A compound, conjugate, composition, and their uses

Abstract

The present disclosure relates to the field of medicine, in particular to a compound, a conjugate, a composition and their uses. The compound has a structure shown in formula (Ia). The conjugate provided by the present disclosure is formed by conjugating the compound provided by the present disclosure to an oligonucleotide. The conjugate provided by the present disclosure can be highly effectively targeted to the liver and effectively inhibit the expression of a target gene in the liver, and can be used for treating and / or preventing a liver-derived disease, has higher in vivo activity than a control vector, more stable and persistent pharmacodynamic effect and is expected to have excellent safety and low animal level toxicity.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of medicine, in particular, the present disclosure relates to a compound, conjugate, composition and their uses. BACKGROUND

[0002] Small molecule nucleic acid drugs such as small interference RNA (siRNA), antisense oligodeoxynucleotide (ASODN) and nucleic acid stimulating motif (CpG) play an increasingly important role in gene therapy. Some drugs have been approved by FDA for marketing, and currently, a variety of nucleic acid drugs are in preclinical and clinical trials. Nucleic acid drugs refer to nucleic acid sequences that specifically target pathogenic genes or proteins through binding or cleavage to inhibit / promote the expression of certain genes / proteins, including all human normal genes that can replace defective genes, antisense nucleic acids that block gene expression or single-stranded nucleic acids that promote triple-strand formation, such as siRNA, DNA, MicroRNA or CpG, etc.

[0003] Delivery system is one of the core key technologies in the development of small nucleic acid drugs. The most widely studied delivery system for small nucleic acid delivery system worldwide is the targeted conjugate delivery technology. There is still an urgent need in the art to develop a new drug conjugate with high in vivo delivery efficiency of active drugs, low toxicity and high activity. SUMMARY

[0004] The present disclosure aims to at least partially solve at least one of the technical problems existing in the prior art.

[0005] To this end, the present disclosure provides a compound, conjugate, composition and their uses. The conjugate provided by the present disclosure can be highly effective in targeting the liver and effectively inhibiting the expression of target genes in the liver, and can be used for treating and / or preventing liver diseases, has higher in vivo activity than the control vector, more stable and persistent drug efficacy, and is expected to have excellent safety and low animal level toxicity.

[0006] In a first aspect of the present disclosure, the present disclosure provides a compound having a structure represented by formula (Ia) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0007]

[0008] wherein R1 is selected from H, a hydroxyl protecting group or * represents a linking site for connecting a pharmaceutically active molecule;

[0009] each Z is independently selected from hydroxyl or thiol;

[0010] R2is selected from H, a reactive phosphorus group or R 2b selected from a solid support comprising an amino functional group, R 2a selected from a covalent linker attached to the amino functional group;

[0011] n is selected from 0, 1, 2 or 3;

[0012] each j is independently selected from 1, 2 or 3;

[0013] each L is independently selected from a substituted or unsubstituted C2-C 10 alkylene or wherein each R L is independently selected from a substituted or unsubstituted C1-C5 alkylene, each X is independently selected from O, S, NH or -NH-C(O)-, and m is selected from 1, 2, 3, 4 or 5;

[0014] each R3is independently selected from H, a substituted or unsubstituted C1-C4 alkyl acyl group or a substituted or unsubstituted C5-C7 aryl acyl group, each substituent in R3is independently selected from halogen, C1-C2 alkoxy.

[0015] In some alternative embodiments of the present disclosure, each L is independently selected from a substituted or unsubstituted C2-C 10 alkylene.

[0016] In some alternative embodiments of the present disclosure, each L is independently selected from a substituted or unsubstituted C2-C 10 straight chain alkylene. For example: a substituted or unsubstituted C2-C9 straight chain alkylene, a substituted or unsubstituted C2-C8 straight chain alkylene, a substituted or unsubstituted C2-C7 straight chain alkylene, a substituted or unsubstituted C2-C6 straight chain alkylene, a substituted or unsubstituted C2-C5 straight chain alkylene, a substituted or unsubstituted C2-C4 straight chain alkylene, a substituted or unsubstituted C3-C9 straight chain alkylene, a substituted or unsubstituted C3-C8 straight chain alkylene, a substituted or unsubstituted C3-C7 straight chain alkylene, a substituted or unsubstituted C3-C6 straight chain alkylene, a substituted or unsubstituted C3-C5 straight chain alkylene, a substituted or unsubstituted C3-C4 straight chain alkylene, a substituted or unsubstituted C4-C9 straight chain alkylene, a substituted or unsubstituted C4-C8 straight chain alkylene, a substituted or unsubstituted C4-C7 straight chain alkylene, a substituted or unsubstituted C4-C6 straight chain alkylene, a substituted or unsubstituted C4-C5 straight chain alkylene, a substituted or unsubstituted C4 straight chain alkylene.

[0017] In some embodiments of the disclosure, each L is independently selected from

[0018] In some embodiments of the disclosure, each L is selected from

[0019] In some embodiments of the disclosure, j is selected from 1.

[0020] In some embodiments of the disclosure, each Z is selected from hydroxyl.

[0021] In some embodiments of the disclosure, n is selected from 0, 1 or 2.

[0022] In some embodiments of the disclosure, n is selected from 0.

[0023] In some embodiments of the disclosure, n is selected from 1.

[0024] In some embodiments of the disclosure, n is selected from 2.

[0025] In some embodiments of the disclosure, each R3is independently selected from H, substituted or unsubstituted C1-C4alkylacyl

[0026] (e.g., acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, 2-methylbutyryl, pivaloyl / tert-butyryl) or substituted or unsubstituted C5-C7arylacyl (e.g., benzoyl), each substituent in R3is independently selected from halogen (e.g., F).

[0027] In some embodiments of the disclosure, each R3is independently selected from H,

[0028] In some embodiments of the disclosure, each R3is independently selected from H or

[0029] In some embodiments of the disclosure, each R3is selected from

[0030] In some embodiments of the disclosure, each R3is selected from H.

[0031] In the disclosure, the hydroxyl protecting group can be various hydroxyl protecting groups as long as it can protect the hydroxyl group, and the specific type is not limited. In some embodiments, the hydroxyl protecting group is stable under basic conditions, but can be removed under acidic conditions.

[0032] In some alternative embodiments of the present disclosure, the hydroxyl protecting groups that can be used in the present disclosure include, but are not limited to, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox), trityl (Tr), 4-methoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), and 4,4',4"-trimethoxytrityl (TMTr).

[0033] In some alternative embodiments of the present disclosure, the hydroxyl protecting group is selected from trityl (Tr), 4-methoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), or 4,4',4"-trimethoxytrityl (TMTr).

[0034] In some specific embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr).

[0035] In some alternative embodiments of the present disclosure, selected from * represents a linking site for linking a pharmaceutically active molecule.

[0036] In some alternative embodiments of the present disclosure, the pharmaceutically active molecule is selected from a small molecule drug, an antibody, or an oligonucleotide.

[0037] In some alternative embodiments of the present disclosure, the oligonucleotide is selected from a single-stranded oligonucleotide and a double-stranded oligonucleotide.

[0038] In some alternative embodiments of the present disclosure, the single-stranded oligonucleotide is selected from an antisense oligonucleotide, an aptamer, a ribozyme, a deoxyribozyme, a circular RNA, a sense strand of siRNA, or an antisense strand of siRNA.

[0039] In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide is selected from a small interfering RNA, a double-stranded RNA, a microRNA, a small guide RNA, a small activating RNA, or a short hairpin RNA.

[0040] In some specific embodiments of the present disclosure, the pharmaceutically active molecule is selected from a double-stranded oligonucleotide.

[0041] In some specific embodiments of the present disclosure, the pharmaceutically active molecule is selected from an siRNA.

[0042] In some alternative embodiments of the present disclosure, the solid support is selected from a resin containing an amino functional group (e.g., Polystyrene, abbreviated PS) or a controlled pore glass (CPG) containing an amino functional group.

[0043] In some specific embodiments of the present disclosure, R 2a is selected from

[0044] In some specific embodiments of the present disclosure, R 2b is selected from is selected from a resin or a controlled pore glass;

[0045] In some specific embodiments of the present disclosure, is selected from

[0046] In some alternative embodiments of the present disclosure, each Z is independently selected from

[0047] In some specific embodiments of the present disclosure, each Z is In some specific embodiments of the present disclosure, each Z is In some specific embodiments of the present disclosure, each Z is In some specific embodiments of the present disclosure, each Z is

[0048] In some alternative embodiments of the present disclosure, each is independently selected from

[0049]

[0050] In the present disclosure, a “reactive phosphorus group” refers to a phosphorus-containing group that can be removed by reacting with other compounds.

[0051] In some alternative embodiments of the present disclosure, the reactive phosphorus group is selected from In some specific embodiments of the present disclosure, the reactive phosphorus group is selected from In some alternative embodiments of the present disclosure, the compound has a structural formula shown in Formula (II):

[0052]

[0053] wherein each R4 is independently selected from H, C1-C2 alkyl, or C1-C2 alkoxy;

[0054] each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0055] R1, R2, n, j, Z, R3are as defined above.

[0056] In some embodiments of the present disclosure, each R4is selected from H.

[0057] In some embodiments of the present disclosure, each k is selected from 4.

[0058] In some alternative embodiments of the present disclosure, the compound has a structural formula as shown in formula (III):

[0059]

[0060] wherein R1, R2, n, Z, R3, R4, k are as defined above.

[0061] In some alternative embodiments of the present disclosure, the compound has a structural formula as shown in formula (IV):

[0062]

[0063] wherein R1, R2, n, Z, R3, k are as defined above.

[0064] In some alternative embodiments of the present disclosure, the compound is selected from any one of the following structures:

[0065]

[0066]

[0067]

[0068] In a second aspect of the present disclosure, the present disclosure provides a conjugate having a structure as shown in formula (Ib) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0069]

[0070] wherein Nu represents an oligonucleotide;

[0071] n' is selected from 1, 2, 3, or 4;

[0072] j, Z, L are as defined above.

[0073] In some embodiments of the present disclosure, n' is selected from 1, 2, or 3.

[0074] In some embodiments of the present disclosure, n' is selected from 3.

[0075] In some alternative embodiments of the present disclosure, the conjugate has a structural formula as shown in formula (IIb):

[0076]

[0077] wherein Nu, j, Z, n', R4, k are as defined above.

[0078] In some alternative embodiments of the present disclosure, the conjugate has a structural formula as shown in formula (IIIb):

[0079]

[0080] wherein Nu, Z, n', R4, k are as defined above.

[0081] In some alternative embodiments of the present disclosure, the conjugate has a structural formula as shown in formula (IVb):

[0082]

[0083] wherein Nu, Z, n', k are as defined above.

[0084] In some specific embodiments of the present disclosure, the conjugate has a structural formula selected from

[0085]

[0086] In a third aspect of the present disclosure, the present disclosure provides a composition comprising the conjugate of the second aspect.

[0087] In some alternative embodiments of the present disclosure, the composition further comprises at least one pharmaceutically acceptable carrier.

[0088] In a fourth aspect of the present disclosure, the present disclosure provides a use of any of the following in the preparation of a medicament for the prevention and / or treatment of a disease:

[0089] (I) the compound of the first aspect; and / or

[0090] (II) the conjugate of the second aspect; and / or

[0091] (III) the composition of the third aspect.

[0092] In some embodiments of the present disclosure, the disease is selected from a pathological condition or disease caused by abnormal expression of a target gene in a liver cell. Illustratively, the target gene includes, but is not limited to, at least one of Apoa, ApoB, ApoC, ANGPTL3, PCSK9, SOD1, FVII, p53, C3, C4, C5, AGT, CFB, USP20, ASGR1, FTO, INHBE, HBV and HCV.

[0093] In some embodiments of the present disclosure, the target gene is selected from SOD1.

[0094] In a fifth aspect of the present disclosure, the present disclosure provides a method for reducing expression or activity of a target gene, the method comprising contacting a liver cell with any of:

[0095] (I) the conjugate of the second aspect; and / or

[0096] (II) the composition of the third aspect.

[0097] In some embodiments of the present disclosure, the target gene includes, but is not limited to, at least one of Apoa, ApoB, ApoC, ANGPTL3, PCSK9, SOD1, FVII, p53, C3, C4, C5, AGT, CFB, USP20, ASGR1, FTO, INHBE, HBV and HCV.

[0098] In some embodiments of the present disclosure, the target gene is selected from SOD1.

[0099] Additional aspects and advantages of the present disclosure will be in part apparent and in part pointed out hereinafter.

[0100] Beneficial effects

[0101] The conjugate provided by the present disclosure is formed by conjugating the compound provided by the present disclosure to an oligonucleotide. The conjugate provided by the present disclosure is capable of highly effective targeting to the liver and effectively inhibiting expression of a target gene in the liver, and can be used for treating and / or preventing a liver-derived disease, has higher in vivo activity than a control carrier, more stable and persistent pharmacodynamic effect, and is expected to have excellent safety and low animal level toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 is the inhibitory activity of a target gene in a mouse in vivo after administration of the siRNA conjugate described in Example 2.1;

[0103] Figure 2The inhibitory activity of the target gene in the mouse after administration of the siRNA conjugate described in Example 2.2 is evaluated. Specific embodiments

[0104] A specific embodiment of the present disclosure is described in detail below. It should be understood that the specific embodiment described herein is merely intended for the purpose of illustration and explanation of the present disclosure and is not intended to limit the present disclosure.

[0105] Explanation of terms

[0106] In the context of the present disclosure, the term "alkyl" refers to a chain alkyl group of the general formula The chain alkyl group can be a straight chain alkyl group or a branched chain alkyl group. Among them, the term "C1-C2 alkyl" refers to a chain alkyl group having 1 to 2 carbon atoms, for example, methyl or ethyl.

[0107] In the context of the present disclosure, the term "alkylene" refers to a chain alkylene group of the general formula The chain alkylene group can be a straight chain alkylene group or a branched chain alkylene group. Among them, the term "C2-C10 alkylene" refers to a chain alkylene group having 1 to 10 carbon atoms. 10

[0108] In the context of the present disclosure, the term "NH" refers to an imino group, which has the structural formula

[0109] In the context of the present disclosure, the term "alkanoyl" has the structural formula For example, "C1-C5 alkanoyl" can be acetyl, propionyl, n-butyryl, or isobutyryl, etc.

[0110] In the context of the present disclosure, the term "aroyl" has the structural formula Among them, Ar refers to an aryl group (aryl group), which refers to any functional group or substituent derived from a simple aromatic ring in organic chemistry. The simplest aryl group is phenyl (Phenyl), which is derived from benzene.

[0111] In the context of the present disclosure, the term "alkoxy" has the structural formula Among them, "C1-C2 alkoxy" can be methoxy or ethoxy.

[0112] In the context of the present disclosure, "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salt and pharmaceutically acceptable base addition salt.

[0113] ​In the context of the present disclosure, "pharmaceutically acceptable acid addition salt" refers to salts of a free base that retain the biological effectiveness of the free base and do not impart undesired toxicological effects on the recipient. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, hexanoates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbates, salicylates, 4-aminosalicylates, naphthalene-2- sulfonates, and the like. These salts can be prepared by methods known in the art.

[0114] In the context of the present disclosure, "pharmaceutically acceptable base addition salt" refers to salts of a free acid that retain the biological effectiveness of the free acid and do not impart undesired toxicological effects on the recipient. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0115] In the context of the present disclosure, "oligonucleotide" is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), generally consisting of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as ribonucleic acid interference, ribonuclease-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0116] The term denotes the site of attachment of a group by a covalent bond.

[0117] In the structural formula of the compounds of the present disclosure, the bond “—” represents unspecified configuration. If there is chiral isomerism in the chemical structure, the bond “—” can be , , or both and configurations. Although all the above structural formulae are drawn in certain isomer form for simplicity, the present disclosure can include all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers.

[0118] In the structural formula of the compounds of the present disclosure, the bond “=” represents unspecified configuration. If there is cis-trans isomerism in the chemical structure, the configuration of the bond “=” can be E type, Z type, or both E and Z configurations.

[0119] The following definitions shall apply unless otherwise indicated. For the purposes of this disclosure, chemical elements are identified by their atomic number as specified in the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75thEd., 1994. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference. Unless otherwise indicated, the terms used herein are to be interpreted in accordance with their ordinary meaning in the context of the present disclosure. The articles "a", "an", and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By the use of the term "or" is meant that the term is selected from the group, but that it is also possible to use more than one of the terms selected from the group. By the use of the terms "comprising", "comprises" or "comprised of" is meant that the terms are used to specify the presence of stated features, integers, steps or components but that the disclosure is not limited to who or what exactly is recited. It is further to be understood that the use of relational terms such as first, second, third and fourth, and the like, are used solely to distinguish one from another entity or action without necessarily implying a serial or chronological order of such entities or actions, or a matrix or hierarchy.

[0120] The term "comprising" is an open term, i.e. it includes the stated features but not excluding other features.

[0121] "Stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0122] "Chiral" refers to molecules which have the property of existing as two non-superimposable mirror images. "Achiral" refers to molecules which cannot be superimposed on their mirror image.

[0123] "Enantiomer" refers to two isomers of a compound which are non-superimposable mirror images of one another.

[0124] "Diastereomer" refers to two or more stereoisomers having at least two asymmetric centers which are not mirror images of one another. Diastereomers have different physical properties, even if they have the same molecular formula and structure. Mixtures of diastereomers can be separated by standard techniques, including but not limited to, high resolution analysis operations such as electrophoresis and chromatography, e.g., HPLC.

[0125] The stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0126] As described herein, the compounds of the present disclosure can be optionally substituted with one or more substituents, such as the compounds of the general formulae above, or as specific examples, sub-generic classes, and classes of compounds encompassed by the present disclosure in the Examples.

[0127] In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a particular substituent. Unless otherwise indicated, a substituted group can have a substituent at each substitutable position of the group. When more than one position in the general formula given can be substituted with one or more substituents selected from the indicated group, the substituents can be the same or different at each position.

[0128] The term "unsubstituted" means that the designated group bears no substituents.

[0129] The term "optionally substituted" can be used interchangeably with the term "unsubstituted or substituted" to mean that the structure is either unsubstituted or substituted with one or more substituents as described herein. Substituents as described herein include, but are not limited to, D, F, Cl, Br, I, N3, CN, NO2, OH, SH, NH2, alkyl, haloalkyl, haloalkoxy, haloalkylamino, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like.

[0130] In addition, it should be noted that the description "each independently" used in the present disclosure is interchangeable with "each independently" and "independently" unless explicitly indicated otherwise, and should be interpreted broadly, which means that the specific options expressed by the same symbols in different groups do not affect each other, or the specific options expressed by the same symbols in the same group do not affect each other. For example, R3, the specific options of R3 between the two structures "C1-C50 alkylene optionally substituted by R3" and "-C(O)-NH-C1-50 alkylene optionally substituted by R3" do not affect each other.

[0131] The term "small interfering RNA (siRNA)" is a class of double-stranded RNA, which contains a sense strand and an antisense strand, each strand having a length of 17 to 30 nucleotides. siRNA mediates the targeting and cleavage of RNA transcripts of the RISC pathway by forming a 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 the conversion into proteins.

[0132] In the context of the present disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The "sense strand (or passenger strand)" refers to an iRNA strand containing a sequence substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the iRNA.

[0133] It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest.

[0134] In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), generally consisting of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as RNA interference, ribozyme-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0135] In the context of the present disclosure, "antisense oligonucleotides (ASO)" is a single-stranded oligonucleotide molecule, generally consisting of 10-50 nucleotides. After entering the cell, ASO binds to the complementary target mRNA through base pairing under the action of ribonuclease H1, thereby inhibiting the expression of the target gene.

[0136] In the context of the present disclosure, capital letters A, U, G, C, T represent the base composition of nucleotides, lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorinated modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0137] In the context of the present disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (such as antibacterial agent, antifungal agent), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for any conventional carrier incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0138] In the context of the present disclosure, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except for any conventional excipient incompatible with the siRNA of the present disclosure, for example, any adverse biological effects produced or any other components of the pharmaceutically acceptable composition interacted in a harmful way, their use is also considered within the scope of the present disclosure.

[0139] In the context of the present disclosure, "subject" refers to any animal, such as a mammal or a marsupial. The subject of the present disclosure includes but is not limited to humans, non-human primates (e.g., monkeys), mice, pigs, horses, donkeys, cows, sheep, and any kind of poultry.

[0140] In the context of 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, therapeutic benefits. A “therapeutic benefit” means the eradication or improvement of the underlying disorder being treated. Here, a therapeutic 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.

[0141] In the context of this disclosure, “prevention” and “avoidance” are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” a 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.

[0142] In the context of this disclosure, unless otherwise stated, the reagent ratios described in the embodiments of this disclosure are calculated on a volume ratio (v / v).

[0143] Unless otherwise stated, all raw materials and reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. Details of some of the reagents used in this disclosure are shown in Table 1.

[0144] Table 1 Reagent Details

[0145]

[0146] Unless otherwise stated, the reagents and consumables (Table 2) and instruments and equipment (Table 3) used in this disclosure are all commercially available products from the following manufacturers.

[0147] Table 2 Main Reagents and Consumables

[0148]

[0149]

[0150] Table 3 Main Instruments and Equipment

[0151] Name Manufacturer Full-automatic nucleic acid extractor Zhejiang Hanwei Science and Technology Co., Ltd. High-speed refrigerated centrifuge Eppendorf NANODROP OneC Thermo Fisher Scientific Gradient PCR amplifier Eppendorf Fluorescent quantitative PCR instrument ABI StepOne Plus Gel imaging instrument Shanghai Tian Neng Life Science Co., Ltd. Electrophoresis instrument Beijing Liuyi Instrument Factory Tissuelyser II full-automatic tissue homogenizer Shanghai Jingxin Industrial Development Co., Ltd.

[0152] Preparation Example 1: Synthesis of Compound CR01018

[0153] In this preparation example, the synthetic route of compound CR01018 is as follows:

[0154]

[0155] (1-1) Synthesis of compound CR01018-2

[0156] Methanesulfonamide (1.28 g, 13.5 mmol) was dissolved in a mixture of 60 ml of tert-butyl alcohol and 30 ml of water at 25 °C, AD-mix-β (21 g) was added, the reaction system was cooled to 0 °C with an ice water bath, and compound CR01018-1 (2.85 g, 13.5 mmol, tert-butyl 4-vinylpiperidine-1-carboxylate) was added at 0 °C. The reaction system was warmed to 25 °C and stirred at 25 °C for 16 hours. After the reaction was completed, the reaction system was cooled to 0 °C with an ice water bath, and sodium thiosulfate pentahydrate (33.48 g, 135 mmol) was added at 0 °C. It was extracted with 100 ml of ethyl acetate three times (3 x 100 ml), the organic phase was combined, washed with 50 ml of saturated sodium chloride aqueous solution once (1 x 50 ml), dried over anhydrous sodium sulfate, and filtered. It was concentrated and purified by column chromatography (elution gradient: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01018-2 (3.31 g, yield 96.6%) in the form of a white solid.

[0157] (1-2) Synthesis of compound CR01018-3

[0158] Compound CR01018-2 (3.31 g, 13.5 mmol) was dissolved in 15 ml of 1,4-dioxane at 25 °C, and hydrogen chloride solution in 1,4-dioxane (15 mL, 4 mol / L) was added. The reaction was stirred for 2 hours. After the reaction was completed, the reaction solution was directly evaporated to dryness to obtain compound CR01018-3 (2.45 g, yield 100%) in the form of a white solid.

[0159] (1-3) Synthesis of compound CR01018-4

[0160] Compound GAL-5 (1.83 g, 4.1 mmol) was dissolved in 10 ml of N,N-dimethylformamide (DMF) at 25 °C, N,N-diisopropylethylamine (826.5 mg, 6.4 mmol), benzotriazol-1-yl-oxy-tris-(dimethylamino-phosphonium hexafluorophosphate (2.33 g, 6.2 mmol), and compound CR01018-3 (2.45 g, 13.5 mmol) were added, and the reaction was stirred for 3 hours. After the reaction was completed, the reaction solution was added to 100 ml of saturated sodium bicarbonate aqueous solution, and then extracted with 50 ml of ethyl acetate three times (3 x 50 ml). The organic phase was combined, washed with 50 ml of saturated sodium chloride aqueous solution once (1 x 50 ml), dried over anhydrous sodium sulfate, and filtered. It was concentrated and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 2 / 1, v / v) to obtain compound CR01018-4 (2.1 g, yield 90.6%) in the form of a yellow solid. ESI-MS (m / z) = 575.3 [M+H]+ .

[0161] Synthesis of compound CR01018-5

[0162] Compound CR01018-4 (2.13 g, 3.9 mmol) was dissolved in 20 ml of pyridine at 25 °C, the reaction system was cooled to 0 °C with an ice water bath, and 4,4'-dimethoxytrityl chloride (1.58 g, 4.7 mmol) was added in batches at 0 °C. The reaction was stirred at 0 °C for 1 h, and then methanol was added to quench the reaction. After the reaction was completed, the reaction solution was directly evaporated to dryness, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 2, v / v) to obtain compound CR01018-5 (3.08 g, yield 90.3%) in the form of a yellow solid. ESI-MS (m / z) = 877.6 [M+H] + .

[0163] Synthesis of compound CR01018

[0164] Compound CR01018-5 (800 mg, 0.91 mmol) was dissolved in 8 ml of dichloromethane at 25 °C, 4,5-dicyanoimidazole (87.0 mg, 0.73 mmol), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (306.3 mg, 1.09 mmol) were added, and the reaction was stirred at 25 °C for 1 h under nitrogen replacement for three times. After the reaction was completed, the reaction solution was washed with 10 ml of saturated aqueous sodium bicarbonate solution twice (2 x 10 ml) and 20 ml of saturated aqueous sodium chloride solution once (1 x 20 ml), dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 3, v / v) to obtain compound CR01018 (678 mg, yield 69.0%) in the form of a white solid. ESI-MS (m / z) = 1076.3 [M+H] + .

[0165] 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 9.2 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.35 - 7.18 (m, 7H), 6.88 (t, J = 8.0 Hz, 4H), 5.21 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.02 (s, 3H), 3.86 (dt, J = 16.7, 8.9 Hz, 3H), 3.73 (d, J = 2.7 Hz, 7H), 3.71 - 3.45 (m, 4H), 3.11 (s, 1H), 2.76 (t, J = 5.8 Hz, 1H), 2.61 - 2.53 (m, 1H), 2.23 (s, 2H), 2.08 (d, J = 9.0 Hz, 5H), 1.99 (s, 3H), 1.89 (s, 4H), 1.75 (s, 3H), 1.46 (s, 6H), 1.13 (t, J = 7.1 Hz, 9H), 1.02 (d, J = 6.7 Hz, 4H).

[0166] Preparation Example 2: Synthesis of compound CR01018Z

[0167] In this preparation example, the synthesis route of compound CR01018Z is as follows:

[0168]

[0169] (2-1) Synthesis of compound CR01018-6

[0170] Compound CR01018-5 (120 mg, 0.14 mmol) was dissolved in 2 ml of dichloromethane at 25 °C, triethylamine (34.6 mg, 0.35 mmol), 4-dimethylaminopyridine (1.67 mg, 0.01 mmol) and succinic anhydride (20.5 mg, 0.21 mmol) were added, and the reaction was stirred at 25 °C for 16 hours. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 1, v / v) to obtain compound CR01018-6 (108 mg, yield 80.8%) in yellow oil. ESI-MS (m / z) = 977.3 [M+H] + .

[0171] (2-2) Synthesis of compound CR01018Z

[0172] Compound CR01018-6 (50 mg, 0.05 mmol) was dissolved in 10 ml acetonitrile at 25 °C, and benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (24.2 mg, 0.07 mmol), N, N-diisopropyl ethylamine (13.2 mg, 0.1 mmol) and amino CPG (1.1 g, 100-200 mesh, loading capacity 80 umol / g) were added, and the reaction was stirred at 25 °C for 16 hours. After the reaction was completed, the reaction solution was directly filtered, the filter cake was first washed with 50 ml dichloromethane twice (2 x 50 ml), 50 ml acetonitrile three times (3 x 50 ml), and 50 ml ethyl acetate once (1 x 50 ml), and then vacuum dried.

[0173] Cap1 (5 ml), Cap2 (0.56 ml) and 4-dimethylaminopyridine (6.25 mg) were added to the dried filter cake, and the reaction was stirred at 25 °C for 5 hours. After the reaction was completed, the reaction solution was directly filtered, the filter cake was first washed with 50 ml acetonitrile three times (3 x 50 ml), and then vacuum dried to obtain compound CR01018Z (1.03 g, loading capacity 30 umol / g).

[0174] Cap1 and Cap2 are cap reagents, Cap1 is a 20% (by volume) N-methylimidazole solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20% (by volume) acetic anhydride solution in acetonitrile.

[0175] Preparation Example 3: Synthesis of reference compound CR01014

[0176] In this preparation example, the synthesis route of compound CR01014 is as follows:

[0177]

[0178] (3-1) Synthesis of compound CR01014-3

[0179] Compound CR01014-1 (5.0 g, maleic anhydride), compound CR01014-2 (12.1 g, N- (methoxymethyl)-N-(trimethylsilylmethyl)benzylamine) and trifluoroacetic acid (0.58 g) were dissolved in 35 ml dichloromethane, and nitrogen was replaced for 3 times, and the reaction was stirred at 25 °C for 3 hours. After the reaction was completed, the reaction solution was washed with 10 ml purified water once (1 x 10 ml) and 10 ml saturated sodium chloride aqueous solution once (1 x 10 ml) in sequence, the organic phases were combined, dried with anhydrous sodium sulfate and filtered, and concentrated to obtain compound CR01014-3 (11 g), which was directly used in the next step without purification. MS-ESI (m / z) = 232.0 [M+H] + .

[0180] Synthesis of compound CR01014-4

[0181] Compound CR01014-3 (4.1 g) was dissolved in 20 ml of tetrahydrofuran, replaced with nitrogen for 3 times, the reaction system was cooled to 0 °C with an ice water bath, and a tetrahydrofuran solution (1.0 mol / L, 17.7 ml) of LiAlH4was added dropwise at 0 °C. The reaction was stirred at 0 °C for 1 hour. After the reaction was completed, 32 ml of purified water was added dropwise to the reaction solution, followed by the addition of 24 ml of a 1.0 mol / L aqueous sodium hydroxide solution, and the filtrate was obtained by filtration. The filtrate was evaporated under reduced pressure to obtain compound CR01014-4 (6.0 g), which was directly used in the next step without purification. MS-ESI (m / z) = 222.3 [M+H] + .

[0182] Synthesis of compound CR01014-5

[0183] Compound CR01014-4 (5.0 g) was dissolved in 50 ml of methanol, wet palladium on carbon (0.5 g, 10 mass% loading) and palladium hydroxide on carbon (0.5 g, 10 mass% loading) were added respectively, the reaction system was replaced with hydrogen for 3 times, and the reaction was stirred at 40 °C for 16 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was evaporated under reduced pressure to obtain compound CR01014-5 (2.9 g). MS-ESI (m / z) = 132.18 [M+H] + .

[0184] Synthesis of compound CR01014-7

[0185] Compound CR01014-5 (4.0 g), compound CR01014-6 (2.8 g, N-benzyloxy carbonyl-4-aminobutyric acid), and N,N-diisopropyl ethylamine (15.6 g) were dissolved in 20 ml of N,N-dimethyl formamide, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (17.2 g) was added, the reaction system was replaced with nitrogen for 3 times, and the reaction was stirred at 25 °C for 3 hours. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01014-7 (1.46 g, yield 35.6%). MS-ESI (m / z) = 367.41 [M+H] + .

[0186] Synthesis of compound CR01014-8

[0187] Compound CR01014-7 (1.46 g) was dissolved in 10 ml of methanol, wet palladium carbon (0.15 g, 10 mass%) was added, and the reaction was stirred at 25 °C for 16 hours after hydrogen replacement for 3 times. After the reaction was completed, the reaction solution was filtered, and the filtrate was evaporated under reduced pressure to obtain compound CR01014-8 (1.06 g), which was directly used in the next reaction without purification. MS-ESI (m / z) = 233.28 [M+H] + .

[0188] (3-6) Synthesis of compound CR01014-9

[0189] Compound CR01014-8 (1.02 g) and compound GAL-5 (1.41 g) were dissolved in 14 ml of N,N-dimethylformamide, N,N-diisopropylethylamine (0.81 g) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.8 g) were added, and the reaction was stirred at 25 °C for 3 hours after nitrogen replacement for 3 times. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 2 / 1, v / v) to obtain compound CR01014-9 (1.28 g, yield 63.5%). MS-ESI (m / z) = 648.68 [M+H] + .

[0190] (3-7) Synthesis of compound CR01014-10

[0191] Compound CR01014-9 (1.18 g) was dissolved in 12 ml of pyridine, and 4,4'-dimethoxytrityl chloride (0.98 g) was added in batches, and the reaction was stirred at 25 °C for 3 hours after nitrogen replacement for 3 times. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 2, v / v) to obtain compound CR01014-10 (1.0 g, yield 61.1%). MS-ESI (m / z) = 951.0 [M+H] + .

[0192] (3-8) Synthesis of compound CR01014

[0193] Compound CR01014-10 (300 mg) was dissolved in 6 ml of dichloromethane, and bis (diisopropylamino) (2-cyanoethoxy) phosphine (152 mg) and 4, 5-dicyanoimidazole (30 mg) were added in batches, replaced with nitrogen for 3 times, and stirred at 25 °C for 3 hours. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 3, v / v) to obtain compound CR01014 (270 mg, yield 74.4%). MS-ESI (m / z) = 1151.27 [M+H] + .

[0194] 1H NMR (400 MHz, DMSO-d6) d: 0.92 - 1.03 (d, J = 6.7 Hz, 8H), 1.04 - 1.13 (d, J = 6.8 Hz, 7H), 1.16 - 1.29 (m, 4H), 1.38 - 1.53 (dq, J = 7.2, 14.6 Hz, 4H), 1.55 - 1.68 (dt, J = 9.0, 15.1 Hz, 2H), 1.75 - 1.82 (s, 3H), 1.86 - 1.94 (s, 3H), 1.97 - 2.07 (s, 6H), 2.09 - 2.15 (s, 3H), 2.15 - 2.29 (m, 2H), 2.64 - 2.76 (d, J = 5.5 Hz, 3H), 2.99 - 3.16 (dt, J = 6.7, 14.4 Hz, 4H), 3.20 - 3.30 (d, J = 14.8 Hz, 1H), 3.37 - 3.53 (tt, J = 7.1, 14.6 Hz, 6H), 3.53 - 3.66 (dt, J = 8.4, 17.5 Hz, 3H), 3.74 - 3.78 (s, 6H), 4.00 - 4.09 (s, 3H), 6.84 - 6.96 (d, J = 8.3 Hz, 4H), 7.19 - 7.28 (t, J = 7.6 Hz, 5H), 7.28 - 7.41 (dt, J = 7.8, 22.9 Hz, 4H), 7.70 - 7.88 (d, J = 5.9 Hz, 2H).

[0195] Preparation Example 4: Synthesis of Reference Compound CR01014Z

[0196] In this preparation example, the synthesis route of compound CR01014Z is as follows:

[0197]

[0198] (4-1) Synthesis of compound CR01014-11

[0199] Compound CR01014-10 (100 mg) was dissolved in 2 ml of dichloromethane, succinic anhydride (15.7 mg), 4-dimethylaminopyridine (1.2 mg) and triethylamine (19.7 mg) were added, and the reaction was stirred at 25 °C for 16 hours after nitrogen replacement for 3 times. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (C18 column, elution gradient: water / acetonitrile = 1 / 1, v / v) to obtain compound CR01014-11 (73 mg, yield 66.7%). MS-ESI (m / z) = [M+H] + .

[0200] Synthesis of compound CR01014Z

[0201] Compound CR01014-11 (50 mg), amino CPG (1.19 g), benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (27 mg) and N,N- diisopropylethylamine (12 mg) were mixed, and the reaction was carried out on a shaker for 16 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with 10 ml of acetonitrile once (1x10 ml) and then vacuum dried.

[0202] The dried filter cake, 4-dimethylaminopyridine (3 mg), Cap1 (10 ml) and Cap2 (10 ml) were mixed, and the reaction was carried out on a shaker for 6 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with 10 ml of acetonitrile once (1x10 ml) and then vacuum dried to obtain compound CR01014Z (1.03 g, loading 20-30 umol / g).

[0203] wherein Cap1 and Cap2 are cap reagents, Cap1 is a 20% (v / v) N-methylimidazole solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; and Cap2 is a 20% (v / v) acetic anhydride solution in acetonitrile.

[0204] Reference compound L96-PS

[0205] Compound L96-PS was purchased from Kalley Medicine Group (Tianjin) Co., Ltd., and the loading was 120±12 μmol / g (detection method: UV / HPLC).

[0206] The structural formula of compound L96-PS is as follows:

[0207]

[0208] wherein PS represents a polystyrene (Polystyrene) resin solid phase carrier.

[0209] Preparation Example 5: Preparation of siRNA conjugate

[0210] (5-1) Synthesis of sense strand (SS)

[0211] The nucleotide monomers are connected one by one in the order of 3'-5' according to the nucleotide sequence by the method of phosphoramidite nucleic acid solid phase synthesis, starting with the above-mentioned compound (i.e. CR01018Z, CR01014Z, L96-PS) linked to the solid phase carrier. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0212] The nucleotide monomers are prepared into an acetonitrile solution of nucleotide monomers with a concentration of 0.1 M.

[0213] The conditions of deprotection reaction in each step are the same. The conditions of deprotection reaction are as follows: temperature is 25°C, reaction time is 70 seconds, deprotection reagent is dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0214] The conditions of coupling reaction in each step are the same. The conditions of coupling reaction are as follows: temperature is 25°C, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleotide monomer is 1:10, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the coupling reagent is 1:65, reaction time is 600 seconds, the coupling reagent is 5-ethylthio-1H-tetrazole in acetonitrile solution with a concentration of 0.5 M, and the sulfur reagent is a mixture of acetonitrile / pyridine with a volume ratio of 1:1, in which the concentration of hydrogenated xanthine is 0.2 mol / L.

[0215] The conditions of capping reaction in each step are the same. The conditions of capping reaction are as follows: temperature is 25°C, reaction time is 2 minutes, and the capping reagent solution is a mixture of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is N-methylimidazole in pyridine / acetonitrile mixed solution with a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is acetic anhydride in acetonitrile solution with a concentration of 20% by volume; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence connected on the solid phase carrier is 1:1:1.

[0216] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature is 25°C; reaction time is 3 seconds; the concentration of the oxidizing agent is 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfurization reaction are: temperature is 25°C; reaction time is 360 seconds; the concentration of the sulfurizing agent is 0.2M pyridine solution of hydrogen xanthate, the molar ratio of the sulfurizing agent to the nucleic acid sequence attached to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0217] After the last nucleotide monomer is attached, the nucleic acid sequence attached to the solid support is sequentially subjected to cleavage, deprotection, purification, desalting, and then lyophilization to obtain the sense strand, wherein:

[0218] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence attached to the solid support is added to 25% ammonia water by mass, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and vacuum concentration is carried out to dryness. After ammonia water treatment, the product is dissolved in 0.4 ml / μmol N-methylpyrrolidine, followed by the addition of 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trifluoromethanesulfonate, and the 2'-O-TBDMS protection on the ribose is removed.

[0219] The purification and desalting conditions are as follows: the nucleic acid is purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 is 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1); the elution gradient is eluent 1:eluent 2=(100:0)-(50:50). After the product eluate is collected, desalting is carried out using a reverse phase chromatography purification column, and the desalting conditions include desalting using a dextran gel column, the filler is dextran gel G25, and elution is carried out using deionized water.

[0220] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using liquid chromatography-mass spectrometry (LC-MS), and the measured value of the molecular weight is compared with the theoretical value; if the measured value and the theoretical value are consistent, it indicates that the compound is conjugated to the 3' end of the sense strand of the siRNA.

[0221] In the synthesis of the sense strand, the following targeting ligands (carriers) are synthesized, respectively:

[0222] The structural formula of the two-cluster CR01018 (denoted as (CR01018) x 2) is:

[0223]

[0224] The structural formula of the three-cluster CR01018 (denoted as (CR01018) x 3) is:

[0225]

[0226] The structural formula of the two-cluster CR01014 (denoted as (CR01014) x 2) is:

[0227]

[0228] The structural formula of the three-cluster CR01014 (denoted as (CR01014) x 3) is:

[0229]

[0230] (5-2) Synthesis of antisense strand (AS)

[0231] The antisense strand was synthesized using a general solid-phase carrier. The deprotection, coupling, capping, oxidation or sulfuration reaction conditions, cleavage and deprotection conditions, purification and desalination conditions and steps in the solid-phase synthesis method of the antisense strand are the same as those in step (5-1) for synthesizing the sense strand.

[0232] Detection: purity detection was performed using ion exchange chromatography (IEX-HPLC); molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS), and the measured value was compared with the theoretical value. If the measured value and the theoretical value are consistent, it is indicated that the siRNA antisense strand is obtained.

[0233] (5-3) Synthesis of siRNA conjugate

[0234] The sense strand synthesized in step (5-1) and the antisense strand synthesized in step (5-2) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. Slow cooling to room temperature was performed and maintained at room temperature for 10 minutes, so that the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonding, thereby obtaining an siRNA conjugate having the sense strand and the antisense strand shown in Table 4.

[0235] Sense strand: UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm, as shown in SEQ ID NO: 1;

[0236] Antisense strand: UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm, as shown in SEQ ID NO: 2.

[0237] When the carrier is tri-cluster CR01018, the structural formula of the siRNA conjugate is as follows:

[0238]

[0239] When the carrier is tri-cluster CR01014, the structural formula of the siRNA conjugate is as follows:

[0240]

[0241] When the carrier is L96, the structural formula of the siRNA conjugate is as follows:

[0242]

[0243] wherein, represents siRNA, and the targeting ligand (carrier) is conjugated to the 3' end of the sense strand of siRNA.

[0244] Table 4 Sequence information of siRNA conjugates

[0245]

[0246] Unless otherwise specified, the base composition and modification described in each embodiment of the present disclosure are as follows: capital letters A, U, G, C, T represent the base composition of nucleotides, and lowercase letters m represent that the nucleotide represented by the previous letter is a methoxy-modified nucleotide; lowercase letters f represent that the nucleotide represented by the previous letter is a fluorine-modified nucleotide; and lowercase letters s represent that the nucleotide represented by the previous and subsequent letters is connected by a phosphorothioate bond.

[0247] Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Bexin Biotechnology Co., Ltd.; the PCR primers used in the present disclosure are synthesized by Beijing Qikexin Biotechnology Co., Ltd.; and the experimental animal C57BL / 6J mice used in the present disclosure are purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0248] Table 5 Detection results of siRNA conjugates

[0249]

[0250]

[0251] As can be seen from the data in Table 5, the sense strand (SS) and the antisense strand (AS) can be well connected to the ligand while maintaining a high purity.

[0252] General experiments

[0253] Experimental Example 1 In vivo toxicity experiment of siRNA conjugate

[0254] C57BL / 6J mice were randomly divided into 3 groups (i.e. RZ599001 experimental group, RZ899043 experimental group, RZ899027 experimental group), 2 mice in each group, half male and half female, and the mice in each experimental group were administered with siRNA conjugate at a single dose of 300 mg / kg mouse body weight (calculated based on siRNA) by subcutaneous injection, and observed continuously for 14 days. No animal death occurred, and no clinical symptoms related to drug adverse reactions were observed. After the observation, the mice were subjected to gross autopsy, and no abnormalities were found. Therefore, the above results show that the siRNA conjugate of the present disclosure is safe and has low animal level toxicity.

[0255] Example 2 Method for evaluating target gene inhibition activity in vivo in mice

[0256] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight (all female). The mice in each group were calculated for drug dose according to body weight, and administered with a single dose by subcutaneous injection in the abdomen. Each siRNA conjugate was prepared into a solution of corresponding concentration (calculated based on siRNA) with PBS solution for administration, and the administration volume was 5 ml / kg mouse body weight (calculated based on siRNA). The PBS control group was administered with PBS solution (without drug conjugate) at 5 ml / kg mouse body weight. The day of administration was recorded as day 1 (D1), and at the pre-set time after administration, for example, day 8 (D8), day 15 (D15) and day 29 (D29), 5 mice in each group were sacrificed. The sacrificed mice were subjected to gross autopsy, and the liver tissues of each sacrificed mouse were collected, cut into small pieces of about 2 mm3, and stored with RNA later.

[0257] The liver tissue samples at different time points in different experimental groups were taken from the above RNA later, and the liver tissue samples were crushed in a Tissuelyser II type automatic tissue homogenizer for 60 s. Then, total RNA was extracted using a full-automatic nucleic acid extractor (purchased from Zhejiang Hanwei Science and Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Science and Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0258] Take 1 μg of total RNA, use reverse transcription kit (Promega Company, Reverse Transcription System, A3500) and select Oligo (dT) 15 reverse transcription primer, according to the method recorded in the reverse transcription kit instruction book. Configure 20 μL reverse transcription system and complete reverse transcription reaction. After the reaction, add 80 μL RNase-Free water to the reverse transcription system to obtain cDNA solution. Then use real-time fluorescence quantitative PCR kit (ABI Company, SYBR TM Select MasterMix, Catalog number: 4472908) to detect the expression amount of target gene mRNA in liver tissue. In this real-time fluorescence quantitative PCR method, the primers for target genes and the primers for internal reference genes are used to detect target genes and internal reference genes, respectively. According to the method recorded in the real-time fluorescence quantitative PCR kit instruction book, configure 20 μL Real-time PCR reaction system for each PCR detection hole, which contains 5 μL cDNA solution obtained by the above reverse transcription reaction, 10 μL SYBR TM Select Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. Place the prepared reaction system on a real-time fluorescence quantitative PCR instrument (ABI Company, StepOnePlus TM ) and use three-step method for Real-time PCR amplification, with amplification program of 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, repeating the process of denaturation, annealing and extension for 40 cycles. In this real-time fluorescence quantitative PCR method, the ΔΔCt method is used to calculate the expression level and inhibition rate of target gene mRNA in each test group, and the calculation method is as follows:

[0259] ΔCt(test group) = Ct(test group target gene) - Ct(test group internal reference gene)

[0260] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0261] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0262] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0263] Wherein, ACt(control group average) is the arithmetic mean of the ACt(control group) of each of the 5 mice in the control group at the same time point. Therefore, each mouse in the test group and the control group corresponds to a ACt value.

[0264] The mRNA expression level of the target gene in the test group was normalized based on the control group, and the mRNA expression level of the target gene in the control group was defined as 100%.

[0265] The relative expression level of the target gene mRNA in the test group = 2-ACt(test group) x 100%

[0266] The mRNA expression inhibition rate of the target gene in the test group = (1- relative expression level of the target gene mRNA in the test group) x 100%

[0267] Unless otherwise specified, the in vivo activity experimental data are expressed as X ± SD, and the experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0268] Example 2.1 In vivo activity evaluation of tricistronic CR01018 vector conjugated superoxide dismutase 1 (SOD1) target gene

[0269] In this experimental example, the siRNA sequence RZ599001 conjugated with L96 carrier at the 3' end of the sense strand of siRNA and the siRNA sequence RZ899043 conjugated with tricistronic CR01018 carrier at the 3' end of the sense strand of siRNA were evaluated for their inhibitory activity on the target gene SOD1 in mice using the method for evaluating the inhibitory activity of target genes in mice. RZ899043 and RZ599001 have exactly the same nucleic acid sequence and chemical modification, and the only difference is the delivery carrier.

[0270] 6-8 week old C57BL / 6j mice were randomly divided into groups according to body weight, 15 mice in each group, a total of 3 groups. Each group of mice was given PBS solution and the above siRNA conjugate by subcutaneous administration in the abdomen, wherein the dose given to each mouse in the PBS control group was 5 ml / kg, and the dose given to each mouse in the siRNA conjugate experimental group was 3 mg / kg (calculated as siRNA), and the volume of the dose was 5 mL / kg. The day of administration is recorded as the first day (D1), and 5 mice from each group were sacrificed on the 8th day (D8), the 29th day (D29) and the 54th day (D54) after administration. The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ACt method.

[0271] Table 6 Primer sequence list of this example

[0272] The mRNA expression level of the target gene in the test group was normalized based on the control group, and the mRNA expression level of the target gene in the control group was defined as 100%.

[0265] The relative expression level of the target gene mRNA in the test group = 2-ACt(test group) x 100%

[0266] The mRNA expression inhibition rate of the target gene in the test group = (1- relative expression level of the target gene mRNA in the test group) x 100%

[0267] Unless otherwise specified, the in vivo activity experimental data are expressed as X ± SD, and the experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0268] Example 2.1 In vivo activity evaluation of tricistronic CR01018 vector conjugated superoxide dismutase 1 (SOD1) target gene

[0269] In this experimental example, the siRNA sequence RZ599001 conjugated with L96 carrier at the 3' end of the sense strand of siRNA and the siRNA sequence RZ899043 conjugated with tricistronic CR01018 carrier at the 3' end of the sense strand of siRNA were evaluated for their inhibitory activity on the target gene SOD1 in mice using the method for evaluating the inhibitory activity of target genes in mice. RZ899043 and RZ599001 have exactly the same nucleic acid sequence and chemical modification, and the only difference is the delivery carrier.

[0270] 6-8 week old C57BL / 6j mice were randomly divided into groups according to body weight, 15 mice in each group, a total of 3 groups. Each group of mice was given PBS solution and the above siRNA conjugate by subcutaneous administration in the abdomen, wherein the dose given to each mouse in the PBS control group was 5 ml / kg, and the dose given to each mouse in the siRNA conjugate experimental group was 3 mg / kg (calculated as siRNA), and the volume of the dose was 5 mL / kg. The day of administration is recorded as the first day (D1), and 5 mice from each group were sacrificed on the 8th day (D8), the 29th day (D29) and the 54th day (D54) after administration. The liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ACt method.

[0271] Table 6 Primer sequence list of this example

[0272]

[0273] Experimental results are as follows Figure 1 As shown in Table 4, the in vivo inhibitory effect and sustained efficacy of the siRNA conjugate RZ899043 conjugated with the tri-cluster CR01018 on the target gene are comparable to, and slightly better than, those of the siRNA conjugate RZ599001 conjugated with the L96.

[0274] Table 7 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in this example.

[0275]

[0276] Example 2.2 In vivo activity assessment of the superoxide dismutase 1 (SOD1) target gene conjugated to the tri-cluster CR01014 vector

[0277] This experiment evaluated the inhibitory activity of siRNA sequences RZ599001 (3' end conjugated to the L96 vector) and RZ899027 (3' end conjugated to the CR01014 vector) on the target gene SOD1 in mice using in vivo target gene inhibitory activity assessment methods. RZ899043 and RZ899027 have identical nucleic acid sequences and chemical modifications, differing only in their delivery vectors.

[0278] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of 20 mice each, based on body weight. Each group was administered PBS solution and the aforementioned siRNA conjugate subcutaneously via abdominal administration. In the PBS control group, the dose was 5 ml / kg body weight 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 body weight. The day of administration was designated as day 1 (D1). Five mice from each group were sacrificed on days 15 (D15), 29 (D29), 43 (D43), and 57 (D57). Liver tissue was collected for RNA extraction, reverse transcription, and Real-time PCR detection. The relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0279] The primer sequences for this embodiment are shown in Table 6 of Example 2.1.

[0280] Experimental results are as follows Figure 2As shown in Table 5, the in vivo inhibitory effect of siRNA conjugate RZ899027 conjugated with tri-cluster CR01014 on the target gene was equivalent to that of siRNA conjugate RZ599001 conjugated with L96 on D15, and the inhibitory effect was significantly weaker than that of siRNA conjugate RZ599001 conjugated with L96 on D29, D43 and D57.

[0281] Table 8 shows the inhibitory activity of siRNA conjugates described in this example on the target gene in mice

[0282]

[0283] As can be seen from Examples 2.1 and 2.2, the in vivo inhibitory effect and the pharmacodynamic duration of action of siRNA conjugate RZ899043 conjugated with tri-cluster CR01018 on the target gene were significantly superior to those of siRNA conjugate RZ899027 conjugated with tri-cluster CR01014.

[0284] It can be understood that the above embodiments are only exemplary embodiments adopted for illustrating the principles of the present disclosure, and the present disclosure is not limited thereto. Various modifications and improvements can be made by those of ordinary skill in the art without departing from the spirit and essential characteristics of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A compound having the structure of Formula (IVa) or a stereoisomer, a pharmaceutically acceptable salt thereof: wherein R1is selected from H, 4,4'-dimethoxytrityl or * represents a linking site for attachment of siRNA; each Z is independently selected from hydroxyl or thiol; R2is selected from H, or R 2b is selected from , is selected from a resin or a controlled pore glass sphere, R 2a is selected from ; n is selected from 0, 1, 2, or 3; each k is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3is independently selected from H or .

2. A compound characterized in that, the compound is selected from any one of the following structures: 。 3. A conjugate having the structure of Formula (IVb) or a stereoisomer, a pharmaceutically acceptable salt thereof: wherein, Nu represents a siRNA; n' is selected from 3; Z, k are as defined in claim 1.

4. A conjugate, characterized in that, the conjugate is selected from 。 5. A composition characterized in that, the composition comprises the conjugate of claim 4; the composition further comprises at least one pharmaceutically acceptable carrier.

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