Sirna for inhibiting CFB gene expression and use thereof.

Abstract

Disclosed in the present invention are a siRNA for inhibiting CFB gene expression and a use thereof. The siRNA and nucleic acid conjugate in the present invention can effectively prevent and / or treat complement factor B-mediated associated diseases.

Description

siRNA for inhibiting expression of CFB gene and use thereof

[0001] Priority information

[0002] The present application claims priority to and the benefit of Chinese Patent Application Nos. 2024112535311, 2024114424417, 2025101179820, 2025105265098, 2025109904476, filed on September 6, 2024, October 15, 2024, January 24, 2025, April 24, 2025, and July 17, 2025, respectively, and the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to siRNA and nucleic acid conjugates for inhibiting the expression of complement factor B and uses thereof. BACKGROUND

[0004] The complement system is part of the host's innate immune system involved in lysing foreign cells, enhancing phagocytosis of antigens, agglutinating agents carrying antigens, and attracting macrophages and neutrophils. The complement system is divided into three activation pathways: the classical pathway, the lectin pathway, and the alternative pathway, which converge at component C3 to generate an enzyme complex called C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to C3 convertase, which is regulated by complement factor B (CFB), and causes the generation of C5 convertase, which cleaves C5 into C5a and C5b, which activates the membrane attack pathway, leading to the formation of a membrane attack complex (MAC) comprising components C5b, C6, C7, C8, and C9. The membrane attack complex (MAC) forms a transmembrane channel and disrupts the phospholipid bilayer of the target cell, leading to cell lysis.

[0005] Abnormal activation of the complement system CFB is the cause of proliferation and / or initiation of lesions in many diseases. Therefore, the development of complement factor B inhibitors has great clinical value and market prospects. SUMMARY

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

[0007] In a first aspect, the present application provides an siRNA. According to embodiments of the present 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 set forth in any one of SEQ ID NO: 1-70 and SEQ ID NO: 141-143, or at least 15 contiguous nucleotides thereof; the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and SEQ ID NO: 144-146, or at least 15 contiguous nucleotides thereof; each nucleotide in the sense strand and / or the antisense strand is independently a modified or unmodified nucleotide. The siRNA according to embodiments of the present application can inhibit the expression of CFB, and thus can be effective in preventing and / or treating complement factor B-mediated related diseases.

[0008] According to embodiments of the present application, the siRNA described above can further include at least one of the following technical features:

[0009] According to embodiments of the present application, the sense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 1-70 and SEQ ID NO: 141-144, or has no more than 5 nucleotide differences therefrom.

[0010] According to embodiments of the present application, the sense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 1-70 and SEQ ID NO: 141-144, or has no more than 4, 3, 2, or 1 nucleotide differences therefrom.

[0011] According to embodiments of the present application, the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and SEQ ID NO: 145-148, or has no more than 5 nucleotide differences therefrom.

[0012] According to embodiments of the present application, the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and SEQ ID NO: 145-148, or has no more than 4, 3, 2, or 1 nucleotide differences therefrom.

[0013] According to embodiments of the present application, the sense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof; and the antisense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof.

[0014] According to embodiments of the present application, the siRNA comprises the sense strand and the antisense strand of any one of the groups in Table A (see the section of detailed description of the present specification), or has no more than 5 nucleotides difference from the sense strand and / or the antisense strand, respectively, preferably has no more than 4, no more than 3, no more than 2 or no more than 1 nucleotide difference.

[0015] According to embodiments of the present application, the siRNA can be selected from any one of the following:

[0016] a) both the 3' end and the 5' end of the sense strand are blunt ends, and both the 3' end and the 5' end of the antisense strand are blunt ends;

[0017] b) the sense strand contains a 3' overhang or a 5' overhang, and both the 3' end and the 5' end of the antisense strand are blunt ends; or

[0018] the antisense strand contains a 3' overhang or a 5' overhang, and both the 3' end and the 5' end of the sense strand are blunt ends;

[0019] c) the sense strand contains a 3' overhang or a 5' overhang, and the antisense strand contains a 3' overhang or a 5' overhang;

[0020] d) the sense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof;

[0021] e) the antisense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof;

[0022] f) the length of the sense strand and the antisense strand is independently 17-25 nucleotides, preferably 19-25 nucleotides.

[0023] According to embodiments of the application, the sense strand and / or the antisense strand further comprises a 3' overhang and / or a 5' overhang, the 3' overhang or 5' overhang comprising at least one nucleotide or analogue thereof, preferably comprising at least two nucleotides or analogues thereof.

[0024] In some alternative embodiments of the application, in the siRNA one of the sense strand or the antisense strand comprises at least one nucleotide overhang at one end, and the other strand is blunt at both ends.

[0025] In some alternative embodiments of the application, in the siRNA the antisense strand comprises at least one nucleotide overhang at one end, and the sense strand is blunt at both ends.

[0026] According to embodiments of the application, the sense strand and / or the antisense strand comprises a 3' overhang, the 3' overhang comprising at least two nucleotides or analogues thereof.

[0027] According to embodiments of the application, each of the nucleotides in the 3' overhang and / or 5' overhang is independently selected from U, T, dU or dT.

[0028] According to embodiments of the application, the 3' overhang and / or 5' overhang of the sense strand and / or the antisense strand comprises two nucleotides or analogues thereof.

[0029] According to embodiments of the application, the 3' overhang and / or 5' overhang of the sense strand and / or the antisense strand is selected from UU, TT, dUdU or dTdT.

[0030] According to embodiments of the application, the antisense strand comprises a 3' overhang, the 3' overhang comprising at least two nucleotides.

[0031] According to embodiments of the application, the 3' overhang of the antisense strand is UU.

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

[0033] According to embodiments of the application, the double-stranded region has a length of 19-21 nucleotides, for example 19, 20 or 21 nucleotides.

[0034] According to embodiments of the application, the sense strand has a length of no more than 23 nucleotides, preferably no more than 21 nucleotides, for example 19, 20 or 21 nucleotides.

[0035] According to embodiments of the application, the antisense strand has a length of no more than 25 nucleotides, preferably no more than 23 nucleotides, for example 21, 22 or 23 nucleotides.

[0036] According to embodiments of the present application, the sense strand and / or the antisense strand comprises at least one modified nucleotide, each of the modified nucleotide is independently selected from at least one of:

[0037] inverted deoxyabasic nucleotide, deoxynucleotide, 2'-fluoro-modified nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C 1～25 alkyl-modified nucleotide, 2'-O-C 1～25 alkyl-modified nucleotide, 2'-methoxyethyl-modified nucleotide, locked nucleotide, conformationally restricted nucleotide, 2'-allyl-modified nucleotide, abasic nucleotide, morpholino nucleotide, tetrahydropyranyl-modified nucleotide, 1,5-anhydrohexitol-modified nucleotide, cyclohexenyl-modified nucleotide, PEG-modified nucleotide, 5'-phosphoramidate-modified nucleotide, 5'-phosphorothioate-modified nucleotide, 5'-methylphosphonate-modified nucleotide, 5'-phosphate mimic-modified nucleotide, 5'-methylated cytosine-modified nucleotide, nucleic acid analog.

[0038] According to embodiments of the present application, the modified nucleotide is independently selected from at least one of inverted deoxyabasic nucleotide, 2'-methoxy-modified nucleotide, 2'-fluoro-modified nucleotide and 5'-phosphorothioate-modified nucleotide.

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

[0040] According to embodiments of the present application, in the sense strand and / or the antisense strand, the 2'-fluoro-modified nucleotide is present at the following positions:

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

[0042] In an alternative embodiment of the present application, in the sense strand and / or the antisense strand, the 2'-fluoro-modified nucleotide is present at the following positions:

[0043] the nucleotide at the 5' end of the sense strand is the 9th, 11thand 13thposition from the start; and / or, the nucleotide at the 5' end of the antisense strand is the 2nd, 7th, 12th, 14thand 16thposition from the start.

[0044] According to an embodiment of the present application, in the antisense strand, 5'-phosphorothioate group-modified nucleotides are present at the following positions:

[0045] The nucleotides at the 5' end of the antisense strand are at least one of the 1st and 2nd positions from the start point, and / or the nucleotides at the 3' end of the antisense strand are at least one of the 1st and 2nd positions from the start point.

[0046] In an alternative embodiment of the present application, in the antisense strand, 5'-phosphorothioate group-modified nucleotides are present at the following positions:

[0047] The nucleotides at the 5' end of the antisense strand are the 1st and 2nd positions from the start point, and / or the nucleotides at the 3' end of the antisense strand are the 1st and 2nd positions from the start point.

[0048] According to an embodiment of the present application, in the sense strand and / or in the antisense strand, 2'-methoxy-modified nucleotides are present at the following positions:

[0049] The nucleotides at the 5' end of the sense strand are at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions from the start point; and / or the nucleotides at the 5' end of the antisense strand are at least one of the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions from the start point.

[0050] In an alternative embodiment of the present application, in the sense strand and / or in the antisense strand, 2'-methoxy-modified nucleotides are present at the following positions:

[0051] 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 from the start point; and / or 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 from the start point.

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

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

[0054] the 9th, 11th, and 13th nucleotides from the 5' terminus of the sense strand; and, the 2nd, 7th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand;

[0055] 5'-phosphorothioate modified nucleotides are present at the following positions:

[0056] the 1st and 2nd nucleotides from the 5' terminus of the antisense strand, and, the 1st and 2nd nucleotides from the 3' terminus of the antisense strand;

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

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

[0059] According to embodiments of the application, the 1st nucleotide from the 3' terminus of the antisense strand is EVpu, EVpa, cPrpu, or cPrpa.

[0060] According to embodiments of the application, the double-stranded region has 21 nucleotides, and the modified nucleotides of the sense strand and the antisense strand are as follows:

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

[0062] the 9th, 11th, and 13th nucleotides from the 5' terminus of the sense strand; and, the 2nd, 7th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand;

[0063] 5'-phosphorothioate modified nucleotides are present at the following positions:

[0064] the 1st and 2nd nucleotides from the 5' terminus of the antisense strand, and, the 1st and 2nd nucleotides from the 3' terminus of the antisense strand;

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

[0066] the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st nucleotides from the 5' terminus of the sense strand; and, the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides from the 5' terminus of the antisense strand;

[0067] the 1st nucleotide from the 3' terminus of the antisense strand is EVpu, EVpa, cPrpu, or cPrpa.

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

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

[0070] It is to be noted that "nucleotide at the 5' terminus of the sense strand is the starting point" means that the first nucleotide at the 5' end of the sense strand is the starting point, wherein "the Xth nucleotide from the 5' terminus of the sense strand is the starting point" means that the position of the Xth nucleotide is counted from the first nucleotide at the 5' end of the sense strand as the starting point.

[0071] It is to be noted that "nucleotide at the 3' terminus of the sense strand is the starting point" means that the first nucleotide at the 3' end of the sense strand is the starting point, wherein "the Xth nucleotide from the 3' terminus of the sense strand is the starting point" means that the position of the Xth nucleotide is counted from the first nucleotide at the 3' end of the sense strand as the starting point.

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

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

[0074] According to an embodiment of the present application, one or more inverted abasic nucleotides are added to the 3' end and / or 5' end of the sense strand.

[0075] According to an embodiment of the present application, one inverted abasic nucleotide is added to the 3' end and / or 5' end of the sense strand.

[0076] According to an embodiment of the present application, the first nucleotide at the 3' end of the sense strand is connected to the inverted abasic nucleotide through a 5'-thiophosphodiester group.

[0077] According to an embodiment of the present application, the first nucleotide at the 5' end of the sense strand is connected to the inverted abasic nucleotide through a 5'-thiophosphodiester group.

[0078] In the second aspect of the present application, an siRNA is provided. According to an embodiment of the present application, the siRNA comprises the sense strand and the antisense strand of any one of the groups in Table B (see the specific implementation method section of the present specification for details), or has no more than 5 nucleotide differences, preferably no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide difference, from the sense strand and / or the antisense strand.

[0079] According to an embodiment of the present application, the siRNA comprises the sense strand and the antisense strand of any one of the groups in Table C (see the specific implementation method section of the present specification for details), or has no more than 5 nucleotide differences, preferably no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide difference, from the sense strand and / or the antisense strand.

[0080] In the third aspect of the present application, a nucleic acid conjugate is provided. According to an embodiment of the present application, the nucleic acid conjugate comprises the siRNA of the first aspect or the second aspect, and a ligand conjugated to the siRNA. The above-mentioned nucleic acid conjugate of the present application can facilitate the targeting and delivery of nucleic acid drugs, connect the siRNA to the desired cells and / or tissues in the body, so as to target and silence the inhibition of complement factor B in the target cells and / or tissues, thereby effectively preventing and / or treating the complement factor B-mediated related diseases.

[0081] According to an embodiment of the present application, the above-mentioned nucleic acid conjugate can further comprise at least one of the following technical features:

[0082] According to embodiments of the present application, the ligand is one or more, and the ligand is used to target any cell, preferably the ligand is selected from the group consisting of a lipid, a carbohydrate, an aptamer, a vitamin and / or a peptide that specifically binds to a cell membrane or a cell surface.

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

[0084] wherein, represents the point of attachment of the ligand to the siRNA.

[0085] According to embodiments of the present application, the ligand is attached to the 3' end or 5' end of the sense strand via a phosphorothioate bond or a phosphate bond.

[0086] In this context, without further specification, in the sense strand and / or the antisense strand used for conjugating the ligand, the end of the sense strand and / or the antisense strand to which the ligand is attached comprises a phosphorothioate bond or a phosphate, i.e. the phosphorothioate bond or the phosphate is located at the 3' end or 5' end of the sense strand and / or the antisense strand, which is used for conjugating the ligand.

[0087] According to embodiments of the present application, the nucleic acid conjugate comprises the sense strand and the antisense strand of any one of the groups in Table D (see the specific implementation section of the present specification for details).

[0088] In a fourth aspect of the present application, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the siRNA of the first aspect or the second aspect, or the nucleic acid conjugate of the third aspect. As previously described, the aforementioned siRNA and nucleic acid conjugate can degrade the mRNA encoding CFB, and inhibit the expression or activity of the CFB gene. Therefore, the pharmaceutical composition of the present application can be effective in treating and preventing diseases related to complement factor B.

[0089] According to embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0090] In a fifth aspect of the present application, the present application provides the use of the siRNA of the first aspect or the second aspect, the nucleic acid conjugate of the third aspect, or the pharmaceutical composition of the fourth aspect, and the use comprises at least one of the following:

[0091] inhibiting the expression or activity of the complement factor B gene;

[0092] preparing a medicament for inhibiting the expression or activity of the complement factor B gene;

[0093] manufacture of a medicament for preventing and / or treating a complement factor B-mediated related disease;

[0094] preventing and / or treating a complement factor B-mediated related disease.

[0095] The method of the present application can be used to inhibit the expression or activity of a CFB gene. For example, the expression or activity of a CFB gene can be inhibited in vivo in an animal; or the expression or activity of a CFB gene can be inhibited for non-disease treatment purposes, for example, the expression or activity of a CFB gene of an isolated cell can be inhibited in vitro for subsequent research.

[0096] According to embodiments of the present application, the complement factor B-mediated related disease comprises nephropathy, systemic lupus erythematosus (SLE).

[0097] According to embodiments of the present application, the complement factor B-mediated related disease is selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy (e.g., C3 glomerulopathy), age-related macular degeneration, atypical hemolytic-uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0098] According to embodiments of the present application, the complement factor B-mediated related disease is selected from glomerulopathy, systemic lupus erythematosus.

[0099] According to embodiments of the present application, the complement factor B-mediated related disease is selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease.

[0100] In a sixth aspect of the present application, the siRNA of the first or second aspect, the nucleic acid conjugate of the third aspect, or the pharmaceutical composition of the fourth aspect is used for at least one of: inhibiting the expression or activity of a complement factor B gene; preventing and / or treating a complement factor B-mediated related disease.

[0101] In a seventh aspect of the present application, a method for preventing and / or treating a complement factor B-mediated related disease is provided. According to embodiments of the present application, the method comprises administering to a subject having a complement factor B-mediated related disease a pharmaceutically acceptable dose of the siRNA of the first or second aspect, the nucleic acid conjugate of the third aspect, or the pharmaceutical composition of the fourth aspect.

[0102] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. DETAILED DESCRIPTION

[0103] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not intended to be limiting of the present application.

[0104] It should be noted that the terms "first", "second" are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or a quantity of the specified technical features. Thus, features defined with "first", "second" can include one or more of the features implicitly or explicitly. Further, in the description of the present application, the meaning of "a plurality of" is two or more unless otherwise specified.

[0105] In this document, the terms "comprising" or "including" are open-ended terms, i.e., the inclusion of the recited elements is not intended to be limiting, but rather, the description is intended to be illustrative of the described aspects.

[0106] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0107] In this document, the term "small interfering RNA (siRNA)" is a double-stranded RNA comprising a sense strand and an antisense strand. The siRNA mediates the targeted cleavage of RNA transcripts of the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through the RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins. For example, siRNA can inhibit the expression of CFB gene.

[0108] In this document, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence, such as mRNA encoding CFB. The "sense strand (or passenger strand)" refers to an iRNA strand that contains a sequence substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior of the molecule or in the terminal regions, wherein the most tolerated mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the iRNA.

[0109] It is noted that an "antisense strand is at least partially substantially complementary to an mRNA" means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest, e.g., an mRNA encoding CFB. Alternatively, an antisense strand is complementary to at least a portion of an mRNA encoding CFB if a polynucleotide is substantially non-interruptedly complementary to a portion of an mRNA encoding CFB.

[0110] In the present context, the term "target sequence" or "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a CFB-encoding gene, including mRNA that is a processing product of the primary transcription product.

[0111] In the present context, the term "inhibiting expression of a CFB gene" includes inhibition of a CFB gene at any level, e.g., at least partial inhibition of CFB gene expression, such as 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%. Wherein, the expression of a CFB gene can be assessed based on the level of any variable associated with CFB gene expression, e.g., the level of an mRNA encoding CFB or the level of CFB. Inhibition can be assessed by a decrease 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 utilized in the art, e.g., a baseline level prior to administration, or a level determined from a similar subject, cell, or sample that has never been treated or has been treated with a control, e.g., a control with only a buffer or an inactive agent.

[0112] In the present context, the term "blunt end" refers to the end of a sense strand or an antisense strand in an siRNA that does not have unpaired nucleotides, i.e., does not have a nucleotide overhang. An siRNA that is "blunt ended" refers to an siRNA that is double stranded throughout its length, i.e., both ends (3' end and 5' end) of the sense strand and the antisense strand of the siRNA do not have a nucleotide overhang.

[0113] In the present context, the term "a sense strand or an antisense strand having at least one end with a nucleotide overhang" refers to a sense strand or an antisense strand that has one end with a nucleotide overhang and the other end that is blunt ended, or a sense strand or an antisense strand that has both ends with a nucleotide overhang.

[0114] In the present context, the term "one strand of the sense or antisense strand... the other strand..." includes two scenarios, namely 1) one strand is the sense strand and the other strand is the antisense strand, or 2) one strand is the antisense strand and the other strand is the sense strand.

[0115] In the present context, the terms "nucleotide overhang" and "overhang" are synonymous and refer to at least one unpaired nucleotide overhanging the duplex region structure of the siRNA. For example, a nucleotide overhang is present when the 3' end of one strand of the sense and / or antisense strand overhangs the 5' end of the other strand, or when the 5' end of one strand of the sense and / or antisense strand overhangs the 3' end of the other strand. The overhang can comprise at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides or more. The nucleotide overhang can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on any one or any combination of the sense and antisense strands. Furthermore, the nucleotides of the overhang can be on the 5'-end, the 3'-end or both ends of the antisense or sense strand.

[0116] In the present context, the terms "nucleotide" and "nucleic acid" refer to A, T / U, C, G; the terms "nucleotide analog" and "nucleic acid analog" refer to a structure that has a similarity in chemical structure to a nucleotide (A, T / U, C, G).

[0117] In the present context, "overhang" as referred to by the present application includes 3' overhang and 5' overhang, unless otherwise indicated.

[0118] In the present context, the "length of the sense strand" refers to the full length of the sense strand; when the sense strand contains an overhang, the "length of the sense strand" = the length of the duplex region + the length of the overhang; when the sense strand does not contain an overhang, the "length of the sense strand" = the length of the duplex region. The "length of the antisense strand" refers to the full length of the antisense strand; when the antisense strand contains an overhang, the "length of the antisense strand" = the length of the duplex region + the length of the overhang; when the antisense strand does not contain an overhang, the "length of the antisense strand" = the length of the duplex region.

[0119] In the present context, the term "C 1～25 Alkyl includes alkyl groups containing 1, 2, 3, 4,... 24 or 25 C atoms. For example, 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. For example, C 1～3Alkyl includes methyl, ethyl, n-propyl, and i-propyl.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 1～10 Alkyl-modified nucleotides, 2'-C 1～5 Alkyl-modified nucleotides or 2'-C 1～3 Alkyl-modified nucleotides. Illustratively, can be 2'-methyl-modified nucleotides, 2'-ethyl-modified nucleotides, 2'-n-propyl-modified nucleotides, or 2'-i-propyl-modified nucleotides.

[0120] 2'-O-C 1～25 Alkyl-modified nucleotides can be 2'-O-C 1～20 Alkyl-modified nucleotides, 2'-O-C 1～16 Alkyl-modified nucleotides, 2'-O-C 1～10 Alkyl-modified nucleotides, 2'-O-C 1～5 Alkyl-modified nucleotides or 2'-O-C 1～3 Alkyl-modified nucleotides. Illustratively, can be 2'-O-methyl-modified nucleotides, 2'-O-ethyl-modified nucleotides, 2'-O-n-propyl-modified nucleotides, or 2'-O-i-propyl-modified nucleotides.

[0121] As known to those skilled in the art, a ribonucleotide (herein simply referred to as a nucleotide) is composed of a phosphate, a ribose, and a base. In this document, "modified nucleotide" means that, compared to an A, U, C, or G nucleotide, there is a change in the group attached to a different C atom on the ribose, a change in the phosphate group, or a deletion of the base. In this document, "modified nucleotide" and "modified nucleotide" are synonymous.

[0122] As known to those skilled in the art, a nucleotide is composed of a phosphate, a ribose, and a base. "Natural nucleotide" or "nucleotide with a natural base" means a nucleotide with a base of adenine A, uracil U, guanine G, or cytosine C. In some alternative embodiments of the present application, the modified nucleotide has one or more modifications.

[0123] It is intended that the application cover all alternatives, modifications and equivalents thereof which fall within the scope of the application as defined by the claims. It will be apparent to those skilled in the art that many methods and materials other than those described herein can be used to practice the present application. Nothing in the present application should be taken as a restriction on the scope of the application. To the extent necessary to the apprehension of the various benefits and advantages of the application, the disclosure as illustrated in the description, examples, and figures will be deemed adequate. Any one of the foregoing documents, patents or similar materials, to the extent they are inconsistent with the present application, including but not limited to defined terms, term application, described techniques, and the like, are hereby superseded by the present application.

[0124] It will be further understood that various features of the application, which are, for clarity, described in the context of separate embodiments or implementations, can also be provided in combination in a single embodiment or implementation. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment or implementation, can also be provided separately or in any suitable

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents, publications, and other documents cited herein are hereby incorporated by reference in their entirety.

[0126] The compounds of the present application can contain asymmetric or chiral centers, and as such can exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the present application, including but not limited to, diastereomeric, enantiomeric, and atropisomeric forms, and mixtures thereof such as racemic mixtures, are within the scope of the present application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing compounds, the prefixes R or S are used to denote the absolute configuration of the molecule, with respect to a specific chiral center (or multiple chiral centers). The prefixes d and 1 or (+) and (-) are used to designate the sign of the rotation of plane-polarized light by the compound, with (-) or 1 meaning 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 one another. Specific stereoisomers are also referred to as enantiomers when they are mirror images of each other. A mixture of such isomers is often referred to as the racemic mixture or racemic modification. A racemic mixture is a 50:50 mixture of ( ) and (-) isomers.

[0127] Depending on the choice of starting materials and methods, the compounds of the application can be present in the form of one of the possible isomers or as a mixture of them, for example, as a racemic mixture, depending on the number of asymmetric carbon atoms. The optically active isomers (R)- or (S)- can be prepared using chiral synthons or chiral reagents, or by resolution of the racemic compounds using conventional techniques. If the compound contains a double bond, the substituents can be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl, the substituents on the cycloalkyl can be in the cis- or trans- (or syn- or anti-) configuration.

[0128] The compounds of the application can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the application, including but not limited to, diastereomeric, enantiomeric, and atropisomeric forms, and geometric (or conformational) isomeric forms, and mixtures thereof, such as racemic mixtures, are within the scope of the application.

[0129] Unless otherwise stated, structures depicted are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, atropisomeric, and geometric (or conformational) isomeric) forms of the structure; for example, structures having asymmetrically substituted atoms, such as enantiomeric (R) and (S) configurations, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the application.

[0130] Any asymmetric atom (e.g., carbon) of the compounds of the application can exist in the racemic or enantiomerically enriched form, e.g., in the (R)-, (S)-, or (R,S)-configurational form. In certain embodiments, each asymmetric atom is in the (R)- or (S)-configurational form with at least a 50% enantiomeric excess, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess, or at least a 99% enantiomeric excess. If possible, substituents on atoms having an unsaturated double bond can exist in the cis-(Z)- or trans-(E)-form.

[0131] Thus, as described herein, the compounds of the application can exist in the form of one of the possible isomers, rotamers, atropisomers, tautomers, or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] In the description of the functional groups of this invention It is used to describe the position where a group is substituted or the connection site with other groups.

[0137] In the chemical structure of the ligands or compounds described in this disclosure, the bonds... This indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include 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.

[0138] In the present text, the terms "optionally substituted", "optionally substituted" and "substituted or unsubstituted" are used interchangeably. In general, the term "optionally" whether or not preceded by the term "substituted" means that one or more hydrogen atoms in the given structure are replaced by a particular substituent. Unless otherwise indicated, an optional substituent group can be substituted at each substitutable position of the group.

[0139] In the present text, the term "one or more" (e.g. in the definition of substituents of the compounds of the general formula of the present application) means "one, two, three, four or five, especially one, two, three or four, more especially one, two or three, more especially one or two".

[0140] Further, it is to be understood that the descriptive terms "each independently" and "each of" and "independently" as employed in the present application are interchangeable, unless explicitly stated otherwise, and are to be interpreted broadly, meaning that the specific options expressed by the same symbol between different groups do not influence each other, as well as the specific options expressed by the same symbol between the same groups do not influence each other.

[0141] In the present text, "pharmaceutical composition" can refer to the treatment of a disease, but also to the in vitro cultivation experiments of cells. 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 one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the auxiliary ingredient(s) that make up the one or more accessory ingredients. Typically, the compositions are prepared by uniformly and intimately bringing the active siRNA into association with a liquid auxiliary ingredient, a finely divided solid auxiliary ingredient, or both.

[0142] In the present text, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal being treated with it. Preferably, "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0143] In the present text, the term "pharmaceutically acceptable auxiliary ingredient" can include any solvent, diluent or other liquid vehicle, etc. suitable for the particular target dosage form. Except insofar as any conventional auxiliary ingredient is incompatible with the siRNA of the present application, for example, by producing any undesirable biological effect or interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated to be within the scope of this application.

[0144] As used herein, the term "treatment" refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed or diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. As used herein, "treatment" covers any administration of a drug or siRNA to an individual to treat, cure, relieve, alleviate, alter, remedy, improve, lessen, or inhibit a disease in an individual, including, but not limited to, administration of a drug comprising a siRNA or siRNA conjugate described herein to an individual in need thereof.

[0145] Detailed description of siRNA, siRNA conjugate, composition and use of the present application

[0146] The present application provides a siRNA, a siRNA conjugate, a cell and a pharmaceutical composition, and their use, a method for expressing complement factor B and a method for preventing and / or treating a complement factor B-mediated related disease, which will be described in detail respectively as follows.

[0147] siRNA

[0148] In a first aspect of the present application, the present application provides a siRNA. According to an embodiment of the present 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 set forth 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; the antisense strand comprises a nucleotide sequence as set forth 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 15 consecutive nucleotides thereof; each nucleotide in the sense strand and / or the antisense strand is independently a modified or unmodified nucleotide. The siRNA according to an embodiment of the present application can inhibit the expression of CFB, thereby effectively preventing and / or treating a complement factor B-mediated related disease.

[0149] The present application provides an siRNA. According to an embodiment of the present 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 set forth in any one of SEQ ID NO: 1-70 and 141-144, or at least 10 contiguous nucleotides thereof; the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and 145-148, or at least 10 contiguous nucleotides thereof; each of the nucleotides in the sense strand and / or the antisense strand is independently a modified or unmodified nucleotide. The inventors have designed the above suitable small interfering RNA (siRNA) to form a silencing complex (RNA-induced silencing complex, RISC) and to be complementary to the sequence of the mRNA of the target gene (CFB gene), so as to degrade the mRNA encoding CFB and inhibit the expression of CFB, thereby effectively preventing and / or treating diseases related to complement factor B.

[0150] In the present application, the term "or at least 15 contiguous nucleotides thereof" and "or at least 10 contiguous nucleotides thereof" refers to the aforementioned nucleotide sequence. For example, "the sense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 1-70 and 141-144, or at least 15 contiguous nucleotides thereof" refers to the sense strand comprising a nucleotide sequence as set forth in any one of SEQ ID NO: 1-70 and 141-144, or at least 15 contiguous nucleotides of the nucleotide sequence as set forth in any one of SEQ ID NO: 1-70 and 141-144. For example, "the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and 145-148, or at least 10 contiguous nucleotides thereof" refers to the antisense strand comprising a nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and 145-148, or at least 10 contiguous nucleotides of the nucleotide sequence as set forth in any one of SEQ ID NO: 71-140 and 145-148.

[0151] In the present disclosure, the term "no more than 5 nucleotides" or "having no more than 5 nucleotide differences" means that there can be 0, 1, 2, 3, 4 or 5 nucleotide differences compared to the target nucleotide sequence (mRNA encoding CFB gene), which includes but is not limited to deletion of nucleotides, insertion of nucleotides (which can be inserted to the 3' end, 5' end or between any two nucleotides of the nucleotide sequence), substitution of nucleotides, etc. In "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 has no more than 5 nucleotide differences therefrom", "therefrom" 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; in "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 has no more than 5 nucleotide differences therefrom", "therefrom" 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.

[0152] It should be noted that in the present disclosure, the 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, and / or the 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, are located in the double-stranded region of the siRNA.

[0153] In some alternative embodiments of the present disclosure, the antisense strand has a double-stranded region with at least 85% base pairing, or at least 90% base pairing, or at least 95% base pairing with the sense strand.

[0154] According to embodiments of the present disclosure, the siRNA comprises the sense strand and the antisense strand of any one of the groups in Table A, or has no more than 5 nucleotide differences, preferably no more than 4 nucleotide differences, no more than 3 nucleotide differences, no more than 2 nucleotide differences, no more than 1 nucleotide difference, with the sense strand and / or the antisense strand;

[0155] Table A

[0156] According to embodiments of the present application, the 5'-phosphate mimic modified nucleotide is selected from the group consisting of EVpu, EVpa, cPrpu, cPrpa. In this context, the structures of EVpu, EVpa, cPrpu and cPrpa are shown as follows:

[0157] In some alternative embodiments of the present application, the modification comprises at least one of a 2'-methoxy modification, a 2'-fluoro modification and a 5'-phosphorothioate modification.

[0158] It is noted that when a modified nucleotide is selected from the group consisting of 2'-alkyl modified nucleotide and 5'-phosphorothioate modified nucleotide, it means that the modified nucleotide has both 2'-alkyl modification and 5'-phosphorothioate modification.

[0159] In some alternative embodiments of the present application, one or more inverted abasic residues (invAb) are added to the 3' end of the sense strand and / or the antisense strand. In some alternative embodiments of the present application, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand and / or the antisense strand. In some alternative embodiments of the present application, one or more inverted abasic residues are inserted between the ligand and the nucleotide sequence of the sense strand and / or the antisense strand. In some alternative embodiments of the present application, the inverted abasic residues can be linked via a phosphate, a phosphorothioate or other internucleoside linkage.

[0160] In this context, the structure of the inverted abasic modification and the inverted abasic nucleotide are shown as follows:

[0161] wherein a is a linkage towards the 5' end of the sense strand or the antisense strand, and b is a linkage towards the 3' end of the sense strand or the antisense strand.

[0162] In this context, "phosphoramidite" refers to a starting material in the solid phase synthesis of oligonucleotides, which connects a modified or unmodified nucleotide or a linker or a conjugate group to the oligonucleotide to form a phosphate or a phosphorothioate.

[0163] In some alternative embodiments of the present application, the modification comprises a deoxynucleotide.

[0164] In this context, "deoxynucleotide" refers to a nucleotide with deoxyhydroxyl in the pentose sugar of the nucleotide, which can be 2'-OH or 3'-OH.

[0165] In some alternative embodiments of the present application, the deoxynucleotides comprise 3'-deoxy-modified nucleotides and 2'-deoxy-modified nucleotides.

[0166] In the present context, "2'-deoxy-modification" means that the hydroxyl group (2'-OH) in the pentose sugar of a nucleotide is replaced by hydrogen (2'-H), and "3'-deoxy-modification" means that the hydroxyl group (3'-OH) in the pentose sugar of a nucleotide is replaced by hydrogen (3'-H).

[0167] In the present context, "2'-X modification" means that the hydroxyl group (2'-OH) in the pentose sugar of a nucleotide is replaced by X (2'-X). For example, "2'-fluoro modification" means that the hydroxyl group (2'-OH) in the pentose sugar of a nucleotide is replaced by fluorine (2'-F), "2'-amino modification" means that the hydroxyl group (2'-OH) in the pentose sugar 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 sugar 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 sugar 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 sugar of a nucleotide is replaced by an alkoxy group (2'-alkoxy).

[0168] In some alternative embodiments of the present application, the modifications comprise locked nucleotides.

[0169] In the present context, "locked nucleotide" means a nucleotide in which the 2' and 4' carbons of the pentose sugar are linked together.

[0170] In the present context, "5'-X modification" means that the phosphate ester (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by X (5'-X). For example, "5'-phosphoramidate modification" means that the phosphate ester group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a phosphoramidate group, "5'-phosphorothioate group modification" means that the phosphate ester group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a phosphorothioate group, "5'-methylphosphonate group modification" means that the phosphate ester group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a methylphosphonate group, and "5'-phosphate mimic modification" means that the phosphate ester group (5'-PO(OH)2) in the pentose sugar of a nucleotide is replaced by a phosphate mimic.

[0171] In some alternative embodiments of the present application, the 2'-C 1～25 Alkyl-modified nucleotides include 2'-methoxy-modified nucleotides and 2'-methoxyethyl-modified nucleotides.

[0172] In the present context, "2'-methoxy modification" means that the hydroxyl group (2'-OH) in the pentose sugar of a nucleotide is replaced by a methoxy group (2'-OCH3).

[0173] In the present context, "2'-methoxyethyl modification" means that the hydroxyl group (2'-OH) in the pentose sugar of a nucleotide is replaced by a methoxyethyl group (2'-OCH2CH2OCH3).

[0174] In some alternative embodiments of the present application, the modified nucleotide is a 5'-methylated cytosine modified nucleotide.

[0175] In the present context, "5'-methylated cytosine modification" means methylation at the 5th carbon atom of cytosine.

[0176] In some alternative embodiments of the present application, the modified nucleotide is a conformationally restricted nucleotide.

[0177] In the present context, "conformationally restricted nucleotides" (CRN) means nucleotide analogs, in particular nucleotide analogs with linkers attached to the C2' and C4' carbons of the ribose or to the C3' and C5' carbons of the ribose that place the oxygen in a position of optimal stability and affinity, reducing the puckering of the ribose ring. CRNs lock the ribose ring in a stable conformation and increase the hybridization affinity to mRNA. Methods for the preparation of certain CRNs include, but are not limited to, US Patent Publication No. US2013 / 0190383 and PCT Publication WO 2013 / 036868. It is noted that the entire contents of US2013 / 0190383 and WO 2013 / 036868 are incorporated herein by reference.

[0178] The present application provides an siRNA. According to embodiments of the present application, the siRNA comprises the sense and antisense strands of any one of the groups in Table B, or differs from the sense and / or antisense strands by no more than 5 nucleotides, preferably by no more than 4, no more than 3, no more than 2 nucleotides, no more than 1 or no more than 0 nucleotides;

[0179] Table B

[0180] The present application provides an siRNA. According to embodiments of the present application, the siRNA comprises the sense and antisense strands of any one of the groups in Table C, or differs from the sense and / or antisense strands by no more than 5 nucleotides, preferably by no more than 4, no more than 3, no more than 2 nucleotides, no more than 1 or no more than 0 nucleotides;

[0181] Table C

[0182] In Table B, Table C and Table D hereinafter, the lower case letters c, g, u, a, t are 2'-methoxy modified nucleotides (i.e. the ribosyl 2'-OH of the nucleotide represented by the corresponding upper case letter is replaced by a methoxy group); f indicates that the nucleotide represented by the letter preceding f is a 2'-fluoro modified nucleotide (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 nucleotide residues flanking s are linked by a phosphorothioate (i.e. the 5'-phosphate group of the nucleotide represented by the letter preceding s is replaced by a 5'-phosphorothioate group).

[0183] In the present application, the siRNA in Table B, Table C and Table D hereinafter can have the same nucleotide sequence as the natural sequence in Table A or Table B, or the sense strand and / or the antisense strand of the siRNA can have not more than 5 (preferably not more than 1 or 2, more preferably 1) nucleotide difference(s) from the nucleotide sequence in Table A or Table B, and the specific type is not limited.

[0184] Nucleic acid conjugate

[0185] In the second aspect of the present application, a nucleic acid conjugate is provided. According to an embodiment of the present application, the nucleic acid conjugate comprises the siRNA of the first aspect, and a ligand conjugated to the siRNA. The nucleic acid conjugate of the present application can facilitate the targeting and delivery of the nucleic acid drug, and link the siRNA to the desired cells and / or tissues in vivo, so as to target the silencing of the inhibition of complement factor B in the target cells and / or tissues, thereby effectively preventing and / or treating the complement factor B mediated related diseases. As used herein, the terms "nucleic acid conjugate", "siRNA conjugate" and "conjugate" can be used interchangeably.

[0186] In an alternative embodiment of the present application, the ligand is directly or indirectly conjugated to the oligonucleotide (such as siRNA) through a linker.

[0187] The structure of the ligand is not particularly limited, and any ligand capable of delivering the siRNA to the cells or tissues can be used, and preferably the ligand can target the liver cells and / or tissues.

[0188] The connection mode of the ligand to the siRNA is also not particularly limited, and the ligand can be directly connected to the siRNA through a group without using an additional linker / connection group, or can be indirectly connected to the siRNA through a linker.

[0189] The way of connecting the ligand to the sense strand and / or the antisense strand of the siRNA is also not particularly limited. Preferably, the ligand is connected to the siRNA via a phosphorothioate bond or a phosphate bond. Preferably, the ligand is connected to the 5' end or the 3' end of the sense strand. Preferably, the ligand is connected to the 5' end or the 3' end of the antisense strand.

[0190] The limitations of the oligonucleotide, the ligand and the way of connecting them in the nucleic acid conjugate can also be found in the fourth aspect of the summary of the application.

[0191] According to embodiments of the present application, the ligand can also have the structure as 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; P4; P5; P6, the preparation method of which is described in WO2015 / 168514A1.

[0192] The nucleic acid conjugate of the present application can further improve the degradation of the mRNA encoding the target protein by adding the ligand, which can effectively prevent and / or treat the related diseases.

[0193] According to embodiments of the present application, the ligand can also have the structure as described above L1, L2, L3, L4, L5, L6.

[0194] Therefore, the use of the above ligand can promote the targeting and delivery of the oligonucleotide, so that the oligonucleotide is connected to the desired cells and / or tissues in vivo, so as to target the silencing of the target gene in the target cells and / or tissues, thereby effectively preventing and / or treating the related diseases.

[0195] According to embodiments of the present application, the nucleic acid conjugate has the following structure:

[0196] wherein, represents the siRNA described herein; X represents O or S.

[0197] According to embodiments of the present application, the 5'-end of the sense strand of the siRNA has the structure of NH2-alkyl group (such as NH2-C6 alkylene-group), which contains the linker moiety structure (such as -NH-(B 10alkyl group having an NH2-alkyl group, the terminal amino group of which can subsequently be conjugated to a ligand represented by Formula (II) as follows, for example, to form a nucleic acid conjugate. In some embodiments, the 5'-terminal of the sense strand of the siRNA disclosed herein has one or more alkynyl groups, the terminal alkynyl groups of which can subsequently be reacted with a group of a ligand described herein to form a conjugate.

[0198] In the present context, the siRNA comprises a sense strand and an antisense strand, and a phosphorothioate bond or a phosphate for connection to a linker in the ligand structure described below. It is known from the foregoing that the NH2-alkyl group mentioned above belongs to the ligand, which is pre-connected to the sense strand of the siRNA for the convenience of synthesizing the nucleic acid conjugate of the present application.

[0199] In some preferred embodiments of the present application, the nucleic acid conjugate is as shown in Table D.

[0200] Table D

[0201] L1 has the following structure, which is conjugated to a nucleic acid sequence by compound I-1:

[0202] NAG37 has the structure:

[0203] The preparation method thereof is described in WO2023 / 070082A2.

[0204] Pharmaceutical composition

[0205] The present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the aforementioned siRNA or the aforementioned nucleic acid conjugate. As described previously, both the aforementioned siRNA and the nucleic acid conjugate can degrade the mRNA encoding CFB, inhibit the expression or activity of the CFB gene. Therefore, the pharmaceutical composition of the present application can effectively treat and prevent the related diseases mediated by complement factor B.

[0206] According to an embodiment of the present application, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier or excipient.

[0207] Method for inhibiting the expression or activity of complement factor B

[0208] The present application provides a method for inhibiting the expression or activity of complement factor B. According to an embodiment of the present application, the method comprises introducing the aforementioned siRNA or the aforementioned nucleic acid conjugate into a cell. As previously described, either the aforementioned siRNA or the aforementioned nucleic acid conjugate can degrade the mRNA encoding CFB, inhibit the expression or activity of CFB gene. Therefore, the method of the present application can be used to inhibit the expression or activity of CFB gene. For example, the expression or activity of CFB gene can be inhibited in vivo in an animal; or the expression of CFB gene can be inhibited for non-disease treatment purposes, for example, the expression or activity of CFB gene can be inhibited in isolated cells in vitro for subsequent research.

[0209] According to an embodiment of the present application, the cell is a hepatocyte and / or a renal cell.

[0210] Use

[0211] The present application provides a use of the aforementioned siRNA, the aforementioned nucleic acid conjugate or the aforementioned pharmaceutical composition, the use comprising at least one of the following: inhibiting the expression or activity of complement factor B gene; preparing a medicament for inhibiting the expression or activity of complement factor B gene; preparing a medicament for preventing and / or treating a complement factor B-mediated related disease. The method of the present application can be used to inhibit the expression or activity of CFB gene; prevent and / or treat a complement factor B-mediated related disease. For example, the expression or activity of CFB gene can be inhibited in vivo in an animal; or the expression of CFB gene can be inhibited for non-disease treatment purposes, for example, the expression or activity of CFB gene can be inhibited in isolated cells in vitro for subsequent research.

[0212] According to an embodiment of the present application, the complement factor B-mediated related disease comprises nephropathy, systemic lupus erythematosus (SLE), for example, lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy (for example, C3 glomerulopathy), age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0213] According to an embodiment of the present application, the complement factor B-mediated related disease is selected from glomerulopathy and systemic lupus erythematosus.

[0214] According to an embodiment of the present application, the complement factor B-mediated related disease is selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease.

[0215] Method for preventing and / or treating a complement factor B-mediated related disease

[0216] The present application provides a method for preventing and / or treating a complement factor B-mediated related disease. According to an embodiment of the present application, the method comprises administering to a subject a pharmaceutically acceptable dose of the aforementioned siRNA, the aforementioned nucleic acid conjugate, or the aforementioned pharmaceutical composition. According to an embodiment of the present application, the method is effective for preventing and / or treating a complement factor B-mediated related disease.

[0217] The effective amount of the siRNA, the nucleic acid conjugate or the pharmaceutical composition according to the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by one of ordinary skill in the art according to various factors (e.g., through clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

[0218] According to an embodiment of the present application, the complement factor B-mediated related disease includes nephropathy, systemic lupus erythematosus (SLE) such as lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy (e.g., C3 glomerulopathy), age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0219] According to an embodiment of the present application, the complement factor B-mediated related disease is selected from glomerulopathy, systemic lupus erythematosus.

[0220] According to an embodiment of the present application, the complement factor B-mediated related disease is selected from lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease.

[0221] The scheme of the present application will be explained below with reference to examples. Those skilled in the art will appreciate that the examples below are for illustration only and should not be taken as limiting the scope of the present application. Unless otherwise indicated, the techniques and conditions in the examples are as described in the literature or as per the product manual. Unless otherwise indicated, the reagents or instruments used are commercially available conventional products.

[0222] Preparation Example 1: Preparation of Ligand I-1 to Ligand I-3

[0223] 1. Preparation of Ligand I-1

[0224] The synthetic route is shown below:

[0225] Step 1: Synthesis of ethyl 4-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)butanoate (01B)

[0226] Into a 500 mL single-neck flask, was added N-tert-butoxycarbonyl-1,3- propanediamine (30.00 g, 172.2 mmol), acetonitrile (250 mL) and potassium carbonate (47.59 g, 344.3 mmol) successively, the mixture was cooled to 0 °C, and 4-bromobutanoic acid ethyl ester (26.87 g, 137.8 mmol) was added dropwise. After the addition was completed, the temperature was allowed to rise to room temperature and the reaction was allowed to proceed for 16 h. The reaction solution was filtered, and di-tert-butyl dicarbonate (56.36 g, 258.2 mmol) was added to the filtrate, and the reaction was allowed to proceed 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 obtain ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butanoate (01B) (45.00 g, yield 84.08%).

[0227] LC-MS, M / Z (ESI): 289.4 [M-99] +

[0228] Step 2: Synthesis of 4-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)butanoic acid (01C)

[0229] Into a 500 mL single-neck flask, was added ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butanoate (45.00 g, 115.8 mmol), methanol (200 mL) and an aqueous solution of lithium hydroxide (5.55 g, 231.8 mmol) (50 mL), and the mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure, water (50 mL) was added, 1 N hydrochloric acid was added to adjust the pH to 6, and ethyl acetate (150 mL x 3) was added for extraction. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butanoic acid (01C) (24.00 g, yield 57.49%).

[0230] 1H NMR (400 MHz, DMSO-d6) δ 6.73 (s, 1H), 3.08 (q, J = 8.6, 8.1 Hz, 4H), 2.85 (q, J = 6.6 Hz, 2H), 2.12 (t, J = 7.3 Hz, 2H), 1.67 - 1.60 (m, 2H), 1.57 - 1.49 (m, 2H), 1.36 (s, 9H), 1.35 (s, 9H).

[0231] LC-MS, M / Z (ESI): 383.4 [M+Na] +

[0232] Third Step: Synthesis of tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (01E)

[0233] Into a 500 mL single-necked flask, 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 successively. The mixture was cooled to 0 °C, and 5-bromo-1-pentene (20.53 g, 137.8 mmol) was added dropwise slowly. After the addition was completed, the mixture was allowed to warm to room temperature and stirred for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give the 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%).

[0234] 1 H NMR (400 MHz, DMSO-d6) δ 6.78 (t, J = 5.0 Hz, 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 = 6.7 Hz, 2H), 2.43 (t, J = 7.0 Hz, 4H), 2.01 (q, J = 7.3 Hz, 2H), 1.49 - 1.40 (m, 4H), 1.34 (s, 9H).

[0235] LC-MS, M / Z (ESI): 243.3 [M+H] +

[0236] Fourth Step: 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)

[0237] Into a 500 mL single necked flask, was added 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)butanoic acid (23.80 g, 66.03 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18.98 g, 99.01 mmol), 1-hydroxybenzotriazole (8.92 g, 66.01 mmol) and triethylamine (20.04 g, 198.0 mmol) successively. The mixture was stirred at room temperature for 16 h under nitrogen atmosphere. The reaction mixture was washed successively with 0.5 N hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate solution (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product which 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%).

[0238] 1 H NMR (400 MHz, DMSO-d6) δ 6.91 - 6.60 (m, 2H), 5.84 - 5.72 (m, 1H), 5.06 - 4.89 (m, 2H), 3.22 - 3.14 (m, 4H), 3.12 - 3.06 (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 = 3.8 Hz, 27H).

[0239] LC-MS, M / Z (ESI): 607.6 [M+Na] +

[0240] Fifth Step: Synthesis of N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N- (pent-4-en-1-yl)butanamide (trifluoroacetate salt) (01G)

[0241] To a 250 mL single necked flask was added tert-butyl (3-((tert- butyloxycarbonyl)amino)propyl) (4-((3-((tert-butyloxycarbonyl)amino)propyl) (pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (5.00 g, 8.55 mmol), dichloromethane (200 mL) and trifluoroacetic acid (13.09 mL, 171.1 mmol) and the mixture was stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure to give N-(3- aminopropyl)-4-((3-aminopropyl)amino)-N-(pent-4-en-1-yl)butanamide (trifluoroacetate salt) (01G) (5.34 g, 99.7% yield).

[0242] LC-MS, M / Z (ESI): 285.46 [M+H] +

[0243] Sixth Step: 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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10- (pent-4-en-1-yl)-6,10,15,19-tetraazadocosa-1,24-diyl)bis(oxy))bis(5-acetamido-2- (acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (01I)

[0244] To a 250 mL single necked flask was added N-(3-aminopropyl)-4-((3- aminopropyl)amino)-N-(pent-4-en-1-yl)butanamide (trifluoroacetate) (01G) (3.30 g, 5.27 mmol), dichloromethane (100 mL), 5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (8.26 g, 18.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.05 g, 26.3 mmol), 1-hydroxybenzotriazole (2.14 g, 15.8 mmol) and triethylamine (5.34 g, 52.8 mmol) successively under nitrogen atmosphere. The mixture was stirred at room temperature for 16 h. The reaction mixture was washed successively with 0.5 N hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, 84.5% yield)

[0245] 1 H NMR (600 MHz, DMSO-d6) δ 7.85 - 7.77 (m, 4H), 7.71 - 7.69 (m, 1H), 5.83 - 5.77 (m, 1H), 5.19 (d, J = 3.3 Hz, 3H), 5.05 - 4.91 (m, 5H), 4.46 (dd, J = 8.4, 4.1 Hz, 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).

[0246] LC-MS, M / Z (ESI): 787.26 [M+2H]2+

[0247] Step 7: Synthesis of 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)- 3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido) propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic acid (01J)

[0248] Into a 250 mL single-necked flask, (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-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazadocosa-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, 4.45 mmol), dichloromethane (30 mL), acetonitrile (30 mL), water (48 mL) were added successively, and after stirring uniformly, sodium periodate (4.76 g, 22.3 mmol) and ruthenium trichloride (18.5 mg, 89.0 μmol) were added. The mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, saturated aqueous sodium bicarbonate solution was added to the filtrate, and the aqueous phase was adjusted to pH 7-8. After separation, the aqueous phase was washed with dichloromethane (50 mL x 2), 1 N hydrochloric acid was added to adjust the pH to about 5-6, and dichloromethane (100 mL x 3) was added for extraction. 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-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic acid (01J) (3.50 g, yield 49.4%).

[0249] 1H NMR (600 MHz, DMSO-d6) δ 12.12 (s, 1H), 7.84 - 7.81 (m, 4H), 7.74 - 7.71 (m, 1H), 5.19 (d, J = 3.2 Hz, 3H), 4.94 (dd, J = 11.2, 3.3 Hz, 3H), 4.47 (d, J = 8.4 Hz, 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).

[0250] LC-MS, M / Z (ESI): 796.60 [M+2H] 2+

[0251] Eighth Step: 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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-10-(4-(4- nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadotriaconta-1,24- diyl)bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4- triyl)tetraacetate (1-1)

[0252] Into a 100 mL single necked flask was added 4-(4-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N- (3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic 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 obtained filtrate was concentrated to obtain a 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-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-(4-nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5-acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-1) (850 mg, yield 87.8%).

[0253] 1H NMR (400 MHz, DMSO-d6) δ 8.29 (dd, J = 9.2, 2.7 Hz, 2H), 7.82 - 7.67 (m, 4H), 7.74 - 7.66 (m, 1H), 7.44 (dd, J = 9.1, 2.1 Hz, 2H), 5.19 (d, J = 3.3 Hz, 3H), 4.94 (dd, J = 11.2, 3.4 Hz, 3H), 4.46 (d, J = 8.4 Hz, 3H), 4.00 (s, 9H), 3.85 (q, J = 9.2 Hz, 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).

[0254] LC-MS, M / Z (ESI): 856.5 [M+2H] 2+

[0255] 2. Preparation of Ligand I-2

[0256] The synthetic route is as follows:

[0257] First Step: Synthesis of methyl 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3- ((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02B)

[0258] Into a 100 mL single necked flask was added methyl 3-bromo-2- (bromomethyl)propanoate (2.00 g, 7.69 mmol), N-tert-butoxycarbonyl-1,3- propanediamine (5.36 g, 30.8 mmol) and acetonitrile (30 mL) successively, stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure, dichloromethane (30 mL), di-tert-butyl dicarbonate (6.72 g, 30.8 mmol) and N,N-diisopropyl ethylamine (3.98 g, 30.8 mmol) were added, stirred at room temperature for 16 h. Water 50 mL was added to the reaction, extracted with dichloromethane (50 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was 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)propanoate (02B) (3.50 g, yield 70.7%).

[0259] Second Step: Synthesis of 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoic acid (02C)

[0260] Into a 100 mL single necked flask was added methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (3.50 g, 5.44 mmol), methanol (30 mL) and aqueous lithium hydroxide (389 mg, 16.2 mmol) (10 mL) successively, stirred at room temperature for 16 h. The organic solvent was removed by concentration under reduced pressure, water 50 mL was added, the pH was adjusted to 6 using 1 N hydrochloric acid, extracted with ethyl acetate (150 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was 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)propanoic acid (02C) (3.00 g, yield 87.6%).

[0261] LC-MS, M / Z (ESI): 533.5 [M-99] + .

[0262] Step 3: Synthesis of benzyl 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02D)

[0263] Into a 500 mL single necked flask, was added 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)methyl)propanoic 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) successively, and stirred at room temperature for 16 h. The reaction was filtered, and the filtrate was added with water 30 mL, and extracted with ethyl acetate (60 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) to give benzyl 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02D) (3.00 g, yield 87.5%).

[0264] 1 H NMR (400 MHz, DMSO-d6) d 7.39 - 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).

[0265] Step 4: Synthesis of benzyl 3-((3-aminopropyl)amino)-2-(((3- aminopropyl)amino)methyl)propanoate tetra(trifluoroacetate) salt (02E)

[0266] Into a 250 mL single necked flask was added 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)methyl)propanoic acid benzyl ester (4.50 g, 6.22 mmol), dichloromethane (60 mL), trifluoroacetic acid (21.3 g, 187 mmol), stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure to give 3-((3- aminopropyl)amino)-2-(((3-aminopropyl)amino)methyl)propanoic acid benzyl ester tetra(trifluoroacetic acid) salt (02E) (4.85 g).

[0267] LC-MS, M / Z (ESI): 323.04 [M+H] + .

[0268] Fifth Step: 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-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12- ((benzyloxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5- oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran- 6,3,4-triyl)tetraacetate (02F)

[0269] To a 250 mL single necked flask was added 3-((3-aminopropyl)amino)-2-(((3- aminopropyl)amino)methyl)benzyl propanoate tetra(trifluoroacetate) salt (4.85 g, 6.23 mmol), dichloromethane (100 mL), 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (12.55 g, 28.05 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.17 g, 37.4 mmol), 1-hydroxybenzotriazole (3.37 g, 24.9 mmol), and N,N-diisopropylethylamine (12.1 g, 93.5 mmol) sequentially. The reaction was stirred at room temperature for 16 h under nitrogen. The reaction was washed with 0.5 N aqueous hydrochloric acid (100 mL) and saturated sodium bicarbonate solution (100 mL) sequentially. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to give (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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19- dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (02F) (9.00 g, 70.8% yield)

[0270] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 - 7.64 (m, 6H), 7.37 - 7.25 (m, 5H), 5.17 (d, J = 3.3 Hz, 4H), 4.98 (d, J = 10.7 Hz, 2H), 4.93 (dd, J = 11.2, 3.3 Hz, 4H), 4.45 (d, J = 8.4 Hz, 4H), 3.98 (s, 12H), 3.84 (q, J = 9.2 Hz, 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), 2.06 (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).

[0271] LC-MS, M / Z (ESI): 1020.9 [M+2H] 2+ .

[0272] Step 6: Synthesis of 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-2-((5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2- yl)oxy)pentanamido)propyl)pentanamido)methyl)propanoic acid (02G)

[0273] To a 100 mL single necked flask was added (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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19- dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)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 added sequentially. The mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature for 16 h. The reaction was filtered through celite, the filter cake was washed with methanol, and the filtrates were combined. The filtrates were concentrated under reduced pressure to give 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- 2-((5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanamido)propyl)pentanamido)methyl)propanoic acid (02G) (0.12 g, 70% yield).

[0274] 1H NMR (400 MHz, DMSO-d6) δ 7.83 - 7.81 (m, 5H), 7.72 - 7.68 (m, 1H), 5.21 (d, J = 3.3 Hz, 4H), 4.96 (dd, J = 11.2, 3.3 Hz, 4H), 4.49 (dd, J = 8.4, 3.3 Hz, 4H), 4.04 - 3.99 (m, 12H), 3.87 (q, J = 10.5, 9.8 Hz, 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).

[0275] LC-MS, M / Z (ESI): 975.9 [M+2H] 2+ .

[0276] 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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((4-nitrophenoxy)carbonyl)- 5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2- (acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-2)

[0277] To a 100 mL single necked flask was added 3-(5-(((2R,3R,4R,5R,6R)-3- acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5- (((2R,3R,4R,5R,6R)-3-acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)pentanamido)propyl)pentanamido)-2-((5-(((2R,3R,4R,5R,6R)-3-acylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)- 3-acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido) propyl)pentanamido)methyl)propanoic acid (200 mg, 0.103 mmol), dichloromethane (10 mL), 4-nitrophenol (21.4 mg, 0.154 mmol) and N,N'-dicyclohexylcarbodiimide (31.7 mg, 0.154 mmol), the mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, and the obtained filtrate was concentrated to give a crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 1:0-8:2) gave (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3- acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((4- nitrophenoxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5- oxopentane-5,1-diyl))bis(oxy))bis(5-acylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran- 6,3,4-triyl)tetraacetate (I-2) (120 mg, yield 56.5%).

[0278] 1H NMR (400 MHz, DMSO-d6) δ 8.33 - 8.29 (m, 2H), 7.79 - 7.69 (m, 6H), 7.43 - 7.31 (m, 2H), 5.18 (d, J = 3.3 Hz, 4H), 4.94 (dd, J = 11.2, 3.3 Hz, 4H), 4.45 (d, J = 8.4 Hz, 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 = 2.7 Hz, 12H), 2.04 - 2.01 (m, 4H), 1.96 (d, J = 2.0 Hz, 12H), 1.86 (s, 12H), 1.74 (s, 12H), 1.65 - 1.39 (m, 20H).

[0279] 3. Preparation of Ligand I-3

[0280] The synthesis route is as follows:

[0281] Step 1: 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- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)- 4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (03B)

[0282] To a 100 mL single necked flask was added 4-(4-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N- (3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido) butanoic acid (01J, 1.0 g, 0.63 mmol), dichloromethane (20 mL), (3R,5S)-5-{[bis(4- methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidin-3-ol (03A, 0.29 g, 0.69 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (376 mg, 0.991 mmol), 1- hydroxybenzotriazole (255 mg, 1.89 mmol), and N,N-diisopropylethylamine (244 mg, 1.89 mmol) were added successively under nitrogen protection. The reaction was stirred at room temperature for 16 h. The reaction was washed with saturated sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give a crude product. The product was purified by column chromatography (dichloromethane (2% triethylamine) / methanol (V / V) = 1:0-9:1) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1- yl)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (03B) (1.0 g, 80% yield).

[0283] 1H NMR (400 MHz, DMSO-d6) δ 7.86 - 7.65 (m, 5H), 7.32 - 7.29 (m, 4H), 7.21 - 7.17 (m, 5H), 6.90 - 6.86 (m, 4H), 5.21 (s, 3H), 5.03 - 4.89 (m, 4H), 4.48 (d, J = 8.3 Hz, 3H), 4.36 (m, 1H), 4.16 (s, 1H), 4.02 (s, 9H), 3.87 (q, J = 9.8 Hz, 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).

[0284] Step 2: Synthesis of 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3- acylamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3- (5-(((2R,3R,4R,5R,6R)-3-acylamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acylamido-4,5-diacetoxy)-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butyryl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3- yl)oxy)-4-oxobutanoic acid (I-3)

[0285] To a 100 mL single neck flask was added (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- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-10-(4-((2S,4R)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-4- oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)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) and stirred at room temperature for 16 h under nitrogen. The reaction was concentrated under reduced pressure and the crude product was purified by reverse phase preparative HPLC (column: Waters Xbridge Prep C18 (150 mm x 40 cm x 10 pm), mobile phase: A = water (0.1% ammonium bicarbonate), B = acetonitrile; gradient: 15% - 45%) to give 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy)-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4- oxobutanoic acid (I-3) (630 mg, 75.0% yield).

[0286] 1H NMR (400 MHz, DMSO-d6) δ 7.90 - 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, 4H), 5.37 - 5.28 (m, 1H), 5.21 (d, J = 2.9 Hz, 3H), 4.97 (dd, J = 11.2, 3.0 Hz, 3H), 4.49 (d, J = 8.4 Hz, 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).

[0287] Example 1: Design and synthesis of siRNA

[0288] The siRNA sequences were designed according to the mRNA encoding CFB as the target sequence, and finally the siRNA sequences in Tables A, B and C were obtained. The synthesis of siRNA sequences without conjugated groups (siRNA for short) is as follows:

[0289] 1) Synthesis of single-stranded oligoribonucleotides: The oligoribonucleotides were synthesized according to the phosphoramidite solid-phase synthesis technique (Kunkun 48-channel synthesizer). The synthesis was carried out on universal controllable multi-well glass CPG or ligand-coupled CPG. All 2'-modified RNA, DNA, ligand phosphoramidites and auxiliary reagents were commercially available reagents (Tianyuan Pharmaceutical Co., Ltd.). All phosphoramidites were dissolved in anhydrous acetonitrile and added with molecular sieves The coupling time was 200 seconds using 5-ethylthio-1H-tetrazole (ETT, Suzhou Kelun Biotech Co., Ltd.) as the activator, and the coupling ligand time was 10 minutes. A 50 mM iodine (Suzhou Kelun Biotech Co., Ltd.) pyridine (National Pharmaceutical) / water solution was used to generate phosphates, with a reaction time of 5 minutes. A 0.2 M hydrogen xanthate (Shanghai Zilve Science and Technology Development Co., Ltd.) pyridine (National Pharmaceutical) solution was used to generate phosphorothioate bonds, with a reaction time of 3 minutes. All sequences were completed after the DMT group was removed at the end of the synthesis.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] Example 2: Design and synthesis of siRNA conjugates

[0294] 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:

[0295] 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 activator (Suzhou Colema) was 8-12 minutes. The phosphate bond was constructed using 0.05M iodine solution (dissolved in pyridine / water = 9:1, Suzhou Colema); the phosphorothioate bond was generated using 0.2M xanthate (Suzhou Colema) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) solution, and the reaction time was 5 minutes. All sequences were completed after the last removal of the DMT group.

[0296] 2) Cleavage and deprotection of oligoribonucleotides bound on CPG: After the termination of solid-phase synthesis, the protecting group was removed by treating with 20% diethylamine (Guojia) in acetonitrile for 10 minutes. The resulting CPG carrier was subjected to heated ammonolysis with concentrated ammonia water (Guojia) to remove the protecting groups on the carrier and bases, and a solution containing the product was obtained after filtration.

[0297] 3) Purification of single-stranded oligoribonucleotides: The oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 20mM sodium hydroxide solution; and buffer B, containing 20mM sodium hydroxide solution, 3M sodium chloride, the target product was separated. The target product obtained was desalted by gel column (Cytiva).

[0298] 4) Coupling and purification of conjugated groups: The purified nucleic acid sequence containing NH2-C6 at the end (prepared and purified using conventional methods in the art) was first dissolved in 100mM PB buffer, and the ligand (such as compound I-1) of Preparation Example 1 was dissolved in DMF or DMSO and then added to the nucleic acid solution, and the reaction was carried out at room temperature for 4-16h. The reaction was monitored by mass spectrometry, 2 times the volume of the reaction solution of AMA solution (25% ammonia water: 40% methylamine aqueous solution = 1:1) was added, stirred at room temperature for 0.5h, and quenched with water. The mixed solution was desalted to remove organic reagents and then purified, and finally the nucleic acid chain with conjugated groups at the end was obtained.

[0299] 5) The sense strand and antisense strand obtained by chemical synthesis were base complementary paired according to the molar ratio of 1:1, the reaction condition was 70℃, 10min, and slowly recovered to room temperature, and finally the product was obtained.

[0300] Among them, the nucleotide sequences of the sense strand and antisense strand of the siRNA conjugate obtained by the present application are shown in Table D.

[0301] The structures of EVpu and EVpa are as follows:

[0302] The siRNA and conjugate obtained in Examples 1 and 2 will be verified for inhibitory activity, and the sequence of the positive control used in the experiment is as follows:

[0303] SS: (NAG37)s(invAb)sgcugugguGfUfCfugaguacuuas(invAb),

[0304] AS: usAfsaguaCfucagAfcAfcUfacagsc.

[0305] Test Example 1: Inhibition of CFB mRNA expression in Hep3B cells by siRNA and its conjugates

[0306] siRNA and its conjugates: 20 mM stock solution was prepared with Nuclease-Free Water. Hep3B cells were provided by Chengdu Pharmaron New Drug Development 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), 1% penicillin-streptomycin (HyClone, SV30010).

[0307] 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 plate (Costar-3599). The target gene CFB (Thermo, Assay ID-Hs00156060_m1) primer probe was purchased from Thermo Fisher Scientific Inc, and the Taqman primer probe of the internal reference gene GAPDH was designed and provided by Shanghai Pharmaron New Drug Development Co., Ltd.

[0308] Hep3B cells (2 x 10 4 cells / well) were inoculated into a 96-well cell plate, and siRNA was transfected into cells with Lipofectamine™ RNAiMAX at the same time, 9 concentration points of siRNA were tested (with 10 nM as the starting concentration, 6-fold dilution), 3 replicate wells, and Lipofectamine TMThe no compound control group of RNAiMAX was cultured in a 5% CO2 incubator at 37°C for 48 hours. Some siRNAs were tested at a concentration of 1 nM. After 48 hours of transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® Plus Universal 96 Kit (QIAGEN-74182) according to the kit instructions. cDNA was synthesized using the FastKing RT kit (with gDNase) / FastKing cDNA First-Strand Synthesis Kit (TIANGEN-KR116-03) according to the instructions.

[0309] The target gene cDNA will be detected by qPCR, and GAPDH cDNA will be detected as an internal control for parallel detection. Add 8 μL of prepared PCR reaction solution and 2 μL of sample cDNA to a 384-well plate. The qPCR reaction program is as follows: preheat at 50°C for 2 minutes, heat at 95°C for 10 minutes, then enter the cycle mode, heat at 95°C for 15 seconds, then at 60°C for 1 minute, for a total of 40 cycles.

[0310] The expression level of the target gene mRNA of each sample is calculated by the ΔΔCT relative quantification method. The relative expression of the target gene is represented by 2-ΔΔCT.

[0311] The calculation formula is as follows:

[0312] ΔCT = average CT value of target gene - average CT value of GAPDH gene;

[0313] ΔΔCt = ΔCT (sample group) - ΔCT (Lipofectamine TM RNAiMAX control group);

[0314] Relative expression of target gene = 2-ΔΔCT;

[0315] Inhibition rate % = (1 - sample relative expression / Lipofectamine TM RNAiMAX control average expression) x 100;

[0316] GraphPad Prism software was used for graphing and analysis. The test results are shown in the following table.

[0317] Table 1

[0318] Table 2

[0319] Table 3

[0320] The results show that the siRNA and conjugate obtained by the application have a significant silencing effect on the expression of CFB gene in Hep3B cells.

[0321] Test Example 2: Inhibition of hCFB expression in Tg-hCFB mice by siRNA conjugate

[0322] A 6-8 week old male Tg-hCFB transgenic mouse model (purchased from Bio-Swift Biotech Co., Ltd.) was used to evaluate the in vivo efficacy of the CFB siRNA conjugate.

[0323] The test sample was prepared with normal saline (0.4 mg / mL) on the day of administration, stored at room temperature, and the remaining test sample was stored in a -20℃ refrigerator. The animals were divided into a normal control group and a test drug group, with 5 mice in each group. The mice were grouped according to the hCFB protein level in the serum (day-3 blood collection), and the test drug group was given a single subcutaneous injection of the test drug, and the normal control group was given normal saline of the same volume. The day of administration is Day 0. Starting from one day before administration, the mice were weighed once a week.

[0324] Each mouse was taken about 200 μL of blood from the medial canthus one day before administration (Day-1), Day 7, Day 14, Day 21, Day 28, and Day 35 after administration. The whole blood sample was centrifuged at 3000r for 10 minutes after 37℃ water bath for 1 hour. The supernatant was collected as a freshly obtained serum sample, and the hCFB protein was detected by ELISA method. At the end of the test, the mouse liver was taken, and the hCFB mRNA expression in the liver tissue was detected by qPCR.

[0325] The test data is expressed as mean ± standard deviation (Mean ± SD), and the data is analyzed by GraphPad Prism 8.3 analysis software. T-test is used for data statistical analysis, and P<0.05 indicates statistical significance. The hCFB level of each animal at a certain time point is divided by the pre-administration (Day-1) level of the animal to determine the expression remaining rate. The expression remaining rate of a single animal in the test drug group at a certain time point is divided by the average expression remaining rate of all mice in the control group to obtain the "normalized expression remaining rate", and the average value of each group is obtained.

[0326] The results show that the siRNA and conjugate obtained by the application have a significant and persistent silencing effect on the expression of hCFB in Tg-hCFB mice. The test results of different batches of samples are shown in Table 4-1 and Table 4-2.

[0327] Table 4-1

[0328] Table 4-2 “-” in the table means not tested.

[0329] Test Example 3: Inhibition of CFB mRNA expression by free uptake of siRNA conjugates by primary hepatocytes of cynomolgus monkeys The prepared siRNA conjugates were configured into corresponding concentration stock solutions with Nuclease-Free Water.

[0330] The main reagents and consumables used in this experiment include: AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-P8), 2x Color SYBR Green qPCR Master Mix (ROX2) (EZB-A0012-R2-L), EZ-Press 96 RNA Purification Kit (EZB-EZ4001-L), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323), and 96-well cell culture plates (Cellpro-803096). The primers and probes of the target gene CFB and the reference gene β-actin were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. Primary hepatocytes of cynomolgus monkeys (PCH, Catalog No.: CCH100CY-V10088) were provided by Chengdu Pharmaron New Drug Development Co., Ltd.

[0331] PCH cells (4.5x10 4Cells were plated at 10,000 cells / well into pre-collagen coated 96-well cell culture plates, plating was done in the presence of test siRNA compounds, 8 concentration points of test siRNA were tested, starting at 200nM concentration, 5-fold dilution, 2 replicates, a no compound Nuclease-Free Water control group was also set up, cells were incubated at 37℃ in a 5% CO2 incubator for 48 hours. After 48 hours, the culture medium was removed and cells were collected for RNA extraction. Some compounds were tested at 100nM concentration. After 48 hours of incubation, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using EZ-Press 96 RNA Purification Kit (EZB-EZ4001-L) according to the kit instructions. cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323) according to the instructions. Target gene CFB will be detected by SYBR qPCR, while detecting β-actin cDNA as an internal control for parallel detection. 8 μL of prepared PCR reaction solution and 2 μL of sample cDNA were added to a 384-well plate. The qPCR reaction program was as follows: heating at 95℃ for 5 minutes, then entering the cycle mode, heating at 95℃ for 10 seconds, then at 60℃ for 30 seconds, for a total of 40 cycles.

[0332] The expression level of each sample target gene mRNA was calculated by ΔΔCT relative quantification method. The relative expression of the target gene was expressed using 2 -ΔΔCT The calculation formula is as follows:

[0333] ΔCT = average CT value of target gene - average CT value of internal reference gene

[0334] ΔΔCT = ΔCT (sample group) - ΔCT (control group)

[0335] Relative expression of target gene = 2 -ΔΔCT

[0336] Inhibition rate % = (1 - relative expression of sample / average relative expression of control group) x 100

[0337] The test results are shown in Tables 5 and 6.

[0338] Table 5

[0339] Table 6

[0340] The results show that the siRNA and conjugates of the present application have a significant silencing effect on the expression of CFB gene in cynomolgus monkey primary hepatocytes under free uptake conditions.

[0341] Test Example 4: Inhibition of CFB mRNA expression by free uptake of siRNA conjugates in human primary hepatocytes

[0342] The siRNA conjugates prepared in Example 2 were configured into corresponding concentration stock solutions with Nuclease-Free Water.

[0343] The diluted siRNA conjugates were added to collagen-coated 96-well cell plates, followed by the addition of a primary human hepatocyte (PHH) (from Chengdu Pharming) cell suspension. At the same time, an siRNA conjugate-free Nuclease-Free Water control group was set up. Incubation was carried out in a 5% CO2, 37°C incubator for 48 h.

[0344] After 48 h of free uptake, the culture medium was removed, the cell plates were washed once with PBS, and cell lysis solution was added. Total RNA was extracted using an RNA extraction kit (Qiagen-74182). After gDNA removal using a reverse transcription kit (HiScript III RT SuperMix for qPCR, Vazyme-R323-01), cDNA was synthesized using random primers.

[0345] Target gene cDNA was detected by qPCR, and the corresponding internal reference gene (GAPDH cDNA) was detected in parallel. The qPCR reaction program (TapMan Probe) was as follows: 50°C for 2 min, 95°C for 10 min, then enter the cycle mode, 95°C for 10 s, then 60°C for 1 min, a total of 40 cycles.

[0346] The expression level of the target gene mRNA of each sample was calculated by the ΔΔCT quantitative method. The relative expression of the target gene was represented by 2-ΔΔCT.

[0347] The calculation formula is as follows:

[0348] ΔCT = average Ct value of target gene - average Ct value of internal reference gene;

[0349] ΔΔCT = ΔCT (drug group) - ΔCT (Nuclease-Free Water control group);

[0350] Relative expression of target gene = 2-ΔΔCT;

[0351] Inhibition rate of target gene = (1 - sample relative expression / Nuclease-Free Water control average expression) x 100%;

[0352] The test results are shown in Table 7.

[0353] Table 7

[0354] The results show that the siRNA and conjugate of the application have a significant silencing effect on the CFB gene expression in human primary hepatocytes under free uptake conditions.

[0355] Test Example 5: In vitro dual luciferase psiCHECK-2 vector HEK293 cell screening

[0356] In a 96-well cell plate, 3 μL of siRNA prepared in Example 1 was added to each well, with two replicates for each siRNA, then 2 μL of psiCHECK-2 vector plasmid (referred to as plasmid, from Tsingke Biotech) carrying the CFB gene with a concentration of 100 ng / μL was added to each well, 20 μL of Opti-MEM medium was added to obtain a mixture containing siRNA and plasmid. 0.4 μL of Lipofectamine 2000 (Invitrogen, item number 11668-019) was added to 24.6 μL of Opti-MEM medium (Gibco, item number 31985-070), incubated for 5 minutes, and then added to the mixture containing siRNA and plasmid in each well. After incubation at room temperature for 20 minutes, 100 μL of Dulbecco's Modified Eagle Medium (Gibco, item number C11995500BT) containing 5×10 4 HEK293 cells was added to the mixture containing siRNA, plasmid and Lipofectamine 2000 transfection reagent in each well. After incubating the cells for 48 hours, firefly luciferase and sea pansy luciferase were measured (Novizhan, DD1205-02). The test concentration range was 10 nM to 25.6 fM, with a five-fold concentration stepwise dilution.

[0357] The measurement results of some siRNAs at some concentrations in this test example are shown in Table 8.

[0358] Table 8

[0359] The results show that the siRNA and conjugate of the application have a significant silencing effect on the CFB gene expression.

[0360] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0361] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A siRNA, characterized in that, a double-stranded region comprising a sense strand and an antisense strand having at least partial base pairing with the sense strand; wherein the sense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof; the antisense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof; each nucleotide in the sense strand and / or antisense strand is independently a modified or unmodified nucleotide.

2. The siRNA of claim 1, wherein the siRNA comprises one or more of the following conditions: a) the sense strand comprises a nucleotide sequence as set forth 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 the same by no more than 5, 4, 3, 2, 1 nucleotides; b) the antisense strand comprises a nucleotide sequence as set forth 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 the same by no more than 5, 4, 3, 2, 1 nucleotides; c) the siRNA comprises the sense strand and antisense strand of any one of the groups in Table A, or differs from the same by no more than 5, 4, 3, 2, 1 nucleotides, respectively; Table A d) the sense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof; e) the antisense strand comprises a nucleotide sequence as set forth 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 contiguous nucleotides thereof; f) the length of the sense strand, antisense strand is independently 17-25 nucleotides, preferably 19-25 nucleotides.

3. The siRNA of claim 1, wherein the siRNA comprises one or more of the following conditions: 1) the double-stranded region has a length of 19-23 nucleotides; 2) the length of the sense strand is no more than 23 nucleotides, preferably no more than 21 nucleotides; 3) the length of the antisense strand is no more than 25 nucleotides, preferably no more than 23 nucleotides; 4) the siRNA is selected from any one of: 4-1) the 3' end and 5' end of the sense strand are both blunt ends, and the 3' end and 5' end of the antisense strand are both blunt ends; 4-2) the sense strand contains a 3' overhang or a 5' overhang, and both the 3' end and the 5' end of the antisense strand are blunt ends; or the antisense strand contains a 3' overhang or a 5' overhang, and both the 3' end and the 5' end of the sense strand are blunt ends; 4-3) the sense strand contains a 3' overhang or a 5' overhang, and the antisense strand contains a 3' overhang or a 5' overhang; 5) each nucleotide in the 3' overhang and / or the 5' overhang of the sense strand and / or the 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, which is independently selected from at least one of the following: inverted deoxy nucleotides, 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-0-allyl modified nucleotides, 2'-C 1～25 alkyl modified nucleotides, 2'-0-C 1～25 alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, locked nucleotides, configuration restricted nucleotides, 2'-allyl modified nucleotides, abasic nucleotides, morpholino nucleotides, tetrahydropyranyl modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, PEG modified nucleotides, 5'-aminophosphoramidate modified nucleotides, 5'-phosphorothioato modified nucleotides, 5'-methylphosphonate modified nucleotides, 5'-phosphate mimic modified nucleotides, 5'-methylated cytosine modified nucleotides, nucleic acid analogs.

4. The siRNA according to claim 1 or 3, characterized in that, The siRNA comprises one or more of the following conditions: 1) the double-stranded region has a length of 19-21 nucleotides; 2) the 3' overhang and / or the 5' overhang comprises 2 nucleotides or analogs thereof; 3) the 3' overhang and / or the 5' overhang is selected from UU, TT, dUdU, or dTdT; 4) the modified nucleotide is independently selected from at least one of inverted deoxyabasic nucleotide, 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, and 5'-phosphorothioate modified nucleotide; 5) all nucleotides in the sense strand are modified nucleotides; 6) all nucleotides in the antisense strand are modified nucleotides.

5. The siRNA of claim 4, wherein The siRNA comprises one of the following conditions: 1) in the sense strand and / or the antisense strand, 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 from the start; 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 from the start; 2) in the antisense strand, 5'-phosphorothioate modified nucleotide is present at the following positions: the nucleotide at the 5' end of the antisense strand is at least one of the 1st and 2nd positions from the start, and / or, the nucleotide at the 3' end of the antisense strand is at least one of the 1st and 2nd positions from the start; 3) the 1st 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, 2'-methoxy modified nucleotide is present at the following positions: the nucleotide at the 5' end of the sense strand is at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, and 21st positions from the start; and / or, the nucleotide at the 5' end of the antisense strand is at least one of the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions from the start; 5) one or more inverted abasic nucleotides are added to the 3' end and / or 5' end of the sense strand.

6. The siRNA of claim 5, wherein The siRNA comprises one of the following conditions: 1) the double-stranded region has 21 nucleotides, the modified nucleotides of the sense strand and the antisense strand are as follows: 2'-fluoro-modified nucleotides are present at the following positions: the 9th, 11th and 13th nucleotides from the 5' end of the sense strand; and, the 2nd, 7th, 12th, 14th and 16th nucleotides from the 5' end of the antisense strand; 5'-phosphorothioate-modified nucleotides are present at the following positions: the 1st and 2nd nucleotides from the 5' end of the sense strand; and, the 1st and 2nd nucleotides from the 3' end of the antisense strand; 2'-methoxy-modified nucleotides are present at the following positions: the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st nucleotides from the 5' end of the sense strand; and, the 1st, 3rd, 4th, 5th, 6th, 8th, 9th, 10th, 11th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st nucleotides from the 5' end of the antisense strand; 2) one inverted abasic nucleotide is added to the 3' end and 5' end of the sense strand; 3) the first nucleotide of the 3' end of the sense strand is linked to the inverted abasic nucleotide via a 5'-phosphorothioate group; 4) the first nucleotide of the 5' end of the sense strand is linked to the inverted abasic nucleotide via a 5'-phosphorothioate group.

7. The siRNA of claim 1, wherein The siRNA comprises the sense strand and the antisense strand of any one of the groups in Table B or Table C, or differs from the sense strand and / or the antisense strand by no more than 5, 4, 3, 2, 1 nucleotides; Table B Table C 8. A nucleic acid conjugate, characterized in that, comprises: the siRNA of any one of claims 1 to 7, and a ligand conjugated to the siRNA.

9. The nucleic acid conjugate of claim 8, wherein, satisfies one or more of the following conditions: 1) the ligand is one or more of: 2) the ligand is coupled to the sense strand and / or the antisense strand of the siRNA; preferably to the sense strand; 3) the ligand is selected from the group consisting of a lipid, a carbohydrate, an aptamer, a vitamin and / or a peptide that specifically binds to a cell membrane or a cell surface; 4) the ligand has one or more of the following structures: wherein, represents the point of attachment of the ligand to the siRNA.

10. The nucleic acid conjugate of claim 8, wherein, The nucleic acid conjugate comprises the sense strand and the antisense strand of any one of the groups in Table D; Table D 11. A pharmaceutical composition, characterized by, comprises: the siRNA of any one of claims 1 to 7 or the nucleic acid conjugate of any one of claims 8 to 10; optionally, the pharmaceutical composition further comprises: a pharmaceutically acceptable carrier.

12. Use of the siRNA of any one of claims 1 to 7, the nucleic acid conjugate of any one of claims 8 to 10 or the pharmaceutical composition of claim 11, the use comprising at least one of the following: inhibiting expression or activity of a complement factor B gene; manufacturing a medicament for inhibiting expression or activity of a complement factor B gene; manufacturing a medicament for preventing and / or treating a complement factor B-mediated related disease; preventing and / or treating a complement factor B-mediated related disease.

13. Use according to claim 12, characterized in that, The complement factor B-mediated related disease includes nephropathy, systemic lupus erythematosus; Preferably, the complement factor B-mediated related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy, 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 related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease.

14. The siRNA of any one of claims 1 to 7, the nucleic acid conjugate of any one of claims 8 to 10, or the pharmaceutical composition of claim 11, for at least one of: inhibiting expression or activity of a complement factor B gene; preventing and / or treating a complement factor B-mediated related disease.

15. The siRNA of any one of claims 1 to 7, the nucleic acid conjugate of any one of claims 8 to 10, or the pharmaceutical composition of claim 11, for at least one of: inhibiting expression or activity of a complement factor B gene; preventing and / or treating a complement factor B-mediated related disease.

15. The siRNA of any one of claims 1 to 7, the nucleic acid conjugate of any one of claims 8 to 10, or the pharmaceutical composition of claim 11, for at least one of: inhibiting expression or activity of a complement factor B gene; preventing and / or treating a complement factor B-mediated related disease. The complement factor B-mediated related disease includes nephropathy, systemic lupus erythematosus; Preferably, the complement factor B-mediated related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy, 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 related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease. A subject having a complement factor B-mediated related disease is administered a pharmaceutically acceptable dose of the siRNA of any one of claims 1 to 7, the nucleic acid conjugate of any one of claims 8 to 10, or the pharmaceutical composition of claim 11. The complement factor B-mediated related disease includes nephropathy, systemic lupus erythematosus; Preferably, the complement factor B-mediated related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease, membranous nephropathy, glomerulopathy, age-related macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

16. A method of preventing and / or treating a complement factor B mediated related disease, comprising administering a therapeutically effective amount of the compound of claim 1 to a subject in need thereof. Preferably, the complement factor B-mediated related disease is selected from the group consisting of lupus nephritis, IgA nephropathy, diabetic nephropathy, polycystic kidney disease.

17. The method of claim 16, wherein, ​ ​ ​

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