SIRNA, SIRNA-containing conjugates, pharmaceutical compositions and their uses

Abstract

The present invention relates to siRNA, such as double-stranded ribonucleic acid (dsRNA), which interferes with or inhibits the expression of a target gene. The present invention further relates to conjugates or pharmaceutical compositions containing siRNA, and to the use of siRNA, conjugates and the pharmaceutical compositions thereof in the manufacture of drugs for treating and / or preventing diseases or disorders.

Description

[Technical Field]

[0001] This disclosure relates to nucleic acids capable of inhibiting the expression of the complement component C3 gene, pharmaceutical compositions containing the nucleic acid, and siRNA conjugates, and belongs to the field of nucleic acid drugs. This disclosure further relates to methods for producing the nucleic acid, pharmaceutical compositions, and siRNA conjugates, and uses thereof. [Background technology]

[0002] The complement pathway is part of the host's innate immune system against invading pathogens. Complement consists primarily of a set of proteins that circulate in the bloodstream in precursor forms or reside on the cell membrane. Complement activation triggers a cascade of enzymatic reactions, forming potent anaphylatoxins and inducing a range of physiological responses, including chemotaxis and cell death. Complement protein C3 is the link in the complement cascade. When C3 is hydrolyzed, a highly reactive C3b is formed that binds to the cell surface. Such binding leads to the sequential formation of multiple complexes, thereby triggering autoamplification of the so-called "alternative pathway (AP)," ultimately causing an inflammatory response, leading to the attraction and opsonization of phagocytic cells, thereby removing pathogens, immune complexes, and cellular debris. Inappropriate complement activation is associated with the transmission and / or induction of numerous diseases, including C3-related disorders (e.g., C3 glomerulosis), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. Therefore, therapeutic agents that can inhibit the complement system (including those that inhibit C3 activity) may prove beneficial. Currently, therapies available for the treatment of complement component C3-related disorders are limited, and these therapies are time-consuming, invasive, and costly. Therefore, there is a need in this field to provide alternative therapies for subjects suffering from complement component C3-related disorders.

[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 specificity of target inhibition and achieving the objective of disease treatment. Of course, reducing the expression of the C3 gene by regulating C3 mRNA levels is an effective pathway for blocking complement protein production, maintaining normal immune function, and preventing abnormal immune responses.

[0004] The key to developing siRNA drugs that inhibit C3 gene expression lies in finding siRNA sequences with appropriate target selectivity, suitable modifications for chemical stability, and effective delivery systems. Because C3 expression is primarily limited to hepatocytes before it is secreted into the bloodstream, it is an ideal target for siRNA therapy. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide siRNA for inhibiting the expression of the C3 gene, a pharmaceutical composition containing the siRNA as a pharmacoactive ingredient, and an siRNA conjugate, a method for inhibiting the expression of the C3 gene 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] In other words, the present invention achieves the above objective by providing the following technical solutions.

[0007] In one embodiment, the present invention provides an siRNA agent having a 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, which differ by three or fewer nucleotides from any one of the nucleotide sequences of SEQ ID NO:1 nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716, and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

[0008] In one embodiment, the first chain 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 one of the nucleotide sequences of SEQ ID NO:1 nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716 by two or fewer nucleotides, preferably by one or fewer nucleotides, and more preferably without any nucleotide differences.

[0009] In one embodiment, the present invention provides an siRNA agent having a 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, and it is a 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, 112, 11 It differs from any one of the following nucleotides by three or fewer nucleotides: 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.

[0010] In one embodiment, the siRNA agent is a nucleic acid, where the first strand sequence is 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, 1 The sequences include 16, 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, and 188, respectively, where the second strand sequence is 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, Includes the array 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.

[0011] One aspect of the present invention relates to an siRNA reagent, wherein the first and second strands are located on a single strand of nucleic acid, and the first and second strands can hybridize with each other to form a double-stranded nucleic acid having a double-stranded region.

[0012] In one embodiment, the first and second chains form double-stranded regions having a nucleotide length of 15 to 25, preferably 15 to 23, more preferably 15 to 19, even more preferably 17 to 19, and most preferably 15, 16, 17, 18, 19, 20, 21, 22, and 23 nucleotides.

[0013] In one embodiment, the first and second strands of the nucleic acid are separate strands. The two separate strands preferably have a nucleotide length of 15 to 25 nucleotides each, more preferably 17 to 25 nucleotides each. The lengths of the two strands may be the same or different. The first strand may have a nucleotide length of 17 to 25 nucleotides, preferably 18 to 24 nucleotides, and may have 18, 19, 20, 21, 22, 23, or 24 nucleotides. Most preferably, the first strand has a nucleotide length of 19 nucleotides. The second strand may independently have a nucleotide length of 17 to 25 nucleotides, preferably 18 to 24 nucleotides, and may have 18, 19, 20, 21, 22, 23, or 24 nucleotides. More preferably, the second strand has a nucleotide length of 18, 19, or 20 nucleotides, and most preferably 19 nucleotides.

[0014] In one embodiment, the siRNA agent is a nucleic acid, where the first strand sequence is 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, 1 The sequence has nucleotide sequences represented by 18, 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, and 188, respectively, where the second strand is 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, 111, 1 It has a nucleotide sequence represented by 13, 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.

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

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

[0017] In one embodiment, the first strand has a nucleotide sequence represented by SEQ ID NO:20 and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96 and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16 and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150 and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32 and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100 and the second strand has a nucleotide sequence shown in SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID The first strand has the nucleotide sequence shown in 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:136, and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

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

[0019] As used herein, “substantially all nucleotides are modified” means that most are modified but not all are modified, and that the nucleotides contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

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

[0021] In one embodiment, all nucleotides in the first chain include modifications in the following pattern: the pentoses of nucleotide residues 7, 9, 10 and 11 from the 5' end are modified by 2'-fluoro substitution; the pentoses of nucleotide residues 2, 4, 6, 8, 12, 14, 16 and 18 from the 5' end are modified by 2'-methoxy substitution; and the pentoses of nucleotide residues 1, 3, 5, 13, 15, 17 and 19 from the 5' end are modified by 2'-methoxy or 2'-fluoro substitution. All nucleotides in the second chain have modifications in the following pattern: the pentoses of nucleotide residues 2, 6, 8, 14, and 16 from the 5' end are modified by 2'-fluoro substitution; the pentoses of nucleotide residues 1, 3, 5, 7, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; and residues 4, 9, 10, and 18 from the 5' end are modified by either 2'-methoxy modification or 2'-fluoro substitution.

[0022] In one embodiment, all nucleotides in the first chain include modifications in the following pattern: the pentoses of the 5th, 7th, 8th and 9th nucleotide residues from the 5' end are modified by 2'-fluoro substitution; the pentoses of the 1st, 2nd, 4th, 6th, 10th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy substitution; and the pentoses of the 3rd, 11th and 14th nucleotide residues from the 5' end are modified by 2'-methoxy or 2'-fluoro substitution. All nucleotides in the second chain have modifications in the following patterns: pentoses at nucleotide residues 2, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; pentoses at nucleotide residues 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy substitution; and pentoses at nucleotide residues 4, 8, and 10 from the 5' end are modified by 2'-methoxy or 2'-fluoro substitution.

[0023] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 7, 9, 10 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 6, 8, 9, 14 and 16 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 from the 5' end.

[0024] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 3, 5, 7, 8, and 9 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 2, 4, 14, 16, and 18 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end.

[0025] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0026] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 4, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0027] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0028] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 2, 4, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0029] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 3, 5, 7, 8, and 9 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 2, 10, 14, 16, and 18 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 from the 5' end.

[0030] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 3, 5, 7, 8, and 9 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16, and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end.

[0031] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 10, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0032] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end. All nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses at nucleotide residues 2, 10, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0033] In one embodiment, the first chain includes two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends, and / or the second chain includes two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends.

[0034] In some embodiments, the first chain includes two phosphorothioate nucleotide interlinks at its 3' end, and / or the second chain includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end.

[0035] In some embodiments, the first chain includes two phosphorothioate nucleotide interlinks at its 5' end, and / or the second chain includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end.

[0036] In some embodiments, the first strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and / or the second strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end.

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

[0038] In the specification of this invention, “same or common modification” refers to the same modification to any nucleotide, meaning the modification of A, G, C, or U by a group, for example, a methyl group (2'-OMe) or a fluoro group (2'-F). For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, and 2'-F-dG are all considered the same or common modification, as are 2'-OMe-rU, 2'-OMe-rA, 2'-OMe-rC, and 2'-OMe-rG. Conversely, a 2'-F modification is a different modification from a 2'-OMe modification.

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

[0040] In some embodiments, the siRNA agent further comprises a targeted ligand that targets liver tissue.

[0041] In one embodiment, the targeted ligand is a GalNAc derivative.

[0042] In one embodiment, the targeted ligand is a GalNAc conjugate.

[0043] In one embodiment, the targeting ligand is conjugated to the 3' or 5' end of the first chain 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 embodiment, the siRNA agent is (i) comprising a targeting ligand conjugated to the 3' or 5' terminal nucleotide of the first strand, (ii) Having a phosphorothioate bond between three nucleotides at the other end of the first chain opposite one end that is conjugated with the targeted ligand, (iii) The second chain has phosphorothioate bonds between the three 3' nucleotides at the end and the three 5' nucleotides at the end, (iv) Optionally, all remaining bonds between nucleotides in the first and / or second chains are phosphodiester bonds.

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

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

[0048] In one embodiment, 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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:217), or The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUfGmGmUmUmGmUmC*mU*mU (SEQ ID NO:222), 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), the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:217), or, The first strand has a nucleotide sequence represented by GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO:223), 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), the second strand has a nucleotide sequence represented by (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:217), Here, 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, "*" is a phosphorothioate nucleotide internucleotide linkage, "(E)-VP" is the (E)-vinylphosphonate moiety at the 5'-end, and "GN" is a targeting ligand.

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

[0050] In one aspect, each linker in the figure is optionally the same or different.

[0051] In one aspect, the linkers in the figure are independently (1) -(CH2)x-C(O)NH-(CH2)y-C(O)-, (2) -(CH2)x-C(O)NH-(CH2)y-NHC(O)-(CH2)z-C(O)-, (3) -NH-(CH2)y-C(O)-, (4) -C(O)-(CH2)z-C(O)-, where x, y, and z are independently selected from 1 to 10, preferably x and y are independently selected from 2 to 8, 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 is conjugated to a ligand as shown in the following figure, [Chemical formula] where X is O or S, [Chemical formula] is [Chemical formula] and said [Chemical formula] is

Chem.

Chem.

Chem.

Chem.

[0053] [[ID=2८]] In one aspect, the siRNA agent is conjugated to a ligand as shown in the figure below,

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0054] In one aspect, the siRNA agent is conjugated to a ligand as shown in the figure below,

Chem.

Chem.

[0055] It should be noted that there may be some inaccuracies in the chemical formula part in the original text, and the translation is based on the content provided. If there are specific requirements for chemical formula translation, it may need to be adjusted according to relevant chemical naming rules. In some embodiments, the GalNAc conjugate is one or more GalNAc derivatives, which are optionally conjugated with a double-stranded RNA agent via a linker or carrier, and the one or more GalNAc derivatives are optionally conjugated with a double-stranded RNAi agent via a linker or carrier.

[0056] In some embodiments, the siRNA agent is conjugated with a ligand, as shown in the figure below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Here, X is either O or S.

[0057] In some embodiments, the siRNA agent is conjugated with a ligand, as shown in the figure below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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

[0059] In one embodiment, 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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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 as (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO:239), and the second strand has a nucleotide sequence represented as (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:233), Here, 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; "*" indicates an internucleotide linkage of phosphorothioate; and "(E)-VP" is the (E)-vinylphosphonate moiety at the 5' end.

[0060] In one embodiment, the pharmaceutical composition comprises an 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 embodiment, the pharmaceutical composition comprises an siRNA agent disclosed herein and another therapeutic agent selected from oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides.

[0062] In one embodiment, the pharmaceutical composition comprises reagents and pharmaceutically acceptable carriers disclosed herein.

[0063] Another aspect of the present disclosure provides a method for inhibiting the expression of the C3 gene in cells (e.g., nerve cells or photoreceptor cells), the method comprising the steps of (a) contacting cells with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, and (b) inhibiting the expression of the C3 gene in the cells by maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the C3 gene.

[0064] Another aspect of the present disclosure provides a method for treating a subject suffering from a disease or disorder that would benefit from reduced expression of complement component C3, the method comprising treating the subject by administering a therapeutically effective amount of the siRNA agent or the pharmaceutical composition to the subject.

[0065] The diseases or disorders treated with the nucleic acids or compositions disclosed herein are complement-mediated diseases, disorders, or syndromes, preferably C3-related diseases or disorders.

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

[0067] The siRNA according to the present invention can sequence-specifically mediate the inhibition of C3 gene expression. The gene may be located within a cell, for example, within a subject's cell, for example, within a human cell. Without being bound by theory, it is believed that combinations or subcombinations of the above properties and specific target sites or modifications in these iRNAs give the iRNAs of the present invention improved therapeutic effects, stability, efficacy, persistence, and safety. [Brief explanation of the drawing]

[0068] [Figure 1] Table 2A shows the results of detecting the inhibitory effect of siRNA on C3 mRNA expression levels. [Figure 2] Figure 2A shows the dose-response curves of the C3 mRNA knockdown effect of nine selected siRNAs in HepG2 cells. Figure 2B shows the detection results of the C3 mRNA knockdown effect of the nine selected siRNAs in HepG2 cells at concentrations of 100 nM and 0.16 nM. [Figure 3] Figure 3A shows the detection results of the C3 mRNA knockdown effect of nine selected GalNAcs and conjugated siRNAs in PHH cells at concentrations of 1000 nM and 1 nM. Figure 3B shows the dose-response curves of the C3 mRNA knockdown effect of four selected GalNAcs and conjugated siRNAs in PHH cells. [Figure 4]Figure 4A shows the dose-response curve of the C3 mRNA knockdown effect of 20 selected GalNAcs and conjugated siRNAs in PHH cells. Figure 4B shows the dose-response curve of the C3 mRNA knockdown effect of 20 selected GalNAcs and conjugated siRNAs in PHH cells. Figure 4C shows the dose-response curve of the C3 mRNA knockdown effect of 20 selected GalNAcs and conjugated siRNAs in PHH cells. Figure 4D shows the dose-response curve of the C3 mRNA knockdown effect of 20 selected GalNAcs and conjugated siRNAs in PHH cells. [Figure 5] Figure 5A shows the detection results of the serum human C3 protein reduction effect of two selected GalNAc-conjugated siRNAs in human C3 transgenic mice (male). Figure 5B shows the detection results of the serum human C3 protein reduction effect of two selected GalNAc-conjugated siRNAs in human C3 transgenic mice (female). [Figure 6] The results of detecting the reduction effect of human C3 protein by the tested conjugated siRNA are shown. [Figure 7] The results of detecting the reduction effect of human C3 protein by different doses of GalNAc and conjugated siRNA tested are shown. [Modes for carrying out the invention]

[0069] The present invention will be further described by the following specific description.

[0070] The present invention provides siRNA agents that affect the cleavage of the RNA transcript of the complement component C3 (C3) gene mediated by the RNA-induced silencing complex (RISC). The gene may be located within a cell, for example, within a subject's cell, for example, within a human cell. These iRNAs can be used to target and degrade the mRNA of the relevant gene (C3) in mammals.

[0071] The siRNA agent of the present invention is designed to target the human complement component C3 (C3) gene, which includes a portion of the gene conserved in C3 orthologues of other mammalian species. The siRNA agent of the present invention has improved therapeutic efficacy, stability, efficacy, duration of action, and safety.

[0072] I. Nucleic acid sequences A first aspect of the present invention is an siRNA agent having a 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, and it differs by three or fewer nucleotides from any one nucleotide sequence of the first strand in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, or 3F.

[0073] Preferably, the first chain 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 one of the first chain sequences shown in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, or 3F by three or fewer nucleotides, preferably two or fewer nucleotides, more preferably one or fewer nucleotides, and most preferably no nucleotide differences whatsoever.

[0074] The present invention further provides an siRNA agent having a 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 and differs by 3 or fewer nucleotides from any one nucleotide sequence of the first strand in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F, and the second strand sequence comprises at least 15 consecutive nucleotides and differs by 3 or fewer nucleotides from any one nucleotide sequence of the second strand in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F.

[0075] Preferably, the first chain 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 one of the first chain sequences shown in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by only 3 or fewer nucleotides, preferably 2 or fewer nucleotides, more preferably 1 or fewer nucleotides, and most preferably without any nucleotide differences. The second chain 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 one of the second chain sequences shown in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F by only 3 or fewer nucleotides, preferably 2 or fewer nucleotides, more preferably 1 or fewer nucleotides, and most preferably without any nucleotide differences.

[0076] The present invention further provides an siRNA agent having a 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 one nucleotide sequence of the first strand in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F, and the second strand comprises a nucleotide sequence selected from any one nucleotide sequence of the second strand in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, and 3F.

[0077] For example, although the sequences in Table 1 are not described as modified or conjugated sequences, it should be understood that the dsRNA of the siRNA agent of the present invention (e.g., the present invention's dsRNA) may include any one of the sequences shown in any one of Tables 1, 2A, 3A, 3B, 3C, 3D, 3E, or 3F, and may be unmodified, unconjugated, or modified or conjugated in a manner different from those described herein. In other words, the present invention covers the dsRNAs in Tables 2-7 that are unmodified, unconjugated, modified, or conjugated as described herein.

[0078] 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 is 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, The sequence includes at least 15 consecutive nucleotide sequences that differ by three or fewer nucleotides from any one of the sequences 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, where the second strand sequence is 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, 111, 113, 115, 117, 119, 121, 1 It contains a sequence of at least 15 consecutive nucleotides that differs by three or fewer nucleotides from any one of the following sequences: 23, 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.

[0079] In some embodiments, the first chain sequence contains at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 consecutive nucleotides, which are 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, 8 6, 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 The second strand sequence differs from any one of the sequences 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 by three or fewer nucleotides, preferably two or fewer nucleotides, more preferably one or fewer nucleotides, and most preferably without any nucleotide differences, and the second strand sequence contains at least 16, more preferably at least 17, even more preferably at least 18, most preferably all 19 consecutive nucleotides, and it is SEQ IDNO: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, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 , differs from any one of the sequences 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 by three or fewer nucleotides, preferably two or fewer nucleotides, more preferably one or fewer nucleotides, and most preferably there is no difference in any nucleotides.

[0080] In some embodiments, the siRNA agent is a nucleic acid, where the first strand sequence is 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, 1 The sequences include 16, 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, and 188, respectively, where the second strand sequence is 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, Includes the array 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.

[0081] II. Modified SIRNA agents A second aspect of the present invention is a specific modification pattern for a polynucleic acid molecule, wherein the polynucleic acid molecule is a double-stranded RNA (dsRNA) comprising a first strand and a second strand.

[0082] In one embodiment, 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 and second strands each comprise at least one modified nucleotide, preferably substantially all nucleotides of the first strand are modified nucleotides, substantially all nucleotides of the second strand are modified nucleotides, and more preferably all nucleotides of the first strand are modified nucleotides, and all nucleotides of the second strand are modified nucleotides.

[0083] As used herein, “substantially all nucleotides are modified” means that most are modified but not all are modified, and that the nucleotides contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0084] 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 all nucleotides of the first strand are modified with 2′-fluoro substitutions of pentoses at nucleotide residues 7, 9, 10 and 11 from the 5' end, pentoses at nucleotide residues 2, 4, 6, 8, 12, 14, 16 and 18 from the 5' end, and pentoses at nucleotide residues 1, 3, 5, 13, 15, 17 and 19 from the 5' end The modification pattern involves the nucleotide being modified by 2′-methoxy modification or 2′-fluoro substitution, and all nucleotides in the second chain are modified by 2′-fluoro substitution of the pentoses at nucleotide residues 2, 6, 8, 14 and 16 from the 5' end, by 2′-methoxy modification of the pentoses at nucleotide residues 1, 3, 5, 7, 11, 12, 13, 15, 17 and 19 from the 5' end, and by 2′-methoxy modification or 2′-fluoro substitution of residues 4, 9, 10 and 18 from the 5' end.

[0085] 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 all nucleotides of the first strand are modified with 2′-fluoro substitutions of the pentoses at nucleotide residues 5, 7, 8 and 9 from the 5' end, 2′-methoxy substitutions of the pentoses at nucleotide residues 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18 and 19 from the 5' end, and 2′-methoxy substitutions of the pentoses at nucleotide residues 3, 11 and 14 from the 5' end The modifications include a pattern of modification consisting of toxic modification or 2′-fluoro substitution, where all nucleotides in the second chain have a pattern of modification consisting of pentoses at nucleotide residues 2, 14, 16 and 18 from the 5' end being modified by 2′-fluoro substitution, pentoses at nucleotide residues 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17 and 19 from the 5' end being modified by 2′-methoxy modification, and pentoses at nucleotide residues 4, 8 and 10 from the 5' end being modified by 2′-methoxy modification or 2′-fluoro substitution.

[0086] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0087] 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 all nucleotides of the first strand contain modifications in the following pattern: pentoses of nucleotide residues 5, 7, 8 and 9 from the 5' end are modified by 2'-fluoro substitution; pentoses of nucleotide residues 1, 2, 4, 6, 10, 12, 13, 15, 16, 17, 18 and 19 from the 5' end are modified by 2'-methoxy substitution; and pentoses of nucleotide residues 3, 11 and 14 from the 5' end are modified by 2'-methoxy or 2'-fluoro substitution. Furthermore, all nucleotides in the second strand include modifications in the following pattern: pentoses at nucleotide residues 2, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; pentoses at nucleotide residues 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy substitution; and pentoses at nucleotide residues 4, 8, and 10 from the 5' end are modified by 2'-methoxy or 2'-fluoro substitution, wherein the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent is SEQ The first strand has a nucleotide sequence represented by ID NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16, and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150, and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32, and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100, and the second strand has a nucleotide sequenceThe molecule has the nucleotide sequence shown in 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:136 and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0088] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 7, 9, 10 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 6, 8, 9, 14 and 16 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 from the 5' end.

[0089] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 3, 5, 7, 8, and 9 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 4, 14, 16, and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end.

[0090] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0091] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 4, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0092] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0093] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 4, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0094] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 3, 5, 7, 8 and 9 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 10, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0095] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 3, 5, 7, 8 and 9 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 8, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0096] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 10, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0097] In some embodiments, all nucleotides in the first chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 5, 7, 8, 9 and 14 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18 and 19 from the 5' end, and all nucleotides in the second chain include modifications in the pattern of 2′-fluoro substitution of the pentoses of nucleotide residues 2, 10, 14, 16 and 18 from the 5' end, and 2′-methoxy modification of the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17 and 19 from the 5' end.

[0098] In some embodiments, all nucleotides in the first strand of the double-stranded RNA (dsRNA) preparation have a modification pattern in which the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end are modified by 2'-fluoro substitution, and the pentoses of nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18 and 19 from the 5' end are modified by 2'-methoxy substitution, and all nucleotides in the second strand have a modification pattern in which the pentoses of nucleotide residues 5, 7, 8, 9 and 11 from the 5' end are modified by 2'-methoxy substitution, and all nucleotides in the second strand have a modification pattern in which the pentoses of nucleotide residues 5, 7 and 8 and 9 from the 5' end The modification pattern includes modifications where the pentoses at nucleotide residues 8, 14, 16, and 18 are modified by 2′-fluoro substitutions, and the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2′-methoxy modifications, where the target genes of the double-stranded RNA (dsRNA) agent are complement component C3 (C3), complement component C5 (C5), complement factor B (CFB), PCSK9, TTR, AGT, LPA, and Agtr 1, ALK, VEGF, ANGPTL3, ANGPTL4, ANGPTL8, APOA, APOC3, ASGR1, CIDEB, COL1A1, COL3A1, CTGF, DGAT2, DMPK, DNAJC15 / MCJ, DPP4, factor VI II, Factor X, Factor IX, Factor XI, Factor A component selected from 1L1, PNPLA3, ASGR1, SCAP, SERPINA1, SERPINF2, 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 preferred embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: 21, or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and the second strand has a nucleotide sequence represented by SEQ ID NO: 97, or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and the second strand has a nucleotide sequence represented by SEQ ID NO: 17, or the first strand has a nucleotide sequence represented by SEQ ID NO: 150 and the second strand has a nucleotide sequence represented by SEQ ID NO: 151, or the first strand has a nucleotide sequence represented by SEQ ID NO: 32 and the second strand has a nucleotide sequence represented by SEQ ID NO: 33, or the first strand has a nucleotide sequence represented by SEQ ID NO: 100 and the second strand has a nucleotide sequence represented by SEQ ID The first strand has a nucleotide sequence represented by NO:101, or the first strand has a nucleotide sequence represented by SEQ ID NO:110 and the second strand has a nucleotide sequence represented by SEQ ID NO:111, or the first strand has a nucleotide sequence represented by SEQ ID NO:134 and the second strand has a nucleotide sequence represented by SEQ ID NO:135, or the first strand has a nucleotide sequence represented by SEQ ID NO:136 and the second strand has a nucleotide sequence represented by SEQ ID NO:137.

[0099] 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'-nnxnNfnNfNfNfnxnnxnnnnn-3' and the second strand comprises 5'-nNfnxnnnxnxnnnNfnNfn-3', where "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.

[0100] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0101] 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'-nnxnNfnNfNfNfnxnnxnnnnn-3', and the second strand comprises 5'-nNfnxnnnxnxnnnNfnNfn-3', where "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 double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO:20, and the second strand has SEQ ID The first strand has a nucleotide sequence represented by NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96 and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16 and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence represented by SEQ ID NO:150 and the second strand has a nucleotide sequence represented by SEQ ID NO:151, or the first strand has a nucleotide sequence represented by SEQ ID NO:32 and the second strand has a nucleotide sequence represented by SEQ ID NO:33, or the first strand has a nucleotide sequence represented by SEQ ID NO:100 and the second strand has a nucleotide sequence represented by SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID NO:110 and the second strand has a nucleotide sequence represented by SEQ ID The molecule has a nucleotide sequence represented by NO:111, or the first strand has a nucleotide sequence represented by SEQ ID NO:134 and the second strand has a nucleotide sequence represented by SEQ ID NO:135, or the first strand has a nucleotide sequence represented by SEQ ID NO:136 and the second strand has a nucleotide sequence represented by SEQ ID NO:137.

[0102] 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'-nnNfnNfnNfNfNfnnnnnnnn-3' and the second strand comprises 5'-nNfnnnnnnnnNfnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and "n" represents a 2'-O-methyl-modified nucleotide.

[0103] 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'-nnNfnNfnNfNfNfnnnnnnnn-3' and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0104] 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'-nnNfnNfnNfNfNfnnnnnnnn-3' and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0105] 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'-nnnnNfnNfNfnNfnNfnnnnnn-3' and the second strand comprises 5'-nNfnnnnnnnnNfnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0106] 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'-nnnnNfnNfNfnNfnnnnnn-3' and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and "n" represents a 2'-O-methyl-modified nucleotide.

[0107] 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'-nnnnNfnNfNfnNfnNfnnnnnn-3' and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and "n" represents a 2'-O-methyl-modified nucleotide.

[0108] 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'-nnnnNfnNfNfNfnnnnNfnnnnn-3' and the second strand comprises 5'-nNfnnnnnnnNfnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0109] 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'-nnnnNfnNfNfNfnnnnNfnnnnn-3' and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0110] 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'-nnnnNfnNfNfNfnnnnNfnnnnn-3' and the second strand comprises 5'-nNfnNfnnnnnnnnnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and "n" represents a 2'-O-methyl-modified nucleotide.

[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'-nnnnnnNfnNfNfNfnnnnnn-3' and the second strand comprises 5'-nNfnnnNfnNfNfnnnnNfnNfnnn-3', where "Nf" represents a 2'-fluoro-modified nucleotide and where "n" represents a 2'-O-methyl-modified nucleotide.

[0112] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[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'-nnnnNfnNfNfnNfnNfnnnnnn-3', and the second strand comprises 5'-nNfnnnnnNfnnnnnNfnNfnNfn-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, and the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), preferably the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID The first strand has a nucleotide sequence represented by NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16 and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown by SEQ ID NO:150 and the second strand has a nucleotide sequence shown by SEQ ID NO:151, or the first strand has a nucleotide sequence shown by SEQ ID NO:32 and the second strand has a nucleotide sequence shown by SEQ ID NO:33, or the first strand has a nucleotide sequence shown by SEQ ID NO:100 and the second strand has a nucleotide sequence shown by SEQ ID NO:101, or the first strand has a nucleotide sequence shown by SEQ ID NO:110 and the second strand has a nucleotide sequence shown by SEQ ID NO:111, or the first strand has a nucleotide sequence shown by SEQ ID NO:134 and the second strand has a nucleotide sequence It has the nucleotide sequence shown in NO:135, or the first strand has the nucleotide sequence shown in SEQ ID NO:136 and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0114] In some embodiments, the first strand includes two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends, and / or the second strand includes two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends.

[0115] In some embodiments, the first chain includes two phosphorothioate nucleotide interlinks at its 3' end, and / or the second chain includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end.

[0116] In some embodiments, the first chain includes two phosphorothioate nucleotide interlinks at its 5' end, and / or the second chain includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end.

[0117] In some embodiments, the first strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and / or the second strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end.

[0118] In some embodiments, the siRNA agent further comprises a targeted ligand that targets liver tissue.

[0119] In some embodiments, the targeted ligand is a GalNAc derivative. In one embodiment, the targeted ligand is a GalNAc conjugate.

[0120] In some embodiments, the targeted ligand is conjugated to the 3' or 5' end of the first chain of the siRNA agent.

[0121] In some embodiments, the targeting ligand is conjugated to the 3' end of the first chain, the first chain having two phosphorothioate nucleotide interbonds at its 5' end, the second chain having two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and optionally, all remaining bonds between nucleotides of the first and / or second chains are phosphodiester bonds.

[0122] In some embodiments, the targeting ligand is conjugated to the 5' end of the first chain, the first chain having two phosphorothioate nucleotide interbonds at its 3' end, and the second chain having two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and optionally, all remaining bonds between nucleotides of the first and / or second chains are phosphodiester bonds.

[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'-n*n*xnNfnNfNfNfnxnnxnnnnn(GN)-3', and the second strand comprises 5'-n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, "x" represents a 2'-fluoro or 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide linkage, and where "GN" represents a targeting ligand.

[0124] 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)nnxnNfnNfNfNfnxnnxnnn*n*n-3', and the second strand comprises 5'-n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, "x" represents a 2'-fluoro or 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide linkage, and where "GN" represents a targeting ligand.

[0125] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0126] 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*NfnNfnNfNfNfnnnnnnnn(GN)-3', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0127] 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', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0128] 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', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0129] 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*nnNfnNfNfNfnnnnNfnnnnn(GN)-3', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0130] 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*nnNfnNfNfNfnnnnNfnnnnn(GN)-3', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0131] 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)nnNfnNfnNfNfNfnnnnnn*n*n-3', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0132] 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)nnnnNfnNfNfnNfnNfnnnn*n*n-3', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0133] 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)nnnnNfnNfNfnNfnNfnnnn*n*n-3', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0134] 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', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0135] 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', and the second strand comprises 5'-n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, and where "GN" represents a targeting ligand.

[0136] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0137] 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', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, where the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent comprises SEQ ID The first strand has a nucleotide sequence represented by NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16, and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150, and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32, and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100, and the second strand has a nucleotide sequence shown in SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID The first strand has the nucleotide sequence shown in 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:136, and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[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'-(GN)nnnnNfnNfNfNfnNfnnnn*n*n-3', and the second strand comprises 5'-n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, where the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent comprises SEQ ID The first strand has a nucleotide sequence represented by NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16, and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150, and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32, and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100, and the second strand has a nucleotide sequence shown in SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID The first strand has the nucleotide sequence shown in 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:136, and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0139] In some embodiments, the second chain contains a terminal 5'(E)-vinylphosphonate nucleotide at its 5' end.

[0140] 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*xnNfnNfNfNfnxnnxnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, "x" represents a 2'-fluoro or 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-(GN)nnxnNfnNfNfNfnxnnxnnn*n*n-3' and the second strand comprises 5'-(E-VP)n*Nf*nxnnnxnxnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, "n" represents a 2'-O-methyl-modified nucleotide, "x" represents a 2'-fluoro or 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[0142] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0143] 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*NfnNfnNfNfNfnnnnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-n*n*nnNfnNfNfnNfnNfnnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-n*n*nnNfnNfNfnNfnNfnnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-n*n*nnNfnNfNfNfnnnnNfnnnnn(GN)-3', and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[0148] 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)nnNfnNfnNfNfNfnnnnnn*n*n-3' and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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'-(GN)nnnnNfnNfNfnNfnNfnnnn*n*n-3' and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[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)nnnnNfnNfNfnNfnNfnnnn*n*n-3' and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[0151] 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' and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[0152] 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' and the second strand comprises 5'-(E-VP)n*Nf*nNfnnnnnnnnnNfnNfn*Nf*n-3', where "Nf" represents a 2'-fluoro-modified nucleotide, where "n" represents a 2'-O-methyl-modified nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, and where "E-VP" represents an (E)-vinylphosphonate nucleotide.

[0153] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0154] 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' and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" is a 2'-fluoromodified nucleo Here, "n" represents a nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, where "E-VP" represents an (E)-vinylphosphonate nucleotide, where the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent is SEQ The first strand has a nucleotide sequence represented by ID NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16, and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150, and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32, and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100, and the second strand has a nucleotide sequence shown in SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID The first strand has the nucleotide sequence shown in 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:136, and the second strand has SEQ IDIt has the nucleotide sequence shown in NO:137.

[0155] 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)nnnnNfnNfNfnNfnNfnnnn*n*n-3' and the second strand comprises 5'-(E-VP)n*Nf*nnnnnNfnnnnnNfnNfn*Nf*n-3', where "Nf" is a 2'-fluoromodified nucleo Here, "n" represents a nucleotide, where "*" represents a phosphorothioate nucleotide bond, where "GN" represents a targeting ligand, where "E-VP" represents an (E)-vinylphosphonate nucleotide, where the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3), and preferably, the first strand of the double-stranded RNA (dsRNA) agent is SEQ The first strand has a nucleotide sequence represented by ID NO:20, and the second strand has a nucleotide sequence represented by SEQ ID NO:21, or the first strand has a nucleotide sequence represented by SEQ ID NO:96, and the second strand has a nucleotide sequence represented by SEQ ID NO:97, or the first strand has a nucleotide sequence represented by SEQ ID NO:16, and the second strand has a nucleotide sequence represented by SEQ ID NO:17, or the first strand has a nucleotide sequence shown in SEQ ID NO:150, and the second strand has a nucleotide sequence shown in SEQ ID NO:151, or the first strand has a nucleotide sequence shown in SEQ ID NO:32, and the second strand has a nucleotide sequence shown in SEQ ID NO:33, or the first strand has a nucleotide sequence shown in SEQ ID NO:100, and the second strand has a nucleotide sequence shown in SEQ ID NO:101, or the first strand has a nucleotide sequence represented by SEQ ID The first strand has the nucleotide sequence shown in 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:136, and the second strand has SEQ IDIt has the nucleotide sequence shown in NO:137.

[0156] 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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:217), Here, 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; "*" indicates a phosphorothioate nucleotide bond; "(E)-VP" is the 5' terminal (E)-vinylphosphonate moiety; and "GN" is the targeted ligand.

[0157] III. SIRNA Conjugates In a third aspect of the present invention, the siRNA reagent is coupled or conjugated with one or more targeting moieties. In some cases, the selection of targeting moieties is based on the ability of the conjugate molecules described herein to selectively or suitably target a desired cell population, tissue, or organ. In some cases, the targeting moiety targets cells, tissues, or organs expressing the appropriate binding partner of the targeting moiety (e.g., the appropriate receptor or ligand). For example, a polynucleotide molecule conjugated with N-acetylgalactosamine (GalNAc) can target hepatocytes expressing asialoglycoprotein (ASGP-R). Any suitable GalNAc molecule known in the art to be used as a targeting moiety is conceivable. Exemplary GalNAc molecules include tribranched GalNAc (e.g., L96).

[0158] In one embodiment, 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.

[0159] In one embodiment, 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 as shown in the figure below, and the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, (ii) a linker that conjugates at least one GalNAc moiety or derivative thereof with a nucleic acid, (iii) a bicyclic group that links GalNAc and siRNA, or (iv) a tetrafunctional group that links the GalNAc moiety. [ka] Here, X is O and S.

[0160] In one embodiment, [ka] teeth, [ka] And, [ka] teeth, [ka] [ka] [ka] [ka] And, In one embodiment, the siRNA agent is conjugated with a ligand as shown in the figure below. [ka] The tetrafunctional group is, [ka] Selected from.

[0161] In one embodiment, the linker in the figure is independent, (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) Selected from -C(O)-(CH2)yC(O)-, Here, each linker in the figure is either 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, z is selected from 1 to 6, more preferably x and y are independently selected from 3 to 6, z is selected from 1 to 4.

[0162] In one embodiment, the siRNA agent is conjugated with a ligand as shown in the figure below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Here, X is either O or S.

[0163] In some embodiments, the target genes of the above 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, XDH, LDHA, ALDH2, DMD, EPHA2, KIF11, BCL2L12, APOA1, TRPV1, CASP2, KRAS, TMPRSS6, STAT3, PRDM14, PTGS2, CTGF or DDIT4.

[0164] In some embodiments, the target gene of the double-stranded RNA (dsRNA) agent is complement component C3 (C3).

[0165] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a sequence containing at least 15 consecutive nucleotides, which differ by three or fewer nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, specifically nucleotides 3123-3141, 5019-5037, 2304-2322, 494-512, 2643-2661, 2244-2262, 589-607, 2482-2500, or 4698-4716, and the second strand contains a nucleotide sequence that is at least partially complementary to the first strand.

[0166] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence 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 a nucleotide sequence represented by SEQ ID NO: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively.

[0167] In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: 21, or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and the second strand has a nucleotide sequence represented by SEQ ID NO: 97, or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and the second strand has a nucleotide sequence represented by SEQ ID NO: 17, or the first strand has a nucleotide sequence shown in SEQ ID NO: 150 and the second strand has a nucleotide sequence shown in SEQ ID NO: 151, or the first strand has a nucleotide sequence shown in SEQ ID NO: 32 and the second strand has a nucleotide sequence shown in SEQ ID NO: 33, or the first strand has a nucleotide sequence shown in SEQ ID NO: 100 and the second strand has a nucleotide sequence The molecule has the nucleotide sequence shown in 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:136 and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0168] In some embodiments, the double-stranded RNA (dsRNA) agent is as shown in the figure below. [ka] Here, the target gene of the above double-stranded RNA (dsRNA) agent is complement component C3 (C3).

[0169] In some embodiments, the double-stranded RNA (dsRNA) agent is as shown in the figure below. [ka] Here, the first strand has a sequence containing at least 15 consecutive nucleotides, which differs by no more than 3 nucleotides from any one 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, and the second strand contains a nucleotide sequence that is at least partially complementary to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence 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 NOs: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively.In preferred embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: 21, or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and the second strand has a nucleotide sequence represented by SEQ ID NO: 97, or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and the second strand has a nucleotide sequence represented by SEQ ID NO: 17, or the first strand has a nucleotide sequence shown in SEQ ID NO: 150 and the second strand has a nucleotide sequence shown in SEQ ID NO: 151, or the first strand has a nucleotide sequence shown in SEQ ID NO: 32 and the second strand has a nucleotide sequence shown in SEQ ID NO: 33, or the first strand has a nucleotide sequence shown in SEQ ID NO: 100 and the second strand has a nucleotide sequence The molecule has the nucleotide sequence shown in 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:136 and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0170] In some embodiments, the double-stranded RNA (dsRNA) agent is as shown in the figure below. [ka] Here, the first strand has a sequence containing at least 15 consecutive nucleotides, which differs by no more than 3 nucleotides from any one 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, and the second strand contains a nucleotide sequence that is at least partially complementary to the first strand. In some embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence 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 NOs: 21, 97, 17, 151, 33, 101, 111, 135, and 137, respectively.In preferred embodiments, the first strand of the double-stranded RNA (dsRNA) agent has a nucleotide sequence represented by SEQ ID NO: 20 and the second strand has a nucleotide sequence represented by SEQ ID NO: 21, or the first strand has a nucleotide sequence represented by SEQ ID NO: 96 and the second strand has a nucleotide sequence represented by SEQ ID NO: 97, or the first strand has a nucleotide sequence represented by SEQ ID NO: 16 and the second strand has a nucleotide sequence represented by SEQ ID NO: 17, or the first strand has a nucleotide sequence shown in SEQ ID NO: 150 and the second strand has a nucleotide sequence shown in SEQ ID NO: 151, or the first strand has a nucleotide sequence shown in SEQ ID NO: 32 and the second strand has a nucleotide sequence shown in SEQ ID NO: 33, or the first strand has a nucleotide sequence shown in SEQ ID NO: 100 and the second strand has a nucleotide sequence The molecule has the nucleotide sequence shown in 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:136 and the second strand has the nucleotide sequence shown in SEQ ID NO:137.

[0171] 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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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 as (GalNAc-3)-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO:239), and the second strand has a nucleotide sequence represented as (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO:233), Here, 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; "*" indicates an internucleotide linkage of phosphorothioate; and "(E)-VP" is the (E)-vinylphosphonate moiety at the 5' end.

[0172] definition Before describing the present invention in detail below, it will be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, as they may differ. Furthermore, it will be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, and the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains.

[0173] For interpretation of this specification, the following definitions shall apply, and wherever appropriate, a singular term shall also include a plural form, and vice versa. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0174] As used herein, the terms “complement component 3” and “C3” are used interchangeably and 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, acylation-stimulated protein cleavage product, CPAMD1, complement C3, C3 and PZP-like α-2-macroglobulin domain-containing protein 1, complement component C3 and AHUS5.

[0175] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a complement component C3 gene, and includes mRNA, which is the RNA processed product of the main transcript. The target portion of the sequence is at least long enough to be used as a substrate for iRNA-directed cleavage on or near the nucleotide sequence portion of the mRNA molecule formed during the transcription of a complement component C3 gene.

[0176] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant” are interchangeable herein and refer to reagents containing RNA as defined herein, which mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs induce sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNAs regulate (e.g., inhibit) the expression of the C3 gene in cells (e.g., cells in a test subject, e.g., cells in a mammalian test subject).

[0177] The term "mRNA (messenger RNA)" refers to the RNA molecule that serves as a protein translation template in vivo, transferring genetic coding information from DNA to protein products. The term "RNA interference" or "RNAi" refers to the post-transcriptional regulation of gene expression in living organisms. This phenomenon is caused by the specific degradation of target mRNA mediated by single-stranded or double-stranded RNA. For details on the RNAi regulatory mechanism, please refer to Biotech.Adv.2008,26(3):202 and other literature.

[0178] In this invention, unless otherwise specified, the terms "small interference RNA" or "siRNA" refer to RNA molecules capable of sequence-specific induction of RNAi phenomena, which consist of two single-stranded RNAs with a length of 15 to 27 nucleotides and have a partially or completely complementary double-stranded structure. In the siRNA of this invention, the length of the complementary double-stranded structure may be 16 to 25, 17 to 22, or 18 to 21 base pairs. The siRNA described in this invention may be a blunt-ended double-stranded RNA structure consisting of two single-stranded RNAs with a length of 15 to 27 nucleotides, or it may have a 3′ overhang consisting of 1 to 3 consecutive nucleotides at at least one end of the double-stranded structure.

[0179] In the present invention, unless otherwise specified, the term “first single strand” or “sense strand” refers to one of the two single strands of siRNA, which has the same nucleotide sequence as some or all of the nucleotide sequence of the site of action in the siRNA target mRNA, while the term “second single strand” or “antisense strand” refers to the other of the two single strands of siRNA, which has a nucleotide sequence complementary to some or all of the nucleotide sequence of the site of action in the siRNA target mRNA. The first single strand (or sense strand) and the corresponding second single strand (or antisense strand) of siRNA referred to in the present invention can form a partially or completely complementary double-stranded structure.

[0180] The “complementary” sequences used herein may further include base pairs formed by non-Watson-Crick base pairs and / or non-naturally modified nucleotides, or may be formed entirely from base pairs formed by non-Watson-Crick base pairs and / or non-naturally modified nucleotides, provided that they satisfy the above requirements regarding hybridization ability. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen base pairings.

[0181] In this invention, unless otherwise specified, the terms “suppress / suppressing” and “inhibit / inhibiting” refer to situations in which mRNA degradation mediated by siRNA or other small interfering nucleic acid (siNA) inhibitors causes significant downregulation of target gene expression. “Significant downregulation” refers to situations in which target gene expression is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or 100%, compared to normal or pre-treatment levels.

[0182] As used herein, the phrase "contacting cells with an RNAi agent (e.g., dsRNA)" includes contacting cells in any possible manner. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or in vivo. Contact may be direct or indirect. For example, the individual performing the method may physically bring the RNAi agent into contact with cells, or may place the RNAi agent in an environment that allows or causes subsequent contact with cells. For example, in vitro cell contact may be achieved by incubating cells with the RNAi agent. For example, in vivo cell contact may be achieved by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region (e.g., the central nervous system (CNS)), and optionally, the reagent may then be delivered to the tissue where the contacting cells are located by intrathecal, intravitreous, or other injection, or by injection into the bloodstream or subcutaneous space.

[0183] "G," "C," "A," and "U" typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it should be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, such as those described in more detail below, or to substitutional portions. Those skilled in the art will understand that guanine, cytosine, adenine, and uracil can be substituted with other portions without significantly altering the base-pairing properties of oligonucleotides containing nucleotides with substitutional portions.

[0184] In this invention, the chemical modification performed on siRNA is 1) Modification of phosphodiester bonds that link nucleotide residues in the main chain structure of RNA chains, 2) Modification of ribose in the main chain structure of RNA chains, 3) This may be one chemical modification or a combination of multiple chemical modifications selected from chemical modifications, which are modifications of bases in RNA nucleotide residues.

[0185] The naturally occurring nucleotide bond in siRNA is a 3′ to 5′ phosphodiester bond. RNA chains with one or more modified (i.e., unnaturally occurring) nucleotide bonds are usually preferred over RNA chains with naturally occurring nucleotide 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.

[0186] Oligonucleotides having modified internucleotide bonds include internucleotide bonds that retain a phosphorus atom and internucleotide bonds that do not contain a phosphorus atom. Typical phosphorus-containing internucleotide bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates.

[0187] RNAi agents may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphonate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinyl phosphonate (5'-VP), a 5'-terminal methyl phosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. If the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), then 5'-VP may be a 5'-E-VP isomer phosphonate, an isomer (i.e., cis-vinyl phosphonate), or a mixture thereof.

[0188] In the present invention, the RNAi agent comprises one or more modified internucleotide bonds. In some embodiments, the modified internucleotide bonds are phosphorothioate bonds. In some embodiments, the RNAi agent comprises a 5'-E-VP in the second strand.

[0189] In this invention, modification of the ribose group refers to modification of the 2′-hydroxy group (2′-OH) on the ribose group. By introducing several substituents, such as a methoxy group or a fluoro group, at the 2′-hydroxy group position of the ribose group, the stability of nucleic acids is improved by making it more difficult for ribonucleases in serum to digest the nucleic acids, thereby giving the nucleic acids stronger resistance to hydrolysis by nucleases. Modification of the 2′-hydroxy group on the pentose of a nucleotide includes 2′-fluoro modification, 2′-methoxy modification, 2′-methoxyethoxy modification, 2′-2,4-dinitrophenol modification (2′-DNP modification), loc nucleic acid modification (LNA modification), 2′-amino modification, and 2′-deoxy modification.

[0190] In this invention, base modification refers to modification of a base on a nucleotide group. Examples include 5′-bromouracil modification and 5′-iodouracil modification, where introducing bromine or iodine at the 5-position of uracil is a common method of base modification. Further modifications, such as N3-methyluracil modification and 2,6-diaminopurine modification, may also be used.

[0191] 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, which are optionally conjugated with the double-stranded RNAi agent via a linker or carrier.

[0192] In some embodiments, the conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be linked via a linker, for example, a divalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated with an iRNA agent (e.g., the 3' end of the sense strand) via a linker (e.g., a linker as described herein). In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated with an iRNA agent (e.g., the 5' end of the sense strand) via a linker (e.g., a linker as described herein).

[0193] 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 linker. In some embodiments, GalNAc or a GalNAc derivative is linked to the iRNA agent of the present invention via a divalent linker. 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 linker. 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 linker.

[0194] The single-stranded RNA siRNA of the present invention can be synthesized by solid-phase or liquid-phase nucleic acid synthesis methods. These methods include four processing steps: 1) oligonucleotide synthesis, 2) deprotection, 3) purification and separation, and 4) desalting. The technical details of these four steps are well known to those skilled in the art and will not be described in detail here.

[0195] The siRNA of the present invention can be obtained not only by chemical synthesis but also by expression of plasmids and / or viral vectors. For example, a DNA sequence having a length of 50 to 90 nucleotides is designed, and two different restriction enzyme cleavage sites, such as BamHI and EcoRI enzyme cleavage sites, are added to both ends. The intermediate segment sequence of the RNA transcript encoded by the designed DNA can form a loop structure, while the sequences at both ends of the loop after a U-turn can form a complementary double-stranded structure. By cloning techniques, the designed DNA is inserted into an expression vector digested with the appropriate restriction enzyme. The expression vector is introduced into cells, and the RNA transcript produced from the designed DNA sequence can be processed into mature siRNA by a cell-specific siRNA processing mechanism. This allows the siRNA to be expressed transiently or stably in cells.

[0196] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. When carrying out or testing the RNAi agents and methods of the present invention, similar or equivalent methods and materials as those described herein may be used, but suitable methods and materials are described below. All publications, patent applications, patents and other references referenced herein are incorporated herein by reference as a whole. In case of any inconsistency, the provisions of this specification (including definitions) shall prevail. The materials, methods and examples are used for illustrative purposes only and are not intended to limit the scope of the invention.

[0197] Examples The present invention will be described in conjunction with the following embodiments. Unless otherwise specifically described herein, sources of reagents and other experimental materials can be obtained from any molecular biological reagent supplier, provided that their quality / purity meets the standards for molecular biological applications.

[0198] Example 1. Design of small interfering nucleotides of the human-derived C3 gene The 19 bp nucleotide sequence was selected from the mRNA sequences of human-derived C3 genes within the range of 1–5231 bp, and the sequence is relatively conservative (Genbank accession number NM_000064, SEQ ID NO:1).

[0199] Table 1 lists the sequences of the sense strand and complementary antisense strand 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 the same as 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 were numbered sequentially in the same manner as siRNA C3-1.

[0200] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0201] Example 2: Verification of the inhibitory effect of siRNA on the expression of the C3 gene. The siRNAs designed in Table 2A were chemically synthesized by Thermo Fisher Scientific Inc. or GenScript. Complementary oligoribonucleotides can be annealed to form double-stranded RNA using the method described in "Molecular Cloning: A Laboratory Manual".

[0202] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0203] (1) HepG2 cell culture HepG2 cells were transfected using a custom-designed silencer siRNA library (C3). RQ values ​​were standardized to empty RNAiMAX-transfected HepG2 cells. Three measurements were performed for each sample and / or probe. (n=3) (2) HepG2 cell transfection method The day before transfection, 17,000 HepG2 parental cells per well were inoculated into 96-well tissue culture plates. siRNA transfection was prepared using the Lipofectamine® RNAiMAX transfection kit (catalog number #13778030) with 20 nM siRNA and 0.3 RNAiMAX reagent per well. Cells were harvested 72 hours after 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 qPCR reaction template, and TaqMan® gene expression master mix and custom TaqMan® gene expression assay probes specific to the target genes β-actin (Hs01060665_g1) and human C3 (Hs00163811_m1) were used. β-actin was used as an endogenous control. Samples were run on a Quant Studio® 12K Flex real-time PCR system. Data were analyzed using a comparative CT method, as shown in Table 2B and Figure 1. Relative C3 mRNA (%) = 2 -(サンプル ΔCt(C3のCt-ハウスキーピング遺伝子のCt)-モックΔCt(C3のCt-ハウスキーピング遺伝子のCt)) X 100%

[0204] The results showed that the exemplary siRNA agents tested effectively reduced levels of human C3 messenger RNA.

[0205] [Table 3-1] [Table 3-2]

[0206] Example 3. C3 mRNA knockdown effect in HepG2 cells transfected with selected siRNAs ranging from 0.128 pM to 100 nM (dose-response curve experiment).

[0207] After transfection of HepG2 cells with siRNAs ranging from 0.128 pM to 100 nM, the C3 knockdown effects of 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 confirmed. siRNA sequence information is shown in Table 2A. The results are shown in Figure 2A. 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 in Figure 2B.

[0208] HepG2 cell transfection method Cells were inoculated into 96-well clear F-bottom TC-treated plates (VWR #734-2327) at a density of 13,300 cells / well. siRNA transfection was performed using Lipofectamine® RNAiMAX transfection reagent (Invitrogen Life Technology) according to the manufacturer's protocol. Dose-response experiments were conducted using eight different concentrations of C3 siRNA, starting at 100 nM and diluting fivefold to 0.128 pM. Mock cells were those not treated with siRNA.

[0209] 72 hours after transfection, RNA was extracted using the Dynabeads® mRNA DIRECT® purification kit (Invitrogen Life Technology) according to the manufacturer's instructions. In short, after removing the medium from the cells, 100 μL of lysis buffer was added. The cell culture plate was placed in a shaker and shaken at 300 rpm for 30 minutes at room temperature. Dynabeads was then added to purify the mRNA in the lysate. Immediate reverse transcription was performed after RNA extraction using the SuperScript VILO cDNA synthesis kit (Invitrogen Life Technology). C3 and TBP mRNA were identified using the Quantstudio 5 real-time PCR system (Invitrogen Life Technology) with TaqMan qPCR primers, C3-FAM (Assay ID: Hs00163811_m1) and TBP-VIC (Assay ID: Hs00427620_m1). The activity of a given C3 siRNA was expressed as the percentage of the remaining C3 mRNA normalized to TBP mRNA in treated cells, relative to the C3 mRNA normalized to TBP mRNA (mock) in untreated cells. Dose-response curves were constructed using a 4-parameter logistic model in GraphPad Prism version 9. The data shown in Figures 2A and 2B are derived from three biological replicates. The data in Figure 2B are derived from the 0.16 nM and 100 nM data in Figure 2A.

[0210] Example 4. Chemical modification of siRNA To enhance the stability of small interfering nucleotides and improve the hepatic targeting of drug administration, chemical modification designs were performed on 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-derived C3 gene. The design principle is as follows: Nucleotides were numbered sequentially starting from the first nucleotide at the 5' end of the first or second single strand.

[0211] Modification pattern of the first single strand (sense strand): SS7: The pentoses of nucleotide residues 7, 9, 10, and 11 are modified by 2′-fluoro substitution, and the pentoses of nucleotide residues 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2′-methoxy modification.

[0212] SS1: The pentoses at nucleotide residues 3, 5, 7, 8, and 9 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 2, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2′-methoxy modification.

[0213] SS2: The pentoses at nucleotide residues 5, 7, 8, 9, and 11 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 2, 3, 4, 6, 10, 12, 13, 14, 15, 16, 17, 18, and 19 are modified by 2′-methoxy modification.

[0214] SS3: Pentoses at nucleotide residues 5, 7, 8, 9, and 14 are modified by 2′-fluoro substitution, and pentoses at nucleotide residues 1, 2, 3, 4, 6, 10, 11, 12, 13, 15, 16, 17, 18, and 19 are modified by 2′-methoxy modification.

[0215] Modification pattern of the second single strand (antisense strand): AS8: The pentoses at nucleotide residues 2, 6, 8, 9, 14 and 16 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18 and 19 are modified by 2′-methoxy modification.

[0216] AS4: The pentoses at nucleotide residues 2, 10, 14, 16, and 18 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 17, and 19 are modified by 2′-methoxy modification.

[0217] AS5: The pentoses at nucleotide residues 2, 8, 14, 16, and 18 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2′-methoxy modification.

[0218] AS6: The pentoses at nucleotide residues 2, 4, 14, 16, and 18 are modified by 2′-fluoro substitution, and the pentoses at nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 are modified by 2′-methoxy modification.

[0219] siRNA includes modification patterns of SS7 / AS8, SS1 / AS4, SS1 / AS5, SS1 / AS6, SS2 / AS4, SS2 / AS5, SS2 / AS6, SS3 / AS4, SS3 / AS5, or SS3 / AS6.

[0220] A phosphorothioate (represented by * in Tables 3A, 3B, 3C, 3D, 3E, and 3F) was introduced between the last three nucleotides at the 5' and 3' ends of the second single strand (antisense strand). A phosphorothioate was also introduced between the last three nucleotides at either the 5' or 3' end of the first single strand (sense strand). (E)-vinylphosphonate (represented by "(E)-VP" in Tables 3A, 3B, 3C, 3D, 3E, and 3F) was used at the 5' end of the second single strand (antisense strand).

[0221] In Tables 3A, 3B, and 3C, "GN" refers to a targeted ligand containing N-acetylgalactosamine (GalNAc).

[0222] Modified siRNAs C3-144 to C3-152 include the modification pattern SS7 / AS8. The sequence information for modified siRNAs C3-144 to C3-152 is shown in Table 3A. Modified siRNAs C3-104 to C3-113 include the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. The sequence information for modified siRNAs C3-104 to C3-113 is shown in Table 3B. Modified siRNAs C3-114 to C3-123 include the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. The sequence information for modified siRNAs C3-114 to C3-123 is shown in Table 3C.

[0223] [Table 4]

[0224] [Table 5]

[0225] [Table 6]

[0226] Specifically, GalNAc-1, GalNAc-3, and GalNAc-7 are conjugated with dsRNA and used in in vitro and in vivo screening. siRNA agents are conjugated with GalNAc-1, GalNAc-3, and GalNAc-7, as shown in the figure below. [ka]

[0227] Modified siRNAs C3-95 to C3-103 include the modification pattern SS7 / AS8. The sequence information for modified siRNAs C3-95 to C3-103 is shown in Table 3D. Modified siRNAs C3-124 to C3-133 include the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. The sequence information for modified siRNAs C3-124 to C3-133 is shown in Table 3E. Modified siRNAs C3-134 to C3-143 include the modification patterns SS1 / AS6, SS2 / AS5, SS2 / AS6, SS3 / AS5, and SS3 / AS6. The sequence information for modified siRNAs C3-134 to C3-143 is shown in Table 3F.

[0228] [Table 7]

[0229] [Table 8]

[0230] [Table 9]

[0231] Chemically modified small interfering nucleotides were synthesized by Wuxi AppTec. The targeted ligand (GalNAc) may be conjugated stepwise to the 3' or 5' end of an oligonucleotide as a single (tribranched) molecule or in a 1+1+1 trivalent form. To realize the tribranched GalNAc conjugate or 1+1+1 assembly, GalNAc amidates and / or GalNAc solid supports should be synthesized, respectively. These GalNAc amidates and / or GalNAc solid supports were then used as building blocks in solid-phase oligonucleotide synthesis.

[0232] GalNAc conjugate phosphoramidite: The synthesis of all GalNAc conjugate phosphoramidites followed the method described in Example 5.

[0233] GalNAc conjugate succinate and loading onto a solid carrier: The synthesis of GalNAc conjugate succinate and its loading onto a solid support were carried out according to the method described in Example 5.

[0234] General methods for siRNA preparation 1. Loading: Pre-loaded CPG (0.19 g, 500 A, ~10 μmol) of GalNAc- or nucleoside into a 5 mL syringe.

[0235] 2. Washing: Acetonitrile (2.0 mL, 0.3 minutes, repeated twice, at room temperature).

[0236] 3. Detritylation: 3% trichloroacetic acid in dichloromethane solution (2.0 mL, 0.7 minutes, repeated four times at room temperature).

[0237] 4. Washing: Acetonitrile (2.0 mL, 0.3 minutes, repeat twice, at room temperature).

[0238] 5. Coupling: A solution of 0.067 M reaction monomer in acetonitrile (1.0 mL) and a solution of 0.30 M 5-(benzylthio)-1H-tetrazole (BTT) in acetonitrile (1.0 mL) were used as activators (repeated three times for 7.0 minutes at room temperature). 6. Sulfidation or oxidation: N,N-dimethyl-N'-(3-thio-3H-1,2,4-diazole-5-yl)formamidine (DDTT, 4.80 M, pyridine / acetonitrile = 2 / 1, 2.0 mL, 1 minute, twice, room temperature) or solution of 0.05 M I2 in pyridine / H2O = 80 / 20 (v / v) (2.0 mL, 1 minute, twice, room temperature).

[0239] 7. Blocking: 1-Methylimidazole (NMI) / acetonitrile = 15 / 85 (v / v) (2.0 mL) and acetic anhydride / acetonitrile = 20 / 80 (v / v) (0.9 mL, 1 minute, once, room temperature).

[0240] 8. Washing: Acetonitrile (2.6 mL, 0.3 minutes, repeated twice, at room temperature).

[0241] 9. The synthesis procedure was automatically cycled 19 times.

[0242] 10. The solid support was immersed in 10% DEA for 20 minutes. The solid support was suspended in NH3·H2O (5 mL) and stirred in a 48 mL sealed tube at 40°C for 16 hours. The reaction mixture was cooled to 25°C. The solid support was then filtered, and the aqueous phase was concentrated under vacuum to obtain a yellow solution.

[0243] 11. Add 40 mL of ethanol to the filtrate (10 mL), and then add 0.3 mL of NaCl (3 M). The centrifuge tube was left to stand at -20°C for 20 minutes. Then the tube was centrifuged. Discard the supernatant and collect the remaining solid.

[0244] 12. The white solid was purified by preparative HPLC (column: O-C18 150*40mm*10μm, mobile phase: [0.1 M TEAB-ACN], B%: 14%~24%, 30 minutes).

[0245] 13. After freeze-drying, the compound was obtained as a desired white solid.

[0246] Annealing step: 1. Calculate the molar value of the double-stranded compound (for example, 2 mg of double-stranded compound, molar value = 2 mg / double-stranded compound MW, free acid), which is the molar value of the single-stranded compound.

[0247] 2. At 25°C, the sense and antisense strands were dissolved in ultrapure water (DNase / RNase-free, sterilized). The oligomer concentration was quantified by ultraviolet-visible light (UV-vis).

[0248] 3. The two solutions were mixed in a 1:1 molar ratio.

[0249] 4. The mixed sample was left at room temperature for 10 minutes. HPLC was used to monitor whether the mixing ratio was appropriate.

[0250] 5. The solution was dispensed into test tubes and freeze-dried to obtain siRNA samples.

[0251] Example 5: Chemical synthesis of GalNAc ligand. [ka]

[0252] Compound 1-2: Compound 1-1 (5 g, 12.84 mmol) was dissolved in anhydrous 1,2-dichloroethane (30 mL), stirred at 0°C, and within 10 minutes, TMSOTf (3.43 g, 15.41 mmol, 2.78 mL) was added dropwise, 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 obtain compound 1-2 (4.23 g, yield 99%), a yellow oily substance, which was usable without further purification.

[0253] C 14 H 19 Calculated mass of NO8: 329.1, Measured mass: 330.1 [M+H] + ,ESI.

[0254] Compound 1-3: Compound 1-2 (4.23 g, 12.85 mmol) was dissolved in anhydrous 1,2-dichloroethane (20 mL) and stirred at room temperature for 5 minutes with a 4 Å molecular weight sieve (4.7 g). 5-Hexen-1-ol (1.42 g, 14.13 mmol) was added and stirring continued for 30 minutes. TMSOTf (1.43 g, 6.42 mmol, 1.16 mL) was added dropwise at 0°C and stirring continued at room temperature for 2 hours. 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 obtain compound 1-3 (5.5 g, 99% yield), a yellow oily substance, which was usable without further purification.

[0255] C 20 H 31 Calculated mass of NO9: 429.2, Measured mass: 430.2 [M+H] + ,ESI.

[0256] Compound 1-4: To the solutions of compounds 1-3 (5.5 g, 12.81 mmol) in DCM (35 mL) and MeCN (35 mL), 4.0 mol equivalents of sodium (meth)periodate (10.96 g, 51.24 mmol) in water (45.5 mL) were added. The mixture was cooled to 0°C in an ice bath and stirred for 15 minutes. Ruthenium chloride trihydrate (110.5 mg, 423 μmol) was added to the cold reaction mixture. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water (90 mL), and solid NaHCO3 was added to adjust the pH to 7.5. The DCM layer was removed, the aqueous layer was washed with DCM (30 mL x 2), and the organic extract was discarded. Citric acid was added to adjust the pH of the aqueous layer to 3, and carboxylic acids 1-4 were extracted in DCM (50 mL x 3). The organic layer was stirred 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. Each layer was separated, and the organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to obtain compound 1-4 (2.3 g, 40% yield), which was a white solid. It was usable without further purification.

[0257] C 19 H 29 NO 11 Mass calculated value of: 447.2, measured: 448.2 [M+H] + , ESI. 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J=9.2 Hz, 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).

[0258] 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) were added HBTU (2.92 g, 7.71 mmol) and DIPEA (1.99 g, 15.42 mmol, 2.69 mL). 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 successively washed with water (100 mL x 3) and brine (100 mL). After drying over anhydrous Na2SO4, the solvent was evaporated under reduced pressure to obtain compound 1 - 6 (4.3 g, crude product) as a yellow oil, which could be used without further purification.

[0259] C 29 H 48 N2O 12 Mass calculated value of: 616.3, measured: 617.4 [M+H] + , ESI.

[0260] Compound 1 - 7: Compound 1-6 (assuming 4.23 g, 6.86 mmol) was added to formic acid (30 mL), and the mixture was stirred overnight at room temperature. The completion of the reaction was monitored by LC-MS. The mixture was evaporated under reduced pressure and purified by silica gel chromatography (DCM:MeOH = 10:1) to obtain compound 1-7 (2.88 g, 2-step yield 75%), a yellow oily substance.

[0261] C 25 H 40 N2O 12 Calculated mass: 560.3, measured mass: 561.3 [M+H] + ,ESI.

[0262] 1 H NMR (400MHz,DMSO-d6) δ12.02 (s,1H),7.83 (d,J=9.2Hz,1H),7.25 (t,J=5.6Hz,1H),5.21 (d,J=3.2Hz,1H),4.96 (dd,J=11.2,3.6Hz,1H),4.47 (d,J=8.4Hz,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=7.2Hz,2H),2.10 (s,3H),2.02 (t,J=7.2Hz,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). [ka]

[0263] 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, extracted with EA (1 L), washed with NaHCO3 solution, washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was recrystallized with MeOH to obtain the desired product 2-1 (247 g, 68% yield). 1 H NMR (400MHz,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).

[0264] Compound 2-2: To a suspension of t-BuOK (137.27 g, 1.43 mol) in anhydrous DMF (700 mL), diisopropyl malonate (268.85 g, 1.43 mol) was added dropwise (while maintaining a temperature below 70°C), and then compound 2-1 (250 g, 714.19 mmol) was added. The resulting reaction mixture was heated at 140°C for 6 hours. After cooling, saturated NH4Cl solution (1.5 L) was added, and the mixture was extracted with hexane (500 mL x 3). The combined organic extract was 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 obtain pure product 2-2 (194 g, yield 68%), which was a white solid.

[0265] C 21 H 28 Calculated mass of O6: 376.2, measured mass: 377.2 [M+H] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.45-7.31 (m,5H),5.45 (s,1H),5.02-4.93 (m,2H),3.96 (d,J=11.2Hz,2H),3.77 (d,J=11.1Hz,2H),2.55 (s,2H),2.13 (s,2H),1.23-1.15 (m,12H).

[0266] Compound 2-3: To a solution of compound 2-2 (155 g, 411.7 mmol) in MeOH (750 mL), 10% Pd / C (31 g) was added, and the resulting suspension was hydrogenated with H2 at 5 atm at ambient temperature for 48 hours with stirring. The catalyst was filtered, and the solvent was removed by vacuum to obtain compound 2-3 (118 g, 99% yield), a colorless oily substance that could be used in the next step without further purification.

[0267] C 21 H 28 Calculated mass of O6: 288.2, measured mass: 289.4 [M+H] + ,ESI.

[0268] Compound 2-4: To a solution of compound 2-3 (140 g, 485.5 mmol) in dichloromethane (840 mL), methanesulfonyl chloride (155.7 g, 1.36 mol) was added. The resulting mixture was cooled to -30°C, and triethylamine (323.9 g, 3.2 mol) was added dropwise. After the addition was complete, the reaction mixture was heated to ambient temperature, stirred for 12 hours, and 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 obtain compound 2-4 (125 g, yield 58%).

[0269] C 21 H 28 Calculated mass of O6: 444.1, measured mass: 445.2 [M+H] + ,ESI. 1H NMR (400MHz,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).

[0270] Compound 2-5: A solution of compound 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 SiO2 (600 mL). The combined organic phase was washed with 10% aqueous citric acid (600 mL) and brine (600 mL), dried over sodium sulfate, evaporated under vacuum, and recrystallized with isopropyl ether to obtain compound 2-5 (48 g, yield 80%).

[0271] C 21 H 28 The calculated value for O6 is 423.2, while the measured value is 424.2 [M+H]. + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.67 (d,J=8.2Hz,2H),7.45 (d,J=8.0Hz,2H),4.45 (t,J=5.4Hz,2H ),3.60 (s,4H),3.14 (d,J=5.7Hz,4H),2.43 (s,3H),1.67 (s,4H).

[0272] Compound 2-6: At -20°C, a solution of compound 2-5 (58.7 g, 138.60 mmol) in THF (100 mL) was added to a solution of lithium borohydride (2 M) in THF (263.82 mL). The resulting mixture was stirred at room temperature for 16 hours. The mixture was slowly added to ice water (1 L). The pH was adjusted to 7 with citric acid aqueous solution. The mixture was extracted with EA (500 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain compound 2-6 (40 g, 93% yield).

[0273] C21 H 28 Calculated mass of O6: 311.1, measured mass: 312.1 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-D6) δ 7.67 (d,J=8.2Hz,2H),7.45 (d,J=8.0Hz,2H),4.45 (t,J=5.4Hz,2H ),3.60 (s,4H),3.14 (d,J=5.7Hz,4H),2.43 (s,3H),1.67 (s,4H).

[0274] Compound 2-7: Compound 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). The mixture was concentrated to dry to obtain compound 2-7 (2.55 g, crude product), which was a yellow solid. The crude product was usable without further purification.

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

[0276] Compound 2-8: Compound 2-7 (assuming 2.55 g, 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 the addition of Na2CO3 (saturated, 20 mL). The reaction was stirred at 30°C for 3 hours. LC-MS showed complete conversion. The reaction mixture was extracted using EA (20 mL x 3). The organic phase was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH=97:3) to obtain a white solid 2-8 (2.75 g, 2-step yield 44.7%).

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

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

[0279] C 44 H 43 Calculated mass of NO6: 681.3, measured mass: 682.3 [M+H] + ,ESI.

[0280] Compound 2-10: Compound 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 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH = 95:5) to obtain the yellow solid 2-10 (0.5 g, yield 33%).

[0281] C 29 H 33 Calculated mass of NO4: 459.2, measured mass: 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 minutes, and then cooled to 0°C. DIPEA (211 mg, 1.6 mmol) was added, followed by compound 2-9 (300 mg, 0.65 mmol), and the mixture was stirred at room temperature for 4 hours. LC-MS showed complete conversion. NaHCO3 (10 mL saturated solution) was added. The reaction mixture was extracted using DCM (5 mL x 3). The organic phase was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH = 95:5) to obtain crude compound 2-11 (350 mg, 88% purity at 210 nm, 46% yield), which was a white solid. Purification by preparative HPLC: 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 rate 42%). 54 H 71 N3O 15 Calculated mass: 1001.5, measured mass: 1024.5 [M+Na] + ,ESI.

[0283] 1 H NMR (400MHz,DMSO) δ 7.82 (d,J=9.2Hz,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=3.3Hz,1H),5.00-4.90 (m,1H),4.66-4.63 (m,1H),4.48 (d,J=8.4Hz,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). [ka]

[0284] Compound 2-12: To a solution of compound 2-11 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (32.0 mg, 0.27 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (108 mg, 0.36 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 2-12 (177 mg, 49% yield), a white solid, was produced.

[0285] C 63 H 88 N5O 16 Calculated mass of P: 1201.60, measured mass: 1202.6 [M+H] + ,ESI. 1 HNMR (600MHz,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=6.0Hz,1H),4.93-4.91 (m,1H),4.44 (d,J=6.0Hz,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 (242MHz,CD3CN) δ 147.37,147.30.

[0286] 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 LC-MS showed that the starting materials were completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), and then with brine (3 mL x 4). The organic layer was concentrated to obtain compound 2-13 (85 mg, 97% yield), which was a white solid.

[0287] C 58 H 75 N3O 18 Calculated mass: 1101.50, measured mass: 1100.4 [MH] - ,ESI. 1 HNMR (600MHz,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=6.0Hz,1H),4.97-4.95 (m,1H),4.48 (d,J=12.0Hz,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).

[0288] 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 dried overnight under high vacuum. DIPEA (30 mg, 0.23 mmol) was added to a solution of succinate ester 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 minutes. Then, natural amino-LCAA-CPG (250 mg, loading 75 μmol / g) was added, and the suspension was shaken at room temperature for 20 hours. After filtration, the mixture was washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until no eluate spots were visible at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (260 mg). It was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the solid support, and washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until TLC showed no eluate spots at 254 nm. The solid support was vacuum-dried for 15 hours to obtain solid supports 2-14 (260 mg). To calculate the loading, 6.5 mg of dry loading CPG was taken and 25 mL of 3% DCA-containing DCM solution was added. The solution was shaken and the UV absorbance at 500 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. 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 added DCA (mL) = 25 mL Abs value at 500nm = 0.7721 Number of mg of CPG taken = 6.5 mg Loading (μmol / g) = ((25) * (0.7721) * 1000) / (76 * (6.5)) = 39 μmol / g. Method 2: [ka]

[0289] Compound 2-2: Compound 2-1 (11 g, 26.0 mmol) was dissolved in 100 mL of anhydrous THF in a 250 mL flask. LiAlH4 (1.97 g, 52 mmol) was added gradually at 0°C for 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, water (2 mL) was slowly added, then NaOH (10%, 2 mL) was added, and then water (6 mL) was added. The mixture was filtered, and the filtrate was concentrated to dry to obtain compound 2-2 (6.5 g, yield 80%), a white solid. The crude product was usable without further purification.

[0290] C 15 H 21 NO4S: 311.1, Measured: 312.1 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO) δ 7.67 (d,J=8.0Hz,2H),7.48 (d,J=8.0Hz,2H),4.46 (t,J=5.4Hz,2H),3.60 (s,4H),3.31 (d,J=5.6Hz,4H),2.43 (s,3H),1.67 (s,4H).

[0291] Compound 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). The mixture was concentrated to dry to obtain compound 2-3 (2.55 g, crude product), which was a yellow solid. The crude product was usable without further purification.

[0292] C8H 15 Calculated mass of NO2: 157.1, measured mass: 158.1 [M+H] + ,ESI.

[0293] Compound 2-4: Compound 2-3 (assuming 2.55 g, 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 the addition of Na2CO3 (saturated, 20 mL). The reaction was stirred at 30°C for 3 hours. LC-MS showed complete conversion. The reaction mixture was extracted using EA (20 mL x 3). The organic phase was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH=97:3) to obtain a white solid 2-4 (2.75 g, 2-step yield 44.7%).

[0294] C 23 H 25 Calculated mass of NO4: 379.2, measured mass: 380.2 [M+H] + ,ESI.

[0295] Compound 2-5: Compound 2-4 (2.7 g, 7.1 mmol) and pyridine (20 mL) were mixed in a flask, and DMTr-Cl (2.4 g, 7.1 mmol) was added gradually. The reaction was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The reaction mixture was extracted using EA (20 mL x 3). The organic phase was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH = 97:3) to obtain a yellow solid 2-5 (2.8 g, 58% yield).

[0296] C 44 H 43 Calculated mass of NO6: 681.3, measured mass: 682.3 [M+H] + ,ESI.

[0297] Compound 2-6: Compound 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 hours. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH = 95:5) to obtain a yellow solid 2-6 (0.5 g, yield 33%).

[0298] C 29 H 33 Calculated mass of NO4: 459.2, measured mass: 460.2 [M+H] + ,ESI.

[0299] Compound 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 minutes, 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 hours. LC-MS showed complete conversion. NaHCO3 (10 mL solution of saturated solution) was added. The reaction mixture was extracted using DCM (5 mL x 3). The organic phase was concentrated and purified by high-performance silica gel column chromatography (DCM:MeOH = 95:5) to obtain crude compound 2-7 (350 mg, 88% purity at 210 nm, 46% yield) as a white solid. Purification by preparative HPLC: 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, measured mass: 1024.5 [M+Na] + ,ESI.

[0300] 11H 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).

[0301] Compound 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 Compound 2 - 7 (300.0 mg, 0.30 mmol) in anhydrous DCM (3.0 mL), and the mixture was stirred for 1 hour. LCMS indicated that the SM was completely consumed. The solution was quenched with water (100 mL), washed with brine (300 mL x 3), and dried over Na2SO4. Then the solution was 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, increasing to CH3CN / H2O = 1 / 0 within 20 minutes, eluted product was collected at CH3CN / H2O = 3 / 2, detector: UV 254 nm). Compound 2 - 8 (177 mg, 0.15 mmol, 97.0% purity, 49% yield), a white solid, was obtained.

[0302] C 63 H 88 N5O 16 Calculated mass for P: 1201.60, found: 120I.6 [M + H] + , ESI. 11H-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), 6.44 - 6.43 (m, 1H), 5.20 - 5.19 (d, J = 6.0 Hz, 1H), 4.93 - 4.91 (m, 1H), 4.44 - 4.43 (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 13C NMR (242 MHz, CD3CN) δ 147.37, 147.29.

[0303] Compound 2-9: A solution of Compound 2-7 (80 mg, 0.080 mmol) in anhydrous DCM (1.0 mL) was added with DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol), and then succinic anhydride (20 mg, 0.2 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and LCMS indicated that the starting material was completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), and then washed with brine (3 mL x 4). The organic layer was concentrated to obtain Compound 2-9 (85 mg, 0.077 mmol, purity 97%, yield 97%) as a white solid, C 58 H 75 N3O 18 Calculated mass for: 1101.50, Found: 1100.4 [M-H] - , ESI. 1H-NMR (600MHz,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=6.0Hz,1H),4.97-4.95 (m,1H),4.49-4.47 (d,J=12.0Hz,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).

[0304] 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.

[0305] To a solution of succinate ester 2-9 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL), DIPEA (30 mg, 0.23 mmol) was added, and the reaction mixture was shaken at room temperature for 10 minutes. Then, natural amino-LCAA-CPG (250 mg, loading 75 μmol / g) was added, and the suspension was shaken at room temperature for 20 hours. 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 no spots of eluate were observed at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (260 mg). The solid support was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the solid support, and washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until the eluate spot disappeared at 254 nm by TLC. The solid support was vacuum-dried for 15 hours to obtain solid support 2-10 (260 mg). To calculate the loading, 6.5 mg of dry loading CPG was taken and 25 mL of 3% DCA-containing DCM solution was added. The solution was shaken and the UV absorbance at 500 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. 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 added DCA (mL) = 25 mL Abs value at 500nm = 0.7721 Number of mg of CPG taken = 6.5 mg Loading (μmol / g) = ((25) * (0.7721) * 1000) / (76 * (6.5)) = 39 μmol / g. [ka]

[0306] Compound 3-2: To a solution of compound 3-1 (40 g, 211.36 mmol) in NH3 / MeOH (7 M, 192 mL), 2-ethyl cyanoethyl (47.82 g, 422.71 mmol) was added dropwise, and the mixture was stirred overnight. LC-MS showed complete conversion, and the reaction mixture was filtered. The filtered cake was ground in PE (100 mL) and filtered. The filtered cake was dried to obtain solid compound 3-2 (41 g, 44% yield).

[0307] Calculated mass of C18H18N4O2: 322.1, measured mass: 323.1 [M+H] + ,ESI.

[0308] 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 hours. LC-MS showed complete conversion, and 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 dry to obtain crude compound 3-3 (72 g, 95% yield). The crude product could be used directly without further purification.

[0309] Calculated mass of C16H21NO4: 291.2, Measured mass: 292.3 [M+H] + ,ESI.

[0310] Compound 3-4: Compound 3-3 (72.3 g of crude product) was stirred at 85°C for 4 hours 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, concentrated to dry, and purified by silica gel column (0-25% EA in PE) to obtain compound 3-4 (62.5 g, 2-step yield 78%).

[0311] C 18 H 25 Calculated mass of NO4: 319.2, measured mass: 320.3 [M+H] + ,ESI.

[0312] Compound 3-5: At 0°C, LiAlH4 (14.3g, 375.1 mmol) was gradually added to a solution of compound 3-4 (40g, 125.2 mmol) in THF (295 mL), and the mixture was stirred for 1 hour. LC-MS showed complete conversion, and ethyl acetate (100 mL) was added to the reaction mixture, followed by 10% NaOH (aqueous solution) to adjust the pH to 9-10. The mixture was then filtered and washed with ethyl acetate (50 mL x 2). The combined filtrate was concentrated to dry to obtain crude compound 3-5 (23g, 83% yield). The crude product could be used in the next step without further purification.

[0313] Calculated mass of C16H25NO2: 263.2, measured mass: 264.3 [M+H] + ,ESI.

[0314] Compound 3-6: Compound 3-5 (20 g, 75.94 mmol), Boc2O (16.57 g, 75.94 mmol), and Pd / C (10% palladium-on-carbon, wet, ~55% water) were placed in MeOH (150 mL) and stirred overnight at room temperature with H2 at 1 atm. LC-MS showed complete conversion, and the reaction mixture was filtered and concentrated to obtain oily compound 3-6 (20 g, 96% yield), which was usable without further purification.

[0315] C 14 H 27 The calculated mass of NO4 is 273.2, while the measured mass is 274.1 [M+H]. + ,ESI.

[0316] Compound 3-7: To a solution of compound 3-6 (58 g, 212.17 mmol) in triethylamine (85.88 g, 848.67 mmol, 118.37 mL) and DCM (1.16 L), MsCl (72.91 g, 636.51 mmol) was added. The reaction was stirred overnight at room temperature. LC-MS showed complete conversion (detection by EtOH quench and diether). The reaction mixture was diluted with DCM (1 L), washed with 10% citrate, then with NaHCO3 (saturated aqueous solution), and dried to obtain the oily compound 3-7 (87 g, 95% yield), which was used directly in the next step without further purification.

[0317] C 16 H 31 Calculated mass of NO8S2: 429.2, measured mass: 430.3 [M+H] + ,ESI.

[0318] Compound 3-8: Under nitrogen gas, 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 minutes to form a clarified solution. KI (1.55 g, 9.31 mmol) was added, followed by compound 3-7 (20 g, 46.56 mmol). The reaction mixture was stirred at 70°C for 30 minutes, and then at 140°C for a further 30 minutes. 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 (solution in PE with 0-10% EA) to obtain compound 3-8 (3.55 g, yield 18%), a colorless oily substance.

[0319] C 23 H 39 Calculated mass of NO6: 425.3, measured mass: 426.2 [M+H] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 4.96-4.90 (m,2H),3.27 (br,4H),1.86 (t,J=10.8Hz,4H),1.38 (br,13H),1.28 (s,4H),1.17-1.16 (m,12H).

[0320] Compound 3-9: Compound 3-8 (7.5 g, 17.62 mmol) was added to LiBH4 (172 mL, 1 M, in THF) 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 NaOH (10% aqueous solution, 20 mL), and the mixture was filtered. The filtrate was diluted with water (200 mL), extracted with ethyl acetate (50 mL x 3), and concentrated to obtain compound 3-9 (6.35 g, 98% yield), a colorless oily substance that could be used directly in the next step without further purification.

[0321] C 17 H 31 Calculated mass of NO4: 313.2, measured mass: 314.2 [M+H] + ,ESI.

[0322] Compound 3-10: Compound 3-9 (6.35 g, 20.26 mmol) was 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 dry and azeotropically dried with toluene to obtain compound 3-10 (7 g, 99% yield), which was ready for direct use in the next step without further purification.

[0323] C 12 H 23 Calculated mass of NO2 (free base): 213.2, measured mass: 214.2 [M+H] + ,ESI. 1 H NMR (400MHz,D2O) δ 3.39 (br,4H),3.18 (t,J=11.6Hz,4H),1.59 (t,J=12.0Hz,4H),1.36-1.33 (m,4H),1.25 (t,J=12.4Hz,4H).

[0324] Compound 3-11: The solutions of compound 3-10 (5g, 20.02 mmol), DIEA (6.47g, 50.04 mmol, 8.72 mL), HOBT (3.25g, 24.02 mmol), and EDCI (4.60g, 24.02 mmol) in DCM (150 mL) were stirred at 0°C for 30 minutes, and then Fmoc-6-aminocaproic acid (5.66g, 16.01 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 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, concentrated to obtain the crude product, which was purified by silica gel column (solution in PE of 0-10% EA) to obtain the gel-like compound 3-11 (3.65 g, LC-MS purity 92%, yield 33%).

[0325] C 33 H 44 Calculated mass of N2O5: 548.3, measured mass: 549.3 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.88 (d,J=7.5Hz,2H),7.84 (d,J=7.5Hz,2H),7.41 (t,J=7.3Hz,2H),7.34 (t,J=7.3Hz,2H),6.65 (t,J=5.3Hz,1H),6.28 (s,2H),4.36-4.18 (m,3H),3.36 (dt,J=19.9,5.2Hz,4H),3.24 (s,4H),2.92-2.87 (m,2H),2.24 (t,J=7.4Hz,2H),1.49-1.26 (m,18H).

[0326] 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 concentrated and purified by silica gel column (eluting impurities with a solution of 10-40% EA in PE, and then eluting impurities with a solution of 5-10% MeOH and 0.1% NH3·H2O in DCM) to obtain compound 3-12 (2.75 g, yield 69%). 1 H NMR (400MHz,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=5.0Hz,1H),4.28 (d,J=6.8Hz,2H),4.20 (t,J=6.8Hz,1H),3.72 (s,8H),3.40-3.39 (m,2H),3.32-3.27 (m,4H),2.96 (q,J=6.3Hz,2H),2.22 (t,J=7.9Hz,2H),1.48-0.86 (m,18H).

[0327] 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 azeotropically dried with ditoluene. The residue was purified by silica gel column chromatography (solution of 0-10% MeOH and 0.1% NH3·H2O in DCM) to obtain the gel compound 3-13 (1.1 g, yield 87%).

[0328] C 39 H 52 Calculated mass of N2O5: 628.4, measured mass: 629.4 [M+H] + ,ESI.

[0329] Compound 3-14: The solution of compound 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 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 in another DCM (20 mL), washed with NaHCO3 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to obtain compound 3-14 (700 mg, yield 44%).

[0330] C 58 H 79 N3O 15 Calculated mass: 1057.6, measured mass: 1080.5 [M+Na] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.82 (d,J=9.2Hz,1H),7.69 (t,J=5.5Hz,1H),7.40-7.38 (m,2H),7.32-7.19 (m,7H),6.89-6.87 (m,4H),5.21 (d,J=3.3Hz,1H),4.97 (dd,J=11.2,3.4Hz,1H),4.48 (d,J=8.5Hz,1H),4.40 (t,J=5.0Hz,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=6.6Hz,2H),2.87 (s,2H),2.22 (t,J=7.3Hz,2H),2.10 (s,3H),2.03 (t,J=7.0Hz,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).

[0331] Compound 3-15: To a solution of compound 3-14 (670.0 mg, 0.61 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 3-15 (600 mg, 83% yield), a white solid, was produced.

[0332] C 67 H 96 N5O 16 Calculated mass of P: 1257.66, measured mass: 1204.5 [M-CH2CH2CN] - ,ESI.

[0333] 1 HNMR (600MHz,DMSO-d6) δ 7.81-7.67 (m,2H),7.40-7.19 (m,9H),6.88-6.86 (m,4H),5.21 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 146.21,146.19. [ka]

[0334] Compound 4-2: At 0°C, 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). 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 concentrated to obtain crude product 4-2 (27 g, 95% yield), which was usable in the next step without further purification.

[0335] C 12 H 21 Calculated mass of NO4: 243.2, measured mass: 244.1 [M+H] + ,ESI.

[0336] Compound 4-3: To a solution of compound 4-2 (35 g, 143.86 mmol) in methanol (28 ml), sodium borohydride (11.97 g, 316.48 mmol) was added within 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 saturated ammonium chloride solution, concentrated, extracted three times with ethyl acetate (EA), washed with brine, and recrystallized in an EA:PE ratio of 1:3 to obtain compound 4-3 (19.6 g, 54% yield).

[0337] C 12 H 23 Calculated mass of NO4: 245.2, measured mass: 146.1 [M+H-Boc] + ,ESI.

[0338] 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 the mixture was cooled to -30°C. MsCl (35.02 g, 305.73 mmol) was 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% aqueous citric acid solution, and dried over anhydrous sodium sulfate. After concentration, the residue was purified by silica gel column chromatography to obtain compound 4-4 (40 g, yield 81%), a pale yellow oily liquid.

[0339] C 14 H 27 Calculated mass of NO8S2: 401.1, measured mass: 302.1 [M+H-Boc] + ,ESI.

[0340] Compound 4-5: Under nitrogen gas, diisopropyl malonate (14.06 g, 74.72 mmol) was slowly added to a mixture of t-BuONa (9.57 g, 99.63 mmol) in DMA (50 mL). After stirring for 20 minutes, 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 adding saturated ammonium chloride and extracted with ethyl acetate. After concentration, the residue was purified by silica gel column (PE:EA) to obtain compound 4-5 (6 g, 30% yield).

[0341] C 21 H 35 Calculated mass of NO6: 397.3, measured mass: 398.2 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO) δ 4.98-4.90 (m,2H),3.22-3.20 (m,4H),2.25 (s,4H),1.42 (t,J=5.6Hz,4H),1.38 (s,9H),1.17 (d,J=6.0Hz,12H).

[0342] Compound 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. Ethyl acetate (200 mL) was then added to the reaction mixture, extracted with water (150 mL x 3), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain crude product 4-6 (6.52 g, 93% yield), which was ready for use in the next step without further purification.

[0343] C 15 H 27 Calculated mass of NO4: 285.2, measured mass: 186.2 [M + H - Boc] + ,ESI.

[0344] Compound 4-7: At 0°C, a solution of product 4-6 (7.52 g, 26.35 mmol) in DCM (100 mL) was added to HCl (4 M in 1,4-dioxane, 21.52 mL) and stirred for 15 minutes. The reaction mixture was heated to room temperature and stirred for 3 hours. The reaction mixture was filtered, and the filter cake was washed with dichloromethane (x3) to obtain crude product 4-7 (3.7 g, 76% yield).

[0345] C 10 H 19 Calculated value of NO2 (free base): 185.1 m / z, measured value: 186.2 [M+H] + ,ESI.

[0346] Compound 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 a 10 mL solution of DCM was stirred for 30 minutes at room temperature. 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 to obtain compound 4-8 (1.35 g, yield 58%).

[0347] C 31 H 40 Calculated mass of N2O5: 520.3, measured mass: 521.2 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO) δ 7.88 (d,J=7.5Hz,2H),7.68 (d,J=7.4Hz,2H),7.39 (d,J=7.5Hz,4H),7.34-7.18 (m,10H),6.89 (d,J=8.8Hz,4H),4.64 (t,J=4.7Hz,1H),4.28 (d,J=6.8Hz,2H),4.20 (t,J=6.8Hz,1H),3.73 (s,6H),3.44 (d,J=5.0Hz,2H),3.30-3.10 (m,4H),3.01 (s,2H),3.00-2.90 (m,2H),2.21 (t,J=7.2Hz,2H),1.56 (d,J=11.9Hz,2H),1.46-1.30 (m,8H),1.21-1.00 (m,4H).

[0348] Compound 4-9: A mixture of DMTr-Cl (488.07 mg, 1.44 mmol) and compound 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, dried with alumina, and purified by column chromatography on alumina (0-10% MeOH, 0.1% TEA in DCM) to obtain compound 4-9 (0.39 g, yield 32.9%). 1 H NMR (400MHz,DMSO) δ 7.39 (d,J=7.3Hz,2H),7.34-7.18 (m,7H),6.89 (d,J=8.8Hz,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=7.4Hz,2H),1.57 (d,J=12.1Hz,2H),1.47-1.08 (m,12H).

[0349] Compound 4-10: Compound 4-9 (1.4 g, 1.70 mmol) was dissolved in MeOH (40 mL), and piperidine (4 mL) was added. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum. Piperidine residue was further removed by azeotropic drying with ditoluene. The residue was then purified by silica gel column chromatography (DCM:MeOH = 10:1, methanol containing 10% aqueous ammonia) to obtain compound 4-10 (1 g, 97% yield).

[0350] C 37 H 48 Calculated mass of N2O5: 600.4, measured mass: 601.4 [M+H] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.40-7.39 (m,2H),7.33-7.21 (m,7H),6.89 (d,J=8.8Hz,4H),4.63 (s,1H),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=12.2Hz,2H),1.45-1.33 (m,7H),1.27-1.21 (m,4H),1.09 (s,1H).

[0351] 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 10 mL of DCM was stirred for 20 minutes, and then added to a solution of compound 4-10 (920 mg, 1.53 mmol) in 5 mL of DCM. The mixture was stirred overnight at room temperature. Water was added to quench the reaction, the organic layer was separated and concentrated. The residue was purified by preparative HPLC to obtain compound 4-11 (1 g, 63% yield).

[0352] C 56 H 75 N3O 15 Calculated mass: 1029.5, measured mass: 1052.3 [M+Na] + ,ESI.

[0353] 1H NMR (400MHz,DMSO-d6) δ 7.82 (d,J=9.2Hz,1H),7.79 (t,J=5.0Hz,1H),7.40-7.19 (m,9H),6.90-6.88 (m,2H),5.21 (d,J=3.2Hz,1H),4.97 (dd,J=11.2,3.3Hz,1H),4.62 (t,J=4.9Hz,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=7.3Hz,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). [ka]

[0354] Compound 4-12: To a solution of compound 4-11 (750.0 mg, 0.75 mmol) in anhydrous DCM (7.0 mL) at room temperature, 4,5-dicyanoimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (270 mg, 0.90 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column, C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). A white solid of 4-12 (500 mg, yield 53%) was produced.

[0355] C 65 H 92 N5O 16 Calculated mass of P: 1229.63, measured mass: 1129.0 [MN(CH(CH3)2)2] +,ESI. 1 HNMR (600MHz,DMSO-d6): δ 7.44-7.18 (m,9H),6.85-6.83 (m,4H),6.52-6.44 (m,2H),5.26 (d,J=6.0Hz,1H),4.99-4.96 (m,1H),4.49 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 147.17,147.16.

[0356] Compound 4-13: To a solution of compound 4-11 (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 LC-MS showed that the starting materials were completely consumed. The reaction mixture was then diluted with DCM, washed with water (3 mL x 4), and then with brine (3 mL x 4). The organic layer was concentrated to obtain compound 4-13 (85 mg, 97% yield), which was a white solid.

[0357] C 60 H 79 N3O 18 Calculated mass: 1129.54, measured mass: 1128.6 [MH] - ,ESI. 1HNMR (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), 4.12 - 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).

[0358] Compound 4-14: A solution of compound 4-13 (85 mg, 0.077 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL) was mixed with DIPEA (30 mg, 0.23 mmol). The reaction mixture was shaken at room temperature for 10 minutes, then natural amino-ICAA-CPG (250 mg, loading 75 μmol / g) was added. The suspension was shaken at room temperature for 20 hours, filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until no eluate spots were visible at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (260 mg). The solid support was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the support, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until the eluate spot disappeared at 254 nm by TLC. The solid support was vacuum-dried for 15 hours to obtain solid support 4-14 (260 mg). To calculate the loading, 5.97 mg of dry loading CPG was taken and a solution in 25 mL of 3% DCA in DCM was added. The solution was shaken and the ultraviolet absorbance at 500 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. 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 added DCA (mL) = 25 mL Abs value at 500nm = 0.6689 The amount of magnesium in the CPG taken was 5.97 mg. Loading (μmol / g) = ((25) * (0.6689) * 1000) / 76 * (5.97) = 36.85 μmol / g. [ka]

[0359] Compound 5-2: At 0°C under nitrogen gas, compound 5-1 (10 g, 41.78 mmol) was added to a solution of methoxymethyl(triphenyl)phosphonium chloride (12.89 g, 37.61 mmol) and KOtBu (4.69 g, 41.79 mmol) in THF (150 mL). 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 high-performance column chromatography (solution in PE with 0-10% EA) to obtain oily compound 5-2 (2.92 g, yield 37%).

[0360] C 15 H 25 Calculated mass of NO3: 267.2, measured mass: 268.2 [M+H] + ,ESI.

[0361] Compound 5-3: A mixture of compound 5-2 (8.2 g, 30.67 mmol) in MeCN (220 mL) was mixed with a solution of 2,2,2-trichloroacetic acid (6.31 g, 38.64 mmol) in water (70 mL), and the mixture was stirred overnight. TLC showed complete conversion. 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 obtain the oily compound 5-3 (7.8 g). The crude product could be used directly in the next step without further purification.

[0362] C 14 H 23 Calculated mass of NO3: 253.2, measured mass: 154.2 [M + H - Boc] + ,ESI.

[0363] 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 mixed with 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 (solution in 0-5% MeOH in DCM) to obtain solid compound 5-4 (1.5 g, 2-step yield 96%).

[0364] C 15 H 25 Calculated mass of NO4: 283.2, measured mass: 184.2 [M + H - Boc] + ,ESI.

[0365] 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), concentrated to dry, and the residue was extracted by DCM and filtered. The filtrate was concentrated to obtain compound 5-5 (2.95 g, 73% yield), which was used directly in the next step without further purification.

[0366] C 15 H 27 Calculated mass of NO4: 285.2, measured mass: 271.2 [M + ACN + H - tBu] + ,ESI.

[0367] Compound 5-6: Compound 5-5 (4 g, 14.02 mmol) was mixed with HCl (4 M solution in dioxane, 63.5 mL). The reaction mixture was stirred for 3 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to obtain compound 5-6 (3.5 g), which was used directly in the next step without further purification.

[0368] C 10 H19 Calculated mass of NO2 (free base): 185.1, measured mass: 186.1 [M+H] + ,ESI.

[0369] Compound 5-7: A solution of Fmoc-6-aminocaproic 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 minutes, 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 in 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 (solution in DCM with 0-5% MeOH) to obtain compound 5-7 (3.5 g, yield 34%).

[0370] C 31 H 40 Calculated mass of N2O5: 520.3, measured mass: 521.2 [M+H] + ,ESI. 1 H NMR (400MHz,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=6.6Hz,1H),3.69 (s,2H),3.43 (s,2H),3.22 (t,J=5.6Hz,4H),2.02-1.98 (m,3H),1.55-1.52 (t,J=6.6Hz,4H),2.26-2.23 (m,4H),1.24-1.16 (m,8H).

[0371] Compound 5-8: DMTrCl (1.43 g, 4.23 mmol) was added to a solution of compound 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. LC-MS showed partial conversion. 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 (solution of 10-40% EA and 0.1% aqueous ammonia in PE, and solution of 0-10% MeOH and 0.1% aqueous ammonia in DCM) to obtain compound 5-8 (3.0 g, yield 86%).

[0372] C 52 H 58 Calculated mass of N2O7: 822.4, measured mass: 845.3 [M+Na] + ,ESI. 1H NMR (400MHz,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=7.6Hz,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).

[0373] Compound 5-9: Piperidine (9 mL) was added to a solution of compound 5-8 (3 g, 3.65 mmol) in MeOH (90 mL). The reaction mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was concentrated and then azeotropically distilled with ditoluene. The residue was purified by silica gel column chromatography (solution in DCM of 0-10% MeOH and 1% TEA) to obtain compound 5-9 (1.1 g, 91% yield).

[0374] C 37 H 48Calculated mass of N2O5: 600.4, measured mass: 601.3 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.40-7.21 (m,9H),6.89 (d,J=7.2Hz,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=7.4Hz,2H),1.51-1.21 (m,16H).

[0375] Compound 5-10: The solutions 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) were stirred at 0°C for 30 minutes, and then compounds 5-9 (2 g, 3.33 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (C18, water / ACN) to obtain compound 5-10 (1.0 g, yield 15%).

[0376] C 56 H 75 N3O 15 Calculated mass: 1029.5, measured mass: 1052.3 [M+Na] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.83 (d,J=9.2Hz,1H),7.71 (t,J=5.2Hz,1H),7.40-7.38 (m,2H),7.32-7.19 (m,5H),6.90-9.87 (m,2H),5.22 (d,J=3.4Hz,1H),4.97 (dd,J=11.2,3.4Hz,1H),4.49 (d,J=8.5Hz,1H),4.42 (t,J=5.0Hz,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=12.7,6.6Hz,2H),2.85 (d,J=9.6Hz,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).

[0377] Compound 5-11: To a solution of compound 5-10 (690.0 mg, 0.66 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 5-11 (460 mg, 56% yield), a white solid, was produced.

[0378] C 65 H 92 N5O 16 Calculated mass of P: 1229.63, measured mass: 1230.3 [M+H] + ,ESI.

[0379] 1HNMR (600MHz,DMSO-d6) δ 7.81-7.67 (m,2H),7.40-7.19 (m,9H),6.88-6.86 (m,4H),5.21 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 146.33,146.26. [ka]

[0380] 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 stirred at 0°C for 30 minutes. 2,3-Dibromopropene (20.96 g, 104.88 mmol) was slowly added to the reaction mixture. The reaction was stirred at room temperature for 2 hours. 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 high-performance silica gel column chromatography to obtain compound 6-2 (31 g, 97% yield), a colorless oil.

[0381] C 13 H 19 Calculated mass of BrO4: 318.0, measured mass: 319.0 [M+H] + ,ESI. 1 H NMR (400MHz,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=7.2Hz,2H),1.26 (t,J=6.8Hz,6H).

[0382] 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 an N2 atmosphere. The reaction was cooled to room temperature, filtered, vacuum concentrated, and purified by high-performance silica gel column chromatography to obtain compound 6-3 (21 g, 91% yield), a colorless oil. 13 H 18 Calculated mass of O4: 238.1, Measured mass: 239.2 [M+H] + ,ESI. 1 H NMR (400MHz,CDCl3) δ 5.39 (t,J=2Hz,2H),4.95 (t,J=1.6Hz,2H),4.18 (q,J=7.2Hz,4H),3.03 (t,J=7.2Hz,4H),1.24 (t,J=7.2Hz,6H).

[0383] 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 within 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 high-performance silica gel column chromatography to obtain compound 6-4 (28 g, 80% yield), a pale yellow oily substance.

[0384] C 13 H 18 Calculated mass of Br2O4: 396.0, measured mass: 397.0 [M+H] + ,ESI. 1H NMR (400MHz,CDCl3) δ:4.21 (q,J=7.2Hz,4H),4.00 (s,4H),3.19 (s,4H),1.26 (t,J=7.2Hz,6H).

[0385] 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 minutes. The 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 hour. 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 high-performance silica gel column chromatography to obtain compound 6-5 (22 g, yield 76%), a colorless solid.

[0386] C 20 H 25 Calculated mass of NO6S: 407.1, measured mass: 408.2 [M+H] + ,ESI. 1 H NMR (400MHz,CDCl3) δ 7.70-7.68 (m,2H),7.32-7.30 (m,2H),4.17 (q,J=7.2Hz,4H),3.96 (s,4H),2.89 (s,4H),2.42 (s,3H),1.22 (t,J=7.2Hz,6H).

[0387] 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 was stirred at room temperature for 1 hour. 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 high-performance silica gel column chromatography to obtain compound 6-6 (14.8 g, yield 89%), a white solid.

[0388] C 16 H 21 Calculated mass of NO4S: 323.1, Measured mass: 324.1 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.70-7.68 (m,2H),7.43-7.41 (m,2H),4.56 (t,J=5.2Hz,2H),3.26 (d,J=4.8Hz,4H),2.84 (s,4H),2.39 (s,3H),1.95 (s,4H).

[0389] Compound 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). At 0°C, a solution of DMTrCl (4.30 g, 12.68 mmol) in DCM (80 mL) was slowly added to the reaction mixture. The reaction was stirred at room temperature for 1 hour. 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 on Al2O3 by high-performance column chromatography (0-3% MeOH) to obtain compound 6-7 (6.39 g, yield 80%) as a white foamy solid.

[0390] C 37 H 39 Calculated mass of NO6S: 625.25, measured mass: 648.25 [M+Na] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.68 (d,J=8.0Hz,2H),7.41 (d,J=8.0Hz,2H),7.33 (d,J=4.0Hz,2H),7.31-7.18 (m,7H),6.85 (d,J=8.0Hz,4H),4.68 (t,J=4.8Hz,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).

[0391] Compound 6-8: Compound 6-7 (6.0 g, 9.59 mmol) was dissolved in MeOH (300 mL), and 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 on Al2O3 by high-performance column chromatography (0-15% MeOH) to obtain compound 6-8 (1.2 g, yield 26.6%) as a white foamy solid.

[0392] C 30 H 33 Calculated mass of NO4: 471.24, measured mass: 472.20 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.37 (d,J=8.0Hz,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).

[0393] Compound 6-9: Compound 6-8 (2.4 g, 5.09 mmol) and Fmoc-6-aminocaproic acid (1.80 g, 5.09 mmol) were dissolved in DCM (60 mL), and 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 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 on Al2O3 by high-performance column chromatography (0-10% MeOH) to obtain compound 6-9 (3.1 g, yield 75%), a white foamy solid. 1 H NMR(400MHz,DMSO-d6) δ 7.88 (d,J=7.4Hz,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=8.9Hz,4H),4.75 (t,J=4.8Hz,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=4.6Hz,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).

[0394] Compound 6-10: Compound 6-9 (3.1 g, 3.84 mmol) was dissolved in MeOH (60 mL), and piperidine (6 mL) was added to the mixture. The reaction 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 on Al2O3 by high-performance column chromatography (0-20% MeOH) to obtain compound 6-10 (1.45 g, yield 64%) as a white foamy solid.

[0395] Calculated mass of C36H44N2O5: 584.33, measured mass: 585.4 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.39-7.39 (m,2H),7.37-7.28 (m,2H),7.25-7.19 (m,5H),6.88 (d,J=8.8Hz,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).

[0396] 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 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 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 on Al2O3 by high-performance column chromatography (0-10% MeOH) to obtain compound 6-11 (2 g, yield 76%), a white foamy solid.

[0397] C 55 H 71 N3O 15 Calculated mass: 1013.49, measured mass: 1014.4 [M+H] + ,ESI. 1H NMR(400MHz,DMSO-d6) δ 7.82 (d,J=9.2Hz,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,5H),6.90-6.87 (m,4H),5.22 (d,J=3.4Hz,1H),4.97 (dd,J=11.2,3.4Hz,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).

[0398] Compound 6-12: To a solution of compound 6-11 (790.0 mg, 0.78 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 6-12 (680 mg, 71% yield), a white solid, was produced.

[0399] C 64 H 88 N5O 16 Calculated mass of P: 1213.60, measured mass: 1159.5 [M-CH2CH2CN] - ,ESI.

[0400] 1 HNMR (600MHz,DMSO-d6) δ 7.82-7.68 (m,2H),7.38-7.19 (m,9H),6.90-6.86 (m,4H),5.21 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 146.51,146.47. [ka]

[0401] Compound 7-2: At 0°C, 54.78 g, 159.80 mmol of methoxymethyl(triphenyl)phosphonium chloride was added to a solution in 600 mL of THF, and KOtBu (19.92 g, 177.55 mmol) was added and the mixture was stirred for 0.5 hours. Then, at 0°C, compound 7-1 (20 g, 88.78 mmol) was added and the mixture was stirred overnight, and the reaction was allowed 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 dry. The residue was purified by silica gel column chromatography (solution in PE with 0-5% EA) to obtain oily compound 7-2 (15.5 g, yield 68%).

[0402] C 14 H 23 Calculated mass of NO3: 253.2, measured mass: 239.1 [M-tBu+ACN+H] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 5.99 (pent,J=2.1Hz,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).

[0403] 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 obtain crude compound 7-3 (4.7 g, 99% yield), an oily substance, which could be used directly in the next step without further purification.

[0404] C 13 H 21 Calculated mass of NO3: 239.2, measured mass: 225.1 [M-tBu+ACN+H] + ,ESI.

[0405] Compound 7-4: Formalin (15.9 g, 37% formaldehyde aqueous solution, 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 (solution in 0-3% MeOH in DCM) to obtain oily compound 7-4 (5.2 g, 98% yield).

[0406] C 14 H 23 Calculated mass of NO4: 269.2, measured mass: 170.1 [M-Boc+H] + ,ESI.

[0407] Compound 7-5: NaBH4 (5.25 g, 138.86 mmol) was added to a solution of compound 7-4 (17 g, 63.12 mmol) in MeOH (190 mL). 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 by DCM. The organic phase was concentrated and purified by silica gel column (solution in DCM with 0-5% MeOH) to obtain compound 7-5 (14.5 g, yield 84%).

[0408] C 14 H 25 Calculated mass of NO4: 271.2, measured mass: 172.1 [M-Boc+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 4.53 (t,J=5.3Hz),3.37-3.32 (m,2H),3.27 (d,J=5.3Hz,2H),3.19 (d,J=5.3Hz,2H),3.07 (dd,J=10.78,3.5Hz,2H),1.60 (dd,J=13.4,7.9Hz,2H) 1.39 (s,9H),1.27 (dd,J=13.5,6.3Hz,2H).

[0409] Compound 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 hours. LC-MS showed complete conversion. The reaction mixture was concentrated to obtain crude compound 7-6 (11 g), which could be used directly in the next step without further purification.

[0410] C9H 17 Calculated mass of NO2 (free base): 171.1, measured mass: 172.1 [M-Boc+H] + ,ESI.

[0411] Compound 7-7: A mixture of Fmoc-6-aminocaproic 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 minutes, and then compound 7-6 (10.8 g, 52.00 mmol) was added. The reaction mixture was stirred overnight and then heated to room temperature. LC-MS showed complete conversion. The reaction mixture was diluted in DCM (300 mL) and washed with NaHCO3 (saturated aqueous solution) and water. The organic phase was concentrated and purified by silica gel column (solution in DCM with 0-10% MeOH) to obtain the gel compound 7-7 (19.6 g, yield 74%).

[0412] C 30 H 38 Calculated mass of N2O5: 506.3, measured mass: 507.3 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.90-7.68 (m,4H),7.44-7.33 (m,5H),4.55 (s,2H),4.29 (d,J=6.8Hz,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=7.6Hz,2H),1.63-1.23 (m,12H).

[0413] Compound 7-8: At 0°C, DMTrCl (13.98 g, 41.25 mmol) was gradually added to a solution of compound 7-7 (19 g, 37.50 mmol), N,N-dimethylpyridine-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 by-product di-DMTr. The reaction mixture was concentrated and purified by silica gel column (20-100% EA, 0.2% TEA-containing PE solution) to obtain compounds 7-8a and 7-8b (17.9 g, 47% yield), which are mixtures of the endo-isomer and exo-isomer.

[0414] The isomers were separated by preparative HPLC (C18 column, water / ACN) to obtain compound 7-8a (exo-isomer, 7.5 g) and compound 7-8b (endo-isomer, 5.7 g).

[0415] 7-8a: C 51 H 56 Calculated mass of N2O7: 808.4, measured mass: 832.3 [M+Na] + ,ESI.

[0416] 1 H NMR (400MHz,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=6.7Hz,1H),3.73 (s,6H),3.47 (dd,J=9.9,8.2Hz,1H),3.28 (br,2H),3.11 (td,J=12.0,4.3Hz,2H),2.99-2.91 (m,4H),2.66-2.54 (m,2H),2.19-2.06 (m,2H),1.67 (pent,J=6.8Hz,2H),1.51-1.17 (m,10H). 7-8b: C 51 H 56Calculated mass of N2O7: 808.4, measured mass: 832.3 [M+Na] + ,ESI.

[0417] 1 H NMR (400MHz,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=12.7,6.5Hz,2H),2.86 (s,2H),2.44-2.29 (m,2H),2.15 (t,J=7.1Hz,2H),1.64-1.18 (m,12H). [ka]

[0418] Compound 7-9a: The solution of compound 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 (20-30% MeOH, 1% TEA-containing DCM solution) to obtain compound 7-9a (4.4 g, 94% yield).

[0419] C 36 H 46 Calculated mass of N2O5: 586.3, measured mass: 587.3 [M+H] + ,ESI.

[0420] Compound 7-10a: The solutions 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) were stirred at 0°C for 30 minutes, 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 in DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column (solution in DCM of 0-10% MeOH containing 1% TEA) to obtain compound 7-10a (3.8 g, yield 51%).

[0421] C 55 H 73 N3O 15 Calculated mass: 1015.5, measured mass: 1039.1 [M+Na] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.85 (d,J=9.2Hz,1H),7.74 (t,J=5.3Hz,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=2.8Hz,1H),4.97 (dd,J=11.2,2.8Hz,1H),4.65 (t,J=4.8Hz,1H),4.49 (d,J=8.4Hz,1H),4.03 (s,3H),3.88 (dd,J=19.5,9.1Hz,1H),3.73-3.70 (m,7H),3.50-3.39 (m,2H),3.34-3.31 (m,1H),3.27 (d,J=4.5Hz,2H),3.17 (d,J=5.2Hz,1H),3.15-3.09 (m,2H),3.00 (dd,J=12.4,6.3Hz,2H),2.94 (br,1H),2.67-2.53 (m,2H),2.19-2.08 (m,5H),2.04 (t,J=6.9Hz,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).

[0422] Compound 7-11a: To a solution of compound 7-10a (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). This produced a white solid, 7-11a (390 mg, 47% yield).

[0423] C 64 H 90 N5O16 P Calculated mass: 1215.61, measured mass: 1161.3 [M-CH2CH2CN] - ,ESI.

[0424] 1 HNMR (600MHz,DMSO-d6) δ 7.81-7.69 (m,2H),7.40-7.19 (m,9H),6.88-6.86 (m,4H),5.21 (d,J=6.0Hz,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(242MHz,DMSO-d6) δ 146.70. [ka]

[0425] Compound 7-9b: The solutions of compound 7-8b (5.70 g, 7.05 mmol) in MeOH (200 mL) and piperidine (20 mL) were stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by silica gel column (10-20% MeOH, 1% TEA-containing DCM solution) to obtain compound 7-9b (4.1 g, 99% yield).

[0426] C 36 H 46 Calculated mass of N2O5: 586.3, measured mass: 587.3 [M+H] + ,ESI.

[0427] Compound 7-10b: The solutions 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) were stirred at 0°C for 30 minutes, 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 in DCM (150 mL) and washed with water. The organic phase was concentrated and purified by silica gel column (solution in DCM of 0-10% MeOH containing 1% TEA) to obtain 4.4 g of crude product, which was further purified by preparative HPLC (C18, water / ACN) to obtain compound 7-10b (2.0 g, yield 28%).

[0428] C 55 H 73 N3O 15 Calculated mass: 1015.5, measured mass: 1039.1 [M+Na] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.84 (d,J=9.2Hz,1H),7.72 (t,J=5.5Hz,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=3.3Hz,1H),4.97 (dd,J=11.2,3.3Hz,1H),4.66 (t,J=4.9Hz,1H),4.49 (d,J=8.5Hz,1H),4.03 (s,3H),3.88 (dd,J=20.2,8.8Hz,1H),3.74-3.69 (m,7H),3.48-3.38 (m,4H),3.34-3.31 (m,1H),3.21 (dd,J=10.4,4.2Hz,1H),3.14 (dd,J=12.0,4.6Hz,1H),3.00 (dd,J=12.7,6.6Hz,2H),2.86 (s,2H),2.46-2.27 (m,2H),2.16 (t,J=6.7Hz,2H),2.11 (s,3H),2.04 (t,J=7.0Hz,2H),2.00 (s,3H),1.89 (s,3H),1.78 (s,3H),1.63-1.12 (m,14H).

[0429] Compound 7-11b: To a solution of compound 7-10b (680.0 mg, 0.67 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (96.0 mg, 0.81 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (324 mg, 1.08 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 7-11b (390 mg, 47% yield), a white solid, was produced.

[0430] C 64 H90 N5O 16 Calculated mass of P: 1215.61, measured mass: 1161.6 [M-CH2CH2CN] - ,ESI.

[0431] 1 HNMR (600MHz,DMSO-d6) δ 7.81-7.69 (m,2H),7.40-7.19 (m,9H),6.88-6.86 (m,4H),5.21 (d,J=6.0Hz,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(242MHz,DMSO-d6) δ 146.22. [ka]

[0432] Compound 8-2: At 0°C, 9.66 g (86.12 mmol) of methoxymethyl(triphenyl)phosphonium chloride (17.71 g, 51.67 mmol) was added to a solution in THF (120 mL) and stirred for 0.5 hours. Then, at 0°C, compound 8-1 (9.7 g, 43.06 mmol) was added and stirred overnight, and the reaction was raised 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 dry. The residue was purified by silica gel column (solution in PE with 0-5% EA) to obtain oily compound 8-2 (6.0 g, yield 55%). 1 H NMR (400MHz,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).

[0433] Compound 8-3: To a solution of compound 8-2 (5.85 g, 23.09 mmol) in THF (50 mL), hydrochloric acid (3.28 g, 90.00 mmol, 2N, 45 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. TLC showed complete conversion. The reaction mixture pH was adjusted to 7 by adding NaHCO3 (saturated aqueous solution), and it was extracted with EA. The organic phase was concentrated to obtain crude compound 8-3 (5.5 g), an oily substance, which could be used directly in the next step without further purification. 1 H NMR (400MHz,DMSO-d6) δ 9.62 (pent,J=1.2Hz,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).

[0434] 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 obtain compound 8-4 (6.7 g), which could be used directly in the next step without further purification.

[0435] Compound 8-5: NaBH4 (2.65 g, 70.17 mmol) was added to a solution of compound 8-4 (6.3 g, 23.39 mmol) in MeOH (50 mL). 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 by DCM. The organic phase was concentrated and purified by silica gel column (solution in DCM with 0-5% MeOH) to obtain compound 8-5 (1.4 g, yield 22%).

[0436] C 14 H 25 Calculated mass of NO4: 271.18, measured mass: 257.24 [M-tBu+CH3CN+H] + ,ESI. 1 H NMR (400MHz,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).

[0437] Compound 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 obtain crude product 8-6 (1.3 g), an oily substance, which could be used in the next step without further purification.

[0438] C9H 17 Calculated mass of NO2: 171.1, measured mass: 172.2 [M+H] + ,ESI.

[0439] Compound 8-7: Z-6-aminocaproic 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 was stirred for 20 minutes, then compound 8-6 (1.3 g, 6.26 mmol) was added, and the mixture was stirred for a further 2 hours. 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 on a silica gel column by high-performance column chromatography (DCM:MeOH = 20:1) to obtain compound 8-7 (1.5 g, 3.58 mmol, yield 57%) as a white foamy solid. 23 H 34Calculated mass of N2O5: 418.3, measured mass: 419.3 [M+H] + ,ESI.

[0440] 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 on Al2O3 by high-performance column chromatography (DCM:MeOH:TEA = 20:1:0.5%) to obtain compound 8-8 (850 mg, yield 41%), a white foamy solid.

[0441] C 44 H 52 Calculated mass of N2O7: 720.4, measured mass: 721.4 [M+H] + ,ESI. 1 H NMR (400MHz,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=8.9Hz,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=4.5Hz,2H),3.00-2.95 (m,4H),2.92-2.84 (m,1H),2.62 (q,J=11Hz,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).

[0442] Compound 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 under a hydrogen gas atmosphere for 3 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude product 8-9 (550 mg, yield 84%), a white foamy solid, which could be used in the next step without further purification.

[0443] C 36 H 46 Calculated mass of N2O5: 586.3, measured mass: 587.3 [M+H] + ,ESI.

[0444] 1 H NMR (400MHz,DMSO-d6) δ 7.40-7.38 (m,2H),7.31 (t,J=7.7Hz,2H),7.29-7.18 (m,5H),6.89 (d,J=8.9Hz,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). [ka]

[0445] Compound 9-2: Methoxymethyl(triphenyl)phosphonium 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 a further 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 to adjust the pH 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 high-performance silica gel column chromatography (solution of 10-30% EA in PE) to obtain the oily compound 9-2 (24 g, 52% yield).

[0446] C 13 H 21 Calculated mass of NO3: 239.3, measured mass: 225.1 [M-tBu+ACN+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 5.88 (t,J=2.4Hz,1H),3.83 (s,4H),3.47 (s,3H),2.79-2.74 (m,4H),1.41 (s,9H).

[0447] 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, and then the reaction was quenched with saturated NaHCO3 to adjust the pH 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 obtain crude product 9-3 (13 g, 92% yield), an oily substance, which could be used in the next step without further purification.

[0448] C 12 H 19 Calculated mass of NO3: 225.3, measured mass: 211.1 [M-tBu+ACN+H] + ,ESI.

[0449] 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, solution in water) were added to the mixture. The reaction was stirred overnight at room temperature, and then the reaction was quenched with saturated citric acid to adjust the pH 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 obtain crude product 9-4 (14 g, yield 95%).

[0450] C 13 H 21 Calculated mass of NO4: 255.3, measured mass: 241.1 [M-tBu+ACN+H] + ,ESI.

[0451] 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 gradually. 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). It was washed with brine (100 mL), dried over Na2SO4, concentrated under vacuum, and purified on silica gel by high-performance column chromatography (0-5% MeOH) to obtain compound 9-5 (7.05 g, 50% yield).

[0452] C 13 H 23 Calculated mass of NO4: 257.3, measured mass: 243.1 [M-tBu+ACN+H] + ,ESI. 1 H NMR (400MHz,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).

[0453] Compound 9-6: Compound 9-5 (13.1 g, 50.91 mmol) was dissolved in HCl 1,4-dioxane (4 M, 131 mL). The reaction mixture was stirred at room temperature for 1 hour and then concentrated under vacuum to obtain crude product 9-6 (9.8 g), which was ready for use in the next step without further purification.

[0454] C8H 13 Calculated mass of NO2Cl: 157.2, measured mass: 158.2 [M+H] + ,ESI.

[0455] Compound 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 on silica gel by high-performance column chromatography (0-10% MeOH) to obtain compound 9-7 (6.7 g, 2-step yield 25%).

[0456] C 30 H 36 Calculated mass of N2O5: 492.3, measured mass: 493.3 [M+H] + ,ESI. 1H NMR (400MHz,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).

[0457] Compound 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 gradually added to the reaction mixture 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 on Al2O3 by high-performance column chromatography (50-100% EA) to obtain compound 9-8 (4.3 g, 40% yield).

[0458] C 51 H 56 Calculated mass of N2O8: 824.4, measured mass: 848.3 [M+Na] + ,ESI. 1 H NMR (400MHz,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).

[0459] Compound 9-9: Compound 9-8 (4.2 g, 5.09 mmol) was dissolved in MeOH (126 mL), and piperidine (12.6 mL) was added to the mixture. The reaction mixture was stirred overnight at room temperature, and then the reaction mixture was concentrated under vacuum. It was purified on Al2O3 by high-performance column chromatography (0-10% MeOH) to obtain compound 9-9 (3 g, 97% yield), which was a white foam.

[0460] C 36 H 46 Calculated mass of N2O6: 602.3, measured mass: 603.4 [M+H] + ,ESI.

[0461] Compound 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 obtain compound 9-10 (610 mg, yield 12%).

[0462] C 55 H 73 N3O 16 Calculated mass: 1031.5, measured mass: 1054.5 [M+Na] + ,ESI.

[0463] 1H NMR (400MHz,DMSO-d6) δ 7.82-7.73 (m,2H),7.24-6.88 (m,12H),5.22 (s,1H),4.97 (d,J=10.8Hz,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). [ka]

[0464] Compound 9-11: To a solution of compound 9-10 (1.0 g, 0.97 mmol) in anhydrous DCM (3.0 mL) at room temperature, 4,5-dicyanoimidazole (32.0 mg, 0.27 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (108 mg, 0.36 mmol) were added, and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column: C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 9-11 (680 mg, yield 56%), a white solid, was produced.

[0465] C 64 H 90 N5O 17 Calculated mass of P: 1231.61, measured mass: 1177.1 [M-CH2CH2CN] - ,ESI. 1HNMR (600MHz,DMSO-d6) δ 7.81-7.67 (m,2H),7.26-7.23 (m,6H),6.88-6.86 (m,6H),5.22 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 146.82,146.70.

[0466] Compound 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 LC-MS showed that the starting materials were completely consumed. The reaction mixture was diluted with DCM (10 mL), washed with H2O (3 mL x 4), and then with brine (3 mL x 4). The organic layer was concentrated to obtain compound 9-12 (85 mg, 90% yield), which was a white solid.

[0467] C 59 H 77 N3O 19 Calculated mass: 1131.52, measured mass: 1130.2 [MH] - ,ESI. 1HNMR (600MHz,DMSO-d6) δ 7.87-7.72 (m,2H),7.24-7.21 (m,6H),6.90-6.87 (m,6H),5.21 (d,J=6.0Hz,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).

[0468] 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.

[0469] To a solution of succinate ester 9-12 (85 mg, 0.075 mmol) and HBTU (53 mg, 0.14 mmol) in anhydrous DMF (1.5 mL), DIPEA (30 mg, 0.23 mmol) was added, the reaction mixture was shaken at room temperature for 10 minutes, then natural amino-LCAA-CPG (300 mg, loading 75 μmol / g) was added, the suspension was shaken at room temperature for 20 hours, filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until no eluate spots were observed at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (300 mg). The solid support was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the solid support, and washed with DMF (20 mL x 5), CAN (20 mL x 5), and DCM (20 mL x 5) until the eluate spot disappeared at 254 nm by TLC. The solid support was vacuum-dried for 15 hours to obtain solid support 9-13 (300 mg). To calculate the loading, 5.00 mg of dry loading CPG was taken and a solution in 25 mL of 3% DCA in DCM was added. The solution was shaken and the UV absorbance at 482 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. Loading (μmol / g) = (Total volume of added DCA (mL)) * (Abs value at 482 nm) * 1000) / (78.3 * (Number of mg of CPG taken)) Total volume of added DCA (mL) = 25 mL Abs value at 482nm = 0.577 Magnesium content of the extracted CPG = 5.00 mg Loading (μmol / g) = ((25) * (0.577) * 1000) / 78.3 * (5.00) = 36.8 μmol / g. [ka]

[0470] 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), stirred at room temperature for 30 minutes, and then compound 2-10 (412.34 mg, 0.897 mmol) was added and stirred at room temperature for 1 hour. LC-MS showed complete conversion. The reaction mixture was concentrated and purified by preparative HPLC (C18 column, eluate A: water, eluate B: ACN) to obtain compound 10-2 (580 mg, yield 79%), a white solid.

[0471] C 120 H 179 N 11 O 42 Calculated mass: 2446.22, measured mass: 1247.61 [M+2Na] 2+ ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.86-7.82 (m,6H),7.75 (t,J=5.5Hz,3H),7.39-7.29 (m,4H),7.26-7.19 (m,5H),7.00 (br,1H),6.89 (dd,J=8.9,3.7Hz,4H),5.21 (d,J=3.4Hz,3H),4.96 (dd,J=11.2,3.4Hz,3H),4.66-4.64 (m,1H),4.78 (d,J=8.4Hz,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=5.8Hz,12H),2.90 (s,2H),2.27 (t,J=6.2Hz,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).

[0472] 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 LC-MS showed that the starting materials were completely consumed. The reaction mixture was diluted with DCM, washed with water (3 mL x 4), and then with brine (3 mL x 4). The organic layer was concentrated to obtain compound 10-3 (130 mg, 97% yield), which was a white solid.

[0473] C 124 H 183 N 11 O 45 Calculated mass: 2546.24, measured mass: 1273.5 [M+2H] 2+ ,ESI. 1 HNMR (600MHz,DMSO-d6):7.84-7.73(m,9H),7.37-7.21 (m,9H),6.98-6.89 (m,5H),5.21 (d,J=6.0Hz,3H),4.97-4.94 (m,3H),4.48 (d,J=12.0Hz,3H),4.07-3.66 (m,26H),3.55-3.37 (m,17H),3.04-2.95 (m,14H),2.45 (d,J=6.0Hz,4H),2.29-2.25 (m,6H),2.11-1.72 (m,50H),1.51-1.12 (m,38H).

[0474] Compound 10-4: 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), DIPEA (30 mg, 0.23 mmol) was added. The reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-ICAA-CPG (300 mg, loading 75 μmol / g) was added. The suspension was shaken at room temperature for 20 hours, filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until no spots of eluate were observed at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (300 mg). The mixture was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the support, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until the eluate spot disappeared at 254 nm by TLC. The solid support was vacuum-dried for 15 hours to obtain 10-4 (300 mg) of solid support. To calculate the loading, 5.19 mg of dry loading CPG was taken and a solution in 20 mL of 3% DCA in DCM was added. The solution was shaken and the UV absorbance at 500 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. 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 500nm = 0.335 The amount of magnesium in the CPG taken was 5.19 mg. Loading (μmol / g) = ((20) * (0.335) * 1000) / 76 * (5.19) = 17 μmol / g. [ka]

[0475] Compound 11-1: To a solution of compound 2-10 (1.27 g, 3.59 mmol) in DCM (15 mL), TEA (495.41 mg, 4.90 mmol) and HATU (1.35 g, 3.59 mmol) were added. After stirring for 15 minutes, Fmoc-6-hexanoic acid (1.5 g, 3.26 mmol) was added to the reaction mixture, and the mixture was continuously stirred for 4 hours. The mixture was divided into DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography and eluted at DCM / MeOH = 20 / 1 to obtain 600 mg of compound 11-1 (yield 23.2%). 1 H NMR (400MHz,DMSO-d6) δ 7.87 (d,J=7.6Hz,2H),7.68 (d,J=7.4Hz,2H),7.44-7.35 (m,4H),7.35-7.28 (m,4H),7.27-7.17 (m,6H),6.88 (d,J=7.6Hz,4H),4.64 (td,J=5.04,1.6Hz,1H),4.28 (d,J=6.9Hz,2H),4.20 (t,J=6.8Hz,1H),3.96 (s,1H),3.73 (d,J=3.8Hz,6H),3.66 (d,J=10.4Hz,2H),3.50 (s,1H),3.39 (t,J=5.9Hz,2H),2.96 (t,J=6.6Hz,2H),2.91(s,2H),2.06-1.74 (m,6H),1.47-1.31(m,4H),1.26-1.13 (m,2H).

[0476] 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 and eluted at DCM / MeOH / NH3OH = 100 / 10 / 1 to obtain 360 mg of compound 11-2 (82% yield).

[0477] C 35 H 44 Calculated mass of N2O5: 572.3, measured mass: 573.4 [M+H] +,ESI. 1H NMR (400MHz,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=6.5Hz,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).

[0478] Compound 11-4: A mixture solution 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 purging with nitrogen gas. The mixture was then heated at 60 °C for 6 hours. The mixture was concentrated. The residue was purified by column chromatography and eluted at DCM / MeOH / NH4OH = 95 / 5 / 1 to obtain 8 g of compound 11-4 (yield 48%), which was a yellow solid.

[0479] C 12 Calculated mass of H9N3: 195.1, measured mass: 196.2 [M+H] + ,ESI.

[0480] Compound 11-5: Compound 11-4 (10 g, 51.22 mmol) was dissolved in tert-butanol, the mixture was heated to 50°C, and KOH (574.84 mg, 10.24 mmol) and MeOH (5 mL) were added. Then, tert-butyl acrylate (7.22 g, 56.35 mmol) was slowly added to the reaction mixture and the mixture was stirred for 5 hours. The mixture was concentrated under vacuum. The residue was separated into ethyl acetate (250 mL) and water (150 mL), 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 and eluted at PE / EA = 1 / 2 to obtain 11 g of compound 11-5 (yield 66%).

[0481] C 19 H 21 Calculated mass of N3O2: 323.2, measured mass: 324.2 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 8.65 (d,J=5.6Hz,4H),7.50 (d,J=6.0Hz,4H),2.82 (t,J=7.8Hz,2H),2.23 (t,J=7.8Hz,2H),1.35 (s,9H).

[0482] 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 obtain 0.7 g of compound 11-6 (yield 69%).

[0483] C 19 H 25 Calculated mass of N3O2: 327.2, measured mass: 328.3 [M+H] + ,ESI.

[0484] 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 the 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 divided into DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography and eluted at DCM / MeOH = 9 / 1 to obtain 2.5 g of compound 11-7 (yield 71%), which was a yellow oily substance. C 30 H 44 Calculated mass of N4O5: 540.3, measured mass: 541.4 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 8.48 (d,J=5.9Hz,4H),7.45 (t,J=6.6Hz,1H),7.14 (d,J=6.1Hz,4H),6.73 (t,J=5.4Hz,1H),3.88 (d,J=6.1Hz,2H),2.85 (q,J=6.6Hz,2H),2.31 (t,J=8.0Hz,2H),1.94 (q,J=8.0Hz,4H),1.36 (s,9H),1.32 (s,9H),1.31-1.23(m,4H),1.1-0.9(m,2H).

[0485] Compound 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 hours. 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 vacuum dried. The residue was dissolved in THF, and the mixture was filtered. The filtrate was evaporated under reduced pressure to obtain 4 g of compound 11-8, a colorless oily substance, with a yield of 87%.

[0486] C 30 H 56 Calculated mass of N4O5: 552.4, measured mass: 553.4 [M+H] + ,ESI.

[0487] Compound 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 minutes, compound 11-8 (1 g, 1.18 mmol) was added to the reaction mixture and stirring continued for 5 hours. The mixture was divided into DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography and eluted at DCM / MeOH = 15 / 1 to obtain 0.4 g of compound 11-9 (yield 22%) as a white solid.

[0488] C 52 H 94 N6O 11 Calculated mass: 978.7, measured mass: 979.8 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.41(t,J=6.0Hz,1H),6.78-6.70 (m,3H),3.88 (d,J=11.4Hz,2H),3.02(d,J=6.5Hz,6H),2.92-2.82 (m,8H),2.25 (t,J=7.4Hz,4H),2.15 (t,J=8.1Hz,2H),2.16 (t,J=7.2Hz,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).

[0489] Compound 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 obtain 850 mg of compound 11-10, a colorless oil, in a yield of 95%.

[0490] C 33 H 62 Calculated mass of N6O5: 622.5, measured mass: 623.6 [M+H] + ,ESI.

[0491] 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 using a reversed-phase column (C18 column) to obtain 1.3 g of compound 11-11 as a white solid, with a yield of 47%.

[0492] C 90 H 143 N9O 35 Calculated mass: 1909.9, measured mass: 956.0 [M+2H] 2+ ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 12.0 (br,1H),7.83 (d,J=9.2Hz,3H),7.70 (t,J=5.5Hz,3H),7.50-7.40 (m,1H),5.20 (d,J=3.4Hz,3H),4.96 (dd,J=11.24,3.4Hz,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=7.3Hz,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).

[0493] Compound 11-12: Compound 11-11 (1.4 g, 0.73 mmol) was dissolved in DCM (15 mL), and 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 divided into DCM (50 mL) and water (35 mL), the DCM extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by reverse-phase column (C18 column) to obtain 0.8 g of compound 11-12 as a white powder with a yield of 44%.

[0494] C 125 H 185 N 11 O 39 Calculated mass: 2464.3, measured mass: 1255.6 [M+2Na] 2+ ,ESI.

[0495] 1H NMR (400MHz,DMSO-d6) δ 7.86-7.76 (m,4H),7.71 (t,J=5.3Hz,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=3.3Hz,3H),5.02-4.90 (dd,J=11.2,3.4Hz,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). [ka]

[0496] Compound 11-13: To a solution of compound 11-12 (2.0 g, 0.8 mmol) in anhydrous DCM (7.0 mL) at room temperature, 4,5-dicyanoimidazole (80.0 mg, 0.67 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (270 mg, 0.90 mmol) were added and the mixture was stirred for 1 hour. LC-MS 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 fast preparative HPLC under the following conditions (column, C18 silica gel, mobile phase: CH3CN / H2O=1 / 1, increased to CH3CN / H2O=1 / 0 within 20 minutes, detector: UV 254 nm). Compound 11-13 (1.0 g, yield 45%) was produced as a white solid.

[0497] C 134 H 202 N 13 O 40 Calculated mass of P: 2664.39, measured mass: 1132.5 [M-DMTr-N(CH(CH3)2)2+4H] 2+ ,ESI.

[0498] 1 HNMR (600MHz,DMSO-d6) δ 7.82-7.69 (m,6H),7.44-7.19 (m,9H),6.90-6.87 (m,4H),5.21 (d,J=6.0Hz,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 (242MHz,DMSO-d6) δ 146.79,146.67. [ka]

[0499] Compounds 11-14: To a solution of compound 11-12 (150 mg, 0.06 mmol) in anhydrous DCM (1.0 mL), DMAP (5 mg, 0.04 mmol) and TEA (24 mg, 0.24 mmol) were added, and succinic anhydride (20 mg, 0.2 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and LCMS showed that the starting materials were 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 obtain compound 11-14 (135 mg, yield 97%), which was a white solid.

[0500] C 129 H 189 N 11 O 42 Calculated mass: 2564.30, measured mass: 1132.5 [M-DMTr+2H] 2+ ,ESI. 1 H NMR (600MHz,DMSO-d6): 7.84-7.71(m,6H),7.45-7.21 (m,10H),6.92-6.89 (m,4H),5.21 (d,J=6.0Hz,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).

[0501] Compound 11-15: 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), DIPEA (30 mg, 0.23 mmol) was added. The reaction mixture was shaken at room temperature for 10 minutes, and then natural amino-ICAA-CPG (300 mg, loading 75 μmol / g) was added. The suspension was shaken at room temperature for 20 hours, filtered, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until no eluate spots were visible at 254 nm by TLC. The solid support was vacuum-dried for 2 hours to obtain a solid support (300 mg). The solid support was stirred with Ac2O / pyridine / N-methylimidazole (90 μL / 1.0 mL / 80 μL) at room temperature for 1 hour to block unreacted amine on the support, and washed with DMF (20 mL x 5), ACN (20 mL x 5), and DCM (20 mL x 5) until the eluate spot disappeared at 254 nm by TLC. The solid support was vacuum-dried for 15 hours to obtain solid supports 11-15 (300 mg). To calculate the loading, 5.00 mg of dry loading CPG was taken and a solution in 20 mL of 3% DCA in DCM was added. The solution was shaken and the UV absorbance at 500 nm was measured. To ensure that the signal did not saturate, the absorbance value was kept below 1.0 unit. The following formula was then used. 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 500nm = 0.464 The amount of magnesium in the CPG taken was 5.0 mg. Loading (μmol / g) = ((20) * (0.464) * 1000) / 76 * (5.0) = 24 μmol / g. [ka]

[0502] 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. The mixture was then concentrated under vacuum and purified by high-performance silica gel column chromatography (PE / EA = 5 / 1) to obtain compound 12-1 (1.5 g, yield 84%), a white foamy solid.

[0503] C 20 H 29 Calculated mass of NO4: 347.2, measured mass: 348.3 [M+H] + ,ESI.

[0504] 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). At 0°C, the solution of DMTrCl (1.48 g, 4.37 mmol) in DCM (20 mL) was slowly added to the reaction mixture. The reaction 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 on Al2O3 by high-performance column chromatography (0-3% MeOH) to obtain compound 12-4 (1.9 g, yield 67%), a white foamy solid.

[0505] C41 H 47 Calculated mass of NO6: 649.34, measured mass: 650.34 [M+H] + ,ESI. 1 H NMR (400MHz,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).

[0506] Compound 12-3: Compound 12-2 (0.7 g, 1.08 mmol) was dissolved in MeOH (30 mL), and Pd / C (140 mg) was added to the solution. The reaction mixture was stirred at room temperature under a hydrogen gas atmosphere for 3 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude compound 12-3 (520 mg), a white foamy solid, which was ready for use in the next step without further purification.

[0507] C 33 H 41 Calculated mass of NO4: 515.30, measured mass: 516.30 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.38 (d,J=8.8Hz,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).

[0508] Compound 12-4: Compound 10-1 (1.56 g, 0.756 mmol) was dissolved in DCM (20 mL). At 0°C, 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. The reaction was stirred at 0°C for 30 minutes. Compound 12-3 (400 mg, 0.756 mmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-8% MeOH) to obtain compound 12-4 (1 g, yield 51%), a white foamy solid.

[0509] C 124 H 187 N 11 O 42 Calculated mass: 2503.28, measured mass: 1101.6 [M-DMTr+2H + ] 2+ ,ESI.

[0510] 1H NMR (400MHz,DMSO-d6) δ 7.85-7.81 (m,6H),7.73 (t,J=5.6Hz,3H),7.39 (d,J=7.32Hz,2H),7.32-7.19 (m,8H),6.99 (s,1H),6.88 (d,J=8.9Hz,4H),5.21-5.20 (m,3H),4.98-4.94 (m,3H),4.48 (d,J=8.44Hz,3H),4.42 (t,J=5.0Hz,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=7.4Hz,2H),2.10 (s,8H),2.05 (t,J=6.96Hz,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). [ka]

[0511] 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 the 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 high-performance silica gel column chromatography (0-5% MeOH) to obtain compound 13-1 (1.95 g, yield 87%), a white foamy solid.

[0512] C 18 H 25 Calculated mass of NO4: 319.2, measured mass: 320.2 [M+H] + ,ESI.

[0513] 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 was stirred at 0°C for 1 hour. 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 on Al2O3 by high-performance column chromatography (PE / EA = 0-40%) to obtain compound 13-2 (2.6 g, yield 66%) as a white foamy solid.

[0514] Compound 13-3: Compound 13-2 (600 mg, 0.96 mmol) was dissolved in MeOH (20 mL), and Pd / C was added to the reaction mixture. The reaction mixture was stirred at room temperature under a hydrogen gas atmosphere for 3 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude product 13-3 (470 mg, crude product), a white foamy solid, which could be used in the next step without further purification.

[0515] C 31 H 37 Calculated mass of NO4: 487.3, measured mass: 488.3 [M+H] + ,ESI.

[0516] Compound 13-4: Compound 10-1 (1.23 g, 0.61 mmol) was dissolved in DCM (20 mL). At 0°C, 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. The reaction was stirred at 0°C for 30 minutes. Compound 13-3 (300 mg, 0.61 mmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-8% MeOH) to obtain compound 13-4 (1 g, yield 66%) as a white foamy solid.

[0517] 1 H NMR (400MHz,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=3.2Hz,3H),4.97 (dd,J=3.2,11.2Hz,3H),4.63-4.61(m,1H),4.48 (d,J=8.4Hz,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,12H),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=14.8Hz,2H),2.10 (s,9H),2.04 (t,J=6.8Hz,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). [ka]

[0518] 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 the 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 high-performance silica gel column chromatography (0-2% MeOH) to obtain compound 14-1 (5 g, yield 81%), a white foamy solid.

[0519] C 18 H 25 Calculated mass of NO4: 319.2, measured mass: 320.2 [M+H] + ,ESI. 1 H NMR (400MHz,DMSO) δ 7.39-7.28 (m,5H),5.01 (s,2H),4.27 (t,J=5.4Hz,2H),3.61-3.50 (m,4H),3.22 (d,J=5.3Hz,4H),1.55 (t,J=6.1Hz,4H),1.25-1.22 (m,8H).

[0520] 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 was stirred at 0°C for 3 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 on Al2O3 by high-performance column chromatography (PE / EA = 0-60%) to obtain compound 14-2 (5.5 g, yield 81%), a white foamy solid. 1H NMR (400MHz,DMSO-d6) δ 7.39-7.18 (m,14H),6.88 (d,J=8.8Hz,4H),5.00 (s,2H),4.39 (t,J=4.9Hz,1H),3.73 (s,6H),3.58-3.35 (m,6H),2.83 (s,2H),1.29-1.15 (m,8H).

[0521] Compound 14-3: Compound 14-2 (254 mg, 408.52 μmol) was dissolved in MeOH (10 mL), and Pd / C was added to the reaction mixture. The reaction mixture was stirred at room temperature under a hydrogen gas atmosphere for 2 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude product 14-3 (199 mg, crude product), a white foamy solid, which could be used in the next step without further purification.

[0522] C 31 H 37 Calculated mass of NO4: 487.3, measured mass: 488.3 [M+H] + ,ESI.

[0523] Compound 14-4: Compound 10-1 (820 mg, 408.51 μmol) was dissolved in DCM (20 mL). At 0°C, DIEA (158 mg, 1.23 mmol), EDCI (157 mg, 817 μmol), and HOBT (110 mg, 817 μmol) were added to the mixture. The reaction was stirred at 0°C for 30 minutes. Compound 14-3 (199 mg, 408.51 μmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-6% MeOH) to obtain compound 14-4 (540 mg, yield 53%) as a white foamy solid.

[0524] 1H NMR (400MHz,DMSO-d6) δ 7.89-7.80 (m,6H),7.75 (t,J=5.5Hz,3H),7.37 (d,J=7.3Hz,2H),7.30 (t,J=7.7Hz,2H),7.23-7.17 (m,5H),6.99 (s,1H),6.87 (d,J=8.4Hz,4H),5.21 (d,J=3.4Hz,3H),4.96 (dd,J=10.8,3.4Hz,3H),4.47(d,J=8.5Hz,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=9.4Hz,2H),2.27 (t,J=6.3Hz,6H),2.09 (s,9H),2.03 (t,J=7.0Hz,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). [ka]

[0525] 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 obtain a white, foamy crude product 15-1 (3.46 g, crude product).

[0526] C 17 H 23 Calculated mass of NO4: 305.2, measured mass: 306.2 [M+H] + ,ESI.

[0527] 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 was stirred at 0°C for 3 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 high-performance column chromatography (PE / EA = 0-60%) to Al2O3 and by preparative HPLC to obtain compound 15-2a (1.04 g, yield 34%) and compound 15-2b (1.78 g, yield 58%), which were white foamy solids. 15-2a: 1 H NMR (400MHz,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=8.9Hz,4H),5.02 (d,J=6.1Hz,2H),6.98 (s,1H),4.61 (t,J=5.0Hz,1H),3.71 (s,6H),3.46-3.35 (m,2H),3.27 (d,J=5.0Hz,2H),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 (400MHz,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=8.9Hz,4H),5.03 (s,2H),6.98 (s,1H),4.64 (t,J=5.1Hz,1H),3.73 (s,6H),3.43-3.34 (m,4H),3.19-3.09 (m,2H),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).

[0528] Compound 15-3: Compound 15-2a (210 mg, 350.48 μmol) was dissolved in MeOH (10 mL), and Pd / C was added to the solution. The reaction mixture was stirred at room temperature under a hydrogen gas atmosphere for 2 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude product 15-3a (166 mg, crude product), a white foamy solid, which could be used in the next step without further purification.

[0529] C 30 H 35 Calculated mass of NO4: 473.3, measured mass: 474.3 [M+H] + ,ESI.

[0530] Compound 15-2b (610 mg, 1.00 mmol) was dissolved in MeOH (10 mL), and Pd / C was added to the solution. The reaction mixture was stirred at room temperature under a hydrogen gas atmosphere for 2 hours. The reaction mixture was filtered and concentrated under vacuum to obtain crude product 15-3b (475 mg, crude product), a white foamy solid, which could be used in the next step without further purification.

[0531] Compound 15-4: Compound 10-1 (700 mg, 348.92 μmol) was dissolved in DCM (20 mL). At 0°C, DIEA (135 mg, 1.05 mmol), EDCI (134 mg, 698 μmol), and HOBT (94 mg, 698 μmol) were added to the mixture. The reaction was stirred at 0°C for 30 minutes. Compound 15-3a (199 mg, 408.51 μmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-6% MeOH) to obtain compound 15-4a (550 mg, yield 64%) as a white foamy solid. 1H NMR (400MHz,DMSO-d6) δ 7.88-7.80 (m,6H),7.75 (t,J=5.4Hz,3H),7.38-7.34 (m,2H),7.28 (t,J=7.6Hz,2H),7.24-7.17 (m,5H),7.00 (s,1H),6.87 (d,J=8.9Hz,4H),5.21 (d,J=3.4Hz,3H),4.96 (dd,J=11.3,3.4Hz,3H),4.64-4.60 (m,1H),4.47 (d,J=8.5Hz,3H),4.05-3.98 (m,9H),3.92-3.83 (m,3H),3.76-3.65 (m,9H),3.62-3.45 (m,13H),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=6.2Hz,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).

[0532] Compound 10-1 (1 g, 500.29 μmol) was dissolved in DCM (20 mL). At 0°C, DIEA (194 mg, 1.50 mmol), EDCI (192 mg, 1.00 mmol), and HOBT (135 mg, 1.00 mmol) were added to the mixture. The reaction was stirred at 0°C for 30 minutes. Compound 15-3b (238 mg, 502.53 μmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-6% MeOH) to obtain compound 15-4b (550 mg, yield 44%) as a white foamy solid.

[0533] 1H NMR (400MHz,DMSO-d6) δ 7.87-7.80 (m,4H),7.74 (t,J=5.6Hz,3H),7.40-7.36 (m,2H),7.31 (t,J=7.7Hz,2H),7.27-7.18 (m,5H),6.99 (s,1H),6.91-6.87 (m,4H),5.22 (d,J=3.4Hz,3H),4.96 (dd,J=11.3,3.4Hz,3H),4.66-4.62 (m,1H),4.48 (d,J=8.4Hz,3H),4.05-3.98 (m,9H),3.92-3.83 (m,3H),3.75-3.67 (m,9H),3.57-3.47 (m,11H),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). [ka]

[0534] 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 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 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 on Al2O3 by high-performance column chromatography (0-10% MeOH) to obtain compound 16-1 (772 mg, yield 77%) as a white foamy solid.

[0535] C 121 H 179 N 11 O 42 Calculated mass value: 2458.22, Actual mass value: 1079.2 [M-DMTr+2H] 2+ 、ESI.

[0536] 1 H NMR (400MHz, 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=8.0Hz,4H),5.21 (d,J=4.0Hz,3H),4.97 (dd,J=3.2,8.0Hz,3H),4.80-4.70 (m,1H),4.48 (d,J=8.4Hz,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=5.2Hz,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).

change

[0537] Compound 17-1: Compound 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, and then compound 3-12 (493.56 mg, 0.78 mmol) was added. 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 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to obtain compound 17-1 (1.14 g, yield 58%).

[0538] C 129 H 193 N 11 O 39 Calculated mass: 2520.35, measured mass: 1109.35 (M-DMTr+2H) 2+ ,ESI.

[0539] 1 H NMR (400MHz,DMSO-d6) δ 7.83 (d,J=9.2Hz,4H),7.73 (t,J=9.2Hz,3H),7.47-7.39 (m,3H),7.33-7.19 (m,7H),6.88 (d,J=8.92Hz,4H),5.21 (d,J=3.3Hz,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). [ka]

[0540] Compound 18-1: At 0°C, compound 11-11 (1.01 g, 0.45 mmol) was dissolved in DCM (20 mL) and 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. The reaction was stirred at 0°C for 30 minutes. Compound 4-10 (300 mg, 0.49 mmol) was added to the reaction 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 on Al2O3 by high-performance column chromatography (0-8% MeOH) to obtain compound 18-1 (500 mg, yield 46%) as a white foamy solid.

[0541] C 127 H 189 N 11 O 39 Calculated mass: 2493.31, measured mass: 1096.7 [M-DMTr+2H] 2+ ,ESI.

[0542] 1H NMR (400MHz,DMSO-d6) δ 7.84-7.78 (m,2H),7.72 (t,J=5.4Hz,3H),7.45 (br,1H),7.40-7.39 (m,2H),7.33-7.30 (m,2H),7.28-7.20 (m,5H),5.22 (d,J=3.4Hz,3H),4.99-4.95 (m,3H),4.64-4.62 (m,3H),4.49 (d,J=8.4Hz,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). [ka]

[0543] Compound 19-1: Compounds 11-11 (1.59 g, 832.24 μmol) (3.2 g, 7.16 mmol) and 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 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 obtain compound 19-1 (840 mg, 36% yield).

[0544] C 127 H 189 N 11 O 39Calculated mass: 2492.3, measured mass: 1258.4 [M+2Na] 2+ ,ESI.

[0545] 1 H NMR (400MHz,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=8.8Hz,4H),5.22 (d,J=3.2Hz,3H),4.97 (d,J=11.6Hz,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). [ka]

[0546] Compound 20-1a: Compound 11-11 (554 mg, 289.73 μmol) was dissolved in DCM (20 mL). At 0°C, DIEA (112 mg, 869.18 μmol), EDCI (222 mg, 1.16 mmol), and HOBT (157 mg, 1.16 mmol) were added to the mixture. The reaction was stirred at 0°C for 30 minutes. Compound 7-9a (170 mg, 289.73 μmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. 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 obtain compound 20-1a (640 mg, yield 89%) as a white foamy solid. 1H NMR (400MHz,DMSO-d6) δ 7.86-7.76 (m,4H),7.72 (t,J=5.4Hz,3H),7.49-7.42 (m,1H),7.38 (d,J=8.6Hz,2H),7.31 (t,J=7.7Hz,2H),7.27-7.19 (m,5H),6.88 (d,J=8.9Hz,4H),5.21 (d,J=3.4Hz,3H),4.96 (dd,J=11.2,3.4Hz,3H),4.65 (t,J=5.0Hz,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).

[0547] Compound 20-1b: Compound 11-11 (1.3 g, 681.71 μmol) was dissolved in DCM (20 mL). At 0°C, DIEA (176 mg, 1.36 mmol), EDCI (261 mg, 1.36 mmol), and HOBT (138 mg, 1.02 mmol) were added to the mixture. The reaction was stirred at 0°C for 30 minutes. Compound 7-9b (400 mg, 681.71 μmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours.

[0548] 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 obtain compound 20-1b (610 mg, yield 36%), a white foamy solid.

[0549] 1H NMR(400MHz,DMSO-d6) δ:7.87-7.76 (m,4H),7.72 (t,J=5.3Hz,3H),7.46 (s,1H),7.35(d,J=7.4Hz,2H),7.28 (t,J=7.6Hz,2H),7.25-7.18 (m,5H),6.86 (d,J=8.9Hz,4H),5.21 (d,J=3.4Hz,3H),4.96 (dd,J=11.2,3.4Hz,3H),4.62 (t,J=5.0Hz,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). [ka]

[0550] 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 2 hours, and after concentration, it was purified by preparative HPLC to obtain compound 21-1 (1.2 g, 53% yield).

[0551] C 126 H 187 N 11 O 39 Calculated mass: 2478.3, measured mass: 1089.6 [M-DMTr+2H] 2+, ESI.

[0552] 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).

Chem.

[0553] 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 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 on Al2O3 by high-performance column chromatography (0-20% MeOH) to obtain compound 22-1 (1.2 g, yield 69%), a white foamy solid.

[0554] C 126 H 185 N 11 O 39 Calculated mass: 2476.28, measured mass: 1088.2 [M-DMTr+2H] 2+ ,ESI.

[0555] 1 H NMR (400MHz,DMSO-d6) 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=8.84Hz,4H),5.22 (d,J=3.4Hz,2H),4.97 (d,J=11.2Hz,3H),4.75 (t,J=4.4Hz,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). [ka]

[0556] Compound 23-1: A solution of N-Boc-4-piperidone (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 hours. LC-MS showed complete conversion. The reaction mixture was washed with water (50 mL), dried over Na2SO4, concentrated, and purified by silica gel column (solution in PE with 0-10% EA) to obtain the gel compound 23-1 (6.18 g, 98% yield).

[0557] C 13 H 17 Calculated mass of O2N3: 247.1 MH, Measured mass: 246.2 MH - ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 3.53 (t,J=11.6Hz,4H),2.69 (d,J=11.6Hz,4H),7.41 (t,J=7.3Hz,2H),1.427 (s,9H).

[0558] Compound 23-2: A solution of compound 23-1 (7.6 g, 30.73 mmol) in MeOH (76 mL) was stirred at 0°C. NaBH4 (1.16 g, 30.73 mmol) was gradually added to the stirred solution and stirred for 20 minutes. The mixture was concentrated under vacuum. The residue was separated into ethyl acetate (250 mL) and water (150 mL), 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 and eluted at PE / EA = 1 / 5 to obtain the gel-like compound 23-2 (4.6 g, yield 60%).

[0559] C 13 H 19 Calculated mass of O2N3: 249.2 MH, measured mass: 248.3 MH - ,ESI. 1 H NMR (400MHz,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).

[0560] 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 divided into ethyl acetate (100 mL x 3) and water (150 mL x 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 and eluted at PE / EA = 10 / 1 to obtain the gel-like compound 23-3 (32.87 g, yield 87%).

[0561] C 20 H 31 Calculated mass of O4N3: 377.2, measured mass: 319.4 [M-Boc+MeCN+H] + ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 4.06 (d,J=11.2Hz,2H),2.75 (s,2H),2.55-2.53 (m,1H),2.35-2.31 (m,3H),1.89 (d,J=12.4Hz,2H),1.43 (s,9H),1.41 (s,9H),1.27-1.19 (m,3H).

[0562] Compound 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), and sodium borohydride (32.07 g, 847.73 mmol) was slowly added to the reaction mixture at -20°C and stirred for 1 hour. The mixture was then stirred at room temperature for 1 hour. The reaction mixture was concentrated until dry and azeotropically dried over DMF to obtain crude compound 23-4, which could be used directly in the next step without further purification.

[0563] C 20 H 39 Calculated mass of O4N3: 385.5, measured mass: 386.5 [M+H] + ,ESI.

[0564] Compound 23-5: A solution of Z-6-aminocaproic 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 minutes. 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 mixture. The mixture was filtered, and the filtrate was divided into ethyl acetate (100 mL x 3) and water (150 mL x 3). The ethyl acetate extract was washed with brine (100 mL), dried over sodium sulfate, and concentrated under vacuum. The residue was purified by preparative HPLC (C18 column, ACN / water) to obtain the gel compound 23-5 (8.3 g, yield 23%).

[0565] C 48 H 72 O 11 Calculated mass of N4: 879.5, measured mass: 880.1 [M+H] + ,ESI. 1H NMR (400MHz,DMSO-d6) δ 7.73 (t,J=12.4Hz,2H),7.38-7.30 (m,10H),7.23 (t,J=10.8Hz,2H),5.00 (s,4H),4.06-3.60 (m,4H),3.00-2.97 (m,8H),2.23-2.19 (m,4H),1.99 (s,2H),1.58-1.48 (m,6H),1.42-1.38 (m,25H),1.25-1.23 (m,4H).

[0566] Compound 23-6: Under a hydrogen gas atmosphere, a mixture of compound 23-5 (3.8 g, 4.31 mmol), NH3.H2O (3.5 mL), and Pd / C (760 mg) in MeOH (38 mL) was stirred at room temperature for 2 hours. LC-MS showed complete conversion. The mixture was filtered, and the filtrate was concentrated under vacuum to obtain crude compound 23-6, which could be used directly in the next step without further purification.

[0567] C 32 H 60 Calculated mass of N4O7: 611.8, measured mass: 612.8 [M+H] + ,ESI.

[0568] Compound 23-7: A mixture of compounds 1-4 (2.46 g, 5.49 mmol), TEA (757.89 mg, 7.49 mmol), and HATU (2.35 mg, 6.24 mmol) in DCM (15 mL) was stirred at room temperature for 30 minutes. Compound 23-6 (1.53 g, 2.5 mmol) was added, and the mixture was stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to obtain compound 23-7 (3.14 g, 85% yield). 1H NMR (400MHz,DMSO-d6) δ 7.82 (d,J=9.2Hz,2H),7.71 (s,4H),5.52 (d,J=0.4Hz,2H),4.99-4.95 (m,2H),4.48 (d,J=8.8Hz,2H),4.03 (s,8H),3.95-3.84 (m,2H),3.72-3.70 (m,2H),3.44-3.38 (m,2H),3.01-2.99 (m,8H),2.23-2.19 (m,2H),2.11-2.07 (m,10H),2.05-2.00 (m,10H),1.90 (s,6H),1.78 (s,6H),1.58-1.49 (m,14H),1.39-1.38 (m,25H),1.24-1.23 (m,8H).

[0569] Compound 23-8: A solution of compound 23-7 (3.0 g, 2.04 mmol) and TFA (12 mL) in DCM (30 mL) was stirred at room temperature for 4 hours. LC-MS showed complete conversion. The solution was concentrated under vacuum, and then basement was performed with TEA. Compound 23-8 was ready for the next step without further purification.

[0570] C 61 H 99 O 24 Calculated mass of N7: 1313.4, measured mass: 1314.4 [M+H] + ,ESI.

[0571] Compound 23-9: The solutions of compound 1-7 (2.68 g, 2.04 mmol), TEA (412.62.89 mg, 4.08 mmol), and HATU (769.19 mg, 2.04 mmol) in DCM (30 mL) were stirred at room temperature for 4 hours. Then, the solutions were added to compound 23-8 (2.68 g, 2.04 mmol) and stirred overnight at room temperature. LC-MS showed complete conversion. The reaction mixture was diluted with DCM (20 mL), washed with water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ACN / water) to obtain compound 23-9 (1.76 g, 47% yield).

[0572] C 86 H 137 O 35 Calculated mass of N9: 1857.0, measured mass: 929.0 [M+2H] 2+ ,ESI. 1 H NMR (400MHz,DMSO-d6) δ 7.94 (d,J=9.2Hz,2H),7.88-7.79 (m,6H),5.21 (d,J=3.2Hz,3H),4.99-4.95 (m,3H),4.51-4.48 (m,4H),4.05-4.00 (m,9H),3.92-3.84 (m,4H),3.73-3.37 (m,3H),2.99 (s,10H),2.81 (t,J=23.2Hz,1H),2.48-2.44 (m,2H),2.34-2.16 (m,6H),2.11-2.09 (m,13H),2.05-2.00 (m,16H),1.89 (s,9H),1.78 (s,9H),1.49-1.35 (m,31H),1.24-1.23 (m,6H).

[0573] Compound 23-10: A mixture of compound 23-9 (1.76 g, 0.94 mmol), DIEA (367.47 mg, 2.84 mmol), HOBT (256.12 mg, 1.90 mmol), and EDCI (363.37 mg, 1.90 mmol) in DCM (20 mL) was stirred at 0°C for 1 hour, and then compound 11-2 (542.80 mg, 0.94 mmol) was added. The mixture was stirred at room temperature overnight. LC-MS showed complete conversion. The reaction mixture was diluted by adding DCM (20 mL), washed with NaHCO3 (saturated aqueous solution, 50 mL) and water (50 mL), concentrated, and purified by preparative HPLC (C18 column, ...

Claims

1. An siRNA agent having a 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, and the consecutive nucleotides differ by three or fewer nucleotides from any one nucleotide sequence among 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, and the second strand comprises a nucleotide sequence that is at least partially complementary to the first strand.

2. The siRNA agent according to claim 1, wherein the first chain sequence comprises at least 16, preferably at least 17, more preferably at least 18, and most preferably all 19 consecutive nucleotides, and the consecutive nucleotides differ by two or fewer nucleotides from any one 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, preferably by one or fewer nucleotides, and more preferably without any nucleotide differences.

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

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

5. The first chain has the nucleotide sequence shown in SEQ ID NO: 20, and the second chain has the nucleotide sequence shown in SEQ ID NO: 21, or the first chain has the nucleotide sequence shown in SEQ ID NO: 96, and the second chain has the nucleotide sequence shown in SEQ ID NO: 97, or the first chain has the nucleotide sequence shown in SEQ ID NO: 16, and the second chain has the nucleotide sequence shown in 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 An siRNA agent according to any one of claims 1 to 4, wherein the first strand has the nucleotide sequence shown in 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: 136, and the second strand has the nucleotide sequence shown in SEQ ID NO:

137.

6. The siRNA agent according to any one of claims 1 to 5, wherein the siRNA agent comprises at least one modified nucleotide, preferably not all of which are modified, and comprises 5, 4, 3, 2 or 1 or fewer unmodified nucleotides, and more preferably, wherein all nucleotides in the first chain are modified nucleotides, and all nucleotides in the second chain are modified nucleotides.

7. At least one modified nucleotide is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-methoxy-modified nucleotide, a 2'-fluoro-modified nucleotide, or a 2'-OCF nucleotide. ２ The siRNA agent according to claim 6, selected from H-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and nucleotides containing a 5'-(E)-vinylphosphonate.

8. (i) All nucleotides in the first strand are The pentoses of the 5th, 7th, 8th, and 9th nucleotide residues from the 5' end are modified by 2'-fluoro substitution. The pentoses of the 1st, 2nd, 4th, 6th, 10th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified with 2'-methoxy modifications. This modification pattern includes modifications in which the pentoses of the 3rd, 11th, and 14th nucleotide residues from the 5' end are modified by 2'-methoxy or 2'-fluoro substitutions. All nucleotides in the second strand are The pentoses at the 2nd, 14th, 16th, and 18th nucleotide residues from the 5' end are modified by 2'-fluoro substitution. The pentoses of the 1st, 3rd, 5th, 6th, 7th, 9th, 11th, 12th, 13th, 15th, 17th, and 19th nucleotide residues from the 5' end are modified with 2'-methoxy modification. The modification pattern includes modifications in which the pentoses of the 4th, 8th, and 10th nucleotide residues from the 5' end are modified by 2'-methoxy modification or 2'-fluoro substitution, or (ii) All nucleotides of the first strand are The pentoses of the 7th, 9th, 10th, and 11th nucleotide residues from the 5' end are modified by 2'-fluoro substitution. The pentoses at the 2nd, 4th, 6th, 8th, 12th, 14th, 16th and 18th nucleotide residues from the 5' end are modified with 2'-methoxy modifications. This modification pattern includes modifications in which the pentoses of the 1st, 3rd, 5th, 13th, 15th, 17th, and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification or 2'-fluoro substitution. All nucleotides in the second strand are The pentoses of the 2nd, 6th, 8th, 14th, and 16th nucleotide residues from the 5' end are modified by 2'-fluoro substitution. The pentoses of the 1st, 3rd, 5th, 7th, 11th, 12th, 13th, 15th, 17th and 19th nucleotide residues from the 5' end are modified with 2'-methoxy modifications. The siRNA agent according to claim 6 or 7, comprising a modification pattern in which the 4th, 9th, 10th, and 18th positions from the 5' end are modified with 2'-methoxy or 2'-fluoro substitutions.

9. (i) All nucleotides in the first chain have a modification pattern in which the pentoses of the 5th, 7th, 8th, 9th and 11th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification, All nucleotides in the second chain include modifications in the following pattern: the pentoses of nucleotide residues 2, 8, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; and the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; or (ii) All nucleotides in the first chain have a modification pattern in which the pentoses of the 3rd, 5th, 7th, 8th and 9th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification, All nucleotides in the second chain include modifications in the following pattern: the pentoses of nucleotide residues 2, 4, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; and the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; or (iii) All nucleotides in the first chain have a modification pattern in which the pentoses of the 5th, 7th, 8th, 9th and 11th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 3rd, 4th, 6th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification, All nucleotides in the second chain include modifications in the following pattern: the pentoses of nucleotide residues 2, 4, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; and the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; or (iv) All nucleotides in the first chain have a modification pattern in which the pentoses of the 5th, 7th, 8th, 9th and 14th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification. All nucleotides in the second chain include modifications in the following pattern: the pentoses of nucleotide residues 2, 8, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; and the pentoses of nucleotide residues 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; or (v) All nucleotides in the first chain have a modification pattern in which the pentoses of the 5th, 7th, 8th, 9th and 14th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification. All nucleotides in the second chain include modifications in the following pattern: the pentoses of nucleotide residues 2, 4, 14, 16, and 18 from the 5' end are modified by 2'-fluoro substitution; and the pentoses of nucleotide residues 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, and 19 from the 5' end are modified by 2'-methoxy modification; or (vi) All nucleotides in the first chain have a modification pattern in which the pentoses of the 7th, 9th, 10th and 11th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification. The siRNA agent according to claim 8, wherein all nucleotides of the second strand include modifications in the following pattern: the pentoses of the 2nd, 6th, 8th, 9th, 14th and 16th nucleotide residues from the 5' end are modified by 2'-fluoro substitution, and the pentoses of the 1st, 3rd, 4th, 5th, 7th, 10th, 11th, 12th, 13th, 15th, 17th, 18th and 19th nucleotide residues from the 5' end are modified by 2'-methoxy modification.

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

11. The first strand contains two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends, and / or the second strand contains two phosphorothioate nucleotide interlinks at its 5' and / or 3' ends. Preferably, (i) The first strand contains two phosphorothioate nucleotide interlinks at its 3' end, The second strand contains two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, or (ii) The first strand contains two phosphorothioate nucleotide interlinks at its 5' end, The siRNA agent according to any one of claims 1 to 10, wherein the second strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end.

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

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

14. The targeted ligand is conjugated to the 5' end of the first chain, The first strand contains two phosphorothioate nucleotide interlinks at its 3' end, The second strand contains two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end. Optionally, all remaining bonds between the nucleotides of the first and / or second strands are phosphodiester bonds. Or, The targeted ligand is conjugated to the 3' end of the first chain, The first strand contains two phosphorothioate nucleotide interlinks at its 5' end, The second strand contains two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end. The siRNA agent according to claim 12 or 13, wherein optionally, all remaining bonds between nucleotides of the first and / or second strands are phosphodiester bonds.

15. 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 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*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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 as GN-mCmCmGmAfGmAfGfCfAmUmGmGmUfUmGmUmC*mU*mU (SEQ ID NO: 223), and the second strand has a nucleotide sequence represented as (E)-VP-mA*fA*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 217), Herein, 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; "*" is a phosphorothioate nucleotide bond; "(E)-VP" is the 5' terminal (E)-vinylphosphonate moiety; and "GN" is a targeted ligand, as described in any one of claims 12 to 14.

16. The siRNA agent is conjugated with a ligand as shown in the figure below, and the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, (ii) a linker that conjugates at least one GalNAc moiety or derivative thereof with a nucleic acid, (iii) a bicyclic group that links GalNAc and siRNA, or (iv) a tetrafunctional group that links the GalNAc moiety. 【Chemistry 1】 Here, X is O or S, the siRNA agent according to any one of claims 12 to 15.

17. As shown in the figure below, the siRNA agent is conjugated with the ligand, 【Chemistry 2】 Here, X is either O or S, The aforementioned 【Transformation 3】 teeth, 【Chemistry 4】 And, The aforementioned 【Transformation 5】 teeth, 【Transformation 6】 【Transformation 7】 【Transformation 8】 And, Or, 【Chemistry 9】 Here, X is either O or S, The aforementioned 【Chemistry 10】 teeth, 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 The siRNA agent according to claim 16.

18. The linker in the above figure is independent, (1)-(CH ２ )x-C(O)NH-(CH ２ )y-C(O)-、 (2)-(CH) ２ )x-C(O)NH-(CH ２ )y-NHC(O)-(CH ２ )z-C(O)-、 (3)-NH-(CH ２ )y-C(O)-、 (4)-C(O)-(CH ２ ) Selected from y-C(O)-, Here, each linker in the figure is either 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, the siRNA agent according to claim 16 or 17.

19. As shown in the figure below, the siRNA agent is conjugated with the ligand, 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 The siRNA agent according to any one of claims 12 to 18, wherein X is O or S.

20. 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 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*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*mGfAmCmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmCmCmAmUmGmCfUmCfUmC*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*mGmAmCmAmAfCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*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*mGfAmCmAmAmCmCmAmUmGmCfUmCfUmC*fG*mG (SEQ ID NO: 233), Herein, mA, mC, mG, and mU represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively, and fA, fC, gG, and fU represent 2'-fluoroadenosine, cytidine, guanosine, or uridine, respectively, and "*" is a phosphorothioate nucleotide bond, and "(E)-VP" is the 5' terminal (E)-vinylphosphonate moiety, according to any one of claims 12 to 19.

21. A pharmaceutical composition comprising an siRNA agent according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. A method for inhibiting the expression of the complement component C3 gene in cells, wherein the method is: (a) a step of contacting the cells with the siRNA reagent according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 21, A method comprising step (a) inhibiting the expression of the complement component C3 gene in the cells by maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the complement component C3 gene.

23. A method for treating a subject suffering from a disease or disorder that would benefit from reduced expression of complement component C3, the method comprising treating the subject by administering to the subject a therapeutically effective amount of a siRNA agent according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21.

24. The method according to claim 23, wherein the disease or disorder is a complement-mediated disease, disorder or syndrome, preferably a C3-related disease or disorder, and more preferably the disease or disorder is selected from IgA nephropathy (IgAN), C3 glomerulopathy (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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