Modified nucleoside compound and use thereof in oligonucleotide

Abstract

The present invention provides a novel nucleoside phosphoramidite compound. The compound is used for preparing an oligonucleotide. The nucleoside phosphoramidite compound of the present invention can be incorporated into the end and / or the middle of the oligonucleotide, so that the modified oligonucleotide hybridizes with a part of a target mRNA, thereby causing loss or downregulation of the normal function of the target mRNA.

Description

A modified nucleoside compound and its application in oligonucleotides

[0001] This application is based on and claims priority to the applications CN application number 202411448743.5 filed on October 17, 2024 and CN application number 202510720779.2 filed on May 30, 2025, the disclosures of the aforementioned applications are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of biopharmaceuticals, specifically to a modified nucleoside compound and oligonucleotides prepared therefrom. Background Technology

[0003] Oligonucleotides are polymers of nucleotides that, as nucleic acid inhibitor molecules, can regulate the levels of target mRNAs within cells and have shown early promise in the treatment of genetic diseases, metabolic diseases, inflammatory diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate mRNA expression through a variety of mechanisms, including RNA interference (RNAi).

[0004] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target mRNA with a complementary sequence to the dsRNA. In one typical RNAi pathway, the longer dsRNA is cleaved by a nuclease (Dicer) into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with the nuclease, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0005] Various double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 6 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which are cleaved in vivo by endonucleases into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules in which at least one end of at least one strand extends beyond the double-stranded target region of the molecule, one of the strands comprising a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). These structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0006] In recent years, many modified nucleosides have been used for RNAi, such as common modifications like 2'-fluorine, 2'-methoxy, 2'-methoxyethyl, UNA, LNA, BNA, GNA, and 5'-(E)-vinyl phosphate. In some cases, chemical modifications are introduced into nucleic acid inhibitor molecules to obtain potentially desired properties after in vivo administration. These modifications include those designed to, for example, antagonize nucleases or other enzymes that interfere with the structure or activity of oligonucleotides, increase cellular uptake of oligonucleotides, or improve the pharmacokinetic properties of oligonucleotides. Introducing modified nucleosides into the corresponding oligonucleotide sequences improves the pharmacokinetic properties and bioavailability of oligonucleotide drugs in vivo. Further research is needed on the chemical modification of oligonucleotide drugs to obtain oligonucleotide drugs with superior properties in all aspects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to develop a 4'-modified threonine compound with more stable conformation and metabolic stability. When applied to oligonucleotide molecules (including but not limited to siRNA, antisense nucleic acids, saRNA, miRNA aptamers, and lncRNA), it can modify the pharmacokinetics of oligonucleotides while improving their activity and metabolic stability. Furthermore, this type of modified nucleoside compound with long-chain alkane conjugation enables the delivery of oligonucleotides to the liver, CNS, adipose tissue, and muscle tissue.

[0008] One of the objectives of this invention is to provide a nucleoside compound (nucleoside phosphoramide compound, used to modify nucleotides, i.e., 4'-modified threonine compound) as shown in Formula 1.

[0009] A nucleoside phosphoramidide compound as shown in Formula 1a, and its stereoisomers and deuterated compounds:

[0010] in:

[0011] R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl.

[0012] R 4 It is a hydroxyl protecting group; preferably, R 4 For DMTr and MMTr;

[0013] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0014] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0015] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0016] X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10;

[0017] X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5;

[0018] M is P(OR) 10 )(N(R 11)2) P(R 10 )(N(R 11 )2); where R 10 R 11 Each is independently selected from cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl, substituted or unsubstituted C1-C25 alkoxy;

[0019] L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15 )-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 Independently hydrogen or unsubstituted C1-C6 alkyl;

[0020] In some embodiments of the present invention, the compound represented by formula 1a has the structure represented by formula 2a:

[0021] in:

[0022] R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl.

[0023] R 4 It is a hydroxyl protecting group; preferably, R 4 For DMTr and MMTr;

[0024] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0025] R 17 It is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy);

[0026] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0027] X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10;

[0028] X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5;

[0029] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0030] L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15 )-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 It is independently hydrogen or an unsubstituted C1-C6 alkyl group.

[0031] In some embodiments of the present invention, the compound represented by formula 1a has the structure represented by formula 3a:

[0032] in:

[0033] R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl.

[0034] R 4 It is a hydroxyl protecting group; preferably, R 4 For DMTr and MMTr;

[0035] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0036] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0037] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0038] R 17 It is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

[0039] In some embodiments of the present invention, the compound represented by Formula 1a has the structure represented by Formula 4a:

[0040] in:

[0041] R 4 It is a hydroxyl protecting group; preferably, R 4 For DMTr and MMTr;

[0042] R 7It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0043] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0044] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0045] R 17 It is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

[0046] In some embodiments of the present invention, the compound represented by formula 1a has the structure represented by formula 5a:

[0047] in:

[0048] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0049] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0050] R 17 It is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

[0051] In some embodiments of the present invention, the compound represented by Formula 1a has the structure represented by Formula 6a:

[0052] R 7It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0053] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

[0054] In some embodiments of the present invention, the compound represented by Formula 1a has the structure represented by Formula 7a:

[0055] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0056] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

[0057] In some embodiments of the present invention, the compound represented by formula 1a has the following characteristics as shown in formula 8a:

[0058] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0059] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

[0060] In some embodiments of the present invention, the compound represented by formula 1a has a structure as shown in formulas 8a-2:

[0061] R 7 It can be cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0062] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

[0063] In some embodiments of the present invention, the Base is a natural nucleobase, a modified nucleobase, a universal base, or an aryl group, or may not exist. Further, it includes, but is not limited to, the following structures:

[0064] In some embodiments of the present invention, the nucleoside compound represented by Formula 1a includes, but is not limited to, the following nucleoside phosphoramide compounds:

[0065] The present invention also provides the use of the above-mentioned nucleoside phosphoramide compound in the preparation of oligonucleotides.

[0066] The present invention also provides the use of the above-mentioned nucleoside phosphoramide compound as an intermediate in the preparation of oligonucleotides.

[0067] Furthermore, the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamer, and lncRNA.

[0068] The present invention also provides an oligonucleotide comprising at least one structure shown in Formula 9a:

[0069] in:

[0070] R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl.

[0071] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0072] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0073] Z represents -O-, -S-, -Se-, -NR8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0074] X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10;

[0075] X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5;

[0076] M 2 for Or H, where R 3 The alkyl group is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2;

[0077] L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15)-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 It is independently hydrogen or an unsubstituted C1-C6 alkyl group.

[0078] In some embodiments of the present invention, the oligonucleotide comprises at least one structure shown in Formula 10a:

[0079] in:

[0080] R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl.

[0081] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0082] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0083] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0084] M 2 for Or H, where R 3 The alkyl group is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2;

[0085] In some embodiments of the present invention, the oligonucleotide comprises at least one structure shown in formula 11a:

[0086] in:

[0087] R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0088] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0089] Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0090] M 2 for Or H, where R 3 The alkyl group is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2;

[0091] In some embodiments of the present invention, the oligonucleotide comprises at least one structure shown in formula 12a:

[0092] in:

[0093] R 7It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl.

[0094] Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist.

[0095] M 2 for Or H, where R 3 Y is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25 alkyl (e.g., methyl), substituted or unsubstituted C1-C25 alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2.

[0096] Furthermore, the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamer, and lncRNA.

[0097] Furthermore, the oligonucleotide is selected from siRNA, wherein the siRNA is a double-stranded oligonucleotide composed of one sense strand and one antisense strand, which can mediate the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway, achieving specific degradation of the target mRNA sequence. For example, it can inhibit the expression of target genes in cells, wherein the cells are cells in an organism, such as cells in a mammal.

[0098] The siRNA sense strand has a length of 10-40 nucleotides; the antisense strand has a length of 10-40 nucleotides. The sense and antisense strands together form a double-stranded siRNA molecule. The double-stranded region of the siRNA molecule can be of any length, as long as it can degrade the target mRNA through the RNA-induced silencing complex process. The length of its double-stranded region can vary from 10 to 40 base pairs, for example, approximately 15-40 base pairs, such as approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 base pairs, such as approximately 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 1... 5-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19- 24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pair lengths. Ranges and lengths between the above ranges are also considered part of this invention.

[0099] The modified nucleoside compounds described above can be conjugated to siRNA sequences, having the structure described in Formula 9a, and can be conjugated to the ends or middle of each nucleic acid single strand of the siRNA. For example, the modified nucleoside compounds mentioned above can be conjugated to various positions on the positive strand of the siRNA. When the siRNA has a sense strand with a length of 21 nucleotides and an antisense strand with a length of 23 nucleotides, the above-mentioned modified nucleoside compounds can be synthesized in a solid-phase manner at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 on the sense strand of the siRNA, and positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 on the antisense strand of the siRNA (the first nucleotide position at the 5' end of both the sense and antisense strands is designated as position 1).

[0100] Nucleoside phosphoramidides (e.g., LCT-051 to LCT-070, etc., modified nucleoside compounds conjugated with long-chain alkanes) as described above can be conjugated to siRNA sequences. These compounds have the structure described in Formula 9a within the siRNA and can be conjugated to the ends or middle of each nucleic acid single strand of the siRNA. For example, the aforementioned modified nucleoside compounds can be conjugated to various positions on the sense strand of the siRNA. When the siRNA has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, the aforementioned modified nucleoside compounds can be synthesized in a solid-phase manner at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 on the sense strand of the siRNA, and positions 1, 2, 3, 4, and 5 on the antisense strand of the siRNA. Positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (the positions of the sense and antisense strands are defined by the first nucleotide position at the 5' end of each nucleic acid single strand as position 1), wherein the siRNA conjugated with the nucleoside compound shown in Formula 9a can achieve targeted delivery to the liver and extrahepatic tissues, degrading the target mRNA in the liver and extrahepatic tissues. The extrahepatic targeted delivery, for example, targets CNS tissue, muscle tissue, adipose tissue, eyes, etc., and the siRNA conjugate conjugated with LCT-modified nucleosides has the following general structural formulas (Formulas 1 to 3):

[0101] As described above, nucleoside phosphoramidide compounds (e.g., modified nucleoside compounds such as LCT-051 to LCT-070 that are conjugated with long-chain alkanes) can be conjugated to siRNA sequences, having the structure described in Formula 9a in the siRNA. They can be conjugated to the end or middle of each nucleic acid single strand of the siRNA to achieve extrahepatic delivery of siRNA by LCT compounds (e.g., fat, muscle, heart, eye, lung, kidney, CNS tissues). To enhance the extrahepatic delivery efficiency of LCT compounds conjugated with long-chain alkane modifications, at least one ligand group can be conjugated to each of the two nucleic acid single strands of the siRNA. The ligand group includes: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths, and fatty acid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as permeabilizing peptides, targeting peptides (lipid-targeting polypeptides; muscle-targeting polypeptides; CNS-targeting polypeptides, etc.); aptamers; antibodies; carbohydrates, such as lactose, polylactose, mannose, galactose, and N-acetylgalactosamine (GalNAc); folic acid; or receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, integrin receptors, aminoglycosides, and gentamicin-like molecules. The conjugates connected with LCT-modified molecules (the modified nucleoside phosphoramidides compounds as described in claims 1-11) and ligand groups have the following general structural formula:

[0102] The M1, M2, M3, M 4、 M5, M6, M7, M8, M9, T1, T2, T3, T4, T5, T6, T7, T8, and T9 can be independently selected from one or more of the following structural units: -N(R 20 )-, -O-, -S-, -C(O)-, -N(R 20 )C(O)-、-C(O)N(R 21 )-、-N(R 20 )C(O)N(R 21 -, -C(O)O-, -OC(O)-, -N(R) 20 )C(O)O-、-OC(O)N(R 21 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 22 )-O-、-OP(S)(R 22 )-O-、-OP(O)(NR 20 R 21 -N-、-OP(S)(NR)20 R 21 )-N-、-OP(O)(NR 20 R 21 )-O-、-OP(S)(NR 20 R 21 )-O-、-P(O)(NR 20 R 21 -N-、-P(S)(NR) 20 R 21 -N-、-P(O)(NR) 20 R 21 )-O-、-P(S)(NR 20 R 21 -O-, -SS-, triazolyl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; wherein each R 20 R 21 and R 22 It is independently hydrogen or an unsubstituted C1-C6 alkyl group; the substitution is preferably deuterated or halogenated.

[0103] Furthermore, the ligand group, i.e., the "Ligand" molecular fragment, includes, but is not limited to, the following structures:

[0104] Among them, R 30 It is hydrogen or a C1-C6 alkyl group; R 31 It is protected by H or an amino group;

[0105] In this invention, "terminal" refers to the 3' terminal and the 5' terminal; "non-terminal" refers to sites other than the 3' terminal and the 5' terminal.

[0106] As described above, nucleoside phosphoramidide compounds (e.g., modified nucleoside compounds such as LCT-051 to LCT-070 that are conjugated with long-chain alkanes) can be conjugated to siRNA sequences, having the structure described in Formula 9a in the siRNA, enabling extrahepatic delivery and degradation of target genes via RNA-induced silencing complex (RISC). The target genes mentioned include: adrenaline receptor β1 (ADRB1); calcium voltage-gated channel subunit α1C (CACNA1C); calcium voltage-gated channel subunit α1G (CACNA1G) (T-type calcium channel); angiotensin II receptor type 1 (AGTR1); sodium voltage-gated channel α subunit 2 (SCN2A); hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (HCN3); potassium voltage-gated channel subfamily A member 5 (KCNA5); potassium inward rectifier channel subfamily J member 3 (KCNJ3); potassium inward rectifier channel subfamily J member 4 (KCNJ4); phosphatidylinositol (PLN); calcium / calmodulin-dependent protein kinase II delta (CAMK2D); or phosphodiesterase 1 (PDE1); Delta 4-Desaturase, sphingolipid 1 (DEGS1); leptin; fibroblast growth factor (FLCN); zinc finger protein 423 (ZFP423); cell cycle-dependent kinase 6 (CDK6); regulatory proteins related to mammalian target of rapamycin complex 1 (RPTOR); mammalian target of rapamycin kinase (mTOR); forkhead box P1 (FOXP1); phosphodiesterase 3B (PDE3B); and activin A receptor type 1 (ACVR1C); myosin (MSTN); cholinergic receptor nicotinic α1 subunit (CHRNA1); cholinergic receptor nicotinic acid The target genes of CNS tissues, adipose tissues, and muscle tissues include: β1 subunit (CHRNB1); cholinergic receptor δ subunit (CHRND); cholinergic receptor ε subunit (CHRNE); cholinergic receptor γ subunit (CHRNG); collagen XIII α1 chain (COL13A1); docking protein 7 (DOK7); low-density lipoprotein receptor-associated protein 4 (LRP4); muscle-associated receptor tyrosine kinase (MUSK); synaptic-associated protein (RAPSN); sodium voltage-gated channel α subunit 4 (SCN4A); and dual homeobox 4 (DUX4).

[0107] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0108] The carbon, hydrogen, oxygen, sulfur, nitrogen, or F, Cl, Br, I mentioned in the groups and compounds described in this application include their isotopes, and the carbon, hydrogen, oxygen, sulfur, or nitrogen mentioned in the groups and compounds described in this application may optionally be further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C 13 C and 14 C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called superheavy hydrogen), and the isotopes of oxygen include 16 O、 17 O and 18 O, isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, isotopes of fluorine include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Cl, isotopes of bromine include 79 Br and 81 Br.

[0109] In this invention, "oligonucleotide" refers to a polymer of linked nucleosides, each nucleoside being independently modified or unmodified, comprising an oligonucleotide sequence of approximately 10-50 single-stranded or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a nucleobase sequence at least partially complementary to the core sequence of a target gene expressed in cells. In some embodiments, the oligonucleotide, upon delivery to a cell expressing the gene, regulates the expression of the corresponding target gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), non-coding RNA (lncRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0110] In this invention, "nucleic acid drug" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0111] Nucleic acid molecules can exert their effects 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 "nucleic acid drug" as used in this invention is considered to primarily exert its effects through RNA interference mechanisms, the nucleic acid drug is not limited to or restricted to any particular pathway or mechanism of action. Types of nucleic acid drug molecules include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), non-coding RNA (lncRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The nucleic acid drug of this invention comprises an oligonucleotide chain having at least a partial complementarity to the mRNA serving as the target. In some embodiments, the nucleic acid drug of this invention is double-stranded and comprises an antisense strand and a sense strand at least partially complementary to the antisense strand.

[0112] 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 nucleic acid drug molecules described in this invention, compared to administration into cells, tissues, organs, or animals that have not been so treated.

[0113] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0114] 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.

[0115] 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.

[0116] The term "complementarity," used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of a nucleic acid drug or target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded antisense strand of a nucleic acid 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 base pairs and a double-stranded or double-helix 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.

[0117] 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.

[0118] 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.

[0119] The terms “optional” or “optionally” mean that the event or environment described below may, but does not have to, occur, and the description includes the possibility that the event or environment may or may not occur. 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.

[0120] 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, either 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). The substituents can be halogen atoms, amino groups, mercapto groups, cyano groups, etc.

[0121] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～ C b Alkyl groups indicate any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0122] 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. For example, C22 alkyl refers to C... 22 H 45 - The alkyl group 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 group.

[0123] "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, Ca-b Alkenyl 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.

[0124] "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 those hydrocarbon groups having one triple bond and one double bond. For example, C 2-6 The term "alkynyl" is intended to include ethynyl, propynyl, etc.

[0125] "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.

[0126] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0127] "Halogenated alkyl" refers to an alkyl group that is substituted with one or more halogens, wherein the alkyl group is as defined above.

[0128] "Ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0129] "Acyl" refers to a compound containing a -C(O)R group, where R is an alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group.

[0130] 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.

[0131] "Heteroaryl" refers to an aromatic ring group having a conjugated planar ring system and containing heteroatoms. It can be a 3- to 8-membered (e.g., 3, 4, 5, 6, 7, 8-membered) monocyclic, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O, or S. Non-limiting examples of heteroaryl include triazolyl, pyridyl, furanyl, thiophene, pyranyl, pyrroloyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridyl, and pyrrolopyridyl. The heteroaryl group may optionally be further substituted by one or more substituents.

[0132] "Heterocyclic group" or "heterocycle" refers to a saturated or unsaturated aromatic heterocycle or a non-aromatic heterocycle. When it is an aromatic heterocycle, its definition is the same as the definition of "heteroaryl" above. When it is a non-aromatic heterocycle, it can be a 3- to 10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic, a 4- to 12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O, or S, preferably a 3- to 8-membered heterocyclic group. Non-limiting examples of "heterocyclic group" or "heterocycle" include oxoheterobutyl, azaheterobutyl, thioheterobutyl, 1,3-dioxopentyl, 1,4-dioxopentyl, 1,3-dioxahexacycloyl, azaheptanyl, oxoheptanyl, thioheptanyl, triazolyl, pyridyl, piperidinyl, furanyl, thiophene, pyranyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thiaxylyl, 1,3-dithiaalkyl, dihydrofuranyl, dithiapentylyl Cycloyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydropyrroleyl, tetrahydroimidazoyl, tetrahydrothiazoyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxapentyl, pyrazolinyl, dithiaalkyl, dithiamonyl, dihydrothiophenyl, pyrazolyl, imidazolinyl, imidazolinyl, 1,2,3,4-tetrahydroisoquinolinyl. The "heterocyclic group" or "heterocycle" may optionally be further substituted with one or more substituents.

[0133] 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.

[0134] 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).

[0135] 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.

[0136] 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.

[0137] 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. Attached Figure Description

[0138] Figure 1: Male cynomolgus monkeys were administered conjugate 35 subcutaneously at a dose of 3.0 mg / kg. The relative expression levels of adipose-derived ALK7 mRNA at different time points were detected by qPCR. Detailed Implementation

[0139] Unless otherwise specified, the instruments used in this invention are not conventional instruments, and the reagents used are all conventional reagents.

[0140] The structure of a compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0141] NMR shifts (δ) are given in units of 10⁻⁶ (ppm). NMR measurements were performed using a Broker Avance III 400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), and deuterated methanol (CD₃OD) as solvents, and tetramethylsilane (TMS) as the internal standard. MS measurements were performed using Agilent 6120B (ESI) and Agilent 6120B (APCI). Thin-layer chromatography (TLC) used Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The TLC plates used had a diameter of 0.15 mm–0.20 mm, and the purified products used had a diameter of 0.4 mm–0.5 mm.

[0142] Example

[0143] Synthesis of compound LCT-063:

[0144] Synthesis of Compound 2: Substrate 1 (150.0 g, 789.5 mmol) was dissolved in anhydrous pyrine (1500 mL) in a dry 3000 mL single-necked flask. Then, under nitrogen protection, TrtCl (242.0 g, 868.4 mmol) and DMAP (9.6 g, 78.9 mmol) were added. After the addition was complete, the reaction mixture was stirred at room temperature for 6 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 1000 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with water (1000 mL x 3) and then with saturated brine (1000 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude product 2 (320 g, 740.7 mmol). ESI-LCMS m / z 431.2 [MH] - .

[0145] Synthesis of Compound 3: Substrate 2 (320 g, 740.7 mmol) was dissolved in anhydrous THF (3200 mL) in a dry 10000 mL single-necked flask. Then, under nitrogen protection, BnBr (152.0 g, 888.8 mol) and NaI (11.2 g, 74.1 mmol) were added. Finally, NaH (59.2 g, 1.48 mol, 60% w) was added in portions at 0 °C. After the addition was complete, the reaction system was stirred at 0 °C for 1 hour, then brought to room temperature and stirred for another 15 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was diluted with 5000 mL of ethyl acetate, quenched with saturated NH4Cl solution and washed (5000 mL * 3), and then washed with saturated brine (2000 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude product 3 (402.0 g). ESI-LCMS m / z 521.3 [MH] - .

[0146] Synthesis of Compound 4: Compound 3 (402.0 g) dissolved in TBME (1600 mL) was added to a dry 10000 mL single-necked flask. Formic acid (2400 mL) was then added at 0 °C. After the addition was complete, the reaction mixture was stirred at 0 °C for 30 minutes. TLC and LCMS confirmed the complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was then diluted with 2000 mL of ethyl acetate, quenched with saturated NaHCO3 solution and washed (4000 mL * 3), followed by washing 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 = 20:1), and concentrated to give a light brown oily product 4 (100.0 g, 357.1 mmol, 45% three-step yield). ESI-LCMS m / z 279.3 [MH] - .

[0147] Synthesis of Compound 5: Compound 4 (100.0 g, 357.1 mmol) was dissolved in anhydrous DMF (1000 mL) in a dry 3000 mL single-necked flask, followed by the addition of bromo-22-alkyl (153.0 g, 392.8 mmol), and finally, NaH (21.5 g, 535.6 mmol, 60% w) was added in portions at 0 °C. After the addition was complete, the reaction system was stirred at 0 °C for 1 hour, then brought to room temperature and stirred for another 15 hours. The reaction was considered complete when the starting material was completely eliminated by TLC and LCMS. The reaction solution was then diluted with 1000 mL of ethyl acetate, quenched with saturated NH4Cl solution and washed (3000 mL * 3), and washed with saturated brine (1000 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude compound 5 (195.0 g, 331.1 mmol). ESI-LCMS m / z 587.3 [MH] - .

[0148] Synthesis of Compound 6: Substrate 5 (195.0 g, 331.1 mmol) was dissolved in glacial acetic acid (2000 mL) in a dry 5000 mL single-necked flask. Ac₂O (195 mL) was then slowly added at 0 °C, and concentrated sulfuric acid (19.5 mL) was added dropwise while maintaining the temperature at 0 °C. After the addition was complete, the reaction system was stirred at room temperature for 20 minutes. The reaction was considered complete when the starting material was completely eliminated by TLC and LCMS. The reaction solution was then diluted with 300 mL of ethyl acetate, quenched with saturated NaHCO₃ solution and washed (4000 mL * 3), and washed 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 = 20:1) and concentrated to give a colorless oily product 6 (102.0 g, 161.3 mmol, 45% two-step yield). ESI-LCMS m / z 631.3 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ739-7.27(m,5H),7.46–7.15(m,9H),6.43–6.10(m,1H),5.33–5.23(m,1H),4.80–4.56(m,2H) ,4.25–3.95(m,1H),3.78–3.36(m,4H),2.11–2.02(m,6H),1.62–1.49(m,2H),1.34–1.21(m,38H),0.92–0.88(m,3H).

[0149] Synthesis of Compound 7: Substrate 6 (21.0 g, 33.2 mmol) was dissolved in anhydrous ACN (210 mL) in a dry 250 mL single-necked flask, followed by the addition of BSA (20.2 g, 99.6 mmol) and Uracil (4.1 g, 36.5 mmol). The reaction mixture was stirred at 50 °C for half an hour. Then, TMSOTf (11.1 g, 49.8 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was stirred at 80 °C for 5 hours. LC-MS analysis showed complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was then added to the reaction mixture, followed by washing with water (100 mL x 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 = 6:1) and concentrated to give a pale yellow solid, product 7 (19.0 g, 27.8 mmol, 84% yield). ESI-LCMS m / z 683.2 [MH] - .

[0150] Synthesis of Compound 8: Substrate 7 (19.0 g, 27.8 mmol) was dissolved in anhydrous DCM (700 mL) in a dry 3000 mL single-necked flask. Then, BCl3 (139 mL, 1 M, 139.1 mmol) was slowly added at -78 °C. After the addition was complete, the reaction mixture was stirred at -40 °C for 5 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 30 mL of methanol was added to the reaction mixture, and the mixture was stirred at -40 °C for at least half an hour. The mixture was then diluted with ethyl acetate (1000 mL), washed with water (1000 mL x 3), and washed with saturated brine (1000 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 = 3:1) and concentrated to give a white solid product 8 (11.5 g, 19.4 mmol, 69% yield). 1 H NMR(400MHz,DMSO-d6)δ11.36(s,1H),7.81–7.71(m,1H),5.97–5.91(m,1H),5.85–5.78(m,1H),5.70–5.62(m,1H),4.94(s,1H),4.1 7–4.08(m,2H),3.75–3.55(m,2H),3.47–3.37(m,2H),2.14–2.02(m,3H),1.62–1.49(m,2H),1.34–1.21(m,38H),0.92–0.88(m,3H). ESI-LCMS: m / z 593.2[MH] - .

[0151] Synthesis of Compound 9: Substrate 8 (11.5 g, 19.4 mmol) dissolved in anhydrous DCM (220 mL) was added to a dry 500 mL single-necked flask. Then, under nitrogen protection, collidine (18.8 g, 155.2 mmol), DMTrCl (9.8 g, 29.1 mmol), and AgNO3 (6.0 g, 34.9 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 15 hours. LCMS analysis indicated the reaction was complete when the starting material had completely disappeared. The mixture was filtered, and 200 mL of dichloromethane was added to the filtrate. The mixture was washed with water (300 mL x 3) and then with saturated brine (300 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude product 9 (20.0 g). ESI-LCMS m / z 896.2 [MH] - .

[0152] Synthesis of Compound 10: Substrate 9 (20.0 g) dissolved in ammonia in methanol (200 mL) was added to a dry 500 mL single-necked flask. After addition, the reaction mixture was stirred at room temperature for half an hour. The reaction was considered complete when the starting material was completely eliminated by TLC and LCMS. The reaction solution was directly concentrated to obtain a crude product, which was then purified by column chromatography (petroleum ether: ethyl acetate = 4:1). Concentration yielded a pale yellow solid, product 10 (7.5 g, 8.77 mmol, 45% two-step yield). ESI-LCMS m / z 854.2 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.27(s,1H),7.92–7.82(m,1H),7.44–7.16(m,9H),6.89–6.79(m,4H),5.52–5.40(m,3H),4.16–4 .11(m,1H),3.72(s,6H),3.62–3.48(m,2H),3.46–3.38(m,3H),1.60–1.49(m,2H),1.39–1.15(m,38H),0.87–0.78(m,3H).

[0153] Synthesis of compound LCT-063: Substrate 10 (600 mg, 0.70 mmol) was dissolved in anhydrous DCM (10 mL) in a dry 50 mL single-necked flask. Then, under nitrogen protection, DCI (70.5 mg, 0.60 mmol) and 10a (238.6 mg, 1.73 mmol) were added. 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, 50 mL of dichloromethane 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 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 LCT-063 (500 mg, 0.49 mmol, 70% yield). 1 H NMR(400MHz,DMSO-d6)δ7.92-7.85(m,1H),7.46–7.15(m,9H),6.88–6.78(m,4H),5.76–5.46(m,2H),4.43–4.21(m,1H),3.95 –3.68(m,7H),3.33(s,6H),3.21–3.12(m,3H),1.59–1.49(m,2H),1.38–1.16(m,38H),1.08–0.98(m,11H),0.85–0.77(m,3H). 31 P NMR (400MHz, DMSO-d6) δ152.18,149.71. ESI-LCMS:m / z 1014.2[MH] - .

[0154] Synthesis of compound LCT-066:

[0155] Synthesis of Compound 11: Substrate 6 (25.0 g, 39.5 mmol) and ABz (11.3 g, 47.4 mmol) dissolved in anhydrous ACN (500 mL) were added to a dry 1 L single-necked flask, followed by the addition of BSA (24.1 g, 118.5 mmol). After the addition was complete, the reaction mixture was stirred at 50 °C for 1 hour. The reaction mixture became clear, and was cooled in an ice-water bath. Then, TMSOTf (13.2 g, 59.2 mmol) was added to the reaction mixture. After the addition was complete, the reaction mixture was stirred at 80 °C overnight. LCMS analysis showed that the starting material had completely disappeared, indicating the end of the reaction. Then, ethyl acetate (500 mL) was added to the reaction solution and quenched with sodium bicarbonate solution. The solution was washed with water (1.0 L * 3) and then with saturated brine (500 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 (dichloromethane:methanol = 30:1) and concentrated to obtain product 11 (14.5 g, 17.8 mmol, 45% yield). 1 H NMR (400MHz, DMSO-d6): δ11.18(s,1H),8.71(s,1H),8.37(s,1H),8.05-8.02(m,2H),7.60 -7.58(m,1H),7.52-7.48(m,2H),7.45-7.20(m,5H),6.28(s,1H),5.70(s,1H),4.77-4.70( m,1H),4.60-4.53(m,1H),4.38-4.37(m,1H),4.22-4.20(m,1H),3.82-3.62(m,2H),3.40- 3.33(m,2H),2.08-2.07(m,3H),1.43-1.41(m,2H),1.25-1.15(m,38H),0.81-0.78(m,3H). ESI-LCMS: m / z 812.6[M+H] + .

[0156] Synthesis of Compound 12: Substrate 11 (14.5 g, 17.8 mmol) was dissolved in anhydrous DCM (700 mL) in a dry 1 L single-necked flask. Then, 1 M BCl3 in DCM (89 mL, 89.0 mmol) was slowly added dropwise at -78 °C. After the addition was complete, the reaction mixture was stirred at -40 °C for 6 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. The mixture was then quenched with methanol and sodium bicarbonate solution, washed with water (500 mL), and then washed with saturated brine (500 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 (dichloromethane:methanol = 30:1) to obtain product 12 (5.0 g, 6.9 mmol, 38% yield). 1H NMR (400MHz, DMSO-d6): δ11.22(s,1H),8.75(s,1H),8.57(s,1H),8.06-8.04(m,2H),7.64-7.60(m,1H),7.55-7.50(m,2H),6.40- 6.12(m,2H),5.33(s,1H),4.28(s,2H),3.79-3.66(m,2H),3.40-3.66(m,2H),2.10(s,3H),1.46-1.11(m,40H),0.84-0.81(m,3H). ESI-LCMS: m / z 722.6[M+H] + .

[0157] Synthesis of Compound 13: Substrate 12 (5.0 g, 6.9 mmol) was dissolved in DCM (50 mL) in a dry 100 mL single-necked flask, followed by the addition of DMTrCl (4.7 g, 13.8 mmol), AgNO3 (2.1 g, 12.4 mmol), and 2,4,6-collidine (5.0 g, 41.4 mmol). After the addition was complete, the reaction system was stirred at 35 °C for 16 hours. The reaction was considered complete when the starting material was detected by LCMS. Then, DCM (100 mL) was added to the reaction solution, and the mixture was washed with water (200 mL x 3) 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 (dichloromethane:methanol = 50:1) and concentrated to obtain product 13 (4.0 g, 3.9 mmol, 56% yield). 1 H NMR (400MHz, DMSO-d6): δ11.20(s,1H),8.70(s,1H),8.56(s,1H),8.05-8.04(m,2H), 7.73-7.68(m,1H),7.63-7.52(m,2H),7.50-7.40(m,2H),7.30-7.24(m,7H),6.90-6.8 7(m,4H),6.04-6.03(m,1H),5.63-5.60(m,1H),4.71-4.68(m,1H),3.73(s,6H),3.73 -3.32(m,5H),1.99(s,3H),1.62-1.48(m,2H),1.40-1.20(m,38H),0.84-0.81(m,3H). ESI-LCMS: m / z 1024.4[M+H] + .

[0158] Synthesis of Compound 14: Substrate 13 (4.0 g, 3.9 mmol) dissolved in THF (40 mL) was added to a dry 50 mL single-necked flask, followed by the addition of a 1 M NaOH solution in water / methanol (1 / 4) (40 mL). After the addition was complete, the reaction system was stirred at 0 °C for 1 hour. The reaction was considered complete when the starting material was detected by LCMS. Then, ethyl acetate (100 mL) was added to the reaction solution, and the mixture was washed with water (200 mL * 3) and saturated brine. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (dichloromethane:methanol = 30:1) and concentrated to obtain product 14 (3.4 g, 3.5 mmol, 90% yield). 1 H NMR (400MHz, DMSO-d6): δ11.19(s,1H),8.71(s,1H),8.57(s,1H),8.06-8.04(m,2H),7.63-7. 60(m,1H),7.63-7.52(m,2H),7.50-7.40(m,2H),7.30-7.19(m,7H),6.82-6.77(m,4H),5.86- 5.83(m,2H),4.40-4.30(m,1H),4.33-4.27(m,1H),3.81-3.70(m,6H),3.68-3.62(m,1H),3.6 1-3.50(m,2H),3.42-3.34(m,2H),1.62-1.48(m,2H),1.40-1.20(m,38H),0.84-0.81(m,3H). ESI-LCMS: m / z 982.3[M+H] + .

[0159] Synthesis of compound LCT-066: Substrate 14 (3.4 g, 3.5 mmol) dissolved in anhydrous DCM (40 mL) was added to a dry 50 mL single-necked flask. Then, under nitrogen protection, DIPEA (496 mg, 3.8 mmol) and 15 (1.1 g, 4.2 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. LCMS showed complete disappearance of the starting material, yielding compound 16. Anhydrous ethanol (243 mg, 3.8 mmol) and 0.25 M ETT acetonitrile solution (8 mL) were then added to the reaction solution. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. LCMS showed complete disappearance of compound 16. The solution was then concentrated to obtain the crude product, which was then subjected to reverse-phase C18 (CH3CN / DCM = 2 / 1) and concentrated to obtain product LCT-066 (2.5 g, 2.2 mmol, 62% yield). 1H NMR (400MHz, DMSO-d6): δ11.13(s,1H),8.67-8.61(m,1H),8.60-8.47(m,1H),8.10-7.97(m ,2H),7.62-7.52(m,1H),7.50-7.42(m,2H),7.40-7.31(m,2H),7.30-7.07(m,7H),6.82-6.4 1(m,4H),6.05-5.92(m,1H),4.50-4.10(m,2H),3.90-3.75(m,1H),3.74-3.54(m,8H),3.54- 3.37(m,7H),1.59-1.45(m,2H),1.39-1.11(m,40H),1.03-0.87(m,12H),0.83-0.76(m,3H). 31 P NMR (162MHz, DMSO-d6): δ149.53, 148.01. ESI-LCMS: m / z 1157.5[M+H] + .

[0160] Synthesis of compound LCT-068:

[0161] Synthesis of Compound 17: Substrate 10 (5.6 g, 6.55 mmol) dissolved in anhydrous DCM (60 mL) was added to a dry 100 mL single-necked flask. Then, under nitrogen protection, DIPEA (0.93 g, 7.20 mmol) and 15 (2.26 g, 8.51 mmol), along with activated molecular sieves, were added. After the addition was complete, the reaction system was stirred at room temperature under nitrogen atmosphere for 1 hour. LC-MS analysis showed complete disappearance of the starting material, indicating the end of the reaction. No further processing was required; it can be directly used in the next step.

[0162] Synthesis of compound LCT-068: ETT (15 mL, 0.25 M, 3.93 mmol) and EtOH (330 mg, 7.20 mmol) were added to the reaction solution used in the synthesis of compound 17 above under nitrogen protection. After addition, 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 then 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 LCT-068 (4.0 g, 3.88 mmol, 59% yield). 1HNMR(400MHz,DMSO-d6)δ11.36-11.24(m,1H),7.93–7.75(m,1H),7.48–6.12(m,9H),6.89–5.71(m,4H),5.73–5.39(m,2H),4.40–4 .26(m,1H),4.01–3.40(m,14H),3.31–3.18(m,1H),1.61–1.44(m,2H),1.42–1.12(m,38H),1.10–0.90(m,15H),0.84-0.76(m,3H). 31 P NMR (400MHz, DMSO-d6) δ149.73,147.33. ESI-LCMS:m / z 1028.3[M+H] - .

[0163] Synthesis of compound LCT-342:

[0164] Synthesis of Compound 18-1: Substrate 6 (22.5 g, 35.60 mmol) was dissolved in anhydrous ACN (220 mL) in a dry 1000 mL single-necked flask, followed by BSA (21.73 g, 106.80 mmol) and 6Cl-G (7.2 g, 42.72 mmol). The reaction mixture was stirred at 50 °C for half an hour. Then, TMSOTf (11.9 g, 53.40 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was stirred at 80 °C for 15 hours. LCMS analysis showed complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was then added to the reaction mixture, followed by washing with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude product 18-1 (28.0 g, 35.60 mmol). ESI-LCMS: m / z 740.5 [MH] - .

[0165] Synthesis of compound 18-2: Substrate 18-1 (28.0 g, 35.60 mmol) was dissolved in anhydrous pyridine (280 mL) in a dry 1000 mL single-necked flask. Then, iBuCl (4.2 g, 39.16 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. LCMS analysis showed complete disappearance of the starting material, indicating the end of the reaction. The mixture was diluted with ethyl acetate (200 mL), washed with water (200 mL x 3), and then washed with saturated brine (200 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude 18-2 (30.5 g, 35.60 mmol). ESI-LCMS: m / z 810.6 [MH] - .

[0166] Synthesis of compound 18-3: Substrate 18-2 (30.5 g, 35.60 mmol) dissolved in anhydrous DMF (300 mL) was added to a dry 1000 mL single-necked flask, followed by the addition of TEA (10.8 g, 106.80 mmol), CsOAc (20.5 g, 106.80 mmol), and DBACO (4.8 g, 42.72 mmol). 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 detected by LCMS. Then, 200 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 = 4:1) and concentrated to give a yellow solid product 18-3 (16.0 g, 20.18 mmol, 56.7% three-step yield). 1 H NMR(400MHz,DMSO-d6)δ12.11(s,1H),11.67(s,1H),7.96(s,1H),7.45–7.25(m,6H),5.98 (d,J=1.9Hz,1H),5.56(t,J=1.7Hz,1H),4.80(d,J=11.8Hz,1H),4.59(d,J=11.9Hz,1H),4. 46–4.16(m,2H),3.69(ddd,J=41.1,10.6,5.5Hz,2H),3.40(td,J=6.5,3.9Hz,2H),2.81–2 .74(m,1H),2.10(s,3H),1.51–1.41(m,3H),1.21(d,J=7.6Hz,45H),0.85(t,J=6.6Hz,3H). ESI-LCMS: m / z 792.7[MH] - .

[0167] Synthesis of Compound 19: Substrate 18-3 (16.0 g, 20.18 mmol) was dissolved in anhydrous DCM (100 mL) in a dry 500 mL single-necked flask. Then, BCl3 (166 mL, 1 M, 166.40 mmol) was slowly added at -78 °C. After the addition was complete, the reaction mixture was stirred at -40 °C for 6 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 30 mL of methanol was added to the reaction mixture, and the mixture was stirred at -40 °C for at least half an hour. The mixture was then diluted with ethyl acetate (100 mL), washed with water (100 mL x 3), and washed 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 = 3:1) and concentrated to obtain a white solid product 19 (4.2 g, 5.96 mmol). ESI-LCMS: m / z 704.5 [M+H] + .

[0168] Synthesis of Compound 20: Substrate 19 (4.2 g, 5.96 mmol) was dissolved in anhydrous DCM (80 mL) in a dry 100 mL single-necked flask. Then, under nitrogen protection, collidine (5.8 g, 47.68 mmol), DMTrCl (4.0 g, 11.92 mmol), and AgNO3 (1.8 g, 10.73 mmol) were added. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. 50 mL of dichloromethane was then added to the reaction mixture, followed by washing with water (100 mL x 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 = 5:1) and concentrated to give a yellow solid, product 20 (5.0 g, 4.97 mmol). ESI-LCMS: m / z 1006.5 [M+H] + .

[0169] Synthesis of Compound 21: Substrate 20 (5.0 g, 4.97 mmol) dissolved in methanol (10 mL) and THF (10 mL) was added to a dry 100 mL single-necked flask, followed by the addition of water (20 mL). Then, 2N NaOH solution (10 mL) was slowly added dropwise at 0 °C. After the addition was complete, the reaction system was stirred at 0 °C for half an hour. The reaction was considered complete when the starting material was completely eliminated by TLC and LCMS. The reaction solution was diluted with 50 mL of ethyl acetate, quenched with saturated NH4Cl solution and washed (100 mL * 3), and washed 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 = 3:1) and concentrated to obtain a white solid product 21 (3.0 g, 3.11 mmol). 1 H NMR(400MHz,DMSO-d6)δ12.10(s,1H),11.64(s,1H),7.49–7.37(m,2H),7.34– 7.16(m,7H),6.80(t,J=8.7Hz,4H),5.58(dd,J=24.0,4.2Hz,2H),3.72(d,J=1. 6Hz,6H),3.68–3.57(m,3H),3.45–3.37(m,2H),1.52(q,J=6.8Hz,2H),1.22(d ,J=2.7Hz,38H),1.12(dd,J=7.9,6.8Hz,6H),0.88–0.81(m,3H).ESI-LCMS:m / z 964.6[MH] - .

[0170] Synthesis of compound LCT-342: Substrate 21 (3.0 g, 3.11 mmol) was dissolved in anhydrous DCM (30 mL) in a dry 100 mL single-necked flask. Then, under nitrogen protection, DCI (312 mg, 2.64 mmol) and CEP[N(iPr)2]2 (1.12 g, 3.73 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 8 hours. The reaction was considered complete when the starting material was completely eliminated by LCMS. Then, 50 mL of dichloromethane was added to the reaction solution, and the mixture was washed with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The product was then processed using a reversed-phase C242 flask. 18 (CH3CN / H2O(0.05% NH4HCO3)=1 / 0), concentrated to give a white solid product LCT-342 (3.1g, 3.11mmol). 1H NMR (600MHz, DMSO-d6) δ12.10(s,1H),11.52(d,J=12.0Hz,1H),8.18(d,J=48.0Hz,1H),7.54–7.43(m,2H),7 .41–7.24(m,7H),6.93–6.82(m,4H),5.74(d,J=60.0Hz,1H),4.52(dt,J=17.4,5.5Hz,2H),3.76–3.69(m,7H) ,3.64–3.35(m,5H),3.32(d,J=11.2Hz,3H),2.78(pd,J=6.9,2.0Hz,1H),2.57(dq,J=16.5,6.1,5.6Hz,1H),1 .59–1.45(m,2H),1.21(d,J=2.8Hz,38H),1.11(td,J=7.0,2.1Hz,6H),1.09–0.98(m,9H),0.87–0.77(m,6H). 31 P NMR (243MHz, DMSO-d6) δ151.87,148.88. ESI-LCMS:m / z 1164.6[MH] - .

[0171] Synthesis of compound LCT-344:

[0172] Synthesis of Compound 23: Substrate 9 (7.0 g, 7.8 mmol) was dissolved in anhydrous ACN (100 mL) in a dry 250 mL single-necked flask. Then, TPSCl (3.6 g, 11.7 mmol) and DMAP (1.9 g, 15.6 mmol) were added to the reaction mixture at 0 °C, and the mixture was stirred at room temperature for 2 hours. Ammonia water (35 mL) was then added, and the mixture was stirred at room temperature for another 2 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Ethyl acetate (100 mL) was then added to the reaction mixture, followed by washing with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain crude product 23 (8.0 g, 7.8 mmol), which was used in the next step. ESI-LCMS: m / z 896.8 [M+H] + .

[0173] Synthesis of Compound 24: Substrate 23 (8.0 g, 7.8 mmol) dissolved in anhydrous DCM (80 mL) was added to a dry 250 mL single-necked flask. Then, pyridine (5.3 g, 78.0 mmol) and B2Cl (1.4 g, 9.3 mmol) were added to the reaction mixture under ice bath conditions. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was then concentrated to obtain crude product 24 (8.8 g, 7.8 mmol), which was used in the next reaction. ESI-LCMS: m / z 998.7 [MH] - .

[0174] Synthesis of Compound 25: Substrate 24 (8.8 g, 7.8 mmol) was dissolved in THF (90 mL) in a dry 250 mL single-necked flask, followed by the addition of 44 mL of 1 M NaOH (water:methanol = 1:4). After the addition was complete, the reaction system was stirred at 0 °C for 1 hour. The reaction was considered complete when the starting material was detected by LCMS. Then, ethyl acetate (100 mL) was added to the reaction solution, and the mixture was washed with water (200 mL * 3) and saturated brine. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (dichloromethane:methanol = 50:1) and concentrated to obtain product 25 (3.5 g, 3.6 mmol, 46% yield in three steps). 1 H NMR(400MHz,DMSO-d6)δ:11.22(s,1H),8.40(m,1H),8.11–7.92(m,2H),7.62 –7.58(m,1H),7.52–7.48(m,2H),7.40(m,1H),7.32(m,2H),7.29–7.10(m,7H) ,6.82–6.75(m,4H),5.55–5.50(m,2H),4.10–3.91(m,2H),3.83–3.54(m,8H), 3.45–3.43(m,3H),1.57–1.53(m,2H),1.30–1.15(m,38H),0.84–0.81(m,3H). ESI-LCMS: m / z 958.7 [M+H] + .

[0175] Synthesis of compound LCT-344: Substrate 25 (3.5 g, 3.6 mmol) was dissolved in anhydrous DCM (40 mL) in a dry 50 mL single-necked flask. Then, under nitrogen protection, DCI (361 mg, 3.0 mmol) and CEP[N(iPr)2]2 (1.6 g, 5.4 mmol) were added. 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 detected by LCMS. The mixture was then washed with water (100 mL x 1) and 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 processed using a reversed-phase C244 flask. 18 (CH3CN / THF=1 / 0~3 / 1), concentrated to give product LCT-344 (3.5g, 3.0mmol, 83% yield). 1 H NMR(600MHz,DMSO-d6)δ:11.28–11.19(d,J=54.2Hz,1H),8.44–8.34(d,J=60.0Hz,1H),8 .00(m,2H),7.68–7.03(m,13H),6.88–6.79(m,4H),5.69–5.66(d,J=18.2Hz,1H),4.19(s, 1H),4.09–3.59(m,11H),3.56–3.37(m,5H),2.68–2.65(m,1H),2.31–2.27(m,1H),1.53–1 .49(m,2H),1.33–1.19(m,38H),1.07–0.98(m,9H),0.98–0.90(m,3H),0.85–0.79(m,3H). 31 P NMR (162MHz, DMSO-d6): δ149.46, 149.10. ESI-LCMS: m / z 1158.8[M+H] + .

[0176] Synthesis of compound LCT-333:

[0177] Synthesis of compound 28: Substrate 27 (12.0 g, 40.67 mmol) was dissolved in anhydrous DCM (120 mL) in a dry 250 mL single-necked flask, followed by the addition of C under nitrogen protection. 16 H 33CHO (11.4 g, 44.74 mmol) and DIPEA (15.7 g, 120.03 mmol) were added. After addition, the reaction system was stirred at room temperature for 1 hour. The reaction was considered complete when the starting material was completely eliminated by LCMS. 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 = 4:1) and concentrated to give 28 (17.5 g, 33.85 mmol, 83% yield). 1 H NMR (400MHz, DMSO-d6) δ7.44-7.27(m,5H),7.44–6.72(m,1H),5.89–5.87(m,1H),4.76–7.64(m,2H),4.52–4.47(m,1H),4.32–4.02(m, 3H),3.91–3.89(m,1H),2.26–2.20(m,1H),2.18–2.08(m,1H),1.47–1.32(m,5H),1.28–1.18(m,27H),0.89–0.83(m,3H).ESI-LCMS: m / z 518.3[M+H] + .

[0178] Synthesis of Compound 29: Substrate 28 (17.5 g, 33.85 mmol) was dissolved in anhydrous DCE (180 mL) in a dry 500 mL single-necked flask. Then, under nitrogen protection, HCHO (1.5 g, 50.77 mmol) and AcOH (2.0 g, 33.85 mmol) were added. After the additions, the reaction mixture was stirred at room temperature for half an hour. Then, NaCNBH3 (4.3 g, 67.71 mmol) was added at 0 °C, and the reaction mixture was stirred at room temperature for 15 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with saturated NaHCO3 (100 mL x 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 = 5:1) and concentrated to obtain 29 (3.5 g, 6.56 mmol, 19% yield). ESI-LCMS: m / z 534.3 [M+H] + .

[0179] Synthesis of Compound 30: Substrate 29 (3.5 g, 6.56 mmol) was dissolved in glacial acetic acid (35 mL) in a dry 100 mL single-necked flask. Ac₂O (3500 μL) was then slowly added at 0°C, and concentrated sulfuric acid (350 μL) was added dropwise while maintaining the temperature at 0°C. After the addition was complete, the reaction mixture was stirred at room temperature for 20 minutes. The reaction was considered complete when the starting material was completely eliminated by TLC and LCMS. The reaction solution was then diluted with 200 mL of ethyl acetate, quenched with saturated NaHCO₃ solution and washed (200 mL x 3), followed by washing with saturated brine (20 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 30 (2.2 g, 3.81 mmol, 58% yield). ESI-LCMS: m / z 578.7 [M+H] + .

[0180] Synthesis of Compound 31: Substrate 30 (2.2 g, 3.81 mmol) was dissolved in anhydrous acetonitrile (22 mL) in a dry 250 mL single-necked flask, followed by the addition of BSA (2.3 g, 11.43 mmol) and Uracil (512 mg, 4.57 mmol). The reaction mixture was stirred at 50 °C for half an hour. Then, TMSOTf (1.3 g, 5.71 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was stirred at 80 °C for 8 hours. LC-MS analysis showed complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was then added to the reaction mixture, followed by washing with water (100 mL x 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 = 3:1) and concentrated to obtain product 31 (720 mg, 1.14 mmol, 30% yield). ESI-LCMS: m / z 630.3 [M+H] + .

[0181] Synthesis of Compound 32: Substrate 31 (720 mg, 1.14 mmol) was dissolved in anhydrous DCM (28 mL) in a dry 100 mL single-necked flask. Then, BCl3 (5 mL, 1 M, 5.70 mmol) was slowly added at -78 °C. After the addition was complete, the reaction system was stirred at -40 °C for 6 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 10 mL of methanol was added to the reaction solution, and the mixture was stirred at -40 °C for at least half an hour. The solution was then diluted with ethyl acetate (100 mL), washed with water (100 mL x 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 product was then processed using a reversed-phase C2O4 solution. 18(CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) was purified and concentrated to give product 32 (530 mg, 0.98 mmol, 85% yield). ESI-LCMS: m / z 540.2 [M+H] + .

[0182] Synthesis of Compound 33: Substrate 32 (530 mg, 0.98 mmol) was dissolved in anhydrous DCM (10 mL) in a dry 50 mL single-necked flask. Then, under nitrogen protection, trimethylpyridine (717 mg, 5.88 mmol), DMTrCl (498 mg, 1.47 mmol), and AgNO3 (300 mg, 1.76 mmol) were added. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. The reaction was considered complete when the starting material was detected by LCMS. The mixture was filtered, and the filtrate was diluted with ethyl acetate (100 mL), washed with water (100 mL x 3), and then washed 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 = 5:1) and concentrated to give a yellow solid product 33 (420 mg, 0.50 mmol, 50% yield). 1 H NMR(400MHz, DMSO-d6)δ7.84-7.82(m,1H),7.35–7.18(m,9H),6.95–6.81(m,5H),5.67–5.63(m,2H),4.82–4.75(m,1H),4.51–4.42(m, 1H),3.79–3.64(m,8H),2.65–2.50(m,5H),2.05–1.95(m,3H),1.48–1.39(m,2H),1.26–1.20(m,26H),0.87–0.85(m,3H).ESI-LCMS: m / z 840.3[MH] - .

[0183] Synthesis of Compound 34: Substrate 33 (420 mg, 0.50 mmol) dissolved in ammonia in methanol (4 mL) was added to a dry 100 mL single-necked flask. After addition, the reaction system was stirred at room temperature for half an hour. The reaction was considered complete when the starting material was detected by TLC and LCMS. The reaction solution was directly concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 3:1). The concentrated crude product was then purified by reverse-phase C24 chromatography. 18 (CH3CN / H2O(0.05% NH4HCO3)=1 / 0) was purified and concentrated to obtain crude product, which was then separated by SFC and concentrated to obtain product 34 (350 mg, 0.44 mmol, 87% yield). 1H NMR(400MHz,DMSO-d6)δ7.84-7.82(m,1H),7.42–7.18(m,9H),6.90–6.75(m,4H),5.56–5.35(m,3H),4.09–3.80(m,4H),3 .72(s,6H),3.20(s,1H),2.65–2.50(m,5H),2.05–1.95(m,3H),1.48–1.39(m,2H),1.26–1.20(m,26H),0.87–0.85(m,3H). ESI-LCMS: m / z 798.2[MH] - .

[0184] Synthesis of compound LCT-333: Substrate 34 (350 mg, 0.44 mmol) was dissolved in anhydrous DCM (4 mL) in a dry 100 mL single-necked flask. Then, under nitrogen protection, DCI (44 mg, 0.37 mmol) and CEP[N(iPr)2]2 (160 mg, 0.53 mmol) were added. 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, 50 mL of dichloromethane was added to the reaction solution, and the mixture was washed with water (100 mL x 3) and then 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 processed using a reversed-phase C2SO4 solution. 18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) was purified and concentrated to give the product LCT-333 (350 mg, 0.35 mmol, 79% yield). 1 H NMR (400MHz, DMSO-d6) δ11.43–11.22(m,1H),7.94–7.78(m,1H),7.44–7.12(m,9H),6.93–6.77(m,4H),5.64–5.41(m,2H),4.22–4.04(m,2H) ,4.00–3.38(m,13H),11.43–11.22(m,1H),2.72–2.34(m,7H),1.49–1. 36(m,2H),1.28–1.14(m,26H),1.13–0.96(m,12H),0.90–0.78(m,3H). 31 P NMR (400MHz, DMSO-d6) δ150.29,149.67. ESI-LCMS:m / z 998.4[MH] - .

[0185] Synthesis of compound LCT-341:

[0186] Synthesis of compound LCT-341: Substrate 14 (7.2 g, 7.3 mmol) was dissolved in anhydrous DCM (80 mL) in a dry 50 mL single-necked flask. Then, under nitrogen protection, DCI (732 mg, 6.2 mmol) and CEP[N(iPr)2]2 (3.3 g, 10.9 mmol) were added. 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 detected by LCMS. The mixture was then washed with water (100 mL x 1) and 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 / THF = 1 / 0–3 / 1) to obtain product LCT-341 (7.5 g, 6.3 mmol, 86.3% yield). 1 H NMR(600MHz, DMSO-d6)δ11.19(s,1H),8.69–8.67(d,J=12.0Hz,1H),8.62–8.53(d,J=54.2Hz,1H),8.05–8.04(m,2H),7.63–7.60(m, 1H),7.53–7.50(m,2H),7.37–7.33(m,2H),7.20–7.14(m,7H),6.78–6.71(m,4H),6.10–6.00(d,J=60.2Hz,1H),4.65–4.23(m,2H),3. 83(m,1H),3.69–3.55(m,10H),3.54–3.36(m,4H),2.65–2.62(m,1H),2.55–2.40(m,1H),1.68–1.44(m,2H),1.34–1.20(m,38H),1.1 2–1.09(d,J=18.0Hz,3H), 1.04–1.01(d,J=18.4Hz,3H), 0.98–0.97(d,J=6.4Hz,3H), 0.89–0.88(d,J=6.0Hz,3H), 0.83–0.80(m,3H). 31 P NMR (162MHz, DMSO-d6): δ150.06, 149.23. ESI-LCMS: m / z 1182.8[M+H] + .

[0187] Synthesis of compound LCT-343:

[0188] Synthesis of compound LCT-343: Substrate 10 (12.5 g, 14.6 mmol) dissolved in anhydrous DCM (120 mL) was added to a dry 50 mL single-necked flask. Then, under nitrogen protection, DCI (1.4 g, 12.4 mmol) and CEP[N(iPr)2]2 (6.6 g, 21.9 mmol) were added. 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 detected by LCMS. The mixture was then washed with water (100 mL x 1) and 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 processed using a reversed-phase C243 flask. 18 (CH3CN / THF=1 / 0~3 / 1), concentrated to give product LCT-343 (12.5g, 11.8mmol, 80.8% yield). 1 H NMR (600MHz, DMSO-d6) δ11.36–11.33 (d, J=18.4Hz, 1H), 7.89–7.85 (m, 1H), 7.42–7.36 (m, 2H), 7.30–7. 13(m,7H),6.88–6.71(m,4H),5.67–5.66(d,J=6.0Hz,1H),5.57–5.43(m,1H),4.35–4.20(m,1H),4.00– 3.83(m,1H),3.71–3.60(m,8H),3.59–3.46(m,4H),3.44–3.36(m,3H),2.68–2.66(m,1H),2.51–2.24(m ,1H),1.53–1.48(m,2H),1.38–1.17(m,38H),1.09–1.06(m,6H),1.01–0.97(m,6H),0.82–0.80(m,3H). 31 P NMR (162MHz, DMSO-d6): δ150.71,148.64. ESI-LCMS: m / z 1055.8[M+H] + .

[0189] Experimental Example

[0190] Experimental Example 1: In vitro evaluation of siRNA sequence synthesis

[0191] The synthesis of siRNA is no different from the usual phosphorus amide solid-phase synthesis. When synthesizing nucleotides modified at various positions of the SS chain, the original nucleotides in the parent sequence are replaced with the LCT phosphorus amide monomer synthesized above.

[0192] The synthesis process is briefly described as follows: For the sense and antisense strands of the siRNA sequence of the present invention, as well as the sense and antisense strands of the modified double strands, CPG is used as a solid-phase carrier; GalNAc-modified CPG is used as the starting cycle for the synthesis of the sense strand, and universal CPG is used as the starting cycle for the synthesis of the antisense strand.

[0193] Using a YB-192S synthesizer, a solid-phase synthesis method of phosphoramidite was employed. Starting with a solid support, nucleoside monomers were sequentially linked in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0194] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0195] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C or room temperature (25 °C) for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. After cooling to room temperature, the crude sequence was obtained by ethanol precipitation.

[0196] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0197] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The product was then freeze-dried to obtain the target product.

[0198] The modification schemes used in Table 1 are as follows:

[0199] In this context, uppercase letters A, C, G, U, I, and T represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, inosine-3'-phosphate, and 5-methyluracil-3'-phosphate, respectively; lowercase letter m indicates that the nucleotide adjacent to the right of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide adjacent to the right of f is 2'-fluoro modified; the two nucleotides adjacent to the left and right of "*" are linked by thiophosphate groups; LCT indicates that the nucleotide at this location is one of the above-mentioned modified nucleosides; GalNAc is L96; and da indicates that the nucleotide adjacent to the left of da is 2'-OC. 22 H 45 Modified nucleotide; eVP indicates that the adjacent nucleotide on the right is 5'-(E)-vinyl phosphate;

[0200] Some of the LCT phosphoramidone monomers, after synthesis via Oligo and subsequent ammonolysis, have the following structures in siRNA, as shown below for LCT-063 and LCT-066 as examples:

[0201] Table 0: Naked siRNA Sequences

[0202] For the modified siRNA conjugates listed in Table 1 below, the base sequences of the sense and antisense strands of each modified siRNA conjugate before modification are listed in the table below.

[0203] Table 1: siRNA conjugates linked to LCT-modified nucleosides

[0204] The following are the structural characterization methods and results of the conjugates in Table 1b:

[0205] Representative LC-MS test method: When the test sample is subjected to denaturing IP·RP-LC detection, the complementary double strands are untied into single strands (sense and antisense strands). Then, the parent ions of the sense and antisense strands are fragmented by tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the software CONFIRM Sequence. The sequence of the test sample is consistent with the theoretical sequence, that is, the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.05%. The results are shown in Table 2.

[0206] Table 1b: Molecular weight (MW) of modified siRNA conjugates

[0207] Experimental Example 2: In vivo activity assay of SOD1 target mRNA and siRNA containing LCT-modified nucleosides

[0208] Evaluation of siRNA sequence activity in wild-type mice:

[0209] The activity of siRNA with LCT-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0210] C57BL / 6 mice aged 6-8 weeks were used. Before administration, the mice were acclimatized in the facility for more than 3 days and divided into groups of 3 mice per group according to their weight. The mice were administered a single injection of the compound (compounds in Table 1) subcutaneously or intravenously. The mice were euthanized before administration and on days 7, 28 and 84 after administration. The SOD1 mRNA in the inguinal adipose tissue, epididymal adipose tissue, scapular subcutaneous adipose tissue, perirenal adipose tissue, heart, liver, quadriceps femoris muscle and kidney was detected.

[0211] Table 2: Testing protocol for conjugates in C57BL / 6 mice

[0212] Table 3: Residual SOD1 mRNA expression levels in various tissues at Day 7

[0213] Testing revealed that LCT-063 nucleosides conjugated to the positive strand of SOD1 siRNA (conjugates 2, 3, 4, and 5) exhibited inhibition rates exceeding 80% against the SOD1 target gene in inguinal fat, periepididymal fat, and subcutaneous fat of the scapula in wild-type mice. Furthermore, the inhibition rate of SOD1 mRNA in the liver was 46%–70%, significantly lower than that of Yangshen compound (conjugate 1). When LCT-063 molecules are conjugated to siRNA, they enable siRNA delivery to both fat and muscle.

[0214] Table 4: Testing protocol for conjugates in C57BL / 6 mice

[0215] Table 5: Residual SOD1 mRNA expression levels in various tissues at Day 7

[0216] Testing revealed that LCT-068 nucleosides conjugated to the positive strand of SOD1 siRNA (conjugates 10, 11, 12, and 13) exhibited inhibition rates exceeding 80% against the SOD1 target gene in inguinal fat, periepididymal fat, and subcutaneous fat of the scapula in wild-type mice. Furthermore, the inhibition rate of SOD1 mRNA in the liver was 39%–69%, significantly lower than that of Yangshen compound (conjugate 1). When LCT-068 molecules are conjugated to siRNA, they enable siRNA delivery to both fat and muscle.

[0217] Table 6. Testing protocols for conjugates in C57BL / 6 mice

[0218] Table 7: Residual SOD1 mRNA expression levels in various tissues at Day 7

[0219] Table 8: Testing protocol for conjugates in C57BL / 6 mice

[0220] Table 9: Residual SOD1 mRNA expression levels in various tissues at Day 7

[0221] Tests showed that LCT-066 and LCT-068 molecules, when attached to different positions on the positive strand of different SOD1 sequences, knocked down the SOD1 target gene in inguinal fat, periepididymal fat, subcutaneous fat of the scapula, and perirenal fat by 60-80%. The LCT-066 and LCT-068 molecules are universally applicable to other SOD1 sequences.

[0222] Table 10. Testing protocols for conjugates in C57BL / 6 mice

[0223] Table 11: Residual SOD1 mRNA expression levels in various tissues at Day 7 after 2 mpk subcutaneous administration.

[0224] Table 12: Residual SOD1 mRNA expression levels in various tissues at Day 28 after 2 mpk subcutaneous administration.

[0225] Testing revealed that LCT-063 is conjugated at position 16 of the positive strand of the SOD1 sequence. Compared to Yangshen molecular conjugate 1, conjugate 5 achieved a comparable (~90%) mRNA KD in inguinal fat, periepididymal fat, scapular subcutaneous fat, and perirenal fat at D7 and D28, while achieving ~80% mRNA KD in the heart and quadriceps muscle. Surprisingly, its mRNA KD was almost completely lost in the liver, which indirectly verifies that LCT-063 can achieve low knockdown of target genes in the liver while delivering fat and muscle, thus reducing the potential liver toxicity caused by the compound.

[0226] Table 13: Residual SOD1 mRNA expression levels in various tissues at Day 7 after 5 mpk subcutaneous administration.

[0227] Table 14: Residual SOD1 mRNA expression levels in various tissues at Day 84 after 5 mpk subcutaneous administration.

[0228] Testing revealed that conjugate 5 (LCT-modified nucleoside conjugated at position 16 of the siRNA positive strand) exhibited better mRNA KD in inguinal subcutaneous fat and demonstrated better long-lasting efficacy compared to conjugate 1 (Alnylam C22 conjugated at position 6 of the siRNA positive strand).

[0229] Experimental Example 3: In vivo activity assay of SOD1 target mRNA and siRNA containing LCT-modified nucleosides

[0230] 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 the table 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. The injection time and needle retention time after needle insertion are 30-40 seconds. The administration volume is 30uL, and the dosage is 0.9mg. The day of administration is defined as Day 1.

[0231] Fourteen 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.

[0232] 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 residual expression level ± SEM (standard error). One-way ANOVA was used for analysis, and Tukey's test was used for statistical significance testing 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. The residual expression levels of SOD1 mRNA in various CNS tissues of compounds conjugated with LCT-modified nucleosides are shown in the table.

[0233] Table 15: Residual SOD1 mRNA expression levels in various tissues at Day 15 after IT administration.

[0234] Tests showed that when compound LCT-063 was conjugated to the sixth position of the positive strand of SOD1 siRNA, after administration to rats via intraperitoneal administration (IT), on day 15, it resulted in 60%–90% SOD1 target gene knockdown in CNS tissues such as the spinal cord, hippocampus, frontal cortex, cortex, and midbrain. Simultaneously, it resulted in 30%–50% SOD1 target gene knockdown in the striatum, hypothalamus, and remaining brain tissues. This indicates that compound LCT-063 can deliver siRNA to various CNS tissues.

[0235] Experimental Example 4: In vivo activity assay of ALK7 target mRNA and siRNA containing LCT-modified nucleosides

[0236] Evaluation of siRNA sequence activity in wild-type mice:

[0237] The activity of siRNA with LCT-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0238] C57BL / 6 mice aged 6-8 weeks were used. Before administration, the mice were acclimatized in the facility for more than 3 days. They were divided into groups of 3 mice each according to their weight. The mice were administered a single injection of the compound (compounds in Table 2) at doses of 1 mg / kg, 2 mg / kg, or 5 mg / kg subcutaneously or intravenously. The mice were euthanized before administration and on days 7 and 21 after administration. The ALK7 mRNA in the inguinal adipose tissue, scapular subcutaneous adipose tissue, and perirenal adipose tissue of the mice was detected.

[0239] Table 16: Testing protocol for conjugates in C57BL / 6 mice

[0240] Table 17: Residual ALK7 mRNA expression levels in various tissues at Day 7 after subcutaneous administration (5 mpk).

[0241] Testing revealed that conjugate 31 (ALK7 siRNA sequence conjugated to LCT-066-b) was more effective than conjugate 30 (ALK7 siRNA sequence conjugated to Alnylam-C22) in achieving mRNA delivery to groin and scapular subcutaneous fat. Furthermore, LCT-066-b was more effective than Alnylam-C22 in delivering siRNA to fat.

[0242] Experimental Example 5: In vivo activity assay of adiponectin target mRNA and siRNA containing LCT-modified nucleosides

[0243] Evaluation of siRNA sequence activity in wild-type mice:

[0244] The activity of siRNA with LCT-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0245] C57BL / 6 mice aged 6-8 weeks were used. Before administration, the mice were placed in the facility for acclimatization for more than 3 days. They were divided into groups of 3 mice each according to their weight. The mice were administered a single injection of the compound (compounds in Table 2) at doses of 1 mg / kg, 2 mg / kg, or 5 mg / kg subcutaneously or intravenously. Blood samples were collected from the mice before administration and on days 0, 7, 14, 21, and 28 after administration to detect adiponectin protein in the mouse serum.

[0246] Table 18: Testing protocol for conjugates in C57BL / 6 mice

[0247] Table 19: Residual expression levels of adiponectin protein in mouse serum after 0.5 mpk administration.

[0248] Table 20: Residual expression level of adiponectin protein in mouse serum after 1.0 mpk administration.

[0249] Tests have shown that LCT-modified nucleoside compounds can achieve effective delivery to adipose tissue, enabling efficient and long-lasting silencing of target genes. Furthermore, when LCT-modified nucleosides are attached to multiple positions on the positive strand of siRNA (the first nucleotide at the 5' end of the positive strand is counted as position 1), they can achieve more effective delivery to adipose tissue than Alnylam C22, resulting in superior target gene silencing efficiency.

[0250] Experimental Example 6: In vivo activity assay of SOD1 target mRNA and siRNA containing LCT-modified nucleosides

[0251] Evaluation of siRNA sequence activity in wild-type mice:

[0252] The activity of siRNA with LCT-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0253] C57BL / 6 mice aged 6-8 weeks were used. Before administration, the mice were acclimatized in the facility for more than 3 days and divided into groups of 3 mice per group according to their weight. The mice were administered a single injection of the compound (compounds in Table 1) subcutaneously or intravenously. The mice were euthanized before administration and on days 7, 28 and 84 after administration. The SOD1 mRNA in the inguinal adipose tissue, epididymal adipose tissue, scapular subcutaneous adipose tissue, perirenal adipose tissue, heart, liver, quadriceps femoris muscle and kidney was detected.

[0254] Table 21: Testing protocol for conjugates in C57BL / 6 mice

[0255] Testing revealed that conjugate 5 (LCT-modified nucleoside conjugated at position 16 of the siRNA positive strand) achieved better mRNA KD in adipose and muscle tissues than conjugates 36, 37, 38, 39, 40, and 41. This demonstrates that LCT (C22 alkane conjugated) modified nucleosides can achieve better delivery efficiency to extrahepatic tissues such as adipose and muscle tissues compared to patented or literature reference molecules (Ref1–Ref6).

[0256] Experiment 7: In vivo activity assay of ALK7 target mRNA and siRNA containing LCT-modified nucleosides

[0257] Healthy male cynomolgus macaques were selected during their environmental acclimatization period based on body weight, hematology, and blood biochemistry levels. They were randomly divided into groups of three. Abdominal fat samples were collected at baseline (before drug administration) via fat biopsy. One to two weeks after recovery, the animals were administered the drug subcutaneously. The conjugate 35 single-dose group received the drug subcutaneously on Day 0 at a dose of 3.0 mg / kg. Abdominal fat samples were collected via fat biopsy on Day 14, Day 28, Day 42, Day 56, and Day 84. The relative expression level of ALK7 mRNA in adipose tissue at different time points was detected using qPCR, as shown in Figure 1.

Claims

1. A nucleoside phosphoramidite compound as shown in Formula 1a and stereoisomers, deuterated compounds thereof: ###0001### Formula 1a in: R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl. R 4 is a hydroxyl protecting group; preferably, R 4 is DMTr, MMTr; R 7 is cyclopropanyl, cyclobutanyl, C1-C30alkyl, substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl; X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10; X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5; M is P(OR 10 )(N(R 11 )2), P(R 10 )(N(R 11 )2); wherein R 10 , R 11 are each independently selected from the group consisting of cyclopropane, cyclobutane, substituted or non-substituted C1-C25 alkyl, substituted or non-substituted C1-C25 alkoxy; L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15 )-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 It is independently hydrogen or an unsubstituted C1-C6 alkyl group.

2. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound of Formula 1a has a structure of Formula 2a: in: R 1 R 2 R 5 R 6 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 ynyl or substituted C2-C6 ynyl. R 4 is a hydroxyl protecting group; preferably, R 4 is DMTr, MMTr; R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; R 17 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy); Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z represents -O-, -S-, -Se-, -NR 8 or -CR 8 R 9 , where R 8 and R 9 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl; X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10; X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5; L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15 )-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 It is independently hydrogen or an unsubstituted C1-C6 alkyl group.

3. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in Formula 1a has a structure as shown in Formula 3a: in: R 1 , R 2 , R 5 , R 6 each independently is selected from hydrogen, halogen, cycloalkyl, C1-C6alkyl, substituted C1-C6alkyl, C1-C6alkoxy, substituted C1-C6alkoxy, C2-C6alkenyl, substituted C2-C6alkenyl, C2-C6alkynyl, or substituted C2-C6alkynyl; R 4 is a hydroxyl protecting group; preferably, R 4 is DMTr, MMTr; R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z is -0-, -S-, -Se-, -NR 8 or -CR 8 R 9 wherein R 8 and R 9 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; R 17 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

4. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in Formula 1a has a structure as shown in Formula 4a: in: R 4 is a hydroxyl protecting group; preferably, R 4 is DMTr, MMTr; R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z is -0-, -S-, -Se-, -NR 8 or -CR 8 R 9 wherein R 8 and R 9 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; R 17 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

5. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in formula 1a has a structure as shown in formula 5a: in: R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. R 17 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy).

6. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in Formula 1a has a structure as shown in Formula 6a: R 7 It is a cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

7. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in formula 1a has a structure as shown in formula 7a: R 7 It is cyclopropane, cyclobutane, C1-C30 alkyl (e.g., C22 alkyl), substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 ynyl or substituted C2-C30 ynyl. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

8. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in formula 1a has a structure as shown in formula 8a: R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

9. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in Formula 1a has a structure as shown in Formula 8a-2: R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl, Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or it may not exist.

10. The nucleoside phosphoramidide compound according to claims 1-9, characterized in that: said Base is selected from the following structures:

11. The nucleoside phosphoramidite compound of claims 1-10, including but not limited to the following structures:

12. Use of the nucleoside phosphoramidide compound according to claims 1-11 in the preparation of oligonucleotides.

13. Use of the nucleoside phosphoramide compound of claims 1-12 as an intermediate in the preparation of oligonucleotides.

14. Use according to claims 12-13, characterized in that: The oligonucleotides are selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamers, and lncRNA.

15. An oligonucleotide comprising at least one structure according to Formula 9a: in: R 1 , R 2 , R 5 , R 6 each independently is selected from hydrogen, halogen, cycloalkyl, C1-C6alkyl, substituted C1-C6alkyl, C1-C6alkoxy, substituted C1-C6alkoxy, C2-C6alkenyl, substituted C2-C6alkenyl, C2-C6alkynyl, or substituted C2-C6alkynyl; R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z is -0-, -S-, -Se-, -NR 8 or -CR 8 R 9 wherein R 8 and R 9 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X1 and X2 are each independently selected from -(CH2)nO- or -(CH2)nS-, where n is any integer from 0 to 10; X3 is selected from -(CH2)nO-, -(CH2)nS-, -(CH2)nNH(CO)- or -(CH2)nO(CH2)nA-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 5; M 2 For or H, wherein R 3 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2; L is absent or selected from one or more structural units and combinations thereof; wherein the structural units include: -N(R) 14 )-, -O-, -S-, -C(O)-, -N(R 14 )C(O)-、-C(O)N(R 15 )-、-N(R 14 )C(O)N(R 15 -, -C(O)O-, -OC(O)-, -N(R) 14 )C(O)O-、-OC(O)N(R 15 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 16 )-O-、-OP(S)(R 16 )-O-、-OP(O)(NR 14 R 15 -N-、-OP(S)(NR) 14 R 15 )-N-、-OP(O)(NR 14 R 15 )-O-、-OP(S)(NR 14 R 15 )-O-、-P(O)(NR 14 R 15 -N-、-P(S)(NR) 14 R 15 -N-、-P(O)(NR) 14 R 15 )-O-、-P(S)(NR 14 R 15 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 14 R 15 and R 16 It is independently hydrogen or an unsubstituted C1-C6 alkyl group.

16. An oligonucleotide comprising at least one structure represented by Formula 10a: in: R 1 , R 2 , R 5 , R 6 each independently is selected from hydrogen, halogen, cycloalkyl, C1-C6alkyl, substituted C1-C6alkyl, C1-C6alkoxy, substituted C1-C6alkoxy, C2-C6alkenyl, substituted C2-C6alkenyl, C2-C6alkynyl, or substituted C2-C6alkynyl; R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z is -0-, -S-, -Se-, -NR 8 or -CR 8 R 9 wherein R 8 and R 9 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; M 2 For or H, wherein R 3 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2.

17. An oligonucleotide comprising at least one structure represented by Formula 11a: in: R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. Z is -0-, -S-, -Se-, -NR 8 or -CR 8 R 9 wherein R 8 and R 9 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; M 2 for or H, wherein R 3 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2.

18. An oligonucleotide comprising at least one structure represented by Formula 12a: in: R 7 is cyclopropane, cyclobutane, C1-C30alkyl (e.g., C22alkyl), substituted C1-C30alkyl, C1-C30alkoxy, substituted C1-C30alkoxy, C2-C30alkenyl, substituted C2-C30alkenyl, C2-C30alkynyl, or substituted C2-C30alkynyl; Base may be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or may not exist. M 2 For or H, wherein R 3 is cyclopropane, cyclobutane, substituted or unsubstituted C1-C25alkyl (e.g., methyl), substituted or unsubstituted C1-C25alkoxy (e.g., methoxy, ethoxy, cyanoethoxy), OH, SH; Y is S, O, BH2.

19. Use of the nucleoside phosphoramidide compound according to claims 1-11 in the preparation of siRNA conjugates, wherein the nucleoside phosphoramidide compound is conjugated to siRNA, for example, to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21 of the sense strand or positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23 of the antisense strand (the first nucleotide at the 5' end of the sense strand or antisense strand is position 1), having the following structural schematic diagram: in, Formula 9a as set forth in claim 15 is Formula 10a as set forth in claim 16 is Formula 11a according to claim 17 is Formula 12a of claim 18 is Each variable in Equations 9a, 10a, 11a and 12a is defined as claimed in any one of claims 15 to 18.

20. The use as described in claim 19, wherein when the nucleoside phosphoramide compound of claims 1-11 is conjugated to siRNA, it enables siRNA to target CNS tissues, adipose tissues, muscles, eyes, etc., thereby achieving degradation of the target mRNA in extrahepatic tissues.

21. The use of claim 19 or 20, wherein the nucleoside phosphoramidite compounds of claims 1-11, when conjugated to siRNA, are used simultaneously with a targeting ligand group Ligand to enhance the targeting of siRNA to CNS tissues, adipose tissues, muscle, eye, etc., to achieve the degradation of the target mRNA in extrahepatic tissues, wherein the targeting ligand group Ligand comprises: Lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths, and fatty acid molecules of different chain lengths; polymers, such as polyethylene glycol; Polypeptides, such as transmembrane peptides and targeting peptides; aptamers; antibodies; carbohydrates, such as lactose, polylactose, mannose, galactose, and N-acetylgalactosamine (GalNAc); folic acid; or receptor ligands expressed by hepatocytes, such as desialyl glycoproteins, desialyl sugar residues, lipoproteins (such as high-density lipoprotein and low-density lipoprotein), glucagon, neurotransmitters (such as adrenaline), growth factors, and transferrin. The conjugates of the nucleoside phosphoramidide compound and the ligand group Ligand according to claims 1-11 have the following general structural formula: Wherein, formula 9a as described in claim 15 is Formula 10a as described in claim 16 is Formula 11a as described in claim 17 is Formula 12a as described in claim 18 is The variables in Equations 9a, 10a, 11a, and 12a are defined as claimed in any one of claims 15 to 18. The M1, M2, M3, M 4、 M5, M6, M7, M8, M9, T1, T2, T3, T4, T5, T6, T7, T8, and T9 can be independently selected from one or more of the following structural units: -N(R 20 )-, -O-, -S-, -C(O)-, -N(R 20 )C(O)-、-C(O)N(R 21 )-、-N(R 20 )C(O)N(R 21 -, -C(O)O-, -OC(O)-, -N(R) 20 )C(O)O-、-OC(O)N(R 21 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 22 )-O-、-OP(S)(R 22 )-O-、-OP(O)(NR 20 R 21 -N-、-OP(S)(NR) 20 R 21 )-N-、-OP(O)(NR 20 R 21 )-O-、-OP(S)(NR 20 R 21 )-O-、-P(O)(NR 20 R 21 -N-、-P(S)(NR) 20 R 21 -N-、-P(O)(NR) 20 R 21 )-O-、-P(S)(NR 20 R 21 -O-, -SS-, triazolyl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; wherein each R 20 R 21 and R 22 It is independently hydrogen or an unsubstituted C1-C6 alkyl group; the substitution is preferably deuterated or halogenated.

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