Extrahepatic delivery ligand and conjugate thereof

Abstract

The present invention belongs to the technical field of oligonucleotide drugs, and specifically relates to a ligand compound having the ability to improve delivery capacity of an oligonucleotide to an extrahepatic tissue such as the nervous system or kidney, and a conjugate formed by linking the ligand compound to double-stranded oligonucleotides. Provided in the present invention are an extrahepatic delivery ligand and a conjugate thereof. The ligand compound provided in the present invention has the function of improving the delivery of the oligonucleotide to the extrahepatic tissue, and can effectively deliver an siRNA drug to a target tissue, thereby facilitating the alleviation, prevention and / or treatment of a target gene-mediated disease or disorder.

Description

Extrahcpatic delivery ligands and conjugates thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of oligonucleotide drugs, and specifically relates to a ligand compound having the ability to improve the delivery of oligonucleotides to extrahcpatic tissues such as the nervous system or the kidney, and a conjugate formed by connecting the ligand compound to a double-stranded oligonucleotide. BACKGROUND

[0002] Small nucleic acid drugs have been proven to have wide application potential in various disease fields. However, small nucleic acid drugs also face a series of technical and application challenges, for example, how to effectively deliver siRNA drugs to target tissues, especially extrahcpatic tissues (such as the kidney or central nervous system). Therefore, there is still a need to develop more suitable delivery systems to more effectively deliver siRNA to extrahcpatic tissue cells. SUMMARY

[0003] In view of this, the present application provides an extrahcpatic delivery ligand and a conjugate thereof.

[0004] In a first aspect of the present disclosure, the present disclosure provides a ligand compound, characterized in that the ligand compound is selected from or comprises a structure represented by formula (I), or a tautomer thereof, or a stereoisomer thereof:

[0005] wherein A is selected from a 5-8 membered oxygen-containing heterocycle;

[0006] In some optional embodiments of the present disclosure, A is selected from a 5-8 membered saturated oxygen-containing heterocycle;

[0007] In some optional embodiments of the present disclosure, A is selected from a six-membered saturated heterocycle containing one oxygen atom;

[0008] In some optional embodiments of the present disclosure, A is selected from

[0009] In some optional embodiments of the present disclosure, the compound is selected from or comprises a structure represented by formula (II), or a tautomer thereof, or a stereoisomer thereof:

[0010] p and q are each independently selected from 0, 1, 2, 3 or 4;

[0011] R1is selected from H, a hydroxyl protecting group or wherein * represents a linking site for linking a pharmaceutically active molecule; Z is selected from a hydroxyl group or a thiol group;

[0012] R2is selected from H, a reactive phosphorus group or wherein R 2bR is selected from a solid support comprising an amino functional group 2a R is selected from a covalent linker or a chemical bond to said amino functional group;

[0013] L1is selected from

[0014] R3is selected from H, hydroxyl, amino, halogen, C1-C6alkyl, or C1-C6alkoxy;

[0015] L2is selected from substituted or unsubstituted C1-C 20 alkylene, substituted or unsubstituted C2-C 20 alkenylene, substituted or unsubstituted C2-C 20 alkynylene, or substituted or unsubstituted wherein j is selected from an integer from 1 to 10; each L 2a is independently selected from C1-C5alkylene; each L 2b is independently selected from -O-, -S-, -NH-, -NH-C(O)-, -C(O)-NH-, -C(O)-, -C(O)-O-, -O-C(O)-, -NH-C(O)-O-, or -O-C(O)-NH-; or L 2a -L 2b consisting of (-CH2-CH2-O-) n units, n is an integer from 0 to 20 (preferably 1 to 10); L 2c is selected from C1-C5alkylene.

[0016] L3is selected from -O-, -NH-, -NHC(O)-, -(NH)C(O)O-, -(NH)O-, -C(O)-, -C(O)NH-, or -C(O)O-.

[0017] R4is selected from a targeting ligand group capable of binding to a target cell surface receptor;

[0018] The target cell is selected from a kidney cell, a central nervous system cell, a skeletal muscle cell, an ocular cell, a cardiac muscle cell, an adipocyte, or a lung cell.

[0019] In a second aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate comprising a sense strand and an antisense strand, the sense strand and / or the antisense strand conjugated to at least one targeted delivery group comprising a structure represented by Formula (i), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0020] A, p, q, n, R3, R4, L1, L2, L3are as defined in the first aspect of the present disclosure.

[0021] In some alternative embodiments of the present disclosure, the sense strand is conjugated to at least one targeted delivery moiety.

[0022] In some alternative embodiments of the present disclosure, each of the targeted delivery moieties is independently located at the 5' end or the 3' end of the sense strand.

[0023] In some alternative embodiments of the present disclosure, the sense strand is conjugated to one targeted delivery moiety, and the targeted delivery moiety is located at the 5' end or the 3' end of the sense strand.

[0024] In some alternative embodiments of the present disclosure, the sense strand is conjugated to two targeted delivery moieties, and the two targeted delivery moieties are linked by a phosphodiester bond or a phosphorothioate bond, and the linked two targeted delivery moieties are located at the 5' end or the 3' end of the sense strand.

[0025] Alternatively, the sense strand is conjugated to two targeted delivery moieties, and the two targeted delivery moieties are located at the 5' end and the 3' end of the sense strand, respectively.

[0026] In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide conjugate is selected from siRNA conjugates.

[0027] In a third aspect of the present disclosure, the present disclosure provides a composition, characterized in that the composition comprises the double-stranded oligonucleotide conjugate according to the second aspect of the present disclosure.

[0028] In a fourth aspect of the present disclosure, the present disclosure provides the use of any of the following in the preparation of a medicament for treating and / or preventing a disease or a symptom associated with the mRNA level disorder of a target gene expression:

[0029] (1) the ligand compound according to the first aspect of the present disclosure; and / or

[0030] (2) the double-stranded oligonucleotide conjugate according to the second aspect of the present disclosure; and / or

[0031] (3) the composition according to the third aspect of the present disclosure.

[0032] In a fifth aspect of the present disclosure, the present disclosure provides the use of any of the following in the preparation of a medicament for reducing the expression or activity of a target gene:

[0033] (1) the ligand compound according to the first aspect of the present disclosure; and / or

[0034] (2) the double-stranded oligonucleotide conjugate according to the second aspect of the present disclosure; and / or

[0035] (3) the composition according to the third aspect of the present disclosure.

[0036] In a sixth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any one of the following, and optionally a pharmaceutically acceptable carrier or excipient:

[0037] (1) the ligand compound of the first aspect of the present disclosure; and / or

[0038] (2) the double-stranded oligonucleotide conjugate of the second aspect of the present disclosure; and / or

[0039] (3) the composition of the third aspect of the present disclosure.

[0040] In a seventh aspect of the present disclosure, the present disclosure provides a method of reducing expression or activity of a target gene, characterized in that the method comprises contacting a cell with any one of the following:

[0041] (1) the ligand compound of the first aspect of the present disclosure; and / or

[0042] (2) the double-stranded oligonucleotide conjugate of the second aspect of the present disclosure; and / or

[0043] (3) the composition of the third aspect of the present disclosure; and / or

[0044] (4) the pharmaceutical composition of the sixth aspect of the present disclosure.

[0045] In some optional embodiments of the present disclosure, the cell includes, but is not limited to, a kidney cell, a central nervous system cell, a skeletal muscle cell, an ocular cell, a cardiac muscle cell, an adipocyte, and a lung cell.

[0046] In an eighth aspect of the present disclosure, the present disclosure provides a method of treating and / or preventing a disease or condition associated with dysregulation of mRNA levels of a target gene, characterized in that any one of the following is administered to a subject:

[0047] (1) the ligand compound of the first aspect of the present disclosure; and / or

[0048] (2) the double-stranded oligonucleotide conjugate of the second aspect of the present disclosure; and / or

[0049] (3) the composition of the third aspect of the present disclosure; and / or

[0050] (4) the pharmaceutical composition of the sixth aspect of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 shows the relative residual expression levels of a target gene of interest in the brain of a mouse after administration of the siRNA conjugate.

[0052] Figure 2 shows the relative residual expression levels of a target gene of interest in the lumbar region of a rat after administration of the siRNA conjugate.

[0053] Figure 3 Relative remaining expression levels of target genes in thoracic segments of rats after administration of the siRNA conjugate.

[0054] Figure 4 Relative remaining expression levels of target genes in cervical segments of rats after administration of the siRNA conjugate.

[0055] Figure 5 Relative remaining expression levels of target genes in cerebellum of rats after administration of the siRNA conjugate.

[0056] Figure 6 Relative remaining expression levels of target genes in hippocampus of rats after administration of the siRNA conjugate.

[0057] Figure 7 Relative remaining expression levels of target genes in cortex of rats after administration of the siRNA conjugate.

[0058] Figure 8 Relative remaining expression levels of target genes in liver and kidney of rats after administration of the siRNA conjugate.

[0059] Figure 9 Relative remaining expression levels of target genes in kidney cortex of mice after administration of the siRNA conjugate.

[0060] Figure 10 Relative remaining expression levels of target genes in kidney of mice after administration of the siRNA conjugate.

[0061] Figure 11 Relative remaining expression levels of target genes in mice after administration of the siRNA conjugate.

[0062] Figure 12 Relative remaining expression levels of target genes in mice after administration of the siRNA conjugate.

[0063] Figure 13 Relative remaining expression levels of target genes in kidney cortex of mice after administration of the siRNA conjugate.

[0064] Figure 14 Relative remaining expression levels of target genes in kidney medulla of mice after administration of the siRNA conjugate.

[0065] Figure 15 Relative remaining expression levels of target genes in mice after administration of the siRNA conjugate.

[0066] Figure 16 Relative remaining expression levels of target genes in mice after administration of the siRNA conjugate. DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present application will be described clearly and completely below, and those skilled in the art can implement the process parameters by referring to the content herein and making appropriate improvements.

[0068] TERMS EXPLANATION

[0069] 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 this 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.

[0070] For example, the "C1~C" described in this disclosure 10 Alkylene, C2-C 10 "alkenyl" and "C2~C" 10 "Iso-ynyl group" refers to the group with the C1 to C2 groups removed. 10 Alkyl, C2-C 10 alkenyl and C2~C 10 The alkylene group is a divalent group formed by the other hydrogen atom of the alkynyl group, and can be substituted or unsubstituted. Unsubstituted alkylene groups include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), etc. Substituted alkylene groups, for example, those substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- )wait.

[0071] As used in this disclosure, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within a reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including zwitterionic forms of the compounds.

[0072] This disclosure includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule to two positions. A compound is not limited to any particular tautomer, but is intended to encompass all tautomer forms.

[0073] The compounds of the present invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomer forms, such as enantiomers and / or diastereomers.

[0074] In the present disclosure, the term "comprising" or "including" is an open term, used in the present disclosure to mean "including, but not limited to," and is synonymous with the phrase "including, but not limited to," and is used in the present disclosure in the same manner as the phrase "including, but not limited to."

[0075] In the present disclosure, the term "optionally," "optional" or "optional" generally means that the subsequently described event or circumstance can or can not occur, and that the description includes situations where the event or circumstance occurs and situations where it does not.

[0076] 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 of the RISC pathway by forming a silencing complex (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 conversion into proteins.

[0077] In the present disclosure, the term "sequence" and "nucleotide sequence" refer to a series of nucleobases or nucleotides. As used in the present disclosure, "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes the purine bases adenine and guanine, as well as the pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified. The synthesis of modified nucleobases, including phosphoramidite compounds of modified nucleobases, is known in the art.

[0078] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by partial or complete base pairing of two oligonucleotides, including a sense strand and an antisense strand, which can or can not be the same in length, as long as there is at least a region of partial base pairing to form a duplex region. An oligonucleotide having a double-stranded structure is within the scope of the present disclosure. In the present disclosure, the nucleotides in the double-stranded oligonucleotide can be modified or unmodified nucleotides. When referring to modified nucleotides, the modifications referred to in the present disclosure are not specific to the site of modification, unless otherwise specified. In the present disclosure, the double-stranded oligonucleotide can further comprise a linker between the modified nucleotides. A double-stranded oligonucleotide comprising a linker between the modified nucleotides is within the scope of the present disclosure. In the present disclosure, the double-stranded oligonucleotide can further comprise a compound molecule or a modification acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as a conjugate formed by a ligand.

[0079] In the present disclosure, the term "antisense strand (or called guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or called passenger strand)" refers to an iRNA strand that contains a sequence that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior of the molecule or within the terminal regions, with the most tolerated mismatches existing within the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5'- and / or 3' terminus of the iRNA. It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

[0080] 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. Generally, 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. The term "linked" as used herein when referring to the linkage between two molecules means that the two molecules are connected directly or indirectly by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0081] In the present disclosure, the term "targeting ligand" refers to a polypeptide that has affinity for a receptor present on the surface of a kidney cell; the term "ligand unit" refers to a targeting ligand linked to a linker; the term "ligand unit molecule" refers to a compound that is reacted with an oligonucleotide in a molecular form.

[0082] In the present disclosure, the term "linked" or "conjugated" when referring to the linkage between two compounds or molecules means that the two molecules are connected by a covalent bond or are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise specified, the terms "linked" and "conjugated" as used in the present disclosure can refer to the linkage between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0083] In the present disclosure, a linking group is one or more atoms that link one molecule or portion of a molecule to a second molecule or a second portion of a molecule. A linking group can comprise any number of atoms or functional groups. In some embodiments, a linking group is used only to link two biologically active molecules.

[0084] Unless otherwise specified, the symbols as used in the present disclosure mean that any group or groups can be attached thereto, consistent with the scope of the application as described in the present disclosure.

[0085] In the present disclosure, Indicates the site of attachment of a group by a covalent bond. As used herein, Indicates the 3' carbon or corresponding position of attachment to the next nucleotide or nucleotide analog by a phosphate group, a thiophosphate group, or other linking group.

[0086] A "reactive phosphorus group" refers to a phosphorus-containing group comprised in a nucleotide unit or a nucleotide analog unit which can react with a hydroxyl or amine group comprised in another molecule, in particular another nucleotide unit or another nucleotide analog, by a nucleophilic attack reaction. Typically, such a reaction results in an ester type internucleosidic linkage connecting said first nucleotide unit or said first nucleotide analog unit with said second nucleotide unit or said second nucleotide analog unit. A reactive phosphorus group can be selected from a phosphoramidite, a H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimic.

[0087] A "hydroxyl protecting group" refers to a group capable of avoiding a hydroxyl group from undergoing a chemical reaction, and which can be removed under certain conditions to restore the hydroxyl group. It mainly includes silyl-type protecting groups, acyl-type protecting groups or ether-type protecting groups, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxybenzhydryl.

[0088] According to the general knowledge in the art, a peptide is a compound which results from the linkage of two or more amino acids by amide bonds. Here, the individual amino acids are linked in a certain order (sequence) into a chain. An amino acid is a compound which carries at least one amino group and at least one carboxyl group. It includes natural (in vivo protein- generating amino acids), unnatural amino acids or prepared amino acids which can exist in a living organism.

[0089] In the peptides of the present disclosure, the amino acid units can be present in the D- or L-form, except where specifically noted.

[0090] As used in the present disclosure, the term "standard amino acid" refers to the following twenty amino acids: alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0091] As used herein, the term "non-standard amino acid" refers to an amino acid other than a "standard amino acid" as defined herein. "Non-standard amino acids" include, but are not limited to, N-formylmethionine, hydroxyproline, selenomethionine, isovaline, citrulline (Cit), ornithine, a-methyl-aspartate (aMeD), a-methyl-leucine (aMeL), N-methylalanine, N-methyl-glycine (NMe G), N-methyl-leucine (NMe L), O-cyclohexyl-alanine (Cha), N-ethylalanine, N,N-epsilon-dimethyllysine (K(Me)2), dimethylarginine (R(Me)2), n-alkylated L-a amino acids, and other amino acid analogs or mimetics that function in a similar manner to naturally occurring amino acids.

[0092] As used herein and as understood by one skilled in the art, a polyethylene glycol (PEG) unit refers to a repeating unit of the formula (CH2CH2O). It will be understood that in the chemical structures disclosed herein, a PEG unit can be depicted as (CH2CH2O), (OCH2CH2), or (CH2OCH2). It will further be understood that the number representing the number of repeating PEG units can be placed on either side of the parentheses representing the PEG unit. It will further be understood that a terminal PEG unit can be capped with an atom (e.g., a hydrogen atom) or some other moiety.

[0093] In the present disclosure, the term "cyclic peptide" means that two cysteines in a polypeptide chain are connected by a disulfide bond, such that an intramolecular ring is formed to form a cyclic polypeptide chain. In the present disclosure, the disulfide bond between the two cysteines in the "cyclic peptide" is represented by " ".

[0094] In the present disclosure, the term "pharmaceutical composition" or "composition" can refer to the use for the treatment of a disease, and can also be used for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the auxiliary ingredient or ingredients that make up the one or more additional ingredients. Typically, the compositions are prepared by uniformly and intimately bringing the active siRNA into association with a liquid auxiliary ingredient, a finely divided solid auxiliary ingredient, or both.

[0095] In the present disclosure, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved or approvable by a regulatory agency of the Federal or a state government or the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0096] 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.

[0097] In the publication, "aliphatic ring" refers to a structure with a cyclic carbon skeleton. The term "5- to 8-membered aliphatic ring" refers to a single-ring structure with 5 to 8 carbon atoms in the cyclic carbon skeleton. The term "saturated aliphatic ring" refers to a cyclic carbon skeleton composed entirely of carbon-carbon single bonds.

[0098] In the publication, "5- to 8-membered aliphatic heterocycles" refers to a ring in which one or more carbon atoms are replaced by heteroatoms. The term "saturated aliphatic heterocycles" refers to a ring in which one or more carbon atoms are replaced by heteroatoms.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] Extrahepatic delivery of ligand compounds

[0105] In a first aspect, this disclosure provides a ligand compound, characterized in that the ligand compound is selected from or includes the structure shown in formula (I), or its tautomers or stereoisomers:

[0106] In some alternative embodiments of this disclosure, A is selected from 5- to 8-membered oxygen-containing heterocycles.

[0107] In some alternative embodiments of this disclosure, A is selected from 5- to 8-membered saturated oxygen-containing heterocycles.

[0108] In some alternative embodiments of this disclosure, A is selected from a six-membered saturated heterocycle containing one oxygen atom.

[0109] In some alternative embodiments of this disclosure, A is selected from

[0110] In some alternative embodiments of this disclosure, the compound is selected from or includes the structure shown in formula (II), or its tautomers or stereoisomers:

[0111] In some alternative embodiments of this disclosure, p and q are each independently selected from 0, 1, 2, 3 or 4.

[0112] In some alternative embodiments of this disclosure, p and q are each independently selected from 0 or 1;

[0113] In some alternative embodiments of this disclosure, p = 1 and q = 1, or p = 1 and q = 0, or p = 0 and q = 1, or p = 0 and q = 0.

[0114] In some alternative embodiments of this disclosure, p = 1 and q = 0.

[0115] In some alternative embodiments of this disclosure, R1 is selected from H, a hydroxyl protecting group, or... In this context, * represents the linking site used to connect the active drug molecule; Z is selected from hydroxyl or thiol groups.

[0116] In some alternative embodiments of this disclosure, the hydroxyl protecting group is selected from triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, or 4,4',4”-trimethoxytriphenyl.

[0117] In some alternative embodiments of this disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytriphenylmethyl.

[0118] In some alternative embodiments of this disclosure, the pharmaceutically active molecule is selected from small molecule drugs, antibodies, or oligonucleotides.

[0119] In some alternative embodiments of this disclosure, the oligonucleotide is selected from single-stranded oligonucleotides and double-stranded oligonucleotides.

[0120] In some alternative embodiments of this disclosure, the pharmaceutically active molecule is selected from double-stranded oligonucleotides.

[0121] In some alternative embodiments of this disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0122] In some alternative embodiments of this disclosure, R2 is selected from H, reactive phosphorus groups, or... Among them, R 2b Selected from solid supports containing amino functional groups, R 2a Selected from covalent linkages or chemical bonds connected to the amino functional group.

[0123] In some alternative embodiments of this disclosure, the solid support is selected from resins containing amino functional groups or glass beads with controllable pore size containing amino functional groups.

[0124] In some alternative embodiments of this disclosure, the reactive phosphorus group is selected from...

[0125] In some alternative embodiments of this disclosure, the reactive phosphorus group is selected from...

[0126] In some alternative embodiments of this disclosure, R 2b Selected from Represents resin or glass beads with controllable pore size.

[0127] In some alternative embodiments of this disclosure, R 2a Selected from

[0128] In some alternative embodiments of this disclosure, Selected from

[0129] In some alternative embodiments of this disclosure, n is selected from integers from 1 to 10.

[0130] In some alternative embodiments of this disclosure, L1 is selected from

[0131] In some alternative embodiments of this disclosure, the compound is selected from the structure shown in formula (III), or its tautomers, or its stereoisomers:

[0132] And / or, the compound is selected from the structure shown in formula (IV), or its tautomers, or its stereoisomers:

[0133] In some alternative embodiments of this disclosure, R3 is selected from H, hydroxyl, amino, halogen, C1-C6 alkyl, or C1-C6 alkoxy.

[0134] In some alternative embodiments of this disclosure, R3 is selected from H or C1-C6 alkoxy groups.

[0135] In some alternative embodiments of this disclosure, R3 is selected from H or methoxy.

[0136] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 20 alkyne group, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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-; or L 2a -L 2b Composition (-CH2-CH2-O-) n Unit, n is an integer from 0 to 20 (preferably 1 to 10); L 2c Selected from C1 to C5 alkylene groups;

[0137] If L2 has substituents, then each substituent of L2 is independently selected from halogens, amino groups, hydroxyl groups, and C1-C2 groups. 20 Alkoxy, C2-C20 Alkyl, C2-C 20 alkenyl or C2-C 20 Alkyne group; or substituents on one or more carbon atoms in L2 can be linked to form a saturated or unsaturated ring.

[0138] In some alternative embodiments of this disclosure, the substituents of L2 are each independently selected from halogens, amino groups, hydroxyl groups, and C1-C2 groups. 10 Alkoxy, C2-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 Alkyne group; or substituents on one or more carbon atoms in L2 can be linked to form a saturated or unsaturated ring.

[0139] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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 2c Selected from C1 to C5 alkylene groups.

[0140] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 15 Alkylene, or substituted or unsubstituted Where j is selected from integers from 2 to 6; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b Each is independently selected from -O- or -C(O)-NH-; L 2c Selected from C1 to C5 alkylene groups.

[0141] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 15 alkylene or Where j is selected from integers from 2 to 6.

[0142] In some alternative embodiments of this disclosure, L2 is selected from any of the following structures:

[0143] In some alternative embodiments of this disclosure, L3 is selected from -O-, -NH-, -NHC(O)-, -(NH)C(O)O-, -(NH)O-, -C(O)-, -C(O)NH- or -C(O)O-.

[0144] In some alternative embodiments of this disclosure, L3 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-.

[0145] In some alternative embodiments of this disclosure, R4 is selected from a targeting ligand group capable of binding to a receptor on the surface of a target cell.

[0146] In some alternative embodiments of this disclosure, the target cells are selected from kidney cells, central nervous system cells, skeletal muscle cells, eye cells, cardiomyocytes, adipocytes, or lung cells.

[0147] In some alternative embodiments of this disclosure, R4 is selected from substituted or unsubstituted C. 12 ~C 30 Alkyl, substituted or unsubstituted C 12 ~C 30 Alkenyl, substituted or unsubstituted C2-C 30 Alkyne group, CB1 ligand group, TrkB ligand group, αvβ6 ligand group, or polypeptide targeting ligand group; if R4 contains a substituent, the substituent is independently selected from amino, hydroxyl, halogen, C1-C1 groups. 20 Alkoxy, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0148] In some alternative embodiments of this disclosure, R4 is selected from substituted or unsubstituted C. 15 ~C 30 Alkyl, substituted or unsubstituted C 15 ~C 30 Alkenyl, substituted or unsubstituted C2-C 30 Alkyne group, CB1 ligand group, TrkB ligand group, αvβ6 ligand group, or polypeptide targeting ligand group; if R4 contains a substituent, the substituent is independently selected from amino, hydroxyl, halogen, C1-C1 groups. 20 Alkyl group.

[0149] In some alternative embodiments of this disclosure, the CB1 ligand group includes, but is not limited to,

[0150] In some alternative embodiments of this disclosure, the TrkB ligand group includes, but is not limited to,

[0151] In some alternative embodiments of this disclosure, the αvβ6 ligand group includes, but is not limited to,

[0152] In some optional embodiments of this disclosure, the polypeptide targeting ligand group includes, but is not limited to, a kidney-targeting polypeptide ligand group, a CNS-targeting polypeptide ligand group, or a lung-targeting polypeptide ligand group;

[0153] In some alternative embodiments of this disclosure, the kidney-targeting polypeptide ligand group comprises at least one of the amino acid sequences shown in (1) to (19) in the direction from the amino terminus to the carboxyl terminus:

[0154] (1)-KKEEE-KKEEE-KKEEE-K-

[0155] (2)-SHSNTQTLAKAPEHTGC-

[0156] (3)-CKKEEE-KKEEE-KKEEE-K-

[0157] (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC-

[0158] (5)-CLPVASC-

[0159] (6) Cyclic peptide

[0160] (7)-CYFQNCPRG-

[0161] (8)

[0162] (9)-KKKEEKKKEEKKKEEK-

[0163] (10)-KKEEEOOEEEKKEEE-K-

[0164] (11)-OKEEEOKEEEOKEEE-O-

[0165] (12)-KKEEERREEEKKEEE-K-

[0166] (13)-RKEEERKEEERKEEE-R-

[0167] (14)-KKEEDKKEEDKKEED-K-

[0168] (15)-KKEEEKKQQQKKEEE-K-

[0169] (16)-KKEEQKKEEQKKEEQ-K-

[0170] (17)-CKKEEEKKEEEKKEEE-KC-

[0171] (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K-

[0172] (19)-CRPPR-;

[0173] in, The thiol groups on the C-side chains of the two cysteine ​​residues in this amino acid sequence form disulfide bonds, thus forming a cyclic peptide structure.

[0174] In some alternative embodiments of this disclosure, the kidney-targeting polypeptide ligand group is selected from at least one of (1) to (19):

[0175] (1)-KKEEE-KKEEE-KKEEE-K-NH2;

[0176] (2)-SHSNTQTLAKAPEHTGC-NH2;

[0177] (3)-CKKEEE-KKEEE-KKEEE-K-NH2;

[0178] (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC-NH2;

[0179] (5)-CLPVASC-NH2;

[0180] (6)

[0181] (7)-CYFQNCPRG-NH2;

[0182] (8)

[0183] (9)-KKKEEKKKEEKKKEEK-NH2

[0184] (10)-KKEEEOOEEEKKEEE-K-NH2

[0185] (11)-OKEEEOKEEEOKEEE-O-NH2

[0186] (12)-KKEEERREEEKKEEE-K-NH2

[0187] (13)-RKEEERKEEERKEEE-R-NH2

[0188] (14)-KKEEDKKEEDKKEED-K-NH2

[0189] (15)-KKEEEKKQQQKKEEE-K-NH2

[0190] (16)-KKEEQKKEEQKKEEQ-K-NH2

[0191] (17)-CKKEEEKKEEEKKEEE-KC-NH2

[0192] (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K-NH2

[0193] (19)-CRPPR-NH2;

[0194] In some alternative embodiments of this disclosure, the ligand compound is selected from any of the following structures:

[0195] Double-stranded oligonucleotide conjugates

[0196] In a second aspect, this disclosure provides a double-stranded oligonucleotide conjugate, characterized in that the double-stranded oligonucleotide conjugate comprises a sense strand and an antisense strand, wherein the sense strand and / or the antisense strand is conjugated to at least one targeting delivery group, the targeting delivery group comprising the structure shown in formula (i), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0197] Among them, A is selected from 5- to 8-membered oxygen-containing heterocycles;

[0198] In some alternative embodiments of this disclosure, A is selected from 5- to 8-membered saturated oxygen-containing heterocycles;

[0199] In some alternative embodiments of this disclosure, A is selected from a six-membered saturated heterocycle containing one oxygen atom;

[0200] In some alternative embodiments of this disclosure, A is selected from

[0201] In some alternative embodiments of this disclosure, the targeted delivery group comprises the structure shown in formula (ii), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0202] p and q are each independently selected from 0, 1, 2, 3 or 4;

[0203] In some alternative embodiments of this disclosure, p and q are each independently selected from 0 or 1;

[0204] In some alternative embodiments of this disclosure, p = 1 and q = 1, or p = 1 and q = 0, or p = 0 and q = 1, or p = 0 and q = 0;

[0205] In some alternative embodiments of this disclosure, p = 1 and q = 0.

[0206] n is an integer selected from 1 to 10;

[0207] L1 is selected from

[0208] In some alternative embodiments of this disclosure, the targeted delivery group comprises the structure shown in formula (iii), or a tautomer thereof, or a stereoisomer thereof:

[0209] And / or, the targeted delivery group comprises the structure shown in formula (iv), or a tautomer thereof, or a stereoisomer thereof:

[0210] R3 is selected from H, hydroxyl, amino, halogen, C1-C6 alkyl, or C1-C6 alkoxy;

[0211] In some alternative embodiments of this disclosure, R3 is selected from H or C1-C6 alkoxy groups;

[0212] In some alternative embodiments of this disclosure, R3 is selected from H or methoxy.

[0213] L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 20 alkyne group, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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-; or L 2a -L 2b Composition (-CH2-CH2-O-) n Unit, n is an integer from 0 to 20 (preferably 1 to 10); L 2c Selected from C1 to C5 alkylene groups.

[0214] If L2 has substituents, then each substituent of L2 is independently selected from halogens, amino groups, hydroxyl groups, and C1-C2 groups. 20 Alkoxy, C2-C 20 Alkyl, C2-C 20 alkenyl or C2-C 20 Alkyne group; or substituents on one or more carbon atoms in L2 can be linked to form a saturated or unsaturated ring.

[0215] In some alternative embodiments of this disclosure, the substituents of L2 are each independently selected from halogens, amino groups, hydroxyl groups, and C1-C2 groups. 10 Alkoxy, C2-C 10 Alkyl, C2-C 10 alkenyl or C2-C 10 Alkyne group; or substituents on one or more carbon atoms in L2 can be linked to form a saturated or unsaturated ring.

[0216] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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 2c Selected from C1 to C5 alkylene groups.

[0217] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 15 Alkylene, or substituted or unsubstituted Where j is selected from integers from 2 to 6; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b Each is independently selected from -O- or -C(O)-NH-; L 2c Selected from C1 to C5 alkylene groups.

[0218] In some alternative embodiments of this disclosure, L2 is selected from substituted or unsubstituted C1 to C2. 15 alkylene or Where j is an integer selected from 2 to 6;

[0219] In some alternative embodiments of this disclosure, L2 is selected from any of the following structures:

[0220] In some alternative embodiments of this disclosure, L3 is selected from -O-, -NH-, -NHC(O)-, -(NH)C(O)O-, -(NH)O-, -C(O)-, -C(O)NH- or -C(O)O-.

[0221] In some alternative embodiments of this disclosure, L3 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-.

[0222] In some alternative embodiments of this disclosure, R4 is selected from a targeting ligand group capable of binding to a receptor on the surface of a target cell.

[0223] The target cells are selected from kidney cells, central nervous system cells, skeletal muscle cells, eye cells, cardiomyocytes, fat cells, or lung cells.

[0224] In some alternative embodiments of this disclosure, R4 is selected from substituted or unsubstituted C. 15 ~C 30 Alkyl, substituted or unsubstituted C 15 ~C 30 Alkenyl, substituted or unsubstituted C2-C 30 Alkyne group, CB1 ligand group, TrkB ligand group, αvβ6 ligand group, or polypeptide targeting ligand; if R4 contains a substituent, the substituent is independently selected from amino, hydroxyl, halogen, C1-C1 groups. 20 Alkoxy, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0225] In some alternative embodiments of this disclosure, the CB1 ligand group includes, but is not limited to,

[0226] In some alternative embodiments of this disclosure, the TrkB ligand group includes, but is not limited to,

[0227] In some alternative embodiments of this disclosure, the αvβ6 ligand group includes, but is not limited to,

[0228] In some optional embodiments of this disclosure, the polypeptide targeting ligand group includes, but is not limited to, kidney-targeting polypeptide ligand groups, CNS-targeting polypeptide ligand groups, or lung-targeting polypeptide ligand groups;

[0229] In some alternative embodiments of this disclosure, the kidney-targeting polypeptide ligand group comprises at least one of the amino acid sequences shown in (1) to (19) in the direction from the amino terminus to the carboxyl terminus:

[0230] (1)-KKEEE-KKEEE-KKEEE-K-

[0231] (2)-SHSNTQTLAKAPEHTGC-

[0232] (3)-CKKEEE-KKEEE-KKEEE-K-

[0233] (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC-

[0234] (5)-CLPVASC-

[0235] (6) Cyclic peptide

[0236] (7)-CYFQNCPRG-

[0237] (8)

[0238] (9)-KKKEEKKKEEKKKEEK-

[0239] (10)-KKEEEOOEEEKKEEE-K-

[0240] (11)-OKEEEOKEEEOKEEE-O-

[0241] (12)-KKEEERREEEKKEEE-K-

[0242] (13)-RKEEERKEEERKEEE-R-

[0243] (14)-KKEEDKKEEDKKEED-K-

[0244] (15)-KKEEEKKQQQKKEEE-K-

[0245] (16)-KKEEQKKEEQKKEEQ-K-

[0246] (17)-CKKEEEKKEEEKKEEE-KC-

[0247] (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K-

[0248] (19)-CRPPR-;

[0249] in, The thiol groups on the C-side chains of the two cysteine ​​residues in this amino acid sequence form disulfide bonds, thus forming a cyclic peptide structure.

[0250] In some alternative embodiments of this disclosure, the kidney-targeting peptide targeting ligand group is selected from at least one of (1) to (19):

[0251] (1)-KKEEE-KKEEE-KKEEE-K-NH2;

[0252] (2)-SHSNTQTLAKAPEHTGC-NH2;

[0253] (3)-CKKEEE-KKEEE-KKEEE-K-NH2;

[0254] (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC-NH2;

[0255] (5)-CLPVASC-NH2;

[0256] (6)

[0257] (7)-CYFQNCPRG-NH2;

[0258] (8)

[0259] (9)-KKKEEKKKEEKKKEEK-NH2

[0260] (10)-KKEEEOOEEEKKEEE-K-NH2

[0261] (11)-OKEEEOKEEEOKEEE-O-NH2

[0262] (12)-KKEEERREEEKKEEE-K-NH2

[0263] (13)-RKEEERKEEERKEEE-R-NH2

[0264] (14)-KKEEDKKEEDKKEED-K-NH2

[0265] (15)-KKEEEKKQQQKKEEE-K-NH2

[0266] (16)-KKEEQKKEEQKKEEQ-K-NH2

[0267] (17)-CKKEEEKKEEEKKEEE-KC-NH2

[0268] (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K-NH2

[0269] (19)-CRPPR-NH2;

[0270] In some alternative embodiments of this disclosure, the targeted delivery group comprises any of the following structures, or tautomers, or stereoisomers, or pharmaceutically acceptable salts thereof:

[0271] In some alternative embodiments of this disclosure, the positive strand of the double-stranded oligonucleotide conjugate is conjugated with at least one targeted delivery group.

[0272] In some alternative embodiments of this disclosure, the targeted delivery groups are each independently located at the 5' end or 3' end of the positive chain.

[0273] In some alternative embodiments of this disclosure, the positive chain is fused with a targeting delivery group, one of which is located at the 5' or 3' end of the positive chain.

[0274] In some alternative embodiments of this disclosure, the positive chain is conjugated with two target delivery groups, which are connected by a phosphodiester bond or a thiophosphate diester bond, and the two connected target delivery groups are located at the 5' end or the 3' end of the positive chain;

[0275] Alternatively, the positive chain may be conjugated with two targeting delivery groups, which are located at the 5' end and 3' end of the positive chain, respectively.

[0276] In some alternative embodiments of this disclosure, the double-stranded oligonucleotide conjugate is selected from siRNA conjugates.

[0277] Composition

[0278] In a third aspect of this disclosure, a composition is provided, characterized in that the composition comprises the double-stranded oligonucleotide conjugate described in the second aspect of this disclosure.

[0279] Uses in disease treatment

[0280] In a fourth aspect of this disclosure, the following are provided for use in the preparation of medicaments for treating and / or preventing diseases or symptoms associated with dysregulation of mRNA levels of target gene expression:

[0281] (1) The ligand compounds described in the first aspect of this disclosure; and / or

[0282] (2) The double-stranded oligonucleotide conjugates described in the second aspect of this disclosure; and / or

[0283] (3) The composition described in the third aspect of this disclosure.

[0284] Uses of reducing the expression or activity of target genes in extrahepatic cells

[0285] In a fifth aspect of this disclosure, the following are provided for use in the preparation of a medicament for reducing the expression or activity of a target gene:

[0286] (1) The ligand compounds described in the first aspect of this disclosure; and / or

[0287] (2) The double-stranded oligonucleotide conjugates described in the second aspect of this disclosure; and / or

[0288] (3) The composition described in the third aspect of this disclosure.

[0289] Pharmaceutical Composition

[0290] In a sixth aspect, this disclosure provides a pharmaceutical composition comprising any one of the following, and optionally a pharmaceutically acceptable carrier or excipient:

[0291] (1) The ligand compounds described in the first aspect of this disclosure; and / or

[0292] (2) The double-stranded oligonucleotide conjugates described in the second aspect of this disclosure; and / or

[0293] (3) The composition described in the third aspect of this disclosure.

[0294] Methods to reduce the expression or activity of target genes in extrahepatic cells

[0295] In a seventh aspect of this disclosure, a method for reducing the expression or activity of a target gene is provided, characterized in that the method comprises contacting the cell with any of the following:

[0296] (1) The ligand compounds described in the first aspect of this disclosure; and / or

[0297] (2) The double-stranded oligonucleotide conjugates described in the second aspect of this disclosure; and / or

[0298] (3) The compositions described in the third aspect of this disclosure; and / or

[0299] (4) The pharmaceutical composition described in the sixth aspect of this disclosure.

[0300] In some alternative embodiments of this disclosure, the cells include, but are not limited to, kidney cells, central nervous system cells, skeletal muscle cells, eye cells, cardiomyocytes, fat cells, and lung cells.

[0301] Disease treatment methods

[0302] In an eighth aspect of this disclosure, a method for treating and / or preventing diseases or symptoms associated with dysregulation of mRNA levels in the expression of a target gene is provided, characterized in that any of the following is administered to a subject:

[0303] (1) The ligand compounds described in the first aspect of this disclosure; and / or

[0304] (2) The double-stranded oligonucleotide conjugates described in the second aspect of this disclosure; and / or

[0305] (3) The compositions described in the third aspect of this disclosure; and / or

[0306] (4) The pharmaceutical composition described in the sixth aspect of this disclosure.

[0307] 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.

[0308] Unless otherwise stated, the reagent ratios described in the embodiments of this disclosure are calculated on a volume ratio (v / v).

[0309] Unless otherwise stated, Cap1 and Cap2 are capping reagents used in this disclosure; wherein, Cap1 is a 20 vol% N-methylimidazole pyridine / acetonitrile mixed solution with a pyridine to acetonitrile volume ratio of 3:5; and Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

[0310] 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).

[0311] Unless otherwise stated, all raw materials and reagents used in the preparation of the compounds disclosed herein are commercially available. The main reagents and consumables used in this disclosure are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0312] Table 1 Main Reagents and Consumables

[0313] Table 2 Main Instruments and Equipment

[0314] Preparation Example 1: Preparation of Compound NM041

[0315] In this preparation example, the synthetic route of compound NM041 is shown below:

[0316] (1-1) Synthesis of compound NM041-2

[0317] Compound NM041-1 (25.0 g, 1.0 eq, 1,3,4,6-tetraacetoxy-alpha-D-glucopyranose, CAS No. 4292-12-0) was dissolved in 250 ml of toluene, heated to 110 °C, and then tributyltin hydride (17.7 g, 1.0 eq, CAS No. 688-73-3) and azobisisobutyronitrile (1 g, 0.1 eq, CAS No. 78-67-1) were added. The mixture was then refluxed at 110 °C for 2 hours. After the reaction was complete, the reaction solution was cooled to 25°C, and ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml water, 3.0 eq) were added. The mixture was stirred at 25°C for 2 hours. The reaction solution was filtered, and the organic phase was separated. The aqueous phase was washed twice with 50 ml of ethyl acetate (50 ml × 2). The organic phases were combined and washed once with 50 ml of saturated sodium chloride aqueous solution (50 ml × 1). The solution was dried over anhydrous sodium sulfate and filtered. The concentration yielded a pale yellow oily compound of formula NM041-2 (18.7 g, yield 87.9%). MS ESI (m / z) = 333.1 [M + H] + .

[0318] (1-2) Synthesis of compound NM041-3

[0319] Compound NM041-2 (18.7 g, 1 eq) was dissolved in anhydrous methanol (50 ml), and sodium methoxide (0.288 g, 0.1 eq) was added. The mixture was stirred at 25 °C for 1 hour. After the reaction was complete, the pH of the reaction solution was adjusted to 6-7 with a 4 mol / L hydrogen chloride solution of 1,4-dioxane under ice bath conditions. The solution was concentrated, condensed twice with acetonitrile, and then dried under vacuum to obtain a white solid, compound NM041-3 (9.4 g, 100% yield).

[0320] (1-2) Synthesis of compound NM041-4

[0321] Compound NM041-3 (9.4 g, 1.0 eq) was dissolved in 150 mL of acetonitrile. Benzaldehyde dimethyl acetal (30 mL, 3 eq, CAS No. 1125-88-8) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq, CAS No. 5872-08-2) were added separately. The mixture was stirred at 25 °C for 5 hours. Then, 3 mL of triethylamine was added, and the mixture was stirred at 25 °C for 30 min. After the reaction was complete, the reaction solution was concentrated. 100 mL of water was added to the concentrated solution, and the mixture was extracted twice with 100 mL of ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (elution: ethyl acetate / petroleum ether = 57 / 43, v / v) to give compound NM041-4 (8.6 g, yield 59.5%) as a white solid. MS ESI(m / z) = 252.2 [M+H] + .

[0322] (1-3) Synthesis of compound NM041-5

[0323] Compound NM041-4 (4.3 g, 1.0 eq) was dissolved in 40 mL of DMF. Sodium hydride (2.7 g, 4 eq) was added under ice bath conditions, and the reaction was continued under ice bath conditions for 30 minutes. Then, 3-bromopropyne (8.1 g, 4 eq, CAS No. 106-96-7) was added, and the mixture was stirred at 25 °C for 2 hours. The reaction was then quenched with 20 mL of water. After the reaction was complete, the reaction solution was extracted three times with 50 mL of ethyl acetate (50 mL × 3). The organic phases were combined, washed five times with 20 mL of saturated sodium chloride solution (20 mL × 5), dried over anhydrous sodium sulfate, filtered, and concentrated to give a brown oily compound NM041-5 (100% yield). MS ESI (m / z) = 329.2 [M+H] + .

[0324] (1-4) Synthesis of compound NM041-6

[0325] Compound NM041-5 (5.6 g, 1.0 eq) was dissolved in 30 mL of dichloromethane (DCM), and 300 mL of 70% (w / w) aqueous acetic acid solution was added. The reaction was carried out at 70 °C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to give compound NM041-6 (100% yield) as a yellow oil. MS ESI (m / z) = 241 [M+H]+.

[0326] (1-5) Synthesis of compound NM041-7

[0327] Compound NM041-6 (5.6 g, 1.0 eq) was dissolved in 50 mL of pyridine. 4,4'-dimethoxytriphenylchloromethane (10.2 g, 30.16 mmol, 1.3 eq, abbreviation DMTrCl, CAS No. 40615-36-9) was added under ice bath conditions. The mixture was purged with nitrogen three times, stirred at 25 °C for 3 hours, and quenched with 50 mL of methanol. After the reaction was complete, the reaction solution was concentrated, and 50 mL of water was added. The mixture was extracted three times with 50 mL of ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (elution: ethyl acetate / ether = 16 / 84, v / v) to give compound NM041-7 (5 g, yield 39.6%) as a pale yellow solid. MS ESI (m / z) = 543.1 [M+H] + .

[0328] (1-6) Synthesis of compound NM041

[0329] Compound NM041-7 (2.0 g, 1.5 eq) was dissolved in 20 mL of anhydrous dichloromethane. 4,5-Dicyanoimidazole (347.4 mg, 0.8 eq, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.22 g, 1.1 eq, CAS No. 102691-36-1) were added separately. The mixture was purged with nitrogen three times and stirred at 25 °C for 2 hours. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 mL of dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v). The solution was dried under vacuum for 12 hours to obtain a white powder, compound NM041 (2 g, yield 73.09%). MS ESI (m / z): 743.2 [M+H] + .

[0330] 1H NMR(400MHz, Acetonitrile-d3)δ7.53–7.47(m,2H),7.36(tt,J=9.6,3.4Hz,6H),7.28–7.22(m,1H),6.94–6 .86(m,4H),4.45(d,J=2.4Hz,1H),4.35(dt,J=6.1,1.9Hz,2H),4.18(ddd,J=11.2,5.4,2.6Hz,1H),3.81(s,7 H),3.79–3.20(m,8H),3.07(dt,J=10.5,7.0Hz,1H),2.79(q,J=2.6Hz,1H),2.72(dt,J=5.1,2.4Hz,1H),2.69 –2.63(m,1H),2.48–2.34(m,1H),2.17(d,J=1.0Hz,2H),1.08(dd,J=6.8,4.1Hz,10H),0.90(d,J=6.8Hz,2H).

[0331] Preparation Example 2: Synthesis of Compound NM064

[0332] In this preparation example, the synthetic route of compound NM064 is shown below:

[0333] (2-1) Synthesis of compound NM064-2

[0334] 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 separately. The mixture was stirred at 25 °C for 5 hours. Then, 3 mL of triethylamine was added, and the mixture was stirred at 25 °C for 30 min. After the reaction was complete, the reaction solution was concentrated, 100 mL of water was added, and the mixture was extracted twice with 100 mL of ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column purification (elution: ethyl acetate / petroleum ether = 57 / 43, v / v) to give compound NM064-2 (11.5 g, yield 60.8%) as a white solid. MS ESI (m / z) = 283 [M+H] + .

[0335] (2-2) Synthesis of compound NM064-3

[0336] 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 bath conditions, and the reaction was continued for 30 minutes under ice bath conditions. Then, 3-bromopropyne (8.1 g, 4 eq) was added, and the mixture was stirred at 25 °C for 2 hours. The reaction was then quenched with 20 mL of water. After the reaction was complete, the reaction solution was extracted three times with 50 mL of ethyl acetate (50 mL × 3). The organic phases were combined and washed five times with 20 mL of saturated sodium chloride solution (20 mL × 5). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a brown oily compound NM064-3 (6.3 g, 100% yield). MS ESI (m / z) = 359 [M+H] + .

[0337] (2-3) Synthesis of compound NM064-4

[0338] Compound NM064-3 (6.3 g, 17 mmol, 1.0 eq) was dissolved in 30 mL of dichloromethane, and 300 mL of 70% (w / w) aqueous acetic acid solution was added. The reaction was carried out at 70 °C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to give a yellow oily compound NM064-4 (4.78 g, 100% yield). MS ESI (m / z) = 271 [M+H] + .

[0339] (2-4) Synthesis of compound NM064-5

[0340] Compound NM064-4 (4.78 g, 17.7 mmol, 1.0 eq) was dissolved in 50 mL of pyridine. DMTTrCl (7.8 g, 23.0 mmol, 1.3 eq) was added under ice bath conditions. The mixture was purged with nitrogen three times, stirred at 25 °C for 3 hours, and quenched with 50 mL of methanol. After the reaction was complete, the reaction solution was concentrated, 50 mL of water was added, and the mixture was extracted three times with 50 mL of ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (elution: ethyl acetate / petroleum ether = 16 / 84, v / v) to give compound NM064-5 (6.7 g, yield 66.3%) as a pale yellow solid. MS ESI (m / z) = 573 [M+H] + .

[0341] (2-5) Synthesis of compound NM064

[0342] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 mL of anhydrous dichloromethane. DCI (330.4 mg, 0.8 eq, 4,5-dicyanimidazole) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 eq) were added separately. The mixture was purged with nitrogen three times and stirred at 25 °C for 2 hours. After the reaction was complete, 20 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 mL of dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (elution: acetonitrile / water = 72 / 28, v / v). The solution was dried under vacuum for 12 hours to obtain a white powder, compound NM064 (2 g, yield 74.07%). MS ESI (m / z) = 774 [M+H] + .

[0343] 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).

[0344] Preparation Example 3: Synthesis of Compound LD300

[0345] In this preparation example, the synthetic route of compound LD300 is shown below:

[0346] (3-1) Synthesis of compound LD300-2

[0347] Compound LD300-1 (5 g, 28.4 mmol, 1.0 eq, methyl 1H-indazole-3-carboxylate, CAS No. 43120-28-1) and N,N-dimethylformamide (50 ml) were added to a 100 ml reactor. The mixture was warmed to 0 °C and sodium hydride (1.19 g, 29.8 mmol, 1.05 eq) was slowly added at 0 °C. The mixture was purged with nitrogen three times, and the reaction system was stirred at room temperature under a nitrogen atmosphere for 0.5 hours. 5-Bromo-1-pentene (4.44 g, 29.8 mmol, 1.05 eq, CAS No. 1119-51-3) was slowly added, and the mixture was purged with nitrogen three times. The reaction system was stirred at room temperature under a nitrogen atmosphere for 2 hours. After the reaction was monitored by HPLC, purified water (50 ml) and ethyl acetate (50 ml) were added to the reaction solution for extraction. The organic phase was separated and then washed, dried, concentrated, and purified by column chromatography to obtain compound LD300-2 (3.2 g, yield 46.2%). MS ESI (m / z) = 245.0 [M+H] + .

[0348] (3-2) Synthesis of compound LD300-3

[0349] Compound LD300-2 (3.2 g, 13.1 mmol, 1.0 eq) and methanol (30 ml) were added to a 100 ml reaction vessel, followed by 22 ml of 3 M sodium hydroxide aqueous solution. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS until completion. The reaction solution was concentrated, the pH was adjusted to 4 with 1 M hydrochloric acid, filtered, and the filter cake was dried to obtain compound LD300-3 (2.7 g, 90% yield). MS ESI (m / z) = 231.0 [M+H] + .

[0350] (3-3) Synthesis of compound LD300-4

[0351] Compound LD300-3 (1 g, 4.35 mmol, 1.0 eq) and N,N-dimethylformamide (15 ml) were added to a 100 ml reaction vessel. Then, 1-hydroxybenzotriazole (646 mg, 4.79 mmol, 1.1 eq, CAS No. 2592-95-2), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.25 g, 6.54 mmol, 1.5 eq, CAS No. 7084-11-9), and N,N-diisopropylethylamine (2 g, ...) were added. 15.5 mmol (3.5 eq) and L-tert-leucine methyl ester hydrochloride (864 mg, 4.77 mmol, 1.1 eq, CAS No. 63038-27-7) were added to the reaction mixture. The mixture was purged with nitrogen three times, and the reaction system was stirred at room temperature under a nitrogen atmosphere for 3 hours. After the reaction was completed, purified water (20 ml) and ethyl acetate (50 ml) were slowly added to the reaction solution for extraction. The organic phase was separated, washed, dried, concentrated, and purified by column chromatography to give compound LD300-4 (0.6 g, yield 38.7%). MS ESI (m / z) = 358 [M+H] + .

[0352] (3-4) Synthesis of compound LD300-5

[0353] Compound LD300-4 (0.6 g, 1.68 mmol, 1.0 eq) and methanol (6 ml) were added to a 100 ml reaction vessel, followed by 6 ml of 3 M sodium hydroxide aqueous solution. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS until completion. The reaction solution was concentrated, the pH was adjusted to 4 with 1 M hydrochloric acid, filtered, and the filter cake was dried to obtain compound LD300-5 (0.5 g, yield 87.7%). MS ESI (m / z) = 344.0 [M+H] + .

[0354] (3-5) Synthesis of compound LD300

[0355] Compound LD300-5 (300 mg, 0.87 mmol, 1.0 eq) and N,N-dimethylformamide (3 ml) were added to a 100 ml reaction vessel. 1-Hydroxybenzotriazole (153 mg, 1.14 mmol, 1.3 eq, CAS No. 2592-95-2), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (250 mg, 1.31 mmol, 1.5 eq, CAS No. 7084-11-9), N,N-diisopropylethylamine (564 mg, 4.37 mmol, 5 eq) and 1-amino-11-azido-3,6,9-trioxaundecanane (286 mg, 1.31 mmol, 1.5 eq, CAS No. 134179-38-7) were added. The mixture was purged with nitrogen three times, and the reaction system was stirred at room temperature under a nitrogen atmosphere for 3 hours. The reaction was monitored by LCMS until completion. The reaction solution was purified by reverse-phase chromatography to obtain compound LD300 (200 mg, yield 42.1%). MS ESI (m / z) = 544 [M+H] + .

[0356] Preparation Example 4: Synthesis of Compound LD301

[0357] In this preparation example, the synthetic route of compound LD301 is shown below:

[0358] Compound LD300-5 (200 mg, 0.58 mmol, 1.0 eq) and N,N-dimethylformamide (2 ml) were added to a 100 ml reaction vessel. Then, 1-hydroxybenzotriazole (102 mg, 0.758 mmol, 1.3 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (167 mg, 0.875 mmol, 1.5 eq), N,N-diisopropylethylamine (376 mg, 2.9 mmol, 5 eq), and 6-azidohexylamine (124 mg, 0.875 mmol, 1.5 eq, CAS No. 349553-73-7) were added. The mixture was purged with nitrogen three times, and the reaction system was stirred at room temperature for 3 hours under a nitrogen atmosphere. The reaction was monitored by LCMS until completion. The mixture was purified by reverse phase to give compound LD301 (120 mg, yield 44.1%). MS ESI (m / z) = 468 [M+H] + .

[0359] Preparation Example 5: Synthesis of Compound LD308

[0360] In this preparation example, the synthetic route of compound LD308 is shown below:

[0361] Trimethyl 1,3,5-benzenetricarboxylate (1 g, 15.3 mmol, 1.0 eq, CAS No. 2672-58-4) and 1-amino-11-azido-3,6,9-trioxaundecanane (765 mg, 1.0 eq, CAS No. 134179-38-7) were mixed, heated to 150 °C, and stirred at 150 °C for 2 hours. After cooling to room temperature, ethanolamine (2.42 g, 10 eq, CAS No. 141-43-5) was added; the mixture was then heated to 150 °C and stirred at 150 °C for 3 hours. After the reaction was complete, water (10 ml) was added to the reaction solution, and the mixture was purified by reverse phase (elution buffer: acetonitrile / water = 20 / 80, v / v) to give compound LD308 (140 mg). MS ESI (m / z) = 497 [M+H] + .

[0362] Preparation Example 6: Synthesis of Compound LD309

[0363] In this preparation example, the synthetic route of compound LD309 is shown below:

[0364] (6-1) Synthesis of compound LD309-1

[0365] 2,3,4-Trihydroxyacetophenone (3 g, 17.9 mmol, 1 eq, CAS No. 528-21-2) was dissolved in DMF (30 mL), and potassium carbonate (14.8 g, 107.1 mmol, 6 eq), BnBr (10.7 g, 62.5 mmol, 3.5 eq, CAS No. 100-39-0), and KI (444.6 mg, 2.68 mmol, 0.15 eq) were added. The mixture was heated to 50 °C and stirred at 50 °C for 16 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (150 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phase was washed with saturated sodium chloride aqueous solution (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to give a yellow solid compound LD309-1 (7.24 g, yield 92.5%). MS ESI(m / z) = 461.1 [M + Na] + .

[0366] (6-2) Synthesis of compound LD309-2

[0367] Compound LD309-1 (5.73 g, 13.1 mmol, 1 eq) was dissolved in methanol (230 mL), and 4-carboxybenzaldehyde (2.94 g, 19.6 mmol, 1.5 eq, CAS No. 619-66-9) and NaOH (2.1 g, 52.3 mmol, 4 eq) were added. The mixture was heated to 60 °C and stirred at 60 °C for 48 hours. After the reaction was complete, the reaction solution was concentrated to at least the amount of solvent, and the pH was adjusted to 2-3 with 4M hydrochloric acid. A yellow precipitate appeared, which was filtered to obtain a yellow solid crude compound LD309-2 (7.6 g). This crude compound was not purified and proceeded directly to the next reaction. MS ESI (m / z) = 571.1 [M+H] + .

[0368] (6-3) Synthesis of compound LD309-3

[0369] Compound LD309-2 (7.6 g, 13.3 mmol, 1 eq) was dissolved in toluene (80 mL), and trifluoroacetic acid (15 mL, TFA, CAS No. 76-05-1) was added. The mixture was stirred at 25 °C for 72 hours. After the reaction was complete, the reaction solution was concentrated to remove the solvent, and ethyl acetate was added. A yellow precipitate formed, which was filtered to obtain a yellow solid crude compound LD309-3 (3.79 g). This crude compound was not purified and proceeded directly to the next step of the reaction. MS ESI (m / z) = 481.3 [M+H] + .

[0370] (6-4) Synthesis of compound LD309-4

[0371] Compound LD309-3 (3.79 g, 7.90 mmol, 1 eq) was dissolved in dimethyl sulfoxide (20 mL), and iodine (200.4 mg, 0.79 mmol, 0.1 eq) was added. The mixture was heated to 120 °C and stirred at 120 °C for 16 hours. After the reaction was complete, water was added to the reaction solution, resulting in a white precipitate. The precipitate was filtered to obtain a white solid crude compound LD309-4 (3.7 g), which was directly proceeded to the next reaction without purification. MS ESI (m / z) = 479.5 [M+H] + .

[0372] (6-5) Synthesis of compound LD309-5

[0373] Compound LD309-4 (3.3 g, 6.90 mmol, 1 eq) was dissolved in tetrahydrofuran (100 mL), and N-hydroxysuccinimide (1.19 g, 10.4 mmol, 1.5 eq, abbreviated as NHS, CAS No. 6066-82-6) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.33 g, 10.4 mmol, 1.5 eq, abbreviated as EDCI, CAS No. 7084-11-9) were added. The mixture was heated to 40 °C and stirred at 40 °C for 16 hours. After the reaction was complete, the reaction solution was evaporated to dryness to remove the solvent, and isopropanol was added and stirred until a white precipitate appeared. The precipitate was filtered to obtain a white solid crude compound LD309-5 (3.9 g), which was directly proceeded to the next step without purification. MS ESI (m / z) = 576.3 [M+H] + .

[0374] (6-6) Synthesis of compound LD309-6

[0375] Compound LD309-5 (500 mg, 869.2 μmol, 1 eq) was dissolved in TFA (5 mL), heated to 80 °C, and stirred at 80 °C for 72 hours. After the reaction was complete, the reaction solution was evaporated to dryness to remove the solvent, yielding a yellow solid crude compound LD309-6 (350 mg). MS ESI (m / z) = 396.1 [M+H] + .

[0376] (6-7) Synthesis of compound LD309

[0377] Compound LD309-6 (70 mg, 177.2 μmol, 1 eq) was dissolved in DCM (1 mL), and H2N-PEG5-N3 (65.1 mg, 212.6 μmol, 1.2 eq, CAS No. 516493-93-9) and triethylamine (35.8 mg, 354.3 μmol, 2 eq) were added. The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (5 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 5 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (2 × 3 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution: acetonitrile / water = 2 / 3, v / v) to give compound LD309 (24 mg, yield 23.1%) as a white solid. MS ESI (m / z) = 587.4 [M+H] + .

[0378] Preparation Example 7: Synthesis of Compound LD003

[0379] In this preparation example, the synthetic route of compound LD003 is shown below:

[0380] (7-1) Synthesis of compound 4BBA

[0381] In this preparation example, the synthetic route of compound 4BBA is shown below:

[0382] (7-1-1) Synthesis of compound 4BBA-02

[0383] Compound 4BBA-01 (35 g, 157.68 mmol, 4-bromonaphthol, CAS No. 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 reaction was carried out at 0 °C for 10 min, then heated to 25 °C and reacted at 25 °C for 12 h. The reaction was monitored by LC-MS until completion. The reaction solution was quenched in purified water (500 mL), extracted twice with ethyl acetate (500 mL each time), and the organic phases were combined. The organic phase was washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane) to give compound 4BBA-02 (28 g) as a white solid. MS ESI (m / z) = 313.01 [M+H] + .

[0384] (7-1-2) Synthesis of compound 4BBA

[0385] Compound 4BBA-02 (24.7 g, 79.16 mmol) was added to THF (250 ml), purged with nitrogen three times, cooled to -78 °C, and a tetrahydrofuran solution of n-BuLi (33.07 ml, 118.75 mmol) was added dropwise. After the addition was complete, the reaction was allowed to proceed for 1 h. Triisopropyl borate (23 g, 118.75 mmol) was added dropwise. After the addition was complete, the reaction system was allowed to rise naturally to 25 °C and reacted for 1 h. The reaction was monitored by TCL until completion (electrolyte: ethyl acetate, Rf = 0.9 for the product, Rf = 0.4 for the starting material). The reaction solution was quenched in an ice-water mixture (300 ml) of purified water. The pH was adjusted to 3 with 12N hydrochloric acid. Extraction was performed three times with methyl tert-butyl ether (300 ml each time). The organic phases were combined and washed once with a 10% NaCl aqueous solution (500 ml each time). The mixture was 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 mixed solution 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 for slurry preparation, yielding a white solid compound 4BBA (13 g). MS ESI (m / z) = 278.11 [M+H] + .

[0386] (7-2) Synthesis of compound PEG5

[0387] In this preparation example, the synthetic route of compound PEG5 is as follows:

[0388] (7-2-1) Synthesis of compound PEG5-02

[0389] 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). The mixture was cooled to 0 °C, and 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 mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C under a nitrogen atmosphere for 2 hours. The reaction was monitored by TLC until it ended. The reaction solution was washed twice with purified water (200 ml each time) and once with saturated sodium chloride aqueous solution (200 ml each time). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give the oily compound PEG5-02 (13 g, yield 52.6%). MS ESI (m / z) = 393.0 [M+H] + .

[0390] (7-2-2) Synthesis of compound PEG5-03

[0391] Compound PEG5-02 (12 g, 30.5 mmol), trimethyl azidosilane (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 mixture was heated to 60 °C and reacted at 60 °C for 1.5 h. The reaction was monitored by LCMS until 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, washed three times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound PEG5-03 (6.37 g, yield 79%). MS ESI (m / z) = 264.0 [M+H] + .

[0392] (7-2-3) Synthesis of compound PEG5

[0393] 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), cooled to 0 °C, and p-toluenesulfonyl chloride (4.3 g, 22.7 mol) was added at 0 °C. The mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C under a nitrogen atmosphere for 16 hours. The reaction was monitored by TLC until completion. The reaction solution was washed twice with purified water (50 mL each time), once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound PEG5 (7.2 g, yield 76%). MS ESI (m / z) = 418 [M+H] + .

[0394] (7-3) Synthesis of compound IM003

[0395] In this preparation example, the synthetic route of compound IM003 is shown below:

[0396] (7-3-1) Synthesis of compound IM003-02

[0397] 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. The mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C for 16 hours under a nitrogen atmosphere. The reaction was monitored by TLC until the reaction ended. The reaction solution was concentrated, and extracted with ethyl acetate (160 ml) and purified water (160 ml). The organic phase was separated, washed three times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound IM003-02 (13 g). This compound was directly used for the next reaction without purification. MS ESI (m / z) = 205.0 [M+H] + .

[0398] (7-3-2) Synthesis of compound IM003-03

[0399] Compound IM003-02 (8.0 g, 39.22 mmol) and platinum dioxide (270 mg, 1.19 mmol) were added to ethanol (160 mL), and the mixture was purged with hydrogen three times. The reaction system was then incubated at 25 °C for 16 hours under a hydrogen atmosphere. The reaction was monitored by TLC until completion. The reaction solution was filtered and concentrated to give compound IM003-03 (8.0 g, yield 97.7%). MS ESI (m / z) = 210.0 [M+H]+ .

[0400] (7-3-3) Synthesis of compound IM003-04

[0401] Compound IM003-03 (8.0 g, 38.1 mmol) was added to trifluoroacetic acid (64 mL), purged three times with nitrogen, and reacted at 25 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by TLC until completion. The pH of the reaction solution was adjusted to alkaline with saturated sodium bicarbonate aqueous solution, and the mixture was extracted twice with dichloromethane (50 mL each time). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound IM003-04 (6.1 g, 97.1% yield). MS ESI (m / z) = 165.0 [M+H] + .

[0402] (7-3-4) Synthesis of compound IM003-05

[0403] Ammonia (5.23 g, 73.93 mmol), sodium acetate (6.17 g, 73.93 mmol), and purified water (61 ml) were mixed and heated to 60 °C. The mixture was stirred at 60 °C for 0.5 hours. A methanol (60 ml) solution of compound IM003-04 (6.1 g, 36.97 mmol) was added dropwise. The mixture was purged with nitrogen three times, and the reaction was carried out at 60 °C under a nitrogen atmosphere for 2 hours. The reaction was monitored by TLC until completion. 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 give compound IM003-05 (4.5 g, yield 67.7%). MS ESI (m / z) = 180.0 [M+H] + .

[0404] (7-3-5) Synthesis of compound IM003

[0405] Compound IM003-05 (4.5 g, 25.14 mmol) and zinc powder (9 g) were added to trifluoroacetic acid (90 ml). The mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C for 20 minutes under a nitrogen atmosphere. The reaction was monitored by TLC until completion. A 3M sodium hydroxide aqueous solution (450 ml) was added to the reaction solution, and the mixture was cooled to 0 °C. Dichloromethane (400 ml) was added, and the mixture was shaken thoroughly. Diatomaceous earth was added, and the mixture was filtered. The organic phase was separated, 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] + .

[0406] (7-4) Synthesis of compound LD003-01

[0407] Compound IM001 (1.0 g, 2.4 mmol, (S)-3-(6-bromopyridin-3-yl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate, purchased from Kanglong Pharmaceutical Technology Co., Ltd.), compound 4BBA (1.33 g, 4.8 mmol), palladium acetate (27 mg, 0.12 mmol, CAS No. 3375-31-3), and 2-dicyclohexylphosphine-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, purged with nitrogen three times, and heated to 70 °C. The reaction system was maintained at 70 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by LCMS until completion. 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 aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LD003-01 (1.0 g, yield 73%). MS ESI (m / z) = 570 [M+H] + .

[0408] (7-5) Synthesis of compound LD003-02

[0409] Compound LD003-01 (1.0 g, 1.75 mmol), wet palladium on carbon (0.1 g, 10% w / w, abbreviated as Pd / C), and methanol (10 mL) were mixed, purged three times with hydrogen, and reacted at 25 °C for 16 hours under a hydrogen atmosphere. The reaction solution was filtered and concentrated to give compound LD003-02 (0.78 g, yield 92.8%). MS ESI (m / z) = 480 [M+H] + .

[0410] (7-6) Synthesis of compound LD003-03

[0411] 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, purged three times with nitrogen, and heated to 100 °C. The reaction system was maintained at 100 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by TLC until completion. Ethyl acetate (30 ml) and purified water (20 ml) were added to the reaction solution for extraction. The organic phase was separated, washed three times with saturated sodium chloride aqueous 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] + .

[0412] (7-7) Synthesis of compound LD003-04

[0413] Compound LD003-03 (0.73 g, 1 mmol), a 1,4-dioxane solution in 4 M hydrochloric acid (3.65 mL), and dichloromethane (3.65 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the reaction solution was concentrated, and dichloromethane (20 mL) was added and concentrated (this step was repeated twice) to obtain compound LD003-04 (0.6 g, yield 95.2%). MS ESI (m / z) = 625 [M+H] + .

[0414] (7-8) Synthesis of compound LD003-05

[0415] 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 mixture 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, and the reaction was monitored by LC-MS until completion. The reaction solution was washed with purified water (2 mL), the organic phase was separated, dried with anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD003-05 (100 mg, yield 57.1%). MS ESI (m / z) = 814 [M+H] + .

[0416] (7-9) Synthesis of compound LD003

[0417] Compound LD003-05 (100 mg, 0.12 mmol), 1 M sodium hydroxide aqueous solution (0.3 mL), and methanol (0.3 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction was carried out at 25 °C for 1 hour under a nitrogen atmosphere. The reaction was monitored by LC-MS until completion. The pH of the reaction solution was adjusted to 2 with 1 M hydrogen chloride aqueous solution. The reaction solution was concentrated, and dichloromethane (2 mL) and methanol (2 mL) were added. The mixture 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] + .

[0418] 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).

[0419] Preparation Example 8: Synthesis of Compound LD006

[0420] In this preparation example, the synthetic route of compound LD006 is shown below:

[0421] (8-1) Synthesis of compound 3BBA

[0422] In this preparation example, the synthetic route of compound 3BBA is shown below:

[0423] (8-1-1) Synthesis of compound 3BBA-02

[0424] 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, abbreviated as DMF), cooled to 0 °C, and potassium carbonate (31.1 g, 225.30 mmol) was added at 0 °C and reacted at 0 °C for 10 min. The temperature was then raised to 25 °C and... The reaction was carried out at 25°C for 12 hours, and the reaction was monitored by liquid chromatography-mass spectrometry (LC-MS) until completion. The reaction solution was quenched in purified water (500 ml), extracted twice with ethyl acetate (500 ml each time), and the organic phases were combined. The organic phase was washed twice with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane, CAS number 142-82-5) to give a pale yellow oily compound 3BBA-02 (21 g). MS ESI (m / z) = 313.01 [M+H] + .

[0425] (8-1-2) Synthesis of compound 3BBA

[0426] Compound 3BBA-02 (16 g, 51.28 mmol) was added to tetrahydrofuran (160 ml, THF), purged with nitrogen three times, cooled to -78 °C, and a tetrahydrofuran solution of n-butyllithium (30.8 ml, 76.92 mmol, n-BuLi, CAS No. 109-72-8) was added dropwise. After the addition was complete, the reaction was allowed to proceed for 1 h. Triisopropyl borate (14.7 g, 76.92 mmol, B(OiPr)3, CAS No. 5419-55-6) was added dropwise. After the addition was complete, the reaction system was allowed to rise naturally to 25 °C and reacted at 25 °C for 1 h. The reaction was monitored by thin-layer chromatography (TCL) (developing solvent: Hep / EA = 5:1, v / v, Rf = 0.80 for the product, Rf = 0.50 for the reactants). The reaction solution was quenched in an ice-water mixture (500 ml) of purified water. The pH was adjusted to 3 with 12N hydrochloric acid. Extraction was performed 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% (w / w) NaCl aqueous solution (500 ml each time). The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: n-heptane / ethyl acetate = 5 / 1, v / v) to give a white solid, 3BBA (9.7 g). MS ESI (m / z) = 279.11 [M+H] + .

[0427] (8-2) Synthesis of compound LD006-01

[0428] 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. The mixture was purged with nitrogen three times, and the temperature was raised to 70 °C. The reaction system was stirred at 70 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by LC-MS until completion. The reaction system was cooled to 25 °C, and ethyl acetate (50 ml) was added to the reaction solution for extraction. The organic phase was separated, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LD006-01 (2.36 g, yield 86.1%). MS ESI (m / z) = 570 [M+H] + .

[0429] (8-3) Synthesis of compound LD006-02

[0430] Compound LD006-01 (1.3 g, 2.28 mmol), wet palladium on carbon (130 mg, 10% w / w loading), and methanol (13 mL) were mixed, purged three times with hydrogen, and the reaction mixture was stirred at 25 °C for 16 hours under a hydrogen atmosphere. The mixture was then filtered and concentrated to obtain compound LD006-02 (1 g, 91.7% yield). MS ESI (m / z) = 480 [M+H] + .

[0431] (8-4) Synthesis of compound LD006-03

[0432] 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, purged three times with nitrogen, and heated to 100 °C. The reaction system was stirred at 100 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by TLC until 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 aqueous 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] + .

[0433] (8-5) Synthesis of compound LD006-04

[0434] Compound LD006-03 (1.3 g, 1.79 mmol), a 4M hydrogen chloride solution of 1,4-dioxane (6.5 mL), and dichloromethane (6.5 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction mixture was stirred at 25 °C for 1 hour under a nitrogen atmosphere. The reaction solution was concentrated, and dichloromethane (10 mL) was added and the mixture was concentrated (this step was repeated twice) to give compound LD006-04 (1 g, 91% yield). MS ESI (m / z) = 625 [M+H] + .

[0435] (8-6) Synthesis of compound LD006-05

[0436] 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 CR02006-04 (400 mg, 0.64 mmol) was added, and the mixture was purged with nitrogen three times. The reaction system was stirred at 25 °C under a nitrogen atmosphere for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was washed with purified water (8 mL), and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LD006-05 (320 mg, yield 61.5%). MS ESI (m / z) = 813 [M+H] + .

[0437] (8-7) Synthesis of compound LD006

[0438] Compound LD006-05 (320 mg, 0.394 mmol), 1 M sodium hydroxide aqueous solution (0.96 mL), and methanol (0.96 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. The reaction was monitored by LC-MS until completion. The pH of the reaction solution was adjusted to 2 with 1 M hydrogen chloride aqueous solution. The reaction solution was concentrated, and dichloromethane (5 mL) and methanol (5 mL) were added. The mixture 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] + .

[0439] 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).

[0440] Preparation Example 9: Synthesis of Compound LD008

[0441] In this preparation example, the synthetic route of compound LD008 is shown below:

[0442] (9-1) Synthesis of compound 5BBA

[0443] In this preparation example, the synthetic route of compound 5BBA is shown below:

[0444] (9-1-1) Synthesis of compound 5BBA-02:

[0445] 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 a saturated aqueous solution of sodium nitrite (12 g, 0.17 mol) was added. The reaction was continued at 0 °C for 1 h. Potassium iodide (65.2 g, 0.39 mol) was added, and the mixture was heated to 25 °C and reacted at 25 °C. The reaction was monitored by TLC until completion (developing solvent: Hep / EA = 5 / 1, v / v, Rf = 0.75 for the product, Rf = 0.25 for the reactants). 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 (elution buffer: Hep / EA = 5 / 1, v / v) to give a brown solid compound 5BBA-02 (18 g). MS ESI (m / z) = 270.96 [M+H] + .

[0446] (9-1-2) Synthesis of compound 5BBA-03

[0447] 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 BnBr (9.3 g, 0.054 mol). The reaction was carried out at 25 °C, and the reaction was monitored by TLC until completion (developing solvent: Hep / EA = 20 / 1, v / v, product Rf = 0.75, starting material Rf = 0.2). The reaction mixture was quenched with 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 (elution: Hep / EA = 80 / 1, v / v) to give a pale yellow solid, compound 5BBA-03 (14 g). MS ESI (m / z) = 361.00 [M+H] + .

[0448] (9-1-3) Synthesis of compound 5BBA

[0449] Compound 5BBA-03 (14 g, 0.039 mol) was added to THF (140 ml), purged with nitrogen three times, cooled to -78 °C, and a THF solution of n-BuLi (23.3 ml, 0.058 mol) was added dropwise. The reaction was allowed to proceed for 1 h. Triisopropyl borate (11.0 g, 0.058 mol) was then added dropwise, the temperature was raised to 25 °C, and the reaction was carried out at 25 °C. The reaction was monitored by TLC until completion (developing solvent: Hep / EA = 2 / 1, v / v, product Rf = 0.75, reactant Rf = 0.50). The reaction system was cooled to 5–10 °C, quenched with purified water (150 ml), and the pH was adjusted to 3 with 1 M HCl aqueous solution. The mixture was extracted three times with methyl tert-butyl ether (150 ml each time). The organic phases were combined, washed once with a 10% 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 and the mixture was stirred to obtain a white powdery compound 5BBA. MS ESI (m / z) = 2279.11 [M+H] + .

[0450] (9-2) Synthesis of compound LD008-01

[0451] 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. The mixture was purged with nitrogen three times, and the temperature was raised to 70 °C. The reaction system was stirred at 70 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by LC-MS until completion. The reaction system was then cooled to 25 °C, and ethyl acetate (60 ml) was added to the reaction solution for extraction. The organic phase was separated, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LD008-01 (2.0 g, 73% yield). MS ESI (m / z) = 570 [M+H] + .

[0452] (9-3) Synthesis of compound LD008-02

[0453] Compound LD008-01 (2.0 g, 3.5 mmol), wet palladium on carbon (0.2 g, 10% w / w loading), and methanol (20 mL) were mixed, purged three times with hydrogen, and the reaction mixture was stirred at 25 °C for 16 hours under a nitrogen atmosphere. The mixture was then filtered and concentrated to obtain compound LD008-02 (1.56 g, 92.8% yield). MS ESI (m / z) = 480 [M+H]+ .

[0454] (9-4) Synthesis of compound LD008-03

[0455] 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, purged three times with nitrogen, and heated to 100 °C. The reaction system was stirred at 100 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by TLC until 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 aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound LD008-03 (300 mg, yield 41.4%). MS ESI (m / z) = 725 [M+H] + .

[0456] (9-5) Synthesis of compound LD008-04

[0457] Compound LD008-03 (150 mg, 0.207 mmol), a 4M hydrogen chloride solution of 1,4-dioxane (0.75 mL), and dichloromethane (0.75 mL) were mixed. The mixture was purged with nitrogen three times, and 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 the mixture was concentrated (this step was repeated twice) to obtain compound LD008-04 (100 mg, yield 77.5%). MS ESI (m / z) = 625 [M+H] + .

[0458] (9-6) Synthesis of compound LD008-05

[0459] 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 mixture was purged with nitrogen three times. The reaction system was stirred at 25 °C under a nitrogen atmosphere for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was washed with purified water (2 ml), 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, yield 46.2%). MS ESI (m / z) = 813 [M+H] + .

[0460] (9-7) Synthesis of compound LD008

[0461] Compound LD008-05 (60 mg, 0.074 mmol), 1 M sodium hydroxide aqueous solution (0.18 mL), and methanol (0.18 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. The reaction was monitored by LC-MS until completion. 1 M hydrogen chloride aqueous solution was added to the reaction solution to adjust the pH to 2. The mixture was concentrated, and dichloromethane (2 mL) and methanol (2 mL) were added. The mixture 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] + .

[0462] 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).

[0463] Preparation Example 10: Preparation of Compound LD002

[0464] In this preparation example, the synthetic route of compound LD002 is as follows:

[0465] (10-1) Synthesis of compound LD002-1:

[0466] Compound IM001 (1.8 g, 4.3 mmol), compound 4BBA (2.4 g, 8.6 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (103 mg, 0.22 mmol, abbreviation 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 mL) and water (13.5 mL). The mixture was purged with nitrogen three times, heated to 70 °C, and stirred for 16 hours until the reaction was complete. After the reaction solution cooled to room temperature, 20 mL of water was added, 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 give compound LD002-1 (1.1 g, yield 44.7%) as a pale yellow solid. ESI-MS (m / z) = 569.3 [M+H] + .

[0467] (10-2) Synthesis of compound LD002-2:

[0468] 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 mixture was purged with hydrogen three times and stirred at room temperature for 16 hours until the reaction was complete. The reaction solution was filtered and concentrated to give compound LD002-2 (850 mg, yield 91.7%) as a white, bubbly solid. ESI-MS (m / z) = 479.2 [M+H] + .

[0469] (10-3) Synthesis of compound LD002-3:

[0470] 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 until the reaction was complete. 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 give compound LD002-3 (600 mg, yield 46.6%) as a pale yellow bubbly solid. ESI-MS (m / z) = 725.3 [M+H] + .

[0471] Synthesis of compound LD002-4 (10-4):

[0472] Compound LD002-3 (600 mg, 0.83 mmol), dichloromethane (6 mL), and a 4M hydrogen chloride solution of 1,4-dioxane (0.6 mL) were added to a reaction vessel and stirred for 1 hour until the reaction was complete. The reaction solution was concentrated to give compound LD002-4 (500 mg, yield 96.7%) as a pale yellow bubbly solid. ESI-MS (m / z) = 625.4 [M+H] + .

[0473] Synthesis of compound LD002-5 (10-5):

[0474] 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 h. Compound LD002-4 (500 mg, 0.8 mmol) was then added, and the mixture was stirred for 3 h until the reaction was complete. 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 (elution solvent: methanol / dichloromethane = (0 / 100) to (10 / 90), v / v) to give compound LD002-5 (290 mg, yield 45.3%) as a white bubbly solid. ESI-MS (m / z) = 801.3 [M+H] + .

[0475] (10-6) Synthesis of compound LD002:

[0476] Compound LD002-5 (290 mg, 1 mmol) was dissolved in methanol (3 ml), and 1 M sodium hydroxide aqueous solution (3 ml) was added. The mixture was stirred at room temperature for 1 hour. Under ice bath conditions, the pH of the reaction solution was adjusted to 6-7 with 1 M hydrochloric acid aqueous solution. The solution was concentrated, 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] + .

[0477] Preparation Example 11: Preparation of Compound LD005

[0478] In this preparation example, the synthetic route of compound LD005 is as follows:

[0479] (11-1) Synthesis of compound LD005-1:

[0480] 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) were added to a 100 ml reaction vessel. The mixture was purged with nitrogen three times, heated to 70 °C, and stirred for 16 hours until 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 aqueous solution, dried, and concentrated. The solution was purified by column chromatography to give compound LD005-1 (600 mg, yield 87.3%). MS ESI (m / z) = 570 [M+H] + .

[0481] (11-2) Synthesis of compound LD005-2:

[0482] Compound LD005-1 (600 mg, 1.053 mmol), wet palladium on carbon (60 mg, 10% w / w, abbreviated Pd / C), and methanol (6 ml) were added to a 50 ml reaction vessel. The mixture was purged with hydrogen three times and stirred for 16 hours until 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] + .

[0483] (11-3) Synthesis of compound LD005-3:

[0484] 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) were added to a 100 ml reaction vessel. The mixture 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 until the reaction was complete. 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 aqueous solution, dried, concentrated, and purified by column chromatography to obtain compound LD005-3 (500 mg, yield 78.7%). MS ESI (m / z) = 725 [M+H] + .

[0485] (11-4) Synthesis of compound LD005-4:

[0486] Compound LD005-3 (500 mg, 0.69 mmol), a 1,4-dioxane solution in 4M hydrochloric acid (2.5 ml), and dichloromethane (2.5 ml) were added to a 50 ml reaction vessel. The mixture was purged with nitrogen three times and stirred at room temperature for 1 hour until the reaction was complete. The reaction solution was directly concentrated, and then concentrated again with dichloromethane (10 ml) to obtain compound LD005-4 (420 mg, 98% yield). MS ESI (m / z) = 625 [M+H] + .

[0487] (11-5) Synthesis of compound LD005-5:

[0488] Compound LD005-4 (420 mg, 0.672 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea 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) were added to a 25 ml reaction vessel. The mixture was purged with nitrogen three times and stirred at room temperature for 2 hours until the reaction was complete. The reaction solution 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, yield 91.7%). MS ESI (m / z) = 813 [M+H] + .

[0489] (11-6) Synthesis of compound LD005:

[0490] Compound LD005-5 (500 mg, 0.615 mmol), 1 M sodium hydroxide aqueous solution (1.5 ml), and methanol (1.5 ml) were added to a 25 ml reaction vessel. The mixture was purged with nitrogen three times and stirred at room temperature for 1 hour until the reaction was complete. The pH of the reaction solution was adjusted to 2 by adding 1 M hydrogen chloride aqueous solution. The solution was concentrated, and dichloromethane (5 ml) and methanol (5 ml) were added. The mixture was filtered, concentrated, and purified by reverse phase to obtain compound LD005 (220 mg, yield 44.9%). MS ESI (m / z) = 800 [M+H] + .

[0491] 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).

[0492] Preparation Example 12: Synthesis of Compound LD007

[0493] (12-1) Synthesis of compound LD007-01

[0494] 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 mixture was purged with nitrogen three times. The reaction system was stirred at 25 °C under a nitrogen atmosphere for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was washed with purified water (5 ml), and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound LD007-01 (100 mg, yield 52.1%). MS ESI (m / z) = 802 [M+H] + .

[0495] (12-2) Synthesis of compound LD007

[0496] Compound LD007-01 (100 mg, 0.125 mmol), 1 M sodium hydroxide aqueous solution (0.3 mL), and methanol (0.3 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. The reaction was then stopped by LC-MS. 1 M hydrogen chloride aqueous solution was added to the reaction solution to adjust the pH to 2. The mixture was concentrated, and dichloromethane (2 mL) and methanol (2 mL) were added. The mixture 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] + .

[0497] 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).

[0498] Preparation Example 13: Synthesis of Compound LD009

[0499] (13-1) Synthesis of compound IM002

[0500] In this preparation example, the synthetic route of compound IM002 is as follows:

[0501] (13-1-1) Synthesis of compound IM002-03

[0502] 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, purged with nitrogen three times, and heated to 100 °C. The reaction system was stirred at 100 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored by TLC until it ended. 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, washed three times with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound IM002-03 (2.2 g, yield 27.7%). MS ESI (m / z) = 252.0 [M+H] + .

[0503] (13-1-2) Synthesis of compound IM002

[0504] Compound IM002-03 (2.2 g, 8.76 mmol) was mixed with a 1,4-dioxane solution (11 ml) in 4 M hydrochloric acid. The reaction mixture was incubated at 25 °C for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was concentrated to give compound IM002 (1.27 g, 96% yield). MS ESI (m / z) = 152.0 [M+H] + .

[0505] (13-2) Synthesis of compound LD009-01

[0506] 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 mixture was purged with nitrogen three times. The reaction system was stirred at 25 °C under a nitrogen atmosphere for 2 hours, and the reaction was monitored by LC-MS until completion. The reaction solution was washed with purified water (2 mL), 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 LD009-01 (60 mg, yield 66.7%). MS ESI (m / z) = 802 [M+H] + .

[0507] (13-3) Synthesis of compound LD009

[0508] Compound LD009-01 (60 mg, 0.074 mmol), 1 M sodium hydroxide aqueous solution (0.18 mL), and methanol (0.18 mL) were mixed. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 1 hour under a nitrogen atmosphere. The reaction was monitored by LC-MS until completion. 1 M hydrogen chloride aqueous solution was added to the reaction solution to adjust the pH to 2. The mixture was concentrated, and dichloromethane (2 mL) and methanol (2 mL) were added. The mixture 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] + .

[0509] 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).

[0510] Preparation Example 14: Peptide-based Targeting Ligands

[0511] Unless otherwise stated, all peptide-based targeted delivery ligands listed in Table 3 of this disclosure were synthesized by Hangzhou Zhuntai Biotechnology Co., Ltd.

[0512] Table 3 Molecular structure information of peptide-based targeted delivery ligands

[0513] K(Ac) represents the acetylation of the amino group on the lysine side chain.

[0514] Among them, K(CO-CH2CH2-PEG2-N3) is the same as Lys(CO-CH2CH2-PEG2-N3), and its structural formula is:

[0515] 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).

[0516] In this context, the letter O represents ornithine, and its structure is as follows:

[0517] The letter K stands for lysine;

[0518] The letter E stands for glutamic acid;

[0519] The letter S stands for serine;

[0520] The letter H represents histidine;

[0521] The letter N represents asparagine;

[0522] The letter T stands for threonine;

[0523] The letter Q stands for glutamine;

[0524] The letter L represents leucine;

[0525] The letter A represents alanine;

[0526] The letter P stands for proline;

[0527] The letter C stands for cysteine;

[0528] The letter G stands for glycine;

[0529] The letter R represents arginine;

[0530] The letter V stands for valine;

[0531] The letter Y represents tyrosine.

[0532] During the synthesis process, the compound Each can be considered as a single amino acid monomer.

[0533] In this preparation example, the specific preparation method of the targeted ligand includes the following steps:

[0534] (14-1) 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., piperazine and DMF volume ratio of 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.

[0535] (14-2) 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)-Liner-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;

[0536] Subsequently, step (14-2) needs to be repeated for each amino acid monomer to obtain the amino acid sequence (side chain protecting group) - Liner-MBHA Resin.

[0537] (14-3) 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.

[0538] (14-4) Cutting: 6 times the volume of the cutting solution (the cutting solution is 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) and shaken on a shaker for 2 hours. The resin was filtered off, and the filtrate was precipitated with anhydrous diethyl ether. The precipitate was washed three times with anhydrous diethyl ether. Finally, the precipitate was placed in a vacuum drying oven and dried at room temperature for 24 hours. The precipitate was purified by HPLC to obtain the targeted delivery ligands N3-PEG2-CH2CH2CO-amino acid sequence-NH2 or N3-PEG2-CH2CH2CO-amino acid sequence-NHCH3 with a purity of over 95%.

[0539] Preparation Example 15: Synthesis of siRNA conjugates

[0540] In this preparation example, the method for preparing the siRNA conjugate includes the following steps:

[0541] (15-1) Synthesis of the Justice Chain (SS) and the Antisense Chain (AS)

[0542] Starting with a carrier compound (such as a CPG carrier or a PS carrier), nucleoside monomers are linked one by one along the 3'-5' direction according to the nucleotide sequence using a phosphoramide solid-phase synthesis method. During the synthesis, compounds NM041 and NM064 are each considered as a single nucleotide monomer.

[0543] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

[0544] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0545] 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 dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0546] 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).

[0547] 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 of Cap1 and Cap2, Cap1 being a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 being 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.

[0548] 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).

[0549] 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:

[0550] 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 deprotect the 2'-O-TBDMS protection on the ribose.

[0551] 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 of 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 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 dextran gel) and elution with deionized water.

[0552] 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.

[0553] Compounds NM041 and NM064 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 phosphodiester bonds. In some specific embodiments, these compounds are linked to the 3' end or 5' end of the sense chain via phosphodiester bonds.

[0554] When a compound NM041 is attached to the 3' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: When two compounds NM041 are linked to the 3' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: When a compound NM041 is attached to the 5' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: When two compounds NM041 are linked to the 5' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: in, Represents the Chain of Justice (SS).

[0555] 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:

[0556] When two compounds NM064 are linked to the 3' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: When a compound NM064 is attached to the 5' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: When two compounds NM064 are linked to the 5' end of the positive chain via a phosphodiester bond, the structural formula of the positive chain is as follows: in, Represents the Chain of Justice (SS).

[0557] (15-2) Synthesis of SS conjugates

[0558] 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:

[0559] (15-2-1) 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 (15-1) in the mixed solvent to obtain a positive chain solution with a concentration of 1.0 eq.

[0560] (15-2-2) The ligand compound (6.0 eq) was dissolved in DMF (70 μL) to obtain a ligand compound solution.

[0561] (15-2-3) Mix the positive chain solution from step (15-2-1) and the ligand compound solution from step (15-2-2) to obtain a reaction mixture.

[0562] (15-2-4) 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 (15-2-3) above and vortexed to obtain an intermediate product mixture. The pH of the intermediate product mixture was measured to be 8.

[0563] (15-2-5) Take 25.0 eq sodium ascorbate and quickly add it to the intermediate product mixture of step (15-2-4), and vortex it. React at 40℃ for 1 h to obtain the product mixture.

[0564] (15-2-6) 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.

[0565] (15-3) Synthesis of siRNA conjugates

[0566] The AS from step (15-1) and the SS conjugate from step (15-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was 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.

[0567] For example, when the ligand compound is LD002, the structural formula of the siRNA conjugate is shown below:

[0568] For example, when the ligand compound is LD003, the structural formula of the siRNA conjugate is shown below:

[0569] For example, when the ligand compound is LD005, the structural formula of the siRNA conjugate is shown below:

[0570] For example, when the ligand compound is LD006, the structural formula of the siRNA conjugate is shown below:

[0571] For example, when the ligand compound is LD007, the structural formula of the siRNA conjugate is shown below:

[0572] For example, when the ligand compound is LD008, the structural formula of the siRNA conjugate is shown below:

[0573] For example, when the ligand compound is LD009, the structural formula of the siRNA conjugate is shown below:

[0574] According to the above method, the present disclosure obtains the siRNA conjugates shown in Table 5, wherein the unmodified nucleotide sequence information of the siRNA conjugates shown in Table 5 is shown in Table 4.

[0575] Table 4. Information on the unmodified nucleotide sequences forming siRNA conjugates.

[0576] Table 5 Sequence information of siRNA conjugates

[0577] Unless otherwise specified, the base composition and modifications described in the embodiments of this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-methyl; lowercase letter f indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-fluoro; (moe) indicates that the nucleotide represented by the uppercase letter to its left is a nucleotide modified with 2'-O-methoxyethyl; lowercase letter s indicates that the two nucleotides to its left and right are connected by a phosphate thioester bond. VP indicates that the 5' end of the antisense strand in siRNA is modified with 5'-(E)-vinylphosphonate (5'-(E)-VP).

[0578] The structural formula of VPUm is:

[0579] The structural formula of the nucleotide modified with 2'-O-methyl is

[0580] The structural formula of the 2'-fluorinated nucleotide is

[0581] The structural formula of the nucleotide modified with 2'-O-methoxyethyl is:

[0582] Where Base represents the nucleobases A, U, G, C, and T.

[0583] Biological testing experiments

[0584] Unless otherwise stated, the siRNA sequences used in this disclosure were synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Beijing Xuanjing Rui Pharmaceutical Technology Co., Ltd.; the synthesis of PCR primers was commissioned to Beijing Qingke Biotechnology Co., Ltd.; and the experimental animals, C57BL / 6J mice, were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0585] siRNA conjugate mouse lateral ventricle administration activity assessment test

[0586] Six- to eight-week-old C57BL / 6j mice were randomly divided into groups according to body weight. Each group of mice was administered the siRNA conjugate via lateral ventricle administration, with the dosage per mouse determined based on the specific experiment. Using the Bregma point in the mouse brain as the zero point, the coordinates of the right ventricle were located using a stereotaxic instrument and marked on the skull surface (lateral ventricle location: AP (anteroposterior): -0.58 mm, ML (mediolateral): -1.2 mm, DV (dorsoventral): -2.2 mm). A hole was drilled in the skull above the target location using a dental drill. The drug was then injected into the lateral ventricle using a microinfusion pump, with a total injection volume of 5 μL over 10 minutes. The needle was left in place for 5 minutes after injection, then slowly withdrawn. When the DV reached -1.0 mm, the needle was left in place for 1 minute, and then slowly withdrawn until completely removed. The needle holes were then sealed with bio-adhesive, and the head skin was glued in place. After the animals regained consciousness, they were returned to the animal house for continued care. The PBS control group received the same volume of PBS solution without siRNA conjugate. The day of administration was designated as day 0 (D0). Mice were euthanized at the observation point after administration. The left and right brains were harvested and preserved in RNAlaters, respectively.

[0587] siRNA conjugate rat intrathecal administration activity assessment test

[0588] Six- to eight-week-old SD rats were randomly divided into groups according to body weight. Each group of rats was administered the siRNA conjugate intrathecally at a dose of 900 μg per rat. The needle was inserted into the L5-L6 segment of the rat spinal cord, with a dosage of 40 μL. The needle was left in place for 1 minute after injection, and then slowly withdrawn. The PBS control group received the same volume of PBS solution without the siRNA conjugate. The day of administration was designated as day 0 (D0). Rats were sacrificed at the observation point after administration. Different segments of the spinal cord and different regions of the brain were collected and preserved in RNAlater.

[0589] Methods for assessing the inhibitory activity of siRNA conjugates on target genes in mice

[0590] Six- to eight-week-old C57BL / 6J mice (all female) were randomly divided into groups according to body weight. The drug dosage for each group was calculated based on body weight and administered via subcutaneous abdominal injection or tail vein injection. Each siRNA conjugate was prepared into a solution of the appropriate concentration (based on siRNA) using PBS solution for administration, with an administration 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 drug conjugates). The day of administration was designated as day 0 (D0). At a predetermined time after administration, five mice from each group were sacrificed. Gross dissection was performed on the sacrificed mice, and renal cortex, medulla, heart, liver, and duodenum tissues were collected from each mouse. The tissues were cut into pieces approximately 2 mm in size. 3 Small pieces, stored using RNA Later.

[0591] mRNA expression level detection

[0592] 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 seconds in a Tissuelyser II automated tissue homogenizer. After brief centrifugation and incubation at room temperature for 10 minutes, 200 μL of chloroform was added, and the mixture was vortexed and incubated at room temperature for 3 minutes. The samples were then centrifuged at 12000 rpm for 10 minutes at 4°C. 400 μL of the supernatant was transferred to a centrifuge tube containing 400 μL of isopropanol, mixed, and incubated at room temperature for 10 minutes. The samples were then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was discarded. 1 mL of 75% ethanol was added, and the centrifuge tube was inverted to wash the precipitate. The samples were then centrifuged at 12000 rpm for 5 minutes at 4°C, the supernatant was removed, and the samples were air-dried at room temperature to extract total RNA.

[0593] Take 1 μg of total RNA, and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) with Oligo(dT)15 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the 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). TMSelectMaster Mix (Catalog number: 4472908) was used to detect the expression level of target gene mRNA in tissues. 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. A 20 μL Real-time PCR reaction system was prepared for each PCR detection well according to the instructions of the real-time quantitative PCR kit. Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction and 10 μL of SYBR Green. TM Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of Nase-Free H2O. Place the prepared reaction mixture in a real-time quantitative PCR instrument (ABI, StepOnePlus). TM Real-time PCR amplification was performed using a three-step method on a 7500T ( / 7500) plate. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated for 40 cycles. In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of the target gene mRNA in each test group. The calculation method is as follows:

[0594] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0595] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0596] ΔCt(test group) = ΔCt(test group) – ΔCt(control group average)

[0597] ΔCt(control group) = ΔCt(control group) – ΔCt(control group average)

[0598] Here, ΔCt (control group mean) is the arithmetic mean of the ΔCt (control group) values ​​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.

[0599] The relative expression level of the target gene mRNA in the test group was 2. -ΔΔCt( (Test group) × 100%

[0600] 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%.

[0601] 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

[0602] Unless otherwise stated, in vivo activity data are presented in [year]. It is noted that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0603] Example 1: Evaluation of the inhibitory activity of NM064-linked small molecule conjugate sequence on the target gene superoxide dismutase 1 (SOD1) by intraventricular administration in mice.

[0604] In this embodiment, the inhibitory activity of the target gene SOD1 by the siRNA sequence RZ899123, which uses NM064 as the linker structure and is conjugated to LD300 at the 3' end of the positive strand, and the siRNA sequence RZ899124, which is conjugated to LD301 at the 3' end of the positive strand, was evaluated in different regions of the mouse brain using the mouse lateral ventricle drug delivery target gene inhibitory activity assessment method.

[0605] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of five mice each, based on body weight. Each group of mice was administered the aforementioned siRNA conjugate via lateral ventricle administration, with a dose of 150 μg per mouse. The PBS control group received the same volume of PBS solution without the siRNA conjugate. The day of administration was designated as day 0 (D0), and mice were sacrificed on day 5. The left and right hemispheres were preserved in RNAlaters, respectively. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed using real-time fluorescence. Gene expression differences were calculated using the ΔΔCt method described above.

[0606] Table 6 Primer sequence list

[0607] The results of Example 1 showed that after administration, the LD300 conjugate RZ899123 and the LD301 conjugate RZ899124 at the 3' end of the D5 sense chain had a certain degree of inhibition in both the left and right hemispheres, with the inhibitory activity reaching 70% or more on the administration side (right side) (Figure 1, Table 7).

[0608] Table 7 shows the inhibitory activity of the target gene in the mouse brain after administration of the siRNA conjugate described in this example.

[0609] Example 2. Evaluation of the inhibitory activity of NM064-linked small molecule conjugate sequence against SOD1 in rats via intrathecal administration.

[0610] This embodiment uses a rat intrathecal drug delivery target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ899124, which is conjugated to the LD301 terminal of the positive strand with NM064 as the linker structure, on the target gene SOD1 in the rat spinal cord and brain.

[0611] Six- to eight-week-old SD rats were randomly divided into two groups of 15 rats each, based on body weight. Each group of rats was administered the aforementioned siRNA conjugate intrathecally at a dose of 900 μg per rat, in a volume of 40 μL. The PBS control group received the same volume of PBS solution without the siRNA conjugate. The day of administration was designated D0. Rats were sacrificed on D14, D28, and D56. Tissues from different spinal cord segments (lumbar, thoracic, and cervical), cerebral cortex, hippocampus, cerebellum, liver, and kidneys were collected and preserved in RNAlater. RNA was extracted from each tissue, reverse transcribed, and quantitatively analyzed using real-time fluorescence. Gene expression differences were calculated using the ΔΔCt method described above.

[0612] Table 8 Primer sequence list

[0613] The results of Example 2 showed that at D14, RZ899124 maintained an inhibitory activity of over 90% in the lumbar and thoracic segments, and approximately 70% in the cervical segment, with over 40% activity in the cerebellum and cortex; at D28, the activity in the lumbar and thoracic segments remained above 80%, reaching 65% in the cervical segment, and over 40% in the cerebellum and cortex; at D56, the inhibitory activity in the lumbar and thoracic segments remained above 80%, and the cervical segment was consistent with D28 at around 67%, with the cerebellum still maintaining over 50% inhibitory activity, and no inhibitory activity in the liver and kidneys (Figures 2-8, Table 9).

[0614] Table 9 shows the inhibitory activity of the target gene in rats after administration of the siRNA conjugate described in this example.

[0615] Example 3. Evaluation of the inhibitory activity of the NM064-LD105 conjugate sequence on the target gene URAT1 in mice.

[0616] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ891003, which is conjugated with two LD105 clusters at the 3' end of the positive strand with NM064 as the linker structure, on the target gene URAT1 in mice after different doses were administered.

[0617] Six- to eight-week-old C57BL / 6J mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received a single subcutaneous intraperitoneal administration of the aforementioned siRNA sequence. The PBS control group received 5 mL / kg of siRNA per mouse. The three experimental groups received 10 mg / kg, 20 mg / kg, and 50 mg / kg (based on siRNA), respectively, with an administration volume of 5 mL / kg. The day of administration was designated D0. Five mice from each group were sacrificed on D7, D14, and D28. 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, with primers shown in the table below. Gene expression differences were calculated using the ΔΔCt method.

[0618] Table 10 Primer Sequence List

[0619] The results of Example 3 showed that the siRNA sequence RZ891003, which is conjugated to two clusters of LD105 at the 3' ends of the D7 and D14 positive strands, had a certain inhibitory effect on target genes in the renal cortex, and the inhibitory activity was dose-dependent (Figure 9, Table 11).

[0620] Table 11 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this embodiment.

[0621] Example 4. Evaluation of the inhibitory activity of the NM041-LD105 conjugate sequence on SOD1 in mice.

[0622] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ899103, which is conjugated with two LD105 clusters at the 3' end of the positive strand with NM041 as the linker structure, and the control sequence RZ899056 without vector conjugation on the target gene SOD1 in mice.

[0623] 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 sequence subcutaneously via abdominal administration. In the PBS control group, the dose was 5 mL / kg per mouse, while in the experimental groups, the dose was 3 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. Administration continued for three consecutive days. The day of administration was designated D0. On day 14 (D16) after the last administration, five mice from each group were sacrificed. Gross dissection was performed, and renal cortex, renal medulla, heart, liver, and duodenum tissues were collected and cut into several 2 mm sections. 3Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, with primers shown in Table 6 of Example 1. Gene expression differences were calculated using the ΔΔCt method.

[0624] The results of Example 4 showed that the siRNA sequence RZ899103, which uses NM041 as the linker structure and is conjugated with two clusters of LD105 at the 3' end of the positive strand, exhibited a better inhibitory effect in the renal cortex than the unconjugated siRNA sequence RZ899056. Meanwhile, no target gene inhibition effect was observed in the heart, liver, duodenum, and other tissues, indicating a kidney-specific targeting effect (Figure 10, Figure 11, Table 12).

[0625] Table 12 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this embodiment.

[0626] Example 5. Evaluation of the inhibitory activity of the NM064-ligated LD100 conjugate against the target gene URAT1 in mice.

[0627] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ891002, which is conjugated with two LD100 clusters at the 3' end of the positive strand with NM041 as the linker structure, and the vector-free siRNA sequence RX891001, on the target gene URAT1 in mice.

[0628] 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. In the PBS control group, the dose was 5 mL / kg per mouse, while in the experimental groups, the dose was 3 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. Administration continued for three consecutive days. The day of administration was designated D0. On day 6 (D8) after the last administration, five mice from each group were sacrificed. Gross dissection was performed, and the renal cortex and medulla were collected and cut into several 2 mm sections. 3 The small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, with primers shown in Table 10 of Example 3. Gene expression differences were calculated using the ΔΔCt method.

[0629] The results of Example 5 show that the siRNA sequence RZ891002, which is conjugated with two clusters of LD100 at the 3' end of the positive strand with NM041 as the linker structure, has a certain inhibitory effect on target genes in both the renal cortex and medulla, and shows a better inhibitory effect in the renal cortex. In contrast, the siRNA sequence RX891001 without conjugation has virtually no inhibitory activity in the kidney (Figure 12, Table 13).

[0630] Table 13 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this embodiment.

[0631] Example 6. Evaluation of the inhibitory activity of the NM041-LD100 conjugate sequence on the target gene SOD1 in mice.

[0632] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the following siRNA sequences at the SOD1 target site in mice: RZ899063 (with NM041 as the linker structure, two clusters at the 3' end of the LD100 positive strand), RZ899064 (with two clusters at the 5' end of the LD100 positive strand), RZ899065 (with four clusters at the 3' end of the LD100 positive strand), and RZ899056 (without vector conjugation).

[0633] Six- to eight-week-old C57BL / 6j mice were randomly divided into five groups of ten mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the PBS control group, the dose was 5 mL / kg per mouse, while in the siRNA conjugate experimental group, the dose was 3 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. Administration continued for three consecutive days. The day of administration was designated D0. On day 14 (D16) and day 21 (D23) after the last administration, five mice from each group were sacrificed. Gross dissection was performed, and renal cortex and medulla tissue were collected and cut into several 2 mm sections. 3 Small fragments were preserved using RNAlater. RNA extraction and Real-time PCR detection were performed as described above, with primers shown in Table 6 of Example 1. Gene expression differences were calculated using the ΔΔCt method.

[0634] The results of Example 6 showed that, compared with the carrier-free conjugate RZ899056, the LD100-conjugated siRNA sequences RZ899063, RZ899064, and RZ899065 all exhibited higher inhibitory activity in the renal cortex. Among them, the LD100-conjugated siRNA sequence RZ899063, which has two clusters conjugated at the 3' end of the positive strand, showed better inhibitory effect and longer duration of action in the kidney (Figures 13-14, Table 14).

[0635] Table 14 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example.

[0636] Example 7: Evaluation of the inhibitory activity of NM064-linked small molecule ligand conjugate sequence targeting integrins on the target gene superoxide dismutase 1 (SOD1).

[0637] In this embodiment, an in vivo mouse activity assessment assay was used to evaluate the inhibitory activity of the sequences RZ699168, RZ699173, RZ699174, RZ699175, RZ699176, and RZ699177, which are conjugated with two clusters of small molecule ligands targeting integrins at the 3' end of the positive strand using NM064 as a linker, on the target gene SOD1.

[0638] Six- to eight-week-old C57BL / 6j mice were randomly divided into seven groups of five mice each, based on body weight. Each group of mice was administered the aforementioned siRNA conjugate via tail vein injection at a dose of 5 mg / kg per mouse, with a volume of 5 mL / kg. The day of administration was designated D0, and mice were sacrificed on D7. Quadriceps muscle, heart, liver, and kidney samples were collected and stored in RNAlater. RNA extraction, reverse transcription, and real-time quantitative PCR were performed on each tissue using the methods described above. Primers are shown in Table 6 of Example 1. Differences in target gene expression were calculated using the ΔΔCt method described above.

[0639] The results of Example 7 showed that, on day 7 after drug administration, the small molecule conjugate sequences RZ699168, RZ699173, RZ699174, RZ699175, RZ699176, and RZ699177, which target integrin, exhibited superior inhibitory activity in muscle, with an inhibitory effect of approximately 80% or higher. However, their activity in tissues such as the heart, liver, kidney, and lung was all below 50%, demonstrating good muscle targeting. (Figure 15, Table 15) Table 15: Inhibitory activity of the target gene in mice after administration of the siRNA conjugates described in this example.

[0640] Example 8: Evaluation of the inhibitory activity of the NM064-linked LRP2-targeting peptide ligand conjugate sequence against the target gene SOD1 in mice.

[0641] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequences RZ699246-RZ699256, which are conjugated with two clusters of peptide ligands targeting kidney LRP2 at the 3' end of the positive strand using NM064 as the linker, on the target gene SOD1.

[0642] Six- to eight-week-old C57BL / 6j mice were randomly divided into 12 groups (n=5 per group) according to body weight. Each group of mice was administered the aforementioned siRNA conjugate subcutaneously via abdominal administration. In the PBS control group, the administration volume was 5 mL / kg per mouse, while in the siRNA conjugate experimental group, the dosage was 3 mg / kg (based on siRNA) per mouse, administered at a volume of 5 mL / kg. Administration continued for 3 consecutive days. The day of administration was designated as day 0 (D0). On day 14 (D16) after the last administration, mice in each group were sacrificed, and the animals were grossly dissected. 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, with primers shown in Table 6 of Example 1. Gene expression differences were calculated using the ΔΔCt method.

[0643] The results of Example 8 showed that, after administration on day 16, all siRNA sequences conjugated with ligand peptides targeting LPR2 exhibited certain target gene inhibitory activity in the renal cortex. Among them, RZ699252, RZ699254, and RZ699256 showed higher inhibitory activity, at 50% or above. (Figure 16, Table 16)

[0644] Table 16 shows the inhibitory activity of the mouse target gene after administration of the siRNA conjugate described in this embodiment.

[0645] 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. A ligand compound, characterized in that, The ligand compound is selected from or includes the structure shown in formula (I), its tautomers, or its stereoisomers: Among them, A is selected from 5- to 8-membered oxygen-containing heterocycles; p and q are each independently selected from 0, 1, 2, 3 or 4; R1 is selected from H, a hydroxyl protecting group, or... Wherein, * represents the linking site used to connect the active drug molecule; Z is selected from hydroxyl or thiol groups; R2 is selected from H, reactive phosphorus groups, or... Among them, R 2b Selected from solid supports containing amino functional groups, R 2a Selected from covalent linkages or chemical bonds connected to the amino functional group; R3 is selected from H, hydroxyl, amino, halogen, C1-C6 alkyl or C1-C6 alkoxy; n is an integer selected from 1 to 10; L1 is selected from L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 20 alkyne group, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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-; or L 2a -L 2b Composition (-CH2-CH2-O-) n Unit, n is an integer from 0 to 20; L 2c Selected from C1 to C5 alkylene groups; If L2 has substituents, then each substituent of L2 is independently selected from halogens, amino groups, hydroxyl groups, and C1-C2 groups. 20 Alkoxy, C2-C 20 Alkyl, C2-C 20 alkenyl or C2-C 20 Alkynyl group; or substituents on one or more carbon atoms in L2 can be linked together to form a saturated or unsaturated ring; L3 is selected from -O-, -NH-, -NHC(O)-, -(NH)C(O)O-, -(NH)-O-, -C(O)-, -C(O)NH- or -C(O)O-; R4 is selected from a targeting ligand group that can bind to receptors on the surface of target cells.

2. The ligand compound according to claim 1, characterized in that, A is selected from 5- to 8-membered saturated oxygen-containing heterocycles; Optionally, A is selected from a six-membered saturated heterocycle containing one oxygen atom; Optionally, A is selected from Optionally, the compound is selected from or includes the structure shown in formula (II), its tautomers, or its stereoisomers: Optionally, p and q are each independently selected from 0 or 1; preferably, p = 1 and q = 1, or p = 1 and q = 0, or p = 0 and q = 1, or p = 0 and q = 0; Optionally, p = 1 and q = 0; Optionally, R3 is selected from H or C1-C6 alkoxy groups; Optionally, R3 is selected from H or methoxy; Optionally, the hydroxyl protecting group is selected from triphenylmethyl, 4-methoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl or 4,4',4”-trimethoxytriphenyl; Optionally, the active pharmaceutical molecule is selected from double-stranded oligonucleotides; Optionally, the double-stranded oligonucleotide is selected from siRNA; Optionally, the solid support is selected from resins containing amino functional groups or glass beads with controllable pore size containing amino functional groups.

3. The ligand compound according to any one of claims 1-2, characterized in that, The ligand compound is selected from the structure shown in formula (III), or its tautomers, or its stereoisomers: And / or, the compound is selected from the structure shown in formula (IV), or its tautomers, or its stereoisomers: The definitions of each substituent are the same as those in claim 1 or 2.

4. The ligand compound according to any one of claims 1-3, characterized in that, L2 is selected from substituted or unsubstituted C1 to C2. 20 Alkylene, or substituted or unsubstituted Where j is selected from integers from 1 to 10; each L 2a Each is independently selected from C1 to C5 alkylene groups; each L 2b 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 2c Selected from C1 to C5 alkylene groups; Optionally, L2 is selected from substituted or unsubstituted C1 to C2. 15 alkylene or Where j is selected from integers from 2 to 6.

5. The ligand compound according to claims 1-4, characterized in that, L3 is selected from -O-, -NH-, -NHC(O)-, -C(O)- or -C(O)NH-.

6. The ligand compound according to claims 1-5, characterized in that, The target cells are selected from kidney cells, central nervous system cells, skeletal muscle cells, eye cells, cardiomyocytes, fat cells, or lung cells; Optionally, R4 is selected from substituted or unsubstituted C. 12 ~C 30 Alkyl, substituted or unsubstituted C 12 ~C 30 Alkenyl, substituted or unsubstituted C2-C 30 Alkyne group, CB1 ligand group, TrkB ligand group, αvβ6 ligand group, or polypeptide targeting ligand group; if R4 contains a substituent, the substituent is independently selected from amino, hydroxyl, halogen, C1-C1 groups. 20 Alkoxy, C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group; Optionally, the CB1 ligand group includes, but is not limited to, the following: Optionally, the TrkB ligand group includes, but is not limited to, the following: Optionally, the αvβ6 ligand group includes, but is not limited to, the following: Optionally, the polypeptide targeting ligand group includes a kidney-targeting polypeptide ligand group, a CNS-targeting polypeptide ligand group, or a lung-targeting polypeptide ligand group; Optionally, the kidney-targeting polypeptide ligand group comprises at least one of the amino acid sequences shown in (1) to (19) from the amino terminus to the carboxyl terminus: (1)-KKEEE-KKEEE-KKEEE-K- (2)-SHSNTQTLAKAPEHTGC- (3)-CKKEEE-KKEEE-KKEEE-K- (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC- (5)-CLPVASC- (6) Cyclic peptide (7)-CYFQNCPRG- (8) (9)-KKKEEKKKEEKKKEEK- (10)-KKEEEOOEEEKKEEE-K- (11)-OKEEEOKEEEOKEEE-O- (12)-KKEEERREEEKKEEE-K- (13)-RKEEERKEEERKEEE-R- (14)-KKEEDKKEEDKKEED-K- (15)-KKEEEKKQQQKKEEE-K- (16)-KKEEQKKEEQKKEEQ-K- (17)-CKKEEEKKEEEKKEEE-KC- (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K- (19)-CRPPR- in, The thiol groups on the C-side chains of the two cysteine ​​residues in this amino acid sequence form disulfide bonds, thus forming a cyclic peptide structure. Optionally, the kidney-targeting polypeptide ligand group is selected from at least one of (1) to (19): (1)-KKEEE-KKEEE-KKEEE-K-NH2 (2)-SHSNTQTLAKAPEHTGC-NH2 (3)-CKKEEE-KKEEE-KKEEE-K-NH2 (4)-SHSNTQTLA-K(CO-CH2CH2-PEG2-N3)-APEHTGC-NH2 (5)-CLPVASC-NH2 (6) (7)-CYFQNCPRG-NH2 (8) (9)-KKKEEKKKEEKKKEEK-NH2 (10)-KKEEEOOEEEKKEEE-K-NH2 (11)-OKEEEOKEEEOKEEE-O-NH2 (12)-KKEEERREEEKKEEE-K-NH2 (13)-RKEEERKEEERKEEE-R-NH2 (14)-KKEEDKKEEDKKEED-K-NH2 (15)-KKEEEKKQQQKKEEE-K-NH2 (16)-KKEEQKKEEQKKEEQ-K-NH2 (17)-CKKEEEKKEEEKKEEE-KC-NH2 (18)-K(Ac)KEEQK(Ac)KEEQK(Ac)KEEE-K-NH2 (19)-CRPPR-NH2.

7. The ligand compound according to any one of claims 1-6, characterized in that, The ligand compound is selected from any of the following compounds:

8. A double-stranded oligonucleotide conjugate, characterized in that, The double-stranded oligonucleotide conjugate comprises a sense strand and an antisense strand, wherein the sense strand and / or the antisense strand is conjugated to at least one targeting delivery group, the targeting delivery group comprising the structure shown in formula (i), or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Wherein, A, p, q, R3, n, L1, L2, L3, and R4 are as defined in any one of claims 1-7.

9. The double-stranded oligonucleotide conjugate according to claim 8, characterized in that, The targeted delivery group comprises the structure shown in formula (ii), or its tautomer, or its stereoisomer, or its pharmaceutically acceptable salt: Optionally, the targeted delivery group comprises the structure shown in formula (iii), or a tautomer thereof, or a stereoisomer thereof: And / or, the targeted delivery group comprises the structure shown in formula (iv), or a tautomer thereof, or a stereoisomer thereof:

10. The double-stranded oligonucleotide conjugate according to any one of claims 8-9, characterized in that, The positive chain is conjugated with at least one targeting delivery group; optionally, each of the targeting delivery groups is independently located at the 5' end or the 3' end of the positive chain; Optionally, the positive chain is conjugated with a targeting delivery group, one of which is located at the 5' end or 3' end of the positive chain; Optionally, the positive chain is conjugated with two targeting delivery groups, which are connected by a phosphate diester bond or a thiophosphate diester bond, and the two connected targeting delivery groups are located at the 5' end or the 3' end of the positive chain; Alternatively, the positive chain may be conjugated with two targeting delivery groups, which are located at the 5' end and 3' end of the positive chain, respectively.

11. The double-stranded oligonucleotide conjugate according to any one of claims 8-10, characterized in that, The double-stranded oligonucleotide conjugate is selected from siRNA conjugates.

12. A composition, characterized in that, The composition comprises the double-stranded oligonucleotide conjugate according to any one of claims 8-11.

13. Any of the following uses in the preparation of medicaments for the treatment and / or prevention of diseases associated with dysregulation of mRNA levels of target gene expression: (I) The ligand compound as described in any one of claims 1-7; and / or (II) The double-stranded oligonucleotide conjugate as described in any one of claims 8-11; and / or (III) The composition as described in claim 12.

14. The use of any of the following in the preparation of a drug for reducing the expression or activity of a target gene: (I) The ligand compound as described in any one of claims 1-7; and / or (II) The double-stranded oligonucleotide conjugate as described in any one of claims 8-11; and / or (III) The composition as described in claim 12.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the following and a pharmaceutically acceptable carrier or excipient: (I) The ligand compound as described in any one of claims 1-7; and / or (II) The double-stranded oligonucleotide conjugate as described in any one of claims 8-11; and / or (III) The composition as described in claim 12.

16. A method for reducing the expression or activity of a target gene, characterized in that, The method includes contacting the cells with any of the following: (I) The ligand compound as described in any one of claims 1-7; and / or (II) The double-stranded oligonucleotide conjugate as described in any one of claims 8-11; and / or (III) The composition as described in claim 12; and / or (IV) The pharmaceutical composition as described in claim 15; Optionally, the cells include, but are not limited to, kidney cells, central nervous system cells, skeletal muscle cells, eye cells, cardiomyocytes, fat cells, and lung cells.

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