SiRNA for inhibiting CFB gene expression and application thereof

Abstract

The invention discloses siRNA (small interfering Ribonucleic Acid) for inhibiting CFB (circulating fluid bed) gene expression and application of the siRNA. The siRNA and the nucleic acid conjugate provided by the invention can effectively prevent and / or treat complement factor B mediated related diseases.

Description

[0001] Priority information

[0002] This application claims priority and benefit to patent applications filed with the China National Intellectual Property Administration on September 6, 2024, October 15, 2024, January 24, 2025, April 24, 2025, and July 17, 2025, with patent application numbers 2024112535311, 2024114424417, 2025101179820, 2025105265098, and 2025109904476, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedical technology, specifically to siRNA and nucleic acid conjugates for inhibiting complement factor B expression and their uses. Background Technology

[0004] The complement system is part of the host's innate immune system, involved in lysing foreign cells, enhancing antigen phagocytosis, agglutinating antigen-carrying agents, and attracting macrophages and neutrophils. The complement system operates via three activation pathways: the classical pathway, the lectin pathway, and the alternative pathway. The activation pathway focuses on component C3 to produce an enzyme complex called C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to C3 convertase, regulated by complement factor B (CFB), leading to the production of C5 convertase, which cleaves C5 into C5a and C5b. C5 convertase activates the membrane attack pathway, resulting in the formation of the membrane attack complex (MAC), which comprises components C5b, C6, C7, C8, and C9. The MAC forms transmembrane channels and disrupts the phospholipid bilayer of the target cell, leading to cell lysis.

[0005] Aberrant activation of complement factor B (CFB) is the cause of proliferation and / or initiation of lesions in many diseases. Therefore, the development of complement factor B inhibitors has enormous clinical value and market potential. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides an siRNA for inhibiting complement factor B expression, which can suppress the expression of the complement factor B gene and effectively prevent and / or treat complement factor B-mediated diseases.

[0007] In a first aspect, this application provides an siRNA. According to embodiments of this application, the siRNA comprises a sense strand and an antisense strand, the antisense strand having a double-stranded region with at least partial base pairing with the sense strand; wherein the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1–SEQ ID NO:70 and SEQ ID NO:141–SEQ ID NO:143, or at least 15 consecutive nucleotides thereof; the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71–SEQ ID NO:140 and SEQ ID NO:144–SEQ ID NO:146, or at least 15 consecutive nucleotides thereof; each nucleotide in the sense strand and / or antisense strand is independently a modified or unmodified nucleotide. The siRNA according to embodiments of the present invention can inhibit the expression of CFB, thereby effectively preventing and / or treating complement factor B-mediated diseases.

[0008] According to embodiments of this application, the siRNA may further include at least one of the following technical features:

[0009] According to embodiments of this application, the positive chain comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or differs from it by no more than 5 nucleotides.

[0010] According to embodiments of this application, the positive chain comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or differs from it by no more than 4, 3, 2, or 1 nucleotides.

[0011] According to embodiments of this application, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or differs from it by no more than 5 nucleotides.

[0012] According to embodiments of this application, the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or differs from it by no more than 4, 3, 2, or 1 nucleotides.

[0013] According to an embodiment of the present invention, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 10 consecutive nucleotides thereof; the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 10 consecutive nucleotides thereof.

[0014] According to an embodiment of the present invention, the siRNA includes any one of the sense and antisense strands in Table A (see the Specific Implementation Methods section of this specification), or has a difference of no more than 5 nucleotides from the sense and / or antisense strands, preferably having a difference of no more than 4, 3, 2 or 1 nucleotides.

[0015] According to embodiments of the present invention, the siRNA may be selected from any of the following:

[0016] a) Both the 3' and 5' ends of the justice chain are blunt ends, and both the 3' and 5' ends of the antisense chain are blunt ends;

[0017] b) The justice chain has a 3' or 5' protrusion, and both the 3' and 5' ends of the antisense chain are blunt ends; or

[0018] The antisense chain has a 3' protruding end or a 5' protruding end, and the 3' end and 5' end of the justice chain are both blunt ends;

[0019] c) The justice chain has a 3' protrusion or a 5' protrusion, and the antisense chain has a 3' protrusion or a 5' protrusion;

[0020] d) The positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 17, 19 or 21 consecutive nucleotides thereof;

[0021] e) The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 17, 19 or 21 consecutive nucleotides thereof;

[0022] f) The lengths of the sense and antisense strands are independently 17-25 nucleotides, preferably 19-25 nucleotides.

[0023] According to an embodiment of the present invention, the sense strand and / or antisense strand further comprises a 3' overhang and / or a 5' overhang, wherein the 3' overhang or the 5' overhang comprises at least one nucleotide or the like thereof, preferably comprising at least two nucleotides or the like thereof.

[0024] In some optional embodiments of the present invention, in the siRNA, one of the sense strands or the antisense strand has at least one nucleotide overhang, and both ends of the other strand are blunt ends.

[0025] In some optional embodiments of the present invention, the siRNA has at least one nucleotide overhang in the antisense strand and both ends of the sense strand are blunt ends.

[0026] According to an embodiment of the present invention, the sense strand and / or antisense strand includes a 3' overhang, the 3' overhang comprising at least two nucleotides or the like thereof.

[0027] According to embodiments of the present invention, each nucleotide in the 3' and / or 5' protrusions is independently selected from U, T, dU, or dT.

[0028] According to an embodiment of the present invention, the 3' and / or 5' protrusions of the sense strand and / or antisense strand comprise two nucleotides or the like.

[0029] According to an embodiment of the present invention, the 3' protrusion and / or 5' protrusion of the sense chain and / or antisense chain are selected from UU, TT, dUdU or dTdT.

[0030] According to an embodiment of the present invention, the antisense strand includes a 3' overhang, the 3' overhang containing at least two nucleotides.

[0031] According to an embodiment of the present invention, the 3' protrusion of the antisense chain is UU.

[0032] According to embodiments of the present invention, the double-stranded region has a length of 19-23 nucleotides, for example, 19, 20, 21, 22 or 23 nucleotides.

[0033] According to an embodiment of the present invention, the double-stranded region has a length of 19-21 nucleotides, for example, 19, 20 or 21 nucleotides.

[0034] According to an embodiment of the present invention, the length of the positive chain is no more than 23 nucleotides, preferably no more than 21 nucleotides, for example 19, 20 or 21 nucleotides.

[0035] According to an embodiment of the present invention, the length of the antisense strand is no more than 25 nucleotides, preferably no more than 23 nucleotides, for example 21, 22 or 23 nucleotides.

[0036] According to an embodiment of the present invention, the sense strand and / or the antisense strand comprises at least one modified nucleotide, each of the modified nucleotides being independently selected from at least one of the following:

[0037] Reverse debasing nucleotides, deoxynucleotides, 2'-fluorinated nucleotides, 2'-amino nucleotides, 2'-O-allyl nucleotides, 2'-C 1～25 Alkyl-modified nucleotides, 2'-OC 1～25 Alkyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, locked nucleotides, conformation-restricted nucleotides, 2'-allyl-modified nucleotides, base-free nucleotides, morpholinyl nucleotides, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate-modified nucleotides, 5'-thiophosphate-modified nucleotides, 5'-methylphosphonate-modified nucleotides, 5'-phosphate mimicry-modified nucleotides, 5'-methylcytosine-modified nucleotides, and nucleic acid analogs.

[0038] According to embodiments of the present invention, the modified nucleotide is independently selected from at least one of reverse debased nucleotides, 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, and 5'-thiophosphate modified nucleotides.

[0039] According to embodiments of the present invention, all nucleotides in the sense strand are modified nucleotides; and / or, all nucleotides in the antisense strand are modified nucleotides.

[0040] According to an embodiment of the present invention, in the sense strand and / or the antisense strand, the 2'-fluoro-modified nucleotide is present at the following position:

[0041] The nucleotide at the 5' end of the sense strand is at least one of the 9th, 11th, and 13th positions of the start point; and / or, the nucleotide at the 5' end of the antisense strand is at least one of the 2nd, 7th, 12th, 14th, and 16th positions of the start point.

[0042] In an optional embodiment of the present invention, the 2'-fluoromodified nucleotide is present at the following position in the sense strand and / or the antisense strand:

[0043] The nucleotides at the 5' end of the sense strand are the 9th, 11th, and 13th positions of the start point; and / or, the nucleotides at the 5' end of the antisense strand are the 2nd, 7th, 12th, 14th, and 16th positions of the start point.

[0044] According to an embodiment of the present invention, in the antisense strand, the nucleotide modified with a 5'-thiophosphate group is present at the following position:

[0045] The nucleotide at the 5' end of the antisense strand is at least one of the first and second positions of the start point, and / or the nucleotide at the 3' end of the antisense strand is at least one of the first and second positions of the start point.

[0046] In an optional embodiment of the present invention, the nucleotide modified with a 5'-thiophosphate group is present at the following position in the antisense strand:

[0047] The nucleotides at the 5' end of the antisense strand are the first and second positions of the start point, and / or the nucleotides at the 3' end of the antisense strand are the first and second positions of the start point.

[0048] According to an embodiment of the present invention, in the sense strand and / or the antisense strand, the 2'-methoxy modified nucleotide is present at the following position:

[0049] The 5' terminal nucleotide of the sense strand is at least one of the following positions: position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 10, position 12, position 14, position 15, position 16, position 17, position 18, position 19, position 20, and position 21 of the start point; and / or, the 5' terminal nucleotide of the antisense strand is at least one of the following positions: position 1, position 3, position 4, position 5, position 6, position 8, position 9, position 10, position 11, position 13, position 15, position 17, position 18, position 19, position 20, and position 21 of the start point.

[0050] In an optional embodiment of the present invention, the 2'-methoxy modified nucleotide in the sense strand and / or the antisense strand is present at the following position:

[0051] The 5' terminal nucleotides of the sense strand are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions of the start point; and / or, the 5' terminal nucleotides of the antisense strand are the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions of the start point.

[0052] According to an embodiment of the present invention, the double-stranded region has 21 nucleotides, and the modifying nucleotides of the sense strand and the antisense strand are as follows:

[0053] 2'-Fluorinated nucleotides are present at the following positions:

[0054] The nucleotides at the 5' end of the sense strand are the 9th, 11th, and 13th positions of the start point; and the nucleotides at the 5' end of the antisense strand are the 2nd, 7th, 12th, 14th, and 16th positions of the start point;

[0055] Nucleotides modified with 5'-thiophosphate groups are present at the following positions:

[0056] The nucleotides at the 5' end of the antisense strand are the first and second positions of the start point, and the nucleotides at the 3' end of the antisense strand are the first and second positions of the start point;

[0057] 2'-methoxy modified nucleotides are present at the following positions:

[0058] The nucleotides at the 5' end of the sense strand are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions of the start point; and the nucleotides at the 5' end of the antisense strand are the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions of the start point.

[0059] According to an embodiment of the present invention, the first nucleotide at the 3' end of the antisense strand is EVpu, EVpa, cPrpu, or cPrpa.

[0060] According to an embodiment of the present invention, the double-stranded region has 21 nucleotides, and the modifying nucleotides of the sense strand and the antisense strand are as follows:

[0061] 2'-Fluorinated nucleotides are present at the following positions:

[0062] The nucleotides at the 5' end of the sense strand are the 9th, 11th, and 13th positions of the start point; and the nucleotides at the 5' end of the antisense strand are the 2nd, 7th, 12th, 14th, and 16th positions of the start point;

[0063] Nucleotides modified with 5'-thiophosphate groups are present at the following positions:

[0064] The nucleotides at the 5' end of the antisense strand are the first and second positions of the start point, and the nucleotides at the 3' end of the antisense strand are the first and second positions of the start point;

[0065] 2'-methoxy modified nucleotides are present at the following positions:

[0066] The 5' terminal nucleotides of the sense strand are at positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, and 21 of the start point; and the 5' terminal nucleotides of the antisense strand are at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21 of the start point;

[0067] The first nucleotide at the 3' end of the antisense strand is EVpu, EVpa, cPrpu, or cPrpa.

[0068] In this article, the term "5' end" refers to the position of the first nucleotide at the 5' end of a nucleotide sequence. The term "3' end" refers to the position of the first nucleotide at the 3' end of a nucleotide sequence.

[0069] In this article, the term "5' terminal nucleotide" or "5' terminal nucleotide" refers to the first nucleotide at the 5' end of a nucleotide sequence. The term "3' terminal nucleotide" or "3' terminal nucleotide" refers to the first nucleotide at the 3' end of a nucleotide sequence.

[0070] It should be noted that "the nucleotide at the 5' end of the positive strand as the starting point" means that the first nucleotide at the 5' end of the positive strand is the starting site, and "the Xth position of the nucleotide at the 5' end of the positive strand as the starting point" means the position of the Xth nucleotide counted from the first nucleotide at the 5' end of the positive strand as the starting site.

[0071] It should be noted that "the nucleotide at the 3' end of the positive strand as the starting point" means that the first nucleotide at the 3' end of the positive strand is the starting site, and "the Xth position of the nucleotide at the 3' end of the positive strand as the starting point" means the position of the Xth nucleotide counted from the first nucleotide at the 3' end of the positive strand as the starting site.

[0072] It should be noted that "the nucleotide at the 5' end of the antisense strand as the starting point" means that the first nucleotide at the 5' end of the antisense strand is the starting site, and "the Xth position of the nucleotide at the 5' end of the antisense strand as the starting point" means the position of the Xth nucleotide counted from the first nucleotide at the 5' end of the antisense strand as the starting site.

[0073] It should be noted that "the nucleotide at the 3' end of the antisense strand as the starting point" means that the first nucleotide at the 3' end of the antisense strand is the starting site, and "the Xth position of the nucleotide at the 3' end of the antisense strand as the starting point" means the position of the Xth nucleotide counted from the first nucleotide at the 3' end of the antisense strand as the starting site.

[0074] According to an embodiment of the present invention, one or more reverse debasing nucleotides are added to the 3' end and / or 5' end of the positive strand.

[0075] According to an embodiment of the present invention, a reverse debase nucleotide is added to the 3' end and / or 5' end of the positive strand.

[0076] According to an embodiment of the present invention, the first nucleotide at the 3' end of the positive strand is linked to the reverse debased nucleotide via a 5'-thiophosphate group.

[0077] According to an embodiment of the present invention, the first nucleotide at the 5' end of the positive strand is linked to the reverse debased nucleotide via a 5'-thiophosphate group.

[0078] In a second aspect of this application, an siRNA is proposed. According to embodiments of this application, the siRNA comprises any one of the sense and antisense strands in Table B (see the Specific Implementation section of this specification), or differs from the sense and / or antisense strands by no more than 5 nucleotides, preferably by no more than 4, 3, 2, or 1 nucleotide.

[0079] According to an embodiment of the present invention, the siRNA includes any one of the sense and antisense strands in Table C (see the Specific Implementation Methods section of this specification), or has a difference of no more than 5 nucleotides from the sense and / or antisense strands, preferably having a difference of no more than 4, 3, 2 or 1 nucleotides.

[0080] In a third aspect, this application proposes a nucleic acid conjugate. According to embodiments of this application, the nucleic acid conjugate comprises: the siRNA described in the first or second aspect, and a ligand conjugated to the siRNA. The nucleic acid conjugate of the present invention can facilitate the targeting and delivery of nucleic acid drugs, enabling siRNA to connect to desired cells and / or tissues in vivo to target and silence complement factor B inhibitors in target cells and / or tissues, thereby effectively preventing and / or treating complement factor B-mediated diseases.

[0081] According to embodiments of the present invention, the above-mentioned nucleic acid conjugate may further include at least one of the following technical features:

[0082] According to embodiments of the present invention, the ligand is one or more, the ligand being used to target any cell, preferably the ligand being selected from lipids, carbohydrates, aptamers, vitamins and / or peptides that specifically bind to cell membranes or cell surfaces.

[0083] According to embodiments of the present invention, the ligand may further have one or more of the following structures:

[0084]

[0085]

[0086]

[0087]

[0088] in, This indicates the junction point between the ligand and the siRNA.

[0089] According to an embodiment of the present invention, the ligand is connected to the 3' or 5' end of the positive chain via a thiophosphate bond or a phosphate bond.

[0090] Unless otherwise specified herein, in the sense and / or antisense strands used for conjugating ligands, the end of the sense and / or antisense strand connected to the ligand contains a thiophosphate bond or phosphate bond, that is, the thiophosphate bond or phosphate bond is located at the 3' or 5' end of the sense and / or antisense strand, which is used for conjugation with the ligand.

[0091] According to an embodiment of the present invention, the nucleic acid conjugate includes any one of the sense and antisense strands in Table D (see the Specific Implementation Methods section of this specification for details).

[0092] In a fourth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the siRNA described in the first or second aspect, or the nucleic acid conjugate described in the third aspect. As previously mentioned, both the aforementioned siRNA and nucleic acid conjugate can degrade the mRNA encoding CFB, inhibiting the expression or activity of the CFB gene. Therefore, the pharmaceutical composition of the present invention can effectively treat and prevent complement factor B-mediated diseases.

[0093] According to embodiments of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0094] In a fifth aspect of the invention, the invention provides for use of the siRNA described in the first or second aspect, the nucleic acid conjugate described in the third aspect, or the pharmaceutical composition described in the fourth aspect, said use including at least one of the following:

[0095] Inhibit the expression or activity of complement factor B gene;

[0096] Prepare drugs for inhibiting the expression or activity of complement factor B gene;

[0097] To prepare drugs for the prevention and / or treatment of complement factor B-mediated diseases;

[0098] Prevention and / or treatment of diseases mediated by complement factor B.

[0099] The method of this invention can be used to inhibit the expression or activity of the CFB gene. For example, it can inhibit the expression or activity of the CFB gene in animals; or it can inhibit the expression or activity of the CFB gene for non-disease treatment purposes, such as inhibiting the expression or activity of the CFB gene in vitro for subsequent research.

[0100] According to embodiments of the present invention, the diseases mediated by complement factor B include nephropathy and systemic lupus erythematosus (SLE).

[0101] According to embodiments of the present invention, the complement factor B-mediated related diseases are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerular diseases (e.g., C3 glomerulonephropathy), age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0102] According to an embodiment of the present invention, the diseases mediated by complement factor B are selected from glomerular diseases and systemic lupus erythematosus.

[0103] According to embodiments of the present invention, the diseases mediated by complement factor B are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, and polycystic kidney disease.

[0104] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Detailed Implementation

[0105] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0106] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0107] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0108] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0109] In this paper, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA consisting of a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through RNA interference (RNAi), inhibiting the translation of mRNA into amino acids and its conversion into proteins. For example, siRNA can suppress the expression of the CFB gene.

[0110] In this paper, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence, such as mRNA encoding CFB. "Sense strand (or follower strand)" refers to an iRNA strand containing a sequence substantially complementary to the antisense strand. The term "substantially complementary" means fully complementary or at least partially complementary, for example, the antisense strand being fully or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the molecule or in terminal regions, with the most tolerant mismatches occurring in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-terminus of the iRNA.

[0111] It should be noted that "at least partially substantially complementary" to mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding CFB). Alternatively, if a polynucleotide is substantially non-discontinuously complementary to a portion of the mRNA encoding CFB, then the antisense strand is complementary to at least a portion of the mRNA encoding CFB.

[0112] In this paper, the terms “target sequence” or “target marker sequence” refer to the continuous portion of the nucleotide sequence of the mRNA molecule formed during transcription of the CFB gene, including mRNA of the RNA processing product of the primary transcription product.

[0113] In this document, the term "inhibition of CFB gene expression" includes any level of inhibition of the CFB gene, such as at least partial inhibition of CFB gene expression, including inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. CFB gene expression can be evaluated based on the level of any variable associated with CFB gene expression, such as the level of mRNA encoding CFB or the CFB level. Inhibition can be evaluated by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as baseline level before administration, or level measured in similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a control with only buffer or a control without active agent).

[0114] In this paper, the term "blunt end" refers to an siRNA whose sense or antisense strand has no unpaired nucleotides at its ends, i.e., no nucleotide overhangs. "Blunt end" siRNA means that the entire length of the siRNA is a double-stranded region, meaning that neither end (3' nor 5') of the sense or antisense strands of the siRNA has nucleotide overhangs.

[0115] In this paper, the term "a sense strand or antisense strand containing at least one nucleotide overhang" means that one end of the sense strand or antisense strand is a nucleotide overhang and the other end is a blunt end, or that both ends of the sense strand or antisense strand are nucleotide overhangs.

[0116] In this article, the term “one of the chains of justice or antisense, the other chain…” includes two cases: 1) one chain is a justice chain and the other chain is an antisense chain, or 2) one chain is an antisense chain and the other chain is a justice chain.

[0117] In this document, the terms "nucleotide overhang" and "overhang" are synonymous, both referring to at least one unpaired nucleotide protruding from the double-stranded region of siRNA. For example, a nucleotide overhang occurs when the 3' end of one strand of the sense and / or antisense strand extends beyond the 5' end of the other strand, or when the 5' end of one strand of the sense and / or antisense strand extends beyond the 3' end of the other strand. The overhang may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The nucleotide overhang may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be located on either the sense or antisense strand, or in any combination of the two strands. Furthermore, the nucleotide at the overhang may be located at the 5' end, 3' end, or both ends of the antisense or sense strand.

[0118] In this article, the terms "nucleotide" and "nucleic acid" refer to A, T / U, C, and G; the terms "nucleotide analog" and "nucleic acid analog" refer to structures that are chemically similar to nucleotides (A, T / U, C, and G).

[0119] Unless otherwise specified herein, the term "protruding end" as used in this invention includes the 3' protruding end and the 5' protruding end.

[0120] In this article, "length of the justice chain" refers to the total length of the justice chain; when the justice chain has a protruding end, "length of the justice chain" = length of the double-chain region + length of the protruding end; when the justice chain has no protruding end, "length of the justice chain" = length of the double-chain region. "Length of the antisense chain" refers to the total length of the antisense chain; when the antisense chain has a protruding end, "length of the antisense chain" = length of the double-chain region + length of the protruding end; when the antisense chain has no protruding end, "length of the antisense chain" = length of the double-chain region.

[0121] In this article, the term "C" 1～25 "Alkyl" includes alkyl groups containing 1, 2, 3, 4...24 or 25 carbon atoms. For example, it can be C... 1～20 Alkyl, C 1～19 Alkyl, C 1～18 Alkyl, C 1～17 Alkyl, C 1～16 Alkyl, C 1～15 Alkyl, C 1～10 Alkyl, C 1～5 Alkyl or C 1～3 Alkyl group. Exemplarily, C 1～3 Alkyl groups include methyl, ethyl, n-propyl, and isopropyl. 2'-C 1～25 Alkyl-modified nucleotides can be 2'-C 1～20 Alkyl-modified nucleotides, 2'-C 1～16 Alkyl-modified nucleotides, 2'-C 1～10Alkyl-modified nucleotides, 2'-C 1～5 Alkyl-modified nucleotides or 2'-C 1～3 Alkyl-modified nucleotides. Exemplarily, they may be 2'-methyl-modified nucleotides, 2'-ethyl-modified nucleotides, 2'-n-propyl-modified nucleotides, or 2'-isopropyl-modified nucleotides.

[0122] 2'-OC 1～25 Alkyl-modified nucleotides can be 2'-OC 1～20 Alkyl-modified nucleotides, 2'-OC 1～16 Alkyl-modified nucleotides, 2'-OC 1～10 Alkyl-modified nucleotides, 2'-OC 1～5 Alkyl-modified nucleotides or 2'-OC 1～3 Alkyl-modified nucleotides. Exemplarily, they may be 2'-O-methyl-modified nucleotides, 2'-O-ethyl-modified nucleotides, 2'-O-n-propyl-modified nucleotides, or 2'-O-isopropyl-modified nucleotides.

[0123] As will be known to those skilled in the art, ribonucleotides (hereinafter referred to as nucleotides) are composed of phosphate, ribose, and bases. In this document, a "modified nucleotide" refers to a nucleotide that, compared to an A, U, C, or G nucleotide, has a change in the group attached to a different C atom on the ribose, a change in the phosphate group, or a deletion of a base. In this document, "modified nucleotide" and "modified nucleotide" are synonymous.

[0124] As can be seen from the above, nucleotides are composed of phosphate, ribose, and a base. "Natural nucleotide" or "nucleotide with a natural base" refers to a nucleotide whose base is adenine (A), uracil (U), guanine (G), or cytosine (C). In some optional embodiments of the present invention, the modified nucleotide has one or more modifications.

[0125] It should be noted that the structural and chemical formula descriptions of the embodiments or implementations of this invention are intended to cover all alternatives, modifications, and equivalent technical solutions, all of which are within the scope of this invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice this invention. This invention is by no means limited to the methods and materials described herein. In the event that one or more of the linked documents, patents, and similar materials differ from or contradict this application (including but not limited to defined terminology, terminology application, described techniques, etc.), this invention shall prevail.

[0126] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments or implementations, but may also be provided in combination in a single embodiment or implementation. Conversely, various features of the invention, for brevity, have been described in a single embodiment or implementation, but may also be provided individually or in any suitable sub-combination.

[0127] Unless otherwise indicated, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and unless otherwise indicated, all patent publications cited in the entirety of this disclosure are incorporated herein by reference.

[0128] The compounds of this invention may contain asymmetric or chiral centers, and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of this invention, including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof such as racemic mixtures, are also included within the scope of this invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to the chiral centers (or multiple chiral centers) in the molecule. The prefixes d and l or (+) and (-) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers can also be called enantiomers, and mixtures of such isomers are usually referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or method.

[0129] Depending on the choice of raw materials and methods, the compounds of this invention can exist as one or a mixture of possible isomers, for example, as pure optical isomers, or as mixtures of isomers, such as as racemic and non-corresponding isomer mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral formulations, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may be cis or trans (cis- or trans-) configuration.

[0130] The compounds of this invention may contain asymmetric or chiral centers, and thus exist in different stereoisomer forms. It is contemplated that all stereoisomer forms of the compounds of this invention, including but not limited to diastereomers, enantiomers, atropisomers, and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of this invention.

[0131] Unless otherwise stated, the structures described in this invention also represent all isomers including this structure (e.g., enantiomers, diastereotropic atropisomers, and geometric (or conformational) forms; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers). Therefore, individual stereochemical isomers of the compounds of this invention, as well as mixtures of enantiomers, diastereomeric mixtures, and mixtures of geometric isomers (or conformational isomers), are all within the scope of this invention.

[0132] Any asymmetric atom (e.g., carbon) in the compounds of this invention can exist in a racemic or enantiomerically enriched form, such as (R)-, (S)-, or (R,S)- configuration. In some embodiments, each asymmetric atom has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% enantiomeric excess in the (R)- or (S)- configuration. If possible, substituents on atoms having unsaturated double bonds can be present in cis-(Z)- or trans-(E)- form.

[0133] Therefore, as described in this invention, the compounds of this invention can exist in the form of one of the possible isomers, rotational isomers, tautomers, tautomers, or mixtures thereof, for example, in the form of essentially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0134] Any mixture of isomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, and racemates based on the physicochemical differences of the components, for example by chromatography and / or stepwise crystallization.

[0135] Racemic derivatives of any resulting end product or intermediate can be separated into optical enantiomers using known methods familiar to those skilled in the art, such as by separating salts of their diastereomers. Racemic products can also be separated by chiral chromatography, such as high-performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared via asymmetric synthesis.

[0136] In this document, the terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerization is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. Unless otherwise indicated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0137] In this document, the term "pharmaceutically acceptable salt" refers to both organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art.

[0138] The "group description of this invention" "or" "" is used to describe the position where a group is substituted or the connection site with other groups.

[0139] In the chemical structures of the ligands or compounds described in this disclosure, the "-" sign indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the "-" sign can be "...". "

[0140] , ", or both contain " "and" "Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as: tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers."

[0141] In this document, the terms “optionally substituted,” “optionally substituted,” and “substituted or unsubstituted” are used interchangeably. Generally, the term “optionally,” whether or not it precedes the term “substituted,” indicates that one or more hydrogen atoms in the given structure are substituted by a specific substituent. Unless otherwise indicated, an optional substituent group may be substituted at each substituted position of the group.

[0142] In this document, the term "one or more" (e.g., in the definition of substituents in compounds of the general formula of the present invention) means "one, two, three, four or five, especially one, two, three or four, more especially one, two or three, and even more especially one or two".

[0143] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive terms “each…independently is”, “…each independently is”, and “…independently is” used in this invention are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0144] In this document, "pharmaceutical composition" can refer to a drug used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a drug in unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, the composition is prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0145] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0146] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this invention, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this invention.

[0147] In this document, the term "treatment" refers to the administration of a drug or siRNA to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a disease in individuals who are susceptible but have not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or siRNA to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing the siRNA or siRNA conjugate described herein to an individual in need.

[0148] Detailed description of siRNA, siRNA conjugates, compositions and uses in this application

[0149] This invention proposes siRNA, siRNA conjugates, cell and pharmaceutical compositions, and their uses, methods for expression of complement factor B, and methods for prevention and / or treatment of complement factor B-mediated diseases, which will be described in detail below.

[0150] siRNA

[0151] In a first aspect, this application provides an siRNA. According to embodiments of this application, the siRNA comprises a sense strand and an antisense strand, the antisense strand having a double-stranded region with at least partial base pairing with the sense strand; wherein the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1–SEQ ID NO:70 and SEQ ID NO:141–SEQ ID NO:144, or at least 15 consecutive nucleotides thereof; the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71–SEQ ID NO:140 and SEQ ID NO:145–SEQ ID NO:148, or at least 15 consecutive nucleotides thereof; each nucleotide in the sense strand and / or antisense strand is independently a modified or unmodified nucleotide. The siRNA according to embodiments of the present invention can inhibit the expression of CFB, thereby effectively preventing and / or treating complement factor B-mediated diseases.

[0152] This invention proposes an siRNA. According to embodiments of the invention, the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand has a double-stranded region with at least partial base pairing with the sense strand; wherein the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 10 consecutive nucleotides thereof; the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 10 consecutive nucleotides thereof; each nucleotide in the sense strand and / or antisense strand is independently a modified or unmodified nucleotide. Through experimental design, the inventors obtained the above-mentioned suitable small interfering RNA (siRNA), which forms an RNA-induced silencing complex (RISC) that pairs complementaryly with the mRNA sequence of the target gene (CFB gene), causing the mRNA encoding CFB to degrade and inhibiting the expression of CFB. This can effectively prevent and / or treat diseases mediated by complement factor B.

[0153] In this document, the "it" in the terms "or at least 15 consecutive nucleotides thereof" and "or at least 10 consecutive nucleotides thereof" refers to the aforementioned nucleotide sequence. For example, "the positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 15 consecutive nucleotides thereof" means that the positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 15 consecutive nucleotides in such a nucleotide sequence. For example, "the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 10 consecutive nucleotides thereof" means that the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 10 consecutive nucleotides in a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148.

[0154] In this article, the terms "different from no more than 5 nucleotides" or "having a difference of no more than 5 nucleotides" refer to differences of 0, 1, 2, 3, 4, or 5 nucleotides compared to the target nucleotide sequence (mRNA encoding the CFB gene). These differences include, but are not limited to, nucleotide deletions, nucleotide insertions (which may be inserted at the 3' end, 5' end, or between any two nucleotides in the nucleotide sequence), and nucleotide substitutions. For example, in the statement "The sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or differs from it by no more than 5 nucleotides", "it" refers to one of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144; and in the statement "The antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or differs from it by no more than 5 nucleotides", "it" refers to one of the nucleotide sequences shown in SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148.

[0155] It should be noted that, in this invention, the nucleotide sequences shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, and / or the nucleotide sequences shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148 are all located in the double-stranded region of the siRNA.

[0156] In some alternative embodiments of the invention, the antisense strand and the sense strand have a double-stranded region with at least 85%, or at least 90%, or at least 95% base pairing.

[0157] According to an embodiment of the present invention, the siRNA includes any one of the sense and antisense strands in Table A, or has a difference of no more than 5 nucleotides from the sense and / or antisense strands, preferably having a difference of no more than 4, no more than 3, no more than 2 nucleotides, or no more than 1 nucleotide.

[0158] Table A

[0159]

[0160]

[0161] According to embodiments of the present invention, the nucleotide modified with the 5'-phosphate mimic is selected from EVpu, EVpa, cPrpu, and cPrpa. The structures of EVpu, EVpa, cPrpu, and cPrpa are shown below:

[0162]

[0163] In some alternative embodiments of the present invention, the modification includes at least one of 2'-methoxy modification, 2'-fluorination modification and 5'-thiophosphate modification.

[0164] It should be noted that when a modified nucleotide is selected from nucleotides modified with 2'-alkyl and nucleotides modified with 5'-thiophosphate, it means that the modified nucleotide has both 2'-alkyl and 5'-thiophosphate modifications.

[0165] In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 3' end of the sense strand and / or antisense strand. In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 5' end of the sense strand and / or antisense strand. In some alternative embodiments of the invention, one or more inverted abasic residues are inserted between the ligand and the nucleotide sequence of the sense strand and / or antisense strand. In some alternative embodiments of the invention, the inverted abasic residues may be linked via phosphate esters, thiophosphate esters, or other nucleoside bonds.

[0166] In this paper, the structural formulas of "reverse debase modification" and "reverse debase nucleotide" are shown below:

[0167] Here, 'a' is the 5' end of the chain pointing toward the justice chain or the antisense chain, and 'b' is the 3' end of the chain pointing toward the justice chain or the antisense chain.

[0168] In this article, "phosphoramidite" refers to the starting material in oligonucleotide solid-phase synthesis by linking modified or unmodified nucleotides, linkers, or conjugation groups to oligonucleotides to form phosphate esters or thiophosphate esters.

[0169] In some alternative embodiments of the invention, the modification includes deoxynucleotides.

[0170] In this article, "deoxynucleotide" refers to the nucleotide after the hydroxyl group in the pentose of the nucleotide is deoxygenated, and the deoxygenation position can be 2'-OH or 3'-OH.

[0171] In some alternative embodiments of the present invention, the deoxynucleotide includes 3'-deoxy modified nucleotides and 2'-deoxy modified nucleotides.

[0172] In this paper, "2'-deoxygenation modification" refers to the deoxygenation of the hydroxyl group (2'-OH) in the pentose of a nucleotide to hydrogen (2'-H), and "3'-deoxygenation modification" refers to the deoxygenation of the hydroxyl group (3'-OH) in the pentose of a nucleotide to hydrogen (3'-H).

[0173] In this paper, "2'-X modification" refers to the substitution of the hydroxyl group (2'-OH) in the pentose of a nucleotide by X (2'-X). For example, "2'-fluorination modification" means that the hydroxyl group (2'-OH) in the pentose of a nucleotide is replaced by fluorine (2'-F); "2'-amino modification" means that the hydroxyl group (2'-OH) in the pentose of a nucleotide is replaced by an amino group (2'-NH2); "2'-O-allyl modification" means that the hydroxyl group (2'-OH) in the pentose of a nucleotide is replaced by an allyloxy group (2'-OCH2CH=CH2); "2'-alkyl modification" means that the hydroxyl group (2'-OH) in the pentose of a nucleotide is replaced by an alkyl group (2'-alkyl); and "2'-O-alkyl modification" means that the hydroxyl group (2'-OH) in the pentose of a pentose of a nucleotide is replaced by an alkoxy group (2'-alkoxy).

[0174] In some alternative embodiments of the invention, the modification includes locked nucleotides.

[0175] In this article, "locked nucleotide" refers to a nucleotide obtained by modifying the 2' and 4' carbons of the pentose sugar of a nucleotide by linking them together.

[0176] In this paper, "5'-X modification" refers to the substitution of the phosphate group (5'-PO(OH)2) in the pentose sugar of a nucleotide by X (5'-X). For example, "5'-aminophosphate modification" means that the phosphate group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by an aminophosphate group; "5'-thiophosphate modification" means that the phosphate group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a thiophosphate group; "5'-methylphosphonate modification" means that the phosphate group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a methylphosphate group; and "5'-phosphate mimicry modification" means that the phosphate group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a phosphate mimicry.

[0177] In some alternative embodiments of the present invention, the 2'-C 1～25 Alkyl-modified nucleotides include 2'-methoxy-modified nucleotides and 2'-methoxyethyl-modified nucleotides.

[0178] In this paper, "2'-methoxy modification" refers to the substitution of the hydroxyl group (2'-OH) in the pentose of the nucleotide by the methoxy group (2'-OCH3).

[0179] In this paper, "2'-methoxyethyl modification" refers to the substitution of the hydroxyl group (2'-OH) in the pentose of the nucleotide by methoxyethyl (2'-OCH2CH2OCH3).

[0180] In some alternative embodiments of the present invention, the modified nucleotide is a nucleotide modified with 5'-methylcytosine.

[0181] In this paper, "5'-methylated cytosine modification" refers to methylation at the 5th carbon atom of cytosine.

[0182] In some alternative embodiments of the present invention, the modified nucleotide is a conformation-restricted nucleotide.

[0183] In this document, "conformationally restricted nucleotides" (CRNs) refer to nucleotide analogs, specifically nucleotide analogs with linkers attached to the C2' and C4' carbons or the C3' and C5' carbons of the ribose. These linkers position oxygen in an optimal position for stability and affinity, thereby reducing ribose ring wrinkling. CRNs lock the ribose ring in a stable conformation and increase hybridization affinity with mRNA. Methods for preparing certain CRNs include, but are not limited to, US Patent Publication No. US2013 / 0190383 and PCT Publication No. WO 2013 / 036868. It should be noted that the entire contents of US2013 / 0190383 and WO 2013 / 036868 are incorporated herein by reference.

[0184] This application proposes an siRNA. According to embodiments of the present invention, the siRNA comprises the sense and antisense strands of any group in Table B, or differs from the sense and / or antisense strands by no more than 5 nucleotides, preferably by no more than 4, 3, 2, 1, or 0 nucleotides.

[0185] Table B

[0186]

[0187]

[0188]

[0189] This application proposes an siRNA. According to embodiments of the present invention, the siRNA comprises the sense and antisense strands of any group in Table C, or differs from the sense and / or antisense strands by no more than 5 nucleotides, preferably by no more than 4, 3, 2, 1, or 0 nucleotides.

[0190] Table C

[0191]

[0192]

[0193]

[0194] In Tables B and C of this paper, and Table D below, the lowercase letters c, g, u, a, and t all represent nucleotides modified with 2'-methoxy groups (i.e., c, g, u, a, and t respectively indicate that the ribosyl 2'-OH of the nucleotide represented by the corresponding uppercase letter is replaced by a methoxy group); f indicates that the nucleotide represented by the letter preceding f is a nucleotide modified with 2'-fluoride (i.e., the 2'-OH of the nucleotide represented by the letter preceding f is replaced by a fluorine atom); s indicates that the two adjacent nucleotide residues of s are thiophosphate esters (i.e., the 5'-phosphate ester group of the nucleotide represented by the letter preceding s is replaced by a 5'-thiophosphate ester group).

[0195] In this document, the siRNAs in Tables B, C, and D below may have the same nucleotide sequence as the corresponding natural sequence in Table A or B, or their positive and / or antisense sequences may have no more than 5 (preferably no more than 1 or 2, more preferably 1) nucleotide differences from the nucleotide sequence in Table A or B. The specific types are not limited.

[0196] Nucleic acid conjugates

[0197] In a second aspect of this application, a nucleic acid conjugate is proposed. According to embodiments of this application, the nucleic acid conjugate comprises: the siRNA described in the first aspect, and a ligand conjugated to the siRNA. The nucleic acid conjugate of the present invention can facilitate the targeting and delivery of nucleic acid drugs, enabling siRNA to connect to desired cells and / or tissues in vivo to target and silence complement factor B inhibitors in target cells and / or tissues, thereby effectively preventing and / or treating complement factor B-mediated diseases. As used herein, the terms "nucleic acid conjugate," "siRNA conjugate," and "conjugate" are used interchangeably.

[0198] In an alternative embodiment of the invention, the ligand is directly or indirectly conjugated to the oligonucleotide (such as siRNA) via a linker.

[0199] There are no particular limitations on the ligand structure; any ligand capable of delivering siRNA into cells or tissues can be used, with a preference for ligands that can target liver cells and / or tissues.

[0200] There are no particular restrictions on the way the ligand is connected to the siRNA. The ligand can be directly connected to the siRNA without using a separate adapter / connector group, or it can be indirectly connected to the siRNA through an adapter.

[0201] There are no particular restrictions on the way the ligand is linked to the sense and / or antisense strands of the siRNA. Preferably, the ligand is linked to the siRNA via a thiophosphate bond or a phosphate bond. Preferably, the ligand is linked to the 5' or 3' end of the sense strand. More preferably, the ligand is linked to the 5' or 3' end of the antisense strand.

[0202] For further details regarding the oligonucleotides, ligands, and the ways in which they are linked in nucleic acid conjugates, please refer to the fourth aspect of the invention description in this specification.

[0203] According to embodiments of the present invention, the ligand may also have the structure described above: P1, the preparation method of which is described in WO2023 / 070082A2; P2; P3 (i.e., L96), the preparation method of which is described in WO2014 / 025805A1;

[0204] For P4, P5, and P6, please refer to WO2015 / 168514A1 for their preparation methods.

[0205] The nucleic acid conjugates of the present invention, by adding ligands, can further enhance the degradation of mRNA encoding target proteins, and can effectively prevent and / or treat related diseases.

[0206] According to embodiments of the present invention, the ligand may also have the structures shown as L1, L2, L3, L4, L5, and L6 as described above.

[0207] Therefore, the use of the above-mentioned ligands can promote the targeting and delivery of oligonucleotides, enabling the oligonucleotides to be linked to desired cells and / or tissues in the body, so as to target and silence target genes in target cells and / or tissues, thereby effectively preventing and / or treating related diseases.

[0208] According to an embodiment of the present invention, the nucleic acid conjugate has the following structure:

[0209]

[0210]

[0211]

[0212] in, This indicates the siRNA described in this article; X represents O or S.

[0213] According to an embodiment of the present invention, the 5'-terminus of the siRNA positive strand has an NH2-alkyl group (such as an NH2-C6 alkylene group), which comprises a linker portion structure in the following ligand structures (such as -NH-(B 10 Alkylene). In some embodiments, the siRNA having an NH2-alkyl group at its terminal amino group can then bind to a ligand, for example, that shown in formula (II) below, to form a nucleic acid conjugate. In some embodiments, the 5'-terminus of the siRNA disclosed herein has one or more alkynyl groups, which can then react with groups of ligands, for example, those described in this invention, to form a conjugate.

[0214] In this document, siRNA comprises a sense strand and an antisense strand, and a thiophosphate bond or phosphate ester for attachment to the linker in the ligand structure described below. As previously known, the aforementioned NH2-alkyl group is a ligand, and this NH2-alkyl group is pre-attached to the sense strand of siRNA for the convenience of synthesizing the nucleic acid conjugates of this invention.

[0215] In some preferred embodiments of the present invention, the nucleic acid conjugates are shown in Table D.

[0216] Table D

[0217]

[0218] L1 has the following structure, which is obtained by conjugating compound I-1 with a nucleic acid sequence:

[0219]

[0220] NAG37 has the following structure:

[0221] For its preparation method, please refer to WO2023 / 070082A2.

[0222] Pharmaceutical Composition

[0223] This invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises either the aforementioned siRNA or the aforementioned nucleic acid conjugate. As previously stated, both the aforementioned siRNA and nucleic acid conjugate can degrade the mRNA encoding CFB, inhibiting the expression or activity of the CFB gene. Therefore, the pharmaceutical composition of this invention can effectively treat and prevent complement factor B-mediated diseases.

[0224] According to embodiments of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0225] Methods to inhibit complement factor B expression or activity

[0226] This invention provides a method for inhibiting the expression or activity of complement factor B. According to embodiments of the invention, the method includes introducing the aforementioned siRNA or the aforementioned nucleic acid conjugate into cells. As previously mentioned, both the aforementioned siRNA and the aforementioned nucleic acid conjugate can degrade the mRNA encoding CFB, inhibiting the expression or activity of the CFB gene. Therefore, the method of this invention can be used to inhibit the expression or activity of the CFB gene. For example, it can inhibit the expression or activity of the CFB gene in vivo; or it can inhibit the expression or activity of the CFB gene for non-disease treatment purposes, such as inhibiting the expression or activity of the CFB gene in vitro for subsequent research.

[0227] According to an embodiment of the present invention, the cells are hepatocytes and / or kidney cells.

[0228] use

[0229] This invention proposes the use of the aforementioned siRNA, the aforementioned nucleic acid conjugate, or the aforementioned pharmaceutical composition, said use including at least one of the following: inhibiting the expression or activity of the complement factor B gene; preparing a drug for inhibiting the expression or activity of the complement factor B gene; preparing a drug for preventing and / or treating complement factor B-mediated diseases. The method of this invention can be used to inhibit the expression or activity of the CFB gene; to prevent and / or treat complement factor B-mediated diseases. For example, the expression or activity of the CFB gene can be inhibited in vivo in animals; or the expression of the CFB gene can be inhibited for non-disease treatment purposes, such as inhibiting the expression or activity of the CFB gene in vitro for subsequent studies.

[0230] According to embodiments of the present invention, the complement factor B-mediated related diseases include nephropathy, systemic lupus erythematosus (SLE), such as lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulonephropathy (e.g., C3 glomerulonephropathy), age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0231] According to an embodiment of the present invention, the diseases mediated by complement factor B are selected from glomerular diseases and systemic lupus erythematosus.

[0232] According to embodiments of the present invention, the diseases mediated by complement factor B are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, and polycystic kidney disease.

[0233] Methods for preventing and / or treating complement factor B-mediated diseases

[0234] This invention provides a method for preventing and / or treating complement factor B-mediated diseases. According to embodiments of the invention, the method includes administering a pharmaceutically acceptable dose of the aforementioned siRNA, the aforementioned nucleic acid conjugate, or the aforementioned pharmaceutical composition to a subject. According to embodiments of the invention, this method is an effective way to prevent and / or treat complement factor B-mediated diseases.

[0235] The effective amount of the siRNA, nucleic acid conjugate, or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0236] According to embodiments of the present invention, the complement factor B-mediated related diseases include nephropathy, systemic lupus erythematosus (SLE), such as lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulonephropathy (e.g., C3 glomerulonephropathy), age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0237] According to an embodiment of the present invention, the diseases mediated by complement factor B are selected from glomerular diseases and systemic lupus erythematosus.

[0238] According to embodiments of the present invention, the diseases mediated by complement factor B are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, and polycystic kidney disease.

[0239] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0240] Preparation Example 1: Preparation of ligands I-1 to I-3

[0241] 1. Preparation of ligand I-1

[0242] The synthesis route is shown below:

[0243]

[0244] Step 1: Synthesis of ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyrate (01B)

[0245]

[0246] N-tert-butoxycarbonyl-1,3-propanediamine (30.00 g, 172.2 mmol), acetonitrile (250 mL), and potassium carbonate (47.59 g, 344.3 mmol) were added sequentially to a 500 mL single-necked flask. The mixture was cooled to 0 °C, and ethyl 4-bromobutyrate (26.87 g, 137.8 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature and reacted for 16 h. The reaction solution was filtered, and di-tert-butyl dicarbonate (56.36 g, 258.2 mmol) was added to the filtrate. The mixture was reacted at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-7:3) to give ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyrate (01B) (45.00 g, yield 84.08%).

[0247] LC-MS, M / Z (ESI): 289.4 [M-99] +

[0248] Step 2: Synthesis of 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyric acid (01C)

[0249]

[0250] Ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyrate (45.00 g, 115.8 mmol), methanol (200 mL), and lithium hydroxide (5.55 g, 231.8 mmol) aqueous solution (50 mL) were added sequentially to a 500 mL single-necked flask. The mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure, water (50 mL) was added, the pH was adjusted to 6 with 1 N hydrochloric acid, and ethyl acetate (150 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyric acid (01C) (24.00 g, yield 57.49%).

[0251] 1 H NMR(400MHz, DMSO-d6)δ6.73(s,1H),3.08(q,J=8.6,8.1Hz,4H),2.85(q,J=6.6Hz,2H), 2.12(t,J=7.3Hz,2H),1.67–1.60(m,2H),1.57–1.49(m,2H),1.36(s,9H),1.35(s,9H).

[0252] LC-MS, M / Z (ESI): 383.4 [M+Na] +

[0253] Step 3: Synthesis of tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (01E)

[0254]

[0255] N-tert-butyloxycarbonyl-1,3-propanediamine (30.00 g, 172.2 mmol), acetonitrile (250 mL), and potassium carbonate (47.59 g, 344.3 mmol) were added sequentially to a 500 mL single-necked flask. The mixture was cooled to 0 °C, and 5-bromo-1-pentene (20.53 g, 137.8 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature and stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-20:1) to give tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (01E) (16.00 g, yield 38.34%).

[0256] 1H NMR(400MHz,DMSO-d6)δ6.78(t,J=5.0Hz,1H),5.84–5.74(m,1H),5.00–4.95(m,1H),4.93–4.89(m,1H),3.14 (s,1H),2.92(q,J=6.7Hz,2H),2.43(t,J=7.0Hz,4H),2.01(q,J=7.3Hz,2H),1.49–1.40(m,4H),1.34(s,9H). LC-MS,M / Z(ESI):243.3[M+H] +

[0257] Step 4: Synthesis of tert-butyl(3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)(pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (01F)

[0258]

[0259] To a 500 mL single-necked flask, tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (16.00 g, 66.02 mmol), dichloromethane (200 mL), 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butyric acid (23.80 g, 66.03 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18.98 g, 99.01 mmol), monohydroxybenzotriazole (8.92 g, 66.01 mmol), and triethylamine (20.04 g, 198.0 mmol) were added sequentially. The mixture was reacted at room temperature for 16 h under nitrogen protection. The reaction solution was washed sequentially with 0.5N hydrochloric acid (100 mL) and saturated sodium bicarbonate aqueous solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give tert-butyl(3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)(pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (01F) (19.00 g, yield 49.21%).

[0260] 1H NMR(400MHz,DMSO-d6)δ6.91–6.60(m,2H),5.84–5.72(m,1H),5.06–4.89(m,2H),3.22–3.14(m,4H),3.12–3.0 6(m,4H),2.90–2.83(m,4H),2.23–2.10(m,2H),1.98–1.92(m,2H),1.47–1.66(m,8H),1.35(d,J=3.8Hz,27H).

[0261] LC-MS, M / Z (ESI): 607.6 [M+Na] +

[0262] Step 5: Synthesis of N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N-(pent-4-en-1-yl)butyramide (trifluoroacetate) (01G)

[0263]

[0264] 5.00 g (8.55 mmol) of tert-butyl(3-((tert-butoxycarbonyl)amino)propyl)(4-((3-(((tert-butoxycarbonyl)amino)propyl)(pent-4-en-1-yl)amino)-4-oxobutyl)carbamate, 200 mL of dichloromethane, and 13.09 mL (171.1 mmol) of trifluoroacetic acid were added to a 250 mL single-necked flask and stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure to give N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N-(pent-4-en-1-yl)butyramide (trifluoroacetate) (0.1 g) (5.34 g, 99.7% yield).

[0265] LC-MS, M / Z (ESI): 285.46 [M+H] +

[0266] Step 6: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazaeicosaecan-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (01I)

[0267]

[0268] N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N-(pent-4-en-1-yl)butyramide (trifluoroacetate) (01G) (3.30 g, 5.27 mmol), dichloromethane (100 mL), 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valerate (8.26 g, 18.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.05 g, 26.3 mmol), monohydroxybenzotriazole (2.14 g, 15.8 mmol), and triethylamine (5.34 g, 52.8 mmol) were added sequentially to a 250 mL single-necked flask. The mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was washed sequentially with 0.5N hydrochloric acid (100 mL) and saturated sodium bicarbonate aqueous solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to obtain (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetyl) Amino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazaeicosano-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, yield 84.5%)

[0269] 1 H NMR(600MHz,DMSO-d6)δ7.85–7.77(m,4H),7.71–7.69(m,1H),5.83–5.77(m,1H),5.19(d,J =3.3Hz,3H),5.05–4.91(m,5H),4.46(dd,J=8.4,4.1Hz,3H),4.02–3.99(m,9H),3.89–3.82( m,3H),3.70–3.68(m,3H),3.40–3.37(m,3H),3.25–3.16(m,8H),3.04–2.95(m,4H),2.30–2 .17(m,4H),2.08(s,9H),2.05–1.94(m,15H),1.87(s,9H),1.75(s,9H),1.61–1.43(m,20H).

[0270] LC-MS, M / Z (ESI): 787.26 [M+2H] 2+

[0271] Step 7: Synthesis of 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)pentanoylamino)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)butyrylamino)butyric acid (01J)

[0272]

[0273] Add (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15, 19-Tetraazaeicosano-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, 4.45 mmol), dichloromethane (30 mL), acetonitrile (30 mL), and water (48 mL) were stirred until homogeneous. Sodium periodate (4.76 g, 22.3 mmol) and ruthenium trichloride (18.5 mg, 89.0 μmol) were then added. The mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, and a saturated sodium bicarbonate aqueous solution was added to the filtrate to adjust the pH of the aqueous phase to 7–8. After separation, the aqueous phase was washed with dichloromethane (50 mL × 2), and the pH was adjusted to approximately 5–6 with 1 N hydrochloric acid. Extraction was then performed with dichloromethane (100 mL × 3). The obtained organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3 ... Acyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)pentanoyl)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)butyrylamino)butyric acid (01J) (3.50 g, yield 49.4%).

[0274] 1H NMR(600MHz,DMSO-d6)δ12.12(s,1H),7.84–7.81(m,4H),7.74–7.71(m,1H),5.19(d,J=3.2Hz, 3H),4.94(dd,J=11.2,3.3Hz,3H),4.47(d,J=8.4Hz,3H),4.00–3.99(m,9H),3.88–3.83(m,3H) ,3.71–3.66(m,3H),3.40–3.37(m,3H),3.22–3.17(m,8H),3.04–2.95(m,4H),2.30–2.13(m,6H ),2.08(s,9H),2.04–2.01(m,4H),1.98(s,9H),1.87(s,9H),1.75(s,9H),1.69–1.43(m,20H).

[0275] LC-MS, M / Z (ESI): 796.60 [M+2H] 2+

[0276] Step 8: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valeryl)-10-(4-(4-nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazaeicosaecan-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-1)

[0277]

[0278] Add the following ingredients sequentially to a 100 mL single-necked flask: 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)pentanoamide)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)pentanoamide)-N-(3-(5-((( 2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoylamino)propyl)butyrylamino)butyric acid (900 mg, 0.566 mmol), dichloromethane (20 mL), 4-nitrophenol (94.5 mg, 0.679 mmol), N,N'-dicyclohexylcarbodiimide (175.11 mg, 0.85 mmol), the mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to obtain (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-

[0279] (((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valeryl)-10-(4-(4-nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazaeicosano-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-1) (850 mg, yield 87.8%).

[0280] 1H NMR (400MHz, DMSO-d6) δ8.29(dd,J=9.2,2.7Hz,2H),7.82–7.67(m,4H),7.74–7.66(m,1H),7.44(dd,J=9.1,2. 1Hz, 2H), 5.19 (d, J = 3.3Hz, 3H), 4.94 (dd, J = 11.2, 3.4Hz, 3H), 4.46 (d, J = 8.4Hz, 3H), 4.00 (s, 9H), 3.85 (q, J = 9. 2Hz,3H),3.72–3.65(m,3H),3.41–3.37(m,3H),3.24–3.19(m,8H),3.06–2.92(m,4H),2.77–2.49(m,2H),2.36– 2.15(m,4H),2.07–2.08(m,9H),2.03–2.02(m,4H),1.97(s,9H),1.87(s,9H),1.75(s,9H),1.67–1.37(m,20H).

[0281] LC-MS, M / Z (ESI): 856.5 [M+2H] 2+

[0282] 2. Preparation of ligand I-2

[0283] The synthesis route is as follows:

[0284]

[0285] Step 1: Synthesis of methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionate (02B)

[0286]

[0287] Methyl 3-bromo-2-(bromomethyl)propionate (2.00 g, 7.69 mmol), N-tert-butoxycarbonyl-1,3-propanediamine (5.36 g, 30.8 mmol), and acetonitrile (30 mL) were added sequentially to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and dichloromethane (30 mL), di-tert-butyl dicarbonate (6.72 g, 30.8 mmol), and N,N-diisopropylethylamine (3.98 g, 30.8 mmol) were added. The mixture was stirred at room temperature for 16 h. 50 mL of water was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-3:2) to give methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionate (02B) (3.50 g, yield 70.7%).

[0288] Step 2: Synthesis of 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionic acid (O2C)

[0289]

[0290] Methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionate (3.50 g, 5.44 mmol), methanol (30 mL), and lithium hydroxide (389 mg, 16.2 mmol) aqueous solution (10 mL) were added sequentially to a 100 mL single-necked flask and stirred at room temperature for 16 h. The organic solvent was removed by vacuum concentration, 50 mL of water was added, the pH was adjusted to 6 using 1 N hydrochloric acid, and ethyl acetate (150 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionic acid (O2C) (3.00 g, yield 87.6%).

[0291] LC-MS, M / Z (ESI): 533.5 [M-99] + .

[0292] Step 3: Synthesis of 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)benzyl propionate (02D)

[0293]

[0294] To a 500 mL single-necked flask, 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionic acid (3.00 g, 4.74 mmol), N,N-dimethylformamide (30 mL), potassium carbonate (1.97 g, 14.3 mmol), and benzyl bromide (0.93 g, 5.4 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 h. The reaction solution was filtered, and 30 mL of water was added to the filtrate. The solution was extracted with ethyl acetate (60 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Column chromatography purification (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) yielded 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionate (02D) (3.00 g, yield 87.5%).

[0295] 1 H NMR(400MHz,DMSO-d6)δ7.39–7.31(m,5H),6.79–6.68(m,2H),5.07(s,2H),3.28–3.25(m,4H),3 .15–3.05(m,3H),2.98–2.92(m,2H),2.90–2.82(m,4H),1.54–1.50(m,4H),1.37–1.35(m,36H).

[0296] Step 4: Synthesis of tetra(trifluoroacetic acid) salt of 3-((3-aminopropyl)amino)-2-(((3-aminopropyl)amino)methyl)propionate (02E)

[0297]

[0298] Benzyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propionate (4.50 g, 6.22 mmol), dichloromethane (60 mL), and trifluoroacetic acid (21.3 g, 187 mmol) were added sequentially to a 250 mL single-necked flask, and the mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure to obtain tetra(trifluoroacetic acid) salt (02E) of benzyl 3-((3-aminopropyl)amino)methyl)propionate (4.85 g).

[0299] LC-MS, M / Z (ESI): 323.04 [M+H] + .

[0300] Step 5: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatriane-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (02F)

[0301]

[0302] Add 4.85 g (6.23 mmol) tetra(trifluoroacetic acid) salt of 3-((3-aminopropyl)amino)methyl)propionate, 100 mL of dichloromethane, 12.55 g (28.05 mmol) of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valerate, 7.17 g (37.4 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 3.37 g (24.9 mmol) of 1-hydroxybenzotriazole, and 12.1 g (93.5 mmol) of N,N-diisopropylethylamine to a 250 mL single-necked flask in sequence. Stir at room temperature for 16 h under nitrogen protection. The reaction solution was washed sequentially with 100 mL of 0.5 N hydrochloric acid aqueous solution and 100 mL of saturated sodium bicarbonate solution. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) yielded (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl) 12-((benzyloxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatriane-10,14-diyl)bis(5-oxopentane-5,1-diyl)bis(oxy)bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (02F) (9.00 g, yield 70.8%)

[0303] 1 H NMR (400MHz, DMSO-d6) δ7.82–7.64(m,6H),7.37–7.25(m,5H),5.17(d,J=3.3Hz,4H),4.98(d ,J=10.7Hz,2H),4.93(dd,J=11.2,3.3Hz,4H),4.45(d,J=8.4Hz,4H),3.98(s,12H),3.84(q,J =9.2Hz,4H),3.69-3.64(m,4H),3.57–3.36(m,8H),3.10–2.90(m,9H),2.20–2.11(m,4H),2.0 6(s,12H),2.03–1.98(m,4H),1.95(s,12H),1.85(s,12H),1.73(s,12H),1.58–1.37(m,20H).

[0304] LC-MS, M / Z (ESI): 1020.9 [M+2H] 2+ .

[0305] Step 6: 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-2-(( Synthesis of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)methyl)propionic acid (02G)

[0306]

[0307] Add (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19-dioxo-6,10,14 1,8-Tetraazatriane-10,14-diyl)bis(5-oxopentane-5,1-diyl)bis(oxy)bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (0.18 g, 88 μmol), methanol (5 mL), ethyl acetate (5 mL), and 10% palladium on carbon (18 mg) were mixed and stirred at room temperature for 16 h under a hydrogen atmosphere (1 atm). The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrates were combined. Concentrating the filtrate under reduced pressure yielded 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-2-((5-(( (2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)methyl)propionic acid (02G) (0.12g, yield 70%).

[0308] 1H NMR (400MHz, DMSO-d6) δ7.83–7.81(m,5H),7.72–7.68(m,1H),5.21(d,J=3.3Hz,4H),4.96(dd,J =11.2,3.3Hz,4H),4.49(dd,J=8.4,3.3Hz,4H),4.04–3.99(m,12H),3.87(q,J=10.5,9.8Hz,4H), 3.75–3.66(m,4H),3.59–3.39(m,8H),3.27–3.13(m,3H),3.08–2.90(m,6H),2.34–2.15(m,4H), 2.10(s,12H),2.07–2.02(m,4H),1.99(s,12H),1.88(s,12H),1.77(s,12H),1.61–1.43(m,20H).

[0309] LC-MS, M / Z (ESI): 975.9 [M+2H] 2+ .

[0310] Step 7: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((4-nitrophenoxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatriane-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-2)

[0311]

[0312] Add the following ingredients sequentially to a 100 mL single-necked flask: 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-2-((5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxy) A mixture of 200 mg (0.103 mmol) of tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-((((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)methyl)propionic acid, 21.4 mg (0.154 mmol) dichloromethane (10 mL), 4-nitrophenol (21.4 mg (0.154 mmol) and N,N'-dicyclohexylcarbodiimide (31.7 mg (0.154 mmol)) was stirred at room temperature for 16 h under nitrogen protection. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product. Column chromatography purification (dichloromethane / methanol (V / V) = 1:0-8:2) yielded (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy) -12-((4-nitrophenoxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatriane-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-2) (120 mg, yield 56.5%).

[0313] 1H NMR (400MHz, DMSO-d6) δ8.33–8.29(m,2H),7.79–7.69(m,6H),7.43–7.31(m,2H),5.18(d,J=3.3Hz,4 H),4.94(dd,J=11.2,3.3Hz,4H),4.45(d,J=8.4Hz,4H),3.99(s,12H),3.89–3.80(m,4H),3.69–3.61 (m,5H),3.49–3.31(m,7H),3.29–3.13(m,5H),3.09–2.94(m,4H),2.35–2.17(m,4H),2.07(d,J=2.7H z,12H),2.04–2.01(m,4H),1.96(d,J=2.0Hz,12H),1.86(s,12H),1.74(s,12H),1.65–1.39(m,20H).

[0314] 3. Preparation of ligand I-3

[0315] The synthesis route is as follows:

[0316]

[0317] Step 1: (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valeryl)-10-(4-((2S,4R)-2-(( Synthesis of bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazaeicosano-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetic acid ester (03B)

[0318]

[0319] Add 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-2) H-pyran-2-yl)oxy)pentamido)propyl)butamido)butyric acid (O1J, 1.0 g, 0.63 mmol), dichloromethane (20 mL), (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidine-3-ol (O3A, 0.29 g, 0.69 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (376 mg, 0.991 mmol), 1-hydroxybenzotriazole (255 mg, 1.89 mmol), and N,N-diisopropylethylamine (244 mg, 1.89 mmol) were stirred at room temperature for 16 h under nitrogen protection. The reaction solution was washed with saturated sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. Column chromatography purification (dichloromethane (2% triethylamine) / methanol (V / V) = 1:0-9:1) yielded (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-10-(4-(( 2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazaeicosaecan-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetic acid ester (03B) (1.0 g, 80% yield).

[0320] 1H NMR (400MHz, DMSO-d6) δ7.86–7.65(m,5H),7.32–7.29(m,4H),7.21–7.17(m,5H),6.90–6.86(m,4 H),5.21(s,3H),5.03–4.89(m,4H),4.48(d,J=8.3Hz,3H),4.36(m,1H),4.16(s,1H),4.02(s,9H) ,3.87(q,J=9.8Hz,3H),3.73–3.68(m,9H),3.64–3.45(m,7H),3.19–2.93(m,12H),2.35–2.14(m, 6H),2.09(s,9H),2.06–2.02(m,4H),1.99(s,9H),1.88(s,9H),1.76(s,9H),1.74–1.25(m,22H).

[0321] Step 2: 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl) Synthesis of 1-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)butamido)butyryl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-yl)oxy)-4-oxobutyric acid (I-3)

[0322]

[0323] Add (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valeryl)-10-(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-4- (O-butyl)-5,11,20-trioxo-6,10,15,19-tetraazaeicosano-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (800 mg, 0.402 mmol), dichloromethane (30 mL), succinic anhydride (321 mg, 3.21 mmol), and 4-dimethylaminopyridine (12.3 mg, 0.101 mmol) were stirred at room temperature for 16 h under nitrogen protection. The reaction solution was concentrated under reduced pressure, and the crude product was prepared by reverse reaction (column: Waters Xbridge Prep). C18 (150mm×40cm×10μm), mobile phase: A = water (0.1% ammonium bicarbonate), B = acetonitrile; gradient: 15%-45%), yielding 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)pentamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)butamido)butyryl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-yl)oxy)-4-oxobutyric acid (I-3) (630 mg, yield 75.0%).

[0324] 1H NMR(400MHz,DMSO-d6)δ7.90–7.79(m,4H),7.76–7.69(m,1H),7.31-7.27(m,4H),7.23–7.17(m,5H),6.89–6.87(m,4 H),5.37–5.28(m,1H),5.21(d,J=2.9Hz,3H),4.97(dd,J=11.2,3.0Hz,3H),4.49(d,J=8.4Hz,3H),4.21(s,1H),4.02( s,9H),3.91–3.84(m,3H),3.73–3.70(m,9H),3.56–3.39(m,7H),3.23–3.19(m,8H),3.04–2.97(m,4H),2.49–2.40(m ,4H),2.29–2.17(m,6H),2.09(s,9H),2.05–2.04(m,4H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.72–1.40(m,22H).

[0325] Example 1: Design and Synthesis of siRNA

[0326] The siRNA sequences were designed based on the mRNA encoding CFB as the target sequence, resulting in the siRNA sequences shown in Tables A, B, and C. The synthesis of siRNA sequences without conjugation groups (hereinafter referred to as siRNA) is shown below:

[0327] 1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramide solid-phase synthesis technology (Lingkun 48-channel synthesizer). This was achieved using a general-purpose controllable porous glass CPG system. Synthesized on CPG after ligand coupling. All 2'-modified RNA, DNA, phosphoramidite ligands, and auxiliary reagents were commercially available (Tangzhi Pharmaceutical). All phosphoramidite was dissolved in anhydrous acetonitrile and sieved with molecular sieves. The coupling time using 5-ethylthio-1H-tetrazole (ETT, Suzhou Kelama Biotechnology Co., Ltd.) as the activator was 200 seconds, and the coupling ligand time was 10 minutes. Phosphate esters were generated using a pyridine (Sinopharm) / aqueous solution containing 50 mM iodine (Suzhou Kelama Biotechnology Co., Ltd.) for 5 minutes. Thiophosphate ester bonds were generated using a pyridine (Sinopharm) solution containing 0.2 M hydroflavin (Shanghai Zhaowei Technology Development Co., Ltd.) for 3 minutes. The synthesis was completed after the final removal of the DMT group from all sequences.

[0328] 2) Cleavage and deprotection of oligonucleotides bound to CPG: After solid-phase synthesis, the dried solid support was treated with ammonia solution at 55°C for 16 hours. Some of the oligonucleotides were deprotected using a mixture of DMSO and triethylamine hydrogen fluoride (Beijing Bailingwei Technology Co., Ltd.) (5:1) at 25°C for 4 hours. The solution was evaporated and the solid residue was redissolved in water.

[0329] 3) Purification of single-stranded oligonucleotides: The crude product was purified by reversed-phase HPLC using a Waters XBridge C18 column and an Autotide 100 system (Insys). Buffer A was a 100 mM aqueous solution of triethylamine acetate, pH 7.5, containing 5% acetonitrile, and buffer B was 100% acetonitrile. UV traces were recorded at 260 nm, and appropriate fractions were collected.

[0330] 4) Annealing of single-stranded oligonucleotides to produce siRNA: The single-stranded oligonucleotides to be annealed were prepared to 200 μM using sterile RNase-free water (free of RNase). The annealing reaction system was set up as follows: 10 nmol of the 100 μL mixture was placed in a 95°C water bath for 5 minutes (≥100 nmol requires 20 minutes at high temperature). The mixture was then quickly placed in a 60°C water bath and allowed to cool naturally to 20–30°C. The annealed solution should not be stored at high temperatures. By combining equimolar amounts of the single-stranded oligonucleotide solutions to form complementary strands, the siRNAs shown in Tables A, B, and C were finally obtained. The molecular weight of the siRNAs was determined using liquid chromatography-mass spectrometry (LC-MS). Comparing the measured and theoretical molecular weights, the results showed that the measured values ​​were approximately equal to the theoretical values, confirming the availability of the siRNAs listed in the table.

[0331] Example 2: Design and synthesis of siRNA conjugates

[0332] 1.1 The synthetic steps of siRNA conjugates with conjugation groups attached to the 5' end of the siRNA positive strand (i.e., the siRNA conjugates in Table D), taking D1 in Table D as an example, are as follows:

[0333] 1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramide solid-phase synthesis technology. This was achieved using a universally controllable porous glass CPG... The synthesis was performed using a Linkon 48-channel synthesizer. All phosphoramidite monomers (from Tangzhi Pharmaceutical & Shanghai Zhaowei) and auxiliary reagents were commercially available. All phosphoramidites were dissolved in anhydrous acetonitrile (Suzhou Kelama) and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT) as an activator (Suzhou Kelama) was 8–12 minutes. Phosphate ester bonds were constructed using a 0.05 M iodine solution (dissolved in pyridine / water = 9:1, Suzhou Kelama); thiophosphate ester bonds were generated using a 0.2 M hydroflavin (Suzhou Kelama) solution in anhydrous acetonitrile / pyridine (v / v = 1 / 1), with a reaction time of 5 minutes. The synthesis was complete after the final removal of the DMT group from all sequences.

[0334] 2) Cleavage and deprotection of oligonucleotides bound to CPG: After the solid-phase synthesis was terminated, the protecting groups were removed by treating with an acetonitrile solution containing 20% ​​diethylamine (Sinopharm) for 10 minutes. The obtained CPG carrier was then subjected to ammonolysis with concentrated ammonia (Sinopharm) to remove the protecting groups on the carrier and bases. After filtration, a solution containing the product was obtained.

[0335] 3) Purification of single-stranded oligonucleotides: Oligomers were obtained by HPLC purification using NanoQ anion exchange. Buffer A was a 20 mM sodium hydroxide solution; and buffer B contained 20 mM sodium hydroxide solution and 3 M sodium chloride, from which the target product was separated. The obtained target product was then desalted by gel column chromatography (Cytiva).

[0336] 4) Coupling and purification of the conjugated group: The purified nucleic acid sequence with NH2-C6 at the end (prepared and purified using conventional methods in the art) was first dissolved in 100 mM PB buffer. The ligand from Preparation Example 1 (such as compound I-1) was dissolved in DMF or DMSO and then added to the nucleic acid solution. The reaction was carried out at room temperature for 4–16 h. Mass spectrometry was used to monitor the completeness of the reaction. Two volumes of AMA solution (25% ammonia: 40% methylamine aqueous solution = 1:1) were added, and the mixture was stirred at room temperature for 0.5 h, followed by quenching with water. The mixed solution was desalted to remove organic reagents and then purified to obtain the nucleic acid chain with the conjugated group at the end.

[0337] 5) The sense and antisense chains obtained by chemical synthesis were subjected to base complementation in a molar ratio of 1:1. The reaction conditions were 70℃ for 10 min, and then slowly restored to room temperature to finally obtain the product.

[0338] The nucleotide sequences of the sense and antisense strands of the siRNA conjugates obtained in this invention are shown in Table D.

[0339] The structures of EVpu and EVpa are shown below:

[0340]

[0341] The inhibitory activity of the siRNAs and conjugates obtained in Examples 1 and 2 will be verified below. The sequences of the positive controls used in the experiments are shown below:

[0342] SS:(NAG37)s(invAb)sgcugugguGfUfCfugaguacuuas(invAb),

[0343] AS:usAfsaguaCfucagAfcAfcUfacagsc.

[0344] Test Example 1: Inhibitory effect of siRNA and its conjugates on CFB mRNA expression in Hep3B cells

[0345] siRNA and its conjugates: A 20 μM stock solution was prepared using Nuclease-Free Water. Hep3B cells were provided by Chengdu WuXi AppTec Co., Ltd. Hep3B cells were cultured in EMEM medium (Gibco A58412DJ) containing 10% fetal bovine serum (FBS, ExCell Bio, FSP500), 1% glutamine (GlutaMAX, Gibco, 35050061), 1% NEAA (Gibco, 11140050), and 1% penicillin-streptomycin (HyClone, SV30010).

[0346] Main reagents and their sources: Lipofectamine TM RNAiMAX transfection reagent (INVITROGEN, -13778150), FastKing RT kit (with gDNase) / FastKing cDNA first strand synthesis kit (TIANGEN-KR116-03), RNA extraction kit (QIAGEN-74182), FastStart Universal Probe Master (Rox) (ROCHE-04914058001), and 96-well plates (Costar-3599). Primers and probes for the target gene CFB (Thermo, Assay ID-Hs00156060_m1) were purchased from Thermo Fisher Scientific Inc., and Taqman primers and probes for the internal control gene GAPDH were designed and provided by Shanghai WuXi AppTec Co., Ltd.

[0347] Seed Hep3B cells (2×10⁻⁶) 4 Cells were plated into 96-well cell culture plates, and siRNA was simultaneously transfected into the cells using Lipofectamine™ RNAiMAX. Nine concentration points were measured for siRNA (starting at 10 nM, 6-fold dilution), with three replicates. A Lipofectamine-containing solution was also added to each well. TMThe RNAiMAX compound-free control group was cultured at 37°C in a 5% CO2 incubator for 48 hours. A portion of the siRNA was tested at a concentration of 1 nM. 48 hours after transfection, the culture medium was removed and cells were collected for RNA extraction. Use according to the kit instructions. Total RNA was extracted using a 96-kit (QIAGEN-74182). cDNA was synthesized using the FastKing RT kit (with gDNase) / FastKing cDNA first-strand synthesis kit (TIANGEN-KR116-03) according to the manufacturer's instructions.

[0348] The target gene cDNA will be detected by qPCR, while GAPDH cDNA will be detected as an internal control in parallel. 8 μL of prepared PCR reaction solution and 2 μL of sample cDNA will be added to each of 384 wells. The qPCR program is as follows: preheat at 50°C for 2 minutes, heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0349] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0350] The calculation formula is as follows:

[0351] ΔCT = Average CT value of target gene - Average CT value of GAPDH gene;

[0352] ΔCt = ΔCT (sample group) - ΔCT (Lipofectamine) TM RNAiMAX control group);

[0353] Relative expression level of target gene = 2 - ΔΔCT;

[0354] Inhibition rate % = (1 – relative expression level of sample / Lipofectamine) TM (RNAiMAX control average expression level) × 100;

[0355] GraphPad Prism software was used for graph analysis. The test results are shown in the table below.

[0356] Table 1

[0357]

[0358] Table 2

[0359]

[0360] Table 3

[0361]

[0362]

[0363] The results showed that the siRNA and conjugates obtained in this invention had a significant silencing effect on the expression of the CFB gene in Hep3B cells.

[0364] Test Example 2: Inhibitory effect of siRNA conjugates on hCFB expression in Tg-hCFB mice

[0365] The in vivo potency of CFB siRNA conjugates was evaluated using a 6-8 week old male Tg-hCFB transgenic mouse model (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.).

[0366] The test sample was prepared with physiological saline (0.4 mg / mL) on the day of administration, stored at room temperature, and administered within 2 hours. Any remaining test sample was stored at -20°C. Animals were divided into a normal control group and a test drug group, with 5 mice in each group. Grouping was based on the hCFB protein level in the mouse serum (blood was collected on day 3). The test drug group received a single subcutaneous injection of the test drug, while the normal control group received an equal volume of physiological saline. The day of administration was recorded as Day 0. Starting one day before administration, mice were weighed weekly.

[0367] Blood samples of approximately 200 μL were collected from the inner canthus of each mouse on Day 1 (the day before drug administration) and on Day 7, Day 14, Day 21, Day 28, and Day 35 after drug administration. Before centrifugation, the whole blood samples were incubated at 37°C for 1 hour and then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected as the fresh serum sample, and hCFB protein was detected using ELISA. At the endpoint of the experiment, mouse livers were harvested, and hCFB mRNA expression in the liver tissue was detected using qPCR.

[0368] Experimental data are expressed as mean ± standard deviation (Mean ± SD), and data were plotted and analyzed using GraphPad Prism 8.3 software. Statistical analysis was performed using the t-test, with P < 0.05 indicating statistical significance. The residual expression rate was determined by dividing the hCFB level of each animal at a given time point by its pre-drug (Day-1) level. The normalized residual expression rate was obtained by dividing the residual expression rate of a single animal in the test drug group at a given time point by the average residual expression rate of all mice in the control group, and the mean value for each group was calculated.

[0369] The results showed that the siRNA and its conjugates obtained in this invention had a significant and durable silencing effect on hCFB expression in Tg-hCFB mice. The test results for different batches of samples are shown in Tables 4-1 and 4-2.

[0370] Table 4-1 Table 4-2

[0371]

[0372] The “-” in the table indicates that it has not been tested yet.

[0373] Test Example 3: Inhibitory effect of free uptake of siRNA conjugates by cynomolgus monkey primary hepatocytes on CFB mRNA expression

[0374] The prepared siRNA conjugates were prepared into stock solutions of the appropriate concentration using Nuclease-Free Water.

[0375] The main reagents and consumables used in this experiment included: AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-P8), 2×Color SYBR Green qPCR Master Mix (ROX2) (EZB-A0012-R2-L), EZ-Press 96RNA Purification Kit (EZB-EZ4001-L), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323), and a 96-well cell culture plate (Cellpro-803096). Primers and probes for the target gene CFB and the internal reference gene β-actin were synthesized by Shanghai Sangon Biotech Co., Ltd. Primary hepatocytes of cynomolgus monkeys (PCH, catalog number: CCH100CY-V10088) were provided by Chengdu WuXi AppTec Co., Ltd.

[0376] PCH cells (4.5 × 10⁻⁶) were seeded. 4Cells (per well) were plated into pre-collagen-coated 96-well cell culture plates, with the test siRNA compound added simultaneously. Eight concentrations of the test siRNA were tested, starting at 200 nM, with 5-fold dilutions and two replicates. A nuclease-free water control group without the compound was also included. Cells were incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the culture medium was removed and cells were collected for RNA extraction. Some compounds were tested at a concentration of 100 nM. Total RNA was extracted using the EZ-Press 96RNA Purification Kit (EZB-EZ4001-L) according to the kit instructions. cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme-R323) according to the kit instructions. The target gene CFB was detected by SYBR qPCR, and β-actin cDNA was detected as an internal control in parallel assays. Add 8 μL of the prepared PCR reaction solution and 2 μL of sample cDNA to a 384-well plate. The qPCR reaction program is as follows: heat at 95°C for 5 minutes, then enter the cycling mode, heat at 95°C for 10 seconds, followed by 60°C for 30 seconds, for a total of 40 cycles.

[0377] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression level of the target gene was calculated using a 2-1T / T method. -ΔΔCT The calculation formula is as follows:

[0378] ΔCT = Average CT value of target gene - Average CT value of internal reference gene

[0379] ΔCT = ΔCT (sample group) - ΔCT (control group)

[0380] Target gene relative expression level = 2 -ΔΔCT

[0381] Inhibition rate % = (1 - relative expression level of sample / average relative expression level of control group) × 100

[0382] The test results are shown in Tables 5 and 6.

[0383] Table 5

[0384]

[0385] Table 6

[0386]

[0387] The results showed that the siRNA and conjugates of the present invention had a significant silencing effect on the expression of the CFB gene in primary hepatocytes of cynomolgus monkeys under free intake conditions.

[0388] Test Example 4: Inhibitory effect of free uptake of siRNA conjugates by human primary hepatocytes on CFB mRNA expression

[0389] The siRNA conjugate prepared in Example 2 was prepared into a stock solution of the corresponding concentration using Nuclease-Free Water.

[0390] The diluted siRNA conjugate was added to collagen-coated 96-well cell plates, followed by a suspension of primary human hepatocytes (PHH) (from WuXi AppTec, Chengdu). A control group containing nuclease-free water (without siRNA conjugate) was also included. The plates were incubated at 37°C with 5% CO2 for 48 hours.

[0391] After 48 hours of free uptake, the culture medium was removed, the cell plates were washed once with PBS, and cell lysis buffer was added. Total RNA was extracted using an RNA extraction kit (Qiagen-74182). gDNA was removed using a reverse transcription kit (HiScript III RT SuperMix for qPCR, Vazyme-R323-01), and cDNA was synthesized using random primers.

[0392] The target gene cDNA will be detected by qPCR, while the corresponding internal reference gene (GAPDH cDNA) will be detected in parallel. The qPCR reaction program (TapMan Probe) is as follows: 50°C for 2 minutes, 95°C for 10 minutes, then enter the cycling mode, 95°C for 10 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0393] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT quantification method. The relative expression level of the target gene was expressed using 2-ΔΔCT.

[0394] The calculation formula is as follows:

[0395] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene;

[0396] ΔCT = ΔCT (drug-treated group) - ΔCT (Nuclease-Free Water control group);

[0397] Relative expression level of target gene = 2 - ΔΔCT;

[0398] Target gene inhibition rate % = (1 - relative expression level of sample / average expression level of Nuclease-Free Water control) × 100%;

[0399] The test results are shown in Table 7.

[0400] Table 7

[0401]

[0402] The results showed that the siRNA and conjugates of the present invention had a significant silencing effect on the expression of the CFB gene in human primary hepatocytes under free uptake conditions.

[0403] Test Example 5: In vitro screening of HEK293 cells using dual-luciferase psiCHECK-2 vector

[0404] In a 96-well cell plate, 3 μL of the siRNA prepared in Example 1 was added to each well, with two replicates for each siRNA. Then, 2 μL of the psiCHECK-2 vector plasmid carrying the CFB gene (from Tsingke Biotech) at a concentration of 100 ng / μL was added to each well, along with 20 μL of Opti-MEM medium, to obtain a mixture containing siRNA and plasmid. 0.4 μL of Lipofectamine 2000 (Invitrogen, catalog number 11668-019) was added to 24.6 μL of Opti-MEM medium (Gibco, catalog number 31985-070), and after incubation for 5 minutes, it was added to each well containing the siRNA and plasmid mixture. After incubation at room temperature for 20 minutes, 100 μL of a mixture containing 5 × 10⁵ ng / μL of siRNA and plasmid was added to each well. 4 Dulbecco's Modified Eagle Medium (Gibco, catalog number C11995500BT) was added to each well of a mixture containing siRNA, plasmid, and Lipofectamine 2000 transfection reagent. After incubating the cells for 48 hours, firefly luciferase and Renilla luciferase (Novizan, DD1205-02) were measured. The test concentration range was 10 nM to 25.6 fM, with stepwise five-fold dilutions.

[0405] The measurement results for a portion of the exemplary siRNA concentrations in this test case are shown in Table 8.

[0406] Table 8

[0407]

[0408] The results showed that the siRNA and its conjugates of the present invention had a significant silencing effect on CFB gene expression.

[0409] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0410] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A siRNA, characterized in that, It includes a sense strand and an antisense strand, wherein the antisense strand has a bistranded region with at least partial base pairing with the sense strand; The positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:143, or at least 15 consecutive nucleotides thereof; The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:144 to SEQ ID NO:146, or at least 15 consecutive nucleotides thereof; Each nucleotide in the sense strand and / or antisense strand is independently a modified or unmodified nucleotide.

2. The siRNA according to claim 1, characterized in that, The siRNA includes one or more of the following conditions: a) The positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or differs from it by no more than 5, 4, 3, 2, or 1 nucleotides; b) The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or differs from it by no more than 5, 4, 3, 2, or 1 nucleotides; c) The siRNA includes the sense strand and antisense strand of any group in Table A, or differs from the sense strand and / or antisense strand by no more than 5, 4, 3, 2, or 1 nucleotides, respectively. Table A d) The positive strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:70 and SEQ ID NO:141 to SEQ ID NO:144, or at least 17, 19 or 21 consecutive nucleotides thereof; e) The antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NO:71 to SEQ ID NO:140 and SEQ ID NO:145 to SEQ ID NO:148, or at least 17, 19 or 21 consecutive nucleotides thereof; f) The lengths of the sense and antisense strands are independently 17-25 nucleotides, preferably 19-25 nucleotides.

3. The siRNA according to claim 1, characterized in that, The siRNA includes one or more of the following conditions: 1) The double-stranded region has a length of 19-23 nucleotides; 2) The length of the positive strand does not exceed 23 nucleotides, preferably not exceeding 21 nucleotides; 3) The length of the antisense strand does not exceed 25 nucleotides, preferably not more than 23 nucleotides; 4) The siRNA is selected from any of the following: 4-1) The 3' and 5' ends of the justice chain are both blunt ends, and the 3' and 5' ends of the antisense chain are both blunt ends; 4-2) The positive chain has a 3' protruding end or a 5' protruding end, and the 3' end and the 5' end of the negative chain are both blunt ends; or the negative chain has a 3' protruding end or a 5' protruding end, and the 3' end and the 5' end of the positive chain are both blunt ends; 4-3) The justice chain has a 3' protrusion or a 5' protrusion, and the antisense chain has a 3' protrusion or a 5' protrusion; 5) Each nucleotide in the 3' and / or 5' overhangs of the sense strand and / or antisense strand is independently selected from U, T, dU, or dT; 6) The sense strand and / or the antisense strand comprises at least one modified nucleotide, each of which is independently selected from at least one of the following: Reverse debasing nucleotides, deoxynucleotides, 2'-fluorinated nucleotides, 2'-amino nucleotides, 2'-O-allyl nucleotides, 2'-C 1～25 Alkyl-modified nucleotides, 2'-OC 1～25 Alkyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, locked nucleotides, configuration-restricted nucleotides, 2'-allyl-modified nucleotides, base-free nucleotides, morpholinyl nucleotides, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate-modified nucleotides, 5'-thiophosphate-modified nucleotides, 5'-methylphosphonate-modified nucleotides, 5'-phosphate mimicry-modified nucleotides, 5'-methylcytosine-modified nucleotides, and nucleic acid analogs.

4. The siRNA according to claim 1 or 3, characterized in that, The siRNA includes one or more of the following conditions: 1) The double-stranded region has a length of 19-21 nucleotides; 2) The 3' and / or 5' protrusions comprise two nucleotides or analogues; 3) The 3' protruding end and / or 5' protruding end are selected from UU, TT, dUdU or dTdT; 4) The modified nucleotide is independently selected from at least one of the following: reverse debased nucleotide, 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, and 5'-thiophosphate modified nucleotide; 5) All nucleotides in the positive strand are modified nucleotides; 6) All nucleotides in the antisense strand are modified nucleotides.

5. The siRNA according to claim 4, characterized in that, The siRNA includes one of the following conditions: 1) In the sense strand and / or the antisense strand, the 2'-fluoro-modified nucleotide is present at the following positions: The nucleotide at the 5' end of the sense strand is at least one of the 9th, 11th, and 13th positions of the start point; and / or, the nucleotide at the 5' end of the antisense strand is at least one of the 2nd, 7th, 12th, 14th, and 16th positions of the start point; 2) In the antisense strand, the nucleotide modified with the 5'-thiophosphate group is located at the following positions: The nucleotide at the 5' end of the antisense strand is at least one of the first and second positions of the start point, and / or the nucleotide at the 3' end of the antisense strand is at least one of the first and second positions of the start point; 3) The first nucleotide at the 3' end of the antisense strand is EVpu, EVpa, cPrpu, or cPrpa; 4) In the sense strand and / or the antisense strand, the 2'-methoxy modified nucleotide is present at the following position: The 5' terminal nucleotide of the sense strand is at least one of the following positions: position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 10, position 12, position 14, position 15, position 16, position 17, position 18, position 19, position 20, and position 21 of the start point; and / or, the 5' terminal nucleotide of the antisense strand is at least one of the following positions: position 1, position 3, position 4, position 5, position 6, position 8, position 9, position 10, position 11, position 13, position 15, position 17, position 18, position 19, position 20, and position 21 of the start point; 5) One or more reverse debase nucleotides are added to the 3' end and / or 5' end of the positive strand.

6. The siRNA according to claim 5, characterized in that, The siRNA includes one of the following conditions: 1) The double-stranded region has 21 nucleotides, and the modifying nucleotides of the sense strand and the antisense strand are as follows: 2'-Fluorinated nucleotides are present at the following positions: The nucleotides at the 5' end of the sense strand are the 9th, 11th, and 13th positions of the start point; and the nucleotides at the 5' end of the antisense strand are the 2nd, 7th, 12th, 14th, and 16th positions of the start point; Nucleotides modified with 5'-thiophosphate groups are present at the following positions: The nucleotides at the 5' end of the antisense strand are the first and second positions of the start point, and the nucleotides at the 3' end of the antisense strand are the first and second positions of the start point; 2'-methoxy modified nucleotides are present at the following positions: The 5' terminal nucleotides of the sense strand are at positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, and 21 of the start point; and the 5' terminal nucleotides of the antisense strand are at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21 of the start point; 2) A reverse debasing nucleotide is added to the 3' and 5' ends of the positive strand; 3) The first nucleotide at the 3' end of the positive strand is linked to the reverse debased nucleotide via a 5'-thiophosphate group; 4) The first nucleotide at the 5' end of the positive strand is linked to the reverse debased nucleotide via a 5'-thiophosphate group.

7. The siRNA according to claim 1, characterized in that, The siRNA includes the sense and antisense strands of any group in Table B or Table C, or differs from the sense and / or antisense strands by no more than 5, 4, 3, 2, or 1 nucleotides. Table B Table C 8. A nucleic acid conjugate, characterized in that, include: The siRNA according to any one of claims 1 to 7, and the ligand conjugated to the siRNA.

9. The nucleic acid conjugate according to claim 8, characterized in that, One or more of the following conditions must be met: 1) The ligand may be one or more; 2) The ligand is coupled to the sense strand and / or the antisense strand of the siRNA; preferably coupled to the sense strand; 3) The ligands are selected from lipids, carbohydrates, aptamers, vitamins and / or peptides that specifically bind to cell membranes or cell surfaces; 4) The ligand has one or more of the following structures: in, This indicates the junction point between the ligand and the siRNA.

10. The nucleic acid conjugate according to claim 8, characterized in that, The nucleic acid conjugates include the sense and antisense strands of any one of the groups in Table D; Table D 11. A pharmaceutical composition, characterized in that, include: The siRNA according to any one of claims 1 to 7 or the nucleic acid conjugate according to any one of claims 8 to 10; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

12. Use of the siRNA according to any one of claims 1 to 7, the nucleic acid conjugate according to any one of claims 8 to 10, or the pharmaceutical composition according to claim 11, wherein the use includes at least one of the following: Inhibit the expression or activity of complement factor B gene; Prepare drugs for inhibiting the expression or activity of complement factor B gene; To prepare drugs for the prevention and / or treatment of complement factor B-mediated diseases; Prevention and / or treatment of diseases mediated by complement factor B.

13. The use according to claim 12, characterized in that, The diseases mediated by complement factor B include nephropathy and systemic lupus erythematosus; Preferably, the complement factor B-mediated related diseases are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulonephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis; Preferably, the complement factor B-mediated diseases are selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, and polycystic kidney disease.

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