 and 2'

[0125] Compound 2-7 (10 mg, 16.12 µmol) was added to N,N-dimethylacetamide (0.5 mL), followed by lithium chloride (3.42 mg, 80.58 µmol, 1.65 µL). The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was cooled to room temperature and diluted with acetonitrile (2 mL). The crude reaction mixture was purified by preparative high-performance liquid chromatography (pHPLC) (column: Xtimate C18100). 30mm 3µm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile%: 40%-60%, 8 min) to give compound 2 (retention time 3.205 min) and compound 2' (retention time 3.301 min).

[0126] Compound 2 (retention time 3.205 minutes). 1 ¹H 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] + .

[0127] Compound 2' (retention time 3.301 minutes) 1 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] + .

[0128] Example 3

[0129] Step 1: Synthesis of compound 3-2

[0130] Compound 3-1 (66 g, 383.43 mmol) was dissolved in dichloromethane (460 mL), and N,N-dimethylformamide (280.27 mg, 3.83 mmol, 295.02 µL) was added. Oxaloyl chloride (73.00 g, 575.15 mmol, 50.35 mL) was added dropwise to the reaction solution. After the addition was complete, the reaction solution was stirred at 20 °C for 30 minutes, and then concentrated to dryness under reduced pressure. The 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 with stirring. The reaction solution was stirred at 20 °C for 1 hour. Add water (100 mL), separate the layers, and extract the aqueous phase with dichloromethane (50 mL × 2). Combine the organic phases and wash with dilute hydrochloric acid (0.2 M, 50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and saturated brine (50 mL), respectively. Dry the mixture with anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness under reduced pressure to give compound 3-2. 1 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).

[0131] Step 2: Synthesis of compound 3-3

[0132] 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 addition was complete, the reaction mixture was heated to 20 °C and stirred for 2 hours. The reaction mixture was quenched with 1 M hydrochloric acid, the pH was adjusted to 7, and the mixture was extracted with ethyl acetate (100 mL × 2). The organic phases were combined and washed with dilute hydrochloric acid (0.2 M, 50 mL), saturated sodium bicarbonate solution (50 mL), and saturated brine (50 mL), respectively. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain compound 3-3. 1 ¹H 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).

[0133] Step 3: Synthesis of compounds 3-4

[0134] Compound 3-3 (15 g, 88.15 mmol) was dissolved in pyridine (90 mL), and selenium dioxide (19.56 g, 176.31 mmol) was added. The reaction mixture was stirred at 110 °C for 12 hours. The reaction mixture 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 the product was 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 ¹H NMR (400 MHz, deuterated methanol) δ 7.64–7.68 (m, 1H), 7.30–7.32 (m, 1H), 2.52 (s, 3H).

[0135] Step 4: Synthesis of compounds 3-5

[0136] Compound 3-4 (15 g, 74.95 mmol) was dissolved in dichloromethane (60 mL) and methanol (60 mL) at 0–20 °C. Trimethylsilyldiazomethane (2 M, 44.97 mL) was added dropwise, and the reaction mixture was stirred at 20 °C for 2 hours. Then, acetic acid (3 mL) was added, and the mixture was stirred for 5 minutes. The reaction mixture was concentrated to dryness under reduced pressure, and water (50 mL) was added. The mixture was extracted with dichloromethane (50 mL × 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–20%) to give compound 3-5. 1 HNMR (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).

[0137] Step 5: Synthesis of compounds 3-6

[0138] Compound 3-5 (5 g, 23.35 mmol) was dissolved in 1,2-dichloroethane (50 mL), and N-bromosuccinimide (8.31 g, 46.69 mmol) and azobisisobutyronitrile (383.37 mg, 2.33 mmol) were added. The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature and washed with saturated sodium sulfite solution (20 mL), water (20 mL), and saturated brine (20 mL), respectively. The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–5%) to give compound 3-6. 1 ¹H 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).

[0139] Step 6: Synthesis of compounds 3-7

[0140] Sodium dihydrogen phosphate (11.52 g, 96.05 mmol) was dissolved in water (60 mL), then acetonitrile (30 mL) was added, followed by 3-(3-pyridyldiselenoyl)pyridine (3.62 g, 11.53 mmol), and zinc powder (1.88 g, 28.82 mmol) was added in portions. The reaction mixture was stirred at 20 °C for 30 min. Compound 3-6 (5.63 g, 19.21 mmol) was added, and the reaction mixture was stirred at 20 °C for 3 h. The reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–60%) to give compound 3-7. MS (ESI) m / z: 373.8 [M+H] + .

[0141] Step 7: Synthesis of compounds 3-8

[0142] Compounds 3-7 (4.2 g, 11.28 mmol) were dissolved in dichloromethane (80 mL), and Dys-Martin periodide (7.18 g, 16.93 mmol) was added. The reaction mixture was stirred at 20 °C for 12 hours. A saturated sodium sulfite solution (30 mL) was added to the reaction mixture, and the mixture was stirred for 5 minutes. The mixture was extracted with dichloromethane (30 mL × 2), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–50%) to give compounds 3-8. MS (ESI) m / z: 371.9 [M+H] + .

[0143] Step 8: Synthesis of compounds 3-9

[0144] Compounds 3-8 (2.9 g, 7.83 mmol) were dissolved in tetrahydrofuran (16 mL), and an aqueous sodium hydroxide solution (626.67 mg, 15.67 mmol, 4 mL) was added. The reaction 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 1 N hydrochloric acid. The solid phase was filtered, and the filter cake was dried under reduced pressure to give compounds 3-9. MS (ESI) m / z: 357.9 [M+H] + .

[0145] Step 9: Synthesis of compounds 3-10

[0146] 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 separately. The reaction mixture 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 mixture, and the mixture was stirred for 5 minutes. The mixture was filtered, and the filtrate was separated. The aqueous phase was extracted with dichloromethane (10 mL). The combined organic phases were washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–50%) to give compound 3-10. MS (ESI) m / z: 311.8 [M+H] + .

[0147] Step 10: Synthesis of compound 3-11

[0148] Compound 3-10 (390 mg, 1.26 mmol) was dissolved in isopropanol (8 mL), and sodium borohydride (95.14 mg, 2.51 mmol) was added. The reaction mixture was stirred at 20 °C for 1 hour. The pH was adjusted to 7 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (15 mL × 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, ethyl acetate ratio: 0–50%) to give compound 3-11. MS (ESI) m / z: 313.8 [M+H] + .

[0149] Step 11: Synthesis of Compounds 3-12

[0150] Compound 3-11 (330 mg, 1.06 mmol) was dissolved in dichloromethane (6 mL), and thionyl chloride (251.53 mg, 2.11 mmol, 153.37 µL) was added. The reaction mixture was stirred at 20 °C for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure to obtain compound 3-12, which was used directly in the next reaction step.

[0151] Step 12: Synthesis of compounds 3-13 and 3-13'

[0152] Compounds 2-6 (340 mg, 1.04 mmol) were dissolved in acetonitrile (6 mL), and compounds 3-12 (343.41 mg, 1.04 mmol) and cesium carbonate (676.86 mg, 2.08 mmol) were added. The reaction mixture was stirred at 60 °C for 12 hours. The reaction mixture was cooled to room temperature, and water (5 mL) was added. The mixture was extracted with ethyl acetate (5 mL × 3). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (methanol / dichloromethane, methanol ratio: 0–5%). The obtained compounds were detected by supercritical fluid chromatography (analytical method: column model: 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 increased from 5% to 40% over 2 min, held at 40% for 1.2 min, then held at 5% for 0.8 min) Analyzed as a mixture using chiral separation (column model: DAICEL CHIRALPAK AD (250 mm)). 30 mm, 10 µm); Mobile phase: [A: carbon dioxide, B: 0.1% ammonia / ethanol]; Gradient: mobile phase B maintained at 40%), the chiral isomers 3-13 (retention time 1.831 min, ee = 96.1%) and 3-13' (retention time 2.031 min, ee = 100%) were separated.

[0153] Step 13: Synthesis of Compound 3

[0154] Compound 3-13 (6 mg, 9.65 µmol) was added to N,N-dimethylacetamide (1 mL), followed by lithium chloride (2.05 mg, 48.27 µmol). The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction mixture was purified by preparative high-performance liquid chromatography (pHPLC) (column: Phenomenex Gemini-NX C1875). 30 mm 3 µm; Mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; Gradient: acetonitrile%: 30%-53%, 5 min) to obtain compound 3. 1 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 = 10.54Hz, 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] + .

[0155] Step 14: Synthesis of compound 3'

[0156] Compound 3-13' (5 mg, 8.05 µmol) was added to N,N-dimethylacetamide (1 mL), followed by lithium chloride (1.71 mg, 40.23 µmol). The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction mixture was purified by preparative high-performance liquid chromatography (pHPLC) (column: Phenomenex Gemini-NX C1875). 30 mm 3 µm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile %: 30%-53%, 5 min) to obtain compound 3'. 1 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] + .

[0157] Example 4

[0158] Compound 3 (130.00 mg, 244.65 µmol) was added to N,N-dimethylacetamide (2 mL), followed by 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). The reaction mixture was stirred at 70 °C for 3 hours. The reaction mixture was cooled to room temperature, and water (10 mL) was added. The mixture was extracted with ethyl acetate (10 mL × 2), and the organic phase was washed with saturated brine (10 mL × 4). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 20:1) to give compound 4. 1 H 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] + .

[0159] The absolute configuration of compound 4 was confirmed by single-crystal X-ray diffraction (SXRD) as follows: .

[0160] Example 5

[0161] Compound 2 (30.00 mg, 56.56 µmol) was added to N,N-dimethylacetamide (1 mL), followed by 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). The reaction mixture was stirred at 70 °C for 3 hours. The reaction mixture was cooled to room temperature, and water (3 mL) was added. Extraction was performed with ethyl acetate (3 mL × 2). The organic phase was washed with saturated brine (3 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by preparative thin-layer chromatography using silica gel plates (dichloromethane:methanol = 10:1) to give compound 5. 1 ¹H 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=10.54Hz, 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] + .

[0162] Example 6

[0163] Step 1: Synthesis of Compound 6-2

[0164] Under ice bath conditions, a trimethylsilane-diazomethane solution (2 M, 13.61 mL, 27.22 mmol) was added dropwise to a methanol (8 mL) and tetrahydrofuran (32 mL) solution of compound 6-1 (3.35 g, 13.61 mmol). After the addition was complete, the reaction mixture was heated to 20 °C and stirred for 1 hour. A saturated citric acid solution (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined and washed successively with a saturated sodium bicarbonate solution (100 mL) and a saturated brine solution (100 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain crude compound 6-2, which was used directly in the next reaction.

[0165] Step 2: Synthesis of Compound 6-3

[0166] Compound 6-2 (3.98 g, 15.29 mmol), tert-butyl hydrazinoate (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 mixture was reacted at 60 °C for 12 hours. The reaction mixture was cooled to room temperature, and water (200 mL) was added. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with water (200 mL) and saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:2) to give compound 6-3.

[0167] Step 3: Synthesis of Compound 6-4

[0168] 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 mixture was reacted at 50 °C for 12 hours. The reaction mixture was concentrated and evaporated to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:2, v / v) to give compound 6-4.

[0169] Step 4: Synthesis of compounds 6-5

[0170] An ethyl acetate solution (4 M, 10 mL) was added to a 20 mL solution of compound 6-4 (1.6 g, 3.47 mmol), and the reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was then concentrated under reduced pressure to obtain crude hydrochloride 6-5, which was used directly in the next reaction step.

[0171] Step 5: Synthesis of Compound 6-6

[0172] 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 mixture was stirred at 25 °C for 1 hour. Methanol (30 mL) was added, the mixture was concentrated, and evaporated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 0:1, v / v, followed by dichloromethane:methanol = 10:1 to 0:1) to obtain compound 6-6.

[0173] Step 6: Synthesis of compounds 6-7

[0174] Compound 6-6 (3 g, 9.14 mmol) was dissolved in dimethyl sulfoxide (30 mL) and water (3 mL), and sodium chloride (1.07 g, 18.27 mmol) was added. The reaction mixture was stirred at 90 °C for 12 hours. The reaction mixture was diluted with water (100 mL), extracted with dichloromethane (100 mL × 3), washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to give compound 6-7.

[0175] Step 7: Synthesis of compounds 6-8

[0176] Compounds 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), and 1,8-diazabicyclo[5.4.0]undec-7-ene (506.93 mg, 3.33 mmol) was added. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was diluted with water (30 mL), extracted with ethyl acetate (30 mL × 3), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to give compounds 6-8. MS (ESI) m / z: 297.3 [M+H] + .

[0177] Step 8: Synthesis of compounds 6-9

[0178] Compounds 6-8 (70 mg, 236.23 µmol) and 2-5 (73.51 mg, 236.23 µmol) were added to ethyl acetate (2 mL), followed by tri-n-propylcyclic phosphoric 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). The reaction mixture was stirred at 77 °C for 3 hours. The reaction mixture was cooled to room temperature, and water (10 mL) was added. The mixture was extracted with ethyl acetate (5 mL × 2). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (dichloromethane:methanol = 1:0 to 10:1) to give compound 6-9. MS (ESI) m / z: 591.1 [M+H] + .

[0179] Step 9: Synthesis of compounds 6-9A and 6-9B

[0180] Compounds 6-9 were analyzed as racemic compounds by supercritical fluid chromatography (analytical 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%: from 5% to 40% over 4 min, then held for 2.5 min; then held at 5% for 1.5 min). Chiral separation (column type: DAICEL CHIRALCEL OD-H (25 mm × 30 mm, 5 µm); mobile phase: [A: carbon dioxide, B: 0.1% ammonia / ethanol]; gradient: B%: 40%-40%) yielded compounds 6-9A (retention time 4.024 min) and 6-9B (retention time 4.447 min).

[0181] Step 10: Synthesis of Compound 6

[0182] 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). The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was cooled to room temperature and diluted with acetonitrile (2 mL). The crude product was purified by preparative high-performance liquid chromatography (HPLC) (column type: Xtimate C18 100×30 mm×3 µm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 50%-70%, 5 min) to obtain compound 6. 1¹H 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] + .

[0183] Step 11: Synthesis of compound 6'

[0184] 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). The reaction mixture was stirred at 80 °C for 12 hours. After cooling to room temperature, the mixture was diluted with acetonitrile (2 mL). The crude product was purified by preparative high-performance liquid chromatography (HPLC) (column: Ultimate C18 100×30 mm×3 µm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile %: 50%-70%, 5 min) to obtain compound 6'. 1 ¹H 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] + .

[0185] Biological test data

[0186] Experiment Example 1: Influenza Virus Cytopathic Effect (CPE) Experiment

[0187] By determining the half-maximal effective concentration (EC50) of the compound. 50 The antiviral activity of compounds against influenza virus (IFV) is evaluated using the cytopathic effect (CPE) assay. The CPE assay is widely used to determine the protective effect of compounds against virus-infected cells, reflecting the compound's antiviral activity.

[0188] Influenza virus CPE experiment

[0189] MDCK cells were seeded at a density of 2,000 cells per well in black 384-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator. The compound was diluted using an Echo555 non-contact nano-level ultrasonic pipetting system and added to the cell wells (4-fold serial 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 dose (TCID90) per well, with a final DMSO concentration of 0.5% in the culture medium. Virus control wells (DMSO and virus added, no compound), cell control wells (DMSO added, no compound and virus added), and culture medium control wells (culture medium only, no cells) were set up. Cytotoxicity and antiviral activity assays were performed in parallel, with all experimental conditions identical to the antiviral activity assay except for the absence of virus. Cell plates were incubated at 37°C in a 5% CO2 incubator for 5 days. Cell viability was assessed using a CCK8 cell viability assay kit after 5 days of incubation. The raw data were used to calculate the antiviral activity and cytotoxicity of the compounds.

[0190] The antiviral activity and cytotoxicity of the compounds are expressed as the percentage (%) of inhibition of the viral-induced cellular effects by the compounds. The calculation formula is as follows:

[0191] The inhibition rate and cytotoxicity of the compound were analyzed using nonlinear fitting with GraphPad Prism software to obtain the EC50 of the compound. 50 Values. The experimental results are shown in Table 3.

[0192] Table 3. Inhibitory activity of compounds against influenza virus A / PR / 8 / 34 (H1N1)

[0193] Conclusion: The compounds of this invention showed positive effects in the cellular-level inhibition of influenza virus replication.

[0194] Experimental Example 2: In vivo drug efficacy study

[0195] Experimental objective: To evaluate the efficacy of the compound in a mouse model of H1N1 influenza A virus infection.

[0196] Experimental protocol: Mice were infected with influenza A virus A / PR / 8 / 34 (H1N1) via intranasal instillation. Treatment with the compound began 48 hours post-infection and was administered orally twice daily for 7 consecutive days. The compound's anti-H1N1 activity in this model was evaluated by observing changes in mouse body weight and survival rate.

[0197] SPF-grade BALB / c mice, 6-7 weeks old and female, were used in the experiment. Mice were allowed at least 3 days to acclimatize after arriving at the BSL-2 animal facility before the experiment began. The day of infection was designated as day 0 of the experiment. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg / kg, 10 mL / kg). After the animals were deeply anesthetized, they were infected with A / PR / 8 / 34 (H1N1) virus via intranasal instillation, 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 48 hours post-infection. The mice were observed daily, and their weight and survival rate were recorded. On day 14, all surviving animals were euthanized.

[0198] Experimental results: The animal survival rate and weight loss rate were measured. Weight loss rate = (body weight on day 0 – body weight on day N) / body weight on day 0 100%. The results are shown in Table 4: Compound 5 achieved a maximum weight loss rate of 13.77% in the protected animals on day 7, and then began to recover, with a mouse survival rate of 100% by the end of the experiment; Compound 4 achieved a maximum weight loss rate of 7.03% in the protected animals on day 3, and then began to recover, with a mouse survival rate of 100% by the end of the experiment.

[0199] Table 4 Results of animal survival rate and weight loss rate

[0200] Conclusion: The compounds of this invention exhibit excellent weight protection and early recovery time in animal pharmacodynamic models.

[0201] Experiment Example 3: Cytopathic Effect (CPE) Experiment on Baloxavir-Resistant A / PR / 8 / 34 (H1N1) I38T Influenza Virus Strain

[0202] Experimental objective: To determine the half-maximal effective concentration (EC50) of a compound. 50 The antiviral activity of the compound against the Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T influenza virus strain was evaluated using the 40-400 saturation value.

[0203] Experimental protocol: MDCK cells were seeded at a density of 15,000 cells per well in 96-well cell culture plates and cultured overnight at 37°C in a 5% CO2 incubator. The next day, a compound solution (3-fold serial dilution, 8 concentration points, triplet wells) and Baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strain were added, with a final DMSO concentration of 0.5% in the cell culture medium. Cells were cultured for 5 days in a 5% CO2 incubator at 37°C until cytopathic effect (CPE) reached 80-95% in the virus-infected control wells without the compound. Cell viability was then assessed using a CCK8 assay. If the cell viability in the wells containing the compound was higher than that in the virus-infected control wells (i.e., CPE was reduced), it indicated that the compound had an inhibitory effect on the tested virus.

[0204] Experimental results: The antiviral activity of a compound is expressed as the percentage (%) of its inhibitory activity against the cellular viral effect induced by the virus. The calculation formula is as follows:

[0205] EC 50 The inhibitory activity and cell viability of the compounds were analyzed using nonlinear fitting with GraphPad Prism (version 5) software. The fitting method was "log(inhibitor) vs. response -- variable slope". The experimental results are shown in Table 5.

[0206] Table 5. Inhibitory activity of the compounds of the present invention against Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T influenza virus strain

[0207] Conclusion: The compounds of this invention showed a positive effect in inhibiting the replication of Baloxavir-resistant A / PR / 8 / 34 (H1N1) influenza virus strains at the cellular level.

[0208] Experimental Example 4: In vivo drug efficacy study

[0209] Experimental Objective: To evaluate the efficacy of the compound in a mouse infection model of drug-resistant influenza A virus H1N1.

[0210] Experimental protocol: Mice were infected with Baloxavir-resistant influenza A virus strain A / PR / 8 / 34 (H1N1) I38T via intranasal instillation. Treatment with the compound began 2 hours before infection, followed by oral administration for 7 consecutive days, twice daily. The effect of the compound against influenza A virus H1N1 in this model was evaluated by observing changes in mouse body weight and survival rate.

[0211] SPF-grade BALB / c mice, 6-7 weeks old and female, were used in the experiment. Mice were allowed at least 3 days to acclimatize after arriving at the BSL-2 animal facility before the experiment began. The day of infection was designated as day 0 of the experiment. Mice were deeply anesthetized by intraperitoneal injection of salbutamol / xylazine hydrochloride, and then infected with Baloxavir-resistant A / PR / 8 / 34 (H1N1) I38T virus strain via nasal instillation, with an infection volume of 50 μL. From day 2 to day 8, the test compound was administered orally twice daily at 15 mg / kg or 50 mg / kg (administration volume 10 mL / kg). The first administration was 2 hours before infection. The mice were observed daily, and their weight and survival rate were recorded. On day 14, all surviving animals were euthanized.

[0212] Experimental results: The animal survival rate and weight loss rate were measured. Weight loss rate = (body weight on day 0 – body weight on day N) / body weight on day 0 100%. The experimental results are shown in Table 6 below. Compound 4 caused almost no decrease in mouse body weight at a dose of 50 mg / kg, and the survival rate of mice was 100% by the end of the experiment.

[0213] Table 6 Results of animal survival rate and weight loss rate

[0214] Conclusion: The compounds of this invention exhibit excellent weight protection and early recovery time in animal pharmacodynamic models.

[0215] Experimental Example 5: Plasma Protein Binding Rate Test of Compounds

[0216] Experimental objective: To evaluate the protein binding rate of the compounds of this invention in CD-1 mice, SD rats and human plasma using the balanced dialysis method.

[0217] Experimental protocol: The test compounds were diluted with dialysis buffer into the plasma of the five species 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 a control compound. The concentrations of the test compounds and warfarin in plasma and buffer were determined by LC-MS / MS.

[0218] Experimental results: The results are shown in Table 7.

[0219] Table 7. Results of plasma protein binding rates of the compounds of this invention.

[0220] Note: H represents human, R represents rat, M represents mouse, D represents dog, and C represents cynomolgus monkey.

[0221] Conclusion: The compounds of this invention exhibited moderate plasma protein binding rates in the plasma of all five species, indicating that the free drug concentration ratio of the test compounds was moderate in the plasma of the above five species, and that they possessed good drug-like properties.

[0222] Experiment Example 6: Rat Pharmacokinetic Study

[0223] Experimental objective: To investigate the plasma pharmacokinetics of the compound of the present invention in male SD rats after single intravenous injection and gavage administration.

[0224] Experimental animals: Male SD rats, 6-8 weeks old, weighing 200-300 grams; Experimental procedure: Injection (iv), dose was 1 mpk, concentration was 0.50 mg / mL, solvent was 40% DMAC + 40% PG + 20% (20% HP-β-CD + water); Oral administration (po), dose was 10 mpk, concentration was 1 mg / mL, solvent was 3% DMSO + 10% Solutol HS + 87% water.

[0225] Sample Collection: At each time point, 0.03 mL of blood was collected from the experimental animals via saphenous vein puncture, and the actual blood collection time was recorded. All blood samples were added to 1.5 mL commercially available EDTA-K2 anticoagulant tubes. After blood collection, DDV was added to the plasma matrix as a stabilizer, with a plasma:DDV solution ratio of 40:1. The DDV solution was a 1:1 solution of 40 mM DDV in acetonitrile / water. Within half an hour, the supernatant plasma was centrifuged at 4 ℃ and 3000 g for 10 minutes, and the sample was quickly placed on dry ice and stored at -80 ℃ for LC-MS / MS analysis.

[0226] Data Analysis: Plasma concentrations were processed using a non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetic software. Pharmacokinetic parameters, including Cl (apparent clearance) and T, were calculated using the linear logarithmic trapezoidal method. 1 / 2 (Time required to clear half of the compound), C max (Peak concentration), AUC 0-last (0 - concentration integral area during the last sampling time), the results are shown in Table 8.

[0227] Table 8. Rat PK results of the compounds of the present invention.

[0228] Experimental conclusion: The compound of this invention has a high oral plasma exposure and good pharmacokinetic properties.

[0229] Experimental Example 7: Pharmacokinetic Study of Beagle Dogs

[0230] Experimental objective: To investigate the plasma pharmacokinetics of male beagle dogs after a single intravenous injection and gavage administration of the compound of the present invention.

[0231] Laboratory animals: Male beagle dogs, ≥6 months old, weighing 6-12 kg; Experimental procedure: Injection (iv), dose was 1 mpk, concentration was 1 mg / mL, solvent was 10% DMAC + 90% (20% HP-β-CD + water); Oral administration (po), dose was 10 mpk, concentration was 2 mg / mL, solvent was 3% DMSO + 10% Solutol HS + 87% water.

[0232] Sample Collection: At each time point, 0.8 mL of blood was collected from the experimental animals via saphenous vein puncture, and the actual blood collection time was recorded. All blood samples were added to 1.5 mL commercially available EDTA-K2 anticoagulant tubes. After blood collection, DDV was added to the plasma matrix as a stabilizer, with a plasma:DDV solution ratio of 40:1. The DDV solution was a 1:1 solution of 40 mM DDV in acetonitrile / water. Within half an hour, the supernatant plasma was centrifuged at 4 ℃ and 3000 g for 10 minutes, and the sample was quickly placed on dry ice and stored at -80 ℃ for LC-MS / MS analysis.

[0233] Data Analysis: Plasma concentrations were processed using a non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetic software, and pharmacokinetic parameters Cl were calculated using the linear logarithmic trapezoidal method. , T 1 / 2 C max AUC 0-last The results are shown in Table 9.

[0234] Table 9. Beagle PK results of the compounds of this invention.

[0235] Experimental conclusion: The compound of this invention has low clearance rate, long half-life, high oral plasma exposure, and good pharmacokinetic properties.

Claims

1. A compound of formula (IV-A) or a pharmaceutically acceptable salt thereof, ， in, R5 and R6 are independently selected from H, F, Cl, Br, I, OH, NH2, and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R groups. b replace; R7 is selected from H and ; R8 is selected from C 1-3 Alkyl and The C 1-3 Alkyl and Choose 1, 2, or 3 Rs a replace; E1 is Se, p is selected from 0 and 1, E2 is selected from S and O; q is selected from 0 and 1; Each R a and R b They are each independently selected from H, F, Cl, Br, and I; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer." 2. The compound represented by formula (V-1-A) or a pharmaceutically acceptable salt thereof, ， in, R5 and R6 are independently selected from H, F, Cl, Br, I, OH, NH2, and C, respectively. 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R groups. b replace; R7 is H; q is selected from 0 and 1; R b Independently selected from H, F, Cl, Br, and I; bring" "The carbon atom is a chiral carbon atom, existing as a single enantiomer (R) or (S) or rich in one enantiomer." 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R8 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and The CH3, CH2CH3, CH2CH2CH3, CH(CH3)2 and Choose 1, 2, or 3 Rs a replace.

4. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein, R8 is selected from CH3, CH2CH3, CH(CH3)2 and .

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, R7 is selected from H, , , and .

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, E1 is Se, and E2 is O.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from, , , and , in, R1 and R2 are independently selected from H, F, Cl, Br, I, OH and NH2, respectively; m is selected from 0 and 1; R5, R6 and R8 are as defined in claim 1.

8. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from, and , in, q, R5, R6 and R7 are as defined in claim 2.

9. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are F.

10. The compound according to claim 7 or 8, or a pharmaceutically acceptable salt thereof, wherein, R5 and R6 are F.

11. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from, and , in, R1, R2, R5, R6 and R8 are as defined in claim 7.

12. The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from, and , in, R5, R6 and R7 are as defined in claim 8.

13. A compound of the following formula or a pharmaceutically acceptable salt thereof, and .

14. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 in the preparation of a medicament for treating diseases related to influenza virus.

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