SIRNA, conjugate containing SIRNA, pharmaceutical composition and application thereof

Abstract

The present invention relates to siRNAs, e.g., double-stranded ribonucleic acids (dsRNAs), that interfere with or inhibit the expression of a target gene. The invention also relates to a conjugate or a pharmaceutical composition containing the siRNA. And uses of siRNA, conjugates and pharmaceutical compositions thereof in the preparation of medicaments for the treatment and / or prevention of diseases or conditions.

Description

Technical Field

[0001] This disclosure relates to a nucleic acid capable of inhibiting the expression of the complement component C3 gene, a pharmaceutical composition containing the nucleic acid, and an siRNA conjugate, belonging to the field of nucleic acid drugs. This disclosure also relates to methods for preparing the nucleic acid, the pharmaceutical composition, and the siRNA conjugate, and their uses. Background Technology

[0002] The complement pathway is part of the host's innate immune system, which defends against invading pathogens. Complement is primarily composed of a series of proteins that circulate in the bloodstream in their precursor form or are present on the cell membrane. Activation of complement leads to a cascade of enzymatic reactions that form potent anaphylatoxins, triggering a series of physiological responses, including chemotaxis and cell death. Complement protein C3 is the linker of the complement cascade. C3 hydrolysis forms highly reactive C3b that binds to the cell surface. This binding leads to the sequential formation of multiple complexes, triggering the autoproliferation of the so-called "alternative pathway (AP)," ultimately resulting in an inflammatory response, phagocytic attraction, opsonization, and the clearance of pathogens, immune complexes, and cellular debris. Inappropriate activation of complement is associated with the spread and / or onset of many diseases, including C3-related diseases (e.g., C3 glomerulonephropathy), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. Therefore, therapeutics that inhibit the complement system, including those that inhibit C3 activity, can prove beneficial. Currently, there are limited therapies available for treating complement component C3-related diseases, and these therapies are time-consuming, invasive, and costly. Therefore, there is a need in the art to provide alternative therapies for subjects suffering from complement component C3-related diseases.

[0003] Small interfering RNAs (siRNAs) based on RNA interference (RNAi) mechanisms can inhibit or block the expression of target genes in a sequence-specific manner, thereby achieving targeted inhibition and therapeutic effects. Clearly, reducing C3 gene expression by regulating C3 mRNA levels is an effective way to block complement protein production, maintain normal immune function, and prevent abnormal immune responses.

[0004] The key to developing siRNA drugs that inhibit C3 gene expression lies in finding suitable target-selective siRNA sequences, appropriate modifications for chemical stability, and efficient delivery systems. Since C3 expression is primarily confined to hepatocytes before being secreted into the bloodstream, it is an ideal target for siRNA therapy. Summary of the Invention

[0005] The present invention aims to provide an siRNA for inhibiting C3 gene expression, a pharmaceutical composition containing the siRNA as a pharmaceutically active ingredient and an siRNA conjugate, a method for inhibiting C3 gene expression using the siRNA, the pharmaceutical composition or the siRNA conjugate, and the use of the siRNA, the pharmaceutical composition or the siRNA conjugate in the treatment and / or prevention of C3-related diseases.

[0006] That is, the present invention achieves the above objectives by providing the following technical solutions.

[0007] In one aspect, the present invention provides an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the dsRNA comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides that differ from any nucleotide sequence of nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

[0008] In one aspect, the first strand sequence comprises at least 16, preferably at least 17, more preferably at least 18, and most preferably all 19 consecutive nucleotides, which differ from any of the nucleotide sequences of SEQ ID NO: 1, namely nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716, by no more than 2 nucleotides, preferably by no more than 1 nucleotide, and more preferably by no nucleotide difference.

[0009] In one aspect, the present invention provides an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 consecutive nucleotides, which corresponds to the sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110. The difference between any of the following nucleotides is no more than 3 nucleotides: 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188.

[0010] In one respect, the siRNA agent is a nucleic acid, wherein: the first-strand sequence contains SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, respectively. The sequences 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second chain sequence contains SEQ ID. NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 ,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,10 9, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, The sequence of 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

[0011] One aspect of the present invention relates to an siRNA reagent in which a first strand and a second strand are present on a single strand of nucleic acid, and the first strand and the second strand are capable of hybridizing to form a double-stranded nucleic acid having a double-stranded region.

[0012] In one aspect, the first and second strands form a double-stranded region of 15-25 nucleotides in length; preferably, a double-stranded region of 15-23 nucleotides in length; more preferably, a double-stranded region of 15-19 nucleotides in length; even more preferably, a double-stranded region of 17-19 nucleotides in length; and most preferably, a double-stranded region of 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0013] In one aspect, the first and second strands of the nucleic acid are separate strands. Preferably, each of the two separate strands is 15-25 nucleotides long, more preferably 17-25 nucleotides long. The lengths of the two strands can be the same or different. The first strand can be 17-25 nucleotides long, preferably 18-24 nucleotides long, and can be 18, 19, 20, 21, 22, 23, or 24 nucleotides long. Most preferably, the first strand is 19 nucleotides long. The second strand can independently be 17-25 nucleotides long, preferably 18-24 nucleotides long, and can be 18, 19, 20, 21, 22, 23, or 24 nucleotides long. More preferably, the second strand is 18, 19, or 20 nucleotides long, and most preferably, it is 19 nucleotides long.

[0014] In one respect, the siRNA agent is a nucleic acid, wherein: the first strand sequence has the following SEQ ID NOs respectively: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 11 4, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 nucleotide sequences; and optionally, wherein the second strand has nucleotide sequences represented by SEQ ID. NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 1 The nucleotide sequences represented by 11, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 are nucleotide sequences.

[0015] In one aspect, the first strand has nucleotide sequences represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136, respectively.

[0016] In one aspect, the second strand, complementary to the first strand, has nucleotide sequences represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, 137, respectively.

[0017] In one aspect, the first chain has the nucleotide sequence represented by SEQ ID NO: 20 and the second chain has the nucleotide sequence represented by SEQ ID NO: 21; or the first chain has the nucleotide sequence represented by SEQ ID NO: 96 and the second chain has the nucleotide sequence represented by SEQ ID NO: 97; or the first chain has the nucleotide sequence represented by SEQ ID NO: 16 and the second chain has the nucleotide sequence represented by SEQ ID NO: 17; or the first chain has the nucleotide sequence represented by SEQ ID NO: 150 and the second chain has the nucleotide sequence represented by SEQ ID NO: 151; or the first chain has the nucleotide sequence represented by SEQ ID NO: 32 and the second chain has the nucleotide sequence represented by SEQ ID NO: 33; or the first chain has the nucleotide sequence represented by SEQ ID NO: 100 and the second chain has the nucleotide sequence represented by SEQ ID NO: 101; or the first chain has the nucleotide sequence represented by SEQ ID NO: 110 and the second chain has the nucleotide sequence represented by SEQ ID NO: 111; or the first chain has the nucleotide sequence represented by SEQ ID NO: 134 and the second chain has the nucleotide sequence represented by SEQ ID NO: 135; or the first chain has the nucleotide sequence represented by SEQ ID NO: 20. The nucleotide sequence shown in NO: 136 and the second strand have the nucleotide sequence shown in SEQ ID NO: 137.

[0018] In one aspect, the siRNA agent comprises at least one modified nucleotide, preferably wherein substantially all nucleotides of the first strand are modified nucleotides and substantially all nucleotides of the second strand are modified nucleotides; more preferably, wherein all nucleotides of the first strand are modified nucleotides and all nucleotides of the second strand are modified nucleotides.

[0019] As used in this article, “virtually all nucleotides are modified” means that most, but not all, nucleotides are modified, including no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0020] In one respect, the modified nucleotide is selected from 3'-terminal deoxythymidine (dT) nucleotides, 2'-OCF2H-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, nucleotides containing a 5'-thiophosphate group, and nucleotides containing a 5'-(E)-vinylphosphonate.

[0021] In one aspect, all nucleotides of the first strand contain modifications in the following pattern: the pentose sugars at positions 7, 9, 10, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 2, 4, 6, 8, 12, 14, 16, and 18 from the 5' end are modified with 2′-methoxy; and the pentose sugars at positions 1, 3, 5, 13, 15, 17, and 19 from the 5' end are modified with either 2′-methoxy or 2′-fluorine substitution. All nucleotides of the second strand contain modifications in the following pattern: the pentose sugars at positions 2, 6, 8, 14, and 16 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 7, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy; and positions 4, 9, 10, and 18 from the 5' end are modified with either 2′-methoxy or 2′-fluorine substitution.

[0022] In one aspect, all nucleotides of the first strand contain modifications in the following pattern: the pentose sugars at positions 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy; and the pentose sugars at positions 3, 11, and 14 from the 5' end are modified with either 2′-methoxy or 2′-fluorine substitution. The second strand contains all nucleotides modified in the following pattern: the pentose sugars at positions 2, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy; and the pentose sugars at positions 4, 8, and 10 from the 5' end are modified with either 2′-methoxy or 2′-fluorine substitution.

[0023] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 7, 9, 10, and 11 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 6, 8, 9, 14, and 16 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, and 19 from the 5' end are modified with 2′-methoxy.

[0024] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0025] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0026] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0027] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0028] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0029] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0030] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0031] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0032] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0033] In one aspect, the first strand contains two phosphate thioester nucleotide bonds at the 5' end and / or the 3' end; and / or the second strand contains two phosphate thioester nucleotide bonds at the 5' end and / or the 3' end.

[0034] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 3' end; and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0035] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 5' end; and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0036] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end, and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0037] In one aspect, the second strand contains a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end.

[0038] In the specification of this invention, "identical or common modification" refers to identical modifications of any nucleotide, namely, A, G, C, or U modified with a group such as methyl (2'-OMe) or fluorine (2'-F). For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, and 2'-F-dG are all considered identical or common modifications, as are 2'-OMe-rU, 2'-OMe-rA, 2'-OMe-rC, and 2'-OMe-rG. Conversely, 2'-F modification is a different modification compared to 2'-OMe modification.

[0039] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, such as a nucleotide modified with 2'-F or 2'-OMe.

[0040] In some implementations, the siRNA agent also contains a targeting ligand that targets liver tissue.

[0041] In one respect, the targeting ligand is a GalNAc derivative.

[0042] In one respect, the targeting ligand is a GalNAc conjugate.

[0043] In one respect, the targeting ligand is conjugated to the 3' or 5' end of the first strand of the siRNA agent.

[0044] In some embodiments, the targeting ligand is conjugated to the 3' end of the first strand of the siRNA agent. In some embodiments, the targeting ligand is conjugated to the 5' end of the first strand of the siRNA agent.

[0045] In one respect, siRNA agents: (i) Contains a targeting ligand conjugated to the 3' or 5' nucleotide of the first strand; (ii) There are phosphate thioester bonds between three nucleotides at the other end of the first strand opposite one end that is conjugated to the target ligand; (iii) A phosphate thioester bond exists between the three terminal 3' nucleotides and the three terminal 5' nucleotides of the second strand; and (iv) Optionally, all remaining bonds between nucleotides of the first and / or second strand are phosphodiester bonds.

[0046] In some embodiments, the targeting ligand is conjugated to the 3' end of the first chain; the first chain contains two phosphate thioester nucleotide bonds at the 5' end, and the second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; optionally, all remaining bonds between the nucleotides of the first chain and / or the second chain are phosphodiester bonds.

[0047] In some embodiments, the targeting ligand is conjugated to the 5' end of the first chain; the first chain contains two phosphate thioester nucleotide bonds at the 3' end, and the second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; optionally, all remaining bonds between the nucleotides of the first chain and / or the second chain are phosphodiester bonds.

[0048] In one aspect, the first strand has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide inter-linked bond of thiophosphate; "(E)-VP" is the (E)-vinylphosphonate moiety at the 5' end; and "GN" is the targeting ligand.

[0049] In one aspect, the siRNA agent is conjugated with a ligand as shown in the figure below: the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to a nucleic acid, and (iii) a bicyclic group linking GalNAc and siRNA, or (iv) a tetrafunctional group linking the GalNAc moieties, wherein X is O or S. or .

[0050] In one respect, each joint in the figure may be identical or different.

[0051] In one respect, the joints in the diagram are independently selected from: (1) -(CH2)xC(O)NH-(CH2)yC(O)-; (2) -(CH2)xC(O)NH-(CH2)y-NHC(O)-(CH2)zC(O)-; (3) -NH-(CH2)yC(O)-; (4) –C(O)-(CH2)zC(O)-; x, y and z are independently selected from 1 to 10; preferably, x and y are independently selected from 2 to 8, and z is selected from 1 to 6; more preferably, x and y are independently selected from 3 to 6, and z is selected from 1 to 4.

[0052] In one aspect, the siRNA agent conjugates with a ligand, as shown in the figure below: , Where X is O or S; yes: and The yes: and .

[0053] In one aspect, the siRNA agent conjugates with a ligand, as shown in the figure below: ; Where X is O or S. yes: and .

[0054] In one aspect, the siRNA agent conjugates with a ligand, as shown in the figure below: ; The four sensory groups are selected from: and .

[0055] In some embodiments, the GalNAc conjugate is one or more GalNAc derivatives, optionally conjugated to a double-stranded RNA agent via a linker or vector, wherein one or more GalNAc derivatives are optionally conjugated to a double-stranded RNA agent via a linker or vector.

[0056] In some implementations, the siRNA agent is conjugated with a ligand, as shown in the figure below: , Where X is O or S.

[0057] In some implementations, the siRNA agent is conjugated with a ligand, as shown in the figure below: GalNAc-32 GalNAc-33 GalNAc-34 GalNAc-35 GalNAc-36 GalNAc-37 GalNAc-38.

[0058] In a preferred embodiment, the siRNA agent is conjugated with a ligand, as shown in the figure below: .

[0059] In one aspect, the first strand has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID N: 228), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 232), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233). Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide bond between thiophosphates; and "(E)-VP" represents the (E)-vinylphosphonate moiety at the 5' end.

[0060] In one aspect, the pharmaceutical composition comprises the siRNA agent disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent (preferably water) and / or a delivery carrier and / or a physiologically acceptable excipient and / or a pharmaceutically acceptable carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative.

[0061] In one aspect, the pharmaceutical composition comprises the siRNA agent disclosed herein and another therapeutic agent selected from oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

[0062] In one respect, the pharmaceutical composition comprises the reagents disclosed herein and a pharmaceutically acceptable carrier.

[0063] Another aspect of this disclosure provides a method for inhibiting the expression of the C3 gene in cells (such as nerve cells or eye cells), the method comprising: (a) contacting the cells with a double-stranded RNAi agent of this disclosure or a pharmaceutical composition of this disclosure; and (b) maintaining the cells produced in step (a) for a sufficient time to allow degradation of the mRNA transcript of the C3 gene, thereby inhibiting the expression of the C3 gene in the cells.

[0064] Another aspect of this disclosure provides a method for treating a subject suffering from a disease or condition in which reduced expression of complement component C3 would benefit, the method comprising administering to the subject a therapeutically effective amount of the aforementioned siRNA agent or the aforementioned pharmaceutical composition, thereby treating the subject.

[0065] The disease or condition to be treated with the nucleic acid or composition disclosed herein is a complement-mediated disease, condition or syndrome, preferably a C3-related disease or condition.

[0066] The diseases or conditions for which the nucleic acids or compositions disclosed herein are to be treated are preferably selected from IgA nephropathy (IgAN), C3 glomerulonephropathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), and myasthenia gravis (MG).

[0067] The siRNA provided by this invention can sequence-specifically mediate the inhibition of C3 gene expression. This gene can be located intracellularly, for example, within the cells of a subject, such as human cells. Without being bound by theory, it is believed that combinations or sub-combinations of the above-mentioned properties, as well as specific target sites or modifications in these iRNAs, confer improved efficacy, stability, potency, durability, and safety to the iRNAs of this invention. Attached Figure Description

[0068] Figure 1 The results of the detection of the inhibitory effect of siRNA on C3 mRNA expression level are shown in Table 2A.

[0069] Figure 2A The dose-response curves show the C3 mRNA knockdown effects of nine selected siRNAs in HepG2 cells.

[0070] Figure 2B The results show the detection of C3 mRNA knockdown effects of nine selected siRNAs in HepG2 cells at concentrations of 100 nM and 0.16 nM.

[0071] Figure 3A The results show the detection of C3 mRNA knockdown effects of nine selected GalNAc-conjugated siRNAs in PHH cells at concentrations of 1000 nM and 1 nM.

[0072] Figure 3B The dose-response curves show the C3 mRNA knockdown effects of four selected GalNAc-conjugated siRNAs in PHH cells.

[0073] Figure 4A The dose-response curves show the C3 mRNA knockdown effects of twenty selected GalNAc-conjugated siRNAs in PHH cells.

[0074] Figure 4B The dose-response curves show the C3 mRNA knockdown effects of twenty selected GalNAc-conjugated siRNAs in PHH cells.

[0075] Figure 4C The dose-response curves show the C3 mRNA knockdown effects of twenty selected GalNAc-conjugated siRNAs in PHH cells.

[0076] Figure 4D The dose-response curves show the C3 mRNA knockdown effects of twenty selected GalNAc-conjugated siRNAs in PHH cells.

[0077] Figure 5A The results show the detection effect of two selected GalNAc-conjugated siRNAs on the reduction of serum human C3 protein in male human C3 transgenic mice.

[0078] Figure 5B The results show the detection effect of two selected GalNAc-conjugated siRNAs on the reduction of serum human C3 protein in female human C3 transgenic mice.

[0079] Figure 6 The results show the detection results of the effect of the conjugated siRNA on reducing human C3 protein.

[0080] Figure 7 The results show the detection results of the effect of different doses of GalNAc-conjugated siRNA on reducing human C3 protein. Detailed Implementation

[0081] The present invention will be further described in detail below.

[0082] This invention provides an siRNA agent that affects the cleavage of the RNA transcript of the complement component C3 (C3) gene mediated by the RNA-induced silencing complex (RISC). This gene can be located intracellularly, for example, within the cells of a subject, such as human cells. Using these iRNAs enables targeted degradation of the mRNA of the corresponding gene (C3) in mammals.

[0083] The siRNA agent of this invention is designed to target the human complement component C3 (C3) gene, including the conserved portion of this gene in C3 orthologs of other mammalian species. The siRNA agent of this invention exhibits improved efficacy, stability, potency, durability, and safety.

[0084] I. Nucleic acid sequence The first aspect of the present invention is an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the sequence of the first strand comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the first strand in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides.

[0085] Preferably, the first strand sequence comprises at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 consecutive nucleotides, which differ from any of the first strand sequences shown in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference.

[0086] The present invention also provides an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the first strand in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides; and the second strand sequence comprises at least 15 consecutive nucleotides, which differs from any nucleotide sequence of the second strand in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides.

[0087] Preferably, the first strand sequence comprises at least 16, more preferably at least 17, still more preferably at least 18, and most preferably all 19 consecutive nucleotides, which differ from any first strand sequence shown in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference; the second strand sequence comprises at least 16, more preferably at least 17, still more preferably at least 18, and most preferably all 19 consecutive nucleotides, which differ from any second strand sequence shown in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference.

[0088] The present invention also provides an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a nucleotide sequence selected from any nucleotide sequence of the first strand in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F; and the second strand comprises a nucleotide sequence selected from any nucleotide sequence of the second strand in any of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F.

[0089] It should be understood that although the sequences in Table 1, for example, are not described as modified or conjugated sequences, the dsRNA of the siRNA agents of the present invention (e.g., the dsRNA of the present invention) may comprise any of the sequences shown in Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F, which may be unmodified, unconjugated, or modified or conjugated differently from those described herein. In other words, the present invention covers unmodified, unconjugated, modified, or conjugated dsRNAs in Tables 2-7, as described herein.

[0090] Furthermore, the present invention provides an siRNA agent having double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the first strand sequence comprises a sequence of at least 15 consecutive nucleotides differing from any of the following sequences by no more than 3 nucleotides: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 ,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second strand sequence comprises a sequence of at least 15 consecutive nucleotides differing by no more than 3 nucleotides from any of the following sequences: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 5 7, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 1 09, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

[0091] In some embodiments, the first-strand sequence comprises at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 consecutive nucleotides, differing from any of the following sequences by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 ,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and the second strand sequence comprises at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 consecutive nucleotides, differing from any of the following sequences by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotide difference: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 5 7, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 1 09, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

[0092] In some implementations, the siRNA agent is a nucleic acid, wherein: the first-strand sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, The sequences 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188; and optionally, wherein the second chain sequence contains SEQ ID. NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 ,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,10 9, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, The sequence of 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189.

[0093] II. Modified siRNA agents A second aspect of the invention is a specific modification pattern for a polynucleotide molecule, which is a double-stranded RNA (dsRNA) comprising a first strand and a second strand.

[0094] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand and the second strand contain at least one modified nucleotide, preferably wherein substantially all nucleotides of the first strand are modified nucleotides and substantially all nucleotides of the second strand are modified nucleotides; more preferably, wherein all nucleotides of the first strand are modified nucleotides and all nucleotides of the second strand are modified nucleotides.

[0095] As used in this article, “virtually all nucleotides are modified” means that most, but not all, nucleotides are modified, including no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0096] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein all nucleotides of the first strand are modified in the following pattern: the pentose sugars at positions 7, 9, 10, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 2, 4, 6, 8, 12, 14, 16, and 18 from the 5' end are modified with 2′-methoxy; and the pentose sugars at positions 1, 3, 5, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution. All nucleotides of the second strand are modified in the following pattern: the pentose sugars at positions 2, 6, 8, 14, and 16 from the 5' end are modified with 2′-fluorine substitution; and the pentose sugars at positions 1, 3, 5, 7, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy. The 4th, 9th, 10th and 18th positions from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution.

[0097] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein all nucleotides of the first strand are modified in the following pattern: the pentose sugar at positions 5, 7, 8, and 9 from the 5' end is modified with 2′-fluorine substitution; the pentose sugar at positions 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18, and 19 from the 5' end is modified with 2′-methoxy; the nucleotide at positions 3, 11, and 14 from the 5' end... The pentose sugars of the nucleotide residues are modified with 2′-methoxy or 2′-fluorine substitution; all nucleotides of the second strand contain the following modification patterns: the pentose sugars of the nucleotide residues at positions 2, 14, 16, and 18 from the 5′ end are modified with 2′-fluorine substitution; the pentose sugars of the nucleotide residues at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, and 19 from the 5′ end are modified with 2′-methoxy; the pentose sugars of the nucleotide residues at positions 4, 8, and 10 from the 5′ end are modified with 2′-methoxy or 2′-fluorine substitution.

[0098] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0099] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein all nucleotides of the first strand are modified in the following pattern: the pentose sugars at positions 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy; the pentose sugars at positions 3, 11, and 14 from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution; the second strand... All nucleotides contain the following modification patterns: the pentose sugars at positions 2, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy; the pentose sugars at positions 4, 8, and 10 from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution; wherein, the target gene of the above double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the following structure: SEQ The nucleotide sequence represented by SEQ ID NO: 20 and the second chain having the nucleotide sequence represented by SEQ ID NO: 21; or the first chain having the nucleotide sequence represented by SEQ ID NO: 96 and the second chain having the nucleotide sequence represented by SEQ ID NO: 97; or the first chain having the nucleotide sequence represented by SEQ ID NO: 16 and the second chain having the nucleotide sequence represented by SEQ ID NO: 17; or the first chain having the nucleotide sequence shown in SEQ ID NO: 150 and the second chain having the nucleotide sequence shown in SEQ ID NO: 151; or the first chain having the nucleotide sequence shown in SEQ ID NO: 32 and the second chain having the nucleotide sequence shown in SEQ ID NO: 33; or the first chain having the nucleotide sequence shown in SEQ ID NO: 100 and the second chain having the nucleotide sequence shown in SEQ ID NO: 101; or the first chain having the nucleotide sequence shown in SEQ ID NO: 110 and the second chain having the nucleotide sequence shown in SEQ ID NO: 111; or the first chain having the nucleotide sequence shown in SEQ ID NO: 134 and the second chain having the nucleotide sequence shown in SEQ ID NO: 135; or the first chain having SEQ ID NO: 20 and the second chain having the nucleotide sequence shown in SEQ ID NO: 21; or the first chain having the nucleotide sequence shown in SEQ ID NO: 134 and the second chain having the nucleotide sequence shown in SEQ ID NO: 135; or the first chain having the nucleotide sequence shown in SEQ ID NO: 150 and the second chain having the nucleotide sequence shown in SEQ ID NO: 151; or the first chain having the nucleotide sequence shown in SEQ ID NO: 32 and the second chain having the nucleotide sequence shown in SEQ ID NO: 33; or the first chain having the nucleotide sequence shown in SEQ ID NO: 100 and the second chain having the nucleotide sequence shown in SEQ ID NO: 101; or The nucleotide sequence shown in NO: 136 and the second strand have the nucleotide sequence shown in SEQ ID NO: 137.

[0100] In some embodiments, all nucleotides of the first strand are modified in the following pattern: the pentose sugars at positions 7, 9, 10, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. All nucleotides of the second strand are modified in the following pattern: the pentose sugars at positions 2, 6, 8, 9, 14, and 16 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, and 19 from the 5' end are modified with 2′-methoxy.

[0101] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0102] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0103] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0104] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0105] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0106] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0107] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 3, 5, 7, 8, and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0108] In some embodiments, the entire nucleotide chain of the first strand comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire nucleotide chain of the second strand comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0109] In some embodiments, the entire first strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 14 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy. The entire second strand of nucleotides comprises modifications in the following pattern: the pentose sugars at positions 2, 10, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy.

[0110] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent comprises all nucleotides modified in the following pattern: the pentose sugars at positions 5, 7, 8, 9, and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end are modified with 2′-methoxy substitution; the second strand comprises all nucleotides modified in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution. Substitution modification; the pentose sugars at nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxy groups; among them, the target genes of the double-stranded RNA (dsRNA) agent are selected from complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, and A SGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, Factor VIII, Factor X, Factor IX, Factor XI, Factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG-1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, S CAP, SERPINA1, SERPINAF2, SREBF2, HMGCR, TGFB1, COX-2, TP53, CD4, CD8, CD40, CD71, DUX4, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4; preferably, complement component C3 (C3).In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 16 and the second strand has the nucleotide sequence represented by SEQ ID NO: 17; or the first strand has the nucleotide sequence represented by SEQ ID NO: 150 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151; or the first strand has the nucleotide sequence represented by SEQ ID NO: 32 and the second strand has the nucleotide sequence represented by SEQ ID NO: 33; or the first strand has the nucleotide sequence represented by SEQ ID NO: 100 and the second strand has the nucleotide sequence represented by SEQ ID NO: 101; or the first strand has the nucleotide sequence represented by SEQ ID NO: 110 and the second strand has the nucleotide sequence represented by SEQ ID NO: 111; or the first strand has the nucleotide sequence represented by SEQ ID NO: 20. The nucleotide sequence represented by NO: 134 and the second strand having the nucleotide sequence represented by SEQ ID NO: 135; or the first strand having the nucleotide sequence represented by SEQ ID NO: 136 and the second strand having the nucleotide sequence represented by SEQ ID NO: 137.

[0111] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnxnNfnNfNfNfnxnnxnnnnnn-3'; and the second strand comprises 5'-nNfnxnnnxnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide, "n" represents a 2'-O-methylmodified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methylmodified nucleotide.

[0112] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0113] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnxnNfnNfNfNfnxnnxnnnnnn-3'; the second strand comprises 5'-nNfnxnnnxnxnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoro-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methyl-modified nucleotide; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 20. The nucleotide sequence represented by NO: 16 and the second chain having the nucleotide sequence represented by SEQ ID NO: 17; or the first chain having the nucleotide sequence represented by SEQ ID NO: 150 and the second chain having the nucleotide sequence represented by SEQ ID NO: 151; or the first chain having the nucleotide sequence represented by SEQ ID NO: 32 and the second chain having the nucleotide sequence represented by SEQ ID NO: 33; or the first chain having the nucleotide sequence represented by SEQ ID NO: 100 and the second chain having the nucleotide sequence represented by SEQ ID NO: 101; or the first chain having the nucleotide sequence represented by SEQ ID NO: 110 and the second chain having the nucleotide sequence represented by SEQ ID NO: 111; or the first chain having the nucleotide sequence represented by SEQ ID NO: 134 and the second chain having the nucleotide sequence represented by SEQ ID NO: 135; or the first chain having the nucleotide sequence represented by SEQ ID NO: 136 and the second chain having the nucleotide sequence represented by SEQ ID NO: 137.

[0114] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfNfnnnnnnnnnn-3'; and the second strand comprises 5'-nNfnnnnnnnnNfnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0115] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfNfnnnnnnnnnn-3'; and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0116] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnNfnNfnNfNfNfnnnnnnnnnn-3'; and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0117] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnnnnnnn-3'; and the second strand comprises 5'-nNfnnnnnnnnNfnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0118] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnNfnnnnnn-3'; and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0119] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfnNfnnnnnnnn-3'; and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0120] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfNfnnnnNfnnnn-3'; and the second strand comprises 5'-nNfnnnnnnnNfnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0121] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfNfnnnnNfnnnn-3'; and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0122] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnNfnNfNfNfnnnnNfnnnn-3'; and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0123] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-nnnnnnNfnNfNfNfnnnnnnnn-3'; and the second strand comprises 5'-nNfnnnNfnNfNfnnnnNfnnn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide.

[0124] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0125] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand contains 5'-nnnnNfnNfNfnNfnnNfnnnnnn-3'; the second strand contains 5'-nNfnnnnnNfnnnnnNfnNfn-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and wherein "n" represents a 2'-O-methylmodified nucleotide; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 16 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21. The nucleotide sequence represented by NO: 17; or the first chain having the nucleotide sequence shown in SEQ ID NO: 150 and the second chain having the nucleotide sequence shown in SEQ ID NO: 151; or the first chain having the nucleotide sequence shown in SEQ ID NO: 32 and the second chain having the nucleotide sequence shown in SEQ ID NO: 33; or the first chain having the nucleotide sequence shown in SEQ ID NO: 100 and the second chain having the nucleotide sequence shown in SEQ ID NO: 101; or the first chain having the nucleotide sequence shown in SEQ ID NO: 110 and the second chain having the nucleotide sequence shown in SEQ ID NO: 111; or the first chain having the nucleotide sequence shown in SEQ ID NO: 134 and the second chain having the nucleotide sequence shown in SEQ ID NO: 135; or the first chain having the nucleotide sequence shown in SEQ ID NO: 136 and the second chain having the nucleotide sequence shown in SEQ ID NO: 137.

[0126] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and / or the 3' end; and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and / or the 3' end.

[0127] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 3' end; and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0128] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 5' end; and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0129] In some embodiments, the first strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end, and / or the second strand contains two phosphate thioester nucleotide inter-bonds at the 5' end and two phosphate thioester nucleotide inter-bonds at the 3' end.

[0130] In some implementations, the siRNA agent also contains a targeting ligand that targets liver tissue.

[0131] In some implementations, the targeting ligand is a GalNAc derivative. In another aspect, the targeting ligand is a GalNAc conjugate.

[0132] In some implementations, the targeting ligand is conjugated to the 3' or 5' end of the first strand of the siRNA agent.

[0133] In some embodiments, the targeting ligand is conjugated to the 3' end of the first chain; the first chain contains two phosphate thioester nucleotide bonds at the 5' end, and the second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; optionally, all remaining bonds between the nucleotides of the first chain and / or the second chain are phosphodiester bonds.

[0134] In some embodiments, the targeting ligand is conjugated to the 5' end of the first chain; the first chain contains two phosphate thioester nucleotide bonds at the 3' end, and the second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; optionally, all remaining bonds between the nucleotides of the first chain and / or the second chain are phosphodiester bonds.

[0135] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*xnNfnNfNfnxnnxnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide, "n" represents a 2'-O-methylmodified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0136] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnxnNfnNfNfnxnnxnnn*n*n-3'; the second strand comprises 5'-n*Nf*nxnnnxnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide, "n" represents a 2'-O-methylmodified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0137] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0138] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*NfnNfnNfNfNfnnnnnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0139] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0140] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0141] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfNfnnnnNfnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0142] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfNfnnnnNfnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0143] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnNfnNfnNfNfNfnnnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0144] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnNfnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0145] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0146] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0147] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and wherein "GN" represents a targeting ligand.

[0148] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0149] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnNfnnnnnn(GN)-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and "GN" represents a target ligand; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 96. The nucleotide sequence represented by SEQ ID NO: 97; or the first chain has the nucleotide sequence represented by SEQ ID NO: 16 and the second chain has the nucleotide sequence represented by SEQ ID NO: 17; or the first chain has the nucleotide sequence shown in SEQ ID NO: 150 and the second chain has the nucleotide sequence shown in SEQ ID NO: 151; or the first chain has the nucleotide sequence shown in SEQ ID NO: 32 and the second chain has the nucleotide sequence shown in SEQ ID NO: 33; or the first chain has the nucleotide sequence shown in SEQ ID NO: 100 and the second chain has the nucleotide sequence shown in SEQ ID NO: 101; or the first chain has the nucleotide sequence shown in SEQ ID NO: 110 and the second chain has the nucleotide sequence shown in SEQ ID NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

[0150] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a phosphate thioester nucleotide bond, and "GN" represents a target ligand; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 96. The nucleotide sequence represented by SEQ ID NO: 97; or the first chain has the nucleotide sequence represented by SEQ ID NO: 16 and the second chain has the nucleotide sequence represented by SEQ ID NO: 17; or the first chain has the nucleotide sequence shown in SEQ ID NO: 150 and the second chain has the nucleotide sequence shown in SEQ ID NO: 151; or the first chain has the nucleotide sequence shown in SEQ ID NO: 32 and the second chain has the nucleotide sequence shown in SEQ ID NO: 33; or the first chain has the nucleotide sequence shown in SEQ ID NO: 100 and the second chain has the nucleotide sequence shown in SEQ ID NO: 101; or the first chain has the nucleotide sequence shown in SEQ ID NO: 110 and the second chain has the nucleotide sequence shown in SEQ ID NO: 111; or the first chain has the nucleotide sequence shown in SEQ ID NO: 134 and the second chain has the nucleotide sequence shown in SEQ ID NO: 135; or the first chain has the nucleotide sequence shown in SEQ ID NO: 136 and the second chain has the nucleotide sequence shown in SEQ ID NO: 137.

[0151] In some implementations, the second chain contains a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end.

[0152] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*xnNfnNfNfNfnxnnxnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide, "n" represents a 2'-O-methylmodified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond, wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0153] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnxnNfnNfNfNfnxnnxnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide, "n" represents a 2'-O-methylmodified nucleotide, and "x" represents a 2'-fluoro or 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond, wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0154] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0155] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*NfnNfnNfNfNfnnnnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0156] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnNfnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0157] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnnNfnnnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0158] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfNfnnnnNfnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0159] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfNfnnnnNfnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0160] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnNfnNfnNfNfNfnnnnnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0161] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnNfnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0162] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnNfnnnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0163] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0164] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting target gene expression, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfNfnnnnNfnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond; wherein "GN" represents a targeting ligand; and wherein "E-VP" represents (E)-vinylphosphonate nucleotide.

[0165] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0166] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-n*n*nnNfnNfNfnNfnNfnnnnnn(GN)-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents (E)-vinylphosphonate nucleotide; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3; preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 21. The nucleotide sequence represented by NO: 96 and the second chain having the nucleotide sequence represented by SEQ ID NO: 97; or the first chain having the nucleotide sequence represented by SEQ ID NO: 16 and the second chain having the nucleotide sequence represented by SEQ ID NO: 17; or the first chain having the nucleotide sequence shown in SEQ ID NO: 150 and the second chain having the nucleotide sequence shown in SEQ ID NO: 151; or the first chain having the nucleotide sequence shown in SEQ ID NO: 32 and the second chain having the nucleotide sequence shown in SEQ ID NO: 33; or the first chain having the nucleotide sequence shown in SEQ ID NO: 100 and the second chain having the nucleotide sequence shown in SEQ ID NO: 101; or the first chain having the nucleotide sequence shown in SEQ ID NO: 110 and the second chain having the nucleotide sequence shown in SEQ ID NO: 111; or the first chain having the nucleotide sequence shown in SEQ ID NO: 134 and the second chain having the nucleotide sequence shown in SEQ ID NO: 135; or the first chain having the nucleotide sequence shown in SEQ ID NO: 136 and the second chain having the nucleotide sequence shown in SEQ ID NO: 137.

[0167] In some embodiments, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, comprising a first strand and a second strand, wherein the first strand comprises 5'-(GN)nnnnNfnNfNfnNfnnnnNfnnnn*n*n-3'; the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3'; wherein "Nf" represents a 2'-fluoromodified nucleotide and "n" represents a 2'-O-methylmodified nucleotide; wherein "*" represents a thiophosphate nucleotide bond, wherein "GN" represents a targeting ligand; wherein "E-VP" represents (E)-vinylphosphonate nucleotide; wherein the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3); preferably, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 21. The nucleotide sequence represented by NO: 96 and the second chain having the nucleotide sequence represented by SEQ ID NO: 97; or the first chain having the nucleotide sequence represented by SEQ ID NO: 16 and the second chain having the nucleotide sequence represented by SEQ ID NO: 17; or the first chain having the nucleotide sequence shown in SEQ ID NO: 150 and the second chain having the nucleotide sequence shown in SEQ ID NO: 151; or the first chain having the nucleotide sequence shown in SEQ ID NO: 32 and the second chain having the nucleotide sequence shown in SEQ ID NO: 33; or the first chain having the nucleotide sequence shown in SEQ ID NO: 100 and the second chain having the nucleotide sequence shown in SEQ ID NO: 101; or the first chain having the nucleotide sequence shown in SEQ ID NO: 110 and the second chain having the nucleotide sequence shown in SEQ ID NO: 111; or the first chain having the nucleotide sequence shown in SEQ ID NO: 134 and the second chain having the nucleotide sequence shown in SEQ ID NO: 135; or the first chain having the nucleotide sequence shown in SEQ ID NO: 136 and the second chain having the nucleotide sequence shown in SEQ ID NO: 137.

[0168] In some embodiments, the first strand has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first strand has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first strand has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide inter-linked bond of thiophosphate; "(E)-VP" is the (E)-vinylphosphonate moiety at the 5' end; and "GN" is the targeting ligand.

[0169] III. SIRNA conjugates In a third aspect of the invention, the siRNA reagent is coupled or conjugated to one or more targeting moieties. In some cases, the targeting moieties are chosen based on their ability to selectively or preferably target the conjugated molecules described herein to a desired cell population, tissue, or organ. In other cases, the targeting moieties target cells, tissues, or organs expressing a corresponding binding partner (e.g., a corresponding receptor or ligand). For example, a polynucleotide molecule conjugated to N-acetylgalactosamine (GalNAc) can target hepatocytes expressing sialic acid glycoprotein (ASGP-R). Any suitable GalNAc molecule known in the art as a targeting moiety is conceivable. Exemplary GalNAc molecules include tribranched GalNAc (e.g., L96).

[0170] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, wherein the siRNA agent is conjugated with a ligand.

[0171] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, wherein the siRNA agent is conjugated to a ligand as shown in the figure below: the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or a derivative thereof to a nucleic acid, and (iii) a bicyclic group linking GalNAc and siRNA, or (iv) a tetrafunctional group linking the GalNAc moieties. or ; Where X represents O and S.

[0172] In one respect, yes: and ; yes: and ; In one aspect, the siRNA agent conjugates with a ligand, as shown in the figure below: , ; The four sensory groups are selected from: and .

[0173] In one respect, the joints in the diagram are independently selected from: (1)-(CH2)xC(O)NH-(CH2)yC(O)-; (2)-(CH2)xC(O)NH-(CH2)y-NHC(O)-(CH2)zC(O)-; (3) -NH-(CH2)yC(O)-; (4) –C(O)-(CH2)yC(O)-; In this figure, each connector may optionally be the same or different; x, y and z are independently selected from 1 to 10; preferably, x and y are independently selected from 2 to 8, and z is selected from 1 to 6; more preferably, x and y are independently selected from 3 to 6, and z is selected from 1 to 4.

[0174] In one aspect, the siRNA agent conjugates with a ligand, as shown in the figure below: GalNAc-32 GalNAc-33 GalNAc-34 GalNAc-35 GalNAc-36 GalNAc-37 GalNAc-38 Where X is O or S.

[0175] In some embodiments, the target genes of the above-mentioned double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, Agtr1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VIII, factor X, factor IX, factor XI, factor XII, GPR146, GPR75, GRB10 / 14, TLR7 / 8 / RIG. -1, HSD17B13, INHBE, ITGV6, KHK, KLK1, MASP2, MTARC1, MUC5B, NPC1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, SREBF2, HMGCR, TGFB1, COX-2, TP53 , CD4, CD8, CD40, CD71, DUX4,

[0176] In some implementations, the target gene of the above-mentioned double-stranded RNA (dsRNA) agent is complement component C3 (C3).

[0177] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a sequence comprising at least 15 consecutive nucleotides, which differ from any of the nucleotide sequences 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

[0178] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has nucleotide sequences represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, and 136, respectively. In some embodiments, the second strand, complementary to the first strand, has nucleotide sequences represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively.

[0179] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 16 and the second strand has the nucleotide sequence represented by SEQ ID NO: 17; or the first strand has the nucleotide sequence represented by SEQ ID NO: 150 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151; or the first strand has the nucleotide sequence represented by SEQ ID NO: 32 and the second strand has the nucleotide sequence represented by SEQ ID NO: 33; or the first strand has the nucleotide sequence represented by SEQ ID NO: 100 and the second strand has the nucleotide sequence represented by SEQ ID NO: 101; or the first strand has the nucleotide sequence represented by SEQ ID NO: 110 and the second strand has the nucleotide sequence represented by SEQ ID NO: 111; or the first strand has the nucleotide sequence represented by SEQ ID NO: 134 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151. The nucleotide sequence shown in NO: 135; or the first strand having the nucleotide sequence shown in SEQ ID NO: 136 and the second strand having the nucleotide sequence shown in SEQ ID NO: 137.

[0180] In some implementations, the double-stranded RNA (dsRNA) agent is shown in the figure below: or , The target gene of the aforementioned double-stranded RNA (dsRNA) agent is complement component C3 (C3).

[0181] In some implementations, the double-stranded RNA (dsRNA) agent is shown in the figure below: , The first strand has a sequence comprising at least 15 consecutive nucleotides, differing from any one of the nucleotide sequences 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises at least partially complementary nucleotide sequences to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has nucleotide sequences represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, and 136, respectively. In some embodiments, the second strand complementary to the first strand has nucleotide sequences represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively. In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 16 and the second strand has the nucleotide sequence represented by SEQ ID NO: 17; or the first strand has the nucleotide sequence represented by SEQ ID NO: 150 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151; or the first strand has the nucleotide sequence represented by SEQ ID NO: 32 and the second strand has the nucleotide sequence represented by SEQ ID NO: 33; or the first strand has the nucleotide sequence represented by SEQ ID NO: 100 and the second strand has the nucleotide sequence represented by SEQ ID NO: 101; or the first strand has the nucleotide sequence represented by SEQ ID NO: 110 and the second strand has the nucleotide sequence represented by SEQ ID NO: 111; or the first strand has the nucleotide sequence represented by SEQ ID NO: 134 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151. The nucleotide sequence shown in NO: 135; or the first strand having the nucleotide sequence shown in SEQ ID NO: 136 and the second strand having the nucleotide sequence shown in SEQ ID NO: 137.

[0182] In some implementations, the double-stranded RNA (dsRNA) agent is shown in the figure below: , The first strand has a sequence comprising at least 15 consecutive nucleotides, differing from any one of the nucleotide sequences 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence at least partially complementary to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has nucleotide sequences represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, and 136, respectively. In some embodiments, the second strand complementary to the first strand has nucleotide sequences represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively. In a preferred embodiment, the first strand of the double-stranded RNA (dsRNA) agent has the nucleotide sequence represented by SEQ ID NO: 20 and the second strand has the nucleotide sequence represented by SEQ ID NO: 21; or the first strand has the nucleotide sequence represented by SEQ ID NO: 96 and the second strand has the nucleotide sequence represented by SEQ ID NO: 97; or the first strand has the nucleotide sequence represented by SEQ ID NO: 16 and the second strand has the nucleotide sequence represented by SEQ ID NO: 17; or the first strand has the nucleotide sequence represented by SEQ ID NO: 150 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151; or the first strand has the nucleotide sequence represented by SEQ ID NO: 32 and the second strand has the nucleotide sequence represented by SEQ ID NO: 33; or the first strand has the nucleotide sequence represented by SEQ ID NO: 100 and the second strand has the nucleotide sequence represented by SEQ ID NO: 101; or the first strand has the nucleotide sequence represented by SEQ ID NO: 110 and the second strand has the nucleotide sequence represented by SEQ ID NO: 111; or the first strand has the nucleotide sequence represented by SEQ ID NO: 134 and the second strand has the nucleotide sequence represented by SEQ ID NO: 151. The nucleotide sequence shown in NO: 135; or the first strand having the nucleotide sequence shown in SEQ ID NO: 136 and the second strand having the nucleotide sequence shown in SEQ ID NO: 137.

[0183] In some embodiments, the first strand has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID N: 228), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second strand has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first strand has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 232), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first strand has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233). Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide bond between thiophosphates; and "(E)-VP" represents the (E)-vinylphosphonate moiety at the 5' end.

[0184] definition Before detailing the invention below, it will be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they may differ. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0185] For the interpretation of this specification, the following definitions will apply, and where appropriate, terms used in the singular form may also include the plural form, and vice versa. It will be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0186] As used herein, the term “complement component 3” is used interchangeably with the term “C3” to refer to well-known genes and polypeptides, also known in the art as ARMD9, C3a anaphylatoxin, ASP, complement component C3a, C3a, complement component C3b, C3b, prepro-C3, acylated stimulating protein cleavage product, CPAMD1, complement C3, C3 and protein 1 containing a PZP-like α-2-macroglobulin domain, complement component C3 and AHUS5.

[0187] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during transcription of the complement component C3 gene, including the mRNA of the RNA processing product, which is the major transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage on or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the complement component C3 gene.

[0188] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably herein and refer to an agent containing RNA as defined herein that mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of the C3 gene in cells (e.g., cells within a subject, such as cells within a mammalian subject).

[0189] The term "mRNA (messenger RNA)" refers to RNA molecules that serve as templates for protein translation in vivo, transferring gene-encoding information from DNA to protein products. The term "RNA interference" or "RNAi" refers to the post-transcriptional regulation of gene expression in organisms. This phenomenon is caused by the specific degradation of target mRNA mediated by single-stranded or double-stranded RNA. For detailed information on the RNAi regulatory mechanism, please refer to Biotech.Adv.2008, 26(3):202 and other literature.

[0190] In this invention, unless otherwise stated, the terms "small interference RNA" or "siRNA" refer to RNA molecules capable of sequence-specifically inducing RNAi, consisting of two single-stranded RNA molecules of 15-27 nucleotides in length, and having a partially or completely complementary double-stranded structure. In the siRNA of this invention, the length of the complementary double-stranded structure can be 16-25, 17-22, or 18-21 base pairs. The siRNA described in this invention can be a blunt-ended double-stranded RNA structure composed of two single-stranded RNA molecules of 15-27 nucleotides in length, or a structure having a 3′ overhang of 1-3 consecutive nucleotides at at least one end of the double-stranded structure.

[0191] In this invention, unless otherwise stated, the term "first single strand" or "sense strand" refers to one of the two single strands of siRNA, having a nucleotide sequence that is partially or entirely identical to the nucleotide sequence of the siRNA's site of action in the target mRNA; while the term "second single strand" or "antisense strand" refers to the other of the two single strands of siRNA, having a nucleotide sequence that is partially or entirely complementary to the nucleotide sequence of the siRNA's site of action in the target mRNA. The first single strand (or sense strand) and the corresponding second single strand (or antisense strand) of siRNA mentioned in this invention can form a partially or completely complementary double-stranded structure.

[0192] The “complementary” sequences used in this article may also include non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, or base pairs formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, as long as they meet the above requirements regarding hybridization ability. Such non-Watson-Crick base pairs include, but are not limited to, G:U swinging or Hoogsteen base pairings.

[0193] In this invention, unless otherwise stated, the terms "suppressed" and "inhibited" refer to a situation where mRNA degradation mediated by siRNA or other small interfering nucleic acid (siNA) inhibitors leads to a significant downregulation of target gene expression. "Significant downregulation" means a reduction in target gene expression of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, or 100%, compared to normal or pre-treatment levels.

[0194] As used herein, the phrase “contacting cells with an RNAi agent (such as dsRNA)” includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells in vitro with the RNAi agent or contacting cells in vivo with the RNAi agent. Contact can be direct or indirect. Thus, for example, an individual performing this method may physically contact the RNAi agent with the cells, or the RNAi agent may be placed in an environment that allows or results in subsequent contact with the cells. For example, in vitro cell contact can be achieved by incubating the cells with the RNAi agent. In vivo cell contact can be achieved, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another area (e.g., the central nervous system (CNS)), optionally via intrathecal, intravitreal, or other injection, or injection into the bloodstream or subcutaneous space, such that the agent subsequently reaches the tissue where the cells to be contacted are located.

[0195] “G,” “C,” “A,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to modified nucleotides, as further detailed below, or to alternative substitutions. Those skilled in the art will fully understand that guanine, cytosine, adenine, and uracil can be substituted with other parts without significantly altering the base-pairing properties of the oligonucleotide containing the nucleotide with such substitutions.

[0196] The chemical modification of siRNA in this invention can be selected from one of the following chemical modifications, or a combination of several of the following chemical modifications: 1) Modification of the phosphodiester bonds that link nucleotide residues in the RNA backbone structure; 2) Modification of ribose in the RNA backbone structure; 3) Modification of bases in the nucleotide residues of RNA.

[0197] The naturally occurring nucleotide internucleotide bonds of siRNA are 3′ to 5′ phosphodiester bonds. RNA strands with one or more modified (i.e., non-naturally occurring) nucleotide internucleotide bonds are generally preferred over RNA strands with naturally occurring nucleotide internucleotide bonds because they possess desirable properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0198] Oligonucleotides with modified nucleoside bonds include those retaining a phosphorus atom and those without a phosphorus atom. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphate diesters, phosphate triesters, methylphosphonates, aminophosphates, and thiophosphates.

[0199] RNAi agents may contain a phosphorus-containing group at the 5' end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group can be a 5'-phosphate ester (5'-P), a 5'-thiophosphate ester (5'-PS), a 5'-dithiophosphate ester (5'-PS2), a 5'-vinylphosphonate ester (5'-VP), a 5'-methylphosphonate ester (MePhos), or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is a 5'-vinylphosphonate ester (5'-VP), 5'-VP can be a 5'-E-VP isomer phosphate ester, an isomer (i.e., cis-vinylphosphonate), or a mixture thereof.

[0200] In this invention, the RNAi agent comprises one or more modified nucleoside internucleotide bonds. In some embodiments, the modified nucleoside internucleotide bond is a phosphate thioester bond. In some embodiments, the RNAi agent contains 5'-E-VP in the second strand.

[0201] In this invention, modification of the ribose group refers to the modification of the 2′-hydroxyl group (2′-OH) on the ribose group. Introducing certain substituents, such as methoxy or fluorine groups, at the 2′-hydroxyl position of the ribose group makes it more difficult for serum ribonuclease to digest nucleic acids, thereby improving the stability of nucleic acids and giving them a stronger resistance to nuclease hydrolysis. Modification of the 2′-hydroxyl group on the pentose of the nucleotide includes 2′-fluorination modification, 2′-methoxy modification, 2′-methoxyethoxy modification, 2′-2,4-dinitrophenol modification (2′-DNP modification), locked nucleic acid modification (LNA modification), 2′-amino modification, and 2′-deoxy modification, etc.

[0202] In this invention, base modification refers to the modification of bases on nucleotide groups. For example, 5′-bromouracil modification and 5′-iodouracil modification, which introduce bromine or iodine at the 5-position of uracil, are common base modification methods. Other modifications, such as N3-methyluracil modification and 2,6-diaminopurine modification, can also be used.

[0203] In some embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a GalNAc conjugate. In some embodiments, the GalNAc conjugate is one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or vector.

[0204] In some embodiments, the conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, such as a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the positive sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 3' end of the positive sense strand) via a linker (e.g., the linker described herein). In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the positive sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 5' end of the positive sense strand) via a linker (e.g., the linker described herein).

[0205] In some embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a monovalent adapter. In some embodiments, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a bivalent adapter. In other embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a trivalent adapter. In other embodiments of the present invention, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a tetravalent adapter.

[0206] One single strand of the siRNA of the present invention can be synthesized by solid-phase or liquid-phase nucleic acid synthesis. These methods include four processing steps: 1) oligonucleotide synthesis; 2) deprotection; 3) purification and separation; and 4) desalting. The technical details of the above four steps are well known to those skilled in the art, and therefore will not be described in detail here.

[0207] In addition to chemical synthesis, the siRNA of this invention can also be obtained through expression using plasmids and / or viral vectors. For example, a DNA sequence of 50-90 nucleotides in length is designed, and two different restriction enzyme sites, such as BamHI and EcoRI, are added to both ends of it. The middle segment sequence of the designed DNA-encoded RNA transcript can form a circular structure, while the sequences at both ends of the loop after the U-turn can form a complementary double-stranded structure. Using cloning technology, the designed DNA is inserted into an expression vector digested with the corresponding restriction enzymes. The expression vector is introduced into cells, and the RNA transcript generated from the designed DNA sequence can be processed into mature siRNA through the cell's inherent siRNA processing mechanism. Thus, the siRNA can be expressed transiently or stably in cells.

[0208] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in carrying out or testing the RNAi agents and methods of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0209] Example This invention will be described in conjunction with the examples described below. Unless otherwise specified herein, such reagents and other experimental materials may be obtained from any molecular biology reagent supplier, provided that their quality / purity meets the standards for molecular biology applications.

[0210] Example 1. Design of small interfering nucleotides in the human C3 gene The 19 bp nucleotide sequence was selected from the mRNA sequence of the human C3 gene in the range of 1-5231 bp, and its sequence is relatively conserved (Genbank accession number NM_000064, SEQ ID NO: 1).

[0211] Table 1 lists the sequences of the sense and antisense strands of each siRNA. Specifically, for example, the first strand (sense strand) of siRNA C3-1 has the sequence shown in SEQ ID NO: 2, which is identical to the corresponding target site sequence in the C3 mRNA sequence; and the second strand (antisense strand) has the sequence shown in SEQ ID NO: 3, which is complementary to the corresponding target site sequence in the C3 mRNA sequence. The sequences of the two single strands of each other siRNA are numbered sequentially in the same manner as siRNA C3-1.

[0212] Table 1 Example 2: Verification of the inhibitory effect of siRNA on C3 gene expression The siRNAs designed in Table 2A were chemically synthesized by Thermo Fisher Scientific Inc. or GenScript. Complementary oligonucleotides were annealed to form double-stranded RNA according to the method described in "Molecular Cloning: A Laboratory Manual".

[0213] Table 2A (1) HepG2 cell culture HepG2 cells were transfected using a custom-designed silencer siRNA library (C3). RQ values ​​were normalized to empty RNAiMAX transfected HepG2 cells. Each sample and / or probe was assayed three times. (n=3) (2) Methods for transfecting HepG2 cells One day prior to transfection, 17,000 cells / well of HepG2 parental cells were seeded in 96-well tissue culture plates. siRNA transfection was prepared using the Lipofectamine™ RNAiMAX Transfection Kit (catalog number #13778030), with 20 nM siRNA and 0.3g RNAiMAX reagent per well. Cells were harvested 72 hours post-transfection and analyzed by RT-qPCR. RNA was isolated from siRNA-treated and untreated cells. RNA was extracted using the TaqMan® Gene Expression Cells-to-Ct Kit (#4399002). The generated cDNA was used as a template for qPCR reactions using the TaqMan® Gene Expression Master Mixture and custom TaqMan® gene expression assay probes specific to the target genes β-actin (Hs01060665_g1) and human C3 (Hs00163811_m1). β-actin was used as an endogenous control. Samples were run on a Quant Studio™ 12K Flex real-time PCR system. The data were analyzed using comparative CT methods, as shown in Table 2B and Figure 1 As shown.

[0214] The results showed that the exemplary siRNA agent tested effectively reduced the level of human C3 messenger RNA.

[0215] Table 2B Example 3. C3 mRNA knockdown effect in HepG2 cells transfected with 0.128 pM–100 nM selected siRNA (dose-response curve experiment).

[0216] After transfecting HepG2 cells with 0.128 pM–100 nM siRNA, the C3 knockdown effects of the selected siRNAs C3-8-dTdT, C3-10-dTdT, C3-16-dTdT, C3-48-dTdT, C3-50-dTdT, C3-55-dTdT, C3-67-dTdT, C3-68-dTdT, and C3-75-dTdT were determined. The siRNA sequence information is shown in Table 2A. Results are as follows: Figure 2A As shown. After transfection, all siRNAs showed dose-dependent C3 mRNA knockdown. The most effective siRNAs were C3-48-dTdT, C3-16-dTdT, and C3-8-dTdT at a concentration of 100 nM, as shown. Figure 2B As shown.

[0217] HepG2 cell transfection method Cells were seeded at a density of 13,300 cells / well in 96-well clear F-bottom TC-treated plates (VWR #734-2327). siRNA transfection was performed using Lipofectamine™ RNAiMAX transfection reagent (Invitrogen lifetechnology) according to the manufacturer's protocol. Dosage-response experiments were conducted using eight concentrations of C3 siRNA, starting at 100 nM and diluted 5-fold to 0.128 pM. Mock cells were untreated cells.

[0218] 72 hours post-transfection, RNA was extracted using the Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen Life Technology) according to the manufacturer's protocol. Briefly, after removing the medium from the cells, 100 μL of lysis buffer was added to the cells. The cell culture plate was placed in a shaker and vortexed at 300 rpm for 30 minutes at room temperature. Dynabeads were then added to purify the mRNA from the lysate. Immediately after RNA extraction, reverse transcription was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Technology). C3 and TBP mRNAs were identified using the Quantstudio 5 real-time PCR system (Invitrogen Life Technology) using TaqMan qPCR primers, C3-FAM (assay ID: Hs00163811_m1), and TBP-VIC (assay ID: Hs00427620_m1). The activity of a given C3 siRNA is expressed as the percentage of C3 mRNA normalized to TBP mRNA in treated cells relative to the C3 mRNA normalized to TBP mRNA in untreated cells (simulated). Dose-response curves were generated using a four-parameter logistic model with GraphPad Prism version 9. Figure 2A and Figure 2B The data shown comes from three biological replicates. Figure 2B The data in the middle comes from Figure 2A The 0.16 nM and 100 nM data are shown.

[0219] Example 4. Chemical modification of siRNA To enhance the stability of small interfering nucleotides and improve the liver-targeting of drug administration, chemical modifications were designed for the small interfering nucleotides C3-8, C3-10, C3-16, C3-48, C3-50, C3-55, C3-67, C3-68, and C3-75 of the human C3 gene. The design principle is as follows: nucleotides are numbered sequentially starting from the first nucleotide at the 5' end of the first or second single strand.

[0220] Modification pattern of the first single chain (justice chain): SS7: The pentose sugars of nucleotide residues 7, 9, 10 and 11 are modified with 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18 and 19 are modified with 2′-methoxy substitution.

[0221] SS1: The pentose sugars of nucleotide residues 3, 5, 7, 8 and 9 are modified by 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 are modified by 2′-methoxy substitution.

[0222] SS2: The pentose sugars of nucleotide residues 5, 7, 8, 9 and 11 are modified by 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 are modified by 2′-methoxy substitution.

[0223] SS3: The pentose sugars of nucleotide residues 5, 7, 8, 9 and 14 are modified by 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 are modified by 2′-methoxy substitution.

[0224] Modification pattern for the second single chain (antisense chain): AS8: The pentose sugars of nucleotide residues 2, 6, 8, 9, 14 and 16 are modified with 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 are modified with 2′-methoxy substitution.

[0225] AS4: The pentose sugars of nucleotide residues 2, 10, 14, 16 and 18 are modified with 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 are modified with 2′-methoxy substitution.

[0226] AS5: The pentose sugars of nucleotide residues 2, 8, 14, 16 and 18 are modified with 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 are modified with 2′-methoxy substitution.

[0227] AS6: The pentose sugars of nucleotide residues 2, 4, 14, 16 and 18 are modified with 2′-fluorine substitution; the pentose sugars of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 are modified with 2′-methoxy substitution.

[0228] The siRNA contains modification patterns of SS7 / AS8, SS1 / AS4, SS1 / AS5, SS1 / AS6, SS2 / AS4, SS2 / AS5, SS2 / AS6, SS3 / AS4, SS3 / AS5, or SS3 / AS6.

[0229] A thiophosphate ester (* in Tables 3A, 3B, 3C, 3D, 3E, and 3F) is introduced between the last three nucleotides at the 5' and 3' ends of the second single strand (antisense strand). A thiophosphate ester is also introduced between the last three nucleotides at either the 5' or 3' end of the first single strand (sense strand). (E)-vinylphosphonate (“(E)-VP” in Tables 3A, 3B, 3C, 3D, 3E, and 3F indicates (E)-vinylphosphonate) is used at the 5' end of the second single strand (antisense strand).

[0230] “GN” in Tables 3A, 3B and 3C refers to a targeting ligand containing N-acetylgalactosamine (GalNAc).

[0231] Modified siRNAs C3-144 to C3-152 contain the modification pattern SS7 / AS8. Sequence information for modified siRNAs C3-144 to C3-152 is shown in Table 3A. Modified siRNAs C3-104 to C3-113 contain the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. Sequence information for modified siRNAs C3-104 to C3-113 is shown in Table 3B. Modified siRNAs C3-114 to C3-123 contain the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. Sequence information for modified siRNAs C3-114 to C3-123 is shown in Table 3C.

[0232] Table 3A Table 3B Table 3C Specifically, GalNAc-1, GalNAc-3, and GalNAc-7 are conjugated with dsRNA for in vitro and in vivo screening. The siRNA agents conjugated with GalNAc-1, GalNAc-3, and GalNAc-7 are shown in the figure below: Modified siRNAs C3-95 to C3-103 contain the modification pattern SS7 / AS8. Sequence information for modified siRNAs C3-95 to C3-103 is shown in Table 3D. Modified siRNAs C3-124 to C3-133 contain the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. Sequence information for modified siRNAs C3-124 to C3-133 is shown in Table 3E. Modified siRNAs C3-134 to C3-143 contain the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. Sequence information for modified siRNAs C3-134 to C3-143 is shown in Table 3F.

[0233] Table 3D Table 3E Table 3F Chemically modified small interfering nucleotides were synthesized by Wuxi AppTec. The targeting ligand (GalNAc) can be conjugated to the 3' or 5' end of the oligonucleotide as a single (tri-branched) or stepwise in a 1+1+1 trivalent configuration. To achieve tri-branched GalNAc conjugation or 1+1+1 assembly, GalNAc amidates and / or GalNAc solid supports should be synthesized separately. The GalNAc amidates and / or GalNAc solid supports are then used as building blocks for solid-phase oligonucleotide synthesis.

[0234] GalNAc-conjugated phosphoramides: The synthesis of all GalNAc-conjugated phosphoramids followed the method described in Example 5.

[0235] GalNAc-conjugated succinate and its loading onto a solid support: The synthesis and loading of GalNAc-conjugated succinates onto a solid support followed the method described in Example 5.

[0236] General methods for siRNA preparation 1. Sample loading: Load 0.19 g, 500A, ~10 µmol of pre-loaded CPG (GalNAc- or nucleoside) into a 5 mL syringe.

[0237] 2. Washing: Acetonitrile (2.0 mL, 0.3 min, repeat twice, room temperature).

[0238] 3. Detrimethylation: 3% trichloroacetic acid in dichloromethane solution (2.0 mL, 0.7 min, repeated four times at room temperature).

[0239] 4. Washing: Acetonitrile (2.0 mL, 0.3 min, repeat twice, room temperature).

[0240] 5. Coupling: A solution of 0.067 M of the reactant monomer in acetonitrile (1.0 mL) and a solution of 0.30 M of 5-(benzylthio)-1H-tetrazole (BTT) in acetonitrile (1.0 mL) were used as activators (7.0 min, repeated three times at room temperature). 6. Sulfidation or oxidation: N,N-dimethyl-N'-(3-thio-3H-1,2,4-diazol-5-yl)formamidin (DDTT, 4.80 M, in pyridine / acetonitrile = 2 / 1, 2.0 mL, 1 min, twice, at room temperature) or 0.05 M I2 in pyridine / H2O = 80 / 20 (v / v) solution (2.0 mL, 1 min, twice, at room temperature).

[0241] 7. Capping: 1-Methylimidazole (NMI) / acetonitrile = 15 / 85 (v / v) (2.0 mL) and acetic anhydride / acetonitrile = 20 / 80 (v / v) (0.9 mL, 1 min, once, at room temperature).

[0242] 8. Washing: Acetonitrile (2.6 mL, 0.3 min, repeat twice, room temperature).

[0243] 9. The synthesis program automatically loops 19 times.

[0244] 10. Immerse the solid support in 10% DEA for 20 minutes. Suspend the solid support in NH3·H2O (5 mL) and stir in a 48 mL sealed tube at 40°C for 16 hours. Cool the reaction mixture to 25°C. Then filter the solid support and concentrate the aqueous phase under vacuum to obtain a yellow solution.

[0245] 11. Add 40 mL of ethanol to the filtrate (10 mL), followed by 0.3 mL of NaCl (3M). Incubate the centrifuge tube at -20°C for 20 minutes. Then centrifuge the tube. Discard the supernatant and collect the remaining solid.

[0246] 12. The white solid was purified by preparative HPLC (column: O-C18 150*40 mm *10 µm; mobile phase: [0.1 MTEAB-ACN]; B%: 14%-24%, 30 min).

[0247] 13. After freeze-drying, the desired white solid compound is obtained.

[0248] Annealing steps: 1. Calculate the molar value of the duplex (e.g., 2 mg of duplex, molar value = 2 mg / duplex MW, free acid), which is the molar value of the single chain.

[0249] 2. The sense and antisense chains were dissolved in ultrapure water (DNase / RNase-free, sterile) at 25°C. The concentration of the oligomers was quantified by ultraviolet-visible light (UV-vis).

[0250] 3. Mix the two solutions in a 1:1 molar ratio.

[0251] 4. Let the mixed sample stand at room temperature for 10 minutes. Use HPLC to monitor the suitability of the mixture.

[0252] 5. Aliquot the solution into test tubes and freeze-dry to obtain siRNA samples.

[0253] Example 5: Chemical synthesis of GalNAc ligands. Compounds 1-2: Compound 1-1 (5 g, 12.84 mmol) was dissolved in anhydrous 1,2-dichloroethane (30 mL) and stirred at 0 °C. TMSOTf (3.43 g, 15.41 mmol, 2.78 mL) was added dropwise over 10 minutes, and stirring continued overnight at room temperature. The reaction mixture was quenched with cold saturated NaHCO3 solution (200 mL), and the organic layer was separated. The product was extracted with dichloromethane (60 mL x 2); the combined organic layers were washed with water, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure to give compound 1-2 (4.23 g, 99% yield) as a yellow oil, which was ready for use without further purification.

[0254] C 14 H 19 The calculated mass of NO8 is 329.1; the actual measured mass is 330.1 [M+H]. + , ESI.

[0255] Compounds 1-3: Compounds 1-2 (4.23 g, 12.85 mmol) were dissolved in anhydrous 1,2-dichloroethane (20 mL) and stirred with 4 Å molecular sieve (4.7 g) at room temperature for 5 min. 5-Hexen-1-ol (1.42 g, 14.13 mmol) was added, and stirring continued for 30 min. TMSOTf (1.43 g, 6.42 mmol, 1.16 mL) was added dropwise at 0 °C, and stirring continued for 2 h at room temperature. The reaction mixture was quenched with cold saturated NaHCO3 solution (100 mL), and the organic layer was separated. The product was extracted with dichloromethane (60 mL x 2); the combined organic layers were washed with water, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure to give compounds 1-3 (5.5 g, 99% yield) as a yellow oil, which was ready for use without further purification.

[0256] C 20 H 31 The calculated mass of NO9 is 429.2; the actual measured mass is 430.2 [M+H]. + , ESI.

[0257] Compounds 1-4: To a solution of compounds 1-3 (5.5 g, 12.81 mmol) in DCM (35 mL) and MeCN (35 mL), add a solution of sodium metaperiodate (10.96 g, 51.24 mmol) in water (45.5 mL). Cool the mixture to 0 °C in an ice bath and stir for 15 min. Add ruthenium chloride trihydrate (110.5 mg, 423 μmol) to the cooled reaction mixture. Stir the reaction mixture at room temperature for 4 h. Dilute the reaction mixture with water (90 mL) and adjust the pH to 7.5 by adding solid NaHCO3. Remove the DCM layer, wash the aqueous layer with DCM (30 mL x 2), and discard the organic extract. Adjust the pH of the aqueous layer to 3 by adding citric acid, and extract carboxylic acids 1-4 into DCM (50 mL x 3). The organic layer was stirred together with saturated brine (50 mL x 1), and then Na₂S₂O₃ solution (50 mL x 1) was added dropwise until the dark green organic phase turned pale yellow. The layers were separated, and the organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to give compounds 1-4 as white solids (2.3 g, 40% yield). No further purification was required for use.

[0258] C 19 H 29 NO 11 Calculated mass: 447.2; Actual measurement: 448.2 [M+H] + , ESI.

[0259] 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 9.2Hz, 1H), 5.84-5.73 (m,1H), 5.21 (d, J = 3.3 Hz, 1H), 4.98-4.93 (m, 3H), 4.48 (d, J = 8.5 Hz, 1H),4.04-4.00 (m, 3H), 3.88-3.83 (m, 1H), 3.73-3.68 (m, 1H), 3.40-3.36 (m, 1H),2.10 (s, 3H), 2.03-2.02 (m, 2H), 2.00 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 1.43-1.32 (m, 4H). Compounds 1-6: To a solution of compounds 1-5 (1.16 g, 6.17 mmol) and acid 1-4 (2.3 g, 5.14 mmol) in DMF (30 mL), HBTU (2.92 g, 7.71 mmol) and DIPEA (1.99 g, 15.42 mmol, 2.69 mL) were added. The reaction was stirred at room temperature for 43 hours and diluted with water (150 mL). The mixture was extracted with ethyl acetate (60 mL x 3). The combined organic layers were washed continuously with water (100 mL x 3) and brine (100 mL). After drying with anhydrous Na₂SO₄, the solvent was evaporated under reduced pressure to give compounds 1-6 as a yellow oil (4.3 g, crude product), which was ready for use without further purification.

[0260] C 29 H 48 N2O 12 Calculated mass: 616.3; Actual measurement: 617.4 [M+H] + , ESI.

[0261] Compounds 1-7: Compounds 1-6 (4.23 g, assumed 6.86 mmol) were added to formic acid (30 mL), and the mixture was stirred overnight at room temperature. The reaction was monitored by LC-MS to indicate completion. The mixture was evaporated under reduced pressure and purified by silica gel chromatography (DCM:MeOH = 10:1) to give compounds 1-7 as a yellow oil (2.88 g, 75% yield in two steps).

[0262] C 25 H 40 N2O 12 Calculated mass: 560.3; Actual measurement: 561.3 [M+H] + , ESI.

[0263] 1 H NMR (400 MHz, DMSO-d6) δ12.02 (s, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.25 (t, J = 5.6 Hz, 1H), 5.21 (d, J = 3.2 Hz, 1H), 4.96 (dd, J = 11.2, 3.6Hz, 1H), 4.47 (d, J= 8.4 Hz, 1H), 4.04-4.00 (m, 3H), 3.90-3.83 (m, 1H), 3.73-3.68 (m, 1H), 3.42-3.36 (m, 1H), 3.02-2.97(m, 1H), 2.18 (t, J = 7.2 Hz,2H), 2.10 (s, 3H), 2.02 (t, J = 7.2 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77(s, 3H), 1.54-1.41 (m, 7H), 1.27-1.21 (m, 3H). Compound 2-1: A mixture of 2,2-bis(bromomethyl)propane-1,3-diol (270 g, 1.03 mol), benzaldehyde (114.86 g, 1.08 mol), and TsOH (17.74 g, 340.55 mmol) was refluxed in toluene (1 L) for 6 hours. The mixture was cooled and extracted with EA (1 L), washed with NaHCO3 solution, then washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was recrystallized from MeOH to give the desired product 2-1 (247 g, 68% yield).

[0264] 1 H NMR (400 MHz, DMSO-d6) δ 7.47-7.41 (m, 2H), 7.40-7.36 (m, 3H), 5.48 (s, 1H), 4.09-3.93 (m, 6H), 3.46 (s, 2H). Compound 2-2: Diisopropyl malonate (268.85 g, 1.43 mol) was added dropwise to a suspension of t-BuOK (137.27 g, 1.43 mol) in anhydrous DMF (700 mL) (temperature maintained below 70 °C), followed by compound 2-1 (250 g, 714.19 mmol). The resulting reaction mixture was heated at 140 °C for 6 hours. After cooling, a saturated NH4Cl solution (1.5 L) was added, and the mixture was extracted with hexane (500 mL x 3). The combined organic extracts were dried over sodium sulfate and concentrated under vacuum. The solid product was separated from the liquid residue by filtration, washed with hexane (100 mL x 2), and dried to give pure product 2-2 as a white solid (194 g, yield 68%).

[0265] C 21 H 28 The calculated mass of O6 is 376.2; the actual measured mass is 377.2 [M+H]. + , ESI.

[0266] 1 H NMR (400 MHz, DMSO-d6) δ 7.45-7.31 (m, 5H), 5.45 (s, 1H), 5.02-4.93 (m, 2H), 3.96 (d, J = 11.2 Hz, 2H), 3.77 (d, J = 11.1 Hz, 2H), 2.55 (s,2H), 2.13 (s, 2H), 1.23-1.15 (m, 12H). Compounds 2-3: 10% Pd / C (31 g) was added to a MeOH (750 mL) solution of compound 2-2 (155 g, 411.7 mmol). The resulting suspension was hydrogenated at ambient temperature with stirring under H2 pressure of 5 atm for 48 hours. The catalyst was filtered and the solvent was removed under vacuum to give compound 2-3 as a colorless oil (118 g, 99% yield), which could be used for the next step without further purification.

[0267] C 21 H 28 The calculated mass of O6 is 288.2; the actual measured mass is 289.4 [M+H]. + , ESI.

[0268] Compounds 2-4: Methanesulfonyl chloride (155.7 g, 1.36 mol) was added to a solution of compound 2-3 (140 g, 485.5 mmol) in dichloromethane (840 mL). The resulting mixture was cooled to -30 °C, and then triethylamine (323.9 g, 3.2 mol) was added dropwise. After the addition was complete, the reaction mixture was heated to ambient temperature and stirred for 12 hours, then washed with water (1000 mL), 10% citric acid aqueous solution (1000 mL), and brine (1000 mL). The organic phase was dried over sodium sulfate and evaporated under reduced pressure to give compound 2-4 (125 g, 58% yield).

[0269] C 21 H 28 The calculated mass of O6 is 444.1; the actual measured mass is 445.2 [M+H]. + , ESI.

[0270] 1H NMR (400 MHz, DMSO-d6) δ 5.02-4.93 (m, 2H), 4.20 (s, 4H), 3.23 (s,6H), 2.45 (s, 4H), 1.22-1.14 (m, 12H). Compounds 2-5: A solution of compounds 2-4 (63 g, 141.73 mmol), potassium carbonate (100.88 g, 729.89 mmol), and p-toluenesulfonamide (25.48 g, 148.81 mmol) in DMSO (300 mL) was heated at 85 °C for 12 hours. After cooling, water (300 mL) was added, and the mixture was extracted with EtOAc (600 mL). The combined organic phases were washed with 10% citric acid aqueous solution (600 mL) and brine (600 mL), dried over sodium sulfate and evaporated under vacuum, and then recrystallized from isopropyl ether to give compounds 2-5 (48 g, 80% yield).

[0271] C 21 H 28 O6 was calculated to be 423.2; actual measurement: 424.2 [M+H] + , ESI.

[0272] 1 H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J = 8.2 Hz, 2H), 7.45 (d, J =8.0 Hz, 2H), 4.45 (t, J = 5.4 Hz, 2H), 3.60 (s, 4H), 3.14 (d, J = 5.7 Hz,4H), 2.43 (s, 3H), 1.67 (s, 4H). Compounds 2-6: At -20 °C, a solution of lithium borohydride (2 M) in THF (263.82 mL) was added to a solution of compound 2-5 (58.7 g, 138.60 mmol) in THF (100 mL). The resulting mixture was stirred at room temperature for 16 hours. The mixture was then slowly added to ice water (1 L). The pH was adjusted to 7 with aqueous citric acid. The mixture was extracted with EA (500 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give compound 2-6 (40 g, 93% yield).

[0273] C 21 H 28The calculated mass of O6 is 311.1; the actual measured mass is 312.1 [M+H]. + , ESI.

[0274] 1 H NMR (400 MHz, DMSO-D6) δ 7.67 (d, J = 8.2 Hz, 2H), 7.45 (d, J =8.0 Hz, 2H), 4.45 (t, J = 5.4 Hz, 2H), 3.60 (s, 4H), 3.14 (d, J = 5.7 Hz,4H), 2.43 (s, 3H), 1.67 (s, 4H). Compounds 2-7: Compounds 2-6 (5 g, 16.1 mmol) and Mg (powder, 3.47 g, 144.6 mmol) were mixed in MeOH (anhydrous, 60 mL) and stirred overnight at room temperature. LC-MS showed complete conversion, and water (30 mL) was added. A white precipitate formed. The mixture was filtered, and the filtrate was adjusted to pH 6 with HCl (4 M, aqueous solution) and concentrated to dryness to give compounds 2-7 (2.55 g, crude product) as a yellow solid. The crude product was ready for use without further purification.

[0275] C8H 15 Calculated NO2 mass: 157.1; Actual measured: 158.1 [M+H] + , ESI.

[0276] Compounds 2-8: Compound 2-7 (2.55 g, assumed to be 16.2 mmol) was dissolved in dioxane (20 mL), and FmocCl (4.60 g, 17.8 mmol) was slowly added at 0 °C, followed by saturated Na₂CO₃ (20 mL). The reaction was stirred at 30 °C for 3 h. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 97:3) to give a white solid 2-8 (2.75 g, 44.7% yield in two steps).

[0277] C 23 H 25 Calculated NO4 mass: 379.2; Actual measured: 380.2 [M+H] + , ESI.

[0278] Compounds 2-9: Compound 2-8 (2.7 g, 7.1 mmol) and pyridine (20 mL) were mixed in a flask, and then DMTr-Cl (2.4 g, 7.1 mmol) was added in portions. The reaction was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 97:3) to give a yellow solid 2-9 (2.8 g, 58% yield).

[0279] C 44 H 43 The calculated mass of NO6 is 681.3; the actual measured mass is 682.3 [M+H]. + , ESI.

[0280] Compounds 2-10: Compounds 2-9 (2.3 g, 6.8 mmol), piperidine (5 mL), and MeOH (anhydrous, 45 mL) were mixed in a flask and stirred at 30 °C for 3 h. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 95:5) to give a yellow solid 2-10 (0.5 g, yield 33%).

[0281] C 29 H 33 Calculated NO4 mass: 459.2; Actual measured: 460.2 [M+H] + , ESI.

[0282] Compound 2-11: Compounds 1-7 (325 mg, 0.58 mmol), HOBT (105 mg, 0.77 mmol), and EDCI (150 mg, 0.78 mmol) were dissolved in DCM (6 mL), stirred at room temperature for 15 min, and then cooled to 0 °C. DIPEA (211 mg, 1.6 mmol) was added, followed by compounds 2-9 (300 mg, 0.65 mmol), and the mixture was stirred at room temperature for 4 h. LC-MS showed complete conversion. NaHCO3 (10 mL saturated solution) was added. The reaction mixture was extracted with DCM (5 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 95:5) to give crude compound 2-11 (350 mg, 88% purity at 210 nm, 46% yield) as a white solid. Preparative HPLC purification: Crude compound 2-7 (1.8 g, purity 88% at 210 nm) was purified by preparative HPLC (C-18 column, water / ACN, 10%-80% ACN) to obtain compound 2-11 (650 mg, recovery 42%). 54 H 71 N3O 15 Calculated mass: 1001.5; Actual measured mass: 1024.5 [M+Na] + , ESI.

[0283] 1 H NMR (400 MHz, DMSO) δ 7.82 (d, J = 9.2 Hz, 1H), 7.75-7.69 (br,1H), 7.40-7.35 (m, 2H), 7.37-7.21 (m, 7H), 6.95-6.88 (m, 4H), 5.21 (d, J =3.3 Hz, 1H), 5.00-4.90 (m, 1H), 4.66-4.63 (m, 1H), 4.48 (d, J = 8.4 Hz, 1H),4.08-4.00 (br, 4H), 3.91-3.83(m, 1H), 3.73 (s, 6H), 3.72-3.66 (m, 3H), 3.51(s, 1H), 3.43-3.37 (m, 3H), 3.03-2.96 (m, 2H), 2.91 (s, 2H), 2.10 (s, 3H), 2.00-1.76 (m, 17H), 1.49-1.30 (m, 8H), 1.23-1.19 (m, 2H). Compound 2-12: At room temperature, 4,5-dicyanimidazolium (32.0 mg, 0.27 mmol) and 2-cyanoethyl were added to a solution of compound 2-11 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL). N , N , N ′, N ′-Tetraisopropylphosphonic diamide (108 mg, 0.36 mmol) was stirred for 1 h. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH3CN / H2O = 1 / 1, increased to CH3CN / H2O = 1 / 0 over 20 min; detector: UV 254 nm). Compound 2-12 was obtained as a white solid (177 mg, yield 49%).

[0284] C 63 H 88 N5O 16 Calculated mass P: 1201.60; Actual measured mass: 1202.6 [M+H] + , ESI.

[0285] 1 HNMR (600 MHz, CD3CN) δ 7.37-7.34 (m, 2H), 7.24-7.20 (m, 6H), 7.14-7.12 (m, 1H), 6.79-6.77 (m, 4H), 6.57-6.54 (m, 1H), 6.44-6.43 (m, 1H), 5.20(d, J = 6.0 Hz, 1H), 4.93-4.91 (m, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.04-3.83(m, 5H), 3.73-3.39 (m, 16H), 3.04-2.91 (m, 4H), 2.52-2.50 (m, 2H), 2.03-1.75(m, 24H), 1.53-1.33 (m, 9H), 1.21-1.04 (m, 15H). 31 PNMR (242 MHz, CD3CN) δ 147.37, 147.30. Compound 2-13: To a solution of compound 2-11 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL), DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added, followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed complete consumption of the starting material. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), then washed with brine (3 mL x 4), and the organic layer was concentrated to give compound 2-13 as a white solid (85 mg, 97% yield).

[0286] C 58 H 75 N3O 18 Calculated mass: 1101.50; Actual measurement: 1100.4 [MH] - , ESI.

[0287] 1 HNMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.84-7.81 (m, 1H), 7.71-7.69 (m, 1H), 7.37-7.16 (m, 9H), 6.92-6.89 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H),4.97-4.95 (m, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.07-4.01 (m, 6H), 3.89-3.84(m, 1H), 3.73-3.65 (m, 9H), 3.49 (s, 1H), 3.41-3.38 (m, 1H), 3.01-2.95 (m,4H), 2.45-2.44 (m, 4H), 2.09-1.76 (m, 20H), 1.48-1.19 (m, 10H). Solid carrier 2-14: Natural amino-LCAA-CPG (loading value: 75 μmol / g, 1000 Å) was washed with ACN (100 mL x 2), DMF (100 mL x 2), and DCM (100 mL x 2), and then dried overnight under high vacuum. DIPEA (30 mg, 0.23 mmol) was added to a solution containing succinate 2-13 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL), and the reaction mixture was shaken at room temperature for 10 min. Then, natural amino-LCAA-CPG (250 mg, loading 75 μmol / g) was added, and the suspension was shaken at room temperature for 20 h. The mixture was then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was dried under vacuum for 2 h to obtain a solid support (260 mg). The solid support was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL) to cap unreacted amines. The solid support was then washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 2-14 (260 mg). For sample loading calculation, 6.5 mg of dried CPG was added to 25 mL of DCM solution containing 3% DCA. The solution was shaken, and the absorbance was measured at 500 nm. The absorbance value was ensured to be below 1.0 unit to prevent signal saturation. The following formula was then applied: Sample loading (µmol / g) = (Total volume of added DCA (mL)) * (Abs value at 500 nm) * 1000) / (76 * (Number of mg of CPG taken)) Total volume of DCA added (mL) = 25 mL Abs value at 500 nm = 0.7721 The amount of CPG taken was 6.5 mg. Loading sample (µmol / g) = ((25)*(0.7721)*1000) / (76*(6.5)) = 39 µmol / g.

[0288] Method 2: Compound 2-2: Compound 2-1 (11 g, 26.0 mmol) was dissolved in 100 mL anhydrous THF in a 250 mL flask. LiAlH4 (1.97 g, 52 mmol) was added in portions at 0 °C over 15 minutes. The reaction was then stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction was cooled to 0 °C, and water (2 mL) was slowly added, followed by NaOH (10%, 2 mL), and then water (6 mL). The mixture was filtered, and the filtrate was concentrated to dryness to give compound 2-2 (6.5 g, 80% yield) as a white solid. The crude product was ready for use without further purification.

[0289] C 15 H 21 NO4S value: 311.1; Actual measurement: 312.1 [M+H] + , ESI.

[0290] 1 H NMR (400 MHz, DMSO) δ 7.67 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0Hz, 2H), 4.46 (t, J = 5.4 Hz, 2H), 3.60 (s, 4H), 3.31 (d, J = 5.6 Hz, 4H), 2.43 (s, 3H), 1.67 (s, 4H). Compounds 2-3: Compound 2-2 (5 g, 16.1 mmol) and Mg (powder, 3.47 g, 144.6 mmol) were mixed in MeOH (anhydrous, 60 mL) and stirred overnight at room temperature. LC-MS showed complete conversion, and water (30 mL) was added. A white precipitate formed. The mixture was filtered, and the filtrate was adjusted to pH 6 with HCl (4 M aqueous solution) and concentrated to dryness to give compound 2-3 (2.55 g, crude product) as a yellow solid. The crude product was ready for use without further purification.

[0291] C8H 15 Calculated NO2 mass: 157.1; Actual measured: 158.1 [M+H] + , ESI.

[0292] Compounds 2-4: Compounds 2-3 (2.55 g, assumed to be 16.2 mmol) were dissolved in dioxane (20 mL), and FmocCl (4.60 g, 17.8 mmol) was slowly added at 0 °C, followed by saturated Na₂CO₃ (20 mL). The reaction was stirred at 30 °C for 3 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 97:3) to give a white solid 2-4 (2.75 g, 44.7% yield in two steps).

[0293] C 23 H 25 Calculated NO4 mass: 379.2; Actual measured: 380.2 [M+H] + , ESI.

[0294] Compounds 2-5: Compounds 2-4 (2.7 g, 7.1 mmol) and pyridine (20 mL) were mixed in a flask, and then DMTr-Cl (2.4 g, 7.1 mmol) was added in portions. The reaction was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction mixture was extracted with EA (20 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 97:3) to give a yellow solid 2-5 (2.8 g, 58% yield).

[0295] C 44 H 43 The calculated mass of NO6 is 681.3; the actual measured mass is 682.3 [M+H]. + , ESI.

[0296] Compounds 2-6: Compounds 2-5 (2.3 g, 6.8 mmol), piperidine (5 mL), and MeOH (anhydrous, 45 mL) were mixed in a flask and stirred at 30 °C for 3 h. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 95:5) to give yellow solid 2-6 (0.5 g, yield 33%).

[0297] C 29 H 33 Calculated NO4 mass: 459.2; Actual measured: 460.2 [M+H] + , ESI.

[0298] Compounds 2-7: Compounds 1-7 (325 mg, 0.58 mmol), HOBT (105 mg, 0.77 mmol), and EDCI (150 mg, 0.78 mmol) were dissolved in DCM (6 mL), stirred at room temperature for 15 min, and then cooled to 0 °C. DIPEA (211 mg, 1.6 mmol) was added, followed by compound 2-6 (300 mg, 0.65 mmol), and the mixture was stirred at room temperature for 4 h. LC-MS showed complete conversion. NaHCO3 (10 mL saturated solution) was added. The reaction mixture was extracted with DCM (5 mL x 3). The organic phase was concentrated and purified by rapid silica gel column chromatography (DCM:MeOH = 95:5) to give crude compound 2-7 (350 mg, 88% purity at 210 nm, 46% yield) as a white solid. Preparative HPLC purification: Crude compound 2-7 (1.8 g, purity 88% at 210 nm) was purified by preparative HPLC (C-18 column, water / ACN, 10%-80% ACN) to obtain compound 2-7 (650 mg, purity 96% at 210 nm, recovery 42%). 54 H 71 N3O 15 Calculated mass: 1001.5; Actual measured mass: 1024.5 [M+Na] + , ESI.

[0299] 1 H NMR (400 MHz, DMSO) δ 7.82 (d, J = 9.2 Hz, 1H), 7.75-7.69 (br,1H), 7.40-7.35 (m, 2H), 7.37-7.21 (m, 7H), 6.95-6.88 (m, 4H), 5.21 (d, J =3.3 Hz, 1H), 5.00-4.90 (m, 1H), 4.66-4.63 (m, 1H), 4.48 (d, J = 8.4 Hz, 1H),4.08-4.00 (br, 4H), 3.91-3.83(m, 1H), 3.73 (s, 6H), 3.72-3.66 (m, 3H), 3.51(s, 1H), 3.43-3.37 (m, 3H), 3.03-2.96 (m, 2H), 2.91 (s, 2H), 2.10 (s, 3H), 2.00-1.76 (m, 17H), 1.49-1.30 (m, 8H), 1.23-1.19 (m, 2H). Compounds 2-8: At room temperature, DCI (32.0 mg, 0.27 mmol) and CEP[N(iPr)2]2 (108 mg, 0.36 mmol) were added to a solution of compounds 2-7 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL), and the mixture was stirred for 1 h until LCMS showed complete consumption of the SM. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. The solution was then concentrated under reduced pressure, and the residue was purified by MPLC under the following conditions (column: C18 silica gel; mobile phase: CH3CN / H2O = 1 / 1, increased to CH3CN / H2O = 1 / 0 over 20 min; eluted product collected at CH3CN / H2O = 3 / 2; detector: UV 254 nm). Compounds 2-8 were produced as white solids (177 mg, 0.15 mmol, 97.0% purity, 49% yield).

[0300] C 63 H 88 N5O 16 Calculated mass P: 1201.60; Actual measured mass: 1202.6 [M+H] + , ESI.

[0301] 1 H-NMR (600 MHz, CD3CN): δ = 7.37-7.34 (m, 2H), 7.24-7.20 (m, 6H), 7.14-7.12 (m, 1H), 6.79-6.77 (m, 4H), 6.57-6.54 (m, 1H), 3.73-3.39 (m, 16H), 3.04-2.91 (m, 4H), 2.52-2.50 (m,2H), 2.03-1.75 (m, 24H), 1.53-1.33 (m, 9H), 1.21-1.04 (m, 15H); 31 PNMR (242 MHz, CD3CN) δ 147.37, 147.29. Compounds 2-9: To a solution of compounds 2-7 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL), DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added, followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed complete consumption of the starting material. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), then washed with brine (3 mL x 4), and the organic layer was concentrated to give compounds 2-9 as a white solid (85 mg, 0.077 mmol, purity 97%, yield 97%), C 58 H 75 N3O 18 Calculated mass: 1101.50; Actual measurement: 1100.4 [MH] - , ESI.

[0302] 1 H-NMR (600 MHz, DMSO-d6): δ = 12.22 (s, 1H), 7.84-7.81 (m, 1H), 7.71-7.69 (m, 1H), 7.37-7.16 (m, 9H), 6.92-6.89 (m, 4H), 5.21-5.20 (d, J =6.0 Hz, 1H), 4.97-4.95 (m, 1H), 4.49-4.47 (d, J = 12.0 Hz, 1H), 4.07-4.01 (m,6H), 3.89-3.84 (m, 1H), 3.73-3.65 (m, 9H), 3.49 (s, 1H), 3.41-3.38 (m, 1H), 3.01-2.95 (m, 4H), 2.45-2.44 (m, 4H), 2.09-1.76 (m, 20H), 1.48-1.19 (m, 10H). Solid carrier 2-10: Natural amino-LCAA-CPG (loading value: 75 μmol / g, 1000 Å) was washed with ACN (100 mL x 2), DMF (100 mL x 2), and DCM (100 mL x 2), and then dried overnight under high vacuum.

[0303] DIPEA (30 mg, 0.23 mmol) was added to a solution of succinate 2-9 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL). The reaction mixture was shaken at room temperature for 10 min, followed by the addition of natural amino-LCAA-CPG (250 mg, 75 μmol / g loading). The suspension was shaken at room temperature for 20 h, then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 2 h to obtain a solid support (260 mg). The solid support was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL) to cap unreacted amines. The solid support was then washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 2-10 (260 mg). For loading calculation, 6.5 mg of dried CPG was added to 25 mL of DCM solution containing 3% DCA. The solution was shaken, and the absorbance was measured at 500 nm. The absorbance value was ensured to be below 1.0 unit to prevent signal saturation. The following formula was then applied: Loading (µmol / g) = (Total volume of added DCA (mL)) * (Abs value at 500 nm) * 1000) / (76 * (Number of mg of CPG taken)) Total volume of DCA added (mL) = 25 mL Abs value at 500 nm = 0.7721 The amount of CPG taken was 6.5 mg. Loading sample (µmol / g) = ((25)*(0.7721)*1000) / (76*(6.5)) = 39 µmol / g. Compound 3-2: Ethyl 2-cyanoethyl (47.82 g, 422.71 mmol) was added dropwise to a solution of compound 3-1 (40 g, 211.36 mmol) in NH3 / MeOH (7 M, 192 mL), and the mixture was stirred overnight. LC-MS showed complete conversion, and the reaction mixture was filtered. The filter cake was ground in PE (100 mL) and filtered. The filter cake was dried to give compound 3-2 as a solid (41 g, 44% yield).

[0304] The calculated mass of C18H18N4O2 is 322.1; the actual measured mass is 323.1 [M+H]. + , ESI.

[0305] Compound 3-3: Compound 3-2 (80 g, 248.17 mmol) was dissolved in sulfuric acid solution (concentrated H₂SO₄ / water = 1:1, v / v, 160 mL) and stirred at 120 °C for 5 h. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 9-10 by adding NaOH (30% aqueous solution) and then readjusted to pH 4-5 by adding 1 M HCl solution. The reaction mixture was then filtered, and the filtrate was concentrated to dryness to give crude compound 3-3 (72 g, 95% yield). The crude product was ready for use without further purification.

[0306] The calculated mass of C16H21NO4 is 291.2; the actual measured mass is 292.3 [M+H]. + , ESI.

[0307] Compounds 3-4: Compound 3-3 (72.3 g crude product) was stirred at 85 °C for 4 h in MeOH (1600 mL) and H2SO4 (160 mL, concentrated H2SO4 / water = 1:1, v / v). LC-MS showed complete conversion. The reaction mixture was filtered and concentrated to dryness, and purified by silica gel column chromatography (0-25% EA in PE) to give compound 3-4 (62.5 g, 78% yield in 2 steps).

[0308] C 18 H 25 Calculated NO4 mass: 319.2; Actual measured: 320.3 [M+H] + , ESI.

[0309] Compounds 3-5: LiAlH4 (14.3 g, 375.1 mmol) was added fractionally to a THF (295 mL) solution of compounds 3-4 (40 g, 125.2 mmol) at 0 °C, and the mixture was stirred for 1 hour. LC-MS showed complete conversion. Ethyl acetate (100 mL) was added to the reaction mixture, and the pH was adjusted to 9-10 with 10% NaOH (aqueous solution). The mixture was then filtered and washed with ethyl acetate (50 mL x 2). The combined filtrates were concentrated to dryness to give crude compounds 3-5 (23 g, 83% yield). The crude product was ready for use in the next step without further purification.

[0310] The calculated mass of C16H25NO2 is 263.2; the actual measured mass is 264.3 [M+H].+ , ESI.

[0311] Compounds 3-6: Compounds 3-5 (20 g, 75.94 mmol), Boc₂O (16.57 g, 75.94 mmol), and Pd / C (10% carbon-supported palladium, wet, ~55% water) were placed in MeOH (150 mL) and stirred overnight at room temperature under H₂ at 1 atm. LC-MS showed complete conversion. The reaction mixture was filtered and concentrated to give oily compounds 3-6 (20 g, 96% yield), which were ready for use without further purification.

[0312] C 14 H 27 The calculated mass of NO4 is 273.2; the actual measured mass is 274.1 [M+H]. + , ESI.

[0313] Compounds 3-7: MsCl (72.91 g, 636.51 mmol) was added to a solution of compounds 3-6 (58 g, 212.17 mmol) in triethylamine (85.88 g, 848.67 mmol, 118.37 mL) and DCM (1.16 L). The reaction mixture was stirred overnight at room temperature. LC-MS showed complete conversion (by EtOH quenching and diether detection). The reaction mixture was diluted with DCM (1 L), washed with 10% citric acid, then washed with NaHCO3 (saturated aqueous solution), and dried to give compounds 3-7 as an oil (87 g, 95% yield), which was used directly for the next step without further purification.

[0314] C 16 H 31 The calculated mass of NO8S2 is 429.2; the actual measured mass is 430.3 [M+H]. + , ESI.

[0315] Compounds 3-8: Under nitrogen atmosphere, NaH (60% mineral oil, 3.72 g, 93.12 mmol) was suspended in anhydrous DMF (60 mL), and diisopropyl malonate (8.76 g, 46.56 mmol) was added. The reaction mixture was stirred at room temperature for 30 min to form a clear solution. KI (1.55 g, 9.31 mmol) was added, followed by compounds 3-7 (20 g, 46.56 mmol). The reaction mixture was stirred at 70 °C for 30 min, then at 140 °C for another 30 min. LC-MS showed complete conversion. The reaction was cooled to room temperature, diluted with ethyl acetate (300 mL), and washed with citric acid (10% aqueous solution, 200 mL x 2) and NaHCO3 (saturated aqueous solution, 200 mL). The organic phase was concentrated and purified by silica gel column chromatography (0-10% EA solution in PE) to give compounds 3-8 as a colorless oil (3.55 g, 18% yield).

[0316] C 23 H 39 Calculated mass of NO6: 425.3; Actual measured mass: 426.2 [M+H] + , ESI.

[0317] 1 H NMR (400 MHz, DMSO-d6) δ 4.96-4.90 (m, 2H), 3.27 (br, 4H), 1.86(t, J = 10.8 Hz, 4H), 1.38 (br, 13H), 1.28 (s, 4H), 1.17-1.16 (m, 12H). Compounds 3-9: LiBH4 (172 mL, 1 M, in THF) was added to compounds 3-8 (7.5 g, 17.62 mmol) at 0 °C, and the mixture was stirred overnight. LC-MS showed complete conversion. Ethyl acetate (10 mL) was added to the reaction mixture, followed by 20 mL of NaOH (10% aqueous solution) and filtration. The filtrate was diluted with water (200 mL), extracted with ethyl acetate (50 mL x 3), and concentrated to give compounds 3-9 (6.35 g, 98% yield) as a colorless oil, which could be used directly for the next step without further purification.

[0318] C 17 H 31 Calculated NO4 mass: 313.2; Actual measured: 314.2 [M+H] + , ESI.

[0319] Compounds 3-10: Compounds 3-9 (6.35 g, 20.26 mmol) were mixed with HCl (63.5 mL, 4 M in dioxane) and stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to dryness and azeotropically dried over toluene to give compound 3-10 (7 g, 99% yield), which could be used directly for the next step without further purification.

[0320] C 12 H 23 The calculated mass of NO2 (free bases) is 213.2; the measured mass is 214.2 [M+H]. + , ESI.

[0321] 1 H NMR (400 MHz, D2O) δ 3.39 (br, 4H), 3.18 (t, J = 11.6 Hz, 4H), 1.59(t, J = 12.0 Hz, 4H), 1.36-1.33 (m, 4H), 1.25 (t, J = 12.4 Hz, 4H). Compound 3-11: A solution of compounds 3-10 (5 g, 20.02 mmol), DIEA (6.47 g, 50.04 mmol, 8.72 mL), HOBT (3.25 g, 24.02 mmol), and EDCI (4.60 g, 24.02 mmol) in DCM (150 mL) was stirred at 0 °C for 30 min, then Fmoc-6-aminocaproic acid (5.66 g, 16.01 mmol) was added, and the mixture was stirred at room temperature for 1 h. LC-MS showed complete conversion. The reaction mixture was diluted with another DCM (100 mL), washed with citric acid (10% aqueous solution, 200 mL), NaHCO3 (saturated aqueous solution, 200 mL) and water (200 mL), dried over Na2SO4, and concentrated to give a crude product, which was purified by silica gel column chromatography (0-10% EA solution in PE) to give a colloidal compound 3-11 (3.65 g, LC-MS purity 92%, yield 33%).

[0322] C 33 H 44 The calculated mass of N₂O₅ is 548.3; the actual measured mass is 549.3 [M+H]. + , ESI.

[0323] 1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 7.5 Hz, 2H), 7.84 (d, J =7.5 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.34 (t, J = 7.3 Hz, 2H), 6.65 (t, J =5.3 Hz, 1H), 6.28 (s, 2H), 4.36-4.18 (m, 3H), 3.36 (dt, J = 19.9, 5.2 Hz,4H), 3.24 (s, 4H), 2.92-2.87 (m, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.49-1.26 (m,18H). Compound 3-12: A solution of compound 3-11 (2.55 g, 4.65 mmol), 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (1.57 g, 4.65 mmol), DIEA (600.6 mg, 4.65 mmol), and DMAP (56.8 mg, 0.47 mmol) in DCM (51 mL) was stirred overnight at room temperature. LC-MS showed partial conversion. The reaction mixture was then concentrated and purified by silica gel column chromatography (eluting impurities with 10–40% EA in PE, followed by elution with 5–10% MeOH and 0.1% NH3•H2O in DCM) to give compound 3-12 (2.75 g, 69% yield).

[0324] 1 H NMR (400 MHz, DMSO-d6) δ 7.88-7.87 (m, 2H), 7.69-7.68 (m, 2H), 7.41-7.37 (m, 4H), 7.33-7.25 (m, 9H), 6.89-6.80 (m, 5H), 4.41 (t, J = 5.0 Hz, 1H), 4.28 (d, J = 6.8 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 3.72 (s, 8H), 3.40-3.39 (m, 2H), 3.32-3.27 (m, 4H), 2.96 (q,J = 6.3 Hz, 2H), 2.22 (t, J = 7.9Hz, 2H), 1.48-0.86 (m, 18H). Compound 3-13: To a solution of compound 3-12 (1.7 g, 2.00 mmol) in MeOH (51 mL), piperidine (5.1 mL) was added, and the mixture was stirred at room temperature for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and dried over xylene azeotropic drying. The residue was purified by silica gel column chromatography (0-10% MeOH and 0.1% NH3•H2O in DCM) to give a gel-like compound 3-13 (1.1 g, 87% yield).

[0325] C 39 H 52 The calculated mass of N₂O₅ is 628.4; the actual measured mass is 629.4 [M+H]. + , ESI.

[0326] Compound 3-14: A solution of compounds 3-13 (0.94 g, 1.49 mmol), DIEA (482.98 mg, 3.74 mmol), HOBT (242.38 mg, 1.79 mmol), and EDCI (343.87 mg, 1.79 mmol) in DCM (29 mL) was stirred at 0 °C, and then compound 1-4 (936.36 mg, 2.09 mmol) was added. The mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was diluted with another 20 mL of DCM, washed with NaHCO3 (50 mL saturated aqueous solution) and water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to give compound 3-14 (700 mg, 44% yield).

[0327] C 58 H 79 N3O 15 Calculated mass: 1057.6; Actual measured mass: 1080.5 [M+Na] + , ESI.

[0328] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 9.2 Hz, 1H), 7.69 (t, J=5.5 Hz, 1H), 7.40-7.38 (m, 2H), 7.32-7.19 (m, 7H), 6.89-6.87 (m, 4H), 5.21(d, J = 3.3 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.40 (t, J = 5.0 Hz, 1H), 4.02 (s, 3H), 3.91-3.83 (m, 1H), 3.73-3.68 (m,7 H), 3.43-3.39 (m, 3H), 3.32-3.27 (m, 2H), 2.99 (q, J = 6.6 Hz, 2H), 2.87(s, 2H), 2.22 (t, J = 7.3 Hz, 2H), 2.10 (s, 3H), 2.03 (t, J = 7.0 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.53-1.03 (m, 20H), 0.90-0.83 (m, 2H). Compound 3-15: At room temperature, 4,5-dicyanimidazolium (96.0 mg, 0.81 mmol) and 2-cyanoethyl were added to a solution of compound 3-14 (670.0 mg, 0.61 mmol) in anhydrous DCM (3.0 mL). N , N , N ′, N ′-Tetraisopropylphosphonic diamide (324 mg, 1.08 mmol) was stirred for 1 hour. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH3CN / H2O = 1 / 1, increased to CH3CN / H2O = 1 / 0 over 20 min; detector: UV 254 nm). Compounds 3-15 were produced as a white solid (600 mg, yield 83%).

[0329] C 67 H 96 N5O 16Calculated mass of P: 1257.66; Actual measured mass: 1204.5 [M-CH2CH2CN] - , ESI.

[0330] 1 HNMR (600 MHz, DMSO- d 6) δ 7.81-7.67 (m, 2H), 7.40-7.19 (m, 9H), 6.88-6.86 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.94 (m, 1H), 4.49 (d, J = 6.0Hz, 1H), 4.04-3.83 (m, 4H), 3.73-3.32 (m, 15H), 3.01-2.89 (m, 4H), 2.73-2.70(m, 2H), 2.24-2.20 (m, 2H), 2.11-1.76 (m, 15H), 1.49-0.91 (m, 34H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.21, 146.19. Compound 4-2: Potassium carbonate (8.10 g, 58.61 mmol) was added to a solution of MeOH (80 mL), H₂O (60 mL), formaldehyde (3.52 g, 117.22 mmol), and compound 4-1 (25 g, 117.22 mmol) at 0 °C. The reaction mixture was stirred overnight at 0 °C. The reaction mixture was concentrated to 70% of its volume, and the residue was extracted three times with ethyl acetate. The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, and then concentrated to give crude product 4-2 (27 g, 95% yield), which could be used in the next step without further purification.

[0331] C 12 H 21 The calculated mass of NO4 is 243.2; the actual measured mass is 244.1 [M+H]. + , ESI.

[0332] Compound 4-3: A solution of compound 4-2 (35 g, 143.86 mmol) in methanol (28 ml) was added to sodium borohydride (11.97 g, 316.48 mmol) over approximately 5 minutes at 0 °C. The reaction mixture was stirred at 0 °C, then heated to room temperature and stirred for 5 hours. The reaction was quenched with a saturated ammonium chloride solution, then concentrated, extracted three times with ethyl acetate (EA), washed with brine, and recrystallized from EA:PE = 1:3 to give compound 4-3 (19.6 g, 54% yield).

[0333] C 12 H 23 Calculated NO4 mass: 245.2; Actual measured: 146.1 [M+H-Boc] + , ESI.

[0334] Compound 4-4: Compound 4-3 (30 g, 122.29 mmol), triethylamine (30.94 g, 305.73 mmol, 42.64 mL), and DCM (300 mL) were mixed and cooled to -30 °C. MsCl (35.02 g, 305.73 mmol) was then slowly added. After stirring at room temperature for 1.5 hours, the reaction mixture was extracted with 400 mL of water, washed with 100 mL of 10% citric acid aqueous solution, and dried over anhydrous sodium sulfate. The residue was concentrated and purified by silica gel column chromatography to give compound 4-4 as a pale yellow oily liquid (40 g, 81% yield).

[0335] C 14 H 27 The calculated mass of NO8S2 is 401.1; the actual measured mass is 302.1 [M+H-Boc] + , ESI.

[0336] Compounds 4-5: Diisopropyl malonate (14.06 g, 74.72 mmol) was slowly added to a mixture of t-BuONa (9.57 g, 99.63 mmol) and DMA (50 mL) under nitrogen atmosphere. After stirring for 20 min, compound 4-4 (20 g, 49.81 mmol) and KI (4.13 g, 24.91 mmol) were added. The reaction mixture was heated to 140 °C and stirred overnight. The reaction was quenched by the addition of saturated ammonium chloride and extracted with ethyl acetate. After concentration, the residue was purified by silica gel column chromatography (PE:EA) to give compound 4-5 (6 g, 30% yield).

[0337] C 21 H 35Calculated mass of NO6: 397.3; Actual measured mass: 398.2 [M+H] + , ESI.

[0338] 1 H NMR (400 MHz, DMSO) δ 4.98-4.90 (m, 2H), 3.22-3.20 (m, 4H), 2.25(s, 4H), 1.42 (t, J = 5.6 Hz, 4H), 1.38 (s, 9H), 1.17 (d, J = 6.0 Hz, 12H). Compounds 4-6: At 0 °C, lithium borohydride (10.69 g, 490.56 mmol) was added to a solution of compound 4-5 (9.75 g, 24.53 mmol) in anhydrous THF (200 mL). The reaction mixture was slowly heated to room temperature and stirred overnight. Then, ethyl acetate (200 mL) was added to the reaction mixture, and the mixture was extracted with water (150 mL x 3), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give crude product 4-6 (6.52 g, 93% yield), which could be used for the next step without further purification.

[0339] C 15 H 27 The calculated mass of NO4 is 285.2; the actual measured mass is 186.2 [M+H-Boc] + , ESI.

[0340] Compounds 4-7: HCl (4M in 1,4-dioxane, 21.52 mL) was added to a solution of product 4-6 (7.52 g, 26.35 mmol) in DCM (100 mL) at 0 °C over 15 minutes. The reaction mixture was then heated to room temperature and stirred for 3 hours. The reaction mixture was filtered, and the filter cake was washed with dichloromethane (x3) to give crude product 4-7 (3.7 g, 76% yield).

[0341] C 10 H 19 NO2 calculated (free bases): 185.1 m / z; measured: 186.2 [M+H] + , ESI.

[0342] Compounds 4-8: A mixture of Fmoc-6-aminocaproic acid (1.91 g, 5.41 mmol), 1-hydroxybenzotriazole (HOBT, 609.41 mg, 4.51 mmol), EDCI (864.59 mg, 4.51 mmol), and DIEA (582.89 mg, 4.51 mmol, 785.56 μL) in 10 mL DCM solution was stirred at room temperature for 30 minutes. Then, a solution of compound 4-7 (1 g, 4.51 mmol) in DCM was added, and the mixture was stirred for 4 hours. The reaction mixture was washed with saturated sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by silica gel column chromatography to give compound 4-8 (1.35 g, 58% yield).

[0343] C 31 H 40 The calculated mass of N₂O₅ is 520.3; the actual measured mass is 521.2 [M+H]. + , ESI.

[0344] 1 H NMR (400 MHz, DMSO) δ 7.88 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.4Hz, 2H), 7.39 (d, J = 7.5 Hz, 4H), 7.34-7.18 (m, 10H), 6.89 (d, J = 8.8 Hz, 4H), 4.64 (t, J = 4.7 Hz, 1H), 4.28 (d, J = 6.8 Hz, 2H), 4.20 (t, J = 6.8 Hz,1H), 3.73 (s, 6H), 3.44 (d, J = 5.0 Hz, 2H), 3.30-3.10 (m, 4H), 3.01 (s, 2H), 3.00-2.90 (m, 2H), 2.21 (t, J = 7.2 Hz, 2H), 1.56 (d, J = 11.9 Hz, 2H), 1.46-1.30 (m, 8H), 1.21-1.00 (m, 4H). Compounds 4-9: The mixture of DMTr-Cl (488.07 mg, 1.44 mmol) and compounds 4-8 (0.75 g, 1.44 mmol) in a pyridine (6 mL) solution was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum. The residue was redissolved in ethyl acetate containing 1% pyridine and dried over alumina for column chromatography purification on alumina (0-10% MeOH, 0.1% TEA in DCM) to give compounds 4-9 (0.39 g, 32.9% yield).

[0345] 1 H NMR (400 MHz, DMSO) δ 7.39 (d, J = 7.3 Hz, 2H), 7.34-7.18 (m, 7H), 6.89 (d, J = 8.8 Hz, 4H), 4.63 (s, 1H), 3.73 (s, 6H), 3.43 (s, 2H), 3.30-3.18(m, 6H), 3.05 (s, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.57 (d, J = 12.1 Hz, 2H),1.47-1.08 (m, 12H). Compounds 4-10: Compound 4-9 (1.4 g, 1.70 mmol) was dissolved in MeOH (40 mL), and then piperidine (4 mL) was added. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum. Piperidine residues were further removed by azeotropic drying with xylene. The residues were then purified by silica gel column chromatography (DCM:MeOH = 10:1, methanol containing 10% ammonia) to give compound 4-10 (1 g, 97% yield).

[0346] C 37 H 48 The calculated mass of N₂O₅ is 600.4; the actual measured mass is 601.4 [M+H]. + , ESI.

[0347] 1 H NMR (400 MHz, DMSO-d6) δ 7.40-7.39 (m, 2H), 7.33-7.21 (m, 7H),6.89 (d, J =8.8 Hz, 4H), 4.63 (s, 1HH), 3.73 (s, 6H), 3.44-3.16 (m, 8H), 3.01-3.00 (m, 2H), 2.24-2.21 (m, 2H), 1.57 (d, J = 12.2 Hz, 2H), 1.45-1.33(m, 7H), 1.27-1.21 (m, 4H), 1.09 (s, 1H). Compound 4-11: A mixture of compounds 1-4 (959.23 mg, 2.14 mmol), HOBt (289.68 mg, 2.14 mmol), EDCI (410.98 mg, 2.14 mmol), and DIEA (395.82 mg, 3.06 mmol, 533.45 μL) in DCM (10 mL) was stirred for 20 minutes. This mixture was then added to a solution of compound 4-10 (920 mg, 1.53 mmol) in DCM (5 mL), and the mixture was stirred overnight at room temperature. The reaction was quenched with water, the organic layer was separated, and concentrated. The residue was purified by preparative HPLC to give compound 4-11 (1 g, 63% yield).

[0348] C 56 H 75 N3O 15 Calculated mass: 1029.5; Actual measured mass: 1052.3 [M+Na] + , ESI.

[0349] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 9.2 Hz, 1H), 7.79 (t, J =5.0 Hz, 1H), 7.40-7.19 (m, 9H), 6.90-6.88 (m, 2H), 5.21 (d, J = 3.2 Hz, 1H), 4.97 (dd, J = 11.2, 3.3 Hz, 1H), 4.62 (t, J = 4.9 Hz, 1H), 4.48 (d, J = 8.4Hz, 1H), 4.02 (s, 3H), 3.87 (dd, J= 20.4, 9.6Hz, 1H), 3.97-3.67 (m, 7H), 3.45-3.38 (m, 3H), 3.30-3.13 (m, 4H), 3.05-2.96 (m, 4H), 2.22 (t, J = 7.3 Hz, 2H), 2.10 (s, 3H), 2.04-1.99 (m, 5H), 1.89 (s, 3H), 1.77 (s, 3H), 1.59-1.21 (m, 18H). Compound 4-12: To a solution of compound 4-11 (750.0 mg, 0.75 mmol) in anhydrous DCM (7.0 mL), 4,5-dicyanimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphonium diamide (270 mg, 0.90 mmol) were added and stirred for 1 h. LC-MS showed complete consumption of the starting material. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na₂SO₄. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH₃CN / H₂O = 1 / 1 increased to CH₃CN / H₂O = 1 / 0 over 20 min; detector: UV 254 nm). 4-12 was produced as a white solid (500 mg, 53% yield).

[0350] C 65 H 92 N5O 16 The calculated mass of P is 1229.63; the measured mass is 1129.0 [MN(CH(CH3)2)2] + , ESI.

[0351] 1 HNMR (600 MHz, DMSO- d 6): δ 7.44-7.18 (m, 9H), 6.85-6.83 (m, 4H), 6.52-6.44 (m, 2H), 5.26 (d, J = 6.0 Hz, 1H), 4.99-4.96 (m, 1H), 4.49 (d, J=6.0 Hz, 1H), 4.13-3.89 (m, 4H), 3.79-3.55 (m, 13H), 3.44-3.48 (m, 1H), 3.35-3.07 (m, 8H), 2.58-2.56 (m, 2H), 2.24-1.89 (m, 16H), 1.62-1.11 (m, 31H). 31 PNMR (242 MHz, DMSO- d 6) δ 147.17, 147.16. Compound 4-13: DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added to a solution of compound 4-11 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours. LCMS showed that the starting material had been completely consumed. The reaction mixture was then diluted with DCM, washed with water (3 mL x 4), followed by washing with brine (3 mL x 4), and the organic layer was concentrated to give compound 4-13 as a white solid (85 mg, 97% yield).

[0352] C 60 H 79 N3O 18 Calculated mass: 1129.54; Actual measured mass: 1128.6 [MH] - , ESI.

[0353] 1 HNMR (600 MHz, DMSO- d 6) δ 12.22 (s, 1H), 7.84-7.81 (m, 1H), 7.71-7.69(m, 1H), 7.37-7.16 (m, 9H), 6.92-6.89 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H),4.97-4.95 (m, 1H), 4.48 (d, J= 12.0 Hz, 1H), 4.12-4.01 (m, 3H), 3.89-3.84(m, 1H), 3.73-3.65 (m, 7H), 3.41-3.38 (m, 2H), 3.28-3.15 (m, 4H), 3.15-2.98(m, 4H), 2.46-2.44 (m, 4H), 2.22-2.20 (m, 2H), 2.09-1.76 (m, 14H), 1.65-1.60(m, 2H), 1.48-1.06 (m, 19H). Compound 4-14: DIPEA (30 mg, 0.23 mmol) was added to a solution of compound 4-13 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL). The reaction mixture was shaken at room temperature for 10 min, and then natural amino-lcaa-CPG (250 mg, 75 μmol / g) was added. The suspension was shaken at room temperature for 20 h, then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 2 h to obtain a solid support (260 mg). The mixture was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL). Unreacted amines on the support were capped and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 4-14 (260 mg). For sample loading calculation, 5.97 mg of dried sample CPG was added to 25 mL of 3% DCA in DCM. The solution was shaken, and the UV absorbance was measured at 500 nm. The absorbance value was ensured to be below 1.0 unit to prevent signal saturation. The following formula was then applied: Sample loading (µmol / g) = (Total volume of DCA added (mL)) * (Abs value at 500 nm) * 1000) / (76 * (number of mg of CPG taken)) Total volume of DCA added (mL) = 25 mL Abs value at 500 nm = 0.6689 The amount of Mg in the CPG sample was 5.97 mg. Loading sample (µmol / g) = ((25)*(0.6689)*1000) / 76*(5.97) = 36.85 μmol / g. Compound 5-2: Compound 5-1 (10 g, 41.78 mmol) was added to a solution of methoxymethyl (triphenyl)phosphorus chloride (12.89 g, 37.61 mmol) and KOtBu (4.69 g, 41.79 mmol) in THF (150 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred overnight at 25 °C. TLC showed complete conversion. The reaction was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with ethyl acetate (EA). The organic phase was concentrated and purified by rapid column chromatography (0-10% EA solution in PE) to give compound 5-2 (2.92 g, 37% yield) as an oil.

[0354] C 15 H 25 Calculated mass of NO3: 267.2; Actual measured mass: 268.2 [M+H] + , ESI.

[0355] Compound 5-3: A solution of 2,2,2-trichloroacetic acid (6.31 g, 38.64 mmol) in water (70 mL) was added to a mixture of compound 5-2 (8.2 g, 30.67 mmol) in MeCN (220 mL), and the mixture was stirred overnight. Complete conversion was achieved by TLC. The reaction was then quenched by adjusting the pH to 7-8 with NaHCO3 (saturated aqueous solution), and extracted with ethyl acetate. The organic phase was concentrated to give compound 5-3 (7.8 g) as an oil. The crude product was used directly in the next step without further purification.

[0356] C 14 H 23 The calculated mass of NO3 is 253.2; the actual measured mass is 154.2 [M+H-Boc] + , ESI.

[0357] Compound 5-4: A mixture of compound 5-3 (1.35 g, 5.33 mmol) and NaOH (21.32 mg, 532.89 μmol, 29.38 μL) in MeOH (30 mL) was added to formaldehyde solution (454.47 mg, 15.13 mmol, 37% aqueous solution, 0.91 g). The reaction mixture was stirred at 25 °C for 36 hours. The reaction mixture was concentrated to remove most of the methanol and extracted with ethyl acetate. The organic phase was concentrated and purified by column chromatography (0-5% MeOH solution in DCM) to give compound 5-4 as a solid (1.5 g, 96% yield in two steps).

[0358] C 15 H 25 The calculated mass of NO4 is 283.2; the actual measured mass is 184.2 [M+H-Boc] + , ESI.

[0359] Compound 5-5: Sodium borohydride (866.50 mg, 22.90 mmol) was added to a solution of compound 5-4 (2.95 g, 10.41 mmol) in MeOH (90 mL). The reaction mixture was stirred for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 with citric acid (10% aqueous solution), then concentrated to dryness. The residue was extracted with DCM and filtered. The filtrate was concentrated to give compound 5-5 (2.95 g, 73% yield), which was used directly for the next step without further purification.

[0360] C 15 H 27 The calculated mass of NO4 is 285.2; the actual measured mass is 271.2 [M+ACN+H-tBu]. + , ESI.

[0361] Compounds 5-6: To compound 5-5 (4 g, 14.02 mmol), HCl (4 M solution in dioxane, 63.5 mL) was added. The reaction mixture was stirred for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to give compound 5-6 (3.5 g), which was used directly for the next step without further purification.

[0362] C 10 H 19 The calculated mass of NO2 (free bases) is 185.1; the measured mass is 186.1 [M+H]. + , ESI.

[0363] Compounds 5-7: A solution of Fmoc-6-aminohexanoic acid (3.19 g, 9.02 mmol), DIEA (2.91 g, 22.55 mmol, 3.93 mL), HOBT (1.46 g, 10.82 mmol), and EDCI (2.08 g, 10.82 mmol) in DCM (12 mL) was stirred at 0 °C for 30 min, and then compound 5-6 (2 g, 9.02 mmol) was added. The reaction mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (12 mL) and washed with citric acid (10% aqueous solution, 20 mL) and water. The organic phase was concentrated and purified by silica gel column chromatography (0-5% MeOH solution in DCM) to give compound 5-7 (3.5 g, 34% yield).

[0364] C 31 H 40 The calculated mass of N₂O₅ is 520.3; the actual measured mass is 521.2 [M+H]. + , ESI.

[0365] 1 H NMR (400 MHz, DMSO-d6) δ 7.91-7.68 (m, 4H), 7.44-7.28 (m, 5H), 4.32-4.28 (m, 4H), 4.20 (t, J = 6.6 Hz, 1H), 3.69 (s, 2H), 3.43 (s, 2H), 3.22(t, J = 5.6 Hz, 4H), 2.02-1.98 (m, 3H), 1.55-1.52 (t, J = 6.6 Hz, 4H), 2.26-2.23 (m, 4H), 1.24-1.16 (m, 8H). Compounds 5-8: DMTrCl (1.43 g, 4.23 mmol) was added to a solution of compounds 5-7 (2.2 g, 4.23 mmol), DMAP (51.6 mg, 0.423 mmol), and DIEA (1.09 g, 8.46 mmol) in DCM (66 mL). The reaction mixture was stirred overnight at room temperature. Partial conversion was observed by LC-MS. The reaction mixture was filtered through an alumina pad and washed with DCM (50 mL). The filtrate was concentrated and purified by silica gel column chromatography (10–40% EA in 0.1% ammonia in PE, then 0–10% MeOH in 0.1% ammonia in DCM) to give compounds 5-8 (3.0 g, 86% yield).

[0366] C 52 H 58 The calculated mass of N₂O₇ is 822.4 g; the actual measured mass is 845.3 g [M + Na]. + , ESI.

[0367] 1H NMR (400 MHz, DMSO-d6) δ 7.90-7.67 (m, 4H), 7.42-7.39 (m, 4H), 7.41-7.24 (m, 14H), 6.89-6.87 (m, 4H), 4.44 (t, J = 7.6 Hz, 1H), 4.24-4.19(m, 1H), 4.06-4.01 (m, 1H), 3.74-3.73 (m, 6H), 3.65-3.59 (m, 2H), 3.42-3.39(m, 3H), 3.32 (br, 1H), 3.00-2.83 (m, 4H), 1.99-1.96 (m, 4H), 1.45-1.16 (m,16H). Compounds 5-9: To a solution of compounds 5-8 (3 g, 3.65 mmol) in MeOH (90 mL), piperidine (9 mL) was added. The reaction mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was concentrated and then azeotropically distilled with xylene. The residue was purified by silica gel column chromatography (0-10% MeOH and 1% TEA in DCM) to give compounds 5-9 (1.1 g, 91% yield).

[0368] C 37 H 48 The calculated mass of N₂O₅ is 600.4; the actual measured mass is 601.3 [M+H]. + , ESI.

[0369] 1 H NMR (400 MHz, DMSO-d6) δ 7.40-7.21 (m, 9H), 6.89 (d, J = 7.2 Hz, 4H), 3.74 (s, 6H), 3.67-3.61 (m, 2H), 3.39-3.33 (m, 2H), 2.86-2.62 (m, 4H), 1.99 (t, J = 7.4 Hz, 2H), 1.51-1.21 (m, 16H). Compounds 5-10: A solution of compounds 1-4 (2.09 g, 4.66 mmol), DIEA (1.08 g, 8.32 mmol, 1.45 mL), HOBT (539.77 mg, 3.99 mmol), and EDCI (765.80 mg, 3.99 mmol) in DCM (60 mL) was stirred at 0 °C for 30 min, and then compound 5-9 (2 g, 3.33 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (C18, water / ACN) to give compound 5-10 (1.0 g, 15% yield).

[0370] C 56 H 75 N3O 15 Calculated mass: 1029.5; Actual measured mass: 1052.3 [M+Na] + , ESI.

[0371] 1 H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J = 9.2 Hz, 1H), 7.71 (t, J =5.2 Hz, 1H), 7.40-7.38 (m, 2H), 7.32-7.19 (m, 5H), 6.90-9.87 (m, 2H), 5.22(d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.49 (d, J = 8.5 Hz, 1H), 4.42 (t, J= 5.0 Hz, 1H), 4.03 (s, 2H), 3.91-3.84 (m, 2H), 3.74 (s, 6H), 3.67-3.61 (m, 1H), 3.44-3.37 (m, 4H), 3.00 (dd, J = 12.7, 6.6 Hz, 2H), 2.85(d, J = 9.6 Hz, 2H), 2.11 (s, 3H), 2.05-1.96 (m, 7H), 1.90 (s, 3H), 1.78 (s,3H), 1.52-1.18 (m, 18H). Compound 5-11: To a solution of compound 5-10 (690.0 mg, 0.66 mmol) in anhydrous DCM (3.0 mL), 4,5-dicyanimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphonium diamide (324 mg, 1.08 mmol) were added and stirred for 1 h. LC-MS showed complete consumption of the starting material. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na₂SO₄. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH₃CN / H₂O = 1 / 1 increased to CH₃CN / H₂O = 1 / 0 over 20 min; detector: UV 254 nm). Compound 5-11 was produced as a white solid (460 mg, yield 56%).

[0372] C 65 H 92 N5O 16 Calculated mass P: 1229.63; Actual measured mass: 1230.3 [M+H] + , ESI.

[0373] 1 HNMR (600 MHz, DMSO- d 6) δ 7.81-7.67 (m, 2H), 7.40-7.19 (m, 9H), 6.88-6.86 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.94 (m, 1H), 4.49 (d, J= 6.0Hz, 1H), 4.04-3.83 (m, 4H), 3.73-3.32 (m, 18H), 3.01-2.83 (m, 4H), 2.72-2.69(m, 2H), 2.10-1.76 (m, 16H), 1.52-1.04 (m, 30H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.33, 146.26. Compound 6-2: Compound 6-1 (20 g, 99.88 mmol) was dissolved in THF (200 mL). NaH (4.79 g, 119.86 mmol, 60% purity) was added to the reaction mixture, and the mixture was stirred at 0 °C for 30 min. 2,3-Dibromopropene (20.96 g, 104.88 mmol) was slowly added to the reaction mixture, and the reaction was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NH4Cl solution (100 mL), extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography to give compound 6-2 as a colorless oil (31 g, 97% yield).

[0374] C 13 H 19 The calculated mass of BrO4 is 318.0; the actual measured mass is 319.0 [M+H]. + , ESI.

[0375] 1 H NMR (400 MHz, CDCl3) δ 5.71-5.58 (m, 3H), 5.14-5.10 (m, 2H), 4.25-4.13 (m, 4H), 3.14 (s, 2H), 2.7 (d, J = 7.2 Hz, 2H), 1.26 (t, J = 6.8 Hz, 6H). Compound 6-3: Compound 6-2 (31 g, 97.12 mmol), Pd(OAc)2 (2.18 g, 9.71 mmol), PPh3 (5.09 g, 19.42 mmol), and AgOAc (19.45 g, 116.54 mmol) were dissolved in CAN (600 mL). The reaction mixture was stirred at 85 °C for 3 hours under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, filtered, concentrated under vacuum, and purified by rapid silica gel column chromatography to give compound 6-3 as a colorless oil (21 g, 91% yield). 13 H 18 The calculated mass of O4 is 238.1; the actual measured mass is 239.2 [M+H]. + , ESI.

[0376] 1 H NMR (400 MHz, CDCl3) δ 5.39 (t, J = 2 Hz, 2H), 4.95 (t, J = 1.6Hz, 2H), 4.18 (q, J = 7.2 Hz, 4H), 3.03 (t, J = 7.2 Hz, 4H), 1.24 (t, J = 7.2Hz, 6H). Compound 6-4: Compound 6-3 (21 g, 88.13 mmol) was dissolved in DCM (300 mL) and cooled to -78 °C. Br2 (14.08 g, 88.13 mmol, 4.51 mL) was dissolved in DCM (100 mL) and added to the reaction mixture over 1 hour. The mixture was stirred at -78 °C for 1 hour. The reaction mixture was quenched with saturated Na2SO3 solution (100 mL), extracted with DCM (200 mL x 3), washed with brine (100 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography to give compound 6-4 as a pale yellow oil (28 g, 80% yield).

[0377] C 13 H 18 The calculated mass of Br2O4 is 396.0; the measured mass is 397.0 [M+H]. + , ESI.

[0378] 1 H NMR (400 MHz, CDCl3) δ:4.21 (q, J= 7.2 Hz, 4H), 4.00 (s, 4H), 3.19 (s, 4H), 1.26 (t, J = 7.2 Hz, 6H). Compound 6-5: 4-Methylbenzenesulfonamide (12.05 g, 70.35 mmol) was dissolved in DMF (260 mL). NaH (6.19 g, 154.77 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. A solution of compound 6-4 (28 g, 70.35 mmol) in DMF (50 mL) was slowly added to the reaction mixture. The mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated NH4Cl solution (500 mL), extracted with EA (200 mL x 3), washed with water (500 mL x 3) and brine (300 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography to give compound 6-5 as a colorless solid (22 g, 76% yield).

[0379] C 20 H 25 NO6S mass calculated as: 407.1; Actual measured: 408.2 [M+H] + , ESI.

[0380] 1 H NMR (400 MHz, CDCl3) δ 7.70-7.68 (m, 2H), 7.32-7.30 (m, 2H), 4.17(q, J = 7.2 Hz, 4H), 3.96 (s, 4H), 2.89 (s, 4H), 2.42 (s, 3H), 1.22 (t, J =7.2 Hz, 6H). Compound 6-6: Compound 6-5 (22 g, 54.05 mmol) was dissolved in THF (300 mL). LiBH4 (11.8 g, 540.5 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water (100 mL), extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography to give compound 6-6 as a white solid (14.8 g, 89% yield).

[0381] C 16 H 21The calculated mass of NO4S is 323.1; the actual measured mass is 324.1 [M+H]. + , ESI.

[0382] 1 H NMR (400 MHz, DMSO-d6) δ 7.70-7.68 (m, 2H), 7.43-7.41 (m, 2H),4.56 (t, J = 5.2 Hz, 2H), 3.26 (d, J = 4.8 Hz, 4H), 2.84 (s, 4H), 2.39 (s, 3H), 1.95 (s, 4H). Compounds 6-7: Compound 6-6 (4.1 g, 12.68 mmol) and DIEA (4.92 g, 3.83 mmol) were dissolved in DCM (20 mL). A solution of DMTTrCl (4.30 g, 12.68 mmol) in DCM (80 mL) was slowly added to the reaction mixture at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated NaHCO3 solution (20 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-3% MeOH) on Al2O3 to give compound 6-7 as a white foamy solid (6.39 g, 80% yield).

[0383] C 37 H 39 The calculated mass of NO6S is 625.25; the actual measured mass is 648.25 [M+Na]. + , ESI.

[0384] 1 H NMR (400 MHz, DMSO- d 6) δ 7.68 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0Hz, 2H), 7.33 (d, J = 4.0 Hz, 2H), 7.31-7.18 (m, 7H), 6.85 (d, J = 8.0 Hz, 4H), 4.68 (t, J = 4.8 Hz, 1H), 3.83 (s, 4H), 3.72 (s, 6H), 3.37 (d, J= 2.0Hz, 2H), 2.94 (s, 2H), 2.39 (s, 3H), 2.07-2.03 (m, 2H), 1.92-1.89 (m, 2H). Compounds 6-8: Compounds 6-7 (6.0 g, 9.59 mmol) were dissolved in MeOH (300 mL), and then Mg (18 g) was added to the reaction mixture at 0 °C. The mixture was stirred overnight at room temperature. The reaction mixture was quenched with H₂O, extracted with DCM, washed with brine (50 mL), dried over Na₂SO₄, concentrated under vacuum, and purified by rapid column chromatography (0-15% MeOH) on Al₂O₃ to give compounds 6-8 as a white, foamy solid (1.2 g, yield 26.6%).

[0385] C 30 H 33 Calculated NO4 mass: 471.24; Actual measured: 472.20 [M+H] + , ESI.

[0386] 1 H NMR (400 MHz, DMSO- d 6) δ 7.37 (d, J = 8.0 Hz, 2H), 7.32-7.28 (m,2H), 7.25-7.19 (m, 5H), 6.89-6.87 (m, 4H), 4.76 (s, 1H), 3.83 (s, 1H), 3.73(s, 6H), 3.47 (s, 3H), 3.45 (s, 3H), 3.00 (s, 2H), 2.13-2.09 (m, 2H), 1.97-1.93 (m, 2H). Compounds 6-9: Compounds 6-8 (2.4 g, 5.09 mmol) and Fmoc-6-aminohexanoic acid (1.80 g, 5.09 mmol) were dissolved in DCM (60 mL), and then DIEA (1.97 g, 15.27 mmol), EDCI (1.95 g, 10.18 mmol), and HOBT (1.38 g, 10.18 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO3 solution (200 mL), extracted with DCM (60 mL x 3), washed with brine (60 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-10% MeOH) on Al2O3 to give compounds 6-9 as a white foamy solid (3.1 g, 75% yield).

[0387] 1 H NMR (400 MHz, DMSO-) d 6) δ 7.88 (d, J = 7.4 Hz, 2H), 7.68 (d, J = 7.4Hz, 2H), 7.43-7.37 (m, 4H), 7.34-7.28 (m, 5H), 7.26-7.21 (m, 5H), 6.88 (d, J = 8.9 Hz, 4H), 4.75 (t, J = 4.8 Hz, 1H), 4.30-4.28 (m, 2H), 4.22-4.19 (m,1H), 4.11-3.91 (m, 2H), 3.91-3.85 (m, 2H), 3.73 (s, 6H), 3.49-3.48 (d, J =4.6 Hz, 2H), 3.02-2.95 (m, 4H), 2.20-2.14 (m, 4H), 2.00-1.97 (m, 2H), 1.52-1.46 (m, 2H), 1.44-1.36 (m, 2H), 1.24-1.29 (m, 2H). Compounds 6-10: Compounds 6-9 (3.1 g, 3.84 mmol) were dissolved in MeOH (60 mL), and piperidine (6 mL) was added to the mixture. The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was quenched with water, extracted with DCM (100 mL x 3), washed with brine (60 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-20% MeOH) on Al2O3 to give compounds 6-10 as a white foamy solid (1.45 g, 64% yield).

[0388] The calculated mass of C36H44N2O5 is 584.33; the actual measured mass is 585.4 [M+H]. + , ESI.

[0389] 1 H NMR (400 MHz, DMSO- d 6) δ 7.39-7.39 (m, 2H), 7.37-7.28 (m, 2H), 7.25-7.19 (m, 5H), 6.88 (d, J = 8.8 Hz, 2H), 4.76 (s, 1H), 4.04 (m, 2H), 3.86(m, 2H), 3.78 (s, 6H), 3.49 (s, 2H), 3.02 (s, 2H), 2.55 (m, 2H), 2.22-2.14(m, 4H), 2.01-1.97 (m, 2H), 1.53-1.46 (m, 2H), 1.37-1.25 (m, 5H). Compound 6-11: Compounds 6-10 (1.5 g, 2.57 mmol) and 1-4 (1.15 g, 2.57 mmol) were dissolved in DCM (60 mL), and then DIEA (1.66 g, 12.83 mmol), EDCI (1.10 g, 6.41 mmol), and HOBT (519.92 mg, 3.85 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO3 solution (200 mL), extracted with DCM (60 mL x 3), washed with brine (60 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-10% MeOH) on Al2O3 to give compound 6-11 as a white foamy solid (2 g, yield 76%).

[0390] C 55 H 71 N3O 15Calculated mass: 1013.49; Actual measurement: 1014.4 [M+H] + , ESI.

[0391] 1 H NMR (400 MHz, DMSO-) d 6) δ 7.82 (d, J = 9.2 Hz, 1H), 7.72 (t, J = 5.6Hz, 1H), 7.39-7.37 (m, 2H), 7.32-7.28 (m, 2H), 7.26-7.19 (m, 5 H), 6.90-6.87(m, 4H), 5.22 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.50-4.48(m, 1H), 4.03-4.00 (m, 5H), 3.91-3.84 (m, 3H), 3.74 (s, 7H), 3.49 (d, J = 4.6Hz, 1H), 3.43-3.38 (m,1H), 3.02-2.98 (m, 4H), 2.21-2.15 (m, 4H), 2.10-2.08(m, 3H), 2.05-2.00 (m, 7H), 1.89 (s, 3H), 1.78 (s, 3H), 1.52-1.35 (m, 9H), 1.27-1.24 (m, 3H). Compounds 6-12: To a solution of compound 6-11 (790.0 mg, 0.78 mmol) in anhydrous DCM (3.0 mL), 4,5-dicyanimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphonium diamide (324 mg, 1.08 mmol) were added at room temperature and stirred for 1 h. LC-MS showed complete consumption of the starting material. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na₂SO₄. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH₃CN / H₂O = 1 / 1 increased to CH₃CN / H₂O = 1 / 0 over 20 min; detector: UV 254 nm). Compound 6-12 was produced as a white solid (680 mg, 71% yield).

[0392] C64 H 88 N5O 16 Calculated mass of P: 1213.60; Actual measured mass: 1159.5 [M-CH2CH2CN] - , ESI.

[0393] 1 HNMR (600 MHz, DMSO- d 6) δ 7.82-7.68 (m, 2H), 7.38-7.19 (m, 9H), 6.90-6.86 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.94 (m, 1H), 4.48 (d, J = 12.0Hz, 1H), 4.04-3.99 (m, 4H), 3.90-3.37 (m, 16H), 3.09-2.87 (m, 4H), 2.71-2.68(m, 2H), 2.19-1.76 (m, 20H), 1.52-1.05 (m, 22H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.51, 146.47. Compound 7-2: KOtBu (19.92 g, 177.55 mmol) was added to a solution of methoxymethyl (triphenyl)phosphorus chloride (54.78 g, 159.80 mmol) in THF (600 mL) at 0 °C and stirred for 0.5 h. Then, compound 7-1 (20 g, 88.78 mmol) was added at 0 °C and stirred overnight, allowing the reaction to rise to room temperature. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and concentrated to dryness. The residue was purified by silica gel column chromatography (0-5% EA solution in PE) to give compound 7-2 (15.5 g, 68% yield) as an oil.

[0394] C 14 H 23 The calculated mass of NO3 is 253.2; the actual measured mass is 239.1 [M-tBu+ACN+H]. + , ESI.

[0395] 1H NMR (400 MHz, DMSO-d6) δ 5.99 (pent, J = 2.1 Hz, 1H), 3.48 (s,3H), 3.44-3.36 (m, 2H), 2.99-2.91 (m, 2H), 2.59 (br, 2H), 2.44-2.36 (m, 2H),2.04-1.97 (m, 2H), 1.38 (s, 9H). Compound 7-3: To a solution of compound 7-2 (5 g, 19.74 mmol) in MeCN (150 mL), 2,2,2-trichloroacetic acid (4.06 g, 24.87 mmol, in 50 mL of water) was added. The reaction mixture was stirred overnight at room temperature. TLC showed complete conversion. The reaction mixture was adjusted to pH 7 by adding NaHCO3 (saturated aqueous solution) and extracted with EA. The organic phase was concentrated to give crude compound 7-3 as an oil (4.7 g, 99% yield), which could be used directly for the next step without further purification.

[0396] C 13 H 21 The calculated mass of NO3 is 239.2; the actual measured mass is 225.1 [M-tBu+ACN+H]. + , ESI.

[0397] Compound 7-4: Formalin (15.9 g, 37% aqueous formaldehyde, 55.78 mmol) was added to a mixture of compound 7-3 (4.7 g, 19.64 mmol) and K₂CO₃ (542.87 mg, 3.93 mmol) in MeOH (150 mL). The reaction mixture was stirred overnight at room temperature. TLC showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with EA. The organic phase was concentrated and purified by silica gel column chromatography (0-3% MeOH in DCM) to give compound 7-4 (5.2 g, 98% yield) in an oily state.

[0398] C 14 H 23 The calculated mass of NO4 is 269.2; the actual measured mass is 170.1 [M-Boc+H]. + , ESI.

[0399] Compound 7-5: To a solution of compound 7-4 (17 g, 63.12 mmol) in MeOH (190 mL), NaBH4 (5.25 g, 138.86 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and extracted with DCM. The organic phase was concentrated and purified by silica gel column chromatography (0-5% MeOH in DCM) to give compound 7-5 (14.5 g, 84% yield).

[0400] C 14 H 25 The calculated mass of NO4 is 271.2; the actual measured mass is 172.1 [M-Boc+H]. + , ESI.

[0401] 1 H NMR (400 MHz, DMSO-d6) δ 4.53 (t, J = 5.3 Hz), 3.37-3.32 (m, 2H), 3.27 (d, J = 5.3 Hz, 2H), 3.19 (d, J = 5.3 Hz, 2H), 3.07 (dd, J = 10.78, 3.5Hz, 2H), 1.60 (dd, J = 13.4, 7.9 Hz, 2H) 1.39 (s, 9H), 1.27 (dd, J = 13.5,6.3 Hz, 2H). Compounds 7-6: A solution of HCl in dioxane (4 M, 140 mL) was added to a reaction flask containing compound 7-5 (14.5 g, 53.44 mmol). The reaction mixture was stirred at room temperature for 1.5 h. LC-MS showed complete conversion. The reaction mixture was concentrated to give crude compound 7-6 (11 g), which could be used directly for the next step without further purification.

[0402] C9H 17 The calculated mass of NO2 (free bases) is 171.1; the measured mass is 172.1 [M-Boc+H]. + , ESI.

[0403] Compound 7-7: A mixture of Fmoc-6-aminohexanoic acid (18.38 g, 52.00 mmol), DIEA (16.80 g, 130.00 mmol, 22.64 mL), HOBT (8.43 g, 62.40 mmol), and EDCI (11.96 g, 62.40 mmol) in DCM (300 mL) was stirred at 0 °C for 30 min, and then compound 7-6 (10.8 g, 52.00 mmol) was added. The reaction mixture was stirred overnight and allowed to warm to room temperature. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (300 mL) and washed with NaHCO3 (saturated aqueous solution) and water. The organic phase was concentrated and purified by silica gel column chromatography (0-10% MeOH solution in DCM) to give compound 7-7 (19.6 g, 74% yield) in a gel form.

[0404] C 30 H 38 Calculated mass of N₂O₅: 506.3; Actual measured mass: 507.3 [M+H] + , ESI.

[0405] 1 H NMR (400 MHz, DMSO-d6) δ 7.90-7.68 (m, 4H), 7.44-7.33 (m, 5H), 4.55 (s, 2H), 4.29 (d, J = 6.8 Hz, 2H), 4.23-4.18 (m, 1H), 3.28-3.21 (m, 6H), 2.97-2.96 (m, 2H), 2.61-2.53 (m, 2H), 2.17 (t, J =7.6 Hz, 2H), 1.63-1.23 (m,12H). Compounds 7-8: At 0 °C, DMTrCl (13.98 g, 41.25 mmol) was added fractionally to a solution of compound 7-7 (19 g, 37.50 mmol), N,N-dimethylpyridin-4-amine (458.17 mg, 3.75 mmol), and DIEA (7.27 g, 56.25 mmol, 9.80 mL) in DCM (590 mL). The reaction mixture was stirred overnight. LC-MS showed partial conversion, forming two isomers and the byproduct di-DMTr. The reaction mixture was concentrated and purified by silica gel column chromatography (20–100% EA, PE solution containing 0.2% TEA) to give compounds 7-8a and 7-8b, a mixture of endogenous and exogenous isomers (17.9 g, 47% yield).

[0406] The isomers were separated by preparative HPLC (C18 column, water / ACN) to obtain compounds 7-8a (exo-isomer, 7.5 g) and 7-8b (endo-isomer, 5.7 g).

[0407] 7-8a: C 51 H 56 The calculated mass of N₂O₇ is 808.4; the actual measured mass is 832.3 [M+Na]. + , ESI.

[0408] 1 H NMR (400 MHz, DMSO-d6) δ 7.90-7.88 (m, 2H), 7.69-7.67 (m, 2H), 7.43-8.18 (m, 13H), 6.88-6.85 (m, 4H), 4.62 (br, 1H), 4.37-4.28 (m, 2H), 4.20(t, J = 6.7 Hz, 1H), 3.73 (s, 6H), 3.47 (dd, J = 9.9, 8.2 Hz, 1H), 3.28 (br,2H), 3.11 (td, J = 12.0, 4.3 Hz, 2H), 2.99-2.91 (m, 4H), 2.66-2.54 (m, 2H), 2.19-2.06 (m, 2H), 1.67 (pent, J = 6.8 Hz, 2H), 1.51-1.17 (m, 10H). 7-8b: C 51 H 56 The calculated mass of N₂O₇ is 808.4; the actual measured mass is 832.3 [M+Na]. + , ESI.

[0409] 1 H NMR (400 MHz, DMSO-d6) δ 7.90-7.84 (m, 4H), 7.44-7.20 (m, 13H), 6.91-6.89 (m, 4H), 6.66 (br, 1H), 6.29 (s, 2H), 3.74 (s, 6H), 3.36-3.31 (m,3H), 3.22-3.12 (m, 3H), 2.90 (dd, J = 12.7, 6.5 Hz, 2 H), 2.86 (s, 2H), 2.44-2.29 (m, 2H), 2.15 (t,J = 7.1 Hz, 2H), 1.64-1.18 (m, 12H). Compounds 7-9a: A solution of compounds 7-8a (6.5 g, 8.03 mmol) in MeOH (200 mL) and piperidine (20 mL) was stirred overnight at room temperature, and LC-MS showed complete conversion. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (20-30% MeOH, DCM solution containing 1% TEA) to give compounds 7-9a (4.4 g, 94% yield).

[0410] C 36 H 46 The calculated mass of N₂O₅ is 586.3; the actual measured mass is 587.3 [M+H]. + , ESI.

[0411] Compounds 7-10a: A solution of compounds 1-4 (4.70 g, 10.50 mmol), DIEA (2.42 g, 18.75 mmol, 3.27 mL), HOBT (1.22 g, 9.00 mmol), and EDCI (1.73 g, 9.00 mmol) in DCM (132 mL) was stirred at 0 °C for 30 min, and then compound 7-9a (4.4 g, 7.50 mmol) was added. The reaction mixture was stirred overnight. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column chromatography (0-10% MeOH solution in DCM containing 1% TEA) to give compound 7-10a (3.8 g, 51% yield).

[0412] C 55 H 73 N3O 15 Calculated mass: 1015.5; Actual measured mass: 1039.1 [M+Na] + , ESI.

[0413] 1 H NMR (400 MHz, DMSO-d6) δ 7.85 (d, J = 9.2 Hz, 1H), 7.74 (t, J= 5.3 Hz, 1H), 7.38 - 7.36 (m, 2H), 7.31 - 7.27 (m, 2H), 7.24 - 7.19 (m, 5H), 6.88 - 6.86 (m, 4H), 5.22 (d, J = 2.8 Hz, 1H), 4.97 (dd, J = 11.2, 2.8 Hz, 1H), 4.65 (t, J = 4.8 Hz, 1H), 4.49 (d, J = 8.4 Hz, 1H), 4.03 (s, 3H), 3.88 (dd, J = 19.5, 9.1 Hz, 1H), 3.73 - 3.70 (m, 7H), 3.50 - 3.39 (m, 2H), 3.34 - 3.31 (m, 1H), 3.27 (d, J = 4.5 Hz, 2H), 3.17 (d, J = 5.2 Hz, 1H), 3.15 - 3.09 (m, 2H), 3.00 (dd, J = 12.4, 6.3 Hz, 2H), 2.94 (br, 1H), 2.67 - 2.53 (m, 2H), 2.19 - 2.08 (m, 5H), 2.04 (t, J = 6.9 Hz, 2H), 2.00 (s, 3H), 1.89 (s, 3H), 1.78 (s, 3H), 1.70 - 1.63 (m, 2H), 1.53 - 1.16 (m, 14H). Compound 7 - 11a: To a solution of compound 7-10a (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL), 4,5-dicyanimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphonium diamide (324 mg, 1.08 mmol) were added and stirred for 1 h. LC-MS showed complete consumption of the starting material. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na₂SO₄. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH₃CN / H₂O = 1 / 1 increased to CH₃CN / H₂O = 1 / 0 over 20 min; detector: UV 254 nm). This yielded 7-11a as a white solid (390 mg, 47% yield).

[0414] C 64 H 90 N5O16 P Calculated mass: 1215.61; Actual measured mass: 1161.3 [M-CH2CH2CN] - , ESI.

[0415] 1 HNMR (600 MHz, DMSO- d 6) δ 7.81-7.69 (m, 2H), 7.40-7.19 (m, 9H), 6.88-6.86 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.94 (m, 1H), 4.49 (d, J = 6.0Hz, 1H), 4.02-3.83 (m, 4H), 3.73-3.32 (m, 18H), 3.01-2.97 (m, 6H), 2.71-2.68(m, 4H), 2.12-1.99 (m, 18H), 1.49-1.04 (m, 24H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.70. Compounds 7-9b: A solution of compounds 7-8b (5.70 g, 7.05 mmol) in MeOH (200 mL) and piperidine (20 mL) was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by silica gel column chromatography (10-20% EA, DCM solution containing 1% TEA) to give compounds 7-9b (4.1 g, 99% yield).

[0416] C 36 H 46 The calculated mass of N₂O₅ is 586.3; the actual measured mass is 587.3 [M+H]. + , ESI.

[0417] Compounds 7-10b: A solution of compounds 1-4 (4.38 g, 9.78 mmol), DIEA (2.26 g, 17.47 mmol, 3.04 mL), HOBT (1.13 g, 8.39 mmol), and EDCI (1.61 g, 8.39 mmol) in DCM (120 mL) was stirred at 0 °C for 30 min, and then compound 7-9b (4.10 g, 6.99 mmol) was added. The reaction mixture was stirred overnight. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column chromatography (0-10% MeOH in DCM containing 1% TEA) to give 4.4 g of crude product, which was further purified by preparative HPLC (C18, water / ACN) to give compound 7-10b (2.0 g, 28% yield).

[0418] C 55 H 73 N3O 15 Calculated mass: 1015.5; Actual measured mass: 1039.1 [M+Na] + , ESI.

[0419] 1H NMR (400 MHz, DMSO-d6) δ 7.84 (d, J = 9.2 Hz, 1H), 7.72 (t, J =5.5 Hz, 1H), 7.40-7.38 (m, 2H), 7.33-7.29 (m, 2H), 7.28-7.20(m, 5H), 6.91-6.88 (m, 4H), 5.22 (d, J = 3.3 Hz, 1H), 4.97 (dd, J = 11.2, 3.3 Hz, 1H), 4.66(t, J = 4.9 Hz, 1H), 4.49 (d, J = 8.5 Hz, 1H), 4.03 (s, 3H), 3.88 (dd, J =20.2, 8.8 Hz, 1H), 3.74-3.69 (m, 7H), 3.48-3.38 (m, 4H), 3.34-3.31 (m, 1H), 3.21 (dd, J = 10.4, 4.2 Hz, 1H), 3.14 (dd, J = 12.0, 4.6 Hz, 1H), 3.00 (dd, J= 12.7, 6.6 Hz, 2H), 2.86 (s, 2H), 2.46-2.27 (m, 2H), 2.16 (t, J = 6.7 Hz,2H), 2.11 (s, 3H), 2.04 (t, J = 7.0 Hz, 2H), 2.00 (s, 3H), 1.89 (s, 3H), 1.78(s, 3H), 1.63-1.12 (m, 14H). Compound 7-11b: At room temperature, 4,5-dicyanimidazolium (96.0 mg, 0.81 mmol) and 2-cyanoethyl were added to a solution of compound 7-10b (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL). N , N , N ′, N′-Tetraisopropylphosphonic diamide (324 mg, 1.08 mmol) was stirred for 1 h. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH3CN / H2O = 1 / 1 increased to CH3CN / H2O = 1 / 0 over 20 min; detector: UV 254 nm). Compounds 7-11b were produced as a white solid (390 mg, yield 47%).

[0420] C 64 H 90 N5O 16 Calculated mass of P: 1215.61; Actual measured mass: 1161.6 [M-CH2CH2CN] - , ESI.

[0421] 1 HNMR (600 MHz, DMSO- d 6) δ 7.81-7.69 (m, 2H), 7.40-7.19 (m, 9H), 6.88-6.86 (m, 4H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.94 (m, 1H), 4.49 (d, J = 6.0Hz, 1H), 4.02-3.83 (m, 4H), 3.73-3.32 (m, 18H), 3.01-2.97 (m, 6H), 2.71-2.68(m, 4H), 2.12-1.99 (m, 18H), 1.49-1.04 (m, 24H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.22. Compound 8-2: KOtBu (9.66 g, 86.12 mmol) was added to a solution of methoxymethyl (triphenyl)phosphorus chloride (17.71 g, 51.67 mmol) in THF (120 mL) at 0 °C and stirred for 0.5 h. Then, compound 8-1 (9.7 g, 43.06 mmol) was added at 0 °C and stirred overnight, allowing the reaction to reach room temperature. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and concentrated to dryness. The residue was purified by silica gel column chromatography (0-5% EA solution in PE) to give compound 8-2 (6.0 g, 55% yield) as an oil.

[0422] 1 H NMR (400 MHz, DMSO-d6) δ 6.01 (s, 1H), 3.48 (s, 3H), 3.43-3.33 (m,2H), 2.82-2.74 (m, 2H), 2.35-2.26 (m, 2H), 1.90-1.79 (m, 4H), 1.38 (s, 9H). Compound 8-3: Hydrochloric acid (3.28 g, 90.00 mmol, 2N, 45 mL) was added to a solution of compound 8-2 (5.85 g, 23.09 mmol) in THF (50 mL). The reaction mixture was stirred at room temperature for 3 hours. TLC showed complete conversion. The reaction mixture was adjusted to pH 7 by adding NaHCO3 (saturated aqueous solution) and extracted with EA. The organic phase was concentrated to give crude compound 8-3 as an oil (5.5 g), which could be used directly for the next step without further purification.

[0423] 1 H NMR (400 MHz, DMSO-d6) δ 9.62 (pent, J = 1.2 Hz, 1H), 3.42-3.33(m, 3H), 2.82-2.72 (m, 2H), 2.03-1.93 (m, 1H), 1.92-1.87 (m, 2H), 1.75-1.62(m, 1H), 1.51-1.44 (m, 1H), 1.38 (s, 9H). Compound 8-4: Formalin (6.59 g, 37% aqueous solution, 219.38 mmol) was added to a mixture of compound 8-3 (5.25 g, 21.94 mmol) and K₂CO₃ (660.39 mg, 4.39 mmol) in MeOH (30 mL). The reaction mixture was stirred overnight at room temperature. TLC showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution) and extracted with EA. The organic phase was concentrated to give compound 8-4 (6.7 g), which could be used directly in the next step without further purification.

[0424] Compound 8-5: To a solution of compound 8-4 (6.3 g, 23.39 mmol) in MeOH (50 mL), NaBH4 (2.65 g, 70.17 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was adjusted to pH 6 by adding citric acid (10% aqueous solution), filtered, and extracted with DCM. The organic phase was concentrated and purified by silica gel column chromatography (0-5% MeOH in DCM) to give compound 8-5 (1.4 g, 22% yield).

[0425] C 14 H 25 Calculated NO4 mass: 271.18; Actual measured: 257.24 [M-tBu+CH3CN+H] + , ESI.

[0426] 1 H NMR (400 MHz, DMSO-d6) δ 7.16-7.55 (m, 1H), 4.52-4.48 (m, 2H), 3.36-3.27 (m, 5H), 2.74-2.50 (m, 2H), 1.98-1.84 (m 2H), 1.54-1.50 (m 2H),1.49 (s, 9H), 1.05-0.97 (m, 2H). Compounds 8-6: Compound 8-5 (1.35 g, 4.97 mmol) was dissolved in DCM (5 mL), and HCl / dioxane (4 M, 10 mL) was added to the mixture at room temperature. The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under vacuum to give crude product 8-6 (1.3 g), which was an oil and could be used in the next step without further purification.

[0427] C9H 17 Calculated NO2 mass: 171.1; Actual measured: 172.2 [M+H] +, ESI.

[0428] Compounds 8-7: Z-6-aminohexanoic acid (1.99 g, 7.51 mmol), HATU (4.72 g, 12.52 mmol), and DIPEA (2.43 g, 18.78 mmol, 3.27 mL) were dissolved in DCM (20 mL). The reaction mixture was stirred for 20 min, then compound 8-6 (1.3 g, 6.26 mmol) was added, and the mixture was stirred for another 2 h. The reaction mixture was quenched with H2O (20 mL), washed with saturated NaHCO3 aqueous solution (30 mL), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (DCM:MeOH = 20:1) on a silica gel column to give compound 8-7 as a white, foamy solid (1.5 g, 3.58 mmol, yield 57%). 23 H 34 The calculated mass of N₂O₅ is 418.3; the actual measured mass is 419.3 [M+H]. + , ESI.

[0429] Compound 8-8: Compound 8-7 (1.2 g, 2.87 mmol) was dissolved in DCM (20 mL). DMTrCl (971.49 mg, 2.87 mmol) and DIPEA (741.12 mg, 5.73 mmol, 998.81 μL) were added. The reaction mixture was stirred at room temperature for 2 hours. The reaction was then quenched with water, extracted with DCM (50 mL x 2), and washed with brine (50 mL). The organic phase was collected, dried, concentrated under vacuum, and purified by rapid column chromatography (DCM:MeOH:TEA = 20:1:0.5%) on Al2O3 to give compound 8-8 as a white, foamy solid (850 mg, 41% yield).

[0430] C 44 H 52 The calculated mass of N₂O₇ is 720.4 g; the actual measured mass is 721.4 g [M+H]. + , ESI.

[0431] 1 H NMR (400 MHz, DMSO-d6) δ 7.41-7.36 (m, 3H), 7.35-7.29 (m, 6H),7.27-7.20 (m, 5H), 6.89 (d, J = 8.9 Hz, 4H), 5.00 (s, 2H), 4.67 (t,J = 4.9Hz, 1H), 3.75 (s, 6H), 3.55-3.47 (m, 2H), 3.39 (d, J = 4.5 Hz, 2H), 3.00-2.95(m, 4H), 2.92-2.84 (m, 1H), 2.62 (q, J = 11 Hz, 1H), 2.16-2.10 (m, 2H), 1.95-1.70 (m, 2H), 1.62-1.58 (m, 1H), 1.51-1.35 (m, 5H), 1.27-1.23 (m, 3H), 1.12-1.00 (m, 2H). Compounds 8-9: Compound 8-8 (0.8 g, 1.11 mmol) was dissolved in MeOH (10 mL). Pd / C (80 mg) was added. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude product 8-9 as a white, foamy solid (550 mg, 84% yield), which could be used in the next step without further purification.

[0432] C 36 H 46 The calculated mass of N₂O₅ is 586.3; the actual measured mass is 587.3 [M+H]. + , ESI.

[0433] 1 H NMR (400 MHz, DMSO-d6) δ 7.40-7.38 (m, 2H), 7.31 (t, J = 7.7 Hz,2H), 7.29-7.18 (m, 5H), 6.89 (d, J = 8.9 Hz, 4H), 3.74 (s, 6H), 3.54-3.43 (m,2H), 3.34-3.21 (m, 2H), 2.96 (s, 2H), 2.94-2.85 (m, 2H), 2.67-2.55 (m, 1H),2.17-2.10 (m, 2H), 1.99-1.74 (m, 2H), 1.63-1.58 (m, 1H), 1.53-1.42 (m, 3H),1.36-1.24 (m, 5H), 1.16-1.10 (m, 1H), 1.05-0.98 (m, 1H). Compound 9-2: Methoxymethyl (triphenyl)phosphorus chloride (116.8 g, 340.72 mmol) was dissolved in THF (1200 mL). KOtBu (46.74 g, 416.56 mmol) was added to the mixture at 0 °C. The reaction mixture was stirred at 0 °C for another 30 minutes. Compound 9-1 (40 g, 189.34 mmol) was added to the mixture. The mixture was heated to room temperature and stirred overnight at room temperature. The reaction was quenched with saturated citric acid and the pH was adjusted to 6-7. The reaction mixture was extracted with EA (500 mL x 3), washed with brine (200 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography (10-30% EA solution in PE) to give compound 9-2 as an oil (24 g, 52% yield).

[0434] C 13 H 21 The calculated mass of NO3 is 239.3; the actual measured mass is 225.1 [M-tBu+ACN+H]. + , ESI.

[0435] 1 H NMR (400 MHz, DMSO-d6) δ 5.88 (t, J = 2.4 Hz, 1H), 3.83 (s, 4H), 3.47 (s, 3H), 2.79-2.74 (m, 4H), 1.41 (s, 9H). Compound 9-3: Compound 9-2 (15 g, 62.68 mmol) was dissolved in ACN (450 mL). A solution of 2,2,2-trichloroacetic acid (12.90 g, 78.98 mmol) in water (150 mL) was added to the reaction mixture. The reaction was stirred overnight at room temperature, then quenched with saturated NaHCO3 and the pH was adjusted to 7-8. The mixture was extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na2SO4, and concentrated under vacuum to give crude product 9-3 as an oil (13 g, 92% yield), which could be used for the next step without further purification.

[0436] C 12 H 19 The calculated mass of NO3 is 225.3; the actual measured mass is 211.1 [M-tBu+ACN+H]. + , ESI.

[0437] Compound 9-4: Compound 9-3 (13 g, 57.71 mmol) was dissolved in MeOH (400 mL). K₂CO₃ (1.60 g, 11.54 mmol) and formaldehyde (4.92 g, 163.88 mmol, in aqueous solution) were added to the mixture. The reaction was stirred overnight at room temperature, then quenched with saturated citric acid and the pH was adjusted to 6-7. The mixture was concentrated under vacuum to remove MeOH. The residue was extracted with EA (200 mL x 3), washed with brine (100 mL), dried over Na₂SO₄, and concentrated under vacuum to give crude product 9-4 (14 g, 95% yield).

[0438] C 13 H 21 The calculated mass of NO4 is 255.3; the actual measured mass is 241.1 [M-tBu+ACN+H]. + , ESI.

[0439] Compound 9-5: Compound 9-4 (14 g, 54.84 mmol) was dissolved in MeOH (420 mL). NaBH4 (4.56 g, 120.64 mmol) was added in portions. The reaction was stirred at room temperature for 1 hour, and then quenched with water. The pH was adjusted to 6-7 with saturated citric acid. The mixture was concentrated under vacuum to remove MeOH, and the residue was extracted with DCM (200 mL x 3), washed with brine (100 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-5% MeOH) on silica gel to give compound 9-5 (7.05 g, 50% yield).

[0440] C 13 H 23 The calculated mass of NO4 is 257.3; the actual measured mass is 243.1 [M-tBu+ACN+H]. + , ESI.

[0441] 1 H NMR (400 MHz, DMSO-d6) δ 4.51-4.11 (m, 2H), 3.83-3.75 (m, 4H), 3.25-3.17 (m, 4H), 2.01-1.91 (m, 4H), 1.36 (s, 9H). Compound 9-6: Compound 9-5 (13.1 g, 50.91 mmol) was dissolved in HCl in a solution of 1,4-dioxane (4 M, 131 mL). The reaction mixture was stirred at room temperature for 1 hour, and then concentrated under vacuum to give crude product 9-6 (9.8 g), which could be used in the next step without further purification.

[0442] C8H 13 The calculated mass of NO2Cl is 157.2; the actual measured mass is 158.2 [M+H]. + , ESI.

[0443] Compounds 9-7: Compound 9-6 (9.8 g, 50.60 mmol) was dissolved in DCM (300 mL). DIEA (22.89 g, 177.10 mmol, 30.85 mL), HOBT (8.20 g, 60.72 mmol), EDCI (11.64 g, 60.72 mmol), and Fmoc-6-hexanoic acid (17.88 g, 50.60 mmol) were added to the reaction mixture. The reaction was stirred overnight at room temperature, then quenched with water, washed with 10% citric acid (50 mL), saturated NaHCO3 solution (50 mL), and brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-10% MeOH) on silica gel to give compound 9-7 (6.7 g, 25% yield in two steps).

[0444] C 30 H 36 The calculated mass of N₂O₅ is 492.3; the actual measured mass is 493.3 [M+H]. + , ESI.

[0445] 1 H NMR (400 MHz, DMSO-d6) δ 7.91-7.68 (m, 4H), 7.44-7.28 (m, 4H), 6.76 (s, 1H), 6.68-6.66 (m, 1H), 6.29 (s, 2H), 4.01 (s, 2H), 3.74 (s, 2H),3.69 (s, 2H), 3.27 (s, 4H), 2.92-2.87 (m, 2H), 1.98-1.91 (m, 4H), 1.46-1.18(m, 6H). Compounds 9-8: Compound 9-7 (6.7 g, 13.60 mmol), DMAP (166.15 mg, 1.36 mmol), and DIEA (2.64 g, 20.40 mmol, 3.18 mL) were dissolved in DCM (180 mL). DMTrCl (5.52 g, 14.96 mmol) was added to the reaction mixture in portions at 0 °C. The reaction was stirred overnight at room temperature and then quenched with water. The mixture was extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (50-100% EA) on Al2O3 to give compound 9-8 (4.3 g, 40% yield).

[0446] C 51 H 56 The calculated mass of N₂O₈ is 824.4 g; the actual measured mass is 848.3 g [M + Na]. + , ESI.

[0447] 1 H NMR (400 MHz, DMSO-d6) δ 7.91-7.68 (m, 4H), 7.44-7.28 (m, 4H), 7.75-7.71 (m, 6H), 6.90-6.88 (m, 6H), 6.67-6.65 (m, 1H), 6.29 (s, 2H), 3.96(s, 1H), 3.74 (s, 9H), 3.69-3.67 (m, 2H), 3.53 (s, 1H), 3.39-3.37 (m, 2H), 2.92-2.89 (m, 4H), 2.03-1.79 (m, 8H), 1.46-1.18 (m, 6H). Compound 9-9: Compound 9-8 (4.2 g, 5.09 mmol) was dissolved in MeOH (126 mL), and then piperidine (12.6 mL) was added to the mixture. The reaction mixture was stirred overnight at room temperature, and then concentrated under vacuum and purified by rapid column chromatography (0-10% MeOH) on Al2O3 to give compound 9-9 as a white foam (3 g, 97% yield).

[0448] C 36 H 46 The calculated mass of N₂O₆ is 602.3; the actual measured mass is 603.4 [M+H]. + , ESI.

[0449] Compounds 9-10: Compounds 1-4 (3.2 g, 7.16 mmol) and 9-9 (3 g, 5.11 mmol) were dissolved in DCM (130 mL). DIEA (1.65 g, 12.78 mmol, 2.17 mL), HOBT (807.01 mg, 6.13 mmol), and EDCI (1.18 g, 6.13 mmol) were added to the mixture. The reaction was stirred overnight at room temperature, then quenched with water (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na₂SO₄, concentrated under vacuum, and purified by HPLC to give compound 9-10 (610 mg, 12% yield).

[0450] C 55 H 73 N3O 16 Calculated mass: 1031.5; Actual measured mass: 1054.5 [M+Na] + , ESI.

[0451] 1 H NMR (400 MHz, DMSO-d6) δ 7.82-7.73 (m, 2H), 7.24-6.88 (m, 12H), 5.22 (s, 1H), 4.97 (d, J = 10.8 Hz, 1H), 4.64-4.48 (m, 2H), 4.05-3.85 (m,4H), 3.71-3.69 (m, 12H), 3.52 (s, 1H), 3.39-3.38 (m, 1H), 3.04-2.91 (m, 4H), 2.11-1.78 (m, 19H), 1.46-1.18 (m, 14H). Compounds 9-11: Add 4,5-dicyanimidazolium (32.0 mg, 0.27 mmol) and 2-cyanoethyl to a solution of compound 9-10 (1.0 g, 0.97 mmol) in anhydrous DCM (3.0 mL) at room temperature. N , N , N ′, N′-Tetraisopropylphosphonic diamide (108 mg, 0.36 mmol) was stirred for 1 h. LCMS showed that the starting material was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column: C18 silica gel; mobile phase: CH3CN / H2O = 1 / 1, increased to CH3CN / H2O = 1 / 0 over 20 min; detector: UV 254 nm). Compounds 9-11 were produced as a white solid (680 mg, yield 56%).

[0452] C 64 H 90 N5O 17 Calculated mass of P: 1231.61; Actual measured mass: 1177.1 [M-CH2CH2CN] - , ESI.

[0453] 1 HNMR (600 MHz, DMSO- d 6) δ 7.81-7.67 (m, 2H), 7.26-7.23 (m, 6H), 6.88-6.86 (m, 6H), 5.22 (d, J = 6.0 Hz, 1H), 4.98-4.95 (m, 1H), 4.48 (d, J = 12.0Hz, 1H), 4.04-3.96 (m, 4H), 3.89-3.84 (m, 1H), 3.73-3.38 (m, 20H), 3.01-2.93(m, 4H), 2.73-2.71 (m, 2H), 2.10-1.75 (m, 20H), 1.51-1.07 (m, 22H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.82, 146.70. Compounds 9-12: To a solution of compound 9-10 (100 mg, 0.097 mmol) in anhydrous DCM (1.0 mL), DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added, followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed complete consumption of the starting material. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), then washed with brine (3 mL x 4), and the organic layer was concentrated to give compound 9-12 as a white solid (85 mg, 90% yield).

[0454] C 59 H 77 N3O 19 Calculated mass: 1131.52; Actual measurement: 1130.2 [MH] - , ESI.

[0455] 1 HNMR (600 MHz, DMSO- d 6) δ 7.87-7.72 (m, 2H), 7.24-7.21 (m, 6H), 6.90-6.87 (m, 6H), 5.21 (d, J = 6.0 Hz, 1H), 4.98-4.95 (m, 1H), 4.49 (d, J = 12.0Hz, 1H), 4.06-3.98 (m, 6H), 3.90-3.83 (m, 1H), 3.73-3.50 (m, 13H), 3.02-2.94(m, 4H), 2.45-2.44 (m, 4H), 2.11-1.76 (m, 20H), 1.48-1.18 (m, 12H). Solid carriers 9-13: Natural amino-LCAA-CPG (loading value: 75 μmol / g, 1000 Å) was washed with ACN (100 mL x 2), DMF (100 mL x 2), and DCM (100 mL x 2), and then dried overnight under high vacuum.

[0456] DIPEA (30 mg, 0.23 mmol) was added to a solution of succinate 9-12 (85 mg, 0.075 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL). The reaction mixture was shaken at room temperature for 10 min, followed by the addition of natural amino-LCAA-CPG (300 mg, 75 μmol / g loading). The suspension was shaken at room temperature for 20 h, then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 2 h to obtain a solid support (300 mg). The solid support was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL) to cap unreacted amines on the solid support. The solid support was then washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 9-13 (300 mg). For sample loading calculation, 5.00 mg of dried sample CPG was added to 25 mL of 3% DCA in DCM. The solution was shaken, and the UV absorbance at 482 nm was measured. The absorbance value was ensured to be below 1.0 unit to prevent signal saturation. The following formula was then applied: Sample loading (µmol / g) = (Total volume of DCA added (mL)) * (Abs value at 482 nm) * 1000) / (78.3 * (number of mg of CPG taken)) Total volume of DCA added (mL) = 25 mL The Abs value at 482 nm is 0.577. The amount of Mg in the CPG sample was 5.00 mg. Loading sample (µmol / g) = ((25)*(0.577)*1000) / 78.3*(5.00) = 36.8 μmol / g. Compound 10-2: Compound 10-1 (0.6 g, 0.299 mmol), DIEA (96.63 mg, 0.748 mmol, 130.23 μL), HOBT (48.49 mg, 0.359 mmol), and EDCI (68.80 mg, 0.359 mmol) were mixed in DCM (18 mL) and stirred at room temperature for 30 min. Then, compound 2-10 (412.34 mg, 0.897 mmol) was added, and the mixture was stirred at room temperature for 1 h. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by preparative HPLC (C18 column, eluent A: water, eluent B: ACN) to give compound 10-2 as a white solid (580 mg, 79% yield).

[0457] C 120 H 179 N 11 O 42 Calculated mass: 2446.22; Actual measured mass: 1247.61 [M+2Na] 2+ , ESI.

[0458] 1 H NMR (400 MHz, DMSO-d6) δ 7.86-7.82 (m, 6H), 7.75 (t, J = 5.5 Hz,3H), 7.39-7.29 (m, 4H), 7.26-7.19 (m, 5H), 7.00 (br, 1H), 6.89 (dd, J = 8.9, 3.7 Hz, 4H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.66-4.64 (m, 1H), 4.78 (d, J = 8.4 Hz, 3H), 4.04-3.96 (m, 10H), 3.90-3.83 (m,3H), 3.73-3.67 (m, 11H), 3.55-3.50 (m, 13H), 3.43-3.37 (m, 5H), 3.02 (pent, J = 5.8 Hz, 12H), 2.90 (s, 2H), 2.27 (t, J= 6.2 Hz, 6H), 2.10 (s, 9H) 2.07-2.00 (m, 10H), 1.99 (s, 9H), 1.96-1.92 (m, 2H), 1.89 (s, 9H), 1.85-1.80 (m,2H), 1.77 (s, 9H), 1.54-1.37 (m, 22H), 1.22-1.20 (m, 12H). Compound 10⁻³: To a solution of compound 10⁻² (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL), DMAP (14 mg, 0.12 mmol) and TEA (24 mg, 0.24 mmol) were added, followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed complete consumption of the starting material. The reaction mixture was diluted with DCM, washed with water (3 mL x 4), then with brine (3 mL x 4), and the organic layer was concentrated to give compound 10⁻³ (130 mg, 97% yield) as a white solid.

[0459] C 124 H 183 N 11 O 45 Calculated mass: 2546.24; Actual measurement: 1273.5 [M+2H] 2+ , ESI.

[0460] 1 HNMR (600 MHz, DMSO- d 6): 7.84-7.73(m, 9H), 7.37-7.21 (m, 9H), 6.98-6.89(m, 5H), 5.21 (d, J = 6.0 Hz, 3H), 4.97-4.94 (m, 3H), 4.48 (d, J = 12.0 Hz,3H), 4.07-3.66 (m, 26H), 3.55-3.37 (m, 17H), 3.04-2.95 (m, 14H), 2.45 (d, J =6.0 Hz, 4H), 2.29-2.25 (m, 6H), 2.11-1.72 (m, 50H), 1.51-1.12 (m, 38H). Compound 10⁻⁴: DIPEA (30 mg, 0.23 mmol) was added to a solution of compound 10⁻³ (130 mg, 0.05 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL). The reaction mixture was shaken at room temperature for 10 min, and then natural amino-lcaa-CPG (300 mg, 75 μmol / g) was added. The suspension was shaken at room temperature for 20 h, then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 2 h to obtain a solid support (300 mg). The mixture was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL). Unreacted amines on the support were capped and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 10⁻⁴ (300 mg). For sample loading calculation, 5.19 mg of dried sample CPG was added to 20 mL of 3% DCA in DCM. The solution was shaken, and the UV absorbance at 500 nm was measured. The absorbance value was ensured to be below 1.0 unit to ensure no signal saturation. Then the following formula was applied: Sample loading (µmol / g) = (Total volume of DCA added (mL)) * (Abs value at 500 nm) * 1000) / (76 * (number of mg of CPG taken)) Total volume of DCA added (mL) = 20 mL The Abs value at 500 nm is 0.335. The amount of Mg in the CPG sample was 5.19 mg. Loading sample (µmol / g) = ((20)*(0.335)*1000) / 76*(5.19) = 17 µmol / g. Compound 11-1: TEA (495.41 mg, 4.90 mmol) and HATU (1.35 g, 3.59 mmol) were added to a solution of compound 2-10 (1.27 g, 3.59 mmol) in DCM (15 mL). After stirring for 15 min, Fmoc-6-hexanoic acid (1.5 g, 3.26 mmol) was added to the reaction mixture, and stirring was continued for 4 h. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with DCM / MeOH = 20 / 1 to give 600 mg of compound 11-1 (yield 23.2%).

[0461] 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (d, J = 7.6 Hz, 2H), 7.68 (d, J =7.4 Hz, 2H), 7.44-7.35 (m, 4H), 7.35-7.28 (m, 4H), 7.27-7.17 (m, 6H), 6.88(d, J = 7.6 Hz, 4H), 4.64 (td, J = 5.04, 1.6 Hz, 1H), 4.28 (d, J = 6.9 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 3.96 (s, 1H), 3.73 (d, J = 3.8 Hz, 6H), 3.66(d, J = 10.4 Hz, 2H), 3.50 (s, 1H), 3.39 (t, J = 5.9 Hz, 2H), 2.96 (t, J =6.6 Hz, 2H), 2.91(s, 2H), 2.06-1.74 (m, 6H), 1.47-1.31(m, 4H), 1.26-1.13 (m, 2H). Compound 11-2: Piperidine (0.4 mL) was added to a solution of compound 11-1 (600 mg, 0.75 mmol) in MeOH (4 mL). After stirring overnight, the mixture was concentrated under vacuum. The residue was purified by column chromatography, eluting with DCM / MeOH / NH3OH = 100 / 10 / 1 to give 360 ​​mg of compound 11-2 (yield 82%).

[0462] C 35 H 44 The calculated mass of N₂O₅ is 572.3; the actual measured mass is 573.4 [M+H]. + , ESI.

[0463] 1H NMR (400 MHz, DMSO-d6) δ 7.27-7.21 (m, 4H), 6.91-6.85 (m, 9H), 3.97 (s, 1H), 3.73 (s, 6H), 3.68 (s, 2H), 3.47 (d, J = 6.5 Hz, 2H), 3.16 (s,2H), 2.90 (s, 2H), 2.66-2.62 (m, 2H), 2.54-2.51 (m, 2H), 1.49-1.17 (m, 12H). Compound 11-4: A mixture of compound 11-3 (10 g, 84.65 mmol), 4-bromopyridine (14.71 g, 93.11 mmol), Pd(PPh3)2Cl2 (4 g, 5.70 mmol), and t-BuOK (19.00 g, 169.30 mmol) in 1,4-dioxane (200 mL) was degassed by nitrogen purging. The mixture was then heated at 60 °C for 6 hours. The mixture was concentrated. The residue was then purified by column chromatography, eluting with DCM / MeOH / NH4OH = 95 / 5 / 1 to give 8 g of compound 11-4 as a yellow solid (yield 48%).

[0464] C 12 The calculated mass of H9N3 is 195.1; the actual measured mass is 196.2 [M+H]. + , ESI.

[0465] Compound 11-5: Compound 11-4 (10 g, 51.22 mmol) was dissolved in tert-butanol and heated to 50 °C, followed by the addition of KOH (574.84 mg, 10.24 mmol) and MeOH (5 mL). Tert-butyl acrylate (7.22 g, 56.35 mmol) was then slowly added to the reaction mixture, and the mixture was stirred for 5 hours. The mixture was concentrated under vacuum. The residue was partitioned between ethyl acetate (250 mL) and water (150 mL), and the ethyl acetate extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with PE / EA = 1 / 2 to give 11 g of compound 11-5 (66% yield).

[0466] C 19 H 21 The calculated mass of N3O2 is 323.2; the actual measured mass is 324.2 [M+H]. + , ESI.

[0467] 1 H NMR (400 MHz, DMSO-d6) δ 8.65 (d, J = 5.6 Hz, 4H), 7.50 (d, J =6.0 Hz, 4H), 2.82 (t, J = 7.8 Hz, 2H), 2.23 (t, J = 7.8 Hz, 2H), 1.35 (s, 9H). Compound 11-6: Cobalt chloride hexahydrate (1.47 g, 6.18 mmol) was added to a solution of compound 11-5 (1 g, 3.09 mmol) in MeOH (20 mL). Then, sodium borohydride (1.17 g, 30.92 mmol) was slowly added to the reaction mixture at -6 °C. The mixture was heated to room temperature and stirred for 5 hours. The mixture was concentrated under vacuum. The residue was dissolved in EA, filtered through a diatomaceous earth bed, and washed with MeOH. The filtrate was concentrated to give 0.7 g of compound 11-6 (69% yield).

[0468] C 19 H 25 The calculated mass of N3O2 is 327.2; the actual measured mass is 328.3 [M+H]. + , ESI.

[0469] Compound 11-7: Compound 11-6 (2.12 g, 9.16 mmol) was dissolved in DCM (40 mL). Then, TEA (925.42 mg, 9.16 mmol) and HATU (3.46 g, 9.16 mmol) were added to this solution. After stirring for 20 minutes, Boc-6-aminocaproic acid (3 g, 9.16 mmol) was added to the reaction mixture, and the mixture was stirred for 5 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with DCM / MeOH = 9 / 1 to give 2.5 g of compound 11-7 as a yellow oil (71% yield).

[0470] C 30 H 44 The calculated mass of N4O5 is 540.3; the actual measured mass is 541.4 [M+H]. + , ESI.

[0471] 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J = 5.9 Hz, 4H), 7.45 (t, J =6.6 Hz, 1H), 7.14 (d, J = 6.1 Hz, 4H), 6.73 (t, J = 5.4 Hz, 1H), 3.88 (d, J =6.1 Hz, 2H), 2.85 (q, J = 6.6 Hz, 2H), 2.31 (t, J = 8.0 Hz, 2H), 1.94 (q, J =8.0 Hz, 4H), 1.36 (s, 9H), 1.32 (s, 9H), 1.31-1.23(m, 4H), 1.1-0.9(m, 2H). Compounds 11-8: Platinum oxide (2.2 g, 9.69 mmol) was added to a solution of compound 11-7 (4.5 g, 8.32 mmol) in AcOH (15 mL). The mixture was hydrogenated at ~0.4 MPa for 56 h. The catalyst was filtered through a diatomaceous earth bed and washed with MeOH. The mixture was concentrated under vacuum. The pH was adjusted to 7 by adding concentrated NaHCO3 aqueous solution, and then dried under vacuum. The residue was dissolved in THF, and the mixture was filtered. The filtrate was evaporated under reduced pressure to give 4 g of compound 11-8 as a colorless oil, in 87% yield.

[0472] C 30 H 56 The calculated mass of N4O5 is 552.4; the actual measured mass is 553.4 [M+H]. + , ESI.

[0473] Compounds 11-9: TEA (402.72 mg, 3.98 mmol) and HATU (1.50 g, 3.98 mmol) were added to a solution of Boc-6-aminocaproic acid (920.49 mg, 3.98 mmol) in DCM (10 mL). After stirring the solution for 20 min, compound 11-8 (1 g, 1.18 mmol) was added to the reaction mixture and stirring was continued for 5 h. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with DCM / MeOH = 15 / 1 to give 0.4 g of compound 11-9 as a white solid (yield 22%).

[0474] C 52 H 94 N6O 11 Calculated mass: 978.7; Actual measurement: 979.8 [M+H] + , ESI.

[0475] 1 H NMR (400 MHz, DMSO-d6) δ 7.41(t, J = 6.0 Hz, 1H), 6.78-6.70 (m,3H), 3.88 (d, J = 11.4 Hz, 2H), 3.02(d, J = 6.5 Hz, 6H), 2.92-2.82 (m, 8H), 2.25 (t, J = 7.4 Hz, 4H), 2.15 (t, J= 8.1 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H),1.70-1.50 (m, 6H), 1.49-1.38 (m, 10H), 1.38 (s, 9H), 1.36 (s, 27H), 1.34-1.15(m, 14H). Compounds 11-10: TFA (2 mL) was added to a solution of compound 11-9 (1.4 g, 1.43 mmol) in DCM (6 mL), and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated under vacuum to give 850 mg of compound 11-10 as a colorless oil in 95% yield.

[0476] C 33 H 62 The calculated mass of N6O5 is 622.5; the actual measured mass is 623.6 [M+H]. + , ESI.

[0477] Compound 11-11: TEA (584.84 mg, 5.78 mmol) and HATU (1.64 g, 4.33 mmol) were added to a solution of compound 1-4 (2.13 g, 4.77 mmol) in DMF (10 mL). After stirring for 20 minutes, compound 11-10 (0.9 g, 1.44 mmol) was added to the reaction mixture and stirring was continued for 5 hours. The mixture was purified by reversed-phase column chromatography (C18 column) to give 1.3 g of compound 11-11 as a white solid, in a yield of 47%.

[0478] C 90 H 143 N9O 35 Calculated mass: 1909.9; Actual mass: 956.0 [M+2H] 2+ , ESI.

[0479] 1 H NMR (400 MHz, DMSO-d6) δ 12.0 (br, 1H), 7.83 (d, J = 9.2 Hz, 3H), 7.70 (t, J = 5.5 Hz, 3H), 7.50-7.40 (m, 1H), 5.20 (d, J = 3.4 Hz, 3H), 4.96(dd, J= 11.24, 3.4 Hz, 3H), 4.50-4.40 (m, 5H), 4.08-3.96 (m, 9H), 3.92-3.82(m, 5H), 3.76-3.66 (m, 3H), 3.46-3.36 (m, 4H), 3.15-2.75 (m, 11H), 2.40-2.30(m, 2H), 2.25 (t, J = 7.3 Hz, 4H), 2.10 (s, 9H), 2.08-2.00 (m, 8H), 1.99 (s,9H), 1.98 (s, 9H), 1.77 (s, 9H), 1.60-1.08 (m, 42H). Compounds 11-12: Compound 11-11 (1.4 g, 0.73 mmol) was dissolved in DCM (15 mL), and then HOBT (148.47 mg, 1.10 mmol), EDCI (210.10 mg, 1.10 mmol), and DIEA (236.69 mg, 1.83 mmol) were added to the solution. After stirring for 15 minutes, compound 11-2 (503.47 mg, 0.88 mmol) was added to the reaction mixture and stirring was continued for 4 hours. The mixture was partitioned between DCM (50 mL) and water (35 mL), and the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by reversed-phase column chromatography (C18 column) to give 0.8 g of compound 11-12 as a white powder, in a yield of 44%.

[0480] C 125 H 185 N 11 O 39 Calculated mass: 2464.3; Actual measured mass: 1255.6 [M+2Na] 2+ , ESI.

[0481] 1 H NMR (400 MHz, DMSO-d6) δ 7.86-7.76 (m, 4H), 7.71 (t, J = 5.3 Hz, 3H), 7.45 (s, 1H), 7.41-7.35 (m, 2H), 7.34-7.28 (m, 2H), 7.27-7.18 (m, 5H), 6.93-6.86 (m, 4H), 5.27-5.15 (d, J= 3.3 Hz, 3H), 5.02-4.90 (dd, J = 11.2,3.4 Hz, 3H), 4.69-4.60 (m, 1H), 4.57-4.33 (m, 5H), 4.11-3.98 (m, 9H), 3.97(s, 1H), 3.93-3.81 (m, 5H), 3.73 (s, 6H), 3.72-3.62 (m, 5H), 3.51 (s, 1H), 3.46-3.36 (m, 5H), 3.14-2.73 (m, 14H), 2.43-2.29 (m, 2H), 2.29-2.19 (m, 4H), 2.01 (s, 9H), 2.06-1.97 (m, 19H), 1.96-1.91 (m, 3H), 1.87 (s, 9H), 1.86-1.80(m, 2H), 1.77 (s, 9H), 1.70-1.55 (m, 5H), 1.54-1.13 (m, 44H). Compounds 11-13: To a solution of compounds 11-12 (2.0 g, 0.8 mmol) in anhydrous DCM (7.0 mL), 4,5-dicyanimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphonium diamide (270 mg, 0.90 mmol) were added and stirred for 1 h. LC-MS showed complete consumption of the starting material. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na₂SO₄. The solution was then concentrated under reduced pressure, and the residue was purified by rapid preparative HPLC under the following conditions (column, C18 silica gel; mobile phase: CH₃CN / H₂O = 1 / 1 increased to CH₃CN / H₂O = 1 / 0 over 20 min; detector: UV 254 nm). Compounds 11-13 were produced as a white solid (1.0 g, 45% yield).

[0482] C 134 H 202 N 13 O 40 Calculated mass of P: 2664.39; Actual mass: 1132.5 [M-DMTr-N(CH(CH3)2)2+4H] 2+ , ESI.

[0483] 1 HNMR (600 MHz, DMSO-d 6) δ 7.82-7.69 (m, 6H), 7.44-7.19 (m, 9H), 6.90-6.87 (m, 4H), 5.21 (d, J = 6.0 Hz, 3H), 4.98-4.94 (m, 3H), 4.49-4.44 (m, 5H), 4.04-3.37 (m, 35H), 3.02-2.71 (m, 15H), 2.33-1.75 (m, 56H), 1.64-1.07 (m,57H). 31 PNMR (242 MHz, DMSO- d 6) δ 146.79, 146.67. Compounds 11-14: DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added to a solution of compounds 11-12 (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL), followed by succinic anhydride (20 mg, 0.2 mmol). The reaction mixture was stirred at room temperature for 3 hours. LCMS showed that the starting material was completely consumed. The reaction mixture was diluted with DCM, washed with water (3 mL x 4), and then washed with brine (3 mL x 4). The organic layer was concentrated to give compounds 11-14 (135 mg, 97% yield) as a white solid.

[0484] C 129 H 189 N 11 O 42 Calculated mass: 2564.30; Actual measurement: 1132.5 [M-DMTr+2H] 2+ , ESI.

[0485] 1 H NMR (600 MHz, DMSO- d 6): 7.84-7.71(m, 6H), 7.45-7.21 (m, 10H), 6.92-6.89 (m, 4H), 5.21 (d, J= 6.0 Hz, 3H), 4.98-4.94 (m, 3H), 4.49-4.44 (m, 5H), 4.07-3.37 (m, 36H), 3.16-2.84 (m, 14H), 2.44-1.77 (m, 62H), 1.45-1.21 (m,50H). Compounds 11-15: DIPEA (30 mg, 0.23 mmol) was added to a solution of compounds 11-14 (135 mg, 0.05 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL). The reaction mixture was shaken at room temperature for 10 min, and then natural amino-lcaa-CPG (300 mg, 75 μmol / g) was added. The suspension was shaken at room temperature for 20 h, then filtered and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 2 h to obtain a solid support (300 mg). The mixture was stirred at room temperature for 1 hour with Ac2O / pyridine / N-methylimidazole (90 µL / 1.0 mL / 80 µL). Unreacted amines on the support were capped and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no spots in the eluent at 254 nm. The solid support was vacuum dried for 15 hours to obtain solid support 11-15 (300 mg). For sample loading calculation, 5.00 mg of dried sample CPG was added to 20 mL of 3% DCA in DCM. The solution was shaken, and the UV absorbance at 500 nm was measured. The absorbance value was ensured to be below 1.0 unit to prevent signal saturation. The following formula was then applied: Sample loading (µmol / g) = (Total volume of DCA added (mL)) * (Abs value at 500 nm) * 1000) / (76 * (number of mg of CPG taken)) Total volume of DCA added (mL) = 20 mL Abs value at 500 nm = 0.464 The amount of Mg in the CPG sample was 5.0 mg. Loading sample (µmol / g) = ((20)*(0.464)*1000) / 76*(5.0) = 24 μmol / g. Compound 12-1: Compound 3-4 (1.1 g, 5.16 mmol) was dissolved in H₂O (10 mL) and THF (20 mL). K₂CO₃ (1.43 g, 10.31 mmol) and benzyl chloroformate (967 mg, 5.67 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. It was then concentrated under vacuum and purified by silica gel rapid column chromatography (PE / EA = 5 / 1) to give compound 12-1 as a white foamy solid (1.5 g, 84% yield).

[0486] C 20 H 29 Calculated NO4 mass: 347.2; Actual measured: 348.3 [M+H] + , ESI.

[0487] Compound 12-2: Compound 12-1 (1.52 g, 4.37 mmol), DMAP (147.10 mg, 1.20 mmol), and DIEA (3.11 g, 24.08 mmol, 4.19 mL) were dissolved in DCM (20 mL). A solution of DMTrCl (1.48 g, 4.37 mmol) in DCM (20 mL) was slowly added to the reaction mixture at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0–3% MeOH) on Al2O3 to give compound 12-4 as a white, foamy solid (1.9 g, 67% yield).

[0488] C 41 H 47 Calculated mass of NO6: 649.34; Actual measured mass: 650.34 [M+H] + , ESI.

[0489] 1 H NMR (400 MHz, DMSO-d6) δ 7.41-7.19 (m, 15H), 6.90-6.87 (m, 3H), 5.06-5.05 (m, 2H), 4.42-4.40 (m, 1H), 3.76 (s, 6H), 3.40-3.38 (m, 6H), 2.88(s, 2H), 1.39-1.11 (m, 12H). Compound 12-3: Compound 12-2 (0.7 g, 1.08 mmol) was dissolved in MeOH (30 mL), and then Pd / C (140 mg) was added to the solution. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude compound 12-3 as a white foamy solid (520 mg), which could be used for the next step without further purification.

[0490] C 33 H 41 Calculated NO4 mass: 515.30; Actual measured: 516.30 [M+H] + , ESI.

[0491] 1 H NMR (400 MHz, DMSO-d6) δ 7.38 (d, J = 8.8 Hz, 2H),7.32-7.20 (m,7H), 6.89-6.87 (m, 4H), 4.51-4.49 (m, 1H), 3.73 (s, 6H), 2.89-2.86 (m, 8H),1.52-1.51 (m, 2H), 1.19-1.12 (m, 7H), 0.91-0.88 (m, 2H). Compound 12-4: Compound 10-1 (1.56 g, 0.756 mmol) was dissolved in DCM (20 mL). DIEA (300.74 mg, 2.33 mmol), EDCI (297.39 mg, 1.55 mmol), and HOBT (209.62 mg, 1.55 mmol) were added to the mixture at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. Compound 12-3 (400 mg, 0.756 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-8% MeOH) on Al2O3 to give compound 12-4 as a white, foamy solid (1 g, 51% yield).

[0492] C 124 H 187 N 11 O 42 Calculated mass: 2503.28; Actual measurement: 1101.6 [M-DMTr+2H] + ] 2+ , ESI.

[0493] 1 H NMR (400 MHz, DMSO-d6) δ 7.85-7.81 (m,6H), 7.73 (t, J = 5.6 Hz, 3H), 7.39 (d, J = 7.32 Hz, 2H), 7.32-7.19 (m, 8H), 6.99 (s, 1H), 6.88 (d, J =8.9 Hz, 4H), 5.21-5.20 (m, 3H), 4.98-4.94 (m, 3H), 4.48 (d, J = 8.44 Hz, 3H), 4.42 (t, J = 5.0 Hz, 1H), 4.05-4.00 (m, 9H), 3.90-3.83 (m, 3H), 3.73-3.67 (m,9H), 3.55-3.52 (m, 11H), 3.43-3.38 (m, 6H), 3.22-3.21 (m, 6H), 3.06-3.00 (m,11H), 2.29-2.24 (m, 5H), 2.22 (t, J = 7.4 Hz, 2H), 2.10 (s, 8H), 2.05 (t, J =6.96 Hz, 8H), 1.99-1.98 (m, 9H), 1.89 (s, 8H), 1.77 (s, 8H), 1.52-1.44 (m, 22H), 1.30-1.22 (m, 24H). Compound 13-1: Compound 4-7 (1.3 g, 7.01 mmol) was dissolved in THF (30 mL) and H2O (15 mL). K2CO3 (2.91 g, 21.05 mmol) was added to this solution. Benzyl chloroformate (1.32 g, 7.72 mmol) was added to the reaction mixture at 0 °C. The mixture was stirred at room temperature for 3 hours. The reaction mixture was extracted with EA (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography (0-5% MeOH) to give compound 13-1 as a white foamy solid (1.95 g, 87% yield).

[0494] C 18 H25 Calculated NO4 mass: 319.2; Actual measured: 320.2 [M+H] + , ESI.

[0495] Compound 13-2: Compound 13-1 (2 g, 6.26 mmol) and DIEA (2.43 g, 18.79 mmol, 4.19 mL) were dissolved in DCM (60 mL). DMTrCl (2.02 g, 5.95 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (PE / EA = 0-40%) on Al2O3 to give compound 13-2 as a white, foamy solid (2.6 g, 66% yield).

[0496] Compound 13-3: Compound 13-2 (600 mg, 0.96 mmol) was dissolved in MeOH (20 mL), and then Pd / C was added to the reaction mixture. The reaction mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude product 13-3 as a white, foamy solid (470 mg, crude product), which could be used for the next step without further purification.

[0497] C 31 H 37 Calculated NO4 mass: 487.3; Actual measured mass: 488.3 [M+H] + , ESI.

[0498] Compound 13-4: Compound 10-1 (1.23 g, 0.61 mmol) was dissolved in DCM (20 mL). DIEA (239 mg, 1.85 mmol, 0.32 mL), EDCI (236 mg, 1.23 mmol), and HOBT (166 mg, 1.23 mmol) were added to the mixture at 0 °C. The reaction was stirred at 0 °C for 30 min. Compound 13-3 (300 mg, 0.61 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-8% MeOH) on Al2O3 to give compound 13-4 as a white, foamy solid (1 g, yield 66%).

[0499] 1 H NMR (400 MHz, DMSO-d6) δ 7.85-7.82(m, 6H), 7.75-7.73 (m, 3H), 7.40-7.38 (m, 2H), 733-7.29 (m, 2H), 7.27-7.25 (m, 4H), 7.23-7.19 (m, 1H),6.98 (s, 1H), 6.90-6.88 (m, 4H), 5.21 (d, J = 3.2 Hz, 3H), 4.97 (dd, J = 3.2,11.2 Hz, 3H), 4.63-4.61(m, 1H), 4.48 (d, J = 8.4 Hz, 3H), 4.04-4.00 (m, 9H), 3.91-3.83 (m, 3H), 3.73 (s, 6H), 3.71-3.67 (m, 3H), 3.55-3.52 (m, 12HH), 3.44-3.37 (m, 5H), 3.27-3.15 (m, 5H), 3.06-3.01 (m, 14H), 2.27 (t, J = 12.8Hz, 6H), 2.21 (d, J = 14.8 Hz, 2H), 2.10 (s, 9H), 2.04 (t, J= 6.8 Hz, 8H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.58-1.33 (m, 30H), 1.29-1.23 (m, 12H). Compound 14-1: Compound 5-6 (3.6 g, 19.27 mmol) was dissolved in THF (30 mL) and H2O (15 mL). K2CO3 (5.33 g, 38.54 mmol) was added to this solution. Benzyl chloroformate (3.62 g, 21.20 mmol) was added to the reaction mixture at 0 °C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EA (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid silica gel column chromatography (0-2% MeOH) to give compound 14-1 as a white foamy solid (5 g, 81% yield).

[0500] C 18 H 25 Calculated NO4 mass: 319.2; Actual measured: 320.2 [M+H] + , ESI.

[0501] 1 H NMR (400 MHz, DMSO) δ 7.39-7.28 (m, 5H), 5.01 (s, 2H), 4.27 (t, J = 5.4 Hz, 2H), 3.61-3.50 (m, 4H), 3.22 (d, J = 5.3 Hz, 4H), 1.55 (t, J = 6.1Hz, 4H), 1.25-1.22 (m, 8H). Compound 14-2: Compound 14-1 (3.5 g, 10.96 mmol) and DIEA (2.12 g, 16.44 mmol) were dissolved in DCM (60 mL). DMTrCl (3.34 g, 9.86 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (PE / EA = 0-60%) on Al2O3 to give compound 14-2 as a white, foamy solid (5.5 g, 81% yield).

[0502] 1 H NMR (400 MHz, DMSO-d6) δ 7.39-7.18 (m, 14H), 6.88 (d, J = 8.8 Hz,4H), 5.00 (s, 2H), 4.39 (t, J = 4.9 Hz, 1H), 3.73 (s, 6H), 3.58-3.35 (m, 6H), 2.83 (s, 2H), 1.29-1.15 (m, 8H). Compound 14-3: Compound 14-2 (254 mg, 408.52 μmol) was dissolved in MeOH (10 mL), and then Pd / C was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude product 14-3 as a white, foamy solid (199 mg, crude product), which could be used for the next step without further purification.

[0503] C 31 H 37 Calculated NO4 mass: 487.3; Actual measured mass: 488.3 [M+H] + , ESI.

[0504] Compound 14-4: Compound 10-1 (820 mg, 408.51 μmol) was dissolved in DCM (20 mL). DIEA (158 mg, 1.23 mmol), EDCI (157 mg, 817 μmol), and HOBT (110 mg, 817 μmol) were added to the mixture at 0 °C. The reaction was stirred at 0 °C for 30 min. Compound 14-3 (199 mg, 408.51 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-6% MeOH) on Al2O3 to give compound 14-4 as a white foamy solid (540 mg, 53% yield).

[0505] 1 H NMR (400 MHz, DMSO-d6) δ 7.89-7.80 (m, 6H), 7.75 (t, J = 5.5 Hz, 3H), 7.37 (d, J = 7.3 Hz, 2H), 7.30 (t, J = 7.7 Hz, 2H), 7.23-7.17 (m, 5H), 6.99 (s, 1H), 6.87 (d, J = 8.4 Hz, 4H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 10.8, 3.4 Hz, 3H), 4.47(d, J = 8.5 Hz, 3H), 4.41(t, J = 4.9Hz, 1H), 4.04-3.98 (m, 9H), 3.91-3.82 (m, 3H), 3.74-3.48 (m, 23H), 3.43-3.36 (m, 6H), 3.06-2.98 (m, 12H), 2.83 (d, J = 9.4 Hz, 2H), 2.27 (t, J = 6.3Hz, 6H), 2.09 (s,9H), 2.03 (t, J= 7.0 Hz, 8H), 2.00-1.95 (m, 12H), 2.01 (s, 9H), 1.89 (s,9H), 1.77 (s, 9H), 1.54-1.37 (m, 22H), 1.31-1.14 (m, 20H). Compound 15-1: Compound 7-6 (3.8 g, 18.30 mmol) was dissolved in THF (30 mL) and H2O (15 mL). K2CO3 (5.06 g, 36.59 mmol) was added to the reaction mixture. Benzyl chloroformate (3.43 g, 20.13 mmol) was added to the reaction mixture at 0 °C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with EA (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, and concentrated under vacuum to give crude product 15-1, a white foam (3.46 g, crude product).

[0506] C 17 H 23 Calculated NO4 mass: 305.2; Actual measured: 306.2 [M+H] + , ESI.

[0507] Compound 15-2: Compound 15-1 (3.07 g, 10.06 mmol) and DIEA (1.95 g, 15.09 mmol) were dissolved in DCM (60 mL). DMTrCl (3.41 g, 10.06 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (PE / EA = 0-60%) on Al2O3 and preparative HPLC to give compound 15-2a as a white frothy solid (1.04 g, 34% yield) and compound 15-2b (1.78 g, 58% yield).

[0508] 15-2a: 1 H NMR (400 MHz, DMSO-d6) δ 7.38-7.36 (m, 1H), 7.36-7.31 (m, 5H), 7.31-7.20 (m, 7H), 7.20-7.15 (m, 1H), 6.85 (d,J = 8.9 Hz, 4H), 5.02 (d, J =6.1 Hz, 2H), 6.98 (s, 1H), 4.61 (t, J = 5.0 Hz, 1H), 3.71 (s, 6H), 3.46-3.35(m, 2H), 3.27 (d, J = 5.0 Hz, 2H H), 3.13-3.05 (m, 2H), 2.93 (s, 2H), 2.64-2.55 (m, 2H), 1.72-1.62 (m, 2H), 1.30-1.21 (m, 2H). 15-2b: 1 H NMR (400 MHz, DMSO-d6) δ 7.40-7.27 (m, 9H), 7.27-7.22 (m, 4H), 7.22-7.16 (m, 1H), 6.89 (d, J = 8.9 Hz, 4H), 5.03 (s, 2H), 6.98 (s, 1H), 4.64(t, J = 5.1 Hz, 1H), 3.73 (s, 6H), 3.43-3.34 (m, 4H), 3.19-3.09 (m, 2H H), 3.13-3.05 (m, 2H), 2.84 (s, 2H), 2.41-2.31 (m, 2H), 1.62-1.54 (m, 2H), 1.38-1.30 (m, 2H). Compound 15-3: Compound 15-2a (210 mg, 350.48 μmol) was dissolved in MeOH (10 mL), and then Pd / C was added to the solution. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude product 15-3a as a white, foamy solid (166 mg, crude product), which could be used for the next step without further purification.

[0509] C 30 H 35 Calculated NO4 mass: 473.3; Actual measured: 474.3 [M+H] + , ESI.

[0510] Compound 15-2b (610 mg, 1.00 mmol) was dissolved in MeOH (10 mL), and then Pd / C was added to the solution. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction mixture was filtered and concentrated under vacuum to give crude product 15-3b as a white, foamy solid (475 mg, crude product), which could be used for the next step without further purification.

[0511] Compound 15-4: Compound 10-1 (700 mg, 348.92 μmol) was dissolved in DCM (20 mL). DIEA (135 mg, 1.05 mmol), EDCI (134 mg, 698 μmol), and HOBT (94 mg, 698 μmol) were added to the mixture at 0 °C. The reaction was stirred at 0 °C for 30 min. Compound 15-3a (199 mg, 408.51 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-6% MeOH) on Al2O3 to give compound 15-4a as a white, foamy solid (550 mg, 64% yield).

[0512] 1 H NMR (400 MHz, DMSO-d6) δ 7.88-7.80 (m, 6H), 7.75 (t, J = 5.4 Hz,3H), 7.38-7.34 (m, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.24-7.17 (m, 5H), 7.00 (s,1H), 6.87 (d, J = 8.9 Hz, 4H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.3,3.4 Hz, 3H), 4.64-4.60 (m, 1H), 4.47 (d, J= 8.5 Hz, 3H), 4.05-3.98 (m, 9H), 3.92-3.83 (m, 3H), 3.76-3.65 (m, 9H), 3.62-3.45 (m, 13HH), 3.45-3.37 (m, 6H), 3.28-3.23 (m, 2H), 3.17-3.09 (m, 2H), 3.07-2.98 (m, 12H), 2.97-2.89 (m, 2H), 2.69-2.53 (m, 2H), 2.27 (t, J = 6.2 Hz, 6H), 2.16-2.07 (m,11H), 2.06-1.96 (m,17H), 1.88 (s, 9H), 1.78 (s, 9H), 1.57-1.36 (m, 22H), 1.26-1.15 (m, 14H). Compound 10-1 (1 g, 500.29 μmol) was dissolved in DCM (20 mL). DIEA (194 mg, 1.50 mmol), EDCI (192 mg, 1.00 mmol), and HOBT (135 mg, 1.00 mmol) were added to the mixture at 0 °C. The reaction was stirred at 0 °C for 30 min. Compound 15-3b (238 mg, 502.53 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-6% MeOH) on Al2O3 to give compound 15-4b as a white foamy solid (550 mg, 44% yield).

[0513] 1 H NMR (400 MHz, DMSO-d6) δ 7.87-7.80 (m, 4H), 7.74 (t, J = 5.6 Hz,3H), 7.40-7.36 (m, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.27-7.18 (m, 5H), 6.99 (s,1H), 6.91-6.87 (m, 4H), 5.22 (d, J = 3.4 Hz, 3H), 4.96 (dd, J= 11.3, 3.4 Hz,3H), 4.66-4.62 (m, 1H), 4.48 (d, J = 8.4 Hz, 3H), 4.05-3.98 (m, 9H), 3.92-3.83 (m, 3H), 3.75-3.67 (m, 9H), 3.57-3.47 (m, 11HH), 3.46-3.36 (m, 6H),3.34-3.27 (m,4H), 3.23-3.09 (m, 2H), 3.07-2.98 (m, 12H), 2.85 (s, 2H), 2.46-2.31 (m, 2H), 2.30-2.24 (m, 6H), 2.18-2.12 (m, 2H), 2.09 (s, 9H), 2.06-2.01(m, 8H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.55-1.37 (m, 22H), 1.33-1.14 (m, 14H). Compound 16-1: Compounds 6-8 (190 mg, 0.403 mmol) and 10-1 (809 mg, 0.403 mmol) were dissolved in DCM (10 mL), and then DIEA (156.35 mg, 1.21 mmol), EDCI (154.61 mg, 0.81 mmol), and HOBT (108.97 mg, 0.81 mmol) were added to the mixture. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-10% MeOH) on Al2O3 to give compound 16-1 as a white foamy solid (772 mg, 77% yield).

[0514] C 121 H 179 N 11 O 42 Calculated mass: 2458.22; Actual measurement: 1079.2 [M-DMTr+2H] 2+ , ESI.

[0515] 1 H NMR (400 MHz, DMSO-) d6) δ 7.85-7.82 (m, 6H), 7.75-7.72 (m, 3H), 7.38-7.36 (m, 2H), 7.29-7.27 (m, 2H), 7.25-7.22 (m, 5H), 6.98 (s, 1H), 6.87 (d, J = 8.0 Hz, 4H), 5.21 (d, J = 4.0 Hz, 3H), 4.97 (dd, J = 3.2, 8.0 Hz, 3H),4.80-4.70 (m, 1H), 4.48 (d, J = 8.4 Hz, 3H), 4.02-4.01 (m, 11H), 3.88-3.86(m, 5H), 3.73-3.69 (m, 9H), 3.55-3.52 (m, 12H), 3.46-3.36 (m, 5H), 3.17 (d, J = 5.2 Hz, 1H), 3.04-3.00 (m, 14H), 2.29-2.26 (m, 10H), 2.10-2.07 (m, 8H), 2.06-2.02 (m, 19H), 1.99 (s, 9H), 1.77 (s, 9H), 1.52-1.46 (m, 22H), 1.45-1.22(m, 12H). Compound 17-1: Compounds 11-11 (1.5 g, 0.78 mmol), DIEA (302.32 mg, 2.35 mmol), HOBT (212.11 mg, 1.57 mmol), and EDCI (300.92 mg, 1.57 mmol) were dissolved in DCM (15 mL) at 0 °C, followed by the addition of compound 3-12 (493.56 mg, 0.78 mmol). The mixture was stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (20 mL), washed with NaHCO3 (50 mL saturated aqueous solution) and water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to give compound 17-1 (1.14 g, 58% yield).

[0516] C 129 H 193 N 11 O 39Calculated mass: 2520.35, Actual measurement: 1109.35 (M-DMTr+2H) 2+ , ESI.

[0517] 1 H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J = 9.2 Hz, 4H), 7.73 (t, J =9.2 Hz, 3H), 7.47-7.39 (m, 3H), 7.33-7.19 (m, 7H), 6.88 (d, J = 8.92 Hz, 4H), 5.21 (d, J = 3.3 Hz, 3H), 4.98-4.94 (m, 3H), 4.49-4.42 (m, 5H), 4.02 (s, 9H), 3.91-3.86 (m, 5H), 3.73-3.68 (m, 9H), 3.43-3.39 (m, 5H), 3.34-3.30 (m, 3H), 3.02-2.99 (m, 10H), 2.87 (s, 4H), 2.34-2.33 (m, 2H), 2.25-2.21 (m, 6H), 2.10 (s, 9H), 2.10-2.08 (m, 8H), 2.0 (s, 9H), 1.99 (s, 3H), 1.89 (s, 9H), 1.77 (s,9H), 1.45-1.24 (m, 64H). Compound 18-1: At 0 °C, DIEA (193.61 mg, 1.85 mmol, 0.32 mL), EDCI (191.45 mg, 0.99 mmol), and HOBT (134.95 mg, 0.99 mmol) were added to a solution of compound 11-11 (1.01 g, 0.45 mmol) in DCM (20 mL). The reaction mixture was stirred at 0 °C for 30 min. Compound 4-10 (300 mg, 0.49 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-8% MeOH) on Al2O3 to give compound 18-1 as a white foamy solid (500 mg, 46% yield).

[0518] C 127 H 189 N 11 O 39 Calculated mass: 2493.31; Actual measurement: 1096.7 [M-DMTr+2H] 2+ , ESI.

[0519] 1 H NMR (400 MHz, DMSO-d6) δ 7.84-7.78 (m, 2H), 7.72 (t, J = 5.4 Hz,3H), 7.45 (br, 1H), 7.40-7.39 (m, 2H), 7.33-7.30 (m, 2HH), 7.28-7.20 (m, 5H),5.22 (d, J = 3.4 Hz, 3H), 4.99-4.95 (m, 3H), 4.64-4.62 (m, 3H), 4.49 (d, J =8.4 Hz, 2H), 4.03 (s, 9H), 3.91-3.84 (m, 5H), 3.74 (s, 6H), 3.72-3.68 (m,3H), 3.45-3.41 (m, 2H), 3.40-3.34 (m, 4H), 3.28-3.16 (m, 4H), 3.01-2.99 (m,11H), 2.85-2.67 (m, 4H), 2.34-2.32 (m, 2H), 2.27-2.22 (m, 6H), 2.07-2.00 (m,17H), 1.89 (s, 9H), 1.78 (s, 9H), 1.64-1.56 (m, 4H), 1.55-1.34 (m, 36H),1.28-1.21(m, 16H). Compound 19-1: Compound 11-11 (1.59 g, 832.24 μmol) (3.2 g, 7.16 mmol) and compound 5-9 (500 mg, 832.24 μmol) were dissolved in DCM (50 mL). DIEA (215.12 mg, 1.66 mmol, 289.92 μL), EDCI (239.31 mg, 1.25 mmol), and HOBT (112.45 mg, 832.24 μmol) were added to the mixture. The reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched with water (40 mL), extracted with DCM (50 mL x 2), washed with brine (50 mL), dried over Na2SO4, concentrated under vacuum, and purified by HPLC to give compound 19-1 (840 mg, 36% yield).

[0520] C 127 H 189 N 11 O 39 Calculated mass: 2492.3; Actual measured mass: 1258.4 [M+2Na] 2+ , ESI.

[0521] 1 H NMR (400 MHz, DMSO-d6) δ 7.85-7.71 (m, 7H), 7.49 (s, 1H), 7.37-7.20 (m, 9H), 6.82-6.87 (m, J = 8.8 Hz, 4H), 5.22 (d, J = 3.2 Hz, 3H), 4.97(d, J = 11.6 Hz, 3H), 4.49-4.46 (m, 6H), 4.05 (s, 9H), 3.91-3.85 (m, 5H), 3.77-3.62 (m, 11H), 3.41 (s, 6H), 3.01-2.71 (m, 14H), 2.32-2.25 (m, 6H), 2.11-1.78 (m, 48H), 1.62-1.34 (m, 37H), 1.26-1.21 (m, 20H). Compound 20-1a: Compound 11-11 (554 mg, 289.73 μmol) was dissolved in DCM (20 mL). DIEA (112 mg, 869.18 μmol), EDCI (222 mg, 1.16 mmol), and HOBT (157 mg, 1.16 mmol) were added to the mixture at 0 °C. The reaction was stirred at 0 °C for 30 min. Compound 7-9a (170 mg, 289.73 μmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated, and purified by preparative HPLC (C18 column, ACN / water) to give compound 20-1a as a white foamy solid (640 mg, 89% yield).

[0522] 1 H NMR (400 MHz, DMSO-d6) δ 7.86-7.76 (m, 4H), 7.72 (t, J = 5.4 Hz,3H), 7.49-7.42 (m, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H),7.27-7.19 (m, 5H), 6.88 (d, J = 8.9 Hz, 4H), 5.21 (d, J = 3.4 Hz, 3H), 4.96(dd, J = 11.2, 3.4 Hz, 3H), 4.65 (t, J = 5.0 Hz, 1H), 4.50-4.41(m, 5H), 4.05-3.99 (m, 9H), 3.91-3.82 (m, 5H), 3.76-3.66 (m, 9H), 3.49-3.36 (m, 6H), 3.32-3.28 (m, 1H), 3.23-3.09 (m, 2H), 3.08-2.91(m, 10H), 2.84 (s, 2H), 2.44-2.21(m, 8H), 2.20-1.92 (m, 30H), 1.88 (s, 9H), 1.77 (s, 9H), 1.70-1.08 (m, 54H). Compound 20-1b: Compound 11-11 (1.3 g, 681.71 μmol) was dissolved in DCM (20 mL). DIEA (176 mg, 1.36 mmol), EDCI (261 mg, 1.36 mmol), and HOBT (138 mg, 1.02 mmol) were added to the mixture at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. Compound 7-9b (400 mg, 681.71 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h.

[0523] The reaction mixture was quenched with saturated NaHCO3 solution (40 mL), extracted with DCM (30 mL x 2), washed with brine (30 mL), dried over Na2SO4, concentrated, and purified by preparative HPLC (C18 column, ACN / water) to give compound 20-1b as a white foamy solid (610 mg, yield 36%).

[0524] 1 H NMR (400 MHz, DMSO-d6) δ: 7.87-7.76 (m, 4H), 7.72 (t, J = 5.3 Hz,3H), 7.46 (s, 1H), 7.35(d, J = 7.4 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.25-7.18 (m, 5H), 6.86 (d, J = 8.9 Hz, 4H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.62 (t, J= 5.0 Hz, 1H), 4.50-4.38 (m, 5H), 4.07-3.98(m, 9H), 3.92-3.82 (m, 5H), 3.75-3.66 (m, 9H), 3.51-3.36 (m, 6H), 3.29-3.23(m, 2H), 3.15-3.07 (m, 2H), 3.01-2.94 (m, 10H), 2.93-2.88 (m, 2H), 2.87-2.78(m, 2H), 2.68-2.60 (m, 1H), 2.37-2.23 (m, 2H), 2.29-2.19 (m, 4H), 2.13-2.08(m, 10H), 2.07-1.98 (m, 18H), 1.88 (s, 9H), 1.77 (s, 9H), 1.71-1.58 (m, 6H), 1.53-1.13 (m, 48H). Compound 21-1: Compound 11-11 (1.76 g, 920.31 μmol), HOBt (248.70 mg, 1.84 mmol), and EDCI (529.27 mg, 2.76 mmol) were dissolved in DCM (20 mL). The mixture was stirred for 30 minutes, and then compound 8-9 (540 mg, 920.31 μmol) was added. The reaction mixture was stirred for another 2 hours, then concentrated and purified by preparative HPLC to give compound 21-1 (1.2 g, 53% yield).

[0525] C 126 H 187 N 11 O 39 Calculated mass: 2478.3; Actual measurement: 1089.6 [M-DMTr+2H] 2+ , ESI.

[0526] 1 H NMR (400 MHz, DMSO-d6) δ 7.84-7.80 (m, 4H), 7.72 (t, J = 5.4 Hz, 3H), 7.39 (s, 1H), 7.40-7.37 (m, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.27-7.20(m, 5H), 6.98 (d, J= 8.9 Hz, 4H), 5.22 (d, J = 3.4 Hz, 3H), 4.97 (dd, J =3.4, 7.8 Hz, 3H), 4.67 (t, J = 4.8 Hz, 1H), 4.48 (d, J = 8.5 Hz, 3H), 4.47-4.42 (m, 2H), 4.05-3.98 (m, 9H), 3.92-3.83 (m, 5H), 3.73 (s, 6H), 3.72-3.67(m, 3H), 3.55-3.45 (m, 2H), 3.44-3.35 (m, 5H), 3.05-2.93 (m, 11H), 2.91-2.80(m, 3H), 2.65-2.55 (m, 2H), 2.38-2.20 (m, 9H), 2.10 (s, 9H), 2.08-2.01 (m,9H), 2.00 (s, 9H), 1.89 (s, 9H), 1.78 (s, 9H), 1.69-1.52 (m, 6H), 1.47-0.98(m, 48H). Compound 22-1: Compounds 6-10 (410 mg, 0.71 mmol) and 11-11 (1.34 g, 0.71 mmol) were dissolved in DCM (60 mL), and then DIEA (453.09 mg, 3.51 mmol), EDCI (299.57 mg, 1.75 mmol), and HOBT (236.85 mg, 1.75 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched with saturated NaHCO3 solution (100 mL), extracted with DCM (60 mL x 3), washed with brine (60 mL), dried over Na2SO4, concentrated under vacuum, and purified by rapid column chromatography (0-20% MeOH) on Al2O3 to give compound 22-1 as a white frothy solid (1.2 g, yield 69%).

[0527] C 126 H 185 N 11 O 39 Calculated mass: 2476.28; Actual measurement: 1088.2 [M-DMTr+2H] 2+ , ESI.

[0528] 1 H NMR (400 MHz, DMSO- d 6) δ 7.85-7.81 (m, 3H), 7.75-7.72 (m, 3H), 7.47(s, H), 7.39-7.37 (m, 2H), 7.32-7.25 (m, 2H),7.24-7.19 (m, 5H), 6.88 (d, J =8.84 Hz, 4H), 5.22 (d, J = 3.4 Hz, 2H), 4.97 (d, J = 11.2 Hz, 3H), 4.75 (t, J = 4.4 Hz, 1H), 4.50-4.48 (m, 5H), 4.03 (s, 11H), 3.94-3.80 (m, 7H), 3.77-3.66(m, 9H), 3.50-3.41 (m, 6H), 3.02-3.00 (m, 12H), 2.92-2.79 (m, 2H), 2.43-2.30(m, 2H), 2.29-2.22 (m, 2H), 2.21-2.19 (m, 3H), 2.11-2.08 (m, 11H), 2.05-2.02(m, 8H), 2.00 (s, 9H), 1.90 (s, 9H), 1.78 (s, 9H), 1.62-1.23 (m, 48H). Compound 23-1: N A solution of 4-piperidinone (5 g, 25.09 mmol), malononitrile (2.49 g, 37.64 mmol), AcNH2 (3.87 g, 50.19 mmol), and AcOH (4.52 g, 75.28 mmol) in toluene (50 mL) was stirred at 110 °C for 2 h. LC-MS showed complete conversion. The reaction mixture was washed with water (50 mL), dried over Na2SO4, concentrated, and purified by silica gel column chromatography (0–10% EA solution in PE) to give compound 23-1 (6.18 g, 98% yield) in a gel form.

[0529] C 13 H 17 The calculated mass of O2N3 is 247.1 mmol; the actual measured mass is 246.2 mmol [MH]. - , ESI.

[0530] 1 H NMR (400 MHz, DMSO-d6) δ 3.53 (t, J = 11.6 Hz, 4H), 2.69 (d, J =11.6 Hz, 4H), 7.41 (t, J = 7.3 Hz, 2H), 1.427 (s, 9H). Compound 23-2: Compound 23-1 (7.6 g, 30.73 mmol) was stirred in a solution of MeOH (76 mL) at 0 °C. NaBH4 (1.16 g, 30.73 mmol) was added in portions to the stirred solution, and the mixture was stirred for 20 min. The mixture was concentrated under vacuum. The residue was partitioned between ethyl acetate (250 mL) and water (150 mL), and the ethyl acetate extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with PE / EA = 1 / 5 to give a gel-like compound 23-2 (4.6 g, 60% yield).

[0531] C 13 H 19 The calculated mass of O2N3 is 249.2; the actual measured mass is 248.3 [MH]. - , ESI.

[0532] 1 H NMR (400 MHz, DMSO-d6) δ 4.04-4.01 (m, 2H), 2.75 (s, 2H), 2.31-2.27 (m, 1H), 1.82-1.78 (m, 2H), 1.04 (s, 9H), 1.27-1.16 (m, 3H). Compound 23-3: A mixture of compound 23-2 (25 g, 100.28 mmol), tert-butyl 3-bromopropionate (41.93 g, 200.56 mmol), t-BuOK (22.50 g, 200.56 mmol), and KI (33.29 g, 200.56 mmol) in DMSO (250 mL) was stirred overnight at 60 °C. LC-MS showed complete conversion. The mixture was concentrated under vacuum. The residue was partitioned between ethyl acetate (100 mL × 3) and water (150 mL × 3), the ethyl acetate extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography, eluting with PE / EA = 10 / 1 to give compound 23-3 (32.87 g, 87% yield) in a gel.

[0533] C 20 H 31 The calculated mass of O4N3 is 377.2; the actual measured mass is 319.4 [M-Boc+MeCN+H]. + , ESI.

[0534] 1 H NMR (400 MHz, DMSO-d6) δ 4.06 (d, J = 11.2 Hz, 2H), 2.75 (s, 2H), 2.55-2.53 (m, 1H), 2.35-2.31 (m, 3H), 1.89 (d, J = 12.4 Hz, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.27-1.19 (m, 3H). Compounds 23-4: Cobalt chloride hexahydrate (40.34 g, 169.55 mmol) was added to a solution of compound 23-3 (16 g, 42.39 mmol) in MeOH (400 mL). Then, sodium borohydride (32.07 g, 847.73 mmol) was slowly added to the reaction mixture at -20 °C, and the mixture was stirred for 1 hour. The mixture was then stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness and azeotropically dried with DMF to give crude compound 23-4, which could be used directly for the next step without further purification.

[0535] C 20 H 39 The calculated mass of O4N3 is 385.5; the actual measured mass is 386.5 [M+H]. + , ESI.

[0536] Compound 23-5: A solution of Z-6-aminohexanoic acid (27.53 g, 103.75 mmol), TEA (12.6 g, 124.50 mmol), and HATU (46.97 g, 124.50 mmol) in DMF (240 mL) was stirred at room temperature for 30 min. Compound 23-4 (16 g, 41.50 mmol) was added, and the mixture was stirred overnight at room temperature. LC-MS showed complete conversion. Water (50 mL) and EA (50 mL) were added to the reaction mixtur...

Claims

1. A siRNA agent comprising double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C3 (C3), wherein the dsRNA comprises a first strand and a second strand, wherein the first strand sequence comprises at least 15 consecutive nucleotides, the consecutive nucleotides differing from any one of the nucleotide sequences 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500 or 4698-4716 of SEQ ID NO: 1 by no more than 3 nucleotides; and the second strand comprises a nucleotide sequence at least partially complementary to the first strand.

2. The siRNA agent according to claim 1, wherein the first strand sequence comprises at least 16, preferably at least 17, more preferably at least 18, and most preferably all 19 consecutive nucleotides, wherein the consecutive nucleotides differ from any of the nucleotide sequences 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 of SEQ ID NO: 1 by no more than 2 nucleotides, preferably by no more than 1 nucleotide, and more preferably by no nucleotide difference.

3. The siRNA agent according to claim 1 or 2, wherein the first strand and the second strand form a double-stranded region of 15-25 nucleotides in length; preferably, a double-stranded region of 15-23 nucleotides in length; more preferably, a double-stranded region of 15-19 nucleotides in length; even more preferably, a double-stranded region of 17-19 nucleotides in length; and most preferably, a double-stranded region of 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

4. The siRNA agent according to any one of claims 1-3, wherein the first strand has nucleotide sequences represented by SEQ ID NO: 20, 96, 16, 150, 32, 100, 110, 134, 136 respectively; and / or the second strand complementary to the first strand has nucleotide sequences represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, 137 respectively.

5. The siRNA agent according to any one of claims 1-4, wherein the first strand has the nucleotide sequence shown in SEQ ID NO: 20 and the second strand has the nucleotide sequence shown in SEQ ID NO: 21; or the first strand has the nucleotide sequence shown in SEQ ID NO: 96 and the second strand has the nucleotide sequence shown in SEQ ID NO: 97; or the first strand has the nucleotide sequence shown in SEQ ID NO: 16 and the second strand has the nucleotide sequence shown in SEQ ID NO: 17; or the first strand has the nucleotide sequence shown in SEQ ID NO: 150 and the second strand has the nucleotide sequence shown in SEQ ID NO: 151; or the first strand has the nucleotide sequence shown in SEQ ID NO: 32 and the second strand has the nucleotide sequence shown in SEQ ID NO: 33; or the first strand has the nucleotide sequence shown in SEQ ID NO: 100 and the second strand has the nucleotide sequence shown in SEQ ID NO: 101; or the first strand has the nucleotide sequence shown in SEQ ID NO: 110 and the second strand has the nucleotide sequence shown in SEQ ID NO: 111; or the first strand has the nucleotide sequence shown in SEQ ID NO: 134 and the second strand has the nucleotide sequence shown in SEQ ID NO: 135; or the first strand has the nucleotide sequence shown in SEQ ID NO: 20 and the second strand has the nucleotide sequence shown in SEQ ID NO: 21; or the first strand has the nucleotide sequence shown in SEQ ID NO: 134 and the second strand has the nucleotide sequence shown in SEQ ID NO: 135; or the first strand has the nucleotide sequence shown in SEQ ID NO: 16 and the second strand has the nucleotide sequence shown in SEQ ID NO: 17; or the first strand has the nucleotide sequence shown in SEQ ID NO: 150 and the second strand has the nucleotide sequence shown in SEQ ID NO: 151; or the first strand has the nucleotide sequence shown in The nucleotide sequence shown in NO: 136 and the second strand have the nucleotide sequence shown in SEQ ID NO:

137.

6. The siRNA agent according to any one of claims 1-5, wherein the siRNA agent comprises at least one modified nucleotide; preferably, wherein not all of them are modified and comprises no more than 5, 4, 3, 2 or 1 unmodified nucleotide; more preferably, wherein all nucleotides of the first chain are modified nucleotides and all nucleotides of the second chain are modified nucleotides.

7. The siRNA agent according to claim 6, wherein at least one modified nucleotide is selected from 3'-terminal deoxythymidine (dT) nucleotide, 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 2'-OCF2H modified nucleotide, nucleotide containing a 5'-thiophosphate group, and nucleotide containing a 5'-(E)-vinylphosphonate.

8. The siRNA agent according to claim 6 or 7, wherein: (i) All nucleotides of the first chain contain modifications in the following pattern: The pentose sugars at positions 5, 7, 8, and 9 of the nucleotide sequence starting from the 5' end are modified with 2′-fluorine substitution. The pentose sugars at positions 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18 and 19 of the nucleotide from the 5' end are modified by 2′-methoxylation. The pentose sugars at positions 3, 11, and 14 of the nucleotides starting from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution. The second strand contains all nucleotides modified in the following pattern: The pentose sugars at positions 2, 14, 16, and 18 of the nucleotide sequence starting from the 5' end are modified with 2′-fluorine substitution. The pentose sugars at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17 and 19 of the nucleotide sequence starting from the 5' end are modified by 2′-methoxylation. The pentose sugar at positions 4, 8, and 10 of the 5' end is modified with 2′-methoxy or 2′-fluorine substitution; or (ii) All nucleotides of the first chain contain modifications in the following pattern: The pentose sugars at positions 7, 9, 10, and 11 of the nucleotides from the 5' end are modified with 2′-fluorine substitution. The pentose sugars at positions 2, 4, 6, 8, 12, 14, 16, and 18 of the nucleotide sequence starting from the 5' end are modified by 2′-methoxylation. The pentose sugars at positions 1, 3, 5, 13, 15, 17, and 19 of the nucleotide from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution. The second strand contains all nucleotides modified in the following pattern: The pentose sugars at positions 2, 6, 8, 14, and 16 of the nucleotide sequence starting from the 5' end are modified with 2′-fluorine substitution. The pentose sugars at positions 1, 3, 5, 7, 11, 12, 13, 15, 17, and 19 of the nucleotide sequence starting from the 5' end are modified by 2′-methoxy groups. The 4th, 9th, 10th and 18th positions from the 5' end are modified with 2′-methoxy or 2′-fluorine substitution.

9. The siRNA agent according to claim 8, wherein: (i) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 5, 7, 8, 9 and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. The second strand comprises all nucleotides modified in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxylation; or (ii) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 3, 5, 7, 8 and 9 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. All nucleotides in the second strand contain modifications in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxylation; or (iii) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 5, 7, 8, 9 and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. The second strand comprises all nucleotides modified in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxylation; or (iv) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 5, 7, 8, 9 and 14 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. The second strand comprises all nucleotides modified in the following pattern: the pentose sugars at positions 2, 8, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxylation; or (v) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 5, 7, 8, 9 and 14 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. The second strand comprises all nucleotides modified in the following pattern: the pentose sugars at positions 2, 4, 14, 16, and 18 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified with 2′-methoxylation; or (vi) All nucleotides of the first chain are modified in the following pattern: the pentose sugars at positions 7, 9, 10 and 11 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end are modified with 2′-methoxy. The second strand contains all nucleotides modified in the following pattern: the pentose sugars at positions 2, 6, 8, 9, 14 and 16 from the 5' end are modified with 2′-fluorine substitution; the pentose sugars at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 from the 5' end are modified with 2′-methoxy.

10. The siRNA agent according to any one of claims 1-9, wherein the second strand comprises a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end.

11. The siRNA agent according to any one of claims 1-10, wherein: The first chain contains two phosphate thioester nucleotide bonds at the 5' end and / or the 3' end; and / or the second chain contains two phosphate thioester nucleotide bonds at the 5' end and / or the 3' end; Preferably, (i) The first chain contains two phosphate thioester nucleotide bonds at its 3' end; And the second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; or (ii) The first chain contains two phosphate thioester nucleotide bonds at its 5' end; The second chain contains two thiophosphate nucleotide inter-bonds at the 5' end and two thiophosphate nucleotide inter-bonds at the 3' end.

12. The siRNA agent according to any one of claims 1-11, further comprising a targeting ligand; preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) conjugate or a derivative thereof.

13. The siRNA agent of claim 12, wherein the targeting ligand is conjugated to the 3' end or 5' end of the first strand of the siRNA agent.

14. The siRNA agent according to any one of claims 12 or 13, wherein: The targeting ligand is conjugated to the 5' end of the first chain; The first chain contains two phosphate thioester nucleotide bonds at its 3' end; The second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; Optionally, all remaining bonds between nucleotides in the first chain and / or the second chain are phosphodiester bonds; or The targeting ligand is conjugated to the 3' end of the first chain; The first chain contains two phosphate thioester nucleotide bonds at its 5' end; The second chain contains two phosphate thioester nucleotide bonds at the 5' end and two phosphate thioester nucleotide bonds at the 3' end; Optionally, all remaining bonds between nucleotides in the first chain and / or the second chain are phosphodiester bonds.

15. The siRNA agent according to any one of claims 12-14, wherein: The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 208) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-GN (SEQ ID NO: 210) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-GN (SEQ ID NO: 212) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by GN-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 213) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 214) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 211); or The first chain has a nucleotide sequence represented by GN-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 215) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 209); or The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 216) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-GN (SEQ ID NO: 218) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-GN (SEQ ID NO: 220) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first chain has a nucleotide sequence represented by GN-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 221) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 222) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); or The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 219); or The first chain has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223) and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 217); Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide bond between thiophosphates; "(E)-VP" represents the (E)-vinylphosphonate moiety at the 5' end; and "GN" represents the targeting ligand.

16. The siRNA agent according to any one of claims 12-15, wherein the siRNA agent is conjugated with a ligand as shown in the figure below: the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or a derivative thereof to a nucleic acid, and (iii) a bicyclic group linking GalNAc and siRNA, or (iv) a tetrafunctional group, wherein the GalNAc moieties are linked... or ， Where X represents O and S.

17. The siRNA agent according to claim 16, wherein the siRNA agent is conjugated to the ligand as shown in the figure below: ， Where X is O or S; The yes: and ; The yes: and ; or ， Where X is O or S; The yes and .

18. The siRNA agent according to any one of claims 16 or 17, wherein the adapters in the figure are independently selected from: (1)-(CH2)xC(O)NH-(CH2)yC(O)-; (2)-(CH2)xC(O)NH-(CH2)y-NHC(O)-(CH2)zC(O)-; (3) -NH-(CH2)yC(O)-; (4) –C(O)-(CH2)yC(O)-; in, Each connector in the figure may optionally be the same or different; x, y and z are independently selected from 1 to 10; preferably, x and y are independently selected from 2 to 8, and z is selected from 1 to 6; more preferably, x and y are independently selected from 3 to 6, and z is selected from 1 to 4.

19. The siRNA agent according to any one of claims 12-18, wherein the siRNA agent is conjugated to the ligand as shown in the figure below: ， X is either O or S.

20. The siRNA agent according to any one of claims 12-19, wherein: The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first chain has a nucleotide sequence represented by mC*mG*fGmUfCmAfUfCfGmCmUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 224) and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCfUmGmUmGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 226), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID N: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first chain has a nucleotide sequence represented by mC*mG*mGmUfCmAfUfCfGmCmUmGmUfGmCmAmUmUmA-(GalNAc-7) (SEQ ID NO: 228), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGfGmUfCmAfUfCfGmCmUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 229), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCfUmGmUmGmCmAmU*mU*mA (SEQ ID NO: 230), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAmUmGmCmAfCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 227); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmGmGmUfCmAfUfCfGmCmUmGmUfGmCmAmU*mU*mA (SEQ ID NO: 231), and the second chain has a nucleotide sequence represented by (E)-VP-mU*fA*mAfUmGmCmAmCmAmGmCmGmAfUmGfAmC*fC*mG (SEQ ID NO: 225); or The first chain has a nucleotide sequence represented by mC*mC*fGmAfGmAfGfCfAmUmGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 232), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUfGmGmUmUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 234), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first chain has a nucleotide sequence represented by mC*mC*mGmAfGmAfGfCfAmUmGmGmUfUmGmUmCmUmU-(GalNAc-7) (SEQ ID NO: 236), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCfGmAfGmAfGfCfAmUmGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 237), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO: 238), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 235); or The first chain has a nucleotide sequence represented by (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 239), and the second chain has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmCfUmC*fG*mG (SEQ ID NO: 233). Where mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively; "*" represents the nucleotide inter-linking bond of thiophosphate; and "(E)-VP" represents the (E)-vinylphosphonate moiety at the 5' end.

21. A pharmaceutical composition comprising the siRNA agent of any one of claims 1-20 and a pharmaceutically acceptable carrier.

22. A method for inhibiting the expression of the complement component C3 gene in cells, the method comprising: (a) Contacting the cells with the siRNA reagent of any one of claims 1-20 or the pharmaceutical composition of claim 21; as well as (b) The cells produced in step (a) are maintained for a period of time sufficient to allow degradation of the mRNA transcript of the complement component C3 gene, thereby inhibiting the expression of the complement component C3 gene in the cells.

23. A method of treating a subject suffering from a disease or condition in which the expression of complement component C3 is reduced, the method comprising administering to the subject a therapeutically effective amount of the siRNA agent of any one of claims 1-20 or the pharmaceutical composition of claim 21, thereby treating the subject.

24. The method of claim 23, wherein the disease or condition is a complement-mediated disease, condition, or syndrome, preferably a C3-related disease or condition, more preferably selected from IgA nephropathy (IgAN), C3 glomerulonephropathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), and myasthenia gravis (MG).

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