Modified siRNA with reduced off-target activity

Abstract

The present invention relates to a modified siRNA with reduced off-target activity. The siRNA comprises a sense strand and an antisense strand containing a chemical modification represented by formula (I) or a tautomer modification thereof at at least one nucleotide position from positions 2 to 8 in the 5' region of the sense strand. The present invention also relates to a complex, pharmaceutical composition, cell, or reagent kit containing the siRNA, and pharmaceutical uses of the siRNA, the complex, and / or the pharmaceutical composition. The present invention also relates to compounds represented by formula (II) and formula (III) or tautomers thereof, and methods for preparing the same.

Description

[Technical Field]

[0001] This application claims priority to Chinese patent applications CN202010772542.6, filed on August 4, 2020; CN202110244977.8, filed on March 5, 2021; and CN202110361502.7, filed on April 2, 2021. This application cites the original texts of the aforementioned Chinese patent applications.

[0002] This disclosure relates to siRNA that suppresses the expression of target genes, and more specifically, to modified siRNA with reduced off-target activity. [Background technology]

[0003] RNA interference (RNAi) is an efficient method for silencing gene expression. Statistics show that over 80% of disease-related proteins in the human body are currently untargetable by conventional small molecule drugs and large molecule formulations, making drug discovery impossible. RNA interference technology allows for the design of appropriate siRNAs using the mRNA encoding these proteins, specifically targeting and degrading the target mRNA, thereby suppressing the production of the associated proteins. Therefore, siRNA holds significant potential for drug development.

[0004] However, siRNAs often exhibit various off-target effects. One such off-target effect is miRNA-like off-target activity, specifically the inhibitory activity against mRNA resulting from complete or incomplete pairing between the seed region (positions 2-8 at the 5' end) of the siRNA antisense strand (also known as the AS strand) and the target mRNA. The off-target effect of a single siRNA molecule can affect multiple mRNAs. Consequently, this can lead to unpredictable toxicity and side effects, which is a contributing factor to the toxicity and side effects of siRNA drugs (Janas, MM, Schlegel, MK, Harbison, CE et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 9, 723 (2018)). [Overview of the Initiative]

[0005] siRNA This disclosure provides an siRNA that maintains (and ultimately enhances) siRNA on-target activity while suppressing or reducing siRNA off-target activity by introducing chemical modifications to its seed region.

[0006] This disclosure provides an siRNA comprising a sense strand and an antisense strand, each strand having 15 to 35 nucleotides, wherein the antisense strand includes a chemical modification represented by formula (I) or a tautomer thereof at at least one nucleotide position in its 5' region from position 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8), [ka] (I) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. J2 is H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. Q1 is [ka] Q2 is R2, or Q1 is R2, Q2 is [ka] And, Eventually, R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. J1 is H or a C1-C6 alkyl group. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue. Of these, the chemical modification shown by formula (I) above is [ka] isn't it.

[0007] In some embodiments, the antisense strand includes a chemical modification represented by formula (I-1) or a tautomer modification thereof at at least one nucleotide position in its 5' region from position 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8), [ka] (I-1) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. Each of J1 and J2 is independently H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring.

[0008] In some embodiments, the antisense strand includes a chemical modification represented by formula (I-2) or a tautomer thereof at at least one nucleotide position in its 5' region from position 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8), [ka] (I-2) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C6 alkyl group. R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) qSelected from R7, among which R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, q = 1, 2 or 3, R2 is selected from H, C1-C6 alkyl group, C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r Selected from R8, among which R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, r = 1, 2 or 3, Optionally, R1 and R2 are directly linked to form a ring.

[0009] In some embodiments, the nucleotide containing the chemical modification represented by formula (I) or its tautomeric modification is a nucleotide containing the chemical modification represented by formula (I') or its tautomeric modification, [[ID=X]] (I’) Among which, Y is selected from O, NH and S, Each X is independently selected from CR4(R4’), S, NR5 and NH-CO, among which, R4, R4’ and R5 are each independently H or a C1-C6 alkyl group, J2 is H or a C1-C6 alkyl group, n = 0, 1 or 2, m = 0, 1 or 2, s = 0 or l, [[ID=X]] R3 is selected from H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p Selected from R6, among which R6 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, p = 1, 2 or 3, Q 1’ is Q 2’ is R2, or Q 1’ R2, Q 2’ teeth [ka] And, Of these, R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. J1 is H or a C1-C6 alkyl group. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. M is either O or S. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue. Of these, the chemical modification shown by formula (I') above is: [ka] isn't it.

[0010] In some embodiments, the antisense strand includes a chemical modification represented by formula (I-3) or a tautomer thereof at at least one nucleotide position in its 5' region from position 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8), [ka] (I-3) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. Each of J1 and J2 is independently H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. M is either O or S. R1 and R2 are arbitrarily connected directly to form a ring.

[0011] In some embodiments, the antisense strand includes a chemical modification represented by formula (I-4) or a tautomer modification thereof at at least one nucleotide position in its 5' region from position 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8), [ka] (I-4) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C6 alkyl group. R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 consists of H, C1-C6 alkyl, C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. M is either O or S. R1 and R2 are arbitrarily connected directly to form a ring.

[0012] In some embodiments, the above chemical modification is [ka] isn't it.

[0013] In some embodiments, when X is NH-CO, R1 is not H.

[0014] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are each independently H or C1-C3 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C3 alkyl group. R3 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2 or 3. R1 consists of H, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and q = 1, 2 or 3. R2 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) rR8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0015] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) p R6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0016] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) pR6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0017] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2 and NH. n=0 or 1, m=0 or 1, s=0 or 1, Each of J1 and J2 is independently H. R1 is selected from H, a methyl group, and CH2OH. R2 is selected from H, OH, NH2, a methyl group, and CH2OH, R3 is selected from H, OH, NH2, a methyl group, and CH2OH, Optionally, R1 and R2 are directly linked to form a ring, B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0018] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2, and NH, n = 0 or 1, m = 0 or 1, s = 0 or 1, Each J1 and J2 is independently H, R1 is selected from H, a methyl group, and CH2OH, R2 is selected from H, a methyl group, and CH2OH, R3 is selected from H, OH, NH2, a methyl group, and CH2OH, Optionally, R1 and R2 are directly linked to form a ring, B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0019] In some embodiments, the chemical modification represented by the above formula (I) is

Chemical formula

[0020] In some embodiments, B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0021] In some other embodiments, B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0022] In some embodiments, B is a base at the corresponding position in the 5' region of the antisense strand, from position 2 to position 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8).

[0023] In some specific embodiments, B is a native base at the corresponding position in the 5' region of the antisense strand, from position 2 to position 8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8).

[0024] In some embodiments, the chemical modification represented by formula (I) above is [ka] B is selected from among a base or a base analogue, for example, from purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0025] In some embodiments, B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0026] In some other embodiments, B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0027] In some embodiments, the chemical modification represented by formula (I) above is [ka] B is selected from among a base or a base analogue, for example, from purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0028] In some embodiments, B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0029] In some other embodiments, B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

[0030] In some other embodiments, B is selected from adenine, guanine, cytosine, uracil and thymine.

[0031] In some embodiments, the chemical modification represented by the above formula (I) is

Chem.

Chem.

[0032] [[ID=२५]] In some embodiments, B is selected from purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, purine modified at C2, purine modified at N8, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, pyrimidine modified at C5, thymine, indole, 5-nitroindole and 3-nitropyrrole.

[0033] In some other embodiments, B is selected from purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.

[0034] In some embodiments, the chemical modification represented by formula (I) above is [ka] Selected from among, of which M is either O or S, B is a base or a base analogue, selected from, for example, purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0035] In some embodiments, B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0036] In some other embodiments, B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0037] In some embodiments, the chemical modification represented by formula (I) above is [ka] Selected from among, of which M is either O or S, B is a base or a base analogue, selected from, for example, purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0038] In some embodiments, B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0039] In some other embodiments, B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0040] In some other embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0041] In some embodiments, the chemical modification represented by formula (I) above is [ka] This includes, but is not limited to, those in which adenine in their structure is substituted with guanine, cytosine, uracil, or thymine.

[0042] In some embodiments, the antisense strand includes a chemical modification represented by formula (I) or a tautomer thereof at at least one nucleotide position in its 5' region between positions 2 to 8, 3 to 8, 4 to 8, 5 to 8, and 5 to 7.

[0043] In some embodiments, the antisense strand includes a chemical modification represented by formula (I) or a tautomer thereof at the nucleotide position 5, 6, or 7 of its 5' region.

[0044] In some embodiments, the antisense strand includes a chemical modification represented by formula (I) or a tautomer thereof at the nucleotide position 7 of its 5' region.

[0045] In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

[0046] In some embodiments, the lengths of the sense strand and the antisense strand are homologous or different, with the sense strand having 19 to 23 nucleotides and the antisense strand having 19 to 26 nucleotides. The sense strand to antisense strand length ratio of the siRNA provided in this disclosure may be 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the sense strand-to-antisense strand length ratio of the siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0047] In some embodiments, the antisense strand is at least partially inversely complementary to the target sequence in order to mediate RNA interference; in some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer mismatches between the antisense strand and the target sequence; and in some embodiments, the antisense strand is completely inversely complementary to the target sequence.

[0048] In some embodiments, the sense strand is at least partially inversely complementary to the antisense strand in order to form a double-stranded region; in some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, and 1 or fewer mismatches between the sense strand and the antisense strand; and in some embodiments, the sense strand is completely inversely complementary to the antisense strand.

[0049] This disclosure further provides a modified siRNA wherein, in addition to a nucleotide comprising the chemical modification represented by formula (I) or a tautomer thereof, at least one of the sense strand and / or antisense strand further comprises at least one other modified nucleotide.

[0050] In some embodiments, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides, with the exception of the nucleotide containing the chemical modification represented by formula (I) or its tautomer modification.

[0051] In some embodiments, the other modified nucleotides are independently selected from deoxy-nucleotides, 3'-terminal deoxythymine nucleotides, nucleotides modified with a 2'-O-methyl group, nucleotides modified with a 2'-fluoro group, nucleotides modified with a 2'-deoxy- group, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, nucleotides modified with a 2'-amino- group, nucleotides modified with a 2'-O-allyl- group, nucleotides modified with a 2'-C-alkyl- group, nucleotides modified with a 2'-hydroxy- group, nucleotides modified with a 2'-methoxyethyl group, nucleotides modified with a 2'-O-alkyl- group, morpholinyl nucleotides, aminophosphates, non-natural base-containing nucleotides, nucleotides modified with tetrahydropyranyl, nucleotides modified with 1,5-anhydrohexitol, nucleotides modified with a cyclohexenyl group, thiophosphate-containing nucleotides, methylphosphate-containing nucleotides, 5'-phosphate-containing nucleotides, and 5'-phosphate mimetic-containing nucleotides.

[0052] In some embodiments, the other modified nucleotides are independently selected from nucleotides modified with a 2'-alkoxy group, nucleotides modified with a 2'-substituted alkoxy group, nucleotides modified with a 2'-alkyl group, nucleotides modified with a 2'-substituted alkyl group, nucleotides modified with a 2'-amino group, nucleotides modified with a 2'-substituted amino group, nucleotides modified with a 2'-fluoro group, 2'-deoxynucleotides, nucleotides modified with a 2'-deoxy-2'-fluoro group, 3'-deoxy-thymine nucleotides, isonucleotides, LNA, ENA, cET, UNA, and GNA.

[0053] In some embodiments, the other modified nucleotides are independently selected from nucleotides modified with a 2'-methoxy group, nucleotides modified with a 2'-fluoro group, and nucleotides modified with a 2'-deoxy group.

[0054] In the context of this disclosure, a fluoromodified nucleotide is a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with a fluoropolymer. In some embodiments, a 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe). In some embodiments, a 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl-modified nucleotide (2'-MOE) or a 2'-amino-modified nucleotide (2'-NH2).

[0055] In some embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0056] In some embodiments, the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0057] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0058] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0059] In some embodiments, at least one phosphate group in the sense strand and / or antisense strand is a phosphate group having a modifying group. The modifying group provides the siRNA with improved stability in biological samples or the environment.

[0060] In some embodiments, the phosphate group having the above-mentioned modifying group is a thiophosphate group. Specifically, a thiophosphate group is a phosphodiester group modified by substituting one non-bridged oxygen atom with a sulfur atom.

[0061] In some embodiments, the thiophosphate group is Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 3' end of the sense strand mentioned above, Between the second and third nucleotides at the 3' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, and Between the second and third nucleotides at the 3' end of the antisense strand mentioned above, It is located in at least one position selected from the given positions.

[0062] In some embodiments, the sense chain is 5'-N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a N a N a N ahaving a nucleotide sequence represented by the formula -3', wherein each N a and N b each independently represents a modified nucleotide or an unmodified nucleotide, and the modifications in N a and N b are different, and / or, the antisense strand is 5'-N a 'N b 'N a 'X'N a 'N b 'W'N a 'N b 'N a 'N a 'N b 'N a 'N b 'N a 'Y'N a 'X'N a 'N a 'N a ' -3' having a nucleotide sequence represented by the formula, wherein each N a ' and N b ' each independently represents a modified nucleotide or an unmodified nucleotide, among which the modifications in N a ' and N b ' are different, each X' independently represents N a ' or N b ', and Y' is N a ' or N b ', and W' represents a nucleotide containing the chemical modification represented by the above formula (I) or its tautomeric modification.

[0063] In some embodiments, the sense strand is 5'-N a N a N a N a N b N a N b N b N b N a N a N a Na N a N a N a N a N a N a having a nucleotide sequence represented by the formula -3', wherein each N a and N b each independently represents a modified nucleotide or an unmodified nucleotide, and the modifications in N a and N b are different, and / or the antisense strand is 5'-N a 'N b 'N a 'X'N a 'W'N a 'N a 'N b 'N a 'N a 'N b 'N a 'N b 'N a 'Y'N a 'X'N a 'N a 'N a ' -3' having a nucleotide sequence represented by the formula, wherein each N a ' and N b ' each independently represents a modified nucleotide or an unmodified nucleotide, wherein the modifications in N a ' and N b ' are different, each X' independently represents N <​​​​​​​​​​​​​​​​​​​​​​​a N b N b N b N a N a N a N a N a N a N a N a N a N a It has a nucleotide sequence represented by the formula -3', Of those, each N a and N b Each of these independently represents a modified nucleotide or an unmodified nucleotide, and N a and N b The modifications in these are different, and / or The antisense chain is, 5'-N a 'N b 'N a 'X'W'N b 'N a 'N a 'N b 'N a 'N a 'N b 'N a 'N b 'N a 'Y'N a 'X'N a 'N a 'N a It has a nucleotide sequence represented by the formula '-3', Of those, each N a 'and N b ' indicates independently modified nucleotides or unmodified nucleotides, of which N a 'and N b The modifications in ' are different, and each X' is independently N a 'or N b ' is N a 'or N b 'W' represents a nucleotide containing the chemical modification shown in formula (I) above or its tautomer modification.

[0065] In some embodiments, Na N is a nucleotide modified with a 2'-methoxy group, b These are nucleotides modified with 2'-fluoro or 2'-deoxy.

[0066] In some embodiments, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' is a nucleotide modified with 2'-fluoro or 2'-deoxy-.

[0067] In some embodiments, at least one phosphate group in the sense strand and / or antisense strand is a phosphate group having a modifying group, and the modifying group provides the siRNA with improved stability in a biological sample or environment.

[0068] In some embodiments, the phosphate group having the above-mentioned modifying group is a thiophosphate group. Specifically, a thiophosphate group is a phosphodiester group modified by substituting one non-bridged oxygen atom with a sulfur atom.

[0069] In some embodiments, the thiophosphate group is Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 3' end of the sense strand mentioned above, Between the second and third nucleotides at the 3' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, and Between the second and third nucleotides at the 3' end of the antisense strand mentioned above, It is located in at least one position selected from the given positions.

[0070] In some embodiments, the sense chain is It has a nucleotide sequence represented by the formula 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-2', Of these, Nm represents any nucleotide modified with a methoxy group, for example, C, G, U, A, T modified with a methoxy group, Nf represents any nucleotide modified with a fluoro group, for example, C, G, U, A, T modified with a fluoro group, the lowercase s indicates that two nucleotides adjacent to the s are linked by a thiophosphate group, and / or The antisense chain is, It has a nucleotide sequence represented by the formula 5'-Nms'Nfs'Nm'Nm'Nm'Nf'W'Nm'Nm'Nm'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nm'Nms'Nms'Nm'-3', Of these, Nm' represents any nucleotide modified with a methoxy group, for example, C, G, U, A, and T modified with a methoxy group; Nf' represents any nucleotide modified with a fluoro group, for example, C, G, U, A, and T modified with a fluoro group; the lowercase s indicates that two nucleotides adjacent to the s are linked by a thiophosphate group; and W' represents a nucleotide containing the chemical modification shown in formula (I) above or its tautomer modification.

[0071] In some embodiments, the nucleotides at positions 2, 6, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0072] In some embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0073] In some embodiments, the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0074] In some embodiments, the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0075] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0076] In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0077] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0078] In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0079] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 10, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-deoxynucleotides or 2'-fluoronucleotides.

[0080] In some embodiments, the nucleotides at positions 2, 6, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are independently nucleotides modified with 2'-fluoropolymers.

[0081] In some embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand, in the direction from the 5' end to the 3' end, are independently nucleotides modified with 2'-fluoropolymers.

[0082] In some embodiments, the nucleotides at positions 2, 6, 12, and 14 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-fluoropolymers.

[0083] In some embodiments, the nucleotides at positions 2, 4, 6, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-fluoropolymers.

[0084] In some embodiments, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-fluoropolymers.

[0085] In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand, in the direction from the 5' end to the 3' end, are each independently nucleotides modified with 2'-fluoropolymers.

[0086] In some embodiments, the sense strand of the siRNA described herein is 5'-N a N a N a N a XN a N b N b N b N a N a N a N a N a N a N a N a N a N a It has a nucleotide sequence represented by the formula -3', Of those, each N a and N b Each of these independently represents a modified nucleotide or an unmodified nucleotide, and N a and N b The modifications in each case are different, and each X is independently N a or N b That is the case.

[0087] In some embodiments, the antisense strand of the siRNA described herein is 5'-N a 'N b 'N a 'X'N a 'N b 'W'N a 'X'Y'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a It has a nucleotide sequence represented by the formula '-3', Of those, each N a 'and N b ' indicates independently modified nucleotides or unmodified nucleotides, of which N a 'and N b The modifications in ' are different, and each X' is independently N a 'or N b ' is Na 'or N b 'W' represents a nucleotide comprising a chemical modification represented by any one of the formulas (I) relating to this disclosure or a tautomer thereof.

[0088] In some embodiments, the modifications in X' and Y' are different.

[0089] In some embodiments, N a N is a nucleotide modified with a 2'-methoxy group, b These are nucleotides modified with 2'-fluoro or 2'-deoxy.

[0090] In some embodiments, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' is a nucleotide modified with 2'-fluoro or 2'-deoxy-.

[0091] In some specific embodiments, N a N is a nucleotide modified with a 2'-methoxy group, b This is a nucleotide modified with 2'-fluoropolymer.

[0092] In some specific embodiments, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' is a nucleotide modified with 2'-fluoro.

[0093] In some embodiments, the antisense strand of the siRNA described herein is 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'X'Y'N a 'N b 'N a 'N b 'Na 'N b 'N a 'N b 'N a 'N a 'N a It has a nucleotide sequence represented by the formula '-3', Of these, each X' is independently N a 'or N b ' is N a 'or N b 'and the modifications in X' and Y' are different, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' represents a nucleotide modified with 2'-fluoro, and W' represents a nucleotide comprising a chemical modification represented by any one of the formulas (I) relating to this disclosure or a tautomer thereof.

[0094] In some embodiments, the sense strand of the siRNA described herein is 5'-N a N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a -3', or 5'-N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a N a Na N a It has a nucleotide sequence represented by the formula -3', Eventually, N a N is a nucleotide modified with a 2'-methoxy group. b This is a nucleotide modified with 2'-fluoropolymer.

[0095] In some embodiments, the antisense strand of the siRNA described herein is 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a '-3', or, 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N b 'N a 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a It has a nucleotide sequence represented by the formula '-3', Eventually, N a N is a nucleotide modified with a 2'-methoxy group, b is a nucleotide modified with 2'-fluoro, and / or N a' is a nucleotide modified with a 2'-methoxy group, N b ' is a nucleotide modified with 2'-fluoro.

[0096] W' represents a nucleotide comprising a chemical modification represented by any one of the formulas (I) relating to this disclosure or a tautomer thereof.

[0097] In some specific embodiments, W' represents a nucleotide comprising a chemical modification or a tautomer modification thereof, where the chemical modification is [ka] B is selected from among guanine, adenine, cytosine, or uracil, and in some specific embodiments, B is selected from the base at the corresponding position at position 7 of the 5' region of the antisense strand.

[0098] In some specific embodiments, W' represents a nucleotide comprising a chemical modification or a tautomer modification thereof, where the chemical modification is [ka] A base is selected from among, of which M is O or S, and of which B is selected from guanine, adenine, cytosine or uracil, and in some specific embodiments, B is selected from the base at the corresponding position at position 7 of the 5' region of the antisense chain.

[0099] In some specific embodiments, M is S. In some specific embodiments, M is O.

[0100] In some embodiments, at least one phosphate group in the sense chain and / or antisense chain is a phosphate group having a modifying group, and the modifying group provides the siRNA with improved stability in biological samples or the environment, and in some embodiments, the phosphate group having the modifying group is a thiophosphate group. Specifically, a thiophosphate group is a phosphodiester group modified by substituting one non-bridged oxygen atom with a sulfur atom.

[0101] In some embodiments, the thiophosphate group is Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, The first nucleotide end of the 3' end of the sense strand mentioned above, Between the first and second nucleotides at the 3' end of the sense strand mentioned above, Between the second and third nucleotides at the 3' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, The first nucleotide end of the 3' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, and Between the second and third nucleotides at the 3' end of the antisense strand mentioned above, It is located in at least one position selected from the given positions.

[0102] In some embodiments, the sense chain and / or antisense chain comprises a plurality of thiophosphate groups, the thiophosphate groups are Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, Between the second and third nucleotides at the 3' end of the antisense strand mentioned above, Optionally, the first nucleotide end of the 3' end of the sense strand, and / or Selectively, between the first and second nucleotides at the 3' end of the sense strand, It is located there.

[0103] In some embodiments, the sense chain is 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-2', ..., 5'-NmsNmsNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNm-2', ..., 5'-NmsNmsNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNms-2', ..., 5'-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNms-2', ..., 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNm-2', ..., 5'-NmsNmsNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNm-2', ..., 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNms-2', or, 5'-NmsNmsNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmsNms-3' Selected from the nucleotide sequences represented by the formula, Of these, Nm represents any nucleotide modified with a 2'-methoxy group, for example, C, G, U, A, and T modified with a 2'-methoxy group, and Nf represents any nucleotide modified with a 2'-fluoro group, for example, C, G, U, A, and T modified with a 2'-fluoro group. When a lowercase 's' is between uppercase letters, it indicates that the two nucleotides adjacent to that 's' are linked by a thiophosphate group. When a lowercase 's' is at the first position of the 3' end, it indicates that the single nucleotide end adjacent to the left of that 's' is a thiophosphate group.

[0104] In some embodiments, the antisense chain is 5'-Nms'Nfs'Nm'Nf'Nm'Nf'W'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nms'Nms'Nm'-3', or 5'-Nms'Nfs'Nm'Nf'Nm'Nf'W'Nm'Nf'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nms'Nms'Nm'-3' It has a nucleotide sequence represented by the formula, Of these, Nm' represents any nucleotide modified with a 2'-methoxy group, for example, C, G, U, A, and T modified with a 2'-methoxy group, and Nf' represents any nucleotide modified with a 2'-fluoro group, for example, C, G, U, A, and T modified with a 2'-fluoro group. When a lowercase 's' is between uppercase letters, it indicates that the two nucleotides adjacent to that letter 's' are linked by a thiophosphate group. When a lowercase 's' is at the first position of the 3' end, it indicates that the nucleotide end adjacent to the left of that letter 's' is a thiophosphate group. W' represents a nucleotide containing a chemical modification or a tautomer modification thereof, and the above chemical modification is [ka] B is selected from among guanine, adenine, cytosine, or uracil, and in some embodiments, is selected from the base at the corresponding position at position 7 of the 5' region of the antisense chain.

[0105] In some specific embodiments, W' represents a nucleotide comprising a chemical modification or a tautomer modification thereof, where the chemical modification is [ka] A base is selected from among, of which M is O or S, and of which B is selected from guanine, adenine, cytosine or uracil, and in some specific embodiments, B is selected from the base at the corresponding position at position 7 of the 5' region of the antisense chain.

[0106] In some specific embodiments, M is S. In some specific embodiments, M is O.

[0107] In some embodiments, the siRNA comprises a sense strand selected from Table 5.

[0108] In some embodiments, the siRNA comprises any one antisense strand selected from Table 5.

[0109] In some embodiments, the siRNA comprises any one sense strand selected from Table 8.

[0110] In some embodiments, the siRNA comprises any one antisense strand selected from Table 8.

[0111] In some embodiments, the siRNA comprises any one antisense strand selected from Table 9.

[0112] In some embodiments, the siRNA comprises any one sense strand selected from Table 13.

[0113] In some embodiments, the siRNA comprises any one antisense strand selected from Table 13.

[0114] In some embodiments, the siRNA comprises any one sense strand selected from Table 15.

[0115] In some embodiments, the siRNA comprises any one antisense strand selected from Table 15.

[0116] In some embodiments, the siRNA comprises any one sense strand selected from Table 24.

[0117] In some embodiments, the siRNA comprises any one antisense strand selected from Table 24.

[0118] In some embodiments, the siRNA comprises any one sense strand selected from Table 25.

[0119] In some embodiments, the siRNA comprises any one antisense strand selected from Table 25.

[0120] In some embodiments, the siRNA comprises any one sense strand selected from Table 26.

[0121] In some embodiments, the siRNA comprises any one antisense strand selected from Table 26.

[0122] In some embodiments, the siRNA comprises any one sense strand selected from Table 66.

[0123] In some embodiments, the siRNA comprises any one antisense strand selected from Table 66.

[0124] In some embodiments, when the siRNA comes into contact with cells expressing a target gene, as measured by methods such as psiCHECK activity screening and luciferase reporter gene detection, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods like Western Blot or flow cytometry, the siRNA suppresses the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0125] In some embodiments, when the siRNA comes into contact with cells expressing a target gene, the excess expression percentage of the target gene mRNA by the siRNA is measured by methods such as psiCHECK activity screening and luciferase reporter gene detection, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods like Western Blot or flow cytometry, and is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0126] In some embodiments, when siRNA containing the chemical modifications according to the Disclosure comes into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods such as Western Blot or flow cytometry, the siRNA containing the chemical modifications according to the Disclosure, for example, the chemical modifications represented by formula (I) or formula (II), retains on-target activity while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0127] In some embodiments, when siRNA containing the chemical modifications according to the Disclosure comes into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods such as Western Blot or flow cytometry, the siRNA containing the chemical modifications according to the Disclosure, for example, the chemical modifications represented by formula (I) or formula (II), reduces on-target activity by at least 20%, at least 19%, at least 15%, at least 10%, at least 5%, or more than 1%, and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0128] In some embodiments, when the siRNA comprising the chemical modification according to this disclosure comes into contact with cells expressing a target gene, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as methods based on PCR or branched DNA (bDNA), or protein-based methods, such as Western When measured by immunofluorescence analysis methods such as Blot or flow cytometry, siRNAs containing the chemical modifications relating to this disclosure, for example, the chemical modification represented by formula (I) or formula (II), increase on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0129] complex This disclosure further provides an siRNA complex comprising any one of the above-mentioned siRNAs and a complex group linked to the above-mentioned siRNA.

[0130] In some embodiments, the complex group comprises a pharmaceutically acceptable target ligand and an optional linker, and the siRNA, the linker, and the target ligand are covalently or noncovalently linked in order.

[0131] In some embodiments, the linker is ligated to the 3' end of the sense strand of the siRNA.

[0132] This disclosure further provides an siRNA complex comprising any one of the above-mentioned siRNAs and a target ligand linked to the above-mentioned siRNA.

[0133] In some embodiments, the siRNA is covalently or noncovalently bound to the target ligand.

[0134] In some embodiments, the target ligand is ligated to the 3' end of the sense strand of the siRNA.

[0135] In some embodiments, the target ligand targets the liver.

[0136] In some embodiments, the target ligand is linked to an asial glycoprotein receptor (ASGPR).

[0137] In some embodiments, the target ligand is selected from a galactose cluster or a galactose derivative cluster, and the galactose derivative is selected from N-acetyl-galactoseamine, N-trifluoroacetylgalactoseamine, N-propionylgalactoseamine, Nn-butyrylgalactoseamine, or N-isobutyrylgalactoseamine.

[0138] In some embodiments, a lipophilic group such as cholesterol can be introduced to the end of the siRNA sense strand to promote the entry of siRNA into cells. The lipophilic group covalently binds to small interfering RNA, and by introducing, for example, cholesterol, lipoprotein, or vitamin E to the end, it contributes to interaction with intracellular mRNA via the cell membrane, which is composed of a lipid bilayer. siRNA may also be modified non-covalently; for example, by binding to phospholipid molecules, polypeptides, cationic polymers, etc., via hydrophobic or ionic bonds, its stability and biological activity can be improved.

[0139] In some embodiments, the target ligand is ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0140] In some embodiments, the target ligand is indirectly ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0141] In some embodiments, the target ligand is directly ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0142] In some embodiments, the target ligand is directly ligated to the siRNA terminus via a phosphate group or a thiophosphate group.

[0143] In some embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0144] In some embodiments, the target ligand structure is as shown by formula (IV) below, [ka] Of these, T is the target part, E is the branching base, L1 is the linker part, and L2 is the anchoring part between the target part and the branching base, and i is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0145] In some embodiments, i is an integer selected from 2 to 8.

[0146] In some embodiments, i is an integer selected from 3 to 5.

[0147] In some embodiments, L1 is [ka] And, R 9 and R 10Each of these groups is independently selected from -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -CH2O-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, -NH(CO)NH-, and 3- to 12-membered heterocyclyl groups, wherein the above -CH2- is optionally substituted with substituents selected from halogens, alkyl groups, alkoxy groups, and alkylamino groups, and the above alkyl groups are optionally further substituted with substituents selected from hydroxyl groups, amino groups, and halogens. R 11 The alkyl groups are selected from deuterium, halogens, alkyl groups, amino groups, cyano groups, nitro groups, alkenyl groups, alkynyl groups, carboxyl groups, hydroxyl groups, mercapto groups, alkylmercapto groups, alkoxy groups, alkylamino groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH2, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, -S(O)ONH2, and -S(O)ONH-alkyl groups, and the alkyl groups, alkenyl groups, alkynyl groups, alkylmercapto groups, alkyloxy groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, and -S(O)ONH-alkyl groups are optionally further substituted with substituents selected from halogens, hydroxyl groups, amino groups, and mercapto groups. The above k is selected from 0, 1, 2, 3, 4. The above j is an integer selected from 1 to 20 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0148] In some embodiments, L1 is [ka] And among them, R 11The alkyl groups are selected from deuterium, halogens, alkyl groups, amino groups, cyano groups, nitro groups, alkenyl groups, alkynyl groups, carboxyl groups, hydroxyl groups, mercapto groups, alkylmercapto groups, alkoxy groups, alkylamino groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH2, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, -S(O)ONH2, and -S(O)ONH-alkyl groups, and the alkyl groups, alkenyl groups, alkynyl groups, carboxyl groups, alkylmercapto groups, alkyloxy groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, and -S(O)ONH-alkyl groups are optionally further substituted with substituents selected from halogens, hydroxyl groups, amino groups, and mercapto groups. The above k is selected from 0, 1, 2, 3, 4. In some embodiments, L1 is [ka] That is the case.

[0149] In some embodiments, L1 is [ka] That is the case.

[0150] In some embodiments, L1 is [ka] That is the case.

[0151] In some embodiments, E in the target ligand is [ka] And, The above R 12 , R 13 , R 14 and R15 Each of these is independently selected from -C(O)NH- and -C(O)-, and the carbonyl group is optionally further substituted with an alkyl group, and the alkyl group is optionally further substituted with a group selected from alkyl groups, hydroxyl groups, -C(O)O-, -C(O)O-alkyl-, and -C(O)NH-. The above X 2 , X 3 , X 4 and X 5 These are integers independently selected from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0152] In some embodiments, E in the target ligand is [ka] And, The above R 12 , R 13 , R 14 and R 15 Each of these is independently selected from -C(O)NH- and -C(O)-, and the above -C(O)NH- and -C(O)- are optionally further substituted with alkyl groups, and the above alkyl groups are optionally further substituted with groups selected from alkyl groups, hydroxyl groups, -C(O)O-, -C(O)O-alkyl-, and -C(O)NH-. The above X 2 , X 3 , X 4 and X 5 These are integers independently selected from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0153] In some embodiments, E in the target ligand is [ka] And, The above R 12 , R 13 , R 14 and R 15 These are -C(O)NH- and -C(O)-, respectively, independently. [ka] Selected from the above X 2 , X 3 , X 4 and X 5 These are integers independently selected from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0154] In some embodiments, E in the target ligand is [ka] That is the case.

[0155] In some embodiments, E in the target ligand is [ka] They are selected from among them.

[0156] In some embodiments, E in the target ligand is [ka] They are selected from among them.

[0157] In some embodiments, E in the target ligand is [ka] They are selected from among them.

[0158] In some embodiments, E in the target ligand is [ka] Therefore, L1 is, [ka] It was chosen based on this structure, R 9and R 10 Each of these is independently selected from -S-, -NH-, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -CH2O-, -NH-C(O)-CH2-, -C(O)-CH2-NH-, -NH(CO)NH-, and a 3- to 12-membered heterocyclyl group, wherein the above -CH2- is optionally substituted with a substituent selected from halogens, alkyl groups, alkoxy groups, and alkylamino groups, and the above alkyl groups are optionally further substituted with substituents selected from hydroxyl groups, amino groups, and halogens. R 11 The alkyl groups are selected from deuterium, halogens, alkyl groups, amino groups, cyano groups, nitro groups, alkenyl groups, alkynyl groups, carboxyl groups, hydroxyl groups, mercapto groups, alkylmercapto groups, alkoxy groups, alkylamino groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH2, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, -S(O)ONH2, and -S(O)ONH-alkyl groups, and the alkyl groups, alkenyl groups, alkynyl groups, alkylmercapto groups, alkyloxy groups, -C(O)-alkyl groups, -C(O)-O-alkyl groups, -CONH-alkyl groups, -OC(O)-alkyl groups, -NH-C(O)-alkyl groups, -S(O)O-alkyl groups, and -S(O)ONH-alkyl groups are optionally further substituted with substituents selected from halogens, hydroxyl groups, amino groups, and mercapto groups. The above k is selected from 0, 1, 2, 3, 4. The above j is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0159] In some embodiments, E in the target ligand is [ka] Selected from, L1 is [ka] They are selected from among them.

[0160] In some embodiments, E in the target ligand is [ka] Selected from, L1 is [ka] Selected from among, that is, the E-L1 is [ka] That is the case.

[0161] In some embodiments, E in the target ligand is [ka] Selected from, L1 is [ka] Selected from among, that is, the E-L1 is [ka] That is the case.

[0162] In some embodiments, E in the target ligand is [ka] Selected from, L1 is [ka] Selected from among, that is, the E-L1 is [ka] That is the case.

[0163] In some embodiments, E in the target ligand is [ka] Selected from, L1 is [ka] Selected from among, that is, the E-L1 is [ka] That is the case.

[0164] In this disclosure, L2 is an anchoring portion between the target portion and the branching base, and L2 plays a connecting and separating role between the branching base and each target portion.

[0165] In some embodiments, L2 is directly connected at one end to the target ligand and at the other end to the branching group E.

[0166] In some embodiments, L2 is directly linked at one end to the target ligand and indirectly linked at the other end to the branching group E.

[0167] In some embodiments, L2 is indirectly connected at one end to a target ligand and at the other end to a branching group E.

[0168] In some embodiments, the target ligands disclosed herein include two L2s and two target moieties.

[0169] In some embodiments, the target ligands disclosed herein include three L2s and three target moieties.

[0170] In some embodiments, the target ligands disclosed herein include four L2s and four target moieties.

[0171] In some embodiments, the target ligands disclosed herein include multiple L2s and multiple target moieties.

[0172] In some embodiments, L2 in this disclosure is a combination of one or two to twenty covalent bonds selected from the following groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), i.e., [ka] It is a combination of covalent bonds between substituted or unsubstituted cycloalkyl groups (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl groups (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptene, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl groups (e.g., phenyl, naphthyl, binaphthyl, anthryl, etc.), substituted or unsubstituted heteroaryl groups (e.g., pyridyl, pyrimidine, pyrrolyl, imidazolyl, furanyl, benzofuranyl, indolyl, etc.), and substituted or unsubstituted heterocyclyl groups (e.g., tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, pyrrolidinyl, etc.).

[0173] In some embodiments, L2 in this disclosure is a combination of one or two to twenty covalent bonds selected from the following groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), i.e., [ka] .

[0174] In some embodiments, the target ligand is [ka] It includes L2 having the following structure, of which x 6is an integer between 1 and 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0175] In some embodiments, the target ligand is [ka] It includes L2 which has the following structure.

[0176] In some embodiments, the target ligand is [ka] It includes L2 which has the following structure.

[0177] In some embodiments, the target ligand is [ka] It includes L2 which has the following structure.

[0178] In some embodiments, the target ligand is [ka] It includes L2 which has the following structure.

[0179] In some embodiments, the target ligand is [ka] It includes L2 which has the following structure.

[0180] In some embodiments, the target ligand is [ka] It includes L2 having the following structure, of which x 7is an integer between 1 and 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Z is [ka] That is the case.

[0181] In some embodiments, the target ligand is [ka] It has L2, which has the following structure.

[0182] In some embodiments, the target ligand is [ka] It has an L2 structure, Eventually, x 8 is an integer between 1 and 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Z is [ka] That is the case.

[0183] In some embodiments, the target ligand is [ka] L2 has the following structure, and of which, x 9 and X 10 Each of the integers is independently selected from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and Z is [ka] That is the case.

[0184] In some embodiments, the target ligand is [ka] It has L2, which has the following structure.

[0185] In some embodiments, the target ligand is [ka] It has an L2 structure, Eventually, x 7 and X 8 Each of the integers is independently selected from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and Z is [ka] That is the case.

[0186] In some specific embodiments, the target ligand is [ka] or [ka] It has the following structure.

[0187] In some specific embodiments, the target ligand is [ka] or [ka] It has the following structure.

[0188] In some specific embodiments, the target ligand is [ka] It has the following structure.

[0189] In some specific embodiments, the target ligand is [ka] or [ka] It has the following structure.

[0190] In some embodiments, the target portion of the target ligand consists of one or more target groups, and the target ligand cooperates to guide the therapeutic reagent to which it is linked to be delivered to a desired target site. In some cases, the target portion can bind to cells or cell receptors and activate endocytosis to facilitate the entry of the therapeutic reagent into the cell. The target portion may include compounds that have affinity for cell receptors, cell surface molecules, or antibodies. Various target ligands containing the target portion can be linked to therapeutic reagents and other compounds so that the reagent targets cells and specific cell receptors.

[0191] In some embodiments, the type of target moiety includes carbohydrates, cholesterol and cholesteryl groups, or steroids. Target moieties capable of binding to cell receptors include sugars, such as galactose, galactose derivatives (e.g., N-acetyl-galactoseamine, N-trifluoroacetylgalactoseamine, N-propionylgalactoseamine, Nn-butyrylgalactoseamine, N-isobutyrylgalactoseamine), mannose, and mannose derivatives.

[0192] The target moiety that binds to the asialocrypoprotein receptor (ASGPR) is known to be particularly useful for inducing the delivery of oligomers to the liver. The asialocrypoprotein receptor is highly expressed in liver cells (hepatocytes). The target moiety of ASCPR-targeting cell receptors includes galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters consisting of 2, 3, 4, or 4 or more N-acetyl-galactoseamines (GalNAc or NAG), can promote the uptake of certain compounds in hepatocytes. The GalNAc cluster coupled to the oligomer compound is intended to guide the composition to the liver, where the N-acetyl-galactoseamine sugar can bind to the asialocrypoprotein receptor on the surface of liver cells. Binding to the asialocrypoprotein receptor is thought to promote the entry of compounds into the cell by initiating receptor-mediated endocytosis.

[0193] In some embodiments, the target ligand may contain two, three, four, or more target moieties.

[0194] In some embodiments, the target ligands disclosed herein may contain one, two, three, four, or more target moieties linked to a branching group via L2.

[0195] In some embodiments, the target ligand is in the form of a galactose cluster.

[0196] In some embodiments, each target moiety comprises a galactoseamine derivative, specifically N-acetyl-galactoseamine. Other sugars available as target moieties and having affinity for the asialocrypoprotein receptor may be selected from galactose, galactoseamine, N-formyl-galactoseamine, N-acetyl-galactoseamine, N-propionyl-galactoseamine, Nn-butyryl-galactoseamine, and N-isobutyryl-galactoseamine.

[0197] In some embodiments, the target ligand in this disclosure comprises N-acetylgalactoseamine as the target moiety. [ka] .

[0198] In some embodiments, the target ligand comprises three terminal galactoseamines or galactoseamine derivatives (e.g., N-acetyl-galactoseamines) each having affinity for the asialoclycoprotein receptor. In some embodiments, the target ligand comprises three terminal N-acetyl-galactoseamines (GalNAc or NAG) as the target moiety.

[0199] In some embodiments, the target ligand comprises four terminal galactoseamines or galactoseamine derivatives (e.g., N-acetyl-galactoseamines) each having affinity for the asialoclycoprotein receptor. In some embodiments, the target ligand comprises four terminal N-acetyl-galactoseamines (GalNAc or NAG) as the target moiety.

[0200] When referring to three terminal N-acetyl-galactoseamines, commonly used terms in this field include tri-antennary, tri-valent, and trimer.

[0201] When referring to four-terminal N-acetyl-galactoseamines, commonly used terms in this field include tri-antennary, tri-valent, and tetramer.

[0202] In some specific embodiments, the target ligand of this disclosure is [ka] or [ka] It has the following structure.

[0203] In some specific embodiments, the target ligand provided herein is [ka] or [ka] It has the following structure.

[0204] In some specific embodiments, the target ligand provided herein is [ka] It has the following structure.

[0205] In some specific embodiments, the target ligand provided herein is [ka] or [ka] It has the following structure.

[0206] In some embodiments, the siRNA relating to the Disclosure is linked to a target ligand relating to the Disclosure, [ka] Forms an siRNA complex having Of these, T is the target region, E is the branching group, L1 is the linker region, and L2 is the anchoring region between the target region and the branching group. Of these, x is an integer selected from 1 to 10, and D is an siRNA relating to one of the above configurations.

[0207] In some embodiments, D is an siRNA that targets ApoC3.

[0208] In some embodiments, D is an siRNA that targets HBV-X.

[0209] In some embodiments, D is an siRNA that targets F11.

[0210] In some embodiments, D is an siRNA that targets HBV-S.

[0211] In some embodiments, D is an siRNA that targets angiopoietin-like protein-3 (ANGPTL3).

[0212] In some embodiments, D is an siRNA that targets the thyroxine transport protein (TTR) gene.

[0213] In some embodiments, D is any one of the siRNAs relating to this disclosure.

[0214] In some embodiments, L1 is ligated to the 3' end of the sense strand of the siRNA.

[0215] In some embodiments, the target ligand is ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0216] In some embodiments, the target ligand is indirectly ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0217] In some embodiments, the target ligand is directly ligated to the siRNA terminus via a phosphate group, a thiophosphate group, or a phosphate group.

[0218] In some embodiments, the target ligand is directly ligated to the siRNA terminus via a phosphate group or a thiophosphate group.

[0219] In some embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0220] In some specific embodiments, the siRNA complex described herein is [ka] or [ka] As shown, D is an siRNA relating to one of the above forms.

[0221] In some embodiments, D is an siRNA that targets ApoC3.

[0222] In some embodiments, D is an siRNA that targets HBV-X.

[0223] In some embodiments, D is an siRNA that targets F11.

[0224] In some embodiments, D is an siRNA that targets HBV-S.

[0225] In some embodiments, D is an siRNA that targets angiopoietin-like protein-3 (ANGPTL3).

[0226] In some embodiments, D is an siRNA that targets the thyroxine transport protein (TTR) gene.

[0227] In some specific embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0228] In some specific embodiments, the siRNA complex described herein is [ka] or [ka] As shown, D is an siRNA relating to one of the above forms.

[0229] In some embodiments, D is an siRNA that targets ApoC3.

[0230] In some embodiments, D is an siRNA that targets HBV-X.

[0231] In some embodiments, D is an siRNA that targets F11.

[0232] In some embodiments, D is an siRNA that targets HBV-S.

[0233] In some embodiments, D is an siRNA that targets angiopoietin-like protein-3 (ANGPTL3).

[0234] In some embodiments, D is an siRNA that targets the thyroxine transport protein (TTR) gene.

[0235] In some specific embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0236] In some specific embodiments, the siRNA complex described herein is [ka] or [ka] As shown, D is an siRNA relating to one of the above forms.

[0237] In some embodiments, D is an siRNA that targets ApoC3.

[0238] In some embodiments, D is an siRNA that targets HBV-X.

[0239] In some embodiments, D is an siRNA that targets F11.

[0240] In some embodiments, D is an siRNA that targets HBV-S.

[0241] In some embodiments, D is an siRNA that targets angiopoietin-like protein-3 (ANGPTL3).

[0242] In some embodiments, D is an siRNA that targets the thyroxine transport protein (TTR) gene.

[0243] In some specific embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0244] In some specific embodiments, the siRNA complex described herein is [ka] As shown, D is an siRNA relating to one of the above forms.

[0245] In some embodiments, D is an siRNA that targets ApoC3.

[0246] In some embodiments, D is an siRNA that targets HBV-X.

[0247] In some embodiments, D is an siRNA that targets F11.

[0248] In some embodiments, D is an siRNA that targets HBV-S.

[0249] In some embodiments, D is an siRNA that targets angiopoietin-like protein-3 (ANGPTL3).

[0250] In some embodiments, D is an siRNA that targets the thyroxine transport protein (TTR) gene.

[0251] In some specific embodiments, the target ligand is directly ligated to the 3' end of the siRNA sense strand via a phosphate group or a thiophosphate group.

[0252] In some specific embodiments, L1 and D are linked via a phosphate group, a thiophosphate group, or a phosphate group.

[0253] In some specific embodiments, L1 and the 3' end of the D sense chain are linked via a phosphate group, a thiophosphate group, or a phosphate group.

[0254] In some specific embodiments, L1 and the 3' end of the D sense chain are directly linked via a phosphate group or a thiophosphate group.

[0255] In some specific embodiments, L1 and the 3' end of the D sense strand are indirectly linked via a phosphate group or a thiophosphate group. In some embodiments, to promote the entry of siRNA into cells, a lipophilic group such as cholesterol can be introduced to the end of the siRNA sense strand. This lipophilic group covalently binds to small interfering RNA, and by introducing, for example, cholesterol, lipoprotein, or vitamin E to the end, it contributes to interaction with intracellular mRNA via the cell membrane, which is composed of a lipid bilayer. Furthermore, siRNA may be modified non-covalently, for example, by binding to phospholipid molecules, polypeptides, cationic polymers, etc., via hydrophobic or ionic bonds to improve stability and biological activity.

[0256] composition Another aspect of the present disclosure provides a composition comprising the composite and one or more pharmaceutically acceptable excipients, such as carriers, transporters, diluents, and / or delivery polymers.

[0257] This disclosure further provides pharmaceutical compositions comprising siRNA or siRNA complexes relating to this disclosure.

[0258] In some embodiments, the pharmaceutical composition may further contain pharmaceutically acceptable additives and / or adjuvants, which may be one or more of the various formulations or compounds commonly used in the art. For example, the pharmaceutically acceptable additives may include at least one of pH buffers, protective agents, and osmotic pressure modifiers.

[0259] Uses and methods Another aspect of this disclosure provides the use of the complex or a composition containing the complex in the preparation of a drug for treating a target disease, in some embodiments the disease is selected from liver-derived diseases.

[0260] Another aspect of this disclosure provides a method for treating a disease of interest, comprising administering the complex or composition to the subject.

[0261] Another aspect of this disclosure provides a method for suppressing mRNA expression in a subject's body, comprising administering the subject the complex or composition.

[0262] Another aspect of this disclosure provides a method for delivering an oligomeric compound that suppresses expression in the body to the liver, comprising administering the complex or composition to a subject.

[0263] The complexes, compositions, and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues, or subjects, and include administering a therapeutically effective amount of the expression-repressive oligomer compound described herein to the subject, wherein the expression-repressive oligomer compound is linked to a target ligand, thereby suppressing the expression of target mRNA in the subject.

[0264] In some embodiments, the subject has already been identified as having a pathogenic upregulation of the target gene in the target cells or tissues.

[0265] The subjects described in this disclosure are those suffering from a disease or condition that would benefit from the reduction or suppression of target mRNA expression.

[0266] Delivery may be by local administration (e.g., direct injection, implantation, or local administration), systemic administration, or by subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., ventricular, parenchymal, and intrathecal), intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or local (including oral, cheek, and sublingual) administration.

[0267] In a selective embodiment, the pharmaceutical compositions provided herein may be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0268] In a selective embodiment, the complex may be packaged in a reagent kit. In some embodiments, when the siRNA complex or pharmaceutical composition comes into contact with cells expressing a target gene, for example, when measured by immunofluorescence analysis methods such as Western Blot or flow cytometry, in addition to psiCHECK activity screening and luciferase reporter gene detection, the siRNA complex or pharmaceutical composition suppresses the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0269] In some embodiments, when the siRNA complex or pharmaceutical composition comes into contact with cells expressing a target gene, the excess expression percentage of the target gene mRNA by the siRNA complex or pharmaceutical composition is measured by methods such as psiCHECK activity screening and luciferase reporter gene detection, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods such as Western Blot or flow cytometry, and is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0270] In some embodiments, when the siRNA complex or pharmaceutical composition comes into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods like Western Blot or flow cytometry, the siRNA complex retains on-target activity while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0271] In some embodiments, when an siRNA complex or pharmaceutical composition comes into contact with cells expressing a target gene, as measured by methods such as psiCHECK activity screening and luciferase reporter gene detection, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis including Western Blot and flow cytometry, the siRNA complex reduces on-target activity by at least 20%, at least 19%, at least 15%, at least 10%, at least 5%, or more than 1%, while also reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0272] In some embodiments, when an siRNA complex or pharmaceutical composition comes into contact with cells expressing a target gene, for example, by immunofluorescence analysis such as Western Blot or flow cytometry, in addition to psiCHECK activity screening and luciferase reporter gene detection, the siRNA complex increases on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0273] This disclosure further provides a method for silencing a target gene or mRNA of a target gene in a cell, comprising the step of introducing an siRNA, siRNA complex, and / or pharmaceutical composition relating to this disclosure into the cell.

[0274] This disclosure provides a method for silencing a target gene or the mRNA of a target gene in cells in vivo or in vitro, further comprising the step of introducing an siRNA, siRNA complex, and / or pharmaceutical composition according to this disclosure into the cells.

[0275] This disclosure further provides a method for suppressing the expression of a target gene or the mRNA of a target gene, comprising administering an effective amount or effective dose of an siRNA, siRNA complex and / or pharmaceutical composition according to this disclosure to a subject requiring such suppression.

[0276] In some embodiments, administration is carried out by methods of administration including intramuscular, intrabronchial, intrathoracic, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal fluid, or a combination thereof.

[0277] In some embodiments, the effective amount or effective dose of siRNA, siRNA conjugate and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0278] In some embodiments, the target gene is the hepatitis B virus (HBV) gene, the angiopoietin-like protein-3 (ANGPTL3) gene, or the thyroxine transport protein (TTR) gene.

[0279] This disclosure further provides the use of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of agents for the prevention and / or treatment of pathological conditions and diseases caused by hepatitis B virus.

[0280] This disclosure further provides applications of the above-mentioned siRNA and / or pharmaceutical compositions and / or siRNA complexes in the preparation of agents for the prevention and / or treatment of hepatitis B.

[0281] This disclosure further provides a method for treating hepatitis B, comprising administering the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex to a patient in need.

[0282] This disclosure further provides a method for suppressing HBV gene expression in hepatitis cells infected with chronic HBV, comprising introducing an effective amount or effective dose of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex into the hepatitis cells infected with chronic HBV.

[0283] This disclosure further provides the use of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of agents for the prevention and / or treatment of pathological conditions and diseases caused by abnormal expression of the ANGPTL3 gene or TTR gene in mammals (e.g., humans).

[0284] This disclosure further provides a method for treating pathological conditions and diseases caused by abnormal expression of the ANGPTL3 gene or the TTR gene, comprising administering an effective amount or effective dose of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex.

[0285] Pathological conditions and diseases resulting from abnormal expression of the ANGPTL3 gene include cardiovascular and / or metabolic diseases, such as hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, obesity, diabetes, and / or ischemic heart disease.

[0286] Pathological conditions and diseases resulting from abnormal TTR gene expression include sensory neuropathy (e.g., distal limb numbness, hypoesthesia), autonomic neuropathy (e.g., gastrointestinal dysfunction such as gastric ulcers, or orthostatic hypotension), motor neuropathy, epileptic seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic deficiency, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantial decrease in mBMI (change in body mass index), cranial nerve dysfunction, and lattice keratopathy.

[0287] In some embodiments, the siRNA and / or pharmaceutical composition and / or siRNA complex exhibit superior on-target activity and reduced off-target activity in regulating genes expressed in the liver or in treating pathological conditions or diseases caused by abnormal gene expression in hepatocytes. Genes expressed in the liver include, but are not limited to, genes such as ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. In some embodiments, the specific genes are selected from hepatitis B virus genes, angiopoietin-like protein 3 genes, or apolipoprotein C3 genes. Accordingly, the diseases are selected from chronic liver disease, hepatitis, hepatic fibrosis, hepatic proliferative disorders, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0288] In some embodiments, the siRNA, siRNA complexes, and / or pharmaceutical compositions described above may be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, hematological disorders, metabolic disorders, and inflammatory disorders. Proliferative disorders of the liver may be benign or malignant, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma. Hepatic or inflammatory diseases may be diseases related to blood coagulation factors, complement-mediated inflammation, or fibrosis. Metabolic diseases of the liver include dyslipidemia and irregularities in glucose regulation.

[0289] host cell This disclosure further provides cells comprising siRNA or siRNA complex relating to this disclosure.

[0290] Reagent kit This disclosure further provides reagent kits containing siRNA or siRNA complexes relating to this disclosure.

[0291] intermediate This disclosure further provides a compound represented by formula (II) or a tautomer thereof, [ka] (II) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. J2 is H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. Q'1 is [ka] Q2 is R2, or Q1 is R2, Q'2 is [ka] And, Of these, R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. J1 is H or a C1-C6 alkyl group. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) rR8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group, and Z is a phosphorus-containing active reactive group.

[0292] In some embodiments, W is an MMTr or a DMTr.

[0293] In some embodiments, Z is [ka] That is the case.

[0294] In some embodiments, the above compound is [ka] Rather, W, B, and Z are defined as described above.

[0295] In some embodiments, when X is NH-CO, R1 is not H.

[0296] In some embodiments, the above compound is [ka] isn't it.

[0297] In some embodiments, the above compound is [ka] Rather, Z is as defined above.

[0298] In some embodiments, the compound represented by formula (II) or its tautomer is specifically the compound represented by formula (II-1) or its tautomer, [ka] (II-1) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. Each of J1 and J2 is independently H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group, and Z is a phosphorus-containing active reactive group.

[0299] In some embodiments, W is an MMTr or a DMTr.

[0300] In some embodiments, Z is [ka] That is the case.

[0301] In some embodiments, the above compound is [ka] Rather, W, B, and Z are defined as described above.

[0302] In some embodiments, when X is NH-CO, R1 is not H.

[0303] In some embodiments, the above compound is [ka] isn't it.

[0304] In some embodiments, the above compound is [ka] Rather, Z is as defined above.

[0305] In some embodiments, the compound represented by formula (II) or its tautomer is specifically the compound represented by formula (II-2) or its tautomer, [ka] (II-2) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C6 alkyl group. R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 consists of H, C1-C6 alkyl, C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group, and Z is a phosphorus-containing active reactive group.

[0306] In some embodiments, W is an MMTr or a DMTr.

[0307] In some embodiments, Z is [ka] That is the case.

[0308] In some embodiments, the above compound is [ka] Rather, W, B, and Z are defined as described above.

[0309] In some embodiments, when X is NH-CO, R1 is not H.

[0310] In some embodiments, the above compound is [ka] isn't it.

[0311] In some embodiments, the above compound is [ka] Rather, Z is as defined above.

[0312] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are each independently H or C1-C3 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C3 alkyl group. R3 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2 or 3. R1 consists of H, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and q = 1, 2 or 3. R2 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0313] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) p R6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0314] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) p R6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0315] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2 and NH. n=0 or 1, m=0 or 1, s=0 or 1, Each of J1 and J2 is independently H. R1 is selected from H, a methyl group, and CH2OH. R2 is selected from H, OH, NH2, methyl group and CH2OH. R3 is selected from H, OH, NH2, methyl group and CH2OH. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0316] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2 and NH. n=0 or 1, m=0 or 1, s=0 or 1, Each of J1 and J2 is independently H. R1 is selected from H, a methyl group, and CH2OH. R2 is selected from H, a methyl group, and CH2OH. R3 is selected from H, OH, NH2, methyl group and CH2OH. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0317] In some other embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0318] In some embodiments, the above compound is [ka] This includes, but is not limited to, compounds in which adenine is substituted with guanine, cytosine, uracil, or thymine.

[0319] This disclosure further provides a compound represented by formula (III) or a tautomer thereof, [ka] (III) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. J2 is H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. Q''1 is [ka] Q2 is R2, or Q1 is R2, Q''2 is [ka] And, Of these, R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) qR7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. J1 is H or a C1-C6 alkyl group. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group.

[0320] In some embodiments, W is an MMTr or a DMTr.

[0321] In some embodiments, the above compound is [ka] Rather, W and B are as defined above.

[0322] In some embodiments, when X is NH-CO, R1 is not H.

[0323] In some embodiments, the above compound is [ka] It is not one or more of the compounds mentioned above.

[0324] In some embodiments, the compound represented by formula (III) or its tautomer is specifically the compound represented by formula (III-1) or its tautomer, [ka] (III-1) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. Each of J1 and J2 is independently H or a C1-C6 alkyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group.

[0325] In some embodiments, W is an MMTr or a DMTr.

[0326] In some embodiments, the above compound is [ka] Rather, W and B are as defined above.

[0327] In some embodiments, when X is NH-CO, R1 is not H.

[0328] In some embodiments, the compound represented by formula (III) is [ka] It is not one or more of the compounds mentioned above.

[0329] In some embodiments, the compound represented by formula (III) or its tautomer is specifically the compound represented by formula (III-2) or its tautomer, [ka] (III-2) Among them, Y is selected from O, NH and S. Each X is independently selected from CR4(R4'), S, NR5 and NH-CO, of which R4, R4', and R5 are independently H or C1-C6 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C6 alkyl group. R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) pR6 is selected from among OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2, or 3. R1 consists of H, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and q = 1, 2 or 3. R2 consists of H, C1-C6 alkyl, C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is a base or a base analogue, and W is a leaving group.

[0330] In some embodiments, W is an MMTr or a DMTr.

[0331] In some embodiments, the above compound is [ka] Rather, W and B are as defined above.

[0332] In some embodiments, when X is NH-CO, R1 is not H.

[0333] In some embodiments, the above compound is [ka] It is not one or more of the compounds mentioned above.

[0334] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are each independently H or C1-C3 alkyl groups. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently H or a C1-C3 alkyl group. R3 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C6 alkenyl group and C2-C6 alkynyl group, and p=1, 2 or 3. R1 consists of H, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group and (CH2) q R7 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and q = 1, 2 or 3. R2 consists of H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8 is selected from OH, halogen, methoxy group, ethoxy group, N3, C2-C4 alkenyl group and C2-C4 alkynyl group, and r=1, 2 or 3. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purine bases, pyrimidine bases, indoles, 5-nitroindole, and 3-nitropyrrole.

[0335] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) p R6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purines, N8-modified purines, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0336] In some embodiments, each X is independently selected from CR4(R4'), S, NR5 and NH-CO, where R4, R4', and R5 are independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. n=0, 1, or 2, m=0, 1, or 2, s=0 or 1, Each of J1 and J2 is independently either an H or a methyl group. R3 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) p R6 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and p=1 or 2. R1 is H, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group and (CH2) q R7 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and q=1 or 2. R2 consists of H, OH, F, Cl, NH2, methyl group, ethyl group, n-propyl group, isopropyl group, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, vinyl group, allyl group, ethynyl group, propargyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino group, -O-ethylamino group and (CH2) r R8 is selected from OH, F, Cl, methoxy group, ethoxy group, N3, vinyl group, allyl group, ethynyl group, and propargyl group, and r=1 or 2. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0337] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2 and NH. n=0 or 1, m=0 or 1, s=0 or 1, Each of J1 and J2 is independently H. R1 is selected from H, a methyl group, and CH2OH. R2 is selected from H, OH, NH2, methyl group and CH2OH. R3 is selected from H, OH, NH2, methyl group and CH2OH. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0338] In some embodiments, Y is O or NH, and each X is independently selected from NH-CO, CH2 and NH. n=0 or 1, m=0 or 1, s=0 or 1, Each of J1 and J2 is independently H. R1 is selected from H, a methyl group, and CH2OH. R2 is selected from H, a methyl group, and CH2OH. R3 is selected from H, OH, NH2, methyl group and CH2OH. R1 and R2 are arbitrarily connected directly to form a ring. B is selected from purines, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0339] In some other embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0340] In some embodiments, the above compound is [ka] And compounds in which adenine is substituted with guanine, cytosine, uracil, or thymine, and [ka] [ka] [ka] [ka] [ka] This includes, but is not limited to, compounds in which a base or base analogue among them is substituted with purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, or 3-nitropyrrole.

[0341] This disclosure further provides siRNA or siRNA complexes characterized by a 2'-methoxy group modification that replaces a chemical modification represented by formula (I) or a tautomer modification thereof of the antisense chain of any one siRNA or siRNA complex relating to this disclosure.

[0342] This disclosure further provides siRNA or siRNA complexes characterized in that the chemical modification represented by formula (I) in the antisense chain of any one siRNA or siRNA complex relating to this disclosure is a 2'-methoxy group modification. This disclosure further provides siRNA or siRNA complexes wherein the antisense chain includes a modification that is a chemical modification represented by formula (I) or a tautomer modification in at least one nucleotide between positions 2 and 8 of its 5' region.

[0343] This disclosure further provides siRNA or siRNA complexes characterized by substituting base T for one or more bases U of any one siRNA or siRNA complex relating to this disclosure, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, or 10 bases U.

[0344] This disclosure further provides a method for preparing the above-mentioned siRNA or siRNA complex, comprising the steps of (1) synthesizing a compound represented by formula (II) or a tautomer thereof, and (2) synthesizing the above-mentioned siRNA or siRNA complex using the compound or tautomer thereof from step (1).

[0345] In some embodiments, a compound represented by formula (III) or a tautomer thereof is used to synthesize a compound represented by formula (II) or a tautomer thereof.

[0346] This disclosure further provides applications for the compound represented by formula (II) above or its tautomers in suppressing or reducing siRNA off-target activity.

[0347] This disclosure further provides applications of the compound represented by formula (II) above or its tautomers in the preparation of siRNA.

[0348] term To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0349] Unless otherwise specified, the compounds relating to this disclosure may have a particular geometric or stereoisomeric form. This disclosure includes all cis-trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures rich in enantiomers or diastereomers, and all such compounds are intended to be within the scope of this disclosure. Substituents such as alkyl groups may have other chiral carbon atoms. Any such isomers and mixtures thereof are within the scope of this disclosure.

[0350] Furthermore, the compounds and intermediates of this disclosure may exist in different tautomer forms, and all such forms are included within the scope of this disclosure.

[0351] The term "tautomer" or "tautomer form" refers to structural isomers of different energies that can be interconverted over a low energy barrier. For example, proton tautomers (also called proton transfer tautomers) include interconversion by protrisis, such as keto-enol and imine-enamine, and lactam-lactim isomerization. An example of lactam-lactim equilibrium is between A and B shown below.

[0352] [ka] .

[0353] All compounds in this disclosure can be depicted as either type A or type B. All tautomer forms are within the scope of this disclosure. The nomenclature of the compounds does not exclude any tautomer.

[0354] The compounds relating to this disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise specified, all stereoisomers include, for example, enantiomers and diastereomers. Compounds containing an asymmetric carbon atom relating to this disclosure can be separated in the form of optically active pures or racemates. The optically active pures may be separated from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

[0355] Optically active (R)- and (S)- isomers and D- and L isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain one enantiomer of a compound in this disclosure, preparation may be carried out by asymmetric synthesis or induction with a chiral aid, where the resulting diastereomer mixture is separated and the group splitting is assisted to provide the required pure enantiomer. Alternatively, if the molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxyl group), a salt of the diastereomer is formed with a suitable optically active acid or base, and the diastereomer is divided by conventional methods known in the art, after which the obtained pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomers is generally completed by chromatography, which employs a chiral stationary phase and is optionally combined with chemical induction methods (e.g., purifying carbamates from amines).

[0356] This disclosure further includes several compounds of the disclosure that are isotopically labeled, the same as those described herein, but in which one or more atoms are substituted with atoms having atomic weights or mass numbers different from those commonly found in nature. Examples of isotopes that can be bound to the compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, respectively 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Examples include Cl.

[0357] Unless otherwise specified, where one position is specifically designated as deuterium (D), that position should be understood to be deuterium having an abundance at least 1000 times higher than the natural abundance of deuterium (0.015%) (i.e., incorporating at least 10% deuterium). In the examples, compounds having an abundance higher than the natural abundance of deuterium may be deuterium with an abundance of at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even greater. This disclosure further includes compounds of formula I in various deuterated forms. Each available hydrogen atom bonded to a carbon atom may be independently substituted with a deuterium atom. Those skilled in the art can synthesize compounds of formula I in deuterated forms by referring to relevant literature. When preparing the deuterated form of the compound of formula I, commercially available deuterated starting materials may be used, or it may be synthesized using deuterated reagents by conventional techniques. Deuterated reagents include, but are not limited to, borane deuterated, borane tetrahydrofuran trihydrogenated solution, lithium aluminum hydride deuterated, iodoethane deuterated, and iodomethane deuterated.

[0358] "Optionally" or "optionally" means that the event or situation described thereafter may or may not occur, and this description includes both cases in which the event or situation occurs and cases in which it does not occur. For example, "C where C is optionally substituted with a halogen or cyano group." 1-6 The term "alkyl group" means that a halogen or cyano group may or may not be present, and this explanation includes cases where the alkyl group is substituted with a halogen or cyano group, and cases where the alkyl group is not substituted with a halogen or cyano group.

[0359] In the chemical structure of the compounds described in this disclosure, [ka]

[0360] Unless otherwise specified, the “chemical modifications,” “compounds,” “ligands,” “complexes,” and “nucleic acids” of this disclosure may exist independently in the form of salts, mixed salts, or unsalted substances (e.g., free acids or free bases). If they exist in the form of salts or mixed salts, they may be pharmaceutically acceptable salts.

[0361] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0362] A "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that can retain the bioavailability of the free base without other side effects. Inorganic salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, and phosphate salts. Organic salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacinate, adipine, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, and naphthalenedisulfonate salts. These salts can be prepared by methods known in this art.

[0363] A "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamide resins. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0364] The term "covalent linkage" refers to a connection between two molecules, whether by covalent bonds or non-covalent bonds (e.g., hydrogen bonds or ionic bonds), and includes both direct and indirect linkages.

[0365] The term "direct linkage" refers to a linkage between a first compound or group and a second compound or group without any intervening atoms or groups of atoms.

[0366] The term "indirect linkage" refers to a linkage between a first compound or group and a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0367] As used herein, in the case of RNA-mediated gene silencing, the sense strand (also called the significant strand, SS, or SS strand) of siRNA refers to the strand containing the same or essentially the same sequence as the target mRNA sequence, and the antisense strand (also called the AS, or AS strand) of siRNA refers to the strand containing a sequence complementary to the target mRNA sequence.

[0368] As used herein, the terms “complementary” and “reverse complementary” are interchangeable and have meanings known to those skilled in the art, where, in a double-stranded nucleic acid molecule, a base in one strand pairs complementaryly with a base in the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. When adenine in one strand always pairs with thymine (or uracil) in the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strands can be inferred from the sequence of the complementary strands. Accordingly, in this field, "mismatch" means that in double-stranded nucleic acids, the bases at corresponding positions do not exist in a complementary pairing.

[0369] The term "base" includes any known DNA and RNA bases and base analogues, such as purines and pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogues.

[0370] In this disclosure, base analogs are generally purine or pyrimidine bases and do not include the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3β-D-ribofuranose-(2,6-diaminopyrimidine)(K), 3-β-D-ribofuranose-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione)(P), isocytosine (iso-C), isoguanine (iso-G), and 1-β-D-ribofuranose-(5- Nitroindole), 1-β-D-ribofuranose-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-aldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl group, 4-fluoro-6-methyl This includes tilbenzimidazole, 4-methylbenzimidazole, 3-methylhydroxyisoquinolinyl group, 5-methylhydroxyisoquinolinyl group and 3-methyl-7-propynylhydroxyisoquinolinyl group, 7-azaindole group, 6-methyl-7-azaindole group, imidazopyridyl group, 9-methylimidazopyridyl group, pyrrolopyrazinyl group, hydroxyisoquinolinyl group, 7-propynylhydroxyisoquinolinyl group, propynyl-7-azaindole group, 2,4,5-trimethylphenyl group, 4-methylindole group, 4,6-dimethylindole group, phenyl group, naphthyl group, anthryl group, phenanthryl group, pyrenyl group, stilbenzyl group, naphthacenyl group, pentacenyl group and their structural derivatives. The base analogs may also be universal bases.

[0371] As used herein, the term “universal base” refers to a heterocyclic moiety at the 1′ position of the nucleotide sugar moiety in a modified nucleotide or at an equivalent position in a substituted nucleotide sugar moiety, which, when present in a nucleic acid duplex, can be positioned opposite one or more bases and does not alter the double helix structure (e.g., the structure of the phosphate backbone). Furthermore, the universal base does not disrupt the ability of the single-stranded nucleic acid and target nucleic acid to form a duplex. The ability of a single-stranded nucleic acid and target nucleic acid to form a duplex containing a universal base can be measured by methods apparent to those skilled in the art (e.g., UV absorbance, circular dichroism, gel shift method, single-stranded nuclease sensitivity, etc.). The stability or formation of the duplex can be determined by changing the conditions under which duplex formation is observable; for example, temperature, such as the melting temperature (Tm), is related to the stability of the nucleic acid duplex. Compared to a reference single-stranded nucleic acid that is precisely complementary to the target nucleic acid, a single-stranded nucleic acid containing universal bases has a lower Tm (transformation meter) in the double-stranded nucleic acid formed with the target nucleic acid than in the double-stranded nucleic acid formed with the complementary nucleic acid. However, compared to a reference single-stranded nucleic acid in which a single mismatch occurs due to the substitution of a universal base, a single-stranded nucleic acid containing universal bases has a higher Tm in the double-stranded nucleic acid formed with the target nucleic acid than in the double-stranded nucleic acid formed with the mismatched base.

[0372] Some universal bases can achieve base pairing by forming hydrogen bonds with any of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pairing conditions. Universal bases are not bases that form base pairs with only a single complementary base. In a double-stranded nucleotide, a universal base may not form hydrogen bonds with its counterpart G, C, A, T, and U on the opposing strand of the double-stranded nucleotide, or it may form one hydrogen bond or one or more hydrogen bonds with each of them. Preferably, a universal base does not interact with its counterpart on the opposing strand of the double-stranded nucleotide. In a double-stranded nucleotide, base pairing with a universal base does not alter the double-helix structure of the phosphate backbone. Universal bases may also interact with bases in adjacent nucleotides on the same nucleic acid chain through stacking interactions. Such stacking interactions can stabilize the diploid, particularly when the universal base does not form any hydrogen bonds with the base positioned opposite it on the opposing strand of the diploid. Non-limiting examples of universally binding nucleotides include inosine, 1-β-D-ribofuranose-5-nitroindole, and / or 1-β-D-ribofuranose-3-nitropyrrole.

[0373] As used herein, “chemical modification” or “modification” includes all chemical changes to a nucleotide, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety with another.

[0374] In the chemical structural formulas of this disclosure, the wavy line " [ka] The symbol " indicates a linkage, and the asterisk "*" indicates a chiral center.

[0375] In the context of this disclosure, [ka] Based on the premise [ka] The portion can be replaced with any group that can be linked to an adjacent nucleotide.

[0376] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is linear or branched and contains 1 to 20 carbon atoms, such as alkyl groups containing 1 to 12 carbon atoms or alkyl groups containing 1 to 6 carbon atoms. Non-exclusive examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, n-propyl groups, isopropyl groups, n-butyl groups, isobutyl groups, tert-butyl groups, sec-butyl groups, n-pentyl groups, 1,1-dimethylpropyl groups, 1,2-dimethylpropyl groups, 2,2-dimethylpropyl groups, 1-ethylpropyl groups, 2-methylbutyl groups, 3-methylbutyl groups, n-hexyl groups, 1-ethyl-2-methylpropyl groups, 1,1,2-trimethylpropyl groups, 1,1-dimethylbutyl groups, 1,2-dimethylbutyl groups, 2,2-dimethylbutyl groups, 1,3-dimethylbutyl groups, 2-ethylbutyl groups, 2-methylpentyl groups, 3-methylpentyl groups, 4-methylpentyl groups, or 2,3-dimethylbutyl groups.

[0377] The term "alkoxy group" refers to an -O-alkyl group, the definition of an alkyl group being as described above. Non-exclusive examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. C1-C6 alkoxy groups may be optionally substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, carboxyl groups, or carboxylic acid ester groups.

[0378] The term "alkenyl group" refers to a hydrocarbon group containing at least one double bond. Non-exclusive examples of alkenyl groups include, but are not limited to, vinyl groups, 1-propenyl groups, 2-propenyl groups, 1-butenyl groups, or 2-butenyl groups and their various branched isomers.

[0379] The term "alkynyl group" refers to a hydrocarbon group containing at least one triple bond. Non-exclusive examples of alkynyl groups include, but are not limited to, the ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, or 2-butynyl group and their various branched isomers.

[0380] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0381] In this disclosure, the "ring" in "directly linked so that R1 and R2 form a ring" may be a "cycloalkyl group" or a "heterocycloalkyl group".

[0382] The term "cycloalkyl group" may also be called "carbocyclic group," and refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, where a cycloalkyl ring contains 3 to 20 carbon atoms, 3 to 7 carbon atoms in some embodiments, and 5 to 6 carbon atoms in some embodiments. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cyclohexadienyl groups, while polycyclic cycloalkyl groups include cycloalkyl groups of spirocycles, fused rings, and crosslinked rings. Cycloalkyl groups may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available linking point, independently of halogens, deuterium, hydroxyl groups, oxo, nitro, cyano, and C in some embodiments. 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0383] The above cycloalkyl ring may be condensed with an aryl group or a heteroaryl group, and the ring linked to the parent structure is a cycloalkyl group, and non-limiting examples include an indanyl group, a tetrahydronaphthyl group, a benzocycloheptanyl group, etc. The cycloalkyl group may be optionally substituted or not substituted, and if substituted, the substituent may, in some embodiments, be independently a halogen, deuterium, a hydroxyl group, an oxo, a nitro group, a cyano group, or C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0384] The term "heterocyclyl group" is also called "heterocyclyl" or "heterocyclyl group" and refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more ring atoms are nitrogen, oxygen, or S(O). mA heteroatom selected from (where m is an integer from 0 to 2), but without the -OO-, -OS-, or -SS- ring portion, and the remaining ring atoms are carbon. In some embodiments, selected from those containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and in some embodiments, selected from those containing 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl group, imidazolidinyl group, tetrahydrofuranyl group, tetrahydrothienyl group, dihydroimidazolyl group, dihydrofuranyl group, dihydropyrazolelyl group, dihydropyrrolyl group, piperidinyl group, piperazinyl group, morpholinyl group, thiomorpholinyl group, homopiperazinyl group, etc. Polycyclic heterocycloalkyl groups include heterocycloalkyl groups of spiro rings, fused rings, and crosslinked rings. Non-limiting examples of "heterocycloalkyl groups" are: [ka] This includes, among others.

[0385] The above heterocycloalkyl ring may be condensed with an aryl group or a heteroaryl group, and the ring linked to the parent structure is a heterocycloalkyl group, and non-limiting examples thereof are: [ka] This includes, among others.

[0386] The heterocycloalkyl group may be optionally substituted or left unsubstituted. If substituted, the substituents may, in some embodiments, be a halogen, deuterium, hydroxyl group, oxo, nitro group, cyano group, or C13. 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0387] In the context of this disclosure, Bz represents a benzoyl protecting group, MMTr represents a methoxyphenyldiphenylmethyl group, and DMTr represents a dimethoxytriphenylmethyl protecting group.

[0388] Unless otherwise specified, in the context of this disclosure, uppercase C, G, U, A, and T indicate the base composition of a nucleotide; lowercase d indicates that the nucleotide adjacent to the right of d is a deoxyribonucleotide; lowercase m indicates that the nucleotide adjacent to the left of m is a methoxy-modified nucleotide; lowercase f indicates that the nucleotide adjacent to the left of f is a fluoro-modified nucleotide; and lowercase s indicates that the two nucleotides adjacent to both sides of s are linked by thiophosphate groups.

[0389] As used herein, the term “fluoro-modified nucleotide” refers to a nucleotide formed by substituting a fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide, and “non-fluoro-modified nucleotide” refers to a nucleotide or nucleotide analogue formed by substituting a non-fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide. “Nucleotide analogue” refers to a group that can substitute a nucleotide in a nucleic acid but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acid (abbreviated as BNA), or acyclic nucleotides. Methoxy-modified nucleotides refer to nucleotides formed by substituting a methoxy group for the 2'-hydroxyl group of the ribosyl group. Isonucleotides refer to compounds formed by changing the position of a base in a nucleotide on the ribose ring. In some embodiments, isonucleotides may be compounds formed by moving a base from the 1'- position to the 2'- or 3'- position of the ribose ring. BNA refers to a restricted or inaccessible nucleotide. BNA may include a shrunk crosslink structure having a "fixed" C3'-heparanase in a 5-membered, 6-membered, or 7-membered ring. Typically, the crosslink is incorporated at the 2'-,4'-positions of this ribose to provide a single 2',4'-BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc. Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides may be unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0390] As used herein, the term “suppression” is interchangeable with “reduction,” “silencing,” “downregulation,” “inhibition,” and other similar terms, and includes any level of suppression. Suppression can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level may be any type of control level used in this art, for example, a pre-administration baseline level, or a similar level determined from untreated or control-treated subjects, cells, or samples (e.g., buffer control or inactivator control only). For example, the amount of mRNA excess expression can represent the degree of suppression of target gene expression by siRNA. For instance, mRNA excess expression levels are 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The suppression rate of target gene expression is detected by the Dual-Glo® Luciferase Assay System, and the chemiluminescence values ​​for fireflies and sea urchins (Renilla) are read. The relative value Ratio = Ren / Fir, and the suppression rate (%) = 1 - (Ratio + siRNA / Ratioreporter only) × 100% can be calculated. In this disclosure, the excess mRNA expression ratio (or excess activity %) = 100% - suppression rate (%).

[0391] "Effective dose" refers to the amount of drug, compound, or pharmaceutical composition required to obtain any one or more beneficial or necessary therapeutic outcomes. With respect to prophylactic use, beneficial or necessary outcomes include the elimination or reduction of risk, reduction of severity, or delay of episodes of the disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the progression of the disease. With respect to therapeutic applications, beneficial or necessary outcomes include clinical outcomes such as a reduction in the incidence of various target gene, target mRNA, or target protein-related disease conditions of this disclosure, improvement of one or more symptoms of such conditions, a reduction in the dose of other drugs required to treat the disease, an improvement in the therapeutic effect of another drug, and / or a delay in the progression of a patient's target gene, target mRNA, or target protein-related disease conditions of this disclosure.

[0392] As used herein, the term “angiopoietin-like protein-3” (also known as “ANGPTL3” or “ANGPTL3”) may refer to any nucleic acid or protein of ANGPTL3. The sequence registration number for human ANGPTL3 is NP_055310. “ANGPTL3 expression” refers to the level of mRNA transcribed from the gene encoding ANGPTL3, or the level of protein translated from that mRNA.

[0393] As used herein, the term “thyroxine transport protein” (“TTR”) may also be ATTR, HsT2651, PALB, prealbumin, TBPA, and trans thyretin (prealbumin, amyloidosis type I), and may represent any nucleic acid or protein of TTR. The sequence registration number for the human TTR mRNA transcript is NM_000371. “TTR expression” refers to the level of mRNA transcribed from the gene encoding TTR, or the level of protein translated from the mRNA.

[0394] A "pharmaceutical composition" comprises the siRNA or siRNA complex relating to this disclosure and a pharmaceutically acceptable additive and / or adjuvant, the additive being one or more of the various formulations or compounds commonly used in the art. For example, the pharmaceutically acceptable additive may include at least one of a pH buffer, a protective agent, and an osmotic pressure modifier.

[0395] As used herein, “patient,” “subject,” or “individual” are interchangeable and may include humans or non-human animals such as mammals, e.g., humans or monkeys.

[0396] Various drug delivery systems are known and applicable to the siRNA or siRNA conjugates of this disclosure, such as packaging into liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated cell endocytosis, reverse transcription viruses, or the construction of nucleic acids that become part of other vectors.

[0397] The siRNAs provided in this disclosure can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Of these, solid-phase synthesis is already available as a commercially available customized service. Modified nucleotide groups can be introduced into the siRNAs described in this disclosure using nucleoside monomers having the corresponding modifications, and methods for preparing nucleoside monomers having the corresponding modifications, and methods for introducing modified nucleotide groups into siRNAs, are well known to those skilled in the art. [Brief explanation of the drawing]

[0398] [Figure 1] [Figure 1A] to [Figure 1L] show the results of off-target activity experiments for siRNAs containing different compounds awaiting measurement. [Figure 2] [Figure 2A] to [Figure 2G] show the results of off-target activity experiments of siRNA2 containing different compounds awaiting measurement. [Figure 3][Figure 3A] to [Figure 3G] show the results of off-target activity experiments for siRNA3, including compounds awaiting measurement. [Figure 4] This describes the inhibitory activity of siRNA conjugated with an aminogalactose molecular cluster on the mTTR gene in primary mouse hepatocytes. [Figure 5] This describes the in vivo suppressive activity of siRNA complexed with an aminogalactose molecular cluster against the mouse mTTR gene. [Figure 6] This describes the long-lasting in vivo inhibitory activity of siRNA complexed with an aminogalactose molecular cluster on the mouse mTTR gene. [Figure 7] This study describes the effect of siRNA reagents on total cholesterol levels in Apoc3 transgenic mice. [Figure 8] This study examines the effects of siRNA reagents on triglyceride levels in Apoc3 transgenic mice. [Figure 9] This study examines the effects of siRNA reagents on Apoc3 protein levels in Apoc3 transgenic mice. [Modes for carrying out the invention]

[0399] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify concrete conditions will generally follow normal conditions or conditions recommended by the raw material or product manufacturer. In the case of reagents for which the specific source is not specified, such reagents may be obtained from any molecular biological reagent supplier in a quality / purity suitable for molecular biological applications. [Examples]

[0400] 1. Preparation of chemical modifications and evaluation of activity Example 1 Preparation of chemical modification 1.1 Synthesis of Compound 1-1a and Compound 1-1b [ka] Compound 1 (500 mg, 3.42 mmol) and triethylamine (Et3N, 692 mg, 6.84 mmol, 0.95 mL) were dissolved in dichloromethane (DCM, 10 mL). A solution of 4-toluenesulfonyl chloride (TsCl, 717 mg, 3.76 mmol) in dichloromethane (10 mL) was added dropwise under ice bath. After the addition was complete, the mixture was allowed to react overnight at room temperature with stirring. After the reaction was complete, the mixture was quenched with water, and the aqueous phase was extracted three times with dichloromethane (15 mL). The combined organic phase was first washed with saturated sodium bicarbonate aqueous solution (10 mL), then with saturated brine (20 mL), and finally the solvent was evaporated under reduced pressure to obtain crude product 2 (820 mg, 80%), which was then used directly in the next reaction. MS m / z:C 14 H 21 O5S, [M+H] + Theoretical: 301.10, Measured: 301.2.

[0401] [ka] Compound 3 (239 mg, 1.22 mmol) is dissolved in dimethylformamide (DMF, 10 mL), and a solution of NaH (60% dissolved in mineral oil, 93 mg, 2.33 mmol) is added under ice bath. The reaction is carried out with stirring for 30 minutes, then compound 2 (350 mg, 1.16 mmol) is added dropwise. After addition is complete, the mixture is reacted with stirring at 60°C for 5 hours. After the reaction is complete, water is added to quench the reaction. The aqueous phase is extracted three times with ethyl acetate (15 mL), and the combined organic phase is washed three times with water (10 mL) and then with saturated brine (10 mL). The solvent is evaporated under reduced pressure, and the mixture is subjected to reverse-phase preparative HPLC (C). 18 The reaction proceeded under the following conditions: 5%~50% (A: H2O, B: CH3CN), flow rate: 70 mL / min), followed by lyophilization to obtain 220 mg of compound 4. MS m / z: C 19 H 21 N5O3Na, [M+Na] + Theoretical value: 390.16, Measured value: 390.3.

[0402] [ka] At room temperature, dissolve compound 4 (1.50 g, 4.08 mmol) in a 20 mL mixture of acetic acid and water (4:1), stir at 60°C for 30 minutes, and after the reaction is complete, evaporate the solvent under reduced pressure and perform reverse-phase preparative HPLC (C). 18 The reaction proceeded under the following conditions: 5%~25% (A: H2O, B: CH3CN), flow rate: 70 mL / min), followed by lyophilization to obtain 1.10 g of compound 5. MS m / z: C 16 H 18 N5O3, [M+H] + Theoretical value: 328.13, Measured value: 328.4.

[0403] [ka] Compound 5 (1.00 g, 3.05 mmol) is dissolved in pyridine (Py, 10 mL), and a solution of 4,4'-dimethoxytrityl chloride (DMTrCl, 1.50 g, 4.58 mmol) in pyridine (5 mL) is added dropwise under ice bath. After addition is complete, the mixture is allowed to react overnight at room temperature with stirring. After the reaction is complete, the mixture is quenched with water, the solvent is evaporated under reduced pressure, and the mixture is subjected to reverse-phase preparative HPLC (C). 18 The reaction proceeded under the following conditions: 5%~80% (A: H2O, B: CH3CN), flow rate: 70 mL / min), followed by lyophilization to obtain 1.00 g of compound 6. MS m / z: C 37 H 36 N5O5, [MH] + Theoretical: 630.26, experimental: 630.5. Racemic compound 6 was divided by chiral column (Daicel CHIRALPAK® IE 250 mm*4.6 mm, 5 μm, A: n-hexane, B: ethanol) to obtain 410 mg of 6A(-) and 435 mg of 6B(+).

[0404] [ka] Compound 6A(-) (200 mg, 0.32 mmol), tetrazolium (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (144 mg, 0.48 mmol) was added at room temperature, and the mixture was stirred overnight at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated sodium bicarbonate aqueous solution (10 mL), then further washed with saturated saline solution (20 mL). The filtrate was concentrated and dried, and reverse-phase preparative HPLC (C) was performed. 18 The mixture was prepared under the following conditions: 5%~100% (A: water, B: CH3CN), flow rate: 70 mL / min, and then freeze-dried to obtain 200 mg of compound 1-1a. MS m / z: C 40 H 39 N6O7P, [M-diisopropyl+OH] + Theory: 747.26, Actual measurement: 747.6. 1H NMR (400 MHz, acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

[0405] [ka] Compound 6B(+) (200 mg, 0.32 mmol), tetrazolium (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (144 mg, 0.48 mmol) was added at room temperature, and the mixture was stirred overnight at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated sodium bicarbonate aqueous solution (10 mL), then further washed with saturated saline solution (20 mL). The filtrate was concentrated and dried, and reverse-phase preparative HPLC (C) was performed. 18The mixture was then subjected to a reaction under the following conditions: 5%~100% (A: water, B: CH3CN), flow rate: 70 mL / min, followed by lyophilization to obtain 200 mg of compound 1-1b. MS m / z: C 40 H 39 N6O7P, [M-diisopropyl+OH] + Theoretical value: 747.26, Measured value: 747.5.

[0406] 1.2 Synthesis of Compounds 1-2 [ka] Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was gradually added under argon gas protection at room temperature. After stirring at room temperature for 2 hours, Compound 2 (3.3 g, 16.72 mmol) was added to the reaction mixture. After stirring at room temperature for 12 hours, the reaction mixture was concentrated, and the residue was recrystallized with ethanol (EtOH, 50 mL) to obtain target product 3A (1.3 g, yield 44.0%) (dichloromethane:ethyl acetate = 2:1, Rf = 0.2) and target product 3B (0.6 g, mixture of Compound 1) (dichloromethane:ethyl acetate = 2:1, Rf = 0.18).

[0407] [ka] Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 4 mL) and DCM (20 mL), then stirred at room temperature for 12 hours. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C). 18 The target product 4 (1 g, yield 91.44%) was obtained by adding H2O and acetonitrile. MS m / z:C 39 H 38 N6O6,[M+H] + : 687.5.

[0408] [ka] The compound (D-Threoninol 5, 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL), and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was gradually added. After stirring at room temperature for 16 hours, the reaction mixture was quenched with H2O (10 mL) and concentrated. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C). 18 The target product 6 (4.0 g, yield 86.0%) was obtained by adding H2O and acetonitrile. MS m / z:C 25 H 29 NO4,[M+Na] + : 430.4.

[0409] [ka] Compound 6 (600 mg, 2.02 mmol), Compound 4 (822.5 mg, 2.02 mmol), and dihydroquinoline (EEDQ, 998.2 mg, 4.04 mmol) were dissolved in DCM (10 mL) and methanol (MeOH, 5 mL). The mixture was stirred at room temperature for 16 hours, the solid was removed by filtration, and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with H2O (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography (C 18 The target product 7 (780 mg, yield 56.3%) was obtained by adding H2O and acetonitrile. MS m / z:C 39 H 38 N6O6,[M+H] + : 687.5.

[0410] [ka] Compound 7 (780 mg, 1.13 mmol), tetrazolium (39.8 mg, 0.57 mmol), and N-methylimidazole (18.7 mg, 0.23 mmol) were dissolved in CH3CN (10 mL), and a 3A molecular sieve (700 mg) was added. After stirring at room temperature for 5 minutes under argon gas protection, compound 8 (513.5 mg, 1.70 mmol) was added. After stirring at room temperature for 1 hour, the molecular sieve was removed by filtration, and the solid was rinsed three times with DCM (30 mL). The filtrates were washed with saturated NaHCO3 aqueous solutions (30 mL x 4) and H2O (30 mL x 4), respectively, and the organic phase was concentrated at 30°C. The resulting residue was purified by reverse-phase column chromatography (C 18 After H2O + acetonitrile, acetonitrile (90%), and lyophilization, target compounds 1-2 (700 mg, yield 69.5%) were obtained. MS m / z:C 48 H 55 N8O7P, [M-cyanoethyl-diisopropyl+OH] - :749.3.

[0411] 1.3 Synthesis of Compounds 1-3 [ka] Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL), and NaH (0.37 g, 9.2 mmol, 60% dissolved in mineral oil) was gradually added under argon gas protection at room temperature. After stirring at room temperature for 2 hours, Compound 2 (3.3 g, 16.72 mmol) was added to the reaction mixture. After stirring at room temperature for 12 hours, the reaction mixture was concentrated, and the residue was recrystallized with EtOH (50 mL) to obtain target product 3A (1.3 g, yield 44.0%) (dichloromethane:ethyl acetate = 2:1, Rf = 0.2) and target product 3B (0.6 g, mixture of Compound 1) (dichloromethane:ethyl acetate = 2:1, Rf = 0.18).

[0412] [ka] Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of TFA (4 mL) and DCM (20 mL), then stirred at room temperature for 12 hours. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C). 18 The target product 4 (1 g, yield 91.44%) was obtained by adding H2O and acetonitrile. MS m / z:C 39 H 38 N6O6,[M+H] + : 687.5.

[0413] [ka] The compound L-Threoninol (5 g, 11.4 mmol) was dissolved in pyridine (10 mL), and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was gradually added. After stirring at room temperature for 16 hours, the reaction mixture was quenched with H2O (10 mL) and concentrated. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C). 18 The target product 6 (4.0 g, yield 86.0%) was obtained by adding H2O and acetonitrile. MS m / z:C 25 H 29 NO4,[M+Na] + : 430.4.

[0414] [ka] Compound 6 (600 mg, 2.02 mmol), Compound 4 (822.5 mg, 2.02 mmol), tetramethyluronium hexafluorophosphate (HATU, 1.15 g, 3.03 mmol), and diisopropylethylamine (DIEA, 1 mL, 6.05 mmol) were dissolved in DMF (10 mL). After stirring at room temperature for 16 hours, the reaction mixture was filtered, and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with H2O (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography (C 18After adding H2O + acetonitrile (60% acetonitrile) and lyophilizing, target compound 7 (1.0 g, yield 72.1%) was obtained. MS m / z:C 39 H 38 N6O6,[M+H] + : 687.5.

[0415] [ka] Compound 7 (1.2 g, 1.75 mmol), tetrazolium (61.2 mg, 0.87 mmol), and N-methylimidazole (28.7 mg, 0.35 mmol) were dissolved in CH3CN (10 mL), and a 3A molecular sieve (700 mg) was added. After stirring at room temperature for 5 minutes under argon gas protection, compound 8 (0.79 g, 2.62 mmol) was added. After stirring at room temperature for 1 hour, the molecular sieve was removed by filtration, and the solid was rinsed three times with DCM (30 mL). The filtrates were washed with saturated NaHCO3 aqueous solution (30 mL × 4) and H2O (30 mL × 4), respectively. The organic phase was concentrated at 30°C. The resulting residue was purified by reverse-phase column chromatography (C 18 After H2O + acetonitrile, 90% acetonitrile, and lyophilization, target compounds 1-3 (1.2 g, 77.4% yield) were obtained. MS m / z:C 48 H 55 N8O7P, [M-cyanoethyl-diisopropyl+OH] - :749.3.

[0416] 1.4 Synthesis of Compounds 1-4a and 1-4b [ka] Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL), and NaH (60%, 1.24 g, 30.95 mmol) was gradually added under argon gas protection. After stirring the mixture at room temperature for 30 min, the reaction solution was placed in a solution of tetrahydrofuran (THF, 60 mL) containing tetrakis(triphenylphosphin)palladium (Pd(PPh3)4, 1.95 g, 1.69 mmol), triphenylphosphine (PPh3, 0.74 g, 2.81 mmol), and compound 1 (4.0 g, 28.14 mmol). After stirring the reaction solution at 55°C for 16 h, the solid was filtered off and the mixture was washed three times with DCM (60 mL). The filtrate was concentrated, and the resulting residue was purified by normal-phase column (first with ethyl acetate, and then the column was washed with ethyl acetate:methanol (12:1)) to obtain target product 2 (7 g, crude).

[0417] [ka] Compound 2 (8 g, crude product) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). The mixture was stirred at room temperature for 16 hours, then quenched with water (80 mL) and concentrated. The resulting residue was purified by reverse-phase column chromatography (C). 18 After lyophilization with water + acetonitrile, target compound 3 (13 g, yield 83.7%) was obtained.

[0418] [ka] Compound 3 (5 g, 8.02 mmol) was dissolved in methanol (MeOH, 20 mL) and aqueous ammonia (6 mL). The mixture was stirred at room temperature for 16 hours, and then the reaction solution was concentrated. The resulting residue was purified using a normal-phase column (DCM:MeOH = 20:1) to obtain target compound 4 (4 g, yield 96.0%).

[0419] [ka] Under argon gas protection, 38.54 mL of borane (BH3) tetrahydrofuran solution (dissolved in THF at 1.0 M, 38.54 mmol) was added dropwise to 12 mL of THF solution containing compound 4 (4.00 g, 7.71 mmol). The compound was stirred under argon gas protection at 0°C for 6 hours, after which 27 mL of H2O was added dropwise. Subsequently, 52 mL of 3 M aqueous NaOH solution (156 mmol) was added dropwise to the reaction mixture at 0°C, followed by 10 mL of 30% aqueous H2O2 solution, and then EtOH. The reaction mixture was stirred at room temperature for 48 hours, after which saturated Na2S2O3 was gradually added at 0°C until no bubbles were generated. H2O (300 mL) was added to the reaction mixture and extracted with DCM (4 × 200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography (C 18 After lyophilization with acetonitrile + H2O (50%), target product 5a (730 mg, yield 17.6%) and target product 5b (1.1 g, 26.6%) were obtained.

[0420] [ka] Compound 5a (730 mg, 1.36 mmol) was dissolved in pyridine (8 mL), and TMSCl (0.67 g, 6.14 mmol) was added under argon gas protection and at room temperature. After stirring at room temperature for 1 hour, BzCl (0.29 mL, 2.46 mmol) was added to the reaction mixture. After stirring at room temperature for 16 hours, the reaction mixture was quenched with H2O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL), and tetrabutylammonium fluoride (TBAF, 1 mL) was added. After stirring at room temperature for 1 hour, aqueous ammonia (0.5 mL) was added, and after stirring at room temperature for 5 hours, the reaction mixture was diluted with ethanol (EA, 100 mL) and washed 5 times with saturated saline (30 mL). The organic phase was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C18, H2O + acetonitrile, 60% acetonitrile) and lyophilized to obtain the target product 6a (480 mg, yield 74.8%). MS m / z:C 38 H35 N5O5,[M+H] + : 642.6.

[0421] [ka] Compound 5b (1.1 g, 2.05 mmol) was dissolved in pyridine (20 mL), and TMSCl (1.34 g, 1.28 mmol) was added under argon gas protection and at room temperature. After stirring at room temperature for 1 hour, benzoyl chloride (BzCl, 0.59 mL, 5.92 mmol) was added to the reaction mixture. After stirring at room temperature for 16 hours, the reaction mixture was quenched with H2O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL), and TBAF (2 mL) was added. After stirring at room temperature for 1 hour, aqueous ammonia (0.5 mL) was added, and after stirring at room temperature for 5 hours, the reaction mixture was diluted with EA (100 mL) and washed 5 times with saturated brine (30 mL). The organic phase was concentrated, and the resulting residue was purified by reverse-phase column chromatography (C18, H2O + acetonitrile, 60% acetonitrile) and lyophilized to obtain the target product 6b (1.4 g, yield 82.1%). MS m / z:C 38 H 35 N5O5,[M+H] + : 642.5.

[0422] [ka] Compound 6a (700 mg, 1.04 mmol), tetrazolium (26.2 mg, 0.37 mmol), and N-methylimidazole were dissolved in CH3CN (10 mL), and a 3A molecular sieve (500 mg) was added. After stirring at room temperature for 5 minutes under argon gas protection, compound 7 (470.4 mg, 1.56 mmol) was added. After stirring at room temperature for 1 hour, the molecular sieve was removed by filtration, and the solid was rinsed three times with DCM (50 mL). The filtrates were washed with saturated NaHCO3 aqueous solution (50 mL x 4) and H2O (50 mL x 4). The organic phase was concentrated at 30°C. The resulting residue was purified by reverse-phase column chromatography (C 18After H2O + acetonitrile, 90% acetonitrile, and lyophilization, target compound 1-4A (600 mg, yield 66.1%) was obtained. MS m / z:C 47 H 52 N7O6P, [M-cyanoethyl-diisopropyl+OH] - :704.3.

[0423] [ka] Compound 6b (1.3 g, 2.03 mmol), tetrazolium (71.0 mg, 1.01 mmol), and N-methylimidazole (33.3 mg, 0.41 mmol) were dissolved in CH3CN (20 mL), and a 3A molecular sieve (700 mg) was added. After stirring at room temperature for 5 minutes under argon gas protection, compound 7 (0.92 g, 3.04 mmol) was added. After stirring at room temperature for 1 hour, the molecular sieve was removed by filtration, and the solid was rinsed three times with DCM (50 mL). The filtrates were washed with saturated NaHCO3 aqueous solution (50 mL x 4) and H2O (50 mL x 4). The organic phase was concentrated at 30°C. The resulting residue was purified by reverse-phase column chromatography (C 18 After H2O + acetonitrile, 90% acetonitrile, and lyophilization, target compounds 1-4b (1.4 g, yield 82.1%) were obtained. MS m / z:C 47 H 52 N7O6P, [M-cyanoethyl-diisopropyl] - :704.3.

[0424] 1.5 Synthesis of Compounds 1-5 [ka] Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL), and NaH (60%, 1.24 g, 30.95 mmol) was gradually added under argon gas protection. After stirring the mixture at room temperature for 30 min, the reaction solution was added to a THF (60 mL) solution containing Pd(PPh3)4 (1.95 g, 1.69 mmol), PPh3 (0.74 g, 2.81 mmol), and compound 1 (4.0 g, 28.14 mmol). After stirring the reaction solution at 55°C for 16 h, the solid was filtered off and washed three times with DCM (60 mL). The filtrate was concentrated, and the resulting residue was purified by normal-phase column (first with ethyl acetate, and then the column was washed with ethyl acetate:methanol (12:1)) to obtain target solid 2 (7 g, crude product).

[0425] [ka] Compound 2 (8 g, crude product) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). The mixture was stirred at room temperature for 16 hours, then quenched with water (80 mL) and concentrated. The resulting residue was purified by reverse-phase column chromatography (C). 18 After lyophilization with water + acetonitrile, target compound 3 (13 g, yield 83.7%) was obtained.

[0426] [ka] Compound 3 (1 g, 1.60 mmol), KHCO3 (0.48 g, 4.81 mmol), and ethylene glycol (0.40 g, 6.41 mmol) were dissolved in acetone (50 mL), and KMnO4 (40% dissolved in water, 0.67 g, 1.68 mmol) was gradually added at -30°C. After stirring at -30°C for 1 hour, the reaction was quenched with saturated sodium thiosulfate aqueous solution (30 mL). The mixture was extracted four times with DCM (30 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by reverse-phase column chromatography (C18, H2O + acetonitrile, 60% acetonitrile) and freeze-dried to obtain target product 4 (600 mg, yield 56.9%). MS m / z:C 38 H 35 N5O6,[M+H] + : 658.5.

[0427] [ka] In a 250 mL round-bottom flask, add reaction 4 (5.0 g, 7.601 mmol), NaIO4 and 1,4-dioxane / water (50 mL / 5 mL), and react at room temperature for 2 hours. After removing the solvent under reduced pressure, a white solid (6.0 g) is obtained. Then, dissolve in methanol (50 mL), add sodium borohydride (1.62 g, 38 mmol), and stir at room temperature for 2 hours. After adding 10 mL of 10% ammonium chloride solution and removing the solvent under reduced pressure, product P1 and colorless oily substance 5 (2.0 g, 3.0315 mmol, 39%) are obtained by C18 column chromatography (water / acetonitrile: 5%~95%). LCMS, MS+, [M+H] + The value was 660.

[0428] [ka] Compound 5 (1.7 g, 2.58 mmol) and DBU (0.77 mL, 5.15 mmol) were dissolved in DCM (20 mL), and BzCl (0.5 M in DCM, 0.8 mL) was added dropwise to the reaction at -70°C under argon gas protection. The reaction solution was left at -70°C for 1 hour, and then quenched with ethanol (5 mL). The quenched reaction solution was diluted with DCM (100 mL), washed three times with water (30 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by normal-phase column (DCM:EA = 1:1) to obtain a white solid 6 (80 mg, yield 4.14%). MS m / z:C 45 H 41 N5O7,[M+H] + : 764.5.

[0429] [ka] Compound 4 (380 mg, 0.50 mmol), tetrazolium (17.43 mg, 0.25 mmol), and N-methylimidazole (8.17 mg, 0.10 mmol) were dissolved in CH3CN (10 mL), and a 3A molecular sieve (500 mg) was added. After stirring at room temperature for 5 minutes under argon gas protection, compound 7 (224.95 mg, 0.75 mmol) was added. After stirring at room temperature for 1 hour, the molecular sieve was removed by filtration, and the solid was rinsed three times with DCM (50 mL). The filtrates were washed with saturated NaHCO3 aqueous solution (50 mL x 4) and H2O (50 mL x 4). The organic phase was concentrated at 30°C. The resulting residue was purified by reverse-phase column chromatography (C 18 After adding H2O + acetonitrile (90% acetonitrile) and lyophilizing, target products 1-5 (330 mg, 68.8% yield) were obtained. MS m / z:C 54 H 58 N7O8P, [M-cyanoethyl-diisopropyl] - :826.3.

[0430] 1.6 Synthesis of Compounds 1-6a [ka] Compound 1 (10 g, 68.404 mmol), Compound 2 (15 g, 62.186 mmol), and triphenylphosphine (32.62 g, 124.371 mmol) were dissolved in anhydrous THF (30 mL), and DIAD (24.656 mL, 124.371 mmol) was gradually added dropwise at 0°C. The reaction mixture was allowed to react at 25°C for 12 hours, and LC-MS confirmed completion of the reaction. The reaction mixture was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried, and the filtrate was concentrated. The resulting residue was purified by normal-phase column chromatography (DCM / MeOH = 10 / 1) to obtain target product 3 (20 g).

[0431] [ka] Compound 3 (20 g, 28.585 mmol) was dissolved in acetic acid (24 mL, 426.016 mmol) and H2O (12 mL) and stirred at 60°C for 1 hour. The reaction mixture was then concentrated and dried, and THF (12 mL) and H2O (12 mL) were added, and the mixture was stirred at 80°C for 7 hours. LC-MS confirmed the completion of the reaction. Ethyl acetate (200 mL) and water (100 mL) were added to the reaction mixture for extraction, and sodium carbonate solid was added to the aqueous phase until a large amount of solid precipitated in the aqueous phase. The solid was filtered, washed with water, and the filter cake was dried by suction using an oil pump to obtain target compound 5 (9 g).

[0432] [ka] Under nitrogen gas protection, compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL), and TMSCl (14.250 mL, 111.489 mmol) was gradually added at 0°C, and the mixture was stirred for 2 hours. Then, Isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0°C, and the mixture was stirred at 25°C for 1 hour. LC-MS confirmed the completion of the reaction. The mixture was extracted with dichloromethane (200 mL) and water (200 mL), and the organic phase was concentrated and dried. The sample was then mixed and purified using a normal-phase column (DCM:MeOH = 10:1), and the column was run (a peak was observed at 4.8%) to obtain yellow oily compound 6 (12 g).

[0433] [ka] Under nitrogen gas protection, compound 6 (5.5 g, 12.392 mmol) was dissolved in pyridine (30 mL), and molecular sieve 4A 1 / 16 (7 g, 12.392 mmol) was added. Then, DMTrCl (5.04 g, 14.870 mmol) solid was added in several portions at 0°C, and the reaction was carried out at 25°C for 2 hours. TLC (PE:SiO = 1:1, Rf = 0.69) indicated that the reaction was already complete. The reaction solution was treated together with TJN200879-040-P1. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), and the organic phase was concentrated and dried. The sample was then mixed and purified by normal-phase column (PE:SiO column, with a peak appearing at 84%) to obtain yellow oily compound 7 (12 g).

[0434] [ka] Compound 7 (12 g, 15.389 mmol) was dissolved in SiO2 (140 mL), and wet palladium-carbon Pd / C (7 g, 15.389 mmol) was added. The reaction mixture was reacted at 25°C under hydrogen gas (15 Psi) for 2 hours. TLC (PE:SiO2 = 0:1, Rf = 0.09) showed that the reaction was already complete. The reaction mixture was filtered, and the filter cake was washed three times with ethyl acetate (30 mL), after which the filtrate was collected. After concentrating and drying the filtrate, 50 mL of dichloromethane and 2 mL of triethylamine were added, and the sample was mixed and purified by normal-phase column chromatography (DCM:MeOH = 10:1, with a peak at 0.5%) to obtain 9 g (yellow foamy solid). The resulting racemic compound was separated by SFC to obtain the target compound 7A(-) (3.9 g) and target compound 7B(+) (3.8 g).

[0435] [ka] Compound 7A(-) (3.30 g, 5.40 mmol), tetrazolium (190 mg, 2.70 mmol), 1-methylimidazole (90 mg, 1.10 mmol), and 3A molecular sieve (500 mg) were dissolved in 30 mL of acetonitrile. Compound 8 (2.50 g, 8.10 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, and the mixture was washed with DCM (150 mL) and saturated sodium bicarbonate aqueous solution (30 mL x 3). Further washing was performed with saturated saline solution (30 mL), and the filtrate was concentrated and dried to obtain 1-6a (2.9 g, 66%) after reverse-phase preparative HPLC (C18, conditions: 5%~100% (A: water, B: CH3CN), flow rate: 70 mL / min). After lyophilization, 1-6a (2.9 g, 66%) was obtained. MS m / z: C 43 H 55N7O7P [M+H]+, theoretical: 812.38, measured: 812.5. 1H NMR (400 MHz, acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

[0436] 1.7 Synthesis of Compounds 1-7a [ka] Under nitrogen gas protection, compound 1 (5 g, 23.1272 mmol), compound 2 (6.76 g, 46.254 mmol), and triphenylphosphine (7.28 g, 27.753 mmol) were dissolved in 30 mL of dioxane, and DEAD (5.502 mL, 27.753 mmol) was gradually added dropwise at 0°C. After the addition was complete, the reaction was gradually heated to 25°C and continued for 1 hour. 100 mL of H2O and 100 mL of pharmaceutically acceptable phosphate were added to the reaction mixture for extraction. The organic phases were combined, dried, filtered, and concentrated. The sample was then mixed and subjected to column chromatography, and purified using a normal-phase column (PE:siRNA = 1:1) to obtain the target product (4 g).

[0437] [ka] Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and H2O (4 mL), heated in an oil bath at 60°C for 0.5 hours, the reaction mixture was concentrated and dried, and the resulting residue was purified by normal-phase column chromatography (PE:HCl = 0:1) to obtain target product 4 (3 g).

[0438] [ka] Compound 4 (3 g, 8.873 mmol) was dissolved in 5 mL of pyridine, and 10 mL of a pyridine solution of DMTrCl (3.91 g, 11.535 mmol) was gradually added dropwise at 0°C under nitrogen gas protection. After the addition was complete, the reaction was raised to 25°C and continued for 1 hour. The reaction mixture was extracted with 50 mL of water and 100 mL of ethyl acetate. The aqueous phase was further extracted three times with 100 mL of ethyl acetate, and the organic phases were combined, dried, filtered, concentrated, and purified by normal-phase column chromatography (PE:HCl = 2:1). The target product 5 (4 g) was obtained.

[0439] [ka] Compound 5 (4 g, 5.769 mmol) was dissolved in methanol (10 mL), saturated NH3 methanol solution (40 mL) was added, and the reaction was carried out at 0°C for 6 hours. The reaction mixture was concentrated and dried, and purified by normal-phase column (PE:SiO = 0:1) to obtain 2.4 g of a racemic compound. This compound was then separated by SFC to obtain target product 6A (750 mg, 100% purity) and target product 6B (400 mg, 99.16% purity).

[0440] [ka] Compound 6A(-) (700 mg, 1.40 mmol), tetrazolium (50 mg, 0.70 mmol), 1-methylimidazole (23 mg, 0.28 mmol), and 3A molecular sieve (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (630 mg, 2.10 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, and the mixture was washed with DCM (50 mL) and saturated sodium bicarbonate aqueous solution (10 mL x 3). Further washing was performed with saturated saline solution (20 mL), and the filtrate was concentrated and dried to obtain 1-7a (700 mg, 72%) after reverse-phase preparative HPLC (C18, conditions: 5%~100% (A: water, B: CH3CN), flow rate: 70 mL / min). After lyophilization, 1-7a (700 mg, 72%) was obtained. MS m / z: C 38 H 47N4O7PNa[M+Na]+, theoretical: 725.32, measured: 725.5.

[0441] 1.8 Synthesis of Compounds 1-8a [ka] Compound 1 (8.5 g, 76.508 mmol) and Compound 2 (30.64 g, 91.809 mmol) were dissolved in DMF (150 mL), CS2CO3 (29.91 g, 91.809 mmol) was added, and the reaction was carried out at 90°C for 12 hours under nitrogen gas protection. Completion of the reaction was detected by LC-MS. The reaction mixture was filtered, concentrated and dried using an oil pump, and purified by normal-phase column chromatography (80 g, DCM / MeOH = 10 / 1 to 5 / 1) to obtain target product 3 (13.5 g, 80% purity).

[0442] [ka] Compound 3 (10.5 g, 35.105 mmol) was dissolved in pyridine (65 mL) and CH3CN (65 mL), and BzCl (4.894 mL, 42.126 mmol) was added dropwise to the solution. The reaction was allowed to proceed at 25°C for 2 hours. LC-MS detected that most of the starting materials had reacted. H2O (100 mL) was added to quench the mixture, and the solution was extracted with HCl (100 mL x 3). The mixture was concentrated and dried, and separated by column (combined with TJN200872-101) for purification (80 g, PE / HCl = 10 / 1 to 0 / 1, DCM / MeOH = 10 / 1) to obtain target product 4 (14 g, 90% purity).

[0443] [ka] Compound 4 (14 g, 36.694 mmol) was dissolved in HOAc (56 mL, 314.796 mmol) and H2O (14 mL), and the reaction was carried out at 60°C for 2 hours. LC-MS confirmed the completion of the reaction. The mixture was concentrated using an oil pump and separated by normal-phase column chromatography (40 g, DCM / MeOH = 1 / 0 to 5 / 1) to obtain target product 5 (8.4 g, 90% purity & 2.4 g, 80% purity).

[0444] [ka] Compound 5 (7.4 g, 21.957 mmol), DMAP (0.54 g, 4.391 mmol), and MOLECULAR SIEVE 4A (11.1 g, 2.967 mmol) were dissolved in pyridine (60 mL), stirred under ice bath for 10 minutes, then DMTrCl (8.93 g, 26.348 mmol) was added, and the reaction was carried out with stirring for 1.8 hours. LC-MS detected approximately 19% of the starting material and approximately 60% of the target material as MS. This was purified together with (TJN200872-105&106). H2O (50 mL) was added to the reaction mixture, extracted with DCM (50 mL x 3), dried, concentrated and dried, and separated by column (120 g, PE / (EA:DCM:TEA=1:1:0.05)=1 / 0~0 / 1, so DCM / MeOH=10 / 1) to obtain yellow solid compound 6 (11 g, 89% purity, TJN200872-105&106&107), and the starting material (3.0 g, 70% purity) was recovered.

[0445] [ka] Compound 6 (15 g, 22.041 mmol) was separated by SFC (DAICEL CHIRALPAK AD (250 mm*50 mm, 10 μm), 0.1% NH3H2O ​​EtOH, B: 45%~45%, 200 mL / min) to obtain target product 6A (5.33 g, 94.29% purity) and target product 6B (6.14 g, 97.91% purity), and 1.0 g of compound 6 was recovered.

[0446] [ka] Compound 6B(-) (5.4 g, 8.92 mmol), tetrazolium (312 mg, 4.46 mmol), 1-methylimidazole (146 mg, 1.78 mmol), and 3A molecular sieve (500 mg) were dissolved in 40 mL of acetonitrile. Compound 7 (4 g, 13.4 mmol) was added at room temperature, and the mixture was stirred for 2 hours at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, and the mixture was washed with DCM (200 mL) and saturated sodium bicarbonate aqueous solution (30 mL x 3). Further washing was performed with saturated saline solution (50 mL), and the filtrate was concentrated and dried to obtain 1-8a (5.8 g, 80%) after reverse-phase preparative HPLC (C18, conditions: 5%~100% (A: water, B: CH3CN), flow rate: 70 mL / min). After lyophilization, 1-8a (5.8 g, 80%) was obtained. MS m / z: C 45 H 51 N5O7P, [M+H]+, Theoretical: 804.36, Measured: 804.4.

[0447] Example 2 Synthesis of siRNA The synthesis of siRNA was no different from the usual phosphoramidite solid-phase synthesis method. When synthesizing the nucleotide modified at position 7 of the AS chain 5', the parent sequence nucleotide was substituted with the synthesized phosphoramidite monomer described above.

[0448] The synthesis process is briefly described below. Using a Dr. Oligo48 synthesizer (Biolytic), nucleoside phosphoramidite monomers were linked one by one using a synthesis program, starting with the Universal CPG vector. Except for the nucleoside phosphoramidite monomer at position 7 of the 5' of the AS chain described above, other nucleoside monomer raw materials such as 2'-F RNA and 2'-O-methyl RNA were purchased from Shanghai Zhaowei or Suzhou Jima. 5-ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M acetonitrile solution), a solution of 0.22 M PADS dissolved in acetonitrile and trimethylpyridine (Suzhou Kema) in a 1:1 volume ratio was used as a vulcanizing agent, and iodopyridine / aqueous solution (Kema) was used as an oxidizing agent.

[0449] After solid-phase synthesis was complete, the oligo-ribonucleotides were dissolved from the solid support and immersed in a 3:1 28% aqueous ammonia and ethanol solution at 50°C for 16 hours. The mixture was then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated. Purification was performed by C18 reverse-phase chromatography, with DMTr removed using 0.1 M TEAA and acetonitrile as the mobile phase, and 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LC-MS, and further quantified by UV (260 nm).

[0450] The obtained single-stranded oligonucleotides were paired complementaryally in equimolar ratios, annealed, and finally the resulting double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment for use.

[0451] Example 3: psiCHECK activity screening experiment 3.1 Materials and equipment for the experiment The synthesis of the siRNA sample was as described above, and the plasmid was sourced from Bioengineering (Shanghai) Co., Ltd. The consumables, reagents, and equipment used in the psiCHECK experiment are shown in Tables 1 and 2. [Table 1]

[0452] [Table 2]

[0453] 3.2 psiCHECK Activity Screening Experiment Procedure The process involves seeding cells onto plates and transfection of cells. The specific amounts of the transfection complex are shown in Table 3. [Table 3] According to Table 4, the fluids were diluted to different concentrations according to the needs of different experiments and prepared for use as working fluids.

[0454] [Table 4] After 24 hours of transfection, detection was performed according to the experimental procedure of the Dual-Glo® Luciferase Assay System detection reagent kit. For Dual-Glo® Luciferase Assay System detection, the Firfly and Renilla chemiluminescence values ​​were read using the Dual luciferase reporter gene assay kit (Promega, cat.E2940), the relative value = Ren / Fir was calculated, and the inhibition rate = 1 - (Ratio + siRNA / Ratioreporter only) × 100% = inhibition rate (%) was calculated. In this disclosure, the ratio of excess mRNA expression is 100% minus the suppression rate (%).

[0455] Example 4: On-target and off-target activity experiments of siRNAs with different chemical modifications. Using the compound from Example 1, the following siRNAs were synthesized according to the method of Example 2, and the on-target and off-target activities of each siRNA were verified according to the method of Example 3. Each siRNA used the same sense strand, and the 7th position of the 5' end of the antisense strand had the following modified nucleotides / chemical modifications: [ka] .

[0456] Of these, nucleotides synthesized using 2-hydroxymethyl-1,3-propanediol as a starting material are defined as hmpNA. TJ-NA019(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-2 in Example 1.1. TJ-NA020(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-3 in Example 1.1. TJ-NA026(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-4a in Example 1.1. TJ-NA027(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-4b in Example 1.1. TJ-NA038(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-5 in Example 1.1. (+)hmpNA(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-1b in Example 1.1, and its absolute configuration is (S)-hmpNA(A). (-)hmpNA(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-1a in Example 1.1, and its absolute configuration is (R)-hmpNA(A). Similarly, by substituting the base types of hmpNA and obtaining the following structures via solid-phase synthesis, the absolute configuration was confirmed: (+)hmpNA(G) has an absolute configuration of (S)-hmpNA(G), (-)hmpNA(G) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-6a in Example 1.6, and its absolute configuration is (R)-hmpNA(G). (+)hmpNA(C) has an absolute configuration of (S)-hmpNA(C), (-)hmpNA(C) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-8a in Example 1.8, and its absolute configuration is (R)-hmpNA(C). (+)hmpNA(U) has an absolute configuration of (R)-hmpNA(U), (-)hmpNA(U) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-7a in Example 1.7, and its absolute configuration is (S)-hmpNA(U).

[0457] The absolute configurations of (S)-hmpNA(G), (R)-hmpNA(G), (S)-hmpNA(C), (R)-hmpNA(C), (S)-hmpNA(U), and (R)-hmpNA(U) were confirmed by X-ray diffraction from their intermediates or derivatives.

[0458] The structure of the intermediate or derivative is as follows: [ka] TJ-NA067: The detected crystal is colorless and blocky (0.30 mm³ × 0.10 mm³ × 0.04 mm³) and belongs to the monoclinic P21 space group. Cell parameters are a=16.0496(5)Å, b=4.86260(10)Å, c=16.4686(5)Å, α=90°, β=118.015(4)°, γ=90°, V=1134.65(7)ų, Z=4. Calculated density Dc=1.389 g / cm³, number of electrons in the unit cell F(000)=504.0, linear absorption coefficient of the unit cell μ(Cu Kα)=0.840 mm⁻¹, diffraction experiment temperature T=150.00(11)K.

[0459] [ka] 6A(+): The detected crystal is colorless and blocky (0.30 mm³ × 0.20 mm³ × 0.10 mm³) and belongs to the monoclinic P21 space group. Cell parameters are a=22.6688(7)Å, b=8.5595(2)Å, c=23.3578(5)Å, α=90°, β=113.876(3)°, γ=90°, V=4144.3(2)ų, Z=2. Calculated density Dc=0.999 g / cm³, number of electrons in the unit cell F(000)=1318.0, linear absorption coefficient μ(Cu Kα) of the unit cell=0.570 mm⁻¹, diffraction experiment temperature T=100.01(18)K.

[0460] [ka] TJ-NA048: The detected crystal is colorless, needle-shaped (0.30 mm³ × 0.04 mm³ × 0.04 mm³) and belongs to the monoclinic P1 space group. Cell parameters are a=7.6165(4)Å, b=11.3423(5)Å, c=17.3991(8)Å, α=85.007(4)°, β=88.052(4)°, γ=70.532(4)°, V=1411.75(12)ų, Z=2. Calculated density Dc=1.366 g / cm³, number of electrons in the unit cell F(000)=620.0, linear absorption coefficient μ(Cu Kα) of the unit cell=0.856 mm⁻¹, diffraction experiment temperature T=150.00(13)K.

[0461] [ka] TJ-NA092: The detected crystal is colorless and prismatic (0.30 mm³ × 0.10 mm³ × 0.10 mm³) and belongs to the monoclinic P1 space group. Cell parameters are a=5.17960(10)Å, b=8.0667(2)Å, c=12.4077(2)Å, α=93.146(2)°, β=101.266(2)°, γ=96.134(2)°, V=503.993(18)ų, Z=2. Calculated density Dc=1.412 g / cm³, number of electrons in the unit cell F(000)=228.0, linear absorption coefficient μ(Cu Kα) of the unit cell=0.945 mm⁻¹, diffraction experiment temperature T=100.00(10)K.

[0462] [Table 5] The results of the on-target activity experiments are shown in Table 6, and the results of the off-target activity experiments are shown in Table 7 and Figures 1A to 1L. Currently, all of the experimental compounds show activity equivalent to or slightly better than the parent sequence in the test sequence, explaining that the modifications do not affect the on-target activity. Among them, the compounds with the best activity were GNA / Abasic / Id and TJ-NA019, respectively. (A) TJ-NA020 (A) TJ-NA026 (A) These are siRNAs containing (+)hmpNA(A) and (-)hmpNA(A). The parent sequence has significant off-target activity, and each modification shows a significant inhibitory effect on off-target activity, particularly TJ-NA027. (A) No off-target activity was observed in siRNAs containing (+)hmpNA(A) and (-)hmpNA(A).

[0463] [Table 6]

[0464] [Table 7]

[0465] Example 5 Sequence-dependent experiments of siRNAs with different chemical modifications Since basic modifications are known to be siRNA sequence-dependent, the inventors tested the compounds awaiting measurement according to this disclosure on several different sequences. Using siRNAs targeting four different gene mRNAs (ANGPTL3, HBV-S, HBV-X, TTR) (sequences are shown in Table 8), the compounds TJ-NA020(A) and TJ-NA027 of Example 1 were tested. (A)(+) hmpNA(A), (-) hmpNA(A), and GNA as a control. (A The 7th position of the 5' end of the AS chain (the sequence is shown in Table 9) was modified with an Id compound, and the on-target and off-target activities were compared with the parent sequence.

[0466] [Table 8]

[0467] [Table 9-1] [Table 9-2] The results of the on-target activity experiment are shown in Table 10, and GNA (A) The compound exhibits significant sequence dependence, and the on-target activity of different sequences is clearly different. The compound awaiting measurement according to this disclosure does not exhibit significant sequence dependence and has greater general applicability. Furthermore, similar activity effects have been obtained simply by changing the 9th position of the 5' end of the AS chain to a 2'-F modification and the 10th position to a 2'-OMe modification; that is, the compound awaiting measurement according to this disclosure does not exhibit significant sequence dependence.

[0468] [Table 10] The results of off-target activity experiments for siRNA2 and siRNA3 are shown in Table 11, Figures 2A-2G (targeting HBV-S), and Figures 3A-3G (targeting HBV-X). It can be seen that the compounds awaiting measurement according to this disclosure significantly reduced the off-target activity of siRNA compared to the parent sequence. Furthermore, similar off-target activity effects were obtained simply by changing the 9th position of the 5' end of the AS chain to a 2'-F modification and the 10th position to a 2'-OMe modification, meaning that the off-target activity of siRNA can be similarly significantly reduced.

[0469] [Table 11]

[0470] 2. Preparation and activity evaluation of target ligands [Table 12] Example 6: Aminogalactose compound 1-t linked to a solid-phase vector [ka] The synthesis route is as follows:

[0471] 1) Synthesis route of compound 1-g [ka] 2) Synthesis route of compound 1-h [ka] 3) Synthesis route of compound 1-l [ka] 4) Synthesis of compound 1-q [ka] 5) Synthesis of aminogalactose compound 1-t linked to a solid-phase vector [ka] Step 1 Starting materials 1-a (297 g, 763 mmol) and 1-b (160 g, 636 mmol) were dissolved in 960 mL of DCE. Under conditions of 15°C, Sc(OTf)3 (15.6 g, 31.8 mmol) was added, and the reaction temperature was raised to 85°C. The reaction was carried out for 2 hours with stirring. After the reaction was complete, 1.5 L of saturated NaHCO3 was added to stop the reaction, the organic phase was separated, and the solution was washed with 1.5 L of saturated brine. The organic phase was dried over anhydrous Na2SO4, and the filtered solution was distilled under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate 5:1~0:1) to obtain a pale yellow oily product 1-c (328 g, 544 mmol, yield 85.5%, purity 96.4%). 1 HNMR:(400 MHz, CDCl3) δ 7.44-7.29 (m, 5H), 5.83 (d, J = 8.8 Hz, 1H), 5.40-5.23 (m, 2H), 5.18-5.06 (m, 2H), 4.86 (s, 1H), 4.66 (d, J = 8.4 Hz, 1H), 4.21-4.07 (m, 2H), 4.04-3.77 (m, 3H), 3.51-3.45 (m, 1H), 3.31-3.11 (m, 2H), 2.18 (d, J = 2.0 Hz, 1H), 2.14 (s, 3H), 2.06 (s, 3H), 2.03-1.99 (m, 3H), 1.95 (s, 3H), 1.64-1.46 (m, 4H), 1.43-1.29 (m, 4H). MS, C 28 H 40 N2O 11 , measured M + 581.3.

[0472] Step 2 The compound obtained in Step 1 was carried out in parallel in two parts: each reaction involved adding compound 1-c (72.0 g, 124 mmol) to 432 mL of THF, adding Pd / C (20.0 g, 10% purity) under argon gas protection, and then adding TFA (14.1 g, 124 mmol, 9.18 mL). Hydrogen gas was passed through the reaction solution, the gas pressure was maintained at 30 Psi, and the mixture was heated to 30°C and stirred for 16 hours. After the reaction was complete, the two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. After drying by suction under reduced pressure, the target compound 1-d (139 g) was obtained. 1 HNMR(400 MHz, DMSO-d6)δ 7.85 (d, J = 9.2 Hz, 1H), 7.74 (s, 3H), 5.21 (d, J = 3.6 Hz, 1H), 4.97 (dd, J = 2.8, 10.8 Hz, 1H), 4.48 (d, J = 8.8 Hz, 1H), 4.06-3.98 (m, 3H), 3.93-3.82 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.56 (m, 1H), 3.43-3.38 (m, 1H), 2.82-2.71 (m, 2H), 2.13-2.09 (m, 3H), 2.01-1.97 (m, 3H), 1.91-1.87 (m, 3H), 1.77 (s, 3H), 1.76-1.73 (m, 1H), 1.52-1.44 (m, 4H), 1.28 (s, 4H).

[0473] Step 3 Compound 1-d (139 g, 247 mmol) and compound 1-e (75.3 g, 223 mmol) were added to DMF solution (834 mL). At 0°C, DIPEA (41.6 g, 322 mmol, 56.1 mL), HOBt (36.8 g, 272 mmol), and EDCI (52.2 g, 272 mmol) were added, and the mixture was allowed to react with stirring at 15°C for 16 hours. After the reaction was complete, the reaction solution was diluted with dichloromethane (400 mL), and then washed sequentially with saturated ammonium chloride solution (1 L), saturated NaHCO3 (1.00 L), and saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 0:1) to obtain the target compound 1-f (108 g, yield 56.8%). 1 HNMR(40 (400 MHz, DMSO-d6) δ 7.89-7.78 (m, 2H), 7.41-7.27 (m, 6H), 5.21 (d, J = 3.2 Hz, 1H), 5.08-4.92 (m, 3H), 4.48 (d, J = 8.4 Hz, 1H), 4.07-3.99 (m, 3H), 3.97-3.81 (m, 2H), 3.75-3.64 (m, 1H), 3.42-3.37 (m, 1H), 3.13-2.93 (m, 2H), 2.20 (t, J = 8.0 Hz, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.87-1.79 (m, 1H), 1.76 (s, 3H), 1.74-1.64 (m, 1H), 1.48-1.41 (m, 2H), 1.38 (s, 12H), 1.29-1.20 (m, 4H), 1.19-1.14 (m, 1H). MS, C 37 H 55 N3O 14 , measured value M + 766.4.

[0474] Step 4 The compound 1-f obtained above was carried out in parallel in two parts: each reaction involved adding compound 6 (47.0 g, 61.3 mmol) to 280 mL of THF, adding Pd / C (15.0 g, 10% purity) under argon gas protection, and then adding TFA (7.00 g, 61.3 mmol, 4.54 mL). Hydrogen gas was passed through the reaction solution, and the gas pressure was maintained at 30 Psi. The mixture was heated to 30°C and stirred for 16 hours. After the reaction was complete, the two parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. After suction drying under reduced pressure, the target compound 1-g (94.0 g, crude) was obtained. 1 HNMR(400 MHz, DMSO-d6)δ 8.38 (s, 1H), 8.10 (s, 3H), 7.83 (d, J = 9.2 Hz, 1H), 5.21 (d, J = 3.2 Hz, 1H), 4.96 (dd, J = 3.6, 11.2 Hz, 1H), 4.47 (d, J = 8.4 Hz, 1H), 4.06-3.98 (m, 3H), 3.92-3.82 (m, 1H), 3.75-3.67 (m, 2H), 3.60 (s, 1H), 3.43-3.37 (m, 1H), 3.18-3.04 (m, 2H), 2.30-2.24 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.95-1.90 (m, 2H), 1.89 (s, 3H), 1.78-1.75 (m, 3H), 1.49-1.41 (m, 3H), 1.40 (s, 9H), 1.26 (s, 4H).

[0475] Step 5 The compound 1-f obtained above was carried out in two parallel parts: each reaction involved adding compound 1-f (46.0 g, 60 mmol) to HCl-siRNA (2.00 M, 276 mL) and reacting with stirring at 15°C for 16 hours. After the reaction was complete, the two reaction solutions were combined, concentrated by distillation under reduced pressure, and the residue was diluted with dichloromethane and concentrated under reduced pressure, repeating this process three times. After drying by suction under reduced pressure, a pale red compound 1-h (91.0 g, crude product) was obtained. 1 HNMR(400 MHz, DMSO-d6)δ 7.91-7.80 (m, 2H), 7.42-7.26 (m, 6H), 5.21 (d, J = 3.2 Hz, 1H), 5.07-4.92 (m, 4H), 4.48 (d, J = 8.4 Hz, 1H), 4.06-3.98 (m, 3H), 3.98-3.82 (m, 3H), 3.73-3.65 (m, 1H), 3.44-3.35 (m, 1H), 3.12-2.94 (m, 2H), 2.22 (t, J = 8.0 Hz, 2H), 2.10 (s, 3H), 2.01-1.97 (m, 4H), 1.94-1.90 (m, 1H), 1.89 (s, 3H), 1.87-1.79 (m, 2H), 1.76 (s, 3H), 1.74-1.67 (m, 1H), 1.49-1.40 (m, 2H), 1.40-1.32 (m, 2H), 1.24 (d, J = 4.0 Hz, 4H), 1.19-1.13 (m, 1H). MS, C 33 H 47 N3O 14 , measured M + 710.3.

[0476] Step 6 Two reactions were carried out in parallel: each reaction involved adding compound 1-g (45.0 g, 60.3 mmol) and compound 1-h (38.5 g, 54.3 mmol) to 270 mL of DMF, followed by the addition of DIPEA (10.1 g, 78.4 mmol, 13.6 mL) at 0°C, and then HOBt (8.97 g, 66.3 mmol) and EDCI (12.7 g, 66.3 mmol). The reaction was carried out at 15°C for 16 hours with stirring. After the reaction was complete, the two reaction solutions were combined, diluted with 300 mL of DCM, washed with saturated ammonium chloride (800 mL), saturated NaHCO3 (800 mL), and saturated brine (800 mL), and the organic phase was dried over anhydrous Na2SO4. After filtration, the solution was concentrated by evaporation under pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 0:1) to obtain white compound 1-i (66.0 g, 47.4 mmol, yield 39.3%, purity 95.1%). 1 HNMR(400 MHz, DMSO-d6) δ 7.96-7.78 (m, 5H), 7.41-7.25 (m, 6H), 5.21 (d, J = 3.6 Hz, 2H), 5.05-4.92 (m, 4H), 4.48 (d, J = 8.8 Hz, 2H), 4.22-4.12 (m, 1H), 4.02 (s, 6H), 3.94-3.80 (m, 3H), 3.74-3.64 (m, 2H), 3.45-3.35 (m, 2H), 3.11-2.92 (m, 4H), 2.20-2.12 (m, 4H), 2.10 (s, 6H), 1.99 (s, 6H), 1.89 (s, 6H), 1.82-1.79 (m, 2H), 1.76 (s, 6H), 1.74-1.63 (m, 2H), 1.44 (d, J = 6.0 Hz, 4H), 1.37 (s, 12H), 1.24 (s, 9H). MS:C 62 H 94 N6O 25 The measured value was 1323.8 m / z.

[0477] Step 7 The reaction was carried out in 11 separate steps: compound 1-i (5.00 g, 3.78 mmol) and toluene (300 mL) were added to each step, followed by silica gel (45.0 g). The reactions were carried out at 100°C for 40 hours with stirring, and after the reactions were complete, the 11 reaction mixtures were combined. After removing the solvent by distillation under reduced pressure, isopropanol and dichloromethane were added to the residue, and the mixture was stirred for 20 minutes. The mixture was filtered to remove insoluble matter, and the filter cake was washed with isopropanol until no product was eluted. The solvent was removed from the resulting solution, and it was dried by suction to obtain a pale yellow compound 1-j (43.2 g, 34.0 mmol, yield 82.0%). 1 HNMR: (400 MHz, DMSO-d6)δ 8.01 (d, J = 7.6 Hz, 1H), 7.93-7.79 (m, 2H), 7.39-7.27 (m, 3H), 5.21 (d, J = 3.2 Hz, 1H), 5.06-4.91 (m, 2H), 4.48 (d, J = 8.0 Hz, 1H), 4.07-3.97 (m, 3H), 3.94-3.82 (m, 2H), 3.73-3.65 (m, 1H), 3.45-3.36 (m, 2H), 3.10-2.94 (m, 2H), 2.15 (d, J = 7.6 Hz, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.86-1.79 (m, 1H), 1.77 (s, 3H), 1.74-1.65 (m, 1H), 1.44 (s, 2H), 1.37 (d, J = 5.2 Hz, 2H), 1.24 (s, 4H). MS:C 58 H 86 N6O 25 The measured m / z value was 1267.8.

[0478] Step 8 This step was carried out in two parallel reactions: each reaction involved adding compound 1-d (11.8 g, 21.0 mmol) and compound 1-j (21.3 g, 16.8 mmol) to 70 mL of DMF, followed by the addition of DIPEA (3.54 g, 27.3 mmol, 4.77 mL) at 0°C, and then HOBt (3.13 g, 23.1 mmol) and EDCI (4.44 g, 23.1 mmol). The reaction was carried out at 15°C for 16 hours with stirring. After the reaction was complete, the two reaction solutions were combined, diluted with 500 mL of DCM, washed with saturated ammonium chloride (1.5 mL), saturated NaHCO3 (1.5 mL), and saturated brine (1.5 mL) in order, and the organic phase was dried over anhydrous Na2SO4. After filtration, the mixture was concentrated by evaporation under pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 50:1 to 10:1) to obtain pale yellow compound 1-k (54.0 g, 31.8 mmol, yield 75.6%). 1 HNMR(400 MHz, DMSO-d6) δ 7.91 (d, J = 7.6 Hz, 1H), 7.87-7.78 (m, 5H), 7.73 (t, J = 5.2 Hz, 1H), 7.42-7.24 (m, 6H), 5.21 (d, J = 3.6 Hz, 3H), 5.06-4.92 (m, 5H), 4.48 (d, J = 8.4 Hz, 3H), 4.19-4.09 (m, 2H), 4.07-3.97 (m, 10H), 3.94-3.80 (m, 4H), 3.76-3.64 (m, 3H), 3.42-3.37 (m, 4H), 3.08-2.94 (m, 6H), 2.20-2.12 (m, 2H), 2.10 (s, 9H), 2.08-2.01 (m, 2H), 1.99 (s, 9H), 1.89 (s, 9H), 1.87-1.79 (m, 2H), 1.77 (s, 9H), 1.74-1.63 (m, 2H), 1.44 (d, J = 5.6 Hz, 6H), 1.40-1.31 (m, 6H), 1.24 (s, 13H). MS:C 78 H 118N8O 33 The measured value was m / z = 1696.1.

[0479] Step 9 This step was carried out in three parallel reactions: in each reaction, compound 1-k (17.0 g, 10.0 mmol) and THF (100 mL) were added, followed by Pd / C (5.0 g, 10% purity) under argon gas protection, and then TFA (1.14 g, 10.0 mmol, 742 μL). Hydrogen gas was passed through the reaction solution, and the gas pressure was maintained at 15 Psi. The mixture was heated to 30°C and stirred for 4 hours. After the reaction was complete, the three parallel reactions were combined, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure three times. The residue was purified by preparative liquid chromatography (C18, mobile phase A: 0.1% TFA-water, mobile phase B: 10%~40% CAN, 20 min) to obtain the white compound 1-l (17.3 g, 10.2 mmol, yield 34.0%). 1 HNMR: (400 MHz, DMSO-d6) δ 8.45 (t, J = 5.2 Hz, 1H), 8.14 (d, J = 5.2 Hz, 3H), 7.97 (t, J = 5.2 Hz, 1H), 7.90-7.77 (m, 4H), 5.21 (d, J = 2.8 Hz, 3H), 4.96 (dd, J = 3.2, 11.6 Hz, 3H), 4.47 (d, J = 8.4 Hz, 3H), 4.20-4.10 (m, 1H), 4.02 (s, 8H), 3.87 (q, J = 9.6 Hz, 3H), 3.75-3.61 (m, 4H), 3.46-3.34 (m, 3H), 3.21-2.93 (m, 6H), 2.21 (s, 2H), 2.14-2.02 (m, 11H), 1.99 (s, 9H), 1.96-1.82 (m, 12H), 1.80-1.65 (m, 10H), 1.44 (d, J = 5.6 Hz, 8H), 1.36 (d, J = 6.4 Hz, 4H), 1.30-1.17 (m, 12H) MS:C70 H 112 N8O 31 The measured value is m / 2z = 781.8.

[0480] Step 10 Compound 1-m (2 g, 12.64 mmol) was dissolved in pyridine (10 mL), and a pyridine (10 mL) solution of DMTrCl (4.71 g, 13.90 mmol) was added dropwise at room temperature. The reaction was carried out with stirring at room temperature for 5 hours until the reaction was complete. After the reaction was complete, the mixture was quenched with methanol and concentrated under reduced pressure to obtain the crude product. The crude product was purified using silica gel (eluted with petroleum ether:ethyl acetate = 10:1), the product eluent was collected, and the solvent was evaporated under reduced pressure to obtain 4 g of compound 1-n. MS m / z:C 29 H 32 O5, [M+H] + Actual measurement: 461.3.

[0481] Step 11 Compound 1-n (2 g, 4.34 mmol), N,N-diisopropylethylamine (DIEA, 1.43 mL, 8.68 mmol), and HATU (2.47 g, 6.51 mmol) were dissolved in DMF (10 mL). At room temperature, a solution of compound 1-o in DMF (5 mL) was added, and the reaction was carried out with stirring at room temperature for 8 hours. After the reaction was complete, water was added to quench the reaction, the aqueous phase was extracted with ethyl acetate, and the combined organic phase was washed first with water, then with saturated brine (20 mL). The solvent was evaporated under reduced pressure, and the mixture was subjected to reverse-phase preparative HPLC (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 25%~80% (A: water 0.075% NH3). . Compound 1-p was obtained by lyophilization after being administered via H2O (B:CH3CN) at a flow rate of 55 mL / min. MS m / z:C 33 H 39 NO7, [M+H] + Actual measurement: 562.4.

[0482] Step 12 Compound 1-p (2.4 g, 4.27 mmol) was dissolved in 15 mL of methanol and water (2:1) mixture, and LiOH (0.36 g, 8.54 mmol) was added and stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated under reduced pressure and reversed-phase preparative HPLC was performed (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 25%~75% (A: water 0.075% NH3) . After lyophilization following a reaction with H2O (B:CH3CN) at a flow rate of 55 mL / min, 2 g of compound 1-q was obtained. MS m / z:C 32 H 37 NO7, [M+H] + Actual measurement: 548.6.

[0483] Step 13 Compound 1-q (0.37 g, 0.69 mmol), DIEA (0.19 mL, 1.15 mmol), and HATU (0.32 g, 0.86 mmol) were dissolved in 2 mL of DMF. At room temperature, a solution of compound 1-l (0.9 g, 0.69 mmol) in 2 mL of DMF was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was diluted with dichloromethane (10 mL), and then washed sequentially with saturated NaHCO3 (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and then concentrated under reduced pressure. Reverse-phase preparative HPLC (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 25%~65% (A: water 0.075% NH3) . The compound was purified using H2O (B:CH3CN) at a flow rate of 45 mL / min, and then freeze-dried to obtain 0.5 g of compound 1-r. MS m / z:C 102 H 147 N9O 37 [MH] + Actual measurement: 2088.5.

[0484] Step 14 Compound 1-r (300 mg, 0.14 mmol) and succinic anhydride (28.70 mg, 0.28 mmol) were dissolved in tetrahydrofuran, and DMAP (3.50 mg, 0.028 mmol) was added to the reaction mixture and stirred overnight at 40°C. After the reaction was complete, methanol (18.8 mg) was added and the reaction was continued for 10 minutes with stirring. The reaction mixture was then diluted with dichloromethane (3 mL) and washed twice with saturated NaHCO3 (5 mL). The organic phase was concentrated under reduced pressure until dry, and reverse-phase preparative HPLC (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 25%~65% (A: water 0.075% NH3)) was performed. . The compound 1-s was purified using H2O (B:CH3CN) at a flow rate of 35 mL / min, and then freeze-dried to obtain 140 mg of compound 1-s. MS m / z:C 106 H 151 N9O 40 [MH] + Actual measurement: 2189.4.

[0485] Step 15 Compound 1-r (140 mg, 64 μmol) obtained in the previous step was added to acetonitrile (5 mL), then HBTU (48.7 mg, 128 μmol) was added, followed by the addition of a solid support (CPG-NH2, 2.3 g) with an amino group-modified surface, and then DIEA (41.5 mg, 320 μmol, 55 μL). The reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (8 mL × 4) and dichloromethane (8 mL × 4). The solid was then added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL) and the reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (8 mL × 4) and dichloromethane (8 mL × 4). 2.1 g of compound 1-t linked to a solid vector was obtained.

[0486] Example 7: Aminogalactose compound 2-e linked to a solid-phase vector [ka] The synthesis route is as follows: 1) Synthesis of compound 2-b [ka] 2) Synthesis of compound 2-e [ka] Step 1 Compound 2-a (1.00 g, 2.37 mmol) was added to THF (7.5 mL) and H2O (7.5 mL), and then LiOH.H2O (109 mg, 2.60 mmol) was added. The mixture was allowed to react at 16°C for 16 hours with stirring. After the reaction was complete, the solvent was evaporated and removed under reduced pressure, and the residue was lyophilized to obtain the white compound 2-b (960 mg, 2.32 mmol, yield 97.8%). 1 HNMR: (400 MHz, DMSO-d6) δ 7.44 (d, J = 8.4 Hz, 2H), 7.34-7.23 (m, 6H), 7.22-7.15 (m, 1H), 6.86 (d, J = 8.0 Hz, 4H), 3.73 (s, 6H), 3.66 (d, J = 6.4 Hz, 1H), 3.32 (d, J = 12.0 Hz, 1H), 3.11 (dd, J = 2.0, 9.2 Hz, 1H), 2.85 (t, J = 8.8 Hz, 1H). MS m / z:C 24 H 24 O6, measured m / z: 407.2.

[0487] Step 2 Compound 1-l (500 mg, 0.30 mmol) was added to dichloromethane (3 mL) and the reaction was carried out at 15°C. Compound 2-b (0.14 g, 0.34 mmol) was then added to the reaction, and the reaction was carried out at 0°C. HBTU (142 mg, 375 μmol) and DIEA (115 mg, 895 μmol) were added, and the reaction was continued at 15°C for 16 hours. After the reaction was complete, the reaction solution was diluted with dichloromethane (10 mL), and then washed sequentially with saturated NaHCO3 (20 mL) and saturated saline (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (column: Welch Xtimate C18 250 mm*70 mm#10 μm, mobile phase: [water-ACN], B%: 40%~66%, 18 min) to obtain compound 2-c. MS m / z:C 94 H 134 N8O 36 [MH]+Measured value: 1952.1.

[0488] Step 3 Compound 2-c (230 mg, 0.12 mmol) and succinic anhydride (23.5 mg, 0.26 mmol) were dissolved in dichloromethane solution (2 mL). DMAP (43.1 mg, 0.35 mmol) was added to the reaction mixture, and the mixture was stirred at 15°C for 16 hours. After the reaction was complete, methanol (18.8 mg) was added, and the reaction was continued for 10 minutes with stirring. The reaction mixture was then diluted with dichloromethane (3 mL) and washed twice with saturated NaHCO3. The reaction mixture was concentrated under reduced pressure and dried by suction to obtain compound 2-d (240 mg, crude product). MS m / z:C 106 H 151 N9O 40 [MH]+Measured: m / 2z: 2070.2

[0489] Step 4 Compound 2-d (240 mg, 116 μmmol) obtained in the previous step was added to acetonitrile (8 mL), then HBTU (88.7 mg, 233 μmol) was added, followed by the addition of a solid support (CPG-NH2, 4 g) with a surface modified with amino groups, and then DIEA (75.5 mg, 584 μmol, 101 μL). The reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (8 mL × 4) and dichloromethane (8 mL × 4). The solid was then added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL) and the reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (8 mL × 4) and dichloromethane (8 mL × 4). 3.7 g of compound 2-e, with the target product linked to a solid vector, was obtained.

[0490] Example 8: Aminogalactose compound 3-n linked to a solid-phase vector [ka] The synthesis route is as follows: 1) Synthesis of compound 3-d [ka] 2) Synthesis of compound 3-g [ka] 3) Synthesis of compound 3-n [ka] [ka] Step 1 Starting materials 3-a (78.8 g, 202 mmol) and 3-b (40 g, 168 mmol) were dissolved in DCE (250 mL), and under conditions of 15°C, CF3SO3H (4.15 g, 8.43 mmol) was added. The reaction temperature was then raised to 75°C, and the reaction was carried out for 2 hours with stirring. After the reaction was complete, 1 L of saturated NaHCO3 was added to stop the reaction, the organic phase was separated, and the mixture was further washed with 1 L of saturated brine. The organic phase was dried over anhydrous Na2SO4, and the filtered solution was distilled under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate 5:1~0:1) to obtain the target product 3-c (63.2 g, 107 mmol, yield 63.5%). 1 HNMR:(400 MHz, CDCl3) δ 7.35-7.26 (m, 5H), 5.88 (s, 1H), 5.34-5.25 (m, 2H), 4.65 (d, J = 8.4 Hz, 1H), 4.16-4.13 (m, 2H), 3.92-3.87 (m, 3H), 3.18-3.17 (m, 1H), 3.15-3.14 (m, 2H), 2.16-1.91 (m, 15H), 1.58-1.50 (m, 5H), 1.49-1.36 (m, 2H). MS m / z:C 24 H 40 N2O 11 Actual measured m / z: 567.4.

[0491] Step 2 Compound 3-c (60.0 g, 10⁶ mmol) obtained above was added to 360 mL of THF, and under argon gas protection, Pd / C (15.0 g, 10% purity) was added, followed by TFA (12.1 g, 10⁶ mmol, 7.84 mL). Hydrogen gas was passed through the reaction solution, and the gas pressure was maintained at 30 Psi. The mixture was heated to 30°C and stirred for 16 hours. After the reaction was complete, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure repeatedly, repeating this process three times (500 mL x 3). After suction drying under reduced pressure, pale yellow compound 3-d (44 g, 10⁶ mmol, yield 96.1%) was obtained.

[0492] Step 3 Compound 3-e (60.0 g, 447 mmol) was dissolved in DMF (300 mL), K2CO3 (92.7 g, 671 mmol) was added, and BnBr (115 g, 671 mmol, 79.7 mL) was added dropwise under 0°C conditions. The reaction was allowed to proceed at 25°C for 6 hours with stirring. The reaction mixture was placed on crushed ice and extracted with ethyl acetate (100 mL x 6). The organic phase was then washed sequentially with water (100 mL x 2) and saturated brine (100 mL x 3). After drying the organic phase over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1 to 0:1) to obtain the white solid compound 3-f (60.3 g, 269 mmol, yield 60.1%). 1 HNMR: (400 MHz, CDCl3) δ 7.37-7.26 (m, 5H), 5.18 (d, J = 4.4 Hz, 2H), 3.95-3.90 (m, 2H), 3.75-3.71 (m, 2H), 1.08 (s, 1H). MS m / z:C 12 H 16 O4, measured m / z: 223.5.

[0493] Step 4 Compound 3-f (50.0 g, 223 mmol) was dissolved in dichloromethane (300 mL), pyridine (73.5 g, 929 mmol, 75 mL) and p-nitrophenyl chloroformate (180 g, 892 mmol) dissolved in dichloromethane (50 mL) were added, and the reaction was carried out under nitrogen gas protection at 25°C for 24 hours with stirring. After the reaction was complete, the mixture was diluted with dichloromethane (250 mL), then washed sequentially with NaHSO4 solution (30 mL x 3) and saturated brine (30 mL x 2), the organic phase was dried over MgSO4, filtered, and the solvent was removed by evaporation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain the target compound 3-g (37.0 g, 66.7 mmol, yield 29.9%). MS m / z:C 26 H 22 N2O 12 Actual measured m / z: 553.4

[0494] Step 5 Compound 3-g (22.0 g, 39.7 mmol) was added to acetonitrile (120 mL), and under nitrogen gas protection, triethylamine (24.1 g, 238 mmol, 33.1 mL) was added. The reaction mixture was cooled to 0°C, and compound 3-d (42.1 g, 40 mmol) dissolved in acetonitrile (120 mL) was added dropwise. The temperature was raised to 25°C, and the reaction was carried out with stirring for 1 hour. After the reaction was complete, the solvent was concentrated and removed under reduced pressure, and then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the target compound 3-h (37.0 g, 12.0 mmol, yield 30.2%). MS m / z:C 52 H 76 N4O 24 Actual measured m / z: 1141.8.

[0495] Step 6 Compound 3-h (11.0 g, 9.64 mmol) was dissolved in ethyl acetate (60 mL), Pd / C (2.00 g, 10% purity) was added, hydrogen gas was passed through the reaction solution, and the reaction was carried out at 25°C for 8 hours with stirring while maintaining a gas pressure of 40 Psi. After the reaction was complete, the solution was filtered and evaporated under reduced pressure until dry to obtain the target compound 3-i (10.0 g, 9.42 mmol, yield 97.7%). 1 HNMR: (400 MHz, DMSO-d6) δ 7.79 (d, J = 9.2 Hz, 2H), 7.10 (s, 2H), 5.74 (t, J = 1.6 Hz, 2H), 5.21 (d, J = 3.6 Hz, 2H), 4.98-4.95 (m, 2H), 4.48 (d, J = 8.4 Hz, 2H), 4.02 (d, J = 4.8 Hz, 11H), 3.87-3.84 (m, 2H), 3.69-3.67 (m, 2H), 3.41-3.39 (m, 2H), 2.94-2.90 (m, 4H), 2.10 (s, 5H), 1.99 (s, 7H), 1.89 (s, 6H), 1.77 (s, 6H), 1.47-1.35 (m, 8H), 1.26-1.24 (m, 4H), 1.23-1.08 (m, 3H). MS m / z:C 45 H 70 N4O 24 Actual measured m / z: 1051.4

[0496] Step 7 Compound 3-i (5.00 g, 4.76 mmol) was added to a mixed solvent of dichloromethane (30 mL) and DMF (30 mL), followed by compound 33 (312 mg, 2.38 mmol), HBTU (1.80 g, 4.76 mmol) and DIEA (615 mg, 4.76 mmol). The mixture was allowed to react at 25°C for 12 hours with stirring. After the reaction was complete, the reaction solution was placed in ethyl acetate (100 mL), washed with saturated brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed by evaporation under reduced pressure. The residue was purified by preparative HPLC to obtain the target compound 3-k (2.1 g, 956 μmol, yield 20.1%). 1 HNMR: (400 MHz, DMSO-d6) δ 7.84-7.81 (m, 5H), 7.12-7.07 (m, 3H), 5.21 (d, J = 3.6 Hz, 4H), 4.99-4.96 (m, 4H), 4.49 (d, J = 8.4 Hz, 4H), 4.06-4.00 (m, 24H), 3.88-3.86 (m, 4H), 3.55-3.52 (m, 4H), 3.49-3.43 (m, 4H), 3.25-3.05 (m, 4H), 2.94-2.93 (m, 8H), 2.11 (s, 12H), 2.00 (s, 16H), 1.90 (s, 12H), 1.78 (s, 12H), 1.46-1.44 (m, 8H), 1.38-1.35 (m, 8H), 1.26-1.24 (m, 8H), 1.18-1.16 (m, 6H), 1.09-0.99 (m, 2H). MS m / z:C 96 H 153 N 11 O 46 Actual measured m / z: 2197.5.

[0497] Step 8 Compound 3-k (100 mg, 45.5 μmol) was added to DMF (1 mL), followed by compound 2-b (21.1 mg, 54 μmol), then HBTU (21.8 mg, 57.3 μmol) and DIEA (17.7 mg, 136 μmol). The reaction was allowed to proceed at 15°C for 16 hours. After the reaction was complete, the reaction solution was diluted with dichloromethane (10 mL), washed sequentially with saturated NaHCO3 and saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (column: Phenomenex Gemini-NX 150 mm*30 mm*5 μm, mobile phase: [water-ACN], B%: 35%~75%, 12 min) to obtain compound 3-1. MS m / z: C 120 H 175 N 11 O 51 Actual measurement: 2586.9.

[0498] Step 9 Compound 3-l (14 mg, 5.4 μmol) and succinic anhydride (1.08 mg, 10.8 μmol) were dissolved in dichloromethane solution (1 mL). DMAP (2.0 mg, 16 μmol) and TEA (1.1 mg, 10.8 μmol, 1.5 μL) were added to the reaction mixture, and the mixture was stirred at 15°C for 16 hours. After the reaction was complete, methanol (0.9 mg) was added, and the reaction was continued with stirring for 10 minutes. The reaction mixture was then diluted with dichloromethane and washed twice with saturated NaHCO3. The reaction mixture was concentrated under reduced pressure and dried by suction to obtain compound 3-m (18 mg). MS m / z:C 124 H 179 N 11 O 54 Actual measurement: 2687.2.

[0499] Step 10 Compound 3-m (18 mg, 6.7 μmol) obtained in the previous step was added to acetonitrile (3 mL), then HBTU (5.1 mg, 13.4 μmol) was added, followed by a solid support (CPG-NH2, 200 mg) with a surface modified with amino groups, and then DIEA (4.3 mg, 33.5 μmol, 5.8 μL). The reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (2 mL × 4) and dichloromethane (2 mL × 4). The solid was then added to pyridine:anhydride acetate (v:v=4:1, 2 mL) and the reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol and dichloromethane. 200 mg of compound 3-n, with the target product linked to a solid vector, was obtained.

[0500] Example 9: Aminogalactose compound 4-c linked to a solid-phase vector [ka] The synthesis route is as follows: 1) Synthesis of compound 4-c [ka] Step 1 Compound 3-k (149.5 mg, 68 μmmol), DIEA (141.0 mg, 1.09 mmol), 3A molecular sieve (500 mg), and DEPBT (163.4 mg, 0.55 mmol) were dissolved in 5 mL of DCM. Compound 1-q (400 mg, 0.18 mmol) was added at room temperature, and the mixture was stirred overnight at room temperature. After the reaction was complete, the molecular sieve was removed by filtration, and the filtrate was concentrated and dried to obtain compound 4-a (118 mg, 32 μmmol, yield 62.6%) through reverse-phase preparative HPLC (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 5%~50% (A: water, B: CH3CN), flow rate: 45 mL / min). After lyophilization, compound 4-a (118 mg, 32 μmmol, yield 62.6%) was obtained. MS m / z:C128 H 188 N 12 O 52 , Actual measurement [M+HCOO - ] = 2770.6.

[0501] Step 2 Compound 4-a (110 mg, 4.0 μmol), DMAP (7.4 mg, 40 μmol), 3A molecular sieve (100 mg), and succinic anhydride (11.9 mg, 120 μmol) were dissolved in 5 mL of THF and stirred at 40°C for 4 hours under argon gas protection. After the reaction was complete, the molecular sieve was removed by filtration, and the filtrate was concentrated and dried. The filtrate was then purified by reverse-phase preparative HPLC (Column: Boston Green ODS 150 mm*30 mm*5 μm, conditions: 5%~50% (A: water, B: CH3CN), flow rate: 45 mL / min), and after lyophilization, compound 4-b (80 mg, 28.3 μmmol, yield 70.8%) was obtained. MS m / z:C 132 H 192 N 12 O 55 [MH] + Actual measurement: 2824.6.

[0502] Step 3 Compound 38 (71 mg, 25 μmol) obtained in the previous step was added to acetonitrile (5 mL), then HBTU (19.0 mg, 50 μmol) was added, followed by the addition of a solid support (CPG-NH2, 0.86 g) with an amino group-modified surface, and then DIEA (16.2 mg, 125 μmol, 21.6 μL). The reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol (5 mL × 4) and dichloromethane (5 mL × 4). The solid was then added to pyridine:acetic anhydride (v:v=4:1, 6.0 mL) and the reaction was carried out at 30°C for 16 hours with shaking. After the reaction was complete, the mixture was filtered and washed sequentially with methanol and dichloromethane. 0.74 g of compound 4-c linked to a solid vector was obtained.

[0503] Example 10. Preparation of control compound L96 [ka] Control compound L96 was prepared according to the method described in patent WO2014025805A1.

[0504] Example 11 Synthesis of siRNA complexed with aminogalactose molecular cluster The siRNA used for testing, siRNA targeting the mouse TTR gene mRNA (Molecular Therapy Vol. 26 No 3 March 2018), is covalently linked to the aminogalactose molecular cluster M at the 3' end of the SS chain, as shown below. SS chain (5'-3'): CmsAmsGmUmGfUmUfCfUfUmGmCmUmCmUmAmUmAm Am-M AS chain (5'-3'): UmsUfsAmUmAmGfAmGmCmAmAmGmAmAfCm AfCmUmGmsUmsUm Referring to the phosphoramidite solid-phase synthesis method described above, this method differs in that, when synthesizing the disulfide chain, the Universal-CPG vector is replaced with a CPG vector to which aminogalactose clusters are linked.

[0505] A brief explanation follows. Using the Dr. Oligo48 synthesizer (Biolytic), nucleoside phosphoramidite monomers were linked one by one using a synthesis program, starting with the CPG vector linked to the above-synthesized aminogalactose. The nucleoside phosphoramidite monomers, such as 2'-F RNA and 2'-O-methyl RNA, which are the raw materials for the nucleoside monomers, were purchased from Shanghai Zhaowei or Suzhou Jima. 5-ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M acetonitrile solution), a solution of 0.22 M PADS dissolved in acetonitrile and trimethylpyridine (Suzhou Kema) in a 1:1 volume ratio was used as a vulcanizing agent, and iodopyridine / aqueous solution (Kema) was used as an oxidizing agent.

[0506] After solid-phase synthesis was complete, the oligo-ribonucleotides were dissolved from the solid support and immersed in a 3:1 28% aqueous ammonia and ethanol solution at 50°C for 16 hours. The mixture was then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated. Purification was performed by C18 reverse-phase chromatography, with DMTr removed using 0.1 M TEAA and acetonitrile as the mobile phase, and 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LC-MS, and further quantified by UV (260 nm).

[0507] The obtained single-stranded oligonucleotides were paired complementaryally in equimolar ratios, annealed with AS chains, and finally the resulting double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment.

[0508] siRNA was synthesized in complex with an aminogalactose cluster, and the experimental siRNA targeted mouse TTR mRNA. [ka] NAG1 [ka] NAG2 [ka] NAG3 [ka] NAG4 [ka] L96 (control compound).

[0509] [Table 13]

[0510] Example 12: Suppression of mRNA expression in primary hepatic cells by siRNA complexed with an aminogalactose molecular cluster. Fresh primary mouse hepatocytes were isolated using a method reported by Severgini et al. (Cytotechnology. 2012; 64(2):187-195.).

[0511] After isolating primary hepatocytes, they were inoculated into 24-well plates at a concentration of 100,000 / well. The siRNAs awaiting measurement were then added to achieve final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. The primary hepatocytes were then cultured for 24 hours at 37°C in a 5% CO2 environment. After 24 hours, the mRNA expression level of mTTR was detected by qPCR.

[0512] As shown in Figure 4, S-1, S-2, S-3, and S-4 all exhibit excellent mTTR gene expression repression efficiency. IC of S-1 and S-4 50 The value was lower than the other two groups, and the IC of the control group S-L96 was lower. 50 Compared to the value of 0.280 nM, the IC of S-1 50 The value is 0.131 nM, S-4 IC 50 The value of 0.135 nM indicates that the efficiency of free uptake of siRNA conjugated with S-1 and S-4 compounds by primary hepatocytes in vitro is superior to that of the control group, revealing that S-1 and S-4 compounds can more effectively mediate the entry of siRNA into primary hepatocytes.

[0513] Example 13: Inhibition of mRNA expression in the body by siRNA complexed with an aminogalactose molecular cluster. Eight-week-old C57BL / 6 mice (Shōgen organism, SPF grade, female) were used to deliver the above-mentioned siRNAs by subcutaneous injection. On day 1, 100 μL of the solution was administered subcutaneously into the loose skin of the mouse's neck and shoulders. This solution contained PBS or PBS-prepared 1 mg / kg (mpk), 0.2 mpk doses of the corresponding siRNA (S-L96, S-3, S-2, S-4, or S-1). Six mice were injected into each group.

[0514] Three days after administration, the mice were sacrificed by cervical dislocation, and the mRNA expression level of mTTR in the mouse liver tissue was detected by qPCR.

[0515] As shown in Figure 5, S-1, S-2, S-3, and S-4 all exhibit excellent mTTR gene expression repression efficiency. Of these, S-2, S-3, and S-4 have activity levels at 1 mpk and 0.2 mpk that are similar to the control group S-L96. S-1, however, exhibits superior activity levels at 1 mpk and 0.2 mpk compared to the control group S-L96.

[0516] Example 14: Long-term effect experiment on the suppression of mRNA expression in the body by siRNA complexed with an aminogalactose molecular cluster. Two groups of siRNA compounds, S-1-2 and S-L96-2 (SS and AS chains are shown in Table 13), were synthesized again according to the synthesis method in Example 11 and used for intracellular administration to mice. Eight-week-old C57BL / 6 mice (Shōgen organism, SPF grade, female) were used, and the siRNAs conjugated with the above aminogalactose molecular clusters were delivered by subcutaneous injection. On day 0, 100 μL of the solution was administered subcutaneously into the loose skin of the neck and shoulders of the mice. This solution contained PBS (referred to as the Mock group, i.e., the blank control group) or PBS-prepared PBS-prepared siRNAs (S-1 and S-L96) conjugated with the corresponding aminogalactose molecular clusters at a dose of 1 mg / kg (mpk). Nine mice were injected into each group.

[0517] Three mice were each sacrificed by cervical vertebral dislocation 7, 14, and 28 days after administration. Two liver tissue samples were taken from each mouse, and the mRNA expression level of mTTR in the mouse liver tissue was detected by qPCR.

[0518] On days 7, 14, and 28 after administration, the mRNA ratios of S-1-2 to the PBS group were 0.13, 0.12, and 0.21, respectively, while the mRNA ratios of S-L96-2 to the PBS group were 0.17, 0.13, and 0.29, respectively.

[0519] Figure 6 also shows the mRNA expression levels in mouse liver tissue on days 7, 14, and 28 after administration of compounds S-1-2 and S-L96-2.

[0520] As a result, the administered siRNA showed an efficient mRNA suppression rate even on day 28, and it was found that the suppression rate of S-1-2 was higher compared to the control group S-L96-2.

[0521] III. Verification of activity Example 15 Synthesis of siRNA complex The nucleotides were linked one by one to nucleoside monomers from the 3'-5' direction according to the sequence order of the nucleotides using the solid-phase phosphoramidite method. Linking to a single nucleoside monomer involves a four-step reaction: deprotection, coupling, capping, and oxidation or sulfidation. The same synthetic conditions were used for both the sense and antisense strands.

[0522] Oligonucleotide synthesis equipment and model numbers: Biolytic Dr. Oligo 48 oligonucleotide solid-phase synthesizer, GE oligo pilot 100 oligonucleotide solid-phase synthesizer.

[0523] [Table 14] The detection method is as follows: The purity of the sense and antisense strands was detected and their molecular weight analyzed using Waters Acquity UPLC-SQD2 LCMS (column: ACQUITY UPLC BEH C18). When the measured values ​​match the theoretical values, it is clear that the synthesized product consists of a 3'-terminal complex molecule with both sense and antisense strands. The above siRNA has the sense and antisense strands shown in Table 15.

[0524] [Table 15-1] [Table 15-2] Of these, nucleotides synthesized using 2-hydroxymethyl-1,3-propanediol as a starting material are defined as hmpNA. (-)hmpNA(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-1a in Example 1.1. (-)hmpNA(G) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-6a in Example 1.6. (-)hmpNA(C) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-8a in Example 1.8. (-)hmpNA(U) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomers 1-7a in Example 1.7. In Table 15, the NAG1 structure is as shown in Example 11.

[0525] Example 16: Verification of siRNA activity and off-target levels In HEK293A cells, in vitro molecular-level on-target and off-target screening for the compounds disclosed herein was simulated.

[0526] Corresponding on-target and off-target sequences for the siRNA sequence were constructed and inserted into the psiCHECK-2 plasmid. This plasmid contained the sea urchin luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the siRNA target sequence was inserted into the 3'UTR region of the sea urchin luciferase gene. The activity of the siRNA against the target sequence could be reflected by detecting the expression status of sea urchin luciferase calibrated with firefly luciferase, and a Dual-Luciferase Reporter Assay System (Promega, E2940) was used for detection.

[0527] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. 24 hours prior to transfection, HEK293A cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0528] Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions, with 0.2 μL of Lipofectamine 2000 used per well. The plasmid transfection rate was 10 ng / well. For on-target and off-target sequence plasmids, the siRNA had a total of 5 or 11 concentration points. Under the 5-concentration-point condition, the maximum concentration at transfection was 10 nM, diluted with a 10-fold gradient. Under the 11-concentration-point condition, the final concentration at the maximum concentration at transfection was 20 nM, diluted with a 3-fold gradient. Off-target levels were detected 24 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, E2940).

[0529] The results in Tables 16 to 19 show that the compounds TRD006890 and TRD006924 of this disclosure have high on-target activity and low off-target activity, and both are significantly superior to the positive control AD81890.

[0530] The results in Table 20 show that GNA modification exhibits significant sequence site dependence. When the GNA modification site is located at position 7 (TRD006912) or position 6 (AD81890) of the AS chain 5', both off-target activity is relatively high, suggesting relatively high toxicity. Furthermore, the on-target activity of TRD006912 is further reduced (from 0.68 to 0.96) compared to AD81890.

[0531] [Table 16]

[0532] [Table 17]

[0533] [Table 18]

[0534] [Table 19]

[0535] [Table 20]

[0536] Example 17: Evaluation of in vitro anti-HBV activity of siRNA compounds using HepG2.2.15 cells On day 1, HepG2.2.15 cells were inoculated into 96-well plates, with 20,000 cells per well. Simultaneously with cell inoculation, different concentrations of siRNA were transferred to the HepG2.2.15 cells using RNAiMax. On day 4, the cell culture supernatant was collected, and HBsAg was detected by ELISA (the remaining supernatant was cryopreserved for later use). Finally, the cells were collected, intracellular RNA was extracted, and total HBV RNA (containing 3.5 kb + 2.4 kb + 2.1 kb + 0.7 kb of RNA) and 3.5 kb of HBV RNA (containing pgRNA + preCore RNA) were detected by RT-PCR, along with the detection of GAPDH gene RNA as an internal reference. Five concentration points were available for measurement, and two parallel wells were measured in parallel. The final concentration of DMSO in the culture medium was 0.5%.

[0537] The formula for calculating the suppression percentage is as follows: %HBsAg inhibition rate = (1 - HBsAg content in sample / HBsAg content in DMSO control group) × 100 % HBV RNA suppression rate = (1 - HBV RNA content in the sample / HBV RNA content in the DMSO control group) × 100 %Cell viability = (Sample absorbance - Culture medium control absorbance) / (DMSO control absorbance - Culture medium control absorbance) × 100.

[0538] Using Graphpad Prism software analysis (four-parameter logistic equations) for EC 50 The value was calculated.

[0539] [Table 21] As shown in Table 21, when comparing the detection indicators for antiviral activity with the control compounds AD66810 and AD81890, the test compounds TRD006890, TRD006894, TRD006895, TRD006896, TRD006897, TRD006899, TRD006900, TRD006905, TRD006906, TRD006907, and TRD006908 showed excellent antiviral activity in HepG2.2.15 cells.

[0540] Example 18 Evaluation of in vitro anti-HBV infection activity of siRNA compounds using human primary hepatocytes On day 0, primary human hepatocytes were inoculated into 48-well plates, with 120,000 cells per well. Along with cell inoculation, the compounds awaiting measurement were added, and siRNA was transferred to the primary human hepatocytes ad libitum. The initial siRNA concentration was 200 nM, which was then diluted five-fold to seven different concentrations. On day 1, primary human hepatocytes were infected with HBV type D. On days 2, 4, and 6, the culture medium was replaced with fresh medium, and the final DMSO concentration in the culture medium was 2%. On day 8, the cell culture supernatant was collected, HBV DNA was detected by qPCR, and HBeAg and HBsAg were detected by ELISA. Both the compounds awaiting measurement and the control compounds were measured at seven concentration points, with two parallel wells measured in parallel.

[0541] [Table 22] As shown in Table 22, when comparing the detection indicators for antiviral activity with that of the control compound AD81890, the test compound TRD006894 exhibits remarkably superior antiviral activity in human primary hepatocytes.

[0542] Example 19: Intracellular anti-HBV activity of siRNA compounds On day 28, mice (C57BL / 6, male) were injected with rAAV8-1.3HBV via the tail vein. On days 14 and 21 after viral injection, all experimental mice were collected from the submandibular vein for plasma collection. The plasma was to be tested for HBV DNA, HBeAg, and HBsAg content.

[0543] On day 28 after viral injection, mice were randomly divided into groups based on the results of plasma samples taken on days 14 and 21 after viral injection.

[0544] All mice received a single subcutaneous dose of 3 mg / kg starting 28 days after viral injection. Plasma was collected from the submandibular region of all mice before administration to detect HBV DNA, HBeAg, HBsAg, and ALT. The day of administration was designated as day 0. For detection, plasma was collected from the submandibular region of all mice on days 7, 14, and 21 after administration.

[0545] Plasma HBV DNA was quantified by qPCR. Plasma HBeAg and HBsAg were quantified by ELISA.

[0546] On day 7, compared to the control compound AD81890, the test compound TRD006894 showed superior antiviral activity in the mice. The test compound TRD006894 was able to maintain its activity in the body for a relatively long period, effectively suppressing viral activity even on day 14 and day 21.

[0547] [Table 23]

[0548] Example 20: Design and Synthesis of Human ApoC3 siRNA 1) For siRNA design, the human ApoC3 gene (NM_000040.3) was used as the target gene, and a 19 / 21nt siRNA was designed to satisfy the general rules for active siRNA. The sequences of the unmodified sense strand and antisense strand are shown in detail in Table 14, and of these, neither the SS strand nor the AS strand of the unmodified siRNA was modified. 2) siRNA was synthesized in 200 nanomoles (nmol) using a universal solid support (Shenzhen Dosen Bio)-mediated phosphoramidite chemical in a Dr. Oligo 48 synthesizer (Biolytic). The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and further quantified by UV (260 nm).

[0549] When synthesizing the nucleotide modified at position 7 of the 5' of the AS chain, the parent sequence nucleotide was substituted with the phosphoramidite monomer synthesized in Example 1. The sequence of the antisense strand modified at position 7 of the 5' of the AS chain is shown in detail in Table 14, of which W' is [ka] Selected from, Of these, M was either O or S, and of these, B was selected from the native bases at the corresponding positions in Table 24.

[0550] The sequences of the sense and antisense strands of ApoC3 siRNA modified with 2'-fluoro and 2'-methoxy groups are shown in detail in Table 25, and the sequences of the sense and antisense strands of the ApoC3 siRNA complex are shown in detail in Table 26.

[0551] The sense and antisense strands synthesized by the above steps were annealed in equimolar ratios to form a double-stranded structure via hydrogen bonding. Finally, the resulting double-stranded siRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment.

[0552] [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4]

[0553] [Table 25-1] [Table 25-2] [Table 25-3]

[0554] [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] In Tables 25-26, nucleotides synthesized using 2-hydroxymethyl-1,3-propanediol as a starting material are defined as hmpNA, and hmpNA has a racemic structure. (-)hmpNA(A) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-1a in Example 1.1, and (+)hmpNA(A) is an optical isomer. (-)hmpNA(G) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-3a in Example 1.6, and (+)hmpNA(G) is an optical isomer. (-)hmpNA(C) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-8a in Example 1.8, and (+)hmpNA(C) is an optical isomer. (-)hmpNA(U) was obtained by solid-phase synthesis from nucleoside phosphoramidite monomer 1-7a in Example 1.7, and (+)hmpNA(U) is an optical isomer.

[0555] The lowercase letter 'm' indicates that the nucleotide adjacent to the left of the letter 'm' is modified with a 2'-methoxy group, and the lowercase letter 'f' indicates that the nucleotide adjacent to the left of the letter 'f' is modified with a 2'-fluoro group. When a lowercase 's' is between uppercase letters, it indicates that the linkage between the two nucleotides adjacent to the 's' is via a thiophosphate group. If the lowercase letter 's' is at the first position of the 3' end, it indicates that the nucleotide end adjacent to the left of that letter 's' is a thiophosphate group.

[0556] In Table 26, the NAG1 structure is as shown in Example 11.

[0557] Example 21: Inhibition of human ApoC3 by siRNA in Huh7 cells - Screening of inhibitory activity at a single concentration point. The effect of siRNA targeting human ApoC3 on human ApoC3 mRNA expression levels was tested in vitro. Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0558] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, 13778150), and the final transfected concentration of siRNA was 10 nM. After 24 hours of treatment, TaqMan TM Fast Advanced Cells-to-CT TM Cell lysis, one-step reverse transcription, and quantitative real-time PCR detection were performed using Kit (ThermoFisher, A35378) to measure human ApoC3 mRNA levels, and these levels were corrected using ACTIN internal reference gene levels.

[0559] For materials and equipment used in the 96-well plate cell activity screening (Cells-to-CT) experiment, refer to Tables 1 and 2 in Example 3.1.

[0560] The experimental procedure for 96-well plate cell activity screening (Cells-to-CT) is as follows:

[0561] (1) This was a cell transfection experiment, and the amounts of transfection complex components used are as shown in Table 27.

[0562] [Table 27] (ii) Cell RNA extraction experiments and cell RNA reverse transcription experiments using the Cells-to-CT method. The reverse transcription reaction system is shown in Table 29, and the reaction conditions are shown in Table 30.

[0563] [Table 28]

[0564] [Table 29]

[0565] [Table 30] Once reverse transcription is complete, the sample can be stored in a 4°C refrigerator for Taqman Q-PCR or in a -40°C refrigerator (for 6 months).

[0566] (3) Taqman probe Q-PCR detection experiment 1. The reaction reagent kit (ThermoFisher TaqMan Fast Advanced Master Mix (4444964)) was tested for its expiration date, the components of the reagent kit were stored in a refrigerator at -40°C, and after dissolution and use, were stored in a refrigerator at 4°C. 2. The following reaction mixture (Table 32) was prepared in a microtube, and the working concentration of the primer was 10 μM.

[0567] [Table 31]

[0568] [Table 32] The samples were placed in an RT-PCR instrument and reacted according to the reaction program in Table 33 (40 recurring reactions).

[0569] [Table 33] Note: TaqMan® FastAdvanced Master Mix is ​​a registered trademark of ROX TM Includes reference dye.

[0570] 3. Results analysis method After the Taqman probe Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to a threshold automatically set in the system, and the expression of a certain gene can be relatively quantified by comparing the Ct values: Ct comparison means calculating the difference in gene expression by the difference value from the internal reference gene Ct value, 2 -△△Ct Also known as △△Ct, the formula is △△Ct = [(Target gene in Ct experimental group - Internal standard in Ct experimental group) - (Target gene in Ct control group - Internal standard in Ct control group)]. The suppression rate (%) = (1 - Excess expression amount of target gene) × 100%.

[0571] The experimental results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA, and the results are shown in Table 34.

[0572] [Table 34-1] [Table 34-2]

[0573] Example 22: Inhibition of human ApoC3 by siRNA in Huh7 cells - Inhibitory activity at 5 concentration points siRNAs were screened in Huh7 cells using five concentration gradients. The initial final concentration of each siRNA sample before transfection was 10 nM, and it was diluted with a 10-fold gradient, resulting in five concentration points.

[0574] Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0575] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, 13778150), with final transfection concentrations of siRNA being 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM. After 24 hours of treatment, TaqMan TM Fast Advanced Cells-to-CT TM Cell lysis, one-step reverse transcription, and quantitative real-time PCR detection were performed using Kit (ThermoFisher, A35378) to measure human ApoC3 mRNA levels, and these levels were corrected using ACTIN internal reference gene levels.

[0576] The results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 35.

[0577] The experimental procedure refers to the 96-well plate cell activity screening (Cells-to-CT) experimental procedure in Example 21.

[0578] [Table 35-1] [Table 35-2]

[0579] Example 23 Verification of siRNA on-target activity and off-target levels using psiCHECK. In Huh 7 cells, 11 concentration gradients were used to simulate in vitro molecular-level on-target and off-target screening of siRNA. The results showed that the siRNA disclosed herein has high activity and low off-target properties.

[0580] Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well. Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions for use, with 0.2 μL of Lipofectamine 2000 used per well. The plasmid transfection rate was 10 ng / well. For on-target and off-target plasmids, siRNA was diluted with a 3-fold gradient, with a total of 11 concentration points established, and the final concentration at the highest concentration point being 40 nM (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.494 nM, 0.165 nM, 0.0549 nM, 0.0183 nM, 0.00609 nM, 0.00203 nM, and 0.000677 nM). Off-target levels were detected 24 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, E2940). The results are shown in Tables 37 to 40.

[0581] In the Huh7 cell line, psi-CHECK screening experiments were performed to detect the on-target and off-target activities of the relatively active siRNAs listed in Table 35.

[0582] The Psi-CHECK plasmid was purchased from Suzhou Hongxun Biotechnology Co., Ltd. and Biotechnology (Shanghai) Co., Ltd.

[0583] The materials and equipment used in the experiment are detailed in Tables 1 and 2 of Example 3.1, and the experimental results are detailed in Tables 37 to 40. The psiCHECK activity screening experimental procedure is as described in Example 3.2, and the multi-concentration dilution plan for the siRNA sample is shown in Table 36. The results are shown in Tables 37 to 40.

[0584] [Table 36]

[0585] [Table 37]

[0586] [Table 38-1] [Table 38-2] [Table 39] [Table 40]

[0587] Example 24: Inhibition of human ApoC3 by siRNA in Huh7 cells - Inhibitory activity at 11 concentration points In Huh7 cells, 11 concentration gradients were used, and siRNAs that showed 80% or more in vitro suppression (20% or less mRNA excess expression level) were subjected to off-target modification (AS chain 7-position modification), followed by Huh7 cell activity screening. The initial final concentration of each siRNA sample after transfection was 40 nM, and it was diluted with a 3-fold gradient, resulting in 11 concentration points.

[0588] Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0589] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, 13778150). The final transfected concentrations of siRNA were 40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.494 nM, 0.165 nM, 0.0549 nM, 0.0183 nM, 0.00609 nM, 0.00203 nM, and 0.000677 nM. After 24 hours of treatment, TaqMan TM Fast Advanced Cells-to-CT TM Cell lysis, one-step reverse transcription, and quantitative real-time PCR detection were performed using Kit (ThermoFisher, A35378) to measure human ApoC3 mRNA levels, and these levels were corrected using ACTIN internal reference gene levels.

[0590] The results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 41.

[0591] The experimental procedure refers to the 96-well plate cell activity screening (Cells-to-CT) experimental procedure in Example 21.

[0592] [Table 41-1] [Table 41-2]

[0593] Example 25 Verification of siRNA on-target activity and off-target levels using psiCHECK. In HEK293A cells, siRNA modification (modification at position 7 of the AS chain) was performed using 11 concentration gradients, and then on-target and off-target activity screening was simulated at the in vitro molecular level. As a result, the siRNA disclosed herein was found to have high activity and low off-target properties. The experimental procedure was as described in Example 16, and for the antisense strand of the siRNA, a GSSM-5 hits off-target plasmid was constructed to improve detection sensitivity, i.e., five identical GSSM sequences were linked by TTCC.

[0594] The results are shown in Tables 43 to 46. As a result, the in vitro on-target inhibitory activity levels of siRNA were all high (GSCM IC). 50 The value is less than 0.3 nM, indicating no significant off-target phenomena. psiCHECK activity screening experimental procedure In the HEK293A cell line, siRNA activity was detected by performing a psi-CHECK activity experiment. The materials and equipment used in the experiment are detailed in Tables 1 and 2 of Example 3.1, and the experimental results are detailed in Tables 43 to 46.

[0595] The psiCHECK activity screening experiment procedure is as described in Example 3.2, and the proposed high-concentration dilution of the siRNA sample is shown in Table 42.

[0596] [Table 42]

[0597] [Table 43-1] [Table 43-2] Note: Due to the internal synthesis process, the transfection efficiency is low at the highest concentration (40 nM), so experimental data corresponding to the highest concentration (40 nM) was discarded during data processing.

[0598] [Table 44]

[0599] [Table 45]

[0600] [Table 46]

[0601] Example 26: Inhibition of human ApoC3 by siRNA in Huh7 cells - Inhibitory activity at 11 concentration points In Huh7 cells, siRNA was modified (at position 7 of the AS chain) using 11 concentration gradients, and then Huh7 cells were screened for activity. The initial final concentration of each siRNA sample after transfection was 20 nM, and it was diluted with a 3-fold gradient, resulting in 11 concentration points.

[0602] Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0603] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final transfected concentrations of siRNA were 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM. After 24 hours of processing, total cellular RNA was extracted using a high-throughput cell RNA extraction reagent kit, and RNA reverse transcription and quantitative real-time PCR detection were performed to measure the mRNA level of human ApoC3. The mRNA level of human ApoC3 was then corrected using the ACTIN internal reference gene level.

[0604] The results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 53.

[0605] The materials used for the 96-well plate cell activity screening experiment (using a nucleic acid extraction device) are shown in Tables 1 and 2.

[0606] The experimental procedure for 96-well plate cell activity screening (using a nucleic acid extraction device) is as follows:

[0607] I. Cell transfection experiments The procedure was performed referring to the cell transfection experiment in Example 21.

[0608] The amounts used for the transfection complex components are shown in Table 47.

[0609] [Table 47] II. Cell RNA extraction experiment using nucleic acid extraction device (magnetic bead method) 1. Experiment preparation: High-throughput cell RNA extraction reagent kit (FG0417-L / FG0418-XL, magnetic bead method).

[0610] [Table 48]

[0611] 2. The following reagents were added to each of the six deep-well plates.

[0612] [Table 49] Note: The recommended amount of anhydrous ethanol, as indicated on the bottle label, was added to buffer WB1 and WB2, respectively.

[0613] Preparation of cell lysate: 200 μL of lysate LB + 3.5 μL of 1M DTT solution was used. The supernatant of the culture medium in a 96-well plate was aspirated, 200 μL / well was added, and the cells were dissolved for 5 minutes.

[0614] DNase I Mix solution: 3.4 μL of DNase I + 5 μL of DNase diluent + 41.6 μL of 0.1% DEPC water (50 μL / well). Mix thoroughly and homogeneously to prepare DNase I Mix, then place on ice.

[0615] Instrument program selection: Cell RNA 96.

[0616] 3. Place the six deep-well plates in the corresponding six locking grooves of the nucleic acid extraction device and mark them accordingly. Then, insert the magnetic rod into the 96-deep-well plate 3, start the device to run the cell RNA extraction program, pause the program after 35 minutes, remove the 96-deep-well plate 2, add 220 μL of buffer WB1 to it, and continue running the cell RNA extraction program.

[0617] 4. After nucleic acid extraction is complete, measure the concentration, then seal the 96-deep-well plate with aluminum foil sealing film, mark it completely, and store it in a refrigerator at 4°C for reverse transcription experiments, or store it in a refrigerator at -40°C.

[0618] III. Cellular RNA Reverse Transcription Experiment 1. Experiment preparation: (1) Reverse transcription reagent kit (Takara PrimeScript TM II. The expiration date of the 1st Strand cDNA Synthesis Kit (6210A) and reagent kit was checked, and all components of the reagent kit were stored in a refrigerator at -40°C.

[0619] [Table 50]

[0620] 2. The following reaction mixture (Mix1) was prepared in a microtube.

[0621] [Table 51] The samples were incubated at 65°C for 5 minutes, then rapidly cooled on ice for 2 minutes. (Note: The above treatment can denature the template RNA and improve reverse transcription efficiency.)

[0622] 3. The following reverse transcription reaction solution (Mix 2) was prepared in a microtube.

[0623] [Table 52] 10 μL of Mix2 reaction solution was taken and added to Mix1, for a total volume of 20 μL, and reverse transcription was performed according to the following process: 42°C 45 min - 95°C 5 min - 4°C Forever.

[0624] 4. After reverse transcription is complete, replenish each tube with 80 μL of DEPC water (final concentration 10 ng / μL). The samples can then be stored in a refrigerator at 4°C for Taqman Q-PCR or stored in a refrigerator at -40°C.

[0625] IV. In the Taqman probe Q-PCR detection experiment, the experimental procedure was as described in Example 16, and the experimental results are shown in Table 53.

[0626] [Table 53] Example 27 Verification of siRNA on-target activity and off-target levels using psiCHECK. In HEK293A cells, 11 concentration gradients were used to simulate in vitro molecular-level on-target and off-target screening of compounds awaiting measurement. The results showed that the siRNAs disclosed herein have high activity and low off-target properties. The Psi-CHECK plasmids were purchased from Suzhou Hongxun Biotechnology Co., Ltd. and Biotechnology (Shanghai) Co., Ltd. The experimental procedure was as described in Example 16. For the antisense strand of the siRNA, a GSSM-5 hits off-target plasmid was constructed to improve detection sensitivity, i.e., five identical GSSM sequences were ligated together using TTCC.

[0627] As a result, all six siRNAs showed high levels of in vitro on-target inhibitory activity (GSCM IC). 50 The values ​​are smaller than 0.3 nM. The results of the siRNA off-target evaluation (GSSM-5 hits, PSCM, PSSM) show that there are no significant off-target phenomena in the five siRNAs.

[0628] In the HEK293A cell line, the activity of six siRNAs was detected by psi-CHECK activity testing. The experimental results are shown in detail in Tables 54 to 57.

[0629] [Table 54-1] [Table 54-2]

[0630] [Table 55]

[0631] [Table 56]

[0632] [Table 57]

[0633] Example 28: Inhibition of siRNA against human ApoC3 in Hep3B cells - Inhibitory activity at 11 concentration points In Hep3B cells, 11 concentration gradients were used to screen for the activity of compounds awaiting measurement in Hep3B cells. The initial final concentration of each siRNA sample after transfection was 20 nM, and it was diluted with a 3x gradient, resulting in 11 concentration points.

[0634] Hep3B cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Twenty-four hours before transfection, Hep3B cells were inoculated into 96-well plates at an inoculation density of 10,000 cells / well, with 100 μL of medium per well.

[0635] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final transfected concentrations of siRNA were 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM. After 24 hours of processing, total cellular RNA was extracted using a high-throughput cell RNA extraction reagent kit, and RNA reverse transcription and quantitative real-time PCR detection were performed to measure the mRNA level of human ApoC3. The mRNA level of human ApoC3 was then corrected using the ACTIN internal reference gene level.

[0636] The results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 58.

[0637] [Table 58]

[0638] Example 29: Inhibition of human ApoC3 in human primary hepatocytes (PHH) by siRNA - Inhibitory activity at 11 concentration points Human primary hepatocyte (PHH) activity screening was performed on compounds awaiting measurement using 11 concentration gradients in human primary hepatocytes (PHH). The initial final concentration of each siRNA sample after transfection was 20 nM, and it was diluted with a 3x gradient, resulting in 11 concentration points.

[0639] Human primary hepatocytes (PHH) were cryopreserved in liquid nitrogen and resuscitated 24 hours before transfection. They were then inoculated into 96-well plates at an inoculation density of 40,000 cells / well, with 100 μL of culture medium per well.

[0640] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final gradient concentrations of siRNA transfection were 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM. After 24 hours of processing, total cellular RNA was extracted using a high-throughput cell RNA extraction reagent kit, and RNA reverse transcription and quantitative real-time PCR detection were performed to measure the mRNA level of human ApoC3. The mRNA level of human ApoC3 was then corrected using the ACTIN internal reference gene level.

[0641] The results are shown as the excess percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 59, and all of them can effectively suppress human ApoC3 mRNA expression.

[0642] [Table 59]

[0643] Example 30: Inhibition of siRNA against monkey ApoC3 in primary monkey hepatocytes - Inhibitory activity at 11 concentration points In primary monkey hepatocytes, 11 concentration gradients were used to screen for the activity of compounds awaiting measurement in primary monkey hepatocytes. The initial final concentration of each siRNA sample after transfection was 20 nM, and it was diluted with a 3-fold gradient, resulting in 11 concentration points.

[0644] Referring to the product instructions, siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final gradient concentrations of siRNA transfection were 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM. Treatment solutions of the above concentrations were prepared in advance and added to 96-well plates. Primary monkey hepatocytes were cryopreserved in liquid nitrogen, and after resuscitation, they were inoculated into 96-well plates (containing the siRNA samples) at an inoculation density of 30,000 cells / well, with 100 μL of culture medium in each well.

[0645] After 24 hours following reverse transfection, the culture medium was changed and the cells were incubated for another 24 hours. Total cellular RNA was then extracted using a high-throughput cell RNA extraction reagent kit, followed by RNA reverse transcription and quantitative real-time PCR detection. The mRNA levels of monkey ApoC3 were measured, and the mRNA levels of monkey ApoC3 were corrected using the GAPDH internal reference gene level.

[0646] The results are shown as the excess percentage of monkey ApoC3 mRNA expression in cells treated with control siRNA. IC of suppression rate 50 The results are shown in Table 61, and all methods effectively suppress monkey ApoC3 mRNA expression in primary monkey hepatocytes.

[0647] [Table 60]

[0648] [Table 61]

[0649] Example 31: In vivo testing of siRNA reagent in Apoc3 transgenic mice To evaluate and evaluate the in vivo effects of a particular ApoC3 siRNA reagent, we purchased and used ApoC3 transgenic mice (The Jackson Laboratory, 006907-B6, CBA-Tg(APOC3)3707Bres / J). For the ApoC3 transgenic mice, we performed experiments according to the manufacturer's recommendations for the reagent kit (Roche Cobas C311:CHOL2 & TRIGL, MSD Human ApoC3 antibody set (B21ZV-3)) to measure human ApoC3 protein, triglycerides, and total cholesterol in the serum.

[0650] For normalization, the expression ratio "relative to pre-treatment normalization" was measured by dividing the ApoC3 protein, triglycerides, and total cholesterol at each animal's time point by the pre-treatment expression level of that animal.

[0651] ApoC3 protein, triglycerides, and total cholesterol may be measured at time points before and after administration of the ApoC3 siRNA reagent. Unless otherwise specified herein, blood samples were collected from the submandibular region of mice and placed in a centrifuge tube containing heparin sodium. After thoroughly mixing the blood sample with heparin sodium, the plasma was separated by centrifugation at 3,000 × g for 5 minutes and stored at 4°C.

[0652] The above ApoC3 transgenic mouse model was used. On day 0, each mouse received a single subcutaneous dose of either the corresponding siRNA reagent dissolved in PBS (1x) or the control (PBS (1x)) (i.e., the Vehicle group), which included the dose groups shown in Table 62 below.

[0653] [Table 62] ApoC3 siRNA reagent was injected between the skin and muscle (i.e., subcutaneously). Six mice per group (n=6) were tested. Serum was collected on day 2 (blood was collected before administration if the mice were fasted overnight), and on days 7, 14, 21, 28, 35, and 42. Mice were fasted overnight before each collection. Serum ApoC3 protein, triglycerides, and total cholesterol were measured using instruments according to the reagent manufacturer's recommendations.

[0654] The ApoC3 protein level, triglyceride level, and total cholesterol level were normalized for each animal. Normalization was performed by dividing the ApoC3 protein, triglyceride, and total cholesterol levels at each animal's time point by the pre-treatment expression level in that animal (in this case, on day -2), thereby measuring the expression ratio "relative to pre-treatment normalization."

[0655] The experimental data are shown in Tables 63-65 and Figures 7-9 below. Each of the ApoC3 siRNA reagents in each treatment group (i.e., Groups 2-6) showed a significant reduction in ApoC3 protein levels, triglyceride levels, and total cholesterol levels compared to the control group (Group 1).

[0656] [Table 63]

[0657] [Table 64]

[0658] [Table 65]

[0659] Example 32: Evaluation of different modifications at positions 9 and 10 of the AS chain. This experiment investigated the repressive efficiency of the siRNA complexes modified with different site-2'-fluoros in this disclosure on the mRNA expression levels of target genes in vivo.

[0660] Male C57BL / 6 mice aged 6 to 8 weeks were randomly divided into groups of 6 mice each, with 3 mice at each time point. Each group of mice was administered the test conjugate (TRD007047 and TRD006870), the comparison conjugate (TRD002218), and PBS, respectively.

[0661] For all animals, the dosage was calculated based on body weight and administered as a single subcutaneous injection. The siRNA complex dose (amount of siRNA) was 1 mg / kg, and the administration volume was 5 mL / kg. Seven days after administration, the mice were sacrificed, their livers were collected and stored in RNA Later (Sigma Aldrich). Subsequently, the liver tissue was homogenized using a tissue homogenizer, and then total RNA from the liver tissue was extracted using a tissue RNA extraction reagent kit (Fanzhi Medical Technology, FG0412) according to the operating steps described in the instruction manual. The total RNA was reverse transcribed into cDNA, and the expression level of TTR mRNA in the liver tissue was detected by real-time fluorescence quantitative PCR. In this fluorescence quantitative PCR, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and the mRNA expression levels of TTR and GAPDH were detected using Taqman probe primers for TTR and GAPDH, respectively.

[0662] TTR mRNA expression levels were calculated using the following formula.

[0663] TTR mRNA expression level = {(Test group TTR mRNA expression level / Test group GAPDH mRNA expression level) / (Control group TTR mRNA expression level / Control group GAPDH mRNA expression level)} × 100% Compound information is shown in Table 66, grouping information for compounds awaiting measurement in mice is shown in Table 67, and the sequences of the detection primers are shown in Table 68.

[0664] [Table 66]

[0665] [Table 67]

[0666] [Table 68] Table 69 shows the inhibitory efficiency of siRNA complexes modified at different site F of this disclosure on target gene mRNA expression levels in the body 28 days after administration. Referring to the positive control compound TRD002218, siRNA compounds modified at different site F showed higher inhibition of TTR mRNA expression 28 days after administration than the reference positive compound. Both the 9F and 10F modification methods showed high inhibitory efficiency with no significant difference, indicating that the 9F and 10F modification methods can mediate more effective siRNA inhibitory efficiency.

[0667] [Table 69]

Claims

1. The molecule comprises a sense strand and an antisense strand, each strand having 15 to 35 nucleotides, wherein the antisense strand includes a chemical modification represented by formula (I) or a tautomer thereof at at least one nucleotide position between positions 2 and 8 of its 5' region. Here, the chemical modification represented by formula (I) is given by the following formula: 【Chemistry 1】 It is one of the options selected from this structure, Of these, B is a base. siRNA.

2. The siRNA according to claim 1, wherein the antisense strand includes a chemical modification represented by formula (I) as defined in claim 1 or a tautomer modification thereof at the nucleotide position 7 of its 5' region.

3. In addition to the nucleotides comprising the chemical modification represented by formula (I) or its tautomer modification as defined in claim 1, the remaining nucleotides in the sense strand and / or antisense strand are other modified nucleotides. The siRNA according to claim 1, wherein the other modified nucleotides are independently selected from nucleotides modified with a 2'-methoxy group, nucleotides modified with a 2'-fluoro group, and nucleotides modified with a 2'-deoxy group.

4. From the 5' end to the 3' end, the nucleotides at positions 2, 4, 6, 9, 12, 14, 16 and 18 of the antisense strand are each independently modified with 2'-fluoropolymers. From the 5' end to the 3' end, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand are each independently modified with 2'-fluoropolymers. The siRNA according to claim 1.

5. The aforementioned sense chain, 5'-N ａ N ａ N ａ N ａ XN ａ N ｂ N ｂ N ｂ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ having a nucleotide sequence represented by the formula -3' Among them, each X independently becomes N ａ or N ｂ And each N ａ and N ｂ However, each independently represents a modified nucleotide or an unmodified nucleotide, and N ａ and N ｂ The modifications in are different, and / or, The aforementioned antisense chain, 5'-N ａ 'N ｂ 'N ａ 'X'N ａ 'N ｂ 'W'N ａ 'X'Y'N ａ 'X'N ａ 'N ｂ 'N ａ 'X'N ａ 'X'N ａ 'N ａ 'N ａ It has a nucleotide sequence represented by the formula '-3', Of these, each X' independently becomes N ａ 'or N ｂ ' is N ａ 'or N ｂ ' and each N ａ 'and N ｂ ' indicates independently modified nucleotides or unmodified nucleotides, among which N ａ 'and N ｂ The modifications in ' are different, and W' represents a nucleotide comprising a chemical modification represented by formula (I) or a tautomer thereof as defined in claim 1, N ａ This is a nucleotide modified with a 2'-methoxy group, N ｂ is a nucleotide modified with 2'-fluoro, and / or N ａ ' is a nucleotide modified with a 2'-methoxy group, N ｂ ' is a nucleotide modified with 2'-fluoro, The siRNA according to claim 1.

6. The aforementioned antisense chain, 5'-N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'W'N ａ 'X'Y'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ａ 'N ａ It has a nucleotide sequence represented by the formula '-3', Of these, each X' independently becomes N ａ 'or N ｂ ' is N ａ 'or N ｂ 'and, N ａ ' is a nucleotide modified with a 2'-methoxy group, N ｂ ' is a nucleotide modified with 2'-fluoro, W' is as defined in claim 5, The siRNA according to claim 5.

7. The aforementioned sense chain, 5'-N ａ N ａ N ａ N ａ N ａ N ａ N ｂ N ｂ N ｂ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ -3', or, 5'-N ａ N ａ N ａ N ａ N ｂ N ａ N ｂ N ｂ N ｂ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ It has a nucleotide sequence represented by the formula -3'. Eventually, N ａ This is a nucleotide modified with a 2'-methoxy group, N ｂ This is a nucleotide modified with 2'-fluoro. The siRNA according to claim 5.

8. The aforementioned antisense chain, 5'-N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'W'N ａ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ａ 'N ａ '-3', or, 5'-N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'W'N ａ 'N ｂ 'N ａ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ａ 'N ａ It has a nucleotide sequence represented by the formula '-3', Eventually, N ａ ' is a nucleotide modified with a 2'-methoxy group, N ｂ ' is a nucleotide modified with 2'-fluoro, W' is, 【Chemistry 2】 This shows nucleotides containing chemical modifications or tautomer modifications selected from the structure, Of these, B is the base at the position corresponding to the 7th position in the 5' region of the antisense strand. The siRNA according to claim 6.

9. The siRNA according to any one of claims 1 to 8, wherein at least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a thiophosphate group.

10. The siRNA described in any one of claims 1 to 9, The complex group linked to the siRNA comprises, The aforementioned complex group comprises a pharmaceutically acceptable target ligand and a linker. The target ligand comprises a galactose cluster or a galactose derivative cluster, wherein the galactose derivative is selected from N-acetyl-galactoseamine, N-trifluoroacetyl-galactoseamine, N-propionyl-galactoseamine, N-n-butyryl-galactoseamine, or N-isobutyryl-galactoseamine. siRNA complex.

11. siRNA according to any one of claims 1 to 9, or The siRNA complex according to claim 10, Pharmaceutical composition.

12. This includes administering an effective amount or dose of siRNA, siRNA complex, and / or pharmaceutical composition to a subject requiring it. A siRNA according to any one of claims 1 to 9, an siRNA complex according to claim 10, or a pharmaceutical composition according to claim 11, for use in suppressing the expression of a target gene or its mRNA.

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