Phosphoramidite lipid molecule and use thereof in rnai molecule

Abstract

The present invention belongs to the technical field of biochemistry, and specifically relates to a phosphoramidite lipid molecule and a use thereof in an RNAi molecule. In the present invention, a series of lipid molecules containing phosphoramidite are prepared and reacted with oligonucleotides to prepare an RNAi molecule. The RNAi molecule can effectively target extrahepatic tissues, while not interfering with the effect of inhibiting a target gene. The lipid molecule prepared in the present invention has good prospects in extrahepatic delivery vectors, and the RNAi molecule prepared in the present invention has important value for the research and development and clinical application of RNAi drugs.

Description

A phosphoramidite lipid molecule and its use in RNAi molecules Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a phosphoramidite lipid molecule and its use in RNAi molecules. Background Technology

[0002] In recent years, significant progress has been made in the development of siRNA drugs. siRNA is now widely known as an active pharmaceutical ingredient. RNA interference (RNAi) is a mechanism mediated by endonuclease complexes, through which siRNA exerts its effects. At the gene level, siRNA can treat almost all diseases, including tumors, infectious diseases, and genetic disorders, making it a hot area of ​​biomedical research. Theoretically, siRNA therapy can target genes in any pathogenic genome, exhibiting potent and long-lasting efficacy, significantly reducing dosing frequency, improving patient adherence, and thus achieving better therapeutic results.

[0003] However, RNAi therapies have also encountered unique challenges in their development. Targeted delivery of oligonucleotides, potential off-target effects, and in vivo stability have been major challenges in the development of RNAi drugs. Among these, achieving efficient RNAi drug delivery has become the primary constraint on the clinical application of such drugs.

[0004] To address these challenges, a range of technologies have been developed to overcome the delivery difficulties of RNAi drugs. Among these, the most important is the development of N-acetylgalactosamine (GalNAc)-siRNA conjugates, which successfully delivered siRNA to liver tissue. Although several biotechnology companies have successfully developed their extrahepatic delivery vectors, clinical studies have shown that different vectors exhibit varying degrees of effectiveness in delivering RNAi drugs. Furthermore, some delivery vectors even interfere with the pharmacological effects of RNAi.

[0005] Extrahepatic delivery of nucleic acids still faces many challenges. The development and application of novel extrahepatic delivery vectors are of great value for the research and clinical application of RNAi drugs. Therefore, developing a targeted delivery method for RNAi drugs to extrahepatic tissues remains a problem in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a phosphate backbone lipid compound and its use in RNAi molecules, with the aim of improving the tissue-targeted distribution of RNAi drug molecules in vivo.

[0007] This invention provides an oligonucleotide, wherein the structure shown in Formula I is attached to an internal position or end of the oligonucleotide;

[0008] in,

[0009] X1 is selected from S or O;

[0010] X2 is selected from O, S, CH2 or NH;

[0011] L is selected from cleavable or non-cleavable chemical linkers, wherein the chemical linkers are selected from -SS-, -CF2-, and -(CH2). b -, -NHCONH-, -NHCOO-, -NHCOCH2-, -NHCO-, -CONH-, -COO-, -OOC-, C3-C 10 cycloalkyl, C6-C 20 At least one of aryl and -NHSO2-, where b is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0012] R1 is selected from substituted or unsubstituted C1-C. 30 Alkyl, substituted or unsubstituted C2-C 30 alkenyl, substituted or unsubstituted C2-C 30 acetylinyl Substituents are selected from halogens, deuterium, C1-C 30 Straight-chain alkyl, C3-C 10 cycloalkyl, C1-C 30 Halogenated alkyl groups;

[0013] R5 is selected from unmodified or modified nucleosides, wherein the oxygen at the 3' end of the pentose sugar or the 3' end of the tetraose sugar in the nucleoside is linked to P, and the modified nucleoside is selected from the nucleoside of modified nucleotide a, wherein the modified nucleotide a is selected from threonucleotides, C6-C 20 Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-C1-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides.

[0014] q is selected from 0, 1, 2, 3, 4, 5.

[0015] Preferably, the oligonucleotide is composed of nucleotides, wherein the nucleotides are selected from unmodified nucleotides or modified nucleotide b, and the modified nucleotide b is selected from threonucleotides, C6-C... 20 Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-C1-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, 3'-OMe nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides.

[0016] Preferably, the connection is accomplished by replacing the phosphate ester group between two adjacent nucleotides in the oligonucleotide.

[0017] Preferably, the oligonucleotide consists of 14-45 nucleotides.

[0018] Preferably, the structures shown in Formula I are used to replace the 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, and 25- in the oligonucleotides. Phosphate groups between nucleotides at positions 26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, and 44-45, wherein the first nucleotide at the 5' end of the oligonucleotide is position 1.

[0019] Preferably, the oligonucleotide consists of 21-23 nucleotides.

[0020] Preferably, the phosphate ester groups between the nucleotides at positions 4-5, 5-6, 6-7, 15-16, and 16-17 in the oligonucleotide are replaced by the structure shown in Formula I, wherein the first nucleotide at the 5' end of the oligonucleotide is position 1.

[0021] Preferably, the oligonucleotide consists of 21 nucleotides, wherein the phosphate ester group between the 5th-6th or 15th-16th nucleotides in the oligonucleotide is replaced by the structure shown in Formula I.

[0022] Preferably, in Formula I The structure is selected from at least one of the following structures:

[0023] Where m, n, and k are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, respectively.

[0024] Preferably, in Formula I The structure is selected from at least one of the following structures:

[0025] Preferably, the oligonucleotide comprises at least one of the following structures:

[0026] in,

[0027] A and B are independently selected from O, S, -CH2-, and Se atoms, respectively;

[0028] Base1 and Base2 are independently selected from A, T, G, C, I or unconventional bases, respectively;

[0029] R' and R” are independently selected from H, F, OMOE, C1-C4 alkyl, and C1-C4 alkoxy, respectively;

[0030] Preferably, the oligonucleotide comprises nucleotides with the following structure:

[0031] And / or, the unconventional bases are selected from the following structures:

[0032] Preferably, the structure shown in Formula I is selected from at least one of the following structures:

[0033] Preferably, it is selected from the following oligonucleotides: oligonucleotide 3, oligonucleotide 4, oligonucleotide 5, oligonucleotide 6, oligonucleotide 7, oligonucleotide 8, oligonucleotide 9, oligonucleotide 10, oligonucleotide 11, oligonucleotide 12, oligonucleotide 14, and oligonucleotide 17.

[0034] This invention provides a method for preparing the oligonucleotides described in any one of the preceding claims, comprising:

[0035] Unmodified nucleotides or modified nucleotide b are reacted with phosphoramidite lipid molecules in an automated apparatus for synthesizing oligonucleotides to obtain the product.

[0036] The structure of the phosphoramidite lipid molecule is shown in Formula II:

[0037] in,

[0038] X2 is selected from O, S, CH2 or NH;

[0039] L represents a chemical linker selected from -SS-, -CF2-, and -(CH2). b -, -NHCONH-, -NHCOO-, -NHCOCH2-, -NHCO-, -CONH-, -COO-, -OOC-, C3-C 10 cycloalkyl, C6-C 20 At least one of aryl and -NHSO2-, where b is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0040] R1 is selected from substituted or unsubstituted C1-C. 30 Alkyl, substituted or unsubstituted C2-C 30 alkenyl, substituted or unsubstituted C2-C 30 acetylinyl Substituents are selected from halogens, deuterium, C1-C 30 Straight-chain alkyl, C3-C 10 cycloalkyl, C1-C 30 Halogenated alkyl groups;

[0041] R2, R3, and R4 are independently selected from hydrogen and -NR7R8, respectively, and R7 and R8 are independently selected from C1-C. 30 alkyl;

[0042] R5 is selected from unmodified or modified nucleosides, wherein the oxygen at the 5' end of the pentose sugar or the 4' end of the tetraose sugar in the nucleoside is linked to R6, and the oxygen at the 3' end of the pentose sugar or the 3' end of the tetraose sugar in the nucleoside is linked to P. The modified nucleoside is selected from the modified nucleotide a, and the modified nucleotide a is selected from threonucleotides, C6-C... 20Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-C1-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides.

[0043] R6 is selected from a protecting group;

[0044] q is selected from 0, 1, 2, 3, 4, 5.

[0045] Preferably, the mixing process also requires the addition of an activator, which is selected from 5-benzylthio-1H-tetrazole, tetrazolium, 5-phenylthiotetrazole, 5-ethyltetrazole, and 5-methyltetrazole, and the molar ratio of the phosphoramidite lipid molecule to the activator is 0.04-0.06:0.28-0.36;

[0046] And / or, after the reaction in the automated equipment for synthesizing oligonucleotides, the oligonucleotides may need to be cleaved and deprotected from the solid support.

[0047] The present invention provides an RNAi molecule comprising any of the oligonucleotides described above; wherein the RNAi molecule is single-stranded or double-stranded.

[0048] Preferably, it is a double-stranded siRNA molecule.

[0049] Preferably, the positive strand of the siRNA molecule contains the oligonucleotide.

[0050] Preferably, it is composed of oligonucleotide 2 and oligonucleotide 3;

[0051] And / or, it is composed of oligonucleotide 2 and oligonucleotide 4;

[0052] And / or, it is composed of oligonucleotide 2 and oligonucleotide 5 acid;

[0053] And / or, it is composed of oligonucleotide 2 and oligonucleotide 6;

[0054] And / or, it is composed of oligonucleotide 2 and oligonucleotide 7;

[0055] And / or, it is composed of oligonucleotide 2 and oligonucleotide 8;

[0056] And / or, it is composed of oligonucleotide 2 and oligonucleotide 9;

[0057] And / or, it is composed of oligonucleotide 2 and oligonucleotide 10;

[0058] And / or, it is composed of oligonucleotide 2 and oligonucleotide 12;

[0059] And / or, it is composed of oligonucleotide 2 and oligonucleotide 14;

[0060] And / or, it is composed of oligonucleotide 15 and oligonucleotide 17.

[0061] The present invention provides the use of the oligonucleotides and RNAi molecules described in any of the above claims in the preparation of RNAi drugs.

[0062] Preferably, the RNAi drug is delivered to an extrahepatic tissue.

[0063] Preferably, the extrahepatic tissues include central nervous system tissues, muscle tissues, and adipose tissues.

[0064] The present invention provides an RNAi drug comprising any of the oligonucleotides and any of the RNAi molecules described above.

[0065] In some embodiments, the H atoms in sp3 CH and sp2 CH in the compounds of Formula I can be replaced by D (deuterium) atoms or F (fluorine) atoms.

[0066] In some embodiments, the compound of formula I, R1 is C1-C 30 Alkyl, C2-C 30 alkenyl or C2-C 30 Alkyne group, where C1-C 30 Alkyl, C2-C 30 alkenyl or C2-C 30 The hydrogen atom in the alkynyl group can be optionally replaced by a halogen, deuterium, straight-chain alkyl, cycloalkyl, aryl, or haloalkyl.

[0067] The present invention also provides a pharmaceutical composition comprising the siRNA described in any one of the preceding claims and a pharmaceutically acceptable carrier.

[0068] The present invention also provides a kit comprising the siRNA described in any of the above claims, and / or the above pharmaceutical composition and / or the above siRNA conjugate.

[0069] The present invention also provides the use of any of the above-described siRNAs, and / or the above-described pharmaceutical compositions, and / or the above-described siRNA conjugates, and / or the above-described kits in the preparation of pharmaceuticals.

[0070] On the other hand, the present invention also provides a method for inhibiting the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention.

[0071] The modified nucleotides of this invention can enhance the silencing effect of RNAi drug molecules on target genes and improve the drug-likeness of siRNA, thereby enabling more effective treatment of diseases caused by target gene mRNA.

[0072] The modified nucleotides described in this invention can improve the druggability of siRNA drug molecules. In some embodiments, the modified nucleotides described in this invention can enhance the effect of siRNA in degrading target genes. Therefore, the modified nucleotides described in this invention can enhance the pharmacological efficacy of siRNA silencing.

[0073] The siRNA conjugate provided by this invention is composed of siRNA and a conjugate molecule, comprising the siRNA of this invention and a conjugate group conjugated to the siRNA. The conjugate group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery helper group. The siRNA, the linker, and the targeting group or the delivery helper group are covalently or non-covalently linked in sequence. Each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery helper group is selected from groups capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue.

[0074] In some embodiments, the siRNA drug comprising the modified nucleotides described in this invention is double-stranded. With regard to the double-stranded siRNA drug, the length of the sense and antisense strands of the siRNA drug is independently 15-45 nucleotides. In some embodiments, the double-stranded siRNA drug comprises a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary) to each other. The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands may be the same length or different lengths.

[0075] In some embodiments, the length of the sense strand is about 19 nucleotides, while the length of the antisense strand is about 21 nucleotides. In other embodiments, the length of the sense strand is about 21 nucleotides, while the length of the antisense strand is about 23 nucleotides. The perfect or partially complementary region between the sense and antisense strands is typically 15-25 nucleotides in length and is located at or near the 5' end of the antisense strand (e.g., this region is 1, 2, 3, or 4 nucleotides from the 5' end of the antisense strand, and is imperfectly or partially complementary).

[0076] In the case of double-stranded siRNA drugs, if other sense strand nucleotides are present, they may be identical or different from the corresponding sequence in the target mRNA. If other antisense strand nucleotides are present, they may be complementary or non-complementary to other nucleotides of the corresponding sense strand (if present).

[0077] In some embodiments, the sense and antisense strands of the double-stranded siRNA drug comprising the modified nucleotides of the present invention contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the siRNA drug of the present invention contain different numbers of nucleotides.

[0078] In some implementations, both the sense and antisense chains contain 1 to 4 thiophosphate linkages.

[0079] In some embodiments, each nucleotide in the siRNA of the present invention is a modified or unmodified nucleotide. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide whose bases are modified. The modified nucleotide does not result in a significant reduction or loss of the siRNA's function in regulating gene expression. For example, the modified nucleotides disclosed in JKWatts, G.G. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov. Today, 2008, 13(19-20):842-55 may be selected.

[0080] In some embodiments, the siRNA drug comprising the modified nucleotide inhibits the expression of target mRNA in cells, tissues, or in vivo. In some embodiments, a therapeutically effective amount of the siRNA drug comprising the modified nucleotide described herein is administered in vivo to inhibit the expression of target mRNA in vivo.

[0081] In some embodiments, the siRNA drug is used to treat, prevent, or control clinical manifestations related to the expression of a target mRNA. In some embodiments, a therapeutically or preventively effective amount of one or more siRNA drugs is given to a subject in need of such treatment, prevention, or control.

[0082] The siRNA drugs comprising the modified nucleotides described in this invention, as well as compositions comprising the siRNA drugs described in this invention, can be delivered to cells, tumors, tissues, or objects using oligonucleotide delivery technologies known in the art. Generally, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) in the art can be applied to the siRNA drugs comprising one or more modified nucleotides described in this invention.

[0083] In some embodiments of siRNA drugs that are double-stranded, a targeting ligand or targeting group, a linker group, or a delivery vector is covalently linked to the sense strand to form a conjugate. In some embodiments, the targeting ligand, linker group, and / or delivery vector are directly or indirectly linked to the 3' or 5' end of the sense strand via a linker.

[0084] In some embodiments, the targeting ligand or targeting group contains two to four terminal N-acetylgalactosamines (GalNAc), which are linked to an siRNA drug comprising one or more of the modified nucleotides. It is known that the triantralized (GalNAc) form has a greater affinity for ASGPR than the diantralized or monoantralized forms (Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0085] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0086] In this invention, the term "derivative group" refers to the substitution of hydrogen atoms or groups of atoms in a simple compound by other atoms or groups of atoms, resulting in a more complex product.

[0087] In this invention, unless otherwise specified, uppercase letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; m indicates that the nucleotide adjacent to the right of the identifier m is a 2'-methoxynucleotide; f indicates that the nucleotide adjacent to the right of the identifier f is a 2'-fluoronucleotide; d indicates that the nucleotide adjacent to the right of the identifier d is a 2'-deoxynucleotide; gn indicates that the nucleotide adjacent to the right of the identifier gn is a glycerol nucleotide (GNA); the identifier * indicates that the two nucleotides adjacent to the identifier * on the left and right are linked by a thiophosphate group (or between the nucleotide and the linker-targeting ligand portion); eVP indicates that the nucleotide adjacent to its right is a (E)-vinyl phosphate modified nucleotide; and iab indicates a reverse debasement residue.

[0088] In this invention, "oligonucleotide" or "oligomeric nucleotide" refers to a polymer formed by the linkage of nucleotides, wherein each nucleotide may be independently modified or unmodified, and the oligonucleotide sequence comprises about 10-50 single-stranded or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a nucleobase sequence that is at least partially complementary to the core sequence of a target gene expressed in the cell. In some embodiments, the oligonucleotide, upon delivery to a cell expressing the gene, can regulate the expression of the corresponding target gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" or "oligomeric nucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0089] In this invention, the positions of nucleotides in oligonucleotides or oligonucleotides are numbered sequentially from the 5' end of the oligonucleotide or oligonucleotide towards the 3' end, starting from 1. For example, in an oligonucleotide sequence such as "5'-AUGC-3'", the first nucleotide is adenosine, the second nucleotide is uridine, the third nucleotide is guanosine, and the fourth nucleotide is cytidine. The order of nucleotides in oligonucleotides or oligonucleotides is determined by their position within the oligonucleotide, with the position closer to the 5' end being the first and the position closer to the 3' end being the last.

[0090] In this invention, "siRNA" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains RNA or RNA-like (e.g., chemically modified RNA) molecules that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0091] siRNAs can function through RNA interference mechanisms (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells) or any other mechanism or pathway. While the term siRNA drug as used in this invention is considered to function primarily through RNA interference mechanisms, the siRNA drug is not limited to any particular pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of this invention consists of an oligonucleotide chain having at least a partial complementarity to the mRNA that is the target. In some embodiments, the siRNA drug of this invention is double-stranded and consists of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0092] The terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” refer to a reduction or decrease in the expression level of a given gene when it is directly administered into cells, tissues, organs, or animals treated with the siRNA drug molecules described in this invention, compared to administration into cells, tissues, organs, or animals that have not been so treated.

[0093] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0094] The term "heterocyclic base" is a nucleobase or modified nucleobase as defined in this invention. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine, as well as a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety may include one or more protecting groups.

[0095] In this invention, "unmodified nucleotides" refers to naturally occurring nucleotides, including adenosine monophosphate, guanylic acid, cytidine monophosphate, and uridine monophosphate.

[0096] In this invention, "unmodified nucleosides" refers to the nucleoside portion (i.e., without phosphate groups) of naturally occurring nucleotides, including adenosine, guanosine, cytidine, and uridine.

[0097] In this invention, "nucleotide base" or "nucleobase" refers to heterocyclic pyrimidine or purine compounds, wherein "conventional nucleoside base" includes the bases of conventional nucleotides such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Nucleobases can be modified or substituted with novel unconventional nucleobases. Nucleotide bases include naturally occurring nucleotide bases as well as non-naturally occurring nucleotide bases. It should be obvious to those skilled in the art that various nucleotide bases previously considered "non-naturally occurring" have later been discovered in nature. Therefore, "nucleotide base" includes not only known purine and pyrimidine heterocycles but also their heterocyclic analogs and tautomers. Exemplary examples of nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4,N4-bridged ethylidene cytosine, N6,N6-bridged ethylidene-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolpyridine, isocytosine, isoguanine, inosine, and the “non-naturally occurring” nucleotide bases described in U.S. Patent No. 5,432,272 to Benner et al. The term “nucleotide base” includes each and all of these examples and their analogues and tautomers. Particularly important nucleotide bases include adenine, guanine, thymine, cytosine, and uracil, which are considered to be naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0098] The term "complementarity," used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of an siRNA drug or the target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded antisense strand of an siRNA drug), refers to the ability of an oligonucleotide or oligonucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or oligonucleotide containing the second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form a base pair and a double helix or double-stranded structure. A complementary sequence contains either a Watson-Crick base pair or a non-Watson-Crick base pair and contains native or modified nucleotides or nucleotide analogs to a degree sufficient to satisfy the hybridization requirements described above. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0099] The term "sense strand" refers to the nucleotide sequence on an RNA molecule that carries the information of the amino acids that encode the protein. It is also called the coding strand, sense strand, or positive strand, while the other nucleotide sequence that is complementary to it is called the antisense strand.

[0100] The term "antisense strand" refers to a nucleotide sequence in the mRNA of the target gene that is substantially anticomplementary or substantially anticomplementary to the antisense strand.

[0101] The terms “optional,” “optionally,” and “any one” mean that the event or situation described below may, but does not have to, occur, and the description includes the possibility or absence of such event or situation. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may, but does not have to, be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0102] The term "substitution" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, being independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can be substituted, whether experimentally or theoretically, by means of existing technology and experimental conditions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0103] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a -C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, C1-C6 alkyl refers to straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0104] Alkyl refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-. The alkyl group can also be part of other groups, such as C1-C6 alkoxy groups or C1-C6 alkylamino groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and n-hexyl groups. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl, or carboxylic acid ester groups.

[0105] "Alkenyl" refers to a straight-chain or branched hydrocarbon group having at least two carbon atoms and at least one vinyl unsaturation site (>C=C<). For example, C a -C bAlkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0106] "Alynyl" refers to a straight-chain monovalent hydrocarbon group or a branched monovalent hydrocarbon group containing at least one triple bond. The term "alkynyl" is also intended to include hydrocarbon groups having one triple bond and one double bond. For example, C2-C6 alkynyl is intended to include ethynyl, propynyl, etc.

[0107] "Alkoxy" refers to -O- (alkyl) or -O- (cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0108] "Cycloalkyl" refers to a saturated or partially saturated cyclic group having a specified number of carbon atoms, no heteroatoms, and a single ring.

[0109] "Aryl" refers to an unsaturated alkane group having a specified number of carbon atoms, no heteroatoms, and at least one aromatic ring.

[0110] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0111] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0112] "Ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0113] "Acyl" refers to a compound containing a -C(O)R group, where R is an alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group.

[0114] "Aromatic acyl" refers to the aryl group through... When connected to a linking group, the number of carbon atoms indicates the number of carbon atoms in the aryl group.

[0115] A heteroatom is an atom in an organic compound that is other than carbon and hydrogen. It generally refers to an atom that replaces carbon in the molecular skeleton (especially in ring systems). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, and iodine are common heteroatoms. If an organic compound contains a ring that includes heteroatoms, it is called a heterocyclic compound.

[0116] In this invention, a "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl, and thiol groups) from undergoing undesirable reactions during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect other reactive sites in the reaction and are subsequently removed to release unprotected groups, making them available for further reactions. In some embodiments, "substituted" groups or substituents contain protecting groups.

[0117] Representative hydroxyl protecting groups used in this invention are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, all of which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0118] The compounds and compositions described in this invention may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is situated. Therefore, as used in this invention, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, can be protonated or deprotonated. The disclosure of this invention is intended to cover the compounds and compositions described above, regardless of their protonation state based on environmental pH, as will be readily understood by those skilled in the art.

[0119] The terms "salt" and "available salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds, or their stereoisomers, with an appropriate (e.g., equimolar) amount of acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, or by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium.

[0120] This invention prepares a series of lipid molecules containing phosphoramidite groups, reacts them with oligonucleotides to obtain RNAi molecules, and further modifies these lipid molecules with phosphoramidite groups at different positions within the oligonucleotides to obtain RNAi molecules with superior efficacy. These RNAi molecules can effectively target extrahepatic tissues while maintaining their ability to inhibit target genes. The lipid molecules prepared in this invention show promising potential as extrahepatic delivery vectors, and the RNAi molecules prepared in this invention have significant value for the research and clinical application of RNAi drugs.

[0121] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0122] Figure 1: The inhibitory effect of LDR0182177 and positive compound LDR0182176 on target gene hAPP mRNA in brain tissue after 28 days at a dose of 0.3 mg; aCSF in the figure represents artificial cerebrospinal fluid as a blank control.

[0123] Figure 2: At a dose of 0.3 mg, LDR0162028 showed the same inhibitory effect on the target gene SDO1 mRNA in brain tissue as the positive compound LDR0162001 after 28 days; at a dose of 0.9 mg, LDR0162028 showed the same inhibitory effect on the target gene SDO1 mRNA in brain tissue after 28 days; aCSF in the figure represents artificial cerebrospinal fluid as a blank control.

[0124] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0125] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0126] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0127] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0128] The compounds disclosed in this invention can be prepared using the following synthetic methods.

[0129] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available. All procedures, including nucleic acid electrophoresis and real-time PCR, were performed according to standard protocols. For example, they could be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0130] In the following description, exemplary embodiments will be provided to aid in understanding this disclosure. However, the following embodiments are provided merely for the purpose of facilitating understanding of this disclosure and are not intended to limit it.

[0131] Example 1: Synthesis of XY-021-12 phosphorusamide monomer

[0132] The XY-021-12 phosphorus amide monomer in this embodiment was prepared by the following method:

[0133] In substrate XY-021-12-1, DMTr refers to the 4,4'-dimethoxytriphenylmethyl (Dimethoxytrityl) protecting group.

[0134] In a dry 100 mL single-necked flask, substrate XY-021-12-1 (3.0 g, 5.3 mmol) was added and dissolved in anhydrous DCM (30 mL, dichloromethane). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (760.0 mg, 5.83 mmol, N,N-diisopropylethylamine), and XY-021-12-1a (1.8 g, 6.9 mmol) were added. After the additions were complete, the reaction mixture was kept at room temperature for 1 h. Then, XY-021-12-2a (1.6 g, 6.4 mmol) and a 0.25 M ETT (2-mercaptoethanethiol) acetonitrile solution (12.9 mL, 3.2 mmol) were added. After the additions were complete, the reaction mixture was kept at room temperature for 2 h. LCMS analysis showed that XY-021-12-2 had completely disappeared. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated, and the crude product was purified by MPLC (medium-pressure preparative chromatography). The crude product was then concentrated to give a white solid product XY-021-12 (2.8 g, 3.0 mmol, 56% yield).

[0135] The characterization data of product XY-021-12 are as follows:

[0136] ESI-LCMS m / z 931.0 [M+H] -

[0137] 1 H NMR (400MHz, DMSO-d6) δ11.41 (s, 1H), 7.81-7.80 (m, 1H), 7.40-7.23 (m, 9H), 6.90-6.8 7(m,4H), 5.87-5.83(m,1H), 4.40-4.30(m,1H), 4.39–4.30(m,1H), 4.10–4.06(m,1H), 3 .94–3.91(m,28H), 3.80-3.38(m,14H), 3.32–3.28(m,1H), 1.53–1.45(m,1H), 1.40–1.3 6(m,1H), 1.28–1.15(m,26H), 1.12–1.08(m,9H), 1.00–0.97(m,3H), 0.85–0.83(m,3H).

[0138] 31 P NMR (400MHz, DMSO-d6) δ148.48, 148.12.

[0139] Example 2: Synthesis of XY-021-13 phosphorusamide monomer

[0140] The XY-021-13 phosphorus amide monomer in this embodiment was prepared by the following method:

[0141] 1. Synthesis of compound XY-021-13-3

[0142] In a dry 100 mL single-necked flask, substrate XY-021-13-1 (2.5 g, 8.33 mmol) was dissolved in DCM / THF at a ratio of 1:1 (25 mL). Then, under nitrogen protection, CDI (1.5 g, 9.21 mmol) was added. After the addition was complete, the reaction mixture was kept at room temperature for 1 h. LC-MS analysis showed that XY-021-13-1 had completely disappeared, and XY-021-13-2 had been formed. Then, XY-021-13-2a (1.8 g, 9.21 mmol) was added to the reaction mixture. After the addition was complete, the reaction mixture was kept at room temperature for 15 h. LC-MS analysis showed that XY-021-13-2 had completely disappeared, indicating the reaction was complete. Then, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (100 mL * 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) and concentrated to obtain a white solid product XY-021-13-3 (4.0 g, 7.59 mmol, 91% yield).

[0143] 2. Synthesis of compound XY-021-13

[0144] In a dry 100 mL single-necked flask, substrate XY-021-13-5 (3.4 g, 6.05 mmol) was dissolved in anhydrous DCM (34 mL). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (861.5 mg, 6.60 mmol), and XY-021-13-5a (2.1 g, 7.90 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. Next, XY-021-13-3 (3.2 g, 6.00 mmol) and a 0.25 M ETT acetonitrile solution (36.4 mL, 9.11 mmol) were added to the reaction mixture, and the reaction mixture was kept at room temperature for 1 h. LC-MS analysis showed that XY-021-13-3 had completely disappeared. The mixture was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-13 (2.1 g, 1.72 mmol, 28% yield).

[0145] The characterization data of product XY-021-13 are as follows:

[0146] ESI-LCMS: m / z 1216.2 [MH] - .

[0147] 1 H NMR(400MHz,DMSO-d6)δ11.41(s,1H),7.87–7.56(m,2H),7.45–7.17(m,9H),6.9 5–6.78(m,4H),5.86–5.78(m,1H),5.37–5.23(m,1H),4.83–4.64(m,2H),4.44–4 .27(m,1H),4.13–3.86(m,2H),3.73(s,6H),3.64–3.33(m,7H),3.32–3.24(m,2H ),3.03–2.95(m,2H),1.43–1.35(m,2H),1.31–1.06(m,27H),1.01–0.91(m,3H).

[0148] 31 P NMR(400MHz,DMSO-d6)δ148.41,148.14.

[0149] Example 3: Synthesis of phosphorusamide monomer XY-021-14

[0150] The XY-021-14 phosphorusamide monomer in this embodiment was prepared by the following method:

[0151] 1. Synthesis of compound XY-021-14-3

[0152] In a dry 100 mL single-necked flask, substrate XY-021-14-1 (2.0 g, 10.00 mmol) was dissolved in DCM / THF at a ratio of 1:1 (20 mL). Then, under nitrogen protection, CDI (1.8 g, 11.00 mmol) was added. After the addition was complete, the reaction system was maintained at room temperature for 1 h. LCMS analysis showed that XY-021-14-1 had completely disappeared, and XY-021-14-2 had been formed. Then, XY-021-14-2a (2.2 g, 11.00 mmol) was added to the reaction solution. After the addition was complete, the reaction system was maintained at room temperature for 15 h. LCMS analysis showed that XY-021-14-2a had completely disappeared, indicating the reaction was complete. Then, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (100 mL * 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether: ethyl acetate = 1:1) and concentrated to obtain a white solid product XY-021-14-3 (3.5 g, 8.20 mmol, 82% yield).

[0153] 2. Synthesis of compound XY-021-14

[0154] In a dry 100 mL single-necked flask, substrate XY-021-14-4 (3.2 g, 5.71 mmol) was dissolved in anhydrous DCM (32 mL). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (810 mg, 6.31 mmol), and XY-021-14-4a (1.68 g, 7.41 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. Next, XY-021-14-3 (2.44 g, 5.71 mmol) and 0.25 M ETT acetonitrile solution (13.7 mL, 3.41 mmol) were added to the reaction mixture, and the reaction mixture was kept at room temperature for 1 h. LC-MS analysis showed complete disappearance of XY-021-14-5. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-14 (2.3 g, 2.05 mmol, 36% yield).

[0155] The characterization data of product XY-021-14 are as follows:

[0156] ESI-LCMS: m / z 1116.3 [MH] - .

[0157] 1 H NMR(400MHz,DMSO-d6)δ11.41(s,1H),7.87–7.56(m,2H),7.45–7.17(m,9H),6.9 5–6.78(m,4H),5.86–5.78(m,1H),5.37–5.23(m,1H),4.85–4.64(m,2H),4.44–4 .27(m,1H),4.13–3.86(m,2H),3.73(s,6H),3.64–3.33(m,7H),3.32–3.24(m,2H ),3.03–2.95(m,2H),1.55–1.17(m,20H),1.15–1.05(m,9H),1.01–0.91(m,3H).

[0158] 31 P NMR(400MHz,DMSO-d6)δ148.42,148.14.

[0159] Example 4: Synthesis of XY-021-16 phosphorusamide monomer

[0160] The XY-021-16 phosphorusamide monomer in this embodiment was prepared by the following method:

[0161] 1. Synthesis of compound XY-021-16-2a

[0162] In a dry 100 mL single-necked flask, substrate XY-021-16-1a (4.0 g, 19.90 mmol) dissolved in 22 mL of DMF was added. Then, under nitrogen protection, EDCI (4.6 g, 24.08 mmol), HOBT (3.2 g, 23.70 mmol), DIPEA (5.1 g, 26.08 mmol), DMAP (243 mg, 2.00 mmol), and XY-021-16-1b (6.9 g, 21.80 mmol) were added, one after the other. After the additions were complete, the reaction system was maintained at room temperature for 15 h. LCMS analysis showed that XY-021-16-1a had completely disappeared, indicating the end of the reaction. Then, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (100 mL * 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether: ethyl acetate = 2:1) and concentrated to obtain a white solid product XY-021-16-2a (2.0 g, 4.02 mmol, 20% yield).

[0163] 2. Synthesis of compound XY-021-16

[0164] In a dry 100 mL single-necked flask, substrate XY-021-16-3a (2.2 g, 3.93 mmol) was dissolved in anhydrous DCM (22 mL). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (557.5 mg, 4.31 mmol), and XY-021-16-3b (1.36 g, 5.11 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. Next, XY-021-16-2a (2.0 g, 3.91 mmol) and 0.25 M ETT acetonitrile solution (9.6 mL, 2.42 mmol) were added to the reaction mixture, and the reaction mixture was kept at room temperature for 1 h. LCMS analysis showed complete disappearance of XY-021-16-4. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-16 (2.0 g, 1.77 mmol, 45% yield).

[0165] The characterization data for XY-021-16 are as follows:

[0166] ESI-LCMS: m / z 1185.4 [MH]-. 1H NMR(400MHz,DMSO-d6)δ11.41(s,1H),9.55–9.38(m,1H),7.90–7.68(m,1H),7.4 5–7.17(m,9H),6.95–6.78(m,4H),5.86–5.78(m,1H),5.37–5.23(m,1H),4.44–4 .27(m,1H),4.13–3.86(m,2H),3.73(s,6H),3.64–3.33(m,7H),3.32–3.24(m,2H ),3.23–3.13(m,2H),1.55–1.35(m,4H),1.34–1.03(m,25H),1.01–0.94(m,3H). 31 P NMR(400MHz,DMSO-d6)δ148.42,148.15.

[0167] Example 5: Synthesis of phosphorous amide monomer XY-021-19

[0168] The XY-021-19 phosphorus amide monomer in this embodiment was prepared by the following method:

[0169] In a dry 100 mL single-necked flask, substrate XY-021-19-1 (2.2 g, 3.93 mmol) was dissolved in anhydrous DCM (22 mL). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (557.5 mg, 4.31 mmol), and XY-021-19-1a (1.36 g, 5.11 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. Next, XY-021-19-2a (2.1 g, 4.31 mmol) and 0.25 M ETT acetonitrile solution (9.6 mL, 2.41 mmol) were added, and the reaction mixture was kept at room temperature for 2 h. LCMS analysis showed complete disappearance of XY-021-19-2. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-19 (2.0 g, 1.69 mmol, 43% yield).

[0170] The characterization data for XY-021-19 are as follows:

[0171] ESI-LCMS m / z 1178.4 [MH] - . 1H NMR(400MHz,DMSO-d6)δ11.41(s,1H),7.86-7.70(m,1H),7.44-7.13(m,9H) ,6.97-6.78(m,4H),5.87-5.74(m,1H),5.38-5.18(m,1H),4.45-4.25(m,1H ),4.16–3.85(m,2H),3.71(s,6H),3.66–3.35(m,7H),3.32-3.22(m,2H),2. 24–2.01(m,2H),1.54–1.43(m,3H),1.36–1.04(m,24H),0.99–0.91(m,3H). 31 P NMR(400MHz,DMSO-d6)δ148.52,148.14.

[0172] Example 6: Synthesis of XY-021-21 phosphoridamide monomer

[0173] The XY-021-21 phosphorus amide monomer in this embodiment was prepared by the following method:

[0174] 1. Synthesis of compound XY-021-21-2

[0175] In a dry 500 mL single-necked flask, substrate XY-021-21-1 (12.0 g, 63.11 mmol) was dissolved in anhydrous ACN (120 mL), followed by the addition of PPh3 (17.4 g, 66.33 mmol) under nitrogen protection. After the addition was complete, the reaction system was stirred under reflux for 15 h. The reaction was considered complete when the starting material was completely eliminated by TLC. The reaction solution was directly concentrated to obtain crude product XY-021-21-2 (24.0 g), which was used directly in the next step.

[0176] 2. Synthesis of compound XY-021-21-3

[0177] Substrate 1a (30.0 g, 148.55 mmol) was added to a dry 500 mL single-necked flask and dissolved in THF (300 mL). Then, NaH (8.9 g, 222.77 mmol) was added at 0 °C, and the reaction mixture was stirred at 0 °C for 1.0 h. Subsequently, BnBr (24.1 g, 141.11 mmol) was added, and the reaction mixture was stirred at room temperature for 16 h. The reaction was considered complete when the starting material was observed to have disappeared relative to the reaction mixture by TLC. Then, saturated ammonium chloride solution (100 mL) was added at 0 °C, and the mixture was extracted with ethyl acetate (500 mL x 3) and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 4:1), and concentrated to give product XY-021-21-3 (12.0 g, 41.09 mmol, 28% yield).

[0178] 3. Synthesis of compound XY-021-21-4

[0179] Substrate XY-021-21-3 (12.0 g, 41.09 mmol) was added to a dry 250 mL single-necked flask and dissolved in DMSO (120 mL). Then, EDCI (31.5 g, 164.92 mmol) and Pyridine (6.5 g, 82.20 mmol) were added at room temperature, followed by the dropwise addition of TFA (4.7 g, 41.11 mmol) at 0 °C. After the addition was complete, the reaction system was stirred at room temperature for 1 h. The reaction was considered complete when the starting material was completely eliminated by TLC. Water (100 mL) was then added at 0 °C, and the mixture was extracted with ethyl acetate (500 mL x 3) and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude product XY-021-21-4 (12.0 g).

[0180] 4. Synthesis of compound XY-021-21-5

[0181] In a dry 50 mL single-necked flask, 24.0 g of substrate XY-021-21-2 was dissolved in 240 mL of anhydrous THF. Then, under nitrogen protection, 75.77 mmol of LiHMDS (75.7 mL, 1 M in THF) was added at -78 °C. After the addition was complete, the reaction mixture was stirred at -78 °C for 2 hours. Next, 12.0 g of XY-021-21-4 was dissolved in anhydrous THF and added to the reaction mixture at -78 °C. After the addition was complete, the reaction mixture was stirred at -78 °C for 2 hours. TLC analysis showed complete disappearance of the starting material, indicating the reaction was complete. Then, 50 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (100 mL * 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 80:1) and concentrated to obtain product XY-021-21-5 (6.5 g, 16.93 mmol, 40% yield).

[0182] 5. Synthesis of compound XY-021-21-6

[0183] In a dry 250 mL single-necked flask, substrate XY-021-21-5 (6.5 g, 16.93 mmol) was dissolved in anhydrous methanol (65 mL). Then, under nitrogen protection, 10% Pd / C (3.3 g) was added. After the addition was complete, the mixture was replaced with a hydrogen balloon, and the reaction system was stirred at room temperature under hydrogen atmosphere for 1 hour. The mixture was filtered, and Pd / C(OH)₂ (3.3 g, 50% TCI) was added. The reaction was then stirred at room temperature under hydrogen atmosphere for 5 hours. TLC analysis showed complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was filtered, and the filtrate was concentrated to obtain product XY-021-21-6 (3.2 g, 10.81 mmol, 64% yield).

[0184] 6. Synthesis of compound XY-021-21

[0185] In a dry 100 mL single-necked flask, substrate XY-021-21-1a (3.0 g, 5.35 mmol) was dissolved in anhydrous DCM (30 mL). Then, under nitrogen protection, molecular sieve (3.0 g), DIPEA (760.2 mg, 5.91 mmol), and 2a (1.9 g, 7.01 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. Next, XY-021-21-6 (1.7 g, 5.90 mmol) and 0.25 M ETT in acetonitrile solution (12.8 mL, 3.21 mmol) were added, and the reaction mixture was kept at room temperature for 2 h. LC-MS analysis showed complete disappearance of XY-021-21-2a. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-21 (1.5 g, 1.52 mmol, 28% yield).

[0186] The characterization data for XY-021-21 are as follows:

[0187] ESI-LCMS: m / z 984.5 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.41(s,1H),7.81–7.74(m,1H),7.40–7.20(m,9H),6.92–6 .85(m,4H),5.83–5.80(m,1H),5.32–5.25(m,1H),4.41–4.29(m,1H),4.10–4.08(m,1 H), 3.95–3.90(m,1H),3.73(s,6H),3.65–3.44(m,3H),3.41–3.36(m,4H),2.27–2.13 (m,2H),2.30–2.08(m,2H),1.3–1.37(m,4H),1.33–1.03(m,31H),1.00–0.92(m,3H). 31 P NMR(600MHz,DMSO-d6)δ148.46,148.12.

[0188] Example 7 Synthesis of phosphorusamide monomer XY-021-23

[0189] The XY-021-23 phosphorus amide monomer in this embodiment was prepared by the following method:

[0190] 1. Synthesis of compound XY-021-23-2

[0191] In a dry 500 mL single-necked flask, substrate XY-021-23-1 (25.0 g, 152.41 mmol) was dissolved in acetonitrile (300 mL). Then, under nitrogen protection, triphenylphosphine (40.3 g, 153.89 mmol) was added. After the addition was complete, the reaction mixture was refluxed and stirred overnight at 80 °C. After cooling, the reaction solution was concentrated to obtain crude product XY-021-23-2 (124.4 g), which was directly used in the next step.

[0192] 2. Synthesis of compound XY-021-23-4

[0193] In a dry 500 mL single-necked flask, substrate XY-021-23-3 (11.0 g, 45.41 mmol) was dissolved in acetonitrile (150 mL). Then, under nitrogen protection, triphenylphosphine (12.0 g, 45.89 mmol) was added. After the addition was complete, the reaction mixture was refluxed and stirred overnight at 80 °C. After cooling, the reaction solution was concentrated to obtain crude product XY-021-23-4 (26.8 g), which was used directly in the next step.

[0194] 3. Synthesis of compound XY-021-23-6

[0195] In a dry 3.0L single-necked flask, substrate XY-021-23-5 (180.0 g, 1249.11 mmol) was dissolved in pyridine (1.8 L). After the addition was complete, the mixture was purged with nitrogen and cooled to 0°C. Then, TBDPSCl (412.0 g, 1498.9 mmol) was slowly added dropwise. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. The reaction was considered complete when the starting material was completely eliminated by LCMS. Then, 3000 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (3000 mL * 3) and then with saturated brine (2000 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether: ethyl acetate = 50:1 to 10:1) and concentrated to obtain a white oily substance XY-021-23-6 (150.0 g, 392.55 mmol, 31% yield).

[0196] 4. Synthesis of compound XY-021-23-7

[0197] In a dry 250 mL single-necked flask, substrate XY-021-23-6 (70.0 g, 183.21 mmol) was dissolved in anhydrous DMSO (70 mL). Then, pyridine (28.9 g, 366.41 mmol) and EDCI (140.5 g, 732.82 mmol) were added. After the addition was complete, the mixture was purged with nitrogen and cooled to 0 °C. Trifluoroacetic acid (20.9 g, 183.21 mmol) was then slowly added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. The reaction was considered complete when the starting material was completely eliminated by TLC. Then, 1000 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with saturated sodium bicarbonate (1000 mL x 1) and then with saturated brine (1000 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude product XY-021-23-7 (74.0 g), which was used directly in the next step.

[0198] 5. Synthesis of compound XY-021-23-8

[0199] In a dry 2L single-necked flask, substrate XY-021-23-2 (124.4 g, 292.11 mmol) was dissolved in anhydrous THF (1.3 L), and the mixture was purged with nitrogen. The temperature was then lowered to -78°C, and LiHMDS (234 mL, 233.61 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at 0°C for 1 hour. The temperature was then lowered to -78°C, and XY-021-23-7 (74.0 g, 194.71 mmol) was dissolved in anhydrous THF (100 mL) and added to the reaction mixture. The mixture was stirred at 0°C for 1 hour. TLC analysis showed complete disappearance of the starting material, indicating the reaction was complete. The reaction solution was then extracted with 1000 mL of saturated ammonium chloride aqueous solution using ethyl acetate (1000 mL), washed with water (1000 mL * 2), and washed with saturated brine (1000 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product which was purified by column chromatography (petroleum ether) and concentrated to obtain a transparent oily product XY-021-23-8 (32.0 g, 71.38 mmol, 36% yield).

[0200] 6. Synthesis of compound XY-021-23-9

[0201] In a dry 1L single-necked flask, substrate XY-021-23-9 (21.5 g, 48.00 mmol) was dissolved in THF (200 mL), followed by the addition of TBAF (72 mL, 72.00 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction was considered complete when the starting material was completely eliminated by TLC. The crude product XY-021-23-9 was directly concentrated and purified by column chromatography (petroleum ether: ethyl acetate 80:1–20:1). The purified product was then concentrated to give a colorless liquid product XY-021-23-9 (9.6 g, 33.21 mmol, 69% yield).

[0202] 7. Compound XY-021-23-10

[0203] In a dry 1L single-necked flask, substrate XY-021-23-9 (21.5 g, 56.31 mmol) was dissolved in anhydrous DMSO (200 mL), followed by the addition of pyridine (8.9 g, 112.61 mmol) and EDCI (43.2 g, 225.22 mmol). After the addition was complete, the mixture was purged with nitrogen and cooled to 0°C. Trifluoroacetic acid (6.4 g, 56.31 mmol) was then slowly added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. The reaction was considered complete when the starting material was completely eliminated by TLC. Then, 200 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with saturated sodium bicarbonate (200 mL x 1), washed with water (200 mL x 2), and washed with saturated brine (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude product XY-021-23-10 (13.5 g), which was used directly in the next step.

[0204] 8. Synthesis of compound XY-021-23-11

[0205] In a dry 1L single-necked flask, substrate XY-021-23-4 (26.8 g, 53.31 mmol) was dissolved in anhydrous THF (250 mL) and purged with nitrogen. The mixture was then cooled to -78°C, and LiHMDS (42 mL, 42.61 mmol) was slowly added dropwise. After the addition was complete, the reaction system was stirred at 0°C for 1 hour. The temperature was then lowered to -78°C, and XY-021-23-10 (13.5 g, 35.55 mmol) was dissolved in anhydrous THF (20 mL) and added to the reaction system. The mixture was stirred at 0°C for 1 hour. TLC analysis showed complete disappearance of the starting material, indicating the reaction was complete. The reaction solution was then added to 200 mL of saturated ammonium chloride aqueous solution and extracted with ethyl acetate (200 mL). The solution was washed with water (200 mL * 2) and then with saturated brine (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether) and concentrated to obtain a transparent oily product XY-021-23-8 (5.0 g, 14.11 mmol, 26% yield).

[0206] 9. Synthesis of compound XY-021-23-12

[0207] In a dry 250 mL single-necked flask, substrate XY-021-23-11 (5.0 g) was dissolved in anhydrous methanol (100 mL), followed by Pd / C (2.5 g). After the addition was complete, the reaction system was stirred at room temperature for 0.5 hours. The reaction solution was then filtered, and Pd(OH)2 / C (2.0 g) was added to purge with hydrogen gas, and stirring was continued for 3 hours. The reaction was considered complete when the starting material was completely eliminated by TLC. The organic phase was filtered and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether) and concentrated to obtain a transparent oily product XY-021-23-12 (2.3 g, 5.11 mmol, 39% yield).

[0208] 10. Synthesis of compound XY-021-23

[0209] In a dry 50 mL single-necked flask, substrate XY-021-23-2a (1.5 g, 2.67 mmol) was dissolved in anhydrous DCM (15 mL). Then, under nitrogen protection, molecular sieve (1.5 g), DIPEA (387 mg, 3.00 mmol), and XY-021-23-1b (1.8 g, 6.90 mmol) were added, and the reaction mixture was kept at room temperature for 1 h. LC-MS analysis showed complete disappearance of XY-021-23-2a. Next, XY-021-23-12 (1.1 g, 4.10 mmol) and 0.25 METT acetonitrile solution (16 mL, 1.60 mmol) were added to the reaction mixture. The reaction mixture was kept at room temperature for 2 h. LC-MS analysis showed complete disappearance of XY-021-23-2c. The product was then filtered through diatomaceous earth, and the organic phase was collected. The organic phase was concentrated to obtain a crude product, which was purified by MPLC and concentrated to give a white solid product XY-021-23 (1.5 g, 1.56 mmol, 58% yield).

[0210] The characterization data for XY-021-23 are as follows:

[0211] ESI-LCMS: m / z 957.5 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.41(s,1H),7.81–7.75(m,1H),7.40–7.25(m,9H),6.8 9–6.87(m,4H),5.83–5.81(m,1H),5.32–5.26(m,1H),4.40–4.29(m,1H),4.09–4 .07(m,1H),3.95–3.90(m,1H),3.73–3.40(m,14H),3.32–3.26(m,H),1.66–1.62 (m,1H),1.51(s,1H),1.37–1.08(m,32H),0.98–0.96(m,3H),0.86–0.78(m,6H). 31 P NMR(400MHz,DMSO-d6)δ148.43,148.09.

[0212] Example 8: Synthesis of the phosphoramidite monomer XY-021-31

[0213] The XY-021-31 phosphorus amide monomer in this embodiment was prepared by the following method:

[0214] 1. Synthesis of compound XY-021-31-2a

[0215] In a dry 100 mL single-necked flask, substrate XY-021-31-1a (5.0 g, 7.54 mmol) dissolved in anhydrous DCM (50 mL) was added. Then, under nitrogen protection, DIPEA (1.1 g, 8.29 mmol) and XY-021-31-1b (2.6 g, 9.80 mmol), along with activated molecular sieves, were added. After the additions were complete, the reaction system was stirred at room temperature under a nitrogen atmosphere for 1 hour. LC-MS analysis showed complete disappearance of the starting material, indicating the reaction was complete. No further processing was required; the mixture could be directly used in the next step.

[0216] 2. Synthesis of compound XY-021-31

[0217] In the reaction solution of XY-021-31-2a, ETT (18 mL, 0.25 M, 4.52 mmol) and XY-021-31-2b (2.2 g, 8.05 mmol) were added under nitrogen protection. After the addition was complete, the reaction system was stirred at room temperature under nitrogen atmosphere for 2 hours. The reaction was considered complete when the starting material was completely eliminated by LCMS. The mixture was filtered, and the filtrate was diluted with 100 mL of dichloromethane, washed with water (100 mL * 3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was then concentrated using a reversed-phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) product to obtain the white solid product XY-021-31 (2.5 g, 2.42 mmol, 32% yield).

[0218] The characterization data for XY-021-31 are as follows:

[0219] ESI-LCMS: m / z 1033.5 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.32(s,1H),8.79–8.42(m,1H),8.14–7.96(m,2H),7.64–7.11(m,13H),6.92–6.78(m,4H),5.98–5.81(m,1H),4.53–4.3 4(m,1H),4.23–4.15(m,1H),3.93–3.79(m,1H),3.79–3.33(m,15H),1.55 –1.34(m,2H),1.30–1.04(m,35H),1.02–0.98(m,3H),0.85–0.79(m,3H). 31 P NMR (162MHz, DMSO-d6) δ148.56,147.55.

[0220] Example 9: siRNA Synthesis

[0221] The SOD1 / hAPP gene and siRNA used in this embodiment were commercially available. The specific synthesis method is as follows.

[0222] For the sense and antisense strands of the siRNA duplex and the sense and antisense strands of the modified duplex of the present invention, deoxynucleoside CPG is used as a solid support, the sense strand is synthesized using the solid support, and the antisense strand is synthesized using universal CPG.

[0223] Sequence synthesis was performed using a 48-channel synthesizer at a scale of 0.2 μmol. A phosphoramide monomer at a concentration of 0.05 M and a 0.3 M BTT (5-benzylthio-1H-tetrazole) activator were used. The phosphoramide monomers prepared in the above examples were used to synthesize modified siRNAs. The phosphoramide monomers were used to replace the phosphate ester groups between adjacent nucleotides in the oligonucleotides.

[0224] Sequence cutting and deprotection were performed in 1.5 ml tubes. The first step used AMA (Ammonium Hydroxide / Methylamine), and the second step used triethylamine trifluoride to remove the protecting group at position 2. For sequences containing complete modifications at position 2, ammonia hydrolysis was performed. The cut and deprotected sequences were precipitated using an acetone:ethanol (80:20) mixture and dissolved in RNase-free water. Sequence accuracy was determined by LC-MS, quantification by spectrophotometry, and purity was determined by HPLC.

[0225] After HPLC purification, lyophilization, and quality control, the salt was replaced by sodium acetate alcohol precipitation, and then desalted using a 3KD ultrafiltration tube. After desalting, the sense and antisense strands were quantitatively determined by spectrophotometer, and then mixed and annealed at a 1:1 ratio to form siRNA duplexes.

[0226] For the modified siRNA conjugates listed in Tables 1 and 2 below, the base sequences of the sense and antisense strands of each modified siRNA conjugate before modification are listed in the tables below.

[0227] In this embodiment, the siRNA was modified based on the sequences in Table 1 to obtain the sequences in Table 2.

[0228] Table 1

[0229] Table 2

[0230] Wherein, C, G, U, A, and T represent the base composition of the nucleotide, including modified and unmodified nucleotides; m indicates that the nucleotide adjacent to the right of the m identifier is a 2'-methoxynucleotide; i2F indicates that the nucleotide adjacent to the right of the i2F identifier is a 2'-fluoronucleotide; the asterisk (*) indicates that the two nucleotides adjacent to the asterisk (or between the nucleotide and the linker-target ligand) are linked by a phosphate thioester group; hd indicates a nucleotide modified with 2'O-C16; da indicates a nucleotide modified with 2'O-C22; gn indicates GNA modification; eVP represents... The nucleotide adjacent to the right of the identifier eVP is a (E)-vinyl phosphate modified nucleotide; there is no symbol between directly adjacent nucleotides; XY-021-12, XY-021-13, XY-021-14, XY-021-16, XY-021-19, XY-021-21, XY-021-23, XY-021-36, and XY-021-31 respectively represent the structures of the phosphoramidite monomer synthesized in the above embodiments of the present invention in siRNA after synthesis by Oligo and ammonolysis, as follows:

[0231] The technical solution of the present invention will be further illustrated by the following experiments. The sample RNAi molecules used in the following experimental examples were prepared by the methods described in the above embodiments.

[0232] Example 1: In vivo activity test of SOD1 gene in SD rats

[0233] 1. Experimental Methods

[0234] In this experiment, all animals were randomly assigned to groups (4 male SD rats per group, with 3 additional rats as reserves). All animals were weighed and grouped the day before drug administration (Day 0). Day 1 was the day of drug administration. Grouping and drug administration information are shown in Table 3 below. All animals had free access to water and food during the experiment. Rats were anesthetized with 2.5% isoflurane. After maintaining the depth of anesthesia, the rats were placed in a prone position, and the hair on their lower back was shaved. The puncture site was wiped with alcohol. The L5-6 interspinous space was used as the puncture point, maintaining a vertical distance >3 cm between the puncture point and the highest point of the rat's back. The experimenter's left hand reached the hip tubercle; its horizontal position was the L5-6 interspinous space of the rat. The left thumb and middle finger were placed on both sides of the L5-6 interspinous space of the rat, and the skin was stretched taut outwards. Using the index finger as the reference point, hold the microsyringe in your right hand and slowly insert it vertically into the gap. Observe the rat's tail while inserting the needle. When the tail flicks, gently pull back the syringe plunger. When you see cerebrospinal fluid reflux, determine the injection site and administer intrathecal injection (IT). The injection time and needle retention time after needle insertion are 30-40 seconds. The administration volume is 30uL, and the single dose is 0.9mg. The day of administration is defined as Day 1.

[0235] Fourteen days after administration, rats were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. The lumbar vertebrae, cervical vertebrae, frontal cortex, and hippocampus were then dissected and tissues were collected. These tissues were rinsed with pre-cooled physiological saline at 2–8°C, blotted dry with filter paper, and aliquoted into two tubes of 30–50 mg each for preservation. The tissues were then immersed in 5 times their volume of RNAlater at 4°C overnight, followed by storage at -80°C. The final samples will be used for Q-PCR detection.

[0236] The relative expression abundance of the target gene SOD1 in different tissues was detected using the 2-ΔΔCt method. Body weight was recorded and the weight and rate of change data were analyzed. Data were processed using Office Excel 2013 and GraphPad Prism 9.0, and are expressed as Mean ± SEM (standard error). One-way ANOVA was used for analysis, and Tukey's test was used to test for significant differences between groups. When comparing two groups, a two-tailed t-test was used. A p-value < 0.05 was considered statistically significant between the two groups.

[0237] 2. Experimental Results

[0238] The expression levels of the SOD1 gene are shown in Table 3. Several compounds with phosphate backbone-coupled lipids exhibited better inhibitory effects on the target gene than the Yangshen sequence LDR0162001. Furthermore, the inhibitory effects on gene expression varied significantly depending on the position of the lipid molecule containing the phosphoramidite group modified within the oligonucleotide.

[0239] Table 3. Expression levels of the SOD1 gene

[0240] Experiment Example 2: In vivo activity test of the hAPP gene in transgenic mice

[0241] 1. Experimental Methods

[0242] After maintaining the depth of anesthesia with 2.5% isoflurane inhalation, the animals were placed in a prone position, and the hair on their waists was shaved. The puncture site was wiped with alcohol. The L5-6 interspinous space was chosen as the puncture point, maintaining a vertical distance of >3cm between the puncture point and the highest point of the mouse's back. The experimenter's left hand reached the hip tubercle; its horizontal position was the L5-6 interspinous space of the rat. The left thumb and middle finger were placed on both sides of the L5-6 interspinous space of the mouse, and the skin was stretched outwards. With the index finger positioned, the right hand held a microsyringe and slowly inserted the needle vertically into the interspinous space. The rat's tail was observed during needle insertion. When a tail-flicking motion was observed, the syringe plunger was gently pulled back. When cerebrospinal fluid reflux was observed, the injection site was determined, and intrathecal injection was performed. The injection time and needle retention time were 30-40 seconds after needle insertion. The administration volume was 10uL, and the single dose was 0.3mg. The day of administration was defined as Day 0.

[0243] Seven days after administration (Day 7), mice were anesthetized with isoflurane and euthanized by exsanguination from the abdominal aorta. Dissection was performed to collect tissues from the lumbar spinal cord (administration site), cervical spinal cord, frontal cortex, midbrain, hippocampus, striatum, hypothalamus, and remaining brain tissue. The remaining brain tissue was flash-frozen in liquid nitrogen and then stored at -80°C for later testing. The remaining tissues were rinsed with pre-cooled physiological saline at 2–8°C, blotted dry with filter paper, and aliquoted into two tubes (10–20 mg per tube) for storage. All tissues were then immersed in 5 times their volume of RNAlater overnight at 4°C before being transferred to -80°C for storage. The final samples will be used for Q-PCR detection.

[0244] 2. Experimental Results

[0245] The test results are shown in Figure 1. At a dose of 0.3 mg, after 28 days, the inhibitory effect of LDR0182177 on the target gene hAPP mRNA in brain tissue was significantly better than that of the positive compound LDR0182176.

[0246] Experimental Example 3: Long-term effect test in SD rats

[0247] This experiment measured the inhibition efficiency of the SOD1 gene in SD rats.

[0248] 1. Experimental Methods

[0249] In this experiment, all animals were randomly divided into 3 groups (4 male SD rats in each group, with 3 additional rats as backup). The drug administration groups were LDR0182001, LDR0182028, and PBS. All animals were weighed and grouped the day before drug administration (Day 0). The day of drug administration was Day 1. All animals had free access to water and food during the experiment. Rats were anesthetized with 2.5% isoflurane. After maintaining the depth of anesthesia, the rats were placed in a prone position, and the hair on their lower back was shaved. The puncture site was wiped with alcohol. The L5-6 interspinous space was used as the puncture point, maintaining a vertical distance >3 cm between the puncture point and the highest point of the rat's back. The experimenter's left hand reached the hip tubercle; its horizontal position was the L5-6 interspinous space of the rat. The left thumb and middle finger were placed on both sides of the L5-6 interspinous space of the rat, and the skin was stretched taut outwards. Using the index finger as the reference point, hold the microsyringe in your right hand and slowly insert it vertically into the gap. Observe the rat's tail while inserting the needle. When the tail flicks, gently pull back the syringe plunger. When you see cerebrospinal fluid reflux, determine the injection site and administer intrathecal injection. The injection time and needle retention time after needle insertion are 30-40 seconds. The administration volume is 30uL. The single administration doses are 0.3mg and 0.9mg, respectively. The day of administration is defined as Day 1.

[0250] Twenty-eight days after administration, rats were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. Lumbar vertebrae, cervical vertebrae, frontal cortex, and hippocampus tissues were then dissected and rinsed with pre-cooled physiological saline at 2–8°C. After rinsing, the tissues were blotted dry with filter paper. Each tissue sample was aliquoted into two tubes, 30–50 mg per tube, for preservation. The tissues were then immersed in 5 times their volume of RNAlater overnight at 4°C, followed by storage at -80°C. The final samples will be used for Q-PCR detection.

[0251] Twenty-eight days after a single injection of different doses of iodine (IT) into rats, the relative expression abundance of the target gene SOD1 in different tissues was detected using the 2-ΔΔCt method. Body weight was recorded and the weight and rate of change were analyzed. Data were processed using Office Excel 2013 and GraphPad Prism 9.0, and are expressed as Mean ± SEM (standard error). One-way ANOVA was used for analysis, and Tukey's test was used for statistical significance testing between groups. For comparisons between two groups, a two-tailed t-test was used; a p-value < 0.05 was considered statistically significant between the two groups.

[0252] 2. Experimental Results

[0253] The test results are shown in Figure 2. At a dose of 0.3 mg, after 28 days, LDR0162028 showed a similar inhibitory effect on the target gene SDO1 mRNA in brain tissue to the positive compound LDR0162001. At a dose of 0.9 mg, after 28 days, LDR0162028 showed a significantly better inhibitory effect on the target gene SDO1 mRNA in brain tissue than the positive compound, while its inhibitory effect on the heart was weaker than that of the positive compound, which may indicate better safety.

[0254] Experiment 4: In vivo tolerance evaluation in SD rats

[0255] 1. Experimental Methods

[0256] SD rats were administered different doses of LDR0182177 and the control group LDR0182176 via intrathecal injection for a single week (6-8 weeks). After drug withdrawal, the rats were observed for 2 weeks to assess acute toxicity and its reversibility or possible delayed toxicity. The tolerance of SD rats to different doses of LDR0182177 and LDR0182176 was evaluated through clinical observation and testing, clinicopathology, nerve conduction function testing, safety pharmacology, biodistribution, and histopathology.

[0257] Preparation of the test sample: Thaw the pre-prepared stock solution of the test sample at 2–8°C and vortex to mix before use. The operation should be performed in a clean bench (aseptic and protected from light): Pipette the required volume of solvent into a sterile EP tube, add the required volume of the stock solution of the test sample (which can be diluted stepwise), and vortex to mix to obtain the test sample formulation of the required concentration. The test sample formulation should be temporarily stored at 2–8°C and transported under moist ice and protected from light for administration. For long-term storage, store in a -20°C freezer.

[0258] The grouping and dosage are shown in Table 4 below:

[0259] Table 4

[0260] 2. Experimental Results

[0261] The experimental results are shown in Table 5 below.

[0262] Table 5

[0263] As can be seen from the above embodiments and experimental examples, this invention prepares a series of lipid molecules containing phosphoramidite groups, reacts them with oligonucleotides to obtain RNAi molecules; by modifying the lipid molecules containing phosphoramidite groups to different positions in the oligonucleotides, RNAi molecules with better effects are obtained. This RNAi molecule can effectively target extrahepatic tissues while maintaining its inhibitory effect on the target gene. The lipid molecules prepared by this invention show promising potential as extrahepatic delivery vectors, and the RNAi molecules prepared by this invention have significant value for the research and development and clinical application of RNAi drugs.

Claims

1. An oligonucleotide, characterized in that: the structure of formula I is attached at an internal position or at a terminus of an oligonucleotide; in, X1 is selected from S or O; X2 is selected from O, S, CH2 or NH; L is selected from a cleavable or non-cleavable chemical linker selected from -SS-, -(CF2) b - -(CH2) b - -NHCONH-, -NHCOO-, -NHCOCH2-, -NHCO-, -CONH-, -COO-, -OOC-, C3-C 10 cycloalkyl, C6-C 20 aryl, -NHSO2- at least one, b is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R1is selected from substituted or unsubstituted C1-C 30 substituted or unsubstituted C2-C 30 substituted or unsubstituted C2-C 30 substituted or unsubstituted C2-C substituents are selected from the group consisting of halogen, deuterium, Ci-C 30 linear alkyl, C3-C 10 cycloalkyl, Ci-C 30 haloalkyl; R5 is selected from unmodified or modified nucleosides, wherein the oxygen at the 3' end of the pentose sugar or the 3' end of the tetraose sugar in the nucleoside is linked to P. The modified nucleoside is selected from the nucleoside of modified nucleotide a, wherein the modified nucleotide a is selected from threonucleotides, C6-C... 20 Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-Cl-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides. q is selected from 0, 1, 2, 3, 4, 5.

2. The oligonucleotide according to claim 1, characterized in that: The oligonucleotide is composed of nucleotides, which are selected from unmodified nucleotides or modified nucleotide b, wherein the modified nucleotide b is selected from threonucleotides, C6-C... 20 Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-Cl-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, 3'-OMe nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides.

3. The oligonucleotide according to claim 2, characterized in that: The connection is accomplished by replacing the phosphate ester group between two adjacent nucleotides in the oligonucleotide.

4. The oligonucleotide according to claim 3, characterized in that: The oligonucleotides consist of 14-45 nucleotides.

5. The oligonucleotide according to claim 4, characterized in that: Replace the numbers 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, and 25-26 in the oligonucleotides with the structure shown in Formula I. Phosphate groups between nucleotides 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, and 44-45, wherein the first nucleotide at the 5' end of the oligonucleotide is position 1.

6. The oligonucleotide of claim 3, wherein: The oligonucleotide consists of 21-23 nucleotides.

7. The oligonucleotide according to claim 6, characterized in that: Replace the phosphate ester groups between the nucleotides at positions 4-5, 5-6, 6-7, 15-16, and 16-17 in the oligonucleotide with the structure shown in Formula I, wherein the first nucleotide at the 5' end of the oligonucleotide is position 1.

8. The oligonucleotide according to claim 7, characterized in that: The oligonucleotide consists of 21 nucleotides, with the phosphate ester group between the 5th-6th or 15th-16th nucleotides in the oligonucleotide replaced by the structure shown in Formula I.

9. The oligonucleotide of claim 1, wherein: the structure of formula I is selected from at least one of the following structures: ​ Where m, n, and k are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, respectively.

10. The oligonucleotide of claim 1, wherein: the structure of formula I is selected from at least one of the following structures: ​ 11. The oligonucleotide of claim 1, wherein: The oligonucleotide comprises at least one of the following structures: in, A and B are independently selected from O, S, -CH2-, and Se atoms, respectively; Base1 and Base2 are independently selected from A, T, G, C, I or unconventional bases, respectively; R' and R" are independently selected from H, F, OMOE, C1-C4 alkyl, and C1-C4 alkoxy, respectively.

12. The oligonucleotide according to claim 11, characterized in that, The oligonucleotide comprises nucleotides of the following structure: and / or the unconventional base is selected from the following structures:

13. The oligonucleotide of claim 1, wherein The structure shown in Formula I is selected from at least one of the following structures:

14. The oligonucleotide according to claim 13, characterized in that, It is selected from the following oligonucleotides: oligonucleotide 3, oligonucleotide 4, oligonucleotide 5, oligonucleotide 6, oligonucleotide 7, oligonucleotide 8, oligonucleotide 9, oligonucleotide 10, oligonucleotide 11, oligonucleotide 12, oligonucleotide 14, and oligonucleotide 17.

15. A method of producing an oligonucleotide according to any one of claims 1 to 14, characterized in that, include: Unmodified nucleotides or modified nucleotide b are reacted with phosphoramidite lipid molecules in an automated apparatus for synthesizing oligonucleotides to obtain the product. The structure of the phosphoramidite lipid molecule is shown in Formula II: in, X2 is selected from O, S, CH2 or NH; L is a chemical linker selected from -SS-, -CF2-, -(CH2) b - NHCONH-, -NHCOO-, -NHCOCH2-, -NHCO-, -CONH-, -COO-, -OOC-, C3-C 10 cycloalkyl, C6-C 20 aryl, -NHSO2- at least one, b is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R1is selected from substituted or unsubstituted C1-C 30 unsubstituted C2-C 30 unsubstituted C2-C 30 unsubstituted C2-C substituents are selected from the group consisting of halogen, deuterium, Ci-C 30 linear alkyl, C3-C 10 cycloalkyl, Ci-C 30 haloalkyl; R2, R3, and R4 are independently selected from hydrogen and -NR7R8, respectively, and R7 and R8 are independently selected from C1-C. 30 alkyl; R5 is selected from unmodified or modified nucleosides, wherein the oxygen at the 5' end of the pentose sugar or the 4' end of the tetraose sugar in the nucleoside is linked to R6, and the oxygen at the 3' end of the pentose sugar or the 3' end of the tetraose sugar in the nucleoside is linked to P. The modified nucleoside is selected from the modified nucleotide a, and the modified nucleotide a is selected from threonucleotides, C6-C... 20 Nucleotides with acyl-modified bases, 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides, glycol nucleic acid nucleotides, 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotides, baseless nucleotides, ribitol, reverse nucleotides, reverse baseless nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-C1-C 30 At least one of the following: alkyl-modified nucleotides, morpholinonucleotides, 2'-amino-modified nucleotides, nucleotides containing a 5'-thiophosphate group, and aminophosphate-modified nucleotides. R6 is selected from a protecting group; q is selected from 0, 1, 2, 3, 4, 5.

16. The method of claim 15, wherein: The mixing process also requires the addition of an activator, which is selected from 5-benzylthio-1H-tetrazole, tetrazolium, 5-phenylthiotetrazole, 5-ethyltetrazole, and 5-methyltetrazole. The molar ratio of the phosphoramidite lipid molecule to the activator is 0.04-0.06:0.28-0.

36. And / or, after the reaction in the automated equipment for synthesizing oligonucleotides, the oligonucleotides may need to be cleaved and deprotected from the solid support.

17. An RNAi molecule, characterized in that: It includes the oligonucleotides according to any one of claims 1-14; wherein the RNAi molecule is single-stranded or double-stranded.

18. The RNAi molecule according to claim 17, wherein: It is a double-stranded siRNA molecule.

19. The RNAi molecule according to claim 18, characterized in that: The positive strand of the siRNA molecule contains the oligonucleotide.

20. The RNAi molecule according to claim 19, wherein: It is composed of oligonucleotide 2 and oligonucleotide 3; And / or, it is composed of oligonucleotide 2 and oligonucleotide 4; And / or, it is composed of oligonucleotide 2 and oligonucleotide 5; And / or, it is composed of oligonucleotide 2 and oligonucleotide 6; And / or, it is composed of oligonucleotide 2 and oligonucleotide 7; And / or, it is composed of oligonucleotide 2 and oligonucleotide 8; And / or, it is composed of oligonucleotide 2 and oligonucleotide 9; And / or, it is composed of oligonucleotide 2 and oligonucleotide 10; And / or, it is composed of oligonucleotide 2 and oligonucleotide 12; And / or, it is composed of oligonucleotide 2 and oligonucleotide 14; And / or, it consists of oligonucleotide 15 and oligonucleotide 17.

21. Use of the oligonucleotide according to any one of claims 1-14 and the RNAi molecule according to any one of claims 17-20 in the preparation of RNAi drugs.

22. Use according to claim 21, characterized in that: The RNAi drug is delivered to extrahepatic tissues.

23. Use according to claim 22, characterized in that: The extrahepatic tissues include central nervous system tissues, muscle tissues, and adipose tissues.

24. An RNAi drug, characterized in that: It comprises the oligonucleotides according to any one of claims 1-14 and the RNAi molecules according to any one of claims 17-20.

25. A lipid-modified phosphoramidite selected from the group consisting of:

Images