Ligand targeting integrin αvβ6, conjugate and composition, and uses thereof

Abstract

The present disclosure relates to the technical field of targeted delivery of small nucleic acid drugs, and particularly provides a ligand targeting integrin αvβ6, a conjugate and a composition, and uses thereof. The ligand provided by the present disclosure can target integrin αvβ6, and by introducing the ligand into a double-stranded oligonucleotide, functional groups can be specifically delivered to cells and / or tissues expressing integrin αvβ6, thereby achieving the purpose of disease diagnosis and / or treatment.

Description

A ligand, conjugate, composition targeting integrin αvβ6 and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent applications with application numbers 202311787123.X filed with the State Intellectual Property Office of China on December 22, 2023 and 202411630802.0 filed with the State Intellectual Property Office of China on November 14, 2024, the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure relates to the technical field of small nucleic acid drug targeted delivery, and specifically discloses a ligand, conjugate, and composition targeting integrin αvβ6, and uses thereof. Background Art

[0004] Integrins are transmembrane receptors that mediate the connection between cells and their external environment. During signal transduction, integrins deliver information about the chemical composition of the extracellular matrix, among other things, to cells. Integrin αvβ6 is an epithelial cell-specific integrin, a transmembrane heterodimer composed of α and β transmembrane subunits non-covalently bound together. It is primarily expressed in malignant epithelial tumors, with lower expression in healthy tissue and benign tumors.

[0005] However, when tissue is damaged or inflamed, the expression of integrin αvβ6 increases, promoting epithelial cell proliferation and migration to the damaged site, thereby helping to rebuild epithelial tissue.

[0006] Integrin αvβ6 is an endocytic receptor expressed in various cell types. This study aims to develop a ligand that specifically binds to integrin αvβ6 to achieve the goal of specifically targeting drug molecules to cells expressing integrin αvβ6. Summary of the Invention

[0007] The present disclosure provides a ligand, conjugate, composition, and use thereof targeting integrin αvβ6. The ligand provided herein is capable of targeting integrin αvβ6. By introducing the ligand into a double-stranded oligonucleotide, a functional group can be specifically delivered to cells and / or tissues expressing integrin αvβ6, thereby achieving the purpose of disease diagnosis and / or treatment.

[0008] In a first aspect, the present disclosure provides a ligand targeting integrin αvβ6, wherein the ligand is selected from the structure represented by formula (I), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0009] Wherein, R1 is selected from

[0010] L1 is selected from optionally substituted C 10 -C 30 Alkylene or wherein Z is selected from O or S, j is selected from an integer from 1 to 5, and k is selected from an integer from 1 to 10. 10 -C 30 The alkylene group contains a substituent, the substituent is selected from halogen or C1-C3 alkoxy;

[0011] R2 is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C5-C 10 Aryl or wherein each R2' is independently selected from an optionally substituted C1-C6 alkyl group; if the optionally substituted C1-C6 alkyl group contains a substituent, the substituent is selected from halogen, C5-C 10 Aryl or C1-C3 alkoxy; if optionally substituted C5-C 10 If the aryl group contains a substituent, the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy;

[0012] m is an integer selected from 1 to 5;

[0013] Each R3 is independently selected from H or an amino acid residue;

[0014] Y is selected from CH2 or NH;

[0015] R6 is selected from Among them, R 4a and R 4b are each independently selected from H, OH, SH, NH2, halogen, C1-C3 alkyl or C1-C3 alkoxy; R 6a 、R 6b 、R 6c and R 6d Each independently selected from H or C1-C3 alkyl;

[0016] n is an integer selected from 1-4.

[0017] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0018] In some embodiments, the ligand is selected from the structure shown in formula (I-1), or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0019] In other embodiments, the ligand is selected from the structure shown in formula (I-2), or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0020] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0021] In some embodiments, the ligand is selected from the structure shown in formula (IIA), or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0022] In other embodiments, the ligand is selected from the structure shown in formula (IIB), or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0023] In other embodiments, the ligand is selected from the structure shown in formula (IIC), or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0024] In a second aspect, the present disclosure provides a conjugate having a structure represented by formula (VI), or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof:

[0025] Among them, Nu represents the active drug molecule;

[0026] t is an integer selected from 1 to 6;

[0027] Each M is independently selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0028] L2 is selected from wherein Z' is selected from O or S, j1 is selected from an integer of 1 to 5, j2 is selected from an integer of 1 to 5, j3 is selected from an integer of 1 to 10, and j4 is selected from an integer of 1 to 5;

[0029] L3 is selected from wherein e is selected from an integer of 2-6, and f is selected from an integer of 4-8;

[0030] a1 is selected from 1, 2, 3 or 4; a2 is selected from 1 or 2; a3 is selected from an integer selected from 1 to 5;

[0031] X1 is selected from hydroxyl or thiol;

[0032] p and q are each independently selected from 0, 1, 2, 3 or 4;

[0033] R5 is selected from H or C1-C3 alkoxy;

[0034] X2 is selected from O, S, or NH;

[0035] Each M' is independently selected from the structure represented by formula (i), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt:

[0036] R1' is selected from L1, m, R3 and R6 are as defined in the first aspect.

[0037] In a third aspect, the present disclosure provides a composition comprising the ligand as described in the first aspect and / or the conjugate as described in the second aspect.

[0038] In a fourth aspect, the present disclosure provides use of the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect in the preparation of a medicament for preventing and / or treating a disease or symptom associated with abnormal expression of a target gene in a cell expressing integrin αvβ6.

[0039] In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the ligand described in the first aspect, and / or the conjugate described in the second aspect, and / or the composition described in the third aspect. Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0040] In a sixth aspect, the present disclosure provides a method for reducing the expression or activity of a target gene in a cell expressing integrin αvβ6, comprising contacting the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect, or the pharmaceutical composition of the fifth aspect with a cell expressing integrin αvβ6.

[0041] In a seventh aspect, the present disclosure provides a method for preventing and / or treating a pathological condition or disease caused by abnormal expression of a target gene in a cell expressing integrin αvβ6, characterized in that the method comprises administering to a subject a pharmaceutically acceptable dose of the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect, or the pharmaceutical composition of the fifth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the relative expression levels of target genes in rat lung tissue after administration of the siRNA conjugates RZ895001, RZ895003, RZ895004, and RZ895006 in Example 1;

[0043] FIG2 shows the relative expression levels of target genes in rat lung tissue after administration of the siRNA conjugates RZ895001, RZ895009 and siRNA sequence RX895001 in Example 2;

[0044] FIG3 shows the relative expression levels of target genes in mouse lung tissue after administration of the siRNA conjugates RZ895003, RZ895007, RZ895001 and the siRNA sequence RX895001 in Example 3;

[0045] FIG4 shows the relative expression levels of target genes in mouse lung tissue after administration of the siRNA conjugates RZ895001, RZ895009 and siRNA sequence RX895001 in Example 4;

[0046] FIG5 shows the relative expression levels of target genes in mouse lung tissue after administration of the siRNA conjugates RZ895009, RZ895010, RZ895001 and the siRNA sequence RX895001 in Example 5;

[0047] FIG6 shows the relative expression levels of target genes in mouse lung tissue after administration of the siRNA conjugates RZ899012 and RZ899014 in Example 6;

[0048] FIG. 7 shows the relative expression levels of target genes in mouse lung tissue after administration of the siRNA conjugates RZ895011, RZ895001 and siRNA sequence RX895001 in Example 7. FIG.

[0049] FIG8 shows the relative expression levels of target genes in different tissues of mice after administration of the siRNA conjugates R699168, R699173, R699174, R699175, R699176, and R699177 in Example 8. DETAILED DESCRIPTION

[0050] This disclosure discloses a ligand, conjugate, and composition targeting integrin αvβ6, as well as their uses. Those skilled in the art can refer to the disclosure herein and appropriately modify process parameters to achieve these results. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by this disclosure. The methods and applications of this disclosure have been described through preferred embodiments, and it is apparent that those skilled in the art can modify, alter, and combine the methods and applications described herein to implement and apply the disclosed technology without departing from the content, spirit, and scope of this disclosure.

[0051] Explanation of terms

[0052] In the present disclosure, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0053] In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0054] In this disclosure, the term "optionally substituted" is used to define a variable that can be unsubstituted or substituted.

[0055] In this disclosure, the term "unsubstituted" means that the designated group bears no substituents.

[0056] In this disclosure, the terms "substituted," "substituted," and "substituted" are used interchangeably to indicate that any one or more hydrogen atoms in a given structure are replaced by a specified substituent (e.g., C 1-3 Alkyl, C 1-3 alkoxy or halogen), provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structural formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each substitutable position.

[0057] In the present disclosure, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0058] In the present disclosure, the term "stereoisomers" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0059] In this disclosure, the term "chiral" refers to a molecule that has the property of being non-superimposable on its mirror image; whereas "achiral" refers to a molecule that is superimposable on its mirror image.

[0060] In this disclosure, the term "enantiomers" refers to two non-superimposable isomers of a compound that are mirror images of each other.

[0061] In this disclosure, the term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0062] In this disclosure, the term "tautomer" refers to functional group isomers that arise from the rapid shift of an atom in a molecule between two positions. For example, a compound containing a carbonyl group exhibits enol tautomerism if there is a hydrogen atom adjacent to the carbonyl carbon (the α position).

[0063] In the present disclosure, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0064] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base complementary pairing. The two oligonucleotides include a sense chain and an antisense chain. The lengths of the sense chain and the antisense chain may be the same or different. As long as there is at least a partial base complementary pairing region to form a duplex region, the oligonucleotide having a double-stranded structure belongs to the double-stranded oligonucleotide described in the present disclosure. The nucleotides constituting the double-stranded oligonucleotide in the present disclosure may be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modification described in the present disclosure does not specifically refer to the site of modification. The double-stranded oligonucleotide in the present disclosure is modified except for the nucleotides, and the connecting bonds between the nucleotides may also be modified. The double-stranded oligonucleotide containing the connecting bonds between the modified nucleotides also belongs to the double-stranded oligonucleotide described in the present invention. The double-stranded oligonucleotide in the present disclosure is removed from the nucleotide portion and may also contain compound molecules or modifiers acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as connecting ligands to form conjugates.

[0065] In the present disclosure, the term "ligand" or "conjugate group" refers to an atom or group of atoms that is bound to an oligonucleotide or other oligomer. In general, a conjugate group modifies one or more properties of the compound to which it is attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties. When referring to a connection between two molecules, the term "connection" as used herein refers to the direct or indirect connection of the two molecules by a covalent bond, or the association of the two molecules by a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0066] In this disclosure, the term "pharmaceutical composition" or "composition" may refer to a composition for use in the treatment of a disease, as well as for use in in vitro cell culture experiments. When used in the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0067] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" in the present disclosure means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0068] In the present disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0069] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts in the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and protein.

[0070] In this disclosure, the terms "treat," "treat," or "ameliorate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0071] In this disclosure, the terms "prevention" and "prevent" are used interchangeably to refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0072] In the present disclosure, the term "administer" generally refers to introducing the disclosed pharmaceutical formulation into the body of a subject by any introduction or delivery route. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug can be used. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous or oral administration. The daily dose may be divided into one, two or more doses in a suitable form for administration at one, two or more times during a certain time period.

[0073] As used herein, the term "modulate gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated so that the expression, level, or activity is greater or less than that observed in the absence of the modulator. For example, the term "modulate" may mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[0074] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0075] Ligands targeting integrin αvβ6:

[0076] In a first aspect, the present disclosure provides a ligand targeting integrin αvβ6, wherein the ligand is selected from the structure represented by formula (I), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0077] Wherein, R1 is selected from

[0078] L1 is selected from optionally substituted C 10 -C 30 Alkylene or wherein Z is selected from O or S, j is selected from an integer from 1 to 5, and k is selected from an integer from 1 to 10. 10 -C 30 The alkylene group contains a substituent, the substituent is selected from halogen or C1-C3 alkoxy;

[0079] R2 is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C5-C 10 Aryl or wherein each R2' is independently selected from an optionally substituted C1-C6 alkyl group; if the optionally substituted C1-C6 alkyl group contains a substituent, the substituent is selected from halogen, C5-C 10 Aryl or C1-C3 alkoxy; if optionally substituted C5-C 10 If the aryl group contains a substituent, the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy;

[0080] m is an integer selected from 1 to 5;

[0081] Each R3 is independently selected from H or an amino acid residue;

[0082] Y is selected from CH2 or NH;

[0083] R6 is selected from Among them, R 4a and R 4b are each independently selected from H, OH, SH, NH2, halogen, C1-C3 alkyl or C1-C3 alkoxy; R 6a 、R 6b 、R 6c and R 6d Each independently selected from H or C1-C3 alkyl;

[0084] n is an integer selected from 1-4.

[0085] In some optional embodiments, L1 is selected from Furthermore, Selected from

[0086] In some embodiments, Z is selected from O.

[0087] In some alternative embodiments, j is selected from an integer from 1 to 3. In some specific embodiments, j is selected from 2.

[0088] In some optional embodiments, L1 is selected from

[0089] In some alternative embodiments, k is selected from an integer from 2 to 8; for example, 2, 3, 4, 5, 6, 7 or 8.

[0090] In some embodiments, L1 is selected from

[0091] In some embodiments, R1 is selected from

[0092] In some embodiments, Selected from

[0093] In some embodiments, R2 is selected from H;

[0094] In some embodiments, m is selected from 1.

[0095] In some embodiments, R3 is selected from H;

[0096] In some alternative embodiments, n is selected from an integer between 2 and 4. In some specific embodiments, n is selected from 3.

[0097] In some embodiments, R 4a and R 4b All selected from H.

[0098] In some optional embodiments, R 6a 、R 6c 、R 6b and R 6d Each is independently selected from H or methyl. In some embodiments, R 6a 、R 6c and R 6d are all selected from H, and R 6b Selected from methyl.

[0099] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0100] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0101] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0102] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0103] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0104] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0105] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0106] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0107] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0108] In some optional embodiments, the ligand is selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0109] Conjugates targeting integrin αvβ6:

[0110] In a second aspect, the present disclosure provides a conjugate having a structure represented by formula (VI), or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof:

[0111] Among them, Nu represents the active drug molecule;

[0112] t is an integer selected from 1 to 6;

[0113] Each M is independently selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0114] L2 is selected from wherein Z' is selected from O or S, j1 is selected from an integer of 1 to 5, j2 is selected from an integer of 1 to 5, j3 is selected from an integer of 1 to 10, and j4 is selected from an integer of 1 to 5;

[0115] L3 is selected from wherein e is selected from an integer of 2-6, and f is selected from an integer of 4-8;

[0116] a1 is selected from 1, 2, 3 or 4; a2 is selected from 1 or 2; a3 is selected from an integer selected from 1 to 5;

[0117] X1 is selected from hydroxyl or thiol;

[0118] p and q are each independently selected from 0, 1, 2, 3 or 4;

[0119] R5 is selected from H or C1-C3 alkoxy;

[0120] X2 is selected from O, S, or NH;

[0121] Each M' is independently selected from the structure represented by formula (i), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt:

[0122] R1' is selected from L1, m, R3 and R6 are as defined in the first aspect.

[0123] In some alternative embodiments, the active drug molecule is selected from a double-stranded oligonucleotide, a single-stranded oligonucleotide, or a small molecule chemical drug. The double-stranded oligonucleotide is selected from an siRNA. The single-stranded oligonucleotide is selected from an ASO. In some specific embodiments, Nu represents a double-stranded oligonucleotide.

[0124] In some optional embodiments, each M is independently conjugated to the 3' end, the 5' end, or a position intermediate between the 3' end and the 5' end of the sense strand of the double-stranded oligonucleotide.

[0125] In some optional embodiments, t is selected from 1 or 2; each M is independently conjugated to the 3' end or the 5' end of the sense strand of the double-stranded oligonucleotide.

[0126] In some optional embodiments, t is selected from 1, and one M is conjugated to the 5' end or the 3' end of the sense strand of the double-stranded oligonucleotide. In some specific embodiments, t is selected from 1, and one M is conjugated to the 5' end of the sense strand of the double-stranded oligonucleotide. In other specific embodiments, t is selected from 1, and one M is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0127] In some embodiments, Z' is selected from O.

[0128] In some embodiments, j1 is selected from 1.

[0129] In some embodiments, j2 is selected from 2.

[0130] In some embodiments, j3 is selected from 2.

[0131] In some embodiments, j4 is selected from 2.

[0132] In some embodiments, L2 is selected from

[0133] In some embodiments, e is selected from 3.

[0134] In some embodiments, f is selected from 6.

[0135] In some embodiments, L3 is selected from

[0136] In some optional embodiments, each M' is independently selected from any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0137] In some optional embodiments, each M is independently selected from any of the following structures, or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0138] In some optional embodiments, the conjugate is selected from the structure represented by formula (VIIA) or (VIIB), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt:

[0139] in, stands for double-stranded oligonucleotide; SS stands for the sense strand of the double-stranded oligonucleotide; AS stands for the antisense strand of the double-stranded oligonucleotide.

[0140] In some optional embodiments, the conjugate is selected from any of the following structures, or its tautomers, or its stereoisomers, or its pharmaceutically acceptable salts:

[0141] Compositions targeting integrin αvβ6:

[0142] In a third aspect, the present disclosure provides a composition comprising the ligand as described in the first aspect and / or the conjugate as described in the second aspect.

[0143] use:

[0144] In a fourth aspect, the present disclosure provides use of the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect in the preparation of a medicament for preventing and / or treating a disease or symptom associated with abnormal expression of a target gene in a cell expressing integrin αvβ6.

[0145] Pharmaceutical composition:

[0146] In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the ligand described in the first aspect, and / or the conjugate described in the second aspect, and / or the composition described in the third aspect.

[0147] Furthermore, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers or excipients.

[0148] Methods for reducing the expression or activity of a target gene in a cell expressing integrin αvβ6:

[0149] In a sixth aspect, the present disclosure provides a method for reducing the expression or activity of a target gene in a cell expressing integrin αvβ6, comprising contacting the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect, or the pharmaceutical composition of the fifth aspect with a cell expressing integrin αvβ6.

[0150] In some optional embodiments, the cell is selected from but not limited to at least one of alveolar epithelial cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells, epithelial tumor cells and muscle cells (e.g., skeletal muscle cells). In some specific embodiments, the cell is selected from muscle cells.

[0151] Methods for preventing and / or treating pathological conditions or diseases caused by abnormal expression of a target gene in cells expressing integrin αvβ6:

[0152] In a seventh aspect, the present disclosure provides a method for preventing and / or treating a pathological condition or disease caused by abnormal expression of a target gene in a cell expressing integrin αvβ6, characterized in that the method comprises administering to a subject a pharmaceutically acceptable dose of the ligand of the first aspect, and / or the conjugate of the second aspect, and / or the composition of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0153] In some optional embodiments, the disease or symptom includes but is not limited to pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, pneumonia and muscle-related diseases. In some specific embodiments, the disease or symptom is selected from muscle-related diseases.

[0154] In some optional embodiments, the cell is selected from but not limited to at least one of alveolar epithelial cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells, epithelial tumor cells and muscle cells (e.g., skeletal muscle cells). In some specific embodiments, the cell is selected from muscle cells.

[0155] In some optional embodiments, the disease or symptom includes but is not limited to pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, pneumonia and muscle-related diseases. In some specific embodiments, in some specific embodiments, the disease or symptom is selected from muscle-related diseases.

[0156] In some alternative embodiments, the subject is a human.

[0157] In order to make the purpose, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to examples.

[0158] Preparation of compounds

[0159] Preparation Example 1: Synthesis of Compound 3BBA

[0160] In this preparation example, the synthetic route of compound 3BBA is as follows:

[0161] (1-1) Synthesis of Compound 3BBA-02

[0162] Compound 3BBA-01 (25 g, 112.63 mmol, 1-bromo-3-hydroxynaphthalene, CAS No. 5498-31-7) and benzyl bromide (28.9 g, 168.94 mmol, BnBr, CAS No. 100-39-0) were added to N,N-dimethylformamide (125 ml, DMF), cooled to 0°C, potassium carbonate (31.1 g, 225.30 mmol) was added at 0°C and reacted at 0°C for 10 min, then heated to 25°C and stirred. The reaction was allowed to proceed at 25°C for 12 hours, and completion was monitored by liquid chromatography-mass spectrometry (LCMS). The reaction solution was quenched by adding purified water (500 ml) and extracted twice with ethyl acetate (500 ml each time). The organic phases were combined, washed twice with saturated aqueous NaCl, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane, CAS No. 142-82-5) to obtain compound 3BBA-02 (21 g) as a light yellow oil. MS ESI (m / z) = 313.01 [M+H] + .

[0163] (1-2) Synthesis of compound 3BBA

[0164] Compound 3BBA-02 (16 g, 51.28 mmol) was added to tetrahydrofuran (160 ml, THF), replaced with nitrogen three times, and the temperature was lowered to -78°C. A solution of n-butyllithium (30.8 ml, 76.92 mmol, n-BuLi, CAS No. 109-72-8) in tetrahydrofuran was added dropwise, and the mixture was allowed to react for 1 h. Triisopropyl borate (14.7 g, 76.92 mmol, B(OiPr)3, CAS No. 5419-55-6) was added dropwise, and the reaction system was naturally warmed to 25°C and reacted at 25°C for 1 h. The completion of the reaction was monitored by thin layer chromatography (TCL) (developing solvent: Hep / EA = 5:1, v / v, Rf value of the product = 0.80, Rf value of the raw material = 0.50). The reaction solution was quenched by adding it to a mixture of purified water and ice (500 ml), adjusted to pH 3 with 12N hydrochloric acid, and extracted twice with methyl tert-butyl ether (CAS No. 1634-04-4) (300 ml each time). The organic phases were combined and washed once with a 10% (by mass) aqueous solution of NaCl (500 ml each time), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane / ethyl acetate = 5 / 1, volume ratio v / v) to obtain compound 3BBA (9.7 g) as a white solid. MS ESI (m / z) = 279.11 [M+H] + .

[0165] Preparation Example 2: Synthesis of Compound 4BBA

[0166] In this preparation example, the synthetic route of compound 4BBA is as follows:

[0167] (2-1) Synthesis of Compound 4BBA-02

[0168] Compound 4BBA-01 (35 g, 157.68 mmol, 4-bromonaphthol, CAS number 571-57-3) and BnBr (53.94 g, 315.36 mmol) were added to DMF (175 ml), cooled to 0°C, and potassium carbonate (43.58 g, 315.36 mmol) was added at 0°C. The mixture was reacted at 0°C for 10 min, then raised to 25°C and reacted at 25°C for 12 hours. The reaction was monitored by LCMS. The reaction solution was quenched by addition of purified water (500 ml) and extracted twice with ethyl acetate (500 ml each). The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane) to afford compound 4BBA-02 (28 g) as a white solid. MS ESI (m / z) = 313.01 [M+H]+ .

[0169] (2-2) Synthesis of compound 4BBA

[0170] Compound 4BBA-02 (24.7 g, 79.16 mmol) was added to THF (250 ml), replaced with nitrogen three times, and cooled to -78°C. A tetrahydrofuran solution of n-BuLi (33.07 ml, 118.75 mmol) was added dropwise, and the reaction was allowed to proceed for 1 h. Triisopropyl borate (23 g, 118.75 mmol) was added dropwise, and the reaction system was naturally warmed to 25°C after the addition was completed, and the reaction was allowed to proceed for 1 h. The completion of the reaction was monitored by TLC (developing solvent: ethyl acetate, Rf value of the product = 0.9, Rf value of the raw material = 0.4). The reaction mixture was quenched by adding it to a mixture of purified water and ice (300 ml), adjusted to pH 3 with 12N hydrochloric acid, and extracted three times with 300 ml of methyl tert-butyl ether. The organic phases were combined, washed once with 500 ml of a 10% (by mass) aqueous solution of NaCl, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 5 / 1, v / v) to obtain a crude product (15 g). A mixture of 1-[2-(2-hydroxyethoxy)ethyl]piperazine (Hep, CAS No. 13349-82-1) and ethyl acetate (EA) (Hep / EA = 10 / 1, v / v) was added to the crude product and beaten to obtain compound 4BBA (13 g) as an off-white solid. MS ESI (m / z) = 278.11 [M+H] + .

[0171] Preparation Example 3: Synthesis of Compound 5BBA

[0172] In this preparation example, the synthetic route of compound 5BBA is as follows:

[0173] (3-1) Synthesis of compound 5BBA-02:

[0174] Compound 5BBA-01 (25 g, 0.16 mol, 1-amino-5-naphthol, CAS No. 83-55-6) was added to acetonitrile (1500 ml), followed by p-toluenesulfonic acid monohydrate (89.6 g, 0.47 mol, CAS No. 6192-52-5). The mixture was cooled to 0°C and then added with a saturated aqueous solution of sodium nitrite (12 g, 0.17 mol). The mixture was reacted at 0°C for 1 h. Potassium iodide (65.2 g, 0.39 mol) was then added, and the mixture was heated to 25°C and allowed to react at 25°C. The reaction was monitored for completion by TLC (developing solvent: Hep / EA = 5 / 1, v / v, Rf value of the product = 0.75, Rf value of the starting material = 0.25). The reaction mixture was diluted with ethyl acetate (2 L), washed once with purified water (800 mL), and the aqueous and organic phases were separated. The aqueous phase was extracted three times with ethyl acetate (400 mL each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: Hep / EA = 5 / 1, v / v) to obtain compound 5BBA-02 (18 g) as a brown solid. MS ESI (m / z) = 270.96 [M+H] + .

[0175] (3-2) Synthesis of Compound 5BBA-03

[0176] Compound 5BBA-02 (15 g, 0.055 mol) and cesium carbonate (18.9 g, 0.058 mol) were added to DMF (150 ml), followed by the addition of BnBr (9.3 g, 0.054 mol). The reaction was allowed to proceed at 25°C. Completion of the reaction was monitored by TLC (developing solvent: Hep / EA = 20 / 1, v / v, Rf of the product = 0.75, Rf of the starting material = 0.2). The reaction solution was quenched by the addition of water (300 ml), extracted with ethyl acetate, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: Hep / EA = 80 / 1, v / v) to afford compound 5BBA-03 (14 g) as a light yellow solid. MS ESI (m / z) = 361.00 [M+H] + .

[0177] (3-3) Synthesis of Compound 5BBA

[0178] Compound 5BBA-03 (14 g, 0.039 mol) was added to THF (140 ml), the atmosphere was replaced with nitrogen three times, the temperature was lowered to -78°C, and a THF solution of n-BuLi (23.3 ml, 0.058 mol) was added dropwise. The reaction was allowed to react for 1 h. Triisopropyl borate (11.0 g, 0.058 mol) was added dropwise. The temperature was raised to 25°C and the reaction was continued at 25°C. The reaction was monitored for completion by TLC (developing solvent: Hep / EA = 2 / 1, v / v, Rf = 0.75 for the product and 0.50 for the starting material). The reaction system was cooled to 5-10°C and quenched by the addition of purified water (150 ml). The pH was adjusted to 3 with 1M HCl aqueous solution, and the mixture was extracted three times with 150 ml of methyl tert-butyl ether. The organic phases were combined, washed once with 10% (by mass) sodium chloride aqueous solution, and concentrated to obtain the crude product. A mixed solution of Hep and DCM (Hep / DCM = 10 / 3, v / v) was added to the crude product for slurrying to obtain compound 5BBA as a white powder. MS ESI (m / z) = 2279.11 [M+H] + .

[0179] Preparation Example 4: Synthesis of Compound PEG5

[0180] In this preparation example, the synthetic route of compound PEG5 is as follows:

[0181] (4-1) Synthesis of Compound PEG5-02

[0182] Compound PEG5-01 (15 g, 63.03 mmol, pentaethylene glycol, CAS No. 4792-15-8), silver oxide (22.0 g, 94.5 mmol, 1.5 eq) and potassium iodide (11.5 g, 69.33 mmol, 1.1 eq) were added to dichloromethane (150 ml), and the temperature was lowered to 0°C. A solution of p-toluenesulfonyl chloride (12.0 g, 63.03 mmol, 1.0 eq, CAS No. 98-59-9, abbreviated as TosCl) in dichloromethane (150 ml) was added dropwise at 0°C. The atmosphere was replaced with nitrogen three times, and the reaction solution was reacted at 25°C in a nitrogen atmosphere for 2 hours. The completion of the reaction was monitored by TLC. The reaction solution was washed twice with purified water (200 ml each time) and once with a saturated sodium chloride solution (200 ml each time). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the oily compound PEG5-02 (13 g, yield 52.6%). MS ESI (m / z) = 393.0 [M+H] + .

[0183] (4-2) Synthesis of Compound PEG5-03

[0184] Compound PEG5-02 (12 g, 30.5 mmol), trimethylsilyl azide (10.53 g, 91.6 mmol, CAS No. 4648-54-8, abbreviated as TMSN3), and potassium fluoride (5.3 g, 91.6 mmol) were added to N,N-dimethylformamide (96 ml). The temperature was raised to 60°C and the reaction was allowed to react at 60°C for 1.5 hours. The reaction was monitored by LCMS for completion. The reaction system was cooled to 25°C, the reaction solution was concentrated, and extracted with ethyl acetate (100 ml) and purified water (100 ml). The organic phase was separated and washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound PEG5-03 (6.37 g, 79% yield). MS ESI (m / z) = 264.0 [M+H] + .

[0185] (4-3) Synthesis of compound PEG5

[0186] Compound PEG5-03 (6 g, 22.7 mmol), triethylamine (4.6 g, 45.5 mmol), and 4-dimethylaminopyridine (27.7 mg, 0.23 mmol) were added to dichloromethane (66 ml). The temperature was lowered to 0°C, and p-toluenesulfonyl chloride (4.3 g, 22.7 mol) was added at 0°C. The atmosphere was replaced with nitrogen three times. The reaction system was allowed to react at 25°C under a nitrogen atmosphere for 16 hours. The reaction was monitored by TLC. The reaction solution was washed twice with purified water (50 ml each time) and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound PEG5 (7.2 g, 76% yield). MS ESI (m / z) = 418 [M+H] + .

[0187] Preparation Example 5: Synthesis of Compound IM002

[0188] In this preparation example, the synthetic route of compound IM002 is as follows:

[0189] (5-1) Synthesis of Compound IM002-03

[0190] Compound IM002-01 (3.5 g, 31.53 mmol, 2-fluoro-4-methylpyridine, CAS No. 461-87-0), compound IM002-02 (25.2 g, 157.5 mmol, N-tert-butoxycarbonyl-1,2-ethylenediamine, CAS No. 57260-73-8), cesium carbonate (20.5 g, 62.88 mmol) and N,N-dimethylformamide (70 ml) were mixed, replaced with nitrogen three times, and heated to 100°C. The reaction system was stirred at 100°C in a nitrogen atmosphere for 16 hours. The completion of the reaction was monitored by TLC. The reaction system was cooled to 25°C, and ethyl acetate (100 ml) and purified water (100 ml) were added to the reaction solution for extraction. The organic phase was separated and washed three times with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound IM002-03 (2.2 g, yield 27.7%). MS ESI (m / z) = 252.0 [M+H] + .

[0191] (5-2) Synthesis of Compound IM002

[0192] Compound IM002-03 (2.2 g, 8.76 mmol) was mixed with a 4M hydrochloric acid solution in 1,4-dioxane (11 ml). The reaction system was allowed to react at 25°C for 2 hours. LCMS monitored the reaction completion. The reaction solution was concentrated to obtain compound IM002 (1.27 g, 96% yield). MS ESI (m / z) = 152.0 [M+H] + .

[0193] Preparation Example 6: Synthesis of Compound IM003

[0194] In this preparation example, the synthetic route of compound IM003 is as follows:

[0195] (6-1) Synthesis of Compound IM003-02

[0196] Compound IM003-01 (8.0 g, 66.12 mmol, 2-amino-3-pyridinecarboxaldehyde, CAS No. 7521-41-7), 1,1-dimethoxyacetone (10.1 g, 85.59 mmol, CAS No. 6342-56-9), sodium hydroxide (3.43 g, 85.75 mmol), purified water (28 ml), and ethanol (160 ml) were mixed and the atmosphere was replaced with nitrogen three times. The reaction system was allowed to react at 25°C under a nitrogen atmosphere for 16 hours. Completion of the reaction was monitored by TLC. The reaction solution was concentrated and extracted with ethyl acetate (160 ml) and purified water (160 ml). The organic phase was separated and washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound IM003-02 (13 g), which was directly used in the next step without purification. MS ESI (m / z) = 205.0 [M+H] + .

[0197] (6-2) Synthesis of Compound IM003-03

[0198] Compound IM003-02 (8.0 g, 39.22 mmol) and platinum dioxide (270 mg, 1.19 mmol) were added to ethanol (160 ml). The mixture was replaced with hydrogen three times. The reaction system was allowed to react at 25°C under a hydrogen atmosphere for 16 hours. Completion of the reaction was monitored by TLC. The reaction solution was filtered and concentrated to obtain compound IM003-03 (8.0 g, yield 97.7%). MS ESI (m / z) = 210.0 [M+H] + .

[0199] (6-3) Synthesis of Compound IM003-04

[0200] Compound IM003-03 (8.0 g, 38.1 mmol) was added to trifluoroacetic acid (64 ml), and the atmosphere was replaced with nitrogen three times. The reaction system was allowed to react at 25°C under a nitrogen atmosphere for 2 hours. The reaction was monitored by TLC. The pH of the reaction solution was adjusted to alkaline with saturated aqueous sodium bicarbonate solution, and the solution was extracted twice with dichloromethane (50 ml each time). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound IM003-04 (6.1 g, yield 97.1%). MS ESI (m / z) = 165.0 [M+H] + .

[0201] (6-4) Synthesis of Compound IM003-05

[0202] Ammonia (5.23 g, 73.93 mmol), sodium acetate (6.17 g, 73.93 mmol), and purified water (61 ml) were mixed, heated to 60°C, and stirred at 60°C for 0.5 hour. A solution of compound IM003-04 (6.1 g, 36.97 mmol) in methanol (60 ml) was added dropwise. The atmosphere was purged with nitrogen three times. The reaction system was allowed to react at 60°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by TLC. The reaction solution was concentrated and extracted twice with dichloromethane (60 ml each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound IM003-05 (4.5 g, yield 67.7%). MS ESI (m / z) = 180.0 [M+H] + .

[0203] (6-5) Synthesis of Compound IM003

[0204] Compound IM003-05 (4.5 g, 25.14 mmol) and zinc powder (9 g) were added to trifluoroacetic acid (90 ml). The atmosphere was replaced with nitrogen three times, and the reaction system was allowed to react at 25°C under a nitrogen atmosphere for 20 minutes. Completion of the reaction was monitored by TLC. A 3M aqueous solution of sodium hydroxide (450 ml) was added to the reaction solution, which was cooled to 0°C and then added with dichloromethane (400 ml). The mixture was shaken until uniform, filtered through celite, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound IM003 (3.9 g, yield 94.5%). MS ESI (m / z) = 165.0 [M+H] + .

[0205] Preparation Example 7: Synthesis of Compound LD003

[0206] In this preparation example, the synthetic route of compound LD003 is as follows:

[0207] (7-1) Synthesis of Compound LD003-01

[0208] Compound IM001 (1.0 g, 2.4 mmol, (S)-3-(6-bromopyridin-3-yl)-3-(2-((tert-butyloxycarbonyl)amino)acetylamino)propionic acid methyl ester, purchased from Kanglong Chemical New Drug Technology Co., Ltd.), compound 4BBA (1.33 g, 4.8 mmol), palladium acetate (27 mg, 0.12 mmol, CAS No. 3375-31-3), 2-dicyclohexylphosphino-2',4',6 '-Triisopropylbiphenyl (57 mg, 0.12 mmol, CAS No. 564483-18-7, abbreviated as X-PHOS), potassium phosphate (1.53 g, 7.2 mmol, CAS No. 7778-53-2), tetrahydrofuran (22.5 ml), and purified water (7.5 ml) were mixed, replaced with nitrogen three times, and heated to 70°C. The reaction system was allowed to react at 70°C under a nitrogen atmosphere for 16 hours, and the reaction was monitored for completion by LCMS. The reaction system was cooled to 25°C, and ethyl acetate (30 ml) was added to the reaction solution for extraction. The organic phase was separated, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD003-01 (1.0 g, 73% yield). MS ESI (m / z) = 570 [M+H] + .

[0209] (7-2) Synthesis of Compound LD003-02

[0210] Compound LD003-01 (1.0 g, 1.75 mmol), wet palladium on carbon (0.1 g, 10% w / w loading, referred to as Pd / C), and methanol (10 ml) were mixed and the atmosphere was replaced with hydrogen three times. The reaction system was allowed to react at 25°C under a hydrogen atmosphere for 16 hours. The reaction solution was filtered and concentrated to obtain compound LD003-02 (0.78 g, 92.8% yield). MS ESI (m / z) = 480 [M+H] + .

[0211] (7-3) Synthesis of Compound LD003-03

[0212] Compound LD003-02 (0.78 g, 1.63 mmol), compound PEG5 (0.91 g, 2.1 mmol), potassium carbonate (0.67 g, 4.9 mmol), and N,N-dimethylformamide (20 ml) were mixed, replaced with nitrogen three times, and heated to 100°C. The reaction system was allowed to react at 100°C under a nitrogen atmosphere for 16 hours. Completion of the reaction was monitored by TLC. Ethyl acetate (30 ml) and purified water (20 ml) were added to the reaction solution for extraction. The organic phase was separated and washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD003-03 (0.73 g). MS ESI (m / z) = 725 [M+H] + .

[0213] (7-4) Synthesis of Compound LD003-04

[0214] Compound LD003-03 (0.73 g, 1 mmol), a 4M hydrochloric acid solution in 1,4-dioxane (3.65 ml), and dichloromethane (3.65 ml) were mixed and the atmosphere was replaced with nitrogen three times. The reaction system was allowed to react at 25°C for 1 hour under a nitrogen atmosphere. After completion of the reaction, the reaction solution was concentrated, dichloromethane (20 ml) was added, and the reaction solution was concentrated (this step was repeated twice) to obtain compound LD003-04 (0.6 g, yield 95.2%). MS ESI (m / z) = 625 [M+H] + .

[0215] (7-5) Synthesis of Compound LD003-05

[0216] Compound IM003 (70 mg, 0.43 mmol), N,N'-carbonyldiimidazole (52.5 mg, 0.33 mmol, CAS No. 530-62-1), and dichloromethane (2.5 ml) were mixed, cooled to 0°C, and reacted at 0°C for 1 hour. Compound LD003-04 (135 mg, 0.22 mmol) was added, the atmosphere was purged with nitrogen three times, and the temperature was raised to 25°C. The reaction system was reacted at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LCMS. Purified water (2 ml) was added to the reaction solution for washing, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD003-05 (100 mg, 57.1% yield). MS ESI (m / z) = 814 [M+H] + .

[0217] (7-7) Synthesis of Compound LD003

[0218] Compound LD003-05 (100 mg, 0.12 mmol), a 1M aqueous solution of sodium hydroxide (0.3 ml), and methanol (0.3 ml) were mixed and replaced with nitrogen three times. The reaction system was allowed to react at 25°C under a nitrogen atmosphere for 1 hour. Completion of the reaction was monitored by LCMS. The pH of the reaction solution was adjusted to 2 with a 1M aqueous solution of hydrogen chloride. The reaction solution was concentrated, and dichloromethane (2 ml) and methanol (2 ml) were added. The solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD003 (75 mg, yield 76.5%). MS ESI (m / z) = 800 [M+H] + .

[0219] 1 H NMR(400MHz, DMSO-d6)δ13.05(s,1H),δ8.84–8.62(m,2H),8.27(m,2H),8.16–8.07(m,2H),7.86(dd,J=8.2,2.4Hz,1H),7.58( d,J=8.0Hz,1H),7.56–7.47(m,3H),7.28(dd,J=7.9,2.1Hz,2H),6.77(s,1H),6.20(d,J=7.3Hz,1H),5.27(q,J=7.2Hz,1H),4. 35(t,J=4.6Hz,2H),3.93(t,J=4.5Hz,2H),3.71–3.65(m,5H),3.66–3.54(m,6H),3.26(t,J=4.9Hz,3H),3.22(d,J=6.1Hz,3H) ,2.82(dd,J=6.9,3.5Hz,2H),2.71(t,J=7.8Hz,2H),2.60(t,J=6.3Hz,2H),2.46(dd,J=7.9,3.0Hz,2H),1.73(p,J=6.1Hz,2H).

[0220] Preparation Example 8: Synthesis of Compound LD006

[0221] (8-1) Synthesis of Compound LD006-01

[0222] Compound IM001 (2 g, 4.8 mmol), compound 3BBA (2.66 g, 9.6 mmol), palladium acetate (54 mg, 0.24 mmol), X-PHOS (114 mg, 0.24 mmol), potassium phosphate (3.06 g, 14.4 mmol), tetrahydrofuran (45 ml), and purified water (15 ml) were mixed and replaced with nitrogen three times. The temperature was raised to 70°C and the reaction system was stirred at 70°C under a nitrogen atmosphere for 16 hours. The reaction was monitored for completion by LC-MS. The reaction system was cooled to 25°C and extracted with ethyl acetate (50 ml). The organic phase was separated and washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD006-01 (2.36 g, yield 86.1%). MS ESI (m / z) = 570 [M+H] + .

[0223] (8-2) Synthesis of Compound LD006-02

[0224] Compound LD006-01 (1.3 g, 2.28 mmol), wet palladium carbon (130 mg, 10% w / w loading), and methanol (13 ml) were mixed and replaced with hydrogen three times. The reaction system was stirred at 25°C under a hydrogen atmosphere for 16 hours, filtered, and concentrated to obtain compound LD006-02 (1 g, yield 91.7%). MS ESI (m / z) = 480 [M+H] + .

[0225] (8-3) Synthesis of Compound LD006-03

[0226] Compound LD006-02 (1 g, 2.08 mmol), compound PEG5 (1.17 g, 2.7 mmol), potassium carbonate (860 mg, 6.25 mmol), and N,N-dimethylformamide (25 ml) were mixed and replaced with nitrogen three times. The temperature was raised to 100°C, and the reaction system was stirred at 100°C under a nitrogen atmosphere for 16 hours. The reaction was monitored by TLC to determine completion. The reaction system was cooled to 25°C, and ethyl acetate (20 ml) and purified water (10 ml) were added to the reaction solution for extraction. The organic phase was separated and washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography. Compound LD006-03 (1.3 g, yield 86.7%) was obtained. MS ESI (m / z) = 725 [M+H] + .

[0227] (8-4) Synthesis of Compound LD006-04

[0228] Compound LD006-03 (1.3 g, 1.79 mmol), a 4 M hydrogen chloride solution in 1,4-dioxane (6.5 ml), and dichloromethane (6.5 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. The reaction solution was concentrated, and dichloromethane (10 ml) was added and concentrated (this step was repeated twice) to obtain compound LD006-04 (1 g, yield 91%). MS ESI (m / z) = 625 [M+H] + .

[0229] (8-5) Synthesis of Compound LD006-05

[0230] Compound IM003 (208 mg, 1.28 mmol), N,N'-carbonyldiimidazole (155 mg, 0.96 mmol, CAS No. 530-62-1), and dichloromethane (8 ml) were mixed, cooled to 0°C, and stirred at 0°C for 1 hour. Compound LD006-04 (400 mg, 0.64 mmol) was added, and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LC-MS. Purified water (8 ml) was added to the reaction solution for washing. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD006-05 (320 mg, 61.5% yield). MS ESI (m / z) = 813 [M+H] + .

[0231] (8-6) Synthesis of Compound LD006

[0232] Compound LD006-05 (320 mg, 0.394 mmol), a 1M aqueous solution of sodium hydroxide (0.96 ml), and methanol (0.96 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. Completion of the reaction was monitored by LC-MS. The pH of the reaction solution was adjusted to 2 with a 1M aqueous solution of hydrogen chloride. The reaction solution was concentrated, and dichloromethane (5 ml) and methanol (5 ml) were added. The reaction solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD006 (240 mg, yield 76.4%). MS ESI (m / z) = 800 [M+H] + .

[0233] 1H NMR(500MHz, DMSO-d6)δ13.05(s,1H),δ8.84–8.62(m,2H),8.27(m,2H),8.16–8.07(m,2H),7.86(dd,J=8.2,2.4Hz,1H),7.58( d,J=8.0Hz,1H),7.56–7.47(m,3H),7.28(dd,J=7.9,2.1Hz,2H),6.77(s,1H),6.20(d,J=7.3Hz,1H),5.27(q,J=7.2Hz,1H),4. 35(t,J=4.6Hz,2H),3.93(t,J=4.5Hz,2H),3.71–3.65(m,5H),3.66–3.54(m,6H),3.26(t,J=4.9Hz,3H),3.22(d,J=6.1Hz,3H) ,2.82(dd,J=6.9,3.5Hz,2H),2.71(t,J=7.8Hz,2H),2.60(t,J=6.3Hz,2H),2.46(dd,J=7.9,3.0Hz,2H),1.73(p,J=6.1Hz,2H).

[0234] Preparation Example 9: Synthesis of Compound LD007

[0235] (9-1) Synthesis of Compound LD007-01

[0236] Compound IM002 (72.5 mg, 0.48 mmol), N,N'-carbonyldiimidazole (58.3 mg, 0.36 mmol), and dichloromethane (3 ml) were mixed, cooled to 0°C, and stirred at 0°C for 1 hour. Compound LD006-04 (150 mg, 0.24 mmol) was added, and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LC-MS. Purified water (5 ml) was added to the reaction solution for washing, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD007-01 (100 mg, yield 52.1%). MS ESI (m / z) = 802 [M+H] + .

[0237] (9-2) Synthesis of Compound LD007

[0238] Compound LD007-01 (100 mg, 0.125 mmol), a 1M aqueous solution of sodium hydroxide (0.3 ml), and methanol (0.3 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. The reaction was complete by LC-MS. 1M aqueous hydrogen chloride solution was added to the reaction solution to adjust the pH to 2. The solution was concentrated, and dichloromethane (2 ml) and methanol (2 ml) were added. The solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD007 (60 mg, yield 61.2%). MS ESI (m / z) = 788 [M+H] + .

[0239] 1 H NMR (500MHz, DMSO-d6) δ13.05(s,1H),8.56(d,J=8.3Hz,1H),8.27(dd,J=7.8,1.8Hz,1H),8.20(t,J=5.9Hz,1H),7.81–7.73(m,2H),7. 53(dqd,J=8.2,6.8,1.5Hz,2H),7.45(d,J=8.0Hz,2H),7.39(d,J=8.0Hz,2H),7.32(d,J=7.9Hz,1H),7.05(d,J=8.0Hz,1H),6.80(s,1H) ,6.67(d,J=6.4Hz,1H),5.30(q,J=7.5Hz,1H),4.33(t,J=4.6Hz,2H),3.99–3.87(m,2H),3.77(d,J=5.8Hz,2H),3.69(dd,J=5.9,3.7Hz, 2H),3.62–3.49(m,10H),3.49–3.34(m,3H),3.30(m,2H),2.79(d,J=7.3Hz,2H),2.29(s,3H),1.81(p,J=7.2Hz,2H),1.36–1.13(m,2H).

[0240] Preparation Example 10: Synthesis of Compound LD008

[0241] (10-1) Synthesis of Compound LD008-01

[0242] Compound IM001 (2.0 g, 4.8 mmol), compound 5BBA (2.66 g, 9.6 mmol), palladium acetate (54 mg, 0.24 mmol), X-PHOS (114 mg, 0.24 mmol), potassium phosphate (3.06 g, 14.4 mmol), tetrahydrofuran (45 ml), and purified water (15 ml) were mixed, replaced with nitrogen three times, and heated to 70°C. The reaction system was stirred at 70°C under a nitrogen atmosphere for 16 hours. Completion of the reaction was monitored by LC-MS. The reaction system was cooled to 25°C, and ethyl acetate (60 ml) was added to the reaction solution for extraction. The organic phase was separated and washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD008-01 (2.0 g, yield 73%). MS ESI (m / z) = 570 [M+H] + .

[0243] (10-2) Synthesis of Compound LD008-02

[0244] Compound LD008-01 (2.0 g, 3.5 mmol), wet palladium carbon (0.2 g, 10% w / w loading), and methanol (20 ml) were mixed and replaced with hydrogen three times. The reaction system was stirred at 25° C. under a nitrogen atmosphere for 16 hours, filtered, and concentrated to obtain compound LD008-02 (1.56 g, yield 92.8%). MS ESI (m / z) = 480 [M+H] + .

[0245] (10-3) Synthesis of Compound LD008-03

[0246] Compound LD008-02 (500 mg, 1.04 mmol), compound PEG5 (565 mg, 1.35 mmol), potassium carbonate (432 mg, 3.13 mmol), and N,N-dimethylformamide (12.5 ml) were mixed, replaced with nitrogen three times, and heated to 100°C. The reaction system was stirred at 100°C under a nitrogen atmosphere for 16 hours. The reaction was monitored by TLC to determine completion. The reaction system was cooled to 25°C, and ethyl acetate (20 ml) and purified water (10 ml) were added to the reaction solution for extraction. The organic phase was separated and washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD008-03 (300 mg, 41.4% yield). MS ESI (m / z) = 725 [M+H] + .

[0247] (10-4) Synthesis of Compound LD008-04

[0248] Compound LD008-03 (150 mg, 0.207 mmol), a 4M hydrogen chloride solution in 1,4-dioxane (0.75 ml), and dichloromethane (0.75 ml) were mixed and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C for 1 hour under a nitrogen atmosphere. The reaction solution was concentrated, and dichloromethane (10 ml) was added and concentrated (this step was repeated twice) to obtain compound LD008-04 (100 mg, yield 77.5%). MS ESI (m / z) = 625 [M+H] + .

[0249] (10-5) Synthesis of Compound LD008-05

[0250] Compound IM003 (52 mg, 0.32 mmol), N,N'-carbonyldiimidazole (38.9 mg, 0.24 mmol), and dichloromethane (2 ml) were mixed, cooled to 0°C, and stirred at 0°C for 1 hour. Compound LD008-04 (100 mg, 0.16 mmol) was added, and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LC-MS. Purified water (2 ml) was added to the reaction solution for washing, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD008-05 (60 mg, 46.2% yield). MS ESI (m / z) = 813 [M+H] + .

[0251] (10-6) Synthesis of Compound LD008

[0252] Compound LD008-05 (60 mg, 0.074 mmol), 1M aqueous sodium hydroxide solution (0.18 ml), and methanol (0.18 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. Completion of the reaction was monitored by LC-MS. 1M aqueous hydrogen chloride solution was added to the reaction solution to adjust the pH to 2. The solution was concentrated, and dichloromethane (2 ml) and methanol (2 ml) were added. The solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD008-05 (40 mg, yield 67.8%). MS ESI (m / z) = 800 [M+H] + .

[0253] 1H NMR(400MHz, DMSO-d6)δ13.05(s,1H),δ8.84–8.62(m,2H),8.27(m,2H),8.16–8.07(m,2H),7.86(dd,J=8.2,2.4Hz,1H),7.58( d,J=8.0Hz,1H),7.56–7.47(m,3H),7.28(dd,J=7.9,2.1Hz,2H),6.77(s,1H),6.20(d,J=7.3Hz,1H),5.27(q,J=7.2Hz,1H),4. 35(t,J=4.6Hz,2H),3.93(t,J=4.5Hz,2H),3.71–3.65(m,5H),3.66–3.54(m,6H),3.26(t,J=4.9Hz,3H),3.22(d,J=6.1Hz,3H) ,2.82(dd,J=6.9,3.5Hz,2H),2.71(t,J=7.8Hz,2H),2.60(t,J=6.3Hz,2H),2.46(dd,J=7.9,3.0Hz,2H),1.73(p,J=6.1Hz,2H).

[0254] Preparation Example 11: Synthesis of Compound CR02009

[0255] (11-1) Synthesis of Compound LD009-01

[0256] Compound IM002 (70 mg, 0.11 mmol), N,N'-carbonyldiimidazole (27.2 mg, 0.168 mmol), and dichloromethane (1.4 ml) were mixed, cooled to 0°C, and stirred at 0°C for 1 hour. Compound LD008-04 (33.8 mg, 0.22 mmol) was added, and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LC-MS. Purified water (2 ml) was added to the reaction solution for washing. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD009-01 (60 mg, yield 66.7%). MS ESI (m / z) = 802 [M+H] + .

[0257] (11-2) Synthesis of Compound LD009

[0258] Compound LD009-01 (60 mg, 0.074 mmol), a 1M aqueous solution of sodium hydroxide (0.18 ml), and methanol (0.18 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. Completion of the reaction was monitored by LC-MS. A 1M aqueous solution of hydrogen chloride was added to the reaction solution to adjust the pH to 2. The solution was concentrated, and dichloromethane (2 ml) and methanol (2 ml) were added. The solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD009 (50 mg, yield 84.7%). MS ESI (m / z) = 788 [M+H] + .

[0259] 1 H NMR (400MHz, DMSO-d6) δ13.05(s,1H),8.56(d,J=8.3Hz,1H),8.27(dd,J=7.8,1.8Hz,1H),8.20(t,J=5.9Hz,1H),7.81–7.73(m,2H),7. 53(dqd,J=8.2,6.8,1.5Hz,2H),7.45(d,J=8.0Hz,2H),7.39(d,J=8.0Hz,2H),7.32(d,J=7.9Hz,1H),7.05(d,J=8.0Hz,1H),6.80(s,1H) ,6.67(d,J=6.4Hz,1H),5.30(q,J=7.5Hz,1H),4.33(t,J=4.6Hz,2H),3.99–3.87(m,2H),3.77(d,J=5.8Hz,2H),3.69(dd,J=5.9,3.7Hz, 2H),3.62–3.49(m,10H),3.49–3.34(m,3H),3.30(m,2H),2.79(d,J=7.3Hz,2H),2.29(s,3H),1.81(p,J=7.2Hz,2H),1.36–1.13(m,2H).

[0260] Preparation Example 12: Synthesis of Compound LD010

[0261] (12-1) Synthesis of Compound LD010-01

[0262] Compound IM005 (94 mg, 0.576 mmol, 5,6,7,8-tetrahydro-1,8-naphthyridine-2-propionic acid, CAS No. 658712-81-3), N,N'-carbonyldiimidazole (70 mg, 0.432 mmol), and dichloromethane (3.6 ml) were mixed, cooled to 0°C, and stirred at 0°C for 1 hour. Compound LD008-04 (180 mg, 0.288 mmol) was added, and the atmosphere was replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 2 hours. Completion of the reaction was monitored by LC-MS. The reaction solution was washed with purified water (3 ml), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD010-01 (110 mg, 47% yield). MS ESI (m / z) = 813 [M+H] + .

[0263] (12-2) Synthesis of Compound LD010

[0264] Compound LD010-01 (110 mg, 0.135 mmol), 1M aqueous sodium hydroxide solution (0.33 ml), and methanol (0.33 ml) were mixed and replaced with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 1 hour. The reaction was monitored by LC-MS. 1M aqueous hydrogen chloride solution was added to the reaction solution to adjust the pH value to 2. The reaction solution was concentrated, and dichloromethane (2 ml) and methanol (2 ml) were added. The solution was filtered, concentrated, and purified by reverse phase column chromatography to obtain compound LD010 (60 mg, yield 55.6%). MS ESI (m / z) = 800 [M+H] + .

[0265] 1H NMR(500MHz,DMSO-d6)δ8.74–8.62(m,2H),8.27(m,2H),8.16–8.07(m,2H),7.86(dd,J=8.2,2.4Hz,1H),7.58(d,J=8.0Hz,1H), 7.56–7.47(m,3H),7.08(dd,J=7.9,2.1Hz,2H),6.87(s,1H),6.30(d,J=7.3Hz,1H),5.27(q,J=7.2Hz,1H),4.35(t,J=4.6Hz,2H) ,3.93(t,J=4.5Hz,2H),3.81–3.65(m,5H),3.61–3.54(m,6H),3.52(d,J=3.7Hz,6H),3.36(t,J=4.9Hz,3H),3.22(d,J=6.1Hz,3H ),2.82(dd,J=6.9,3.5Hz,2H),2.71(t,J=7.8Hz,2H),2.60(t,J=6.3Hz,2H),2.46(dd,J=7.9,3.0Hz,2H),1.73(p,J=6.1Hz,2H).

[0266] Preparation Example 13: Synthesis of Reference Compound LD000

[0267] (13-1) Synthesis of Compound LD000-02

[0268] Compound LD000-01 (2.42 g, 10 mmol, (S)-3-amino-3-(4-bromophenyl)propanoic acid, CAS No. 275826-36-3) was added to 30 mL of methanol. The temperature was lowered to 0°C, and thionyl chloride (3.57 g, 30 mmol) was slowly added dropwise at 0°C. The temperature was raised to 25°C and stirred at 25°C for 12 hours. After the reaction, the reaction solution was concentrated to obtain compound LD000-02 (2.4 g, 95% yield) as a white solid powder. This was used directly in the next reaction without purification. ESI-MS (m / z) = 258.1 [M+H] + .

[0269] (13-2) Synthesis of Compound LD000-03

[0270] Compound LD000-02 (2 g, 7.8 mmol), Boc-glycine (1.6 g, 9.36 mmol, CAS No. 4530-20-5), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.54 g, 9.36 mmol, CAS No. 148893-10-1, abbreviated as HATU) were added to anhydrous dichloromethane (50 ml), and N,N-diisopropylethylamine (3 g, 23.4 mmol) was slowly added dropwise. After the addition was complete, the reaction system was stirred at 25°C for 2 hours. After the reaction, purified water (30 ml) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 ml each time). The organic phases were combined, washed once with saturated sodium chloride solution (20 ml each time), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution solvent: ethyl acetate / petroleum ether = 0 / 100 to 50 / 50 (v / v) gradient elution) to obtain compound LD000-03 as a white solid powder (2.57 g, 80% yield). ESI-MS (m / z): 415.1 [M+H].

[0271] (13-3) Synthesis of Compound LD000-04

[0272] Compound LD000-03 (2 g, 4.8 mmol) was dissolved in a 4 M solution of hydrogen chloride in 1,4-dioxane (10 ml), and the reaction system was stirred at 25°C for 2 hours. After the reaction, the reaction solution was concentrated to obtain compound LD000-04 (1.6 g, 100% yield) as a pale yellow foamy solid, which was used directly in the next reaction without purification.

[0273] (13-4) Synthesis of Compound LD000-05

[0274] Compound LD000-04 (1.67 g, 4.8 mmol), compound IM006 (1.6 g, 5.76 mmol, CAS No. 2320478-13-3), and HATU (2.2 g, 5.76 mmol) were added to anhydrous dichloromethane (30 ml), followed by the slow dropwise addition of N,N-diisopropylethylamine (2.2 g, 16.8 mmol). The reaction system was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 ml each time). The organic phases were combined, washed once with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution solvent: methanol / dichloromethane = 0 / 100 to 50 / 50 (v / v) gradient elution) to obtain compound LD000-05 (2.16 g, 78% yield) as a white solid powder. ESI-MS (m / z) = 577.2 [M+H]+ .

[0275] (13-5) Synthesis of Compound LD000-06

[0276] Compound LD000-05 (1 g, 1.74 mmol), compound 4BBA (0.96 g, 3.84 mmol), X-PHOS (41 mg, 0.087 mmol), palladium acetate (20 mg, 0.087 mmol) and potassium phosphate (1.1 g, 5.22 mmol) were respectively added to a mixed solvent of tetrahydrofuran (37.5 ml) and water (12.5 ml), the atmosphere was replaced with argon three times, the temperature was raised to 70°C, and the reaction system was stirred at 70°C in an argon atmosphere for 16 hours. After the reaction, the reaction system was cooled to 25°C, the reaction solution was concentrated, purified water (20 ml) was added, and the mixture was extracted three times with ethyl acetate (30 ml each time). The organic phases were combined; the organic phases were washed with saturated sodium chloride aqueous solution (10 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution solvent: methanol / dichloromethane = 0:100 to 50:50 (v / v) gradient elution) to obtain compound LD000-06 (968 mg, yield 78%) as a light yellow solid powder. ESI-MS (m / z) = 745.4 [M+H] + .

[0277] (13-6) Synthesis of Compound LD000-07

[0278] Compound LD000-06 (0.968 g, 1.3 mmol) was dissolved in anhydrous methanol (10 ml), and wet palladium carbon (200 mg, 0.26 mmol) with a 10% mass loading was added. The atmosphere was purged with hydrogen three times, and the reaction system was stirred at 25°C for 12 hours. After completion of the reaction, the reaction solution was filtered and concentrated to obtain compound LD000-07 (870 mg, 100% yield) as an off-white solid powder. ESI-MS (m / z) = 655.3 [M+H] + .

[0279] (13-7) Synthesis of Compound LD000-08

[0280] Compound LD000-07 (0.87 g, 1.3 mmol), compound PEG5 (0.72 g, 1.7 mmol), and potassium carbonate (0.55 g, 3.9 mmol) were added to DMF (15 ml). The reaction system was heated to 100°C and stirred at 100°C for 6 hours. After the reaction was completed and the reaction system was cooled to 25°C, water (20 ml) and ethyl acetate (40 ml) were added to the reaction solution for extraction. The organic phase was separated and washed once with saturated sodium chloride solution (15 ml), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution solvent: methanol / dichloromethane = 0 / 100 to 50 / 50 (v / v) gradient elution) to obtain compound LD000-08 (0.96 g, 80% yield) as a light yellow foamy solid. ESI-MS (m / z) = 900.4 [M+H] + .

[0281] (13-8) Synthesis of Compound LD000-09

[0282] Compound LD000-08 (0.96 g, 1 mmol) was dissolved in methanol (5 ml), and 1 M aqueous NaOH (5 ml) was added. The reaction solution was stirred at 25°C for 1 hour. The pH of the reaction solution was adjusted to 6-7 with 1 M aqueous hydrogen chloride in an ice bath. Dichloromethane (20 ml) was added to the reaction solution for extraction, and the organic phase was separated; the organic phase was washed with saturated aqueous sodium chloride (15 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound LD000-09 (0.9 g, 95% yield) as a white solid. ESI-MS (m / z) = 886.4 [M+H] + .

[0283] (13-9) Synthesis of Compound LD000

[0284] Compound LD000-09 (0.9 g, 1 mmol) was dissolved in a 4 M solution of hydrogen chloride in 1,4-dioxane (5 ml). The reaction mixture was stirred at 25°C for 2 hours. After the reaction, the reaction mixture was concentrated to obtain compound LD000 (0.78 g, 100% yield) as a light yellow foamy solid. ESI-MS (m / z) = 786.4 [M+H] + .

[0285] 1H NMR (500MHz, DMSO-d6) δ13.05(s,1H),8.56(d,J=8.3Hz,1H),8.27(dd,J=7.8,1.8Hz,1H),8.20(t,J=5.9Hz,1H),7.81–7.73(m,2H),7. 53(dqd,J=8.2,6.8,1.5Hz,2H),7.45(d,J=8.0Hz,2H),7.39(d,J=8.0Hz,2H),7.32(d,J=7.9Hz,1H),7.05(d,J=8.0Hz,1H),6.80(s,1H) ,6.67(d,J=6.4Hz,1H),5.30(q,J=7.5Hz,1H),4.33(t,J=4.6Hz,2H),3.99–3.87(m,2H),3.77(d,J=5.8Hz,2H),3.69(dd,J=5.9,3.7Hz, 2H),3.62–3.49(m,10H),3.49–3.34(m,4H),3.30(m,2H),2.79(d,J=7.3Hz,2H),2.29(s,3H),1.81(p,J=7.2Hz,2H),1.36–1.13(m,2H).

[0286] Preparation Example 14: Preparation of Compound LD002:

[0287] In this preparation example, the synthetic route of compound LD002 is as follows:

[0288] (14-1) Synthesis of compound LD002-1:

[0289] Compound IM001 (1.8 g, 4.3 mmol), compound 4BBA (2.4 g, 8.6 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (103 mg, 0.22 mmol, abbreviated as X-Phos, CAS No. 564483-18-7), palladium acetate (49 mg, 0.22 mmol, CAS No. 3375-31-3) and potassium phosphate (2.75 g, 12.9 mmol) were added to a mixed solvent of tetrahydrofuran (40.5) and water (13.5 ml), the atmosphere was replaced with nitrogen three times, the temperature was raised to 70°C, and the mixture was stirred for 16 hours to complete the reaction. After the reaction solution was cooled to room temperature, 20 ml of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 mL each time). The organic phases were combined, washed with 20 ml of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD002-1 (1.1 g, yield 44.7%) as a light yellow solid. ESI-MS (m / z) = 569.3 [M+H]+ .

[0290] (14-2) Synthesis of compound LD002-2:

[0291] Compound LD002-1 (1.1 g, 1.9 mmol), methanol (11 ml), and wet palladium on carbon (0.11 g, 10% w / w) were added to a reaction vessel. The atmosphere was replaced with hydrogen three times and stirred at room temperature for 16 hours. The reaction was completed. The reaction solution was filtered and concentrated to obtain compound LD002-2 (850 mg, 91.7% yield) as an off-white foamy solid. ESI-MS (m / z) = 479.2 [M+H] + .

[0292] (14-3) Synthesis of compound LD002-3:

[0293] Compound LD002-2 (850 mg, 1.77 mmol), PEG5 (962 mg, 2.3 mmol), potassium carbonate (735 mg, 5.32 mmol), and DMF (17 ml) were added to a reaction vessel, heated to 100°C, and stirred for 16 hours to complete the reaction. After the reaction solution cooled to room temperature, 20 ml of ethyl acetate and 20 ml of purified water were added for extraction. The organic phase was separated and washed three times with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD002-3 (600 mg, yield 46.6%) as a light yellow foamy solid. ESI-MS (m / z) = 725.3 [M+H] + .

[0294] (14-4) Synthesis of compound LD002-4:

[0295] Compound LD002-3 (600 mg, 0.83 mmol), dichloromethane (6 ml), and a 4M hydrogen chloride solution in 1,4-dioxane (0.6 ml) were added to a reaction vessel and stirred for 1 hour. The reaction was complete. The reaction solution was concentrated to obtain compound LD002-4 (500 mg, 96.7% yield) as a light yellow foamy solid. ESI-MS (m / z) = 625.4 [M+H] + .

[0296] (14-5) Synthesis of compound LD002-5:

[0297] Compound IM002 (242 mg, 1.6 mmol), N,N'-carbonyldiimidazole (259 mg, 1.6 mmol), and triethylamine (162 mg, 1.6 mmol) were added to dichloromethane (5 ml), cooled to 0°C, and stirred for 0.5 hours. Compound LD002-4 (500 mg, 0.8 mmol) was added and stirred for 3 hours. The reaction was completed. The reaction solution was washed with 5 ml of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluting solvent, methanol / dichloromethane = (0 / 100) to (10 / 90), v / v) to obtain compound LD002-5 (290 mg, yield 45.3%) as an off-white foamy solid. ESI-MS (m / z) = 801.3 [M+H] + .

[0298] (14-6) Synthesis of compound LD002:

[0299] Compound LD002-5 (290 mg, 1 mmol) was dissolved in methanol (3 ml), and a 1 M aqueous sodium hydroxide solution (3 ml) was added. The mixture was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to 6-7 with a 1 M aqueous hydrochloric acid solution under ice-cooling, and the mixture was concentrated. The mixture was washed with dichloromethane (5 ml), filtered, and the filtrate was concentrated to obtain compound LD002 (250 mg, yield 89.3%). ESI-MS (m / z) = 787.4 [M+H] + .

[0300] Preparation Example 15: Preparation of Compound LD005:

[0301] In this preparation example, the synthetic route of compound LD005 is as follows:

[0302] (15-1) Synthesis of compound LD005-1:

[0303] To a 100 ml reaction vessel were added compound IM001 (500 mg, 1.2 mmol), compound 3BBA (668 mg, 2.4 mmol), palladium acetate (13.5 mg, 0.06 mmol), X-Phos (28.6 mg, 0.06 mmol), potassium phosphate (764 mg, 3.6 mmol), tetrahydrofuran (11.25 ml), and purified water (3.75 ml). The atmosphere was replaced with nitrogen three times, the temperature was raised to 70°C, and the reaction was stirred for 16 hours. The reaction was complete. The reaction system was cooled to room temperature under a nitrogen atmosphere, and ethyl acetate (20 ml) was added to the reaction solution for extraction. The organic phase was washed once with saturated sodium chloride solution, dried, concentrated, and purified by column chromatography to obtain compound LD005-1 (600 mg, yield 87.3%). MS ESI (m / z) = 570 [M+H]+ .

[0304] (15-2) Synthesis of compound LD005-2:

[0305] Compound LD005-1 (600 mg, 1.053 mmol), wet palladium on carbon (60 mg, 10% w / w, abbreviated as Pd / C), and methanol (6 ml) were added to a 50 ml reactor. The atmosphere was replaced with hydrogen three times and stirred for 16 hours. The reaction was complete. The reaction solution was filtered and concentrated to obtain compound LD005-2 (420 mg, yield 83.2%). MS ESI (m / z) = 480 [M+H] + .

[0306] (15-3) Synthesis of compound LD005-3:

[0307] To a 100 ml reaction vessel were added compound LD005-2 (420 mg, 0.877 mmol), compound PEG5 (491 mg, 1.14 mmol), potassium carbonate (363 mg, 2.63 mmol), and N,N-dimethylformamide (10.5 ml). The atmosphere was purged with nitrogen three times, and the temperature was raised to 100°C. The reaction system was stirred at 100°C under a nitrogen atmosphere for 16 hours, upon completion of the reaction. Ethyl acetate (20 ml) and purified water (10 ml) were added to the reaction solution for extraction, and the organic phase was separated. The organic phase was washed three times with saturated sodium chloride solution, dried, concentrated, and purified by column chromatography to yield compound LD005-3 (500 mg, 78.7% yield). MS ESI (m / z) = 725 [M+H] + .

[0308] (15-4) Synthesis of compound LD005-4:

[0309] To a 50 ml reaction vessel, compound LD005-3 (500 mg, 0.69 mmol), a 4 M hydrochloric acid solution in 1,4-dioxane (2.5 ml), and dichloromethane (2.5 ml) were added. The atmosphere was replaced with nitrogen three times and stirred at room temperature for 1 hour. The reaction was complete. The reaction solution was directly concentrated, dichloromethane (10 ml) was added, and the solution was concentrated again to obtain compound LD005-4 (420 mg, 98% yield). MS ESI (m / z) = 625 [M+H] + .

[0310] (15-5) Synthesis of compound LD005-5:

[0311] To a 25 ml reaction vessel were added compound LD005-4 (420 mg, 0.672 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (306 mg, 0.806 mmol, CAS No. 148893-10-1), N,N-diisopropylethylamine (433 mg, 3.36 mmol), compound IM005 (166 mg, 0.806 mmol), and dichloromethane (8.4 ml). The atmosphere was purged with nitrogen three times and stirred at room temperature for 2 hours. The reaction was complete. The reaction mixture was washed with purified water (20 ml), separated, and the organic phase was dried, concentrated, and purified by column chromatography to obtain compound LD005-5 (500 mg, 91.7% yield). MS ESI (m / z) = 813 [M+H] + .

[0312] (15-6) Synthesis of compound LD005:

[0313] Compound LD005-5 (500 mg, 0.615 mmol), 1 M aqueous sodium hydroxide solution (1.5 ml), and methanol (1.5 ml) were added to a 25 ml reactor. The atmosphere was replaced with nitrogen three times and stirred at room temperature for 1 hour. The reaction was complete. 1 M aqueous hydrogen chloride solution was added to the reaction solution to adjust the pH to 2. The reaction solution was concentrated, and dichloromethane (5 ml) and methanol (5 ml) were added. The solution was filtered, concentrated, and purified by reverse phase chromatography to obtain compound LD005 (220 mg, 44.9% yield). MS ESI (m / z) = 800 [M+H] + .

[0314] 1H NMR (500MHz, DMSO-d6) δ13.05(s,1H), δ8.74–8.62(m,2H),8.27(m,2H),8.16–8.07(m,2H),7.86(dd,J=8.2,2.4Hz,1H),7.58(d,J=8 .0Hz,1H),7.56–7.47(m,3H),7.08(dd,J=7.9,2.1Hz,2H),6.87(s,1H),6.30(d,J=7.3Hz,1H),5.27(q,J=7.2Hz,1H),4.35(t,J=4.6 Hz,2H),3.93(t,J=4.5Hz,2H),3.81–3.65(m,5H),3.61–3.54(m,6H),3.52(d,J=3.7Hz,6H),3.36(t,J=4.9Hz,3H),3.22(d,J=6.1Hz ,3H),2.82(dd,J=6.9,3.5Hz,2H),2.71(t,J=7.8Hz,2H),2.60(t,J=6.3Hz,2H),2.46(dd,J=7.9,3.0Hz,2H),1.73(p,J=6.1Hz,2H).

[0315] Preparation Example 16: Preparation of Compound NM064:

[0316] In this preparation example, the synthetic route of compound NM064 is as follows:

[0317] (16-1) Synthesis of compound NM064-2:

[0318] Compound NM064-1 (13 g, 1.0 eq, methyl β-D-glucopyranoside, CAS No. 709-50-2) was dissolved in 150 ml of acetonitrile. Benzaldehyde dimethyl acetal (30 ml, 3.0 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, respectively, and stirred at 25°C for 5 hours. 3 ml of triethylamine was added, and the reaction was stirred at 25°C for 30 minutes. The reaction mixture was concentrated, water (100 ml) was added, and extraction was completed twice with ethyl acetate (100 ml each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified using a normal phase column (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM064-2 (11.5 g, 60.8% yield) as a white solid. MS ESI (m / z) = 283 [M+H] + .

[0319] (16-2) Synthesis of compound NM064-3:

[0320] Compound NM064-2 (5 g, 1.0 eq) was dissolved in 40 ml of N,N-dimethylformamide. Sodium hydride (2.7 g, 4 eq) was added under ice-cooling and the mixture was allowed to react for 30 minutes. 3-Bromopropyne (8.1 g, 4 eq) was added and stirred at 25°C for 2 hours. The mixture was quenched with 20 ml of water. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate (50 ml each time). The organic phases were combined and washed five times with saturated sodium chloride solution (20 ml each time). The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound NM064-3 (6.3 g, 100% yield) as a brown oil. MS ESI (m / z) = 359 [M+H] + .

[0321] (16-3) Synthesis of compound NM064-4:

[0322] Compound NM064-3 (6.3 g, 17 mmol, 1.0 eq) was dissolved in 30 ml of dichloromethane, and 300 ml of 70% acetic acid solution was added. The mixture was allowed to react at 70°C for 1 hour until completion. The reaction solution was directly concentrated to obtain compound NM064-4 (4.78 g, 100% yield) as a yellow oil. MS ESI (m / z) = 271 [M+H] + .

[0323] (16-4) Synthesis of compound NM064-5:

[0324] Compound NM064-4 (4.78 g, 17.7 mmol, 1.0 eq) was dissolved in 50 ml of pyridine. 4,4'-dimethoxytriphenylmethane (7.8 g, 23.0 mmol, 1.3 eq, abbreviated as DMTrCl, CAS No. 40615-36-9) was added under ice-cooling. The atmosphere was replaced with nitrogen three times and stirred at 25°C for 3 hours. The reaction was quenched with 50 ml of methanol. The reaction mixture was concentrated, 50 ml of water was added, and the mixture was extracted with ethyl acetate three times (50 ml each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM064-5 (6.7 g, yield 66.3%) as a pale yellow solid. MS ESI (m / z) = 573 [M+H] + .

[0325] (16-5) Synthesis of compound NM064:

[0326] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and 4,5-dicyanoimidazole (330.4 mg, 0.8 eq, abbreviated as DCI, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 eq, CAS No. 102691-36-1) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 ml each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 72 / 28, v / v). The mixture was dried in vacuo for 12 hours to obtain compound NM064 (2 g, 74.07% yield) as a white powder. MS ESI (m / z) = 774 [M+H] + .

[0327] 1 H NMR(400MHz, DMSO-d6)δ7.45–7.39(d,J=7.8Hz,2H),7.36–7.18(tt,J=14.5,8.5Hz,7 H),6.95–6.84(d,J=7.5Hz,4H),5.01–4.96(s,1H),4.43–4.28(s,4H),3.81–3.70(s,8 H),3.65–3.35(m,12H),3.28–3.18(dt,J=14.5,7.2Hz,1H),3.06–2.97(t,J=9.5Hz,1 H),2.75–2.68(m,1H),1.07–0.95(q,J=7.4,6.8Hz,10H),0.84–0.78(d,J=6.6Hz,2H).

[0328] Preparation Example 17: Preparation of siRNA conjugates

[0329] (17-1) Synthesis of sense and antisense chains:

[0330] The sense strand (SS) or antisense strand (AS) of the siRNA to be synthesized was introduced into the ABI394 synthesizer. Using Universal CPG / PS as a carrier, the nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide sequence through phosphoramidite nucleic acid solid-phase synthesis method (in the synthesis process, the 5'-AMINO-MODIFIER C6-TFA phosphoramidite monomer and compound NM064 were each considered a nucleoside monomer). Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given as follows:

[0331] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0332] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support.

[0333] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 mol / L solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0334] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0335] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0336] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain or antisense chain, wherein:

[0337] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to a solid support was added to 0.5 ml / μmol of 25% ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0338] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0339] Table 1 Sequence information of sense strand and antisense strand

[0340] The siRNAs disclosed herein are all obtained by modifying the nucleotide sequences shown in the table below.

[0341] Table 1a Sequence information of unmodified sense and antisense strands

[0342] Unless otherwise indicated, in the sequences disclosed herein, capital letters A, U, G, C, and T represent the base composition of nucleotides, wherein U represents uracil, T represents thymine, C represents cytosine, A represents adenine, and G represents guanine.

[0343] The lowercase letter m indicates that the nucleotide represented by the capital letter to the left of the letter m is a 2'-O-methyl modified nucleotide. The structural formula of the 2'-O-methyl modified nucleotide is

[0344] The lowercase letter f indicates that the nucleotide represented by the capital letter to the left of the letter f is a 2'-fluoro modified nucleotide. The structural formula of the 2'-fluoro modified nucleotide is

[0345] The combination symbol (moe) indicates that the nucleotide represented by the capital letter adjacent to the left side of the combination symbol (moe) is a 2'-O-methoxyethyl modified nucleotide. The structural formula of the 2'-O-methoxyethyl modified nucleotide is

[0346] Unless otherwise indicated, Base used in the present disclosure represents a nucleoside base, such as uracil U, thymine T, cytosine C, adenine A or guanine G.

[0347] (invAb) indicates that the nucleotide at this position is an inverted abasic nucleotide. The structural formula of (invAb) is It is composed of nucleoside monomers (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, purchased from Shanghai Zhaowei Technology Development Co., Ltd.) is formed after participating in the synthesis of siRNA.

[0348] VP stands for 5'-(E)-vinylphosphonate modification, which is a 5'-terminal phosphorylation modification. Wherein, VPUm represents the structural formula of the nucleotide at this site is

[0349] A lowercase letter s indicates that the internucleoside bond between the two nucleotides adjacent to the letter s is a phosphorothioate diester bond.

[0350] The structural formula of (NH2-C6) is It is conjugated to the 5' end of the sense strand. (NH2-C6) is composed of 5'-AMINO-MODIFIER C6-TFA phosphoramidite monomer (CAS No. 133975-85-6) participates in the synthesis of the positive chain. Among them, the structural formula of the 5' end conjugated (NH2-C6) of the positive chain is

[0351] The structural formula of NM064 in the sequence is It is conjugated to the 3' end of the sense chain. The structural formula of the 3' end of the sense chain conjugated with NM064 is

[0352] (17-2) Synthesis of siRNA conjugate (siRNA-LK004 + ligand*3):

[0353] (17-2-1) siRNA synthesis:

[0354] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was then slowly cooled to 25°C and maintained at 25°C for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonding, thereby producing siRNA. (14-2) Synthesis of siRNA Conjugate (siRNA-LK004 + Ligand*3):

[0355] (17-2-2) Synthesis of siRNA-LK004:

[0356] The specific synthesis process is carried out according to Example 2 of WO2019010274A1, the entire content of which is incorporated herein by reference and will not be repeated here.

[0357] The structural formula of LK004 is as follows:

[0358] The structural formula of siRNA-LK004 is shown below:

[0359] (17-2-3) Synthesis of siRNA-LK004+ligand*3:

[0360] 150 μL of H 2 O, 70 μL of 0.2 M carbonate buffer solution (pH = 9.2) and 70 μL of N,N-dimethylformamide (DMF) were mixed to obtain a mixed solvent; siRNA-LK004 was dissolved in the mixed solvent to obtain a siRNA-LK004 solution with a concentration of 1.0 eq.

[0361] 6.0 eq of the ligand compound was dissolved in 70 μL of DMF to obtain a ligand compound solution.

[0362] The siRNA-LK004 solution and the ligand compound solution were mixed to obtain a reactant mixture, wherein the molar ratio of siRNA-LK004 to the ligand compound was 1:6.

[0363] 10.0 eq of tris(3-hydroxypropyltriazolemethyl)amine (CAS No. 760952-88-3, abbreviated as THPTA) and 3.0 eq of CuSO4·5H2O were mixed in a volume ratio of THPTA:CuSO4·5H2O = 10:3. After shaking at 40°C for 5 minutes, 37 μL of the mixture was added to the above reaction mixture and vortexed to obtain an intermediate product mixture. The pH of the intermediate product mixture was measured to be 8. 25.0 eq of sodium ascorbate was then quickly added to the intermediate product mixture and vortexed. The mixture was reacted at 40°C for 1 hour to obtain a product mixture.

[0364] 3 μL of the product mixture was diluted with a mixture of DMF and HO (DMF:HO volume ratio of 1:5), and then separated and purified by HPLC. The HPLC process employed a C18 column, an ammonium bicarbonate buffered solution as the mobile phase, and a gradient elution method. The purified product after HPLC was lyophilized to obtain the conjugate product.

[0365] Each conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water analyzer, resistivity 18.2 MΩ*cm (25°C)). Molecular weights were then determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugates were the intended siRNA conjugates.

[0366] Wherein, when the ligand compound is LD000, the structural formula of the siRNA conjugate is as follows:

[0367] When the ligand compound is LD002, the structural formula of the siRNA conjugate is as follows:

[0368] When the ligand compound is LD003, the structural formula of the siRNA conjugate is as follows:

[0369] When the ligand compound is LD005, the structural formula of the siRNA conjugate is as follows:

[0370] When the ligand compound is LD006, the structural formula of the siRNA conjugate is as follows:

[0371] When the ligand compound is LD007, the structural formula of the siRNA conjugate is as follows:

[0372] When the ligand compound is LD008, the structural formula of the siRNA conjugate is as follows:

[0373] When the ligand compound is LD009, the structural formula of the siRNA conjugate is as follows:

[0374] When the ligand compound is LD010, the structural formula of the siRNA conjugate is as follows:

[0375] (17-3) Synthesis of siRNA conjugate (siRNA-NM064 + ligand*2):

[0376] (17-3-1) Synthesis of SS-NM064+ligand*2:

[0377] The synthesis method of SS-NM064+ligand*2 is to connect the ligand compound to the sense chain NM064 through an azide-alkyne cycloaddition reaction. The synthesis method specifically includes the following steps:

[0378] 150 μL of H2O, 70 μL of 0.2 mol / L carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) were mixed to obtain a mixed solvent; then, the positive chain of step (14-1) was dissolved in the mixed solvent to obtain a positive chain solution with a concentration of 1.0 eq.

[0379] The ligand compound (6.0 eq) was dissolved in DMF (70 μL) to obtain a ligand compound solution.

[0380] The sense chain solution and the ligand compound solution are mixed to obtain a reactant mixture.

[0381] 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O were mixed and shaken at 40°C for 5 min. 37 μL of the mixture was added to the above reactant mixture and vortexed to obtain an intermediate product mixture. The pH of the intermediate product mixture was measured to be 8.

[0382] 25.0 eq of sodium ascorbate was quickly added to the intermediate product mixture, and the mixture was vortexed and reacted at 40° C. for 1 h to obtain a product mixture.

[0383] Purification: 3 μL of the product mixture was diluted with a mixture of DMF and HO (1:5 volume ratio), and then purified by HPLC. The HPLC process used a C18 column, an ammonium bicarbonate buffered mobile phase, and a gradient elution method. The purified product was lyophilized.

[0384] (17-3-2) Synthesis of siRNA-NM064+ligand*2:

[0385] SS-NM064+ligand*2 and AS were mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, so that SS-NM064+ligand*2 and AS formed a double-stranded siRNA conjugate through hydrogen bonding.

[0386] Wherein, when the ligand compound is LD000, the structural formula of the siRNA conjugate is as follows:

[0387] When the ligand compound is LD002, the structural formula of the siRNA conjugate is as follows:

[0388] When the ligand compound is LD003, the structural formula of the siRNA conjugate is as follows:

[0389] When the ligand compound is LD005, the structural formula of the siRNA conjugate is as follows:

[0390] When the ligand compound is LD006, the structural formula of the siRNA conjugate is as follows:

[0391] When the ligand compound is LD007, the structural formula of the siRNA conjugate is as follows:

[0392] When the ligand compound is LD008, the structural formula of the siRNA conjugate is as follows:

[0393] When the ligand compound is LD009, the structural formula of the siRNA conjugate is as follows:

[0394] When the ligand compound is LD010, the structural formula of the siRNA conjugate is as follows:

[0395] The present disclosure adopts the above method to prepare the siRNA conjugates shown in Table 2.

[0396] Table 2 Sequence information of siRNA conjugates

[0397] Table 3 Detection results of siRNA conjugates

[0398] Unless otherwise stated, the siRNAs used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.

[0399] Unless otherwise stated, the siRNA conjugates used in the present disclosure were synthesized by Beijing Xuanjingrui Pharmaceutical Technology Co., Ltd.

[0400] Biological detection experiments

[0401] Unless otherwise specified, the experimental animals C57BL / 6J mice and SD rats used in the present disclosure were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0402] Unless otherwise stated, the synthesis of PCR primers used in this disclosure was commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0403] Unless otherwise specified, all reagents, consumables, and instruments used in this disclosure were commercially available. The main reagents and consumables are shown in Table 4, and the main instruments and equipment are shown in Table 5.

[0404] Table 4 Main reagents and consumables

[0405] Table 5 Main instruments and equipment

[0406] Evaluation of the activity of siRNA conjugates in rat lung spray administration

[0407] Male mice aged 6-8 weeks were randomly divided into experimental and blank control groups based on body weight. Each group of mice was administered via pulmonary spray, with each siRNA / siRNA conjugate prepared in PBS at the corresponding concentration (calculated as siRNA). The experimental groups were administered the siRNA / siRNA conjugate solution, with the dosage and volume determined for each mouse according to the specific experiment. The blank control group was administered a PBS solution without the siRNA / siRNA conjugate.

[0408] The specific procedure for pulmonary aerosolization was to anesthetize mice using an R540IE small animal anesthesia machine. The mice were secured on an airway intubation platform and kept upright. Using a laryngoscope, a pulmonary endotracheal (liquid) drug delivery device was inserted into the mouse's trachea and aerosolized for 1-2 seconds. The day of drug administration was designated as day 0 (D0). After drug administration, mice were sacrificed at the observation point. Whole lungs were minced and stored in RNAlater.

[0409] RNA extraction: The whole lung tissue samples were removed from RNAlater and disrupted in a Tissuelyser II fully automated tissue homogenizer for 60 seconds. Total RNA was extracted from each tissue sample using a fully automated nucleic acid extractor and nucleic acid extraction kit from Zhejiang Hanwei Technology Co., Ltd. according to the instructions.

[0410] Reverse transcription: 1 μg of total RNA from each tissue sample was extracted and used with the Promega Reverse Transcription System (A3500) using the Oligo(dT)15 reverse transcription primer. A 20 μL reverse transcription system was prepared according to the kit instructions and the reverse transcription reaction was completed. After the reaction, 80 μL of RNase-free water was added to the reverse transcription system to obtain the cDNA solution for real-time PCR analysis.

[0411] Real-time PCR assay: A 20 μL real-time PCR reaction system was prepared per PCR well using ABI SYBR™ Select Master Mix (Catalog number: 4472908) according to the kit instructions. Each reaction system contained 5 μL of cDNA template obtained from the reverse transcription reaction, 10 μL of SYBR™ Select Master Mix, 0.5 μL of a 10 μM upstream primer, 0.5 μL of a 10 μM downstream primer, and 4 μL of RNase-Free H2O. The prepared reaction system was placed on an ABI StepOnePlus thermal cycler for real-time PCR amplification using a three-step protocol: a 95°C denaturation for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Gene expression differences were calculated using the ΔΔCt method.

[0412] In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to perform relative quantitative calculation of the target gene mRNA expression level and inhibition rate in each test group. The calculation method is as follows: ΔCt(test group) = Ct(test group target gene) – Ct(test group internal reference gene) ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene) ΔΔCt(test group) = ΔCt(test group) – ΔCt(control group average) ΔΔCt(control group) = ΔCt(control group) – ΔCt(control group average)

[0413] Where ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) values ​​of the animals sacrificed at the same time point in the control group. Therefore, each animal in the test group and the control group corresponds to a ΔΔCt value. The relative expression level of the target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0414] The relative expression level of the target gene mRNA in the control group was used as the benchmark to normalize the relative expression level of the target gene mRNA in the test group, and the relative expression level of the target gene mRNA in the control group was defined as 100%. The expression inhibition rate of the target gene mRNA in the test group = (1 – relative expression level of the target gene mRNA in the test group) × 100%

[0415] Unless otherwise stated, the in vivo activity data are based on The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0416] Example 1 Evaluation of the inhibitory activity of ligands LD003 and LD006 conjugated to siRNA against the target gene advanced glycosylation end-product specific receptor (RAGE)

[0417] This example uses a rat lung spray administration activity evaluation test to evaluate the inhibitory activity of siRNA conjugates RZ895003 with LD003 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895006 with LD006 conjugated to the 5' end of the sense chain, and siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain against the target gene RAGE in the lung tissue of SD rats.

[0418] 6-8 week old SD rats were randomly divided into 4 groups according to body weight, with 5 rats in each group, for a total of 4 groups (including 3 experimental groups and 1 blank control group). Among them, the rats in each experimental group were given siRNA conjugate solution by single pulmonary spray administration, with each dose of 0.8 mg (in terms of siRNA) / kg (in terms of rat body weight) and a dosing volume of 200 μL. The blank control group was given a PBS solution without siRNA / siRNA conjugate, with a dosing volume of 200 μL. The day of administration was recorded as day 0 (D0), and the rats were killed on D7 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and the gene expression differences were calculated by the above ΔΔCt method.

[0419] Table 6 Primer sequence list used in Example 1

[0420] The results of Example 1 showed that the inhibitory effects of LD003 conjugate RZ895003 and LD006 conjugate RZ895006 on the target gene at D7 were better than those of LD000 conjugate RZ895001; the inhibition rates of the target genes could reach 90% or above ( Figure 1 , Table 7 ).

[0421] Table 7 Inhibitory activity of RZ895001, RZ895003, and RZ895006 on target genes in rat lung tissue

[0422] Example 2 Evaluation of the inhibitory activity of ligand LD008 conjugated siRNA on the target gene RAGE

[0423] This example uses a rat lung spray drug administration activity evaluation test to evaluate the inhibitory activity of siRNA conjugates RZ895009 with LD008 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain, and siRNA sequence RX895001 without ligand compound on the target gene RAGE in SD rat lung tissue.

[0424] 6-8 week old SD rats were randomly divided according to body weight, with 5 rats in each group, for a total of 8 groups (including 3 0.1 mg / kg dose test groups and 1 0.1 mg / kg dose blank control group, and 3 0.8 mg / kg dose test groups and 1 0.8 mg / kg dose blank control group). The rats in each 0.1 mg / kg dose test group were administered a single pulmonary spray of siRNA / siRNA conjugate solution at a dose of 0.1 mg (as siRNA) / kg (as rat body weight) in a 200 μL dose volume. The rats in the 0.1 mg / kg dose blank control group were administered a single pulmonary spray of PBS solution without siRNA / siRNA conjugate in a 200 μL dose volume. Each rat in the 0.8 mg / kg dose test group was given a single pulmonary spray of siRNA / siRNA conjugate solution, with a dose of 0.8 mg (in terms of siRNA) / kg (based on rat body weight) per rat, and a dosing volume of 200 μL; the 0.8 mg / kg dose blank control group was given a single pulmonary spray of PBS solution without siRNA / siRNA conjugate, and a dosing volume of 200 μL. The day of administration was recorded as day 0 (D0), and the rats were killed on D7 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection were performed for each tissue according to the above method, and gene expression differences were calculated by the above-mentioned ΔΔCt method.

[0425] The primers used in Example 2 are shown in Table 6, the primer sequence table used in Example 1.

[0426] The results of Example 2 showed that at D7, the inhibitory effects of the small molecule LD008 conjugate RZ895009 at different dosages on the target gene were superior to those of the siRNA (RX895001) without small molecule conjugation at the same dose; the inhibitory effect of the small molecule LD008 conjugate RZ895009 on the target gene at a dosage of 0.8 mg / kg was superior to that of the reference small molecule LD000 conjugate RZ895001, and the inhibition rate of RZ895009 and the reference conjugate RZ895001 on the target gene at a dosage of 0.1 mg / kg was the same ( Figure 2 , Table 8).

[0427] Table 8 Inhibitory activity of RZ895001, RZ895009, and RX895001 on target genes in rat lung tissue

[0428] Example 3 Evaluation of the inhibitory activity of ligands LD003 and LD007 conjugated to siRNA sequences on the target gene RAGE

[0429] In this example, a mouse lung spray administration activity evaluation test was used to evaluate the inhibitory activity of siRNA conjugates RZ895003 with LD003 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895007 with LD007 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain, and siRNA sequence RX895001 without a ligand compound against the target gene RAGE in the lung tissue of C57BL / 6j mice.

[0430] 6-8 week old C57BL / 6j mice were randomly divided into 5 groups according to body weight, with 4 mice in each group, for a total of 5 groups (including 4 experimental groups and 1 blank control group). The rats in each experimental group were given siRNA / siRNA conjugate solution by single pulmonary spray administration, with each dose of 0.6 mg (in terms of siRNA) / kg (in terms of mouse body weight) and a dosing volume of 50 μL. The blank control group was given a PBS solution without siRNA / siRNA conjugate by single pulmonary spray administration, with a dosing volume of 50 μL. The day of administration was recorded as day 0 (D0), and the mice were killed on D14 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and the gene expression differences were calculated by the above ΔΔCt method.

[0431] Table 9 Primer sequence list used in Example 3

[0432] The results of Example 3 showed that the LD003 conjugate, RZ895003, exhibited superior inhibitory effects on target genes compared to the reference LD000 conjugate, RZ895001, and the siRNA sequence RX895001, which was conjugated to a ligand-free compound. The inhibition rate in the entire mouse lung was 79.91%, 22.58% higher than that of the siRNA sequence conjugated to a ligand-free compound (Figure 3, Table 10).

[0433] Table 10 Inhibitory activity of RZ895003, RZ895007, RZ895001, and RX895001 on target genes in mouse lung tissue

[0434] Example 4 Evaluation of the inhibitory activity of ligand LD008 conjugated to siRNA sequence on the target gene RAGE

[0435] In this example, a mouse lung spray drug administration activity evaluation test was used to evaluate the inhibitory activity of the siRNA conjugate RZ895009 with LD008 conjugated to the 5' end of the sense chain, the siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain, and the siRNA sequence RX895001 without a ligand compound against the target gene RAGE in the lung tissue of C57BL / 6j mice.

[0436] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 4 mice in each group, for a total of 4 groups (including 3 experimental groups and 1 blank control group). The rats in each experimental group were given siRNA / siRNA conjugate solution by single pulmonary spray administration, with each dose of 0.6 mg (in terms of siRNA) / kg (in terms of mouse body weight) and a dosing volume of 50 μL. The blank control group was given a PBS solution without siRNA / siRNA conjugate by single pulmonary spray administration, with a dosing volume of 50 μL. The day of administration was recorded as day 0 (D0), and the mice were killed on D14 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and the gene expression differences were calculated by the above ΔΔCt method.

[0437] The primers used in Example 4 are shown in Table 9, the primer sequence table used in Example 3.

[0438] The results of Example 4 showed that at D14, the small molecule LD008 conjugate RZ895009 had a better inhibitory effect on the target gene than the reference small molecule LD000 conjugate RZ895001 and the siRNA sequence RX895001 without small molecule conjugation ( Figure 4 , Table 11 ).

[0439] Table 11 Inhibitory activity of RZ895001, RZ895009, and RX895001 on target genes in mouse lung tissue

[0440] Example 5 Evaluation of the inhibitory activity of ligands LD008 and LD009 conjugated to siRNA sequences on the target gene RAGE

[0441] In this example, a mouse lung spray administration activity evaluation test was used to evaluate the inhibitory activity of siRNA conjugates RZ895009 with LD008 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895010 with LD009 conjugated to the 5' end of the sense chain, siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain, and siRNA sequence RX895001 without a ligand compound against the target gene RAGE in the lung tissue of C57BL / 6j mice.

[0442] 6-8 week old C57BL / 6j mice were randomly divided into 5 groups according to body weight, with 4 mice in each group, for a total of 5 groups (including 4 experimental groups and 1 blank control group). The rats in each experimental group were given siRNA / siRNA conjugate solution by single pulmonary spray administration, with each dose of 0.6 mg (in terms of siRNA) / kg (in terms of mouse body weight) and a dosing volume of 50 μL. The blank control group was given a PBS solution without siRNA / siRNA conjugate by single pulmonary spray administration, with a dosing volume of 50 μL. The day of administration was recorded as day 0 (D0), and the mice were killed on D7 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and the gene expression differences were calculated by the above ΔΔCt method.

[0443] The primers used in Example 5 are shown in Table 9, which is the sequence table of primers used in Example 3.

[0444] The results of Example 5 showed that on day 7, the small molecule LD008 conjugate RZ895009 had a better inhibitory effect on the target gene than the reference small molecule LD000 conjugate RZ895001 and the siRNA without small molecule conjugation (RX895001) ( Figure 5 , Table 12 ).

[0445] Table 12 Inhibitory activity of RZ895009, RZ895010, RZ895001, and RX895001 on target genes in mouse lung tissue

[0446] Example 6 Evaluation of the inhibitory activity of ligand LD006 conjugated to siRNA sequence on the target gene superoxide dismutase 1 (SOD1)

[0447] This example uses a mouse lung spray administration activity evaluation test to evaluate the inhibitory activity of siRNA conjugates RZ899014 with LD006 conjugated to the 5' end of the sense chain and siRNA conjugate RZ899012 with LD000 conjugated to the 5' end of the sense chain against the target gene RAGE in the lung tissue of C57BL / 6j mice.

[0448] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups (including 2 experimental groups and 1 blank control group) by body weight. The rats in each experimental group were given siRNA conjugate solution by pulmonary spray administration, with a frequency of administration once a day for two consecutive days, and a dose of 1.5 mg (in terms of siRNA) / kg (in terms of mouse body weight) per dose per time, and a volume of 50 μL per dose. The blank control group was given a PBS solution without siRNA conjugate by pulmonary spray administration, with a frequency of administration once a day for two consecutive days, and a volume of 50 μL per dose per time. The day of the first administration was recorded as day 0 (D0), and the mice were killed on D6 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and gene expression differences were calculated by the above ΔΔCt method.

[0449] Table 13 Sequence list of primers used in Example 6

[0450] The results of Example 6 showed that the inhibitory effect of the LD006 conjugate RZ899014 on the target gene SOD1 was better than that of the reference LD000 conjugate RZ899012, with an inhibitory activity 15.68% higher ( FIG. 6 , Table 14 ).

[0451] Table 14 Inhibitory activity of RZ899012 and RZ899014 on target genes in mouse lung tissue

[0452] Example 7 Evaluation of the inhibitory activity of the ligand LD0010 conjugated siRNA sequence on the target gene RAGE

[0453] In this example, a mouse lung spray drug administration activity evaluation test was used to evaluate the inhibitory activity of the siRNA conjugates RZ895011 with LD010 conjugated to the 5' end of the sense chain, the siRNA conjugate RZ895001 with LD000 conjugated to the 5' end of the sense chain, and the siRNA sequence RX895001 without a ligand compound against the target gene RAGE in the lung tissue of C57BL / 6j mice.

[0454] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 4 mice in each group, for a total of 4 groups (including 3 experimental groups and 1 blank control group). The rats in each experimental group were given siRNA / siRNA conjugate solution by single pulmonary spray administration, with each dose of 0.6 mg (in terms of siRNA) / kg (in terms of mouse body weight) and a dosing volume of 50 μL. The blank control group was given a PBS solution without siRNA / siRNA conjugate by single pulmonary spray administration, with a dosing volume of 50 μL. The day of administration was recorded as day 0 (D0), and the mice were killed on D7 after administration. The whole lung tissue was minced and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection of each tissue were performed according to the above method, and the gene expression differences were calculated by the above ΔΔCt method.

[0455] The primers used in Example 7 are shown in Table 9, which is the sequence table of primers used in Example 3.

[0456] The results of Example 7 showed that at D7, the inhibitory effect of the small molecule LD010 conjugate RZ895011 on the target gene was basically the same as that of the siRNA without small molecule conjugation (RX895001), and weaker than the control small molecule LD000 conjugate RZ895001 ( Figure 7 , Table 15 ).

[0457] Table 15 Inhibitory activity of RZ895011, RZ895001, and RX895001 on target genes in mouse lung tissue

[0458] In vivo activity evaluation test of siRNA conjugates in mice

[0459] 6-8 week old C57BL / 6j mice were randomly divided into groups according to body weight, with 5 mice per group. Among them, the mice in the experimental group were given PBS buffer containing siRNA conjugates by tail vein administration, with each mouse receiving a dose of 5 mg (as siRNA) / kg (as mice) and a dosing volume of 5 ml / kg (as mice); the mice in the blank control group were given PBS buffer without siRNA conjugates by tail vein administration, with each mouse receiving a dosing volume of 5 ml / kg (as mice). The day of administration was recorded as the first day (D0). After administration, 5 mice in each group were sacrificed on the seventh day (D7). The animals were grossly dissected, and tissues such as quadriceps, heart, liver, and kidney were collected, cut into several 2 mm3 small pieces, and stored in RNAlater.

[0460] RNA extraction: The above tissue samples were removed from RNAlater and disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Total RNA from each tissue sample was extracted using the Trizol method.

[0461] Reverse transcription: 1 μg of total RNA from each tissue sample was extracted and used with the Oligo(dT)15 reverse transcription primer in the Promega Reverse Transcription System (A3500). A 20 μL reverse transcription system was prepared according to the kit's instructions and the reverse transcription reaction was completed. After the reaction, 80 μL of RNase-free water was added to the reverse transcription system to obtain the cDNA solution for real-time PCR analysis.

[0462] Real-time PCR assay: Using ABI SYBR™ Select Master Mix (Catalog number: 4472908), a 20 μL real-time PCR reaction system was prepared per PCR well according to the kit instructions. Each reaction system contained 5 μL of cDNA template obtained from the reverse transcription reaction, 10 μL of SYBR™ Select Master Mix, 0.5 μL of a 10 μM upstream primer, 0.5 μL of a 10 μM downstream primer, and 4 μL of RNase-free H2O. The prepared reaction system was placed on an ABI StepOnePlus thermal cycler for real-time PCR amplification using a three-step protocol: a 95°C initial denaturation for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Gene expression differences were calculated using the ΔΔCt method.

[0463] In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to perform relative quantitative calculation of the target gene mRNA expression level and inhibition rate in each test group. The calculation method is as follows: ΔCt(test group) = Ct(test group target gene) – Ct(test group internal reference gene) ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene) ΔΔCt(test group) = ΔCt(test group) – ΔCt(control group average) ΔΔCt(control group) = ΔCt(control group) – ΔCt(control group average)

[0464] Wherein, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of the animals sacrificed at the same time point in the control group. Therefore, each animal in the test group and the control group corresponds to a ΔΔCt value. The relative expression level of the target gene mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0465] The target gene mRNA expression level of the test group was normalized with the control group as the benchmark. The target gene mRNA expression inhibition rate of the test group = (1 – relative expression level of the target gene mRNA of the test group) × 100%

[0466] Unless otherwise stated, the in vivo activity experimental data were expressed as X±STDEV, and the experimental data were graphed and analyzed using GraphPadprism 8.0 software.

[0467] Example 8 evaluates the inhibitory activity of different ligand-conjugated siRNA sequences targeting integrin αvβ6 on the target gene superoxide dismutase 1 (SOD1):

[0468] This example uses an in vivo mouse activity assessment assay to evaluate the inhibitory activity of sequences R699168, R699173, R699174, R699175, R699176, and R699177, which are conjugated to different ligands targeting integrin αvβ6, against the target gene SOD1. Six- to eight-week-old C57BL / 6j mice were randomly divided according to body weight, with five mice per group, into six experimental groups and one blank control group. The day of administration was designated D0, and mice were sacrificed on D7 after administration. Quadriceps muscle, heart, liver, and kidney were collected and stored in RNAlater. RNA extraction, reverse transcription, and real-time fluorescence quantitative detection were performed for each tissue according to the above-described methods, and the expression difference of the target gene was calculated using the above-described ΔΔCt method.

[0469] Table 16 Sequence information of primers in Example 8

[0470] [Corrected 11.12.2024 according to Rule 91] The results of Example 8 showed that at D7 after administration, the sequences R699168, R699173, R699174, R699175, R699176, and R699177 conjugated with different ligand small molecules targeting integrin αvβ6 all had excellent inhibitory activity in muscle, with inhibitory effects of about 80% or above, while the activities in tissues such as the heart, liver, kidney, and lung were all 50% or below; this indicates that the ligands targeting integrin αvβ6 disclosed herein all exhibited good muscle targeting ( Figure 8 , Table 17).

[0471] Table 17 Inhibitory activity of target genes in mice after administration of siRNA conjugates R699168, R699173, R699174, R699175, R699176, and R699177 in Example 8

[0472] The above specific embodiments are merely illustrative of the present invention and do not limit the present invention. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present invention, and such modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A ligand targeting integrin αvβ6, characterized in that The ligand is selected from the structure shown in formula (I), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: Wherein R1 is selected from L1 is selected from optionally substituted C 10 -C 30 Alkylene or wherein Z is selected from O or S, j is selected from an integer from 1 to 5; k is selected from an integer from 1 to 10; if the optionally substituted C 10 -C 30 The alkylene group contains a substituent, and the substituent is selected from halogen or C1-C3 alkoxy; R2 is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C5-C 10 Aryl or wherein each R2′ is independently selected from an optionally substituted C1-C6 alkyl group; if the optionally substituted C1-C6 alkyl group contains a substituent, the substituent is selected from halogen, C5-C 10 aryl or C1-C3 alkoxy; if the optionally substituted C5-C 10 The aryl group contains a substituent, and the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy; m is an integer selected from 1 to 5; Each R3 is independently selected from H or an amino acid residue; Y is selected from CH2 or NH; R6 is selected from Among them, R 4a and R 4b Each is independently selected from H, OH, SH, NH2, halogen, C1-C3 alkyl or C1-C3 alkoxy; R 6a , R 6b , R 6c and R 6d Each independently selected from H or C1-C3 alkyl; n is an integer selected from 1-4.

2. The ligand according to claim 1, characterized in that L1 is selected from Optionally, Selected from Optionally, Z is selected from O; Optionally, j is selected from an integer from 1 to 3; Optionally, L1 is selected from Optionally, k is selected from an integer from 2 to 8; Optionally, R1 is selected from Optionally, R2 is selected from H; Optionally, m is selected from 1; Optionally, R3 is selected from H; Optionally, n is selected from an integer of 2-4; Optionally, R 4a and R 4b All selected from H; Optionally, R 6a , R 6b , R 6c and R 6d Each is independently selected from H or methyl.

3. The ligand according to any one of claims 1 to 2, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

4. The ligand according to any one of claims 1 to 2, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof: Optionally, the ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

5. The ligand according to any one of claims 1 to 2, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof: Optionally, the ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

6. The ligand according to any one of claims 1 to 2, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof: Optionally, the ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

7. The ligand according to any one of claims 1 to 2, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof: Optionally, the ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

8. The ligand according to claim 1, characterized in that The ligand is selected from any of the following structures, or tautomers thereof, or stereoisomers thereof, or pharmaceutically acceptable salts thereof:

9. A conjugate, characterized in that The conjugate has a structure represented by formula (VI), or a tautomer, or a stereoisomer, or a pharmaceutically acceptable salt thereof: Among them, Nu represents the active drug molecule; t is an integer selected from 1 to 6; Each M is independently selected from any of the following structures, or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: L2 is selected from wherein Z' is selected from O or S, j1 is selected from an integer of 1 to 5, j2 is selected from an integer of 1 to 5, j3 is selected from an integer of 1 to 10, and j4 is selected from an integer of 1 to 5; L3 is selected from wherein e is selected from an integer of 2-6, and f is selected from an integer of 4-8; a1 is selected from 1, 2, 3 or 4; a2 is selected from 1 or 2; a3 is selected from an integer selected from 1-5; X1 is selected from hydroxyl or thiol; p and q are each independently selected from 0, 1, 2, 3 or 4; R5 is selected from H or C1-C3 alkoxy; X2 is selected from O, S, or NH; Each M' is independently selected from the structure represented by formula (i), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: R1' is selected from L1, m, R3 and R6 are as defined in any one of claims 1-8.

10. [Corrected 11.12.2024 according to Rule 91] The conjugate according to claim 9, characterized in that Each M is independently conjugated to the 3' end, 5' end or a position in the middle between the 3' end and 5' end of the sense strand of the double-stranded oligonucleotide; Optionally, t is selected from 1 or 2; each M is independently conjugated to the 3' end of the sense strand or the 5' end of the sense strand of the double-stranded oligonucleotide; Optionally, t is selected from 1, one of the M is conjugated to the 5' end of the sense strand of the double-stranded oligonucleotide; Optionally, t is selected from 1, and one of the M is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide; Optionally, Z' is selected from O; Optionally, j1 is selected from 1; Optionally, j2 is selected from 2; Optionally, j3 is selected from 2; Optionally, j4 is selected from 2; Optionally, L2 is selected from Optionally, e is selected from 3; Optionally, f is selected from 6; L3 is selected from 11. The conjugate according to claim 9, characterized in that Each M' is independently selected from any of the following structures, or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: Optionally, each M is independently selected from any of the following structures, or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt:

12. The conjugate according to any one of claims 9 to 11, characterized in that The conjugate is selected from the structure represented by formula (VIIA) or (VIIB), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: in, represents Nu double-stranded oligonucleotide; SS represents the sense strand of Nu double-stranded oligonucleotide; AS represents the antisense strand of Nu double-stranded oligonucleotide; Optionally, the conjugate is selected from any of the following structures, or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt:

13. A composition, characterized in that The composition comprises the ligand according to any one of claims 1 to 8, and / or the conjugate according to any one of claims 9 to 12.

14. Use of the ligand according to any one of claims 1 to 8, and / or the conjugate according to claims 9 to 12, and / or the composition according to claim 13 in the preparation of a medicament for preventing and / or treating a disease or symptom associated with abnormal expression of a target gene in a cell expressing integrin αvβ6.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the ligand according to any one of claims 1 to 8, and / or the conjugate according to any one of claims 9 to 12, and / or the composition according to claim 13; Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

16. A method for reducing the expression or activity of a target gene in a cell expressing integrin αvβ6, characterized in that: The method comprises contacting the ligand of any one of claims 1 to 8, or the conjugate of any one of claims 9 to 12, or the composition of claim 13, or the pharmaceutical composition of claim 15 with a cell expressing integrin αvβ6.

17. A method for preventing and / or treating a pathological condition or disease caused by abnormal expression of a target gene in a cell expressing integrin αvβ6, characterized in that: The method comprises: administering to a subject a pharmaceutically acceptable dose of the ligand according to any one of claims 1 to 8, or the conjugate according to any one of claims 9 to 12, or the composition according to claim 13, or the pharmaceutical composition according to claim 15.

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