Oligonucleotide conjugates containing kidney delivery vectors

Abstract

The present disclosure provides an oligonucleotide conjugate, which is obtained by covalently linking a kidney-targeting polypeptide as a ligand to an oligonucleotide molecule capable of inhibiting the expression of a target gene via a linker group. The oligonucleotide conjugate has affinity to a cell receptor present on a kidney target cell, and can selectively and effectively reduce or inhibit the expression of a target gene in the kidney of a subject (e.g., a human or an animal). The oligonucleotide conjugate or pharmaceutical composition provided by the present disclosure can effectively treat and / or prevent a pathological condition or disease caused by abnormal expression of a specific gene in kidney tissue cells.

Description

Technical Field

[0001] This disclosure belongs to the field of nucleic acid drug technology, specifically relating to a double-stranded oligonucleotide conjugate that can target kidney tissue or cells and its uses. Background Technology

[0002] Currently, oligonucleotide therapy (carrying N-acetylgalactosamine (GalNAc) delivery carriers) has achieved success in treating hepatogenic diseases, but efficient delivery carriers are lacking for delivery to non-hepatic tissues (such as the kidneys). Although existing technologies have reported the use of ligand groups targeting renal cell surface factors to facilitate the delivery of oligonucleotides to renal cells, there is still a need to develop more delivery carriers or therapeutic oligonucleotide conjugates for the kidneys, particularly suitable for delivery to proximal tubular epithelial cells and / or podocytes. Summary of the Invention

[0003] This disclosure relates to oligonucleotide conjugates, which use a kidney-targeting polypeptide as a ligand and are covalently linked to an oligonucleotide molecule capable of inhibiting target gene expression via a linker group. This disclosure also describes pharmaceutical compositions comprising said conjugates, said pharmaceutical compositions including at least one pharmaceutically acceptable carrier or excipient. Furthermore, this disclosure relates to the use of said oligonucleotide conjugates and pharmaceutical compositions in the treatment of kidney-related diseases or conditions.

[0004] In a first aspect, this disclosure provides an oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, said oligonucleotide conjugate having the structure shown in Formula I: (I) Nu refers to single-stranded or double-stranded oligonucleotide molecules that have preventive or therapeutic effects on kidney-related diseases or symptoms.

[0005] Nu is an siRNA molecule that has preventive or therapeutic effects on kidney-related diseases or symptoms. It contains a sense strand or antisense strand consisting of 15-25 modified or unmodified nucleotides. The sense strand and antisense strand form a complementary or substantially complementary double-stranded region. The antisense strand sequence is at least partially complementary to the mRNA of a target gene in target cells in kidney tissue. The surface of the target cells has a receptor capable of binding to the ligand group Lg.

[0006] t is an integer selected from 1 to 3.

[0007] m is selected from 1, 2, or 3.

[0008] Each Z is independently either OH or SH.

[0009] A is selected from or .

[0010] p and q are each independently selected from 0, 1, or 2.

[0011] R1 is selected from H, hydroxyl, amino, halogen, C1-6 alkyl or C1-6 alkoxy (preferably methoxy).

[0012] L a Selected from substituted or unsubstituted straight or branched C1-C 15 An alkylene group, wherein one or more methylene units (-CH2-) in the alkylene group are optionally replaced by one or more substituents selected from the group consisting of: -C(O)-, -OC(O)-, -NHC(O)-, -NH-, -O-, -S-, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6 alkylene group, C6-C 10 aryl or C3-C 10 Heterocyclic group.

[0013] L b Selected from chemical bonds, C1-C3 alkyl, amide bonds, -O-, -S-, substituted or unsubstituted 3-8 membered cycloalkyl, 3-6 membered heterocycloalkyl, C5-C 10 The aryl group or 3-6 heteroaryl group, or a fused ring group or spiro ring group consisting of several aryl groups and / or heteroaryl groups and / or heterocyclic alkyl groups and / or cycloalkyl groups with a carbon number not exceeding 30, or a combination of the above groups.

[0014] L c Selected from substituted or unsubstituted straight-chain or branched C1-C5 alkylene groups, -(PEG) 1-5 - or any combination thereof, wherein one or more methylene (-CH2-) units in the alkylene group or one or more PEG units in the PEG chain may be replaced by one or more substituents selected from the following groups: -C(O)-, -OC(O)-, -NHC(O)-, -NH-, -O-, -S-, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6 alkylene, C6-C 10 aryl or C3-C 10 Heterocyclic group.

[0015] Among them, when La, L b When Lc has substituents, the substituents are selected from: hydroxyl, amino, halogen, C1-C6 alkyl, C6 ... 10 Aryl, C5-C 10 Any group selected from heteroaryl, C1-C5 alkoxy, C1-C5 alkylphenyl, nitro, -CONH2, -C(O)-, C1-C6 alkyl, -SO2NH2, or any combination of the above substituents.

[0016] Lg is selected from ligand groups targeting tissues or cells in the kidneys of mammals. Each Lg group may be the same or different, and each Lg is independently selected from the structure shown in Formula II: (II) Ar is selected from substituted or unsubstituted C6-C. 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Monocyclic heteroaryl or fused-ring heteroaryl, wherein the heteroaryl is preferably a nitrogen-containing aromatic heterocyclic group; when substitution is present, the substituent is selected from hydroxyl, amino, halogen or C1-C3 alkyl; Preferably, the fused-ring heteroaryl group is selected from substituted or unsubstituted benzocycloalkyl or benzoheteroaryl groups.

[0017] In some embodiments of this disclosure, Ar is selected from indolyl or pyridyl.

[0018] R is selected from hydroxyl, amino, halogen, straight-chain or branched C1-C6 alkyl (preferably straight-chain or branched C2-C4 alkyl), C3-C6 cycloalkyl (preferably cyclopropane), -CH2-(C3-C6 cycloalkyl), C6-C 10 aryl or heteroaryl, wherein the straight-chain or branched C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 10 The aryl or heteroaryl groups can be substituted or unsubstituted, and when substituted, the substituents are selected from hydroxyl, amino, halogen or C1-C3 alkyl groups; t1, t2, and t3 are each independently selected from integers 1 to 3.

[0019] In some embodiments of this disclosure, R is selected from -CH2-, cyclopropane, or -CH2-CH(CH3)2.

[0020] In some embodiments of this disclosure, the Lg is selected from any of the following groups: , , , or .

[0021] In some embodiments of this disclosure, at least one or all of the ligand groups Lg are connected to the end of the positive chain, such as the 3' end or the 5' end, via a linking group.

[0022] In some embodiments of this disclosure, the oligonucleotide conjugate or its pharmaceutically acceptable salt or its stereoisomer has the structure shown in Formula I-a: (Ⅰ-a) Where t is selected from 1 or 2.

[0023] i is an integer between 0 and 3.

[0024] Lb is selected from chemical bonds, amide bonds, .

[0025] Lc is selected from -L1-L2-, where L1 is selected from substituted or unsubstituted (CH2CH2O). n substituted or unsubstituted C1-C 20 Alkylene or substituted or unsubstituted Where n and j are selected from integers from 1 to 10; each L 1a Each is independently selected from C1-C5 alkylene groups; each L 1b Each is independently selected from -O-, -S-, -NH-, -NH-C(O)-, -C(O)-NH-, -C(O)-, -C(O)-O-, -OC(O)-, -NH-C(O)-O-, or -OC(O)-NH-; L 1c Selected from C1-C5 alkylene groups.

[0026] In some embodiments of this disclosure, L1 is selected from (CH2CH2O). n C1-C 10 Alkylene (preferably C1-C5 alkylene), where n is an integer selected from 1 to 3.

[0027] In some embodiments of this disclosure, L1 is selected from j is an integer selected from 1 to 6.

[0028] L2 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-.

[0029] More preferably, the -Lb-Lc- is selected from... k1 is selected from integers from 1 to 3, and k2 is selected from integers from 0 to 6.

[0030] The definitions of the remaining substituents are the same as those described above.

[0031] In some embodiments of this disclosure, the structure of formula I-a further has the following structure: (III) The definitions of Lc and Lg are the same as above.

[0032] Furthermore, in some embodiments of this disclosure, the oligonucleotide conjugate or its pharmaceutically acceptable salt or its stereoisomer has the following structure: .

[0033] In some embodiments of this disclosure, the oligonucleotide conjugate or its pharmaceutically acceptable salt or its stereoisomer has the structure shown in Formula I-b: (Ⅰ-b) t is an integer selected from 1 to 3.

[0034] x is selected from an integer from 1 to 10 (preferably 1 to 5).

[0035] y is selected from 0 or 1 (preferably 0).

[0036] Lb' is selected from chemical bonds, C1-C6 alkylene groups, or... .

[0037] Lc' is selected from -L3-L4-, where L3 is selected from substituted or unsubstituted (CH2CH2O). n substituted or unsubstituted C1-C 20 Alkylene or substituted or unsubstituted Where n and j' are selected from integers from 1 to 10; each L 3a Each is independently selected from C1-C5 alkylene groups; each L 3b Each is independently selected from -O-, -S-, -NH-, -NH-C(O)-, -C(O)-NH-, -C(O)-, -C(O)-O-, -OC(O)-, -NH-C(O)-O-, or -OC(O)-NH-; L 3c Selected from C1-C5 alkylene groups.

[0038] In some embodiments of this disclosure, L3 is selected from (CH2CH2O). n C1-C 15 Alkylene, where n is an integer selected from 1 to 5.

[0039] In some embodiments of this disclosure, L3 is selected from j' is an integer selected from 1 to 5.

[0040] L4 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-.

[0041] More preferably, the -Lb'-Lc'- is selected from... k3 is selected from integers from 1 to 3, and k4 is selected from integers from 0 to 6.

[0042] The definitions of the remaining substituents are the same as those described above.

[0043] In some embodiments of this disclosure, the oligonucleotide conjugate or its pharmaceutically acceptable salt or its stereoisomer has the following structure: Equation (a); Equation (b); Equation (c).

[0044] The Lg group is defined as described above.

[0045] Optionally, the Lg group in formula (a), formula (b) or formula (c) is selected from any of the following groups: or Further, optionally, the Lg group in formula (a), formula (b) or formula (c) is selected from: .

[0046] In a second aspect of this disclosure, a pharmaceutical composition is provided comprising the oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, a metabolite or a prodrug described in the first aspect of this disclosure.

[0047] In some alternative embodiments of this disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0048] In a third aspect of this disclosure, this disclosure provides the use of the oligonucleotide conjugate described in the first aspect of this disclosure or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, or the pharmaceutical composition described in the second aspect of this disclosure, in the preparation of a medicament for treating kidney-related diseases or conditions.

[0049] In some alternative embodiments of this disclosure, the disease is one associated with the expression of receptors on the surface of kidney cells.

[0050] In some alternative embodiments of this disclosure, the kidney-related disease or condition is chronic kidney disease; In some alternative embodiments of this disclosure, the kidney-related diseases or conditions are selected from: hypertension-related kidney damage, diabetic nephropathy, hyperuricemia, gout, hyperuricemic kidney damage, hepatitis B virus-related kidney damage, myeloma-related kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulonephropathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremia syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

[0051] In a fourth aspect of this disclosure, this disclosure provides a method for treating kidney-related diseases or conditions, the method comprising administering to a subject the oligonucleotide conjugate described in the first aspect of this disclosure or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a pharmaceutical composition described in the second aspect of this disclosure.

[0052] In some alternative embodiments of this disclosure, the kidney-related disease or condition is chronic kidney disease; In some alternative embodiments of this disclosure, the kidney-related diseases or conditions are selected from: hypertension-related kidney damage, diabetic nephropathy, hyperuricemia, gout, hyperuricemic kidney damage, hepatitis B virus-related kidney damage, myeloma-related kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulonephropathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremia syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

[0053] In a fifth aspect of this disclosure, this disclosure provides a method for reducing the expression or activity of a target gene, the method comprising contacting a cell with an oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof as described in the first aspect of this disclosure or a pharmaceutical composition as described in the second aspect of this disclosure; In some alternative embodiments of this disclosure, the cells are selected from kidney cells.

[0054] In some alternative embodiments of this disclosure, the target gene is selected from target genes related to chronic kidney disease (CKD) and renal fibrosis, target genes related to hereditary kidney diseases (polycystic kidney disease (PKD), renal diabetes, Alport syndrome, etc.), target genes related to abnormal renal ion transport and metabolism, or target genes specifically related to diabetic nephropathy (DKD).

[0055] In some alternative embodiments of this disclosure, the target gene is selected from the SLC5A1 gene or the SLC5A2 gene.

[0056] In a sixth aspect of this disclosure, this disclosure provides a cell or kit comprising the oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof as described in the first aspect of this disclosure, or a pharmaceutical composition as described in the second aspect of this disclosure.

[0057] In some alternative embodiments of this disclosure, the cells are selected from kidney cells or mammalian cells.

[0058] This disclosure provides an oligonucleotide conjugate that has an affinity for cellular receptors present on kidney target cells, and can selectively and effectively reduce or inhibit the expression of kidney target genes in a subject (e.g., human or animal). The oligonucleotide conjugate or pharmaceutical composition provided in this disclosure can effectively treat and / or prevent pathological conditions or diseases caused by the abnormal expression of specific genes in kidney tissue cells. Attached Figure Description

[0059] Figure 1 The relative expression levels of the target gene in mice after administration of the siRNA conjugate described in Example 1.

[0060] Figure 2 The relative expression levels of the target gene in mice after administration of the siRNA conjugate described in Example 2.

[0061] Figure 3 The relative expression levels of the target gene in mice after administration of the siRNA conjugate described in Example 2.

[0062] Figure 4 The relative expression levels of the target gene in mice after administration of the siRNA conjugate described in Example 3.

[0063] Figure 5 The relative expression levels of the target gene in mice after administration of the siRNA conjugate described in Example 4. Detailed Implementation

[0064] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0065] Terminology Explanation Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0066] In this disclosure, "alkyl" refers to a fully saturated (i.e., free of double or triple bonds) straight-chain or branched alkane chain. "C1-C6 alkyl" indicates an alkyl chain having 1 to 6 carbon atoms (wherever it appears in the text, numerical ranges such as "1 to 6" refer to each integer within a given range; for example, "1 to 6 carbon atoms" means that an alkyl chain can consist of 1, 2, 3, 4, 5, or 6 carbon atoms, but this definition also covers the term "alkyl" where no numerical range is specified). Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc. Alkyl groups can be substituted or unsubstituted.

[0067] In this disclosure, "alkoxy" refers to the formula -OR, where R is an alkyl group as defined above, such as "C1-C6 alkoxy", including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0068] In this disclosure, the term "chemical modification" or "modification" includes all alterations to nucleic acids by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0069] In this disclosure, "base" includes any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, hypoxanthine, and natural analogues.

[0070] In this disclosure, the structural formulas of "compound", "ligand" and "support" contain the bond " "" indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the bond " "can be " " ", or both contain " " "Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as: tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers."

[0071] In this disclosure, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans) isomers, blocked isomers, and so on.

[0072] In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0073] In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0074] In this 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 targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0075] In this disclosure, the terms "sequence" and "nucleotide sequence" refer to a sequence of nucleobases or nucleotides. As used herein, "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes purine bases adenine and guanine, and pyrimidine bases cytosine, thymine, and uracil. Nucleobases may be further modified. The synthesis of modified nucleobases (including phosphorous amide compounds of modified nucleobases) is known in the art.

[0076] In this disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be of the same length. As long as at least some base-pairing regions exist to form a double-stranded region, the oligonucleotide having a double-stranded structure is considered a double-stranded oligonucleotide as described in this disclosure. The nucleotides constituting the double-stranded oligonucleotide in this disclosure can be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modification does not specifically refer to the modified site. In addition to the modification of the nucleotides, the linking bonds between the nucleotides in the double-stranded oligonucleotide in this disclosure may also be modified. Double-stranded oligonucleotides containing modified linking bonds between nucleotides are also considered double-stranded oligonucleotides as described in this invention. Besides the nucleotide portion, the double-stranded oligonucleotide in this disclosure may also contain compounds or modifiers acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as linking ligands to form conjugates.

[0077] In this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The term "sense strand (or follower strand)" refers to an RNAi strand containing a sequence substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand being completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may be present within the internal or terminal regions of the molecule, wherein the most tolerant mismatches are present in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3' ends of the iRNA. It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide sequence substantially complementary to a continuous portion of the mRNA of interest.

[0078] In this disclosure, and unless otherwise stated, the term “complementary” when used to describe a first nucleobase or nucleotide sequence (e.g., the positive / antisense strand of an RNAi agent or a targeting mRNA) relative to a second nucleobase or nucleotide sequence means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) and form a double-stranded or double-helical structure under certain standard conditions.

[0079] In this disclosure, "complete complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, all (100%) bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases of the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence. "Partial complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, a majority, such as at least 70% (but not all), of the bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases of the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0080] In this disclosure, "substantially complementary" means that in the hybridized nucleobase or nucleotide sequence pair, the vast majority, such as at least 85% or up to three nucleotide differences (but not all), of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

[0081] In this disclosure, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used to refer to nucleobase or nucleotide matching between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of the target mRNA.

[0082] In this disclosure, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule that is formed during transcription of a target gene, including mRNA that is a product of RNA processing as a primary transcription product. The target gene may be intracellular, for example, in the cells of a subject.

[0083] In this disclosure, the terms "ligand" or "conjugation group" refer to an atom or group of atoms that binds to an oligonucleotide or other oligomer. Generally, a conjugation group modifies one or more properties of the compound to which it is linked, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. When referring to a link between two molecules, the term "link" as used herein means that the two molecules are directly or indirectly connected by a covalent bond, or that the two molecules are associated by a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0084] In this disclosure, the term "targeting ligand" refers to a polypeptide that has affinity for receptors present on the surface of kidney cells; the term "ligand unit" refers to a targeting ligand that links to a linker; and the term "ligand unit molecule" refers to a compound that is reactively linked to an oligonucleotide in molecular form.

[0085] In this disclosure, the terms “link” or “association” refer to the connection between two compounds or molecules, meaning that the two molecules are linked by a covalent bond or associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise stated, the terms “link” and “association” as used in this disclosure may refer to a link between a first compound and a second compound, with or without any inserted atoms or groups of atoms.

[0086] In this disclosure, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another second molecule or a second portion of a molecule. A linking group can contain any number of atoms or functional groups. In some embodiments, the linking group is used solely to link two bioactive molecules.

[0087] Unless otherwise stated, any symbol used in this disclosure refers to any group or groups that may be attached thereto, which is consistent with the scope of the invention as described in this disclosure.

[0088] According to common knowledge in the art, a peptide is a compound formed by the linkage of two or more amino acids via amide bonds. Here, individual amino acids are linked in a specific sequence to form a chain. An amino acid is a compound carrying at least one amino group and at least one carboxyl group. This includes naturally occurring amino acids (amino acids that form proteins in vivo), non-natural amino acids, or prepared amino acids that can be found in organisms.

[0089] In the peptides disclosed herein, unless otherwise specified, the amino acid units may be in the D- or L- form.

[0090] As used in this disclosure and as understood by those skilled in the art, a polyethylene glycol (PEG) unit refers to a repeating unit of the formula (CH2CH2O). It should be understood that in the chemical structures disclosed in this disclosure, a PEG unit can be described as (CH2CH2O), (OCH2CH2), or (CH2OCH2). It should also be understood that the number representing the number of repeating PEG units can be placed on either side of the brackets representing the PEG unit. It should be further understood that terminal PEG units can be capped by atoms (e.g., hydrogen atoms) or some other part.

[0091] In this disclosure, the terms "pharmaceutical composition" or "composition" can refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0092] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0093] In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0094] In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0095] In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0096] In this disclosure, the term "administration" generally refers to the introduction of a pharmaceutical preparation of this disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a period of time.

[0097] In this disclosure, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to limit this disclosure to any particular type of subject. In some embodiments of this disclosure, humans are preferred subjects, while in other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0098] As in this disclosure, the term "regulation of 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 such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" may mean "inhibition," but the use of the word "regulation" is not limited to this definition.

[0099] In addition to any conventional excipients, the use of any range of siRNAs incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0100] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to examples.

[0101] Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Beijing Xuanjingrui Biomedical Technology Co., Ltd.; all PCR primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and all experimental animals, C57BL / 6J mice, were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0102] Unless otherwise stated, the CPG vector (loading capacity 80 μmol / g) used in this disclosure was purchased from Beijing Coupling Technology Co., Ltd. The CPG vector is denoted as […]. ; This refers to a glass sphere with a controllable aperture (CPG).

[0103] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main reagents and consumables are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0104] Table 1 Main Reagents and Consumables Table 2 Main Instruments and Equipment Preparation Example 1: Synthesis of Compound NM064 In this preparation example, the synthetic route of compound NM064 is shown below: The specific synthesis steps of NM064 were prepared by referring to the relevant embodiments in the Chinese invention patent with publication number CN119019354A, which was first published by the applicant on November 26, 2024.

[0105] Preparation Example 2: Synthesis of LD100 The structure of compound LD100 is: N3-CH2CH2O-CH2CH2O-CH2CH2CO-KKEEE-KKEEE-KKEEE-K-NH 2， In this context, the letter K represents lysine; and the letter E represents glutamic acid. The Fmoc solid-phase peptide synthesis method was used to connect amino acid monomers one by one from the carboxyl terminus to the amino terminus according to the amino acid sequence (denoted as Fmoc-amino acid (side chain protecting group)-OH).

[0106] The specific preparation method of LD100 includes the following steps: (2-1) Take 2.0 g of Fmoc-Linker-MBHA Resin (degree of substitution approximately 0.5 mmol / g, total 1.0 mmol reaction sites) for peptide synthesis, swell with N,N-dimethylformamide (DMF) for 20 minutes; then add 3 times the resin volume of 20% Pip / DMF mixed solution (i.e., the volume ratio of piperidine to DMF is 1:4), purge with nitrogen for 30 minutes, dry under vacuum and wash (wash 5 times with 2 times the resin volume of DMF) to obtain H2N-Linker-MBHA Resin.

[0107] (2-2) Take 3.0 mmol of amino acid monomer, 6.0 mmol of N,N-diisopropylethylamine (DIPEA, CAS No. 7087-68-5), 2.85 mmol of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, CAS No. 94790-37-1), and an appropriate amount of DMF solvent. React for 30 minutes, dry under vacuum and wash (wash 3 times with 2 times the resin volume of DMF) to obtain Fmoc-amino acid (side chain protecting group)-Linker-MBHA Resin; then, add 3 times the resin volume of 20% Pip / DMF mixed solution, purge with nitrogen for 30 minutes, dry under vacuum and wash (wash 5 times with 2 times the resin volume of DMF) to obtain H2N-amino acid (side chain protecting group)-Liner-MBHA Resin; Afterward, step (2-2) needs to be repeated for each amino acid monomer: to obtain the amino acid sequence (side chain protecting group) - Linker - MBHA Resin.

[0108] (2-3) Take 3.0 mmol N3-PEG2-CH2CH2COOH (CAS No. 1312309-63-9), 6.0 mmol DIPEA, 2.85 mmol HBTU, and an appropriate amount of DMF solvent and react for 30 minutes. Dry the mixture and wash it (wash with methanol 3 times) to obtain N3-PEG2-CH2CH2CO-amino acid sequence (side chain protecting group)-Liner-MBHA Resin.

[0109] (2-4) Cutting: 6 times the volume of the resin was used to prepare the cutting solution (the cutting solution was prepared by mixing trifluoroacetic acid, anisole, 1,2-ethylenedithiol, phenol and water in a volume ratio of 87.5:5:2.5:2.5:2.5). The solution was shaken on a shaker for 2 hours, the resin was filtered off, the filtrate was precipitated with anhydrous diethyl ether, and the precipitate was washed 3 times with anhydrous diethyl ether. Finally, the precipitate was placed in a vacuum drying vessel and dried at room temperature for 24 hours. The precipitate was purified by HPLC to obtain compound LD100 with a purity of over 95%.

[0110] Preparation Example 3: Synthesis of LD154 (3-1) Synthesis of LD154-1 (3-1-1) Take 2.0 g of Wang Resin resin (degree of substitution approximately 0.5 mmol / g, total 1.0 mmol reaction sites) for peptide synthesis and swell it in dichloromethane (DCM) for 20 minutes; then, take 3.0 mmol of Fmoc-Thr(tBu)-OH, 6.0 mmol of N,N'-diisopropylcarbodiimide (DIC, CAS No. 693-13-0), and 1.5 mmol of 4-dimethylaminopyridine (DMAP, CAS No. 1122-58-3), and react them in an appropriate amount of DCM solvent for 60 minutes. Dry the mixture and wash it (wash three times with 2 times the resin volume of DCM, and then wash three times with 2 times the resin volume of DMF) to obtain Fmoc-Thr(tBu)-Wang Resin was then added; subsequently, 3 times the resin volume of a 20% Pip / DMF mixed solution (i.e., the volume ratio of piperidine to DMF was 1:4), nitrogen was purged for 30 minutes, the mixture was dried and washed (washed 5 times with 2 times the resin volume of DMF) to obtain H2N-Thre(tBu)-Wang Resin.

[0111] (3-1-2) Take 3.0 mmol of the Fmoc protected amino acid monomer corresponding to the target sequence, 9.0 mmol of DIC, and 9.0 mmol of 1-hydroxybenzotriazole (HOBt, CAS No. 2592-95-2), react with an appropriate amount of DMF solvent for 45 minutes, dry under vacuum and wash (wash 3 times with 2 times the resin volume of DMF) to obtain Fmoc-amino acid (side chain protecting group)-Lys(Boc)-WangResin; then, add 3 times the resin volume of 20% Pip / DMF mixed solution, purge with nitrogen for 30 minutes, dry under vacuum and wash (wash 5 times with 2 times the resin volume of DMF) to obtain H2N-amino acid (side chain protecting group)-Thre(tBu)-Wang Resin; repeat step (3-1-2) to sequentially couple the amino acid monomers. In this step, the amino acid linkage order is as follows: Fmoc-Dab(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-3Pal-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, resulting in the amino acid sequence (side chain protecting group) -Wang Resin.

[0112] (3-1-3) Take 3.0 mmol N3-PEG2 propionic acid (a carboxylic acid derivative with the corresponding structure), 9.0 mmol DIPEA, 9.0 mmol 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, CAS No. 148893-10-1), add an appropriate amount of DMF solvent and react for 30 minutes. Dry and wash (wash 3 times with 2 times the resin volume of DMF, and then wash 3 times with 2 times the resin volume of methanol) to obtain N3-PEG2-CH2CH2CO-amino acid sequence (side chain protecting group)-Wang Resin.

[0113] (3-1-4) Cutting: 6 times the volume of the cutting fluid (the cutting fluid is prepared by trifluoroacetic acid, phenol, water and triisopropylsilane in a volume ratio of 92.5:2.5:2.5:2.5), shake on a shaker for 0.5 hours, filter out the resin, pour the filtrate into ice-cold ether to precipitate, centrifuge and wash the precipitate 3 times with ice-cold ether, finally place the precipitate in a vacuum drying oven and dry at room temperature for 24 hours, and purify by HPLC to obtain the targeted delivery ligand LD154-1 with a purity of over 95%.

[0114] (3-2) Synthesis of LD154 Take NM154-1 (1.0 mmol) and DMF (150 mL), stir at room temperature for 30 min to completely dissolve; cool the system to 0℃, add EDCI (1.2 mmol), HOBt (1.2 mmol) and NMM (3.0 mmol) sequentially under nitrogen protection, and maintain the reaction at 0℃ with stirring for 15 h; after the reaction is completed, remove DMF by rotary evaporation under reduced pressure, add 10 mL of mixed deprotecting agent (TFA: phenol: H2O: triisopropylsilane = 92.5:2.5:2.5:2.5, volume ratio) and react for 1 h; slowly pour the reaction solution into ice-cold ether (500 mL) pre-cooled to 0-5℃, and a large amount of white solid precipitates; centrifuge at 8000 rpm for 15 min at 4℃, discard the supernatant, wash the solid with ice-cold ether (20 mL × 3), and vacuum dry to obtain crude product. Purification was performed by HPLC (mobile phase: 0.1% TFA aqueous solution - acetonitrile, gradient elution: acetonitrile volume fraction 10% → 90%, 30 min; flow rate: 5 mL / min; detection wavelength: 220 nm); the eluent of the target peak was collected and freeze-dried to obtain pure NM154 in 44.0% yield. The structure was confirmed by HPLC and ESI-MS.

[0115] LD155, LD156, and LD157 can be prepared using the methods described above, and their structures are as follows: The structure of LD162 is as follows: .

[0116] Example 4: Preparation of compound NM155 The synthetic route for compound NM155 is as follows: (4-1) Synthesis of compound NM155-3 Compound NM155-2 (1.7 g, 10.8 mmol, 1.0 eq) was dissolved in DMF (10 mL), and HOBt (2.64 g, 16.2 mmol, 1.5 eq), EDCI (3.72 g, 16.2 mmol, 1.5 eq), and DIEA (5.0 g, 32.4 mmol, 3.0 eq) were added. The mixture was stirred for 10 min, and then compound NM155-1 (5 g, 10.8 mmol, 1 eq) was added. The reaction was carried out for 2 hours. After the reaction was completed, the product was purified by reverse chromatography (ACN / H2O = 2:1) to give 3.7 g of compound 2. Yield: 60%. MS ESI ( m / z =592 [M + Na] + . (4-2) Synthesis of compound NM155 Compound NM155-3 (3.7 g, 1.0 eq) was dissolved in 20 mL of DCM, and DCI (600 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.95 g, 1.2 eq) were added. The reaction was carried out at room temperature for 2 hours. After reverse purification (ACN / H2O = 9:1), 3 g of compound NM155 as a white solid was obtained. Yield: 60%. MS ESI ( m / z = 792 [M + Na] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.67 (d, J = 7.8 Hz, 1H), 7.43 – 7.36 (m,2H), 7.34 – 7.18 (m, 7H), 6.93 – 6.83 (m, 4H), 3.85 – 3.61 (m, 10H), 3.51(dq, J = 16.9, 6.9 Hz, 3H), 2.89 – 2.67 (m, 5H), 2.16 (td,J = 7.0, 2.7 Hz, 2H), 2.05 (t, J = 7.3 Hz, 2H), 1.71 – 1.37 (m, 8H), 1.09 (dd, J = 35.4, 6.7 Hz, 14H). Preparation Example 5: Synthesis of siRNA conjugates In this preparation example, the method for preparing the siRNA conjugate includes the following steps: (5-1) Synthesis of the justice chain SS and the antisense chain AS Starting with a carrier compound (such as a CPG carrier or a PS carrier), nucleoside monomers are linked sequentially along the 3'-5' direction using a phosphoramide solid-phase nucleic acid synthesis method. During the synthesis, compounds NM064 and NM155 are considered as a single nucleotide monomer.

[0117] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below: The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0118] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloroacetic acid solution in dichloromethane (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0119] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0120] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio mixture of Cap1 and Cap2, where Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0121] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0122] After the last nucleoside monomer is ligated, the nucleic acid sequence ligated on the solid-phase support is sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the sense or antisense strand, wherein: The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to remove the 2'-O-TBDMS protection from the ribose.

[0123] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); eluent 2 was 1.5 M sodium chloride and 20 mM sodium phosphate (pH=8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0124] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the sense and antisense strands of siRNA were obtained.

[0125] Compounds NM064 and NM155 can be linked to any position on the sense chain (e.g., the 3' end, 5' end, or any site between the 3' and 5' ends) or any position on the antisense chain (e.g., the 3' end, 5' end, or any site between the 3' and 5' ends) via a phosphodiester bond. In some specific embodiments, these compounds are linked to the 3' end of the sense chain via a phosphodiester bond.

[0126] When a compound NM064 is attached to the 3' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is shown below: in, Represents the Chain of Justice (SS).

[0127] Alternatively, as an example, when another compound NM155 is attached to the 3' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is shown below: in, Represents the Chain of Justice (SS).

[0128] (5-2) Synthesis of SS conjugates The synthesis of SS conjugates involves attaching a targeted delivery ligand to the sense chain via an azide-yne ​​cycloaddition reaction. The specific steps of the synthesis include: (5-2-1) Take 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) and mix them to obtain a mixed solvent; then, dissolve the positive chain from step (5-1) in the mixed solvent to obtain a positive chain solution with a concentration of 1.0 eq.

[0129] (5-2-2) The targeting ligand compound (6.0 eq) was dissolved in DMF (70 μL) to obtain a solution of the targeting ligand compound.

[0130] (5-2-3) Mix the positive chain solution from step (5-2-1) with the target ligand compound solution from step (3-2) or (6-2) to obtain a reactant mixture.

[0131] (5-2-4) After mixing 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O, the mixture was shaken at 40℃ for 5 min. Then, 37 μL of the mixture was added to the reaction mixture in step (5-2-3) above and vortexed to obtain an intermediate product mixture. The pH of the intermediate product mixture was measured to be 8.

[0132] (5-2-5) Take 25.0 eq sodium ascorbate and quickly add it to the intermediate product mixture of step (5-2-4), and vortex it. React at 40℃ for 1 h to obtain the product mixture.

[0133] (5-2-6) Purification: Take 3 μL of the product mixture obtained in step S3, dilute it with a mixed solution of DMF and H2O (DMF to H2O volume ratio of 1:5), and then separate and purify it using HPLC. The HPLC process uses a C18 column, an ammonium bicarbonate buffer solution as the mobile phase, and a gradient elution method. The purified product from HPLC is then lyophilized.

[0134] (5-3) Annealing of siRNA The AS from step (5-1) and the SS conjugate from step (5-2) are mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture is then slowly cooled to room temperature and kept at room temperature for 10 minutes, allowing the SS conjugate and AS to form a double-stranded siRNA conjugate through hydrogen bonds.

[0135] For example, when the ligand compound is LD100, the siRNA conjugate structure of the conjugation vector (NM064+LD100*2) is as follows: Specifically, the azide groups in the two LD100s and the two alkynyl groups in the siRNA terminal linker structure (NM064) are converted into triazole groups through click chemistry to achieve covalent linkage between the two ligand compounds LD100 and siRNA.

[0136] Correspondingly, the other supports also adopt the above representation of the corresponding ligand compounds; for example, (NM064+LD156*2) represents a support that covalently links two LD156 ligand compounds through a Linker structure (NM064).

[0137] Alternatively, as an example, when another compound NM155+ targeting ligand is attached to the 3' end of the sense chain via a phosphodiester bond, the sense chain comprises the following structural formula: Where * indicates a covalently linked site with the ligand. Represents the Chain of Justice (SS).

[0138] Preparation Example 6: Synthesis of LD162 The structural formula of compound LD162 is: N3-PEG2-CH2CH2CO-Cyclo(Dab-Lys-DTrp-Leu-Lys-Lys-Thr) (6-1) Synthesis of Compound 1 First, Fmoc-Thr(tBu)-OH was coupled to CTC resin under DIEA (4 eq) and DMF conditions. Subsequently, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-DTrp-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Dab(Dde)-OH were coupled sequentially under DIC (3 eq), HOBt (3 eq), and DMF conditions. Then, N3-PEG2 propionic acid was coupled to the above products under DIEA (3 eq), HATU (3 eq), and DMF conditions. During the coupling process, the Fmoc protecting groups of all amino acids were cleaved using a 20% Pip / DMF solution. After the above steps were completed, the Dde protecting group of the Dab(Dde) side chain was removed using a 5% N2H4·H2O / DMF solution. Finally, the peptide was lysed from CTC Resin for 1 h at room temperature using HFIP / DCM (1:4 v / v) solution. The lysate was then filtered, and the filtrate was poured into ice-cold ether, where the peptide precipitated. After centrifugation, crude peptide was obtained, which was then purified by reversed-phase HPLC (0.1% TFA-acetonitrile) on a C18 preparative column to obtain pure peptide (yield: 30.5%). The peptide was finally validated by HPLC and MS.

[0139] (6-2) Synthesis of LD162 Compound 1 (1 eq) was dissolved in 150 mL of DMF, and the temperature was then adjusted to 0 °C. EDCI (1 eq), HOBt (3 eq), and NMM (3 eq) were added, and the reaction was carried out overnight at 0 °C. After the reaction was complete, the DMF was evaporated, and 6 mL of a TFA / phenol / H2O / triisopropylsilane (92.5% / 2.5% / 2.5% / 2.5%) solution was added and reacted for 1 h. Then, 500 mL of icy diethyl ether was added, and a large amount of solid precipitated. After centrifugation and drying, LD162 was prepared by reverse phase reaction (yield: 35.6%).

[0140] According to the above method, the present disclosure obtains the siRNA conjugates shown in Table 4, wherein the unmodified nucleotide sequence information forming the siRNA conjugates is shown in Table 3.

[0141] Table 3 Sequence information of unmodified siRNA Table 4 Sequence information of siRNA conjugates Unless otherwise stated, the base composition and modifications in this disclosure have the following meanings: Uppercase letters A, U, G, C, and T represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to its left is 2'-O-methyl modified; lowercase letter f indicates that the nucleotide represented by the uppercase letter to its left is 2'-fluoro modified; (moe) indicates that the nucleotide represented by the uppercase letter to its left is 2'-O-methoxyethyl modified; lowercase letter s indicates that the two nucleotides represented by the two adjacent letters to its left and right are linked by a phosphate dithioester bond. VP indicates that the 5' end of the antisense strand in siRNA is modified with 5'-(E)-vinylphosphonate (5'-(E)-VP). The contents of parentheses “()” in the above-mentioned sense strand represent the vector portion; the number outside the parentheses indicates the number of vector clusters, for example, “( ) ×2” indicates that the number of vector clusters is 2; the number inside the parentheses indicates the number of ligand groups, for example, *2 indicates that the linker group connects 2 ligand compounds.

[0142] For example, (NM155+LD162)×2 represents the following structure: .

[0143] The structural formula of VPUm is: or .

[0144] The structural formula of the nucleotide modified with 2'-O-methyl is: .

[0145] The structural formula of the 2'-fluorinated nucleotide is: .

[0146] The structural formula of the nucleotide modified with 2'-O-methoxyethyl is: .

[0147] Where Base represents the nucleobases A, U, G, C, and T.

[0148] Biological testing experiments Methods for assessing the inhibitory activity of siRNA conjugates on target genes in mice Six- to eight-week-old C57BL / 6J mice (all female) were randomly divided into groups based on body weight. The dosage for each group was calculated based on body weight and administered via subcutaneous abdominal injection for three consecutive days. Each siRNA conjugate was prepared into a solution of the appropriate concentration (based on siRNA) using PBS for administration, at a volume of 5 mL (based on siRNA) / kg (based on mice). The PBS control group received 5 mL / kg (based on mice) of PBS solution (without the drug conjugate). The day of administration was designated as day 0 (D0). At a predetermined time after administration, five mice from each group were sacrificed. The sacrificed mice were grossly dissected, and renal cortical tissue was collected from each mouse. The tissue was cut into pieces approximately 2 mm thick. 3 Small pieces, stored using RNA Later.

[0149] Tissue samples from different experimental groups were taken from the RNA later sample. 1 mL of Trizol solution was added, and the tissue samples were homogenized for 120 s in a Tissuelyser II automated tissue homogenizer. After brief centrifugation and standing at room temperature for 10 min, 200 μL of chloroform was added, and the mixture was vortexed and incubated at room temperature for 3 min. The mixture was then centrifuged at 4 ℃, 12000 rpm for 10 min. 400 μL of the supernatant was transferred to a centrifuge tube containing 400 μL of isopropanol, mixed, and incubated at room temperature for 10 min. The supernatant was discarded. 1 mL of 75% ethanol was added, and the centrifuge tube was inverted to wash the precipitate. The mixture was then centrifuged at 4 ℃, 12000 rpm for 5 min to remove the supernatant, and the samples were air-dried at room temperature to extract total RNA.

[0150] mRNA expression level detection: Take 1 μg of total RNA and use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) with Oligo (dT)18 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the kit's instructions and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution. Then, use a real-time quantitative PCR kit (ABI, SYBR™ Select Master Mix, Catalog number: 4472908) to detect the expression level of the target gene mRNA in kidney tissue. In this real-time quantitative PCR method, primers targeting the target gene and primers targeting the internal reference gene were used to detect the target gene and the internal reference gene, respectively. Prepare a 20 μL Real-time PCR reaction system for each PCR well according to the instructions of the Real-time PCR kit. Each reaction system contains 5 μL of cDNA solution obtained from the reverse transcription reaction, 10 μL of SYBR™ Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-Free H2O. Place the prepared reaction system on a Real-time PCR instrument (ABI, StepOnePlus™) and perform Real-time PCR amplification using a three-step method. The amplification program is: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. Repeat the denaturation, annealing, and extension process for 40 cycles. In this Real-time PCR method, the ΔΔCt method is used to calculate the relative quantitative expression level and inhibition rate of the target gene mRNA in each test group. The calculation method is as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) – ΔCt(control group average) ΔCt(control group) = ΔCt(control group) – ΔCt(control group average) In cell experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from several replicates in the control group. In animal experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from each of the five mice sacrificed at the same time point in the control group. Therefore, each sample in both the test and control groups corresponds to a ΔCt value.

[0151] The relative expression level of the target gene mRNA in the test group = 2 -ΔΔCt( (Test group) × 100% Using the control group as a baseline, the expression level of the target gene mRNA in the test group was normalized, and the expression level of the target gene mRNA in the control group was defined as 100%.

[0152] The inhibition rate of target gene mRNA expression in the test group (%) = 1 – the relative expression level of target gene mRNA in the test group Unless otherwise stated, all in vivo activity data are expressed as X±STDEV, and all data were plotted and analyzed using GraphPadprism 8.0 software.

[0153] Example 1 The LD154-157 conjugate sequence inhibits the activity of the target gene superoxide dismutase 1 protein in mice. Evaluation of the inhibitory activity of (Superoxide dismutase 1, SOD1) In this embodiment, the inhibitory activity of the LD154-LD157 conjugated siRNA sequence R699310-R699313 and the LD100 conjugated control sequence R699256 on the target gene SOD1 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0154] Six- to eight-week-old C57BL / 6j mice were randomly divided into six groups of five mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. Mice in the PBS control group received 5 mL / kg of siRNA, while mice in the siRNA conjugate experimental group received 3 mg / kg (based on siRNA) of siRNA. Administration was repeated for three consecutive days at a dose of 5 mL / kg. Day 0 (D0) was designated as the day of administration. On day 14 (D16) after the last administration, five mice from each group were sacrificed. The animals were then grossly dissected, and the renal cortex was collected and cut into several 2 mm segments. 3 Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method.

[0155] Table 5 Primer sequence listing Table 6. Repressive activity of target genes in mice after administration of siRNA conjugates. The results of Example 1 showed that the LD154-157 conjugated sequences R699310-R699313 and the LD100 conjugated control sequences both exhibited comparable or even superior inhibitory activity, with R699312 showing the best inhibitory effect on renal cortical target genes, achieving an inhibition rate of up to 60%. Figure 1 (Table 6) Example 2 The LD162 conjugate sequence inhibits the activity of the target gene superoxide dismutase 1 protein in mice. Evaluation of inhibitory activity of dismutase 1 (SOD1) In this embodiment, the inhibitory activity of the LD162 vector conjugate R699330 on the target gene SOD1 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0156] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of ten mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. The PBS control group received 5 mL / kg of siRNA per mouse, while the siRNA conjugate experimental group received 9 mg / kg (based on siRNA) per mouse, administered as a single dose of 5 mL / kg. Day 0 (D0) was designated as the day of administration. Five mice from each group were sacrificed on days 14 (D14) and 28 (D28) after the last administration. The animals were grossly dissected, and the renal cortex and medulla were collected, cut into several 2 mm³ pieces, and preserved using RNAlater. RNA extraction and Real-time PCR were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primer sequences are shown in Table 5.

[0157] Table 7. Inhibitory activity of target genes in the renal cortex of mice after administration of siRNA conjugates. Table 8. Inhibitory activity of target genes in the renal medulla of mice after administration of siRNA conjugates. The results of Example 1 show that the siRNA sequence R6999330 conjugated to the LD162 vector, compared to R6999312 conjugated to the LD156 vector, exhibits strong inhibitory activity in the renal cortex on D14. Furthermore, R6999330 maintains a similar gene-inhibiting effect to D14 on D28, demonstrating a longer duration of action. Figure 2-3 (Table 7-8) Example 3. Evaluation of the inhibitory activity of siRNA conjugates on the target gene aldehyde dehydrogenase 2 family member (ALDH2) in mice. In this embodiment, the inhibitory activity of conjugates R381094 and R381095 of the LD162 vector with different cluster numbers on the target gene ALDH2 in mice was evaluated using the in vivo target gene inhibitory activity assessment method.

[0158] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of five mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. The PBS control group received 5 mL / kg of siRNA per mouse, while the siRNA conjugate experimental group received 9 mg / kg (based on siRNA) per mouse, administered as a single dose of 5 mL / kg. Day 0 (D0) was designated as the day of administration. On day 14 (D14) after the last administration, five mice from each group were sacrificed. The animals were grossly dissected, and the renal cortex was collected and cut into several 2 mm segments. 3 Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method.

[0159] Table 9 Primer sequence listing The results of Example 3 showed that the siRNA sequence R381094 conjugated with two LD162 vectors and the siRNA sequence R381095 conjugated with three LD162 vectors both exhibited high inhibitory activity on the renal cortex at D14. Figure 4 (Table 10).

[0160] Table 10. Inhibitory activity of the target genes in the renal cortex of mice after administration of the siRNA conjugate described in this example. Example 4. Evaluation of the inhibitory activity of LD156-conjugated siRNAs with different cluster numbers on the target gene ALDH2 in mice. In this embodiment, the inhibitory activity of conjugates R381043 and R381044 of the LD156 vector with different cluster numbers on the target gene ALDH2 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0161] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of five mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. The PBS control group received 5 mL / kg of siRNA per mouse, while the siRNA conjugate experimental group received 9 mg / kg (based on siRNA) per mouse, administered as a single dose of 5 mL / kg. Day 0 (D0) was designated as the day of administration. On day 14 (D14) after the last administration, five mice from each group were sacrificed. The animals were grossly dissected, and the renal cortex was collected and cut into several 2 mm segments.3 Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method.

[0162] The results of Example 4 showed that the siRNA sequence R381043 conjugated with two LD156 vectors and the siRNA sequence R381044 conjugated with three LD156 vectors both exhibited high inhibitory activity on the renal cortex at D14. Figure 5 (Table 11).

[0163] Table 11. Inhibitory activity of the target genes in the renal cortex of mice after administration of the siRNA conjugate described in this example. The above specific embodiments are merely illustrative of the present invention and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. An oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, said conjugate having the structure shown in Formula I: （Ⅰ） Nu refers to single-chain or double-chain oligonucleotide molecules that have preventive or therapeutic effects on kidney-related diseases or symptoms; t is an integer selected from 1 to 3; Each Z is independently either OH or SH; R1 is selected from H, hydroxyl, amino, halogen, C1-6 alkyl or C1-6 alkoxy; A is selected from or ; p and q are each independently selected from 0, 1 or 2; m is selected from 1, 2, or 3; L a Selected from substituted or unsubstituted straight or branched C1-C 15 An alkylene group, wherein one or more methylene units (-CH2-) in the alkylene group are optionally replaced by one or more substituents selected from the group consisting of: -C(O), -OC(O)-, -NHC(O)-, -NH-, -O-, -S-, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6 alkylene, C6-C 10 aryl or C3-C 10 Heterocyclic groups; L b Selected from chemical bonds, C1-C3 alkyl, amide bonds, -O-, -S-, substituted or unsubstituted 3-8 membered cycloalkyl, 3-6 membered heterocycloalkyl, C5-C 10 The aryl or 3-6 heteroaryl group, or a fused ring group or spiro ring group with no more than 30 carbon atoms composed of several aryl and / or heteroaryl and / or heterocycloalkyl and / or cycloalkyl groups, or a combination of the above groups; L c Selected from substituted or unsubstituted straight-chain or branched C1-C5 alkylene groups, -(PEG) 1-5 - or any combination thereof, wherein one or more methylene (-CH2-) units in the alkylene group or one or more PEG units in the PEG chain may be replaced by one or more substituents selected from the following groups: -C(O)-, -OC(O)-, -NHC(O)-, -NH-, -O-, -S-, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6 alkylene, C6-C 10 aryl or C3-C 10 Heterocyclic groups; in, When L a L b or L c When substituents are present, the substituents are selected from: hydroxyl, amino, halogen, C1-C6 alkyl, C6 ... 10 Aryl, C5-C 10 Any group selected from heteroaryl, C1-C5 alkoxy, C1-C5 alkylphenyl, nitro, -CONH2, -C(O)C1-C6 alkyl, -SO2NH2, or any combination of the above substituents; Each Lg group can be the same or different, and each Lg is independently selected from the structure shown in Formula II: （Ⅱ） Ar is selected from substituted or unsubstituted C6-C. 10 Cycloalkyl, substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted C5-C 10 Monocyclic heteroaryl or fused-ring heteroaryl, wherein the heteroaryl is preferably a nitrogen-containing aromatic heterocyclic group; when substitution is present, the substituent is selected from hydroxyl, amino, halogen or C1-C3 alkyl; R is selected from hydroxyl, amino, halogen, straight-chain or branched C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 10 aryl or heteroaryl, wherein the straight-chain or branched C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 10 The aryl or heteroaryl groups can be substituted or unsubstituted, and when substituted, the substituents are selected from hydroxyl, amino, halogen or C1-C3 alkyl groups; t1, t2, and t3 are each independently selected from integers 1 to 3.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the conjugate has the structure shown in formula Ia: (I-a) in, t is selected from 1 or 2; i is an integer between 0 and 3; Lb is selected from chemical bonds, amide bonds, or... ; Lc is selected from -L1-L2-, where L1 is selected from substituted or unsubstituted (CH2CH2O). n substituted or unsubstituted C1-C 20 Alkylene or substituted or unsubstituted Where n and j are selected from integers from 1 to 10; each L 1a Each is independently selected from C1-C5 alkylene groups; each L 1b Each is independently selected from -O-, -S-, -NH-, -NH-C(O)-, -C(O)-NH-, -C(O)-, -C(O)-O-, -OC(O)-, -NH-C(O)-O-, or -OC(O)-NH-; L 1c Selected from C1-C5 alkylene groups; Optionally, L1 is selected from (CH2CH2O). n C1-C 10 Alkylene (preferably C1-C5 alkylene) or Where j is an integer from 1 to 6, and n is an integer from 1 to 3; L2 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-; Preferably, -Lb-Lc- is selected from k1 is selected from integers from 1 to 3, and k2 is selected from integers from 0 to 6; Preferably, the structure of formula I-a further has the following structure: （Ⅲ） The remaining substituents are defined as in claim 1.

3. The conjugate as claimed in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the conjugate has the structure shown in formula Ib: (I-b) t is an integer selected from 1 to 3; x is an integer selected from 1 to 10; y is selected from 0 or 1; Lb' is selected from chemical bonds, C1-C6 alkylene groups, or... ; Lc' is selected from -L3-L4-; in, L3 is selected from substituted or unsubstituted (CH2CH2O). n substituted or unsubstituted C1-C 20 Alkylene or substituted or unsubstituted Where n and j' are selected from integers from 1 to 10; each L 3a Each is independently selected from C1-C5 alkylene groups; each L 3b Each is independently selected from -O-, -S-, -NH-, -NH-C(O)-, -C(O)-NH-, -C(O)-, -C(O)-O-, -OC(O)-, -NH-C(O)-O-, or -OC(O)-NH-; L 3c Selected from C1-C5 alkylene groups; Optionally, selected from (CH2CH2O). n C1-C 15 alkylene or , where n and j' are selected from integers from 1 to 5; L4 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-; Preferably, -Lb'-Lc'- is selected from k3 is selected from integers 1-3, and k4 is selected from integers 0-6; The remaining substituents are defined as in claim 1.

4. The conjugate or its pharmaceutically acceptable salt or its stereoisomer as described in any one of claims 1-3, wherein the Lg is selected from any of the following groups: ， ， , or 。 5. The conjugate or its pharmaceutically acceptable salt or its stereoisomer as described in any one of claims 1-2 or 4, having any of the following structures: 。 6. The conjugate or its pharmaceutically acceptable salt or its stereoisomer as described in any one of claims 1 or 3-4, having any of the following structures: Equation (a); Equation (b); Equation (c); Nu and Lg are defined as in the foregoing claims; Optionally, the Lg group in formula (a), formula (b) or formula (c) is selected from any of the following groups: or Further, optionally, the Lg group in formula (a), formula (b) or formula (c) is selected from: 。 7. The conjugate or its pharmaceutically acceptable salt or its stereoisomer as described in any one of claims 1-6, wherein Nu is an siRNA molecule having a preventive or therapeutic effect on kidney-related diseases or symptoms, comprising a sense strand or antisense strand composed of 15-25 modified or unmodified nucleotides; wherein the sense strand and antisense strand form a complementary or substantially complementary double-stranded region; in, The antisense strand sequence is at least partially complementary to the mRNA of the target gene in the target cells of the kidney tissue, and the surface of the target cells has a receptor capable of binding to the ligand group Lg. Optionally, at least one or all of the ligand groups Lg are connected to the end of the positive chain via a linker group.

8. A pharmaceutical composition comprising the oligonucleotide conjugate of any one of claims 1-7 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, a metabolite or a prodrug, and a pharmaceutically acceptable excipient.

9. Use of the oligonucleotide conjugate of any one of claims 1-7 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, or the pharmaceutical composition of claim 8, in the preparation of a medicament for treating kidney-related diseases or conditions; Optionally, the kidney-related disease or condition is chronic kidney disease; Optionally, the kidney-related diseases or conditions are selected from: hypertension-related kidney damage, diabetic nephropathy, hyperuricemia, gout, hyperuricemic kidney damage, hepatitis B virus-related kidney damage, myeloma-related nephropathy, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulonephropathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremia syndrome, or systemic lupus erythematosus (SLE)-related kidney disease.

10. A cell or kit comprising an oligonucleotide conjugate of any one of claims 1-7 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, or a pharmaceutical composition of claim 8.

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